WO2022238381A2 - Constructions d'immunothérapie pour le traitement d'une maladie - Google Patents

Constructions d'immunothérapie pour le traitement d'une maladie Download PDF

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WO2022238381A2
WO2022238381A2 PCT/EP2022/062600 EP2022062600W WO2022238381A2 WO 2022238381 A2 WO2022238381 A2 WO 2022238381A2 EP 2022062600 W EP2022062600 W EP 2022062600W WO 2022238381 A2 WO2022238381 A2 WO 2022238381A2
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unit
seq
cancer
construct
targeting
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PCT/EP2022/062600
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WO2022238381A3 (fr
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Agnete Brunsvik Fredriksen
Audun Trygge Haugen BERSAAS
Stine GRANUM
Pierre DILLARD
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Nykode Therapeutics ASA
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Publication of WO2022238381A3 publication Critical patent/WO2022238381A3/fr

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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/0005—Vertebrate antigens
    • A61K39/0011—Cancer antigens
    • 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
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/385—Haptens or antigens, bound to carriers
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • 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
    • A61K2039/58—Medicinal preparations containing antigens or antibodies raising an immune response against a target which is not the antigen used for immunisation
    • A61K2039/585—Medicinal preparations containing antigens or antibodies raising an immune response against a target which is not the antigen used for immunisation wherein the target is cancer
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/60—Medicinal preparations containing antigens or antibodies characteristics by the carrier linked to the antigen
    • A61K2039/6031—Proteins
    • 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
    • A61K2039/64—Medicinal preparations containing antigens or antibodies characterised by the architecture of the carrier-antigen complex, e.g. repetition of carrier-antigen units
    • 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
    • C12N2710/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA dsDNA viruses
    • C12N2710/00011—Details
    • C12N2710/20011—Papillomaviridae
    • C12N2710/20034—Use of virus or viral component as vaccine, e.g. live-attenuated or inactivated virus, VLP, viral protein

Definitions

  • the present disclosure relates to constructs for immunotherapy useful in the treatment of cancer or prevention and/or treatment of infectious diseases.
  • Both B cell (humoral/antibody mediated) and system cell responses are important components of protective responses against infections 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) inducing 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.
  • 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 requires additional support from T-helper (Th1 and Th2) lymphocytes.
  • Cytotoxic T lymphocytes also play a significant role (F. Sheperd et al., Int J Mol Sci 21, 2020, 6144) with e.g. intracellular pathogens where MHC class l-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 (Th1) and Th2 cells.
  • Th1 and Th2 cells After binding specific T cell epitopes on the surface of antigen-presenting cells (APCs), Th1 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.
  • a vaccine may provide sufficient protection against infection with 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.
  • cancer immune therapies targeting cancer cells with the help of the patient's own immune system i.e. cancer vaccines, have attracted interest because such therapies may reduce or even eliminate some of the side-effects seen in the traditional cancer treatment.
  • the foundation of immunology is based on self/non-self discrimination. Most of the pathogens inducing infectious diseases contain molecular signatures that can be recognized by the host and trigger immune responses. However tumor cells are derived from normal cells, and do not generally express any molecular signatures, making them more difficult to be distinguished from normal cells.
  • tumor antigens a class of tumor antigens
  • tumor associated antigens i.e. antigens expressed at low levels in normal tissues and expressed at a much higher level in tumor tissue.
  • tumor-associated antigens have been the target for cancer vaccines for the last decade.
  • Tumor neoantigens arise due to one or more mutations in the tumor genome leading to a change in the amino acid sequence of the protein in question.
  • the present inventors have realized that by targeting at least one T cell epitope to antigen presenting cells (APCs) such as dendritic cells, the APCs are then able to mediate immune responses against these T cell epitopes.
  • APCs antigen presenting cells
  • the inventors have devised a construct with a targeting unit, a first joint region, an antigenic unit comprising at least one T cell epitope, a second joint region and a second targeting unit.
  • the construct will be targeted and taken up by APCs, processed, and the T cell epitopes will be presented to the immune system. This induces immune responses in T cells recognizing the displayed epitopes.
  • the construct will activate T cells, which will recognize and target cells expressing such cancer antigens or pathogens comprising such surface antigens.
  • the construct will thus be of use in the treatment of cancer or infectious diseases caused by pathogens.
  • the construct of the disclosure may be administered to a subject in the form of a polynucleotide (e.g. a DNA plasmid) comprising a nucleotide sequence encoding a polypeptide.
  • a polynucleotide e.g. a DNA plasmid
  • the polypeptide is expressed and multiple polypeptides, for example two polypeptides, form a multimeric protein, for example a dimeric protein, by being linked via their joint regions in the manner described below in more detail.
  • the multimeric protein comprises multiple targeting units, it is a versatile protein; in the following the term “multimeric protein” is used for such a protein.
  • a dimeric protein comprises two targeting units, and is a dual-targeting dimeric protein.
  • the term “dimeric protein” is used for such a protein.
  • the present disclosure provides an immunotherapy construct comprising: i) a polynucleotide comprising a nucleotide sequence encoding an immunomodulating polypeptide, the immunomodulating polypeptide comprising, in the specified order: a. a first targeting unit, a first joint region; b. an antigenic unit comprising at least one T cell epitope; c. a second joint region; and d.
  • a second targeting unit ii) an immunomodulating polypeptide encoded by the nucleotide sequence as defined in i); or iii) a multimeric protein consisting of multiple immunomodulating polypeptides as defined in ii), such as a dimeric protein consisting of two polypeptides as defined in ii).
  • an immunotherapy construct comprising: i) a polynucleotide comprising a nucleotide sequence encoding an immunomodulating polypeptide, the immunomodulating polypeptide comprising, in the specified order: a. a first targeting unit, a first joint region; b. an antigenic unit comprising at least one T cell epitope; c. a second joint region; and d. a second targeting unit; ii) an immunomodulating polypeptide encoded by the nucleotide sequence as defined in i); or iii) a multimeric protein consisting of multiple immunomodulating polypeptides as defined in ii).
  • an immunotherapy construct comprising: i) a polynucleotide comprising a nucleotide sequence encoding an immunomodulating polypeptide, the immunomodulating polypeptide comprising, in the specified order: a. a first targeting unit, a first joint region; b. an antigenic unit comprising at least one T cell epitope; c. a second joint region; and d. a second targeting unit; ii) an immunomodulating polypeptide encoded by the nucleotide sequence as defined in i); or iii) a dimeric protein consisting of two immunomodulating polypeptides as defined in ii).
  • immunomodulating refers to the modulation of the immune system, i.e. the stimulation of the immune system.
  • the immune response may be induced or amplified.
  • the present disclosure provides a pharmaceutical composition
  • a pharmaceutical composition comprising the polynucleotide, the immunomodulating polypeptide or the multimeric protein, for example the dimeric protein, as described herein, and a pharmaceutically acceptable carrier.
  • composition comprising the polynucleotide, the immunomodulating polypeptide or the multimeric protein as described herein, and a pharmaceutically acceptable carrier.
  • a pharmaceutical composition comprising the polynucleotide, the immunomodulating polypeptide or the dimeric protein as described herein, and a pharmaceutically acceptable carrier.
  • the present disclosure provides a method of preparing a pharmaceutical composition, said method comprising: a) providing a polynucleotide, an immunomodulating polypeptide or a multimeric protein, for example the dimeric protein, as described herein; and b) combining the polynucleotide, the immunomodulating polypeptide or the multimeric protein with a pharmaceutically acceptable carrier.
  • Also disclosed herein is a method of preparing a pharmaceutical composition, said method comprising: a) providing a polynucleotide, an immunomodulating polypeptide or a multimeric protein as described herein; and b) combining the polynucleotide, the immunomodulating polypeptide or the multimeric protein with a pharmaceutically acceptable carrier.
  • Also disclosed herein is a method of preparing a pharmaceutical composition, said method comprising: a) providing a polynucleotide, an immunomodulating polypeptide or a dimeric protein as described herein; and b) combining the polynucleotide, the immunomodulating polypeptide or the dimeric protein with a pharmaceutically acceptable carrier.
  • the present disclosure provides a pharmaceutical composition as described herein for use as a medicament.
  • the present disclosure provides a pharmaceutical composition as described herein for use in the treatment of disease, such as for use in the treatment of cancer or prevention and/or treatment of an infectious disease.
  • the present disclosure provides a vector comprising the polynucleotide as described herein.
  • the present disclosure provides a host cell comprising the vector as described herein or comprising the polynucleotide, the immunomodulating polypeptide or the multimeric protein, such as a dimeric protein, as described herein. Also disclosed is a host cell comprising the vector as described herein or comprising the polynucleotide, the immunomodulating polypeptide or the multimeric protein as described herein.
  • Also disclosed is a host cell comprising the vector as described herein or comprising the polynucleotide, the immunomodulating polypeptide or the dimeric protein, as described herein.
  • the present disclosure provides a method of preparing an immunomodulating polypeptide or a multimeric protein, for example a dimeric protein, said method comprising: a) transfecting or transducing a cell with the vector as described herein or the polynucleotide as described herein; b) culturing the cell, c) collecting and optionally purifying the multimeric protein and/or the immunomodulating polypeptide expressed by the cell.
  • Also disclosed herein is a method of preparing an immunomodulating polypeptide or a multimeric protein, said method comprising: a) transfecting or transducing a cell with the vector as described herein or the polynucleotide as described herein; b) culturing the cell, c) collecting and optionally purifying the multimeric protein and/or the immunomodulating polypeptide expressed by the cell.
  • Also disclosed herein is a method of preparing an immunomodulating polypeptide or a dimeric protein, said method comprising: a) transfecting or transducing a cell with the vector as described herein or the polynucleotide as described herein; b) culturing the cell, c) collecting and optionally purifying the dimeric protein and/or the immunomodulating polypeptide expressed by the cell.
  • the present disclosure provides a method for treating cancer or an infectious disease, said method comprising administering the polynucleotide, the immunomodulating polypeptide or the multimeric protein, for example a dimeric protein, as described herein, the vector as described herein, the host cell as described herein, or the pharmaceutical composition as described herein, to a subject in need thereof.
  • Also disclosed is a method for treating cancer or an infectious disease comprising administering the polynucleotide, the immunomodulating polypeptide or the multimeric protein as described herein, the vector as described herein, the host cell as described herein, or the pharmaceutical composition as described herein, to a subject in need thereof.
  • Also disclosed is a method for treating cancer or an infectious disease comprising administering the polynucleotide, the immunomodulating polypeptide or the dimeric protein, as described herein, the vector as described herein, the host cell as described herein, or the pharmaceutical composition as described herein, to a subject in need thereof.
  • the figure in the top illustrates the construct on the basis of the polypeptide.
  • the lower part of the figure shows an embodiment of a dimeric protein formed by two polypeptides linked via their respective first and second joint regions.
  • A shows a first targeting unit B shows a second targeting unit
  • C shows an antigenic unit comprising at least one T cell epitope D illustrates the flexibility rendered to the targeting unit due to the presence of the flexible unit
  • A.A shows a first joint region
  • B.A shows a second joint region.
  • the figure shows an embodiment of the joint region.
  • A shows three covalent bonds that are formed between the covalent binding units comprised in each of the two polypeptide chains.
  • FIG. B shows how the flexible unit is located between the binding unit and the targeting unit, providing flexibility to the targeting unit, as shown by arrow D in figure 1.
  • the figure shows another embodiment of the joint region.
  • A shows the dimerization of the two polypeptide chains by hydrophobic interactions between the non-covalent binding units comprised in each of the polypeptides.
  • FIG. B shows how the flexible unit is located between the binding unit and the targeting unit, providing flexibility to the targeting, as shown by arrow D in figure 1.
  • Expression and secretion levels of proteins encoded by a DNA plasmid Shows the protein expression and secretion levels of the polypeptides encoded by DNA plasmids VB4217, TECH002-CV009 and TECH002-CV002 detected in the supernatant of Expi293F cells transfected with said DNA plasmids by the enzyme- linked immunosorbent assay (ELISA) using rat anti-mouse GM-CSF capture antibody (MAB415), and goat anti-human CCL3/MIP-1 alpha biotinylated detection antibody (BAF270).
  • ELISA enzyme- linked immunosorbent assay
  • MAB415 rat anti-mouse GM-CSF capture antibody
  • BAF270 goat anti-human CCL3/MIP-1 alpha biotinylated detection antibody
  • an embodiment of the present disclosure relates to an immunotherapy construct comprising: i) a polynucleotide comprising a nucleotide sequence encoding an immunomodulating polypeptide, the immunomodulating polypeptide comprising, in the specified order: a. a first targeting unit, a first joint region; b. an antigenic unit comprising at least one T cell epitope; c. a second joint region; and d.
  • a second targeting unit ii) an immunomodulating polypeptide encoded by the nucleotide sequence as defined in i); or iii) a multimeric protein, such as a dimeric protein, consisting of multiple polypeptides as defined in ii), such as of two polypeptides as defined in ii).
  • the present disclosure relates to an immunotherapy construct comprising: i) a polynucleotide comprising a nucleotide sequence encoding an immunomodulating polypeptide, the immunomodulating polypeptide comprising, in the specified order: a. a first targeting unit, a first joint region; b. an antigenic unit comprising at least one T cell epitope; c. a second joint region; and d. a second targeting unit; ii) an immunomodulating polypeptide encoded by the nucleotide sequence as defined in i); or iii) a multimeric protein consisting of multiple polypeptides as defined in ii).
  • the present disclosure relates to an immunotherapy construct comprising: i) a polynucleotide comprising a nucleotide sequence encoding an immunomodulating polypeptide, the immunomodulating polypeptide comprising, in the specified order: a. a first targeting unit, a first joint region; b. an antigenic unit comprising at least one T cell epitope; c. a second joint region; and d. a second targeting unit; ii) an immunomodulating polypeptide encoded by the nucleotide sequence as defined in i); or iii) a dimeric protein consisting of two polypeptides as defined in ii).
  • an “immunotherapy 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, preferably a human.
  • a subject may be a patient, i.e. a human suffering from an infectious disease or a cancer and who is in need of a treatment, or it may be a subject in need of prevention from being infected with an infectious disease or developing a cancer, or it may be a subject suspected of suffering from an infectious disease or cancer.
  • the terms “subject” and “individual” are used interchangeably herein.
  • a “cancer” refers to a broad group of various diseases characterized by the uncontrolled growth of abnormal cells in the body.
  • a “cancer” or “cancer tissue” includes a tumor, and as used herein, encompasses both a solid tumor as well as tumor cells found in a bodily fluid such as blood, and includes metastatic cancer. Unregulated cell division and growth results in the formation of malignant tumors that can invade neighboring tissues and can also metastasize to distant parts of the body through the lymphatic system or bloodstream. Following metastasis, the distal tumors can be said to be "derived from” a pre-metastasis tumor.
  • cancer antigen includes neoantigens, patient-present shared cancer antigens or shared cancer antigens, as described 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 a therapeutic treatment.
  • a prophylactic treatment is a treatment administered to a subject who does not (or not yet) display signs or symptoms of, or displays only early signs or symptoms of, a cancer or an infectious disease, such that treatment is administered for the purpose of preventing or decreasing the risk of developing the cancer or the infectious disease and/or symptoms associated with these.
  • a prophylactic treatment functions as a preventative treatment against a cancer or an infectious disease, or as a treatment that inhibits or reduces further development or enhancement of the disease and/or its associated symptoms.
  • 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 or a cancer, in which treatment is administered to the subject for the purpose of diminishing or eliminating those signs or symptoms.
  • a “part” of an antigen is a fragment or portion of an antigen; preferably, the part or fragment of the antigen is immunogenic. These terms will be used throughout interchangeably.
  • 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 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, dimeric protein or multimeric protein as amino acid sequences.
  • the units of the construct are mainly explained in relation to the polypeptide/dimeric protein/multimeric protein, i.e. on the basis of their amino acid sequences.
  • the immunomodulating polypeptide 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 immunomodulating polypeptides are linked to each other via their joint regions. In some embodiments, the multimeric protein of the disclosure is one where the multiple polypeptides are linked to each other via their joint regions. In some embodiments, the multimeric protein is a dimeric protein where the two polypeptides are linked to each other via their joint regions.
  • the multimeric protein of the disclosure is a dimeric protein where the immunomodulating 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 antigenic unit and the targeting unit. Any amino acid sequence that is capable of joining the multiple polypeptides (for embodiments relating to a multimeric protein), for example capable of joining the two polypeptides, but at the same time providing flexibility and appropriate protein conformation to the multimeric/dimeric protein is a suitable joint region.
  • the joint regions provide flexibility to the multimeric/dimeric protein, such that 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.
  • the joint regions join the multiple monomeric polypeptides into a multimeric protein.
  • the joint regions join the two monomeric polypeptides into a dimeric protein. Any amino acid sequence that fulfils any one or more of these requirements is a suitable joint region.
  • the joint region comprises a flexible unit which provides flexibility and a binding unit which joins the multiple polypeptides to form a multimer, for example which joins the two polypeptides to form a dimer.
  • the joint region comprises 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 comprised in the joint region is closest to the targeting unit and the binding unit is closest to the antigenic unit.
  • 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 multiple molecules, for example the two 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 multiple molecules, for example the two 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.
  • two polypeptide molecules are linked to each other via their respective first joint regions and via their respective second joint regions, forming a dimeric protein.
  • first joint region and the second joint region are the same.
  • the binding unit comprised in the first joint region of one polypeptide molecule is able to bind to the binding unit comprised in either the first joint region or the second joint region of another polypeptide molecule. The same applies to the binding unit comprised in the second joint region.
  • first and second joint regions are the same, the first and second targeting units either are different, but bind to the same surface molecule on the APCs, or the first and second targeting units are identical.
  • the amino acid sequence of the first and/or second joint regions comprises at least one naturally occurring sequence or consists of a naturally occurring sequence. In some embodiments, the amino acid sequence of the first and/or second joint region comprises at least one artificial sequence or consists of an artificial sequence.
  • the binding unit is a covalent binding unit, in other embodiments, the binding unit is a non-covalent binding unit.
  • the amino acid sequence of one or both joint regions is a non- immunogenic sequence, preferably, the sequences of both joint regions are non- immunogenic sequences.
  • the joint regions comprise a flexible unit and a binding unit.
  • the flexible unit is between the targeting unit and the binding unit.
  • FIG. 1 The structure of the construct is illustrated in figures 1-3 on the basis of the polypeptide and an embodiment of a dimeric protein, formed by two polypeptides linked via their respective first and second joint regions.
  • the polypeptide (figure 1, top) comprises, in the specified order, a first targeting unit (A), a first joint region (A. A), an antigenic unit (C), a second joint region (B.A) and a second targeting unit (B).
  • Figure 1 shows how the flexible unit comprised in the second joint region provides flexibility to the second targeting unit (arrow D).
  • Embodiments of the joint region comprised in the dimeric protein are illustrated in figures 2 and 3.
  • joint region illustrated in figure 2 comprises a flexible unit (B) closest to the targeting unit and a covalent binding unit adjacent to it, which is closest to the antigenic unit.
  • the covalent binding unit of figure 2 shows three covalent bonds (A) that are formed between the two polypeptide chains.
  • FIG 3 An embodiment of a joint region with a non-covalent binding unit is illustrated in figure 3.
  • the joint region illustrated in figure 3 (joint region 1 or joint region 2) comprises a flexible unit (B) closest to the targeting unit and a non-covalent binding unit adjacent to it, which is closest to the antigenic unit.
  • the non-covalent binding unit of figure 3 facilitates the dimerization of the two polypeptide chains by, for example, hydrophobic interactions (A).
  • the amino acid sequence of the flexible unit is a non- immunogenic sequence. In some embodiments, the amino acid sequence of the flexible unit is a naturally occurring peptide sequence. In some embodiments, the flexible unit is derived from an immunoglobulin. In some embodiments, the flexible unit is a hinge region of an immunoglobulin, which hinge region does not comprise cysteine residues.
  • the amino acid sequence of 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.
  • 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 flexible unit in aqueous solutions by forming hydrogen bonds with the water molecules, and therefore reduces the unfavorable interaction between the flexible unit and antigens.
  • the flexible unit is an artificial sequence, e. g a serine (S) and/or glycine (G) rich linker, i.e. a linker comprising several serine and/or several glycine residues.
  • S serine
  • G glycine
  • GGGGS SEQ ID NO: 7
  • GGGSS SEQ ID NO: 8
  • GGGSG SEQ ID NO: 9
  • GGGGS SEQ ID NO: 7
  • SGSSGS SEQ ID NO: 70
  • GGGGSGGGGS SEQ ID NO: 10
  • GGGGS GGGSm
  • GGGSS GGGGSm
  • SEQ ID NO: 72 GGGGSG
  • GGGSGG SEQ ID NO: 74
  • SGSSGS SEQ ID NO: 75
  • m is an integer from 1 to 5, e.g., 1, 2, 3, 4, or 5, In preferred embodiments, m is 2.
  • 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: 11), GLGGS (SEQ ID NO: 12), GGLGS (SEQ ID NO: 13), GGGLS (SEQ ID NO: 14) or GGGGL (SEQ ID NO: 15).
  • the flexible unit comprises or consists LGGSG (SEQ ID NO: 16), GLGSG (SEQ ID NO: 17), GGLSG (SEQ ID NO: 18), GGGLG (SEQ ID NO: 19) or GGGSL (SEQ ID NO: 20).
  • the flexible unit comprises or consists of LGGSS (SEQ ID NO: 21), GLGSS (SEQ ID NO: 22) or GGLSS (SEQ ID NO: 23).
  • the flexible unit comprises or consists of LGLGS (SEQ ID NO: 24), GLGLS (SEQ ID NO: 25), GLLGS (SEQ ID NO: 26), LGGLS (SEQ ID NO: 27) or GLGGL (SEQ ID NO: 28).
  • the flexible unit comprises or consists of LGLSG (SEQ ID NO: 29), GLLSG (SEQ ID NO: 30), GGLSL (SEQ ID NO: 31), GGLLG (SEQ ID NO: 32) or GLGSL (SEQ ID NO: 33).
  • the flexible unit comprises or consists of LGLSS (SEQ ID NO: 34), or GGLLS (SEQ ID NO: 35).
  • the flexible unit is a 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: 36), GLGGSGGGGS (SEQ ID NO: 37), GGLGSGGGGS (SEQ ID NO: 38), GGGLSGGGGS (SEQ ID NO: 39) or GGGGLGGGGS (SEQ ID NO: 40).
  • the flexible unit comprises or consists of LGGSGGGGSG (SEQ ID NO: 41), GLGSGGGGSG (SEQ ID NO: 42), GGLSGGGGSG (SEQ ID NO: 43), GGGLGGGGSG (SEQ ID NO: 44) or GGGSLGGGSG (SEQ ID NO: 45).
  • the flexible unit comprises or consists of LGGSSGGGSS (SEQ ID NO: 46), GLGSSGGGSS (SEQ ID NO: 47), GGLSSGGGSS (SEQ ID NO: 48), GGGLSGGGSS (SEQ ID NO: 49) or GGGSLGGGSS (SEQ ID NO: 50).
  • the flexible unit comprises or consists of LGGGSLGGGS (SEQ ID NO: 51), GLGGSGLGGS (SEQ ID NO: 52), GGLGSGGLGS (SEQ ID NO: 53), GGGLSGGGLS (SEQ ID NO: 54) or GGGGLGGGGL (SEQ ID NO: 55).
  • the flexible unit comprises or consists of LGGSGLGGSG (SEQ ID NO: 56), GLGSGGLGSG (SEQ ID NO: 57), GGLSGGGLSG (SEQ ID NO: 58), GGGLGGGGLG (SEQ ID NO: 59) or GGGSLGGGSL (SEQ ID NO: 60).
  • the flexible unit comprises or consists of LGGSSLGGSS (SEQ ID NO: 61), GLGSSGLGSS (SEQ ID NO: 62) or GGLSSGGLSS (SEQ ID NO: 63).
  • the flexible unit comprises or consists of GSGGGA (SEQ ID NO: 76), GSGGGAGSGGGA (SEQ ID NO: 77), GSGGGAGSGGGAGSGGGA (SEQ ID NO: 78), GSGGGAGSGGGAGSGGGAGSGGGA (SEQ ID NO: 79) or GENLYFQSGG (SEQ ID NO: 80).
  • the flexible unit comprises or consists of SGGGSSGGGS (SEQ ID NO: 81), GGGGSGGGGS (SEQ ID NO: 82),
  • GSGSGSGSGSGSGS (SEQID NO: 85), GGGSSGGGSG (SEQ ID NO: 86; corresponding to amino acids 121-130 of SEQ ID NO: 1), GGGSSS (SEQ ID NO: 87), GGGSSGGGSSGGGSS (SEQ ID NO: 88) or GLGGLAAA (SEQ ID NO: 89).
  • the flexible unit comprises or consists of the sequence TQKSLSLSPGKGLGGL (SEQ ID NO: 64). In other embodiments, the flexible unit comprises or consists of the sequence SLSLSPGKGLGGL (SEQ ID NO: 65). In other embodiments, the flexible unit comprises or consists of AAY or GPGPG (SEQ ID NO: 112).
  • 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: 67) 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
  • the flexible unit is not a target of proteases.
  • the flexible unit consists of up to 20 amino acids, such as at up to 15 amino acids, such as 14 amino acids, such as 13 amino acids, such as 12 amino acids such as 11 amino acids or such as 10 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 is hinge exon hi of lgG3.
  • 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 an amino acid sequence 16-23 of SEQ ID NO: 4, 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: 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 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 is the lower hinge region of lgG1.
  • the flexible unit comprises or consists of the amino acid sequence 16-23 of SEQ ID NO: 4.
  • the joint region as described herein 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 of the respective first and second joint regions.
  • the covalent binding unit consists of or comprises a cysteine rich sequence. In some embodiments, 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 the 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: 114; corresponding to amino acids 106-120 of SEQ ID NO: 1).
  • the amino acid sequence of the covalent binding unit is or comprises a non-immunogenic sequence.
  • the amino acid sequence of the covalent binding unit is an artificial sequence.
  • the amino acid sequence of the covalent binding unit is a naturally occurring peptide 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,
  • the covalent binding unit consists of 15 amino acids. In more 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 lgG3 or the middle hinge region of lgG1.
  • the hinge region may be Ig derived, such as derived from IgG, e.g. lgG2 or lgG3.
  • the hinge region is derived from IgM, e.g. comprising or consisting of the nucleotide sequence with SEQ ID NO: 97 or an amino acid sequence encoded by said nucleic acid 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 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 or such as no more than 1 amino acid.
  • the covalent binding unit comprises or is hinge exon h4 of lgG3.
  • the covalent binding unit 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: 4, 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: 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 5 amino acids have been so altered, such as no more than 4 amino acids, such as no more than 3 amino acids, such as no more than 2 amino acids or such as no more than 1 amino acid.
  • the covalent binding unit is the middle hinge region of lgG1.
  • the covalent binding unit consists of or comprises the amino acid sequence 5-15 of SEQ ID NO: 4. In other embodiments, the covalent binding unit consists of or comprises the amino acid sequence of SEQ ID NO: 118.
  • the joint region as described herein comprises a non-covalent binding unit.
  • the non-covalent binding unit contributes to multimerization, such as to dimerization, through non-covalent interactions, e.g. hydrophobic interactions. In some embodiments, the non-covalent binding unit has the ability to form multimers, such as dimers, via non-covalent interactions. In some embodiments, the non-covalent binding unit contributes to multimerization through non-covalent interactions, e.g. hydrophobic interactions. In some embodiments, the non-covalent binding unit has the ability to form multimeric proteins via non-covalent interactions. In other embodiments, the non- covalent binding unit contributes to dimerization through non-covalent interactions, e.g. hydrophobic interactions. In some embodiments, the non-covalent binding unit has the ability to form dimeric proteins 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 amino acid sequence of the non-covalent binding unit is an artificial sequence.
  • the non-covalent binding unit is or comprises an immunoglobulin domain, such as an immunoglobulin constant domain (C domain), such as a carboxyterminal C domain (i.e. a CH3 domain), a CH1 domain or a CH2 domain, or a sequence that is substantially identical to the C domain or a variant thereof.
  • the non-covalent binding unit is a CH3 domain derived from IgG, such as derived from lgG3 or lgG1, preferably derived from lgG1.
  • 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 lgG3 with an amino acid sequence having at least 80 % sequence identity to the amino acid sequence 131-237 of SEQ ID NO: 1.
  • the non-covalent binding unit comprises or consists of a CH3 domain derived from lgG3 with an amino acid sequence having at least 85% sequence identity to the amino acid sequence 131-237 of SEQ ID NO: 1, 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 derived from lgG3 with the amino acid sequence 131-237 of SEQ ID NO: 1.
  • the non-covalent binding unit comprises or consists of a carboxyterminal C domain derived from lgG3 with the amino acid sequence 131-237 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 21 amino acids have been so altered, 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
  • the non-covalent binding unit comprises or consists of a CH3 domain derived from lgG1 with an amino acid sequence having at least 80 % sequence identity to the amino acid sequence of SEQ ID NO: 6.
  • the non-covalent binding unit comprises or consists of a CH3 domain from lgG1 with an amino acid sequence having at least 85% sequence identity to the amino acid sequence according to SEQ ID NO: 6, 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 lgG1 with the amino acid sequence of SEQ ID NO: 6, wherein any one of the amino acids of the flexible unit has been substituted, deleted or inserted, with the proviso that no more than 21 amino acids have been so altered, 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
  • the non-covalent binding unit is or comprises CH3 of lgG1, such as comprising or consisting of the amino acid sequence according SEQ ID NO: 6.
  • 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 protein is 60 to 80 amino acids in length with a highly conserved DNA binding basic region in the N-terminal part and a more diversified leucine zipper dimerization region in the C-terminal part.
  • 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 Jun/Fos-based leucine zipper.
  • the non-covalent binding unit is or comprises a ATF6-based leucine zipper.
  • the non-covalent binding unit is or comprises a PAR-based leucine zipper.
  • the non-covalent binding unit is or comprises a C/EBPa-based leucine zipper.
  • 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: 5).
  • the non-covalent binding unit comprises or consists of an amino acid sequence having at least 85% sequence identity to the amino acid sequence of SEQ ID NO: 5, 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 the amino acid sequence of SEQ ID NO: 5, 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.
  • 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 nucleic acid sequence with SEQ ID NO: 95, or an amino acid sequence encoded by said nucleic acid 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: 96, or a nucleic acid 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 nucleic acid sequence with SEQ ID NO: 92, or an amino acid sequence encoded by said nucleic acid 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. In some embodiments, 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 or consists of hinge exon hi and hinge exon h4 of lgG3.
  • 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
  • the joint region is hinge exon hi and hinge exon h4 of lgG3. In other embodiments, the joint region consists of or comprises the amino acid sequence 94-120 of SEQ ID NO: 1.
  • 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 targeting unit.
  • the joint region comprises or consists of the middle and lower hinge regions of lgG1.
  • the joint region comprises or consists of an amino acid sequence having at least 40% sequence identity to the amino acid sequence 5-23 of SEQ ID NO: 4, 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 an amino acid sequence having at least 40% sequence identity to the amino acid sequence of SEQ ID NO: 118, 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: 4, 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.
  • 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
  • the joint region comprises or consists of the amino acid sequence of SEQ ID NO: 118, 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.
  • 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
  • the joint region is the middle and lower hinge region of lgG1. In other preferred embodiments, the joint region consists of or comprises the amino acid sequence 5-23 of SEQ ID NO: 4. In some embodiments, the joint region consists of or comprises the amino acid sequence 1-23 of SEQ ID NO: 4.
  • 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 first targeting unit.
  • the joint region comprises hinge exon hi and hinge exon h4 of lgG3 and/or the join region comprising the middle and lower hinge region of lgG1 may further comprise a non-covalent biding region, e.g. the afore-described non-covalent binding units, preferably an immunoglobulin constant domain.
  • a non-covalent biding region e.g. the afore-described non-covalent binding units, preferably an immunoglobulin constant domain.
  • the immunomodulating polypeptide of the disclosure comprises a first and a second targeting unit.
  • the first targeting unit and the second targeting unit are independently selected among the below described targeting units, bearing in mind that pairs are preferred, as will be detailed below.
  • the first and the second targeting units are connected to the first and second joint regions as described herein, respectively.
  • targeting unit refers to a unit that delivers the immunomodulating polypeptide/multimeric protein/dimeric protein of the disclosure to an antigen- presenting cell either for MHC class ll-restricted presentation to CD4+ T cells, for providing cross presentation to CD8+ T cells by MHC class I restriction or for stimulating the antigen-presenting cell.
  • APCs include dendritic cells (DCs) and subsets thereof.
  • DCs dendritic cells
  • the construct of the disclosure is administered to a subject (e.g. by intramuscular administration) in the form of the polynucleotide (e.g. a DNA plasmid) comprising the nucleotide sequence encoding the immunomodulating polypeptide
  • the host cells e.g. muscle cells of the subject
  • multiple polypeptides form a multimeric protein (for example two polypeptides form a dimeric protein), by being linked via their joint regions in the manner described herein.
  • the multimeric protein comprises multiple of the same first targeting units and multiple of the same second targeting units.
  • the dimeric protein comprises two of the same first targeting units and two of the same second targeting units.
  • the advantage of having multiple, for example two, of the same first or second targeting units is that when they bind to the same APC, cross-linking is achieved which results in increased avidity and thus increased response.
  • the dimeric protein comprising two of the same first targeting units and two of the same second targeting units, this effect is further increased.
  • the multimeric proteins comprising multiples of the same first targeting units and multiples of the same second targeting units, this effect is even further increased.
  • the first and the second targeting units are identical. In some embodiments, the first and the second targeting units are different.
  • the skilled person can design an immunomodulating polypeptide eliciting an improved immune response by choosing a different second targeting unit which allows to utilize other signals/exploit other ways to improve immune response than with the first targeting unit.
  • chemokines are small, secreted proteins that bind to G-protein coupled receptors (GPCR). Such class of receptors, despite being able to function as monomers, forms also homo or heterodimers. Dimerization events are key parameters in the emergence of cooperative interactions between receptors and signaling pathways. These interactions modulate the amplification, duration, avidity and synergy of the response in a cell-dependent manner.
  • APCs targeting units By including two or multiple APCs targeting units into the construct of the disclosure, the probability of occurrence of such multimerization events, for example dimerization events, increases greatly.
  • phenotypical rerouting of APCs is a multi-dependency process that requires the integration of several activation signals that follows time and spatial constraints.
  • cDCI In addition to cDCI’s ability to cross present tumor antigens in draining lymph nodes, their roles encompass the attraction, re-stimulation, expansion and support of tumor infiltrating lymphocytes. Their abundance in the tumor microenvironment is considered as a favorable prognostic marker.
  • the skilled person may further choose to design an immunomodulating polypeptide comprising targeting units that target the same surface molecules, e.g. identical first and second targeting units or different first and second targeting units, which both target the same surface molecule, whereby a high avidity is achieved.
  • the targeting unit comprises a moiety that interacts with surface molecules on the antigen-presenting cells, e.g. binds to surface molecules on the APCs.
  • the surface molecules are present on the same cell.
  • the binding of the first and the second targeting units results in crosslinking of the targeted surface molecules. In some embodiments, the binding of the targeting units stimulates the cell. In some embodiments, the binding of the targeting units causes internalization of the multimeric protein. In other embodiments, the binding of the targeting units causes internalization of the dimeric protein.
  • the multimeric protein/dimeric protein of the disclosure may attract DCs, neutrophils and other immune cells.
  • the multimeric protein/dimeric protein 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 the construct of the disclosure.
  • the stimulation can result in attraction, activation, maturation and/or proliferation of the APCs.
  • at least one of the targeting units promotes attraction or activation of antigen-presenting cells or promotes the maturation or proliferation of antigen presenting cells.
  • the targeting unit is designed to target the dimeric protein/multimeric protein of the present disclosure to surface molecules expressed on the APCs, such as molecules expressed exclusively on subsets of DCs.
  • HLA HLA
  • CD14 cluster of differentiation 14
  • CD40 cluster of differentiation 40
  • CLEC9A GM-CSF-receptor
  • LT-3R IL- 15R
  • IL- 15R a TNF receptor
  • 4-1BB/4-1BBL CD70
  • ICOSL chemokine receptors
  • TLRs Toll-like receptors
  • 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 comprises or consists of an antibody binding region with specificity for HLA, CD14, CD40, CLEC9A or GM-CSF-receptor, LT-3R, IL-15R, a TNF receptor, 4-1BB/4-1BBL, CD70, ICOSL, chemokine receptors or Toll- like receptors.
  • the targeting unit comprises or consists of a synthetic or natural ligand.
  • chemokines such as their human forms, e.g. chemokine ligand 5, also called C-C motif ligand 5 (CCL5 or RANTES), macrophage inflammatory protein alpha and its isoforms, including mouse (CCL3 or MIP-10) and human isoforms hCCL3, hCCL3L1, hCCL3L2 and hCCL3L3, chemokine ligand 4 (CCL4), chemokine ligand 19 (CCL19), chemokine ligand 20 (CCL20), chemokine ligand 21 (CCL21), chemokine motif ligand 1 or 2 (XCL1 orXCL2) and bacterial antigens like for example flagellin.
  • chemokines such as their human forms, e.g. chemokine ligand 5, also called C-C motif ligand 5 (CCL5 or RANTES), macrophage inflammatory protein alpha and its isoforms, including mouse (CCL3 or MIP-
  • the targeting unit is or comprises a moiety that interacts with soluble, MIR-1b (CCL4), MIR-3a (CCL20), PAMPs such as flagellin, DAMPS, HMGB1 or HSPs, GM-CSF, FLT-3L, IL-15, 4-1BB (CD137)/4-1BBL, CD27, ICOS.
  • the first targeting unit is or comprises MIP-1a, preferably human MIP-1a.
  • the first targeting unit is or comprises MIP-1a and the second targeting unit is or comprises any of soluble CD40 ligand, CLEC9A, RANTES, XCL1, XCL2, MIR-1b (CCL4), MIR-3a (CCL20), PAMPs such as flagellin, DAMPS, HMGB1, HSPs, GM-CSF, FLT-3L, IL-15, 4-1 BB (CD137)/4-1 BBL, CD27, ICOS, anti-HLA-DP, anti-HLA-DR, anti-pan HLA class II, anti-CD40, anti-TLR-2, anti-TLR-4 or anti-TLR-5.
  • soluble CD40 ligand such as flagellin, DAMPS, HMGB1, HSPs, GM-CSF, FLT-3L, IL-15, 4-1 BB (CD137)/4-1 BBL, CD27, ICOS, anti-HLA-DP, anti-HLA-DR, anti-pan HLA class II, anti-CD40,
  • the targeting unit has affinity for an MHC class II protein.
  • the targeting unit comprises or consists of an antibody-binding region, such as the antibody variable domains (VL and VH) with specificity for MHC class II proteins, such as those selected from the group consisting of anti-HLA-DP, anti-HLA-DR and anti-pan HLA class II.
  • VL and VH antibody variable domains
  • the targeting unit has affinity for a surface molecule selected from the group consisting of CD14, CLEC9A, CD40, TLR-2, TLR-4 and TLR-5.
  • the targeting unit comprises or consists of an antibody-binding region, such as the antibody variable domains (VL and VH) with specificity for CD14, CD40, TLR-2, TLR4 or TLR-5, such as anti-CD14, anti-CD40, anti-TLR-2, anti-TLR-4 or anti-TLR-5.
  • 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 CLEC9 peptide ligand, e.g. a CLEC9 ligand comprising or consisting of the nucleotide sequence with SEQ ID NO: 94 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, CCR 6 and CCR7.
  • 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: 99) or an amino acid sequence encoded by said nucleotide sequence,, or CCL21, such as the human forms of CCL19 or CCL21.
  • the targeting unit comprises or consists of chemokine human macrophage inflammatory protein alpha (human MIP-1a (hMIP-1a), also called I_078b or CCL3L1), which binds to its cognate receptors, CCR1 and CCR5 expressed on the cell surface of APCs.
  • chemokine human macrophage inflammatory protein alpha human MIP-1a (hMIP-1a), also called I_078b or CCL3L1
  • the targeting unit is an antibody-binding region with specificity for a dendritic cell receptor selected from the group consisting of CLEC9A, CD11c, CD80, CD86, CD141, CD172a, CD11b, 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.
  • CCL5 is human CCL5.
  • FLT3L is human FLT3L, such as the targeting unit comprises or consists of the amino acid sequence of SEQ ID NO: 120.
  • GM-CSF is human GM-CSF.
  • 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 first targeting unit is CCL3 and the second targeting unit is CCL5. In some embodiments, the first targeting unit is CCL3 and the second targeting unit is CCL7. In some embodiments, the first targeting unit is CCL3 and the second targeting unit is CCL4. In some embodiments, the first targeting unit is CCL3 and the second targeting unit is XCL1. In some embodiments, the first targeting unit is CCL5 and the second targeting unit is CCL7. In some embodiments, the first targeting unit is CCL5 and the second targeting unit is XCL1. In some embodiments, the first targeting unit is CCL5 and the second targeting unit is CCL4. In some embodiments, the first targeting unit is CCL3 and the second targeting unit is CD11c.
  • the first targeting unit is CCL3 and the second targeting unit is CD80. In some embodiments, the first targeting unit is CCL5 and the second targeting unit is CD11c. In some embodiments, the first targeting unit is CCL5 and the second targeting unit is CD80. In some embodiments, the first targeting unit is CCL5 and the second targeting unit is GM-CSF. In some embodiments, the first targeting unit is CCL3 and the second targeting unit is FLT3L. In some embodiments, the first targeting unit is CCL3 and the second targeting unit is 4-1 BBL. In some embodiments, the first targeting unit is CCL3 and the second targeting unit is GM-CSF. In some embodiments, the first targeting unit is CCL3 and the second targeting unit is CD40L.
  • the first targeting unit is FLT3L and the second targeting unit is GM-CSF. In some embodiments, the first targeting unit is FLT3L and the second targeting unit is CD205. In some embodiments, the first targeting unit is 4-1 BBL and the second targeting unit is CD40L. In some embodiments, the first targeting unit is 4-1 BBL and the second targeting unit is GM-CSF. In some embodiments, the first targeting unit is GM-CSF and the second targeting unit is CD205.
  • the first targeting unit is CCL3L1 and the second targeting unit is CCL5.
  • the first targeting unit is CCL3L1 and the second targeting unit is CCL7.
  • the first targeting unit is CCL3L1 and the second targeting unit is CCL4.
  • the first targeting unit is CCL3L1 and the second targeting unit is XCL1.
  • the first targeting unit is CCL3L1 and the second targeting unit is CD11c.
  • the first targeting unit is CCL3L1 and the second targeting unit is CD80.
  • the first targeting unit is CCL3L1 and the second targeting unit is FLT3L.
  • the first targeting unit is CCL3L1 and the second targeting unit is 4-1 BBL. In some embodiments, the first targeting unit is CCL3L1 and the second targeting unit is GM- CSF. In some embodiments, the first targeting unit is CCL3L1 and the second targeting unit is CD40L.
  • the binding of a targeting unit to its cognate receptor leads to internalization of the dimeric protein/multimeric protein 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 an immune response against the T cell epitopes of the antigenic unit. If the T cell epitopes are cancer epitopes, a cancer specific immune response is induced. Once stimulated, and with help from activated CD4+ T cells,
  • CD8+ T cells will target and kill cancer cells expressing the same antigens.
  • 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-1a, preferably hMIP-1a.
  • MIP-1a 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 Th1 CD4+ responses and CD8+ T cell responses.
  • MIP-1a is also capable of supporting the induction of antibody responses, in particular lgG2a, 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, 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 24-93 of SEQ ID NO: 1.
  • 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 , such as consisting of the amino acid sequence 26-93 of SEQ I D NO: 1 or comprising the amino acid sequence 28-93 of SEQ ID NO: 1.
  • 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, 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 24-93 of SEQ ID NO: 1.
  • the targeting unit comprises the amino acid sequence 24-93 of SEQ ID NO: 1, 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.
  • An embodiment of such a targeting unit is one comprising the amino acid sequence 26-93 of SEQ ID NO: 1 or one comprising the amino acid sequence 28-93 of SEQ ID NO: 1.
  • the targeting unit consists of the amino acid sequence 24-93 of SEQ ID NO: 1 , 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.
  • An embodiment of such a targeting unit is one consisting of the amino acid sequence 26-93 of SEQ ID NO: 1 or one consisting of the amino acid sequence 28-93 of SEQ ID NO: 1.
  • the targeting unit is described as a nucleic acid, thus the nucleic acid sequence is capable of encoding a polypeptide functioning as a targeting unit.
  • the targeting unit comprises a nucleic acid sequence having at least 80% sequence identity to the nucleic acid sequence with SEQ ID NO: 100.
  • the targeting unit comprises a nucleic acid sequence having at least 85% sequence identity to the nucleic acid sequence with SEQ ID NO: 100, 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 nucleic acid sequence of SEQ ID NO: 100.
  • the targeting unit consists of a nucleic acid sequence having at least 80% sequence identity to the nucleic acid sequence with SEQ ID NO: 100.
  • the targeting unit consists of a nucleic acid sequence having at least 85% sequence identity to the nucleic acid sequence 70-277 of 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 targeting unit has the nucleic acid sequence of SEQ ID NO: 100.
  • the first targeting unit (or second targeting unit) is human MIP1a and the second targeting unit (or the first targeting unit) is human FLT3L.
  • the present disclosure relates to immunotherapy constructs useful in the prevention or treatment of cancer and in the prevention or treatment of infectious diseases.
  • the antigenic unit of the immunomodulating polypeptides of the present disclosure comprises at least one T cell epitope derived from a cancer antigen or a pathogen antigen.
  • T cell epitope refers to a single T cell epitope or a part, fragment or region of an antigen containing multiple T cell epitopes, e.g. hotspot(s) of minimal epitopes.
  • a hotspot of minimal epitopes is a region that contains several minimal epitopes (e.g. having a length of from 7-15 amino acids) that are predicted to be presented by different HLA alleles to cover a broad range of world population.
  • T cell epitopes suitable for inclusion into the antigenic unit may be known in the art, i.e. have been studied, proposed and/or verified to be involved and of relevance for a certain immune disease and published in the literature.
  • the at least one T cell epitope comprised in the antigenic unit of the immunomodulating polypeptide of the disclosure has a length of from 7 to about 200 amino acids, with the longer T cell epitopes possibly including hotspots of minimal epitopes.
  • the 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 9 to 100 amino acids or from 15 to 100 amino acids or from 9 to 60 amino acids or from 9 to 30 amino acids or from 15 to 60 of from 15 to 30 or from 20 to 75 amino acids or from 25 to 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 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 antigenic unit, with a 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 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 15 amino acids for MHC class II presentation. In some embodiments, 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 some preferred 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 antigenic unit may vary, and depends on the length and number of other elements included in the antigenic unit, e.g. T cell epitope linkers as described in this application.
  • the antigenic unit comprises from about 21 to about 2000 amino acids, preferably from about 30 amino acids to about a 1500 amino acids, more preferably from about 50 to about 1000 amino acids, such as from about 100 to about 500 amino acids or from about 100 to about 400 amino acids or from about 100 to about 300 amino acids.
  • the 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 T cell epitope may be comprised in any of the pathogen proteins, e.g. surface proteins but also structural 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,
  • 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. Finding conserved regions across the genome of betacoronavirus genus indicates that these conserved regions are needed to maintain essential structures or functions, thus it can be assumed that future mutations will take place in the less-conserved regions. 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.
  • the antigenic unit comprises 1 to 10 T cell epitopes such as 1,
  • 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 subunit comprises 1 to 3 T cell epitopes or 1 to 5 T cell epitopes or 3 to 6 T cell epitopes or 5 to 15 T cell epitopes or 7 to 17 T cell epitopes or 9 to 19 T cell epitopes.
  • the T cell epitopes are randomly arranged in the antigenic unit. In other embodiments, one or more of the following methods for arranging them in the antigenic unit may be 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 end of the antigenic unit.
  • the most hydrophobic T cell epitope(s) may be positioned substantially in the middle of the antigenic unit and the most hydrophilic T cell epitope(s) is/are positioned closest to the joint regions.
  • the skilled person will have no difficulty predicting antigenicity/immunogenicity and/or hydrophilicity/hydrophobicity by methods known in the art and as described herein.
  • the term “substantially” in this context refers to 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.
  • an antigenic unit comprises 5 T cell epitopes, which are arranged as follows: 1-2-3 -5; with 1, 2, 3*, 4 and 5 each being a different T cell epitope, e.g. a neoepitope, and - being a T cell epitope linker and * indicating the most hydrophobic T cell epitope, which is positioned in the middle of the antigenic unit.
  • an antigenic unit comprises 6 T cell epitopes, which are arranged as follows: 1-2-3 -5-6 or, alternatively, as follows: 1-2-4-3*-5-6; with 1, 2, 3*, 4, 5 and 6 each being a different T cell epitope, e.g. a neoepitope, and - being a T cell epitope linker and * indicating the most hydrophobic T cell epitope, which is positioned substantially in the middle of the 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 sequences encoding T cell epitopes are arranged such that there is at least one non-GC rich 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.
  • the antigenic unit comprises multiple T cell epitopes
  • the epitopes are preferably separated by T cell epitope linkers. This ensures that each T cell epitope is presented in an optimal way to the immune system.
  • the antigenic unit comprises n T cell epitopes, it preferably comprises n-1 T cell epitope linkers, separating each T cell epitope from one or two other T cell epitopes, where n is an integer equal to or greater than 1.
  • the T cell epitope linker is designed to be non-immunogenic. It 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. Alternatively, 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 epitope linker is preferably also a flexible linker, which allows for presenting the T cell epitope in an optimal manner to the immune system, even if the antigenic unit comprises a large number of T cell epitopes.
  • the T cell epitope linker is a peptide consisting of from 440 amino acids, e.g. 35, 30, 25 or to 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 other preferred embodiments, the T cell epitope linker consists of 10 amino acids.
  • All T cell epitope linkers comprised in the antigenic unit are preferably identical. If, however, one or more of the T cell epitopes comprise 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 immunogenic 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.
  • small, non-polar (e.g. glycine, alanine or leucine) or polar (e.g. serine or threonine) amino acids e.g. serine or threonine
  • 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 linker is a serine (S) and/or glycine (G) rich linker, i.e.
  • GGGGS (SEQ ID NO: 7), GGGSS (SEQ ID NO: 8), GGGSG (SEQ ID NO: 9), GGSGG (SEQ ID NO: 74), GGGGS (SEQ ID NO: 7), SGSSGS (SEQ ID NO: 70) or multiple variants thereof such as GGGGSGGGGS (SEQ ID NO: 10), (GGGGS)m (SEQ ID NO: 71), (GGGSS)m (SEQ ID NO: 72), (GGSGG)m (SEQ ID NO: 74), (GGGSG)m (SEQ ID NO: 73) or (SGSSGS)m (SEQ ID NO: 75), where m is an integer from 1 to 5, e.g., 1, 2, 3, 4, or 5, In preferred embodiments, m is 2.
  • 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 T cell epitope linker comprises or consists of LGGGS (SEQ ID NO: 11), GLGGS (SEQ ID NO: 12), GGLGS (SEQ ID NO: 13), GGGLS (SEQ ID NO: 14) or GGGGL (SEQ ID NO: 15).
  • the T cell epitope linker comprises or consists of LGGSG (SEQ ID NO: 16), GLGSG (SEQ ID NO: 17), GGLSG (SEQ ID NO: 18), GGGLG (SEQ ID NO: 19) or GGGSL (SEQ ID NO: 20).
  • the T cell epitope linker comprises or consists of LGGSS (SEQ ID NO: 21), GLGSS (SEQ ID NO: 22) or GGLSS (SEQ ID NO: 23).
  • the T cell epitope linker comprises or consists of LGLGS (SEQ ID NO: 24), GLGLS (SEQ ID NO: 25), GLLGS (SEQ ID NO: 26), LGGLS (SEQ ID NO: 27) or GLGGL (SEQ ID NO: 28).
  • the T cell epitope linker comprises or consists of LGLSG (SEQ ID NO: 29), GLLSG (SEQ ID NO: 30), GGLSL (SEQ ID NO: 31), GGLLG (SEQ ID NO: 32) or GLGSL (SEQ ID NO: 33).
  • the T cell epitope linker comprises or consists of LGLSS (SEQ ID NO: 34), or GGLLS (SEQ ID NO: 35).
  • 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: 36), GLGGSGGGGS (SEQ ID NO: 37), GGLGSGGGGS (SEQ ID NO: 38), GGGLSGGGGS (SEQ ID NO: 39) or GGGGLGGGGS (SEQ ID NO: 40).
  • the T cell epitope linker comprises or consists of LGGSGGGGSG (SEQ ID NO: 41), GLGSGGGGSG (SEQ ID NO: 42), GGLSGGGGSG (SEQ ID NO: 43), GGGLGGGGSG (SEQ ID NO: 44) or GGGSLGGGSG (SEQ ID NO: 45).
  • the T cell epitope linker comprises or consists of LGGSSGGGSS (SEQ ID NO: 46), GLGSSGGGSS (SEQ ID NO: 47), GGLSSGGGSS (SEQ ID NO: 48), GGGLSGGGSS (SEQ ID NO: 49) or GGGSLGGGSS (SEQ ID NO: 50).
  • the T cell epitope linker comprises or consists of LGGGSLGGGS (SEQ ID NO: 51), GLGGSGLGGS (SEQ ID NO: 52), GGLGSGGLGS (SEQ ID NO: 53), GGGLSGGGLS (SEQ ID NO: 54) or GGGGLGGGGL (SEQ ID NO: 55).
  • the T cell epitope linker comprises or consists of LGGSGLGGSG (SEQ ID NO: 56), GLGSGGLGSG (SEQ ID NO: 57), GGLSGGGLSG (SEQ ID NO: 58), GGGLGGGGLG (SEQ ID NO: 59) or GGGSLGGGSL (SEQ ID NO: 60).
  • the T cell epitope linker comprises or consists of LGGSSLGGSS (SEQ ID NO: 61), GLGSSGLGSS (SEQ ID NO: 62) or GGLSSGGLSS (SEQ ID NO: 63).
  • the T cell linker comprises or consists of GSGGGA (SEQ ID NO: 76), GSGGGAGSGGGA (SEQ ID NO: 77), GSGGGAGSGGGAGSGGGA (SEQ ID NO: 78), GSGGGAGSGGGAGSGGGAGSGGGA (SEQ ID NO: 78) or GENLYFQSGG (SEQ ID NO: 80).
  • the T cell linker comprises or consists of SGGGSSGGGS (SEQ ID NO: 81), GGGGSGGGGS (SEQ ID NO: 82),
  • GSGSGSGSGSGSGS (SEQID NO: 85), GGGSSGGGSG (SEQ ID NO: 86), GGGSSS (SEQ ID NO: 87), GGGSSGGGSSGGGSS (SEQ ID NO: 88) or GLGGLAAA (SEQ ID NO: 85), GGGSSGGGSG (SEQ ID NO: 86), GGGSSS (SEQ ID NO: 87), GGGSSGGGSSGGGSS (SEQ ID NO: 88) or GLGGLAAA (SEQ ID NO:
  • 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: 107), AEAAAKEAAAKA (SEQ ID NO: 108), (EAAAK)m (SEQ ID NO:
  • the T cell epitope linker comprises or consists of the sequence TQKSLSLSPGKGLGGL (SEQ ID NO: 64). In other embodiments, the T cell epitope linker comprises or consists of the sequence SLSLSPGKGLGGL (SEQ ID NO: 65). In other embodiments, the T cell epitope linker comprises or consists of the sequence AAY or GPGPG (SEQ ID NO: 112).
  • the T cell epitope linker is a GSAT (SEQ ID NO: 66) 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: 67) 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 GGSGGGSEGGGSEGGGSEGGGSEGGGSEGGGSGGGS (SEQ ID NO: 68) or
  • ELKTPLGDTTHT (SEQ ID NO: 69; corresponding to amino acids 94-105 of SEQ ID NO: 1).
  • 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.
  • Antigenic unit of individualized anticancer immunotherapy constructs An immunomodulating polypeptide encoded by the polynucleotide of individualized anticancer immunotherapy constructs according to the disclosure comprises an antigenic unit, which is designed specifically and only for the patient who is treated with the immunotherapy construct.
  • the antigenic unit of such an immunomodulating polypeptide comprises at least one T cell epitope comprised in a patient-specific antigen, such antigen being either a neoantigen or a patient-present shared cancer antigen. If the at least one T cell epitope is comprised in a neoantigen, such T cell epitopes are called neoepitopes.
  • Patient-present shared cancer antigen is used herein to describe an amino acid sequence, or a nucleotide sequence encoding same, comprised in a patient-present shared cancer antigen or shared tumor antigen that has been identified to be present in the patient’s tumor cells, wherein the patient-present shared cancer antigen or shared tumor antigen comprises one or more immunogenic mutations, i.e. mutations which is are known to be immunogenic or which are predicted to be immunogenic.
  • Neoantigen or patient-specific cancer antigen is used herein to describe a cancer antigen or tumor antigen found in a patient’s tumor cells that comprises one or more mutations compared to the same patient’s normal (i.e. healthy, non-cancerous) cells.
  • Neoepitope or patient-specific cancer epitope is used herein to describe an amino acid sequence, or a nucleotide sequence encoding same, of a T cell epitope comprised in a neoantigen, which comprises one or more mutations and which is predicted to be immunogenic.
  • the disclosure provides an immunotherapy construct comprising: i) a polynucleotide comprising a nucleotide sequence encoding an immunomodulating polypeptide, the immunomodulating polypeptide comprising, in the specified order: a. a first targeting unit, a first joint region; b. an antigenic unit comprising at least one T cell epitope; wherein the T cell epitope is comprised in a patient-specific cancer antigen; c. a second joint region; and d.
  • a second targeting unit ii) an immunomodulating polypeptide encoded by the nucleotide sequence as defined in i); or iii) a multimeric protein, such as a dimeric protein, consisting of multiple polypeptides as defined in ii), such as of two polypeptides as defined in ii).
  • the present disclosure relates to an immunotherapy construct comprising: i) a polynucleotide comprising a nucleotide sequence encoding an immunomodulating polypeptide, the immunomodulating polypeptide comprising, in the specified order: a. a first targeting unit, a first joint region; b. an antigenic unit comprising at least one T cell epitope; wherein the T cell epitope is comprised in a patient-specific cancer antigen; c. a second joint region; and d. a second targeting unit; ii) an immunomodulating polypeptide encoded by the nucleotide sequence as defined in i); or iii) a multimeric protein consisting of multiple polypeptides as defined in ii).
  • the present disclosure relates to an immunotherapy construct comprising: i) a polynucleotide comprising a nucleotide sequence encoding an immunomodulating polypeptide, the immunomodulating polypeptide comprising, in the specified order: a. a first targeting unit, a first joint region; b. an antigenic unit comprising at least one T cell epitope; wherein the T cell epitope is comprised in a patient-specific cancer antigen; c. a second joint region; and d. a second targeting unit; ii) an immunomodulating polypeptide encoded by the nucleotide sequence as defined in i); or iii) a dimeric protein consisting of two polypeptides as defined in ii).
  • the antigenic unit of the construct above comprises at least one T cell epitope, i.e. one or more T cell epitopes, which are comprised in one or more patient-present shared cancer antigens and/or which are neoepitopes.
  • the antigenic unit comprises at least one T cell epitope, i.e. one or more T cell epitopes, comprised in one or more patient-present shared cancer antigens.
  • the antigenic unit comprises at least one neoepitope, i.e. one or more neoepitopes.
  • the antigenic unit comprises at least one T cell epitope, i.e. one or more T cell epitopes, comprised in one or more patient-present shared cancer antigens and one or more neoepitopes.
  • T cell epitope i.e. one or more T cell epitopes
  • Antigenic units comprising one or more T cell epitopes, which are comprised in one or more patient-present shared cancer antigens and/or which are neoepitopes are described in detail in PCT/EP2021/059353, the content of which is included herein by reference. Any of such antigenic units can be used as antigenic unit in an immunotherapy construct of the disclosure for use in individualized anticancer therapy.
  • Antigenic unit of individualized anticancer immunotherapy constructs comprising one or more neoantigens or parts or fragments thereof Cancers develop from the patient’s normal tissue by one or a few cells starting an abnormal, uncontrolled proliferation of the cells due to mutations. Although the cancer cells are mutated, most of the genome is intact and identical to the remaining cells in the patient.
  • One approach of attacking a tumor is based on the knowledge that any tumor in any patient is unique: patient-specific mutations lead to expression of patient- specific mutated proteins, i.e. neoantigens, that are unique for the particular patient. These neoantigens are not identical to any proteins in the normal cells of the patient. Therefore, such neoantigens are suitable targets for a therapeutic anticancer immunomodulating polypeptide which is manufactured specifically and only for the patient in question, i.e. an individualized anticancer immunotherapy construct.
  • the mutation may be any mutation leading to a change in at least one amino acid.
  • the mutation may be one of the following:
  • chromosomal rearrangements that give rise to a chimeric protein with a tumor- specific epitope at the junction of the two proteins.
  • the tumor-specific epitope can arise from a change in at least one amino acid or from a combination of two in-frame coding sequences.
  • the antigenic unit comprises one or more neoepitopes and more preferably several neoepitopes.
  • neoepitopes may be selected for inclusion into antigenic unit according to their predicted therapeutic efficacy, see WO 2017/118695A1, the disclosure of which is incorporated herein by reference.
  • the disclosure provides an immunotherapy construct comprising: i) a polynucleotide comprising a nucleotide sequence encoding an immunomodulating polypeptide, the immunomodulating polypeptide comprising, in the specified order: a. a first targeting unit, a first joint region; b. an antigenic unit comprising at least one neoepitope; c. a second joint region; and d.
  • a second targeting unit ii) an immunomodulating polypeptide encoded by the nucleotide sequence as defined in i); or iii) a multimeric protein consisting of multiple polypeptides as defined in ii), such as a dimeric protein consisting of two polypeptides as defined in ii).
  • the present disclosure relates to an immunotherapy construct comprising: i) a polynucleotide comprising a nucleotide sequence encoding an immunomodulating polypeptide, the immunomodulating polypeptide comprising, in the specified order: a. a first targeting unit, a first joint region; b. an antigenic unit comprising at least one neoepitope; c. a second joint region; and d. a second targeting unit; ii) an immunomodulating polypeptide encoded by the nucleotide sequence as defined in i); or iii) a multimeric protein consisting of multiple polypeptides as defined in ii).
  • the present disclosure relates to an immunotherapy construct comprising: i) a polynucleotide comprising a nucleotide sequence encoding an immunomodulating polypeptide, the immunomodulating polypeptide comprising, in the specified order: a. a first targeting unit, a first joint region; b. an antigenic unit comprising at least one neoepitope; c. a second joint region; and d. a second targeting unit; ii) an immunomodulating polypeptide encoded by the nucleotide sequence as defined in i); or iii) a dimeric protein consisting of two polypeptides as defined in ii).
  • the neoepitope preferably has a length suitable for presentation by HLA (human leukocyte antigen) molecules.
  • HLA human leukocyte antigen
  • the neoepitope has a length of from 7 to 30 amino acids. More preferred are neoepitopes having a length of from 7 to 10 amino acids or of from 15 to 30 amino acids, e.g. from 20 to 30 amino acids, e.g. 27 amino acids.
  • HLA is a major histocompatibility complex (MHC) in humans.
  • MHC class I and MHC II There are two primary classes of MHC molecules, MHC class I and MHC II.
  • MHC class I and MHC class II are interchangeably used herein with HLA class I and HLA class II, the terms HLA allele and HLA molecule are also interchangeably used herein.
  • the antigenic unit comprises a plurality of different neoepitopes, i.e. several different neoepitopes. In other embodiments, the antigenic unit comprises multiple copies of the same neoepitope. In yet other embodiments, the antigenic unit comprises several different neoepitopes and multiple copies of the same neoepitope.
  • a preferred approach is to include as many neoepitopes as possible in the antigenic unit (i.e. different and/or multiple copies of the same neoepitope) to thereby attack the cancer efficiently without compromising the immunomodulating polypeptide’s ability to activate T cells against the neoepitopes due to dilution of the desired T cell effect.
  • all neoepitope-encoding nucleotide sequences are comprised in a continuous polynucleotide chain resulting in the expression of a protein comprising all the neoepitopes instead of expressing each neoepitope as a discrete peptide.
  • the patient’s tumor exome is analyzed to identify neoantigens.
  • the sequences of the most immunogenic neoepitopes from one or more neoantigens are selected for inclusion into the antigenic unit.
  • the antigenic unit comprises at least 1 neoepitope, preferably at least 3 neoepitopes, more preferably at least 5 neoepitopes, such as 7 neoepitopes.
  • the antigenic unit comprises at least 10 neoepitopes.
  • the antigenic unit comprises at least 15 neoepitopes, such as at least 20 or at least 25 or at least 30 or at least 35 or at least 40 or at least 45 neoepitopes.
  • Antigenic units comprising one or more neoepitopes are described in detail in WO 2017/118695A1. Any of such antigenic units can be used as antigenic unit in an immunotherapy construct of the disclosure for use in individualized anticancer therapy.
  • Antigenic unit of individualized anticancer immunotherapy constructs comprising one or more patient-present shared cancer antigens or parts or fragments thereof
  • Shared tumor antigens are expressed by many tumors, either across patients with the same cancer type, or across patients and cancer types.
  • An example is the HPV16 antigen, a viral antigen that is expressed in about 50% of all patients with squamous cell carcinoma of the head and neck, but also in patients with other cancers such as cervical cancer and vulvar squamous cell carcinoma.
  • Many of these shared antigens have previously been characterized as immunogenic and/or are known, i.e. their immunogenicity has been confirmed by appropriate methods and the results have been published, e.g. in a scientific publication. Others have already been predicted to be presented on specific HLA class I or class II alleles, e.g. by algorithms known in the art and their predicted immunogenicity has been published, e.g. in a scientific publication, without having confirmed their immunogenicity by appropriate methods.
  • the antigenic unit comprises one or more T cell epitopes comprised in one or more patient-present shared cancer antigen, which are known to be immunogenic, have known expression patterns and/or are known or have already been predicted to bind to specific HLA class I and class II alleles.
  • T cells specific to patient-present shared cancer antigens can travel to the tumor and affect the tumor microenvironment, thus increasing the likelihood that additional tumor-specific T cells are able to attack the cancer.
  • the disclosure provides an immunotherapy construct comprising: i) a polynucleotide comprising a nucleotide sequence encoding an immunomodulating polypeptide, the immunomodulating polypeptide comprising, in the specified order: a. a first targeting unit, a first joint region; b. an antigenic unit comprising at least one T cell epitope comprised in a patient- present shared cancer antigen; c. a second joint region; and d.
  • a second targeting unit ii) an immunomodulating polypeptide encoded by the nucleotide sequence as defined in i); or iii) a multimeric protein consisting of multiple polypeptides as defined in ii), such as a dimeric protein consisting of two polypeptides as defined in ii).
  • the present disclosure relates to an immunotherapy construct comprising: i) a polynucleotide comprising a nucleotide sequence encoding an immunomodulating polypeptide, the immunomodulating polypeptide comprising, in the specified order: a. a first targeting unit, a first joint region; b. an antigenic unit comprising at least one T cell epitope comprised in a patient- present shared cancer antigen; c. a second joint region; and d. a second targeting unit; ii) an immunomodulating polypeptide encoded by the nucleotide sequence as defined in i); or iii) a multimeric protein consisting of multiple polypeptides as defined in ii).
  • the present disclosure relates to an immunotherapy construct comprising: i) a polynucleotide comprising a nucleotide sequence encoding an immunomodulating polypeptide, the immunomodulating polypeptide comprising, in the specified order: a. a first targeting unit, a first joint region; b. an antigenic unit comprising at least one T cell epitope comprised in a patient- present shared cancer antigen; c. a second joint region; and d. a second targeting unit; ii) an immunomodulating polypeptide encoded by the nucleotide sequence as defined in i); or iii) a dimeric protein consisting of two polypeptides as defined in ii).
  • the antigenic unit comprises at least one T cell epitope, i.e. one or more T cell epitopes, comprised in one or more patient-present shared cancer antigens.
  • the patient-present shared cancer antigen is selected from the group consisting of overexpressed cellular proteins, aberrantly expressed cellular proteins, cancer testis antigens, viral antigens, differentiation antigens, mutated oncogenes and mutated tumor suppressor genes, oncofetal antigens, shared fusion antigens, shared intron retention antigens, dark matter antigens and shared antigens caused by spliceosome mutations or frameshift mutations.
  • the patient-present shared cancer antigen is an overexpressed or aberrantly expressed human cellular protein, i.e. a cellular protein found at increased levels in tumors compared with normal, healthy cells and tissues.
  • overexpressed or aberrantly expressed cellular proteins include tumor protein D52, Her-2/neu, hTERT (telomerase) and survivin.
  • the patient-present shared cancer antigen is a cancer testis antigen whose expression occurswhich is normally expressed in male germ cells in the testis but not in adult somatic tissues. In some cases, such antigens are also expressed in ovary and trophoblast. In malignancy, this gene regulation is disrupted, resulting in antigen expression in a proportion of tumors of various types in human malignancies as well as in normal testicular tissue.
  • cancer testis antigens include MAGE-A, MAGE-B, GAGE, PAGE-1, SSX, HOM-MEL-40 (SSX2), NY-ESO-1, LAGE-1 and SCP-1.
  • the patient-present shared cancer antigen is a differentiation antigen, for example tyrosinase.
  • the patient-present shared antigen is a viral antigen.
  • viral antigens include human papilloma virus (HPV), hepatitis B virus (HBV), Epstein-Barr virus (EBV), Kaposi's sarcoma-associated herpesvirus (KSHV), Merkel cell polyomavirus (MCV or MCPyV), human cytomegalovirus (HCMV) and human T-lymphotropic virus (HTLV).
  • the patient-present shared cancer antigen is a mutated oncogene.
  • mutated oncogenes include KRAS, CALR and TRP-2.
  • the patient-present shared cancer antigen is a mutated tumor suppressor gene. Examples include mutated p53, mutated pRB, mutated BCL2 and mutated SWI/SNF.
  • the patient-present shared cancer antigen is an oncofetal antigen, for example alpha-fetoprotein or carcinoembryonic antigen.
  • the patient-present shared antigen is a shared intron retention antigen or shared antigen caused by frameshift mutation, for example CDX2 or CALR.
  • the patient-present shared antigen is a shared antigen caused by spliceosome mutations.
  • An example is an antigen caused by mutations like SF3B1 mut.
  • an anticancer vaccine should specifically trigger immune response to the antigens incorporated in the vaccine.
  • the peripheral immune tolerance to the selected antigens may be weak or strong.
  • a vaccine comprising such antigenic unit elicits an immune response which is strong and broad enough to affect the tumor microenvironment and change the patient’s immune response against the tumor from a suppressive/tolerated type to a pro-inflammatory type. This may help to break tolerance to several other antigens, thus representing a considerable clinical benefit for the patient.
  • the afore-described concept may be referred to as tipping the cancer immunity set point.
  • the antigenic unit comprises one or more patient-present shared cancer antigens or parts or fragments thereof that is a human cellular protein, preferably an overexpressed or aberrantly expressed human cellular protein or a differentiation antigen.
  • the patient-present shared cancer antigen may be detected in the tissue or body fluid of the patient by methods known in the art, including:
  • RT-PCR e.g. to detect the presence of viral antigens or known mutations in oncogenes
  • RNA-seq data to identify e.g. shared viral antigens
  • RNA-seq of the patient’s tumor samples with either patient’s own healthy tissue or a cohort/database (e.g. TCGA) versus consensus transcript expression, such as GTEX/HPA gene expression data.
  • TCGA TCGA
  • the antigenic unit comprises one or more patient-present shared cancer antigens or part(s) or fragments of such antigen(s) that is known to be immunogenic, e.g. has previously been described to elicit an immune response in other patients, or has been predicted to bind to the patient’s HLA class I and/or class II alleles.
  • the antigenic unit comprises one or more parts or fragments of one or more patient-present shared cancer antigens, e.g. one or more epitopes that are known to be immunogenic or have been predicted to bind to the patient’s HLA class I and/or class II alleles.
  • the antigenic unit comprises one or more patient-present shared cancer epitopes. In preferred embodiments, such epitopes have a length suitable for presentation by the patient’s HLA alleles.
  • the antigenic unit comprises one or more patient-present shared cancer epitopes having a length suitable for specific presentation on HLA class I or HLA class II.
  • the epitope has a length of from 7 to 11 amino acids for HLA class I presentation. In other embodiments, the epitope has a length of from 13 to 30 amino acids for HLA class II presentation.
  • the antigenic unit comprises one or more patient-present shared cancer epitopes having a length of from 7 to 30 amino acids, e.g. from 7 to 10 amino acids (such as 7, 8, 9 or 10 amino acids) or from 13 to 30 amino acids (such as 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29 or 30 amino acids), such as 7, 8, 9, 10, 11, 12, 13, 14 or 15 amino acids.
  • 7 to 30 amino acids e.g. from 7 to 10 amino acids (such as 7, 8, 9 or 10 amino acids) or from 13 to 30 amino acids (such as 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29 or 30 amino acids), such as 7, 8, 9, 10, 11, 12, 13, 14 or 15 amino acids.
  • the antigenic unit may comprise one or more patient-present shared cancer antigens either in full length or one or more parts or fragments thereof.
  • the antigenic unit comprises one patient-present shared cancer antigen in full length. In other embodiments, the antigenic unit comprises several patient-present shared cancer antigens, each of them in full length.
  • the antigenic unit comprises one or more parts or fragments of a patient-present shared cancer antigen, e.g. one or more patient-present shared cancer epitopes. In yet other embodiments, the antigenic unit comprises a one or more parts or fragments of several patient-present shared cancer antigens, e.g. one or more epitopes of several patient-present shared cancer antigens.
  • the antigenic unit comprises one or more patient-present shared antigens in full length and one or more parts or fragments of one or more patient-present shared cancer antigens. Examples include:
  • antigenic units comprising one patient-present shared antigen in full length and one or more epitopes of one patient-present shared cancer antigen; • antigenic units comprising several patient-present shared cancer antigens, each of them in full length, and one or more epitopes of one patient-present shared cancer antigen;
  • antigenic units comprising one patient-present shared antigen in full length and one or more epitopes of several patient-present shared cancer antigens
  • antigenic units comprising several patient-present shared cancer antigens, each of them in full length, and one or more epitopes of several patient-present shared cancer antigens.
  • the aforementioned epitopes are already known to be immunogenic (e.g. have been described to be immunogenic in the literature) or have already been predicted to bind to the patient’s HLA class I and class II alleles (e.g. as described in the literature), preferably have already been predicted to bind to the patient’s HLA class I alleles.
  • the immunogenicity of the aforementioned epitopes is predicted, e.g.
  • HLA class I and/or HLA class II molecules are predicted by methods known in the art, such as those disclosed in WO 2021/205027 A1, the disclosures of which is incorporated herein by reference, or those described in the section “Methods for designing an antigenic unit of an individualized anticancer vaccine for use in the invention” included herein.
  • the antigenic unit comprises 1 to 10 patient-present shared antigens in full length.
  • the antigenic unit comprises 1 to 30 parts or fragments of one or more patient-present shared antigens, wherein these parts or fragments include one or multiple epitopes that are predicted to bind to a patient’s HLA class I or class II alleles. In yet other embodiments, the antigenic unit comprises 1 to 50 patient-present shared cancer epitopes, optionally epitopes that are predicted to bind to the patient’s HLA class I or class II alleles.
  • Antigenic units comprising one or more T cell epitopes, which are comprised in one or more patient-present shared cancer antigens are described in detail in PCT/EP2021/059353, the content of which is included herein by reference. Any of such antigenic units can be used as antigenic unit in an immunotherapy construct of the disclosure for use in individualized anticancer therapy.
  • Antigenic units of individualized anticancer immunotherapy constructs comprising one or more patient-present shared cancer antigens or parts or fragments thereof and one or more neoantigens or parts or fragments thereof
  • antigenic units of individualized anticancer immunotherapy constructs comprise multiple T cell epitopes comprised in one or more patient-present shared cancer antigens and one or more neoepitopes.
  • Such antigenic units are a combination of all of the afore-described embodiments relating to antigenic units, which comprise one or more T cell epitopes comprised in one or more patient-present shared cancer antigens and all of the afore-described embodiments relating to antigenic units, which comprise one or more neoepitopes.
  • the disclosure provides an immunotherapy construct comprising: i) a polynucleotide comprising a nucleotide sequence encoding an immunomodulating polypeptide, the immunomodulating polypeptide comprising, in the specified order: a. a first targeting unit, a first joint region; b. an antigenic unit comprising at least one neoepitope and at least one T cell epitope comprised in a patient-present shared cancer antigen; c. a second joint region; and d.
  • a second targeting unit ii) an immunomodulating polypeptide encoded by the nucleotide sequence as defined in i); or iii) a multimeric protein consisting of multiple polypeptides as defined in ii), such as a dimeric protein, consisting of two polypeptides as defined in ii).
  • the present disclosure relates to an immunotherapy construct comprising: i) a polynucleotide comprising a nucleotide sequence encoding an immunomodulating polypeptide, the immunomodulating polypeptide comprising, in the specified order: a. a first targeting unit, a first joint region; b. an antigenic unit comprising at least one neoepitope and at least one T cell epitope comprised in a patient-present shared cancer antigen; c. a second joint region; and d. a second targeting unit; ii) an immunomodulating polypeptide encoded by the nucleotide sequence as defined in i); or iii) a multimeric protein consisting of multiple polypeptides as defined in ii).
  • the present disclosure relates to an immunotherapy construct comprising: i) a polynucleotide comprising a nucleotide sequence encoding an immunomodulating polypeptide, the immunomodulating polypeptide comprising, in the specified order: a. a first targeting unit, a first joint region; b. an antigenic unit comprising at least one neoepitope and at least one T cell epitope comprised in a patient-present shared cancer antigen; c. a second joint region; and d. a second targeting unit; ii) an immunomodulating polypeptide encoded by the nucleotide sequence as defined in i); or iii) a dimeric protein consisting of two polypeptides as defined in ii).
  • the antigenic unit comprises at least one T cell epitope, i.e. one or more T cell epitopes, comprised in one or more patient-present shared cancer antigens and one or more neoepitopes.
  • Antigenic units comprising one or more neoepitopes and T cell epitopes, which are comprised in one or more patient-present shared cancer antigens are described in detail in PCT/EP2021/059353, the content of which is included herein by reference. Any of such antigenic units can be used as antigenic unit in an immunotherapy construct of the disclosure for use in individualized anticancer therapy.
  • the patient-present shared cancer antigens and neoantigens identified in a particular patient are preferably further processed to find those T cell epitopes that will render the immunomodulating polypeptide most effective, when being included into the antigenic unit.
  • the way and order in which such processing is done depends on the data that form the basis for such processing.
  • the processing and selecting of the T cell epitopes to be included in the antigenic unit is carried out as follows:
  • a search in the literature and/or in one or more databases is carried out to retrieve information about and sequences of shared cancer antigens and preferably information about their expression pattern, immunogenicity or predicted immunogenicity, epitopes and/or HLA presentation. Such search is also carried out to determine whether the identified antigen is a patient-present shared cancer antigen or a neoantigen.
  • the sequence thereof is studied to identify T cell epitopes, preferably all T cell epitopes, that are predicted to bind to the patient’s HLA class I/ll alleles.
  • the prediction may be carried out by using prediction tools known in the art, e.g. prediction software known in the art, such as NetMHCpan and similar software.
  • the most promising, sequences of the patient-present shared cancer antigen which are most immunogenic or predicted to be most immunogenic, i.e. those that show predicted binding to one or more of the patient’s HLA class I/ll alleles, are selected for inclusion into the antigenic unit.
  • minimal epitopes are selected, e.g. if only a few promising T cell epitopes were identified in step 2 or if longer stretches of non-immunogenic sequences are present between the epitopes.
  • a longer sequence is selected which comprises several T cell epitopes that bind to the patient’s specific HLA alleles.
  • the full-length sequence of the antigen is selected for inclusion into the antigenic unit.
  • Tumor mutations are discovered by sequencing of tumor and normal tissue and comparing the obtained sequences from the tumor tissue to those of the normal tissue.
  • a variety of methods is available for detecting the presence of a particular mutation or allele in a patient’s DNA or RNA. Such methods include dynamic allele-specific hybridization (DASH), microplate array diagonal gel electrophoresis (MADGE), pyrosequencing, oligonucleotide- specific ligation, the TaqMan system as well as various DNA "chip” technologies such as the Affymetrix SNP chips.
  • DASH dynamic allele-specific hybridization
  • MADGE microplate array diagonal gel electrophoresis
  • pyrosequencing oligonucleotide- specific ligation
  • the TaqMan system as well as various DNA "chip” technologies such as the Affymetrix SNP chips.
  • mutations may be identified by direct protein sequencing. Out of the maybe hundreds or thousands of mutations in the tumor exome, the most promising sequences are selected in silico based on
  • the intention is to identify all relevant epitopes and after a ranking or scoring, determine the sequences to be included in the antigenic unit.
  • Methods known in the art may suitable for scoring, ranking and selecting neoepitopes include those disclosed in WO 2020/065023A1 and WO 2020/221/783A1.
  • any suitable algorithm for such scoring and ranking may be used, including the following:
  • Each mutation is scored with respect to its antigenicity or immunogenicity, and the most antigenic or immunogenic neoepitopes are selected and preferably optimally arranged in the antigenic unit, e.g. as described herein.
  • Antigenic unit of non-individualized anticancer immunotherapy construct comprises an antigenic unit, which comprises one or more T cell epitopes comprised in shared cancer antigens.
  • Shared cancer antigen or “shared tumor antigen” is used herein to describe an antigen that has been described to be expressed by many tumors, either across patients with the same cancer type, or across patients and cancer types.
  • Shared cancer epitope is used herein to describe an amino acid sequence comprised in a shared cancer antigen, which is known or has been predicted to be immunogenic.
  • the antigenic unit of an immunotherapy construct preferably includes hotspots of minimal T cell epitopes, i.e. one or more regions of an antigen that contain multiple minimal epitopes (e.g. having a length of from 7-15 amino acids) that are predicted to be presented by different HLA alleles to cover a broad range of subjects, e.g. an ethnic population or even a world population.
  • hotspots of minimal T cell epitopes i.e. one or more regions of an antigen that contain multiple minimal epitopes (e.g. having a length of from 7-15 amino acids) that are predicted to be presented by different HLA alleles to cover a broad range of subjects, e.g. an ethnic population or even a world population.
  • the antigenic unit of such non-individualized anticancer vaccines comprises one or more T cell epitopes comprised in shared cancer antigens, which are known to be immunogenic, have known expression patterns and/or are known or have been predicted to bind to specific HLA class I and class II molecules.
  • the disclosure provides an immunotherapy construct comprising: i) a polynucleotide comprising a nucleotide sequence encoding an immunomodulating polypeptide, the immunomodulating polypeptide comprising, in the specified order: a. a first targeting unit, a first joint region; b. an antigenic unit comprising at least one T cell epitope comprised in a shared cancer antigen; c. a second joint region; and d.
  • a second targeting unit ii) an immunomodulating polypeptide encoded by the nucleotide sequence as defined in i); or iii) a multimeric protein, such as a dimeric protein, consisting of multiple polypeptides as defined in ii), such as of two polypeptides as defined in ii).
  • the present disclosure relates to an immunotherapy construct comprising: i) a polynucleotide comprising a nucleotide sequence encoding an immunomodulating polypeptide, the immunomodulating polypeptide comprising, in the specified order: a. a first targeting unit, a first joint region; b. an antigenic unit comprising at least one T cell epitope comprised in a shared cancer antigen; c. a second joint region; and d. a second targeting unit; ii) an immunomodulating polypeptide encoded by the nucleotide sequence as defined in i); or iii) a multimeric protein consisting of multiple polypeptides as defined in ii).
  • the present disclosure relates to an immunotherapy construct comprising: i) a polynucleotide comprising a nucleotide sequence encoding an immunomodulating polypeptide, the immunomodulating polypeptide comprising, in the specified order: a. a first targeting unit, a first joint region; b. an antigenic unit comprising at least one T cell epitope comprised in a shared cancer antigen; c. a second joint region; and d. a second targeting unit; ii) an immunomodulating polypeptide encoded by the nucleotide sequence as defined in i); or iii) a dimeric protein consisting of two polypeptides as defined in ii).
  • the antigenic unit comprises at least one T cell epitope, i.e. one or more T cell epitopes, comprised in one or more shared cancer antigens.
  • shared cancer antigens include those disclosed under the heading “Antigenic unit of individualized anticancer immunotherapy constructs comprising one or more patient-present shared cancer antigens or parts or fragments thereof” and scFvs derived from a monoclonal Ig produced by myeloma or lymphoma, also called the myeloma/lymphoma M component in patients with B cell lymphoma or multiple myeloma, telomerase, e.g. hTERT, HIV derived sequences like e. g.
  • the antigenic unit comprises an amino acid sequence of at least 8 amino acids, corresponding to at least about 24 nucleotides, in a nucleotide sequence encoding such antigenic unit.
  • the antigenic unit comprises one or more parts or fragments of a shared cancer antigen, e.g. one or more shared cancer epitopes. In yet another embodiment, the antigenic unit comprises one or more parts or fragments of several shared cancer antigens, e.g. one or more epitopes of several shared cancer antigens. In yet another embodiment, the antigenic unit comprises one or more shared antigens in full length and one or more parts or fragments of one or more shared cancer antigens. Examples include:
  • antigenic units comprising one shared antigen in full length and one or more epitopes of one shared cancer antigen; and ⁇ antigenic units comprising several shared cancer antigens, each of them in full length and one or more epitopes of one shared cancer antigen;
  • antigenic units comprising one shared antigen in full length and one or more epitopes of several shared cancer antigens; and antigenic units comprising several shared cancer antigens, each of them in full length, and one or more epitopes of several shared cancer antigens.
  • the antigenic unit is designed to include those T cell epitopes that are likely to render the immunomodulating polypeptide effective in a variety of patients, e.g. patients having a certain type of cancer.
  • the selection of the T cell epitopes to be included in the antigenic unit is carried out by performing a search in the literature and/or in one or more databases to retrieve information about and sequences of shared cancer antigens and preferably information about their expression pattern, immunogenicity or predicted immunogenicity, epitopes and/or HLA presentation. T cell epitopes are then identified that are known or predicted to bind to a variety of HLA class I/ll alleles of many patients or that bind a certain subset of HLA class I/ll alleles which is dominant in a certain cancer indication and/or a certain patient population across different cancer indications. Preferably, the most promising, i.e. the T cell epitopes of the shared cancer antigen which are most immunogenic or predicted to be most immunogenic are selected for inclusion into the antigenic unit.
  • the antigenic unit comprises T cell epitopes from multiple different cancer antigens from the same cancer. In a related embodiment, the antigenic unit comprises T cell epitopes from cancer antigens present in multiple different cancer types.
  • Antigenic units of immunomodulating polypeptides comprising one or more infectious antigens or parts or fragments thereof
  • the immunomodulating polypeptide of the disclosure comprises an antigenic unit, which is designed for the treatment or prevention of an infectious disease and the immunomodulating polypeptide is for use in the treatment or prevention of an infectious disease.
  • the antigenic unit comprises one or more T cell epitopes comprised in antigens which are relevant for infectious diseases, e.g. antigens derived from pathogens.
  • the 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. comprised in the same or different proteins of the pathogen.
  • the multiple T cell epitopes are of multiple different pathogens, i.e. comprised in protein 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.
  • antigens derived from pathogens are such of bacterial origin, e.g. tuberculosis antigens and OMP31 from brucellosis, or viral origin, more specifically HIV derived sequences like e.g. gp120 derived sequences, glycoprotein D from HSV-2, and influenza virus antigens like hemagglutinin, nucleoprotein and M2.
  • the antigen may be HPV derived such as E1, E2, E6, E7, L1, L2 E6 or E7 of HPV16 or HPV18.
  • the antigenic unit comprises one or more T cell epitopes comprised in betacoronavirus antigens, e.g. in a SARS-CoV or SARS-CoV-2 antigen.
  • the T cell epitope is comprised in a part of spike protein or the membrane protein or the envelope protein or the nucleocapsid protein or the ORF1a/b or ORF3a protein. In other embodiments, the T cell epitope is comprised in proteins: NCAP, AP3A, spike, ORF1a/b, ORF3a, VME1 and VEMP.
  • the infectious disease treated by the construct of the disclosure is selected from the list consisting of influenza, Herpes, CMV, HPV, HBV, brucellosis, HIV, HSV-2 and tuberculosis.
  • the immunotherapy construct of the disclosure for the treatment of infectious disease is ideal for fighting pandemics and epidemics as it can induce a rapid, strong immune response.
  • Such an immunotherapy construct for the treatment of infectious disease is designed to induce an antigenic effect through selected T cell epitopes.
  • Antigenic units of immunomodulating polypeptides comprising one or more T- cell epitope of an infectious antigen
  • the antigenic unit of the immunomodulating polypeptide comprises at least one T cell epitope from a pathogen.
  • conserveed parts of the genome among many pathogens comprise T cell epitopes capable of initiating immune responses.
  • the at least one T cell epitope is from a conserved region of a pathogen, i.e. conserved between several subgenera, species or strains of respective pathogens.
  • the T cell epitope may be encoded by a nucleic acid 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 epitopes may be comprised in any of the pathogen’s proteins, i.e. in surface proteins but also in the internal proteins such as nucleocapsid protein or replicase polyproteins or in other structural and non-structural proteins; in other words, the T cell epitope may be found in the proteins naturally present in said pathogen.
  • An antigenic unit comprising T cell epitopes from conserved regions of pathogens will provide protection against several species/strains of the pathogen. Such an antigenic unit will also provide protection against multiple variants of a pathogen, which is important for the efficacy of such an immunomodulating polypeptide against future mutated pathogens.
  • Viruses are known to mutate, e.g. undergo viral antigen drift or antigen shift. The finding of conserved regions across a viral genus makes it likely that these conserved regions are needed to maintain essential structures or functions, thus it is anticipated that future mutations will take place in the less-conserved regions. By raising an immune response against the conserved regions, the individual treated with the immunomodulating polypeptide will be, or is at least expected to be, protected also against mutated (and thus novel) strains of the future.
  • the antigenic unit is therefore designed to evoke a cell-mediated immune response through activation of T cells against epitopes of the infectious antigen.
  • T cells recognize epitopes when they have been processed and presented complexed to an MHC molecule.
  • 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.
  • the T cell epitope is selected based on the predicted ability to bind to HLA class I/ll alleles.
  • the antigenic unit includes multiple T cell epitopes that are predicted to bind to HLA class I/ll alleles. The T cell epitopes are selected in silico on the basis of predictive H LA- binding algorithms. After having identified all relevant epitopes, the epitopes are ranked according to their ability to bind to HLA class I/ll alleles and the epitopes that are predicted to bind best are selected to be included in the antigenic unit.
  • Antigenic units comprising T cell epitopes for use in a construct of the disclosure for the prophylactic and therapeutic treatment of betacoronavirus infections and methods which are generally applicable for selecting T cell epitopes for constructs of the disclosure used in the prophylactic and therapeutic treatment of infectious diseases are disclosed in detail in PCT/EP2021/061602, the disclosure of which is incorporated herein by reference.
  • 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: 28) or GLSGL (SEQ ID NO: 142).
  • the unit linker comprises or consists of GGGGS (SEQ ID NO: 21), GGGGSGGGGS (SEQ ID NO: 97), (GGGGS)m (SEQ ID NO: 71), EAAAK (SEQ ID NO: 143), (EAAAK)mGS (SEQ ID NO: 109), (EAAK)mGS (SEQ ID NO: 145) where m is an integer greater than or equal to 1, GPSRLEEELRRRLTEPG (SEQ ID NO: 144), AAY or HEYGAEALERAG (SEQ ID NO: 93).
  • the immunotherapy construct of the disclosure is a polynucleotide which further comprises a nucleotide sequence encoding a signal peptide.
  • the signal peptide is designed to allow secretion of the immunomodulating polypeptide encoded by the nucleic acid comprised in the polynucleotide in the cells transfected with said polynucleotide.
  • Any suitable signal peptide may be used.
  • suitable signal peptides are an Ig VH signal peptide, a human TPA signal peptide, such as SEQ ID NO: 3, and a human MIP1-a signal peptide.
  • the signal peptide is that which is naturally present at the N-terminus of any of the targeting units described herein.
  • the polynucleotide may comprise one nucleotide sequence encoding one signal peptide (for one targeting unit) or two nucleotide sequences encoding two signal peptides (for both targeting units).
  • the polynucleotide comprises a nucleotide sequence encoding a human MIP1-a signal peptide (corresponding to amino acids 1-23 of SEQ ID NO: 1) and preferably comprises a nucleotide sequence encoding a human MIP1-a targeting unit (corresponding to amino acids 24-93 of SEQ ID NO: 1).
  • 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 1-23 of SEQ ID NO: 1.
  • the polynucleotide comprises a nucleotide sequence encoding a signal peptide that comprises the amino acid sequence 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 comprises a nucleotide sequence encoding a signal peptide that comprises the amino acid sequence 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 1-23 of SEQ ID NO: 1.
  • the polynucleotide comprises a nucleotide sequence encoding a signal peptide with the amino acid sequence 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 nucleic acid sequence with SEQ I D NO: 113.
  • the polynucleotide comprises a nucleotide sequence encoding a signal peptide, wherein said nucleotide sequence has at least 85% sequence identity to the nucleic acid sequence with SEQ ID NO: 113, 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: 113.
  • the polynucleotide comprises a nucleotide sequence encoding an Ig VH signal peptide (nucleotides 1-57 of SEQ ID NO: 91) and preferably further comprises a nucleotide sequence encoding an anti-pan HLA class II targeting unit (nucleotides 58-780 of SEQ ID NO: 91).
  • 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 of SEQ ID NO: 2.
  • the polynucleotide comprises a nucleotide sequence encoding a signal peptide that comprises the amino acid sequence of SEQ ID NO: 2, 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 of SEQ ID NO: 2.
  • 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 of SEQ ID NO: 2.
  • the polynucleotide comprises a nucleotide sequence encoding a signal peptide with the amino acid sequence 1-19 of SEQ ID NO: 2.
  • the polynucleotide comprises a nucleotide sequence encoding a signal peptide, wherein said nucleotide sequence has at least 80% sequence identity to the nucleic acid sequence corresponding to nucleotides 1-57 of SEQ ID NO: 91.
  • the polynucleotide comprises a nucleotide sequence encoding a signal peptide, wherein said nucleotide sequence has at least 85% sequence identity to the nucleic acid sequence corresponding to nucleotides 1-57 of SEQ ID NO: 91, 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 nucleotides 1-57 of SEQ ID NO: 91.
  • 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 J., 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.
  • Another preferred mathematical algorithm utilized for the comparison of sequences is the algorithm of Myers and Miller, CABIOS (1989). Such an algorithm is incorporated into the ALIGN program (version 2.0) which is part of the FASTA sequence alignment software package (Pearson WR, Methods Mol Biol, 2000, 132:185-219). Align calculates sequence identities based on a global alignment. AlignO does not penalize to gaps in the end of the sequences. When utilizing the ALIGN and AlignO program for comparing amino acid 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 the immunogenic construct, 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, naphthylalanine 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-l- 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 immunotherapy construct of the disclosure may be in the form of a polynucleotide, e.g. DNA or RNA, including genomic DNA, cDNA and mRNA, either double-stranded or single-stranded.
  • the immunotherapy construct is a DNA immunotherapy construct, i.e. the polynucleotide is a DNA.
  • a further aspect of the disclosure is a polynucleotide comprising a nucleotide sequence encoding an immunomodulating polypeptide comprising, in the specified order: a. a first targeting unit, a first joint region; b. an antigenic unit comprising at least one T cell epitope; c. a second joint region; and d. a second targeting unit.
  • the polynucleotide may be a DNA or an RNA polynucleotide, 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.
  • the polynucleotide is optimized to the species of the subject to which it is administered.
  • the polynucleotide sequence is human codon optimized.
  • Polypeptides and multimeric proteins such as dimeric proteins
  • the immunotherapy construct of the disclosure may be in the form of an immunomodulating polypeptide encoded by the polynucleotide as described above.
  • the immunomodulating polypeptide may be expressed in vitro for production of the immunotherapy construct, or the immunomodulating polypeptide may be expressed in vivo as a result of the administration of the polynucleotide to a subject, as described above.
  • a further aspect of the disclosure is a multimeric protein consisting of multiple polypeptides, such as a dimeric protein consisting of two polypeptides, each of which comprising, in the specified order, a) a first targeting unit, b) a first joint region, c) an antigenic unit comprising at least one T cell epitope, d) a second joint region and e) a second targeting unit, wherein the multiple polypeptides, for example the two polypeptides, are linked to each other via their respective first joint regions and via their respective second joint regions.
  • the multimeric/dimeric protein may be prepared by expression of the polypeptide in vitro.
  • a further aspect of the disclosure is a method for preparing a multimeric protein consisting of multiple polypeptides, such as a dimeric protein consisting of two polypeptides; each of which comprising, in the specified order, a. a first targeting unit, a first joint region; b. an antigenic unit comprising at least one T cell epitope; c. a second joint region; and d.
  • the method comprises: 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 purifying the fraction of multimeric proteins, such as the fraction of dimeric proteins, wherein the polypeptides are linked to each other via their respective first joint regions and via their respective second joint regions.
  • Isolation of the multimeric protein, such as a 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 purification is optional. In other embodiments, the purification is required.
  • the polynucleotide may be comprised in a plasmid for transfection or a vector for transduction.
  • a further aspect of the disclosure is a multimeric protein consisting of multiple polypeptides, each of which comprising, in the specified order, a) a first targeting unit, b) a first joint region, c) an antigenic unit comprising at least one T cell epitope, d) a second joint region and e) a second targeting unit, wherein the multiple polypeptides are linked to each other via their respective first joint regions and via their respective second joint regions.
  • the multimeric protein may be prepared by expression of the polypeptide in vitro.
  • a further aspect 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 first targeting unit, a first joint region; b. an antigenic unit comprising at least one T cell epitope; c. a second joint region; and d.
  • the method comprises: 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 multimeric proteins, wherein the multiple polypeptides are linked to via their respective first joint regions and via their respective second joint regions.
  • Isolation of the multimeric protein in step d) and the purification can be carried out by methods known in the art, including precipitation, differential solubilization and chromatography.
  • the purification is optional. In other embodiments, the purification is required.
  • the polynucleotide may be comprised in a plasmid for transfection or a vector for transduction.
  • 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 first targeting unit, a first joint region; b. an antigenic unit comprising at least one T cell epitope; c. a second joint region; and d.
  • the method comprises: 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 dimeric proteins, wherein the two polypeptides are linked to each other via their respective first joint regions and via their respective second joint regions.
  • 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 polynucleotide may be comprised in a plasmid for transfection or a vector for transduction.
  • the above-described dimeric protein or multimeric protein may be used as the active ingredient in a protein vaccine for the prophylactic or therapeutic treatment of cancerous and/or infectious diseases.
  • the polynucleotide sequence of the immunotherapy construct may be a DNA polynucleotide comprised in a vector suitable for transfecting or transducing a host cell and expression of an immunomodulating polypeptide or multimeric/dimeric protein encoded by the polynucleotide, i.e. an expression vector, such as a DNA plasmid or viral vector, such as a DNA plasmid.
  • the vector is suitable for transfecting a host cell and expression of an mRNA encoding for the polypeptide/multimeric protein.
  • the vector allows for easy exchange of the various units described above, particularly the antigenic unit in case of individualized immunotherapy constructs.
  • the disclosure provides a vector comprising a polynucleotide comprising a nucleotide sequence encoding an immunomodulating polypeptide, the immunomodulating polypeptide comprising, in the specified order: a. a first targeting unit, a first joint region; b. an antigenic unit comprising at least one T cell epitope; c. a second joint region; and d. a second targeting unit.
  • the vector is a DNA plasmid and the polynucleotide is DNA.
  • the present disclosure provides a host cell comprising a vector as described herein.
  • 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 further aspect of the disclosure is a vector comprising:
  • A polynucleotide comprising a nucleotide sequence encoding a polypeptide comprising, in the specified order, a. a first targeting unit, a first joint region; b. an antigenic unit comprising at least one T cell epitope; c. a second joint region; and d. a second targeting unit; and
  • the one or more immunostimulatory compounds can 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. nucleic acid 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.
  • a co-expression element 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.
  • 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 nucleic acid 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 promotors, i.e. the polycistronic plasmid comprises a promoter for each of the nucleic acid 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.
  • the polycistronic plasmid of the present disclosure comprises one or more nucleic acid 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) orXCRI (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
  • XCRI
  • the immunostimulatory compound is selected from the list consisting of MIP-1a, preferably human MIP-1a (hMIP-1a or I_078b or CCL3L1), RANTES (CCL5), MIR-1b (CCL4), MIP-3a (CCL20), CCL19, CCL 21, XCL1 orXCL2.
  • 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), CD137 (4-1 BB), CD27, ICOSL (CD275) or RANK.
  • a receptor of the TNF receptor superfamily including CD40 (cluster of differentiation 40), CD137 (4-1 BB), CD27, ICOSL (CD275) or RANK.
  • Such immunostimulatory compounds may be selected from the list consisting of CD40L (CD40 ligand, CD154), CD137L (4-1 BBL, 4-1 BB 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.
  • 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 A1.
  • PAMPs/DAMPs include those can be included as a nucleic acid 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 A1), 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,
  • the immunostimulatory compound is a growth factor, such as GM-CSF (granulocyte-macrophage colony-stimulating factor), FLT-3L, IL-15 or IL-4.
  • GM-CSF granulocyte-macrophage colony-stimulating factor
  • FLT-3L granulocyte-macrophage colony-stimulating factor
  • IL-15 IL-15
  • the polycistronic DNA plasmid comprises nucleic acid sequences encoding 2, 3, 4, 5, 6, 7 or 8 immunostimulatory compounds.
  • the DNA plasmid comprises nucleic acid 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 nucleic acids 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 further embodiment of the disclosure is a host cell comprising i) a polynucleotide comprising a nucleotide sequence encoding an immunomodulating polypeptide, the immunomodulating polypeptide comprising, in the specified order: a) a first targeting unit, a first joint region; b) an antigenic unit comprising at least one T cell epitope; c) a second joint region; and d) a second targeting unit; or ii) a vector comprising the polynucleotide.
  • the vector may be the polycistronic vector described herein.
  • Suitable host cells include prokaryotes, yeast, insect or higher eukaryotic cells.
  • compositions comprising a construct, polynucleotide, polypeptide, multimeric protein or dimeric protein as disclosed herein, and one or more pharmaceutically acceptable carriers.
  • 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.
  • pharmaceutically acceptable adjuvants include, but are not limited to poly-ICLC, 1018 ISS, aluminum salts, Amplivax, AS 15, BCG, CP-870,893, CpG7909, CyaA, dSLIM, GM-CSF, IC30, IC31, Imiquimod, ImuFact EV1 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, Pam3
  • the composition can be administered without additional adjuvant; thus, in some embodiments, the composition does not comprise an adjuvant.
  • the 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® P188, 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,
  • 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 immunotherapy construct of the disclosure may be administered to a subject as a vaccine, i.e. a pharmaceutical composition comprising the construct, e.g. in the form of a polynucleotide or dimeric/multimeric protein and pharmaceutically acceptable carrier.
  • a further aspect of the disclosure is a vaccine, comprising a pharmaceutically acceptable carrier and the immunotherapy construct.
  • 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 NaCI, 6 mM KCI, 3 mM CaCI2, 2 mM MgCI2, 10 mM 4-(2-hydroxyethyl)-1-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 ISS, aluminum salts, Amplivax, AS 15, BCG, CP-870,893, CpG7909, CyaA, dSLIM, GM-CSF, IC30, IC31, Imiquimod, ImuFact EV1 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, Pam3Cy
  • 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® P188, 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 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. such as a liquid formulation for injection, e.g. for intradermal or intramuscular injection.
  • the vaccine 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 oral, enteral or intravesicular (to the bladder).
  • the vaccine comprises a polynucleotide as described herein, preferably a polynucleotide and a pharmaceutically acceptable carrier, and is administered by intramuscular or intradermal injection.
  • the vaccine 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 vaccine of the disclosure typically comprises the polypeptide/dimeric protein/multimeric protein in the range of from 5 pg to 5 mg.
  • the amount of polynucleotide/polypeptide/multimeric/dimeric 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.
  • the disclosure relates to a method for preparing a vaccine comprising the multimeric/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 present disclosure provides a method of preparing an immunomodulating polypeptide, multimeric protein or dimeric protein, said method comprising: a) transfecting or transducing a cell with the vector as described herein or the polynucleotide as described herein; b) culturing the cell, whereby the cell expresses an immunomodulating polypeptide encoded by said polynucleotide; and c) collecting and purifying the multimeric protein and/or the immunomodulating polypeptide expressed by the cell.
  • the present disclosure provides a method of preparing an immunomodulating polypeptide or a multimeric protein, said method comprising: a) transfecting or transducing a cell with the vector as described herein or the polynucleotide as described herein; b) culturing the cell, whereby the cell expresses an immunomodulating polypeptide encoded by said polynucleotide; and c) collecting and purifying the multimeric protein and/or the immunomodulating polypeptide expressed by the cell.
  • the present disclosure provides a method of preparing an immunomodulating polypeptide or a dimeric protein, said method comprising: a) transfecting or transducing a cell with the vector as described herein or the polynucleotide as described herein; b) culturing the cell, whereby the cell expresses an immunomodulating polypeptide encoded by said polynucleotide; and c) collecting and purifying the dimeric protein and/or the immunomodulating polypeptide expressed by the cell.
  • the present disclosure provides a pharmaceutical composition
  • a pharmaceutical composition comprising the polynucleotide, the immunomodulating polypeptide, the dimeric protein or the multimeric protein as described herein and a pharmaceutically acceptable carrier.
  • the acceptable carrier may be any carrier as described herein.
  • the pharmaceutical composition may be formulated into any way suitable administration to a subject, e.g. patient, 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, e.g. patient, of either an immunomodulating polypeptide/protein or a polynucleotide, such as administered by intradermal, intramuscular, intranodal or subcutaneous injection, or by mucosal or epithelial application, such as intranasal, oral, enteral or intravesicular (to the bladder) administration.
  • an immunomodulating polypeptide/protein or a polynucleotide such as administered by intradermal, intramuscular, intranodal or subcutaneous injection, or by mucosal or epithelial application, such as intranasal, oral, enteral or intravesicular (to the bladder) administration.
  • the pharmaceutical composition comprises a polynucleotide, preferably a polynucleotide and a pharmaceutically acceptable carrier and is administered by intramuscular or intradermal injection.
  • the pharmaceutical composition typically comprises the polynucleotide in a range of 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,
  • the pharmaceutical composition typically comprises the polypeptide/dimeric protein/multimeric protein in the range of from 5 pg to 5 mg.
  • the present disclosure provides a pharmaceutical composition as described herein for use as a medicament.
  • the present disclosure provides a pharmaceutical composition as described herein for use in the treatment of disease, such as an infectious disease.
  • the infectious disease is a disease caused by a pathogen.
  • the pathogen is selected from the group consisting of bacteria, viruses (such as betacoronaviruses or influenza viruses or HIV), fungi and parasites.
  • the pathogen is any of the pathogens described previously in this disclosure.
  • the present disclosure provides a method for treating infectious diseases, said method comprising administering the polynucleotide, immunomodulating polypeptide, the dimeric protein or the multimeric protein as described herein, the vector as described herein or the pharmaceutical composition as described herein to a subject in need thereof.
  • the present disclosure provides a pharmaceutical composition as described herein for use in the treatment of cancer.
  • the cancer is a solid or liquid cancer.
  • the cancer is selected from the group consisting of breast cancer, ovarian cancer, colon cancer, prostate cancer, bone cancer, colorectal cancer, gastric cancer, lymphoma, malignant melanoma, liver cancer, small cell lung cancer, non-small cell lung cancer, pancreatic cancer, thyroid cancers, kidney cancer, cancer of the bile duct, brain cancer, cervical cancer, bladder cancer, esophageal cancer, Hodgkin's disease and adrenocortical cancer.
  • the present disclosure provides a method for treating cancer and/or infectious diseases, said method comprising administering the polynucleotide, polypeptide, the dimeric protein or the multimeric protein as described herein, the vector as described herein, the host cell as described herein, and/or the pharmaceutical composition as described herein, to a subject in need thereof.
  • the disclosure provides a method for treating a subject having or suspected of having cancer and/or an infectious disease or being in need of prevention of cancer and/or an infectious disease, the method comprising administering to the subject a vaccine comprising a pharmaceutically acceptable carrier and: an immunotherapy construct comprising: i) a polynucleotide comprising a nucleotide sequence encoding an immunomodulating polypeptide, the immunomodulating polypeptide comprising, in the specified order: a. a first targeting unit, a first joint region; b. an antigenic unit comprising at least one T cell epitope; c. a second joint region; and d.
  • a second targeting unit ii) an immunomodulating polypeptide encoded by the nucleotide sequence as defined in i); or iii) a multimeric protein, such as a dimeric protein, consisting of multiple polypeptides as defined in ii).
  • the disclosure provides a method for treating a subject having or suspected of having cancer and/or an infectious disease or being in need of prevention of cancer and/or an infectious disease, the method comprising administering to the subject a vaccine comprising a pharmaceutically acceptable carrier and: an immunotherapy construct comprising: i) a polynucleotide comprising a nucleotide sequence encoding an immunomodulating polypeptide, the immunomodulating polypeptide comprising, in the specified order: a. a first targeting unit, a first joint region; b. an antigenic unit comprising at least one T cell epitope; c. a second joint region; and d.
  • the disclosure provides a method for treating a subject having or suspected of having cancer and/or an infectious disease or being in need of prevention of cancer and/or an infectious disease, the method comprising administering to the subject a vaccine comprising a pharmaceutically acceptable carrier and: an immunotherapy construct comprising: i) a polynucleotide comprising a nucleotide sequence encoding an immunomodulating polypeptide, the immunomodulating polypeptide comprising, in the specified order: a.
  • a first targeting unit a first joint region; b. an antigenic unit comprising at least one T cell epitope; c. a second joint region; and d. a second targeting unit; ii) an immunomodulating polypeptide encoded by the nucleotide sequence as defined in i); or iii) a dimeric protein consisting of two polypeptides as defined in ii).
  • an immunomodulating construct comprising: i) a polynucleotide comprising a nucleotide sequence encoding an immunomodulating polypeptide, the immunomodulating polypeptide comprising, in the specified order: a. a first targeting unit, a first joint region; b. an antigenic unit comprising at least one T cell epitope; c. a second joint region; and d.
  • a second targeting unit ii) an immunomodulating polypeptide encoded by the nucleotide sequence as defined in i); or iii) a multimeric protein consisting of multiple immunomodulating polypeptides as defined in ii), such as two polypeptides, for use in a method of treatment of a subject suffering or suspected of suffering from a cancer and/or an infectious disease or for use in a method of treatment or prevention of an infectious disease.
  • an immunotherapy construct comprising: i) a polynucleotide comprising a nucleotide sequence encoding an immunomodulating polypeptide, the immunomodulating polypeptide comprising, in the specified order: a. a first targeting unit, a first joint region; b. an antigenic unit comprising at least one T cell epitope; c. a second joint region; and d.
  • a second targeting unit ii) an immunomodulating polypeptide encoded by the nucleotide sequence as defined in i); or iii) a multimeric protein consisting of multiple immunomodulating polypeptides as defined in ii), for use in a method of treatment of a subject suffering or suspected of suffering from a cancer and/or an infectious disease or for use in a method of treatment or prevention of an infectious disease.
  • an immunotherapy construct comprising: i) a polynucleotide comprising a nucleotide sequence encoding an immunomodulating polypeptide, the immunomodulating polypeptide comprising, in the specified order: a. a first targeting unit, a first joint region; b. an antigenic unit comprising at least one T cell epitope; c. a second joint region; and d.
  • a second targeting unit ii) an immunomodulating polypeptide encoded by the nucleotide sequence as defined in i); or iii) a dimeric protein consisting of two immunomodulating polypeptides as defined in ii), for use in a method of treatment of a subject suffering or suspected of suffering from a cancer and/or an infectious disease or for use in a method of treatment or prevention of an infectious disease.
  • the vaccine, the immunotherapy construct, the immunomodulating polypeptide, the multimeric/dimeric protein and the different units and regions are described in detail earlier in this application.
  • a vaccine comprising: i) a polynucleotide comprising a nucleotide sequence encoding an immunomodulating polypeptide, the immunomodulating polypeptide comprising, in the specified order: a. a first targeting unit, a first joint region; b. an antigenic unit comprising at least one T cell epitope; c. a second joint region; and d.
  • a second targeting unit ii) an immunomodulating polypeptide encoded by the nucleotide sequence as defined in i); or iii) a multimeric protein consisting of multiple immunomodulating polypeptides as defined in ii), such as two polypeptides, for the manufacture of a medicament for the treatment of a subject suffering from a cancer or for the treatment or prevention of an infectious disease in a subject, wherein the medicament is administered to said subject.
  • a vaccine comprising: i) a polynucleotide comprising a nucleotide sequence encoding an immunomodulating polypeptide, the immunomodulating polypeptide comprising, in the specified order: a. a first targeting unit, a first joint region; b. an antigenic unit comprising at least one T cell epitope; c. a second joint region; and d.
  • a second targeting unit ii) an immunomodulating polypeptide encoded by the nucleotide sequence as defined in i); or iii) a multimeric protein consisting of multiple immunomodulating polypeptides as defined in ii), for the manufacture of a medicament for the treatment of a subject suffering from a cancer or for the treatment or prevention of an infectious disease in a subject, wherein the medicament is administered to said subject.
  • a vaccine comprising: i) a polynucleotide comprising a nucleotide sequence encoding an immunomodulating polypeptide, the immunomodulating polypeptide comprising, in the specified order: a. a first targeting unit, a first joint region; b. an antigenic unit comprising at least one T cell epitope; c. a second joint region; and d.
  • a second targeting unit ii) an immunomodulating polypeptide encoded by the nucleotide sequence as defined in i); or iii) a dimeric protein consisting of two immunomodulating polypeptides as defined in ii), for the manufacture of a medicament for the treatment of a subject suffering from a cancer or for the treatment or prevention of an infectious disease in a subject, wherein the medicament is administered to said subject.
  • the vaccine, the immunotherapy construct, the immunomodulating polypeptide, the multimeric/dimeric protein and the different units and regions are described in detail earlier in this application.
  • a vaccine comprising: i) a polynucleotide comprising a nucleotide sequence encoding an immunomodulating polypeptide, the immunomodulating polypeptide comprising, in the specified order: a. a first targeting unit, a first joint region; b. an antigenic unit comprising at least one T cell epitope; c. a second joint region; and d.
  • a second targeting unit ii) an immunomodulating polypeptide encoded by the nucleotide sequence as defined in i); or iii) a multimeric protein consisting of multiple immunomodulating polypeptides as defined in ii), such as a dimeric protein consisting of two polypeptides, for the treatment of a subject suffering or suspected of suffering from a cancer or for the treatment or prevention of an infectious disease in a subject, wherein the vaccine is administered to said subject.
  • a vaccine comprising: i) a polynucleotide comprising a nucleotide sequence encoding an immunomodulating polypeptide, the immunomodulating polypeptide comprising, in the specified order: a. a first targeting unit, a first joint region; b. an antigenic unit comprising at least one T cell epitope; c. a second joint region; and d.
  • a second targeting unit ii) an immunomodulating polypeptide encoded by the nucleotide sequence as defined in i); or iii) a multimeric protein consisting of multiple immunomodulating polypeptides as defined in ii), for the treatment of a subject suffering or suspected of suffering from a cancer or for the treatment or prevention of an infectious disease in a subject, wherein the vaccine is administered to said subject.
  • a vaccine comprising: i) a polynucleotide comprising a nucleotide sequence encoding an immunomodulating polypeptide, the immunomodulating polypeptide comprising, in the specified order: a. a first targeting unit, a first joint region; b. an antigenic unit comprising at least one T cell epitope; c. a second joint region; and d.
  • a second targeting unit ii) an immunomodulating polypeptide encoded by the nucleotide sequence as defined in i); or iii) a dimeric protein consisting of two immunomodulating polypeptides as defined in ii), for the treatment of a subject suffering or suspected of suffering from a cancer or for the treatment or prevention of an infectious disease in a subject, wherein the vaccine is administered to said subject.
  • the vaccine, the immunotherapy construct, the immunomodulating polypeptide, the multimeric/dimeric protein and the different units and regions are described in detail earlier in this application.
  • a vaccine comprising: i) a polynucleotide comprising a nucleotide sequence encoding an immunomodulating polypeptide, the immunomodulating polypeptide comprising, in the specified order: a. a first targeting unit, a first joint region; b. an antigenic unit comprising at least one T cell epitope; c. a second joint region; and d.
  • a second targeting unit ii) an immunomodulating polypeptide encoded by the nucleotide sequence as defined in i); or iii) a multimeric protein consisting of multiple immunomodulating polypeptides as defined in ii), such as a dimeric protein consisting of two polypeptides, when used in the treatment of cancer and/or in the treatment or prevention of an infectious disease.
  • a vaccine comprising: i) a polynucleotide comprising a nucleotide sequence encoding an immunomodulating polypeptide, the immunomodulating polypeptide comprising, in the specified order: a. a first targeting unit, a first joint region; b. an antigenic unit comprising at least one T cell epitope; c. a second joint region; and d. a second targeting unit; ii) an immunomodulating polypeptide encoded by the nucleotide sequence as defined in i); or iii) a multimeric protein consisting of multiple immunomodulating polypeptides as defined in ii), when used in the treatment of cancer and/or in the treatment or prevention of an infectious disease.
  • a vaccine comprising: i) a polynucleotide comprising a nucleotide sequence encoding an immunomodulating polypeptide, the immunomodulating polypeptide comprising, in the specified order: a. a first targeting unit, a first joint region; b. an antigenic unit comprising at least one T cell epitope; c. a second joint region; and d. a second targeting unit; ii) an immunomodulating polypeptide encoded by the nucleotide sequence as defined in i); or iii) a dimeric protein consisting of two immunomodulating polypeptides as defined in ii), when used in the treatment of cancer and/or in the treatment or prevention of an infectious disease.
  • the vaccine, the immunotherapy construct, the immunomodulating polypeptide, the multimeric/dimeric protein and the different units and regions are described in detail earlier in this application.
  • a vaccine comprising: i) a polynucleotide comprising a nucleotide sequence encoding an immunomodulating polypeptide, the immunomodulating polypeptide comprising, in the specified order: a. a first targeting unit, a first joint region; b. an antigenic unit comprising at least one T cell epitope; c. a second joint region; and d.
  • a second targeting unit ii) an immunomodulating polypeptide encoded by the nucleotide sequence as defined in i); or iii) a multimeric protein consisting of multiple immunomodulating polypeptides as defined in ii), such as two polypeptides, for treatment of a cancer and/or for treatment or prevention of an infectious disease.
  • a vaccine comprising: i) a polynucleotide comprising a nucleotide sequence encoding an immunomodulating polypeptide, the immunomodulating polypeptide comprising, in the specified order: a. a first targeting unit, a first joint region; b. an antigenic unit comprising at least one T cell epitope; c. a second joint region; and d. a second targeting unit; ii) an immunomodulating polypeptide encoded by the nucleotide sequence as defined in i); or iii) a multimeric protein consisting of multiple immunomodulating polypeptides as defined in ii), for treatment of a cancer and/or for treatment or prevention of an infectious disease.
  • a vaccine comprising: i) a polynucleotide comprising a nucleotide sequence encoding an immunomodulating polypeptide, the immunomodulating polypeptide comprising, in the specified order: a. a first targeting unit, a first joint region; b. an antigenic unit comprising at least one T cell epitope; c. a second joint region; and d. a second targeting unit; ii) an immunomodulating polypeptide encoded by the nucleotide sequence as defined in i); or iii) a dimeric protein consisting of two immunomodulating polypeptides as defined in ii), for treatment of a cancer and/or for treatment or prevention of an infectious disease.
  • the vaccine, the immunotherapy construct, the immunomodulating polypeptide, the multimeric/dimeric protein and the different units and regions are described in detail earlier in this application.
  • a medicament for the treatment of cancer in a subject having or suspected of having cancer, or for the therapeutic or prophylactic treatment of an infectious disease in a subject by administering to the subject a vaccine comprising: i) a polynucleotide comprising a nucleotide sequence encoding an immunomodulating polypeptide, the immunomodulating polypeptide comprising, in the specified order: a. a first targeting unit, a first joint region; b. an antigenic unit comprising at least one T cell epitope; c. a second joint region; and d.
  • a second targeting unit ii) an immunomodulating polypeptide encoded by the nucleotide sequence as defined in i); or iii) a multimeric protein consisting of multiple immunomodulating polypeptides as defined in ii), such as a dimeric protein consisting of two polypeptides.
  • a medicament for the treatment of cancer in a subject having or suspected of having cancer, or for the therapeutic or prophylactic treatment of an infectious disease in a subject by administering to the subject a vaccine comprising: i) a polynucleotide comprising a nucleotide sequence encoding an immunomodulating polypeptide, the immunomodulating polypeptide comprising, in the specified order: a.
  • a first targeting unit a first joint region; b. an antigenic unit comprising at least one T cell epitope; c. a second joint region; and d. a second targeting unit; ii) an immunomodulating polypeptide encoded by the nucleotide sequence as defined in i); or iii) a multimeric protein consisting of multiple immunomodulating polypeptides as defined in ii).
  • a medicament for the treatment of cancer in a subject having or suspected of having cancer, or for the therapeutic or prophylactic treatment of an infectious disease in a subject by administering to the subject a vaccine comprising: i) a polynucleotide comprising a nucleotide sequence encoding an immunomodulating polypeptide, the immunomodulating polypeptide comprising, in the specified order: a. a first targeting unit, a first joint region; b. an antigenic unit comprising at least one T cell epitope; c. a second joint region; and d. a second targeting unit; ii) an immunomodulating polypeptide encoded by the nucleotide sequence as defined in i); or iii) a dimeric protein consisting of two immunomodulating polypeptides as defined in ii).
  • the vaccine, the immunotherapy construct, the immunomodulating polypeptide, the multimeric/dimeric protein and the different units and regions are described in detail earlier in this application. Examples
  • Example 1 Design and production of DNA plasmids VB4212, TECH002-CV008,
  • TECH002-CV001 that encode immunotherapy constructs with CCL3L1 and mGM- CSF as targeting units and CT26 derived point mutation epitopes as antigenic unit
  • VB1026 (SEQ ID NO: 1) comprising the following units (Signal peptide, 1 st targeting unit, 1 st joint region) (SEQ ID NO: 115, 116, 69, 114, 86 and 117) as described in Table 1 does not encode any antigenic units or 2 nd targeting unit. This plasmid was used as negative control in the in vivo studies.
  • DNA plasmids comprising 8 CT26 derived point mutation epitopes:
  • Exome sequencing and RNA sequencing of the mouse colon cancer cell line CT26 revealed hundreds to thousands of tumor-specific non-synonymous mutations compared to the wild type Balb/c mouse strain exome.
  • In silico methods were used to identify potential immunogenic sequences, i.e. epitopes comprising a mutation, and 8 of them (T able 2) were chosen for inclusion into the antigenic unit of the first polypeptide encoded by the above-mentioned DNA plasmids.
  • the epitopes in said antigenic unit are separated by glycine-serine linkers ((GGGGS)2, SEQ ID NO: 10), i.e. all epitopes but the terminal epitope are arranged in subunits, each subunit consisting of one epitope and one (GGGGS) 2 linker.
  • Each of these DNA plasmids is a model of a DNA plasmid encoding for an individualized polypeptide, i.e. one that comprises an antigenic unit comprising several patient-specific epitopes, e.g. several neoepitopes and/or several patient-present shared cancer epitopes, with the patient-present shared cancer antigens being mutated patient-present shared cancer antigens, or a model of a DNA plasmid encoding for a non-individualized polypeptide, i.e. one that comprises an antigenic unit comprising several shared cancer epitopes, with the shared cancer antigens being mutated shared cancer antigens.
  • Table 2 The sequence of 8 epitopes with mutations from CT26 cell line included in plasmid DNA constructs
  • Example 2 In vitro assessment of expression and secretion of proteins encoded by immunotherapy constructs VB4212, TECH002-CV008, TECH002-CV001 that comprises CCL3L1 and mGM-CSF as targeting units and CT26 derived point mutation epitopes as antigenic unit
  • the purpose of this study was to characterize the protein expression and secretion post transient transfection of mammalian cells with the VB4212, TECH002-CV008, and TECH002-CV001 DNA plasmids by measuring the presence of proteins in the cell supernatant by an ELISA assay using antibodies detecting the targeting and dimerization units.
  • the Expi293F 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 CO2 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 mouse anti-human IgG CH3 domain antibody (capture antibody, 100 pl/well, 2 pg/ml, MCA878G, Bio-Rad) and goat anti-human CCL3/MIP-1 alpha antibody (secondary biotinylated antibody, 100 pl/well, 0.2 mg/ml, BAF270, R&D systems).
  • mouse anti-human IgG CH3 domain antibody capture antibody, 100 pl/well, 2 pg/ml, MCA878G, Bio-Rad
  • goat anti-human CCL3/MIP-1 alpha antibody secondary biotinylated antibody, 100 pl/well, 0.2 mg/ml, BAF270, R&D systems.
  • Figure 4 shows that all constructs with different designs were expressed and secreted as proteins from transfected Expi293F cells.
  • Example 3 Assessment of T cell responses induced against 8 T-cell epitopes encoded by immunotherapy constructs VB4212, TECH002-CV008, TECH002- CV001 that comprises CCL3L1 and mGM-CSF as targeting units and CT26 derived point mutation epitopes as antigenic unit
  • 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.
  • VB1026 was used as a negative control.
  • a final dose of 6 pg DNA plasmid dissolved in sterile PBS was administered by intramuscular needle injection to each tibialis anterior (2 x 25 mI, 120 pg/ml), followed by electroporation with AgilePulse in vivo electroporation system (BTX, USA).
  • the spleens were collected 11 days after vaccination and mashed in a 70 pm cell strainer to obtain a single cell suspension.
  • the red blood cells were lysed using ammonium- chloride-potassium (ACK) lysing buffer.
  • the splenocytes were counted using the NucleoCounter NC-202 (ChemoMetec, Denmark) and resuspended to a final concentration of 6x10 6 cells/ml.
  • the splenocytes were then seeded 6x10 5 cells/well and re-stimulated with 4 pg/ml of single peptides corresponding to plasmid encoded T cell epitopes (Table 2) for 22 hours. No-peptide-stimulation was used as negative control.
  • the stimulated splenocytes were analyzed for IFN-y responses using the IFN-y FluoroSpot kit (Mabtech AB, Sweden). Spot-forming cells were measured in an IRIS Fluorospot and ELISpot plate reader (Mabtech AB) and analyzed using the Apex software (Mabtech AB). Results are shown as the mean number of IFN-y+ spots/10 6 splenocytes.
  • mice administered with the negative control VB1026 has very low basal immunogenicity, whereas all the immunotherapy constructs tested; VB4212, TECH002-CV008, and TECH002-CV001 that comprises CCL3L1 and mGM- CSF as the first and second targeting units, respectively, induced strong T cell responses against the encoded T cell epitopes described in Table 2.
  • Example 4 Design and production of DNA plasmid VB4166 that encode immunotherapy constructs with CCL3L1 and human FLT3L as targeting units and CT26 derived point mutation epitopes as antigenic unit
  • the gene sequence of construct VB4166 was synthesized and cloned into the expression vector pUMVC4a and were ordered from Genscript (Genscript Biotech B.V., Netherlands).
  • Genscript Genscript Biotech B.V., Netherlands.
  • the DNA plasmid comprising nucleotide sequences encoding different units/parts are described in Table 3 and 2.
  • VB1026 (SEQ ID NO: 1) comprising the following units (Signal peptide, 1 st targeting unit, 1 st joint region) (SEQ ID NO: 115, 116, 69, 114, 86 and 117) as described in Table 1 and does not encode any antigenic units or 2 nd targeting unit.
  • This plasmid was used as negative control in in vivo studies.
  • Example 5 In vitro assessment of expression and secretion of protein encoded by immunotherapy construct VB4166 that comprises CCL3L1 and mFLT3L as targeting units and CT26 derived point mutation epitopes as antigenic unit
  • the purpose of this study was to characterize the protein expression and secretion post transient transfection of mammalian cells with the VB4166 DNA plasmid by measuring the presence of protein in the cell supernatant by an ELISA assay using antibodies detecting the targeting and dimerization units.
  • HEK293 cells ATCC were transiently transfected with the above-mentioned DNA plasmids.
  • the transfected cells were then maintained for 6 days at 37°C with 5% CO2, then the cell supernatant was collected for characterization of the expression and secretion of the proteins encoded by the plasmids by sandwich ELISA of the supernatant using antibodies specific for mouse anti-human IgG CH3 domain antibody (capture antibody, 100 pl/well, 2 pg/ml, MCA878G, Bio-Rad) and goat anti-human CCL3/MIP-1 alpha antibody (secondary biotinylated antibody, 100 pl/well, 0.2 mg/ml, BAF270, R&D systems).
  • mouse anti-human IgG CH3 domain antibody capture antibody, 100 pl/well, 2 pg/ml, MCA878G, Bio-Rad
  • goat anti-human CCL3/MIP-1 alpha antibody secondary biotinylated antibody, 100 pl/well, 0.2 mg/ml, BAF270, R&D systems.
  • Figure 6 shows that VB4166 (Table 3) was expressed and secreted as a protein from transfected HEK293 cells.
  • Example 6 Assessment of T cell responses induced against 8 T cell epitopes encoded by immunotherapy construct VB4166 that comprises CCL3L1 and mFLT3L as targeting units and CT26 derived point mutation epitopes as antigenic unit
  • 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 construct comprising an antigenic unit, whereas 3 mice/group were used for the negative control.
  • a final dose of 6 pg DNA plasmid of VB4166 and VB1026 (negative control) dissolved in sterile PBS was administered by intramuscular needle injection to each tibialis anterior (2 x 25 mI, 120 pg/ml), followed by electroporation with AgilePulse in vivo electroporation system (BTX, USA).
  • the spleens were collected 10 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.
  • the splenocytes were counted using the NucleoCounter NC-202 (ChemoMetec, Denmark) and resuspended to a final concentration of 6x10 6 cells/ml.
  • the splenocytes were then seeded 6x10 5 cells/well and re-stimulated with 4 pg/ml of single peptides corresponding to plasmid encoded T cell epitopes (Table 2) for 24 hours. No-peptide-stimulation was used as negative control.
  • the stimulated splenocytes were analyzed for IFN-y responses using the IFN-y FluoroSpot kit (Mabtech AB, Sweden). Spot-forming cells were measured in an IRIS Fluorospot and ELISpot plate reader (Mabtech AB) and analyzed using the Apex software (Mabtech AB). Results are shown as the mean number of IFN-y+ spots/10 6 splenocytes.
  • mice administered with the negative control VB1026 have very low basal immunogenicity, whereas the immunotherapy construct tested; VB4166 that comprises CCL3L1 and mFLT3L as the first and second targeting units, respectively, induced strong T cell responses against the plasmid encoded T cell epitopes described in Table 2.
  • Example 7 Design and production of DNA plasmids VB4217, TECH002-CV009 and TECH002-CV002 that encode immunotherapy constructs with CCL3L1 and mGM-CSF as targeting units and HPV16 E6 and E7 as antigenic unit
  • Example 8 In vitro assessment of expression and secretion of proteins encoded by immunotherapy constructs VB4217, TECH002-CV009 and TECH002-CV002 that comprises CCL3L1 and mGM-CSF as targeting units and HPV16 E6 and E7 as antigenic unit
  • the purpose of this study was to characterize the protein expression and secretion post transient transfection of mammalian cells with the VB4217, TECH002-CV009 and TECH002-CV002 DNA plasmids by measuring the presence of proteins in the cell supernatant by an ELISA assay using antibodies detecting the targeting and dimerization units.
  • the Expi293F 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 CO2 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 rat anti-mouse GM-CSF antibody (capture antibody, 100 mI/well, 1 pg/ml, MAB415, R&D systems) and goat anti-human CCL3/MIP-1 alpha antibody (secondary biotinylated antibody, 100 mI/well, 0.2 mg/ml, BAF270, R&D systems).
  • Figure 8 shows that all immunotherapy constructs were expressed and secreted as proteins from transfected Expi293F cells.
  • Example 9 Design and production of DNA plasmids TECH002-CV014, TECH002- CV015, and TECH002-CV016 that encode immunotherapy constructs with mCCL5 and mGM-CSF as targeting units and HPV16 E6 and E7 as antigenic unit
  • Example 10 In vitro assessment of expression and secretion of proteins encoded by immunotherapy constructs TECH002-CV014, TECH002-CV015, and TECH002-CV016 that comprises mCCL5 and mGM-CSF as targeting units and HPV16 E6 and E7 as antigenic unit
  • the purpose of this study was to characterize the protein expression and secretion post transient transfection of mammalian cells with the TECH002-CV014, TECH002-CV015, and TECH002-CV016 DNA plasmids by measuring the presence of proteins in the cell supernatant by an ELISA assay using antibodies detecting the targeting and dimerization units.
  • the Expi293F 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 CO2 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 rat anti-mouse CCL5/RANTES antibody (capture antibody, 100 mI/well, 2 pg/ml, MAB4781, R&D systems) and goat anti-mouse GM-CSF biotinylated antibody (secondary antibody, 100 mI/well, 0.2 mg/ml, BAF415, R&D systems).
  • Figure 9 shows that all immunotherapy constructs were expressed and secreted as proteins from transfected Expi293F cells. Sequence overview SEQ ID NO: 1
  • 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)
  • Trimerization unit is the C-terminal domain of T4 fibritin GYIPEAPRDGQAYVRKDGEWVLLSTFL SEQ ID NO: 97
  • SEQ ID NO 100 Nucleotide sequence encoding amino acids 24-93 of SEQ ID NO: 1 GCACCACTTGCTGCTGACACGCCGACCGCCTGCTGCTTCAGCTACACCTCCCGA CAGATTCCACAGAATTTCAT AGCT GACT ACTTT GAGACGAGCAGCCAGTGCTCCA AGCCCAGT GTCATCTTCCT AACCAAGAGAGGCCGGCAGGTCT GTGCT GACCCCA GT GAGGAGT GGGTCCAGAAAT ACGTCAGT GACCTGGAGCT GAGTGCC
  • SEQ ID NO: 103 Nucleotide sequence encoding amino acids 94-120 of SEQ ID NO: 1
  • SEQ ID NO 104 Nucleotide sequence encoding amino acids 121-130 of SEQ ID NO: 1 GGCGGTGGAAGCAGCGGAGGTGGAAGTGGA
  • Nucleotide sequence encoding amino acids 94-237 of SEQ ID NO: 1 GAGCTCAAAACCCCACTTGGTGACACAACTCACACAGAGCCCAAATCTTGTGACA CACCTCCCCCGTGCCCAAGGTGCCCAGGCGGTGGAAGCAGCGGAGGTGGAAGT GGAGGACAGCCCCGAGAACCACAGGTGTACACCCTGCCCCCATCCCGGGAGGA GAT G ACCAAG AACCAGGTCAGCCT G ACCTGCCT GGTCAAAGGCTTCT ACCCC AG CGACATCGCCGTGGAGTGGGAGAGCAGCGGGCAGCCGGAGAACAACTACAACA CCACGCCTCCCATGCTGGACTCCGACGGCTCCTTCTTCCTCTACAGCAAGCTCAC CGTGGACAAGAGCAGGTGGCAGCAGGGGAACATCTTCTCATGCTCCGTGATGCA TGAGGCTCTGCACAACCGCTTCACGCAGAAGAGCCTCTCCCTGTCCGGGTAAA
  • SEQ ID NO: 120 human FLT3L TQDCSFQHSPISSDFAVKIRELSDYLLQDYPVTVASNLQDEELCGGLWRLVLAQRWM
  • KPWITRQNFSRCLELQCQPDSSTLPPPWSPRPLEATAPTAPQP SEQ ID NO: 121 Native leader sequence for mouse CCL5 M KISAAALTI I LTAAALCTPAPA
  • An immunotherapy construct comprising: i) a polynucleotide comprising a nucleotide sequence encoding an immunomodulating polypeptide, the immunomodulating polypeptide comprising, in the specified order: a. a first targeting unit, a first joint region; b. an antigenic unit comprising at least one T cell epitope; c. a second joint region; and d. a second targeting unit; ii) an immunomodulating polypeptide encoded by the nucleotide sequence as defined in i); or iii) a multimeric protein consisting of multiple polypeptides as defined in ii), such as a dimeric protein consisting of two polypeptides as defined in ii).
  • the immunotherapy construct of item 1 wherein the construct is the dimeric protein and said dimeric protein consists of two immunomodulating polypeptides that are linked to each other via their joint regions, preferably via their respective first joint regions and via their respective second joint regions. 3.
  • first- and/or second joint regions comprise a binding unit which is a non-covalent binding unit.
  • first- and/or second joint regions comprise a binding unit which is a covalent binding unit.
  • first- and/or second joint regions comprise or consist of an artificial sequence.
  • first and second joint regions comprise a covalent binding unit which comprises cysteine residues.
  • the covalent binding unit comprises at least 2 cysteine residues.
  • non-covalent binding unit is or comprises a leucine zipper selected from the group consisting of Jun/Fos-based leucine zipper, PAR-based leucine zipper, Oasis-based leucine zipper, ATF6-based leucine zipper and C/EBPa-based leucine zipper
  • any item 37 wherein the surface molecule is selected from the group consisting of HLA, CD14, CD40, GM-CSF-receptor, FLT- 3RJL-15R, a TNF receptor, 4-1BB/4-1BBL, CD70, ICOSL, chemokine receptors, such as CCR1, CCR5 orXCRI, and Toll-like receptors, such as TLR2, TLR4 or TLR5.
  • the surface molecule is selected from the group consisting of HLA, CD14, CD40, GM-CSF-receptor, FLT- 3RJL-15R, a TNF receptor, 4-1BB/4-1BBL, CD70, ICOSL, chemokine receptors, such as CCR1, CCR5 orXCRI, and Toll-like receptors, such as TLR2, TLR4 or TLR5.
  • the targeting unit is a ligand selected from the group consisting of soluble CD40 ligand, RANTES, MIP-1a, XCL1, XCL2, MIP-1h (CCL4), MIP-3a (CCL20), PAMPs such as flagellin, DAMPS, HMGB1 or HSPs, GM-CSF, FLT-3L, IL-15, 4-1 BB (CD137)/4-1 BBL, CD27, ICOS, anti-HLA-DP, anti-HLA-DR, anti-pan HLA class II, anti-CD40, anti-TLR-2, anti-TLR-4 or anti-TLR-5.
  • the targeting unit is a ligand selected from the group consisting of soluble CD40 ligand, RANTES, MIP-1a, XCL1, XCL2, MIP-1h (CCL4), MIP-3a (CCL20), PAMPs such as flagellin, DAMPS, HMGB1 or HSPs, GM-CSF, FLT-3L
  • the second targeting unit is one selected from the group consisting of soluble CD40 ligand, RANTES, XCL1 , XCL2, MIP-1h (CCL4), MIP-3a (CCL20), PAMPs such as flagellin, DAMPS, HMGB1 , HSPs, GM-CSF, FLT-3L, IL-15, 4-1 BB (CD137)/4-1 BBL, CD27, ICOS, anti-HLA-DP, anti-HLA-DR, anti-pan HLA class II, anti-CD40, anti-TLR-2, anti- TLR-4 or anti-TLR-5, preferably FLT-3L. 51.
  • the construct is the polynucleotide, which further comprises a nucleotide sequence encoding a signal peptide.
  • a multimeric protein such as a dimeric protein, as defined in any of the preceding items, wherein the multiple immunomodulating polypeptides, such as the two immunomodulating polypeptides, are linked to each other via their respective first joint regions and via their respective second joint regions.
  • a method of preparing a pharmaceutical composition comprising: a) providing a polynucleotide, an immunomodulating polypeptide or the multimeric or dimeric protein according to any of items 1-52; and b) combining the polynucleotide, the immunomodulating polypeptide or the multimeric or dimeric protein with a pharmaceutically acceptable carrier.
  • a pharmaceutical composition comprising the polynucleotide, the immunomodulating polypeptide or the multimeric or dimeric protein of any of items 1-52 and a pharmaceutically acceptable carrier.
  • composition of item 54 for use as a medicament.
  • the pharmaceutical composition of item 54 for use in the treatment of disease such as a disease selected from the group consisting of a cancer or an infectious disease.
  • composition of item 54 for use in the treatment of cancer wherein the cancer is a solid or liquid cancer.
  • the pharmaceutical composition of items 57 wherein the cancer is selected from the group consisting of breast cancer, ovarian cancer, colon cancer, prostate cancer, bone cancer, colorectal cancer, gastric cancer, lymphoma, malignant melanoma, liver cancer, small cell lung cancer, non-small cell lung cancer, pancreatic cancer, thyroid cancers, kidney cancer, cancer of the bile duct, brain cancer, cervical cancer, bladder cancer, esophageal cancer, Hodgkin's disease and adrenocortical cancer.
  • the pharmaceutical composition of item 54 for use in the treatment of an infectious disease, wherein the infectious disease is a condition caused by a pathogen and the treatment is a prophylactic or therapeutic treatment.
  • a vector comprising the polynucleotide of any of items 1-51.
  • a host cell comprising the vector of item 60.
  • a method of preparing an immunomodulating polypeptide or multimeric or dimeric protein comprising: a) transfecting or transducing a cell with the vector as defined in item 60 or the polynucleotide according to any of items 1-51 ; b) culturing the cell, whereby the cell expresses an immunomodulating polypeptide encoded by said polynucleotide; and c) obtaining and optionally purifying the multimeric/dimeric protein and/or the immunomodulating polypeptide expressed by the cell.
  • step c comprises the step of purifying the fraction containing the multimeric protein, such as the dimeric protein, wherein two immunomodulating polypeptides are linked to each other via their respective first joint regions and via their respective second joint regions.
  • a method for treating cancer and/or infectious disease comprising administering the polynucleotide, the immunomodulating polypeptide or the multimeric or dimeric protein according to any of items 1-53, the vector according to item 60, or the pharmaceutical composition according to any one of items 54-59, to a subject in need thereof.

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Abstract

La présente invention concerne des constructions d'immunothérapie pour induire une réponse immunitaire dirigée contre des antigènes spécifiques d'un patient chez un hôte pour traiter un cancer ou des maladies infectieuses, par exemple par ciblage de la construction sur des cellules présentatrices d'antigène (CPA). L'invention concerne en outre des polynucléotides, des vecteurs, des cellules hôtes et des compositions pharmaceutiques comprenant ladite construction d'immunothérapie.
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