Ubiquitin vaccine components

Abstract

Provided herein is a vaccine component comprising a ubiquitin covalently bonded to an antigen, wherein the vaccine component does not comprise a cell permeability tag; as well as vaccines comprising such a vaccine component. The vaccine component may be useful in the activation of dendritic cells. Also provided are combinations comprising such a vaccine component or vaccine and a biologically active substance; and formulations comprising such a vaccine component, vaccine, or combination. The vaccine component, vaccine, combination, or formulation may be used as a medicament, for example in the treatment of a condition selected from a cancer and an autoimmune disease.

Description

Ubiquitin Vaccine Components

[0001] This invention relates to a vaccine component, in particular to a vaccine component comprising a ubiquitin covalently bonded to an antigen. The invention also provides vaccines comprising said vaccine components, as well as combinations and formulations thereof. The invention also provides uses of said vaccine components, vaccines, combinations and formulations.BACKGROUND

[0002] Dendritic cells (DCs) are attractive targets for vaccination against a range of pathogens and diseases for which cellular immunity is a crucial part of the immune response. Known cellbased immunotherapy with ex vivo generated DCs loaded with antigens suffers from various drawbacks, namely being laborious and expensive.

[0003] There remains a need in the art for improved vaccination strategies that target dendritic cells.BRIEF SUMMARY OF THE DISCLOSURE

[0004] In a first aspect, the invention provides a vaccine component comprising a ubiquitin covalently bonded to an antigen, wherein the vaccine component does not comprise a cell permeability tag.

[0005] In embodiments of the first aspect, residues 75-76 of the ubiquitin are GG.

[0006] Ubiquitin (Ub) is a small protein tag involved in numerous cellular processes, such as protein degradation, cell division and differentiation, and signal transduction. It is a 76-amino acid post-translational modifier that is covalently attached to target proteins in a highly regulated process called ubiquitination. This process is coordinated by an enzymatic cascade involving ubiquitin-activating (E1), ubiquitin-conjugating (E2), and ubiquitin-ligating (E3) enzymes, resulting in the covalent attachment of the C-terminal glycine residue of ubiquitin to the N-terminus or lysine residues of target proteins. Ubiquitin molecules can also be conjugated to each other at one of the seven internal lysine residues or at the N-terminal methionine residue. This results in the formation of polyubiquitin chains with different linkage types.

[0007] It is known that unconjugated ubiquitin is physiologically ubiquitous and well-tolerated such that no physiological response would be expected.

[0008] The inventors have surprisingly found that a monoubiquitin moiety covalently bonded to an antigen can provide a highly functional dendritic cell-targeted vaccine component that demonstrates an enhanced immune response and surprising vaccination potential in vitro and in vivo. Without wishing to be bound by theory, it is thought that the monoubiquitin moiety (without a cell permeability tag) is surprisingly efficiently taken up by dendritic cells, then processed by deubiquitinating enzymes (DUBs), which are considered to be active in the cytosol. It is thought that the DUBs cleave the ubiquitin moiety from the antigen moiety after the uptake, providing a released antigen that generates an adaptive immune response.

[0009] The vaccine component of the invention also achieves improved T cell activation compared to current state-of-the-art synthetic long peptides.

[0010] In a second aspect, the invention provides a vaccine comprising a vaccine component of the first aspect. The vaccine component may be encapsulated, for example to provide encapsulated nanoparticles.

[0011] In a third aspect, the invention provides a combination comprising a vaccine component of the first aspect or a vaccine of the second aspect, and a biologically active substance.

[0012] In a fourth aspect, the invention provides a formulation comprising the vaccine component of the first aspect, the vaccine of the second aspect, or the combination of the third aspect, and optionally a pharmaceutically acceptable carrier.

[0013] In a fifth aspect, the invention provides a vaccine component of the first aspect, a vaccine of the second aspect, a combination of the third aspect, or a formulation the fourth aspect, for use as a medicament.

[0014] In a sixth aspect, the invention provides a vaccine component of the first aspect, a vaccine of the second aspect, a combination of the third aspect, or a formulation the fourth aspect, for use in the treatment of a condition selected from a cancer and an autoimmune disease.

[0015] In a seventh aspect, the invention provides a method for the treatment of a cancer or an autoimmune disease, comprising administration of an effective amount of a vaccine component of the first aspect, a vaccine of the second aspect, a combination of the third aspect, or a formulation the fourth aspect, to a subject in need thereof.

[0016] In an eighth aspect, the invention provides a use of the vaccine component of the first aspect in the activation of dendritic cells.BRIEF DESCRIPTION OF THE DRAWINGS

[0017] Embodiments of the invention are further described hereinafter with reference to the accompanying drawings, in which:Figure 1 shows the effects a monoubiquitin-antigen conjugate of the invention (with and without a FR dipeptide linker motif) on T-cell activation in vitro. (A) Structures of the “linear” monoubiquitin-antigen conjugates Ub-OT-I and Ub-FR-OT-I (with and without a FR dipeptide linker motif) (B) Schematic overview of assay. GM-CSF BMDCs were generated and pulsed with 10-100-1000 nM Ub-OT-I or Ub-FR-OT-I for 2 h. at 37 °C before setting up a 1 :5 (BMDC:OT-I) co-culture. After 3 days, OT-I activation was assessed by FACS and ELISA analyses. (C-E) FACS analysis of CD25 (C), 4-1 BB (D), and CD44 (E) expression. Data (N = 4, technical duplicates) are shown as normalized MFI to positive control ±SD. (F,G) ELISA analysis of IL-2 (F) and IFN-y (G) secretion. Data (N = 4, technical duplicates) are shown as relative concentration to positive control ±SD.Figure 2 shows the effects of iso-peptide conjugated ubiquitin-antigen fusions on T cell activation compared to known peptide vaccine OVA24 in vitro. (A) Structures of iso-peptide conjugated ubiquitin - peptide epitope fusions at the epitope intrinsic lysine position (UbiQ-368) and epitope extrinsic N-terminally added lysine (UbiQ-369). (B) Schematic overview of assay. Flt3L BMDCs were generated and pulsed with 10-100-1000 nM vaccine peptide (OVA24 is a standard OT-I epitope containing synthetic long peptide vaccine:DEVSGLEQLESIINFEKLAAAAAK, SEQ ID NO: 14) for 2 h. at 37 °C before setting up a 1 :5 (BMDC:OT-I) co-culture. After 3 days, OT-I activation was assessed by flow cytometry. (C) FACS analysis of proliferation (division index was calculated for different conditions based on CTV signal), CD25, 4-1 BB and CD44 expression. Data is depicted as mean±SD (N = 3, technical duplicates).Figure 3 shows the effects of monoubiquitin-antigen conjugates of the invention vs Fab-Ubz-antigen conjugates on B16-OVA cancer cells. (A) Schematic overview of B16-OVA killing assay. 1e5 Flt3L BMDCs are generated and pulsed with 100 nM vaccine for 2h. at 37 °C. Subsequently, the BMDCs were washed and a 1:5 co-culture with 0.5e6 OT-I cells was set-up and incubated overnight. The following day, activation of OT-I cells was measured by FACS and ELISA analysis, and the OT-I cell co-culture was added in 5:1 (OT-I celktumor cells) ratio to 1e4 B16-OVA. The co-culture was incubated overnight and following day specific tumor killing was assessed by FACS. (B) Specific B16-OVA lysis was calculated by (1-(freq. viable B16-OVA / freq. viable B16-OVA in neg. control)) x 100%. C,D) Activation of OT-I cells at day 1 as measured by IL-2 ELISA analysis (C) and CD69 / CD25 double positive expression (D). (E,F) ELISA analysis of supernatant killing assay for TNFa (E) and IFNy (F). All data is depicted as mean±SD (N = 5, technical duplicates). Statistical significance is performed using one-way ANOVA with Tukey multiple comparison correction and depicted as ****P<0.0001 , ***P<0.001, **P<0.01, *P<0.05, ns P>0.05.Figure 4 shows the effects of a monoubiquitin-antigen conjugate of the invention, Fab-Ub2-antigen conjugates and known sortagged vaccines on T cell responses in vivo after intravenous administration. (A) Schematic overview of in vivo OT-I cell activation assay. Mice (C57BL / 6) received 1e6 CTV-labelled OT-I cells on day 0, followed by 5 pmol vaccine conjugate or non-conjugated + 10 pg LPS on day 1. Spleens were harvested on day 3. (B) Division index obtained by flow cytometry analysis (n = 4) of OT-I cells isolated from spleen. Data are shown as mean ±SD. Unpaired T tests, p-values are noted in the figure ****P<0.0001, **P<0.01. (C) Representative histograms of OT-I cell proliferation in spleen. (D) Division index obtained by flow cytometry analysis (n = 4) of OT-I cells isolated from inguinal lymph nodes. Data are shown as mean ±SD. Paired T tests, ****P<0.0001, ***P<0.001, *P<0.05. (E) Representative histograms of OT-I cell proliferation in inguinal lymph nodes.Figure 5 shows the effect of exemplary monoubiquitin-antigen conjugates of the invention compared ubi-tagged targeted vaccines on the in vivo immune response followingsubcutaneous vaccination. (A) Schematic overview assay. In short, mice received 1e6 CTV-labeled OT-I cells and the following day 20 pmol vaccine s.c.. After 2 days, inguinal lymph nodes were harvested and proliferation was measured. (B) Proliferation, (C) CD25 expression, (D) CD69 expression and E) CD137 expression of OT-I cells isolated from inguinal lymph nodes is depicted as mean±SD (N = 2x2, individual lymph nodes shown). (N = 2x2, individual lymph nodes shown).Figure 6 shows the effects of an exemplary Ub-gp100 human cancer-associated epitope vaccine of the invention on T-cell activation in vitro. (A) Schematic representation of primary TCR-transfected CD8+T cell activation assay. HLA-A*02:01+moDCs were generated and pulsed with 0.1 -0.5-1 pM vaccine conjugate or 1 pM control condition (unconjugated gp100 peptide epitope) for 1 h. at 37 °C. TCR-transfected CD8+T cells were added in 1:5 ratio and after 3 days, supernatant and cells were harvested for analysis. (B) Flow cytometry analysis of CD8+T cells. Proliferation (Division index was calculated for different conditions based on CTV signal), CD25 expression, CD69 expression, CD127 expression, TNFa secretion, IFN-y secretion and IL2 secretion are depicted as mean±SD (n = 4).Figure 7 shows the effects of ubiquitin-helper T cell epitope conjugates on T cell activation in vitro. (A-D) FACS analysis of Flt3L BMDC-mediated OT-II activation assay.Vaccines / peptides were provided in a 2 h. pulse at 100 nM followed by 3 day incubation of the BMDC-OT-II (1:5 ratio) co-culture. Data is depicted as mean (N = 2, technical duplicates) for the division index (A), CD25 (B), 4-1 BB (C), and CD44 (D).Figure 8 shows the effects of mutating C-terminal DUB cleavage-site in ubiquitinepitope vaccines (where the ubiquitin C-terminal has mutated from GG to W). (A,B) DUB-cleavage assay using 50 nM UCH-L3 (A) or 500 nM UCH-L1 (B) on Ub-Ag and Ub(W)-Ag. Cleavage is observed as assessed by ESI-TOF for Ub-Ag, but not for Ub(W)-Ag. (C-F) Flt3L BMDC-mediated OT-I activation assay. BMDCs were pulsed with 100 nM vaccine for 2 h. at 37 °C, washed and a 1:5 (BMDC:OT-I) co-culture was set-up. After 3 days, FACS and ELISA analyses were performed. Data is depicted as mean±SD (N = 4, technical duplicates) for proliferation (C), CD25 expression (D), 4-1 BB expression (E), and IFNy secretion (F). Statistical significance using one-way ANOVA with Tukey multiple comparison correction is depicted as ****P<0.0001, ***P<0.001, **P<0.01, *P<0.05, ns P>0.05.Figure 9 shows the synthesis of isopeptide-linked Ub-peptide conjugates SIINFEK(Ub)L (UbiQ-368) and Ac-K(Ub)SIINFEKL (UbiQ-369). (i) Ub-MESNa, 6M Gdn-HCI, 200 mM sodium phosphate, 150 mM methyl thioglycolate, 10 mM TCEP, pH 7.6, 37°C. (ii) 75 mM glutathione, 75 mM VA-044 and 150 mM TCEP, pH 7, 37°C.Figure 10 shows the general synthesis of Ub-antigen conjugates of the invention, where the C-terminus of the ubiquitin is attached to a cysteine, serine, threonine or tyrosine residue on the antigen.DETAILED DESCRIPTION

[0018] Throughout the description and claims of this specification, the words “comprise” and “contain” and variations of them mean “including but not limited to”, and they are not intended to (and do not) exclude other moieties, additives, components, integers or steps. Throughout the description and claims of this specification, the singular encompasses the plural unless the context otherwise requires. In particular, where the indefinite article is used, the specification is to be understood as contemplating plurality as well as singularity, unless the context requires otherwise.

[0019] Unless specifically excluded, embodiments in the above specification that recite “comprising” various components are also contemplated as “consisting of’ or “consisting essentially of’ the recited components; embodiments in the specification that recite “consisting of” various components are also contemplated as “comprising” or “consisting essentially of’ the recited components; and embodiments in the specification that recite “consisting essentially of” various components are also contemplated as “consisting of’ or “comprising” the recited components (this interchangeability does not apply to the use of these terms in the claims).

[0020] Features, integers, characteristics, compounds, chemical moieties or groups described in conjunction with a particular aspect, embodiment or example of the invention are to be understood to be applicable to any other aspect, embodiment or example described herein unless incompatible therewith. All of the features disclosed in this specification (including any accompanying claims, abstract and drawings), and / or all of the steps of any method or process so disclosed, may be combined in any combination, except combinations where at least some of such features and / or steps are mutually exclusive. The invention is not restricted to the details of any foregoing embodiments. The invention extends to any novel one, or any novel combination, of the features disclosed in this specification (including any accompanying claims, abstract and drawings), or to any novel one, or any novel combination, of the steps of any method or process so disclosed.

[0021] The reader's attention is directed to all papers and documents which are filed concurrently with or previous to this specification in connection with this application and which are open to public inspection with this specification, and the contents of all such papers and documents are incorporated herein by reference.

[0022] All publications, patent applications, patents, and other references mentioned herein are incorporated by reference in their entirety. In case of conflict, the present specification, including definitions, will control.DEFINITIONS

[0023] The following explanations of terms and methods are provided to better describe the present disclosure and to guide those of ordinary skill in the art in the practice of the present disclosure.

[0024] "Percentage of sequence identity" is determined by comparing two optimally aligned sequences over a comparison window, where the portion of the polynucleotide sequence orpolypeptide sequence in the comparison window may comprise additions or deletions (i.e., gaps) as compared to the reference sequence (which does not comprise additions or deletions) for optimal alignment of the two sequences. The percentage is calculated by determining the number of positions at which the identical nucleic acid base or amino acid residue occurs in both sequences to yield the number of matched positions, dividing the number of matched positions by the total number of positions in the window of comparison, and multiplying the result by 100 to yield the percentage of sequence identity. The percentage of identity may be determined using NCBI Basic Local Alignment Search Tool (BLAST) (Altschul et aL, J. Mol. Biol., (1990) 215, 403-10). BLAST is available from several sources, including the National Center for Biological Information (NCBI, National Library of Medicine, 8600 Rockville Pike, Bethesda, MD, 20894, USA) and on the Internet, for use in connection with the sequence analysis programs blastp, blastn, blastx, tblastn and tblastx. Additional information can be found at the NCBI web site (https: / / www.ncbi.nlm.nih.gov / ).

[0025] The term "substantial identity" of amino acid sequences (and of polypeptides or proteins having these amino acid sequences) normally means sequence identity of at least 75% compared to a reference sequence as determined using a standard program; preferably BLAST using standard parameters.

[0026] Preferred percent identity of amino acids can be any integer from 75% to 100%. More preferred embodiments include amino acid sequences that have at least 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity compared to a reference sequence. Polypeptides that are "substantially identical" may share amino acid sequences as noted above except that residue positions which are not identical may differ by conservative amino acid changes. Conservative amino acid substitutions refer to the interchangeability of residues having similar side chains. For example, a group of amino acids having aliphatic side chains is glycine, alanine, valine, leucine, and isoleucine; a group of amino acids having aliphatic hydroxyl side chains is serine and threonine; a group of amino acids having amide-containing side chains is asparagine and glutamine; a group of amino acids having aromatic side chains is phenylalanine, tyrosine, and tryptophan; a group of amino acids having basic side chains is lysine, arginine, and histidine; and a group of amino acids having sulfur-containing side chains is cysteine and methionine. Preferred conservative amino acids substitution groups are: valine-leucine-isoleucine, phenylalanine-tyrosine, lysine-arginine, alanine-valine, aspartic acid-glutamic acid, and asparagine-glutamine. Polypeptides that are “substantially identical” to a reference sequence may also differ by specified point mutations, where the specified point mutation or point mutations may or may not represent a conservative substitution; such mutations include lysine to arginine, lysine to alanine and lysine to cysteine.

[0027] For the avoidance of doubt, the term “amino acid” in the context of the invention may refer to a natural or unnatural amino acid, or a derivative thereof. Accordingly, an amino acid sequence referred to herein may include one or more unnatural amino acids, such as unnaturalamino acids comprising a chemical handle. The chemical handle may be biorthogonal, e.g. an azide moiety. A (biorthogonal) chemical handle may allow specific bioconjugation of various functionalities, such as a probe; e.g. an azide moiety allows for bioconjugation by click chemistry or Staudinger ligation. An example of an unnatural amino acid that may be present in certain embodiments is azidolysine.

[0028] The term “ubiquitin” includes reference to a (functional) amino acid sequence that is substantially identical to SEQ ID NO: 1. Ubiquitin may have at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 1. The ubiquitin may be a wild type ubiquitin, e.g. of SEQ ID NO: 1. The ubiquitin may comprise 1 , 2, 3, 4, 5, 6, 7 or 8 conservative substitutions. It may be that one or more substitutions are at physiological conjugation sites. It may be that one or substitutions are made to improve solubility. It may be that at least one substitution is replacing a lysine with an arginine. Without wishing to be bound by theory, it is thought that this modification helps to reduce polyubiquitination. It may be that at least one substitution is replacing a residue (e.g. a lysine residue) with an unnatural amino acid. Each unnatural amino acid may be an amino acid substituted with a probe or with a reactive group capable of participating in a click chemistry reaction. Each reactive group may be independently selected from: azido, alkynyl, alkenyl, cycloalkenyl, cycloalkynyl, and azinyl comprising at least 3 nitrogen atoms. Exemplary reactive groups include: -N3, -CHCH, -CHCCH3, tetrazine, trans-cyclooctene (TCO), dibenzocyclooctyne (DBCO), and bicyclononyne (BCN). For example, each unnatural amino acid may be selected from azido-lysine and azido-ornithine.

[0029] It may be that residues 66-74 of the ubiquitin are conserved, i.e. not replaced with another residue. In other words, it may be that residues 66-74 of the ubiquitin are TLHLVLRLR.

[0030] A ubiquitin may be a translated ubiquitin, such as a ubiquitin that is produced by a cell (e.g. a CHO cell or a hybridoma cell), optionally as part of a fusion protein. A ubiquitin may be a synthetic ubiquitin, such as a ubiquitin prepared by total linear synthesis using solid phase peptide synthesis, e.g. as described in El Oualid et al., Angew. Chem. Int. Ed. Engl., (2010), 49(52), 10149-53; or Hameed et al., Bioconjug Chem., (2017), 28(3), 805-15. A synthetic ubiquitin may comprise 1, 2, 3, 4, 5, 6, 7 or 8 unnatural amino acid substitutions in addition to or instead of conservative substitutions. A synthetic ubiquitin may comprise a probe, for example a synthetic ubiquitin may be substituted with a probe. An example of such a synthetic ubiquitin is a synthetic ubiquitin covalently bonded to an antigen, which further comprises a probe.

[0031] The term “isolated” means a biological component (such as a nucleic acid molecule or protein) that has been substantially separated or purified away from other biological components in the cell of the organism in which the component naturally occurs, i.e., other chromosomal and extrachromosomal DNA and RNA, and proteins. Nucleic acids and proteinsthat have been "isolated" include nucleic acids and proteins purified by standard purification methods. The term also embraces nucleic acids and proteins prepared by recombinant expression in a host cell as well as chemically synthesized nucleic acids, proteins and peptides.

[0032] Variants, fragments or fusion proteins: The disclosed proteins and polypeptides include variants, fragments, and fusions thereof.

[0033] The term “antibody” or “antibodies” include monoclonal, polyclonal, chimeric, single chain, bispecific, human and humanized antibodies as well as active multivalent fragments thereof. Examples of active multivalent fragments of molecules that bind to known antigens and are useful include F(ab')2, F(ab')3, diabodies, triabodies, scFv-Fc and di-scFv and minibodies, including the products of a Fab immunoglobulin expression library and epitope-binding multivalent fragments of any of the antibodies and multivalent fragments mentioned above.

[0034] In a particular example, the antibody may be a monoclonal antibody (Mab). As used herein, the term “monoclonal antibody” refers to an antibody that is mass produced in the laboratory from a single clone and that recognizes only one antigen. Monoclonal antibodies may be generated by any appropriate technique known in the art (e.g. by production in HEK or insect cells, or by generation of B cell hybridomas). Examples of production systems for recombinant antibodies are set out in Schirrmann T., et al., Front Biosci. (2008), 13:4576-94. Review.Monoclonal antibodies may also be produced, for example by production in hybridoma cells, using methods disclosed in, e.g.: Kohler, G. & Milstein, C., Nature (1975) 256, 495—497;Cheong, T.-C., etal., Nat. Common., (2016) 7, 10934; Pogson, M., etal., Nat. Common., (2016), 7, 12535; or Mason, D. M. et al. High-throughput antibody engineering in mammalian cells by CRISPR / Cas9-mediated homology-directed mutagenesis. Nocleic Acids Res. (2018). doi:10.1093 / nar / gky550.

[0035] The term “deubiquitinating enzyme” (DUB) refers to a peptidase that cleaves the peptide bond between a ubiquitin and the moiety to which it is attached. A given ubiquitin linkage (e.g. to an antigen via a linker or bond) may be cleaved by an appropriate DUB.

[0036] The term “probe” includes reference to a payload or a label. Where a moiety is substituted with a probe, the probe may be a payload. The probe may be a label.

[0037] The term “payload” includes reference to an immunomodulating agent that may be conjugated to a targeting agent. Exemplary payloads include TLR-agonists (which may act as vaccine adjuvants). Exemplary TLR-agonists include monophosphoryl lipid A (MPL), imiquimod, CpG oligodeoxynucleotides (ODNs), Polyriboinosinic-polyribocytidylic acid (Poly l:C), flagellin, Bacillus Calmette-Guerin (BCG), Cadi-05, mobilan.

[0038] The term “label” includes reference to a chemical moiety or protein that is directly or indirectly detectable (e.g. due to its spectral properties, conformation or activity). The label can be directly detectable (e.g. dye or fluorophore) or indirectly detectable (e.g. hapten or enzyme). Such labels include, but are not limited to, radiolabels that can be measured with radiationcounting devices; pigments, dyes or other chromogens that can be visually observed ormeasured with a spectrophotometer; spin labels that can be measured with a spin label analyser; and fluorescent labels (fluorophores), where the output signal is generated by the excitation of a suitable molecular adduct and that can be visualized by excitation with light that is absorbed by the dye or can be measured with standard fluorometers or imaging systems. The label may be a chemiluminescent substance, where the output signal is generated by chemical modification of the signal compound; a metal-containing substance; or an enzyme, where there occurs an enzyme-dependent secondary generation of signal, such as the formation of a coloured product from a colourless substrate.

[0039] The term label can also refer to a “tag” or hapten that can bind selectively to a conjugated molecule such that the conjugated molecule, when added subsequently along with a substrate, is used to generate a detectable signal. For example, one can use a peptide tag (e.g. FLAG tag, His tag) or biotin as a tag and then use an avidin or streptavidin conjugate of horseradish peroxidate (HRP) to bind to the tag, and then use a colorimetric substrate (e.g., tetramethylbenzidine (TMB)) or a fluorogenic substrate such as Amplex Red reagent (Molecular Probes, Inc.) to detect the presence of HRP. Numerous labels are known by those of skill in the art and include, but are not limited to, particles, fluorophores, haptens, enzymes and their colorimetric, fluorogenic and chemiluminescent substrates and other labels. Preferred labels include fluorophores, and haptens.

[0040] Alternatively, the term label can refer to a “tag” that can bind selectively to a conjugated molecule such that the conjugated molecule, when added subsequently along with a substrate, is used as an affinity tag in purification methods or in non-covalent conjugation methods. For example, one can use a peptide tag (e.g. FLAG tag, His tag) then use an avidin or streptavidin conjugate of horseradish peroxidate (HRP) to bind to the tag.

[0041] The vaccine components of the present invention may contain unnatural proportions of atomic isotopes at one or more of the atoms that constitute part of the vaccine component or part of a label on said vaccine component. For example, the vaccine components may be radiolabeled with radioactive isotopes, such as for example tritium (3H), iodine-125 (125l) or carbon-14 (14C). In addition, the vaccine components may be labeled with stable isotopes that have a relatively low natural abundance, such as deuterium (2H), carbon-13 (13C), or nitrogen-15 (15N). Similarly, isotopic variants of O (e.g.17O or18O) may be utilised. All isotopic variations of the vaccine components of the present invention, whether radioactive or not, are encompassed within the scope of the present invention.

[0042] The invention concerns amongst other things the treatment of a disease. The term “treatment”, and the therapies encompassed by this invention, include the following and combinations thereof: (1) hindering, e.g. delaying initiation and / or progression of, an event, state, disorder or condition, for example arresting, reducing or delaying the development of the event, state, disorder or condition, or a relapse thereof in case of maintenance treatment or secondary prophylaxis, or of at least one clinical or subclinical symptom thereof; (2) preventingor delaying the appearance of clinical symptoms of an event, state, disorder or condition developing in an animal (e.g. human) that may be afflicted with or predisposed to the state, disorder or condition but does not yet experience or display clinical or subclinical symptoms of the state, disorder or condition; and / or (3) relieving and / or curing an event, state, disorder or condition (e.g., causing regression of the event, state, disorder or condition or at least one of its clinical or subclinical symptoms, curing a patient or putting a patient into remission). The benefit to a patient to be treated may be either statistically significant or at least perceptible to the patient or to the physician. It will be understood that a medicament will not necessarily produce a clinical effect in each patient to whom it is administered; thus, in any individual patient or even in a particular patient population, a treatment may fail or be successful only in part, and the meanings of the terms “treatment”, “prophylaxis” and “inhibitor” and of cognate terms are to be understood accordingly. The compositions and methods described herein are of use for therapy and / or prophylaxis of the mentioned conditions.

[0043] The term “prophylaxis” includes reference to treatment therapies for the purpose of preserving health or inhibiting or delaying the initiation and / or progression of an event, state, disorder or condition, for example for the purpose of reducing the chance of an event, state, disorder or condition occurring. The outcome of the prophylaxis may be, for example, preservation of health or delaying the initiation and / or progression of an event, state, disorder or condition. It will be recalled that, in any individual patient or even in a particular patient population, a treatment may fail, and this paragraph is to be understood accordingly.

[0044] The term “inhibit” (and “inhibiting”) includes reference to delaying, stopping, reducing the incidence of, reducing the risk of and / or reducing the severity of an event, state, disorder or condition. Inhibiting an event, state, disorder or condition may therefore include delaying or stopping initiation and / or progression of such, and reducing the risk of such occurring.Vaccine Components

[0045] The invention provides vaccine components as previously described. Vaccine components of the invention comprise ubiquitin covalently bonded to an antigen. The vaccine component of the invention does not comprise a cell permeability tag.

[0046] It may be that residues 75-76 of the ubiquitin are GG.

[0047] The vaccine component may comprise a fusion protein comprising the ubiquitin and the antigen.

[0048] For the avoidance of doubt, the ubiquitin conjugates employed in the present invention are typically chemically synthesised ubiquitin conjugates, rather than recombinantly expressed protein-peptide fusions.

[0049] Typically, the vaccine component is a monoubiquitin component, i.e. comprises a ubiquitin covalently bound to an antigen, and does not comprise any other ubiquitin.

[0050] It may be that the ubiquitin covalently bound to the antigen is of formula (I):U-A1-B (I)whereinU is an ubiquitin;A1is a bond or a linker; andB is an antigen.

[0051] It may be that the vaccine component is of formula (I):U-A1-B (I)whereinU is an ubiquitin;A1is a bond or a linker; andB is an antigen.

[0052] In embodiments, the U-A1-B moiety may be or may comprise a fusion protein.

[0053] The fusion protein may further comprise a probe. The probe may be or comprise one or more detection tags, e.g. dyes. The probe may be or comprise one or more payloads, e.g. immunomodulating agents. Suitable immunomodulating agents would be well known to the skilled person in the art.

[0054] The fusion protein may comprise at least one probe. For example, the fusion protein may comprise 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 probes. Each probe may be or comprise one or more detection tags, e.g. dyes. Each probe may be or comprise one or more payloads, e.g. immunomodulating agents. Suitable immunomodulating agents would be well known to the skilled person in the art.

[0055] The one or more probes may be substituted on the ubiquitin. It may be that the one or more probes are each substituted onto the sidechain of an amino acid residue in the ubiquitin sequence.

[0056] It may be that the one or more probes are present at one or more of residues 1 , 11 , 33, and 63 of the ubiquitin. In these embodiments, it may be that the one or more probes are payloads, e.g. immunomodulating agents.

[0057] Alternatively, it may be that one or more residues of the ubiquitin is functionalised with one or more reactive groups, wherein the reactive group is independently selected at each occurrence from: azido, alkynyl, alkenyl, cycloalkenyl and cycloalkynyl. Exemplary reactive groups include: -N3, -C=CH, -C=CCH3, tetrazine, trans-cyclooctene (TCO), dibenzocyclooctyne (DBCO), and bicyclononyne (BCN). It may be that the one or more residues are selected from residues 1 , 11 , 33 and 63 of the ubiquitin.

[0058] It may be that the ubiquitin is further functionalised at the N-terminus with a moiety comprising at least one amino acid and at least one linker group (e.g. polyethylene glycol). For example, the N-terminus of the ubiquitin may be bound to at least one amino acid via a polyethylene glycol linker. It may be that the moiety comprising at least one amino acid and at least one polyethylene glycol comprises a plurality of amino acids and polyethylene glycol groups. For example, it may be that the moiety comprises two or three amino acids and linkergroups (e.g. polyethylene glycol). The at least one amino acid may be an unnatural amino acid. For example, each amino acid may be functionalised by a probe or by a reactive group independently selected from azido, alkynyl, alkenyl, cycloalkenyl, cycloalkynyl, and azinyl comprising at least 3 nitrogen atoms.

[0059] It may be that the ubiquitin is functionalised at the N-terminus with a moiety comprising at least one group independently selected from: -PEG-azido-lysine and -PEG-azido-ornithine.

[0060] U may be a ubiquitin comprising at least 80% sequency identity to SEQ ID NO: 1. U may be a ubiquitin comprising at least 85% sequency identity to SEQ ID NO: 1. U may be a ubiquitin comprising at least 90% sequency identity to SEQ ID NO: 1. U may be a ubiquitin comprising at least 95% sequency identity to SEQ ID NO: 1. U may be a ubiquitin comprising at least 98% sequency identity to SEQ ID NO: 1.

[0061] It may be that one or more residues (e.g. one or more of residues 1 , 11 , 33 and 63) in the ubiquitin are unnatural amino acids. Each unnatural amino acid may be an amino acid substituted with a reactive group capable of participating in a click chemistry reaction. Each reactive group may be independently selected from: azido, alkynyl, alkenyl, cycloalkenyl, cycloalkynyl, and azinyl comprising at least 3 nitrogen atoms. For example, each unnatural amino acid may be selected from azido-lysine and azido-ornithine.

[0062] It may be that U is a ubiquitin of SEQ ID NO: 1.

[0063] In embodiments, U may be a ubiquitin as defined herein (e.g. of at least a specified sequence identity to SEQ ID NO:1), wherein residues 75-76 are GG.

[0064] It may be that U is a ubiquitin of SEQ ID NO: 34. In these embodiments, it may be that one or more of residues X1, X2, X3and X4comprises a probe and / or a reactive group. Each probe may be a payload, for example, an independently selected immunomodulating agent. Each reactive group may be a functional group capable of participating in a click chemistry reaction. For example, each reactive group may be independently selected from: azido, alkynyl, alkenyl, cycloalkenyl, cycloalkynyl, and azinyl comprising at least 3 nitrogen atoms. Exemplary reactive groups include: -N3, -C=CH, -CHCCHS, tetrazine, trans-cyclooctene (TCO), dibenzocyclooctyne (DBCO), and bicyclononyne (BCN).

[0065] It may be that one or more of X1, X2, X3and X4is a clickable amino acid residue. For the avoidance of doubt, the term ‘clickable amino acid’ refers to an unnatural amino acid having a group capable of participating in a click chemistry reaction. For example, it may be that one or more of X1, X2, X3and X4is an amino acid residue substituted with one or more reactive groups selected from: azido, alkynyl, alkenyl, cycloalkenyl, cycloalkynyl, and azinyl comprising at least 3 nitrogen atoms. It may be that one or more of X1, X2, X3and X4is substituted with a reactive group selected from: azido, alkynyl or cycloalkynyl. It may be that one or more of X1, X2, X3and X4is substituted with an azido group.

[0066] X1may be selected from: methionine, norleucine, lysine and ornithine, optionally substituted with a reactive group selected from: azido, alkynyl, alkenyl, cycloalkenyl,cycloalkynyl, and azinyl comprising at least 3 nitrogen atoms. X1may be selected from: methionine, norleucine, lysine and ornithine, optionally substituted with a reactive group selected from: azido, alkynyl, and cycloalkynyl. X1may be selected from: methionine, norleucine, lysine and ornithine, optionally substituted with an azido group. X1may be selected from: methionine and norleucine, optionally substituted with an azido group. X1may be methionine, optionally substituted with an azido group.

[0067] It may be that X1is substituted at the N-terminus by at least one PEG-linked lysine or PEG-linked ornithine. It may be that each lysine and / or ornithine is substituted by a reactive group, e.g. azido, alkynyl, alkenyl, cycloalkenyl, cycloalkynyl, and azinyl comprising at least 3 nitrogen atoms. For example, it may be that X1is substituted at the N-terminus by at least one PEG-linked azido-lysine and / or PEG-linked azido-ornithine.

[0068] Exemplary X1groups include: methionine, norleucine, norleucine substituted with at least one PEG-linked azido-lysine, norleucine substituted with at least one PEG-linked azidoornithine, azido-lysine and azido-ornithine.

[0069] X2may be selected from lysine and ornithine, optionally substituted with a reactive group selected from: azido, alkynyl, alkenyl, cycloalkenyl, cycloalkynyl, and azinyl comprising at least 3 nitrogen atoms. X2may be selected from lysine and ornithine, optionally substituted with a reactive group selected from: azido, alkynyl and cycloalkynyl. X2may be selected from lysine and ornithine, optionally substituted with an azido group.

[0070] It may be that X2is lysine. It may be that X2is azidolysine.

[0071] X3may be selected from lysine and ornithine, optionally substituted with a reactive group selected from: azido, alkynyl, alkenyl, cycloalkenyl, cycloalkynyl, and azinyl comprising at least 3 nitrogen atoms. X3may be selected from lysine and ornithine, optionally substituted with a reactive group selected from: azido, alkynyl and cycloalkynyl. X3may be selected from lysine and ornithine, optionally substituted with an azido group.

[0072] It may be that X3is lysine. It may be that X3is azidolysine.

[0073] X4may be selected from lysine and ornithine, optionally substituted with a reactive group selected from: azido, alkynyl, alkenyl, cycloalkenyl, cycloalkynyl, and azinyl comprising at least 3 nitrogen atoms. X4may be selected from lysine and ornithine, optionally substituted with a reactive group selected from: azido, alkynyl and cycloalkynyl. X4may be selected from lysine and ornithine, optionally substituted with an azido group.

[0074] It may be that X4is lysine. It may be that X4is azidolysine.

[0075] It may be that each of X2, X3and X4is independently selected from ornithine and lysine, wherein at least one of X2, X3and X4is substituted by a reactive group independently selected from: azido, alkynyl and cycloalkynyl. It may be that each of X2, X3and X4is independently selected from ornithine and lysine, wherein at least one of X2, X3and X4is substituted by an azido group.

[0076] It may be that A1is bond.

[0077] It may be that A1is a peptide linker. For example, it may be that A1is a peptide linker comprising or consisting of a sequence selected from SEQ ID NOs: 12 and 13.

[0078] It may be that A1is attached to the C-terminus of the ubiquitin. For the avoidance of doubt, in these embodiments, A1is between the C-terminus of the ubiquitin and the N-terminus of the antigen or an amino acid side chain of the antigen. For example, it may be that the C-terminus of the ubiquitin it attached to the antigen via a lysine, cysteine, tyrosine, serine or threonine side chain of the antigen.

[0079] It may be that the ubiquitin covalently bound to the antigen is of any of formulae (l-a) to (l-f):wherein U and B are as defined herein.

[0080] The antigen may comprise an epitope. The epitope may be selected from: gp100, NY-ESO-1, MAGE, Melan, OT-1, 2W1S, FluM1, OVA 257-264, OVA 323-339 and Casp5. The epitope may be selected from: gp100, NY-ESO-1, MAGE, Melan, FluM1, and Casp5.

[0081] The antigen may be selected from a tumour-associated antigen, a tumour neoantigen, a pathogen derived antigen and a self-antigen. The antigen may be a tumour-associated antigen. The antigen may be a tumour neoantigen. The antigen may be a pathogen derived antigen. The antigen may be a self-antigen.

[0082] In embodiments, the antigen comprises a tumour-associated antigen for CD8+ and I or CD4+ T cell activation. It may be that the antigen comprises a tumour-associated antigen for CD8+ T cell activation. It may be that the antigen comprises a tumour-associated antigen for CD4+ T cell activation.

[0083] In embodiments, the tumour-associated antigen is or comprises a MHCI epitope or a MHCII epitope.

[0084] The tumour-associated antigen may be a MHCI epitope selected from gp100, NY-ESO-1, MAGE-1 and MART-1. The tumour-associated antigen may be a MHCI epitope selected from gp100 and NY-ESO-1. In these embodiments, the tumour-associated antigen is for CD8+ T cell activation.

[0085] The tumour-associated antigen may be a MHCII epitope. In these embodiments, the tumour-associated antigen is for CD4+ T cell activation.

[0086] It may be that the tumour-associated antigen is selected from gp100, NY-ESO-1, MAGE and Melan.

[0087] The tumour neoantigen may be a frame shift mutation-derived neoantigen; such as that occurring in Lynch syndrome, e.g., Casp5.

[0088] The pathogen derived antigen may be or may comprise a pathogen derived T cell epitope. The pathogen derived antigen may be influenza matrix protein M1.

[0089] B may be an antigen comprising at least 80% sequence identity to a sequence selected from SEQ ID Nos: 2 - 11. B may be an antigen comprising at least 85% sequence identity to a sequence selected from SEQ ID Nos: 2 - 11. B may be an antigen comprising at least 90% sequence identity to a sequence selected from SEQ ID Nos: 2 - 11. B may be an antigen comprising at least 95% sequence identity to a sequence selected from SEQ ID Nos: 2 - 11. B may be an antigen comprising at least 99% sequence identity to a sequence selected from SEQ ID Nos: 2 - 11. B may be an antigen comprising a sequence selected from SEQ ID Nos: 2 - 11.

[0090] B may be an antigen comprising at least 80% sequence identity to a sequence selected from SEQ ID Nos: 6- 11. B may be an antigen comprising at least 85% sequence identity to a sequence selected from SEQ ID Nos: 6 - 11. B may be an antigen comprising at least 90% sequence identity to a sequence selected from SEQ ID Nos: 6 - 11. B may be an antigen comprising at least 95% sequence identity to a sequence selected from SEQ ID Nos: 6- 11. B may be an antigen comprising at least 99% sequence identity to a sequence selected from SEQ ID Nos: 6 - 11. B may be an antigen comprising a sequence selected from SEQ ID Nos: 6- 11.

[0091] B may be or may comprise a tumour-associated antigen. The tumour-associated antigen may have at least 80% sequence identity to a sequence selected from SEQ ID Nos: 6- 9. The tumour-associated antigen may have at least 85% sequence identity to a sequence selected from SEQ ID Nos: 6- 9. The tumour-associated antigen may have at least 90% sequence identity to a sequence selected from SEQ ID Nos: 6 - 9. The tumour-associated antigen may have at least 95% sequence identity to a sequence selected from SEQ ID Nos: 6 - 9. The tumour-associated antigen may have at least 99% sequence identity to a sequence selected from SEQ ID Nos: 6- 9.Vaccines

[0092] The invention provides a vaccine comprising a vaccine component as described herein.

[0093] The vaccine component may be encapsulated. For example, the vaccine component may be encapsulated to provide encapsulated nanoparticles. The vaccine component may be encapsulated in any suitable material, such as a (pharmaceutically acceptable) polymeric or lipid component, optionally as encapsulated nanoparticles. For example, the vaccine component may be encapsulated in poly(D,L-lactic-co-glycolic acid) (PLGA), optionally as encapsulated nanoparticles.

[0094] It may be that the vaccine component is not encapsulated.

[0095] The vaccine may be an infectious disease vaccine, a mucosal vaccine, an autoimmune disease vaccine or an anti-cancer vaccine. It may be that the vaccine is an autoimmune disease vaccine or an anti-cancer vaccine. It may be that the vaccines is an autoimmune disease vaccine. It may be that the vaccine is an anti-cancer vaccine.

[0096] In embodiments, the infectious disease vaccine is a vaccine used to treat or prevent viral, bacterial, fungal, protozoa and / or parasite infections. In embodiments, the vaccine is used to prevent and / or treat the infectious disease by presenting one or more pathogen-derived antigens (e.g. pathogen-derived protein, peptide, or fragment thereof) to the subject’s immune system.

[0097] In embodiments, the vaccine is used to prevent and / or treat cancer by presenting one or more tumour-associated antigens or tumour neoantigens to the subject’s immune system.

[0098] In embodiments, the vaccine is used to prevent and / or treat an autoimmune disease by presenting one or more self-antigens to the subject’s immune system. Without wishing to be bound by theory, it is thought that enhancing antigen (epitope) delivery to antigen-presenting cells (e.g. dendritic cells) is beneficial for the induction of antigen-specific tolerance.Furthermore, it is known that targeting antigens to antigen-presenting cells, in particular dendritic cells, in the absence of immune stimulating adjuvants will result in a tolerogenic response. This can be further pushed by co-delivery of tolerance inducing immunomodulating molecules (e.g., dexamethazone or vitamin D3). This is discussed in Pishesha et al., Nature Biomedical Engineering, 5, 1389-1401, (2021).Combinations

[0099] The invention provides a combination comprising a vaccine component or vaccine as described herein, and a biologically active substance.

[0100] The biologically active substance may comprise any such substance that may be included in a vaccine. Examples of relevant biologically active substances include an adjuvant, an antibody or a fragment thereof, and a conjugate of an antibody or fragment thereof with a probe.Formulations

[0101] Vaccine components, vaccines or combinations of the invention may be administered topically, intravenously, subcutaneously, buccally, rectally, dermally, nasally, tracheally, bronchially, by any other parenteral route, by injection, as an oral or nasal spray or via inhalation. The vaccine components, vaccines or combinations may be administered in the form of pharmaceutical preparations comprising the vaccine component either as a free compound or, for example, a pharmaceutically acceptable non-toxic organic or inorganic acid or base addition salt, in a pharmaceutically acceptable dosage form. Depending upon the disorder and patient to be treated and the route of administration, the compositions may be administered at varying doses.

[0102] Typically, therefore, the vaccine components, vaccines or combinations of the invention may be administered topically, or parenterally (“parenterally” as used herein, refers to modes of administration which include intravenous, intramuscular, intraperitoneal, intrasternal, subcutaneous and intraarticular injection and infusion) to a host. In the case of larger animals,such as humans, the vaccine components, vaccines or combinations may be administered alone or as compositions in combination with pharmaceutically acceptable diluents, excipients or carriers.

[0103] Actual dosage levels of active ingredients in the pharmaceutical formulations and pharmaceutical compositions of this invention may be varied so as to obtain an amount of the active vaccine component, vaccine or combination(s) that is effective to achieve the desired therapeutic response for a particular patient, compositions and mode of administration. The selected dosage level will depend upon the activity of the particular compound, the route of administration, the severity of the condition being treated and the condition and prior medical history of the patient being treated. However, it is within the skill of the art to start doses of the vaccine components, vaccines or combinations at levels lower than required to achieve the desired therapeutic effect and to gradually increase the dosage until the desired effect is achieved.

[0104] According to a further aspect of the invention there is thus provided a pharmaceutical formulation or composition including a vaccine component, vaccine or combination of the invention, optionally in admixture with a pharmaceutically acceptable adjuvant, diluents or carrier.

[0105] Exemplary adjuvants include: mineral salts, emulsions, liposomes, virosomes, toll- like receptor (TLR) agonists, STING agonists, costimulatory ligands, cytokines and chitosan.

[0106] The carrier may be a nanocarrier. The nanocarrier may be selected from: a biogenic nanocarrier, a semi-biogenic nanocarrier or a synthetic nanocarrier. Exemplary nanocarriers include: outer membrane vesicles (OMVs), exosomes, self-assembled structural proteins (VLPs), endogenous protein-based nanocarriers, cell membrane-coated nanocarriers, liposomes, polymer-based nanoparticles (e.g. PLGA nanoparticles), inorganic nanocarriers (e.g. inorganic nanoparticles), nanogels, lipid nanoparticles. The carrier may be PLGA nanoparticles.

[0107] Suitable adjuvant, diluent or carriers are well known to the skilled person in the art. Exemplary adjuvants, diluent or carriers that may be employed in the present invention are described in Gurunathan etal. (Biomedicine & Pharmacotherapy, 170, (2024), 115992);Priyanka et al. (Biomedicine & Pharmacotherapy, 162, (2023), 115597); and Silva etal. (Human Vaccines & Immunotherapeutics, 2016, vol. 12, no. 4, 1056-1069).Uses

[0108] Vaccine components of the invention may be useful in the activation of dendritic cells.

[0109] Without wishing to be bound by theory, we have found that said vaccine components are surprisingly (specifically) taken up by dendritic cells. After this, the vaccine components are processed by the cells such that the antigen moieties (or epitope comprising portions thereof) are presented by the dendritic cells, thereby boosting the immune response.

[0110] According to another aspect of the invention, there is thus provided a use of the vaccine component as described herein in the activation of dendritic cells. It may be that said activation is conducted in vitro.

[0111] In an embodiment, the vaccine component, vaccine, combination or formulation of the invention is for use as a medicament.

[0112] In another embodiment, the vaccine component, vaccine, combination or formulation of the invention is for use in the treatment of a condition selected from a cancer and an autoimmune disease. The condition may be selected from melanoma, lung cancer, Lynch syndrome, type 1 diabetes, and rheumatoid arthritis. The condition may be selected from melanoma and lung cancer.

[0113] In another embodiment, there is provided a method for the treatment of a cancer or an autoimmune disease, comprising administration of an effective amount of a vaccine component, vaccine, combination or formulation of the invention to a subject in need thereof. The cancer or autoimmune disease may be selected from melanoma, lung cancer, Lynch syndrome, type 1 diabetes, and rheumatoid arthritis.Material and Methods

[0114] LC-MS measurements were performed on a system equipped with a Waters 2795 Seperation Module (Alliance HT), Waters XSelect™ CSHTMC18 (4.6 x 100mm, 5 pm) and Waters LCT Premier XE Mass Spectrometer. Samples were run (column temperature= 40°C, flow= 0.8 mL / min) using 2 mobile phases: A= 1% CH3CN and 0.1% formic acid in water and B= 1% water and 0.1% formic acid in CH3CN. Gradient: 0.0 - 1.4 min: 5 - 30%B, 1.4 - 4.2 min: 30 - 80%B, 4.2 - 4.4 min: 80 - 95%B, 4.4 - 5.0 min: 95%B, 5 - 5.3 min: 95 - 5%B, 5.3 - 6 min: 5%B. RP-HPLC purifications were performed on a system equipped with a Waters Dual Absorbance Detector 2487, Waters 600 controller and pump and XBridge Prep C185pm OBD 30x250mm column. Mobile phases: A= 0.05% aq. TFA and 5% CH3CN, and B= 0.05% TFA in CH3CN with 5% H2O. flow rate= 25 mL / min. Data processing was performed using Waters MassLynx Mass Spectrometry Software 4.1.

[0115] The C-terminally linked Ub-peptide conjugates were prepared as follows: Solid-phase peptide synthesis (SPPS) of the Ub-epitopeZ-peptide conjugates was performed on a Syro II Multisyntech Automated Peptide synthesizer (SYRO robot; Part Nr: S002PS002; MultiSyntech GmbH, Germany) on a 25 pmol scale using standard 9-fluorenylmethoxycarbonyl (Fmoc) based solid phase peptide chemistry. Ub was synthesized based on the procedure described by (El Oualid et al. 2010) using a fourfold excess of amino acids relative to pre-loaded Fmoc amino acid trityl resin (between 0.17 and 0.20 mmol / g, Rapp Polymere, Germany). The fused epitopes / peptides were fused by continued SPPS at the C-terminus of Ub. All synthetic products were purified by RP-HPLC on a Waters preparative RP-HPLC system equipped with a Waters C18-Xbridge 5 pm OBD (10 x 150 mm) column. The purified products were lyophilized and assayed for purity by high resolution mass spectrometry on a Waters Acquity H-class UPLC with XEVO-G2 XS Q-TOF mass spectrometer and by SDS-PAGE analysis.

[0116] The y-thiolysine (thi0K) peptides SIINFE(thi0K)L (SEQ ID NO: 17) and Ac-(thi0K)SIINFEKL (SEQ ID NO: 18) were synthesized on a Syro II MultiSyntech Automated Peptide synthesizerusing standard 9-fluorenylmethoxycarbonyl (Fmoc) based solid phase peptide chemistry (25 umol scale, Merkx et al. 2013).

[0117] Starting with pre-loaded Fmoc-Leu-PEG-PS resin (Rapp Polymere GmbH), each successive amino acid was double coupled in NMP with HATU (4 equiv) and DiPEA (8 equiv). Deprotection of the Fmoc group was achieved with 20% piperidine in NMP. N-terminal acetylation as performed by treating the resin for 45 min with 5 equiv. AC2O and 10 equiv.DiPEA in 3 mL CH2CI2. After each step, the resin was washed with NMP and CH2CI2. After completion, the resin was treated for 2 hrs with TFA / H2O / iPr3SiH (92 / 5 / 3 v / v / v, 3 mL) and the crude peptide was precipitated in cold Et20. The precipitated peptide was washed 3x with cold Et20 (40 mL, pellet peptide by centrifugation for 10 min at 4000 rpm), dissolved in a mixture of H2O / CH3CN / formic acid (50 / 50 / 1 v / v / v) and lyophilized. The crude product was dissolved in a minimal amount of warm DMSO (1 -2 mL), diluted by addition of HPLC buffer A and purified by RP-HPLC. Gradient: 30^50% B over 20 min. Pure fractions as judged by LC-MS were pooled and lyophilized (repeated 2x in H2O / CH3CN / formic acid, 50 / 50 / 1 v / v / v).

[0118] The isopeptide linked Ub-peptide conjugates were prepared as follows:• Ub-MESNa (20 mg, 2.3 umol) and peptide SIINFE(thi0K)L (SEQ ID NO: 17) (12.4 mg, 11.5 umol) are dissolved in 2 mL 6M Gdn-HCI, 200 mM sodium phosphate, 150 mM methyl thioglycolate, 10 mM TCEP, pH 8. Next, the pH is checked with a micro pH probe and confirmed to be correct for the ligation (±7.6), after which the reaction mixture is incubated overnight at 37°C.• Ub-MESNa (20 mg, 2.3 umol) and peptide Ac-(thi0K)SIINFEKL• (SEQ ID NO: 18) (11.1 mg, 9.2 umol) are dissolved in 2 mL 6M Gdn-HCI, 200 mM sodium phosphate, 150 mM methyl thioglycolate, 10 mM TCEP, pH 8. Next, the pH is checked with a micro pH probe and confirmed to be suitable for the ligation (±7.6), after which the reaction mixture is incubated overnight at 37°C.

[0119] After LC-MS analysis confirmed total consumption of UbMESNa and formation of ligation product, each reaction is diluted with 18 mL 6M Gdn-HCI, 200 mM sodium phosphate pH 7.4. Next, 75 mM glutathione (1.5 mmol, 460 mg), 75 mM 2,2'-azobis[2-(2-imidazolin-2-yl)propane] dihydrochloride (VA-044, 1.5 mmol, 484 mg) and 150 mM TCEP (3.0 mmol, 0.86 gr) are added. To dissolve all reagents, 1 mL 10N NaOH is added followed by pH adjustment to ±7 with 10N NaOH, resulting in a clear solution. Incubating the reaction mixture overnight at 37°C resulted in complete desulfurization, as judged by LC-MS analysis. The crude Ub-peptide conjugates (UbiQ-368 and UbiQ-369) were purified by RP-HPLC. Gradient: 30^40% B over 20 min. Pure fractions as judged by LC-MS were pooled and lyophilized (repeated 3x in H2O / CH3CN / formic acid, 50 / 50 / 1 v / v / v).Yield: SIINFEK(Ub)L (UbiQ-368, SEQ ID NO: 15): 13 mg, 1.4 umol, 59%, white powder.Yield: Ac-K(Ub)SIINFEKL (UbiQ-369, SEQ ID NO: 16): 15 mg, 1.5 umol, 67%, white powder.

[0120] Table 1 below provides a list of synthesized Ub-peptide conjugates. (m)= mouse, (h)= human, Ub= ubiquitin, linkers are underlined, epitope / antigen is highlighted. K(Ub)= Lys linked to Ub via an isopeptide bond. K(N3)= 6-azido-Lys, Abu= 2-aminobutanoic acid.Table 1ASSAYS

[0121] All mice were purchased from Charles River Laboratories, France. Female C57BL / 6 WT, OT-I (Tg(TcraTcrb)l 10OMjb / Crl) and OT-II (C57BL / 6-Tg(TcraTcrb)425Cbn / Crl) between 8-12 weeks of age and 18-25 g body weight were used for in vitro and in vivo experiments. Mice were sacrificed by cervical dislocation.Cell lines culture conditions

[0122] B 16-0 VA|B16-OVA cells were cultivated in a 5% CO2 humidified incubator at 37 °C in complete RPMI medium (RPMI1640 (ThermoFischer, Gibco™, 11875093) supplemented with 10% heat-inactivated fetal bovine serum (HyClone, SV35959), 2 mM stable glutamine (Capricorn Scientific, STA-B), 1x antibiotic-antimycotic (Thermo Fisher, Gibco™, 15240062), 1 mg / mL Geneticin (Gibco, 11811064), 60 pg / mL Hygromycin B (Gibco, 10687010) and 50 pM 2-mercaptoethanol (Sigma-Aldrich, M6250-100mL)) in T75 flasks (VWR, Greiner Bio-One, 658170). Cells were passaged 1:10 upon reaching >70% confluency using 0.25% Trypsin -0.03% EDTA to loosen the adherent population.OT-I, OT-I I, and CD11c+splenocyte isolation

[0123] OT-I cells were obtained from transgenic 6-8 weeks old female C57BL / 6 Tg(TcraTcrb)1100Mjb / Crl (Charles River), OT-II were obtained from transgenic 6-8 weeks old female C57BL / 6-Tg(TcraTcrb)425Cbn / Crl (Charles River), CD11c+splenocytes were obtained from 8-24 weeks old female C57BL / 6J (Charles River). Spleens were harvested and prepared for magnetic-assisted cell sorting (MACS) according to manufacturer’s protocol. For OT-I, OT-II, and CD11c+splenocytes, we used respectively CD8a T cell isolation kits (130-104-075, Miltenyi Biotec), CD4 T cell isolation kit s(130-104-454. Miltenyi Biotec), and CD11c MicroBeads UltraPure isolation kits (130-125-835, Miltenyi Biotec). For in vivo T cell activation studies, cells were stained with CellTrace™ Violet (ThermoFischer) before injection.GM-CSF BMDCs generation

[0124] Hindlegs of C57BL / 6 (Charles River) were dissected. Tibia and femur were cleaned and cut open with a scalpel. Bone marrow cells were flushed out and collected in a petri-dish. 10 mL complete RPMI 1640 medium (Thermo Fisher) supplemented with 50 pM 2-mercaptoethanol and 25 ng / mL GM-CSF was added per 10e6 cells. On day 3, 5 mL fresh media (+ 50 pM 2-mercaptoethanol, 25 ng / mL GM-CSF) was added. On day 8, non-adherent dendritic cells were harvested.Flt3L bone-marrow dendritic cells generation

[0125] Hindlegs of C57BL / 6 (Charles River) were dissected. Tibia and femur were harvested from 6-8 weeks old female C57BL / 6J mice (Charles River) and bone-marrow was flushed out. After ammonium-chloride-potassium (ACK) lysis, 15-25e6 cells were seeded in a T75 flask in complete RPMI medium supplemented with 200 ng / mL hFlt3-ligand (Miltenyi Biotec, 130-096-479). After 8 days, Flt3L BMDCs were harvested and resuspended in complete RPMI medium for experiments.Ex vivo OT-I activation assays

[0126] 10,000-20,000 Flt3L BMDCs were plated in 25 pL cRPMI and pulsed for 2 h at 37 °C with vaccine candidates at concentrations given in the figures, supplemented with 0.3 pg / mL LPS final concentration if indicated. Afterwards, BMDCs were washed and CellTrace™ violet-stained OT-I cells (5:1 OT-l:BMDCs) were added. The BMDC-OT-I co-culture was incubated for 72 h at 37 °C in 200 pL complete RPMI medium. Then, supernatant was collected for ELISA analysis and cells were prepared for FACS analysis using a FACSLyric™ (BD Biosciences) or FACSVerse™ (BD Biosciences).In vivo OT-I activation assay

[0127] CellTrace™ violet-stained OT-I cells were obtained as described below. 6-8 weeks old female C57BL / 6 (Charles River, Fra) were injected intravenously with 1e6 OT-I cells in 200 pL PBS. The following day mice were injected intravenously with 5 pmol vaccine constructs (±0.25 pg, ±12.5 ng / g) and 10 pg LPS in 200 pL PBS. Two days after, mice were killed by cervical dislocation and spleens and lymph nodes were harvested. Cells were prepared for FACS analysis and analyzed using a FACSLyric™ (BD Biosciences).Ex vivo OT-I I activation assay

[0128] Flt3L BMDCs and OT-II cells were prepared as described above. Flt3L BMDCs were plated in 25 pL at 20,000 cells per condition. The cells were incubated for 2 h at 37 °C with 10-100-1000 nM vaccine conjugates and 0.3 pg / mL LPS final concentration. Afterwards, cells were washed with PBS and 100,000 CellTrace™ violet-stained OT-II cells were added. The BMDC-OT-II co-culture was incubated for 72 h at 37 °C in 200 pL complete RPMI medium. Then, supernatant was collected for ELISA analysis and cells were prepared for FACS analysis using a FACSVerse™ (BD Biosciences).B16-OVA in vitro killing assay

[0129] Flt3L bone-marrow dendritic cells (BMDCs) were prepared as described above. Flt3L BMDCs were harvested and seeded in 50 pL complete RPMI at 100,000 cells per condition. Vaccine conjugates were added in 50 pL PBS at a final concentration of 100 nM and 0.3 pg / mL LPS. The cells were incubated for 2 h in a 5% CO2 humidified incubator at 37 °C. Meanwhile, OT-I cells were prepared as described herein. The BMDCs were washed and transferred to a 12-well plate, in which a 1:5 co-culture was set up by adding 500,000 OT-I cells. This co-culture was incubated overnight in a 5% CO2 humidified incubator at 37 °C. In parallel, B16-OVA cells were pulsed overnight with 100 ng / mL IFNy. The following day, B16-OVA cells were harvested, labeled with CellTrace™ Violet (ThermoFischer) for 20 min at 37 °C, washed, and seeded in 100 pL complete RPMI at 10,000 cells per condition. The cells were left to attach for 2-3 h. Meanwhile, OT-I cells were harvested and the supernatant was stored for ELISA analysis. Part of the OT-I cells was analyzed using flow cytometry, while the other part was resuspended to be added to the B16-OVA cells. The co-culture with B16-OVA cells was incubated for 24 h in a 5% CO2 humidified incubator at 37 °C. After incubation, supernatant was stored for analysis and cells were harvested using Trypsin-EDTA and analyzed by flow cytometry using a FACSVerse™ (Biosciences). Percentage killing was calculated using the following formula: %killing = (1 -(frequency condition viable target cells / frequency negative control viable targeted cells)) x 100%.EXAMPLESExample 1: In vitro study of linear mono-ubiquitin vaccines on dendritic cell mediated OT-I T cell activation.

[0130] Mono-ubiquitin vaccines for dendritic cell (DC)-targeted antigen delivery were investigated. The ovalbumin epitope SIINFEKL (OVAp, OVA 257-264; SEQ ID NO: 2) was used and attached to the C-terminus of ubiquitin directly (Ub-OT-I) or via FR dipeptide motif (Ub-FR-OT-I), via solid-phase peptide synthesis (Fig. 1A).

[0131] After synthesis and purification, the OT-I epitope conjugates were tested for their ability to induce antigen cross-presentation by DCs, as measured by their ability to activate SHNFEKL-specific (SEQ ID NO: 2) OT-I T cells in vitro (Fig. 1B). High levels of activation markers CD25, 4-1 BB and CD44 were observed on OT-I cells, as well as secretion of the pro-inflammatory cytokines interferon y (I FNy) and interleukin-2 (IL-2) in the Ub-OT-I conjugate conditions, irrespective of the presence of the FR-motif (Fig. 1B-F).Example 2: In vitro study of isopeptide-linked mono-ubiquitin vaccines on dendritic cell mediated OT-I T cell activation

[0132] Mono-ubiquitin vaccines with epitopes conjugated via isopeptide-linkage for dendritic cell (DC)-targeted antigen delivery was investigated. The ovalbumin epitope SIINFEKL (OVAp, OVA257-264; SEQ ID NO: 2) was used and attached to ubiquitin via the ‘intra-epitope’-lysine (UbiQ-368) or via an additional lysine attached to the C-terminus of the epitope (UbiQ-369) (Fig. 2A).

[0133] After synthesis and purification, the OT-I epitope conjugates were tested for their ability to induce antigen cross-presentation by DCs, as measured by their ability to activate SHNFEKL-specific (SEQ ID NO: 2) OT-I T cells in vitro (Fig. 2B). The activity was compared to OVA24 (the standard OT-I epitope containing synthetic long peptide vaccine DEVSGLEQLESIINFEKLAAAAAK, SEQ ID NO: 14) and linear Ub-OT-I (OT-I pep (SIINFEKL ‘short peptide’; SEQ ID NO: 2) was used as assay positive control condition). High levels of T cell activation markers CD25, 4-1 BB and CD44 were observed on OT-I cells in the ubiquitin conjugate conditions, irrespective of linkage type (Fig. 2C). All ubiquitin conjugates outperform the classical long peptide vaccine (OVA24). It is thought that the slightly reduced activity of UbiQ-369 is because the additional lysine has to be removed by amino peptidases to release the epitope and to facilitate loading into MHC class I molecules.Example 3: Mono-ubiquitin and ubi-tagged ubiquitin - epitope conjugates elicit potent T cell mediated ex vivo B16-OVA cancer cell killing.

[0134] The ubiquitin - OT-I epitope conjugates were tested for their ability to induce DC antigen cross-presentation mediated antigen-specific T cell activation and target cell (B16-OVA melanoma cell line) killing (Fig. 3A). Mono-ubiquitin-epitope fusions were compared to anti-DEC205 Fab targeted di-ubiquitin conjugates (OT-I pep (SIINFEKL ‘short peptide’; SEQ ID NO:2) was used as assay positive control condition). Potent antigen-specific target cell killing was observed for all ubiquitin vaccines (Fig. 3B), as well as robust OT-I T cell activation (Fig. 3C-F).Example 4: In vivo studies of Ub-antigen conjugates on OT-I activation after intravenous vaccination

[0135] The ability of Ub-OVAp, Ub-FR-OVAp, Fab-Ub2-OVAp, Fab-Ub2-FR-OVAp and Fab-Srt-FR-OVAp conjugates to induce OT-I activation were investigated in vivo after intravenous vaccination (NOTE: OVAp = OT-I epitope = SIINFEKL, SEQ ID NO: 2).

[0136] The day after adoptive transfer of CellTrace Violet (CTV)-labeled OT-I cells, mice were intravenously injected with a low dose (5 pmol, ±12.5 ng / g) of either conjugated anti-DEC205 Fab-Ub2-OVAp, Fab-Ub2-FR-OVAp, Fab-Srt-FR-OVAp, ora combination of unconjugated Fab-Ub(K48R)donand Ubacc-OVAp (= Ub-OT-I) or Ubacc-FR-OVAp (= Ub-FR-OT-I) (Fig. 4A). Two days after vaccination, the progressive dilution of CTV in the OT-I cells in the spleens and inguinal lymph nodes was evaluated (Fig. 4B-E). Strong OT-I cell proliferation in the mice treated with the ubitagged di-ubiquitin conjugates, as well as with Ub-OT-I monoubiquitin vaccine was observed, whereas the sortagged conjugate induced minimal OT-I proliferation at the dose used in this experiment. This experiment demonstrates the superiority of the mono-ubiquitin-epitope conjugate over the state-of-the-art DC targeted sortagged vaccine.Example 5: In vivo studies of Ub-antigen conjugates on OT-I activation after subcutaneous vaccination

[0137] The ability of Ub-OT-I, Fab-Ub2-OT-I and PLGA nanovaccine encapsulated Ub-OT-I to induce OT-I activation were investigated in vivo after subcutaneous vaccination.

[0138] The day after adoptive transfer of CellTrace Violet (CTV)-labeled OT-I cells, mice were subcutaneously injected with a low dose (20 pmol) of either conjugated anti-DEC205 Fab-Ub2-OT-I, Ub-OT-I or PLGA NP encapsulated Ub-OT-I (Fig. 5A). Two days after vaccination, the progressive dilution of CTV and activation marker expression in the OT-I cells in the inguinal lymph nodes was evaluated (Fig. 5B and Fig. 5C-E, respectively). Strong OT-I cell proliferation and activation in the mice treated with the ubiquitin vaccine was observed, with the monoubiquitin vaccine outperforming the anti-DEC205 Fab conjugated di-ubiquitin vaccine. The PLGA NP encapsulated Ub-OT-I is also able to induce T cell activation, albeit with lower efficacy as determined at the current timepoint. It is thought that this may be due to the slow release kinetics of PLGA nanovaccines. This experiment demonstrates the efficacy of the mono-ubiquitin-epitope conjugate to induce antigen-specific T cell activation upon subcutaneous vaccination.Example 6: Primary TCR-transfected T cell activation assay

[0139] Glycoprotein 100 (gp100) is considered a melanoma tumor-associated antigen of which gp100280-288 (gplOOp) has been identified as an HLA-A*0201 binding epitope.

[0140] Primary CD8 T cells from HLA-A*02:01+ donors were transfected with a gplOOp specific T cell receptor (TCR) to assess the efficacy of the ubiquitin conjugates to induce HLA-A*02:01+ moDC-mediated activation of primary human T cells (Fig. 6A). Proliferation, activation and cytokine secretion (Fig. 6B) of the T cells were assessed and showed a more potent dosedependent increase in the conditions where moDCs were pulsed with Ub-gp100 compared to the non-targeted conjugate. In summary, the results obtained demonstrate that ubi-tagged nanobody-peptide epitope conjugates are worthy of further examination as DC-targeted vaccine vehicles in a human setting.Example 7: In vitro evaluation of ubiquitin-helper T cell epitope conjugates on T cell activation

[0141] Mono-ubiquitin vaccines for dendritic cell (DC)-targeted MHCII epitope delivery were investigated. The ovalbumin epitope ISQAVHAAHAEINEAGR (OT-II, OVA 323-339; SEQ ID NO: 3) was used and attached to the C-terminus of ubiquitin (Ub-OT-I I).

[0142] After synthesis and purification, the OT-II epitope conjugates were tested for their ability to induce MHCII antigen presentation by DCs, as measured by their ability to activate antigen-specific OT-II T cells in vitro. The activity was compared to unconjugated OT-II peptide (ISQAVHAAHAEINEAGR, SEQ ID NO: 3) and Fab-Ub2-OT-ll, the anti-DEC205 Fab conjugated di-ubiquitin vaccine. Strong OT-II T cell proliferation (Fig. 7A) and high levels of T cell activation markers CD25, 4-1 BB and CD44 were observed on OT-II cells, with both ubiquitin vaccines outperforming the non-conjugated OT-II peptide (Fig. 2B-D). This demonstrates that monoubiquitin conjugation improves the efficacy of MHCII peptide epitope presentation by DCs.Example 8: Effects of ubiquitin mutation on the performance of ubiquitin-antigen vaccines

[0143] The importance of the C-terminal ubiquitin DUB cleavage site for vaccine efficacy is investigated. Mono-ubiquitin-OT-l conjugate is synthesized in which glycine-75 and glycine-76 are mutated to valine (Ub(W)-OT-l).

[0144] After synthesis and purification, Ub-OT-I and Ub(W)-OT-l were treated with the DUBs UCH-L3 and UCH-L1 (Fig. 8A and Fig. 8B, respectively). DUB treatment of Ub-OT-I resulted in the detection of a product with the molecular weight of 8564 Da, which corresponds to free ubiquitin (calculated mass: 8564), while no or reduced Ub-OT-I could be detected (calculated mass: 9510). DUB treatment of Ub(W)-OT-l did not induce cleavage, with no mass detected corresponding to Ub(W) (calculated mass: 8648), but only the mass of Ub(W)-0T-l (calculated mass: 9594). The OT-I epitope ubiquitin mutant conjugates were tested fortheir ability to induce MHCI antigen cross-presentation by DCs, as measured by their ability toactivate antigen-specific OT-I T cells in vitro. The activity of the mono-ubiquitin conjugates was compared to the anti-DEC205 Fab conjugated di-ubiquitin analogues. All conditions induced OT-I T cell proliferation (Fig. 8C), which is an extremely sensitive readout with minimal crosspresentation required to reach the plateau of the assay. T cell activation marker expression and cytokine secretion are better reporters for differential efficacy (Fig. 8D-F). Here it becomes evident that in particular for the efficacy of the mono-ubiquitin-epitope conjugate the integrity of the C-terminal di-glycine DUB cleavage motif is of crucial importance. This is in contrast with the anti-DEC205 Fab conjugated di-ubiquitin derivates, which are impacted less by ubiquitin mutation. This demonstrates that mono-ubiquitin-epitopes are differentially processed, potentially via an alternative intracellular routing compared to Fab conjugated di-ubiquitin derivates.SEQUENCES

[0145] The following sequences are provided:

[0146] SEQ ID NO:1 Ubiquitin (Ub), amino acids 1 to 76 of UBB_HUMAN MQIFVKTLTGKTITLEVEPSDTIENVKAKIQDKEGIPPDQQRLIFAGKQLEDGRTLSDYN IQKESTLHLVLRLRGG

[0147] SEQ ID NO:2: OVAp or OT-I (OVA 257-264)SIINFEKL

[0148] SEQ ID NO:3: OVA 323-339 or OT-IIISQAVHAAHAEINEAGR

[0149] SEQ ID NO:4: 2W1SEAWGALANWAVDSA

[0150] SEQ ID NO:5: gp100YLEPGPVTA

[0151] SEQ ID NO:6: NY-ES-O1SLLMWITQAbu

[0152] SEQ ID NO:7: MAGEKVLEYVIKV

[0153] SEQ ID NO:8: MelanELAGIGILTV

[0154] SEQ ID NO:9: Casp5FLIIWQNTM

[0155] SEQ ID NQ:10: FluM1GILGFVFTL

[0156] SEQ ID NO:11:FR

[0157] SEQ ID NO:12: K*FRK(N3)FR

[0158] SEQ ID NO:13: SrtLPESGG

[0159] SEQ ID NO: 14 : OVA24 DEVSGLEQLESIINFEKLAAAAAK

[0160] SEQ ID NO:15 : UbiQ-368 SIINFEK(Ub)L

[0161] SEQ ID NO:16 : UbiQ-369Ac-K(Ub)SIINFEKL

[0162] SEQ ID NO:17 :SIINFE(thi0K)L

[0163] SEQ ID NO:18 :Ac-(thi0K)SIINFEKL

[0164] SEQ ID NO:19 : Ub-OT-IUb-SIINFEKL

[0165] SEQ ID NQ:20 : Ub-FR-OT-I Ub-FRSIINFEKL

[0166] SEQ ID NO:21 : Ub-K*FR-OVAp Ub-K(N3)FRSIINFEKL

[0167] SEQ ID NO:22 : Ub-OVA24Ub-DEVSGLEQLESIINFEKLAAAAAK

[0168] SEQ ID NO:23 : Ub-OVA 323-339 Ub-ISQAVHAAHAEINEAGR

[0169] SEQ ID NO:24 : Ub-21WSUb-EAWGALANWAVDSA

[0170] SEQ ID NO:25 : Ub-gp100Ub-YLEPGPVTA

[0171] SEQ ID NO:26 : Ub-NY-ESO-1 Ub-SLLMWITQAbu

[0172] SEQ ID NO:27 : Ub-Casp5Ub-FLIIWQNTM

[0173] SEQ ID NO:28 : Ub-MAGEUb-KVLEYVIKV

[0174] SEQ ID NO:29 : Ub-FluM1Ub-GILGFVFTL

[0175] SEQ ID NQ:30 : Ub-MelanUb-ELAGIGILTV

[0176] SEQ ID NO:31 : N3-Ub-FR-OTIIOrn(N3)-PEG-Nle- QIFVKTLTGKTITLEVEPSDTIENVKAKIQDKEGIPPDQQRLIFAGKQLEDGRTLSDYNIQKESTLH LVLRLRGGFRISQAVHAAHAEINEAGR

[0177] SEQ ID NO:32 : mN3-Ub-FR-OTIOrn(N3)-PEG-Orn(N3)-PEG-Orn(N3)-PEG-Nle- QIFVKTLTGKTITLEVEPSDTIENVKAKIQDKEGIPPDQQRLIFAGKQLEDGRTLSDYNIQKESTLH LVLRLRGGFRSIINFEKL

[0178] SEQ ID NO:33 : N3-Ub-FR-OTIOrn(N3)-PEG-Nle-QIFVKTLTGKTITLEVEPSDTIENVKAKIQDKEGIPPDQQRLIFAGKQLEDGRTLSDYNIQKESTLH LVLRLRGGFRSIINFEKL

[0179] SEQ ID NO: 34 : Modified Ub X1QIFVKTLTGX2TITLEVEPSDTIENVKAKIQDX3EGIPPDQQRLIFAGKQLEDGRTLSDYNIQ X4ESTLHLVLRLRGG

Claims

CLAIMS1. A vaccine component comprising a ubiquitin covalently bonded to an antigen, wherein the vaccine component does not comprise a cell permeability tag.

2. The vaccine component of claim 1 , wherein residues 75-76 of the ubiquitin are GG.

3. The vaccine component of claim 1 , wherein the vaccine component comprises a fusion protein comprising the ubiquitin and the antigen.

4. The vaccine component of claim 1 , wherein the vaccine component is of formula I:U-A1-B (I)whereinU is an ubiquitin;A1is a bond or a linker; andB is an antigen.

5. The vaccine component of claim 4, wherein U-A1-B is or comprises a fusion protein.

6. The vaccine component of claim 4 or claim 5, wherein U is a ubiquitin comprising at least 80% sequency identity to SEQ ID NO: 1.

7. The vaccine component of any preceding claim, wherein the antigen is selected from a tumour-associated antigen, a tumour neoantigen, a pathogen-derived antigen and a selfantigen.

8. The vaccine component of any preceding claim, wherein the antigen comprises an epitope.

9. The vaccine component of any preceding claim, wherein the antigen comprises a tumour-associated antigen for CD8+ and I or CD4+ T cell activation;optionally, wherein the tumour-associated antigen is or comprises a MHCI epitope or a MHCII epitope; and / oroptionally wherein the tumour-associated antigen is selected from gp100, NY-ESO-1, MAGE and Melan.

10. The vaccine component of any of claims 1 to 7, wherein the antigen is selected from OT-1 and 2W1S.

11. The vaccine component of any of claims 4 to 10, wherein B comprises a tumour-associated antigen, optionally having at least 80% sequence identity to a sequence selected from SEQ ID Nos: 2 - 11.

12. The vaccine component of any of claims 4 to 11, wherein A1is a bond.

13. The vaccine component of any of claims 4 to 11, wherein A1is a peptide linker, optionally comprising or consisting of a sequence selected from SEQ ID NOs: 12 and 13.

14. The vaccine component of any of claims 4 to 13, wherein A1is attached to the C-terminus of the ubiquitin.

15. The vaccine component of any of claims 4 to 14, wherein the ubiquitin covalently bound to the antigen is of any of formulae (l-a) to (l-f):

16. The vaccine component of any of claims 1 to 15, further comprising an adjuvant;optionally wherein the adjuvant is covalently attached to the ubiquitin or antigen.

17. The vaccine component of claim 16, wherein the ubiquitin covalently bound to the antigen is of formula (ll-a), (ll-b) or (ll-c):D-A2-U-A1-B (ll-a) U-A1-B-A2-D (ll-b) U-A2-D-A1-B (ll-c), whereinD is an adjuvant; andeach A2is independently a bond or a linker.

18. A vaccine, comprising a vaccine component of any preceding claim, optionally wherein the vaccine component is encapsulated.

19. The vaccine of claim 18, wherein the vaccine component is encapsulated to provide encapsulated nanoparticles.

20. A combination comprising a vaccine component of any of claims 1 -17 or a vaccine of claim 18 or claim 19, and a biologically active substance.

21. A formulation comprising the vaccine component of any of claims 1 to 17, the vaccine of claim 18 or claim 19, or the combination of claim 19, and optionally a pharmaceutically acceptable carrier and / or excipient.

22. A vaccine component of any of claims 1 to 17, vaccine of claim 18 or claim 19, combination of claim 20, or formulation of claim 21 , for use as a medicament.

23. A vaccine component of any of claims 1 to 17, vaccine of claim 18 or claim 19, combination of claim 20, or formulation of claim 21 , for use in the treatment of a condition selected from a cancer and an autoimmune disease.

24. The vaccine component, vaccine, combination, or formulation for use in the treatment of a condition of claim 23, wherein the condition is selected from melanoma, lung cancer, Lynch syndrome, type 1 diabetes, and rheumatoid arthritis.

25. A method for the treatment of a cancer or an autoimmune disease, comprising administration of an effective amount of a vaccine component of any of claims 1 to 17, vaccine of claim 18 or claim 19, combination of claim 20, or formulation of claim 21 , to a subject in need thereof.

26. The method of claim 25, wherein the cancer or autoimmune disease is selected from melanoma, lung cancer, Lynch syndrome, type 1 diabetes, and rheumatoid arthritis.

27. Use of the vaccine component of any of claims 1 to 17 in the activation of dendritic cells, optionally wherein the activation of dendritic cells is conducted in vitro.

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