Vaccine compositions

Abstract

This invention relates to a cancer vaccine conjugate that comprises a CRM197 carrier protein and an immunogenic peptide covalently linked by a linker to residues C186 and 0201 of the CRM197 carrier. Suitable immunogenic peptides include glycopeptides, for example glycopeptides comprising tumor-associated carbohydrate antigens (TACAs), such as GalNAc, Galb(1,3)GalNAc, or Neu5Acα(2-6)GalNAc. Cancer vaccine conjugates and methods for their production and use are provided.

Description

[0001] Vaccine Compositions Field The present invention relates to vaccines, in particular conjugate vaccines, and methods for their production. Background Mucin-1 (MUC1) is a highly O-glycosylated glycoprotein expressed on the surface of epithelial cells. Its extracellular domain is comprised by tandem repeats of 20 amino acids (AHGVTSAPDTRPAPGSTAPP) containing five potential O-glycosylation sites (shown as bold letters in the peptide sequence).(1, 2) While this protein bears complex oligosaccharides in healthy tissues, MUC1 is decorated with simple and truncated carbohydrates in tumor cells, where its expression is dramatically increased(3-5) due to malfunction or translocation of GalNAc-transferases(2) or mutations in COSMC, a chaperone required for glycosyltransferase C1GalT activity.(6) As a result, the immunogenic epitope APDTRP(7, 8) and several ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^O-GalNAc-Ser / Thr, hereinafter Tn- Ser and Tn-Thr, respectively) become exposed and may elicit a weak immune response.(9) .(9) The development of MUC1-based vaccines has been influenced by the discovery that cancer patients can generate anti-MUC1 antibodies in the early stages of the disease.(10, 11) These vaccines typically contain the full sequence of MUC1 glycosylated at one or more sites with Tn or other TACAs conjugated to protein carriers, liposomes, or nanoparticles, among others.(12-18) This strategy favours the multipresentation of the antigen but, on the other hand, leads to a heterogeneous conjugate that may result in poorly reproducible formulations. Despite these synthetic efforts, there have been no successful clinical applications,(19) likely because the aberrantly glycosylated proteins can be present even at low concentrations on healthy cells (PMID: 34526666), leading to immune system tolerance and, consequently, poor immune response in pre- clinical models in mice. Summary The present inventors have developed a cancer vaccine conjugate that generates robust immune responses against tumours and can be produced with high reproducibility in a homogeneous form. A first aspect of the invention provides a cancer vaccine conjugate comprising; a CRM197 carrier protein, an immunogenic peptide and a linker that covalently links the immunogenic peptide to residues C186 and C201 of the CRM197 carrier, and the salts, solvates and protected forms thereof. Suitably, the cancer vaccine conjugate may have the formula: Z-L-D wherein: Z is the CRM197 carrier protein, L is the linker, and D is the immunogenic peptide. In some preferred embodiments, the cancer vaccine conjugate of the first aspect may stimulate immune responses against cancer cells expressing Mucin-1 (MUC1). For example, the immunogenic peptide may be a natural or synthetic MUC1 glycopeptide. A second aspect of the invention provides a method of producing a cancer vaccine conjugate, providing a CRM197 carrier protein covalently linked to a first connector, wherein the first connector is covalently linked to residues C186 and C201 of CRM197 carrier protein and comprises a first reactive group, providing an immunogenic peptide covalently linked to a second connector, wherein the second connector comprises a second reactive group, reacting the first and second reactive groups to covalently link the first and second connectors and attach the immunogenic peptide to the CRM197 carrier protein, thereby producing the cancer vaccine conjugate. In some preferred embodiments, the first reactive group may be an azido group and the second reactive group may be a cyclooctyne group. A CRM197 carrier protein may be covalently linked to a first connector for use in methods of the second aspect by a method comprising; providing a CRM197 carrier protein, selectively reducing the C186 - C201 disulfide linkage of the CRM197 carrier protein to generate free thiol groups at residues C186 and C201 of the CRM197 carrier protein, providing a first connector compound comprising first, third and fourth reactive groups, and reacting the third and fourth reactive groups of the first connector compound with the free thiol groups at C186 and C201, respectively, to covalently link the first connector to residues C186 and C201 of the CRM197 carrier protein, thereby covalently linking the CRM197 carrier protein to the first connector. In some preferred embodiments, the third and fourth reactive groups may be halide groups, such as chloride groups. The immunogenic peptide of the first and second aspects may comprise a tumour specific antigen or a tumour associated antigen (TAA), preferably a tumor-associated carbohydrate antigen (TACA). A third aspect of the invention provides a kit for producing a cancer vaccine conjugate comprising; a CRM197 carrier protein covalently linked to a first connector, wherein the first connector is covalently linked to residues C186 and C201 of CRM197 carrier protein and comprises a first reactive group. The kit may be suitable for use in a method of the second aspect. A fourth aspect of the invention provides a pharmaceutical composition comprising a cancer vaccine conjugate of the first aspect and a pharmaceutically acceptable excipient. A fifth aspect of the invention provides a method of treatment of cancer comprising administering a cancer vaccine conjugate of the first aspect or a pharmaceutical composition of the fourth aspect to an individual in need thereof. A sixth aspect of the invention provides a cancer vaccine conjugate of the first aspect or a pharmaceutical composition of the fourth aspect for use in the treatment of cancer. A seventh aspect of the invention provides the use of a cancer vaccine conjugate of the first aspect or a pharmaceutical composition of the fourth aspect in the manufacture of a medicament for use in the treatment of cancer. Other aspects and embodiments of the invention are described in more detail below. Brief Description of the Figures Figure 1 shows the present approach for preparation of cancer vaccines based on unnatural MUC1-derived glycopeptides. (a) schematic of the cancer vaccine conjugate. (b) structure of MUC-1 derived glycopeptide (c) structure of N-terminal moiety R (1’). Figure 2 shows (a) Optimized conditions used in this work to selectively modify cysteines 186 and 201 of protein CRM197 ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^197-linker-azide. (c) CD spectra of CRM197 and CRM197-linker- azide Figure 3 shows (a) Optimized conditions used in this work to prepare the vaccine candidate CRM197-linker-1’. ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^197-linker-1’. (c) Representative ensembles obtained from MD simulations. The protein is shown in white ribbons and carbon atoms of the linker, MUC1 and GalNAc are represented as blue, green and orange sticks, respectively. The E-oxime and 1,5-triazole adduct was considered in these calculations. Figure 4 shows a) Vaccine administration scheme used in this work. (b) Total level of IgG antibodies elicited by CRM197-linker-1’, CRM197 and PBS at different stages, as determined by ELISA assay. (d) IgG isotypes detected after the 4thimmunization by ELISA assay. (d) Total level of IgM antibodies elicited by CRM197- linker-1’, CRM197 and PBS after 4thimmunization, as determined by ELISA assay. Flow cytometry analysis to study the binding to (e) HEK293T cells (negative control) and (f) TD47 cells that express TA-MUC1 on their surface. Black line – serum from mice injected with PBS; Blue line – commercial anti-MUC1 antibody; Pink line – serum from mice immunized with CRM197; Red line – serum from mice immunized with CRM197- MUC1. (e) Statistical significance was determined by an unpaired T-test Figure 5 shows (a) Tumor weight determined at day 15 post tumor induction (MC38-MUC1 cells). Mice were first treated with the different formulations before the tumor induction. differences not statistically significant. (b) Tumor volume measured after tumor induction with the different formulations. (c) Circulating Th1 cytokine levels, after tumor induction, in mice treated with the different conjugates. (d) Circulating Th2 cytokine levels in mice, after tumor induction, treated with the different conjugates. Differences not statistically significant (e) Cytotoxic T Lymphocyte assay. T-cells isolated from the spleens of the immunized mice were co-cultured with MC38-MUC1 cells for 24h at 90:1 ratio (T-cells:MC38-MUC1 cells). A decrease in MC38-MUC1 cells viability was observed. Statistical significance was determined by an unpaired T-test. Data represents Mean+SEM. (f) Probability of survival of mice after inducing of a tumor (MC38-MUC1 cells) and being treated with the corresponding conjugates. Figure 6 shows that CRM197-linker-1’ delays tumor growth and prolongs survival when administered therapeutically to a pancreatic cancer mouse model. Tumor volume measured after tumor induction with the different formulations (*p= 0.0197) (left panel), together with probability of survival (right panel) of mice after inducing of a tumor and being treated with the corresponding formulations (*p=0.1092). Figure 7 shows CRM197-linker-1’ delays tumor growth and prolongs survival when administered therapeutically in a combination treatment with a checkpoint inhibitor. Tumor volume measured after tumor induction (MC38-MUC1 –upper panel, p=0,0362– or Panc02-MUC1 –lower panel, p=0,0201– cell lines) with the different formulations, along with probability of survival of mice under these conditions (MC38-MUC1 – upper right, *p=0.1017– or Panc02-MUC1 –lower right, *p= 0.0163– cell lines). Figure 8 shows CRM197-linker-1’ endotoxin levels. Conjugate samples were subjected to endotoxin levels determination prior to animal administration. Two independent samples had levels below 1 EU / mL (0,844100013 and 0,946199968). Endotoxin levels are considered safe for mice administration if below 1.5 EU / mL. Figure 9 shows Maximum tolerated dose (MTD) evaluation. The MTD of the CRM197-linker-1’ was evaluated by injecting different doses of the vaccine, in normal Balb / c mice, in a total of 4 subcutaneous (SC) injections administered with 2 days Interval. Signs of toxicity were checked both at the macroscopic level (such as mouse weight loss and signs of pain / discomfort) and at the microscopic level (by histological analysis of the different organs). No significant weight loss was observed neither any abnormalities in the mice tissues, indicating no signs of toxicity at the tested doses of vaccine. Detailed Description This invention relates to cancer vaccines that comprise a CRM197 carrier protein and an immunogenic peptide that is covalently attached by a linker to residues C186 and C201 of the CRM197 carrier. Populations of cancer vaccine conjugates described herein may be homogeneous i.e. all of the molecular species within the population may have the same chemically defined structure. Preferably, a single immunogenic peptide is covalently attached to each CRM197 carrier protein in the cancer vaccine conjugates described herein i.e., the stoichiometric ratio of CRM197 carrier protein to immunogenic peptide in a cancer vaccine conjugate may be 1: 1. The production of cancer vaccine conjugates as described herein may be useful in reducing batch to batch variation in vaccine production. The cancer vaccine conjugates may elicit antibodies that recognize selectively the naturally occurring antigen corresponding to the immunogenic peptide in patient’s samples. In addition, cancer vaccine conjugates may activate the Th1 immune response and generate T cells that promote cytotoxic T lymphocyte (CTL) responses. The cancer vaccine conjugate may exert an anti-tumour effect in patients. The cancer vaccine conjugates may also display reduced enzymatic degradation and reduced tolerance in vivo compared to vaccines employing natural antigens. A cancer vaccine conjugate described herein comprises an immunogenic peptide. The immunogenic peptide may elicit an immune response against cancer cells in an individual. For example, the immunogenic peptide may comprise one or more epitopes from a tumour antigen that is expressed by cancer cells in an individual. Tumour antigens are well-known in the art and may include protein antigens and tumor-associated carbohydrate antigens (TACAs), such as Survivin, p53, PSA, MUC-1, MUC-4, MUC- 16, and CEA. Suitable epitopes may include peptide and carbohydrate epitopes. Preferably, the immunogenic peptide may be a glycopeptide. Suitable glycopeptides may include natural or artificial glycopeptides. For example, suitable glycopeptides may include glycopeptides that comprise one or more glycosylation sites, for example O-, S- , Se- or N-glycosylation sites. Glycosylation sites may include serine residues, threonine residues or unnatural amino acid residues with a side chain comprising a hydroxyl (R-OH) group, a thiol (R-SH) or disulfide (S-S) group, or a selenol (R-SeH) or diselenium (Se-Se) group. In some preferred embodiments, the immunogenic peptide may be a mucin 1 (MUC-1) glycopeptide. The MUC-1 glycopeptide may be a natural mucin 1 (MUC-1) glycopeptide or more preferably a synthetic artificial mucin 1 (MUC-1) glycopeptide.). A synthetic artificial mucin 1 (MUC-1) glycopeptide may comprise one or more unnatural or non-canonical amino acid residues i.e. amino acid residues that are not encoding by the genetic code of naturally occurring organisms. Unnatural amino acid residues may include ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ A synthetic artificial MUC-1 glycopeptide may further comprise a carbohydrate antigen, such as GalNAc (Tn ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ antigen derived from one of these. The carbohydrate antigen may be attached to a glycosylation site within the peptide sequence of the glycopeptide by a glycosidic linkage. For example, a carbohydrate antigen, such as Tn, T or sTn, may be linked to the peptide sequence by an O-glycoside linkage at a Ser, Thr or unnatural amino acid containing R-OH. A carbohydrate antigen may be linked to the peptide sequence by an S- glycoside linkage at an unnatural amino acid containing R-SH. For example, an immunogenic glycopeptide ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ linked to the peptide sequence by a Se-glycoside linkage at an unnatural amino acid containing R-SeH. In some preferred embodiments, the immunogenic glycopeptide may be a MUC-1 glycopeptide and may ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ comprise the amino acid sequence AX1DX2RP, where X1 is 4S ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^2 is S ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ thiothreonine (SEQ ID NO: 1), or a variant thereof. In some preferred embodiments, a MUC-1 glycopeptide may comprise the amino acid sequence HGVTSAX1DX2RPAPGSTAPPA, where X1 is 4S ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ and X2 is S ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ In some embodiments, the immunogenic glycopeptide may comprise two or more glycosylation sites. Carbohydrate antigens may be attached to the glycopeptide at the two or more of the glycosylation sites. The carbohydrate antigens attached to the two or more glycosylation sites may be the same or different. For ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ may be attached to the glycopeptide at a both a first and a second glycosylation site or one of GalNAc (Tn ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ glycopeptide at a first glycosylation site and another of GalNAc (Tn antigen) , Galb(1,3)GalNAc (T antigen), ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ Cross-reactive material 197 (CRM197) is a diphtheria toxin (DT) that is detoxified by the presence of a Gly to Glu mutation at a position corresponding to position 52 of the wild type CRM197 sequence shown in SEQ ID NO: 3 (Broker et al (2011) Biologicals 39(4) 195-204). CRM197 is widely used as a carrier protein for polysaccharides and haptens and has been used successfully in vaccines against Haemophilus influenzae, pneumococcus, and meningococcus (e.g. Menveo®, a tetravalent conjugate vaccine against Neisseria meningitidis serogroups A-C-W135-Y) (25-27) The CRM197 carrier protein may comprise the amino acid sequence of SEQ ID NO: 3 or a variant thereof. A variant of a reference sequence set out herein, such as a reference immunogenic glycopeptide or CRM197 sequence, may comprise an amino acid sequence having at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, or at least 98% sequence identity to the reference sequence. Particular amino acid sequence variants may differ from a reference sequence shown herein by insertion, addition, substitution, or deletion of 1 amino acid, 2, 3, 4, 5, 6, 7, 8, 9, or 10 or more than 10 amino acids. Sequence similarity and identity are commonly defined with reference to the algorithm GAP (Wisconsin Package, Accelerys, San Diego USA). GAP uses the Needleman and Wunsch algorithm to align two complete sequences that maximizes the number of matches and minimizes the number of gaps. Generally, default parameters are used, with a gap creation penalty = 12 and gap extension penalty = 4. Use of GAP may be preferred but other algorithms may be used, e.g. BLAST (which uses the method of Altschul et al. (1990) J. Mol. Biol. 215: 405-410), FASTA (which uses the method of Pearson and Lipman (1988) PNAS USA 85: 2444-2448), or the Smith-Waterman algorithm (Smith and Waterman (1981) J. Mol Biol. 147: 195- 197), or the TBLASTN program, of Altschul et al. (1990) supra, generally employing default parameters. In particular, the psi-Blast algorithm (Nucl. Acids Res. (1997) 253389-3402) may be used. CRM197 may be produced by recombinant means using established techniques or obtained from commercial suppliers. In the vaccine conjugates described herein, the immunogenic glycopeptide is connected to residues C186 and C201 of the CRM197 carrier protein by a covalent linker. The linker forms a bridge between residues C186 and C201 of the CRM197 carrier protein and connects the immunogenic glycoprotein to the CRM197 carrier protein. The linker may be covalently attached to (i) residue C186 of the CRM197 carrier, (ii) residue C201 of the CRM197 carrier and (iii) the N terminus of the MUC-1 glycopeptide. The linker, -L-, is a group that attaches the CRM197 carrier protein, Z-, to the immunogenic glycopeptide, -D. It will be appreciated that any suitable linker may be used to connect the CRM197 carrier protein to the immunogenic glycopeptide. In some embodiments, the linker may comprise one or more groups selected from a polyethylene glycol moiety, an amino acid residue, an alkylenediamine moiety, an arylene-containing moiety, a heteroarylene- containing moiety, a heterocyclyl-containing moiety, a cycloalkyl-containing moiety and combinations thereof. Suitably, the linker comprises between 1 and 20 of the above-mentioned groups. More suitably, the linker comprises between 1 and 15 of the above-mentioned groups. Even more suitably, the linker comprises between 1 and 10 of the above-mentioned groups. Suitably, the linker comprises one or more groups selected from a polyethylene glycol moiety, an amino acid residue, an alkylenediamine moiety and combinations thereof. The polyethylene glycol moiety will be understood to be a functional grouping that comprises one or more alkylene glycol moieties, such as, one or more ethylene glycol moieties. In certain embodiments, the linker comprises between 1 and 20 alkylene glycol moieties (e.g., ethylene glycol moieties), preferably between 1 and 10 alkylene glycol moieties (e.g. ethylene glycol moieties), and most preferably between 2 and 8 alkylene glycol moieties (e.g. ethylene glycol moieties). The alkylenediamine moiety will be understood to be a functional grouping that contains or is derived from an alkylenediamine moiety, such as, an ethylenediamine. The arylene-containing moiety will be understood to be a functional grouping that comprises one or more arylene groups. Suitably, the arylene will be a 6-membered arylene such as phenylene. In certain embodiments, the arylene-containing moiety is a functional grouping that includes a 1,3,5-benzyl group. The heteroarylene-containing moiety will be understood to be a functional grouping that comprises one or more heteroarylene groups. Suitably, the heteroarylene will be a functional grouping that includes a 6- membered heteroaryl such as triazine. In certain embodiments, the arylene-containing moiety is a functional grouping that includes a 1,3,5 triazine group. The heterocyclyl-containing moiety will be understood to be a functional grouping that comprises one or more heterocycle groups. Suitably, the heterocycle is a 6-membered heterocycle such as triazinane. In certain embodiments, the heterocyclyl-containing moiety is a functional grouping that includes a 1,3,5 triazinane group. The cycloalkyl-containing moiety will be understood to be a functional grouping that comprises one or more cycloalkyl groups. Suitably, the cycloalkyl is a 6-membered cycloalkyl such as cyclohexane. In certain embodiments, the cycloalkyl-containing moiety is a functional grouping that includes a 1,3,5 cyclohexane. The amino acid residues will be understood to constitute both natural and unnatural amino acid residues. Suitably, the amino acid residue will be a natural amino acid residue. The amino acid residue may also comprise two or more amino acids, such as, for example, dipeptide and tripeptide moieties. Suitable amino acid residues include Phe, Lys, Val, Ala, Cit, Leu, Ile, Arg, and Trp residues. In some embodiments, the amino acid residue is a dipeptide residue. The amino acids in the dipeptide may be any combination of natural amino acids and non-natural amino acids. In some embodiments, the dipeptide comprises natural amino acids. In certain embodiments the linker is a cathepsin labile linker, and the dipeptide is the site of action for cathepsin-mediated cleavage. The dipeptide then is a recognition site for cathepsin. In some embodiments, the amino acid residue is selected from the group consisting of: NH-Phe-Lys-C=O, NHVal-Ala-C=O, NHVal-Lys-C=O, NHAla-Lys-C=O, NH-Val-Cit-C=O, NH-Phe-Cit-C=O, NH-Leu-Cit-C=O, NH-Ile-Cit-C=O, NH-Phe-Arg-C=O, and NH-Trp-Cit-C=O; where Cit is citrulline, and NH and C=O are amino and carboxy terminals of the amino acid residues which are in turn connected to the remainder of the linker. is selected from the group consisting of: NH-Val-Cit-C=O. Most preferably, the amino acid residue is selected fromNH-Phe-Lys-C=O,NH-Val-Cit-C=OorNH-Val-Ala-C=O. Other dipeptide combinations may be used, including those described by Dubowchik et al., Bioconjugate Chemistry, 2002, 13,855-869, which is incorporated herein by reference. In some embodiments, the amino acid residue is a tripeptide residue. The amino acids in the tripeptide may be any combination of natural amino acids and non-natural amino acids. In some embodiments, the tripeptide comprises natural amino acids. Where the linker is a cathepsin labile linker, the tripeptide is the site of action for cathepsin-mediated cleavage. The tripeptide then is a recognition site for cathepsin. ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ An amino acid side chain may be chemically protected, where appropriate. Protected amino acid sequences may be enzymatically cleavable. For example, a dipeptide sequence comprising a Boc side chain-protected Lys residue is cleavable by cathepsin. Protecting groups for the side chains of amino acids are well known in the art and are described in the Novabiochem Catalogue. In certain embodiments, the linker -L- comprises one or more groups that are susceptible to enzymatic cleavage (e.g., proteolytic or peptidase cleavage, such as cathepsin cleavage, sulfatase cleavage or galactosidase cleavage). Thus, in some embodiments, the linker -L- comprises one or more amino-acid residues, dipeptide residues or tripeptide residues, one or more aryl sulfates, one or more aryl galactosides or combinations thereof. Suitably, the linker -L- comprises one or more amino-acid residues, dipeptide residues or tripeptide residues, wherein the amino-acid residues, dipeptide residues or tripeptide residues are as described below. In certain embodiments, the linker -L- is or comprises one or more groups that are susceptible to chemical cleavage (e.g. a disulfide or a hydrazone). In other embodiments, the linker may be a non-cleavable linker. Such non-cleavable linkers are well known in the art of antibody drug conjugates. Thus, in some embodiments, the linker -L- is or comprises one or more groups selected from a C1- C20alkylene, a (poly)ethylene glycol moiety, an alkylenediamine moiety, an amino acid residue and combinations thereof. In certain embodiments, the linker is a below: p Formula V wherein: ZAis a protein linking group; Q is a spacer group; p indicates the position where the linker is attached to the CRM197 carrier protein; and the position where the linker is attached to the immunogenic glycopeptide. The protein linking group ZAmay be any functional group that can link the CRM197 carrier protein to the linker. The protein linking group ZAis thus a functional grouping that is capable of forming a covalent attachment to sulfur atoms present on the CRM197 carrier protein. Suitably, the sulfur atoms are sulfur atoms from residues C186 and C201 of the CRM197 carrier protein. It will therefore be appreciated that the linker serves to re- bridge a reduced interchain disulfide bond between residues C186 and C201 of the CRM197 carrier protein. Functional groups capable of linking the CRM197 carrier protein to the linker in this way are well known in the art. Thus, the skilled person will be able to select suitable protein linking groups to use with the invention. Examples of suitable protein linking groups, ZA, include, for instance, those described in, for example, Xu 2021 (ChemRxiv. Cambridge: Cambridge Open Engage; 2021, 1-17), Stieger 2021 (Angew. Chemie Int. Ed., 2021, 60, 1-7), Walsh 2019 (Chem. Sci., 2019, 10, 694–700), Walsh 2020 (Org. Biomol. Chem., 2020, 18, 4224–4230), Badescu 2014 (Bioconjug. Chem., 2014, 25, 1124–1136), Koniev 2018 (Medchem comm, 2018, 9, 827–830), Robinson 2017 (RSC Adv., 2017, 7, 9073–9077), Behrens 2015 (Mol. Pharm., 2015, 12, 3986–3998) and WO2019 / 011078. In certain embodiments, ZAis selected from one of the following protein linking groups: , p p , , Q where: p indicates the position ZAis attached to the CRM197 carrier protein; and Q indicates the position where ZAis attached to the spacer group, Q. In certain embodiments, ZAis the following protein linking group: where: p indicates the position ZAis attached to the CRM197 carrier protein; and Q indicates the position where ZAis attached to the spacer group, Q In some embodiments, Q is a spacer comprising one or more groups selected from a polyethylene glycol moiety, an amino acid residue, an alkylenediamine moiety, an arylene-containing moiety, a heteroarylene- containing moiety, a heterocyclyl-containing moiety, a cycloalkyl-containing moiety and combinations thereof. Each of the polyethylene glycol moiety, the amino acid residue, the alkylenediamine moiety, the arylene- containing moiety, the heteroarylene-containing moiety, the heterocyclyl-containing moiety, and the cycloalkyl-containing moiety may preferably be any of those groups defined hereinabove. In certain preferred embodiments, Q is a spacer comprising one or more groups selected from a C1- C20alkylene, a polyethylene glycol moiety, an amino acid residue, an alkylenediamine moiety and combinations thereof. As outlined below, the linker may be formed by covalently linking a first connector and a second connector. The first connector may be covalently linked to the CRM197 carrier protein, and the second connector may be covalently linked to the immunogenic glycopeptide. The covalent linkage formed between the first connector and second connector results in the formation of the linker, -L-, that covalently links the CRM197 carrier protein and immunogenic glycopeptide. Thus, in certain embodiments, the spacer group, Q, is group of Formula VI shown below: -Q1-FG-Q2- Formula VI where: Q1 is a first spacing group covalently linked to the CRM197 carrier protein; Q2 is a second spacing group covalently linked to the immunogenic glycopeptide; and FG is a functional group linking Q1 to Q2. It will be appreciated that the first spacing group, Q1, and the second spacing group, Q2, may be any of the groups defined herein in association with spacer group Q or in association with linker, L. In certain preferred embodiments, the first spacing group, Q1, and the second spacing group, Q2, independently comprise one or more groups selected from a C1-C20alkylene, a (poly)ethylene glycol moiety, an alkylenediamine moiety, an amino acid residue and combinations thereof. In certain preferred embodiments, first spacing group, Q1, is a group of the formula: ZA wherein: n from 1 to 6, preferably 1 to 4 and most preferably 2; ZA indicates the position where the first spacing group, Q1, is attached to the protein ZA; and FG indicates the position where the first spacing group, Q1, is attached to the functional group, FG. In certain preferred embodiments, the second spacing group, Q2, is a group of the formula: O O O wherein: m is an integer from 1 to 8, preferably 2 to 6 and most preferably 4; indicates the position where the second spacing group, -Q2-, is attached to the functional group, FG; and indicates the position where the second spacing group, -Q2-, is attached to the immunogenic glycopeptide, D. In certain embodiments, FG is a bond or a functional group formed from the reaction between: i) a ketone or aldehyde and an alkoxyamine (e.g. hydroxylamine) or hydrazine; ii) an azide and an alkyne; iii) an amine and an acyl halide or carboxylic acid; iv) an electron-rich dienophile (e.g. a 1,3-nitrone alkene) and an electron- poor diene (e.g. tetrazine); and iii) a strained alkene or alkyne (e.g. norbornene or cyclooctyne) and a tetrazine. Suitably, FG is a bond or a functional group formed from the reaction between an azide and an alkyne or an amine and an acyl halide or carboxylic acid. Most suitably, FG is a functional group formed from the reaction between an azide and an alkyne. Thus, in some embodiments FG is a functional group selected from a bond, , , , , -NH(=O)C- and -C(=O)NH-. In some embodiments, FG is selected from , . In certain preferred embodiments, the linker is a group of the formula: where; n is an integer selected from 1 to 4 (preferably 2); m is an integer selected from 2 to 8 (preferably 4); p are residues C186 and C201 of the CRM197 carrier protein where the linker is attached to the carrier protein; and pep indicates the position where the linker is attached to the immunogenic glycopeptide (e.g. the N terminus of the MUC-1 glycopeptide). A method of producing a cancer vaccine conjugate as described herein may comprise; providing a CRM197 carrier protein covalently linked to a first connector, wherein the first connector is covalently linked to residues C186 and C201 of CRM197 carrier protein and comprises a first reactive group, providing an immunogenic glycopeptide covalently linked to a second connector, wherein the second connector comprises a second reactive group, and reacting the first and second reactive groups to covalently link the immunogenic glycopeptide to the CRM197 carrier protein, thereby producing the cancer vaccine. The first and second reactive groups may be any functional groups that may be reacted together to covalently link the immunogenic glycopeptide to the CRM197 carrier protein. In some embodiments, the first and second reactive groups may react to form a covalent bond through a click chemistry reaction. In certain embodiments, the first and second reactive groups are selected from the following pairings: i) an azide and an alkyne ii) a ketone or aldehyde and an alkoxyamine (e.g. hydroxylamine) or hydrazine; iii) an amine and an acyl halide or carboxylic acid; iv) an electron-rich dienophile (e.g. a 1,3-nitrone alkene) and an electron-poor diene (e.g. tetrazine); and v) a strained alkene or alkyne (e.g. norbornene or cyclooctyne) and a tetrazine. In the above pairings, the first reactive group will be one functional group from the pairing, whilst the second reactive group will be the other (paired) functional group from the pairing. In certain preferred embodiments, one of the first and second reactive groups is an azide and the other of the first and second reactive groups is an alkyne. Preferably, the first reactive group is an azide (azido group) and the second reactive group is an alkyne (alkynyl group). Most preferably, the first reactive group is an azide and the second reactive group is a cyclooctyne, such as a dibenzocyclooctyne. Thus, in certain embodiments, the first connector is group of Formula VI shown below: p Formula VI where: Q1 is a first spacer group covalently linked to the CRM197 carrier protein; RG1 is the first reactive group (e.g., an azide); and p are residues C186 and C201 of the CRM197 carrier protein where the linker is to the CRM197 carrier protein. In some embodiments, the second connector is group of Formula VII shown below: where: Q2 is a second spacer group covalently linked to the immunogenic glycopeptide; and RG2 is the second reactive group (e.g., an alkyne); and pep indicates the position where the linker is attached to the immunogenic glycopeptide (e.g. MUC-1 glycopeptide). It will be appreciated that the first spacer group, Q1, and the second spacer group, Q2, of the first and second connectors may be any of the groups defined hereinabove in association with Q1 and Q2. In certain preferred embodiments, the first connector covalently linked to the CRM197 carrier protein is a group of the formula: p where: n from 1 to 4, preferably 2; and p are residues C186 and C201 of the CRM197 carrier protein where the linker is attached to the CRM197carrier protein. In other preferred embodiments, the second connector covalently linked to the immunogenic glycopeptide is a group of the formula: pep where: m is an integer from 2 to 8, preferably 4; and indicates the position where the linker is attached to the immunogenic glycopeptide (e.g. MUC-1 glycopeptide). The CRM197 carrier protein may be covalently linked to a first connector by a method comprising; providing a CRM197 carrier protein, selectively reducing the C186 - C201 disulfide linkage of the CRM197 carrier protein to generate free thiol groups at residues C186 and C201 of the CRM197 carrier protein, providing a first connector compound comprising first, third and fourth reactive groups, and reacting the third and fourth reactive groups of the first connector compound with the free thiol groups at C186 and C201, respectively, to covalently link the first connector compound to residues C186 and C201 of the CRM197 carrier protein, thereby covalently linking the CRM197 carrier protein to the first connector. The first connector compound may be any compound comprising reactive groups (i.e. third and fourth reactive groups) that are capable of forming a covalent attachment to sulfur atoms from residues C186 and C201 of the CRM197 carrier protein. It will also be appreciated that by forming covalent attachments to the sulfur atoms from residues C186 and C201 of the CRM197 carrier protein, the first connector compound serves to re-bridges the reduced interchain disulfide bond between residues C186 and C201 of the CRM197 carrier protein. Reactive groups capable of linking the first connector compound to the sulfur atoms from residues C186 and C201 of the CRM197 carrier protein are well known in the art. Thus, the skilled person will be able to select suitable reactive groups (third and fourth reactive groups) to use with the invention. Examples of suitable reactive groups, include, for instance, those described in, for example, Xu 2021 (ChemRxiv. Cambridge: Cambridge Open Engage; 2021, 1-17), Stieger 2021 (Angew. Chemie Int. Ed., 2021, 60, 1-7), Walsh 2019 (Chem. Sci., 2019, 10, 694–700), Walsh 2020 (Org. Biomol. Chem., 2020, 18, 4224–4230), Badescu 2014 (Bioconjug. Chem., 2014, 25, 1124–1136), Koniev 2018 (Medchemcomm, 2018, 9, 827–830), Robinson 2017 (RSC Adv., 2017, 7, 9073–9077), Behrens 2015 (Mol. Pharm., 2015, 12, 3986–3998) and WO2019 / 011078. In certain embodiments, the first connector compound is a compound of the general formula:WQ1RG1where: Q1 is a first spacer group covalently linked to the CRM197 carrier protein, as defined herein; RG1 is the first reactive group (e.g., an azide); and W is a functional group comprising the third and fourth reactive groups. In other embodiments, the first connector compound is a compound of the general formula: where: n is an integer selected from 1 to 4 (e.g. 2); RG1 is the first reactive group (e.g., an azide); and W is a functional group comprising the third and fourth reactive groups. Suitably, the functional group comprising the third and fourth reactive groups, W-, is selected from one of the functional groups shown below: Q1 , wherein: the position where the functional group comprising the third and fourth reactive groups, W-, is attached to the remainder of the first connector compound. In certain embodiments, the third and fourth reactive groups are halide groups, such as fluoro, chloro, bromo or iodo groups, preferably chloro groups. In certain preferred embodiments, the first connector compound is a compound of the formula: where n is an integer selected from 1 to 4 (e.g. 2). The C186 - C201 disulphide linkage of the CRM197 carrier protein may be selectively reduced, such that the C461-C471 disulphide linkage is not reduced. For example, the C186 - C201 disulfide linkage may be selectively reduced using TCEP. The invention also provides reagents and kits for producing a cancer vaccine conjugate as described herein. For example, a kit for producing a cancer vaccine conjugate may comprise a CRM197 carrier protein covalently linked to a first connector, wherein the first connector is covalently linked to residues C186 and C201 of CRM197 carrier protein and comprises a first reactive group. Examples of salts of the compounds (e.g., conjugates and first connector compounds) of the invention include all pharmaceutically acceptable salts, such as, without limitation, acid addition salts of strong mineral acids such as HCI and HBr salts and addition salts of strong organic acids such as a methanesulfonic acid salt. Further examples of salts include sulphates and acetates such as trifluoroacetate or trichloroacetate. A reference to a compound (e.g., conjugates and first connector compounds) described herein, is also a reference to a solvate of that compound. Examples of solvates include hydrates. A compound (e.g., conjugates and first connector compounds) described herein, includes a compound where an atom is replaced by a naturally occurring or non-naturally occurring isotope. In one embodiment the isotope is a stable isotope. Thus, a compound described here includes, for example deuterium containing compounds and the like. For example, H may be in any isotopic form, including1H,2H (D), and3H (T); C may be in any isotopic form, including12C,13C, and14C; O may be in any isotopic form, including16O and18O; and the like. Certain compounds (e.g. conjugates and first connector compounds), may exist in one or more particular geometric, optical, enantiomeric, diasteriomeric, epimeric, atropic, stereoisomeric, tautomeric, conformational, or anomeric forms, including but not limited to, cis- and trans-forms; E- and Z-forms; c-, t-, and r- forms; endo- and exo-forms; R-, S-, and meso-forms; D- and L-forms; d- and l-forms; (+) and (-) forms; ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ equatorial forms; boat-, chair-, twist-, envelope-, and halfchair-forms; and combinations thereof, hereinafter collectively referred to as “isomers” (or “isomeric forms”). Note that, except as discussed below for tautomeric forms, specifically excluded from the term “isomers,” as used herein, are structural (or constitutional) isomers (i.e., isomers which differ in the connections between atoms rather than merely by the position of atoms in space). For example, a reference to a methoxy group, OCH3, is not to be construed as a reference to its structural isomer, a hydroxymethyl group, CH2OH. Similarly, a reference to ortho-chlorophenyl is not to be construed as a reference to its structural isomer, meta-chlorophenyl. However, a reference to a class of structures may well include structurally isomeric forms falling within that class (e.g., C16alkyl includes n-propyl and iso-propyl; butyl includes n, iso-, sec-, and tert-butyl; methoxyphenyl includes ortho-, meta-, and para-methoxyphenyl). Unless otherwise specified, a reference to a particular compound (e.g. conjugates and first connector compounds) includes all such isomeric forms, including mixtures (e.g., racemic mixtures) thereof. Methods for the preparation (e.g., asymmetric synthesis) and separation (e.g., fractional crystallisation and chromatographic means) of such isomeric forms are either known in the art or are readily obtained by adapting the methods taught herein, or known methods, in a known manner. In certain embodiments, the compounds (e.g., conjugates and first connector compounds) described herein may be in substantially purified form and / or in a form substantially free from contaminants. In some embodiments, the substantially purified form is at least 50% by weight, e.g., at least 60% by weight, e.g., at least 70% by weight, e.g., at least 80% by weight, e.g., at least 90% by weight, e.g., at least 95% by weight, e.g., at least 97% by weight, e.g., at least 98% by weight, e.g., at least 99% by weight. Unless specified, the substantially purified form refers to the compound in any stereoisomeric or enantiomeric form. For example, in one embodiment, the substantially purified form refers to a mixture of stereoisomers, i.e., purified with respect to other compounds. In one embodiment, the substantially purified form refers to one stereoisomer, e.g., optically pure stereoisomer. In one embodiment, the substantially purified form refers to a mixture of enantiomers. In one embodiment, the substantially purified form refers to an equimolar mixture of enantiomers (i.e., a racemic mixture, a racemate). In one embodiment, the substantially purified form refers to one enantiomer, e.g., optically pure enantiomer. In some embodiments, the contaminants represent no more than 50% by weight, e.g., no more than 40% by weight, e.g., no more than 30% by weight, e.g., no more than 20% by weight, e.g., no more than 10% by weight, e.g., no more than 5% by weight, e.g., no more than 3% by weight, e.g., no more than 2% by weight, e.g., no more than 1% by weight. Unless specified, the contaminants refer to other compounds, that is, other than stereoisomers or enantiomers. In one embodiment, the contaminants refer to other compounds and other stereoisomers. In one embodiment, the contaminants refer to other compounds and the other enantiomer. In some embodiments, the substantially purified form is at least 60% optically pure (i.e., 60% of the compound, on a molar basis, is the desired stereoisomer or enantiomer, and 40% is the undesired stereoisomer or enantiomer), e.g., at least 70% optically pure, e.g., at least 80% optically pure, e.g., at least 90% optically pure, e.g., at least 95% optically pure, e.g., at least 97% optically pure, e.g., at least 98% optically pure, e.g., at least 99% optically pure. Compounds of the invention, such as the cancer vaccine conjugates described herein, may be provided in a protected form. Here, reactive functionality, such as amino functionality, may be masked in order to prevent its reaction during a synthesis step. A protecting group is provided to mask the reactive functionality, and this protecting groups may be removed at a later stage of the synthesis to reveal the original reactive functionality. For example, amino, hydroxyl, carboxyl and thiol functionality present in the conjugate may be protected with a protecting group, such as described herein. In one embodiment, the protected form is a compound where amino, hydroxyl, thiol, and / or carboxyl functionality is protected (masked) by a protecting group. In one embodiment, the protected form is a compound where the side chain functionality of the amino acids residues with the compound are protected. Protecting groups, such as those for amino acid residues, are well known and well described in the art. Amino acids having side group protection, optionally together with amino and carboxy protection, are commercially available. Thus, a protected conjugate compound may be prepared from appropriately protected amino acid starting materials. Where a protecting group is used it is removable under conditions that do not substantially disrupt the structure of the conjugate, for example conditions that do not alter the stereochemistry of the amino acid residues, or do not cause release of the protected ortho-quinone. In some embodiments, the protecting groups are acid-labile, base labile, or are removable under reducing conditions. Example protecting groups for amino functionality include Boc (tert-butoxycabonyl), Bn (benzyl, Bzl), CbZ (Z), 2-CL-Z (2-chloro), Dde (1-[4,4-dimethyl-2,6-dioxocylcohex-1-ylidene]-3-methylbutyl), Fmoc (fluorenylmethyloxycarbonyl), HSO3-Fmoc (sulfonylated Fmoc, such as 2-sulfo-Fmoc, as described in e.g. Schechter et al, J.Med Chem 2002, 45(19) 4264), ivDde (1-[4,4-dimethyl-2,6-dioxocylcohex-1-ylidene]ethyl), Mmt (4-methoxytrityl), Mtt (4-methyltrityl), Nvoc (6-nitroveratroyloxycarbonyl), and Tfa (trifluroacetyl). Example protecting groups for aromatic nitrogen functionality includes Boc, Mtt, Trt and Dnp (dinitrophenyl). In some embodiments, the protecting group for amino functionality is selected from Boc, CbZ, Bn and Fmoc and HSO3-Fmoc. In some embodiments, the protecting group for amino functionality is Boc, Fmoc or CbZ. Example protecting groups for hydroxyl functionality include Trt (trityl), Bn (benzyl), tBu (tert-butyl), and 2- ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ In one embodiment, the protecting group for amino functionality is Trt. Further example protecting groups include silyl ether protecting groups, such as TMS, TES, TBS, TIPS, TBDMS, and TBDPS, and ethers such as THP. Such protecting groups are removable with TBAF, for example. Example protecting groups for carboxyl functionality include Bn (benzyl, Bz), tBu (tert-butyl), TMSET (trimethylsilylethyl) and Dmab ({1-[4,4-dimethyl-2,6-dioxocylcohex-1-ylidene]-3-methylbutyl}amino benzyl). Example protecting groups for aromatic nitrogen functionality include Boc, Mtt, Trt and Dnp (dinitrophenyl). In some embodiments, only some types of functionalities are protected. For example, only amino groups may be protected, such as amino groups in the side chain of an amino acid residue. In some embodiments, amino groups and hydroxyl groups are protected. The invention also provides pharmaceutical compositions comprising a cancer vaccine conjugate as described above and a pharmaceutically acceptable excipient. A method may further comprise admixing a cancer vaccine conjugate as described herein with a pharmaceutically acceptable excipient to produce a pharmaceutical composition. The term “pharmaceutically acceptable” relates to compounds, materials, compositions, and / or dosage forms which are, within the scope of sound veterinary or medical judgement, suitable for use in contact with the tissues of a subject (e.g., human or other mammal) without excessive toxicity, irritation, allergic response, or other problem or complication, commensurate with a reasonable benefit / risk ratio. Each carrier, excipient, etc. must also be “acceptable” in the sense of being compatible with the other ingredients of the formulation. Suitable excipients and carriers include, without limitation, water, saline, buffered saline, phosphate buffer, alcoholic / aqueous solutions, emulsions or suspensions. Other conventionally employed diluents, adjuvants, and excipients may be added in accordance with conventional techniques. Such carriers can include ethanol, polyols, and suitable mixtures thereof, vegetable oils, and injectable organic esters. Buffers and pH- adjusting agents may also be employed, and include, without limitation, salts prepared from an organic acid or base. Representative buffers include, without limitation, organic acid salts, such as salts of citric acid (e.g., citrates), ascorbic acid, gluconic acid, carbonic acid, tartaric acid, succinic acid, acetic acid, phthalic acid, Tris, trimethylamine hydrochloride, or phosphate buffers. Parenteral carriers can include sodium chloride solution, Ringer's dextrose, dextrose, trehalose, sucrose, lactated Ringer's, or fixed oils. Intravenous carriers can include fluid and nutrient replenishers, electrolyte replenishers, such as those based on Ringer's dextrose, and the like. Preservatives and other additives such as, for example, antimicrobials, antioxidants, chelating agents (e.g., EGTA; EDTA), inert gases, and the like may also be provided in the pharmaceutical carriers. The pharmaceutical compositions described herein are not limited by the selection of the carrier. The preparation of these pharmaceutically-acceptable compositions, from the above-described components, having appropriate pH, isotonicity, stability and other conventional characteristics, is within the skill of the art. Suitable carriers, excipients, etc. may be found in standard pharmaceutical texts, for example, Remington’s Pharmaceutical Sciences and The Handbook of Pharmaceutical Excipients, 4th edit., eds. R. C. Rowe et al, APhA Publications, 2003. The pharmaceutical composition of the invention may be a vaccine composition. The pharmaceutical composition or vaccine composition may further comprise an adjuvant. An adjuvant is a compound that enhances or augments the immune response to an immunogenic compound. Suitable adjuvants for use in vaccine compositions include but are not limited to inorganic compounds, e.g. aluminium salt, oils, bacterial products, e.g. toxoids, and cytokines, e.g. IL-1 and IL-2. Other suitable adjuvants include squalene-based oil-in-water nano-emulsions, such as MF-59 and AddaVax™. Vaccines and pharmaceutical compositions may conveniently be presented in unit dosage form and may be prepared by any methods well-known in the art of pharmacy. Such methods include the step of bringing the one or more isolated conjugates / immunogenic polypeptides into association with a carrier or excipient as described above which may constitute one or more accessory ingredients. In general, the formulations are prepared by uniformly and intimately bringing into association the active compound with liquid carriers or finely divided solid carriers or both. Vaccines and pharmaceutical compositions may be made in the form of sterile aqueous solutions or dispersions, suitable for injectable use, or made in lyophilized forms using freeze-drying techniques. Lyophilized pharmaceutical compositions are typically maintained at about 4°C, and can be reconstituted in a stabilizing solution, e.g., saline or HEPES, with or without adjuvant. Vaccines and pharmaceutical compositions may be presented in unit-dose or multi-dose sealed containers, for example, ampoules and vials, and may be stored in a freeze-dried (lyophilised) condition requiring only the addition of the sterile liquid carrier, for example water for injections immediately prior to use. Vaccines and therapeutic pharmaceutical compositions according to the present invention may be formulated for administration by a number of routes, including but not limited to, parenteral, intravenous, intra-arterial, intramuscular, intratumoural, oral and nasal. Cancer vaccine conjugates comprising an immunogenic glycopeptide linked to a CRM197 polypeptide as described herein may be internalised by B cells and may be useful, for example in the generation of B- and T-cell mediated immune responses against the immunogenic glycopeptide. Also provided herein are methods of stimulating an immune response in a subject, wherein the method comprises administering a conjugate or pharmaceutical composition described herein to a subject in need thereof; and conjugates or pharmaceutical compositions described herein for use in such methods. Immune responses include B cell mediated immune responses and T cell mediated immune responses. Also provided herein are methods of treatment of cancer comprising administering a cancer vaccine conjugate or pharmaceutical composition described herein to an individual in need thereof; a cancer vaccine conjugate or pharmaceutical composition described herein or use in the treatment of cancer and the use of a cancer vaccine conjugate or pharmaceutical composition described herein in the manufacture of a medicament for use in the treatment of cancer. Cancer is characterised by the abnormal proliferation of malignant cancer cells. Cancer may be of any cancer type, including leukaemia, such as AML, CML, ALL and CLL, lymphoma, such as Hodgkin lymphoma, non-Hodgkin lymphoma and multiple myeloma, and solid cancers such as sarcomas, skin cancer, melanoma, bladder cancer, brain cancer, such as glioma, breast cancer, such as triple-negative breast cancer and metastatic breast carcinoma, uterus cancer, oral cancer, ovary cancer, prostate cancer, lung cancer, colorectal cancer, such as colon carcinoma, cervical cancer, liver cancer, head and neck cancer, oesophageal cancer, pancreas cancer, renal cancer, adrenal cancer, stomach cancer, testicular cancer, cancer of the gall bladder and biliary tracts, thyroid cancer, thymus cancer, neuroblastoma, cancer of bone, and cerebral cancer. In some preferred embodiments, the cancer may be pancreatic, breast, ovarian, gastric, lung, gallbladder, or colorectal cancer. Cancer cells of a cancer suitable for treatment as described herein may express a tumour antigen that is recognised by an immune response elicited by the immunogenic peptide of the cancer vaccine conjugate. Suitable tumour antigens are known in the art and include Mucin-1 (MUC1). A cancer cell suitable for treatment as described herein may overexpress MUC1. For example, a cancer cell may express 1.5-fold or more, 2-fold or more, 3-fold or more, 4-fold or more, 5-fold or more, 6-fold or more, 7-fold or more, 8-fold or more, 9-fold or more, or 10-fold or more MUC1 than a normal cell. A cancer cell suitable for treatment as described herein may express MUC1 with aberrant glycosylation. For example, an aberrantly glycosylated MUC1 may be recognised by an immune response elicited by an immunogenic glycopeptide comprising the epitope of SEQ ID NO: 1. An individual suitable for treatment with a cancer vaccine conjugate or pharmaceutical composition described herein, may be a mammal, such as a rodent (e.g. a guinea pig, a hamster, a rat, a mouse), murine (e.g. a mouse), canine (e.g. a dog), feline (e.g. a cat), equine (e.g. a horse), a primate, simian (e.g. a monkey or ape), a monkey (e.g. marmoset, baboon), an ape (e.g. gorilla, chimpanzee, orang-utan, gibbon), or a human. In some preferred embodiments, the individual is a human. In other preferred embodiments, non-human mammals, especially mammals that are conventionally used as models for demonstrating therapeutic efficacy in humans (e.g., murine, primate, porcine, canine, or rabbit animals) may be employed. In some embodiments, the individual may have minimal residual disease (MRD) after an initial cancer treatment. An individual with a cancer may display at least one identifiable sign, symptom, or laboratory finding that is sufficient to make a diagnosis of cancer in accordance with clinical standards known in the art. Examples of such clinical standards can be found in textbooks of medicine such as Harrison’s Principles of Internal Medicine, 15th Ed., Fauci AS et al., eds., McGraw-Hill, New York, 2001. In some instances, a diagnosis of a cancer in an individual may include identification of a particular cell type (e.g. a cancer cell) in a sample of a body fluid or tissue obtained from the individual. The term “treatment”, as used herein in the context of treating a condition, pertains generally to treatment and therapy in which some desired therapeutic effect is achieved, for example, the inhibition of the progress of the condition, and includes a reduction in the rate of progress, a halt in the rate of progress and amelioration of the condition, and cure of the condition. Treatment may be any treatment and therapy, whether of a human or an animal (e.g. in veterinary applications), in which some desired therapeutic effect is achieved, for example, the inhibition or delay of the progress of the condition, and includes a reduction in the rate of progress, a halt in the rate of progress, amelioration of the condition, cure or remission (whether partial or total) of the condition, preventing, delaying, abating or arresting one or more symptoms and / or signs of the condition or prolonging survival of a subject or patient beyond that expected in the absence of treatment. Treatment as a prophylactic measure (i.e. prophylaxis) is also included. For example, an individual susceptible to or at risk of the occurrence or re-occurrence of cancer may be treated as described herein. Such treatment may prevent or delay the occurrence or re-occurrence of cancer in the individual. The cancer vaccine conjugates, and pharmaceutical compositions described herein may therefore be useful in the therapeutic treatment of an individual with cancer or the prophylactic treatment of an individual to prevent or delay the occurrence or re-occurrence of cancer. In particular, treatment may include inhibiting cancer growth, including complete cancer remission, and / or inhibiting cancer metastasis. Cancer growth generally refers to any one of a number of indices that indicate change within the cancer to a more developed form. Thus, indices for measuring an inhibition of cancer growth include a decrease in cancer cell survival, a decrease in tumor volume or morphology (for example, as determined using computed tomographic (CT), sonography, or other imaging method), a delayed tumor growth, a destruction of tumor vasculature, improved performance in delayed hypersensitivity skin test, an increase in the activity of cytolytic cancer cells, and a decrease in levels of tumor-specific antigens. In some preferred embodiments, a therapeutic agent, such as cancer vaccine conjugate or pharmaceutical composition described herein may be administered to the individual without other concomitant cancer therapy, such as cytotoxic chemotherapy or radiotherapy i.e. the therapeutic agent may be administered alone. In other embodiments, a cancer vaccine conjugate or pharmaceutical composition described herein may be administered in combination with one or more other therapies, such as cytotoxic chemotherapy or radiotherapy. When the therapeutic agents are used in combination with additional therapeutic agents, the compounds may be administered either sequentially or simultaneously by any convenient route. When a therapeutic agent is used in combination with an additional therapeutic agent active against the same disease, the dose of each agent in the combination may differ from that when the therapeutic agents are used alone. Appropriate doses will be readily appreciated by those skilled in the art. In some preferred embodiments, a cancer vaccine conjugate or pharmaceutical composition described herein may be administered in combination with an immune checkpoint inhibitor. An immune checkpoint inhibitor is an agent capable of inhibiting cellular signalling mediated by an immune checkpoint molecule. Immune checkpoint molecules may include PD-1, CTLA-4, LAG-3, TIM-3, TIGIT and BTLA. Immune checkpoint inhibitors are reviewed in e.g. Darvin P et al., Experimental & Molecular Medicine, 2018, 50:165; de Miguel M & Calvo E, Cancer Cell, 2020, 38:326; and Marin-Acevedo JA et al., J Hematol Oncol. 2021, 14:45. Suitable immune checkpoint inhibitors are available in the art and include for example ipilimumab, tremelimumab, nivolumab, pembrolizumab, atezolizumab, avelumab, durvalumab, cemiplimab, and dostarlimab. In some embodiments, the immune checkpoint inhibitor may inhibit PD-1-mediated cell signalling. The agent may be a PD-L1-targeted agent, or more preferably a PD-1-targeted agent. For example, the agent may be an antibody that specifically binds to PD-L1 or more preferably PD-1 and inhibits PD-1-mediated signalling. In some embodiments, the agent may be an anti-PD-L1 antibody or fragment thereof (e.g. an antagonist anti-PD-L1 antibody or fragment thereof), or an anti-PD-1 antibody or fragment thereof (e.g. an antagonist anti-PD-1 antibody or fragment thereof). Suitable anti-PD1 antibodies include nivolumab, pembrolizumab, dostarlimab and cemiplimab. Suitable anti-PD-L1 antibodies include atezolizumab, avelumab, and durvalumab. Administration of a cancer vaccine conjugate or pharmaceutical composition described herein, can be effected in one dose, continuously or intermittently (e.g., in divided doses at appropriate intervals) throughout the course of treatment. Methods of determining the most effective means and dosage of administration are well known to those of skill in the art and will vary with the formulation used for therapy, the purpose of the therapy, the target cell being treated, and the subject being treated. Single or multiple administrations can be carried out with the dose level and pattern being selected by the treating physician. Other aspects and embodiments of the invention provide the aspects and embodiments described above with the term “comprising” replaced by the term “consisting of” and the aspects and embodiments described above with the term “comprising” replaced by the term ”consisting essentially of”. It is to be understood that the application discloses all combinations of any of the above aspects and embodiments described above with each other, unless the context demands otherwise. Similarly, the application discloses all combinations of the preferred and / or optional features either singly or together with any of the other aspects, unless the context demands otherwise. Modifications of the above embodiments, further embodiments and modifications thereof will be apparent to the skilled person on reading this disclosure, and as such, these are within the scope of the present invention. All documents and sequence database entries mentioned in this specification are incorporated herein by reference in their entirety for all purposes. “and / or” where used herein is to be taken as specific disclosure of each of the two specified features or components with or without the other. For example, “A and / or B” is to be taken as specific disclosure of each of (i) A, (ii) B and (iii) A and B, just as if each is set out individually herein. Experimental Materials and Methods Synthesis of glycopeptide 1’. The synthesis was performed automatically with Rink Amide MBHA resin (0.05 mmol) and an automated synthesizer. The glycosylated amino acid building block (2.0 equiv) was synthesized as described in the literature.(24) This residue and the cyclooctyne derivate were manually coupled using HBTU [(2-(1H- benzotriazol-1-yl)-1,1,3,3-tetramethyluronium hexafluorophosphate], while the other fluorenylmethyloxycarbonyl (Fmoc) amino acids (5.0 equiv) were automatically coupled using oxyma pure / DIC (N,N'-diisopropylcarbodiimide). 20% (v / v) solution of piperidine in dimethylformamide (DMF) was used for Fmoc deprotection. O-acetyl groups of the carbohydrate were deprotected with 5 mL (3 × 15 min) of ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ 1’ was detached from the resin and all acid sensitive sidechain protecting groups were simultaneously removed using TFA 95%, TIS (triisopropylsilane) 2.5% and H2O 2.5%, followed by precipitation with cold diethyl ether. The crude ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ × 21.2 mm) and a dual absorbance detector, with a flow rate of 10 or 20 mL / min.. Synthesis of CRM197-linker-azide. 20 mg of 1,3-dichloroacetone (0.16 mmol) was reacted with 40 mg of aminooxy-PEG3-azide (0.17 mmol, 1.1 equiv.) in 1 mL of DMF at 21 ºC for 24 h. The reaction product was then purified through a silica column by applying an increasing gradient of ethyl acetate in hexane (40%-100%), followed by 20% methanol in ethyl acetate and 100% methanol. A total of 13 mg of compound 2 (0.04 mmol, 25% yield) was obtained and a stock of 40 mM in DMF was prepared. Next, CRM197 ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ mg / mL stock of CRM197 ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ compound 2 ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ 2) at 21 ºC for 5 d. The resulting modified protein was then passed through a ZebaSpin column (for buffer exchange to H2O) and analyzed by LC-MS. Synthesis of CRM197-linker-1’ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^197 ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ derivative 1’ (10 mg / mL in PBS, 20 equiv.) at 25 ºC for 20 h. At the end, the reaction mixtures were purified on Zeba spin columns and analyzed by mass spectrometry and SDS-PAGE and western blot. Circular Dichroism (CD) Spectroscopy Proteins were desalted by using a PD-minitrap G25 column (following manufacturer instructions) and further used at 5 µM (in PBS buffer pH 7.4). Far-UV circular dichroism (CD) data were acquired on a Jasco J-815 CD spectropolarimeter (Hachioji, Tokyo, Japan) using quartz cuvettes of 1.0 mm (Hellma GmbH & Co, Müllheim, Germany). All spectra were collected at 25 ºC, between 195 and 300 nm, with sampling velocity of 200 nm·min-1, data pitch of 0.5 nm, data integration time of 1 s, and 1 nm bandwidth. Each spectrum represents an average of 5 scans. Absorbance spectra were also monitored to control the light scattering and the signal detection saturation. In addition to blank subtraction, experimental instrument-related baseline drift was corrected by subtracting the average of the signal between 250 and 260 nm to each spectrum. The ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^2·dmol-1). All conditions were measured independently and in duplicate. Mass spectrometry analysis ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ MS was performed on a Waters Acquity UPLC system equipped with a QDa single quadrupole mass ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ mm) that was maintained at 40 °C. Solvents A (0.1% v / v formic acid in bidistilled water) and B (0.01% v / v formic acid in acetonitrile) were used as mobile phases. The gradients of solvents A and B were applied according to the following method: 10% of B for 5 min, followed by a slow gradient to 100% of B in 15 min. At 20.1 min B was returned to 10% and remained at this percentage for more 7.9 min to recondition the LC column. The total run time was 28 min and the flow rate 0.2 mL / min. The electrospray source was operated with a capillary voltage of 1.5 kV and a cone voltage of 20 V. Nitrogen was used as the desolvation gas at a total flow of 800 L / h with cone volume at 1 L / h. Desolvation temperature was set to 400 ºC. Total mass spectra were reconstructed from the ion series using the MaxEnt algorithm preinstalled on MassLynx software (version 4.1 from Waters). To obtain the ion series described, the protein peak(s) of the chromatogram were selected for integration and further analysis. SDS-PAGE Conjugation was confirmed by running the conjugates, WT CRM197 and glycopeptide 1’ in non-reducing conditions at 100V for 1.5 h in 12% acrylamide gels in a Tris-Glycine / SDS running buffer (25 mM Tris base, 190 mM glycine, 0.1% SDS, pH 8.3). PageRuler pre-stained protein ladder was used as molecular marker. The gels were further used for SDS-PAGE analysis, by staining them with InstantBlue Coomassie Protein Stain from Abcam (following the manufacturer instructions). Molecular dynamics (MD) simulations The crystal structure of CRM197 was used as starting coordinates for the protein.(25) It is important to note that although only one copy of glycopeptide 1’ is presented by the protein, up to 4 possible isomers could be expected (Z / E-oxime and 1,4 / 1,5-triazole adducts). Simulations were performer for the different isomers with AMBER 20 package (36), implemented with ff14SB(37) and gaff2(38) force fields. The LEaP module of AMBER 20 was used to generate the topology and coordinate files for the MD simulations, which were carried out using the CUDA version of the PMEMD module of the AMBER simulation package. Each conjugate was immersed in a water box with a 10 Å buffer of TIP3P water molecules(39) and the system was neutralized by adding explicit counter ions (Na+). A two-stage geometry optimization approach was performed with the PMEMD module. The first stage minimizes only the positions of solvent molecules and ions, using a 50 kcal·mol·-1·Å-2harmonic potential, and the second stage is an unrestrained minimization of all the atoms in the simulation cell. In both stages, 2500 steps of steepest descent minimization were followed by 2500 steps of conjugate gradient minimization. The systems were then heated by incrementing the temperature from 0 to 300 K under a constant pressure of 1 atm and periodic boundary conditions for 2 ns. Harmonic restraints of 10 kcal·mol-1were applied to the solute, and the Andersen temperature coupling scheme(40) was used to control and equalize the temperature. The time step was kept at 1 fs during the heating stages. The SHAKE algorithm was applied to constrain all bonds involving hydrogen atoms.(41) Long-range electrostatic effects were modelled using the particle-mesh Ewald method.(42) A sharp cut-off of 8 Å was applied to Lennard-Jones interactions. Each system was equilibrated for 2 ns with a 2-fs time step at a constant volume and temperature of 300 K. Production trajectories were then run for additional 0.5 µs under the same simulation conditions. MUC1.Tg mice genotyping MUC1.Tg breading pairs were purchased from The Jackson Laboratory and breading was setup at iMM-JLA Animal Facility. Ear samples were collected from the pups by the animal house technicians. Samples were then used for gDNA extraction through the NaOH gDNA extraction method. Briefly, samples were incubated ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ Taq DNA Polymerase (recombinant) (5 U / µL) from ThermoScientific. The reaction mixture was composed ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^2 ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ºC 30 s, 3-60 ºC 30 s, 4- 72 ºC 1 min (40 cycles of steps 2-4), 72 ºC 10min. Results from the PCR reactions were confirmed by agarose gel electrophoresis (2% gels). PCR were considered positive when 2 bands were present (transgene and internal control) and negative when only 1 band was present (internal control). Mice immunization protocol 12–14-week-old MUC1.Tg mice that express human MUC1 at the physiological level (breed in house) received a total of 4 IP injections of the vaccine candidates, administered with 21 days interval. A total of 3 groups of mice (with 5 mice per group) were used: CRM197-linker-1’ (total of 2 µg MUC1) – Group 1; CRM197 (47 µg of protein, same amount as injected in group 1) – Group 2; PBS – Group 3. Mice were injected with a total of 100µL, using the Squalene-oil-in-water Addavax adjuvant as part of the formulation (50 µL of adjuvant + 50 µL of vaccine candidate). Pre-immune and 5 days after each boost blood samples were collected by cheek puncture and five days after last boost the whole blood was collected by heart puncture. Sera was recovered from the blood by centrifugation (4 min, 4000 rpm) of the blood samples. Immunized mice were further used for tumor challenge with MC38-MUC1 cells. Antibody titers were determined by ELISA assay 96 well Maxisorb ELISA plates (Nunc) were coated with either the unnatural glycopeptide 1’ or CRM197 in coating buffer (4 ºC, overnight). Once blocked with ELISA Blocking Buffer, the plates were incubated with 1:100 dilutions of the sera from the first and second bleeds compared to the pre-immune sera. Bound antibodies were detected by the use of the anti-mouse secondary antibody IgG HRP (Invitrogen, 1:3000), IgG1-HRP (1:1000, eBioscience), IgG2a-HRP (1:50.000, Abcam), IgG2b-HRP (1:50.000, Abcam), IgG3- HRP (1:50.000, Abcam) and IgM-HRP (1:50.000, Abcam), All incubations were performed at 25 ºC for 1 h, with 3 washes with ELISA washing buffer between each incubation. The assays were developed with TMB 1x solution (eBioscience) for 10 min at 25 ºC. The TMB kinetic reaction was stopped with ELISA Stop Solution which resulted in a color change from blue to yellow that was possible to read at 450 nm by using an Infinite M200 plate reader. Background absorbance values (absorbance obtained for wells incubated only with the secondary antibody - blank) were subtracted before the representation of the raw data. Tumor induction in mice A localized model of colon cancer was established in the immunized MUC1.Tg mice by inoculating 1x106MC38-MUC1 cells (commercially available in Kerafast) subcutaneously in the flank 6 days after the last immunization. Tumor growth was monitored over time, by performing bilateral vernier caliper measurements every 3 days and mean tumor volumes were calculated using the formula (length x width2) / 2. At day 15 post tumor induction, animals were euthanized for collection of sera, tumors, spleens and draining lymph nodes. No signs of animal suffering, or discomfort were observed, including no weight loss. The data collected was analyzed using GraphPad Prism8. Cytokine and chemokine levels determination Cytokine levels in the sera of the immunized mice was determined through the Mouse High Sensitivity T-Cell Discovery Array 18-plex (MDHSTC18) assay from Eve Technologies. This assay allowed to quantify the ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ T-cells isolation for flow cytometry and CTL assay Mice were euthanized by isoflurane overdose and tumor, inguinal lymph nodes (iLNs) and spleens were collected for T-cell isolation. While iLNs and spleens where smashed against a 100 µm cell strainer and diluted in 2 mL and 8 mL of DMEM complete medium (containing 0.05 mM 2-mercaptoethanol) respectively, tumors were cut into small pieces (in 5 mL tubes) and incubated with 1 mL of digestion media (0.4 mg / mL collagenase I, 1mg / mL collagenase IV and 10 µg / mL DNase I for 30 min at 37 ºC, 220rpm. After digestion, cell suspensions were diluted in 4 mL ice cold DMEM complete medium, filtered with 100 µm cell strainers and centrifuged for 5 min at 500x g. Cell pellets were incubated with 1 mL of red blood cell lysis buffer and immediately centrifuged for 5 min at 500x g. At this point, cell pellets were resuspended in 2 mL of complete DMEM and counted for staining or cytotoxic T Lymphocyte (CTL) assay. Cell staining for T-cell determination by Flow cytometry Approximately 2x106cells were transferred to a round bottom 96-well plate and centrifuged for 3 min at 500x g to remove supernatant. Cell pellets were incubated with 20 µL of Fc Block (1:100) in FACS buffer for 10 min on ice. After washing cells with 100 µL FACS buffer and centrifuged (as previously), cells were incubated with 20 µL of extracellular antibody mix for approximately 45 min on ice. The extracellular antibody mix was composed by CD62L-FITC (1:500), CD44-PE (1:500), CD4-APC (1:500), CD45-BV510 (1:100), CD8a-SB600 (1:500) and CD3-BV711 (1:100). 7aad viability dye (5 µL / 1x106cells) was used to determine viable cells. Samples were acquired using a BD LSR Fortessa and analyzed with FlowJo (version 6.3.4). Magnetic-activated Cell Sorting (MACs) for isolation of T-cells from spleens Once smashed and passed through a 100 µm cell strainer, spleen cells were centrifuged for 5 min at 500x g. Cell pellets were incubated with red blood cell lysis buffer and immediately centrifuged again (500x g, 5 min). Supernatant was removed and cell pellets were incubated with anti-CD3-APC antibody (1:100 in MACS buffer) on ice for 15 min. After incubation, cells were washed with MACS buffer and centrifuged (5 min, 500x g). After removing supernatant, the cells were incubated with APC nano-beads solution (1:10 µL, around 10 µL per 107cells), on ice for 15 min. Once finished this incubation step, a washing step was performed with MACS buffer (centrifuged for 3 min at 500x g). Supernatant was removed and cells were resuspended in 2.5 mL MACS buffer and placed in the magnet stand for 5 min. Unlabelled fraction was discarded and the attached fraction was resuspended in 2.5 mL MACS buffer and the tube was put again in the magnetic stand. This step was repeated one more time (3x total). After the last step cells were counted and used for CTL assay. Cytotoxic T-lymphocyte Assay (CTL) To determine the CTL activity, MC38-MUC1 and MC38 cells were used together with the T-cells isolated from the spleens of the immunized mice. MC38-MUC1 and MC38 cells were cultured as indicated by the manufacturer (using complete DMEM in the case of MC38 and complete DMEM with gentamycin and Geneticin antibiotics in the case of MC38-MUC1 cells). Cells were seeded at 10.000 cells per well and allowed to adhere for 24 h. T-cells isolated from the mice were then added at 3:1, 10:1 and 90:1 effector-to- target ratios (T-cells: cancer cells). After 24 h the cell viability was determined by using CellTiter Blue (Promega) and M200 microplate reader. Cytotoxicity was calculated as percentage of viable cells in the presence of T-cells in comparison to cells without the presence of T-cells. Flow Cytometry analysis of Sera binding The binding affinity of the antibodies generated by the CRM197-linker-1’ vaccine candidate was determined by Flow Cytometry analysis. Briefly, T47D cells (human breast cancer cells with high expression of TA- MUC1) and HEK293T cells (with no expression of the human TA-MUC1) were fixed with an ice-cold solution of 4% paraformaldehyde for 10min. After fixation, the cells were permeabilized with 0.1% Triton-X100 in PBS for 15min, followed by a blocking step with 10% FBS in PBS for 30min. The cells were then incubated with with a 1:50 dilution of mice sera, followed by an incubation with the Goat anti-Mouse polyclonal IgG H&L Alexa Fluor 488 (1:2000) secondary antibody from Abcam. All the incubation steps were performed at 25 ºC for 1h and were followed by washing steps with PBS. Samples were acquired using a BD LSR Fortessa set up with a 488 nm laser and a 530 / 30 nm band-pass filter (combination used for Alexa488 detection) and analyzed with FlowJo (version 6.3.4). Results The previous studied unnatural glycopeptide 1(24) (Fig. 1) has been equipped with a cyclooctyne moiety at the N-terminus to react with an azido group selectively introduced into the CRM197 protein (see below). The treatment of CRM197 with an excess of tris(2-carboxyethyl)phosphine (TCEP) at 25 ºC for 2 h allowed us to selectively reduce the C186–C201 bond (Fig. 2a).(29) The resulting protein was then reacted with 20 equiv. of compound 2 (Materials and Methods) in a PBS buffer (pH 7.4) at 21 ºC for 5 days to give the desired modified protein (CRM197-linker-azide, Fig. 2b) upon purification using a Zebra spin column. Liquid chromatography–mass spectrometry (LC-MS) analysis showed a single peak at 58705 Da which corresponds to modification of one disulfide and subsequent re-bridging to introduce the azide handle (Fig. 2b). The impact of the installation of compound 2 into CRM197 was studied by circular dichroism (CD, Fig. 2c). We observed that CD spectrum of CRM197-linker-azide was identical to that of CRM197, (29, 30) which indicated that the secondary structure was preserved upon the site-selective chemical modification. The conjugate CRM197-linker-azide was subsequently treated with glycopeptide 1’ (1:20 protein / glycopeptide ratio) at 25 ºC for 20 h (Fig. 3a). The reaction mixture was then purified on Zebra spin column and analyzed by LC-MS (Fig. 3b), which confirmed successful conjugation and formation of a homogenous CRM197-linker- 1’. Molecular dynamics (MD) simulations performed with this conjugate showed that the addition of the glycopeptide did not change the 3D structure of the protein (Fig. 3c). These calculations also showed the flexibility of the linker and how it can favour the exposition of the glycopeptide (antigen) to the solvent. It is worth noting that although only one copy of glycopeptide 1' was presented by the protein, up to 4 possible isomers could be expected (Z / E-oxime and 1,4 / 1,5-triazole adducts). The conjugation procedure was amenable to scale up and enables production of homogenous vaccine CRM197-linker-1’ for immunization studies in mice. We then tested the immunogenic potential of CRM197-linker-1’ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ MUC1.Tg mice was immunized with an initial dose followed by three equal booster doses of the vaccine candidate with 21-day intervals, while one control group was treated with unconjugated CRM197 and another with only phosphate-buffered saline (PBS). Serum from the immunized mice was collected 5 days after each injection to examine the levels of anti-MUC1 antibodies (Fig. 4a). For this, an ELISA assay was performed loading the plates with glycopeptide 1 (Fig. 1). As shown in Fig. 4b, anti-MUC1 antibodies were be detected after the 2ndinjection. Antibody titers were higher after the 3thand 4thimmunizations, which suggests a boost effect. Importantly, the antibodies elicited by our vaccine candidate in mice were mainly IgG antibodies (Fig. 4b and 4c), while the levels of IgM antibodies were significantly lower (Fig. 4d). This result suggests a mature response with recombination of class switches. IgG antibodies were further examined with an additional ELISA assay to determine their isotypes (Fig. 4c). While all IgG isotypes were detected, the triggered antibodies were mostly of the IgG2 type, which suggests vaccine triggered Th1 response. It is worth nothing that antibodies against CRM197 were also observed. Next, we checked whether the generated antibodies were able to recognize human tumor-associated MUC1 (TA-MUC1), which is overexpressed in cancer cells. Therefore, serum from the immunized mice was incubated with T47D(33) (used as a positive control) and with HEK293T(34) (as a negative control), and a binding analysis was assessed by flow cytometry. These experiments showed that the antibodies generated by our vaccine were able to bind to T47D cells, which express TA-MUC1 on their surface. In contrast, negligible binding was observed in HEK293T cells, which is consistent with the absence of MUC1 on their surface (compare Fig. 4e and 4f). Having demonstrated that the CRM197-linker-1’ vaccine generates antibodies that selectively recognize human TA-MUC1, we next assessed the ability of these antibodies to promote protection against cancer. For this purpose, tumors were induced in the immunized mice 6 days after the last injection, by inoculating MC38-MUC1 cells in mice. We use MC38-MUC1 cell line, which derives from a murine colon adenocarcinoma which was further engineered to express human MUC1.(35) Tumor growth was followed until day 15 and then the studied mice were sacrificed. A delay in tumor growth was observed in animals immunized with CRM197-linker-1’ relative to the controls immunized with CRM197 or PBS (Fig. 5a and 5b). The tumor size of the treated mice was decreased by approximately 55% at day 15 relative to control. Moreover, one mouse in the treated group did not develop a detectable a tumor up to day 15 post inoculation of the cancer cells. An evaluation of circulating cytokine levels was also performed one the tumor was induced. Notably, while an increased levels of Th1 related cytokines, such as IL-2 and IFN-gamma and TNF-alpha, was detected (Fig. 5c), no significant amount of Th2 associated cytokines, such as IL-4, IL-10 and IL13 was observed (Fig. 5d). This aspect corroborates that the immune response triggered by the vaccine candidate was a Th1 type response, as suggested by the isotype of the IgGs detected in the sera (Fig. 4c). We also evaluated the efficacy of T-cells to promote cytotoxic T Lymphocyte (CTL) responses. Briefly, CD3+T-cells (which include CD4+and CD8+T-cells) were isolated from the spleens of the mice from both the ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ decrease in MC38-MUC1 cells viability was observed when these cells were co-cultured with the T-cells isolated from the spleens of the mice group treated with CRM197-linker-1’ in comparison to those obtained from the control groups (Fig. 5e). This experiment suggests that T-cells from the treatment group can recognize tumor-associated MUC1 expressed in the MC38-MUC1 cells to induce cancer cell death. Hence, CRM197-linker-1’ can retard tumor proliferation, potentially by enhancing, along with other factors, the activity of cytotoxic T-cells directed at cancer cells exhibiting human TA-MUC1. In the final phase of our study, we conducted a critical experiment to evaluate the efficacy of our homogeneous conjugate as a potential therapeutic vaccine for reducing tumor growth (Fig. 5f). To achieve this, we induced tumors in mice (MC38-MUC1 cells or Panc02-MUC1 cells) that had not previously been treated with the vaccine. The MC38-MUC1 cell line represents a murine colon adenocarcinoma that expresses TA-MUC1 on its surface. The Panc02-MUC1 cell line corresponds to a murine pancreatic cancer model that expresses the human MUC1 sequence on its surface. Treatment with CRM197-linker-1’ was started once the tumors reached an average size of 100 mm3. Treatments were initiated with the CRM197- linker-1’ either as sole therapy (Figures 5f and 6) or in combination with a checkpoint inhibitor (Figure 7). Four doses, identical to those administered in the previous in vivo experiments, were given at 2-day intervals. The mice were closely monitored, and once the tumors reached a size of 1000 mm3, they were sacrificed for analysis. The results of this experiment were highly promising, as all the mice treated with the vaccine survived beyond day 10, while the untreated mice were sacrificed due to the tumor size. In the MC38-MUC1 cancer model, tumor size in treated mice decreased by approximately 40% by day 7, while in the Panc02-MUC1 model it decreased by approximately 75% by day 25, days when the first control animals reached the endpoint (Figures 5b and 6, respectively). In terms of survival probability, all vaccine-treated mice in the MC38-MUC1 model survived beyond day 10, in contrast to the non-treated mice, whose tumors reached 1000 mm³ by day 10. The survival probability of the treated mice was about 40% until day 15 (Figure 5f). Although tumors generally grow more slowly in the Panc02-MUC1 model, similar results were observed, with delayed tumor growth and prolonged survival in the treated animals compared to the controls. In this case, the survival probability of treated mice on day 54 was 25% (Figure 6, right panel). These results indicate that our homogeneous vaccine has a potential therapeutic effect on tumor growth, as evidenced by the prolonged survival time. We administered our homogenous vaccine in combination with checkpoint inhibitors to study the combined effect of both treatments in one therapeutic formulation. In this combined therapeutic approach, four identical subcutaneous (SC) doses of the vaccine were administered 2 days apart, regardless of the cancer cell line used (Figure 7), while the anti-PD1 antibody was administered via three intraperitoneal (IP) injections 3 days apart, with an IgG2a isotype antibody used as a control. The mice were closely monitored and sacrificed once tumors reached a size of 1000 mm3. Of note, this therapy resulted in a significant reduction in tumor size. In the MC38-MUC1 cancer model, there was an approximately 70% reduction in tumor size in the treated mice group compared to the PBS-treated mice by day 10 (when the first control mouse reached the endpoint). Additionally, mice treated with CRM197-linker-1’ and anti-PD1 showed a survival rate of approximately 80% by day 20. Remarkably, one mouse showed a complete regression of the tumor at day 65 when it was euthanized (Figure 7, upper panel). Similar results were obtained with the pancreatic cancer model. In this case, tumor growth was reduced approximately 84% by day 25 (day of death of the first control animal) in mice treated with the combination therapy, and more than 20% of the mice survived at day 60 (Figure 7, lower panel). In summary, we have produced the first homogeneous cancer vaccine based on a MUC1-like glycopeptide containing an unnatural fluorinated proline and a surrogate of the Tn antigen, which has an S-glycosidic linkage. This antigen was site-selectively conjugated to the protein CRM197, the most used immunogenic carrier in vaccine design for human vaccination campaigns. For this, the most reactive of the two disulfide bridges in the protein (C186–C201) was selectively reduced in TCEP and re-bridged with a linker containing an azido group. The copper-free click reaction between this group and the cyclooctyne moiety installed at the N-terminus of the antigen allowed the production of a vaccine with a single copy of the antigen, as confirmed by LC-MS assays. MD simulations performed with the conjugate showed that the 3D structure of CRM197 was preserved upon the addition of the antigen and that this determinant is exposed to the solvent, which is essential for recognition by immune system cells. The vaccine was tested in mice, and a significant immune response was elicited in vivo, producing mainly IgG2b antibodies. We also inoculated mice with human colon adenocarcinoma cells and observed the reduction in tumor size in mice treated with the vaccine compared with those exposed to a negative control. Additionally, we found that the vaccine can stimulate the production of T cells, which play a crucial role in promoting a potent cytotoxic T lymphocyte (CTL) response that effectively curtailed the growth of the inoculated tumors in mice. Remarkably, the vaccine exhibited also an excellent therapeutic effect against this type of cancer, prolonging the survival time of mice treated with the vaccine post-tumor inoculation. The vaccine was also shown to significantly delay tumor growth and enhance survival in mouse models of both colon adenocarcinoma and pancreatic cancer. Importantly, its efficacy is further amplified when combined with the PD-1 immune checkpoint inhibitor, suggesting a synergistic potential that leads to improved survival rates in murine models. In addition, the strategy presented in the present work can avoid batch-to-batch variation in vaccine production, which is critical for the development of new chemically defined and effective glycopeptide-based cancer vaccines. Finally, and despite a prevailing dogma wherein glycoconjugate vaccines require the display of multiple copies of antigens on a protein carrier, our work demonstrates that the use of a single copy of a non-natural, potent antigen precisely displayed on a protein carrier can induce strong immune responses in vivo.

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[0003] Sequences AX1DX2RP wherein X1 is 4S ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^2 is S ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ SEQ ID NO: 1 HGVTSAX1DX2RPAPGSTAPPA wherein X1 is 4S ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^2 is S ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ SEQ ID NO: 2. GADDVVDSSKSFVMENFSSYHGTKPGYVDSIQKGIQKPKSGTQGNYDDDWKEFYSTDNKYDAAGYSVDNENPLSGKAGGV VKVTYPGLTKVLALKVDNAETIKKELGLSLTEPLMEQVGTEEFIKRFGDGASRVVLSLPFAEGSSSVEYINNWEQAKALS VELEINFETRGKRGQDAMYEYMAQACAGNRVRRSVGSSLSCINLDWDVIRDKTKTKIESLKEHGPIKNKMSESPNKTVSE EKAKQYLEEFHQTALEHPELSELKTVTGTNPVFAGANYAAWAVNVAQVIDSETADNLEKTTAALSILPGIGSVMGIADGA VHHNTEEIVAQSIALSSLMVAQAIPLVGELVDIGFAAYNFVESIINLFQVVHNSYNRPAYSPGHKTQPFLHDGYAVSWNT VEDSIIRTGFQGESGHDIKITAENTPLPIAGVLLPTIPGKLDVNKSKTHISVNGRKIRMRCRAIDGDVTFCRPKSPVYVG NGVHANLHVAFHRSSSEKIHSNEISSDSIGVLGYQKTVDHTKVNSKLSLFFEIKS SEQ ID NO: 3 – CRM197 amino acid sequence

Claims

Claims 1. A cancer vaccine conjugate comprising; a CRM197 carrier protein, an immunogenic peptide and a linker that covalently links the immunogenic peptide to residues C186 and C201 of the CRM197 carrier, and the salts, solvates and protected forms thereof.

2. A cancer vaccine conjugate according to claim 1 wherein the cancer vaccine conjugate stimulates an immune response against cancer cells.

3. A cancer vaccine conjugate according to claim 1 or claim 2 wherein the immunogenic peptide comprises a tumour associated antigen (TAA).

4. A cancer vaccine conjugate according to any one of claims 1 to 3 wherein the immunogenic peptide is a glycopeptide.

5. A cancer vaccine conjugate according to any one of claims 1 to 4 wherein the immunogenic glycopeptide comprises a tumor-associated carbohydrate antigen (TACA).

6. A cancer vaccine conjugate according to any one of claims 1 to 5 wherein the tumor-associated ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ 7. A cancer vaccine conjugate according to any one of claims 1 to 6 wherein the tumor-associated carbohydrate antigen (TACA) is attached to the immunogenic glycopeptide via a glycosidic bond.

8. A cancer vaccine conjugate according to any one of claims 1 to 7 wherein the glycosidic bond is an O-glycosidic bond, an S-glycosidic bond or an Se-glycosidic bond.

9. A cancer vaccine conjugate according to any one of claims 1 to 8 wherein the immunogenic glycopeptide comprises two or more TACAs.

10. A cancer vaccine conjugate according to any one of claims 1 to 9 wherein the two or more TACAs are the same or different.

11. A cancer vaccine conjugate according to any one of claims 1 to 10 wherein the immunogenic glycopeptide is natural or synthetic MUC1 glycopeptide.

12. A cancer vaccine conjugate according to claim 11 wherein the immunogenic glycopeptide comprises the amino acid sequence AX1DX2RP, where X1 is 4S ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^2 is S ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ thiothreonine (SEQ ID NO: 1) or a variant thereof.

13. A cancer vaccine conjugate according to claim 12 wherein the immunogenic glycopeptide comprises the amino acid sequence HGVTSAX1DX2RPAPGSTAPPA, where X1 is 4S ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^2 is S ^ ^ ^ ^ D-GalNAc)-thiothreonine (SEQ ID NO: 2) or a variant thereof.

14. A cancer vaccine conjugate according to any one of the preceding claims wherein the CRM197 carrier protein comprised the amino acid sequence of SEQ ID NO:3 or a variant thereof.

15. A cancer vaccine conjugate according to any one of the preceding claims wherein the linker comprises one or more groups selected from a C1-C20alkylene, a (poly)ethylene glycol moiety, an alkylenediamine moiety, an amino acid residue and combinations thereof.

16. A cancer vaccine conjugate according to any one of the preceding claims wherein the linker is a group of the formula:where; n is an integer selected from 1 to 4; m is an integer selected from 2 to 8; pare residues C186 and C201 of the CRM197 carrier protein where the linker is attached to the CRM197 carrier protein; and pep indicates the position where the linker is attached to the immunogenic glycopeptide 17. A method of producing a cancer vaccine conjugate comprising; providing a CRM197 carrier protein covalently linked to a first connector, wherein the first connector is covalently linked to residues C186 and C201 of CRM197 carrier protein and comprises a first reactive group, providing an immunogenic glycopeptide covalently linked to a second connector, wherein the second connector comprises a second reactive group, reacting the first and second reactive groups to covalently link the first and second connectors and attach the immunogenic glycopeptide to the CRM197 carrier protein, thereby producing the cancer vaccine conjugate.

18. A method according to claim 17 wherein the first and second reactive groups are selected from the following pairings: i) an azide and an alkyne ii) a ketone or aldehyde and an alkoxyamine (e.g. hydroxylamine) or hydrazine; iii) an amine and an acyl halide or carboxylic acid; iv) an electron-rich dienophile (e.g. a 1,3-nitrone alkene) and an electron-poor diene (e.g. tetrazine);and (v) a strained alkene or alkyne (e.g. norbornene or cyclooctyne) and a tetrazine.

19. A method according to claim 17 or claim 18 wherein one of the first and second reactive groups is an azide (azido group) and the other of the first and second reactive groups is an alkyne (alkynyl group).

20. A method according to any one of claims 17 to 19 wherein the first connector covalently linked to the CRM197 carrier protein is a group of the formula:where: n is an integer from 1 to 4, preferably 2; and pare residues C186 and C201 of the CRM197 carrier protein where the linker is attached to the CRM197 carrier protein. 21 A method according to any one of claims 17 to 20 wherein the second connector covalently linked to the immunogenic glycopeptide is a group of the formula: pepwhere: m is an integer from 2 to 8, preferably 4; andthe position where the linker is attached to the immunogenic glycopeptide (e.g. MUC-1.

22. A method according to any one of claims 17 to 21 wherein the CRM197 carrier protein is covalently linked to the first connector by a method comprising; providing a CRM197carrier protein, selectively reducing the C186 - C201 disulfide linkage of the CRM197 carrier protein to generate free thiol groups at residues C186 and C201 of the CRM197 carrier protein, providing a first connector compound comprising first, third and fourth reactive groups, and reacting the third and fourth reactive groups of the first connector with the free thiol groups at C186 and C201, respectively, to covalently link the first connector to residues C186 and C201 of the CRM197 carrier protein, thereby covalently linking the CRM197 carrier protein to the first connector.

23. A method according to claim 22 wherein the third and fourth reactive groups are halide groups, such as chloride groups.

24. A method according to claim 22 or claim 23 wherein the first connector compound is a compound of the formula:where n is an integer selected from 1 to 4 (e.g. 2).

25. A kit for producing a cancer vaccine conjugate comprising; a CRM197 carrier protein covalently linked to a first connector, wherein the first connector is covalently linked to residues C186 and C201 of CRM197 carrier protein and comprises a first reactive group.

26. A kit according to claim 25 wherein the CRM197 carrier protein is as defined in claim 14 and the first connector is as defined in claim 20.

27. A pharmaceutical composition comprising a cancer vaccine conjugate according to any one of claims 1 to 16 and a pharmaceutically acceptable excipient.

28. A pharmaceutical composition according to claim 27 further comprising an adjuvant.

29. A method of treatment of cancer comprising administering a cancer vaccine conjugate according to any one of claims 1 to 16 or a pharmaceutical composition according to any one of claims 27 to 28 to an individual in need thereof.

30. A cancer vaccine conjugate according to any one of claims 1 to 16 or a pharmaceutical composition according to any one of claims 27 to 28 for use in the treatment of cancer.

31. Use of a cancer vaccine conjugate according to any one of claims 1 to 16 or a pharmaceutical composition according to any one of claims 27 to 28 in the manufacture of a medicament for use in the treatment of cancer.

32. A method according to claim 29, cancer vaccine conjugate or pharmaceutical composition for use according to claim 30 or use according to claim 31, wherein the immunogenic peptide is a natural or synthetic MUC1 glycopeptide and the cancer is characterised by the overexpression of Mucin-1 or the expression of an aberrantly glycosylated Mucin-1.

33. A method according to claim 29 or 32, cancer vaccine conjugate or pharmaceutical composition for use according to claim 30 or 32 or use according to claim 31 or 32, wherein the cancer vaccine conjugate is administered in combination with an immune checkpoint inhibitor.

34. A method according to claim 33, cancer vaccine conjugate or pharmaceutical composition for use according to claim 33 or use according to claim 33, wherein the immune checkpoint inhibitor is a PD-1 immune checkpoint inhibitor.

35. A method according to claim 34, cancer vaccine conjugate or pharmaceutical composition for use according to claim 34 or use according to claim 34, wherein the PD-1 immune checkpoint inhibitor is an anti- PD-1 antibody.

36. A method according to claim 29 or 32 to 35, cancer vaccine conjugate or pharmaceutical composition for use according to claim 30 or 32 to 35 or use according to claim 31 to 35, wherein the cancer vaccine conjugate is administered for therapeutic treatment of the cancer in the individual.

37. A method according to claim 29 or 32 to 35, cancer vaccine conjugate or pharmaceutical composition for use according to claim 30 or 32 to 35 or use according to claim 31 to 35, wherein the cancer vaccine conjugate is administered for prophylactic treatment of the cancer in the individual.

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