Vaccine composition

Abstract

This invention relates to CRM 197 Carrier proteins and CRM 197 This invention relates to a cancer vaccine conjugate comprising an immunogenic peptide covalently linked to residues C186 and C201 of a carrier by a linker. Suitable immunogenic peptides include glycopeptides, such as those containing tumor-associated glycan antigens (TACAs), including GalNAc, Galb(1,3)GalNAc, or Neu5Acα(2-6)GalNAc. The invention also provides a cancer vaccine conjugate, as well as a method for producing and using the same.

Description

[Technical Field]

[0001] This invention relates to vaccines, particularly conjugate vaccines, and methods for producing them. [Background technology]

[0002] Mucin-1 (MUC1) is a highly O-glycosylated glycoprotein expressed on the surface of epithelial cells. Its extracellular domain consists of 20 amino acids containing five potential O-glycosylation sites (shown in bold in the peptide sequence). It is composed of a tandem repeat of TIFF2026521572000001.tif7170 (1,2). In healthy tissues, this protein retains complex oligosaccharides, but in tumor cells, MUC1 is modified with simple and shortened glycans, and its expression dramatically increases due to dysfunction or translocation of GalNAc transferase (2) or mutations in COSMC, a chaperone required for the activity of glycosyltransferase C1GalT (6) (3-5). As a result, the immunogenic epitope APDTRP (7,8) and several tumor-associated glycan antigens (TACAs), such as Tn antigen (α-O-GalNAc-Ser / Thr, hereafter referred to as Tn-Ser and Tn-Thr, respectively), can be exposed, potentially inducing a weak immune response (9). The development of MUC1-based vaccines has been influenced by the discovery that cancer patients can produce anti-MUC1 antibodies in the early stages of the disease (10,11). These vaccines typically contain the entire sequence of MUC1, glycosylated at one or more sites by Tn or other TACAs, conjugated particularly on protein carriers, liposomes, or nanoparticles (12-18). While this strategy promotes multivalent antibody presentation, it can lead to heterogeneous conjugation and consequently low formulation reproducibility. Despite these synthetic efforts, no successful clinical applications exist (19), likely because abnormally glycosylated proteins may be present at low concentrations even in healthy cells (PMID:34526666), leading to immune tolerance and consequently an insufficient immune response in preclinical mouse models. [Overview of the project]

[0003] The inventors have developed a cancer vaccine conjugate that can generate a robust immune response against tumors and can be manufactured with high reproducibility in a homogeneous form.

[0004] A first aspect of the present invention is: CRM 197 Carrier proteins, Immunogenic peptides, Immunogenic peptides in CRM197 A linker that is covalently linked to residues C186 and C201 of the support, The present invention provides cancer vaccine conjugates containing the same, as well as salts, solvates, and protective forms thereof.

[0005] Preferably, the cancer vaccine conjugate is of the formula: ZLD (In the formula, Z is CRM 197 It is a carrier protein, L is a linker, and D is an immunogenic peptide. It may have.

[0006] In some preferred embodiments, the cancer vaccine conjugate of the first embodiment may stimulate an immune response against cancer cells expressing mucin-1 (MUC1). For example, the immunogenic peptide may be a natural or synthetic MUC1 glycopeptide.

[0007] A second aspect of the present invention is a method for producing a cancer vaccine conjugate, CRM covalently connected to the first connector 197 To provide a carrier protein, Note that the first connector is CRM 197 It is covalently linked to residues C186 and C201 of the carrier protein and contains a first reactive group; To provide an immunogenic peptide covalently linked to a second connector, Furthermore, the second connector contains a second reactive group; The first and second reactive groups are reacted to covalently link the first and second connectors, and the immunogenic peptide is converted to CRM 197 To attach it to a carrier protein and thereby produce a cancer vaccine conjugate, This provides a method that includes [something].

[0008] In some preferred embodiments, the first reactive group may be an azide group and the second reactive group may be a cyclooctyne group.

[0009] CRM 197 The carrier protein is CRM 197 providing a carrier protein, CRM 197 selectively reducing the C186-C201 disulfide bond of the carrier protein to generate free thiol groups at residues C186 and C201 of the CRM 197 carrier protein, providing a first linker compound comprising a first reactive group, a third reactive group and a fourth reactive group, reacting the third reactive group and the fourth reactive group of the first linker compound with the free thiol groups of C186 and C201 respectively to covalently link the first linker to the residues C186 and C201 of the CRM 197 carrier protein, thereby covalently linking the CRM 197 carrier protein to the first linker, and can be covalently linked to a first linker for use in the method of the second aspect by a method comprising

[0010] In some preferred embodiments, the third reactive group and the fourth reactive group may be halide groups, such as chloride groups.

[0011] The immunogenic peptides of the first aspect and the second aspect may comprise a tumor-specific antigen or a tumor-associated antigen (TAA), preferably a tumor-associated carbohydrate antigen (TACA).

[0012] A third aspect of the present invention is a kit for manufacturing a cancer vaccine conjugate, CRM covalently linked to a first linker 197 carrier protein comprising, wherein the first linker is CRM 197The kit provides a first reactive group that is covalently linked to residues C186 and C201 of a carrier protein.

[0013] This kit may be suitable for use in the manner of the second embodiment.

[0014] A fourth aspect of the present invention provides a pharmaceutical composition comprising a cancer vaccine conjugate according to the first aspect and a pharmaceutically acceptable excipient.

[0015] A fifth aspect of the present invention provides a method for treating cancer, comprising administering a cancer vaccine conjugate of the first aspect or a pharmaceutical composition of the fourth aspect to an individual in need of cancer treatment.

[0016] A sixth aspect of the present 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.

[0017] A seventh aspect of the present 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 pharmaceutical for use in the treatment of cancer.

[0018] Other aspects and embodiments of the present invention will be described in more detail below. [Brief explanation of the drawing]

[0019] [Figure 1] This figure shows the approach to preparing cancer vaccines based on non-natural MUC1-derived glycopeptides. (a) Schematic diagram of the cancer vaccine conjugate. (b) Structure of the MUC-1-derived glycopeptide. (c) Structure of the N-terminal portion R(1'). [Figure 2] (a) The optimization conditions used in this study to selectively modify cysteine ​​186 and 201 of the protein CRM197, (b) the ESI-MS spectrum of CRM197-linker-azide, and (c) the CD spectra of CRM197 and CRM197-linker-azide. [Figure 3] (a) The optimization conditions used in this study to prepare the vaccine candidate CRM197-linker-1', (b) the ESI-MS spectrum of CRM197-linker-1', and (c) a representative ensemble obtained from MD simulations. Proteins are shown as white ribbons, and the carbon atoms of the linker, MUC1, and GalNAc are represented as blue, green, and orange sticks, respectively. E-oxime and 1,5-triazole adducts were considered in these calculations. [Figure 4] This figure shows (a) the vaccine administration scheme used in this study, (b) the total levels of IgG antibodies induced by CRM197-linker-1', CRM197, and PBS at different stages as determined by ELISA assay, (d) the IgG isotypes detected by ELISA assay after the fourth immunization, and (d) the total levels of IgM antibodies induced by CRM197-linker-1', CRM197, and PBS as determined by ELISA assay after the fourth immunization. Flow cytometry analysis was performed to examine (e) the binding of TA-MUC1 to HEK293 T cells (negative control) and (f) TD47 cells expressing TA-MUC1 on their surface. Black line - serum of mice injected with PBS; blue line - commercially available anti-MUC1 antibody; pink line - serum of mice immunized with CRM197; red line - serum of mice immunized with CRM197-MUC1. (e) Statistical significance was determined by unpaired t-tests. [Figure 5](a) Tumor weight determined on day 15 after tumor induction (MC38-MUC1 cells) (mice were first treated with different formulations before tumor induction. Differences are not statistically significant), (b) Tumor volume measured after tumor induction using different formulations, (c) Serum Th1 cytokine levels after tumor induction in mice treated with different conjugates, (d) Serum Th2 cytokine levels after tumor induction in mice treated with different conjugates (differences are not statistically significant), (e) Cytotoxic T lymphocyte assay (T cells isolated from the spleen of immunized mice were co-cultured with MC38-MUC1 cells in a 90:1 ratio (T cells:MC38-MUC1 cells) for 24 hours. A decrease in the viability of MC38-MUC1 cells was observed. Statistical significance was determined by an unpaired t-test. Data represent mean + SEM), (f) Survival probability of mice after tumor induction (MC38-MUC1 cells) and treatment with the corresponding conjugate. [Figure 6] This figure shows that when CRM197-Linker-1' is therapeutically administered to a mouse model of pancreatic cancer, it delays tumor growth and extends survival. Tumor volume measured after tumor induction with different formulations (*p=0.0197) (left panel) is shown along with the survival rate of mice after tumor induction and treatment with the corresponding formulations (*p=0.1092) (right panel). [Figure 7] This figure shows that when CRM197-linker-1' is therapeutically administered in combination with checkpoint inhibitors, it delays tumor growth and extends survival. Tumor volume measured after tumor induction with different formulations (MC38-MUC1-upper panel, p=0.0362- or Panc02-MUC1-lower panel, p=0.0201- cell lines) is shown together with the survival rate of mice under these conditions (MC38-MUC1-upper right, *p=0.1017- or Panc02-MUC1-lower right, *p=0.0163- cell lines). [Figure 8]This figure shows the endotoxin levels of CRM197-Linker-1'. Conjugated samples were subjected to endotoxin level determination before administration to animals. The levels of two independent samples were less than 1 EU / mL (0.844100013 and 0.946199968). Endotoxin levels less than 1.5 EU / mL are considered safe for administration to mice. [Figure 9] This figure shows the maximum tolerated dose (MTD) assessment. The MTD of CRM197-Linker-1' was assessed by injecting normal Balb / c mice with different doses of the vaccine via subcutaneous (SC) injection at 2-day intervals for a total of four times. Signs of toxicity were confirmed at both the macroscopic level (e.g., signs of mouse weight loss and pain / discomfort) and the microscopic level (by histological analysis of different organs). No significant weight loss was observed, and no abnormalities were observed in mouse tissue, indicating that there were no signs of toxicity at the tested vaccine doses. [Modes for carrying out the invention]

[0020] This invention relates to CRM 197 Carrier proteins and CRM 197 This invention relates to a cancer vaccine comprising an immunogenic peptide covalently attached to residues C186 and C201 of a carrier by a linker.

[0021] The population of cancer vaccine conjugates described herein may be homogeneous, that is, all molecular species within the population may have the same chemically defined structure. Preferably, a single immunogenic peptide is present in each CRM in the cancer vaccine conjugates described herein. 197 It is covalently attached to the carrier protein, i.e., CRM in cancer vaccine conjugates. 197 The stoichiometric ratio of the carrier protein to the immunogenic peptide may be 1:1. The production of cancer vaccine conjugates described herein may be useful in reducing batch-to-batch variability in vaccine production.

[0022] Cancer vaccine conjugates can induce antibodies that selectively recognize naturally occurring antigens corresponding to immunogenic peptides in patient samples. In addition, cancer vaccine conjugates can activate the Th1 immune response and generate T cells that promote the cytotoxic T lymphocyte (CTL) response. Cancer vaccine conjugates may exert antitumor effects in patients.

[0023] Cancer vaccine conjugates may also exhibit reduced enzymatic degradation and reduced tolerance in vivo compared to vaccines using natural antigens.

[0024] The cancer vaccine conjugates described herein contain immunogenic peptides.

[0025] Immunogenic peptides can induce an immune response against cancer cells in an individual. For example, an immunogenic peptide may contain one or more epitopes from tumor antigens expressed by cancer cells in an individual. Tumor antigens are known in the art and may include protein antigens and tumor-associated glycosylation antigens (TACAs), such as Survivin, p53, PSA, MUC-1, MUC-4, MUC-16, and CEA. Preferred epitopes may include peptides and glycosylation epitopes.

[0026] Preferably, the immunogenic peptide may be a glycopeptide. Suitable glycopeptides may include natural or artificial glycopeptides. For example, suitable glycopeptides may include glycopeptides containing one or more glycosylation sites, such as O-, S-, Se-, or N-glycosylation sites. The glycosylation sites may include serine residues, threonine residues, or unnatural amino acid residues having side chains containing a hydroxyl (R-OH) group, a thiol (R-SH) or disulfide (SS) group, or a selenol (R-SeH) or diselenium (Se-Se) group.

[0027] In some preferred embodiments, the immunogenic peptide may be mucin 1 (MUC-1) glycopeptide.

[0028] MUC-1 glycopeptides may be natural mucin 1 (MUC-1) glycopeptides or, more preferably, synthetic artificial mucin 1 (MUC-1) glycopeptides. Synthetic artificial mucin 1 (MUC-1) glycopeptides may contain one or more non-natural or non-standard amino acid residues, i.e., amino acid residues not encoded by the genetic code of naturally occurring organisms. Non-natural amino acid residues may include thiothreonine (SThr) and 4-fluoro-L-proline.

[0029] Synthetic artificial MUC-1 glycopeptides may further contain glycan antigens, such as GalNAc(Tn antigen), Galb(1,3)GalNAc(T antigen), or Neu5Acα(2-6)GalNAc(sTn antigen), or unnatural glycan antigens derived from one of these. Glycan antigens may attach to glycosylation sites in the peptide sequence of the glycopeptide by glycosidic bonds. For example, glycan antigens, such as Tn, T, or sTn, may bind to the peptide sequence by O-glycosidic bonds in unnatural amino acids containing Ser, Thr, or R-OH. Glycan antigens may bind to the peptide sequence by S-glycosidic bonds in unnatural amino acids containing R-SH. For example, immunogenic glycopeptides may contain S-(α-D-GalNAc)-thiothreonine (SThr) or its derivatives. Glycan antigens may bind to the peptide sequence by Se-glycosidic bonds in unnatural amino acids containing R-SeH.

[0030] In some preferred embodiments, the immunogenic glycopeptide may be a MUC-1 glycopeptide and may contain S-(α-D-GalNAc)-thiothreonine (SThr) or a derivative thereof. A preferred MUC-1 glycopeptide may include the amino acid sequence AX1DX2RP (wherein X1 is 4S-4-fluoro-L-proline and X2 is S-(α-D-GalNAc)-thiothreonine) (SEQ ID NO: 1), or a variant thereof. In some preferred embodiments, the MUC-1 glycopeptide may include the amino acid sequence HGVTSAX1DX2RPAPGSTAPPA (wherein X1 is 4S-4-fluoro-L-proline and X2 is S-(α-D-GalNAc)-thiothreonine) (SEQ ID NO: 2), or a variant thereof.

[0031] In some embodiments, the immunogenic glycopeptide may contain two or more glycosylation sites. Glycan antigens may attach to the glycopeptide at two or more glycosylation sites. The glycan antigens attached to the two or more glycosylation sites may be the same or different. For example, one of GalNAc(Tn antigen), Galb(1,3)GalNAc(T antigen), or Neu5Acα(2-6)GalNAc(sTn antigen) may be attached to the glycopeptide at both the first and second glycosylation sites, or one of GalNAc(Tn antigen), Galb(1,3)GalNAc(T antigen), or Neu5Acα(2-6)GalNAc(sTn antigen) may be attached to the glycopeptide at the first glycosylation site, and another one of GalNAc(Tn antigen), Galb(1,3)GalNAc(T antigen), or Neu5Acα(2-6)GalNAc(sTn antigen) may be attached to the glycopeptide at the second glycosylation site.

[0032] Cross-reactive substance 197 (CRM) 197 ) is the wild-type CRM shown in Sequence ID No. 3. 197This is diphtheria toxin (DT) detoxified by the presence of a Gly-to-Glu mutation at the position corresponding to the 52nd position of the sequence (Broker et al (2011) Biologicals 39(4) 195-204). CRM 197 It is widely used as a carrier protein for polysaccharides and haptens, and is also effectively used in vaccines against Haemophilus influenzae, Streptococcus pneumoniae, and Neisseria meningitidis (for example, Menveo®, a tetravalent conjugate vaccine against Neisseria meningitidis serogroup AC-W135-Y) (25-27).

[0033] CRM 197 The carrier protein may contain the amino acid sequence of SEQ ID NO: 3 or a variant thereof.

[0034] Reference sequences shown herein, for example, reference immunogenic glycopeptides or CRM 197 Sequence variants may include amino acid sequences 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 with respect to the reference sequence. A particular amino acid sequence variant may differ from the reference sequence shown herein by the insertion, addition, substitution, or deletion of one, two, three, four, five, six, seven, eight, nine, or ten, or more than ten, amino acids.

[0035] Sequence similarity and identity are generally defined by referring to the GAP algorithm (Wisconsin Package, Accelerys, San Diego, USA). GAP uses the Needleman and Wunsch algorithms to align two complete sequences, maximizing the number of matches and minimizing the number of gaps. Generally, default parameters are used with a gap formation penalty of 12 and a gap expansion penalty of 4. While the use of GAP may be preferred, other algorithms, such as BLAST (using the method described in Altschul et al. (1990) J. Mol. Biol. 215: 405-410), FASTA (using the method described in 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 from Altschul et al. (1990) (cited above), may generally be used with default parameters. In particular, the psi-Blast algorithm (Nucl. Acids Res. (1997) 25 3389-3402) may also be used.

[0036] CRM 197 These may be manufactured by recombinant means using established technologies or obtained from commercial suppliers.

[0037] In the vaccine conjugates described herein, the immunogenic glycopeptide is linked to the CRM by a covalent linker. 197 It is connected to residues C186 and C201 of the carrier protein.

[0038] Linker is CRM 197 It forms a bridge between residues C186 and C201 of the carrier protein, and the immunogenic glycoprotein CRM 197 It connects to a carrier protein. The linker is (i)CRM 197 Carrier residue C186, (ii)CRM197 (iii) The carrier residue C201 and the N-terminus of the MUC-1 glycopeptide can be covalently attached to it.

[0039] Linker, -L- is CRM 197 This is the group that attaches the carrier protein, Z-, to the immunogenic glycopeptide, -D.

[0040] Use any suitable linker for CRM 197 It will be understood that carrier proteins can be linked to immunogenic glycopeptides.

[0041] In some embodiments, the linker may comprise one or more groups selected from polyethylene glycol moieties, amino acid residues, alkylenediamine moieties, arylene-containing moieties, heteroarylene-containing moieties, heterocyclyl-containing moieties, cycloalkyl-containing moieties, and combinations thereof.

[0042] Preferably, the linker contains between 1 and 20 of the above-mentioned groups. More preferably, the linker contains between 1 and 15 of the above-mentioned groups. Even more preferably, the linker contains between 1 and 10 of the above-mentioned groups.

[0043] Preferably, the linker comprises one or more groups selected from polyethylene glycol moieties, amino acid residues, alkylenediamine moieties, and combinations thereof.

[0044] The polyethylene glycol portion will be understood as a functional group comprising one or more alkylene glycol portions, for example, one or more ethylene glycol portions. In certain embodiments, the linker comprises between 1 and 20 alkylene glycol portions (e.g., ethylene glycol portions), preferably between 1 and 10 alkylene glycol portions (e.g., ethylene glycol portions), and most preferably between 2 and 8 alkylene glycol portions (e.g., ethylene glycol portions).

[0045] The alkylenediamine moiety will be understood to be a functional group containing or derived from an alkylenediamine moiety, such as ethylenediamine.

[0046] The arylene-containing portion will be understood as a functional group containing one or more arylene groups. Preferably, the arylene is a six-membered arylene such as phenylene. In certain embodiments, the arylene-containing portion is a functional group containing a 1,3,5-benzyl group.

[0047] The heteroarylene-containing portion will be understood as a functional group containing one or more heteroarylene groups. Preferably, the heteroarylene is a functional group containing a six-membered heteroaryl such as a triazine. In certain embodiments, the arylene-containing portion is a functional group containing a 1,3,5-triazine group.

[0048] The heterocyclyl-containing portion will be understood as a functional group containing one or more heterocyclic groups. Preferably, the heterocycle is a six-membered heterocycle such as a triazinan. In certain embodiments, the heterocyclyl-containing portion is a functional group containing a 1,3,5-triazinan group.

[0049] The cycloalkyl-containing portion will be understood as a functional group containing one or more cycloalkyl groups. Preferably, the cycloalkyl group is a six-membered cycloalkyl group such as cyclohexane. In certain embodiments, the cycloalkyl-containing portion is a functional group containing 1,3,5-cyclohexane.

[0050] The amino acid residues will be understood to consist of both natural and non-natural amino acid residues. Preferably, the amino acid residues are natural amino acid residues. The amino acid residues may also contain two or more amino acids, such as dipeptide and tripeptide moieties. Preferred amino acid residues include Phe, Lys, Val, Ala, Cit, Leu, Ile, Arg, and Trp residues.

[0051] In some embodiments, the amino acid residue is a dipeptide residue. The amino acids in the dipeptide may be any combination of natural and non-natural amino acids. In some embodiments, the dipeptide contains natural amino acids.

[0052] In a particular embodiment, the linker is a linker unstable to cathepsin, and the dipeptide is the site of action for cathepsin-mediated cleavage. In this case, the dipeptide is the recognition site of cathepsin.

[0053] In some embodiments, amino acid residues are NH -Phe-Lys- C=O , NH -Val-Ala- C=O , NH -Val-Lys- C=O , NH -Ala-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 Selected from the group consisting of, Here, Cit is citrulline, and NH and C=O are the amino and carboxyl terms of an amino acid residue, which are connected to the rest of the linker.

[0054] Preferably, the amino acid residues are NH -Phe-Lys- C=O, NH -Val-Ala- C=O , NH -Val-Lys- C=O , NH -Ala-Lys- C=O , and, NH -Val-Cit- C=O It is selected from the group consisting of the following.

[0055] Most preferably, the amino acid residue is NH -Phe-Lys- C=O , NH -Val-Cit- C=O or NH -Val-Ala- C=O Selected from.

[0056] Other dipeptide combinations may also be used, including those described in Dubowchik et al., Bioconjugate Chemistry, 2002, 13, 855-869, which are incorporated herein by reference.

[0057] In some embodiments, the amino acid residues are tripeptide residues. The amino acids in the tripeptide may be any combination of native and non-native amino acids. In some embodiments, the tripeptide contains native amino acids.

[0058] If the linker is unstable to cathepsin, the tripeptide is the site of action for cathepsin-mediated cleavage. In that case, the tripeptide is the recognition site for cathepsin.

[0059] References to amino acids and amino acid residues generally refer to α-amino acids.

[0060] The amino acid side chains may be chemically protected where appropriate. The protected amino acid sequence may be enzymatically cleavable. For example, a dipeptide sequence containing a Lys residue with a protected Boc side chain can be cleaved by cathepsin.

[0061] Protecting groups for amino acid side chains are known in the art and are listed in the Novabiochem catalog.

[0062] In certain embodiments, linker-L- comprises one or more groups that are susceptible to enzymatic cleavage (e.g., proteolysis or peptidase cleavage, e.g., cathepsin cleavage, sulfatase cleavage, or galactosidase cleavage). Therefore, in some embodiments, 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 a combination thereof. Preferably, linker-L- comprises one or more amino acid residues, dipeptide residues or tripeptide residues, where the amino acid residues, dipeptide residues or tripeptide residues are as described below.

[0063] In certain embodiments, the linker-L- is one or more groups (e.g., disulfide or hydrazone) that are susceptible to chemical cleavage, or includes such groups.

[0064] In other embodiments, the linker may be a non-cleaving linker. Such non-cleaving linkers are known in the art of antibody-drug conjugates.

[0065] Therefore, in some embodiments, linker-L- is C1~C 20 One or more groups selected from alkylene, (poly)ethylene glycol moiety, alkylenediamine moiety, amino acid residues, and combinations thereof, or comprising the same.

[0066] In a particular embodiment, the linker is given by the formula V shown below: TIFF2026521572000002.tif35170 formula V (In the formula: Z A It is a protein linking group; Q is a spacer base; TIFF2026521572000003.tif14170 is a linker for CRM 197 It indicates the position where the support protein attaches; and, TIFF2026521572000004.tif15170 is the base (indicating the position where the linker attaches to the immunogenic glycopeptide).

[0067] Protein linking group Z A CRM 197 Any functional group capable of linking a carrier protein to the linker may be used. Therefore, the protein linking group Z A CRM 197 This is a functional group capable of forming covalent attachment to sulfur atoms present on a carrier protein. Preferably, the sulfur atom is CRM 197 These are sulfur atoms from residues C186 and C201 of the carrier protein. Therefore, the linker is CRM. 197 It will be understood that this plays a role in re-crosslinking the reduced interchain disulfide bond between residues C186 and C201 of the carrier protein.

[0068] This is how CRM 197 Functional groups capable of linking a carrier protein to a linker are known in the art. Therefore, those skilled in the art will be able to select a protein linking group suitable for use in the present invention. Suitable protein linking groups, Z AExamples include, for instance, 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., Examples include those described in 2017, 7, 9073-9077), Behrens 2015 (Mol. Pharm., 2015, 12, 3986-3998), and International Publication No. 2019 / 011078.

[0069] In a particular embodiment, Z A The following protein linking groups: Selected from one of TIFF2026521572000005.tif131170, During the ceremony: TIFF2026521572000006.tif14170 is Z A CRM 197 It indicates the position where the support protein attaches; and, TIFF2026521572000007.tif14170 is Z A This indicates the position where the spacer base attaches to Q.

[0070] In a particular embodiment, Z A The following protein linking groups: The filename is TIFF2026521572000008.tif35170. During the ceremony: TIFF2026521572000009.tif14170 is Z A CRM 197It indicates the position where the support protein attaches; and, TIFF2026521572000010.tif14170 is Z A This indicates the position where the spacer base attaches to Q.

[0071] In some embodiments, Q is a spacer comprising one or more groups selected from polyethylene glycol moieties, amino acid residues, alkylenediamine moieties, arylene-containing moieties, heteroarylene-containing moieties, heterocyclyl-containing moieties, cycloalkyl-containing moieties, and combinations thereof.

[0072] Each of the polyethylene glycol portion, amino acid residue, alkylenediamine portion, arylene-containing portion, heteroarylene-containing portion, heterocyclyl-containing portion, and cycloalkyl-containing portion may preferably be any of the groups defined above.

[0073] In a particular preferred embodiment, Q is C1~C 20 The spacer comprises one or more groups selected from alkylene, polyethylene glycol moieties, amino acid residues, alkylenediamine moieties, and combinations thereof.

[0074] As outlined below, the linker may be formed by covalently connecting a first connector and a second connector. The first connector is CRM 197 The first connector may be covalently linked to a carrier protein, and the second connector may be covalently linked to an immunogenic glycopeptide. The covalent linkage formed between the first connector and the second connector is CRM 197 This leads to the formation of a linker, the -L-, which covalently links the carrier protein and the immunogenic glycopeptide.

[0075] Therefore, in a particular embodiment, the spacer base, Q, is given by the formula VI shown below: -Q1-FG-Q2- Equation VI (In the formula: Q1 is a first spacer group covalently linked to a carrier protein; 197 and Q2 is a second spacer group covalently linked to an immunogenic glycopeptide; and FG is a functional group that links Q1 to Q2). It will be understood that the first spacer group, Q1, and the second spacer group, Q2, may each be any of the groups defined herein in relation to the spacer group Q or in relation to the linker, L.

[0076] In certain preferred embodiments, the first spacer group, Q1, and the second spacer group, Q2, independently comprise one or more groups selected from C₁-C

[0077] alkylene, (poly)ethylene glycol moieties, alkylenediamine moieties, amino acid residues, and combinations thereof. 20 In certain preferred embodiments, the first spacer group, Q1, has the formula:

[0078] TIFF2026521572000011.tif20170 (wherein: n is an integer from 1 to 6, preferably from 1 to 4, most preferably 2; TIFF2026521572000012.tif14170 indicates the position where the first spacer group, Q1, attaches to the protein linking group, Z and TIFF2026521572000013.tif14170 indicates the position where the first spacer group, Q1, attaches to the functional group, FG). A In certain preferred embodiments, the second spacer group, Q2, has the formula:

[0079] TIFF2026521572000014.tif31170 (wherein, m is an integer from 1 to 8, preferably from 2 to 6, most preferably 4; TIFF2026521572000014.tif31170 indicates the position where the second spacer group, Q2, attaches to the functional group, FG). ​TIFF2026521572000015.tif13170 indicates the position where the second spacing group, -Q2-, attaches to the functional group, FG; and, TIFF2026521572000016.tif15170 is the group of the second spacing group, -Q2- (indicating the position where it attaches to the immunogenic glycopeptide, D).

[0080] In certain embodiments, FG is a bond or functional group formed from the reaction between i) a ketone or aldehyde and an alkoxyamine (e.g., hydroxyamine) 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., 1,3-nitrone alkene) and an electron-deficient diene (e.g., tetrazine); and iii) a strained alkene or alkyne (e.g., norbornene or cyclooctin) and tetrazine. Preferably, FG is a bond or functional group formed from the reaction between an azide and an alkyne or an amine and an acyl halide or carboxylic acid. Most preferably, FG is a functional group formed from the reaction between an azide and an alkyne.

[0081] Therefore, in some embodiments, FG is bonded, TIFF2026521572000017.tif62170 is a functional group selected from -NH(=O)C- and -C(=O)NH-.

[0082] In some embodiments, FG is TIFF2026521572000018.tif71170 Selected from.

[0083] Preferably, FG is TIFF2026521572000019.tif38170 Selected from.

[0084] In a particular preferred embodiment, the linker is of the formula: TIFF2026521572000020.tif40170 (where; n is an integer selected from 1 to 4 (preferably 2); m is an integer selected from 2 to 8 (preferably 4); TIFF2026521572000021.tif15170 is where the linker is CRM 197 CRM attached to the carrier protein 197 are the residues C186 and C201 of the carrier protein; and, TIFF2026521572000022.tif15170 is a group indicating the position where the linker is attached to an immunogenic glycopeptide (e.g., the N-terminus of a MUC-1 glycopeptide).

[0085] The method for producing the cancer vaccine conjugate described herein is providing a CRM covalently linked to a first connector 197 carrier protein, and where the first connector is a CRM 197 covalently linked to the residues C186 and C201 of the carrier protein and containing a first reactive group; providing an immunogenic glycopeptide covalently linked to a second connector, and where the second connector contains a second reactive group; reacting the first reactive group and the second reactive group to covalently link the immunogenic glycopeptide to the CRM 197 carrier protein, thereby producing a cancer vaccine, and may include.

[0086] The first reactive group and the second reactive group can be any functional groups that can react with each other to covalently link the immunogenic glycopeptide to the CRM 197 carrier protein.<000055​​​​

[0088] In a particular embodiment, the first reactive group and the second reactive group are paired as follows: i) Azid and Alkyn; ii) Ketones or aldehydes with alkoxyamines (e.g., hydroxylamines) or hydrazines; iii) Amines and acyl halides or carboxylic acids; iv) electron-rich dienophiles (e.g., 1,3-nitrone alkenes) and electron-deficient dienes (e.g., tetrazine); and, v) Distorted alkenes or alkynes (e.g., norbornene or cyclooctyne) and tetrazine Selected from.

[0089] In the above pairing, the first reactive group is one of the functional groups in the pairing, while the second reactive group is the other (paired) functional group in the pairing.

[0090] In a particular preferred embodiment, one of the first reactive group and the second reactive group is an azide, and the other of the first reactive group and the second reactive group is an alkyne.

[0091] Preferably, the first reactive group is an azide (azide 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 cyclooctin, for example, dibenzocyclooctin.

[0092] Therefore, in a particular embodiment, the first connector is given by formula VI shown below: TIFF2026521572000023.tif23170 Formula VI (In the formula: Q1 is CRM 197 It is the first spacer group covalently linked to the carrier protein; RG1 is the first reactive group (e.g., azide); and, TIFF2026521572000024.tif14170 is a linker for CRM197 CRM attached to the carrier protein 197 It is the base of the carrier protein residues C186 and C201.

[0093] In some embodiments, the second connector is given by formula VII shown below: TIFF2026521572000025.tif24170 Formula VII (In the formula: Q2 is a second spacer group covalently linked to the immunogenic glycopeptide; and, RG2 is a second reactive group (e.g., an alkyne); and, TIFF2026521572000026.tif15170 is a group of linkers (indicating the position where the linker attaches to an immunogenic glycopeptide (e.g., MUC-1 glycopeptide)).

[0094] It will be understood that the first spacer base, Q1, and the second spacer base, Q2 of the first and second connectors may be any of the bases defined above in relation to Q1 and Q2.

[0095] In a particular preferred embodiment, CRM 197 The first connector, covalently linked to the carrier protein, is given by formula: TIFF2026521572000027.tif39170(in the formula: n is an integer from 1 to 4, preferably 2; and, TIFF2026521572000028.tif15170 is a linker for CRM 197 CRM attached to the carrier protein 197 It is the base of the carrier protein residues C186 and C201.

[0096] In another preferred embodiment, a second connector covalently linked to the immunogenic glycopeptide is given by formula: TIFF2026521572000029.tif45170(in the formula: m is an integer between 2 and 8, preferably 4; and, TIFF2026521572000030.tif14170 is a group of linkers (indicating the position where the linker is attached to an immunogenic glycopeptide (e.g., MUC-1 glycopeptide)).

[0097] CRM 197 Carrier proteins are, CRM 197 To provide a carrier protein, CRM 197 Selective reduction of the C186-C201 disulfide bond of the carrier protein, CRM 197 The formation of free thiol groups at residues C186 and C201 of the carrier protein, To provide a first connector compound comprising a first reactive group, a third reactive group, and a fourth reactive group, The third and fourth reactive groups of the first connector compound are reacted with the free thiol groups of C186 and C201, respectively, to obtain the CRM. 197 The residues C186 and C201 of the carrier protein are covalently linked, thereby enabling CRM 197 The carrier protein is covalently linked to the first connector, The first connector can be covalently connected by a method including the following:

[0098] The first connector compound is CRM 197 It may also be any compound containing reactive groups (i.e., a third reactive group and a fourth reactive group) capable of forming covalent attachments to sulfur atoms from residues C186 and C201 of the carrier protein. 197 By forming covalent attachments from residues C186 and C201 of the carrier protein to the sulfur atom, the first connector compound is linked to CRM 197 It will also be understood that this role involves re-crosslinking the reduced interchain disulfide bond between residues C186 and C201 of the carrier protein.

[0099] The first connector compound is CRM 197 Reactive groups capable of linking to sulfur atoms from residues C186 and C201 of the carrier protein are known in the art. Therefore, those skilled in the art will be able to select reactive groups (the third and fourth reactive groups) suitable for use in the present invention. For example, suitable reactive groups include 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 Examples include those described in Adv., 2017, 7, 9073-9077), Behrens 2015 (Mol. Pharm., 2015, 12, 3986-3998), and International Publication No. 2019 / 011078.

[0100] In a particular embodiment, the first connector compound has a general formula: TIFF2026521572000031.tif8170(in the formula: Q1 is the CRM as defined herein. 197 It is the first spacer group covalently linked to the carrier protein; RG1 is the first reactive group (e.g., azide); and, W is a compound of a functional group containing a third and a fourth reactive group.

[0101] In other embodiments, the first connector compound is given by the general formula: TIFF2026521572000032.tif15170 (in the formula: n is an integer selected from 1 to 4 (for example, 2); RG1 is the first reactive group (e.g., azide); and, W is a compound of a functional group containing a third and a fourth reactive group.

[0102] Preferably, a functional group containing a third reactive group and a fourth reactive group, where W- is the functional group shown below: TIFF2026521572000033.tif135170 (in the formula: TIFF2026521572000034.tif14170 is selected from one of the following (indicating the position where the functional group containing the third and fourth reactive groups, W-, attaches to the residual portion of the first connector compound).

[0103] In certain embodiments, the third and fourth reactive groups are halogen groups, such as a fluoro group, a chloro group, a bromo group, or an iodo group, and are preferably chloro groups.

[0104] In a particular preferred embodiment, the first connector compound is of formula: The compound is TIFF2026521572000035.tif35170 (wherein n is an integer selected from 1 to 4 (e.g., 2)).

[0105] CRM 197 The C186-C201 disulfide linkage of carrier proteins can be selectively reduced so as not to reduce the C461-C471 disulfide linkage. For example, the C186-C201 disulfide linkage can be selectively reduced using TCEP.

[0106] The present invention also provides reagents and kits for producing cancer vaccine conjugates as described herein. For example, a kit for producing cancer vaccine conjugates comprises a CRM covalently coupled to a first connector. 197 It may contain a carrier protein, where the first connector is CRM 197It is covalently linked to residues C186 and C201 of the carrier protein and contains a first reactive group.

[0107] Examples of salts of the compounds of the present invention (e.g., the conjugate and the first connector compound) include, but are not limited to, all pharmaceutically acceptable salts, such as acid addition salts of strong inorganic acids like HCl and HBr salts, and addition salts of strong organic acids like methanesulfonates. Further examples of salts include sulfates and acetates, such as trifluoroacetate or trichloroacetate.

[0108] References to compounds described herein (e.g., conjugates and first connector compounds) also refer to solvates of those compounds. Examples of solvates include hydrates.

[0109] The compounds described herein (e.g., conjugates and first connector compounds) include compounds in which atoms are replaced with naturally occurring or non-naturally occurring isotopes. In one embodiment, the isotopes are stable isotopes. Therefore, the compounds described herein include, for example, deuterium-containing compounds. For example, H is, 1 H, 2 H(D), and 3 It may be any isotopic form including H(T); C is 12 C, 13 C, and 14 It may be any isotopic form containing C; O is 16 O and 18 Any isotopic form containing O may, etc.

[0110] A particular compound (e.g., a conjugate and a first connector compound) may exist in one or more specific geometric, optical, enantiomer, diastereomer, epimer, atrop, stereoisomer, tautomer, conformation, 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; keto, enol, and enolate forms; syn and anti forms; syncrinal and anticrinal forms; α and β forms; axial and equatorial forms; boat, chair, twisted, envelope, and semi-chair forms; and combinations thereof (hereinafter collectively referred to as "isomers" (or "isomer forms")).

[0111] Except as discussed below regarding tautomer forms, it should be noted that the term “isomer” as used herein explicitly excludes structural (or constitutive) isomers (i.e., isomers that differ not only in the spatial position of atoms but also in the connections between atoms). For example, a reference to the methoxy group, -OCH3, should not be interpreted as a reference to its structural isomer, the hydroxymethyl group, -CH2OH. Similarly, a reference to ortho-chlorophenyl should not be interpreted as a reference to its structural isomer, meta-chlorophenyl. However, a reference to a certain class of structures may naturally include the structural isomer forms that belong to that class (e.g., C 1~6 Alkyl compounds include n-propyl and isopropyl; butyl compounds include n-, iso-, sec-, and tert-butyl; and methoxyphenyl compounds include ortho-, meta-, and para-methoxyphenyl.

[0112] Unless otherwise specified, references to specific compounds (e.g., conjugates and first connector compounds) include all such isomeric forms, including mixtures thereof (e.g., racemic mixtures). Methods for the preparation (e.g., asymmetric synthesis) and separation (e.g., fractional crystallization and chromatographic means) of such isomeric forms are known in the art or readily obtainable by methods taught herein or by adapting known methods in known ways.

[0113] In certain embodiments, the compounds described herein (e.g., the conjugate and the first connector compound) may be in a substantially purified form and / or a substantially impurity-free form.

[0114] In some embodiments, the substantially purified form is at least 50% by weight, for example, at least 60% by weight, for example, at least 70% by weight, for example, at least 80% by weight, for example, at least 90% by weight, for example, at least 95% by weight, for example, at least 97% by weight, for example, at least 98% by weight, for example, at least 99% by weight.

[0115] Unless otherwise specified, a substantially purified form refers to a compound in any stereoisomer or enantiomer form. For example, in one embodiment, a substantially purified form refers to a mixture of stereoisomers, i.e., a form purified relative to another compound. In one embodiment, a substantially purified form refers to one stereoisomer, e.g., an optically pure stereoisomer. In one embodiment, a substantially purified form refers to a mixture of enantiomers. In one embodiment, a substantially purified form refers to an equimolar mixture of enantiomers (i.e., a racemic mixture, a racemate). In one embodiment, a substantially purified form refers to one enantiomer, e.g., an optically pure enantiomer.

[0116] In some embodiments, impurities represent 50% by weight or less, for example, 40% by weight or less, for example, 30% by weight or less, for example, 20% by weight or less, for example, 10% by weight or less, for example, 5% by weight or less, for example, 3% by weight or less, for example, 2% by weight or less, for example, 1% by weight or less.

[0117] Unless otherwise specified, impurities refer to compounds other than stereoisomers or enantiomers. In one embodiment, impurities refer to other compounds and other stereoisomers. In one embodiment, impurities refer to other compounds and other enantiomers.

[0118] In some embodiments, the substantially purified form is at least 60% optically pure (i.e., 60% of the compound is the desired stereoisomer or enantiomer on a molar basis, and 40% is the undesirable stereoisomer or enantiomer), for example, at least 70% optically pure, for example, at least 80% optically pure, for example, at least 90% optically pure, for example, at least 95% optically pure, for example, at least 97% optically pure, for example, at least 98% optically pure, for example, at least 99% optically pure.

[0119] The compounds of the present invention, such as cancer vaccine conjugates described herein, may be provided in a protected form. In this case, reactive functional groups, such as amino functional groups, may be masked to prevent reactions during the synthesis steps. Protecting groups are provided to mask the reactive functional groups, and these protecting groups can be removed at a later stage of synthesis to expose the original reactive functional groups.

[0120] For example, amino, hydroxyl, carboxyl, and thiol functional groups present in the conjugate can be protected with protecting groups as described herein.

[0121] In one embodiment, the protected form is a compound in which amino, hydroxyl, thiol, and / or carboxyl functional groups are protected (masked) by a protecting group. In one embodiment, the protected form is a compound in which the side chain functional groups of amino acid residues in the compound are protected.

[0122] Protecting groups for amino acid residues, such as protecting groups, are known and well-explained in the art.

[0123] Amino acids with side-chain protection and optional amino and carboxyl protection are commercially available. Therefore, protected conjugate compounds can be prepared from appropriately protected amino acid starting materials.

[0124] If a protecting group is used, it can be removed under conditions that do not substantially disrupt the structure of the conjugate, for example, without altering the stereochemistry of the amino acid residue or causing the release of the protected ortho-quinone.

[0125] In some embodiments, the protecting group is acid-unstable, base-unstable, or can be removed under reducing conditions.

[0126] Exemplary protecting groups for amino functional groups include Boc (tert-butoxycarbonyl), Bn (benzyl, Bzl), CbZ (Z), 2-Cl-Z (2-chloro), Dde (1-[4,4-dimethyl-2,6-dioxocyclohexa-1-ylidene]-3-methylbutyl), Fmoc (fluorenylmethyloxycarbonyl), HSO3-Fmoc (e.g., sulfonylated Fmoc such as 2-sulfo-Fmoc described in Schechter et al, J. Med Chem 2002, 45(19) 4264), ivDde (1-[4,4-dimethyl-2,6-dioxocyclohexa-1-ylidene]ethyl), Mmt (4-methoxytrityl), Mtt (4-methyltrityl), Nvoc (6-nitroveratroyloxycarbonyl), and Tfa (trifluoroacetyl).

[0127] Exemplary protecting groups for aromatic nitrogen functional groups include Boc, Mtt, Trt, and Dnp (dinitrophenyl).

[0128] In some embodiments, the protecting group for the amino functional group is selected from Boc, CbZ, Bn, Fmoc, and HSO3-Fmoc.

[0129] In some embodiments, the protecting group for the amino functional group is Boc, Fmoc, or CbZ.

[0130] Exemplary protecting groups for hydroxyl functional groups include Trt (trityl), Bn (benzyl), tBu (tert-butyl), and 2-acetamido-2-deoxy-3,4,6-tri-O-acetyl-α-galactopyranosyl.

[0131] In one embodiment, the protecting group for the amino functional group is Trt.

[0132] Further exemplary protecting groups include silyl ether protecting groups such as TMS, TES, TBS, TIPS, TBDMS, and TBDPS, as well as ethers such as THP. Such protecting groups can be removed, for example, with TBAF.

[0133] Exemplary protecting groups for carboxyl functional groups include Bn (benzyl, Bz), tBu (tert-butyl), TMSET (trimethylsilylethyl), and Dmab ({1-[4,4-dimethyl-2,6-dioxocyclohexa-1-ylidene]-3-methylbutyl}aminobenzyl).

[0134] Exemplary protecting groups for aromatic nitrogen functional groups include Boc, Mtt, Trt, and Dnp (dinitrophenyl).

[0135] In some embodiments, only certain types of functional groups are protected. For example, only amino groups, such as the amino groups of the side chains of amino acid residues, may be protected.

[0136] In some embodiments, the amino group and the hydroxyl group are protected. The present invention also provides a pharmaceutical composition comprising the above-described cancer vaccine conjugate and a pharmaceutically acceptable excipient.

[0137] The method may further include mixing the cancer vaccine conjugate described herein with a pharmaceutically acceptable excipient to produce a pharmaceutical composition.

[0138] The term "pharmaceutically acceptable" refers to compounds, materials, compositions, and / or dosage forms that, within the bounds of sound veterinary or medical judgment, are suitable for use in contact with the tissues of a subject (e.g., human or other mammal) without excessive toxicity, irritation, allergic reactions, or other problems or complications, in proportion to a reasonable benefit-risk ratio. Each carrier, excipient, etc., must be "acceptable" in the sense that it is compatible with the other components of the formulation.

[0139] Suitable excipients and carriers include, but are not limited to, water, physiological saline, buffered physiological saline, phosphate buffer, alcohol / aqueous solution, emulsions, or suspensions. Other conventionally used diluents, adjuvants, and excipients may be added according to the prior art. Such carriers may include ethanol, polyols, and suitable mixtures thereof, vegetable oils, and injectable organic esters. Buffers and pH adjusters may also be used, which include, but are not limited to, salts prepared from organic acids or bases. Typical buffers include, but are not limited to, organic acid salts such as citrates (e.g., citrates), ascorbic acid, gluconic acid, carbonic acid, tartaric acid, succinic acid, acetic acid, phthalic acid, Tris, trimethylamine hydrochloride, or phosphate buffer. Parenteral carriers may include sodium chloride solution, ringer's dextrose, dextrose, trehalose, sucrose, Ringer's lactate solution, or fixative oils. Intravenous carriers may contain fluids and nutritional supplements, electrolyte supplements such as those based on ringer's dextrose, etc. Preservatives and other additives, such as antimicrobial agents, antioxidants, chelating agents (e.g., EGTA; EDTA), inert gases, and the like, may be provided in the pharmaceutical carrier. The pharmaceutical compositions described herein are not limited by the choice of carrier. The preparation of these pharmaceutically acceptable compositions from the above components having appropriate pH, isotonicity, stability, and other conventional properties is within the scope of the art.

[0140] Suitable carriers, excipients, etc., can be found in standard pharmaceutical textbooks, such as Remington's Pharmaceutical Sciences and The Handbook of Pharmaceutical Excipients, 4th edit., eds. RC Rowe et al, APhA Publications, 2003.

[0141] The pharmaceutical composition of the present invention may also be a vaccine composition. The pharmaceutical composition or vaccine composition may further contain an adjuvant.

[0142] An adjuvant is a compound that enhances or increases the immune response to an immunogenic compound. Suitable adjuvants for use in vaccine compositions include, but are not limited to, inorganic compounds such as aluminum salts, oils, bacterial products such as toxoids, and cytokines such as IL-1 and IL-2. Other suitable adjuvants include squalene-based oil-in-water nanoemulsions such as MF-59 and AddaVax®.

[0143] Vaccines and pharmaceutical compositions may, for convenience, be provided in unit dosage forms and may be prepared by any method known in the field of pharmaceutics. Such methods include the step of conjugating one or more isolated conjugate / immunogenic polypeptides with one or more carriers or excipients that may constitute auxiliary components. Generally, formulations are prepared by uniformly and closely conjugating the active compound with a liquid carrier, a finely divided solid carrier, or both.

[0144] Vaccines and pharmaceutical compositions may be prepared in the form of sterile aqueous solutions or dispersions suitable for injection, or in lyophilized form using freeze-drying technology. Lyophilized pharmaceutical compositions are typically maintained at about 4°C and can be reconstituted in a stabilizing solution, such as saline or HEPES, with or without adjuvants.

[0145] Vaccines and pharmaceutical compositions may be provided in sealed containers for single or multiple doses, such as ampoules and vials, and can be stored in a freeze-dried state, requiring only the addition of a sterile liquid carrier, such as water for injection, immediately before use.

[0146] The vaccines and therapeutic pharmaceutical compositions according to the present invention can be formulated for administration by many routes, including, but not limited to, parenteral, intravenous, intra-arterial, intramuscular, intratumor, oral, and nasal.

[0147] CRM as described herein 197Cancer vaccine conjugates containing immunogenic glycopeptides linked to polypeptides can be internalized by B cells and may be useful, for example, in generating B cell and T cell-mediated immune responses to the immunogenic glycopeptides.

[0148] This specification also provides a method for stimulating an immune response in a subject, the method comprising administering a conjugate or pharmaceutical composition described herein to a subject requiring stimulation of an immune response; and conjugates or pharmaceutical compositions described herein for use in such a method.

[0149] Immune responses include B cell-mediated immune responses and T cell-mediated immune responses.

[0150] This specification also provides a method for treating cancer, comprising administering a cancer vaccine conjugate or pharmaceutical composition described herein to an individual in need of treatment for cancer; a cancer vaccine conjugate or pharmaceutical composition described herein for use in the treatment of cancer; and the use of a cancer vaccine conjugate or pharmaceutical composition described herein in the manufacture of a drug for use in the treatment of cancer.

[0151] Cancer is characterized by the abnormal proliferation of malignant cancer cells. Cancer may be any type of cancer, including leukemia such as AML, CML, ALL, and CLL; lymphomas such as Hodgkin lymphoma, non-Hodgkin lymphoma, and multiple myeloma; and sarcoma, skin cancer, melanoma, bladder cancer, glioma, and other brain cancers; breast cancer such as triple-negative breast cancer and metastatic breast cancer; uterine cancer, oral cancer, ovarian cancer, prostate cancer, lung cancer, colon cancer, and other colorectal cancers; cervical cancer, liver cancer, head and neck cancer, esophageal cancer, pancreatic cancer, kidney cancer, adrenal cancer, gastric cancer, testicular cancer, gallbladder and bile duct cancer, thyroid cancer, thymic cancer, neuroblastoma, bone cancer, and cerebral cancer. In some preferred embodiments, cancer may be pancreatic cancer, breast cancer, ovarian cancer, gastric cancer, lung cancer, gallbladder cancer, or colorectal cancer.

[0152] Cancer cells suitable for the treatments described herein may express tumor antigens that are recognized by an immune response induced by the immunogenic peptides of the cancer vaccine conjugate.

[0153] Suitable tumor antigens are known in the art and include mucin-1 (MUC1). Cancer cells suitable for the treatments described herein may overexpress MUC1. For example, cancer cells may express 1.5 times, 2 times, 3 times, 4 times, 5 times, 6 times, 7 times, 8 times, 9 times, or 10 times more MUC1 than normal cells. Cancer cells suitable for the treatments described herein may express MUC1 with abnormal glycosylation. For example, abnormally glycosylated MUC1 may be recognized by an immune response induced by an immunogenic glycopeptide containing the epitope of SEQ ID NO: 1.

[0154] Individuals suitable for treatment with the cancer vaccine conjugates or pharmaceutical compositions described herein may be rodents (e.g., guinea pigs, hamsters, rats, mice), murids (e.g., mice), canids (e.g., dogs), felines (e.g., cats), equids (e.g., horses), primates, apes (e.g., monkeys or apes), primates (e.g., marmosets, baboons), apes (e.g., gorillas, chimpanzees, orangutans, gibbons), or mammals such as humans.

[0155] In some preferred embodiments, the individual is human. In other preferred embodiments, non-human mammals, particularly mammals conventionally used as models to demonstrate therapeutic efficacy in humans (e.g., mice, primates, pigs, dogs, or rabbits), may be used.

[0156] In some embodiments, individuals may have minimal residual disease (MRD) after initial cancer treatment.

[0157] An individual with cancer may present with at least one identifiable sign, symptom, or laboratory finding sufficient to make a diagnosis of cancer according to clinical criteria known in the art. Examples of such clinical criteria can be found in medical textbooks such as Harrison's Principles of Internal Medicine, 15th Ed., Fauci AS et al., eds., McGraw-Hill, New York, 2001. In some cases, the diagnosis of cancer in an individual may involve the identification of a specific cell type (e.g., cancer cells) in a sample of bodily fluids or tissue obtained from the individual.

[0158] As used herein in the context of treating a disease, the term “treatment” generally refers to treatments and therapies that achieve some desired therapeutic effect, such as inhibiting the progression of a disease, including a reduction in the rate of progression, cessation of the rate of progression, improvement of the disease, and cure of the disease.

[0159] The treatment may be any treatment or therapy, whether in humans or animals (e.g., for veterinary use), that achieves any desired therapeutic effect, such as inhibiting or delaying the progression of a disease, including but not limited to reducing the rate of progression, halting the rate of progression, improving the condition, curing or relieving the condition (whether partially or completely), preventing, delaying, mitigating or inhibiting one or more symptoms and / or signs of the condition, or extending the survival of a subject or patient beyond the survival expected without treatment.

[0160] This also includes preventative measures (i.e., prophylactic methods). For example, individuals susceptible to or at risk of developing or recurring cancer may be treated as described herein. Such treatment may prevent or delay the development or recurrence of cancer in the individual. Therefore, the cancer vaccine conjugates and pharmaceutical compositions described herein may be useful in the therapeutic treatment of individuals with cancer, or in the prophylactic treatment of individuals to prevent or delay the development or recurrence of cancer.

[0161] In particular, treatment may include suppression of cancer growth, including complete remission of the cancer, and / or suppression of cancer metastasis. Cancer growth generally refers to one of many indicators that show a change to a more developed morphology within the tumor. Therefore, indicators for measuring suppression of cancer growth include a decrease in cancer cell viability, a decrease in tumor volume or morphology (e.g., determined using computed tomography (CT), ultrasound, or other imaging techniques), a delay in tumor growth, destruction of the tumor vascular system, improved performance in delayed-type hypersensitivity skin tests, increased activity of cytolytic cancer cells, and a decrease in tumor-specific antigen levels.

[0162] In some preferred embodiments, therapeutic agents such as cancer vaccine conjugates or pharmaceutical compositions described herein may be administered to an individual without other combination cancer therapies such as cytotoxic chemotherapy or radiotherapy; that is, the therapeutic agent may be administered alone.

[0163] In other embodiments, the cancer vaccine conjugates or pharmaceutical compositions described herein may be administered in combination with one or more other therapies, such as cytotoxic chemotherapy or radiotherapy.

[0164] When a therapeutic agent is used in combination with an additional therapeutic agent, these compounds may be administered sequentially or simultaneously by any convenient route. When a therapeutic agent is used in combination with an additional therapeutic agent effective for the same disease, the dose of each drug in combination may differ from the dose when the therapeutic agent is used alone. Appropriate doses will be readily understood by those skilled in the art.

[0165] In some preferred embodiments, the cancer vaccine conjugates or pharmaceutical compositions described herein may be administered in combination with immune checkpoint inhibitors.

[0166] Immune checkpoint inhibitors are drugs that can inhibit cellular signaling mediated by immune checkpoint molecules. Immune checkpoint molecules may include PD-1, CTLA-4, LAG-3, TIM-3, TIGIT, and BTLA. Immune checkpoint inhibitors are outlined, for example, in 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, semiprimab, and dostallimab.

[0167] In some embodiments, immune checkpoint inhibitors can inhibit PD-1-mediated cellular signaling. The agent may be a PD-L1-targeting agent, or more preferably a PD-1-targeting 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 signaling. In some embodiments, the agent may be an anti-PD-L1 antibody or a fragment thereof (e.g., an antagonist anti-PD-L1 antibody or a fragment thereof), or an anti-PD-1 antibody or a fragment thereof (e.g., an antagonist anti-PD-1 antibody or a fragment thereof). Suitable anti-PD-1 antibodies include nivolumab, pembrolizumab, dostallimab, and semiprimab. Suitable anti-PD-L1 antibodies include atezolizumab, avelumab, and durvalumab.

[0168] The cancer vaccine conjugates or pharmaceutical compositions described herein may be administered as single doses, sequential doses, or intermittently (e.g., divided doses at appropriate intervals) during the treatment period. Methods for determining the most effective means of administration and dosage are known to those skilled in the art and vary depending on the formulation used for treatment, the therapeutic purpose, the target cells being treated, and the subject being treated. Single or multiple doses may be administered, and the dose level and administration pattern are selected by the attending physician.

[0169] Other aspects and embodiments of the present invention provide the aspects and embodiments described above with the terms "comprising" replaced by the term "consisting of", and the aspects and embodiments described above with the terms "comprising" replaced by the term "consisting essentially of".

[0170] It is understood that this application discloses all aspects and all combinations of the embodiments described above, unless the context should be interpreted otherwise. Similarly, this application discloses all combinations of preferred and / or any features, either individually or with any other aspects, unless the context should be interpreted otherwise.

[0171] Modifications of the above embodiments, further embodiments, and variations thereof will become apparent to those skilled in the art by reading this disclosure, and they themselves fall within the scope of the present invention.

[0172] All documents and sequence database entries referenced herein constitute part of this specification by reference in their entirety for all purposes.

[0173] As used herein, “and / or” shall be understood as a specific disclosure that includes or excludes each of two expressed features or components. For example, “A and / or B” shall be understood as if each of (i) A, (ii) B, and (iii) A and B were described separately herein.

[0174] experiment Materials and methods Synthesis of glycopeptide 1' The synthesis was carried out automatically using Rink Amide MBHA resin (0.05 mmol) and an automated synthesizer. The glycosylated amino acid building block (2.0 equivalents) was synthesized as described in reference (24). This residue and the cyclooctin derivative were manually coupled using HBTU [(2-(1H-benzotriazole-1-yl)-1,1,3,3-tetramethyluronium hexafluorophosphate)], while the other fluorenylmethyloxycarbonyl (Fmoc) amino acid (5.0 equivalents) was automatically coupled using oxyma pure / DIC (N,N'-diisopropylcarbodiimide). A 20% (v / v) piperidine solution in dimethylformamide (DMF) was used to deprotect Fmoc. The O-acetyl group of the sugar chain was deprotected with 5 mL (3 × 15 min) of hydrazine / MeOH (7:3) solution. After synthesis, glycopeptide 1' was cleaved from the resin, and all acid-sensitive side-chain protecting groups were simultaneously removed using 95% TFA, 2.5% TIS (triisopropylsilane), and 2.5% H2O, followed by precipitation with cold diethyl ether. The crude glycopeptide was processed using Phenomenex. Purification was performed using a semi-preparative HPLC equipped with a Luna C18(2) column (10 μm, 250 mm × 21.2 mm) and a dual-wavelength absorbance detector at a flow rate of 10 mL / min or 20 mL / min.

[0175] CRM 197 -Linker-Azide Synthesis 20 mg of 1,3-dichloroacetone (0.16 mmol) was reacted with 40 mg of aminooxy-PEG3-azide (0.17 mmol, 1.1 equivalents) in 1 mL of DMF at 21°C for 24 hours. The reaction product was then purified by passing it through a silica column and applying an increasing gradient of ethyl acetate in hexane (40% to 100%), followed by 20% methanol and 100% methanol in ethyl acetate. A total of 13 mg of compound 2 (0.04 mmol, 25% yield) was obtained, and a 40 mM stock solution in DMF was prepared. Next, CRM... 197 Reduced with 12 equivalents of TCEP: 5 μL of 47 mg / mL CRM 197 The stock solution (final concentration 40 μM) and 2.4 μL of 6 mg / mL TCEP stock solution were mixed in PBS pH 7.4 (final volume 99 μL) at 21°C. After stirring for 2 hours, the protein was mixed with 20 equivalents of compound 2 (2 μL of a 40 mM solution of compound 2) at 21°C for 5 days. The resulting modified protein was then passed through a ZebaSpin column (for buffer exchange to H2O) and analyzed by LC-MS.

[0176] CRM 197 - Linker-1' synthesis 20 μL CRM 197 Linker-azide (41 μM in PBS) was incubated with 8.5 μL of derivative 1' (10 mg / mL, 20 equivalents in PBS) at 25°C for 20 hours. Finally, the reaction mixture was purified using a Zeba spin column and analyzed by mass spectrometry, SDS-PAGE, and Western blotting.

[0177] Circular dichroism (CD) spectroscopy Proteins were desalted using a PD-minitrap G25 column (according to the manufacturer's instructions) and then diluted to 5 μM (in PBS buffer, pH 7.4). Far-ultraviolet circular dichroism (CD) data were acquired using a 1.0 mm quartz cuvette (Hellma GmbH & Co, Mülheim, Germany) with a JASCO J-815 CD spectropolarizer (Hachioji, Tokyo, Japan). All spectra were obtained at 25°C, between 195 nm and 300 nm, with a sampling rate of 200 nm·min.-1 Data was collected with a data pitch of 0.5 nm, a data integration time of 1 second, and a bandwidth of 1 nm. Each spectrum represents the average value of 5 scans. Absorbance spectra were also monitored to control light scattering and signal detection saturation. In addition to blank subtraction, baseline drift related to the experimental apparatus was corrected by subtracting the average value of the signal between 250 nm and 260 nm for each spectrum. The raw ellipticity θ was then calculated, followed by the molar ellipticity ([θ]; deg·cm). 2 ·dmol -1 The data was converted to units. All conditions were measured independently and in pairs.

[0178] mass spectrometry Modified proteins were analyzed by liquid chromatography-mass spectrometry (LC-MS). Protein LC-MS was performed on a Waters Acquity UPLC system equipped with a QDa single quadrupole mass detector. Separation was performed using an Xbridge BEH C4 column (Waters, 3.5 μm, pore size 300 Å, 2.1 × 100 mm) maintained at 40°C. Solvents A (0.1% (v / v) formic acid in redistilled water) and B (0.01% (v / v) formic acid in acetonitrile) were used as mobile phases. The gradient of solvents A and B was applied according to the following method: a gradual gradient of 10% B for 5 minutes, followed by 100% B over 15 minutes. At 20.1 minutes, B was returned to 10%, and this percentage was maintained for a further 7.9 minutes to recondition the LC column. The total runtime was 28 minutes, and the flow rate was 0.2 mL / min. The electrospray ion 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, with a total flow rate of 800 L / h and a cone flow rate of 1 L / h. The desolvation temperature was set to 400 °C. The total mass spectrum was reconstructed from the ion series using the MaxEnt algorithm pre-installed in MassLynx software (Waters, version 4.1). To obtain the described ion series, protein peaks (multiple peaks may be present) from the chromatogram were selected and integrated and further analyzed.

[0179] SDS-PAGE Conjugation, Conjugate, Wild-type (WT) CRM 197 The glycopeptide 1' was identified by electrophoresis on a 12% acrylamide gel in Tris-glycine / SDS electrophoresis buffer (25 mM Tris base, 190 mM glycine, 0.1% SDS, pH 8.3) under non-reducing conditions at 100V for 1.5 hours. A PageRuler-stained protein ladder was used as a molecular marker. The gel was further used for SDS-PAGE analysis by staining it with Abcam's InstantBlue Coomassie Protein Stain (according to the manufacturer's instructions).

[0180] Molecular dynamics (MD) simulation CRM 197 The crystal structure of was used as the starting coordinate for the protein (25). It is important to note that although only one copy of the glycopeptide 1' presented by the protein is present, up to four possible isomers (Z / E-oxime and 1,4 / 1,5-triazole adducts) can be assumed. Simulations were performed for different isomers using the AMBER 20 package (36) with ff14SB (37) and gaff2 (38) force fields implemented. Topology and coordinate files for MD simulations were generated using the LEaP module of AMBER 20, which was executed using the CUDA version of the PMEMD module of the AMBER simulation package. Each conjugate was immersed in a water box containing 10 Å TIP3P water molecule (39) buffer, and explicit counterions (Na) were used. + The system was neutralized by adding ). A two-step structural optimization approach was performed using the PMEMD module. In the first step, 50 kcal·mol -1 · Å -2Using the harmonic potential, only the positions of the solvent molecules and ions were minimized in the first step, and in the second step, unconstrained minimization of all atoms in the simulation cell was performed. In both steps, a 2500-step minimization was performed using the steepest descent method, followed by a 2500-step minimization using the conjugate gradient method. Then, under constant pressure of 1 atmosphere and periodic boundary conditions, the system was heated by raising the temperature from 0K to 300K over 2ns. The solute was 10 kcal·mol. -1 Harmonic constraints were applied, and the temperature was controlled and homogenized using the Andersen temperature coupling scheme (40). During the heating phase, the time step was kept at 1 fs. The SHAKE algorithm was applied to constrain all bonds involving hydrogen atoms (41). Long-range electrostatic effects were modeled using the particle-mesh Ewald method (42). A sharp cutoff of 8 Å was applied to the Lennard-Jones interaction. Each system was equilibrated for 2 ns with a constant volume and a temperature of 300 K using a time step of 2 fs. Then, under the same simulation conditions, a further 0.5 μs generation orbital was performed.

[0181] MUC1.Tg mouse genotyping MUC1.Tg breeding pairs were purchased from The Jackson Laboratory and breeding was initiated at the iMM-JLA animal facility. Ear samples were collected from the pup mice by the animal house technicians. The samples were then used for gDNA extraction using the NaOH gDNA extraction method. Briefly, the samples were incubated with 75 μL of alkaline solubilant at 95°C for 1 hour. After incubation, the samples were vortexed and 75 μL of neutralizing reagent was added. The samples were centrifuged at 4000 rpm for 3 minutes, and 2 μL of the sample was immediately used for PCR. The PCR reaction was performed using primers from ThermoScientific and Taq DNA polymerase (recombinant) (5 U / μL). The reaction mixture consisted of 2.5 μL of 10x buffer containing KCl, 1 μL of primer, 1 μL of 25 mM MgCl2, 0.5 μL of 10 mM dNTP mixture, 2 μL of template DNA, 0.25 μL of Taq, and water (maximum 25 μL). The reaction conditions were: 1°C to 95°C for 3 minutes, 2°C to 95°C for 30 seconds, 3°C to 60°C for 30 seconds, 4°C to 72°C for 1 minute (40 cycles of steps 2-4), and 72°C for 10 minutes. The PCR reaction results were confirmed by agarose gel electrophoresis (2% gel). The presence of two bands (transgene and internal standard) was considered PCR positive, and the presence of only one band (internal standard) was considered negative.

[0182] Mouse immunization protocol MUC1.Tg mice (auto-bred) aged 12-14 weeks expressing human MUC1 at physiological levels were intraperitoneally injected with a vaccine candidate a total of four times at 21-day intervals. Three groups of mice (5 mice per group) were used: CRM 197 -Linker-1' (MUC1 total 2μg)-Group 1; CRM 197(47 μg of protein, the same amount injected in Group 1) - Group 2; PBS - Group 3. Mice were injected with a total of 100 μL (50 μL adjuvant + 50 μL vaccine candidate) using squalene-oil-in-water Addavax adjuvant as part of the formulation. Blood samples were collected by buccal puncture before immunization and 5 days after each boost, and whole blood was collected by cardiac puncture 5 days after the final boost. Serum was recovered from the blood by centrifugation (4 minutes, 4000 rpm) of the blood samples. Immunized mice were further used for tumor challenge using MC38-MUC1 cells.

[0183] Antibody titers were determined by ELISA assay. A 96-well Maxisorb ELISA plate (Nunc) is coated with non-natural glycopeptide 1' or CRM in the coating buffer. 197 The plates were coated with one of the following (4°C, overnight). After blocking with ELISA blocking buffer, the plates were incubated with 1:100 dilutions of serum from the first and second blood collections and compared with preimmune serum. Bound antibodies were detected using the following anti-mouse secondary antibodies: IgG HRP (Invitrogen, 1:3000), IgG1-HRP (1:1000, eBioscience), IgG2a-HRP (1:50000, Abcam), IgG2b-HRP (1:50000, Abcam), IgG3-HRP (1:50000, Abcam), and IgM-HRP (1:50000, Abcam). All incubations were performed at 25°C for 1 hour, and the plates were washed three times with ELISA washing buffer between incubations. The assay was developed using TMB 1x solution (eBioscience) at 25°C for 10 minutes. The kinetic reaction of TMB was stopped in ELISA stop solution, and the resulting color change from blue to yellow could be read at 450 nm using an Infinite M200 plate reader. Before displaying the raw data, the background absorbance value (absorbance obtained in wells incubated with only the secondary antibody minus the blank) was subtracted.

[0184] Tumor induction in mice In immunized MUC1.Tg mice, a local model of colon cancer was established by subcutaneous inoculation of 1 × 10 6 MC38-MUC1 cells (commercially available from Kerafast) into the flank on day 6 after the final immunization. Tumor growth was monitored over time by performing two-way measurements with calipers every 3 days, and the mean tumor volume was calculated using the formula (length × width 2 ) / 2. On day 15 after tumor induction, animals were euthanized to collect serum, tumors, spleens, and draining regional lymph nodes. No signs of animal distress or discomfort, including weight loss, were observed. The data collected were analyzed using GraphPad Prism8.

[0185] Determination of cytokine and chemokine levels Cytokine levels in the serum of immunized mice were determined by the Mouse High Sensitivity T-Cell Discovery Array 18-plex (MDHSTC18) assay from Eve Technologies. This assay enabled quantification of the following cytokines: GM-CSF, IFNγ, IL-1α, IL-1β, IL-2, IL-4, IL-5, IL-6, IL-7, IL-10, IL-12(p70), IL-13, IL-17A, KC / CXCL1, LIX, MCP-1, MIP-2, TNF-α.

[0186] T cell isolation for flow cytometry and CTL assay Mice were euthanized by overdose of isoflurane, and tumors, inguinal lymph nodes (iLNs), and spleens were harvested for T cell isolation. The iLNs and spleens were mashed by pressing against a 100-μm cell strainer and diluted with 2 mL and 8 mL of complete DMEM 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 medium (0.4 mg / mL collagenase I, 1 mg / mL collagenase IV, and 10 μg / mL DNase I) at 37 °C and 220 rpm for 30 min. After digestion, the cell suspension was diluted with 4 mL of ice-cold complete DMEM medium, filtered through a 100-μm cell strainer, and centrifuged at 500×g for 5 min. The cell pellet was incubated with 1 mL of red blood cell lysis buffer and immediately centrifuged at 500×g for 5 min. At this point, the cell pellet was resuspended in 2 mL of complete DMEM and counted for staining or cytotoxic T lymphocyte (CTL) assay.

[0187] Cell staining for T cell determination by flow cytometry Approximately 2×10 6 cells were transferred to a round-bottom 96-well plate and centrifuged at 500×g for 3 min to remove the supernatant. The cell pellet was incubated with 20 μL of Fc block (1:100) in FACS buffer on ice for 10 min. The cells were washed with 100 μL of FACS buffer, centrifuged (as described above), and then incubated with 20 μL of extracellular antibody mix on ice for approximately 45 min. The extracellular antibody mix consisted of 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). A 7-aad viability staining reagent (5 μL / 1×10 6 cells) was used to determine viable cells. Samples were acquired using a BD LSR Fortessa and analyzed with FlowJo (version 6.3.4).

[0188] Isolation of T cells from spleen by magnetic-activated cell sorting (MACs) The spleen cells were crushed and passed through a 100 μm cell strainer, then centrifuged at 500 × g for 5 minutes. The cell pellet was incubated with erythrocyte lysis buffer and immediately centrifuged again (500 × g, 5 minutes). The supernatant was removed, and the cell pellet was incubated with anti-CD3-APC antibody (1:100 in MACS buffer) on ice for 15 minutes. After incubation, the cells were washed with MACS buffer and centrifuged (5 minutes, 500 × g). After removing the supernatant, the cells were sterilized with APC nanobead solution (1:10 μL, 10 7 The cells were incubated on ice for 15 minutes with approximately 10 μL of MACS buffer per cell. After this incubation step, a washing step with MACS buffer was performed (centrifugation at 500 × g for 3 minutes). The supernatant was removed, the cells were resuspended in 2.5 mL of MACS buffer, and the tube was placed on a magnetic stand for 5 minutes. The unlabeled fraction was discarded, the adhered fraction was resuspended in 2.5 mL of MACS buffer, and the tube was placed on the magnetic stand again. This step was repeated one more time (a total of three times). After the final step, the cells were counted and used for the CTL assay.

[0189] Cytotoxic T lymphocyte (CTL) assay To determine CTL activity, MC38-MUC1 and MC38 cells were used along with T cells isolated from the spleen of immunized mice. MC38-MUC1 and MC38 cells were cultured according to the manufacturer's instructions (complete DMEM was used for MC38 cells, and complete DMEM containing gentamicin and geneticin antibiotics was used for MC38-MUC1 cells). Cells were seeded at 10,000 cells per well and allowed to adhere for 24 hours. Then, T cells isolated from mice were added at effector-to-target ratios of 3:1, 10:1, and 90:1 (T cells:cancer cells). After 24 hours, cell viability was determined using CellTiter Blue (Promega) and an M200 microplate reader. Cytotoxicity was calculated as the percentage of viable cells in the presence of T cells compared to cells without T cells.

[0190] Serum binding analysis by flow cytometry CRM 197 The binding affinity of antibodies produced by the -linker-1' vaccine candidate was determined by flow cytometry analysis. Briefly, T47D cells (human breast cancer cells highly expressing TA-MUC1) and HEK293T cells (cells not expressing human TA-MUC1) were fixed in ice-cold 4% paraformaldehyde solution for 10 minutes. After fixation, the cells were permeabilized with 0.1% Triton-X100 in PBS for 15 minutes, followed by a blocking step of 30 minutes with 10% FBS in PBS. The cells were then incubated with 1:50 dilution mouse serum, followed by incubation with Abcam's goat anti-mouse polyclonal IgG H&L Alexa Fluor 488 (1:2000) secondary antibody. All incubation steps were performed at 25°C for 1 hour, followed by a washing step with PBS. Samples were acquired using a BD LSR Fortessa equipped with a 488nm laser and a 530 / 30nm bandpass filter (the combination used for Alexa488 detection) and analyzed with FlowJo (version 6.3.4).

[0191] result Previously investigated non-natural glycopeptide 1(24) (Figure 1) contains CRM 197 A cyclooctin moiety was added to the N-terminus to allow it to react with azide groups selectively introduced into the protein (see below). CRM 197 By treating the compound with an excess amount of tris(2-carboxyethyl)phosphine (TCEP) at 25°C for 2 hours, the C186-C201 bond could be selectively reduced (Figure 2a) (29). The resulting protein was then reacted with 20 equivalents of compound 2 (materials and methods) in PBS buffer (pH 7.4) at 21°C for 5 days, and the desired modified protein (CRM) was purified using a Zebra spin column. 197A linker-azide (Figure 2b) was obtained. Liquid chromatography-mass spectrometry (LC-MS) analysis showed a single peak at 58705 Da, which corresponds to the modification of one disulfide and the subsequent introduction of an azide handle by re-crosslinking (Figure 2b).

[0192] CRM 197 The effect of introducing compound 2 to the sample was investigated by circular dichroism (CD, Figure 2c). We used CRM 197 -Linker-Azide's CD spectrum is CRM 197 It was observed that it was identical to that of (29,30), indicating that the secondary structure was preserved even after site-selective chemical modification.

[0193] After that, Conjugate CRM 197 -Linker-azide was treated with glycopeptide 1' (protein / glycopeptide ratio 1:20) at 25°C for 20 hours (Figure 3a). The reaction mixture was then purified using a Zebra spin column and analyzed by LC-MS (Figure 3b), which confirmed successful conjugation and homogeneous CRM. 197 The formation of the -linker-1' was confirmed. Molecular dynamics (MD) simulations performed using this conjugate showed that the addition of the glycopeptide did not alter the three-dimensional structure of the protein (Figure 3c).

[0194] These calculations also demonstrated the flexibility of the linker and how it can facilitate the exposure of the glycopeptide (antigen) to the solvent. It is noteworthy that although only one copy of the glycopeptide 1' is presented by the protein, up to four possible isomers (Z / E-oxime and 1,4 / 1,5-triazole adducts) can be assumed. This conjugation procedure is scalable and can be used to create a homogeneous vaccine CRM for immunological studies in mice. 197 - Enables the manufacture of Linker-1'.

[0195] Next, we investigated CRM in mice. 197The immunogenicity potential of -linker-1' was tested. For this purpose, a group of five MUC1.Tg mice were immunized with an initial dose, followed by three equal escalator doses of the vaccine candidate at 21-day intervals. One control group received a non-conjugate CRM. 197 One group was treated with [a specific solution], and the other group was treated with phosphate-buffered saline (PBS) alone. Serum was collected from the immunized mice 5 days after each injection, and the level of anti-MUC1 antibody was examined (Figure 4a). For this purpose, glycopeptide 1 (Figure 1) was loaded onto plates and an ELISA assay was performed. As shown in Figure 4b, anti-MUC1 antibody was detected after the second injection. Antibody titers increased after the third and fourth immunizations, suggesting a boosting effect. Importantly, the antibodies induced in mice by our vaccine candidate were mainly IgG antibodies (Figures 4b and 4c), while IgM antibody levels were significantly lower (Figure 4d). This result suggests a mature response with class-switching recombination. For IgG antibodies, further investigation was performed with additional ELISA assays to determine their isotype (Figure 4c). Although all IgG isotypes were detected, the induced antibodies were mostly IgG2 type, suggesting that the vaccine induced a Th1 response. CRM 197 The observation of antibodies against it is noteworthy.

[0196] Next, we verified whether the generated antibodies could recognize human tumor-associated MUC1 (TA-MUC1), which is overexpressed in cancer cells. Therefore, serum from immunized mice was incubated with T47D (33) (used as a positive control) and HEK293T (34) (used as a negative control), and binding analysis was evaluated by flow cytometry. These experiments showed that the antibodies generated by our vaccine could bind to T47D cells expressing TA-MUC1 on their surface. In contrast, consistent with the absence of MUC1 on the surface of HEK293T cells, only negligible binding was observed (compare Figures 4e and 4f).

[0197] CRM 197After demonstrating that the -linker-1' vaccine generates antibodies that selectively recognize human TA-MUC1, we then evaluated the ability of these antibodies to promote protection against cancer. For this purpose, tumors were induced in immunized mice by inoculating them with MC38-MUC1 cells 6 days after the final injection. We used an MC38-MUC1 cell line derived from mouse colon adenocarcinoma and further modified to express human MUC1 (35). Tumor growth was tracked up to day 15, after which the mice under study were sacrificed. 197 Or compared to a control immunized with PBS, CRM 197 In animals immunized with -linker-1', delayed tumor growth was observed (Figures 5a and 5b). Tumor size in treated mice was reduced by approximately 55% compared to controls at day 15. Furthermore, in one mouse in the treated group, no detectable tumor developed up to day 15 after inoculation with cancer cells.

[0198] After inducing tumors, serum cytokine levels were also evaluated. Notably, elevated levels of Th1-related cytokines such as IL-2, IFN-γ, and TNF-α were detected (Figure 5c), while no significant levels of Th2-related cytokines such as IL-4, IL-10, and IL-13 were observed (Figure 5d). This aspect supports the idea that the immune response induced by the vaccine candidate was a Th1-type response, as suggested by the IgG isotypes detected in the serum (Figure 4c).

[0199] We also evaluated the effectiveness of T cells that promote cytotoxic T lymphocyte (CTL) responses. Briefly, we evaluated CD3 from the spleen of mice in both the treatment and control groups. + T cells (CD4 + and CD8 + T cells (including) were isolated (materials and methods) and used in a CTL assay. Interestingly, these cells were found in CRM 197When MC38-MUC1 cells were co-cultured with T cells isolated from the spleens of mice treated with -linker-1', the viability of MC38-MUC1 cells was observed to be approximately 20% lower compared to cells obtained from the control group (Figure 5e). This experiment suggests that T cells in the treatment group can recognize tumor-associated MUC1 expressed on MC38-MUC1 cells and induce cancer cell death. Therefore, CRM 197 Linker-1', along with other factors, can potentially delay tumor growth by enhancing the activity of cytotoxic T cells directed towards cancer cells presenting human TA-MUC1.

[0200] In the final stage of our research, we conducted a crucial experiment to evaluate the efficacy of our homogeneous conjugate as a potential therapeutic vaccine for suppressing tumor growth (Figure 5f). To achieve this, we induced tumors (MC38-MUC1 cells or Panc02-MUC1 cells) in mice that had not been previously treated with the vaccine. The MC38-MUC1 cell line is representative of mouse colon adenocarcinoma expressing TA-MUC1 on its surface. The Panc02-MUC1 cell line corresponds to a mouse pancreatic cancer model expressing the human MUC1 sequence on its surface. The tumors averaged 100 mm. 3 Once it reaches the size, CRM 197 -Initiated treatment with Linker-1'. Treatment is CRM 197 Linker-1' was initiated either as monotherapy (Figures 5f and 6) or in combination with a checkpoint inhibitor (Figure 7).

[0201] The same four doses administered in previous in vivo experiments were given at two-day intervals. Mice were closely monitored, and when the tumor grew to 1000 mm², 3 Once the mice reached a certain size, they were sacrificed for analysis.

[0202] The results of this experiment are very promising, as all vaccinated mice survived beyond day 10, while untreated mice were sacrificed due to tumor size. In the MC38-MUC1 cancer model, the tumor size of treated mice decreased by approximately 40% by day 7, while in the Panc02-MUC1 model, it decreased by approximately 75% by day 25, the day the first control animal reached the endpoint (Figures 5b and 6, respectively). Regarding survival probability, in contrast to all vaccinated mice in the MC38-MUC1 model surviving beyond day 10, the tumor size of untreated mice was 1000 mm by day 10. 3 The survival rate of treated mice was approximately 40% up to day 15 (Figure 5f). Although tumor growth is generally slower in the Panc02-MUC1 model, similar results were observed, with delayed tumor growth and extended survival in treated animals compared to the control group. In this case, the survival rate of treated mice at day 54 was 25% (Figure 6, right panel). These results, as evidenced by the extended survival rate, suggest that our homogeneous vaccine has potential therapeutic effects against tumor growth.

[0203] To study the combined effects of both treatments in a single therapeutic formulation, we administered our homogeneous vaccine in combination with a checkpoint inhibitor. In this combination therapy approach, regardless of the cancer cell line used, four identical subcutaneous (SC) doses of the vaccine were administered at 2-day intervals (Figure 7), while the anti-PD-1 antibody was administered by three intraperitoneal (IP) injections at 3-day intervals, with an IgG2a isotype antibody used as a control. Mice were closely monitored, and when the tumor reached 1000 mm 3 The tumors were sacrificed once they reached a certain size. Notably, this therapy resulted in a significant reduction in tumor size. In the MC38-MUC1 cancer model, by day 10 (when the first control mice reached the endpoint), the tumor size of the treated mice was reduced by approximately 70% compared to the mice treated with PBS. In addition, CRM 197Mice treated with -linker-1' and anti-PD-1 showed a survival rate of approximately 80% by day 20. Notably, one mouse showed complete tumor regression at day 65, when it was euthanized (Figure 7, upper panel). Similar results were obtained in a pancreatic cancer model. In this case, tumor growth was suppressed by approximately 84% by day 25 (the day of death of the first control animal) in mice treated with the combination therapy, and more than 20% of the mice were still alive at day 60 (Figure 7, lower panel).

[0204] In summary, we have manufactured the first homogeneous cancer vaccine based on a MUC1-like glycopeptide containing a non-naturally fluorinated proline and a Tn antigen substitute with an S-glycoside linkage. This antigen is based on the protein CRM, the most commonly used immunogenic carrier in vaccine design for human vaccination campaigns. 197 Site-selective conjugation was performed. For this purpose, the most reactive of the two disulfide crosslinks in the protein (C186-C201) was selectively reduced in TCEP and re-crosslinked with an azide-containing linker. A copper-free click reaction between this group and the cyclooctin moiety introduced at the N-terminus of the antigen enabled the production of a vaccine with a single copy of the antigen, as confirmed by LC-MS assay. MD simulations performed using this conjugate showed that CRM was effective even with the addition of the antigen. 197 This demonstrates that the three-dimensional structure is preserved and that this determinant is exposed to the solvent, which is essential for recognition by immune system cells.

[0205] The vaccine was tested in mice, and a remarkable immune response was induced in vivo, mainly producing IgG2b antibodies. We also inoculated human colon adenocarcinoma cells into mice, and in the mice treated with the vaccine, a reduction in tumor size was observed compared to the mice exposed to the negative control. Additionally, we found that this vaccine plays a crucial role in stimulating the production of T cells and promoting a potent cytotoxic T lymphocyte (CTL) response that effectively suppresses the growth of the inoculated tumors in mice. Notably, this vaccine also demonstrated excellent therapeutic efficacy against this type of cancer, extending the survival period of the mice treated with the vaccine after tumor inoculation. Moreover, this vaccine was also shown to significantly delay tumor growth and increase the survival rate in both mouse models of colon adenocarcinoma and pancreatic cancer. Importantly, its efficacy is further amplified by combination with a PD-1 immune checkpoint inhibitor, suggesting a synergistic potential leading to improved survival rates in the mouse model. In addition, the strategy presented in this study can avoid batch-to-batch variations in vaccine production, which is extremely important for the development of a chemically newly defined and effective glycopeptide-based cancer vaccine. Finally, despite the conventional doctrine that glycoconjugate vaccines need to present multiple copies of antigens on protein carriers, our study has demonstrated that the use of a single copy of a non-natural and potent antigen precisely presented on a protein carrier can induce a strong immune response in vivo.

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Stefanetti et al., Sugar-Protein Connectivity Impacts on the Immunogenicity of Site-Selective Salmonella O-Antigen Glycoconjugate Vaccines. Angew. Chem. Int. Ed. 54, 13198-13203 (2015). 32. Q.-Y. Hu, F. Berti, R. Adamo, Towards the next generation of biomedicines by site-selective conjugation. Chem. Soc. Rev. 45, 1691-1719 (2016). 33. K. B. Horwitz, M. B. Mockus, B. A. Lessey, Variant T47D human breast cancer cells with high progesterone-receptor levels despite estrogen and antiestrogen resistance. Cell 28, 633-642 (1982). 34. V. M. Kavsan, A. V. Iershov, O. V. Balynska, Immortalized cells and one oncogene in malignant transformation: old insights on new explanation. BMC Cell Biol. 12, 23 (2011). 35. J. Akagi et al., Therapeutic Antitumor Response After Immunization with an Admixture of Recombinant Vaccinia Viruses Expressing a Modified MUC1 Gene and the Murine T-Cell Costimulatory Molecule B7. J. Immunother. 20, 38-39 (1997). 36. Case DA, Belfon K, Ben-Shalom IY, Brozell SR, Cerutti DS, Cheatham TE, Cruzeiro VWD, Darden TA, Duke RE, Giambasu G, Gilson MK, Gohlke H, Goetz AW, Harris R, Izadi S, Iz- mailov SA, Kasavajhala K, Kovalenko A, Krasny R, Kurtzman T, Lee TS, LeGrand S, Li, C, Liu, J, Luchko, T, Luo, V, Man, KM, Merz, Y, Miao, O, Mikhailovskii, G, Monard, H, Nguyen, A, Onufriev, F, Pan, S, Pantano, R, Qi, DR, Roe, A, Roitberg; Sagui C, Schott-Verdugo S, Shen J, Simmerling CL, Skrynnikov NR, Smith J, Swails J, Walker RC, Wang J, Wilson L, Wolf RM, Wu X, Xiong Y, Xue Y, York DM and Kollman PA (2020). 37. JA Maier et al., ff14SB: Improving the Accuracy of Protein Side Chain and Backbone Parameters from ff99SB. J. Chem. Theory Comput. 11, 3696–3713 (2015). 38. J. Wang, RM Wolf, JW Caldwell, PA Kollman, DA Case, Development and testing of a general amber force field. J. Comput. Chem. 25, 1157-1174 (2004). 39. WL Jorgensen, J. Chandrasekhar, JD Madura, RW Impey, ML Klein, Comparison of simple potential functions for simulating liquid water. J. Chem. Phys. 79, 926-935 (1983). 40. HC Andersen, Molecular dynamics simulations at constant pressure and / or temperature. J. Chem. Phys. 72, 2384-2393 (1980). 41. S. Miyamoto, PA Kollman, Settle: An analytical version of the SHAKE and RATTLE algorithm for rigid water models. J. Comp. Chem. 13, 952-962 (1992). 42. T. Darden, D. York, L. Pedersen, Particle mesh Ewald: An N log(N) method for Ewald sums in large systems. J. of Chem. Phys. 98, 10089-10092 (1993). [Sequence List]

[0207] AX1DX2RP Here, X1 is 4S-4-fluoro-L-proline and X2 is S-(α-D-GalNAc)-thiothreonine (SEQ ID NO: 1). HGVTSAX1DX2RPAPGSTAPPA Here, X1 is 4S-4-fluoro-L-proline and X2 is S-(α-D-GalNAc)-thiothreonine (SEQ ID NO: 2). GADDVVDSSKSFVMENFSSYHGTKPGYVDSIQKGIQKPKSGTQGNYDDDWKEFYSTDNKYDAAGYSVDNENPLSGKAGGVVKVTYPGLTKVLALKVDNAETIKKELGLSLTEPLMEQVGTEEFIKRFGDGASR VVLSLPFAEGSSSVEYINNWEQAKALSVELEINFETRGKRGQDAMYEYMAQACAGNRVRRSVGSSLSCINLDWDVIRDKTKTKIESLKEHGPIKNKMSESPNKTVSEEKAKQYLEEFHQTALEHPELSELKTVT GTNPVFAGANYAAWAVNVAQVIDSETADNLEKTTAALSILPGIGSVMGIADGAVHHNTEEIVAQSIALSSLMVAQAIPLVGELVDIGFAAYNFVESIINLFQVVHNSYNRPAYSPGHKTQPFLHDGYAVSWNTV EDSIIRTGFQGESGHDIKITAENTPLPIAGVLLPTIPGKLDVNKSKTHISVNGRKIRMRCRAIDGDVTFCRPKSPVYVGNGVHANLHVAFHRSSSEKIHSNEISSDSIGVLGYQKTVDHTKVNSKLSLFFEIKS Sequence ID 3 - CRM 197 amino acid sequence

Claims

1. CRM １９７ Carrier proteins, Immunogenic peptides, The immunogenic peptide is used in the CRM １９７ A linker that is covalently linked to residues C186 and C201 of the support, Cancer vaccine conjugates containing the same, as well as salts, solvates, and protected forms thereof.

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

3. The cancer vaccine conjugate according to claim 1 or 2, wherein the immunogenic peptide comprises a tumor-associated antigen (TAA).

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

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

6. The cancer vaccine conjugate according to any one of claims 1 to 5, wherein the tumor-associated glycan antigen (TACA) is GalNAc, Galb(1,3)GalNAc, or Neu5Acα(2-6)GalNAc.

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

8. The 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 a Se-glycosidic bond.

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

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

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

12. The immunogenic glycopeptide has the amino acid sequence AX １ DX ２ Including RP, here, X １ is 4S-4-fluoro-L-proline, and X ２ The cancer vaccine conjugate according to claim 11, wherein is S-(α-D-GalNAc)-thiothreonine (SEQ ID NO: 1) or a variant thereof.

13. The immunogenic glycopeptide has the amino acid sequence HGVTSAX １ DX ２ contains RPAPGSTAPP, where X １ is 4S-4-fluoro-L-proline, X ２ is S-(α-D-GalNAc)-thioserine (SEQ ID NO: 2) or a variant thereof, the cancer vaccine conjugate according to claim 12.

14. The CRM １９７ The cancer vaccine conjugate according to any one of claims 1 to 13, wherein the carrier protein comprises the amino acid sequence of SEQ ID NO: 3 or a variant thereof.

15. The linker is C １ ~C ２０ A cancer vaccine conjugate according to any one of claims 1 to 14, comprising one or more groups selected from alkylene, (poly)ethylene glycol moiety, alkylenediamine moiety, amino acid residues, and combinations thereof.

16. The aforementioned linker is, formula: (In the formula; n is an integer selected from 1 to 4; m is an integer selected from 2 to 8; The linker is the CRM １９７ The CRM attached to the carrier protein １９７ These are residues C186 and C201 of the carrier protein; and, The cancer vaccine conjugate according to any one of claims 1 to 15, wherein the linker is a base (indicating the position where the linker is attached to the immunogenic glycopeptide).

17. A method for manufacturing cancer vaccine conjugates, CRM covalently connected to the first connector １９７ To provide a carrier protein, The first connector is CRM １９７ It is covalently linked to residues C186 and C201 of the carrier protein and contains a first reactive group; To provide an immunogenic glycopeptide covalently linked to a second connector, Furthermore, the second connector includes a second reactive group; The first reactant and the second reactant are reacted to covalently link the first connector and the second connector, and the immunogenic glycopeptide is used in the CRM １９７ To attach it to a carrier protein, thereby producing the cancer vaccine conjugate, Methods that include...

18. The first reactive group and the second reactive group are paired as follows: i) Azid and Alkyn; ii) Ketones or aldehydes and alkoxyamines (e.g., hydroxylamines) or hydrazines; iii) Amines and acyl halides or carboxylic acids; iv) electron-rich dienophiles (e.g., 1,3-nitrone alkenes) and electron-deficient dienes (e.g., tetrazine); and, v) Distorted alkenes or alkynes (e.g., norbornene or cyclooctyne) and tetrazine The method according to claim 17, selected from the following.

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

20. The CRM １９７ The first connector, covalently linked to the carrier protein, is given by formula: (In the formula: n is an integer from 1 to 4, preferably 2; and, The linker is the CRM １９７ The CRM attached to the carrier protein １９７ The method according to any one of claims 17 to 19, wherein the group is the residues C186 and C201 of the carrier protein.

21. The second connector, covalently linked to the immunogenic glycopeptide, is given by formula: (In the formula: m is an integer between 2 and 8, preferably 4; and, The method according to any one of claims 17 to 20, wherein the linker is a group of (indicating the position where the linker is attached to the immunogenic glycopeptide (e.g., MUC-1 glycopeptide).

22. The CRM １９７ Carrier proteins, CRM １９７ To provide a carrier protein, The CRM １９７ The C186-C201 disulfide bond of the carrier protein is selectively reduced, and the CRM １９７ To generate free thiol groups at residues C186 and C201 of the carrier protein, To provide a first connector compound comprising a first reactive group, a third reactive group, and a fourth reactive group, The third and fourth reactive groups of the first connector are reacted with the free thiol groups of C186 and C201, respectively, to form the first connector into the CRM １９７ The residues C186 and C201 of the carrier protein are covalently linked, thereby the CRM １９７ The carrier protein is covalently linked to the first connector, The method according to any one of claims 17 to 21, comprising covalently connecting to the first connector by a method including the above.

23. The method according to claim 22, wherein the third reactive group and the fourth reactive group are halogen groups such as chloride groups.

24. The first connector compound is given by formula: The method according to claim 22 or 23, wherein the compound is an integer selected from 1 to 4 (for example, 2).

25. A kit for manufacturing cancer vaccine conjugates, CRM covalently connected to the first connector １９７ Carrier protein This includes, where the first connector is CRM １９７ A kit comprising a first reactive group covalently linked to residues C186 and C201 of a carrier protein.

26. The CRM １９７ The kit according to claim 25, wherein the 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. The pharmaceutical composition according to claim 27, further comprising an adjuvant.

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

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

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

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

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

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

36. The method according to any one of claims 29 or 32-35, wherein the cancer vaccine conjugate is administered for the therapeutic treatment of cancer in the individual; the cancer vaccine conjugate or pharmaceutical composition for use according to any one of claims 30 or 32-35; or the use according to any one of claims 31-35.

37. The method according to any one of claims 29 or 32-35, wherein the cancer vaccine conjugate is administered for the prophylactic treatment of cancer in the individual; the cancer vaccine conjugate or pharmaceutical composition for use according to any one of claims 30 or 32-35; or the use according to any one of claims 31-35.

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