Novel glycoconjugates as vaccines and diagnostic tools

Abstract

Novel glycoconjugates as vaccines and diagnostic tools are described herein. The novel glycoconjugates are produced by conjugating carbohydrate antigen intermediates to free amine groups of a carrier material (e.g., a carrier protein). The intermediate comprises a linker having a first end and a second end, the first end being conjugated to a carbohydrate antigen via a thioether bond, and the second end comprising a functional group capable of reacting with a free amine group. Upon coupling, the carbohydrate antigen is covalently bound to the carrier material via an amide, carbamate, sulfamoyl, urea or thiourea bond to produce a new glycoconjugate. The use of the novel glycoconjugates as antigens, immunogens, vaccines and in diagnostics is also described. In particular, the use of (novel) glycoconjugates as vaccine candidates and other therapeutic tools to combat cancers, viruses such as SARS-CoV-2 and other diseases characterized by aberrant glycosylation expression is also described.

Description

[0001] This application is a divisional application of the application filed on September 18, 2020, with application number 202080081353.2 and invention title "Novel Glycoconjugates as Vaccines and Diagnostic Tools". Technical Field

[0002] This specification relates to novel glycoconjugates that can be used as immunogens and as therapeutic / diagnostic tools. More specifically, this specification relates to conjugations between carbohydrate antigens and free amine groups of carrier materials (e.g., immunogenic and antigenic carrier peptides and proteins). This specification also relates to improved methods for generating novel glycoconjugates for use as antigens, immunogens, vaccines, and in diagnostics. Furthermore, this specification relates to glycoconjugates as vaccine candidates and other therapeutic tools against cancers and viruses (e.g., SARS-CoV-2) associated with aberrant glycosylation.

[0003] This specification references several documents, the contents of which are incorporated herein by reference in their entirety. Background Technology

[0004] The ultimate goal of immunotherapy is to treat diseases such as infections or cancers by modulating the innate and adaptive responses of the immune system to improve its ability to fight foreign substances such as bacteria, viruses, and cancer cells. Innate immunity is considered the first line of immune defense triggered in the early stages of exposure to pathogens. Cells involved include natural killer (NK) cells, dendritic cells (DCs), macrophages, monocytes, γδ T cells, and natural killer T (NKT) cells. Unlike the innate immune system, adaptive immunity develops more slowly upon initial exposure to foreign antigens but produces a highly specific response and generates immune memory to provide long-term protection. It involves clonal expansion of T cells and B cells, as well as their humoral and cellular regulatory factors, cytokines, and antibodies. The main interface between the innate and adaptive immune responses is professional antigen-presenting cells (pAPCs); macrophages, B cells, and especially dendritic cells (DCs). pAPCs are able to process endogenous and exogenous antigens and present them to T cells. They recognize microorganisms through pattern recognition receptors (PRRs) such as Toll-like receptors (TLRs). After recognizing microbial surface determinants or aberrations and non-natural antigens, microorganisms or tumors and their associated antigenic markers can be phagocytosed by pAPCs via endocytosis. In endocytosis, these markers are typically degraded into peptide fragments, and the released antigens bind to intracellular MHC class I or II molecules (pMHC). pAPCs undergo maturation and activation, leading to the redistribution of pMHC complexes from intracellular compartments to the cell surface, secretion of cytokines and chemokines. Apart from pAPCs, all nucleated cell types display endogenous peptides only on the cell membrane. In contrast to pAPCs, these peptides, derived from within the cell itself (including viruses and intracellular pathogens), are displayed by MHC class I molecules coupled to β2-microglobulins. APCs typically do not express MHC class II.

[0005] Peptides displayed on MHC class II molecules are typically recognized by T cell antigen receptors (TCRs) on CD4+ T helper cells. These cells, upon receiving co-stimulatory signals from pAPCs, subsequently undergo functional maturation into distinct subsets, such as Th1 or Th2 cells. Th1 cells lead the primary pro-inflammatory response through the secretion of IFN-γ and TNF-α, while Th2 cells secrete typical cytokines. Although Th1 cells are primarily associated with cell-mediated responses, both types of Th cells support B cell antibody production, thereby influencing antibody isotypes and function. For example, IL-12 and TNF-α are involved in Th1 cell differentiation and the production of type 1 IgG subclasses, while IL-6 and other Th2 cytokines contribute to the production of type 2 IgG subclasses (IgG1).

[0006] APCs displaying peptides on MHC class I molecules are recognized by the TCRs of CD8+ cytotoxic T cells. Additional interactions between co-stimulatory molecules expressed by both cell types trigger the activation of cytotoxic T cells into effector cells, while strong and durable memory T cells are generated through the interaction of dendritic cells with both activated helper T cells and cytotoxic T cells. Once activated, T cells undergo clonal selection and expansion with the aid of cytokines. This increases the number of cells specific to dysregulated target antigens, which can then migrate throughout the body to seek out dysregulated antigen-positive somatic cells. Upon docking with target cells, activated cytotoxic T cells release a payload of cytotoxins, such as perforin and granzymes. Through the action of perforin, granzymes penetrate the target cell, and their proteases induce cell death. Cytotoxic T cells can also trigger target cell death via the FAS signaling pathway. Therefore, it is desirable to modulate vaccine-induced immunity into an appropriate response to treat target pathogens or tumor antigens.

[0007] Unlike proteins and peptides, carbohydrates are T-cell-independent antigens that do not properly trigger Th cell involvement and therefore cannot induce immune cell proliferation, antibody class switching, or affinity / specificity maturation. The major early advances initially encountered by carbohydrate-based vaccines were supported by the finding that bacterial capsular polysaccharides, as T-cell-dependent epitopes, can acquire the immunochemical capacity required to generate opsonized phagocytic antibodies when properly conjugated to carrier proteins.

[0008] Traditionally, strategies for conjugating carbohydrate antigens to carrier proteins rely on the reductive amination of aldehyde-derived sugars at the ε-amino group of lysine residues, or simple amide coupling reactions. In both cases, partial and random carbohydrate antigen conjugation typically occurs. Furthermore, if all amide conjugates (the amine of lysine or the acid of glutamic acid / aspartic acid) are used for carbohydrate conjugation, too much carbohydrate antigen attaches to the carrier protein, thus masking potential essential T-cell peptide epitopes and inherently reducing / eliminating immunogenicity. Therefore, current strategies for preparing glycoconjugated vaccines are inadequate and face significant regulatory and / or commercial barriers because these formulations lack the necessary homogeneity in their carbohydrate distribution and reproducibility (i.e., the sugar attachment sites on the protein are randomly distributed and vary in density between batches). Therefore, there is a strong need for glycoconjugated vaccines with higher carbohydrate antigen homogeneity, more precisely characterized structures, and batch-to-batch reproducibility.

[0009] Initial vaccine development efforts for SARS-CoV-2 primarily focused on the protein antigens and epitopes present on the spike (S) glycoprotein, which mediate SARS-CoV-2 entry into host cells and membrane fusion. However, global health experts strongly recommend that scientists simultaneously explore different strategies to develop therapeutic interventions against SARS-CoV-2 to mitigate the potential failures or complications of single-strategy approaches. Therefore, while focusing on the protein antigens present on the SARS-CoV-2 S protein, it remains necessary to develop vaccines and other therapeutic tools against SARS-CoV-2. Summary of the Invention

[0010] In a first aspect, this document describes a method for generating novel glycoconjugates, the method comprising: (a) providing a novel carbohydrate antigen or a novel carbohydrate antigen intermediate comprising a linker having a first end and a second end, wherein the first end is conjugated to the carbohydrate antigen via a thioether bond, and the second end comprises a functional group capable of reacting with a free amine group, said functional group being COX, SO2X, OC(O)X, N=C=O, or N=C=S, wherein X is a leaving group; (b) providing a carrier material (e.g., a carrier protein or peptide) having one or more free amine groups; and (c) performing a coupling reaction to conjugate one or more purified said novel carbohydrate antigens or novel carbohydrate antigen intermediates to said one or more free amine groups of said carrier material (e.g., a carrier protein or peptide) via an amide, carbamate, aminosulfonyl, urea, or thiourea bond, thereby generating novel glycoconjugates.

[0011] In some embodiments, prior to step (a), the novel carbohydrate antigen or novel carbohydrate antigen intermediate in (a) is prepared by a method comprising: (i) providing a carbohydrate antigen (alkenyl carbohydrate antigen) covalently linked to a terminal alkene, the terminal alkene being directly conjugated to a thiol group via a thiol-alkene reaction; (ii) providing a thiol-containing linker comprising a first functional group at a first end and a second functional group at a second end, the first functional group being a free thiol group and the second functional group being a carboxyl group, a sulfinic acid group, a carbonate group, an isocyanate group, or a thiocyanate group; (iii) performing a photocatalytic thiol-alkene reaction to directly conjugate the alkenyl carbohydrate antigen to the first end of the thiol-containing linker, thereby producing a novel carbohydrate antigen comprising the carbohydrate antigen at the first end and the second functional group at the second end; (iv) When the second functional group is a carboxyl group, a sulfinic acid group, or a carbonate group, the novel carbohydrate antigen is converted into a novel carbohydrate antigen intermediate by replacing the terminal hydroxyl group of the carboxyl group, sulfinic acid group, or carbonate group with a better leaving group for conjugation with the free amine group of the polypeptide; and (v) purifying the novel carbohydrate antigen or the novel carbohydrate antigen intermediate.

[0012] On the other hand, this article describes novel carbohydrate antigens or novel carbohydrate antigen intermediates comprising a linker having a first end and a second end, wherein the first end is conjugated to the carbohydrate antigen via a thioether bond, and the second end comprises a functional group capable of reacting with a free amine group, wherein the functional group is COX, SO2X, OC(O)X, N=C=O, or N=C=S, where X is a leaving group.

[0013] On the other hand, this article describes the synthesis of novel glycoconjugates comprising a connector having a first end and a second end, wherein the first end is conjugated to a carbohydrate antigen via a thioether bond, and the second end is conjugated to one or more free amine groups in a carrier protein or peptide via an amide, carbamate, sulfamyl, urea, or thiourea bond.

[0014] On the other hand, this article describes synthetic novel glycoconjugates comprising one or more carbohydrate antigens (CAs) conjugated to one or more amine groups of a carrier protein or peptide (CP-NH) via a linker, said synthetic novel glycoconjugates having the following structure:

[0015] Where: X is O, S, NR1, or CH2; R1 is H, COH (formamide), COME, or COEt; m is 1, 2, 3, 4, or 5; Y is -(CH2). n -or-(OCH2CH2O) n-; n is 0, 1, 2, 3, 4, or 5; o is 0, 1, 2, 3, 4, or 5; or o is 0 and Z is -CO- and Y is -(OCH2CH2O) n -; or o is 0 and Z is -SO2- and Y is -(OCH2CH2O). n -; Z is -CO-, -NR2SO2-, -OCO-, -NR2CO-, or -NR2CS-; R2 is H, Me, or Et; and p is an integer (e.g., p = 1 to 50) corresponding to the total number of carbohydrate antigens conjugated to one or more amine groups of the carrier protein or peptide. In embodiments, the carrier protein or peptide in the synthetic novel glycoconjugate has a native or non-denatured conformation, and the conjugation of the carbohydrate antigen to the carrier protein or peptide increases the immunogenicity of the carbohydrate antigen after administration to a subject compared to the corresponding administration of the unconjugated carbohydrate antigen.

[0016] On the other hand, this document describes a method for generating novel glycoconjugate vaccines or immune response-triggered compositions. The method may include formulating a novel glycoconjugate as described herein or prepared by methods described herein with pharmaceutically acceptable excipients and / or adjuvants.

[0017] On the other hand, this document describes novel glycoconjugate vaccines or adaptive immune response triggering compositions that, through the methods described herein, produce and / or contain novel glycoconjugates as described herein and pharmaceutically acceptable excipients and / or adjuvants as described herein.

[0018] In some respects, this document describes a method for immunizing, vaccinating, or treating a subject, including administering to the subject a novel glycoconjugate produced by the method described herein, a synthetic novel glycoconjugate as described herein, a novel glycoconjugate vaccine or adaptive immune response triggering composition produced by the method described herein, or a novel glycoconjugate vaccine as described herein.

[0019] In some embodiments, this document describes novel glycoconjugates produced by methods as described herein, synthetic novel glycoconjugates as described herein, novel glycoconjugate vaccines or adaptive immune response triggering compositions produced by methods as described herein, or novel glycoconjugate vaccines as described herein, for use in immunization, vaccination or treatment of a subject with a disease, or for detecting the presence of antibodies that specifically bind to said novel glycoconjugate or for detecting said immunization, vaccination or treatment (e.g., in a biological sample from said subject).

[0020] In some embodiments, this document describes novel glycoconjugates produced as described herein, synthetic novel glycoconjugates as described herein, or adaptive immune response triggering compositions produced as described herein, for use in manufacturing vaccines for immunizing or treating subjects with a disease, or for detecting the presence of antibodies that specifically bind to said novel glycoconjugates, or for detecting said immunization or treatment (e.g., in biological samples from said subjects).

[0021] In some embodiments, this document describes novel glycoconjugates produced by methods as described herein, synthetic novel glycoconjugates as described herein, novel glycoconjugate vaccines or adaptive immune response triggering compositions produced by methods as described herein, or novel glycoconjugate vaccines as described herein, for use in treating subjects with a disease associated with increased expression of the said carbohydrate antigens.

[0022] In some embodiments, this document describes novel glycoconjugates produced by methods as described herein, synthetic novel glycoconjugates as described herein, novel glycoconjugate vaccines or adaptive immune response triggering compositions produced by methods as described herein, or novel glycoconjugate vaccines as described herein, for detecting or screening the presence of antibodies that specifically bind to the carbohydrate antigen or circulating tumor cells containing the carbohydrate antigen, or for detecting the presence of antibodies generated by immunization or vaccination with the carbohydrate antigen.

[0023] In other aspects, this document describes methods for treating subjects, including administering a novel glycoconjugate or novel glycoconjugate immunogen as defined herein or generated by methods described herein to generate an immune response against a carbohydrate antigen in the subject, and optionally screening for the presence of antibodies that specifically bind to the carbohydrate antigen in biological samples from the subject.

[0024] In other aspects, this document describes glycoconjugates for use in immunizing a subject against SARS-CoV-2, for triggering the production of anti-SARS-CoV-2 antibodies in a subject, or for detecting the presence of anti-SARS-CoV-2 antibodies in a sample from a subject, said glycoconjugates comprising a carbohydrate antigen conjugated to a suitable carrier material (e.g., a carrier protein or peptide), said carbohydrate antigen comprising, or consisting of, sialylated Thomsen-Friedenreich (TF) antigen, non-sialylated TF antigen, sialylated Tn antigen, non-sialylated Tn antigen, or any combination thereof.

[0025] In other respects, this article describes SARS-CoV-2 vaccines comprising one or more glycoconjugates as described herein, and pharmaceutically acceptable excipients and / or adjuvants.

[0026] In other respects, this article describes methods for vaccinating subjects against SARS-CoV-2 or for triggering the production of anti-SARS-CoV-2 antibodies in subjects, the methods including administering glycoconjugates or SARS-CoV-2 vaccines as described herein.

[0027] In other respects, this article describes compositions for protecting subjects from SARS-CoV-2 virus infection or for treating COVID-19, the compositions comprising one or more ligands (e.g., antibodies, antibody fragments, or lectins) that bind to O-linked glycans expressed on the SARS-CoV-2 S protein, the O-linked glycans comprising sialylated TF antigens (monosialized or disialylated TF antigens), nonsialylated TF antigens, sialylated Tn antigens, nonsialylated Tn antigens, or any combination thereof.

[0028] In other aspects, this document describes a complex comprising: (a) a SARS-CoV-2 S protein or a fragment thereof expressing an O-linked glycan comprising sialylated TF antigen (monosialized or disialylated TF antigen), nonsialylated TF antigen, sialylated Tn antigen, nonsialylated Tn antigen, or any combination thereof; and (b) a ligand as described herein, said ligand binding to said O-linked glycan of said SARS-CoV-2 S protein or a fragment thereof.

[0029] General definition The use of headings and other identifiers, such as (a), (b), (i), (ii), etc., is solely for the purpose of facilitating the reading of the specification and claims. The use of headings or other identifiers in the specification or claims does not necessarily require that the steps or elements be performed in alphabetical or numerical order or in the order in which they are presented.

[0030] When used in conjunction with the term “comprising” in the claims and / or specification, the word “a” or “an” may mean “one” or “a type”, but it is also consistent with the meaning of “one or more types”, “at least one type” and “one or more types”.

[0031] The term "about" is used to indicate the standard deviation of a value, including the error of the equipment or method used to determine that value. Generally, the term "about" refers to a possible variation of up to 10%. Therefore, variations of 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, and 10% of a value are included in the term "about." Unless otherwise stated, the use of the term "about" before a range applies to both ends of the range.

[0032] As used in this specification and claims, the terms “comprising” (and any form of inclusion, such as “comprise” and “comprises”), “having” (and any form of having, such as “have” and “has”), “including” (and any form of inclusion, such as “includes” and “include”), or “containing” (and any form of containing, such as “contains” and “contain”) are inclusive or open-ended and do not exclude additional, unlisted elements or method steps.

[0033] As used herein, the term "protein" (e.g., in the expression "carrier protein") refers to any amino acid chain linked by a peptide, which may or may not contain any type of modification (e.g., chemical or post-translational modifications, such as acetylation, phosphorylation, glycosylation, sulfation, SUMOylation, isopentenylation, ubiquitination, etc.) as long as the modification does not impair the immunogenicity of the novel glycoconjugate immunogen and the novel glycoconjugate vaccine described herein. For further clarity, the terms "protein" and "carrier protein" as used herein encompass both peptides and polypeptides, although these two embodiments may be described together, for example, in the expression "carrier protein or peptide".

[0034] As used herein, the term "novel glycoconjugate" refers to a carbohydrate antigen (e.g., an antigenic monosaccharide, disaccharide, oligosaccharide, or polysaccharide, preferably a natural antigen) conjugated to a carrier protein or peptide to enhance the immunogenicity of the carbohydrate antigen in a target subject. The expressions "carbohydrate antigen" and "glycoantigen" have the same meaning as used herein. The term "immunogen" refers to a substance (agent) capable of being specifically bound by components of the immune system (e.g., by antibodies and / or lymphocytes) and generating a humoral and / or cell-mediated immune response in a target subject. As used herein, the term "immunogen" in expressions such as "novel glycoconjugate immunogen" refers to the ability (i.e., physical characteristics or properties) of the novel glycoconjugate, without limiting the novel glycoconjugate itself to a particular use (e.g., as an immunogen for generating an immune response in a subject). For example, in some embodiments, the novel glycoconjugate immunogen described herein can be used in diagnostic assays or methods (e.g., in vitro methods) to detect the presence or absence of antibodies that bind to the novel glycoconjugate immunogen in a biological sample (e.g., from a subject). In some implementations, the novel glycoconjugate immunogens described herein can be used to screen, identify, or evaluate antibodies that specifically bind to the novel glycoconjugate immunogen (e.g., monoclonal antibodies suitable for diagnosis or treatment).

[0035] As used in this article, the term "synthetic" refers to a compound that is not a natural product but is produced through human intervention.

[0036] As used herein, the term "conjugable" refers to the ability or capacity of at least two molecules (e.g., a carbohydrate antigen and a thiol-containing linker; or a novel carbohydrate antigen and a carrier protein or peptide) to covalently bond to each other through a chemical reaction, regardless of whether the molecules are actually covalently bonded. In contrast, the term "conjugated" refers to at least two molecules covalently bonded to each other (e.g., a carbohydrate antigen and a thiol-containing linker, or a novel carbohydrate antigen and a carrier protein or peptide).

[0037] As used herein, the term “administration” can include routes of administration such as parenteral (e.g., subcutaneous, intradermal, intramuscular, or intravenous), oral, transdermal, or intranasal, as long as the route of administration results in an immune response in the subject.

[0038] As used herein, the term "subject" generally refers to an organism (e.g., an animal or human) capable of generating an immune response to the novel glycoconjugate described herein, preferably resulting in the production of antibodies and / or lymphocytes and / or cells that specifically bind to the novel glycoconjugate. In some embodiments, the subject described herein may be a patient seeking therapeutic treatment (e.g., through vaccination with the novel glycoconjugate immunogen described herein) or a means that can be used to generate tools (e.g., antibodies) for research, diagnostic, and / or therapeutic purposes.

[0039] Other objects, advantages, and features of this specification will become more apparent upon reading the following non-limiting description of specific embodiments given by way of example only with reference to the accompanying drawings. Attached Figure Description

[0040] In the attached diagram: Figure 1 Examples of typical chemical structures of key tumor-associated carbohydrate antigens (TACAs) (e.g., derived from human mucin glycoproteins (MUCs)) are shown.

[0041] Figure 2 The reaction protocols for synthesizing allyl Tn antigen and allyl TF antigen intermediates as described in Examples 2-6 are shown.

[0042] Figure 3 shows allyl 2-acetamido-3,6-di-O-neovaleroyl-2-deoxy-α-D-glucopyranoside (compound 2). 1 H-NMR ( Figure 3A )and 13 C-NMR ( Figure 3B ) spectral and mass spectrometry results ( Figure 3C and 3D ).

[0043] Figures 4A-4D This shows that allyl 2-acetamido-2-deoxy-α-D-galactopyranoside (allylTn) 1 H-NMR ( Figure 4A )and 13 C-NMR ( Figure 4B ) spectral and mass spectrometry results ( Figure 4C and 4D ).

[0044] Figures 5A-5C The product shown is allyl 2-acetamido-4,6-O-benzyl-2-deoxy-α-D-galactopyranoside (compound 4). 1 H-NMR ( Figure 5A ) spectral and mass spectrometry results ( Figure 5B and 6C ).

[0045] Figures 6A-6D The product shown is allyl(2,3,4,6-tetra-O-benzoyl-β-D-galactopyranosyl)-(1→3)-2-acetamido-4,6-O-benzylidene-2-deoxy-α-D-galactopyranoside (compound 6). 1 H-NMR ( Figure 6A )and 13 C-NMR ( Figure 6B ) spectral and mass spectrometry results ( Figure 6C and6D ).

[0046] Figures 7A-7D Compound 7 was shown. 1 H-NMR ( Figure 7A )and 13 C-NMR ( Figure 7B ) spectral and mass spectrometry results ( Figure 7C and 7D ).

[0047] Figures 8A-8D It shows allyl(β-D-galactopyranosyl)-(1→3)-2-acetamido-2-deoxy-α-D-galactopyranoside (allyl TF). 1 H-NMR ( Figure 8A )and 13 C-NMR ( Figure 8B ) spectral and mass spectrometry results ( Figure 8C and 8D ).

[0048] Figure 9 The reaction protocol for generating a novel glycoconjugate immunogen from allyl Tn is shown.

[0049] Figure 10 Compound 8 was shown. 1 H-NMR and 13 C-NMR spectral results.

[0050] Figure 11A-11B Compound 9 was shown. 1 H-NMR and 13 C-NMR ( Figure 11A ) Spectroscopy and mass spectrometry (LC-MS) results ( Figure 11B ).

[0051] Figure 12 The reaction protocol for generating a novel glycoconjugate immunogen from allyl Tn is shown.

[0052] Figure 13 Compound 12 was shown. 1 H-NMR and 13 C-NMR spectra.

[0053] Figure 14 Compound 13 was shown 1 H-NMR and 13 C-NMR spectra.

[0054] Figures 15A-15E The results showed that Tn recognizes the lectin in Vitex longifolia (Vigna pubescens). Vicia Villosa (VVA) for BSA-Tn( Figures 15A-15D ) and CRM197-Tn ( Figure 15E The reactivity of the conjugate. Figure 15A Western blots were displayed, showing different BSA-Tn conjugates generated in buffer solutions ranging from pH 6 to 10. Figure 15B The corresponding ELISA analysis is shown in the image. Figure 15C This is a protein blot showing the titration of PFP-Tn (compound 9) when conjugated with BSA at the optimal pH 8. Figure 15D The corresponding ELISA analysis is shown in the image. Figure 15E Western blot analysis of CRM197-Tn is shown. For the three conjugation ratios of PFP-Tn tested (i.e., 3.4, 9.7, and 15), the blot showed a VVA reaction band within the molecular weight range of CRM197 (approximately 58.4 kDa), with the most reactive of the three producing the highest PFP-Tn ratio at 15 equivalents.

[0055] Figure 16A and 16B The reactivity of the BSA-TF conjugate to the TF-specific lectin peanut agglutinin (PNA) and the dosage of galactose derived from the conjugated disaccharide TF antigen were shown. Figure 16A The protein imprinting showed a major band in response to PNA within the expected molecular weight range of the BSA monomer, indicating the coupling of TF with BSA. Figure 16B The corresponding ELISA analysis using PNA-hrp is shown. The conjugation of TF to BSA was also demonstrated by measuring the galactose associated with BSA using the Dubois method. Figure 16B (Bar chart in the image).

[0056] Figures 17A-17C The conjugation of COOH-Tn and COOH-TF with carrier proteins CRM197 and dTT was shown. Figure 17A The SDS-PAGE gels showing Coomassie staining indicate that the conjugated proteins (“-Tn” and “-TF”) appear to have a slightly increased molecular weight relative to their corresponding unconjugated proteins (“crm” and “dTT”). Figure 17B The corresponding Western blots shown demonstrate the specific reactivity of each -Tn and -TF protein conjugate to VVA and PNA lectins, respectively. Specific reactivity to the same pattern of lectins was also observed by ELISA. Figure 17C ).

[0057] Figure 18 The normalized reactivity of serum from mice immunized three times with dTT-TF to various TF and Tn screening antigens was demonstrated by ELISA.

[0058] Figures 19A-19BThe responsiveness of serum titrations from mice immunized five times with dTT-TF and 6 weeks after the fifth immunization to the screening antigen BSA-TF was shown. Figure 19A Results from mouse "A1" are shown, and Figure 19B The results from mouse "A2" are shown.

[0059] Figure 20 The relative abundance of all O-glycosylated forms of the peptide VQPTESIVR (SEQ ID NO: 3) on the recombinant S1 protein of SARS-CoV-2 is shown, with protein exceeding 75 kDA analyzed by high-resolution LC-MS / MS.

[0060] Figure 21 The relative abundance of all O-glycosylation forms of the peptide VQPTESIVR (SEQ ID NO: 3) on the recombinant S1 protein of SARS-CoV-2 is shown, with protein samples ranging from 75 to 100 kDA analyzed by high-resolution LC-MS / MS.

[0061] Figure 22 The reactivity of anti-TF (JAA-F11 IgG and SPM320 IgM) and anti-Tn (Tn218 IgM) monoclonal antibodies to culture supernatants from mammalian cells transfected with either the SARS-CoV-2 S1 protein or the full-length S protein was demonstrated by ELISA. Results showed a fold increase relative to exposure to the same monoclonal antibodies to culture supernatants from the corresponding mammalian cells transfected with the empty vector.

[0062] Figure 23 The reactivity of a group of lectins to culture supernatants from mammalian cells transfected with either the SARS-CoV-2 S1 protein or the full-length S protein was shown. Results showed a fold increase relative to exposure to the same monoclonal antibody from culture supernatants from the corresponding mammalian cells transfected with an empty vector.

[0063] Figure 24 This study demonstrates the effect of different anticarbohydrate ligands on inhibiting the infectivity of pseudotyped lenti-luc-SARS-CoV-2-S virus on host cells expressing human angiotensin-converting enzyme 2. "PNA" and "AIA" (jac) are lectins with different carbohydrate antigen binding specificities; the latter is known to bind specifically to both sialylated and non-sialylated forms of TF and Tn antigens. "JAA-F11" is a monoclonal antibody that binds only to the non-sialylated form of TF antigen.

[0064] sequence list This application contains a sequence list in a computer-readable form, created on September 17, 2020, which is approximately 12 Kb in size. This computer-readable form is incorporated herein by reference. Detailed Implementation

[0065] This specification relates to novel glycoconjugates applicable to, for example, immunogens, vaccines, diagnostics, or for generating analytical or therapeutic tools (e.g., generating novel antibodies against novel glycoconjugates), and improved methods for generating them.

[0066] The conjugation of carbohydrate antigens ending with terminal acid functional groups to the amine groups of carrier proteins has traditionally been accomplished through random activation using succinimide or carbodiimide reagents. One of the main drawbacks of this carbohydrate antigen-carrier protein conjugation method is the uncontrolled / undesirable self-crosslinking that occurs within the carrier protein itself, where the side chains of aspartic / glutamate residues of the carrier protein are coupled to the ε-amine groups of lysine residues of the carrier protein. This method leads to perturbation or disruption of the native structure of the carrier protein, often resulting in significant loss of key peptide sequences that would otherwise be highly immunogenic, and potentially undesirable crosslinking of the carrier protein. Furthermore, the carbohydrate antigen-carrier protein conjugation methods described in the art often use linkers such as squaric acids, which may trigger an immune response against the linker itself, not just against the carbohydrate antigens they are conjugated to. Additionally, the carbohydrate antigen-carrier protein conjugation methods described in the art do not adequately control the degree of glycosylation of the carrier protein, often resulting in heteroglycoconjugates, a significant obstacle to the production of human therapeutics.

[0067] In contrast, the carbohydrate antigen-carrier protein conjugation strategies described herein differ from those previously described. First, in some embodiments, a carbohydrate antigen with an alkenyl functional group is coupled to a non-immunogenic linker via a reagent-free photolytic thiol-ene reaction to generate the novel carbohydrate antigen described herein; this upstream step does not affect the structure of the carrier protein and peptide. Second, in some embodiments, the novel carbohydrate antigen or its intermediate described herein is terminated with a better leaving group, such as an active ester group, and purified before conjugation to the carrier protein or peptide, thereby improving conjugation efficiency and simultaneously avoiding self-crosslinking within the carrier protein or peptide. Third, the efficacy and stoichiometry of the reaction coupler in the carbohydrate antigen-carrier protein conjugation strategies described herein allow for more precise control of the amount of carbohydrate antigen conjugated to the carrier protein or peptide, thereby avoiding potential masking of key immunogenic peptide sequences.

[0068] In a first aspect, this specification relates to an improved method for generating novel glycoconjugates. The method generally includes providing a carbohydrate antigen (alkenyl carbohydrate antigen) covalently linked to a terminal alkene, the terminal alkene being directly conjugated to a thiol group via a thiol-alkene reaction. The method further includes providing a thiol-containing linker comprising a first functional group at a first end and a second functional group at a second end, the first functional group being a free thiol group and the second functional group being a group such as a carboxyl group, a sulfinic acid group, a carbonate group, an isocyanate group, or a thiocyanate group. A photocatalytic thiol-alkene reaction is then performed to directly conjugate the alkenyl carbohydrate antigen to the first end of the thiol-containing linker, thereby generating a novel carbohydrate antigen comprising the carbohydrate antigen at the first end and the second functional group at the second end. When the second functional group is a carboxyl group, a sulfinic acid group, or a carbonate group, the method herein further includes converting the novel carbohydrate antigen into a novel carbohydrate antigen intermediate by replacing the terminal hydroxyl group of the carboxyl group, sulfinic acid group, or carbonate group with a better leaving group for conjugation to a free amine group of a polypeptide. The novel carbohydrate antigen or intermediate can then be purified and subsequently used for a coupling reaction with a carrier material (e.g., a carrier protein or peptide) having one or more free amine groups. The coupling reaction conjugates one or more purified novel carbohydrate antigens or intermediates to the one or more free amine groups of the carrier material (e.g., via amide, carbamate, sulfanilic, urea, or thiourea bonds), thereby producing a novel glycoconjugate.

[0069] On the other hand, this document describes a method for generating novel glycoconjugates, the method comprising: providing a novel carbohydrate antigen or a novel carbohydrate antigen intermediate comprising a linker having a first end and a second end, wherein the first end is conjugated to the carbohydrate antigen via a thioether bond, and the second end comprises a functional group capable of reacting with a free amine group, said functional group being COX, SO2X, OC(O)X, N=C=O, or N=C=S, wherein X is a leaving group. The method further comprises providing a carrier material (e.g., a carrier protein or peptide) having one or more free amine groups; and performing a coupling reaction to conjugate one or more purified novel carbohydrate antigens or novel carbohydrate antigen intermediates to said one or more free amine groups of said carrier material via an amide, carbamate, sulfanyl, urea, or thiourea bond, thereby generating novel glycoconjugates.

[0070] On the other hand, this document describes a novel carbohydrate antigen or a novel carbohydrate antigen intermediate. In some embodiments, the novel carbohydrate antigen or novel carbohydrate antigen intermediate comprises a linker having a first end and a second end, wherein the first end is conjugated to the carbohydrate antigen via a thioether bond, and the second end comprises a functional group capable of reacting with a free amine group, said functional group being COX, SO2X, OC(O)X, N=C=O, or N=C=S, wherein X is a leaving group.

[0071] On the other hand, this paper describes novel synthetic sugar conjugates comprising a connector having a first end and a second end, wherein the first end is conjugated to a carbohydrate antigen via a thioether bond, and the second end is conjugated to one or more free amine groups in a carrier material (e.g., a carrier protein or peptide) via an amide, carbamate, aminosulfonyl, urea, or thiourea bond.

[0072] On the other hand, this article describes novel synthetic glycoconjugates comprising one or more carbohydrate antigens (CAs) conjugated to one or more amine groups of a carrier protein or peptide (CP-NH) via a linker, the novel synthetic glycoconjugates having the following structure:

[0073] Where: X is O, S, NR1, or CH2; R1 is H, COH (formamide), COME, or COEt; m is 1, 2, 3, 4, or 5; Y is -(CH2). n -or-(OCH2CH2O) n -; n is 0, 1, 2, 3, 4, or 5; o is 0, 1, 2, 3, 4, or 5; or o is 0 and Z is -CO- and Y is -(OCH2CH2O) n -, or o is 0 and Z is -SO2- and Y is -(OCH2CH2O). n -; Z is -CO-, -NR2SO2-, -OCO-, -NR2CO-, or -NR2CS-; R2 is H, Me, or Et; and p is an integer representing the total number of carbohydrate antigens conjugated to one or more amine groups of the carrier protein or peptide.

[0074] On the other hand, this article describes novel synthetic glycoconjugates comprising one or more carbohydrate antigens (CAs) conjugated to one or more amine groups of a carrier protein or peptide (CP-NH) via a linker, the novel synthetic glycoconjugates having the following structure: , Where p is an integer corresponding to the total number of carbohydrate antigens conjugated to one or more amine groups of the carrier protein or peptide.

[0075] In some embodiments, p is an integer of at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, or 50, up to the total number of amine groups available for conjugation on a given carrier protein or peptide (e.g., the total number of lysine residues, such as the total number of solvent-accessible lysine residues).

[0076] In some implementations, the carrier protein or peptide in the synthetic novel glycoconjugate has a native or non-denatured conformation, and the conjugation of the carbohydrate antigen to the carrier protein or peptide increases the immunogenicity of the carbohydrate antigen after administration to the subject, compared to the corresponding administration of the unconjugated carbohydrate antigen.

[0077] In some embodiments, the thiol-ene reaction described herein, which directly conjugates alkenyl carbohydrate antigens to thiol-linked sites, can be carried out under reaction conditions that minimize or avoid disruption or perturbation of the structure and / or antigenicity of the carbohydrate antigen (e.g., which may lead to undesirable immune responses and / or the generation of antibodies against undesirable carbohydrate antigens with perturbed structures).

[0078] In some embodiments, the thiol-ene reaction described herein can be a photocatalytic thiol-ene reaction, including irradiation under ultraviolet light. In some embodiments, the photocatalytic thiol-ene reaction described herein can include irradiation under short-wavelength ultraviolet light (e.g., about 254 nm) or long-wavelength ultraviolet light (e.g., about 355 nm or 365 nm). In some embodiments, the thiol-ene conjugation reaction described herein can have the versatility to be performed under both short-wavelength and long-wavelength ultraviolet light.

[0079] In some embodiments, the thiol-ene reaction described herein can be carried out in the presence of a catalyst. For example, in some embodiments, the photocatalytic thiol-ene reaction described herein can be carried out under long-wave ultraviolet light in the presence of a catalyst, or under short-wave ultraviolet light in the absence of a catalyst, to further simplify the process. In the context of the thiol-ene reaction as used herein, the terms “catalyst,” “photoinitiator,” and “activator” are used interchangeably to refer to substances that accelerate the conjugation of carbohydrate antigens to free thiol groups containing thiol linkers via photocatalytic thiol-ene reaction. In some embodiments, the catalyst may be a water-soluble catalyst, such as a water-soluble radical-generating azo compound; 2,2'-azobis[2-(2-imidazolin-2-yl)propane] dihydrochloride (Vazo 44 or VA-044); 2,2'-azobis(2-amidinylpropane) dihydrochloride (AAPH); lithium phenyl-2,4,6-trimethylbenzoylphosphine (LAP); a metal or metal ion having photoinitiator activity; a peroxide; tert-butyl hydroperoxide; benzoyl peroxide; ammonium persulfate; or any derivative thereof having photoinitiator activity. In some embodiments, the catalyst may be a water-insoluble catalyst, such as a water-insoluble radical-generating azo compound, 2,2-dimethoxy-2-phenylacetophenone (DMPA), azobisisobutyronitrile (AIBN), 2,2'-azobis(2-methylpropionitrile), 4,4'-azobis(4-cyanopentanoic acid) (ACVA), 1,1'-azobis(cyanocyclohexane) (ACHN), diazepine dicarboxylic acid bis( N,N Dimethylamide (TMAD); azodicarboxylic acid dipiperidide (ADD), or any of its derivatives having photoinitiator activity.

[0080] In some embodiments, the photocatalytic thiol-ene reaction described herein may include reacting each free thiol group of the thiol-containing linker with 1 to 200 or 1 to 100 molar equivalents of the alkenyl carbohydrate antigen. In some embodiments, the photocatalytic thiol-ene reaction described herein may be carried out for 10 to 300, 10 to 270, 10 to 240, 10 to 210, 10 to 180, 10 to 150, 10 to 120, 10 to 90, 10 to 60, or 10 to 30 minutes.

[0081] In some embodiments, the photocatalytic thiol-ene reaction described herein can be carried out at a pH between about 3.0, 3.1, 3.2, 3.3, 3.4, 3.5, 3.6, 3.7, 3.8, 3.9, or 4.0 and about 4.5, 4.6, 4.7, 4.8, 4.9, 5.0, 5.5, 6.0, 6.5, 7.0, 7.5, 8.0, 8.5, 9.0, 9.5, or 10. In some embodiments, the photocatalytic thiol-ene reaction described herein can be carried out at a pH that minimizes or avoids disturbance or destruction of carbohydrate antigens.

[0082] In some embodiments, the carbohydrate antigen described herein can be chemically modified to link (directly or indirectly via a linker or spacer) to a terminal alkene (e.g., via a glycosidic bond or via reductive amination between an allylamine or alkenylamine and a reducing sugar, preferably using NaBH4 and / or NaBH3CN), wherein the terminal alkenyl group of the alkenyl carbohydrate antigen can be conjugated to a free thiol-containing linker via a thiol-alkene reaction (e.g., a photocatalytic thiol-alkene reaction). The terminal alkenyl group of the alkenyl carbohydrate antigen can be a monosubstituted alkene, a vinyl group, or an allyl group.

[0083] In some embodiments, the carbohydrate antigens described herein may be covalently linked to the terminal olefin via a glycosidic bond, such as an O-glycosidic bond, S-glycosidic bond, N-glycosidic bond, or C-glycosidic bond, or via a bond obtained, for example, by reductive amination between an allylamine and a reducing sugar (including bacterial CPS). As used herein, a "glycosidic bond" may include one or more of an S-glycosidic bond, an N-glycosidic bond, an O-glycosidic bond, or a C-glycosidic bond, or a bond obtained by reductive amination of a reducing sugar (e.g., using NaBH4 or preferably NaBH3CN). In some embodiments, the glycosidic bond may be a glycosidic bond that cannot be cleaved by the subject's endogenous enzymes (e.g., glycerol hydrolases). Such an uncleavable glycosidic bond can result in a longer half-life of the novel glycoconjugate immunogen after administration to the subject, which in turn can generate a more favorable immune response for therapeutic and / or antibody-generating purposes. In some embodiments, the glycosidic bond can be an S-glycosidic bond, an N-glycosidic bond, or a C-glycosidic bond, or a bond obtained by, for example, reductive amination between allylamine and a reducing sugar.

[0084] In some embodiments, the thiol-containing connector described herein may include the following structure:

[0085] Where: Y is -(CH2) n -or-(OCH2CH2O) n-; Z is -CO2H, -SO2H, -OC(O)-H, -N=C=O, or -N=C=S; O is 0, 1, 2, 3, 4, or 5; O is 0 and Z is -CO- and Y is -(OCH2CH2O). n -; or O is 0 and Z is -SO2- and Y is -(OCH2CH2O). n -

[0086] In some embodiments, the methods described herein and the novel glycoconjugates preferably use unprotected carbohydrate antigens, which improve the water solubility of the carbohydrate antigens themselves and avoid the step of subsequently removing the protecting groups of the carbohydrate antigens. Therefore, in some embodiments, the carbohydrate antigen, alkenyl carbohydrate antigen, novel carbohydrate antigen, novel carbohydrate antigen intermediate, and / or the carbohydrate portion of the novel glycoconjugate remains unprotected throughout the methods described herein.

[0087] In some embodiments, when the second functional group of the novel carbohydrate antigen described herein is a group such as a carboxyl group, a sulfinic acid group, or a carbonate group, the method herein may further include converting the novel carbohydrate antigen into a novel carbohydrate antigen intermediate by replacing the terminal hydroxyl group of the carboxyl group, sulfinic acid group, or carbonate group with a better leaving group for conjugation with the free amine group of the polypeptide. As used herein, a "better leaving group" means a leaving group that provides improved reaction efficiency and / or specificity (i.e., improved conjugation with the free amine group of the polypeptide) compared to the corresponding functional group before replacement with the leaving group. In some embodiments, the leaving group used herein may be an active ester group (e.g., a fluorophenyl group (e.g., OPhF5, OPhF4 (para-SO3Na)), or a succinimide group). The novel carbohydrate antigen intermediate can then be purified and subsequently used for coupling reactions with a carrier protein or peptide having one or more free amine groups. The coupling reaction conjugates one or more purified novel carbohydrate antigen intermediates to one or more free amine groups of the carrier protein or peptide (e.g., via amide, carbamate, sulfamyl, urea, or thiourea bonds) to produce the novel glycoconjugates described herein.

[0088] In some embodiments, the novel carbohydrate antigen-carrier protein coupling reaction described herein can advantageously minimize or avoid self-crosslinking between the side chains of aspartic acid / glutamate residues present in the carrier protein or peptide itself and ε-lysine amine.

[0089] In some embodiments, the novel carbohydrate antigen-carrier protein conjugation reaction described herein can be controlled by efficacy and / or stoichiometry of the reactants (e.g., the molar ratio of the carrier protein or peptide to the novel carbohydrate antigen or the novel carbohydrate antigen intermediate). In some embodiments, the novel carbohydrate antigen-carrier protein conjugation reaction described herein may include reacting each carrier protein or peptide with 1 to 500, 1 to 400, 1 to 300, 1 to 200, 5 to 500, 5 to 400, 5 to 300, or 5 to 200 molar equivalents of the novel carbohydrate antigen or the novel carbohydrate antigen intermediate. In some embodiments, this specification relates to compositions comprising novel glycoconjugate immunogens having about or at least 70%, 75%, 80%, 85%, 90%, or 95% homogeneity in terms of the type of carbohydrate conjugate (e.g., at least 90% of the novel glycoconjugate types / molecules in the composition have the same number of carbohydrate antigens conjugated to the carrier protein).

[0090] In some embodiments, the (novel) carbohydrate antigen described herein may contain B-cell epitopes and / or induce a humoral immune response in a subject. In some embodiments, the (novel) carbohydrate antigen described herein may contain T-cell epitopes and / or induce a cell-mediated immune response in a subject. In some embodiments, the (novel) carbohydrate antigen described herein may contain both B-cell and T-cell epitopes and / or induce both a humoral immune response and a cell-mediated immune response in a subject. In some embodiments, the carbohydrate antigen described herein may be or contain tumor-associated carbohydrate antigens (TACAs), such as Tn, S-Tn, etc. Thomsen-Friedenreich (TF)、(2,3)-S-TF、(2,6)-S-TF( Figure 1 ), Globo H, PSA, GD2, GD3, GM2, GM3, N-glycoyl-GM3, fucosyl GM1, Le a ,sLe a Le x ,sLe x Le y Or any combination thereof. In some embodiments, the carbohydrate antigens described herein may comprise viral polysaccharide antigens or bacterial capsular polysaccharides (CPS) (e.g., pneumococcal and / or streptococcal polysaccharide serotypes, meningococcal CPS; influenza (e.g., influenza A or B) CPS).

[0091] In some embodiments, the novel carbohydrate antigen-carrier protein coupling reaction described herein can conjugate at least two identical carbohydrate antigens or more than one type of carbohydrate antigen to a carrier protein or peptide via one or more types of thiol-containing linkers, thereby generating a multivalent novel glycoconjugate. In some embodiments, the multivalent novel glycoconjugate may comprise 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15 or more identical or different types of carbohydrate antigens conjugated to a carrier protein or peptide. In some embodiments, the novel glycoconjugate immunogen described herein may comprise any combination selected from the following TACAs: Tn, S-Tn, ... Thomsen-Friedenreich (TF), (2,3)-S-TF, (2,6)-S-TF, Globo H, PSA, GD2, GD3, GM2, GM3, N-ethanolyl-GM3, fucose-based GM1, Le a ,sLe a Le x ,sLe x and Le y In some embodiments, sialylated or non-sialylated Tn and TF antigens (and their analogues) can be synthesized as described herein or, for example, as described below: Ress et al., 2005; Wu et al., 2019; Thompson et al., 2015; and Yang et al., 2010.

[0092] In some implementations, the ratio of each TACA in the combination can vary depending on the tumor being targeted and can include molar ratios from 1 to 20. In this way, the novel glycoconjugate immunogens described herein can be tailored to specific cancer forms, for example, those associated with increased expression of specific combinations of multiple TACAs, as shown in the table below. Distribution of tumor-associated carbohydrate antigens (TACA) in different cancers

[0093] In some embodiments, the polyvalent neoglycoconjugate immunogen described herein may comprise more than one (novel) carbohydrate antigen conjugated to a single free amine group on a carrier protein (e.g., via a branched linker). In some embodiments, the polyvalent neoglycoconjugate immunogen described herein may comprise multiple (e.g., at least 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15 or more) (novel) carbohydrate antigens conjugated to thiol-containing linkers (e.g., via linkers with extensive branching) prior to attachment as dendritic macromolecules to a carrier protein or peptide.

[0094] In some embodiments, the carrier protein or peptide described herein comprises one or more free amine groups. As used herein, "free amine" or "free amine group" refers to a carrier protein or peptide having one or more amino groups (e.g., with respect to carbohydrate antigens described herein, such as solvent-accessible lysine residues that tend to be exposed to the periphery of the carrier protein) that can be chemically modified and / or conjugated. In some embodiments, it may be advantageous to avoid conjugating too many (new) carbohydrate antigens to adjacent positions on the carrier protein. In some embodiments, the carrier protein or peptide may preferably lack lysine-rich domains (e.g., fragments containing at least 4, 5, 6, 7, 8, 9, or 10 consecutive amino acids comprising at least 50% lysine residues).

[0095] In some embodiments, the carrier protein may comprise at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60. The total number of lysine residues may be 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100. In some embodiments, the carrier protein may contain 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30.

[0096] In some embodiments, the carrier protein or peptide comprises one or more lysine residues having one or more free amine groups, or optionally is engineered to add one or more additional lysine residues, for example, at the amino terminus, carboxyl terminus, or solvent-accessible position of the carrier protein or peptide. In some embodiments, the carrier protein comprises a T-cell epitope and / or induces a cell-mediated immune response in a subject. In some embodiments, the carrier protein or peptide comprises a B-cell epitope and / or induces a humoral immune response in a subject. In some embodiments, the carrier protein comprises both a B-cell epitope and a T-cell epitope, and / or induces both a humoral immune response and a cell-mediated immune response in a subject.

[0097] Preferably, the carrier protein described herein may be a protein that has been regulatoryly (e.g., FDA) approved for use in human subjects (e.g., in an approved vaccine). In some embodiments, the carrier protein is, is derived from, or comprises: tetanus toxoid (TT), diphtheria toxoid (DT), cross-reactive material 197 (CRM197), meningococcal outer membrane protein complex (OMPC), Haemophilus influenzae protein D (HiD), virus-like particles (VLP), cytokines, immunogenic peptides such as tetanus toxin 831-844 (SEQ ID NO: 1 or 2), albumin (e.g., bovine serum albumin or human serum albumin), keyhole hemocyanin (KLH), or immunogenic fragments thereof.

[0098] In some implementations, the carrier protein or peptide is exogenous to the subject to be administered, preferably having no (close) orthologs in the subject. In the context of human vaccine production, the carrier protein referred to herein means a “carrier protein suitable for human use” or simply a “suitable carrier protein,” which means a carrier protein whose antigenicity differs from that of human proteins such that the carrier protein will not be considered a “self-antigen” in humans. Using a carrier protein that is too similar in antigenicity to the corresponding human protein may result in the carrier protein being considered a “self-antigen,” which may not be ideal in human vaccines. For example, novel glycoconjugate immunogens consisting of TF antigens composed of ε-amino groups of lysine residues randomly conjugated to bovine serum albumin (BSA) have previously been described and characterized (e.g., Demian et al., 2014; Rittenhouse-Diakun et al., 1998; Heimburg et al., 2006; Tati et al., 2017). However, not only are the carbohydrate levels at the 59 lysine residues of BSA random and inefficient (each BSA molecule conjugates no more than 4 to 6 TF antigens), but BSA is also unsuitable as a carrier protein in human vaccines because its antigenicity is too similar to human albumin. In some implementations, the carrier protein is not albumin (e.g., bovine serum albumin).

[0099] In some embodiments, the novel glycoconjugates described herein may be novel glycoconjugate immunogens, wherein the carrier protein or peptide is immunogenic when administered to a subject, and the carbohydrate antigen, conjugated to the carrier protein or peptide via the thiol-containing linker, enhances the immunogenicity of the carbohydrate antigen after administration to the subject compared to the corresponding administration of an unconjugated carbohydrate antigen.

[0100] In some embodiments, the thiol-containing linkers described herein are non-immunogenic to subjects receiving the novel glycoconjugate, such that administration of the novel glycoconjugate immunogen to the subject does not trigger antibodies against the thiol-containing linkers contained in the novel glycoconjugate immunogen. In some embodiments, the thiol-containing linkers described herein lack synthetic chemical groups / functional groups (i.e., chemical groups / functional groups not naturally found in the subject). In some embodiments, the thiol-containing linkers described herein contain only natural chemical groups / functional groups, i.e., functional groups naturally present in the subject. In this regard, some carbohydrate antigen-protein linkers used in the art, such as squaric acids, may elicit an immune response against the linker itself, rather than only against the carbohydrate antigen coupled to it, which may be related to the "foreign" and / or antigenic nature of their chemical groups / functional groups.

[0101] In some implementations, the carbohydrate antigen or novel carbohydrate antigen, after being conjugated with a carrier protein or peptide, cannot be cleaved from the carrier protein or peptide by the subject's endogenous enzymes.

[0102] In some implementations, the carrier protein or peptide described herein may contain T-cell epitopes and / or induce a cell-mediated immune response in a subject upon administration.

[0103] In some implementations, the synthetic novel glycoconjugate immunogens described herein can induce a cell-mediated immune response against (novel) carbohydrate antigens upon administration to a subject.

[0104] On the other hand, this document describes a method for generating novel glycoconjugate vaccines or immune response-triggered compositions. The method may include formulating a novel glycoconjugate as described herein or prepared by methods described herein with pharmaceutically acceptable excipients and / or adjuvants. In some embodiments, the adjuvant is or comprises: inorganic compounds, mineral oils, microbial derivatives, plant derivatives, cytokines, squalene, alum, aluminum hydroxide, aluminum phosphate, calcium phosphate hydroxide, toll-like receptor agonists, immunostimulatory polynucleotides (e.g., CPG), immunostimulatory lipids, Freund's adjuvant, RIBI adjuvant, QS-21, muramyl dipeptide, TiterMax. TM Stevino TM Stimune TM Or any combination thereof.

[0105] Taking into account factors such as the recipient animal's age, sex, weight, species, and condition, as well as the route of administration, the vaccine composition can be administered at doses and techniques well known to those skilled in the medical or veterinary field. Routes of administration can be percutaneous, via mucous membranes (e.g., oral, nasal, ocular), or via parenteral routes (e.g., intradermal, intramuscular, subcutaneous). The vaccine composition can be administered alone, or co-administered or sequentially with other treatments or therapies. Forms of administration can include suspensions and formulations intended for parenteral, subcutaneous, intradermal, or intramuscular administration (e.g., injectable administration), such as sterile suspensions or emulsions. The vaccine can be administered as a spray or mixed with food and / or water, or delivered by mixing with suitable carriers, diluents, or excipients such as sterile water, saline, glucose, etc. Depending on the route of administration and the desired formulation, the composition may contain excipients such as wetting agents or emulsifiers, pH buffers, adjuvants, gelling or thickening additives, preservatives, flavoring agents, colorants, etc. Suitable formulations can be prepared by referring to standard drug texts, such as "Remington's Pharmaceutical Sciences," 1990, without having to conduct excessive experiments.

[0106] On the other hand, this document describes novel glycoconjugate vaccines or adaptive immune response triggering compositions that generate and / or contain novel glycoconjugates as described herein and pharmaceutically acceptable excipients and / or adjuvants as described herein, generated by the methods described herein. In embodiments, the novel glycoconjugate vaccine may be a prophylactic vaccine or a therapeutic vaccine. In embodiments, the vaccine composition described herein may contain one or more TACAs, and the vaccine composition may be an anticancer vaccine against cancers expressing TACAs. In embodiments, the cancer may be B-cell lymphoma, breast cancer, colon cancer, non-small cell lung cancer, melanoma, neuroblastoma, ovarian cancer, prostate cancer, sarcoma, small cell lung cancer, or gastric cancer.

[0107] In some respects, this document describes a method for immunizing, vaccinating, or treating a subject, including administering to the subject a novel glycoconjugate produced by the method described herein, a synthetic novel glycoconjugate as described herein, a novel glycoconjugate vaccine or adaptive immune response triggering composition produced by the method described herein, or a novel glycoconjugate vaccine as described herein.

[0108] In some embodiments, this document describes novel glycoconjugates produced by methods as described herein, synthetic novel glycoconjugates as described herein, novel glycoconjugate vaccines or adaptive immune response triggering compositions produced by methods as described herein, or novel glycoconjugate vaccines as described herein, for use in immunization, vaccination or treatment of a subject with a disease, or for detecting the presence of antibodies that specifically bind to said novel glycoconjugate or for detecting said immunization, vaccination or treatment (e.g., in a biological sample from said subject).

[0109] In some embodiments, this document describes novel glycoconjugates produced as described herein, synthetic novel glycoconjugates as described herein, or adaptive immune response triggering compositions produced as described herein, for use in manufacturing vaccines for immunizing or treating subjects with a disease, or for detecting the presence of antibodies that specifically bind to said novel glycoconjugates, or for detecting said immunization or treatment (e.g., in biological samples from said subjects).

[0110] In some embodiments, this document describes novel glycoconjugates produced by methods as described herein, synthetic novel glycoconjugates as described herein, novel glycoconjugate vaccines or adaptive immune response triggering compositions produced by methods as described herein, or novel glycoconjugate vaccines as described herein, for use in treating subjects with a disease associated with increased expression of the said carbohydrate antigens.

[0111] In some embodiments, this document describes novel glycoconjugates generated as described herein, synthetic novel glycoconjugates as described herein, novel glycoconjugate vaccines or adaptive immune response triggering compositions generated as described herein, or novel glycoconjugate vaccines as described herein, for detecting or screening the presence of antibodies that specifically bind to the carbohydrate antigen or circulating tumor cells containing the carbohydrate antigen, or for detecting the presence of antibodies generated by immunization or vaccination with the carbohydrate antigen. In some embodiments, detection or screening can be performed by any suitable detection method, such as immunoadsorption assay, ELISA, microarray, or Western blot analysis.

[0112] In other aspects, this document describes methods for treating subjects, including administering a novel glycoconjugate or novel glycoconjugate immunogen as defined herein or generated by methods described herein to generate an immune response against a carbohydrate antigen in the subject, and optionally screening for the presence of antibodies that specifically bind to the carbohydrate antigen in biological samples from the subject.

[0113] In other respects, this article describes glycoconjugates for use as therapeutic and / or diagnostic tools related to SARS-CoV-2. More specifically, this article describes a glycoconjugate for use in immunizing a subject against SARS-CoV-2, for triggering the production of anti-SARS-CoV-2 antibodies in a subject, for inducing a cell-mediated immune response against SARS-CoV-2 in a subject, or any combination thereof. This article also describes glycoconjugates for use in detection / diagnostic tools related to SARS-CoV-2. For example, this article describes the use of a glycoconjugate for detecting the presence of anti-SARS-CoV-2 antibodies in samples from a subject.

[0114] As used herein, the term "anti-SARS-CoV-2 antibody" refers to an antibody capable of binding to antigens of its natural conformation (e.g., carbohydrate antigens), such as antigens expressed on natural recombinant proteins and / or present on assembled viral particles. In contrast, antibodies that bind only to denatured antigens (e.g., under denaturing conditions, such as after SDS-PAGE) but not to the same antigens of their natural conformation are excluded from the term "anti-SARS-CoV-2 antibody." In some embodiments, the glycoconjugates or vaccines described herein can induce the production of antibodies with neutralizing activity. As used herein, the term "neutralizing activity" refers to the ability of a ligand (e.g., an antibody) to bind to SARS-CoV-2 viral particles and inhibit their infection of susceptible host cells.

[0115] In some embodiments, the glycoconjugates described herein may comprise carbohydrate antigens conjugated to a suitable carrier material (e.g., a carrier protein or peptide, or a non-protein polymer material), wherein the carbohydrate antigen comprises, or is composed of, sialylated Thomsen-Friedenreich (TF) antigen, non-sialylated TF antigen, sialylated Tn antigen, non-sialylated Tn antigen, or any combination thereof. In some embodiments, the carbohydrate antigen comprises a monosialylated TF antigen, such as (2,3)-S-TF, and / or a disialylated TF antigen, such as disialic acid core 1. These carbohydrate antigens were detected by quantitative high-resolution mass spectrometry at positions 4 and 6 corresponding to the peptide fragment VQPTESIVR (SEQ ID NO: 3) on the recombinantly expressed SARS-CoV-2 spike (S) protein and / or its S1 fragment (Example 16;). Figure 21 and 21 In addition, Examples 17 and Figure 22 and 23 The results shown demonstrate that at least some of these carbohydrate antigens can be used for ligand binding (e.g., with lectins and / or antibodies), and Examples 18 and Figure 24The results shown demonstrate that ligands binding to these carbohydrate antigens inhibit the ability of pseudotyped viral particles expressing the SARS-CoV-2 S protein to infect host cells expressing human angiotensin-converting enzyme 2 (ACE2, the receptor for S-binding to enter host cells). These results were unexpected because multiple reports have indicated that the SARS-CoV-2 S protein is largely overshielded by N-linked glycans, and that O-linked glycans (if detected, as reports are contradictory) represent a minor component of the overall glycosylation profile of the SARS-CoV-2 S protein, which may not be available for ligand binding in the case of pathogenic viral particles (Watanabe et al., 2020; Shajahan et al., 2020; Grant et al., 2020).

[0116] In some embodiments, the carbohydrate antigens described herein can be conjugated to a carrier material containing B-cell epitopes or T-cell epitopes, depending on whether a humoral immune response and a cell-mediated immune response are required. In some embodiments, the carbohydrate antigens can be covalently conjugated to the SARS-CoV-2 S protein fragment of SEQ ID NO: 3 or 4, for example, at positions 4 and / or 6 of SEQ ID NO: 3 or positions 323, 325, and / or 678 of SEQ ID NO: 4. In particular, position 678 of SEQ ID NO: 4 (near the furin cleavage site at R682 of the spike protein) has reportedly been O-glycosylated by core-1 and core-2 structures.

[0117] In some embodiments, the carrier protein or peptide may comprise an immunogenic fragment of the SARS-CoV-2 S protein sequence of SEQ ID NO: 4, the fragment containing one or more carbohydrate antigens conjugated to positions 323, 325, and / or 678 of SEQ ID NO: 4. In some embodiments, the carbohydrate antigens may be covalently conjugated to variants of the SARS-CoV-2 S protein fragment of SEQ ID NO: 3, such as variants in which residues at positions 4 and / or 6 can be replaced with lysine and / or cysteine ​​residues, which can facilitate chemical conjugation with the carbohydrate antigens. In some embodiments, the carrier protein or peptide may comprise an immunogenic fragment of a variant of the SARS-CoV-2 S protein sequence of SEQ ID NO: 4 having lysine or cysteine ​​at positions 323, 325, and / or 678, the fragment containing one or more carbohydrate antigens conjugated to lysine or cysteine ​​residues at positions 323, 325, and / or 678 of SEQ ID NO: 4. In the case of lysine residues, carbohydrate antigens can be conjugated to the carrier protein by the conjugation methods described herein or by other conjugation methods known in the art. In the case of cysteine ​​residues, carbohydrate antigens can be conjugated to the carrier protein by, for example, the conjugation methods described in WO / 2019 / 178699 or US 10,610,576 or by other conjugation methods known in the art. Therefore, in some embodiments, the carrier material described herein may comprise, or consist of, the peptide of SEQ ID NO: 3, or a variant of the peptide of SEQ ID NO: 3 containing cysteine ​​or lysine at positions 4 and / or 6. In some embodiments, the peptide of SEQ ID NO: 3 or a peptide variant thereof may be included in the carrier material (e.g., recombinantly engineered into the amino acid sequence) or may be fused into the carrier material (e.g., as a fusion protein).

[0118] In some embodiments, the carrier material is, is derived from, or comprises: tetanus toxoid (TT), diphtheria toxoid (DT), cross-reactive material 197 (CRM197), meningococcal outer membrane protein complex (OMPC), Haemophilus influenzae protein D (HiD), virus-like particles (VLP), cytokines, immunogenic peptides such as tetanus toxoid 831-844 (SEQ ID NO: 1 or 2), albumin (e.g., bovine serum albumin or human serum albumin), keyhole hemocyanin (KLH), or immunogenic fragments thereof.

[0119] In some embodiments, the glycoconjugate may be: (i) a novel glycoconjugate generated by the methods described herein or as defined in the methods described herein; (ii) a novel carbohydrate antigen described herein; (iii) a novel synthetic glycoconjugate described herein; or (iv) a novel glycoconjugate vaccine or adaptive immune response triggering composition described herein.

[0120] In some embodiments, the glycoconjugate can be produced by methods described as in WO / 2019 / 178699 or US 10,610,576. Briefly, in some embodiments, the method may include: (a) providing a water-soluble carbohydrate antigen (alkenyl carbohydrate antigen) covalently linked to a terminal alkene, said terminal alkene being directly conjugated to a thiol group via a thiol-alkene reaction, and said alkenyl carbohydrate antigen being an unprotected water-soluble alkenyl carbohydrate antigen; (b) providing a carrier material having one or more free thiol groups; and (c) performing a photocatalytic thiol-alkene reaction to directly conjugate the carbohydrate antigen to said one or more free thiol groups of said material, thereby producing the glycoconjugate. In other embodiments, the method may include one or more features described in items 51 to 53 listed below.

[0121] In some respects, this article describes SARS-CoV-2 or COVID-19 vaccines or adaptive immune response inducing compositions comprising one or more glycoconjugates as defined herein, and pharmaceutically acceptable excipients and / or adjuvants. Glycoconjugates typically contain one or more carbohydrate antigens expressed on SARS-CoV-2 viral particles, such as carbohydrate antigens expressed on the S (or S1) protein of SARS-CoV-2. As described herein, carbohydrate antigens suitable for SARS-CoV-2 vaccines are carbohydrate antigens with anomalous glycosylation patterns, i.e., those not expressed on normal or healthy cells and tissues of subjects, to reduce the risk of triggering an autoimmune response in vaccine-administered subjects. Analysis of the N-linked and O-linked glycosylation profiles of the SARS-CoV-2 S protein revealed that most N-linked glycans expressed on the SARS-CoV-2 S protein may not be ideal candidates for glycoconjugate vaccine development because they may resemble carbohydrate antigens present on normal or healthy cells and tissues of human subjects. In contrast, analysis of the O-linked glycosylation profile of the SARS-CoV-2 S protein revealed several anomalous O-linked glycans that may be suitable for glycoconjugate vaccine development. In some embodiments, the carbohydrate antigens described herein may comprise those of Example 16 and... Figure 20 and 21 One or more O-linked glycans identified in [the study].

[0122] In some embodiments, the glycoconjugates or SARS-CoV-2 vaccines described herein induce the production of antibodies that bind to SARS-CoV-2 viral particles, and preferably have neutralizing activity (e.g., the ability to inhibit SARS-CoV-2 viral particles from infecting susceptible host cells).

[0123] In some respects, this article describes methods for vaccinating subjects against SARS-CoV-2 or for triggering the production of anti-SARS-CoV-2 antibodies in subjects, the methods comprising administering one or more of the glycoconjugates or SARS-CoV-2 vaccines described herein.

[0124] In some aspects, this document describes compositions for protecting subjects from SARS-CoV-2 virus infection (or for reducing severity) or for treating COVID-19 (or for reducing complications caused by COVID-19), comprising one or more ligands (e.g., antibodies, antibody fragments, or lectins) that bind to O-linked glycans expressed on the SARS-CoV-2 S protein. In some embodiments, the O-linked glycan may comprise sialylated TF antigens (monosialized or disialylated TF antigens), nonsialylated TF antigens, sialylated Tn antigens, nonsialylated Tn antigens, or any combination thereof. In some embodiments, one or more ligands may comprise recombinant monoclonal antibodies (e.g., JAA-F11 or humanized JAA-F11). In some embodiments, the ligand may have binding affinity for sialylated TF and / or nonsialylated Tn. In some embodiments, the ligand may have binding affinity for both sialylated and nonsialylated TF. In some embodiments, the ligand may be a lectin such as Jacalin or a Jacalin-associated lectin. In this regard, Examples 18 and... Figure 24 The study indicated that the ligand with the strongest inhibitory effect on the ability of pseudovirus particles expressing the SARS-CoV-2 S protein to infect host cells was lectin Jacalin, a jackfruit tree species isolated from the seeds of Jackfruit (Aralia elata). Artocarpus integrifolia Jacalin (AIA). Interestingly, it is known that Jacalin binds specifically to both sialylated and non-sialylated forms of TF and Tn antigens (Jeyaprakash et al., 2002). This paper also demonstrates inhibitory effects on pseudoviral infectivity for ligands that bind only to non-sialylated TF antigens such as lectin PNA and the anti-TF monoclonal antibody JAA-F11. Figure 24 In some embodiments, the above composition can be formulated as an intranasal composition.

[0125] In some aspects, this document describes a complex comprising: (a) a SARS-CoV-2 S protein or a fragment thereof expressing an O-linked glycan comprising sialylated TF antigen (monosialized or disialylated TF antigen), nonsialylated TF antigen, sialylated Tn antigen, nonsialylated Tn antigen, or any combination thereof; and (b) a ligand as defined herein, said ligand binding to said O-linked glycan of said SARS-CoV-2 S protein or a fragment thereof. In some embodiments, the complex may be contained within the SARS-CoV-2 S protein in an intact SARS-CoV-2 viral particle. In some embodiments, such a complex may be formed in vitro or in vivo.

[0126] project This article describes one or more of the following projects.

[0127] 1. A method for generating a novel glycoconjugate, the method comprising: (a) providing a novel carbohydrate antigen or a novel carbohydrate antigen intermediate comprising a linker having a first end and a second end, wherein the first end is conjugated to the carbohydrate antigen via a thioether bond, and the second end comprises a functional group capable of reacting with a free amine group, said functional group being COX, SO2X, OC(O)X, N=C=O, or N=C=S, wherein X is a leaving group; (b) providing a carrier protein or peptide having one or more free amine groups; and (c) performing a coupling reaction to conjugate one or more purified novel carbohydrate antigens or novel carbohydrate antigen intermediates to said carrier protein or peptide via an amide, carbamate, sulfamyl, urea, or thiourea bond, thereby generating a novel glycoconjugate.

[0128] 2. The method according to Project 1, wherein prior to step (a), the novel carbohydrate antigen or novel carbohydrate antigen intermediate in (a) is prepared by a method comprising: (i) providing a carbohydrate antigen (alkenyl carbohydrate antigen) covalently linked to a terminal alkene, the terminal alkene being directly conjugated to a thiol group via a thiol-alkene reaction; (ii) providing a thiol-containing linker comprising a first functional group at a first end and a second functional group at a second end, the first functional group being a free thiol group and the second functional group being a carboxyl group, a sulfinic acid group, a carbonate group, an isocyanate group, or a thiocyanate group; (iii) performing a photocatalytic thiol-alkene reaction to directly conjugate the alkenyl carbohydrate antigen to the first end of the thiol-containing linker, thereby producing a novel carbohydrate antigen comprising the carbohydrate antigen at the first end and the second functional group at the second end; (iv) When the second functional group is a carboxyl group, a sulfinic acid group, or a carbonate group, the novel carbohydrate antigen is converted into a novel carbohydrate antigen intermediate by replacing the terminal hydroxyl group of the carboxyl group, sulfinic acid group, or carbonate group with a better leaving group for conjugation with the free amine group of the polypeptide; and (v) purifying the novel carbohydrate antigen or the novel carbohydrate antigen intermediate.

[0129] 3. The method according to Project 2, wherein the photocatalytic thiol-ene reaction in (iii) is carried out under reaction conditions that preserve the antigenicity and / or structure of the carbohydrate antigen.

[0130] 4. The method according to Project 2 or 3, wherein the photocatalytic thiol-ene reaction is carried out in the presence of a catalyst, wherein the catalyst is: a water-soluble catalyst, such as a water-soluble radical generating azo compound; 2,2'-azobis[2-(2-imidazolin-2-yl)propane] dihydrochloride (Vazo 44 or VA-044); 2,2'-azobis(2-amidinylpropane) dihydrochloride (AAPH); lithium phenyl-2,4,6-trimethylbenzoylphosphine (LAP); a metal or metal ion with photoinitiator activity; a peroxide; tert-butyl hydroperoxide; benzoyl peroxide; ammonium persulfate; or any derivative thereof with photoinitiator activity; or a water-insoluble catalyst, such as a water-insoluble radical-generating azo compound, 2,2-dimethoxy-2-phenylacetophenone (DMPA), azobisisobutyronitrile (AIBN), 2,2'-azobis(2-methylpropionitrile), 4,4'-azobis(4-cyanopentanoic acid) (ACVA), 1,1'-azobis(cyanocyclohexane) (ACHN), diazepine dicarboxylic acid bis( N,N -dimethylamide (TMAD); dipiperidine azodiacarboxylic acid (ADD), or any of its derivatives having photoinitiator activity; 5. The method according to any one of items 2 to 4, wherein the photocatalytic thiol-ene reaction comprises irradiation under ultraviolet light (e.g., short-wave ultraviolet light, such as about 254 nm, or long-wave ultraviolet light, such as about 355 nm or 365 nm).

[0131] 6. The method according to any one of items 2 to 5, wherein: the photocatalytic thiol-ene reaction comprises reacting each free thiol group of the thiol-containing linker with 1 to 200 or 1 to 100 molar equivalents of the alkenyl carbohydrate antigen; the photocatalytic thiol-ene reaction is carried out at 10 to 300, 10 to 270, 10 to 240, 10 to 210, 10 to 180, 10 to 150, 10 to 120, Performed at a pH of 10 to 90, 10 to 60, or 10 to 30 minutes; at a pH of about 3.0, 3.1, 3.2, 3.3, 3.4, 3.5, 3.6, 3.7, 3.8, 3.9, or 4.0 to about 4.5, 4.6, 4.7, 4.8, 4.9, 5.0, 5.5, 6.0, 6.5, 7.0, 7.5, 8.0, 8.5, 9.0, 9.5, or 10; or any combination thereof.

[0132] 7. The method according to any one of items 2 to 6, wherein the carbohydrate antigen is preferably linked to the terminal alkene via a glycosidic bond, such as an O-glycosidic bond, S-glycosidic bond, N-glycosidic bond or C-glycosidic bond, or via a bond obtained, for example, by reductive amination between an allylamine and a reducing sugar.

[0133] 8. The method according to any one of items 2 to 7, wherein the thiol-containing connector in (ii) comprises the following structure:

[0134] Where: Y is -(CH2) n -or-(OCH2CH2O) n -; Z is -CO2H, -SO2H, -OC(O)-H, -N=C=O, or -N=C=S; O is 1, 2, 3, 4, or 5; O is 0 and Z is -CO- and Y is -(OCH2CH2O). n -; or O is 0 and Z is -SO2- and Y is -(OCH2CH2O). n -

[0135] 9. The method according to any one of items 1 to 8, wherein the carbohydrate antigen, the alkenyl carbohydrate antigen, the novel carbohydrate antigen, the novel carbohydrate antigen intermediate, and / or the carbohydrate portion of the novel glycoconjugate remains unprotected throughout the method.

[0136] 10. The method according to any one of items 1 to 9, wherein the leaving group is an active ester group (e.g., a fluorophenyl group (e.g., OPhF5, OPhF4 (para-SO3Na)) or a succinimide group).

[0137] 11. The method according to any one of items 1 to 10, wherein the method avoids self-crosslinking of the carrier protein or peptide between aspartic acid / glutamate residues and ε-lysine amine present in the same carrier protein or peptide.

[0138] 12. The method according to any one of items 1 to 11, wherein the amount of the novel carbohydrate antigen conjugated with the carrier protein or peptide is controlled by the stoichiometry of efficacy and / or reactants (e.g., the molar ratio of the carrier protein or peptide to the novel carbohydrate antigen or the intermediate of the novel carbohydrate antigen).

[0139] 13. The method according to any one of items 1 to 12, wherein the carbohydrate antigen is or comprises: tumor-associated carbohydrate antigen (TACA) (e.g., Tn, S-Tn, ...). Thomsen-Friedenreich (TF), (2,3)-S-TF, (2,6)-S-TF, Globo H, PSA, GD2, GD3, GM2, GM3, N-ethanolyl-GM3, fucose-based GM1, Le a ,sLe a Le x ,sLe x Le y (or any combination thereof); viral polysaccharide antigens; or bacterial capsular polysaccharides (CPS) (e.g., CPS derived from or containing pneumococcal and / or streptococcal polysaccharide serotypes, meningococcal CPS, or influenza CPS (e.g., influenza A or B CPS).

[0140] 14. The method according to any one of items 1 to 19, wherein the coupling reaction in (c) conjugates at least two identical or more than one type of the novel carbohydrate antigen to the carrier protein or peptide to produce a polyvalent novel glycoconjugate (e.g., a polyvalent novel glycoconjugate comprising 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15 or more identical or different types of novel carbohydrate antigens conjugated to the carrier protein or peptide).

[0141] 15. The method according to any one of items 1 to 14, wherein the carrier protein or peptide is a protein or peptide engineered to have one or more additional lysine residues added, for example at the amino terminus, carboxyl terminus, or solvent-accessible position of the carrier protein or peptide.

[0142] 16. The method according to any one of items 1 to 15, wherein the carrier protein or peptide is, is derived from, or comprises: tetanus toxoid (TT), diphtheria toxoid (DT), cross-reactive material 197 (CRM197), meningococcal outer membrane protein complex (OMPC), Haemophilus influenzae protein D (HiD), cytokines, immunogenic peptides such as tetanus toxin 831-844 (SEQ ID NO: 1 or 2), albumin (e.g., bovine serum albumin or human serum albumin), keyhole hemocyanin (KLH), or immunogenic fragments thereof.

[0143] 17. The method according to any one of items 1 to 16, wherein the novel glycoconjugate has the following structure:

[0144] Wherein: CA is or contains the carbohydrate antigen; CP-NH is the carrier protein or peptide having one or more amine groups; X is O, S, NR1, or CH2; R1 is H, COH (formamide), COME, or COEt; m is 1, 2, 3, 4, or 5; Y is -(CH2). n -or-(OCH2CH2O) n -; n is 0, 1, 2, 3, 4, or 5; o is 0, 1, 2, 3, 4, or 5; or o is 0 and Z is -CO- and Y is -(OCH2CH2O) n -; or o is 0 and Z is -SO2- and Y is -(OCH2CH2O). n -; Z is -CO-, -NR2SO2-, -OCO-, -NR2CO- or -NR2CS-; R2 is H, Me or Et; and p is 1 indicating 50.

[0145] 18. The method according to any one of items 1 to 18, wherein the novel glycoconjugate is a novel glycoconjugate immunogen, the carrier protein or peptide is immunogenic when administered to a subject, and the carbohydrate antigen, conjugated to the carrier protein or peptide via the thiol-containing linker, enhances the immunogenicity of the carbohydrate antigen after administration to the subject compared to the corresponding administration of an unconjugated carbohydrate antigen.

[0146] 19. The method of claim 18, wherein the thiol-containing linker is non-immunogenic to the subject, such that administration of the novel glycoconjugate immunogen to the subject does not trigger antibodies against the thiol-containing linker contained in the novel glycoconjugate immunogen.

[0147] 20. The method according to item 18 or 19, wherein the novel carbohydrate antigen, after being conjugated with the carrier protein or peptide, cannot be cleaved from the carrier protein or peptide by the subject's endogenous enzymes.

[0148] 21. The method according to any one of items 18 to 20, wherein the novel carbohydrate antigen comprises a B-cell epitope and / or induces a humoral immune response in the subject; and / or comprises a T-cell epitope and / or induces a cell-mediated immune response in the subject.

[0149] 22. The method according to any one of items 18 to 21, wherein the carrier protein or peptide comprises a human T cell epitope and / or induces a cell-mediated immune response in the subject.

[0150] 23. The method according to any one of items 18 to 22, wherein the novel glycoconjugate immunogen induces a cell-mediated immune response to the carbohydrate antigen after administration to the subject.

[0151] 24. A novel carbohydrate antigen or a novel carbohydrate antigen intermediate comprising a linker having a first end and a second end, wherein the first end is conjugated to the carbohydrate antigen via a thioether bond, and the second end comprises a functional group capable of reacting with a free amine group, said functional group being COX, SO2X, OC(O)X, N=C=O, or N=C=S, wherein X is a leaving group.

[0152] 25. The novel carbohydrate antigen or novel carbohydrate antigen intermediate according to item 24, wherein: the carbohydrate antigen is not protected; the leaving group is as defined in item 10; the carbohydrate antigen is as defined in item 13; or any combination thereof.

[0153] 26. A synthetic novel glycoconjugate comprising a linker having a first end and a second end, wherein the first end is conjugated to a carbohydrate antigen via a thioether bond, and the second end is conjugated to one or more free amine groups in a carrier protein or peptide via an amide, carbamate, sulfamyl, urea, or thiourea bond.

[0154] 27. A synthetic novel glycoconjugate comprising one or more carbohydrate antigens (CAs) conjugated to one or more amine groups of a carrier protein or peptide (CP-NH) via a linker, said synthetic novel glycoconjugate having the following structure:

[0155] Where: X is O, S, NR1, or CH2; R1 is H, COH (formamide), COME, or COEt; m is 1, 2, 3, 4, or 5; Y is -(CH2).n -or-(OCH2CH2O) n -; n is 0, 1, 2, 3, 4, or 5; o is 0, 1, 2, 3, 4, or 5; or o is 0 and Z is -CO- and Y is -(OCH2CH2O) n -; or o is 0 and Z is -SO2- and Y is -(OCH2CH2O). n -; Z is -CO-, -NR2SO2-, -OCO-, -NR2CO- or -NR2CS-; R2 is H, Me or Et; and p is 1 indicating 50.

[0156] 28. A synthetic novel glycoconjugate according to item 26 or 27, wherein: the carbohydrate antigen is not protected; the carbohydrate antigen is as defined in item 13; the novel glycoconjugate is a multivalent novel glycoconjugate as defined in item 14; the carrier protein or peptide is as defined in items 15, 16, 18 or 22; the novel glycoconjugate has a structure as defined in item 17; the linker is as defined in item 19; the novel carbohydrate antigen is as defined in item 20 or 21; the synthetic novel carbohydrate is produced by any one of items 1 to 25; or any combination thereof.

[0157] 29. A method for generating a novel glycoconjugate vaccine or an adaptive immune response triggering composition, the method comprising formulating a novel glycoconjugate prepared by any one of items 1 to 23 or as defined in any one of items 24 to 28 with a pharmaceutically acceptable excipient and / or adjuvant.

[0158] 30. The method according to Item 29, wherein the adjuvant is or comprises: an inorganic compound, mineral oil, microbial derivative, plant derivative, cytokine, squalene, alum, aluminum hydroxide, aluminum phosphate, calcium hydroxide, toll-like receptor agonist, immunostimulatory polynucleotide (e.g., CPG), immunostimulatory lipid, Freund's adjuvant, RIBI adjuvant, QS-21, muramyl dipeptide, TiterMax, Stevine, Stimune, or any combination thereof.

[0159] 31. A novel glycoconjugate vaccine or adaptive immune response triggering composition, produced by the method described in Item 29 or 30, and / or comprising a novel glycoconjugate as defined in any one of Items 1 to 28 and a pharmaceutically acceptable excipient and / or adjuvant.

[0160] 32. The novel glycoconjugate vaccine according to item 31, wherein the novel glycoconjugate vaccine is a prophylactic vaccine or a therapeutic vaccine (e.g., against cancers expressing tumor-associated carbohydrate antigens, such as breast cancer, prostate cancer, gastric cancer, B-cell lymphoma, colon cancer, lung cancer, melanoma, neuroblastoma, ovarian cancer, sarcoma, small cell lung cancer; or against viruses or bacteria expressing carbohydrate antigens).

[0161] 33. A method for immunizing, vaccinating, or treating a subject, comprising administering to the subject a novel glycoconjugate produced by any one of items 1 to 23, a synthetic novel glycoconjugate produced by any one of items 26 to 28, a novel glycoconjugate vaccine or adaptive immune response triggering composition produced by any one of items 29 or 30, or a novel glycoconjugate vaccine described in item 31 or 32.

[0162] 34. A novel glycoconjugate produced by any one of items 1 to 23, a synthetic novel glycoconjugate produced by any one of items 26 to 28, a novel glycoconjugate vaccine or adaptive immune response triggering composition produced by any one of items 29 or 30, or a novel glycoconjugate vaccine described in item 31 or 32, for use in immunization, vaccination, or treatment of a subject with a disease (e.g., cancer expressing tumor-associated carbohydrate antigens, such as breast cancer, prostate cancer, gastric cancer, B-cell lymphoma, colon cancer, lung cancer, melanoma, neuroblastoma, ovarian cancer, sarcoma, small cell lung cancer; or a virus or bacteria expressing carbohydrate antigens), or for detecting the presence of antibodies that specifically bind to said novel glycoconjugate or for detecting said immunization, vaccination, or treatment (e.g., in a biological sample from said subject).

[0163] 35. Use of the novel glycoconjugate produced by any one of items 1 to 23, the synthetic novel glycoconjugate produced by any one of items 26 to 28, the novel glycoconjugate vaccine or adaptive immune response triggering composition produced by the method of item 29 or 30, or the novel glycoconjugate vaccine of item 31 or 32 for immunization, vaccination or treatment of subjects with a disease (e.g., cancers expressing tumor-associated carbohydrate antigens, such as breast cancer, prostate cancer, gastric cancer, B-cell lymphoma, colon cancer, lung cancer, melanoma, neuroblastoma, ovarian cancer, sarcoma, small cell lung cancer; or viruses or bacteria expressing carbohydrate antigens), or for detecting the presence of antibodies that specifically bind to said novel glycoconjugate or for detecting said immunization, vaccination or treatment (e.g., in biological samples from said subjects).

[0164] 36. Use of novel glycoconjugates produced by any one of items 1 to 23, synthetic novel glycoconjugates produced by any one of items 26 to 28, or adaptive immune response triggering compositions produced by any one of items 29 or 30, for the manufacture of vaccines for the immunization or treatment of subjects with a disease (e.g., cancers expressing tumor-associated carbohydrate antigens, such as breast cancer, prostate cancer, gastric cancer, B-cell lymphoma, colon cancer, lung cancer, melanoma, neuroblastoma, ovarian cancer, sarcoma, small cell lung cancer; or viruses or bacteria expressing carbohydrate antigens), or for the detection of the presence of antibodies specifically binding to said novel glycoconjugates or for the detection of said immunization or treatment (e.g., in biological samples from said subjects).

[0165] 37. Use of a novel glycoconjugate produced by any one of items 1 to 23, a synthetic novel glycoconjugate produced by any one of items 26 to 28, a novel glycoconjugate vaccine or adaptive immune response triggering composition produced by any one of items 29 or 30, or a novel glycoconjugate vaccine described in item 31 or 32, for the treatment of a disease associated with increased expression of the said carbohydrate antigen (e.g., cancers such as breast cancer, prostate cancer, gastric cancer, B-cell lymphoma, colon cancer, lung cancer, melanoma, neuroblastoma, ovarian cancer, sarcoma, small cell lung cancer; or viruses or bacteria expressing carbohydrate antigens).

[0166] 38. Use of a novel glycoconjugate produced by any one of items 1 to 23, a synthetic novel glycoconjugate produced by any one of items 26 to 28, a novel glycoconjugate vaccine or adaptive immune response triggering composition produced by any one of items 29 or 30, or a novel glycoconjugate vaccine described in item 31 or 32, for the purpose of generating antibodies that specifically bind to the novel glycoconjugate, or for the purpose of detecting antibodies that specifically bind to the immunogen of the novel glycoconjugate.

[0167] 39. Use of a novel glycoconjugate produced by any one of items 1 to 23, a synthetic novel glycoconjugate produced by any one of items 26 to 28, a novel glycoconjugate vaccine or adaptive immune response triggering composition produced by any one of items 29 or 30, or a novel glycoconjugate vaccine described in item 31 or 32, for detecting or screening the presence of antibodies that specifically bind to the carbohydrate antigen or of circulating tumor cells containing the carbohydrate antigen, or for detecting the presence of antibodies produced by immunization or vaccination with the carbohydrate antigen.

[0168] 40. As described in item 39, the detection or screening is performed by any suitable detection method, such as immunoadsorption assay, ELISA, microarray or immunoblotting analysis.

[0169] 41. A method of treating a subject, comprising administering a novel glycoconjugate or novel glycoconjugate immunogen as defined in any of the preceding items or generated by a method defined in any of the preceding items to generate an immune response against a carbohydrate antigen in the subject, and optionally screening for the presence of antibodies specifically binding to the carbohydrate antigen in a biological sample from the subject.

[0170] 42. A glycoconjugate for use in immunizing a subject against SARS-CoV-2, for triggering the production of anti-SARS-CoV-2 antibodies in a subject, or for detecting the presence of anti-SARS-CoV-2 antibodies in a sample from a subject, said glycoconjugate comprising a carbohydrate antigen conjugated to a suitable carrier material (e.g., a carrier protein or peptide), said carbohydrate antigen comprising, or consisting of, sialylated Thomsen-Friedenreich (TF) antigen, non-sialylated TF antigen, sialylated Tn antigen, non-sialylated Tn antigen, or any combination thereof.

[0171] 43. The glycoconjugate used according to item 42, wherein the carbohydrate antigen comprises or is composed of a sialylated TF antigen (e.g., (2,3)-S-TF and / or disialic acid core 1).

[0172] 44. The glycoconjugate used according to item 42 or 43, wherein the carbohydrate antigen comprises or is composed of Tn (e.g., sialylated and / or non-sialylated Tn).

[0173] 45. The glycoconjugate used in any one of items 42 to 44, wherein the carrier material comprises a peptide that is a B-cell epitope or a T-cell epitope.

[0174] 46. ​​The glycoconjugate used in any of items 42 to 45, wherein the carbohydrate antigen is conjugated to the peptide of SEQ ID No: 3 or to position 4 and / or 6 of a peptide variant of SEQ ID No: 3 containing cysteine ​​or lysine at position 4 and / or 6.

[0175] 47. The glycoconjugate used according to item 46, wherein the peptide or peptide variant of SEQ ID No: 3 is contained in or fused with the carrier material.

[0176] 48. The glycoconjugate used in any one of items 42 to 47, wherein the carrier material is, is derived from, or comprises: tetanus toxoid (TT), diphtheria toxoid (DT), cross-reactive material 197 (CRM197), meningococcal outer membrane protein complex (OMPC), Haemophilus influenzae protein D (HiD), virus-like particles (VLP), cytokines, immunogenic peptides such as tetanus toxoid 831-844 (SEQ ID NO: 1 or 2), albumin (e.g., bovine serum albumin or human serum albumin), keyhole hemocyanin (KLH), or immunogenic fragments thereof.

[0177] 49. A glycoconjugate as defined in any one of items 42 to 48, wherein the glycoconjugate is: (i) a novel glycoconjugate produced by any one of items 1 to 23 or as defined in any one of items 1 to 23; (ii) a novel carbohydrate antigen as described in item 24 or 25; (iii) a novel synthetic glycoconjugate as described in item 27 or 28; or (iv) a novel glycoconjugate vaccine or adaptive immune response triggering composition as defined in or produced by the method of item 29 or 30 or as defined in item 31 or 32.

[0178] 50. A glycoconjugate as defined in any one of items 42 to 48, which is produced by a method comprising: (a) providing a water-soluble carbohydrate antigen (alkenyl carbohydrate antigen) covalently linked to a terminal alkene, the terminal alkene being directly conjugated to a thiol group via a thiol-alkene reaction, and wherein the alkenyl carbohydrate antigen is an unprotected water-soluble alkenyl carbohydrate antigen; (b) providing a carrier material having one or more free thiol groups; and (c) performing a photocatalytic thiol-alkene reaction to directly conjugate the carbohydrate antigen to the one or more free thiol groups of the material, thereby producing the glycoconjugate.

[0179] 51. The glycoconjugate according to claim 50, wherein: the photocatalytic thiol-ene reaction is carried out under reaction conditions that avoid denaturation of the support material and / or retain the activity, antigenicity, and / or structure of the support material; the photocatalytic thiol-ene reaction is carried out in the absence of any organic solvent, or wherein the photocatalytic thiol-ene reaction is carried out in the presence of an organic solvent at a sufficiently low concentration to avoid denaturation of the support material; the photocatalytic thiol-ene reaction is carried out in the presence of a catalyst, wherein the catalyst is: a water-soluble catalyst, such as a water-soluble radical-derived azo compound; 2,2'-azobis[2-(2-imidazolin-2-yl)propane] dihydrochloride (Vazo 44 or VA-044); 2,2'-azobis(2-amidinylpropane) dihydrochloride (AAPH); lithium phenyl-2,4,6-trimethylbenzoylphosphine (LAP); a metal or metal ion with photoinitiator activity; a peroxide; tert-butyl hydroperoxide; benzoyl peroxide; ammonium persulfate; or any derivative thereof with photoinitiator activity; or a water-insoluble catalyst, such as a water-insoluble radical-generating azo compound, 2,2-dimethoxy-2-phenylacetophenone (DMPA), azobisisobutyronitrile (AIBN), 2,2'-azobis(2-methylpropionitrile), 4,4'-azobis(4-cyanopentanoic acid) (ACVA), 1,1'-azobis(cyanocyclohexane) (ACHN), diazepine dicarboxylic acid bis( N,N-dimethylamide (TMAD); dipiperidine azodicarboxylate (ADD), or any derivative thereof having photoinitiator activity; the photocatalytic thiol-ene reaction comprises irradiation under ultraviolet light; the photocatalytic thiol-ene reaction comprises reacting each free thiol group of the support material with 1 to 200 molar equivalents of the alkenyl carbohydrate antigen; and / or wherein the photocatalytic thiol-ene reaction is carried out for 10 to 300, 10 to 270, 10 to 240, 10 to 210, 10 to 180, 10 to 150, 10 to 120, 10 to 90, 10 to 60, or 10 to 30 minutes; and / or for a sufficient time to reduce the total free thiol concentration in the support material by at least 5, 10, 15, 20, 25, 30, 35, 40, 45, or 50 times; the photocatalytic thiol-ene reaction avoids The reaction is carried out at a denaturing pH of the carrier material; after conjugation with the carrier material, the photocatalytic thiol-ene reaction produces a carbohydrate antigen that cannot be cleaved from the carrier material by the subject's endogenous enzymes; the alkenyl carbohydrate antigen is covalently linked to a terminal ene, and / or the carbohydrate antigen is conjugated to the carrier material via an O-glycosidic bond, S-glycosidic bond, N-glycosidic bond, or C-glycosidic bond, or via a bond obtained through reductive amination between an allylamine and a reducing sugar; the photocatalytic thiol-ene reaction conjugates more than one type of carbohydrate antigen to the carrier material; the carbohydrate antigen in (a) is linked to the terminal ene via a linker; and / or the carrier material provided in step (b) is: (i) a carrier material comprising one or more cysteine ​​residues having one or more free thiol groups, (ii) The carrier material is engineered to add one or more additional cysteine ​​residues at a solvent-accessible location; (iii) the carrier material is treated with a thiolating agent; (iv) the carrier material is treated with a reducing agent; or (v) any combination of (i) to (iv).

[0180] 52. A glycoconjugate as defined in any of items 42 to 48, wherein the glycoconjugate is: (a) Synthetic sugar conjugates having the following structure:

[0181] Wherein: CA is the carbohydrate antigen; S-CM is the carrier material having z sulfur atoms available for conjugation, where z is at least 1; X is O, S, NR1, or CH2; R1 is -H, -COH, -COCH3, or -COEt; n is 0, 1, 2, 3, 4, or 5; and R2 is H or Me; or a stereoisomer thereof; or (b) Synthetic sugar conjugates having the following structures:

[0182] Wherein: CA is the carbohydrate antigen; S-CM is the carrier material having z sulfur atoms available for conjugation, wherein z is at least 1; X is S, NR1, CH2, or O; R1 is -H, -COH, -COMe, or -COEt; n is 0, 1, 2, 3, 4, or 5; R2 is H or Me; q is 1, 2, 3, 4, or 5; R3 and R4 are each hydrogen atoms and m is 1, 2, 3, 4, or 5, or R3 and R4 together form a group -CO-CH2- or a group -CO-CH2-CH2-, wherein the carbonyl group is attached to a nitrogen atom and m is 1; or a stereoisomer thereof; or (c) Synthetic sugar conjugates having the following structures:

[0183] Wherein: CA is the carbohydrate antigen; y is at least 1; and when y is greater than 1, CA are the same or different; [S] z -CM is a carrier material having z sulfur atoms available for concatenation, where z is at least equal to y; and L is a connector selected from the group consisting of connectors having the following structures:

[0184] Where: X is O, S, NR1, or CH2; R1 is -H, -COH, -COCH3, or -COEt; n is 0, 1, 2, 3, 4, or 5; and R2 is H or Me, and when y is greater than 1, L is the same or different; or a stereoisomer thereof; or (d) Synthetic sugar conjugates having the following structures:

[0185] Wherein: CA is the carbohydrate antigen; y is at least 1; and when y is greater than 1, CA are the same or different; S-CM is a carrier material having z sulfur atoms available for conjugation, where z is at least 1 and at least equal to y; and L is a linker selected from the group consisting of linkers having the following structures:

[0186] Where: X is S, NR1, CH2, or O; R1 is -H, -COH, -COMe, or -COEt; n is 0, 1, 2, 3, 4, or 5; R2 is H or Me; q is 1, 2, 3, 4, or 5; R3 and R4 are each hydrogen atoms and m is 1, 2, 3, 4, or 5, or R3 and R4 together form the group -CO-CH2- or the group -CO-CH2-CH2-, wherein the carbonyl group is attached to a nitrogen atom, and m is 1; and when y is greater than 1, L are the same or different; or stereoisomers thereof; or (e) Synthetic sugar conjugates having the following structures:

[0187] Wherein: CA is the carbohydrate antigen; y is at least 1; and when y is greater than 1, CA are the same or different; [S] z -CM is a carrier material having z sulfur atoms available for concatenation, where z is at least equal to y; and L is a connector selected from the group consisting of connectors having the following structures:

[0188] Where: X is S, NR1, CH2, or O; R1 is -H, -COH, -COMe, or -COEt; n is 0, 1, 2, 3, 4, or 5; R2 is H or Me; q is 1, 2, 3, 4, or 5; r is 1, 2, 3, 4, or 5; R5 is S-CM, a covalent bond, or a group with the following structure:

[0189] R3 and R4 are each hydrogen atoms and m is 1, 2, 3, 4 or 5, or R3 and R4 together form a group -CO-CH2- or a group -CO-CH2-CH2-, wherein the carbonyl group is attached to a nitrogen atom and m is 1; and L is the same or different when y is greater than 1; or stereoisomers thereof.

[0190] 53. The sugar conjugate as described in item 52: - Having the structure defined in (e), wherein the connector has the following structure:

[0191] Where: X is S, NR1, CH2, or O; R1 is -H, -COH, -COMe, or -COEt; n is 0, 1, 2, 3, 4, or 5; R2 is H or Me; q is 1, 2, 3, 4, or 5; and r is 1, 2, 3, 4, or 5. - Having the structure defined in (e), wherein the connector has the following structure: or

[0192] Where: X is S, NR1, CH2, or O; R1 is -H, -COH, -COMe, or -COEt; n is 0, 1, 2, 3, 4, or 5; R2 is H or Me; q is 1, 2, 3, 4, or 5; and r is 1 or 2; - The carrier material is or comprises a polymer, polypeptide, carrier protein, solid support, particles or any other material having at least one or more free thiol groups suitable for conjugation with the carbohydrate antigen by photocatalytic thiol-ene reaction; - wherein the conjugate material is coupled to at least two identical carbohydrate antigens or more than one type of carbohydrate antigen to produce a multivalent synthetic sugar conjugate; - wherein the carbohydrate antigen is not cleaved from the carrier protein by the subject's endogenous enzymes; or - Any combination thereof.

[0193] 54. A SARS-CoV-2 vaccine comprising one or more glycoconjugates as defined in any one of items 42 to 53, and pharmaceutically acceptable excipients and / or adjuvants.

[0194] 55. The SARS-CoV-2 vaccine as described in Project 54 comprises at least two different glycoconjugates, each glycoconjugate comprising a carrier material conjugated to at least two different carbohydrate antigens selected from sialylated TF antigen (monosialized or disialylated TF antigen), nonsialylated TF antigen, sialylated Tn antigen, and nonsialylated Tn antigen.

[0195] 56. A glycoconjugate as defined in any of items 42 to 53, or a SARS-CoV-2 vaccine as described in item 54 or 55, wherein the glycoconjugate or vaccine induces the production of antibodies that bind to SARS-CoV-2 virus particles, and preferably has neutralizing activity.

[0196] 57. A method for vaccinating a subject against SARS-CoV-2 or for triggering the production of anti-SARS-CoV-2 antibodies in a subject, the method comprising administering any one of items 42 to 53 or the glycoconjugate of item 56, or the SARS-CoV-2 vaccine of any one of items 54 to 56.

[0197] 58. A composition for protecting a subject from SARS-CoV-2 virus infection or for treating COVID-19, the composition comprising one or more ligands (e.g., antibodies, antibody fragments, or lectins) that bind to an O-linked glycan expressed on the SARS-CoV-2 S protein, said O-linked glycan comprising sialylated TF antigen (monosialized or disialylated TF antigen), nonsialylated TF antigen, sialylated Tn antigen, nonsialylated Tn antigen, or any combination thereof.

[0198] 59. The composition used in Item 58, wherein one or more ligands comprise a recombinant monoclonal antibody (e.g., JAA-F11 or humanized JAA-F11).

[0199] 60. The composition used according to item 58, wherein the one or more ligands comprise a lectin (e.g., a lectin that binds to both sialylated and non-sialylated TF antigen forms).

[0200] 61. The composition used according to any one of items 58 to 60, wherein the lectin is Jacalin or a Jacalin-associated lectin.

[0201] 62. The composition used in any one of items 58 to 61 is formulated as an intranasal composition.

[0202] 63 A complex comprising: (a) a SARS-CoV-2 S protein or a fragment thereof expressing an O-linked glycan comprising sialylated TF antigen (monosialized or disialylated TF antigen), nonsialylated TF antigen, sialylated Tn antigen, nonsialylated Tn antigen, or any combination thereof; and (b) a ligand as defined in any one of items 58 to 61, said ligand binding to said O-linked glycan of said SARS-CoV-2 S protein or a fragment thereof.

[0203] 64. The complex according to item 63, wherein the SARS-CoV-2 S protein or a fragment thereof is contained within an intact SARS-CoV-2 viral particle.

[0204] Example Example 1: General Method The reaction was carried out under an argon atmosphere using commercially available HPLC-grade reagents. Commercially available reagents (Sigma Aldrich) were used without further purification. N Acetyl-D-galactosamine and N-acetylneuraminic acid were supplied by Rose Scientific Ltd., Alberta, Canada. Fmoc-β-Ala-Wang resin and Fmoc amino acids were commercially available from Peptide Technologies Ltd., Pierrefonds, Qc, Canada. Use silica gel 60F. 254The reaction progress was monitored by thin-layer chromatography on coated plates (E. Merck). Conjugation via click thiol-ene photoconjugation was carried out in quartz cuvettes (10×10 mm path length, Fisher Scientific Canada, catalog 14958-130) placed between two handheld UV 365 nm lamps (UV-AC handheld lamps, dual 254 / 365 nm UV; 115V-60 Hz, 0.16 amps, VWR Canada, catalog 89131-492) placed between two handheld UV 365 nm lamps (UV-AC handheld lamps, dual 254 / 365 nm UV; 115V-60 Hz, 0.16 amps, VWR Canada, catalog 89131-492). ZEOprep from Canadian Life Science was used. TM Rapid chromatography was performed on silica gel 60 (40-63 µm). Detection was carried out under UV light or by spraying 20% ​​ethanol-sulfuric acid, molybdate, or KMnO4 solution followed by heating. (Bruker ULTRASHIELD) TM NMR spectra were recorded on a 300 MHz and a Bruker Avance™ III HD 600 MHz spectrometer. Proton and carbon chemical shifts ( ) relative to 7.26 ppm ( 1 H) and 77.16 ppm ( 13 C) The chemical shift of residual CHCl3 is reported in ppm. Coupling constant ( J Reporting is in Hertz (Hz), and peak multiplicity is abbreviated as follows: singlet (s), doublet (d), doublet (dd), doublet with equal coupling constant (t). ap Three-peak (t) and multi-peak (m) patterns were observed. Analysis and partitioning were performed using COSY (correlation spectroscopy) and HSQC (heteronuclear single quantum coherence) experiments. High-resolution mass spectrometry (HRMS) measurements were performed using an Agilent Technologies LC-MS-TOF (liquid chromatography-mass spectrometry time-of-flight) instrument in positive and / or negative electrospray ionization modes via the UQAM analytical platform. Protonated ions (M+H) were also measured. + Or sodium adduct (M+Na) + For empirical formula validation. Natural TT and TT conjugates were dialyzed using 2000 kDa benzoylation dialysis tubes (Sigma-Aldrich (Ontario, Canada)). The thiol content of natural and conjugated TT was determined at 412 nm by Ellman assay (Ellman, GL). Arch. Biochem. Biophys. 1959, 82, 70-77). The total sugar content of the TT-conjugate was determined by a colorimetric DuBois test at 492 nm using UV / VIS spectroscopy (Dubois, M.; Gilles, KA; Hamilton, JK; Rebers, PA; Smith, F. Colorimetric Method for Determination of Sugars and Related Substances). Anal. Chem ., 1956, 28 (350-356). Dynamic light scattering (DLS) and particle size distribution were measured in PBS using a Zetasizer Nano S90 from Malvern. Mouse monoclonal IgG3 antibody JAA-F11 was generated as previously described in Rittenhouse-Diakun et al., 1998.

[0205] General Solid Phase Peptide Synthesis (SPPS) Procedure Solid-phase peptide synthesis (SPPS) was performed according to a literature procedure (Papadopoulos et al., 2012) and started with Fmoc-β-Ala-Wang resin (650 mg, 0.34 mmol, 1.0 equivalent; 100-200 mesh, loading = 0.52 mmol / g). The reaction was carried out by rotary stirring in a 1.5 × 14 cm (20 mL) disposable Econo-Pac column (Bio-Rad Laboratories, ON, Canada). The resin swelled in CH₂Cl₂ for 1 h, then filtered and reduced in DMF for 1 h. The Fmoc protecting groups of commercially available resins or amino acids were removed with a 20% piperidine DMF solution (5 mL, 2 × 5 min then 1 × 10 min). Solvents and reagents were removed by filtration, and the resin was washed with DMF, CH₂Cl₂, and MeOH (3X each). The presence of free amino groups was verified by the Kaiser test or TNBS test. The free amines on the resin were treated with a deactivated Fmoc amino acid solution: at 4°C, 3 equivalents of amino acids and 3 equivalents of HBTU in DMF were added. N,N,N′,N′ -Tetramethyl- O -(1 H1-Benzotriazole-1-yl)uronium hexafluorophosphate) and catalytic amount of HOBt (1-hydroxybenzotriazole) (10 min). Then DIPEA (diisopropylamine, 9 equivalents) was added to the mixture and stirred at room temperature for 1 hour 30 minutes. The completion of coupling was confirmed by Kaiser or TNBS colorimetric assay. After filtration, the resin was washed and the Fmoc removal procedure was repeated again. At the end of the synthetic sequence, the last free amine was capped by acetylation (Ac2O / DIPEA / DMF 1:1:8, 1 h). After filtration, the solution was drained, the resin was dried under vacuum and lysed for 3 h using trifluoroacetic acid / water / ethylenedithiol / triisopropylsilane (94.0 / 2.5 / 2.5 / 1.0). The resulting peptide was precipitated with methyl tert-butyl ether and separated from the resin beads by centrifugation (20 min, 2000 rpm, 3X). The precipitate was carefully dried with an air jet stream. The crude peptide was dissolved in water to separate it from the resin. The solution is then freeze-dried to provide the desired peptides.

[0206] Purification of tetanus toxoid monomers Tetanus toxoid (TT) monomers were obtained by gel filtration chromatography prior to conjugation. One mL of a liquid formulation containing 4.5 mg / mL protein (determined by a modified Lowry protein assay) was loaded into an XK16-100 column packed with Superdex® 200 Prep Grade (GE Healthcare Life Sciences, Uppsala, Sweden), equilibrated in PBS (20 mM NaHPO4 [pH 7.2], 150 mM NaCl), and eluted with the same buffer. The protein eluted from the column as two peaks: an earlier elution peak containing the oligotoxoid, and a later elution peak corresponding to 150,000 MR containing the TT monomer. Fractions corresponding to the later (monomer) peak were combined, desalted with deionized water, concentrated using a Centricon® Plus-70 centrifugal filtration system (30K Ultracel PL membrane; Millipore, Billerica, MA), and then lyophilized.

[0207] HPLC analysis of conjugates HPLC analysis of the allyl neosaccharide conjugate formulation was performed using size exclusion chromatography. Chromatographic separation was performed using three tandem 8 × 300 mm Shodex OHpak gel filter columns (two SB-804 and one SB-803) with SB-807G guard columns (Showa Denko). The neosaccharide conjugate immunogen was eluted with 0.1 M NaNO3 at a flow rate of 0.4 mL / min using a Knauer Smartline system equipped with a Type 2300 differential refractometer (RI) detector and a Type 2600 UV detector at a wavelength of 280 nm. 50- The conjugate formulation (8 mg / mL solution in the mobile phase) was introduced via the injection loop. In selected experiments, fractions eluted in an empty volume corresponding to the conjugate fraction were combined, dialyzed with water (Spectra / Por; molecular weight cutoff (MWCO) 12,000 to 14,000 [Spectrum Laboratories]), and lyophilized. This corresponds to a 2:1 fractionation of the conjugate.

[0208] Example 2: Allyl 2-acetamido-3,6-di-O-neovalero-2-deoxy-α-D-glucopyranoside (compound) Object 2)

[0209] Compound 2 refer to Figure 2 Acetyl chloride (2.76 mL, 38.80 mmol, 3.43 equivalents) was added dropwise to allyl alcohol (20.8 mL) at 0 °C under an argon atmosphere. At room temperature, [the volume of the solution was increased]. N -Acetyl-D-glucosamine (Compound 1) (2.50 g, 11.3 mmol, 1.00 equivalent). The reaction mixture was stirred at 70 °C for 3 h, then quenched by adding solid NaHCO3 until pH 7. The suspension was filtered through a diatomaceous earth pad and washed several times with MeOH. The solvent was removed under reduced pressure, and the crude allyl 2-acetamido-2-deoxy-D-glucosamine was precipitated by milling with Et2O / ethanol. The solvent was then removed under reduced pressure several times after milling. Neopentanoyl chloride (3.90 mL, 31.64 mmol, 2.80 equivalent) was then added dropwise to a suspension of the crude allyl 2-acetamido-2-deoxy-α-D-glucanopyranoside intermediate in anhydrous dichloromethane-pyridine (45 mL, v / v, 1:2) mixture under a nitrogen atmosphere at -15 °C. The reaction mixture was stirred for 2 h and warmed to room temperature to give the desired α-anomeric compound (Compound 2) (R f =0.32) and some β-anodes (R f= 0.18); hexane / EtOAc 1:1). The mixture was then diluted with CH2Cl2, and the organic phase was washed several times with HCl (1M), saturated KHSO4 aqueous solution, saturated NaHCO3 solution, and brine. The organic phase was dried over Na2SO4, filtered, and evaporated under reduced pressure. The pale yellow oil was purified by silica gel rapid chromatography (hexane-EtOAc 6:4 to 1:1) to give the desired compound allyl 2-acetamido-3,6-di- O - Neopentanoyl-2-deoxy-α-D-glucopyranoside (compound 2) is a white solid (4.85 g, 6.78 mmol, 60%). R f = 0.32; Hexane / EtOAc 1:1; Figure 3A and 3B : 1 H NMR (CDCl3, 600 MHz): δ 5.87 (dddd, 1H, J H,H = 16.8, 10.5, 6.2, 5.3Hz, OCH2C H =CH2), 5.77 (d, 1H, J NH,H2 = 9.7 Hz, N H ), 5.31-5.25 (m, 1H, OCH2CH=C H 2), 5.22 (dd, 1H, J H,H = 10.4, 1.3 Hz, OCH2CH=C H 2), 5.09 (dd, 1H, J 3,4 = 10.7, J 2,3 = 9.3 Hz, H-3), 4.83 (d, 1H, J 1,2 = 3.7 Hz, H -1), 4.39 (m, 1H, H -6a), 4.35-4.25 (m, 2H, H -6b and H -2), 4.19 (m, 1H, OC H 2), 4.02-3.93 (m, 1H, OC H 2), 3.85 (m, 1H, H -5), 3.59-3.48 (m, 1H, H-4), 3.03 (d, 1H, J 4,OH = 5.1 Hz, O H -4), 1.93 (s, 3H, NHCOC) H 3), 1.23 (s, 9H, tert-butyl) and 1.19 ppm (s, 9H, tert-butyl); 13 CNMR (CDCl3, 150 MHz): δ 179.8, 179.1 ( Uncle -Bu C O), 169.7 (NH C O), 133.2 (OCH2 C H=CH2), 118.1 (OCH2CH= C H2), 96.4 ( C -1), 73.4 ( C -3), 70.5 ( C -5), 69.1 ( C -4).68.2(O C H2), 63.1 ( C -6), 51.4 ( C -2), 39.0, 38.9 (2 x C (CH3)3), 27.2, 27.0 (2 x C( C H3)3) and 23.2 ppm ( C H3). Figure 3C and 3D ESI + -HRMS: M+H + C 21 H 36 Calculated O8N value: 430.2435; measured value: 430.2445. The β-anomeric compound was separated as a white solid (971 mg, 2.26 mmol, 20%). R f = 0.18; Hexane / EtOAc 1:1; 1 H NMR (CDCl3, 300 MHz): δ 6.00 (d, 1H, J NH,H2 = 9.3 Hz, N H ), 5.95-5.75 (m, 1H, OCH2C H=CH2), 5.35-5.03 (m, 3H, OCH2CH=C H 2 and H -3), 4.55 (d,1H, J 1,2 = 8.4 Hz, H -1), 4.47-425 (m, 3H, H -6a, 6b and OC H 2), 4.14-3.90 (m, 2H, OC) H 2 and H -2), 3.65-3.43 (m, 2H, H -5 and H -4), 3.23 (sb, 1H, O H -4), 1.92 (s, 3H, NHCOC) H 3), 1.23 (s, 9H, tert-butyl) and 1.20 ppm (s, 9H, tert-butyl).

[0210] Example 3: Allyl 2-acetamido-2-deoxy-α-D-galactopyranoside (Allyl Tn)

[0211] Allyl Tn See Figure 2 The di- pyridine in an anhydrous dichloromethane-pyridine (126 mL, v / v 20:1) mixture was removed under an argon atmosphere. O - Neopentyl compound (compound 2) (5.50 g, 12.80 mmol, 1.0 equivalent) was cooled to -35°C. Trifluoromethanesulfonic anhydride (2.58 mL, 15.36 mmol, 1.2 equivalent) was then added and the mixture was stirred at this temperature. The temperature was raised to room temperature for 2 hours. Water (12 mL) was then added to the solution. The mixture was heated and refluxed (…). The mixture was stirred overnight (12 hours) at 50°C. After reaching room temperature, the reaction mixture was diluted with dichloromethane and washed several times with 1M HCl aqueous solution. The organic layer was washed with H2O, saturated NaHCO3, and brine. The organic layer was dried over Na2SO4, filtered, and evaporated under reduced pressure. The crude product was treated under Zemplén conditions (40 mL of 1M sodium methoxide in methanol, pH 9). The solution was stirred overnight at 50°C. After cooling to room temperature, the solution was subjected to ion exchange with Amberlite® IR 120 (H2O). + Neutralize, filter, wash with MeOH, and remove solvent under reduced pressure. After lyophilization, allyl T is separated by precipitation in MeOH / EtOAc / hexane. NIt was a white solid (2.36 g, 9.10 mmol, 71%). R f = 0.32; EtOAc / MeOH 4:1; Figure 4A and 4B : 1 H NMR (CD3OD, 600 MHz): δ 5.99-5.88 (m, 1H, OCH2C H =CH2), 5.31 (dd, 1H, J trans = 17.3, J gem = 1.3 Hz, OCH2CH=C H 2), 5.17 (dd, 1H, J cis = 10.5 Hz, OCH2CH=C H 2), 4.86 (d, 1H, J 1,2 = 3.8 Hz, H -1), 4.27 (dd, 1H, J 2,3 = 11.0 Hz, H -2), 4.20 (m, 1H, OC H 2), 4.00 (m, 1H, OC H 2), 3.89 (dd, J 3,4 = J 4,5 = 2.6 Hz, H -4), 3.85-3.77 (m, 2H, H -3 and H -5), 3.72 (m,2H, H -6a and H -6b) and 1.99 ppm (s, 3H, C H 3); 13 C NMR (CD3OD, 150 MHz): δ 172.5(NH C O), 134.2 (OCH2 C H=CH2), 116.1 (OCH2CH= C H2), 96.6 ( C -1), 71.2 ( C -3), 69.0( C -4), 68.3 (C -5).67.8 (O C H2), 61.4 ( C -6), 50.2 ( C -2) and 21.2 ppm ( C H3). Figure 4C and 4D ESI + -HRMS: M+H + C 11 H 20 Calculated value of O6N: 262.1285; Measured value: 262.1294.

[0212] Procedure B: Allyl 2-acetamido-2-deoxy-α-D-galactopyranoside can also be derived from the literature procedure. N Direct preparation of acetylgalactosamine (GalNAc) (Feng et al., 2004: at room temperature) N Add BF3·Et2O (250 µL, 2 mmol, 1.0 equivalent) to an allyl alcohol (8 mL) solution of acetylgalactosamine (442 mg, 2 mmol, 1.0 equivalent) and stir the mixture at 70 °C for 2 hours. Cool the solution to room temperature and remove the solvent under reduced pressure. Dissolve the dried crude product in a minimal amount of EtOH (5 mL). Add the desired allyl T N The product precipitated in diisopropyl ether and separated as a white solid (417 mg, 1.60 mmol, 80%).

[0213] C-allyl GalNAc analogues ( Figure 2 [1-(2'-acetamido-2'-deoxy-α-D-galactopyranosyl)-2-propene] was prepared according to a literature procedure (Cipolla et al., 2000: 3-(2-acetamido-3,4,6-tri- O 1-Acetyl-2-deoxy-α-D-galactopyranosyl)-1-propene (Cui et al., 1998) (371 mg, 1.00 mmol, 1.0 equivalent) was treated under Zemplén conditions (1 M sodium methoxide in methanol, 5 mL, pH 8–9). The solution was stirred at room temperature for 1 h. The reaction mixture was then subjected to ion exchange with an ion exchange resin (Amberlite® IR 120, H... + Neutralize, filter, wash with MeOH, and remove solvent under reduced pressure. C-Allyl T NPurified by silica gel chromatography (EtOAc / MeOH 9:1 to 4:1), followed by crystallization in EtOH to a white solid (213 mg, 0.87 mmol, 87%). f = 0.28; EtOH 4:1; mp 230℃ (Litt. 215-217℃, EtOAc / EtOH); Based on literature NMR data: 1 H NMR (CD3OD, 600 MHz): δ 5.81(m, 1H, 1 CH2C H =CH2), 5.08 (dd, 1H, J trans = 17.2, J gem = 1.7 Hz, 1 CH2CH=C H 2), 5.02(dd, 1H, J cis = 10.2 Hz, 1 CH2CH=C H 2), 4.22 (dd, 1H, J = 9.3, 5.0 Hz, H -2'), 4.14 (dt, 1H, J = 10.0, 5.0 Hz, H -1'), 3.91 (dd, 1H, J = 3.0 Hz, H -4'), 3.82-3.64 (m, 4H, H -3', H -5' and H -6'ab), 2.45 (m, 1H, H -1a), 3.17 (m, 1H, H -1b) and 1.97 ppm (s, C H 3); 13 C NMR (CD3OD, 150 MHz): δ 173.6 (NH C O), 136.2 ( 1 CH2 C H=CH2), 117.1 ( 1 CH2CH= C H2), 72.9 ( C -1'), 69.7 ( C -4'), 69.5 (C -3' and C -5'), 61.8( C -6'), 52.0 ( C -2'), 32.4 ( 1 C H2) and 22.5 ppm ( C H3). ESI + -LCMS: M+H + C 11 H 20 Calculated O5N value: 246.1336; Measured value: 246.1332. CAN / H2O 5 to 95% 1.4 min.

[0214] S-allyl GalNAc analogues ( Figure 2 Prepared according to the literature (Knapp et al., 2002).

[0215] Example 4: Allyl 2-acetamido-4,6-O-benzyl-2-deoxy-α-D-galactopyranoside (compound) 4)

[0216] Compound 4 refer to Figure 2 A catalytic amount of p-toluenesulfonic acid monohydrate was added to a solution of allyl GalNAc (Tn) (2.35 g, 9.0 mmol, 1.0 equivalent) and benzaldehyde dimethyl acetal (6.75 mL, 45.0 mmol, 5.0 equivalent) in anhydrous DMF (20 mL). The mixture was stirred at room temperature. After 5 hours, the mixture was diluted with CHCl3 and washed with a saturated aqueous solution of NaHCO3. The organic layer was separated and washed with water, dried over Na2SO4, and concentrated to give a white solid. Benzylene acetal (compound 4) was separated by precipitation in EtOAc / hexane as a white solid (2.64 g, 7.56 g, 84%). f = 0.21; DCM / MeOH9.0:0.5; Figure 5A : 1 H NMR (CDCl3, 300 MHz): δ 7.59-7.46 (m, 2H, H -ar), 7.43-7.31(m, 3H, H -ar), 5.91 (m, 1H, OCH2C H =CH2), 5.75 (d, 1H, J NH,H2= 9.0 Hz, N H ),5.58 (s, 1H, PhC H ), 5.34-5.17 (m, 2H, OCH2CH=C H 2), 5.01 (d, 1H, J 1,2 = 3.5 Hz, H -1), 4.56-4.42 (ddd, 1H, J 2,3 = 10.9 Hz, J 2,OH = 9.1 Hz, H -2), 4.34 (dd, 1H, J 5,6a = 1.5 Hz, J 6a,6b = 12.5 Hz, H-6a), 4.19 (m, 2H, H -4 and OC H 2), 4.04 (m, 1H,dd, 1H, J 5,6b = 1.6 Hz, J 6a,6b = 12.5 Hz, H-6b), 4.01 (m, OC H 2), 3.86 (dd, 1H, J 3,4 = 10.9 Hz, H -3), 3.71 (sb, 1H, H -5), 2.80 (d, 1H, J 3,OH = 10.7 Hz, O H -3) and 2.05 ppm (s, 3H, C H 3); Figure 5B and 5C ESI + -HRMS: M+H + C 18 H 24 Calculated value of O6N: 350.1598; Measured value: 350.1608.

[0217] Example 5: Allyl(2,3,4,6-tetra-O-benzoyl-β-D-galactopyranosyl)-(1→3)-2-acetyl Amino-4,6-O-benzyl-2-deoxy-α-D-galactopyranoside (Compound 6)

[0218] Compound 6 refer to Figure 2 Compound 4 (2.0 g, 5.72 mmol, 1.0 equivalent) and mercuric cyanide (2.17 g, 8.60 mmol, 1.5 equivalent) were dissolved in anhydrous nitromethane-toluene (100 mL, 3:2, v / v) containing 4 Å molecular sieves under an argon atmosphere. The mixture was stirred at room temperature for 30 minutes. 2,3,4,6-tetra- O -benzoyl-α-D-pyranogalactosyl bromide (compound 5) (5.66 g, 8.58 mmol, 1.5 equivalents) was added to the mixture. The solution was stirred at 70 °C for 5 h, and then stirred overnight (8 h) at room temperature. The solvent was removed under reduced pressure after TLC (DCM / MeOH 9.0:0.5) indicated that the starting material (compound 4) had been completely consumed. The residue was dissolved in EtOAc and then filtered through a diatomaceous earth mat. The filtrate was washed successively with 10% potassium iodide aqueous solution, saturated sodium bicarbonate solution, and water, and then dried over Na2SO4. The solvent was evaporated under reduced pressure to give a white foam. The crude product was purified by silica gel chromatography using a gradient from 100% hexane to hexane / EtOAc 1:2 to give the desired disaccharide (compound 6) as a white solid (4.98, 5.38 mmol, 94%). mp: 110-111 °C, R f = 0.20; Hexane / EtOAc 1:2; Figure 6A and 6B : 1 H NMR (CDCl3, 600 MHz): δ 8.06-7.19 (m, 5H, H -ar), 5.98 (dd, 1H, J 3,4’ = 3.3 Hz, J 4,5’ = 1.0 Hz, H-4 II ), 5.85-5.78 (m, 2H, OCH2C H =CH2 and H-2 II ), 5.60 (dd, 1H, J 2,3’ = 10.2 Hz, J 3,4’ = 3.4 Hz, H-3 II ), 5.48 (sb, 1H,N H ), 5.23 (m, 3H, OCH2CH=CH 2 and H -1), 4.68 (dd, 1H, J 5’,6a’ = 6.9 Hz, J 6a’,6b’ =11.4 Hz, H-6a I ), 4.63-4.58 (m, 1H, H -2), 4.46-4.36 (m, 3H, H -4. H -5 and H -6b II ), 4.14-4.07 (m, 3H, H -6a, OC H 2 and H -3), 3.96 (m, 1H, OC H 2), 3.75 (m, 1H, H -6b), 3.51 (m, 1H, H -5) and 1.40 ppm (s, 3H, CH3); 13 C NMR (CDC 13 , 150 MHz): δ170.0 (NH C O), 166.0, 165.5, 165.4, 165.2 ( C O), 137.6-126.2 (multi, 30 C -arom), 133.2 (OCH2 C H=CH2), 117.8 (OCH2CH= C H2), 102.0 ( C -1 II ), 100.9 (CPh C H), 97,3 ( C -1 I ), 76.1 ( C -3), 75.4 ( C -4), 71.7 ( C -3 II and C -5 II ), 70.2 ( C -2 II ), 69.1( C -6), 68.6 (O C H2), 68.1 ( C -4II ), 62.9 ( C -5), 62.6 ( C -6 I ), 48.2 ( C -2) and 22.5ppm ( C H3). Figure 6C and 6D ESI + -HRMS: M+H + C 52 H 50 O 15 Calculated value of N: 928.3175; Measured value: 928.3133.

[0219] Example 6: Allyl (β-D-galactopyranosyl)-(1→3)-2-acetamido-2-deoxy-α-D-galactopyranosyl Lactoside (allyl TF)

[0220] Allyl TF refer to Figure 2 The solution of compound 6 (1.12 g, 1.20 mmol, 1.0 equivalent) in 1 M sodium methoxide in methanol (12 mL, pH 8–9) was stirred at room temperature until the starting material was consumed. After 1 hour and 30 minutes, the solution was added to an ion exchange resin (Amberlite IR 120, H... + The solution was neutralized, filtered, washed with MeOH, and then suspended in silica gel, filtered, and the solvent was removed under reduced pressure. The silica gel was washed several times with 100% EtOAc, and then washed with a second solution (EtOAc / MeOH / H2O 1:1:0.1). The combined filtrates were evaporated under reduced pressure to give the intermediate (compound 7), a white solid. f = 0.20; CHCl3 / MeOH / H2O 11:6:1; Figure 7A and 7B : 1 H NMR (D2O, 600 MHz): δ 7.47-7.39 (m, 2H, H -ar), 7.37-7.26 (m, 3H, H -ar), 5.82 (m, 1H, OCH2C H =CH2), 5.61 (s, 1H, PhC H ), 5.20-5.09(m, 2H, OCH2CH=C H 2), 4.88 (d, 1H, J1,2 = 3.4 Hz, H -1), 4.47 (dd, H -4), 4.37 - 4.25 (m, 2H, H -2 and H -1 II ), 4.13 - 3.98 (m, 4H, H -3, H -6a, H -6b, OC H 2), 3.96 - 3.88 (m, 1H, H -5), 4.56 - 4.42 (ddd, 1H, J 2,3 = 10.9 Hz, J 2,OH = 9.1 Hz, H -2),4.34 (dd, 1H, J 5,6a = 1.5 Hz, J 6a,6b = 12.5 Hz, H-6a), 4.19 (m, 2H, H -4 and OC H 2), 4.04 (m, 1H, dd, 1H, J 5,6b = 1.6 Hz, J 6a,6b = 12.5 Hz, H-6b), 4.01 (m,OC H 2), 3.86 (dd, 1H, J 3,4 = 10.9 Hz, H -3), 3.71 (sb, 1H, H -5), 2.80 (d, 1H, J 3,OH = 10.7 Hz, O H -3) and 2.05 ppm (s, 3H, C H 3); 3.83 (s, 1H, OC H 2), 3.72 (d,1H, J 3’,4’ = J 4’,5’ = 3.2 Hz, H-4 II), 3.65 - 3.55 (m, 2H, H -6a,b), 3.49, (m, 1H, H -5 II ), 3.43 (dd, 1H, J 2’,3’ = 10.0 Hz, J 3’,4’ = 3.3 Hz, H -3 II ), 3.33 - 3.24 (m, 1H, H -2 II ) and 1.86 ppm (s, 3H, C H 3); 13 C NMR (D2O, 150 MHz): δ 174.6 (NH C O), 136.8( C -arom), 133.6 (OCH2 C H=CH2), 129.9, 128.7, 126.5 ( C -arom), 118.0 (OCH2CH= C H2), 104.9 ( C -1 II ), 101.3 ( C HPh), 97.0 ( C -1), 76.0 ( C -4), 75.0 ( C -3), 75.0( C -5 II ), 72.4 ( C -3 II ), 70.4 ( C -2 II ), 69.0 ( C -6), 68.7 (OCH2CH= C H2), 68.6 ( C -4 II ), 63.0 ( C -5), 61.0 ( C -6 II ), 48.6 ( C -2) and 22.0 ppm ( C H3). R f = 0.38; EtOAc / MeOH / H2O 7:3:0.1; R f= 0.46; ACN / MeOH / H2O 7:2:1. Figure 7C and 7D ESI + -HRMS: M+H + C 24 H 34 O 11 Calculated value of N: 512.2126; Measured value: 512.2119.

[0221] The white solid intermediate was then dissolved in 10 mL of 60% aqueous acetic acid, and the resulting solution was stirred at 60 °C for 1.5 hours. The solvent was removed under reduced pressure, and the residue was lyophilized to give the final allyl TF as a white solid (427 mg, 1.0 mmol, 84%). mp = 230–232 °C; R f = 0.53; CHCl3 / MeOH / H2O 11:6:1; Figure 8A and 8B : 1 HNMR (D2O, 600 MHz): δ 5.80 (m, 1H, OCH2C H =CH2), 5.19 (dd, 1H, J trans = 17.3 Hz, OCH2CH=C H 2), 5.09 (dd, 1H, J cis = 10.4 Hz, OCH2CH=C H 2), 4.77 (d, 1H, J 1,2 = 3.7Hz, H -1), 4.29 (d, 1H, J 1,2 = 3.7 Hz, H -1), 4.29 (d, 1H, J 1,2 = 7.8 Hz, H -1 II ),4.16 (dd, 1H, J 2,3 = 11.2 Hz, J 1,2 = 3.7 Hz, H -2), 4.08-4.01 (m, 2H, H -4 and OCH 2), 3.92-3.82 (m, 3H, H -3, H -5 and OC H 2), 3.73 (dd, 1H, H -4 II ), 3.63-3.52(m, 4H, H-6a,b and H-6'a,b), 3.47 (m, 2H, H -3 II and H -5 II ), 3.39 (dd, 1H, J 2’,3’ =10.0 Hz, J 1’,2’ = 7.7 Hz, H -2 II ) and 1.85 ppm (s, 3H, CH3); 13 C NMR (150 MHz, CDC) 13 ): δ 174.6 (NH C O), 133.7 (OCH2 C H=CH2), 117.9 (OCH2CH= C H2), 104.7 (C-1 II ),96.4 ( C -1), 77.2 ( C -3), 75.0 (C-5 II ), 72.5 (C-3 II ), 70.7 ( C -5), 70.6 (C-2 II ),68.8 ( C -4), 68.6 (C-4 II ), 68.4 (O C H2), 61.2 (C-6 II ), 61.0 ( C -6), 48.6 ( C -2) and 22.0 ppm ( C H3). Figure 8C and 8D ESI + -HRMS: M+Na + C 17 H 29 O 11Calculated NNa value: 446.1633; Measured value: 446.1613.

[0222] Example 7: 3-{[3-(2-acetamido-2-deoxy-α-D-galactopyranosyl)oxypropyl]thio}propionic acid (compound) Object 8)

[0223] Compound 8 refer to Figure 9 A solution of allyl Tn (392 mg, 1.50 mmol, 1.0 equivalence) and 3-mercaptopropionic acid (392 µL, 4.50 mmol, 3.0 equivalence) in degassed 2O / MeOH (3 mL, 1:1, v / v) was stirred overnight at 254 nm (A) or stirred for 15 min at 365 nm with DMPAP (23 mg, 0.03 mmol, 0.06 equivalence) (B). The mixture was then concentrated with silica gel under reduced pressure and purified by rapid silica gel chromatography via a gradient (EtOAc 100% to EtOAc / MeOH / AcOH 3:1:0.01) to give the desired compound 8 as a white foam (from A: 536 mg, 1.46 mmol, 97%); (from B: 82%). f = 0.34; EtOAc / MeOH / AcOH 3:1:0.01; 1 H NMR (CD3OD, 600 MHz): δ 4.82 (d, 1H, J = 3.7 Hz, H-1), 4.25 (dd, 1H, J = 11.0, 3.7 Hz, H-2), 3.89 (d, 1H, J = 3.2 Hz, H-4), 3.85-3.72 (m, 5H, H-3, H-5, H-6a, H-6b,OCH2), 3.49 (dt, 1H, J = 10.0, 6.0 Hz, OCH2), 2.77 (t, 2H, J = 7.1 Hz, CH2), 2.68 (t, 2H, J = 7.2 Hz, CH2), 2.58 (t, 2H, J = 7.1 Hz, CH2), 2.00 (s, 3H, CH3), 1.95-1.83 (m, 2H, CH2); 13C NMR (CD3OD, 150 MHz): δ 174.6 (NH C O), 172.5 ( C O), 97.5 ( C -1), 71.1 ( C -3), 69.0 ( C -4), 68.3 ( C -5), 66.1 (O C H2), 61.4 ( C -6), 50.3 ( C -2), 34.5, 29.1, 28.3, 26.6 (4x C H2) and 21.3 ppm ( C H3). ESI + -HRMS: M+H + C 14 H 26 O8NS calculated value: 368.1374; measured value: 368.1377. Figure 10 ).

[0224] Example 8: Pentafluorobenzene of 3-{[3-(2-acetamido-2-deoxy-α-D-galactopyranosyl)oxypropylthio}propionic acid Basic ester (compound 9)

[0225] Compound 9 refer to Figure 9 The crude reaction of unpurified compound 8 (522.54 mg, 2.0 mmol, 1.0 equivalence), 3-mercaptopropionic acid (522 µL, 6.0 mmol, 3.0 equivalence), and DMPAP (31 mg, 0.12 mmol, 0.06 equivalence) was carried out at room temperature with pentafluorophenol (2.210 g, 12.0 mmol, 6.0 equivalence) and EDC.HCl (1.725 g, 9.0 mmol, 4.5 equivalence) in THF / H2O (10 mL, 4:1, v / v) for one hour. The mixture was concentrated under reduced pressure. The crude product was purified by silica gel rapid chromatography (EtOAc / MeOH 9:1) to give ester compound 9 as a white solid (320 mg, 0.60 mmol, 30%).

[0226] The purified acid of compound 8 (100 mg, 0.27 mmol, 1.0 equivalence) in water (1.0 mL) was treated with pentafluorophenol (200 mg, 1.09 mmol, 2.0 equivalence) in THF (4 mL) and EDC.HCl (104 mg, 0.55 mmol, 2.0 equivalence) at room temperature for one hour. The mixture was concentrated under reduced pressure. The crude product was purified by silica gel rapid chromatography (EtOAc / MeOH 9:1) to give ester compound 9 as a white solid (49 mg, 0.09 mmol, 34%). f = 0.18; EtOAc / MeOH 9:1; 1 H NMR (CD3OD, 600 MHz): δ 4.73 (d, 1H, J = 3.7 Hz, H-1), 4.25 (dd, 1H, J =11.0, 3.7 Hz, H-2), 3.79 (d, 1H, J = 3.2 Hz, H-4), 3.74-3.54 (m, 5H, H-3, H-5, H-6a, H-6b, OCH2), 3.41 (dt, 1H, J = 10.0, 6.0 Hz, OCH2), 2.95 (t, 2H, J =6.9 Hz, CH2), 2.81 (t, 2H, J = 6.9 Hz, CH2), 2.64 (t, 2H, J = 7.2 Hz, CH2),1.89 (s, 3H, CH3), 1.80 (q, 2H, J = 6.7 Hz, CH2); 13 C NMR (CD3OD, 150 MHz): δ172.5 (NH C O), 168.1 ( C O), 97.5 ( C -1), 71.1 ( C -3), 69.0 ( C -4), 68.3 ( C -5), 66.0 (O C H2), 61.4 ( C -6), 50.3 ( C -2), 33.6, 29.0, 28.3, 26.0 (4x CH2) and 21.2ppm ( C H3); 19 F NMR (CD3OD, 564 MHz): δ -(154.86-156.03, m), -(160.90-161.58,m), -(165.33-166.16, m); ESI + -LC-MS: M+H + C 20 H 25 Calculated value of O8NSF5: 534.1216; Measured value: 534.1222, 6.69 min. Figure 11A and 11B ).

[0227] Example 9: 3-{[3-(2,3,4,6-four- O -benzoyl-β-D-galactopyranosyl)-(1→3)-2-acetyl Amino-4,6- O [-benzyl-2-deoxy-α-D-galactopyranosyl]oxypropyl]thiopropionic acid (compound 10)

[0228] Compound 10 refer to Figure 12 A solution of protected allyl TF (compound 6) (659 mg, 0.71 mmol, 1.0 equivalence) and 3-mercaptopropionic acid (186 µL, 2.13 mmol, 3.0 equivalence) in degassed THF (3 mL) was stirred with DMPAP (11 mg, 0.43 mmol, 0.06 equivalence) for 15 min at 365 nm irradiation at room temperature. The mixture was then concentrated under reduced pressure and purified by silica gel rapid chromatography (DCM / MeOH / AcOH 98:2:0.01) to give the desired compound 10 as a white foam (712 mg, 0.69 mmol, 97%); R f = 0.25; MeOH / MeOH / AcOH 98:2:0.01; 1 H NMR (CDCl3, 600 MHz): δ 8.05-7.14 (m, 25H), 5.92 (t, 1H, J = 7.2 Hz, 1H), 5.85-5.76 (m,1H), 5.75-5.65 (m, 1H), 5.53-5.47 (m, 1H), 5.45 (d, 1H, J = 9.5 Hz), 5.16(dd, 1H, J = 19.2, 11.5 Hz), 5.10-5.02 (m, 1H), 4.71 (d, 1H,J = 3.5 Hz), 4.69-4.65 (m, 1H), 4.65-4.60 (m, 1H), 4.50 (m, 1H), 4.43-4.29 (m, 1H), 4.12-3.97 (m, 1H), 3.88 (td, 1H, J = 10.7, 3.3 Hz), 3.67 (ddd, 1H, J = 19.3, 12.1,5.3 Hz), 3.40-3.38 (m, 1H), 3.25 (td, 1H, J = 9.5, 2.7 Hz), 2.91-2.82 (m,1H), 2.73-2.45 (m, 1H) and 1.64 ppm (s, 1H); 13 C NMR (CDC 13 , 150 MHz): δ177.07, 175.59, 170.46, 137.73, 133.51, 133.47, 133.41, 133.21, 133.17,129.90, 129.64, 129.61, 128.57, 128.50, 128.19, 128.03, 125.99, 102.57,100.45, 98.87, 76.44, 75.58, 72.02, 71.38, 69.41, 69.01, 68.17, 66.82, 63.15,62.45, 52.49, 36.62, 31.48, 29.54, 28.68, 25.96, 22.55, 20.05 and 14.03 ppm; ESI + -HRMS: M+Na + C 55 H 55 O 17 Calculated NSNa value: 1056.3083; Measured value: 1056.3116.

[0229] Example 10: 3-{[3-(β-D-galactopyranosyl)-(1→3)-2-acetamido-2-deoxy-α-D-galactopyranosyl] Lactosyl)oxypropyl]thio]propionic acid (compound 12)

[0230] Compound 12 refer to Figure 12A solution of allyl TF (compound 11) (85 mg, 0.2 mmol, 1.0 equivalent) and 3-mercaptopropionic acid (52 µL, 0.6 mmol, 3.0 equivalent) in degassed H₂O / MeOH (3 mL, 1:2, v / v) was stirred overnight at 254 nm under room temperature. The mixture was then concentrated with silica gel under reduced pressure and purified by rapid silica gel chromatography via a gradient (EtOAc 100% to EtOAc / MeOH / H₂O 7:2:1) to give the desired compound 12 as a white foam (87 mg, 0.16 mmol, 82%).

[0231] A solution of compound 10 (672 mg, 0.65 mmol, 1.0 equivalent) in 1 M sodium methoxide in methanol (6 mL, pH 8–9) was stirred at room temperature until the starting material was consumed. After 1 hour and 30 minutes, the solution was added via ion exchange resin (Amberlite IR 120, H...). + The solution was neutralized to pH 4, washed with methanol, and concentrated under reduced pressure. The crude white foam intermediate was then treated in 6 mL of 60% aqueous acetic acid at 60°C for 1.5 h. The solvent was removed under reduced pressure, and the residue was purified by rapid silica gel chromatography via a gradient (EtOAc 100% to EtOAc / MeOH / H2O 7:2:1) to give the desired compound 13 as a white foam (306 mg, 0.58 mmol, 89%). f = 0.34; EtOAc / MeOH / AcOH 3:1:0.01; 1 HNMR (CD3OD, 600 MHz): δ 4.83 (d, 1H, J = 3.7 Hz, H-1), 4.43 (d, 1H, J = 7.6Hz, H-1'), 4.41 (dd, 1H, J = 11.0, 3.7 Hz, H-2), 4.18 (d, 1H, J = 2.0 Hz, H-4'), 3.91 (dd, 1H, J = 11.1, 3.1 Hz, H-3), 3.87-3.67 and 3.58-3.43 (m, 11H, H-2', H-3', H-4, H-5, H-5', H-6a, H-6a', H-6b, H-6b', OCH2), 2.92 (t, 2H, J=7.2 Hz, CH2), 2.77 (t, 2H, J = 7.4 Hz, CH2), 2.68 (t, 2H, J = 7.2 Hz, CH2), 2.53 (t, 2H, J = 7.4 Hz, CH2), 1.98 (s, 3H, CH3), 1.89 (m, 2H, CH2); 13 C NMR (CD3OD, 150 MHz): δ 174.0 ( C O), 106.2 ( C -1'), 98.9 ( C -1), 78.9, 76.7, 74.7, 72.49, 72.0 ( C -3), 70.2 ( C -4), 70.0, 67.4 ( C -5), 62.7 (O C H2), 62.5 ( C -6), 50.3 ( C -2), 37.4, 35.1, 30.5, 29.5, 28.6 ( C H2) and 22.8 ppm ( C H3). ESI + -HRMS: M+H + C 20 H 36 O 13 Calculated NS value: 530.1902; Measured value: 530.1909. Figure 13 ).

[0232] Example 11: 3-{[3-(β-D-galactopyranosyl)-(1→3)-2-acetamido-2-deoxy-α-D-galactopyranosyl] Pentafluorophenyl lactose propionate (compound 13)

[0233] Compound 13 Purified acid compound 12 (159 mg, 0.30 mmol, 1.0 equivalent) in water (1.0 mL) was treated with pentafluorophenol (331 mg, 1.80 mmol, 6.0 equivalent) in acetonitrile (4 mL) and EDC.HCl (259 mg, 1.35 mmol, 4.5 equivalent) for 1 h at room temperature. The mixture was concentrated with silica gel under reduced pressure. The crude product was purified by rapid silica gel chromatography via a gradient (EtOAc 100% to EtOAc / MeOH 6:4) to give ester compound 13 as a white solid (62 mg, 0.09 mmol, 30%). f = 0.50; EtOAc / MeOH 6:4; 1 H NMR (CD3OD, 600 MHz): δ 4.45 (d, 1H, J =7.5 Hz, H-1'), 4.41 (ddd, 1H, J = 12.6, 6.1, 2.8 Hz), 4.19 (d, 1H, J = 3.0Hz), 3.92 (dd, 1H, J = 11.0, 3.1 Hz), 3.89-3.77 (m, 3H), 3.74 (m, 4H), 3.68(s, 1H), 3.52 (m, 4H), 3.06 (t,1H, J = 6.9 Hz), 2.91 (t, 1H, J = 6.9 Hz), 2.77 (dt, 2H, J = 15.0, 7.1 Hz), 2.68 (t, 1H, J = 7.1 Hz), 2.63 (t, 1H, J =7.0 Hz), 1.99 (s, 1H) and 1.95-1.84 ppm (m, 2H); 13 C NMR (CD3OD, 150 MHz): δ172.7 (NH C O), 168.1 ( C O), 104.7 ( C -1'), 97.6 ( C-1), 77.5, 75.3, 73.3, 71.1, 70.6, 69.0, 68.7, 66.0, 61.4, 61.2, 50.9, 48.9, 34.2, 33.6, 29.0, 28.2, 28.2, 26.4, 26.1, 21.5 and 21.5 ppm; 19 F NMR (CD3OD, 564 MHz): δ -155.12 (m), -161.21(m), -165.63 (m); ESI + -HRMS: M+H + C 26 H 35 O 13 Calculated NSF5 value: 696.1744; Measured value: 696.1739. Figure 14 ).

[0234] Example 12: PFP-Tn conjugation to BSA and CRM197 refer to Figure 9 The conjugation of PFP-Tn (compound 9) to the protein was demonstrated using BSA and CRM197. BSA (fatty acid-free, low endotoxin; Sigma) was dissolved at a concentration of 1 mg / mL in each of five buffers with increasing pH values: (1) 0.1 M MES and 150 mM NaCl pH 6; (2) phosphate-buffered saline (PBS) pH 7; (3) PBS pH 8; (4) 0.1 M sodium carbonate and 150 mM NaCl pH 9; and (5) 0.1 M sodium carbonate and 150 mM NaCl pH 10. The conjugation of Tn and BSA was initiated by combining 400 µL (6 nmol) of BSA solution with 60 µL of each buffer (20 mM in water) containing 5, 15, 50, or 200 equivalents of PFP-Tn (compound 9) relative to BSA and gently vortexing for 90 min. The obtained BSA-Tn conjugate was then obtained by centrifugation and filtration (MWCO 10 kDa, Amicon) followed by washing with PBS pH 7.4. Protein concentration was measured using the Bradford assay. Under similar conditions, the conjugate CRM197-Tn was obtained by incubating up to 15 molar equivalents of PFP-Tn (compound 9) with 4 mg / mL purified CRM197 in 0.4 M sodium phosphate buffer pH 7.2 for 2 hours.

[0235] The reactivity of protein-Tn conjugates to the Tn-specific lectin *Vicia pubescens* (VVA) was analyzed by Western blotting and / or ELISA.

[0236] Figure 15 illustrates the reactivity of BSA-Tn conjugates generated in buffer solutions at pH 6 to 10 and at a ratio of 50 equivalents of PFP-Tn to BSA to VVA. Figure 15A Western blot analysis revealed a dominant reactive band in the molecular weight region of BSA (approximately 66.5 kDa), indicating that Tn conjugation occurred in all five buffers from pH 6 to 10. A stronger and slightly slower-migrating band was detected at pH 8, indicating a higher level of Tn conjugation to BSA, likely due to the more efficient reaction at pH 8 compared to other pH values. The increased reactivity of BSA-TF at pH 8 to VVA was also confirmed by ELISA. Figure 15B ).

[0237] Figure 15C The titration of PFP-Tn (compound 9) with BSA conjugation at the optimal pH 8 is shown. VVA-reactive BSA-Tn material generated using a minimum of 15 molar equivalents of PFP-Tn relative to BSA was detected by Western blotting. Increasing the ratio of PFP-Tn to BSA to 50 and 200 molar equivalents resulted in correspondingly increased band intensity and slower migration on the gel, due to the higher levels of Tn conjugation with BSA. A correlation between the amount of PFP-Tn and the reactivity of the resulting BSA-Tn conjugate was also observed by ELISA. Figure 15D ELISA further demonstrated that the correlation of PFP-T equivalents from 5 to 200 was linear, indicating that there was no saturation of PFP-Tn conjugate sites on BSA.

[0238] Figure 15E Western blot analysis of CRM197-Tn is shown. The blot results show a VVA reaction band in the molecular weight range of CRM197 (approximately 58.4 kDa) for the three conjugation ratios of the tested PFP-Tn (i.e., 3.4, 9.7, and 15), with the most reactive substance producing the highest PFP-Tn ratio at 15 equivalents.

[0239] Further mass analysis of CRM197 conjugated with 15 equivalents of PFP-Tn was performed by mass spectrometry, and the conjugation was confirmed (data not shown).

[0240] Example 13: Conjugation of PFP-TF to BSA The conjugation of PFP-TF (compound 13) to proteins was demonstrated using BSA. BSA (fatty acid-free, low endotoxin; Sigma) was dissolved at a concentration of 1 mg / mL in PBS pH 8. 400 µL (6 nmol) of the BSA was mixed with 60 µL of PBS pH 8 containing 5, 15, 50, or 200 equivalents of PFP-TF (20 mM in water), and then gently vortexed for 90 min. The resulting BSA-TF conjugate was washed with PBS pH 7.4 after centrifugation (MWCO 10 kDa, Amicon). Protein concentration was measured using the Bradford assay.

[0241] Figure 16 shows the responsiveness of the obtained BSA-TF conjugate to the TF-specific lectin peanut agglutinin (PNA) and the dosage of galactose from the conjugated disaccharide TF antigen. Figure 16A Western blot analysis revealed a dominant band for PNA reaction within the expected molecular weight range of the BSA monomer, indicating TF coupling with BSA. Gel blotting also showed that increasing the ratio of PFP-TF to BSA to 50 and 200 molar equivalents resulted in correspondingly increased band intensity and slower migration on the gel, due to the higher levels of TF conjugation to BSA. A linear correlation was also observed by ELISA between 15–200 molar equivalents of PFP-TF and the reactivity of the resulting BSA-Tn conjugates. Figure 16B The results indicate that the PFP-TF conjugate sites on BSA are unsaturated under these conditions. The conjugation of TF to BSA was also confirmed by measuring the galactose associated with BSA using the Dubois method. Figure 16B The bar graph shows that the amount of galactose increases with the PNA reactivity of BSA-TF, and reaches a ratio of 45 per BSA when conjugated with 200 equivalents of PFP-TF.

[0242] Example 14: Conjugation of COOH-Tn and COOH-TF with CRM197 and dTT The conjugation of COOH-Tn and COOH-TF to proteins was demonstrated using CRM197 and dTT. First, COOH-Tn (compound 8) and COOH-TF (compound 12) were succinimidized by combining a carbohydrate antigen dissolved in water at 0.1 M with two equivalents of each of an aqueous solution of 0.1 M N-(3-dimethylaminopropyl)-N'-ethylcarbodiimide (EDC, Sigma-Aldrich) and N-hydroxysuccinimide (NHS, Sigma-Aldrich) and vortexing at room temperature for at least 30 min. The resulting chemically reactive antigens were then diluted up to 4-fold with PBS pH 8, and conjugation with proteins was initiated by adding 50 equivalents at a concentration of 10 mg / mL to the protein solution in PBS pH 8. The solutions were vortexed for 30–120 min, and the resulting protein-antigen conjugates were washed with PBS pH 7.4 through a membrane filter (Amicon, MWCO 30,000 Da). Protein concentrations were measured using the Bradford assay.

[0243] Figure 17 shows the migration of individual proteins and the resulting conjugates by gel electrophoresis, and their reactivity to lectins VVA (Tn-specific) and PNA (TF-specific) by Western blotting and ELISA. Coomassie-stained SDS-PAGE gels of the conjugates ( Figure 17A The Western blot showed that the conjugated proteins migrated similarly to the native unconjugated proteins, indicating that there was no aggregation or degradation due to protein conjugation to Tn or TF. Figure 17B The specific reactivity of the protein conjugate to VVA or PNA lectins indicates coupling with Tn or TF. Specific reactivity to the same pattern of lectins was also observed by ELISA. Figure 17C ).

[0244] Example 15: Characterization of immunoreactivity from serum of mice immunized with the novel glycoconjugate dTT-TF The glycoconjugate dTT-TF was prepared as described in Example 14, diluted to 1 mg / mL with PBS pH 7.4, and then mixed with an equal volume of TiterMax. TM Gold (Sigma Aldrich) emulsification. 25 μL of the formulation was injected intramuscularly into the left and right thighs of 8-week-old female BALB / c mice every 2 weeks, for a total of 12 mice undergoing 5 immunizations. Blood was collected from the mice before immunization to obtain pre-immunization serum samples, and then blood was collected 1 week after each immunization.

[0245] Serum titers of anti-TF or anti-Tn antibodies were assessed by ELISA. 96-well plates (Nunc, Maxisorp™) were coated with the specified screening antigens: BSA-Tn and BSA-TF at a concentration of 1 µg / 100 µL in PBS pH 7.4 (as described in Example 14), or polyacrylamide [PAA]-Tn and PAA-TF (GlycoTech, USA). After incubation at room temperature for 1 hour, the coated wells were washed with 0.05% PBS-Tween (PBS-T) and blocked with PBS-T 0.05% + 1% BSA for 30 minutes. After washing with PBS-Tween 0.05%, the wells were incubated with serum diluted 1 / 200 in PBS-Tween 0.05% for 1 hour. The wells were then washed and incubated with goat anti-mouse IgG-HRP diluted 1 / 1000 in PBS-Tween 0.05% for 30 minutes. The binding of mouse antibodies was measured by adding the HRP colorimetric substrate ultra-TMB (Thermo) followed by an equal volume of 0.5 M sulfuric acid to terminate the reaction. The results were measured in OD values ​​using a plate reader. 450 Read the plate. Of the 12 mice that received at least five dTT-TF immunizations, significant levels of anti-TF antibodies were detected in the serum of 6 mice by ELISA.

[0246] Table 1: Reactivity of serum from mice immunized with dTT-TF to TF and Tn screening antigens.

[0247] * (Immune OD) 450 ) / (Pre-immune OD 450 ) Table 1 and Figure 18 ELISA results from sera from two mice (A1 and A2) after three immunizations with dTT-TF are shown, along with normalized data based on their respective pre-immunization sera. The results indicate that sera from mouse A1 reacted with all TF and Tn selection antigens (based on a 6.5 to 32.5-fold increase in the corresponding pre-immunization sera), with the highest reactivity observed for BSA-TF (based on a 32.5-fold increase in the corresponding pre-immunization sera). Sera from mouse A2 showed reactivity with two TF selection antigens (BSA-TF and PAA-TF), but only with one Tn antigen conjugated to a carrier protein (Tn-BSA). These results demonstrate that immunization with the novel glycoconjugate dTT-TF successfully induced antibody production against TF carbohydrate antigens, independent of the carrier. Furthermore, these results suggest that at least some of the generated anti-TF antibodies cross-react with the Tn antigen, which is a hidden epitope within the TF antigen (…). Figure 1 ).

[0248] Figure 19A and 19B The ELISA results are shown for serially diluted serum (1:100 to 1:1,000,000) from two mice collected after the fifth immunization with dTT-TF and 6 weeks after the fifth immunization, using BSA-TF as the selection antigen. The curves show that the antibody titer did not decrease 6 weeks after the last immunization, indicating a prolonged antibody half-life. Similar results were observed when PAA-TF was used as the selection antigen (data not shown).

[0249] Example 16: Quantitative analysis of O-glycosylation of SARS-CoV-2 spike protein subunit S1 by high-resolution mass spectrometry Chemical spectrum S1 protein preparation Recombinant SARS-CoV-2 S1 subunits with C-terminal His-tagged cells were isolated from 27 µL of serum-free cell culture supernatant of transfected HEK293 cells (RayBiotech, USA) in reduced sample buffer by electrophoresis on 10% SDS-PAGE. The gel was stained with Coomassie Blue G-250. Lane portions corresponding to 75 kDa and higher were cut into 10 pieces under a clean bench, each piece further cut into 1 mm pieces. 3 piece.

[0250] Gel block preparation The gel block was first washed with water for 5 minutes, then destained twice with destaining buffer (100 mM sodium thiosulfate, 30 mM potassium ferricyanide), 15 minutes each time. After destaining with ammonium bicarbonate buffer (50 mM), it was washed for an additional 5 minutes. The gel block was then dehydrated with acetonitrile (ACN). The protein cysteine ​​disulfide groups were reduced at 40°C for 30 minutes by adding reducing buffer (10 mM dithiothreitol (DTT), 100 mM ammonium bicarbonate). The resulting free thiol groups were then alkylated to S-carboxamide methyl groups by adding alkylation buffer (55 mM iodoacetamide, 100 mM ammonium bicarbonate) and incubating in the dark at 40°C for 20 minutes. The gel block was then dehydrated and washed at 40°C with acetonitrile for 5 minutes, after which all reagents were discarded.

[0251] Protein hydrolysis and peptide extraction steps The gel blocks were dried at 40°C for 5 minutes and then rehydrated at 4°C for 40 minutes with trypsin solution (6 ng / µL trypsin [sequencing grade] from Promega, 25 mM ammonium bicarbonate). Protein digestion was performed at 58°C for 1 hour and then stopped by adding 15 µL of 1% formic acid / 2% acetonitrile. The supernatant was then transferred to 96-well plates and peptide extraction was performed using extraction buffer (1% formic acid / 50% ACN) at room temperature for two 30-minute extraction steps. All peptide extracts were completely dried in a vacuum centrifuge.

[0252] LC-MS / MS analysis Prior to LC-MS / MS, the protein digest was redissolved in 10 µL of 1% ACN / 0.5% formic acid with stirring for 15 min. A 15 cm long, 75 µm id Self-Pack PicoFrit™ fused silica capillary column (New Objective, Woburn, MA) was packed with C18 Jupiter™ (5 µm, 300 Å) reversed-phase material (Phenomenex, Torrance, CA). The column was mounted on an Easy-nLC™ II system (Proxeon Biosystems, Odense, Denmark) and coupled to an Orbitrap Fusion™ (Thermo-Fisher Scientific, Bremen, Germany) equipped with a Nanospray Flex™ ion source (Thermo-Fisher Scientific). The buffers used for chromatography were 0.2% formic acid (buffer A) and 100% acetonitrile / 0.2% formic acid (buffer B). Peptides were eluted with a bislope gradient at a flow rate of 250 nL / min. Solvent B was first increased from 1% to 35% over 75 minutes, and then from 35% to 86% over 15 minutes. Nanospray and S-lens voltages were set to 1.3–1.7 kV and 50 V, respectively. The capillary temperature was set to 225 °C. Full-scan MS spectra (360–1560 nm) in profile mode were obtained using Orbitrap. m / zThe resolution was 120,000, and the target value was set to 8e5. Data-dependent MS / MS analysis was performed using a 3-second cycle time, during which the selected precursor ion fragmented in an HCD (high-energy C-trap dissociation) collision cell and analyzed in Orbitrap at a resolution of 30,000, a target value of 7e4, and a normalized collision energy of 28 V. Subsequent MS / MS analysis was performed in Orbitrap using CID (collision-induced dissociation) after the detection of oxonium ions. A list of all known forms of peptides 320-328, also used for SARS-CoV-2, was included. A second series of analyses of these peptides was performed using SIM (single ion monitoring) and targeted MS2 methods.

[0253] Data Analysis A protein database search was performed in the Uniprot protein database (April 11, 2017) using Mascot 2.6 (Matrix Science). The mass tolerances for the precursor and fragment ions were set to 10 ppm and 0.6 Da, respectively. The specified enzyme was trypsin, allowing for one missed cleavage. Cysteine ​​ureidomethylation was specified as a fixed modification, and methionine oxidation was specified as a variable modification. A second series search of SARS-CoV-2 sequences was performed using Mascot 2.6 and Byos 3.8 (ProteinMetrics). The specified enzyme was cysteine ​​trypsin, allowing for one missed cleavage. Cysteine ​​ureidomethylation was specified as a fixed modification. Methionine oxidation and peptides 320-328 in all known O-glycosylated forms were used as variable modifications.

[0254] Figure 20 The quantitative O-glycosylation spectrum of peptide VQPTESIVR (SEQ ID NO: 3) from SARS-CoV-2 S1 protein, characterized by high-resolution LC-MS / MS of a protein greater than 75 kDa, is shown. Figure 21 The same analysis of proteins ranging from 75 to 100 kDa by high-resolution LC-MS / MS is shown. The notation for the graphical representation of individual glycans is as follows (Varki et al., 2015): yellow squares = GalNAc, yellow circles = Gal, purple rhombuses = Neu5Ac (sialic acid). TFs are formed from the disaccharide Gal-GalNAc. These figures show different patterns of glycosylated peptides, with the dominant type being disialic acid-TF, followed by TF.

[0255] Example 17: Detection of anti-Tn and anti-TF ligands against recombinant SARS-CoV-2 S1 by ELISA and Western blotting Reactivity with S protein The wells of a 96-well plate were coated for 1 hour with 100 µL of serum-free culture supernatant from mammalian cells in PBS (pH 7.4). The mammalian cells were transfected with an empty vector or with DNA encoding recombinant SARS-CoV-2 S1 (S1 subdomain of the spike protein; RayBiotech, USA, catalog 230-20407) or S (full-length spike protein; NRC, Canada) with a C-terminal His tag. The wells were then washed with 0.05% PBS-Tween and blocked with 0.05% PBS-T + 1% BSA for 30 minutes. The wells were then washed with 0.05% PBS-T and incubated for 1 hour with the specified anti-TF (JAA-F11 IgG, 1 µg / mL; SPM320 IgM, Abnova, catalog number MAB13207, 0.2 mg / mL, 1:100 dilution) or anti-Tn (Tn218 IgM, Abnova, catalog number MAB6198, 1:100 dilution) antibody in 0.01% PBS-T. This was followed by incubation for 30 minutes with an appropriate HRP-conjugated secondary antibody (goat anti-mouse IgG-HRP or goat anti-mouse IgM-HRP; Jackson Immuno, 1 / 1000) in PBS-T. Ligand binding was revealed using the HRP colorimetric substrate ultra-TMB (Thermo). The reaction was terminated by adding an equal volume of 0.5M sulfuric acid, and the optical density was measured at 450 nm on a Biotek plate reader. Figure 22 The results shown are for each antibody, compared with the OD of the supernatant from HEK cells transfected with the empty vector. 450 In comparison, the OD of the supernatant from HEK cells expressing recombinant S1 or S protein was higher. 450 The fold increases are shown. The fold increases represented are the averages of at least six individual experiments, and the error bars represent the standard error of the average. The experiment was also repeated using recombinant S1 protein expressed from insect (Sf9) cells (S1-His, SinoBiological, catalog number 40591-V08B1), and the results showed that the mean fold increase for JAA-F11 was 3.32 ± 1.35, for SPM320 it was 5.50 ± 1.80, and for Tn218 it was 1.77 ± 0.64 (mean of three experiments ± SEM).

[0256] Serum-free culture supernatant from HEK293 cells transfected with an empty vector, DNA encoding the S1 protein, or DNA encoding the full-length S protein of SARS-CoV-2 was used to perform a similar ELISA assay as described above using a set of HRP-conjugated lectins. The lectin set included lectins from the following sources: peanut (…). Arachis hypogaea)(PNA; Catalog No.: H-2301-1), long-haired wild pea (VVA; Catalog No.: H-4601-1), clary sage ( Salvia sclarea (SSA; Catalog No.: H-3501-1), Mountain Locust ( Maackia amurensis (MAA; Catalog No.: H-7801-1), Mulberry ( Maclura pomifera (MPA; Catalog No.: H-3901-1). All lectins were purchased from EY Laboratories (CA, USA; 1 mg / mL, HRP-conjugated) and used at 10 µg / mL in PBS-Tween 0.01%. Figure 23 The results shown are for each lectin, compared with the OD of the supernatant from HEK cells transfected with empty vector. 450 In comparison, the OD of the supernatant from HEK cells expressing recombinant S1 or S protein was higher. 450 The fold increases are shown. The fold increases represented by the figures represent the average of at least seven individual experiments, and the error bars represent the standard error of the average. The experiment was also repeated using recombinant S1 protein expressed from insect (Sf9) cells (S1-His, SinoBiological, catalog number 40591-V08B1), and the results showed that the mean fold increase for PNA was 22.67 ± 12.52, for VVA 22.51 ± 9.71, for SSA 3.97 ± 1.09, for MAA 4.30 ± 1.23, and for MPA 7.51 ± 3.64.

[0257] For Western blotting, 2 µL of serum-free culture supernatant from HEK293 cells transfected with DNA encoding SARS-CoV-2 S1 with a C-terminal His-tag was separated from the sample buffer by SDS-PAGE on a 10% polyacrylamide gel. The proteins were then transferred to a PVDF membrane and analyzed using anti-glycan lectin, antibody, and serum. The reactivity of the anti-glycan was compared to that of the anti-His-tag antibody under the corresponding conditions as a control. Bands detected with PNA, VVA, and JAA-F11 mAb were similar to those detected with the control anti-His-tag Ab, indicating that they recognize the same S1 protein (data not shown). S1 was also strongly detected in mouse A1 immunized serum (Example 15) compared to the pre-immunization serum bank.

[0258] In summary, the results in this embodiment consistently demonstrate that carbohydrate antigens Tn and TF are present on recombinant SARS-CoV-2 S1 and S proteins produced by different expression systems, and that these antigens can be bound by anti-Tn and anti-TF ligands.

[0259] Example 18: Inhibition of cellular infectivity of O-glycan ligands against pseudoviruses expressing SARS-CoV-2 S Figure 24 The relative infectivity of pseudotype lenti-luc-SARS-CoV-2-S virus to 293T cells expressing human angiotensin-converting enzyme 2 (293T-ACE2) (the receptor for S-binding) was demonstrated. The virus was first pre-incubated at 37°C for 1 hour with serially diluted anti-glucan agglutinin PNA, AIA (jac), [jacalin (Paramyxophylla)], and monoclonal anti-TF antibody JAA-F11. The mixture was then added to 293T cells seeded in 96-well plates and incubated for 2 hours. The medium was then replaced with fresh medium and cultured for another 2 days. Luciferase activity was then measured. Figure 24 The study showed that lectins PNA and AIA inhibited the infection of 293T-ACE2 cells by pseudo-lenti-luc-SARS-CoV-2-S virus in a concentration-dependent manner. Only the highest concentration of JAA-F11 showed some inhibitory effect on infectivity.

[0260] Interestingly, AIA (jacalin) lectins are known to bind specifically to both sialylated and non-sialylated forms of TF and Tn antigens, while PNA lectins (which have a binding affinity for terminal β-galactose) are known to bind only to the non-sialylated form of TF antigen (Li et al., 2010). Similarly, JAA-F11 antibodies are known to bind only to the non-sialylated form of TF antigen. Therefore, with Figure 23 Compared to PNA and JAA-F11, AIA showed a more effective inhibitory effect. Figure 20 and Figure 21 The distribution of O-glycosylation forms detected on the SARS-CoV-2 S1 protein is shown in the figure. Figure 22 and Figure 23 The relative reactivity of the different anti-Tn and anti-TF ligands shown is consistent.

[0261] The results in this embodiment demonstrate that the SARS-CoV-2-S protein expressed in the context of pseudovirus particles does indeed contain surface TF and Tn carbohydrate antigens, which can be bound by anti-TF and / or anti-Tn ligands. The results in this embodiment further suggest that ligand binding of O-glycans on the surface of the S protein expressed in a viable pseudovirus particle background—particularly the binding of lectins PNA and AIA—may be a promising strategy for preventative and / or therapeutic interventions against COVID-19.

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Claims

1. A neo-glycoconjugate vaccine or adaptive immune response triggering composition comprising a synthetic neo-glycoconjugate and a pharmaceutically acceptable excipient and / or adjuvant, wherein the synthetic neo-glycoconjugate comprises one or more carbohydrate antigens (CA) conjugated to one or more amine groups of a carrier protein or peptide (CP-NH) via a linker, the synthetic neo-glycoconjugate having the following structure: wherein p is 1 to 50 and corresponds to the total number of carbohydrate antigens conjugated to the carrier protein or peptide at the one or more amine groups.

2. The neo-glycoconjugate vaccine or adaptive immune response triggering composition according to claim 1, wherein the carrier protein or peptide is a protein or peptide engineered to add one or more additional lysine residues.

3. The neo-glycoconjugate vaccine or adaptive immune response triggering composition according to claim 1 or 2, wherein the carrier protein or peptide is, from or comprises: cross-reacting material 197 (CRM197), tetanus toxoid (TT), diphtheria toxoid (DT), meningococcal outer membrane protein complex (OMPC), Haemophilus influenzae protein D (HiD), a cytokine, an immunogenic peptide tetanus toxin 831-844 (SEQ ID NO: 1 or 2), albumin, keyhole limpet hemocyanin (KLH), or an immunogenic fragment thereof.

4. The neo-glycoconjugate vaccine or adaptive immune response triggering composition according to claim 1 or 2, wherein the carrier protein or peptide is, from or comprises: CRM197, TT, or an immunogenic fragment thereof.

5. The neo-glycoconjugate vaccine or adaptive immune response triggering composition according to any one of claims 1 to 4, which is a prophylactic vaccine.

6. The neo-glycoconjugate vaccine or adaptive immune response triggering composition according to any one of claims 1 to 4, which is a therapeutic vaccine. Thomsen-Friedenreich 7. The neoglycoconjugate vaccine or adaptive immune response triggering composition according to any one of claims 1 to 6, wherein the carbohydrate antigen is 8. The neo-glycoconjugate vaccine or adaptive immune response triggering composition according to any one of claims 1 to 7, wherein the one or more carbohydrate antigens comprise a non-sialylated TF antigen, a non-sialylated Tn antigen, or an analog thereof. (TF), Tn, S-Tn, (2,3)-S-TF, (2,6)-S-TF, Globo H, PSA, GD2, GD3, GM2, GM3, N-glycolyl-GM3, fucosyl GM1, Le a , sLe a , Le x , sLe x , Le y or any combination thereof.

9. The neo-glycoconjugate vaccine or adaptive immune response triggering composition according to any one of claims 1 to 8, wherein the carbohydrate antigen is or comprises a tumor associated carbohydrate antigen (TACA).

10. The neo-glycoconjugate vaccine or adaptive immune response triggering composition according to any one of claims 1 to 6, wherein the carbohydrate antigen is or comprises a viral polysaccharide antigen.

11. The neo-glycoconjugate vaccine or adaptive immune response triggering composition according to any one of claims 1 to 6, wherein the carbohydrate antigen is or comprises a bacterial capsular polysaccharide (CPS).

12. The neo-glycoconjugate vaccine or adaptive immune response triggering composition as defined in any one of claims 1 to 11 for use in immunizing, vaccinating or treating a subject having a disease, or for use in detecting the presence of an antibody that specifically binds to the neo-glycoconjugate. ​ 13. Use of a neoglycoconjugate vaccine or adaptive immune response triggering composition as defined in claim 9 for the manufacture of a medicament for immunizing, vaccinating or treating a subject suffering from a cancer expressing said TACA.

14. Use of a neoglycoconjugate vaccine or adaptive immune response triggering composition as defined in claim 10 for the manufacture of a medicament for immunizing or vaccinating a subject against a virus expressing said viral polysaccharide antigen.

15. Use of a neoglycoconjugate vaccine or adaptive immune response triggering composition as defined in claim 11 for the manufacture of a medicament for immunizing or vaccinating a subject against a bacterium expressing said CPS.

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