Precision glycoconjugates as therapeutic tools

Abstract

The present invention relates to glycoconjugates, glycoconjugate immunogens and glycoconjugate vaccines comprising carbohydrate antigens coupled to immunogenic carrier proteins, or materials for detecting and screening the resulting antibodies. Improved methods for more directly and accurately conjugating carbohydrate antigens to free thiol groups of immunogenic carrier proteins are described, including "click chemistry" methods based on photocatalyzed thiol-ene reactions.

Description

[0001] This specification relates to glycoconjugate therapeutic tools. More specifically, the invention relates to carbohydrate antigens non-randomly coupled to free thiol groups of immunogenic and antigenic carrier peptides and proteins, and improved methods for producing them using, for example, photocatalyzed thiol-ene "click chemistry" reactions. Use as antigens, immunogens, vaccines, and in diagnostics is also described.

[0002] This specification refers to a number of documents, the entire contents of which are incorporated herein by reference in their entirety. Background Art

[0003] The ultimate goal of immunotherapy is to trigger the innate and adaptive responses of the immune system in a manner similar to that produced during infection or tumor progression. The main interface between the innate and adaptive immune responses is the antigen presenting cell (APC), especially the dendritic cell (DC). APCs are able to recognize microorganisms through pattern recognition receptors (PRRs), such as Toll-like receptors (TLRs). After recognizing microbial surface determinants or abnormal and non-native antigens, APCs undergo maturation and activation, resulting in the redistribution of MHC molecules from intracellular compartments to the cell surface and the secretion of cytokines and chemokines. Microorganisms or tumors and their associated antigenic markers can be engulfed by APCs through the endocytic pathway, where they are usually degraded. Peptides and covalently linked antigens released by protein processing are then displayed on class II MHC molecules and recognized by CD4+ T cells, which in turn undergo functional maturation after receiving costimulatory signals from APCs, transforming into different subclasses, such as Th1 or Th2 cells. Th1 cells lead to a significant proinflammatory response and secrete 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 production of antibodies, which in turn influences the isotype and function of the antibodies. For example, IL-12 and TNF-α are associated with the differentiation of Th1 cells and the production of type 1 IgG subclass, while IL-6 and other Th2 cytokines contribute to the production of type 2 IgG subclass (IgG1). Therefore, it is desirable to be able to modulate vaccine-induced immunity into an appropriate response to address the target pathogen or tumor antigen.

[0004] In contrast to proteins and peptides, carbohydrates are T-cell-independent antigens that are not properly assembled to trigger the engagement of Th cells and are therefore unable to induce immune cell proliferation, antibody class switching, and affinity / specificity maturation. The major early advances initially encountered with carbohydrate-based vaccines were supported by the discovery that bacterial capsular polysaccharides, when properly conjugated to protein carriers that serve as T-cell-dependent epitopes, are able to acquire the desired immunochemical capacity to generate opsonophagocytic antibodies.

[0005] Traditionally, strategies for conjugating carbohydrate antigens to carrier proteins have relied on reductive amination of aldehyde-derivatized sugars on the ε-amino group of lysine residues, or simply amide coupling reactions. In both cases, partial and random carbohydrate antigen conjugation typically occurs. In addition, if all amide partners (the amine of lysine or the acid of glutamic acid / aspartic acid) are used for carbohydrate conjugation, too much carbohydrate antigen is attached to the carrier protein, resulting in masking of potential essential T cell peptide epitopes and inherently reducing or eliminating immunogenicity. Therefore, current strategies for preparing glycoconjugate vaccines are inadequate and face significant regulatory and / or commercial obstacles because these preparations lack the necessary homogeneity in their carbohydrate distribution and reproducibility (i.e., the attachment points of sugars on the protein are randomly distributed and the density varies from batch to batch). Therefore, there is a great need for glycoconjugate vaccines with greater carbohydrate antigen homogeneity, more precisely characterizable structures, and reproducibility from batch to batch. Summary of the invention

[0006] The present specification relates to glycoconjugate immunogens, comprising carbohydrate antigens directly coupled to precise (non-random) positions of an immunogenic carrier protein. More particularly, the carrier protein comprises one or more free thiol groups (e.g., corresponding to the side chains of cysteine ​​residues), and the carbohydrate antigen is conjugated to one or more of these free thiol groups of the carrier protein. The present specification also relates to improved methods for synthesizing glycoconjugate immunogens / vaccines, involving, for example, direct conjugation of carbohydrate antigens to free thiol groups of the carrier protein using a "click chemistry" method (e.g., a photocatalyzed thiol-ene reaction). The improved conjugation methods described herein can be performed under sufficiently mild conditions (e.g., using only water-soluble reagents, in the absence of organic solvents, or using organic solvents at concentrations sufficiently low (e.g., <5%) to avoid denaturation of the carrier protein, and / or at relatively neutral pH values) to avoid destroying the activity, antigenicity and / or structure of the carrier protein (e.g., cleavage and / or denaturation of native disulfide bonds) without affecting the specificity of the conjugation. Furthermore, the photocatalytic thiol-ene reaction described herein can be carried out under ultraviolet light (eg, 355 nm or 365 nm) in the presence of a catalyst or under short-wave ultraviolet light (eg, 254 nm) in the absence of a catalyst, further simplifying the process.

[0007] In some aspects, the disclosure relates to a method for producing a glycoconjugate immunogen, the method comprising: (a) providing a carbohydrate antigen covalently linked to a terminal olefin (alkenyl carbohydrate antigen), wherein the terminal olefin is directly conjugable to a thiol group via a thiol-ene reaction; (b) providing a carrier protein having one or more free thiol groups; and (c) performing a photocatalyzed thiol-ene reaction to directly conjugate the carbohydrate antigen to the one or more free thiol groups of the carrier protein, thereby producing a glycoconjugate immunogen, wherein the carrier protein is immunogenic upon administration to a subject, and wherein conjugation of the carbohydrate antigen to the carrier protein increases the immunogenicity of the carbohydrate antigen upon administration to the subject compared to administration of the unconjugated carbohydrate antigen.

[0008] In some embodiments, the photocatalytic thiol-ene reaction is performed under reaction conditions that avoid denaturation of the carrier protein. In some embodiments, the alkenyl carbohydrate antigen is water-soluble, and the photocatalytic thiol-ene reaction is performed under reaction conditions that preserve the activity, antigenicity, and / or structure of the carrier protein (e.g., in the absence of any organic solvent, or in the presence of an organic solvent at a sufficiently low concentration (e.g., <5%) to avoid denaturation of the carrier protein). The photocatalytic thiol-ene reaction can be performed in the presence of a catalyst (e.g., a water-soluble or water-insoluble catalyst) under irradiation with short-wave (e.g., 254 nm) and / or long-wave (e.g., 355 or 365 nm) ultraviolet light.

[0009] In some embodiments, the photocatalyzed thiol-ene reaction comprises reacting each free thiol of the carrier protein with 1 to 200 molar equivalents of an ene carbohydrate antigen; and / or the photocatalyzed thiol-ene reaction is carried out at a pH of about 3 to 10, 3.5 to 9.5, 4 to 9, 4.5 to 8.5, 5 to 8, 5.5 to 8, 6 to 8, or 6.5 to 7.5.

[0010] In some embodiments, after conjugation to the carrier protein, the carbohydrate antigen cannot be cleaved from the carrier protein by endogenous enzymes of the subject. In some embodiments, the alkenyl carbohydrate antigen is covalently linked to the terminal olefin, and / or the carbohydrate antigen is conjugated to the carrier protein via a glycosidic bond such as an O-glycosidic bond, an S-glycosidic bond, an N-glycosidic bond, or a C-glycosidic bond, or a bond obtained by, for example, reductive amination between an allylamine and a reducing sugar (including bacterial CPS). In some embodiments, the carbohydrate antigen comprises a B cell epitope, and / or induces a humoral immune response in a subject. In some embodiments, the carbohydrate antigen comprises a T cell epitope, and / or induces a cell-mediated immune response in a subject. In some embodiments, the carbohydrate antigen comprises both a B cell epitope and a T cell epitope, and / or induces both a humoral and a cell-mediated immune response in a subject. In some embodiments, the carbohydrate antigen is or comprises a tumor-associated carbohydrate antigen (TACA), such as Tn, S-Tn, Thomsen-Friedenreich (TF), (2,3)-S-TF, (2,6)-S-TF, Globo H, GD2, GD3, GM2, GM3, N-glycolyl-GM3, Lea, sLea, Lex, sLex, or any combination thereof. In some embodiments, a photocatalyzed thiol-ene reaction conjugates at least two identical carbohydrate antigens or more than one type of carbohydrate antigen to a carrier protein, thereby generating a multivalent glycoconjugate immunogen (e.g., comprising 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). In some embodiments, the carbohydrate antigen is or comprises a viral polysaccharide antigen, or a bacterial capsular polysaccharide (CPS) (e.g., pneumococcal and / or streptococcal polysaccharide serotypes, meningococcal CPS; influenza (e.g., influenza type a or b) CPS).

[0011] In some embodiments, the alkenyl carbohydrate antigen is linked to the terminal olefin via a linker (eg, a linker described herein).

[0012] In some embodiments, the carrier protein comprises one or more cysteine ​​residues having one or more free thiol groups. In some embodiments, the carrier protein comprises a T cell epitope, and / or induces a cell-mediated immune response in a subject.

[0013] In some embodiments, the carrier protein 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 and a cell-mediated immune response in a subject. In some embodiments, the carrier protein is, is derived from or comprises: tetanus toxoid (TT), diphtheria toxoid (DT), cross-reactive material 197 (CRM197), outer membrane protein complex (OMPC) of meningococcus, 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), or an immunogenic fragment thereof. In some embodiments, the carrier protein is a protein having one or more disulfide bonds, and wherein: (i) after the photocatalytic thiol-ene reaction, the one or more disulfide bonds remain unaffected; or (ii) the carrier protein is pretreated with a reducing agent to expose one or more additional free thiol groups for conjugation with a carbohydrate antigen.

[0014] In some embodiments, the total number of carbohydrate antigens included in the glycoconjugate immunogen is equal to the number of free thiol groups available on the carrier protein prior to conjugation. In some embodiments, the glycoconjugate immunogen induces a cell-mediated immune response to the carbohydrate antigen upon administration to a subject.

[0015] In some embodiments, the methods described herein further comprise: (d) purifying the glycoconjugate immunogen.

[0016] In some aspects, the description relates to a method for producing a glycoconjugate vaccine, the method comprising formulating a glycoconjugate immunogen prepared by the method described herein with a pharmaceutically acceptable excipient and / or adjuvant (e.g., an inorganic compound, mineral oil, a microbial derivative, a plant derivative, a cytokine, squalene, alum, aluminum hydroxide, aluminum phosphate, calcium phosphate hydroxide, a toll-like receptor agonist, an immunostimulatory polynucleotide (e.g., CPG), an immunostimulatory lipid, Freund's adjuvant, RIBI adjuvant, QS-21, muramyl dipeptide, or any combination thereof).

[0017] In some aspects, the specification relates to a synthetic glycoconjugate immunogen comprising one or more carbohydrate antigens and an immunogenic carrier protein having one or more solvent accessible cysteine ​​residues, wherein the one or more carbohydrate antigens are linked to the one or more solvent accessible cysteine ​​residues of the immunogenic carrier protein, and wherein the conjugation of the one or more carbohydrate antigens to the immunogenic carrier protein increases the immunogenicity of the one or more carbohydrate antigens after administration to a subject compared to administration of the unconjugated carbohydrate antigen. In some embodiments, the one or more carbohydrate antigens are linked to the one or more solvent accessible cysteine ​​residues via a linker as described herein, and the one or more carbohydrate antigens are as described herein. In some embodiments, the synthetic glycoconjugate immunogen is a multivalent glycoconjugate immunogen as described herein, and a carrier protein as described herein is used. In some embodiments, the total number of carbohydrate antigens contained in the glycoconjugate immunogen is equal to the number of solvent accessible cysteine ​​residues on the carrier protein. In some embodiments, the synthetic glycoconjugate immunogen induces a cell-mediated immune response against the carbohydrate antigen after administration to a subject. In some embodiments, the synthetic glycoconjugate immunogen is prepared by the method described herein.

[0018] In some aspects, the description relates to a glycoconjugate vaccine produced by a method as described herein, and / or comprising a synthetic glycoconjugate immunogen as described herein and a pharmaceutically acceptable excipient and / or adjuvant. In some embodiments, the glycoconjugate vaccine is a prophylactic vaccine or a therapeutic vaccine.

[0019] In some aspects, the description relates to a method of immunizing, vaccinating or treating a subject, comprising administering to the subject a glycoconjugate immunogen produced by a method as described herein, a glycoconjugate vaccine produced by a method as described herein, a synthetic glycoconjugate immunogen as described herein, or a glycoconjugate vaccine as described herein. In some embodiments, the description relates to a synthetic glycoconjugate immunogen or glycoconjugate vaccine as described herein for use in immunizing, vaccinating or treating a subject having a disease, or for detecting the presence of an antibody that specifically binds to the glycoconjugate, or for detecting the immunization, vaccination or treatment (e.g., in a biological sample from the subject).

[0020] In some aspects, the present description relates to a synthetic glycoconjugate immunogen comprising one or more saccharide antigens covalently linked to one or more free thiol groups of a carrier protein via a linker, wherein the synthetic glycoconjugate immunogen has the following structure or a stereoisomer thereof:

[0021]

[0022] in

[0023] -y is at least 1;

[0024] - SA is selected from the group consisting of carbohydrate antigens or immunogenic portions thereof, and when y is greater than 1, SA are identical or different;

[0025] -[S] z - PC is a carrier protein having one or more sulfur atoms derived from one or more free sulfhydryl groups;

[0026] - z is at least 1 and at least equal to y; and

[0027] -L is a linker selected from the group consisting of linkers having the following structures:

[0028]

[0029] in:

[0030] -X is O, S, NR1 or CH2;

[0031] -R1 is -H, -COH, -COCH3 or -COEt;

[0032] -n is 1, 2, 3, 4, or 5; and

[0033] - R2 is H or Me; and

[0034] When y is greater than 1, L are the same or different.

[0035] In some aspects, the present description relates to a synthetic glycoconjugate immunogen comprising one or more saccharide antigens covalently linked to one or more free thiol groups of a carrier protein via a linker, wherein the synthetic glycoconjugate immunogen has the following structure or a stereoisomer thereof:

[0036]

[0037] in

[0038] -y is at least 1;

[0039] - SA is selected from the group consisting of carbohydrate antigens or immunogenic portions thereof, and when y is greater than 1, SA are identical or different;

[0040] -[S] z - PC is a carrier protein having one or more sulfur atoms derived from one or more free sulfhydryl groups;

[0041] - z is at least 1 and at least equal to y; and;

[0042] -L is a linker selected from the group consisting of linkers having the following structures:

[0043]

[0044] in:

[0045] -X is S, NR1, CH2 or O;

[0046] -R1 is -H, -COH, -COMe or -COEt;

[0047] -n is 1, 2, 3, 4, or 5;

[0048] -R2 is H or Me;

[0049] -q is 1, 2, 3, 4, or 5;

[0050] - R3 and R4 are each a hydrogen atom 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- in which the carbonyl group is attached to the nitrogen atom and m is 1; and

[0051] When y is greater than 1, L are the same or different.

[0052] In some aspects, the present description relates to a synthetic glycoconjugate immunogen comprising one or more saccharide antigens covalently linked to one or more free thiol groups of a carrier protein via a linker, wherein the synthetic glycoconjugate immunogen has the following structure or a stereoisomer thereof:

[0053]

[0054] in

[0055] -y is at least 1;

[0056] - SA is selected from the group consisting of carbohydrate antigens or immunogenic portions thereof, and when y is greater than 1, SA are the same or different

[0057] -[S] z - PC is a carrier protein having one or more sulfur atoms derived from one or more free sulfhydryl groups;

[0058] - z is at least 1 and at least equal to y; and

[0059] -L is a linker selected from the group consisting of linkers having the following structures:

[0060]

[0061] in

[0062] -X is S, NR1, CH2 or O;

[0063] -R1 is -H, -COH, -COMe or -COEt;

[0064] -n is 1, 2, 3, 4, or 5;

[0065] -R2 is H or Me;

[0066] -q is 1, 2, 3, 4, or 5;

[0067] -r is 1, 2, 3, 4, or 5;

[0068] -R5 is S-PC, a covalent bond, or a group having the following structure:

[0069]

[0070] wherein R3 and R4 are each a hydrogen atom 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- in which the carbonyl group is attached to the nitrogen atom and m is 1; and

[0071] When y is greater than 1, L are the same or different.

[0072] In some embodiments, the description relates to a synthetic glycoconjugate immunogen as defined above, wherein the linker has the following structure:

[0073]

[0074] in

[0075] -X is S, NR1, CH2 or O;

[0076] -R1 is -H, -COH, -COMe or -COEt;

[0077] -n is 1, 2, 3, 4, or 5;

[0078] -R2 is H or Me;

[0079] -q is 1, 2, 3, 4, or 5; and

[0080] -r is 1, 2, 3, 4, or 5.

[0081] In some embodiments, the description relates to a synthetic glycoconjugate immunogen as defined above, wherein the linker has the following structure:

[0082]

[0083] in

[0084] -X is S, NR1, CH2 or O;

[0085] -R1 is -H, -COH, -COMe or -COEt;

[0086] -n is 1, 2, 3, 4, or 5;

[0087] -R2 is H or Me;

[0088] -q is 1, 2, 3, 4, or 5; and

[0089] -r is 1 or 2.

[0090] In some embodiments, the description relates to a synthetic glycoconjugate immunogen as defined above, wherein the linker has the following structure:

[0091]

[0092] in

[0093] -X is S, NR1, CH2 or O;

[0094] -R1 is -H, -COH, -COMe or -COEt;

[0095] -n is 1, 2, 3, 4, or 5;

[0096] -R2 is H or Me;

[0097] -q is 1, 2, 3, 4, or 5; and

[0098] -r is 1 or 2.

[0099] In some embodiments, the description relates to a synthetic glycoconjugate immunogen designed as above, wherein y is an integer from 1 to 50, 1 to 40, 1 to 30, 1 to 20, 1 to 10, 1 to 5, or 1 to 3.

[0100] In some aspects, the present description relates to synthetic glycoconjugate immunogens comprising one or more saccharide antigens covalently linked to one or more free thiol groups of a carrier protein via a linker, wherein each linker has the following structure or a stereoisomer thereof:

[0101]

[0102] wherein: SA is a carbohydrate antigen or a portion thereof; S-PC is a carrier protein; X is O, S, NR1 or CH2; R1 is -H, -COH, -COCH3 or -COEt; n is 1, 2, 3, 4 or 5; and R2 is H or Me.

[0103] In some aspects, the present description relates to synthetic glycoconjugate immunogens comprising one or more saccharide antigens covalently linked to one or more free thiol groups of a carrier protein via a linker, wherein each linker has the following structure or a stereoisomer thereof:

[0104]

[0105] wherein: SA is a carbohydrate antigen or a portion thereof; S-PC is a carrier protein; X is S, NR1, CH2 or O; R1 is -H, -COH, -COMe or -COEt; n is 1, 2, 3, 4 or 5; R2 is H or Me; q is 1, 2, 3, 4 or 5; R3 and R4 are each a hydrogen atom 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-, in which the carbonyl group is attached to the nitrogen atom and m is 1.

[0106] In some embodiments, the saccharide antigen is a carbohydrate antigen as described herein, the carrier protein is as described herein, and the synthetic glycoconjugate immunogen is a multivalent glycoconjugate immunogen as described herein.

[0107] In some aspects, the description relates to vaccines comprising a synthetic glycoconjugate immunogen as described herein, and a pharmaceutically acceptable excipient and / or adjuvant as described herein.

[0108] In some aspects, the description relates to the use of a glycoconjugate immunogen prepared by a method as described herein or a synthetic glycoconjugate immunogen as described herein for the manufacture of a vaccine.

[0109] In some aspects, the invention relates to the use of a glycoconjugate immunogen prepared by a method as described herein, a glycoconjugate vaccine produced by a method as described herein, a synthetic glycoconjugate immunogen as described herein, or a vaccine as described herein for treating a subject suffering from a disease associated with increased expression of said one or more carbohydrate antigens or saccharide antigens.

[0110] General Definition

[0111] Headings and other identifiers, such as (a), (b), (i), (ii), etc., are provided only to facilitate 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.

[0112] When used in conjunction with the word "comprising" in the claims and / or the specification, an unqualified noun may mean "a", but also conforms to the meaning of "one or more", "at least one" and "one or more than one".

[0113] The term "about" is used to indicate that a value includes the standard deviation of error for the device or method used to determine the value. In general, the term "about" is intended to indicate a possible variation of up to 10%. Thus, variations of 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, and 10% of a value are included in the term "about". Unless otherwise indicated, the use of the term "about" before a range applies to both endpoints of the range.

[0114] As used in this specification and claims, the words "comprising" (and any forms of comprising, such as "comprise" and "comprises"), "having" (and any forms of having, such as "have" and "has"), "including" (and any forms of including, such as "includes" and "include"), or "containing" (and any forms of containing, such as "contains" and "contain") are inclusive or open-ended, and do not exclude additional unrecited elements or method steps.

[0115] As used herein, the term "protein" (e.g., in the expression "carrier protein") refers to any peptide-linked amino acid chain, which may or may not contain any type of modification (e.g., chemical or post-translational modification, such as acetylation, phosphorylation, glycosylation, sulfation, sumoylation, prenylation, ubiquitination, etc.), as long as the modification does not destroy the immunogenicity of the glycoconjugate immunogens and glycoconjugate vaccines described herein. For further clarity, the terms "protein" and "carrier protein" as used herein encompass peptides and polypeptides.

[0116] As used herein, the term "administering" may include routes of administration such as parenteral (e.g., subcutaneous, intradermal, intramuscular or intravenous), oral, transdermal, intranasal, etc., as long as the administration by this route results in an immune response in the subject.

[0117] As used herein, "subject" generally refers to mammals, including primates, and particularly humans.

[0118] Other objects, advantages and features of the present specification will become more apparent from a reading of the following non-limiting description of specific embodiments thereof, which are given by way of example only with reference to the accompanying drawings. BRIEF DESCRIPTION OF THE DRAWINGS

[0119] In the attached picture:

[0120] Figure 1Examples of typical chemical structures of key tumor-associated carbohydrate antigens (TACAs) from human glycoprotein (MUC) are shown.

[0121] Figure 2 Shown is a proposed "click chemistry" approach for the direct conjugation of carbohydrate antigens (B-cell epitopes) to available thiol groups of immunogenic carrier proteins (eg, T-cell epitopes such as tetanus toxoid).

[0122] Figure 3 Shown are examples of "precision glycoconjugates" prepared by conjugating Tn or TF antigens to six native "free" cysteine ​​residues on the carrier protein tetanus toxoid via a photocatalyzed thiol-ene reaction.

[0123] Figure 4 Examples of "precision glycoconjugates" prepared by linking Tn or TF antigens to thiol-specific iodoacetamide or maleimidyl groups followed by conjugation to "free" cysteine ​​residues on carrier proteins via reactions below pH 8 (iodoacetamide or maleimidyl) or thiol-ene reactions (maleimidyl) are shown.

[0124] Figure 5 Reaction schemes for the synthesis of allyl Tn antigen and allyl TF antigen ready for conjugation to carrier protein as described in Examples 2-6 are shown.

[0125] Figures 6A-6D Allyl 2-acetylamino-3,6-di-O-pivaloyl-2-deoxy-α-D-glucopyranoside (Compound 2) is shown. 1 H-NMR( Fig. 6A )and 13 C-NMR( Figure 6B ) spectrum and mass spectrometry analysis results ( Figure 6C and 6D ).

[0126] Figures 7A-7D Allyl 2-acetylamino-2-deoxy-α-D-galactopyranoside (allyl Tn) is shown. 1 H-NMR( Fig. 7A )and 13 C-NMR( Figure 7B ) spectrum and mass spectrometry analysis results ( Figure 7C and 7D ).

[0127] Figures 8A-8C Allyl 2-acetylamino-4,6-O-benzylidene-2-deoxy-α-D-galactopyranoside (Compound 4) is shown. 1 H-NMR( Fig. 8A) spectrum and mass spectrometry analysis results ( Figure 8B and 8C ).

[0128] Figures 9A-9D The structure of allyl(2,3,4,6-tetra-O-benzoyl-β-D-galactopyranosyl)-(1→3)-2-acetylamino-4,6-O-benzylidene-2-deoxy-α-D-galactopyranoside (Compound 6) was shown. 1 H-NMR( Fig. 9A )and 13 C-NMR( Fig. 9B ) spectrum and mass spectrometry analysis results ( Fig. 9C and 9D ).

[0129] Figures 10A-10D The compound 7 1 H-NMR( Fig. 10A )and 13 C-NMR( Fig. 10B ) spectrum and mass spectrometry analysis results ( Fig. 10C and 10D ).

[0130] Figures 11A-11D Allyl (β-D-galactopyranosyl)-(1→3)-2-acetylamino-2-deoxy-α-D-galactopyranoside (allyl TF) is shown. 1 H-NMR( Fig.11A )and 13 C-NMR( Fig. 11B ) spectrum and mass spectrometry analysis results ( Fig. 11C and 11D ).

[0131] Fig.12 Shown are the results of dynamic light scattering (DLS) analysis of TF-TT glycoconjugate alone (upper left panel), peanut agglutinin alone (upper right panel), or a combination of both (lower panel).

[0132] Fig.13 Shown is the thiol concentration of peptide dTT831-840 as a function of time in the photocatalyzed thiol-ene conjugation reactions with O-allyl-Tn and O-allyl-TF.

[0133] Fig.14 Shown are the fold reduction of the thiol groups of peptide dTT831-840 in the photocatalyzed thiol-ene conjugation reaction with O-allyl-Tn as a function of the cysteine ​​position of the peptide and the presence of AAPH or DMPA.

[0134] Fig.15Shown is the reactivity of the lectin towards the dTT831-840 peptide conjugated to Tn via a photocatalyzed thiol-ene conjugation reaction with O-allyl-Tn as a function of the cysteine ​​position of the peptide and the presence of AAPH or DMPA.

[0135] Fig.16 Shown are the kinetics of the thiol concentration of peptide dTT831-840 in the photocatalyzed thiol-ene conjugation reaction with O-allyl-Tn in the presence of AAPH or DMPA at different wavelengths.

[0136] Fig.17 Reactivity of Vicia Villosa lectin towards peptide dTT831-840 conjugated to Tn via photocatalyzed thiol-ene conjugation reaction with O-allyl-Tn in the presence of AAPH or DMPA at different wavelengths.

[0137] Fig.18 Shown are the kinetics of the thiol concentration of peptide dTT831-840 in photocatalyzed thiol-ene conjugation reactions with C-allyl and O-allyl sugars.

[0138] Fig.19 Shown are the reactivity of lectins and anti-TF antibodies to peptide dTT831-840 conjugated via a photocatalyzed thiol-ene conjugation reaction with O-allyl-Tn.

[0139] Fig. 20 The reactivity of anti-TF antibodies towards reduced dTT or reduced dTT conjugated via a photocatalyzed thiol-ene conjugation reaction with O-allyl-TF is shown.

[0140] Fig.21 Shown are the molar ratios of thiol groups of dTT during the various steps of the photocatalyzed thiol-ene conjugation reaction and the subsequent reduction of the conjugated dTT.

[0141] Fig. 22 The reactivity of dTT conjugated via a photocatalyzed thiol-ene conjugation reaction with O-allyl-Tn towards lectin and anti-TF mAb is shown.

[0142] Fig.23 Shown are the molar ratios of thiol groups of chemically thiolated dTT that were conjugated or unconjugated via a photocatalyzed thiol-ene conjugation reaction with O-allyl-Tn in the presence of low or high amounts of AAPH.

[0143] Fig.24 Shown is the reactivity of Vicia villosa lectin towards chemically thiolated dTT conjugated by a photocatalyzed thiol-ene conjugation reaction with O-allyl-Tn in the presence of low or high amounts of AAPH at two different times.

[0144] Fig.25 Shown are the reactivity of lectin and anti-TF antibodies to reduced BSA conjugated via a photocatalyzed thiol-ene conjugation reaction with O-allyl-Tn.

[0145] Fig.26 The relationship between the reducing conditions and the molar ratio of the thiol groups of the reduced BSA is shown.

[0146] Fig. 27 Shown are the reactivity of Tn-specific Vicia villosa lectin and anti-TF antibodies towards BSA having various free thiol molar ratios and conjugated via a photocatalyzed thiol-ene conjugation reaction with various amounts of O-allyl-Tn.

[0147] Fig.28 Shown is the relationship between the reduction conditions, the molar ratio of the thiol groups of the reduced BSA, and the TF reactivity of the reduced BSA conjugated via the photocatalyzed thiol-ene conjugation reaction with O-allyl-TF.

[0148] Fig.29 Shown are the molar ratios of thiol adducts in BSA reacted by chemical thiolation alone or by simultaneous thiolation and by photocatalyzed thiol-ene conjugation in the presence or absence of AAPH and O-allyl-Tn as a function of time.

[0149] Fig.30 Shown is the reactivity of Tn-specific Vicia villosa lectin with thiolated BSA or BSA simultaneously thiolated and conjugated via a photocatalyzed thiol-ene conjugation reaction in the presence or absence of AAPH and O-allyl-Tn.

[0150] Fig.31 Shown is the relationship between the thiol molar ratio as a function of time and the reactivity of BSA conjugated via a photocatalyzed thiol-ene conjugation reaction with O-allyl-Tn towards the Tn-specific Vicia villosa lectin.

[0151] Fig.32 The reactivity of the Tn-specific Vicia villosa lectin towards thiolated BSA, either natively or conjugated via a photocatalyzed thiol-ene conjugation reaction with O-allyl-Tn, in the presence of low or high amounts of AAPH and at two wavelengths is shown.

[0152] Fig.33 Shown is the relationship between the galactose molar ratio and the reactivity of anti-TF mAbs toward BSA in different steps of thiolation and photocatalyzed thiol-ene conjugation reactions with O-allyl-Tn under low or high amounts of AAPH.

[0153] Fig.34 Shown are the chemical structures of dTT831-844-Cys-βAla and Cys-dTT831-844-βAla with cysteine ​​residues at the C- and N-termini, respectively, and their tabulated LC-MS data.

[0154] Fig.35 Detailed LC-MS data for peptide dTT831-844-Cys-βAla are shown.

[0155] Fig.36 Detailed LC-MS data for the peptide Cys-dTT831-844-βAla are shown.

[0156] Fig.37 Shown is the photolytic AAPH-catalyzed thiol-ene reaction of O-allyl Tn on the C-terminal peptide dTT831-844-Cys-βAla.

[0157] Fig.38 The LC-MS spectrum of O-allyl Tn on the C-terminal peptide is shown

[0158] Fig.39 Shown is the photolytic AAPH-catalyzed thiol-ene reaction of O-allyl Tn on the N-terminal peptide Cys-dTT831-844-βAla.

[0159] Fig.40 The LC-MS spectrum of O-allyl Tn on the N-terminal peptide is shown.

[0160] Sequence Listing

[0161] This application contains a sequence listing in computer readable form, which was created on March 19, 2019 and is approximately 4 KB in size. This computer readable form is incorporated herein by reference. DETAILED DESCRIPTION OF THE INVENTION

[0163] The present specification relates to glycoconjugate immunogens (e.g., for use as vaccines, for diagnosis, or for the production of diagnostic or therapeutic tools, such as specific anti-glycoconjugate antibodies). The glycoconjugate immunogens described herein generally comprise a carbohydrate antigen coupled to an immunogenic carrier protein. More particularly, the carrier protein comprises one or more free thiol groups (e.g., corresponding to the side chains of cysteine ​​residues), and the carbohydrate antigen is conjugated to one or more of these free thiol groups of the carrier protein. The present specification also relates to improved methods for synthesizing glycoconjugate immunogens / vaccines, which involve, for example, direct conjugation of carbohydrate antigens to free thiol groups of the carrier protein using a "click chemistry" method (e.g., a photocatalyzed thiol-ene reaction). The improved conjugation methods described herein can be performed under sufficiently mild conditions (e.g., using only water-soluble reagents, in the absence of organic solvents, using organic solvents at sufficiently low concentrations (e.g., <5%) to avoid denaturation of the carrier protein, and / or at relatively neutral pH values) to avoid destroying the activity, antigenicity and / or structure of the carrier protein (e.g., cleavage and / or denaturation of native disulfide bonds) without affecting the specificity of the conjugation. Furthermore, the photocatalytic thiol-ene reaction described herein can be carried out under ultraviolet light (eg, 355 nm or 365 nm) in the presence of a catalyst or under short-wave ultraviolet light (eg, 254 nm) in the absence of a catalyst, further simplifying the process.

[0164] Because the conjugation methods described herein provide greater precision, the glycoconjugate immunogens / vaccines described herein can have greater homogeneity (in terms of their carbohydrate distribution), reproducibility, and can be more easily characterized than glycoconjugates that rely on random coupling to other amino acid side chains (e.g., amines from lysine or acids from glutamic acid / aspartic acid residues). Such features can facilitate regulatory approval and / or commercialization of glycoconjugate vaccines, both of which have historically proven difficult based on traditional methods.

[0165] In some aspects, the present specification relates to methods for producing glycoconjugate immunogens (e.g., for administration to a subject). As used herein, the term "subject" generally refers to an organism (e.g., an animal or human) that is capable of producing an immune response to a glycoconjugate immunogen as described herein, preferably resulting in the production of antibodies that specifically bind to the glycoconjugate immunogen. In some embodiments, the subject described herein can be a patient to be treated therapeutically (e.g., by vaccination with a glycoconjugate immunogen as described herein), or can be used as a means of producing tools (e.g., antibodies) for research, diagnostic and / or therapeutic purposes. Methods for producing glycoconjugate immunogens generally include providing a carbohydrate antigen having (e.g., chemically modified to include) a sulfhydryl-specific functional group; providing a carrier protein having one or more free sulfhydryl groups (e.g., one or more solvent accessible cysteine ​​residues and / or cysteines that are not involved in disulfide bonds); and reacting the carbohydrate antigen with the carrier protein, thereby coupling the carbohydrate antigen to the carrier protein at one or more predictable (non-random) attachment points corresponding to the positions of the free sulfhydryl groups of the carrier protein. In some embodiments, the method may further comprise purifying or isolating the glycoconjugate immunogen, which may then be formulated into a vaccine (eg, including an adjuvant).

[0166] As used herein, the term "glycoconjugate" refers to a carbohydrate antigen (e.g., an antigenic monosaccharide, disaccharide, oligosaccharide, or polysaccharide) coupled to a carrier protein to enhance the immunogenicity of the carbohydrate antigen in a target subject. As used herein, the expressions "carbohydrate antigen" and "sugar antigen" have the same meaning. The term "immunogen" refers to a substance that is capable of specifically binding to components of the immune system (e.g., through antibodies and / or lymphocytes) in a target subject and producing a humoral immune response and / or a cell-mediated immune response. As used herein, the term "immunogen" in expressions such as "glycoconjugate immunogen" refers to the ability (i.e., physical characteristics or properties) of the glycoconjugate without limiting the glycoconjugate itself to a specific use (e.g., as an immunogen for producing an immune response in a subject). For example, in some embodiments, the glycoconjugate immunogens described herein can be used in a diagnostic assay or method (e.g., an in vitro method) to detect the presence or absence of antibodies that bind to the glycoconjugate immunogen in a biological sample (e.g., from a subject). In some embodiments, the glycoconjugate immunogens described herein can be used to screen, identify, or evaluate antibodies that specifically bind to the glycoconjugate immunogens (eg, monoclonal antibodies that can be used diagnostically or therapeutically).

[0167] In some aspects, the present description relates to the use of a "click chemistry type" approach for directly conjugating carbohydrate antigens to free sulfhydryl groups of carrier proteins. More specifically, as Figure 2As shown, a photocatalyzed thiol-ene reaction is preferred, wherein a carbohydrate antigen (R2) is modified to contain a terminal olefin functional group (olefinic carbohydrate antigen) suitable for direct covalent attachment to a cysteine ​​thiol (—SH) group of a carrier protein (R1) using a photocatalyzed thiol reaction. Figure 3 Carbohydrate antigens containing B cell epitopes (e.g., tumor-associated carbohydrate antigens Tn and TF) can be photochemically conjugated to immunogenic carrier proteins, such as tetanus toxoid (TT), which is known to have six free thiol groups due to its six available cysteine ​​residues (excluding the four cysteine ​​residues involved in disulfide bonds), via a thiol-ene reaction. Figure 3 In the case of the tetanus toxoid carrier protein shown, the conjugation methods described herein preferably result in the conjugation of exactly six carbohydrate antigens at predictable (non-random) attachment points. The methods described herein are applicable to any natural or synthetic monosaccharide, oligosaccharide and polysaccharide, which may terminate in an olefin group (terminal olefin).

[0168] In some embodiments, the carbohydrate antigens described herein can be chemically modified to be linked (directly or interdigitated via a linker or spacer) to a terminal olefin (e.g., via a glycosidic bond or via a bond obtained by, for example, reductive amination between allylamine and a reducing sugar (preferably using NaBH4 and / or NaBH3CN)), wherein the terminal olefinic group of the alkenyl carbohydrate antigen can be conjugated to a free thiol group (e.g., one or more solvent accessible cysteine ​​residues and / or a cysteine ​​not involved in a disulfide bond) of a carrier protein via a thiol-ene reaction (e.g., a photocatalyzed thiol-ene reaction). The terminal olefinic group of the alkenyl carbohydrate antigen can be a monosubstituted olefin, a vinyl group, or an allyl group. In some preferred embodiments, the alkenyl carbohydrate antigen is water soluble, enabling its use in the water-based thiol-ene reaction described herein.

[0169] As used herein, "glycosidic bonds" may comprise one or more of the following: 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 bond that is not cleavable by endogenous enzymes of the subject to be administered (e.g., glycerol hydrolases). Such non-cleavable glycosidic bonds may result in a longer half-life of the glycoconjugate immunogen after administration to the subject, which in turn may produce a more favorable immune response for therapeutic and / or antibody production purposes. In some embodiments, the glycosidic bond may be an S-glycosidic bond, an N-glycosidic bond, or a C-glycosidic bond, or a bond obtained by reductive amination, for example, between allylamine and a reducing sugar.

[0170] In some embodiments, the use of a "linker" or "spacer" is preferred, and such terms are used herein to refer to a chemical linkage that provides sufficient physical separation of the carbohydrate antigen from the carrier protein to which it is conjugated, thereby allowing the carbohydrate antigen to be recognized by the subject's immune system (as opposed to being masked by the carrier protein). In some embodiments, the linker may comprise a chain of at least 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 consecutive residues selected from C, S, N, and O.

[0171] As used herein, the term "conjugable" refers to the ability of at least two molecules (e.g., a carbohydrate antigen and a carrier protein) to covalently bond to each other through a chemical reaction, regardless of whether these molecules are actually covalently bonded to each other. Conversely, the term "conjugated" refers to at least two molecules (e.g., a carbohydrate antigen and a carrier protein) that are covalently bonded to each other.

[0172] In some embodiments, different types of carbohydrate antigens can be made to have terminal olefinic groups that can be directly conjugated to sulfhydryl groups via sulfhydryl-olefin reactions. For example, carbohydrate antigens with terminal olefinic groups can be synthesized by adapting the methods described in Examples 2-7 herein for synthesizing allyl Tn and allyl TF reactants.

[0173] In some embodiments, carbohydrate antigens with terminal alkenyl groups can be conjugated to free thiol groups of a carrier protein using a photocatalytic thiol-ene reaction, for example, by adapting the methods described in Examples 8 and 9 herein for synthesizing Tn-TT and TF-TT conjugates. For example, one or more alkenyl carbohydrate antigens dissolved in an aqueous solvent (e.g., a buffer such as PBS) are mixed with a carrier protein also dissolved in an aqueous solvent (e.g., a buffer such as PBS) in a container suitable for UV irradiation (e.g., a quartz cell). The mixture is then irradiated under short-wave, medium-wave, or long-wave UV light (e.g., having a peak wavelength at about 254 nm, at about 355 nm, or at about 365 nm) in the presence or absence of a catalyst (e.g., a photoinitiator or activator). As used herein, in the context of a thiol-ene reaction, the terms "catalyst," "photoinitiator," and "activator" are used interchangeably to refer to substances that accelerate the conjugation of carbohydrate antigens to carrier proteins at one or more free thiol groups via a photocatalytic thiol-ene reaction.

[0174] In some embodiments, the catalyst can be a water-soluble photoinitiator, such as a water-soluble free radical generating azo compound; 2,2'-azobis[2-(2-imidazolin-2-yl)propane] dihydrochloride (Vazo 44 or VA-044); 2,2'-azobis(2-amidinopropane) dihydrochloride (AAPH); a metal or metal ion having photoinitiating activity; or any derivative thereof having photoinitiating activity in the photocatalytic thiol-ene reaction described herein. In some embodiments, the catalyst can be a water-soluble photoinitiator, such as a water-soluble peroxide, such as tert-butyl hydroperoxide or benzoyl peroxide or ammonium persulfate, or other suitable catalyst.

[0175] In some embodiments, the catalyst can be a water-insoluble photoinitiator, such as a water-insoluble free radical generating azo compound; 2,2-dimethoxy-2-phenylacetophenone (DMPA), azobisisobutyronitrile (AIBN), 2,2'-azobis(2-methylpropionitrile), 4,4'-azobis(4-cyanovaleric acid) (ACVA), 1,1'-azobis(cyanocyclohexane) (ACHN), diazonium dicarboxylate bis(N,N-dimethylamide) (TMAD); dipiperidin azodicarboxylate (ADD), or any derivative thereof having photoinitiating activity in the photocatalyzed thiol-ene reaction described herein.

[0176] In some embodiments, the photocatalyzed thiol-ene reactions described herein may include controlling the ratio of molar equivalents of an alkenyl carbohydrate antigen (i.e., a carbohydrate antigen comprising a terminal olefin) corresponding to each free thiol group of a carrier protein, for example, to reduce, minimize, or avoid sugar polymerization. In some embodiments, the photocatalyzed thiol-ene reactions described herein may include reacting each free thiol group of a carrier protein with 1 to 300, 1 to 250, 1 to 200, 1 to 100, or 1 to 10, 1 to 5, or 1 to 2 molar equivalents of a carbohydrate antigen.

[0177] In some embodiments, the photocatalyzed thiol-ene reaction described herein is carried out for a sufficient time to achieve a reduction in the total free thiol concentration in the carrier protein by at least 2, 3, 4, 5, 10, 15, 20, 25, 30, 35, 40, 45, or 50 fold (e.g., as determined by an Ellman assay). In some embodiments, the photocatalyzed thiol-ene reaction described herein 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.

[0178] In some embodiments, the photocatalytic thiol-ene reactions described herein can be carried out 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. In some embodiments, the photocatalytic thiol-ene reactions described herein can be carried out at a pH that avoids denaturation of the carrier protein. In some embodiments, the photocatalytic thiol-ene reactions described herein can be carried out at a pH of 4 to 5, preferably at pH 4.4 (e.g., in acetate buffer).

[0179] In some embodiments, since the thiol-ene conjugation reactions described herein can be performed using only aqueous reagents and aqueous solvents, it may be advantageous to use a water-soluble photoinitiator (relative to photoinitiators that require organic solvents) because organic solvents may promote carrier protein denaturation and undesirable / unpredictable conjugation to non-free cysteine ​​residues (e.g., cysteines involved in intramolecular and / or intermolecular disulfide bonds) as described in Dondoni et al., 2009 and Dondoni et al., 2012.

[0180] In alternative embodiments, a water-insoluble photoinitiator may be used with an organic solvent to dissolve it, as desired. Preferably, the presence or concentration of the organic solvent should not promote denaturation of the carrier protein, and undesired / unpredictable conjugation with non-free cysteine ​​residues (e.g., cysteine ​​involved in intramolecular and / or intermolecular disulfide bonds). For example, such a photoinitiator may be a photoinitiator such as 2,2-dimethoxy-2-phenylacetophenone (DMPA).

[0181] In an alternative embodiment of the thiol-ene method described herein, the carbohydrate antigens described herein can be modified to have one or more thiol-specific functional groups, such as iodoacetamide, maleimide, benzyl halide, or bromomethylketone, which react to produce a stable thioether product by S-alkylation of the thiol group (e.g., at a relatively low pH (e.g., below 9, 8, 7.5, or 7 and above 5, 5.5, or 6), which can avoid unwanted random conjugation of carbohydrate antigens to lysine residues). Figure 4 Examples of carbohydrate antigens that have been modified to include thiol-specific functional groups (e.g., iodoacetamide and maleimidyl groups) are shown in In some embodiments, the maleimidyl group qualifies as a terminal olefin of a thiol that can be conjugated to a carrier protein via a thiol-ene reaction.

[0182] In some embodiments, the carbohydrate antigens described herein may comprise one or more B cell epitopes, and / or may induce a humoral immune response, and / or may comprise T cell epitopes, and / or induce a cell-mediated immune response in a subject following administration. In some embodiments, the glycoconjugate immunogens described herein may induce at least a cell-mediated immune response (e.g., in addition to a humoral response) to the carbohydrate antigen following administration to a subject.

[0183] In some embodiments, the carbohydrate antigen described herein can be or include, for example, a tumor-associated carbohydrate antigen (TACA). The glycoproteins and glycolipids of the outer cell membrane of cancer cells overexpress specific O-glycans. These glycoproteins constitute a family of proteins collectively referred to as mucins (MUCs), of which MUC1 represents the most extensive research. Due to active glycosyltransferases that lead to complex glycosylation patterns, MUCs on normal cells are largely O-glycosylated. In cancer cells, downregulation of key glycosyltransferases triggers the accumulation of shorter glycans, resulting in very limited O-glycosylation on mucins. The result of these changes in glycosylation patterns between normal cells and cancer cells is the excessive accumulation of TACAs, which are otherwise hidden (masked) by complex glycosylation on normal tissues. The most common TACAs include Tn antigens (monosaccharides with N-acetylgalactosamine (GalNAc) structures), TF antigens (Thomsen-Friedenreich antigens, disaccharides with Galβ1-3GalNAcα1 structures) and their respective homologous sialylated analogs. Overexpression of both Tn and TF antigens on the cell surface of various types of tumor cells contributes to cancer cell adhesion and severe metastasis to sites containing receptor lectins (lungs, liver, lymph nodes, etc.). Therefore, in some embodiments, the TACA described herein is, is derived from or comprises: Tn antigen, STn antigen, Thomsen-Friedenreich (TF) antigen, (2,3)-S-TF, (2,6)-S-TF, Globo H, GD2, GD3, GM2, GM3, N-glycolyl-GM3, Lea, sLea, Lex, sLex or any combination thereof. The structures of some common TACAs are shown in Figure 1 As used herein, the expression "derived from" when used in the context of a carbohydrate antigen refers to a carbohydrate variant derived from a known carbohydrate antigen, wherein the variant at least retains the antigenicity of the known carbohydrate antigen. For those skilled in the art, the synthesis of alkenyl-terminated TACAs can be performed by chemical and chemoenzymatic methods, as described in Danishefsky et al., 2015.

[0184] In some embodiments, the carbohydrate antigens described herein may be or comprise a carbohydrate antigen associated with an infectious agent (e.g., but not limited to, bacteria and / or viruses) or associated with an infection (e.g., but not limited to, bacterial infection and / or viral infection). In some embodiments, the carbohydrate antigens described herein may be or comprise, for example, a viral polysaccharide antigen or a bacterial capsular polysaccharide (CPS). In some embodiments, the bacterial CPS is, is derived from, or comprises a polysaccharide serotype of pneumococcus and / or streptococcus, a meningococcal CPS, or an influenza (e.g., influenza type a or b) CPS.

[0185] In some embodiments, the conjugation methods described herein may conjugate the same or more than one type of carbohydrate antigen to a carrier protein, thereby generating a multivalent glycoconjugate immunogen. In some embodiments, the multivalent glycoconjugate immunogens described herein may comprise 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15 or more carbohydrate antigens of the same or different types conjugated to a carrier protein. In some embodiments, the multivalent glycoconjugate immunogens described herein may comprise more than one carbohydrate antigen conjugated to a single free thiol group on a carrier protein (e.g., via a branched linker). In some embodiments, the multivalent glycoconjugate immunogens described herein may comprise multiple (e.g., at least 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15 or more) carbohydrate antigens conjugated to a single free thiol group on a carrier protein as a dendrimer (e.g., via a linker with extensive branches). In some embodiments, the carrier protein described herein comprises one or more free thiol groups. As used herein, "free thiol" or "free thiol groups" refers to a carrier protein having one or more thiol groups that can be used for chemical modification and / or conjugation (e.g., with a carbohydrate antigen described herein). In some embodiments, the free thiol concentration of a given carrier protein can be measured, for example, using an Ellman test using a cysteine ​​standard curve, as described in the Examples herein. The free thiol concentration of a given carrier protein can be expressed as a fold reduction in the free thiol concentration. In some embodiments, after the conjugation methods described herein, the free thiol concentration of the glycoconjugates described herein can be reduced by at least 50, 45, 40, 35, 30, 25, 20, 15, 10, 5, 4, 3, or 2 times (e.g., as measured by an Ellman test).

[0186] In some embodiments, the carrier proteins described herein can be engineered to add one or more additional cysteine ​​residues, such as at the amino terminus, carboxyl terminus, or any solvent accessible position therebetween on the carrier protein (as opposed to a position that is solvent inaccessible or "buried" in the three-dimensional structure of the protein carrier). In some embodiments, the free thiol groups of the protein carriers described herein can be defined as cysteine ​​residues that can be readily conjugated to an ene carbohydrate antigen via a photocatalyzed thiol-ene reaction when the reaction is carried out under conditions that are mild enough not to destroy the immunogenicity, structure, or activity of the carrier protein. In some embodiments, the term "activity" as used herein with respect to a carrier protein refers to the ability of the carrier protein to maintain the immunogenicity of a carbohydrate antigen (e.g., to an antibody known to specifically bind to the carbohydrate antigen).

[0187] In some embodiments, free thiol groups may refer to one or more solvent accessible cysteine ​​residues of a carrier protein, and / or cysteine ​​that is not involved in a disulfide bond, which in some cases may be important for maintaining the structure of the carrier protein. In some embodiments, the carrier protein may be a protein with one or more disulfide bonds, and wherein after conjugation with a carbohydrate antigen, one or more disulfide bonds remain unaffected (i.e., intact). In some embodiments, the carrier protein described herein does not include free thiol groups at its N-terminus and / or C-terminus. Alternatively, in some embodiments, the carrier protein described herein may include or be engineered to include free thiol groups at its N-terminus and / or C-terminus. In some embodiments, the carrier protein may be a protein with one or more disulfide bonds, and wherein the carrier protein may be pretreated with a reducing agent (e.g., dithiothreitol (DTT), 2-mercaptoethanol, tris(2-carboxyethyl)phosphine (TCEP) or 2-mercaptoethylamine-HCl) to expose one or more additional free thiol groups for conjugation with a carbohydrate antigen. Such pretreatment may be useful, for example, when the denatured (reduced) carrier protein is more immunogenic in the subject than the native (non-reduced) carrier protein.

[0188] In some embodiments, the carrier protein may be pretreated with a reducing agent (e.g., as described herein) prior to or in conjunction with the photocatalytic thiol-ene reaction. The pretreatment may expose additional free thiols that may be used for conjugation. In some embodiments, the carrier protein may be pretreated with a thiolating agent (e.g., 2-iminothiolane, N-hydroxysuccinimidyl dithiopropionate (DPS)) prior to or in conjunction with the photocatalytic thiol-ene reaction. The pretreatment may be used to increase the number of free thiols available for conjugation. In some embodiments, the carrier protein may be pretreated with a thiolating agent and then pretreated with a reducing agent prior to or in conjunction with the photocatalytic thiol-ene reaction.

[0189] In some embodiments, it may be advantageous to avoid conjugating too many multiple carbohydrate antigens to adjacent positions on the carrier protein. In some embodiments, the carrier protein may preferably lack a cysteine-rich domain (e.g., a fragment of at least 4, 5, 6, 7, 8, 9, or 10 consecutive amino acids comprising at least 50% cysteine ​​residues).

[0190] In some embodiments, the carrier protein may comprise a total of 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 cysteine ​​residues. In some embodiments, the carrier protein may comprise a total of 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 free cysteine ​​residues.

[0191] In some embodiments, the total number of carbohydrate antigens included in the glycoconjugate immunogens described herein is equal to the number of free sulfhydryl groups available on the carrier protein before conjugation. In some embodiments, the glycoconjugate immunogens described herein may include 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 carbohydrate antigens per carrier protein. In some embodiments, the present description relates to a composition comprising a glycoconjugate immunogen having about or at least 70%, 75%, 80%, 85%, 90% or 95% homogeneity in terms of carbohydrate conjugated species (e.g., at least 90% of the glycoconjugate immunogen species / molecules in the composition have the same number of carbohydrate antigens conjugated to a carrier protein).

[0192] In some embodiments, the carrier protein described herein is a protein (e.g., a peptide or polypeptide) that is preferably immunogenic to a subject to which the glycoconjugate immunogen is administered. Preferably, conjugation of the carbohydrate antigen to the carrier protein increases the immunogenicity of the carbohydrate antigen after administration to a subject, compared to administration of the unconjugated carbohydrate antigen (i.e., administration of the carbohydrate antigen alone to the subject).

[0193] In some embodiments, the carrier protein may comprise a T cell epitope, and / or induce a cell-mediated immune response in a subject following administration.

[0194] In some embodiments, the carrier protein is a protein that is foreign to the subject to be administered, which preferably has no (close) orthologs in the subject. In the case of human vaccine production, the carrier protein described herein refers to a "carrier protein suitable for human use" or simply a "suitable carrier protein", which refers to a carrier protein that is antigenically different from a human protein, so that the carrier protein will not be regarded as a "self-antigen" in humans. The use of a carrier protein that is too similar to the corresponding human protein antigenically can result in the carrier protein being regarded as a "self-antigen", which may not be ideal in human vaccines. For example, a glycoconjugate immunogen consisting of a TF antigen randomly conjugated to the ε-amino group of lysine residues of bovine serum albumin (BSA) has been previously 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 is the carbohydrate level on the 59 lysine residues of BSA random and inefficient (no more than 4 to 6 TF antigens are conjugated per BSA molecule), but BSA is not suitable as a carrier protein in human vaccines because its antigenicity is too similar to human albumin. In some embodiments, the carrier protein is not albumin (e.g., bovine serum albumin).

[0195] Preferably, the carrier protein described herein may be a protein that has been approved by a regulatory agency (e.g., FDA) for administration to 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), outer membrane protein complex of meningococcus (OMPC), Haemophilus influenzae protein D (HiD), a cytokine, or an immunogenic fragment (or variant) thereof.

[0196] In some embodiments, the carrier protein may be TT, which comprises 10 cysteine ​​residues, 4 of which are involved in disulfide bonds, thereby generating a glycoconjugate immunogen with 6 conjugated carbohydrate antigens. In some embodiments, the carrier protein may be CRM197, which comprises 4 cysteine ​​residues, thereby generating a glycoconjugate immunogen with 4 conjugated carbohydrate antigens.

[0197] In some embodiments, the carrier protein may be engineered to introduce additional cysteine ​​residues in order to introduce additional free thiol groups for conjugation to carbohydrate antigens. In some embodiments, cysteine ​​residues may be engineered in solvent exposed portions of the carrier protein.

[0198] In some aspects, the present specification relates to methods for producing a glycoconjugate vaccine or a composition for triggering an immune response. The method may include formulating the glycoconjugate immunogen described herein with a pharmaceutically acceptable excipient and / or adjuvant. In some embodiments, the adjuvant is or comprises: an inorganic compound, a mineral oil, a microbial derivative, a plant derivative, a cytokine, squalene, alum, aluminum hydroxide, aluminum phosphate, calcium phosphate hydroxide, a toll-like receptor agonist, an immunostimulatory polynucleotide (e.g., CPG), an immunostimulatory lipid, Freund's adjuvant, RIBI adjuvant, QS-21, muramyl dipeptide, TiterMax TM 、Steviune TM , Stimune TM or any combination thereof.

[0199] In some aspects, the present specification relates to a synthetic glycoconjugate immunogen comprising one or more carbohydrate antigens and an immunogenic carrier protein having one or more solvent accessible cysteine ​​residues, wherein the one or more carbohydrate antigens are linked to the one or more solvent accessible cysteine ​​residues of the immunogenic carrier protein. The conjugation of the one or more carbohydrate antigens to the immunogenic carrier protein increases the immunogenicity of the one or more carbohydrate antigens after administration to a subject compared to administration of the unconjugated carbohydrate antigen. As used herein, the term "synthetic" refers to a compound that is not a natural product, but is produced by human intervention.

[0200] In some embodiments, the glycoconjugate immunogens described herein may include more than one carbohydrate antigen (e.g., more than one type of TACA) conjugated to the same carrier protein. For example, the glycoconjugate immunogens described herein may include at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15 or more types or species of carbohydrate antigens (e.g., TACAs). In some embodiments, the glycoconjugate immunogens described herein may include any combination of TACAs selected from the group consisting of Tn, S-Tn, Thomsen-Friedenreich (TF), (2,3)-S-TF, (2,6)-S-TF, Globo H, GD2, GD3, GM2, GM3, N-glycolyl-GM3, Lea, sLea, Lex, and sLex. In some embodiments, the ratio of each TACA in the combination may vary depending on the target tumor and may include a 1 to 20 molar ratio. In this way, the glycoconjugate immunogens described herein can be tailored, for example, to specific forms of cancer associated with increased expression of specific combinations of multiple TACAs.

[0201] In some aspects, the specification relates to a glycoconjugate vaccine produced by the methods described herein, and / or comprising a synthetic glycoconjugate immunogen as described herein, and further comprising a pharmaceutically acceptable excipient and / or adjuvant. As used herein, the term "vaccine" refers to a composition comprising a glycoconjugate immunogen as described herein, which is administered to a subject to provide a therapeutic benefit to the subject. In some embodiments, the glycoconjugate vaccine described herein may be a prophylactic vaccine or a therapeutic vaccine.

[0202] The vaccine composition can be administered according to dosages and techniques well known to medical or veterinary technicians, taking into account factors such as the age, sex, weight, species and illness of the recipient animal and the route of administration. The route of administration can be transdermal, administered through mucosal membranes (e.g., oral, nasal, ocular) or by parenteral routes (e.g., intradermal, intramuscular, subcutaneous). The vaccine composition can be administered alone, or can be co-administered or sequentially administered with other treatments or therapies. The form of administration can include suspensions and preparations 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 in food and / or water, or mixed and delivered with a suitable carrier, diluent or excipient (e.g., sterile water, saline, glucose, etc.). The composition can include auxiliary substances, such as wetting agents or emulsifiers, pH buffers, adjuvants, gelling or viscosity enhancing additives, preservatives, flavoring agents, coloring agents, etc., depending on the route of administration and the desired preparation. Standard pharmaceutical texts, such as "Remington's Pharmaceutical Sciences," 1990, may be consulted to prepare suitable formulations without undue experimentation.

[0203] In some aspects, the disclosure relates to methods of immunizing, vaccinating or treating a subject, comprising administering to the subject a glycoconjugate immunogen or glycoconjugate vaccine described herein.

[0204] In some aspects, the specification relates to carbohydrate antigens that are chemically modified to be linked to a sulfhydryl-specific (thiol-specific) functional group. In some embodiments, the carbohydrate antigen is chemically modified to be linked to a terminal olefin (e.g., via a glycosidic bond), wherein the terminal olefin group can be conjugated to a free sulfhydryl group of a carrier protein via a sulfhydryl-ene reaction. In some embodiments, the carbohydrate antigen and the terminal olefin group are linked via a linker as described herein. In some embodiments, the terminal olefin group can be a vinyl or an allyl group.

[0205] In some aspects, the present description relates to synthetic glycoconjugate immunogens comprising one or more saccharide antigens covalently linked to one or more free thiol groups of a carrier protein via a linker, wherein each linker has the following structure or a stereoisomer (e.g., a diastereomer) thereof:

[0206]

[0207] wherein: SA is a carbohydrate antigen or a portion thereof; S-PC is a carrier protein; X is O, S, NR1 or CH2; R1 is -H, -COH, -COCH3 or -COEt; n is 1, 2, 3, 4 or 5; and R2 is H or Me.

[0208] In some aspects, the present description relates to synthetic glycoconjugate immunogens comprising one or more saccharide antigens covalently linked to one or more free thiol groups of a carrier protein via a linker, wherein each linker has the following structure or a stereoisomer (e.g., a diastereomer) thereof:

[0209]

[0210] wherein: SA is a carbohydrate antigen or a portion thereof; S-PC is a carrier protein; X is S, NR1, CH2 or O; R1 is -H, -COH, -COMe or -COEt; n is 1, 2, 3, 4 or 5; R2 is H or Me; q is 1, 2, 3, 4 or 5; R3 and R4 are each a hydrogen atom 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 the nitrogen atom, and m is 1. In some embodiments, when X is O, the carbohydrate antigen does not comprise Tn or STn.

[0211] In some aspects, the present description relates to a synthetic glycoconjugate immunogen comprising one or more saccharide antigens covalently linked to one or more free thiol groups of a carrier protein via a linker, wherein the synthetic glycoconjugate immunogen has the following structure or a stereoisomer thereof:

[0212]

[0213] in

[0214] -y is at least 1;

[0215] - SA is selected from the group consisting of carbohydrate antigens or immunogenic portions thereof, and when y is greater than 1, SA are identical or different;

[0216] -[S] z - PC is a carrier protein having one or more sulfur atoms derived from one or more free sulfhydryl groups;

[0217] - z is at least 1 and at least equal to y; and

[0218] -L is a linker selected from the group consisting of linkers having the following structures:

[0219]

[0220] in:

[0221] -X is O, S, NR1 or CH2;

[0222] -R1 is -H, -COH, -COCH3 or -COEt;

[0223] -n is 1, 2, 3, 4, or 5; and

[0224] - R2 is H or Me; and

[0225] When y is greater than 1, L are the same or different.

[0226] In some aspects, the present description relates to a synthetic glycoconjugate immunogen comprising one or more saccharide antigens covalently linked to one or more free thiol groups of a carrier protein via a linker, wherein the synthetic glycoconjugate immunogen has the following structure or a stereoisomer thereof:

[0227]

[0228] in

[0229] -y is at least 1;

[0230] - SA is selected from the group consisting of carbohydrate antigens or immunogenic portions thereof, and when y is greater than 1, SA are identical or different;

[0231] -[S] z - PC is a carrier protein having one or more sulfur atoms derived from one or more free sulfhydryl groups;

[0232] - z is at least 1 and at least equal to y; and;

[0233] -L is a linker selected from the group consisting of linkers having the following structures:

[0234]

[0235] in:

[0236] -X is S, NR1, CH2 or O;

[0237] -R1 is -H, -COH, -COMe or -COEt;

[0238] -n is 1, 2, 3, 4, or 5;

[0239] -R2 is H or Me;

[0240] -q is 1, 2, 3, 4, or 5;

[0241] - R3 and R4 are each a hydrogen atom 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- in which the carbonyl group is attached to the nitrogen atom and m is 1; and

[0242] When y is greater than 1, L are the same or different.

[0243] In some aspects, the present description relates to a synthetic glycoconjugate immunogen comprising one or more saccharide antigens covalently linked to one or more free thiol groups of a carrier protein via a linker, wherein the synthetic glycoconjugate immunogen has the following structure or a stereoisomer thereof:

[0244]

[0245] in

[0246] -y is at least 1;

[0247] - SA is selected from the group consisting of carbohydrate antigens or immunogenic portions thereof, and when y is greater than 1, SA are the same or different

[0248] -[S] z - PC is a carrier protein having one or more sulfur atoms derived from one or more free sulfhydryl groups;

[0249] - z is at least 1 and at least equal to y; and

[0250] -L is a linker selected from the group consisting of linkers having the following structures:

[0251]

[0252] in

[0253] -X is S, NR1, CH2 or O;

[0254] -R1 is -H, -COH, -COMe or -COEt;

[0255] -n is 1, 2, 3, 4, or 5;

[0256] -R2 is H or Me;

[0257] -q is 1, 2, 3, 4, or 5;

[0258] -r is 1, 2, 3, 4, or 5;

[0259] -R5 is S-PC, a covalent bond, or a group having the following structure:

[0260]

[0261] wherein R3 and R4 are each a hydrogen atom 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- in which the carbonyl group is attached to the nitrogen atom and m is 1; and

[0262] When y is greater than 1, L are the same or different.

[0263] In some embodiments, the description relates to a synthetic glycoconjugate immunogen as defined above, wherein the linker has the following structure:

[0264]

[0265] in

[0266] -X is S, NR1, CH2 or O;

[0267] -R1 is -H, -COH, -COMe or -COEt;

[0268] -n is 1, 2, 3, 4, or 5;

[0269] -R2 is H or Me;

[0270] -q is 1, 2, 3, 4, or 5; and

[0271] -r is 1, 2, 3, 4, or 5.

[0272] In some embodiments, the description relates to a synthetic glycoconjugate immunogen as defined above, wherein the linker has the following structure:

[0273]

[0274] in

[0275] -X is S, NR1, CH2 or O;

[0276] -R1 is -H, -COH, -COMe or -COEt;

[0277] -n is 1, 2, 3, 4, or 5;

[0278] -R2 is H or Me;

[0279] -q is 1, 2, 3, 4, or 5; and

[0280] -r is 1 or 2.

[0281] In some embodiments, the description relates to a synthetic glycoconjugate immunogen as defined above, wherein the linker has the following structure:

[0282]

[0283] in

[0284] -X is S, NR1, CH2 or O;

[0285] -R1 is -H, -COH, -COMe or -COEt;

[0286] -n is 1, 2, 3, 4, or 5;

[0287] -R2 is H or Me;

[0288] -q is 1, 2, 3, 4, or 5; and

[0289] -r is 1 or 2.

[0290] In some embodiments, the description relates to a synthetic glycoconjugate immunogen as described above, wherein y is an integer from 1 to 50, 1 to 40, 1 to 30, 1 to 20, or 1 to 10, 1 to 5, or 1 to 2.

[0291] In some embodiments, the saccharide antigen can be a carbohydrate antigen as described herein, and / or the carrier protein can be a carrier protein as described herein. In some embodiments, the synthetic saccharide conjugate immunogen can be a multivalent saccharide conjugate immunogen, for example comprising 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15 or more carbohydrate antigens of the same or different types conjugated to a carrier protein. In some embodiments, after administration to a subject, the saccharide conjugate immunogen can induce a cell-mediated immune response against the carbohydrate antigen.

[0292] In some aspects, the description relates to vaccines comprising a synthetic glycoconjugate immunogen as described herein, and a pharmaceutically acceptable excipient and / or adjuvant (eg, an adjuvant as described herein).

[0293] In some aspects, the description relates to the use of a glycoconjugate immunogen prepared by a method as described herein or a synthetic glycoconjugate immunogen as described herein for the manufacture of a vaccine.

[0294] In some aspects, the description relates to the use of a glycoconjugate immunogen prepared by a method as described herein, a glycoconjugate vaccine prepared by a method as described herein, a synthetic glycoconjugate immunogen as described herein, or a vaccine as described herein for treating a subject having a disease associated with increased expression of one or more carbohydrate antigens or saccharide antigens.

[0295] In some aspects, the description relates to the use of a glycoconjugate immunogen prepared by a method as described herein, a glycoconjugate vaccine prepared by a method as described herein, a synthetic glycoconjugate immunogen as described herein, or a vaccine as described herein for producing antibodies that specifically bind to a glycoconjugate immunogen.

[0296] In some aspects, the description relates to the use of a glycoconjugate immunogen prepared by a method as described herein, a glycoconjugate vaccine prepared by a method as described herein, a synthetic glycoconjugate immunogen as described herein, or a vaccine as described herein for detecting antibodies that specifically bind to a glycoconjugate immunogen.

[0297] In some aspects, the present specification relates to a method for producing a glycoconjugate, the method comprising: (a) providing a carbohydrate antigen covalently linked to a terminal olefin (alkenyl carbohydrate antigen), the terminal olefin being directly conjugable to a thiol group via a thiol-ene reaction; (b) providing a conjugate material suitable for conjugation with the carbohydrate antigen via a thiol-ene reaction as described herein, the conjugate material having one or more free thiols; and (c) performing a photocatalytic thiol-ene reaction to conjugate the carbohydrate antigen directly to the one or more free thiols of the conjugate material, thereby producing a glycoconjugate. In some embodiments, the conjugate material is or comprises a polymer, a polypeptide, a carrier protein as defined herein, a solid support, a particle, or any other material having a free thiol group suitable for conjugation with the carbohydrate antigen via a thiol-ene reaction as described herein.

[0298] In some aspects, the present description relates to the use of glycoconjugates produced by the methods described herein for detecting or screening for the presence of antibodies that specifically bind to carbohydrate antigens or tumor circulating cells containing carbohydrate antigens, or for detecting the presence of antibodies produced by immunization or vaccination with carbohydrate antigens. In some embodiments, the detection or screening is performed by any suitable detection method such as immunosorbent assay, ELISA, microarray or immunoblot analysis.

[0299] In some aspects, the description relates to a method of treating a subject comprising administering a glycoconjugate or glycoconjugate immunogen as defined herein or produced by a method as defined herein to generate an immune response to a carbohydrate antigen in said subject, and screening a biological sample from said subject for the presence of antibodies that specifically bind to a carbohydrate antigen.

[0300] Other objects, advantages and features of the present specification will become more apparent from a reading of the following non-limiting description of specific embodiments thereof, which are given by way of example only with reference to the accompanying drawings.

[0301] Terms

[0302] 1. A method for producing a glycoconjugate immunogen for administration to a subject, the method comprising: (a) providing a carbohydrate antigen covalently linked to a terminal olefin (alkenyl carbohydrate antigen), the terminal olefin being directly conjugable to a thiol group via a thiol-ene reaction; (b) providing a carrier protein having one or more free thiol groups; and (c) performing a photocatalyzed thiol-ene reaction to directly conjugate the carbohydrate antigen to the one or more free thiol groups of the carrier protein, thereby producing a glycoconjugate immunogen, wherein the carrier protein is immunogenic to the subject, and wherein conjugation of the carbohydrate antigen to the carrier protein increases the immunogenicity of the carbohydrate antigen after administration to the subject compared to administration of the unconjugated carbohydrate antigen.

[0303] 2. The method according to clause 1, wherein the alkenyl carbohydrate antigen is water soluble.

[0304] 3. The method according to clause 1 or 2, wherein the photocatalytic thiol-ene reaction is carried out under reaction conditions that preserve the activity, antigenicity and / or structure of the carrier protein.

[0305] 4. The method according to any one of clauses 1 to 3, wherein the photocatalytic thiol-ene reaction is carried out in the absence of any organic solvent.

[0306] 5. The method according to any one of clauses 1 to 4, wherein the photocatalytic thiol-ene reaction is performed in the presence of a catalyst.

[0307] 6. The method according to clause 5, wherein the catalyst is a water-soluble catalyst.

[0308] 7. The method according to any one of clauses 1 to 6, wherein the photocatalytic thiol-ene reaction comprises irradiation under short-wave ultraviolet light.

[0309] 8. The method of clause 7, wherein the photocatalytic thiol-ene reaction comprises irradiation at about 254 nm.

[0310] 9. The method according to any one of clauses 1 to 6, wherein the photocatalytic thiol-ene reaction comprises irradiation under long-wave ultraviolet light.

[0311] 10. The method of clause 9, wherein the photocatalytic thiol-ene reaction comprises irradiation at about 365 nm.

[0312] 11. A method according to any one of clauses 1 to 10, wherein the photocatalyzed thiol-ene reaction comprises reacting each free thiol group of the carrier protein with 1 to 10 molar equivalents of the ene carbohydrate antigen.

[0313] 12. A method according to any one of clauses 1 to 11, wherein the photocatalytic thiol-ene reaction is carried out 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.

[0314] 13. The method according to any one of clauses 1 to 12, wherein following conjugation to the carrier protein, the carbohydrate antigen cannot be cleaved from the carrier protein by endogenous enzymes of the subject.

[0315] 14. A method according to any one of clauses 1 to 13, wherein the alkenyl carbohydrate antigen is covalently linked to the terminal olefin and / or the carbohydrate antigen is conjugated to the carrier protein via a glycosidic bond, such as an O-glycosidic bond, an S-glycosidic bond, an N-glycosidic bond or a C-glycosidic bond, or a bond obtained by reductive amination, such as between allylamine and a reducing sugar.

[0316] 15. A method according to any one of clauses 1 to 14, wherein the carbohydrate antigen comprises a B cell epitope, and / or induces a humoral immune response in the subject.

[0317] 16. A method according to any one of clauses 1 to 15, wherein the carbohydrate antigen is or comprises a tumour-associated carbohydrate antigen (TACA).

[0318] 17. A method according to clause 16, wherein the TACA is, is derived from or comprises: Tn, S-Tn, Thomsen-Friedenreich (TF), (2,3)-S-TF, (2,6)-S-TF, Globo H, GD2, GD3, GM2, GM3 or any combination thereof.

[0319] 18. A method according to any one of clauses 1 to 17, wherein the photocatalysed thiol-ene reaction conjugates more than one type of carbohydrate antigen to the carrier protein, thereby generating a multivalent glycoconjugate immunogen.

[0320] 19. A method according to clause 18, wherein the multivalent glycoconjugate immunogen comprises 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15 or more different types of carbohydrate antigens conjugated to the carrier protein.

[0321] 20. A method according to any one of clauses 1 to 19, wherein the carbohydrate antigen is or comprises a viral polysaccharide antigen or a bacterial capsular polysaccharide (CPS).

[0322] 21. A method according to clause 20, wherein the bacterial CPS is, is derived from or comprises a polysaccharide serotype of Pneumococcus and / or Streptococcus.

[0323] 22. A method according to any one of clauses 1 to 21, wherein the carbohydrate antigen in (a) is linked to the terminal olefin via a linker.

[0324] 23. A method according to any one of clauses 1 to 22, wherein the carrier protein comprises one or more cysteine ​​residues having one or more free thiol groups.

[0325] 24. A method according to any one of clauses 1 to 23, wherein the carrier protein comprises a human T-cell epitope, and / or induces a cell-mediated immune response in the subject.

[0326] 25. A method according to any one of clauses 1 to 24, wherein 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), a cytokine or an immunogenic fragment thereof.

[0327] 26. A method according to any one of clauses 1 to 25, wherein the carrier protein is a protein having one or more disulfide bonds, and wherein: (i) after the photocatalyzed thiol-ene reaction, the one or more disulfide bonds remain unaffected; or (ii) the carrier protein is pretreated with a reducing agent to expose one or more additional free thiol groups for conjugation with the carbohydrate antigen.

[0328] 27. A method according to any one of clauses 1 to 26, wherein the total number of carbohydrate antigens contained in the glycoconjugate immunogen is equal to the number of free thiol groups available on the carrier protein prior to conjugation.

[0329] 28. The method according to any one of clauses 1 to 27, wherein the glycoconjugate immunogen induces a cell-mediated immune response against the carbohydrate antigen upon administration to the subject.

[0330] 29. The method according to any one of clauses 1 to 28, further comprising: (d) purifying the glycoconjugate immunogen.

[0331] 30. A method for producing a saccharide conjugate vaccine, the method comprising formulating a saccharide conjugate immunogen prepared by the method of any one of clauses 1 to 29 together with a pharmaceutically acceptable excipient and / or adjuvant.

[0332] 31. A method according to clause 30, wherein the adjuvant is or comprises: an inorganic compound, a mineral oil, a microbial derivative, a plant derivative, a cytokine, squalene, alum, aluminum hydroxide, aluminum phosphate, calcium phosphate hydroxide, a toll-like receptor agonist, an immunostimulatory polynucleotide, an immunostimulatory lipid, Freund's adjuvant, RIBI adjuvant, QS-21, muramyl dipeptide or any combination thereof.

[0333] 32. A synthetic glycoconjugate immunogen comprising one or more carbohydrate antigens and an immunogenic carrier protein having one or more solvent accessible cysteine ​​residues, wherein the one or more carbohydrate antigens are linked to the one or more solvent accessible cysteine ​​residues of the immunogenic carrier protein, and wherein conjugation of the one or more carbohydrate antigens to the immunogenic carrier protein increases the immunogenicity of the one or more carbohydrate antigens after administration to a subject compared to administration of unconjugated carbohydrate antigens.

[0334] 33. A synthetic glycoconjugate immunogen according to clause 32, wherein the one or more carbohydrate antigens are linked to the one or more solvent accessible cysteine ​​residues via a linker.

[0335] 34. A synthetic glycoconjugate immunogen according to clause 32, wherein the one or more carbohydrate antigens are linked to the linker via a bond as defined in clause 13 or 14.

[0336] 35. A synthetic glycoconjugate immunogen according to any one of clauses 32 to 34, wherein the one or more carbohydrate antigens are as defined in clause 2, 15, 16, 17, 20 or 21.

[0337] 36. The synthetic glycoconjugate immunogen according to any one of clauses 32 to 35, which is a multivalent glycoconjugate immunogen as defined in clause 18 or 19.

[0338] 37. A synthetic glycoconjugate immunogen according to any one of clauses 32 to 36, wherein the carrier protein is as defined in clause 23, 24, 25 or 26.

[0339] 38. A synthetic glycoconjugate immunogen according to any one of clauses 32 to 37, wherein the total number of carbohydrate antigens contained in the glycoconjugate immunogen is equal to the number of solvent accessible cysteine ​​residues on the carrier protein.

[0340] 39. A synthetic glycoconjugate immunogen according to any one of clauses 32 to 38 which, upon administration to the subject, induces a cell-mediated immune response to the carbohydrate antigen.

[0341] 40. A synthetic glycoconjugate immunogen according to any one of clauses 32 to 39, prepared by the method of any one of clauses 1 to 29.

[0342] 41. A glycoconjugate vaccine produced by the method of clause 30 or 31, and / or comprising the synthetic glycoconjugate immunogen of any one of clauses 32 to 40 and a pharmaceutically acceptable excipient and / or adjuvant.

[0343] 42. The saccharide conjugate vaccine according to clause 41, which is a preventive vaccine or a therapeutic vaccine.

[0344] 43. A method of immunizing, vaccinating or treating a subject, comprising administering to the subject a glycoconjugate immunogen produced by the method of any one of clauses 1 to 29, a glycoconjugate vaccine produced by the method of clauses 30 or 31, a synthetic glycoconjugate immunogen of any one of clauses 32 to 40, or a glycoconjugate vaccine of clauses 41 or 42.

[0345] 44. A synthetic glycoconjugate immunogen according to any one of clauses 32 to 40 or a glycoconjugate vaccine according to clause 41 or 42 for use in immunizing, vaccinating or treating a subject suffering from a disease.

[0346] 45. A synthetic glycoconjugate immunogen comprising one or more saccharide antigens covalently linked to one or more free thiol groups of a carrier protein via a linker, wherein each linker has the following structure or a stereoisomer thereof:

[0347]

[0348] in:

[0349] -SA is a carbohydrate antigen or a portion thereof;

[0350] -PC is a carrier protein;

[0351] -X is O, S, NR1 or CH2;

[0352] -R1 is -H, -COH, -COCH3 or -COEt;

[0353] -n is 1, 2, 3, 4, or 5; and

[0354] -R2 is H or Me.

[0355] 46. ​​A synthetic glycoconjugate immunogen comprising one or more saccharide antigens covalently linked to one or more free thiol groups of a carrier protein via a linker, wherein each linker has the following structure or a stereoisomer thereof:

[0356]

[0357] in:

[0358] -SA is a carbohydrate antigen or a portion thereof;

[0359] -PC is a carrier protein;

[0360] -X is S, NR1, CH2 or O;

[0361] -R1 is -H, -COH, -COMe or -COEt;

[0362] -n is 1, 2, 3, 4, or 5;

[0363] -R2 is H or Me;

[0364] -q is 1, 2, 3, 4, or 5;

[0365] - R3 and R4 are each a hydrogen atom 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- in which the carbonyl group is attached to the nitrogen atom and m is 1.

[0366] 47. A synthetic glycoconjugate immunogen according to clause 45 or 46, wherein the saccharide antigen is a carbohydrate antigen as defined in clause 15, 16, 17, 20 or 21.

[0367] 48. A synthetic glycoconjugate immunogen according to any one of clauses 45 to 47, wherein the carrier protein is a carrier protein as defined in clause 23, 24 or 25.

[0368] 49. The synthetic glycoconjugate immunogen according to any one of clauses 45 to 49 which is a multivalent glycoconjugate immunogen.

[0369] 50. A synthetic glycoconjugate immunogen according to clause 49, wherein the multivalent glycoconjugate immunogen comprises 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15 or more different types of carbohydrate antigens conjugated to the carrier protein.

[0370] 51. A synthetic glycoconjugate immunogen according to any one of clauses 45 to 50, wherein the glycoconjugate immunogen induces a cell-mediated immune response to the carbohydrate antigen upon administration to a subject.

[0371] 52. A vaccine comprising the synthetic glycoconjugate immunogen of any one of clauses 45 to 51 and a pharmaceutically acceptable excipient and / or adjuvant.

[0372] 53. The vaccine of clause 52, wherein the adjuvant is as defined in clause 31.

[0373] 54. Use of a glycoconjugate immunogen prepared by the method of any one of clauses 1 to 29 or a synthetic glycoconjugate immunogen as defined in any one of clauses 32 to 40 and 45 to 51 for the manufacture of a vaccine.

[0374] 55. Use of a glycoconjugate immunogen prepared by the method of any one of clauses 1 to 29, a glycoconjugate vaccine produced by the method of clause 30 or 31, a synthetic glycoconjugate immunogen as defined in any one of clauses 32 to 40 and 45 to 51, or a vaccine of clause 52 or 63 for treating a subject suffering from a disease associated with increased expression of said one or more carbohydrate antigens or saccharide antigens. Example

[0375] Example 1: General Method

[0376] Reactions were performed under nitrogen atmosphere using commercial HPLC grade. Commercial reagents (Sigma Aldrich) were used without further purification. N-acetyl-D-galactosamine and N-acetylneuraminic acid were provided by Rose Scientific Ltd. Alberta, Canada. Fmoc-β-Ala-Wang resin and Fmoc amino acids are commercially available from Peptide Technologies Ltd, Pierrefonds, Qc, Canada. Silica gel 60F 254 Coated plates (E. Merck) were monitored by thin layer chromatography for reaction progress. Conjugation by click thiol-ene photoreaction was performed in a quartz cuvette (10x10 mm pathlength, Fisher Scientific Canada, catalog number 14-958-130) placed between two handheld UV 365 nm lamps (UV-AC handheld lamp, dual 254 / 365 nm UV; 115 V-60 Hz, 0.16 amps, VWR Canada, catalog number 89131-492). ZEOprep from Canadian Life Science was used. TMFlash chromatography was performed on silica gel (40-63 μm). Detection was performed under UV light or by spraying with 20% ethanolic sulfuric acid or molybdate or KMnO4 solution followed by heating. TM 300MHz and Bruker Avance TM NMR spectra were recorded on a III HD 600 MHz spectrometer. 1 H) and 77.16ppm( 13 The chemical shifts of residual CHCl3 in C) are reported in ppm for protons and carbons (δ). Coupling constants (J) are reported in Hertz (Hz), and the following abbreviations are used for peak multiplicities: singlet (s), doublet (d), doublet of doublets (dd), doublets with equal coupling constants (t ap ), triplet (t), multiplet (m). COZY (Correlated SpectroscopY) and HSQC (Heteronuclear Single Quantum Coherence) experiments were used for analysis and distribution. High-resolution mass spectrometry (HRMS) was measured using an LC-MS-TOF (liquid chromatography mass spectrometry time of flight) instrument from Agilent technologies in positive and / or negative electrospray mode via the UQAM analytical platform. Protonated ions (M+H) + or sodium adduct (M+Na) +For empirical formula confirmation. Native TT and TT-conjugates were dialyzed using 2000 KDa benzoylated dialysis tubing (Sigma-Aldrich (Ontario, Canada)). The sulfhydryl content of both native and conjugated TT was determined by the Ellman test at 412 nm (Ellman, GL Arch. Biochem. Biophys. 1959, 82, 70-77). The total sugar content of TT-conjugates was determined by the colorimetric DuBois test (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) measured at 492 nm using a UV / VIS Ultrospec 100prot spectrophotometer (Biochrom, USA). Dynamic light scattering (DLS) particle size distribution was measured in PBS using a Zetasizer Nano S90 from Malvern.The mouse monoclonal IgG3 antibody JAA-F11 was generated as previously described in Rittenhouse-Diakun et al., 1998.

[0377] General Solid Phase Peptide Synthesis (SPPS) Procedure

[0378] The solid phase peptide synthesis (SPPS) procedure was performed according to the literature procedure (Papadopoulos et al., 2012) and starting from Fmoc-β-Ala-Wang resin (650 mg, 0.34 mmol, 1.0 equiv; 100-200 mesh, loading = 0.52 mmol / g). The reaction was carried out by rotary stirring in an Econo-Pac disposable column 1.5 x 14 cm (20 mL) (Bio-Rad Laboratories, ON, Canada). The resin was swollen in CH2Cl2 for 1 hour, then filtered and reconditioned in DMF during i 1. The Fmoc protecting groups of commercial resins or amino acids were removed with a solution of 20% piperidine in DMF (5 mL, 2x 5 min, then 1x 10 min). The solvent and reagents were removed by filtration, and the resin was washed with DMF, CH2Cl2 and MeOH (3X for each solvent). The presence of free amino groups was verified by Kaiser test or TNBS test. The free amines on the resin were treated with a preactivated Fmoc amino acid solution at 4°C (10 min): 3 equivalents of amino acid, 3 equivalents of HBTU (N,N,N′,N′-tetramethyl-O-(1H-benzotriazol-1-yl)uronium hexafluorophosphate) and a catalytic amount of HOBt (1-hydroxybenzotriazole) in DMF. DIPEA (diisopropylamine, 9 equivalents) was then added to the mixture and stirred at room temperature for 1 hour and 30 minutes. The completion of the coupling was determined using a Kaiser or TNBS colorimetric test. After filtration, the resin was washed and the Fmoc removal step was repeated again. At the end of the synthetic sequence, the last free amine was capped by acetylation (Ac2O / DIPEA / DMF 1:1:8, 1h). After filtration, the solution was drained, the resin was dried in vacuo, and cleaved using trifluoroacetic acid / water / ethanedithiol / triisopropylsilane (94.0 / 2.5 / 2.5 / 1.0) for 3 hours. 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 air flow. The crude peptide was dissolved in water to separate from the resin. The solution was then freeze-dried to obtain the desired peptide.

[0379] dTT831-844-Cys-βAla. Starting from Fmoc-β-Ala-Wang resin (650 mg, 0.34 mmol, 1.0 equiv; 100-200 mesh, loading = 0.52 mmol / g), the desired peptide dTT831-844-Cys-βAla (62 mg, 0.034 mmol, 10%) was isolated as a white powder. ESI + -LC-MS:C 83 H 135 O 24 N 19 S[M+2H]+2 Calculated value, 906.9819; Found value, 906.9849; CAN / H2O 5 to 95% 5.42 min.

[0380] Cys-dTT831-844-βAla. Starting from Fmoc-β-Ala-Wang resin (650 mg, 0.34 mmol, 1.0 equiv; 100-200 mesh, loading = 0.52 mmol / g), the desired peptide Cys-dTT831-844-βAla (62 mg, 0.034 mmol, 10%) was isolated as a white powder. ESI + -LC-MS:C 83 H 135 O 24 N 19 S[M+2H] +2 Calculated value, 906.9819; Found, 906.9844; CAN / H2O 5 to 95% 5.32 min.

[0381] Example 2: Allyl 2-acetylamino-3,6-di-O-pivaloyl-2-deoxy-α-D-pyranoglucoside (Chemical Compound 2)

[0382]

[0383] Reference Figure 5, acetyl chloride (2.76mL, 38.80mmol, 3.43 equivalents) was added dropwise to allyl alcohol (20.8mL) at 0°C under an argon atmosphere. At room temperature, N-acetyl-D-glucosamine (Compound 1) (2.50g, 11.3mmol, 1.00 equivalents) was added. The reaction mixture was stirred at 70°C for 3 hours and then quenched by adding solid NaHCO 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 crude allyl 2-acetylamino-2-deoxy-D-glucosamine was precipitated by grinding with EtO / ethanol. After grinding, the solvent was then removed under reduced pressure several times. Then, under nitrogen atmosphere at -15 ° C, pivaloyl chloride (3.90 mL, 31.64 mmol, 2.80 equiv) was added dropwise to a suspension of the crude allyl 2-acetamido-2-deoxy-α-D-pyranoglucopyranoside intermediate in a mixture of anhydrous dichloromethane-pyridine (45 mL, v / v, 1:2). The reaction mixture was stirred for 2 hours and warmed to room temperature to give the desired α-anomer (Compound 2) (Rf = 0.32) and some β-anomer (Rf = 0.18); hexane / EtOAc 1: 1. The mixture was then diluted with CH2Cl2, and the organic phase was washed with HCl (1M) several times, a saturated aqueous solution of KHSO4, a saturated solution of NaHCO3, and brine. The organic phase was dried over Na2SO4, filtered and evaporated under reduced pressure. The yellowish oil was purified by flash chromatography on silica gel (hexane-EtOAc 6:4 to 1:1) to give the desired compound allyl 2-acetamido-3,6-di-O-pivaloyl-2-deoxy-α-D-pyranoglucopyranoside (Compound 2) as a white solid (4.85 g, 6.78 mmol, 60%). Rf = 0.32; hexane / EtOAc 1:1; Fig. 6A and B: 1 H NMR(CDCl3,600MHz): δ5.87(dddd,1H,J H,H =16.8,10.5,6.2,5.3Hz,OCH2CH=CH2),5.77(d,1H,J NH,H2 =9.7Hz,NH),5.31-5.25(m,1H,OCH2CH=CH2),5.22(dd,1H,J H,H =10.4,1.3Hz,OCH2CH=CH2),5.09(dd,1H,J 3,4 =10.7,J 2,3 =9.3Hz,H-3),4.83(d,1H,J 1,2=3.7Hz,H-1),4.39(m,1H,H-6a),4.35-4.25(m,2H,H-6b and H-2),4.19(m,1H,OCH2) ,4.02-3.93(m,1H,OCH2),3.85(m,1H,H-5),3.59-3.48(m,1H,H-4),3.03(d,1H,J 4,OH =5.1 Hz, OH-4), 1.93 (s, 3H, NHCOCH3), 1.23 (s, 9H, tert-butyl) and 1.19 ppm (s, 9H, tert-butyl); 13 C NMR (CDCl3, 150MHz): δ179.8, 179.1 (tert-BuCO), 169.7 (NHCO), 133.2 (OCH2CH=CH2), 118.1 (OCH2CH=CH2), 96.4 (C-1), 73.4 (C-3), 70.5 (C-5), 69.1 (C-4). 68.2 (OCH2), 63.1 (C-6), 51.4 (C-2), 39.0, 38.9 (2x C(CH3)3), 27.2, 27.0 (2x C(CH3)3) and 23.2ppm (CH3). Fig.6C and 6D: ESI + -HRMS:C 21 H 36 O8N's [M+H] + Calculated, 430.2435; found, 430.2445. The β-anomer was isolated as a white solid (971 mg, 2.26 mmol, 20%). Rf = 0.18; Hexane / EtOAc 1:1; 1 H NMR(CDCl3,300MHz):δ6.00(d,1H,J NH,H2 =9.3Hz, NH), 5.95-5.75 (m, 1H, OCH2CH=CH2), 5.35-5.03 (m, 3H, OCH2CH=CH2 and H-3), 4.55 (d, 1H, J 1,2 =8.4 Hz, H-1), 4.47-425 (m, 3H, H-6a, 6b and OCH2), 4.14-3.90 (m, 2H, OCH2 and H-2), 3.65-3.43 (m, 2H, H-5 and H-4), 3.23 (sb, 1H, OH-4), 1.92 (s, 3H, NHCOCH3), 1.23 (s, 9H, tert-butyl) and 1.20 ppm (s, 9H, tert-butyl).

[0384] Example 3: Allyl 2-acetylamino-2-deoxy-α-D-galactopyranoside (allyl Tn)

[0385]

[0386] Reference Figure 5 , under argon atmosphere, di-O-pivaloyl compound (compound 2) (5.50g, 12.80mmol, 1.0 equivalent) in a mixture of anhydrous dichloromethane-pyridine (126mL, v / v 20:1) was cooled to -35°C. Then trifluoromethanesulfonic anhydride (2.58mL, 15.36mmol, 1.2 equivalents) was added, and the mixture was stirred at this temperature. The temperature was warmed to room temperature for 2 hours. Then water (12mL) was added to the solution. The mixture was heated and stirred overnight (12 hours) at reflux (about 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 Zemplen conditions (1M methanol solution of sodium methoxide, 40mL, pH 9). The solution was stirred at 50°C overnight. After cooling to room temperature, the solution was stirred by adding ion exchange resin ( IR 120,H + ), filtered, washed with MeOH, and the solvent removed under reduced pressure. Allyl T was isolated by precipitation in MeOH / EtOAc / hexane N , after freeze-drying, it was a white solid (2.36 g, 9.10 mmol, 71%) Rf = 0.32; EtOAc / MeOH 4:1; Fig. 7A and 7B : 1 H NMR (CD3OD, 600MHz): δ5.99-5.88(m,1H,OCH2CH=CH2),5.31(dd,1H,J trans =17.3,J gem =1.3Hz,OCH2CH=CH2),5.17(dd,1H,J cis =10.5Hz,OCH2CH=CH2),4.86(d,1H,J 1,2 =3.8Hz,H-1),4.27(dd,1H,J 2,3 =11.0Hz,H-2),4.20(m,1H,OCH2),4.00(m,1H,OCH2),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, CH3); 13C NMR (CD3OD, 150MHz): δ172.5 (NHCO), 134.2 (OCH2CH=CH2), 116.1 (OCH2CH=CH2), 96.6 (C-1 ),71.2(C-3),69.0(C-4),68.3(C-5).67.8(OCH2),61.4(C-6),50.2(C-2) and 21.2ppm(CH3). Figure 7C and 7D :ESI + -HRMS:C 11 H 20 O6N's [M+H] + Calculated value, 262.1285; measured value, 262.1294.

[0387] Procedure B: Allyl 2-acetylamino-2-deoxy-α-D-galactopyranoside can also be prepared directly from N-acetylgalactosamine (GalNAc) according to the literature procedure (Feng et al., 2004): To a solution of N-acetylgalactosamine (442 mg, 2 mmol, 1.0 eq.) in allyl alcohol (8 mL) was added BF3.Et2O (250 μL, 2 mmol, 1.0 eq.) at room temperature and the mixture was stirred at 70°C for 2 hours. The solution was cooled to room temperature and the solvent was removed under reduced pressure. The dried crude product was dissolved in a minimum amount of EtOH (5 mL). The desired allyl T was precipitated in diisopropyl ether. N The product was isolated as a white solid (417 mg, 1.60 mmol, 80%).

[0388] The C-allylGalNAc analog ( Figure 5 )[1-(2'-acetylamino-2'-deoxy-α-D-galactopyranosyl)-2-propene]: 3-(2-acetylamino-3,4,6-tri-O-acetyl-2-deoxy-α-D-galactopyranosyl)-1-propene (Cui et al., 1998) (371 mg, 1.00 mmol, 1.0 equiv) 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 separated by adding an ion exchange resin ( IR 120,H + ), filtered, washed with MeOH, and the solvent was removed under reduced pressure. C-allyl T was purified by silica gel chromatography (EtOAc / MeOH 9:1 to 4:1). N, then crystallized in EtOH as a white solid (213 mg, 0.87 mmol, 87%). Rf = 0.28; EtOH 4:1; mp 230 ° C (Litt. 215-217 ° C, EtOAc / EtOH); According to literature NMR data: 1 H NMR(CD3OD,600MHz): δ5.81(m,1H, 1 CH2CH=CH2),5.08(dd,1H,J trans =17.2,J gem =1.7Hz, 1 CH2CH=CH2),5.02(dd,1H,J cis =10.2Hz, 1 CH2CH=CH2),4.22(dd,1H,J=9.3,5.0Hz,H-2'),4.14(dt,1H,J=10.0,5.0Hz,H-1'),3.91(dd,1H,J=3.0H z,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 1.97ppm (s, CH3); 13 C NMR(CD3OD,150MHz): δ173.6(NHCO),136.2( 1 CH2CH=CH2),117.1( 1 CH2CH=CH2), 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 CH2) and 22.5ppm(CH3).ESI + -LCMS:C 11 H 20 O5N's [M+H] + Calculated value, 246.1336; Found, 246.1332; CAN / H2O 5 to 95% 1.4 min.

[0389] According to Knapp et al., 2002, S-allyl GalNAc analogs ( Figure 5 ).

[0390] Example 4: Allyl 2-acetylamino-4,6-O-benzylidene-2-deoxy-α-D-galactopyranoside (compound 4)

[0391]

[0392] Reference Figure 5, to a solution of allylGalNAc(Tn) (2.35 g, 9.0 mmol, 1.0 equiv) and benzaldehyde dimethyl acetal (6.75 mL, 45.0 mmol, 5.0 equiv) in anhydrous DMF (20 mL) was added a catalytic amount of p-toluenesulfonic acid monohydrate. The mixture was stirred at room temperature. After 5 h, the mixture was diluted with CHCl3 and washed with saturated aqueous NaHCO3. The organic layer was separated and washed with water, dried over Na2SO4, and concentrated to give a white solid. The benzylidene acetal (4) was isolated by precipitation in EtOAc / hexane as a white solid (2.64 g, 7.56, 84%). Rf = 0.21; DCM / MeOH 9.0:0.5; Fig.8A: 1 H NMR (CDCl3, 300MHz): δ7.59-7.46(m,2H,H-ar),7.43-7.31(m,3H,H-ar),5.91(m,1H,OCH2CH=CH2),5.75(d,1H,J NH,H2 =9.0Hz,NH),5.58(s,1H,PhCH),5.34-5.17(m,2H,OCH2CH=CH2),5.01(d,1H,J 1,2 =3.5Hz,H-1),4.56-4.42(ddd,1H,J 2,3 =10.9Hz,J 2,OH =9.1Hz,H-2),4.34(dd,1H,J 5,6a =1.5Hz,J 6a,6b =12.5 Hz, H-6a), 4.19 (m, 2H, H-4 and OCH2), 4.04 (m, 1H, dd, 1H, J 5,6b =1.6Hz,J 6a,6b =12.5Hz,H-6b),4.01(m,OCH2),3.86(dd,1H,J 3,4 =10.9Hz,H-3),3.71(sb,1H,H-5),2.80(d,1H,J 3,OH =10.7Hz,OH-3) and 2.05ppm(s,3H,CH3); Fig.8B&8C:ESI + -HRMS:C 18 H 24 O6N's [M+H] + Calculated value, 350.1598; measured value, 350.1608.

[0393] Example 5: Allyl (2,3,4,6-tetra-O-benzoyl-β-D-galactopyranosyl)-(1→3)-2-acetyl Amino-4,6-O-benzylidene-2-deoxy-α-D-galactopyranoside (Compound 6)

[0394]

[0395] Refer to Figure 5 , under a nitrogen atmosphere, compound 4 (2.0 g, 5.72 mmol, 1.0 equiv) and mercuric cyanide (2.17 g, 8.60 mmol, 1.5 equiv) were dissolved in a mixture of anhydrous nitromethane-toluene (100 mL, 3:2, v / v) containing molecular sieve. The mixture was stirred at room temperature for 30 minutes. 2,3,4,6-Tetra-O-benzoyl-α-D-galactopyranosyl bromide (compound 5) (5.66 g, 8.58 mmol, 1.5 equiv) was added to the mixture. The solution was stirred at 70 °C for 5 hours and then continued to be stirred at room temperature overnight (8 hours). After all the starting material (compound 4) was consumed as indicated by TLC (DCM / MeOH 9.0:0.5), the solvent was removed under reduced pressure. The residue was dissolved in EtOAc and then filtered through a pad of diatomaceous earth. The filtrate was washed successively with 10% aqueous potassium iodide 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 of 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, Rf = 0.20; hexane / EtOAc 1:2; Fig. 9A and 9B : 1 1H NMR (CDCl3, 600 MHz): δ8.06-7.19 (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, OCH2CH=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, NH), 5.23 (m, 3H, OCH2CH=CH2 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, OCH2 and H-3), 3.96 (m, 1H, OCH2), 3.75 (m, 1H, H-6b), 3.51 (m, 1H, H-5) and 1.40 ppm (s, 3H, CH3); 13 C NMR (CDC 13 ,150MHz): δ170.0(NHCO),166.0,165.5,165.4,165.2(CO),137.6-126.2(multi,30C-arom),133.2(OCH2CH=CH2),117.8(OCH2CH=CH2),102.0(C-1 II ),100.9(CPhCH),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(OCH2),68.1(C-4 II ),62.9(C-5),62.6(C-6 I ), 48.2(C-2) and 22.5ppm(CH3).Fig.9C&9D:ESI + -HRMS:C 52 H 50 O 15 N's [M+H] + Calculated value, 928.3175; measured value, 928.3133.

[0396] Example 6: Allyl (β-D-galactopyranosyl)-(1→3)-2-acetylamino-2-deoxy-α-D-galactopyranosyl Lactoside (allyl TF)

[0397]

[0398] Reference Figure 5 A solution of compound 6 (1.12 g, 1.20 mmol, 1.0 equiv) 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 stirred by adding an ion exchange resin (Amberlite IR 120, H +), filtered, washed with MeOH, and the solution was suspended with silica gel, filtered, and the solvent was removed under reduced pressure. The silica gel was washed several times with 100% EtOAc and then 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) as a white solid. Rf = 0.20; CHCl3 / MeOH / H2O 11:6:1; Fig. 10A and 10B : 1 H NMR (D2O, 600MHz): δ7.47-7.39(m,2H,H-ar),7.37-7.26(m,3H,H-ar),5.82(m,1 H,OCH2CH=CH2),5.61(s,1H,PhCH),5.20-5.09(m,2H,OCH2CH=CH2),4.88(d,1H,J 1,2 =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,OCH2),3.96-3.88(m,1H,H-5),4.56-4.42(ddd,1H,J 2,3 =10.9Hz,J 2,OH =9.1Hz,H-2),4.34(dd,1H,J 5,6a =1.5Hz,J 6a,6b =12.5 Hz, H-6a), 4.19 (m, 2H, H-4 and OCH2), 4.04 (m, 1H, dd, 1H, J 5,6b =1.6Hz,J 6a,6b =12.5Hz,H-6b),4.01(m,OCH2),3.86(dd,1H,J 3,4 =10.9Hz,H-3),3.71(sb,1H,H-5),2.80(d,1H,J 3,OH =10.7Hz,OH-3) and 2.05ppm(s,3H,CH3); 3.83(s,1H,OCH2),3.72(d,1H,J 3’,4’ =J 4’,5’ =3.2Hz,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’ =10.0Hz,J 3’,4’ =3.3Hz,H-3 II),3.33-3.24(m,1H,H-2 II ) and 1.86ppm(s,3H,CH3); 13 C NMR (D2O, 150MHz): δ174.6 (NHCO), 136.8 (C-arom), 133.6 (OCH2CH=CH2), 129.9, 128.7, 126.5 (C-arom), 118.0 (OCH2CH=CH2), 104.9 (C-1 II ),101.3(CHPh),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=CH2),68.6(C-4 II ),63.0(C-5),61.0(C-6 II ), 48.6 (C-2) and 22.0ppm (CH3). Rf = 0.38; EtOAc / MeOH / H2O 7:3:0.1; Rf = 0.46; ACN / MeOH / H2O7:2:1. Fig. 10C and 10D :ESI + -HRMS:C 24 H 34 O 11 N's [M+H] + Calculated value, 512.2126; measured value, 512.2119.

[0399] 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; Rf = 0.53; CHCl3 / MeOH / H2O 11:6:1; Fig.11A and 11B : 1 H NMR (D2O, 600MHz): δ5.80(m,1H,OCH2CH=CH2),5.19(dd,1H,J trans =17.3Hz,OCH2CH=CH2),5.09(dd,1H,J cis =10.4Hz,OCH2CH=CH2),4.77(d,1H,J 1,2 =3.7Hz,H-1),4.29(d,1H,J1,2 =3.7Hz,H-1),4.29(d,1H,J 1,2 =7.8Hz,H-1 II ),4.16(dd,1H,J 2,3 =11.2Hz,J 1,2 =3.7 Hz, H-2), 4.08-4.01 (m, 2H, H-4 and OCH2), 3.92-3.82 (m, 3H, H-3, H-5 and OCH2), 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’ =10.0Hz,J 1’,2’ =7.7Hz,H-2 II ) and 1.85ppm(s,3H,CH3); 13 C NMR (150MHz, CDC 13 ): δ174.6(NHCO),133.7(OCH2CH=CH2),117.9(OCH2CH=CH2),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(OCH2),61.2(C-6 II ), 61.0(C-6), 48.6(C-2) and 22.0ppm(CH3). Fig. 11C and 11D :ESI + -HRMS:C 17 H 29 O 11 [M+Na] of NNa + Calculated value, 446.1633; measured value, 446.1613.

[0400] Example 7: Purification of Tetanus Toxoid Monomer

[0401] Prior to conjugation, tetanus toxoid (TT) monomer was obtained by gel filtration chromatography. One milliliter of a liquid formulation containing 4.5 mg / ml protein (determined by a modified Lowry protein assay) was loaded onto a 4% tantalum phosphate buffered saline (PBS) filled with 1% tantalum. The protein was eluted in a preparative grade (E Healthcare Life Sciences, Uppsala, Sweden) XK16-100 column equilibrated in PBS (20 mM NaHPO4 [pH 7.2], 150 mM NaCl) with the same buffer. The protein eluted from the column had two peaks: the earlier eluting peak contained the oligomeric toxoid, while the later eluting peak (corresponding to Mr 150,000) contained the TT monomer. The fractions corresponding to the later (monomer) peak were pooled, desalted with deionized water, and purified using Plus-70 centrifugal filter devices (30K Ultracel PL membrane; Millipore, Billerica, MA) were used for concentration and then lyophilization.

[0402] Example 8: Tn-tetanus toxoid conjugate (Tn-TT conjugate)

[0403]

[0404] Procedure A: To a solution of allyl Tn (0.15 mg, 0.56 μmol, 18.0 equiv) in 500 μL PBS in a quartz cell was added 1.5 mL of PBS containing 4.7 mg (0.03 μmol) of tetanus toxoid. After degassing under an argon atmosphere, the solution was irradiated at 254 nm for 7 hours. The solution was dialyzed for 24 hours under several washes of double distilled water and lyophilized to obtain Tn-TT-A conjugate (2.0 mg, 43%) as a white solid. Procedure B: Under the same thiol-ene click reaction, a catalytic amount of DMPA in acetonitrile (100 μL) was added to a solution of allyl Tn (3 mg, 10.8 μmol, 180.0 equiv) containing tetanus toxoid in PBS (2 mL, 4.5 mg / mL, 0.06 μmol). The solution was irradiated at 365 nm for 15 minutes. The solution was then dialyzed for 24 hours and lyophilized to give Tn-TT-B conjugate as a white solid (5.4 mg, 61%).

[0405] Example 9: TF-Tetanus Toxoid Conjugate (TF-TT Conjugate)

[0406]

[0407] Procedure A: To a solution of allyl TF (0.24 mg, 0.56 μmol, 18.0 equiv) in 500 μL PBS in a quartz cell was added 1.5 mL of PBS containing 4.7 mg (0.03 μmol) of tetanus toxoid. After degassed under argon atmosphere, the solution was irradiated at 254 nm for 7 h. The solution was then dialyzed for 24 h and lyophilized to afford TF-TT-A conjugate (2.1 mg, 45%) as a white solid. Procedure B: Under the same thiol-ene click reaction, a catalytic amount of DMPA in acetonitrile (100 μL) was added to a solution of allyl TF (5 mg, 10.8 μmol, 180.0 equiv) and tetanus toxoid in PBS (2 mL, 4.5 mg / mL, 0.06 μmol). The solution was irradiated at 365 nm for 15 min. The solution was then dialyzed for 24 hours and lyophilized to give TF-TT-B conjugate as a white solid (5.3 mg, 60%).

[0408] Example 10: Characterization of Tn-TT and TF-TT conjugates

[0409] Tetanus toxoid (TT) alone and vaccine conjugates (Tn-TT-A; Tn-TT-B; TF-TT-A; and TF-TT-B) were analyzed by SDS gel electrophoresis, colorimetric analysis for the presence of sugar content (Dubois test), thiol content by Ellmann test, dynamic light scattering (DLS) ( Fig.12 ), as well as reactivity with the known mouse monoclonal antibody JAA-F11 using two-way immunodiffusion. The SDS gel electrophoresis results clearly indicated the monomeric form of the conjugate, which also stained positively for the presence of carbohydrate antigens (Tn and TF). Colorimetric analysis (Dubois test) confirmed the presence of carbohydrate antigens (10% by weight) and the absence of residual free thiol groups on the carrier protein after conjugation. Two-way immunodiffusion analysis showed a precipitation band, clearly indicating the cross-reactivity of the new TF-TT conjugate with the anti-TF monoclonal antibody JAA-F11, thereby confirming the presence of the immunogenic carbohydrate antigen (TF) on the TF-TT conjugate, no precipitation band was observed using the carrier protein alone (tetanus toxoid).

[0410] HPLC analysis of conjugates. HPLC analysis of glycoconjugate preparations was performed by size exclusion chromatography. Chromatographic separation was performed using three 8x 300 mm Shodex OHpak gel filtration columns (two SB-804 and one SB-803) connected in series, preceded by a SB-807G guard column (Showa Denko). The glycoconjugate 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 differential refractometer (RI) detector model 2300 and a UV detector model 2600 at a wavelength of 280 nm. The conjugate preparations (8 mg / mL solution in mobile phase) were injected using a 50-μΛ injection loop. In selected experiments, the fractions eluted in the void volume, which corresponded to the conjugate fractions, were combined, dialyzed with water Spectra / Por with a molecular weight cutoff (MWCO) of 12,000 to 14,000 [Spectrum Laboratories]), and lyophilized. This corresponds to a 2:1 fractionated conjugate.

[0411] Example 11: Conjugation of O-allyl sugars to the peptide dTT831-844-Cys-βAla by thiol-ene photoreaction Effect of thiol groups

[0412] The peptide dTT831-844-Cys-βAla (tetanus toxin (831-844); molecular weight: 1813, SEQ ID NO: 2) was dissolved in water at a concentration of 3.7e-3 M, and (500 uL) of the peptide was stirred with (6 uL, 0.1 M, 3.0 equiv) O-allyl-Tn or O-allyl-TF and a water-soluble catalyst AAPH (2,2'-azobis(2-methylpropionitrile, 23 uL, 0.025 M, 3.0 equiv) in a quartz cuvette (optical path length 10x10 mm, Fisher) placed between two handheld UV-visible light sources 2-5 cm away from the cuvette at room temperature. 365 nm lamp (0.16 amp, VWR). UV was turned on immediately after the addition of AAPH. The solution was sampled at 0, 45, 90 and 135 minutes and the free thiol concentration was measured by Ellman's test using a cysteine ​​standard curve. Fig.13 It was shown that the free thiol concentration decreased in a time-dependent manner due to the photothiol-ene reaction.

[0413] Example 12: Location / accessibility of cysteine ​​in dTT831-844-Cys-βAla and type of activator used Effect of thiol type on the conjugation of O-allyl-Tn and the immunoreactivity of the thiol-ene photoreaction products

[0414] Peptide dTT831-844 with N-terminal or C-terminal cysteine ​​was synthesized and dissolved in water at a concentration of 2mM (1mL), and stirred with 200uL, 0.1M, 10 equivalents) of O-allyl-Tn and a catalytic amount (0.2 equivalents) of the activator AAPH in a quartz cuvette (optical path 10x10 mm, Fisher) at room temperature, and the quartz cuvette was placed between two handheld UV 365nm lamps (0.16 amps, VWR) 2-5cm from the cuvette. The UV was turned on immediately after the addition of the activator and allowed to react for 60 minutes. The free thiol concentration was measured by the Ellman test using a cysteine ​​standard curve at time 0 and 60 minutes. The reduction factor of the free thiol corresponds to the thiol concentration of t=0 / t=60.

[0415] The results are shown in Fig.14 , which shows the fold reduction in free thiol concentration of dTT831-844 peptides with N-terminal ("SH-peptide") or C-terminal ("peptide-SH") cysteine ​​in thiol-ene photoreaction conjugation using a water-soluble activator (AAPH) or a water-insoluble activator (DMPA).

[0416] The immunoreactivity of the thiol-ene photoreaction products was measured by enzyme-linked lectin assay (ELLA). 1 μg of peptide in 100 μL PBS pH 7.4 was adsorbed to a 96-well plate (Maxisorp TM , Nunc) for a minimum of 1 hour at room temperature or overnight at 4°C. The wells were then washed with PBS-Tween TM The wells were then washed twice with PBS-T + 1% BSA blocking solution for 30 minutes. The wells were then washed 3 times with PBS-T and filled with 100 μL 1 / 100 dilutions of Vicia villosa lectin (anti-Tn VVA) or peanut lectin (anti-TF PNA) (VVA-hrp, PNA-hrp, EY LAbs) coupled to horseradish peroxidase. After incubation at room temperature for 1 hour with gentle shaking or at 4 ° C for 12 hours, the wells were washed 4 times with PBS-T and 100 μL hrp substrate (Ultra TMB-ELISA, Thermo Scientific) was added to each well. The reaction was terminated by adding 100 μL 0.5N sulfuric acid and the plate was read at OD 450nm in a microplate reader (Biotek EL808).

[0417] The results are shown in Fig.15In the figure, the reactivity of lectins to different Tn peptides (i.e., unconjugated: "peptide-Cys" and "Cys-peptide"; Tn conjugated: "Tn-peptide" and "peptide-Tn"; "unc": uncoated plate) is shown with the use of a water-soluble activator (AAPH) or a water-insoluble activator (DMPA). Interestingly, the TT peptide with a C-terminal cysteine ​​was specifically recognized by the anti-Tn lectin VVA, and the conjugation reaction was most efficient with the water-soluble activator AAPH. None of the Tn glycoconjugates were recognized by the anti-TF lectin (peanut agglutinin, PNA) used as a negative control.

[0418] Example 13: Effect of Wavelength on Thiol-Ene Photoreaction Conjugation of Peptide dTT831-844-Cys-βAla to O-allyl-Tn Impact

[0419] The peptide dTT831-844-Cys-βAla (MW: 1813, SEQ ID NO: 2) was stirred at room temperature with 0.55mM O-allyl-Tn and (1.44mg, 5.5nmol, 10.0 equivalents) AAPH or (0.3mg, 1.1nmol, 2.0 equivalents) DMPA in 1mL water in a quartz cuvette (10x10mm pathlength, Fisher), which was placed between two handheld UV365nm or 355nm lamps (0.16 amps, VWR) 2-5cm from the cuvette. UV was turned on immediately after the addition of AAPH and turned off after (60min) to stop the reaction. The peptide was also conjugated to O-allyl-Tn at UV254nm in the absence of an activator. Free thiol concentrations were measured by Ellman's test using a cysteine ​​standard curve. Fig.16 Shown is the reduction in free thiol groups over time (minutes) for three different conjugation conditions ("355nm+AAPH"; "365nm+DMPA" and "254nm").

[0420] The immunoreactivity of the thiol-ene photoreaction products was measured by enzyme-linked lectin assay (ELLA). The indicated amount of peptides in 100 μL PBS pH 7.4 was adsorbed to a 96-well plate (Maxisorp, Nunc) for a minimum of 1 hour at room temperature or overnight at 4°C. The wells were then washed with PBS-Tween. TMThe wells were washed twice with 0.05% (PBS-T), and the wells were filled with blocking solution for 30 minutes. The wells were then washed 3 times with PBS-T and filled with 100 μL 1 / 100 dilutions of Vicia villosa lectin (VVA-hrp, EY LAbs) coupled to horseradish peroxidase. After incubation for 1 hour or 12 hours at 4 ° C with gentle shaking at room temperature, the wells were washed 4 times with PBS-T, and 100 μL hrp substrate (Ultra TMB-ELISA, Thermo Scientific) was added to each well. The reaction was terminated by adding 100 μL 0.5N sulfuric acid, and the plate was read at 450nm in a microplate reader (Biotek EL808).

[0421] ELLA results are shown in Fig.17 , which shows the reactivity of VVA lectin to various amounts of Tn peptide conjugated via thiol-ene photoreaction in the presence of AAPH or DMPAP at 365 nm, or in the absence of activator at 254 nm. The conjugated product was detected by the lectin, while the unconjugated peptide ("Peptide") or O-allyl-Tn ("Tn") alone did not react. Interestingly, Fig.17 It was shown that conjugation at 254 nm in the absence of an activator produced a glycoconjugate product that was more immunoreactive than the glycoconjugate products produced at 365 nm or 355 nm in the presence of an activator.

[0422] Example 14: C-allyl and O-allyl sugars react with the peptide dTT831-844-Cys-β via thiol-ene photoreaction Conjugation and reactivity of Ala

[0423] The dTT831-844-Cys-βAla peptide (MW: 1813) was dissolved in water at a concentration of 0.55 mM, and 1 mL thereof was stirred at room temperature with O-allyl-Tn, O-allyl-TF or C-allyl-Tn (100 uL, 11 mM, 2.0 equivalents) and AAPH (1.44 mg, 5.5 nmol, 10.0 equivalents) in a quartz cuvette (optical path 10x10 mm, Fisher), which was placed between two handheld UV 365 nm lamps (0.16 amperes, VWR) 2-5 cm from the cuvette. UV was turned on immediately after AAPH was added. The solution was sampled at the specified time, and the free thiol concentration was measured by the Ellman test using a cysteine ​​standard curve. Fig.18 Shown is the reduction in free thiol groups over time (minutes) for the different allyl sugars tested: O-allyl-Tn ("Tn"), O-allyl-TF ("TF"), or C-allyl-Tn ("cTn").

[0424] The immunoreactivity of the thiol-ene photoreaction products was measured by ELISA and ELISA. The indicated amount of peptide conjugates in 100 μL PBS pH 7.4 was adsorbed to a 96-well plate (Maxisorp, Nunc) for a minimum of 1 hour at room temperature or overnight at 4°C. The wells were then washed with PBS-Tween. TM The wells were then washed twice with 0.05% (PBS-T) and then filled with PBS-T + 1% BSA blocking solution for 30 minutes. The wells were then washed 3 times with PBS-T and filled with 100 μL of 1 / 100 dilution of Vicia villosa lectin or peanut agglutinin (VVA-hrp, PNA-hrp, EY LAbs) coupled to horseradish peroxidase or with 0.1 μg / ml purified TF-specific mouse monoclonal antibody JAAF11. After incubation with gentle shaking for 1 hour at room temperature or 12 hours at 4°C, the wells containing JAAF11 were washed with PBS-T and then further incubated with secondary antibody goat anti-mouse IgG (H+L)-hrp (Jackson Immunoresearch) for 60 minutes. The wells were then washed 4 times with PBS-T and 100 μL of hrp substrate (Ultra TMB-ELISA, Thermo Scientific) was added to each well. The reaction was stopped by adding 100 μL 0.5 N sulfuric acid and the plate was read at 450 nm in a microplate reader (Biotek EL808).

[0425] Fig.19 The anti-Tn lectin VVA was shown to recognize only peptide conjugates produced from O-allyl-Tn (and not from C-allyl-Tn). These results suggest that, although conjugated to the peptide, C-allyl-Tn does not have a conformation that allows binding to the lectin.

[0426] Example 15: Conjugation of O-allyl TF with chemically reduced dTT via thiol-ene photoreaction

[0427] Detoxified tetanus toxoid (dTT) was dialyzed in PBS pH 7.4 (MWCO 2,000) and then incubated with 1000eqDTT. After 2 hours of rotation incubation at room temperature, the reduced protein solution was washed in PBS pH 7.4 by centrifugal filtration (MWCO 10,000, Amicon). The reduced protein (1 mL, 3.51 mg / mL, 0.02 nmol) was then stirred with O-allyl-TF (30.0 equivalents) and AAPH (2.0 equivalents) in a quartz cuvette (10x10 mm pathlength, Fisher) with a final volume of 1.0 mL of PBS, and the quartz cuvette was placed between two handheld UV 365 nm lamps (0.16 amperes, VWR) at a distance of 2-5 cm from the cuvette. UV was turned on immediately after adding AAPH to initiate the reaction, and then stopped after 2 hours.

[0428] The free thiol concentration of reduced dTT generated by DTT treatment compared to native dTT was measured on buffer-exchanged dTT samples by Ellman's test using a cysteine ​​standard curve, and protein concentration was measured by Bradford assay using a BSA standard curve.

[0429] To measure the immunoreactivity of dTT samples, 1 μg of protein sample was diluted into 100 μL PBS pH 7.4 and adsorbed to the wells of a 96-well plate (Maxisorp, Nunc) for a minimum of 1 hour at room temperature or overnight at 4°C. The wells were then washed with PBS-Tween TM The wells were then washed twice with 0.05% (PBS-T) and then filled with blocking solution for 30 minutes. The wells were then washed 3 times with PBS-T and filled with 100 μL 0.1 μg / mL purified TF-specific mouse monoclonal antibody JAAF11. After incubation at room temperature with gentle shaking for 1 hour or at 4°C for 12 hours, the plate was washed with PBS-T and then incubated with 100 μL of a 1 / 1000 dilution of the secondary antibody goat anti-mouse IgG (H+L)-hrp (Jackson Immunoresearch) for 60 minutes. The wells were then washed 4 times with PBS-T and 100 μL hrp substrate (Ultra TMB-ELISA, ThermoScientific) was added to each well. The reaction was terminated by adding 100 μL 0.5N sulfuric acid and the plate was read at 450 nm in a microplate reader (Biotek EL808).

[0430] Fig. 20Immunoreactivity of dTT-TF to JAAF11 ("dTT(DTT)-TF") was shown, whereas reduced dTT ("dTT(DTT)") or native dTT (data not shown) were unreactive. This suggests that O-allyl-TF is indeed conjugated to dTT in an immunoreactive conformation via the thiol-ene photoreaction.

[0431] Example 16: Conjugation of O-allyl-Tn with chemically reduced dTT via thiol-ene photoreaction

[0432] DTT was dialyzed in PBS pH 7.4 (MWCO 30,000), and then 0.5 mL (5.8 mg / mL) was incubated with 50 or 500 eq DTT in rotation at room temperature. After 2 hours, the reduced protein solution was washed in PBS pH 7.4 by centrifugal filtration (MWCO10,000, Amicon). The protein (70 uL, 0.5 mmol / mL) reduced with 500 eq DTT was then stirred with 0.1 M O-allyl-TF (60.0 equivalents) and 0.1 M AAPH (4.0 equivalents) in a quartz cuvette (10x10 mm optical path, Fisher), which was placed between two handheld UV 365 nm lamps (0.16 amperes, VWR) at a distance of 2-5 cm from the cuvette. UV was turned on immediately after adding AAPH to initiate the reaction, and then stopped after 45 minutes. To verify whether the free thiol groups of reduced dTT formed a thioether bond with resistance to reduction due to conjugation or were reoxidized to disulfide bonds, the conjugated product was treated with 100 Eq of DTT for 1 hour, then washed by centrifugal filtration, and then the thiol content was measured before and after the reduction treatment. The free thiol dosage was measured by Ellman test using a cysteine ​​standard curve. The protein concentration was measured by Bradford assay using a BSA standard curve.

[0433] Fig.21 The molar ratio of free sulfhydryls in dTT after different conjugation conditions is shown. The results show that the dTT protein is reduced in a dose-dependent manner, and after treatment with 500Eq DTT, 9 free sulfhydryls are reached (in the theoretical value of 10 cysteines in dTT at most). Free sulfhydryls are reduced to a level equal to or lower than that of unreduced dTT by conjugation with O-allyl-Tn or O-allyl-TF through sulfhydryl-ene photoreaction. The resistance of dTT-Tn conjugates to reduction confirms the presence of reduction-resistant thioether bonds formed by sulfhydryl-ene photoreaction.

[0434] To measure the immunoreactivity of dTT samples, 1 μg of protein sample was diluted into 100 μL PBS pH 7.4 and adsorbed to the wells of a 96-well plate (Maxisorp, Nunc) for a minimum of 1 hour at room temperature or overnight at 4°C. The wells were then washed with PBS-TweenTM The wells were then washed twice with 0.05% (PBS-T) and then filled with blocking solution for 30 minutes. The wells were then washed three times with PBS-T and filled with 100 μL of a 1 / 100 dilution of Vicia villosa lectin or peanut agglutinin (VVA-hrp, PNA-hrp, EY LAbs) coupled to horseradish peroxidase or with 0.1 μg / ml of purified TF-specific mouse monoclonal antibody JAAF11. After incubation with gentle shaking at room temperature for 1 hour or at 4°C for 12 hours, the wells containing JAAF11 were washed with PBS-T and then further incubated with 100 μL of a 1 / 1000 dilution of the secondary antibody goat anti-mouse IgG (H+L)-hrp (Jackson Immunoresearch) for 60 minutes. After incubation at room temperature with gentle shaking for 1 hour or at 4°C for 12 hours, the plate was washed 4 times with PBS-T, and 100 μL of hrp substrate (Ultra TMB-ELISA, ThermoScientific) was added to each well. The reaction was terminated by adding 100 μL of 0.5N sulfuric acid and the plate was read at 450 nm in a microplate reader (Biotek EL808).

[0435] Fig. 22 The high reactivity of the dTT-Tn conjugate to lectin VVA was shown compared to unconjugated dTT.

[0436] Native dTT and dTT-Tn conjugates were analyzed by SDS-PAGE electrophoresis on 10% polyacrylamide gels followed by staining with Coomassie Brilliant Blue. Gel electrophoresis results indicated that dTT-Tn conjugates migrated at a higher molecular weight than native non-conjugated dTT (data not shown).

[0437] Example 17: Effect of AAPH concentration on the reactivity of O-allyl-Tn with chemically thiolated dTT via thiol-ene photoreactivity Effect of conjugation

[0438] dTT was dialyzed in PBS pH 8 (MWCO 30,000), and then 100 μL (0.8 mg) was stirred with 200 Eq / protein of 0.1 M 2-iminothiolane hydrochloride (Sigma) in water, 200 Eq / protein of 0.1 M O-allyl-TF in water, and 0.8 Eq / protein or about 450 Eq / protein of AAPH in water in a final volume of 124 μL in a quartz cuvette (optical path 10x10 mm, Fisher), which was placed between two hand-held UV 365 nm lamps (0.16 amps, VWR) at a distance of 2-5 cm from the cuvette. UV was turned on immediately after the addition of AAPH to initiate the reaction. Samples were recovered at 15 minutes during the photoreaction and at the completion of the 45-minute reaction. Sephadex was used TMThe conjugated product was buffer exchanged to PBS pH 7.4 by size exclusion chromatography on a G25-filled mini-spin column. Free thiol concentration was measured by Ellman assay using a cysteine ​​standard curve. Protein concentration was measured by Bradford assay using BSA as standard.

[0439] Fig.23 It was shown that the ratio of free thiols in dTT samples after chemical thiolation with 2-iminothiolane and thiol-ene photoreaction in the absence of O-allyl-Tn reached 25-30, while the presence of O-allyl-Tn reduced the thiol ratio of dTT to about 20 and 5 in a dose-dependent manner related to the AAPH concentration, indicating that the thiol-ene photoreaction formed a thioether bond.

[0440] To measure the immunoreactivity of dTT samples, 1 μg of protein sample was diluted into 100 μL PBS pH 7.4 and adsorbed to the wells of a 96-well plate (Maxisorp, Nunc) for a minimum of 1 hour at room temperature or overnight at 4°C. The wells were then washed with PBS-Tween TM The plates were washed twice with 0.05% (PBS-T) and then filled with blocking solution for 30 minutes. The wells were then washed 3 times with PBS-T and filled with 100 μL 1 / 100 dilutions of Vicia vicifolia lectin (VVA-hrp, EY LAbs) coupled to horseradish peroxidase. After incubation for 1 hour or 12 hours at 4 ° C with gentle shaking at room temperature, the plates were washed 4 times with PBS-T, and 100 μL hrp substrate (Ultra TMB-ELISA, ThermoScientific) was then added to each well. The reaction was terminated by adding 100 μL 0.5N sulfuric acid and the plate was read at 450 nm in a microplate reader (Biotek EL808).

[0441] Fig.24 The reactivity of dTT samples towards lectin VVA-hrp is shown. A higher reactivity of the Tn conjugate compared to native unconjugated dTT was observed. Furthermore, the reactivity correlated with the duration of the photoreaction and the amount of AAPH activator used.

[0442] dTT conjugates (5 μg) were analyzed by SDS-PAGE electrophoresis on 10% polyacrylamide gels, which were stained with Coomassie Brilliant Blue or transferred to membranes (Immobilon-P, Merck Millipore) for western blot analysis. The membranes were blocked in PBS-T + 1% BSA for 1 hour, washed with PBS-T, and then incubated with a 1 / 100 dilution of Vicia villosa lectin conjugated to horseradish peroxidase (VVA-hrp, EY LAbs) in PBS-T for 1 hour at room temperature or overnight at 4°C. The membranes were then washed extensively with PBS-T and bound VVA-hrp was detected with the horseradish peroxidase chromogenic substrate 4-chloro-1-naphthol (0.3 mg / mL in PBS containing 0.03% hydrogen peroxide, Sigma). Results from both Coomassie-stained SDS-PAGE and corresponding Western blots stained with VVA-hrp indicated that dTT-Tn migrated at a higher molecular weight than native dTT and was specifically reactive toward VVA, indicating that the product was indeed conjugated to Tn (data not shown).

[0443] Example 18: Conjugation of O-allyl-Tn and O-allyl-TF with chemically reduced BSA via thiol-ene photoreaction combine

[0444] BSA (7mg in 1mL PBS, pH 7.4) was incubated with 600eq of DTT for 2 hours, then washed in PBS pH 7.4 by centrifugal filtration (MWCO 10,000, Amicon). The reduced protein was then conjugated with O-allyl-Tn or with O-allyl-TF by sulfhydryl-ene photoreaction. Reduced BSA (600uL, 5.3mg / mL, 0.047nmol) was stirred with O-allyl sugar (60eq) and AAPH (4.0 equivalents) and PBS pH 7.4 (720uL) in a quartz cuvette (10x10mm optical path, Fisher), which was placed between two handheld UV 365nm lamps (0.16 amperes, VWR) at a distance of 2-5cm from the cuvette. UV was turned on immediately after AAPH was added to initiate the reaction. After 45 minutes, the product was washed with PBS pH 7.4 by centrifugal filtration (MWCO 10,000, Amicon) to remove any unreacted O-allyl groups and reagents. The free thiol concentration of reduced BSA was measured by Ellman assay using a cysteine ​​standard curve, and protein concentration was measured by Bradford assay using BSA as a standard.

[0445] To measure the immunoreactivity of BSA samples, 1 μg of protein sample was diluted into 100 μL PBS pH 7.4 and adsorbed to the wells of a 96-well plate (Maxisorp, Nunc) for a minimum of 1 hour at room temperature or overnight at 4°C. The wells were then washed with PBS-Tween. TM The wells were then washed twice with 0.05% (PBS-T) and then filled with blocking solution for 30 minutes. The wells were then washed three times with PBS-T and filled with 100 μL of a 1 / 100 dilution of Vicia villosa lectin or peanut agglutinin (VVA-hrp, PNA-hrp, EY LAbs) coupled to horseradish peroxidase or with 0.1 μg / ml of purified TF-specific mouse monoclonal antibody JAAF11. After incubation with gentle shaking for 1 hour at room temperature or 12 hours at 4°C, the wells containing JAAF11 were washed with PBS-T and then further incubated with secondary antibody goat anti-mouse IgG (H+L)-hrp (Jackson Immunoresearch) for 60 minutes. The wells were then washed four times with PBS-T and 100 μL of hrp substrate (Ultra TMB-ELISA, Thermo Scientific) was added to each well. The reaction was stopped by adding 100 μL 0.5 N sulfuric acid and the plate was read at 450 nm in a microplate reader (Biotek EL808).

[0446] Fig.25 It was shown that BSA-TF and BSA-Tn produced under these conditions were highly reactive and specific to the anti-TF JAAF11 monoclonal antibody and the anti-Tn lectin VVA, respectively.

[0447] BSA-Tn conjugates and BSA-TF conjugates (5 μg) were analyzed by SDS-PAGE electrophoresis on 10% polyacrylamide gels stained with Coomassie Brilliant Blue. Reduction of BSA under the conditions used produced a molar ratio of 19.29 thiols, as measured by the Ellman test. After conjugation with either O-allyl sugar, the Ellman test was negative, indicating that the free thiol groups were chemically coupled to the O-allyl sugar via the thiol-ene photoreaction (data not shown). Coomassie-stained SDS-PAGE gels showed that both BSA-conjugates migrated to higher molecular weight species at the expected molecular weight of BSA (data not shown).

[0448] Example 19: Conjugation of O-allyl-Tn to chemically reduced BSA via thiol-ene photoreaction

[0449] BSA (500 μL, 7 mg / mL) was incubated with 400, 200 or 1000 Eq of DTT (0.5 M) at room temperature for 1 hour. The reduced BSA solution was then dialyzed overnight in PBS pH 7.4. The free thiol concentration of the reduced BSA was then measured by the Ellman assay using a cysteine ​​standard curve, and protein concentration was measured by the Bradford assay using BSA as a standard, thus calculating the molar ratio of free thiol / BSA.

[0450] Fig.26 The correlation between the amount of free thiol groups generated and the amount of reducing agent DTT is shown, reaching about 10 thiol groups / BSA when reduction was carried out with 1000 Eq DTT under the conditions used.

[0451] The reduced protein is then conjugated with various ratios of O-allyl-Tn. Reduced BSA (5 nmol) is stirred with 100 Eq of O-allyl-Tn / reduced BSA or 100 Eq of O-allyl-Tn / BSA-sulfhydryl and AAPH and PBS pH 7.4 in a final volume of 500 μL in a quartz cuvette (10x10 mm optical path, Fisher), which is placed between two handheld UV 365 nm lamps (0.16 amperes, VWR) at a distance of 2-5 cm from the cuvette. UV is turned on immediately after AAPH is added to initiate the reaction. After 45 minutes, the product is washed with PBS pH 7.4 by centrifugal filtration (MWCO 10,000, Amicon) to remove unreacted O-allyl and reagents.

[0452] To measure the immunoreactivity of BSA samples, 1 μg of protein sample was diluted into 100 μL PBS pH 7.4 and adsorbed to the wells of a 96-well plate (Maxisorp, Nunc) for a minimum of 1 hour at room temperature or overnight at 4°C. The wells were then washed with PBS-Tween. TMThe wells were then washed twice with 0.05% (PBS-T) and then filled with blocking solution for 30 minutes. The wells were then washed 3 times with PBS-T and filled with 100 μL of a 1 / 100 dilution of Vicia villosa lectin (VVA-hrp, EYLAbs) coupled to horseradish peroxidase or with 0.1 μg / ml purified TF-specific mouse monoclonal antibody JAAF11. After incubation at room temperature with gentle shaking for 1 hour or at 4°C for 12 hours, the wells containing JAAF11 were washed with PBS-T and then further incubated with secondary antibody goat anti-mouse IgG (H+L)-hrp (Jackson Immunoresearch) for 60 minutes. After incubation at room temperature with gentle shaking for 1 hour or at 4°C for 12 hours, the plate was washed 4 times with PBS-T and 100 ul of hrp substrate (Ultra TMB-ELISA, Thermo Scientific) was added to each well. The reaction was stopped by adding 100 μL 0.5 N sulfuric acid and the plate was read at 450 nm in a microplate reader (Biotek EL808).

[0453] Fig. 27 The reactivity of various BSA-Tn conjugates towards the lectin VVA and the anti-TF monoclonal antibody JAAF11 (+ goat anti-mouse IgG-hrp conjugate) is shown. The two BSA-conjugates conjugated with the highest O-allyl ratio were found to be specifically highly reactive towards VVA due to the thiol-ene photoreaction.

[0454] BSA-Tn conjugates (5 μg) were analyzed by SDS-PAGE electrophoresis on 10% polyacrylamide gels stained with Coomassie Brilliant Blue or stained for glycoproteins according to the manufacturer's instructions (Pierce). The results showed that the BSA conjugates migrating at high molecular weight stained positive for glycans, while native unconjugated BSA did not (data not shown).

[0455] Example 19: Conjugation of O-allyl-TF to chemically thiolated BSA via thiol-ene photoreaction

[0456] BSA (1 mL, 0.088 μmol, 5.89 mg / mL, PBS pH 8.0) was incubated with 5, 10 or 20 Eq of 2-iminothiolane (5, 10, 20 μL, 0.1 mmol / mL) for 1 hour. The thiolated BSA was then washed by centrifugal filtration (MWCO 10,000, Amicon) to remove unreacted reagents and then conjugated with O-allyl-TF by thiol-ene photoconjugation. Thiolated BSA (440; 480; 575 μL, 6.8; 6.4; 5.2 mg / mL) was stirred with 3 eq / thiol of O-allyl-Tn and AAPH (0.2 Eq / thiol) and PBS pH 7.4 at a final concentration of 1 mL in a quartz cuvette (10x10 mm pathlength, Fisher), which was placed between two handheld UV 365 nm lamps (0.16 amps, VWR) 2-5 cm from the cuvette. The UV was turned on immediately after the addition of AAPH to initiate the reaction. After 1 hour, the product was washed with PBS pH 7.4 by centrifugal filtration (MWCO10,000, Amicon) to remove unreacted O-allyl groups and other reagents. The free thiol concentration of the BSA samples was measured by the Ellman assay using a cysteine ​​standard curve, and the protein concentration was measured by the Bradford assay using BSA as a standard.

[0457] To measure the immunoreactivity of BSA samples, 1 μg of protein sample was diluted into 100 μL PBS pH 7.4 and adsorbed to the wells of a 96-well plate (Maxisorp, Nunc) for a minimum of 1 hour at room temperature or overnight at 4°C. The wells were then washed with PBS-Tween. TM The wells were then washed twice with 0.05% (PBS-T) and then filled with blocking solution for 30 minutes. The wells were then washed three times with PBS-T and filled with 100 μL of a 1 / 100 dilution of the lectin peanut agglutinin (PNA-hrp, EY LAbs) coupled to horseradish peroxidase or with 0.1 μg / ml of purified TF-specific mouse monoclonal antibody JAAF11. After incubation with gentle shaking for 1 hour at room temperature or 12 hours at 4°C, the wells containing JAAF11 were washed with PBS-T and then further incubated with secondary antibody goat anti-mouse IgG (H+L)-hrp (Jackson Immunoresearch) for 60 minutes. The wells were then washed four times with PBS-T and 100 μL of hrp substrate (Ultra TMB-ELISA, Thermo Scientific) was added to each well. The reaction was stopped by adding 100 μL 0.5 N sulfuric acid and the plate was read at 450 nm in a microplate reader (Biotek EL808).

[0458] exist Fig.28 In Figure 2, the biaxial graph shows the reactivity of BSA-TF conjugates to the anti-TF lectin PNA or mAb JAAF11, which is related to the thiol ratio of each conjugate measured before TF conjugation. These results show that JAAF11 reactivity was detected with the most thiolated BSA.

[0459] Example 20: Conjugation of O-allyl-Tn to chemically thiolated BSA via thiol-ene photoreaction

[0460] BSA (250 μL, 4.3 mg / mL) was incubated with 200 eq of 0.1 M 2-iminothiolane (Sigma) in a final volume of 316 μL in a quartz cuvette (10x10 mm pathlength, Fisher) in the presence or absence of 200 Eq of 0.1 M O-allyl-Tn and approximately 450 Eq of AAPH and PBS pH 8. The quartz cuvette was placed between two handheld UV 365 nm lamps (0.16 amps, VWR) at a distance of 2-5 cm from the cuvette. UV was turned on immediately after the addition of AAPH to initiate the reaction. Samples were taken at designated times during the reaction. The reaction was terminated after 45 minutes and then Sephadex was used. TM The product was buffer exchanged to PBS pH 7.4 by gel filtration chromatography using a G25 mini spin column. The free thiol concentration of the BSA samples was measured by the Ellman assay using a cysteine ​​standard curve, and the protein concentration was measured by the Bradford assay using BSA as a standard.

[0461] Fig.29 A time-dependent increase in free thiol functionality following reaction of BSA with 2-iminothiolane alone or in the presence of 365 nm light is shown, whereas the addition of AAPH to the photoreaction in the presence or absence of O-allyl-Tn prevents the formation of detectable free thiol groups.

[0462] To measure the immunoreactivity of BSA samples, 1 μg of protein sample was diluted into 100 μL PBS pH 7.4 and adsorbed to the wells of a 96-well plate (Maxisorp, Nunc) for a minimum of 1 hour at room temperature or overnight at 4°C. The wells were then washed with PBS-Tween. TMThe wells were then washed twice with 0.05% (PBS-T) and then filled with blocking solution for 30 minutes. The wells were then washed 3 times with PBS-T and filled with 100 μL 1 / 100 dilutions of Vicia villosa lectin coupled to horseradish peroxidase (VVA-hrp, EY LAbs). After incubation for 1 hour at room temperature or 12 hours at 4 ° C, the wells were washed 4 times with PBS-T, and 100 μL hrp substrate (Ultra TMB-ELISA, ThermoScientific) was then added to each well. The reaction was terminated by adding 100 μL 0.5N sulfuric acid and read at 450nm in a microplate reader (Biotek EL808).

[0463] exist Fig.30 In Figure 2, the reactivity of BSA conjugates towards lectin VVA showed that only BSA produced by thiol-ene photoreaction in the presence of O-allyl-Tn was detected by VVA, while the product produced in the absence of O-allyl-Tn was negative in ELLA. These results suggest that in the absence of O-allyl sugars, AAPH and UV365nm photocatalyze the reoxidation of free disulfides to disulfides, which competes with the formation of thioether bonds in the presence of O-allyl-Tn.

[0464] Example 21: Conjugation of O-allyl-Tn to chemically thiolated BSA via thiol-ene photoreaction

[0465] BSA (250 μL, 4.3 mg / mL) was incubated with 200 Eq of 0.1 M 2-iminothiolane (Sigma), 200 Eq of 0.1 M O-allyl-Tn and 0.8 Eq of AAPH or about 450 Eq of AAPH and PBS pH 8 in a final volume of 316 μL in a quartz cuvette (10x10 mm pathlength, Fisher). The effect of the antioxidant vitamin C (about 5 mM) on conjugation was tested under conditions containing large amounts of AAPH. The cuvette was placed between two handheld UV 365 nm lamps (0.16 amps, VWR) at a distance of 2-5 cm from the cuvette. UV was turned on immediately after the addition of AAPH to initiate the reaction. Samples were taken at designated times during the reaction. The reaction was terminated after 45 minutes and then Sephadex was used. TM The product was buffer exchanged to PBS pH 7.4 by gel filtration chromatography using a G25 mini spin column. The free thiol concentration of the conjugated BSA samples was measured by the Ellman assay using a cysteine ​​standard curve, and the protein concentration was measured by the Bradford assay using BSA as a standard.

[0466] To measure the immunoreactivity of BSA samples, 1 μg of protein sample was diluted into 100 μL PBS pH 7.4 and adsorbed to the wells of a 96-well plate (Maxisorp, Nunc) for a minimum of 1 hour at room temperature or overnight at 4°C. The wells were then washed with PBS-Tween. TM The wells were washed twice with 0.05% (PBS-T) and then filled with blocking solution for 30 minutes. The wells were then washed 3 times with PBS-T and filled with 100 μL 1 / 100 dilutions of Vicia vicifolia lectin (VVA-hrp, EY LAbs) coupled to horseradish peroxidase. After incubation for 1 hour or 12 hours at 4 ° C with gentle shaking at room temperature, the wells were washed 4 times with PBS-T, and 100 μL hrp substrate (Ultra TMB-ELISA, ThermoScientific) was then added to each well. The reaction was terminated by adding 100 μL 0.5N sulfuric acid and read at 450nm in a microplate reader (Biotek EL808).

[0467] With dual Y-axis Fig.31 It is shown that as a function of time, the reactivity of the conjugated product towards the lectin VVA increases, and there is a corresponding decrease in free thiol groups due to the thiol-ene photoconjugation of O-allyl-Tn to BSA. These results suggest that the reaction is more efficient at higher concentrations of AAPH. Products produced in the presence of vitamin C are unreactive towards the VVA lectin (not shown).

[0468] BSA-Tn conjugate (5 μg) was analyzed by SDS-PAGE electrophoresis on 10% polyacrylamide gel stained with Coomassie Brilliant Blue. The results showed that the amount of high molecular weight species was higher in the reaction at high AAPH than in the reaction at lower AAPH. Vitamin C prevented the formation of high molecular weight species (data not shown).

[0469] Example 22: Conjugation of O-allyl-Tn to BSA via thiol-ene photoreaction is a function of protein thiolation

[0470] BSA (93 μL, 11.7 mg / mL) was incubated with 200 Eq of 0.1 M 2-iminothiolane (Sigma), 200 Eq of 0.1 M O-allyl-Tn and 0.08 Eq, 0.8 Eq or about 450 Eq of AAPH and PBS pH 8 at a final concentration of 316 μL in a quartz cuvette (optical path of 10x10 mm, Fisher). In some samples, 2-iminothiolane and / or AAPH were omitted and replaced with PBS pH 8. The cuvette was placed between two handheld UV 365 nm or UV254 nm lamps (0.16 amps, VWR) at a distance of 2-5 cm from the cuvette. UV was turned on immediately after the addition of AAPH to initiate the reaction. The reaction was terminated after 45 minutes and then Sephadex was used. TM The product was buffer exchanged into PBS pH 7.4 by gel filtration chromatography using a G25 mini spin column. Protein concentration was measured by Bradford assay using BSA as standard.

[0471] To measure the immunoreactivity of BSA samples, 1 μg of protein sample was diluted into 100 μL PBS pH 7.4 and adsorbed to the wells of a 96-well plate (Maxisorp, Nunc) for a minimum of 1 hour at room temperature or overnight at 4°C. The wells were then washed with PBS-Tween. TM The wells were washed twice with 0.05% (PBS-T) and then filled with blocking solution for 30 minutes. The wells were then washed 3 times with PBS-T and filled with 100 μL 1 / 100 dilutions of Vicia vicifolia lectin (VVA-hrp, EY LAbs) coupled to horseradish peroxidase. After incubation for 1 hour or 12 hours at 4 ° C with gentle shaking at room temperature, the wells were washed 4 times with PBS-T, and 100 μL hrp substrate (Ultra TMB-ELISA, ThermoScientific) was then added to each well. The reaction was terminated by adding 100 μL 0.5N sulfuric acid and read at 450nm in a microplate reader (Biotek EL808).

[0472] Fig.32 It was shown that when the conjugation was performed in the absence of protein thiolation or when the thiolated protein was conjugated in the absence of the activator AAPH, the product was minimally reactive toward VVA. However, in the absence of protein thiolation and AAPH, a VVA reaction product was generated by UV 254 nm.

[0473] Example 23: Absence of BSA-TF reactivity at high conjugation rates

[0474] BSA (93 μL, 11.7 mg / mL) was incubated in a quartz cuvette (optical path 10x10 mm, Fisher) at a final volume of 316 μL on a bench at room temperature or between two hand-held UV 365 nm lamps (0.16 amps, VWR) placed 2-5 cm from the cuvette in the presence or absence of 200 Eq of 0.1 M 2-iminothiolane (Sigma), 200 Eq of 0.1 M O-allyl-TF and 0.8 Eq or about 450 Eq of AAPH and PBS pH 8. The reaction was initiated by turning on UV immediately after the addition of AAPH. The reaction was terminated after 45 minutes and then using Sephadex TM The product was buffer exchanged to PBS pH 7.4 by gel filtration chromatography using a G25 mini spin column. Protein concentration was measured by Bradford assay using BSA as standard. Conjugated galactose of the TF epitope was measured by the Dubois method using galactose standards.

[0475] To measure the immunoreactivity of BSA samples, 1 μg of protein sample was diluted into 100 μL PBS pH 7.4 and adsorbed to the wells of a 96-well plate (Maxisorp, Nunc) for a minimum of 1 hour at room temperature or overnight at 4°C. The wells were then washed with PBS-Tween. TM The wells were then washed twice with 0.05% (PBS-T) and then filled with blocking solution for 30 minutes. The wells were then washed 3 times with PBS-T and filled with 100 μL 0.1 μg / mL dilution of anti-TF monoclonal antibody JAAF11. After incubation at room temperature for 1 hour with gentle shaking or at 4 ° C for 12 hours, the wells were then washed 4 times with PBS-T and further incubated for 60 minutes with 100 μL 1 / 1000 dilution of secondary anti-goat anti-mouse IgG (H+L)-hrp (Jackson Immunoresearch). The wells were then washed 4 times with PBS-T, and 100 μL hrp substrate (Ultra TMB-ELISA, Thermo Scientific) was added to each well. The reaction was terminated by adding 100 μL 0.5N sulfuric acid and the plate was read at 450nm in a microplate reader (Biotek EL808).

[0476] With dual Y-axis Fig.33 The lower immunoreactivity of BSA-TF with high galactose ratio was shown compared to the lower galactose conjugate. The detection of galactose in both conjugates confirmed the conjugation of TF to BSA, but the immunoreactivity of the conjugated TF was reduced at the high ratio.

[0477] Example 24: O-allyl-Tn reacts with the peptide dTT831-844-Cys-(Tn)-βAlaBSA via thiol-ene photoreaction Conjugation

[0478] Fig.34 Shown are the chemical structures of dTT831-844-Cys-βAla and Cys-dTT831-844-βAla having cysteine ​​residues at the C-terminus and N-terminus, respectively, and their tabulated LC-MS data.

[0479] Fig.35 Detailed LC-MS data for peptide dTT831-844-Cys-βAla (C-terminus) are shown.

[0480] Fig.36 Detailed LC-MS data for the peptide Cys-dTT831-844-βAla (N-terminus) are shown.

[0481] Procedure A: To an aqueous solution of the peptide (1 mL, 2 mM) in a UV cuvette was added a solution of O-allyl-Tn in H2O (200 μL, 0.1 M, 10 equiv.) and a catalytic amount of AAPH. The mixture was stirred at room temperature under 365 nm irradiation for 60 min. Ellman's test indicated that the reaction was complete within 45 min. The solution was then lyophilized to afford a white powder, and LC-MS showed the presence of the desired Tn-conjugated peptide. LC-MS: C 94 H 152 O 31 N 20 Calculated value [M] of S, 2089.0653; found, 2089.0780; CAN / H2O 5 to 95% 5.08 min.

[0482] Procedure B in organic solvents: dTT831-844-Cys-(Tn)-βAla ( Fig.34 ). A solution of Tn in H2O (200 μL, 0.1 M, 10 equiv.) and a catalytic amount of DMPAP were added to a MeOH solution of the peptide (1 mL, 2 mM) in a UV cuvette. The mixture was stirred at room temperature under 365 nm irradiation for 60 min. Ellman's test indicated that the reaction was complete within 45 min. The solution was then lyophilized to provide a white powder, and LC-MS indicated the presence of the desired peptide-TN. LC-MS: C 94 H 152 O 31 N 20 Calculated value [M] of S, 2089.0653; found, 2089.0675; CAN / H2O 5 to 95% 5.07 min.

[0483] Fig.37Shown is the photolytic AAPH-catalyzed thiol-ene reaction of O-allyl Tn on the C-terminal peptide dTT831-844-Cys-βAla.

[0484] Fig.38 The LC-MS spectrum of O-allyl Tn on the C-terminal peptide is shown.

[0485] Example 25: Conjugation of O-allyl-Tn to the peptide Cys-(Tn)-dTT831-844-βAla via thiol-ene photoreaction

[0486] Procedure A: Peptide Cys-(Tn)-dTT831-844-βAla ( Fig.33 ) in water (2 mL, 0.482 mM) was added a solution of Tn in H2O (100 μL, 0.1 M, 10 equivalents) and a catalytic amount of AAPH. The mixture was stirred at room temperature under 365 nm irradiation for 60 minutes. Ellman's test showed that the reaction could be completed within 45 minutes. The solution was then lyophilized to provide a white powder, and LC-MS showed the presence of the desired peptide-Tn conjugate. LC-MS: C 94 H 152 O 31 N 20 Calculated [M] for S, 2089.0653; found: 2089.0719; CAN / H2O5 to 95% 5.11 min.

[0487] Procedure B: Cys-(Tn)-dTT831-844-βAla ( Fig.33 ). A solution of Tn in H2O (100 μL, 0.1 M, 10 equiv.) and a catalytic amount of DMPA were added to a methanol solution of the peptide (2 mL, 0.482 mM) in a UV cuvette. The mixture was stirred at room temperature under 365 nm irradiation for 60 min. Ellman's test showed that the reaction could be completed within 45 min. The solution was then lyophilized to provide a white powder, and LC-MS showed the presence of the desired peptide-Tn. LC-MS: C 94 H 152 O 31 N 20 Calculated value of [M] for S: 2089.0653; found: 2089.0817; CAN / H2O5 to 95% 5.10 min.

[0488] Fig.39 Shown is the photolytic AAPH-catalyzed thiol-ene reaction of O-allyl Tn on the N-terminal peptide Cys-dTT831-844-βAla.

[0489] Fig.40 Detailed LC-MS spectrum of O-allyl Tn on the N-terminal peptide is shown.

[0490] References :

[0491] Cipolla et al.,“Stereoselective synthesis of α-C-glycosides of N-acetylgalactosamine”,Tetrahedron Asymm.(2000),11:295-303.

[0492] Cui et al.,“Stereocontrolled allylation of 2-amino-2-deoxy sugarderivatives by a free-radical procedure”,Carbohydr.Res.,(1998),309:319-330.

[0493] Danishefsky et al.,“Development of Globo-H Cancer Vaccine”,Acc.ChemRes.,(2015),48(3):643-652.

[0494] Demian et al.,“Direct targeted glycation of the free sulfhydryl groupof cysteine residue(Cys-34)of BSA.Mapping of the glycation sites of the anti-tumor Thomsen-Friedenreich neoglycoconjugate vaccine prepared by Michaeladdition reaction,”J.Mass Spectrom.,(2014),49:1223-1233.

[0495] Dondoni et al.,“A new ligation strategy for peptide and proteinglycosylation:Photoinduced thiol-ene coupling”,Chem.Eur.J.,(2009),15:11444-11449.

[0496] Dondoni et al.,“Recent applications in thiol-ene coupling as a clickprocess for glycoconjugation”,Chem.Soc.Rev.,(2012),41:573-586.

[0497] Feng et al.,“Chemo-enzymatic synthesis of fluorinated 2-N-acetamidosugar nucleotides using UDP-GlcNAc pyrophosphorylase”,Org.Biomol.Chem.(2004),2:1617-1623.

[0498] Heimburg et al.,“Inhibition of spontaneous breast cancer metastasisby anti-Thomsen-Friedenreich antigen monoclonal antibody JAA-F11.”,Neoplasia,(2006)8(11):939-48.

[0499] Knapp et al.,“Synthesis of α-GalNAc Thioconjugates from an α-GalNAcMercaptan”,J.Org.Chem.(2002),67:2995-2999.

[0500] Papadopoulos,“Diazo transfer and click chemistry in the solid phasesyntheses of lysine-based glycodcndrimcrs as antagonists against Escherichiacoli FimH.”Molecular Pharmaceutics(2012),9(3):394-403.

[0501] Rittenhouse-Diakun et al., "Development and characterization of monoclonal antibody to T-antigen: (gal beta-3GalNAc-alpha-O)", Hybridoma, (1998), 17: 165-173.

[0502] Tati et al., "Humanization of JAA-F11, a Highly Specific Anti-Thomsen-Friedenreich Pancarcinoma Antibody and InVitro Efficacy Analysis.", Neoplasia, (2017), 19(9): 716-733. Sequence Listing <110> KORANEX CAPITAL <120> Precision glycoconjugates as therapeutic tools <130> 18513-3 <140> To be specified <141> 2019-03-22 <150> US 62 / 647,151 <151> 2018-03-23 <160> 2 <170> PatentIn Version 3.5 <210> 1 <211> 14 <212> PRT <213> Clostridium tetani <400> 1 Tyr Ile Lys Ala Asn Ser Lys Phe Ile Gly Ile Thr Glu Leu 1 5 10 <210> 2 <211> 16 <212> PRT <213> Artificial sequence <220> <223> dTT831-844-Cys-beta-Ala <220> <221> MOD_RES <222> (1) <223> Acetylation <220> <221> MOD_RES <222> (16) <223> bA <400> 2 Tyr Ile Lys Ala Asn Ser Lys Phe Ile Gly Ile Thr Glu Leu Cys Ala 1 5 10 15

Claims

1. A method for producing a glycoconjugate immunogen, the method comprising: (a) providing a carbohydrate antigen covalently linked to a terminal olefin (alkenyl carbohydrate antigen), wherein the terminal olefin is directly conjugable to a thiol group via a thiol-ene reaction, wherein the alkenyl carbohydrate antigen is an unprotected water-soluble alkenyl carbohydrate antigen; (b) providing a carrier protein having one or more free thiol groups; and (c) performing a photocatalytic thiol-ene reaction to directly conjugate the carbohydrate antigen to the one or more free thiol groups of the carrier protein, thereby producing a glycoconjugate immunogen, wherein the photocatalytic thiol-ene reaction: (i) carried out at a pH of 6 to 8; (ii) under reaction conditions that avoid denaturation of the carrier protein and / or preserve the activity, antigenicity and / or structure of the carrier protein; and (iii) in the absence of any organic solvent, or in the presence of an organic solvent at a concentration low enough to avoid denaturation of the carrier protein, wherein the carrier protein is immunogenic upon administration to a subject, and wherein conjugation of the carbohydrate antigen to the carrier protein increases the immunogenicity of the carbohydrate antigen upon administration to the subject compared to administration of an unconjugated carbohydrate antigen, and wherein the carbohydrate antigen comprises a B cell epitope and induces a humoral immune response in the subject.

2. The method of claim 1, wherein the photocatalytic thiol-ene reaction is carried out at a pH of 6.5 to 7.

5.

3. The method of claim 1, wherein the photocatalytic thiol-ene reaction is performed in the presence of a catalyst.

4. The method of claim 3, wherein the catalyst is a water-soluble catalyst.

5. The method according to claim 4, wherein the water-soluble catalyst is a water-soluble free radical generating azo compound; 2,2'-azobis[2-(2-imidazolin-2-yl)propane] dihydrochloride (Vazo 44 or VA-044); 2,2'-azobis(2-amidinopropane) dihydrochloride (AAPH); a metal or metal ion with photoinitiating activity; a peroxide; tert-butyl hydroperoxide; benzoyl peroxide; ammonium persulfate; or any derivative thereof with photoinitiating activity.

6. The method of claim 3, wherein the catalyst is a water-insoluble catalyst.

7. The method according to claim 6, wherein the water-insoluble catalyst is a water-insoluble free radical generating azo compound, 2,2-dimethoxy-2-phenylacetophenone (DMPA), azobisisobutyronitrile (AIBN), 2,2′-azobis(2-methylpropionitrile), 4,4′-azobis(4-cyanovaleric acid) (ACVA), 1,1′-azobis(cyanocyclohexane) (ACHN), diazonium dicarboxylate bis(N,N-dimethylamide) (TMAD); dipiperidin azodicarboxylate (ADD) or any derivative thereof having photoinitiator activity.

8. The method of claim 1, wherein the photocatalytic thiol-ene reaction comprises irradiation under short-wave or long-wave ultraviolet light.

9. The method of claim 1, wherein the photocatalyzed thiol-ene reaction comprises reacting each free thiol group of the carrier protein with 1 to 200 molar equivalents of the ene carbohydrate antigen; and / or wherein the photocatalyzed thiol-ene reaction is performed 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 achieve a reduction in the total free thiol concentration in the carrier protein by at least 5, 10, 15, 20, 25, 30, 35, 40, 45, or 50 times.

10. The method of claim 1, wherein after conjugation to the carrier protein, the carbohydrate antigen cannot be cleaved from the carrier protein by endogenous enzymes of the subject.

11. The method of claim 1, wherein the alkenyl carbohydrate antigen is covalently linked to the terminal olefin and / or the carbohydrate antigen is conjugated to the carrier protein via an O-glycosidic bond, an S-glycosidic bond, an N-glycosidic bond, or a C-glycosidic bond, or a bond obtained by reductive amination between an allylamine and a reducing sugar.

12. The method of claim 1, wherein the carbohydrate antigen is or comprises a tumor-associated carbohydrate antigen (TACA), a viral polysaccharide antigen, or a bacterial capsular polysaccharide (CPS).

13. The method of claim 12, wherein the TACA is, is derived from or comprises: Tn, S-Tn, Thomsen-Friedenreich (TF), (2,3)-S-TF, (2,6)-S-TF, Globo H, GD2, GD3, GM2, GM3, N-glycolyl-GM3, Lea, sLea, Lex, sLex or any combination thereof; and wherein the bacterial CPS is, is derived from or comprises a polysaccharide serotype of pneumococcus and / or streptococcus, a meningococcal CPS or an influenza CPS.

14. The method of claim 1, wherein the photocatalyzed thiol-ene reaction conjugates at least two identical carbohydrate antigens or more than one type of carbohydrate antigen to the carrier protein, thereby generating a multivalent glycoconjugate immunogen.

15. The method of claim 1, wherein the carbohydrate antigen in (a) is linked to the terminal olefin via a linker.

16. The method of claim 1, wherein the carrier protein comprises one or more cysteine ​​residues with one or more free thiol groups, or is engineered to add one or more additional cysteine ​​residues at the amino terminus, carboxyl terminus, or solvent accessible position of the carrier protein.

17. The method of claim 1, wherein the carrier protein comprises a human T cell epitope and / or induces a cell-mediated immune response in the subject.

18. The method of claim 1, wherein the carrier protein is, is derived from or comprises: tetanus toxoid (TT), diphtheria toxoid (DT), cross-reactive material 197 (CRM197), outer membrane protein complex of meningococcus (OMPC), Haemophilus influenzae protein D (HiD), cytokines, immunogenic peptides, tetanus toxin 831-844 (SEQ ID NO: 1 or 2), albumin, or an immunogenic fragment thereof.

19. A synthetic glycoconjugate immunogen prepared by the method of any one of claims 1 to 18, comprising one or more carbohydrate antigens and an immunogenic carrier protein having one or more solvent accessible cysteine ​​residues, wherein the one or more carbohydrate antigens are linked at the one or more solvent accessible cysteine ​​residues of the immunogenic carrier protein, and wherein conjugation of the one or more carbohydrate antigens to the immunogenic carrier protein increases the immunogenicity of the one or more carbohydrate antigens after administration to a subject compared to administration of unconjugated carbohydrate antigens.

20. The synthetic glycoconjugate immunogen of claim 19, wherein the synthetic glycoconjugate immunogen is: (a) A synthetic glycoconjugate immunogen comprising one or more saccharide antigens covalently linked to one or more free thiol groups of a carrier protein via a linker, wherein each linker has the following structure or a stereoisomer thereof: in: -SA is a carbohydrate antigen or a portion thereof; -S-PC is a carrier protein; -X is O, S, NR1 or CH2; -R1 is -H, -COH, -COCH3 or -COEt; -n is 1, 2, 3, 4, or 5; and -R2 is H or Me, or (b) a synthetic glycoconjugate immunogen comprising one or more saccharide antigens covalently linked to one or more free thiol groups of a carrier protein via a linker, wherein each linker has the following structure or a stereoisomer thereof: in: -SA is a carbohydrate antigen or a portion thereof; -S-PC is a carrier protein; -X is S, NR1, CH2 or O; -R1 is -H, -COH, -COMe or -COEt; -n is 1, 2, 3, 4, or 5; -R2 is H or Me; -q is 1, 2, 3, 4, or 5; - R3 and R4 are each a hydrogen atom 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- in which the carbonyl group is attached to the nitrogen atom and m is 1, or (c) a synthetic glycoconjugate immunogen comprising one or more saccharide antigens covalently linked to one or more free thiol groups of a carrier protein via a linker, wherein the synthetic glycoconjugate immunogen has the following structure or a stereoisomer thereof: in -y is at least 1; - SA is selected from the group consisting of one or more carbohydrate antigens or immunogenic portions thereof, and when y is greater than 1, SA are the same or different; -[S] z - PC is a carrier protein having one or more sulfur atoms derived from one or more free sulfhydryl groups; - 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: in: -X is O, S, NR1 or CH2; -R1 is -H, -COH, -COCH3 or -COEt; -n is 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 (d) a synthetic glycoconjugate immunogen comprising one or more saccharide antigens covalently linked to one or more free thiol groups of a carrier protein via a linker, wherein the synthetic glycoconjugate immunogen has the following structure or a stereoisomer thereof: in -y is at least 1; - SA is selected from the group consisting of carbohydrate antigens or immunogenic portions thereof, and when y is greater than 1, SA are identical or different; -[S] z - PC is a carrier protein with one or more sulfur atoms derived from z free sulfhydryl groups; -z is at least equal to y; and -L is a linker selected from the group consisting of linkers having the following structures: in: -X is S, NR1, CH2 or O; -R1 is -H, -COH, -COMe or -COEt; -n is 1, 2, 3, 4, or 5; -R2 is H or Me; -q is 1, 2, 3, 4, or 5; - R3 and R4 are each a hydrogen atom 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- in which the carbonyl group is attached to the nitrogen atom and m is 1; and When y is greater than 1, L is the same or different, or (e) a synthetic glycoconjugate immunogen comprising one or more saccharide antigens covalently linked to one or more free thiol groups of a carrier protein via a linker, wherein the synthetic glycoconjugate immunogen has the following structure or a stereoisomer thereof: in -y is at least 1; - SA is selected from the group consisting of carbohydrate antigens or immunogenic portions thereof, and when y is greater than 1, SA are the same or different; -[S] z - PC is a carrier protein with one or more sulfur atoms derived from z free sulfhydryl groups; -z is at least equal to y; and -L is a linker selected from the group consisting of linkers having the following structures: in -X is S, NR1, CH2 or O; -R1 is -H, -COH, -COMe or -COEt; -n is 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-PC, a covalent bond, or a group having the following structure: wherein R3 and R4 are each a hydrogen atom 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- in which the carbonyl group is attached to the nitrogen atom and m is 1; and When y is greater than 1, L are the same or different.

21. The synthetic glycoconjugate immunogen according to claim 20, wherein the linker of the synthetic glycoconjugate immunogen (e) has the following structure: in -X is S, NR1, CH2 or O; -R1 is -H, -COH, -COMe or -COEt; -n is 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.

22. The synthetic glycoconjugate immunogen according to claim 20, wherein the linker of the synthetic glycoconjugate immunogen (e) has the following structure: in -X is S, NR1, CH2 or O; -R1 is -H, -COH, -COMe or -COEt; -n is 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.

23. A glycoconjugate vaccine comprising the synthetic glycoconjugate immunogen according to any one of claims 19 to 22 and a pharmaceutically acceptable excipient and / or adjuvant.

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