Vaccine against Klebsiella pneumoniae

Abstract

The present invention relates to novel immunogenic compounds having at least one antigen of formula (I), particularly immunogenic compounds of formula (II), and their use as pharmaceuticals, particularly vaccines. The invention also relates to related aspects, including intermediates for the preparation of said immunogenic compounds and processes for their preparation. Furthermore, the invention relates to pharmaceutical compositions comprising said immunogenic compounds and to the use of antigens of formula (I) in biological assays. [Formula 1] JPEG2025541816000108.jpg87155

Description

[Technical Field]

[0001] This invention was made with U.S. government support under IDSEP160030-01, IDSEP160030-02, and IDSEP160030-03 awarded by HHS / ASPR. The U.S. government has certain rights in this invention.

[0002] The present invention relates to novel immunogenic compounds having at least one antigen of formula (I), particularly immunogenic compounds of formula (II), and their use as pharmaceuticals, particularly vaccines. The invention also relates to related aspects, including intermediates and methods for preparing said immunogenic compounds. Furthermore, the invention relates to pharmaceutical compositions comprising said immunogenic compounds and to the use of antigens of formula (I) in biological assays. [Background technology]

[0003] Klebsiella pneumoniae (or K. pneumoniae) is a Gram-negative, facultative anaerobic bacillus that primarily colonizes the respiratory, intestinal, and urinary tracts, as well as the skin, causing K. pneumoniae infections (KPIs). This bacterium primarily acts as an opportunistic pathogen. KPIs are a major cause of hospital-acquired infections and primarily affect immunocompromised patients. Infections caused by K. pneumoniae have become a significant challenge in healthcare settings due to the emergence and worldwide spread of strains resistant to almost all available antimicrobial agents. Infections caused by K. pneumoniae cause high morbidity and mortality. Therefore, prevention of infections caused by K. pneumoniae is highly desirable, and vaccination is the most cost-effective and powerful means of combating KPIs.

[0004] K. pneumoniae is an encapsulated bacterium that expresses lipopolysaccharide (LPS) and capsular polysaccharide (CPS, K antigen) on its outer membrane, which contribute to the virulence of this species.

[0005] LPS consists of three components: a lipid A moiety that functions as a membrane anchor, a core oligosaccharide covalently linked to lipid A, and a terminal antigenic polysaccharide with repeating sugar units forming the O antigen covalently linked to the core oligosaccharide. Extracted LPS has been shown to be pyrogenic, toxic, and capable of causing tissue damage. LPS can be coated by CPS and is usually less exposed on the surface than CPS.

[0006] CPSs are composed of repeating sugar units and form a layer on the bacterial outer surface. CPSs are usually complex, linear or branched, and have a larger molecular weight than LPS. Their high immunogenicity and surface exposure make them attractive targets for vaccine strategies. For example, WO2016156338 discloses conjugates of synthetic oligosaccharides related to carbapenem-resistant K. pneumoniae CPS.

[0007] However, Klebsiella CPS is highly diverse. Serologically, over 77 different CPS types, so-called K types, K serotypes, or K antigens, have been identified, with at least 141 K types. These additional K types are identified based on the capsule gene locus (cps-locus) or K gene locus (K-locus) and are referred to as the KL series.

[0008] On the other hand, LPS variability is low, and the currently known so-called O types, O serotypes, or O antigens are limited to 11 major groups: O1, O2a (formerly known as Gal-I), O2ac, O2afg (formerly known as Gal-III), O2aeh (formerly O9), O3 (including subserotypes O3, O3a, and O3b), O4, O5, O7, O8, and O12. In addition to the 11 species listed above, additional O types have been reported based on the O locus, known as the OL series. Although O antigens are less immunogenic and less exposed on the membrane surface than K antigens, they have also been considered in vaccine strategies. Recent large-scale surveys of clinical isolates have revealed the relative prevalence of CPS and LPS serotypes, particularly among multidrug-resistant isolates (e.g., Lam et al., Microbial Genomics 2022;8:000800; DOI 10.1099 / mgen.0.000800).

[0009] O-type O2afg is produced by many multidrug-resistant Klebsiella pneumoniae strains, such as ST258. These strains are widespread worldwide and are highly drug-resistant, e.g., carbapenem-resistant. The repeating unit structure of O2afg is disclosed, for example, in Kelly et al., J. of Bacteriol., 178(17), 1996, 5205-5214:

[0010] [ka]

[0011] O-type O2a is one of the five most frequently occurring O-antigens in clinical isolates. O2a is also a common source of antibiotic resistance. The repeating unit structure of O2a is disclosed, for example, in Kelly et al., J. of Bacteriol., 178(17), 1996, 5205-5214:

[0012] [ka]

[0013] The O2afg serotype is not recognized by O2a-specific antibodies, and vice versa (Szijarto et al., Int J Med Microbiol 2016;306(2):89–98; PMID:26723873; http: / / dx.doi.org / 10.1016 / j.ijmm.2015.12.002).

[0014] Purely isolated bacterial polysaccharides are known to be thymus-independent antigens, activating B cells without the help of T cells. As a result, the immune response to carbohydrates is a primary immune response, with antibodies consisting primarily of low-affinity IgG, lacking affinity maturation / isotype switching, and IgGs that are less stable and shorter-lived. Typical thymus-independent antigens include CPS from Streptococcus pneumoniae and Haemophilus influenzae type b.

[0015] To overcome the limitations of thymus-independent antigens, the O-antigen of CPS or LPS can be covalently conjugated to a carrier protein. Immunization with these polysaccharide-protein conjugates (glycoconjugate vaccines) results in T cell-dependent B cell activation and can induce long-lasting immunity even in children. For example, WO2019106201 discloses conjugates of synthetic oligosaccharides related to the O-polysaccharides of K. pneumoniae serotypes O1, O2, O2ac, and O8 and the O-polysaccharide of carbapenem-resistant K. pneumoniae ST258.

[0016] The mechanism by which conjugate vaccines are presented to T cells remains controversial. Polysaccharide-protein conjugates are recognized and internalized by the polysaccharide-specific B cell receptor (BCR) of follicular B cells. The protein portion of the glycoconjugate is processed and presented on MHC-II molecules on the cell surface of B cells. Recognition of the MHC-II-peptide complex by peptide-specific T cells then leads to cognate T cell / B cell interaction, and the B cell receives an activation signal from the T cell. Recent studies suggest that after binding to the BCR and subsequent internalization into endosomes, glycoconjugates are also processed into glycopeptide fragments. The peptide portion can then bind to MHC-II molecules, while the carbohydrate (glycan) is exposed to the T cell receptor and can interact with carbohydrate-specific CD4+ T cells.

[0017] When two different synthetic glycan antigens, e.g., glycan "A" and glycan "B," are covalently linked to the same carrier protein molecule, the number of glycan "A"- and glycan "B"-specific B cells present and the affinity of the BCRs expressed on these B cells determine the production of glycan-specific antibodies (Abs). For example, if fewer B cells are present for glycan "A" and express low-affinity BCRs on their cell surface, the Ab response elicited following vaccination will be dominated by glycan "B"-specific Abs. Currently, it is not possible to predict the occurrence and strength of immunodominance of glycoconjugates.

[0018] It remains difficult and unpredictable which CPS or LPS may be suitable candidate or model sequences for a vaccine, and in particular which short oligosaccharides are suitable for generating the desired immune response in vivo. In particular, the immunodominance of glycoconjugates remains unpredictable.

[0019] To date, there is no approved vaccine available against K. pneumoniae, which highlights the difficulties involved in developing such a vaccine.

[0020] Surprisingly, it has now been found that novel oligosaccharide-antigens have superior properties as potential vaccines against Klebsiella pneumoniae. Due to their specific non-natural hybrid structure that targets multiple serotypes, the amount of carrier protein per vaccination can be reduced, thereby reducing undesirable carrier-induced epitope suppression and simultaneously reducing production costs.

[0021] Text description of the illustration image022.gif. In the following, the terms "Gal-I" and "O2a" are considered synonymous, as are the terms "Gal-III" and "O2afg". [Brief explanation of the drawings]

[0022] [Figure 1] Figure 1: HPLC-SEC characterization of D13-CRM197*, D17-CRM197*, and D20-CRM197* glycoconjugates compared to CRM197. [Figure 2] Figure 2: SDS-PAGE of D13-CRM197*, D17-CRM197* and D20-CRM197* glycoconjugates compared to CRM197 and Marker (protein size marker is GelCode™ Blue Safe Protein Stain (Thermo Scientific)). [Figure 3]Figure 3 shows immunogenicity studies in ZiKa rabbits (6 rabbits per group) using a 2 μg antigen dose per rabbit and per immunization on days 0, 14, and 28 (D13-CRM197*) or on days 0, 14, and 34 (D17-CRM197* and D20-CRM197*); Figure 3A shows ELISA against Gal-III LPS isolated from strain PCM27 (Polish Collection of Microorganisms) using an LPS extraction kit (JH Science); Figure 3B shows ELISA against O2a LPS isolated from strain NCTC9148 using an LPS extraction kit (JH Science); sera were diluted 1:100. [Figure 4] Figure 4 shows immunogenicity studies in ZiKa rabbits (6 rabbits per group) using a 2 μg antigen dose per rabbit and per immunization on days 0, 14, and 28 (D13-CRM197*) or on days 0, 14, and 34 (D17-CRM197* and D20-CRM197*); Figure 4A shows ELISA against inactivated PCM27 bacteria (Gal-III); Figure 4B shows ELISA against inactivated NCTC9148 (O2a) bacteria; sera were diluted 1:500 (panel A) or 1:100 (panel B). [Figure 5]Figure 5 shows survival data from a challenge experiment in mice. CD-1 mice were intraperitoneally injected twice at −24 h and −1 h prior to infection with rabbit antisera generated with D13-CRM197* (obtained by immunization with a 2 μg D13-CRM197* antigen dose per rabbit and per immunization on days 0, 14, and 28, and harvested on day 35) (Figures 5A and 5B), or rabbit antisera generated with D17-CRM197* or D20-CRM197* (obtained by immunization with a 2 μg D17-CRM197* or D20-CRM197* antigen dose per rabbit and per immunization on days 0, 14, and 34, and harvested on day 41) (Figure 5C), or with control serum from naive rabbits. At 0 h, mice were infected with a lethal dose of Klebsiella pneumoniae O2a-expressing strain NCTC9163 (Figure 5A) or O2afg-expressing strain ST258 (Figures 5B and 5C) by intraperitoneal injection, along with galactosamine treatment (20 mg / mouse). Mice were monitored for survival for 24 h. The survival curves shown indicate statistically significant differences with the indicated P values ​​(Log-rank (Mantel-Cox test)). The number of mice per group was: 8 (Figures 5A and 5C) or 10 (Figure 5B). Summary of the Invention

[0023] Detailed Description of the Invention 1) In a first aspect, the present invention provides an immunogenic compound having at least one oligosaccharide hybrid antigen having formula (I):

[0024] [ka]

[0025] (In the formula, R is OH or

[0026] [ka]

[0027] and; m is 3, 4, 5, 6, 7 or 8; n is 1, 2, 3, 4, 5 or 6; or a pharmaceutically acceptable salt thereof.

[0028] The dotted line " ** " denotes the point of attachment. This means that at this position the antigen is attached to a carrier protein via a linker and / or spacer. The oligosaccharide hybrid antigen of formula (I) is responsible for immunogen selectivity, i.e., targeted (specific) antibody responses against multiple O-serotypes of Klebsiella pneumoniae, especially O2a and O2afg.

[0029] The expression "at least one antigen" means that the immunogenic compound may comprise one or more antigens according to formula (I). For example, the immunogenic compound comprises 1 to 28 antigens according to formula (I).

[0030] In some embodiments, the immunogenic compound may comprise a mixture of different antigens according to formula (I). Preferred immunogenic compounds are those that comprise the same antigen according to formula (I), i.e., only one particular type of antigen according to formula (I).

[0031] The term "hybrid" means that the antigen has two different parts of the O-serotype, namely O2afg (Gal-III) and O2a (Gal-I), and the O2afg part is connected at the attachment point " ** " and O2a is distal to the attachment point " ** " is proximal to

[0032] The oligosaccharides of the present invention are galactans, i.e. - Beta-D-galactofuranose / β-D-Galf:

[0033] [ka]

[0034] (Dotted lines indicate points of attachment (i.e., C1 and C3)); - Alpha-D-Galactopyranose / α-D-Galp:

[0035] [ka]

[0036] (Dotted lines indicate points of attachment (i.e., C1 and C3)); It consists of:

[0037] The definitions set forth herein apply uniformly to compounds of formula (I), (Ia), (II), (IIa), (IIb), (IIc), (III), and (IV) as defined in any one of embodiments 1) to 81), and apply mutatis mutandis throughout the specification and claims, unless a broader or narrower definition is given by a specific definition. It is to be understood that any definition or preferred definition of a term may independently (and together with) define and replace the respective term in any or all other terms or preferred definitions defined herein.

[0038] The oligosaccharide portion (i.e., antigen or epitope) of compounds of formulas (I), (Ia), (II), (IIa), (IIb), (IIc), (III), and (IV) consists of D-galacto-pyranosides and D-galacto-furanosides, respectively. The configuration of each anomeric center is either alpha or beta. The configuration of the anomeric center contributes to a mixture of anomers, and the anomers are synthesized in either alpha or beta form, preferably as pure alpha or beta anomers. Anomeric mixtures may be separated by methods known to those skilled in the art.

[0039] 2) A further embodiment relates to an immunogenic compound according to embodiment 1), or a pharmaceutically acceptable salt thereof, wherein m is 3, 4, 5 or 6 and n is 2, 3 or 4.

[0040] 3) A further embodiment relates to an immunogenic compound according to embodiment 1), or a pharmaceutically acceptable salt thereof, wherein m is 3, 4, 5 or 6 and n is 2 or 3, for example 2.

[0041] 4) A further embodiment relates to an immunogenic compound or a pharmaceutically acceptable salt thereof according to any one of embodiments 1), 2) or 3), wherein R is OH.

[0042] 5) A further aspect is m is 4, n is 2, and R is OH; m is 4, n is 2, and R is

[0043] [ka]

[0044] or m is 4, n is 3, and R is OH; The immunogenic compound according to embodiment 1) or a pharmaceutically acceptable salt thereof.

[0045] 6) A further embodiment relates to an immunogenic compound according to embodiment 1), or a pharmaceutically acceptable salt thereof, wherein m is 4, n is 2 and R is OH.

[0046] 7) A further embodiment relates to an immunogenic compound or a pharmaceutically acceptable salt thereof according to any one of embodiments 1), 2), 3), 4), 5) or 6), wherein the immunogenic compound further comprises a carrier protein. The carrier protein is preferably non-toxic and suitable for inducing immunogenicity. Thus, the carrier protein is preferably a non-toxic carrier protein suitable for inducing immunogenicity.

[0047] 8) A further embodiment relates to an immunogenic compound or a pharmaceutically acceptable salt thereof according to any one of embodiments 1), 2), 3), 4), 5) or 6), wherein said immunogenic compound further comprises a carrier protein selected from the group consisting of: CRM 197 diphtheria toxoid; tetanus toxoid; cholera toxin B subunit; Neisseria meningitidis outer membrane protein (OMP); bacteriophage Qβ capsid protein; oligomers or virus-like particles produced using bacteriophage Qβ capsid protein; Pseudomonas aeruginosa detoxified exotoxin A (EPA); maltose-binding protein (MBP); tetanus toxin Hc fragment (TetHc); Staphylococcus aureus detoxified hemolysin A; Staphylococcus aureus clumping factor A (ClfA) and clumping factor B (ClfB); Escherichia coli FimH; Escherichia coli FimHC; Escherichia Detoxified mutants of Escherichia coli heat-labile enterotoxin; detoxified mutants of cholera toxin; Escherichia coli Sat protein; the passenger domain of Escherichia coli Sat protein; detoxified mutants of Streptococcus pneumoniae pneumolysin; Campylobacter jejuni AcrA; Pseudomonas PcrV protein; Campylobacter jejuni native glycoprotein; bovine serum albumin (BSA); group B Streptococcus pilus protein GBS80; Escherichia coli heat-labile enterotoxin; tetanus toxin; cholera toxin; and Streptococcus pneumoniae pneumolysin.

[0048] CRM 197 The term " refers to Cross Reactive Material 197, which is a non-toxic variant of diphtheria toxin, in which a single amino acid substitution of glycine (Gly, G) at position 52 to glutamic acid (Glu, E) detoxifies the protein. This is described in more detail in embodiment 17).

[0049] The term "diphtheria toxoid" refers to a formalin-inactivated form of diphtheria toxin having SEQ ID NO:2 (Uniprot ID: P00587). The present invention encompasses proteins having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 99.9% identity to the amino acid sequence of SEQ ID NO:2 (preferably, 95%, 96%, 97%, 98%, 99%, or 99.9% identity to the amino acid sequence of SEQ ID NO:2). Diphtheria toxoid can be prepared, for example, as described by Glenny et al. in Br J Exp Pathol. 1923 Oct;4(5):283-8 (PMCID: PMC2047731).

[0050] The term "tetanus toxoid" refers to a formalin-inactivated form of tetanus toxin having SEQ ID NO:3 (Uniprot ID: P04958). The present invention encompasses proteins having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 99.9% identity to the amino acid sequence of SEQ ID NO:3 (preferably, 95%, 96%, 97%, 98%, 99%, or 99.9% identity to the amino acid sequence of SEQ ID NO:3). Tetanus toxoid can be prepared, for example, as described in G. Ramon et al., CR Soc Biol, 93 (1925), pp. 508-509.

[0051] The term "cholera toxin B subunit" refers to the protein having SEQ ID NO:4 (Uniprot ID: P01556). The present invention encompasses proteins having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 99.9% identity to the amino acid sequence of SEQ ID NO:4 (preferably, 95%, 96%, 97%, 98%, 99%, or 99.9% identity to the amino acid sequence of SEQ ID NO:4).

[0052] The term "meningococcal outer membrane protein" (OMP) refers to the protein having SEQ ID NO:5 (Uniprot ID: Q51229). The present invention encompasses proteins having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 99.9% identity to the amino acid sequence of SEQ ID NO:5 (preferably 95%, 96%, 97%, 98%, 99% or 99.9% identity to the amino acid sequence of SEQ ID NO:5).

[0053] The term "bacteriophage Qβ capsid protein" refers to the protein having SEQ ID NO:6 (Uniprot ID: P03615). The present invention encompasses proteins having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 99.9% identity to the amino acid sequence of SEQ ID NO:6 (preferably, 95%, 96%, 97%, 98%, 99%, or 99.9% identity to the amino acid sequence of SEQ ID NO:6).

[0054] 9) A further embodiment relates to an immunogenic compound or a pharmaceutically acceptable salt thereof according to any one of embodiments 1), 2), 3), 4), 5) or 6), wherein said immunogenic compound further comprises a carrier protein selected from the group consisting of: CRM 197diphtheria toxoid; tetanus toxoid; cholera toxin B subunit; meningococcal outer membrane protein (OMP); and the capsid protein of bacteriophage Qβ.

[0055] The preferred carrier protein is CRM 197 is.

[0056] 10) A further embodiment is that the immunogenic compound further comprises a non-immunogenic linker and / or spacer, the non-immunogenic linker and / or spacer comprising on one side: ** - covalently bound at the binding site to said antigen and at the other side to said carrier protein; relates to an immunogenic compound according to any one of embodiments 7), 8) or 9).

[0057] 11) In a second aspect, the present invention provides an immunogenic compound of formula (Ia):

[0058] [ka]

[0059] (In the formula, R is OH or

[0060] [ka]

[0061] and; m is 3, 4, 5, 6, 7 or 8; preferably 3, 4, 5 or 6; n is 1, 2, 3, 4, 5 or 6; preferably 2, 3 or 4; i is at least 1, preferably a number from 1 to a maximum of 90% of the number of lysine residues contained in the carrier protein CP; -LT- represents a linker L and a spacer T that together form a bridge having a covalently linked backbone of 5 to 25 atoms in length, said length forming the shortest distance between the oxygen at C1 of the reducing end of the oligosaccharide and the nitrogen of the amino group of a lysine residue of the carrier protein CP; the atoms of the backbone are selected from the group consisting of carbon, nitrogen, oxygen, and sulfur; The CP is a carrier protein selected from the group consisting of: CRM 197 ;Diphtheria toxoid;Tetanus toxoid;Cholera toxin B subunit;Meningococcal outer membrane protein (OMP);Bacteriophage Qβ capsid protein;Oligomers or virus-like particles produced using bacteriophage Qβ capsid protein;Detoxified Pseudomonas aeruginosa exotoxin A (EPA);Maltose-binding protein (MBP);Hc fragment of tetanus toxin (TetHc);Detoxified Staphylococcus aureus hemolysin A;Staphylococcus aureus clumping factor A (ClfA) and clumping factor B (ClfB);Escherichia coli FimH;Escherichia coli FimHC;Detoxified mutants of Escherichia coli heat-labile enterotoxin;Detoxified mutants of cholera toxin;Escherichia coli Sat protein;Passenger region of Escherichia coli Sat protein;Streptococcus detoxified mutants of Campylobacter pneumoniae pneumolysin; Campylobacter jejuni AcrA; Pseudomonas PcrV protein; Campylobacter jejuni native glycoprotein; bovine serum albumin (BSA); Group B Streptococcus fimbria protein GBS80; Escherichia coli heat-labile enterotoxin; tetanus toxin; cholera toxin; and Streptococcus pneumoniae pneumolysin. or a pharmaceutically acceptable salt thereof.

[0062] Preferably, i is a number from 1 to a maximum of 90% of the number of lysine residues contained in the carrier protein CP; more preferably, i is a number from 1 to a maximum of 75% of the number of lysine residues contained in the carrier protein CP; and even more preferably, i is a number from 1 to a maximum of 40% of the number of lysine residues contained in the carrier protein CP. For example, if the carrier protein CP has 39 lysine residues, the range of "a number from 1 to a maximum of 40% of the number of lysine residues contained in the carrier protein CP" means that i is in the range of 1 to 16.

[0063] Explicit reference should also be made to the definitions of carrier proteins disclosed in embodiments 8) and 17).

[0064] For the avoidance of any doubt, the terms "lysine residue" and "lysine site" are used interchangeably throughout this application.

[0065] The immunogenic compounds according to the present invention are oligosaccharide-carrier protein conjugates, and the term "immunogenic compound" is used synonymously with "oligosaccharide-carrier protein conjugate."

[0066] The immunogenic compound of embodiment 11) may alternatively be described as an oligosaccharide-carrier protein conjugate of formula (Ia):

[0067] The term "hybrid" means that the antigen has two different parts of the O-serotype, namely O2afg (Gal-III) and O2a (Gal-I), with the O2afg part being distal to the carrier protein and O2a being proximal to the carrier protein.

[0068] 12) A further embodiment relates to an immunogenic compound according to embodiment 11), or a pharmaceutically acceptable salt thereof, wherein R is OH.

[0069] 13) A further aspect is m is 4, n is 2, and R is OH; m is 4, n is 2, and R is

[0070] [ka]

[0071] or m is 4, n is 3, and R is OH; The immunogenic compound according to embodiment 11) or a pharmaceutically acceptable salt thereof.

[0072] 14) A further embodiment relates to an immunogenic compound according to embodiment 11), or a pharmaceutically acceptable salt thereof, wherein m is 4, n is 2 and R is OH.

[0073] 15) A further embodiment relates to an immunogenic compound or a pharmaceutically acceptable salt thereof according to embodiment 11), 12), 13) or 14), wherein the CP is a carrier protein selected from the group consisting of: CRM 197 diphtheria toxoid; tetanus toxoid; cholera toxin B subunit; meningococcal outer membrane protein (OMP); and capsid proteins of bacteriophage Qβ (especially CRM 197 ).

[0074] 16) The immunogenic compound according to embodiment 11), 12), 13), 14) or 15) may have a linker-spacer-LT- as disclosed in any one of embodiments 17) to 47), where the linker-spacer-LT- of formula (Ia) is a CRM 197 The linker-spacer-LT- is meant to be the same as that described for the carrier protein CP. Accordingly, the same descriptions and definitions apply mutatis mutandis to the carrier protein CP.

[0075] 17) In a further aspect, the present invention provides an immunogenic compound of formula (II):

[0076] [ka]

[0077] (In the formula, R is OH or

[0078] [ka]

[0079] and; m is 3, 4, 5, 6, 7 or 8; n is 1, 2, 3, 4, 5 or 6; i is 1 to 28; -LT- represents a linker L and a spacer T that together form a bridge having a covalently linked backbone of 5 to 25 atoms in length, the length being determined by the distance between the oxygen at C1 of the reducing end of the oligosaccharide and the carrier protein CRM. 197 form the shortest distance between the nitrogen of the amino group of the lysine residue; The atoms of the backbone are selected from the group consisting of carbon, nitrogen, oxygen, and sulfur; or a pharmaceutically acceptable salt thereof.

[0080] The term "essentially", when used in terms such as "essentially pure", is understood in the context of the present invention to particularly mean that at least 90, particularly at least 95, and especially at least 99 percent by weight of the immunogenic compound / oligosaccharide / oligosaccharide-linker compound / oligosaccharide-linker-spacer compound / glycoconjugate is pure immunogenic compound / oligosaccharide / oligosaccharide-linker compound / oligosaccharide-linker-spacer compound / glycoconjugate, respectively.

[0081] Whenever a substituent is described as optional, such substituent may be absent (i.e., the respective residue is unsubstituted with respect to such optional substituent), in which case all sites having a free valence (e.g., in an aromatic ring, ring carbon atoms and / or ring nitrogen atoms having a free valence, to which such optional substituent could be attached) are replaced with hydrogen, as the case may be. Similarly, when the term "optional" is used with respect to (ring) heteroatoms, this term means that each optional heteroatom, etc. is absent (i.e., a group has no heteroatoms / is a carbocyclic ring / etc.) or that each optional heteroatom, etc. is present as explicitly defined.

[0082] CRM 197 " refers to cross-reacting substance 197, which is a non-toxic variant of diphtheria toxin, in which a single amino acid substitution of glycine (Gly, G) at position 52 to glutamic acid (Glu, E) detoxifies the protein.

[0083] CRM 197 CRM is produced by C. diphtheriae infected with the nontoxigenic phage β197tox, which was generated by nitrosoguanidine mutagenesis of the toxigenic corynephage beta (Uchida et al., J. Biol. Chem., 1973, Vol. 245, No. 11, pp. 3838-3844). 197 Proteins are safe and effective T cell-dependent carriers of sugars. 197 CRM is described, for example, by Giannini et al. in Nucleic Acids Research, Vol. 12, No. 10, 1984, pp. 4063-4069. 197 Further details regarding the preparation of CRM can be found, for example, in US Pat. No. 5,614,382, which is incorporated herein by reference. 197may be produced in various expression systems, such as Corynebacterium diphtheriae, Escherichia coli or Pseudomonas fluorescens (Hickey et al., J. Pharm. Sci., 2018, 107, 1806-1819).

[0084] In the present invention, "CRM 197 The term "protein" encompasses proteins having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.8% or 99.9% identity to the amino acid sequence SEQ ID NO:1 (preferably at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 99.9% identity to the amino acid sequence SEQ ID NO:1; especially at least 95%, 96%, 97%, 98%, 99% or 99.9% identity to the amino acid sequence SEQ ID NO:1), optionally with an additional methionine (Met, M) at the N-terminus and / or optionally with a CRM at the lysine site. 197 and the residue may be in a capped (i.e., inactivated) form.

[0085] CRM at the lysine site 197 The expression "residue resulting from functionalization of CRM" 197 is functionalized at a lysine site with a functional group suitable for forming a covalent bond to an antigen, i.e., a linker and / or spacer moiety attached to the oligosaccharide-linker portion of the conjugate. 197 are known to those skilled in the art. The above functional groups are particularly suitable for binding to thiols or for performing click chemistry. For example, such functional groups are groups having bromoacetamide, iodoacetamide, maleimide, azide or alkyne groups. 197optionally comprises a lysine residue functionalized with a bromo-acetamide, iodo-acetamide, maleimide, azide or alkyne group (preferably a bromo-acetamide, iodo-acetamide, maleimide group), which may be in capped form.

[0086] Preferred Functionalized CRM 197 CRM has a group bearing a bromoacetamide, iodoacetamide, or maleimide group, all of which are suitable for reaction with a thiol group provided by the oligosaccharide / linker moiety. 197 The unreacted functional groups of may then be quenched with a pharmaceutically acceptable thiol, such as L-cysteine ​​or cysteamine (2-aminoethane-1-thiol), to give the "capped form."

[0087] Preferred Lysine-Functionalized CRMs 197 is selected from the group consisting of:

[0088] [ka]

[0089] In a preferred embodiment, the CRM 197 is not functionalized as described above, meaning that the "native" lysine residue, i.e., the unmodified amino group of the lysine residue, is used to directly attach the oligosaccharide / linker / spacer moiety thereto, rather than being "pre-functionalized."

[0090] CRM 197 The amino acid sequence of is known to those skilled in the art and is outlined below as SEQ ID NO:1.

[0091] CRM for the synthesis of glycoconjugates 197 Use of CRM 197The above preferred binding sites have been reported (e.g., Moeginger et al., Sci. Rep. 6, 20488; doi:10.1038 / srep20488(2016)), which is incorporated herein by reference.

[0092] "-LT-" represents a linker L and a spacer T that together form a bridge having a covalently linked backbone of 5 to 25 atoms in length, the length being determined by the distance between the oxygen at C1 of the reducing end of the oligosaccharide and the carrier protein CRM. 197 and the atoms of said backbone are selected from the group consisting of carbon, nitrogen, oxygen and sulfur, and the backbone may be saturated, unsaturated, unsubstituted or may be oxo, (C 1-4 ) alkyl, fluoro and (C 1-2 The ring structure may be saturated, unsaturated, or an aromatic 3- to 8-membered ring, including fused ring systems of 2 to 4 rings, and the ring atoms of the ring structure are selected from carbon, nitrogen, oxygen, and sulfur (particularly carbon and nitrogen), and the ring may be unsubstituted or may be substituted with oxo, (C 1-4 ) alkyl, halogen and (C 1-2 ) alkoxy; and optionally substituted by one or more (particularly 1, 2, 3 or 4) substituents (particularly oxo).

[0093] For the avoidance of any doubt, the number 5 to 25 atoms relates to the number of atoms in the backbone and not to the number of atoms in the bridge.

[0094] Examples of the optional ring structures that may be part of the backbone are pyrrolidine-2,5-dione, cyclobut-3-ene-1,2-dione, triazole, isoindolin-1-one, 8,9-dihydro-1H-dibenzo[b,f][1,2,3]triazolo[4,5-d]azocine, cyclohexane, and benzene, as shown below:

[0095] [ka]

[0096] The introduction of these rings or ring systems is known to those skilled in the art of linker chemistry.

[0097] The phrase "the backbone may be unsaturated" means that the backbone chain may have one or more double bonds, which may or may not be part of a ring system.

[0098] For example, in a bridge having a saturated skeleton with three oxo substituents, and the skeleton being part of a ring system, the atoms are numbered as follows:

[0099] [ka]

[0100] That is, the number of atoms forming the skeleton starts from the first atom after the oxygen of C1 and continues until the CRM 197 It ends with the last atom attached to the lysine nitrogen.

[0101] An oxygen atom in a saturated chain is preferably separated from another oxygen atom by one or more (especially 2, 3, 4 or 5, especially 2) carbon atoms.

[0102] The sulfur atoms in the saturated chain are preferably separated from another sulfur atom by one or more (especially 1, 2, 3, 4 or 5) carbon atoms.

[0103] The term "halogen" means fluorine, chlorine or bromine, preferably fluorine or chlorine, more preferably fluorine.

[0104] The term "oxo" refers to the functional group =O, ie, a substituent oxygen atom attached to another atom (preferably a carbon atom) by a double bond.

[0105] The term "alkyl," used alone or in combination, means a straight or branched saturated hydrocarbon chain having from 1 to 4 carbon atoms. x-y The term (C )alkyl (x and y are each integers) refers to an alkyl group as defined above having x to y carbon atoms. For example, (C 1-4 ) The alkyl group has 1 to 4 carbon atoms. 1-4 ) Examples of alkyl groups are methyl, ethyl, n-propyl, iso-propyl, n-butyl, iso-butyl, sec-butyl and tert-butyl. (C 1-2 ) Examples of alkyl groups are methyl and ethyl.

[0106] The term "alkoxy", used alone or in combination, means an alkyl-O- group, wherein the alkyl group is as previously defined. x-y The term "alkoxy" (x and y are each integers) refers to an alkoxy group as defined above having x to y carbon atoms. For example, (C 1-2 ) alkoxy group is "(C 1-2 )alkyl” has the meaning previously described; 1-2 ) alkyl-O- group. 1-2 ) Examples of alkoxy groups are methoxy and ethoxy.

[0107] As used herein, the term "oligosaccharide-carrier protein conjugate" is considered synonymous with the term "glycoconjugate." Furthermore, the "immunogenic compounds" described herein are "oligosaccharide-carrier protein conjugates."

[0108] The term "hybrid" means that the antigen has two different parts of the O-serotype, namely O2afg (Gal-III) and O2a (Gal-I), with the O2afg part being distal to the carrier protein and O2a being proximal to the carrier protein.

[0109] 18) A further embodiment relates to an immunogenic compound according to embodiment 17), or a pharmaceutically acceptable salt thereof, wherein m is 3, 4, 5 or 6 and n is 2, 3 or 4.

[0110] 19) A further embodiment relates to an immunogenic compound according to embodiment 17), or a pharmaceutically acceptable salt thereof, wherein m is 3, 4, 5 or 6 and n is 2 or 3, for example 2.

[0111] 20) A further embodiment relates to an immunogenic compound or a pharmaceutically acceptable salt thereof according to any one of embodiments 17), 18) or 19), wherein R is OH.

[0112] 21) A further aspect is m is 4, n is 2, and R is OH; m is 4, n is 2, and R is

[0113] [ka]

[0114] or m is 4, n is 3, and R is OH; The immunogenic compound according to embodiment 17) or a pharmaceutically acceptable salt thereof.

[0115] 22) A further embodiment relates to an immunogenic compound according to embodiment 17), or a pharmaceutically acceptable salt thereof, wherein m is 4, n is 2, and R is OH.

[0116] 23) A further embodiment relates to an immunogenic compound or a pharmaceutically acceptable salt thereof according to any one of embodiments 11) to 15) and any one of embodiments 17), 18), 19), 20), 21) or 22), wherein the bridge does not have an aromatic or heteroaromatic ring.

[0117] 24) In a further embodiment, -LT- represents a linker L and a spacer T that together form a bridge having a covalently linked backbone length of 5 to 25 atoms, the length being such that the oxygen at C1 of the reducing end of the oligosaccharide and the carrier protein CRM are separated. 197 (or CP, if applicable) and the nitrogen of the amino group of the lysine residue, and the backbone has at most one double bond; the atoms of the backbone are selected from the group consisting of carbon, nitrogen, oxygen, and sulfur; The above skeleton is oxo, (C 1-4 ) alkyl, fluoro and (C 1-2 ) optionally substituted by one or more (particularly 1, 2, 3 or 4) substituents independently selected from alkoxy (particularly oxo); A part of the skeleton is optionally

[0118] [ka]

[0119] may be a 4-, 5- or 6-membered ring moiety selected from: The present invention relates to an immunogenic compound or a pharmaceutically acceptable salt thereof according to any one of aspects 11) to 15) and any one of aspects 17), 18), 19), 20), 21) or 22).

[0120] Here, the "maximum one double bond" is preferably a double bond of the cyclobut-3-ene-1,2-dione ring.

[0121] 25) A further embodiment is a linker in which -LT- represents a linker L and a spacer T which together form a bridge, said bridge consisting of a backbone which is a saturated chain of 5 to 25 atoms selected from the group consisting of carbon, nitrogen, oxygen and sulfur (particularly carbon, nitrogen and oxygen), said chain being unsubstituted or having oxo, (C 1-4 ) alkyl, fluoro and (C 1-2 and an immunogenic compound or a pharmaceutically acceptable salt thereof according to any one of embodiments 11) to 15), and any one of embodiments 17), 18), 19), 20), 21) or 22), wherein the bridge is optionally substituted by one or more (particularly 1, 2, 3 or 4) substituents (particularly oxo) independently selected from oxo, (C )alkoxy, wherein the bridge consists of a saturated chain of 5 to 25 atoms selected from the group consisting of carbon, nitrogen, oxygen and sulfur (particularly carbon, nitrogen and oxygen), and wherein the chain is unsubstituted or 1-4 ) alkyl, fluoro and (C 1-2 )alkoxy (especially oxo). For the avoidance of doubt, in this embodiment the bridge does not comprise a ring structure.

[0122] 26) A further embodiment is a linker in which -LT- represents a linker L and a spacer T which together form a bridge, said bridge consisting of a backbone which is a saturated chain of 5 to 25 atoms selected from the group consisting of carbon, nitrogen and oxygen (particularly carbon and nitrogen), said chain being unsubstituted or having oxo, (C 1-4 ) alkyl, fluoro and (C 1-2 and an immunogenic compound or a pharmaceutically acceptable salt thereof according to any one of embodiments 11) to 15), and any one of embodiments 17), 18), 19), 20), 21) or 22), wherein the bridge consists of a saturated chain of 5 to 25 atoms selected from the group consisting of carbon, nitrogen and oxygen (particularly carbon and nitrogen), and wherein the chain can be unsubstituted or can be substituted with oxo, (C 1-4) alkyl, fluoro and (C 1-2 )alkoxy (especially oxo). For the avoidance of doubt, in this embodiment the bridge does not comprise a ring structure.

[0123] 27) A further embodiment is a crosslinker in which the backbone of the crosslinker has a length of 8 to 20, preferably 8 to 16, atoms covalently linked together, the length being such that the oxygen at C1 of the reducing end of the oligosaccharide and the carrier protein CRM are separated. 197 (or, if applicable, CP), forming the shortest distance between the nitrogen of the amino group of the lysine residue of

[0124] 28) A further aspect is L, * -(C 2-10 ) alkylene-NH-; * -(CH2CH2O) b -CH2CH2NH- (b is 1, 2 or 3); * -CH2CH2S-CH2CH2NH-; * -(C 2-10 ) fluoroalkylene-NH-; * -(CH2) c NHC(O)(CH2) c’ -NH- (c and c' are independently 2 to 6); * -(CH2) d NHC(O)NH(CH2) d’ -NH- (d and d' are independently 2 to 6); * -(C 1-10 ) alkylene-C(O)-NH-(C 2-10 ) alkylene-NH-; or * -(C 2-10 ) alkylene-O-NH-; represents; T, -C(O)-(C 0-10 ) alkylene-C(O)-; -C(O)-CH2CH2-(OCH2CH2) j -C(O)- (j is 1 to 5); -C(O)-CH2(CH2) k -(SCH2(CH2) k’ ) k’’ —C(O)— (k is 0 or 1, k′ is 0 or 1, and k″ is 1, 2, or 3);

[0125] [ka]

[0126] represents; or LT, * -(C 2-10 ) Alkylene-SR 1 ; represents; R 1 teeth,

[0127] [ka]

[0128] and an immunogenic compound or a pharmaceutically acceptable salt thereof according to any one of embodiments 11) to 15) and any one of embodiments 17), 18), 19), 20), 21), 22) or 27).

[0129] The " * " means that the linker is attached to the oligosaccharide at this position.

[0130] " attached to the spacer T *" means that the spacer is bonded to the linker L at this position.

[0131] R 1 The "♯" attached to the R 1 means that the bond is bonded to the sulfur.

[0132] "-(C x-y The term (C) alkylene- (x and y are each integers), used alone or in combination, refers to a bivalently bound, straight or branched saturated hydrocarbon chain having x to y carbon atoms. For example, (C 2-10 ) alkylene group has 2 to 10 carbon atoms, (C 0-10 A straight chain -(C x-y ) alkylene-, i.e., -(CH2) x-y - is preferred. (C 2-10 Representative examples of alkylene groups are ethylene, propylene, butylene, pentylene, hexylene, heptylene, octylene, nonylene, and decylene (particularly 1,2-ethylene, 1,3-propylene, 1,4-butylene, 1,5-pentylene, 1,6-hexylene, 1,7-heptylene, 1,8-octylene, 1,9-nonylene, and 1,10-decylene).

[0133] "(C x-y The term "fluoroalkylene" (x and y are each integers), used alone or in combination, refers to a two-bonded, straight-chain or branched saturated hydrocarbon group having x to y carbon atoms in which one or more (and in some cases all) hydrogen atoms have been replaced by fluorine. x-y ) Fluoroalkylene- is preferred.

[0134] For the avoidance of any doubt, in these embodiments, the backbone length of -LT- is 5 to 25 atoms, 8 to 20 atoms, or 8 to 16 atoms. This is because the linker L and spacer T, where applicable, are 1 together form a bridge having a backbone length of 5 to 25 (8 to 20 or 8 to 16) atoms covalently linked together, said length being such that the oxygen at C1 of the reducing end of said oligosaccharide and said carrier protein CRM 197 This means forming the shortest distance between the amino nitrogen of the lysine residue of the CP (or CP, if applicable).

[0135] 29) A further aspect is L, * -(CH2) a -NH-; (a is 2 to 10); * -(CH2CH2O) b -CH2CH2NH- (b is 1, 2 or 3); * -CH2CH2S-CH2CH2NH-; * -(C 2-10 ) fluoroalkylene-NH- (fluoroalkylene is a saturated linear chain); * -(CH2) c NHC(O)(CH2) c’ -NH- (c and c' are independently 2 to 6); * -(CH2) d NHC(O)NH(CH2) d’ -NH- (d and d' are independently 2 to 6); * -(CH2) e -C(O)-NH-(CH2) e’ -NH-; (e is 1 to 10, and e' is 2 to 10); or * -(CH2) f -O-NH- (f is 2 to 10); represents; or LT, * -(CH2) g SR 1 (g is 2 to 10); 28) or a pharmaceutically acceptable salt thereof;

[0136] 30) A further aspect is L, * -(CH2) a -NH-; (a is 2 to 10); * -(CH2CH2O) b -CH2CH2NH- (b is 1, 2 or 3); * -(C 2-10 ) fluoroalkylene-NH- (fluoroalkylene is a saturated linear chain); * -(CH2) e -C(O)-NH-(CH2) e’ -NH-; (e is 1 to 10, and e' is 2 to 10); or * -(CH2) f -O-NH- (f is 2 to 10); represents; or LT, * -(CH2) g SR 1 (g is 2 to 10); or a pharmaceutically acceptable salt thereof according to embodiment 28), which represents:

[0137] 31) A further aspect is L, * -(CH2) a -NH-; (a is 2 to 10, preferably 2 to 6); * -(CH2CH2O) b -CH2CH2NH- (b is 1, 2 or 3, preferably 1 or 2); * -(CH2)e -C(O)-NH-(CH2) e’ -NH-; (e is 1 to 10, preferably 1 to 6, and e' is 2 to 10, preferably 2 to 6); or * -(CH2) f -O-NH- (f is 2 to 10, preferably 2 to 6); represents; or LT, * -(CH2) g SR 1 (g is 2 to 10, preferably 2 to 6); or a pharmaceutically acceptable salt thereof according to embodiment 28), which represents:

[0138] 32) A further aspect is L, * -(CH2) a -NH-; (a is 2 to 10, preferably 2 to 6); * -(CH2CH2O) b -CH2CH2NH- (b is 1, 2 or 3, preferably 1 or 2); or * -(CH2) f -O-NH- (f is 2 to 10, preferably 2 to 6); or a pharmaceutically acceptable salt thereof according to embodiment 28), which represents:

[0139] 33) A further aspect is L, * -(CH2) a -NH-; (a is 2 to 10, preferably 2 to 6); or * -(CH2CH2O) b -CH2CH2NH- (b is 1, 2 or 3, preferably 1 or 2); or a pharmaceutically acceptable salt thereof according to embodiment 28), which represents:

[0140] 34) A further aspect is that L is * -(CH2) a -NH-; (a is 2 to 10, preferably 2 to 6), or a pharmaceutically acceptable salt thereof according to embodiment 28).

[0141] 35) A further embodiment is where L is * -(CH2)2-NH-, * -(CH2)3-NH-, * -(CH2)4-NH-, * -(CH2)5-NH- or * or a pharmaceutically acceptable salt thereof according to embodiment 28), wherein -(CH2)6-NH- represents

[0142] 36) A further aspect is that L is * or a pharmaceutically acceptable salt thereof according to embodiment 28), wherein -(CH2)5-NH- represents

[0143] 37) A further aspect is that L is * -(CH2CH2O) b The immunogenic compound according to embodiment 28) or a pharmaceutically acceptable salt thereof, wherein b represents -CH2CH2NH- (b is 1 or 2; preferably 1).

[0144] 38) A further aspect is T, -C(O)-(CH2) h -C(O)- (h is 0 to 10); -C(O)-CH2CH2-(OCH2CH2) j -C(O)- (j is 1 to 5); -C(O)-CH2(CH2) k -(SCH2(CH2) k’ ) k’’ —C(O)— (k is 0 or 1, k′ is 0 or 1, and k″ is 1, 2, or 3);

[0145] [ka]

[0146] or a pharmaceutically acceptable salt thereof according to any one of embodiments 28) to 37), which represents:

[0147] 39) A further aspect is T, -C(O)-(CH2) h —C(O)— (h is 0 to 10, preferably 0 to 6); -C(O)-CH2CH2-(OCH2CH2) j —C(O)— (j is 1 to 5, preferably 1 to 3, and more preferably 1); -C(O)-CH2(CH2) k -(SCH2(CH2) k’ ) k’’ -C(O)- (k is 0 or 1, k' is 0 or 1, and k'' is 1, 2 or 3, preferably 1); or

[0148] [ka]

[0149] or a pharmaceutically acceptable salt thereof according to any one of embodiments 28) to 37), which represents:

[0150] 40) A further aspect is T, -C(O)-(CH2) h -C(O)- (h is 0 to 6); -C(O)-CH2CH2-(OCH2CH2) j -C(O)- (j is 1 or 2); or -C(O)-CH2(CH2) k -(SCH2(CH2) k’ ) k’’—C(O)— (k is 0 or 1, k′ is 0 or 1, and k″ is 1, preferably k and k′ are 0 and k″ is 1); or a pharmaceutically acceptable salt thereof according to any one of embodiments 28) to 37), which represents:

[0151] 41) A further aspect is T, -C(O)-(CH2) h -C(O)- (h is 0, 1, 2, 3, 4, 5 or 6, preferably 4); or -C(O)-CH2CH2-(OCH2CH2) j -C(O)- (j is 1 or 2); or a pharmaceutically acceptable salt thereof according to any one of embodiments 28) to 37), which represents:

[0152] 42) A further embodiment is where T is -C(O)-(CH) h The present invention relates to an immunogenic compound or a pharmaceutically acceptable salt thereof according to any one of aspects 28) to 37), wherein:

[0153] 43) A further aspect is T,

[0154] [ka]

[0155] or a pharmaceutically acceptable salt thereof according to any one of embodiments 28) to 37), which represents:

[0156] 44) A further aspect is T,

[0157] [ka]

[0158] or a pharmaceutically acceptable salt thereof according to any one of embodiments 28) to 37), which represents:

[0159] 45) A further aspect is R 1 but,

[0160] [ka]

[0161] or a pharmaceutically acceptable salt thereof, according to any one of embodiments 28), 29), 30), 38), 43) or 44), which represents:

[0162] Preferably, R 1 teeth:

[0163] [ka]

[0164] Represents.

[0165] 46) A further aspect is L, * -(CH2)2-NH-, * -(CH2)3-NH-, * -(CH2)4-NH-, * -(CH2)5-NH- or * -(CH2)6-NH-; represents; T, -C(O)-C(O)-, -C(O)-CH2-C(O)-, -C(O)-(CH2)2-C(O)-, -C(O)-(CH2)3-C(O)-, -C(O)-(CH2)4-C(O)-, -C(O)-(CH2)5-C(O)- or -C(O)-(CH2)6-C(O)-; represents; The present invention relates to an immunogenic compound or a pharmaceutically acceptable salt thereof according to any one of aspects 11), 12), 13), 14), 15), 17), 18), 19), 20), 21) or 22).

[0166] 47) A further aspect is L * represents -(CH2)5-NH-, and T represents -C(O)-(CH2)4-C(O)-; The present invention relates to an immunogenic compound or a pharmaceutically acceptable salt thereof according to any one of aspects 11), 12), 13), 14), 15), 17), 18), 19), 20), 21) or 22).

[0167] 48) A preferred embodiment is an immunogenic compound having the following formula (II):

[0168] [ka]

[0169] (In the formula, m is 4, n is 2, and R is OH; m is 4, n is 2, and R is

[0170] [ka]

[0171] or m is 4, n is 3, and R is OH; i is 1 to 28; L is * -(CH2)2-NH-, * -(CH2)3-NH-, * -(CH2)4-NH-, * -(CH2)5-NH- or * represents -(CH2)6-NH-; T is -C(O)-C(O)-, -C(O)-CH2-C(O)-, -C(O)-(CH2)2-C(O)-, -C(O)-(CH2)3-C(O)-, -C(O)-(CH2)4-C(O)-, -C(O)-(CH2)5-C(O)- or -C(O)-(CH2)6-C(O)-); represents. ); or a pharmaceutically acceptable salt thereof.

[0172] 49) A further aspect is m is 4, n is 2, and R is OH; L, * -(CH2)2-NH-, * -(CH2)3-NH-, * -(CH2)4-NH-, * -(CH2)5-NH- or * -(CH2)6-NH- (preferably * -(CH2)5-NH-); T, -C(O)-C(O)-, -C(O)-CH2-C(O)-, -C(O)-(CH2)2-C(O)-, -C(O)-(CH2)3-C(O)-, -C(O)-(CH2)4-C(O)-, -C(O)-(CH2)5-C(O)- or -C(O)-(CH2)6-C(O)- (preferably -C(O)-(CH2)4-C(O)-); i is 1 to 28; preferably 6 to 15; The immunogenic compound according to embodiment 48) or a pharmaceutically acceptable salt thereof.

[0173] 50) A further embodiment relates to an immunogenic compound or a pharmaceutically acceptable salt thereof according to any one of embodiments 11) to 49), wherein i is 1 to 28, 1 to 25, 1 to 23; 1 to 20, 1 to 18, 3 to 25, 3 to 23, 3 to 20, 3 to 18, 5 to 23, 5 to 20, 5 to 18, 6 to 23, 6 to 20, 6 to 18, 6 to 15.

[0174] The variable i is the CRM 197The loading of the antigen, i.e., the oligosaccharide hybrid, on the protein carrier is described and is an integer per single molecule. However, it should be noted that if the glycoconjugate is considered as a product of more than one single molecule, the loading can be described as a statistical distribution, i.e., essentially a Gaussian distribution. The chemical process for producing the product results in a mixture of molecules with such a statistical distribution of the loadings, and in this case, the loading will be given as the average of the statistical distribution, particularly the Gaussian distribution.

[0175] When i is 2 or more, the carrier protein CP or CRM is 197 The m and / or n of the two or more oligosaccharides each bound to may be the same or different. Preferably, all of the i oligosaccharides are represented by the same combination of m and n (i.e., have the same structure), or all of the i oligosaccharides are represented by a first combination of m and n or a second combination of m and n (i.e., have one of two structures); most preferably, all of the i oligosaccharides are represented by the same combination of m and n. The linker-spacer unit -LT- is the same among the i oligosaccharides of a particular oligosaccharide-carrier protein conjugate.

[0176] In other words, preferred compounds are those that have the same oligosaccharide / linker / spacer residues, i.e., a carrier protein, preferably a CRM. 197 The oligosaccharides contain only one specific type of oligosaccharide / linker / spacer residue attached to the oligosaccharide.

[0177] 51) A further embodiment relates to an immunogenic compound or a pharmaceutically acceptable salt thereof according to any one of embodiments 11) to 49), wherein i is 6 to 15.

[0178] 52) A preferred embodiment is an immunogenic compound selected from the group consisting of:

[0179] [ka]

[0180] (wherein i is 1 to 28); or a pharmaceutically acceptable salt thereof.

[0181] For the avoidance of any doubt, the immunogenic compounds of formula (IIa), (IIb) and (IIc) according to this embodiment may also be depicted schematically as follows:

[0182] [ka]

[0183] (wherein i is 1 to 28); or a pharmaceutically acceptable salt thereof. 197 ' is a CRM as defined herein 197 The only difference is that in formulas (IIa'), (IIb') and (IIc'), the amino group of the lysine residue is specifically shown as the attachment point for the linker / spacer moiety -LT-.

[0184] Preferred values ​​of i are those disclosed in embodiment 50) or especially 51).

[0185] 53) A further preferred embodiment is an immunogenic compound of formula (IIa):

[0186] [ka]

[0187] (wherein i is 1 to 28); or a pharmaceutically acceptable salt thereof.

[0188] Preferred values ​​of i are those disclosed in embodiment 50) or especially 51).

[0189] 54) A further preferred embodiment is an immunogenic compound selected from the group consisting of:

[0190] [ka]

[0191] (wherein i is 6 to 15); or a pharmaceutically acceptable salt thereof.

[0192] 55) A further preferred embodiment is an immunogenic compound of formula (IIa):

[0193] [ka]

[0194] (wherein i is 6 to 15); or a pharmaceutically acceptable salt thereof.

[0195] The present invention therefore relates, according to each sub-relation, to compounds of formula (I) as defined in embodiment 1) and to such compounds further defined by the feature of any one of embodiments 2) to 10), to compounds of formula (Ia) as defined in embodiment 11) and to such compounds further defined by the feature of any one of embodiments 12) to 16), to compounds of formula (II) as defined in embodiment 17) and to such compounds further defined by the feature of any one of embodiments 18) to 55), to pharmaceutically acceptable salts thereof, and to the uses of such compounds as further described below. In particular, compounds of formulae (Ia) and (II) are sub-forms of formula (I), and compounds of formulae (III) and (IV) are intermediates for preparing compounds of formulae (I), (Ia) and (II), respectively. The embodiments for the definition of LT specified in embodiments 23) to 55) for compounds of formula (II) shall conversely also apply as the definition of LT for compounds of formula (Ia) according to any one of embodiments 11) to 15).

[0196] For the avoidance of any doubt, the following embodiments in particular with respect to compounds of formula (I) and (II) are possible, contemplated, and are specifically disclosed herein as individual forms: 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45+44+36+28+20+17, 45+44+36+28+21+17, 45+44+36+28+22+17, 45+44+36+28+27+17, 45+44+37+28+17, 45+44+37+28+18+17, 45+44+3 7+28+19+17, 45+44+37+28+20+17, 45+44+37+28+21+17, 45+44+37+28+22+17, 45+44+37+28+27+17, 46+11, 46+12+11, 46+13+11, 46+14+11, 46+15+11, 46+15 +12+11, 46+15+13+11, 46+15+14+11, 46+17, 46+18+17, 46+19+17, 46+20+17, 46+20+18+17, 46+20+19+17, 46+21+17, 46+22+17, 47+11, 47+12+11, 47+13+11, 47+14+11, 47+15+11, 47+15+12+11, 47+15+13+11, 47+15+14+11, 47+17, 47+18+17, 47+19+17, 47+20+17, 47+20+18+17, 47+20+19+17, 47+21+17 and 47+22+17;, In the above list, the numbers refer to the embodiments corresponding to the number, and "+" indicates a dependency from another embodiment. The various embodiments are individually separated by commas. In other words, for example, "4+2+1" means embodiment 4) which is dependent on embodiment 2) which is dependent on embodiment 1), i.e. embodiment "4+2+1" corresponds to the compound of embodiment 1) further limited by the characteristics of embodiments 2) and 4).

[0197] The ranges of i set forth in embodiments 50) and 51) shall be considered to be expressly disclosed for each of the above combinations.

[0198] When the plural forms are used for compounds, conjugates, salts, pharmaceutical compositions, diseases, etc., they are intended to refer to the singular compound, conjugate, salt, pharmaceutical composition, disease, etc.

[0199] Any reference to compounds of formula (I), (Ia), (II), (IIa), (IIb), (IIc), (III) and (IV) as defined in any one of aspects 1) to 81) will be understood to also refer to salts (particularly pharmaceutically acceptable salts) of such compounds, where appropriate and appropriate.

[0200] The term "pharmaceutically acceptable salt" refers to a salt that retains the desired biological activity of the subject compound and exhibits minimal undesired toxic effects. Such salts include inorganic or organic acid and / or base addition salts, depending on the presence of basic and / or acidic groups in the subject compound. They may be used for stabilization in the form of a buffer or for lyophilized products containing a buffer. For reference, see, for example, "Handbook of Pharmaceutical Salts. Properties, Selection and Use," P. Heinrich Stahl, Camille G. Wermuth (Eds.), Wiley-VCH, 2008; and "Pharmaceutical Salts and Co-crystals," Johan Wouters and Luc Quere (Eds.), RSC Publishing, 2012.

[0201] This embodiment also includes isotopically labeled, especially 2 Also included are H (deuterium)-labeled compounds of formula (I), (Ia), (II), (IIa), (IIb) and (IIc), which are identical to compounds of formula (I), (Ia), (II), (IIa), (IIb) and (IIc) except that one or more atoms have been replaced, respectively, by atoms having the same atomic number but an atomic mass different from that normally found in nature. Isotopically labeled compounds, especially 2 H (deuterium) labeled compounds of formula (I), (Ia), (II), (IIa), (IIb) and (IIc) and salts thereof are included within the scope of this embodiment. 2Substitution with H (deuterium) can increase metabolic stability, e.g., prolong in vivo half-life, reduce dosage requirements, or alleviate inhibition of cytochrome P450 enzymes, thereby improving safety profiles, for example. In one embodiment, compounds of formulas (I), (Ia), (II), (IIa), (IIb), and (IIc) are not isotopically labeled, or they are labeled only with one or more deuterium atoms. In a subembodiment, compounds of formulas (I), (Ia), (II), (IIa), (IIb), and (IIc) are not isotopically labeled at all. Isotopically labeled compounds of formulas (I), (Ia), (II), (IIa), (IIb), and (IIc) can be prepared in a manner similar to that described below, except for using appropriate isotopic species of the appropriate reagents or starting materials. For example, the labeling can be carried out in the linker L and / or the spacer T.

[0202] The compounds of formulae (I), (Ia), (II), (IIa), (IIb) and (IIc) as defined in any one of aspects 1) to 55) and pharmaceutically acceptable salts thereof can be used as medicines, for example in the form of pharmaceutical compositions for parenteral, enteral (such as oral) or nasal administration, in particular for parenteral administration such as intramuscular, subcutaneous and intradermal injection.

[0203] 56) Thus, one aspect of the present invention relates to a pharmaceutical composition having as an active ingredient an immunogenic compound or a pharmaceutically acceptable salt thereof according to any one of embodiments 1) to 55), in particular embodiments 52), 53), 54) and 55), and further comprising at least one therapeutically inactive excipient.

[0204] Pharmaceutical compositions can be prepared in a manner well known to anyone skilled in the art (see, for example, Remington, The Science and Practice of Pharmacy, 23rd Edition (2021), published by Elsevier Inc., ISBN: 978-0-12-820007-0; Vaccine Development and Manufacturing, 1st edition (2014), published by John Wiley & Sons, ISBN: 9780470261941). The compounds of formula (I), (Ia), (II), (IIa), (IIb) and (IIc) or pharmaceutically acceptable salts thereof can be combined with, optionally, other therapeutically beneficial substances, and formulated into a dosage form together with suitable non-toxic, inert, therapeutically compatible solid or liquid carrier materials and, optionally, conventional pharmaceutical adjuvants.

[0205] The pharmaceutical composition may, in addition to an immunogenic compound according to any one of aspects 1) to 55), comprise one or more (preferably 1, 2, 3 or 4; more preferably 2, 3 or 4; most preferably 2 or 3) other immunogenic compounds (oligosaccharide-carrier protein conjugates) that are immunogenic against one or more other K. pneumoniae serotypes, in particular against O1, O2ac, O2aeh, O3, O3a, O3b, O4, O5, O7, O8, and / or O12; especially against O1, O3, O3b and / or O5.

[0206] The pharmaceutical compositions are suitable for inducing a protective immune response in a human and / or animal (particularly mammalian (including human)) host and are therefore useful for the prevention and / or treatment of diseases associated with Klebsiella pneumoniae. Preferably, the pharmaceutical compositions are suitable for use in humans.

[0207] The terms "prevention," "preventing," and / or "prophylaxis" are used interchangeably and refer to inhibiting the initial onset of a pathological process (i.e., preventing the onset of a disease, disorder, or condition in a prophylactic manner) so that the pathological process that may ultimately lead to the onset of symptoms does not progress or so that the symptoms develop at a less severe and less dangerous intensity.

[0208] The pharmaceutical compositions of the present invention are suitable for administration to animal (particularly human) patients and therefore include both human and veterinary uses. The pharmaceutical compositions may be used in a method of raising an immune response in a patient, such method comprising administering the composition to the patient.

[0209] The pharmaceutical compositions of the present invention may be administered before a subject is exposed to Klebsiella pneumoniae and / or after a subject is exposed to Klebsiella pneumoniae, and are preferably used before a subject is exposed to Klebsiella pneumoniae.

[0210] The pharmaceutical compositions are preferably in aqueous form, particularly at the time of administration, but may also be provided in non-aqueous or dried form, such as gelatin capsules or lyophilized products. For example, solid powders obtained by spray drying, spray freeze drying, vacuum or air drying, or lyophilization may be reconstituted before use. When a solid powder is required, lyophilization is preferred.

[0211] The pharmaceutical composition may contain one or more therapeutically inactive excipients, which may be selected from the group consisting of citric acid monohydrate, sodium citrate, sodium citrate dihydrate, acetic acid, sodium hydroxide, tromethamine, tromethamine hydrochloride (for pH adjustment), cholesterol, sorbitan trioleate, DSPC (1,2-distearoyl-sn-glycero-3-phosphocholine) and (4-hydroxybutyl)azanediylbis(hexane-6,1-diyl)bis(2-hexyldecanoate), polydimethylsiloxane (antifoaming agent), and ascorbic acid (antioxidant).

[0212] Excipients may also serve to adjust osmolality, for example sodium chloride (NaCl) may be present at 1-20 mg / ml. Other salts that may be present include potassium chloride, potassium dihydrogen phosphate, disodium phosphate anhydrous, magnesium chloride, calcium chloride, etc.

[0213] The pharmaceutical composition may contain one or more excipients that function as preservatives, which may be selected from the group consisting of 2-phenoxyethanol, benzethonium chloride, EDTA (ethylenediaminetetraacetic acid), formaldehyde, phenol, and thiomersal (thimerosal). Mercury-free compositions are preferred, and preservative-free vaccines can be produced.

[0214] The pharmaceutical composition may comprise one or more excipients that function as surfactants, which may be selected from the group consisting of polysorbate 20 (polyoxyethylene (20) sorbitan monolaurate), polysorbate 80 (polyoxyethylene (80) sorbitan monooleate), nonylphenol ethoxylate, octoxynol-10, and sodium deoxycholate.

[0215] The pharmaceutical compositions may contain the compound (with or without insoluble metal salts) in plain water (e.g., water for injection, WFI), but typically contain one or more buffers. Typical buffers include phosphate buffer; Tris buffer; borate buffer; succinate buffer; histidine buffer (especially with aluminum hydroxide adjuvant); or citrate buffer. Buffer salts are typically present in the range of 5-20 mM.

[0216] Pharmaceutical compositions typically have a pH between 5.0 and 9.5, for example a pH between 6.0 and 8.0.

[0217] The pharmaceutical composition may further comprise one or more stabilizers.

[0218] The pharmaceutical composition is preferably sterile and gluten-free.

[0219] 57) A further embodiment of the invention relates to a pharmaceutical composition according to embodiment 56), further comprising an adjuvant.

[0220] As used herein, the term "adjuvant" refers to an immunological adjuvant, i.e., a substance used in a vaccine composition that modifies or increases the effectiveness of a vaccine by enhancing the immune response to an antigen contained in the vaccine without antigenically contributing to it. Examples of immunological adjuvants known to those skilled in the art in the classical sense include: aluminum or calcium salt-based adjuvants, saponin or saponin-based adjuvants (e.g., Matrix-M), CpG oligodeoxynucleotide-based adjuvants (e.g., CpG1018), oil-in-water emulsions (e.g., Freund's adjuvant, MF59), activators of natural killer T cells (NKT cells) or invariant NKT cells (e.g., glycosphingolipids such as KRN7000), Toll-like receptor 1 / 2 (TLR-1 / 2) agonists (e.g., Pam3CSK4), TLR-3 agonists (e.g., Poly(I:C)), TLR-4 agonists (e.g., lipopolysaccharide), TLR-5 agonists (e.g., flagellin), TLR-7 / 8 agonists (e.g., resiquimod), immunomodulatory proteins (e.g., Escherichia coli), Detoxified heat-labile enterotoxin from coli (dmLT), TLR-4 agonist glucopyranosyl lipid adjuvant-stable emulsion (GLA-SE) and monophosphoryl lipid A (MPL), non-ionic block polymers, cytokines (e.g., type 1 interferons (IFNs), granulocyte-macrophage colony-stimulating factors (GM-CSFs), interleukins), papain-like cysteine ​​proteases, and many others (e.g., AS04, AS03, AS01). Band formulations of the above adjuvants as liposomes or nanoparticles made with lipids such as DOPC (1,2-dioleoyl-sn-glycero-3-phosphocholine), DSPC (1,2-distearoyl-sn-glycero-3-phosphocholine), cholesterol and / or ALC-0315, formulations as virus-like particles, and co-formulations of the above adjuvants, particularly co-formulations with aluminum or calcium salt-based adjuvants.

[0221] The adjuvant, "aluminum," "aluminum-based adjuvant," or "aluminum salt-based adjuvant" is one or more of the following: amorphous aluminum hydroxyphosphate sulfate (AAHS), aluminum hydroxide, aluminum phosphate, and potassium aluminum sulfate (Alum).

[0222] An example of a calcium-based or calcium salt-based adjuvant is calcium phosphate.

[0223] Matrix-M is a saponin-based adjuvant consisting of nanoparticles of saponin extracted from the Quillaja saponaria (soapbark) tree, cholesterol, and phospholipids.

[0224] CpG-based adjuvants are immunostimulatory oligodeoxynucleotides bearing one or more CpG motifs (CpG ODNs), which are unmethylated cytosine-guanine dinucleotides. The methylation status of the CpG immunostimulatory motif generally relates to the cytosine residue in the dinucleotide. Immunostimulatory oligonucleotides with at least one unmethylated CpG dinucleotide have a 5' unmethylated cytosine linked to a 3' guanine by a phosphate bond and activate the immune system through binding to Toll-like receptor 9 (TLR-9).

[0225] Freund's adjuvant is a mineral oil-based oil-in-water adjuvant.

[0226] MF59 is an oil-in-water emulsion with 4.3% w / v squalene, 0.5% w / v polysorbate 80 (Tween 80) and 0.5% w / v sorbitan trioleate (Span 85).

[0227] Glycosphingolipids are a class of lipids that stimulate the unconventional invariant T-cell receptor on NKT cells or iNKT cells when they are presented on MHC class I-associated molecules such as CD1d.

[0228] Pam3CSK4 (Pam3CysSerLys4) is a synthetic triacylated lipopeptide that is a ligand for TLR-1 and TLR-2 and mimics the acylated amino terminus of bacterial lipopeptides.

[0229] Poly(I:C) is a polymer consisting of one strand of inosinic acid and one strand of cytidylic acid, an analog of double-stranded RNA. It stimulates TLR-3 and simulates viral infection.

[0230] Lipopolysaccharide (LPS) is a membrane component of Gram-negative bacteria and a stimulator of TLR-4.

[0231] Flagellin is a globular protein that forms the filaments of bacterial flagella and activates TLR-5 and TLR-11.

[0232] Resiquimod (R848; 1-[4-amino-2-(ethoxymethyl)-1H-imidazo[4,5-c]quinolin-1-yl]-2-methylpropan-2-ol) is an immune response modifier and small molecule that activates TLR-7 and TLR-8.

[0233] dmLT is a double mutant (and thereby detoxified) form of the heat-labile enterotoxin from Escherichia coli, and is an effective mucosal and systemic adjuvant.

[0234] GLA-SE is an oil-in-water emulsion adjuvant made by combining aqueous glucopyranosyl lipid A (GLA), a TLR-4 agonist, with squalene.

[0235] MPL (monophosphoryl lipid A), a truncated form of LPS, is a clinically used TLR-4 agonist.

[0236] Nonionic block polymers (NBPs) suitable as adjuvants are simple copolymers of polyoxyethylene (POE) and hydrophobic polyoxypropylene (POP), which vary in molecular weight, POE ratio, and bonding mode between POE and POP groups.

[0237] Cytokines are small proteins secreted by cells that affect cell-cell interactions and communication. Typically, cytokines activate target cells, triggering the secretion of additional cytokines and signaling cascades. Cytokines are involved in the induction of innate and adaptive immunity. As adjuvants, cytokines can be used as recombinant proteins or encoded on DNA molecules such as plasmids.

[0238] Papain-like cysteine ​​proteases are derived from viruses, bacteria, yeast, protozoa, plants, or animals and contain a cysteine ​​thiol at the active site. This class of proteases can stimulate a Th2-type immune response.

[0239] AS04 (Adjuvant System 04) is a complex of MPL (3-O-desacyl-4'-monophosphoryl lipid A) and aluminum hydroxide or aluminum phosphate.

[0240] AS03 (Adjuvant System 03) is a squalene-in-water emulsion containing DL-alpha-tocopherol (vitamin E) and polysorbate 80.

[0241] AS01 B is a mixture of 3-O-desacyl-4'-monophosphoryl lipid A (MPL) and saponin QS-21.

[0242] Preferred adjuvants are aluminum-based adjuvants, especially aluminum hydroxide.

[0243] 58) A further aspect of the present invention relates to an immunogenic compound or a pharmaceutically acceptable salt thereof according to any one of embodiments 1) to 55), in particular embodiments 52), 53), 54) and 55), for use as a medicament, in particular as a vaccine. In other words, the present invention relates to a vaccine comprising an immunogenic compound or a pharmaceutically acceptable salt thereof according to any one of embodiments 1) to 55), in particular embodiments 52), 53), 54) and 55). Preferably, said vaccine is used for active vaccination.

[0244] 59) A further aspect of the present invention relates to an immunogenic compound or a pharmaceutically acceptable salt thereof according to any one of embodiments 1) to 55), in particular embodiments 52), 53), 54) and 55), for use in the prevention and / or treatment of K. pneumoniae infections.

[0245] 60) A further aspect of the invention relates to an immunogenic compound or a pharmaceutically acceptable salt thereof according to any one of aspects 1) to 55), in particular aspects 52), 53), 54) and 55), for use in the prophylaxis and / or treatment of K. pneumoniae infections in individuals 50 years of age or older; hospital-acquired (i.e. nosocomial) K. pneumoniae infections, such as nosocomial pneumonia, nosocomial bloodstream infections and nosocomial urinary tract infections; community-acquired K. pneumoniae infections; and all pneumonias, bronchitis, meningitis, urinary tract infections, intra-abdominal infections, wound infections, blood infections, osteomyelitis, bacteremia, sepsis, liver abscesses and inflammatory bowel disease (IBD) caused by K. pneumoniae infections.

[0246] A population-based vaccination strategy for individuals aged 50 years and older against K. pneumoniae infection is desirable because this population is particularly susceptible to K. pneumoniae infection, especially individuals aged 60 years and older who are at risk of exposure to K. pneumoniae and / or who are expected to have weakened immunity.

[0247] K. pneumoniae is a notorious pathogen that frequently causes nosocomial (i.e., hospital-acquired) respiratory and urinary tract infections and is the second most common cause of Gram-negative bacteremia. Drug-resistant isolates are associated with high mortality rates (over 50% in some studies) and significantly prolong hospital stays, which is particularly problematic in ICUs.

[0248] Therefore, it is desirable to prevent nosocomial (i.e., hospital-acquired) K. pneumoniae infections, especially in populations at high risk of exposure, such as patients undergoing elective surgery (e.g., joint replacement) with hospitalization exceeding 72 hours, and immunocompromised and immunosuppressed patients (e.g., solid organ transplant recipients, patients receiving chemotherapy after non-urgent solid tumor surgery). In these populations, vaccination 2 to 8 weeks before surgery, followed by an optional booster vaccination, may be indicated.

[0249] Additionally, prevention of community-acquired infections in specific target populations, such as healthcare workers or the elderly (ages 60 and older) in long-term care facilities or nursing homes, is desirable. The term "community-acquired K. pneumoniae infection" refers to K. pneumoniae infections acquired in the general community, as opposed to nosocomial (hospital-acquired) infections.

[0250] Furthermore, the immunogenic compound of the present invention or a pharmaceutically acceptable salt thereof according to any one of Embodiments 1) to 55), particularly Embodiments 52), 53), 54) and 55), may be used in the prevention and / or treatment of all pneumonias, bronchitis, meningitis, urinary tract infections, intra-abdominal infections, wound infections, blood infections, osteomyelitis, bacteremia, sepsis, liver abscesses and inflammatory bowel diseases (IBD) caused by K. pneumoniae infection.

[0251] 61) A further aspect of the present invention relates to an immunogenic compound or a pharmaceutically acceptable salt thereof according to any one of aspects 1) to 55), in particular aspects 52), 53), 54) and 55), for use in the prevention and / or treatment of K. pneumoniae infections as described in aspects 59) and 60) above, wherein the K. pneumoniae is selected from the O-serotype, which includes O2a and O2afg.

[0252] 62) For the avoidance of any doubt, the immunogenic compounds or pharmaceutically acceptable salts thereof according to any one of embodiments 1) to 55), in particular embodiments 52), 53), 54) and 55), as well as the pharmaceutical compositions according to embodiment 56) or 57), and the vaccines according to embodiment 58) are likewise suitable for the prevention and / or treatment of K. pneumoniae infections as described in any one of embodiments 59), 60) and 61).

[0253] 63) Preferably, the immunogenic compound or pharmaceutically acceptable salt thereof according to any one of embodiments 1) to 55), in particular embodiments 52), 53), 54) and 55), as well as the pharmaceutical composition according to embodiment 56) or 57), and the vaccine according to embodiment 58) are suitable for the prevention of K. pneumoniae infections as described in any one of embodiments 59), 60) and 61).

[0254] 64) A further aspect of the present invention relates to a method for inducing an immune response against K. pneumoniae in a human and / or animal (in particular a mammalian (including human)) host, comprising administering to said human and / or animal an effective amount of an immunogenic compound or a pharmaceutically acceptable salt thereof according to any one of aspects 1) to 55), in particular aspects 52), 53), 54) and 55). The dosage is preferably between 0.05 μg and 30 μg glycan per immunization of a human patient. The term "glycan" refers to the antigen, i.e., the oligosaccharide excluding the linker L and the spacer T. In some cases, more than one immunization may be necessary.

[0255] 65) Similarly, an embodiment of the present invention relates to a method for inducing an immune response against K. pneumoniae in a human and / or animal (in particular a mammalian (including human)) host, comprising administering to said human and / or animal an effective amount of a composition according to embodiment 56) or 57) and a vaccine according to embodiment 58).

[0256] 66) For the avoidance of any doubt, where an immunogenic compound or a pharmaceutically acceptable salt thereof according to any one of embodiments 1) to 55), in particular embodiments 52), 53), 54) and 55), is stated to be useful for the prevention and / or treatment of K. pneumoniae infections according to any one of embodiments 59), 60) and 61), such immunogenic compound is likewise suitable for use in the manufacture of a medicament for the prevention and / or treatment of said K. pneumoniae infections according to any one of embodiments 59), 60) and 61).

[0257] 67) A further aspect of the present invention relates to a multivalent vaccine comprising an immunogenic compound according to any one of embodiments 1) to 55), preferably an immunogenic compound according to embodiments 52), 53), 54) and 55), or a pharmaceutically acceptable salt thereof.

[0258] In this regard, the term "multivalent vaccine" refers to a vaccine that comprises antigens against two or more different K. pneumoniae strains, in particular two or more pathogenic K. pneumoniae strains.

[0259] 68) A further aspect of the present invention is an intermediate compound for producing an immunogenic compound according to any one of embodiments 28) to 55), the intermediate compound having formula (III):

[0260] [ka]

[0261] (In the formula, R is OH or

[0262] [ka]

[0263] and; m is 3, 4, 5, 6, 7 or 8, preferably 3, 4, 5 or 6; n is 1, 2, 3, 4, 5 or 6, preferably 2, 3 or 4; L 1 teeth, * -(C 2-10 ) alkylene-NH2; * -(CH2CH2O) b -CH2CH2NH2 (b is 1, 2 or 3); * -CH2CH2S-CH2CH2NH2; * -(C 2-10 ) Fluoroalkylene-NH2; * -(CH2) c NHC(O)(CH2) c’ -NH2 (c and c' are, independently, 2 to 6); * -(CH2) dNHC(O)NH(CH2) d’ -NH2 (d and d' are independently 2 to 6); * -(C 1-10 ) alkylene-C(O)-NH-(C 2-10 ) alkylene-NH2; * -(C 2-10 ) alkylene-O-NH2; or * -(C 2-10 ) alkylene-SH; represents. ); or a pharmaceutically acceptable salt thereof.

[0264] 69) A further aspect is m is 4, n is 2, and R is OH; m is 4 and n is 2,

[0265] [ka]

[0266] or m is 4, n is 3, and R is OH; The intermediate compound according to embodiment 68) or a pharmaceutically acceptable salt thereof.

[0267] 70) A further aspect is L 1 but, * -(CH2) a -NH2; (a is 2 to 10); * -(CH2CH2O) b -CH2CH2NH2 (b is 1, 2 or 3); * -CH2CH2S-CH2CH2NH2; * -(C 2-10 ) Fluoroalkylene-NH2 (Fluoroalkylene is a saturated straight chain); * -(CH2) c NHC(O)(CH2) c’ -NH2 (c and c' are, independently, 2 to 6); * -(CH2) d NHC(O)NH(CH2) d’ -NH2 (d and d' are independently 2 to 6); * -(CH2) e -C(O)-NH-(CH2) e’ -NH2; (e is 1 to 10, and e' is 2 to 10); * -(CH2) f -O-NH2 (f is 2 to 10); or * -(CH2) g -SH (g is 2 to 10); or a pharmaceutically acceptable salt thereof according to embodiment 68) or 69), which represents

[0268] Similarly, embodiments 30) to 37) include further preferred L having a terminal amino- or SH-group as shown in embodiment 68). 1 shall be disclosed.

[0269] 71) A further aspect is L 1 but, * -(CH2)2-NH2, * -(CH2)3-NH2, * -(CH2)4-NH2, * -(CH2)5-NH2 or * -(CH2)6-NH2; preferably * -(CH2)5-NH2; The intermediate compound according to embodiment 68) or 69) or a pharmaceutically acceptable salt thereof.

[0270] 72) A further aspect is m is 4, n is 2, and R is OH; L 1 but, * -(CH2)2-NH2, * -(CH2)3-NH2, * -(CH2)4-NH2, * -(CH2)5-NH2 or * -(CH2)6-NH2; preferably * -(CH2)5-NH2; The intermediate compound according to embodiment 69) or a pharmaceutically acceptable salt thereof.

[0271] 73) A further aspect of the present invention is an intermediate compound for producing an immunogenic compound according to any one of embodiments 28) to 55), the intermediate compound having formula (IV):

[0272] [ka]

[0273] (In the formula, R is OH or

[0274] [ka]

[0275] and; m is 3, 4, 5, 6, 7 or 8, preferably 3, 4, 5 or 6; n is 1, 2, 3, 4, 5 or 6, preferably 2, 3 or 4; L is * -(C 2-10 ) alkylene-NH-; * -(CH2CH2O) b -CH2CH2NH- (b is 1, 2 or 3); * -CH2CH2S-CH2CH2NH-; * -(C 2-10 ) fluoroalkylene-NH-; * -(CH2)c NHC(O)(CH2) c’ -NH- (c and c' are independently 2 to 6); * -(CH2) d NHC(O)NH(CH2) d’ -NH- (d and d' are independently 2 to 6); * -(C 1-10 ) alkylene-C(O)-NH-(C 2-10 ) alkylene-NH-; or * -(C 2-10 ) alkylene-O-NH-; represents; T 1 teeth, -C(O)-(C 0-10 ) alkylene-C(O)X; -C(O)-CH2CH2-(OCH2CH2) j -C(O)X (j is 1 to 5); -C(O)-CH2(CH2) k -(SCH2(CH2) k’ ) k’’ -C(O)X (k is 0 or 1, k' is 0 or 1, and k'' is 1, 2, or 3);

[0276] [ka]

[0277] represents; -C(O)X represents -C(O)OH or an activated ester; Y represents Me, Et, Bu or -(CH2CH2O)3CH3 (in particular Me, Et, n-Bu or -(CH2CH2O)3CH3); or a pharmaceutically acceptable salt thereof.

[0278] The term "activated ester" refers to the CRM 197By "carboxylic acid" is meant a functionalized carboxylic acid that has enhanced reactivity towards amines (compared to carboxylic acids) for reaction with the amino groups of lysine residues of .

[0279] 74) A further aspect is m is 4, n is 2, and R is OH; m is 4, n is 2, and R is

[0280] [ka]

[0281] or m is 4, n is 3, and R is OH; The intermediate compound according to embodiment 73) or a pharmaceutically acceptable salt thereof.

[0282] 75) A further aspect is L, * -(CH2) a -NH-; (a is 2 to 10); * -(CH2CH2O) b -CH2CH2NH- (b is 1, 2 or 3); * -CH2CH2S-CH2CH2NH-; * -(C 2-10 ) fluoroalkylene-NH- (fluoroalkylene is a saturated linear chain); * -(CH2) c NHC(O)(CH2) c’ -NH- (c and c' are independently 2 to 6); * -(CH2) d NHC(O)NH(CH2) d’ -NH- (d and d' are independently 2 to 6); * -(CH2) e -C(O)-NH-(CH2)e’ -NH-; (e is 1 to 10, and e' is 2 to 10); or * -(CH2) f -O-NH- (f is 2 to 10); represents; T 1 but, -C(O)-(CH2) h -C(O)X (h is 0-10); -C(O)-CH2CH2-(OCH2CH2) j -C(O)X (j is 1 to 5); -C(O)-CH2(CH2) k -(SCH2(CH2) k’ ) k’’ -C(O)X (k is 0 or 1, k' is 0 or 1, and k'' is 1, 2, or 3);

[0283] [ka]

[0284] represents; -C(O)X represents -C(O)OH or an activated ester; Y represents Me, Et, Bu or -(CH2CH2O)3CH3 (in particular Me, Et, n-Bu or -(CH2CH2O)3CH3); The intermediate compound according to embodiment 73) or 74) or a pharmaceutically acceptable salt thereof.

[0285] 76) A further aspect is L, * -(CH2)2-NH-, * -(CH2)3-NH-, * -(CH2)4-NH-, * -(CH2)5-NH- or * -(CH2)6-NH-; preferably * -(CH2)5-NH-; represents; T 1 but, -C(O)-C(O)X, -C(O)-CH2-C(O)X, -C(O)-(CH2)2-C(O)X, -C(O)-(CH2)3-C(O)X, -C(O)-(C H2)4-C(O)X, -C(O)-(CH2)5-C(O)X or -C(O)-(CH2)6-C(O)X; preferably -C(O)-(CH2)4-C(O)X; represents; -C(O)X represents -C(O)OH or an activated ester; The intermediate compound according to embodiment 73) or 74) or a pharmaceutically acceptable salt thereof.

[0286] 77) A further aspect is m is 4, n is 2, and R is OH; L, * -(CH2)2-NH-, * -(CH2)3-NH-, * -(CH2)4-NH-, * -(CH2)5-NH- or * -(CH2)6-NH-; preferably * -(CH2)5-NH-; represents; T 1 but, -C(O)-C(O)X, -C(O)-CH2-C(O)X, -C(O)-(CH2)2-C(O)X, -C(O)-(CH2)3-C(O)X, -C(O)-(C H2)4-C(O)X, -C(O)-(CH2)5-C(O)X or -C(O)-(CH2)6-C(O)X; preferably -C(O)-(CH2)4-C(O)X; represents; -C(O)X represents -C(O)OH or an activated ester; The intermediate compound according to embodiment 76) or a pharmaceutically acceptable salt thereof.

[0287] In embodiments 73), 74), 75), 76) and 77), preferably X is

[0288] [ka]

[0289] Represents.

[0290] Embodiments 30) to 37) are intended to disclose further preferred L's, which are encompassed within this embodiment. Similarly, embodiments 39) to 44) are intended to disclose further preferred T's, which are T's. 1 In the above embodiment, the terminal "C(O)-" is replaced with "C(O)X" and, in the case of squaric acid, a carrier protein, e.g., CRMP 197 The attachment point to 1 is H. These preferred T 1 is deemed to be expressly disclosed.

[0291] 78) A further aspect of the present invention relates to an immunoassay having an oligosaccharide hybrid antigen of formula (I):

[0292] [ka]

[0293] (In the formula, R is OH or

[0294] [ka]

[0295] and; m is 3, 4, 5, 6, 7 or 8; n is 1, 2, 3, 4, 5 or 6.

[0296] In this embodiment, the dotted line " ** " refers to the point of attachment to the array surface (preferably via a linker and / or spacer). The oligosaccharide hybrid antigen of formula (I) can be attached to any suitable carrier surface as an array or microarray, with or without the presence of a linker and / or spacer.

[0297] 79) A preferred embodiment of the invention relates to an immunoassay according to embodiment 78), which comprises a compound of formula (Ia):

[0298] [ka]

[0299] (In the formula, R is OH or

[0300] [ka]

[0301] and; m is 3, 4, 5, 6, 7 or 8, preferably 3, 4, 5 or 6; n is 1, 2, 3, 4, 5 or 6, preferably 2, 3 or 4; i is at least 1, preferably a number from 1 to a maximum of 90% of the number of lysine residues contained in the carrier protein CP; -LT- represents a linker L and a spacer T as disclosed in any one of embodiments 17) or 23) to 47); CP is a carrier protein suitable for immunological assays, in particular ELISA. Preferred CPs are described in embodiments 8) and 9). A particularly preferred carrier protein is BSA.

[0302] The synthesis of the compound of the antigen of formula (Ia) conjugated to BSA is described and exemplified in the experimental section, and it should be understood that this synthesis applies equally to all antigens of formula (Ia) and therefore can be prepared by one skilled in the art.

[0303] The test (assay) of this embodiment is suitable for detecting antibodies against K. pneumoniae O2a and O2afg strains.

[0304] 80) A further aspect of the present invention is a method for producing an immunogenic compound comprising administering to a subject a compound of formula (IV)197 By conjugating to the lysine residue of:

[0305] [ka]

[0306] (In the formula, R is OH or

[0307] [ka]

[0308] and; m is 3, 4, 5, 6, 7 or 8, preferably 3, 4, 5 or 6; n is 1, 2, 3, 4, 5 or 6, preferably 2, 3 or 4; L is * -(C 2-10 ) alkylene-NH-; * -(CH2CH2O) b -CH2CH2NH- (b is 1, 2 or 3); * -CH2CH2S-CH2CH2NH-; * -(C 2-10 ) fluoroalkylene-NH-; * -(CH2) c NHC(O)(CH2) c’ -NH- (c and c' are independently 2 to 6); * -(CH2) d NHC(O)NH(CH2) d’ -NH- (d and d' are independently 2 to 6); * -(C 1-10 ) alkylene-C(O)-NH-(C 2-10 ) alkylene-NH-; or * -(C 2-10) alkylene-O-NH-; represents; a) T 1 teeth, -C(O)-(C 0-10 ) alkylene-C(O)X; -C(O)-CH2CH2-(OCH2CH2) j -C(O)X (j is 1 to 5); -C(O)-CH2(CH2) k -(SCH2(CH2) k’ ) k’’ -C(O)X (k is 0 or 1, k' is 0 or 1, and k'' is 1, 2, or 3);

[0309] [ka]

[0310] represents; -C(O)X represents -C(O)OH or an activated ester; Y represents Me, Et, Bu or -(CH2CH2O)3CH3; Or, b) a compound of formula (IV), 1 but,

[0311] [ka]

[0312] represents; or LT 1 but, * -(C 2-10 ) alkylene-SH; a compound of formula (IV) representing 197 By combining with;

[0313] [ka]

[0314] The present invention relates to an immunogenic compound or a pharmaceutically acceptable salt thereof according to any one of aspects 28) to 55), which can be obtained or produced.

[0315] Preferably, X is

[0316] [ka]

[0317] Represents.

[0318] Embodiments 29) to 37) disclose further preferred L's, and embodiments 73) and 75) to 77) disclose further preferred T's. 1 All of these are included in this embodiment. Furthermore, similarly, embodiments 39) to 44) disclose further preferred T, which are T 1 In the above embodiment, the terminal "C(O)-" is replaced with "C(O)X" and, in the case of squaric acid, a carrier protein, e.g., CRMP 197 The attachment point to 1 is H. These preferred T 1 is deemed to be expressly disclosed.

[0319] 81) A further aspect of the present invention relates to a method for producing an immunogenic compound or a pharmaceutically acceptable salt thereof according to any one of aspects 28) to 55), said method comprising the steps of: The compound of formula (IV) is 197 to a lysine residue of:

[0320] [ka]

[0321] (In the formula, R is OH or

[0322] [ka]

[0323] and; m is 3, 4, 5, 6, 7 or 8, preferably 3, 4, 5 or 6; n is 1, 2, 3, 4, 5 or 6, preferably 2, 3 or 4; L is * -(C 2-10 ) alkylene-NH-; * -(CH2CH2O) b -CH2CH2NH- (b is 1, 2 or 3); * -CH2CH2S-CH2CH2NH-; * -(C 2-10 ) fluoroalkylene-NH-; * -(CH2) c NHC(O)(CH2) c’ -NH- (c and c' are independently 2 to 6); * -(CH2) d NHC(O)NH(CH2) d’ -NH- (d and d' are independently 2 to 6); * -(C 1-10 ) alkylene-C(O)-NH-(C 2-10 ) alkylene-NH-; or * -(C 2-10 ) alkylene-O-NH-; represents; a) T 1 teeth, -C(O)-(C 0-10 ) alkylene-C(O)X; -C(O)-CH2CH2-(OCH2CH2) j -C(O)X (j is 1 to 5); -C(O)-CH2(CH2) k -(SCH2(CH2) k’ ) k’’-C(O)X (k is 0 or 1, k' is 0 or 1, and k'' is 1, 2, or 3);

[0324] [ka]

[0325] represents; -C(O)X represents an activated ester; Y represents Me, Et, Bu or -(CH2CH2O)3CH3; or b) a compound of formula (IV), 1 but,

[0326] [ka]

[0327] represents; or LT 1 but, * -(C 2-10 ) alkylene-SH; a compound of formula (IV) representing 197 and combining the

[0328] [ka]

[0329] Preferably, X is

[0330] [ka]

[0331] Represents.

[0332] Embodiments 29) to 37) disclose further preferred L's, and embodiments 73) and 75) to 77) disclose further preferred T's. 1All of these are included in this embodiment. Furthermore, similarly, embodiments 39) to 44) disclose further preferred T, which are T 1 In the above embodiment, the terminal "C(O)-" is replaced with "C(O)X" and, in the case of squaric acid, a carrier protein, e.g., CRMP 197 The attachment point to 1 is H. These preferred T 1 is deemed to be expressly disclosed.

[0333] Whenever the words "between" or "to" are used to describe a range of numerical values, the endpoints of the stated range are expressly disclosed and are intended to be included within that range. This means, for example, that when a temperature range is stated to be between 40°C and 80°C (or 40°C to 80°C), the endpoints of 40°C and 80°C are meant to be included within the range; or, when a variable is defined as an integer between 1 and 4 (or 1 to 4), it means that the variable is the integer 1, 2, 3, or 4.

[0334] However, for the avoidance of any doubt, the term "covalently linked bridge having a backbone length of 5 to 25 atoms, said length being such that the oxygen at C1 of the reducing end of said oligosaccharide is bonded to said carrier protein CRM" is also used. 197 The definition of "the shortest distance between the nitrogen of the amino group of the lysine residue and the oxygen in C1 and CRM" is 197 This means that the nitrogen of the amino group of lysine is not counted in the backbone numbering thus defined.

[0335] When not used in reference to temperature, the term "about" (or "in the vicinity of") before a numerical value "X" refers in this application to a temperature between 10% of XX and 10% of X+X, preferably between 5% of XX and 5% of X+X. In the specific case of temperatures, the term "about" (or "in the vicinity of") before a temperature "Y" refers in this application to a temperature between Y-10°C and Y+10°C, preferably between Y-5°C and Y+5°C. Furthermore, the term "room temperature" as used herein means a temperature of about 25°C.

[0336] Preparation of Compounds of Formulae (I), (Ia), (II), (IIa), (IIb), (IIc), (III) and (IV) A further aspect of the present invention is a method for preparing compounds of formula (I), (Ia), (II), (IIa), (IIb), (IIc), (III) and (IV). Compounds according to formula (I), (Ia), (II), (IIa), (IIb), (IIc), (III) and (IV) of the present invention can be prepared from commercially available or known starting materials according to the methods described in the experimental section; by analogous methods; or according to the general reaction sequence outlined below, in which L, T, L 1 , T 1 , X and Y are as defined for formulas (I), (Ia), (II), (IIa), (IIb), (IIc), (III) and (IV). Other abbreviations used herein are either explicitly defined or as defined in the experimental section.

[0337] The synthesis of the compounds of the present invention requires a protecting group strategy. Such protecting group strategies can be quite sophisticated, but the use of protecting groups is well known in the art (see, for example, "Protective Groups in Organic Synthesis," T.W. Greene, P.G.M. Buts, Wiley-Interscience, 1999). The resulting compounds may be converted into salts, particularly pharmaceutically acceptable salts thereof, by methods known per se.

[0338] Common manufacturing routes: Antigen display

[0339] [ka]

[0340] Scheme 1: AG-CRM using the NHS-ester method 197 Synthesis of the conjugate

[0341] [ka]

[0342] [ka]

[0343] Antigen AG-L in a suitable solvent (e.g., DMSO) 1 1' is treated in a vial at rt with activated bis-NHS ester 2' of a diacid (e.g., bis-NHS adipic acid ester, which is commercially available or can be prepared by one skilled in the art using the corresponding bis-acid and N-hydroxysuccinic acid) (Odom, OW, Biochemistry, Vol. 29, No. 48, 1990) (5-20 equivalents) in DMSO in the presence of triethylamine and stirred at rt for 3 h. The antigen-NHS ester 3' is precipitated by adding EtOAc and centrifuged. The precipitate is then washed with EtOAc and dried under vacuum before use in the next step. Antigen-NHS ester 3' (25-100 equivalents) and CRM 197 The resulting antigen-CRM solution is stirred at room temperature for 20-24 hours. 197 The conjugate 4' is washed, purified and stored in an appropriate buffer solution.

[0344] In the above synthesis route, CRM 197 may be replaced by any one of the carrier proteins according to aspects 8) or 9).

[0345] Scheme 2: AG-CRM using the squaric acid ester (squarate) method197 Synthesis of the conjugate

[0346] [ka]

[0347] Antigen AG-L in a suitable solvent (e.g., HO-EtOH, buffer) 1 1' is treated with the desired alkyl squarate 5' (e.g., 3,4-dibutoxy-3-cyclobutene-1,2-dione, 3,4-(di(2-(2-(2-methoxyethoxy)ethoxy)ethoxy)-3-cyclobutene-1,2-dione) (Ganesh et al., JACS, 2014, 136, 16260-16269 and Xu et al., Carbhydr. Res, 2018, 456, 24-29) in a vial at rt and stirred at rt and an appropriate pH (7-8). The reaction mixture is neutralized with acetic acid and then concentrated under vacuum (or lyophilized). The crude product is purified using a C18 (or SEC) column with water-acetonitrile as the eluent. The product-containing fractions are frozen and lyophilized to give 6'. The antigen-squarate ester 6' (25-100 equivalents) and CRMP are then mixed. 197 The resulting antigen-CRM was stirred in a 0.5 M borate buffer solution at pH 9 at room temperature for 24 to 72 hours (S. Hou et al., Carbhydr. Res., 2008, 343, 196-210). 197 The conjugate 7' is washed, purified and stored in an appropriate buffer solution.

[0348] In the above synthesis route, CRM 197 may be replaced by any one of the carrier proteins according to aspects 8) or 9).

[0349] Scheme 3: Synthesis of antigen-thiol

[0350] [ka]

[0351] Antigen AG-L in a suitable solvent (e.g., DMSO) 1 1' is treated with 8' (e.g., DSP (dithiobis(succinimidyl propionate)) or DTSSP (3,3'-dithiobis(sulfosuccinimidyl propionate)) in a vial at rt to give the corresponding disulfide, which is then reduced with DTT (dithiothreitol) or TCEP (tris(2-carboxyethyl)phosphine) to give the antigen-thiol 9'. Scheme 4: Antigen-thiol and functionalized CRM 197 AG-CRM using 197 Synthesis of the conjugate

[0352] [ka]

[0353] a) AG-CRM using the antigen-thiol-maleimide method 197 Synthesis of the conjugate Antigen-thiol 9' (25-100 equivalents) and maleimide-functionalized CRM 197 10' (e.g., CRM 197 (This can be prepared by those skilled in the art by treating with 3-maleimido-propionic acid succinimidyl ester or any other suitable NHS ester with maleimide) (Robert M.F. van der Put et al., ACS Cent. Sci. 2022, 8, 4, 449-460) in a buffer solution containing RM is stirred at room temperature for 20-24 hours. L-cysteine ​​in the buffer is then added to the RM and stirred at room temperature for 1 hour to quench the excess maleimide groups. The resulting antigen-CRM 197 The -thio-maleimide conjugate 11' is washed, purified and stored in an appropriate buffer solution.

[0354] In the above synthesis route, CRM 197 may be replaced by any one of the carrier proteins according to aspects 8) or 9).

[0355] b) AG-CRM using the antigen-thiol-ether method 197 Synthesis of the conjugate Antigen - Protein functionalized with thiol 9' (25-100 equivalents) and α-bromoacetate ester 10' (e.g., CRM 197 -BAP, CRM 197 A buffer solution containing RM and SBAP (N-succinimidyl 3-(2-bromoacetamido)propanoate) or any other suitable NHS ester with α-bromoacetate (Schumann, B. et al., Chem. Sci., 2014, 5, 1992-2002) is stirred at room temperature for 24 hours. L-cysteine ​​in the buffer is then added to the RM and stirred at room temperature for 1 hour to quench the excess α-bromoacetate groups. The resulting antigen-CRM 197 The -thio-ether conjugate 11' is washed, purified and stored in an appropriate buffer solution.

[0356] In the above synthesis route, CRM 197 may be replaced by any one of the carrier proteins according to aspects 8) or 9).

[0357] A general retrosynthetic approach to AG-hybrid-antigen RS-1 Antigen RS-1 can be synthesized using functionalized building blocks as shown in Scheme 5. Fully deprotected antigen RS-1 has a linker L1 at its reducing end, which is essential for conjugation to a protein carrier. Linker L1 is as disclosed in embodiments 28) and 29) to 47). RS-1 can be obtained by deprotection of fully protected RS-2. Deprotection strategies may include (acidic or basic) hydrolysis, hydrogenolysis, Birch reduction, or removal of esters, amides, imides, or carbamates via reduction of the azide group to an amine. The deprotection sequence varies depending on the protecting groups and their compatibility with the reaction conditions. Those skilled in the art can successfully accomplish this. RS-2 can be obtained from glycosylation of RS-3 as the donor and RS-4 as the acceptor. RS-4 can be obtained from intermediate RS-5 and RS-6 equipped with an appropriate linker handle (Lx). The RS-9 donor can be treated with a linker handle selected from the various linker handles listed in Table A to give RS-6. RS-5 was synthesized using RS-7 and RS-8, and RS-9 can also be obtained from repeating unit RS-8. The common intermediate RS-8 can be obtained from monosaccharide building blocks RS-10 and RS-11.

[0358] Scheme 5: Retrosynthetic approach to antigen RS-1

[0359] [ka]

[0360] [ka]

[0361] Scheme 6: Introduction of the linker handle Lx

[0362] [ka]

[0363] The linker nucleophile Ln (e.g., 5-azidopentan-1-ol) and RS-9 donor are taken up in a RBF and azeotropically dried under vacuum with dry toluene. The mixture is taken up in an appropriate solvent (e.g., DCM) at room temperature, 4 Å molecular sieves are added, and the mixture is stirred for 30-45 min under a N2 atmosphere. The RM is cooled to an appropriate temperature (e.g., 0 °C to -20 °C), and an activating agent (e.g., TMSOTf, TfOH) is added to the RM. The RM is stirred for 20 min. The RM is then allowed to warm slowly to room temperature over 1 h. The reaction completion is monitored by TLC. The RM is quenched (e.g., with saturated NaHCO3, Na2SO3 solution) and extracted with a solvent (e.g., DCM, EtOAc). The combined organic portions are washed with water, brine, dried, and evaporated under vacuum to give the crude product. The crude product is purified by silica column chromatography using EA / cyclohexane as the eluent. The fractions containing the product are evaporated and dried under vacuum to give the product RS-6.

[0364] [Table 1] [Example]

[0365] Experimental section: Abbreviations (used in this section and above): AcOH acetic acid aq. aqueous solution Bn Benzyl BSA Bovine serum albumin CDCl3 deuterated chloroform Cs2CO3 Cesium Carbonate Cy Cyclohexane D2O deuterium oxide DCM dichloromethane DDQ 2,3-dichloro-5,6-dicyano-1,4-benzoquinone DMAP 4-(dimethylamino)pyridine DMF N,N-dimethylformamide DMSO dimethyl sulfoxide ELISA enzyme-linked immunosorbent assay equiv equivalent ESI Electrospray Ionization Et3N(TEA) Triethylamine EtOAc(EA) ethyl acetate EtOH ethanol EtSH Ethanethiol Fr fraction h time H2 Hydrogen H2O Water H2SO4 Sulfuric Acid HCl Hydrochloric acid HPLC High Performance Liquid Chromatography HPLC-SEC High Performance Liquid Chromatography-Size Exclusion Chromatography I2 Iodine ICU intensive care unit IPA Isopropanol LPS lipopolysaccharide M molar concentration MeOH Methanol Min MS Molecular Sieves N2 nitrogen Na sodium Na2S2O3 Sodium thiosulfate Na2SO4 Sodium Sulfate NaCl Sodium chloride NaHCO3 Sodium Bicarbonate NaOMe Sodium methoxide NaPi buffer sodium phosphate buffer NH2NH2 Hydrazine NIS N-iodosuccinimide NMR nuclear magnetic resonance spectroscopy PBS Phosphate-buffered saline PBS-T: Phosphate-buffered saline containing 0.1% (v / v) Tween-20 Pd(OH)2 palladium hydroxide Pd / C Palladium Carbon py pyridine RBF Round Bottom Flask RM reaction mixture rt room temperature sat. saturation SDS-PAGE sodium dodecyl sulfate-polyacrylamide gel electrophoresis SM starting material sol. solution TBAF Tetrabutylammonium Fluoride TBS Tris-buffered saline TDS Dimethyl-Thexylsilylchlorid TLC thin layer chromatography TMB 3,3',5,5'-tetramethylbenzidine TMSOTf Trimethylsilyl trifluoromethanesulfonate UV ultraviolet light I. Chemistry The following examples illustrate the preparation of biologically active compounds of the present invention but are not intended to limit its scope in any way.

[0366] General Information : All reagents and solvents were used as purchased, and solvents used in reactions were anhydrous. Except for reactions involving water as a solvent, all reactions were performed under a N atmosphere in dry glassware (purchased from VWR and ROTH). It is highly recommended to azeotropically dry the acceptor and donor twice with anhydrous toluene before glycosylation. A Heidolph magnetic stirrer was used to perform the experiments. Thin-layer chromatography (TLC) was performed on silica gel 60 F254 glass plates (Merck) or aluminum plates (VWR). Developed TLC plates were visualized under a short-wave UV lamp by heating the plates immersed in a sugar stain (3-methoxyphenol (0.225 mL), H2SO4 (6 mL), and EtOH (200 mL)). All automated flash chromatography purifications on silica gel (FlashPure Silica 40 μm irregular: BUCHI columns) were performed on Biotage Isolera and Biotage Select. A BUCHI rotary evaporator was used to evaporate solvents. Combinations of dry ice and acetone and ice / water were used to cool the reaction mixture to the desired temperature. All NMR experiments were performed on a BRUKER 400 MHz instrument.

[0367] Temperatures are given in degrees Celsius (°C). In mixtures, the proportions of solvents or eluents or mixtures of reagents in liquid form are given as volumetric relationships (v / v) unless otherwise specified.

[0368] Characterization methods used: HPLC-SEC: The glycoconjugates used for immunization were analyzed by HPLC-SEC to determine the bound and unbound CRM. 197 Mass differences between proteins were observed. Samples were diluted with 50 mM Tris, 20 mM NaCl, pH 7.2, and run on an Agilent 1100 HPLC system equipped with a Tosoh TSK G2000 column (SWx1, 7.8 mm x 30 cm, 5 μm) and a Tosoh TSK Gel Guard column (SWx1 6.0 mm x 4 cm, 7 μm). The flow rate was maintained at 1 mL / min.

[0369] SDS-PAGE: Samples were diluted in Laemmli loading buffer and heated to 95°C for 5 min. After cooling to RT for 5 min, approximately 2-2.5 μg of sample was loaded into the wells of a 10% polyacrylamide gel along with approximately 5 μL of protein size marker. Samples were run at a constant voltage of 120 V for approximately 30-45 min. Staining was performed using GelCode™ Blue Safe Protein Stain according to the manufacturer's instructions. Gels were washed overnight with deionized water and scanned.

[0370] Synthesis of disaccharide diol D1:

[0371] [ka]

[0372] To a solution of disaccharide A3 (see WO2019106201, p. 164), 68 g, 61 mmol) in HPLC-grade DCM (305 mL) was added EtSH (27.1 mL, 366 mmol) and TsOH·HO (3.15 g, 18.29 mmol). After stirring at room temperature for 40 min, TLC analysis (EtOAC / hexane, 1 / 1) showed the disappearance of the starting material and the presence of a new spot. The reaction mixture was then quenched with triethylamine (2.55 mL, 18.29 mmol) and concentrated under reduced pressure. The residue was purified by flash silica gel column chromatography (gradient DCM / MeOH, 0-10%). The product-containing fractions were concentrated under vacuum and dried under high vacuum to give D1 as a colorless oil (55 g, 88%). HRMS C 59 H 70 NO 14 Si + [M+NH4] + The calculated value was 1044.4560 and the measured value was 1044.460.

[0373] Synthesis of disaccharide acceptor D2:

[0374] [ka]

[0375] Benzoic anhydride (12.51 g, 55.3 mmol) and triethylamine (38.9 g, 53.6 mL) were added to a solution of diol D1 (49.4 g, 48.1 mmol) in anhydrous DCM (385 mL), and the reaction was stirred overnight at room temperature. The reaction mixture was transferred to a separatory funnel and washed with saturated aqueous NaHCO3 (150 mL). The layers were separated, and the aqueous layer was extracted with DCM (150 mL). The organic layers were combined, dried over Na2SO4, and the solvent was concentrated on a rotavapor. The residue was purified using an automated purification system (Cy / EtOAc, gradient 0-100%). The product-containing test tubes were combined, and the solvent was evaporated to give product D2 as a white foam (48.5 g, 89%). HRMS C 66 H 74 NO 15 Si + [M+NH4] + The calculated value was 1148.4822 and the measured value was 1148.487.

[0376] Synthesis of protected trisaccharide D3:

[0377] [ka]

[0378] To a solution of phenyl 4,6-di-O-benzoyl-2,3-di-O-benzyl-1-thio-β-D-galactopyranoside donor (25.5 g, 38.6 mmol) and acceptor D2 (32 g, 28.3 mmol) in toluene:dioxane (3:1, 515 mL) was added freshly activated 4 Å MS, and the mixture was stirred at room temperature for 45 min. NIS (10.18 g, 45.3 mmol) was then added, and the reaction mixture was cooled to 0 °C. TMSTOf (0.51 mL, 2.83 mmol) was added, and the reaction mixture was stirred at 0 °C for 1.5 h. The reaction was filtered, quenched with NaHCO3 (150 mL), diluted with ethyl acetate (150 mL), and extracted with 0.1 M aqueous Na2S2O3, saturated aqueous NaHCO3, and brine. The organic layer was dried over Na2SO4, and the solvent was concentrated on a rotavapor. Purification on an automated purification system (Cy / EtOAc, gradient 0-100%) gave the product D3 as a white foam (43.8 g, 92%) after evaporation of the solvent. HRMS C 100 H 104 NO 22 Si + [M+NH4] + The calculated value was 1698.6814 and the measured value was 1699.695.

[0379] Synthesis of trisaccharide alcohol D4:

[0380] [ka]

[0381] To a solution of NAP-protected trisaccharide D3 (60 g, 35.7 mmol) in DCM:MeOH (9:1, 350 mL) in a 500 mL RBF was added DDQ (10.12 g, 44.6 mmol) at 0 °C. The reaction mixture was warmed to room temperature and stirred for 2.5 h. The reaction was monitored by TLC (EtOAc:Cy, 3:1). The reaction was diluted with DCM (100 mL) and quenched with saturated aqueous NaHCO3 (100 mL). The organic layer was washed with NaHCO3 (2 x 100 mL) and brine (100 mL). The organic layer was dried over Na2SO4, filtered, and the filtrate was concentrated in vacuo to give the crude product. The crude product was purified by automated flash column chromatography (Cy / EtOAc, gradient 0-100%). Concentration of the solvent gave the product D4 as a white foam (43.5 g, 79%). HRMS C 89 H 96 NO 22 Si + [M+NH4] + The calculated value was 1558.6188 and the measured value was 1559.633.

[0382] Synthesis of Thexyl dimethylsilyl 4,6-di-O-benzoyl-2,3-di-O-benzyl-α-D-galactopyranosyl-(1→4)-6-O-benzoyl-2-O-benzyl-3-O-levulinoyl-α-D-galactopyranosyl-(1→3)-2,5,6-tri-O-benzoyl-β-D-galactofuranoside D5:

[0383] [ka]

[0384] The trisaccharide starting material D4 (30 g, 19.46 mmol) was dissolved in anhydrous DCM (195 mL) and LevOH (9.04 g, 78 mmol), EDCI (14.92 g, 78 mmol), and DMAP (3.82 g, 68.1 mmol) were added sequentially. The reaction mixture was stirred at room temperature and monitored by TLC. After 72 h, the mixture was partitioned between DCM and brine. The organic layer was dried over NaSO, filtered, and evaporated to give the crude product. The crude material was loaded onto isolute and purified using an automated purification system with Cy / EtOAc (0–100%) to give the product D5 as a white foam (28.4 g, 89%). HRMS C 94 H 102 NO 24 Si + [M+NH4] +The calculated value is 1656.6556 and the measured value is 1657.664. 1 H NMR (400MHz, CDCl3) δ 7.92-7.86(m, 4H), 7.86-7.76(m, 6H), 7.67-7.62(m, 2H), 7.45-7.14(m, 20H), 7.11-6.90(m, 13H), 5.80(d, J=2.0Hz, 1H), 5.74-5.67(m, 1H), 5.33(s, 1H), 5.21(dd, J=10.8, 2.6Hz, 1H), 5.18(s, 1H), 5.12(d, J=3.7Hz, 1H), 4.78-4.71(m, 2H), 4.57-4.29(m, 13H), 4.14(d, J=2 .4Hz, 1H), 4.10 (d, J=5.5Hz, 1H), 4.07-4.00 (m, 1H), 3.99-3.94 (m, 1H), 3.82 (dd, J=10.7, 3.5Hz, 1H), 3.74 (dd, J=10.1, 3.2Hz, 1H), 2.6 3-2.53 (m, 2H), 2.53-2.39 (m, 2H), 2.00 (s, 3H), 1.51-1.45 (m, 1H), 0.71 (dd, J=6.8, 0.9Hz, 6H), 0.68 (s, 6H), 0.03 (s, 3H), 0.00 (s, 3H). 13 C NMR (101MHz, CDCl3) δ 206.3, 172.5, 166.3, 166.1, 166.1, 165.8, 165.8, 165.3, 138.2, 138.1, 138.0, 133.3, 133.2, 133.1, 133.0, 130.1, 1 30.0, 129.9, 129.9, 129.8, 129.7, 129.6, 129.4, 128.8, 128.6, 128.5, 128.5, 128.5, 128.5, 128.4, 128.4, 128.4, 128 .3, 128.1, 128.0, 127.9, 127.7, 127.5, 101.1, 101.0, 99.7, 85.1, 83.5, 81.8, 77.4, 76.5, 74.5, 73.8, 73.3, 72.8, 72.1, 71.7, 70.7, 69.4, 68.4, 67.8, 63.9, 62.7, 62.1, 38.0, 34.2, 29.9, 28.3, 24.9, 20.2, 20.0, 18.7, 18.6, -2.2, -3.3.

[0385] Synthesis of trisaccharide hemiacetal D6:

[0386] [ka]

[0387] The TDS-protected trisaccharide starting material D5 (28.4 g, 17.32 mmol) was dissolved in anhydrous DCM (139 mL). AcOH (20.5 mL, 358 mmol) was added. The solution was stirred for 5 min, and TBAF (350 mL, 1 M in THF) was added. The reaction mixture was stirred at room temperature and monitored by TLC. After 16 h, the mixture was diluted with water (50 mL) and DCM (100 mL). The reaction mixture was quenched with saturated aqueous NaHCO3 (150 mL). The organic layer was separated and washed with brine (150 mL). The organic layer was dried over Na2SO4, filtered, and evaporated to give the crude product. The crude material was loaded onto isolute and purified using an automated purification system with Cy / EtOAc (0-100%) to give the product D6 as a white foam (25 g, 96%). HRMS C 86 H 84 NO 24 + [M+NH4] + The calculated value was 1514.5378 and the measured value was 1514.542.

[0388] Synthesis of trisaccharide imidate donor D7:

[0389] [ka]

[0390] The trisaccharide hemiacetal starting material D6 (14.5 g, 9.68 mmol) was dissolved in anhydrous DCM (97 mL). CsCO (9.46 g, 29.0 mmol) and 2,2,2-trifluoro-N-phenylacetimidoyl chloride (4.02 g, 19.36 mmol) were added. The reaction mixture was stirred at room temperature and monitored by TLC. After 4.5 h, the reaction mixture was filtered through Celite. The solvent was evaporated to give the crude product. The crude material was loaded onto isolute and purified using an automated purification system with Cy / EtOAc (0-100%, containing 0.1% triethylamine) to give the product D7 as a white foam (13.5 g, 84%). HRMS C 94 H 84 F3NNaO 24 + [M+Na] + The calculated value was 1690.5228 and the measured value was 1691.536.

[0391] Synthesis of the undecasaccharide D8:

[0392] [ka]

[0393] To a solution of donor D7 (0.44 g, 0.26 mmol) and acceptor A17 (see WO2019106201, p. 248), 0.83 g, 0.22 mmol) in anhydrous DCM (9 mL) was added 4 Å MS, and the mixture was stirred for 30 min. The reaction mixture was cooled to 0 °C. TMSOTf (0.008 mL, 0.044 mmol) was added, and the reaction mixture was stirred at the same temperature for 30 min. The reaction mixture was diluted with DCM (10 mL), filtered, and quenched by the addition of saturated aqueous NaHCO3 solution (5 mL). The organic layer was separated, dried over Na2SO4, and filtered. The solvent was evaporated to give an oil residue. The crude reaction mixture was purified using an automated purification system using Cy / EtOAc (0-100%) to give product D8 (0.88 g, 78%). MALDI-TOF C 307 H 281 N3NaO 80 +[M+Na] + The calculated value was 5311.7904 and the measured value was 5315.83.

[0394] Synthesis of undecasaccharide acceptor D9:

[0395] [ka]

[0396] To a solution of the Lev-protected undecasaccharide D8 (1.95 g, 0.375 mmol) in DCM (10 mL) was added a solution of hydrazine hydrate (0.12 mL, 3.75 mmol) in AcOH (0.8 mL) and Py (1.2 mL). The resulting reaction mixture was stirred at room temperature for 2 h. The reaction was quenched by the addition of acetone (1 mL), and the solvent was removed in vacuo to give the crude product. The crude product was purified by automated flash column chromatography using Cy / EtOAc (0–100%) as the eluent. Concentration of the solvent in vacuo from the test tube containing the product D9 (based on TLC) gave a white foam (1.88 g, 98%). MALDI-TOF C 302 H 275 NNaO 78 + [M + Na - N2] + The calculated value was 5185.7475 and the measured value was 5187.08.

[0397] 5-Azido-pentyl 4,6-di-O-benzoyl-2,3-di-O-benzyl-α-D-galactopyranosyl-(1→4)-6-O-benzoyl-2-O-benzyl-3-O-levulinoyl-α-D-galactopyranosyl-(1→3)-2,5,6-tri-O-benzoyl-β-D-galactofuranosyl-(1→3)-4-[4,6-di-O-benzoyl-2,3-di-O-benzyl-α-D-galactopyranosyl-(1→)]-6-O-benzoyl-2-O-benzyl-α-D-galactopyranosyl-(1→3)-2,5,6-tri-O-benzoyl-β-D-galactofuranosyl-(1→3)-6-O-benzoyl-2,4-di-O-benzyl-α-D-galac Synthesis of topyranosyl-(1→3)-2,5,6-tri-O-benzoyl-β-D-galactofuranosyl-(1→3)-6-O-benzoyl-2,4-di-O-benzyl-α-D-galactopyranosyl-(1→3)-2,5,6-tri-O-benzoyl-β-D-galactofuranosyl-(1→3)-6-O-benzoyl-2,4-di-O-benzyl-α-D-galactopyranosyl-(1→3)-2,5,6-tri-O-benzoyl-β-D-galactofuranosyl-(1→3)]-6-O-benzoyl-2,4-di-O-benzyl-α-D-galactopyranosyl-(1→3)-2,5,6-tri-O-benzoyl-β-D-galactofuranoside D10:

[0398] [ka]

[0399] Acceptor D9 (1.45 g, 0.28 mmol) was coevaporated twice with toluene and dissolved in anhydrous toluene (8 mL). Freshly activated 4 Å MS was added and stirred for 30 min. The reaction mixture was cooled to 0 °C, and TMSOTf (5 μL, 0.028 mmol) was added dropwise. Donor D7 (0.56 g, 0.33 mmol) was coevaporated twice with toluene and dissolved in toluene (3 mL). The donor was added dropwise to the reaction mixture over 10 min. Toluene (1 mL) was added to wash the flask containing the donor, and this solution was added dropwise to the reaction mixture. The reaction mixture was slowly warmed to 10 °C for 1.5 h. TLC indicated complete consumption of the acceptor. The reaction was filtered, diluted with ethyl acetate (10 mL), and quenched by the addition of saturated aqueous NaHCO3 (10 mL). The organic layer was separated, dried over Na2SO4, and filtered. Evaporation of the solvent gave an oily residue. The crude reaction mixture was purified on an automated purification system using Cy / EtOAc (0-65%) to give product D10 (1.4 g, 75%). MALDI-TOF C 388 H 354 N3O 101 + [M+H] + The calculated value was 6670.2651 and the measured value was 6670.32. 1 H NMR (400MHz, CDCl3) δ 8.14-7.57(m, 63H), 7.51-7.38(m, 24H), 7.36-7.27(m, 30H), 7.24-7.02(m, 61H), 6.99-6.61(m, 32H), 6.05(s, 1H), 5.89-5.58(m, 12H), 5.55-5.44(m, 4H), 5.22-5. 11(m, 4H), 5.11-3.65(m, 140H), 3.62-3.53(m, 1H), 3.33-3.25(m, 1H), 3.10(t, J= 6.9Hz, 2H), 2.24-1.84(m, 4H), 1.71(s, 3H), 1.52-1.39(m, 4H), 1.33-1.23(m, 2H).

[0400] Synthesis of tetradecasaccharide D11:

[0401] [ka]

[0402] To a solution of the Lev-protected tetradecasaccharide D10 (1.3 g, 0.195 mmol) in DCM (5 mL) was added a solution of hydrazine hydrate (0.06 mL, 1.95 mmol) in acetic acid (0.4 mL) and pyridine (0.6 mL). The resulting reaction mixture was stirred at room temperature for 2 h. The reaction was quenched by the addition of acetone (1 mL), and the solvent was removed in vacuo to give the crude product. The crude product was purified by automated flash column chromatography using Cy / EtOAc (0-100%) as the eluent. Concentration of the solvent in vacuo from the test tube containing the product D11 (based on TLC) gave a white foam (1.08 g, 84%). MALDI-TOF C 383 H 347 NNaO 99 + [M + Na - N2] + The calculated value was 6566.2041 and the measured value was 6568.51.

[0403] Synthesis of partially protected tetradecasaccharide D12:

[0404] [ka]

[0405] To a solution of hexadecasaccharide D11 (275 mg, 0.042 mmol) in THF (5 mL) at rt, excess NaOMe in 0.5 M methanol (2.93 mL, 1.464 mmol) was added. The reaction mixture was warmed to 55 °C and stirred for 18 h. The solvent was then evaporated to dryness under vacuum. Water was added to the reaction mixture and neutralized with acetic acid. The aqueous layer was extracted with EtOAc (3 × 10 mL). The combined organic portions were washed with saturated NaHCO (2 × 10 mL), brine (10 mL), dried (NaSO), and evaporated under vacuum to give the crude product. SEC purification was performed on an LH-20 column using 50% CHCl / MeOH as the eluent. Fractions containing the sugar dye active spot were collected, evaporated, and dried under vacuum (125 mg). 1H NMR and MALDI-TOF analysis indicated that a small amount of benzoyl group was still present in the molecule. The substrate (125 mg, 0.031 mmol) was taken up in THF (5 mL) at rt, and a 0.5 M solution of NaOMe in methanol (1.53 mL, 0.766 mmol) was added to the resulting solution. The reaction mixture was stirred at 60 °C for 18 h. The reaction solvent was evaporated to dryness under vacuum. Water was added to the residue and neutralized with acetic acid. The aqueous layer was extracted with EtOAc (3 × 10 mL). The combined organic portions were washed with saturated NaHCO (2 × 10 mL), brine (10 mL), dried (NaSO), and evaporated under vacuum to give the crude product. SEC purification was performed on LH-20 using 30% CHCl / MeOH as the eluent. Fractions containing the sugar dye active spot were collected, evaporated, and dried under vacuum to give a pale yellow fluffy solid D12 (90 mg, 59%). MALDI-TOF C 187 H 239 N4O 71 + [M+NH4] + The calculated value was 3676.5209 and the actual value was 3677.00.

[0406] Synthesis of 5-amino-pentyl α-D-galactopyranosyl-(1→4)-α-D-galactopyranosyl-(1→3)-β-D-galactofuranosyl-(1→3)-4-[α-D-galactopyranosyl-(1→)]-α-D-galactopyranosyl-(1→3)-β-D-galactofuranosyl-(1→3)-α-D-galactopyranosyl-(1→3)-β-D-galactofuranosyl-(1→3)-α-D-galactopyranosyl-(1→3)-β-D-galactofuranosyl-(1→3)-α-D-galactopyranosyl-(1→3)-β-D-galactofuranoside D13:

[0407] [ka]

[0408] Debenzoylated tetradecasaccharide D12 (50 mg) was taken up in a mL mixture of tBuOH:DCM:PBS (3:0.75:0.38). Pd / C (100 mg) was added and hydrogenated under a 5 bar H atmosphere for 20 h. The reaction mixture was filtered through a PTFE filter (3 × 6 mL) using 50% aqueous methanol. The filtrate was concentrated under vacuum to give the crude product as a white solid. The crude product was purified using a Sep-Pak C18 (0.5 g) column with a water-acetonitrile gradient as the eluent to give the desired product as a fluffy white solid after lyophilization. The product was further purified on an SEC column of LH-20 resin with water as the eluent. Fractions containing product D13 were combined, frozen, and lyophilized to give a fluffy white solid (19 mg, 59%). 1 H NMR (400MHz, D2O) δ 5.20(s, 5H), 5.11(d, J=3.8Hz, 1H), 5.09-5.05(m, 6H), 5.03(d, J=1.5Hz, 1H), 4.95(d, J=3.8Hz, 1H) ), 4.43-4.37(m, 4H), 4.35-3.53(m, 82H), 3.03-2.96(m, 2H), 1.74-1.60(m, 4H), 1.50-1.38(m, 2H). MALDI-TOF C 89 H 153 NNaO 71 + [M+Na] + The calculated value was 2394.8285 and the measured value was 2394.83.

[0409] 5-Azido-pentyl 2,3,5,6-Tetra-O-benzoyl-β-D-galactofuranosyl-(1→3)-4-[4,6-di-O-benzoyl-2,3-di-O-benzyl-α-D-galactopyranosyl-(1→)]-6-O-benzoyl-2-O-benzyl-α-D-galactopyranosyl-(1→3)-2,5,6-tri-O-benzoyl-β-D-galactofuranosyl-(1→3)-4-[4,6-di-O-benzoyl-2,3-di-O-benzyl-α-D-galactopyranosyl-(1→)]-6-O-benzoyl-2-O-benzyl-α-D-galactopyranosyl-(1→3)-2,5,6-tri-O-benzoyl-β-D-galactofuranosyl-(1→3)-6-O-benzoyl-2,4- Synthesis of di-O-benzyl-α-D-galactopyranosyl-(1→3)-2,5,6-tri-O-benzoyl-β-D-galactofuranosyl-(1→3)-6-O-benzoyl-2,4-di-O-benzyl-α-D-galactopyranosyl-(1→3)-2,5,6-tri-O-benzoyl-β-D-galactofuranosyl-(1→3)-6-O-benzoyl-2,4-di-O-benzyl-α-D-galactopyranosyl-(1→3)-2,5,6-tri-O-benzoyl-β-D-galactofuranosyl-(1→3)]-6-O-benzoyl-2,4-di-O-benzyl-α-D-galactopyranosyl-(1→3)-2,5,6-tri-O-benzoyl-β-D-galactofuranoside D15:

[0410] [ka]

[0411] Acceptor D11 (0.25 g, 0.038 mmol) was coevaporated twice with toluene and dissolved in anhydrous toluene (3 mL). Freshly activated 4 Å MS was added and stirred for 30 min. The reaction mixture was cooled to 0 °C, and TMSOTf (1.34 μL, 0.038 mmol, 0.1 mL solution in toluene) was added dropwise. Donor D14 (see WO2019106201, p. 198) (0.088 g, 0.114 mmol) was coevaporated twice with toluene and dissolved in DCM (1.5 mL). The donor was added dropwise to the reaction mixture over 10 min. The reaction mixture was slowly warmed to 10 °C for 1.5 h. TLC indicated complete consumption of the acceptor. The reaction was filtered, diluted with ethyl acetate (10 mL), and quenched by the addition of saturated aqueous NaHCO3 (10 mL). The organic layer was separated, dried over Na2SO4, and filtered. The solvent was evaporated to give the crude product. The crude reaction mixture was purified on an automated purification system using Cy / EtOAc (0-100%) to give the product D15 as a white foam (0.17 g, 62%). MALDI-TOF C 417 H 374 N3O 108 + [M+H] + The calculated value was 7150.3860 and the measured value was 7150.75. 1 H NMR (400MHz, CDCl3) δ 8.16-7.27(m, 128H), 7.26-6.71(m, 102H), 6.04(s, 1H), 5.91(s, 1H), 5.87-5.73(m, 8H), 5.71(s , 1H), 5.66-5.57(m, 2H), 5.57-5.41(m, 5H), 5.17-5.10(m, 4H), 5.10-4.91(m, 13H), 4.89-4.78( m, 2H), 4.75-4.17(m, 68H), 4.11-3.94(m, 20H), 3.88-3.74(dd, J=23.9, 10.4Hz, 8H), 3.67-3.53 (m, 1H), 3.39-3.23(m, 1H), 3.13(t, J=6.9Hz, 2H), 1.58-1.39(m, 4H), 1.28(q, J=7.7, 7.2Hz, 2H).

[0412] Synthesis of partially protected hexasaccharide D16:

[0413] [ka]

[0414] A 0.5 M solution of sodium methoxide in MeOH (3 mL, 1.5 mmol) was added to a solution of the decadecasaccharide D15 (160 mg, 0.022 mmol) in THF (3 mL). The reaction was stirred at 60 °C overnight. The reaction solvent was evaporated to give the crude product. The crude product was washed with HO (2 × 2 mL) and AcOH (0.1 mL in 1 mL of HO). The solid was dissolved in MeOH and evaporated on a rotavap. The residue was washed with cyclohexane and then purified by SEC using LH-20 CHCl:MeOH (1:2) to give the product D16 as a white solid (70 mg, 82%). MALDI-TOF C 193 H 246 N3O 76 + [M+H] + The calculated value was 3821.5471 and the measured value was 3821.09.

[0415] 5-amino-pentyl Synthesis of β-D-galactofuranosyl-(1→3)-4-[α-D-galactopyranosyl-(1→)]-α-D-galactopyranosyl-(1→3)-β-D-galactofuranosyl-(1→3)-4-[α-D-galactopyranosyl-(1→)]-α-D-galactopyranosyl-(1→3)-β-D-galactofuranosyl-(1→3)-α-D-galactopyranosyl-(1→3)-β-D-galactofuranosyl-(1→3)-α-D-galactopyranosyl-(1→3)-β-D-galactofuranosyl-(1→3)-α-D-galactopyranosyl-(1→3)-β-D-galactofuranoside D17:

[0416] [ka]

[0417] The debenzoylated hexasaccharide D16 (40 mg, 0.010 mmol) was dissolved in a mixture of DCM:tBuOH:HO (2:8:1, 3.5 mL). Pd / C (70 mg) was added, and the reaction mixture was purged with hydrogen (5 times) and stirred under hydrogen pressure (5 bar) for 20 h. The reaction mixture was then filtered through a PTFE filter with HO:ACN (1:1), the organic solvent was evaporated in a rotavapor, and the crude material was lyophilized. The crude material was purified by Sep-Pak C18 followed by SEC LH-20 with miliQ HO and lyophilized to give the product D17 as a white solid (12.9 mg, 49%). MALDI-TOF C 95 H 163NNaO 76 + [M+Na] + The calculated value was 2556.8813 and the measured value was 2558.40. 1 H NMR (400MHz, D2O) δ 5.20(s, 6H), 5.14-4.96(m, 9H), 4.43-4.36(m, 4H), 4.35-3.54(m, 88H), 2.99(t, J=7.5, 6.4Hz, 2H), 1.75-1.56(m, 4H), 1.50-1.36(m, 2H).

[0418] 5-Azido-pentyl 4,6-di-O-benzoyl-2,3-di-O-benzyl-α-D-galactopyranosyl-(1→4)-6-O-benzoyl-2-O-benzyl-3-O-levulinoyl-α-D-galactopyranosyl-(1→3)-2,5,6-tri-O-benzoyl-β-D-galactofuranosyl-(1→3)-4-[4,6-di-O-benzoyl-2,3-di-O-benzyl-α-D-galactopyranosyl] 1→)]-6-O-benzoyl-2-O-benzyl-α-D-galactopyranosyl-(1→3)-2,5,6-tri-O-benzoyl-β-D-galactofuranosyl-(1→3)-4-[4,6-di-O-benzoyl-2,3-di-O-benzyl-α-D-galactopyranosyl-(1→)]-6-O-benzoyl-2-O-benzyl-α-D-galactopyranosyl-(1→3)-2,5, 6-Tri-O-benzoyl-β-D-galactofuranosyl-(1→3)-6-O-benzoyl-2,4-di-O-benzyl-α-D-galactopyranosyl-(1→3)-2,5,6-tri-O-benzoyl-β-D-galactofuranosyl-(1→3)-6-O-benzoyl-2,4-di-O-benzyl-α-D-galactopyranosyl-(1→3)-2,5,6-tri-O-benzoyl-β-D- Synthesis of galactofuranosyl-(1→3)-6-O-benzoyl-2,4-di-O-benzyl-α-D-galactopyranosyl-(1→3)-2,5,6-tri-O-benzoyl-β-D-galactofuranosyl-(1→3)]-6-O-benzoyl-2,4-di-O-benzyl-α-D-galactopyranosyl-(1→3)-2,5,6-tri-O-benzoyl-β-D-galactofuranoside D18:

[0419] [ka]

[0420] Acceptor D11 (0.55 g, 0.084 mmol) was coevaporated twice with toluene and dissolved in anhydrous toluene (4 mL). Freshly activated 4 Å MS was added and stirred for 30 min. The reaction mixture was cooled to 0 °C, and TMSOTf (3.02 μL, 0.017 mmol, 0.1 mL solution in toluene) was added dropwise. Donor D7 (0.28 g, 0.167 mmol) was coevaporated twice with toluene and dissolved in DCM (2 mL). The donor was added dropwise to the reaction mixture over 10 min. The reaction mixture was slowly warmed to 10 °C for 1.5 h. The reaction was filtered, diluted with ethyl acetate (10 mL), and quenched by the addition of saturated aqueous NaHCO3 (10 mL). The organic layer was separated, dried over Na2SO4, and filtered. The solvent was evaporated in vacuo to give the crude product. The crude reaction mixture was purified on an automated purification system using Cy / EtOAc (0-100%) to give the product D18 as a white foam (0.46 g, 68%). 469 H 426 N3O 122 + [M+H] + The calculated value was 8050.7217 and the measured value was 8050.19. 1H NMR (400MHz, CDCl3) δ 8.15-7.62(m, 76H), 7.55-7.27(m, 86H), 7.22-6.75(m, 93H), 6.12(s, 1H), 6.07-5.98(d , J=12.2Hz, 2H), 5.95-5.62(m, 17H), 5.58-5.48(d, J=11.3Hz, 5H), 5.26-5.14(m, 6H), 5 .14-4.91(m, 18H), 4.90-3.72(m, 148H), 3.66-3.54(m, 1H), 3.33(d, J=9.7Hz, 1H), 3.14 (t, J=6.9Hz, 2H), 2.23-1.81(m, 4H), 1.71(s, 3H), 1.57-1.45(m, 4H), 1.37-1.29(m, 2H).

[0421] Synthesis of partially protected heptasaccharide D19:

[0422] [ka]

[0423] A 0.5 M solution of sodium methoxide in MeOH (4 mL, 2 mmol) was added to a solution of heptasaccharide D18 (160 mg, 0.022 mmol) in THF (3 mL). The reaction was stirred at 60 °C overnight. The reaction solvent was evaporated to give the crude product. The crude product was washed with HO (2 × 2 mL) and AcOH (0.1 mL in 1 mL of HO). The solid was dissolved in MeOH and the solvent was evaporated to dryness under vacuum. The residue was washed with cyclohexane and then purified by SEC using LH-20 CHCl:MeOH (1:2) to give the product D19 as a white solid (220 mg, 87%). MALDI-TOF C 226 H 284 N3O 86 + [M+H] + The calculated value was 4415.7936 and the measured value was 4415.43.

[0424] 5-Aminopentyl α-D-galactopyranosyl-(1→4)-α-D-galactopyranosyl-(1→3)-β-D-galactofuranosyl-(1→3)-4-[α-D-galactopyranosyl-(1→)]-α-D-galactopyranosyl-(1→3)-β-D-galactofuranosyl-(1→3)-4-[α-D-galactopyranosyl-(1→)]-α-D-galactopyranosyl-(1→3)-β-D-galac Synthesis of tofuranosyl-(1→3)-α-D-galactopyranosyl-(1→3)-β-D-galactofuranosyl-(1→3)-α-D-galactopyranosyl-(1→3)-β-D-galactofuranosyl-(1→3)-α-D-galactopyranosyl-(1→3)-β-D-galactofuranosyl-(1→3)-α-D-galactopyranosyl-(1→3)-β-D-galactofuranoside D20:

[0425] [ka]

[0426] The debenzoylated heptasaccharide D19 (37 mg, 0.084 mmol) was dissolved in a mixture of DCM:tBuOH:HO (2:8:1, 2.75 mL). Pd / C (40 mg) was added, and the reaction mixture was purged with hydrogen (5 times) and stirred under hydrogen pressure (5 bar) for 20 h. The reaction mixture was then filtered through a PTFE filter with HO:ACN (1:1), the solvent was evaporated on a rotavapor, and the crude material was lyophilized. The crude material was purified on a Sep-Pak C18 column followed by SEC LH-20 with miliQ HO and lyophilized to give the product D20 as a white solid (5.3 mg, 22%). MALDI-TOF C 107 H 183 NNaO 86 + [M+Na] + The calculated value was 2880.9869 and the measured value was 2882.40. 1 H NMR (400MHz, D2O) δ 5.25-5.17(m, 6H), 5.13-4.98(m, 10H), 4.94(d, J=3.5Hz, 1H), 4.41(s, 4H), 4.33-4.03(m, 40H), 3. 95-3.80(m, 32H), 3.76-3.56(m, 28H), 2.98(t, J=7.2Hz, 2H), 1.75-1.58(m, 4H), 1.51-1.36(m, 2H).

[0427] Synthesis of the conjugate: Synthesis of NHS ester of compound D13 (D13-adipate-NHS)

[0428] [ka]

[0429] Compound D13 (4 mg, 1.686 μmol) was dissolved in DMSO-HO (100 μL-10 μL) in a 15 mL falcon tube at room temperature. Triethylamine (8 μL, 0.059 mmol) was added thereto. Adipate-NHS ester (bis(2,5-dioxopyrrolidin-1-yl)adipate) (11.5 mg, 0.034 mmol) in DMSO (100 μL) was added and stirred at room temperature for 2 h. D13-adipate-NHS ester was precipitated by adding 5 mL of EtOAc, centrifuged, and the precipitate was washed with EtOAc (3 mL × 2) and dried under vacuum to give a white solid (4 mg, 91%), which was used in the next step.

[0430] CRM 197 D13 conjugate (D13-adipate-CRM 197 or D13-CRM 197 * ) synthesis Compound D13-adipate-NHS ester (4 mg, 1.54 μmol) was dissolved in 0.1 M NaPi buffer (pH 7.0, 150 μL) in a 15 mL falcon tube. Freshly washed CRM in 0.1 M NaPi buffer (pH 7.0, 150 μL) in a vial 197 (obtained from EirGenix, Inc., Taiwan, expression system E. coli) (2 mg, 0.034 μmol) was added dropwise thereto. The vial was rinsed with 0.1 M NaPi buffer (pH 7.0, 50 μL) and transferred to the reaction mixture in the falcon tube, which was then stirred at room temperature for 20 h. The resulting D13-adipate-CRM 197The solution was transferred to an Amicon Ultra vial (10 kDa, MWCO) and centrifuged at 2-8°C for 5 minutes. 300 μL of 0.1 M NaPi was added to the reaction falcon tube, rinsed, transferred to the filter, and centrifuged again. Five additional washes were performed with TBS buffer solution (pH 7.4). After the final wash, the conjugate was sterile filtered and stored in 1.0 mL of TBS (pH 7.4) at 2-8°C. The loading obtained using MALDI-TOF MS was 12.3. The conjugate was analyzed using SDS-PAGE, BCA, SEC-HPLC, and also for endotoxin.

[0431] BSA conjugates (D13-adipate-BSA or D13-BSA * ) synthesis Compound D13-adipate-NHS (1.7 mg, 0.654 μmol) was dissolved in 0.1 M NaPi buffer (pH 7.0, 150 μL) in a 15 mL falcon tube. Freshly washed BSA (obtained from Sigma-Aldrich, heat shock fraction, pH 7, ≥98%; product number A7906) (3 mg, 0.045 μmol) in 0.1 M NaPi buffer (pH 7.0, 150 μL) in a vial was added dropwise. The vial was rinsed with 0.1 M NaPi buffer (pH 7.0, 50 μL) and transferred to the reaction mixture in the falcon tube. The mixture was stirred at room temperature for 20 h. The resulting D13-adipate-BSA solution was transferred to an Amicon Ultra vial (10 kDa, MWCO) and centrifuged at 2-8 °C for 5 min. 300 μL of 0.1 M NaPi was added to the reaction falcon tube, rinsed, transferred to the filter, and centrifuged again. Five additional washes were performed using 1×PBS solution. After the final wash, the conjugate was sterile filtered and stored in 0.5 mL of PBS (pH 7.4) at 2-8°C. The resulting load using MALDI-TOF MS was 6.96. The conjugate was analyzed using SDS-PAGE and SEC-HPLC.

[0432] Synthesis of NHS ester of compound D17 (D17-adipate-NHS)

[0433] [ka]

[0434] The NHS ester of compound D17 was synthesized using a procedure similar to that previously described for D13-adipate-NHS and dried under vacuum to give a white solid (7 mg, 92%), which was used in the next step.

[0435] CRM 197 D17 conjugate (D17-adipate-CRM 197 or D17-CRM 197 * ) synthesis CRM 197 The D17 conjugate (3 mg) was added to D17-adipate-NHS and CRM 197 D13-adipate-CRM 197 was synthesized using a procedure similar to that previously described for and had a loading of 9.69 using MALDI-TOF MS.

[0436] BSA conjugates (D17-adipate-BSA or D17-BSA * ) synthesis D17 conjugate with BSA (1 mg) was synthesized using D17-adipate-NHS and BSA using a procedure similar to that described above for D13-adipate-BSA, and the resulting loading using MALDI-TOF MS was 12.78.

[0437] Synthesis of NHS ester of compound D20 (D20-adipate-NHS)

[0438] [ka]

[0439] The NHS ester of compound D20 was synthesized using a procedure similar to that previously described for D13-adipate-NHS and dried under vacuum to give a white solid (7 mg, 93%), which was used in the next step.

[0440] CRM 197 D20 conjugate (D20-adipate-CRM 197 or D20-CRM 197 * ) synthesis CRM 197 The D20 conjugate (3 mg) was added to D20-adipate-NHS and CRM 197 D13-adipate-CRM 197 was synthesized using a procedure similar to that previously described for and had a loading of 7.45 using MALDI-TOF MS.

[0441] BSA conjugates (D20-adipate-BSA or D20-BSA * ) synthesis The D17 conjugate with BSA (1 mg) was synthesized using D17-adipate-NHS and BSA using a procedure similar to that described above for D13-adipate-BSA, and the resulting loading using MALDI-TOF MS was 9.41.

[0442] II. Biology material: - ELISA Plate (High-binding, EIA / RIA Plate, 96-well, flat bottom with low evaporation lid, company: Costar® 3361) - Detection antibody: Goat anti-rabbit IgG peroxidase conjugate (Sigma, #A4914) - Blocking solution: Commercially available blocking solution (Roche, cat. no. 11112589001) - Antibody diluent: PBS + 1% BSA (w / v) - Washing buffer: PBS + 0.1% Tween 20 (PBS-T) - Developing solution: 1Step™ Ultra TMB-ELISA developer (ThermoScientific, Cat#: 34028) - Stop solution: 2M sulfuric acid (H2SO4) - Plate reader: FLUOstar Omega (BMG LABTECH) - Software: GraphPad version 7 or higher for data plotting and analysis Alum: Aluminum Hydroxide Adjuvant (Rehydragel® HPA), Chemtrade - QuantiPro(TM) BCA Assay Kit(SIGMA) Product:QPBCA-1KT;Lot#:SLBR7451V;Pcode:1002296464 - Mini-PROTEAN® TGX™ Gels-10%, 10 wells (30 μL / well) Control Nr: 64175708 - GelCode(TM) Blue Safe Protein Stain;ThermoScientific;Ref:1860957;Lot#:TA260266 method : Bacterial strains and LPS Klebsiella pneumoniae strains with different LPS (O antigen) were used to isolate and purify the corresponding LPS. The purified LPS was used as a coating antigen for enzyme-linked immunosorbent assay (ELISA). LPS was isolated using a commercially available LPS extraction kit (JH Science) according to the manufacturer's protocol.

[0443] [Table 2]

[0444] Formulation of vaccine candidates for immunization. All formulations were manufactured under sterile conditions. The drug substance (DS) and buffer (10 mM TRIS-HCl, pH 7.4) were mixed at the appropriate dilution ratio (see below) calculated in advance for the required glycan dose, excluding the required volume of aluminum hydroxide adjuvant (0.25 mg / mL). The DS-buffer mixture was gently mixed, and aluminum hydroxide adjuvant ("Aluminum") stock was added to achieve a final aluminum concentration of 0.250 mg / mL aluminum. The mixture was quickly mixed by gentle pipetting and then mixed on a horizontal shaker at 250 rpm for 2 h at RT. Aliquots were stored in type 1 glass vials at 4°C until further use.

[0445] The vaccine is prepared to contain the intended glycan dose (e.g., 2 μg of glycan per injection) as follows: The average loading factor of the glycan antigen (expressed as moles of antigen per mole of carrier protein) is determined by the CRM 197 The mass of the glycan antigen is determined by MALDI-TOF MS by subtracting the molecular weight (m / z=1) of the glycan antigen from the determined molecular weight of the DS (m / z=1), and then dividing this mass difference by the theoretical molecular weight of the glycan antigen (including the linker (here: alkyl) and spacer (here: adipoyl) moieties). The resulting loading factor is multiplied by the theoretical molecular weight of the glycan antigen excluding the linker and spacer moieties to obtain the average total mass of the glycan attached per DS molecule. The total mass of the glycan is then divided by the determined CRM. 197Dividing by the molecular weight of the protein gives the glycan-to-protein mass ratio of the DS. Multiplying this ratio by the protein concentration of the DS measured by BCA Assay Kit (Sigma) according to the manufacturer's protocol gives the glycan concentration of the DS. To obtain the dilution ratio required to dilute the DS to obtain the intended glycan dose per immunization, the glycan concentration of the DS is divided by the required glycan concentration (e.g., to obtain a glycan dose of 2 μg for rabbits in an injection volume of 500 μL, a glycan concentration of 4 μg / mL). Then, the DS is diluted by this dilution ratio to the final volume of the vaccine preparation.

[0446] Immunization: Female Zika rabbits were immunized via the intramuscular (i.m.) route with an injection volume of 500 μL per dose. Animals were maintained under specific pathogen-free conditions and provided with water and food ad libitum.

[0447] ELISA: Coating of plates with antigen: Isolated LPS was used for coating. LPS was dissolved in isopropanol to a concentration of 10 μg / mL, and 100 μL was used for coating, resulting in 1 μg of LPS per well. The LPS solution was allowed to evaporate overnight at RT in a safety cabinet. Blocking: The plates were blocked with 100 μL of a commercial blocking solution and incubated for 1 h at RT. After blocking, the plates were washed 3x with PBS containing 0.1% (v / v) Tween-20 (PBS-T). Incubation with diluted serum: Pooled or individual sera from different time points were diluted with 1% BSA (w / v) in PBS to form their respective dilutions. 50–100 μL of diluted serum was added to ELISA wells in duplicate and incubated for 1 h at RT. 100 μL / well of 1% BSA (w / v) in PBS was used as a blank. After incubation with serum, the plates were washed 3x with PBS-T. Incubation with detection antibody: Anti-rabbit IgG HRP conjugate was diluted 1:10,000 in 1% BSA (w / v) in PBS, added 100 μL / well, and incubated for 30 min at RT. After incubation with detection antibody, the plates were washed 3x with PBS-T. Addition of substrate: 100 μL of TMB substrate was added to each well and incubated for approximately 15 min. The reaction was stopped by adding 50 μL / well of 2M H2SO4. Absorbance was measured at 450 nm using a plate reader. Absorbance values ​​were analyzed using GraphPad Prism software.

[0448] Challenge experiments: Female CD-1 mice received pooled rabbit post-immune antisera (30-250 μL) intraperitoneally 24 hours and 1 hour before bacterial challenge. Prior to bacterial challenge, mice were treated with 20 mg of galactosamine per animal. Mice were immunized with 2x10 7 Colony-forming units (CFU) of K. pneumoniae strain NCTC9163 (O2a) or 1 x 10 8Mice were infected intraperitoneally with CFU of strain ST258 (Gal-III). Mice were observed for clinical scores for 24 hours and euthanized at a humane endpoint.

[0449] SEQ ID No:1(CRM 197 ) GADDVVDSSK SFVMENFSSY HGTKPGYVDS 30 IQKGIQKPKS GTQGNYDDDW KEFYSTDNKY 60 DAAGYSVDNE NPLSGKAGGV VKVTYPGLTK 90 VLALKVDNAE TIKKELGLSL TEPLMEQVGT 120 EEFIKRFGDG ASRVVLSLPF AEGSSSVEYI 150 NNWEQAKALS VELEINFETR GKRGQDAMYE 180 YMAQACAGNR VRRSVGSSLS CINLDWDVIR 210 DKTKTKIESL KEHGPIKNKM SESPNKTVSE 240 EKAKQYLEEF HQTALEHPEL SELKTVTGTN 270 PVFAGANYAA WAVNVAQVID SETADNLEKT 300 TAALSILPGI GSVMGIADGA VHHNTEEIVA 330 QSIALSSLMV AQAIPLVGEL VDIGFAAYNF 360 VESIINLFQV VHNSYNRPAY SPGHKTQPFL 390 HDGYAVSWNT VEDSIIRTGF QGESGHDIKI 420 TAENTPLPIA GVLLPTIPGK LDVNKSKTHI 450 SVNGRKIRMR CRAIDGDVTF CRPKSPVYVG 480 NGVHANLHVA FHRSSSEKIH SNEISSDSIG 510 VLGYQKTVDH TKVNSKLSLF FEIKS 535 SEQ ID NO:2 (Diphtheria toxin (Uniprot ID:P00587)) GADDVVDSSK SFVMENFSSY HGTKPGYVDS 30 IQKGIQKPKS GTQGNYDDDW KGFYSTDNKY 60 DAAGYSVDNE NPLSGKAGGV VKVTYPGLTK 90 VLALKVDNAE TIKKELGLSL TEPLMEQVGT 120 EEFIKRFGDG ASRVVLSLPF AEGSSSVEYI 150 NNWEQAKALS VELEINFETR GKRGQDAMYE 180 YMAQACAGNR VRRSVGSSLS CINLDWDVIR 210 DKTKTKIESL KEHGPIKNKM SESPNKTVSE 240 EKAKQYLEEF HQTALEHPEL SELKTVTGTN 270 PVFAGANYAA WAVNVAQVID SETADNLEKT 300 TAALSILPGI GSVMGIADGA VHHNTEEIVA 330 QSIALSSLMV AQAIPLVGEL VDIGFAAYNF 360 VESIINLFQV VHNSYNRPAY SPGHKTQPFL 390 HDGYAVSWNT VEDSIIRTGF QGESGHDIKI 420 TAENTPLPIA GVLLPTIPGK LDVNKSKTHI 450 SVNGRKIRMR CRAIDGDVTF CRPKSPVYVG 480 NGVHANLHVA FHRSSSEKIH SNEISSDSIG 510 VLGYQKTVDH TKVNSKLSLF FEIKS 535 SEQ ID NO:3(Tetanus Toxin (Uniprot ID:P04958)) PITINNFRYS DPVNNDTIIM MEPPYCKGLD 30 IYYKAFKITD RIWIVPERYE FGTKPEDFNP 60 PSSLIEGASE YYDPNYLRTD SDKDRFLQTM 90 VKLFNRIKNN VAGEALLDKI INAIPYLGNS 120 YSLLDKFDTN SNSVSFNLLE QDPSGATTKS 150 AMLTNLIIFG PGPVLNKNEV RGIVLRVDNK 180 NYFPCRDGFG SIMQMAFCPE YVPTFDNVIE 210 NITSLTIGKS KYFQDPALLL MHELIHVLHG 240 LYGMQVSSHE IIPSKQEIYM QHTYPISAEE 270 LFTFGGQDAN LISIDIKNDL YEKTLNDYKA 300 IANKLSQVTS CNDPNIDIDS YKQIYQQKYQ 330 FDKDSNGQYI VNEDKFQILY NSIMYGFTEI 360 ​​​​​​​​​​​​​​TLQRITMTNS VDDALINSTK IYSYFPSVIS 600 KVNQGAQGIL FLQWVRDIID DFTNESSQKT 660 TIDKISDVST IVPYIGPALN IVKQGYEGNF 690 IGALETTGVV LLLEYIPEIT LPVIAALSIA 720 ESSTQKEKII KTIDNFLEKR YEKWIEVYKL 750 VKAKWLGTVN TQFQKRSYQM YRSLEYQVDA 780 IKKIIDYEYK IYSGPDKEQI ADEINNLKNK 810 LEEKANKAMI NINIFMRESS RSFLVNQMIN 840 EAKKQLLEFD TQSKNILMQY IKANSKFIGI 870 TELKKLESKI NKVFSTPIPF SYSKNLDCWV 900 DNEEDIDVIL KKSTILNLDI NNDIISDISG 930 FNSSVITYPD AQLVPGINGK AIHLVNNESS 960 EVIVHKAMDI EYNDMFNNFT VSFWLRVPKV 990 SASHLEQYGT NEYSIISSMK KHSLSIGSGW 1020 SVSLKGNNLI WTLKDSAGEV RQITFRDLPD 1050 KFNAYLANKW VFITITNDRL SSANLYINGV 1080 LMGSAEITGL GAIREDNNIT LKLDRCNNNN 1100 QYVSIDKFRI FCKALNPKEI EKLYTSYLSI 1130 TFLRDFWGNP LRYDTEYYLI PVASSSKDVQ 1160 LKNITDYMYL TNAPSYTNGK LNIYYRRLYN 1190 GLKFIIKRYT PNNEIDSFVK SGDFIKLYVS 1200 YNNNEHIVGY PKDGNAFNNL DRILRVGYNA 1230 PGIPLYKKME AVKLRDLKTY SVQLKLYDDK 1260 NASLGLVGTH NGQIGNDPNR DILIASNWYF 1290 NHLKDKILGC DWYFVPTDEG WTND 1314 SEQ ID NO: 4 (Cholera toxin B subunit (Uniprot ID: P01556)) TPQNITDLCA EYHNTQIYTL NDKIFSYTES 30 LAGKREMAII TFKNGAIFQV EVPGSQHIDS 60 QKKAIERMKD TLRIAYLTEA KVEKLCVWNN 90 KTPHAIAAIS MAN 103 SEQ ID NO: 5 (Meningococcal outer membrane protein (OMP) (Uniprot ID: Q51229)) MKKTVFTCAM IALTGTAAAA QELQTANEFT 30 VHTDLSSISS TRAFLKEKHK AAKHISVRAD 60 IPFDANQGIR LEAGFGRSKK NIINLETDEN 90 KLGKTKNVKL PTGVPENRID LYTGYTYTQT 120 LSDSLNFRVG AGLGFESSKD SIKTTKHTLH 150 SSRQSWLAKV HADLLSQLGN GWYINPWSEV 180 KFDLNSRYKL NTGVTNLKKD INQKTNGWGF 210 GLGANIGKKL GESASIEAGP FYKQRTYKES 240 GEFSVTTKSG DVSLTIPKTS IREYGLRVGI 270 KF 272 SEQ ID NO:6 (Capsid protein of bacteriophage Qβ (Uniprot ID: P03615)) AKLETVTLGN IGKDGKQTLV LNPRGVNPTN 30 GVASLSQAGA VPALEKRVTV SVSQPSRNRK 60 NYKVQVKIQN PTACTANGSC DPSVTRQAYA 90 DVTFSFTQYS TDEERAFVRT ELAALLASPL 120 LIDAIDQLNP AY 132

Claims

1. An immunogenic compound having at least one oligosaccharide hybrid antigen having the structure of formula (I): 【Chemistry 1】 (In the formula, R is OH or 【Chemistry 2】 and m is 3, 4, 5, 6, 7 or 8; preferably 3, 4, 5 or 6; n is 1, 2, 3, 4, 5 or 6; preferably 2, 3 or 4; or a pharmaceutically acceptable salt thereof.

2. m is 4, n is 2, and R is OH; m is 4, n is 2, and R is 【Transformation 3】 or m is 4, n is 3, and R is OH; The immunogenic compound of claim 1 or a pharmaceutically acceptable salt thereof.

3. 2. The immunogenic compound of claim 1, wherein m is 4, n is 2, and R is OH; or a pharmaceutically acceptable salt thereof.

4. Immunogenic compounds of formula (II): 【Chemistry 4】 (In the formula, R is OH or 【Transformation 5】 and m is 3, 4, 5, 6, 7 or 8; preferably 3, 4, 5 or 6; n is 1, 2, 3, 4, 5 or 6; preferably 2, 3 or 4; i is 1 to 28; -LT- represents a linker L and a spacer T that together form a bridge with a covalently linked backbone of 5 to 25 atoms in length, the length being determined by the distance between the oxygen at C1 of the reducing end of the oligosaccharide and the carrier protein CRM. １９７ form the shortest distance between the nitrogen of the amino group of the lysine residue; The atoms of the backbone are selected from the group consisting of carbon, nitrogen, oxygen, and sulfur; or a pharmaceutically acceptable salt thereof.

5. m is 4, n is 2, and R is OH; m is 4, n is 2, and R is 【Transformation 6】 or m is 4, n is 3, and R is OH; The immunogenic compound according to claim 4 or a pharmaceutically acceptable salt thereof.

6. 5. The immunogenic compound of claim 4, wherein m is 4, n is 2, and R is OH; or a pharmaceutically acceptable salt thereof.

7. -LT- represents a linker L and a spacer T that together form a bridge having a covalently linked backbone of 5 to 25 atoms in length, the length being determined by the distance between the oxygen at C1 of the reducing end of the oligosaccharide and the carrier protein CRM. １９７ the shortest distance between the nitrogen of the amino group of the lysine residue ... the atoms of the backbone are selected from the group consisting of carbon, nitrogen, oxygen, and sulfur; The skeleton is oxo, (C １－４ ) alkyl, fluoro and (C １－２ ) optionally substituted by one or more (particularly 1, 2, 3 or 4) substituents independently selected from alkoxy (particularly oxo); A part of the skeleton may optionally be: 【Transformation 7】 It may be a 4-, 5- or 6-membered ring moiety selected from: The immunogenic compound according to any one of claims 4 to 6, or a pharmaceutically acceptable salt thereof.

8. The backbone of the bridge has a length of 8 to 20, preferably 8 to 16 atoms covalently linked, and the length is such that the oxygen at C1 of the reducing end of the oligosaccharide and the carrier protein CRM are bonded together. １９７ The immunogenic compound or a pharmaceutically acceptable salt thereof according to any one of claims 4 to 7, wherein the nitrogen of the amino group of the lysine residue of

9. L, ＊ - (CH ２ ) ａ -NH-; (a is 2 to 10); ＊ - (CH ２ CH ２ O) ｂ -CH ２ CH ２ NH- (b is 1, 2 or 3); ＊ -CH ２ CH ２ S-CH ２ CH ２ NH-; ＊ -(C ２－１０ ) fluoroalkylene-NH- (fluoroalkylene is a saturated linear chain); ＊ - (CH ２ ) ｃ NHC(O)(CH ２ ) ｃ’ -NH- (c and c' are each independently 2 to 6); ＊ - (CH ２ ) ｄ NHC(O)NH(CH ２ ) ｄ’ -NH- (d and d' are independently 2 to 6); ＊ - (CH ２ ) ｅ -C(O)-NH-(CH ２ ) ｅ’ -NH-; (e is 1 to 10 and e' is 2 to 10); or ＊ - (CH ２ ) ｆ —O—NH— (f is 2 to 10); represents; Or, L-T, ＊ - (CH ２ ) ｇ -S-R １ (g is 2 to 10); represents; T, —C(O)—(CH ２ ) ｈ —C(O)— (h is 0 to 10); —C(O)—CH ２ CH ２ -(OCH ２ CH ２ ) ｊ —C(O)— (j is 1 to 5); —C(O)—CH ２ (CH ２ ) ｋ - (SCH ２ (CH ２ ) ｋ’ ) ｋ’’ —C(O)— (k is 0 or 1, k′ is 0 or 1, and k″ is 1, 2, or 3); 【Transformation 8】 represents; R １ teeth, 【Chemistry 9】 Represents; 9. The immunogenic compound of any one of claims 4, 5, 6 or 8, or a pharmaceutically acceptable salt thereof.

10. L, ＊ - (CH ２ ) ２ -NH-, ＊ - (CH ２ ) ３ -NH-, ＊ - (CH ２ ) ４ -NH-, ＊ - (CH ２ ) ５ -NH- or ＊ - (CH ２ ) ６ -NH-, preferably ＊ - (CH ２ ) ５ represents —NH—; T, -C(O)-C(O)-, -C(O)-CH ２ -C(O)-, -C(O)-(CH ２ ) ２ -C(O)-, -C(O)-(CH ２ ) ３ -C(O)-, -C(O)-(CH ２ ) ４ -C(O)-, -C(O)-(CH ２ ) ５ -C(O)- or -C(O)-(CH ２ ) ６ —C(O)—, preferably —C(O)—(CH ２ ) ４ represents —C(O)—; 7. The immunogenic compound of claim 4, 5 or 6, or a pharmaceutically acceptable salt thereof.

11. The immunogenic compound or a pharmaceutically acceptable salt thereof according to any one of claims 4 to 10, wherein i is 6 to 15.

12. The immunogenic compound of claim 4 selected from the group consisting of structures of formulas (IIa), (IIb) and (IIc): 【Chemistry 10】 (wherein i is 1 to 28, preferably 6 to 15); or a pharmaceutically acceptable salt thereof.

13. A pharmaceutical composition comprising the immunogenic compound or a pharmaceutically acceptable salt thereof according to any one of claims 1 to 12 as an active ingredient, and further comprising at least one therapeutically inactive excipient.

14. 14. The pharmaceutical composition of claim 13, further comprising an adjuvant.

15. An immunogenic compound or a pharmaceutically acceptable salt thereof according to any one of claims 1 to 12 for use as a medicine, in particular as a vaccine.

16. 13. The immunogenic compound or a pharmaceutically acceptable salt thereof according to any one of claims 1 to 12, for use in the prevention and / or treatment of K. pneumoniae infection.

17. A multivalent vaccine comprising the immunogenic compound or a pharmaceutically acceptable salt thereof according to any one of claims 1 to 12.

18. An intermediate compound for producing the immunogenic compound of any one of claims 4 to 12, having the structure of formula (III): 【Chemistry 11】 (In the formula, R is OH or 【Chemistry 12】 and m is 3, 4, 5, 6, 7 or 8, preferably 3, 4, 5 or 6; n is 1, 2, 3, 4, 5 or 6, preferably 2, 3 or 4; L １ teeth, ＊ -(C ２－１０ ) alkylene-NH ２ , preferably ＊ - (CH ２ ) ａ -NH ２ (a is 2 to 10, more preferably l is 5); ＊ - (CH ２ CH ２ O) ｂ -CH ２ CH ２ NH ２ (b is 1, 2 or 3); ＊ -CH ２ CH ２ S-CH ２ CH ２ NH ２ ; ＊ -(C ２－１０ ) Fluoroalkylene-NH ２ ; ＊ - (CH ２ ) ｃ NHC(O)(CH ２ ) ｃ’ -NH ２ wherein c and c' are independently 2 to 6; ＊ - (CH ２ ) ｄ NHC(O)NH(CH ２ ) ｄ’ -NH ２ wherein d and d' are independently 2 to 6; ＊ -(C １－１０ ) alkylene-C(O)-NH-(C ２－１０ ) alkylene-NH ２ ; ＊ -(C ２－１０ ) alkylene-O-NH ２ or ＊ -(C ２－１０ ) alkylene-SH; or a pharmaceutically acceptable salt thereof.

19. An intermediate compound for producing the immunogenic compound of any one of claims 4 to 12, having the structure of formula (IV): 【Chemistry 13】 (In the formula, R is OH or 【Chemistry 14】 and m is 3, 4, 5, 6, 7 or 8, preferably 3, 4, 5 or 6; n is 1, 2, 3, 4, 5 or 6, preferably 2, 3 or 4; L is, ＊ -(C ２－１０ ) alkylene-NH-, preferably ＊ - (CH ２ ) ａ -NH- (a is 2 to 10, more preferably 5); ＊ - (CH ２ CH ２ O) ｂ -CH ２ CH ２ NH- (b is 1, 2 or 3); ＊ -CH ２ CH ２ S-CH ２ CH ２ NH-; ＊ -(C ２－１０ ) fluoroalkylene-NH-; ＊ - (CH ２ ) ｃ NHC(O)(CH ２ ) ｃ’ -NH- (c and c' are each independently 2 to 6); ＊ - (CH ２ ) ｄ NHC(O)NH(CH ２ ) ｄ’ -NH- (d and d' are independently 2 to 6); ＊ -(C １－１０ ) alkylene-C(O)-NH-(C ２－１０ ) alkylene-NH-; or ＊ -(C ２－１０ ) alkylene-O-NH-; represents; T １ teeth, -C(O)-(C ０－１０ ) alkylene-C(O)X; —C(O)—CH ２ CH ２ -(OCH ２ CH ２ ) ｊ -C(O)X (j is 1 to 5); —C(O)—CH ２ (CH ２ ) ｋ - (SCH ２ (CH ２ ) ｋ’ ) ｋ’’ -C(O)X (k is 0 or 1, k' is 0 or 1, and k'' is 1, 2, or 3); 【Chemistry 15】 represents; -C(O)X represents -C(O)OH or an activated ester; Preferably, X is 【Chemistry 16】 represents; Y is Me, Et, Bu or -(CH ２ CH ２ O) ３ CH ３ or a pharmaceutically acceptable salt thereof.

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