Vaccine against klebsiella pneumoniae

Abstract

The present invention relates to novel oligosaccharide-carrier protein conjugates of Formula (I), in particular oligosaccharide-carrier protein conjugates of Formula (II), and their use as pharmaceuticals, in particular as vaccines. The invention also concerns related aspects including intermediates, as well as processes for the preparation of the immunogenic compounds. Furthermore, the invention relates to pharmaceutical compositions comprising the immunogenic compounds, as well as the use of the oligosaccharide-carrier protein conjugates of Formula (I) in biological assays.

Description

[0001] ID 410A

[0002] Vaccine against Klebsiella pneumoniae

[0003] This invention was made with U. S. Government support under Agreement Number 75A50122C00028, awarded by the U. S. Department of Health and Human Services. The U. S. Government has certain rights in the invention.

[0004] The present invention relates to novel oligosaccharide-carrier protein conjugates of Formula (I), in particular oligosaccharide-carrier protein conjugates of Formula (II), and their use as pharmaceuticals, in particular as vaccines. The invention also concerns related aspects including intermediates, as well as processes for the preparation of the immunogenic compounds. Furthermore, the invention relates to pharmaceutical compositions comprising the immunogenic compounds, as well as the use of the oligosaccharide-carrier protein conjugates of Formula (I) in biological assays.

[0005] Klebsiella pneumoniae (or K. pneumoniae) is a gram-negative, facultative anaerobic, rodshaped bacterium colonizing mainly respiratory, intestinal and urinary tracts as well as the skin and causing K. pneumoniae infections (KPIs). The bacterium mainly acts as an opportunistic pathogen. KPIs are a major cause of nosocomial infections, primarily affecting immunocompromised patients. Infections caused by K. pneumoniae are an important challenge in healthcare settings due to the emergence of strains resistant to almost all available antimicrobial agents and their worldwide dissemination. Infections caused by K. pneumoniae are responsible for high rates of morbidity and mortality. Thus, prevention of infections caused by K. pneumoniae is highly desirable, and vaccination is the most costefficient and the most powerful means to fight KPIs.

[0006] K. pneumoniae is an encapsulated bacterium, expressing lipopolysaccharides (LPS) and capsular polysaccharide (CPS, K-antigen) on their outer membrane, which contribute to the virulence of this species.

[0007] The LPS consists of three components, namely a lipid A moiety which serves as a membrane anchor, a common oligosaccharide core (COC) covalently bound to lipid A (LA), and a terminal antigenic polysaccharide comprising repeating saccharide units forming the O-antigen which is covalently bound to the core oligosaccharide. Extracted LPS has been shown to be pyrogenic, toxic and able to cause tissue damage. LPS can be masked by CPS and is usually less exposed to the surface than CPS. In respect of LPS, currently 11 major groups of O-antigens, also called O-serotypes, are known: 01, 02a (formerly known as Gal-I), 02ac, 02afg (formerly known as Gal-Ill), 02aeh (previously 09), 03 (includes sub-serotypes 03, 03a and 03b), 04, 05, 07, 08, 012 and 013. Other than the above 11 additional O-types have been reported based on the O-locus known as the OL series. Though O-antigens are less immunogenic than K-antigens and are exposed to a lesser extent to the surface of the membrane, they have also been considered for vaccination strategies. In a recent survey a large collection of clinical isolates determined the relative prevalence of CPS and LPS serotypes, particularly in multidrugresistant isolates (e.g. Lam et al., Microbial Genomics 2022;8:000800; DOI 10.1099 / mgen.0.000800).

[0008] The serogroup 03 has three subtypes namely 03, 03a and 03b which vary in the length of the repeating unit (Guachalla et al, Nature, Scientific Reports, 7:6635; DOI:10.1038 / s41598-017-06682-2; Rollenske et al, Nature Immunology, Vol 19, June 2018, 617-624, doi.org / 10.1038 / s41590-018-0106-2; Rollenske, PhD Thesis 2017, Humans naturally acquire cross-specific anti-glycan antibodies (hu-berlin.de)). 03 has a pentasaccharide repeating unit (RU), 03a has a tetrasaccharide RU and 03b has a trisaccharide RU, whereby the RUs exclusively consist of mannoses. The terminal RU terminates with a methyl-phosphate group (indicated below as “MeP”):

[0009] 03:

[0010]

[0011] WO2019106200 discloses synthetic conjugates of mannan oligosaccharides related to K. pneumoniae serotype 05, 03 and 03b. However, no examples for serotype 03b were provided, only non-conjugates oligosaccharides constituting a frameshift of 03b have been synthesized, the frameshift being — >2)-a-D-Man-(1, 3)-a-D-Man-(1, 3)-a-D-Man-(1— »]n.

[0012] Wantuch etal (bioRxiv 2023.12.12.571344) disclose a heptavalent O-antigen bioconjugate vaccine which exhibits differential functional antibody responses against diverse Klebsiella pneumoniae isolates. Besides others, bioconjugates of 03 and 03b antigen, respectively, with the carrier protein EPA (Pseudomonas aeruginosa exotoxin A) have been described. It was confirmed that these polysaccharides matched with the native K. pneumoniae O-antigens, including the terminal caps, i.e. the methyl-phosphate groups.

[0013] Most importantly, it needs to be noted that no significant cross-reactivity of antisera raised with 03b to the 03 antigen was detected, and that no significant cross-reactivity of antisera raised with 03 to the 03b antigen was detected. Consequently, both antigens, 03b and 03, were included in the heptavalent vaccine, in order to cover both serotypes.

[0014] WO2021259743 relates to immunogenic compositions comprising Klebsiella pneumoniae O-antigen polysaccharide bioconjugates, wherein each of the Klebsiella pneumoniae 01 v1, 02a, 02afg and 03b O-antigen polysaccharides were individually conjugated to a EPA as carrier protein.

[0015] Again, most importantly, no cross-reactivity between 03b and 03 was observed.

[0016] Though the quadrivalent vaccine according to WO201259743 was undergoing clinical studies phase l / ll (Safety and immunogenicity of a Klebsiella pneumoniae tetravalent bioconjugate vaccine (Kleb4V), Clinicaltrials.gov2022 [Available from: https: / / clinicaltrials.gov / ct2 / show / NCT04959344?term=limmatech&draw=2&rank=1), there are no approved vaccines available against K. pneumoniae, which demonstrates clearly the challenges associated with the development of such vaccines.

[0017] Given that mannose is ubiquitous in nature, generating a potent immune response remains challenging. Even in case an immune response is generated, the recognition of the antigen may be very weak or in some cases none. Besides this, generating a potent and long-lasting immune response is always a challenge.

[0018] It has now been surprisingly found that new oligosaccharide-carrier protein conjugates have improved properties as potential vaccines against Klebsiella pneumoniae. Specifically, cross-reactivity between two major O-antigens has been found, which makes them able to reduce the amount of carrier protein per vaccination, thereby reducing undesired carrier induced epitope suppression, and at the same time reducing manufacturing costs.

[0019] Description of the Figures

[0020] Figure 1: Characterization of 19-GC in comparison to CRM197 by HPLC-SEC.

[0021] Figure 2: Characterization of 23-GC and 24-GC in comparison to CRM197 by HPLC-SEC. Figure 3: Characterization of 25-GC in comparison to CRM197 by HPLC-SEC.

[0022] Figure 4: SDS-PAGE of 19-GC, 23-GC, 24-GC and 25-GC in comparison to CRM197 and Marker (protein size marker ROTI®Mark TRICOLOR XTRA, 250 µl, 1 x 250 µl (Carl Roth)).

[0023] Figure 5: The figure shows rabbit IgG levels obtained by ELISA with the respective immunogen-BSA conjugate coated. Rabbits were immunized i.m. with CRM197 glycoconjugates of the indicated immunogens. Rabbits received 3 doses of the respective glycoconjugates corresponding to 2 pg glycan per dose, adjuvanted with Aluminum hydroxide adjuvant, at days 0, 14 and 28 via the intramuscular route. Preimmune sera were obtained at day 0 prior to the first immunization. Post-immune sera were obtained 7 or 14 days after the third immunization. The bars show mean ± SEM of n=4 or n=6 rabbits. Day 0 values are at or slightly below 0. Sera were diluted 1:1000.

[0024] Figure 6: The figure shows rabbit IgG levels obtained by ELISA with isolated LPS from 03 or 03b strains coated. Bars in panel A show data for pooled postimmune sera with the control immunogen being a non-03 serogroup glycan that was immunized in parallel. Bars in panels B-D show mean values of n=4 or n=6 rabbits. Sera were diluted 1: 100.

[0025] Figure 7: The figure shows results of competitive ELISA experiments. Pooled postimmune antisera diluted 1:4000 to 1:8000 were pre-incubated with live K. pneumoniae bacteria (03 or 03b serotypes) at an OD600of 0.6 for 1 hour at room temperature. As controls, the same antisera were also pre-incubated with a control K. pneumoniae strain (02afg serotype). After removal of the bacteria by centrifugation, the supernatants were subjected to ELISA experiments with BSA conjugates coated. The bars show inhibition values that were normalized to the respective 02afg controls.

[0026] Detailed Description of the Invention

[0027] 1) In a first aspect, the present invention relates to an oligosaccharide-carrier protein conjugate of formula (I):

[0028]

[0029] wherein

[0030] R represents OH

[0031] m represents 3, 4, 5, 6, 7 or 8;

[0032] i represents an integer from 1 to imax, wherein imax is an integer corresponding to 90% of the number of lysine residues contained in the carrier protein CP;

[0033] -L-T- represents a linker L and a spacer T which together form a bridge having a backbone with a length of 5 to 25 atoms covalently linked together that forms 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 at the carrier protein CP, wherein the atoms of the backbone are selected from the group consisting of carbon, nitrogen, oxygen and sulphur; and

[0034] CP is selected from the group consisting of CRM197; diphtheria toxoid; tetanus toxoid; cholera toxin B subunit; Neisseria meningitidis outer membrane protein (OMP); and capsid protein of bacteriophage Q|3 (especially CRM197);

[0035] or a pharmaceutically acceptable salt thereof.

[0036] For the avoidance of doubt, each of the oligosaccharides of the oligosaccharide-carrier protein conjugate of formula (I), (II) and (Ila) is attached to the carrier protein CP via a linkerspacer group -L-T-, wherein the terminal end of the spacer T is attached to an amino group of a lysine residue at the carrier protein CP or to the amino group of the N-terminal amino acid of the carrier protein CP. Definitions provided herein are intended to apply uniformly to the compounds of Formula (I), (II), (Ila), (III) and (IV) as defined in any one of embodiments 1) to 60), and, mutatis mutandis, throughout the description and the claims unless an otherwise expressly set out definition provides a broader or narrower definition. It is well understood that a definition or preferred definition of a term defines and may replace the respective term independently of (and in combination with) any definition or preferred definition of any or all other terms as defined herein.

[0037] The oligosaccharide part (i.e. the antigen, or epitope) of the compounds of Formula (I), (II), (Ila), (III) and (IV) is composed of alpha-D-mannose-pyranosides:

[0038] alpha-D-mannopyranose / cr-D-Manp:

[0039]

[0040] the dotted lines show the point of attachment, namely C1 and C3

[0041] alpha-D-mannopyranose / cr-D- Manp:

[0042]

[0043] the dotted lines show the point of attachment, namely C1 and C2

[0044] The oligosaccharide-carrier protein conjugate according to the present invention is an immunogenic compound, and the term “oligosaccharide-carrier protein conjugate” is used synonymously to “immunogenic compound” or to “glycoconjugate”.

[0045] The term “essentially”, for example when used in a term such as "essentially pure" is understood in the context of the present invention to mean especially that the respective oligosaccharide-carrier protein conjugate I oligosaccharide I oligosaccharide-linker compound I oligosaccharide-linker-spacer compound I glycoconjugate consists in an amount of at least 90, especially of at least 95, and notably of at least 99 per cent by weight of the respective pure oligosaccharide-carrier protein conjugate / oligosaccharide / oligosaccharide-linker compound / oligosaccharide-linker-spacer compound / glycoconjugate.

[0046] Whenever a substituent is denoted as optional, it is understood that such substituent may be absent (i.e. the respective residue is unsubstituted with regard to such optional substituent), in which case all positions having a free valency (to which such optional substituent could have been attached to; such as for example in an aromatic ring the ring carbon atoms and I or the ring nitrogen atoms having a free valency) are substituted with hydrogen where appropriate. Likewise, in case the term “optionally” is used in the context of (ring) heteroatom(s), the term means that either the respective optional heteroatom(s), or the like, are absent (i.e. a certain moiety does not contain heteroatom(s) I is a carbocycle I or the like), or the respective optional heteroatom (s), or the like, are present as explicitly defined.

[0047] The variable i represents an integer from 1 to imax, wherein imax is an integer corresponding to 90% of the number of lysine residues contained in the carrier protein CP. Preferably, i represents an integer from 3 to imax, wherein imax is an integer corresponding to 90% of the number of lysine residues contained in the carrier protein CP; more preferably, i represents an integer from 3 to 75% of the number of lysine residues contained in the carrier protein CP; even more preferably, i represents an integer from 4 to 40% of the number of lysine residues contained in the carrier protein CP. As an example, in case the carrier protein CP contains 39 lysine residues, the range “from 3 to 40% of the number of lysine residues contained in the carrier protein CP” means that i ranges from 3 to 16. For the avoidance of any doubt, throughout the present application, the term “lysine residue” and “lysine site” are used synonymously.

[0048] The term “CRM197” refers to Cross Reactive Material 197, which is a nontoxic mutant version of the diphtheria toxin, wherein the single amino acid exchange of a glycine (Gly, G) in position 52 to a glutamic acid (Glu, E) renders the protein non-toxic. It is described in more detail in embodiment 5).

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

[0050] The term “Tetanus toxoid” relates to a formalin-inactivated version of tetanus toxin having SEQ ID NO: 3 (Uniprot ID: P04958). The present invention encompasses a protein having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 99.9% identity to amino acid sequence SEQ ID NO: 3 (preferably 95%, 96%, 97%, 98%, 99% or 99.9% identity to amino acid sequence SEQ ID NO: 3). Tetanus toxoid can be prepared as described e.g. by G. Ramon at al., OR Soc Biol, 93 (1925), pp. 508-509.

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

[0052] The term “Neisseria meningitidis outer membrane protein” (OMP) relates to a protein having SEQ ID NO: 5 (Uniprot ID: Q51229). The present invention encompasses a protein having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 99.9% identity to amino acid sequence SEQ ID NO: 5 (preferably 95%, 96%, 97%, 98%, 99% or 99.9% identity to amino acid sequence SEQ ID NO: 5).

[0053] The term “Capsid protein of bacteriophage Q|3” relates to a protein having SEQ ID NO: 6 (Uniprot ID: P03615). The present invention encompasses a protein having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 99.9% identity to amino acid sequence SEQ ID NO: 6 (preferably 95%, 96%, 97%, 98%, 99% or 99.9% identity to amino acid sequence SEQ ID NO: 6).

[0054] 2) A further embodiment relates to the oligosaccharide-carrier protein conjugate according to embodiment 1), or a pharmaceutically acceptable salt thereof, wherein m represents 4, 5 or6.

[0055] 3) A further embodiment relates to the oligosaccharide-carrier protein conjugate according to embodiment 1), or a pharmaceutically acceptable salt thereof, wherein m represents 5.

[0056] 4) The oligosaccharide-carrier protein conjugate according to embodiments 1), 2) or 3) may bear a linker-spacer -L-T- as disclosed in any one of embodiments 8) to 13), or any combination of embodiments 23) to 30), and in particular as disclosed in embodiment 31) or 32). This means that the linker spacer -L-T- of formula (I) is the same as the linker spacer -L-T- described in connection to CRM197. Hence, the same description and definitions apply, mutatis mutantis, to the carrier protein CP. In particular, the combination of embodiment 1) with either embodiment 31) or embodiment 32), the combination of embodiment 2) with either embodiment 31) or embodiment 32), and the combination of embodiment 3) with either embodiment 31) or embodiment 32) are herewith explicitly mentioned.

[0057] 5) In a further aspect, the present invention relates to an oligosaccharide-carrier protein conjugate of formula (II)

[0058]

[0059] wherein

[0060] R represents OH

[0061] m represents 3, 4, 5, 6, 7 or 8;

[0062] i represents an integer from 1 to 28;

[0063] -L-T- represents a linker L and a spacer T which together form a bridge having a backbone with a length of 5 to 25 atoms covalently linked together that forms 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 at the carrier protein CP, wherein the atoms of the backbone are selected from the group consisting of carbon, nitrogen, oxygen and sulphur;

[0064] or a pharmaceutically acceptable salt thereof. “CRM-197” refers to Cross Reactive Material 197, which is a nontoxic mutant version of the diphtheria toxin, wherein the single amino acid exchange of a glycine (Gly, G) in position 52 to a glutamic acid (Glu, E) renders the protein non-toxic.

[0065] CRM-197 is produced by C. diphtheriae infected by the nontoxigenic phage pi97tox created by nitrosoguanidine mutagenesis of the toxigenic corynephage beta (Uchida et al, J. Biol. Chem., 1973, Vol. 245, No. 11, pp. 3838-3844). The CRM197 protein is a safe and effective T-cell dependent carrier for saccharides. CRM197 is for instance described by Giannini et al. in Nucleic Acids Research, Vol 12, No. 10, 1984, pp. 4063-4069. Further details about CRM197 and production thereof can be found e.g. in US5,614,382, which are incorporated herein by reference. CRM197 may be produced in various expression systems, for instance in Corynebacterium diphtheriae, Escherichia coli or Pseudomonas fluorescens (Hickey et al, J. Pharm. Sci., 2018, 107, 1806-1819).

[0066] In the present invention, the term “CRM197” encompasses a protein having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.8% or 99.9% identity to 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 amino acid sequence SEQ ID NO: 1; and notably at least 95%, 96%, 97%, 98%, 99% or 99.9% identity to amino acid sequence SEQ ID NO: 1), which optionally comprises an additional methionine (Met, M) at the N-terminus, and / or optionally includes residues resulting from functionalizing CRM197 at lysine sites, which residues may be in a capped (i.e. deactivated) form.

[0067] The phrase “residues resulting from functionalizing CRM197 at lysine sites” means that CRM197 is functionalized at lysine sites with functional groups suitable for forming a covalent bond to the linker and / or spacer part attached to the antigen, i.e. the oligosaccharide-linker part of the conjugate. Such lysine-functionalized CRM197 is known to the skilled person. The functional groups are particularly suitable for linking thiols or for performing click-chemistry. For instance, such functional groups are groups containing a bromo-acetamide, a iodo-acetamide, a maleimide, an azido, or an alkyne group. This means that CRM197 optionally includes lysine residues functionalized with a bromoacetamide, a iodo-acetamide, a maleimide, an azido, or an alkyne group (preferably a bromo-acetamide, a iodo-acetamide, a maleimide group), which groups may be in a capped form.

[0068] Preferred functionalized CRM197 contains groups carrying bromo-acetamide, iodoacetamide or maleimide groups, all of them being suitable for reaction with thiol-groups provided by the oligosaccharide / linker moiety. Unreacted functional groups at CRM197 may subsequently be quenched with any pharmaceutically acceptable thiol, such as for instance L-cysteine or cysteamine (2-aminoethane-1 -thiol) to give the “capped form”.

[0069] Preferred lysine-functionalized CRM197 is selected from the group consisting of:

[0070]

[0071] wherein Z is Br or I, q is 2 or 3, and

[0072] t is from 1 to 28, preferably form 15-25;

[0073]

[0074] wherein r is 2 or 3, and t’ is from 1 to 28, preferably form 15-25; and

[0075]

[0076] wherein Z is Br or I, and t” is from 1 to 28, preferably form 15-25.

[0077] In a preferred embodiment, CRM197 is not functionalized in the above-described way. This means that there is no “pre-functionalization”, but rather, the “natural” lysine residues, i.e. the non-modified amino groups of the lysine residues, are used for directly attaching the oligosaccharide / linker / spacer part thereto.

[0078] The amino acid sequence of CRM197 is known to the skilled person, and is outlined below as SEQ ID NO:1.

[0079] The use of CRM197 for the synthesis of saccharide conjugates and preferred conjugation sites on CRM197 has been reported (e.g. Möginger et al., Sci. Rep. 6, 20488; doi:10.1038 / srep20488 (2016)), which is incorporated herein by reference.

[0080] The phrase “-L-T- represents a linker L and a spacer T which together form a bridge having a backbone with a length of 5 to 25 atoms covalently linked together that forms 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 at the carrier protein CRM197 (or CP, if applicable), wherein the atoms of the backbone are selected from the group consisting of carbon, nitrogen, oxygen and sulphur” means that the backbone may be saturated, unsaturated, unsubstituted or substituted with one or more (especially 1, 2, 3 or 4) substituents independently selected from the group consisting of oxo, (Ci-4)alkyl, fluoro, and (Ci-2)alkoxy (especially oxo), and optionally a part of a ring structure may be part of the backbone. The ring structure may be a saturated, unsaturated or aromatic 3- to 8-membered ring including condensed ring systems of 2 to 4 rings, wherein the ring atoms are selected from the group consisting of carbon, nitrogen, oxygen and sulphur (especially from carbon and nitrogen), and the ring is unsubstituted or substituted with one or more (especially 1, 2, 3 or 4) substituents independently selected from the group consisting of oxo, (Ci-4)alkyl, halogen, and (Ci-2)alkoxy (especially oxo).

[0081] For avoidance of any doubt, the count of 5 to 25 atoms relates to the count of atoms of the backbone, not of the bridge.

[0082] Examples for 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, / ][1,2,3]triazolo[4,5-c]azocine, cyclohexane, and benzene as follows:

[0083]

[0084] The introduction of these rings or ring systems is known to the skilled person in the field of linker chemistry.

[0085] The phrase “the backbone may be unsaturated” means that the backbone chain may contain one or more double bonds, which may or may not be part of a ring system. For instance, the atom counting in a bridge having a saturated backbone with 3 oxosubstitutions and which backbone is part of a ring system is as follows:

[0086]

[0087] ■=> the count of -L-T- is 16

[0088] Hence, the count of the atoms forming the backbone starts with the first atom after the oxygen at C1 and ends with the last atom attached to a lysine nitrogen of CRM197.

[0089] An oxygen atom in a saturated chain is preferably separated from another oxygen atom by one or more (especially 2, 3, 4 or 5, and notably 2) carbon atoms.

[0090] A sulphur atom in a saturated chain is preferably separated from another sulphur atom by one or more (especially 1, 2, 3, 4 or 5) carbon atoms.

[0091] The term "halogen" means fluorine, chlorine, or bromine, preferably fluorine or chlorine, more preferably fluorine. A halogen residue or substituent is fluoro, chloro, bromo, preferably fluoro or chloro, more preferably fluoro.

[0092] The term “oxo” relates to the functional group =0, i.e. a substituent oxygen atom connected to another atom (preferably a carbon atom) by a double bond.

[0093] The term "alkyl", used alone or in combination, means a straight or branched saturated hydrocarbon chain containing one to four carbon atoms. The term "(Cx-y)alkyl" (x and y each being an integer), refers to an alkyl group as defined before containing xto y carbon atoms. For example a (Ci.4)alkyl group contains from one to four carbon atoms. Examples of (Ci.4)alkyl groups are methyl, ethyl, n-propyl, / so-propyl, n-butyl, / so-butyl, sec.-butyl and tert.-butyl. Examples of (Ci.2)alkyl groups are methyl and ethyl.

[0094] The term "alkoxy", used alone or in combination, refers to an alkyl-O- group wherein the alkyl group is as defined before. The term "(Cx.y)alkoxy" (x and y each being an integer) refers to an alkoxy group as defined before containing xto y carbon atoms. For example a (Ci-2)alkoxy group means a group of the formula (Ci-2)alkyl-0- in which the term "(Ci-2)alkyl" has the previously given significance. Examples of (Ci-2)alkoxy groups are methoxy and ethoxy. The term “oligosaccharide-carrier protein conjugate” as used herein is taken synonymously to the term “immunogenic compound” or “glycoconjugate”.

[0095] 6) A further embodiment relates to the oligosaccharide-carrier protein conjugate according to embodiment 5), or a pharmaceutically accepable salt thereof, wherein m is 4, 5 or 6. 7) A further embodiment relates to the oligosaccharide-carrier protein conjugate according to embodiment 5), or a pharmaceutically accepable salt thereof, wherein m is 5.

[0096] 8) A further embodiment relates to the oligosaccharide-carrier protein conjugate according to any one of embodiments 1), 2) or 3), and to any one of embodiments 5), 6) or 7), or a pharmaceutically accepable salt thereof, wherein the bridge does not contain an aromatic or heteroaromatic ring.

[0097] 9) A further embodiment relates to the oligosaccharide-carrier protein conjugate according to any one of embodiments 1), 2) or 3), and to any one of embodiments 5), 6) or 7), or a pharmaceutically accepable salt thereof, wherein

[0098] -L-T- represents a linker L and a spacer T which together form a bridge having a backbone with a length of 5 to 25 atoms covalently linked together that forms 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 at the carrier protein CRM197 (or CP, if applicable), bearing at most one double bond,

[0099] wherein the atoms of the backbone are selected from the group consisting of carbon, nitrogen, oxygen and sulphur, and

[0100] wherein the backbone may be substituted with one or more (especially 1, 2, 3 or 4) substituents independently selected from the group consisting of oxo, (Ci-4)alkyl, fluoro, and (Ci-2)alkoxy (especially oxo), and

[0101] wherein a part of the backbone optionally may be part of a 4-, 5- or 6-membered ring selected from:

[0102]

[0103] Thereby, the “at most one double bond” is preferably the double bond of the cyclobut-3-ene-1,2-dione ring.

[0104] 10) A further embodiment relates to the oligosaccharide-carrier protein conjugate according to any one of embodiments 1), 2) or 3), and to any one of embodiments 5), 6) or 7), or a pharmaceutically accepable salt thereof, wherein

[0105] -L-T- represents a linker L and a spacer T which together form a bridge which consists of a backbone which is a saturated chain counting from 5 to 25 atoms selected from the group consisting of carbon, nitrogen, oxygen and sulphur (especially carbon, nitrogen and oxygen), which chain may be unsubstituted or substituted with one or more (especially 1, 2, 3 or 4) substituents independently selected from the group consisting of oxo, (Ci-4)alkyl, fluoro and (Ci-2)alkoxy (especially oxo). This means that the bridge consists of a saturated chain counting from 5 to 25 atoms selected from the group consisting of carbon, nitrogen, oxygen and sulphur (especially carbon, nitrogen and oxygen), which chain may be unsubstituted or substituted with one or more (especially 1, 2, 3 or 4) substituents independently selected from the group consisting of oxo, (Ci-4)alkyl, fluoro and (Ci-2)alkoxy (especially oxo). For the avoidance of any doubt, in this embodiment, the bridge does not contain a ring structure.

[0106] 11) A further embodiment relates to the oligosaccharide-carrier protein conjugate according to any one of embodiments 1), 2) or 3), and to any one of embodiments 5), 6) or 7), or a pharmaceutically accepable salt thereof, wherein

[0107] -L-T- represents a linker L and a spacer T which together form a bridge which consists of a backbone which is a saturated chain counting from 5 to 25 atoms selected from the group consisting of carbon, nitrogen and oxygen (especially carbon and nitrogen), which chain may be unsubstituted or substituted with one or more (especially 1, 2, 3 or 4) substituents independently selected from the group consisting of oxo, (Ci-4)alkyl, fluoro, and (Ci-2)alkoxy (especially oxo). This means that the bridge consists of a saturated chain counting from 5 to 25 atoms selected from the group consisting of carbon, nitrogen and oxygen (especially carbon and nitrogen), which chain may be unsubstituted or substituted with one or more (especially 1, 2, 3 or 4) substituents independently selected from the group consisting of oxo, (Ci-4)alkyl, fluoro, and (Ci-2)alkoxy (especially oxo). For the avoidance of any doubt, in this embodiment, the bridge does not contain a ring structure. 12) A further embodiment relates to the oligosaccharide-carrier protein conjugate according to any one of embodiments 1), 2) or 3), and to any one of embodiments 5), 6) or 7), or a pharmaceutically accepable salt thereof, wherein the backbone of the bridge has a length of 5 to 20, preferably 5 to 16, atoms covalently linked together that forms 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 at the carrier protein CRM197 (or CP, if applicable).

[0108] 13) A further embodiment relates to the oligosaccharide-carrier protein conjugate according to any one of embodiments 1), 2) or 3), and to any one of embodiments 5), 6) or 7), or a pharmaceutically accepable salt thereof, wherein

[0109] L represents

[0110] *-(C2-w)alkylene-NH-;

[0111] *-(CH2CH2O)b-CH2CH2NH-, wherein b is 1, 2 or 3;

[0112] *-CH2CH2S-CH2CH2NH-;

[0113] *-(C2-io)fluoroalkylene-NH-;

[0114] *-(CH2)CNHC(O)(CH2)C-NH-, wherein c and c’ are independently from each other from 2 to 6;

[0115] *-(CH2)dNHC(O)NH(CH2)d -NH-, wherein d and d’ are independently from each other from 2 to 6;

[0116] *-(Ci-io)alkylene-C(0)-NH-(C2-io)alkylene-NH-; or

[0117] *-(C2-io)alkylene-0-NH-;

[0118] T represents

[0119] -C(0)-(Co-io)alkylene-C(0)-;

[0120] -C(O)-CH2CH2-(OCH2CH2)j-C(O)-, wherein j is from 1 to 5;

[0121] -C(O)-CH2(CH2)k-(SCH2(CH2)k’)k”-C(O)-, wherein k is 0 or 1, k’ is 0 or 1, and k” is 1, 2, or 3;

[0122]

[0123] , wherein I is 1 or 2; or

[0124]

[0125] , wherein p is from 1 to 4, and p’ is 1 or 2; or

[0126] L-T represents

[0127] *-(C2-io)alkylene-S-R1; and

[0128] R1represents

[0129]

[0130] The appointed in the linker L means that at this location, the linker is attached to the oligosaccharide.

[0131] The appointed in the spacer T means that at this location, the spacer is attached to the linker L.

[0132] The appointed in R1means that at this location, R1is attached to the sulphur.

[0133] The term “-(Cx-y)alkylene-” (x and y each being an integer), used alone or in combination, refers to a bivalently bound saturated straight or branched hydrocarbon chain containing x to y carbon atoms. For example a (C2- )alkylene group contains from two to ten carbon atoms, and a (Co-w)alkylene group is either a bond (i.e. absent, C being zero) or an alkylene group from one to ten carbon atoms. Straight -(Cx-y)alkylene-, i.e. -(CH2)x-y- is preferred. Representative examples of (C2- )alkylene groups are ethylene, propylene, butylene, pentylene, hexylene, heptylene, octylene, nonylene and decylene (especially 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).

[0134] The term “(Cx-y)fluoroalkylene” (x and y each being an integer), used alone or in combination, refers to a bivalently bound saturated straight or branched chain hydrocarbon group containing x to y carbon atoms in which one or more (and possibly all) hydrogen atoms have been replaced with fluorine. Straight -(Cx-y)fluoroalkylene- is preferred.

[0135] For the avoidance of any doubt, in the present embodiments, the length of the backbone of -L-T- is 5 to 25 atoms, 5 to 20 atoms, or 5 to 16 atoms. This means that the linker L and the spacer T, including R1where applicable, together form a bridge having a backbone with a length of 5 to 25 (5 to 20, or 5 to 16) atoms covalently linked together that forms 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 at the carrier protein CRM197 (or CP, if applicable).

[0136] 14) A further embodiment relates to the oligosaccharide-carrier protein conjugate according to embodiment 13), ora pharmaceutically accepable salt thereof, wherein L represents

[0137] *-(CH2)a-NH-; wherein a is from 2 to 10;

[0138] *-(CH2CH2O)b-CH2CH2NH-, wherein b is 1, 2 or 3;

[0139] *-CH2CH2S-CH2CH2NH-;

[0140] *-(C2-io)fluoroalkylene-NH- with fluoroalkylene being a saturated straight chain; *-(CH2)CNHC(O)(CH2)C-NH-, wherein c and c’ are independently from each other from 2 to 6;

[0141] *-(CH2)dNHC(O)NH(CH2)d -NH-, wherein d and d’ are independently from each other from 2 to 6;

[0142] *-(CH2)e-C(O)-NH-(CH2)e’-NH-; wherein e is from 1 to 10 and e’ is from 2 to 10; or *-(CH2)f-O-NH-, wherein f is from 2 to 10; or

[0143] L-T represents

[0144] *-(CH2)g-S-R1, wherein g is from 2 to 10.

[0145] 15) A further embodiment relates to the oligosaccharide-carrier protein conjugate according to embodiment 13), ora pharmaceutically accepable salt thereof, wherein

[0146] L represents *-(CH2)a-NH-; wherein a is from 2 to 10;

[0147] *-(CH2CH2O)b-CH2CH2NH-, wherein b is 1, 2 or 3;

[0148] *-(C2-io)fluoroalkylene-NH- with fluoroalkylene being a saturated straight chain; *-(CH2)e-C(O)-NH-(CH2)e’-NH-; wherein e is from 1 to 10 and e’ is from 2 to 10; or *-(CH2)f-O-NH-, wherein f is from 2 to 10; or

[0149] L-T represents

[0150] *-(CH2)g-S-R1, wherein g is from 2 to 10.

[0151] 16) A further embodiment relates to the oligosaccharide-carrier protein conjugate according to embodiment 13), ora pharmaceutically accepable salt thereof, wherein L represents

[0152] *-(CH2)a-NH-; wherein a is from 2 to 10, preferably from 2 to 6;

[0153] *-(CH2CH2O)b-CH2CH2NH-, wherein b is 1, 2 or 3, preferably 1 or 2;

[0154] *-(CH2)e-C(O)-NH-(CH2)e’-NH-; wherein e is from 1 to 10, preferably from 1 to 6 and e’ is from 2 to 10, preferably from 2 to 6; or

[0155] *-(CH2)f-O-NH-, wherein f is from 2 to 10, preferably from 2 to 6; or

[0156] L-T represents

[0157] *-(CH2)g-S-R1, wherein g is from 2 to 10, preferably from 2 to 6.

[0158] 17) A further embodiment relates to the oligosaccharide-carrier protein conjugate according to embodiment 13), ora pharmaceutically accepable salt thereof, wherein L represents

[0159] *-(CH2)a-NH-; wherein a is from 2 to 10, preferably from 2 to 6;

[0160] *-(CH2CH2O)b-CH2CH2NH-, wherein b is 1, 2 or 3, preferably 1 or 2; or

[0161] *-(CH2)f-O-NH-, wherein f is from 2 to 10, preferably from 2 to 6.

[0162] 18) A further embodiment relates to the oligosaccharide-carrier protein conjugate according to embodiment 13), ora pharmaceutically accepable salt thereof, wherein

[0163] L represents

[0164] *-(CH2)a-NH-; wherein a is from 2 to 10, preferably from 2 to 6; or

[0165] *-(CH2CH2O)b-CH2CH2NH-, wherein b is 1, 2 or 3, preferably 1 or 2; 19) A further embodiment relates to the oligosaccharide-carrier protein conjugate according to embodiment 13), or a pharmaceutically accepable salt thereof, wherein L represents *-(CH2)a-NH-; wherein a is from 2 to 10, preferably from 2 to 6.

[0166] 20) A further embodiment relates to the oligosaccharide-carrier protein conjugate according to embodiment 13), ora pharmaceutically accepable salt thereof, wherein

[0167] L represents *-(CH2)2-NH-, *-(CH2)3-NH-, *-(CH2)4-NH-, *-(CH2)5-NH-, or *-(CH2)6-NH-.

[0168] 21) A further embodiment relates to the oligosaccharide-carrier protein conjugate according to embodiment 13), ora pharmaceutically accepable salt thereof, wherein

[0169] L represents *-(CH2)5-NH-.

[0170] 22) A further embodiment relates to the oligosaccharide-carrier protein conjugate according to embodiment 13), ora pharmaceutically accepable salt thereof, wherein

[0171] L represents *-(CH2CH2O)b-CH2CH2NH-, wherein b is 1 or 2; preferably 1.

[0172] 23) A further embodiment relates to the oligosaccharide-carrier protein conjugate according to embodiment 13) and in combination with L as described in any one of embodiments 14) to 22), or a pharmaceutically accepable salt thereof, wherein

[0173] T represents

[0174] -C(O)-(CH2)h-C(O)-, wherein h is from 0 to 10;

[0175] -C(O)-CH2CH2-(OCH2CH2)j-C(O)-, wherein j is from 1 to 5;

[0176] -C(O)-CH2(CH2)k-(SCH2(CH2)k’)k”-C(O)-, wherein k is 0 or 1, k’ is 0 or 1, and k” is 1, 2, or 3

[0177]

[0178] , wherein I is 1 or 2; or

[0179]

[0180] , wherein p is from 1 to 4, and p’ is 1 or 2.

[0181] 24) A further embodiment relates to the oligosaccharide-carrier protein conjugate according to embodiment 13) and in combination with L as described in any one of embodiments 14) to 22), or a pharmaceutically accepable salt thereof, wherein

[0182] T represents

[0183] -C(O)-(CH2)h-C(O)-, wherein h is from 0 to 10, preferably from 0 to 6;

[0184] -C(O)-CH2CH2-(OCH2CH2)j-C(O)-, wherein j is from 1 to 5, preferably from 1 to 3, more preferably 1;

[0185] -C(O)-CH2(CH2)k-(SCH2(CH2)k’)k”-C(O)-, wherein k is 0 or 1, k’ is 0 or 1, and k” is 1, 2, or 3, preferably 1; or

[0186]

[0187] 25) A further embodiment relates to the oligosaccharide-carrier protein conjugate according to embodiment 13) and in combination with L as described in any one of embodiments 14) to 22), or a pharmaceutically accepable salt thereof, wherein

[0188] T represents

[0189] -C(O)-(CH2)h-C(O)-, wherein h is from 0 to 6;

[0190] -C(O)-CH2CH2-(OCH2CH2)j-C(O)-, wherein j is 1 or 2; or

[0191] -C(O)-CH2(CH2)k-(SCH2(CH2)k’)k”-C(O)-, wherein k is 0 or 1, k’ is 0 or 1, and k” is 1, preferably wherein k and k’ are 0 and k” is 1.

[0192] 26) A further embodiment relates to the oligosaccharide-carrier protein conjugate according to embodiment 13) and in combination with L as described in any one of embodiments 14) to 22), or a pharmaceutically accepable salt thereof, wherein

[0193] T represents -C(O)-(CH2)h-C(O)-, wherein h is 0, 1, 2, 3, 4, 5, or 6, preferably 4; or

[0194] -C(O)-CH2CH2-(OCH2CH2)j-C(O)-, wherein j is 1 or 2.

[0195] 27) A further embodiment relates to the oligosaccharide-carrier protein conjugate according to embodiment 13) and in combination with L as described in any one of embodiments 14) to 22), or a pharmaceutically accepable salt thereof, wherein

[0196] T represents -C(O)-(CH2)h-C(O)-, wherein h is 0, 1, 2, 3, 4, 5, or 6, preferably 4.

[0197] 28) A further embodiment relates to the oligosaccharide-carrier protein conjugate according to embodiment 13) and in combination with L as described in any one of embodiments 14) to 22), or a pharmaceutically accepable salt thereof, wherein

[0198] T represents

[0199]

[0200] , wherein p is from 1 to 4, preferably 1, and p’ is 1 or 2.

[0201] 29) A further embodiment relates to the oligosaccharide-carrier protein conjugate according to embodiment 13) and in combination with L as described in any one of embodiments 14) to 22), or a pharmaceutically accepable salt thereof, wherein

[0202] T represents

[0203]

[0204] , wherein I is 1 or 2.

[0205] 30) A further embodiment relates to the oligosaccharide-carrier protein conjugate according to any one of embodiments 13) to 16), 23), 28), and 29), or a pharmaceutically accepable salt thereof, wherein

[0206] R1represents

[0207]

[0208] Preferably, R1represents:

[0209] , wherein q is 2 or 3; or

[0210]

[0211] 31) A further embodiment relates to the oligosaccharide-carrier protein conjugate according to any one of embodiments 1), 2) or 3), and to any one of embodiments 5), 6) or 7), or a pharmaceutically accepable salt thereof, wherein

[0212] L represents

[0213] *-(CH2)2-NH-, *-(CH2)3-NH-, *-(CH2)4-NH-, *-(CH2)5-NH-, or *-(CH2)6-NH-; and

[0214] T represents

[0215] -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)-.

[0216] 32) A further embodiment relates to the oligosaccharide-carrier protein conjugate according to any one of embodiments 1), 2) or 3), and to any one of embodiments 5), 6) or 7), or a pharmaceutically accepable salt thereof, wherein L represents *-(CH2)5-NH- and T represents -C(O)-(CH2)4-C(O)-.

[0217] 33) A preferred embodiment is the oligosaccharide-carrier protein conjugate with the following formula (II):

[0218]

[0219] wherein

[0220] R represents OH

[0221] m represents 3, 4, 5, 6, 7 or 8;

[0222] i is from 1 to 28;

[0223] L represents

[0224] *-(CH2)2-NH-, *-(CH2)3-NH-, *-(CH2)4-NH-, *-(CH2)5-NH-, or *-(CH2)6-NH-; and

[0225] T represents

[0226] -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)-;

[0227] or a pharmaceutically acceptable salt thereof.

[0228] 34) A further embodiment relates to the oligosaccharide-carrier protein conjugate according to embodiment 33), or a pharmaceutically acceptable salt thereof, wherein R represents OH m represents 4, 5 or 6, preferably 5;

[0229] i is from 1 to 28, preferably from 8 to 15;

[0230] L represents

[0231] *-(CH2)2-NH-, *-(CH2)3-NH-, *-(CH2)4-NH-, *-(CH2)5-NH-, or *-(CH2)6-NH- (preferably *-(CH2)5-NH-); and

[0232] T represents

[0233] -C(O)-C(O)-, -C(O)-CH2-C(O)-, -C(O)-(CH2)2-C(O)-, -C(O)-(CH2)3-C(O)-,

[0234] -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)-).

[0235] 35) A further embodiment relates to the oligosaccharide-carrier protein conjugate according to any one of embodiments 1) to 34), or a pharmaceutically accepable salt thereof, wherein i is from 1 to 28, 1 to 25, 1 to 23; 1 to 20, 1 to 18, 1 to 16, 3 to 25, 3 to 23, 3 to 20, 3 to 18, 3 to 16, 4 to 25, 4 to 23; 4 to 20, 4 to 18, 4 to 16, 5 to 23, 5 to 20, 6 to 20, 6 to 19, 6 to 18, 7 to 18, 7 to 16, and 8 to 15.

[0236] The variable i describes the loading of antigens, i.e. oligosaccharides on the CRM197 protein carrier and is an integer in respect of one single molecule. However, when considering the glycoconjugate as a product of more than one single molecule, it has to be noted that the loading can be described as a statistical distribution, i.e. essentially a Gaussian distribution. The chemical process of producing the product results in a mixture of molecules with such statistical distribution of the loading, and the loading is then provided as the mean of the statistical distribution, in particular the Gaussian distribution.

[0237] It is to be understood that for i > 2, m of the two or more oligosaccharides, that are attached via -L-T- to the carrier protein CP or CRM197, respectively, may be the same or different. Preferably, all i oligosaccharides are represented by the same m (i.e. have identical structures). The linker-spacer unit -L-T- is identical for the i oligosaccharides of a specific oligosaccharide-carrier protein conjugate.

[0238] In other words, preferred compounds are those that have uniform oligosaccharide / linker / spacer residues, i.e. which bear only one specific type of oligosaccharide / linker / spacer residue attached to the carrier protein, preferably to CRM197.

[0239] 36) A further embodiment relates to the oligosaccharide-carrier protein conjugate according to anyone of embodiments 1) to 34), ora pharmaceutically accepable salt thereof, wherein i is from 8 to 15. 37) A preferred embodiment relates to the oligosaccharide-carrier protein conjugate selected from the group consisting of

[0240]

[0241] (Ila);

[0242] wherein R is OH, m is 5, and i is from 1 to 28, or a pharmaceutically acceptable salt thereof. For the avoidance of any doubt, the oligosaccharide-carrier protein conjugates of formulae (Ila) according to this embodiment can also be schematically drawn as follows:

[0243]

[0244] (Ha’): wherein R is OH, m is 5, and i is from 1 to 28, or a pharmaceutically acceptable salt thereof.

[0245] CRM197’ means CRM197 as defined herein, with the only difference in that in formulae (Ila’), the amino-group of the lysine residue is specifically shown as the attachment position of the linker / spacer part -L-T-.

[0246] Preferred values for i are those as disclosed in embodiment 35) or, especially, 36).

[0247] 38) Hence, a particularly preferred embodiment relates to the oligosaccharide-carrier protein conjugate of formula (Ila):

[0248]

[0249] (Ha):

[0250] wherein R is OH, m is 5, and i is from 8 to 15, or a pharmaceutically acceptable salt thereof.

[0251] The invention, thus, relates to compounds of the Formula (I) as defined in embodiment 1), and to such compounds further limited by the characteristics of any one of embodiments 2) to 4), to compounds of Formula (II) as defined in embodiment 5), and to such compounds further limited by the characteristics of any one of embodiments 6) to 38), including compounds of Formula (Ila), under consideration of their respective dependencies; to pharmaceutically acceptable salts thereof; and to the use of such compounds as further described below. Compounds of Formula (III) and (IV) are intermediates to prepare compounds of Formula (I), (II) and (Ila), respectively. It is to be understood that the embodiments relating to the definition of L-T as specified in embodiments 8) to 32) in relation to the compound of Formula (II) also apply vice versa as a definition of L-T in respect of a compound of Formula (I) according to any one of embodiments 1) to 3). For avoidance of doubt, especially the following embodiments relating to the combinations of linker L and spacer T for the compounds of Formula (I) and Formula (II) are herewith specifically disclosed in individualized form:

[0252] 23+14, 23+15, 23+16, 23+17, 23+18, 23+19, 23+20, 23+21, 23+22, 24+14, 24+15, 24+16, 24+17, 24+18, 24+19, 24+20, 24+21, 24+22, 25+14, 25+15, 25+16, 25+17, 25+18, 25+19, 25+20, 25+21, 25+22, 26+14, 26+15, 26+16, 26+17, 26+18, 26+19, 26+20, 26+21, 26+22, 27+14, 27+15, 27+16, 27+17, 27+18, 27+19, 27+20, 27+21, 27+22, 28+14, 28+15, 28+16, 28+17, 28+18, 28+19, 28+20, 28+21, 28+22, 29+14, 29+15, 29+16, 29+17, 29+18, 29+19, 29+20, 29+21, and 29+22;

[0253] in the list above the numbers refer to the embodiments according to their numbering provided hereinabove whereas “+” indicates the combination with the other embodiment. The different individualized combinations are separated by commas. In otherwords, “23+14” for example refers to the combination of T in embodiment 23) with L of embodiment 14).

[0254] It is to be understood that the range of i as described in embodiments 35) and 36) shall be regarded as explicitly disclosed for each of the above-listed combinations, for embodiments 1) to 34), and including embodiments 1), 2), 3), 5), 6), 7), 8), 9), 10), 11), 12), 13), 31), 32), 33) and 34).

[0255] Where the plural form is used for compounds, conjugates, salts, pharmaceutical compositions, diseases or the like, this is intended to mean also a single compound, conjugate, salt, pharmaceutical composition, disease or the like.

[0256] Any reference to a compound of (I), (lb), (II), (Ila), (III) and (IV) as defined in any one of embodiments 1) to 38) is to be understood as referring also to the salts (and especially the pharmaceutically acceptable salts) of such compounds, as appropriate and expedient.

[0257] The term "pharmaceutically acceptable salts" refers to salts that retain the desired biological activity of the subject compound and exhibit minimal undesired toxicological 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 also be used for stabilisation in the form of buffers or lyophilized products including 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. The present embodiments also include isotopically labelled, especially2H (deuterium) labelled compounds of Formula (I), (II), (Ila), (III) and (IV) which compounds are identical to the compounds of Formula (I), (II), (Ila), (III) and (IV) except that one or more atoms have each been replaced by an atom having the same atomic number but an atomic mass different from the atomic mass usually found in nature. Isotopically labelled, especially2H (deuterium) labelled compounds of Formula (I), (II), (Ila), (III) and (IV), and salts thereof, are within the scope of the present embodiments. Substitution of hydrogen with the heavier isotope2H (deuterium) may lead to greater metabolic stability, resulting e.g. in increased in-vivo half-life or reduced dosage requirements, or may lead to reduced inhibition of cytochrome P450 enzymes, resulting e.g. in an improved safety profile. In one embodiment, the compounds of Formula (I), (II), (Ila), (III) and (IV) are not isotopically labelled, or they are labelled only with one or more deuterium atoms. In a sub-embodiment, the compounds of Formula (I), (II), (Ila), (III) and (IV) are not isotopically labelled at all. Isotopically labelled compounds of Formula (I), (II), (Ila), (III) and (IV) may be prepared in analogy to the methods described hereinafter, but using the appropriate isotopic variation of suitable reagents or starting materials. For instance, the labelling may be performed within the linker L and / or spacer T.

[0258] The compounds of formula (I), (II) and (Ila) as defined in any one of embodiments 1) to 38) and their pharmaceutically acceptable salts can be used as medicaments, e.g. in the form of pharmaceutical compositions for parenteral, enteral (such as oral) or nasal administration, in particular parenteral administration such as intramuscular, subcutaneous, and intradermal injections.

[0259] 39) Hence, one aspect of the present invention relates to a pharmaceutical composition comprising, as active principle, an oligosaccharide-carrier protein conjugate according to any one of embodiments 1) to 38), in particular embodiments 33), 34), 35), 36), 37) and 38), especially embodiments 37) and 38), or a pharmaceutically acceptable salt thereof, and at least one therapeutically inert excipient.

[0260] The production of the pharmaceutical compositions can be effected in a manner which will be familiar to any person 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) by bringing the described compounds of Formula (I), (la), (II) and (Ila) or their pharmaceutically acceptable salts, optionally in combination with other therapeutically valuable substances, into a galenical administration form together with suitable, non-toxic, inert, therapeutically compatible solid or liquid carrier materials and, optionally, usual pharmaceutical adjuvants.

[0261] The pharmaceutical composition may comprise in addition to an oligosaccharide-carrier protein conjugate according to any one of embodiments 1) to 38) one or more (preferably 1, 2, 3, or 4; more preferably 2, 3, or 4) other oligosaccharide-carrier protein conjugates that are immunogenic against one or more other K. pneumoniae serotypes, especially against 01, O2ac, O2aeh, 03, 03a, 03b, 04, 05, 07, 08, 012, 013, or K-antigens (CPS) of K. pneumoniae, or O-antigens of other pathogens like E. coli or Shigella; and notably against 01, and / or 05.

[0262] Said pharmaceutical composition is suitable for eliciting a protective immune response in a human and / or animal (especially a mammal (including a human)) host, and therefore is useful for the prevention and / or treatment of diseases associated with Klebsiella pneumoniae bacteria. Preferably, said pharmaceutical composition is suitable for use in human.

[0263] The terms “prevention”, “preventing” and / or “prophylaxis” are used synonymously and refer to inhibiting the initial onset of a pathologic process, such that the pathologic process that could eventually lead to development of symptoms never develops or that symptoms develop in lower, non-dangerous intensity (i.e. preventing the development of a disease, disorder, or condition in a prophylactic manner).

[0264] The present pharmaceutical composition is suitable for administration to animal (and, in particular, human) patients, and thus include both human and veterinary uses. It may be used in a method of raising an immune response in a patient, comprising the step of administering the composition to the patient.

[0265] 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 a Klebsiella pneumoniae. Preferably, it is used before a subject is exposed to Klebsiella pneumoniae.

[0266] Pharmaceutical compositions are preferably in aqueous form, particularly at the point of administration, but they can also be presented in non-aqueous liquid forms or in dried forms e.g. as gelatin capsules, or as lyophilisates, etc.. Solid powders that are obtained e.g. by spray drying, spray-freeze drying, vacuum or air-drying, or lyophilisation, may be reconstituted before use. Lyophilisation is preferred in case solid powders shall be obtained. The pharmaceutical composition may comprise one or more therapeutically inert excipients. Such excipient may be selected from the group consisting of citric acid monohydrate, sodium citrate, sodium citrate dihydrate, acetic acid, sodium hydroxide, tromethamine, tromethamine hydrochloride (to adjust pH), cholesterol, sorbitan trioleate, DSPC (1,2-distearoyl-sn-glycero-3-phosphocholine), and (4-hydroxybutyl)azanediyl) bis(hexane-6,1-diyl)bis(2-hexyldecanoate), polydimethylsiloxane (antifoam), ascorbic acid (antioxidant). The excipient may serve to adjust tonicity, such as sodium chloride (NaCI), which may be present at from 1 to 20 mg / ml. Other salts that may be present include potassium chloride, potassium dihydrogen phosphate, disodium phosphate dehydrate, magnesium chloride, calcium chloride, etc..

[0267] The pharmaceutical composition may include one or more excipients which serve 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 prepared.

[0268] The pharmaceutical composition may include one or more excipients which serve 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.

[0269] The pharmaceutical composition may include compounds (with or without an insoluble metal salt) in plain water (e.g. water for injection, w.f.i.), but will usually include one or more buffers. Typical buffers include: a phosphate buffer; a Tris buffer; a borate buffer; a succinate buffer; a histidine buffer (particularly with an aluminum hydroxide adjuvant); or a citrate buffer. Buffer salts will typically be included in the 5-20 mM range.

[0270] Pharmaceutical compositions typically have a pH between 5.0 and 9.5 e.g. between 6.0 and 8.0.

[0271] The pharmaceutical composition may further include one or more stabilizer(s).

[0272] Pharmaceutical compositions are preferably sterile and gluten free.

[0273] 40) A further embodiment of the present invention relates to the pharmaceutical composition according to embodiment 39), further comprising an adjuvant. The term “adjuvant” as used herein refers to an immunological adjuvant i.e. a material used in a vaccine composition that modifies or augments the effects of said vaccine by enhancing the immune response to a given antigen contained in the vaccine without being antigenically related to it. For the person skilled in the art, classically recognized examples of immunological adjuvants include, but are not restricted to aluminum or calcium salt based adjuvants, saponins or saponin-based adjuvants (e.g. Matrix-M), CpG oligodexynucleotide based adjuvants (e.g. CpG 1018), 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(l: C)), TLR-4 agonists (e.g., lipopolysaccharide), TLR-5 agonists (e.g., flagellin), TLR-7 / 8 agonists (e.g., resiquimod), immunomodulatory proteins (e.g., detoxified heat-labile enterotoxin (dmLT) from Escherichia coli), TLR-4 agonist glucopyranosyl lipid adjuvant-stable emulsion (GLA-SE) and monophosphoryl lipid A (MPL), non-ionic block polymers, cytokines (e.g., type 1 interferon (IFN), granulocyte-macrophage colonystimulating factor (GM-CSF), interleukins), papain-like cysteine proteases, and many others such as e.g. AS04, AS03, AS01 B, and formulations of the above mentioned adjuvants as liposomes or nanoparticles prepared with lipids such as DOPC (1,2-dioleoyl-sn-glycero-3-phosphocholine), DSPC (1,2-distrearoyl-sn-glycero-3-phosphocholine), cholesterol and / or ALC-0315, formulations as virus-like particles, and co-formulations of the abovementioned adjuvants, especially co-formulation including aluminum or calcium salt based adjuvants. 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). An example for a calcium-based or calcium salt-based adjuvant is calcium phosphate.

[0274] Matrix-M is a saponin-based adjuvant composed of nanoparticles from saponins extracted from Quillaja saponaria (soapbark) trees, cholesterol, and phospholipids.

[0275] CpG based adjuvants are immunostimulatory oligodeoxynucleotides bearing one or more CpG motifs (CpG ODN) that are unmethylated cytosine-guanine dinucleotides. The methylation status of the CpG immunostimulatory motif generally refers to the cytosine residue in the dinucleotide. An immunostimulatory oligonucleotide containing at least one unmethylated CpG dinucleotide is an oligonucleotide which contains a 5' unmethylated cytosine linked by a phosphate bond to a 3' guanine, and which activates the immune system through binding to Toll-like receptor 9 (TLR-9). Freund’s adjuvant is an oil-in-water adjuvant based on mineral oil.

[0276] MF59 is an oil-in-water emulsion comprising 4.3% w / v squalene, 0.5% w / v polysorbate 80 (Tween 80), and 0.5% w / v sorbitan trioleate (Span 85).

[0277] Glycosphingolipids are a class of lipids that stimulate unconventional invariant T-cell receptors on NKT cells or iNKT cells, when the glycosphingolipid is presented MHC class I-related molecules such as CD1d.

[0278] Pam3CSK4 (Pam3CysSerLys4) is a synthetic triacylated lipopeptide that is a ligand for TLR-1 and TLR-2. It mimics the acylated amino terminus of bacterial lipopeptides.

[0279] Poly(l: C) is a polymer and analogue of double-stranded RNA, consisting of one strand of a polymer of inosinic acid and one strand of a polymer of cytidylic acid. It stimulates TLR-3 and simulates viral infections.

[0280] Lipopolysaccharide (LPS) is a membrane component of Gram-negative bacteria and a stimulator of TLR-4.

[0281] Flagellin is a globular protein that forms the filaments of bacterial flagella. Flagellin activates TLR-5 and TLR-11.

[0282] Resiquimod (R848; 1-[4-Amino-2-(ethoxymethyl)-1H-imidazo[4,5-c]chinolin-1-yl]-2-methylpropan-2-ol) is an immune response modifier and small molecule that activates TLR-7 and TLR-8.

[0283] dmLT is the double-mutant (thereby detoxified) of heat-labile enterotoxin from Escherichia coli. It is an effective mucosal and systemic adjuvant.

[0284] GLA-SE is an oil-in-water emulsion adjuvant that is prepared by combining aqueous glucopyranosyl lipid A (GLA), a TLR-4 agonist, with squalene.

[0285] MPL (monophosphoryl lipid A), a truncated LPS, is a clinically used TLR-4 agonist, or synthetic MPLA (synthetic monophosphoryl lipid A) such as Phosphorylated HexaAcyl Disaccharide (PHAD®, from Avanti Research, CAS No. 1246298-63-4), Monophosphoryl 3-Deacyl Lipid A (3D-PHAD®, from Avanti Research, described in US9,241,988, CAS No.

[0286] 1699735-79-9), or Monophosphoryl Hexa-acyl Lipid A, 3-Deacyl (3D-(6-acyl)PHAD®, from Avanti Research, CAS No. 252042-59-4). Nonionic Block Polymers (NBPs) suitable as adjuvants are simple copolymers of polyoxyethylene (POE) and hydrophobic polyoxypropylene (POP) and differ in molecular weight, percentage POE and the mode of linkage of POE and POP-groups.

[0287] Cytokines are small proteins secreted by cells that affect the interaction and communication between cells. Typically, cytokines activate the target cell, leading to 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 can be encoded on DNA molecules such as plasmids.

[0288] 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 Th2 type immune responses.

[0289] AS04 (Adjuvant System 04) is a complex of MPL (3-0-desacyl-4'-monophosphoryl lipid A) and aluminum hydroxide or aluminum phosphate.

[0290] AS03 (Adjuvant System 03) is a squalene-in-water emulsion with DL-alpha-tocopherol (vitamin E) and polysorbate 80.

[0291] AS01 B is a mixture of 3-0-desacyl-4'-monophosphoryl lipid A (MPL) and the saponin QS-21.

[0292] Preferred adjuvants are aluminum-based adjuvants, in particular aluminum hydroxide.

[0293] 41) A further aspect of the present invention relates to the oligosaccharide-carrier protein conjugate according to any one of embodiments 1) to 38), in particular embodiments 33), 34), 35), 36), 37) and 38), especially embodiments 37) and 38), or a pharmaceutically acceptable salt thereof, for the use as a medicament, in particular as a vaccine. In other words, the invention relates to a vaccine comprising the oligosaccharide-carrier protein conjugate according to any one of embodiments 1) to 38), in particular embodiments 33), 34), 35), 36), 37) and 38), especially embodiments 37) and 38), or a pharmaceutically acceptable salt thereof. Preferably, the vaccine is used for active vaccination.

[0294] 42) A further aspect of the present invention relates to an oligosaccharide-carrier protein conjugate according to any one of embodiments 1) to 38), in particular embodiments 33), 34), 35), 36), 37) and 38), especially embodiments 37) and 38), or a pharmaceutically acceptable salt thereof, for the use in the prevention and / or treatment of a K. pneumoniae infection. 43) A further embodiment of the present invention relates to an oligosaccharide-carrier protein conjugate according to any one of embodiments 1) to 38), in particular embodiments 33), 34), 35), 36), 37) and 38), especially embodiments 37) and 38), or a pharmaceutically acceptable salt thereof, for the use in the prevention and / or treatment of K. pneumoniae infections in individuals of 50 years or older; hospital acquired (i.e. nosocomial) K. pneumoniae infections, for instance nosocomial pneumonia, nosocomial bloodstream infections and nosocomial urinary tract infections; community-acquired K. pneumoniae infections; as well as pneumonia, bronchitis, meningitis, urinary tract infection, intraabdominal infections, wound infection, infection of blood, osteomyelitis, bacteremia, septicemia, liver abscess, inflammatory bowel disease (IBD), and neonatal sepsis, all caused by K. pneumoniae infection.

[0295] A population-based strategy for vaccination of individuals of 50 years or older against K. pneumoniae infections is desirable, because this population is particularly susceptible to K. pneumoniae infections, in particular individuals of 60 years or older and at risk of exposure to K. pneumoniae and / or anticipated weakened immune system.

[0296] K. pneumoniae is a notorious pathogen frequently responsible for hospital acquired (i.e. nosocomial) respiratory and urinary tract infections. It is the second most common cause of Gram-negative bacteremia. Drug resistant isolates are associated with high mortality (greater than 50% according to some studies), add significantly to hospital stays, and are especially problematic in ICUs.

[0297] Therefore, it is desired to prevent hospital acquired (i.e. nosocomial) K. pneumoniae infections, in particular in populations at high risk of exposure, including patients who will undergo elective surgery with hospital stays longerthan 72 hours (e.g., joint replacements), patients with weakened immune systems and patients who anticipate having weakened immune systems (e.g., those on solid organ transplant wait lists, non-urgent solid tumor surgery followed by chemotherapy). In these populations, vaccination 2-8 weeks prior to surgery, optionally followed by a booster may be applicable.

[0298] Moreover, the prevention of community-acquired infections in specific target groups such as healthcare workers or elderly (60 years or older) in long-term care facilities or nursing homes is desired. The term “community-acquired K. pneumoniae infections” relates to any K. pneumoniae infection acquired in the community. In contrast to a nosocomial (hospital-acquired) infection. Furthermore, the present oligosaccharide-carrier protein conjugate according to any one of embodiments 1) to 38), in particular embodiments 33), 34), 35), 36), 37) and 38), especially embodiments 37) and 38), or a pharmaceutically acceptable salt thereof, may be used in the prevention of neonatal sepsis by treating / vaccinating pregnant mothers.

[0299] Moreover, the present oligosaccharide-carrier protein conjugate according to any one of embodiments 1) to 38), in particular embodiments 33), 34), 35), 36), 37) and 38), especially embodiments 37) and 38), or a pharmaceutically acceptable salt thereof, may be used in the prevention and / or treatment of pneumonia, bronchitis, meningitis, urinary tract infection, intra-abdominal infections, wound infection, infection of blood, osteomyelitis, bacteremia, septicemia, liver abscess, inflammatory bowel disease (IBD), all caused by K. pneumoniae infection.

[0300] 44) A further embodiment of the present invention relates to an oligosaccharide-carrier protein conjugate according to any one of embodiments 1) to 38), in particular embodiments 33), 34), 35), 36), 37) and 38), especially embodiments 37) and 38), or a pharmaceutically acceptable salt thereof, for the use in the prevention and / or treatment of the K. pneumoniae infections as listed in embodiment 42) and 43) above, wherein K. pneumoniae is selected from O-serotypes comprising 03b and 03.

[0301] 45) For any avoidance of doubt, the oligosaccharide-carrier protein conjugate according to any one of embodiments 1) to 38), in particular embodiments 33), 34), 35), 36), 37) and 38), especially embodiments 37) and 38), or a pharmaceutically acceptable salt thereof, as well as the pharmaceutical composition of embodiment 39) or 40), and the vaccine according to embodiment 41) are likewise suitable for the prevention and / or the treatment of the K. pneumoniae infections as listed in any one of embodiments 42), 43) and 44). 46) Preferably, the oligosaccharide-carrier protein conjugate according to any one of embodiments 1) to 38), in particular embodiments 33), 34), 35), 36), 37) and 38), especially embodiments 37) and 38), or a pharmaceutically acceptable salt thereof, as well as the pharmaceutical composition of embodiment 39) or 40), and the vaccine according to embodiment 41) are suitable for the prevention or prophylaxis of the K. pneumoniae infections as listed in any one of embodiments 42), 43) and 44).

[0302] 47) A further aspect of the present invention relates to a method of eliciting an immune response against K. pneumoniae in a human and / or animal (especially a mammal (including a human)) host, comprising administering to the human and / or animal an effective amount of the oligosaccharide-carrier protein conjugate according to any one of embodiments 1) to 38), in particular embodiments 33), 34), 35), 36), 37) and 38), especially embodiments 37) and 38), or a pharmaceutically acceptable salt thereof. The administered amount is preferably from 0.05 pg to 30 pg glycan per immunization of the human patient. The term “glycan” refers to antigen, i.e. oligosaccharide excluding linker L and spacer T. Possibly, more than one immunization is required.

[0303] 48) Likewise, an embodiment of the present invention relates to a method of eliciting an immune response against K. pneumoniae in a human and / or animal (especially a mammal (including a human)) host, comprising administering to the human and / or animal an effective amount of the composition according to embodiment 39) or 40), as well as the vaccine according to embodiment 41).

[0304] 49) For avoidance of any doubt, if oligosaccharide-carrier protein conjugates according to any one of embodiments 1) to 38), in particular embodiments 33), 34), 35), 36), 37) and 38), especially embodiments 37) and 38), or pharmaceutically acceptable salts thereof, are described as useful for the prevention and / or treatment of a K. pneumoniae infection according to any one of embodiments 42), 43) and 44), such oligosaccharide-carrier protein conjugates are likewise suitable for use in the preparation of a medicament for the prevention and / or treatment of said K. pneumoniae infection according to any one of embodiments 42), 43) and 44).

[0305] 50) A further aspect of the present invention relates to a multivalent vaccine comprising the oligosaccharide-carrier protein conjugate according to any one of embodiments 1) to 38), preferably the oligosaccharide-carrier protein conjugate according embodiments 37) and 38), or a pharmaceutically acceptable salt thereof.

[0306] The term “multivalent vaccine” in this respect relates to a vaccine comprising antigens against two or more different K. pneumoniae strains, in particular to two or more pathogenic K. pneumoniae strains, for instance two to seven pathogenic K. pneumoniae strains. 51) A further aspect of the present invention relates to an intermediate compound for preparing the oligosaccharide-carrier protein conjugate according to any one of embodiments 13) to 38), having the formula (III)

[0307]

[0308] wherein

[0309] R represents OH;

[0310] m represents 3, 4, 5, 6, 7 or 8; preferably 4, 5 or 6, most preferred 5;

[0311] L1represents

[0312] *-(C2-10)alkylene-NH2;

[0313] *-(CH2CH2O)b-CH2CH2NH2, wherein b is 1, 2 or 3;

[0314] *-CH2CH2S-CH2CH2NH2;

[0315] *-(C2-10)fluoroalkylene-NH2;

[0316] *-(CH2)cNHC(O)(CH2)c’-NH2, wherein c and c’ are independently from each other from 2 to 6;

[0317] *-(CH2)dNHC(O)NH(CH2)d’-NH2, wherein d and d’ are independently from each other from 2 to 6;

[0318] *-(C1-10)alkylene-C(O)-NH-(C2-10)alkylene-NH2;

[0319] *-(C2-10)alkylene-O-NH2; or

[0320] *-(C2-10)alkylene-SH;

[0321] or a pharmaceutically acceptable salt thereof.

[0322] 52) A further embodiment relates to the intermediate compound according to embodiment 51), or a pharmaceutically accepable salt thereof, wherein

[0323] L1represents

[0324] *-(CH2)a-NH2; wherein a is from 2 to 10;

[0325] *-(CH2CH2O)b-CH2CH2NH2, wherein b is 1, 2 or 3; *-CH2CH2S-CH2CH2NH2;

[0326] *-(C2-io)fluoroalkylene-NH2with fluoroalkylene being a saturated straight chain; *-(CH2)CNHC(O)(CH2)C’-NH2, wherein c and c’ are independently from each other from 2 to 6;

[0327] *-(CH2)dNHC(O)NH(CH2)d -NH2, wherein d and d’ are independently from each other from 2 to 6;

[0328] *-(CH2)e-C(O)-NH-(CH2)e’-NH2; wherein e is from 1 to 10 and e’ is from 2 to 10; *-(CH2)f-O-NH2, wherein f is from 2 to 10; or

[0329] *-(CH2)g-SH, wherein g is from 2 to 10.

[0330] It is to be understood that in an analogous manner, embodiments 15) to 22) disclose further preferred L1which bear terminal amino- or SH-groups as demonstrated in embodiment 51) and 52).

[0331] 53) A further embodiment relates to the intermediate compound according to embodiment 51) or 52), or a pharmaceutically accepable salt thereof, wherein

[0332] L1represents

[0333] *-(CH2)2-NH2, *-(CH2)3-NH2, *-(CH2)4-NH2, *-(CH2)5-NH2, or *-(CH2)6-NH2; preferably *-(CH2)s-NH2.

[0334] 54) A further embodiment relates to the intermediate compound according to embodiment 53), or a pharmaceutically accepable salt thereof, wherein

[0335] R represents OH;

[0336] m represents 5; and

[0337] L1represents

[0338] *-(CH2)2-NH2, *-(CH2)3-NH2, *-(CH2)4-NH2, *-(CH2)5-NH2, or *-(CH2)6-NH2.; preferably *-(CH2)s-NH2.

[0339] 55) A further aspect of the present invention relates to an intermediate compound for preparing the oligosaccharide-carrier protein conjugate according to any one of embodiments 13) to 38), or a pharmaceutically acceptable salt thereof, having the formula (IV):

[0340]

[0341] wherein

[0342] R represents OH;

[0343] m represents 3, 4, 5, 6, 7 or 8; preferably 4, 5 or 6, most preferred 5;

[0344] L represents

[0345] *-(C2-10)alkylene-NH-;

[0346] *-(CH2CH2O)b-CH2CH2NH-, wherein b is 1, 2 or 3;

[0347] *-CH2CH2S-CH2CH2NH-;

[0348] *-(C2-10)fluoroalkylene-NH-;

[0349] *-(CH2)CNHC(O)(CH2)C-NH-, wherein c and c’ are independently from each other from 2 to 6;

[0350] *-(CH2)dNHC(O)NH(CH2)d -NH-, wherein d and d’ are independently from each other from 2 to 6;

[0351] *-(C1-10)alkylene-C(O)-NH-(C2-10)alkylene-NH-; or

[0352] *-(C2-10)alkylene-O-NH-; and

[0353] T1represents

[0354] -C(O)-(C0-10)alkylene-C(O)X;

[0355] -C(O)-CH2CH2-(OCH2CH2)j-C(O)X, wherein j is from 1 to 5;

[0356] -C(O)-CH2(CH2)k-(SCH2(CH2)k’)k”-C(O)X, wherein k is 0 or 1, k’ is 0 or 1, and k” is 1, 2, or 3;

[0357]

[0358] , wherein p is from 1 to 4, preferably 1, and p’ is 1 or 2;

[0359] -C(O)X represents -C(O)OH or an activated ester; and

[0360] Y represents Me, Et, Bu or -(CH2CH2O)3CH3(especially Me, Et, n-Bu or-(CH2CH2O)3CH3).

[0361] The term “activated ester” refers to a functionalized carboxylic acid with enhanced reactivity towards amines (in comparison to a carboxylic acid), for the reaction with the amino group of a lysine residue of CRM197. Suitable “activated ester” groups are known to the person skilled in the art.

[0362] 56) A further embodiment relates to the intermediate compound according to embodiment 55), or a pharmaceutically accepable salt thereof, wherein

[0363] L represents

[0364] *-(CH2)a-NH-; wherein a is from 2 to 10;

[0365] *-(CH2CH2O)b-CH2CH2NH-, wherein b is 1, 2 or 3;

[0366] *-CH2CH2S-CH2CH2NH-;

[0367] *-(c2-io)fluoroalkylene-NH- with fluoroalkylene being a saturated straight chain; *-(CH2)CNHC(O)(CH2)C-NH-, wherein c and c’ are independently from each other from 2 to 6;

[0368] *-(CH2)dNHC(O)NH(CH2)d -NH-, wherein d and d’ are independently from each other from 2 to 6;

[0369] *-(CH2)e-C(O)-NH-(CH2)e’-NH-; wherein e is from 1 to 10 and e’ is from 2 to 10; or *-(CH2)f-O-NH-, wherein f is from 2 to 10; and

[0370] T1represents -C(O)-(CH2)h-C(O)X, wherein h is from 0 to 10;

[0371] -C(O)-CH2CH2-(OCH2CH2)j-C(O)X, wherein j is from 1 to 5;

[0372] -C(O)-CH2(CH2)k-(SCH2(CH2)k’)k”-C(O)X, wherein k is 0 or 1, k’ is 0 or 1, and k” is 1, 2, or 3;

[0373]

[0374] , wherein p is from 1 to 4, preferably 1, and p’ is 1 or 2;

[0375] -C(O)X represents -C(O)OH or an activated ester; and

[0376] Y represents Me, Et, Bu or -(CH2CH2O)3CH3(especially Me, Et, n-Bu or-(CH2CH2O)3CH3).

[0377] 57) A further embodiment relates to the intermediate compound according to embodiment 55) or 56), or a pharmaceutically accepable salt thereof, wherein

[0378] L represents

[0379] *-(CH2)2-NH-, *-(CH2)3-NH-, *-(CH2)4-NH-, *-(CH2)5-NH-, or *-(CH2)6-NH-; preferably *-(CH2)5-NH-;

[0380] T1represents

[0381] -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)-(CH2)4-C(O)X, -C(O)-(CH2)5-C(O)X, or -C(O)-(CH2)6-C(O)X;

[0382] preferably -C(O)-(CH2)4-C(O)X; and

[0383] -C(O)X represents -C(O)OH or an activated ester.

[0384] In embodiments 55), 56) and 57), preferably, X represents

[0385]

[0386] It is to be understood that embodiments 15) to 22) disclose further preferred L which are encompassed in the present embodiment. Moreover, it is to be understood that in an analogous manner, embodiments 25) to 29) disclose further preferred T, which translate to T1, wherein the terminal “C(O)-“ is replaced by “C(O)X”, and in case of squaric acid, the attachment point to the carrier protein, e.g. to CRM197, is denoted as “O-Y”, and R1is H. These preferred T1are to be regarded as explicitly disclosed.

[0387] 58) A further aspect of the present invention relates to an immuno-assay comprising the oligosaccharide-carrier protein conjugate of formula (lb):

[0388]

[0389] wherein

[0390] R represents OH;

[0391] m represents 3, 4, 5, 6, 7 or 8; preferably 4, 5 or 6, most preferred 5;

[0392] i is at least 1, preferably from 1 up to an integer corresponding to 90% of the number of lysine residues contained in the carrier protein CP*;

[0393] -L-T- represents a linker L and a spacer T as disclosed in any one of embodiments 1), 5) and 8) to 38); and CP* is a carrier protein suitable for immunological assays, in particular ELISA. Preferred CP* is bovine serum albumin (BSA) and those carrier proteins described in embodiment 1). A particularly preferred carrier protein is BSA.

[0394] The synthesis of oligosaccharide-carrier protein conjugates of formula (I) conjugated to BSA are described and exemplified in the experimental part. It is to be understood that this synthesis applies analogously to all oligosaccharides relating to formula (I) so that the skilled person can prepare them.

[0395] The assay of this embodiment is suitable for the detection of antibodies against K. pneumoniae 03b and 03 strains.

[0396] 59) A further aspect of the present invention relates to the oligosaccharide-carrier protein conjugate according to any one of embodiments 13) to 38), ora pharmaceutically acceptable salt thereof, wherein the oligosaccharide-carrier protein conjugate is obtainable by or prepared by conjugating the compound of formula (IV):

[0397]

[0398] wherein

[0399] R represents OH;

[0400] m represents 3, 4, 5, 6, 7 or 8; preferably 4, 5 or 6, most preferred 5;

[0401] L represents

[0402] *-(c2-io)alkylene-NH-;

[0403] *-(CH2CH2O)b-CH2CH2NH-, wherein b is 1, 2 or 3;

[0404] *-CH2CH2S-CH2CH2NH-;

[0405] *-(C2-10)fluoroalkylene-NH-;

[0406] *-(CH2)CNHC(O)(CH2)C'-NH-, wherein c and c’ are independently from each other from 2 to 6;

[0407] *-(CH2)dNHC(O)NH(CH2)d -NH-, wherein d and d’ are independently from each other from 2 to 6;

[0408] *-(C1-10)alkylene-C(O)-NH-(C2-10)alkylene-NH-; or

[0409] *-(C2-10)alkylene-O-NH-; and

[0410] a) T1represents

[0411] -C(O)-(C0-10)alkylene-C(O)X;

[0412] -C(O)-CH2CH2-(OCH2CH2)j-C(O)X, wherein j is from 1 to 5;

[0413] -C(O)-CH2(CH2)k-(SCH2(CH2)k’)k”-C(O)X, wherein k is 0 or 1, k’ is 0 or 1, and k” is 1, 2, or 3;

[0414]

[0415] -C(O)X represents -C(O)OH or an activated ester; and

[0416] Y represents Me, Et, Bu or -(CH2CH2O)3CH3

[0417] to a lysine residue of CRM197; or

[0418] b) conjugating the compound of formula (IV) wherein T1represents

[0419]

[0420] , wherein I is 1 or 2; or

[0421]

[0422] , wherein p is from 1 to 4, preferably 1, and p’ is 1 or2; or

[0423] wherein L-T1represents

[0424] *-(c2-io)alkylene-SH;

[0425] to modified CRM197 selected from the group consisting of:

[0426]

[0427] wherein Z is Br or I, q is 2 or 3, and t is from 1 to 28, preferably form 15-25;

[0428]

[0429] wherein r is 2 or 3, and t’ is from 1 to 28, preferably form 15-25; and

[0430]

[0431] wherein Z is Br or I, and t” is from 1 to 28, preferably form 15-25.

[0432] Preferably, X represents

[0433]

[0434]

[0435] It is to be understood that embodiments 14) to 22) disclose further preferred L which are encompassed in the present embodiment. Moreover, it is to be understood that in an analogous manner, embodiments 24) to 29) disclose further preferred T, which translate to T1, wherein the terminal “C(O)-“ is replaced by “C(O)X”, and in case of squaric acid, the attachment point to the carrier protein, e.g. to CRM197, is denoted as “O-Y”, and R1is H. These preferred T1are to be regarded as explicitly disclosed.

[0436] 60) A further aspect of the present invention relates to a process for preparing the oligosaccharide-carrier protein conjugate according to any one of embodiments 13) to 38), or a pharmaceutically acceptable salt thereof, wherein the process comprises conjugating the compound of formula (IV):

[0437]

[0438] wherein

[0439] R represents OH;

[0440] m represents 3, 4, 5, 6, 7 or 8; preferably 4, 5 or 6, most preferred 5;

[0441] L represents

[0442] *-(c2-io)alkylene-NH-;

[0443] *-(CH2CH2O)b-CH2CH2NH-, wherein b is 1, 2 or 3; *-CH2CH2S-CH2CH2NH-;

[0444] *-(C2-10)fluoroalkylene-NH-;

[0445] *-(CH2)CNHC(O)(CH2)C-NH-, wherein c and c’ are independently from each other from 2 to 6;

[0446] *-(CH2)dNHC(O)NH(CH2)d -NH-, wherein d and d’ are independently from each other from 2 to 6;

[0447] *-(C1-10)alkylene-C(O)-NH-(C2-10)alkylene-NH-; or

[0448] *-(C2-10)alkylene-O-NH-; and

[0449] a) T1represents

[0450] -C(O)-(C0-10)alkylene-C(O)X;

[0451] -C(O)-CH2CH2-(OCH2CH2)j-C(O)X, wherein j is from 1 to 5;

[0452] -C(O)-CH2(CH2)k-(SCH2(CH2)k’)k”-C(O)X, wherein k is 0 or 1, k’ is 0 or 1, and k” is

[0453] 1, 2, or 3;

[0454]

[0455] -C(O)X represents an activated ester;

[0456] Y represents Me, Et, Bu or -(CH2CH2O)3CH3;

[0457] to a lysine residue of CRM197; or

[0458] b) conjugating the compound of formula (IV) wherein T1represents

[0459]

[0460] , wherein p is from 1 to 4, preferably 1, and p’ is 1 or2; or

[0461] wherein L-T1represents

[0462] (C2-io)alkylene-SH; to modified CRM197 selected from the group consisting of:

[0463]

[0464] wherein Z is Br or I, q is 2 or 3, and

[0465] t is from 1 to 28, preferably form 15-25;

[0466]

[0467] wherein r is 2 or 3, and t’ is from 1 to 28, preferably form 15-25; and

[0468]

[0469] wherein Z is Br or I, and t” is from 1 to 28, preferably form 15-25.

[0470] Preferably, X represents

[0471]

[0472] It is to be understood that embodiments 14) to 22) disclose further preferred L which are encompassed in the present embodiment. Moreover, it is to be understood that in an analogous manner, embodiments 24) to 29) disclose further preferred T, which translate to T1, wherein the terminal “C(O)-“ is replaced by “C(O)X”, and in case of squaric acid, the attachment point to the carrier protein, e.g. to CRM197, is denoted as “O-Y”, and R1is H. These preferred T1are to be regarded as explicitly disclosed.

[0473] Whenever the word “between” or “to” is used to describe a numerical range, it is to be understood that the end points of the indicated range are explicitly disclosed and included in the range. For example: if a temperature range is described to be between 40 °C and 80 °C (or 40 °C to 80 °C), this means that the end points 40 °C and 80 °C are included in the range; or if a variable is defined as being an integer between 1 and 4 (or 1 to 4), this means that the variable is the integer 1, 2, 3, or 4.

[0474] Whenever the phrase “is a, b,... orx” is used, this is synonym to “and” can be replaced by “is selected from the group consisting of a, b,... and x”. The same applies for the phrase “represents a, b,... orx” as well as “represents a, b,... and x”, which can be replaced by “is selected from the group consisting of a, b,... and x”.

[0475] For the avoidance of any doubt, the definition “a bridge having a backbone with a length of 5 to 25 atoms covalently linked together that forms 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 at the carrier protein CRM?" means that the oxygen at C1 and the nitrogen of the amino group of the lysine at the CRM197 do not count to the numbering of the so-defined backbone.

[0476] Unless used regarding temperatures, the term “about” (or alternatively “around”) placed before a numerical value “X” refers in the current application to an interval extending from X minus 10% of XtoX plus 10% of X, and preferably to an interval extending from X minus 5% of X to X plus 5% of X. In the particular case of temperatures, the term “about” (or alternatively “around”) placed before a temperature “Y” refers in the current application to an interval extending from the temperature Y minus 10 °C to Y plus 10 °C, and preferably to an interval extending from Y minus 5 °C to Y plus 5 °C. Besides, the term “room temperature” as used herein refers to a temperature of about 25°C.

[0477] Preparation of compounds of Formula (I), (II), (Ila), (III) and (IV)

[0478] A further aspect of the invention is a process for the preparation of compounds of Formula (I), (II), (Ila), (III) and (IV). Compounds according to Formula (I), (II), (Ila), (III) and (IV) of the present invention can be prepared from commercially available or well-known starting materials according to the methods described in the experimental part; by analogous methods; or according to the general sequence of reactions outlined below, wherein L, T, L1, T1, X and Y are as defined for Formula (I), (II), (Ila), (III) and (IV). Other abbreviations used herein are explicitly defined, or are as defined in the experimental section.

[0479] The synthesis of the compounds of the present invention requires protection group strategy. Though such protecting group strategy may be sophisticated, 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. Wuts, Wiley-lnterscience, 1999). The compounds obtained may also be converted into salts, especially pharmaceutically acceptable salts thereof in a manner known perse.

[0480] General preparation routes:

[0481] Antigen representation

[0482]

[0483] m = 3, 4, 5, 6, 7, or 8 Scheme 1: Synthesis of AG-CRM197 conjugate using NHS-ester method

[0484]

[0485] L1= — NH2

[0486]

[0487] means all linkers L1as described in embodiments 13) to 23) and L ' — ""“NHj

[0488] 31) to 35), having a terminal amino group

[0489]

[0490] k = 0 or 1, k' = 0 or 1, and k" = 1, 2, or 3 Antigen AG-L1T in appropriate solvent (e.g., DMSO) in a vial at rt is treated with activated Bis-NHS ester of the diacid 2’ (e.g., Bis-NHS adipate, which is commercially available or can be prepared by the person skilled in the art using corresponding Bis-acid and N-hydroxy succinic acid) (Odom, O. W., Biochemistry, Vol.29, No.48, 1990) (5-20 equiv.) in DMSO in presence of triethylamine and stirred for 3 h at rt. The Antigen-NHS ester 3’ is precipitated out by adding EtOAc, and centrifuged, subsequently the precipitate is washed with EtOAc, dried in vacuum before taken for the next step. The buffer solution containing Antigen-NHS ester 3’ (25-100 equiv.) and CRM197 is stirred at rt for 20-24 h. The resulting Antigen-CRM197 conjugate 4’ is washed, purified and stored using appropriate buffer solution.

[0491] In the above synthesis routes, CRM197 may be replaced by any one of the carrier proteins as described in embodiment 1).

[0492] Scheme 2: Synthesis of AG-CRM197 conjugate using the squarate method

[0493]

[0494] Antigen AG-L1T in appropriate solvent (e.g., H2O-EtOH, buffer) in a vial at rt is treated with 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) and stirred at rt in solvent with appropriate pH (7-8). The reaction mixture is neutralized using acetic acid and then concentrated (or lyophilized) in vacuum. The crude is purified using C18 (or SEC) column using water-acetonitrile as eluents. The fractions containing product are frozen and lyophilized to afford 6’. The 0.5 M pH 9 borate buffer solution containing Antigen-squarate ester 6’ (25-100 equiv.) and CRM197 is stirred at rt for 24-72 h (S. Hou etal, Carbhydr. Res, 2008, 343, 196-210). The resulting Antigen-CRMi97conjugate 7’ is washed, purified, and stored using appropriate buffer solution.

[0495] In the above synthesis routes, CRM197 may be replaced by any one of the carrier proteins as described in embodiment 1).

[0496] Scheme 3: Synthesis of Antiqen-Thiol

[0497]

[0498] L1= R1= H or SO3Na

[0499] — NH2

[0500] I =1-2

[0501] Antigen AG-L11’ in appropriate solvent (e.g., DMSO) in a vial at rt is treated with 8’ (e.g., DSP (dithiobis(succinimidylpropionate), or DTSSP (3,3’-dithiobis(sulfo-succinimidylpropionate)), to obtain the corresponding disulfide, which in turn is reduced by DTT (dithiothreitol) or TCEP (tris(2-carboxyethyl)phosphine) to afford the Antigen-thiol 9’. Scheme 4: Synthesis of AG-CRM197 conjugate using Antiqen-Thiol and functionalized CRM197

[0502]

[0503] a) Synthesis of AG-CRM197 conjugate using Antigen-Thiol-Maleimide method

[0504] The buffer solution containing Antigen-thiol 9’ (25-100 equiv.) and CRM197 functionalized with Maleimide 10’ (which can for instance be prepared by treating CRM197 with 3-maleimido-propionic acid succinimidyl ester or any other appropriate NHS ester equipped with maleimide, by person skilled in the art) (Robert M. F. van der Put et al, ACS Cent. Sci. 2022, 8, 4, 449-460) is stirred at rt for 20-24 h. Then excess maleimide moieties are quenched by adding L-cysteine in buffer to the RM and stirring for an hour at rt. The resulting Antigen-CRM197-thio-maleimide conjugate 11’ is washed, purified, and stored using appropriate buffer solution.

[0505] In the above synthesis routes, CRM197 may be replaced by any one of the carrier proteins as described in embodiment 1).

[0506] b) Synthesis of AG-CRM197 conjugate using Antiqen-Thiol-ether method

[0507] The buffer solution containing Antigen-thiol 9’ (25-100 equiv.) and protein functionalized with a-bromoacetate 10’ (e.g., CRM197-BAP, synthesized using CRM197 and SBAP ( / V-succinimidyl 3-(2-bromoacetamido)propanoate) or any other appropriate NHS ester equipped with a-bromoacetate) (Schumann, B. et al, Chem. Sci., 2014, 5, 1992-2002) is stirred at rt for 24 h. Then excess a-bromoacetate moieties are quenched by adding L-cysteine in buffer to the RM and stirring for an hour at rt. The resulting Antigen-CRM197-thio-ether conjugate 11’ is washed, purified, and stored using appropriate buffer solution. In the above synthesis routes, CRM197 may be replaced by any one of the carrier proteins as described in embodiment 1).

[0508] c) Introduction of linker handles:

[0509] Nucleophilic linkers Ln, such as those exemplified in Table 2, can be introduced to the respective donors carrying a leaving group such as e.g. imidate, phosphate, STol, 5-tert-Butyl-o-toluenethiol, SPh or SEt at the C1 carbon atom, according to methods well known to the skilled person in the art (e.g. as shown in W0202412180).

[0510] Table A: List of nucleophilic linkers Ln

[0511]

[0512] Experimental section:

[0513] Abbreviations (as used herein and in the description above): AcOH Acetic acid

[0514] aq. Aqueous

[0515] Bn Benzyl

[0516] BSA Bovine serum albumin

[0517] CDCl3Deuterated chloroform

[0518] Cs2CO3Cesium carbonate

[0519] Cy Cyclohexane

[0520] D2O Deuterium oxide

[0521] DCM Dichloromethane

[0522] DDQ 2,3-dichloro-5,6-dicyano-1,4-benzoquinone DMAP 4-(Dimethylamino)pyridine

[0523] DMF N, N-dimethylformamide

[0524] DMSO Dimethylsulfoxide

[0525] ELISA Enzyme-linked immunosorbent assay

[0526] equiv Equivalents

[0527] ESI Electrospray ionization

[0528] Et3N (TEA) Triethylamine

[0529] EtOAc (EA) Ethyl acetate

[0530] EtOH Ethanol

[0531] EtSH Ethanethiol

[0532] Fr Fraction

[0533] h Hours

[0534] H2Hydrogen

[0535] H2O Water

[0536] H2SO4Sulfuric acid

[0537] HCI Hydrochloric acid

[0538] HPLC High-performance liquid chromatography

[0539] High-performance liquid chromatography-size exclusion HPLC-SEC

[0540] chromatography

[0541] h Iodine

[0542] ICU Intensive Care Unit

[0543] I PA Isopropanol

[0544] LPS Lipopolysaccharide

[0545] M Molar

[0546] MeOH Methanol

[0547] Min Minutes MS Molecular sieves

[0548] N2Nitrogen

[0549] Na Sodium

[0550] Na2S2O3Sodium thiosulfate

[0551] Na2SO4Sodium sulfate

[0552] NaCI Sodium chloride

[0553] NaHCO3Sodium bicarbonate

[0554] NaOMe Sodium methoxide

[0555] NaPi buffer Sodium phosphate buffer

[0556] NH2NH2Hydrazine

[0557] NIS N-iodosuccinimide

[0558] NMR Nuclear magnetic resonance spectroscopy

[0559] PBS Phosphate-buffered saline

[0560] PBS-T Phosphate-buffered saline with 0.1% (v / v) Tween-20 Pd(OH)2Palladium hydroxide

[0561] Pd / C Palladium on carbon

[0562] py Pyridine

[0563] RBF Round bottom flask

[0564] RM Reaction Mixture

[0565] rt Room temperature

[0566] sat. Saturated

[0567] SDS-PAGE Sodium dodecyl sulfate-polyacrylamide gel electrophoresis SM Starting material

[0568] sol. Solution

[0569] TBAF Tetrabutylammonium fluoride

[0570] TBS Tris-buffered saline

[0571] TDS Dimethyl-Thexylsilylchloride

[0572] TLC Thin layer chromatography

[0573] TMB 3,3’,5,5’-Tetramethylbenzidine

[0574] TMSOTf T rimethylsilyl trifluoromethanesulfonate

[0575] UV Ultraviolet

[0576] I. Chemistry

[0577] The following examples illustrate the preparation of biologically active compounds of the invention but do not at all limit the scope thereof. General information:

[0578] All reagents and solvents were used as purchased and solvents used for the reactions were anhydrous. Except reactions containing water as solvent, all reactions were conducted under an atmosphere of N2in dried glassware (purchased from VWR and ROTH). Before glycosylation it is highly recommended to dry acceptor and donor by azeotrope with anhydrous toluene twice. Heidolph magnetic stirrer was used to carry out the experiments. Thin-layer chromatography (TLC) was performed on silica gel 60 F254 glass plates (Merck) or aluminium plates (VWR). Developed TLC plates were visualized under a short-wave UV lamp and by heating plates that were dipped in sugar stain solution (3-methoxy phenol (0.225 mL), H2SO4(6 mL) and EtOH (200 mL)). All automated flash chromatography purifications on silica gel (FlashPure Silica 40 pm irregular: BUCHI columns) were carried out with Biotage Isolera and Biotage Select. BUCHI rotary evaporator was used to evaporate the solvent. Dry ice and acetone and ice / water combination were used for cooling the reaction mixture to get the desired temperatures. All NMR experiments were carried out on BRUKER 400 MHz instrument.

[0579] Temperatures are indicated in degrees Celsius (°C). In mixtures, relations of parts of solvent or eluent or reagent mixtures in liquid form are given as volume relations (v / v), unless indicated otherwise.

[0580] Synthesis of Mannose derivative 1:

[0581]

[0582] S1 (52 g, 93 mmol) (Chayajarus et al, Organic Letters, Vol 6 / lssue 21, September 18, 2004; https: / / pubs.acs.org / doi / 10.1021 / ol048427o) was dissolved in anhydrous dichloromethane (623 mL). Pyridine (60.4 mL, 747 mmol) and FmocCI (48.3 g, 187 mmol) were added at rt under nitrogen atmosphere. The reaction stirred at rt for 1h and RM was monitored by TLC. Reaction mixture was diluted with sat. NaHCO3sol. (500 mL) and extracted with DCM (2 x 250 mL). Combined organic layers were dried over Na2SO4, filtered and concentrated to get the crude product. The purification was performed using automated purification system on silica column using EtOAc and Cyclohexane as eluents. The product fractions were collected and evaporated in vacuum and dried to get 1 as white solid (60 g, 82%). 1H NMR (400 MHz, CDCI3) 57.76 (dd, J= 7.6, 3.0 Hz, 2H), 7.62 (t, J= 7.1 Hz, 2H), 7.47 -7.16 (m, 21 H), 7.06 (d, J= 8.0 Hz, 2H), 5.57 (d, J= 1.6 Hz, 1H), 5.45 (dd, J= 2.8, 1.7 Hz, 1H), 4.93 (d, J= 10.8 Hz, 1H), 4.78 (d, J= 11.4 Hz, 1H), 4.71 -4.61 (m, 2H), 4.58 (d, J= 10.8 Hz, 1H), 4.50 (d, J= 12.0 Hz, 1H), 4.45 -4.20 (m, 4H), 4.07 - 3.95 (m, 2H), 3.88 (dd, J= 10.9, 5.0 Hz, 1H), 3.77 (dd, J= 11.0, 1.9 Hz, 1H), 2.31 (s, 3H).

[0583] Synthesis of Mannose derivative 2:

[0584]

[0585] To the stirred solution of S2 (4.6 g, 9.86 mmol) (Liu et al, Carbohydrate Research 342 (2007) 2818-2825) in anhydrous chloroform (50 mL), (trimethoxymethyl)benzene (5.08 mL, 29.6 mmol) and trifluoroacetic acid (0.076 mL, 0.986 mmol) were added at room temperature under nitrogen. The reaction was allowed to stir at room temperature for 3 h. The reaction was diluted with acetonitrile (50 mL) and H2O (10mL) and stirred for 2 h at rt and the completion of reaction was monitored by TLC. Reaction mixture was diluted with sat. NaHCO3solution (250 mL) and extracted with DCM (2 x250 mL). Combined organic layers were dried over Na2SO4, filtered and concentrated to get the crude product. The purification was performed using automated purification system on silica column using EtOAc and Cyclohexane as eluents. The product fractions were collected and evaporated in vacuum and dried to get 2 as white sticky solid (4.63 g, 82%). 1H NMR (400 MHz, CDCl3) 58.16 - 7.96 (m, 2H), 7.59 (ddt, J = 8.8, 7.1, 1.4 Hz, 1 H), 7.50 - 7.23 (m, 12H), 7.02 (d, J = 9.1 Hz, 2H), 6.80 (d, J = 9.1 Hz, 2H), 5.59 (d, J = 1.9 Hz, 1H), 5.54 (dd, J = 3.4, 1.9 Hz, 1H), 4.83 (d, J = 11.1 Hz, 1H), 4.71 (dd, J = 22.8, 11.5 Hz, 2H), 4.52 (d, J = 11.9 Hz, 1H), 4.49 -4.42 (m, 1H), 4.12 (t, J = 9.6 Hz, 1H), 4.00 (ddd, J = 9.7, 3.7, 1.9 Hz, 1H), 3.92 (dd, J = 11.1, 3.6 Hz, 1H), 3.79- 3.67 (m, 4H), 2.15 (d, J= 5.2 Hz, 1H).

[0586] Synthesis of Disaccharide acceptor 3:

[0587]

[0588] 1 (18.35 g, 23.55 mmol) and 2 (11.2 g, 19.63 mmol) were azeotroped with toluene (100 mL) and then dissolved in dichloromethane anhydrous (196 mL), molecular sieves were added and stirred for 40 min at room temperature. Cooled the RM to 0 °C and added N-iodosuccinimide (5.52 g, 24.53 mmol) and TMSOTf (0.43 mL, 2.35 mmol) and stirred at the same temperature for 30 min and monitored by TLC. After the complete consumption of the SM, triethylamine (54.7 mL, 393 mmol) was added dropwise and left to stir at the same temperature for 2 h till the reaction was complete. RM was filtered to remove molecular sieves and washed with DCM (100 mL x2), and the filtrate was washed with sat. Na2S2O3solution (150 mL) and extracted with DCM (150 mL), combined organic layers are washed with sat. NaHCO3solution (200 mL), dried (Na2SO4), filtered and concentrated. Crude compound was purified on silica using EA / cyclohexane as eluents by automated flash chromatography. The product fractions were evaporated and dried in high vacuum to obtain yellow foamy solid 3 (15.5 g, 79%).1H NMR (400 MHz, CDCI3) δ 8.16 - 7.95 (m, 2H), 7.62 - 7.50 (m, 1 H), 7.47 - 7.05 (m, 27H), 6.98 (d, J = 9.1 Hz, 2H), 6.77 (d, J = 9.1 Hz, 2H), 5.61 (dd, J = 3.2, 2.0 Hz, 1 H), 5.57 (d, J = 2.0 Hz, 1 H), 5.28 (d, J = 1.8 Hz, 1 H), 4.69 (dt, J = 14.4, 11.5 Hz, 4H), 4.61 -4.36 (m, 7H), 4.22 (t, J= 9.7 Hz, 1H), 4.01 -3.84 (m, 5H), 3.79 - 3.66 (m, 5H), 3.61 (d, J= 2.5 Hz, 2H), 2.36 (t, J= 2.0 Hz, 1H). HRMS(QTOF) m / z: Calcd for C61H66NO13+[M+NH4]+1020.4529, found 1020.4627.

[0589] Synthesis of Trisaccharide 5:

[0590]

[0591] 3 (13 g, 12.96 mmol) and 4 (12.13 g, 18.14 mmol) (Kim et al, J. Org. Chem. 2022, 87, 7, 4894-4907; https: / / doi.org / 10.1021 / acs.joc.2c00252) were azeotroped with toluene (100 mL) and then dissolved in anhydrous DCM (260 ml_), molecular sieves were added and stirred for 40 min at room temperature. Cooled the RM to 0 °C and added N-iodosuccinimide (4.08 g, 18.14 mmol) and TMSOTf (0.28 mL, 1.56 mmol) and stirred at the same temperature for 30 min and monitored by TLC. After the complete consumption of the SM, triethylamine (27.1 mL, 194 mmol) was added dropwise and allowed to warm to room temperature and stirred for 1.5 h. RM was filtered to remove molecular sieves and washed with DCM (100 mL x 2), and the filtrate was washed with sat. Na2S2O3solution (150 mL) and extracted with DCM (100 mL), combined organic layers are washed with sat. NaHCO3solution (200 mL), dried (Na2SO4), filtered and concentrated. Crude compound was purified on silica using EA / cyclohexane as eluents by automated flash chromatography. The product fractions were evaporated and dried in high vacuum to obtain yellow foamy solid 5 (16.18 g, 81%).1H NMR (400 MHz, CDCI3) 5 8.02 - 7.94 (m, 4H), 7.61 - 7.43 (m, 2H), 7.37 - 6.95 (m, 39H), 6.90 (d, J = 9.1 Hz, 2H), 6.70 (d, J = 9.1 Hz, 2H), 5.59 (ddd, J = 11.2, 3.2, 2.1 Hz, 2H), 5.53 (d, J = 2.0 Hz, 1H), 5.44 (dd, J = 9.6, 3.2 Hz, 1H), 5.27 (d, J= 1.9 Hz, 1H), 4.94 (d, J = 2.3 Hz, 1H), 4.80 - 4.24 (m, 15H), 4.19 - 3.98 (m, 3H), 3.93 - 3.66 (m, 10H), 3.63 - 3.43 (m, 4H), 2.81 -2.21 (m, 4H), 2.01 (s, 3H). HRMS(QTOF) m / z: Calcd for C93H98NO21+[M+NH4]+1564.6626, found 1564.6821.

[0592] Synthesis of Trisaccharide hemiacetal 6:

[0593]

[0594] Ceric ammonium nitrate (8.78 g, 16.02 mmol) was added to a solution of the starting material 5 (6.2 g, 4.01 mmol) in acetonitrile (100 mL) and DI water (10 mL) at 0 °C. The reaction mixture was slowly warmed to room temperature and stirred for 3 h. TLC analysis showed that reaction is complete with presence of major polar spot. The reaction was diluted with water (100 mL) and extracted with EtOAc (100 mL x 2). Combined organic layer was washed with sat. NaHCO3(100 mL x 2) and brine (50 mL). The organic layer was dried over Na2SO4, filtered and the solvent concentrated in rotavapor (water bath ~ 35 °C). The residue was purified on silica column using automated purification system in ethyl acetate in cyclohexane. The fractions of polar intense spots were combined, and the solvent evaporated to give the product 6 as an orange gummy residue (4.7 g, 81%). HRMS(QTOF) m / z: Calcd for C86H88NaO20+[M+Na]+1463.5761, found 1463.5733. Synthesis of Trisaccharide-RU-imidate donor 7:

[0595]

[0596] Starting material 6 (4.0 g, 2.77 mmol) was taken in anhydrous DCM (50 mL) at rt under N2atmosphere, added (E)-2,2,2-trifluoro-N-phenylacetimidoyl chloride (1.32 mL, 8.32 mmol) and cesium carbonate (2.71 g, 8.32 mmol) to it and stirred for 18 h. RM was filtered through celite to remove the solid, washed the residue with DCM (5 mL x 3). The filtrate was concentrated in vacuum to get dark orange gummy residue. Purified on silica column using 0.1% TEA in Cyclohexane and EtOAc as eluents to get product spot, evaporated and dried vacuum to afford pale brown solid (3.8 g, 85%).

[0597] Synthesis of Trisaccharide with C5-azido linker 9:

[0598]

[0599] Both substrates 7 (0.8 g, 0.50 mmol) and 5-azidopentan-1-ol (8) (0.23 g, 1.74 mmol) were taken in RBF and dried azeotropically using dry toluene in the vacuum. Mixture was taken in anhydrous Toluene (5 mL) at rt, added 4A molecular sieves to it and stirred for 30 min under N2atmosphere. Cooled the RM to -7 °C and added TMS-OTf (18 pL, 0.10 mmol) to the RM, and stirred the RM at -5 °C for 20 min. RM was then allowed to warm slowly to room temp over 1 h. RM was quenched with TEA (0.1 mL), stirred for 10 min and evaporated in vacuum to get crude product. Purified on silica column using cyclohexane and EtOAc as eluents to get fractions containing sugar stain active spot, evaporated and dried vacuum to afford colorless gummy solid 9 (0.61 g, 79%).1H NMR (400 MHz, CDCI3) 58.05 (dt, J = 8.4, 1.4 Hz, 4H), 7.67 - 7.52 (m, 2H), 7.47 - 6.90 (m, 39H), 5.68 (dd, J = 3.2, 2.1 Hz, 1H), 5.51 (dd, J= 9.6, 3.2 Hz, 1H), 5.46 (dd, J = 3.2, 1.9 Hz, 1H), 5.28 (d, J= 1.9 Hz, 1H), 4.99 (dd, J= 17.4, 2.0 Hz, 2H), 4.77 (d, J= 11.4 Hz, 1H), 4.72-4.62 (m, 5H), 4.60 - 4.45 (m, 6H), 4.40 (dd, J = 12.0, 9.1 Hz, 2H), 4.31 (dd, J = 9.5, 3.2 Hz, 1H), 4.17 (t, J = 9.7 Hz, 1 H), 4.08 (t, J = 9.7 Hz, 2H), 3.97 (t, J = 9.6 Hz, 1 H), 3.90 - 3.61 (m, 8H), 3.60 -3.49 (m, 3H), 3.42 (dt, J = 9.6, 6.4 Hz, 1 H), 3.23 (t, J = 7.0 Hz, 2H), 2.86 - 2.32 (m, 4H), 2.09 (s, 3H), 1.59 - 1.49 (m, 4H), 1.43 - 1.32 (m, 2H). HRMS(QTOF) m / z: Calcd for C91H101N4O20+[M+NH4]+1569.7004, found 1569.7159.

[0600] Synthesis of trisaccharide acceptor 10:

[0601]

[0602] A freshly prepared solution of hydrazine mono hydrate (0.12 ml_, 3.93 mmol) dissolved in acetic acid (0.41 ml_, 7.07 mmol) and pyridine (0.60 ml_, 7.46 mmol) was added to a solution of 9 (0.61 g, 0.393 mmol) in DCM (10 mL) at 5 °C. The resulting reaction mixture was stirred at room temperature for 2 h. Reaction was monitored by TLC for completion and was quenched by acetone (1 mL) and stirred the RM for 30 mins. The solvent removed under vacuum to obtain the crude product. The crude was purified on silica column using ethyl acetate and cyclohexane. The fractions containing intense sugar stain active spot were combined and the solvent evaporated to give the product as colorless gummy residue which became crispy solid 10 after drying in vacuum overnight (0.53 g, 93%). HRMS(QTOF) m / z: Calcd for C86H95N4O18+[M+NH4]+1471.6636, found 1471.6601. Synthesis of Hexasaccharide 11:

[0603]

[0604] Both acceptor 10 (0.52 g, 0.357 mmol) and imidate donor 7 (0.67 g, 0.415 mmol) were taken in RBF and dried azeotropically using dry toluene in the vacuum. Mixture was taken in anhydrous toluene, (5 mL) at rt, added 4A molecular sieves to it and stirred for 30 min under N2atmosphere. Cooled the RM to -7 °C and added TMS-OTf (13 pL, 0.071 mmol) to the RM and stirred the RM at -5 °C for 20 mins. RM was then allowed to warm slowly to room temp over 1 h. RM was quenched with TEA (0.10 mL), stirred for 10 mins and evaporated in vacuum to get crude product. Column purification was done using EA / cyclohexane on biotage using silica column. Fractions containing pure product were collected, evaporated in vacuum to get white crispy solid 11 (735 mg, 71%). HRMS(QTOF) m / z: Calcd for C172H181N4O37+[M+NH4]+2894.2399, found 2894.2567. Synthesis of Hexa saccharide acceptor 12:

[0605]

[0606] A freshly prepared solution of hydrazine mono hydrate (0.074 ml_, 2.35 mmol) dissolved in acetic acid (0.24 ml_, 4.22 mmol) and pyridine (0.36 ml_, 4.46 mmol) was added to a solution of 11 (675 mg, 0.235 mmol) in DCM (10 mL) at 5 °C. The resulting reaction mixture was stirred at room temperature for 2 h. Reaction was monitored by TLC for completion and was quenched by acetone (3 mL) and stirred the RM for 30 mins. The solvent removed under vacuum to obtain the crude product. The crude was purified using automated purification system in ethyl acetate in cyclohexane. The fractions containing intense sugar stain active spot were combined and the solvent evaporated to give the product as colorless gummy residue which became crispy solid 12 after drying in vacuum overnight (598 mg, 92%). HRMS(QTOF) m / z: Calcd for C167H175N4O35+[M+NH4]+2796.2031, found 2796.2158. Synthesis of Nonasaccharide 13:

[0607]

[0608] Both acceptor 12 (0.58 g, 0.209 mmol) and imidate donor 7 (0.39 g, 0.240 mmol) were taken in RBF and dried azeotropically using dry toluene in the vacuum. Mixture was taken in anhydrous toluene (5 mL) at rt, added 4A molecular sieves to it and stirred for 30 min under N2atmosphere. Cooled the RM to -7 °C and added TMS-OTf (7.5 pL, 0.042 mmol) to the RM and stirred the RM at -5 °C for 20 mins. RM was then allowed to warm slowly to room temp over 1h. RM was quenched with TEA (0.10 mL), stirred for 10 mins and evaporated in vacuum to get crude product. Column purification was done using EA / cyclohexane on silica column using biotage. Fractions containing pure product were collected, evaporated in vacuum to get colorless gummy solid 13 (713 mg, 81%). HRMS(QTOF) m / z: Calcd for C253H265N5O542+[M+2NH4]2+2118.4067 (4236.8133 / 2 = 2118.4067); found 2118.4202. Synthesis of Nonasaccharide acceptor 14:

[0609]

[0610] A freshly prepared solution of hydrazine mono hydrate (0.052 ml_, 1.67 mmol) dissolved in acetic acid (0.17 ml_, 3.00 mmol) and pyridine (0.26 ml_, 3.16 mmol) was added to a solution of 13 (700 mg, 0.167 mmol) in dichloromethane (10 mL) at 5 °C. The resulting reaction mixture was stirred at room temperature for 2 h. Reaction was monitored by TLC for completion and was quenched by acetone (3 mL) and stirred the RM for 30 mins. The solvent removed under vacuum to obtain the crude product. The crude was purified using silica column on automated purification system using ethyl acetate and cyclohexane. The fractions containing intense sugar stain active spot were combined and the solvent evaporated to give the product as colorless gummy residue which became crispy solid 14 after drying in vacuum overnight (590 mg, 86%). MALDI-TOF Calcd for C248H251N3NaO52+[M+Na]+4125.70, found 4125.85. Synthesis of Dodecasaccharide 15:

[0611]

[0612] Both acceptor 14 (575 mg, 0.140 mmol) and imidate donor 7 (260 mg, 0.161 mmol) were taken in RBF and dried azeotropically using dry toluene in the vacuum. Mixture was taken in anhydrous toluene (5 mL) at rt, added 4A molecular sieves to it and stirred for 30 min under N2 atmosphere. Cooled the RM to -7 °C and added TMS-OTf (5.1 pL, 0.028 mmol) to the RM and stirred the RM at -5 °C for 20 mins. RM was then allowed to warm slowly to room temp over 1h. RM was quenched with TEA (0.10 mL), stirred for 10 mins, and evaporated in vacuum to get crude product. Column purification was done using EA / cyclohexane on silica column using biotage. Fractions containing pure product were collected, evaporated in vacuum to get colorless gummy solid 15 (691 mg, 89%). MALDI-TOF Calcd for C334H338N3O71+[M+H]+5526.29, found 5524.53. Synthesis of Dodecasaccharide acceptor 16:

[0613]

[0614] A freshly prepared solution of hydrazine mono hydrate (38 pL, 1.221 mmol) dissolved in acetic acid (0.13 ml_, 2.197 mmol) and pyridine (0.19 ml_, 2.319 mmol) was added to a solution of 15 (675 mg, 0.122 mmol) in DCM (10 mL) at 5 °C. The resulting reaction mixture was stirred at room temperature for 2 h. Reaction was monitored by TLC for completion and was quenched by acetone (3 mL) and stirred the RM for 30 mins. The solvent removed under vacuum to obtain the crude product. The crude was purified using silica column on automated purification system using ethyl acetate and cyclohexane. The fractions containing intense sugar stain active spot were combined and the solvent evaporated to give the product as colorless gummy residue which became crispy solid 16 after drying in vacuum overnight (610 mg, 92%). MALDI-TOF Calcd for C329H332N3O69+[M+H]+5428.26, found 5426.70. Synthesis of Pentadecasaccharide 17:

[0615]

[0616] Both acceptor 16 (595 mg, 0.110 mmol) and imidate donor 7 (212 mg, 0.131 mmol) were taken in RBF and dried azeotropically using dry toluene in the vacuum. Mixture was taken in anhydrous toluene (5 mL) at rt, added 4A molecular sieves to it and stirred for 30 min under N2atmosphere. Cooled the RM to -7 °C and added TMS-OTf (4.0 pL, 0.022 mmol) to the RM and stirred the RM at -5 °C for 20 mins. RM was then allowed to warm slowly to room temp over one hour. RM was quenched with TEA (0.10 mL), stirred for 10 mins, and evaporated in vacuum to get crude product. Column purification was done using EA / cyclohexane on silica column using biotage. Fractions containing pure product were collected, evaporated in vacuum to get colorless gummy residue which became crispy solid 17 (650 mg, 87%) after drying in vacuum overnight. MALDI-TOF Calcd for C415H418N3O88[M+H]+6850.83, found 6854.45. Synthesis of partially deprotected Pentadecasaccharide 18:

[0617]

[0618] Substrate 17 (130 mg, 0.019 mmol) was taken in anhydrous tetrahydrofuran (5 mL) and methanol (5 mL) at rt and added 0.5 M solution of sodium methoxide (1.33 mL, 0.664 mmol) to it and stirred at rt for 2.5 days. The RM was evaporated in vacuum to dryness. The residue was diluted with water (50 mL) and extracted with EtOAc (20 mL X 3). Combined organic was washed with water (20 mL), dil. acetic acid solution (30 mL), brine solution (30 mL), dried (Na2SO4), filtered and evaporated in vacuum to get crude product as pale yellowish gummy solid. The crude material was purified by size exclusion column using LH-20 resin using 50% CHCl3in methanol. The initial intense sugar stain active spots were collected and evaporated in vacuum and dried under high vacuum to afford white solid layer 18 after drying (100 mg, 92%). MALDI-TOF Calcd for C340H372N3O76+[M+H]+5712.53, found 5711.97. Synthesis of fully deprotected Pentadecasaccharide 5-amino-pentyl cr-D- i-or-D-i l-(1 — >3)-cr-D-i l-(1— >3)-cr-D-

[0619]

[0620] i-or-D-i l-(1 — >3)-cr-D-i

[0621]

[0622] l-(1— >3)-cr-D-

[0623]

[0624]

[0625] l-(1— ►3)-cr-D-mannopyranoside 19:

[0626]

[0627] Substrate 18 (100 mg, 0.017 mmol) was taken in mixture of tert-butanol (5 ml_), DCM (HPLC grade, 2 ml_), and PBS (0.5 ml_), added 10% Pd / C (195 mg) to it and hydrogenated under ~3 bar H2atmosphere for 20 h. RM was filtered through the PTFE filter, washed with 50% methanol in water (6 mL X 3), and with water (6 mL X 2). The filtrate was concentrated under vacuum to get crude product as glassy white solid. 1 H NMR in D2O showed that the reaction was complete. The crude product was purified using C18-sepak column using water-acetonitrile as eluents. All the fractions were frozen and lyophilized over night to get white solids. The water fraction contained pure product 19 (29.7 mg, 67%).1H NMR (400 MHz, D2O) 5 5.38 (d, J = 4.7 Hz, 5H), 5.13 (d, J = 1.8 Hz, 4H), 5.05 (dd, J = 4.4, 1.8 Hz, 5H), 4.84 (d, J= 1.8 Hz, 1H), 4.23 (dt, J= 3.6, 1.9 Hz, 8H), 4.10 (dt, J= 13.5, 2.3 Hz, 7H), 4.01 - 3.62 (m, 76H), 3.55 (dt, J = 9.9, 6.0 Hz, 1 H), 3.01 (t, J = 7.6 Hz, 2H), 1.78 - 1.60 (m, 4H), 1.52 - 1.38 (m, 2H). MALDI-TOF Calcd for C95H163NNaO76+[M+Na]+2556.88, found 2556.20. Synthesis of pentadecasaccharide acceptor 20

[0628]

[0629] A freshly prepared solution of hydrazine monohydrate (16 pL, 0.525 mmol) dissolved in acetic acid (54 pL, 0.945 mmol) and pyridine (81 pL, 0.998 mmol) was added to a solution of 17 (360 mg, 0.053 mmol) in DCM (5 mL) at 5 °C. The resulting reaction mixture was stirred at room temperature for 20 h. Reaction was monitored by TLC for completion and was quenched using acetone (3 mL) and stirred for 30 mins. The solvent was removed under vacuum to obtain the crude product. The crude was purified using silica column on automated purification system using ethyl acetate and cyclohexane. The fractions containing intense sugar stain active spot were combined and the solvent evaporated to give the product 20 as colorless gummy residue which solidified on drying under vacuum overnight (283 mg, 80%). HRMS(QTOF) m / z: Calcd for C410H410N3Na3O863+[M+3Na]3+2273.2498 (6819.7495 / 3 = 2273.2498); found 2273.2450. Synthesis of pentadecasaccharide 21 with terminal methylphosphate

[0630]

[0631] To a solution of 20 (250 mg, 0.037 mmol) in anhydrous pyridine (2.5 ml_), diphenyl phosphite (0.18 ml_, 0.925 mmol) was added and the reaction mixture was stirred at rt under nitrogen for 15 min. The RM was then heated at 50-60 °C for 1 h and then slowly brought to rt and stirred overnight.

[0632] TEAB (2.5 ml_, 2.50 mmol) was added to the reaction mixture at rt. Immediately a white precipitate was observed, and the RM was diluted with DCM (30 ml_), stirred for 1 h and the organic layer was separated, dried over Na2SO4, and concentrated under reduced pressure. The crude product was purified by automated flash column chromatography using cyclohexane, 2% Et3N in EtOAc and 10% MeOH in DCM with 1% Et3N as eluents to give H-phosphonate derivative as white solid after drying (205 mg, 80%).31P NMR (162 MHz, CDCI3) 54.90.

[0633] To a solution of H-phosphonate (200 mg, 0.029 mmol) in anhydrous pyridine (4 ml_), methanol (0.023 ml_, 0.578 mmol), and pivaloyl chloride (0.018 ml_, 0.144 mmol) were added and the reaction mixture was stirred at rt under nitrogen for 4 h. The RM was then cooled to -40 °C, added a freshly made solution of I2(0.037 mg, 0.144 mmol) in pyridine (2 mL) and water (0.2 mL) and stirred at -40 °C for 1 h, and slowly warmed to rt over 20 min. TEAB (6 mL) was added to the reaction mixture at rt and the RM stirred at rt for 10 min. Reaction mixture was diluted with DCM (30 mL), stirred for 10 min and then the organic layer was separated. The separated organic layer was dried over Na2SO4. filtered and concentrated under reduced pressure. The crude product was purified by automated flash column chromatography using cyclohexane, 2% Et3N in EtOAc and 10% MeOH in DCM with 1% Et3N as eluents to give desired product 21 (81 mg, 40.3%).31P NMR (162 MHz, CDCI3) 0 -0.37.

[0634] Synthesis of pentadecasaccharide 23 with terminal methylphosphate

[0635]

[0636] 22, RT = HNEt3

[0637] Dowex-

[0638] water

[0639]

[0640] 23, RT = Na

[0641] Substrate 21 (40 mg, 5.75 pmol) was taken in a mixture of anhydrous tetrahydrofuran (2 mL) and anhydrous methanol (1 ml_). At rt added 0.5 M sodium methoxide solution (0.23 ml_, 0.12 mmol) and stirred for 2 h, and neutralized with acetic acid and evaporated to dryness. The crude was taken 50% CHCl3in methanol and purified using size exclusion chromatography using LH-20 resin. The initial intense sugar stain active spots were collected and evaporated in vacuum and dried under high vacuum to afford the partially deprotected substrate as a colorless layer on drying (25 mg, 73.5%).31P NMR (162 MHz, CDCI3) 53.03.

[0642] The partially deprotected substrate (24 mg, 4.06 pmol) was taken in mixture of tert-butanol (3 ml), dichloromethane (2 mL), and PBS buffer (0.5 mL), added 10% Pd / C (55 mg) to it and hydrogenated under ~3 bar H2 atmosphere for 16 h. RM was filtered through the PTFE filter, washed with methanol (3 mL X 2), 50% methanol in water (3 mL X 2), and water (3 mL X 2). The filtrate was concentrated under vacuum to get crude product as glassy white solid which was purified using C18 (2 g / 20 ml_)-Sepak column using water-acetonitrile as the eluents to obtain sugar stain active spots. All the fractions were frozen and lyophilized to get white fluffy solid and analyzed by HPLC and nmr. The pure product 22 containing fraction was Na+ion exchanged using Dowex-50WX8 resin and water as the eluent. The elute was collected, frozen and lyophilized to afford white fluffy solid 23 (9.2 mg, 83%, a mixture of both 2-methlyphosphate and 3-methlyphosphate compounds).31P NMR (162 MHz, D2O) 5 1.19, 1.01. MALDI-TOF Calcd for C96H165NNa2O79P+[M+Na]+2672.85, found 2672.78.

[0643] Synthesis of pentadecasaccharide 5-amino-pentyl cr-D-mannopyranosyl-(1 --or-D-

[0644]

[0645] mannopyranosyl-f 1 -^3)-or-D-mannopyranosyl-( 1 -^2)-or-D-mannopyranosyl-(1 -^3)-or-D-mannopyranosyl-f 1 -^3)-or-D-mannopyranosyl-( 1 -^2)-or-D-mannopyranosyl-(1 -^3)-or-D-mannopyranosyl-(1 -^3)-or-D-mannopyranosyl-(1 -^2)-or-D-mannopyranosyl-(1 --or-D-

[0646]

[0647] mannopyranosyl-(1 -^3)-or-D-mannopyranosyl-(1 -^2)-or-D-mannopyranosyl-(1 --or-D-

[0648]

[0649] mannopyranosyl-f 1 -^3)-or-D-mannopyranoside 24:

[0650]

[0651] Compound 24 was synthesized based on the protocols described in WO2019106200, pages 135-139 and 142-143, in analogy to the synthesis of compound 85b* (page 145).

[0652] 1H NMR (400 MHz, D2O) 5 5.38 (d, J = 1.7 Hz, 4H), 5.14 (d, J = 1.7 Hz, 1 H), 5.14 - 5.12 (m, 4H), 5.11 (d, J = 1.7 Hz, 1H), 5.04 (d, J = 1.8 Hz, 4H), 4.85 (d, J = 1.8 Hz, 1H), 4.30 -4.18 (m, 9H), 4.13 - 4.05 (m, 6H), 4.04 - 3.61 (m, 76H), 3.56 (dt, J = 9.8, 5.9 Hz, 1 H), 3.01 (t, J = 7.6 Hz, 2H), 1.77 - 1.59 (m, 4H), 1.56 - 1.38 (m, 2H). MALDI-TOF Calcd for C95H163NNaO76+[M+Na]+2556.88, found 2558.05.

[0653] Synthesis of the 03 antigen 25

[0654]

[0655] The 03 antigen compound 25 was synthesized according to W02019106200, compound 86b*, page 146.

[0656] Synthesis of qlycoconjugates:

[0657] Materials:

[0658] CRM197 was obtained from Eirgenix, Inc., Taiwan, expression system E.coli

[0659] BSA was obtained from Sigma / Merck, Cat.no. A7906-1 OG. Synthesis of NHS activated ester (AE) 19-AE

[0660]

[0661] Antigen 19 (6.8 mg, 2.68 pmol) was dissolved in DMSO (150 pL). Added triethylamine (13 pL, 0.094 mmol) to it at rt. Added Di-(N-succinimidyl) adipate (18.25 mg, 0.054 mmol) in DMSO (150 pL) and stirred for 2 h more at rt. The Antigen-NHS ester was precipitated out by adding EtOAc (3.5 mL) and centrifuged, removed the supernatant. Washed the precipitate with EtOAc (3 ml_X2), dried to get 19-NHS ester (19-AE) as white solid in vacuum for 15 minutes (6.8 mg, 92%).

[0662] Synthesis of glycoconjugate (GC) 19-GC: 19-AE

[0663]

[0664] 19-GC

[0665] Dissolved 19-NHS ester (19-AE) (6.8 mg, 2.463 pmol) in 0.1 M NaPi buffer (pH 7, 100 pL) added a stir bar, and stirred with 200 rpm at rt. Added freshly washed ~5.6 mg CRM197 in buffer (~80 pL) to it at rt. And rinsed the CRM197-microtube with 70 pL more of the buffer and transferred it to the reaction vial, the RM was stirred at rt for 20 h. Glyco-conjugate solution was transferred to the Amicon Ultra vial (30kDa MWCO), centrifuged for 5 minutes at room temperature. Added 300 pL of NaPi buffer to the reaction vial, rinsed and transferred to the filter and centrifuged again. Additional washings were done using 1X PBS solution for three-five more times. The last two washings were done using 10 mM T ris-HCI buffer (pH 7.4). After the final wash, the conjugate was sterile-filtered and stored 10 mM Tris-HCI buffer (pH 7.4) at 2-8 °C. The conjugate was analysed using MALDI-TOF MS (loading found was 13.05), SDS-PAGE, protein determination, endotoxin content and SEC-HPLC. Synthesis of glycoconjugate (GC) 19-BSA-GC:

[0666] 19-AE

[0667]

[0668] 19-BSA-GC

[0669] Analogously, the BSA-conjugate was synthesized by dissolving 19-NHS ester (19-AE) (2.96 mg, 1.072 pmol) in 0.1 M NaPi buffer (pH 7, 100 pL) in an Eppendorf tube. Added freshly washed BSA (2.85 mg, 0.043 pmol) in buffer (~80 pL) to it at rt. And rinsed the BSA-microtube with 20 pL more of the buffer and transferred it to the reaction vial, the RM was stirred at rt for 20 h. Glyco-conjugate solution was transferred to the Amicon Ultra vial (30kDa MWCO), centrifuged for 5 minutes at room temperature. Added 300 pL of NaPi buffer to the reaction vial, rinsed and transferred to the filter and centrifuged again. Additional washings were done using 1X PBS solution for five more times. After the final wash, the conjugate was sterile-filtered and stored in PBS buffer (pH 7.4) at 2-8 °C. The conjugate was analysed using MALDI-TOF MS (loading found was 18.13). Synthesis of activated ester 23-AE

[0670]

[0671] 23-AE

[0672] Antigen 23 (6.1 mg, 2.301 pmol) was dissolved in DMSO (150 pL). Added triethylamine (11 pL, 0.081 mmol) to it at rt. Added Di-(N-succinimidyl) adipate (17 mg, 0.050 mmol) in DMSO (150 pL) and stirred for 2 h more at rt. The Antigen-NHS ester was precipitated out by adding EtOAc (3.5 mL) and centrifuged, removed the supernatant. Washed the precipitate with EtOAc (3 ml_X2), dried to get 23-NHS ester (23-AE) as white solid in vacuum for 15 minutes (6.1 mg, 92%).

[0673] Synthesis of glycoconjugate 23-GC: 23-AE

[0674] pH 7.0 Buffer

[0675] CRM197

[0676]

[0677] 23-GC

[0678] Dissolved 23-NHS ester (23-AE) (6.1 mg, 2.121 pmol) in 0.1 M NaPi buffer (pH 7, 100 pL), added a stir bar, and stirred with 200 rpm at rt. Added freshly washed ~4.5 mg CRM197 in buffer (~80 pL) to it at rt. And rinsed the CRM-microtube with 75 pL more of the buffer and transferred it to the reaction vial, the RM was stirred at rt for 20 h. Glyco-conjugate solution was transferred to the Amicon Ultra vial (30kDa MWCO), centrifuged for 5 minutes at room temperature. Added 300 pL of NaPi buffer to the reaction vial, rinsed and transferred to the filter and centrifuged again. Additional washings were done using 1X PBS solution forthree-five more times. The last two washings were done using 10 mM Tris-HCI buffer (pH 7.4). After the final wash the conjugate was sterile-filtered and stored 10 mM Tris-HCI buffer (pH 7.4) at 2-8 °C. The conjugate was analysed using MALDI-TOF MS (loading found was 12.15), SDS-PAGE, protein determination, endotoxin content and SEC-HPLC.

[0679] Synthesis of glycoconjugate 23-BSA-GC:

[0680] Analogously, the BSA-conjugate was synthesized by dissolving 23-NHS ester (23-AE) (2.5 mg, 0.858 pmol) in 0.1 M NaPi buffer (pH 7, 100 pL) in a falcon tube. Added freshly washed BSA (1.5 mg, 0.023 pmol) in buffer (~80 pL) to it at rt. And rinsed the BSA-microtube with 20 pL more of the buffer and transferred it to the reaction vial, the RM was stirred at rt for 20 h. Glyco-conjugate solution was transferred to the Amicon Ultra vial (30kDa MWCO), centrifuged for 5 minutes at room temperature. Added 300 pL of NaPi buffer to the reaction vial, rinsed and transferred to the filter and centrifuged again. Additional washings were done using 1X PBS solution for five more times. After the final wash, the conjugate was sterile-filtered and stored in PBS buffer (pH 7.4) at 2-8 °C. The conjugate was analysed using MALDI-TOF MS (loading found was 10.69).

[0681] Synthesis of activated ester 24-AE

[0682]

[0683] 24-AE

[0684] Antigen 24 (7 mg, 2.76 pmol) was dissolved in DMSO (150 pL). Added triethylamine (13 pL, 0.097 mmol) to it at rt. Added Di-(N-succinimidyl) adipate (18.79 mg, 0.055 mmol) in DMSO (150 pL) and stirred for 2 h more at rt. The Antigen-NHS ester was precipitated out by adding EtOAc (3.5 mL) and centrifuged, removed the supernatant. Washed the precipitate with EtOAc (3 ml_X2), dried to get 24-NHS ester (24-AE) as white solid in vacuum for 15 minutes (7.0 mg, 92%). Synthesis of glycoconjugate 24-GC:

[0685] 24-AE

[0686]

[0687] 24-GC

[0688] Dissolved 24-NHS ester (24-AE) (7.0 mg, 2.54 pmol) in 0.1 M NaPi buffer (pH 7, 100 pL), added a stir bar, and stirred with 200 rpm at rt. Added freshly washed ~5.6 mg CRM197 in buffer (~100 pL) to it at rt. And rinsed the CRM-microtube with 50 pL more of the buffer and transferred it to the reaction vial, the RM was stirred at rt for 20 h. Glyco-conjugate solution was transferred to the Amicon Ultra vial (30kDa MWCO), centrifuged for 5 minutes at room temperature. Added 300 pL of NaPi buffer to the reaction vial, rinsed and transferred to the filter and centrifuged again. Additional washings were done using 1X PBS solution for three-five more times. The last two washings were done using 10 mM T ris-HCI buffer (pH 7.4). After the final wash the conjugate was sterile-filtered and stored 10 mM Tris-HCI buffer (pH 7.4) at 2-8 °C. The conjugate was analysed using MALDI-TOF MS (loading found was 13.05), SDS-PAGE, protein determination, endotoxin content and SEC- HPLC. Synthesis of glycoconjugate 24-BSA-GC:

[0689] Analogously, the BSA-conjugate was synthesized by dissolving 24-NHS ester (24-AE) (3.37 mg, 1.222 pmol) in 0.1 M NaPi buffer (pH 7, 100 pL) in a falcon tube. Added freshly washed BSA (2.85 mg, 0.043 pmol) in buffer (~80 pL) to it at rt. And rinsed the BSA-microtube with 20 pL more of the buffer and transferred it to the reaction vial, the RM was stirred at rt for 20 h. Glyco- conjugate solution was transferred to the Amicon Ultra vial (30kDa MWCO), centrifuged for 5 minutes at room temperature. Added 300 pL of NaPi buffer to the reaction vial, rinsed and transferred to the filter and centrifuged again. Additional washings were done using 1X PBS solution for five more times. After the final wash, the conjugate was sterile-filtered and stored in PBS buffer (pH 7.4) at 2-8 °C. The conjugate was analysed using MALDI-TOF MS (loading found was 8.45).

[0690] Synthesis of activated ester 25-AE

[0691]

[0692] 25-AE Antigen 25 (4.68 mg, 1.85 pmol) was dissolved in DMSO (200 pL). Added triethylamine (9 pL, 0.065 mmol) to it at rt. Added Di-(N-succinimidyl) adipate (12.56 mg, 0.037 mmol) in DMSO (150 pL) and stirred for 2 h more at rt. The Antigen-NHS ester was precipitated out by adding EtOAc (3.5 mL) and centrifuged, removed the supernatant. Washed the precipitate with EtOAc (3 ml_X2), dried to get 25-NHS ester (25-AE) as white solid in vacuum for 15 minutes (4.68 mg, 92%).

[0693] Synthesis of glycoconjugate 25-GC:

[0694] 25-AE

[0695]

[0696] 25-GC Dissolved 25-NHS ester (25-AE) (3.78 mg, 1.37 pmol) in 0.1 M NaPi buffer (pH 7, 120 pL), added a stir bar, and stirred with 200 rpm at rt. Added freshly washed 2.0 mg CRM197 in buffer (~100 pL) to it at rt. And rinsed the CRM-microtube with 50 pL more of the buffer and transferred it to the reaction vial, the RM was stirred at rt for 20 h. Glyco-conjugate solution was transferred to the Amicon Ultra-0.5 vial (30kDa MWCO), centrifuged for 5 minutes at room temperature. Added 300 pL of NaPi buffer to the reaction vial, rinsed and transferred to the filter and centrifuged again. Additional washings were done using 1X PBS solution for three-five more times. The last two washings were done using 10 mM Tris-HCl buffer (pH 7.4). After the final wash the conjugate was sterile-filtered and stored 10 mM Tris-HCl buffer (pH 7.4) at 2-8 °C. The conjugate was analysed using MALDI-TOF MS (loading found was 10.52), SDS-PAGE, protein determination, endotoxin content and SEC-HPLC.

[0697] Synthesis of glycoconjugate 25-BSA-GC:

[0698] Analogously, the BSA-conjugate was synthesized by dissolving 25-NHS ester (0.83 mg, 0.301 pmol) in 0.1 M NaPi buffer (pH 7, 30 pL) in a micro tube. Added freshly washed BSA (0.5 mg, 0.008 pmol) in buffer (~80 pL) to it at rt. And rinsed the BSA-microtube with 30 pL more of the buffer and transferred it to the reaction vial, the RM was stirred at rt for 20 h. Glyco-conjugate solution was transferred to the Amicon Ultra vial (30kDa MWCO), centrifuged for 5 minutes at room temperature. Added 300 pL of NaPi buffer to the reaction vial, rinsed and transferred to the filter and centrifuged again. Additional washings were done using 1X PBS solution for five more times. After the final wash, the conjugate was sterile-filtered and stored in PBS buffer (pH 7.4) at 2-8 °C. The conjugate was analysed using MALDI-TOF MS (loading found was 9.32).

[0699] Characterisation of

[0700]

[0701] HPLC-SEC: The glycoconjugates used for immunizations were analyzed by HPLC-SEC to observe mass differences between conjugated and unconjugated CRM197 proteins. The samples were diluted in 50 mM Tris, 20 mM NaCI, pH 7.2 and run on an Agilent 1100 HPLC system fitted with Tosoh TSK G2000 column (SWxl, 7.8 mm x 30 cm, 5 pm) and a Tosoh TSK gel Guard column (SWxl 6.0 mm x 4 cm, 7 pm). The flow rate was kept at 1 mL / min.

[0702] SDS-PAGE: The samples were mixed in microcentrifuge tubes and heated for 5 min at 95 °C. After cooling at room temperature for 5 min, approximately 2.5 pg of the samples were loaded onto the wells of a 10 % polyacrylamide gel along with 5 pL of the protein size marker (ROTI®Mark TRICOLOR XTRA, 10 to 310 kDa in Tris-Glycine buffer (Carl Roth)). The samples were run at a constant voltage of 120 for approximately 30-45 min. Staining was done using the Gel CodeTM Blue Safe Protein Stain as per manufacturer’s instructions. The gels were washed with deionized water overnight and scanned using a gel documentation system.

[0703] MALDI-TOF MS: Sinapic acid was used as matrix. Samples were measured using a Bruker Autoflex 2 instrument using linear positive mode.

[0704] Materials:

[0705] • ELISA plates (high-binding, EIA / RIA Plate, 96 well, flat bottom with low evaporation lid, company: Costar® 3361)

[0706] • Detection antibody: Goat anti rabbit IgG peroxidase conjugate (Sigma, #A4914) • Blocking solution: Commercial blocking reagent (Roche, cat.no. 11112589001) • Antibody diluent: PBS+1% BSA (w / v)

[0707] • Wash Buffer: PBS+0.1 % (v / v) Tween® 20 (PBS-T)

[0708] • Developing solution: 1 Step™ Ultra TMB-ELISA developer. (ThermoScientific, Cat # 34028)

[0709] • Stop solution- 2M sulphuric acid (H2SO4)

[0710] • Plate reader: FLUOstar Omega (BMG LABTECH)

[0711] • Software: GraphPad Prism version 7 or higher for data plotting and analysis • Alum: Alhydrogel® adjuvant 2%, InvivoGen, Cat. Code vac-alu-50

[0712] • Tween® 80 pure Ph. Eur, Th. Geyer, cat. no. 142050-1 L

[0713] • Pierce™ Modified Lowry Protein Assay Kit, Thermo Fisher Scientific, Cat. No.

[0714] 23240

[0715] • Mini-PROTEAN® TGX™ Gels- 10 %, 10 well (30pL / well) Control Nr:64175708 • GelCode™ Blue Safe Protein Stain; ThermoScientific; Ref: 1860957; Lot#:

[0716] TA260266

[0717] Methods:

[0718] Bacterial Strains and LPS.

[0719] Klebsiella pneumoniae strains differing in their LPS (O-antigen) were used to isolate and purify the corresponding LPS. The purified LPS were used as coating antigen in Enzyme Linked Immunosorbent Assay (ELISA). LPS was isolated using a commercial LPS extraction kit (JH Science) according to the manufacturer’s protocol.

[0720] Table 1. Klebsiella pneumoniae strains used for LPS isolation.

[0721]

[0722] Formulation of vaccine candidates for immunization. All formulations were prepared under sterile conditions. Drug substance (DS) and buffer (10 mM TRIS-HCI, pH 7.4, 140 mM NaCI and 0.03% (v / v) Tween® 80) were mixed in the appropriate pre-calculated dilution factor (see below) for the required glycan dose leaving out the required volume of aluminum hydroxide adjuvant (0.25 mg / mL). The DS-buffer mixture was gently mixed and aluminum hydroxide adjuvant stock was added fora final concentration of 0.250 mg / mL of aluminum. The mixture was immediately mixed by gentle pipetting and then mixed on a horizontal shaker at 250 rpm for at least 1 h at RT. Aliquots were stored in type 1 glass vials at 4 °C until further use.

[0723] The vaccines described above are prepared to contain the intended glycan dose (e.g., 2 pg glycan per injection) as follows. The average loading factor of the glycan antigen (in moles of antigen per mol carrier protein) is determined via MALDI-TOF MS by subtracting the determined molecular weight (m / z = 1) of CRM197 from the determined molecular weight of the DS (m / z = 1), then this mass difference is divided 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, providing the total mass of glycan attached on average per DS molecule. This total mass of glycan is divided by the determined molecular weight of the CRM197 protein to yield the glycan-to-protein mass ratio of the DS. This ratio is multiplied by the determined protein concentration of the DS, as determined by the Modified Lowry Protein Assay Kit (Thermo Fisher) according to the manufacturer’s protocol, to yield the glycan concentration of the DS. To obtain the dilution factor necessary 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., 4 pg / mL glycan concentration for a 2 pg glycan dose for rabbits with an injection volume of 500 pL). The DS is then diluted with this dilution factor relative to the final volume of the vaccine preparation.

[0724] Immunizations: Female Zika rabbits were immunized via the intramuscular (i.m.) route with an injection volume of 500 pL per dose. Animals were kept under specific pathogen-free conditions and were provided with water and food ad libitum.

[0725] Immunogenicity test with ELISA plates coated with BSA-conjugates:

[0726] Rabbits were immunized i.m. with CRM197 glycoconjugates of the indicated immunogens. Rabbits received 3 doses of the respective glycoconjugates corresponding to 2 pg glycan per dose, adjuvanted with Aluminum hydroxide adjuvant, at days 0, 14 and 28 via the intramuscular route. Preimmune sera were obtained at day 0 prior to the first immunization. Post-immune sera were obtained 7 or 14 days after the third immunization. The bars show mean ± SEM of n=4 or n=6 rabbits, obtained by ELISA experiments. To test the immunogenicity by ELISA, 96-well plates were coated with immunogen-BSA conjugates (0.2 pg antigen per well, in PBS, overnight at 4 °C), followed by a blocking step with commercial blocking agent (30 min. at room temperature) and incubation of antisera (diluted 1:1000 in PBS+1% (w / v) BSA), for 1 hour at room temperature. After three washing steps with PBS with 0.1% (v / v) Tween® 20, detection antibody was added (1:10,000 in PBS+1% (w / v) BSA, 30 min. at room temperature). After another 3 washing steps with PBS with 0.1% (v / v) Tween® 20, 100 pL per well of developing solution was and after 15-30 min., the reaction was stopped with 50 pL per well of 2M H2SO4. Finally, absorbance at 450 nm was determined in a plate reader. Day 0 values are at or slightly below 0. Sera were diluted 1:1000.

[0727] ELISA experiments with LPS:

[0728] Coating of plates with antigen: Isolated LPS were used for coating. LPS was dissolved in PBS to a concentration of 10 pg / mL and 50 pL was used for coating so that each well was coated with 0.5 pg of LPS. LPS solutions were subjected to overnight evaporation at RT at 4 °C.

[0729] Blocking: The plates were blocked using 100 pL of commercial blocking solution and incubated for 30 min. at RT. Incubation with diluted sera: Pooled or individual sera from different timepoints were diluted to their respective dilutions using 1% BSA (w / v) in PBS. 50 pL of the diluted sera were added in duplicates to the ELISA wells and incubated for 1 h at RT. 100 pL / well of 1 % BSA (w / v) in PBS served as blank. After incubation with sera, the plates were washed 3X with PBS-T.

[0730] Incubation with detection antibody: Anti-rabbit IgG HRP conjugate was diluted 1: 10,000 in 1% BSA (w / v) in PBS and 50 pL / well were added and incubated for 30 minutes at RT. After the incubation with detection antibody, the plates were washed 3X with PBS-T.

[0731] Substrate addition: To each well, 100 pL of TMB substrate were added and incubated for approx. 15-30 min. The reaction was stopped by adding 50 pL / well of 2M H2SO4. Absorption was measured at 450 nm using a plate reader. The absorption values were analyzed with the GraphPad Prism software.

[0732] Binding to live bacteria:

[0733] Pooled postimmune antisera diluted 1:4000 to 1:8000 in PBS with 1 % (w / v) BSA were preincubated with live K. pneumoniae bacteria (03 or O3b serotypes) obtained from overnight cultures in LB medium, adjusted to an OD600of 0.6 in PBS with 1% (w / v) BSA for 1 hour at room temperature. As controls, the same antisera were also pre-incubated with a control K. pneumoniae strain (02afg serotype). After removal of the bacteria by centrifugation, the supernatants were subjected to ELISA experiments with BSA conjugates coated, as described above. The bars show inhibition vales that were normalized to the respective 02afg controls.

[0734] The following results were obtained:

[0735]

[0736] In summary, it could be shown that the O3b glycoconjugate 19-GC, which does not bear a terminal methyl-phosphate group, is immunogenic, and the antibodies induced by this glycoconjugate recognises the O3b antigen on the surface of O3b serotype bacteria. In addition, and surprisingly, the antibodies elicited by this O3b glyco conjugate 19-GC are also cross reactive to the 03 antigen on the surface of 03 serotype bacteria. The crossreactivity of O3b glyco conjugate 19-GC is strong enough that it can be expected that 19-GC can serve as single compound against both, O3b and 03 strains of Klebsiella pneumoniae.

[0737] SEQ ID NO: 1 (CRM197)

[0738] GADDVVDSSK SFVMENFSSY HGTKPGYVDS 30

[0739] IQKGIQKPKS GTQGNYDDDW KEFYSTDNKY 60

[0740] DAAGYSVDNE NPLSGKAGGV VKVTYPGLTK 90

[0741] VLALKVDNAE TIKKELGLSL TEPLMEQVGT 120

[0742] EEFIKRFGDG ASRVVLSLPF AEGSSSVEYI 150

[0743] NNWEQAKALS VELEINFETR GKRGQDAMYE 180

[0744] YMAQACAGNR VRRSVGSSLS CINLDWDVIR 210

[0745] DKTKTKIESL KEHGPIKNKM SESPNKTVSE 240

[0746] EKAKQYLEEF HQTALEHPEL SELKTVTGTN 270

[0747] PVFAGANYAA WAVNVAQVID SETADNLEKT 300

[0748] TAALSILPGI GSVMGIADGA VHHNTEEIVA 330

[0749] QSIALSSLMV AQAIPLVGEL VDIGFAAYNF 360

[0750] VESIINLFQV VHNSYNRPAY SPGHKTQPFL 390

[0751] HDGYAVSWNT VEDSIIRTGF QGESGHDIKI 420

[0752] TAENTPLPIA GVLLPTIPGK LDVNKSKTHI 450

[0753] SVNGRKIRMR CRAIDGDVTF CRPKSPVYVG 480

[0754] NGVHANLHVA FHRSSSEKIH SNEISSDSIG 510

[0755] 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

[0756] 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 ELGKKFNIKT RLSYFSMNHD PVKIPNLLDD 390 TIYNDTEGFN IESKDLKSEY KGQNMRVNTN 420 AFRNVDGSGL VSKLIGLCKK IIPPTNIREN 450 LYNRTASLTD LGGELCIKIK NEDLTFIAEK 480 NSFSEEPFQD EIVSYNTKNK PLNFNYSLDK 510 IIVDYNLQSK ITLPNDRTTP VTKGIPYAPE 540 YKSNAASTIE IHNIDDNTIY QYLYAQKSPT 570 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

[0757] 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

[0758] SEQ ID NO: 5 (Neisseria meningitidis outer membrane protein (OMP) (Uniprot ID: Q51229))

[0759] 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

[0760] KF 272 SEQ ID NO: 6 (capsid protein of bacteriophage Q|3 (Uniprot ID: P03615)) AKLETVTLGN IGKDGKQTLV LNPRGVNPTN 30 GVASLSQAGA VPALEKRVTV SVSQPSRNRK 60 NYKVQVKIQN PTACTANGSC DPSVTRQAYA 90 DVTFSFTQYS TDEERAFVRT ELAALLASPL 120 LIDAIDQLNP AY 132

Claims

Claims1. An oligosaccharide-carrier protein conjugate of formula (I)whereinR represents OHm represents 3, 4, 5, 6, 7 or 8, preferably 4, 5, or 6;i is at least 1, preferably from 1 up to an integer corresponding 90% of the number of lysine residues contained in the carrier protein CP;-L-T- represents a linker L and a spacer T which together form a bridge having a backbone with a length of 5 to 25 atoms covalently linked together that forms 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 at the carrier protein CP, wherein the atoms of the backbone are selected from the group consisting of carbon, nitrogen, oxygen and sulphur; andCP is a carrier protein selected from the group consisting of CRM197; diphtheria toxoid; tetanus toxoid; cholera toxin B subunit; Neisseria meningitidis outer membrane protein (OMP); and capsid protein of bacteriophage Q|3 (especially CRM197);or a pharmaceutically acceptable salt thereof.

2. The oligosaccharide-carrier protein conjugate according to claim 1, or a pharmaceutically acceptable salt thereof, wherein m is 5.

3. The oligosaccharide-carrier protein conjugate according to claim 1 or 2, or a pharmaceutically acceptable salt thereof, wherein-L-T- represents a linker L and a spacer T which together form a bridge having a backbone with a length of 5 to 25 atoms covalently linked together that forms 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 at the carrier protein CP, bearing at most one double bond,wherein the atoms of the backbone are selected from the group consisting of carbon, nitrogen, oxygen and sulphur, andwherein the backbone may be substituted with one or more (especially 1, 2, 3 or 4) substituents independently selected from the group consisting of oxo, (Ci-4)alkyl, fluoro, and (Ci-2)alkoxy (especially oxo), andwherein a part of the backbone optionally may be part of a 4-, 5- or 6-membered ring selected from:

4. The oligosaccharide-carrier protein conjugate according to any one of claims 1 to 3, or a pharmaceutically acceptable salt thereof, wherein the backbone of the bridge has a length of 5 to 20, preferably 5 to 16, atoms covalently linked together that forms 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 at the carrier protein CP.

5. The oligosaccharide-carrier protein conjugate according to any one of claims 1 to 4, or a pharmaceutically acceptable salt thereof, wherein the carrier protein CP is CRM197.

6. The oligosaccharide-carrier protein conjugate according to any one of claims 1 to 5, or a pharmaceutically acceptable salt thereof, whereinL represents-(C2-io)alkylene-NH-, preferably *-(CH2)a-NH-; wherein a is from 2 to 10;*-(CH2CH2O)b-CH2CH2NH-, wherein b is 1, 2 or 3;*-CH2CH2S-CH2CH2NH-;*-(C2-io)fluoroalkylene-NH-, preferably with fluoroalkylene being a saturated straight chain;*-(CH2)CNHC(O)(CH2)C-NH-, wherein c and c’ are independently from each other from 2 to 6;*-(CH2)dNHC(O)NH(CH2)d -NH-, wherein d and d’ are independently from each other from 2 to 6;*-(Ci-io)alkylene-C(0)-NH-(C2-io)alkylene-NH-; preferably *-(CH2)e-C(O)-NH- (CH2)e’-NH-; wherein e is from 1 to 10 and e’ is from 2 to 10; or*-(c2-io)alkylene-0-NH-, preferably *-(CH2)f-O-NH-, wherein f is from 2 to 10; or L-T represents*-(c2-w)alkylene-S-R1, preferably *-(CH2)g-S-R1, wherein g is from 2 to 10; and T represents-C(0)-(Co-io)alkylene-C(0)-, preferably -C(O)-(CH2)h-C(O)-, wherein h is from 0 to 10;-C(O)-CH2CH2-(OCH2CH2)j-C(O)-, wherein j is from 1 to 5;-C(O)-CH2(CH2)k-(SCH2(CH2)k’)k”-C(O)-, wherein k is 0 or 1, k’ is 0 or 1, and k” is 1, 2, or 3;, wherein p is from 1 to 4, and p’ is 1 or 2; andR1represents7. The oligosaccharide-carrier protein conjugate according to claim 6, or a pharmaceutically acceptable salt thereof, whereinL represents*-(CH2)2-NH-, *-(CH2)3-NH-, *-(CH2)4-NH-, *-(CH2)5-NH-, or *-(CH2)6-NH-, preferably *-(CH2)5-NH-; andT represents-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)-.

8. The oligosaccharide-carrier protein conjugate according to any one of claims 1 to 7, or a pharmaceutically acceptable salt thereof, wherein i is from 1 to 28, preferably from 8 to 15, or a pharmaceutically acceptable salt thereof.

9. The oligosaccharide-carrier protein conjugate according to claim 7, or a pharmaceutically acceptable salt thereof, having the structure of formula (Ila):(Ila);whereinR represents OH;m represents 3, 4, 5, 6, 7 or 8, preferably 4, 5 or 6, more preferably 5; andi is from 1 to 28, preferably from 8 to 15.

10. A pharmaceutical composition comprising, as active principle, an oligosaccharide-carrier protein conjugate according to any one of claims 1 to 9, or a pharmaceutically acceptable salt thereof, and at least one therapeutically inert excipient.

11. The pharmaceutical composition according to claim 10, further comprising an adjuvant.

12. An oligosaccharide-carrier protein conjugate according to any one of claims 1 to 9, or a pharmaceutically acceptable salt thereof, for the use as a medicament, in particular as a vaccine.

13. An oligosaccharide-carrier protein conjugate according to any one of claims 1 to 9, or a pharmaceutically acceptable salt thereof, for the use in the prevention and / or treatment of a K. pneumoniae infection.

14. A multivalent vaccine comprising the oligosaccharide-carrier protein conjugate according to any one of claims 1 to 9, or a pharmaceutically acceptable salt thereof.

15. An intermediate compound for preparing the oligosaccharide-carrier protein conjugate according to any one of claims 6 to 9, having the structure of formula (III):whereinR represents OH;m represents 3, 4, 5, 6, 7 or 8; preferably 4, 5 or 6;L1represents*-(C2-10)alkylene-NH2, preferably *-(CH2)a-NH2; wherein a is from 2 to 10, more preferably a is 5;*-(CH2CH2O)b-CH2CH2NH2, wherein b is 1, 2 or 3;*-CH2CH2S-CH2CH2NH2;*-(C2-10)fluoroalkylene-NH2;*-(CH2)CNHC(O)(CH2)C -NH2, wherein c and c’ are independently from each other from 2 to 6;*-(CH2)dNHC(O)NH(CH2)d -NH2, wherein d and d’ are independently from each other from 2 to 6;*-(C1-10)alkylene-C(O)-NH-(C2-10)alkylene-NH2;*-(C2-10)alkylene-O-NH2; or*-(C2-10)alkylene-SH;or a pharmaceutically acceptable salt thereof.

16. An intermediate compound for preparing the oligosaccharide-carrier protein conjugate according to any one of claims 6 to 9, or a pharmaceutically acceptable salt thereof, having the structure of formula (IV):whereinR represents OH;m represents 3, 4, 5, 6, 7 or 8; preferably 4, 5 or 6;L represents*-(C2-10)alkylene-NH-, preferably *-(CH2)a-NH- wherein a is from 2 to 10, more preferably 5;*-(CH2CH2O)b-CH2CH2NH-, wherein b is 1, 2 or 3;*-CH2CH2S-CH2CH2NH-;*-(C2-io)fluoroalkylene-NH-;*-(CH2)CNHC(O)(CH2)C-NH-, wherein c and c’ are independently from each other from 2 to 6;*-(CH2)dNHC(O)NH(CH2)d -NH-, wherein d and d’ are independently from each other from 2 to 6;*-(C1-10)alkylene-C(O)-NH-(C2-10)alkylene-NH-; or*-(C2-10)alkylene-O-NH-;T1represents-C(O)-(C0-10)alkylene-C(O)X;-C(O)-CH2CH2-(OCH2CH2)j-C(O)X, wherein j is from 1 to 5;-C(O)-CH2(CH2)k-(SCH2(CH2)k’)k”-C(O)X, wherein k is 0 or 1, k’ is 0 or 1, and k” is 1, 2, or 3;, wherein p is from 1 to 4, preferably 1, and p’ is 1 or 2;-C(O)X represents -C(O)OH or an activated ester, wherein preferablyX represents, orY represents Me, Et, Bu or -(CH2CH2O)3CH3.

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