# US 2014/0193416 A1 · Oligosaccharides and oligosaccharide-protein conjugates derived from clostridium difficle polysaccaride ps-i, methods of synthesis and uses thereof, in particular as vaccines and diagnostic tools

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- Number: US 2014/0193416 A1
- Document ID: US20140193416A1
- Status: Published application
- Kind: A1
- Application number: 14/236,620
- Filed: 2012-07-30
- Published: 2014-07-10
- Earliest claimed: 2011-08-02
- Assignees: Max-Planck-Gesellschaft zur Foerderung der Wissenschaften e.V.
- Inventors: Peter H. Seeberger; Christopher E. Martin; Felix Broecker; Chakkumkal Anish
- Web page: https://patentgrep.com/patent/US20140193416A1

## CPC

- C07H 3/06: Oligosaccharides, i.e. having three to five saccharide radicals attached to each other by glycosidic linkages
- A61P 1/12: Antidiarrhoeals
- A61P 31/04: Antibacterial agents
- C07H 3/08: Deoxysugars; Unsaturated sugars (1,2-dideoxy-1-enoses C07D); Osones
- C07H 5/04: to nitrogen
- C07H 13/08: having the esterifying carboxyl radicals directly attached to carbocyclic rings
- C07H 15/04: attached to an oxygen atom of the saccharide radical
- C07H 15/20: Carbocyclic rings
- C07K 16/1282: Clostridium (G)
- G01N 33/56911: {Bacteria}
- C07H: SUGARS; DERIVATIVES THEREOF; NUCLEOSIDES; NUCLEOTIDES; NUCLEIC ACIDS (derivatives of aldonic or saccharic acids C07C, C07D; aldonic acids, saccharic acids C07C59/105, C07C59/285; cyanohydrins C07C255/16; glycals C07D; compounds of unknown constitution C07G; polysaccharides, derivatives thereof C08B; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification C12N15/00; sugar industry C13)
- A61P: SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- C07K: PEPTIDES (peptides in foodstuffs A23; obtaining protein compositions for foodstuffs, working-up proteins for foodstuffs A23J; preparations for medicinal purposes A61K; peptides containing beta-lactam rings C07D; cyclic dipeptides not having in their molecule any other peptide link than those which form their ring, e.g. piperazine-2,5-diones, C07D; ergot alkaloids of the cyclic peptide type C07D519/02; macromolecular compounds having statistically distributed amino acid units in their molecules, i.e. when the preparation does not provide for a specific; but for a random sequence of the amino acid units, homopolyamides and block copolyamides derived from amino acids C08G69/00; macromolecular products derived from proteins C08H1/00; preparation of glue or gelatine C09H; single cell proteins, enzymes C12N; genetic engineering processes for obtaining peptides C12N15/00; compositions for measuring or testing processes involving enzymes C12Q; investigation or analysis of biological material G01N33/00)
- G01N: INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES (measuring or testing apparatus or processes other than immunoassay, involving enzymes or microorganisms C12M, C12Q)

## Abstract

The invention relates to a synthetic oligosaccharide representing part of the repeating unit of the Clostridium difficile glycopolymer PS-I and having the sequence of the pentasaccharide a-L-Rhap-(1→3)-β-D-Glcp-(1→4)-[a-L-Rhap-(1→3]-a-D-Glcp-(1→2)-a-D-Glcp or a synthetic fragment or derivative thereof. Preferably, the claimed synthetic oligosaccharide bears at least one linker L for conjugation to a carrier protein or for immobilization on a surface. Further aspects of the invention relate to advantageous methods for synthesizing said synthetic oligosaccharide and oligosaccharide-protein conjugate as well as to uses thereof, in particular as vaccines and diagnostic tools.

## Claims

### Claim 1

A synthetic oligosaccharide representing part of a repeating unit of a Clostridium difficile glycopolymer PS-I and having a sequence of a pentasaccharide α-L-Rhap-(1→3)-β-D-Glcp-(1→4)-[α-L-Rhap-(1→3)]-α-D-Glcp-(1→2)-α-D-Glcp or a fragment or derivative thereof.

### Claim 2 (depends on 1)

The synthetic oligosaccharide according to claim 1 bearing at least one linker L for conjugation to a carrier protein or for immobilization on a surface.

### Claim 3 (depends on 2)

The synthetic oligosaccharide according to claim 2 wherein the linker L is a member selected from the group consisting of an aliphatic or aromatic residue, comprising a reactive functional group.

### Claim 4 (depends on 3)

The synthetic oligosaccharide according to claim 3 wherein the linker L is (CH2)nNH2, with n being an integer from 2 to 50.

### Claim 5 (depends on 1)

The synthetic oligosaccharide according to claim 1, wherein at least one hydroxyl group is derivatized and/or substituted by other functional groups or atoms.

### Claim 6 (depends on 3)

The synthetic oligosaccharide according to claim 3, represented by structure 1 or 1′
or a fragment 2, 3, 4, 5, 20, 21, 22, 23, 24, 27, 29, 30, 31, 32, 33, 34 35, 36, 37, 38, 39 of 1; or a fragment 2′, 20′, 21′, 22′, 23′, 24′, 30′, 31′, 32′, 33′, 34′, 35′, 36′, 37′, 38′, 39′ of 1′
or a derivative thereof.

### Claim 7 (depends on 1)

The synthetic oligosaccharide according to claim 1 conjugated to a carrier protein.

### Claim 8 (depends on 7)

The synthetic oligosaccharide according to claim 7, wherein the carrier protein is diphtheria toxoid CRM197, tetanus toxoid (TT), outer membrane protein (OMP), bovine serum albumin, (BSA), keyhole limpet hemocyanine (KLH), diphtheria toxoid (DT), cholera toxoid (CT), recombinant Pseudomonas aeruginosa exotoxin A (rEPA), Clostridium difficile toxin A (TcdA), or Clostridium difficile toxin B (TcdB).

### Claim 9 (depends on 1)

A composition comprising a synthetic oligosaccharide as defined in claim 1 in a pharmaceutically acceptable formulation.

### Claim 10 (depends on 9)

The composition according to claim 9, wherein the composition further comprises an immuno-stimulatory component.

### Claim 11 (depends on 1)

An antibody having specificity for an immunogenic determinant which comprises the pentasaccharide of claim 1 or a truncated derivative thereof.

### Claim 12 (depends on 1, 11)

The antibody according to claim 11 which has been raised against the oligosaccharide-protein conjugate according to claim 1 conjugated to a carrier protein.

### Claim 13 (depends on 11)

The antibody according to claim 11 which is a polyclonal or monoclonal antibody.

### Claim 14 (depends on 13)

The antibody of claim 13 which is a monoclonal antibody 2C5, 10A1 or 10D6.

### Claim 15 (depends on 6)

A method for preparing the pentasaccharide of claim 6 having the following formula 1
which comprises
a) assembling the monosaccharide building blocks 2 and 3 or 4 to yield the corresponding disaccharide 21, reacting the disaccharide 21 with building block 4 to form the trisaccharide 23, subjecting the trisaccharide 23 to a bis-glycosylation reaction with 2 molecules of building block 5 to yield the fully protected pentasaccharide 24 and finally, after deprotection, to yield pentasaccharide 1, or
b) assembling the monosaccharide building blocks 2 and 27 to yield the corresponding disaccharide 30, reacting the disaccharide 30 with building block 4 or 29 to form the protected trisaccharide 32, deprotecting the trisaccharide 32 to obtain trisaccharide 33 and subjecting trisaccharide 33 to a bis-glycosylation reaction with 2 molecules of building block 5 to yield the fully protected pentasaccharide 34 and finally, after deprotection, to yield pentasaccharide 1.

### Claim 16 (depends on 6)

A method for preparing the pentasaccharide of claim 6, which is α-L-Rhap-(1→3)-β-D-Glcp-(1→4)-[α-L-Rhap-(1→3)]-α-D-Glcp-(1→2)-α-D-Glcp or a derivative thereof, said method comprising
a) assembling a monosaccharide building block 2′, wherein the specific protected amino linker of building block 2 is replaced by a different protected or unprotected linker L, and building blocks 3 or 4 to yield the corresponding disaccharide 21′, reacting the disaccharide 21′ with building block 4 to form the trisaccharide 23′, subjecting the trisaccharide 23′ to a bis-glycosylation reaction with 2 molecules of building block 5 to yield the corresponding fully protected pentasaccharide 24′ and finally, after deprotection, to yield pentasaccharide 1′, wherein the specific amino linker of pentasaccharide 1 is replaced by a different linker L, or
b) assembling a monosaccharide building block 2′, wherein the specific protected amino linker of building block 2 is replaced by a different protected or unprotected linker L, and building block 27 to yield the corresponding disaccharide 30′, reacting the disaccharide 30′ with building block 4 or 29 to form the corresponding protected trisaccharide 32′, deprotecting the trisaccharide 32′ to obtain trisaccharide 33′, subjecting the trisaccharide 33′ to a bis-glycosylation reaction with 2 molecules of building block 5 to yield the corresponding fully protected pentasaccharide 34′ and finally, after deprotection, to yield pentasaccharide 1′, wherein the specific amino linker of pentasaccharide 1 is replaced by a different linker L.

### Claim 17 (depends on 6)

A method for preparing the pentasaccharide α-L-Rhap-(1→3)-β-D-Glcp-(1→4)-[α-L-Rhap-(1→3)]-α-D-Glcp-(1→2)-α-D-Glcp of claim 6 having the following formula 1
or the pentasaccharide 1′, wherein the specific amino linker of pentasaccharide 1 is replaced by a different linker L, said method comprising an intermediate reaction comprising at least one of molecules, 2, 2′, 3, 4, 5, 20, 21, 22, 23, 24, 27, 29, 30, 30′, 31, 32, 32′, 33, 33′, 34′.

### Claim 18 (depends on 1)

An in vitro method of detecting Clostridium difficile which comprises providing the synthetic oligosaccharide as defined in claim 1 or a mixture thereof, and detecting whether the synthetic oligosaccharide is bound to Clostridium difficile.

### Claim 19 (depends on 1)

A method of identifying a certain strain of Clostridium difficile comprising providing the synthetic oligosaccharide of claim 1 and detecting whether the synthetic oligosaccharide is bound to a certain strain of Clostridium difficile.

### Claim 20 (depends on 1)

An analytical standard for immunoassays comprising the synthetic oligosaccharide as defined in claim 1 or a mixture thereof.

### Claim 21 (depends on 1)

A method for preparing a pharmaceutical composition for the treatment or prevention of a disease caused by the pathogen Clostridium difficile said method comprising providing in the pharmaceutical composition the synthetic oligosaccharide as defined in claim 1 or an antibody thereof.

### Claim 22 (depends on 1)

A method of treating or preventing a disease caused by the pathogen Clostridium difficile which comprises administering to a subject the synthetic oligosaccharide according to claim 1 or an antibody thereof.

### Claim 23 (depends on 1)

A method of inducing an immune response against Clostridium difficile in a subject comprising administering to the subject the synthetic oligosaccharide according to claim 1 or a mixture thereof.

### Claim 24 (depends on 1)

A method of treating or preventing Clostridium difficile infection in a subject comprising administering to the subject the synthetic oligosaccharide according to claim 1 or a composition or a mixture thereof.

### Claim 25 (depends on 1)

A diagnostic method for Clostridium difficile infection comprising contacting a sample with the synthetic oligosaccharide of claim 1 or a composition or a mixture thereof.

### Claim 26 (depends on 7)

A method for preparing the oligosaccharide according to claim 7 which comprises reacting a unique terminal amine of the linker L with one of two NHS-activated esters of Di(N-succinimidyl) adipate to form an amide and subsequent coupling of the amide to the carrier protein.

### Claim 27 (depends on 26)

The method according to claim 26 wherein the carrier protein is diphtheria toxoid CRM197, tetanus toxoid (TT), outer membrane protein (OMP), bovine serum albumin, (BSA), keyhole limpet hemocyanine (KLH), diphtheria toxoid (DT), cholera toxoid (CT), recombinant Pseudomonas aeruginosa exotoxin A (rEPA), Clostridium difficile toxin A (TcdA), or Clostridium difficile toxin B (TcdB).