# US 10,500,263 B2 · Glycoconjugate vaccines comprising basic units of a molecular construct expressing built-in multiple epitopes for the formulation of a broad-spectrum vaccine against infections due to enteropathogenic bacteria

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- Number: US 10,500,263 B2
- Document ID: US10500263B2
- Status: Granted
- Kind: B2
- Application number: 15/329,205
- Filed: 2015-07-24
- Published: 2017-07-27
- Granted: 2019-12-10
- Earliest claimed: 2014-07-25
- Assignees: Biosynth AG
- Applicants: Biosynth S.R.L.
- Inventors: Massimo Porro
- Web page: https://patentgrep.com/patent/US10500263B2

## CPC

- A61K 39/107: {Vibrio}
- A61K 39/0258: {Escherichia}
- A61K 39/0275: {Salmonella}
- A61K 47/646: {the entire peptide or protein drug conjugate elicits an immune response, e.g. conjugate vaccines}
- A61P 31/00: Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
- A61P 31/04: Antibacterial agents
- A61P 37/04: Immunostimulants
- A61K 2039/54: {characterised by the route of administration}
- A61K 2039/545: {characterised by the dose, timing or administration schedule}
- A61K 2039/55505: {Inorganic adjuvants}
- A61K 2039/575: {humoral response}
- A61K 2039/6037: {Bacterial toxins, e.g. diphteria toxoid [DT], tetanus toxoid [TT]}
- A61K 2039/627: {characterised by the linker}
- A61K 2039/70: {Multivalent vaccine}
- Y02A 50/30: Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change
- A61K: PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES (devices or methods specially adapted for bringing pharmaceutical products into particular physical or administering forms A61J3/00; chemical aspects of, or use of materials for deodorisation of air, for disinfection or sterilisation, or for bandages, dressings, absorbent pads or surgical articles A61L; soap compositions C11D)
- A61P: SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- Y02A: TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE

## Abstract

The present invention refers to new glycoconjugate antigens expressing built-in multiple epitopes and to polyvalent glycoconjugate vaccines intended for the protection of mammalians, and particularly for the protection of the human population from enteropathogenic bacteria, such as the Gram-positive anaerobic bacterium Clostridium difficile and the Gram-negative bacteria Salmonella typhi, Escherichia Coli, Vibrio Cholerae, Shigella flexneri, Salmonella typhimurium, Salmonella enteritidis, Salmonella paratyphi A, Shigella sonnei, Shigella dysenteriae, Salmonella cholerasuis, Klebsiella, Enterobacter, Pseudomonas aeruginosa and/or from viral gastrointestinal infections due to human noroviruses.

## Claims

### Claim 1

Antigenic multivalent molecular construct consisting of basic units comprising the helper-T dependent carrier detoxified proteins selected between Enterotoxoid A and Cytotoxoid B from Clostridium difficile covalently bound to a minimum of three carbohydrate structures from enteropathogenic bacteria selected between bacterial polysaccharides or detoxified lipopolysaccharides of different serological specificity, wherein each carbohydrate structure comprises at least one of the repeating basic epitopes consisting of a minimum of five to twelve monosaccharide residues, wherein at least one mole of carrier protein is bound to at least one mole of each of the at least three carbohydrate structure or their molar sum to form carried carbohydrate structures of different serological specificity.

### Claim 2 (depends on 1)

Antigenic multivalent molecular construct according to claim 1, wherein said Enterotoxoid A or Cytotoxoid B originating from Clostridium difficile are detoxified by formalin-treatment or by DNA recombinant technology.

### Claim 3 (depends on 1)

Antigenic multivalent molecular construct according to claim 1, wherein said carried carbohydrate structures of different serological specificity are selected among capsular polysaccharides of Salmonella typhi, Vibrio cholerae, Escherichia coli and Clostridium difficile or a combination thereof.

### Claim 4 (depends on 1)

Antigenic multivalent molecular construct according to claim 1, wherein said detoxified lipopolysaccharide is an Endotoxoid.

### Claim 5 (depends on 1)

Antigenic multivalent molecular construct according to claim 1, wherein said carried carbohydrate structures of different serological specificity are selected among detoxified lipopolysaccharides/Endotoxoids of enteropathogenic bacteria selected among Salmonella typhi, Escherichia coli, Vibrio cholerae, Salmonella enteritidis, Shigella flexneri, Salmonella paratyphi A, Salmonella dysenteriae, Shigella sonnei and Salmonella cholerasuis or a combination thereof.

### Claim 6 (depends on 1)

Antigenic multivalent molecular construct according to claim 1, selected between:
Enterotoxoid A covalently bound to the capsular polysaccharides of Salmonella typhi, Vibrio cholerae and Escherichia coli;
Cytotoxoid B covalently bound to the capsular polysaccharides of Salmonella typhi, Vibrio cholerae and Escherichia coli;
Enterotoxoid A covalently bound to the detoxified lipopolysaccharides/Endotoxoids of Salmonella enteritidis, Salmonella paratyphi A and Salmonella dysenteriae;
Cytotoxoid B covalently bound to the detoxified lipopolysaccharides/Endotoxoids of Salmonella enteritidis, Salmonella paratyphi A and Salmonella dysenteriae.

### Claim 7

A conjugation process for preparing the antigenic multivalent molecular construct according to 1, which comprises the following steps: a) chemical activation of the at least three antigenically different carbohydrate structures selected between:
capsular polysaccharides of Salmonella typhi, Vibrio cholerae, Escherichia coli and Clostridium difficile
or
lipopolysaccharides from Salmonella typhi, Escherichia coli, Vibrio cholerae, Salmonella enteritidis, Shigella jlexneri, Shigella sonnei, Salmonella paratyphi A, Salmonella dysenteriae and Salmonella cholerasuis;
forming imine reduced bonds with an alkyl diamine spacer, then derivatized to active esters; b) simultaneous coupling of the at least three ester-derivative carbohydrate structures to the amino groups of the polyfunctional carrier protein Enterotoxoid A or Cytotoxoid B from Clostridium difficile, through the formation of amide bonds;
wherein at least one mole of protein carrier is reacted with at least one mole of each, or their molar sum, of said antigenically different carbohydrate structures.

### Claim 8 (depends on 7)

A conjugation process according to claim 7, wherein the carbohydrate structures are chemically activated in their corresponding diamine butyric acid derivatives and the active esters are succinimidyl esters.

### Claim 9 (depends on 1)

A conjugation process for preparing the antigenic multivalent molecular construct according to claim 1, which comprises the simultaneous coupling of the amino groups of the polyfunctional carrier protein Enterotoxoid A or Cytotoxoid B from Clostridium difficile with at least three antigenically different carbohydrate structures selected between:
capsular polysaccharides of Salmonella typhi, Vibrio cholera, Escherichia coli and Clostridium difficile or
lipopolysaccharides from Salmonella typhi, Escherichia coli, Vibrio cholerae, Salmonella enteritidis, Shigella flexneri, Shigella sonnei, Salmonella paratyphi A, Salmonella dysenteriae and Salmonella cholerasuis;
via reductive amination forming imine-reduced bond(s), such carbohydrate structures being previously activated to polyfunctionality, with or without molecular spacers, by O-de-hydrogen uncoupling in vicinal hydroxyl groups, via oxidation.

### Claim 10 (depends on 7)

A conjugation process according to claim 7, further comprising an additional step of detoxification of said lipopolysaccharides alternatively by a) cleaving out the Lipid A moiety before or after the coupling reaction is performed, or b) saturation of the Lipid A-binding site through a specific strategy that use the Synthetic Anti-Endotoxin Peptides (SAEP), before or after the coupling reaction is performed;
wherein at least one mole of protein carrier is reacted with at least one mole of each, or their molar sum, of said antigenically different carbohydrate structures.

### Claim 11 (depends on 7)

A conjugation process according to claim 7, wherein the carbohydrate structures of step a) comprise at least one of the repeating basic epitopes consisting of a minimum of five to twelve monosaccharide residues as assessed by molecular mass determination and NMR spectroscopy, said repeating basic epitopes being antigenically assessed by reactivity with type-specific or group-specific polyclonal or monoclonal antibodies through the determination of their respective MIC50 values in the inhibition of their homologous Polysaccharide-Antibody reference system.

### Claim 12 (depends on 7)

The antigenic multivalent molecular construct obtainable by the conjugation process according to claim 7.