# US 6,573,245 B1 · Modified polysaccharide adjuvant-protein antigen conjugates, the preparation thereof and the use thereof

> Agents: this data is available as JSON from the patentgrep API and MCP server — see https://patentgrep.com/llms.txt

- Number: US 6,573,245 B1
- Document ID: US6573245B1
- Status: Granted
- Kind: B1
- Application number: 09/301,115
- Filed: 1999-04-28
- Granted: 2003-06-03
- Earliest claimed: 1998-04-28
- Assignees: Galenica Pharmaceuticals, Inc.
- Inventors: Dante J. Marciani
- Web page: https://patentgrep.com/patent/US6573245B1

## CPC

- A61K 39/39: characterised by the immunostimulating additives, e.g. chemical adjuvants
- A61K 39/385: Haptens or antigens, bound to carriers
- A61P 31/04: Antibacterial agents
- A61P 31/10: Antimycotics
- A61P 31/12: Antivirals
- A61P 31/14: for RNA viruses
- A61P 31/16: for influenza or rhinoviruses
- A61P 35/00: Antineoplastic agents
- A61P 37/04: Immunostimulants
- C07H 3/04: Disaccharides
- C07H 3/06: Oligosaccharides, i.e. having three to five saccharide radicals attached to each other by glycosidic linkages
- A61K 2039/55583: {Polysaccharides}
- A61K 2039/6087: {Polysaccharides; Lipopolysaccharides [LPS]}
- A61K 2039/627: {characterised by the linker}
- 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
- 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)

## Abstract

The present invention is directed to chemical conjugates (herein referred to as polysaccharide adjuvant-antigen conjugates) that have a polysaccharide backbone capable of binding to the cell surface of Antigen Presenting Cells (APCs), to which is covalently attached (a) one or more molecules having a stable carbonyl group (i.e. an aldehyde and ketone group that is capable of reacting with amino groups to form an imine or Schiff base), and (b) one or more polypeptides or peptides that are capable of eliciting an immunogenic response when covalently attached to polysaccharide backbone. Also disclosed are methods for making the conjugates and methods of using the conjugates to enhance the potentiation of an immune response in a mammal. Also disclosed is a method of vaccination, and pharmaceutical and veterinary compositions comprising one or more of the polysaccharide adjuvant-antigen conjugates of the present invention.

## Claims

### Claim 1

A polysaccharide adjuvant-protein antigen conjugate, wherein said conjugate is represented by the formula:
(A—L′)y—P—(L—I)x
or pharmaceutically acceptable salts thereof, where
P is a polysaccharide which will bind to the cell surface of an Antigen Presenting Cell;
each L′ is the same or different and is a covalent bond, or a bifunctional linking molecule;
A is a protein or peptide antigen which will elicit an immunogenic response when covalently attached to a polysaccharide backbone, and wherein when y is greater than 1, each A can be the same or different protein or peptide;
each L is the same or different and is a covalent bond, or a bifunctional linking molecule; and
I is an compound having a stable carbonyl group capable of reacting with an amino group on an Antigen Presenting Cell to form an imine or Schiff's base, said compound having (a) a ketone or aldehyde functionality; and (b) a second functional group which reacts with a complementary functional group present on said polysaccharide or said bifunctional linking molecule, if present, and wherein when x is greater than 1, each I can be the same or different;
x is from 1 to 100 units per 100 glycoside residues; and
y is from 1 to about 20 units per 100 glycoside residues.

### Claim 2 (depends on 1)

The conjugate of claim 1, wherein said imine-forming compound is selected from the group consisting of aromatic aldehydes, aromatic ketones, cycloalkyl aldehydes, cycloalkyl ketones, cycloalkenyl aldehydes, cycloalkenyl ketones, heterocyclic aldehydes, heterocyclic ketones, heteroaromatic ketones, heteroaromatic aldehydes, alkyl aldehydes, alkyl ketones, alkenyl aldehydes, alkenyl ketones, and mixtures thereof.

### Claim 3 (depends on 1)

The conjugate of claim 1, wherein said imine-forming compound is bound to said polysaccharide via a direct covalent bond.

### Claim 4 (depends on 1)

The conjugate of claim 1, wherein said imine-forming compound is bound to said polysaccharide via a bifunctional linker molecule.

### Claim 5 (depends on 1)

The conjugate of claim 1, wherein said imine-forming compound is selected from the group consisting of mono- and di-substituted C6-10 arylaldehydes, C6-10 aryl(C1-4)alkylaldehydes, hydroxy-substituted C1-4alkyl(C6-10)aryl ketones, hydroxy-substituted C6-10 aryl ketones, and mixtures thereof.

### Claim 6 (depends on 5)

The conjugate of claim 5, wherein said imine-forming compound is one of phenyl or naphthyl substituted by a formyl or formyl(C1-4)alkyl substituent, and optionally including one or two additional substituents independently selected from the group consisting of halo, hydroxy, C1-4 alkyl, C1-4 alkoxy, trifluoromethyl, and benzyloxy.

### Claim 7 (depends on 6)

The conjugate of claim 6, wherein said imine-forming compound is one of benzaldehyde and naphthaldehyde, substituted by one or two of hydroxy and halo.

### Claim 8 (depends on 7)

The conjugate of claim 7, wherein said imine-forming compound is one of 2,3-dihydroxybenzaldehyde, 2,4-dihydroxybenzaldehyde, 2,5-dihydroxybenzaldehyde, 3,4-dihydroxybenzaldehyde, 5-chloro-2-hydroxybenzaldehyde, vanillin, ethyl vanillin, naringenin, 3-hydroxybenzaldehyde, 4-hydroxybenzaldehyde, 4-hydroxyphenylacetaldehyde, 2-hydroxyacetophenone, 3-hydroxyacetophenone, 4-hydroxyacetophenone, 6-hydroxy-1,2-naphthoquinone, 4,6-dioxoheptanoic acid or 8,10-dioxoundecanoic acid.

### Claim 9 (depends on 1)

The conjugate of claim 1, wherein said imine-forming compound is selected from the group consisting of C4-10 alkylaldehydes, C4-10 alkenylaldehydes, C4-10 alkylketones, C4-10 alkenylketones, and mixtures thereof, all of which are substituted by one or two of amino, hydroxy or a combination thereof.

### Claim 10 (depends on 1)

The conjugate of claim 1, wherein said imine-forming compound is selected from the group consisting of heteroaryl aldehydes and heteroaryl ketones.

### Claim 11 (depends on 10)

The conjugate of claim 10, wherein said imine-forming compound is one of thiophene, furan, benzothiophene, benzofuran, pyridine, quinoline, pyridazine, pyrimidine, pyrazole, imidazole, 1,2,3-triazole, 1,2,4-triazole, isoxazole, or oxazole, each having a keto, formyl or formyl(C1-4)alkyl substituent, and optionally including an additional halo or hydroxy substituent.

### Claim 12 (depends on 11)

The conjugate of claim 11, wherein said imine-forming compound is one of pyridoxal, 2-thiophenecarboxaldehyde, and 3-thiophenecarboxaldehyde.

### Claim 13 (depends on 1)

The conjugate of claim 1, wherein said polysaccharide comprises a minimum of seven glycosyl residues.

### Claim 14 (depends on 13)

The conjugate of claim 13, wherein said polysaccharide is selected from the group consisting of β-glucans; mannans; pectic polysaccharides; chitin; deacetylated chitin; glycol chitin; carboxymethyl chitin; hydrolyzed chitin; murein; bacterial fructans; xanthans; bacterial heteropolysaccharides; fungal pullulan; and esters, sulfonates, sulfates, phosphates, ethers, and cross-linked derivatives thereof.

### Claim 15 (depends on 14)

The conjugate of claim 14, wherein said polysaccharide is selected from the group consisting of β-glucans; mannans; pectic polysaccharides; and 2-acetamido glucan polysaccharides.

### Claim 16 (depends on 15)

The conjugate of claim 15 wherein said polysaccharide is a β-glucan having a backbone chain of (1→3)-linked β-D-glucopyranosyl units and which has β-D-glucopyranosyl units attached by (1→6) linkages, and a molecular weight of between about 5,000 to about 500,000, and wherein said β-glucan is optionally modified by the addition of one or more anionic, cationic or non-ionic groups.

### Claim 17 (depends on 15)

The conjugate of claim 15, wherein said polysaccharide is a β-mannan comprising (1→4) polymannose having a terminus reducing mannosyl residue, or the acetylation product thereof.

### Claim 18 (depends on 15)

The conjugate of claim 15, wherein said polysaccharide is a pectic polysaccharide selected from the group consisting of homogalacturonans, rhamnogalacturonans, arabinans, galactans, and arabinogalactans.

### Claim 19 (depends on 1)

The conjugate of claim 1, wherein said protein antigen is derived from a virus, bacteria, protozoan, fungi, or mixtures thereof.

### Claim 20 (depends on 19)

The conjugate of claim 19, wherein said protein antigen is derived from a virus, or bacteria that cause influenza, feline leukemia, feline immunodeficiency, HIV-1, HIV-2, rabies, measles, hepatitis B, hoof and mouth disease, anthrax, diphtheria, Lyme disease, tuberculosis, or mixtures thereof.

### Claim 21 (depends on 1)

The conjugate of claim 1, wherein said protein antigen is bound to said polysaccharide via a direct covalent bond.

### Claim 22 (depends on 1)

The conjugate of claim 1, wherein said protein antigen is bound to said polysaccharide via a residue of a bifunctional linker molecule.

### Claim 23 (depends on 1)

The conjugate of claim 1, wherein said protein antigen is selected from the group consisting of carcinoembryonic antigen, prostate-specific membrane antigen, prostate specific antigen, protein MZ2-E, polymorphic epithelial mucin, folate-binding-protein LK26, an epitope of carcinoembryonic antigen, an epitope of prostate-specific membrane antigen, an epitope of prostate specific antigen, an epitope of protein MZ2-E, an epitope of polymorphic epithelial mucin, an epitope of folate-binding-protein LK26 and mixtures thereof.

### Claim 24 (depends on 1)

The conjugate of claim 1, wherein said bifunctional linking molecule is a residue of a compound selected from the group consisting of succinimidyl 4-(p-maleimidophenyl)butyrate, 4-(4-N-maleimidophenyl)butyric acid hydrazide hydrochloride, and maleimidobenzoyl-N-hydroxysuccinimide ester.

### Claim 25 (depends on 1)

The conjugate of claim 1, wherein said bifunctional linking molecule is a residue of a compound selected from the group consisting of:
H2N—(CH2)r—NH2, where r is from 2 to 12;
HO—(CH2)r—NH2, where r is from 2 to 12;
HS—(CH2)r—NH2, where r is from 2 to 12;
amino acids that are optionally carboxy-protected; and
H—(O—CH2—CH2)n—OH, where n is 1-4.

### Claim 26 (depends on 1)

The conjugate of claim 1, wherein said bifunctional linking molecule is a residue of a compound selected from the group consisting of ethylenediamine, 1,4-butanediamine, spermidine, 2,4-diaminobutyric acid, lysine, β-alanine, γ-aminobutyric acid, dialanine, trialanine, 3,3′-diaminodipropylamine, diaminopropionic acid, N-(2-aminoethyl)-1,3-propanediamine, and 2-(4-aminophenyl)ethylamine.

### Claim 27 (depends on 1)

The conjugate of claim 1, wherein said bifunctional linking molecule is selected from the group consisting of:
—NH—(CH2)r—NH—, where r is from 2-5,
—O—(CH2)r—NH—, where r is from 2-5,
—NH—CH2—C(O)—,
—O—CH2—CH2—O—CH2—CH2—O—,
—NH—NH—C(O)—CH2—,
—NH—C(CH3)2—C(O)—,
—S—(CH2)r—C(O)—, where r is from 1-5,
—S—(CH2)r—NH—, where r is from 2-5,
—S—(CH2)r—O—, where r is from 1-5,
—S—(CH2)—CH(NH2)—C(O)—,
—S—(CH2)—CH(COOH)—NH—,
—O—CH2—CH(OH)—CH2—S—CH(CO2H)—NH—,
—O—CH2—CH(OH)—CH2—S—CH(NH2)—C(O)—,
—O—CH2—CH(OH)—CH2—S—CH2—CH2—NH—,
—S—CH2—C(O)—NH—CH2—CH2—NH—, and
—NH—O—C(O)—CH2—CH2—O—P(O2H)—.

### Claim 28 (depends on 1)

A pharmaceutical composition, comprising:
(a) a conjugate of claim 1, and
(b) a pharmaceutically acceptable carrier or diluent.

### Claim 29 (depends on 28)

A method of enhancing an immune response in an animal, comprising administering, to an animal in need of such enhancement, a composition of claim 28 in an amount effective to enhance the immune response of said animal.

### Claim 30

A polysaccharide adjuvant-antigen conjugate, comprising:
(i) a polysaccharide having a minimum of seven glycosyl residues, said polysaccharide selected from the group consisting of a β-glucan; a mannan; a pectic polysaccharide; chitin; deacetylated chitin; glycol chitin; carboxymethyl chitin; hydrolyzed chitin; murein; a bacterial fructan; a xanthan; a bacterial heteropolysaccharide; fungal pullulan; and esters, sulfonates, sulfates, phosphates, ethers, and cross-linked derivatives thereof;
(ii) one or more molecules selected from the group consisting of aromatic aldehydes, aromatic ketones, cycloalkyl aldehydes, cycloalkyl ketones, cycloalkenyl aldehydes, cycloalkenyl ketones, heterocyclic aldehydes, heterocyclic ketones, heteroaromatic ketones, heteroaromatic aldehydes, alkyl aldehydes, alkyl ketones, alkenyl aldehydes, alkenyl ketones, and mixtures thereof;
(iii) a protein antigen capable of eliciting an immune response when covalently attached to said polysaccharide;
wherein one or more molecules (ii) are attached to the polysaccharide (i) through a direct covalent bond or covalently via a bifunctional linker in a manner that keeps the carbonyl group of molecule (ii) intact; and
wherein said protein antigen (iii) are attached to the polysaccharide (i) through a direct covalent bond or covalently via a bifunctional linker.

### Claim 31 (depends on 30)

The polysaccharide adjuvant-antigen conjugate of claim 30, wherein said protein antigen is selected from the group consisting of carcinoembryonic antigen, prostate-specific membrane antigen, prostate specific antigen, protein MZ2-E, polymorphic epithelial mucin, folate-binding-protein LK26, an epitope of carcinoembryonic antigen, an epitope of prostate-specific membrane antigen, an epitope of prostate specific antigen, an epitope of protein MZ2-E, an epitope of polymorphic epithelial mucin, an epitope of folate-binding-protein LK26 and mixtures thereof.