# US 4,446,128 A · Antigen derivatives and processes for their preparation

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- Number: US 4,446,128 A
- Document ID: US4446128A
- Status: Granted
- Kind: A
- Application number: 06/379,404
- Filed: 1982-05-18
- Granted: 1984-05-01
- Earliest claimed: 1978-02-24
- Assignees: Ciba-Geigy Corporation
- Inventors: Gerhard Baschang; Felix M. Dietrich; Roland Gisler; Albert Hartmann; Jaroslav Stanek; Lajos Tarcsay
- Web page: https://patentgrep.com/patent/US4446128A

## CPC

- C07H 9/04: Cyclic acetals
- A61K 39/00: Medicinal preparations containing antigens or antibodies (materials for immunoassay G01N33/53)
- A61K 39/385: Haptens or antigens, bound to carriers
- C07H 23/00: Compounds containing boron, silicon or a metal, e.g. chelates or vitamin B12 (esters with inorganic acids C07H11/00)
- C07K 9/005: {containing within the molecule the substructure [IMAGE cpc-sch-C07K-1009.gif] with m, n > 0 and m+n > 0, A, B, D, E being heteroatoms; X being a bond or a chain, e.g. muramylpeptides}
- A61K 2039/55583: {Polysaccharides}
- A61K 2039/6062: {Muramyl peptides}
- 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
- Y10S 530/806: Antigenic peptides or proteins
- Y10S 530/807: Hapten conjugated with peptide or protein
- 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)
- 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)
- 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)
- Y02A: TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
- Y10S: TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS

## Abstract

The invention relates to novel antigen derivatives comprising an antigen and at least one muramyl-peptide covalently bonded thereto, if appropriate via a spacer, to pharmaceutical preparations which contain such compounds and to their use as a vaccine. The novel antigen derivatives produce a pronounced increase in the immuno-response to the antigen, and in particular also a cell-medicated immunity under clinically acceptable conditions of administration.

## Claims

### Claim 1

A novel antigen derivative, which comprises an antigen radical and one or more muramylpeptide radicals covalently bound thereto, directly or via a spacer, the antigen and muramylpeptide of those radicals each containing at least one functional group characterized in that upon reaction between antigen and muramylpeptide, antigen and spacer or muramylpeptide and spacer one of the below-mentioned covalent linking-structures is formed between the reactants, said muramylpeptide has the formula ##STR10## in which X is a carbonyl, carbonyloxy or sulfonyl group, R.sub.1, R.sub.4 and R.sub.6 independently of one another represent hydrogen, alkyl having from 1 to 18 carbon atoms, benzyl which is unsubstituted or substituted in the aromatic nucleus by lower alkyl, hydroxyl, lower alkoxy, lower alkylenedioxy, mercapto, lower alkylmercapto, trifluoromethyl and/or halogen, or represent alkanoyl having from 2 to 18 carbon atoms, benzoyl or naphthoyl which are unsubstituted or substituted by halogen, lower alkyl, lower alkoxy, trifluoromethyl, hydroxyl or lower alkanoyloxy, or represent an acyl radical of a lower alkanesulfonic acid
or of phenylsulfonic acid, which is unsubstituted or substituted by lower alkyl or halogen, or represent carbamoyl, lower alkyl-carbamoyl or phenyl-carbamoyl, R.sub.2 represents alkyl having 1 to 18 carbon atoms which is unsubstituted or substituted by hydroxy, lower alkoxy, lower alkanoyloxy, halogen, mercapto, lower alkylmercapto, lower alkanoylmercapto, carboxy, lower alkoxycarbonyl and/or carbamoyl, or represents phenyl which is unsubstituted or substituted by lower alkyl, hydroxy, lower alkoxy, lower alkanoyl, lower alkylenedioxy, halogen and/or trifluoromethyl, R.sub.3, R.sub.7, R.sub.9 and R.sub.13 are independently of one another hydrogen or lower alkyl, R.sub.8 is hydrogen, lower alkyl which is unsubstituted or substituted by hydroxy, lower alkoxy, lower alkanoyloxy, mercapto, lower alkylmercapto, lower alkanoylmercapto, amino, lower alkanoylamino, carbamoylamino or cycloalkyl having 5 or 6 carbon atoms, or represents cycloalkyl-lower alkyl, the cycloalkyl radical of which contains 5 or 6 carbon atoms, phenyl or phenyl-lower alkyl, in which the phenyl radicals are unsubstituted or substituted as defined above, or heterocyclic radicals are 5- or 6-membered, contain one or two nitrogen atoms in the ring and may or may not contain a fused phenyl radical, R.sub.7 and R.sub.8 together can also represent alkylene having 3 or 4 carbon atoms, R.sub.10, R.sub.11 and R.sub.12 independently of one another represent carboxyl, lower alkoxycarbonyl or carbamoyl which is unsubstituted or mono- or disubstituted at the nitrogen atom by unsubstituted or carboxy- or carbamoyl-substituted lower alkyl or by phenyl, benzyl, tetramethylene or pentamethylene and R.sub.11 can also be hydrogen, with the proviso that the muramylpeptide and the antigen are bonded together via the covalent linking structures carbonyloxy ##STR11## carbonylimino ##STR12## wherein R is hydrogen or lower alkyl), carbonylthio ##STR13## thiocarbonyloxy ##STR14## and/or thiocarbonylimino ##STR15## wherein R' is hydrogen or lower alkyl) directly or via a spacer, and with the further proviso that the antigen-muramylpeptide derivative may or may not be fixed to a high molecular weight carrier and that the antigen is not a constituent of a vaccine against tumour cells.

### Claim 2 (depends on 1)

A novel antigen derivative according to claim 1, wherein the antigen is the constituent of malaria merozoites, type-specific meningococcal polysaccharides A, B or C, M-proteins of streptococci, influenza virus haemagglutinins, partial sequences of human choriongonadotropin, grass pollen extracts, anti-specific T-cell lymphoblasts and their receptors for antigens, or antigen-specific immunoglobulins.

### Claim 3 (depends on 1)

An antigen derivative according to claim 1 wherein R.sub.13 represents hydrogen.

### Claim 4 (depends on 1)

An antigen derivative according to claim 1, or 3, wherein the antigen is covalently bound to the carrier via the covalent linking structures carbonyloxy ##STR16## carbonylimino ##STR17## wherein R is hydrogen or lower alkyl) or carbonylthio ##STR18## directly or via a spacer, and the muramylpeptide is covalently bound to the antigen and may additionally also be bound to the carrier via the covalent linking structures mentioned in this claim, directly or via a spacer.

### Claim 5 (depends on 1)

An antigen derivative according to claim 1 wherein a spacer is the radical or a .alpha.,.omega.-diamino-lower alkane bound via the nitrogen atoms, lower alkanedicarbonyl or .alpha.-, .beta.- or .gamma.-aminoalkanecarbonyl.

### Claim 6 (depends on 1)

An antigen derivative according to claim 1 whrein the antigen and the muramylpeptide are bound directly together.

### Claim 7 (depends on 6)

An antigen derivative according to claim 6 wherein the muramylpeptide is bound via the substituent R.sub.12 of formula II.

### Claim 8 (depends on 1, 5, 7)

An antigen derivative according to claim 1, 5 or 7, wherein the antigen is a substance including ingredients of vaccines which brings about the specific immunisation of a living organism against infectious pathogens or undesired reactions, which are allergic sensitisation or rejection of transplanted foreign tissue.

### Claim 9 (depends on 1)

An antigen derivative according to claim 1 wherein the antigen is the effective constituent, which represents or comprises the antigenic determinant, of vaccines against parasites, bacteria, viruses or physiological body-innate constituents, their modified forms or sub-units thereof.

### Claim 10 (depends on 7)

An antigen derivative according to claim 7, wherein the antigen represents group C polysaccharide of Neisseria Meningitidis, merozoites of the malaria pathogen Plasmodium knowlesi, T-lymphoblasts, erythrocyte membranes, the active ingredient of food and mouth disease culture vaccine or of rabies vaccine, influenza virus antigen, tetanus toxoid, cholera toxoid, eicosapeptid which is identical with the C-terminal sequence of human choriongonadotropin, or Trypanosoma cruzi parasites, or components or parts of these antigens carrying the desired antigenic determinant.

### Claim 11 (depends on 10)

An antigen derivative according to claim 10, which contains pro 1 mg or pro 10.sup.7 cells respectively of the antigen from 0.01 to 0.5 mg muramylpeptide, these values are derived from the results of the analytical determination of the product by means of the Morgan-Elson-reaction.

### Claim 12 (depends on 1)

An antigen derivative according to claim 1 wherein the antigen and the muramylpeptide are bound together via the covalent linking structures carbonyloxy ##STR19## carbonylimino ##STR20## wherein R is hydrogen or lower alkyl) or carbonylthio ##STR21## directly or via a spacer.

### Claim 13 (depends on 1, 4, 7, 9, 10)

An antigen derivative according to claim 1, 7, 9, 10, or 5 wherein the muramylpeptide is a compound of the formula II wherein R.sub.1, R.sub.3, R.sub.4, R.sub.6, R.sub.7 and R.sub.13 are hydrogen, X is carbonyl and R.sub.2 is lower alkyl which is unsubstituted or substituted by hydroxyl or lower alkoxy, or is phenyl which is unsubstituted or substituted by hydroxyl, lower alkoxy, lower alkyl or halogen, and R.sub.8, R.sub.9, R.sub.10, R.sub.11 and R.sub.12 are as defined in claim 4.

### Claim 14 (depends on 1, 7, 9, 10)

An antigen derivative according to claim 1, 7, 9, 10, or 5 wherein the muramylpeptide is a compound of the formula II wherein R.sub.1, R.sub.4, R.sub.6, R.sub.7 and R.sub.13 are hydrogen, X is carbonyl, R.sub.2 lower alkyl which is unsubstituted or substituted by hydroxyl or lower alkoxy or is phenyl which is unsubstituted or substituted by hydroxyl, lower alkoxy, lower alkyl or halogen, R.sub.3 is methyl and R.sub.8, R.sub.9, R.sub.10, R.sub.11 and R.sub.12 are as defined in claim 1.

### Claim 15 (depends on 1)

An antigen derivative according to claim 1 wherein the muramylpeptide is a compound of the formula II wherein X is carbonyl, R.sub.1, R.sub.4, R.sub.6, R.sub.7, R.sub.9 and R.sub.13 are hydrogen, R.sub.2 is lower alkyl which is unsubstituted or substituted by hydroxyl or methoxy or is phenyl which is unsubstituted or substituted by hydroxyl, methoxy, methyl, ethyl or halogen, R.sub.3 is hydrogen or methyl, R.sub.8 is benzyl, p-hydroxybenzyl, phenyl or lower alkyl which is unsubstituted or substituted by lower alkylmercapto or hydroxy, and R.sub.10, R.sub.11 and R.sub.12 are carboxyl, lower alkoxycarbonyl or carbamoyl and R.sub.11 can also be hydrogen.

### Claim 16 (depends on 1, 7, 9, 10)

An antigen derivative according to claim 1, 7, 9, 10, or 2 wherein the muramylpeptide is a compound of the formula II wherein X is carbonyl, R.sub.1, R.sub.4, R.sub.6 and R.sub.13 are hydrogen, R.sub.2 is lower alkyl which is unsubstituted or substituted by hydroxyl or methoxy or is phenyl which is unsubstituted or substituted by hydroxyl, methoxy, methyl, ethyl or halogen, R.sub.3, R.sub.7 and R.sub.9 are hydrogen or methyl, R.sub.8 is methyl, ethyl, n-propyl, i-propyl, 2-methyl-propyl, methylmercaptomethyl, hydroxymethyl, hydroxyethyl, phenyl, benzyl, or p-hydroxybenzyl and R.sub.10, R.sub.11 and R.sub.12 are carboxyl, lower alkoxycarbonyl or carbamoyl and R.sub.11 can also be hydrogen.

### Claim 17 (depends on 1)

An antigen derivative according to claim 1 wherein the muramylpeptide is a compound of the formula II wherein X is carbonyl, R.sub.1, R.sub.4 R.sub.6 and R.sub.13 are hydrogen, and R.sub.7 and R.sub.8 together are propylene or butylene.

### Claim 18 (depends on 1)

An antigen derivative according to claim 1 containing a muramylpeptide of the formula II wherein R.sub.11 and R.sub.13 is hydrogen.

### Claim 19 (depends on 1)

An antigen derivative according to claim 1 wherein wherein the muramylpeptide is a compound of the formula II wherein X is carbonyl, R.sub.1, R.sub.4, R.sub.6, R.sub.11 and R.sub.13 hydrogen, R.sub.2 is methyl, ethyl or phenyl, R.sub.3 is hydrogen or methyl, R.sub.7 is hydrogen, methyl, ethyl or n-propyl, R.sub.8 is methyl, ethyl or isopropyl, R.sub.9 is hydrogen or methyl, R.sub.10 is carboxyl, carbamoyl, N-(carbamoylmethyl)-carbamoyl or N-(1-carboxy-ethyl)-carbamoyl and R.sub.12 is carboxy or N-(1-carboxy-ethyl)-carbamoyl.

### Claim 20 (depends on 1)

An antigen derivative according to claim 1 containing a muramylpeptide of the formula II wherein R.sub.7 is methyl, ethyl or n-propyl.

### Claim 21 (depends on 1, 7, 9, 10)

An antigen derivative according to claim 1, 7, 9, 10, or 2 wherein the muramylpeptide is a compound of the formula II wherein the muramylpeptide is 2-acetamido-3-O-{[L-1-(D-1-carbamoyl-3-carboxy-propyl)-carbamoyl-ethyl]-ca rbamoyl-methyl}-2-deoxy-D-glucose or 2-acetamido-3-O-{D-1-[L-1-(D-1-carbamoyl-3-carboxy-propyl)-carbamoyl-ethyl ]-carbamoyl-ethyl}-2-deoxy-D-glucose.

### Claim 22 (depends on 1)

An antigen derivative according to claim 1 wherein the muramylpeptide is a compound of the formula II wherein the muramylpeptide is 2-acetamido-3-O-{D-1-[L-1-(D-1-carbamoyl-3-carboxy-propyl)-carbamoyl-propy l]-N-methyl-carbamoyl-ethyl}-2-deoxy-D-glucose.

### Claim 23 (depends on 1)

A pharmaceutical preparation for enteral or parenteral administration for modulation of the immune response to warm-blooded animals including man containing an effective dose of an antigen derivative according to claim 1 together with a significant amount of a pharmaceutically acceptable carrier.

### Claim 24 (depends on 1)

A method of using an antigen derivative according to claim 1 for modulating the immune response of warm-blooded animals including man by enterally or parenterally administrating an effective dose thereof.

### Claim 25 (depends on 1)

An antigen derivative according to claim 1, wherein the antigen is the constituent of a vaccine against parasites, bacteria, viruses, or attenuated or killed forms or part-components thereof, constituent of a vaccine against structures innate in the body or immunological response structures, or the constituent of a vaccine suitable for specific desensitisation in the case of allergies.

### Claim 26 (depends on 1)

An antigen derivative according to claim 1, wherein the antigen is the constituent of a vaccine against malaria, cholera, typhus, meningitis, rheumatic fever as a consequence of streptococcal infections, influenza, rabies or foot and mouth disease, for the induction of immunity against components of the reproductive system, for desensitisation or induction of specific immuno-tolerances to allergies or for the reinstatement of tolerance towards tissue constituents, and circulating molecules, innate in the body.

### Claim 27 (depends on 1)

A method of immunizing warm-blooded animals comprising administering to said animals an effective amount of an antigen derivative according to claim 1.

### Claim 28 (depends on 3)

A novel antigen derivative according to claim 3, wherein the antigen is fixed to a high molecular weight carrier.

### Claim 29 (depends on 28)

A novel antigen derivative according to claim 28, wherein the carrier is a polymer of lactic acid and its derivatives, which carry a free carboxyl group at the chain end, alginic acid, polygalacturonic acid, pectic acid, carboxymethylcellulose, agarose or a basic, neutral or acidic polyaminoacid.

### Claim 30 (depends on 3)

A novel antigen derivative according to claim 3, wherein the antigen is separated from the muramyl-peptide by a bridge member (spacer).

### Claim 31 (depends on 3)

A novel antigen derivative according to claim 3, of the formula ##STR22## in which A is the radical of the antigen, T is the radical of the carrier, MP is the radical of the muramyl-peptide, Z' and Z"' are bivalent spacers and X', X", X"' and X"" are covalent linking structures, and m and n are integers greater than 0.

### Claim 32 (depends on 23)

A pharmaceutical preparation according to claim 23 containing a solution or suspension of the antigen derivative together with carboxymethylcellulose.