# US 4,639,512 A · Conjugates of haptenes and muramyl-peptides, endowed with immunogenic activity and compositions containing them

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- Number: US 4,639,512 A
- Document ID: US4639512A
- Status: Granted
- Kind: A
- Application number: 06/474,361
- Filed: 1983-03-11
- Granted: 1987-01-27
- Earliest claimed: 1982-03-15
- Assignees: Agence Nationale de Valorisation de la Recherche
- Inventors: Francoise Audibert; Claude Carelli; Louis Chedid; Pierre LeFrancier; Michel Level; Jean Choay
- Web page: https://patentgrep.com/patent/US4639512A

## CPC

- A61K 39/385: Haptens or antigens, bound to carriers
- 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 2039/6062: {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 930/13: Luteinizing hormone-releasing hormone; related peptides
- Y10S 930/15: Oxytocin or vasopressin; related peptides
- Y10S 930/16: Somatostatin; related peptides
- 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 an immunogenic principle containing a specific antigen determinant of a predetermined native antigen. This immunogenic principle is constituted by a conjugate between a hapten bearing said antigenic site and a muramyl-peptide, essentially in the absence of supporting macromolecules, this conjugate having an immunogenic effect not only against itself, but also with respect to the native antigen carrying said antigenic site.

## Claims

### Claim 1

An immunogenic and apyrogenic conjugate consisting essentially of a hapten covalently linked to a muramyl peptide, which conjugate comprises a molecular weight which does not exceed 5000,
wherein the hapten is selected from the group consisting of peptides, glycopeptides, oligosaccharides, lipopeptides, glycolipids, lipids and steroids which are incapable of inducing an immunological response in vivo except when bound to an immunological carrier;
and wherein the muramyl peptide part conjugated to the hapten is derived from the muramyl peptide which has the general formula (I): ##STR8## in which the following groups, when not covalently linked to the hapten, have the following meaning:
R.sub.5 is a free carboxyl group or amido group, or a carboxyl group esterified by an aliphatic alcohol possessing from 1 to 10 carbon atoms, or a carboxyl
group substituted by an amino acid possessing itself a free carboxyl group or an amidated group or a carboxyl group esterified by an aliphatic alcohol possessing from 1 to 10 carbon atoms,
R is a hydrogen or a methyl group,
R.sub.2 is an acetamido or a glycoyl-amido group,
R.sub.1 is either --NH.sub.2, or OH, or a radical comprising at the most 10 carbon atoms, corresponding to an alkyl, aryl, arylalkyl or alkylaryl group, optionally bearing hydroxyl-, amido, carboxamido-, sulfonamido-, ether, thioether, ester- or thioester substituents
R.sub.4 is a hydroxyl or an oxyacyl group comprising from 1 to 4 carbon atoms,
X is an aminoacyl residue of the group comprising: alanyl, arginyl, asparagyl, aspartyl, cysteinyl, glutaminyl, glutamyl, glycyl, histidyl, hydroxy-prolyl isoleucyl, leucyl, lysyl, methionyl, phenylalanyl, prolyl, seryl, threonyl, tryptophanyl, tyrosyl, valyl and aminobutyryl,
Z is a group substituting the position at C-6 of muramic acid, and which has the following general formula (II):
--R.sub.6 --(R.sub.7).sub.x
in which x=0 or 1 and in which,
if x=0,
R.sub.6 is hydroxyl or amino and,
if x=1,
R.sub.6 is hydroxyl group or amino, carboxamido, sulfonamide ether or thioether, ester or thioester,
R.sub.7 then being a chain of --CH.sub.2, --CH.dbd.CH--, --S--, --CO-- or NH groups, --CH.sub.2 --; --CH.dbd.CH--, or NH-groups, said groups being optionally replaced or substituted by aryl groups or by carboxlyic, amino, sulfhydryl or hydroxyl groups, or by alkyl groups including up to 5 carbon atoms or by alkylaryl groups, arylalkyl including in all up to 8 carbon atoms or by heterocyclic groups including up to six carbon atoms and one or two oxygen, nitrogen or sulfur atoms, said alkylaryl or arylalkyl groups optionally containing carbonyl groups or being optionally substituted by carboxyl, amino, sulfhydryl or hydroxyl groups, said heterocyclic groups being themselves optionally substituted by alkyl groups which can contain up to 5 carbon atoms, carboxyl, amino, sulfhydryl or hydroxyl, the above said R.sub.7 group having none the less a length not exceeding that of an n-decyl hydrocarbon chain,
Y is a free or amidated carboxyl group, or an ester group comprising up to 10 carbon atoms, or substituted by an amino acid or is a group of formula (III):
--R.sub.8 --(R.sub.9).sub.x (III)
in which x equals 0 or 1 and
in which R.sub.8 is an amide or ester linkage when x is equal to zero, or when x is different from zero, R.sub.8 is a carboxamido-, ester- or thioester-group, R.sub.9 then having the same meaning as R.sub.7 ;
and which conjugate when administered in vivo elicits only antibodies or a cell mediated immunological response involving the hapten.

### Claim 2 (depends on 1)

The conjugate of claim 1, wherein the hapten is a peptide or a glycopeptide.

### Claim 3 (depends on 1)

The conjugate of claim 1, wherein the hapten comprises a hormone or part thereof.

### Claim 4 (depends on 1)

The conjugate of claim 1 which has a molecular weight less than 3,000.

### Claim 5 (depends on 4)

The conjugate of claim 4 which has a molecular weight less than 2,000.

### Claim 6 (depends on 1)

The conjugate of claim 1, wherein the hapten is a peptide of less than 50 aminoacyl residues.

### Claim 7 (depends on 6)

The conjugate of claim 6, wherein the hapten is a peptide of less than 30 aminoacyl residues.

### Claim 8 (depends on 7)

The conjugate of claim 7, wherein the hapten is a peptide of less than 20 aminoacyl residues.

### Claim 9 (depends on 1)

The conjugate of claim 1, wherein the muramyl peptide, when not conjugated with the hapten, is pyrogenic.

### Claim 10 (depends on 1)

The conjugate of claim 1, wherein the hapten comprises an antigenic site which has the structure of a portion of a natural antigen.

### Claim 11 (depends on 1)

The conjugate of claim 1, wherein the antigenic site is a peptide which possess a sequence present in a native peptide antigen.

### Claim 12 (depends on 1)

The conjugate of claim 1, wherein the hapten comprises at least one mono- or polysaccharide.

### Claim 13 (depends on 12)

The conjugate of claim 12, wherein the polysaccharide is a hexose or a pentose.

### Claim 14 (depends on 1, 11, 12)

The conjugate of claim 1, 11 or 12, wherein the hapten is synthetic.

### Claim 15 (depends on 1)

The conjugate of claim 1, wherein X is a glycyl, seryl, valyl, leucyl, aminobutyryl residue, or alanyl.

### Claim 16 (depends on 1)

The conjugate of claim 1, wherein, x being equal to zero, Z is a hydroxyl or amino function.

### Claim 17 (depends on 1)

The conjugate of claim 1, wherein:
x=1,
R.sub.6 is a carboxamide linkage or sulphonamide either or thioether, ester or thioester, and
R.sub.7 represents one of the groups such as those indicated below
(CH.sub.2).sub.n --COOH, particularly with n=1, 2, 3 or 4
(CH.sub.2).sub.n --NH.sub.2, particularly with n=1, 2, 3 or 4 ##STR9## particularly with n=1, 2, 3 or 4 (CH.sub.2).sub.n --C.sub.6 H.sub.5 --NH.sub.2, particularly with n=0 or 1
(CH.sub.2).sub.n --SH, particularly n=2.

### Claim 18 (depends on 1)

The conjugate of claim 1, wherein the R.sub.5 group is a carboxyl function esterified by an alcohol comprising from 1 to 5 carbon atoms and Y is a --CONH.sub.2 group.

### Claim 19 (depends on 1)

The conjugate of claim 1, wherein the group R of the muramyl-peptide group is a methyl group.

### Claim 20 (depends on 1)

The conjugate of claim 1, wherein the group Z of the muramyl-peptide portion is a succinyl group.

### Claim 21 (depends on 1)

The conjugate of claim 1, wherein R.sub.1, R.sub.4 and R.sub.6 are hydroxyls and R.sub.2 is acetamido, R is a methyl group, X is an alanyl group, R.sub.5 is a carboxamide group or an ester group including from 1 to 5 carbon atoms, and Y is a carboxamide function.

### Claim 22 (depends on 1)

The conjugate of claim 1, whrein the muramylpeptide portion is selected from one of the following muramylpeptides:
N-acetyl-muramyl-L-alanyl-D-isoglutamine,
a-n.butyl-ester of N-acetyl-muramyl-L-alanyl-D-glutamine,
6-O-succinylated derivatives of the two foregoing muramylpeptides,
a mono-a- or y-ester or a diester of N-acetyl-muramyl-L-alanyl-D-glutamic acid, wherein ester groups contain from 1 to 5 carbon atoms.

### Claim 23 (depends on 1)

The conjugate of claim 1, wherein Y includes a lysyl group.

### Claim 24 (depends on 15)

The conjugate of claim 15 wherein the muramylpeptide portion is N-acetyl-muramyl-L-alanyl-D-isoglutaminyl-L-lysine.

### Claim 25

The conjugate which consists of (LH--RH)--N--acetyl-muramyl-L-alanyl-D-isoglutamine-L-lysine.