Method of coupling polysaccharides to proteins

Details Method of coupling polysaccharides to proteins Method of coupling polysaccharides to proteins

1999-08-16

2002-03-26

Peselev, Elli (Department: 1623)

Drug, bio-affecting and body treating compositions

Antigen, epitope, or other immunospecific immunoeffector

Conjugate or complex

C424S184100, C514S025000, C530S300000, C530S322000, C530S350000, C530S395000, C536S123100, C536S124000

Reexamination Certificate

active

06361777

ABSTRACT:

The present invention relates to the use of aminothiol compounds as linkers in preparing conjugate vaccines.
Covalent binding of a polysaccharide or other hapten to an immunogenic protein or peptide or other bio-organic molecule has proven to be a suitable method of preparing effective vaccines, for example against pathogenic organisms such as
Haemophilus influenzae
type b (meningitis, otitis media),
Bordetella pertussis
(whooping cough),
Clostridium tetani
(tetanus), meningococci (
Neisseria meningitidis,
meningitis, otitis media) and pneumoccocci (
Streptococcus pneumoniae,
pneumonia, meningitis, otitis media). Such conjugate vaccines have been described e.g. in U.S. Pat. No. 4,762,713. According to this US patent, binding between the polysaccharide and the carrier protein is performed by reductive amination of aldehyde or hemiacetal functions of the poly-saccharides with amino groups in the protein. Another suitable method of covalently binding a polysaccharide to a proteinaceous material is by activating hydroxyl functions to produce a side chain containing a function that can be coupled to the protein. Thus, the polysaccharide can be activated and then coupled to a thiol-bearing group such as cysteamine, which can be coupled to an activated amino acid in the protein. The use of cysteamine for coupling oligosaccharides to proteins has been described by Verheul et al (
Infect. Immun.
59 (1991) 843-851). This use comprises activation of the saccharide by converting a carboxylic group to an N-succinimidyl ester (NSu), according to the following scheme:
Ps—COOH+XONSu→Ps—CO—ONSu+HOX  (1)
Ps—CO—ONSu+H
2
N—CH
2
—CH
2
—S—S—CH
2
—CH
2
—NH
2
→→Ps—CO—NH—CH
2
—CH
2
—S—S—CH
2
—CH
2
—NH
2
+HONSu  (2)
Ps—CO—NH—CH
2
—CH
2
—S—S—CH
2
—CH
2
—NH
2
+DTT-rd→→Ps—CO—NH—CH
2
—CH
2
—SH+HS—CH
2
—CH
2
—NH
2
+DTT-ox  (3)
Ps—CO—NH—CH
2
—CH
2
—SH+Br—CH
2
—CO—NH—Pr→→Ps—CO—NH—CH
2
—CH
2
—S—CH
2
—CO—NH—Pr+HBr  (4)
wherein Ps represents a polysaccharide, Pr represents a protein or peptide, and DTT-rd represents dithiothreitol in its reduced (dithiol) form and DTI-ox in its oxidised (1,2-dithiane) form. This approach, however, requires the presence of carboxyl groups in the polysaccharide, while many biologically interesting polysaccharides do not contain a carboxyl group.
It was found that polysaccharides can be effectively bound to cysteamine-like linkers without the need of other functional being present than hydroxyl groups by cyanogen bromide activation according to the following scheme.
Ps—OH+Br—CN→Ps—O—CN+HBr  (5)
Ps—O—CN+H
2
N—CH
2
—CH
2
—S—S—CH
2
—CH
2
—NH
2
→→Ps—O—C(═NH)—HN—CH
2
—CH
2
—S—S—CH
2
—CH
2
—NH
2
  (6)
Reaction (5) is followed by side reactions including a reaction with a second hydroxyl group to produce a cyclic imidocarbonate which can also result in coupling with an amino group as in reaction (6).
Thus the invention relates to a method of coupling a polysaccharide to another biopolymer wherein the polysaccharide is activated with a cyananting agent such as cyanogen halide and the activated polysaccharide is reacted with and aminothiol linker having formula 1
H
2
N—[(CH
2
)
m
—CHR
1
—CR
2
R
3
—A]
q
—CHR
4
—(CHR
5
)
p
—CHR
6
—S—R
7
wherein
A is a direct bond or a group having the formula
—{Z—(CH
2
)
m
—CHR
1
—CHR
2
R
3
}
n
Z—,
m is an integer from 0 to 5;
n is an integer from 0 to 3;
p is an integer from 0 to 2;
q is the integer 0 or 1;
R
1
is hydrogen or C
1
-C
6
alkyl, wherein the C
1
-C
6
alkyl is optionally substituted by amino, hydroxyl, carboxyl, C
1
-C
4
alkoxycarbonyl, carbamoyl, mono- or di-C
1
-C
4
-alkylcarbamoyl or N-(&agr;-carboxyalkyl)carbamoyl-; or if m ≠0, R
1
is hydroxyl, amino or peptidyl-amino;
R
2
and R
3
are independently hydrogen or C
1
-C
4
alkyl, or together from an oxo group;
R
4
is hydrogen, C
1
-C
4
alkyl, carboxly, C
1
-C
4
alkoxycarbonyl, carbamoly, mono-or di-C
1
-C
4
-alkylcarbamoyl or N-(&agr;-carboxyalkyl) carbamoyl;
R
5
is hydrogen, methyl, hydroxy or C
1
-C
7
acyloxy;
R
6
is hydrogen or methyl;
R
7
is hydrogen or thiol-protecting group or group having the formula
—S—CHR
6
—(CHR
5
)
p
—CHR
4
—[A—CR
2
R
3
—CHR
1
—(CH
2
)
m
]
q
—NH
2
;
 and
Z is imino, methylimino, oxygen or sulphur;
to produce a thiolated polysaccharide having the formula 2
Ps—O—C(═NH)—NH—[(CH
2
)
m
—CHR
1
—CR
2
R
3
—A]
q
—CHR
4
—(CHR
5
)
p
—CHR
6
—S—R
7
  2
wherein Ps represents a polysaccharide residue and A, m, p, q, R
1
, R
2
, R
3
, R
4
, R
5
, R
6
and R
7
are as defined above, followed by optionally removing protecting group R
7
and reacting the thiolated polysaccharide with an activated biopolymer. The preferred linkers are as defined in claims 3-8. A suitable example of the linkers is cysteamine or its oxidised form cystamine.
This method works satisfactorily for the majority of polysaccharides including most bacterial polysaccharides. However, no coupling to a useful degree is found with some polysaccharides such as the 19F type pneumococcal capsular polysaccharide.
Although the present inventors do not wish to be bound by any specific theory, one possible explanation for the incompleteness or failure of the cysteamine coupling is that the cysteamine adduct, once formed, may revert to the original materials by intramolecular displacement.
It has furthermore been found that any insufficient coupling can be solved by using amino-thiol linkers complying with formula 3,(=formula 1 with q=1)
H
2
N—(CH
2
)
m
—CHR
1
—CR
2
R
3
—A—CHR
4
—(CHR
5
)
p
—CHR
6
—S—R
7
wherein
A is a direct bond or a group having the formula
—{Z—(CH
2
)
m
—CHR
1
—CHR
2
R
3
}
n
Z—,
m is an integer from 0 to 5;
n is an integer from 0 to 3, preferably between 0 and 2;
p is an integer from 0 to 2;
q is the integer 0 or 1;
R
1
is hydrogen or C
1
-C
6
alkyl, wherein the C
1
-C
6
alkyl is optionally substituted by amino, hydroxyl, carboxyl, C
1
-C
4
alkoxycarbonyl, carbamoyl, mono- or di-C
1
-C
4
-alkylcarbamoyl or N-(&agr;-carboxyalkyl)carbamoyl; or, if m≠0, R
1
is hydroxyl, amino or peptidyl-amino;
R
2
and R
3
are independently hydrogen or C
1
-C
4
alkyl, or together from an oxo group;
R
4
is hydrogen, C
1
-C
4
alkyl, carboxly, C
1
-C
4
alkoxycarbonyl, carbamoly, mono-or di-C
1
-C
4
-alkylcarbamoyl or N-(&agr;-carboxyalkyl) carbamoyl;
R
5
is hydrogen, methyl, hydroxy or C
1
-C
7
acyloxy;
R
6
is hydrogen or methyl;
R
7
is hydrogen or thiol-protecting group or group having the formula
—S—CHR
6
—(CHR
5
)
p
—CHR
4
—[A—CR
2
R
3
—CHR
1
—(CH
2
)
m
]
q
—NH
2
;
 and
Z is imino, methylimino, oxygen or sulphur, wherein R
2
and R
3
together can form an oxo group.
The amino-thiol linkers according to formula 3 may be straight or branched &agr;,&ohgr;-aminothiol derivatives having at least 4 carbon atoms and optionally one or more heteroatoms in the chain, such as 4-aminobutanethiol, 5-aminopentanethiol, 2-(2-aminoethylaminoethanethiol and the like. Preferred compounds are those wherein H
2
N—(CH
2
)
m
—CHR
1
—CR
2
R
3
— represents an amino acid such as glycine, alanine, &bgr;-alanine, serine, glutamine, &ggr;-aminobutyric acid, lysine and &egr;-aminocaproic acid, or an oligopeptide such as N&agr;-glycyl-lysine and higher homologues of N&agr;-peptidyl-&agr;,&ohgr;-diaminoacids. The group H
2
N—(CH
2
)
m
—CHR
1
—CR
2
R
3
—A— may also represent a linear oligopeptide such as glycylglycine.
In the linkers of both formula 1 and 3, the group A—CHR
4
—(CHR
5
)
p
—CHR
6
—S—R
7
may e.g. be derived from 2-aminoethanethiol (cysteamine), 2-mercaptoethanol, 1,2-ethanedithiol, 2-amino-2-methylpropanethiol, 3-aminopropanethiol, 2-hydroxy-3-aminopropanethiol, monothio- and dithio-threitol or -erythritol, cysteine, homocysteine and their esters or amides, and the like. The most preferred compounds according to formula 3 are N-glycyl-cysteamine and its disulphide precursor N,N′-diglycyl-cystamine.
Most of the compounds com

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