Chemistry: molecular biology and microbiology – Measuring or testing process involving enzymes or... – Involving antigen-antibody binding – specific binding protein...
Reexamination Certificate
1996-07-31
2001-04-03
Wessendorf, T. D. (Department: 1627)
Chemistry: molecular biology and microbiology
Measuring or testing process involving enzymes or...
Involving antigen-antibody binding, specific binding protein...
C424S189100, C424S225100, C424S186100, C424S185100, C424S184100, C530S326000, C514S013800, C435S007900
Reexamination Certificate
active
06210901
ABSTRACT:
FIELD OF THE INVENTION
The invention concerns specific binding substances for antibodies, their use in immunoassays and as vaccines as well as an immunological method of determination using this specific binding substance as a binding partner for the antibody.
BACKGROUND AND PRIOR ART
Antibodies are known to bind their corresponding antigens extremely specifically. If an antibody is directed against a partial amino acid sequence of a protein (an epitope), it is known that this partial sequence can be used alone for its binding.
The specific binding of two biomolecules was compared to the lock and key principle for the first time in the case of enzyme-substrate binding. However, in the 60s Linus Pauling recognized that there was a fundamental difference between enzyme-substrate binding and antibody-antigen binding. The binding site of an enzyme does not completely fit the substrate at all. Rather the binding site of an enzyme appears to be much more similar to the structure of the transition state of a substrate reaction and therefore allows the substrate to have a certain latitude in its structure without the enzyme losing its binding properties for the substrate. In contrast absolute fitting of the antigenic epitope into the binding site of the antibody is required for antigen-antibody binding, which basically excludes any spatial variation of the epitope with regard to antibody binding. It is only by means of this specific epitope/antibody binding that a foreign substance can be specifically eliminated from the organism by an antibody.
Epitopes toward which an antibody is specifically directed are mainly used for two applications. On the one hand, they serve—usually bound to a carrier—as immunogens (vaccines) which cause an organism to produce antibodies which are directed against these epitopes or against antigens which contain these epitopes. On the other hand, such epitopes are used in immunoassays for the specific detection of antibodies in body fluids in which they have been produced by an immune reaction to antigens which contain these epitopes (for example after infection by an infectious organism).
If the epitopes are polypeptides, when they are used in the organism as a vaccine or when they come into contact with body fluids such as serum in diagnostic applications, there is a problem in that the polypeptides are decomposed and lose their function by metabolic degradation, brought about in particular by proteases such as those which occur in body fluids.
It is known that polypeptides which are used for therapeutic purposes, i.e. for binding to enzymes or to hormone receptors in the organism, can be protected from metabolic degradation in the body by modifying the amino acid sequence. A description of such so-called peptide mimetics as therapeutic agents and the production thereof is given inter alia in Giannis, “Angewandte Chemie” 105 (1993) 1303-1326; Lee, Bull Chem. Soc. Jpn 66 (1993) 2006-2010 and Dorsch et al, “Kontakte” (Darmstadt) 1993 (2). However, this modification method did not appear to be applicable to epitopes used for binding antibodies and as immunogens for producing antibodies due to the known high specificity of the epitope-antibody binding.
Recently, at the third European BIAcore Symposium in London 1993, peptide human serum albumin fusion products were proposed for stabilizing such polypeptide epitopes (Integration of Biocore in the Discovery Department S. Reboul et al). However, the coupling of peptide epitopes to human serum albumin merely delays the enzymatic degradation of the polypeptides to a certain extent but does not prevent it.
SUMMARY OF THE INVENTION
The object of the invention is to provide binding substances which function as epitopes for vaccines useful in producing antibodies and in immunoassays for the detection of antibodies which are directed against epitopes with particular natural amino acid sequence. These inventive substances have modified metabolic stability, in particular a modified protease stability, or modified duration of action as an immunogen in an animal organism or as a binding partner in immunoassays using body fluids as the sample material. These inventive substances can, nevertheless, specifically and selectively bind an antibody which is directed against a corresponding natural epitope or produce this antibody in an immune response.
The object is achieved by the invention as characterized herein. It was found, surprisingly that, despite the extremely high specificity of the antigen-antibody reaction, a structural change in a natural epitope polypeptide sequence made by modifying the amino acids in the sequence in the manner according to the invention does not prevent binding of the modified epitope to an antibody which is directed against the unmodified epitope. However, compared to the unmodified epitope, the modified epitope has different metabolic properties when contacted with body fluids or in an animal organ e.g. a modified stability towards endogenous proteases.
DETAILED DESCRIPTION OF PREFERRED EMBODIMENTS
According to the invention the following possibilities are understood as a modification of the amino acids of the amino acid sequence of the natural epitope:
a) Substitution of the —CO—NH group of at least one peptide bond, by a diatomic or triatomic bridge (backbone modification).
A diatomic bridge is preferably understood as an —NH—CO—, —CH
2
—CH
2
—, —CH═CH, —CH
2
—NH—, —NH—CH
2
—, —O—CH
2
—, —CH
2
—O—, —CH
2
—S or —S—CH
2
— group. A triatomic bridge is preferably understood as a —CH
2
—CH
2
—CH
2
group in which a —CH
2
group can also be replaced by an —O— or —NH—, —S— or —CO— group. Diatomic bridges are preferred.
A —CH
2
—CH
2
—, —CH
2
—NH— or —NH—CO group is especially preferred. If a peptide —CO—NH group is for example replaced by a —NH—CO group, one formally obtains a diamine and a dicarboxylic acid from two amino acids of the natural epitope. If a —CO—NH group is for example replaced by a —CH
2
—CH
2
— group, one obtains an aminovaleric acid derivative within the sequence of the binding substance according to the invention.
b) Extension or shortening of the side chains of at least one amino acid of the natural epitope. In this case it is preferable to incorporate an additional —CH
2
—, —S—, —O—, —NH—, —SO
2
—, —CO— group or to omit &bgr;-methylene groups in the side chain. It is especially preferred to carry this out in the side chains that are present in all amino acids of the epitope. If only one side chain is modified, a natural amino acid should not be formed in this process.
c) Substitution of a natural amino acid of the natural epitope by one or several non-biogenic L- or D-amino acids of the formula
HR′N—Y—COOH
wherein
R′ represents hydrogen and
Y represents a (CH
2
)n group in which n=2-8 or a —CH—(CH
2
)m-CR
1
R
2
R
3
group in which m=0−3 in which R
1
, R
2
and R
3
can be the same or different, and may represent hydrogen, a branched or unbranched C
1
-C
4
alkyl residue or a phenyl, napthyl or 5-6-membered heteroaryl residue containing an O, S or N which can be substituted with methyl, halogen, NH
2
, OH or carboxy or in which Y represents a CHR group in which R represents a side chain of a natural amino acid in which a —CH
2
— group is replaced by —S—, —NH—, —CH═, —SO
2
—, —CO— or —O— or in which at least one H atoms on said residue are substituted by CH
3
, NH
2
, carboxyl, SH, halogen or hydroxy,
wherein in all cases Y is a group which does not occur in any natural amino acid or wherein R′ is methyl, ethyl or phenyl and Y represents a CHR group in which R is a side chain of a natural amino acid.
In this case halogen is understood as F, Cl, Br or iodine, in particular Cl. Among heteroaryl residues pyridine, pyrrol, furan and thiophene are particularly preferred.
The binding substance according to the invention binds those antibodies which are directed against an antigen with a corresponding epitope consisting of a natural amino acid sequence. In particular these include antibodies which are directed against an epitope of an inf
Batz Hans-Georg
Herrmann Rupert
Hoess Eva
Seidel Christoph
Fulbright & Jaworski LLP
Roche Diagnostics GmbH
Wessendorf T. D.
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