Chemistry: molecular biology and microbiology – Enzyme – proenzyme; compositions thereof; process for... – Stablizing an enzyme by forming a mixture – an adduct or a...
Reexamination Certificate
1998-10-20
2001-04-17
Russel, Jeffrey E. (Department: 1653)
Chemistry: molecular biology and microbiology
Enzyme , proenzyme; compositions thereof; process for...
Stablizing an enzyme by forming a mixture, an adduct or a...
C530S322000, C530S345000, C530S391500, C530S395000, C530S409000
Reexamination Certificate
active
06218160
ABSTRACT:
BACKGROUND OF THE INVENTION
Enzyme-antibody conjugates are among the most important reagents in biomedical/biochemical research and applications. Numerous reagents and methods for coupling enzymes to antibody molecules (mostly IgG) have been developed. Most of these coupling methods involve the use of cross linking reagents such as homobifunctional cross-linkers, or heterobifunctional cross linking reagents (Wong, S. S.,
Chemistry of Protein Conjugation and Cross
-
Linking
, CRC, Boca Raton, 75-189 (1991); Ishikawa, E., et. al.,
Journal of Immunoassay
4(3), 209-327 (1983); Means, G. E. and Feeney, R. E.,
Bioconjugate Chem.
1, 2-12 (1990)). Most protein cross-linking chemistries are conducted either through lysine (Lys) residues which contain primary amines, aspartic acid (Asp) or glutamic acid (Glu) residues which contain carboxyl groups, or cysteine (Cys) residues which contain sulfhydryl groups. However, Lys, Asp, Glu, and Cys are among the six most frequently appearing amino acid residues in the active sites of proteins (e.g., enzymes) (Becker, W. M.,
World of the Cell
, Benjamin Cummings, Menlo Park, Calif., 147-174 (1986)). In addition, in most cases, the coupling of proteins through cysteines or lysines is not site-specific due to the random distribution of these residues in protein molecules. Consequently, crosslinking enzymes and antibodies through these residues may result in cross-linking at or near the active sites of these molecules impairing the biochemical functions (e.g., the catalytic activity of enzymes, the antigenic binding affinity of antibodies) of the conjugated protein molecules due to the blocking of the active site.
Glutaraldehyde is a typical homobifunctional cross-linking reagent and has long been used to prepare enzyme-antibody conjugates (Avrameas, S.,
Immunochemistry
6, 43-52 (1969); Avrameas, S. and Ternynck, T.,
Immunochemistry,
6, 53-66 (1969)). Glutaraldehyde cross-links proteins by forming a Schiff's base between two aldehyde groups and two lysine residues, one from each of the protein molecules. The disadvantage of this method is that self-conjugation of the protein molecules may occur. Methods which reduce protein self-conjugation may also lead to over polymerization in which three or more protein molecules crosslink to each other due to the excess number of derivatized groups on the proteins (Avrameas, S. and Ternynck, T.,
Immunochemistry
8, 1175-9 (1971); Clyne, D. H., et. al.,
J. Histochem. Cytochem.
21, 233-40 (1973)).
Conjugating proteins utilizing heterobifunctional reagents as crosslinkers, which usually involves the labeling of one or both proteins with the cross-linkers, eliminates the problem of self-coupling between molecules of the same protein. In a very common conjugation method, enzyme molecules are first labeled with a NHS-ester-maleimide heterobifunctional reagent (Yoshitake, S., et al.,
Eur. J. Biochem.
101-395 (1979); Myers, D. E., et al.
J. Immunol. Meth.
121, 129-142 (1989)). IgG has been labeled with a NHS-pyridyldithiol cross-linker (Carlsson, J., et al.,
J. Biochem.
173, 723-737 (1978); Cumber, A. J., et al.,
Meth. Enymol.
112, 207-225 (1985)) which is then reduced to generate free sulfhydryl groups (—SH). The antibody molecules are conjugated to the enzyme molecules through the nucleophilic addition of the —SH groups to the maleimide groups on the enzyme. However, a disadvantage of using heterobifunctional crosslinking reagents is that the site of crosslinking on the proteins is nonspecific and may result in the loss of activity of the proteins.
Glycoproteins can be cross-linked through their carbohydrate moieties. The oligosaccharide moieties of glycoproteins are often located at sites far away from the active or binding sites of the proteins and therefore are not usually involved in binding to ligands. This was demonstrated in IgG where the IgG was crosslinked through the carbohydrate groups, located on the complement fragment (Fc) region. The antigen binding fragment (Fab) of the IgG bound to the antigen almost as well as the intact IgG; (Avrameas, S. and Ternynck, T.,
Immunochemistry
8, 1175-9 (1971)). Results of studies of enzyme mechanisms suggest that carbohydrate moieties are not involved in the binding of the enzymes to their substrates (Becker, W. M., supra).
Enzyme-antibody conjugates joined through carbohydrate groups have been prepared by converting the vicinal hydroxyl groups of the carbohydrate moieties to aldehydes via periodate oxidation (Kawaoi, A. and Nakane, P. K.,
Fed. Proc.
32, 840 (1972)). The conjugation of horseradish peroxidase (BRP) to IgG has been described, wherein the aldehyde groups generated on HRP reacted with the primary amines on IgG to form Schiff's bases, which were subsequently stabilized through reduction by sodium borohydride. This method, however, resulted in self-coupling of the enzyme molecules and subsequent blocking of the active site on the enzyme. Amino-blocking reagents, such as 1-fluoro-2,4-dinitrobenzene, which react with reactive lysine residues, have been used to prevent self-coupling of HRP-aldehyde (Nakane, P. K. and Kawaoi, A.,
J. Histochem. Cytochem.
22, 1084-1091 (1974); Wilson, M. B. and Nakane, P. K.,
Immunofluorescence and Related Staining Techniques
, Elsevier, Amsterdam, 215-224 (1978); Imagawa, M., et. al.,
J. Applied Biochem.
4, 41-57 (1982); Tijssen, P. and Kurstak, E.,
Anal. Biochem.
136, 451-7 (1984)). However, the use of amino-blocking reagents has limited applicability since lysine residues are crucial for maintaining ligand-binding activity among many proteins. Therefore, conjugation chemistry involving the formation of Schiff's bases still presents the disadvantage of blocking enzyme active sites and antibody binding sites.
Hydrazide, hydrazine, or semicarbazide containing compounds, which modify aldehydes generated on glycoproteins through the formation of a stable hydrazone bond between the aldehyde and hydrazide, have been used in an attempt to overcome the various difficulties encountered in Schiff's base chemistry. This methodology has been used to immobilize IgG to a solid matrix. The IgG molecules immobilized through their carbohydrate moieties to hydrazide-containing agarose beads displayed an enhanced antigenic affinity about three times higher than IgG molecules immobilized through lysine residues. (O'Shannessy, D. J. and Hoffman, W. L.,
Biotechnol. Appl. Biochem.
9, 488-496 (1987); Hoffman, W. L. and O'Shannessy, D. J.,
J. Immunol. Method,
112, 113-120 (1988)). A heterobifunctional cross-linking reagent containing a hydrazide group and a pyridyl disulfide moiety was used for coupling antibodies to molecules containing a free sulfhydryl group, wherein the modified antibodies retained full antigen binding ability (Zara, J. J. et. al.,
Anal. Biochem.
194, 156-162 (1991)). A method of crossinking a glycoprotein to a protein containing free sulfhydryl groups using a cross-linking reagent which combines a nucleophilic hydrazide residue with an electrophilic maleimide residue has been described. (U.S. Pat. No. 5,605,791). These two methods, however, require the presence of a free sulfhydryl group on one of the proteins to be conjugated and these conjugation methods may block the active site of one of the proteins.
Therefore, it is an object of the present invention to provide a method of conjugating proteins which not only eliminates protein self-coupling but also provides for optimum orientation of the active sites of the conjugated proteins and minimizes blocking of active sites such that the proteins retain full biological activity.
SUMMARY OF THE INVENTION
In one aspect, the invention concerns a method for the site-specific covalent conjugation of two or more glycoproteins through their carbohydrate residues, comprising: (a) mixing a first oxidized glycoprotein with a dihydrazide or dihydrazine containing reagent to form a glycoprotein-hydrazide or glycoprotein-hydrazine intermediate, wherein a hydrazone bond is formed between the aldehyde group of the first glycoprotei
Gibbons Del Deo Dolan Griffinger & Vecchione
Roche Diagnostics Corporation
Russel Jeffrey E.
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