Chemistry: molecular biology and microbiology – Measuring or testing process involving enzymes or... – Involving antigen-antibody binding – specific binding protein...
Reexamination Certificate
2000-03-02
2003-06-24
Ceperley, Mary E. (Department: 1641)
Chemistry: molecular biology and microbiology
Measuring or testing process involving enzymes or...
Involving antigen-antibody binding, specific binding protein...
C435S960000, C435S974000, C436S503000, C436S546000, C436S811000, C436S829000, C530S408000, C530S409000, C530S812000, C548S545000, C548S546000, C548S547000
Reexamination Certificate
active
06582928
ABSTRACT:
FIELD OF THE INVENTION
The invention is from the field of heterobifunctional cross-linking reagents. More particularly, the invention concerns cross-linking reagents which combine a nucleophilic hydrazide residue with an electrophilic maleimide residue, thereby allowing coupling of aldehydes to free thiols.
BACKGROUND OF THE INVENTION
Bifunctional cross-linking reagents have been extensively used for a variety of purposes including preparation of affinity matrices, modification and stabilization of diverse structures, identification of ligand and receptor binding sites, and structural studies. Homobifunctional reagents which carry two identical functional groups proved to be highly efficient in inducing cross-linking between identical and different macromolecules or subunits of a macromolecule, and linking of polypeptide ligands to their specific binding sites.
Heterobifunctional reagents contain two different functional groups. By taking advantage of the differential reactivities of the two different functional groups, cross-linking can be controlled both selectively and sequentially. The bifunctional cross-linking reagents can be divided according to the specificity of their functional groups, e.g. amino, sulfhydryl, guanidino, indole, carboxyl specific groups. Of these, reagents directed to free amino groups have become especially popular because of their commercial availability, ease of synthesis and the mild reaction conditions under which they can be applied. A majority of heterobifunctional cross-linking reagents contains a primary amine-reactive group and a thiol-reactive group [For review, see Ji, T. H. “Bifunctional Reagents” in:
Meth. Enzymol.
91, 580-609 (1983).]
The development of monoclonal antibody technology has created a new demand for bifunctional reagents that are useful in the synthesis of conjugates between antibodies and other proteins or non-proteinaceous molecules, such as detectable markers, toxins, toxin fragments and cytotoxic drugs. However, primary amino groups are randomly distributed on proteins, and therefore, the derivatization with amino-reactive cross-linking agents may impair the protein function, e.g., the antigen binding function of antibodies [Rodwell, J. D.,
Proc. Natl. Acad. Sci. USA
83, 2632-2636 (1986)]. In contrast, covalent modification of antibodies via their carbohydrate portion offers significant advantages. Chemical or enzymatic oxidation of oligosaccharides to aldehydes yields unique functional groups, capable of selective reaction, for example, with amines, hydrazines, hydrazides and semicarbazides, Since the carbohydrate moieties of antibodies are located distal to the antigen binding sites, it has been proposed that they can be modified without significant impairment of the antigen binding function.
Zara, J. J. et al. [
Anal. Biochem.
194, 156-162 (1991)] describe the synthesis of a heterobifunctional cross-linking reagent, S-(2-thiopyridyl)-L-cysteine hydrazide (TPCH), which contains a hydrazide moiety for coupling to aldehyde groups generated in the carbohydrate residues of antibodies and a pyridyl disulfide moiety for coupling to molecules with a free sulfhydryl (thiol) group. They have demonstrated that derivatization of a human monoclonal IgM antibody against human colon carcinoma cells with 16 TPCH cross-linker molecules did not impair its antigen binding ability.
Heindel, N. D. et al.,
Bioconjugate Chem.
2, 427-430 (1991) describe a maleimide-hydrazide heterobifunctional cross-linking reagent for coupling of thiol groups to formyl groups. Applying this reagent to the coupling of a monoclonal antibody which recognized a nonshed membrane receptor on colon carcinoma, or its Fab′ fragment, to polyaldehyde dextran to which an antineoplastic agent had been attached, they found that high binding avidities for the unshed antigen were retained.
Chemical conjugates of recombinant soluble CD4 (sCD4) with toxins, or with antibodies that activate cytotoxic T cells, are also known in the art.
CD4 is a transmembrane glycoprotein, found on the surface of human T lymphocytes, that acts as the primary receptor for HIV-1 [Dalgleish, A. G., et al.,
Nature
312, 763-766 (1984); Klatzmann, D., et al.,
Nature
312, 767-768 (1984)]. The extracellular portion of CD4 contains four immunoglobulin-like domains (V
1
-V
4
), the first of which (V
1
) is necessary and sufficient for high-affinity binding to gp120, the envelope glycoprotein of HIV-1 [Maddon, P. J., et al.,
Proc. Natl. Acad. Sci. USA
84, 9155-9159 (1987); Richardson, N. E., et al.,
Proc. Natl. Acad. Sci. USA
85, 6102-6106 (1988); Landau, N., et al.,
Nature
334, 159-162 (1988)].
Recombinant, soluble forms of CD4 (sCD4), containing only the extracellular portion of the molecule, have been produced [Smith, D. H., et al.,
Science
238, 1704-1707 (1987); Fisher, R. A., Bertonis, et al.,
Nature
331, 76-78 (1988); Hussey, R. E., et al.,
Nature
331, 78-81 (1988); Deen, K. et al.,
Nature
331, 82-84 (1988); Traunecker, A., et al.,
Nature
331, 84-86 (1988); Berger, E.,
Proc. Natl. Acad. Sci. USA
85, 2357-2361 (1988)]. sCD4 retains high-affinity binding to gp120 and can block the binding of HIV-1 to cell surface CD4 in vitro, thereby inhibiting infection of target cells [reviewed in Capon, D. and Ward, R.,
Curr. Opin. Immunol.
2, 433-438 (1990)].
Several modifications of sCD4 have been made, by either gene fusion or chemical conjugation approaches, to expand its antiviral capabilities. One example is a class of chimeric molecules known as CD4 immunoadhesins, in which genes encoding sCD4 and immunoglobulin heavy-chain are combined, thus creating homodimeric antibody-like molecules with properties of both CD4 and human immunoglobulin [Capon, D. et al.,
Nature
337, 525-531 (1989); Zettlmeissl, G., et al.,
DNA Cell Biol.
9, 347-353 (1990); Traunecker, A. et al.,
Nature
339, 68-70 (1989)]. Other notable examples of modified sCD4 are CD4 peptide-protein conjugates [Ghetie, V.,
Proc. Natl. Acad. Sci. USA
88, 5690-5693 (1991)], CD4 electroinserted into erythrocyte membranes [Zeira, M.,
Proc. Natl. Acad. Sci. USA
88, 4409-4413 (1991)], and sCD4-toxin hybrids, in which sCD4 is attached to a toxin either by gene fusion [Chaudhary, V. K.,
Nature
335, 369-372 (1988); Winkler, G.,
AIDS Res. Hum. Retroviruses
7, 393-401 (1991)], or by chemical crosslinking [Till, M. A.,
Science
242, 1166-1168 (1988)].
Because cells infected actively with HIV express gp120 on their surface, sCD4 can be used to direct toxins to, and thus selectively kill, HIV-infected cells. Similarly, bispecific hybrids of sCD4 and anti-CD3 antibody can be constructed which mediate selective killing of HIV-infected cells by cytotoxic T cells [Berg, J., et al.,
Proc. Natl. Acad. Sci. USA
88, 4723-4727 (1991); Idziorek, T., and Klatzmann, D.,
AIDS Res. Hum. Retroviruses
7, 529-536 (1991)].
These examples represent some of the possible modifications of sCD4 which may improve its efficacy against HIV in vivo.
An object of the present invention is to provide improved carbohydrate-directed heterobifunctional cross-linking reagents.
It is another object, to provide chemical conjugates comprising an aldehyde moiety connected to a thiol group via a heterobifunctional cross-linking reagent.
It is a further object, to provide a method for coupling glycoproteins via their carbohydrate moieties to compounds which either contain thiols, or to which thiol groups can be added.
It is yet another object to derivatize members of the immunoglobulin gene superfamily, and in particular antibodies or CD4 molecules in their carbohydrate portion, and to chemically link them to compounds having thiol functional groups or to which thiol groups can be added.
These and further objects of the invention will be apparent for those skilled in the art.
SUMMARY OF THE INVENTION
In one aspect, the invention concerns a heterobifunctional compound of formula (I)
wherein X is a hydrocarbon spacer of
Ashkenazi Avi J.
Chamow Steven M.
Kogan Timothy P.
Ceperley Mary E.
Conley Deirdre L.
Genentech Inc.
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