Drug – bio-affecting and body treating compositions – Radionuclide or intended radionuclide containing; adjuvant... – Attached to antibody or antibody fragment or immunoglobulin;...
Reexamination Certificate
2001-01-08
2003-03-25
Chan, Christina (Department: 1644)
Drug, bio-affecting and body treating compositions
Radionuclide or intended radionuclide containing; adjuvant...
Attached to antibody or antibody fragment or immunoglobulin;...
C424S001530, C424S178100, C424S179100, C424S275100, C424S194100, C530S387100, C530S391100, C530S391500, C530S391900
Reexamination Certificate
active
06537519
ABSTRACT:
The present invention relates to a method for the preparation of a conjugate comprising a first and a second polypeptide, said method comprising the steps of (a) incubating said first polypeptide in the presence of a heterobifunctional crosslinker comprising an N-hydroxylsuccinimide ester group and a maleimide group linked via a polyethylene oxide spacer; (b) removing excess heterobifunctional crosslinker; and (c) incubating the reaction product of step (b) with said second polypeptide, wherein said second polypeptide comprises at least one sulfhydryl group. Furthermore, the present invention relates to a conjugate obtainable by the method of the present invention. Also described is a pharmaceutical composition comprising the conjugate of the present invention and, optionally, a pharmaceutically acceptable carrier and/or diluent, and the use of the conjugate for the preparation of a pharmaceutical composition for preventing and/or treating an allergic disease or an autoimmune disease.
Immunologic tolerance may be defined as a state of antigen-specific unresponsiveness induced by preexposure to an antigen. If the antigen is an allergen, the immune response is defined as allergy, an adverse reaction with an immunologic basis mediated by IgE immunoglobulin (Sampson (1986), J. Allergy Clin. Immunol. 78:212-219). The immune system may also be a cause of disease or other undesirable consequences, when the principle of self
on-self recognition breaks down and the body's own components are recognized as non-self (autoantigens) in which case autoimmune diseases can ensue.
Interest in immunologic tolerance, discovered by Medawar almost half a century ago (Billingham et al. (1953), Nature 172:603-606), has increased for two main reasons: (1) Several of its mechanisms, such as clonal deletion (Kappler et al. (1987), Cell 49:273-280), anergy (Jenkins and Schwartz (1987), J. Exp. Med. 165:302-319), and regulatory T cells (Gershon and Kondo (1971), Immunology 21:903-914) have been uncovered. (2) Both systemic and oral tolerance (Cremer et al. (1983), J. Immunol. 131:2995-3000; Weiner et al. (1994), Ann. Rev. Immunol. 12:809-837) can be induced to, it is hoped, prevent either autoimmune or allergic diseases. For example, several strategies have been used in trying to prevent allergy, including administration of modified allergen (Lee and Sehon (1977), Nature 267:618-649), allergen linked to nonimmunogenic carriers (Katz et al. (1971), J. Exp. Med. 134:201-203), single peptides (Muckerheide et al. (1977), J. Immunol. 119:1340-1345), or an allergen-antibody complex (Machiels et al. (1990), J. Clin. Invest. 85:1024-1035).
It is known that antigen presentation can influence the type of immune response. Not only haptens (Borel (1989), in “Concepts in Immunopathology”, Cruse and Lewis (Eds.), 7:145-161, Karger, Basel; Sehon (1982), Prog. Allergy 32:161-202) but also proteins covalently linked to a carrier molecule naturally tolerated by the host, such as isologous immunoglobulin, can induce unresponsiveness to these proteins (Filion et al. (1980), Cell Immunol. 54:115-128; Borel and Borel (1990), J. Immunol. Methods 126:159-168).
However, although the above strategies proved to be partially successful, there is still a need for allergen and/or auto-antigen comprising conjugates with improved therapeutic properties.
Thus, the technical problem underlying the present invention was to provide a method for the production of such allergen and/or auto-antigen comprising conjugates.
The solution to this technical problem is achieved by providing the embodiments characterized in the claims.
Accordingly, the present invention relates to a method for the preparation of a conjugate comprising a first and a second polypeptide, said method comprising the steps of:
(a) incubating said first polypeptide in the presence of a heterobifunctional crosslinker comprising an N-hydroxylsuccinimide ester group and a maleimide group linked via a polyethylene oxide spacer;
(b) removing excess heterobifunctional crosslinker; and
(c) incubating the reaction product of step (b) with said second polypeptide, wherein said second polypeptide comprises at least one sulfhydryl group.
It is envisaged in accordance with the present invention that the polyethylene oxide spacer may consist of from 1 to 10 monomer units. Preferably, the spacer consists of from 2 to 5 monomer units.
Unexpectedly, it has been found in accordance with the present invention that due to the crosslinker used conjugates prepared by the method of the present invention show superior features as compared to conjugates prepared by prior art methods. For example, conjugates of ragweed derived antigen (Amba-l) and mouse IgG were prepared by the method of the present invention and a method using as crosslinker N-(&ggr;-maleimidobutyroxy)sulfosuccimimide ester (sulfo-GMBS) a preferred prior art crosslinker (see Example 1, infra). The effects of both conjugates in the treatment of an allergic reaction to ragweed derived antigen were investigated. Surprisingly, it was found that the conjugate of the present invention leads to a significant reduction in airway hyperresponsiveness whereas the prior art conjugate has almost no effect when compared to sensitized animals (see Example 5 and FIG.
3
). Furthermore, the conjugate of the present invention virtually eliminated eosinophils in the bronchoalveolar lavage fluid (BALF). This phenomenon was accompanied by a total suppression of specific anti-Amba-l IgE. In contrast, the prior art conjugate only moderately reduced eosinophils, and specific anti-Amba-l IgEs were only partially suppressed (see Example 5 and FIG.
4
). These results clearly demonstrate that due to the crosslinker used the method of the present invention allows the person skilled in the art to produce conjugates with advantageous immunological properties which render these conjugates for instance suitable for the downregulation of the inflammatory and immunologic reactions of an allergic response.
In a preferred embodiment of the present invention, said at least one sulfhydryl group of said second polypeptide is introduced by:
(i) incubating said second polypeptide in the presence of N-succinimidyl-S-acetylthioacetate (SATA);
(ii) removing excess SATA;
(iii) incubating the reaction product of step (ii) in the presence of hydroxylamine; and
(iv) removing excess hydroxylamine and acetylated hydroxylamine.
This embodiment is of particular importance in cases where said second polypeptide does not comprise at least one endogenous sulfhydryl group that is, for example, provided by a cysteine residue in the amino acid sequence, and which is suitable for crosslinking said second polypeptide with said first polypeptide via a disulfide bond. Sulfhydryl groups that are not suitable for crosslinking may be, for example, sulfhydryl groups that are not readily accessible for the sulfhydryl group of said first polypeptide due to the three dimensional conformation of said second polypeptide, or that are not available due to inter- or intramolecular disulfide bonds. Whether said second polypeptide comprises one or more sulfhydryl groups that allow effective crosslinking to occur or whether sulfhydryl groups have to be introduced into said second polypeptide can be determined by the person skilled in the art without further ado. For example, two polypeptides may be crosslinked by the method of the present invention and the quality and quantity of the obtained conjugate may be analyzed by SDS polyacrylamide gel electrophoresis under non-reducing conditions and immunoblotting with an antibody specific for one of the two crosslinked polypeptides. Preferably, the identity of the conjugate is verified with a second antibody specific for the second of the two polypeptides. For a detailed description of the above outlined experiment, reference is made to Example 1. If the results show that the quality and quantity of the conjugate is not satisfactory as compared to a reference conjugate like, for example, the IgG/Amba-l conjugate produced in Example 1, sulfhydryl groups may
Borel Yves
Gelfand Erwin
Schlegel Werner
Aventis Behring GmbH
Chan Christina
Finnegan, Henderson Farabow, Garrett and Dunner L.L.P.
Huynh Phuong N.
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