Drug – bio-affecting and body treating compositions – Conjugate or complex of monoclonal or polyclonal antibody,... – Conjugated via claimed linking group – bond – chelating agent,...
Reexamination Certificate
1998-05-04
2001-05-29
Wessendorf, T. (Department: 1627)
Drug, bio-affecting and body treating compositions
Conjugate or complex of monoclonal or polyclonal antibody,...
Conjugated via claimed linking group, bond, chelating agent,...
C424S178100, C530S402000, C530S391100, C530S391500, C530S391300, C530S391100
Reexamination Certificate
active
06238667
ABSTRACT:
FIELD OF THE INVENTION
The present invention relates to a method of chemical cross-linking biologically active peptides to antibodies and a composition comprising one or more peptides cross-linked to an antibody. Specifically, the method attaches peptides to a unique affinity site of antibodies using photoreactive derivatives of peptides. These antibody-peptide complexes have immunostimulatory properties for B-cells and T-cells as a molecular adjuvant conjugated antibodies or antibody fragments. In addition the complexes may have biological properties to enhance antibody binding, facilitate membrane transport of antibodies or their fragments, and present B and T-cell epitopes to antigen presenting cells.
BACKGROUND OF THE INVENTION
Antibodies have been praised as “magic bullets” to combat disease. However, the promises made for antibodies were never fully realized. This-is in part due the fact that antibodies represent only one arm of the immune defense, where T-cells provide the other strategy in immune defense. However, antibodies are ideal targeting and delivery devices. They are adapted for long survival in blood, have sites which help vascular and tissue penetration and are functionally linked with a number of defense mechanisms of the innate immunity. One such mechanism is the complement system which helps to destroy pathogens and is involved in the regulation of immune responses. For example the complement fragment C3d binds to the CR2 receptor on B-cells, which is also the binding site for Epstein-Barr virus. Binding of Epstein-Barr virus to CR2 activates B-cells. Accumulated evidence has shown that the CR2 receptor (CD19/Cd20/CD81 complex) has an immuno-stimulatory role and is activated by C3d.
Another example of how antibodies can be used to enhance the immune response has been demonstrated by the work of Zanetti and Bona (Zanetti, M. Nature 355: 466-477, 1992; Zaghouani H.; Anderson S. A., Sperbeer K. E., Daian C. Kennedy R. C., Mayer L. and Bona C. A. 1995 Proc. Nat. Acad. Science U.S. 92: 631-635). These authors have replaced the CDR3 sequence of the Ig heavy chain with a sequence resembling T-cell and B-cell antigens (epitopes) using molecular biology methods and have shown that these modified antibodies induce potent immune response specific for the inserted groups. While this method of CDR replacement or antigenizing is effective, it requires manipulation of the genes encoding the immunoglobulin chains and expression of the modified antibodies with fermentation methods, both of which are expensive and time consuming.
The biological properties of the antibodies can be enhanced with respect to overall avidity for antigen and the ability to penetrate cellular and nuclear membranes. Antigen binding is enhanced by increasing the valency of antibodies such as in pentameric IgM antibodies. Valency and avidity is also increased in certain antibodies which are self binding or homophilic (Kang, C. Y., Cheng, H. L., Rudikoff, s. and Kohler, H. J. Exp. Med. 165:1332, 1987). Xiyun, A. N., Evans, S. V., Kaminki, M. J., fillies, S. F. D., Resifeld, R. A., Noughton, A. N. and Chapman, P. B. J. Immunol. 157: 1582-1588 (1996)). A peptide in the heavy chain variable region was identified which inhibited self-binding (Kang, C. Y. Brunck, T. K., Kiever-Emmons, T., Blalick, J. E. and Kohler, H., Science, 240: 1034-1036, 1988). The insertion of self-binding peptide sequence into an antibody endows the property of self-binding and increases the valency and overall avidity for the antigen.
Similarly the addition of a signal peptide to antibodies facilitates transmembrane transport as demonstrated by Rojas et al, Nature Biotechnology, 16: 370-375 (1998). Rojas et al. have generated a fusion protein which contained a 12 mer peptide and have shown that this protein has cell membrane permeability.
Antibodies have been used as delivery devices for several biologically active molecules, such as toxins, drugs and cytokines. Often fragments of antibodies, Fab or scFv, are preferred because of better tissue penetration and reduced “stickiness”. Two methods to attach these molecules are known. One method is the design of a fusion gene and the expression of the fusion protein. This method requires extensive molecular biology engineering and depends typically on mammalian or bacterial expression systems in large scale fermentation. Another method of generating antibody complexes with other proteins or molecules relies on chemical cross-linking technologies. Usually hetero-bifunctional cross-linkers are used which cross-link the selected molecules at random sites on the immunoglobulin molecule. Hetero-bifunctional cross-linking is associated with two problems. First, the antibody structure is compromised by local protein denaturation at the sites of cross-linking. This leads to changes in half-life in blood and biodistribution and uptake by scavenger cells in lung and liver. The second problem is the potential loss of antigen binding by non-specific cross-linking to the antigen binding site.
A one chemical, site-specific cross-linking method exists (Rodwell, J D; Alvarez, V L; Lee, C; Lopes, A D; Goers, J W; King, H D; Powsner, H J; McKearn, T.,
Proc. Natl. Acad. Sci
., USA, 83:2632-6, 1986) which takes advantage of a unique carbohydrate site in the Fc domain of antibodies. This method has two disadvantages. First, cross-linking to the constant domain carbohydrate restricts this method to full-length antibodies and it cannot be used with variable domain fragments such as Fabs and scFvs. In addition, the method requires rather harsh chemical treatment using periodates for reducing the sugar to a reactive dialdehyde. This chemical reaction can damage other sensitive amino acid side chains, such as in tyrosine, in the Ig molecule leading to undesired changes in biodistribution or loss of antigen binding.
A variation of the carbohydrate site-specific cross-linking has been published by H. Hansen, et al., (Govindan, S V; Goldenberg, D M; Griffiths, G L; Leung, S O; Losman, M J; Hansen, H J.,
Cancer Res.,
55:5721-5725) who has introduced a carbohydrate signal sequence by site-directed mutagenesis in the variable domain of an Ig light chain. This sequence allows the attachment of sugars to a serine residue during synthesis of the mutated antibody. The reducing chemistry to generate dialdehydes is then used with the above described pitfalls. In addition the site-directed insertion of a carbohydrate signal sequence requires molecular biology engineering and expression systems. Furthermore, the site of insertion in the Fv domain has to be carefully selected in order to avoid compromising antigen binding and/or stability of the heavy-light chain dimer structure.
Rajagopolan, et at., (
PNAS,
93:6019-24, 1996) described the affinity site on antibodies for ATP and Adenosine. U.S. Pat. No. 5,596,081 has issued for this site. A method of using azido-adenosine and Azido-ATP has been described by Pavlinkova, et al., (
J. Immun. Methods,
201:77-88,1997). The active binding peptide of C3d (complement fragment) has been described by Lambris, et al., (
PNAS,
82:4235-39, 1985). The synthesis of 5-azido-tryptophan has been described by Miles & Phillips (Miles, E. W. & Phillips, R. S.,
Biochemistry,
24:4694-703, 1985). A method of photo-labeling is reviewed by Potter and Haley (Potter, R. & Haley, B. E.,
Meth. Enzymol,
91:6130633, 1982).
The affinity-site cross-linking chemistry of the present invention overcomes prior art problems in the art and does not require the molecular engineering steps and fusion protein expression, since it allows to cross-link selected peptides to full-length antibodies or antibody fragments in one step using mild photo-reactive chemistry.
SUMMARY OF THE INVENTION
The present invention provides a method of affinity cross-linking a peptide to an antibody comprising the steps of
(a) photo-chemically activating an azido compound in a peptide comprising said azido compound;
(b) adding an antibody to said photochemically activated peptide; and
(c) allowing said photochemically activated pe
Antonelli Terry Stout & Kraus LLP
Wessendorf T.
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