Drug – bio-affecting and body treating compositions – Immunoglobulin – antiserum – antibody – or antibody fragment,... – Structurally-modified antibody – immunoglobulin – or fragment...
Reexamination Certificate
1998-03-02
2003-11-11
Duffy, Patricia A. (Department: 1645)
Drug, bio-affecting and body treating compositions
Immunoglobulin, antiserum, antibody, or antibody fragment,...
Structurally-modified antibody, immunoglobulin, or fragment...
C424S178100, C514S002600, C530S350000, C530S351000, C530S391700, C530S391900, C530S399000
Reexamination Certificate
active
06645490
ABSTRACT:
1. INTRODUCTION
The present invention relates to chimeric proteins with cell-targeting specificity and apoptosis-inducing activities. In particular, the invention is illustrated by a recombinant chimeric protein between human interleukin-2 (IL2) and Bax. The chimeric protein specifically targets IL2 receptor (IL2R)-expressing cells and induces cell-specific apoptosis. In accordance with the invention, chimeric proteins may be generated between any molecule that binds a specific cell type and an apoptosis-inducing protein. Such chimeric proteins are useful for selectively eliminating specific cell types in vitro and in vivo, and may be used in the treatment of autoimmunity, cancer and infectious diseases such as viral infections.
2. BACKGROUND OF THE INVENTION
2.1. IMMUNOTOXINS
The advent of the monoclonal antibody technology and recombinant DNA technology have led to the discovery of numerous cell surface molecules associated with specific cell populations. Based on the expression pattern of these molecules, recombinant immunotoxins have been constructed to specifically target and destroy the cells that express such molecules. Recombinant immunotoxins are a class of targeted molecules designed to recognize and specifically destroy cells expressing specific receptors, such as cancer cells and cells involved in many disorders of the immune system. Generally, immunotoxins utilize a bacterial or plant toxin to destroy the unwanted cells. These molecules are designed and constructed by gene fusion techniques and are composed of both the cell targeting and cell killing moieties, a combination that makes these agents potent molecules for treatment. Examples of immunotoxins are growth factors or antigen-binding domains of antibody, including the Fv portion of an antibody (single-chain immunotoxins) fused to various mutant forms of toxin molecules. However, over the years it has become clear that treatment with such “magic bullets” for targeted immunotherapy possesses still many problems and new approaches are needed to produce improved recombinant immunotoxins.
Each recombinant immunotoxin displays some nonspecific toxicity and at sufficiently high concentrations damages normal cells that do not express the specific target antigen. This non-specific toxicity of immunotoxins is the dose-limiting factor in immunotoxin therapy. Which tissues are affected by nonspecific toxicity is dependent on the particular toxin used for immunotoxin preparation, and the ability of immunotoxins to penetrate into tissues and tumors is largely dependent on the size of the immunotoxins.
Large stable conjugated immunotoxins persist for long periods in blood vessels (T
1/2
5-15 hour), thus endothelial cells are exposed to high toxin concentrations which may lead to endothelial cell damage. Smaller molecules, such as recombinant immunotoxins which rapidly leave the vascular system, would presumably have different toxicity. In humans, immunotoxins made with ricin and other ribotoxins, as well as with Pseudomonas exotoxin A (PE), Diphtheria toxin (DT) and their truncated derivatives have produced a variety of toxicities. These include vascular leak syndrome (mainly ricin immunotoxins) as well as liver toxicity (PE-derived immunotoxins). Vascular leak syndrome observed with ricin immunotoxins in animals and man may be explained by specific binding of ricin A-chain to endothelial cells and subsequent killing of the cells and damage to the vessels. The nonspecific liver-toxicity of PE immunotoxins is likely to be due to easy access and very rapid nonspecific uptake and internalization of proteins by hepatocytes. However, it is also possible that PE contains, in addition to the specific cell-binding site (Domain I) which is removed in most immunotoxins, an additional site which could be recognized with low affinity by hepatocytes, thus accounting for liver toxicity.
Another major impediment with immunotoxins in their clinical application is the human immune response against them, mainly toward the toxin moiety. Bacterial toxins like PE and DT are highly immunogenic and cannot be humanized with standard techniques. Usage of DT-derived immunotoxins is limited because most people in developed countries have been vaccinated against DT and many adults have neutralizing antibodies to DT. Immunogenicity is a problem to which so far no practical solution has been found. Reduced immunogenicity of these molecules would greatly improve the clinical application of immunotoxins.
An example of the successful use of an immunotoxin is the elimination of activated T cells which express high affinity IL2 receptors (IL2R), whereas normal resting T cells and their precursors do not. An immunotoxin made of IL2 could theoretically eliminate IL2R-expressing leukemia cells or IL2R-expressing immune cells involved in various disease states while not destroying IL2R negative normal cells, thereby preserving the full repertoire of antigen receptors required for T cell immune responses.
A chimeric protein, IL2-PE40, was produced and shown to eliminate activated T cells (Lorberboum-Galski et al., 1988, Proc. Natl. Acad. Sci. U.S.A. 85:1922). IL2-PE40 was extremely cytotoxic to IL2R-expressing cell lines of human, ape and murine origin. It was also extremely cytotoxic to Con A-stimulated mouse and rat spleen cells, and had a suppressive effect against antigen-activated mouse cells and the generation of cytotoxic T cells in mixed lymphocyte cultures (Lorberboum-Galski et al., 1988, J. Biol. Chem. 263:18650-18656; Ogata et al., 1988, J. Immunol. 41:4224-4228; Lorberboum-Galski et al., 1990, J. Bio. Chem. 265:16311-16317).
A highly purified IL2-PE40 preparation (Bailon et al., 1988, Biotechnol. 6:1326-1329) was shown to (a) delay and mitigate adjuvant induced arthritis in rats (Case et al., 1989, Proc. Natl. Acad. Sci. U.S.A. 86:287-291), (b) significantly prolong the survival of vascularized heart allograft in mice (Lorberboum-Galski et al., 1989, Proc. Natl. Acad. Sci. U.S.A. 86:1008-1012) and corneal allografts in rats (Herbort et al., 1991, Transplant. 52:470-474), (c) reduce the incidence and severity of experimental autoimmune uveoretinitis in rats (Roberge et al., 1989, J. Immunol. 143:3498-3502), (d) suppress the growth of an IL2R bearing T cell lymphoma in mice (Kozak et al., 1990, J. Immunol. 145:2766-2771) and (e) prevent the development of experimental allergic encephalomyelitis, a T cell mediated disease of the central nervous system, in rats and mice (Beraud et al., 1991, Cell. Immunol. 133:379-389; Rose et al., 1991, J. Neuroimmunol. 32:209-217). However, such immunotoxin still suffers from the same deficiencies outlined above, particularly non-specific toxicity and immunogenicity in the human host.
2.2. APOPTOSIS-INDUCING PROTEINS
The development of multilineage organisms and the maintenance of homeostasis within tissues both require tightly regulated cell death. The ability of an individual cell to execute a suicidal response following a death stimulus varies markedly during its differentiation. Both positive and negative regulators of programmed cell death (apoptosis) have been identified.
A high percentage of follicular lymphomas have a characteristic chromosomal translocation, which places the proto-oncogene, Bcl-2 next to the immunoglobulin heavy chain locus, resulting in deregulation of Bcl-2 expression. Bcl-2 was found to function as a repressor of programmed cell death (Vaux et al., 1988, Nature 334:440-442). Recently, other Bcl-2 homologues were shown to inhibit apoptosis. However, one such homologue, Bax, mediates an opposite effect by accelerating apoptosis. An expanding family of Bcl-2 related proteins has recently been noted to share homology that is principally, but not exclusively, clustered within two conserved regions known as Bcl-2 homology domains 1 and 2 (BH1 and BH2) (Oltvai et al., 1993, Cell 74:609-619; Boise et al., 1993, Cell 74:597-608; Kozopas et al., 1993, Proc. Natl. Acad. Sci. U.S.A. 90:3516-3520; Lin et al., 1993, J. Immunol. 151:1979-1988). Members of the Bcl family include Bax, Bcl-X
L
, Mcl-1, A1
Aqeilan Rami Ishaq
Azar Yehudith
Belostotsky Ruth
Ben-Yehudah Ahmi
Lorberboum-Galski Haya
Duffy Patricia A.
Pennie & Edmonds LLP
Yissum Research Development Company of the Hebrew University of
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