Drug – bio-affecting and body treating compositions – Designated organic active ingredient containing – Peptide containing doai
Reexamination Certificate
2000-06-16
2004-07-06
Nguyen, Dave T. (Department: 1635)
Drug, bio-affecting and body treating compositions
Designated organic active ingredient containing
Peptide containing doai
C530S300000, C424S184100, C424S185100
Reexamination Certificate
active
06759385
ABSTRACT:
BACKGROUND OF THE INVENTION
Ideal treatments for a pathological condition or disease caused by an undesirable immune response would specifically affect antigen-specific T and B cells. Antigen specific tolerization of T cells can be obtained by delivery of the antigen through routes, such as oral, intraperitoneal and nasal administration, that downregulate, rather than activate, CD4+ responses (Matzinger, 1994; Nossal, 1995). Tolerization of T cells by those routes has proven effective for the prevention and/or treatment of CD4+ T cell mediated autoimmune diseases, e.g., experimental autoimmune encephalomyelitis (EAE) (Metzler et al., 1993; Miller et al., 1994; Genain et al., 1996; Al-Sabbagh et al., 1996), collagen-induced arthritis (Al-Sabbagh et al., 1996), and experimental uveitis (Dick et al., 1993). Moreover, the administration of the antigen by these methods reduced or inhibited the immune response specific for the particular antigen administered. For example, aerosol administration of myelin basic protein (MBP) to MBP-immunized rats that had developed relapsing EAE decreased the intensity of the immune response to MBP and the severity of the attacks (Al-Sabbagh et al., 1996). Spleen T cells from rats that had inhaled MBP transferred protection to naive animals (Al-Sabbagh et al., 1996).
It is unclear whether similar approaches could be used for antibody (Ab)-mediated diseases for two reasons. First, while effective at reducing antigen-specific CD4+ responses, administration of antigen through routes that downregulate CD4+ responses may directly stimulate B cells specific for the administered antigen (Kuper et al., 1992; Liu et al., 1993; Husby et al., 1994; Neutra et al., 1996). This stimulation may have disastrous consequences, as has been shown in marmoset EAE (Genain et al., 1996), where intraperitoneal administration of myelin resulted in CD4+ tolerance to myelin, but also in an acute, fatal form of EAE. The fatal form of EAE was characterized by antibody specific for the myelin oligodendrocyte glycoprotein. Second, administration of antigen through routes that stimulate Th2 cells and downregulate pro-inflammatory Thi cells can stimulate antibody synthesis (Neutra et al., 1996; Abbas et al., 1996), and cause exacerbation rather than improvement of antibody-mediated autoimmune diseases.
Short T epitope sequences may be safer for inducing T cell tolerance than the whole antigen molecule, since peptide-specific antibodies very seldom crossreact with the cognate native antigen (Conti-Fine et al., 1996). Peptides have been used with dubious success for oral tolerization in EAE (Karpus et al., 1996; Metzler et al., 1993), although peptides are not ideal for oral tolerization because they are easily digested by gastrointestinal proteases.
Thus, there is a need for an improved method to treat or inhibit antibody-mediated diseases.
SUMMARY OF THE INVENTION
The present invention provides a therapeutic method comprising the administration of an “epitope” peptide comprising a universal and/or immunodominant epitope sequence derived from a particular antigen that is associated with an antibody-mediated disease in a mammal. The method is effective to specifically tolerize, or down regulate the priming and/or activity of, the antigen-specific T cells of said mammal. The sequence of the epitope peptide does not include the entire sequence of the antigen from which it is derived.
Many autoimmune diseases and other pathological conditions are directly caused by antibodies. Such antibodies are directed against proteins or other antigenic components of the host in diseases such as autoimmune diseases, or against exogenous substances in, for example, allergic diseases. The antibodies may also be directed against therapeutic agents, i.e., proteins or other antigenic substances given to the host for therapeutic purposes, such as the administration of factor VIII to treat bleeding in hemophilia A patients. These therapeutic agents may be administered exogenously, or may be synthesized by the host as a result of gene therapy.
Antibody synthesis is controlled by T cells. In mammals there are limited sets of epitopes for each antigen that dominate the T cell response, referred to as immunodominant T cell epitope sequences (hereinafter “immunodominant epitope sequences”). Moreover, in humans, CD4+ cells recognize universal, immunodominant epitope sequences. As T cell epitopes may comprise as few as 7 amino acid residues corresponding to an amino acid sequence present in a particular antigen, peptides having at least about 7 amino acid residues may be useful to tolerize, or down regulate the priming and/or activity of, T cells (e.g., CD4+ cells) specific for the peptide and its corresponding antigen. Thus, immunodominant and/or universal epitope peptides may be administered so as to regulate a mammal's T cell and antibody response.
To determine whether the delivery of a given peptide is useful to inhibit or treat a particular indication or disease in humans, the immunodominant and/or universal epitopes for a relevant antigen are identified. These epitopes are then identified, synthesized and administered to non-human mammals, preferably ones that are models for a particular human indication or disease, to determine whether the epitope peptide is useful to down regulate the T cell and antibody response to a particular antigen. For example, rodents immunized with Torpedo fish ACHR (TAChR) and, thus, susceptible to experimental myasthenia gravis (EMG) are useful to determine whether the administration of acetylcholine receptor (AChR)-derived epitope peptides can result in T cell tolerization. As described hereinbelow, EMG was induced in C57B1/6 (B6) mice by immunization with purified TAChR. The immunized animals have sensitized CD4+ and B cells, and produce high affinity IgG antibodies which cross-react with mouse muscle ACHR. The immunized B6 mice have anti-TAChR CD4+ T cells that recognize primarily epitopes within residues 146-169, 181-200 and 360-378 of the TAChR &agr; subunit. Surprisingly, nasal administration of synthetic sequences of the TAChR &agr; subunit representing epitopes recognized by anti-TAChR CD4+ T helper cells, given before and during immunization with TAChR, resulted in 1) decreased CD4+ responsiveness to those epitopes and to TAChR; 2) reduced synthesis of anti-TAChR antibodies; and 3) an absence of EMG.
In contrast to B6 mice, nasal administration of synthetic ACHR CD4
+
epitopes did not prevent EMG in IL-4 knock out (KO) mice (Example III). Thus, the protective effects of nasal tolerization require presence of Th2 cells, although this procedure also results in the deletion of Th1 cells specific for the administered epitopes.
Moreover, the results obtained with nasal administration of AChR CD4+ epitopes were confirmed using another route of administration, i.e., subcutaneous administration (Example IV). Subcutaneous administration to B6 mice of a pool of solutions of synthetic TAChR CD4
+
epitopes (&agr;150-169, &agr;181-200 and &agr;360-378), or of peptide &agr;150-169 alone, given before and during immunization with TAChR, strongly reduced the synthesis of anti-TAChR Ab and prevented EMG. The peptide treatment reduced the CD4
+
responses in vitro to the administered peptide epitopes, not to the TAChR molecule.
Secretion of cytokines by spleen CD4
+
cells from TAChR-immunized mice, challenged with TAChR in vitro, indicated that in sham-tolerized mice only Th1 cells responded to the TAChR, while in peptide-treated mice the CD4
+
cells that responded to TAChR were, or included, Th2 cells. Peptide-treated mice made anti-peptide Ab, that included a large fraction of Th2-driven IgG1. The subcutaneous treatment had some inhibitory effect on anti-AChR Th2 cells, since the serum anti-TAChR Ab IgG1 were modestly reduced after large doses of tolerizing peptide(s).
To determine directly whether CD4
+
cells have an important pathogenic role in antibody-mediated autoimmune diseases, e.
Angell Jon Eric
Nguyen Dave T.
Regents of the University of Minnesota
Schwegman Lundberg Woessner & Kluth P.A.
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