Chemistry: natural resins or derivatives; peptides or proteins; – Proteins – i.e. – more than 100 amino acid residues
Reexamination Certificate
2002-08-27
2004-11-23
Carlson, Karen Cochrane (Department: 1653)
Chemistry: natural resins or derivatives; peptides or proteins;
Proteins, i.e., more than 100 amino acid residues
C530S350000, C530S300000, C435S007100, C424S192100, C514S002600, C514S012200, C514S021800
Reexamination Certificate
active
06822076
ABSTRACT:
BACKGROUND OF INVENTION
Allergic reactions of the immediate type are characterized in that the patients concerned have formed antibodies of the IgE type against allergens (for example, pollen, house dust, mites, animal hair). These antibodies circulate not only in the blood but also bind to cells present in the tissue exhibiting in the plasma membrane a specific receptor for a portion of the IgE molecule, the Fc fragment (Fishman & Lorberboum-Galski 1997; Hamawy 1997). Cells with the IgE receptors are mastocytes and basophils exclusively. These cells are the cells effecting the allergic reaction of the immediate type. The stored vesicles containing vasoactive amines and prostaglandins, leukotrienes (derivatives of the arachidonic acid), and other effector molecules such as chymase (=effector molecules of the allergic reaction). The secretion process causing the release of these substances and resulting in the degranulation of the mastocytes, occurs through a specific and an unspecific mechanism. Once cells are mechanically destroyed, e.g., by a scratch on the skin, histamine is unspecifically released. At the wound the skin turns red. Nettles (edemas) are formed and the skin itches (triple response). Substances releasing specifically histamine are effective in relatively low concentrations and trigger the following cascade of responses (signal cascade): activation of phospholipase C—formation of the second messengers “diacylglycerol” and “IP3”—mobilization of calcium from cellular depots—fusion of the granules (vesicles) with the cell membrane—exocytosis of the granules without cytolysis—exchange of sodium against the positively charged histamine of the complex with heparin and a basic protein—release of the histamine from the granule matrix.
Provided there is contact between the mastocytes of an allergic person and an allergen, the IgE molecules on the cell surface bind this allergen. Once allergen molecules are bound in sufficient amounts, aggregation of the receptors in the plasma membrane occurs. The aggregation is the specific stimulus for the induction of the above described signal cascade in the interior of the cell. The substances released induce the allergic symptoms (conjunctivitis, rhinitis, asthma, laryngeal edema, urticaria, blood pressure drop up to a pronounced anaphylactic shock). Peptides contained in the toxin of the bee such as the mast cell degranulating peptide (MCD) also effect a degranulation of the mastocytes. Additionally, some pharmaceuticals cause a specific release of histamine as an undesired effect. The release of histamine in humans is described for muscle relaxing agents, dextrans, acetylsalicylic acid (aspirin), morphine, antibiotics, contrast media in radiography, foreign sera etc.
If in the secretion process the fusion of the vesicles with the plasma membrane is successfully inhibited, then there is no release of the amines and arachidonic acid derivatives. Consequently, no allergic reactions are induced. Several proteins (fusion proteins) are involved in the secretion process and the release, respectively, which proteins may be bound to membranes of secretory vesicles and/or to the plasma membrane. Likewise, they may appear in the cytosol. Representatives of these proteins are SNAP 25, synaptobrevin (V AMP), syntaxins and its isoforms, respectively. These proteins form a complex (fusion complex) fixating the secretory vesicles to the inner side of the plasma membrane. The fixation precedes the fusion of the vesicles with the plasma membrane and subsequent release of histamine and other effector molecules. By inactivation of one of these proteins, for example, by proteolytic cleavage, the formation of the complex is inhibited and the secretion process interrupted. As a consequence, the mast cells cannot release anymore the content of the vesicles (amines, arachidonic acid, arachidonic acid derivatives, etc.).
From nerve cells it is known that the fusion proteins (SNAP 25, synaptobrevin and syntaxin) mentioned are the target molecules (substrates) of the light chains of the neurotoxins produced by the bacterium
Clostridium botulinum
in the nerve cells (Ahnert& Bigalke, 1995; Bigalke 2000). At present, seven different types of
botulinum
toxins are known (A, B, C1, D, E, F, and G). The synaptobrevin mentioned additionally is a target molecule for TeNT (Link et al., 1993) produced by
Clostridium tetani
, and also for a protease from
Neisseria gonorrhoeae
(Binscheck et al., 1995). The toxins, apart from the latter, consist of at least two functional domains. The C terminal portion of the protein (heavy chain) is responsible for its binding to the nerve cell whilst the N terminus (light chain) is characterized by the above described highly specific proteolytic activity. The toxins bind to nerve cells via their heavy chain and reach the cytosol via a receptor mediated endocytosis and subsequent translocation, where they cleave one or more of the fusion proteins (SNAP 25, synaptobrevin or syntaxin) which, in turn, are constitutive for the fusion complex. After the cleavage of the respective protein, the secretion of acetylcholine and other transmitters, respectively, from the nerve cells is inhibited (Binscheck and Wellh ö ner,
1997
).
The inhibition of the release of transmitters has been therapeutically used in the past for the treatment of dystonic motor disturbances and for the suppression of excessive parasympathic activities (Benecke and Kessler, 1995). For the
clostridial
neurotoxins biological substrates other than the fusion proteins are not known. The heavy chains have a high affinity for peripheral nerve cells such that the light chains connected to them reach only these cells and become effective only in these cells although other cell types, such as mastocytes and basophils, in which the above described secretion processes occur, possess the above mentioned substrates of these proteases (light chains of the neurotoxins); however, they do not possess a mechanism for the uptake of the protease (Marxen et al., 1989).
To act on the secretory process in mast cells and basophils, it is therefore necessary to substitute a protein, which provides a specific binding to mast cells and basophils, for the heavy chain of the neurotoxin(s).
SUMMARY OF INVENTION
One embodiment of the present invention relates to a hybrid protein, comprising or consisting of
(i) a protein known in the art, said protein binding to mastocytes and/or basophils and/or being taken up (endocyted) by these cells as is known in the art,
(ii) a protease known in the art, said protease cleaving one or several proteins of the secretion process of the mastocytes and/or basophils.
A further embodiment of the present invention relates to a hybrid protein comprising or consisting of
(i) a protein binding to mastocytes and/or basophils and/or being taken up (endocyted) by these cells, wherein the protein (i) is selected from the group consisting of:
IgE;
IgE fragment, in particular, IgE Fc fragment;
antibody against IgE receptor of mastocytes and/or basophils; fragment of the antibody against IgE receptor of mastocytes and/or basophils, in particular Fab fragment; antibody against mastocyte specific potassium channel; and
inactive but binding MCD peptide; and
(ii) a protease, in particular a protease known in the art, cleaving one or several proteins of the secretion process of the mastocytes and/or basophils.
Yet another embodiment of the present invention relates to a hybrid protein comprising or consisting of
(i) a protein, in particular a protein known in the art, said protein binding to mastocytes and/or basophils and/or being taken up (endocyted) by these cells, in particular in a manner known in the art; and
(ii) a protease, said protease cleaving one or several proteins of the secretion process of the mastocytes and/or basophils, wherein the protease (ii) is selected from the group consisting of:
light chain of a
Clostridium botulinum
toxin, in particular, the toxins of type A, B, C1, D, E, F, and G;
proteolytically active fragment of the light chain of a
C
Bigalke Hans
Frevert Jürgen
BioteCon Therapeutics GmbH
Carlson Karen Cochrane
Huckett Gudrun E.
Robinson Hope A.
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