Title:  Hybrid protein for inhibiting the degranulation of mastocytes and the use thereof

United States Patent:  6,822,076

Issued:  November 23, 2004

Inventors:  Bigalke; Hans (Hannover, DE); Frevert; Jurgen (Berlin, DE)

Assignee:  BioteCon Therapeutics GmbH (Potsdam, DE)

Appl. No.:  064903

Filed:  August 27, 2002

Abstract

A hybrid protein contains a protein that binds to a receptor of mastocytes and basophils and is endocyted by them. The protein can be IgE; IgE fragment; IgE Fc fragment; antibody against IgE receptor of mastocytes and basophils; fragment of the antibody against the IgE receptor of mastocytes and basophils; antibody against mastocyte specific potassium channel; and mast cell degranulating peptide. The hybrid protein also contains a protease cleaving proteins of the secretion process of the mastocytes and basophils so as to inhibit the secretion process without killing the mastocytes and basophils. The protease can be light chain Clostridium botulinum toxin; proteolytically active fragment of the light chain of a Clostridium botulinum toxin containing an amino acid sequence His-Xaa-Xaa-Xaa-His-Xaa-Xaa-His wherein Xaa is an amino acid; light chain of the tetanus toxin; proteolytically active fragment of the light chain of the tetanus toxin containing His-Asp-Leu-lIe-His-Val-Leu-His; IgA protease of Neisseria gonorrhoeae; and proteolytic domain of the IgA protease of Neisseria gonorrhoeae.

Description of the Invention

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 o 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 Clostridium botulinum toxin, in particular a toxin of the type A, B, C1, D, E, F, end G, characterized by containing the sequence SEQ ID NO:1 His-Xaa-Xaa-Xaa-His-Xaa-Xaa-His, where Xaa can be any amino acid (for example, Xaa can be Leu, Ile, Val, Arg, Met);

light chain of the tetanus toxin (TeNT);

proteolytically active fragment of the light chain of the tetanus toxin, characterized by containing the sequence SEQ ID NO:2 His-Asp-Leu-lIe-His-Val-Leu-His;

IgA protease of Neisseria gonorrhoeae.

The hybrid protein of the present invention is characterized in that the protein (i) and the protease (ii) are selected from the previous groups of proteins and proteases, respectively.

In the sequence of SEQ ID NO:1 His-Xaa--Xaa-Xaa-His-Xaa-Xaa-His of the proteolytically active fragment of the light chain of a Clostridium botulinum toxin, the first Xaa is preferably Asp or AIa; the second Xaa is preferably Leu or lIe; the third Xaa is preferably lIe, Asn or Tyr and the fourth and fifth Xaa are preferably an amino acid with a non-polar functional group, i.e., amino acids such as Val, Leu, lIe, or AIa, respectively.

The hybrid protein of the present invention is additionally characterized in that the N-terminal portion of the heavy chain of the respective toxin (H_N fragment) or a fragment thereof is part of the hybrid protein, in addition to the light chain of a Clostridium botulinum toxin or of the tetanus toxin.

Finally, one embodiment of the present invention relates to the use of the present hybrid protein to inhibit the degranulation of mastocytes.

If mastocytes were killed, there would exist the danger that an allergic shock would be induced once the dying mastocytes release the stored endogenous amines. Additionally, the drop of the number of mastocytes would stimulate the de novo synthesis of these cells which, in turn, would be available again for allergic reactions. The hybrid protein of the present invention is thus fundamentally different from the IgE pseudomonas exotoxin conjugate inhibiting protein synthesis by its ADP ribosylation activity and thus effecting cell death (Fishman & Lorberboum 1997). Quite conversely, the hybrid protein of the present invention does not serve to kill mastocytes. Rather, the cells remain vital after having been subjected to the hybrid protein of the present invention and have lost no more than their capacity to release vasoconstrictive amines.

A stimulation of the de novo synthesis does not occur. When therapeutically used, conceivable toxic side effects to be expected with a conjugate based on the complete cytotoxic pseudomonas toxin or a comparable cytotoxin are avoided.

Subject matter of the invention is thus a conjugate (hybrid protein) consisting of (i) a protein or peptide (transport protein/peptide) exhibiting a high affinity to mastocytes/basophils and (ii) a specific protease, which conjugate blocks the degranulation and the secretory mechanism, respectively, of the cells. The conjugate is useful for the therapy/prophylaxis of allergic reactions of the immediate type.

(i) Preferred high-affinity mastocyte binding components of the conjugates are immunoglobulins of type E (IgE) and its fragments (e.g., the Fc fragment) respectively. Additionally, antibodies against specific surface molecules of mastocytes/basophils are used, which antibodies selectively bind to the plasma membrane of these cells. Above all, antibodies against the IgE receptor fulfill this purpose. Furthermore, inactive but binding mutants of the mast cell degranulating peptide are to be used as transport peptides/proteins in the hybrid protein. These transport peptides/proteins are useful to channel a protease into the cells. This protease cleaves proteins in the fusion complex of mastocytes in a highly specific manner, which proteins initiate the degranulation mechanism of the cells.

(ii) Useful as a highly specific protease is a metallo-protease, e.g., the light chain of botulinum toxin of type A, B, C1, D, E, F, or G (BoNT/X) and of the tetanus toxin (TeNT) or the IgA protease of Neisseria gonorrhoeae. These proteases cleave the synaptosomal associated protein (M_R 25,000) (SNAP 25), synaptobrevin or syntaxin. If only one of these proteins/peptides is cleaved, the degranulation of the mastocytes is inhibited. As a result, no secretion of histamine, prostaglandins, and leukotrienes will occur, and allergic symptoms cannot occur anymore.

In the present invention the nontoxic light chains of the toxins can be attached to transport proteins exclusively binding to mastocytes and basophils, respectively, and thus, can be taken up only by these cells, wherein the light chains, as if carried along as a passenger, reach the cells. They cannot invade nerve cells and cells of other type of the organism such that the effect is limited to mastocytes and basophils. If one of the substrates is proteolytically destroyed, no allergic symptoms occur subsequent to the contact of these IgE loaded cells with an allergen or with one of the above mentioned pharmaceuticals.

Useful as proteins specifically binding to mastocytes are

1) immunoglobulins of type E and their fragments of the type Fc;

2) antibodies against the IgE receptor;

3) the mast cell degranulating peptide; and

4) an antibody against the mastocyte specific potassium channel.

In regard to the protein listed, reference is made to the following publications:

IgE: Helman (1995)

IgE fragment: Helman (1995)

Antibody against IgE receptor of mastocytes/basophils, antibodies against mastocyte specific potassium channel, Fab fragment of the antibody: these are standard procedures described in: Liddel & Weeks (1995)

MCD peptides: Gmachel & Krell (1995)

Inactive but binding mutant: The mutated peptide is prepared according to standard procedures: Nichol D. S. T. (1995)

Light chains of the various botulinum toxins of type A-G: Binz et al. (1990)

Light chain of tetanus toxin: Eisel et al. (1989)

IgA protease: Bruscheck et al. (1995)

The connection of both components (transport protein and protease) occurs via different routes: First, the light chain of the toxin is chromatographically purified. The light chain is entirely nontoxic because, after its separation from the heavy chain, the neurotropic transport protein, it cannot reach the nerve cells and an extracellular substrate does not exist. The light chain is then chemically bound to one of the four mastocyte binding proteins to form a conjugate which, in turn, is taken up (endocyted) in the cytosol of mastocytes. The light chain cleaves its substrate there, which cleavage inhibits the secretion of histamine and other substances. A second way to prepare the conjugate is to fuse the gene for the light chain and the gene for one of the four mastocyte binding proteins such that a hybrid protein is expressed in suitable host cells. This biotechnologically produced hybrid protein should block the secretory process from mastocytes in analogy to the conjugate prepared from two protein components.

The preparation of hybrid proteins is a procedure known in the art, in particular in the field of tumor therapy (Vogel, 1987; Magerstadt, 1991). In this therapeutic concept an antibody against surface proteins of the tumor cells are attached to a cytotoxic protein, e.g., ricin, diphtheria toxin, to kill cancer cells. The novel aspect in the method of the present invention is the use of specific proteases and proteolytic domains, respectively, in hybrid proteins for the inhibition of the degranulation of mastocytes and, thus, for an anti allergen therapy. These hybrid proteins were not only useful to avoid heavily impairing allergic symptoms (hay fever, asthma, and neurodermitis). They could be administered also prophylactically to avoid allergic reactions during therapies with life saving pharmaceuticals. Moreover, they could avoid allergic symptoms occurring in the course of desensitization.

Claim 1 of 11 Claims

What is claimed is:

1. A hybrid protein comprising:

(i) protein capable of binding to a receptor at least one cell type selected from the group consisting of mastocytes and basophils and of being endocyted by the at least one cell type selected from the group consisting of the mastocytes and basophils:

(ii) a protease capable of cleaning one or more secreted proteins of the at least one cell type selected from the group consisting of the mastocytes and basophils so as to inhibit the secretion process without killing the at least one cell type selected from the group consisting of the mastocytes and basophils, wherein the protease (ii) is selected from the group consisting of:

light chain of a Clostridium botulinum neurotoxin;

proteolytically active fragment of the light chain of a Clostridium botulinum neurotoxin containing an amino acid sequence of SEQ ID NO:1 His-Xaa-Xaa-Xaa-His-Xaa-Xaa-His, wherein Xaa is any amino acid.

light chain of the tetanus toxin (TeNT);

proteolytically active fragment of the light chain of the tetanus toxin containing an amino acid sequence of SEQ ID NO:2 His-Asp-Leu-lIe-His-Val-Leu-His;

IgA protease of Neisseria gonorrhoeae; and

proteolytic domain of the IgA protease of Neisseria gonorrhoeae.

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