Patent Application: US-6490302-A

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:
the invention will now be described in detail with the aid of specific non - limiting examples . synthesis of a hybrid protein from ige and the light chain of bont / a the purified botulinum toxin ( 5 . 0 mg ) of type a was applied , after equilibration in 15 mm sodium tetraborate and 30 mm phosphate ph 8 . 4 , to a qae sephadex column ( 1 . 0 × 3 . 0 cm ), equilibrated with the same buffer . the column was subsequently washed with 10 ml 120 mm dithioerythrol , 2 m urea and 1 mm edta and incubated over night . thereafter , the light chain was eluted from the column by means of 10 mm borate buffer and dialyzed against 20 mm phosphate ph 7 . 0 . immunoglobulin e ( rat ) was purchased . 10 mg of the immunoglobulin were cleaved with 50 μg papain in 1 ml phosphate buffer ( 4 degrees c . over night ). the fc fragment was purified over a gel filtration column ( sephacryl s200 ). 3 . 0 mg of the purified fc fragment were incubated with 3 . 0 mg of a purified light chain of botulinum toxin with 10 mm dithio bis succinimidyl propionate ( bifunctional agent ) in 2 ml na phosphate , ph 7 . 0 , over a period of 16 hours . the hybrid protein thus synthesized was purified via gel filtration ( sephacryl s200 ) and analyzed for its purity via sds gel electrophoresis . the inhibition of the degranulation of the mastocytes is examined in two experimental approaches . in the first approach isolated mastocytes of the rat are incubated with the hybrid molecule . thereafter the release of histamine is stimulated . the stimulation occurs with specific histamine liberators such as the mcd peptide and concanavalin a ( the latter being an experimentally utilized substance ) and by a direct increase of the intracellular calcium concentration , respectively . the latter is achieved by an injection of calcium into distinct mastocytes . thus , one shortcircuits the above described signal cascade as the increase of the calcium concentration is the step during the secretion process which is followed by the fusion of the vesicles . the degranulation of the mastocyte reflecting the release of histamine is followed in the phasecontrast microscope . subsequently , it is possible to quantify released histamine by means of a measurement of the fluorescence . finally , the enlargement of the mastocyte caused by the incorporation of vesicle membranes into the plasma membrane during degranulation can be determined electrophysiologically . in the cells treated with hybrid protein there will , in contrast to control cells , occur ( 1 ) no morphological change , ( 2 ) no enhancement of the fluorescence in the supernatant of the cell , and ( 3 ) no enlargement of the cell . thereby it is possible to prove that the release of histamine is blocked by the hybrid protein . in the second experimental approach the hybrid protein is injected into living rats . the rats are killed after several days and their mastocytes conventionally isolated . the degranulation and release of histamine , respectively , is determined as described above . in this approach it is examined whether the conjugate is able to reach the compartment also in the living animal , in which compartment the mastocytes are located in , and whether the conjugate inactivates the mastocyte in the living animal . production of a recombinant hybrid protein by operably linking the gene encoding the light chain of clostridium botulinum type a to the gene encoding immunoglobulin e and one of its fragments ( fc fragment ), respectively the gene encoding the light chain of botulinum toxin type a is isolated by means of suitable primers via pcr ( polymerase chain reaction ). a culture of clostridium botulinum type a is prepared from which the dna is prepared . from the published sequence of the toxin gene ( binz et al ., 1990 ) a pair of primers is derived and the gene for the light subunit amplified via pcr . thereafter this gene is cloned into a commercial expression vector pqe according to the recipe of the producer . the gene encoding the fc fragment of the human immunoglobulin e ( helman , 1995 l .) was isolated via pcr from a commercial cdna library and fused in the vector construct with the gene light chain of botulinum toxin type a . with this construct competent m15 cells ( e . coli ) are transformed . as in this expression system the inserted genes are equipped with a “ his tag ”. the recombinant protein is purified through affinity chromatography over a ni nta column ( quiagen ). the process of highly purifying the protein is followed by a gel filtration through sephacryl s300 . the measurement of the biological activity was performed again on isolated mastocytes in vitro . preparation of a recombinant hybrid protein by operably linking the gene encoding the light subunit of the tetanus toxin with a mutated gene encoding the mast cell degranulating peptide ( mcd ) the “ sequence for the mast cell degranulating peptide ”, a 22 mer , is known ( gmachl and kreil , 1995 ). based thereon a corresponding oligonucleotide is synthesized . in order to isolate the sequence of the light subunit of the tetanus toxin a culture of c . tetani was prepared and dna recovered therefrom . from the known nucleic acid sequence of the tetanus toxin a primer for pcr and hence the gene for the light subunit of the toxin was obtained . as described in example 1 , both nucleic acid sequences were fused in an expression vector pqu and subsequently expressed in e . coli . the hybrid protein which , in turn , was equipped with a “ his tag ” was purified through affinity chromatography and subsequent gel filtration . the purified gene encoding the mast cell degranulating peptide is chemically synthesized including a point mutation in the active domain of the peptide . the gene is operably linked to the gene encoding the light chain of the tetanus toxin . the hybrid protein is expressed in e . coli and purified . the thus produced hybrid protein is tested in vitro in the mastocyte degranulation assay . preparation of a recombinant hybrid protein by linking the gene encoding the fc fragment of ige to the gene encoding the iga protease the gene encoding the fc fragment of ige was isolated as described in example 1 . the gene encoding the iga protease from n . gonorrhoeae is known . primers were derived therefrom , and the gene encoding the specific protease was recovered by means of pcr from a nucleic acid preparation obtained from n . gonorrhoeae . both nucleic acids were integrated into a commercial vector following the recipe giving by the producer and the hybrid protein purified by affinity chromatography ( see example 2 ). the inhibitory activity is again proven in vitro on isolated mastocytes ( see above ). preparation of a hybrid protein consisting of the fab fragment of an antibody against the ige receptor and the light chain of botulinum toxin type b a monoclonal antibody against the ige receptor on mastocytes was purchased and chromatographically repurified . 0 . 5 mg of the antibody were conjugated to 0 . 4 g of the purified light chain of botulinum toxin f . the light subunit was isolated by cleavage of the neurotoxin and subsequent purification through ion exchange chromatography , once the preparation of the neurotoxin had been performed according to the procedure in example 1 . both proteins ( light subunit of toxin type f and monoclonal antibody ) were linked to each other by using a bifunctional agent . the isolated proteins were incubated with 10 mm maleimido benzoyl n hydroxy succinimide ester for this purpose . the hybrid protein was subsequently purified from nonconjugated proteins by gel filtration over sephacryl s300 . again , isolated mastocytes were used to demonstrate that the hybrid protein synthesized inhibited the secretion of histamine . patent - related documents of interest in this connection are : u . s . pat . no . 4 , 902 , 495 ( ige fc directed delivery systems ) and pct application wo 94 / 21300 ( 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