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Title:  Chimeric toxins

United States Patent:  6,545,126

Issued:  April 8, 2003

Inventors:  Johnson; Eric A. (Madison, WI); Goodnough; Michael C. (Stoughton, WI); Malizio; Carl J. (Madison, WI); Scott; Alan B. (San Francisco, CA)

Assignee:  Wisconsin Alumni Research Foundation (Madison, WI)

Appl. No.:  524841

Filed:  March 14, 2000

Abstract

A chimeric toxin is disclosed. In a preferred embodiment, the chimeric toxin comprises a botulinal neurotoxin heavy chain and non-clostridial toxin chain. A method of creating a chimeric toxin is also disclosed. The chimeric toxin will have utility for pharmacological treatment of neurological disorders.

DETAILED DESCRIPTION OF THE INVENTION

Although botulinum toxin has long been known to be useful in ophthalmic and neurologic clinical practice for therapy of involuntary muscle movements, pain, spasticity and other neuralogic disorders, there are certain drawbacks to its use. One of the most serious drawbacks is a patient's development of only temporary relief and subsequent requirement for periodic injections. The present invention seeks to address some of the drawbacks by providing chimeric botulinal toxins in which the heavy chain of botulinum toxin is conjugated to toxins that lead to neuronal cell death, thereby increasing duration of action.

In practice, injection of a chimeric toxin of the present invention will result in binding to the nerve and incorporation of a lethal toxin into the nerve. Preferably, the nerve cell will be killed. The primary advantage over existing botulinum toxin therapy is increase in the duration of action.

In one embodiment, the present invention is a chimeric toxin comprising a botulinum toxin heavy chain covalently connected to a non-clostridial toxin. In a preferred form of the invention, the chimeric toxin comprises the heavy chain of botulinum toxin type A covalently connected with ricin A chain via the reducible linker.

Typically, the chimeric toxin of the present invention could be produced by molecular biology techniques wherein the enzymatic (non-botulinal) toxin is encoded by DNA that is placed upstream of the codons encoding the botulinal heavy chain on a high copy number plasmid, the expression of which is under the control of an inducible promoter. There may be some difficulties in making these constructs in E. coli in a form in which the expressed produced is biologically active. One difficulty we have encountered is that the gene for type A heavy chain is very A+T rich (up to 90% is some regions) making it difficult to express the gene in organisms such as E. coli, which typically have a much lower A+T content (40-50%). One reason that the high A+T gene is not expressed in E. coli is that the tRNAs responsible for coding the amino acids such as Ile and Leu (high A+T content in the bot gene) are very rare in E. coli. Consequently, when the ribosome goes along the mRNA and encounters one or more of these rare codons (the ribosome stops when it can not find any of the correct tRNSs. This allows the ribosome enough time to dissociate from the mRNA and results in truncated or abbreviated proteins as opposed to full-length transcripts. There is enough flexibility or wobble in the genetic code that the high A+T codons are not used much in E. coli which is what allows it to survive. We propose solving this problem by using a non-toxigenic derivative of C. botulinum that has had the entire toxin gene cluster deleted.

Suitable Botulinum Toxins

We envision that heavy chains isolated from many different botulinum toxins would be suitable for the present invention. The botulinum heavy chain is responsible for targeting and internalization of botulinum toxin light chain into peripheral nerves. Preferably, the heavy chain is isolated from botulinum toxin type A. However, heavy chains isolated from any of the toxins listed in Table 2, above, would be suitable.

One would choose the appropriate heavy chain by the a variety of criteria. One important criteria is ease of purification, and that is why we have chosen the botulinum toxin type A heavy chain. However, other heavy chains may give the chimeric toxin advantageous properties.


Suitable Non-Clostridial Toxins

Preferred Non-clostridial toxins include:

i. ADP-ribosylating toxins, such as brefeldin (Eupenicillium brefeldianum), cholera toxin (Vibrio cholerae), diphtheria toxin (Corynebacterium diphtheriae), pertussis toxin (Bordetella pertussis), and other toxins in this family.

ii. Neurotoxins, such as agatoxin (Agelenopsis aperta), agitoxin (Leiurus quinquestriatus herbraeus), apamin (bee venom), brevetoxin (Plychodiscus brevis), alpha-bungarotoxin, beta-bungarotoxin (Bungarus multicinctus), calcicludine (Dendroaspis angusticeps), cardiotoxins I-IV (Naja naja atra), charybdotoxin agitoxin (Leiurus quinquestriatus herbraeus), cobra venoms (Naja naja), conotoxin (Conus geographus and Conus striatus), crotoxin (Crotalus durissus terrificus), dendrotoxin (Dendroaspis angusticeps), Iberiotoxin (Buthus tamulus), Kailotoxin (Androctonus mauretanicus), Latrotoxin (Latrodectus tredecimguttatus), Maitotoxin (Gambierdiscus toxicus), Myotoxin (Crotalus viridis viridis), Neosaxitoxin (Gymnodiunium catenatum), Notexin (Notechis scutatus), Okadaic acid (Porocentrum concavum), Palytoxin (Palythoa caribaeorum), Picrotoxin (Anamirta cocculin), Resiniferatoxin (Euphorbia poisonii), Saxitoxin (Gonyaulax sps.), Stichodactyla toxin (Stichodactyla helianthus), and Tetrodotoxin (Fugu sps.).

iii. Adenylate cyclase activators, such as forkolin (Coleus forskohlii).

iv. Pore forming toxins such as streptolysin O, Staphylococcal alpha-toxin, Pneumolysin, E. coli hemolysin, aerolysin.

v. Ribosome inactivating proteins (RIPS), including both type I and type II ribosome inactivating proteins. RIPS existing as single chain proteins or glycoproteins are classified as type I RIPS while those that exist as dichain proteins consisting of an A chain have enzymatic activity and a B chain having cell binding properties (particularly those cells showing carbohydrate residues on their surface) are designated type II RIPS. A partial list of some of the RIPS found in nature follows.

                           Plant RIPS.
          Type I                       Type II
          Pokeweed antiviral proteins  Ricin
          Tritin                       Abrin
          Gelonin                      Modecin
          Momordin                     Viscumin
          Saporin                      Volkensin
          Dianthin
          Maize RIP
                         Bacterial RIPS.
          Shiga toxin                  (Shigella dysenteriae)
          Shiga like toxin             (certain E. coli strains)
                           Fungal RIPS.
          alpha-sarcin                 (Aspergillus giganteus)
          mitogillin                   (Aspergillus restrictus)
          restrictocin                 (Aspergillus restrictus)



The purpose of the non-clostridial toxin is neuronal cell death. Therefore, we envision that a variety of toxins would be suitable. We especially envision toxins that would give an increase in duration of therapeutic effect.

A preferred toxin of the present invention is the ricin A chain. Ricin consists of a dichain structure comprising an A chain of 30-32 kD covalently linked to a B chain of 34 kD via a disulfide bond. Following binding to susceptible cells by the B chain of ricin, the A chain is internalized into the cytosol where it irreversibly inactivates the mammalian 28S ribosome by cleaving a single N-glycosidic bond between adenine 4324 and the ribose-phosphate backbone.

Suitable Linkers

In a preferable form of the present invention, the two toxin chains are connected by a covalent bond. Therefore, after one has obtained both the botulinal and the non-clostridial toxins, one must then link the botulinum and non-clostridial toxins together with retention of biological activity. .

Our general idea was to target the sulfhydryl group of the botulinal heavy chain originally involved in the disulfide linkage with the botulinal light chain. By chemically blocking the free sulfhydryls on the neurotoxin prior to separation of the two chains, a single reactive sulfhydryl remained on the heavy chain following chain separation. This avoids mixed disulfide linkage and formation of chimeric constructs with no or very low biological activity.

Conjugation reagents contain at least two reactive groups. Homobifunctional cross-linkers contain two or more identical leaving groups while heterobifunctional cross-linkers contain two or more different leaving groups. Linkers that are reactive with sulfhydryl groups on proteins may do so by generating a reducible disulfide linkage or by generating a non-reducible thioether bond. Common reducing agents for reduction of disulfide bonds including those generated with reducible linkers include dithiothreitol, mercaptoethanol, and reduced glutathione. These agents react with disulfide bonds generating two free sulfhydryl groups per original disulfide bond.

We envision that the non-clostridial toxin will be attached via a disulfide bond. For example, ricin A chain has a single free sulfhydryl which made the use of homobifunctional linkers that are reactive with free thiols the logical choice for specific conjugation of the heterologous chains.

Claim 1 of 5 Claims

We claim:

1. A chimeric toxin comprising

(a) a botulinal neurotoxin heavy chain; and

(b) a non-clostridial toxin chain, wherein the chains are covalently connected with a covalent bond, the covalent bond comprises a reducible disulfide linker, and the toxicity of the toxin is at least 3.3x104 mouse intraperitoneal LD50 /mg of protein.

 


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If you want to learn more about this patent, please go directly to the U.S. Patent and Trademark Office Web site to access the full patent.

 

 

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