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
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
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
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.
Type I Type II
Pokeweed antiviral proteins Ricin
Shiga toxin (Shigella dysenteriae)
Shiga like toxin (certain E. coli strains)
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
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
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
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
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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