U.S. patent application number 10/886557 was filed with the patent office on 2005-12-01 for immunotoxin (mab-ricin) for the treatment of focal movement disorders.
Invention is credited to Dalakas, Marinos C., Hallett, Mark, Hott, Jonathan S., Youle, Richard J..
Application Number | 20050266010 10/886557 |
Document ID | / |
Family ID | 27363013 |
Filed Date | 2005-12-01 |
United States Patent
Application |
20050266010 |
Kind Code |
A1 |
Hott, Jonathan S. ; et
al. |
December 1, 2005 |
Immunotoxin (mAB-RICIN) for the treatment of focal movement
disorders
Abstract
Compositions and methods for treatment of focal muscle spasms.
Immunotoxin conjugates comprise a toxin conjugated to an antibody
reactive to a muscle specific antigen.
Inventors: |
Hott, Jonathan S.;
(Birmingham, MI) ; Youle, Richard J.; (Bethesda,
MD) ; Hallett, Mark; (Bethesda, MD) ; Dalakas,
Marinos C.; (Bethesda, MD) |
Correspondence
Address: |
BERENATO, WHITE & STAVISH, LLC
6550 ROCK SPRING DR.
SUITE 240
BETHESDA
MD
20817
US
|
Family ID: |
27363013 |
Appl. No.: |
10/886557 |
Filed: |
July 9, 2004 |
Related U.S. Patent Documents
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Application
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Filing Date |
Patent Number |
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10886557 |
Jul 9, 2004 |
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10005512 |
Nov 7, 2001 |
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6780413 |
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10005512 |
Nov 7, 2001 |
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09418854 |
Oct 15, 1999 |
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09418854 |
Oct 15, 1999 |
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08937266 |
Sep 15, 1997 |
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60027458 |
Sep 19, 1996 |
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Current U.S.
Class: |
424/178.1 ;
424/731 |
Current CPC
Class: |
A61K 2039/505 20130101;
A61K 47/6819 20170801; A61K 47/6849 20170801; C07K 16/286 20130101;
A61K 47/6827 20170801; A61K 47/6821 20170801 |
Class at
Publication: |
424/178.1 ;
424/731 |
International
Class: |
A61K 039/395; A61K
035/78 |
Claims
1-11. (canceled)
12. An immunotoxin conjugate, comprising an antibody conjugated to
a cellular toxin selected from the group consisting of: ricin and
abrin, wherein said antibody of said immunotoxin conjugate is
selectively reactive, under immunologically reactive conditions, to
a mammalian nicotinic acetylcholine receptor of a muscle cell of a
muscle of a mammal and whereupon said selective reaction said
cellular toxin of said immunotoxin conjugate mediates the death of
said muscle cell.
13. The immunotoxin of claim 12, wherein the antibody is a
monoclonal antibody.
14. The immunotoxin conjugate of claim 12, wherein said mammalian
acetylcholine receptor is a human acetylcholine receptor.
15. The immunotoxin conjugate of claim 12, wherein said toxin is
ricin.
16. The immunotoxin conjugate of claim 12, wherein said muscle
exhibits a focal muscle spasm selected from the group consisting
of: blepharospasm, cervical dystonia, hand dystonia, limb dystonia,
hemifacial spasm, bruxism, strabismus, VI nerve palsy, spasmodic
dysphonia, and oromandibular dystonia.
17. The immunotoxin conjugate of claim 12, wherein said conjugate
is dissolved in a pharmaceutically acceptable carrier suitable for
intramuscular administration to said mammal.
18. An immunotoxin conjugate, comprising an antibody conjugated to
a galactose binding moiety and a cellular toxin selected from the
group consisting of: ricin-A and abrin-A, wherein said antibody of
said immunotoxin conjugate is selectively reactive, under
immunologically reactive conditions, to a mammalian nicotinic
acetylcholine receptor of a muscle cell of a muscle of a mammal and
whereupon said selective reaction said cellular toxin of said
immunotoxin conjugate mediates the death of said muscle cell.
19. The immunotoxin conjugate of claim 18, wherein said galactose
binding moiety is selected from the group consisting of: ricin-B
and abrin-B.
20. The immunotoxin conjugate of claim 18, wherein said antibody is
a monoclonal antibody.
21. The immunotoxin conjugate of claim 18, wherein said mammalian
acetylcholine receptor is a human acetylcholine receptor.
22. immunotoxin conjugate of claim 18, wherein said toxin is
ricin.
23. The immunotoxin conjugate of claim 18, wherein said muscle
exhibits a focal muscle spasm selected from the group consisting
of: blepharospasm, cervical dystonia, hand dystonia, limb dystonia,
hemifacial spasm, bruxism, strabismus, VI nerve palsy, spasmodic
dysphonia, and oromandibular dystonia.
24. The immunotoxin conjugate of claim 18, wherein said conjugate
is dissolved in a pharmaceutically acceptable carrier suitable for
intramuscular administration to said mammal.
Description
TECHNICAL FIELD
[0001] Compositions comprising a toxin conjugated to an antibody
selectively reactive to a muscle specific antigen. Methods of using
the immunotoxin conjugates for treatment of focal muscle spasms are
also provided.
BACKGROUND OF THE INVENTION
[0002] A variety of neurological disorders are characterized by
disabling, involuntary muscular spasms. The most successful
treatment for focal muscle spasm is intramuscular injection of the
botulinum toxin A (BTX), the only pharmaceutical formulation of
botulinum toxin currently on the market. Intramuscular injection of
BTX weakens the muscles and reduces the symptoms. (Jankovic and
Brin, N. Engl. J. Med., (1991) 324:1186-1194; Stell and Moore,
History and current applications of botulinum toxin treatment. In:
Moore P, ed. Handbook of botulinum toxin treatment. Oxford:
Blackwell Science, Inc., 1995:3-15; Report of the Therapeutics and
Technology Assessment Subcommittee of the American Academy of
Neurology. Assessment: the clinical usefulness of botulinum toxin-a
in treating neurologic disorders. Neurology (1990) 40:1332-1336;
Coffield et al. The site and mechanism of action of botulinum
neurotoxin. In: Jankovic J, Hallett M, eds. Therapy with botulinum
toxin. New York: Marcel Dekker, Inc., (1994) 3-14). However, the
therapeutic effect of BTX is transient and as the BTX paralytic
effects wane, patients usually receive additional injections. For
many patients, repeated exposure to BTX has been accompanied with
decreasing efficacy and duration of benefit. Collateral sprouts of
denervated motor-nerve terminals and 2.5 increasing titers of toxin
neutralizing antibodies are two mechanisms of resistance to BTX
(Coffield et al., supra, Jankovic and Schwartz, Neurology (1995)
45:1743-1746). As a result, larger and more frequent doses of BTX
become necessary for relief of the spasm, increasing the risk of
side-effects. Eventually, some patients become completely
refractory to treatment.
[0003] Accordingly, what is needed in the art is a means to treat
focal muscle disorders with greater specificity and duration of
effect. The present invention provides these and other
advantages.
SUMMARY OF THE INVENTION
[0004] In one aspect the present invention is directed to a method
of treating a focal muscle spasm. The method comprises the steps of
administering, by intramuscular injection, a therapeutically
effective dose of an immunotoxin conjugate to a muscle of the focal
muscle spasm. The immunotoxin conjugate comprises an antibody
conjugated to a toxin selected from the group consisting of: ricin
and abrin, and the antibody is selectively reactive, under
immunologically reactive conditions, to a nicotinic acetylcholine
receptor (nAchR). In preferred embodiments the antibody is a
monoclonal antibody. Typically, the mammalian acetylcholine
receptor is a human acetylcholine receptor. In particularly
preferred embodiments the toxin is ricin. Typically the focal
muscle spasm is selected from the group consisting of:
blepharospasm, cervical dystonia, hand dystonia, limb dystonia,
hemifacial spasm, bruxism, strabismus, VI nerve palsy, spasmodic
dysphonia, and oromandibular dystonia. In other embodiments a
therapeutically effective amount of the immunotoxin conjugate is
administered with a therapeutically effective amount of botulinum
toxin, as an immunoconjugate or in unconjugated form.
[0005] In another aspect the present invention relates to a method
of treating a focal muscle spasm. The method comprises the steps of
administering, by intramuscular injection, a therapeutically
effective dose of an immunotoxin conjugate to a muscle of the focal
muscle spasm. The immunotoxin conjugate comprises an antibody
conjugated to a galactose binding moiety and a toxin selected from
the group consisting of: ricin-A and abrin-A, and the antibody is
selectively reactive, under immunologically reactive conditions, to
a nicotinic acetylcholine receptoc (nAchR). In some embodiments the
galactose binding moiety is selected from the group consisting of:
ricin-B and abrin-B. In preferred embodiments the antibody is a
monoclonal antibody. Typically, the mammalian acetylcholine
receptor is a human acetylcholine receptor. In particularly
preferred embodiments the toxin is ricin. Typically the focal
muscle spasm is selected from the group consisting of:
blepharospasm, cervical dystonia, hand dystonia, limb dystonia,
hemifacial spasm, bruxism, strabismus, VI nerve palsy, spasmodic
dysphonia, and oromandibular dystonia.
[0006] In another aspect the present invention relates to an
immunotoxin conjugate, comprising an antibody conjugated to a toxin
selected from the group consisting of: ricin and abrin, where the
antibody is selectively reactive, under immunologically reactive
conditions, to a mammalian nicotinic acetylcholine receptor. In
preferred embodiments the antibody is a monoclonal antibody.
Typically, the mammalian acetylcholine receptor is a human
acetylcholine receptor. In particularly preferred embodiments the
toxin is ricin.
BRIEF DESCRIPTION OF THE DRAWINGS
[0007] FIG. 1 shows the effects of unilateral infusion of either
BTX at {fraction (1/100)} of the LD.sub.50 or ITX at {fraction
(1/100)} of the maximum tolerated dose (MTD) into the gastrocnemius
muscle of female Sprague-Dawley rats compared to control (PBS)
injected rats on rotorod performance. Data points were recorded as
time spent on the rotorod before the first fall. The average time
on the rotorod for three consecutive runs per rat was used as the
score for each rat. Data points represent the average of the
individual scores by rats within each group. Vertical lines
indicate the standard deviation (n=6).
[0008] FIG. 2 shows the effects of different doses of either BTX or
ITX injected into the gastrocnemius muscle on rotorod performance.
Two control groups were utilized, PBS and unconjugated
anti-nicotinic AchR MoAb 35. The doses of BTX-{fraction (1/10)} and
{fraction (1/100)} of the LD.sub.50-were chosen based on the range
of doses used for treatment of humans. BTX was compared to ITX at
doses {fraction (1/100)} and {fraction (1/300)} of the maximum
tolerated dose (MTD). Standard deviation of the mean was calculated
as described in FIG. 1, except 4 rats were used in each
experimental group.
DETAILED DESCRIPTION OF THE INVENTION
[0009] Intramuscular injection of botulinum toxin A (BTX) is often
considered primary therapy of many disorders characterized by
muscular spasms. The utility of BTX, however, is limited by its
short duration of action, the possible development of resistance
after repeated injections, and cross-reactivity with autonomic
neurons. Surprisingly, we have determined an immunotoxin (ITX)
engineered to damage skeletal muscle fibers selectively by
chemically linking a monoclonal antibody against the nicotinic
acetylcholine receptor to the toxin ricin was 20,000-fold more
toxic to myotubes than myoblasts, consistent with the degree of
acetylcholine receptor expression. In vivo, ITX produced
destructive myopathic changes at a dose 300-fold less than the
maximum tolerated dose. Assessment of rat muscle strength after
unilateral gastrocnemius injections showed ITX was more effective
and had a longer duration of action than BTX. Immunotoxins of the
present invention have utility as a tissue culture selection agent
against cells or tissues expressing nicotinic acetylcholine
receptors (nAchR) Immunotoxins of the present invention also have
utility in the treatment of involuntary muscle spasms. Patients
repeatedly exposed to botulinum toxin for the treatment of muscle
spasms frequently become resistant to its use. Consequently,
surgical treatment is often indicated. Intramuscular injection of
the immunotoxin conjugates of the present invention can delay or
prevent the requirement for surgery.
[0010] Definition
[0011] Units, prefixes, and symbols can be denoted in their SI
accepted form. Numeric ranges are inclusive of the numbers defining
the range. The headings provided herein are not limitations of the
various aspects or embodiments of the invention which can be had by
reference to the specification as a whole. Accordingly, the terms
defined immediately below are more fully defined by reference to
the specification as a whole.
[0012] The terms "immunotoxin conjugate" or "immunotoxin" include
reference to a covalent or non-covalent linkage of a toxin to an
antibody. The toxin may be linked directly to the antibody, or
indirectly through, for example, a linker molecule.
[0013] The term "antibody" includes reference to an immunoglobulin
molecule obtained by in vitro or in vivo generation of the humoral
response, and includes both polyclonal and monoclonal antibodies.
The term also includes genetically engineered forms such as
chimeric antibodies (e.g., humanized murine antibodies),
heteroconjugate antibodies (e.g., bispecific antibodies), and
recombinant single chain Fv fragments (scFv). The term "antibody"
also includes antigen binding forms of antibodies (e.g., Fab',
F(ab').sub.2, Fab, Fv, rigG, and, inverted IgG). An antibody
immunologically reactive with a particular antigen can be generated
in vivo or by recombinant methods such as selection of libraries of
recombinant antibodies in phage or similar vectors. See, e.g., Huse
et al. (1989) Science 246:1275-1281; and Ward, et al. (1989) Nature
341:544-546; and Vaughan et al. (1996) Nature Biotechnology,
14:309-314.
[0014] The term "humanized antibody" includes reference to an
antibody which comprises a non-human amino acid sequence but whose
constant region has been altered to reduce immunogenicity in
humans.
[0015] The term "conjugated" includes reference to a covalent or
non-covalent linkage. The linkage may be direct or indirect via an
intermediary molecule.
[0016] The term "ricin" includes reference to the lectin RCA.sub.60
from Ricinus communis (Castor bean). The term also references toxic
variants thereof. See, U.S. Pat. Nos. 5,079,163 and 4,689,401.
Ricinus communis agglutinin (RCA) occurs in two forms designated
RCA.sub.60 and RCA.sub.120 according to their molecular weights of
approximately 65,000 and 120,000, respectively. Nicholson and
Blaustein, J. Biochim. Biophys. Acta, 266:543 (1972). RCA.sub.60,
also referred to as RCA.sub.II', Ricin D or RCL III is extremely
toxic, inhibits protein synthesis and has an affinity for
N-acetyl-D-galactosamine. The toxin is a dimer of an A-chain
(30,000 Da) and B-chain (33,000 Da) joined by a disulfide bond. The
A chain is responsible for inactivating protein synthesis and
killing cells. The B chain binds ricin to cell-surface galactose
residues and facilitates transport of the A chain into the cytosol
(Olsnes et al., Nature, 1974;249:627-631). See, U.S. Pat. No.
3,060,165.
[0017] The term "abrin" includes reference to the toxic lectins
from Abrus precatorius. The toxic principles, abrin a, b, c, and d,
have a molecular weight of from about 63,000 and 67,000 Da and are
composed of two disulfide-linked polypeptide chains A and B. The A
chain inhibits protein synthesis; the B-chain (abrin-b) binds to
O-galactose residues. See, Funatsu et al., The amino acid sequence
of the A-chain of abrin-a and comparison with ricin, Agr. Biol.
Chem. 52:1095 (1988). See also, Olsnes, Methods Enzymol. 50:330-335
(1978).
[0018] The term "selectively reactive" includes reference to the
preferential association of a ligand, in whole or part, with a cell
or tissue bearing a particular target molecule or marker and not to
cells or tissues lacking that target molecule. It is, of course,
recognized that a certain degree of non-specific interaction may
occur between a molecule and a non-target cell or tissue.
Nevertheless, specific binding, may be distinguished as mediated
through specific recognition of the target molecule. Typically
specific binding results in a much stronger association between the
delivered molecule and cells bearing the target molecule than
between the bound molecule and cells lacking the target molecule.
Specific binding typically results in greater than 2 fold,
preferably greater than 5 fold, more preferably greater than 10
fold and most preferably greater than 100 fold increase in amount
of bound ligand (per unit time) to a cell or tissue bearing the
target molecule as compared to a cell or tissue lacking the target
molecule or marker. Specific binding to a protein under such
conditions requires an antibody that is selected for its
specificity for a particular protein. A variety of immunoassay
formats are appropriate for selecting antibodies specifically
immunoreactive with a particular protein. For example, solid-phase
ELISA immunoassays are routinely used to select monoclonal
antibodies specifically immunoreactive with a protein. See Harlow
and Lane (1988) Antibodies, A Laboratory Manual, Cold Spring Harbor
Publications, New York, for a description of immunoassay formats
and conditions that can be used to determine specific
immunoreactivity.
[0019] The term "immunologically reactive conditions" includes
reference to conditions which allow an antibody generated to a
particular epitope to bind to that epitope to a detectably greater
degree than, and/or to the substantial exclusion of, binding to
substantially all other epitopes. Immunologically reactive
conditions are dependent upon the format of the antibody binding
reaction and typically are those utilized in immunoassay protocols.
See Harlow and Lane (1988) Antibodies, A Laboratory Manual, Cold
Spring Harbor Publications, New York, for a description of
immunoassay formats and conditions. Preferably, immunologically
reactive conditions are "physiological conditions" which includes
reference to conditions (e.g., temperature, osmolarity, pH) that
are typical inside a living mammal or a mammalian cell. While it is
recognized that some organs are subject to extreme conditions, the
intra-organismal and intra-cellular environment normally varies
around pH 7 (i.e. from pH 6.0 to pH 8.0, more typically pH 6.5 to
7.5), contains water as the predominant solvent, and exists at a
temperature above 0.degree. C. and below 50.degree. C. Osmolarity
is within the range that is supportive of cell viability and
proliferation.
[0020] The terms "mammalian nicotinic acetylcholine receptor" or
"nAchR" include reference to peripheral, ligand-gated ion channel
which is present on "fast" and "slow" muscles and located at the
post-synaptic membrane of the neuromuscular junction (motor end
plate). Mammalian nicotinic acetylcholine receptors are typically
from primates, preferably from humans.
[0021] The term "focal muscle spasm" includes reference to a brief,
unsustained contraction, or a paroxysmal, spontaneous, prolonged
contraction of one or more muscles. The term references those focal
muscle spasms, the therapeutic treatment of which comprises
selective destruction of one or more muscles at the site of the
focal muscle spasm. Typical focal muscle spasms include
blepharospasm, cervical dystonia, hand dystonia, limb dystonia,
hemifacial spasm, bruxism, strabismus, VI nerve palsy, spasmodic
dysphonia, and oromandibular dystonia. See, Brooke, M. H., A
Clinician's View of Neuromuscular Diseases. Baltimore, Williams
& Wilkins, 1986; and Layzer, R. B., Muscle pain, cramps and
fatigue, in Myology, A G Engel, B Q Banker (eds.). New York,
McGraw-Hill, 1986.
[0022] The term "galactose binding moiety" includes reference to
composition which selectively reacts with cell surface galactose
residues. Typically, the galactose binding moiety is an antibody,
lectin, or lectin derivative (e.g., a subunit thereof). Preferred
lectins or derivatives thereof include: abrin (e.g., abrin-b),
ricin (e.g., ricin-b). Particularly preferred are ricin-b and
abrin-b.
[0023] The terms "effective amount" or "amount effective to" or
"therapeutically effective amount" includes reference to a dosage
sufficient to produce a desired result. Typically, the desired
result is reduction in the severity of a focal muscle spasm.
[0024] Antibodies to Muscle Specific Antigens
[0025] Antibodies of the present invention are selectively
reactive, under immunologically reactive conditions, to a muscle
specific antigen. The term "muscle specific antigen" includes
reference to those antigens whose presence is substantially limited
to the membrane of muscle cells at the localized site at which the
immunotoxin of the present invention is administered. Thus a muscle
specific antigen may be present on non-muscle cells but is
substantially inaccessible to the immunotoxins of the present
invention due to the mode of administration. Preferably, however,
muscle specific antigens are unique to muscle cells.
[0026] Muscle specific antigens are known in the art. For example,
antibodies reactive to N-CAM (neuronal cell adhesion molecule) can
and have been generated and are available commercially (Sigma
Chemical Company, St. Louis, Mo.). Anti-N-CAM monoclonals bind to
the CD56 differentiation antigen specifically expressed on
regenerating or newly denervated muscle fibers (Couvalt and Sanes,
Proc. Natl. Acad. Sci. USA (1985) 82:4544-4548; Cashman et al.,
Ann. Neurol. (1987) 21:481-489; IIIa I, Leon-Monzon M, Dalakas M
C., Ann. Neurol. 1992; 31:46-52). Likewise, the muscle-specific
antigen Leu-19 (Becton Dickinson) can be used to generate
antibodies by standard immunological methods. Antibodies to other
muscle specific antigens, such as monoclonal anti-dystrophin, are
commercially available (Sigma).
[0027] In preferred embodiments the muscle specific antigen is a
nicotinic acetylcholine receptor (nAchR). The nAch receptor and
antibodies generated thereto are readily available from publicly
accessible depositories. Cell line TE671 (equivalent to the RD cell
line) expresses AchR for preparation of anti-human nAchR antibodies
and is available from the ATCC under deposit number CRL 8805. See,
U.S. Pat. Nos. 5,041,389, and 4,789,640, both incorporated herein
by reference. Hybridomas producing a human IgG1 monoclonal antibody
against nAchR is deposited with the Fermentation Research Institute
of Japan under the accession number FERM BP-1798 (U.S. Pat. No.
5,192,684, incorporated herein by reference). Monoclonal antibodies
to acetylcholine receptors are produced by hybridomas having
accession number ATCC Nos.: HB 8987 (mAb 64), HB 189 (mAb 270), and
TIB 175 (mAb 35), all of which are incorporated herein by
reference.
[0028] Many methods of making antibodies are known to persons of
ordinary skill. "Antibody" includes antigen binding forms of
antibodies (e.g., Fab, F(ab).sub.2). The term also refers to a
polypeptide substantially encoded by an immunoglobulin gene or
immunoglobulin genes, or fragments thereof which specifically bind
and recognize an analyte (antigen). The recognized immunoglobulin
genes include the kappa, lambda, alpha, gamma, delta, epsilon and
mu constant region genes, as well as the myriad immunoglobulin
variable region genes. Light chains are classified as either kappa
or lambda. Heavy chains are classified as gamma, mu, alpha, delta,
or epsilon, which in turn define the immunoglobulin classes, IgG,
IgM, IgA, IgD and IgE, respectively.
[0029] An exemplary immunoglobulin (antibody) structural unit
comprises a tetramer. Each tetramer is composed of two identical
pairs of polypeptide chains, each pair having one "light" (about 25
kD) and one "heavy" chain (about 50-70 kD). The N-terminus of each
chain defines a variable region of about 100 to 110 or more amino
acids primarily responsible for antigen recognition. The terms
variable light chain (V.sub.L) and variable heavy chain (V.sub.H)
refer to these light and heavy chains respectively.
[0030] Antibodies exist e.g., as intact immunoglobulins or as a
number of well characterized fragments produced by digestion with
various peptidases. Thus, for example, pepsin digests an antibody
below the disulfide linkages in the hinge region to produce
F(ab)'.sub.2, a dimer of Fab which itself is a light chain joined
to V.sub.H-C.sub.H1 by a disulfide bond. The F(ab)'.sub.2 may be
reduced under mild conditions to break the disulfide linkage in the
hinge region, thereby converting the F(ab)'.sub.2 dimer into an
Fab' monomer. The Fab' monomer is essentially an Fab with part of
the hinge region (see, Fundamental Immunology, Third Edition, W. E.
Paul, ed., Raven Press, N.Y. 1993). While various antibody
fragments are defined in terms of the digestion of an intact
antibody, one of skill will appreciate that such fragments may be
synthesized de novo either chemically or by utilizing recombinant
DNA methodology. Thus, the term antibody, as used herein, also
includes antibody fragments such as single chain Fv, chimeric
antibodies (i.e., comprising constant and variable regions from
different species), humanized antibodies (i.e., comprising a
complementarity determining region (CDR) from a non-human source)
and heteroconjugate antibodies (e.g., bispecific antibodies).
[0031] The following discussion is presented as a general overview
of the techniques available; however, one of skill will recognize
that many variations of the following methods are known.
[0032] A. Antibody Production
[0033] A number of immunogens are used to produce antibodies
specifically reactive with a muscle specific antigen. A
recombinant, synthetic, or native muscle specific antigen of 5
contiguous amino acids in length or greater from a muscle specific
antigen is the preferred immunogen (antigen) for the production of
monoclonal or polyclonal antibodies. The term "recombinant" when
used with reference to a cell, or nucleic acid, or vector, includes
reference to a cell, or nucleic acid, or vector, that has been
modified by the introduction of a heterologous nucleic acid or the
alteration of a native nucleic acid to a form not native to that
cell, or that the cell is derived from a cell so modified. Thus,
for example, recombinant cells express genes that are not found
within the native (non-recombinant) form of the cell or express
native genes that are otherwise abnormally expressed, under
expressed or not expressed at all.
[0034] In a typical procedure, the muscle specific antigen is
injected into an animal capable of producing antibodies. Methods of
producing polyclonal antibodies are known to those of skill in the
art. In brief, an immunogen (antigen), preferably a purified muscle
specific antigen (e.g., nAchR), an muscle specific antigen coupled
to an appropriate carrier (e.g., GST, keyhole limpet hemanocyanin,
etc.), or an muscle specific antigen incorporated into an
immunization vector such as a recombinant vaccinia virus (see, U.S.
Pat. No. 4,722,848) is mixed with an adjuvant and animals are
immunized with the mixture. The animal's immune response to the
immunogen preparation is monitored by taking test bleeds and
determining the titer of reactivity to the muscle specific antigen
of interest. When appropriately high titers of antibody to the
immunogen are obtained, blood is collected from the animal and
antisera are prepared. Further fractionation of the antisera to
enrich for antibodies reactive to the muscle specific antigen is
performed where desired (see, e.g., Coligan (1991) Current
Protocols in Immunology Wiley/Greene, NY; and Harlow and Lane
(1989) Antibodies: A Laboratory Manual Cold Spring Harbor Press,
NY).
[0035] Antibodies, including binding fragments and single chain
recombinant versions thereof, against predetermined fragments of
muscle specific antigen are raised by immunizing animals, e.g.,
with conjugates of the fragments with carrier proteins as described
above. Typically, the immunogen of interest is an muscle specific
antigen of at least about 5 amino acids, more typically the muscle
specific antigen is 10 amino acids in length, preferably, 15 amino
acids in length and more preferably the muscle specific antigen is
20 amino acids in length or greater. The peptides are typically
coupled to a carrier protein (e.g., as a fusion protein), or are
recombinantly expressed in an immunization vector. Antigenic
determinants on peptides to which antibodies bind are typically 3
to 10 amino acids in length.
[0036] Monoclonal antibodies are prepared from cells secreting the
desired antibody. Monoclonals antibodies are screened for binding
to an muscle specific antigen from which the immunogen was derived.
Specific monoclonal and polyclonal antibodies will usually bind
with a K.sub.D of at least about 0.1 mM, more usually at least
about 50 .mu.M, and most preferably at least about 1 .mu.M or
better.
[0037] In some instances, it is desirable to prepare monoclonal
antibodies from various mammalian hosts, such as mice, rodents,
primates, humans, etc. Description of techniques for preparing such
monoclonal antibodies are found in, e.g., Stites et al. (eds.)
Basic and Clinical Immunology (4th ed.) Lange Medical Publications,
Los Altos, Calif., and references cited therein; Harlow and Lane,
Supra; Goding (1986) Monoclonal Antibodies: Principles and Practice
(2d ed.) Academic Press, New York, N.Y.; and Kohler and Milstein
(1975) Nature 256: 495-497. Summarized briefly, this method
proceeds by injecting an animal with an immunogen comprising an
muscle specific antigen. The animal is then sacrificed and cells
taken from its spleen, which are fused with myeloma cells. The
result is a hybrid cell or "hybridoma" that is capable of
reproducing in vitro. The population of hybridomas is then screened
to isolate individual clones, each of which secrete a single
antibody species to the immunogen. In this manner, the individual
antibody species obtained are the products of immortalized and
cloned single B cells from the immune animal generated in response
to a specific site recognized on the immunogenic substance.
[0038] Alternative methods of immortalization include transfection
with Epstein Barr Virus, oncogenes, or retroviruses, or other
methods known in the art. Colonies arising from single immortalized
cells are screened for production of antibodies of the desired
specificity and affinity for the antigen, and yield of the
monoclonal antibodies produced by such cells is enhanced by various
techniques, including injection into the peritoneal cavity of a
vertebrate (preferably mammalian) host. The muscle specific
antigens and antibodies of the present invention are used with or
without modification, and include chimeric antibodies such as
humanized murine antibodies.
[0039] Other suitable techniques involve selection of libraries of
recombinant antibodies in phage or similar vectors (see, e.g., Huse
et al. (1989) Science 246: 1275-1281; and Ward, et al., (1989)
Nature 341: 544-546; and Vaughan et al. (1996) Nature
Biotechnology, 14: 309-314). Alternatively, high avidity human
monoclonal antibodies can be obtained from transgenic mice
comprising fragments of the unrearranged human heavy and light
chain Ig loci (i.e., minilocus transgenic mice). Fishwild et al.,
Nature Biotech., 14:845-851 (1996).
[0040] Also, recombinant immunoglobulins may be produced. See,
Cabilly, U.S. Pat. No. 4,816,567; and Queen et al. (1989) Proc.
Nat'l Acad. Sci. USA 86: 10029-10033.
[0041] B. Human or Humanized (Chimeric) Antibody Production
[0042] The anti-muscle specific antigen antibodies of this
invention can also be administered to a mammal (e.g., a human
patient) for therapeutic purposes (e.g., as targeting molecules
when conjugated or fused to effector molecules such as labels,
cytotoxins, enzymes, growth factors, drugs, etc.). Antibodies
administered to an organism other than the species in which they
are raised are often immunogenic. Thus, for example, murine
antibodies administered to a human often induce an immunologic
response against the antibody (e.g., the human anti-mouse antibody
(HAMA) response) on multiple administrations. The immunogenic
properties of the antibody are reduced by altering portions, or
all, of the antibody into characteristically human sequences
thereby producing chimeric or human antibodies, respectively.
[0043] i) Humanized (Chimeric) Antibodies
[0044] Humanized (chimeric) antibodies are immunoglobulin molecules
comprising a human and non-human portion. More specifically, the
antigen combining region (or variable region) of a humanized
chimeric antibody is derived from a non-human source (e.g., murine)
and the constant region of the chimeric antibody (which confers
biological effector function to the immunoglobulin) is derived from
a human source. The humanized chimeric antibody should have the
antigen binding specificity of the non-human antibody molecule and
the effector function conferred by the human antibody molecule. A
large number of methods of generating chimeric antibodies are well
known to those of skill in the art (see, e.g., U.S. Pat. Nos.
5,502,167, 5,500,362, 5,491,088, 5,482,856, 5,472,693, 5,354,847,
5,292,867, 5,231,026, 5,204,244, 5,202,238, 5,169,939, 5,081,235,
5,075,431, and 4,975,369).
[0045] Detailed methods for preparation of chimeric (humanized)
antibodies can be found in U.S. Pat. No. 5,482,856.
[0046] ii) Human Antibodies
[0047] In another embodiment, this invention provides for fully
human anti-muscle specific antigen antibodies. Human antibodies
consist entirely of characteristically human polypeptide sequences.
The human anti-muscle specific antigen antibodies of this invention
can be produced in using a wide variety of methods (see, e.g.,
Larrick et al, U.S. Pat. No. 5,001,065, for review).
[0048] In preferred embodiments, the human anti-muscle specific
antigen antibodies of the present invention are usually produced
initially in trioma cells.
[0049] Genes encoding the antibodies are then cloned and expressed
in other cells, particularly, nonhuman mammalian cells. The general
approach for producing human antibodies by trioma technology has
been described by Ostberg et al. (1983), Hybridoma 2: 361-367,
Ostberg, U.S. Pat. No. 4,634,664, and Engelman et al., U.S. Pat.
No. 4,634,666. The antibody-producing cell lines obtained by this
method are called triomas because they are descended from three
cells; two human and one mouse. Triomas have been found to produce
antibody more stably than ordinary hybridomas made from human
cells.
[0050] The genes encoding the heavy and light chains of
immunoglobulins secreted by trioma cell lines are cloned according
to methods, including the polymerase chain reaction, known in the
art (see, e.g., Sambrook et al., Molecular Cloning: A Laboratory
Manual, 2nd ed., Cold Spring Harbor, N.Y., 1989; Berger &
Kimmel, Methods in Enzymology, Vol. 152: Guide to Molecular Cloning
Techniques, Academic Press, Inc., San Diego, Calif., 1987; Co et
al. (1992) J. Immunol, 148: 1149). For example, genes encoding
heavy and light chains are cloned from a trioma's genomic DNA or
cDNA produced by reverse transcription of the trioma's RNA. Cloning
is accomplished by conventional techniques including the use of PCR
primers that hybridize to the sequences flanking or overlapping the
genes, or segments of genes, to be cloned.
[0051] Formation of Immunotoxic Conjugates
[0052] Antibodies specifically reactive to muscle specific antigens
are joined via covalent or non-covalent bond to a toxin selected
from the group comprising: ricin, abrin, ricin-a, abrin-a, and
botulinum toxin. Ricin, abrin, and subunits thereof as well as
botulinum toxin A through F, are readily available from commercial
sources (e.g, Sigma Chemical Company, St. Louis, Mo.). Methods of
isolating ricin and abrin are also well known to those of ordinary
skill in the art. See, e.g., Nicholson and Blaustein, J. Biochim.
Biophys. Acta, 266:543 (1972); Tomita et al., Experientia, 28:84
(1972); Wei et al., J. Biol. Chem., 249:3061 (1974); Lin et al.
Toxicon., 19:41 (1981); Oisnes et al. J. Biol. Chem. 249:803
(1974); Wei et al., J. Mol. Biol., 123:707 (1978); Lin and Li, Eur.
J. Biochem., 105:453 (1980); Nicolson et al. Biochemistry, 13:196
(1974); and, Olsnes, Methods Enzymol. 50:330-335 (1978), all of
which are incorporated herein by reference. The molecules may be
attached by any of a number of means well-known to those of skill
in the art. In some embodiments, the immunotoxic conjugates of the
present invention are recombinantly expressed as single-chain
fusion protein comprising both antibody and toxin. Typically the
toxin will be conjugated, either directly or through a linker
(spacer), to the ligand.
[0053] A "linker", as used herein, is a molecule that is used to
join two molecules. The linker is capable of forming covalent bonds
or high-affinity non-covalent bonds to both molecules. Suitable
linkers are well known to those of ordinary skill in the art and
include, but are not limited to, straight or branched-chain carbon
linkers, heterocyclic carbon linkers, or peptide linkers. The
linkers may be joined to the constituent amino acids through their
side groups (e.g., through a disulfide linkage to cysteine).
[0054] The procedure for attaching a toxin to an antibody or other
polypeptide targeting molecule will vary according to the chemical
structure of the toxin. Antibodies contain a variety of functional
groups; e.g., sulfhydryl (--S), carboxylic acid (COOH) or free
amine (--NH.sub.2) groups, which are available for reaction with a
suitable functional group on a toxin. Additionally, or
alternatively, the antibody or toxin can be derivatized to expose
or attach additional reactive functional groups. The derivatization
may involve attachment of any of a number of linker molecules such
as those available from Pierce Chemical Company, Rockford Ill.
[0055] A bifunctional linker having one functional group reactive
with a group on the toxin, and another group reactive with an
antibody, can be used to form a desired immunoconjugate.
Alternatively, derivatization may involve chemical treatment of the
toxin or antibody, e.g., glycol cleavage of the sugar moiety of a
glycoprotein antibody with periodate to generate free aldehyde
groups. The free aldehyde groups on the antibody may be reacted
with free amine or hydrazine groups on the toxin to bind the toxin
thereto. (See U.S. Pat. No. 4,671,958). Procedures for generation
of free sulfhydryl groups on antibodies or antibody fragments, are
also known (See U.S. Pat. No. 4,659,839).
[0056] Many procedures and linker molecules for attachment of
various compounds including toxins are known. See, for example,
European Patent Application No. 188,256; U.S. Pat. Nos. 4,671,958,
4,659,839, 4,414,148, 4,699,784; 4,680,338; 4,569,789; 4,589,071;
and Borlinghaus et al. Cancer Res. 47: 4071-4075 (1987), which are
incorporated herein by reference. In particular, production of
various immunotoxin conjugates is well-known within the art and can
be found, for example in "Monoclonal Antibody-Toxin Conjugates:
Aiming the Magic Bullet," Thorpe et al., Monoclonal Antibodies in
Clinical Medicine, Academic Press, pp. 168-190 (1982), Waldmann,
Science, 252: 1657 (1991), U.S. Pat. Nos. 4,545,985 and 4,894,443
which are incorporated herein by reference. See also, e.g., Birch
and Lennox, Monoclonal Antibodies: Principles and Applications,
Chapter 4, Wiley-Liss, New York, N.Y. (1995); U.S. Pat. Nos.
5,218,112, 5,090,914; Hermanson, Bioconjugate Techniques, Academic
Press, San Diego, Calif. (1996). In preferred embodiments, the
linker molecule is m-Malimidobenzoyl-N-hydroxysuccinimideester
(MBS) which can be used to prepare immunotoxin conjugates as
described, for example, in Youle and Nevelle, Proc. Natl. Acad.
Sci., 77(9):5483-5486 (1980).
[0057] In some circumstances, it is desirable to free the toxin
from the antibody when the immunotoxic conjugate has reached its
target site. Therefore, immunotoxic conjugates comprising linkages
which are cleavable in the vicinity or within the target site may
be used when the toxin is to be released at the target site.
Cleaving of the linkage to release the agent from the ligand may be
prompted by enzymatic activity or conditions to which the
immunoconjugate is subjected either inside the target cell or in
the vicinity of the target site. A number of different cleavable
linkers are known to those of skill in the art. See U.S. Pat. Nos.
4,618,492; 4,542,225, and 4,625,014. SPDP is a reversible
NHS-ester, pyridyl disulfide cross-linker used to conjugate
amine-containing molecules to sulfhydryls. Another chemical
modification reagent is 2-iminothiolane which reacts with amines
and yields a sulfhydryl. Water soluble SPDP analogs, such as
Sulfo-LC-SPDP (Pierce, Rockford, Ill.) are also available. SMPT is
a reversible NHS-ester, pyridyl disulfide cross-linker developed to
avoid cleavage in vivo prior to reaching the antigenic target.
Additionally, the NHS-ester of SMPT is relatively stable in aqueous
solutions.
[0058] Pharmaceutical Compositions and Method of Administration
[0059] Immunotoxic conjugates of the present invention are useful
for the treatment of focal muscle spasms such as, but not limited
to, blepharospasm, cervical dystonia, hand dystonia, limb dystonia,
hemifacial spasm, bruxism, strabismus, VI nerve palsy, spasmodic
dysphonia, and oromandibular dystonia. In preferred embodiments,
the immunotoxin conjugate comprises ricin (RCA.sub.60). While not
bound by theory, it is believed that the use of the galactose
binding ricin B-chain helps prevent diffusion of the immunotoxin
from the site of administration. Additionally, the B-chain
increases the potency of the ricin A-chain toxin.
[0060] The formulations containing therapeutically effective
amounts of the immunotoxin conjugates of the present invention are
either sterile liquid solutions, liquid suspensions or lyophilized
versions and optionally contain stabilizers or excipients.
Lyophilized compositions are reconstituted with suitable diluents,
e.g., water for injection, saline, 0.3% glycine and the like. The
compositions may contain pharmaceutically acceptable auxiliary
substances as required to approximate physiological conditions such
as pH adjusting and buffering agents, toxicity adjusting agents and
the like, for example, sodium acetate, sodium chloride, potassium
chloride, calcium chloride, and the like. Actual methods for
preparing administrable compositions will be known or apparent to
those skilled in the art and are described in more detail in such
publications as Remington's Pharmaceutical Science, 19th ed., Mack
Publishing Company, Easton, Pa. (1995).
[0061] The compositions for administration will commonly comprise a
solution of the immunotoxin conjugate of the present invention
dissolved in a pharmaceutically acceptable carrier, preferably an
aqueous carrier. A variety of aqueous carriers can be used, e.g.,
buffered saline and the like. These solutions are sterile and
generally free of undesirable matter. These compositions may be
sterilized by conventional, well known sterilization
techniques.
[0062] As will be readily understood by the clinician of ordinary
skill in the art, the dose will be dependent upon the properties of
the particular immunotoxin conjugate employed, e.g., its activity
and biological half-life, the concentration of immunotoxin
conjugate in the formulation, the site and rate of dosage, the
clinical tolerance of the patient involved, the disease afflicting
the patient, the severity of the disease, and the like. Preferably,
the pharmaceutical compositions containing the immunotoxin
conjugates will be administered by intramuscular injection in a
therapeutically effective dose ranging from about 1 ng to 200 ng
depending upon the size of the muscle, the severity of the focal
muscle spasm, and the specificity and toxicity of the conjugate.
For example, for a ricin-anti-nAchR immunotoxin conjugate, an eye
muscle will typically require between 5 and 20 ng of conjugate, and
a vocal chord will generally require 1 to 2 ng. Preferably, the
dose is administered at the site of the neuromuscular junctions of
the muscle which is being treated. Those of skill will understand
that the dose may be administered to the various neuromuscular
junctions of the muscle whose activity one wishes to diminish, this
being particularly preferred for muscles whose size allows for such
a mode of administration. Therapeutically effective amounts of
immunotoxin conjugates of the present invention can be administered
alone, in combination, or in conjunction with therapeutically
effective amounts of the unconjugated forms of the toxins (e.g.,
botulinum toxin).
[0063] Preferably, the dosage is administered once but may be
applied periodically until either a therapeutic result is achieved
or until side effects warrant discontinuation of therapy.
Generally, the dose should be sufficient to treat or ameliorate
symptoms or signs of focal muscle spasm without producing
unacceptable toxicity to the patient. An effective amount of the
compound is that which provides either subjective relief of a
symptom(s) or an objectively identifiable improvement as noted by
the clinician or other qualified observer.
[0064] Solutions comprising immunotoxin conjugates of the present
invention will typically have a pH in the range of pH 5 to 9.5,
preferably pH 6.5 to 7.5. The immunotoxin conjugates should be in a
solution having a suitable pharmaceutically acceptable buffer such
as phosphate, tris (hydroxymethyl) aminomethane-HCl, saline, or
citrate and the like. Buffer concentrations should be in the range
of 1 to 100 mM. The solution of antibody may also contain a salt,
such as sodium chloride or potassium chloride in a concentration of
50 to 150 mM. An effective amount of a stabilizing agent such as an
albumin, a globulin, a gelatin, a protamine or a salt of protamine
can also be included to a solution comprising the immunotoxin
conjugate of the present invention. In preferred embodiments the
buffer is a saline solution of 0.9% comprising human serum albumin
of 1 mg/ml. Antibody or immunotoxin may also be administered via
microspheres, liposomes or other microparticulate delivery systems
placed in certain tissues including blood.
[0065] Although the present invention has been described in some
detail by way of illustration and example for purposes of clarity
of understanding, it will be obvious that certain changes and
modifications may be practiced within the scope of the appended
claims.
EXAMPLE 1
ITX Cytotoxic Activity
[0066] A. Protein Purification
[0067] Ricin was purified from seeds of Ricinus communis by elution
from Sepharose columns with N-acetylgalactosamine, as described by
Nicolson et al (Biochemistry (1974) 13:196-204). Onconase was
purified from the eggs of Rana pipiens, as described previously
(Ardelt et al., J. Biol. Chem., (1991) 266:245-251). CRM 107 (a
mutated form of diphtheria toxin with an inactivated binding
domain) was purified as described previously (Greenfield L, Johnson
V G, Youle R J, Science, 1987;238:536-539). The plant lectin,
RCA.sub.120, was purchased from Sigma (St. Louis,. MO). MoAb 35 (an
anti-nicotinic acetylcholine receptor monoclonal antibody) (Tzartos
et al., J. Biol. Chem., (1981) 256:8635-8645; Clementi and Sher,
Eur. J. Cell Biol., (1985) 37:220-228; Tzartos et al., Proc. Natl.
Acad. Sci. USA, 1982;79:188-192) was purified from ascites (mouse)
by ammonium sulfate precipitation and DEAE sepharose.
[0068] B. Immunotoxin Synthesis
[0069] Conjugation of transferrin (tfn) with CRM 107 was
accomplished as described previously (Johnson V G, Wilson D,
Greenfield L, Youle R J., J. Biol. Chem. (1988) 263: 1295-1300).
Conjugation of MoAb 35 to ricin was performed as described
previously (Youle and Neville, Proc. Natl. Acad. Sci. USA (1980)
77:5483-5486), with the following modifications. The antibody was
prepared by adding 71 .mu.l of 1 M dithiothreitol (DTT) in
phosphate-buffered saline (PBS) to 2.8 mg (in 0.5 ml) of antibody.
This mixture was incubated for 30 minutes in order to partially
reduce the antibody. The antibody-DTT mixture was then applied to a
G-25, PD-10 gel filtration column equilibrated with PBS. Peak
antibody fractions were pooled. Ricin, 10 mg in 1.4 ml of PBS was
mixed with 39 .mu.l of dimethylformamide containing 0.15 mg of the
bifunctional cross-linking agent,
m-maleimidobenzoyl-N-hydroxysuccinimidyl (MBS) ester (Pierce
Chemical Co.). The mixture was incubated at room temperature for 30
minutes. Ricin-MBS was immediately reacted with freshly reduced
antibody and incubated at 4.degree. C. overnight.
[0070] C. Immunotoxin Purification
[0071] The conjugate was separated from unreacted ricin by HPLC on
a TSK 3000 SW column (size exclusion) in 0.1 M sodium phosphate
buffer (pH 7.4). The peak fractions from several runs containing
both unreacted antibody and immunotoxin were pooled and loaded onto
an immobilized D-galactose affinity column (Pierce) at 4.degree. C.
After the column was flushed with PBS to remove unreacted antibody,
0.1 M lactose was run over the column to elute the purified
immunotoxin.
[0072] D. Tissue Culture
[0073] The C2 mouse skeletal muscle cells (Yaffe and Saxel, Nature
(1977) 270:725-727; Inestrosa et al., Exp. Cell. Res. (1983)
147:393-405) were maintained as exponentially growing myoblasts in
medium consisting of Dulbecco's modified Eagle medium with high
glucose, supplemented with 0.5% chick embryo extract, 20% fetal
bovine serum, 0.2 M L-glutamine, and 100 .mu.g/ml of
penicillin-streptomycin. The cells were grown in 100 mm sterile
Corning culture dishes under humidified 95% air/5% CO.sub.2
atmosphere at 37.degree. C. Cell stocks were maintained until
reaching 70-80% confluence and then harvested with trypsin and
replated. Both the human rhabdomyosarcoma (RD) (Syapin et al.,
Brain Research, (1982) 231:365-377; Sine, J. Bio. Chem., (1988)
34:18052-18062; Luther et al., The Journal of Neuroscience, (1989)
9:1082-1096) and human glioblastoma multiforme (U251) cell lines
were maintained in Dulbecco's modified Eagle medium supplemented
with 10% fetal calf serum, 1% L-glutamine, non-essential amino
acids, 0.5% sodium pyruvate, and 0.1% gentamycin. Cells were grown
in 100 mm Corning culture dishes. The cells were harvested with
trypsin and replated upon reaching 50-60% confluence.
[0074] E. Cellular Protein Synthesis Assays
[0075] Inhibition of protein synthesis was used to assay the
cytotoxic effect of ITX, ricin, RCA.sub.120, Tfn-CRM 107, and
onconase in C2 myoblasts, myotubes, RD, and U251 cells by methods
similar to those described by Zovickian, et al (J. Neurosurg.,
(1987) 66:850-861). Cells were plated into 96-well microwell plates
at a density of 5.times.10.sup.5/ml in 100 .mu.l of media and
incubated overnight. Fresh media was added to each well prior to
the addition of either serially diluted toxins and/or 0.1 M lactose
or PBS. Cells were then incubated 18-20 hours and the growth media
was aspirated and replaced with leucine-free RPMI media and 0.1
.mu.Ci of (.sup.14C)-labeled leucine. After 2 hours cells were
harvested onto glass fiber filters with a PHD cell harvester. All
cytotoxicity assays were performed 2-5 times in triplicate. Results
were expressed as the percentage of incorporation of radioactivity
compared to either lactose or PBS controls.
[0076] Cytotoxicity assays on myotubes were performed as described
above with slight modifications. After myoblasts reached 80-90%
confluence in the wells, growth media was removed and replaced with
Dulbecco's modified Eagle medium supplemented with 10% horse serum
(fusion medium). Every 24 hours, the fusion medium was replaced.
After 72 hours in fusion medium myotube acetylcholine receptor
expression is at its maximum (Inestrosa et al., Exp. Cell Res.
(1983) 147:393-405) and cells were used in cytotoxicity assays.
[0077] F. ITX Cyto toxicity
[0078] The potency and specificity of ITX was first examined by
comparing activity on C2 muscle cells in undifferentiated and
differentiated states. With decreased serum and withdrawal of
chicken embryo extract, nearly confluent myoblasts which express
undetectable levels of nicotinic acetylcholine receptors are
induced to form multi-nucleated myotubes (Inestrosa et al., Exp.
Cell Res., (1983) 147:393-405). The myotubes often contracted in
the culture dish after 3 days and reportedly express high densities
of nicotinic acetylcholine receptor clusters. Id.
[0079] Toxicity of ricin and ITX to C2 myotubes and myoblasts in
the presence and absence of 0.1 M lactose was measured. The assays
demonstrate a steep dose-response inhibition of protein synthesis.
Myotube protein synthesis was inhibited 5.0% (IC.sub.50) at an ITX
concentration of 2.5.times.10.12 M and at a ricin concentration of
4.5.times.10.sup.-11 M. Lactose,. a competitive inhibitor of ricin
binding to cells (Olsnes et al., Nature (1974) 249:627-631),
blocked ricin toxicity 800-fold, whereas ITX was barely inhibited
by lactose (1.4-fold), indicating that ITX was binding and
inhibiting protein synthesis, not via the ricin receptor but via
the nicotinic acetylcholine receptor. Myotubes were more sensitive
to ITX than ricin, and in the presence of lactose, myotubes were
20,000-fold more sensitive to ITX (3.5.times.10.sup.-12 M) than
native ricin (7.times.10.sup.-8 M).
[0080] To corroborate the nicotinic acetylcholine receptor
specificity of ITX, ricin and ITX activity on C2 myoblasts
(nicotinic acetylcholine receptor-negative) was examined. Myotubes,
were 100-fold more sensitive to ITX than myoblasts. However, ricin
toxicity was essentially identical for both cell types. In the
presence of lactose, myotubes (nicotinic acetylcholine
receptor-positive) were over 14,000-fold more sensitive to ITX than
were myoblasts (nicotinic acetylcholine receptor-negative), whereas
ricin was actually less toxic to myotubes than myoblasts.
[0081] The cytotoxic properties of ITX and ricin on two human
neoplastic cell lines was also compared. RD cells, human
rhabdomyosarcoma cells, are known to express functional human
nicotinic acetylcholine receptors (Syapin et al., Brain Research
(1982) 231:365-377; Sine, J. Biol. Chem. (1988) 34:18052-18062;
Luther et al., The Journal of Neuroscience (1989) 9:1082-1096)
whereas U251 cells, of human glioma origin are not. ITX had a
nearly identical dose-response toxicity profile on RD cells as seen
with myotubes with an IC.sub.50 of 2.4.times.10.sup.-12 M. Addition
of lactose decreased ITX activity only 1.4-fold but inhibited
toxicity of ricin 100-fold. When ricin binding is blocked with
lactose, RD cells are greater than 1000-fold more sensitive to ITX
than ricin alone. On U251 cells ITX had an IC.sub.50 of
2.5.times.10.sup.-10 M, about 100-fold higher than the nicotinic
acetylcholine receptor positive RD cells, whereas ricin was equally
toxic to RD and U251 cells. Thus, non-nicotinic acetylcholine
receptor expressing cell lines (myoblasts and U251 cells) in the
presence of lactose were between 14,000-21,000-fold less sensitive
to ITX than were the nicotinic acetylcholine receptor expressing
cell lines (myotubes and RD cells).
EXAMPLE 2
Cytotoxic Activity Of Other Protein Toxins
[0082] In an effort to identify the most potent and specific
reagent, three other toxins: RCA120, Tfn-CRM 107, and onconase were
investigated. RCA.sub.120 (MW=120,000) is a tetrameric plant lectin
similar to a dimer of ricin (Lin and Li, Eur. J. Biochem. (1980)
105:453-459). Myotubes and myoblasts were nearly equally sensitive
to RCA.sub.120 (IC.sub.50=3.5.times.10.sup.-10 M). Tfn-CRM 107
(MW=150,000) is an immunotoxin (Johnson et al., J. Neurosurg.
(1989) 70:240-248) selective for the transferrin receptor and high
transferrin receptor numbers on myotube cell cultures and high
rates of iron uptake (Sorokin et al., J. Cell Physiol. (1987)
131:342-353) have been observed. Tfn-CRM 107 was nearly as toxic to
myotubes (IC.sub.50=3.times.10.sup.-7 M) as myoblasts
(IC.sub.50=2.times.10.sup.-7 M). However, overall myotoxicity was
lower than predicted. Onconase (MW=12,000), currently in phase III
clinical trials for treatment of pancreatic cancer, was 2.5-fold
more toxic to myotubes than myoblasts at the IC.sub.50
(2.times.10.sup.-6 M and 8.times.10.sup.-7 M, respectively);
however, it was the least toxic to myotubes of all the proteins
examined. Only ITX demonstrated significant differential toxicity
between myotubes and myoblasts.
EXAMPLE 3
Rat Muscle Biopsies
[0083] Female Balb/c mice (16-18 g) received 0.3 ml IP injections
(30 g needle). For each agent, five serial 2-fold dilutions of
toxin were tested. At each dilution three mice were injected and
the experiment was repeated twice. The maximum tolerated dose was
determined to be the maximum dose/kg where all animals survived.
This dose was used as an estimate of the maximum tolerated dose
(MTD) in rats. The maximum tolerated dose of ITX was 2 .mu.g/kg in
mice. Thus, for 250 g rats {fraction (1/100)} and {fraction
(1/300)} of the maximum tolerated dose of ITX was estimated to be 5
ng and 1.7 ng, respectively. These doses of ITX were delivered
intramuscularly to the rats in a volume of 30 .mu.l. Hereafter in
this example, comparable doses of the toxins refers to doses that
are the same fraction of the maximum tolerated dose (or LD.sub.50
in the case of BTX). This comparison yields an estimate of the
therapeutic window, which may be a useful gauge of the toxins
clinical potential.
[0084] Frozen and lyophilized BTX (Allergan) was reconstituted and
diluted in 1.2 ml of sterile PBS to a final concentration of 83
U/ml and immediately used for injections. Thirty microliters of BTX
containing either 0.25 U (0.1 ng) or 2.5 U (1 ng) was injected into
the rat gastrocnemius. These doses correspond to {fraction (1/100)}
and {fraction (1/10)} of the reported rat LD.sub.50, respectively
(Burgen et al., J. Physiol., (1949) 109:10-24). BTX was not diluted
in blue dextran.
[0085] Female Sprague-Dawley rats (225-250 g) from Taconic farms
were anesthetized with ketamine/xylazine (0.1 cc/100 g) IP and the
leg to be injected was immobilized, shaved, and sterilized with
betadine. The midbelly of the gastrocnemius was exposed by
microdissection and five-fold dilutions of toxin or PBS prepared in
25 mg/ml blue dextran were unilaterally injected with a 30 g
needle. To insure reproducible depth of injection, a plastic
stopper was slipped over the needle such that 3.5 mm of the needle
remained exposed. The needle was inserted so the muscle was flush
against the plastic stopper. Toxins were injected from a syringe
pump (KD Scientific) at a rate of 1.0 .mu.l/min for 30 minutes
(total volume=30 .mu.l). After the infusions were completed, the
skin was sutured closed. The gastrocnemius muscles were biopsied
seven days after treatment at the site of blue dextran staining of
the muscle. Muscle specimens were fresh-frozen in isopentane and
cooled in liquid nitrogen. Serial sections were stained with
hematoxylin-eosin (H & E) or modified Gomori trichrome and
examined by light microscopy in a blinded fashion.
[0086] Muscle biopsy from the ITX-injected site seven days after
treatment demonstrated a severe inflammatory response in the
endomysial parenchyma and the perimysium. Inflammatory cells were
invading muscle fibers in a pattern identical to that seen in
primary inflammatory myopathy (Dalakas M C, N. Engl. J. Med. (1991)
325:1487-1498). Necrosis, phagocytosis, and separation of the
muscle fibers, probably due to edematous changes in the
interstitial tissue, were prominent. Inflammation and muscle fiber
destruction was prominent at the site of injection. Areas of
specimen remote from the injection site (beyond 2-3 mm) had minimal
changes. Control-injected (blue dextran diluted in PBS) rats showed
no or minimal response consisting of scattered inflammatory cells
and mild edematous change limited to the perimysium. No primary
invasion by inflammatory cells or destruction of muscle fibers was
detected in the control rats.
[0087] Evidence of muscle weakening by ITX was observable at a dose
{fraction (1/300)} of the maximum tolerated dose. Muscle weakness
induced by Tfn-CRM 107 and RCA.sub.120 (Example 2) was only
noticeable at doses near their maximum tolerated dose, and onconase
had no apparent effect. Histopathological evidence of muscle fiber
damage correlated with observable muscle weakness (Example 4). ITX
at a dose {fraction (1/300)} of the maximum tolerated dose exerted
a significant, selective and focal destruction to the muscle as
assessed histologically seven days after injection, whereas ricin
alone showed nearly undetectable fiber damage at a dose {fraction
(1/16)} of its maximum tolerated dose. This indicates that the
monoclonal antibody to the nicotinic acetylcholine receptor is
indeed binding ITX specifically to the muscle.
EXAMPLE 4
Muscle Strength Assessment
[0088] The efficacy of BTX has been correlated with its muscle
weakening effects. Since no good animal model exists for focal
muscle spasm, a muscle strength test using the rotorod was adapted
to compare ITX to BTX. The rotorod is a preprogrammable, rotating
cylinder suspended 1.5 feet above a plastic platform used for
quantitative measurement of rat motor performance (Janicke et al.,
Ann. N.Y. Acad. Sci. (1988) 515:97-107). Rats were trained to run
on the rotorod daily. The rotorod was programmed to accelerate to
the desired speed in 10 seconds. The rats were considered to be
successfully trained on the rotorod for the first experiment when
they were able to complete the task (25 rpm for 180 seconds) on
three consecutive days. In the second experiment rats were trained
until they were able to complete the task at. 30 rpm for 180
seconds.
[0089] Immediately prior to injections, all rats ran on the rotorod
and were able to complete the training run (25 rpm for 180 s for
the first experiment and 30 rpm for the second rotorod
experiment.). In the first rotorod experiment, rats were randomly
assigned to three groups and injected with either BTX at {fraction
(1/100)} (n=6) of the LD.sub.50 or ITX (n=6) at a dose {fraction
(1/100)} of the maximum tolerated dose, or PBS (n=6). In the second
rotorod experiment there were six groups: BTX at {fraction (1/10)}
and {fraction (1/100)} of the LD.sub.50, ITX at {fraction (1/100)}
and {fraction (1/300)} of the maximum tolerated dose, unconjugated
MoAb 35 at a concentration used in the conjugation of ITX at
{fraction (1/100)} of the maximum tolerated dose, and PBS. All
groups in the second rotorod experiment contained four rats.
Unconjugated ricin at the concentration used in ITX was lethal to
the rats and therefore was unable to be used as a control.
Following injection all rats were tested in three 180-s trials at
30 rpm and times prior to falling were averaged for each rat. The
time spent on the bar before falling for the six rats per group in
the first rotorod experiment and four rats per group in the second
rotorod experiment was averaged and recorded as data points.
[0090] ITX and BTX were compared in two independent experiments
(FIGS. 1,2) evaluating the performance of rats on the rotorod test
as a function of time after injection with toxins. The effect of
extremely low doses of ITX were quick, dramatic and sustained,
while comparable doses of BTX were minimal and transient. After one
day, rats injected with ITX at {fraction (1/100)} of the maximum
tolerated dose showed sufficient weakening such that the rats were
only able to run for an average of 42 s in the first rotorod
experiment (FIG. 1) and 20 s in the second rotorod experiment (FIG.
2). By comparison, control rats ran for an average of 162 s and 172
s in the first and second rotorod experiments, respectively. Rats
treated with ITX at a dose {fraction (1/300)} of the maximum
tolerated dose in the second rotorod experiment (FIG. 2) have
maintained running times between 50-80 s. BTX, at {fraction
(1/100)} of the LD.sub.50, did not affect strength significantly
compared to saline injected rats in either experiment. Rats treated
with BTX at a dose {fraction (1/10)} of the LD.sub.50 in the second
rotorod experiment became observably weak in the injected limb
correlating with their diminished running times on the rotorod.
However, after 4-5 weeks these rats regained strength as their
rotorod times returned to control values. This data confirms the
result of a pilot trial of BTX injected rats at {fraction (1/10)}
of the LD.sub.50 where performance on the rotorod returned to
control values by 6 weeks. In contrast, ITX treated rats have
maintained running times far less than either BTX or control
treated rats throughout the duration of the experimental period.
Observation of rats on the rotorod showed that both ITX and BTX
treated limbs lagged behind the three untreated limbs as the rats
began to fatigue. This was another indication that weakness had
been induced in both ITX and BTX treated rats. Further, it appeared
that the weakness was only in the injected limb. Rats treated with
saline, BTX at {fraction (1/100)} of the LD.sub.50, and
unconjugated MoAb 35 showed no observable limb weakening effects
while running on the rotorod. All rats gained weight, demonstrated
normal grooming practices and were alert when startled, further
indicating the absence of severe systemic toxicity.
[0091] To perform on the rotorod, it is necessary to coordinate
movement and accelerate for the initial ten seconds of the task in
order to achieve the top rate of 30 rpm. The fact that all rats
were able to overcome the initiation and acceleration phase on the
rotorod demonstrates that generalized motor function was not
impaired and the decreased running times on the rotorod reflect
peripheral, not central, effects of the toxins (BTX and ITX).
[0092] When BTX and ITX were examined on the rotorod at comparable
doses ({fraction (1/100)} of the LD.sub.50 and {fraction (1/100)}
of the maximum tolerated dose, respectively), the ITX-injected
animals demonstrated focal muscle weakness while the BTX-injected
animals were unaffected. When the dose of BTX was increased to
{fraction (1/10)} of the LD.sub.50 a significant weakness appeared
but it only lasted for 4-5 weeks. This result is consistent with
leg twitch studies performed by Holds et al on the rat
gastrocnemius muscle after a single injection of 1.0 U of botulinum
toxin in which muscle strength returned to near pre-injection
values between 4-6 weeks (Holds et al., Opthalmic Plast.
Reconstructr. Surg. (1990) 6:252-259). Therefore, based on the
rotorod results, ITX appears to be thirty times more potent than
BTX and yields a dramatically longer response. The results
demonstrate that ITX has excellent potential for a safe and long
lasting focal muscle weakness and may have clinical applications in
the treatment of patients refractory to BTX used alone, in
combination, or even in place of BTX.
[0093] All publications and patents mentioned in this specification
are herein incorporated by reference into the specification to the
same extent as if each, individual publication or patent was
specifically and individually indicated to be incorporated herein
by reference.
* * * * *