U.S. patent application number 10/796522 was filed with the patent office on 2004-07-22 for treatment for central nervous system disorders.
This patent application is currently assigned to Mayo Foundation for Medical Education and Research ,a MN corporation, Mayo Foundation for Medical Education and Research ,a MN corporation. Invention is credited to Curran, Geoffry L., Poduslo, Joseph F..
Application Number | 20040142872 10/796522 |
Document ID | / |
Family ID | 25477805 |
Filed Date | 2004-07-22 |
United States Patent
Application |
20040142872 |
Kind Code |
A1 |
Poduslo, Joseph F. ; et
al. |
July 22, 2004 |
Treatment for central nervous system disorders
Abstract
Compositions that include an A.beta. polypeptide linked to a
non-A.beta. polypeptide are described, as well as methods of using
such compositions.
Inventors: |
Poduslo, Joseph F.;
(Rochester, MN) ; Curran, Geoffry L.; (Rochester,
MN) |
Correspondence
Address: |
FISH & RICHARDSON P.C.
3300 DAIN RAUSCHER PLAZA
60 SOUTH SIXTH STREET
MINNEAPOLIS
MN
55402
US
|
Assignee: |
Mayo Foundation for Medical
Education and Research ,a MN corporation
|
Family ID: |
25477805 |
Appl. No.: |
10/796522 |
Filed: |
March 9, 2004 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
|
10796522 |
Mar 9, 2004 |
|
|
|
09942253 |
Aug 29, 2001 |
|
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Current U.S.
Class: |
424/145.1 ;
514/17.8; 514/5.8; 514/8.4; 530/350; 536/23.5; 800/12 |
Current CPC
Class: |
B82Y 5/00 20130101; A61P
25/28 20180101; A61K 47/62 20170801; A61K 47/6843 20170801; A61K
47/543 20170801; C07K 16/18 20130101; A61K 51/1018 20130101; A61K
47/642 20170801 |
Class at
Publication: |
514/012 ;
536/023.5; 530/350; 800/012 |
International
Class: |
A01K 067/00; C07H
021/04; A61K 038/17 |
Claims
What is claimed is:
1. A composition comprising an amyloid .beta. (A.beta.) polypeptide
and a non-A.beta. polypeptide, wherein said A.beta. polypeptide and
said non-A.beta. polypeptide are linked.
2. The composition of claim 1, wherein said composition further
comprises a pharmaceutically acceptable carrier or excipient.
3. The composition of claim 1, wherein said non-A.beta. polypeptide
is an antibody.
4. The composition of claim 3, wherein said antibody comprises a
Fab fragment.
5. The composition of claim 3, wherein said antibody comprises a
single chain Fv antibody fragment.
6. The composition of claim 3, wherein said antibody comprises a
F(ab).sub.2 fragment.
7. The composition of claim 3, wherein said antibody has specific
binding affinity for amyloid.
8. The composition of claim 3, wherein said antibody is labeled
with a radioisotope or a contrast agent.
9. The composition of claim 3, wherein said antibody is labeled
with a contrast agent.
10. The composition of claim 1, wherein said non-A.beta.
polypeptide is an enzyme or a cytokine.
11. The composition of claim 10, wherein said enzyme is an
antioxidant enzyme.
12. The composition of claim 11, wherein said antioxidant enzyme is
catalase or superoxide dismutase.
13. The composition of claim 1, wherein said non-A.beta.
polypeptide is leptin.
14. The composition of claim 10, wherein said cytokine is an
interferon or an interleukin.
15. The composition of claim 10, wherein said cytokine is a
neurotrophic factor.
16. The composition of claim 1, wherein said A.beta. polypeptide
and said non-A.beta. polypeptide are covalently linked.
17. The composition of claim 1, wherein said A.beta. polypeptide
comprises residues 1-40, 1-42, or 1-43 of SEQ ID NO:1.
18. A method of treating a patient diagnosed with Alzheimer's
disease, said method comprising administering to said patient an
amount of a composition effective to treat Alzheimer's disease,
said composition comprising an A.beta. polypeptide and an antibody
having specific binding affinity for said A.beta. polypeptide.
19. The method of claim 18, wherein said antibody comprises a Fab
fragment.
20. The method of claim 18, wherein said antibody comprises a
single chain Fv-antibody fragment.
21. The method of claim 18, wherein said antibody comprises a
F(ab).sub.2 fragment.
22. A method of treating a patient diagnosed with Alzheimer's
disease, said method comprising administering to said patient an
amount of an antibody effective to treat Alzheimer's disease,
wherein said antibody is polyamine modified and has specific
binding affinity for an A.beta. polypeptide.
23. A method of diagnosing Alzheimer's disease in a patient, said
method comprising a) administering a composition to said patient,
wherein said composition comprises an A.beta. polypeptide and an
antibody having specific binding affinity for amyloid, wherein said
antibody is labeled, and b) detecting the presence or absence of
said antibody bound to amyloid in the brain of said patient,
wherein said patient is diagnosed with Alzheimer's disease based on
the presence of labeled amyloid in the brain of said patient.
24. The method of claim 23, wherein said detecting step comprises
diagnostic imaging.
25. The method of claim 23, wherein said diagnostic imaging
comprises positron emission tomography, gamma-scintigraphy, single
photon emission computerized tomography, magnetic resonance
imaging, functional magnetic resonance imaging, or
magnetoencephalography.
26. The method of claim 23, wherein said diagnostic imaging
comprises magnetic resonance imaging.
27. The method of claim 23, wherein said amyloid comprises
.beta.-amyloid plaques.
28. The method of claim 23, wherein said antibody is labeled with a
contrast agent.
29. The method of claim 28, wherein said contrast agent is selected
from the group consisting of gadolinium, dysprosium, and iron.
30. The method of claim 28, wherein said contrast agent is
gadolinium.
Description
TECHNICAL FIELD
[0001] This invention relates to compositions for treating central
nervous system (CNS) disorders such as Alzheimer's disease (AD),
and more particularly, to compositions that contain a .beta.
amyloid (A.beta.) polypeptide linked to a non-A.beta.
polypeptide.
BACKGROUND
[0002] Both active and passive immunization involving
A.beta.-peptides or specific monoclonal antibodies against these
peptides have been assessed for the treatment and prevention of AD.
Reducing A.beta. accumulation by active immunization improves
cognitive performance in mice. See, for example, Chen et al.,
Nature, 408:975-979 (2000); Janus et al. Nature, 408:979-982
(2000); and Morgan et al., Nature, 408:982-985 (2000). The
mechanism by which host-generated antibodies against A.beta. clear
brain senile plaques is far from being understood. Active
immunization experiments use complete Freund's adjuvant, which, by
itself, induces leakage of serum proteins, including IgG, through
the blood-brain barrier (BBB) 2-3 weeks after injection and cannot
be used as an adjuvant in humans. Passive immunization studies are
confounded by the integrity of the BBB, which restricts passage of
immunoglobulins. The permeability coefficient.times.surface area
(PS) product of IgG has been quantified in rats and found to be
very low (0.03-0.1.times.10.sup.-6 mg/g/sec) and is consistent with
a transport mechanism of passive diffusion or fluid-phase
endocytosis.
SUMMARY
[0003] The invention is based on the discovery that A.beta.-immune
complexes are transported across the BBB via a receptor-mediated
process at a rate greater than that of antibody alone. Thus,
transport of antibodies having specific binding affinity for
A.beta. across the BBB, or other polypeptides that have low
permeability at the BBB, can be enhanced when linked to an A.beta.
polypeptide. As a result, the success of passive immunization and
therapy for AD as well as other CNS disorders is enhanced.
Polyamine modified antibodies having specific binding affinity for
A.beta. also have increased permeability at the BBB and can be used
for passive immunization and treatment of AD.
[0004] In one aspect, the invention features a composition that
includes an A.beta. polypeptide and a non-A.beta. polypeptide,
wherein the A.beta. polypeptide and the non-A.beta. polypeptide are
linked (e.g., covalently). The composition further can include a
pharmaceutically acceptable carrier or excipient. The non-A.beta.
polypeptide can be an antibody or a fragment thereof (e.g., a Fab
fragment, a single chain Fv antibody fragment, or a F(ab).sub.2
fragment). The antibody can be labeled with a radioisotope or a
contrast agent. The antibody can have specific binding affinity for
amyloid. The non-A.beta. polypeptide also can be an enzyme such as
an antioxidant enzyme (e.g., catalase or superoxide dismutase), a
cytokine such as an interferon, an interleukin, or a neurotrophic
factor, or leptin. The A.beta. polypeptide can include residues
1-40, 1-42, or 1-43 of SEQ ID NO:1.
[0005] The invention also features a method of treating a patient
diagnosed with AD. The method includes administering to the patient
an amount of a composition effective to treat AD, wherein the
composition includes an A.beta. polypeptide and an antibody having
specific binding affinity for the A.beta. polypeptide. The antibody
can be a Fab fragment, a single chain Fv antibody fragment, or a
F(ab).sub.2 fragment.
[0006] In another aspect, the invention features a method of
treating a patient diagnosed with AD. The method includes
administering to the patient an amount of an antibody effective to
treat AD, wherein the antibody is polyamine modified and has
specific binding affinity for an AD polypeptide.
[0007] In yet another aspect, the invention features a method of
diagnosing AD in a patient. The method includes administering a
composition to the patient, wherein the composition includes an
A.beta. polypeptide and an antibody having specific binding
affinity for amyloid, wherein the antibody is labeled, and
detecting the presence or absence of the antibody bound to amyloid
in the brain of the patient, wherein the patient is diagnosed with
AD based on the presence of labeled amyloid (e.g., labeled amyloid
deposits such as .beta.-amyloid plaques). The detecting step can
include diagnostic imaging (e.g., positron emission tomography,
gamma-scintigraphy, single photon emission computerized tomography,
magnetic resonance imaging, functional magnetic resonance imaging,
or magnetoencephalography). Magnetic resonance imaging is
particularly useful. The antibody can be labeled with a contrast
agent (e.g., gadolinium, dysprosium, or iron). Gadolinium is a
particularly useful contrast agent.
[0008] Unless otherwise defined, all technical and scientific terms
used herein have the same meaning as commonly understood by one of
ordinary skill in the art to which this invention belongs. Although
methods and materials similar or equivalent to those described
herein can be used to practice the invention, suitable methods and
materials are described below. All publications, patent
applications, patents, and other references mentioned herein are
incorporated by reference in their entirety. In case of conflict,
the present specification, including definitions, will control. In
addition, the materials, methods, and examples are illustrative
only and not intended to be limiting.
[0009] Other features and advantages of the invention will be
apparent from the following detailed description, and from the
claims.
DETAILED DESCRIPTION
[0010] The invention features compositions containing A.beta.
polypeptides that can be used to enhance transport of non-A.beta.
polypeptides across the BBB. As described herein, BBB permeability
of a composition containing A.beta. bound to a monoclonal antibody
was significantly greater than that of the monoclonal antibody
alone. Without being bound by a particular mechanism, A.beta.
itself may be responsible for transporting the antibody across the
BBB. Thus, A.beta. can be used to enhance the permeability of other
polypeptides at the BBB, and as a result, compositions of the
invention can be used in the diagnosis, treatment, and/or
prevention of neurodegenerative disorders such as AD, Parkinson's
disease, frontotemporal dementias (e.g., Pick's disease), and
amyloidotic polyneuropathies, transmissible spongiform
encephalopathies (i.e., prion diseases) such as Creutzfeldt-Jakob
disease (CJD), Gerstmann-Straiussler-Scheinker syndrome, and fatal
familial insomnia, demyelinating diseases such as multiple
sclerosis, and amyotropic lateral sclerosis.
[0011] A.beta. Compositions
[0012] Compositions of the invention include a purified A.beta.
polypeptide linked to a purified non-A.beta. polypeptide. As used
herein, the term "purified" refers to a polypeptide that is
separated from cellular components (e.g., other polypeptides,
lipids, carbohydrates, and nucleic acids) that are naturally
associated with the polypeptide. Thus, a purified polypeptide is
any polypeptide that is removed from its natural environment and is
at least 75% pure (e.g., at least about 80, 85, 90, 95, or 99%
pure). Typically, a purified polypeptide will yield a single major
band on a non-reducing polyacrylamide gel.
[0013] As used herein, "A.beta. polypeptide" refers to 1) the
naturally occurring human A.beta. polypeptide (DAEFRHDSGY
EVHHQKLVFF AEDVGSNKGA IIGLMVGGVV IAT, SEQ ID NO:1) 2) polypeptides
having one or more substitutions or insertions in the amino acid
sequence of the naturally occurring human A.beta. polypeptide that
retain the ability to cross the BBB, and 3) fragments of 1) and 2)
that retain the ability to cross the BBB. Permeability of an
A.beta. polypeptide at the BBB can be assessed according to the
methods of Example 1. See also Poduslo et al., Proc. Natl. Acad.
Sci USA 89:2218-2222 (1992) and Poduslo et al., Neurobiol. Disease
8:555-567 (2001). The naturally-occurring human A.beta. polypeptide
ranges in length from 39 to 43 amino acids (residues 1 to 39, 1 to
40, 1 to 41, 1 to 42, or 1 to 43 of SEQ ID NO:1) and is a
proteolytic cleavage product of the amyloid precursor protein
(APP). Non-limiting examples of amino acid substitutions that can
be introduced into human A.beta. include substitutions at amino
acid residues 5, 10, 13, 19, and 20 of SEQ ID NO:1, or combinations
thereof. In particular, a glycine can be substituted for the
arginine at residue 5, a phenylalanine can be substituted for the
tyrosine at residue 10, or an arginine can be substituted for the
histidine at residue 13. Such substitutions do not alter the
properties of human A.beta. polypeptide. See Fraser et al.,
Biochemistry 31:10716-10723 (1992); and Hilbich et al., Eur. J.
Biochem. 201:61-69 (1992). An isoleucine, leucine, threonine,
serine, alanine, valine, or glycine can be substituted for the
phenylalanine residues at positions 19 and 20.
[0014] Suitable fragments of A.beta. polypeptides are about 6 to 38
amino acid residues in length (e.g., 10 to 36, 10 to 34, 10 to 30,
12 to 28, 14 to 26, 16 to 24, or 18 to 22 amino acid residues in
length) and retain the ability to cross the BBB. For example, an
A.beta. polypeptide may contain residues 1 to 10, 1 to 15, 1 to 20,
5 to 15, 5 to 20, 5 to 25, 10 to 20, 10 to 25, 10 to 30, 15 to 25,
15 to 30, or 15 to 35 of SEQ ID NO:1. Alternatively, an A.beta.
polypeptide may include residues 20 to 30, 20 to 35, 20 to 40, 25
to 35, 25 to 40, 30 to 40, 25 to 42, or 30 to 42 of SEQ ID
NO:1.
[0015] A.beta. polypeptides can be linked to non-A.beta.
polypeptides via covalent links. Covalent cross-linking techniques
are known in the art. See, for example, "Chemistry of Protein
Conjugation and Cross-Linking", Shan S. Wong, CRC Press, Ann Arbor,
1991. Suitable cross-linking reagents do not interfere with the
binding of the A.beta. polypeptide to its cognate receptor and are
chosen for appropriate reactivity, specificity, spacer arm length,
membrane permeability, cleavability, and solubility
characteristics. Similarly, suitable cross-linking reagents do not
interfere with binding of a non-A.beta. polypeptide to its binding
partner (e.g., cognate receptor or epitope on a macromolecule).
Cross-linking reagents are available commercially from many sources
including Pierce Chemical Co., Rockford, Ill.
[0016] An A.beta. polypeptide and a non-A.beta. polypeptide can be
covalently cross-linked using, for example, glutaraldehyde, a
homobifunctional cross-linker, or a heterobifunctional
cross-linker. Glutaraldehyde cross-links polypeptides via their
amino moieties. Homobifunctional cross-linkers (e.g., a
homobifunctional imidoester, a homobifunctional
N-hydroxysuccinimidyl (NHS) ester, or a homobifunctional sulfhydryl
reactive cross-linker) contain two or more identical reactive
moieties and can be used in a one step reaction procedure in which
the cross-linker is added to a solution containing a mixture of the
polypeptides to be linked. Homobifunctional NHS esters and imido
esters cross-link amine containing polypeptides. In a mild alkaline
pH, imido esters react only with primary amines to form
imidoamides, and overall charge of the cross-linked polypeptides is
not affected. Homobifunctional sulfhydryl reactive cross-linkers
include bismaleimidhexane (BMH), 1,5-difluoro-2,4-dinitrobenzene
(DFDNB), and 1,4-di-(3',2'-pyridyldithio) propionamido butane
(DPDPB).
[0017] Heterobifunctional cross-linkers have two or more different
reactive moieties (e.g., an amine reactive moiety and a
sulfhydryl-reactive moiety) and are cross-linked with one of the
polypeptides via the amine or sulfhydryl reactive moiety, then
reacted with the other polypeptide via the non-reacted moiety.
Multiple heterobifunctional. haloacetyl cross-linkers are
available, as are pyridyl disulfide cross-linkers. Carbodiimides
are a classic example of heterobifunctional cross-linking reagents
for coupling carboxyls to amines, which results in an amide
bond.
[0018] Alternatively, an A.beta. polypeptide can be linked to a
non-A.beta. polypeptide such as an antibody via the specific
binding affinity of the antibody for the A.beta. polypeptide.
Purified A.beta. polypeptide and antibody can be incubated together
at 37.degree. C. in an appropriate buffer (e.g., phosphate buffered
saline) to form an immune complex. Such an immune complex
constitutes a composition of the invention.
[0019] A.beta. polypeptides can be linked to any non-A.beta.
polypeptide, and in particular, to any polypeptide that is useful
for diagnosing or treating a disorder of the CNS. Non-A.beta.
polypeptides are at least six amino acid residues in length. For
example, an A.beta. polypeptide can be linked to an enzyme such as
an antioxidant enzyme, which can protect cells against reactive
oxygen species. Non-limiting examples of antioxidant enzymes
include catalase (E.C. 1.11.1.6), superoxide dismutase (E.C.
1.15.1.1), glutathione peroxidase (E.C. 1.6.4.2), and glutathione
reductase (E.C. 1.11.1.9).
[0020] A.beta. polypeptides also can be linked to cytokines such as
an interferon (e.g., interferon .alpha., .beta., or .gamma.),
interleukin (IL) (e.g., IL-1a or b, IL-2, IL-3, IL-4, IL-5, IL-6,
IL-7, IL-8, IL-9, IL-10, IL-11, or IL-12), neurotrophic factors
such as neurotrophins (e.g., nerve growth factor or brain-derived
neurotrophic factor), neuropoietic factors such as cholinergic
differentiation factor, ciliary neurotrophic factor, oncostatin M,
growth-promoting factor, and sweat gland factor, and growth factor
peptides such as glial-cell line-derived neurotrophic factor, or a
hormone such as leptin.
[0021] In addition, A.beta. polypeptides can be linked to an
antibody. For example, an A.beta. polypeptide can be linked to an
antibody having specific binding affinity for amyloid deposits of
A.beta. or of a prion protein (PrP). See U.S. Pat. No. 5,231,000
and U.S. Pat. No. 5,262,332 for examples of antibodies having
specific binding affinity for A.beta.. See Zanusso et al., Proc.
Natl. Acad. Sci. USA, 95:8812-8816 (1998) for examples of
antibodies having specific binding affinity for the protease
resistant form of PrP. As used herein, the term "antibodies"
includes polyclonal or monoclonal antibodies, humanized or chimeric
antibodies, and antibody fragments such as single chain Fv antibody
fragments, Fab fragments, and F(ab).sub.2 fragments. Monoclonal
antibodies are particularly useful. A chimeric antibody is a
molecule in which different portions are derived from different
animal species, such as those having a variable region derived from
a murine monoclonal antibody and a human immunoglobulin constant
region. Chimeric antibodies can be produced through standard
techniques.
[0022] Antibody fragments can be generated by known techniques. For
example, F(ab').sub.2 fragments can be produced by pepsin digestion
of the antibody molecule, and Fab fragments can be generated by
reducing the disulfide bridges of F(ab').sub.2 fragments.
Alternatively, Fab expression libraries can be constructed. See,
for example, Huse et al., Science, 246:1275 (1989). Single chain Fv
antibody fragments are formed by linking the heavy and light chain
fragments of the Fv region via an amino acid bridge (e.g., 15 to 18
amino acids), resulting in a single chain polypeptide. See, for
example, U.S. Pat. No. 4,946,778.
[0023] In some embodiments, the A.beta. polypeptide and/or the
non-A.beta. polypeptide are labeled to facilitate diagnosis of a
CNS disorder. Typical labels that are useful include radioisotopes
and contrast agents used for imaging procedures in humans.
Non-limiting examples of labels include radioisotope such as
.sup.1231I (iodine), .sup.18F (fluorine), .sup.99mTc (technetium),
.sup.111In (indium), and .sup.67Ga (gallium), and contrast agents
such as gadolinium (Gd), dysprosium, and iron. Radioactive Gd
isotopes (.sup.153Gd) also are available and suitable for imaging
procedures in non-human mammals. Polypeptides can be labeled
through standard techniques. For example, polypeptides can be
iodinated using chloramine T or
1,3,4,6-tetrachloro-3.alpha.,6.alpha.-diphenylglyco- uril. For
fluorination, polypeptides are synthesized and fluorine is added
during the synthesis by a fluoride ion displacement reaction. See,
Muller-Gartner, H., TIB Tech., 16:122-130 (1998) and Saji, H.,
Crit. Rev. Ther. Drug Carrier Syst., 16(2):209-244 (1999) for a
review of synthesis of proteins with such radioisotopes.
[0024] Polypeptides also can be labeled with a contrast agent
through standard techniques. For example, polypeptides can be
labeled with. Gd by conjugating low molecular Gd chelates such as
Gd diethylene triamine pentaacetic acid (GdDTPA) or Gd
tetraazacyclododecanetetraacetic (GdDOTA) to the polypeptide. See,
Caravan et al., Chem. Rev. 99:2293-2352 (1999) and Lauffer et al.
J. Magn. Reson. Imaging 3:11-16 (1985). Antibodies can be labeled
with Gd by, for example, conjugating polylysine-Gd chelates to the
antibody. See, for example, Curtet et al., Invest. Radiol.
33(10):752-761 (1998). Alternatively, antibodies can be labeled
with Gd by incubating paramagnetic polymerized liposomes that
include Gd chelator lipid with avidin and biotinylated antibody.
See, for example, Sipkins et al. Nature Med., 4 623-626 (1998).
[0025] Nucleic Acids Encoding A.beta. and Non-A.beta.
Polypeptides
[0026] Isolated nucleic acid molecules encoding A.beta. and
non-A.beta. polypeptides of the invention can be produced by
standard techniques. As used herein, "isolated" refers to a
sequence corresponding to part or all of a gene encoding an A.beta.
or non-A.beta. polypeptide, but free of sequences that normally
flank one or both sides of the wild-type gene in a mammalian
genome. An isolated nucleic acid can be, for example, a recombinant
DNA molecule, provided one or both of the nucleic acid sequences
normally found immediately flanking that DNA molecule in a
naturally-occurring genome is removed or absent. Thus, isolated
nucleic acids include, without limitation, a DNA that exists as a
separate molecule (e.g., a cDNA or genomic DNA fragment produced by
PCR or restriction endonuclease treatment) independent of other
sequences as well as recombinant DNA that is incorporated into a
vector, an autonomously replicating plasmid, a virus (e.g., a
retrovirus, adenovirus, or herpes virus), or into the genomic DNA
of a prokaryote or eukaryote. In addition, an isolated nucleic acid
can include a recombinant DNA molecule that is part of a hybrid or
fusion nucleic acid. A nucleic acid existing among hundreds to
millions of other nucleic acids within, for example, cDNA or
genomic libraries, or gel slices containing a genomic DNA
restriction digest, is not to be considered an isolated nucleic
acid.
[0027] Isolated nucleic acid molecules are at least about 18
nucleotides in length. For example, the nucleic acid molecule can
be about 18 to 20, 20-50, 50-100, or greater than 150 nucleotides
in length. Nucleic acid molecules can be DNA or RNA, linear or
circular, and in sense or antisense orientation.
[0028] Specific point changes can be introduced into the nucleic
acid sequence encoding the naturally-occurring human A.beta.
polypeptide by, for example, oligonucleotide-directed mutagenesis.
In this method, a desired change is incorporated into an
oligonucleotide, which then is hybridized to the wild-type nucleic
acid. The oligonucleotide is extended with a DNA polymerase,
creating a heteroduplex that contains a mismatch at the introduced
point change, and a single-stranded nick at the 5' end, which is
sealed by a DNA ligase. The mismatch is repaired upon
transformation of E. coli or other appropriate organism, and the
gene encoding the modified vitamin K-dependent polypeptide can be
re-isolated from E. coli or other appropriate organism. Kits for
introducing site-directed mutations can be purchased commercially.
For example, Muta-Gene.RTM. in-vitro mutagenesis kits can be
purchased from Bio-Rad Laboratories, Inc. (Hercules, Calif.).
[0029] Polymerase chain reaction (PCR) techniques also can be used
to introduce mutations. See, for example, Vallette et al., Nucleic
Acids Res., 17(2):723-733 (1989). PCR refers to a procedure or
technique in which target nucleic acids are amplified. Sequence
information from the ends of the region of interest or beyond
typically is employed to design oligonucleotide primers that are
identical in sequence to opposite strands of the template to be
amplified, whereas for introduction of mutations, oligonucleotides
that incorporate the desired change are used to amplify the nucleic
acid sequence of interest. PCR can be used to amplify specific
sequences from DNA as well as RNA, including sequences from total
genomic DNA or total cellular RNA. Primers are typically 14 to 40
nucleotides in length, but can range from 10 nucleotides to
hundreds of nucleotides in length. General PCR techniques are
described, for example in PCR Primer: A Laboratory Manual, Ed. by
Dieffenbach, C. and Dveksler, G., Cold Spring Harbor Laboratory
Press, 1995.
[0030] Nucleic acids encoding A.beta. and non-A.beta. polypeptides
also can be produced by chemical synthesis, either as a single
nucleic acid molecule or as a series of oligonucleotides. For
example, one or more pairs of long oligonucleotides (e.g., >100
nucleotides) can be synthesized that contain the desired sequence,
with each pair containing a short segment of complementarity (e.g.,
about 15 nucleotides) such that a duplex is formed when the
oligonucleotide pair is annealed. DNA polymerase is used to extend
the oligonucleotides, resulting in a double-stranded nucleic acid
molecule per oligonucleotide pair, which then can be ligated into a
vector.
[0031] Producing Purified Polypeptides
[0032] Purified A.beta. and non-A.beta. polypeptides of the
invention can be obtained from commercial sources, or
alternatively, can be obtained by extraction from a natural source
(e.g., liver tissue), chemical synthesis, or by recombinant
production in a host cell. In general, recombinant polypeptides are
produced by introducing an expression vector that contains a
nucleic acid encoding the polypeptide of interest operably linked
to regulatory elements necessary for expression of the polypeptide
into a bacterial or eukaryotic host cell (e.g., insect, yeast, or
mammalian cells). Regulatory elements do not typically encode a
gene product, but instead affect the expression of the nucleic acid
sequence. In bacterial systems, a strain of Escherichia coli such
as BL-21 can be used. Suitable E. coli vectors include the pGEX
series of vectors that produce fusion proteins with glutathione
S-transferase (GST). Transformed E. coli are typically grown
exponentially then stimulated with isopropylthiogalactopyranoside
(IPTG) prior to harvesting. Such fusion proteins typically are
soluble and can be purified easily from lysed cells by adsorption
to glutathione-agarose beads followed by-elution in the presence of
free glutathione. The pGEX vectors are designed to include thrombin
or factor Xa protease cleavage sites so that the cloned target gene
product can be released from the GST moiety.
[0033] In eukaryotic host cells, a number of viral-based expression
systems can be utilized to produce the polypeptides of interest. A
nucleic acid encoding a polypeptide of the invention can be cloned
into, for example, a baculoviral vector such as pBlueBac
(Invitrogen, San Diego, Calif.) and then used to co-transfect
insect cells such as Spodoptera frugiperda (Sf9) cells with wild
type DNA from Autographa californica multinuclear polyhedrosis
virus (AcMNPV). Recombinant viruses producing polypeptides of the
invention can be identified by standard methodology. Alternatively,
a nucleic acid encoding a polypeptide of the invention can be
introduced into a SV40, retroviral, or vaccinia based viral vector
and used to infect suitable host cells.
[0034] Mammalian cell lines that stably express a polypeptide of
interest can be produced using an expression vector that contains a
selectable marker and standard techniques. For example, the
eukaryotic expression vector pCR3.1 (Invitrogen, San Diego, Calif.)
can be used to express polypeptides of interest in, for example,
Chinese hamster ovary (CHO) cells, COS-1 cells, human embryonic
kidney 293 cells, NIH3T3 cells, BHK21 cells, MDCK cells, and human
vascular endothelial cells (HUVEC). Following introduction of the
expression vector by electroporation, lipofection, calcium
phosphate or calcium chloride co-precipitation, DEAE dextran, or
other suitable transfection method, stable cell lines are selected,
e.g., by antibiotic resistance to G418, kanamycin, or hygromycin.
Alternatively, a nucleic acid encoding the polypeptide of interest
can be ligated into a mammalian expression vector such as pcDNA3
(Invitrogen, San Diego, Calif.) then transcribed and translated in
vitro using wheat germ extract or rabbit reticulocyte lysate.
[0035] Polypeptides of interest can be purified by known
chromatographic methods including DEAE ion exchange, gel
filtration, and hydroxylapatite chromatography Polypeptides can be
"engineered" to contain an amino acid sequence that allows the
polypeptide to be captured onto an affinity matrix. For example, a
tag such as c-myc, hemagglutinin, polyhistidine, or Flag.TM. tag
(Kodak) can be used to aid polypeptide purification. Such tags can
be inserted anywhere within the polypeptide including at either the
carboxyl or amino termini. Other fusions that could be useful
include enzymes that aid in the detection of the polypeptide, such
as alkaline phosphatase. Immunoaffinity chromatography also can be
used to purify polypeptides of interest.
[0036] Polyamine Modified Antibodies
[0037] As described herein, polyamine modification of an antibody
having specific binding affinity for A.beta. enhances permeability
of the modified antibody at the BBB. In particular,
polyamine-modified monoclonal antibody against A.beta. has a PS
product that is 36 fold higher in the cortex compared to unmodified
antibody and may provide a better approach to passive immunization
for AD. Antibodies having specific binding affinity for A.beta. can
be modified with polyamines that are either naturally occurring or
synthetic. See, for example, U.S. Pat. No. 5,670,477. Useful
naturally occurring polyamines include putrescine, spermidine,
spermine, 1,3-diaminopropane, norspermidine, syn-homospermidine,
thermine, thermospermine, caldopentamine, homocaldopentamine, and
canavalmine. Putrescine, spermidine, and spermine are particularly
useful. Synthetic polyamines are composed of the empirical formula
C.sub.xH.sub.yN.sub.z, and can be cyclic or acyclic, branched or
unbranched, hydrocarbyl chains of 3-12 carbon atoms that further
include 1-6 NR or N(R).sub.2 moieties, wherein R is H,
(C.sub.1-C.sub.4) alkyl, phenyl, or benzyl. Polyamines can be
linked to an antibody using the cross-linking techniques described
above.
[0038] Diagnosis or Treatment of a CNS Disorder
[0039] Compositions of the invention can be formulated with a
pharmaceutically acceptable carrier and administered to a mammal.
For example, a composition of the invention can be administered to
a non-human animal (e.g., a transgenic mouse model of Alzheimer's
disease). or to a human to aid in the diagnosis of a CNS disorder
such as Alzheimer's disease or for treating a human patient that
has been diagnosed with a CNS disorder. As used herein, the term
"treatment" or "treating" refers to administering a composition of
the invention to a patient, regardless of whether the patient
responds to the treatment, with the proviso that when the same
composition is administered to a population of patients, a
statistically significant number of patients within the population
exhibit a clinically recognized improvement or stabilization of one
or more clinical features of the disorder.
[0040] In general, compositions of the invention are administered
intravenously (i.v.), although other parenteral routes of
administration, including subcutaneous, intramuscular,
intra-arterial, intranasal, intracarotid, and intrathecal also can
be used. Formulations for parenteral administration may contain
pharmaceutically acceptable carriers such as sterile water or
saline, polyalkylene glycols such as polyethylene glycol, vegetable
oils, hydrogenated naphthalenes, and the like.
[0041] The dosage of the composition to be administered can be
determined by the attending physician taking into account various
factors known to modify the action of drugs. These include health
status, body weight, sex, diet, time and route of administration,
other medications, and any other relevant clinical factors.
Typically, the dosage is about 1-3000 .mu.g/kg body weight (e.g.,
from about 10-1000 .mu.g/kg body weight or 50-500 .mu.g/kg body
weight). Therapeutically effective dosages may be determined by
either in vitro or in vivo methods.
[0042] Treatment of a CNS disorder can be assessed by determining
if one or more clinical features of the disorder (e.g., cognitive
function, memory, behavior, language skills, motor skills, or
rigidity of the patient) improve or are stabilized in the
patient.
[0043] For diagnosis of a CNS disorder, the composition that is
administered to the patient contains at least one polypeptide that
is labeled as described above. Presence or absence of the labeled
polypeptide (e.g., labeled antibody or labeled A.beta. polypeptide)
is detected in the CNS in vivo (e.g., in the brain of the patient)
using, for example, imaging techniques such as positron emission
tomography (PET), gamma-scintigraphy, magnetic resonance imaging
(MRI), functional magnetic resonance imaging (FMRI),
magnetoencephalography (MEG), and single photon emission
computerized tomography (SPECT). MRI is particularly useful as the
spatial resolution and signal-to-noise ratio provided by MRI (30
microns) is suitable for detecting amyloid deposits, which can
reach up to 200 microns in size. The CNS disorder can be diagnosed
based on the presence, for example, of labeled amyloid (e.g.,
labeled amyloid deposits).
[0044] The invention will be further described in the following
examples, which do not limit the scope of the invention described
in the claims.
EXAMPLES
Example 1
Materials and Methods
[0045] A.beta. Proteins: Human A.beta..sub.1-42 was synthesized by
using f-moc chemistry in a Perkin-Elmer peptide synthesizer in the
Mayo Protein Core Facility. The amino acid sequence of human
A.beta. is provided in SEQ ID NO:1. Purity of the peptide was
evaluated by peptide sequencing and laser desorption mass
spectrometry.
[0046] Monoclonal Antibody Generation: B-cell hybridomas were
generated following the procedure of St. Groths and Scheidegger (J.
Immunol. Methods 35:1 (1980)) in the Mayo Monoclonal Core Facility.
Human A.beta..sub.1-42 that was aggregated and fibrilized by
incubating at 37.degree. C. for 24 hours was used as antigen.
Positive subclones were isotyped and cryopreserved and further
characterized by ELISA and immunohistochemistry labeling of AD
transgenic mouse brain sections. A non-specific, monoclonal
antibody was obtained from ATCC HB96 L227 (anti-human Ia).
[0047] PS/V.sub.p Measurements at the BBB for Radioiodinated
Monoclonal lgG (MoIgG): Aliquots of the proteins (MoIgG or A.beta.)
were labeled with .sup.125I or .sup.131I using the chloramnine T
procedure described by Poduslo et al., Proc. Natl. Acad. Sci. USA
9:5705-5709 (1994). PS/V.sub.p measurements were performed as
described by Poduslo et al., Neurobiol. Diseases, 8:555-567 (2001)
and Poduslo et al., Proc. Natl. Acad. Sci. USA 89:2218-2222 (1992).
The procedure for quantifying BBB permeability of proteins was
adapted from the rat to the mouse and included catheterizing the
femoral artery and vein of the mouse instead of the brachial artery
and vein as for the rat. Because of the smaller blood volume in the
mouse, serial sampling of 20 .mu.l of blood from the femoral artery
was performed and directly TCA precipitated to generate a whole
blood washout curve for the intact protein. Briefly, an I.V. bolus
injection of phosphate-buffered saline (PBS) containing
.sup.125I-MoIgG (100 .mu.C) was rapidly injected into the femoral
vein in pentobarbital-anesthetized mice. Serial blood samples were
collected from the femoral artery over the next 30-120 minutes. At
30-60 seconds before the end of the experiment, the second isotope
of radiolabeled protein (.sup.131I-MoIgG) (100 .mu.C) was
administered intravenously to serve as a V.sub.p indicator.
[0048] After the final blood sample, the animals were sacrificed,
the brain and meninges were removed, and the brain was dissected
into the cortex, caudate-putamen (neostriatum), hippocampus,
thalamus, brain stem, and cerebellum. Tissue was lyophilized, and
dry weights were determined with a microbalance and converted to
respective wet weights with wet weight/dry weight ratios previously
determined. Tissue and plasma samples were assayed for .sup.125I
and .sup.131I radioactivity in a two-channel gamma counter (Packard
COBRA II) with radioactivity corrected for crossover of .sup.131I
activity into the .sup.125I channel and background. Data are
presented as {overscore (x)}.+-.SEM values with statistical
evaluation using ANOVA with significance accepted at the P<0.05
level. The V.sub.p and PS measurements were calculated as described
by Poduslo et al., Neurobiol. Disease, 8:555-567 (2001) and Poduslo
et al., Proc. Natl. Acad. Sci. USA 89:2218-2222 (1992). All
procedures were performed using humane and ethical protocols
approved by the Mayo Clinic Institutional Animal Care and Use
Committee, in accordance with the National Institute of Health's
Guide for the Care and Use of Laboratory Animals. All efforts were
made to minimize both the suffering and the number of animals
used.
[0049] Immune Comlex Preparation: Human A.beta.42 was incubated
with its radioiodinated monoclonal antibody (PC2) or the
radioiodinated non-specific monoclonal antibody (L227) for 1 hour
at 37.degree. C. in PBS at mole ratios of 10:1, 100:1, or 1000:1.
Aliquots were then injected into the femoral vein as an I.V.
bolus.
[0050] Polyamine Modification of Monoclonal IgG: Modification of
the monoclonal antibody (PC2) was performed as described by Poduslo
and Curran, Proc.Natl. Acad. Sci. USA 89:2218-2222 (1992) and
Poduslo and Curran, J. Neurochem. 66:1599-1609 (1996). Putrescine
(PUT) was covalently attached to carboxylic acids using
carbodiimide. Ionization of the carboxylic acid groups was
controlled by pH, which in turn controlled the extent of
modification with the polyamine.
Example 2
Enhanced Permeability of Polyamine Modified Antibody and Immune
Complexes at the BBB
[0051] The BBB permeability of a non-specific monoclonal antibody
(anti-human Ia; L227; IgG.sub.1.kappa.), monoclonal antibody
against human A.beta..sub.1-42 (PC2; IgG.sub.1.kappa.), and the
immune complex [(human A.beta.42)-L227 or (human A.beta.42)-PC2)]
at various mole ratios was determined in the normal adult mouse
(B6SJL) as described in Example 1 by quantifying the permeability
coefficient.times.surface area (PS) product for each protein after
correction for the residual plasma volume (V.sub.p) occupied by the
protein in blood vessels in different brain regions following an
I.V. bolus injection. In these experiments, the V.sub.p value was
determined with a second aliquot of the same protein radioiodinated
with a different isotope of iodine (.sup.125I vs. .sup.131I) given
30-60 seconds before the end of the experiment. Using the same test
substance allows for an accurate determination of the V.sub.p and
corrects for non-specific adherence to capillary walls, which would
be characteristic of the protein tested. Similarly, a dual isotope
approach allows for the determination of the vascular space in each
individual animal. The PS product at the BBB for different
radioiodinated proteins is corrected, therefore, for the V.sub.p
with a second tracer of the same protein.
[0052] The PS product for the non-specific monoclonal antibody
(L227) ranged from 0.5-1.1.times.10.sup.-6 ml/g/sec in six
different brain regions (Table 1). The PS values for the monoclonal
antibody to human A.beta. 1-42 (PC2) ranged from
0.6-1.4.times.10.sup.-6 ml/g/sec in the same brain regions and were
not significantly different. V.sub.p values ranged from 12.8-28.4
.mu.l/g for L227 and from 11.8-28.0 .mu.l/g for PC2 and were not
significantly different (Table 1). The PS values for both
monoclonal antibodies are low and less than that observed for
albumin. Both IgG and albumin are considered to be transported at
the BBB by passive diffusion or fluid phase endocytosis. In
contrast, insulin has very high PS values in mice
(27.7-43.0.times.10.sup.-6 ml/g/sec) and is transported at the BBB
by a receptor-mediated transport. Insulin has a PS product at the
BBB that is approximately 28.3-49.9 fold greater than that of the
monoclonal antibody to human A.beta.42 (PC2). In contrast, the
V.sub.p values for insulin and the monoclonal antibody to human
A.beta.42 (PC2) are similar.
1TABLE 1 BBB Permeability for the Immune Complex [(hA.beta.42)-PC2]
is Greater than the Monoclonal Antibody Alone (PC2) or a
Non-Specific Monoclonal Antibody (L227) (hA.beta.42)-L227
(hA.beta.42)-PC2 L227 100:1 PC2 10:1 100:1 1000:1 RI n = 7 n = 6 n
= 14 n = 6 n = 6 n = 7 100:1 vs PC2 PS: ml/g/sec .times. 10.sup.6
Cortex 0.49 .+-. 0.03 0.95 .+-. 0.15 0.71 .+-. 0.10 1.26 .+-. 0.25
2.87 .+-. 0.27*** 2.74 .+-. 0.31*** 4.0 Caudate-Putamen 0.51 .+-.
0.05 0.63 .+-. 0.15 0.64 .+-. 0.05 1.04 .+-. 0.13* 2.33 .+-.
0.15*** 2.04 .+-. 0.08*** 3.6 Hippocampus 0.59 .+-. 0.05 0.90 .+-.
0.25 0.70 .+-. 0.06 1.15 .+-. 0.32 2.43 .+-. 0.32*** 2.82 .+-.
0.25*** 4.0 Thalamus 0.70 .+-. 0.06 1.05 .+-. 0.24 0.81 .+-. 0.06
1.54 .+-. 0.19* 3.21 .+-. 0.17*** 3.09 .+-. 0.31*** 4.0 Brain Stem
1.10 .+-. 0.05 1.84 .+-. 0.30 1.38 .+-. 0.15 2.70 .+-. 0.48* 4.25
.+-. 0.31*** 4.20 .+-. 0.42*** 3.1 Cerebellum 0.82 .+-. 0.05 1.30
.+-. 0.19 0.98 .+-. 0.10 2.36 .+-. 0.59* 3.58 .+-. 0.24*** 3.89
.+-. 0.54*** 4.0 V.sub.p: .mu.l/g Cortex 21.36 .+-. 1.73 26.62 .+-.
1.52 20.07 .+-. 1.14 24.97 .+-. 0.36 24.70 .+-. 3.00 25.60 .+-.
1.89 1.2 Caudate-Putamen 12.77 .+-. 1.45 16.15 .+-. 1.61 11.78 .+-.
0.57 17.73 .+-. 1.99* 17.54 .+-. 1.70 17.60 .+-. 1.98* 1.5
Hippocampus 20.52 .+-. 2.09 25.31 .+-. 3.05 22.51 .+-. 0.91 27.58
.+-. 1.84 26.06 .+-. 3.28 25.94 .+-. 2.50 1.2 Thalamus 18.47 .+-.
1.15 25.84 .+-. 2.70 17.37 .+-. 0.98 23.17 .+-. 1.28* 26.88 .+-.
3.37** 23.13 .+-. 2.34 1.6 Brain Stem 25.14 .+-. 1.63 29.10 .+-.
2.45 23.68 .+-. 1.72 30.34 .+-. 1.47* 31.11 .+-. 2.80 22.79 .+-.
1.44 1.3 Cerebellum 28.43 .+-. 1.99 34.86 .+-. 2.36 27.99 .+-. 1.85
37.32 .+-. 1.96 33.84 .+-. 3.87 30.73 .+-. 2.36 1.2 {overscore (X)}
.+-. SEM L227: ATCC HB96 (Anti-human Ia) IgG.sub.1.kappa.; BALB/c
PS: Permeability coefficient .times. Surface area product PC2: MoAb
(Anti-human A.beta.42) IgG.sub.1.kappa.; BALB/c V.sub.p: Residual
Plasma Volume RI: Relative increase of immune complex
[(hA.beta.42)-PC2] vs. MoAb (PC2) at mole ratios of 100:1
(hA.beta.42)-L227 {close oversize brace} Immune complex at mole
ratios of 10:1, 100:1, or 1000:1 (hA.beta.42)-PC2 Analysis of
variance followed by Bonferroni multiple comparisons; only
significant differences shown; *P < 0.05, **P < 0.01, **P
< 0.001
[0053] Permeability of immune complexes of human A.beta.42 with its
radioiodinated monoclonal antibody at various mole ratios were
assessed as described above. At a mole ratio of 10:1 [(human
A.beta.42)-PC2], a significant increase in the PS at the BBB in
four of six brain regions was observed compared with the PS values
observed for PC2 alone (Table 1). When the mole ratio was increased
to 100:1, highly significant PS values (2.3-4.3.times.10.sup.-6
ml/g/sec) were obtained in all brain regions (P<0.001). This
represents a 3.1 to 4.0-fold increase in the PS values. In
contrast, when human A.beta.42 was incubated with the non-specific
monoclonal antibody (L227) at the same mole ratio of 100:1, the PS
values obtained were not significantly different from that in the
absence of the antigen (Table 1). When human A.beta.42 was
incubated with PC2 at a mole ratio of 1000:1, there was a
non-significant decrease in the PS values for most of the brain
regions indicating that the receptor for human A.beta.42 at the BBB
was beginning to be saturated (Table 1). In contrast, the V.sub.p
values showed a slight trend toward being increased for the
different mole ratios of immune complex compared to the monoclonal
antibody, and this reached significance in only a few cases. These
studies demonstrate that the BBB permeability for the immune
complex of (human A.beta.42)-PC2 is greater than the monoclonal
antibody alone or the non-specific monoclonal antibody. This
suggests that the mechanism by which this antibody is crossing the
BBB likely involves a receptor for human A.beta. at the BBB.
Example 3
Permeability of Polyamine Modified Antibody at the BBB
[0054] In the following series of experiments, PS values ranging
from 21.5-33.0.times.10.sup.-6 ml/g/sec in six different brain
regions (Table 2) were observed for a polyamine modified monoclonal
antibody to human A.beta. (PC2). These PS values for PUT-PC2 were
highly significant (P<0.0001) and ranged from 22.8-37.9 fold
higher than the antibody (PC2) alone. Polyamine modification of the
monoclonal antibody may allow for better delivery across the BBB.
This approach is not dependant upon circulating A.beta. levels and
may allow for a more dramatic reduction in amyloid burden in the
Alzheimer brain following passive immunization.
2TABLE 2 BBB Permeability of Polyamine-Modified Monoclonal Antibody
(PUT-PC2) is Greater than the Monoclonal Antibody Alone (PC2) PC2
PUT-PC2 n = 14 n = 15 P RI PS: ml/g/sec .times. 10.sup.6 Cortex 0.7
.+-. 0.1 25.1 .+-. 1.5 **** 35.9 Caudate-Putamen 0.6 .+-. 0.1 21.5
.+-. 1.4 **** 35.8 Hippocampus 0.6 .+-. 0.1 26.5 .+-. 1.8 **** 37.9
Thalamus 0.8 .+-. 0.1 27.1 .+-. 1.6 **** 33.9 Brain Stem 1.4 .+-.
0.2 31.9 .+-. 3.3 **** 22.8 Cerebellum 1.0 .+-. 0.1 33.0 .+-. 2.3
**** 33.0 V.sub.p: .mu.l/g Cortex 20.1 .+-. 1.1 17.9 .+-. 0.7 ns
0.9 Caudate-Putamen 11.8 .+-. 0.6 9.8 .+-. 0.4 * 0.8 Hippocampus
22.5 .+-. 1.0 18.3 .+-. 1.0 ns 0.8 Thalamus 17.4 .+-. 1.0 17.0 .+-.
0.8 ns 1.0 Brain Stem 23.7 .+-. 1.7 21.9 .+-. 1.2 ns 0.9 Cerebellum
28.0 .+-. 1.9 23.7 .+-. 0.8 ns 0.8 {overscore (x)} .+-. SEM PC2:
MoAb (Anti-human A.beta.42) IgG1.kappa.; BALB/c PUT-PC2:
Putrescine-modified PC2 RI: Relative increase of immune complex
[(hA.beta.42)-PC2] vs. MoAb (PC2) at mole ratios of 100:1 Analysis
by two-tailed unpaired t-test. Ns, not significant (P > 0.05); *
P < 0.05; **** P < 0.0001 PS: Permeability coefficient
.times. Surface area product V.sub.p: Residual Plasma Volume
Other Embodiments
[0055] It is to be understood that while the invention has been
described in conjunction with the detailed description thereof, the
foregoing description is intended to illustrate and not limit the
scope of the invention, which is defined by the scope of the
appended claims. Other aspects, advantages, and modifications are
within the scope of the following claims.
Sequence CWU 1
1
1 1 43 PRT Homo sapiens 1 Asp Ala Glu Phe Arg His Asp Ser Gly Tyr
Glu Val His His Gln Lys 1 5 10 15 Leu Val Phe Phe Ala Glu Asp Val
Gly Ser Asn Lys Gly Ala Ile Ile 20 25 30 Gly Leu Met Val Gly Gly
Val Val Ile Ala Thr 35 40
* * * * *