U.S. patent application number 10/600862 was filed with the patent office on 2005-02-03 for use of the chaperone receptor-associated protein (rap) for the delivery of therapeutic compounds to the brain and other tissues.
This patent application is currently assigned to BioMarin Pharmaceutical Inc.. Invention is credited to Gabathuler, Reinhard, Starr, Christopher M., Zankel, Todd.
Application Number | 20050026823 10/600862 |
Document ID | / |
Family ID | 34103071 |
Filed Date | 2005-02-03 |
United States Patent
Application |
20050026823 |
Kind Code |
A1 |
Zankel, Todd ; et
al. |
February 3, 2005 |
Use of the chaperone receptor-associated protein (RAP) for the
delivery of therapeutic compounds to the brain and other
tissues
Abstract
This invention provides compounds of conjugates of therapeutic
or active agents with RAP or a RAP polypeptide, their
pharmaceutical compositions and methods for using the such
compounds and compositions in the diagnosis, prophylaxis, or
treatment of diseases and conditions, including particularly
diseases of the central nervous system or lysosomal storage
diseases.
Inventors: |
Zankel, Todd; (San
Francisco, CA) ; Starr, Christopher M.; (Sonoma,
CA) ; Gabathuler, Reinhard; (San Rafael, CA) |
Correspondence
Address: |
Nabeela R. McMillian
MARSHALL, GERSTEIN & BORUN LLP
Sears Tower
233 S. Wacker Drive, Suite 6300
Chicago
IL
60606-6357
US
|
Assignee: |
BioMarin Pharmaceutical
Inc.
Novato
CA
|
Family ID: |
34103071 |
Appl. No.: |
10/600862 |
Filed: |
June 20, 2003 |
Current U.S.
Class: |
424/178.1 ;
514/1.2; 514/17.5; 514/17.7; 514/7.4; 530/350 |
Current CPC
Class: |
A61P 25/02 20180101;
A61P 25/28 20180101; A61P 25/18 20180101; C07K 2319/22 20130101;
A61K 38/00 20130101; A61P 25/00 20180101; A61K 47/64 20170801; C07K
14/705 20130101; C07K 2319/23 20130101; C07K 2319/00 20130101; A61P
9/10 20180101; A61P 35/00 20180101; A61P 25/16 20180101 |
Class at
Publication: |
514/012 ;
530/350 |
International
Class: |
A61K 038/17; C07K
014/705 |
Claims
We claim:
1. A compound comprising Receptor-Associated Protein (RAP) or RAP
polypeptide conjugated to an agent of interest.
2. The compound of claim 1, wherein the agent is selected from the
group comprising a therapeutic agent, diagnostic/investigational
agent, labeled monoclonal antibody which binds a marker of a CNS
pathology, and protein.
3. A method of delivering a therapeutic or
diagnostic/investigational agent into the central nervous system by
increasing transport across the blood brain barrier (BBB) in a
subject in need thereof, said method comprising: administering to
said subject a compound comprising Receptor Associated Protein
(RAP) conjugated to a therapeutic or diagnostic/investigational
agent in an amount effective to increase transport across the
BBB.
4. A method of treating a disease or condition in a subject in need
thereof, said method comprising: administering to said subject a
compound comprising RAP conjugated to a therapeutic agent in an
amount effective to treat said disease.
5. The method of claim 4, wherein the disease or condition is a
neurological or psychological condition or disease.
6. The method of claim 5, wherein the condition or disease is AD,
PD, MS, or ALS.
7. The method of claim 4, wherein the condition is a brain tumor or
tumor metastases in the brain and the therapeutic agent is a
chemotherapeutic agent.
8. A method of diagnosing a disease in a subject in need thereof,
said method comprising: administering to said subject a compound
comprising RAP conjugated to a diagnostic/investigational agent in
an amount effective to diagnose said disease.
9. A method of delivering a therapeutic enzyme to a lysosome in a
cell of a subject, said method comprising: (i) administering to
said subject a compound comprising RAP or RAP polypeptide
conjugated to a therapeutic or diagnostic agent; (ii) transporting
said compound across the cell membrane; (iii) contacting said
compound with an LRP receptor on said cell; (iv) facilitating entry
of said compound into said cell; and (v) delivering said compound
to said lysosome in said cell.
10. A method of treating lysosomal storage diseases in a subject in
need thereof, said method comprising: administering to said subject
a compound comprising RAP or RAP polypeptide conjugated to a
therapeutic agent, wherein said compound crosses the cell membrane,
enters cells and is delivered to lysosomes in an amount effective
to treat said lysosomal storage disease.
11. The method of claim 10, wherein the agent is an enzyme
deficient in the lysosomal storage disease.
12. The method of claim 11, wherein the lysosomal storage disease
is selected from the group consisting of aspartylglucosaminuria,
cholesterol ester storage disease, Wolman disease, cystinosis,
Danon disease, Fabry disease, Farber lipogranulomatosis, Farber
disease, fucosidosis, galactosialidosis types VIII, Gaucher disease
types I/II/III, Gaucher disease, globoid cell leukodystrophy,
Krabbe disease, glycogen storage disease II, Pompe disease,
GM1-gangliosidosis types I/II/III, GM2-gangliosidosis type I, Tay
Sachs disease, GM2-gangliosidosis type II, Sandhoff disease,
GM2-gangliosidosis, .alpha.-mannosidosis types I/II,
.beta.-mannosidosis, metachromatic leukodystrophy, mucolipidosis
type I, sialidosis types I/II mucolipidosis types II/III I-cell
disease, mucolipidosis type IIIC pseudo-Hurler polydystrophy,
mucopolysaccharidosis type I, mucopolysaccharidosis type II, Hunter
syndrome, mucopolysaccharidosis type IIIA, Sanfilippo syndrome,
mucopolysaccharidosis type IIIB, mucopolysaccharidosis type IIIC,
mucopolysaccharidosis type IIID, mucopolysaccharidosis type IVA,
Morquio syndrome, mucopolysaccharidosis type IVB Morquio syndrome,
mucopolysaccharidosis type VI, mucopolysaccharidosis type VII, Sly
syndrome, mucopolysaccharidosis type IX, multiple sulfatase
deficiency, neuronal ceroid lipofuscinosis, CLN1 Batten disease,
Niemann-Pick disease types A/B, Niemann-Pick disease, Niemann-Pick
disease type C1, Niemann-Pick disease type C2, pycnodysostosis,
Schindler disease types I/II, Schindler disease, and sialic acid
storage disease.
13. The method of claim 10, wherein the agent is selected from the
group consisting of aspartylglucosaminidase, acid lipase, cysteine
transporter, Lamp-2, .alpha.-galactosidase A, acid ceramidase,
.alpha.-L-fucosidase, .beta.-hexosaminidase A, GM2-activator
deficiency, .alpha.-D-mannosidase, .beta.-D-mannosidase,
arylsulfatase A, saposin B, neuraminidase,
.alpha.-N-acetylglucosaminidase phosphotransferase,
phosphotransferase .gamma.-subunit, L-iduronidase,
iduronate-2-sulfatase, heparan-N-sulfatase,
.alpha.-N-acetylglucosaminidase, acetylCoA:N-acetyltransferase,
N-acetylglucosamine 6-sulfatase, galactose 6-sulfatase,
.beta.-galactosidase, N-acetylgalactosamine 4-sulfatase,
hyaluronoglucosaminidase, multiple sulfatases, palmitoyl protein
thioesterase, tripeptidyl peptidase I, acid sphingomyelinase,
cholesterol trafficking, cathepsin K, .alpha.-galactosidase B, and
sialic acid transporter.
Description
CROSS-REFERENCE TO RELATED APPLICATIONS
[0001] This application is a continuation-in-part of U.S. patent
application Ser. No. 10/206,448, filed on Jul. 25, 2002, which
claims the benefit of U.S. Provisional Patent Application No.
60/308,002, filed Jul. 25, 2001. The contents of these and all
other U.S. patents cited herein are each hereby incorporated by
reference in their entirety.
FIELD OF THE INVENTION
[0002] The present invention is related to compositions comprising
Receptor-Associated Protein (RAP) linked to a therapeutic and/or
diagnostic/investigational agent, and methods of using such
compounds.
BACKGROUND OF THE INVENTION
[0003] The brain is shielded against potentially harmful substances
by the blood-brain barrier (BBB). The microvascular barrier between
blood and brain is made up of a capillary endothelial layer
surrounded by a basement membrane and tightly associated accessory
cells (pericytes, astrocytes). The brain capillary endothelium is
much less permeable to low-molecular weight solutes than other
capillary endothelia due to an apical band of tight association
between the membranes of adjoining cells, referred to as tight
junctions. In addition to diminished passive diffusion, brain
capillary endothelia also exhibit less fluid-phase pinocytosis than
other endothelial cells. Brain capillaries possess few fenestrae
and few endocytic vesicles, compared to the capillaries of other
organs (see Pardridge, J. Neurovirol. 5: 556-569 (1999)). There is
little transit across the BBB of large, hydrophilic molecules aside
from some specific proteins such as transferrin, lactoferrin and
low-density lipoproteins, which are taken up by receptor-mediated
endocytosis (see Pardridge, 1999); Tsuji and Tamai, Adv.Drug
Deliv.Rev. 36: 277-290 (1999); Kusuhara and Sugiyama, Drug Discov.
Today 6:150-156 (2001); Dehouck, et al. J. Cell. Biol. 138: 877-889
(1997); Fillebeen, et al. J. Biol. Chem. 274: 7011-7017
(1999)).
[0004] The blood-brain barrier (BBB) also impedes access of
beneficial active agents (e.g., therapeutic drugs and diagnostic
agents) to central nervous system (CNS) tissues, necessitating the
use of carriers for their transit. Blood-brain barrier permeability
is frequently a rate-limiting factor for the penetration of drugs
or peptides into the CNS (see Pardridge, 1999); Bickel, et al.,
Adv. Drug Deliv. Rev. 46: 247-279 (2001)). For example, management
of the neurological manifestations of lysosomal storage diseases
(LSDs) is significantly impeded by the inability of therapeutic
enzymes to gain access to brain cell lysosomes. LSDs are
characterized by the absence or reduced activity of specific
enzymes within cellular lysosomes, resulting in the accumulation of
undegraded "storage material" within the intracellular lysosome,
swelling and malfunction of the lysosomes, and ultimately cellular
and tissue damage. Intravenous enzyme replacement therapy (ERT) is
beneficial for LSDs (e.g. MPS I, MPS II). However, the BBB blocks
the free transfer of many agents from blood to brain, and LSDs that
present with significant neurological sequelae (e.g. MPS III, MLD,
GM1) are not expected to be as responsive to intravenous ERT. For
such diseases, a method of delivering the replacement enzyme across
the BBB and into the lysosomes of the affected cells would be
highly desirable.
[0005] Three ways of circumventing the BBB to enhance brain
delivery of an administered active agent include direct
intra-cranial injection, transient permeabilization of the BBB, and
modification of the active agent to alter tissue distribution.
Direct injection of an active agent into brain tissue bypasses the
vasculature completely, but suffers primarily from the risk of
complications (infection, tissue damage) incurred by intra-cranial
injections and poor diffusion of the active agent from the site of
administration. Permeabilization of the BBB entails
non-specifically compromising the BBB concomitant with injection of
intravenous active agent and is accomplished through loosening
tight junctions by hyperosmotic shock (e.g. intravenous mannitol).
High plasma osmolarity leads to dehydration of the capillary
endothelium with partial collapse of tight junctions, little
selectivity in the types of blood-borne substances that gain access
to the brain under these conditions, and damage over the course of
a life-long regimen of treatment.
[0006] The distribution of an active agent into the brain may also
be increased by transcytosis, the active transport of certain
proteins from the luminal space (blood-side) to the abluminal space
(brain-side) of the BBB. Transcytosis pathways are distinct from
other vesicular traffic within the capillary endothelial cell and
transit can occur without alteration of the transported materials.
Transcytosis is a cell-type specific process mediated by receptors
on the BBB endothelial surface. Attachment of an active agent to a
transcytosed protein (vector or carrier) is expected to increase
distribution of the active substance to the brain. In transcytosis,
the vector is presumed to have a dominant effect on the
distribution of the joined pair. Vector proteins include antibodies
directed at receptors on the brain capillary endothelium
(Pardridge, 1999) and ligands to such receptors (Fukuta, et al.,
1994; Broadwell, et al., 1996),). Antibody vectors are transported
through the capillary endothelium by a process of adsorptive
endocytosis (non-specific, membrane-phase endocytosis) and are far
less efficiently transported than actual receptor ligands, which
cross the BBB by a saturable, energy-dependent mechanism
(Broadwell, et al. 1996).
[0007] The lipoprotein receptor-related protein (LRP) receptor
family comprises a group of membrane-spanning, endocytic proteins
with homology to the LDL receptor. Characterized as playing a key
role in lipoprotein metabolism, LRP have subsequently been shown to
bind a variety of ligands present in the blood. (Herz and
Strickland, 2001). LRP ligandsinclude the lipoprotein-associated
proteins ApoE, ApoJ and lipoprotein lipase; proteinases tPA, uPA,
Factor IX and MMP-9; proteinase inhibitors PAI-1, antithrombin III,
alpha-2-macroglobulin and alpha-antitrypsin; the antibacterial
protein lactoferrin; the chaperone receptor-associated protein
(RAP), the hormone thyrotropin, the cofactor cobalamin and the
lysosomal proteins saposin and sphingolipid activator protein. Four
of these ligands, ApoJ (Zlokovic, et al., 1996), thyrotropin
(Marino, et al., 2000), lipoprotein lipase (Obunike, et al. 2001)
and cobalamin (Ramanujam, et al., 1994) have been shown to be
transcytosed across capillary endothelial cells in vitro and in
vivo by LRP family members.
[0008] Taken together, the LRP receptor family comprises a pool of
compositionally and functionally related receptors expressed at
different levels in different tissues, including capillary
endothelium, neurons and astrocytes. LRP family members are
professional endocytic receptors that have also been shown to
transcytose ligands across polarized epithelia.
[0009] A unique LRP ligand is the receptor-associated protein, RAP,
a 39 kD chaperone localized to the endoplasmic reticulum and Golgi
(Bu and Schwartz, Trends Cell. Biol. 8(7):272-6 (1998)). RAP binds
tightly to LRP in these compartments preventing premature
association of the receptor with co-expressed ligands (Herz and
Willnow, Atherosclerosis 118 Suppl:S37-41 (1995)). RAP serves as an
attractive targeting sequence for LRP due to its high affinity for
all members of the LRP receptor family (.about.2 nM) and ability to
out-compete all known LRP ligands. Since RAP is not secreted,
endogenous levels in the blood are low. Endocytosis of RAP by LRP
results in localization to the lysosome and complete degradation of
the protein. Structure-function studies have been performed on RAP,
providing some guidance on minimization of the sequence required to
fulfill the targeting function (Melman, et al., J. Biol. Chem.
276(31): 29338-46 (2001)). It is not known whether RAP is
transcytosed, but Megalin-RAP complexes have been shown to remain
intact as far as the late endosome (Czekay, et al., Mol. Biol.
Cell. 8(3):517-32 (1997)). The integrity of the Megalin-RAP complex
through the Compartment of Uncoupling Ligand from Receptor (CURL)
and into this late endosomal compartment is in contrast to the
observed instability of other LRP-ligand complexes in the early
endosome. The LRP-RAP complex thus appears to have enhanced
resistance to acid-dependent dissociation, a potential indicator of
transcytotic competence. RAP could be engineered to be more
specific for particular members of the LRP family. Such
modifications would allow more selective targeting of RAP fusions
to particular tissues, as dictated by the expression of different
LRP family members on those tissues.
[0010] Futhermore, RAP may be a suitable substitute for the mannose
6-phosphate targeting signal on lysosomal enzymes. The LRP-RAP
system shares many features with the mannose-6-phosphate receptor
(MPR)-mannose 6-phosphate (M6P) system: Both receptor-ligand
complexes, LRP-RAP and MPR-M6P, exhibit dissociation constants in
the 1-2 nM range and are stable in the CURL. Both LRP and MPR are
widely expressed on a variety of tissues and efficiently transport
bound ligand to the lysosome. Both types of ligands are degraded
upon reaching the lysosome. The advantage of RAP targeting over M6P
targeting is that it depends on a protein sequence rather than a
modified carbohydrate. Biosynthetic throughput and quality control
are much higher for an amino acid sequence than for a modified
oligosaccharide, allowing for better drug yield, potency and
safety. The LRP-RAP system may also provide a method of efficiently
targeting other tissues. For example, the high density of the Very
Low Density Lipoprotein Receptor (VLDLR), a member of the LRP
family), as well as LRPI on muscle cells implies that RAP fusions
could be taken up to a significant extent by muscle through LRP
receptor-dependent endocytosis (Takahashi, et al., Proc. Natl.
Acad. Sci. U.S.A. 89(19):9252-6 (1992)).
[0011] There is a need for novel compounds, pharmaceutical
compositions, and methods of administration of such compounds and
compositions that can more effectively deliver active agents to the
brain and other biological compartments. In particular, there is a
need for such novel compounds, pharmaceutical compositions, and
methods of administration which deliver active agents to the brain
and tissues or organs that are set off from the blood compartment
by capillary endothelial cells that are closely sealed by tight
junctions. In particular, there is a need for such novel compounds,
pharmaceutical compositions, and methods of administration, which
efficiently target the delivery of an active agent to a wide
variety of tissues. In particular, there is a need for such novel
compounds, pharmaceutical compositions, and methods of
administration, which target the delivery of an active agent to the
lysosomal compartment of a cell within those tissues. This
invention provides such compounds, pharmaceutical compositions and
methods for their use.
BRIEF SUMMARY OF THE INVENTION
[0012] The present invention relates to the discovery that RAP and
RAP polypeptides selectively bind to LRP receptors and, as carriers
or vectors, RAP serves to increase the transport of therapeutic
and/or diagnostic/investigational agents across the blood brain
barrier and/or deliver agents to lysosomes of cells within and
without the CNS.
[0013] In one aspect, the invention provides compounds comprising
RAP or a RAP polypeptide conjugated to a therapeutic and/or
diagnostic/investigational agent and pharmaceutical compositions of
such compounds. In some embodiments, the RAP or RAP polypeptide
conjugate according to the invention may be modified as desired to
enhance its stability or pharmacokinetic properties (e.g.,
PEGylation of the RAP moiety of the conjugate, mutagenesis of the
RAP moiety of the conjugate). In some preferred embodiments, the
agent is a bioactive protein or peptide covalently linked to the
RAP or RAP polypeptide moiety of the compound. Such conjugates may
be formed by synthetic chemical reactions or joined by linker
groups. In preferred embodiments, when the active agent is a
protein or enzyme, the protein or enzyme is the human enzyme, a
fragment of the human protein or enzyme having a biological
activity of a native protein or enzyme, or a polypeptide that has
substantial amino acid sequence homology with the human protein or
enzyme. In some embodiments, the agent is a protein of human or
mammalian sequence, origin or derivation. In some embodiments, the
compound is a fusion protein of RAP or a RAP polypeptide portion
and an active agent protein or polypeptide portion. The agent
polypeptide portion of the fusion protein may be a substance having
therapeutic activity such as a growth factor, lymphokine or peptide
drug. The agent may be an enzyme or other bioactive protein or
polypeptide. In other embodiments, the agent is an enzyme or
protein whose deficiency causes a human disease such as Pompe's
disease (e.g. alpha-glucosidase). In other embodiments, the enzyme
is selected for its beneficial effect. In other embodiments, the
conjugate is formed by non-covalent bonds between the carrier and
an antibody to which the active agent is attached.
[0014] The RAP or RAP polypeptide can also be of human or mammalian
sequenceorigin or derivation. In yet other embodiments of the
invention, in each of its aspects, the RAP or RAP polypeptide is
identical in amino acid sequence to the corresponding portion of a
human or mammalian RAP polypeptide amino acid sequence. In other
embodiments, the RAP or RAP polypeptide moiety is the native
protein from the human or mammal. In other embodiments, the RAP or
RAP polypeptide is substantially homologous (i.e., at least 80%,
85%, 90%, 95%, 98%, or 99% identical in amino acid sequence) over a
length of at least 25, 50, 100, 150, or 200 amino acids, or the
entire length of the RAP polypeptide, to the native RAP sequence of
human or mammalian RAP. In other embodiments, the subject to which
the conjugate is to be administered is human.
[0015] In preferred embodiments of the invention, when the active
agent conjugated to RAP or RAP polypeptide is a protein or enzyme,
or fragment thereof possessing a biological activity of the protein
or enzyme, the active agent has an amino acid sequence identical to
the amino acid sequence to the corresponding portion of the human
or mammalian protein or enzyme. In other embodiments, the active
agent moiety of the conjugate is a protein or enzyme native to the
species of the human or mammal. In other embodiments, the protein
or enzyme, or fragment thereof, is substantially homologous (i.e.,
at least 80%, 85%, 90%, 95%, more preferably 98%, or most
preferably 99% identical in amino acid sequence over a length of at
least 10, 25, 50, 100, 150, or 200 amino acids, or the entire
length of the active agent) to a native sequence of the
corresponding human or mammal protein or enzyme. In other
embodiments, the subject to which the conjugate is to be
administered is human.
[0016] In a second aspect, the invention provides a method for
delivering therapeutic and/or diagnostic/investigational agents to
the central nervous system using the RAP/LRP carrier system to
transport such agents across the BBB formed by the capillary
endothelial cells which are closely sealed by tight junctions. The
invention thereby provides a novel route of administering agents
with a site of action within the central nervous system. In a
further embodiment, a modulator of LRP is co-administered to
modulate the therapeutic or adverse effects of such a
conjugate.
[0017] In some embodiments, the RAP or RAP polypeptide conjugates
with an active agent comprise more than one therapeutic active
agent useful in treating the same condition or disorder linked to a
single RAP polypeptide. In some embodiments, from about 1 to about
5 or from 2 to 10 molecules of the active agent is attached to one
RAP or RAP polypeptide molecule to be administered to a patient
having the disease, condition or disorder.
[0018] In a third aspect, the invention provides methods for using
the RAP carrier system in the treatment of diseases, disorders, or
conditions. In one group of embodiments, the RAP conjugates may be
used to treat a CNS condition or disorder. In one group of
particularly preferred embodiments to be treated, the CNS condition
or disorder to be treated is a brain tumor or other neoplasia
(e.g., a CNS tumor such as a glioblastoma). Such tumors or
neoplasia may be primary tumors or may be metastases. In these
embodiments, the compounds according to the invention may comprise
RAP or a RAP polypeptide conjugated to a cancer chemotherapeutic
agent. Preferred compounds have from about 1 to about 20 molecules
of the chemotherapeutic agent covalently linked to each RAP or RAP
polypeptide moiety. Such compounds are excellent vehicles for
enhanced delivery of chemotherapeutic agents to brain tumors and
other neoplasia localized in or around the brain, and for improved
treatment of such tumors and neoplasia. In some embodiments, the
cancer chemotherapeutic agents conjugated to a RAP polypeptide may
be the same or different. For instance, from 1 to 3 different
chemotherapeutic agents may be attached in the same or a different
mole RAP polypeptide per mole active agent ratio (e.g., 1:1; 1:2;
1:3; 1:4; and 1:5 to 1:10) with respect to the RAP or RAP
polypeptide moiety of the compound. Preferred chemotherapeutic
agents for such conjugates include, but are not limited to
adriamycin, cisplatin, 5-fluorouracil, camptothecin, and
paclitaxel. In another embodiment, the present invention provides a
method of treating a patient with a brain or CNS tumor or
glioblastoma by administering to the patient a therapeutically
effective amount of RAP or a RAP polypeptide conjugated to the
chemotherapeutic agent. In another embodiment, the present
invention provides for a method for delivering a compound of
interest through the blood-brain barrier of a subject into the
brain parenchyma where the compound is a chemotherapeutic able to
interfere with the division of the tumor cells and are toxic for
dividing cells. These compounds are liberated in the lysosomes
following degradation of the vector and can diffuse thru the
lysosomal membrane and enter the nucleus.
[0019] In another group of embodiments, the present invention
provides compounds, pharmaceutical compositions, and methods for
treating neurologic and psychiatric diseases and CNS diseases,
disorders and conditions, including, but not limited to,
Alzheimer's Disease, Parkinson's Disease, Multiple Sclerosis, and
Amylotrophic Lateral Sclerosis. In some embodiments, the compounds
of the invention comprise RAP or a RAP polypeptide conjugated to a
therapeutic agent for treating such diseases, disorders and
conditions. In a preferred group of embodiments, the therapeutic
agent is a peptide including, but not limited to, Nerve Growth
Factor, other peptide hormones or growth factors, and peptide
neurotransmitters. In another embodiment, the present invention
provides for a method for delivering an active agent through the
blood-brain barrier of a subject into the brain parenchyma where
the active agent is a neurotrophic factors including, but not
limited to, Nerve Growth Factor, Brain-Derived Neurotrophic Factor,
Neurotrophin-3, Neurotrophin-4/5, aFGF, bFGF, CNTF, Leukaemia
Inhibitory Factor, Cardiotrophin-1, TGFb, BMPs, GDFs, Neurturin,
Artemin, Persephin, EGF, TGFa, Neuregulins, IGF-1, IGF-2, ADNF and
PDGFs. Other factors such as caspase inhibitors can also be
conjugated as the active agent member of the compound. In other
embodiments, the active agent is a therapeutic antibody directed
toward a constituent of the CNS. In other embodiments, the active
agent is an antimicrobial agent for treating or preventing a CNS
infection or an immunomodulator such as a lymphokine.
[0020] In some embodiments, the RAP polypeptide active agent
conjugate is administered to treat a disease or condition selected
from the group consisting of neurological diseases including, but
not limited to, conditions such as Alzheimer's Disease, Parkinson's
Disease, schizophrenia, and epilepsy; neurological cancers, such as
primary brain tumors including glioma, meningioma, neurinoma,
pituitary adenoma, medulloblastoma, craniopharyngioma, hemangioma,
epidermoid, sarcoma and intracranial metastasis from other tumor
sources, and neurological infections or neurological inflammatory
conditions.
[0021] In still other aspects, the RAP conjugates of the invention
can be used to treat non-CNS (i.e., non-BBB delimited diseases,
such as cancers, diseases and conditions of non-CNS organs). For
example, conjugated agents can be used to treat conditions
affecting a patient's muscles.
[0022] In other aspects, the invention provides methods of treating
tissues or organs having proportionately greater, preferably more
than two-fold, amounts of LRP receptors on their cells than other
tissues or organs. The selective biodistribution of RAP or
RAP-polypeptide conjugated active agents can enhance the selective
targeting of such conjugated agents to specific organs.
[0023] In a fourth aspect, the invention provides a method for
using the RAP/LRP carrier system in the diagnosis of diseases,
disorders, or conditions. The present invention provides screening
assays for identifying RAP or RAP polypeptide active agent
conjugates that can prevent, amelioriate, or treat a CNS disease or
disorder by measuring the transcytosis of such agents in in vitro
models or by measuring the ability of such conjugates to reach or
bind to the brain parenchyma in vivo. Transcytosis or delivery can
be assessed by labeling the conjugate and then monitoring or
detecting the location or transport of the label in the test
chamber for an in vitro method or in a tissue compartment(s) in an
in vivo method. In addition, a therapeutic effect or other
biological effect of the conjugate can be used to monitor for
passage of the RAP active agent conjugate into the parenchyma of
the central nervous system. In preferred embodiments, the CNS
condition is a brain tumor.
[0024] In a fifth aspect, the invention provides a method of
delivering a therapeutic enzyme to a lysosome in a brain cell of a
subject, comprising: (i) administering a compound comprising RAP
conjugated to the therapeutic enzyme, (ii) transporting such
compound across the capillary endothelium; (iii) contact of such
compound with an LRP receptor on the cell, thereby facilitating
entry of such compound into such cell by endocytosis; and (iv)
delivery to lysosomes within the cell. In certain other aspects,
the invention provides compounds, compositions, and methods for
delivering a therapeutic agent or diagnostic agent to the lysosome
of a cell.
[0025] In a sixth aspect, the invention provides a method of
treating lysosomal storage diseases by administering RAP fused with
a therapeutic enzyme, wherein the RAP-enzyme complex binds to an
LRP receptor and is transported across the cell membrane, enters
the cell and is delivered to the lysosomes within the cell. In some
embodiments, the invention also provides a method of treating a
lysosomal storage disease in a patient by administering RAP or a
RAP polypeptide conjugated to a therapeutic agent which is a
protein or enzyme deficient in the lysosomes of a subject having
such a disease (e.g., enzyme replacement therapy). Such RAP or RAP
polypeptide active agent conjugates are particularly useful, for
example, in the treatment of lysosomal storage diseases such as MPS
I, MPS II, MPS III A, MPS III B, Metachromatic Leukodystrophy,
Gaucher, Krabbe, Pompe, CLN2, Niemann-Pick and Tay-Sachs disease
wherein a lysosomal protein deficiency contributes to the disease
state. In yet other embodiments, the invention also provides a
pharmaceutical composition comprising RAP or RAP polypeptide
covalently linked to a protein or enzyme deficient in a lysosomal
storage disease.
[0026] In some embodiments, the compounds, compositions, and
methods of the invention can be used to treat such lysosomal
storage diseases as Aspartylglucosaminuria, Cholesterol ester
storage disease/Wolman disease, Cystinosis, Danon disease, Fabry
disease, Farber Lipogranulomatosis/Farbe- r disease, Fucosidosis,
Galactosialidosis types I/II, Gaucher disease types I/IIIII Gaucher
disease, Globoid cell leukodystrophy/ Krabbe disease, Glycogen
storage disease II/Pompe disease, GM1-Gangliosidosis types
I/II/III, GM2-Gangliosidosis type I/Tay-Sachs disease,
GM2-Gangliosidosis type II Sandhoff disease, GM2-Gangliosidosis,
alpha-Mannosidosis types I/II, alpha-Mannosidosis, Metachromatic
leukodystrophy, Mucolipidosis type I/Sialidosis types I/II
Mucolipidosis types II/III I-cell disease, Mucolipidosis type IIIC
pseudo-Hurler polydystrophy, Mucopolysaccharidosis type I,
Mucopolysaccharidosis type II Hunter syndrome,
Mucopolysaccharidosis type IIIA Sanfilippo syndrome,
Mucopolysaccharidosis type IIIB Sanfilippo syndrome,
Mucopolysaccharidosis type IIIC Sanfilippo syndrome,
Mucopolysaccharidosis type IIID Sanfilippo syndrome,
Mucopolysaccharidosis type IVA Morquio syndrome,
Mucopolysaccharidosis type IVB Morquio syndrome,
Mucopolysaccharidosis type VI, Mucopolysaccharidosis type VII Sly
syndrome, Mucopolysaccharidosis type IX, Multiple sulfatase
deficiency, Pompe, Neuronal Ceroid Lipofuscinosis, CLN1 Batten
disease, Neuronal Ceroid Lipofuscinosis, CLN2 Batten disease,
Niemann-Pick disease types A/B Niemann-Pick disease, Niemann-Pick
disease type C1 Niemann-Pick disease, Niemann-Pick disease type C2
Niemann-Pick disease, Pycnodysostosis, Schindler disease types I/II
Schindler disease, and Sialic acid storage disease. In particularly
preferred embodiments, the lysosomal storage disease is MPS III,
MLD, or GM1.
[0027] In still another embodiment, the present invention provides
for a method of enzyme replacement therapy by administering a
therapeutically effective amount of a conjugate to a subject in
need of the enzyme replacement therapy, wherein the conjugate
comprises RAP or a RAP polypeptide linked to an enzyme via a
linker, wherein the cells of the patient have lysosomes which
contain insufficient amounts of the enzyme to prevent or reduce
damage to the cells, whereby sufficient amounts of the enzyme enter
the lysosomes to prevent or reduce damage to the cells. The cells
may be within or without the CNS or need not be set off from the
blood by capillary walls whose endothelial cells are closely sealed
to diffusion of an active agent by tight junctions.
[0028] In some embodiments, the RAP or RAP polypeptide conjugates
with an active agent comprising more than one active agent for
treating a lysosomal storage disease linked to a single RAP
polypeptide. In some embodiments, from about 1 to about 5 or from 2
to 10 molecules of the active agent of interest bound to a single
RAP or RAP polypeptide molecule.
[0029] In a particular embodiment, the invention provides compounds
comprising RAP or a RAP polypeptide bound to an active agent having
a biological activity which is reduced, deficient, or absent in the
target lysosome of the subject to which the compound is
administered. In preferred embodiments, the RAP or a RAP
polypeptide is covalently bound to the active agent. Preferred
active agents include, but are not limited to
aspartylglucosaminidase, acid lipase, cysteine transporter, Lamp-2,
alpha-galactosidase A, acid ceramidase, alpha-L-fucosidase,
beta-hexosaminidase A, GM2-activator deficiency,
alpha-D-mannosidase, beta-D-mannosidase, arylsulfatase A, saposin
B, neuraminidase, alpha-N-acetylglucosaminidase phosphotransferase,
phosphotransferase .gamma.-subunit, alpha-L-iduronidase,
iduronate-2-sulfatase, heparan-N-sulfatase,
alpha-N-acetylglucosaminidase, acetylCoA:N-acetyltransferase,
N-acetylglucosamine 6-sulfatase, galactose 6-sulfatase,
alpha-galactosidase, N-acetylgalactosamine 4-sulfatase,
hyaluronoglucosaminidase, palmitoyl protein thioesterase,
tripeptidyl peptidase I, acid sphingomyelinase, cholesterol
trafficking, cathepsin K, beta-galactosidase B,
.alpha.-glucosidase, and sialic acid transporter. In a preferred
embodiment, alpha-L-iduronidase, .alpha.-glucosidase or
N-acetylgalactosamine 4-sulfatase is the enzyme.
[0030] In a seventh aspect, the invention provides screening assays
for identifying RAP or RAP polypeptide active agent conjugates that
can prevent, amelioriate, or treat a lysosomal storage disease by
contacting a cell containing a lysosome with the conjugate and
determining whether the conjugate delivers the agent to the
lysosome. The delivery can be assessed by labeling the conjugate
and then monitoring or detecting the location of the label in the
cell or by determining the effect of the conjugate on the amount of
the storage material found in the lysosome. In a preferred
embodiment, the agent is a protein or enzyme deficient in the
lysosomal storage disease. In another embodiment, the cell is
deficient in the agent conjugated to the RAP or RAP
polypeptide.
[0031] In another embodiment, the present invention provides for a
method for identifying an agent that can prevent, ameliorate or
treat a lysosomal storage disease, by administering RAP or a RAP
polypeptide conjugated enzyme to a cell, wherein absence of the
enzyme causes the lysosomal storage disease; and determining
whether the agent reduces damage to the cell compared to damage to
the cell if the conjugated agent was not administered to the cell.
In certain embodiments, the method is a high throughput assay.
BRIEF DESCRIPTION OF THE DRAWINGS
[0032] FIG. 1. Effect of RAP on [.sup.125I]-p97 transcytosis across
BBCEC monolayers.
[0033] FIG. 2. Preparation of expression constructs endcoding
fusions between human RAP and human glucosidase (GAA),
alpha-L-iduronidase (IDU) and glial-derived neurotrophic factor
(GDNF).
[0034] FIG. 3. Nucleotide and protein sequences of the RAP-GAA
fusion.
[0035] FIG. 4. Nucleotide and protein sequence of RAP-IDU
fusion
[0036] FIG. 5. Nucleotide and protein sequence of RAP-GDNF
fusion.
[0037] FIG. 6. Characterization of the RAP-GAA fusion.
[0038] FIG. 7. Assay for complex oligosaccharides on RAP-GAA.
[0039] FIG. 8. Assay for high-mannose oligosaccharides on
RAP-GAA.
[0040] FIG. 9. Characterization of RAP-IDU fusion.
[0041] FIG. 10. Binding of RAP and RAP-lysosomal enzyme fusion to
LRP.
[0042] FIG. 11. Corrected V.sub.d vs. perfusion time for iodinated
RAP and transferrin at 15 minutes.
[0043] FIG. 12. Distribution of RAP between brain capillary
endothelium and brain parenchyma.
[0044] FIG. 13. RAP-alpha-glucosidase uptake by human Pompe
fibroblasts.
[0045] FIG. 14. Multiple alignment of amino acid sequences of RAP
from different species.
[0046] FIG. 15. SEQ ID NO:1, amino acid sequence of human RAP.
[0047] FIG. 16. SEQ ID NO:2, amino acid sequence of the 28 kD RAP
polypeptide.
DETAILED DESCRIPTION OF THE INVENTION
[0048] The present invention relates to the discovery that RAP and
RAP polypeptides selectively bind to LRP receptors. RAP is a
particularly effective carrier for delivering active agents
conjugated to it across the blood brain barrier, to the lysosomes
within a cell, and to the intracellular compartment of cells
bearing LRP receptors. Compounds comprising RAP polypeptide
conjugated to an active agent are useful in the diagnosis and
treatment of a variety of CNS and non-CNS diseases, conditions, and
disorders, including but not limited to, in particular, cancer and
lysosomal storage diseases.
[0049] I. Definitions
[0050] 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. The
following references provide one of skill with a general definition
of many of the terms used in this invention: Singleton, et al.,
DICTIONARY OF MICROBIOLOGY AND MOLECULAR BIOLOGY (2d ed. 1994); THE
CAMBRIDGE DICTIONARY OF SCIENCE AND TECHNOLOGY (Walker ed., 1988);
THE GLOSSARY OF GENETICS, 5TH ED., R. Rieger, et al. (eds.),
Springer Verlag (1991); and Hale & Marham, THE HARPER COLLINS
DICTIONARY OF BIOLOGY (1991).
[0051] Each publication, patent application, patent, and other
reference cited herein is incorporated by reference in its entirety
to the extent that it is not inconsistent with the present
disclosure.
[0052] It is noted here that as used in this specification and the
appended claims, the singular forms "a," "an," and "the" include
plural reference unless the context clearly dictates otherwise.
[0053] As used herein, the following terms have the meanings
ascribed to them unless specified otherwise.
[0054] "Brain tumors and other neoplasia in or around the brain" as
used herein includes both primary tumors and/or metastases that
develop in or around the brain. It may also mean metastases of
brain tumors that migrate elsewhere in the body, but remain
responsive to RAP or RAP polypeptide conjugates with
chemotherapeutic agents. Many types of such tumors and neoplasia
are known. Primary brain tumors include glioma, meningioma,
neurinoma, pituitary adenoma, medulloblastoma, craniopharyngioma,
hemangioma, epidermoid, sarcoma and others. Fifty percent of all
intracranial tumors are intracranial metastasis. As used herein,
tumors and neoplasia may be associated with the brain and neural
tissue, or they may be associated with the meninges, skull,
vasculature or any other tissue of the head or neck. Such tumors
are generally solid tumors, or they are diffuse tumors with
accumulations localized to the head. Tumors or neoplasia for
treatment according to the invention may be malignant or benign,
and may have been treated previously with chemotherapy, radiation
and/or other treatments.
[0055] The term "effective amount" means a dosage sufficient to
produce a desired result on a health condition, pathology, and
disease of a subject or for a diagnostic purpose. The desired
result may comprise a subjective or objective improvement in the
recipient of the dosage. "Therapeutically effective amount" refers
to that amount of an agent effective to produce the intended
beneficial effect on health.
[0056] "Small organic molecule" refers to organic molecules of a
size comparable to those organic molecules generally used in
pharmaceuticals. The term excludes organic biopolymers (e.g.,
proteins, nucleic acids, etc.). Preferred small organic molecules
range in size up to about 5000 Da, up to about 2000 Da, or up to
about 1000 Da.
[0057] A "subject" of diagnosis or treatment is a human or
non-human animal, including a mammal or a primate.
[0058] "Treatment" refers to prophylactic treatment or therapeutic
treatment or diagnostic treatment.
[0059] A "prophylactic" treatment is a treatment administered to a
subject who does not exhibit signs of a disease or exhibits only
early signs for the purpose of decreasing the risk of developing
pathology. The conjugate compounds of the invention may be given as
a prophylactic treatment to reduce the likelihood of developing a
pathology or to minimize the severity of the pathology, if
developed.
[0060] A "therapeutic" treatment is a treatment administered to a
subject who exhibits signs or symptoms of pathology for the purpose
of diminishing or eliminating those signs or symptoms. The signs or
symptoms may be biochemical, cellular, histological, functional,
subjective or objective. The conjugate compounds of the invention
may be given as a therapeutic treatment or for diagnosis.
[0061] "Diagnostic" means identifying the presence or nature of a
pathologic condition. Diagnostic methods differ in their
specificity and selectivity. While a particular diagnostic method
may not provide a definitive diagnosis of a condition, it suffices
if the method provides a positive indication that aids in
diagnosis.
[0062] "Pharmaceutical composition" refers to a composition
suitable for pharmaceutical use in subject animal, including humans
and mammals. A pharmaceutical composition comprises a
pharmacologically effective amount of a RAP polypeptide conjugated
to an active agent and also comprises a pharmaceutically acceptable
carrier. A pharmaceutical composition encompasses a composition
comprising the active ingredient(s), and the inert ingredient(s)
that make up the carrier, as well as any product which results,
directly or indirectly, from combination, complexation or
aggregation of any two or more of the ingredients, or from
dissociation of one or more of the ingredients, or from other types
of reactions or interactions of one or more of the ingredients.
Accordingly, the pharmaceutical compositions of the present
invention encompass any composition made by admixing a conjugate
compound of the present invention and a pharmaceutically acceptable
carrier.
[0063] "Pharmaceutically acceptable carrier" refers to any of the
standard pharmaceutical carriers, buffers, and excipients, such as
a phosphate buffered saline solution, 5% aqueous solution of
dextrose, and emulsions, such as an oil/water or water/oil
emulsion, and various types of wetting agents and/or adjuvants.
Suitable pharmaceutical carriers and formulations are described in
Remington's Pharmaceutical Sciences, 19th Ed. (Mack Publishing Co.,
Easton, 1995). Preferred pharmaceutical carriers depend upon the
intended mode of administration of the active agent. Typical modes
of administration include enteral (e.g., oral) or parenteral (e.g.,
subcutaneous, intramuscular, intravenous or intraperitoneal
injection; or topical, transdermal, or transmucosal
administration). A "pharmaceutically acceptable salt" is a salt
that can be formulated into a compound for pharmaceutical use
including, e.g., metal salts (sodium, potassium, magnesium,
calcium, etc.) and salts of ammonia or organic amines.
[0064] The term "unit dosage form," as used herein, refers to
physically discrete units suitable as unitary dosages for human and
animal subjects, each unit containing a predetermined quantity of
compounds of the present invention calculated in an amount
sufficient to produce the desired effect in association with a
pharmaceutically acceptable diluent, carrier or vehicle. The
specifications for the novel unit dosage forms of the present
invention depend on the particular conjugate employed and the
effect to be achieved, and the pharmacodynamics associated with
each compound in the host.
[0065] "Modulate," as used herein, refers to the ability to alter,
by increase or decrease (e.g., to act as an antagonist or
agonist).
[0066] "Increasing relative delivery" as used herein refers to the
effect whereby the accumulation at the intended delivery site
(e.g., brain, lysosome) of a RAP-conjugated active agent is
increased relative to the accumulation of the unconjugated active
agent.
[0067] "Therapeutic index" refers to the dose range (amount and/or
timing) above the minimum therapeutic amount and below an
unacceptably toxic amount.
[0068] "Equivalent dose" refers to a dose, which contains the same
amount of active agent.
[0069] "Polynucleotide" refers to a polymer composed of nucleotide
units. Polynucleotides include naturally occurring nucleic acids,
such as deoxyribonucleic acid ("DNA") and ribonucleic acid ("RNA")
as well as nucleic acid analogs. Nucleic acid analogs include those
which include non-naturally occurring bases, nucleotides that
engage in linkages with other nucleotides other than the naturally
occurring phosphodiester bond or which include bases attached
through linkages other than phosphodiester bonds. Thus, nucleotide
analogs include, for example and without limitation,
phosphorothioates, phosphorodithioates, phosphorotriesters,
phosphoramidates, boranophosphates, methylphosphonates,
chiral-methyl phosphonates, 2-O-methyl ribonucleotides,
peptide-nucleic acids (PNAs), and the like. Such polynucleotides
can be synthesized, for example, using an automated DNA
synthesizer. The term "nucleic acid" typically refers to large
polynucleotides. The term "oligonucleotide" typically refers to
short polynucleotides, generally no greater than about 50
nucleotides. It will be understood that when a nucleotide sequence
is represented by a DNA sequence (i.e., A, T, G, C), this also
includes an RNA sequence (i.e., A, U, G, C) in which "U" replaces
"T."
[0070] "cDNA" refers to a DNA that is complementary or identical to
an mRNA, in either single stranded or double stranded form.
[0071] Conventional notation is used herein to describe
polynucleotide sequences: the left-hand end of a single-stranded
polynucleotide sequence is the 5'-end; the left-hand direction of a
double-stranded polynucleotide sequence is referred to as the
5'-direction. The direction of 5' to 3' addition of nucleotides to
nascent RNA transcripts is referred to as the transcription
direction. The DNA strand having the same sequence as an mRNA is
referred to as the "coding strand"; sequences on the DNA strand
having the same sequence as an mRNA transcribed from that DNA and
which are located 5' to the 5'-end of the RNA transcript are
referred to as "upstream sequences"; sequences on the DNA strand
having the same sequence as the RNA and which are 3' to the 3' end
of the coding RNA transcript are referred to as "downstream
sequences."
[0072] "Complementary" refers to the topological compatibility or
matching together of interacting surfaces of two polynucleotides.
Thus, the two molecules can be described as complementary, and
furthermore, the contact surface characteristics are complementary
to each other. A first polynucleotide is complementary to a second
polynucleotide if the nucleotide sequence of the first
polynucleotide is identical to the nucleotide sequence of the
polynucleotide binding partner of the second polynucleotide. Thus,
the polynucleotide whose sequence 5'-TATAC-3' is complementary to a
polynucleotide whose sequence is 5'-GTATA-3'.
[0073] A nucleotide sequence is "substantially complementary" to a
reference nucleotide sequence if the sequence complementary to the
subject nucleotide sequence is substantially identical to the
reference nucleotide sequence.
[0074] "Encoding" refers to the inherent property of specific
sequences of nucleotides in a polynucleotide, such as a gene, a
cDNA, or an mRNA, to serve as templates for synthesis of other
polymers and macromolecules in biological processes having either a
defined sequence of nucleotides (i.e., rRNA, tRNA and mRNA) or a
defined sequence of amino acids and the biological properties
resulting therefrom. Thus, a gene encodes a protein if
transcription and translation of mRNA produced by that gene
produces the protein in a cell or other biological system. Both the
coding strand, the nucleotide sequence of which is identical to the
mRNA sequence and is usually provided in sequence listings, and
non-coding strand, used as the template for transcription, of a
gene or cDNA can be referred to as encoding the protein or other
product of that gene or cDNA. Unless otherwise specified, a
"nucleotide sequence encoding an amino acid sequence" includes all
nucleotide sequences that are degenerate versions of each other and
that encode the same amino acid sequence. Nucleotide sequences that
encode proteins and RNA may include introns.
[0075] "Recombinant polynucleotide" refers to a polynucleotide
having sequences that are not naturally joined together. An
amplified or assembled recombinant polynucleotide may be included
in a suitable vector, and the vector can be used to transform a
suitable host cell. A host cell that comprises the recombinant
polynucleotide is referred to as a "recombinant host cell." The
gene is then expressed in the recombinant host cell to produce,
e.g., a "recombinant polypeptide." A recombinant polynucleotide may
serve a non-coding function (e.g., promoter, origin of replication,
ribosome-binding site, etc.) as well.
[0076] "Expression control sequence" refers to a nucleotide
sequence in a polynucleotide that regulates the expression
(transcription and/or translation) of a nucleotide sequence
operatively linked thereto. "Operatively linked" refers to a
functional relationship between two parts in which the activity of
one part (e.g., the ability to regulate transcription) results in
an action on the other part (e.g., transcription of the sequence).
Expression control sequences can include, for example and without
limitation, sequences of promoters (e.g., inducible or
constitutive), enhancers, transcription terminators, a start codon
(i.e., ATG), splicing signals for introns, and stop codons.
[0077] "Expression vector" refers to a vector comprising a
recombinant polynucleotide comprising expression control sequences
operatively linked to a nucleotide sequence to be expressed. An
expression vector comprises sufficient cis-acting elements for
expression; other elements for expression can be supplied by the
host cell or in vitro expression system. Expression vectors include
all those known in the art, such as cosmids, plasmids (e.g., naked
or contained in liposomes) and viruses that incorporate the
recombinant polynucleotide.
[0078] "Amplification" refers to any means by which a
polynucleotide sequence is copied and thus expanded into a larger
number of polynucleotide molecules, e.g., by reverse transcription,
polymerase chain reaction, and ligase chain reaction.
[0079] "Primer" refers to a polynucleotide that is capable of
specifically hybridizing to a designated polynucleotide template
and providing a point of initiation for synthesis of a
complementary polynucleotide. Such synthesis occurs when the
polynucleotide primer is placed under conditions in which synthesis
is induced, i.e., in the presence of nucleotides, a complementary
polynucleotide template, and an agent for polymerization such as
DNA polymerase. A primer is typically single-stranded, but may be
double-stranded. Primers are typically deoxyribonucleic acids, but
a wide variety of synthetic and naturally occurring primers are
useful for many applications. A primer is complementary to the
template to which it is designed to hybridize to serve as a site
for the initiation of synthesis, but need not reflect the exact
sequence of the template. In such a case, specific hybridization of
the primer to the template depends on the stringency of the
hybridization conditions. Primers can be labeled with, e.g.,
chromogenic, radioactive, or fluorescent moieties and used as
detectable moieties.
[0080] "Probe," when used in reference to a polynucleotide, refers
to a polynucleotide that is capable of specifically hybridizing to
a designated sequence of another polynucleotide. A probe
specifically hybridizes to a target complementary polynucleotide,
but need not reflect the exact complementary sequence of the
template. In such a case, specific hybridization of the probe to
the target depends on the stringency of the hybridization
conditions. Probes can be labeled with, e.g., chromogenic,
radioactive, or fluorescent moieties and used as detectable
moieties.
[0081] A first sequence is an "antisense sequence" with respect to
a second sequence if a polynucleotide whose sequence is the first
sequence specifically hybridizes with a polynucleotide whose
sequence is the second sequence.
[0082] "Hybridizing specifically to" or "specific hybridization" or
"selectively hybridize to", refers to the binding, duplexing, or
hybridizing of a nucleic acid molecule preferentially to a
particular nucleotide sequence under stringent conditions when that
sequence is present in a complex mixture (e.g., total cellular) DNA
or RNA.
[0083] The term "stringent conditions" refers to conditions under
which a probe will hybridize preferentially to its target
subsequence, and to a lesser extent to, or not at all to, other
sequences. "Stringent hybridization" and "stringent hybridization
wash conditions" in the context of nucleic acid hybridization
experiments such as Southern and Northern hybridizations are
sequence dependent, and are different under different environmental
parameters. An extensive guide to the hybridization of nucleic
acids is found in Tijssen (1993) Laboratory Techniques in
Biochemistry and Molecular Biology--Hybridization with Nucleic Acid
Probes part 1 chapter 2 "Overview of principles of hybridization
and the strategy of nucleic acid probe assays", Elsevier, New York.
Generally, highly stringent hybridization and wash conditions are
selected to be about 5.degree. C. lower than the thermal melting
point (Tm) for the specific sequence at a defined ionic strength
and pH. The Tm is the temperature (under defined ionic strength and
pH) at which 50% of the target sequence hybridizes to a perfectly
matched probe. Very stringent conditions are selected to be equal
to the Tm for a particular probe.
[0084] An example of stringent hybridization conditions for
hybridization of complementary nucleic acids which have more than
100 complementary residues on a filter in a Southern or northern
blot is 50% formalin with 1 mg of heparin at 42.degree. C., with
the hybridization being carried out overnight. An example of highly
stringent wash conditions is 0.15 M NaCl at 72.degree. C. for about
15 minutes. An example of stringent wash conditions is a
0.2.times.SSC wash at 65.degree. C. for 15 minutes (see, Sambrook,
et al. for a description of SSC buffer). Often, a high stringency
wash is preceded by a low stringency wash to remove background
probe signal. An example medium stringency wash for a duplex of,
e.g., more than 100 nucleotides, is 1.times.SSC at 45.degree. C.
for 15 minutes. An example low stringency wash for a duplex of,
e.g., more than 100 nucleotides, is 4-6.times.SSC at 40.degree. C.
for 15 minutes. In general, a signal to noise ratio of 2.times. (or
higher) than that observed for an unrelated probe in the particular
hybridization assay indicates detection of a specific
hybridization.
[0085] "Polypeptide" refers to a polymer composed of amino acid
residues, related naturally occurring structural variants, and
synthetic non-naturally occurring analogs thereof linked via
peptide bonds, related naturally occurring structural variants, and
synthetic non-naturally occurring analogs thereof. Synthetic
polypeptides can be synthesized, for example, using an automated
polypeptide synthesizer. The term "protein" typically refers to
large polypeptides. The term "peptide" typically refers to short
polypeptides.
[0086] Conventional notation is used herein to portray polypeptide
sequences: the left-hand end of a polypeptide sequence is the
amino-terminus; the right-hand end of a polypeptide sequence is the
carboxyl-terminus.
[0087] "Conservative substitution" refers to the substitution in a
polypeptide of an amino acid with a functionally similar amino
acid. The following six groups each contain amino acids that are
conservative substitutions for one another:
[0088] 1) Alanine (A), Serine (S), Threonine (T);
[0089] 2) Aspartic acid (D), Glutamic acid (E);
[0090] 3) Asparagine (N), Glutamine (Q);
[0091] 4) Arginine (R), Lysine (K);
[0092] 5) Isoleucine (I), Leucine (L), Methionine (M), Valine (V);
and
[0093] 6) Phenylalanine (F), Tyrosine (Y), Tryptophan (W).
[0094] "Allelic variant" refers to any of two or more polymorphic
forms of a gene occupying the same genetic locus. Allelic
variations arise naturally through mutation, and may result in
phenotypic polymorphism within populations. Gene mutations can be
silent (no change in the encoded polypeptide) or may encode
polypeptides having altered amino acid sequences. "Allelic
variants" also refer to cDNAs derived from mRNA transcripts of
genetic allelic variants, as well as the proteins encoded by
them.
[0095] The terms "identical" or percent "identity," in the context
of two or more polynucleotide or polypeptide sequences, refer to
two or more sequences or subsequences that are the same or have a
specified percentage of nucleotides or amino acid residues that are
the same, when compared and aligned for maximum correspondence, as
measured using one of the following sequence comparison algorithms
or by visual inspection.
[0096] The phrase "substantially homologous" or "substantially
identical"in the context of two nucleic acids or polypeptides,
generally refers to two or more sequences or subsequences that have
at least 40%, 60%, 80%, 90%, 95%, 98% nucleotide or amino acid
residue identity, when compared and aligned for maximum
correspondence, as measured using one of the following sequence
comparison algorithms or by visual inspection. Preferably, the
substantial identity exists over a region of the sequences that is
at least about 50 residues in length, more preferably over a region
of at least about 100 residues, and most preferably the sequences
are substantially identical over at least about 150 residues. In a
most preferred embodiment, the sequences are substantially
identical over the entire length of either or both comparison
biopolymers.
[0097] For sequence comparison, typically one sequence acts as a
reference sequence, to which test sequences are compared. When
using a sequence comparison algorithm, test and reference sequences
are input into a computer, subsequence coordinates are designated,
if necessary, and sequence algorithm program parameters are
designated. The sequence comparison algorithm then calculates the
percent sequence identity for the test sequence(s) relative to the
reference sequence, based on the designated program parameters.
[0098] Optimal alignment of sequences for comparison can be
conducted, e.g., by the local homology algorithm of Smith &
Waterman, Adv. Appl. Math. 2:482 (1981), by the homology alignment
algorithm of Needleman & Wunsch, J. Mol. Biol. 48:443 (1970),
by the search for similarity method of Pearson & Lipman, Proc.
Natl. Acad. Sci. USA 85:2444 (1988), by computerized
implementations of these algorithms (GAP, BESTFIT, FASTA, and
TFASTA in the Wisconsin Genetics Software Package, Genetics
Computer Group, 575 Science Dr., Madison, Wis.), or by visual
inspection (see generally Ausubel, et al., supra).
[0099] One example of a useful algorithm is PILEUP. PILEUP creates
a multiple sequence alignment from a group of related sequences
using progressive, pairwise alignments to show relationship and
percent sequence identity. It also plots a tree or dendogram
showing the clustering relationships used to create the alignment.
PILEUP uses a simplification of the progressive alignment method of
Feng & Doolittle, J. Mol. Evol. 35:351-360 (1987). The method
used is similar to the method described by Higgins & Sharp,
CABIOS 5:151-153 (1989). The program can align up to 300 sequences,
each of a maximum length of 5,000 nucleotides or amino acids. The
multiple alignment procedure begins with the pairwise alignment of
the two most similar sequences, producing a cluster of two aligned
sequences. This cluster is then aligned to the next most related
sequence or cluster of aligned sequences. Two clusters of sequences
are aligned by a simple extension of the pairwise alignment of two
individual sequences. The final alignment is achieved by a series
of progressive, pairwise alignments. The program is run by
designating specific sequences and their amino acid or nucleotide
coordinates for regions of sequence comparison and by designating
the program parameters. For example, a reference sequence can be
compared to other test sequences to determine the percent sequence
identity relationship using the following parameters: default gap
weight (3.00), default gap length weight (0.10), and weighted end
gaps. Another algorithm that is useful for generating multiple
alignments of sequences is Clustal W (Thompson, et al. CLUSTAL W:
improving the sensitivity of progressive multiple sequence
alignment through sequence weighting, positions-specific gap
penalties and weight matrix choice, Nucleic Acids Research 22:
4673-4680 (1994)).
[0100] Another example of algorithm that is suitable for
determining percent sequence identity and sequence similarity is
the BLAST algorithm, which is described in Altschul, et al., J Mol.
Biol. 215:403-410 (1990). Software for performing BLAST analyses is
publicly available through the National Center for Biotechnology
Information (http://www.ncbi.nlm.nih.go- v/). This algorithm
involves first identifying high scoring sequence pairs (HSPs) by
identifying short words of length W in the query sequence, which
either match or satisfy some positive-valued threshold score T when
aligned with a word of the same length in a database sequence. T is
referred to as the neighborhood word score threshold (Altschul et
al, supra). These initial neighborhood word hits act as seeds for
initiating searches to find longer HSPs containing them. The word
hits are then extended in both directions along each sequence for
as far as the cumulative alignment score can be increased.
Cumulative scores are calculated using, for nucleotide sequences,
the parameters M (reward score for a pair of matching residues;
always>0) and N (penalty score for mismatching residues;
always<0). For amino acid sequences, a scoring matrix is used to
calculate the cumulative score. Extension of the word hits in each
direction are halted when: the cumulative alignment score falls off
by the quantity X from its maximum achieved value; the cumulative
score goes to zero or below, due to the accumulation of one or more
negative-scoring residue alignments; or the end of either sequence
is reached. The BLAST algorithm parameters W, T, and X determine
the sensitivity and speed of the alignment. The BLASTN program (for
nucleotide sequences) uses as defaults a wordlength (W) of 11, an
expectation (E) of 10, M=5, N=-4, and a comparison of both strands.
For amino acid sequences, the BLASTP program uses as defaults a
wordlength (W) of 3, an expectation (E) of 10, and the BLOSUM62
scoring matrix (see Henikoff & Henikoff, Proc. Natl. Acad. Sci.
USA 89:10915 (1989)).
[0101] In addition to calculating percent sequence identity, the
BLAST algorithm also performs a statistical analysis of the
similarity between two sequences (see, e.g., Karlin & Altschul,
Proc. Nat'l. Acad. Sci. USA 90:5873-5787 (1993)). One measure of
similarity provided by the BLAST algorithm is the smallest sum
probability (P(N)), which provides an indication of the probability
by which a match between two nucleotide or amino acid sequences
would occur by chance. For example, a nucleic acid is considered
similar to a reference sequence if the smallest sum probability in
a comparison of the test nucleic acid to the reference nucleic acid
is less than about 0.1, more preferably less than about 0.01, and
most preferably less than about 0.001.
[0102] A further indication that two nucleic acid sequences or
polypeptides are substantially identical is that the polypeptide
encoded by the first nucleic acid is immunologically cross reactive
with the polypeptide encoded by the second nucleic acid, as
described below. Thus, a polypeptide is typically substantially
identical to a second polypeptide, for example, where the two
peptides differ only by conservative substitutions. Another
indication that two nucleic acid sequences are substantially
identical is that the two molecules hybridize to each other under
stringent conditions, as described herein.
[0103] "Substantially pure" or "isolated" means an object species
is the predominant species present (i.e., on a molar basis, more
abundant than any other individual macromolecular species in the
composition), and a substantially purified fraction is a
composition wherein the object species comprises at least about 50%
(on a molar basis) of all macromolecular species present.
Generally, a substantially pure composition means that about 80% to
90% or more of the macromolecular species present in the
composition is the purified species of interest. The object species
is purified to essential homogeneity (contaminant species cannot be
detected in the composition by conventional detection methods) if
the composition consists essentially of a single macromolecular
species. Solvent species, small molecules (<500 Daltons),
stabilizers (e.g., BSA), and elemental ion species are not
considered macromolecular species for purposes of this definition.
In some embodiments, the conjugates of the invention are
substantially pure or isolated. In some embodiments, the conjugates
of the invention are substantially pure or isolated with respect to
the macromolecular starting materials used in their synthesis. In
some embodiments, the pharmaceutical composition of the invention
comprises a substantially purified or isolated conjugate of a RAP
polypeptide and the active agent admixed with one or more
pharmaceutically acceptable excipient.
[0104] "Naturally-occurring" as applied to an object refers to the
fact that the object can be found in nature. For example, a
polypeptide or polynucleotide sequence that is present in an
organism (including viruses) that can be isolated from a source in
nature and which has not been intentionally modified by man in the
laboratory is naturally-occurring.
[0105] "Detecting" refers to determining the presence, absence, or
amount of an analyte in a sample, and can include quantifying the
amount of the analyte in a sample or per cell in a sample.
[0106] "Detectable moiety" or a "label" refers to a composition
detectable by spectroscopic, photochemical, biochemical,
immunochemical, or chemical means. For example, useful labels
include .sup.32P, .sup.35S, fluorescent dyes, electron-dense
reagents, enzymes (e.g., as commonly used in an ELISA),
biotin-streptavadin, dioxigenin, haptens and proteins for which
antisera or monoclonal antibodies are available, or nucleic acid
molecules with a sequence complementary to a target. The detectable
moiety often generates a measurable signal, such as a radioactive,
chromogenic, or fluorescent signal, that can be used to quantitate
the amount of bound detectable moiety in a sample. The detectable
moiety can be incorporated in or attached to a primer or probe
either covalently, or through ionic, van der Waals or hydrogen
bonds, e.g., incorporation of radioactive nucleotides, or
biotinylated nucleotides that are recognized by streptavadin. The
detectable moiety may be directly or indirectly detectable.
Indirect detection can involve the binding of a second directly or
indirectly detectable moiety to the detectable moiety. For example,
the detectable moiety can be the ligand of a binding partner, such
as biotin, which is a binding partner for streptavadin, or a
nucleotide sequence, which is the binding partner for a
complementary sequence, to which it can specifically hybridize. The
binding partner may itself be directly detectable, for example, an
antibody may be itself labeled with a fluorescent molecule. The
binding partner also may be indirectly detectable, for example, a
nucleic acid having a complementary nucleotide sequence can be a
part of a branched DNA molecule that is in turn detectable through
hybridization with other labeled nucleic acid molecules. (See,
e.g., P D. Fahrlander and A. Klausner, Bio/Technology (1988)
6:1165.) Quantitation of the signal is achieved by, e.g.,
scintillation counting, densitometry, or flow cytometry.
[0107] "Linker" refers to a molecule that joins two other
molecules, either covalently, or through ionic, van der Waals or
hydrogen bonds, e.g., a nucleic acid molecule that hybridizes to
one complementary sequence at the 5' end and to another
complementary sequence at the 3' end, thus joining two
non-complementary sequences.
[0108] II. LRP
[0109] "LRP" refers to members of the low-density lipoprotein
receptor family including the low-density lipoprotein
receptor-related protein 1 (LRPI). LRP1 is a large protein of 4525
amino acids (600 kDa), which is cleaved by furin to produce two
subunits of 515-(alpha) kDand 85-(13) kDa that remain
non-covalently bound. LRP is expressed on most tissue types. Other
members of the low-density lipoprotein (LDL) receptor family
include LDL-R (132 kDa); LRP/LRP1 and LRP1B (600 kDa); Megalin
((LRP2), 600 kDa); VLDL-R (130 kDa); ER-2 (LRP-8, 130 kDa); Mosaic
LDL-R (LR11, 250 KDa); and other members such as LRP3, LRP6, and
LRP-7. Characteristic features of thefamily include cell-surface
expression; extracellular ligand binding domain repeats
(D.times.SDE); requirement of Ca++ for ligand binding; recognition
of RAP and ApoE; EGF precursor homology domain repeats (YWTD);
single membrane spanning region; internalization signals in the
cytoplasmic domain (FDNPXY); and receptor mediated endocytosis of
various ligands. Some members of the family, including LRP1 and
VLDLR, participate in signal transduction pathways.
[0110] LRP ligands refer to a number of molecules that are known to
bind LRP. These molecules include, for instance, lactoferrin, RAP,
lipoprotein lipase, ApoE, Factor VIII, beta-amyloid precursor,
alpha-2-macroglobulin, thrombospondin 2 MMP-2 (matrix
metalloproteinase-2), MPP-9-TIMP-1 (tissue inhibitor of matrix
metalloproteinase-1); uPA (urokinase plasminogen activator):PAI-I
(plasminogen activator inhibitor-1):uPAR (uPA receptor); and tPA
(tissue plasminogen activator):PAI-1:uPAR.
[0111] LRP1 is believed to be a multifunctional receptor with
clustering of cysteine-rich type repeats. A binding repeat,
resembling those found in the LDL receptor, is the molecular
principle for the ability to bind a variety of ligands that were
previously thought to be unrelated. These include the ligands
described in the previous paragraph in addition to: pseudomonas
exotoxin A, human rhinovirus, lactoferrin and the so-called
receptor associated protein (RAP). See, Meilinger, et al., FEBS
Lett, 360:70-74 (1995). LRP1 is has the GenBank Accession No.: X
13916 and SwissProt Primary Accession No.: Q07954. Alternative
names for the LRP1 gene/protein include: Low-density lipoprotein
receptor-related protein 1 [precursor], LRP, Alpha-2-macroglobulin
receptor, A2MR, Apolipoprotein E receptor, APOER, CD91, LRP1 or
A2MR.
[0112] Members of the LRP family are well expressed on capillary
endothelium and on CNS cell types including neurons and astrocytes
(e.g., LDL receptor, Megalin, LRP). LRP receptors endocytose bound
ligand and have been demonstrated to transcytose ligands across
polarized epithelial cells in the kidney, thyroid and across
capillary endothelial cells in the brain. LRP therefore comprises a
pool of compositionally and functionally related receptors
expressed at different levels in different tissues. In some
embodiments, this invention uses RAP, which binds and thereby
targets members of this pool of related receptors (and particularly
cells, tissues, and organs expressing a member of this pool).
Examples include the VLDLR on muscle tissue, LRP1B on neuronal
tissue, Megalin on both kidney and neuronal tissue and LRP1 on
vascular smooth muscle tissue.
[0113] III. RAP
[0114] "RAP" is a well-known protein of about 39 kDa and 323 amino
acids and is a specialized chaperone for members of the LRP family.
RAP inhibits the binding of ligand to members of the LDL-receptor
family such as LRP (see Bu, G. & Rennke, S. J. Biol. Chem. 271:
22218-2224 (1996); Willnow, T. E, Goldstein, J. L., Orth, K.,
Brown, M. S. & Herz, J. J. Biol. Chem. 267: 26172-26180 (1992);
Bu, G. & Schwartz, A. L. Trends Cell Biol. 8: 272-276 (1998);
and Herz, J. & Strickland, D. K. J. Clin.Invest. 108: 779-784
(2001). See also, Bu and Schwartz, (1998). Further characterization
of RAP, including the complete amino acid sequence of human RAP
(FIG. 15), is found in U.S. Pat. No. 5,474,766 which is
incorporated herein by reference in its entirety and also with
particularity with respect to the RAP amino acid sequences and
fragments disclosed therein. The 28 kDa human C-terminal fragment
(FIG. 16) is an extremely active RAP polypeptide and in preferred
embodiments of the invention, the conjugate comprises this fragment
as the carrier for the active agent.
[0115] RAP polypeptides include, but are not limited to, RAP,
soluble forms of RAP, cleaved RAP, RAP polypeptide fragments,
homologues and analogs of RAP, and the like. RAP polypeptides that
are functional equivalents of RAP with respect to modulation of LRP
receptor binding, transcytosis, or endocytosis can be readily
identified by screening for the ability of the RAP polypeptide to
bind to LRP. In preferred embodiments, the RAP polypeptide is a
homologue of RAP having, for instance, greater than 80%, 90% 95%,
98%, or 99% sequence identity with a naturally occurring, native or
wild type mammalian RAP amino acid sequence of similar length or
over a domain of at least 10 amino acids, 25 amino acids, 50 amino
acids, 100 amino acids, or 200 amino acids, 300 amino acids, or the
entire length of the RAP polypeptide. RAP polypeptides include
allelic variants of RAP, paralogs and orthologs in human, mouse,
rat, chicken, zebrafish, pig, fruit fly, mosquito, and flatworm
native RAP, and derivatives, portions, or fragments thereof
(Genbank accession numbers: P30533 (human), XP132029 (mouse),
Q99068 (rat), CAA05085 (chicken), AAH49517 (zebrafish), AAM90301
(pig), NP649950 (fruit fly), XP313261 (mosquito), NP506187
(flatworm). A multiple alignment of amino acid sequences from
mouse, rat, chicken zebrafish, fruitfly, mosquito, and flatworm and
the consensus sequence is shown in FIG. 14.
[0116] The RAP polypeptide can be in the form of acidic or basic
salts, or in its neutral form. In addition, individual amino acid
residues can be modified, such as by oxidation or reduction.
Moreover, various substitutions, deletions, or additions can be
made to the amino acid or nucleic acid sequences, the net effect of
which is to retain or improve upon the desired biological activity
of RAP. Further characterization of RAP, including the complete
amino acid sequence of RAP, is found in U.S. Pat. No. 5,474,766
which is incorporated herein by reference in its entirety and also
with particularity with respect to the amino acid sequences of the
various RAP polypeptides disclosed therein. Due to code degeneracy,
for example, one of ordinary skill in the art would know of
considerable variations of the nucleotide sequences encoding the
same amino acid sequence.
[0117] Preferred RAP polypeptides share substantial homology with
the native amino acid sequence of a receptor associated protein
(RAP), particularly the native human sequence (SEQ ID NO:1). In
preferred embodiments, the RAP polypeptide is a homologue of RAP
having, for instance, greater than 80%, 90% 95%, 98%, or 99%
sequence identity with a native or wild type mammalian RAP amino
acid sequence of similar length or over a domain or comparison
window of at least 10, amino acids, 25 amino acids, 50 amino acids,
100 amino acids, or 200 amino acids, or 300 amino acids or
more.
[0118] An especially preferred human or mammalian RAP is isolated
RAP or a fragment thereof, such as a soluble polypeptide fragment
of RAP, which contains at least one of the RAP binding sites for
LRP. Substantial guidance exists in the art to which portions of
RAP are important to its LRP binding and modulatory activity and
which portions may be mutated, altered, or deleted without loss of
binding activity (see, Nielsen et al. Proc. Nat. Acad. Sci. USA
94:7521 (1997); and Rall et al. J. Biol. Chem.
273(37):24152(1998)). For instance, RAP's LRP binding function has
been mapped by performing direct binding studies on fusion proteins
representing overlapping domains of RAP (see Willnow et al., J.
Biol. Chem. 267(36):26172-80 (1992). The RAP binding motifs have
also been characterized by use of truncated and site-directed RAP
mutants (see Melman et al. J. Biol. Chem. 276(31):29338-29346
(2001). Particular RAP polypeptide fragments, suitable for use
according to the invention, include fragments (defined from RAP N
terminus amino acid to RAP C-terminus amino acid position) 1-323
(RAP); 1-319; 1-250; 1-110; 91-210; 191-323; 221-323; 1-190; 1-200;
and 1-210. Preferred RAP polypeptides include fragments 1-323
(RAP); 1-319; 191-323; and 1-210. A modified RAP polypeptide having
the C-terminal four amino acid sequence substituted by the sequence
KDEL is also suitable. A modified RAP polypeptide in which the
C-terminal-four amino acid sequence (HNEL) is deleted is also
suitable. Also preferred are RAP polypeptides fragments that
comprise the native sequence of RAP from amino acid 201 to 210.
[0119] Other preferred embodiments, comprise a human or mammalian
RAP polypeptide in which the polypeptide comprises the native amino
acid sequence of RAP over positions 282-289, 201-210, and 311-319.
Mutated and N-terminus or C-terminus truncated variants of RAP
which bind to the LRP receptor are disclosed in Melman et al.,
2001) which is incorporated herein by reference in its entirety and
with particularity to these RAP mutated and truncated variants.
Other preferred RAP polypeptides comprise a native sequence of RAP
between amino acids 85-148 and 178-248. (see Farquhar, et al.,
Proc. Nat. Acad. Sci. USA 91:3161-3162 (1994).
[0120] Thus, many references disclose the binding sites and
structure activity relationships for binding of RAP and RAP
fragments to the LRP receptor. The skilled artisan can readily
adapt a variety of well known techniques in the art in order to
obtain RAP polypeptides that contain a LRP binding site and are
suitable for use as RAP polypeptides according to the invention.
The preferred fragments of RAP are soluble under physiological
conditions. The N-terminus or C-terminus of these polypeptides can
be shortened as desired, provided that the binding capacity for the
LRP particle remains intact. The preferred amino acid sequence of
RAP corresponds to the human protein. Suitable sequences for a RAP
polypeptide can also be derived from the amino acid sequences of
RAP isolated from other mammals or members of the kingdom
Animalia.
[0121] In order to generate fragments of RAP which contains the LRP
binding site, isolated native protein may be converted by enzymatic
and/or chemical cleavage to generate fragments of the whole
protein, for example by reacting RAP with an enzyme such as papain
or trypsin or a chemical such as cyanogen bromide. Proteolytically
active enzymes or chemicals are preferably selected in order to
release the extracellular receptor region. Fragments that contain
the LRP binding site, especially fragments that are soluble under
physiological conditions, can then be isolated using known
methods.
[0122] Alternatively, RAP or a fragment of RAP may be expressed in
a recombinant bacteria, as described, for example, in Williams et
al., J. Biol. Chem. 267:9035-9040 (1992); Wurshawsky et al., J.
Biol. Chem. 269:3325-3330 (1994); Melman et al. J. Biol. Chem.
276(31): 29338-46 (2001).
[0123] RAP can be in the form of acidic or basic salts, or in
neutral forms. In addition, individual amino acid residues can be
modified, such as by oxidation or reduction. Moreover, various
substitutions, deletions, or additions can be made to the amino
acid or nucleic acid sequences, the net effect of which is to
retain or improve upon the desired biological activity of RAP. Due
to code degeneracy, for example, there may be considerable
variation in nucleotide sequences encoding the same amino acid
sequence.
[0124] A RAP fragment as used herein includes, but not limited to,
any portion of RAP or its biologically equivalent analogs that
contains a sufficient portion of the ligand to enable it to bind to
LRP and to be transcytosed, transported across the blood-brain
barrier; or that otherwise retains or improves upon the desired LRP
mediated carrier activities of the ligand.
[0125] FIG. 15 shows the amino acid sequence of human RAP.
[0126] FIG. 16 shows the amino acid sequence of the 28 kd RAP
polypeptide.
[0127] IV. RAP-Conjugates
[0128] A "RAP-conjugate" or "RAP-polypeptide conjugate" each refers
to a compound comprising RAP or a RAP polypeptide, or a fragment
thereof, attached to an active agent. As used herein, the term
"conjugated" means that the therapeutic agent(s) and RAP or the RAP
polypeptide are physically linked by, for example, by covalent
chemical bonds, physical forces such van der Waals or hydrophobic
interactions, encapsulation, embedding, or combinations thereof. In
preferred embodiments, the therapeutic agent(s) and the RAP
polypeptide are physically linked by covalent chemical bonds. As
such, preferred chemotherapeutic agents contain a functional group
such as an alcohol, acid, carbonyl, thiol or amine group to be used
in the conjugation to RAP or the RAP polypeptide. Adriamycin is in
the amine class and there is also the possibility to link through
the carbonyl as well. Paclitaxel is in the alcohol class.
Chemotherapeutic agents without suitable conjugation groups may be
further modified to add such a group. All these compounds are
contemplated in this invention. In the case of multiple therapeutic
agents, a combination of various conjugations can be used.
[0129] In some embodiments, a covalent chemical bond that may be
either direct (no intervening atoms) or indirect (through a linker
e.g., a chain of covalently linked atoms) joins the RAP polypeptide
and the active agent. In preferred embodiments, the RAP or RAP
polypeptide moiety and the active agent moiety of the conjugate are
directly linked by covalent bonds between an atom of the RAP
polypeptide and an atom of the active agent. In some preferred
embodiments, the RAP moiety is connected to the active agent moiety
of the compound according to the invention by a linker which
comprises a covalent bond or a peptide of virtually any amino acid
sequence or any molecule or atoms capable of connecting RAP or the
RAP polypeptide to the active agent.
[0130] In some embodiments, the linker comprises a chain of atoms
from 1 to about 30 atoms or longer, 2 to 5 atoms, 2 to 10 atoms, 5
to 10 atoms, or 10 to 20 atoms long. In some embodiments, the chain
atoms are all carbon atoms. In some embodiments, the chain atoms
are selected from the group consisting of C, O, N, and S. Chain
atoms and linkers may be selected according to their expected
solubility (hydrophilicity) so as to provide a more soluble
conjugate. In some embodiments, the linker provides a functional
group that is subject to enzymatic attack in a lysosome. In some
embodiments, the linker provides a functional group which is
subject to attack by an enzyme found in the target tissue or organ
and which upon attack or hydrolysis severs the link between the
active agent and the RAP polypeptide. In some embodiments, the
linker provides a functional group that is subject to hydrolysis
under the conditions found at the target site (e.g., low pH of a
lysosome). A linker may contain one or more such functional groups.
In some embodiments, the length of the linker is long enough to
reduce the potential for steric hindrance (when an active agent is
large) between one or both of the RAP polypeptide binding site and
the active agent active binding site.
[0131] If the linker is a covalent bond or a peptide and the active
agent is a polypeptide, then the entire conjugate can be a fusion
protein. Such fusion proteins may be produced by recombinant
genetic engineering methods known to one of ordinary skill in the
art. In some embodiments, the RAP fragment degrades quickly to
release the active compound. In other embodiments, the linker is
subject to cleavage under intracellular, or more preferably,
lysosomal environmental conditions to release or separate the
active agent portion from the RAP polypeptide portion.
[0132] The conjugate can comprise one or more active agents linked
to the same RAP polypeptide. For example, conjugation reactions may
conjugate from 1 to 5, about 5, about 1 to 10, about 5 to 10, about
10 to 20, about 20 to30, or 30 or more molecules of an active agent
to the RAP polypeptide. These formulations can be employed as
mixtures, or they may be purified into specific stoichiometric
formulations. Those skilled in the art are able to determine which
format and which stoichiometric ratio is preferred. Further, more
than one type of active agent may be linked to the RAP polypeptide
where delivery of more than one type of an agent to a target site
or compartment is desired. A plurality of active agent species may
be attached to the same RAP polypeptide e.g.,
adriamycin-cisplatinum RAP polypeptide conjugates. Thus, the
conjugates may consist of a range of stoichiometric ratios and
incorporate more than one type of active agent. These, too, may be
separated into purified mixtures or they may be employed in
aggregate.
[0133] The RAP or RAP polypeptide conjugate according to the
invention may be modified as desired to enhance its stability or
pharmacokinetic properties (e.g., PEGylation).Suitable linkers and
their functional groups for conjugating RAP polypeptides and an
active agent, and the synthetic chemical methods readily adaptable
for preparing such, are described in U.S. Patent Application No.
60/395,762 which is assigned to the same assignee as the present
application and herein incorporated by reference in its
entirety.
[0134] The synthesis of these conjugates is efficient and
convenient, producing high yields and drugs with enhanced aqueous
solubility.
[0135] V. Active Agents
[0136] Active agents according to the invention include agents that
can affect a biological process. Particularly preferred active
agents for use in the compounds compositions and methods of the
invention are therapeutic agents, including drugs and diagnostic
agents. The term "drug" or "therapeutic agent" refers to an active
agent that has a pharmacological activity or benefits health when
administered in a therapeutically effective amount. Particularly
preferred agents are naturally occurring biological agents (e.g.,
enzymes, proteins, polynucleotdies, antibodies, polypeptides). In
some embodiments, the active agent conjugated to RAP or RAP
polypeptide is a molecule, as well as any binding portion or
fragment thereof, that is capable of modulating a biological
process in a living host. Examples of drugs or therapeutic agents
include substances that are used in the prevention, diagnosis,
alleviation, treatment or cure of a disease or condition.
[0137] A. Protein Active Agents
[0138] The active agent can be a non-protein or a protein. The
active agent can be a protein or enzyme or any fragment of such
that still retains some, substantially all, or all of the
therapeutic or biological activity of the protein or enzyme. In
some embodiments, the protein or enzyme is one that, if not
expressed or produced or if substantially reduced in expression or
production, would give rise to a disease, including but not limited
to, lysosomal storage diseases. Preferably, the protein or enzyme
is derived or obtained from a human or mouse.
[0139] If the compound is a protein, the compound can be an enzyme,
or any fragment of an enzyme that still retains some, substantially
all, or all of the activity of the enzyme. Preferably, in the
treatment of lysosomal storage diseases, the enzyme is an enzyme
that is found in a cell that if not expressed or produced or is
substantially reduced in expression or production would give rise
to a lysosomal storage disease. Preferably, the enzyme is derived
or obtained from a human or mouse. Preferably, the enzyme is a
lysosomal storage enzyme, such as .alpha.-L-iduronidase,
iduronate-2-sulfatase, heparan N-sulfatase,
.alpha.-N-acetylglucosaminida- se, arylsulfatase A,
galactosylceramidase, acid-alpha-glucosidase, tripeptidyl
peptidase, hexosaminidase alpha, acid sphingomyelinase,
.alpha.-galactosidase, or any other lysosomal storage enzyme.
[0140] In some embodiments, therefore, in the treatment of human
Lysosomal Storage Diseases (LSDs), the RAP polypeptide-active agent
conjugate comprises an active agent protein or enzyme that is
deficient in the lysosomes of a subject or patient to be treated.
Such enzymes, include for example, alpha-L-iduronidase,
iduronate-2-sulfatase, heparan N-sulfatase,
alpha-N-acetylglucosaminidase, Arylsulfatase A,
Galactosylceramidase, acid-alpha-glucosidase, thioesterase,
hexosaminidase A, Acid Spingomyelinase, alpha-galactosidase, or any
other lysosomal storage enzyme. A table of lysosomal storage
diseases and the proteins deficient therein, which are useful as
active agents, follows:
1 Lysosomal Storage Disease Protein deficiency
Mucopolysaccharidosis type I L-Iduronidase Mucopolysaccharidosis
type II Hunter Iduronate-2-sulfatase syndrome Mucopolysaccharidosis
type IIIA Sanfilippo Heparan-N-sulfatase syndrome
Mucopolysaccharidosis type IIIB Sanfilippo
.alpha.-N-Acetylglucosaminidase syndrome Mucopolysaccharidosis type
IIIC Sanfilippo AcetylCoA:N- syndrome acetyltransferase
Mucopolysaccharidosis type IIID Sanfilippo N-Acetylglucosamine
syndrome 6-sulfatase Mucopolysaccharidosis type IVA Morquio
Galactose 6-sulfatase syndrome Mucopolysaccharidosis type IVB
Morquio .beta.-Galactosidase syndrome Mucopolysaccharidosis type VI
N-Acetylgalactosamine 4-sulfatase Mucopolysaccharidosis type VII
Sly .beta.-Glucuronidase syndrome Mucopolysaccharidosis type IX
hyaluronoglucosaminidase Aspartylglucosaminuria
Aspartylglucosaminidase Cholesterol ester storage disease/Wolman
Acid lipase disease Cystinosis Cystine transporter Danon disease
Lamp-2 Fabry disease .alpha.-Galactosidase A Farber
Lipogranulomatosis/Farber disease Acid ceramidase Fucosidosis
.alpha.-L-Fucosidase Galactosialidosis types I/II Protective
protein Gaucher disease types I/IIIII Gaucher disease
Glucocerebrosidase (.beta.-glucosidase) Globoid cell
leukodystrophy/Krabbe disease Galactocerebrosidase Glycogen storage
disease II/Pompe disease .alpha.-Glucosidase GM1-Gangliosidosis
types I/II/III .beta.-Galactosidase GM2-Gangliosidosis type I/Tay
Sachs .beta.-Hexosaminidase A disease GM2-Gangliosidosis type II
Sandhoff .beta.-Hexosaminidase A disease GM2-Gangliosidosis
GM2-activator deficiency .alpha.-Mannosidosis types I/II
.alpha.-D-Mannosidase .beta.-Mannosidosis .beta.-D-Mannosidase
Metachromatic leukodystrophy Arylsulfatase A Metachromatic
leukodystrophy Saposin B Mucolipidosis type I/Sialidosis types I/II
Neuraminidase Mucolipidosis types II/III I-cell disease
Phosphotransferase Mucolipidosis type IIIC pseudo-Hurler
Phosphotransferase polydystrophy .gamma.-subunit Multiple sulfatase
deficiency Multiple sulfatases Neuronal Ceroid Lipofuscinosis, CLN1
Palmitoyl protein Batten disease thioesterase Neuronal Ceroid
Lipofuscinosis, CLN2 Tripeptidyl peptidase I Batten disease
Niemann-Pick disease types A/B Niemann- Acid sphingomyelinase Pick
disease Niemann-Pick disease type C1 Niemann- Cholesterol
trafficking Pick disease Niemann-Pick disease type C2 Niemann-
Cholesterol trafficking Pick disease Pycnodysostosis Cathepsin K
Schindler disease types I/II Schindler disease
.alpha.-Galactosidase B Sialic acid storage disease sialic acid
transporter
[0141] Thus, the lysosomal storage diseases that can be treated or
prevented using the methods of the present invention include, but
are not limited to, Mucopolysaccharidosis I (MPS I), MPS II, MPS
IIIA, MPS IIIB, Metachromatic Leukodystrophy (MLD), Krabbe, Pompe,
Ceroid Lipofuscinosis, Tay-Sachs, Niemnann-Pick A and B, and other
lysosomal diseases.
[0142] Thus, per the above table, for each disease the conjugated
agent would preferably comprise a specific active agent enzyme
deficient in the disease. For instance, for methods involving MPS
I, the preferred compound or enzyme is .alpha.-L-iduronidase. For
methods involving MPS II, the preferred compound or enzyme is
iduronate-2-sulfatase. For methods involving MPS IIIA, the
preferred compound or enzyme is heparan N-sulfatase. For methods
involving MPS IIIB, the preferred compound or enzyme is
.alpha.-N-acetylglucosaminidase. For methods involving
Metachromatic Leukodystropy (MLD), the preferred compound or enzyme
is arylsulfatase A. For methods involving Krabbe, the preferred
compound or enzyme is galactosylceramidase. For methods involving
Pompe, the preferred compound or enzyme is acid
.alpha.-glucosidase. For methods involving CLN, the preferred
compound or enzyme is tripeptidyl peptidase. For methods involving
Tay-Sachs, the preferred compound or enzyme is hexosaminidase
alpha. For methods involving Niemann-Pick A and B the preferred
compound or enzyme is acid sphingomyelinase.
[0143] The RAP or RAP polypeptide active agent conjugate can
comprise one or more agent moieties (e.g., 1 to 10 or 1 to 4 or 2
to 3 moieties) linked to RAP or a RAP polypeptide. For example,
conjugation reactions may conjugate from 1 to 4 or more molecules
of alpha-L-iduronidase to a single RAP polypeptide molecule. These
formulations can be employed as mixtures, or they may be purified
into specific RAP polypeptide-agent stoichiometric formulations.
Those skilled in the art are able to determine which format and
which stoichiometric ratio is preferred. Further, one or more
different active agents may be linked to RAP or the RAP polypeptide
to facilitate a more complete degradation of the stored substrates.
These RAP or RAP polypeptide conjugated agents may consist of a
range of stoichiometric ratios. These, too, may be separated into
purified mixtures or they may be employed in aggregate.
[0144] The RAP or RAP polypeptide conjugated active agents can
enter or be transported into or end up residing in the lysosomes of
a cell within or without the CNS. The rate of passage of the
conjugated agent can be modulated by any compound or protein that
can modulate LRP binding activity. The cell can be from any tissue
or organ system affected by the lysosomal storage disease. The cell
can be, for instance, an endothelial, epithelial, muscle, heart,
bone, lung, fat, kidney, or liver cell. In some embodiments, the
cell is preferably a cell found within the BBB. In some
embodiments, the cell is a neuron or a brain cell. In other
embodiments, the cell is a cell of the periphery or one that is not
isolated from the general circulation by an endothelium such as
that of the BBB.
[0145] B. Drug Active Agents
[0146] Generally, the drug active agent may be of any size.
Preferred drugs are small organic molecules that are capable of
binding to the target of interest. A drug moiety of the conjugate,
when a small molecule, generally has a molecular weight of at least
about 50 D, usually at least about 100 D, where the molecular
weight may be as high as 500 D or higher, but will usually not
exceed about 2000 D.
[0147] The drug moiety is capable of interacting with a target in
the host into which the conjugate is administered during practice
of the subject methods. The target may be a number of different
types of naturally occurring structures, where targets of interest
include both intracellular and extracellular targets, where such
targets may be proteins, phospholipids, nucleic acids and the like,
where proteins are of particular interest. Specific proteinaceous
targets of interest include, without limitation, enzymes, e.g.,
kinases, phosphatases, reductases, cyclooxygenases, proteases and
the like, targets comprising domains involved in protein-protein
interactions, such as the SH2, SH3, PTB and PDZ domains, structural
proteins, e.g., actin, tubulin, etc., membrane receptors,
immunoglobulins, e.g., IgE, cell adhesion receptors, such as
integrins, etc, ion channels, transmembrane pumps, transcription
factors, signaling proteins, and the like.
[0148] In some embodiments, the active agent or drug has a hydroxyl
or an amino group for reacting with the isocyanate reagent or the
active agent is chemically modified to introduce a hydroxyl or an
amino group for reacting with the isocyanate reagent.
[0149] In some embodiments, the active agent or drug comprises a
region that may be modified and/or participate in covalent linkage,
preferably, without loss of the desired biological activity of the
active agent. The drug moieties often comprise cyclical carbon or
heterocyclic structures and/or aromatic or polyaromatic structures
substituted with one or more of the above functional groups. Also
of interest as drug moieties are structures found among
biomolecules, proteins, enzymes, polysaccharides, and polynucleic
acids, including peptides, saccharides, fatty acids, steroids,
purines, pyrimidines, derivatives, structural analogs or
combinations thereof.
[0150] Suitable active agents include, but are not limited to,
psychopharmacological agents, such as (1) central nervous system
depressants, e.g., general anesthetics (barbiturates,
benzodiazepines, steroids, cyclohexanone derivatives, and
miscellaneous agents), sedative-hypnotics (benzodiazepines,
barbiturates, piperidinediones and triones, quinazoline
derivatives, carbamates, aldehydes and derivatives, amides, acyclic
ureides, benzazepines and related drugs, phenothiazines, etc.),
central voluntary muscle tone modifying drugs (anticonvulsants,
such as hydantoins, barbiturates, oxazolidinediones, succinimides,
acylureides, glutarimides, benzodiazepines, secondary and tertiary
alcohols, dibenzazepine derivatives, valproic acid and derivatives,
GABA analogs, etc.), analgesics (morphine and derivatives,
oripavine derivatives, morphinan derivatives, phenylpiperidines,
2,6-methane-3-benzazocaine derivatives, diphenylpropylamines and
isosteres, salicylates, p-aminophenol derivatives, 5-pyrazolone
derivatives, arylacetic acid derivatives, fenamates and isosteres,
etc.) and antiemetics (anticholinergics, antihistamines,
antidopaminergics, etc.), (2) central nervous system stimulants,
e.g., analeptics (respiratory stimulants, convulsant stimulants,
psychomotor stimulants), narcotic antagonists (morphine
derivatives, oripavine derivatives, 2,6-methane-3-benzoxacine
derivatives, morphinan derivatives) nootropics, (3)
psychopharmacologicals, e.g., anxiolytic sedatives
(benzodiazepines, propanediol carbamates) antipsychotics
(phenothiazine derivatives, thioxanthine derivatives, other
tricyclic compounds, butyrophenone derivatives and isosteres,
diphenylbutylamine derivatives, substituted benzamides,
arylpiperazine derivatives, indole derivatives, etc.),
antidepressants (tricyclic compounds, MAO inhibitors, etc.), (4)
respiratory tract drugs, e.g., central antitussives (opium
alkaloids and their derivatives); pharmacodynamic agents, such as
(1) peripheral nervous system drugs, e.g., local anesthetics (ester
derivatives, amide derivatives), (2) drugs acting at synaptic or
neuroeffector junctional sites, e.g., cholinergic agents,
cholinergic blocking agents, neuromuscular blocking agents,
adrenergic agents, antiadrenergic agents, (3) smooth muscle active
drugs, e.g., spasmolytics (anticholinergics, musculotropic
spasmolytics), vasodilators, smooth muscle stimulants, (4)
histamines and antihistamines, e.g., histamine and derivative
thereof (betazole), antihistamines (H.sub.1-antagonists,
H.sub.2-antagonists), histamine metabolism drugs, (5)
cardiovascular drugs, e.g., cardiotonics (plant extracts,
butenolides, pentadienolids, alkaloids from erythrophleum species,
ionophores, adrenoceptor stimulants, etc), antiarrhythmic drugs,
antihypertensive agents, antilipidemic agents (clofibric acid
derivatives, nicotinic acid derivatives, hormones and analogs,
antibiotics, salicylic acid and derivatives), antivaricose drugs,
hemostyptics, (6) blood and hemopoietic system drugs, e.g.,
antianemia drugs, blood coagulation drugs (hemostatics,
anticoagulants, antithrombotics, thrombolytics, blood proteins and
their fractions), (7) gastrointestinal tract drugs, e.g.,
digestants (stomachics, choleretics), antiulcer drugs,
antidiarrheal agents, (8) locally acting drugs; chemotherapeutic
agents, such as (1) anti-infective agents, e.g., ectoparasiticides
(chlorinated hydrocarbons, pyrethins, sulfurated compounds),
anthelmintics, antiprotozoal agents, antimalarial agents,
antiamebic agents, antileiscmanial drugs, antitrichomonal agents,
antitrypanosomal agents, sulfonamides, antimycobacterial drugs,
antiviral chemotherapeutics, etc., and (2) cytostatics, i.e.,
antineoplastic agents or cytotoxic drugs, such as alkylating
agents, e.g., Mechlorethamine hydrochloride (Nitrogen Mustard,
Mustargen, HN2), Cyclophosphamide (Cytovan, Endoxana), Ifosfamide
(IFEX), Chlorambucil (Leukeran), Melphalan (Phenylalanine Mustard,
L-sarcolysin, Alkeran, L-PAM), Busulfan (Myleran), Thiotepa
(Triethylenethiophosphoramide), Carmustine (BiCNU, BCNU), Lomustine
(CeeNU, CCNU), Streptozocin (Zanosar) and the like; plant
alkaloids, e.g., Vincristine (Oncovin), Vinblastine (Velban,
Velbe), Paclitaxel (Taxol), and the like; antimetabolites, e.g.,
Methotrexate (MTX), Mercaptopurine (Purinethol, 6-MP), Thioguanine
(6-TG), Fluorouracil (5-FU), Cytarabine (Cytosar-U, Ara-C),
Azacitidine (Mylosar, 5-AZA) and the like; antibiotics, e.g.,
Dactinomycin (Actinomycin D, Cosmegen), Doxorubicin (Adriamycin),
Daunorubicin (duanomycin, Cerubidine), Idarubicin (Idamycin),
Bleomycin (Blenoxane), Picamycin (Mithramycin, Mithracin),
Mitomycin (Mutamycin) and the like, and other anticellular
proliferative agents, e.g., Hydroxyurea (Hydrea), Procarbazine
(Mutalane), Dacarbazine (DTIC-Dome), Cisplatin (Platinol)
Carboplatin (Paraplatin), Asparaginase (Elspar) Etoposide (VePesid,
VP-16-213), Amsarcrine (AMSA, m-AMSA), Mitotane (Lysodren),
Mitoxantrone (Novatrone), and the like. Preferred chemotherapeutic
agents are those, which in the free form, demonstrate unacceptable
systemic toxicity at desired doses. The general systemic toxicity
associated with therapeutic levels of such agents may be reduced by
their linkage to RAP or a RAP polypeptide. Particularly preferred
are cardiotoxic compounds that are useful therapeutics but are dose
limited by cardiotoxicity. A classic example is adriamycin (also
known as doxorubicin) and its analogs, such as daunorubicin.
Linking RAP or a RAP polypeptide to such drugs may prevent
accumulation and associated cardiotoxicity at the heart.;
[0151] Suitable active agents include, but are not limited to:
Antibiotics, such as: aminoglycosides, e.g., amikacin, apramycin,
arbekacin, bambermycins, butirosin, dibekacin, dihydrostreptomycin,
fortimicin, gentamicin, isepamicin, kanamycin, micronomcin,
neomycin, netilmicin, paromycin, ribostamycin, sisomicin,
spectinomycin, streptomycin, tobramycin, trospectomycin;
amphenicols, e.g., azidamfenicol, chloramphenicol, florfenicol, and
theimaphenicol; ansamycins, e.g., rifamide, rifampin, rifamycin,
rifapentine, rifaximin; beta.-lactams, e.g., carbacephems,
carbapenems, cephalosporins, cehpamycins, monobactams, oxaphems,
penicillins; lincosamides, e.g., clinamycin, lincomycin;
macrolides, e.g., clarithromycin, dirthromycin, erythromycin, etc.;
polypeptides, e.g., amphomycin, bacitracin, capreomycin, etc.;
tetracyclines, e.g., apicycline, chlortetracycline, clomocycline,
etc.; synthetic antibacterial agents, such as
2,4-diaminopyrimidines, nitrofurans, quinolones and analogs
thereof, sulfonamides, sulfones;
[0152] Suitable active agents include, but are not limited to:
Antifungal agents, such as: polyenes, e.g., amphotericin B,
candicidin, dermostatin, filipin, fungichromin, hachimycin,
hamycin, lucensomycin, mepartricin, natamycin, nystatin, pecilocin,
perimycin; synthetic antifungals, such as allylamines, e.g.,
butenafine, naftifine, terbinafine; imidazoles, e.g., bifonazole,
butoconazole, chlordantoin, chlormidazole, etc., thiocarbamates,
e.g., tolciclate, triazoles, e.g., fluconazole, itraconazole,
terconazole;
[0153] Suitable active agents include, but are not limited to:
Antihelmintics, such as: arecoline, aspidin, aspidinol,
dichlorophene, embelin, kosin, napthalene, niclosamide,
pelletierine, quinacrine, alantolactone, amocarzine, amoscanate,
ascaridole, bephenium, bitoscanate, carbon tetrachloride,
carvacrol, cyclobendazole, diethylcarbamazine, etc.;
[0154] Suitable active agents include, but are not limited to:
Antimalarials, such as: acedapsone, amodiaquin, arteether,
artemether, artemisinin, artesunate, atovaquone, bebeerine,
berberine, chirata, chlorguanide, chloroquine, chlorprogaunil,
cinchona, cinchonidine, cinchonine, cycloguanil, gentiopicrin,
halofantrine, hydroxychloroquine, mefloquine hydrochloride,
3-methylarsacetin, pamaquine, plasmocid, primaquine, pyrimethamine,
quinacrine, quinidine, quinine, quinocide, quinoline, dibasic
sodium arsenate;
[0155] Suitable active agents include, but are not limited to:
Antiprotozoan agents, such as: acranil, tinidazole, ipronidazole,
ethylstibamine, pentamidine, acetarsone, aminitrozole, anisomycin,
nifuratel, tinidazole, benzidazole, suramin, and the like.
[0156] Suitable drugs for use as active agents are also listed in:
Goodman & Gilman's, The Pharmacological Basis of Therapeutics
(9th Ed) (Goodman et al. eds) (McGraw-Hill) (1996); and 1999
Physician's Desk Reference (1998).
[0157] Suitable active agents include, but are not limited to:
antineoplastic agents, as disclosed in U.S. Pat. Nos. 5,880,161,
5,877,206, 5,786,344, 5,760,041, 5,753,668, 5,698,529, 5,684,004,
5,665,715, 5,654,484, 5,624,924, 5,618,813, 5,610,292, 5,597,831,
5,530,026, 5,525,633, 5,525,606, 5,512,678, 5,508,277, 5,463,181,
5,409,893, 5,358,952, 5,318,965, 5,223,503, 5,214,068, 5,196,424,
5,109,024, 5,106,996, 5,101,072, 5,077,404, 5,071,848, 5,066,493,
5,019,390, 4,996,229, 4,996,206, 4,970,318, 4,968,800, 4,962,114,
4,927,828, 4,892,887, 4,889,859, 4,886,790, 4,882,334, 4,882,333,
4,871,746, 4,863,955, 4,849,563, 4,845,216, 4,833,145, 4,824,955,
4,785,085, 4,684,747, 4,618,685, 4,611,066, 4,550,187, 4,550,186,
4,544,501, 4,541,956, 4,532,327, 4,490,540, 4,399,283, 4,391,982,
4,383,994, 4,294,763, 4,283,394, 4,246,411, 4,214,089, 4,150,231,
4,147,798, 4,056,673, 4,029,661, 4,012,448;
[0158] psychopharmacological/psychotropic agents, as disclosed in
U.S. Pat. Nos. 5,192,799, 5,036,070, 4,778,800, 4,753,951,
4,590,180, 4,690,930, 4,645,773, 4,427,694, 4,424,202, 4,440,781,
5,686,482, 5,478,828, 5,461,062, 5,387,593, 5,387,586, 5,256,664,
5,192,799, 5,120,733, 5,036,070, 4,977,167, 4,904,663, 4,788,188,
4,778,800, 4,753,951, 4,690,930, 4,645,773, 4,631,285, 4,617,314,
4,613,600, 4,590,180, 4,560,684, 4,548,938, 4,529,727, 4,459,306,
4,443,451, 4,440,781, 4,427,694, 4,424,202, 4,397,853, 4,358,451,
4,324,787, 4,314,081, 4,313,896, 4,294,828, 4,277,476, 4,267,328,
4,264,499, 4,231,930, 4,194,009, 4,188,388, 4,148,796, 4,128,717,
4,062,858, 4,031,226, 4,020,072, 4,018,895, 4,018,779, 4,013,672,
3,994,898, 3,968,125, 3,939,152, 3,928,356, 3,880,834,
3,668,210;
[0159] cardiovascular agents, as disclosed in U.S. Pat. Nos.
4,966,967, 5,661,129, 5,552,411, 5,332,737, 5,389,675, 5,198,449,
5,079,247, 4,966,967, 4,874,760, 4,954,526, 5,051,423, 4,888,335,
4,853,391, 4,906,634, 4,775,757, 4,727,072, 4,542,160, 4,522,949,
4,524,151, 4,525,479, 4,474,804, 4,520,026, 4,520,026, 5,869,478,
5,859,239, 5,837,702, 5,807,889, 5,731,322, 5,726,171, 5,723,457,
5,705,523, 5,696,111, 5,691,332, 5,679,672, 5,661,129, 5,654,294,
5,646,276, 5,637,586, 5,631,251, 5,612,370, 5,612,323, 5,574,037,
5,563,170, 5,552,411, 5,552,397, 5,547,966, 5,482,925, 5,457,118,
5,414,017, 5,414,013, 5,401,758, 5,393,771, 5,362,902, 5,332,737,
5,310,731, 5,260,444, 5,223,516, 5,217,958, 5,208,245, 5,202,330,
5,198,449, 5,189,036, 5,185,362, 5,140,031, 5,128,349, 5,116,861,
5,079,247, 5,070,099, 5,061,813, 5,055,466, 5,051,423, 5,036,065,
5,026,712, 5,011,931, 5,006,542, 4,981,843, 4,977,144, 4,971,984,
4,966,967, 4,959,383, 4,954,526, 4,952,692, 4,939,137, 4,906,634,
4,889,866, 4,888,335, 4,883,872, 4,883,811, 4,847,379, 4,835,157,
4,824,831, 4,780,538, 4,775,757, 4,774,239, 4,771,047, 4,769,371,
4,767,756, 4,762,837, 4,753,946, 4,752,616, 4,749,715, 4,738,978,
4,735,962, 4,734,426, 4,734,425, 4,734,424, 4,730,052, 4,727,072,
4,721,796, 4,707,550, 4,704,382, 4,703,120, 4,681,970, 4,681,882,
4,670,560, 4,670,453, 4,668,787, 4,663,337, 4,663,336, 4,661,506,
4,656,267, 4,656,185, 4,654,357, 4,654,356, 4,654,355, 4,654,335,
4,652,578, 4,652,576, 4,650,874, 4,650,797, 4,649,139, 4,647,585,
4,647,573, 4,647,565, 4,647,561, 4,645,836, 4,639,461, 4,638,012,
4,638,011, 4,632,931, 4,631,283, 4,628,095, 4,626,548, 4,614,825,
4,611,007, 4,611,006, 4,611,005, 4,609,671, 4,608,386, 4,607,049,
4,607,048, 4,595,692, 4,593,042, 4,593,029, 4,591,603, 4,588,743,
4,588,742, 4,588,741, 4,582,854, 4,575,512, 4,568,762, 4,560,698,
4,556,739, 4,556,675, 4,555,571, 4,555,570, 4,555,523, 4,550,120,
4,542,160, 4,542,157, 4,542,156, 4,542,155, 4,542,151, 4,537,981,
4,537,904, 4,536,514, 4,536,513, 4,533,673, 4,526,901, 4,526,900,
4,525,479, 4,524,151, 4,522,949, 4,521,539, 4,520,026, 4,517,188,
4,482,562, 4,474,804, 4,474,803, 4,472,411, 4,466,979, 4,463,015,
4,456,617, 4,456,616, 4,456,615, 4,418,076, 4,416,896, 4,252,815,
4,220,594, 4,190,587, 4,177,280, 4,164,586, 4,151,297, 4,145,443,
4,143,054, 4,123,550, 4,083,968, 4,076,834, 4,064,259, 4,064,258,
4,064,257, 4,058,620, 4,001,421, 3,993,639, 3,991,057, 3,982,010,
3,980,652, 3,968,117, 3,959,296, 3,951,950, 3,933,834, 3,925,369,
3,923,818, 3,898,210, 3,897,442, 3,897,441, 3,886,157, 3,883,540,
3,873,715, 3,867,383, 3,873,715, 3,867,383, 3,691,216,
3,624,126;
[0160] antimicrobial agents as disclosed in U.S. Pat. Nos.
5,902,594, 5,874,476, 5,874,436, 5,859,027, 5,856,320, 5,854,242,
5,811,091, 5,786,350, 5,783,177, 5,773,469, 5,762,919, 5,753,715,
5,741,526, 5,709,870, 5,707,990, 5,696,117, 5,684,042, 5,683,709,
5,656,591, 5,643,971, 5,643,950, 5,610,196, 5,608,056, 5,604,262,
5,595,742, 5,576,341, 5,554,373, 5,541,233, 5,534,546, 5,534,508,
5,514,715, 5,508,417, 5,464,832, 5,428,073, 5,428,016, 5,424,396,
5,399,553, 5,391,544, 5,385,902, 5,359,066, 5,356,803, 5,354,862,
5,346,913, 5,302,592, 5,288,693, 5,266,567, 5,254,685, 5,252,745,
5,209,930, 5,196,441, 5,190,961, 5,175,160, 5,157,051, 5,096,700,
5,093,342, 5,089,251, 5,073,570, 5,061,702, 5,037,809, 5,036,077,
5,010,109, 4,970,226, 4,916,156, 4,888,434, 4,870,093, 4,855,318,
4,784,991, 4,746,504, 4,686,221, 4,599,228, 4,552,882, 4,492,700,
4,489,098, 4,489,085, 4,487,776, 4,479,953, 4,477,448, 4,474,807,
4,470,994, 4,370,484, 4,337,199, 4,311,709, 4,308,283, 4,304,910,
4,260,634, 4,233,311, 4,215,131, 4,166,122, 4,141,981, 4,130,664,
4,089,977, 4,089,900, 4,069,341, 4,055,655, 4,049,665, 4,044,139,
4,002,775, 3,991,201, 3,966,968, 3,954,868, 3,936,393, 3,917,476,
3,915,889, 3,867,548, 3,865,748, 3,867,548, 3,865,748, 3,783,160,
3,764,676, 3,764,677;
[0161] anti-inflammatory agents as disclosed in U.S. Pat. Nos.
5,872,109, 5,837,735, 5,827,837, 5,821,250, 5,814,648, 5,780,026,
5,776,946, 5,760,002, 5,750,543, 5,741,798, 5,739,279, 5,733,939,
5,723,481, 5,716,967, 5,688,949, 5,686,488, 5,686,471, 5,686,434,
5,684,204, 5,684,041, 5,684,031, 5,684,002, 5,677,318, 5,674,891,
5,672,620, 5,665,752, 5,656,661, 5,635,516, 5,631,283, 5,622,948,
5,618,835, 5,607,959, 5,593,980, 5,593,960, 5,580,888, 5,552,424,
5,552,422, 5,516,764, 5,510,361, 5,508,026, 5,500,417, 5,498,405,
5,494,927, 5,476,876, 5,472,973, 5,470,885, 5,470,842, 5,464,856,
5,464,849, 5,462,952, 5,459,151, 5,451,686, 5,444,043, 5,436,265,
5,432,181, RE034918, 5,393,756, 5,380,738, 5,376,670, 5,360,811,
5,354,768, 5,348,957, 5,347,029, 5,340,815, 5,338,753, 5,324,648,
5,319,099, 5,318,971, 5,312,821, 5,302,597, 5,298,633, 5,298,522,
5,298,498, 5,290,800, 5,290,788, 5,284,949, 5,280,045, 5,270,319,
5,266,562, 5,256,680, 5,250,700, 5,250,552, 5,248,682, 5,244,917,
5,240,929, 5,234,939, 5,234,937, 5,232,939, 5,225,571, 5,225,418,
5,220,025, 5,212,189, 5,212,172, 5,208,250, 5,204,365, 5,202,350,
5,196,431, 5,191,084, 5,187,175, 5,185,326, 5,183,906, 5,177,079,
5,171,864, 5,169,963, 5,155,122, 5,143,929, 5,143,928, 5,143,927,
5,124,455, 5,124,347, 5,114,958, 5,112,846, 5,104,656, 5,098,613,
5,095,037, 5,095,019, 5,086,064, 5,081,261, 5,081,147, 5,081,126,
5,075,330, 5,066,668, 5,059,602, 5,043,457, 5,037,835, 5,037,811,
5,036,088, 5,013,850, 5,013,751, 5,013,736, 5,006,542, 4,992,448,
4,992,447, 4,988,733, 4,988,728, 4,981,865, 4,962,119, 4,959,378,
4,954,519, 4,945,099, 4,942,236, 4,931,457, 4,927,835, 4,912,248,
4,910,192, 4,904,786, 4,904,685, 4,904,674, 4,904,671, 4,897,397,
4,895,953, 4,891,370, 4,870,210, 4,859,686, 4,857,644, 4,853,392,
4,851,412, 4,847,303, 4,847,290, 4,845,242, 4,835,166, 4,826,990,
4,803,216, 4,801,598, 4,791,129, 4,788,205, 4,778,818, 4,775,679,
4,772,703, 4,767,776, 4,764,525, 4,760,051, 4,748,153, 4,725,616,
4,721,712, 4,713,393, 4,708,966, 4,695,571, 4,686,235, 4,686,224,
4,680,298, 4,678,802, 4,652,564, 4,644,005, 4,632,923, 4,629,793,
4,614,741, 4,599,360, 4,596,828, 4,595,694, 4,595,686, 4,594,357,
4,585,755, 4,579,866, 4,578,390, 4,569,942, 4,567,201, 4,563,476,
4,559,348, 4,558,067, 4,556,672, 4,556,669, 4,539,326, 4,537,903,
4,536,503, 4,518,608, 4,514,415, 4,512,990, 4,501,755, 4,495,197,
4,493,839, 4,465,687, 4,440,779, 4,440,763, 4,435,420, 4,412,995,
4,400,534, 4,355,034, 4,335,141, 4,322,420, 4,275,064, 4,244,963,
4,235,908, 4,234,593, 4,226,887, 4,201,778, 4,181,720, 4,173,650,
4,173,634, 4,145,444, 4,128,664, 4,125,612, 4,124,726, 4,124,707,
4,117,135, 4,027,031, 4,024,284, 4,021,553, 4,021,550, 4,018,923,
4,012,527, 4,011,326, 3,998,970, 3,998,954, 3,993,763, 3,991,212,
3,984,405, 3,978,227, 3,978,219, 3,978,202, 3,975,543, 3,968,224,
3,959,368, 3,949,082, 3,949,081, 3,947,475, 3,936,450, 3,934,018,
3,930,005, 3,857,955, 3,856,962, 3,821,377, 3,821,401, 3,789,121,
3,789,123, 3,726,978, 3,694,471, 3,691,214, 3,678,169,
3,624,216;
[0162] immunosuppressive agents, as disclosed in U.S. Pat. Nos.
4,450,159, 4,450,159, 5,905,085, 5,883,119, 5,880,280, 5,877,184,
5,874,594, 5,843,452, 5,817,672, 5,817,661, 5,817,660, 5,801,193,
5,776,974, 5,763,478, 5,739,169, 5,723,466, 5,719,176, 5,696,156,
5,695,753, 5,693,648, 5,693,645, 5,691,346, 5,686,469, 5,686,424,
5,679,705, 5,679,640, 5,670,504, 5,665,774, 5,665,772, 5,648,376,
5,639,455, 5,633,277, 5,624,930, 5,622,970, 5,605,903, 5,604,229,
5,574,041, 5,565,560, 5,550,233, 5,545,734, 5,540,931, 5,532,248,
5,527,820, 5,516,797, 5,514,688, 5,512,687, 5,506,233, 5,506,228,
5,494,895, 5,484,788, 5,470,857, 5,464,615, 5,432,183, 5,431,896,
5,385,918, 5,349,061, 5,344,925, 5,330,993, 5,308,837, 5,290,783,
5,290,772, 5,284,877, 5,284,840, 5,273,979, 5,262,533, 5,260,300,
5,252,732, 5,250,678, 5,247,076, 5,244,896, 5,238,689, 5,219,884,
5,208,241, 5,208,228, 5,202,332, 5,192,773, 5,189,042, 5,169,851,
5,162,334, 5,151,413, 5,149,701, 5,147,877, 5,143,918, 5,138,051,
5,093,338, 5,091,389, 5,068,323, 5,068,247, 5,064,835, 5,061,728,
5,055,290, 4,981,792, 4,810,692, 4,410,696, 4,346,096, 4,342,769,
4,317,825, 4,256,766, 4,180,588, 4,000,275, 3,759,921;
[0163] immunomodulatory agents, as disclosed in U.S. Pat. Nos.
4,446,128, 4,524,147, 4,720,484, 4,722,899, 4,748,018, 4,877,619,
4,998,931, 5,049,387, 5,118,509, 5,152,980, 5,256,416, 5,468,729,
5,583,139, 5,604,234, 5,612,060, 5,612,350, 5,658,564, 5,672,605,
5,681,571, 5,708,002, 5,723,718, 5,736,143, 5,744,495, 5,753,687,
5,770,201, 5,869,057, 5,891,653, 5,939,455, 5,948,407, 6,006,752,
6,024,957, 6,030,624, 6,037,372, 6,037,373, 6,043,247, 6,060,049,
6,087,096, 6,096,315, 6,099,838, 6,103,235, 6,124,495, 6,153,203,
6,169,087, 6,255,278, 6,262,044, 6,290,950, 6,306,651, 6,322,796,
6,329,153, 6,344,476, 6,352,698, 6,365,163, 6,379,668, 6,391,303,
6,395,767, 6,403,555, 6,410,556, 6,412,492, 6,468,537, 6,489,330,
6,521,232, 6,525,035, 6,525,242, 6,558,663, 6,572,860;
[0164] analgesic agents, as disclosed in U.S. Pat. Nos. 5,292,736,
5,688,825, 5,554,789, 5,455,230, 5,292,736, 5,298,522, 5,216,165,
5,438,064, 5,204,365, 5,017,578, 4,906,655, 4,906,655, 4,994,450,
4,749,792, 4,980,365, 4,794,110, 4,670,541, 4,737,493, 4,622,326,
4,536,512, 4,719,231, 4,533,671, 4,552,866, 4,539,312, 4,569,942,
4,681,879, 4,511,724, 4,556,672, 4,721,712, 4,474,806, 4,595,686,
4,440,779, 4,434,175, 4,608,374, 4,395,402, 4,400,534, 4,374,139,
4,361,583, 4,252,816, 4,251,530, 5,874,459, 5,688,825, 5,554,789,
5,455,230, 5,438,064, 5,298,522, 5,216,165, 5,204,365, 5,030,639,
5,017,578, 5,008,264, 4,994,450, 4,980,365, 4,906,655, 4,847,290,
4,844,907, 4,794,110, 4,791,129, 4,774,256, 4,749,792, 4,737,493,
4,721,712, 4,719,231, 4,681,879, 4,670,541, 4,667,039, 4,658,037,
4,634,708, 4,623,648, 4,622,326, 4,608,374, 4,595,686, 4,594,188,
4,569,942, 4,556,672, 4,552,866, 4,539,312, 4,536,512, 4,533,671,
4,511,724, 4,440,779, 4,434,175, 4,400,534, 4,395,402, 4,391,827,
4,374,139, 4,361,583, 4,322,420, 4,306,097, 4,252,816, 4,251,530,
4,244,955, 4,232,018, 4,209,520, 4,164,514, 4,147,872, 4,133,819,
4,124,713, 4,117,012, 4,064,272, 4,022,836, 3,966,944;
[0165] cholinergic agents, as disclosed in U.S. Pat. Nos.
5,219,872, 5,219,873, 5,073,560, 5,073,560, 5,346,911, 5,424,301,
5,073,560, 5,219,872, 4,900,748, 4,786,648, 4,798,841, 4,782,071,
4,710,508, 5,482,938, 5,464,842, 5,378,723, 5,346,911, 5,318,978,
5,219,873, 5,219,872, 5,084,281, 5,073,560, 5,002,955, 4,988,710,
4,900,748, 4,798,841, 4,786,648, 4,782,071, 4,745,123,
4,710,508;
[0166] adrenergic agents, as disclosed in U.S. Pat. Nos. 5,091,528,
5,091,528, 4,835,157, 5,708,015, 5,594,027, 5,580,892, 5,576,332,
5,510,376, 5,482,961, 5,334,601, 5,202,347, 5,135,926, 5,116,867,
5,091,528, 5,017,618, 4,835,157, 4,829,086, 4,579,867, 4,568,679,
4,469,690, 4,395,559, 4,381,309, 4,363,808, 4,343,800, 4,329,289,
4,314,943, 4,311,708, 4,304,721, 4,296,117, 4,285,873, 4,281,189,
4,278,608, 4,247,710, 4,145,550, 4,145,425, 4,139,535, 4,082,843,
4,011,321, 4,001,421, 3,982,010, 3,940,407, 3,852,468,
3,832,470;
[0167] antihistamine agents, as disclosed in U.S. Pat. Nos.
5,874,479, 5,863,938, 5,856,364, 5,770,612, 5,702,688, 5,674,912,
5,663,208, 5,658,957, 5,652,274, 5,648,380, 5,646,190, 5,641,814,
5,633,285, 5,614,561, 5,602,183, 4,923,892, 4,782,058, 4,393,210,
4,180,583, 3,965,257, 3,946,022, 3,931,197;
[0168] steroidal agents, as disclosed in U.S. Pat. Nos. 5,863,538,
5,855,907, 5,855,866, 5,780,592, 5,776,427, 5,651,987, 5,346,887,
5,256,408, 5,252,319, 5,209,926, 4,996,335, 4,927,807, 4,910,192,
4,710,495, 4,049,805, 4,004,005, 3,670,079, 3,608,076, 5,892,028,
5,888,995, 5,883,087, 5,880,115, 5,869,475, 5,866,558, 5,861,390,
5,861,388, 5,854,235, 5,837,698, 5,834,452, 5,830,886, 5,792,758,
5,792,757, 5,763,361, 5,744,462, 5,741,787, 5,741,786, 5,733,899,
5,731,345, 5,723,638, 5,721,226, 5,712,264, 5,712,263, 5,710,144,
5,707,984, 5,705,494, 5,700,793, 5,698,720, 5,698,545, 5,696,106,
5,677,293, 5,674,861, 5,661,141, 5,656,621, 5,646,136, 5,637,691,
5,616,574, 5,614,514, 5,604,215, 5,604,213, 5,599,807, 5,585,482,
5,565,588, 5,563,259, 5,563,131, 5,561,124, 5,556,845, 5,547,949,
5,536,714, 5,527,806, 5,506,354, 5,506,221, 5,494,907, 5,491,136,
5,478,956, 5,426,179, 5,422,262, 5,391,776, 5,382,661, 5,380,841,
5,380,840, 5,380,839, 5,373,095, 5,371,078, 5,352,809, 5,344,827,
5,344,826, 5,338,837, 5,336,686, 5,292,906, 5,292,878, 5,281,587,
5,272,140, 5,244,886, 5,236,912, 5,232,915, 5,219,879, 5,218,109,
5,215,972, 5,212,166, 5,206,415, 5,194,602, 5,166,201, 5,166,055,
5,126,488, 5,116,829, 5,108,996, 5,099,037, 5,096,892, 5,093,502,
5,086,047, 5,084,450, 5,082,835, 5,081,114, 5,053,404, 5,041,433,
5,041,432, 5,034,548, 5,032,586, 5,026,882, 4,996,335, 4,975,537,
4,970,205, 4,954,446, 4,950,428, 4,946,834, 4,937,237, 4,921,846,
4,920,099, 4,910,226, 4,900,725, 4,892,867, 4,888,336, 4,885,280,
4,882,322, 4,882,319, 4,882,315, 4,874,855, 4,868,167, 4,865,767,
4,861,875, 4,861,765, 4,861,763, 4,847,014, 4,774,236, 4,753,932,
4,711,856, 4,710,495, 4,701,450, 4,701,449, 4,689,410, 4,680,290,
4,670,551, 4,664,850, 4,659,516, 4,647,410, 4,634,695, 4,634,693,
4,588,530, 4,567,000, 4,560,557, 4,558,041, 4,552,871, 4,552,868,
4,541,956, 4,519,946, 4,515,787, 4,512,986, 4,502,989,
4,495,102;
[0169] the disclosures of all the above of which are herein
incorporated by reference.
[0170] The drug moiety of the conjugate may be the whole drug or a
binding fragment or portion thereof that retains its affinity and
specificity for the target of interest while having a linkage site
for covalent bonding to the vector protein ligand or linker. The
conjugates of such drugs may be used for the same disorders,
diseases, and indications as the drugs themselves.
[0171] C. Preferred Cancer Chemotherapeutic Active Agents
[0172] Preferred cancer chemotherapeutic agents for use in the RAP
or RAP polypeptide conjugates of the invention include all drugs
which may be useful for treating brain tumors or other neoplasia in
or around the brain, either in the free form, or, if not so useful
for such tumors in the free form, then useful when linked to RAP or
a RAP polypeptide. Such chemotherapeutic agents include adriamycin
(also known as doxorubicin), cisplatin, paclitaxel, analogs
thereof, and other chemotherapeutic agents demonstrate activity
against tumours ex vivo and in vivo. Such chemotherapeutic agents
also include alkylating agents, antimetabolites, natural products
(such as vinca alkaloids, epidophyllotoxins, antibiotics, enzymes
and biological response modifiers), topoisomerase inhibitors,
microtubule inhibitors, spindle poisons, hormones and antagonists,
and miscellaneous agents such as platinum coordination complexes,
anthracendiones, substituted ureas, etc. hose of skill in the art
will know of other chemotherapeutic agents.
[0173] Preferred chemotherapeutic agents are those, which in the
free form, demonstrate unacceptable systemic toxicity at desired
doses. The general systemic toxicity associated with therapeutic
levels of such agents is reduced by their linkage to RAP or a RAP
polypeptide. Particularly preferred are cardiotoxic compounds that
are useful therapeutics but are dose limited by cardiotoxicity. A
classic example is adriamycin (also known as doxorubicin) and its
analogs, such as daunorubicin. Linking RAP or a RAP polypeptide to
such drugs accumulation and associated cardiotoxicity at the
heart.
[0174] VI. Methods for Making RAP-Active Agent Conjugates
[0175] The present invention generally provides methods and
compositions comprising RAP or a RAP polypeptide linked to an
active agent.
[0176] In general, RAP-active agent conjugates can be prepared
using techniques known in the art. There are numerous approaches
for the conjugation or chemical crosslinking of compounds to
proteins and one skilled in the art can determine which method is
appropriate for the active agent to be conjugated. The method
employed must be capable of joining the active agent to RAP or the
RAP polypeptide without interfering with the ability of the RAP/RAP
polypeptide to bind to its receptor, preferably without altering
the desired activity of the compound once delivered. Preferred
methods of conjugating RAP to various compounds are set out in the
example section, below. Particularly preferred for linking complex
molecules to RAP is the SATA/sulfo-SMCC cross-linking reaction
(Pierce (Rockford, Ill.)). For linking metals to RAP, preferred
reactions include, but are not limited to, binding to tyrosine
residues through Chloramine T methods, or use of lodo beads
(Pierce) for iodination reactions.
[0177] Methods for conjugating the RAP with the representative
labels set forth above may be readily accomplished by one of
ordinary skill in the art (see, Trichothecene Antibody Conjugate,
U.S. Pat. No. 4,744,981; Antibody Conjugate, U.S. Pat. No.
5,106,951; Fluorogenic Materials and Labeling Techniques, U.S. Pat.
No. 4,018,884; Metal Radionuclide Labeled Proteins for Diagnosis
and Therapy, U.S. Pat. No. 4,897,255; and Metal Radionuclide
Chelating Compounds for Improved Chelation Kinetics, U.S. Pat. No.
4,988,496; see also Inman, Methods In Enzymology, Vol. 34, Affinity
Techniques, Enzyme Purification: Part B, Jakoby and Wichek (eds.),
Academic Press, New York, p.30, 1974; see also Wilchek and Bayer,
"The Avidin-Biotin Complex in Bioanalytical Applications," Anal.
Biochem. 171:1-32, 1988; all incorporated herein by reference in
their entirety for all purposes).
[0178] If the active agent is a protein or a peptide, there are
many crosslinkers available in order to conjugate the active agent
with the RAP or a substance that binds RAP. (See for example,
Chemistry of Protein Conjugation and Crosslinking. 1991, Shans
Wong, CRC Press, Ann Arbor). The crosslinker is generally chosen
based on the reactive functional groups available or inserted on
the therapeutic compound. In addition, if there are no reactive
groups a photoactivatible crosslinker can be used. In certain
instances, it may be desirable to include a spacer between RAP and
the active agent. In one example, RAP and protein therapeutic
compounds can be conjugated by the introduction of a sulfhydryl
group on the RAP and the introduction of a cross-linker containing
a reactive thiol group on to the protein compound through carboxyl
groups (see, Wawizynczak, E. J. and Thorpe, P. E. in
Immunoconjugates: Antibody Conjugates in Radioimaging and Therapy
of Cancer, C. W. Vogel (Ed.) Oxford University Press, 1987, pp.
28-55.; and Blair, A. H. and T. I. Ghose, J. Immunol. Methods
59:129,1983).
[0179] RAP-chemotherapeutic agents can comprise one or more
compound moieties linked to RAP. For example, conjugation reactions
may conjugate from 1 to 10 or more molecules of adriamycin to a
single RAP molecule. Several atoms of gold or iodine can be
conjugated to a single RAP polypeptide. These formulations can be
employed as mixtures, or they may be purified into specific
RAP-compound stoichiometric formulations. Those skilled in the art
are able to determine which format and which stoichiometric ratio
is preferred. Further, mixtures of compounds may be linked to RAP,
such as the RAP adriamycin-cisplatinum composition set out in the
examples. These RAP-active agent conjugates may consist of a range
of stoichiometric ratios of RAP to an active agent (e.g.,
RAP:active agent ratios of 1:1 to 1:4; 1:5 to 1:10; or 1:10 to
1:20). Optionally, a plurality of different active agents (e.g. 2,
3, or 4 such agents) may be each conjugated to the RAP or RAP
polypeptide in its own stoichiometric ratio such that RAP to the
total ratio of such additional active agents is not fewer than 1
RAP per 20 active agents. These, too, may be separated into
purified mixtures or they may be employed in aggregate.
[0180] The linker is preferably an organic moiety constructed to
contain an alkyl, aryl and/or amino acid backbone and which will
contain an amide, ether, ester, hydrazone, disulphide linkage or
any combination thereof. Linkages containing amino acid, ether and
amide bound components will be stable under conditions of
physiological pH, normally 7.4 in serum and 4-5 on uptake into
cells (endosomes). Preferred linkages are linkages containing
esters or hydrazones that are stable at serum pH but hydrolyse to
release the drug when exposed to intracellular pH. Disulphide
linkages are preferred because they are sensitive to reductive
cleavage; amino acid linkers can be designed to be sensitive to
cleavage by specific enzymes in the desired target organ. Exemplary
linkers are set out in Blattler et al. Biochem. 24:1517-1524, 1985;
King et al. Biochem. 25:5774-5779, 1986; Srinivasachar and Nevill,
Biochem. 28:2501-2509, 1989.
[0181] Drug-Linker intermediates are similar to what has been
described above but with either an active ester to react with free
amine groups on the RAP or a maleimide to react with the free
thiols that have been created on RAP through other groups where
persons skilled in the art can attach them to RAP.
[0182] Methods of crosslinking proteins and peptides are well known
to those of skill in the art. Several hundred crosslinkers are
available for conjugating a compound of interest with RAP or with a
substance which binds RAP (see, e.g., Chemistry of Protein
Conjugation and Crosslinking, Shans Wong, CRC Press, Ann Arbor
(1991) and U.S. Pat. No. 5,981,194 and PCT Patent Publication Nos.
WO 02/13843 and WO 01/59459 which are incorporated herein by
reference in their entirety). Many reagents and cross-linkers can
be used to prepare conjugates of an active agent and a RAP
moleculeee, for instance, Hermanson, et al. Bioconjugate
Techniques, Academic Press, (1996). The crosslinker is generally
chosen based on the reactive functional groups available or
inserted on the therapeutic agent. In addition, if there are no
reactive groups, a photoactivatible crosslinker can be used. In
certain instances, it may be desirable to include a spacer between
RAP and the agent. In one embodiment, RAP and the protein
therapeutic agents may be conjugated by the introduction of a
sulfhydryl group on RAP and by the introduction of a crosslinker
containing a reactive thiol group on to the protein compound
through carboxyl groups (Wawizynczak and Thorpe in
Immunoconjugates: Antibody Conjugates in Radioimaging and Therapy
of Cancer, Vogel (Ed.) Oxford University Press, pp. 28-55 (1987);
and Blair and Ghose (1983) J. Immunol. Methods 59:129). In some
embodiments, the linker is vulnerable to hydrolysis at the acidic
pH of the lysosome so as to free the agent from the and/or
linker.
[0183] When a linker is used, the linker is preferably an organic
moiety constructed to contain an alkyl, aryl and/or amino acid
backbone, and containing an amide, ether, ester, hydrazone,
disulphide linkage or any combination thereof. Linkages containing
amino acid, ether and amide bound components are stable under
conditions of physiological pH, normally 7.4 in serum. Preferred
linkages are those containing esters or hydrazones that are stable
at serum pH, but that hydrolyze to release the drug when exposed to
lysosomal pH. Disulphide linkages are preferred because they are
sensitive to reductive cleavage. In addition, amino acid linkers
may be designed to be sensitive to cleavage by specific enzymes in
the desired target organ or more preferably, the lysosome itself.
Exemplary linkers are described in Blattler et al. (1985) Biochem.
24:1517-1524; King et al. (1986) Biochem. 25:5774-5779;
Srinivasachar and Nevill (1989) Biochem. 28:2501-2509.
[0184] In some embodiments, the linker is a polyethylene glycol or
polypropylene glycol. In other embodiments, the linker is from 4 to
20 atoms long. In other embodiments, the linker is from 1 to 30
atoms long with carbon chain atoms that may be substituted by
heteroatoms independently selected from the group consisting of O,
N. or S. In some embodiments, from 1to 4 or up to one-third of the
C atoms are substituted with a heteroatom independently selected
from O, N, S. In other embodiments, the linker contains a moiety
subject to hydrolysis upon delivery to the lysosomal environment
(e.g., susceptible to hydrolysis at the lysosomal pH or upon
contact to a lysosomal enzyme). In some embodiments, the linker
group is preferably hydrophilic to enhance the solubility of the
conjugate in body fluids. In some embodiments, the linker contains
or is attached to the RAP molecule or the protein agent by a
functional group subject to attack by other lysosomal enzymes
(e.g., enzymes not deficient in the target lysosome or a lysosomal
enzyme not conjugated to the RAP carrier). In some embodiments, the
RAP and agent are joined by a linker comprising amino acids or
peptides, lipids, or sugar residues. In some embodiments, the RAP
and agent are joined at groups introduced synthetically or by
post-translational modifications.
[0185] In some embodiments, agent-linker intermediates are similar
to what has been described previously, but comprise, for example,
either an active ester that can react with free amine groups on RAP
or a maleimide that can react with the free thiols created on RAP
via a SATA reaction or through other groups where persons skilled
in the art can attach them to.
[0186] A. Methods for Conjugating a RAP Polypeptide to a Protein or
Enzyme.
[0187] One of ordinary skill in the art would know how to conjugate
an active agent to a protein or peptide. Methods of conjugating
active agents and labels to proteins are well known in the art.
See, for instance, U.S. Pat. No. 5,981,194. Many reagents and cross
linkers can be used to prepare bioconjugates of an active agent and
a biopolymer. See, for instance, Hermanson, et al. Bioconjugate
Techniques, Academic Press, (1996).
[0188] Production of Chimeric Proteins
[0189] In some embodiments of the present invention, the RAP
polypeptide active-agent conjugate is a RAP polypeptide-fusion
protein. Fusion proteins may be prepared using standard techniques
known in the art. Typically, a DNA molecule encoding RAP or a
portion thereof is linked to a DNA molecule encoding the protein
compound. The chimeric DNA construct, along with suitable
regulatory elements can be cloned into an expression vector and
expressed in a suitable host. The resultant fusion proteins contain
RAP or a portion thereof used to the selected protein compound.
RAP-LSD enzyme proteins, RAP-human alpha glucosidase and
RAP-iduronidase, are described in Example VII and FIGS. 3 and 4and
were prepared using standard techniques known in the art.
[0190] The chimeric protein of the present invention can be
produced using host cells expressing a single nucleic acid encoding
the entire chimeric protein or more than one nucleic acid sequence,
each encoding a domain of the chimeric protein and, optionally, an
amino acid or amino acids which will serve to link the domains. The
chimeric proteins can also be produced by chemical synthesis.
[0191] Host Cells
[0192] Host cells used to produce chimeric proteins are bacterial,
yeast, insect, non-mammalian vertebrate, or mammalian cells; the
mammalian cells include, but are not limited to, hamster, monkey,
chimpanzee, dog, cat, bovine, porcine, mouse, rat, rabbit, sheep
and human cells. The host cells can be immortalized cells (a cell
line) or non-immortalized (primary or secondary) cells and can be
any of a wide variety of cell types, such as, but not limited to,
fibroblasts, keratinocytes, epithelial cells (e.g., mammary
epithelial cells, intestinal epithelial cells), ovary cells (e.g.,
Chinese hamster ovary or CHO cells), endothelial cells, glial
cells, neural cells, formed elements of the blood (e.g.,
lymphocytes, bone marrow cells), muscle cells, hepatocytes and
precursors of these somatic cell types. Host cells can include
mutants of CHO cells that do not express LRP such as CHO13-5-1
(FitzGerald, et al. J. Cell Biol. 129(6):1533-41 (1995)).
[0193] Cells that contain and express DNA or RNA encoding the
chimeric protein are referred to herein as genetically modified
cells. Mammalian cells that contain and express DNA or RNA encoding
the chimeric protein are referred to as genetically modified
mammalian cells. Introduction of the DNA or RNA into cells is by a
known transfection method, such as electroporation, microinjection,
microprojectile bombardment, calcium phosphate precipitation,
modified calcium phosphate precipitation, cationic lipid treatment,
photoporation, fusion methodologies, receptor mediated transfer, or
polybrene precipitation. Alternatively, the DNA or RNA can be
introduced by infection with a viral vector. Methods of producing
cells, including mammalian cells, which express DNA or RNA encoding
a chimeric protein are described in co-pending patent applications
U.S. Ser. No. 08/334,797, entitled "In Vivo Protein Production and
Delivery System for Gene Therapy", by Richard F Selden, Douglas A.
Treco and Michael W. Heartlein (filed Nov. 4, 1994); U.S. Ser. No.
08/334,455, entitled "In Vivo Production and Delivery of
Erythropoietin or Insulinotropin for Gene Therapy", by Richard F
Selden, Douglas A. Treco and Michael W. Heartlein (filed Nov. 4,
1994) and U.S. Ser. No. 08/231,439, entitled "Targeted Introduction
of DNA Into Primary or Secondary Cells and Their Use for Gene
Therapy", by Douglas A. Treco, Michael W. Heartlein and Richard F
Selden (filed Apr. 20, 1994). The teachings of each of these
applications are expressly incorporated herein by reference in
their entirety.
[0194] Nucleic Acid Constructs
[0195] A nucleic acid construct used to express the chimeric
protein can be one which is expressed extrachromosomally
(episomally) in the transfected mammalian cell or one which
integrates, either randomly or at a pre-selected targeted site
through homologous recombination, into the recipient cell's genome.
A construct which is expressed extrachromosomally comprises, in
addition to chimeric protein-encoding sequences, sequences
sufficient for expression of the protein in the cells and,
optionally, for replication of the construct. It typically includes
a promoter, chimeric protein-encoding DNA and a polyadenylation
site. The DNA encoding the chimeric protein is positioned in the
construct in such a manner that its expression is under the control
of the promoter. Optionally, the construct may contain additional
components such as one or more of the following: a splice site, an
enhancer sequence, a selectable marker gene under the control of an
appropriate promoter, and an amplifiable marker gene under the
control of an appropriate promoter.
[0196] In those embodiments in which the DNA construct integrates
into the cell's genome, it need include only the chimeric
protein-encoding nucleic acid sequences. Optionally, it can include
a promoter and an enhancer sequence, a polyadenylation site or
sites, a splice site or sites, nucleic acid sequences which encode
a selectable marker or markers, nucleic acid sequences which encode
an amplifiable marker and/or DNA homologous to genomic DNA in the
recipient cell to target integration of the DNA to a selected site
in the genome (targeting DNA or DNA sequences).
[0197] Cell Culture Methods
[0198] Mammalian cells containing the chimeric protein-encoding DNA
or RNA are cultured under conditions appropriate for growth of the
cells and expression of the DNA or RNA. Those cells which express
the chimeric protein can be identified, using known methods and
methods described herein, and the chimeric protein isolated and
purified, using known methods and methods also described herein;
either with or without amplification of chimeric protein
production. Identification can be carried out, for example, through
screening genetically modified mammalian cells displaying a
phenotype indicative of the presence of DNA or RNA encoding the
chimeric protein, such as PCR screening, screening by Southern blot
analysis, or screening for the expression of the chimeric protein.
Selection of cells having incorporated chimeric protein-encoding
DNA may be accomplished by including a selectable marker in the DNA
construct and culturing transfected or infected cells containing a
selectable marker gene under conditions appropriate for survival of
only those cells that express the selectable marker gene. Further
amplification of the introduced DNA construct can be affected by
culturing genetically modified mammalian cells under conditions
appropriate for amplification (e.g., culturing genetically modified
mammalian cells containing an amplifiable marker gene in the
presence of a concentration of a drug at which only cells
containing multiple copies of the amplifiable marker gene can
survive).
[0199] Genetically modified mammalian cells expressing the chimeric
protein can be identified, as described herein, by detection of the
expression product. For example, mammalian cells expressing
chimeric protein in which the carrier is RAP can be identified by a
sandwich enzyme immunoassay. The antibodies can be directed toward
the LRP portion or the active agent portion.
[0200] VII. Labels
[0201] In some embodiments, the RAP polypeptide active agent
conjugate is labeled to facilitate its detection. A "label" or a
"detectable moiety" is a composition detectable by spectroscopic,
photochemical, biochemical, immunochemical, chemical, or other
physical means. For example, labels suitable for use in the present
invention include, for example, radioactive labels (e.g.,
.sup.32P), fluorophores (e.g., fluorescein), electron-dense
reagents, enzymes (e.g., as commonly used in an ELISA), biotin,
digoxigenin, or haptens and proteins which can be made detectable,
e.g., by incorporating a radiolabel into the hapten or peptide, or
used to detect antibodies specifically reactive with the hapten or
peptide.
[0202] As noted above, depending on the screening assay employed,
the active agent, the linker or the RAP polypeptide portion of a
conjugate may be labeled. The particular label or detectable group
used is not a critical aspect of the invention, as long as it does
not significantly interfere with the biological activity of the
conjugate. The detectable group can be any material having a
detectable physical or chemical property. Thus, a label is any
composition detectable by spectroscopic, photochemical,
biochemical, immunochemical, electrical, optical or chemical
means.
[0203] Examples of labels suitable for use in the present invention
include, but are not limited to, fluorescent dyes (e.g.,
fluorescein isothiocyanate, Texas red, rhodamine, and the like),
radiolabels (e.g., .sup.3H, .sup.125I, .sup.35S, .sup.14C, or
.sup.32P), enzymes (e.g., horse radish peroxidase, alkaline
phosphatase and others commonly used in an ELISA), and colorimetric
labels such as colloidal gold or colored glass or plastic beads
(e.g., polystyrene, polypropylene, latex, etc.).
[0204] The label may be coupled directly or indirectly to the
desired component of the assay according to methods well known in
the art. Preferably, the label in one embodiment is covalently
bound to the biopolymer using an isocyanate reagent for conjugating
an active agent according to the invention. In one aspect of the
invention, the bifunctional isocyanate reagents of the invention
can be used to conjugate a label to a biopolymer to form a label
biopolymer conjugate without an active agent attached thereto. The
label biopolymer conjugate may be used as an intermediate for the
synthesis of a labeled conjugate according to the invention or may
be used to detect the biopolymer conjugate. As indicated above, a
wide variety of labels can be used, with the choice of label
depending on sensitivity required, ease of conjugation with the
desired component of the assay, stability requirements, available
instrumentation, and disposal provisions. Non-radioactive labels
are often attached by indirect means. Generally, a ligand molecule
(e.g., biotin) is covalently bound to the molecule. The ligand then
binds to another molecules (e.g., streptavidin) molecule, which is
either inherently detectable or covalently bound to a signal
system, such as a detectable enzyme, a fluorescent compound, or a
chemiluminescent compound.
[0205] The conjugates can also be conjugated directly to signal
generating compounds, e.g., by conjugation with an enzyme or
fluorophore. Enzymes suitable for use as labels include, but are
not limited to, hydrolases, particularly phosphatases, esterases
and glycosidases, or oxidotases, particularly peroxidases.
Fluorescent compounds, i.e., fluorophores, suitable for use as
labels include, but are not limited to, fluorescein and its
derivatives, rhodamine and its derivatives, dansyl, umbelliferone,
etc. Further examples of suitable fluorophores include, but are not
limited to, eosin, TRITC-amine, quinine, fluorescein W, acridine
yellow, lissamine rhodamine, B sulfonyl chloride erythroscein,
ruthenium (tris, bipyridinium), Texas Red, nicotinamide adenine
dinucleotide, flavin adenine dinucleotide, etc. Chemiluminescent
compounds suitable for use as labels include, but are not limited
to, luciferin and 2,3-dihydrophthalazinediones, e.g., luminol. For
a review of various labeling or signal producing systems that can
be used in the methods of the present invention, see U.S. Pat. No.
4,391,904.
[0206] Means of detecting labels are well known to those of skill
in the art. Thus, for example, where the label is a radioactive
label, means for detection include a scintillation counter or
photographic film as in autoradiography. Where the label is a
fluorescent label, it may be detected by exciting the fluorochrome
with the appropriate wavelength of light and detecting the
resulting fluorescence. The fluorescence may be detected visually,
by the use of electronic detectors such as charge coupled devices
(CCDs) or photomultipliers and the like. Similarly, enzymatic
labels may be detected by providing the appropriate substrates for
the enzyme and detecting the resulting reaction product.
Colorimetric or chemiluminescent labels may be detected simply by
observing the color associated with the label. Other labeling and
detection systems suitable for use in the methods of the present
invention will be readily apparent to those of skill in the art.
Such labeled modulators and ligands may be used in the diagnosis of
a disease or health condition.
[0207] VIII. Screening Assays for RAP Polypeptide Active Agent
Conjugates and Modulators of Their Delivery Via the LRP.
[0208] The present invention provides a screening assay for RAP
polypeptide active agents conjugates, wherein the conjugates are
tested for their ability to influence a measurable activity of the
LRP receptor which can be situated in a whole cell, a cell extract,
semi-purified, purified or any other format that allows for
measurement of its activity. The activity can be any activity in
the expression, function or degradation of LRP including, for
example, the amount or timing of such activities. Such activities
include, for example, transcription, transcript processing,
translation or transcript stability of the LRP gene sequence or
mRNA transcript. Such activities include, for example, the
synthesis of new LRP, the sub-cellular localization of LRP and
activation of LRP biological activity. Such activities include, for
example, the ability of LRP to bind substances, adopt
conformations, catalyze reactions, bind known ligands and the like.
Such activities include, for example, the amount or stability of
LRP1, the processing and removal or degradation of LRP and the
like. In preferred embodiments, the LRP receptor for use in
screening is LRP1.
[0209] The invention contemplates a variety of different screening
formats. Some designs are considered low throughput and test only
one or a few compounds in series or in parallel. High throughput
screening assays are suitable for screening tens of thousands or
hundreds of thousands of compounds in a matter of weeks or months.
"In silico" screening formats employ computer-aided rational design
techniques to identify potential modulators of LRP biological
activity.
[0210] A. Modulating Uptake of RAP Conjugated Active Agents by
Modulating LRP Activity
[0211] Those skilled in the art will appreciate that increasing RAP
polypeptide active agent conjugate uptake and delivery to targets
including, but not limited to, the brain or lysosomes is useful and
desirable in situations such as, but not limited to, where the
conjugate is being used to treat a neurological condition and/or a
LSD and increased amounts of delivery would provide therapeutic
benefit. Those skilled in the art will appreciate that decreasing
conjugate uptake and delivery across the blood-brain barrier is
useful and desirable for a variety of reasons including, but not
limited to, where the conjuguate is being used for its potential
cardio-protective effect or used in other (non-CNS) organs and
side-effects of brain uptake are to be avoided.
[0212] Suitable RAP and RAP polypeptdes, active agent conjugates of
RAP and RAP polypeptides, and modulators of LRP activity and
modulators of RAP and RAP polypeptide conjugate delivery can also
be readily identified using a modification of the Transwell
apparatus set out in Example 1 below. In the modified form, a
compound (e.g., RAP polypeptide, RAP polypeptide active agent
conjugate, or modulator) is added to the luminal surface of the
cells in the Transwell apparatus. The compound is then scored
according to how well across the BBCECs to the abluminal side or as
to how well (if a modulator) it increases or decreases the
transport of a RAP conjugate or RAP polypeptide or another LRP
ligand across the BBCECs to the abluminal side. A library of
compounds can be readily screened or tested to identify
pharmacologically superior modulators.
[0213] Other known ligands of the LRP receptor may be screened for
use as modulators of the delivery of the conjugate, or as models
for designing such modulators. These ligands include, but are not
limited to, ApoE, Chylomicron remnants, .beta.-VLDL, activated
.alpha.2-macroglobulin, tPA, Tissue factor inhibitor, Pro-uPA,
PAI-1, Saposin, Gentamycin,Thyroglobuli- n, Polymixin B, Seminal
Vesicle Secretory Protein A, Thrombospondin-1, Lactoferrin,and
P-APP.
[0214] IX. Methods of Using, Pharmaceutical Compositions, and their
Administration
[0215] The conjugates and modulators may be administered by a
variety of routes. For oral preparations, the conjugates can be
used alone or in combination with appropriate additives to make
tablets, powders, granules or capsules, for example, with
conventional additives, such as lactose, mannitol, corn starch or
potato starch; with binders, such as crystalline cellulose,
cellulose derivatives, acacia, corn starch or gelatins; with
disintegrators, such as corn starch, potato starch or sodium
carboxymethylcellulose; with lubricants, such as talc or magnesium
stearate; and if desired, with diluents, buffering agents,
moistening agents, preservatives and flavoring agents.
[0216] The conjugates and modulators can be formulated into
preparations for injection by dissolving, suspending or emulsifying
them in an aqueous or nonaqueous solvent, such as vegetable or
other similar oils, synthetic aliphatic acid glycerides, esters of
higher aliphatic acids or propylene glycol; and if desired, with
conventional additives such as solubilizers, isotonic agents,
suspending agents, emulsifying agents, stabilizers and
preservatives.
[0217] The conjugates, modulators, and LRP ligands can be utilized
in aerosol formulation to be administered via inhalation. The
compounds of the present invention can be formulated into
pressurized acceptable propellants such as dichlorodifluoromethane,
propane, nitrogen and the like.
[0218] Furthermore, the conjugates and modulators can be made into
suppositories by mixing with a variety of bases such as emulsifying
bases or water-soluble bases. The compounds of the present
invention can be administered rectally via a suppository. The
suppository can include vehicles such as cocoa butter, carbowaxes
and polyethylene glycols, which melt at body temperature, yet are
solidified at room temperature.
[0219] Unit dosage forms of the conjugate, modulator, and LRP
ligand for oral or rectal administration such as syrups, elixirs,
and suspensions may be provided wherein each dosage unit, for
example, teaspoonful, tablespoonful, tablet or suppository,
contains a predetermined amount of the composition containing
active agent. Similarly, unit dosage forms for injection or
intravenous administration may comprise the conjugate in a
composition as a solution in sterile water, normal saline or
another pharmaceutically acceptable carrier.
[0220] In practical use, the conjugate, modulator, and LRP ligand
according to the invention can be combined as the active ingredient
in intimate admixture with a pharmaceutical carrier according to
conventional pharmaceutical compounding techniques. The carrier may
take a wide variety of forms depending on the form of preparation
desired for administration, e.g., oral or parenteral (including
intravenous). In preparing the compositions for oral dosage form,
any of the usual pharmaceutical media may be employed, such as, for
example, water, glycols, oils, alcohols, flavoring agents,
preservatives, coloring agents and the like in the case of oral
liquid preparations, such as, for example, suspensions, elixirs and
solutions; or carriers such as starches, sugars, microcrystalline
cellulose, diluents, granulating agents, lubricants, binders,
disintegrating agents and the like in the case of oral solid
preparations such as, for example, powders, hard and soft capsules
and tablets, with the solid oral preparations being preferred over
the liquid preparations.
[0221] With respect to transdermal routes of administration,
methods for transdermal administration of drugs are disclosed in
Remington's Pharmaceutical Sciences, 17th Edition, (Gennaro et al.
Eds. Mack Publishing Co., 1985). Dermal or skin patches are a
preferred means for transdermal delivery of the conjugates,
modulators, and LRP ligands of the invention. Patches preferably
provide an absorption enhancer such as DMSO to increase the
absorption of the compounds. Other methods for transdermal drug
delivery are disclosed in U.S. Pat. Nos. 5,962,012, 6,261,595, and
6,261,595. Each of which is incorporated by reference in its
entirety.
[0222] Pharmaceutically acceptable excipients, such as vehicles,
adjuvants, carriers or diluents, are commercially available.
Moreover, pharmaceutically acceptable auxiliary substances, such as
pH adjusting and buffering agents, tonicity adjusting agents,
stabilizers, wetting agents and the like, are commercially
available.
[0223] Those of skill will readily appreciate that dose levels can
vary as a function of the specific compound, the severity of the
symptoms and the susceptibility of the subject to side effects.
Preferred dosages for a given compound are readily determinable by
those of skill in the art by a variety of means, including, but not
limited to dose response and pharmacokinetic assessments conducted
in patients, test animals, and in vitro.
[0224] In each of these aspects, the compositions include, but are
not limited to, compositions suitable for oral, rectal, topical,
parenteral (including subcutaneous, intramuscular, and
intravenous), pulmonary (nasal or buccal inhalation), or nasal
administration, although the most suitable route in any given case
will depend in part on the nature and severity of the conditions
being treated and on the nature of the active ingredient. Exemplary
routes of administration are the oral and intravenous routes. The
compositions may be conveniently presented in unit dosage form and
prepared by any of the methods well-known in the art of
pharmacy.
[0225] In practical use, the modulators or according to the
invention can be combined as the active ingredient in intimate
admixture with a pharmaceutical carrier according to conventional
pharmaceutical compounding techniques. The carrier may take a wide
variety of forms depending on the form of preparation desired for
administration, e.g., oral or parenteral (including intravenous).
In preparing the compositions for oral dosage form, any of the
usual pharmaceutical media may be employed, such as, for example,
water, glycols, oils, alcohols, flavoring agents, preservatives,
coloring agents and the like in the case of oral liquid
preparations, such as, for example, suspensions, elixirs and
solutions; or carriers such as starches, sugars, microcrystalline
cellulose, diluents, granulating agents, lubricants, binders,
disintegrating agents and the like in the case of oral solid
preparations such as, for example, powders, hard and soft capsules
and tablets, with the solid oral preparations being preferred over
the liquid preparations.
[0226] Because of their ease of administration, tablets and
capsules represent the most advantageous oral dosage unit form in
which case solid pharmaceutical carriers are obviously employed. If
desired, tablets may be coated by standard aqueous or nonaqueous
techniques. The percentage of an active compound in these
compositions may, of course, be varied and may conveniently be
between about 2 percent to about 60 percent of the weight of the
unit. The conjugates, modulators, and ligands of the invention are
useful for therapeutic, prophylactic and diagnostic intervention in
animals, and in particular in humans. As described herein, the
conjugates show preferential accumulation and/or release of the
active agent in any target organ, compartment, or site depending
upon the biopolymer used.
[0227] Compositions of the present invention may be administered
encapsulated in or attached to viral envelopes or vesicles, or
incorporated into cells. Vesicles are micellular particles which
are usually spherical and which are frequently lipidic. Liposomes
are vesicles formed from a bilayer membrane. Suitable vesicles
include, but are not limited to, unilamellar vesicles and
multilamellar lipid vesicles or liposomes. Such vesicles and
liposomes may be made from a wide range of lipid or phospholipid
compounds, such as phosphatidylcholine, phosphatidic acid,
phosphatidylserine, phosphatidylethanolamine, sphingomyelin,
glycolipids, gangliosides, etc. using standard techniques, such as
those described in, e.g., U.S. Pat. No. 4,394,448. Such vesicles or
liposomes may be used to administer compounds intracellularly and
to deliver compounds to the target organs. Controlled release of a
p97-composition of interest may also be achieved using
encapsulation (see, e.g., U.S. Pat. No. 5,186,941).
[0228] Any route of administration that dilutes the RAP polypeptide
active agent conjugate or modulator composition into the blood
stream, or preferably at least outside of the blood-brain barrier,
may be used. Preferably, the composition is administered
peripherally, most preferably intravenously or by cardiac catheter.
Intrajugular and intracarotid injections are also useful.
Compositions may be administered locally or regionally, such as
intraperitoneally or subcutaneously on intramuscularly. In one
aspect, compositions are administered with a suitable
pharmaceutical diluent or carrier.
[0229] Dosages to be administered will depend on individual needs,
on the desired effect, the active agent used, the biopolymer and on
the chosen route of administration. Preferred dosages of a
conjugate range from about 0.2 pmol/kg to about 25 nmol/kg, and
particularly preferred dosages range from 2-250 pmol/kg;
alternatively, preferred doses of the conjugate may be in the range
of 0.02 to 2000 mg/kg. These dosages will be influenced by the
number of active agent or drug moieties associated with the
biopolymer. Alternatively, dosages may be calculated based on the
active agent administered.
[0230] In preferred embodiments the conjugate comprises human RAP.
For instance, doses of RAP-adriamycin comprising from 0.005 to 100
mg/kg of adriamycin are also useful in vivo. Particularly preferred
is a dosage of RAP-adriamycin comprising from 0.05 mg/kg to 20
mg/kg of adriamycin. Those skilled in the art can determine
suitable doses for compounds linked to RAP based in part on the
recommended dosage used for the free form of the compound. RAP
conjugation generally reduces the amount of drug needed to obtain
the same effect.
[0231] The RAP polypeptide conjugates and modulators of the
invention are useful for therapeutic, prophylactic and diagnostic
intervention in animals, and in particular in humans. RAP compounds
may show preferential accumulation in particular tissues. Preferred
medical indications for diagnostic uses include, for example, any
condition associated with a target organ of interest (e.g., lung,
liver, kidney, spleen)
[0232] The subject methods find use in the treatment of a variety
of different disease conditions. In certain embodiments, of
particular interest is the use of the subject methods in disease
conditions where an active agent or drug having desired activity
has been previously identified, but in which the active agent or
drug is not adequately delivered to the target site, area or
compartment to produce a fully satisfactory therapeutic result.
With such active agents or drugs, the subject methods of
conjugating the active agent to RAP or a RAP polypeptide can be
used to enhance the therapeutic efficacy and therapeutic index of
active agent or drug.
[0233] The specific disease conditions treatable by with the
subject conjugates are as varied as the types of drug moieties that
can be present in the conjugate. Thus, disease conditions include
cellular proliferative diseases, such as neoplastic diseases,
autoimmune diseases, cardiovascular diseases, hormonal abnormality
diseases, degenerative diseases, diseases of aging, diseases of the
central nervous system (e.g., Alzheimer's disease, epilepsy,
hyperlipidemias), psychiatric diseases and conditions (e.g.,
schizophrenia, mood disorders such as depression and anxiety),
infectious diseases, enzyme deficiency diseases, lysosomal storage
diseases such as those described above, and the like.
[0234] Treatment is meant to encompass any beneficial outcome to a
subject associated with administration of a conjugate including a
reduced likelyhood of acquiring a disease, prevention of a disease,
slowing, stopping or reversing, the progression of a disease or an
amelioration of the symptoms associated with the disease condition
afflicting the host, where amelioration or benefit is used in a
broad sense to refer to at least a reduction in the magnitude of a
parameter, e.g., symptom, associated with the pathological
condition being treated, such as inflammation and pain associated
therewith. As such, treatment also includes situations where the
pathological condition, or at least symptoms associated therewith,
are completely inhibited, e.g., prevented from happening, or
stopped, e.g., terminated, such that the host no longer suffers
from the pathological condition, or at least the symptoms that
characterize the pathological condition.
[0235] A variety of hosts or subjects are treatable according to
the subject methods. Generally such hosts are "mammals" or
"mammalian," where these terms are used broadly to describe
organisms which are within the class mammalia, including the orders
carnivore (e.g., dogs and cats), rodentia (e.g., mice, guinea pigs,
and rats), and primates (e.g., humans, chimpanzees, and monkeys).
In many embodiments, the hosts will be humans.
[0236] X. Preparation of RAP and RAP Polypeptides.
[0237] RAP and RAP polypeptides for use according to theinvention
include those disclosed in U.S. Pat. No. 5,474,766 that is enclosed
herein by reference in its entirety for the purposes of disclosing
such peptides and how they may be obtained for use in the compounds
and compositions of the present invention.
[0238] XI. Production of RAP Polypeptides
[0239] RAP, and RAP polypeptides, may be produced using any of the
methods and techniques known to those skilled in the art. RAP can
be purified from a naturally occurring source of the protein, can
be isolated from a recombinant host expressing RAP or a RAP
polypeptide, or can be synthesized using well known techniques in
protein synthesis. A skilled artisan can readily adapt a variety of
such techniques in order to obtain RAP or RAP polypeptides
thatcontain the LRP binding site found on RAP. See, for instance,
Melman et al., J. Biol. Chem. 276 (31): 29338-29346 (2001);
Savonen, et al., J Biol Chem. 274(36): 25877-25882 (1999); Nielsen,
et al. Proc. Natl. Acad. Sci. USA 94:7521-7525 (1997); Medved, et
al., J. Biol. Chem. 274(2): 717-727 (1999); Rail, et al., J. Biol.
Chem. 273(37): 24152-24157 (1998); Orlando, et al., Proc. Natl.
Acad. Sci. USA 3161-3163 (1994).
[0240] The isolation of native RAP proteins has been described in
Ashcom, et al., J. Cell. Biol. 110:1041-1048 (1990) and Jensen et
al., FEBS Lett. 255:275-280 (1989). RAP fragments containing the
LRP binding site may be generated from isolated native protein
which is converted by enzymatic and/or chemical cleavage to
generate fragments of the whole protein. Such methods are taught in
U.S. Pat. No. 6,447,775 which is herein incorporated by reference
with particular reference to such methods for obtaining RAP
polypeptides.
[0241] In addition, RAP or a fragment of RAP can be expressed in a
recombinant bacteria, as described, by Williams et al., J. Biol.
Chem. 267:9035-9040 (1992) and Wurshawsky et al., J. Biol. Chem.
269:3325-3330 (1994).
[0242] Procedures for purifying the 39 kDa RAP protein from a
recombinant E.coli strain has been previously described by Herz, et
al., J. Biol. Chem. 266, 21232-21238 (1991). A modified version of
that procedure can be used as described in U.S. Pat. No. 5,474,766
and below.
[0243] Cultures of E. coli strain DH5alpha carrying the expression
plasmid pGEX-39 kDa can be grown to mid-log phase in LB medium with
100 .mu.g/ml ampicillin at 37.degree. C. Cultures can then be
cooled to 30.degree. C. and supplemented with 0.01%
isopropylthio-beta-D-galactoside to induce expression of the
glutathione-S-transferase-39 kDa fusion protein. Following a 4-6
hour induction at 30.degree. C., cultures can be cooled with ice
and recovered by centrifugation. All of the following steps are to
be carried out at 4.degree. C. Cell pellets are lysed in PBS with
1% Triton X-100, 1 .mu.M pepstatin, 2.5 mu.g/ml leupeptin, 0.2 mM
phenylmethylsulfonyl fluoride (PMSF), and 1 .mu.M
ethylenediaminetetraace- tate (EDTA). Sonication of this lysate
with a Branson Model 450 Sonifier with separation of the resulting
membranes and other cellular debris by centrifugation at 15,000 g
for 15 minutes is then followed by retrieval of the supernatant.
The supernatant from this step is incubated overnight with agarose
immobilized glutathione beads (Sigma Chemical Co.) in PBS and 0.1%
sodium azide. The beads can then be washed, and elution of the
fusion protein can be carried out by competition with 5 mM reduced
glutathione (Sigma Chemical Co.). Following dialysis, the fusion
protein can be cleaved by an overnight incubation with 100 ng of
activated human thrombin per 50 .mu.g of fusion protein. The
glutathione-S-transferase epitope can subsequently be removed by
further incubation with agarose immobilized glutathione beads.
[0244] The 28 kDa protein fragment of the 39 kDa protein ("28 kDa
protein") of the present invention has the following amino acid
sequence set forth in the Sequence Listing as SEQ ID NO:2 (FIG.
16).
[0245] The 28 kDa protein has a molecular weight of 28,000 daltons
on SDS-PAGE, is relatively stabile to acid hydrolysis, is soluble
in 1% Triton X-100, and has approximately the same inhibitory
activity (K.sub.i) on t-PA binding to the hepatic receptor as the
39 kDa protein. The 28 kDa protein may be cloned and purified as
further exemplified in U.S. Pat. No. 5,474,766 which is expressly
incorporated herein by reference for such methods of cloning.
[0246] The following examples further illustrate the present
invention. These examples are intended merely to be illustrative of
the present invention and are not to be constructed as being
limiting. The following examples provide exemplary protocols for
assessing transcytosis in vitro and for characterizing the
interaction of RAP and LRP receptor modulators or ligands with the
RAP receptor or the blood-brain barrier.
[0247] EXAMPLES
[0248] Transcytosis experiments were performed as follows. One
insert covered with bovine brain capillary endothelial cells
(BBCECs) was set into a Transwell apparatus containing a six-well
microplate with 2 ml of Ringer/Hepes and pre-incubated for 2 h at
37.degree. C. [.sup.125I]-p97 (250 nM) was added to the upper side
of the filter covered with cells. At various times, the insert was
transferred to avoid re-endocytosis of p97 by the abluminal side of
the BBCECs. At the end of experiment, [.sup.125I-p97 was measured
after TCA precipitation.
[0249] The effect of RAP on transcytosis of .sup.125I-p97was
assessed. In FIG. 1, RAP, a known polypeptide inhibitor of the LRP
family was applied to the cells (25 micrograms/ml). RAP
significantly inhibited the transcytosis of p97, thus directly
implicating the LRP family in transcytosis.
Example II
Construction, Expression, Purification and Characterization of RAP
Fusions
[0250] Expression constructs encoding fusions between the human
receptor-associated protein (RAP) and human alpha-glucosidase
(GAA), alpha-L-iduronidase (IDU) or glial cell-derived neurotrophic
factor (GDNF) were prepared. For this purpose, a sequence that
encodes RAP was fused to the 5'-end of sequences that encode the
different fusion partners. All sequences were obtained by
high-fidelity PCR amplification of human cDNA with the following
primers shown in FIG. 2a. The GDNF fusion was designed for
expression in bacteria. To this end, primer RAPBACF was substituted
for RAPF in the RAP amplification for this construct (FIG. 2b).
[0251] The 5'-end of RAP was truncated to remove the signal peptide
sequence. Instead, an in-frame BamHI site, which encodes the
dipeptide GS, was added for the mammalian expression construct.
Sequence encoding the tetrapeptide MGGS with an NcoI site at the
5'-end was added for the bacterial expression construct. The 3'-end
of RAP was truncated to remove the tetrapeptide HNEL endoplasmic
reticulum retention signal. Instead, the coding sequence for a six
amino-acid spacer (AEAETG) was appended. The last two codons of the
spacer specify an Agel restriction site. The 5'-end of GAA was
truncated to remove the signal peptide and pro-peptide sequences
(Wisselaar, et al., J. Biol. Chem. 268(3):2223-31 (1993)). Instead,
an Agel site was added to permit fusion to the RAP-spacer portion
of the fusion. The 5'-end of IDU was similarly truncated to remove
the signal peptide and introduce the restriction site. The 5'-end
of GDNF was truncated to remove both the signal peptide and
pro-peptide sequences (Lin, et al., Science 260(5111):1130-2
(1993)).
[0252] The open-reading frames encoding the GAA and IDU fusions
were ligated into the expression vector pCINmt using flanking BamHI
and XhoI sites. The vector contains the human melanotransferrin
signal peptide with an in-frame BamHI site at the 3'-end. The
sequences of the resulting fusion proteins are shown in FIGS. 3 and
4. The pCINmt (derived from Invitrogen vector pcDNA3.1) control
sequences consist of the human CMV promoter followed by the rabbit
IVS2 and the rat preproinsulin RNA leader sequence. A bovine growth
hormone terminator sequence is positioned at the 3'-end of the
expression cassette. The vector includes a selectable marker
composed of an attenuated neomycin phosphotransferase gene driven
by the weak HSV-tk promoter (Yenofsky, et al., Proc. Natl. Acad.
Sci. U.S.A. 87(9):3435-9 (1990)). Expression constructs for RAP-GAA
and RAP-IDU were transfected into an Lrp-deficient CHO cell line
(CHO13-5-1) and recombinants selected with 800 .mu.g/mL G418.
[0253] The RAPGDNF fusion (FIG. 5) was cloned into the bacterial
expression vector pBADhisA (Invitrogen) using the flanking NcoI and
XbaI sites. The resulting expression vector was transfected into
BL21 cells and recombinants selected with carbenicillin. Expressed,
purified RAP-GDNF fusion may be assayed for the ability to protect
dopaminergic neurons or other activities as previously described
(Kilic, et al., Stroke 34(5):1304-10 (2003)).
[0254] Expression of RAP Fusions:
[0255] Culture medium was JRH 302 supplemented with 2 mM
L-glutamine, gentamycin, amphotericin, 800 .mu.g/mL G418 and 2.5%
fetal calf serum. Recombinant clones were grown in T225 flasks
prior to seeding into 1 L Corning spinner flasks on Cytopore 1
beads (Amersham) in the presence of serum. Spinner flasks were
maintained in a tissue culture incubator set at 37.degree. C. and
5% CO.sub.2. Medium was replaced every two days with serum-free
medium until serum levels were undetectable. Subsequently, harvests
were collected every two days and medium exchanged.
[0256] Purification of RAP-GAA for Uptake Assay:
[0257] RAP-GAA harvested in the medium from the spinner flasks was
applied to a Blue-Sepharose column (Amersham) in low-salt buffer at
neutral pH. Fusion was eluted with a linear salt gradient, and
fractions containing fusion were loaded to a Heparin-Sepharose
column (Amersham) and again eluted with a linear salt gradient.
Eluted fractions containing activity were pooled and applied to a
Phenyl-Sepharose column (Amersham). RAP-GAA was eluted from the
Phenyl-Sepharose column with a decreasing salt step gradient.
Eluted fractions were run on an SDS-PAGE gel and stained to
determine relative percent purity. Based on gel analysis, peak
activity fractions were about 70% pure. Fractions were pooled,
concentrated using a 30 kD MWCO membrane (Millipore), and exchanged
into phosphate-buffered saline at neutral pH.
[0258] The activity of the lysosomal enzyme in the fusion was
determined to be unaffected by fusion to RAP. Purified human LRP (1
.mu.g, recombinant, binding domain 2) was spotted onto PVDF filters
in a 96-well dot-blot apparatus. Purified RAP-lysosomal enzyme
fusion (RAP-LE) in Tris-buffered saline pH 7.5 with 5 mM CaCl.sub.2
and 3% non-fat dry milk (TBS/Ca/BLOTTO) was overlayed on the
immobilized LRP. Conditioned medium containing the RAP-LE, buffer
alone and RAP alone were similarly incubated with immobilized LRP.
Filters were washed three times to remove unbound protein.
Duplicate filters were probed with anti-LE antibody or anti-RAP
antibody. Blots were developed with chemiluminescent detection. The
activity of the lysosomal enzyme was measured using fluorescent
substrates. It was observed as shown in FIG. 10 that antibodies to
either RAP or to the lysosomal enzyme detect LRP-bound RAP-LE, were
found to bind to the fusion on Western blots, indicating that the
fused proteins were intact and folded. Comparing signal intensity,
it is further observed that the fusion is bound by the immobilized
LRP to a similar extent as RAP alone.
[0259] Characterization of RAP-GAA Fusion:
[0260] Purified RAP-GAA was tested to determine identity, purity
and carbohydrate content. For the identity test, fusion was
resolved on SDS-PAGE, blotted to PVDF and probed with anti-GAA and
anti-RAP antibodies. A single band of about 150 kD cross-reacted
with both antibodies (FIG. 6). Fusion purity was determined by
Coomassie Blue staining of the SDS-PAGE gel and was estimated to be
>95%. Presence of complex oligosaccharides was measured by
digestion with neuraminidase and comparison to undigested samples
on an IEF gel. Neuraminidase digestion resulted in a quantitative
shift in mobility to a more basic pI, consistent with the presence
of complex oligosaccharides (FIG. 7). Endo H digestion was used to
test for the presence of high-mannose oligosaccharides. Unlike
control proteins, no change in molecular weight of the fusion was
observed on SDS-PAGE gels after Endo H digestion. This suggests the
absence of high-mannose oligosaccharides on the fusion (FIG.
8).
[0261] Purification of the RAP-IDU Fusion:
[0262] Blue sepharose 6 Fast Flow resin is used for the first
purification step. The harvest fluid was adjusted to pH 7.0 and
loaded onto a Blue-Sepharose column at a 70 mL/mL resin basis. The
column was equilibrated with 75 mM NaCl, 20 mM Na.sub.2HPO.sub.4 pH
7.0. RAP-IDU eluted off the column at 1.2 M NaCl, 20 mM
Na.sub.2HPO.sub.4 pH 7.0. The eluted fraction containing RAP-IDU
(determined by iduronidase activity assay) was then exchanged into
75 mM NaCl, 20 mM Na.sub.2PO.sub.4 pH 7.0 and loaded onto a Heparin
CL 6B resin. RAP-IDU was eluted from the Heparin column at 0.5 M
NaCl pH 7.0. The eluted fusion was then adjusted to 2M NaCl, 20 mM
Na.sub.2HPO.sub.4 pH 7.0 and loaded directly onto a
Phenyl-Sepharose column. As a final step, RAP-IDU was eluted from
this column at between 0.3 to 0.5M NaCl. Fusion purity was
estimated by SDS-PAGE at >80% (FIG. 9).
Example III
Uptake and Distribution of Unconjugated RAP to the Brain
[0263] The distribution of RAP to brain was measured using a mouse
in situ perfusion model. Volumes of distribution (V.sub.d) for RAP,
the positive control transferrin and the negative control albumin,
were determined over a perfusion interval of 5 minutes. In
addition, the relative quantities of the test proteins in the
vascular and parenchymal fractions of the perfused brain were
determined using the capillary depletion technique (Gutierrez, et
al., J. Neuroimmunology 47(2):169-76 (1993)). The results shown in
FIG. 11 include an observed, corrected K.sub.influx of 1
.mu.L/g/min for transferrin. RAP had an observed, corrected
K.sub.influx of 2.2 .mu.L/g/min. RAP is taken up into brain.
[0264] A separate experiment was carried out at a single, 5-minute
time-point to determine whether RAP is able to traverse the brain
vasculature and enter the parenchyma. Brains were harvested as
before, but were subjected to a capillary depletion procedure to
determine the levels of RAP and albumin in the vascular and
parenchymal spaces. Following harvest, the isolated cortex was
weighed and placed in a Dounce homogenizer on ice. The cortex was
immediately homogenized in 0.7 ml of capillary buffer (10 mM HEPES,
141 mM NaCl, 4 mM KCl, 2.8 mM CaCl.sub.2, 1 mM NaH.sub.2PO.sub.4, 1
mM MgSO.sub.4, 10 mM glucose, pH 7.4) for 10 strokes, after which
1.7 ml of 26% dextran was added and the mixture further homogenized
with an additional 3 strokes on ice. To separate the different
tissue fractions, 1.3 ml of the homogenate was loaded in an
ultracentrifuge tube. The homogenate was centrifuged at 9000 rpm
(5400.times.g) for 15 min at 4.degree. C. in a Beckman TLV-100
swinging-bucket rotor. The parenchymal portion (supernatant) and
the capillary portion (pellet) were than separately counted in a
dual-channel gamma counter. A sample of post-CNS perfusate was also
counted for the V.sub.d calculation. Unlabeled RAP was included as
a competitor in some cases to determine whether uptake into brain
tissue was saturable (5 .mu.g of unlabeled RAP per mouse, about
80-fold excess over labeled RAP). Results were plotted as corrected
V.sub.d (FIG. 11). Each data point is an average derived from 5-6
mice. FIG. 12 shows the distribution of RAP between brain capillary
endothelium and brain parenchyma. These results indicate RAP
crosses the blood-brain barrier to enter brain parenchyma and that
the process of uptake is saturable.
Example IV
Measurement of Specific Uptake of RAP-GAA into Enzyme-Deficient
Patient Fibroblasts
[0265] The uptake of RAGA into cells deficient in GAA was
characterized. The cell line used was GM244 (Coriell Cell
Repository), a primary cell line isolated from a patient with
glycogen storage disorder type II (Pompe's disease). These
fibroblasts take up phosphorylated, recombinant GAA via the
mannose-6-phosphate receptor, but also have LRPI receptors, which
bind RAP. In order to identify the receptors involved in uptake of
different test ligands, samples containing excess free RAP or
mannose-6-phosphate were prepared.
[0266] Dilutions of RAP-GAA were made in the uptake medium
(Dulbecco's Modified Eagle's Medium supplemented with 25 mM HEPES
pH 7.0, 2 mM L-glutamine and 250 pg/mL bovine serum albumin) to
yield fusion protein concentrations of 33, 11, 3.7, 1.2, 0.4, and
0.1 nM. The effect of 3 mM mannose-6-phosphate, 500 nM RAP and a
combination of the two on the uptake of 5 nM RAP-GAA was also
assayed. The GM244 fibroblasts was seeded into 12-well plates and
allowed to grow for 3 days prior to the uptake experiment.
[0267] To initiate uptake, the growth medium was aspirated from the
wells and each sample dispensed into duplicate wells at 1 ml per
well. Plates were incubated for 4 hours at 37.degree. C., 5%
CO.sub.2. Samples were then aspirated from each well, the wells
washed with phosphate-buffered saline (PBS), and pre-warmed 0.25%
trypsin/0.1% EDTA added to each well at 37.degree. for 5 minutes to
release the adherent cells. Released cells were pelleted and rinsed
with chilled PBS. Pre-chilled lysis buffer (phosphate-citrate
buffer, pH 4.0 with 0.15% Triton X-100) was then added and the
pellets resuspended by gentle vortexing. Lysed cells could be
stored at -80.degree. C.
[0268] To measure the levels of GAA activity in the lysed cells,
the frozen lysates were thawed at room temperature. Lysate (50
.mu.l) was added directly to duplicate wells in 96-well opaque
microtiter plates. Pre-warmed GAA fluorescent substrate
(4-methylumbelliferyl-alpha-D-glucos- ide, 100 .mu.L) was added to
each well to initiate the reaction. The plate was incubated at
37.degree. C. for 30 minutes and the reaction terminated by
addition of150 .mu.l glycine/carbonate buffer pH 10. Fluorescence
was measured in a plate reader at an excitation wavelength of 366
nm and an emission wavelength of 446 nm.
[0269] The results in FIG. 13 show that RAP-GAA is taken up by
GM244 fibroblast cells. The K.sub.uptake was .about.19 nM as
determined by a non-linear fit enzymatic algorithm described in the
GraFit software program (Sando and Neufeld 1977). Approximately
60-fold more RAP-GAA gets into the fibroblasts than recombinant GAA
(V.sub.max ratio); 25-fold more at 10 nM. Additionally, 90% of the
RAP-GAA fusion uptake is inhibited by 50 nM RAP while only 20% of
the uptake is inhibited by 3 mM mannose 6-phosphate. The uptake of
the native GAA is almost completely inhibited by mannose
6-phosphate, suggesting alternate receptor pathways for RAP-GAA and
recombinant GAA.
Example V
Measurement of RAP-GAA Uptake and Lysosomal Localization in LRPnull
CHO Cells Expressing Different LRP Receptor Family Members (LRP1B,
LDLR, VLDLR) and into BN Cells Expressing Only LRP2 (Megalin,
gp330)
[0270] Iodine labeling: RAP-GAA or recombinant GAA were
radiolabeled with .sup.125I using the IODO-GEN reagent.
[0271] Cells were seeded in 12-well plates at a density of 200,000
cells/well and used after overnight culture. On the day of the
experiment, cells were rinsed twice in ice-cold ligand binding
buffer (Minimal Eagle's medium containing 0.6% bovine serum albumin
(BSA)), and .sup.125I-RAP-GAA or GAA alone were then added in the
same buffer (0.5 ml/well). The initial ligand concentrations tested
were 10 nM. Binding was carried out at 4.degree. C. for 30 min with
gentle rocking in the presence or absence of unlabeled 500 nM RAP
or 10 mM mannose-6-phosphate to confirm receptor-binding
specificity. Unbound ligand was then removed by washing cell
monolayers three times with ice-cold binding buffer. Ice-cold
stop/strip solution (0.2 M acetic acid, pH 2.6, 0.1 M NaCl) was
then added to one set of plates without warming and kept on ice
prior to counting. Dissociation constants for the receptor-ligand
complexes were determined from the resulting binding data. The
remaining plates were then placed in a 37.degree. C. water bath,
and 0.5 ml of ligand binding buffer prewarmed to 37.degree. C. was
added to the well monolayers to initiate internalization. At each
time point (every 30 seconds for 2 minutes and every 3 minutes
thereafter) the wells were placed on ice, and the ligand-binding
buffer replaced with ice-cold stop/strip solution. Ligand that
remained on the cell surface was stripped by incubation for 20
minutes (0.75 ml for 10 minutes, twice) and counted.
Internalization rates were determined from this data. Cell
monolayers were then solubilized with SDS lysis buffer (62.5 mM
Tris-HCl, pH 6.8, 0.2% SDS, and 10% (v/v) glycerol) and counted.
The sum of ligand that was internalized added to that which
remained on the cell surface after each assay was used as the
maximum potential internalization. The fraction of internalized
ligand after each time point was calculated and plotted.
[0272] Measurement of ligand degradation efficiency (transport to
lysosomes after internalization): Cells were seeded at a density of
200,000 cells/well into 12-well dishes 1 day prior to assays. On
the day of the experiment, pre-warmed assay buffer containing
RAP-GAA or GAA alone was added to cell monolayers in the presence
or absence of unlabeled 500 nM RAP or 10 mM mannose 6-phosphate,
followed by incubation for 4 hours at 37.degree. C. Following
incubation, the medium overlaying the cell monolayers was removed
and proteins were precipitated by addition of BSA to 10 mg/ml and
trichloroacetic acid to a final concentration of 20%. Lysosomal
degradation of ligands was defined as the appearance of radioactive
fragments in the medium that were soluble in 20% trichloroacetic
acid. The protein concentrations of each cell lysate were measured
in parallel dishes that did not contain LRP ligands. The RAP-GAA
and GAA degradation efficiencies were calculated as the value of
degraded radioactive material (soluble cpm/mg cell protein) divided
by the number of cell surface LRP family receptors (as determined
previously by flow cytometry, data not shown).
Example VI
Measurement of Specific Uptake of RAP-LE in to Enzyme-Deficient
Patient Fibroblasts with Concomitant Clearance of Stored
Glycosaminoglycans
[0273] Patient fibroblasts are seeded and grown to confluence in
12-well plates. On the day of the experiment, cells are fed with
fresh medium lacking MgSO.sub.4 and containing 4 .mu.Ci/mL of
Na.sub.2.sup.35SO.sub.4. Cells are also supplemented with RAP-LE
fusion or LE alone in the presence or absence of 500 nM RAP or 10
mM mannose 6-phosphate. Cells are harvested each day for 4 days.
After rinsing with PBS, cells are lysed by freeze-thaw. Stored GAG
is assayed by precipitation with 80% ethanol and quantitated by
scintillation counting. Stored GAG values are normalized to the
protein content of the cell lysates.
Example VII
Measurement of Lysosomal Distribution and Clearance of Storage in
Intravenously-Administered RAP-GAA in GAA-Deficient Mice
[0274] GAA knock out mice (C57B1/6 background) were randomized to
four treatment groups and treated every two days with 100 .mu.l of
either phosphate-buffered saline, 1.3 mg/kg or 0.33 mg/kg RAP-GAA
fusion protein four times via intravenous tail vein injection.
Forty-eight hours after the fourth injection, mice were euthanized
by carbon dioxide inhalation and the brain, heart, diaphragm, upper
and lower body skeletal muscle and liver immediately collected and
flash frozen. Three age-matched wild-type mice were also euthanized
and tissues collected and frozen. Each tissue is prepared for GAA
immunohistochemical staining by embedding in OCT blocks, and for
glycogen staining by fixing in glutaraldehyde and embedding in
paraffin. The remaining tissues were tested for GAA activity using
the fluorescent substrate assay described in Example IV. Serum was
collected at sacrifice and tested for GAA antibody.
2 Dosing Regimen Test Articles Or Dose Dose Group #Animals Vehicle
Articles (mg/kg) #Doses Volume (.mu.l) 1 6 KO PBS -- 4 100 2 6 KO
RAP-GAA 0.33 4 100 3 6 KO RAP-GAA 1.30 4 100 4 6 KO GAA 1.30 4 100
5 3 WT None None None None Study day 0 Inject groups 1-4 Study day
2 Inject groups 1-4 Study day 4 Inject groups 1-4 Study day 7
Inject groups 1-4 Study day 9 Bleed groups 1-4 and Sacrifice groups
1-5, Collect tissues groups 1-5
Example VIII
Treatment of Patients with MPS-I Disorder
[0275] A pharmaceutical composition comprising a conjugated agent
comprising therapeutic enzyme linked to RAP is administered
intravenously. The final dosage form of the fluid includes the
conjugated agent, normal saline, phosphate buffer at pH 5.8 and
human albumin at 1 mg/ml. These are prepared in a bag of normal
saline.
[0276] A preferred composition comprises the conjugated agent
(therapeutic enzyme linked to RAP) in an amount ranging from
0.05-0.5 mg/mL or 12,500-50,000 units per mL; sodium chlorode
solution 150 mM; sodium phosphate buffer 10-50 mM, pH 5.8; human
albumin 1 mg/mL. The composition may be in an intravenous bag of 50
to 250 ml.
[0277] Human patients manifesting a clinical phenotype of
deficiency of lysosomal enzyme, such as in patients with MPS I with
an alpha-L-iduronidase level of less than 1% of normal in
leukocytes and fibroblasts are included in the study. All patients
manifest some clinical evidence of visceral and soft tissue
accumulation of glycosaminoglycans with varying degrees of
functional impairment. Efficacy is determined by measuring the
percentage reduction in urinary GAG excretion over time. The
urinary GAG levels in MPS-I patients are compared to normal
excretion values. There is a wide range of urine GAG values in
untreated MPS-I patients. A greater than 50% reduction in excretion
of undegraded GAGs following therapy with the conjugated agent is a
valid means to measure an individual's response to therapy. For
example, data is collected measuring the leukocyte iduronidase
activity and buccal iduronidase activity before and after therapy
in MPS I patients. Clinical assessment of liver and spleen size is
performed as it is the most widely accepted means for evaluating
successful bone marrow transplant treatment in MPS-I patients
(Hoogerbrugge, et al., Lancet 345:1398 (1995)).
Example IX
Lysosomal Storage Diseases that may be Treated with Corresponding
RAP-LE Conjugates
[0278] The diseases that can be treated or prevented using the
methods of the present invention are: Mucopolysaccharidosis I (MPS
I), MPS II, MPS IIIA, MPS IIIB, Metachromatic Leukodystrophy (MLD),
Krabbe, Pompe, Ceroid Lipofuscinosis, Tay-Sachs, Niemann-Pick A and
B, and other lysosomal diseases. For each disease the conjugated
agent would comprise a specific compound or enzyme. For methods
involving MPS I, the preferred compound or enzyme is
.alpha.-L-iduronidase. For methods involving MPS II, the preferred
compound or enzyme iduronate-2-sulfatase. For methods involving MPS
IIIA, the preferred compound or enzyme is heparan N-sulfatase. For
methods involving MPS IIIB, the preferred compound or enzyme is
.alpha.-N-acetylglucosaminidase. For methods involving
Metachromatic Leukodystropy (MLD), the preferred compound or enzyme
is arylsulfatase A. For methods involving Krabbe, the preferred
compound or enzyme is galactosylceramidase. For methods involving
Pompe, the preferred compound or enzyme is acid
.alpha.-glucosidase. For methods involving CLN, the preferred
compound or enzyme is tripeptidyl peptidase. For methods involving
Tay-Sachs, the preferred compound or enzyme is hexosaminidase
alpha. For methods involving Niemann-Pick A and B the preferred
compound or enzyme is acid sphingomyelinase.
[0279] Each publication, patent application, patent, and other
reference cited in any part of the specification is incorporated
herein by reference in its entirety to the extent that it is not
inconsistent with the present disclosure.
[0280] Based on the invention and examples disclosed herein, those
skilled in the art will be able to develop other embodiments of the
invention. The examples are not intended to limit the scope of the
claims set out below in any way. Although the foregoing invention
has been described in some detail by way of illustration and
example for purposes of clarity of understanding, it will be
readily apparent to those of ordinary skill in the art in light of
the teachings of this invention that certain changes and
modifications may be made thereto without departing from the spirit
or scope of the appended claims.
Sequence CWU 1
1
28 1 323 PRT Homo sapiens 1 Tyr Ser Arg Glu Lys Asn Gln Pro Lys Pro
Ser Pro Lys Arg Glu Ser 1 5 10 15 Gly Glu Glu Phe Arg Met Glu Lys
Leu Asn Gln Leu Trp Glu Lys Ala 20 25 30 Gln Arg Leu His Leu Pro
Pro Val Arg Leu Ala Glu Leu His Ala Asp 35 40 45 Leu Lys Ile Gln
Glu Arg Asp Glu Leu Ala Trp Lys Lys Leu Lys Leu 50 55 60 Asp Gly
Leu Asp Glu Asp Gly Glu Lys Glu Ala Arg Leu Ile Arg Asn 65 70 75 80
Leu Asn Val Ile Leu Ala Lys Tyr Gly Leu Asp Gly Lys Lys Asp Ala 85
90 95 Arg Gln Val Thr Ser Asn Ser Leu Ser Gly Thr Gln Glu Asp Gly
Leu 100 105 110 Asp Asp Pro Arg Leu Glu Lys Leu Trp His Lys Ala Lys
Thr Ser Gly 115 120 125 Lys Phe Ser Gly Glu Glu Leu Asp Lys Leu Trp
Arg Glu Phe Leu His 130 135 140 His Lys Glu Lys Val His Glu Tyr Asn
Val Leu Leu Glu Thr Leu Ser 145 150 155 160 Arg Thr Glu Glu Ile His
Glu Asn Val Ile Ser Pro Ser Asp Leu Ser 165 170 175 Asp Ile Lys Gly
Ser Val Leu His Ser Arg His Thr Glu Leu Lys Glu 180 185 190 Lys Leu
Arg Ser Ile Asn Gln Gly Leu Asp Arg Leu Arg Arg Val Ser 195 200 205
His Gln Gly Tyr Ser Thr Glu Ala Glu Phe Glu Glu Pro Arg Val Ile 210
215 220 Asp Leu Trp Asp Leu Ala Gln Ser Ala Asn Leu Thr Asp Lys Glu
Leu 225 230 235 240 Glu Ala Phe Arg Glu Glu Leu Lys His Phe Glu Ala
Lys Ile Glu Lys 245 250 255 His Asn His Tyr Gln Lys Gln Leu Glu Ile
Ala His Glu Lys Leu Arg 260 265 270 His Ala Glu Ser Val Gly Asp Gly
Glu Arg Val Ser Arg Ser Arg Glu 275 280 285 Lys His Ala Leu Leu Glu
Gly Arg Thr Lys Glu Leu Gly Tyr Thr Val 290 295 300 Lys Lys His Leu
Gln Asp Leu Ser Gly Arg Ile Ser Arg Ala Arg His 305 310 315 320 Asn
Glu Leu 2 209 PRT Homo sapiens 2 Pro Arg Leu Glu Lys Leu Trp His
Lys Ala Lys Thr Ser Gly Lys Phe 1 5 10 15 Ser Gly Glu Glu Leu Asp
Lys Leu Trp Arg Glu Phe Leu His His Lys 20 25 30 Glu Lys Val His
Glu Tyr Asn Val Leu Leu Glu Thr Leu Ser Arg Thr 35 40 45 Glu Glu
Ile His Glu Asn Val Ile Ser Pro Ser Asp Leu Ser Asp Ile 50 55 60
Lys Gly Ser Val Leu His Ser Arg His Thr Glu Leu Lys Glu Lys Leu 65
70 75 80 Arg Ser Ile Asn Gln Gly Leu Asp Arg Leu Arg Arg Val Ser
His Gln 85 90 95 Gly Tyr Ser Thr Glu Ala Glu Phe Glu Glu Pro Arg
Val Ile Asp Leu 100 105 110 Trp Asp Leu Ala Gln Ser Ala Asn Leu Thr
Asp Lys Glu Leu Glu Ala 115 120 125 Phe Arg Glu Glu Leu Lys His Phe
Glu Ala Lys Ile Glu Lys His Asn 130 135 140 His Tyr Gln Lys Gln Leu
Glu Ile Ala His Glu Lys Leu Arg His Ala 145 150 155 160 Glu Ser Val
Gly Asp Gly Glu Arg Val Ser Arg Ser Arg Glu Lys His 165 170 175 Ala
Leu Leu Glu Gly Arg Thr Lys Glu Leu Gly Tyr Thr Val Lys Lys 180 185
190 His Leu Gln Asp Leu Ser Gly Arg Ile Ser Arg Ala Arg His Asn Glu
195 200 205 Leu 3 33 DNA Artificial sequence Synthetic primer 3
ccgcgtggat cccccaggct ggaaaagctg tgg 33 4 35 DNA Artificial
sequence Synthetic primer 4 tcaatgaatt ctcagagttc gttgtgccga gctct
35 5 205 PRT Homo sapiens 5 Pro Arg Leu Glu Lys Leu Trp His Lys Ala
Lys Thr Ser Gly Ile Ser 1 5 10 15 Val Arg Leu Thr Ser Cys Ala Arg
Val Leu His Tyr Lys Glu Lys Ile 20 25 30 His Glu Tyr Asn Val Leu
Leu Asp Thr Leu Ser Arg Ala Glu Glu Gly 35 40 45 Tyr Glu Asn Leu
Leu Ser Pro Ser Asp Met Thr His Ile Lys Ser Asp 50 55 60 Thr Leu
Ala Ser Lys His Ser Glu Leu Lys Asp Arg Leu Arg Ser Ile 65 70 75 80
Asn Gln Gly Leu Asp Arg Leu Arg Lys Val Ser His Gln Leu Arg Pro 85
90 95 Ala Thr Glu Phe Glu Glu Pro Arg Val Ile Asp Leu Trp Asp Leu
Ala 100 105 110 Gln Ser Ala Asn Phe Thr Glu Lys Glu Leu Glu Ser Phe
Arg Glu Glu 115 120 125 Leu Lys His Phe Glu Ala Lys Ile Glu Lys His
Asn His Tyr Gln Lys 130 135 140 Gln Leu Glu Ile Ser His Gln Lys Leu
Lys His Val Glu Ser Ile Gly 145 150 155 160 Asp Pro Glu His Ile Ser
Arg Asn Lys Glu Lys Tyr Val Leu Leu Glu 165 170 175 Glu Lys Thr Lys
Glu Leu Gly Tyr Lys Val Lys Lys His Leu Gln Asp 180 185 190 Leu Ser
Ser Arg Val Ser Arg Ala Arg His Asn Glu Leu 195 200 205 6 3702 DNA
Artificial sequence RAP-GAA fusion sequence 6 cttaccgcca tgcggggtcc
gagcggggct ctgtggctgc tcctggctct gcgcaccgtg 60 ctcggatcct
actcgcggga gaagaaccag cccaagccgt ccccgaaacg cgagtccgga 120
gaggagttcc gcatggagaa gttgaaccag ctgtgggaga aggcccagcg actgcatctt
180 cctcccgtga ggctggccga gctccacgct gatctgaaga tacaggagag
ggacgaactc 240 gcctggaaga aactaaagct tgacggcttg gacgaagatg
gggagaagga agcgagactc 300 atacgcaacc tcaatgtcat cttggccaag
tatggtctgg acggaaagaa ggacgctcgg 360 caggtgacca gcaactccct
cagtggcacc caggaagacg ggctggatga ccccaggctg 420 gaaaagctgt
ggcacaaggc gaagacctct gggaaattct ccggcgaaga actggacaag 480
ctctggcggg agttcctgca tcacaaagag aaagttcacg agtacaacgt cctgctggag
540 accctgagca ggaccgaaga aatccacgag aacgtcatta gcccctcgga
cctgagcgac 600 atcaagggca gcgtcctgca cagcaggcac acggagctga
aggagaagct gcgcagcatc 660 aaccagggcc tggaccgcct gcgcagggtc
agccaccagg gctacagcac tgaggctgag 720 ttcgaggagc ccagggtgat
tgacctgtgg gacctggcgc agtccgccaa cctcacggac 780 aaggagctgg
aggcgttccg ggaggagctc aagcacttcg aagccaaaat cgagaagcac 840
aaccactacc agaagcagct ggagattgcg cacgagaagc tgaggcacgc agagagcgtg
900 ggcgacggcg agcgtgtgag ccgcagccgc gagaagcacg ccctgctgga
ggggcggacc 960 aaggagctgg gctacacggt gaagaagcat ctgcaggacc
tgtccggcag gatctccaga 1020 gctcgcgccg aggcagaaac cggtgcacac
cccggccgtc ccagagcagt gcccacacag 1080 tgcgacgtcc cccccaacag
ccgcttcgat tgcgcccctg acaaggccat cacccaggaa 1140 cagtgcgagg
cccgcggctg ctgctacatc cctgcaaagc aggggctgca gggagcccag 1200
atggggcagc cctggtgctt cttcccaccc agctacccca gctacaagct ggagaacctg
1260 agctcctctg aaatgggcta cacggccacc ctgacccgta ccacccccac
cttcttcccc 1320 aaggacatcc tgaccctgcg gctggacgtg atgatggaga
ctgagaaccg cctccacttc 1380 acgatcaaag atccagctaa caggcgctac
gaggtgccct tggagacccc gcgtgtccac 1440 agccgggcac cgtccccact
ctacagcgtg gagttctccg aggagccctt cggggtgatc 1500 gtgcaccggc
agctggacgg ccgcgtgctg ctgaacacga cggtggcgcc cctgttcttt 1560
gcggaccagt tccttcagct gtccacctcg ctgccctcgc agtatatcac aggcctcgcc
1620 gagcacctca gtcccctgat gctcagcacc agctggacca ggatcaccct
gtggaaccgg 1680 gaccttgcgc ccacgcccgg tgcgaacctc tacgggtctc
accctttcta cctggcgctg 1740 gaggacggcg ggtcggcaca cggggtgttc
ctgctaaaca gcaatgccat ggatgtggtc 1800 ctgcagccga gccctgccct
tagctggagg tcgacaggtg ggatcctgga tgtctacatc 1860 ttcctgggcc
cagagcccaa gagcgtggtg cagcagtacc tggacgttgt gggatacccg 1920
ttcatgccgc catactgggg cctgggcttc cacctgtgcc gctggggcta ctcctccacc
1980 gctatcaccc gccaggtggt ggagaacatg accagggccc acttccccct
ggacgtccaa 2040 tggaacgacc tggactacat ggactcccgg agggacttca
cgttcaacaa ggatggcttc 2100 cgggacttcc cggccatggt gcaggagctg
caccagggcg gccggcgcta catgatgatc 2160 gtggatcctg ccatcagcag
ctcgggccct gccgggagct acaggcccta cgacgagggt 2220 ctgcggaggg
gggttttcat caccaacgag accggccagc cgctgattgg gaaggtatgg 2280
cccgggtcca ctgccttccc cgacttcacc aaccccacag ccctggcctg gtgggaggac
2340 atggtggctg agttccatga ccaggtgccc ttcgacggct tgtggattga
catgaacgag 2400 ccttccaact tcatcagagg ctctgaggac ggctgcccca
acaatgagct ggagaaccca 2460 ccctacgtgc ctggggtggt tggggggacc
ctccaggcgg ccaccatctg tgcctccagc 2520 caccagtttc tctccacaca
ctacaacctg cacaacctct acggcctgac cgaagccatc 2580 gcctcccaca
gggcgctggt gaaggctcgg gggacacgcc catttgtgat ctcccgctcg 2640
acctttgctg gccacggccg atacgccggc cactggacgg gggacgtgtg gagctcctgg
2700 gagcagctcg cctcctccgt gccagaaatc ctgcagttta acctgctggg
ggtgcctctg 2760 gtcggggccg acgtctgcgg cttcctgggc aacacctcag
aggagctgtg tgtgcgctgg 2820 acccagctgg gggccttcta ccccttcatg
cggaaccaca acagcctgct cagtctgccc 2880 caggagccgt acagcttcag
cgagccggcc cagcaggcca tgaggaaggc cctcaccctg 2940 cgctacgcac
tcctccccca cctctacaca ctgttccacc aggcccacgt cgcgggggag 3000
accgtggccc ggcccctctt cctggagttc cccaaggact ctagcacctg gactgtggac
3060 caccagctcc tgtgggggga ggccctgctc atcaccccag tgctccaggc
cgggaaggcc 3120 gaagtgactg gctacttccc cttgggcaca tggtacgacc
tgcagacggt gccaatagag 3180 gcccttggca gcctcccacc cccacctgca
gctccccgtg agccagccat ccacagcgag 3240 gggcagtggg tgacgctgcc
ggcccccctg gacaccatca acgtccacct ccgggctggg 3300 tacatcatcc
ccctgcaggg ccctggcctc acaaccacag agtcccgcca gcagcccatg 3360
gccctggctg tggccctaac caagggtgga gaggcccgag gggagctgtt ctgggacgat
3420 ggagagagcc tggaagtgct ggagcgaggg gcctacacac aggtcatctt
cctggccagg 3480 aataacacga tcgtgaatga gctggtacgt gtgaccagtg
agggagctgg cctgcagctg 3540 cagaaggtga ctgtcctggg cgtggccacg
gcgccccagc aggtcctctc caacggtgtc 3600 cctgtctcca acttcaccta
cagccccgac accaaggtcc tggacatctg tgtctcgctg 3660 ttgatgggag
agcagtttct cgtcagctgg tgttgactcg ag 3702 7 1228 PRT Artificial
sequence RAP-GAA fusion sequence 7 Met Arg Gly Pro Ser Gly Ala Leu
Trp Leu Leu Leu Ala Leu Arg Thr 1 5 10 15 Val Leu Gly Ser Tyr Ser
Arg Glu Lys Asn Gln Pro Lys Pro Ser Pro 20 25 30 Lys Arg Glu Ser
Gly Glu Glu Phe Arg Met Glu Lys Leu Asn Gln Leu 35 40 45 Trp Glu
Lys Ala Gln Arg Leu His Leu Pro Pro Val Arg Leu Ala Glu 50 55 60
Leu His Ala Asp Leu Lys Ile Gln Glu Arg Asp Glu Leu Ala Trp Lys 65
70 75 80 Lys Leu Lys Leu Asp Gly Leu Asp Glu Asp Gly Glu Lys Glu
Ala Arg 85 90 95 Leu Ile Arg Asn Leu Asn Val Ile Leu Ala Lys Tyr
Gly Leu Asp Gly 100 105 110 Lys Lys Asp Ala Arg Gln Val Thr Ser Asn
Ser Leu Ser Gly Thr Gln 115 120 125 Glu Asp Gly Leu Asp Asp Pro Arg
Leu Glu Lys Leu Trp His Lys Ala 130 135 140 Lys Thr Ser Gly Lys Phe
Ser Gly Glu Glu Leu Asp Lys Leu Trp Arg 145 150 155 160 Glu Phe Leu
His His Lys Glu Lys Val His Glu Tyr Asn Val Leu Leu 165 170 175 Glu
Thr Leu Ser Arg Thr Glu Glu Ile His Glu Asn Val Ile Ser Pro 180 185
190 Ser Asp Leu Ser Asp Ile Lys Gly Ser Val Leu His Ser Arg His Thr
195 200 205 Glu Leu Lys Glu Lys Leu Arg Ser Ile Asn Gln Gly Leu Asp
Arg Leu 210 215 220 Arg Arg Val Ser His Gln Gly Tyr Ser Thr Glu Ala
Glu Phe Glu Glu 225 230 235 240 Pro Arg Val Ile Asp Leu Trp Asp Leu
Ala Gln Ser Ala Asn Leu Thr 245 250 255 Asp Lys Glu Leu Glu Ala Phe
Arg Glu Glu Leu Lys His Phe Glu Ala 260 265 270 Lys Ile Glu Lys His
Asn His Tyr Gln Lys Gln Leu Glu Ile Ala His 275 280 285 Glu Lys Leu
Arg His Ala Glu Ser Val Gly Asp Gly Glu Arg Val Ser 290 295 300 Arg
Ser Arg Glu Lys His Ala Leu Leu Glu Gly Arg Thr Lys Glu Leu 305 310
315 320 Gly Tyr Thr Val Lys Lys His Leu Gln Asp Leu Ser Gly Arg Ile
Ser 325 330 335 Arg Ala Arg Ala Glu Ala Glu Thr Gly Ala His Pro Gly
Arg Pro Arg 340 345 350 Ala Val Pro Thr Gln Cys Asp Val Pro Pro Asn
Ser Arg Phe Asp Cys 355 360 365 Ala Pro Asp Lys Ala Ile Thr Gln Glu
Gln Cys Glu Ala Arg Gly Cys 370 375 380 Cys Tyr Ile Pro Ala Lys Gln
Gly Leu Gln Gly Ala Gln Met Gly Gln 385 390 395 400 Pro Trp Cys Phe
Phe Pro Pro Ser Tyr Pro Ser Tyr Lys Leu Glu Asn 405 410 415 Leu Ser
Ser Ser Glu Met Gly Tyr Thr Ala Thr Leu Thr Arg Thr Thr 420 425 430
Pro Thr Phe Phe Pro Lys Asp Ile Leu Thr Leu Arg Leu Asp Val Met 435
440 445 Met Glu Thr Glu Asn Arg Leu His Phe Thr Ile Lys Asp Pro Ala
Asn 450 455 460 Arg Arg Tyr Glu Val Pro Leu Glu Thr Pro Arg Val His
Ser Arg Ala 465 470 475 480 Pro Ser Pro Leu Tyr Ser Val Glu Phe Ser
Glu Glu Pro Phe Gly Val 485 490 495 Ile Val His Arg Gln Leu Asp Gly
Arg Val Leu Leu Asn Thr Thr Val 500 505 510 Ala Pro Leu Phe Phe Ala
Asp Gln Phe Leu Gln Leu Ser Thr Ser Leu 515 520 525 Pro Ser Gln Tyr
Ile Thr Gly Leu Ala Glu His Leu Ser Pro Leu Met 530 535 540 Leu Ser
Thr Ser Trp Thr Arg Ile Thr Leu Trp Asn Arg Asp Leu Ala 545 550 555
560 Pro Thr Pro Gly Ala Asn Leu Tyr Gly Ser His Pro Phe Tyr Leu Ala
565 570 575 Leu Glu Asp Gly Gly Ser Ala His Gly Val Phe Leu Leu Asn
Ser Asn 580 585 590 Ala Met Asp Val Val Leu Gln Pro Ser Pro Ala Leu
Ser Trp Arg Ser 595 600 605 Thr Gly Gly Ile Leu Asp Val Tyr Ile Phe
Leu Gly Pro Glu Pro Lys 610 615 620 Ser Val Val Gln Gln Tyr Leu Asp
Val Val Gly Tyr Pro Phe Met Pro 625 630 635 640 Pro Tyr Trp Gly Leu
Gly Phe His Leu Cys Arg Trp Gly Tyr Ser Ser 645 650 655 Thr Ala Ile
Thr Arg Gln Val Val Glu Asn Met Thr Arg Ala His Phe 660 665 670 Pro
Leu Asp Val Gln Trp Asn Asp Leu Asp Tyr Met Asp Ser Arg Arg 675 680
685 Asp Phe Thr Phe Asn Lys Asp Gly Phe Arg Asp Phe Pro Ala Met Val
690 695 700 Gln Glu Leu His Gln Gly Gly Arg Arg Tyr Met Met Ile Val
Asp Pro 705 710 715 720 Ala Ile Ser Ser Ser Gly Pro Ala Gly Ser Tyr
Arg Pro Tyr Asp Glu 725 730 735 Gly Leu Arg Arg Gly Val Phe Ile Thr
Asn Glu Thr Gly Gln Pro Leu 740 745 750 Ile Gly Lys Val Trp Pro Gly
Ser Thr Ala Phe Pro Asp Phe Thr Asn 755 760 765 Pro Thr Ala Leu Ala
Trp Trp Glu Asp Met Val Ala Glu Phe His Asp 770 775 780 Gln Val Pro
Phe Asp Gly Leu Trp Ile Asp Met Asn Glu Pro Ser Asn 785 790 795 800
Phe Ile Arg Gly Ser Glu Asp Gly Cys Pro Asn Asn Glu Leu Glu Asn 805
810 815 Pro Pro Tyr Val Pro Gly Val Val Gly Gly Thr Leu Gln Ala Ala
Thr 820 825 830 Ile Cys Ala Ser Ser His Gln Phe Leu Ser Thr His Tyr
Asn Leu His 835 840 845 Asn Leu Tyr Gly Leu Thr Glu Ala Ile Ala Ser
His Arg Ala Leu Val 850 855 860 Lys Ala Arg Gly Thr Arg Pro Phe Val
Ile Ser Arg Ser Thr Phe Ala 865 870 875 880 Gly His Gly Arg Tyr Ala
Gly His Trp Thr Gly Asp Val Trp Ser Ser 885 890 895 Trp Glu Gln Leu
Ala Ser Ser Val Pro Glu Ile Leu Gln Phe Asn Leu 900 905 910 Leu Gly
Val Pro Leu Val Gly Ala Asp Val Cys Gly Phe Leu Gly Asn 915 920 925
Thr Ser Glu Glu Leu Cys Val Arg Trp Thr Gln Leu Gly Ala Phe Tyr 930
935 940 Pro Phe Met Arg Asn His Asn Ser Leu Leu Ser Leu Pro Gln Glu
Pro 945 950 955 960 Tyr Ser Phe Ser Glu Pro Ala Gln Gln Ala Met Arg
Lys Ala Leu Thr 965 970 975 Leu Arg Tyr Ala Leu Leu Pro His Leu Tyr
Thr Leu Phe His Gln Ala 980 985 990 His Val Ala Gly Glu Thr Val Ala
Arg Pro Leu Phe Leu Glu Phe Pro 995 1000 1005 Lys Asp Ser Ser Thr
Trp Thr Val Asp His Gln Leu Leu Trp Gly 1010 1015 1020 Glu Ala Leu
Leu Ile Thr Pro Val Leu Gln Ala Gly Lys Ala Glu 1025 1030 1035 Val
Thr Gly Tyr Phe Pro Leu Gly Thr Trp Tyr Asp Leu Gln
Thr 1040 1045 1050 Val Pro Ile Glu Ala Leu Gly Ser Leu Pro Pro Pro
Pro Ala Ala 1055 1060 1065 Pro Arg Glu Pro Ala Ile His Ser Glu Gly
Gln Trp Val Thr Leu 1070 1075 1080 Pro Ala Pro Leu Asp Thr Ile Asn
Val His Leu Arg Ala Gly Tyr 1085 1090 1095 Ile Ile Pro Leu Gln Gly
Pro Gly Leu Thr Thr Thr Glu Ser Arg 1100 1105 1110 Gln Gln Pro Met
Ala Leu Ala Val Ala Leu Thr Lys Gly Gly Glu 1115 1120 1125 Ala Arg
Gly Glu Leu Phe Trp Asp Asp Gly Glu Ser Leu Glu Val 1130 1135 1140
Leu Glu Arg Gly Ala Tyr Thr Gln Val Ile Phe Leu Ala Arg Asn 1145
1150 1155 Asn Thr Ile Val Asn Glu Leu Val Arg Val Thr Ser Glu Gly
Ala 1160 1165 1170 Gly Leu Gln Leu Gln Lys Val Thr Val Leu Gly Val
Ala Thr Ala 1175 1180 1185 Pro Gln Gln Val Leu Ser Asn Gly Val Pro
Val Ser Asn Phe Thr 1190 1195 1200 Tyr Ser Pro Asp Thr Lys Val Leu
Asp Ile Cys Val Ser Leu Leu 1205 1210 1215 Met Gly Glu Gln Phe Leu
Val Ser Trp Cys 1220 1225 8 2937 DNA Artificial sequence RAP-IDU
fusion sequence 8 aagcttaccg ccatgcgggg tccgagcggg gctctgtggc
tgctcctggc tctgcgcacc 60 gtgctcggat cctactcgcg ggagaagaac
cagcccaagc cgtccccgaa acgcgagtcc 120 ggagaggagt tccgcatgga
gaagttgaac cagctgtggg agaaggccca gcgactgcat 180 cttcctcccg
tgaggctggc cgagctccac gctgatctga agatacagga gagggacgaa 240
ctcgcctgga agaaactaaa gcttgacggc ttggacgaag atggggagaa ggaagcgaga
300 ctcatacgca acctcaatgt catcttggcc aagtatggtc tggacggaaa
gaaggacgct 360 cggcaggtga ccagcaactc cctcagtggc acccaggaag
acgggctgga tgaccccagg 420 ctggaaaagc tgtggcacaa ggcgaagacc
tctgggaaat tctccggcga agaactggac 480 aagctctggc gggagttcct
gcatcacaaa gagaaagttc acgagtacaa cgtcctgctg 540 gagaccctga
gcaggaccga agaaatccac gagaacgtca ttagcccctc ggacctgagc 600
gacatcaagg gcagcgtcct gcacagcagg cacacggagc tgaaggagaa gctgcgcagc
660 atcaaccagg gcctggaccg cctgcgcagg gtcagccacc agggctacag
cactgaggct 720 gagttcgagg agcccagggt gattgacctg tgggacctgg
cgcagtccgc caacctcacg 780 gacaaggagc tggaggcgtt ccgggaggag
ctcaagcact tcgaagccaa aatcgagaag 840 cacaaccact accagaagca
gctggagatt gcgcacgaga agctgaggca cgcagagagc 900 gtgggcgacg
gcgagcgtgt gagccgcagc cgcgagaagc acgccctgct ggaggggcgg 960
accaaggagc tgggctacac ggtgaagaag catctgcagg acctgtccgg caggatctcc
1020 agagctcgcg ccgaggcaga aaccggtgag gccccgcacc tggtgcatgt
ggacgcggcc 1080 cgcgcgctgt ggcccctgcg gcgcttctgg aggagcacag
gcttctgccc cccgctgcca 1140 cacagccagg ctgaccagta cgtcctcagc
tgggaccagc agctcaacct cgcctatgtg 1200 ggcgccgtcc ctcaccgcgg
catcaagcag gtccggaccc actggctgct ggagcttgtc 1260 accaccaggg
ggtccactgg acggggcctg agctacaact tcacccacct ggacgggtac 1320
ttggaccttc tcagggagaa ccagctcctc ccagggtttg agctgatggg cagcgcctcg
1380 ggccacttca ctgactttga ggacaagcag caggtgtttg agtggaagga
cttggtctcc 1440 agcctggcca ggagatacat cggtaggtac ggactggcgc
atgtttccaa gtggaacttc 1500 gagacgtgga atgagccaga ccaccacgac
tttgacaacg tctccatgac catgcaaggc 1560 ttcctgaact actacgatgc
ctgctcggag ggtctgcgcg ccgccagccc cgccctgcgg 1620 ctgggaggcc
ccggcgactc cttccacacc ccaccgcgat ccccgctgag ctggggcctc 1680
ctgcgccact gccacgacgg taccaacttc ttcactgggg aggcgggcgt gcggctggac
1740 tacatctccc tccacaggaa gggtgcgcgc agctccatct ccatcctgga
gcaggagaag 1800 gtcgtcgcgc agcagatccg gcagctcttc cccaagttcg
cggacacccc catttacaac 1860 gacgaggcgg acccgctggt gggctggtcc
ctgccacagc cgtggagggc ggacgtgacc 1920 tacgcggcca tggtggtgaa
ggtcatcgcg cagcatcaga acctgctact ggccaacacc 1980 acctccgcct
tcccctacgc gctcctgagc aacgacaatg ccttcctgag ctaccacccg 2040
caccccttcg cgcagcgcac gctcaccgcg cgcttccagg tcaacaacac ccgcccgccg
2100 cacgtgcagc tgttgcgcaa gccggtgctc acggccatgg ggctgctggc
gctgctggat 2160 gaggagcagc tctgggccga agtgtcgcag gccgggaccg
tcctggacag caaccacacg 2220 gtgggcgtcc tggccagcgc ccaccgcccc
cagggcccgg ccgacgcctg gcgcgccgcg 2280 gtgctgatct acgcgagcga
cgacacccgc gcccacccca accgcagcgt cgcggtgacc 2340 ctgcggctgc
gcggggtgcc ccccggcccg ggcctggtct acgtcacgcg ctacctggac 2400
aacgggctct gcagccccga cggcgagtgg cggcgcctgg gccggcccgt cttccccacg
2460 gcagagcagt tccggcgcat gcgcgcggct gaggacccgg tggccgcggc
gccccgcccc 2520 ttacccgccg gcggccgcct gaccctgcgc cccgcgctgc
ggctgccgtc gcttttgctg 2580 gtgcacgtgt gtgcgcgccc cgagaagccg
cccgggcagg tcacgcggct ccgcgccctg 2640 cccctgaccc aagggcagct
ggttctggtc tggtcggatg aacacgtggg ctccaagtgc 2700 ctgtggacat
acgagatcca gttctctcag gacggtaagg cgtacacccc ggtcagcagg 2760
aagccatcga ccttcaacct ctttgtgttc agcccagaca caggtgctgt ctctggctcc
2820 taccgagttc gagccctgga ctactgggcc cgaccaggcc ccttctcgga
ccctgtgccg 2880 tacctggagg tccctgtgcc aagagggccc ccatccccgg
gcaatccatg actcgag 2937 9 972 PRT Artificial sequence RAP-IDU
fusion sequence 9 Met Arg Gly Pro Ser Gly Ala Leu Trp Leu Leu Leu
Ala Leu Arg Thr 1 5 10 15 Val Leu Gly Ser Tyr Ser Arg Glu Lys Asn
Gln Pro Lys Pro Ser Pro 20 25 30 Lys Arg Glu Ser Gly Glu Glu Phe
Arg Met Glu Lys Leu Asn Gln Leu 35 40 45 Trp Glu Lys Ala Gln Arg
Leu His Leu Pro Pro Val Arg Leu Ala Glu 50 55 60 Leu His Ala Asp
Leu Lys Ile Gln Glu Arg Asp Glu Leu Ala Trp Lys 65 70 75 80 Lys Leu
Lys Leu Asp Gly Leu Asp Glu Asp Gly Glu Lys Glu Ala Arg 85 90 95
Leu Ile Arg Asn Leu Asn Val Ile Leu Ala Lys Tyr Gly Leu Asp Gly 100
105 110 Lys Lys Asp Ala Arg Gln Val Thr Ser Asn Ser Leu Ser Gly Thr
Gln 115 120 125 Glu Asp Gly Leu Asp Asp Pro Arg Leu Glu Lys Leu Trp
His Lys Ala 130 135 140 Lys Thr Ser Gly Lys Phe Ser Gly Glu Glu Leu
Asp Lys Leu Trp Arg 145 150 155 160 Glu Phe Leu His His Lys Glu Lys
Val His Glu Tyr Asn Val Leu Leu 165 170 175 Glu Thr Leu Ser Arg Thr
Glu Glu Ile His Glu Asn Val Ile Ser Pro 180 185 190 Ser Asp Leu Ser
Asp Ile Lys Gly Ser Val Leu His Ser Arg His Thr 195 200 205 Glu Leu
Lys Glu Lys Leu Arg Ser Ile Asn Gln Gly Leu Asp Arg Leu 210 215 220
Arg Arg Val Ser His Gln Gly Tyr Ser Thr Glu Ala Glu Phe Glu Glu 225
230 235 240 Pro Arg Val Ile Asp Leu Trp Asp Leu Ala Gln Ser Ala Asn
Leu Thr 245 250 255 Asp Lys Glu Leu Glu Ala Phe Arg Glu Glu Leu Lys
His Phe Glu Ala 260 265 270 Lys Ile Glu Lys His Asn His Tyr Gln Lys
Gln Leu Glu Ile Ala His 275 280 285 Glu Lys Leu Arg His Ala Glu Ser
Val Gly Asp Gly Glu Arg Val Ser 290 295 300 Arg Ser Arg Glu Lys His
Ala Leu Leu Glu Gly Arg Thr Lys Glu Leu 305 310 315 320 Gly Tyr Thr
Val Lys Lys His Leu Gln Asp Leu Ser Gly Arg Ile Ser 325 330 335 Arg
Ala Arg Ala Glu Ala Glu Thr Gly Glu Ala Pro His Leu Val His 340 345
350 Val Asp Ala Ala Arg Ala Leu Trp Pro Leu Arg Arg Phe Trp Arg Ser
355 360 365 Thr Gly Phe Cys Pro Pro Leu Pro His Ser Gln Ala Asp Gln
Tyr Val 370 375 380 Leu Ser Trp Asp Gln Gln Leu Asn Leu Ala Tyr Val
Gly Ala Val Pro 385 390 395 400 His Arg Gly Ile Lys Gln Val Arg Thr
His Trp Leu Leu Glu Leu Val 405 410 415 Thr Thr Arg Gly Ser Thr Gly
Arg Gly Leu Ser Tyr Asn Phe Thr His 420 425 430 Leu Asp Gly Tyr Leu
Asp Leu Leu Arg Glu Asn Gln Leu Leu Pro Gly 435 440 445 Phe Glu Leu
Met Gly Ser Ala Ser Gly His Phe Thr Asp Phe Glu Asp 450 455 460 Lys
Gln Gln Val Phe Glu Trp Lys Asp Leu Val Ser Ser Leu Ala Arg 465 470
475 480 Arg Tyr Ile Gly Arg Tyr Gly Leu Ala His Val Ser Lys Trp Asn
Phe 485 490 495 Glu Thr Trp Asn Glu Pro Asp His His Asp Phe Asp Asn
Val Ser Met 500 505 510 Thr Met Gln Gly Phe Leu Asn Tyr Tyr Asp Ala
Cys Ser Glu Gly Leu 515 520 525 Arg Ala Ala Ser Pro Ala Leu Arg Leu
Gly Gly Pro Gly Asp Ser Phe 530 535 540 His Thr Pro Pro Arg Ser Pro
Leu Ser Trp Gly Leu Leu Arg His Cys 545 550 555 560 His Asp Gly Thr
Asn Phe Phe Thr Gly Glu Ala Gly Val Arg Leu Asp 565 570 575 Tyr Ile
Ser Leu His Arg Lys Gly Ala Arg Ser Ser Ile Ser Ile Leu 580 585 590
Glu Gln Glu Lys Val Val Ala Gln Gln Ile Arg Gln Leu Phe Pro Lys 595
600 605 Phe Ala Asp Thr Pro Ile Tyr Asn Asp Glu Ala Asp Pro Leu Val
Gly 610 615 620 Trp Ser Leu Pro Gln Pro Trp Arg Ala Asp Val Thr Tyr
Ala Ala Met 625 630 635 640 Val Val Lys Val Ile Ala Gln His Gln Asn
Leu Leu Leu Ala Asn Thr 645 650 655 Thr Ser Ala Phe Pro Tyr Ala Leu
Leu Ser Asn Asp Asn Ala Phe Leu 660 665 670 Ser Tyr His Pro His Pro
Phe Ala Gln Arg Thr Leu Thr Ala Arg Phe 675 680 685 Gln Val Asn Asn
Thr Arg Pro Pro His Val Gln Leu Leu Arg Lys Pro 690 695 700 Val Leu
Thr Ala Met Gly Leu Leu Ala Leu Leu Asp Glu Glu Gln Leu 705 710 715
720 Trp Ala Glu Val Ser Gln Ala Gly Thr Val Leu Asp Ser Asn His Thr
725 730 735 Val Gly Val Leu Ala Ser Ala His Arg Pro Gln Gly Pro Ala
Asp Ala 740 745 750 Trp Arg Ala Ala Val Leu Ile Tyr Ala Ser Asp Asp
Thr Arg Ala His 755 760 765 Pro Asn Arg Ser Val Ala Val Thr Leu Arg
Leu Arg Gly Val Pro Pro 770 775 780 Gly Pro Gly Leu Val Tyr Val Thr
Arg Tyr Leu Asp Asn Gly Leu Cys 785 790 795 800 Ser Pro Asp Gly Glu
Trp Arg Arg Leu Gly Arg Pro Val Phe Pro Thr 805 810 815 Ala Glu Gln
Phe Arg Arg Met Arg Ala Ala Glu Asp Pro Val Ala Ala 820 825 830 Ala
Pro Arg Pro Leu Pro Ala Gly Gly Arg Leu Thr Leu Arg Pro Ala 835 840
845 Leu Arg Leu Pro Ser Leu Leu Leu Val His Val Cys Ala Arg Pro Glu
850 855 860 Lys Pro Pro Gly Gln Val Thr Arg Leu Arg Ala Leu Pro Leu
Thr Gln 865 870 875 880 Gly Gln Leu Val Leu Val Trp Ser Asp Glu His
Val Gly Ser Lys Cys 885 890 895 Leu Trp Thr Tyr Glu Ile Gln Phe Ser
Gln Asp Gly Lys Ala Tyr Thr 900 905 910 Pro Val Ser Arg Lys Pro Ser
Thr Phe Asn Leu Phe Val Phe Ser Pro 915 920 925 Asp Thr Gly Ala Val
Ser Gly Ser Tyr Arg Val Arg Ala Leu Asp Tyr 930 935 940 Trp Ala Arg
Pro Gly Pro Phe Ser Asp Pro Val Pro Tyr Leu Glu Val 945 950 955 960
Pro Val Pro Arg Gly Pro Pro Ser Pro Gly Asn Pro 965 970 10 1398 DNA
Artificial sequence RAP-GDNF fusion sequence 10 atggggggtt
cttactcgcg ggagaagaac cagcccaagc cgtccccgaa acgcgagtcc 60
ggagaggagt tccgcatgga gaagttgaac cagctgtggg agaaggccca gcgactgcat
120 cttcctcccg tgaggctggc cgagctccac gctgatctga agatacagga
gagggacgaa 180 ctcgcctgga agaaactaaa gcttgacggc ttggacgaag
atggggagaa ggaagcgaga 240 ctcatacgca acctcaatgt catcttggcc
aagtatggtc tggacggaaa gaaggacgct 300 cggcaggtga ccagcaactc
cctcagtggc acccaggaag acgggctgga tgaccccagg 360 ctggaaaagc
tgtggcacaa ggcgaagacc tctgggaaat tctccggcga agaactggac 420
aagctctggc gggagttcct gcatcacaaa gagaaagttc acgagtacaa cgtcctgctg
480 gagaccctga gcaggaccga agaaatccac gagaacgtca ttagcccctc
ggacctgagc 540 gacatcaagg gcagcgtcct gcacagcagg cacacggagc
tgaaggagaa gctgcgcagc 600 atcaaccagg gcctggaccg cctgcgcagg
gtcagccacc agggctacag cactgaggct 660 gagttcgagg agcccagggt
gattgacctg tgggacctgg cgcagtccgc caacctcacg 720 gacaaggagc
tggaggcgtt ccgggaggag ctcaagcact tcgaagccaa aatcgagaag 780
cacaaccact accagaagca gctggagatt gcgcacgaga agctgaggca cgcagagagc
840 gtgggcgacg gcgagcgtgt gagccgcagc cgcgagaagc acgccctgct
ggaggggcgg 900 accaaggagc tgggctacac ggtgaagaag catctgcagg
acctgtccgg caggatctcc 960 agagctcggg ccgaggcaga aaccggttca
ccagataaac aaatggcagt gcttcctaga 1020 agagagcgga atcggcaggc
tgcagctgcc aacccagaga attccagagg aaaaggtcgg 1080 agaggccaga
ggggcaaaaa ccggggttgt gtcttaactg caatacattt aaatgtcact 1140
gacttgggtc tgggctatga aaccaaggag gaactgattt ttaggtactg cagcggctct
1200 tgcgatgcag ctgagacaac gtacgacaaa atattgaaaa acttatccag
aaatagaagg 1260 ctggtgagtg acaaagtagg gcaggcatgt tgcagaccca
tcgcctttga tgatgacctg 1320 tcgtttttag atgataacct ggtttaccat
attctaagaa agcattccgc taaaaggtgt 1380 ggatgtatct gatctaga 1398 11
463 PRT Artificial sequence RAP-GDNF fusion sequence 11 Met Gly Gly
Ser Tyr Ser Arg Glu Lys Asn Gln Pro Lys Pro Ser Pro 1 5 10 15 Lys
Arg Glu Ser Gly Glu Glu Phe Arg Met Glu Lys Leu Asn Gln Leu 20 25
30 Trp Glu Lys Ala Gln Arg Leu His Leu Pro Pro Val Arg Leu Ala Glu
35 40 45 Leu His Ala Asp Leu Lys Ile Gln Glu Arg Asp Glu Leu Ala
Trp Lys 50 55 60 Lys Leu Lys Leu Asp Gly Leu Asp Glu Asp Gly Glu
Lys Glu Ala Arg 65 70 75 80 Leu Ile Arg Asn Leu Asn Val Ile Leu Ala
Lys Tyr Gly Leu Asp Gly 85 90 95 Lys Lys Asp Ala Arg Gln Val Thr
Ser Asn Ser Leu Ser Gly Thr Gln 100 105 110 Glu Asp Gly Leu Asp Asp
Pro Arg Leu Glu Lys Leu Trp His Lys Ala 115 120 125 Lys Thr Ser Gly
Lys Phe Ser Gly Glu Glu Leu Asp Lys Leu Trp Arg 130 135 140 Glu Phe
Leu His His Lys Glu Lys Val His Glu Tyr Asn Val Leu Leu 145 150 155
160 Glu Thr Leu Ser Arg Thr Glu Glu Ile His Glu Asn Val Ile Ser Pro
165 170 175 Ser Asp Leu Ser Asp Ile Lys Gly Ser Val Leu His Ser Arg
His Thr 180 185 190 Glu Leu Lys Glu Lys Leu Arg Ser Ile Asn Gln Gly
Leu Asp Arg Leu 195 200 205 Arg Arg Val Ser His Gln Gly Tyr Ser Thr
Glu Ala Glu Phe Glu Glu 210 215 220 Pro Arg Val Ile Asp Leu Trp Asp
Leu Ala Gln Ser Ala Asn Leu Thr 225 230 235 240 Asp Lys Glu Leu Glu
Ala Phe Arg Glu Glu Leu Lys His Phe Glu Ala 245 250 255 Lys Ile Glu
Lys His Asn His Tyr Gln Lys Gln Leu Glu Ile Ala His 260 265 270 Glu
Lys Leu Arg His Ala Glu Ser Val Gly Asp Gly Glu Arg Val Ser 275 280
285 Arg Ser Arg Glu Lys His Ala Leu Leu Glu Gly Arg Thr Lys Glu Leu
290 295 300 Gly Tyr Thr Val Lys Lys His Leu Gln Asp Leu Ser Gly Arg
Ile Ser 305 310 315 320 Arg Ala Arg Ala Glu Ala Glu Thr Gly Ser Pro
Asp Lys Gln Met Ala 325 330 335 Val Leu Pro Arg Arg Glu Arg Asn Arg
Gln Ala Ala Ala Ala Asn Pro 340 345 350 Glu Asn Ser Arg Gly Lys Gly
Arg Arg Gly Gln Arg Gly Lys Asn Arg 355 360 365 Gly Cys Val Leu Thr
Ala Ile His Leu Asn Val Thr Asp Leu Gly Leu 370 375 380 Gly Tyr Glu
Thr Lys Glu Glu Leu Ile Phe Arg Tyr Cys Ser Gly Ser 385 390 395 400
Cys Asp Ala Ala Glu Thr Thr Tyr Asp Lys Ile Leu Lys Asn Leu Ser 405
410 415 Arg Asn Arg Arg Leu Val Ser Asp Lys Val Gly Gln Ala Cys Cys
Arg 420 425 430 Pro Ile Ala Phe Asp Asp Asp Leu Ser Phe Leu Asp Asp
Asn Leu Val 435 440 445 Tyr His Ile Leu Arg Lys His Ser Ala Lys Arg
Cys Gly Cys Ile 450 455 460 12 49 DNA Artificial sequence Synthetic
primer 12 gcgataggat cctactcgcg ggagaagaac cagcccaagc cgtccccga 49
13 57 DNA Artificial sequence Synthetic primer 13 gcgataaacc
ggtttctgcc tcggcgcgag ctctggagat cctgccggac aggtcct 57 14 39 DNA
Artificial sequence Synthetic primer 14 gcgataaccg gtgcacaccc
cggccgtccc agagcagtg 39 15 37 DNA Artificial sequence Synthetic
primer 15 gcgatactcg agtcaacacc agctgacgag aaactgc 37 16 46 DNA
Artificial sequence Synthetic primer 16 gcgataaccg gtgaggcccc
ccgcacctgg tgcatgtgga cgcggc 46
17 45 DNA Artificial sequence Synthetic primer 17 gcgatactcg
agtcatggat tgcccgggga tgggggccct cttgg 45 18 33 DNA Artificial
sequence Synthetic primer 18 acagtgaccg gttcaccaga taaacaaatg gca
33 19 38 DNA Artificial sequence Synthetic primer 19 acagtgctcg
agtctagatc agatacatcc acaccttt 38 20 51 DNA Artificial sequence
Synthetic primer 20 acagtggcca tggggggttc ttactcgcgg gagaagaacc
agcccaagcc g 51 21 357 PRT Homo sapiens 21 Met Ala Pro Arg Arg Val
Arg Ser Phe Leu Arg Gly Leu Pro Ala Leu 1 5 10 15 Leu Leu Leu Leu
Leu Phe Leu Gly Pro Trp Pro Ala Ala Ser His Gly 20 25 30 Gly Lys
Tyr Ser Arg Glu Lys Asn Gln Pro Lys Pro Ser Pro Lys Arg 35 40 45
Glu Ser Gly Glu Glu Phe Arg Met Glu Lys Leu Asn Gln Leu Trp Glu 50
55 60 Lys Ala Gln Arg Leu His Leu Pro Pro Val Arg Leu Ala Glu Leu
His 65 70 75 80 Ala Asp Leu Lys Ile Gln Glu Arg Asp Glu Leu Ala Trp
Lys Lys Leu 85 90 95 Lys Leu Asp Gly Leu Asp Glu Asp Gly Glu Lys
Glu Ala Arg Leu Ile 100 105 110 Arg Asn Leu Asn Val Ile Leu Ala Lys
Tyr Gly Leu Asp Gly Lys Lys 115 120 125 Asp Ala Arg Gln Val Thr Ser
Asn Ser Leu Ser Gly Thr Gln Glu Asp 130 135 140 Gly Leu Asp Asp Pro
Arg Leu Glu Lys Leu Trp His Lys Ala Lys Thr 145 150 155 160 Ser Gly
Lys Phe Ser Gly Glu Glu Leu Asp Lys Leu Trp Arg Glu Phe 165 170 175
Leu His His Lys Glu Lys Val His Glu Tyr Asn Val Leu Leu Glu Thr 180
185 190 Leu Ser Arg Thr Glu Glu Ile His Glu Asn Val Ile Ser Pro Ser
Asp 195 200 205 Leu Ser Asp Ile Lys Gly Ser Val Leu His Ser Arg His
Thr Glu Leu 210 215 220 Lys Glu Lys Leu Arg Ser Ile Asn Gln Gly Leu
Asp Arg Leu Arg Arg 225 230 235 240 Val Ser His Gln Gly Tyr Ser Thr
Glu Ala Glu Phe Glu Glu Pro Arg 245 250 255 Val Ile Asp Leu Trp Asp
Leu Ala Gln Ser Ala Asn Leu Thr Asp Lys 260 265 270 Glu Leu Glu Ala
Phe Arg Glu Glu Leu Lys His Phe Glu Ala Lys Ile 275 280 285 Glu Lys
His Asn His Tyr Gln Lys Gln Leu Glu Ile Ala His Glu Lys 290 295 300
Leu Arg His Ala Glu Ser Val Gly Asp Gly Glu Arg Val Ser Arg Ser 305
310 315 320 Arg Glu Lys His Ala Leu Leu Glu Gly Arg Thr Lys Glu Leu
Gly Tyr 325 330 335 Thr Val Lys Lys His Leu Gln Asp Leu Ser Gly Arg
Ile Ser Arg Ala 340 345 350 Arg His Asn Glu Leu 355 22 378 PRT Mus
musculus 22 Met Gly Gly Pro Thr Arg Pro Ser Pro Val Ser Leu Leu Ala
Leu Gln 1 5 10 15 Arg Lys Met Ala Pro Arg Arg Glu Arg Val Ser Thr
Leu Pro Arg Leu 20 25 30 Gln Leu Leu Val Leu Leu Leu Leu Pro Leu
Met Leu Val Pro Gln Pro 35 40 45 Ile Ala Gly His Gly Gly Lys Tyr
Ser Arg Glu Lys Asn Glu Pro Glu 50 55 60 Met Ala Ala Lys Arg Glu
Ser Gly Glu Glu Phe Arg Met Glu Lys Leu 65 70 75 80 Asn Gln Leu Trp
Glu Lys Ala Lys Arg Leu His Leu Ser Pro Val Arg 85 90 95 Leu Ala
Glu Leu His Ser Asp Leu Lys Ile Gln Glu Arg Asp Glu Leu 100 105 110
Asn Trp Lys Lys Leu Lys Val Glu Gly Leu Asp Lys Asp Gly Glu Lys 115
120 125 Glu Ala Lys Leu Ile His Asn Leu Asn Val Ile Leu Ala Arg Tyr
Gly 130 135 140 Leu Asp Gly Arg Lys Asp Ala Gln Met Val His Ser Asn
Ala Leu Asn 145 150 155 160 Glu Asp Thr Gln Asp Glu Leu Gly Asp Pro
Arg Leu Glu Lys Leu Trp 165 170 175 His Lys Ala Lys Thr Ser Gly Lys
Phe Ser Ser Glu Glu Leu Asp Lys 180 185 190 Leu Trp Arg Glu Phe Leu
His Tyr Lys Glu Lys Ile Gln Glu Tyr Asn 195 200 205 Val Leu Leu Asp
Thr Leu Ser Arg Ala Glu Glu Gly Tyr Glu Asn Leu 210 215 220 Leu Ser
Pro Ser Asp Met Ala His Ile Lys Ser Asp Thr Leu Ile Ser 225 230 235
240 Lys His Ser Glu Leu Lys Asp Arg Leu Arg Ser Ile Asn Gln Gly Leu
245 250 255 Asp Arg Leu Arg Lys Val Ser His Gln Gly Tyr Gly Ser Thr
Thr Glu 260 265 270 Phe Glu Glu Pro Arg Val Ile Asp Leu Trp Asp Leu
Ala Gln Ser Ala 275 280 285 Asn Phe Thr Glu Lys Glu Leu Glu Ser Phe
Arg Glu Glu Leu Lys His 290 295 300 Phe Glu Ala Lys Ile Glu Lys His
Asn His Tyr Gln Lys Gln Leu Glu 305 310 315 320 Ile Ser His Gln Lys
Leu Lys His Val Glu Ser Ile Gly Asp Pro Glu 325 330 335 His Ile Ser
Arg Asn Lys Glu Lys Tyr Val Leu Leu Glu Glu Lys Thr 340 345 350 Lys
Glu Leu Gly Tyr Lys Val Lys Lys His Leu Gln Asp Leu Ser Ser 355 360
365 Arg Val Ser Arg Ala Arg His Asn Glu Leu 370 375 23 357 PRT Rat
23 Leu Arg Asp Arg Val Ser Thr Leu Pro Arg Leu Gln Leu Leu Val Leu
1 5 10 15 Leu Leu Leu Pro Leu Leu Leu Val Pro Gln Pro Ile Ala Gly
His Gly 20 25 30 Gly Lys Tyr Ser Arg Glu Lys Asn Glu Pro Glu Met
Ala Ala Lys Arg 35 40 45 Glu Ser Gly Glu Glu Phe Arg Met Glu Lys
Leu Asn Gln Leu Trp Glu 50 55 60 Lys Ala Lys Arg Leu His Leu Ser
Pro Val Arg Leu Ala Glu Leu His 65 70 75 80 Ser Asp Leu Lys Ile Gln
Glu Arg Asp Glu Leu Asn Trp Lys Lys Leu 85 90 95 Lys Val Glu Gly
Leu Asp Gly Asp Gly Glu Lys Glu Ala Lys Leu Val 100 105 110 His Asn
Leu Asn Val Ile Leu Ala Arg Tyr Gly Leu Asp Gly Arg Lys 115 120 125
Asp Thr Gln Thr Val His Ser Asn Ala Leu Asn Glu Asp Thr Gln Asp 130
135 140 Glu Leu Gly Asp Pro Arg Leu Glu Lys Leu Trp His Lys Ala Lys
Thr 145 150 155 160 Ser Gly Lys Phe Ser Ser Glu Glu Leu Asp Lys Leu
Trp Arg Glu Phe 165 170 175 Leu His Tyr Lys Glu Lys Ile His Glu Tyr
Asn Val Leu Leu Asp Thr 180 185 190 Leu Ser Arg Ala Glu Glu Gly Tyr
Glu Asn Leu Leu Ser Pro Ser Asp 195 200 205 Met Thr His Ile Lys Ser
Asp Thr Leu Ala Ser Lys His Ser Glu Leu 210 215 220 Lys Asp Arg Leu
Arg Ser Ile Asn Gln Gly Leu Asp Arg Leu Arg Lys 225 230 235 240 Val
Ser His Gln Gly Tyr Gly Pro Ala Thr Glu Phe Glu Glu Pro Arg 245 250
255 Val Ile Asp Leu Trp Asp Leu Ala Gln Ser Ala Asn Phe Thr Glu Lys
260 265 270 Glu Leu Glu Ser Phe Arg Glu Glu Leu Lys His Phe Glu Ala
Lys Ile 275 280 285 Glu Lys His Asn His Tyr Gln Lys Gln Leu Glu Ile
Ser His Gln Lys 290 295 300 Leu Lys His Val Glu Ser Ile Gly Asp Pro
Glu His Ile Ser Arg Asn 305 310 315 320 Lys Glu Lys Tyr Val Leu Leu
Glu Glu Lys Thr Lys Glu Leu Gly Tyr 325 330 335 Lys Val Lys Lys His
Leu Gln Asp Leu Ser Ser Arg Val Ser Arg Ala 340 345 350 Arg His Asn
Glu Leu 355 24 348 PRT Chicken 24 Met Gly Ala Thr Arg Thr Leu Val
Ala Val Met Ala Ala Phe Leu Ala 1 5 10 15 Val Ser Thr Arg Ala Ser
Lys Tyr Thr Arg Glu Ala Asn Glu Gly Leu 20 25 30 Ala Asp Ala Lys
Arg Arg Glu Ala Gly Glu Phe Arg Val Val Arg Leu 35 40 45 Asn Gln
Val Trp Glu Lys Ala Gln Arg Leu Gln Leu Ser Ala Val Lys 50 55 60
Leu Ala Glu Leu His Ser Asp Leu Lys Ile Gln Glu Lys Asp Glu Leu 65
70 75 80 Ser Trp Lys Lys Leu Lys Ala Glu Gly Leu Gly Glu Asp Gly
Glu Lys 85 90 95 Glu Ala Lys Leu Arg Arg Asn Ile Asn Val Ile Met
Thr Lys Tyr Gly 100 105 110 Met Asn Gly Lys Lys Asp Ser His Leu Thr
Asp Thr Asn Tyr Ile Lys 115 120 125 Asp Gly Thr Glu Ser Asp Thr Leu
Asp Asp Pro Arg Leu Glu Lys Leu 130 135 140 Trp Ser Lys Ala Lys Thr
Ser Gly Lys Phe Ser Asp Glu Glu Leu Asp 145 150 155 160 Lys Leu Trp
Arg Glu Phe Lys His His Lys Glu Lys Ile Arg Glu Tyr 165 170 175 Asn
Ile Leu Leu Glu Thr Val Ser Arg Thr Glu Asp Ile His Lys Lys 180 185
190 Val Ile Asn Pro Ser Glu Glu Asn Pro Val Lys Glu Glu Val Leu His
195 200 205 Asn Lys His Arg Glu Leu Lys Glu Lys Leu Arg Ser Ile Asn
Gln Gly 210 215 220 Phe Glu Arg Leu Arg Lys Val Ser His Gln Gly Tyr
Asp Ala Thr Ser 225 230 235 240 Glu Phe Glu Glu Pro Arg Val Ile Asp
Leu Trp Asp Met Ala Lys Ser 245 250 255 Ala Asn Phe Thr Glu Lys Glu
Leu Glu Ser Phe Arg Glu Glu Leu Lys 260 265 270 His Phe Glu Ala Lys
Ile Glu Lys His His His Tyr Gln Lys Gln Leu 275 280 285 Glu Ile Ser
His Glu Lys Leu Lys His Ile Glu Gly Thr Gly Asp Lys 290 295 300 Glu
His Leu Asn Arg Asn Arg Glu Lys Tyr Ala Met Leu Glu Glu Lys 305 310
315 320 Thr Lys Glu Leu Gly Tyr Lys Val Lys Lys His Leu Gln Asp Leu
Ser 325 330 335 Ser Arg Ile Ser Gln Gly Leu Gln His Asn Glu Leu 340
345 25 331 PRT Zebrafish 25 Met Ala Gly Lys Tyr Ser Lys Glu Met Asn
Glu Lys Asn Ala Ser Asp 1 5 10 15 Lys Ser Asn Asn Gln Val Glu Phe
Arg Ile Ala Lys Leu Asn Gln Val 20 25 30 Trp Glu Lys Ala Ile Arg
Met Gln Leu Ala Pro Val Arg Leu Ser Glu 35 40 45 Leu His Ser Asp
Leu Lys Ile Gln Glu Lys Asp Glu Leu Gln Trp Lys 50 55 60 Lys Leu
Lys Ala Glu Gly Met Asp Glu Asp Gly Glu Arg Glu Ala Lys 65 70 75 80
Leu Arg Arg Asn Phe Asn Ile Ile Leu Ala Lys Tyr Gly Met Asp Gly 85
90 95 Lys Lys Asp Thr Arg Thr Leu Asp Ser Asn Arg Leu Lys Asp His
Glu 100 105 110 Val Lys Ile Gly Asp Thr Phe Asp Asp Pro Lys Leu Asp
Lys Leu Trp 115 120 125 Asn Lys Ala Arg Thr Ser Gly Lys Phe Ser Asp
Glu Glu Leu Gln Thr 130 135 140 Leu His Arg Glu Phe Gln His His Lys
Asp Lys Ile His Glu Tyr Asn 145 150 155 160 Ile Val Met Asp Thr Val
Ser Arg Thr Glu Glu Ile His Lys Asn Val 165 170 175 Ile Ser Pro Leu
Glu Gly Asp Val Lys Glu Asn Val Leu His Gln Lys 180 185 190 His Thr
Asp Leu Lys Gln Arg Met Arg Asp Leu Asn Gln Gly Phe Glu 195 200 205
Arg Leu Arg Lys Ile Thr His Glu Gly Tyr Thr Asp Asp Ser Glu Phe 210
215 220 Arg Glu Pro Arg Val Ile Glu Leu Trp Glu Met Ala Lys Arg Ser
Asn 225 230 235 240 Leu Ser Glu Asp Glu Leu Asp Ser Leu Lys Glu Glu
Leu Arg His Phe 245 250 255 Glu Thr Lys Val Glu Lys His Gln His Tyr
Gln Glu Gln Leu Glu Leu 260 265 270 Ser His Gln Lys Leu Lys His Val
Glu Ala Leu Gly Asp Glu Asp His 275 280 285 Ile Met Arg Asn Lys Glu
Lys Tyr Asn Thr Leu Ala Glu Lys Ala Arg 290 295 300 Glu Met Gly Tyr
Lys Met Lys Lys His Leu Gln Asp Leu Thr Asn Lys 305 310 315 320 Leu
Ser Lys Asn Gly Leu Gln His Asn Glu Leu 325 330 26 379 PRT Fruit
fly 26 Met Val Arg Ser Ala Leu Val Val Ala Ala Ile Ala Leu Ser Val
Leu 1 5 10 15 Ile Ala Leu Gln Gly Val Asp Ala Asp Lys Lys Gln Ser
Lys Lys Tyr 20 25 30 Ser Lys Glu Ala Asn Asp Pro His Phe Gln Gln
Val Lys Gln Glu Lys 35 40 45 Tyr Asp Pro Asp Phe Lys Ser Ile Gln
Arg Pro Phe Arg Met Ala Lys 50 55 60 Leu Asn Leu Val Trp Ala Lys
Ala Gln Asn Arg Leu Thr Glu Pro Lys 65 70 75 80 Leu Lys Ser Leu Tyr
Met Glu Leu Lys Ile His Asp Lys Glu Glu Ile 85 90 95 Ala Trp Lys
Gln Leu Asn Ser Gln His Lys Asp Lys Asp Gly Leu Lys 100 105 110 Ala
Asp Glu Leu Arg Arg Lys Leu Ile Gly Ile Met Ser Ser Tyr Asp 115 120
125 Leu Leu Glu His Phe Asp Asp Thr Gln Asp Thr Glu Lys Leu Lys Pro
130 135 140 Tyr Lys Lys Phe His Asp Ala Glu Glu Arg His Arg Asn Lys
Ser Leu 145 150 155 160 Phe Lys Asp Lys Lys Leu Asn Arg Leu Trp Glu
Lys Ala Glu Ile Ser 165 170 175 Gly Phe Thr Ala Glu Glu Leu Lys Ser
Leu Lys Gln Glu Phe Asp His 180 185 190 His Gln Asp Lys Val Asp Val
Tyr Tyr Ser Leu Leu Glu Asn Ile Gly 195 200 205 Thr Val Asp Thr Asp
Lys His Glu Asn Ala Ile Asn Thr Glu Asp Leu 210 215 220 Asp Thr Tyr
Asn Leu Ile Ser Asn Asp Val Asn Glu Asn Asp Ile Lys 225 230 235 240
Thr His Ala Gln Asn Val Lys Ser Phe Glu Asn Asp Leu Asn Thr Leu 245
250 255 Arg Gly His His Thr Gly Ile Lys Asp His Tyr Asp Arg Leu Glu
Arg 260 265 270 Leu Val Ser Ser Gly Pro His Ser Gln Asp Phe Ile Glu
Pro Lys Val 275 280 285 Gln Gly Leu Trp Arg Val Ala Gln Ala Ser Asn
Phe Thr Val Lys Glu 290 295 300 Leu Glu Ser Ile Lys Thr Glu Leu His
His Phe Glu Ser Arg Leu Leu 305 310 315 320 Lys Leu Arg His Leu His
Ala Glu His Ala Leu Gln Lys Glu Lys Tyr 325 330 335 Lys Gly Glu Lys
Val Lys Asp Lys Ser Ser Arg Phe Glu Glu Met Glu 340 345 350 Asp Gln
Leu Lys Lys Gln Thr Arg Lys Val Glu Lys Leu Gln Glu Asn 355 360 365
Ile Glu Lys Thr Ile Phe Lys His Thr Glu Leu 370 375 27 400 PRT
Mosquito 27 Glu Leu Cys Pro Ile Ala Arg Arg Lys Arg Gly Ile Lys His
Thr Leu 1 5 10 15 Thr Met Pro Leu Phe Thr Arg Leu Cys Val Ile Val
Phe Thr Val Leu 20 25 30 Val Cys Asn His Val Val Gln Ser Glu Lys
Ala His Ser Lys Tyr Ser 35 40 45 Lys His Ala Asn Ala Leu Pro Asp
Ser Glu Ile Tyr Glu Pro Asp Phe 50 55 60 Arg Asn Ile Gln Arg Pro
Phe Arg Met Ala Lys Leu Asn Leu Val Trp 65 70 75 80 Thr Lys Ala Gln
His Arg Leu Thr Glu Pro Lys Leu Lys Ser Leu Tyr 85 90 95 Thr Glu
Leu Lys Leu His Asp Lys Glu Glu Leu Thr Tyr Lys Gln Leu 100 105 110
Lys Glu Lys Asp Lys Asp Gly Leu Lys Glu Ala Glu Leu Arg Asn Lys 115
120 125 Leu Val Ser Ile Met Ser Thr Tyr Gly Leu Leu Glu His Phe Asp
Asp 130 135 140 Thr Gln Asp Pro Glu Lys Tyr Lys Leu Ala Lys Ser Ser
Asp Gly Ala 145 150 155 160 Pro Lys Lys Asp Thr Tyr Lys Asn Lys Ser
Leu Phe Lys Asp Lys Lys 165 170 175 Leu Asn Lys Leu Trp Asp Lys Ala
Glu Ser Ala Gly Phe Thr Lys Glu 180 185 190 Glu Leu Asp Ala Leu Arg
Glu Glu Phe Asp His His Gln Ala Lys Ile 195 200 205 Asp Val Tyr Tyr
Ser Leu Leu
Glu Arg Leu Gly Asp Asp Asp Asp Gly 210 215 220 Gly Ala Ala Gly Gln
Gly Ser Arg Arg Asp Asp Asp Ala Leu Leu Asn 225 230 235 240 Ala Val
Asn Asp Glu Glu His Asp Arg Tyr Asn Glu Val Asp Arg Ala 245 250 255
Glu Glu Thr Asp Arg Ser Gln Pro Gly Ala Asn Lys Gln His Ala Tyr 260
265 270 Leu His Lys Ser Asn Gln Leu Arg Glu Lys His Arg Glu Ile Arg
Asp 275 280 285 Asn Phe Asp Arg Leu Asp Arg Ile Ala Ser Lys Gly Pro
Lys Ser Gln 290 295 300 Asp Phe Val Glu Pro Lys Val Gln Gly Leu Trp
Arg Val Ala Leu Ala 305 310 315 320 Ser Asp Phe Ser Ala Asp Glu Leu
Ala Ser Leu Lys Val Glu Leu Leu 325 330 335 His Tyr Glu Ser Arg Leu
Leu Lys Leu Arg His Met His Ala Glu His 340 345 350 Ala Leu Ser Leu
Glu Lys His Lys His Ser Asp Ala Lys Ala Asp Thr 355 360 365 His Lys
Leu Met Glu Asp Asn Ile Lys Lys Gln Thr Arg Lys Val Glu 370 375 380
Lys Met Gln Glu Glu Val Glu Arg Arg Ile Phe Lys His Ser Glu Leu 385
390 395 400 28 331 PRT Flatworm 28 Met Arg Asn His Phe Ser Phe Leu
Leu Phe Leu Leu Val Ile Gly Ser 1 5 10 15 Ala His Asn Lys Lys Thr
Gln Tyr Arg Thr Glu Arg Ile Asn Phe Ile 20 25 30 Tyr Glu Lys Ala
Leu Gln His Val Thr Asp Arg Gln Asn Leu Ala Arg 35 40 45 Leu Glu
Lys Glu Leu Ser Gly Tyr Asp Ala Ile Tyr Leu Ala Ser Lys 50 55 60
Ser Asn Arg Gln Gly Thr Gln Gly Thr Lys Glu Ile Asp Lys Ile Asp 65
70 75 80 Asp Lys Leu Gly Lys Ile Leu Glu Lys Tyr Gly Leu Glu Lys
Ala Val 85 90 95 Leu Ala Phe Lys Glu Lys Tyr Lys His Lys Asn Leu
Phe Gln Gln Thr 100 105 110 Asp Asp Asn Glu Pro Leu Pro Ser Gly Lys
Phe Thr Asp Gln Asn Leu 115 120 125 Gln Lys Leu Trp Ser Gln Ala Gln
Asn Gly Lys Phe Ser Gln Lys Glu 130 135 140 Leu Asn Ala Leu His Gly
Glu Leu Lys Glu Val Glu Gln Lys Met Arg 145 150 155 160 Val Tyr Glu
Asp Gln Leu Asp Asp Phe Lys Lys Val Pro His Glu Asn 165 170 175 Ser
Ile Gln His Asp Ile Glu Ser Ile Gly Asp Lys Thr Lys Lys Leu 180 185
190 Lys Ala Ala Asn Arg Glu Leu Asn Asp His Leu Asp Glu Val His Arg
195 200 205 Lys Val Thr Ser Glu Glu Phe Ser Pro Phe Asn Glu Pro Arg
Val Lys 210 215 220 Arg Leu Trp Lys Leu Ala Gln Glu Asn Glu Lys Leu
Thr Pro His Glu 225 230 235 240 Leu Ser Val Leu Lys Asp Glu Leu Ser
His Phe Glu Ser Gln Leu Lys 245 250 255 Lys Ile Glu Phe His Lys Val
Phe Phe Phe Val Ala Asn Ser Cys Pro 260 265 270 Lys Arg Gly Lys Asn
Glu Glu Val Ser Arg Leu Gln Glu Asp Ala Glu 275 280 285 Glu Arg Gly
Lys Asp Lys Ser Gln Val Tyr Glu Asn Leu Glu Leu Ser 290 295 300 Ile
Lys His Glu Lys Leu Asn Arg Lys Ala Arg Lys Leu Glu Lys Tyr 305 310
315 320 Ile Glu Glu Lys Ile Ile Ile His Arg Glu Leu 325 330
* * * * *
References