U.S. patent application number 10/501028 was filed with the patent office on 2005-07-21 for use of p97 as an enzyme delivery system for the delivery of therapeutic lysosomal enzymes.
Invention is credited to Starr, Christopher M., Zankel, Todd.
Application Number | 20050158296 10/501028 |
Document ID | / |
Family ID | 23365152 |
Filed Date | 2005-07-21 |
United States Patent
Application |
20050158296 |
Kind Code |
A1 |
Starr, Christopher M. ; et
al. |
July 21, 2005 |
Use of p97 as an enzyme delivery system for the delivery of
therapeutic lysosomal enzymes
Abstract
The present invention provides for compositions and methods for
treating, ameliorating or preventing a lysosomal storage disease by
administering to a patient suffering from a lysosomal storage
disease a P97 conjugated with an enzyme which is capable of
transportation into the lysosomes of cells on either sides of the
blood brain barrier.
Inventors: |
Starr, Christopher M.;
(Sonoma, CA) ; Zankel, Todd; (Novato, CA) |
Correspondence
Address: |
MARSHALL, GERSTEIN & BORUN LLP
233 S. WACKER DRIVE, SUITE 6300
SEARS TOWER
CHICAGO
IL
60606
US
|
Family ID: |
23365152 |
Appl. No.: |
10/501028 |
Filed: |
March 14, 2005 |
PCT Filed: |
January 10, 2003 |
PCT NO: |
PCT/US03/00894 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
|
60347758 |
Jan 11, 2002 |
|
|
|
Current U.S.
Class: |
424/94.4 |
Current CPC
Class: |
A61K 38/47 20130101;
G01N 33/566 20130101; A61K 47/64 20170801; C12Y 302/01076 20130101;
G01N 33/5058 20130101; G01N 33/5008 20130101; G01N 33/5035
20130101; A61K 38/465 20130101; A61P 3/00 20180101; A61K 38/47
20130101; A61K 2300/00 20130101 |
Class at
Publication: |
424/094.4 |
International
Class: |
A61K 038/44 |
Claims
What is claimed is:
1. A method for treating a subject having a lysosomal storage
disease, said method comprising administering a pharmaceutical
composition to the subject wherein the composition comprises a p97
molecule covalently linked to a protein whose deficiency causes the
disease.
2. The method of claim 1, wherein the subject is human.
3. The method of claim 1, wherein the administering is
intravenous.
4. The method of claim 1, wherein the p97 molecule is human
p97.
5. The method of claim 1, wherein the p97 molecule is soluble
p97.
6. The method of claim 1, wherein the protein is
.alpha.-L-iduronidase.
7. The method of claim 1, wherein the p97 molecule is covalently
linked to the protein by a linker from 5 to 20 atoms in length.
8. The method of claim 1, wherein the linker is a polyethylene
glycol.
9. The method of claim 1, wherein the conjugate is a fusion protein
of p97 and the protein.
10. The method of claim 1, wherein the p97 molecule has as sequence
which is 90% identical to the sequence of a corresponding domain of
human p97.
11. The method of claim 1, wherein the composition comprises the
conjugate in a therapeutically effective amount.
12. The method of claim 1, wherein the 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 I/II, Gaucher disease types I/II/III,
Gaucher disease, globoid cell leucodystrophy, 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
leucodystrophy, 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 sulphatase
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 1, wherein the protein 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,
arylsulphatase A, saposin B, neuraminidase,
.alpha.-N-acetylglucosaminidase phosphotransferase,
phosphotransferase .gamma.-subunit, L-iduronidase,
iduronate-2-sulphatase, heparan-N-sulphatase,
.alpha.-N-acetylglucosaminidase, acetylCoA:N-acetyltransferase,
N-acetylglucosamine 6-sulphatase, galactose 6-sulphatase,
.beta.-galactosidase, N-acetylgalactosamine 4-sulphatase,
hyaluronoglucosaminidase, multiple sulphatases, palmitoyl protein
thioesterase, tripeptidyl peptidase I, acid sphingomyelinase,
cholesterol trafficking, cathepsin K, .alpha.-galactosidase B, and
sialic acid transporter.
14. A compound comprising a p97 molecule covalently linked to a
protein whose deficiency causes a lysosomal storage disease.
15. The compound of claim 14, wherein the protein is
.alpha.-L-iduronidase.
16. The compound of claim 14, wherein the p97 molecule is soluble
p97.
17. The compound of claim 14, wherein the compound is a fusion
protein of the p97 molecule and the protein.
18. The compound of claim 14, wherein the p97 molecule is
covalently linked to the protein by a linking group which is 4-20
atoms in length.
19. The compound of claim 14, wherein the conjugate is capable of
passing through the blood-brain barrier and entering a lysosome of
a cell within the central nervous system.
20. The compound of claim 14, wherein the protein 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, arylsulphatase A, saposin B, neuraminidase,
.alpha.-N-acetylglucosaminidase phosphotransferase,
phosphotransferase .gamma.-subunit, L-iduronidase,
iduronate-2-sulphatase, heparan-N-sulphatase,
.alpha.-N-acetylglucosamini- dase, acetylCoA:N-acetyltransferase,
N-acetylglucosamine 6-sulphatase, galactose 6-sulphatase,
.beta.-galactosidase, N-acetylgalactosamine 4-sulphatase,
hyaluronoglucosaminidase, multiple sulphatases, palmitoyl protein
thioesterase, tripeptidyl peptidase I, acid sphingomyelinase,
cholesterol trafficking, cathepsin K, .alpha.-galactosidase B, and
sialic acid transporter.
21. A method of screening a compound for therapeutic activity in
treating a lysosomal storage disease, said method comprising:
contacting a cell having a lysosome with the compound, wherein the
compound comprises p97 covalently linked to a protein deficient in
a lysosomal storage disease; and monitoring delivery of the
compound to the lysosome.
22. The method of claim 21, wherein the compound is labeled and the
monitoring detects the label.
23. The method of claim 21, wherein the cell is human.
24. The method of claim 21, wherein the cell is deficient in the
protein.
25. The method of claim 21, wherein the monitoring is by
determining the effect of the compound on the lysosomal storage
material.
26. The method of claim 21, wherein the cell is not protected by
the blood brain barrier.
27. A pharmaceutical composition comprising a therapeutically
effective amount of compound comprising a p97 molecule covalently
linked to a protein whose deficiency causes a lysosomal storage
disease and a pharmaceutically acceptable excipient.
28. The composition of claim 27, wherein the composition is in unit
dosage format.
29. The composition of claim 27, wherein the protein 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, arylsulphatase A, saposin B, neuraminidase,
.alpha.-N-acetylglucosaminidase phosphotransferase,
phosphotransferase .gamma.-subunit, L-iduronidase,
iduronate-2-sulphatase, heparan-N-sulphatase,
.alpha.-N-acetylglucosamini- dase, acetylCoA:N-acetyltransferase,
N-acetylglucosamine 6-sulphatase, galactose 6-sulphatase,
.beta.-galactosidase, N-acetylgalactosamine 4-sulphatase,
hyaluronoglucosaminidase, multiple sulphatases, palmitoyl protein
thioesterase, tripeptidyl peptidase I, acid sphingomyelinase,
cholesterol trafficking, cathepsin K, .alpha.-galactosidase B, and
sialic acid transporter.
Description
CROSS-REFERENCES TO RELATED APPLICATIONS
[0001] This application claims priority benefit of U.S. Provisional
Application No. 60/347,758 filed on Jan. 11, 2002 which is
incorporated herein by reference in its entirety.
FIELD OF THE INVENTION
[0002] The present invention is related to the field of
pharmaceutics and specifically to treatment of diseases through the
introduction of p97 protein or polypeptide conjugated to a
therapeutic or diagnostic agent to a subject. In particular, this
invention relates to conjugates of p97 and proteins deficient in a
lysosomal storage disease and methods for the treatment,
amelioration or prevention of lysosomal storage diseases by
administration of the conjugates to subjects having such
diseases.
BACKGROUND OF THE INVENTION
[0003] Lysosomal storage diseases (LSDs) result from the absence or
reduced activity of specific enzymes within the lysosomes of a
cell. A large number of these enzymes have been identified and
correlated with their related diseases. Once the missing or
deficient enzyme has been identified, treatment can be reduced to
the problem of delivering replacement enzyme (drug) to a patient's
affected tissues. Within cells, the effect of the missing enzyme
can be seen as an accumulation of undegraded "storage material"
within the intracellular lysosome. This build-up causes lysosomes
to swell and malfunction, resulting in cellular and tissue damage.
As lysosomal storage diseases typically have a genetic etiology,
many tissues will lack the enzyme in question. However, different
tissues suffer the absence of the same enzyme differently. How
adversely a tissue will be affected is determined, to some extent,
by the degree to which that tissue generates the substrate of the
missing enzyme. The types of tissue most burdened by storage, in
turn, dictate how the drug should be administered to the patient.
While intravenous enzyme replacement therapy (ERT) is beneficial
for LSDs (e.g. MPS I, MPS II), means for enhancing the delivery of
the therapeutic enzyme to the lysosome in such diseases would be
advantageous in terms of reduced cost and increased therapeutic
efficacy.
[0004] In addition, the blood-brain barrier (BBB) blocks the free
transfer of many agents from blood to brain. For this reason, LSDs
that present with significant neurological affect (e.g. MPS III,
MLD, GM1) are not expected to be as responsive to intravenous ERT.
For such diseases, a method of delivering the enzyme across the BBB
and into the lysosomes of the affected cells would be highly
desirable.
[0005] In the early 1980's, melanotransferrin (MTf) or p97 was
identified as an oncofetal antigen that was either not expressed,
or only slightly expressed in normal tissues, but was found in much
larger amounts in neoplastic cells (especially malignant melanoma
cells) and fetal tissues (Woodbury, et al., P.N.A.S. USA,
77:2183-2187 (1980)). More recently, there have been additional
reports of human MTf being identified in normal tissues, including
sweat gland ducts, liver endothelial cells and the endothelium and
reactive microglia of the brain (Jefferies, et al., Brain Res.,
712:122-126 (1996); and Rothenberger, et al., Brain Res.,
712:117-121 (1996)). Interestingly, normal serum contains very low
levels of soluble circulating MTf, but increased soluble serum MTf
has been found in patients with advanced Alzheimer's Disease
(Kennard, et al., Nat. Med., 2:1230-1235 (1996); U.S. Pat. No.
5,981,194).
[0006] The biochemical role and metabolism of MTf has proven
difficult to elucidate. Based on appearances, MFt is deceptively
similar to transferrin (Tf) and lactotransferrin (lactoferrin or
Lf). In humans, these proteins share a 37-39% amino acid sequence
homology. In particular, each of these proteins reversibly binds
iron, and their N-terminal iron binding domains are quite similar
(Baker, et al., TIBS, 12:350-353 (1987)).
[0007] However, functional parallels between these proteins have
not been confirmed. For one thing, unlike Tf and Lf, MTf exists in
both a membrane bound form and a serum soluble form. Further, in
contrast to Tf and Lf, no cellular receptor for MTf has been
identified. Serum soluble Tf is known to be taken into cells in an
energy-dependent process mediated by the transferrin receptor
(Tf-R) (Cook, et al., Annu. Rev. Med., 44:63-74) (1993)). Lf
internalization is also likely to be mediated by a receptor
mediated process (Fillebeen, et al., J. Biol. Chem.,
274(11):701-7017 (1999)). Two known receptors for Lf are LRP-1 and
RAGE, although others may exist (Melinger, et al., FEBS Letters,
360:70-74 (1995); Schmidt, J. Biol. Chem., 269(13):9882-9888
(1994).
[0008] With respect to the central nervous system (e.g., brain,
spinal cord), there are at least three ways to enhance delivery:
direct injection, permeabilization of the BBB, and modification of
the drug. Direct injection involves injection of drug into brain
tissue, bypassing the vasculature completely. This method suffers
primarily from the risk of complications (infection, tissue damage)
incurred by intra-cranial injections. This risk is compounded when
considered in the context of a regular treatment regimen applied
over the course of to the patient's life. It is also difficult,
using a limited number of single site injections, to match the
penetration that blood vessels (and hence, potentially, drug) have
throughout the brain.
[0009] The second method entails non-specifically compromising the
BBB with concomitant injection of intravenous drug.
Permeabilization of the BBB is accomplished chemically. This method
suffers from a lack of specificity. All those components in the
blood that are necessarily excluded by the BBB will enter the brain
along with the drug. The brain is left vulnerable under these
conditions and damage would be anticipated over the course of a
life-long regimen of treatment.
[0010] The third means of increasing brain availability of blood
borne drug entails specific functionalization of the drug with
moieties that facilitate transport through an uncompromised BBB.
This method has the advantages of specific BBB infiltration and
convenient intravenous administration. A method of increasing the
ability of a therapeutic agent to cross the blood brain barrier is
taught in U.S. Pat. No. 6,455,494, incorporated herein by reference
its entirety, which discloses the use of p97 as a carrier for
delivering a therapeutic drug across the blood brain barrier.
[0011] p97 (melanotransferrin) is a naturally occurring human
protein. p97 was discovered and characterized as a cell surface
marker for human melanoma (melanoma-associated antigen), but has
more recently been found in other tumor types, as well as in normal
human brain and liver tissue, in trace amounts in other body
tissues and in serum. The role of p97 in the body is unknown, but
based on its structure and binding properties, it is thought to be
involved in the transport of metal ions (e.g. iron) into cells.
Jefferies, et al. have been working with p97 since 1992 (U.S. Pat.
No. 5,981,194). These investigations have focused on p97 as a
diagnostic marker for Alzheimer's disease (AD). Synapse has
developed a blood (serum) test for AD that is based on the finding
that the p97 serum level increases with the progression of the
disease. During the development of this test, it was discovered
that p97 is actively transported from the blood into the brain
tissue of normal individuals. This discovery was the impetus for
the development of p97 as a potential transport system to deliver
molecules from the blood, across the BBB to reach brain
interstitial fluids.
[0012] The key event for the successful delivery of therapeutic
agents into brain is the transport of these large molecules across
the tight network of capillary endothelial cells that comprise the
BBB. During the last few years, it has been demonstrated both in
vitro and in animal models that small synthetic molecules, large
glycoprotein enzymes, and large inorganic particles (5 nm colloidal
gold particles) chemically linked to p97, can be transported across
the BBB to brain cells. Such transport of large molecules across
the BBB involves a process known as transcytosis. This is a
mechanism whereby molecules are picked up from the blood and
transported through the capillary cells of the otherwise intact BBB
to the brain tissue.
[0013] Transcytosis pathways are distinct from other vesicular
traffic within the capillary endothelial cell and transit occurs
without alteration of the transported materials. Transcytosis is a
cell-type specific process mediated by receptors on the BBB
endothelial surface. The transport of p97-conjugates (i.e., p97
chemically linked to macromolecules) across the BBB occurs by
transcytosis. p97 conjugated to the enzyme horseradish peroxidase
(HRP) (an example of an enzyme "payload") can be transported across
the BBB.
[0014] For an effective treatment of LSDs, a therapeutic agent
(e.g., the deficient enzyme, or another enzyme or protein having a
desired therapeutic or missing enzyme activity) must be taken up by
the affected cells and routed to the lysosome where it acts upon
the excessive or harmful amount of storage material residing
therein. Applicants provide below compositions and methods for
treating LSDs involving the use of p97 proteins to target delivery
of therapeutic agents, including proteins or enzymes deficient in
LSDs, to the lysosomes of cells.
BRIEF SUMMARY OF THE INVENTION
[0015] The present invention is related to the discovery that
conjugates of p97 and a therapeutic agent, in which the agent is
covalently linked to p97, or to a fragment or protion thereof, are
excellent vehicles for the enhanced delivery of the agent to
lysosomes of cells within and without the CNS. In a first aspect,
therefore the invention provides a method of delivering a
therapeutic agent to the lysosome of a cell. In a second aspect the
invention provides a method of treating a lysosomal storage disease
in a patient by administering a p97 molecule covalently linked 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 p97-agent conjugates are particularly
useftil, 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 a third aspect, the invention
provides pharmaceutical compositions comprising a p97 molecule
covalently linked to a protein or enzyme deficient in a lysosomal
storage disease. In a fourth aspect, the invention provides methods
for identifying p97 conjugates which are useful in delivering an
agent to a lysosome.
[0016] In some embodiments, the 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/Farber disease, Fucosidosis, Galactosialidosis
types I/II, Gaucher disease types I/IIIII Gaucher disease, Globoid
cell leucodystrophy/Krabbe disease, Glycogen storage disease
II/Pompe disease, GM1-Gangliosidosis types I/III,
GM2-Gangliosidosis type I/Tay Sachs disease, GM2-Gangliosidosis
type II Sandhoff disease, GM2-Gangliosidosis, .alpha.-Mannosidosis
types I/II, .beta.-Mannosidosis, Metachromatic leucodystrophy,
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 sulphatase
deficiency, 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.
[0017] In some embodiments, the p97 conjugates compositions
comprise from about 1 to about 5 molecules of the agent of interest
linked to a single p97 molecule. In some embodiments, more that one
agent of interest may be linked to a single p97 molecule. Selective
biodistribution of p97-agents can enhance the selective targeting
of p97-linked agents to specific organs.
[0018] In addition, the present invention provides screening assays
for identifying p97-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 p97 molecule.
[0019] In some embodiments, the method treats lysosomal storage
diseases wherein the tissues to be treated are isolated from the
circulatory system by the blood brain barrier (e.g., the brain). In
one embodiment, the present invention provides a method for
delivering a compound of interest through the blood-brain barrier
of a subject into a lysosome of a cell of the subject comprising:
administering a conjugated agent to the subject, wherein the
conjugated agent comprises a melanotransferrin linked to the agent
via a linker; whereby the conjugated agent passes through the
blood-brain barrier of the subject, and whereby the agent enters
into a lysosome of a cell of the subject.
[0020] In another embodiment, the present invention provides for a
method for treating, ameliorating, or preventing a lysosomal
storage disease of a subject comprising: administering a conjugated
agent to a subject, wherein the conjugated agent comprises a
melanotransferrin linked to an agent of interest via a linker;
whereby the agent enters into a lysosome of a peripheral cell
(e.g., non-CNS cell) of the subject.
[0021] In still another embodiment, the present invention provides
for a method of enzyme replacement therapy comprising:
administering a conjugated agent to a subject in need of the enzyme
replacement therapy, wherein the conjugated agent comprises a
melanotransferrin 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 the conjugated agent passes through the blood-brain barrier
of the subject, and whereby sufficient amounts of the enzyme enter
the lysosomes to prevent or reduce damage to the cells.
[0022] In even another embodiment, the present invention provides a
method for treating a patient suffering from a lysosomal storage
disease resulting from the absence of an enzyme within the
lysosomes of a cell found in the brain comprising: administering to
the patient a conjugated agent, wherein the conjugated agent
comprises p97 linked to the enzyme via a linker; whereby the
conjugated agent passes through the blood-brain barrier of the
patient, and whereby sufficient amounts of the enzyme enter the
lysosomes to prevent or reduce damage to the cells.
[0023] In yet even another embodiment, the present invention
provides for a method for identifying an agent that can prevent,
ameliorate or treat a lysosomal storage disease, comprising:
administering a p97-conjugated agent 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.
[0024] In a further embodiment, the present invention provides for
a novel composition comprising a conjugated agent, wherein the
conjugated agent comprises a p97 molecule linked to an enzyme via a
linker, wherein the enzyme is an enzyme, such as those set forth
herein, found in the lysosomes of cells contained within the BBB.
The composition can further comprise a suitable pharmaceutical
carrier.
[0025] Further, the present invention provides a lysosome
comprising the conjugated agent. The present invention also
provides a cell comprising a lysosome comprising the conjugated
agent. Preferably, the cell is a cell found surrounded by a
blood-brain barrier. More preferably, the cell is a neuron or a
brain cell.
[0026] In some embodiments, the p97-agent conjugate comprises any
one of the following proteins as the active agent covalently linked
to a p97 molecule: 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,
arylsulphatase A, saposin B, neuraminidase,
.alpha.-N-acetylglucosaminidase phosphotransferase,
phosphotransferase .gamma.-subunit, L-iduronidase,
iduronate-2-sulphatase, heparan-N-sulphatase,
.alpha.-N-acetylglucosaminidase, acetylCoA:N-acetyltransferase,
N-acetylglucosamine 6-sulphatase, galactose 6-sulphatase,
.beta.-galactosidase, N-acetylgalactosamine 4-sulphatase,
hyaluronoglucosaminidase, multiple sulphatases, palmitoyl protein
thioesterase, tripeptidyl peptidase I, acid sphingomyelinase,
cholesterol trafficking, cathepsin K, .alpha.-galactosidase B,
sialic acid transporter. In some embodiments, the agent is a
protein of human or mammalian sequence, origin or derivation. The
p97 protein or fragment can also be of human or mammalian sequence,
origin or derivation.
BRIEF DESCRIPTION OF THE DRAWINGS
[0027] FIG. 1A-1D depict human hNT neurons (derived from human
teratocarcinoma) stained with anti-Cathepsin L and the L235
monoclonal antibody to p97 (see, Example 1 for details).
[0028] FIGS. 2A-2C depict live neurons fed with AlexaFluor 594-p97
and lysosensor green (see, Example 2 for details).
DETAILED DESCRIPTION OF THE INVENTION
[0029] I. Introduction
[0030] The present invention generally provides methods and
compositions for enhanced delivery of the lysosomal storage disease
related agents to the lysosomes of cells affected by a lysosomal
storage disease. The invention relates to the surprising discovery
that MTf or p97 proteins and fragments thereof not only undergo
transcytosis across the blood brain barrier but also are
transported into lysosomes. As a result, p97 molecules which are
demonstrated vehicles for delivering agents across the blood brain
barrier are also surprisingly particularly useful also as a means
of delivering deficient proteins or enzymes to lysosomes for the
treatment of lysosomal storage diseases resulting from such
deficiencies.
[0031] The conjugate agents of the present invention offer many
important advantages in the treatment of lysosomal storage
diseases. The MTf or p97 is a natural compound usually found in
human cells at different levels. Because p97 is a natural protein
of humans, it is unlikely to result in immunological
hyper-responsiveness, as it is frequently the case with the use of
Mab therapies making them is refractory for use in repeated
injections.
[0032] In terms of drug delivery, the p97 system can cross the BBB
quickly (within an hour), and is metabolized within 12 hours, thus
it is efficiently eliminated from tissues. These features provide
the chance of repetitive injections without saturation of the
receptors. Moreover, p97 does not compete with endogenous
transferrin whose amount is estimated to be 10,000 times higher
than serum p97. Thus transferring will not compete with the method
of delivering therapeutic agents by covalently or otherwise linking
them to p97. The protein p97 shows little toxicity if any. The p97
protein is biodegradable and will not circulate for long periods of
time. The protein has a demonstrated capacity to cross the BBB and
due to its affinity for the endothelial cells lining the vascular
bed of the brain, p97 vector is particularly useful for delivering
a conjugated or otherwise bound agent to lysosomes located within
the brain tissue.
[0033] II. Definitions
[0034] 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. 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.
[0035] 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.
[0036] MTf or p97 includes membrane bound p97 (i.e., p97 attached
to a GPI anchor or some other anchor), secreted p97, soluble p97,
cleaved p97, analogs of p97 which are functional equivalents of p97
(having generally greater than 40%, 60%, 80%, 90% or 95% homology
of their corresponding amino acid sequences and including allelic
variants of p97), human, mouse, chicken and/or rabbit p97, and
derivatives, portions, or fragments thereof. The p97 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 p97. Due to code degeneracy, for
example, there may be considerable variation in nucleotide
sequences encoding the same amino acid sequence. Further
characterization of p97, including the complete amino acid sequence
of p97, is found in U.S. Pat. No. 5,981,194.
[0037] A p97 fragment as used herein includes any portion of p97 or
its biologically equivalent analogs that contains a sufficient
portion of p97 to enable it to bind to the MTf or the LRP1 or LRP1B
receptor and to be transported across the blood-barrier; or that
otherwise retains or improves upon the desired biological
activities of p97 in the transcytosis and/or delivery of an agent
to the lysosome.
[0038] Additional teachings regarding the making and using of p97
as diagnostic and therapeutic agents is found in International
Application No. PCT/CA93/00272 and U.S. Pat. No. 5,981,194, the
disclosures of which are incorporated by reference in their
entirety.
[0039] "Modulate," as used herein, refers to the ability to alter,
by increase or decrease (e.g., to act as an antagonist or
agonist).
[0040] "Melanotransferrin receptor" ("MTf-R"), as used herein,
refers to any biological system that specifically or preferentially
binds MTf. This term is intended to include those receptors which
competitively bind Lf and/or .beta.-amyloid protein, but excludes
those receptors which are specific for Tf such as the transferrin
receptor (Tf-R) (which is described at OMIM #*190010, and which is
also known as TFR, TRFR and CD71). A receptor known to specifically
or preferentially bind Lf is herein called a "lactotransferrin
receptor" (Lf-R). Known Lf-Rs include, but are not limited to, the
LDL-related receptors. A known LDL-related receptor is lipoprotein
receptor-related protein/alpha2-macroglobulin receptor ("LRP1").
The term MTf-R specifically includes other receptors found on
endothelial cells that specifically bind both MTf and Lf, but not
Tf. In a preferred embodiment, the MTf-R is the LRP1. In a more
preferred embodiment, the MTf-R is LRP1B.
[0041] 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. Characeristic features of the LDL-R family include
cell-surface expression; extracellular ligand binding domain
repeats (DxSDE); requirement of Ca++ for ligand binding;
recognition of RAP and ApoE; EGF precursor homology domain repeats
(YWTD); single membrane spaning region; internalization signal in
the cytoplasmic domain (FDNPXY); and receptor mediated endocytosis
of various ligands.
[0042] LRP refers to the low density lipoprotein receptor related
protein and members of this receptor family. LRP is a large protein
of 4525 amino acids (600 kDa) which is cleaved by furin to produce
two subunits of 515-(.alpha.) and 85-(.beta.) kDa that remain
non-covalently bound. LRP is mainly expressed in the liver, kidney,
neuron, CNS, BBB, SMC and various cultured cells.
[0043] 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, MPP-9-TIMP-1;
uPA:PAI-I:uPAR; and tPA:PAI-1:uPAR.
[0044] LRP 1B is a recently discovered member of the low density
lipoprotein receptor family. 600 kDa multifunctional cell surface
receptor. See Liu et al., J. Biol. Chem. 276 (31):28889-28896
(2001). See also Liu et al., Genomics 69, 271-274 (2000); and Liu
et al., Cancer Res. 60, 1961-1967 (2000). This receptor is more
closely related to LRP than megalin and shares a 59% homology at
cDNA level and a 52% homology at predicted amino acid level. the
LRP 1B gene is expressed in the brain, thyroid and salivary gland.
Known ligands for LRP 1B include RAP, tPA, PAI-1.
[0045] Mouse LRP1B is accessible through GenBank Accession Nos. XM
143023 XM 130241. Human LRP1B is accessible through GenBank
Accession Nos. XM 015452.
[0046] "Lipoprotein receptor-related protein/alpha2-macroglobulin
receptor" ("LRP1"), as used herein, refers to a multifunctional
receptor. It is believed that the clustering of cysteine-rich type.
A binding repeats, 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: activated
alpha-2-macroglobulin, apolipoprotein E, lipoprotein lipase,
plasminogen activators and complexes with their inhibitor (PA and
PA/PAI-1), lipoprotein(a), pseudomonas exotoxin A, human
rhinovirus, Lf and the so-called receptor associated protein (RAP).
See, Meilinger, et al., FEBS Let., 360:70-74 (1995). LRP1 is
accessible through GenBank Accession No.: X 13916 and Swiss-Prot
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.
[0047] A "p97-conjugate" refers to a composition comprising p97, or
a fragment thereof, covalently conjugated to an agent. The
conjugation may be direct or indirect (i.e., through an extended
linker) so long as it is a chemical conjugation. The general
construct of the composition of the invention is shown in FIG.
3.
[0048] The agent may serve a therapeutic or prophylactic purpose in
the treatment of a lysosomal storage disease in mammals, and
particularly, humans.
[0049] "Increasing relative delivery" as used herein refers to the
effect whereby the accumulation at the lysosome of a p97-conjugated
agent is increased relative to the accumulation of the original
agent.
[0050] "Therapeutic index" refers to the dose range (amount and/or
timing) above the minimum therapeutic amount and below an
unacceptably toxic amount.
[0051] "Equivalent dose" refers to a dose which contains the same
amount of active agent.
[0052] The p97-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
p97. For example, conjugation reactions may conjugate from 1 to 4
or more molecules of alpha-L-iduronidase to a single p97 molecule.
These formulations can be employed as mixtures, or they may be
purified into specific p97:agent (mol:mol) formulations. Those
skilled in the art are able to determine which format and which
mol:mol ratio is preferred. Further, mixtures of agents may be
linked to p97 to facilitate a more complete degradation of the
stored substrates. These p97-agents may consist of a range of
mol:mol ratios. These, too, may be separated into purified mixtures
or they may be employed in aggregate.
[0053] The p97 conjugated 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 a MTf receptor
activity. Methods for identifying or determining such a modulator
are disclosed in U.S. Provisional Patent Application No. 60/308,002
and U.S. patent application Ser. No. 10/206,448, filed on Jul. 25,
2002, the disclosures of which are incorporated by reference in
their entirety. 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 which is not isolated from the
general circulation by an endothelium such as that of the BBB.
[0054] The agent can be a protein or enzyme or any fragment of such
that still retains some, substantially all, or all of the activity
of the 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 lysosomal storage
disease. Preferably, the protein or enzyme is derived or obtained
from a human or mouse. In some embodiments, in the treatment of
human LSDs, the p97-agent conjugate comprises a protein or enzyme
that is deficient in the lysosomal storage enzyme deficient in
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 follows:
1 Lysosomal Storage Disease Protein deficiency
Mucopolysaccharidosis type I L-Iduronidase Mucopolysaccharidosis
type II Hunter syndrome Iduronate-2-sulphatase
Mucopolysaccharidosis type IIIA Sanfilippo syndrome
Heparan-N-sulphatase Mucopolysaccharidosis type IIIB Sanfilippo
syndrome .alpha.-N-Acetylglucosaminidase Mucopolysaccharidosis type
IIIC Sanfilippo syndrome AcetylCoA: N-acetyltransferase
Mucopolysaccharidosis type IIID Sanfilippo syndrome
N-Acetylglucosamine 6-sulphatase Mucopolysaccharidosis type IVA
Morquio syndrome Galactose 6-sulphatase Mucopolysaccharidosis type
IVB Morquio syndrome .beta.-Galactosidase Mucopolysaccharidosis
type VI N-Acetylgalactosamine 4-sulphatase Mucopolysaccharidosis
type VII Sly syndrome .beta.-Glucuronidase Mucopolysaccharidosis
type IX hyaluronoglucosaminidase Aspartylglucosaminuria
Aspartylglucosaminidase Cholesterol ester storage disease/Wolman
disease Acid lipase 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
leucodystrophy/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 disease .beta.-Hexosaminidase A GM2-Gangliosidosis type II
Sandhoff disease .beta.-Hexosaminidase A GM2-Gangliosidosis
GM2-activator deficiency .alpha.-Mannosidosis types I/II
.alpha.-D-Mannosidase .beta.-Mannosidosis .beta.-D-Mannosidase
Metachromatic leucodystrophy Arylsulphatase A Metachromatic
leucodystrophy Saposin B Mucolipidosis type I/Sialidosis types I/II
Neuraminidase Mucolipidosis types II/III I-cell disease
Phosphotransferase Mucolipidosis type IIIC pseudo-Hurler
polydystrophy Phosphotransferase .gamma.-subunit Multiple
sulphatase deficiency Multiple sulphatases Neuronal Ceroid
Lipofuscinosis, CLN1 Batten disease Palmitoyl protein thioesterase
Neuronal Ceroid Lipofuscinosis, CLN2 Batten disease Tripeptidyl
peptidase I Niemann-Pick disease types A/B Niemann-Pick disease
Acid sphingomyelinase Niemann-Pick disease type C1 Niemann-Pick
disease Cholesterol trafficking Niemann-Pick disease type C2
Niemann-Pick disease Cholesterol trafficking Pycnodysostosis
Cathepsin K Schindler disease types I/II Schindler disease
.alpha.-Galactosidase B Sialic acid storage disease sialic acid
transporter
[0055] The melanotransferrin or p97 and the agent are conjugated
directly or indirectly to each other (i.e., through an extended
linker). The linker can comprise a covalent bond or a peptide of
virtually any amino acid sequence or any molecule capable of
conjugating melanotransferrin or p97 and the agent. If the linker
is a covalent bond or a peptide, 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.
[0056] The p97-enzyme conjugate according to the invention may be
modified as desired to enhance its stability or pharmacokinetic
properties (e.g., PEGylation).
[0057] III. Compositions and Preparation Thereof
[0058] In general, p97-conjugates may be prepared using techniques
well known in the art. There are numerous approaches for the
conjugation or chemical crosslinking of agents to a polypeptide
such as p97, and one skilled in the art can determine which method
is most appropriate for conjugating a particular agent. The method
employed must be capable of joining the agent with p97 without
interfering with the ability of p97 to bind to its receptor,
preferably without influencing the biodistribution of the p97-agent
compared to p97 alone, and/or without significantly altering the
desired activity of the agent (be it therapeutic or prophylactic or
the like) once delivered. Preferred methods of conjugating p97 to
various agents are described in the examples section, below. A
particularly preferred method for linking complex molecules to p97
is the SATA/sulfo-SMCC cross-linking reaction (Pierce (Rockford,
Ill.)).
[0059] Methods of cross linking 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 p97 or
with a substance which binds p97 (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 p97
molecule. See, for instance, Hermanson, G T 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
p97 and the agent. In one embodiment, p97 and the protein
therapeutic agents may be conjugated by the introduction of a
sulfhydryl group on p97 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 p97 and/or
linker.
[0060] In some embodiments of the present invention, the p97-agent
conjugate is a p97-fusion protein. Fusion proteins may be prepared
using standard techniques known in the art. Typically, a DNA
molecule encoding p97 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 p97 or a portion thereof used to the
selected protein compound.
[0061] 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.
[0062] 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 which may be substituted by
heteroatoms independently selected from the group consisting of O,
N, or S. In some embodiments, from 1-4 or from 5 to 15 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 p97 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 p97 carrier). In some embodiments, the p97 and
agent are joined by a linker comprising amino acids or peptides
lipids, or sugar residues. In some embodiments, the p97 and agent
are joined at groups introduced synthetically or by
posttranslational modifications.
[0063] 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 p97
or a maleimide that can react with the free thiols created on p97
via a SATA reaction or through other groups where persons skilled
in the art can attach them to p97.
[0064] A. Preparation of p97
[0065] The p97 peptide or molecule for use in the methods and
compositions of the present invention may be obtained, isolated or
prepared from a variety of sources.
[0066] In one aspect, standard recombinant DNA techniques may be
used to prepare p97 or derivatives thereof. Within one embodiment,
DNA encoding p97 may be obtained by polymerase chain reaction (PCR)
amplification of the p97 sequence (see, generally, U.S. Pat. Nos.
4,683,202; 4,683,195; and 4,800,159; see, also, PCR Technology:
Principles and Applications for DNA Amplification, Erlich (ed.),
Stockton Press (1989)). Briefly, double-stranded DNA from cells
which express p97 (e.g., SK-MEL-28 cells) is denatured by heating
in the presence of heat stable Taq polymerase, sequence specific
DNA primers such as 5' GCGGACTTCCTCGG 3' (SEQ ID NO: 1) and 5'
TCGCGAGCTTCCT 3' (SEQ ID NO:2), ATP, CTP, GTP and TTP.
Double-stranded DNA is produced when the synthesis is complete.
This cycle may be repeated many times, resulting in a factorial
amplification of p97 DNA. The amplified p97 DNA may then be readily
inserted into an expression vector as described below.
[0067] Alternatively, DNA encoding p97 may be isolated using the
cloning techniques described by Brown et al. in the UK Patent
Application No. GB 2188 637. Clones which contain sequences
encoding p97 cDNA have been deposited with the American Type
Culture Collection (ATCC) under deposit numbers CRL 8985 (PMTp97b)
and CRL 9304 (pSVp97a).
[0068] Within one embodiment of the present invention, truncated
derivatives of p97 are provided. For example, site-directed
mutagenesis may be performed with the oligonucleotide WJ31
5'CTCAGAGGGCCGCTGCGCCC-3'(- SEQ ID NO:3) in order to delete the
C-terminal hydrophobic domain beyond nucleotide 2219, or with the
oligonucleotide WJ32 5' CCA GCG CAG CTAGCGGGGGCAG 3' (SEQ ID NO:4)
in order to introduce an Nhe I site and a STOP codon in the region
of nucleotides 1146-1166, and thereby also constructing a truncated
form of p97 comprising only the N-terminal domain. Similarly,
mutagenesis may also be performed on p97 such that only the
C-terminal domain is expressed. Within one embodiment, Xho sites
are inserted by mutagenesis with the oligonucleotide WJ
5'-ACACCAGCGCAGCTCGAGGGGCAGCCG 3' (SEQ ID NO:5) into both the
N-terminal and C-terminal domains, allowing subsequent deletion of
the N-terminal domain. Various other restriction enzymes, including
for example, Eco RI, may also be utilized in the context of the
present invention in order to construct deletion or truncation
derivatives of p97.
[0069] Mutations may be introduced at particular loci by
synthesizing oligonucleotides containing a mutant sequence, flanked
by restriction sites enabling the ligation of the mutated fragments
to fragments of the native sequence. Following ligation, the
resulting reconstructed sequence encodes a derivative having the
desired amino acid insertion, substitution, or deletion.
Alternatively, as noted above oligonucleotide-directed
site-specific mutagenesis procedures may be employed to obtain an
altered gene having particular codons altered according to the
desired substitution, deletion, or insertion. Exemplary methods of
making the alterations set forth above are disclosed by Sambrook et
al. Molecular Cloning A Laboratory Manual, 2d Ed., Cold Spring
Harbor Laboratory Press (1989).
[0070] Within a particularly preferred embodiment of the invention,
p97 is cloned into an expression vector as a truncated cDNA with a
deletion of the GPI anchor sequence located in the carboxy terminus
of the protein.
[0071] Briefly, the p97 gene may be generated by polymerase chain
reaction (PCR) using the cloned p97 cDNA as a template. The
truncated p97 is synthesized using WJ47, the 5' PCR primer
encompassing coordinates 36 to 60 (coordinates based on the cDNA
map) and additionally containing a Sna BI restriction site. The
sequence of WJ47 is 5'-GCG CTA CGT ACT CGA GGC CCC AGC CAG CCC CGA
CGG CGC C-3' (SEQ ID NO:6). The 3' primer, WJ48, encompasses
coordinates 2172 to 2193 and additionally contains both a TGA
termination codon and a SnaBI restriction site. The DNA sequence of
WJ48 is 5'-CGC GTA CGT ATG ATC ATC AGC CCG AGC ACT GCT GAG ACG
AC-3' (SEQ ID NO:7). Following amplification, the truncated p97
product is inserted into pNUTAH (obtained from Palmiter (1986) PNAS
83:1261-1265) at the Sma I restriction site. The orientations of
the resulting plasmids may be determined by PCR using one priming
oligonucleotide which anneals to the vector sequence and a second
priming oligonucleotide which anneals to the insert sequence.
Alternatively, appropriate restriction digests can be performed to
verify the orientation. Expression of the amplified sequence
results in the production of a soluble p97 protein lacking the
hydrophobic domain.
[0072] As noted above, the present invention provides recombinant
expression vectors which include either synthetic, or cDNA-derived
DNA fragments encoding p97 or derivatives thereof, which are
operably linked to suitable transcriptional or translational
regulatory elements. Suitable regulatory elements may be derived
from a variety of sources, including, but not limited to,
bacterial, fungal, viral, mammalian, and insect genes. Selection of
appropriate regulatory elements is dependent on the host cell
chosen, and may be readily accomplished by one of ordinary skill in
the art. Examples of regulatory elements include, in particular, a
transcriptional promoter and enhancer or RNA polymerase binding
sequence, a ribosomal binding sequence, including a translation
initiation signal. Additionally, depending on the host cell chosen
and the vector employed, other genetic elements, such as an origin
of replication, additional DNA restriction sites, enhancers,
sequences conferring inducibility of transcription, and selectable
markers, may be incorporated into the expression vector.
[0073] DNA sequences encoding p97 may be expressed by a wide
variety of prokaryotic and eukaryotic host cells, including, but
not limited to, bacterial, mammalian, yeast, fungi, viral, plant,
and insect cells. Methods for transforming or transfecting such
cells for expressing foreign DNA are well known in the art (see,
e.g., Itakura et al., U.S. Pat. No. 4,704,362; Hinnen et al. (1978)
PNAS USA 75:1929-1933; Murray et al., U.S. Pat. No. 4,801,542;
Upshall et al., U.S. Pat. No. 4,935,349; Hagen et al., U.S. Pat.
No. 4,784,950; Axel et al., U.S. Pat. No. 4,399,216; Goeddel et
al., U.S. Pat. No. 4,766,075; and Sambrook et al., supra).
[0074] Promoters, terminators, and methods for introducing
expression vectors of an appropriate type into, for example, plant,
avian, and insect cells may be readily accomplished by those of
skill in the art. Within a particularly preferred embodiment of the
invention, p97 is expressed from baculoviruses (see, e.g., Luckow
and Summers (1988) BioTechnology 6:47; Atkinson et al. (1990)
Petic. Sci. 28:215-224). The use of baculoviruses such as AcMNPV is
particularly preferred since host insect cells express the
GPI-cleaved forms of p97. p97 may be prepared from cultures of the
host/vector systems described above that express the recombinant
p97. Recombinantly produced p97 may be further purified as
described in more detail below.
[0075] The soluble form of p97 may be prepared by culturing cells
containing the soluble p97 through the log phase of the cell's
growth and collecting the supernatant. Preferably, the supernatant
is collected prior to the time at which the cells lose viability.
Soluble p97 may then be purified as described below, in order to
yield isolated soluble p97. Suitable methods for purifying the
soluble p97 can be selected based on the hydrophilic property of
the soluble p97. For example, the soluble p97 may be readily
obtained by Triton X-114 Phase Separation.
[0076] In another embodiment, p97 may be isolated from cultured CHO
cells genetically engineered to express the GPI-anchored p97. The
GPI-anchored protein may be harvested by a brief incubation with an
enzyme capable of cleaving the GPI anchor. Such enzymes are known
in the art (Ferguson (1988) Ann. Rev. Bichem. 57:285-320) and
representative examples are described supra. The cleaved soluble
protein may be recovered from the medium, and the cells may then be
returned to growth medium for further expression of the protein.
Cycles of growth and harvest may be repeated until sufficient
quantities of the protein are obtained. A particularly preferred
GPI enzyme is phospholipase C (PI-PLC) which may be obtained either
from bacterial sources (see, Low "Phospholipase Purification and
Quantification" The Practical Approach Series: Cumulative Methods
Index, Rickwood and Hames, eds. IRC Press, Oxford, N.Y. (1991);
Kupe et al. (1989) Eur. J. Biochem. 185:151-155; Volwerk et al.
(1989) J. Cell. Biochem. 39:315-325) or from recombinant sources
(Koke et al. (1991) Protein Expression and Purification 2:51-58;
and Henner et al. (1986) Nuc. Acids Res. 16:10383).
[0077] p97 and derivatives thereof, including the soluble p97, may
be readily purified according to the methods described herein.
Briefly, p97 may be purified either from supernatants containing
solubilized p97, or from cultured host/vector systems as described
above. A variety of purification steps, used either alone or in
combination, may be utilized to purify p97. For example,
supernatants obtained by solubilizing p97, or from host/vector
cultures as described above, may be readily concentrated using
commercially available protein concentration filters, such as an
Amicon or Millipore Pellicon ultrafiltration unit, or by "salting
out" the protein followed by dialysis. In addition, the
supernatants or concentrates may be applied to an affinity
purification matrix such as an anti-p97 antibody bound to a
suitable support. Alternatively, an anion exchange resin, such as a
matrix or substrate having pendant diethylaminoethyl (DEAE) groups,
may be employed. Representative matrices include acrylamide,
agarose, dextran, cellulose or other types commonly employed in
protein purification. Similarly, cation exchangers which utilize
various insoluble matrices such as sulfopropyl or carboxymethyl
groups may be also used.
[0078] Finally, one or more reversed-phase high performance liquid
chromatography (RP-HPLC) steps using hydrophobic RP-HPLC media,
e.g., silica gel having pendant methyl or other alipathic groups,
can be employed to further purify p97.
[0079] p97 fragments may also be generated using the techniques
described above, with modifications well known in the art. For
example, p97 expression vectors may be modified so that the
expressed protein is a desired fragment of p97. This protein may be
isolated from the expression system (i.e., extracted from cells),
or it may be designed to be secreted into the supernatant of the
expression system, and isolated using techniques described above.
Alternatively, full length p97 protein may be generated and
purified, and p97 fragments may then be generated by cleavage
reactions designed to generate the desired fragment. Chemical
synthesis is an alternative route to obtain the desired p97 protein
or fragment thereof.
[0080] In the context of the present invention, "isolated" or
"purified," as used to define the purity of p97, refer to a protein
that is substantially free of other proteins of natural or
endogenous origin, and that contains less than about 5% and
preferably less than about 1% by mass of protein contaminants due
to the production processes. p97 may be considered "isolated" if it
is detectable as a single protein band upon SDS-PAGE, followed by
staining with Coomassie Blue.
[0081] Production of Fusion/Chimeric Proteins
[0082] The chimeric protein of the present invention can be
produced using host cells expressing a single nucleic acid encoding
the entire fusion 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.
[0083] A. Host Cells
[0084] 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.
[0085] Cells which contain and express DNA or RNA encoding the
chimeric protein are referred to herein as genetically modified
cells. Mammalian cells which 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. patent application 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. patent application
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. patent application 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.
[0086] B. Nucleic Acid Constructs
[0087] 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.
[0088] 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).
[0089] C. Cell Culture Methods
[0090] 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 which express the selectable marker gene. Further
amplification of the introduced DNA construct can be effected 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). 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 p97 can be
identified by a sandwich enzyme immunoassay. The antibodies can be
directed toward the LRP portion or the active agent portion.
[0091] B. Preparation of Antibodies to p97
[0092] Based on the teaching of the instant specification,
antibodies to mouse or human p97 have many uses including, but not
limited to, the use for the isolation and purification of p97, use
in research and identification of p97 both in vitro and in vivo,
and potential diagnostic (e.g., monitoring conjugate dosage levels)
and therapeutic uses (e.g., modulating p97-conjugate dose levels),.
It is, therefore, useful to briefly set forth preferred antibodies
to p97, and methods of producing such antibodies.
[0093] Antibodies reactive against p97 are well known in the art.
Additional anti-p97 antibodies are provided by the present
invention. Representative examples of anti-p97 antibodies include
L235 (ATCC No. HB 8466; see, Real et al. (1985) Cancer Res. 45:4401
4411; see, also, Food et al. (1994) J. Biol. Chem. 269(4):
3034-3040), 4.1, 8.2, 96.5 and 118.1 (see, Brown et al. (1981) J.
Immunol. 127(2):539-546; and Brown et al. (1981) Proc. Natl. Acad.
Sci. USA 78(1):539-543); and HybC (Kennard et al. (1996) Nat. Med.
2(11):1230-1235). Other monoclonal antibodies, including, but not
limited to, 2C7 and 9B6, have been generated at Synapse
Technologies Inc. Antibodies to the mouse p97 include, for example,
a rabbit anti-human p97 polyclonal antibody generated against a
fragment of the mouse p97. In the context of the present invention,
antibodies are understood to include, for example, monoclonal
antibodies, polyclonal antibodies, antibody fragments (e.g., Fab,
and F(ab')2) and recombinantly produced binding partners.
Antibodies are understood to be reactive against p97 if the Ka is
greater than or equal to 10.sup.-7 M.
[0094] Polyclonal antibodies may be readily generated by one of
ordinary skill in the art from a variety of warm-blooded animals.
Monoclonal antibodies may also be readily generated using
conventional techniques (see, e.g., U.S. Pat. Nos. RE 32,011,
4,902,614; 4,543,439; and 4,411,993; see, also, Kennett, McKearn,
and Bechtol (eds.) Monoclonal Antibodies, Hybridomas: A New
Dimension in Biological Analyses, Plenum Press, (1980); and Harlow
and Lane (eds.) Antibodies: A Laboratory Manual, Cold Spring Harbor
Laboratory Press (1988)). Preparation of preferred antibodies is
further described in the examples section, below.
[0095] Labels
[0096] In some embodiments, the p97 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.
[0097] As noted above, depending on the screening assay employed,
the agent, the linker or the p97 molecule 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.
[0098] 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.).
[0099] 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 p97 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 p97 to form a label p97 conjugate without
an active agent attached thereto. The label p97 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 p97
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.
[0100] 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.
[0101] 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.
[0102] Pharmaceutical Compositions, and Methods of
Use/Treatment/Administr- ation
[0103] The diseases that can be treated, ameliorated or prevented
using the methods of the present invention include, but are not
limited to the following: Mucopolysaccharidosis I (MPS I), MPS II,
MPS IIIA, MPS IIIB, Metachromatic Leukodystropy (MLD), Krabbe,
Pompe, CLN2, Tay-Sachs, Niemann-Pick A and B, and other lysosomal
diseases. For each disease the conjugated agent would comprise a
specific compound, protein 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
thioesterase. For methods involving Tay-Sachs, the preferred
compound or enzyme is hexosaminidase A. For methods involving
Niemann-Pick A and B the preferred compound or enzyme is Acid
Spingomyelinase. For methods involving other Glycogenosis disorders
the preferred compound or enzyme is glycolipidoses,
mucopolysaccharidoses, oligosaccharidoses.
[0104] The p97-conjugates of the present invention can be
administered with a "pharmaceutically acceptable carrier." Such
carriers encompass any of the standard pharmaceutical carriers,
buffers and excipients, including phosphate-buffered saline
solution, water, and emulsions (such as an oil/water or water/oil
emulsion), and various types of wetting agents and/or adjuvants.
Suitable pharmaceutical carriers and their formulations are
described in Remington's Pharmaceutical Sciences (Mack Publishing
Co., Easton, 19th ed. 1995). Preferred pharmaceutical carriers
depend upon the intended mode of administration of the active
agent. Typical modes of administration are described below.
[0105] The term "effective amount" means a dosage sufficient to
produce a desired result on a health condition, pathology, 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.
[0106] A "prophylactic treatment" is a treatment administered to a
subject who does not exhibit signs of a disease or exhibits only
early signs of a disease, wherein treatment is administered for the
purpose of decreasing the risk of developing a pathology. The
conjugate conjugates of the invention may be given as a
prophylactic treatment.
[0107] A "therapeutic treatment" is a treatment administered to a
subject who exhibits signs of pathology, wherein treatment is
administered for the purpose of diminishing or eliminating those
pathological signs. The signs may be subjective or objective.
[0108] The term "composition", as in pharmaceutical composition, is
intended to encompass a product 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 p97-agent conjugate of the present
invention and a pharmaceutically acceptable carrier.
[0109] The term "pharmaceutical composition" indicates a
composition suitable for pharmaceutical use in a subject, including
an animal or human. A pharmaceutical composition generally
comprises an effective amount of the p97-conjugate and a
pharmaceutically acceptable carrier.
[0110] The conjugates 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.
[0111] The p97-agent conjugates 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.
[0112] The p97-agent conjugates can be utilized in aerosol
formulation to be administered via inhalation. The conjugates of
the present invention can be formulated into pressurized acceptable
propellants such as dichlorodifluoromethane, propane, nitrogen and
the like.
[0113] Furthermore, the p97-agent conjugates can be made into
suppositories by mixing, with a variety of bases such as
emulsifying bases or water-soluble bases. The conjugates 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.
[0114] Unit dosage forms of the p97-agent conjugates for oral or
rectal administration as, for instance, 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. 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 conjugates 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.
[0115] In practical use, the conjugates 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.
[0116] 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 p97-agent
conjugates of the invention. Patches preferably provide an
absorption enhancer such as DMSO to increase the absorption of the
conjugates. 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.
[0117] The 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.
[0118] Those of skill will readily appreciate that dose levels can
vary as a function of the specific agent, the severity of the
symptoms and the susceptibility of the subject to side effects.
Preferred dosages for a given conjugate are readily determinable by
those of skill in the art by a variety of means.
[0119] 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.
[0120] In practical use, the conjugates 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.
[0121] 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 agent 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.
[0122] The conjugates of the invention are useful for therapeutic,
prophylactic and diagnostic intervention in animals, and in
particular in humans.
[0123] Compositions of the present invention may be administered
encapsulated in or attached to viral envelopes or vesicles.
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 conjugates intracellularly and
to deliver the conjugates 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).
[0124] Any route of administration which brings the conjugates into
contact with the target cells, tissue or organ may be used. The
conjugates can be administered peripherally or centrally. The
conjugates may also be administered intravenously or by
intraperitoneally. The conjugates may be administered locally or
regionally.
[0125] The dosages to be administered will depend on individual
needs and characteristics (age, weight, severity of condition, on
the desired effect, the active agent used, and the chosen route of
administration and treatment regimen). Preferred dosages of
p97-conjugates range from about 0.02 pmol/kg to about 2.5 nmol/kg,
and particularly preferred dosages range from 2-250 pmol/kg;
alternatively, preferred doses of the p97 conjugate may be in the
range of 0.02 to 2000 mg/kg. These dosages will be influenced by
the number of agent moieties associated with each p97 molecule. In
addition, dosages may be calculated based on the agent to be
administered and the severity of the condition to be treated.
Empirical and theoretical methods for determining dose response
relationships and optimizing the dosages employed an individual
patients therapy are will known to one of ordinary skill in the
art.
[0126] The p97-conjugates of the invention are, for example, useful
for therapeutic and prophylactic intervention the treatment of
lysosomal storage diseases in animals, and in particular in humans.
The subject methods find use in the treatment of a variety of
different lysosomal storage diseases. In certain embodiments, of
particular interest is the use of the subject methods in disease
conditions where an active agent having desired activity has been
previously identified, but in which the active agent is not
adequately targeted to the target site, area or compartment. With
such active agent, the subject methods can be used to enhance the
therapeutic efficacy and therapeutic index of active agent.
[0127] Treatment is meant to encompass any beneficial outcome to a
subject associated with administration of a conjugate including a
reduced likelihood 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 severity of the
disease or in a magnitude of a parameter representative of the
severity or presence of the disease, e.g., tissue damage, cell
death, excess or harmful amounts of lysosomal storage materials,
symptoms, associated with the pathological condition being treated,
such as inflammation and pain associated therewith. As such,
treatment also includes, but is not limited to, 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.
[0128] A variety of hosts or subjects are treatable according to
the subject methods. Generally such subjects 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 or subjects will be humans.
[0129] Methods of Screening Conjugates for Therapeutic Activity
[0130] The ability of the conjugates of the present invention to
increase the delivery of a therapeutic agent to a lysosome can be
assessed in vitro by comparing the delivery of a p97 conjugated
agent to a control such as the unconjugated agent. In a preferred
embodiment, the conjugate and agent are administered in vitro to
cells and the localization of the conjugate within lysosomes
determined. The assessment is facilitated by conjugating a label to
the p97 conjugate or unconjugated agent so that its location within
the cell can be more easily determined and quantified. Methods for
monitoring the localization of compounds within a cell are well
known to one of ordinary skill in the art and further exemplified
in Examples 1 and 2. Such methods are also exemplified in U.S.
patent application Ser. No. 10/206,448 filed on Jul. 25, 2002 and
incorporated herein by reference in its entirety.
[0131] In another functional approach, the conjugate with an agent
deficient in a LSD can be contacted in vitro with cells affected by
the LSD and the effect of the conjugate on the amount of the
storage material found within the lysosomes compared to the effect
of an equivalent amount of the unconjugated agent. The cell or
lysosomal volumes may be measured or the stored material directly
quantitated.
[0132] In some embodiments, the invention provides a method of
screening a compound for therapeutic activity in treating a
lysosomal storage disease, by contacting a cell having a lysosome
with the compound, wherein the compound comprises p97 covalently
linked to an enzyme deficient in a lysosomal storage disease; and
then monitoring delivery of the compound to the lysosome. The
monitoring may be by means of a label on the conjugate and
detecting the label within the lysosome or by determining the
effect of the compound on the lysosomal storage material (e.g.,
does it reduce the amount of storage material.) In some
embodiments, the cell is human.
[0133] In a further method, clinical trials are conducted as known
to one of ordinary skill in the art and has exemplified in Example
3. More preferably, the conjugates are administered to test animals
providing an animal model of the LSD of interest. Such animal
models are well known to one of ordinary skill in the art. See, for
example, PCT Patent Publication No: WO 01/83722 which is
incorporated herein by reference.
[0134] 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.
EXAMPLES
Example 1
Immunocytochemical localization of p97
[0135] The focus of our first set of experiments has been to show
that unconjugated p97 is localized to the lysosome in cultured
brain cells. This is accomplished by showing "co-localization" of
p97 with a marker for lysosomes within the cell. Showing lysosomal
"co-localization" is the first step toward validating p97 as a
suitable vehicle for lysosomal enzymes.
[0136] A human neuronal line was used to conduct initial
experiments. Differentiated human NT2 neurons derived from a
teratocarcinoma are available commercially from Stratagene. Human
neuronal lines are the most relevant system since the p97 protein
under study is of human origin and the ultimate target is the
neural tissue within affected patients.
[0137] Immunocytochemical staining of p97 within cells was
accomplished with the L235 monoclonal antibody from Synapse in
conjunction with a fluorescein-conjugated secondary antibody to
localize the L235. The anti-p97 L235 antibody detects endogenous
material (p97 is expressed in normal neuronal cells) in addition to
material that has been taken up from the culture medium. Markers
are necessary to provide an organelle-specific fluorescence pattern
against which the observed p97 fluorescence pattern can be
compared. Overlap of the two patterns confirms the specific
intracellular localization of p97. For this purpose, an antibody
against Cathepsin L, a lysosomal protease, was used in combination
with L235 in the immunofluorescence experiments. This
anti-Cathepsin L antibody was raised against a C-terminal peptide
of murine Cathepsin L and shows strong staining of lysosomes in
human neuronal cells. A Texas red-conjugated secondary antibody was
used to detect the anti-Cathepsin L primary antibody. Human NT2
neurons were grown on glass cover slips and fed for 2 hours with
0.5 mg/ml p97. Cells were then rinsed, fixed with formaldehyde and
permeabilized. Fixed cells were co-stained with primary and
secondary antibodies and mounted on slides. Cells were also treated
with a stain for the nucleus, DAPI. Slides were imaged using
appropriate filter sets to resolve the different markers.
[0138] Experimental Methods
[0139] Cell type: Human neuronal cell lines were primarily used to
conduct these experiments. Differentiated human NT2 neurons derived
from a teratocarcinoma are available commercially from Stratagene.
CRL10742 and CRL10442 immature cortical neurons are available from
ATCC. CRL10742 (designation HCN-2) was developed from a patient
with Rasmussen's encephalitis and stains for neuronal markers but
not for non-neuronal markers. This cell line is covered by U.S.
Pat. No. 5,196,315 for use in screening methods for evaluation of
chemical and biological compounds. CRL10442 (designation HCN-1A) is
a brain cortical neuron derived from a patient suffering unilateral
megalencephaly. Human neuronal lines are the most relevant system
to the current investigations as the p97 protein under study is of
human origin and the ultimate target is the neuronal tissue within
affected patients. Fibroblasts from MPSI patient were obtained from
BioMarin stock of this cell line originally obtained from the
Coriell Cell Bank. Cells were maintained in DMEM with 10% Fetal
Bovine Serum (FBS).
[0140] Antibodies: The mouse anti-p97 monoclonal antibody L235 was
kindly provided by Synapse. An appropriate dilution for
immunohistochemistry was determined by titration of antibody
against fixed DG44 cells until no background signal was visible.
Rabbit anti-Cathepsin L (M-19) antibody was purchased from Santa
Cruz Biotech. Secondary antibodies, Donkey anti-Mouse (DAM) and
Goat Anti-Rabbit (GAR) conjugated with Alexa Fluor 488 or Alexa
Fluor 594 were purchased from Molecular Probes and used at
dilutions recommended by the manufacturer.
[0141] Fluorescent Probes: Molecular Probes Alexa Fluor protein
labeling kits were used to fluorescently tag p97 and Iduronidase.
Fluorescently labeled Transferrin is commercially available from
Molecular Probes. Lysosensor is a marker for acidic organelles
commercially available from Molecular Probes.
[0142] Equipment: Examination of fixed cells was carried out using
a Leica DMIRB with the following filter sets: Leica Filter Cube A
(UV excitation range) Excitation Filter BP340-380/Emission LP425.
Leica Filter Cube 13 (Blue excitation range) Excitation Filter
BP450-490/Emission LP515, used to visualize the Alexa Fluor 488
tag. Leica Filter Cube N2.1, (Green excitation range) Excitation
Filter BP515-560/Emission LP590, used to visualize the Alexa Fluor
594 tag.
[0143] Protein Uptake Conditions: Cells were seeded a day prior to
an uptake experiment on coverslips within six-well plates at a
density of between 2 and 5e5 cells per well. Cells were washed 3
times with serum-free DMEM +I mg/ml BSA. Proteins for uptake were
added to the cells at 60 ug/ml in DMEM+1 mg/ml BSA and incubated in
a 37.degree. C. incubator with 5% CO.sub.2 for the duration of the
uptake period. Cells were then washed 3 times with PBS and fixed
with a commercially available formaldehyde-based fixative available
from CALTAG. Cells were permeabilized by immersion in 70% ethanol.
Antibody staining was carried out in CALTAG permeabilization
solution. All steps were separated by 3 washes in PBS the first of
which contained 0.1 ug/ml DAPI to stain cell nuclei. Coverslips
were mounted in Molecular Probes Antifade for examination.
[0144] Results
[0145] The immunofluorescence images depicted in FIG. 1A-1D show
the results of a co-localization experiment in human NT2 neurons
using the L235 and anti Cathepsin L antibodies. The "Light
Microscopy" frame of FIG. 1A shows a single cultured human neuron
observed under phase-contrast with additional irradiation at the
excitation wavelength of DAPI. The nucleus, which is indicated by
the blue fluorescent signal, is located in the center of the frame.
The cytoplasm of this cell can be seen streaming away from the
nucleus.
[0146] The "Cathepsin Staining" frame (FIG. 1B) is the same cell
viewed under irradiation with light at the excitation wavelength
for Cathepsin L detection. The location of Cathepsin L is
identified by the red fluorescence. The punctate appearance of the
signal pattern seen in this frame is characteristic of
lysosomes.
[0147] The "L235 Staining" frame of (FIG. 1C) is of the same cell
but now irradiated with light at the excitation wavelength for p97
detection. The location of p97 is identified by green fluorescence.
This frame shows the same type of punctate lysosomal staining
pattern as can be seen in the Cathepsin L image. Careful comparison
of the fluorescent patterns in these two frames reveals that they
are coincident. The Cathepsin L and the p97 are localized
identically within the cell.
[0148] Confirmation of the co-localization of the p97 and Cathepsin
L is shown in FIG. 1D ("Overlay"). Combining the two fluorescent
signals yields a orange colored punctate pattern, a combination of
the red and green light from the two different antibodies. These
results have been replicated in the ATCC neuronal line CRL 10742
that is derived from human brain cortical tissue.
Example 2
Intracellular Fluorescence Detection of Tagged p97
[0149] Selective tracking of endocytosed material within a
background of endogenous material requires the use of a second
detection system. For this purpose, cells were fed with p97, which
had been conjugated to the fluorescent marker Alexa Fluor 594.
Observation of marker fluorescence from live cells permits
identification of the signal derived solely from endocytosed p97
with no contribution from endogenous material. A lysosomal marker
for live cells was necessary for co-localization with the
Alexa-Fluor 594 tagged p97. Such a marker is Lysosensor Green, a
dye taken up by living cells that becomes fluorescent upon exposure
to the acidic environment of the late-endosomal and lysosomal
compartments. Similar to the experiment outlined above, cells were
fed for 2 hours with 0.5-mg/ml p97 and washed to remove unbound
material and live cells were imaged using appropriate filter sets
to resolve the different markers.
[0150] Experimental Methods
[0151] See "Experimental Methods" of Example 1.
[0152] Results
[0153] The localization of endocytosed Alexa Fluor 594-p97 was
determined in live cells. Cells were observed directly on a
fluorescence microscope using appropriate filter sets.
[0154] Selective tracking of endocytosed material within a
background of endogenous material requires the use of a second
detection system. For this purpose, cells were fed with p97
conjugated to the fluorescent marker Alexa Fluor 594. Observation
of marker fluorescence from live cells permits identification of
the signal derived solely from endocytosed p97 with no contribution
from endogenous material. A lysosomal marker for live cells was
necessary for co-localization with the Alexa Fluor 594 tagged p97.
Such a marker is Lysosensor Green, a dye taken up by living cells
that becomes fluorescent upon exposure to the acidic environment of
the late endosomal and lysosomal compartments. Similar to the
experiment outlined above, cells were fed for 2 hours with 0.5
mg/ml p97 and washed to remove unbound material and live cells were
imaged using appropriate filter sets to resolve the different
markers.
[0155] The localization of endocytosed Alexa Fluor 594-p97 was
determined in live cells. Cells were observed directly on a
fluorescence microscope using appropriate filter sets (FIGS.
2A-2C). The "Alexa Fluor 594-p97" frame (FIG. 2A) shows a living
hNT neuronal cell fed with the fluorophore-tagged p97 and
Lysosensor Green. The cell in this frame is observed under
irradiation at the excitation wavelength of Alexa Fluor 594. The
location of the endocytosed p97 is identified by the red
fluorescence. The pattern is punctate and perinuclear. The
"Lysosensor Green" frame (FIG. 2B) is the same cell viewed under
irradiation with light at the excitation wavelength of Lysosensor
Green. The locations of the acidified compartments of the cell,
including the lysosomes and late-endosomes, are identified by green
fluorescence. This pattern is also punctate and perinuclear.
Co-localization of the endocytosed p97 and Lysosensor dye is shown
in the third frame ("Overlay") (FIG. 2C). Combining the two
fluorescent signals yields an orange-colored pattern, a combination
of the red and green light from the two different fluorescent
markers.
[0156] The above-experimental data (Examples 1-2) shows that p97 is
localized in the lysosomes and transported from the cell surface to
the lysosomes of cultured cells. The two main transport steps
required for p97-mediated delivery to brain cell lysosomes seem to
occur. Synapse has shown that the p97 molecule delivers its
"payload" across the BBB. Our results have shown that p97 is
transported to the lysosome in cultured brain cells. Taken together
these results indicate that p97 is an effective means to deliver
recombinant enzymes to LSD patients suffering from neurological
manifestations of the disease.
Example 3
Treatment of Patients with MPS-I Disorder
[0157] A pharmaceutical composition comprising a conjugated agent
comprising human .alpha.-L-iduronidase linked to p97 is prepared by
methods well-known to one skilled in the art. It is preferred to
administer the pharmaceutical composition intravenously. The final
dosage form of the fluid comprises 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.
2 Component Composition Conjugated agent 0.05 0.5 mg/mL or
12,500-50,000 (.alpha.-L-iduronidase linked to p97) units per mL
Sodium chloride solution 150 mM in an IV bag, 50-250 cc total
volume Sodium phosphate buffer 10-50 mM, pH 5.8 Human albumin 1
mg/mL
[0158] Human patients manifesting a clinical phenotype of MPS-I
disorder 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. 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 4
Methods and Compositions Linking Agents to the p97 Molecule
[0159] Conjugates and preferred embodiments according to the
present invention include those of the formula 1
[0160] in which A is an active agent and B is a p97 molecule for
targeting or delivery; and X.sub.1 and X.sub.2 are independently N
or O; R is a substituted alkyl or unsubstituted alkyl or
unsubstituted or substituted heteroalkyl from 1 to about 30 atoms
in length or 1 to 50 atoms in length; and n is from 1 to 30. Where
n is greater than 1, the active agents may be the same or
different. Where different, the active agents are useful for the
treatment of the same disease or condition. "Alkyl" encompasses
divalent radicals of alkanes as defined below. Such linkers are
taught in U.S. Provisional Application No. 60/395762 filed on Jul.
12, 2002 and incorporated herein by reference in its entirety.
[0161] In a further embodiment, a label, L, is covalently attached
to a compound of Formula I. The label may be attached to the
conjugate at the active agent portion, the p97 portion, or the
linker joining the active agent to p97: 2
[0162] In some embodiments, the label is preferably attached to the
p97 portion of a conjugate.
[0163] These conjugates have the advantage of release ability. When
an isocyanate reagent according to the invention reacts with a
hydroxy group it forms a carbamate bond, which can be hydrolyzed by
endogenous enzymes (e.g., proteases) in the body of a subject to
which it is administered. The isocyanate reagents according to the
invention react with an amino group to generate an isourea bond,
which can also be hydrolyzed by endogenous enzymes in the body of a
subject to which it is administered. By virtue of attachment to the
p97 moeity, the agent is delivered to the target lysosomal
compartment or site in the body, the protease or other endogenous
enzyme hydrolyzes the carbamate or isourea bond to release the free
drug at the target site or compartment.
[0164] An exemplary conjugate comprises a p97 covalently linked
through functional group, as is well known in the art of PEGylated
peptides and proteins to a PEG moiety which is in turned linked via
a carbamate linkage to the active agent. The term active agent
includes, but is not limited to, proteins or enzymes deficient in a
LSD. In another embodiment, the conjugate is covalently linked
through a carbamate group to a PEG moiety which is in turned linked
via a carbamate linkage to the active agent. In another embodiment,
the conjugate is covalently linked through a carbamate group to a
PEG moiety which is in turned linked via a carbamate linkage to an
alkyl or homoalkyl moiety which is in turned linked via a carbamate
linkage to the active agent.
[0165] These conjugates also have the advantage of being
synthesized with high efficiencies according to the inventive
methods. The inventive reactions between isocyanate groups with
hydroxy and amino are very efficient; and the yields are very high
(usually over 90%). In addition, the new bond formed by the
reaction of an isocyanate group with a hydroxy or an amino group
will increase aqueous solubility of the drug. This property can be
of practical importance.
Example 5
Treatment of Patients with a Lysosomal Storage Disease Disorder
[0166] A pharmaceutical composition comprising a conjugated agent
comprising a human enzyme or protein deficient in a lysosomal
storage disease linked to p97 is prepared by methods well-known to
one skilled in the art. It is preferred to administer the
pharmaceutical composition intravenously. Alternatively, the
composition can be administered locally to the affected organ(s).
The final dosage form of the fluid comprises 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.
3 Component Composition Conjugated agent 0.02 to 2.0 mg/mL
(lysosomal storage disease protein or enzyme linked to p97) Sodium
chloride solution 150 mM in an IV bag, 50-250 cc total volume
Sodium phosphate buffer 10-50 mM, pH 5.8 Human albumin 1 mg/mL
[0167] Human patients manifesting a clinical phenotype of a
lysosomal storage disease or disorder are to be treated with a
conjugate having a protein or enzyme deficient in the particular
disease or disorder. All patients manifest some clinical evidence
of excessive or harmful visceral and soft tissue accumulation of
storage material in their lysosomes as manifested by varying
degrees of functional impairment or worsened health status
associated with a particular lysosomal storage disease or disorder.
Preferably, enzyme levels are monitored in a patient to confirm the
absence or reduced activity of the lysosomal storage disease
protein in their tissues. Efficacy is determined by measuring the
percentage reduction in urinary excretion of the substrate of the
conjugated enzyme over time. The urinary substrate levels in
patients are compared to normal excretion values and or levels in
untreated patients or the same patients before treatment. Efficacy
can also be determined according to the reduced signs and symptoms
of any pathology associated with a lysosomal disease. Efficacy can
be determined by tissue biopsy and examination of cells and or
lysosomes to determine the extent by which substrate or storage
material has been reduced. Efficacy can be determined by functional
assessments which may be objective or subjective (e.g, reduced pain
or difficulty in function, increased muscle strength or stamina,
increased cardiac output, exercise endurance, changes in body mass
or appearance, etc.). A greater than 25% or 50% reduction in
excretion of undegraded substate following therapy with the
conjugated agent is a valid means to measure an individual's
response to therapy. Data may also be collected measuring the
subject conjugated enzyme's activity or presence in tissues before,
after and during therapy. Clinical assessment of organ size may be
performed as a means of assessing therapeutic efficacy (see, for
instance, Hoogerbrugge et al., Lancet 345:1398 (1995)).
[0168] Although the invention has been described with reference to
the presently preferred embodiments, it should be understood that
various modifications can be made without departing from the spirit
of the invention.
[0169] All publications, patents, patent applications; and web
sites are herein incorporated by reference in their entirety to the
same extent as if each individual patent, patent application, or
web site was specifically and individually indicated to be
incorporated by reference in its entirety.
Sequence CWU 1
1
7 1 14 DNA Artificial sequence Synthetic primer 1 gcggacttcc tcgg
14 2 13 DNA Artificial sequence Synthetic primer 2 tcgcgagctt cct
13 3 20 DNA Artificial sequence Synthetic primer 3 ctcagagggc
cgctgcgccc 20 4 22 DNA Artificial sequence Synthetic primer 4
ccagcgcagc tagcgggggc ag 22 5 27 DNA Artificial sequence Synthetic
primer 5 acaccagcgc agctcgaggg gcagccg 27 6 40 DNA Artificial
sequence Synthetic primer 6 gcgctacgta ctcgaggccc cagccagccc
cgacggcgcc 40 7 41 DNA Artificial sequence Synthetic primer 7
cgcgtacgta tgatcatcag cccgagcact gctgagacga c 41
* * * * *