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Patent 2487815 Summary

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(12) Patent Application: (11) CA 2487815
(54) English Title: TARGETED THERAPEUTIC PROTEINS
(54) French Title: PROTEINES THERAPEUTIQUES CIBLEES
Status: Dead
Bibliographic Data
(51) International Patent Classification (IPC):
  • C07K 19/00 (2006.01)
  • A61K 38/16 (2006.01)
  • A61K 38/46 (2006.01)
  • A61P 3/00 (2006.01)
  • C07K 14/65 (2006.01)
  • C12N 9/14 (2006.01)
  • A61K 47/48 (2006.01)
(72) Inventors :
  • LEBOWITZ, JONATHAN H. (United States of America)
  • BEVERLEY, STEPHEN M. (United States of America)
  • SLY, WILLIAM S. (United States of America)
(73) Owners :
  • BIOMARIN PHARMACEUTICAL INC. (Not Available)
(71) Applicants :
  • SYMBIONTICS, INC. (United States of America)
(74) Agent: FASKEN MARTINEAU DUMOULIN LLP
(74) Associate agent:
(45) Issued:
(86) PCT Filing Date: 2003-05-29
(87) Open to Public Inspection: 2003-12-11
Examination requested: 2008-03-26
Availability of licence: N/A
(25) Language of filing: English

Patent Cooperation Treaty (PCT): Yes
(86) PCT Filing Number: PCT/US2003/017211
(87) International Publication Number: WO2003/102583
(85) National Entry: 2004-11-26

(30) Application Priority Data:
Application No. Country/Territory Date
60/384,452 United States of America 2002-05-29
60/386,019 United States of America 2002-06-05
60/408,816 United States of America 2002-09-06
10/272,531 United States of America 2002-10-16
60/445,734 United States of America 2003-02-06

Abstracts

English Abstract




Targeted therapeutics that localize to a specific subcellular compartment such
as the lysosome are provided. The targeted therapeutics include a therapeutic
agent and a targeting moiety that binds a receptor on an exterior surface of
the cell, permitting proper subcellular localization of the targeted
therapeutic upon internalization of the receptor. Nucleic acids, cells, and
methods relating to the practice of the invention are also provided.


French Abstract

L'invention concerne des produits thérapeutiques qui se localisent dans un compartiment infracellulaire spécifique, tel que le lysosome. Les produits thérapeutiques ciblés comprennent un agent thérapeutique et un fragment de ciblage qui se lie à un récepteur sur une surface extérieure de la cellule, ce qui assure une localisation infracellulaire appropriée des produits thérapeutiques après internalisation du récepteur. L'invention concerne en outre des acides nucléiques, des cellules et des méthodes se rapportant à la mise en oeuvre des produits de l'invention.

Claims

Note: Claims are shown in the official language in which they were submitted.





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CLAIMS

1. An underglycosylated targeted therapeutic comprising:
a therapeutic agent that is therapeutically active in a human lysosome; and
a lysosomal targeting domain that binds an extracellular domain of human
cation-
independent mannose-6-phosphate receptor and
(i) does not bind a mutein in which amino acid 1572 of the human cation-
independent mannose-6-phosphate receptor is changed from isoleucine to
threonine; or
(ii) binds the mutein with a dissociation constant at least ten times the
dissociation
constant for binding the extracellular domain of human cation-independent
mannose-6-
phosphate receptor.
2. An underglycosylated targeted therapeutic comprising:
a therapeutic agent that is therapeutically active in a human lysosome; and
a lysosomal targeting domain that is capable of binding a receptor domain
consisting
essentially of repeats 10-15 of the human cation-independent mannose-6-
phosphate receptor.
3. The underglycosylated targeted therapeutic of claim 2, wherein the
lysosomal targeting
domain is capable of binding a receptor domain consisting essentially of
repeats 10-13 of the
human cation-independent mannose-6-phosphate receptor.
4. The underglycosylated targeted therapeutic of claim 3, wherein the
lysosomal targeting
domain binds a receptor domain consisting essentially of repeats 11-12 of the
human cation-
independent mannose-6-phosphate receptor.
5. The underglycosylated targeted therapeutic of claim 4, wherein the
lysosomal targeting
domain binds a receptor domain consisting essentially of repeat 11 of the
human cation-
independent mannose-6-phosphate receptor.
6. The underglycosylated targeted therapeutic of claim 5, wherein the
lysosomal targeting
domain binds a receptor domain consisting essentially of amino acids 1508-1566
of the human
cation-independent mannose-6-phosphate receptor.
7. The underglycosylated targeted therapeutic of claim 2, wherein the
lysosomal targeting
domain binds the receptor domain with a submicromolar dissociation constant at
pH 7.4.




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8. The underglycosylated targeted therapeutic of claim 7, wherein the
dissociation constant
is about 10-7 M.
9. The underglycosylated targeted therapeutic of claim 7, wherein the
dissociation constant
is less than about 10-7 M.
10. An underglycosylated targeted therapeutic comprising:
a therapeutic agent that is therapeutically active in a human lysosome; and
a binding moiety sufficiently duplicative of human IGF-II such that the
binding moiety
binds the human cation-independent mannose-6-phosphate receptor.
11. The underglycosylated targeted therapeutic of claim 10, wherein the
binding moiety is an
organic molecule having a three-dimensional shape representative of at least a
portion of IGF-II.
12. The underglycosylated targeted therapeutic of claim 1, wherein the portion
of IGF-II
comprises amino acids 48-55 of human IGF-II.
13. The underglycosylated targeted therapeutic of claim 11, wherein the
portion of IGF-II
comprises at least three amino acids selected from the group consisting of
amino acids 8, 48, 49,
50, 54, and 55 of human IGF-II.
14. The underglycosylated targeted therapeutic of claim 11, wherein the
organic molecule
has a hydrophobic moiety at a position representative of amino acid 48 of
human IGF-II and has
a positive charge at about pH 7.4 at a position representative of amino acid
49 of human IGF-II.
15. The underglycosylated targeted therapeutic of claim 10, wherein the
binding moiety
comprises a polypeptide comprising the amino acid sequence of IGF-I or of a
mutein of IGF-I in
which
(i) amino acids 55 and 56 are changed,
(ii) amino acids 1-4 are deleted or changed, or
(iii) amino acids 55 and 56 are changed and amino acids 1-4 are deleted or
changed.
16. The underglycosylated targeted therapeutic of claim 10, wherein the
binding moiety
comprises a polypeptide comprising an amino acid sequence at least 60%
identical to human
IGF-II.




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17. The underglycosylated targeted therapeutic of claim 16, wherein the amino
acid sequence
comprises, at positions corresponding to positions 54 and 55 of human IGF-II,
amino acids each
of which are uncharged or negatively charged at pH 7.4.
18. The underglycosylated targeted therapeutic of claim 10 wherein the binding
moiety
comprises a polypeptide having antiparallel alpha-helices separated by not
more than five amino
acids.
19. An underglycosylated targeted therapeutic comprising:
a therapeutic agent that is therapeutically active in a human lysosome; and
a polypeptide comprising the amino acid sequence phenylalanine-arginine-
serine.
20. An underglycosylated targeted therapeutic comprising:
a therapeutic agent that is therapeutically active in a human lysosome; and
a polypeptide comprising an amino acid sequence at least 75% homologous to
amino
acids 48-55 of human IGF-II.
21. An underglycosylated targeted therapeutic comprising:
a therapeutic agent that is therapeutically active in a human lysosome;
amino acids 8-28 of human IGF-II; and
amino acids 41-61 of human IGF-II.
22. The underglycosylated targeted therapeutic of claim 21, wherein amino
acids 8-28 and
41-61 are present in a single polypeptide.
23. An underglycosylated targeted therapeutic comprising:
a therapeutic agent that is therapeutically active in a human lysosome;
amino acids 41-61 of human IGF-II; and
a mutein of amino acids 8-28 of human IGF-II, the mutein differing from human
IGF-II
at a position selected from the group consisting of amino acid 9, amino acid
19, amino acid 26,
and amino acid 27.
24. An underglycosylated therapeutic fusion protein comprising:
a therapeutic domain and
a subcellular targeting domain that binds to an extracellular domain of a
receptor on an
exterior surface of a cell and, upon internalization of the receptor, permits
localization of the




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therapeutic domain to a subcellular compartment where the therapeutic domain
is therapeutically
active.
25. The underglycosylated therapeutic fusion protein of claim 24, wherein the
subcellular
compartment is selected from the group consisting of a lysosome, an endosome,
endoplasmic
reticulum, and golgi complex.
26. The underglycosylated therapeutic fusion protein of claim 25, wherein the
subcellular
compartment is a lysosome.
27. The underglycosylated therapeutic fusion protein of claim 24, wherein the
receptor
undergoes continuous endocytosis.
28. The underglycosylated therapeutic fusion protein of claim 24, wherein the
therapeutic
domain has a therapeutic enzymatic activity.
29. The underglycosylated therapeutic fusion protein of claim 24, wherein the
therapeutic
domain has acid alpha-glucosidase A activity.
30. The underglycosylated therapeutic fusion protein of claim 28, wherein a
cellular or
subcellular deficiency in the enzymatic activity is associated with a human
disease.
31. The underglycosylated therapeutic fusion protein of claim 30, wherein the
human disease
is a lysosomal storage disease.
32. A method of treating a patient, the method comprising administering to the
patient the
underglycosylated therapeutic fusion protein of claim 24.
33. A method of treating a patient, the method comprising:
(i) synthesizing an underglycosylated targeted therapeutic comprising a
therapeutic
agent that is therapeutically active in a mammalian lysosome and a targeting
moiety that binds
human canon-independent mannose-6-phosphate receptor in a mannose-6-phosphate-
independent manner; and
(ii) administering the underglycosylated targeted therapeutic to the patient.
34. The method of claim 33, further comprising, prior to step (i),




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identifying a targeting moiety that binds human cation-independent mannose-6-
phosphate receptor in a mannose-6-phosphate-independent manner.

35. The method of claim 34, wherein the targeting moiety is identified by
screening a nucleic
acid or peptide library.

36. A method of producing a targeted therapeutic, the method comprising the
steps of:
(a) providing a molecular model defining a three-dimensional shape
representative of
at least a portion of human IGF-II;
(b) identifying a candidate IGF-II analog having a three-dimensional shape
corresponding to the three-dimensional shape representative of at least a
portion of human IGF
II; and
(c) producing an underglycosylated therapeutic agent directly or indirectly
bound to
the candidate IGF-II analog, wherein the therapeutic agent is therapeutically
active in a
mammalian lysosome.

37. The method of claim 36, further comprising determining whether the
compound
produced in step c binds to the human cation-independent mannose-6-phosphate
receptor.

Description

Note: Descriptions are shown in the official language in which they were submitted.




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TARGETED THERAPEUTIC PROTEINS
Reference to Related Applications
[0001] This application claims the benefit of U.S. Serial No. 60/384,452,
filed May
29, 2002; U.S. Serial No. 601386,019, filed June 5, 2002; U.S. Serial No.
60/408,816, filed
September 6, 2002; U.S. Serial No. 10/272,531, filed ~ctober 16, 2002; and
U.S. Serial No.
60/445,734, filed February 6, 2003, the contents of each of which are
incorporated by reference
in their entireties. This application is also related to U.S. Serial No.
60/287,531, filed April 30,
2001; U.S. Serial No. 60/304,609, filed July 10, 2001; U.S. Serial No.
60/329,461, filed October
15, 2001, U.S. Serial No. 60/351,276, filed January 23, 2002; U.S. Serial No.
10/136,841, filed
April 30, 3002; and to U.S. Serial No. 10/272,483, filed October 16, 2002, the
contents of each
of which are incorporated by reference in their entireties.
[0002] This invention provides a means for specifically delivering proteins to
a
targeted subcellular compartment of a mammalian cell. The ability to target
proteins to a
subcellular compartment is of great utility in the treatment of metabolic
diseases such as
lysosomal storage diseases, a class of over 40 inherited disorders in which
particular lysosomal
enzymes are absent or deficient.
Background
[0003] Enzyme deficiencies in cellular compartments such as the golgi, the
endoplasmic reticulum, and the lysosome cause a wide variety of human
diseases. For example,
lysyl hydroxylase, an enzyme normally in the lumen of the endoplasmic
reticulum, is required
for proper processing of collagen; absence of the enzyme causes Ehlers-Danlos
syndrome type
VI, a serious connective tissue disorder. GnT II, normally found in the golgi,
is required for
normal glycosylation of proteins; absence of GnT II causes leads to defects in
brain
development. More than forty lysosomal storage diseases (LSDs) are caused,
directly or
indirectly, by the absence of one or more proteins in the lysosome.
[0004] Mammalian lysosomal enzymes are synthesized in the cytosol and traverse
the
ER where they are glycosylated with N-linked, high mannose type carbohydrate.
In the golgi,
the high mannose carbohydrate is modified on lysosomal proteins by the
addition of mannose-6-
phosphate (M6P) which targets these proteins to the lysosome. The M6P-modified
proteins are



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delivered to the lysosome via interaction with either of two M6P receptors.
The most favorable
form of modification is when two M6Ps are added to a high mannose
carbohydrate.
[0005] Enzyme replacement therapy for lysosomal storage diseases (LSDs) is
being
actively pursued. Therapy, except in Gaucher's disease, generally requires
that LSD proteins be
taken up and delivered to the lysosomes of a variety of cell types in an M6P-
dependent fashion.
One possible approach involves purifying an LSD protein and modifying it to
incorporate a
carbohydrate moiety with M6P. This modified material may be taken up by the
cells more
efficiently than unmodified LSD proteins due to interaction with M6P receptors
on the cell
surface. However, because of the time and expense required to prepare, purify
and modify
proteins for use in subcellular targeting, a need for new, simpler, more
efficient, and more cost-
effective methods for targeting therapeutic agents to a cellular compartment
remains.
Summary of the Invention
[OOOG] The present invention facilitates the treatment of metabolic diseases
by
providing targeted protein therapeutics that localize to a subcellular
compartment of a cell where
the therapeutic is needed. The invention simplifies preparation of targeted
protein therapeutics
by reducing requirements for posttranslational or postsynthesis processing of
the protein. For
example, a targeted therapeutic of the present invention can be synthesized as
a fusion protein
including a therapeutic domain and a domain that targets the fusion protein to
a correct
subcellular compartment. ("Fusion protein," as used herein, refers to a single
polypeptide having
at least two domains that are not normally present in the same polypeptide.
Thus, naturally
occurring proteins are not "fusion proteins" as used herein.) Synthesis as a
fusion protein
permits targeting of the therapeutic domain to a desired subcellular
compartment without
complications associated with chemical crosslinking of separate therapeutic
and targeting
domains, for example.
[0007] The invention also permits targeting of a therapeutic to a lysosome in
an M6P-
independent manner. Accordingly, the targeted therapeutic need not be
synthesized in a
mammalian cell, but can be synthesized chemically or in a bacterium, yeast,
protozoan, or other
organism regardless of glycosylation pattern, facilitating production of the
targeted therapeutic
with high yield and comparatively low cost. The targeted therapeutic can be
synthesized as a
fusion protein, further simplifying production, or can be generated by
associating independently-
synthesized therapeutic agents and targeting moieties.
[000] The present invention permits lysosomal targeting of therapeutics
without the
need for M6P addition to high mannose carbohydrate. It is based in part on the
observation that



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one of the 2 M6P receptors also binds other ligands with high affinity. For
example, the cation-
independent mannose-6-phosphate receptor is also known as the insulin-like
growth factor 2
(IGF-II) receptor because it binds IGF-II with high affinity. This low
molecular weight
polypeptide interacts with three receptors, the insulin receptor, the IGF-I
receptor and the
M6P/IGF-II receptor. It is believed to exert its biological effect primarily
through interactions
with the former two receptors while interaction with the cation-independent
M6P receptor is
believed to result predominantly in the IGF-II being transported to the
lysosome where it is
degraded.
[0009] Accordingly, the invention relates in one aspect to a targeted
therapeutic
including a targeting moiety and a therapeutic agent that is therapeutically
active in a mammalian
lysosome. "Therapeutically active," as used herein, encompasses at least
polypeptides or other
molecules that provide an enzymatic activity to a cell or a compartment
thereof that is deficient
in that activity. "Therapeutically active" also encompasses other polypeptides
or other
molecules that are intended to ameliorate or to compensate for a biochemical
deficiency in a cell,
but does not encompass molecules that are primarily cytotoxic or cytostatic,
such as
chemotherapeutics.
[0010] In one embodiment, the targeting moiety is a means (e.g. a molecule)
for
binding the extracellular domain of the human cation-independent M6P receptor
in an M6P-
independent manner when the receptor is present in the plasma membrane of a
target cell. In
another embodiment, the targeting moiety is an unglycosylated lysosomal
targeting domain that
binds the extracellular domain of the human canon-independent M6P receptor. In
either
embodiment, the targeting moiety can include, for example, IGF-II; retinoic
acid or a derivative
thereof; a protein having an amino acid sequence at least 70% identical to a
domain of urokinase-
type plasminogen activator receptor; an antibody variable domain that
recognizes the receptor; or
variants thereof. In some embodiments, the targeting moiety binds to the
receptor with a
submicromolar dissociation constant (e.g. less than 10-8 M, less than 10'9 M,
less than 10-1° M, or
between 10-7 M and 10'11 M) at or about pH 7.4 and with an dissociation
constant at or about pH
5.5 of at least 10'6 M and at least ten times the dissociation constant at or
about pH 7.4. In
particular embodiments, the means for binding binds to the extracellular
domain at least 10-fold
less avidly (i. e. with at least a ten-fold greater dissociation constant) at
or about pH 5.5 than at or
about pH 7.4; in one embodiment, the dissociation constant at or about pH 5.5
is at least 10'6 M.
In a further embodiment, association of the targeted therapeutic with the
means for binding is
destabilized by a pH change from at or about pH 7.4 to at or about pH 5.5.



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[0011] In another embodiment, the targeting moiety is a lysosomal targeting
domain
that binds the extracellular domain of the human cation-independent M6P
receptor but does not
bind a mutein of the receptor in which amino acid 1572 is changed from
isoleucine to threonine,
or binds the mutein with at least ten-fold less affinity (i. e. with at least
a ten-fold greater
dissociation constant). In another embodiment, the targeting moiety is a
lysosomal targeting
domain capable of binding a receptor domain consisting essentially of repeats
10-15 of the
human cation-independent M6P receptor: the lysosomal targeting domain can bind
a protein that
includes repeats 10-15 even if the protein includes no other moieties that
bind the lysosomal
targeting domain. Preferably, the lysosomal targeting domain can bind a
receptor domain
consisting essentially of repeats 10-13 of the human cation-independent
mannose-6-phosphate
receptor. More preferably, the lysosomal targeting domain can bind a receptor
domain
consisting essentially of repeats 11-12, repeat 11, or amino acids 1508-1566
of the human cation-
independent M6P receptor. In each of these embodiments, the lysosomal
targeting domain
preferably binds the receptor or receptor domain with a submicromolar
dissociation constant at
or about pH 7.4. In one preferred embodiment, the lysosomal targeting domain
binds with an
dissociation constant of about 10'7 M. In another preferred embodiment, the
dissociation
constant is less than about 10-7 M.
[0012] In another embodiment, the targeting moiety is a binding moiety
sufficiently
duplicative of human IGF-II such that the binding moiety binds the human canon-
independent
M6P receptor. The binding moiety can be sufficiently duplicative of IGF-II by
including an
amino acid sequence sufficiently homologous to at least a portion of IGF-II,
or by including a
molecular structure sufficiently representative of at least a portion of IGF-
II, such that the
binding moiety binds the ration-independent M6P receptor. The binding moiety
can be an
organic molecule having a three-dimensional shape representative of at least a
portion of IGF-II,
such as amino acids 48-55 of human IGF-II, or at least three amino acids
selected from the group
consisting of amino acids 8, 48, 49, 50, 54, and 55 of human IGF-II. A
preferred organic
molecule has a.hydrophobic moiety at a position representative of amino acid
48 of human IGF-
II and a positive charge at or about pH 7.4 at a position representative of
amino acid 49 of human
IGF-II. In one embodiment, the binding moiety is a polypeptide including a
polypeptide having
antiparallel alpha-helices separated by not more than five amino acids. In
another embodiment,
the binding moiety includes a polypeptide with the amino acid sequence of IGF-
I or of a mutein
of IGF-I in which amino acids 55-56 are changed and/or amino acids 1-4 are
deleted or changed.
In a further embodiment, the binding moiety includes a polypeptide with an
amino acid sequence



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at least 60% identical to human IGF-II; amino acids at positions corresponding
to positions 54
and 55 of human IGF-II are preferably uncharged or negatively charged at or
about pH 7.4.
[0013] In one embodiment, the targeting moiety is a polypeptide comprising the
amino acid sequence phenylalanine-arginine-serine. In another embodiment, the
targeting
moiety is a polypeptide including an amino acid sequence at least 75%
homologous to amino
acids 48-55 of human IGF-II. In another embodiment, the targeting moiety
includes, on a single
polypeptide or on separate polypeptides, amino acids 8-28 and 41-61 of human
IGF-II. In
another embodiment, the targeting moiety includes amino acids 41-61 of human
IGF-II and a
mutein of amino acids 8-28 of human IGF-II differing from the human sequence
at amino acids
9, 19, 26, and/or 27.
[0014] In some embodiments, the association of the therapeutic agent with the
targeting moiety is labile at or about pH 5.5. In a preferred embodiment,
association of the
targeting moiety with the therapeutic agent is mediated by a protein acceptor
(such as imidazole
or a derivative thereof such as histidine) having a plea between 5.5 and 7.4.
Preferably, one of
the therapeutic agent or the targeting moiety is coupled to a metal, and the
other is coupled to a
pH-dependent metal binding moiety.
[0015] In another aspect, the invention relates to a therapeutic fusion
protein
including a therapeutic domain and a subcellular targeting domain. The
subcellular targeting
domain binds to an extracellular domain of a receptor on an exterior surface
of a cell. Upon
internalization of the receptor, the subcellulax targeting domain permits
localization of the
therapeutic domain to a subcellular compartment such as a lysosome, an
endosome, the
endoplasmic reticulum (ER), or the golgi complex, where the therapeutic domain
is
therapeutically active. In one embodiment, the receptor undergoes constitutive
endocytosis. In
another embodiment, the therapeutic domain has a therapeutic enzymatic
activity. The
enzymatic activity is preferably one for which a deficiency (in a cell or in a
particular
compartment of a cell) is associated with a human disease such as a lysosomal
storage disease.
[0016] In further aspects, the invention relates to nucleic acids encoding
therapeutic
proteins and to cells (e.g. mammalian cells, insect cells, yeast cells,
protozoans, or bacteria)
comprising these nucleic acids. The invention also provides methods of
producing the proteins
by providing these cells with conditions (e.g. in the context of ire vitro
culture or by maintaining
the cells in a mammalian body) permitting expression of the proteins. The
proteins can be
harvested thereafter (e.g. if produced ih vitro) or can be used without an
intervening harvesting
step (e.g. if produced in vivo in a patient). Thus, the invention also
provides methods of treating



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a patient by administering a therapeutic protein (e.g. by injection, iu situ
synthesis, or otherwise),
by administering a nucleic acid encoding the protein (thereby permitting ih
vivo protein
synthesis), or by administering a cell comprising a nucleic acid encoding the
protein. In one
embodiment, the method includes synthesizing a targeted therapeutic including
a therapeutic
agent that is therapeutically active in a mammalian lysosome and a targeting
moiety that binds
human cation-independent mannose-6-phosphate receptor in a mannose-6-phosphate-

independent manner, and administering the targeted therapeutic to a patient.
The method can
also include identifying the targeting moiety (e.g. by a recombinant display
technique such as
phage display, bacterial display, or yeast two-hybrid or by screening
libraries for requisite
binding properties). In another embodiment, the method includes providing
(e.g. on a computer)
a molecular model defining a three-dimensional shape representative of at
least a portion of
human IGF-II; identifying a candidate IGF-II analog having a three-dimensional
shape
representative of at least a portion of IGF-II (e.g. amino acids 48-55), and
producing a
therapeutic agent that is active in a mammalian lysosome and directly or
indirectly bound to the
candidate IGF-II analog. The method can also include determining whether the
candidate IGF-II
analog binds to the human cation-independent M6P receptor.
[0017] This invention also provides methods for producing therapeutic proteins
that
are targeted to lysosomes and/or across the blood-brain barrier and that
possess an extended half
life in circulation in a mammal. The methods include producing an
underglycosylated
therapeutic protein. As used herein, "underglycosylated" refers to a protein
in which one or
more carbohydrate structures that would normally be present if the protein
were produced in a
mammalian cell (such as a CHO cell) has been omitted, removed, modified, or
masked, thereby
extending the half life of the protein in a mammal. Thus, a protein may be
actually
underglycosylated due to the absence of one or more carbohydrate structures,
or functionally
underglycosylated by modification or masking of one or more carbohydrate
structures that
promote clearance from circulation. For example, a structure could be masked
(i) by the addition
of one or more additional moieties (e.g. carbohydrate groups, phosphate
groups, alkyl groups,
etc.) that interfere with recognition of the structure by a mannose or
asialoglycoprotein receptor,
(ii) by covalent or noncovalent association of the glycoprotein with a binding
moiety, such as a
lectin or an extracellular portion of a mannose or asialoglycoprotein
receptor, that interferes with
binding to those receptors ih vivo, or (iii) any other modification to the
polypeptide or
carbohydrate portion of a glycoprotein to reduce its clearance from the blood
by masking the
presence of all or a portion of the carbohydrate structure.



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[0018] In one embodiment, the therapeutic protein includes a peptide targeting
moiety (e.g. IGF-I, IGF-II, or a portion thereof effective to bind a target
receptor) that is
produced in a host (e.g. bacteria or yeast) that does not glycosylate proteins
as conventional
mammalian cells (e.g. Chinese hamster ovary (CHO) cells) do. For example,
proteins produced
by the host cell may lack terminal mannose, fucose, and/or N-acetylglucosamine
residues, which
are recognized by the mannose receptor, or may be completely unglycosylated.
In another
embodiment, the therapeutic protein, which may be produced in mammalian cells
or in other
hosts, is treated chemically or enzymatically to remove one or more
carbohydrate residues (e.g.
one or more mannose, fucose, and/or N-acetylglucosamine residues) or to modify
or mask one or
more carbohydrate residues. Such a modification or masking may reduce binding
of the
therapeutic protein to the hepatic mannose and/or asialoglycoprotein
receptors. In another
embodiment, one or more potential glycosylation sites are removed by mutation
of the nucleic
acid encoding the targeted therapeutic protein, thereby reducing glycosylation
of the protein
when synthesized in a mammalian cell or other cell that glycosylates proteins.
Brief Description of the Drawings
[0019] Figure 1 depicts several types of underglycosylation.
[0020] Figure 2 is a map of the human IGF-II open reading frame (SEQ ID NO:1
and its encoded protein (SEQ ID NO:2). Mature IGF-II lacks the signal peptide
and COOH-
cleaved regions. The IGF-II signal peptide and the mature polypeptide can be
fused to the GAA
coding sequence. The IGF-II portion can be modified to incorporated various
mutations that
enhance the selective binding of IGF-II to the IGF-II receptor.
[0021] Figure 3 is a Leishma~ia codon-optimized IGF-II depicted in the XbaI
site of
pIRl-SAT; the nucleic acid is SEQ ID NO:3 and the encoded protein is SEQ ID
N0:4.
[0022] Figure 4 is a depiction of a preferred embodiment of the invention,
incorporating a signal peptide sequence, the mature human (3-glucuronidase
sequence, a bridge
of three amino acids, and an IGF-II sequence. The depicted nucleic acid is SEQ
ID NO:S, and
the encoded protein is SEQ ID N0:6.
[0023] Figure 5 depicts (3-glucuronidase (GUS) activity in human
mucopolysaccharidosis VII skin fibroblast GM4668 cells exposed to GUS, a GUS-
IGF-II fusion
protein (GILT-GUS), GILT-GUS with ol-7 and Y27L mutations in the IGF-II
portion (GILT2-
GUS), or a negative control (DMEM).



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[0024] Figure 6 depicts GUS activity in GM4668 cells exposed to GUS (+~-GUS),
GUS-GILT (+GILT), GUS-GILT in the presence of excess IGF-II (+GILT+IGF-II), or
a
negative control (GM4668).
[0025] Figure 7 is an alignment of human IGF-I (SEQ ID N0:7) and IGF-II (SEQ
ID
N0:8), showing the A, B, C, and D domains.
[002G] Figure 8 depicts GUS in GM4668 cells exposed to GUS, GUS-GILT, GUS-
GILT, GUS=GILT with a deletion of the seven amino-terminal residues (GUS-GILT
dl-7),
GUS-GILT in the presence of excess IGF-II, GUS-GILT ~l-7 in the presence of
excess IGF-II,
or a negative control (Mock).
[0027] Figure 9A depicts one form of a phosphorylated high mannose
carbohydrate
structure linked to a glycoprotein via an asparagine residue, and also depicts
the structures of
mannose and N-acetylglucosamine (GIcNAc). Figure 9B depicts a portion of the
high mannose
carbohydrate structure at a higher level of detail, and indicates positions
vulnerable to cleavage
by periodate treatment. The positions of the sugar residues within the
carbohydrate structure are
labeled with circled, capital letters A-H; phosphate groups are indicated with
a circled capital P.
[0028] Figure 10 depicts SDS-PAGE analysis of GUS~C18-GILTO1-7 (SON), GUS
(HBG), and GUSOC18-GILT~1-7 treated with endoglycosidase Fl (OdN+F1).
[0029] Figure 11 depicts uptake of untagged (3-glucuronidase (M6P), GUSOC 18-
GILTO1-7 (GILT), or GUSOC18-GILTO1-7 treated with endoglycosidase F1 (GILT+Fl)
in the
absence (Enzyme only) of competitor or in the presence of mannose-6-phosphate
(+M6P) or
IGF-II (+Tag).
[0030] Figure 12 depicts results of an experiment measuring the ivy vivo half
life of
GUS~C18-GILT~l-7, untreated, treated with endoglycosidases F1 and F2 (+F1+F2),
or further
treated with dithiothreitol (+F1+F2+DTT).
[0031] Figure 13 depicts results of an experiment measuring accumulation of
infused
untagged (3-glucuronidase (M6P), GUSOC18-GILT~1-7 (GILT), or GUSOC18-GILT~1-7
treated with endoglycosidase Fl (GILT+F1) in the liver, spleen, or bone
marrow.
[0032] Figure 14 depicts results of an experiment measuring accumulation of
infused
untagged [3-glucuronidase (M6P), GUSOC18-GILT~1-7 (GILT), or GUSdCI8-GILT~1-7
treated with endoglycosidase F1 (GILT+Fl) in heart, kidney, or lung tissues.



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[0033] Figure 15 depicts results of an experiment measuring accumulation of
infused
untagged (3-glucuronidase (M6P), GUSOC18-GILT~1-7 (GILT), or GUSOC18-GILT~l-7
treated with endoglycosidase F1 (GILT+F1) in brain tissue.
[0034] Figure 16 depicts histological analysis of liver tissue of animals
infused with
untagged (i-glucuronidase (HGUS), GUSOC18-GILTO1-7 (Dd-l5gilt), or GUSOC18-
GILTO1-7
treated with endoglycosidase F1 (dd15F1).
[0035] Figure 17 depicts histological analysis of kidney tissue of animals
infused
with untagged (3-glucuronidase (HGUS), GUSOC18-GILTO1-7 (Dd-15), or GUS~C18-
GILTO1-
7 treated with endoglycosidase F 1 (F 1 ).
[003G] Figure 18 depicts histological analysis of bone tissue of animals
infused with
untagged (3-glucuronidase (HGUS), GUSOC18-GILTOl-7 (dd-15), or GUSOC18-GILT~l-
7
treated with endoglycosidase F1 (dd-15F1).
[0037] Figure 19 depicts histological analysis of spleen tissue of animals
infused with
untagged (3-glucuronidase (HGUS), GUSOC18-GILTO1-7 (Dd-15), or GUSOC18-GILTO1-
7
treated with endoglycosidase F1 (Dd-15F1:).
[0038] Figure 20 depicts an exemplary GILT-tagged acid a-glucosidase (GAA)
cassette with a targeting portion (GILT), including an optional signal
peptide, fused to the N-
terminus of GAA.
[0039] Figure 21 depicts an exemplary GILT-tagged GAA cassette with a
targeting
portion fused to the C-terminus of GAA.
[0040] Figure 22 shows the GAA cDNA sequence (SEQ ID N0:23) of Human Image
cDNA clone No. 4374238 and its encoded GAA protein (SEQ ID NO:24).
[0041] Figure 23 depicts an SDS-PAGE analysis of GUSOC18-GILT~1-7 produced
in CHO cells (CHO15), Lecl cells (LEC18), or HEK293 cells (HEK) with (+) or
without
endoglycosidase Fl treatment. Lanes marked "P" denote enzymes treated with
PNGase F, which
is expected to completely deglycosylate the enzymes.
[0042] Figure 24 depicts the results of a 24 hour GUS4C18-GILTO1-7
accumulation
experiment in immunotolerant MPSVII mice using endoglycosidase F1-treated CHO-
or Lecl-
produced enzymes and untreated HEK293-produced enzyme.
[0043] Figure 25 depicts the results of a half life experiment in human MPSVII
fibroblasts using untagged human 13-glucuronidase (HBGS), or GUS~C18-GILTO1-7
produced
in CHO or Lecl cells with (+F1) or without endoglycosidase Fl treatment.



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Detailed Description of the Invention
[0044] As used herein and except where otherwise specified, "glycosylation
independent lysosomal targeting" and "GILT" refer to lysosomal targeting that
is mannose-6-
phosphate-independent.
[0045] As used herein, "GILT construct" refers to a construct including a
mannose-6-
phosphate-independent lysosomal targeting portion and a therapeutic portion
effective in a
mammalian lysosome.
[004G] As used herein, "GUS" refers to 13-glucuronidase, an exemplary
therapeutic
portion.
[0047] As used herein, "GUS~C 18" refers to GUS with a deletion of the C-
terminal
18 amino acids, removing a potential proteolysis site.
[0048] As used herein, "GUS-GILT" refers to a GILT construct with GUS coupled
to
an IGF-II targeting portion.
[0049] All references to amino acid positions in IGF-II refer to the positions
in
mature human IGF-II. Thus, for example, positions 1, 2, and 3 are occupied by
alanine, tyrosine,
and arginine, respectively.
[0050] As used herein, GILT~l-7 refers to an IGF-II targeting portion with a
deletion
of the N-terminal 7 amino acids.
[0051] As used herein, GUSOC18-GILTO1-7'refers to a fusion protein in which
GUSOC18 is fused to the N-terminus of GILTO1-7.
[0052] The present invention facilitates treatment of metabolic diseases by
providing
targeted therapeutics that, when provided externally to a cell, enter the cell
and localize to a
subcellular compartment where the targeted therapeutic is active. The targeted
therapeutic
includes at least a therapeutic agent and a targeting moiety, such as a
subcellular targeting
domain of a protein, or, for lysosomal targeting, a means (e.g. a protein,
peptide, peptide analog,
or organic chemical) for binding the human cation-independent mannose-6-
phosphate receptor.
Association between therapeutic agent and targeting moiety
[0053] The therapeutic agent and the targeting moiety are necessarily
associated,
directly or indirectly. In one embodiment, the therapeutic agent and the
targeting moiety are
non-covalently associated. The association is preferably stable at or about pH
7.4. For example,
the targeting moiety can be biotinylated and bind avidin associated with the
therapeutic agent.
Alternatively, the targeting moiety and the therapeutic agent can each be
associated (e.g. as



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fusion proteins) with different subunits of a multimeric protein. In another
embodiment, the
targeting moiety and the therapeutic agent are crosslinked to each other (e.g.
using a chemical
crosslinking agent).
[0054] In a preferred embodiment, the therapeutic agent is fused to the
targeting
moiety as a fusion protein. The targeting moiety can be at the amino-terminus
of the fusion
protein, the carboxy-terminus, or can be inserted within the sequence of the
therapeutic agent at a
position where the presence of the targeting moiety does not unduly interfere
with the therapeutic
activity of the therapeutic agent.
[0055] Where the therapeutic agent is a heteromeric protein, one or more of
the
subunits can be associated with a targeting portion. Hexosaminidase A, for
example, a
lysosomal protein affected in Tay-Sachs disease, includes an alpha subunit and
a beta subunit.
The alpha subunit, the beta subunit, or both can be associated with a
targeting moiety in
accordance with the present invention. If, for example, the alpha subunit is
associated with a
targeting moiety and is coexpressed with the beta subunit, an active complex
is formed and
targeted appropriately (e.g. to the lysosome).
[005G] For targeting a therapeutic to the lysosome, the therapeutic agent can
be
connected to the targeting moiety through an interaction that is disrupted by
decreasing the pH
from at or about 7.4 to at or about 5.5. The targeting moiety binds a receptor
on the exterior of a
cell; the selected receptor is one that undergoes endocytosis and passes
through the late
endosome, which has a pH of about 5.5. Thus, in the late endosome, the
therapeutic agent
dissociates from the targeting moiety and proceeds to the lysosome, where the
therapeutic agent
acts. For example, a targeting moiety can be chemically modified to
incorporate a chelating
agent (e.g. EDTA, EGTA, or trinitrilotriacetic acid) that tightly binds a
metal ion such as nickel.
The targeting moiety (e.g. GUS) can be expressed as a fusion protein with a
six-histidine tag
(e.g. at the amino-terminus, at the carboxy-terminus, or in a surface-
accessible flexible loop). At
or about pH 7.4, the six-histidine tag is substantially deprotonated and binds
metal ions such as
nickel with high affinity. At or about pH 5.5, the six-histidine tag is
substantially protonated,
leading to release of the nickel and, consequently, release of the therapeutic
agent from the
targeting moiety.
Therapeutic agent
[0057] While methods and compositions of the invention are useful for
producing
and delivering any therapeutic agent to a subcellular compartment, the
invention is particularly
useful for delivering gene products for treating metabolic diseases.



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[0058] Preferred LSD genes are shown in Table 1, and preferred genes
associated
with golgi or ER defects are shown in Table 2. In a preferred embodiment, a
wild-type LSD
gene product is delivered to a patient suffering from a defect in the same LSD
gene. In
alternative embodiments, a functional sequence or species variant of the LSD
gene is used. In
further embodiments, a gene coding for a different enzyme that can rescue an
LSD gene defect is
used according to methods of the invention.
Table 1. Lysosomal Storage Diseases and associated enzyme defects
A. Glycogenosis
Disorders


Disease Name Enzyme Defect Substance
Stored


Pompe Disease Acid-al, 4- Glycogen a 1-4
Glucosidase linked
Oligosaccharides



B. Glycolipidosis
Disorders


Disease Name Enzyme Defect Substance
Stored


GM1 Gangliodsidosis(3-GalactosidaseGMl Ganliosides


Tay-Sachs Disease(3-HexosaminidaseGMa Ganglioside
A


GM2 Gangliosidosis:GM2 Activator GMa Ganglioside
AB Variant Protein


Sandhoff Disease (3-HexosaminidaseGMa Ganglioside
A&B


Fabry Disease oc-GalactosidaseGlobosides
A


Gaucher Disease GlucocerebrosidaseGlucosylceramide


Metachromatic Arylsulfatase Sulphatides
Leukodystrophy A


Krabbe Disease GalactosylceramidaseGalactocerebroside


Niemann-Pick, Acid Sphingomyelin
Types Sphingomyelinase
A and B


Niemann-Pick, Cholesterol Sphingomyelin
Type





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C Esterification
Defect


Nieman-Pick, TypeUnknown Sphingomyelin
D


Farber Disease Acid Ceramidase Ceramide


Wolman Disease Acid Lipase Cholesteryl


Esters



C. Mucopolysaccharide
Disorders


Disease Name Enzyme Defect Substance


Stored


Hurler Syndrome a-L-Iduronidase Heparan &


(MPS .IH) Dermatan


Sulfates


Scheie Syndrome a-L-Iduronidase Heparan &


(MPS IS) Dermatan, Sulfates


Hurler-Scheie a-L-Iduronidase Heparan &


(MPS IH/S) Dermatan


Sulfates


Hunter Syndrome Iduronate SulfataseHeparan &


(MPS II) Dermatan


Sulfates


Sanfilippo A Heparan N-SulfataseHeparan


(MPS IIIA) Sulfate


Sanfilippo B a-N- Heparan


(MPS IIIB) AcetylglucosaminidaseSulfate


Sanfilippo C Acetyl-CoA- Heparan


(MPS IIIC) Glucosaminide Sulfate


Acetyltransferase


Sanfilippo D N-AcetylglucosamineHeparan


(MPS IIID) -6-Sulfatase Sulfate


Morquio A Galactosamine-6- Keratan


(MPS IVA) Sulfatase Sulfate





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Morquio B [3-Galactosidase I~eratan
(MPS IVB) Sulfate


Maroteaux-Lamy Arylsulfatase Dermatan
(MPS VI) B Sulfate


Sly Syndrome (3-Glucuronidase
(MPS VII)



D. Oligosaccharide/Glycoprotein
Disorders


Disease Name Enzyme Defect Substance
Stored


a-Mannosidosis a-Mannosidase Mannose/Oligosacchar
ides


(3-Mannosidosis (3-Mannosidase Mannose/Oligosacchar
ides


Fucosidosis a-L-Fucosidase Fucosyl
Oligosaccharides


AsparylglucosaminuriaN-Aspartyl- (3- Asparylglucosamine
Glucosaminidase Asparagines


Sialidosis a-Neuraminidase Sialyloligosaccharides
(Mucolipidosis
I)


GalactosialidosisLysosomal ProtectiveSialyloligosaccharides
(Goldberg Syndrome)Protein Deficiency


Schindler Diseasea-N-Acetyl-
Galactosaminidase



E. Lysosomal Enzyme
Transport Disorders


Disease Name Enzyme Defect Substance
Stored


Mucolipidosis N-Acetylglucosamine-Heparan Sulfate
II (I- 1- Phosphotransferase
Cell Disease)





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Mucolipidosis III Same as ML II
(Pseudo-Hurler
Polydystrophy)
F. Lysosomal Membrane
Transport Disorders


Disease Name Enzyme Defect Substance
~


Stored


Cystinosis Cystine TransportFree Cystine


Protein


Salla Disease Sialic Acid TransportFree Sialic Acid
and


Protein Glucuronic Acid


Infantile Sialic Sialic Acid TransportFree Sialic Acid
Acid and


Storage Disease Protein Glucuronic Acid



C~. Other


Disease Name Enzyme Defect Substance


Stored


Batten Disease Unknown Lipofuscins


(Juvenile Neuronal


Ceroid


Lipofuscinosis)


Infantile NeuronalPalmitoyl-ProteinLipofuscins


Ceroid LipofuscinosisThioesterase


Mucolipidosis Unknown Gangliosides &
IV


Hyaluronic Acid


Prosaposin Saposins A, B,
C or D


Table 2. Diseases of the golgi and ER
Disease Name Gene and Enzyme DefectFeatures



Ehlers-Danlos SyndromePLOD1 lysyl hydroxylaseDefect in lysyl hydroxylation
Type


~I of Collagen; located
in ER





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lumen



Type Ia glycoge storageglucose6 phosphatase Causes excessive


disease accumulation of Glycogen
in


the liver, kidney,
and


Intestinal mucosa;
enzyme is


transmembrane but active
site


is ER lumen


Co~gehital Diso~de~s of Glycosylation
CDG Ic ALG6 Defects in N-glycosylation
ER


a1,3 glucosyltransferaselumen


CDG Id ALG3 Defects in N-glycosylation
ER


a1,3 mannosyltransferasetransmembrane protein


CDG IIa MGAT2 Defects in N-glycosylation


N-acetylglucosaminyl- golgi transmembrane
protein


transferase II


CDG IIb GCS 1 Defect in N glycosylation


a1,2-Glucosidase I ER membrane bound with


lumenal catalytic domain


releasable by proteolysis


[0059] One particularly preferred therapeutic agent is glucocerebrosidase,
currently
manufactured by Genzyme as an effective enzyme replacement therapy for
Gaucher's Disease.
Currently, the enzyme is prepared with exposed mannose residues, which targets
the protein
specifically to cells of the macrophage lineage. Although the primary
pathology in type 1
Gaucher patients are due to macrophage accumulating glucocerebroside, there
can be therapeutic
advantage to delivering glucocerebrosidase to other cell types. Targeting
glucocerebrosidase to
lysosomes using the present invention would target the agent to multiple cell
types and can have
a therapeutic advantage compared to other preparations.
[0060] Another preferred therapeutic agent is acid alpha-glucosidase (GAA), a
lysosomal enzyme deficient in Pompe disease (see discussion in Example 13B).
Pompe disease,



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also known as acid maltase deficiency (AMD), glycogen storage disease type II
(GSDII),
glycogenosis type II, or GAA deficiency, is a lysosomal storage disease
resulting from
insufficient activity of GAA, the enzyme that hydrolyzes the a 1-4 linkage in
maltose and other
linear oligosacchaxides, including the outer branches of glycogen, thereby
breaking down excess
glycogen in the lysosome (Hirschhorn et al. (2001) in The Metabolic and
Molecular Basis of
Inherited Disease, Scriver, et al., eds. (2001), McGraw-Hill: New York, p.
3389-3420). The
diminished enzymatic activity occurs due to a variety of missense and nonsense
mutations in the
gene encoding GAA. Consequently, glycogen accumulates in the lysosomes of all
cells in
patients with Pompe disease. In particular, glycogen accumulation is most
pronounced in
lysosomes of cardiac and skeletal muscle, liver, and other tissues.
Accumulated glycogen
ultimately impairs muscle function. In the most severe form of Pompe disease
death occurs
before two years of age due to cardio-respiratory failure.
[OOG1] Presently, there is no approved treatment available to cure or slow the
progress of Pompe disease. Enzyme replacement therapy currently in clinical
trials requires that
administered recombinant GAA be taken up by the cells in muscle and liver
tissues and be
transported to the lysosomes in those cells. However, recombinant GAA produced
in engineered
CHO cells and in milk of transgenic rabbits currently used in enzyme
replacement therapy
contains extremely little M6P (Van Hove et al. (1996) Proc Natl Acad Sci U S
A, 93(1):65-70;
and U.S. Patent No. 6,537,785). Therefore, M6P-dependent delivery of
recombinant GAA to
lysosomes is not efficient, requiring high dosages and frequent infusions. The
present invention,
in contrast, permits M6P-independent targeting of GAA to patient lysosomes, as
described in
greater detail in Example 13B.
Subcellula~ ta~getihg domains
[OOG2] The present invention permits targeting of a therapeutic agent to a
lysosome
using a protein, or an analog of a protein, that specifically binds a cellular
receptor for that
protein. The exterior of the cell surface is topologically equivalent to
endosomal, lysosomal,
golgi, and endoplasmic reticulum compartments. Thus, endocytosis of a molecule
through
interaction with an appropriate receptors) permits transport of the molecule
to any of these
compartments without crossing a membrane. Should a genetic deficiency result
in a deficit of a
particular enzyme activity in any of these compartments, delivery of a
therapeutic protein can be
achieved by tagging it with a ligand for the appropriate receptor(s).
[OOG3] Multiple pathways directing receptor-bound proteins from the plasma
membrane to the golgi and/or endoplasmic reticulum have been characterized.
Thus, by using a



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targeting portion from, for example, SV40, cholera toxin, or the plant toxin
ricin, each of which
coopt one or more of these subcellular trafficking pathways, a therapeutic can
be targeted to the
desired location within the cell. In each case, uptake is initiated by binding
of the material to the
exterior of the cell. For example, SV40 binds to MHC class I receptors,
cholera toxin binds to
GM1 ganglioside molecules and ricin binds to glycolipids and glycoproteins
with terminal
galactose on the surface of cells. Following this initial step the molecules
reach the ER by a
variety of pathways. For example, SV40 undergoes caveolar endocytosis and
reaches the ER in
a two step process that bypasses the golgi whereas cholera toxin undergoes
caveolar endocytosis
but traverses the golgi before reaching the ER.
[OOG4] If a targeting moiety related to cholera toxin or ricin is used, it is
important
that the toxicity of cholera toxin or ricin be avoided. Both cholera.toxin and
ricin are
heteromeric proteins, and the cell surface binding domain and the catalytic
activities responsible
for toxicity reside on separate polypeptides. Thus, a targeting moiety can be
constructed that
includes the receptor-binding polypeptide, but not the polypeptide responsible
for toxicity. For
example, in the case of ricin, the B subunit possesses the galactose binding
activity responsible
for internalization of the protein, and can be fused to a therapeutic protein.
If the further
presence of the A subunit improves subcellular localization, a mutant version
(mutein) of the A
chain that is properly folded but catalytically inert can be provided with the
B subunit-
therapeutic agent fusion protein.
[DOGS] Proteins delivered to the golgi can be transported to the endoplasmic
reticulum (ER) via the I~DEL receptor, which retrieves ER-targeted proteins
that have escaped to
the golgi. Thus, inclusion of a KDEL motif at the terminus of a targeting
domain that directs a
therapeutic protein to the golgi permits subsequent localization to the ER.
For example, a
targeting moiety (e.g. an antibody, or a peptide identified by high-throughput
screening such as
phage display, yeast two hybrid, chip-based assays, and solution-based assays)
that binds the
cation-independent M6P receptor both at or about pH 7.4 and at or about pH 5.5
permits
targeting of a therapeutic agent to the golgi; further addition of a KDEL
motif permits targeting
to the ER.
Lysosomal targeting moieties
[OOGG] The invention permits targeting of a therapeutic agent to a lysosome.
Targeting may occur, for example, through binding of a plasma membrane
receptor that later
passes through a lysosome. Alternatively, targeting may occur through binding
of a plasma
receptor that later passes through a late endosome; the therapeutic agent can
then travel from the



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late endosome to a lysosome. A preferred lysosomal targeting mechanism
involves binding to
the canon-independent M6P receptor.
Cation-independent M6P receptor
[OOG7] The cation-independent M6P receptor is a 275 kDa single chain
transmembrane glycoprotein expressed ubiquitously in mammalian tissues. It is
one of two
mammalian receptors that bind M6P: the second is referred to as the cation-
dependent M6P
receptor. The cation-dependent M6P receptor requires divalent cations for M6P
binding; the
cation-independent M6P receptor does not. These receptors play an important
role in the
trafficking of lysosomal enzymes through recognition of the M6P moiety on high
mannose
carbohydrate on lysosomal enzymes. The extracellular domain of the cation-
independent M6P
receptor contains 15 homologous domains ("repeats") that bind a diverse group
of ligands at
discrete locations on the receptor.
[OOGB] The cation-independent M6P receptor contains two binding sites for MGP:
one
located in repeats 1-3 and the other located in repeats 7-9. The receptor
binds monovalent M6P
ligands with a dissociation constant in the ~.M range while binding divalent
M6P ligands with a
dissociation constant in the nM range, probably due to receptor
oligomerization. Uptake of IGF-
II by the receptor is enhanced by concomitant binding of multivalent M6P
ligands such as
lysosomal enzymes to the receptor.
[OOG9] The cation-independent M6P receptor also contains binding sites for at
least
three distinct ligands that can be used as targeting moieties. The cation-
independent M6P
receptor binds IGF-II with a dissociation constant of about 14 nM at or about
pH 7.4, primarily
through interactions with repeat 11. Consistent with its function in targeting
IGF-II to the
lysosome, the dissociation constant is increased approximately 100-fold at or
about pH 5.5
promoting dissociation of IGF-II in acidic late endosomes. The receptor is
capable of binding
high molecular weight O-glycosylated IGF-II forms.
[0070] An additional useful ligand for the cation-independent M6P receptor is
retinoic acid. Retinoic acid binds to the receptor with a dissociation
constant of 2.5 nM. Affinity
photolabeling of the cation-independent M6P receptor with retinoic acid does
not interfere with
IGF-II or M6P binding to the receptor, indicating that retinoic acid binds to
a distinct site on the
receptor. Binding of retinoic acid to the receptor alters the intracellular
distribution of the
receptor with a greater accumulation of the receptor in cytoplasmic vesicles
and also enhances
uptake of M6P modified 13-glucuronidase. Retinoic acid has a photoactivatable
moiety that can



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be used to link it to a therapeutic agent without interfering with its ability
to bind to the cation-
independent M6P receptor.
[0071] The cation-independent M6P receptor also binds the urokinase-type
plasminogen receptor (uPAR) with a dissociation constant of 9 ~M. uPAR is a
GPI-anchored
receptor on the surface of most cell types where it functions as an adhesion
molecule and in the
proteolytic activation of plasminogen and TGF-13. Binding of uPAR to the CI-
M6P receptor
targets it to the lysosome, thereby modulating its activity. Thus, fusing the
extracellular domain
of uPAR, or a portion thereof competent to bind the cation-independent M6P
receptor, to a
therapeutic agent permits targeting of the agent to a lysosome.
IGF II
[0072] In a preferred embodiment, the lysosomal targeting portion is a
protein,
peptide, or other moiety that binds the cation independent M6P/IGF-II receptor
in a mannose-6-
phosphate-independent manner. Advantageously, this embodiment mimics the
normal biological
mechanism for uptake of LSD proteins, yet does so in a manner independent of
mannose-6-
phosphate.
[0073] For example, by fusing DNA encoding the mature IGF-II polypeptide to
the 3'
end of LSD gene cassettes, fusion proteins are created that can be taken up by
a variety of cell
types and transported to the lysosome. Alternatively, DNA encoding a precursor
IGF-II
polypeptide can be fused to the 3' end of an LSD gene cassette; the precursor
includes a
carboxyterminal portion that is cleaved in mammalian cells to yield the mature
IGF-II
polypeptide, but the IGF-II signal peptide is preferably omitted (or moved to
the 5' end of the
LSD gene cassette). This method has numerous advantages over methods involving
glycosylation including simplicity and cost effectiveness, because once the
protein is isolated, no
further modifications need be made.
[0074] IGF-II is preferably targeted specifically to the M6P receptor.
Particularly
useful are mutations in the IGF-II polypeptide that result in a protein that
binds the M6P receptor
with high affinity while no longer binding the other two receptors with
appreciable affinity.
IGF-II can also be modified to minimize binding to serum IGF-binding proteins
(Baxter (2000)
Am. J. Physiol Endocrinol Metab. 278(6):967-76) to avoid sequestration of IGF-
II/GILT
constructs. A number of studies have localized residues in IGF-1 and IGF-II
necessary for
binding to IGF-binding proteins. Constructs with mutations at these residues
can be screened
for retention of high affinity binding to the M6P/IGF-II receptor and for
reduced affinity for



CA 02487815 2004-11-26
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-21 -
IGF-binding proteins. For example, replacing PHE 26 of IGF-II with SER is
reported to reduce
affinity of IGF-II for IGFBP-1 and -6 with no effect on binding to the M6P/IGF-
II receptor
(Bach et al. (1993) J. Biol. Chem. 268(13):9246-54). Other substitutions, such
as SER for PHE
19 and LYS for GLU 9, can also be advantageous. The analogous mutations,
separately or in
combination, in a region of IGF-I that is highly conserved with IGF-II result
in large decreases in
IGF-BP binding (Magee et al. (1999) Biochemistry 38(48):15863-70).
[0075] An alternate approach is to identify minimal regions of IGF-II that can
bind
with high affinity to the M6P/IGF-II receptor. The residues that have been
implicated in IGF-II
binding to the M6P/IGF-II receptor mostly cluster on one face of IGF-II
(Terasawa et al. (1994)
EMBO J. 13(23):5590-7). Although IGF-II tertiary structure is normally
maintained by three
intramolecular disulfide bonds, a peptide incorporating the amino acid
sequence on the
M6P/IGF-II receptor binding surface of IGF-II can be designed to fold properly
and have
binding activity. Such a minimal binding peptide is a highly preferred
targeting portion.
Designed peptides based on the region around amino acids 48-55 can be tested
for binding to the
M6P/IGF-II receptor. Alternatively, a random library of peptides can be
screened for the ability
to bind the M6P/IGF-II receptor either via a yeast two hybrid assay, or via a
phage display type
assay.
Blood brain barrier
[0076] One challenge in therapy for lysosomal storage diseases is that many of
these
diseases have significant neurological involvement. Therapeutic enzymes
administered into the
blood stream generally do not cross the blood brain barrier and therefore
cannot relieve
neurological symptoms associated with the diseases. IGF-II, however, has been
reported to
promote transport across the blood brain barrier via transcytosis (Biclcel et
al. (2001) Adv. Drug
Deliv. Rev. 46(1-3):247-79). Thus, appropriately designed GILT constructs
should be capable of
crossing the blood brain barrier, affording for the first time a means of
treating neurological
symptoms associated with lysosomal storage diseases. The constructs can be
tested using GUS
minus mice as described in Example 12. Further details regarding design,
construction and
testing of targeted therapeutics that can reach neuronal tissue from blood are
disclosed in U.S.
Serial No. 60/329,650, filed October 16, 2001, and in U.S. Serial No.
10/136,639, filed April 30,
2002.
Structure of IGF-II
[0077] NMR structures of IGF-II have been solved by two groups (Terasawa et
al.
(1994) EMBO J. 13(23):5590-7; Torres et al. (1995) J. Mol. Biol. 248(2):385-
401) (see, e.g.,



CA 02487815 2004-11-26
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Protein Data Bank record l IGL). The general features of the IGF-II structure
are similar to IGF-
I and insulin. The A and B domains of IGF-II correspond to the A and B chains
of insulin.
Secondary structural features include an alpha helix from residues 11-21 of
the B region
connected by a reverse turn in residues 22-25 to a short beta strand in
residues 26-28. Residues
25-27 appear to form a small antiparallel beta sheet; residues 59-61 and
residues 26-28 may also
participate in intermolecular beta-sheet formation. In the A domain of IGF-II,
alpha helices
spanning residues 42-49 and 53-59 are arranged in an antiparallel
configuration perpendicular to
the B-domain helix. Hydrophobic clusters formed by two of the three disulfide
bridges and
conserved hydrophobic residues stabilize these secondary structure features.
The N and C
termini remain poorly defined as is the region between residues 31-40.
[0078] IGF-II binds to the IGF-II/M6P and IGF-I receptors with relatively high
affinity and binds with lower affinity to the insulin receptor. IGF-II also
interacts with a number
if serum IGFBPs.
Binding to the IGF-II/M6P receptor
[0079] Substitution of IGF-II residues 48-50 (Phe Arg Ser) with the
corresponding
residues from insulin, (Thr Ser Ile), or substitution of residues 54-55 (Ala
Leu) with the
corresponding residues from IGF-I (Arg Arg) result in diminished binding to
the IGF-II/ M6P
receptor but retention of binding to the IGF-I and insulin receptors (Sakano
et al. (1991) J. Biol.
Chem. 266(31):20626-35).
[0080] IGF-I and IGF-II share identical sequences and structures in the region
of
residues 48-50 yet have a 1000-fold difference in affinity for the IGF-II
receptor. The NMR
structure reveals a structural difference between IGF-I and IGF-II in the
region of IGF-II
residues 53-58 (IGF-I residues 54-59): the alpha-helix is better defined in
IGF-II than in IGF-I
and, unlike IGF-I, there is no bend in the backbone around residues 53 and 54
(Torres et al.
(1995) J. Mol. Biol. 248(2):385-401). This structural difference correlates
with the substitution
of Ala 54 and Leu 55 in IGF-II with Arg 55 and Arg 56 in IGF-I. It is possible
either that
binding to the IGF-II receptor is disrupted directly by the presence of
charged residues in this
region or that changes in the structure engendered by the charged residues
yield the changes in
binding for the IGF-II receptor. In any case, substitution of uncharged
residues for the two Arg
residues in IGF-I resulted in higher affinities for the IGF-II receptor
(Cacciari et al. (1987)
Pediatrician 14(3):146-53). Thus the presence of positively charged residues
in these positions
correlates with loss of binding to the IGF-II receptor.



CA 02487815 2004-11-26
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[0081] IGF-II binds to repeat 11 of the cation-independent M6P receptor.
Indeed, a
minireceptor in which only repeat 11 is fused to the transmembrane and
cytoplasmic domains of
the cation-independent M6P receptor is capable of binding IGF-II (with an
affinity
approximately one tenth the affinity of the full length receptor) and
mediating internalization of
IGF-II and its delivery to lysosomes (Grimme et al. (2000) J. Biol. Chem.
275(43):33697-
33703). The structure of domain 11 of the M6P receptor is known (Protein Data
Base entries
1GP0 and 1GP3; Brown et al. (2002) EMBO J. 21(5):1054-1062). The putative IGF-
II binding
site is a hydrophobic pocket believed to interact with hydrophobic amino acids
of IGF-II;
candidate amino acids of IGF-II include leucine 8, phenylalanine 48, alanine
54, and leucine 55.
Although repeat 11 is sufficient for IGF-II binding, constructs including
larger portions of the
cation-independent M6P receptor (e.g. repeats 10-13, or 1-15) generally bind
IGF-II with greater
affinity and with increased pH dependence (see, for example, Linnell et al.
(2001) J. Biol. Chem.
276(26):23986-23991).
Binding to the IGF-I receptor
[0082] Substitution of IGF-II residues Tyr 27 with Leu, Leu 43 with Val or Ser
26
with Phe diminishes the affinity of IGF-II for the IGF-I receptor by 94-, 56-,
and 4-fold
respectively (Tomes et al. (1995) J. Mol. Biol. 248(2):385-401). Deletion of
residues 1-7 of
human IGF-II resulted in a 30-fold decrease in affinity for the human IGF-I
receptor and a
concomitant 12 fold increase in affinity for the rat IGF-II receptor
(Hashimoto et al. (1995) J.
Biol. Chem. 270(30):18013-8). The NMR structure of IGF-II shows that Thr 7 is
located near
residues 48 Phe and 50 Ser as well as near the 9 Cys-47 Cys disulfide bridge.
It is thought that
interaction of Thr 7 with these residues can stabilize the flexible N-terminal
hexapeptide required
for IGF-I receptor binding (Terasawa et al. (1994) EMBO J. 13(23)5590-7). At
the same time
this interaction can modulate binding to the IGF-II receptor. Truncation of
the C-terminus of
IGF-II (residues 62-67) also appear to lower the affinity of IGF-II for the
IGF-I receptor by 5
fold (Roth et al. (1991) Biochem. Biophys. Res. Common. 181(2):907-14).
Deletion mutants of IGF-II
[0083] The binding surfaces for the IGF-I and cation-independent M6P receptors
are
on separate faces of IGF-II. Based on structural and mutational data,
functional cation-
independent M6P binding domains can be constructed that are substantially
smaller than human
IGF-II. For example, the amino terminal amino acids 1-7 and/or the carboxy
terminal residues
62-67 can be deleted or replaced. Additionally, amino acids 29-40 can likely
be eliminated or



CA 02487815 2004-11-26
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-24-
replaced without altering the folding of the remainder of the polypeptide or
binding to the cation-
independent M6P receptor. Thus, a targeting moiety including amino acids 8-28
and 41-61 can
be constructed. These stretches of amino acids could perhaps be joined
directly or separated by a
linker. Alternatively, amino acids 8-28 and 41-61 can be provided on separate
polypeptide
chains. Comparable domains of insulin, which is homologous to IGF-II and has a
tertiary
structure closely related to the structure of IGF-II, have sufficient
structural information to
permit proper refolding into the appropriate tertiary structure, even when
present in separate
polypeptide chains (Wang et al. (1991) Trends Biochem. Sci. 279-281). Thus,
for example,
amino acids 8-28, or a conservative substitution variant thereof, could be
fused to a therapeutic
agent; the resulting fusion protein could be admixed with amino acids 41-61,
or a conservative
substitution variant thereof, and administered to a patient.
Binding to IGF Binding_proteins
[0084] IGF-II and related constructs can be modified to diminish their
affinity for
IGFBPs, thereby increasing the bioavailability of the tagged proteins.
[0085] Substitution of IGF-II residue phenylalanine 26 with serine reduces
binding to
IGFBPs 1-5 by 5-75 fold (Bath et al. (1993) J. Biol. Chem. 268(13):9246-54).
Replacement of
IGF-II residues 48-50 with threonine-serine-isoleucine reduces binding by more
than 100 fold to
most of the IGFBPs (Bath et al. (1993) J. Biol. Chem. 268(13):9246-54); these
residues are,
however, also important for binding to the cation-independent mannose-6-
phosphate receptor.
The Y27L substitution that disrupts binding to the IGF-I receptor interferes
with fornlation of the
ternary complex with IGFBP3 and acid labile subunit (Hashimoto et al. (1997)
J. Biol. Chem.
272(44):27936-42); this ternary complex accounts for most of the IGF-II in the
circulation (Yu et
al. (1999) J. Clin. Lab Anal. 13(4):166-72). Deletion of the first six
residues of IGF-II also
interferes with IGFBP binding (Luthi et al. (1992) Eur. J. Biochem. 205(2):483-
90).
[008G] Studies on IGF-I interaction with IGFBPs revealed additionally that
substitution of serine for phenylalanine 16 did not effect secondary structure
but decreased
IGFBP binding by between 40 and 300 fold (Magee et al. (1999) Biochemistry
38(48):15863-
70). Changing glutamate 9 to lysine also resulted in a significant decrease in
IGFBP binding.
Furthermore, the double mutant lysine 9/ serine 16 exhibited the lowest
affinity for IGFBPs.
Although these mutations have not previously been tested in IGF-II, the
conservation of
sequence between this region of IGF-I and IGF-II suggests that a similar
effect will be observed
when the analogous mutations are made in IGF-II (glutamate 12 lysine/
phenylalanine 19 serine).
IGF-II homolo ~s



CA 02487815 2004-11-26
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- 25 -
[0087] The amino acid sequence of human IGF-II, or a portion thereof affecting
binding to the cation-independent M6P receptor, may be used as a reference
sequence to
determine whether a candidate sequence possesses sufficient amino acid
similarity to have a
reasonable expectation of success in the methods of the present invention.
Preferably, variant
sequences are at least 70% similar or 60% identical, more preferably at least
75% similar or 65%
identical, and most preferably 80% similar or 70% identical to human IGF-II.
[0088] To determine whether a candidate peptide region has the requisite
percentage
similarity or identity to human IGF-II, the candidate amino acid sequence and
human IGF-II are
first aligned using the dynamic programming algorithm described in Smith and
Waterman (1981)
J. Mol. Biol. 147:195-197, in combination with the BLOSUM62 substitution
matrix described in
Figure 2 of Henikoff and Henikoff (1992) PNAS 89:10915-10919. For the present
invention, an
appropriate value for the gap insertion penalty is -12, and an appropriate
value for the gap
extension penalty is -4. Computer programs performing alignments using the
algorithm of
Smith-Waterman and the BLOSUM62 matrix, such as the GCG program suite (Oxford
Molecular Group, Oxford, England), are commercially available and widely used
by those
skilled in the art.
[0089] Once the alignment between the candidate and reference sequence is
made, a
percent similarity score may be calculated. The individual amino acids of each
sequence are
compared sequentially according to their similarity to each other. If the
value in the
BLOSUM62 matrix corresponding to the two aligned amino acids is zero or a
negative munber,
the pairwise similarity score is zero; otherwise the pairwise similarity score
is 1Ø The raw
similarity score is the sum of the pairwise similarity scores of the aligned
amino acids. The raw
score is then normalized by dividing it by the number of amino acids in the
smaller of the
candidate or reference sequences. The normalized raw score is the percent
similarity.
Alternatively, to calculate a percent identity, the aligned amino acids of
each sequence are again
compared sequentially. If the amino acids are non-identical, the pairwise
identity score is zero;
otherwise the pairwise identity score is 1Ø The raw identity score is the
sum of the identical
aligned amino acids. The raw score is then normalized by dividing it by the
number of amino
acids in the smaller of the candidate or reference sequences. The normalized
raw score is the
percent identity. Insertions and deletions are ignored for the purposes of
calculating percent
similarity and identity. Accordingly, gap penalties are not used in this
calculation, although they
are used in the initial alignment.



CA 02487815 2004-11-26
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IGF-II structural analogs
[0090] The known structures of human IGF-II and the cation-independent M6P
receptors permit the design of IGF-II analogs and other cation-independent M6P
receptor
binding proteins using computer-assisted design principles such as those
discussed in U.S. Patent
Nos. 6,226,603 and 6,273,598. For example, the known atomic coordinates of IGF-
II can be
provided to a computer equipped with a conventional computer modeling program,
such as
INSIGHTII, DISCOVER, or DELPHI, commercially available from Biosym,
Technologies Inc.,
or QUANTA, or CHARMM, commercially available from Molecular Simulations, Inc.
These
and other software programs allow analysis of molecular structures and
simulations that predict
the effect of molecular changes on structure and on intermolecular
interactions. For example, the
software can be used to identify modified analogs with the ability to form
additional
intermolecular hydrogen or ionic bonds, improving the affinity of the analog
for the target
receptor.
[0091] The software also permits the design of peptides and organic molecules
with
structural and chemical features that mimic the same features displayed on at
least part of the
surface of the cation-independent M6P receptor binding face of IGF-II. Because
a major
contribution to the receptor binding surface is the spatial arrangement of
chemically interactive
moieties present within the sidechains of amino acids which together define
the receptor binding
surface, a preferred embodiment of the present invention relates to designing
and producing a
synthetic organic molecule having a framework that carries chemically
interactive moieties in a
spatial relationship that mimics the spatial relationship of the chemical
moieties disposed on the
amino acid sidechains which constitute the cation-independent M6P receptor
binding face of
IGF-II. Preferred chemical moieties, include but are not limited to, the
chemical moieties defined
by the amino acid side chains of amino acids constituting the cation-
independent M6P receptor
binding face of IGF-II. It is understood, therefore, that the receptor binding
surface of the IGF-II
analog need not comprise amino acid residues but the chemical moieties
disposed thereon.
[0092] For example, upon identification of relevant chemical groups, the
skilled
artisan using a conventional computer program can design a small molecule
having the receptor
interactive chemical moieties disposed upon a suitable carrier framework.
Useful computer
programs are described in, for example, Dixon (1992) Tibtech 10: 357-363;
Tschinke et al.
(1993) J. Med. Chem 36: 3863-3870; and Eisen el al. (1994) Proteins:
Structure, Function, and
Genetics 19: 199-221, the disclosures of which are incorporated herein by
reference.



CA 02487815 2004-11-26
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-27-
[0093] One particular computer program entitled "CAVEAT" searches a database,
for
example, the Cambridge Structural Database, for structures which have desired
spatial
orientations of chemical moieties (Bartlett et al. (1989) in "Molecular
Recognition: Chemical and
Biological Problems" (Roberts, S. M., ed) pp 182-196). The CAVEAT program has
been used to
design analogs of tendamistat, a 74 residue inhibitor of a-amylase, based on
the orientation of
selected amino acid side chains in the three-dimensional structure of
tendamistat (Bartlett et al.
(1989) supra).
[0094] Alternatively, upon identification of a series of analogs which mimic
the
cation-independent M6P receptor binding activity of IGF-II, the skilled
artisan may use a variety
of computer programs which assist the skilled artisan to develop quantitative
structure activity
relationships (QSAR) and further to assist in the de novo design of additional
morphogen
analogs. Other useful computer programs are described in, for example,
Connolly-Martin (1991)
Methods in Enzymology 203:587-613; Dixon (1992) supra; and Waszkowycz et al.
(1994) J.
Med. Chenm. 37: 3994-4002.
Ta~getihg moiety affinities
[0095] Preferred targeting moieties bind to their target receptors with a
submicromolar dissociation constant. Generally speaking, lower dissociation
constants (e.g. less
than 10-7 M, less than 10-8 M, or less than 10-9 M) are increasingly
preferred. Determination of
dissociation constants is preferably determined by surface plasmon resonance
as described in
Linnell et al. (2001) J. Biol. Chem. 276(26):23986-23991. A soluble form of
the extracellular
domain of the target receptor (e.g. repeats 1-15 of the cation-independent M6P
receptor) is
generated and immobilized to a chip through an avidin-biotin interaction. The
targeting moiety
is passed over the chip, and kinetic and equilibrium constants are detected
and calculated by
measuring changes in mass associated with the chip surface.
Nucleic acids and expression systems
[009G] Chimeric fusion proteins can be expressed in a variety of expression
systems,
including i~ vitro translation systems and intact cells. Since M6P
modification is not a
prerequisite for targeting, a variety of expression systems including yeast,
baculovirus and even
prokaryotic systems such as E. coli that do not glycosylate proteins are
suitable for expression of
targeted therapeutic proteins. In fact, an unglycosylated protein generally
has improved
bioavailability, since glycosylated proteins are rapidly cleared from the
circulation through
binding to the mannose receptor in hepatic sinusoidal endothelium.



CA 02487815 2004-11-26
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- 28 -
[0097] Alternatively, production of chimeric targeted lysosomal enzymes in
mammalian cell expression system produces proteins with multiple binding
determinants for the
ration-independent M6P receptor. Synergies between two or more ration-
independent M6P
receptor ligands (e.g. M6P and IGF-II, or M6P and retinoic acid) can be
exploited: multivalent
ligands have been demonstrated to enhance binding to the receptor by receptor
crosslinking.
[0098] In general, gene cassettes encoding the chimeric therapeutic protein
can be
tailored for the particular expression system to incorporate necessary
sequences for optimal
expression including promoters, ribosomal binding sites, introns, or
alterations in coding
sequence to optimize codon usage. Because the protein is preferably secreted
from the
producing cell, a DNA encoding a signal peptide compatible with the expression
system can be
substituted for the endogenous signal peptide. For example, for expression of
13-glucuronidase
and a-galactosidase A tagged with IGF-II in Leishmania, DNA cassettes encoding
Leishmania
signal peptides (GP63 or SAP) are inserted in place of the DNA encoding the
endogenous signal
peptide to achieve optimal expression. In mammalian expression systems the
endogenous signal
peptide may be employed but if the IGF-II tag is fused at the 5' end of the
coding sequence, it
could be desirable to use the IGF-II signal peptide.
[0099] GHO cells are a preferred mammalian host for the production of
therapeutic
proteins. The classic method for achieving high yield expression from CHO
cells is to use a
CHO cell line deficient in dihydrofolate reductase (DHFR), for example CHO
line DUI~X
(O'Dell et al. (1998) Int. J. Biochem. Cell Biol. 30(7):767-71). This strain
of CHO cells requires
hypoxanthine and thymidine for growth. Co-transfection of the gene to be
overexpressed with a
DHFR gene cassette, on separate plasmids or on a single plasmid, permits
selection for the
DHFR gene and generally allows isolation of clones that also express the
recombinant protein of
choice. For example, plasmid pcDNA3 uses the cytomegalovirus (CMV) early
region regulatory
region promoter to drive expression of a gene of interest and pSV2DHFR to
promote DHFR
expression. Subsequent exposure of cells harboring the recombinant gene
cassettes to
incrementally increasing concentrations of the folate analog methotrexate
leads to amplification
of both the gene copy number of the DHFR gene and of the co-transfected gene.
[0100] A preferred plasmid for eukaryotic expression in this system contains
the gene
of interest placed downstream of a strong promoter such as CMV. An intron can
be placed in the
3' flank of the gene cassette. A DHFR cassette can be driven by a second
promoter from the
same plasmid or from a separate plasmid. Additionally, it can be useful to
incorporate into the



CA 02487815 2004-11-26
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-29-
plasmid an additional selectable marker such as neomycin phosphotransferase,
which confers
resistance to 6418.
[0101] Another CHO expression system (LTlmasov et al.°(2000) PNAS
97(26):14212-
14217) relies on amplification of the gene of interest using 6418 instead of
the
DHFR/methotrexate system described above. A pC~t vector with a slightly
defective
neomycin phosphotransferase driven by a weak promoter (see, e.g., Niwa et al.
(1991) Gene
108:193-200) permits selection for transfectants with a high copy number
(>300) in a single step.
[0102] Alternatively, recombinant protein can be produced in the human HEK 293
cell line using expression systems based on the Epstein-Barr Virus (EBV)
replication system.
This consists of the EBV replication origin oriP and the EBV on binding
protein, EBNA-1.
Binding of EBNA-1 to oriP initiates replication and subsequent amplification
of the
extrachromosomal plasmid. This amplification in turn results in high levels of
expression of
gene cassettes housed within the plasmid. Plasmids containing o~iP can be
transfected into
EBNA-1 transformed HEK 293 cells (commercially available from Invitrogen) or,
alternatively,
a plasmid such as pCEP4 (commercially available from Invitrogen) which drives
expression of
EBNA-1 and contains the EBV oriP can be employed.
[0103] In E. coli, the therapeutic proteins are preferably secreted into the
periplasmic
space. This can be achieved by substituting for the DNA encoding the
endogenous signal
peptide of the LSD protein a nucleic acid cassette encoding a bacterial signal
peptide such as the
ompA signal sequence. Expression can be driven by any of a number of strong
inducible
promoters such as the lac, tip, or tac promoters. One suitable vector is
pBAD/gIII
(commercially available from Invitrogen) which uses the Gene III signal
peptide and the araBAD
promoter.
dh vitro refoldiszg
[0104] One useful IGF-II targeting portion has three intramolecular disulfide
bonds.
GILT fusion proteins (for example GLJS-GILT) in E. coli can be constructed
that direct the
protein to the periplasmic space. IGF-II, when fused to the C-terminus of
another protein, can be
secreted in an active form in the periplasm of E. coli (Wadensten et al.
(1991) Biotechnol. Appl.
Biochem. 13(3):412-21). To facilitate optimal folding of the IGF-II moiety,
appropriate
concentrations of reduced and oxidized glutathione are preferably added to the
cellular milieu to
promote disulfide bond formation. In the event that a fusion protein with
disulfide bonds is
incompletely soluble, any insoluble material is preferably treated with a
chaotropic agent such as
urea to solubilize denatured protein and refolded in a buffer having
appropriate concentrations of



CA 02487815 2004-11-26
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-30-
reduced and oxidized glutathione, or other oxidizing and reducing agents, to
facilitate formation
of appropriate disulfide bonds (Smith et al. (1989) J. Biol. Chem.
264(16):9314-21). For
example, IGF-I has been refolded using 6M guanidine-HCl and 0.1 M tris(2-
carboxyethyl)phosphine reducing agent for denaturation and reduction of IGF-II
(Yang et al.
(1999) J. Biol. Chem. 274(53):37598-604). Refolding of proteins was
accomplished in O.1M
Tris-HCl buffer (pH8.7) containing 1mM oxidized glutathione, 10 mM reduced
glutathione,
0.2M KCl and 1mM EDTA.
U~zderglycosylatioh
[0105] Targeted therapeutic proteins are preferably underglycosylated: one or
more
carbohydrate structures that would normally be present if the protein were
produced in a
mammalian cell is preferably omitted, removed, modified, or masked, extending
the half life of
the protein in a mammal. Underglycosylation can be achieved in many ways,
several of which
are diagrammed in Figure 1. As shown in Figure 1, a protein may be actually
underglycosylated,
actually lacking one or more of the carbohydrate structures, or functionally
underglycosylated
through modification or masking of one or more of the carbohydrate structures.
A protein may
be actually underglycosylated when synthesized, as discussed in Example 14,
and may be
completely unglycosylated (as when synthesized in E. coli), partially
unglycosylated (as when
synthesized in a mammalian system after disruption of one or more
glycosylation sites by site-
directed mutagenesis), or may have a non-mammalian glycosylation pattern.
Actual
underglycosylation can also be achieved by deglycosylation of a protein after
synthesis. As
discussed in Example 14, deglycosylation can be through chemical or enzymatic
treatments, and
may lead to complete deglycosylation or, if only a portion of the carbohydrate
structure is
removed, partial deglycosylation.
Ih vivo expression
[0106] A nucleic acid encoding a therapeutic protein, preferably a secreted
therapeutic protein, can be advantageously provided directly to a patient
suffering from a
disease, or may be provided to a cell ex vivo, followed by adminstration of
the living cell to the
patient. In vivo gene therapy methods known in the art include providing
purified DNA (e.g. as
in a plasmid) , providing the DNA in a viral vector, or providing the DNA in a
liposome or other
vesicle (see, for example, U.S. Patent No. 5,827,703, disclosing lipid
carriers for use in gene
therapy, and U.S. Patent No. 6,281,010, providing adenoviral vectors useful in
gene therapy).



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[0107] Methods for treating disease by implanting a cell that has been
modified to
express a recombinant protein are also well known. See, for example, U.S.
Patent No.
5,399,346, disclosing methods for introducing a nucleic acid into a primary
human cell for
introduction into a human. Although use of human cells for ex vivo therapy is
preferred in some
embodiments, other cells such as bacterial cells may be implanted in a
patient's vasculature,
continuously releasing a therapeutic agent. See, for example, U.S. Patent Nos.
4,309,776 and
5,704,910.
[0108] Methods of the invention are particularly useful for targeting a
protein directly
to a subcellular compartment without requiring a purification step. In one
embodiment, an IGF-
II fusion protein is expressed in a symbiotic or attenuated parasitic organism
that is administered
to a host. The expressed IGF-II fusion protein is secreted by the organism,
taken up by host cells
and targeted to their lysosomes.
[0109] In some embodiments of the invention, GILT proteins are delivered in
situ via
live Leishmahia secreting the proteins into the lysosomes of infected
macrophage. From this
organelle, it leaves the cell and is taken up by adjacent cells not of the
macrophage lineage.
Thus, the GILT tag and the therapeutic agent necessarily remain intact while
the protein resides
in the macrophage lysosome. Accordingly, when GILT proteins are expressed ih
situ, they are
preferably modified to ensure compatibility with the lysosomal environment.
Human 13-
glucuronidase (human "GUS"), an exemplary therapeutic portion, normally
undergoes a C-
terminal peptide cleavage either in the lysosome or during transport to the
lysosome (e.g.
between residues 633 and 634 in GUS). Thus, in embodiments where a GUS-GILT
construct is
to be expressed by Leishmania in a macrophage lysosome human GUS is preferably
modified to
render the protein resistant to cleavage, or the residues following residue
633 are preferably
simply omitted from a GILT fusion protein. Similarly, IGF-II, an exemplary
targeting portion, is
preferably modified to increase its resistance to proteolysis, or a minimal
binding peptide (e.g. as
identified by phage display or yeast two hybrid) is substituted for the
wildtype IGF-II moiety.
~icl~zihistratio~a
[0110] The targeted therapeutics produced according to the present invention
can be
administered to a mammalian host by any route. Thus, as appropriate,
administration can be oral
or parenteral, including intravenous and intraperitoneal routes of
administration. In addition,
administration can be by periodic injections of a bolus of the therapeutic or
can be made more



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continuous by intravenous or intraperitoneal administration from a reservoir
which is external
(e.g., an i.v. bag). In certain embodiments, the therapeutics of the instant
invention can be
pharmaceutical-grade. That is, certain embodiments comply with standards of
purity and quality
control required for administration to humans. Veterinary applications are
also within the
intended meaning as used herein.
[0111] The formulations, both for veterinary and for human medical use, of the
therapeutics according to the present invention typically include such
therapeutics in association
with a pharmaceutically acceptable carrier therefor and optionally other
ingredient(s). The
carriers) can be "acceptable" in the sense of being compatible with the other
ingredients of the
formulations and not deleterious to the recipient thereof. Pharmaceutically
acceptable carriers, in
this regard, are intended to include any and all solvents, dispersion media,
coatings, antibacterial
and antifungal agents, isotonic and absorption delaying agents, and the like,
compatible with
pharmaceutical administration. The use of such media and agents for
pharmaceutically active
substances is known in the art. Except insofar as any conventional media or
agent is
incompatible with the active compound, use thereof in the compositions is
contemplated.
Supplementary active compounds (identified according to the invention and/or
known in the art)
also can be incorporated into the compositions. The formulations can
conveniently be presented
in dosage unit form and can be prepared by any of the methods well known in
the art of
pharmacy/microbiology. In general, some formulations are prepared by bringing
the therapeutic
into association with a liquid carrier or a finely divided solid carrier or
both, and then, if
necessary, shaping the product into the desired formulation.
[0112] A pharmaceutical composition of the invention is formulated to be
compatible
with its intended route of administration. Examples of routes of
administration include oral or
parenteral, e.g., intravenous, intradermal, inhalation, transdermal (topical),
transmucosal, and
rectal administration. Solutions or suspensions used for parenteral,
intradermal, or subcutaneous
application can include the following components: a sterile diluent such as
water for injection,
saline solution, fixed oils, polyethylene glycols, glycerine, propylene glycol
or other synthetic
solvents; antibacterial agents such as benzyl alcohol or methyl paxabens;
antioxidants such as
ascorbic acid or sodium bisulfate; chelating agents such as
ethylenediaminetetraacetic acid;
buffers such as acetates, citrates or phosphates and agents for the adjustment
of tonicity such as
sodium chloride or dextrose. Ph can be adjusted with acids or bases, such as
hydrochloric acid
or sodium hydroxide.



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[0113] Useful solutions for oral or parenteral administration can be prepared
by any
of the methods well lmown in the pharmaceutical art, described, for example,
in Remington's
Pharmaceutical Sciences, (Gennaro, A., ed.), Mack Pub., 1990. Formulations for
paxenteral
administration also can include glycocholate for buccal administration,
methoxysalicylate for
rectal administration, or cutric acid for vaginal administration. The
parenteral preparation can be
enclosed in ampoules, disposable syringes or multiple dose vials made of glass
or plastic.
Suppositories for rectal administration also can be prepared by mixing the
drug with a non-
irritating excipient such as cocoa butter, other glycerides, or other
compositions that are solid at
room temperature and liquid at body temperatures. Formulations also can
include, for example,
polyalkylene glycols such as polyethylene glycol, oils of vegetable origin,
hydrogenated
naphthalenes, and the like. Formulations for direct administration can include
glycerol and other
compositions of high viscosity. Other potentially useful parenteral carriers
for these therapeutics
include ethylene-vinyl acetate copolymer particles, osmotic pumps, implantable
infusion
systems, and liposomes. Formulations for inhalation administration can contain
as excipients,
for example, lactose, or can be aqueous solutions containing, for example,
polyoxyethylene-9-
lauryl ether, glycocholate and deoxycholate, or oily solutions for
administration in the form of
nasal drops, or as a gel to be applied intranasally. Retention enemas also can
be used for rectal
delivery.
[0114] Formulations of the present invention suitable for oral administration
can be
in the form of discrete units such as capsules, gelatin capsules, sachets,
tablets, troches, or
lozenges, each containing a predetermined amount of the drug; in the form of a
powder or
granules; in the form of a solution or a suspension in an aqueous liquid or
non-aqueous liquid; or
in the form of an oil-in-water emulsion or a water-in-oil emulsion. The
therapeutic can also be
administered in the form of a bolus, electuary or paste. A tablet can be made
by compressing or
moulding the drug optionally with one or more accessory ingredients.
Compressed tablets can
be prepaxed by compressing, in a suitable machine, the drug in a free-flowing
form such as a
powder or granules, optionally mixed by a binder, lubricant, inert diluent,
surface active or
dispersing agent. Molded tablets can be made by molding, in a suitable
machine, a mixture of
the powdered drug and suitable caxrier moistened with an inert liquid diluent.
[0115] Oral compositions generally include an inert diluent or an edible
carrier. For
the purpose of oral therapeutic administration, the active compound can be
incorporated with
excipients. Oral compositions prepaxed using a fluid carrier for use as a
mouthwash include the
compound in the fluid carrier and are applied orally and swished and
expectorated or swallowed.



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Pharmaceutically compatible binding agents, and/or adjuvant materials can be
included as part of
the composition. The tablets, pills, capsules, troches and the like can
contain any of the
following ingredients, or compounds of a similar nature: a binder such as
microcrystalline
cellulose, gum tragacanth or gelatin; an excipient such as starch or lactose;
a disintegrating agent
such as alginic acid, Primogel, or corn starch; a lubricant such as magnesium
stearate or Sterotes;
a glidant such as colloidal silicon dioxide; a sweetening agent such as
sucrose or saccharin; or a
flavoring agent such as peppermint, methyl salicylate, or orange flavoring.
[011G] Pharmaceutical compositions suitable for injectable use include sterile
aqueous solutions (where water soluble) or dispersions and sterile powders for
the
extemporaneous preparation of sterile injectable solutions or dispersion. For
intravenous
administration, suitable carriers include physiological saline, bacteriostatic
water, Cremophor
ELTM (BASF, Parsippany, NJ) or phosphate buffered saline (PBS). In all cases,
the
composition can be sterile and can be fluid to the extent that easy
syringability exists. It can be
stable under the conditions of manufacture and storage and can be preserved
against the
contaminating action of microorganisms such as bacteria and fungi. The carrier
can be a solvent
or dispersion medium containing, for example, water, ethanol, polyol (for
example, glycerol,
propylene glycol, and liquid polyetheylene glycol, and the lilee), and
suitable mixtures thereof.
The proper fluidity can be maintained, for example, by the use of a coating
such as lecithin, by
the maintenance of the required particle size in the case of dispersion and by
the use of
surfactants. Prevention of the action of microorganisms can be achieved by
various antibacterial
and antifungal agents, for example, parabens, chlorobutanol, phenol, ascorbic
acid, thimerosal,
and the like. In many cases, it will be preferable to include isotonic agents,
for example, sugars,
polyalcohols such as manitol, sorbitol, and sodium chloride in the
composition. Prolonged
absorption of the injectable compositions can be brought about by including in
the composition
an agent which delays absorption, for example, aluminum monostearate and
gelatin.
[0117] Sterile injectable solutions can be prepared by incorporating the
active
compound in the required amount in an appropriate solvent with one or a
c~mbination of
ingredients enumerated above, as required, followed by filtered sterilization.
Generally,
dispersions are prepared by incorporating the active compound into a sterile
vehicle which
contains a basic dispersion medium and the required other ingredients from
those enumerated
above. In the case of sterile powders for the preparation of sterile
injectable solutions, methods
of preparation include vacuum drying and freeze-drying which yields a powder
of the active



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ingredient plus any additional desired ingredient from a previously sterile-
filtered solution
thereof.
[011] Formulations suitable for infra-articular administration can be in the
form of a
sterile aqueous preparation of the therapeutic which can be in
microcrystalline form, for
example, in the form of an aqueous microcrystalline suspension. Liposomal
formulations or
biodegradable polymer systems can also be used to present the therapeutic for
both infra-articular
and ophthalmic administration.
[0119] Formulations suitable for topical administration, including eye
treatment,
include liquid or semi-liquid preparations such as liniments, lotions, gels,
applicants, oil-in-water
or water-in-oil emulsions such as creams, ointments or pasts; or solutions or
suspensions such as
drops. Formulations for topical administration to the skin surface can be
prepared by dispersing
the therapeutic with a dermatologically acceptable carrier such as a lotion,
cream, ointment or
soap. In some embodiments, useful are carriers capable of forming a film or
layer over the skin
to localize application and inhibit removal. Where adhesion to a tissue
surface is desired the
composition can include the therapeutic dispersed in a fibrinogen-thrombin
composition or other
bioadhesive. The therapeutic then can be painted, sprayed or otherwise applied
to the desired
tissue surface. For topical administration to internal tissue surfaces, the
agent can be dispersed in
a liquid tissue adhesive or other substance known to enhance adsorption to a
tissue surface. For
example, hydroxypropylcellulose or fibrinogen/thrombin solutions can be used
to advantage.
Alternatively, tissue-coating solutions, such as pectin-containing
formulations can be used.
[0120] For inhalation treatments, such as for asthma, inhalation of powder
(self
propelling or spray formulations) dispensed with a spray can, a nebulizer, or
an atomizer can be
used. Such formulations can be in the form of a finely comminuted powder for
pulmonary
administration from a powder inhalation device or self propelling powder-
dispensing
formulations. In the case of self propelling solution and spray formulations,
the effect can be
achieved either by choice of a valve having the desired spray characteristics
(i.e., being capable
of producing a spray having the desired particle size) or by incorporating the
active ingredient as
a suspended powder in controlled particle size. For administration by
inhalation, the therapeutics
also can be delivered in the form of an aerosol spray from a pressured
container or dispenser
which contains a suitable propellant, e.g., a gas such as carbon dioxide, or a
nebulizer. Nasal
drops also can be used.
[0121] Systemic administration also can be by transmucosal or transdermal
means.
For transmucosal or transdermal administration, penetrants appropriate to the
barrier to be



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permeated are used in the formulation. Such penetrants generally are known in
the art, and
include, for example, for transmucosal administration, detergents, bile salts,
and filsidic acid
derivatives. Transmucosal administration can be accomplished through the use
of nasal sprays
or suppositories. For transdermal administration, the therapeutics typically
are formulated into
ointments, salves, gels, or creams as generally lenown in the art.
[0122] In one embodiment, the therapeutics axe prepared with carriers that
will
protect against rapid elimination from the body, such as a controlled release
formulation,
including implants and microencapsulated delivery systems. Biodegradable,
biocompatible
polymers can be used, such as ethylene vinyl acetate, polyanhydrides,
polyglycolic acid,
collagen, polyorthoesters, and polylactic acid. Methods for preparation of
such formulations will
be apparent to those skilled in the art.. The materials also can be obtained
commercially from
Alza Corporation and Nova Pharmaceuticals, Inc. Liposomal suspensions can also
be used as
pharmaceutically acceptable carriers. These can be prepared according to
methods known to
those skilled in the art, for example, as described in LT.S. Pat. No.
4,522,811. Microsomes and
microparticles also can be used.
[0123] Oral or parenteral compositions can be formulated in dosage unit form
for
ease of administration and uniformity of dosage. Dosage unit form refers to
physically discrete
units suited as unitary dosages for the subject to be treated; each unit
containing a predetermined
quantity of active compound calculated to produce the desired therapeutic
effect in association
with the required pharmaceutical carrier. The specification for the dosage
unit forms of the
invention are dictated by and directly dependent on the unique characteristics
of the active
compound and the particular therapeutic effect to be achieved, and the
limitations inherent in the
art of compounding such an active compound for the treatment of individuals.
[0124] Generally, the therapeutics identified according to the invention can
be
formulated for parenteral or oral administration to humans or other mammals,
for example, in
therapeutically effective amounts, e.g., amounts which provide appropriate
concentrations of the
drug to target tissue for a time sufficient to induce the desired effect.
Additionally, the
therapeutics of the present invention can be administered alone or in
combination with other
molecules known to have a beneficial effect on the particular disease or
indication of interest.
By way of example only, useful cofactors include symptom-alleviating
cofactors, including
antiseptics, antibiotics, antiviral and antifungal agents and analgesics and
anesthetics.
[0125] The effective concentration of the therapeutics identified according to
the
invention that is to be delivered in a therapeutic composition will vary
depending upon a number



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of factors, including the final desired dosage of the drug to be administered
and the route of
administration. The preferred dosage to be administered also is likely to
depend on such
variables as the type and extent of disease or indication to be treated, the
overall health status of
the particular patient, the relative biological efficacy of the therapeutic
delivered, the formulation
of the therapeutic, the presence and types of excipients in the formulation,
and the route of
administration. In some embodiments, the therapeutics of this invention can be
provided to an
individual using typical dose units deduced from the earlier-described
mammalian studies using
non-human primates and rodents. As described above, a dosage unit refers to a
unitary, i.e. a
single dose which is capable of being administered to a patient, and which can
be readily handled
and packed, remaining as a physically and biologically stable unit dose
comprising either the
therapeutic as such or a mixture of it with solid or liquid pharmaceutical
diluents or carriers.
[012G] In certain embodiments, organisms are engineered to produce the
therapeutics
identified according to the invention. These organisms can release the
therapeutic for harvesting
or can be introduced directly to a patient. In another series of embodiments,
cells can be utilized
to serve as a carrier of the therapeutics identified according to the
invention.
[0127] Therapeutics of the invention also include the "prodrug" derivatives.
The
term prodrug refers to a pharmacologically inactive (or partially inactive)
derivative of a parent
molecule that requires biotransformation, either spontaneous or enzymatic,
within the organism
to release or activate the active component. Prodrugs are variations or
derivatives of the
therapeutics of the invention which have groups cleavable under metabolic
conditions. Prodrugs
become the therapeutics of the invention which are pharmaceutically active i~
vivo, when they
undergo solvolysis under physiological conditions or undergo enzymatic
degradation. Prodrug
of this invention can be called single, double, triple, and so on, depending
on the number of
biotransformation steps required to release or activate the active drug
component within the
organism, and indicating the number of functionalities present in a precursor-
type form. Prodrug
forms often offer advantages of solubility, tissue compatibility, or delayed
release in the
mammalian organism (see, Bundgard, Design of Prodrugs, pp. 7-9, 21-24,
Elsevier, Amsterdam
1985 and Silverman, The Organic Chemistry of Drug Design and Drug Action, pp.
352-401,
Academic Press, San Diego, Calif., 1992). Moreover, the prodrug derivatives
according to this
invention can be combined with other features to enhance bioavailability.
Examples
Example 1. GILT constructs



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[0128] IGF-II cassettes have been synthesized by ligation of a series of
overlapping
oligos and cloned into Pirl-SAT, a standard Leishmania expression vector. 4
IGF-II cassettes
have been made: one that encodes the wildtype mature polypeptide, one with a
~l-7 deletion,
one with a Y27L mutation, and one with both mutations. These mutations are
reported to reduce
binding of IGF-II to the other receptors while not affecting binding to the
M6P receptor.
[0129] The coding sequence of human IGF-II is shown in Figure 2. The protein
is
synthesized as a pre-pro-protein with a 24 amino acid signal peptide at the
amino terminus and a
89 amino acid carboxy terminal region both of which are removed post-
translationally, reviewed
in (O'Dell et al. (1998) Int. J. Biochem Cell Biol. 30(7):767-71. The mature
protein is 67 amino
acids. A Leishmahia codon optimized version of the mature IGF-II is shown in
Figure 3
(Langford et al. (1992) Exp. Parasitol 74(3):360-1). This cassette was
constructed by annealing
overlapping oligonucleotides whose sequences are shown in Table 3. Additional
cassettes
containing a deletion of amino acids 1-7 of the mature polypeptide (O1-7),
alteration of residue
27 from tyrosine to leucine (Y27L) or both mutations (~l-7,Y27L) were made to
produce IGF-II
cassettes with specificity for only the desired receptor as described below.
To make the wildtype
IGF-II cassette, oligos GILT1-9 were annealed and ligated. To make the Y27L
cassette, oligos
1, 12, 3, 4, 5, 16, 7, 8 and 9 were annealed and ligated. After ligation, the
two cassettes were
column purified. Wildtype and Y27L cassettes were amplified by PCR using
oligos GILT 20
and 10 and the appropriate template. To incorporate the dl-7 deletion, the two
templates were
amplified using oligos GILT 1 l and 10. The resulting 4 IGF-II cassettes
(wildtype, Y27L, O1-7,
and Y27L01-7) were column purified, digested with XbaI, gel purified and
ligated to XbaI cut
Pirl-SAT.
[0130] Gene cassettes were then cloned between the XmaI site (not shown)
upstream
of XbaI in the vector and the AscI site in such a way as to preserve the
reading frame. An
overlapping DAM methylase site at the 3' XbaI site permitted use of the 5'
XbaI site instead of
the XmaI site for cloning. The AscI site adds a bridge of 3 amino acid
residues.



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TABLE 3. Oligonucleotides used in the construction of Pir-GILT vectors.
GILT 1 9 GCGGCGGCGAGCTGGTGGACACGCTGCAGTT 48-97 top strand


CGTGTGCGGCGACCGCGGC


GILT 2 10 TTCTACTTCAGCCGCCCGGCCAGCCGCGTGA 98-147 top
strand


GCCGCCGCAGCCGCGGCAT


GILT 3 11 CGTGGAGGAGTGCTGCTTCCGCAGCTGCGAC 148-197 top
strand


CTGGCGCTGCTGGAGACGT


GILT 4 12 ACTGCGCGACGCCGGCGAAGTCGGAGTAAG 198-237 top
strand


ATCTAGAGCG


GILT 5 13 AGCGTGTCCACCAGCTCGCCGCCGCACAGCG 72-23 bottom


TCTCGCTCGGGCGGTACGC


GILT 6 14 GGCTGGCCGGGCGGCTGAAGTAGAAGCCGC 122-73 bottom


GGTCGCCGCACACGAACTGC


GILT 7 15 GCTGCGGAAGCAGCACTCCTCCACGATGCCG 172-123 bottom


CGGCTGCGGCGGCTCACGC


GILT 8 16 CTCCGACTTCGCCGGCGTCGCGCAGTACGTC 223-173 bottom


TCCAGCAGCGCCAGGTCGCA


GILT 9 17 CCGTCTAGAGCTCGGCGCGCCGGCGTACCGC 1-47 top strand


CCGAGCGAGACGCTGT


GILT 10 18 CGCTCTAGATCTTACTCCGACTTCG 237-202 bottom


GILT 11 19 CCGTCTAGAGCTCGGCGCGCCGCTGTGCGGC 1-67, X23-43
top


GGCGAGCTGGTGGAC


GILT 12 20 TTCCTGTTCAGCCGCCCGGCCAGCCGCGTGA 98-147 (Y27L)
top


GCCGCCGCAGCCGCGGCAT


GILT 16 21 GGCTGGCCGGGCGGCTGAACAGGAAGCCGC 122-73 (Y27L)
bot


GGTCGCCGCACACGAACTGC


GILT 20 22 CCGTCTAGAGCTCGGCGCGCCGGCG 1-25 top strand


[0131] The purpose of incorporating the indicated mutations into the IGF-II
cassette
is to insure that the fusion proteins are targeted to the appropriate
receptor. Human IGF-II has a
high degree of sequence and structural similarity to IGF-I (see, for example
Figure 7) and the B



CA 02487815 2004-11-26
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and A chains of insulin (Terasawa et al. (1994) Embo J. 13(23):5590-7).
Consequently, it is not
surprising that these hormones have overlapping receptor binding
specificities. IGF-II binds to
the insulin receptor, the IGF-I receptor and the cation independent mannose 6-
phosphate/IGF-II
receptor (CIM6P/IGF-II). The CIM6P/IGF-II receptor is a dual activity receptor
acting as a
receptor for IGF-II and as a mannose 6-phosphate receptor involved in sorting
of lysosomal
hydrolases. For a number of years, these two activities were attributed to
separate proteins until
it was determined that both activities resided in a single protein (Morgan et
al. (1987) Nature
329(6137):301-7); (Tong et al. (1988) J. Biol. Chem. 263(6):2585-8).
[0132] The most profound biological effects of IGF-II, such as its mitogenic
effect,
are mediated through the IGF-I receptor rather than the CIM6P/IGF-II receptor,
reviewed in
(Ludwig et al. (1995) Trends in Cell Biolo~y 5:202-206) also see (Korner et
al. (1995) J. Biol.
Chem. 270(1):287-95). It is thought that the primary result of IGF-II binding
to the CIM6P/IGF-
II receptor is transport to the lysosome for subsequent degradation. This
represents an important
means of controlling IGF-II levels and explains why mice carrying null mutants
of the
CIM6P/IGF-II receptor exhibit perinatal lethality unless IGF-II is also
deleted (Lau et al. (1994)
Genes Dev. 8(24):2953-63); (Wang et al. (1994) Nature 372(6505):464-7);
(Ludwig et al. (1996)
Dev. Biol. 177(2):517-35). In methods of the present invention, it is
desirable to have the IGF-II
fusion proteins bind to the CIM6P/IGF-II receptor. The Y27L and 41-7 mutations
reduce IGF-II
binding to the IGF-I and insulin receptors without altering the affinity for
the CIM6P/IGF-II
receptor (Sakano _et al. (1991) J. Biol. Chem. 266(31):20626-35); (Hashimoto
et al. (1995) J.
Biol. Chem. 270(30):18013-8). Therefore, according to the invention, these
mutant forms of
IGF-II should provide a means of targeting fusion proteins specifically to the
CIM6P/IGF-II
receptor.
[0133] In one experiment, 4 different IGF-II cassettes with the appropriate
sequences,
wild type, O1-7, Y27L and O1-7/Y27L are made. 13-GUS cassettes are fused to
IGF-II cassettes
and these constructs are put into parasites. Alpha-galactosidase cassettes are
also fused to the
IGF-II cassettes. GUS fusions have been tested and shown to produce
enzymatically active
protein.
[0134] One preferred construct, shown in Figure 4, includes the signal peptide
of the
L. mexicaaa secreted acid phosphatase, SAP-1, cloned into the XbaI site of a
modified Pirl-SAT
in which the single SaII site has been removed. Fused in-frame is the mature
[3-GUS sequence,
connected to an IGF-II tag by a bridge of three amino acids.



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Example 2. GILT protein pre arp ation
[0135] L. mexicana expressing and secreting 13-GUS were grown at 26°C
in 100 ml
Standard Promastigote medium (M199 with 40 mM HEPES, pH 7.5, 0.1 mM adenine,
0.0005%
hemin, 0.0001% biotin, 5% fetal bovine serum, 5% embryonic fluid, 50 units/ml
penicillin, 50
~,g/ml streptomycin and SO~g/ml nourseothricin). After reaching a density of
approximately 5 x
106 promastigotes/ml, the promastigotes were collected by centrifugation for
10 min. at 1000 x g
at room temperature; these promastigotes were used to inoculate 1 liter of low
protein medium
{M199 supplemented with 0.1 mM adenine, 0.0001% biotin, 50 units/ml penicillin
and 50 ~g/ml
streptomycin) at room temperature. The 1 liter cultures were contained in 2
liter capped flasks
with a sterile stir bar so that the cultures could be incubated at 26°C
with gentle stirring. The 1
liter cultures were aerated twice a day by moving them into a laminar flow
hood, removing the
caps and swirling vigorously before replacing the caps. When the cultures
reached a density of 2-
3 x 107 promastigotes/ml, the cultures were centrifuged as described above
except the
promastigote pellet was discarded and the media decanted into sterile flasks.
The addition of 434
g (NH4)ZSO4 per liter precipitated active GUS protein from the medium; the
salted out medium
was stored at 4°C overnight. Precipitated proteins were harvested
either by centrifugation at
10,500 x g for 30 min. or filtration through Gelman Supor-800 membrane; the
proteins were
resuspended in 10 mM Tris pH 8, 1 mM CaCl2 and stored at -80°C until
dialysis. The crude
preparations from several,liters of medium were thawed, pooled, placed in
dialysis tubing
(Spectra/Por -7, MWCO 25,000), and dialyzed overnight against two 1 liter
volumes of DMEM
with bicarbonate (Dulbecco's Modified Eagle's Medium).
Example 3. GILT uptake assay
[013G] Slcin fibroblast line GM4668 (human, NIGMS Human Genetic Mutant Cell
Repository) is derived from a patient with mucopolysaccharidosis VII; the
cells therefore have
little or no (3-GUS activity. GM4668 cells are therefore particularly useful
for testing the uptake
of GUS-GILT constructs into human cells. GM4668 cells were cultured in 12-well
tissue culture
plates in Dulbecco's modified Eagle's medium (DMEM) supplemented with 15%
(v/v) fetal calf
serum at 37°C in 5% CO2. Fibroblasts were cultured overnight in the
presence of about 150
units of preparations of Leishma~ia-expressed human !3-glucuronidase (GUS),
GUS-IGF-II
fusion protein (GUS-GILT), or mutant GUS-IGF-II fusion protein (GUSD-GILT)
prepared as
described in Example 2. Control wells contained no added enzyme (DMEM media
blank).
After incubation, media was removed from the wells and assayed in triplicate
for GUS activity.



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Wells were washed five times with 1 ml of 37°C phosphate-buffered
saline, then incubated for
15 minutes at room temperature in 0.2 ml of lysis buffer (10 mM Tris, pH7.5,
100 mM NaCI, 5
mM EDTA, 2mM 4-(2-aminoethyl)-benzenesulfonyl fluoride hydrochloride (AEBSF,
Sigma),
and 1 % NP-40). Cell lysates were transferred to microfuge tubes, then spun at
13,000 rpm for 5
minutes to remove cell debris. Three 10 ~,L aliquots of lysate were assayed
for protein
concentration (Pierce Micro BCA protein assay, Pierce, IL).
[0137] Three 38 ~,L aliquots of lysate were assayed for GUS activity using a
standard
fluorometric assay adapted from (Wolfe et al. (1996) Protocols for Gene
Transfer in
Neuroscience: Towards Gene Therapy of Neurological Disorders 263-274). Assays
are done in
disposable fluorimeter cuvettes. 150 ~.1 of reaction mix is added to each
cuvette. 1 ml reaction
mix is 860 ~1 H20, 1001 1M NaAcetate, 40~,125X l3-GUS substrate mix. (25X 13-
GUS
substrate mix is a suspension of 250 mg 4-methylumbelliferyl-13-D glucuronide
in 4.55 ml
ethanol stored at -20°C in a dessicator. 381 of sample are added to the
reaction mix and the
reaction is incubated at 37 °C . Reactions are terminated by addition
of 2 ml stop solution (10.6
g NaaC03, 12.01 g glycine, H20 to 500 ml, pH 10.5). Fluorescence output is
then measured by
fluorimeter.
[0138] Results of the uptake experiment indicate that the amount of cell-
associated
GUS-GILT is 10-fold greater that that of the unmodified GUS (Figure 5). The
double mutant
construct is about 5-fold more effective than unmodified GUS. These results
indicate that the
GILT technology is an effective means of targeting a lysosomal enzyme for
uptake. Uptake can
also be verified using standard immunofluorescence techniques.
Example 4. Competition experiments
[0139] To verify that the GILT -mediated uptake occurs via the IGF-II binding
site
on the cation-independent M6P receptor, competition experiments were performed
using
recombinant IGF-II. The experimental design was identical to that described
above except that
GM4668 fibroblasts were incubated with indicated proteins in DMEM minus serum
+2%BSA
for about 18 hours. Each 13-GUS derivative was added at 150 U per well. 2.85
~.g IGF-II was
added to each well for competition. This represents approximately a 100 fold
molar excess over
GILT-GUS, a concentration sufficient to compete for binding to the M6P/IGF-II
receptor.
[0140] Results of the competition experiment are depicted in Figure 6. In the
absence
of IGF-II over 24 units of GILT-GUS/ mg lysate were detected. Upon addition of
IGF-II, the
amount of cell associated GILT-GUS fell to 5.4 U. This level is similar to the
level of



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unmodified GUS taken up by the fibroblasts. Thus, the bulk of the GILT protein
uptake can be
competed by IGF-II indicating that the uptake is indeed occurring through a
specific receptor-
ligand interaction.
Example 5. Gene Product Expression in serum free media
[0141] Expression products can also be isolated from serum free media. In
general,
the expression strain is grown in medium with serum, diluted into serum free
medium, and
allowed to grow for several generations, preferably 2-5 generations, before
the expression
product is isolated. For example, production of secreted targeted therapeutic
proteins can be
isolated from Leishma~cia mexicaha promastigotes that are cultured initially
in 50 ml 1X M199
medium in a 75 cm2 flask at 27° C. When the cell density reaches 1-3x
107/ml, the culture is
used to inoculate 1.2 L of M199 media. When the density of this culture
reaches about Sx106/ml,
the cells are harvested by centrifugation, resuspended in 180 ml of the
supernatant and used to
inoculate 12 L of "lima" medium in a 16 L spinner flask. The initial cell
density of this culture
is typically about Sx 10$ /ml. This culture is expanded to a cell density of
about 1.0 -1.7 x 10e7
cells/ml. When this cell density is reached, the cells are separated from the
culture medium by
centrifugation and the supernatant is filtered at 4°C through a 0.2 ~,
filter to remove residual
promastigotes. The filtered media was concentrated from 12.0 L to 500 ml using
a tangential
flow filtration device (MILLIPORE Prep/Scale-TFF cartridge).
[0142] Preferred growth media for this method are M199 and "lima" growth
media.
However, other serum containing and serum free media are also useful. M199
growth media is
as follows: (1L batch) = 200 ml SX M199 (with phenol red pH indicator) + 636
ml H2O, 50.0 ml
fetal bovine serum, 50.0 ml EF bovine embryonic fluid, 1.0 ml of 50 mg/ml
nourseothricin, 2.0
ml of 0.25% hemin in 50% triethanolamine , 10 ml of lOmM adenine in SOmM Hepes
pH 7.5,
40.0 ml of 1M Hepes pH 7.5, lml of 0.1% biotin in 95% ethanol, 10.0 ml of
penicillin/streptomycin. All sera used are inactivated by heat. The final
volume =1 L and is
filter sterilized. "lima" modified M199 media is as follows: (20.0 L batch) =
219.2g M199
powder (-)phenol red + 7.Og sodium bicarbonate, 200.0 ml of l OmM adenine in
SOxnM Hepes
pH 7.5, 800.0 ml Of Hepes free acid pH 7.5, 20.0 ml 0.1% biotin in 95%
ethanol, 200.0 ml
penicillin/streptomycin, Final volume = 20.0 L and is filter sterilized.
[0143] The targeted therapeutic proteins are preferably purified by
Concanavalin A
(ConA) chromatography. For example, when a culture reaches a density of > 1.0
x 107
promastigotes/ml, L. mexica~ca are removed by centrifugation, 10 min at 500 x
g. The harvested



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culture medium is passed through a 0.2 ~m filter to remove particulates before
being loaded
directly onto a ConA-agarose column (4% cross-linked beaded agarose, Sigma).
The ConA-
agarose column is pretreated with 1 M NaCI, 20 mM Tris pH 7.4, 5 mM each of
CaCl2, MgCl2
and MnCl2 and then equilibrated with 5 volumes of column buffer (20 mM Tris pH
7.4, 1 mM
CaCl2, and 1 mM MnCl2). A total of 179,800 units (nmol/hr) of GUS activity (in
2 L) in culture
medium is loaded onto a 22 ml ConA agarose column. No activity is detectable
in the flow
through or wash. The GUS activity is eluted with column buffer containing 200
mM methyl
mannopyranoside. Eluted fractions containing the activity peak are pooled and
concentrated.
Uptake and competition experiments were performed as described in Examples 3
and 4, except
that the organisms were grown in serum-free medium and purified with ConA;
about 350-600
units of enzyme were applied to the fibroblasts. Results are shown in Figure
8.
Example 6 Competition experiments using denatured IGF-II as competitor
[0144] The experiment in Example 4 is repeated using either normal or
denatured
IGF-II as competitor. As in Example 4, the amount of cell-associated GUS-GILT
is reduced
when coincubated with normal IGF-II concentrations that are effective for
competition but, at
comparable concentrations, denatured IGF-II has little or no effect.
Example 7. Enzyme assays
[0145] Assays for GUS activity are performed as described in Example 3 andlor
as described below.
[014G] Glass assay tubes are numbered in triplicate, and 100 ~.L of 2x GUS
reaction mix are added to each tube. 2x GUS reaction mix is prepared by adding
100 mg of 4-
methylumbelliferyl-13-D glucuronide to 14.2 mL 200 mM sodium acetate, pH
adjusted to 4.8
with acetic acid. Up to 100 ~,L of sample are added to each tube; water is
added to a final
reaction volume of 200 p,L. The reaction tubes are covered with parafilm and
incubated in a
37°C water bath for 1-2 hours. The reaction is stopped by addition of
1.8 mL of stop buffer
(prepared by dissolving 10.6 g of Na2C03 and 12.01 g of glycine in a final
volume of 500 mL of
water, adjusting the pH to 10.5 and filter-sterilizing into a repeat-
dispensor). A fluorimeter is
then calibrated using 2 mL of stop solution as a blank, and the fluorescence
is read from the
remaining samples. A standard curve is prepared using 1, 2, 5, 10, and 20 ~,L
of a 166 p,M 4-
methylumbelliferone standard in a final volume of 2 mL stop buffer.



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[0147] The 4-methylumbelliferone standard solution is prepared by dissolving
2.5 mg
4-methylumbelliferone in 1 mL ethanol and adding 99 mL of sterile water,
giving a
concentration of approximately 200 nmol/mL. The precise concentration is
determined
spectrophotometrically. The extinction coefficient at 360 nm is 19,000 cm 1
M'1. For example,
100 p.L is added to 900 ~,L of stop buffer, and the absorbance at 360 nm is
read. If the reading is
0.337, then the concentration of the standard solution is 0.337 x 10
(dilution)/19,000 = 177 ~,M,
which can then be diluted to 166 ~M by addition of an appropriate amount of
sterile water.
Example 8. Binding uptake and halflife experiments
[0148] Binding of GUS-GILT proteins to the M6P/IGF-II receptor on fibroblasts
are
measured and the rate of uptake is assessed similar to published methods (York
et al. (1999) J.
Biol. Chem. 274(2):1164-71). GM4668 fibroblasts cultured in 12 well culture
dishes as
described above are washed in ice-cold media minus serum containing 1% BSA.
Ligand, (either
GUS, GUS-GILT or GUS-OGILT, or control proteins) is added to cells in cold
media minus
serum plus 1% BSA. Upon addition of ligand, the plates are incubated on ice
for 30 minutes.
After 30 minutes, ligand is removed and cells are washed quickly 5 times with
ice cold media.
Wells for the 0 time point receive 1 ml ice cold stripping buffer (0.2 M
Acetic acid, pH 3.5, O.SM
NaCI). The plate is then floated in a 37° water bath and 0.5 ml
prewaxmed media is added to
initiate uptake. At every stopping point, 1 ml of stripping buffer is added.
When the experiment
is over, aliquots of the stripping buffer are saved for fluorometric assay of
13-glucuronidase
activity as described in Example 3. Cells are then lysed as described above
and the lysate
assayed for 13-glucuronidase activity. Alternatively, immunological methods
can be used to test
the lysate for the presence of the targeted therapeutic protein.
[0149] It is expected that GUS-GILT is rapidly taken up by fibroblasts in a
matter of
minutes once the temperature is shifted to 37°C (York et al. (1999) J.
Biol. Chem. 274(2):1164-
71) and that the enzyme activity persists in the cells for many hours.
Example 9. Protein production in mammalian cells
CIO cells
[0150] GUS-GILT~1-7 and GUS~C18-GILTOl-7 were expressed in CHO cells
using the system of Ulmasov et al. (2000) PNAS 97(26):14212-14217. Appropriate
gene
cassettes were inserted into the Eco RI site of the pCXN vector, which was
electroporated into



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CHO cells at 50 ~F and 1,200 V in a 0.4-cm cuvette. Selection of colonies and
amplification
was mediated by 400 ~,g/mL 6418 for 2-3 weeks. The CHO cells were propagated
in MEM
media supplemented with 15% FBS, 1.2 mM glutamine, 50 ~,g/mL proline, and 1 mM
pyruvate.
For enzyme production cells were plated in multifloor flasks in MEM. Once
cells reached
confluence, collection medium (Weymouth medium supplemented with 2% FBS, 1.2
mM
glutamine, and 1 mM pyruvate) was applied to the cells. Medium containing the
secreted
recombinant enzyme was collected every 24-72 hours. A typical level of
secretion for one GUS-
GILT01-7 cell line was 4000-5000 units/mL/24 hours.
[0151] A number of GUSOC18-GILTOl-7 CHO lines were assayed for the amount of
secreted enzyme produced. The six highest producers secreted between 8600 and
14900
units/mL/24 hours. The highest producing line was selected for collection of
protein.
HEK 293 cells
[0152] GUS-GILT cassettes were cloned into pCEP4 (Invitrogen) for expression
in
HEK 293 cells. Cassettes used included wild-type GUS-GILT; GUS-GILTOl-7; GUS-
GILTY27L; GUSOC18-GILTO1-7; GILTY27L, and GUS-GILTF19S/E12K.
[0153] HEK 293 cells were cultured to 50-80% confluency in 12-well plates
containing DMEM medium with 4 mM glutamine and 10% FBS. Cells were transfected
with
pCEP-GUS-GILT DNA plasmids using FuGENE 6 (Roche) as described by the
manufacturer.
0.5 ~,g DNA and 2 ~,L of FuGENE 6 were added per well. Cells were removed from
wells 2-3
days post-transfection using trypsin, then cultured in T25 cm2 culture flasks
containing the above
DMEM medium with 100 ~,g/mL hygromycin to select for a stable population of
transfected
cells. Media containing hygromycin were changed every 2-3 days. The cultures
were expanded
to T75 cm2 culture flasks within 1-2 weeks. For enzyme production cells were
plated in
multifloor flasks in DMEM. Once cells reached confluence, collection medium
(Weymouth
medium supplemented with 2% FBS, 1.2 mM glutamine, and 1 mM pyruvate) was
applied to the
cells. This medium has been optimized for CHO cells, not for 293 cells;
accordingly, levels of
secretion with the HEK 293 lines may prove to be significantly higher in
alternate media.



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[0154] Levels of secreted enzyme are shown in Table 4.
Table 4.
Cell line Recombinant Protein Units/mL/24
hours


HEK293 2-1 GUS-GILT 3151


HEK293 2-2 GUS~C18-GILT~l-7 10367


HEK293 2-3 GUS-GILT~l-7 186


HEK293 4-4 GILTY27L 3814


HEK293 3-5 GUS-GILTF19S/E12K 13223


HEK293 3-6 GILTY27L 7948


CHQ 15 GUSOC18-GILTO1-7 18020


Example 10. Purification of GUS-GILT fusion proteins
[0155] Chromoatography, including conventional chromatography and affinity
chromatography, can be used to purify GUS-GILT fusion proteins.
Conventional ch~omatog~aphy
[015G] One procedure for purifying GUS-GILT fusion proteins produced in
Leishmav~ia is described in Example 2. An alternative procedure is described
in the following
paragraph.
[0157] Culture supernatants from Leishma~cia mexicaha cell lines expressing
GUS-
GILT fusions were harvested, centrifuged, and passed through a 0.2 ~ filter to
remove cell
debris. The supernatants were concentrated using a tangential ultrafilter with
a 100,000
molecular weight cutoff and stored at -80°C. Concentrated supernatants
were loaded directly
onto a column containing Concanavalin A (ConA) immobilized to beaded agarose.
The column
was washed with ConA column buffer (50 mM Tris pH 7.4, 1mM CaCl2, 1mM MnCl2)
before
mannosylated proteins including GUS-GILT fusions were eluted using a gradient
of 0-0.2M
methyl-oc-D-pyranoside in the ConA column buffer. Fractions containing
glucuronidase activity
(assayed as described in Example 7) were pooled, concentrated, and the buffer
exchanged to SP
column buffer (25 mM sodium phosphate pH 6, 20 mM NaCI, 1 mM EDTA) in
preparation for
the next column. The concentrated fractions were loaded onto an SP fast flow
column
equilibrated in the same buffer, and the column was washed with additional SP
column buffer.
The GUS-GILT fusions were eluted from the column in two steps: 1) a gradient
of 0-0.15 M
glucuroiuc acid in 25 mM sodium phosphate pH 6 and 10% glycerol, followed by
0.2 M



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glucuronic acid, 25 mM sodium phosphate pH 6, 10% glycerol. Fractions
containing
glucuronidase activity were pooled, and the buffer exchanged to 20 mM
potassium phosphate pH
7.4. These pooled fractions were loaded onto an HA-ultrogel column
equilibrated with the same
buffer. The GUS-GILT fusion proteins were eluted with an increasing gradient
of phosphate
buffer, from 145-340 mM potassium phosphate pH 7.4. The fractions containing
glucuronidase
activity were pooled, concentrated, and stored at -80°C in 20 mM Tris
pH 8 with 25% glycerol.
[0158] A conventional chromatography method for purifying GUS-GILT fusion
proteins produced in mammalian cells is described in the following paragraphs.
[0159] Mammalian cells overexpressing a GUS-GILT fusion protein axe grown to
confluency in Nunc Triple Flasks, then fed with serum-free medium (Waymouth MB
752/1)
supplemented with 2% fetal bovine serum to collect enzyme for purification.
The medium is
harvested and the flasks are refed at 24 hour intervals. Medium from several
flasks is pooled and
centrifuged at 5000 x g for 20 minutes at 4°C to remove detached cells,
etc. The supernatant is
removed and aliquots axe taleen for a ~-GUS assay. The medium can now be used
directly for
purification or frozen at -20°C for later use.
[0160] 1 L of secretion medium is thawed at 37°C (if frozen), filtered
through a 0.2 ~.
filter, and transferred to a 4L beaker. The volume of the medium is diluted 4-
fold by addition of
3 L of dd water to reduce the salt concentration; the pH of the diluted medium
is adjusted to 9.0
using 1 M Tris base. 50 mL of DEAF-Sephacel pre-equilibrated with 10 mM Tris
pH 9.0 is
added to the diluted medium and stirred slowly with a large stirring bar at
4°C for 2 hours. (A
small aliquot can be removed, microfuged, and the supernatant assayed to
monitor binding.)
When binding is complete, the resin is collected on a fritted glass funnel and
washed with 750
mL of 10 mM Tris pH 9.0 in several batches. The resin is transferred to a 2.5
cm column and
washed with an additional 750 mL of the same buffer at a flow rate of 120
mL/hour. The DEAE
column is eluted with a linear gradient of 0-0.4 M NaCI in 10 mM Tris pH 9Ø
The fractions
containing the GUS-GILT fusion proteins are detected by 4-methylumbelliferyl-
13-D glucuronide
assay, pooled, and loaded onto a 600 mL column of Sephacryl S-200 equilibrated
with 25 mM
Tris pH 8, 1 mM (3-glycerol phosphate, 0.15 M sodium chloride and eluted with
the same buffer.
[0161] The fractions containing the GUS-GILT fusion proteins are pooled and
dialyzed with 3 x 4L of 25 mM sodium acetate pH 5.5, 1 mM (3-glycerol
phosphate, 0.025%
sodium azide. The dialyzed enzyme is loaded at a flow rate of 36 mL/hour onto
a 15 mL column
of CM-Sepharose equilibrated with 25 mM sodium acetate pH 5.5, 1 mM [i-
glycerol phosphate,



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0.025% sodium azide. It is then washed with 10 column volumes of this same
buffer. The CM
column is eluted with a linear gradient of 0-0.3 M sodium chloride in the
equilibration buffer.
The fractions containing the GUS-GILT fusion proteins are pooled and loaded
onto a 2.4 x 70
cm (Bed volume = 317 mL) column of Sephacryl S-300 equilibrated with 10 mM
Tris pH 7.5, 1
mM [3-glycerol phosphate, 0.15 M NaCI at a flow rate of 48 mL/hour. The
fractions containing
the fusion proteins are pooled; the pool is assayed for GUS activity and for
protein concentration
to determine specific activity. Aliquots are run on SDS-PAGE followed by
Coomassie or silver
staining to confirm purity. If a higher concentration of enzyme is required,
Amicon
Ultrafiltration Units with an XM-50 membrane (50,000 molecular weight cutoff)
or Centricon C-
30 units (30,000 molecular weight cutoff] can be used to concentrate the
fusion protein. The
fusion protein is stored at -80°C in the 10 mM Tris pH 7.5, 1 mM sodium
(3-glycerol phosphate,
0.15 M NaCI buffer.
Amity ch~omatog~~aphy
[0162] Affinity chromatography conditions are essentially as described in
Islam et al.
(1993) J. Biol. Chem. 268(30):22627-22633. Conditioned medium from mammalian
cells
overexpressing a GUS-GILT fusion protein (collected and centrifuged as
described above for
conventional chromatography) is filtered through a 0.22 ~, filter. Sodium
chloride (crystalline) is
added to a final concentration of O.SM, and sodium azide is added to a final
concentration of
0.025% by adding 1/400 volume of a 10% stock solution. The medium is applied
to a 5 mL
column of anti-human (3-glucuronidase-Affigel 10 (pre-equilibrated with
Antibody Sepharose
Wash Buffer: 10 mM Tris pH 7.5, 10 mM potassium phosphate, 0.5 M NaCI, 0.025%
sodium
azide) at a rate of 25 mL/hour at 4°C. Fractions are collected and
monitored for any GUS
activity in the flow-through. The column is washed at 36 mL/hour with 10-20
column volumes
of Antibody Sepharose Wash Buffer. Fractions are collected and monitored for
GUS activity.
The column is eluted at 36 mL/hour with 50 mL of 10 mM sodium phosphate pH 5.0
+ 3.5 M
MgCl2. 4 mL fractions are collected and assayed for GUS activity. Fractions
containing the
fusion protein are pooled, diluted with an equal volume of P6 buffer (25 mM
Tris pH 7.5, 1 mM
(3-glycerol phosphate, 0.15 mM NaCI, 0.025% sodium azide) and desalted over a
BioGel P6
column (pre-equilibrated with P6 buffer) to remove the MgCl2 and to change the
buffer to P6
buffer for storage. The fusion protein is eluted with P6 buffer, fractions
containing GUS activity
are pooled, and the pooled fractions assayed for GUS activity and for protein.
An SDS-PAGE



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gel stained with Coomassie Blue or silver stain is used to confirm purity. The
fusion protein is
stored frozen at -80°C in P6 buffer for long-term stability.
Example 11 Uptake experiments on mammalian-produced proteins
[0163] Culture supernatants from HEK293 cell lines or CHO cell lines producing
GUS or GUS-GILT constructs were harvested through a 0.2 ~,m filter to remove
cells GM 4668
fibroblasts were cultured in 12-well tissue culture plates in DMEM
supplemented with 15% (v/v)
fetal calf serum at 37°C in 5% CO~. Cells were washed once with uptake
medium (DMEM +
2% BSA (Sigma A-7030)) at 37°C. Fibroblasts were then cultured (3-21
hours) with 1000-4000
units of enzyme per mL of uptake medium. In some experiments, competitors for
uptake were
added. Mannose-6-phosphate (Calbiochem 444100) was added to some media at
concentrations
from 2-8 mM and pure recombinant IGF-II (Cell Sciences OU100) was added to
some media at
2.86 mM, representing a 10-100 fold molar excess depending on the quantity of
input enzyme.
Uptake was typically measured in triplicate wells.
[0164] After incubation, the media were removed from the wells and assayed in
duplicate for GUS activity. Wells were washed five times with 1 mL of
37°C phosphate-
buffered saline, then incubated for 15 minutes at room temperature in 0.2 mL
of lysis buffer (10
mM Tris, pH 7.5, 100 mM NaCI, 5 mM EDTA, and 1 % NP-40). Cell lysates were
transferred to
microfuge tubes and spun at 13,000 rpm for 5 minutes to remove cell debris.
Two 10 p.L
aliquots of lysate were assayed for GUS activity using a standard fluorometric
assay. Three 10
~,L aliquots of lysate were assayed for protein concentration (Pierce Micro
BCA protein assay,
Pierce, IL).
[0165] An initial experiment compared uptake of CHO-produced GUS-GILT~1-7
with CHO-produced GUS~C18-GILTOl-7. As shown in Table 5, the GUSOC18-GILTO1-7
protein, which was engineered to eliminate a potential protease cleavage site,
has significantly
higher levels of uptake levels that can be inhibited by IGF-II and by M6P. In
contrast, the uptake
of a recombinant GUS produced in mammalian cells lacking the IGF-II tag was
unaffected by
the presence of excess IGF-II but was completely abolished by excess M6P. In
this experiment,
uptake was performed for 18 hours.



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Table 5.
Enzyme Input Uptake +IGF-II % IGF-II+M6P % M6P
units (units/mg)(units/mg)inhibition(units/mg)inhibition


CHO GUS-GILT~l-7 982 310+27 8420 73 22336 28


CHO GUS~C18- 1045 704226 25850 63 41279 41
GILT~1-7


CHO GUS 732 35230 33677 5 10.2 99.7


[01GG] A subsequent experiment assessed the uptake of CHO- and HEK293
produced enzymes by human fibroblasts from MPSVII patients. In this
experiment, uptake was
for 21 hours.
TABLE 6.
Enzyme Input Uptake (units/mg)+IGF-II % IGF-II
units Uptake (units/mg)inhibition


CHO GUS~C18-GILT~1-7 2812 40811037 1007132 75


HEK GUS-GILT 2116 1432196


HEK GUSOC18-GILTO1-7 3021 5192320 1207128 77


HEK GUS-GILTY27L 3512 1514203


HEK GUS-GILTF19SE12K 3211 4227371 38896 90.8


HEK GUS-GILTF19S 3169 4733393 43960 90.7


[0167] A further experiment assessed the uptake of selected enzymes in the
presence
of IGF-II, 8mM M6P, or both inhibitors. Uptake was measured for a period of
22.5 hours.
TABLE 7.
Enzyme InputUptake +IGF-II% IGF- +M6P %M6P +IGF-II%IGF-


units(units/ (units/II (units/inhibi- +M6P II+M6P


mg) mg) inhibi- mg) tion (units/inhibi-


tion mg) tion


CHO 1023 1580 47327 70 63961 60 0~1 100


GUS~C18- 150





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GILTO1-7


HEK GUS- 880 1227 222 98.2 846+61 31 0~3 100


GILTF19S 76


E12K


HEK GUS- 912 1594 21717 86 95296 60 152 99.06


GILTF19S 236


[0168] The experiments described above show that CHO and HEK293 production
systems are essentially equivalent in their ability to secrete functional
recombinant proteins. The
experiments also show that the presence of excess IGF-II diminishes uptake of
tagged proteins
by 70-90+%, but does not markedly affect uptake of untagged protein (4.5%),
indicating specific
IGF-II-mediated uptake of the mammalian-produced protein. Unlike Leishma~ia-
produced
proteins, the enzymes produced in mammalian cells are expected to contain M6P.
The presence
of two ligands on these proteins capable of directing uptake through the
M6P/IGF-II receptor
implies that neither excess IGF-II nor excess M6P should completely abolish
uptake.
Furthermore, since the two ligands bind to discrete locations on the receptor,
binding to the
receptor via one ligand should not be markedly affected by the presence of an
excess of the other
competitor.
Example 12. he vivo therapy
[0169] Initially, GUS minus mice can be used to assess the effectiveness of
GUS-
GILT and derivatives thereof in enzyme replacement therapy. GUS minus mice are
generated by
heterozygous coatings of B6.C-H 2bm1/ByBIR-gusmps/+ mice as described by
Birkenmeier et al.
(1989) J. Clin. Invest 83(4):1258-6. Preferably, the mice are tolerant to
human (3-GUS. The
mice may carry a transgene with a defective copy of human (3-GUS to induce
immunotolerance
to the human protein (Sly et al. (2001) PNAS 98:2205-2210). Alternatively,
human [3-GUS (e.g.
as a GUS-GILT protein) can be administered to newborn mice to induce
immunotolerance.
However, because the blood-brain barrier is not formed until about day 15 in
mice, it is simpler
to determine whether GILT-GUS crosses the blood-brain barrier when initiating
injections in
mice older than 15 days; transgenic mice are therefore preferable.
[0170] The initial experiment is to determine the tissue distribution of the
targeted
therapeutic protein. At least three mice receive a CHO-produced GILT-tagged (3-
GUS protein



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referred to herein as GUSOC18-GILTO1-7, in which GUST B, a (3-GUS protein
omitting the last
eighteen amino acids of the protein, is fused to the N-terminus of Ol-7 GILT,
an IGF-II protein
missing the first seven amino acids of the mature protein. Other mice receive
either (3-GUS, a
buffer control, or a GUS~C18-GILTOl-7 protein treated with periodate and
sodium borohydride
as described in Example 14. Generally, preferred doses are in the range of 0.5-
7 mg/kg body
weight. In one example, the enzyme dose is 1 mg/lcg body weight administered
intravenously,
and the enzyme concentration is about 1-3 mg/mL. In addition, at least three
mice receive a dose
of 5 mg/kg body weight of GUSOC18-GILTO1-7 protein treated with periodate and
sodium
borohydride. After 24 hours, the mice are sacrificed and the following organs
and tissues are
isolated: liver, spleen, kidney, brain, lung, muscle, heart, bone, and blood.
Portions of each
tissue are homogenized and the (3-GUS enzyme activity per mg protein is
determined as
described in Sly et al. (2001) PNAS 98:2205-2210. Portions of the tissues are
prepared for
histochemistry and/or histopathology carried out by published methods (see,
e.g., Vogler et al.
(1990) Am J. Pathol. 136:207-217).
[0171] Further experiments include multiple injection protocols in which the
mice
receive weekly injections at a dose of 1 mg/kg body weight. In addition,
measurement of the
half life of the periodate-modified enzyme is determined in comparison with
untreated enzyme
as described in Example 14.
[0172] Two other assay formats can be used. In one format, 3-4 animals are
given a
single injection of 20,OOOU of enzyme in 100 ~1 enzyme dilution buffer (150 mM
NaCI, 10 mM
Tris, pH7.5). Mice are killed 72-96 hours later to assess the efficacy of the
therapy. In a second
format, mice are given weekly injections of 20,000 units over 3-4 weeks and
are killed 1 week
after the final injection. Histochemical and histopathologic analysis of
liver, spleen and brain are
carried out by published methods (Birkenmeier et al. (1991) Blood 78(11):3081-
92; Sands et al.
(1994) J. Clin. Invest 93(6):2324-31; Daly et al. (1999) Proc. Natl. Acad.
Sci. USA 96(5):2296
300). In the absence of therapy, cells (e.g. macrophages and Kupffer cells) of
GUS minus mice
develop large intracellular storage compartments resulting from the buildup of
waste products in
the lysosomes. It is anticipated that in cells in mice treated with GUS-GILT
constructs, the size
of these compartments will be visibly reduced or the compartments will shrink
until they are no
longer visible with a light microscope.
[0173] Similarly, humans with lysosomal storage diseases will be treated using
constructs targeting an appropriate therapeutic portion to their lysosomes. In
some instances,



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treatment will take the form of regular (e.g. weekly) injections of a GILT
protein. In other
instances, treatment will be achieved through administration of a nucleic acid
to permit persistent
ih vivo expression of a GILT protein, or through admiiustration of a cell
(e.g. a human cell, or a
unicellular organism) expressing the GILT protein in the patient. For example,
the GILT protein
can be expressed in situ using a Leislzmania vector as described in U.S.
Patent No. 6,020,144,
issued February 1, 2000; U.S. Provisional Application No. 60/250,446; and U.S.
Provisional
Application Attorney Docket No. SYM-OOSPRA, "Protozoan Expression Systems for
Lysosomal
Storage Disease Genes", filed May 11, 2001.
[0174] Targeted therapeutic proteins of the invention can also be
administered, and
their effects monitored, using methods (enzyme assays, histochemical assays,
neurological
assays, survival assays, reproduction assays, etc.) previously described for
use with GUS. See,
for example, Vogler et al. (1993) Pediatric Res. 34(6):837-840; Sands et al.
(1994) J. Clin.
Invest. 93:2324-2331; Sands et al. (1997) J. Clin. Invest. 99:1596-1605;
O'Connor et al. (1998)
J. Clin. Invest. 101:1394-1400; and Soper et al. (1999) 45(2):180-186.
Example 13 GILT-modified enzyme replacement therapy for Fabry's disease
[0175] The objective of these experiments is to evaluate the efficacy of GILT-
modified alpha-galactosidase A (a-GAL A) as an enzyme replacement therapy for
Fabry's
disease.
[017G] Fabry's disease is a lysosomal storage disease resulting from
insufficient
activity of a-GAL A, the enzyme responsible for removing the terminal
galactose from GL-3
and other neutral sphingolipids. The diminished enzymatic activity occurs due
to a variety of
missense and nonsense mutations in the x-linked gene. Accumulation of GL-3 is
most prevalent
in lysosomes of vascular endothelial cells of the heart, liver, kidneys, skin
and brain but also
occurs in other cells and tissues. GL-3 buildup in the vascular endothelial
cells ultimately leads
to heart disease and kidney failure.
[0177] Enzyme replacement therapy is an effective treatment for Fabry's
disease, and
its success depends on the ability of the therapeutic enzyme to be taken up by
the lysosomes of
cells in which GL-3 accumulates. The Genzyme product, Fabrazyme, is
recombinant a-GAL A
produced in DUI~X Bl 1 CHO cells that has been approved for treatment of
Fabry's patients in
Europe due to its demonstrated efficacy.
[0178] The ability of Fabrazyme to be taken up by cells and transported to the
lysosome is due to the presence of mannose 6-phosphate (M6P) on its N-linked
carbohydrate.



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Fabrazyme is delivered to lysosomes through binding to the mannose-6-
phosphate/IGF-II
receptor, present on the cell surface of most cell types, and subsequent
receptor mediated
endocytosis. Fabrazyme reportedly has three N-linked glycosylation sites at
ASN residues 108,
161, and 184. The predominant carbohydrates at these positions are fucosylated
biantennary
bisialylated complex, monophosphorylated mannose-7 oligomannose, and
biphosphorylated
mannose-7 oligomannose, respectively.
[0179] The glycosylation independent lysosomal targeting (GILT) technology of
the
present invention directly targets therapeutic proteins to the lysosome via a
different interaction
with the IGF-II receptor. A targeting ligand is derived from mature human IGF-
II, which also
binds with high affinity to the IGF-II receptor. In current applications, the
IGF-II tag is provided
as a c-terminal fusion to the therapeutic protein, although other
configurations are feasible
including cross-linking. The competency of GILT-modified enzymes for uptake
into cells has
been established using GILT-modified 13-glucuronidase, which is efficiently
taken up by
fibroblasts in a process that is competed with excess IGF-II. Advantages of
the GILT
modification are increased binding to the M6P/IGF-II receptor, enhanced uptake
into lysosomes
of target cells, altered or improved pharmacokinetics, and expanded, altered
or improved range
of tissue distribution. The improved range of tissue distributions could
include delivery of
GILT-modified a-GAL A across the blood-brain barrier since IGF proteins
demonstrably cross
the blood-brain barrier.
[0180] Another advantage of the GILT system is the ability to produce uptake-
competent proteins in non-mammalian expression systems where M6P modifications
do not
occur. In certain embodiments, GILT-modified protein is produced primarily in
CHO cells. In
certain others, the GILT tag is placed at the c-terminus of a-GAL A, although
the invention is
not so limited.
Example 13B GILT-modified enzyme replacement therapy for Pompe disease
[0181] The objective of these experiments is to evaluate the efficacy of GILT-
modified acid alpha-glucosidase (GAA) as an enzyme replacement therapy for
Pompe disease.
[0182] The glycosylation independent lysosomal targeting (GILT) technology of
the
present invention permits M6P-independent targeting of GAA to patient
lysosomes. In one
embodiment, GAA or a catalytically-active fragment thereof is fused at its N-
terminus to a
targeting moiety including the signal peptide of human IGF-lI. A targeting
moiety including the
signal peptide should improve secretion of GAA from host cells, thereby
achieving high yield



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production and facilitating harvest of the therapeutic GILT-modified GAA. In
one embodiment,
hybrid GAA gene cassettes are generated in which the human IGF-II signal
peptide and mature
IGF-II coding sequences containing desired modifications are fused to the N-
terminus of a GAA
cassette at His 29, a site of signal peptide cleavage observed in COS-1 cells,
or to another
suitable site (Wisselaar et al. (1993) J Biol Chem,. 268(3):2223-31).
Alternatively, the desired
GILT tag can be positioned at sites downstream of the known cleavage sites to
preclude removal
of the GILT tag by possible proteolytic processing. An exemplary N-terminal
GILT-tagged
GAA cassette is depicted in Figure 20.
[0183] In another embodiment, hybrid GAA gene cassettes are generated in which
the human GAA gene is appended to an in frame 3' DNA segment encoding one of a
series of
mature human IGF-II peptide tags. The fusion can occur either after the C-
terminal Cys at
position 952 or at sites that are upstream of Cys952. Placing the GILT tag
upstream from the C-
terminus also removes potential proteolysis sites and two glycosylation sites
that are absent from
the mature GAA protein. Since oligosaccharides participate in clearance via
the mannose
receptor, reducing glycosylation in GAA by placing the GILT tag upstream from
the C-terminus
should increase the half life of GILT-modified GAA. Constructs are designed to
place C
terminal GILT tags every 10 amino acid residues from the C-terminus up to
residue 816 that
marks the upstream limit on the predicted C-terminus of the mature 70 kDa
enzyme. An
exemplary C-terminal GILT-tagged GAA cassette is depicted in Figure 21.
[0184] In yet another embodiment, hybrid GAA gene cassettes can be generated
in
which desired GILT tags are fused to both the N-terminus and C-terminus of GAA
cassette.
[0185] To identify which of the above described GILT-modified GAA constructs
are
most enzymatically active and uptake-competent, the constructs are transfected
into HEK293
cells, CHO cells, or other suitable cells and culture supernatants from pools
of clones axe assayed
for active enzyme and for uptake into Pompe fibroblast cells.
[018G] To test enzymatic activity, culture supernatants are assayed for GAA
activity
using the fluorogenic substrate, 4-methylumbelliferyl-a-D-glucopyranoside
(Reuser et al., (1978)
Am J Hum Genet, 30(2):132-43) or the colorometric substrate p-nitrophenyl-a-D-
glucopyranoside. Total enzyme activity is normalized to cell number to compare
relative levels
of expression achieved by various clonal cell lines. To test uptake
competency, the culture
medium is collected, subjected to filtration, and applied to Pompe fibroblast
cells. To assess
specificity of uptake, cells are incubated for 3-16 hours in the presence or
absence of 5 mM



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M6P, or IGF-II, or both. Pompe fibroblast cells are harvested and the cell
associated GAA
activity is measured.
[0187] The GILT-modified GAA with the most promising uptake characteristics
and/or enzymatic activities are selected for large scale production in CHO
cells or other suitable
expression systems.
[0188] GAA deficient mice (available from Jackson Laboratories) are used to
assess
the ability of GILT-modified GAA to clear accumulated glycogen from tissues
(Bijvoet et al.
(1999) J. Pathol., 189(3):416-24; Raben et al. (1998) J. Biol. Chem.,
273(30):19086-92). One
exemplary protocol includes 4 weekly injections of 3-5 mice with doses of up
to 100 mg/kg.
Evaluation of the injected mice includes biochemical analysis of glycogen and
GAA activity in
selected tissues including various muscle tissues, heart, and liver as well as
histopathological
analysis to visualize glycogen deposits in tissues.
GAA constructs
[0189] Human Image cDNA clone No. 4374238 was obtained from Open
Biosystems. This clone contains a full length cDNA encoding human GAA isolated
from library
NIH MGC 97 made from testis and cloned into pBluescript (pBluescript -GAA).
The sequence
of GAA in clone No. 4374238 is shown in Figure 22 and in SEQ ID NO:23.
Sequence analysis
of GAA in clone No. 4374238 confirmed the presence of a full length cDNA with
four silent
nucleotide changes compared to the GenBank sequence NM 000152. Three of these
have been
noted previously as common single-nucleotide polymorphisms in GAA: 642C/T,
1581G/A,
2133A/G. The residues in bold are present in clone 4374238. The predicted
heterozygosities for
the these three polymorphisms are about 0.23, 0.4, and 0.42, respectively, in
the general
population. The fourth polymorphism is 1665C/A.
[0190] An exemplary C-terminal GILT-tagged GAA cassette is depicted in Figure
21. An initial set of GAA cassettes with a C-terminal GILT tag placed at a
variety of positions
will be constructed by PCR using clone 4374238 as a template. The GAA open
reading frame
(ORF) is 2859 residues long. To minimize PCR-induced mutations, the 5' half of
the ORF is
first amplified by PCR using 5' primer GAA 13 (SEQ ID N0:25) and 3' primer GAA
14 (SEQ
ID N0:26) which anneals at the site of a unique SanDI restriction site at
residue position 1649.
This PCR product is ligated between the EcoRI and XbaI site in pBluescript
creating plasmid
p5' GAA. Next, 3' portions of the GAA ORF are amplified using 5' primer GAA 15
(SEQ ID
N0:27) that resides at the site of the SanDI site and one of a set of 3'
primers that are designed to
specify a series of termini offset by 30 nucleotides, as shown in Table 8.
Each of primers GAA



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16 - GAA 26 contains an AscI site for ligation of the GILT tag and an XbaI
site for cloning into
pBluescript. The primers are shown in Table 8: the lower case portion of
primers GAA 16 -
GAA 26 contains engineered AscI and XbaI sites, whereas the upper case portion
includes GAA
gene sequence. Primer GAA 27 which contains the native stop codon and lacks
the AscI site is
used to generate a full length and untagged clone. The amplified 3' portion is
ligated to p5'GAA
at SanDI and XbaI sites. This creates a series of GAA cassettes to wluch GILT
tags are inserted
using the AscI and XbaI sites. The complete GILT-tagged GAA cassettes are then
cloned into
expression vector pCEP4 for transfection into HEK293 cells.
TABLE 8. Primers for generating IGF-II tagged GAA proteins.
Primers Sequence


GAA 13 (-6-21) ggaattcCAACCATGGGAGTGAGGCACCCGCCC


(SEQ ID NO:25)


GAA 14 (1654-1632) gctctagagcGGGTCCCCCCAACCACCCCAGGC


(SEQ ID N0:26)


GAA 15 (1648-1668) ggaattcacGGGACCCTCCAGGCGGCAACC


(SEQ ID N0:27)


GAA 16 (2448-2429) gctctagacggcgcgccGACGTTGATGGTGTCCAGGG


(SEQ ID N0:28)


GAA 17 (2487-2469) gctctagacggcgcgccAGGGCCCTGCAGGGGGATG


(SEQ ID N0:29)


GAA 18 (2526-2507) gctctagacggcgcgccGGCCATGGGCTGCTGGCGGG


(SEQ ID N0:30)


GAA 19 (2565-2547) gctctagacggcgcgccCCCTCGGGCCTCTCCACCC


(SEQ ID N0:31)


GAA 20 (2604-2584) gctctagacggcgcgccCAGCACTTCCAGGCTCTCTCC


(SEQ ID N0:32)


GAA 21 (2643-2623) gctctagacggcgcgccCCTGGCCAGGAAGATGACCTG


(SEQ ID N0:33)


GAA 22 (2682-2662) gctctagacggcgcgccACTGGTCACACGTACCAGCTC


(SEQ ID N0:34)


GAA 23 (2721-2701) gctctagacggcgcgccCAGGACAGTCACCTTCTGCAG


(SEQ ID N0:35)





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GAA 24 (2760-2739) gctctagacggcgcgccACCGTTGGAGAGGACCTGCTGG


(SEQ ID N0:36)


GAA 25 (2799-2780) gctctagacggcgcgccCTTGGTGTCGGGGCTGTAGG


(SEQ ID N0:37)


GAA 26 (2856-2835) gctctagacggcgcgccACACCAGCTGACGAGAAACTGC


(SEQ ID N0:38)


GAA 27 (2859-2853) gctctagaCTAACACCAGCTGACGAGAAACTGC


(SEQ ID N0:39)


Note: Primers GAA16-GAA27, and GAA14 are bottom strand primers. The lower case
portions of primers (iAA
16-GAA 26 contain engineered AscI and XbaI sites. Primers GAA 13 and GAA 15
are upper strand primers. The
lower case portions of primers GAA 13 and GAA 15 contain an engineered EcoRI
site. The upper case portion of
each primer contains GAA gene sequence.
[0191] An exemplary N-terminal GILT-tagged GAA cassette is depicted in Figure
20. Construction of 5' N-terminal tagged GILT cassettes will be achieved in a
similar fashion.
For example, to construct a GAA cassette with the GILT tag fused to amino acid
29 of GAA (the
site of signal peptide cleavage in COS cells), a 5' GAA cassette is amplified
using a 5' primer
containing an AscI and an EcoRI site and containing GAA coding sequence that
begins at
residue His 29 and a 3' primer that anneals at the site of a unique StuI site
at position 776 in
GAA protein relative to the start site. This fragment is cloned into
pBluescript containing full
length GAA (pBluescript -GAA) at EcoRI and StuI sites replacing the native
amino terminus.
Once this plasmid is made, the desired GILT tag and signal peptide is cloned
between the EcoRI
and AscI sites.
Example 14. Under~lycosylated therapeutic proteins
[0192] The efficacy of a targeted therapeutic can be increased by extending
the serum
half life of the targeted therapeutic. Hepatic mannose receptors and
asialoglycoprotein receptors
eliminate glycoproteins from the circulation by recognizing specific
carbohydrate structures (Lee
et al. (2002) Science 295(5561):1898-1901; Ishibashi et al. (1994) J. Biol.
Chem.
269(45):27803-6). In some embodiments, the present invention permits targeting
of a
therapeutic to lysosomes and/or across the blood brain barrier in a manner
dependent not on a
carbohydrate, but on a polypeptide or an analog thereof. Actual
underglycosylation of these
proteins is expected to greatly increase their half life in the circulation,
by minimizing their
removal from the circulation by the mannose and asialoglycoprotein receptors.
Similarly,



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functional deglycosylation (e.g. by modifying the carbohydrate residues on the
therapeutic
protein, as by periodate/ sodium borohydride treatment) achieves similar
effects by interfering
with recognition of the carbohydrate by one or more clearance pathways.
Nevertheless, because
targeting of the protein relies, in most embodiments, on protein-receptor
interactions rather than
carbohydrate-receptor interactions, modification or elimination of
glycosylation should not
adversely affect targeting of the protein to the lysosome and/or across the
blood brain barrier.
[0193] Any lysosomal enzyme using a peptide targeting signal such as IGF-II
can be
chemically or enzymatically deglycosylated or modified to produce a
therapeutic with the
desirable properties of specific lysosomal targeting plus long serum half
life. In the case of some
lysosomal storage diseases where it might be important to deliver the
therapeutic to macrophage
or related cell types via mannose receptor, fully glycosylated therapeutics
can be used in
combination with underglycosylated targeted therapeutics to achieve targeting
to the broadest
variety of cell types.
Proteins unde~~glycosylated when synthesized
[0194] In some cases it will be preferable to produce the targeted therapeutic
protein
initially in a system that does not produce a fully glycosylated protein. For
example, a targeted
therapeutic protein can be produced in E. coli, thereby generating a
completely unglycosylated
protein. Alternatively, an unglycosylated protein is produced in mammalian
cells treated with
tunicamycin, an inhibitor of Dol-PP-GIcNAc formation. If, however, a
particular targeted
therapeutic does not fold correctly in the absence of glycosylation, it is
preferably produced
initially as a glycosylated protein, and subsequently deglycosylated or
rendered functionally
underglycosylated.
[0195] Underglycosylated targeted therapeutic proteins can also by prepared by
engineering a gene encoding the targeted therapeutic protein so that an amino
acid that normally
serves as an acceptor for glycosylation is changed to a different amino acid.
For example, an
asparagine residue that serves as an acceptor for N-linked glycosylation can
be changed to a
glutamine residue, or another residue that is not a glycosylation acceptor.
This conservative
change is most likely to have a minimal impact on enzyme structure while
eliminating
glycosylation at the site. Alternatively, other amino acids in the vicinity of
the glycosylation
acceptor can be modified, disrupting a recognition motif for glycosylation
enzymes without
necessarily changing the amino acid that would normally be glycosylated.
[0196] In the case of GUS, removal of any one of 4 potential glycosylation
sites
lessens the amount of glycosylation while retaining ample enzyme activity
(Shipley et al. (1993)



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J. Biol. Chem. 268(16):12193-8). Removal of some sets of two glycosylation
sites from GUS
still permits significant enzyme activity. Removal of all four glycosylation
sites eliminates
enzyme activity, as does treatment of cells with tunicamycin, but
deglycosylation of purified
enzyme results in enzymatically active material. Therefore, loss of activity
associated with
removal of the glycosylation sites is likely due to incorrect folding of the
enzyme.
[0197] Other enzymes, however, fold correctly even in the absence of
glycosylation.
For example, bacterial (3-glucuronidase is naturally unglycosylated, and can
be targeted to a
mammalian lysosome and/or across the blood brain barrier using the targeting
moieties of the
present invention. Such enzymes can be synthesized in an unglycosylated state,
rather than, for
example, synthesizing them as glycosylated proteins and subsequently
deglycosylating them.
Deglycosylatioh
[0198] If the targeted therapeutic is produced in a mammalian cell culture
system, it
is preferably secreted into the growth medium, which can be harvested,
permitting subsequent
purification of the targeted therapeutic by, for example, chromatographic
purification protocols,
such as those involving ion exchange, gel filtration, hydrophobic
chromatography, ConA
chromatography, affinity chromatography or immunoaffinity chromatography.
[0199] Chemical deglycosylation of glycoproteins can be achieved in a number
of
ways, including treatment with trifluoromethane sulfonic acid (TFMS), or
treatment with
hydrogen fluoride (HF).
[0200] Chemical deglycosylation by TFMS (Sojar et al. (1989) J. Biol. Chem.
264(5):2552-9; Sojar et al. (1987) Methods Enz~ 138:341-50): 1 mg GILT-GUS is
dried
under vacuum overnight. The dried protein is treated with 150 ~1 TFMS at
0°C for 0.5-2 hours
under nitrogen with occasional shaking. The reaction mix is cooled to below -
20°C in a dry ice-
ethanol bath and the reaction is neutralized by the gradual addition of a
prechilled (-20°C)
solution of 60% pyridine in water. The neutralized reaction mix is then
dialyzed at 4°C against
several changes of NH4HC03 at pH 7Ø Chemical deglycosylation with TFMS can
result in
modifications to the treated protein including methylation, succinimide
formation and
isomerization of aspartate residues (Douglass et al. (2001) J. Protein Chem.
20(7):571-6).
[0201] Chemical deglycosylation by HF (Sojar et al. (1987) Methods Enzymol.
138:341-50): The reaction is carried out in a closed reaction system such as
can be obtained from
Peninsula Laboratories, Inc. 10 mg GILT-GUS is vacuum dried and placed in a
reaction vessel
which is then connected to the HF apparatus. After the entire HF line is
evacuated, 10 mL



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anhydrous HF is distilled over from the reservoir with stirring of the
reaction vessel. The
reaction is continued for 1-2 hours at 0°C. Afterwards, a water
aspirator removes the HF over
15-30 minutes. Remaining traces of HF are removed under high vacuum. The
reaction mixture
is dissolved in 2 mL 0.2M NaOH to neutralize any remaining HF and the pH is
readjusted to 7.5
with cold 0.2M HCI.
[0202] Enzymatic deglycosylation (Thotakura et al. (1987) Methods Enzyrnol.
138:350-9): N-linked carbohydrates can be removed completely from
glycoproteins using
protein N-glycosidase (PNGase) A or F. In one embodiment, a glycoprotein is
denatured prior to
treatment with a glycosidase to facilitate action of the enzyme on the
glycoprotein; the
glycoprotein is subsequently refolded as discussed in the "In vitro refolding"
section above. In
another embodiment, excess glycosidase is used to treat a native glycoprotein
to promote
effective deglycosylation. Some cell types, such as the CHO-derived Lecl cell
line, produce
glycoproteins with reduced or simplified glycosylation, facilitating
subsequent enzymatic
deglycosylation as described in Example 14D.
[0203] In the case of a targeted therapeutic protein that is actually
underglycosylated,
it is possible that the reduced glycosylation will reveal protease-sensitive
sites on the targeted
therapeutic protein, which will diminish the half life of the protein. N-
linked glycosylation is
known to protect a subset of lysosomal enzymes from proteolysis (Kundra et al.
(1999) J. Biol.
Chem. 274(43):31039-46). Such protease-sensitive sites are preferably
engineered out of the
protein (e.g. by site-directed mutagenesis). As discussed below, the risk of
revealing either a
protease-sensitive site or a potential epitope can be minimized by incomplete
deglycosylation or
by modifying the carbohydrate structure rather than omitting the carbohydrate
altogether.
Modification of carbohydrate structure or partial deglycosylatior~
[0204] In some embodiments, the therapeutic protein is partially
deglycosylated. For
example, the therapeutic protein can be treated with an endoglycosidase such
as endoglycosidase
H, which cleaves N-linked high mannose carbohydrate but not complex type
carbohydrate
leaving a single GIcNAc residue linked to the asparagine. A therapeutic
protein treated in this
way will lack high mannose carbohydrate, reducing interaction with the hepatic
mannose
receptor. Even though this receptor recognizes terminal GIcNAc, the
probability of a productive
interaction with the single GIcNAc on the protein surface is not as great as
with an intact high
mannose structure. If the therapeutic protein is produced in mammalian cells,
any complex
carbohydrate present on the protein will remains unaffected by the endoH
treatment and may be
terminally sialylated, thereby diminishing interactions with hepatic
carbohydrate recognizing



CA 02487815 2004-11-26
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receptors. Such a protein is therefore likely to have increased half life. At
the same time, steric
hinderance by the remaining carbohydrate should shield potential epitopes on
the protein surface
from the immune system and diminish access of proteases to the protein surface
(e.g. in the
protease-rich lysosomal environment).
[0205] In other embodiments, glycosylation of a therapeutic protein is
modified, e.g.
by oxidation, reduction, dehydration, substitution, esterification,
alkylation, sialylation, carbon-
carbon bond cleavage, or the like, to reduce clearance of the therapeutic
protein from the blood.
In some preferred embodiments, the therapeutic protein is not sialylated. For
example, treatment
with periodate and sodium borohydride is effective to modify the carbohydrate
structure of most
glycoproteins. Periodate treatment oxidizes vicinal diols, cleaving the carbon-
carbon bond and
replacing the hydroxyl groups with aldehyde groups; borohydride reduces the
aldehydes to
hydroxyls. Many sugar residues include vicinal diols and, therefore, are
cleaved by this
treatment. As shown in Figure 9A, a protein may be glycosylated on an
asparagine residue with
a high mannose carbohydrate that includes N-acetylglucosamine residues near
the asparagine
and mannose residues elsewhere in the structure. As shown in Figure 9B, the
terminal mannose
residues have three consecutive carbons with hydroxyl groups; both of the
carbon-carbon bonds
involved are cleaved by periodate treatment. Some nonterminal mannose residues
also include a
vicinal diol, which would similarly be cleaved. Nevertheless, while this
treatment converts
cyclic carbohydrates into linear carbohydrates, it does not completely remove
the carbohydrate,
minimizing risks of exposing potentially protease-sensitive or antigenic
polypeptide sites.
[020G] The half life of lysosomal enzyme [3-glucuronidase is known to increase
more
than tenfold after sequential treatment with periodate and sodium borohydride
(Houba et al.
(1996) Bioconju~. Chem. 7(5):606-11; Stahl et al (1976) PNAS 73:4045-4049;
Achord et al.
(1977) Pediat. Res. 11:816-822; Achord et al. (1978) Cell 15(1):269-78).
Similarly, ricin has
been treated with a mixture of periodate and sodium cyanoborohydride (Thorpe
et al. (1985) Eur.
J. Biochem. 147:197). After injection into rats, the fraction of ricin
adsorbed by the liver
decreased from 40% (untreated ricin) to 20% (modified ricin) of the injected
dose with chemical
treatment. In contrast the amount of ricin in the blood increased from 20%
(untreated ricin) to
45% (treated ricin). Thus, the treated ricin enjoyed a wider tissue
distribution and longer half
life in the circulation.
[0207] A (3-glucuronidase construct (or other glycoprotein) coupled to a
targeting
moiety of the invention when deglycosylated or modified by sequential
treatment with periodate
and sodium borohydride should enjoy a similar (e.g. more than twofold, more
than fourfold, or



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-64-
more than tenfold) increase in half life while still retaining a high affinity
for the cation-
independent M6P receptor, permitting targeting of the construct to the
lysosome of all cell types
that possess this receptor. The construct is also predicted to cross the blood
brain barrier
efficiently. In contrast, if a (3-glucuronidase preparation that relies on M6P
for lysosomal
targeting is deglycosylated or treated with periodate and sodium borohydride,
it will enjoy an
elevated serum half life but will be unable to target the lysosome since the
M6P targeting signal
will have been modified by the treatment.
[0208] Carbohydrate modification by sequential treatment with periodate and
sodium
borohydride can be performed as follows: Purified GILT-GUS is incubated with
40 mM NaI04
in 50 mM sodium acetate pH 4.5 for 2 hours at 4°C. The reaction is
stopped by addition of
excess ethylene glycol and unreacted reagents are removed by passing the
reaction mix over
Sephadex G-25M equilibrated with PBS pH 7.5. This treatment is followed by
incubation with
40 mM NaBH4 in PBS at pH 7.5 and 37°C for three hours and then for one
hour at 4°C. Passing
the reaction mixture over a Sephadex G-25M column eluted with PBS at pH 7.5
terminates the
reaction.
[0209] Another protocol for periodate and sodium borohydride treatment is
described
in Hickman et al. (1974) BBRC 57:55-61. The purified protein is dialyzed into
O.OlM sodium
phosphate pH 6.0, 0.15 M NaCI. Sodium periodate is added to a final
concentration of O.OlM
and the reaction proceeds at 4°C in the dark for at least six hours.
Treatment of (3-
hexosaminidase with periodate under these conditions is sufficient to prevent
uptake of the
protein by fibroblasts; uptake is normally dependent on M6P moieties on the (3-
hexosaminidase
with the M6P receptor on the fibroblast cell surface. Thus, periodate
oxidation modifies M6P
sufficiently to abolish its ability to interact with the M6P receptor.
[0210] Alternatively, the carbohydrate can be modified by treatment with
periodate
and cyanoborohydride in a one step reaction as disclosed in Thorpe et al.
(1985) Eur. J.
Biochem. 147:197-206.
[0211] The presence of carbohydrate in a partially deglycosylated protein or a
protein
with a modified glycosylation pattern should shield potential polypeptide
epitopes that might be
uncovered by complete absence of glycosylation. In the event that a
therapeutic protein does
provoke an immune response, immunosuppressive therapies can be used in
conjunction with the
therapeutic protein (Brooks (1999) Molecular Genetics and Metabolism 68:268-
275). For
example, it has been reported that about 15% of Gaucher disease patients
treated with
alglucerase developed immune responses (Beutler, et al., in The Metabolic and
Molecular Bases



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of Inherited Disease, 8th ed. (2001), Scriver et al., eds., pp. 3635-3668).
Fortunately, many
(82/142) of the patients that produced antibody against alglucerase became
tolerized by the
normal treatment regimen (Rosenberg et al., (1999) Blood 93:2081-2088). Thus,
to benefit the
small minority of patients who may develop an immune response, a patient
receiving a
therapeutic protein also receives an immunosuppressive therapy in some
embodiments of the
invention.
Testivcg
[0212] To verify that a protein is underglycosylated, it can be tested by
exposure to
ConA. An underglycosylated protein is expected to demonstrate reduced binding
to ConA-
sepharose when compared to the corresponding fully glycosylated protein.
[0213] An actually underglycosylated protein can also be resolved by SDS-PAGE
and compaxed to the corresponding fully-glycosylated protein. For example,
chemically
deglycosylated GUS-GILT can be compared to untreated (glycosylated) GUS-GILT
and to
enzymatically deglycosylated GUS-GILT prepared with PNGase A. The
underglycosylated
protein is expected to have a greater mobility in SDS-PAGE when compared to
the fully
glycosylated protein.
[0214] Underglycosylated targeted therapeutic proteins display uptake that is
dependent on the targeting domain. Underglycosylated proteins should display
reduced uptake
(and, preferably, substantially no uptake) that is dependent on mannose or
M6P. These
properties can be experimentally verified in cell uptake experiments.
[0215] For example, a GUS-GILT protein synthesized in mammalian cells and
subsequently treated with periodate and borohydride can be tested for
functional deglycosylation
by testing M6P-dependent and mannose-dependent uptake. To demonstrate that M6P-
dependent
uptake has been reduced, uptake assays are performed using GM4668 fibroblasts.
In the absence
of competitor, treated and untreated enzyme will each display significant
uptake. The presence
of excess IGF-II substantially reduces uptake of treated and untreated enzyme,
although
untreated enzyme retains residual uptake via a M6P-dependent pathway. Excess
M6P reduces
the uptake of untreated enzyme, but is substantially less effective at
reducing the uptake of
functionally deglycosylated protein. For treated and untreated enzymes, the
simultaneous
presence of both competitors should substantially abolish uptake.
[021G] Uptake assays to assess mannose-dependent uptake are performed using
J774-
E cells, a mouse macrophage-like cell line bearing mannose receptors but few,
if any, M6P
receptors (Diment et al. (1987) J. Leukocyte Biol. 42:485-490). The cells are
cultured in



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DMEM, low glucose, supplemented with 10% FBS, 4 mM glutamine, and antibiotic,
antimycotic
solution (Sigma, A-5955). Uptake assays with these cells are performed in a
manner identical to
assays performed with fibroblasts. In the presence of excess M6P and IGF-II,
which will
eliminate uptake due to any residual M6P/IGF-II receptor, fully glycosylated
enzyme will
display significant uptake due to interaction with the mannose receptor.
Underglycosylated
enzyme is expected to display substantially reduced uptake under these
conditions. The mannose
receptor-dependent uptake of fully glycosylated enzyme can be competed by the
addition of
excess (100 ~,g/mL) mannan.
[0217] Pharmacokinetics of deglycosylated GUS-GILT can be determined by giving
intravenous injections of 20,000 enzyme units to groups of three MPSVII mice
per timepoint.
For each timepoint 50 ~,L of blood is assayed for enzyme activity.
Example 14A. De~ycosylation of GUS~C18-GILTO1-7
[0218] GUS~C18-GILTO1-7 was treated with endoglycosidase Fl to reduce
glycosylation. Specifically, 0.5 mg of GUS~C18-GILT~l-7 were incubated with 25
~.L of
endoglycosidase F1 (Prozyme, San Leandro, CA) for 7.5 hours at 37°C in
a final volume of 250-
300 ~,L of 50 mM phosphate buffer pH 5.5. After incubation, the GUS~C18-GILTO1-
7 was
repeatedly concentrated in a Centricon spin concentrator with a 50 kD
molecular weight cutoff to
separate the GUSOC18-GILTOl-7 from the endoglycosidase F1.
[0219] In a separate experiment, GUSOC18-GILTO1-7 was treated both with
endoglycosidase Fl and with endoglycosidase F2. Specifically, 128 ~L of
GUSOC18-GILTO1-7
(7.79 million units/ mL) were incubated with 12.8 ~,L of each endoglycosidase
(each from
Prozyme) in a final volume of 325 wL of 50 mM phosphate, pH 5.5, for 6 hours
at 37°C,
followed by purification by spin concentrators as described above.
[0220] One to three micrograms of the GUSOC18-GILTO1-7 treated with
endoglycosidase F1 was resolved by electrophoresis through a 7.5%
polyacrylamide gel. The
gel was stained with coomassie brilliant blue dye and is shown in Figure 10.
BAN refers to
untreated GUSOC18-GILT~1-7. HBGS refers to untagged human (3-glucuronidase,
which has a
lower molecular weight and higher mobility than the tagged GUS~C18-GILT~1-7.
The treated
GUS~C18-GILTO1-7 is shown as ~~N+Fl, and has, on average, a modestly increased
mobility
compared to the untreated protein, consistent with at least partial
deglycosylation of the protein.
A further increase in mobility is observed in samples of GUSOC18-GILT~1-7
treated with both



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endoglycosidase F1 and endoglycosidase F2 (data not shown), further suggesting
that
endoglycosidase F1 only partially deglycosylates the protein.



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Example 14B. Ih vitro uptake of de~lycosylated protein
[0221] Cultured mucopolysaccharidosis VII skin fibroblast GM 4668 cells were
used
to assess uptake of GUSOC18-GILT~1-7 with or without treatment with
endoglycosidase F1.
Specifically, the cells were grown in DMEM low glucose + 4 mM glutamine +
antibiotic/antimycotic + 15% FBS (not heat inactivated) at 37°C in 5%
COa. Passaging of cells
was done with a scraper. Cells were cultured to about 50-80% confluency in T75
flasks (75 cm2
surface area). The day before the uptake experiment, the cells were split into
12-well tissue
culture plates (46 cm2total surface area). The cells were permitted to re-
attach for 1-4 hours,
then the medium was changed before permitting the cells to grow overnight.
[0222] Uptake experiments were performed in triplicate. The uptake medium is
DMEM low glucose + 2% BSA (SigmaA-7030) + 4 mM glutamine +
antibiotic/antimycotic.
The final volume of uptake medium + enzyme is 1 niL/well. The cells were
washed in uptake
medium and incubated in uptake medium + [3-glucuronidase (M6P), untreated
GUS~C18-
GILTOl-7 (GILT), or GUSOC18-GILT~l-7 after treatment with endoglycosidase F1
(GILT+F1)
(approximately 4000 units) with or without 2 mM mannose-6-phosphate (+M6P) or
2.86mM
recombinant IGF-II (+Tag) for 3 hours. The medium was then removed, each well
was washed
with 4 x 1mL PBS, and the cells were incubated for 5-10 minutes at room
temperature with 200
~.L lysis buffer (100 mL lysis buffer: 1 mL of 1M Tris pH 7.5, 2 mL NaCI, 1mL
O.SM EDTA, 96
mL water; add 10 ~.L of NP-40 for every mL of lysis buffer immediately prior
to use). All lysate
was transferred into microfuge tubes and spun for 5 minutes at full speed. 10
~,L of lysates were
assayed in duplicate for GUS activity. 10 wL of lysates were assayed in
triplicate for total
protein concentration using Pierces Micro BCA kit. Two p.L of the uptake
medium was also
assayed in duplicate for GUS activity.
[0223] As shown in Figure 11, GUS~C18-GILT~1-7, with or without treatment with
endoglycosidase F1, is taken up efficiently by the fibroblasts, even in the
presence of mannose-6-
phosphate, whereas the uptake of the untagged [3-glucuronidase is essentially
eliminated by the
presence of the mannose-6-phosphate competitor. In contrast, IGF-II
successfully inhibited
uptake of the GUSOC18-GILTO1-7 protein, essentially abolishing uptake of the
protein treated
with endoglycosidase F1, indicating that the uptake of the treated protein is
indeed mediated by
the GILT~l-7 tag.
Example 14C. In vivo uptake experiments



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-69-
[0224] To determine the half life of the treated and untreated proteins in
circulation,
experiments were done using cannulated Sprague-Dawley rats. 100,000-150,000
units of
GUS~C18-GILTO1-7 were administered intravenously, the protein was untreated
(GUSOC18-
GILTO1-7), treated with endoglycosidase Fl and endoglycosidase F2 (+F1+F2), or
treated with
endoglycosidase F1 and endoglycosidase F2 followed by a 45 minute incubation
with 5 mM
dithiothreitol in phosphate-buffered saline at 37°C to disrupt the GILT
tag. Blood was
withdrawn through the cannula at regular intervals thereafter, treated at
60°C for one hour to
inactivate endogenous rat glucuronidase, and assayed for glucuronidase
activity. The results are
shown in Figure 12. The half life of the protein was estimated by fitting the
early time points to
an equation in the form of y=e-kt using I~aleidaGraph v.3.52. The half life of
the untreated
protein is estimated at 8 minutes. Treatment with endoglycosidases F1 and F2
increases the half
life to about 13 minutes, consistent with improved evasion of the
reticuloendothelial clearance
system upon deglycosylation. Treatment with DTT should interfere with uptake
of the tagged
enzyme via the IGF-II receptor and further increases the half life of the
endoglycosidase
F1/endoglycosidase F2 treated protein to about 16 minutes.
[0225] The delivery of untreated GUS~C 18-GILTO1-7 (GILT), treated with
endoglycosidase F1 (GILT+F1) to tissues in vivo was assessed and compared to
delivery of
untagged (3-glucuronidase (M6P). All proteins were produced in CHO cells. 1 mg
of M6P,
GILT, or GILT+F1 per kg of body weight was administered to MPSVII mice by
infusing the
protein into the tail vein. Control animals (Control) were infused with buffer
alone. Twenty-
four hours later the animals were sacrificed and tissue samples were collected
for analysis. The
data depicted in Figures 13-15 result from infusion of 6-7 animals with each
enzyme.
[0226] Figure 13 shows the levels of the various enzymes detected in liver,
spleen,
and bone marrow. Macrophages are abundant in these tissues and clear the
enzymes from the
circulation, primarily through the high mannose receptor. Because GILT+F 1
should have less
lugh mannose carbohydrate than the untreated enzymes, less accumulation in
these tissues is
expected. In the liver and bone marrow, accumulation of the endoglycosidase F1
treated enzyme
was almost as great accumulation of the untreated enzyme. In the spleen,
however, there was a
noticeable reduction in accumulation of GILT+F1. Both the treated and
entreated GUS~C18-
GILT~1-7 proteins reached heart, kidney, and lung tissues efficiently, with
accumulation equal
to or exceeding accumulation of untagged [3-glucuronidase, as shown in Figure
14. Thus, the
GILTOl-7 tag permits targeting to the same tissues as mannose-6-phosphate,
with equal or better



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efficiency. Furthermore, as shown in Figure 15, both the treated and untreated
GUSOC18-
GILTOl-7 proteins reached brain tissue at levels exceeding levels observed for
untagged ~3-
glucuronidase, although accumulation levels in brain tissue were distinctly
lower than the
accumulation levels in other tissues.
[0227] Selected tissues from infused animals were also subjected to histology
to
detect [3-glucuronidase as described in Wolf et al. (1992) Nature 360:749-753.
(3-glucuronidase
is detected as a red stain; nuclei are stained blue. The results of the liver
histology for untagged
(3-glucuronidase (HGUS), untreated GUSOC18-GILT~1-7 (Dd-l5gilt), and GUS~C18-
GILTO1-
7 treated with endoglycosidase F1 (dd15F1) are shown in Figure 16. On the HGUS
slide, the
elongate Kupffer cells stain an intense red, whereas the more compact cells,
primarily
hepatocytes, contain relatively little red stain, and appear in the figure as
a blue nucleus
surrounded by a whitish area. In contrast, the hepatocytes of the other two
slides, containing
untreated or treated GUS~C18-GILTO1-7, stain rather intensely, as evidenced by
the sharply
reduced number of blue nuclei immediately surrounded by white. This helps to
explain why
even the GUSOC18-GILTO1-7 treated with endoglycosidase F1 still localized
significantly to the
liver. The tagged protein reaches a more diverse subset of cell types in the
liver. Thus, while it
may be less subject to clearance by high mannose receptors, the protein
nevertheless reaches the
liver in quantity because it targets a greater variety of cells.
[0228] Additional histology data are shown in Figures 17-19. Figure 17 shows
localization to the glomeruli of the kidney. Although histochemical staining
is not often
quantitative, slides from animals infused with GUS~C18-GILT~1-7 (shown as Dd-
15
(untreated) and F1 (treated with endoglycosidase Fl) reproducibly show a more
intense staining
in the glomeruli than slides from animals infused with the untagged protein.
As shown in
Figures 18 and 19, the tagged protein, whether (dd-15F1) or not (dd-15)
treated with
endoglycosidase F1, appears capable of reaching the same cells that untagged
protein (HGUS)
reaches.
Example 14D. De~lycosylated, Lecl-produced proteins
[0229] GUS~C18-GILT~1-7 protein was produced in Lecl cells, treated with
endoglycosidase F1, and tested ivc vitro and i~c vivo for targeting and
uptake. The Lecl cells,
which were procured from the ATCC (ATCC# CRL1735), are CHO-derived cells
lacking 13-1,2-
N-acetylglucosaminyl transferase I (GIcNAc-T1) activity (Stanley et al.,
(1975) Cell 6(2):121-8;
Stanley et al., (1975) PNAS 72(9):3323-7). GIcNAc-Tl adds N-acetylglucosamine
to the core



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oligosaccharide Mans-GIcNAc2-Asn. This addition is an initial, obligatory step
in the synthesis
of complex and hybrid N-linked oligosaccharides. Thus, glycoproteins from Lecl
cells lack
complex and hybrid oligosaccharides. Although Lecl cells do produce
glycoproteins bearing
high mannose oligosaccharides, these structures are amenable to removal with
endoglycosidase
Fl. Accordingly, production of a GILT-tagged protein in Lecl cells with
subsequent
endoglycosidase F1 treatment should yield a deglycosylated protein. When
injected into the
bloodstream of an animal, such a protein should avoid clearance by mannose
receptors in liver-
resident macrophage and accumulate to greater levels in target tissues.
[0230] A 13-GUSOC18-GILTOl-7 cassette in expression plasmid pCXN was
electroporated into Lecl cells at 50 ~.F and 1,200 V in a 0.4-cm cuvette.
Cells were propagated
in a-MEM supplemented with 15% FBS, 1.2 mM glutamine, 50 ~,g/mL proline, and 1
mM
pyruvate. Selection of colonies and amplification was mediated by 400 ~,g/mL
6418 for 2-3
weeks. Confluent cultures of clonal lines were placed in collection medium
(Weymouth medium
supplemented with 2% FBS, 1.2 mM glutamine, and 1 mM pyruvate). Medium
containing the
secreted recombinant enzyme was collected every 24-72 hours. One line, Lecl-
18, was selected
for enzyme production as it produced the highest yields of recombinant enzyme.
[0231] Recombinant enzyme from Lecl cells was affinity purified using anti-
human
[3-glucuronidase-Affigel 10 resin as described in Example 10. The uptake of
the Lecl-produced,
GILT-tagged enzyme was compared to that of CHO-produced tagged or untagged
enzymes. As
shown in Table 9, the tagged, Lecl-produced enzyme exhibited more M6P-
independent uptake
than the corresponding tagged, CHO-produced enzyme and much more than an
untagged
enzyme. Treatment of the Lecl-produced enzyme with endoglycosidase F1
eliminated M6P-
independent binding but not IGF-II-dependent binding. As shown in Figure 23,
SDS-PAGE of
endoglycosidase Fl treated and untreated Lecl-produced enzymes showed a
distinct mobility
shift approaching the mobility of a PNGase F treated enzyme, which should be
completely
deglycosylated. This suggests treatment of Lecl-produced enzyme with
endoglycosidase F1
causes a significant loss of glycosylation. In contrast, neither the CHO-
produced enzyme nor the
HEIR-produced enzyme showed a marked shift in mobility after treatment with
endoglycosidase
F1.



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TABLE 9
Comparison of uptake
+/- M6P of CHO-produced
untagged human (3-
glucuronidase (HBGS)
and 00 and Lec-1-18-produced
~0 by MPS VII
fibroblasts


non-M6P
Enzyme (cell line) - M6P +M6P Mediated Uptake


HBGS (CHO) 251 7 2.7



0~ (CHO) 207 98 47%



~~ (Lecl-18) 230 150 65%


[0232] The endoglycosidase F1-treated Lecl- and CHO-produced enzymes and the
untreated HEK 293-produced enzyme were each infused into 3 immunotolerant
MPSVII mice
(Sly et al. (2001) PNAS 98:2205-2210) at a dose of 1 mg/kg. 24 hours later,
the mice were
sacrificed and tissues were assayed for enzyme activity. As shown in Figure
24, the Lecl-
produced enzyme accumulated less in the liver and more in the target tissues
of heart, muscle,
and kidney than did the CHO-produced enzyme. HEK 293-produced enzyme showed
slightly
higher accumulation of enzyme in the heart, lcidney and muscle than did the
Lecl-produced
enzyme.
[0233] The measured accumulation of the enzyme in the target tissues depends
both
on the efficiency of targeting to the tissue and on the rate of clearance of
the enzyme from the
tissue. If, upon proper targeting to the lysosome, the Lec-1-produced enzyme
is degraded more
slowly than the CHO-produced enzyme, this could account for the higher
observed levels of
accumulation. On the other hand, a more rapid degradation of the Lec-1-
produced enzyme could
mask a magnified targeting efficacy. To address this possibility, the half
lives of treated and
untreated Lec-l and CHO-produced enzymes were tested in an uptake assay with
MPSVII
fibroblasts. The fibroblasts were incubated with enzyme for 3 hours, the cells
were washed and
placed in fresh media that were changed daily. For each enzyme, duplicate
wells were lysed on
days 0, 3 and 7. The fraction of enzyme remaining was plotted against time and
the data were fit
to an exponential equation. As shown in Figure 25, the endoglycosylase F1-
treated, Lec-1-



CA 02487815 2004-11-26
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produced enzyme was found to have a half life similar to that of HBGS and
slightly less than that
of the CHO-produced enzyme in human fibroblasts.
INCORPORATION BY REFERENCE
[0234] The disclosure of each of the patent documents, scientific
publications, and
Protein Data Bank records disclosed herein, and U.S. Provisional Application
No. 60/250,446,
filed November 30, 2000; U.S. Provisional Application 60/287,531, filed April
30, 2001; U.S.
Provisional Application 60/290,281, filed May 11, 2001; U.S. Provisional
Application
60/304,609, filed July 10. 2001; U.S. Provisional Application No. 60/329,461,
filed October 15,
2001; International Patent Application Serial No. PCT/LJSO1/44935, filed
November 30, 2001;
U.S. Provisional Application No. 60/351,276, filed January 23, 2002; U.S.
Serial Nos.
10/136,841 and 10/136,639, filed April 30, 2002, U.S. Serial No. 60/384,452,
filed May 29,
2002; U.S. Serial No. 60/386,019, filed June 5, 2002; and U.S. Serial No.
60/408,816, filed
September 6, 2002, are incorporated by reference into this application in
their entirety.
We claim:



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SEQUENCE LISTING
<110> Symbiontics, Inc.
LeBowitz, Jonathan H
Beverley, Stephen
Sly, William S.
<120> TARGETED THERAPEUTIC PROTEINS
<130> SYM-009PC
<150> US 60/384,452
<151> 2002-05-29
<150> US 10/272,531
<151> 2002-10-16
<150> US 60/445,734
<151> 2003-02-06
<150> US 60/386,019
<151> 2002-06-05
<150> US 60/408,816
<151> 2002-09-06
<160> 39
<170> PatentIn version 3.1
<210> 1
<211> 543
<212> DNA
<213> Homo sapiens
<220>
<221> CDS
<222> (1)..(540)
<223>
<400>
1 atccca atggggaag tcgatgctg gtgcttctcacc ttcttg 48
ga
at


g g IlePro MetGlyLys SerMetLeu ValLeuLeuThr PheLeu
MetGly


1 5 10 15


gccttc gcctcg tgctgcatt getgettac cgccccagtgag accctg 96


AlaPhe AlaSer CysCysIle AlaAlaTyr ArgProSerGlu ThrLeu


20 25 30


tgcggc ggggag ctggtggac accctccag ttcgtctgtggg gaccgc 144


CysGly GlyGlu LeuValAsp ThrLeuGln PheValCysGly AspArg


35 40 45


ggcttc tacttc agcaggccc gcaagccgt gtgagccgtcgc agccgt 192


GlyPhe TyrPhe SerArgPro AlaSerArg ValSerArgArg SerArg


50 55 60


1





CA 02487815 2004-11-26
WO 03/102583 PCT/US03/17211
ggcatcgtt gaggagtgctgt ttccgcagc tgtgacctg gccctcctg 240


GlyIleVal GluGluCysCys PheArgSer CysAspLeu AlaLeuLeu


65 70 75 80


gagacgtac tgtgetaccccc gccaagtcc gagagggac gtgtcgacc 288


GluThrTyr CysAlaThrPro AlaLysSer GluArgAsp ValSerThr


85 90 95


cctccgacc gtgcttccggac aacttcccc agatacccc gtgggcaag 336


ProProThr ValLeuProAsp AsnPhePro ArgTyrPro ValGlyLys


100 105 110


ttcttccaa tatgacacctgg aagcagtcc acccagcgc ctgcgcagg 384


PhePheGln TyrAspThrTrp LysGlnSer ThrGlnArg LeuArgArg


115 120 125


ggcctgcct gccctcctgcgt gcccgccgg ggtcacgtg ctcgccaag 432


GlyLeuPro AlaLeuLeuArg AlaArgArg GlyHisVal LeuAlaLys


130 135 140


gagctcgag gcgttcagggag gccaaacgt caccgtccc ctgattget 480


GluLeuGlu AlaPheArgGlu AlaLysArg HisArgPro LeuIleAla


145 150 155 160


ctacccacc caagaccccgcc cacgggggc gccccccca gagatggcc 528


LeuProThr GlnAspProAla HisGlyGly AlaProPro GluMetA1a


165 170 175


agcaatcgg aagtga 543


SerAsnArg Lys


180


<210> 2


<211> 180


<212> PRT


<213> Homosapiens


<400> 2
MetGlyIlePro MetGlyLys SerMetLeu ValLeu ThrPheLeu
Leu


1 5 l0 15


AlaPheAlaSer CysCysIle AlaAlaTyr ArgPro GluThrLeu
Ser


20 25 30


CysGlyGlyGlu LeuValAsp ThrLeuGln PheVal GlyAspArg
Cys


35 40 45


GlyPheTyrPhe SerArgPro AlaSerArg ValSer ArgSerArg
Arg


50 55 60


2



CA 02487815 2004-11-26
WO 03/102583 PCT/US03/17211
Gly Ile Val Glu Glu Cys Cys Phe Arg Ser Cys Asp Leu Ala Leu Leu
65 70 75 80
Glu Thr Tyr Cys Ala Thr Pro Ala Lys Ser Glu Arg Asp Val Ser Thr
85 90 95
Pro Pro Thr Val Leu Pro Asp Asn Phe Pro Arg Tyr Pro Val Gly Lys
100 l05 110
Phe Phe Gln Tyr Asp Thr Trp Lys Gln Ser Thr Gln Arg Leu Arg Arg
115 120 125
Gly Leu Pro Ala Leu Leu Arg A1a Arg Arg Gly His Val Leu Ala Lys
130 135 140
Glu Leu Glu Ala Phe Arg Glu Ala Lys Arg His Arg Pro Leu Ile Ala
145 150 155 160
Leu Pro Thr Gln Asp Pro Ala His Gly Gly Ala Pro Pro Glu Met Ala
165 170 175
Ser Asn Arg Lys
180
<210> 3
<211> 237
<2l2> DNA
<213> Artificial Sequence
<220>
<223> Leishmania codon optimized IGF-II
<220>
<221> CDS
<222> (14)..(223)
<223>
<400> 3
ccgtctagag ctc ggc gcg ccg gcg tac cgc ccg agc gag acg ctg tgc 49
Gly Ala Pro Ala Tyr Arg Pro Ser Glu Thr Leu Cys
1 5 10
ggc ggc gag ctg gtg gac acg ctg cag ttc gtg tgc ggc gac cgc ggc 97
Gly Gly Glu Leu Val Asp Thr Leu Gln Phe Val Cys Gly Asp Arg Gly
15 20 25
ttc tac ttc agc cgc ccg gcc agc cgc gtg agc cgc cgc agc cgc ggc 145
Phe Tyr Phe Ser Arg Pro Ala Ser Arg Val Ser Arg Arg Ser Arg Gly
3



CA 02487815 2004-11-26
WO 03/102583 PCT/US03/17211
30 35 40
atc gtg gag gag tgc tgc ttc cgc agc tgc gac ctg gcg ctg ctg gag 193
Ile Val Glu Glu Cys Cys Phe Arg Ser Cys Asp Leu Ala Leu Leu Glu
45 50 55 60
acg tac tgc gcg acg ccg gcg aag tcg gag taagatctag agcg 237
Thr Tyr Cys Ala Thr Pro Ala Lys Ser Glu
65 70
<210> 4
<211> 70
<212> PRT
<2l3> Artificial Sequence
<220>
<223> Leishmania codon optimized IGF-II
<400> 4
Gly Ala Pro Ala Tyr Arg Pro Ser Glu Thr Leu Cys Gly Gly Glu Leu
1 5 10 15
Val Asp Thr Leu Gln Phe Val Cys Gly Asp Arg Gly Phe Tyr Phe Ser
20 25 30
Arg Pro Ala Ser Arg Val Ser Arg Arg Ser Arg Gly Ile Val Glu Glu
35 40 45
Cys Cys Phe Arg Ser Cys Asp Leu Ala Leu Leu Glu Thr Tyr Cys Ala
50 55 60
Thr Pro Ala Lys Ser Glu
65 70
<210> 5
<211> 2169
<212> DNA
<213> Artificial Sequence
<220>
<223> A recombinant DNA sequence incorporating a signal peptide sequenc
e, the mature human beta-glucuronidase sequence, a bridge of thre
a amino acids, and an IGF-II sequence
<220>
<221> CDS
<222> (1)..(2166)
<223>
4



CA 02487815 2004-11-26
WO 03/102583 PCT/US03/17211
<400>



atg gcctct aggctcgtc cgt ctg gcggcc atgctg gca 48
gtg gcc gtt


Met Ser LeuVal Ala Leu
Ala Arg Arg Ala Val
Val Met Ala
Leu
Ala


1 5 10 15


gcg gccgtg tcggtcgac gcgctgcag ggcgggatg ctgtacccc cag 96


Ala AlaVal SerValAsp AlaLeuGln GlyGly LeuTyrPro Gln
Met


20 25 30


gag agcccg tcgcgggag tgcaaggag ctggacggc ctctggagc ttc 144


Glu SerPro SerArgGlu CysLysGlu LeuAspGly LeuTrpSer Phe


35 40 45


cgc gccgac ttctctgac aaccgacgc cggggcttc gaggagcag tgg 192


Arg AlaAsp PheSerAsp AsnArgArg ArgGlyPhe GluGluGln Trp


50 55 60


tac cggcgg ccgctgtgg ,gagtcaggc cccaccgtg gacatgcca gtt 240


Tyr ArgArg ProLeuTrp GluSerGly ProThrVal AspMetPro Val


65 70 75 80


ccc tccagc ttcaatgac atcagccag gactggcgt ctgcggcat ttt 288


Pro SerSer PheAsnAsp IleSerGln AspTrpArg LeuArgHis Phe


85 90 95


gtc ggctgg gtgtggtac gaacgggag gtgatcctg ccggagcga tgg 336


Val GlyTrp Va1TrpTyr GluArgGlu ValIleLeu ProGluArg Trp


100 105 110


acc caggac ctgcgcaca agagtggtg ctgaggatt ggcagtgcc cat 384


Thr GlnAsp LeuArgThr ArgValVal LeuArgIle GlySerAla His


115 120 125


tcc tatgcc atcgtgtgg gtgaatggg gtcgacacg ctagagcat gag 432


Ser TyrAla IleValTrp ValAsnG1y ValAspThr LeuGluHis Glu


130 135 140


ggg ggctac ctccccttc gaggccgac atcagcaac ctggtccag gtg 480


Gly GlyTyr LeuProPhe GluAlaAsp IleSerAsn LeuValGln Val


145 150 155 160


ggg cccctg ccctcccgg ctccgaatc actatcgcc atcaacaac aca 528


Gly ProLeu ProSerArg LeuArgIle ThrIleAla IleAsnAsn Thr


165 170 175


ctc acc accacc ccaccaggg acc caa tacctgact gac 576
ccc ctg atc


Leu Thr ThrThr ProProGly Gln TyrLeuThr Asp
Pro Leu Thr
Ile


180 185 190


acc tcc tac aac ttt gac 624
aag ttt aca
tat gtc tat
ccc cag
aag
ggt


Thr Tyr Tyr Asn Phe Asp
Ser Pro Phe Thr
Lys Lys Val Tyr
G1y Gln


195 200 205


ttt 672
ttc
aac
tac
get
gga
ctg.cag
cgg
tct
gta
ctt
ctg
tac
acg
aca


Phe
Phe
Asn
Tyr
Ala
Gly
Leu
Gln
Arg
Ser
Val
Leu
Leu
Tyr
Thr
Thr


210 215 220


5



CA 02487815 2004-11-26
WO 03/102583 PCT/US03/17211
cccaccacc atcgat atc gtcacc gtg caa 720
tac gac acc acc gag
agc


ProThrThr TyrIleAsp Ile Thr Val
Asp Thr Thr Glu
Val Ser Gln


225 230 235 240


gacagtggg ctggtgaat cag atctctgtc ggcagt ctg 768
tac aag aac


AspSerGly LeuValAsn Gln IleSerVal GlySer Leu
Tyr Lys Asn


245 250 255


ttcaagttg gaagtgcgt cttttg gatgcagaa aacaaagtc gtggcg 816


PheLysLeu GluValArg LeuLeu AspAlaGlu LysVal Ala
Asn Val


260 265 270


aatgggact gggacccag ggccaa cttaaggtg ccaggtgtc agcctc 864


AsnGlyThr GlyThrGln GlyGln LeuLysVal ProGlyVal SerLeu


275 280 285


tggtggccg tacctgatg cacgaa cgccctgcc tatctgtat tcattg 912


TrpTrpPro TyrLeuMet HisGlu ArgProAla TyrLeuTyr SerLeu


290 295 300


gaggtgcag ctgactgca cagacg tcactgggg cctgtgtct gacttc 960


GluValGln LeuThrAla GlnThr SerLeuGly ProValSer AspPhe


305 310 315 320


tacacactc cctgtgggg atccgc actgtgget gtcaccaag agccag 1008


TyrThrLeu ProValGly IleArg ThrValAla ValThrLys SerGln


325 330 335


ttcctcatc aatgggaaa cctttc tatttccac ggtgtcaac aagcat 1056


PheLeuI1e AsnGlyLys ProPhe TyrPheHis GlyValAsn LysHis


340 345 350


gaggatgcg gacatccga gggaag ggcttcgac tggccgctg ctggtg 1104


GluAspAla AspIleArg G1yLys GlyPheAsp TrpProLeu LeuVal


355 360 365


aaggacttc aacctgctt cgctgg cttggtgcc aacgetttc cgtacc 1152


LysAspPhe AsnLeuLeu ArgTrp LeuGlyAla AsnAlaPhe ArgThr


370 375 380


agccactac ccctatgca gaggaa gtgatgcag atgtgtgac cgctat 1200


SerHisTyr ProTyrAla GluGlu ValMetGln MetCysAsp ArgTyr


385 390 395 400


gggattgtg gtcatc gagtgt cccggcgtg ggtctg ctgccg 1248
gat gcg


GlyIleVal ValIleAsp GluCys ProGlyVal GlyLeu LeuPro
Ala


405 410 415


cagttc aacaac tctctg catcaccac atgcag atggaa 1296
ttc gtt gtg


GlnPhe AsnAsn Ser HisHisHis MetGln MetGlu
Phe Val Leu Val


420 425 430


gaa agg aag cac gtcgtg tggtct 1344
gtg gac aac ccc atg
gtg gcg
cgt


Glu Lys HisPro ValVal TrpSer
Val Asn Ala Met
Val
Arg
Arg
Asp


4'35 440 445


gtg tcc getggc tac 1392
gcc cac tac ttg
aac cta
gag gaa
cct tct
gcg


6



CA 02487815 2004-11-26
WO 03/102583 PCT/US03/17211
Val HisLeuGlu Tyr
Ala Ser Leu
Asn Ala
Glu Gly
Pro Tyr
Ala
Ser


450 455 460


aag atggtg aaatccttg ccc cct gtg 1440
atc gac tcc
get cgg
cac
acc


Lys Val Ile LysSerLeu Pro Ser Pro
Met. Ala Asp Arg Val
His
Thr


465 470 475 480


acc tttgtg agcaactct tatgcagca aag ggg ccg tat 1488
aac gac get


Thr PheVal SerAsnSer TyrAlaAla Lys Gly Pro Tyr
Asn Asp Ala


485 990 495


gtg gatgtg atctgtttg agctactac tgg tatcacgac tac 1536
aac tct


Val AspVal IleCysLeu SerTyrTyr Trp TyrHisAsp Tyr
Asn Ser


500 505 510


ggg cacctg gagttgatt cagctgcagctg acc cagtttgag aac 1584
gcc


Gly HisLeu GluLeuIle GlnLeuGlnLeu Thr GlnPheGlu Asn
Ala


515 520 525


tgg tataag aagtatcag aagcccattatt agc gagtatgga gca 1632
cag


Trp TyrLys LysTyrGln LysProIleIle Ser GluTyrGly Ala
Gln


530 535 540


gaa acgatt gcagggttt caccaggatcca ctg atgttcact gaa 1680
cct


Glu ThrIle AlaGlyPhe HisGlnAspPro Leu MetPheThr Glu
Pro


545 550 555 560


gag taccag aaaagtctg ctagagcagtac ctg ggtctggat caa 1728
cat


Glu TyrGln LysSerLeu LeuGluGTnTyr Leu GlyLeuAsp Gln
His


565 570 575


aaa cgcaga aaatatgtg gttggagagctc tgg aattttgcc gat 1776
att


Lys ArgArg LysTyrVal ValGlyGluLeu Trp AsnPheAla Asp
Ile


5g0 585 590


ttc atgact gaacagtca ccgacgagagtg ggg aataaaaag ggg 1824
ctg


Phe MetThr GluGlnSer ProThrArgVal Gly AsnLysLys Gly
Leu


595 600 605


atc ttcact cggcagaga caaccaaaaagt gcg ttccttttg cga 1872
gca


Ile PheThr ArgGlnArg GlnProLysSer Ala PheLeuLeu Arg
Ala


610 615 620


gag agatac tggaagatt gccaatgaaacc tat ccccactca gta 1920
agg


Glu ArgTyr TrpLysIle AlaAsn Tyr ProHisSer Val
Glu
Thr
Arg


625 630 635 640


gcc aagtca caatgtttg gaaaac act ggcgcg gcg 1968
agc ccg
ccg
ttt


Ala LysSer GlnCysLeu Glu Thr GlyAla Ala
Asn Pro
Ser
Pro
Phe


645 650 655


tac agcgagacg ctg gac ctg 2016
cgc tgc acg
ccg ggc
ggc
gag
ctg
gtg


Tyr SerGluThr Leu Asp Leu
Arg Cys Thr
Pro Gly
Gly
Glu
Leu
Val


660 665 670


cag tgc cgc ccg 2064
ttc ggc ggc gcc
gtg gac ttc agc
tac
ttc
agc
cgc


Gln CysGly Arg Pro
Phe Asp Gly Ala
Val Phe Ser
Tyr
Phe
Ser
Arg


7



CA 02487815 2004-11-26
WO 03/102583 PCT/US03/17211
675 680 685
cgc gtg agc cgc cgc agc cgc ggc atc gtg gag gag tgc tgc ttc cgc 2112
Arg Val Ser Arg Arg Ser Arg Gly Ile Val Glu Glu Cys Cys Phe Arg
690 695 . 700
agc tgc gac ctg gcg ctg ctg gag acg tac tgc gcg acg ccg gcg aag 2160
Ser Cys Asp Leu Ala Leu Leu Glu Thr Tyr Cys Ala Thr Pro Ala Lys
705 710 715 720
tcg gag taa 2169
Ser Glu
<210> 6
<211> 722
<212> PRT
<213> Artificial Sequence
<220>
<223> A recombinant DNA sequence incorporating a signal peptide sequenc
e, the mature human beta-glucuronidase sequence, a bridge of thre
a amino acids, and an IGF-II sequence
<400> 6
Met A1a Ser Arg Leu Val Arg Val Leu Ala Ala Ala Met Leu Val Ala
1 5 10 15
Ala A1a Val Ser Val Asp Ala Leu Gln Gly Gly Met Leu Tyr Pro Gln
20 25 30
Glu Ser Pro Ser Arg Glu Cys Lys Glu Leu Asp Gly Leu Trp Ser Phe
35 40 45
Arg Ala Asp Phe Ser Asp Asn Arg Arg Arg Gly Phe Glu Glu Gln Trp
50 55 60
Tyr Arg Arg Pro Leu Trp Glu Ser Gly Pro Thr Val Asp Met Pro Val
65 70 75 80
Pro Ser Ser Phe Asn Asp Ile Ser Gln Asp Trp Arg Leu Arg His Phe
85 90 95
Val G1y Trp Val Trp Tyr Glu Arg Glu Val Ile Leu Pro Glu Arg Trp
100 105 110
Thr Gln Asp Leu Arg Thr Arg Val Val Leu Arg Ile Gly Ser Ala His
115 120 125
8



CA 02487815 2004-11-26
WO 03/102583 PCT/US03/17211
Ser Tyr Ala Ile Val Trp Val Asn Gly Va1 Asp Thr Leu Glu His Glu
130 135 140
Gly Gly Tyr Leu Pro Phe Glu Ala Asp Ile Ser Asn Leu Val Gln Val
145 150 155 160
Gly Pro Leu Pro Ser Arg Leu Arg Ile Thr Ile Ala Ile Asn Asn Thr
165 170 175
Leu Thr Pro Thr Thr Leu Pro Pro Gly Thr Ile Gln Tyr Leu Thr Asp
180 185 190
Thr Ser Lys Tyr Pro Lys Gly Tyr Phe Val Gln Asn Thr Tyr Phe Asp
195 200 205
Phe Phe Asn Tyr Ala Gly Leu G1n Arg Ser Val Leu Leu Tyr Thr Thr
210 215 220
Pro Thr Thr Tyr Ile Asp Asp Ile Thr Val Thr Thr Ser Val Glu Gln
225 230 235 240
Asp Ser Gly Leu Val Asn Tyr Gln Ile Ser Val Lys Gly Ser Asn Leu
245 250 255
Phe Lys Leu Glu Val Arg Leu Leu Asp Ala Glu Asn Lys Val Val Ala
260 265 270
Asn Gly Thr Gly Thr Gln Gly Gln Leu Lys Val Pro Gly Val Ser Leu
275 280 285
Trp Trp Pro Tyr Leu Met His Glu Arg Pro Ala Tyr Leu Tyr Ser Leu
290 295 300
Glu Val Gln Leu Thr A1a Gln Thr Ser Leu Gly Pro Val Ser Asp Phe
305 310 315 320
Tyr Thr Leu Pro Val Gly Ile Arg Thr Val Ala Val Thr Lys Ser Gln
325 330 335
Phe Leu Ile Asn Gly Lys Pro Phe Tyr Phe His Gly Val Asn Lys His
340 345 350
9



CA 02487815 2004-11-26
WO 03/102583 PCT/US03/17211
Glu Asp Ala Asp Ile Arg Gly Lys Gly Phe Asp Trp Pro Leu Leu Val
355 360 365
Lys Asp Phe Asn Leu Leu Arg Trp Leu Gly Ala Asn Ala Phe Arg Thr
370 375 380
Ser His Tyr Pro Tyr Ala Glu Glu Val Met Gln Met Cys Asp Arg Tyr
385 390 395 400
Gly Ile Val Val Ile Asp Glu Cys Pro Gly Val Gly Leu Ala Leu Pro
405 410 415
Gln Phe Phe Asn Asn Val Ser Leu His His His Met Gln Val Met Glu
420 425 430
Glu Val Val Arg Arg Asp Lys Asn His Pro Ala Val Val Met Trp Ser
435 440 445
Val Ala Asn Glu Pro Ala Ser His Leu Glu Ser Ala Gly Tyr Tyr Leu
450 455 460
Lys Met Val Ile Ala His Thr Lys Ser Leu Asp Pro Ser Arg Pro Val
465 470 475 480
Thr Phe Val Ser Asn Ser Asn Tyr Ala A1a Asp Lys Gly Ala Pro Tyr
485 490 495
Val Asp Val Ile Cys Leu Asn Ser Tyr Tyr Ser Trp Tyr His Asp Tyr
500 505 510
Gly His Leu G1u Leu Ile Gln Leu Gln Leu Ala Thr Gln Phe Glu Asn
515 520 525
Trp Tyr Lys Lys Tyr Gln Lys Pro Ile Ile Gln Ser Glu Tyr Gly Ala
530 535 540
Glu Thr Ile Ala Gly Phe His Gln Asp Pro Pro Leu Met Phe Thr Glu
545 550 555 560
Glu Tyr Gln Lys Ser Leu Leu Glu Gln Tyr His Leu Gly Leu Asp Gln
565 570 575



CA 02487815 2004-11-26
WO 03/102583 PCT/US03/17211
Lys Arg Arg Lys Tyr Val Val Gly Glu Leu Ile Trp Asn Phe Ala Asp
580 585 590
Phe Met Thr Glu Gln Ser Pro Thr Arg Val Leu Gly Asn Lys Lys Gly
595 600 605
Ile Phe Thr Arg Gln Arg Gln Pro Lys Ser Ala Ala Phe Leu Leu Arg
610 615 620
Glu Arg Tyr Trp Lys Ile Ala Asn Glu Thr Arg Tyr Pro His Ser Val
625 630 635 640
Ala Lys Ser Gln Cys Leu Glu Asn Ser Pro Phe Thr Gly Ala Pro Ala
645 650 655
Tyr Arg Pro Ser Glu Thr Leu Cys Gly Gly Glu Leu Val Asp Thr Leu
660 665 670
Gln Phe Val Cys Gly Asp Arg Gly Phe Tyr Phe Ser Arg Pro Ala Ser
675 680 685
Arg Val Ser Arg Arg Ser Arg Gly Ile Val Glu Glu Cys Cys Phe Arg
690 695 700
Ser Cys Asp Leu Ala Leu Leu Glu Thr Tyr Cys Ala Thr Pro Ala Lys
705 710 715 720
Ser Glu
<210> 7
<211> 70
<212> PRT
<213> Homo sapiens
<400> 7
Gly Pro Glu Thr Leu Cys Gly Ala Glu Leu Val Asp A1a Leu Gln Phe
1 5 10 15
Val Cys Gly Asp Arg Gly Phe Tyr Phe Asn Lys Pro Thr Gly Tyr Gly
20 25 30
Ser Ser Ser Arg Arg Ala Pro Gln Thr Gly Ile Val Asp Glu Cys Cys
35 40 45
11



CA 02487815 2004-11-26
WO 03/102583 PCT/US03/17211
Phe Arg Ser Cys Asp Leu Arg Arg Leu Glu Met Tyr Cys Ala Pro Leu
50 55 60
Lys Pro Ala Lys Ser Ala
65 70
<210> 8
<211> 67
<212> PRT
<213> Homo sapiens
<400> 8
Ala Tyr Arg Pro Ser Glu Thr Leu Cys Gly Gly Glu Leu Val Asp Thr
1 5 10 15
Leu Gln Phe Val Cys Gly Asp Arg Gly Phe Tyr Phe Ser Arg Pro Ala
20 25 30
Ser Arg Val Ser Arg Arg Ser Arg Gly Ile Val Glu Glu Cys Cys Phe
35 40 45
Arg Ser Cys Asp Leu Ala Leu Leu Glu Thr Tyr Cys Ala Thr Pro Ala
50 55 60
Lys Ser Glu
&5
<210> 9
<211> 50
<212> DNA
<213> Artificial Sequence
<220>
<223> Oligonucleotide GILT 1
<400> 9
gcggcggcga gctggtggac acgctgcagt tcgtgtgcgg cgaccgcggc 50
<210> l0
<211> 50
<212> DNA
<213> Artificial Sequence
<220>
<223> Oligonucleotide GILT 2
12



CA 02487815 2004-11-26
WO 03/102583 PCT/US03/17211
<400> 10
ttctacttca gccgcccggc cagccgcgtg agccgccgca gccgcggcat 50
<210> 11
<211> 50
<212> DNA
<213> Artificial Sequence
<220>
<223> Oligonucleotide GILT 3
<400> 11
cgtggaggag tgctgcttcc gcagctgcga cctggcgctg ctggagacgt 50
<210> 12
<211> 40
<212> DNA
<213> Artificial Sequence
<220>
<223> Oligonucleotide GILT 4
<400> 12
actgcgcgac gccggcgaag tcggagtaag atctagagcg 40
<210> 13
<211> 50
<212> DNA
<213> Artificial Sequence
<220>
<223> Oligonucleotide GILT 5
<400> 13
agcgtgtcca ccagctcgcc gccgcacagc gtctcgctcg ggcggtacgc 50
<210> 14
<21l> 50
<212> DNA
<213> Artificial Sequence
<220>
<223> Oligonucleotide GIZ,T 6
<400> 14
ggctggccgg gcggctgaag tagaagccgc ggtcgccgca cacgaactgc 50
<210> 15
<211> 50
<212> DNA
<213> Artificial Sequence
13



CA 02487815 2004-11-26
WO 03/102583 PCT/US03/17211
<220>
<223> Oligonucleotide GILT 7
<400> 15
gctgcggaag cagcactcct ccacgatgcc gcggctgcgg cggctcacgc 50
<210> 16
<211> 51
<212> DNA
<213> Artificial Sequence
<220>
<223> Oligonucleotide GILT 8
<400> 16
ctccgacttc gccggcgtcg cgcagtacgt ctccagcagc gccaggtcgc a 51
<2l0> 17
<211> 47
<212> DNA
<213> Artificial Sequence
<220>
<223> Oligonucleotide GILT 9
<400> 17
'ccgtctagag ctcggcgcgc cggcgtaccg cccgagcgag acgctgt 47
<210> 18
<211> 25
<212> DNA
<213> Artificial Sequence
<220>
<223> Oligonucleotide GILT 10
<400> 18
cgctctagat cttactccga cttcg 25
<210> 19
<211> 46
<212> DNA
<213> Artificial Sequence
<220>
<223> Oligonucleotide GILT 11
<400> 19
ccgtctagag ctcggcgcgc cgctgtgcgg cggcgagctg gtggac 46
<210> 20
<211> 50
14



CA 02487815 2004-11-26
WO 03/102583 PCT/US03/17211
<212> DNA
<213> Artificial Sequence
<220>
<223> Oligonucleotide GILT 12
<400> 20
ttcctgttca gccgcccggc cagccgcgtg agccgccgca gccgcggcat 50
<210> 21
<211> 50
<212> DNA
<213> Artificial Sequence
<220>
<223> Oligonucleotide GILT 16
<400> 21
ggctggccgg gcggctgaac aggaagccgc ggtcgccgca cacgaactgc 50
<210> 22
<211> 25
<212> DNA
<213> Artificial Sequence
<220>
<223> Oligonucleotide GILT 20
<400> 22
ccgtctagag ctcggcgcgc cggcg 25
<210> 23
<211> 2851
<212> DNA
<213> Homo Sapiens
<220>
<221> CDS
<222> (1)..(2850)
<223>
<400> 23
atg gga gtg agg cac ccg ccc tgc tcc cac cgg ctc ctg gcc gtc tgc 48
Met Gly Val Arg His Pro Pro Cys Ser His Arg Leu Leu A1a Val Cys
1 5 10 15
gcc ctc gtg tcc ttg gca acc get gca ctc ctg ggg cac atc cta ctc 96
Ala Leu Val Ser Leu Ala Thr Ala Ala Leu Leu Gly His Ile Leu Leu
20 25 30
cat gat ttc ctg ctg gtt ccc cga gag ctg agt ggc tcc tcc cca gtc 144
His Asp Phe Leu Leu Val Pro Arg Glu Leu Ser Gly Ser Ser Pro Val
35 40 45



CA 02487815 2004-11-26
WO 03/102583 PCT/US03/17211
ctggag act cacccaget caccag ggagcc cca 192
gag cag agc ggg
aga


LeuGlu Thr ProAla Gln GlyAla Pro
Glu His His Gln Ser Gly
Arg


50 55 60


ccccgg gatgcc caggcacac cccggccgt cccagagca gtgcccaca 240


ProArg Ala GlnAlaHis ProGly ProArg ProThr
Asp Arg Ala
Val


65 70 75 80


cagtgc gacgtc ccccccaac agccgcttc gattgcgcc cctgacaag 288


GlnCys AspVal ProProAsn 5erArgPhe AspCysAla ProAspLys


85 90 95


gccatc acccag gaacagtgc gaggcccgc ggctgctgc tacatccct 336


AlaIle ThrGln GluGlnCys GluAlaArg GlyCysCys TyrIlePro


100 105 110


gcaaag cagggg ctgcaggga gcccagatg gggcagccc tggtgcttc 384


AlaLys GlnGly LeuGlnGly AlaGlnMet GlyGlnPro TrpCysPhe


115 120 125


ttccca cccagc taccccagc tacaagctg gagaacctg agctcctct 432


PhePro ProSer TyrProSer TyrLysLeu GluAsnLeu SerSerSer


130 135 140


gaaatg ggctac acggccacc ctgacccgt accaccccc accttcttc 480


GluMet G1yTyr ThrAlaThr LeuThrArg ThrThrPro ThrPhePhe


145 150 155 160


cccaag gacatc ctgaccctg cggctggac gtgatgatg gagactgag 528


ProLys AspIle LeuThrLeu ArgLeuAsp ValMetMet GluThrGlu


'165 170 175


aaccgc ctccac ttcacgatc aaagatcca getaacagg cgctacgag 576


AsnArg LeuHis PheThrIle LysAspPro AlaAsnArg ArgTyrGlu


180 185 190


gtgccc ttggag accccgcgt gtccacagc cgggcaccg tccccactc 624


ValPro LeuGlu ThrProArg ValHisSer ArgAlaPro SerProLeu


195 200 205


tacagc gtggag ttctctgag gagcccttc ggggtgatc gtgcaccgg 672


TyrSer ValG1u PheSerGlu GluProPhe GlyValIle ValHisArg


210 215 220


cagctg gacggc cgcgtgctg ctgaacacg acggtggcg cccctgttc 720


Gln AspGly ArgVa1Leu LeuAsnThr ThrValAla ProLeuPhe
Leu


225 230 235 240


ttt gac ttc ctg.tcc acc tcg ccc tcgcagtat 768
gcg cag ctt ctg
cag


Phe Asp PheLeu Leu Thr Pro Ser Tyr
Ala Gln Gln Ser Ser G1n
Leu


245 250 255


atc ccc ctc agc agc 816
aca ctg acc
ggc atg
ctc
gcc
gag
cac
ctc
agt


Ile Leu Ser
Thr Ser
Gly Thr
Leu
Ala
G1u
His
Leu
Ser
Pro
Leu
Met


260 265 270


16



CA 02487815 2004-11-26
WO 03/102583 PCT/US03/17211
tgg acc ccc ggt 864
agg atc
acc ctg
tgg aac
cgg gac
ctt gcg
ccc acg


Trp Thr Pro Gly
Arg Ile
Thr Leu
Trp Asn
Arg Asp
Leu Ala
Pro Thr


275 280 285


gcg aac cct gaggac.ggc 912
ctc tac ttc
ggg tct tac
cac ctg
gcg
ctg


Ala Asn Pro GluAsp Gly
Leu Tyr Phe
Gly Ser Tyr
His Leu,Ala
Leu


290 295 300


ggg tcg cac ggg gtg ctg atggat gtg 960
gca ttc cta
aac
agc
aat
gcc


Gly Ser His Gly Val Leu MetAsp Val
Ala Phe Leu
Asn
Ser
Asn
Ala


305 310 315 320


gtc ctg ccg agc cct ctt agc tgg agg tcg ggtggg atc 1008
cag gcc aca


Val Leu Pro Ser Pro Leu Ser Trp Arg Ser GlyGly Ile
Gln Ala Thr


325 330 335


ctg gat tac atc ttc ggc cca gag ccc aag gtggtg cag 1056
gtc ctg agc


Leu Asp Tyr Ile Phe Gly Pro Glu Pro Lys ValVal Gln
Val Leu Ser


340 345 350


cag tac gac gtt gtg tac ccg ttc atg ccg tactgg ggc 1104
ctg gga cca


Gln Tyr Asp Val Val Tyr Pro Phe Met Pro TyrTrp Gly
Leu Gly Pro


355 360 365


ctg ggc cac ctg tgc tgg ggc tac tcc tcc getatc acc 1152
ttc cgc acc


Leu Gly His Leu Cys Trp Gly Tyr Ser Ser AlaIle Thr
Phe Arg Thr


370 375 380


cgc cag gtg gag aac acc agg gcc cac ttc ctggac gtc 1200
gtg atg ccc


Arg Gln Val'Glu Asn Thr Arg Ala His Phe LeuAsp Val
Val Met Pro


385 390 395 400


caa tgg gac ctg gac atg gac tcc cgg agg ttcacg ttc 1248
aac tac gac


Gln Trp Asp Leu Asp Met Asp Ser Arg Arg PheThr Phe
Asn Tyr Asp


405 410 415


aac aag ggc ttc cgg ttc ccg gcc atg gtg gagctg cac 1296
gat gac cag


Asn Lys Gly Phe Arg Phe Pro Ala Met Val GluLeu His
Asp Asp Gln


420 425 430 -


cag ggc cgg cgc tac atg atc gtg gat cct atc 1344
ggc atg gcc agc
agc


Gln Gly Arg Arg Tyr Met Ile Val Asp Pro IleSer Ser
Gly Met Ala


435 440 445


tcg ggc gcc ggg agc agg ccc tac gac gag ctg 1392
cct tac ggt cgg
agg


Ser Gly Ala Gly Ser Arg Pro Tyr Asp Glu Leu
Pro Tyr Gly Arg
Arg


450 455 460


ggg gtt atc acc aac ggc cag ccg ctg ggg 1440
ttc gag acc att aag
gta


Gly Val Ile Thr Asn Gly Gln Pro Leu Gly
Phe Glu Thr Ile Lys
Val


465 470 475 480


tgg ccc gac ttc acc aac 1488
ggg tcc ccc aca gcc
act gcc ctg
ttc ccc


Trp Pro Asp Phe Thr Asn
Gly Ser Pro Thr Ala
Thr Ala Leu
Phe Pro


485 490 495


gcc tgg 1536
tgg gag
gac atg
gtg get
gag ttc
cat gac
cag gtg
ccc ttc


17



CA 02487815 2004-11-26
WO 03/102583 PCT/US03/17211
Ala Trp Trp Glu Asp Met Val Ala Glu Phe His Asp Gln Val Pro Phe
500 505 510


gac ggc atg att gac atg aac gag tcc aac ttc atc agg 1584
tgg cct ggc


Asp Gly Met Ile Asp Met Asn Glu Ser Asn Phe Ile Arg
Trp Pro Gly


515 520 525


tct gag gac tgc ccc aac aat gag gag aac cca ccc tac 1632
ggc ctg gtg


Ser Glu Asp Cys Pro Asn Asn Glu Glu Asn Pro Pro Tyr
Gly Leu Val


530 535 540


cct ggg gtg ggg ggg acc ctc cag gca acc atc tgt gcc 1680
gtt gcg tcc


Pro Gly Val Gly Gly Thr Leu Gln Ala Thr Ile Cys Ala
Val Ala Ser


545 550 555 560


agc cac cag ctc tcc aca cac tac ctg cac aac ctc tac 1728
ttt aac ggc


Ser His Gln Leu Ser Thr His Tyr Leu His Asn Leu Tyr
Phe Asn Gly


565 570 575


ctg acc gaa atc gcc tcc cac agg ctg gtg aag get cgg 1776
gcc gcg ggg


Leu Thr Glu Ile Ala Ser His Arg Leu Val Lys Ala Arg
Ala Ala Gly


580 585 590


aca cgc cca gtg atc tcc cgc tcg ttt get ggc cac ggc 1824
ttt acc cga


Thr Arg Pro Val I1e Ser Arg Ser Phe Ala Gly His Gly
Phe Thr Arg


595 600 605 E


tac gcc ggc tgg acg ggg gac gtg agc tcc tgg gag cag 1872
cac tgg ctc


Tyr Ala G1y Trp Thr Gly Asp Val Ser Sex Trp Glu Gln
His Trp Leu


610 615 620


gcc tcc tcc cca gaa atc ctg cag aac ctg ctg ggg gtg 1920
gtg ttt cct


Ala Ser Ser Pro Glu Ile Leu Gln Asn Leu Leu Gly Val
Val Phe Pro


625 630 635 640


ctg gtc ggg gac gtc tgc ggc ttc ggc aac acc tca gag 1968
gcc ctg gag


Leu Val Gly Asp Val Cys Gly Phe Gly Asn Thr Ser Glu
Ala Leu Glu


645 650 655


ctg tgt gtg tgg acc cag ctg ggg ttc tac ccc ttc atg 2016
cgc gcc cgg


Leu Cys Va1 Trp Thr Gln Leu Gly Phe Tyr Pro Phe Met
Arg Ala Arg


660 665 670


aac cac aac ctg ctc agt ctg ccc gag ccg tac agc ttc 2064
agc cag agc


Asn His Asn Leu Leu Ser Leu Pro
Ser Gln Glu Pro Tyr
Ser Phe Ser


675 680 685


gag ccg gcc 2112
cag cag gcc
atg agg aag
gcc ctc acc
ctg cgc tac
gca


Glu Pro A1a
Gln Gln Ala
Met Arg Lys
Ala Leu Thr
Leu Arg Tyr
Ala


690 695 700


ctc ctc ccc 2160
cac ctc tac
acg ctg ttc
cac cag gcc
cac gtc gcg
ggg


Leu Leu Pro
His Leu Tyr
Thr Leu Phe
His Gln Ala
His Val Ala
Gly


705 710 715 720


gag acc gtg 2208
gcc cgg ccc
ctc ttc ctg
gag ttc,ccc
aag gac tct
agc


Glu Thr Val
Ala Arg Pro
Leu Phe Leu
Glu Phe Pro
Lys Asp Ser
Ser


18



CA 02487815 2004-11-26
WO 03/102583 PCT/US03/17211
725 730 735


acc act cagctc ctgtgg gccctg ctcatc 2256
tgg gtg ggg
gac gag
cac


Thr Thr is GlnLeu LeuTrp Leu LeuIle
Trp Val Gly
Asp Glu
H Ala


740 745 750


accccagtg gggaag gccgaagtg actggctac ttcccc 2304
ctc
cag
gcc


ThrProVal GlyLys AlaGlu ThrGlyTyr PhePro
Leu Val
Gln
Ala


755 760 765


ttgggcaca tac ctgcag acggtgcca atagaggcc cttggc 2352
tgg gac


LeuGlyThr Tyr LeuGln ThrValPro IleGluAla LeuGly
Trp Asp


770 775 780


agcctccca cca cctgcaget ccccgtgag ccagccatc cacagc 2400
ccc


SerLeuPro Pro ProAlaAla ProArgGlu ProAlaIle HisSer
Pro


785 790 795 800


gaggggcag gtg acgctgccg gcccccctg gacaccatc aacgtc 2448
tgg


GluGlyGln Val ThrLeuPro AlaProLeu AspThrIle AsnVal
Trp


805 810 815


cacctccgg ggg tacatcatc cccctgcag ggccctggc ctcaca 2496
get


HisLeuArg Gly TyrIleIle ProLeuGln GlyProGly LeuThr
Ala


820 825 830


accacagag cgc cagcagccc atggccctg getgtggcc ctgacc 2544
tcc


ThrThrGlu Arg GlnGlnPro MetAlaLeu AlaValAla LeuThr
Ser


835 840 845


aagggtgga gcc cgaggggag ctgttctgg gacgatgga gagagc 2592
gag


LysGlyGly Ala ArgGlyGlu LeuPheTrp AspAspGly GluSer
Glu


850 855 860


ctggaagtg gag cgaggggcc tacacacag gtcatcttc ctggcc 2640
ctg


LeuGluVal Glu ArgGlyAla TyrThrGln ValIlePhe LeuAla
Leu


865 870 875 880


aggaataac atc gtgaatgag ctggtacgt gtgaccagt gaggga 2688
acg


ArgAsnAsn Ile ValAsnGlu LeuValArg ValThrSer GluGly
Thr


885 890 895


getggcctg ctg cagaag actgtcctg ggcgtggcc acggcg 2736
cag gtg


AlaGlyLeu Leu G1nLysVal ThrValLeu GlyValAla ThrAla
Gln


900 905 910


ccccag ctc tccaac gtc gtc tccaac acc 2784
cag ggt cct ttc tac
gtc


ProGln Leu Ser Val SerAsn Thr
Gln Asn Phe Tyr
Val Gly
Val
Pro


915 920 925


agc aag gtc gtc 2832
ccc ctg tcg
gac gac ctg
acc atc ttg
tgt atg
gga


Ser Lys ValLeu Va1
Pro Asp Ser
Asp Ile Leu
Thr Cys Leu
Met
Gly


930 935 940


gag agc 2851
cag t
ttt
ctc
gtc


Glu Ser
Gln
Phe
Leu
Val


945 950


19



CA 02487815 2004-11-26
WO 03/102583 PCT/US03/17211
<210> 24
<211> 950
<212> PRT
<213> Homo sapiens
<400> 24
Met Gly Val Arg His Pro Pro Cys Ser His Arg Leu Leu Ala Val Cys
1 5 10 15
Ala Leu Val Ser Leu Ala Thr Ala Ala Leu Leu Gly His Ile Leu Leu
20 25 30
His Asp Phe Leu Leu Val Pro Arg Glu Leu Ser Gly Ser Ser Pro Val
35 40 45
Leu Glu Glu Thr His Pro Ala His Gln Gln Gly Ala Ser Arg Pro Gly
50 55 60
Pro Arg Asp Ala Gln Ala His Pro Gly Arg Pro Arg Ala Val Pro Thr
65 70 75 80
Gln Cys Asp Val Pro Pro Asn Ser Arg Phe Asp Cys Ala Pro Asp Lys
85 90 95
Ala Ile Thr Gln Glu Gln Cys Glu Ala Arg Gly Cys Cys Tyr Ile Pro
100 105 110
Ala Lys Gln Gly Leu Gln Gly Ala Gln Met Gly Gln Pro Trp Cys Phe
115 120 125
Phe Pro Pro Ser Tyr Pro Ser Tyr Lys Leu Glu Asn Leu Ser Ser Ser
130 135 140
Glu Met Gly Tyr Thr Ala Thr Leu Thr Arg Thr Thr Pro Thr Phe Phe
145 150 155 160
Pro Lys Asp Ile Leu Thr Leu Arg Leu Asp Val Met Met Glu Thr Glu
165 170 175
Asn Arg Leu His Phe Thr Ile Lys Asp Pro Ala Asn Arg Arg Tyr Glu
180 185 190



CA 02487815 2004-11-26
WO 03/102583 PCT/US03/17211
Val Pro Leu Glu Thr Pro Arg Val His Ser Arg Ala Pro Ser Pro Leu
195 200 205
Tyr Ser Val Glu Phe Ser Glu Glu Pro Phe Gly Val Tle Val His Arg
210 215 220
Gln Leu Asp Gly Arg Val Leu Leu Asn Thr Thr Val Ala Pro Leu Phe
225 230 235 240
Phe Ala Asp Gln Phe Leu Gln Leu Ser Thr Ser Leu Pro Ser Gln Tyr
245 250 255
Ile Thr Gly Leu Ala Glu His Leu Ser Pro Leu Met Leu Ser Thr Ser
260 265 270
Trp Thr Arg Ile Thr Leu Trp Asn Arg Asp Leu Ala Pro Thr Pro Gly
275 280 285
Ala Asn Leu Tyr Gly Ser His Pro Phe Tyr Leu Ala Leu Glu Asp Gly
290 295 300
Gly Ser Ala His Gly Val Phe Leu Leu Asn Ser Asn Ala Met Asp Val
305 310 315 320
Val Leu Gln Pro Ser Pro Ala Leu Ser Trp Arg Ser Thr Gly Gly Ile
325 330 335
Leu Asp Val Tyr Ile Phe Leu Gly Pro Glu Pro Lys Ser Val Val Gln
340 345 350
Gln Tyr Leu Asp Val Val Gly Tyr Pro Phe Met Pro Pro Tyr Trp Gly
355 360 365
Leu Gly Phe His Leu Cys Arg Trp Gly Tyr Ser Ser Thr Ala Ile Thr
370 ~ 375 380
Arg Gln Val Val Glu Asn Met Thr Arg Ala His Phe Pro Leu Asp Val
385 390 395 400
Gln Trp l~sn Asp Leu Asp Tyr Met Asp Ser Arg Arg Asp Phe Thr Phe
405 410 415
Asn Lys Asp Gly Phe Arg Asp Phe Pro Ala Met Val Gln Glu Leu His
21



CA 02487815 2004-11-26
WO 03/102583 PCT/US03/17211
420 425 430
Gln Gly Gly Arg Arg Tyr Met Met Ile Val Asp Pro Ala Ile Ser Ser
. 435 440 445
Ser Gly Pro Ala Gly Ser Tyr Arg Pro Tyr Asp Glu Gly Leu Arg Arg
450 455 460
Gly Val Phe Ile Thr Asn Glu Thr Gly Gln Pro Leu Ile Gly Lys Val
465 470 475 480
Trp Pro Gly Ser Thr Ala Phe Pro Asp Phe Thr Asn Pro Thr Ala Leu
485 490 495
Ala Trp Trp Glu Asp Met Val Ala Glu Phe His Asp Gln Val Pro Phe
500 505 510
Asp Gly Met Trp Ile Asp Met Asn Glu Pro Ser Asn Phe Ile Arg Gly
515 520 525
Ser Glu Asp Gly Cys Pro Asn Asn Glu Leu Glu Asn Pro Pro Tyr Val
530 535 540
Pro Gly Val Val Gly Gly Thr Leu Gln Ala Ala Thr Ile Cys Ala Ser
545 550 555 560
Ser His Gln Phe Leu Ser Thr His Tyr Asn Leu His Asn Leu Tyr Gly
565 570 575
Leu Thr Glu Ala Ile Ala Ser His Arg Ala Leu Val Lys A1a Arg Gly
580 585 590
Thr Arg Pro Phe Val Ile Ser Arg Ser Thr Phe Ala Gly His Gly Arg
595 600 605
Tyr Ala Gly His Trp Thr Gly Asp Val Trp Ser Ser Trp Glu Gln Leu
610 615 620
Ala Ser Ser Val Pro Glu Ile Leu Gln Phe Asn Leu Leu Gly Val Pro
625 630 635 640
Leu Val Gly Ala Asp Val Cys Gly Phe Leu Gly Asn Thr Ser Glu Glu
645 650 655
22



CA 02487815 2004-11-26
WO 03/102583 PCT/US03/17211
Leu Cys Val Arg Trp Thr Gln Leu Gly Ala Phe Tyr Pro Phe Met Arg
660 665 670
Asn His Asn Ser Leu Leu Ser Leu Pro Gln Glu Pro Tyr Ser Phe Ser
675 680 685
Glu Pro Ala Gln Gln Ala Met Arg Lys Ala Leu Thr Leu Arg Tyr Ala
690 695 700
Leu Leu Pro His Leu Tyr Thr Leu Phe His Gln Ala His Val Ala Gly
705 710 715 720
Glu Thr Val Ala Arg Pro Leu Phe Leu Glu Phe Pro Lys Asp Ser Ser
725 730 735
Thr Trp Thr Val Asp His Gln Leu Leu Trp Gly Glu Ala Leu Leu Ile
740 745 750
Thr Pro Val Leu Gln Ala Gly Lys Ala Glu Val Thr Gly Tyr Phe Pro
755 760 765
Leu Gly Thr Trp Tyr Asp Leu Gln Thr Val Pro Ile Glu Ala Leu Gly
770 775 780
Ser Leu Pro Pro Pro Pro Ala Ala Pro Arg Glu Pro Ala Ile His Ser
785 790 795 800
Glu Gly Gln Trp Val Thr Leu Pro Ala Pro Leu Asp Thr Ile Asn Val
805 810 815
His Leu Arg Ala Gly Tyr Ile Ile Pro Leu Gln Gly Pro Gly Leu Thr
820 825 830
Thr Thr Glu Ser Arg Gln Gln Pro Met Ala Leu Ala Val Ala Leu Thr
835 840 845
Lys Gly Gly Glu Ala Arg Gly Glu Leu Phe Trp Asp Asp Gly Glu Ser
850 855 860
Leu Glu Val Leu Glu Arg Gly Ala Tyr Thr Gln Val Ile Phe Leu Ala
865 870 875 880
23



CA 02487815 2004-11-26
WO 03/102583 PCT/US03/17211
Arg Asn Asn Thr Ile Val Asn Glu Leu Val Arg Val Thr Ser Glu Gly
885 890 895
Ala Gly Leu Gln Leu Gln Lys Val Thr_Val Leu Gly Val Ala Thr Ala
900 905 910
Pro Gln Gln Val Leu Ser Asn Gly Val Pro Val Ser Asn Phe Thr Tyr
915 920 925
Ser Pro Asp Thr Lys Val Leu Asp Ile Cys Val Ser Leu Leu Met Gly
930 935 940
Glu Gln Phe Leu Val Ser
945 , 950
<210> 25
<211> 33
<212> DNA
<213> Artificial Sequence
<220>
<223> 5' PCR primer GAA 13
<400> 25
ggaattccaa ccatgggagt gaggcacccg ccc 33
<210> 26
<211> 33
<212> DNA
<213> Artificial Sequence
<220>
<223> 3' PCR Primer GAA 14
<400> 26
gctctagagc gggtcccccc aaccacccca ggc 33
<210> 27
<211> 30
<212> DNA
<213> Artificial Sequence
<220>
<223> 5' PCR primer GAA 15
<400> 27
ggaattcacg ggaccctcca ggcggcaacc 30
24



CA 02487815 2004-11-26
WO 03/102583 PCT/US03/17211
<210> 28
<211> 37
<212> DNA
<213> Artificial Sequence
<220>
<223> 3' PCR primer GAA 16
<400> 28
gctctagacg gcgcgccgac gttgatggtg tccaggg 37
<210> 29
<2l1> 36
<212> DNA
<213> Artificial Sequence
<220>
<223> 3' PCR primer GAA 17
<400> 29
gctctagacg gcgcgccagg gccctgcagg gggatg 36
<210> 30
<211> 37
<212> DNA
<213> Artificial Sequence
<220>
<223> 3' PCR primer GAA 18
<400> 30
gctctagacg gcgcgccggc catgggctgc tggcggg 37
<210> 31
<211> 36
<212> DNA
<213> Artificial Sequence
<220>
<223> 3' PCR primer GAA 19
<400> 3l
gctctagacg gcgcgccccc tcgggcctct ccaccc 36
<210> 32
<211> 38
<212> DNA
<213> Artificial Sequence
<220>
<223> 3' PCR primer GAA 20
<400> 32



CA 02487815 2004-11-26
WO 03/102583 PCT/US03/17211
gctctagacg gcgcgcccag cacttccagg ctctctcc 38
<210> 33
<211> 38
<212> DNA
<213> Artificial Sequence
<220>
<223> 3' PCR primer GAA 2l
<400> 33
gctctagacg gcgcgcccct ggccaggaag atgacctg 38
<210> 34
<211> 38
<212> DNA
<213> Artificial Sequence
<220>
<223> 3' PCR primer GAA 22
<400> 34
gctctagacg gcgcgccact ggtcacacgt accagctc 38
<210> 35
<211> 38
<212> DNA
<213> Artificial Sequence
<220>
<223> 3' PCR primer GAA 23
<400> 35
gctctagacg gcgcgcccag gacagtcacc ttctgcag 38
<210> 36
<211> 39
<212>, DNA
<213> Artificial Sequence
<220>
<223> 3' PCR primer GAA 24
<400> 36
gctctagacg gcgcgccacc gttggagagg acctgctgg 39
<210> 37
<211> 37
<212> DNA
<213> Artificial Sequence
<220>
26



CA 02487815 2004-11-26
WO 03/102583 PCT/US03/17211
<223> 3' PCR primer GAA 25
<400> 37
gctctagacg gcgcgccctt ggtgtcgggg ctgtagg 37
<210> 38
<211> 39
<212> DNA
<213> Artificial Sequence
<220>
<223> 3' PCR primer GAA 26
<400> 38
gctctagacg gcgcgccaca ccagctgacg agaaactgc 39
<210> 39
<2ll> 33
<212> DNA
<213> Artificial Sequence
<220>
<223> 3' PCR primer GAA 27
<400> 39
gctctagact aacaccagct gacgagaaac tgc 33
27

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Administrative Status

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Administrative Status

Title Date
Forecasted Issue Date Unavailable
(86) PCT Filing Date 2003-05-29
(87) PCT Publication Date 2003-12-11
(85) National Entry 2004-11-26
Examination Requested 2008-03-26
Dead Application 2013-05-29

Abandonment History

Abandonment Date Reason Reinstatement Date
2012-05-29 FAILURE TO PAY APPLICATION MAINTENANCE FEE
2012-06-05 FAILURE TO PAY FINAL FEE

Payment History

Fee Type Anniversary Year Due Date Amount Paid Paid Date
Application Fee $400.00 2004-11-26
Registration of a document - section 124 $100.00 2005-04-06
Maintenance Fee - Application - New Act 2 2005-05-30 $100.00 2005-05-09
Registration of a document - section 124 $100.00 2005-05-25
Registration of a document - section 124 $100.00 2005-05-25
Maintenance Fee - Application - New Act 3 2006-05-29 $100.00 2006-05-12
Maintenance Fee - Application - New Act 4 2007-05-29 $100.00 2007-05-01
Request for Examination $800.00 2008-03-26
Maintenance Fee - Application - New Act 5 2008-05-29 $200.00 2008-05-23
Maintenance Fee - Application - New Act 6 2009-05-29 $200.00 2009-05-12
Maintenance Fee - Application - New Act 7 2010-05-31 $200.00 2010-05-26
Registration of a document - section 124 $100.00 2011-04-01
Maintenance Fee - Application - New Act 8 2011-05-30 $200.00 2011-05-26
Owners on Record

Note: Records showing the ownership history in alphabetical order.

Current Owners on Record
BIOMARIN PHARMACEUTICAL INC.
Past Owners on Record
BEVERLEY, STEPHEN M.
LEBOWITZ, JONATHAN H.
SLY, WILLIAM S.
SYMBIONTICS, INC.
SYMBIOTICS ACQUISITION CORP.
ZYSTOR THERAPEUTICS, INC.
Past Owners that do not appear in the "Owners on Record" listing will appear in other documentation within the application.
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Document
Description 
Date
(yyyy-mm-dd) 
Number of pages   Size of Image (KB) 
Cover Page 2005-02-18 1 31
Abstract 2004-11-26 1 57
Claims 2004-11-26 5 226
Drawings 2004-11-26 30 3,694
Description 2004-11-26 100 5,429
Description 2011-04-21 101 5,480
Claims 2011-04-21 4 144
Drawings 2011-04-21 30 1,101
Assignment 2005-05-25 11 318
Correspondence 2005-02-16 1 26
Prosecution-Amendment 2008-03-26 1 33
Fees 2006-05-12 1 30
Fees 2011-05-26 1 35
PCT 2004-11-26 1 55
Assignment 2004-11-26 4 128
Assignment 2005-04-06 5 166
Fees 2005-05-09 1 26
Correspondence 2005-08-15 1 26
Prosecution-Amendment 2005-09-26 1 33
Prosecution-Amendment 2005-08-12 1 56
Fees 2007-05-01 1 29
Fees 2009-05-12 1 32
Correspondence 2009-12-10 1 20
Correspondence 2009-12-10 1 13
Correspondence 2009-11-30 2 61
Fees 2008-05-23 1 32
Fees 2010-05-26 1 34
Prosecution-Amendment 2010-10-22 5 246
Assignment 2011-04-01 14 550
Prosecution-Amendment 2011-04-21 28 1,272

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