U.S. patent application number 16/904943 was filed with the patent office on 2020-12-03 for mannose-6-phosphate bearing peptides fused to lysosomal enzymes.
The applicant listed for this patent is Shire Human Genetic Therapies, Inc.. Invention is credited to Michael F. CONCINO, Andrea ISKENDERIAN, Lieh Yoon LOW, Dianna LUNDBERG, Muthuraman MEIYAPPAN, Hicham NAIMY, Angela W. NORTON, Alla ROMASHKO, Bettina STRACK-LOGUE, Bohong ZHANG.
Application Number | 20200376095 16/904943 |
Document ID | / |
Family ID | 1000005030682 |
Filed Date | 2020-12-03 |
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United States Patent
Application |
20200376095 |
Kind Code |
A1 |
CONCINO; Michael F. ; et
al. |
December 3, 2020 |
MANNOSE-6-PHOSPHATE BEARING PEPTIDES FUSED TO LYSOSOMAL ENZYMES
Abstract
A targeted therapeutic including a lysosomal enzyme and a
lysosomal targeting moiety that is a peptide containing at least
one N-linked glycosylation site. Methods of producing the targeted
therapeutic may include nucleotide acids encoding the same and host
cells co-expressing GNPT. Pharmaceutical compositions comprising
the targeted therapeutic and methods of using the same to treat a
lysosomal storage disease.
Inventors: |
CONCINO; Michael F.;
(Lexington, MA) ; STRACK-LOGUE; Bettina;
(Lexington, MA) ; MEIYAPPAN; Muthuraman;
(Lexington, MA) ; NORTON; Angela W.; (Lexington,
MA) ; ZHANG; Bohong; (Lexington, MA) ;
ISKENDERIAN; Andrea; (Lexington, MA) ; LOW; Lieh
Yoon; (Lexington, MA) ; LUNDBERG; Dianna;
(Lexington, MA) ; ROMASHKO; Alla; (Lexington,
MA) ; NAIMY; Hicham; (Lexington, MA) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
Shire Human Genetic Therapies, Inc. |
Lexington |
MA |
US |
|
|
Family ID: |
1000005030682 |
Appl. No.: |
16/904943 |
Filed: |
June 18, 2020 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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15503223 |
Feb 10, 2017 |
10722559 |
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PCT/US15/44713 |
Aug 11, 2015 |
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16904943 |
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62036082 |
Aug 11, 2014 |
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Current U.S.
Class: |
1/1 |
Current CPC
Class: |
A61K 38/47 20130101;
C12Y 207/08017 20130101; C12Y 302/0105 20130101; A61K 38/1709
20130101; C07K 14/65 20130101; C07K 2319/06 20130101; C07K 14/4703
20130101; C07K 14/5412 20130101; A61K 38/43 20130101; A61K 38/30
20130101; A61K 38/204 20130101; C12N 9/1288 20130101; C07K 14/4705
20130101 |
International
Class: |
A61K 38/47 20060101
A61K038/47; A61K 38/43 20060101 A61K038/43; A61K 38/17 20060101
A61K038/17; A61K 38/20 20060101 A61K038/20; A61K 38/30 20060101
A61K038/30; C07K 14/47 20060101 C07K014/47; C07K 14/54 20060101
C07K014/54; C07K 14/65 20060101 C07K014/65; C12N 9/12 20060101
C12N009/12 |
Claims
1. A targeted therapeutic comprising: a lysosomal enzyme; and a
lysosomal targeting moiety, wherein the lysosomal targeting moiety
is a peptide containing at least one N-linked glycosylation
site.
2. (canceled)
3. The targeted therapeutic of claim 1, wherein the peptide
contains N-linked glycosylation containing a M6P group.
4. (canceled)
5. The targeted therapeutic of claim 1, wherein the lysosomal
enzyme is an N-Acetylglucosaminidase (Naglu) protein.
6. The targeted therapeutic of claim 1, wherein the Naglu protein
comprises an amino acid sequence at least 80% identical to SEQ ID
NO.: 1.
7.-9. (canceled)
10. The targeted therapeutic of claim 1, wherein the lysosomal
targeting moiety is a peptide selected from the group consisting of
prosaposin, cellular repressor of E1A-stimulate genes, leukemia
inhibitory factor, and fragments thereof.
11.-12. (canceled)
13. The targeted therapeutic of claim 10, wherein the peptide
comprises a sequence at least 95% identical to SEQ ID NO. 4, 6, or
9.
14.-17. (canceled)
18. The targeted therapeutic of claim 1, wherein the lysosomal
targeting moiety and the lysosomal enzyme is fused via a
linker.
19. The targeted therapeutic of claim 1, wherein the linker
comprises a sequence of TABLE-US-00018 (SEQ ID NO.: 15)
GAPGGGGGAAAAAGGGGGGAPGGGGGAAAAAGGGGGGAPGGGGGAAAAAG GGGGGAP.
20. (canceled)
21. The targeted therapeutic of claim 1, wherein the fusion protein
is at least 95% identical to the amino acid sequence of SEQ ID NO.
18, 19 or 20.
22.-24. (canceled)
25. The targeted therapeutic of claim 1, wherein the fusion protein
comprises a sequence at least 95% identical to the amino acid
sequence of SEQ ID NO. 23, 24 or 25.
26. (canceled)
27. A nucleic acid encoding the targeted therapeutic fusion protein
of claim 1.
28. A vector comprising the nucleic acid sequence of claim 27.
29. A host cell comprising the vector of claim 28.
30.-33. (canceled)
34. The host cell of claim 29, wherein the host cell co-expresses
N-acetylglucosamine-1-phosphoTransferase (GNPT).
35. The host cell of claim 34, wherein the host cell comprises an
exogenous nucleic acid encoding GNPT.
36. The host cell of claim 34, wherein the host cell has activated
expression of endogenous GNPT.
37. A method for producing a targeted therapeutic fusion protein,
comprising: a) culturing cells comprising a nucleic acid encoding a
fusion protein of claim 1, wherein the cells co-express
N-acetylglucosamine-1-phosphoTransferase (GNPT); and b) recovering
the fusion protein produced by the cells.
38. A pharmaceutical composition comprising the targeted
therapeutic of claim 1, and a pharmaceutical acceptable
carrier.
39. A method of treating a lysosomal storage disease comprising
administering to a subject in need of treatment a pharmaceutical
composition of claim 38.
40. The method of claim 39, wherein the lysosomal storage disease
is Sanfilippo syndrome type B.
41. (canceled)
Description
CROSS-REFERENCE TO RELATED APPLICATIONS
[0001] This application claims priority to U.S. Provisional Patent
Application No. 62/036,082, filed Aug. 11, 2014, the disclosure of
which is hereby incorporated in its entirety.
BACKGROUND
[0002] More than forty lysosomal storage diseases are caused,
directly or indirectly, by the absence or deficiency of one or more
lysosomal enzymes. Sanfilippo syndrome, or mucopolysaccharidosis
III (MPS III), is one such disease. It is a rare genetic disorder
characterized by the deficiency of enzymes involved in the
degradation of glycosaminoglycans (GAG).
[0003] Four distinct forms of MPS III, designated MPS MA, B, C, and
D, have been identified. Each is characterized by the absence or
deficiency of a different lysosomal enzyme. Mucopolysaccharidosis
type IIIB (MPS IIIB; Sanfilippo B disease) is an autosomal
recessive disorder that is caused by a deficiency of the enzyme
alpha-N-acetyl-glucosaminidase (Naglu), resulting in the
accumulation of heparan sulfate in lysosomes of particularly
neurons and glial cells in the brain, with additional lysosomal
accumulation of heparan sulfate elsewhere. MPS IIIB manifests
itself primarily in the brain.
[0004] Enzyme replacement therapy (ERT) has been used to deliver
enzymes for the treatment of various lysosomal storage diseases.
Normally, lysosomal enzymes are synthesized in the cytosol and then
traverse the endoplasmic reticulum (ER), where they are
glycosylated with N-linked, high mannose type carbohydrates. In the
Golgi apparatus, high mannose carbohydrates on glycoproteins are
then modified by a series of glycotransferases to become mature
N-glycan; one of the modifications is the addition of
mannose-6-phosphate (M6P). Proteins carrying this modification are
then targeted to the lysosome via binding of the M6P moiety to the
cation-independent mannose-6-phosphate receptor (CI-MPR). Efficacy
of enzyme replacement therapy is critically dependent on proper
lysosomal targeting of the replacement enzyme. However,
recombinantly produced Naglu protein is characterized by a dramatic
lack of M6P phosphorylation, making lysosomal targeting of this
enzyme and its effective use for ERT very difficult.
[0005] Therefore, there remains a need to develop alternative
methods for lysosomal targeting to ensure effective enzyme
replacement therapy.
SUMMARY
[0006] The present invention provides a targeted therapeutic
including a lysosomal enzyme and a lysosomal targeting moiety,
wherein the lysosomal targeting moiety is a peptide containing at
least one N-linked glycosylation site.
[0007] In some embodiments, the peptide contains N-linked
glycosylation. In some embodiments, the peptide contains N-linked
glycosylation containing a M6P group.
[0008] In some embodiments, the lysosomal enzyme is selected from
Table 2. In some embodiments, lysosomal enzyme is an
N-Acetylglucosaminidase (Naglu) protein. In some embodiments, the
Naglu protein includes an amino acid sequence at least 80%, 90% or
95% identical to SEQ ID NO.: 1. In some embodiments, the Naglu
protein includes an amino acid sequence identical to SEQ ID NO.:
1.
[0009] In some embodiments, the lysosomal targeting moiety is a
peptide selected from the group consisting of prosaposin, leukemia
inhibitory factor, cellular repressor of E1A-stimulate genes, and
fragments thereof. In some embodiments, the peptide includes a
sequence at least 80%, 90% or 95% identical to SEQ ID NO. 4, 6, or
9. In some embodiments, the peptide includes a sequence identical
to SEQ ID NO. 4, 6, or 9.
[0010] In some embodiments, the targeted therapeutic is a fusion
protein. In some embodiments, the lysosomal targeting moiety is
fused to the N-terminus of the lysosomal enzyme. In some
embodiments, the lysosomal targeting moiety is fused to the
C-terminus of the lysosomal enzyme. In some embodiments, the
lysosomal targeting moiety and the lysosomal enzyme is fused via a
linker. In some embodiments, the linker comprises a sequence of
GAPGGGGGAAAAAGGGGGGAPGGGGGAAAAAGGGGGGAPGGGGGAAAAAGGGGG GAP (SEQ ID
NO.: 15). In some embodiments, the fusion protein is at least 80%
or 95% identical to the amino acid sequence of SEQ ID NO. 18, 19 or
20. In some embodiments, the fusion protein is identical to the
amino acid sequence of SEQ ID NO. 18, 19 or 20.
[0011] In some embodiments, the targeted therapeutic is a fusion
protein. In some embodiments, the lysosomal targeting moiety is
fused to the N-terminus of the lysosomal enzyme. In some
embodiments, the lysosomal targeting moiety is fused to the
C-terminus of the lysosomal enzyme. In some embodiments, the
lysosomal targeting moiety and the lysosomal enzyme arc fused via a
linker. In some embodiments, the linker comprises a sequence of
GAPGGGGGAAAAAGGGGGGAPGGGGGAAAAAGGGGGGAPGGGGGAAAAAGGGGG GAP (SEQ ID
NO.: 15). In some embodiments, the fusion protein is at least 80%
or 95% identical to the amino acid sequence of SEQ ID NO. 23, 24 or
25. In some embodiments, the fusion protein is identical to the
amino acid sequence of SEQ ID NO. 23, 24 or 25.
[0012] In one aspect, the present invention provides a nucleic acid
encoding any of the fusion proteins disclosed herein. In one
aspect, the present invention provides a vector including any one
of the nucleic acids disclosed herein, and a host cell including
said vector. In some embodiments, the host cell is selected from a
bacterial, yeast, insect, mammalian, or human cell. In some
embodiments, the cell is a CHO cell line.
[0013] In some embodiments, the host cell co-expresses
N-acetylglucosamine-1-phosphoTransferase (GNPT). In some
embodiments, the host cell includes an exogenous nucleic acid
encoding GNPT. In some embodiments, the host cell has activated
expression of endogenous GNPT.
[0014] In one aspect, the present invention provides a method for
producing a targeted therapeutic fusion protein, comprising
culturing cells comprising a nucleic acid encoding a fusion protein
described herein, wherein the cells co-express
N-acetylglucosamine-1-phosphoTransferase (GNPT), and recovering the
fusion protein produced by the cells.
[0015] In one aspect, the present invention provides a
pharmaceutical composition including any of the targeted
therapeutics described herein, and a pharmaceutical acceptable
carrier. In one aspect, the the present invention provides a method
of treating a lysosomal storage disease by administering to a
subject in need of treatment any of the pharmaceutical compositions
described herein. In some embodiments, the lysosomal storage
disease is Sanfilippo syndrome type B. In some embodiments, the
pharmaceutical composition is administered intravenously,
subcutaneously, intrathecally and/or combinations thereof.
BRIEF DESCRIPTION OF THE FIGURES
[0016] FIG. 1 shows an M6P receptor in vitro binding assay for
Naglu-IGFII, Naglu-CREG and Naglu-SapDC fusion proteins.
[0017] FIG. 2 shows an M6P receptor in vitro binding assay for
Naglu-IGFII and Naglu-LIF fusion proteins.
[0018] FIG. 3 shows results of an in vitro assay monitoring
M6P-receptor mediated cellular uptake of Naglu-CREG, Naglu-SapDC
and Naglu-LIF fusion proteins and comparison to the cellular uptake
of Naglu-IGFII determined by the same assay.
[0019] FIG. 4 shows Western blot analysis using an anti-M6P
antibody to show the relative content of M6P of acid
alpha-glucosidase (GAA); Naglu-LIF fusion protein (Naglu-LIF);
arylsulfatase A (ARSA); iduronate 2-sulfatase (I2S);
heparan-N-sulfamidase (FINS); and glucocerebrosidase (GCB) protein
produced in wild-type HT1080 cells or an
N-acetylgucosamine-1-phosphotransferase (GNPT) overexpressing cell
line. Western blot analysis was conducted before (-) and after (+)
removal of M6P using cndoglycosidasc H.
[0020] FIG. 5 demonstrates in vitro binding of iduronate
2-sulfatase (I2S) binding to the M6P receptor when produced in
wild-type HT1080 cells or an
N-acetylgucosamine-1-phosphoTransferase (GNPT) overexpressing cell
line.
[0021] FIG. 6 illustrates the in vitro cellular uptake of iduronate
2-sulfatase (I2S) produced in wild-type HT1080 cells or an
N-acetylgucosamine-1-phosphoTransferase (GNPT) overexpressing cell
line.
[0022] FIG. 7 shows the in vitro M6P receptor binding of
recombinant Naglu proteins produced in HT1080 cells or a GNPT
overexpressing cell line (GlcNAc-P-T).
[0023] FIG. 8 shows the in vitro M6P receptor binding of various
Naglu fusion proteins produced in HT1080 cells or a GNPT
overexpressing cell line (GlcNAc-P-T).
[0024] FIG. 9 shows quantification of M6P monosaccharide derived
from Naglu-SapDC and Naglu-LIF by PAD-HPLC. M6P content of I2S was
used as a control.
[0025] FIG. 10 illustrates in vitro cellular uptake of Naglu-SapDC
produced in wild-type HT1080 cells or an
N-acetylgucosamine-1-phosphotransferase (GNPT) overexpressing cell
line. Cellular uptake of Naglu-IGFII was used as a control.
[0026] FIG. 11 illustrates in vitro cellular uptake of Naglu-LIF
produced in wild-type HT1080 cells or an
N-acetylgucosamine-1-phosphotransferase (GNPT) overexpressing cell
line. Cellular uptake of Naglu-IGFII was used as a control.
[0027] FIG. 12A-13 demonstrates in vivo Naglu enzyme activity in
mouse (A) liver and [0028] (B) brain tissue, following intrathecal
delivery of vehicle control or Naglu-SapDC or Naglu-LIF fusion
proteins to Naglu knock-out mice.
[0029] FIG. 13A-F shows immunohistochemical detection of Naglu
protein in tissues. Naglu protein is detectable in neurons of the
cerebral cortex and cerebellum of Naglu-knockout mice having
received three weekly intrathecal injections of vehicle control or
(A) Naglu-SapDC or (C) Naglu-LIF fusion proteins. Naglu stained
neurons in the cerebral cortex of mice injected with Naglu-SapDC
and Naglu-LIF arc circled (B and D, respectively). Similarly, Naglu
was detected in the liver of Naglu-knockout mice following three
weekly intrathecal injections of vehicle control or Naglu-SapDC (E)
or Naglu-LIF (F).
[0030] FIG. 14 demonstrates in vivo levels of glycosaminoglycan
(GAG) in mouse liver following intrathecal delivery of vehicle
control or the Naglu-SapDC and Naglu-LIF fusion proteins to Naglu
knock-out mice.
[0031] FIG. 15 demonstrates in vivo levels of glycosaminoglycan
(GAG) in mouse brain, following two and three weekly intrathecal
injections of vehicle control or Naglu-SapDC and Naglu-LIF fusion
proteins to Naglu knock-out mice.
[0032] FIG. 16 demonstrates in vivo levels of heparan sulfate (HS)
in mouse brain, following two and three weekly intrathecal
injections of vehicle control or the Naglu-SapDC and Naglu-LIF
fusion proteins to Naglu knock-out mice.
[0033] FIGS. 17-19 demonstrate in vivo levels of three San B
biomarkers in mouse brain, following intrathecal delivery of
vehicle control (PBS) or Naglu-SapDC fusion protein to Naglu
knock-out mice.
[0034] FIG. 20 illustrates LAMP-1 immunohistochemical staining in
mouse cerebral cortex tissue, following two or three weekly
intrathecal injections of vehicle control or Naglu-SapDC fusion
protein.
[0035] FIG. 21 illustrates LAMP-1 immunohistochemical staining in
mouse cerebellum tissue, following two or three weekly intrathecal
injections of vehicle control or Naglu-SapDC fusion protein.
[0036] FIG. 22 illustrates LAMP-1 immunohistochemical staining in
mouse thalamus tissue, following two or three weekly intrathecal
injections of vehicle control or Naglu-SapDC fusion protein.
[0037] FIG. 23 illustrates LAMP-1 immunohistochemical staining in
mouse striatum tissue, following two or three weekly intrathecal
injections of vehicle control or Naglu-SapDC fusion protein.
[0038] FIG. 24 illustrates LAMP-1 immunohistochemical staining in
mouse white matter tissue, following two or three weekly
intrathecal injections of vehicle control or Naglu-SapDC fusion
protein.
[0039] FIG. 25 illustrates LAMP-1 immunohistochemical staining in
mouse liver tissue, following three weekly intrathecal injections
of vehicle control or Naglu-SapDC fusion protein.
[0040] FIG. 26 illustrates immunohistochemical staining of GFAP in
mouse cerebrum and cerebellum tissue, following three weekly
intrathecal injections of vehicle control or Naglu-SapDC fusion
protein.
[0041] FIG. 27 illustrates immunohistochemical staining of Iba-1 in
mouse cerebellum and cerebrum tissue, following three weekly
intrathecal injections of vehicle control or Naglu-SapDC fusion
protein.
[0042] FIG. 28 illustrates LAMP-1 immunohistochemical staining in
mouse cerebral cortex tissue following two or three weekly
intrathecal injections of vehicle control or Naglu-LIF fusion
protein.
[0043] FIG. 29 illustrates LAMP-1 immunohistochemical staining in
mouse cerebellum tissue following two or three weekly intrathecal
injections of vehicle control or Naglu-LIF fusion protein.
[0044] FIG. 30 illustrates LAMP-1 immunohistochemical staining in
mouse thalamus tissue following two or three weekly intrathecal
injections of vehicle control or Naglu-LIF fusion protein.
[0045] FIG. 31 illustrates LAMP-1 immunohistochemical staining in
mouse striatum tissue following two or three weekly intrathecal
injections of vehicle control or Naglu-LIF fusion protein.
[0046] FIG. 32 illustrates LAMP-1 immunohistochemical staining in
mouse white matter tissue following two or three weekly intrathecal
injections of vehicle control or Naglu-LIF fusion protein.
[0047] FIG. 33 illustrates LAMP-1 immunohistochemical staining in
mouse liver tissue following three weekly intrathecal injections of
vehicle control or Naglu-LIF fusion protein.
[0048] FIG. 34 illustrates immunohistochemical staining of GFAP in
mouse cerebrum and cerebellum tissue following three weekly
intrathecal injections of vehicle control or Naglu-LIF fusion
protein.
[0049] FIG. 35 illustrates immunohistochemical staining of Iba-1 in
mouse cerebrum and cerebellum tissue following three weekly
intrathecal injections of vehicle control or Naglu-LIF fusion
protein.
DEFINITIONS
[0050] In order for the present invention to be more readily
understood, certain terms are first defined below. Additional
definitions for the following terms and other terms are set forth
throughout the specification.
[0051] Alpha-N-acetyl-glucosaminidase: As used herein, the term
"alpha-N-acetyl-glucosaminidase" refers to enzymes capable of
hydrolyzing terminal non-reducing N-acetyl-D-glucosamine residues
in N-acetyl-alpha-D-glucosaminides. Alpha-N-acetyl-glucosaminidase
is also known as alpha-acety-glucosaminidase,
N-acetyl-alpha-D-glucosaminidase, N-acetyl-alpha-glucosaminidase
and alpha-D-2-acetamido-2-deoxyglucosidase.
[0052] Approximately or about: As used herein, the term
"approximately" or "about," as applied to one or more values of
interest, refers to a value that is similar to a stated reference
value. In certain embodiments, the term "approximately" or "about"
refers to a range of values that fall within 25%, 20%, 19%, 18%,
17%, 16%, 15%, 14%, 13%, 12%, 11%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%,
2%, 1%, or less in either direction (greater than or less than) of
the stated reference value unless otherwise stated or otherwise
evident from the context (except where such number would exceed
100% of a possible value).
[0053] Amelioration: As used herein, the term "amelioration" is
meant the prevention, reduction or palliation of a state, or
improvement of the state of a subject. Amelioration includes, but
does not require complete recovery or complete prevention of a
disease condition. In some embodiments, amelioration includes
increasing levels of relevant protein or its activity that is
deficient in relevant disease tissues.
[0054] Amino Acid: As used herein, term "amino acid," in its
broadest sense, refers to any compound and/or substance that can be
incorporated into a polypeptide chain. In some embodiments, an
amino acid has the general structure H.sub.2N--C(H)(R)--COOH. In
some embodiments, an amino acid is a naturally occurring amino
acid. In some embodiments, an amino acid is a synthetic amino acid;
in some embodiments, an amino acid is a d-amino acid; in some
embodiments, an amino acid is an 1-amino acid. "Standard amino
acid" refers to any of the twenty standard 1-amino acids commonly
found in naturally occurring peptides. "Nonstandard amino acid"
refers to any amino acid, other than the standard amino acids,
regardless of whether it is prepared synthetically or obtained from
a natural source. As used herein, "synthetic amino acid"
encompasses chemically modified amino acids, including but not
limited to salts, amino acid derivatives (such as amides), and/or
substitutions. Amino acids, including carboxy- and/or
amino-terminal amino acids in peptides, can be modified by
methylation, amidation, acetylation, protecting groups, and/or
substitution with other chemical groups that can change the
peptide's circulating half-life without adversely affecting their
activity. Amino acids may participate in a disulfide bond. Amino
acids may comprise one or posttranslational modifications, such as
association with one or more chemical entities (e.g., methyl
groups, acetate groups, acetyl groups, phosphate groups, formyl
moieties, isoprenoid groups, sulfate groups, polyethylene glycol
moieties, lipid moieties, carbohydrate moieties, biotin moieties,
etc.). The term "amino acid" is used interchangeably with "amino
acid residue," and may refer to a free amino acid and/or to an
amino acid residue of a peptide. It will be apparent from the
context in which the term is used whether it refers to a free amino
acid or a residue of a peptide.
[0055] Animal: As used herein, the term "animal" refers to any
member of the animal kingdom. In some embodiments, "animal" refers
to humans, at any stage of development. In some embodiments,
"animal" refers to non-human animals, at any stage of development.
In certain embodiments, the non-human animal is a mammal (e.g., a
rodent, a mouse, a rat, a rabbit, a monkey, a dog, a cat, a sheep,
cattle, a primate, and/or a pig). In some embodiments, animals
include, but are not limited to, mammals, birds, reptiles,
amphibians, fish, insects, and/or worms. In some embodiments, an
animal may be a transgenic animal, genetically-engineered animal,
and/or a clone.
[0056] Biologically active: As used herein, the phrase
"biologically active" (or biological activity) refers to a
characteristic of any agent that has activity in a biological
system, and particularly in an organism. For instance, an agent
that, when administered to an organism, has a biological effect on
that organism, is considered to be biologically active. In
particular embodiments, where a protein or polypeptide is
biologically active, a portion of that protein or polypeptide that
shares at least one biological activity of the protein or
polypeptide is typically referred to as a "biologically active"
portion.
[0057] Cation-independent mannose-6 phosphate receptor (CI-M6PR):
As used herein, the term "cation-independent mannose-6-phosphate
receptor (CI-M6PR)" refers to a cellular receptor that binds
mannose-6-phosphate (M6P) tags on acid hydrolase (e.g.,
alpha-N-acetyl-glucosaminidase) precursors in the Golgi apparatus
that are destined for transport to the lysosome. In addition to
mannose-6-phosphates, the CI-M6PR also binds certain proteins,
including IGF-II. The CI-M6PR is also known as "CI-MPR",
"M6P/IGF-II receptor", "CI-MPR/IGF-II receptor", "CD222", "MPR300",
"IGF-II receptor" or "IGF2 Receptor." These terms and abbreviations
thereof are used interchangeably herein.
[0058] Diluent: As used herein, the term "diluent" refers to a
pharmaceutically acceptable (e.g., safe and non-toxic for
administration to a human) diluting substance useful for the
preparation of a reconstituted formulation. Exemplary diluents
include sterile water, bacteriostatic water for injection (BWFI), a
pH buffered solution (e.g. phosphate-buffered saline), sterile
saline solution, Ringer's solution or dextrose solution.
[0059] Enzyme replacement therapy (ERT): As used herein, the term
"enzyme replacement therapy (ERT)" refers to any therapeutic
strategy that corrects an enzyme deficiency by providing the
missing enzyme. In some embodiments, the missing enzyme is provided
by intrathecal administration. In some embodiments, the missing
enzyme is provided by infusing into bloodsteam. Once administered,
enzyme is taken up by cells and transported to the lysosome, where
the enzyme acts to eliminate material that has accumulated in the
lysosomes due to the enzyme deficiency. Typically, for lysosomal
enzyme replacement therapy to be effective, the therapeutic enzyme
is delivered to lysosomes in the appropriate cells in target
tissues where the storage defect is manifest.
[0060] Expression: As used herein, "expression" of a nucleic acid
sequence refers to one or more of the following events: (1)
production of an RNA template from a DNA sequence (e.g., by
transcription); (2) processing of an RNA transcript (e.g., by
splicing, editing, 5' cap formation, and/or 3' end formation); (3)
translation of an RNA into a polypeptide or protein; and/or (4)
post-translational modification of a polypeptide or protein. In
this application, the terms "expression" and "production," and
grammatical equivalents, are used inter-changeably.
[0061] Glycan: As is known in the art and used herein "glycans" are
sugars. Glycans can be monomers or polymers of sugar residues, but
typically contain at least three sugars, and can be linear or
branched. A glycan may include natural sugar residues (e.g.,
glucose, N-acetylglucosamine, N-acetyl neuraminic acid, galactose,
mannose, fucose, hexose, arabinose, ribose, xylose, etc.) and/or
modified sugars (e.g., 2'-fluororibose, 2'-deoxyribose,
phosphomannose, 6'sulfo N-acetylglucosamine, etc). The term
"glycan" includes homo and heteropolymers of sugar residues. The
term "glycan" also encompasses a glycan component of a
glycoconjugate (e.g., of a glycoprotein, glycolipid, proteoglycan,
etc.). The term also encompasses free glycans, including glycans
that have been cleaved or otherwise released from a
glycoconjugate.
[0062] Glycoconjugate: The term "glycoconjugate", as used herein,
encompasses all molecules in which at least one sugar moiety is
covalently linked to at least one other moiety. The term
specifically encompasses all biomolecules with covalently attached
sugar moieties, including for example N-linked glycoproteins,
O-linked glycoproteins, glycolipids, proteoglycans, etc.
[0063] Glycoprotein: As used herein, the term "glycoprotein" refers
to a protein that contains a peptide backbone covalently linked to
one or more sugar moieties (i.e., glycans). As is understood by
those skilled in the art, the peptide backbone typically comprises
a linear chain of amino acid residues. In certain embodiments, the
peptide backbone spans the cell membrane, such that it comprises a
transmembrane portion, as well as an extracellular portion and/or
an intracellular portion. In certain embodiments, a peptide
backbone of a glycoprotein that spans the cell membrane comprises
an intracellular portion, a transmembrane portion, and an
extracellular portion. The sugar moiety(ies) may be in the form of
monosaccharides, disaccharides, oligosaccharides, and/or
polysaccharides. The sugar moiety(ies) may comprise a single
unbranched chain of sugar residues or may comprise one or more
branched chains. In certain embodiments, sugar moieties may include
sulfate and/or phosphate groups. Alternatively or additionally,
sugar moieties may include acetyl, glycolyl, propyl or other alkyl
modifications. Alternatively or additionally, sugar moieties may be
modified by diacetylation. In certain embodiments, glycoproteins
contain O-linked sugar moieties; in certain embodiments,
glycoproteins contain N-linked sugar moieties. In certain
embodiments, the sugar moiety is specifically a N-linked glycan
containing a mannose-6-phosphate residue. In certain embodiments,
methods disclosed herein comprise a step of analyzing therapeutic
glycoproteins, liberated fragments (e.g., glycopeptides) of cell
surface glycoproteins, cell surface glycans attached to cell
surface glycoproteins, peptide backbones of cell surface
glycoproteins, fragments of such glycoproteins, glycans and/or
peptide backbones, and combinations thereof for their glycosylation
pattern and/or level of M6P phoshorylation.
[0064] High mannose: As used herein, the term "high mannose" is
used herein to describe proteins (e.g., Naglu) containing one or
more Man9(GlcNAc)2, Man8(GlcNAc)2, Man7(GlcNAc)2, Man6(GlcNAc)2,
and/or Man5(GlcNAc)2 N-linked oligosaccharides, or combinations
thereof.
[0065] Homology: As used herein, the term "homology" (or the
corresponding adjective "homogous") refers to the overall
relatedness of polymeric molecules, e.g., of nucleic acid molecules
(e.g., DNA molecules and/or RNA molecules) and/or between
polypeptide molecules. In some embodiments, polymeric molecules are
considered to be "homologous" to one another if their sequences are
at least 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%,
80%, 85%, 90%, 95%, or 99% identical. In some embodiments,
polymeric molecules are considered to be "homologous" to one
another if their sequences are at least 25%, 30%, 35%, 40%, 45%,
50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 99%
similar.
[0066] Identity: As used herein, the term "identity" refers to the
overall relatedness of polymeric molecules, e.g., of nucleic acid
molecules (e.g., DNA molecules and/or RNA molecules) and/or of
polypeptide molecules. Calculation of the percent identity of two
nucleic acid sequences, for example, can be performed by aligning
the two sequences for optimal comparison purposes (e.g., gaps can
be introduced in one or both of a first and a second nucleic acid
sequences for optimal alignment and non-identical sequences can be
disregarded for comparison purposes). In certain embodiments, the
length of a sequence aligned for comparison purposes is at least
30%, at least 40%, at least 50%, at least 60%, at least 70%, at
least 80%, at least 90%, at least 95%, or substantially 100% of the
length of the reference sequence. The nucleotides at corresponding
nucleotide positions are then compared. When a position in the
first sequence is occupied by the same nucleotide as the
corresponding position in the second sequence, then the molecules
are identical at that position. The percent identity between the
two sequences is a function of the number of identical positions
shared by the sequences, taking into account the number of gaps,
and the length of each gap, which needs to be introduced for
optimal alignment of the two sequences. The comparison of sequences
and determination of percent identity between two sequences can be
accomplished using a mathematical algorithm. For example, the
percent identity between two nucleotide sequences can be determined
using the algorithm of Meyers and Miller (CABIOS, 1989, 4: 11-17),
which has been incorporated into the ALIGN program (version 2.0)
using a PAM120 weight residue table, a gap length penalty of 12 and
a gap penalty of 4. The percent identity between two nucleotide
sequences can, alternatively, be determined using the GAP program
in the GCG software package using an NWSgapdna.CMP matrix.
[0067] Improve, increase, or reduce: As used herein, the terms
"improve," "increase" or "reduce," or grammatical equivalents,
indicate values that are relative to a baseline measurement, such
as a measurement in the same individual prior to initiation of the
treatment described herein, or a measurement in a control subject
(or multiple control subject) in the absence of the treatment
described herein. A "control subject" is a subject afflicted with
the same form of disease as the subject being treated, who is about
the same age as the subject being treated.
[0068] In Vitro: As used herein, the term "in vitro" refers to
events that occur in an artificial environment, e.g., in a test
tube or reaction vessel, in cell culture, etc., rather than within
a multi-cellular organism.
[0069] In Vivo: As used herein, the term "in vivo" refers to events
that occur within a multi-cellular organism, such as a human and a
non-human animal. In the context of cell-based systems, the term
may be used to refer to events that occur within a living cell (as
opposed to, for example, in vitro systems).
[0070] Intrathecal administration: As used herein, the term
"intrathecal administration" or "intrathecal injection" refers to
an injection into the spinal canal (intrathecal space surrounding
the spinal cord). Various techniques may be used including, without
limitation, lateral cerebroventricular injection through a burrhole
or cisternal or lumbar puncture or the like. In some embodiments,
"intrathecal administration" or "intrathecal delivery" according to
the present invention refers to IT administration or delivery via
the lumbar area or region, i.e., lumbar IT administration or
delivery. As used herein, the term "lumbar region" or "lumbar area"
refers to the area between the third and fourth lumbar (lower back)
vertebrae and, more inclusively, the L2-S1 region of the spine.
[0071] Linker: As used herein, the term "linker" refers to, in a
fusion protein, an amino acid sequence other than that appearing at
a particular position in the natural protein and is generally
designed to be flexible or to interpose a structure, such as an
a-helix, between two protein moieties. A linker is also referred to
as a spacer.
[0072] Lysosomal enzyme: As used herein, the term "lysosomal
enzyme" refers to any enzyme that is capable of reducing
accumulated materials in mammalian lysosomes or that can rescue or
ameliorate one or more lysosomal storage disease symptoms.
Lysosomal enzymes suitable for the invention include both wild-type
or modified lysosomal enzymes and can be produced using recombinant
and synthetic methods or purified from nature sources. Exemplary
lysosomal enzymes are listed in Table 1.
[0073] Lysosomal enzyme deficiency: As used herein, "lysosomal
enzyme deficiency" refers to a group of genetic disorders that
result from deficiency in at least one of the enzymes that are
required to break macromolecules (e.g., enzyme substrates) down to
peptides, amino acids, monosaccharides, nucleic acids and fatty
acids in lysosomes. As a result, individuals suffering from
lysosomal enzyme deficiencies have accumulated materials in various
tissues (e.g., CNS, liver, spleen, gut, blood vessel walls and
other organs).
[0074] Lysosomal Storage Disease: As used herein, the term
"lysosomal storage disease" refers to any disease resulting from
the deficiency of one or more lysosomal enzymes necessary for
metabolizing natural macromolecules. These diseases typically
result in the accumulation of un-degraded molecules in the
lysosomes, resulting in increased numbers of storage granules (also
termed storage vesicles).
[0075] messenger RNA (mRNA): As used herein, the term "messenger
RNA (mRNA)" refers to a polynucleotide that encodes at least one
polypeptide. mRNA as used herein encompasses both modified and
unmodified RNA. mRNA may contain one or more coding and non-coding
regions.
[0076] Moiety: As used herein, the term "moiety" refers to a part
or functional group of a molecule (e.g., a glycan or a protein).
The "moiety" is used interchangeably with the term "residue"
herein.
[0077] N-acetylglucosamine-1-phosphotransferase (GNPT) protein: As
used herein, the term N-acetylglucosamine-1-phosphotransferase
(GNPT) protein refers to enzymes capable of catalyzing the transfer
of N-acetylglucosamine-1-phosphate from UDP-GlcNAc to mannose
residues on lysosomal hydrolases (e.g.,
alpha-N-acetyl-glucosaminidase).
[0078] Nucleic acid: As used herein, the term "nucleic acid," in
its broadest sense, refers to any compound and/or substance that is
or can be incorporated into a polynucleotide chain. In some
embodiments, a nucleic acid is a compound and/or substance that is
or can be incorporated into a polynucleotide chain via a
phosphodiester linkage. In some embodiments, "nucleic acid" refers
to individual nucleic acid residues (e.g., nucleotides and/or
nucleosides). In some embodiments, "nucleic acid" refers to a
polynucleotide chain comprising individual nucleic acid residues.
In some embodiments, "nucleic acid" encompasses RNA as well as
single and/or double-stranded DNA and/or cDNA. Furthermore, the
terms "nucleic acid," "DNA," "RNA," and/or similar terms include
nucleic acid analogs, i.e., analogs having other than a
phosphodiester backbone. For example, the so-called "peptide
nucleic acids," which are known in the art and have peptide bonds
instead of phosphodiester bonds in the backbone, are considered
within the scope of the present invention. The term "nucleotide
sequence encoding an amino acid sequence" includes all nucleotide
sequences that are degenerate versions of each other and/or encode
the same amino acid sequence. Nucleotide sequences that encode
proteins and/or RNA may include introns. Nucleic acids can be
purified from natural sources, produced using recombinant
expression systems and optionally purified, chemically synthesized,
etc. Where appropriate, e.g., in the case of chemically synthesized
molecules, nucleic acids can comprise nucleoside analogs such as
analogs having chemically modified bases or sugars, backbone
modifications, etc. A nucleic acid sequence is presented in the 5'
to 3' direction unless otherwise indicated. In some embodiments, a
nucleic acid is or comprises natural nucleosides (e.g., adenosine,
thymidine, guanosine, cytidine, uridine, deoxyadenosine,
deoxythymidine, deoxyguanosine, and deoxycytidine); nucleoside
analogs (e.g., 2-aminoadenosine, 2-thiothymidine, inosine,
pyrrolo-pyrimidine, 3-methyl adenosine, 5-methylcytidine, C-5
propynyl-cytidine, C-5 propynyl-uridine, 2-aminoadenosine,
C5-bromouridine, C5-fluorouridine, C5-iodouridine,
C5-propynyl-uridine, C5-propynyl-cytidine, C5-methylcytidine,
2-aminoadenosine, 7-deazaadenosine, 7-deazaguanosine,
8-oxoadenosine, 8-oxoguanosine, O(6)-methylguanine, and
2-thiocytidine); chemically modified bases; biologically modified
bases (e.g., methylated bases); intercalated bases; modified sugars
(e.g., 2'-fluororibose, ribose, 2'-deoxyribose, arabinose, and
hexose); and/or modified phosphate groups (e.g., phosphorothioates
and 5'-N-phosphoramidite linkages). In some embodiments, the
present invention is specifically directed to "unmodified nucleic
acids," meaning nucleic acids (e.g., polynucleotides and residues,
including nucleotides and/or nucleosides) that have not been
chemically modified in order to facilitate or achieve delivery.
[0079] Patient: As used herein, the term "patient" or "subject"
refers to any organism to which a provided composition may be
administered, e.g., for experimental, diagnostic, prophylactic,
cosmetic, and/or therapeutic purposes. Typical patients include
animals (e.g., mammals such as mice, rats, rabbits, non-human
primates, and/or humans). In some embodiments, a patient is a
human. A human includes pre and post natal forms.
[0080] Pharmaceutically acceptable: The term "pharmaceutically
acceptable" as used herein, refers to substances that, within the
scope of sound medical judgment, are suitable for use in contact
with the tissues of human beings and animals without excessive
toxicity, irritation, allergic response, or other problem or
complication, commensurate with a reasonable benefit/risk
ratio.
[0081] Polypeptide: As used herein, a "polypeptide", generally
speaking, is a string of at least two amino acids attached to one
another by a peptide bond. In some embodiments, a polypeptide may
include at least 3-5 amino acids, each of which is attached to
others by way of at least one peptide bond. Those of ordinary skill
in the art will appreciate that polypeptides sometimes include
"non-natural" amino acids or other entities that nonetheless are
capable of integrating into a polypeptide chain, optionally.
[0082] Protein: As used herein, the term "protein" of "therapeutic
protein" refers to a polypeptide (i.e., a string of at least two
amino acids linked to one another by peptide bonds). Proteins may
include moieties other than amino acids (e.g., may be
glycoproteins, proteoglycans, etc.) and/or may be otherwise
processed or modified. Those of ordinary skill in the art will
appreciate that a "protein" can be a complete polypeptide chain as
produced by a cell (with or without a signal sequence), or can be a
characteristic portion thereof. Those of ordinary skill will
appreciate that a protein can sometimes include more than one
polypeptide chain, for example linked by one or more disulfide
bonds or associated by other means. Polypeptides may contain
1-amino acids, d-amino acids, or both and may contain any of a
variety of amino acid modifications or analogs known in the art.
Useful modifications include, e.g., terminal acetylation,
amidation, methylation, etc. In some embodiments, proteins may
comprise natural amino acids, non-natural amino acids, synthetic
amino acids, and combinations thereof. The term "peptide" is
generally used to refer to a polypeptide having a length of less
than about 100 amino acids, less than about 50 amino acids, less
than 20 amino acids, or less than 10 amino acids. In some
embodiments, proteins are antibodies, antibody fragments,
biologically active portions thereof, and/or characteristic
portions thereof.
[0083] Recombinant: As used herein, the term "recombinant" refers
to DNA molecules, peptides, proteins or cells that are generated
using recombinant DNA technology, as the term is commonly
understood by the person gaving ordinary skill in the art.
[0084] Subject: As used herein, the term "subject" refers to a
human or any non-human animal (e.g., mouse, rat, rabbit, dog, cat,
cattle, swine, sheep, horse or primate). A human includes pre- and
post-natal forms. In many embodiments, a subject is a human being.
A subject can be a patient, which refers to a human presenting to a
medical provider for diagnosis or treatment of a disease. The term
"subject" is used herein interchangeably with "individual" or
"patient." A subject can be afflicted with or is susceptible to a
disease or disorder but may or may not display symptoms of the
disease or disorder.
[0085] Substantially: As used herein, the term "substantially"
refers to a an amount greater than 80%, 85%, 90%, 95%, 96%, 97%,
98%, 99% of the total amount.
[0086] Target tissues: As used herein, the term "target tissues"
refers to any tissue that is affected by the lysosomal storage
disease to be treated or any tissue in which the deficient
lysosomal enzyme is normally expressed. In some embodiments, target
tissues include those tissues in which there is a detectable or
abnormally high amount of enzyme substrate, for example stored in
the cellular lysosomes of the tissue, in patients suffering from or
susceptible to the lysosomal storage disease. In some embodiments,
target tissues include those tissues that display
disease-associated pathology, symptom, or feature. In some
embodiments, target tissues include those tissues in which the
deficient lysosomal enzyme is normally expressed at an elevated
level. As used herein, a target tissue may be a brain target
tissue, a spinal cord target tissue and/or a peripheral target
tissue. Exemplary target tissues are described in detail below.
[0087] Therapeutic Glycoprotein: As used herein, the term
"Therapeutic Glycoprotein" refers to any glycoprotein that can act
to replace at least in part the deficient or missing enzyme in a
disease to be treated. In some embodiments, the term therapeutic
glycoprotein, refers to lysosomal proteins (e.g., acid hydrolase)
which may be localized, transported, processed and/or resides in
the lysosome of the cell. In some embodiments, a therapeutic
glycoprotein refers to a glycoprotein with enzyme activity (e.g.,
lysosomal enzyme) which can act to replace at least in part the
deficient or missing lysosomal enzyme in a lysosomal storage
disease to be treated. In some embodiments, a therapeutic
glycoprotein is capable of reducing accumulated materials in
mammalian lysosomes or that can rescue or ameliorate one or more
lysosomal storage disease symptoms. Therapeutic glycoproteins
suitable for the invention include both wild-type, modified or
protein fusion of a lysosomal enzymes and can be produced using
recombinant methods.
[0088] Therapeutically effective amount: As used herein, the term
"therapeutically effective amount" of a therapeutic agent means an
amount that is sufficient, when administered to a subject suffering
from or susceptible to a disease, disorder, and/or condition, to
treat, diagnose, prevent, and/or delay the onset of the symptom(s)
of the disease, disorder, and/or condition. It will be appreciated
by those of ordinary skill in the art that a therapeutically
effective amount is typically administered via a dosing regimen
comprising at least one unit dose.
[0089] Treatment: As used herein, the term "treatment" (also
"treat" or "treating") refers to any administration of a
therapeutic (e.g., lysosomal enzyme) that partially or completely
alleviates, ameliorates, relieves, inhibits, delays onset of,
reduces severity of and/or reduces incidence of one or more
symptoms or features of a particular disease, disorder, and/or
condition (e.g., Hunters syndrome, Sanfilippo B syndrome). Such
treatment may be of a subject who does not exhibit signs of the
relevant disease, disorder and/or condition and/or of a subject who
exhibits only early signs of the disease, disorder, and/or
condition. Alternatively or additionally, such treatment may be of
a subject who exhibits one or more established signs of the
relevant disease, disorder and/or condition.
DETAILED DESCRIPTION
[0090] The present invention provides targeted therapeutics
containing a lysosomal enzyme and a lysosomal targeting moiety
containing a N-linked glycosylation site, as well as methods of
making the same and using the same for effective enzyme replacement
therapy of lysosomal storage diseases.
[0091] In some embodiments, the present invention provides a
lysosomal targeting moiety that is a peptide containing N-linked
glycosylation containing a M6P group. In some embodiments, the
present invention provides a targeted therapeutic wherein the
lysosomal enzyme and the lysosomal targeting moiety are fused via a
linker. In some embodiments, the present invention provides vectors
including a nucleic acid encoding a targeted therapeutic, as well
as host cells containing the same and co-expressing GNPT. In some
embodiments, the lysosomal enzyme is Naglu.
[0092] Various aspects of the invention are described in further
detail in the following subsections. The use of subsections is not
meant to limit the invention. Each subsection may apply to any
aspect of the invention. In this application, the use of "or" means
"and/or" unless stated otherwise.
Therapeutic Lysosomal Enzymes--Naglu Protein
[0093] The present inventors discovered unexpectedly that lysosomal
enzymes could be targeted to the lysosome by connecting them to a
targeting moiety containing a N-linked glycosylation site. The
present invention provides targeted therapeutics containing a
lysosomal enzyme and a lysosomal targeting moiety containing a
N-linked glycosylation site. In some embodiments, a suitable
therapeutic lysosomal enzyme in the context of the present
invention is recombinant human Naglu protein.
Alpha-N-acetyl-glucosaminidasc (Naglu) aids in the stepwise
degradation of the heparan sulfate in lysosomes by removing GlcNAc
(N-acetyl-glucosamine) from the non-reducing end of this
glycosaminoglycan. A lack of Naglu leads to incomplete breakdown of
heparan sulfate and the subsequent accumulation in lysosomes of the
incomplete breakdown products. The absence of Naglu causes the
fatal neurodegenerative disease Sanfilippo Type III B.
[0094] A suitable Naglu protein according to the present invention
can be any molecule that can substitute for naturally-occurring
Naglu protein activity or rescue one or more phenotypes or symptoms
associated with Naglu-deficiency. In some embodiments, a Naglu
protein suitable for the invention is a polypeptide having an
N-terminus and a C-terminus and an amino acid sequence
substantially similar or identical to the mature human Naglu
protein.
[0095] Typically, human Naglu is produced as a precursor molecule
that is processed to a mature form. This process generally occurs
by removing the 23 amino acid signal peptide as the protein enters
the endoplasmic reticulum. Typically, the precursor form is also
referred to as full-length precursor or full-length Naglu protein,
which contains 743 amino acids. The N-terminal 23 amino acids are
cleaved as the precursor protein enters the endoplasmic reticulum,
resulting in a mature form. Thus, it is contemplated that the
N-terminal 23 amino acids are generally not required for Naglu
protein activity. However, the use of the full-length precursor of
the Naglu protein is also contemplated within the scope of the
instant invention. The amino acid sequences of the mature form (SEQ
ID NO:1) and full-length precursor (SEQ ID NO:2) of a typical
wild-type or naturally-occurring human Naglu protein are shown in
Table 1 below.
TABLE-US-00001 TABLE 1 Mature and Full-length Precursor Naglu
Protein Mature
DEAREAAAVRALVARLLGPGPAADFSVSVERALAAKPGLDTYSLGGGGAARVRV Form of
RGSTGVAAAAGLHRYLRDFCGCHVAWSGSQLRLPRPLPAVPGELTEATPNRYRY Naglu
YQNVCTQSYSFVWWDWARWEREIDWMALNGINLALAWSGQEAIWQRVYLALGLT
QAEINEFFTGPAFLAWGRMGNLHTWDGPLPPSWHIKQLYLQHRVLDQMRSFGMT
PVLPAFAGHVPEAVTRVFPQVNVTKMGSWGHENCSYSCSELLAPEDPIFPIIGS
LFLRELIKEFGTDHIYGADTFNEMQPPSSEPSYLAAATTAVYEAMTAVDTEAVW
LLQGWLFQHQPQFWGPAQIRAVLGAVPRGRLLVLDLFAESQPVYTRTASFQGQP
FIWCMLHNFGGNHGLFGALEAVNGGPEAARLFPNSTMVGTGMAPEGISQNEVVY
SLMAELGWRKDPVPDLAAWVTSFAARRYGVSHPDAGAAWRLLLRSVYNCSGEAC
RGHNRSPLVRRPSLQMNTSIWYNRSDVFEAWRLLLTSAPSLATSPAFRYDLLDL
TRQAVQELVSLYYEEARSAYLSKELASLLRAGGVLAYELLPALDEVLASDSRFL
LGSWLEQARAAAVSEAEADFYEQNSRYQLTLWGPEGNILDYANKQLAGLVANYY
TPRWRLFLEALVDSVAQGIPFQQHQFDKNVFQLEQAFVLSKQRYPSQPRGDTVD
LAKKIFLKYYPRWVAGSW (SEQ ID NO.: 1) Full-Length
MEAVAVAAAVGVLLLAGAGGAAGDEAREAAAVRALVARLLGPGPAADFSVSVER Precursor/
ALAAKPGLDTYSLGGGGAARVRVRGSTGVAAAAGLHRYLRDFCGCHVAWSGSQL Full-Length
RLPRPLPAVPGELTEATPNRYRYYQNVCTQSYSFVWWDWARWEREIDWMALNGI Naglu
NLALAWSGQEAIWQRVYLALGLTQAEINEFFTGPAFLAWGRMGNLHTWDGPLPP Protein
SWHIKQLYLQHRVLDQMRSFGMTPVLPAFAGHVPEAVTRVFPQVNVTKMGSWGH
FNCSYSCSFLLAPEDPIFPIIGSLFLRELIKEFGTDHIYGADTFNEMQPPSSEP
SYLAAATTAVYEAMTAVDTEAVWLLQGWLFQHQPQFWGPAQIRAVLGAVPRGRL
LVLDLFAESQPVYTRTASFQGQPFIWCMLHNFGGNHGLFGALEAVNGGPEAARL
FPNSTMVGTGMAPEGISQNEVVYSLMAELGWRKDPVPDLAAWVTSFAARRYGVS
HPDAGAAWRLLLRSVYNCSGEACRGHNRSPLVRRPSLQMNTSIWYNRSDVFEAW
RLLLTSAPSLATSPAFRYDLLDLTRQAVQELVSLYYEEARSAYLSKELASLLRA
GGVLAYELLPALDEVLASDSRFLLGSWLEQARAAAVSEAEADFYEQNSRYQLTL
WGPEGNILDYANKQLAGLVANYYTPRWRLFLEALVDSVAQGIPFQQHQFDKNVF
QLEQAFVLSKQRYPSQPRGDTVDLAKKIFLKYYPRWVAGSW (SEQ ID NO.: 2)
[0096] Thus, in some embodiments, Naglu protein suitable for the
present invention is mature human Naglu protein (SEQ ID NO:1). In
some embodiments, a suitable Naglu protein may be a homologue or an
orthologue of the mature human Naglu protein from a different
species (e.g., mouse, rat, sheep, pig, dog, etc.). In other
embodiments, a suitable Naglu protein may be a functional variant
of the mature human Naglu protein. A functional variant of the
mature human Naglu protein may be a modified mature human Naglu
protein containing one or more amino acid substitutions, deletions,
and/or insertions as compared to a wild-type or naturally-occurring
Naglu protein (e.g., SEQ ID NO:1), while substantially retaining
the biological activity of Naglu protein. Thus, in some
embodiments, a Naglu protein suitable for the present invention is
substantially homologous to mature human Naglu protein (SEQ ID
NO:1). In some embodiments, a Naglu protein suitable for the
present invention has an amino acid sequence at least 50%, 55%,
60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%,
97%, 98%, 99% or more homologous to SEQ ID NO:1. In some
embodiments, a Naglu protein suitable for the present invention is
substantially identical to mature human Naglu protein (SEQ ID
NO:1). In some embodiments, a Naglu protein suitable for the
present invention has an amino acid sequence at least 50%, 55%,
60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%,
97%, 98%, 99% or more identical to SEQ ID NO:1. In some
embodiments, a Naglu protein suitable for the present invention
contains a fragment or a portion of mature human Naglu protein.
[0097] Alternatively, a Naglu protein suitable for the present
invention is full-length Naglu protein. In some embodiments, a
Naglu protein suitable may be a homologue or an orthologue of
full-length human Naglu protein from a different species (e.g.
mouse, rat, sheep, pig, dog, etc.). In some embodiments, a suitable
Naglu protein may be a functional variant of the full-length human
Naglu protein containing one or more amino acid substitutions,
deletions, and/or insertions as compared to a wild-type or
naturally-occurring full-length Naglu protein (e.g., SEQ ID NO:2),
while retaining substantial Naglu protein activity. Thus, In some
embodiments, Naglu protein suitable for the present invention is
substantially homologous to full-length human Naglu protein (SEQ ID
NO:2). In some embodiments, a Naglu protein suitable for the
present invention has an amino acid sequence at least 50%, 55%,
60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%,
97%, 98%, 99% or more homologous to SEQ ID NO:2. In some
embodiments, a Naglu protein suitable for the present invention is
substantially identical to SEQ ID NO:2. In some embodiments, a
Naglu protein suitable for the present invention has an amino acid
sequence at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%,
92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identical to SEQ ID
NO:2. In some embodiments, a Naglu protein suitable for the present
invention contains a fragment or a portion of full-length human
Naglu protein. As used herein, a full-length Naglu protein
typically contains signal peptide sequence.
[0098] As is well known in this art, amino acid or nucleic acid
sequences may be compared using any of a variety of algorithms,
including those available in commercial computer programs such as
BLASTN for nucleotide sequences and BLASTP, gapped BLAST, and
PSI-BLAST for amino acid sequences. Exemplary such programs are
described in Altschul, et al., Basic local alignment search tool,
J. Mol. Biol., 215(3): 403-410, 1990; Altschul, et al., Methods in
Enzymology; Altschul, et al., "Gapped BLAST and PSI-BLAST: a new
generation of protein database search programs", Nucleic Acids Res.
25:3389-3402, 1997; Baxevanis, et al., Bioinformatics: A Practical
Guide to the Analysis of Genes and Proteins, Wiley, 1998; and
Misener, et al., (eds.), Bioinformatics Methods and Protocols
(Methods in Molecular Biology, Vol. 132), Humana Press, 1999; all
of the foregoing of which are incorporated herein by reference. In
addition to identifying homologous sequences, the programs
mentioned above typically provide an indication of the degree of
homology. In some embodiments, two sequences are considered to be
substantially homologous if at least 50%, at least 55%, at least
60%, at least 65%, at least 70%, at least 75%, at least 80%, at
least 85%, at least 90%, at least 91%, at least 92%, at least 93%,
at least 94%, at least 95%, at least 96%, at least 97%, at least
98%, at least 99% or more of their corresponding residues are
homologous over a relevant stretch of residues. In some
embodiments, the relevant stretch is a complete sequence. In some
embodiments, the relevant stretch is at least 10, at least 15, at
least 20, at least 25, at least 30, at least 35, at least 40, at
least 45, at least 50, at least 55, at least 60, at least 65, at
least 70, at least 75, at least 80, at least 85, at least 90, at
least 95, at least 100, at least 125, at least 150, at least 175,
at least 200, at least 225, at least 250, at least 275, at least
300, at least 325, at least 350, at least 375, at least 400, at
least 425, at least 450, at least 475, at least 500 or more
residues.
[0099] The phrase "substantial identity" is used herein to refer to
a comparison between amino acid or nucleic acid sequences. As will
be appreciated by those of ordinary skill in the art, two sequences
are generally considered to be "substantially identical" if they
contain identical residues in corresponding positions. As is well
known in this art, amino acid or nucleic acid sequences may be
compared using any of a variety of algorithms, including those
available in commercial computer programs such as BLASTN for
nucleotide sequences and BLASTP, gapped BLAST, and PSI-BLAST for
amino acid sequences. Exemplary such programs are described in
Altschul, et al., Basic local alignment search tool, J. Mol. Biol.,
215(3): 403-410, 1990; Altschul, et al., Methods in Enzymology;
Altschul et al., Nucleic Acids Res. 25:3389-3402, 1997; Baxevanis
et al., Bioinformatics: A Practical Guide to the Analysis of Genes
and Proteins, Wiley, 1998; and Misener, et al., (eds.),
Bioinformatics Methods and Protocols (Methods in Molecular Biology,
Vol. 132), Humana Press, 1999. In addition to identifying identical
sequences, the programs mentioned above typically provide an
indication of the degree of identity. In some embodiments, two
sequences are considered to be substantially identical if at least
50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%,
95%, 96%, 97%, 98%, 99% or more of their corresponding residues are
identical over a relevant stretch of residues. In some embodiments,
the relevant stretch is a complete sequence. In some embodiments,
the relevant stretch is at least 10, 15, 20, 25, 30, 35, 40, 45,
50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 125, 150, 175, 200,
225, 250, 275, 300, 325, 350, 375, 400, 425, 450, 475, 500 or more
residues.
Alternative Therapeutic Lysosomal Enzymes
[0100] The present inventors discovered unexpectedly that lysosomal
enzymes could be targeted to the lysosome by connecting them to a
targeting moiety containing a N-linked glycosylation site. Thus,
the present invention provides targeted therapeutics containing a
lysosomal enzyme and a lysosomal targeting moiety containing a
N-linked glycosylation site. A suitable therapeutic lysosomal
enzyme in the context of the present invention is any protein which
has been shown to play a role in the development or treatment of a
lysosomal storage disease, in particular those lysosomal storage
diseases having CNS etiology and/or symptoms, including, but not
limited to, aspartylglucosaminuria, cholesterol ester storage
disease, Wolman disease, cystinosis, Danon disease, Fabry disease,
Farber lipogranulomatosis, Farber disease, fucosidosis,
galactosialidosis types I/II, Gaucher disease types I/II/III,
globoid cell leukodystrophy, Krabbe disease, glycogen storage
disease II, Pompe disease, GM1-gangliosidosis types I/II/III,
GM2-gangliosidosis type I, Tay Sachs disease, GM2-gangliosidosis
type II, Sandhoff disease, GM2-gangliosidosis, .alpha.-mannosidosis
types I/II, .beta.-mannosidosis, metachromatic leukodystrophy,
mucolipidosis type I, sialidosis types I/II, mucolipidosis types
II/III, I-cell disease, mucolipidosis type IIIC pseudo-Hurler
polydystrophy, mucopolysaccharidosis type I, mucopolysaccharidosis
type II, mucopolysaccharidosis type IIIA, Sanfilippo syndrome,
mucopolysaccharidosis type IIIB, mucopolysaccharidosis type IIIC,
mucopolysaccharidosis type IIID, mucopolysaccharidosis type IVA,
Morquio syndrome, mucopolysaccharidosis type IVB,
mucopolysaccharidosis type VI, mucopolysaccharidosis type VII, Sly
syndrome, mucopolysaccharidosis type IX, multiple sulfatase
deficiency, neuronal ceroid lipofuscinosis, CLN1 Batten disease,
CLN2 Batten diseae, Niemann-Pick disease types A/B, Niemann-Pick
disease type C1, Niemann-Pick disease type C2, pycnodysostosis,
Schindler disease types I/II, Gaucher disease and sialic acid
storage disease.
[0101] A detailed review of the genetic etiology, clinical
manifestations, and molecular biology of lysosomal storage diseases
are detailed in Scriver et al., eds., The Metabolic and Molecular
Basis of Inherited Disease, 7.sup.th Ed., Vol. II, McGraw Hill,
(1995). The enzymes deficient in the above diseases are generally
known to those of skill in the art; some of these are exemplified
in Table 2 below:
TABLE-US-00002 TABLE 2 Proteins Associated with Lysosomal Storage
Disease Disease Name Enzyme Deficiency Substance Stored Pompe
Disease Acid-a1, 4-Glucosidase Glycogen .alpha.-1-4 linked
Oligosaccharides GM1 Gangliodsidosis .beta.-Galactosidase GM.sub.1
Gangliosides Tay-Sachs Disease .beta.-Hexosaminidase A GM.sub.2
Ganglioside GM2 Gangliosidosis: AB GM.sub.2 Activator Protein
GM.sub.2 Ganglioside Variant Sandhoff Disease .beta.-Hexosaminidase
A&B GM.sub.2 Ganglioside Fabry Disease .alpha.-Galactosidase A
Globosides Gaucher Disease Glucocerebrosidase Glucosylceramide
Metachromatic Arylsulfatase A Sulphatides Leukodystrophy Krabbe
Disease Galactosylceramidase Galactocerebroside Niemann Pick, Types
A & B Acid Sphingomyelinase Sphingomyelin Niemann-Pick, Type C
Cholesterol Esterification Sphingomyelin Defect Niemann-Pick, Type
D Unknown Sphingomyelin Farber Disease Acid Ceramidase Ceramide
Wolman Disease Acid Lipase Cholesteryl Esters Hurler Syndrome
.alpha.-L-Iduronidase Heparan & Dermatan (MPS IH) Sulfates
Scheie Syndrome .alpha.-L-Iduronidase Heparan & Dermatan,
Sulfates (MPS IS) Hurler-Scheie .alpha.-L-Iduronidase Heparan &
Dermatan (MPS IH/S) Sulfates Hunter Syndrome Iduronate Sulfatase
Heparan & Dermatan (MPS II) Sulfates Sanfilippo A Heparan
N-Sulfatase Heparan Sulfate (MPS IIIA) Sanfilippo B .alpha.-N-
Heparan Sulfate (MPS IIIB) Acetylglucosaminidase Sanfilippo C
Acetyl-CoA- Heparan Sulfate (MPS IIIC) Glucosaminide
Acetyltransferase Sanfilippo D N-Acetylglucosamine-6- Heparan
Sulfate (MPS IIID) Sulfatase Morquio B .beta.-Galactosidase Keratan
Sulfate (MPS IVB) Maroteaux-Lamy Arylsulfatase B Dermatan Sulfate
(MPS VI) Sly Syndrome .beta.-Glucuronidase (MPS VII)
.alpha.-Mannosidosis .alpha.-Mannosidase Mannose/Oligosaccharides
.beta.-Mannosidosis .beta.-Mannosidase Mannose/Oligosaccharides
Fucosidosis .alpha.-L-Fucosidase Fucosyl/Oligosaccharides
Aspartylglucosaminuria N-Aspartyl-.beta.- Aspartylglucosamine
Glucosaminidase Asparagines Sialidosis (Mucolipidosis I)
.alpha.-Neuraminidase Sialyloligosaccharides Galactosialidosis
Lysosomal Protective Sialyloligosaccharides (Goldberg Syndrome)
Protein Deficiency Schindler Disease .alpha.-N-Acetyl-
Galactosaminidase Mucolipidosis II (I-Cell N-Acetylglucosamine-1-
Heparan Sulfate Disease) Phosphotransferase Mucolipidosis III
(Pseudo- Same as ML II Hurler Polydystrophy) Cystinosis Cystine
Transport Protein Free Cystine Salla Disease Sialic Acid Transport
Free Sialic Acid and Glucuronic Protein Acid Infantile Sialic Acid
Sialic Acid Transport Free Sialic Acid and Glucuronic Storage
Disease Protein Acid Infantile Neuronal Ceroid Palmitoyl-Protein
Lipofuscins Lipofuscinosis Thioesterase Mucolipidosis IV Unknown
Gangliosides & Hyaluronic Acid Prosaposin Saposins A, B, C or
D
[0102] In some embodiments, a therapeutic lysosomal enzyme suitable
for the present invention is the human wildtype protein. In some
embodiments, a therapeutic lysosomal enzyme may be a homologue or
an orthologue of the human wildtype protein from a different
species (e.g., mouse, rat, sheep, pig, dog, etc.). In other
embodiments, a therapeutic lysosomal enzyme suitable for the
present invention may be a functional variant of the human wildtype
protein. A functional variant of the human wildtype protein may be
a modified human wildtype protein containing one or more amino acid
substitutions, deletions, and/or insertions as compared to a
wild-type or naturally-occurring protein, while substantially
retaining the biological activity of the human wildtype protein.
Thus, in some embodiments, a therapeutic lysosomal enzyme suitable
for the present invention is substantially homologous to the human
wildtype protein. In some embodiments, a therapeutic lysosomal
enzyme suitable for the present invention has an amino acid
sequence at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%,
92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more homologous to the
human wildtype protein. In some embodiments, a therapeutic
lysosomal enzyme suitable for the present invention is
substantially identical to the human wildtype protein. In some
embodiments, a therapeutic lysosomal enzyme suitable for the
present invention has an amino acid sequence at least 50%, 55%,
60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%,
97%, 98%, 99% or more identical to the human wildtype protein. In
some embodiments, a therapeutic lysosomal enzyme suitable for the
present invention contains a fragment or a portion of the human
wildtype protein.
Lysosomal Targeting Moiety
[0103] The present inventors discovered unexpectedly that lysosomal
enzymes could be targeted to the lysosome by connecting them to a
targeting moiety containing a N-linked glycosylation site. Thus,
the present invention provides targeted therapeutics containing a
lysosomal enzyme and a lysosomal targeting moiety containing a
N-linked glycosylation site.
[0104] The present invention contemplates that any peptide may be
used within the scope of the present invention as long as it has a
N-linked glycosylation site. N-linked glycosylation sites may be
predicted by computer algorithms and software, many of which are
generally known in the art. Alternatively, N-linked glycosylation
may be determined experimentally using any one of the many assays
generally known in the art. Non-limiting examples of such assays
are provided in the Examples section herein.
[0105] Suitable targeting moieties may be derived from proteins or
peptides including, but not limited to, IGF-I, Kif, ApoE, TAT, RAP,
p97, Plasminogen, Leukemia Inhibitory Factor Peptide (LIF),
Cellular Repressor of E1A-Stimulated Genes Peptide (CREG), Human
Sortlin-1 Propeptide (SPP), Human Prosaposin peptide (SapDC) and
Progranulin. In some embodiments, a lysosomal targeting moiety is
any protein, peptide, or fragment thereof that binds the CI-M6PR,
in a mannose-6-phosphate-dependent manner. In some embodiments, a
lysosomal targeting moiety is any protein, peptide, or fragment
thereof that binds directly to a region, domain and/or
extracellular portion of CI-M6PR. In some embodiments, a lysosomal
targeting moiety is any protein, peptide, or fragment thereof that
binds directly to a region, domain and/or extracellular portion of
CI-M6PR via a M6P residue.
[0106] In some embodiments, a lysosomal targeting moiety is derived
from human Prosaposin (SEQ ID NO:3). In some embodiments, the
lysosomal targeting moiety is a Prosaposin sequence of a wild-type
or naturally-occurring human Prosaposin protein. In some
embodiments, an amino acid sequence comprising the C terminal
region and D functional domain of Prosaposin is used (SEQ ID
NO:4)
TABLE-US-00003 TABLE 3 Human Prosaposin Human
MYALFLLASLLGAALAGPVLGLKECTRGSAVWCQNVKTA Prosaposin
SDCGAVKHCLQTVWNKPIVKSLPCDTCKDVVTAAGDMLK
DNATEEEILVYLEKTCDWLPKPNMSASCKEIVDSYLPVI
LDIIKGEMSRPGEVCSALNLCESLQKHLAELNHQKQLES
NKIPELDMTEVVAPFMANIPLLLYPQDGPRSKPQPKDNG
DVCQDCIQMVIDIQTAVRINSTFVQALVEHVKEECDRLG
PGMADTCKNYISQYSEIAIQMMMHMQPKETCALVGFCDE
VKEMPMQTLVPAKVASKNVIPALELVEPIKKHEVPAKSD
VYCEVCEFLVKEVTKLIDNNKTEKEILDAFDKMCSKLPK
SLSEECQEVVDTYGSSILSILLEEVSPELVCSMLHLCSG
TRLPALTVHVTQPKDGGFCEVCKKLVGYLDRNLEKNSTK
QEILAALEKGCSFLPDPYQKQCDQFVAEYEPVLIEILVE
VMDPSFVCLKIGACPSAHKPLLGTEKCIWGPSYWCQNTE TAAQCNAVEHCKRHVWN (SEQ ID
NO: 3) Human DGGFCEVCKKLVGYLDRNLEKNSTKQEILAALEKGCSFL Prosaposin
PDPYQKQCDQFVAEYEPVLIEILVEVMDPSFVCLKIGAC peptide
PSAHKPLLGTEKCIWGPSYWCQNTETAAQCNAVEHCKRH (SapDC) VWN (SEQ ID NO:
4)
[0107] In some embodiments, a lysosomal targeting moiety is a
modified human Prosaposin sequence containing amino acid
substitutions, insertions or deletions. In some embodiments, a
lysosomal targeting moiety has a sequence at least 50%, 55%, 60%,
65%, 70%, 75%, 80%, 85%, 90%, 95%, 98%, or 99% identical to the
sequence of human Prosaposin (SEQ ID NO:3). In some embodiments, a
lysosomal targeting moiety is a fragment of human Prosaposin. In
some embodiments, a lysosomal targeting moiety is a modified human
Prosaposin DC peptide (SapDC) sequence containing amino acid
substitutions, insertions or deletions. In some embodiments, a
lysosomal targeting moiety has a sequence at least 50%, 55%, 60%,
65%, 70%, 75%, 80%, 85%, 90%, 95%, 98%, or 99% identical to SapDC
(SEQ ID NO:4). In some embodiments, a lysosomal targeting moiety is
a fragment of SapDC. In some embodiments, a lysosomal targeting
moiety contains human Prosaposin (SEQ ID NO:3) or SapDC (SEQ ID
NO:4) that has one or more N-terminal, C-terminal or internal
deletions. In some embodiments, a lysosomal targeting moiety is a
modified human Prosaposin or SapDC peptide that has diminished
binding affinity for receptors.
[0108] In some embodiments, a lysosomal targeting moiety is derived
from human Leukemia inhibitory Factor (SEQ ID NO:5). In some
embodiments, the lysosomal targeting moiety is a Leukemia
inhibitory Factor sequence of a wild-type or naturally-occurring
human Leukemia inhibitory Factor protein. In some embodiments, an
amino acid sequence comprises the signal peptide, glycosylation
sites, helices A, B, C and/or D of the 4 helix bundle and
combinations thereof, of Leukemia Inhibitory Factor. In some
embodiments, an amino acid peptide sequence comprising the signal
peptide, glycosylation sites, helices A, B, C and D of the 4 helix
bundle and various mutations (LIF) is used (SEQ ID NO:6).
TABLE-US-00004 TABLE 4 Human Leukemia Inhibitory Factor Human
MKVLAAGVVPLLLVLHWKHGAGSPLPITPVNATCAIRHP Leukemia
CHNNLMNQIRSQLAQLNGSANALFILYYTAQGEPFPNNL Inhibitory
DKLCGPNVIDFPPFHANGTEKAKLVELYRIVVYLGTSLG Factor
NITRDQKILNPSALSLHSKLNATADILRGLLSNVLCRLC
SKYHVGHVDVTYGPDTSGKDVFQKKKLGCQLLGKYKQII AVLAQAF (SEQ ID NO: 5)
Human SPLPITPVNATCAIRHPCHNNLMN{A}IR{A}QLA{A}L Leukemia
(N)GSANALFILYYTAQGEPFPNNLDKLCGP(N)VIDFP Inhibitory
PFHA(N)GTEKAKLVELYRIVVYLGTSLG(N)ITRDQKI Factor
LNPSALSLHSKL(N)ATADILRGLLSNVLCRLCSKYHVG Peptide
HVDVTYGPDTSGKDV[[A]]QK[[A]]KLGCQLLGKYKQ IIAVLAQAF (SEQ ID NO:
6)
Sites within LIF Sequence [0109] (N)--denotes glycosylation sites
[0110] { }--denoates mutations made to alter recognition of the
receptor gp130; [[ ]]--denotes mutations made to alter recognition
of the receptor for Leukemia Inhibitory Factor
[0111] In some embodiments, a lysosomal targeting moiety is a
modified human Leukemia inhibitory Factor sequence containing amino
acid substitutions, insertions or deletions. In some embodiments, a
lysosomal targeting moiety has a sequence at least 50%, 55%, 60%,
65%, 70%, 75%, 80%, 85%, 90%, 95%, 98%, or 99% identical to the
sequence of Leukemia inhibitory Factor (SEQ ID NO:5). In some
embodiments, a lysosomal targeting moiety is a fragment of human
Leukemia inhibitory Factor. In some embodiments, a lysosomal
targeting moiety is a modified human LIF sequence (SEQ ID NO:6)
containing amino acid substitutions, insertions or deletions. In
some embodiments, a lysosomal targeting moiety has a sequence at
least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98%, or 99%
identical to LIF (SEQ ID NO:6). In some embodiments, a lysosomal
targeting moiety is a fragment of LIF. In some embodiments, a
lysosomal targeting moiety contains human Leukemia inhibitory
Factor (SEQ ID NO:5) or LIF (SEQ ID NO:6) that has one or more
N-terminal, C-terminal or internal deletions. In some embodiments,
a lysosomal targeting moiety is a modified human Leukemia
inhibitory Factor or LIF peptide that has diminished binding
affinity for the LIF receptor and/or alternative binding proteins,
such as but not limited to, pg130.
[0112] In some embodiments, a lysosomal targeting moiety is derived
from human Cellular Repressor of E1A-stimulated Genes protein (SEQ
ID NO:7). In some embodiments, the lysosomal targeting moiety is a
Cellular Repressor of E1A-stimulated Genes sequence of a wild-type
or naturally-occurring human Cellular Repressor of E1A-stimulated
Genes protein.
[0113] In some embodiments, an amino acid sequence not comprising
the sequence of the signal peptide of Cellular Repressor of
E1A-stimulated Genes, and not comprising the internal peptide of
the sequence DPQS (SEQ ID NO:8) of Cellular Repressor of
E1A-stimulated Genes is used, resulting in Human Repressor of
E1-stimulated Genes Peptide (CREG) (SEQ ID NO:9), is used.
TABLE-US-00005 TABLE 5 Human Repressor of E1-stimulated Genes Human
Cellular MAGLSRGSARALLAALLASTLLALLVSPARGRGGRD Repressor of
HGDWDEASRLPPLPPREDAARVARFVTHVSDWGALA E1-stimulated
TISTLEAVRGRPFADVLSLSDGPPGAGSGVPYFYLS Genes
PLQLSVSNLQENPYATLTMTLAQTNFCKKHGFDPQS
PLCVHIMLSGTVTKVNETEMDIAKHSLFIRHPEMKT
WPSSHNWFFAKLNITNIWVLDYFGGPKIVTPEEYYN VTVQ (SEQ ID NO: 7) Human
Cellular RGGRDHGDWDEASRLPPLPPREDAARVARFVTHVSD Repressor of
WGALATISTLEAVRGRPFADVLSLSDGPPGAGSGVP E1-stimulated
YFYLSPLQLSVSNLQENPYATLTMTLAQTNFCKKHG Genes Peptide
FPLCVHIMLSGTVTKVNETEMDIAKHSLFIRHPEMK (CREG)
TWPSSHNWFFAKLNITNIWVLDYFGGPKIVTPEEYY NVTVQ (SEQ ID NO: 9
[0114] In some embodiments, a lysosomal targeting moiety is a
modified human Cellular Repressor of E1A-stimulated Genes sequence
containing amino acid substitutions, insertions or deletions. In
some embodiments, a lysosomal targeting moiety has a sequence at
least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98%, or 99%
identical to the sequence of human Cellular Repressor of
E1A-stimulated Genes (SEQ ID NO:7). In some embodiments, a
lysosomal targeting moiety is a fragment of human Cellular
Repressor of E1A-stimulated Genes. In some embodiments, a lysosomal
targeting moiety is a modified human Cellular Repressor of
E1A-stimulated Genes sequence containing amino acid substitutions,
insertions or deletions. In some embodiments, a lysosomal targeting
moiety has a sequence at least 50%, 55%, 60%, 65%, 70%, 75%, 80%,
85%, 90%, 95%, 98%, or 99% identical to human Cellular Repressor of
E1A-stimulated Genes Peptide (CREG) (SEQ ID NO:9). In some
embodiments, a lysosomal targeting moiety is a fragment of CREG. In
some embodiments, a lysosomal targeting moiety contains human
Cellular Repressor of E1A-stimulated Genes (SEQ ID NO:7) or CREG
(SEQ ID NO:9) that has one or more N-terminal, C-terminal or
internal deletions. In some embodiments, a lysosomal targeting
moiety is a modified human Cellular Repressor of E1A-stimulated
Genes or CREG that has diminished binding affinity for
receptors.
[0115] Various additional lysosomal targeting moieties are known in
the art and can be used to practice the present invention. For
example, certain peptide-based lysosomal targeting moieties are
described in U.S. Pat. Nos. 7,396,811, 7,560,424, and 7,629,309;
U.S. Application Publication Nos. 2003-0082176, 2004-0006008,
2003-0072761, 20040005309, 2005-0281805, 2005-0244400, and
international publications WO 03/032913, WO 03/032727, WO
02/087510, WO 03/102583, WO 2005/078077, WO/2009/137721, the entire
disclosures of which arc incorporated herein by reference.
[0116] In some embodiments, a lysosomal targeting moiety is any
polypeptide that is M6P phosphorylated by the cell. In some
embodiments, the polypeptide is capable of binding to the CI-M6PR.
In some embodiments, the polypeptide is an amino acid sequence
found within a protein selected from the group consisting of
Cathepsin B, Cathepsin D, Cathepsin L, Beta-Glucuroidase,
Beta-Mannosidase, Alpha-Fucosidase, Beta-Hexosaminidase,
Arylsulfatase, Beta-Galactosidase, Phosphomannan, Latent TGFbeta,
Leukemia Inhibitory Factor, Proliferin, Prorcnin, Herpes Simplex
Virus, PI-LLC cleaved GPI anchor, Rctinoic Acid, IGFII,
Plasminogen, Thyroglobulin, TGFbetaR-V, CD87, GTP-binding Proteins
(Gi-1, Gi-2 and Gi-3), HA-I Adaptin, HA-II Adaptin and combinations
thereof. In some embodiments, the amino acid sequence includes a
domain, fragment, region or segment of one or more proteins
selected from the group consisting of Cathepsin B, Cathepsin D,
Cathepsin L, Beta-Glucuroidase, Beta-Mannosidase, Alpha-Fucosidase,
Beta-Hexosaminidase, Arylsulfatase, Beta-Galactosidase,
Phosphomannan, Latent TGFbeta, Leukemia Inhibitory Factor,
Proliferin, Prorenin, Herpes Simplex Virus, PI-LLC cleaved GPI
anchor, Retinoic Acid, IGFII, Plasminogen, Thyroglobulin,
TGFbetaR-V, CD87, GTP-binding Proteins (Gi-1, Gi-2 and Gi-3), HA-I
Adaptin, HA-II Adaptin and combinations thereof. In some
embodiments, the polypeptide is produced synthetically. In some
embodiments, the polypeptide is produced recombinantely. Both
approaches are widely used in the art and described, for example,
in Sambrook et al., Molecular Cloning: A Laboratory Manual, Cold
Spring Harbor Laboratory Press (2001).
Linking the Lysosomal Targeting Moiety With the Lysosomal
Enzyme--Fusion Proteins
[0117] The lysosomal targeting moiety may be coupled to the
lysosomal enzyme in any of the ways generally known in the art.
Non-limiting examples include creating a fusion protein by genetic
means or by chemical coupling. In some embodiments, the lysosomal
targeting moiety is fused to the N-terminus of the lysosomal
enzyme, and in other embodiments the lysosomal targeting moiety is
fused to the C-terminus of the lysosomal enzyme. In some
embodiments, the lysosomal targeting moiety is located internally,
i.e., within the lysosomal enzyme polypeptide. Creating fusion
proteins is standard in the art described in detail in Sambrook et
al., Molecular Cloning: A Laboratory Manual, Cold Spring Harbor
Laboratory Press (2001). It is contemplated that within in the
scope of the invention more than one lysosomal targeting moiety may
be coupled with a suitable lysosomal enzyme.
[0118] In some embodiments of the present invention, the targeted
therapeutic is a fusion protein. A fusion protein is a polypeptide
that is generated from two or more smaller polypeptides. Fusion
proteins may be made by recombinant DNA technology, which is
standard in the art and described in detail in Sambrook et al.
[0119] Non-limiting examples of fusion proteins within the scope of
the present invention are disclosed in the Examples section herein.
In some embodiments, the fusion proteins have one of the following
amino acid sequences: SEQ ID NO: 23, 24, 25 or 26. In some
embodiments, the fusion protein is at least 70, 75, 80, 85, 90, 95
or 100% identical to any one of these sequences.
Linker or Spacer
[0120] A lysosomal targeting moiety may be fused to the N-terminus
or C-terminus of a suitable lysosomal enzyme polypeptide, or
inserted internally. The lysosomal targeting moiety may be fused
directly to the lysosomal enzyme polypeptide or may be separated
from the lysosomal enzyme polypeptide by a linker or a spacer. An
amino acid linker or spacer is generally designed to be flexible or
to interpose a structure, such as an alpha-helix, between the two
protein moieties. A linker or spacer may be relatively short, such
as a poly "GAG" sequence GGGGGAAAAGGGG (SEQ ID NO:10), a "GAP"
sequence of GAP (SEQ ID NO:11), a "PolyGP" sequence of GGGGGP (SEQ
ID NO:12), or can be longer, such as, for example, 10-50 (e.g.,
10-20, 10-25, 10-30, 10-35, 10-40, 10-45, 10-50) amino acids in
length. In some embodiments, various short linker sequences can be
present in tandem repeats. For example, a suitable linker may
contain the "GAG" amino acid sequence of GGGGGAAAAGGGG (SEQ ID
NO:10) present in tandem repeats. In some embodiments, such as
linker may further contain one or more "GAP" sequences, that frames
the "GAG" sequence of GGGGGAAAAGGGG (SEQ ID NO:10). For example, in
some embodiments a GAG2 linker may be used, which contains two
tandem "GAG" repeats, each framed by a "GAP" sequence, such as
GAPGGGGGAAAAGGGGGAPGGGGGAAAAGGGGGAP (SEQ ID NO:13). In some
embodiments a GAG3 linker may be used, which contains three tandem
"GAG" repeats, each framed by two "GAP" sequences, such as
GAPGGGGGAAAAAGGGGGGAPGGGGGAAAAAGGGGGGAPGGGGGAAAAAGGGGG GAP (SEQ ID
NO:14). In some embodiments, a suitable linker or spacer may
contain a sequence at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%,
90%, 95%, 98%, or 99% identical to the sequence of SEQ ID
NO:14.
[0121] In some embodiments, a suitable linker or spacer may contain
the following sequence:
GAPGGGGGAAAAAGGGGGGAPGGGGGAAAAAGGGGGGAPGGGGGAAAAAGGGGG GAP (SEQ ID
NO.: 15). In some embodiments, a suitable linker or spacer may
contain a sequence at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%,
90%, 95%, 98%, or 99% identical to the sequence of SEQ ID
NO:15.
Vectors
[0122] In one aspect, the invention provides a nucleic acid
encoding any of the fusion proteins disclosed herein. In one
aspect, the present invention provides a vector including said
nucleic acid. The term nucleic acid is described in detail in the
definition section herein. Vectors for expression of the proteins
described herein and/or other components of the invention include
plasmid vectors, as well as double or single-stranded RNA or DNA
viral vectors. Vectors may be introduced directly into cells as DNA
or through indirect use of phages and viruses. Vectors are commonly
known in th art and described in detail in Sambrook et al.,
Molecular Cloning: A Laboratory Manual, Cold Spring Harbor
Laboratory Press (2001).
Host Cells
[0123] In one aspect, the present invention provides a host cell
including a vector. Suitable host cells can be derived from a
variety of organisms, including, but not limited to, mammals,
plants, birds (e.g., avian systems), insects, yeast, and bacteria.
In some embodiments, host cells are mammalian cells. Any mammalian
cell susceptible to cell culture, and to expression of
polypeptides, may be utilized in accordance with the present
invention as a host cell. Non-limiting examples of mammalian cells
that may be used in accordance with the present invention include
human embryonic kidney 293 cells (HEK293), HeLa cells; BALB/c mouse
myeloma line (NSW, ECACC No: 85110503); human retinoblasts (PER.C6
(CruCell, Leiden, The Netherlands)); monkey kidney CV1 line
transformed by SV40 (COS-7, ATCC CRL 1651); human fibrosarcomacell
line (e.g., HT-1080); human embryonic kidney line (293 or 293 cells
subcloned for growth in suspension culture, Graham et al., J. Gen
Virol., 36:59 (1977)); baby hamster kidney cells (BHK, ATCC CCL
10); Chinese hamster ovary cells +/-DHFR (CHO, Urlaub and Chasin,
Proc. Natl. Acad. Sci. USA, 77:4216 (1980)); mouse sertoli cells
(TM4, Mather, Biol. Reprod., 23:243-251 (1980)); monkey kidney
cells (CV1 ATCC CCL 70); African green monkey kidney cells
(VERO-76, ATCC CRL-1 587); human cervical carcinoma cells (HeLa,
ATCC CCL 2); canine kidney cells (MDCK, ATCC CCL 34); buffalo rat
liver cells (BRL 3A, ATCC CRL 1442); human lung cells (W138, ATCC
CCL 75); human liver cells (Hep G2, HB 8065); mouse mammary tumor
(MMT 060562, ATCC CCL51); TRI cells (Mather et al., Annals N.Y.
Acad. Sci., 383:44-68 (1982)); MRC 5 cells; FS4 cells; a human
hepatoma line (Hep G2), Chinese hamster ovary cells (CHO cells),
human cell line CAP and AGE1.HN, and Glycotope's panel.
[0124] In some embodiments, host cells are non-mammalian cells.
Suitable non-mammalian cells include prokaryotes, archae, yeast and
other eukaryotic cells. Non-limiting examples of non-mammalian host
cells suitable for the present invention include yeast cells and
cell lines derived from Pichia pastoris, Pichia methanolica, Pichia
angusta, Schizosacccharomyces pombe, Saccharomyces cerevisiae, and
Yarrowia lipolytica; insect cells and cell lines derived from
Sodoptera frugiperda, Trichoplusis ni, Drosophila melanogaster and
Manduca sexta; and cells nd cell lines derived from Escherichia
coli, Salmonella typhimurium, Bacillus subtilis, Bacillus
licheniformis, Bacteroides fragilis, Clostridia perfringens,
Clostridia difficile; and amphibian cells and cell lines derived
Xenopus Laevis.
GNPT
[0125] In some embodiments of the present invention, the host cell
co-expresses GNPT. Typically, therapeutic glycoproteins, such as
lysosomal enzymes, are heavily influenced by a post-translational
modification in order to ensure proper function. As described
throughout the specification, lysosomal proteins belong to a class
of enzymes known as acid hydrolases and are synthesized as soluble
or membrane-integrated glycoproteins at the rough endoplasmic
reticulum. The N-linked oligosaccharides of the soluble lysosomal
enzymes are modified along the transit to and through the Golgi
complex to expose mannose-6-phosphate (M6P) recognition markers.
These M6P recognition markers are recognized in the trans-Golgi
network (TGN) by cation mannose-6-phosphate receptors (CI-M6PR),
which results in clustering and packaging into AP1-clathrin-coated
vesicles. After fusion with endosomes, the receptors release their
ligands, delivering the lysosmal enzymes to a newly formed
lysosome. In the event lysosomal proteins fail to bind M6P
receptors in the trans-Golgi network, such as in the case of I-cell
disease in which there is a deficiency in the phosphotrasferase,
the proteins are secreted from the cell resulting in excessive
amount of lysomal enzymes in body fluids such as plasma,
cerebrospinal fluid, tears and urine. Since cation
mannose-6-phosphate receptors are important for the recycling and
retrograde movement of lysosomal enzymes to and from the cell
surface, the CI-M6PR can serve as an important target for
lysosmal-mediated cellular delivery of a therapeutic glycoprotein
in enzyme replacement therapy.
[0126] Given the cellular importance of protein targeting,
modification of N-linked oligosaccharides to expose
mannose-6-phosphate recognition tags, as in the case of lysosomal
enzymes, is tightly regulated. The M6P residues are generated in a
two-step process. First, N-acetylglucosamine 1-phosphate is
attached to the C6-hydroxyl group of mannose, followed by removal
of N-acetylglucosamine. The first reaction is catalyzed by the
UDP-GlcNAc:lysosomal enzyme N-acetylgucosamine-1-phosphotransferase
(GNPT), while the second is by an alpha-N-acetylglucosaminidase.
GNPT enzyme is a hexameric complex containing three distinct
subunits (.alpha., .beta., .gamma.). The lysosomal protein binds
within the active cleft of the molecule, where the enzyme catalyzes
the addition of N-acetylglucosamine 1-phosphate to the protein;
making use of uridine diphosphate N-acetylglucosamine (UDP-GlcNAc)
as the molecular substrate.
[0127] The ability of a therapeutic glycoprotein (i.e., lysosomal
enzyme) to traffic in and out of the cell is typically positively
correlated with its level of M6P phosphorylation. For example, the
glycosylation level of one or more therapeutic glycoproteins (i.e.
-lysosomal enzymes) and their ability to traffic may vary based on
their level of M6P phosphorylation (mono- or
bis-phosphorylation).
[0128] In some embodiments, a GNPT enzyme suitable for the present
invention is human GNPT protein (SEQ ID NO:16 and/or 17). In some
embodiments, a suitable GNPT enzyme may be a homologue or an
ortholog of mature human GNPT protein. For example, a homologue or
an ortholog of mature human GNPT protein may be a modified mature
human GNPT protein containing one or more amino acid substitutions,
deletions, and/or insertions as compared to a wild-type or
naturally-occurring GNPT protein (e.g., SEQ ID NO:16 and/or 17),
while retaining substantial GNPT protein activity. Thus, in some
embodiments, a GNPT enzyme suitable for the present invention is
substantially homologous to human GNPT protein (SEQ ID NO:16 and/or
17). In some embodiments, a GNPT enzyme suitable for the present
invention has an amino acid sequence at least 50%, 55%, 60%, 65%,
70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%,
99% or more homologous to SEQ ID NO:16 and/or 17. In some
embodiments, a GNPT enzyme suitable for the present invention is
substantially identical to mature human GNPT protein (SEQ ID NO:16
and/or 17). In some embodiments, a GNPT enzyme suitable for the
present invention has an amino acid sequence at least 50%, 55%,
60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%,
97%, 98%, 99% or more identical to SEQ ID NO:16 and/or 17. In some
embodiments, a GNPT enzyme suitable for the present invention
contains a fragment or a portion of mature human GNPT protein.
TABLE-US-00006 TABLE 6 Human GNPT Protein Human N-acetyl-
MLFKLLQRQTYTCLSHRYGLYVCFLGVVVTIVSAFQFGEVVLEWSRDQYHVLFD
gucosamine-1-
SYRDNIAGKSFQNRLCLPMPIDVVYTWVNGTDLELLKELQQVREQMEEEQKAMR phospho-
EILGKNTTEPTKKSEKQLECLLTHCIKVPMLVLDPALPANITLKDLPSLYPSFH transferase
SASDIFNVAKPKNPSTNVSVVVFDSTKDVEDAHSGLLKGNSRQTVWRGYLTTDK (Alpha and
Beta EVPGLVLMQDLAFLSGFPPTFKETNQLKTKLPENLSSKVKLLQLYSEASVALLK
subunits) LNNPKDFQELNKQTKKNMTIDGKELTISPAYLLWDLSAISQSKQDEDISASRFE
*Underlined DNEELRYSLRSIERHAPWVRNIFIVTNGQIPSWLNLDNPRVTIVTHQDVFRNLS
Region-TGRQLK-
HLPTFSSPAIESHIHRIEGLSQKFIYLNDDVMFGKDVWPDDFYSHSKGQKVYLT is
alpha/beta WPVPNCAEGCPGSWIKDGYCDKACNNSACDWDGGDCSGNSGGSRYIAGGGGTGS
cleavage site
IGVGQPWQFGGGINSVSYCNQGCANSWLADKFCDQACNVLSCGFDAGDCGQDHF
HELYKVILLPNQTHYIIPKGECLPYFSFAEVAKRGVEGAYSDNPIIRHASIANK
WKTIHLIMHSGMNATTIHFNLTFQNTNDEEFKMQITVEVDTREGPKLNSTAQKG
YENLVSPITLLPEAEILFEDIPKEKRFPKFKRHDVNSTRRAQEEVKIPLVNISL
LPKDAQLSLNTLDLQLEHGDITLKGYNLSKSALLRSFLMNSQHAKIKNQAIITD
ETNDSLVAPQEKQVHKSILPNSLGVSERLQRLTFPAVSVKVNGHDQGQNPPLDL
ETTARFRVETHTQKTIGGNVTKEKPPSLIVPLESQMTKEKKITGKEKENSRMEE
NAENHIGVTEVLLGRKLQHYTDSYLGFLPWEKKKYFQDLLDEEESLKTQLAYFT
DSKNTGRQLKDTFADSLRYVNKILNSKFGFTSRKVPAHMPHMIDRIVMQELQDM
FPEEFDKTSFHKVRHSEDMQFAFSYFYYLMSAVQPLNISQVFDEVDTDQSGVLS
DREIRTLATRIHELPLSLQDLTGLEHMLINCSKMLPADITQLNNIPPTQESYYD
PNLPPVTKSLVTNCKPVTDKIHKAYKDKNKYRFEIMGEEEIAFKMIRTNVSHVV
GQLDDIRKNPRKFVCLNDNIDHNHKDAQTVKAVLRDFYESMFPIPSQFELPREY
RNRFLHMHELQEWRAYRDKLKFWTHCVLATLIMFTIFSFFAEQLIALKRKIFPR
RRIHKEASPNRIRV (SEQ ID NO: 16) Human N-acetyl-
MAAGLARLLLLLGLSAGGPAPAGAAKMKVVEEPNAFGVNNPFLPQASRLQAKRD
gucosamine-1-
PSPVSGPVHLFRLSGKCFSLVESTYKYEFCPFHNVTQHEQTFRWNAYSGILGIW phospho-
HEWEIANNTFTGMWMRDGDACRSRSRQSKVELACGKSNRLAHVSEPSTCVYALT transferase
FETPLVCHPHALLVYPTLPEALQRQWDQVEQDLADELITPQGHEKLLRTLFEDA (Gamma
Subunit) GYLKTPEENEPTQLEGGPDSLGFETLENCRKAHKELSKEIKRLKGLLTQHGIPY
TRPTETSNLEHLGHETPRAKSPEQLRGDPGLRGSL (SEQ ID NO: 17)
[0129] Overexpression of enzymes, including GNPT, in host cells and
tissue culture is routine and described in detail in Sambrook et
al.
Culture Conditions
[0130] The present invention provides a method of producing
recombinant targeted therapeutic glycoproteins using a GNPT cell
culture system. In some embodiments, the cell culture system is
used for microscale production of a therapeutic glycoprotein. In
some embodiments, the cellular culture system is used for large
scale production. In some embodiments the therapeutic glycoprotein
produced is a recombinant lysosomal enzyme. Typical large-scale
procedures for producing a recombinant polypeptide of interest
include batch cultures and fed-batch cultures. Batch culture
processes traditionally comprise inoculating a large-scale
production culture with a seed culture of a particular cell
density, growing the cells under conditions (e.g., suitable culture
medium, pH, and temperature) conducive to cell growth, viability,
and/or productivity, harvesting the culture when the cells reach a
specified cell density, and purifying the expressed polypeptide.
Fed-batch culture procedures include an additional step or steps of
supplementing the batch culture with nutrients and other components
that are consumed during the growth of the cells. In some
embodiments, a large-scale production method according to the
present invention uses a fed-batch culture system.
Bioreactors
[0131] The invention also provides bioreactors that are useful for
producing recombinant therapeutic glycoproteins. Bioreactors may be
perfusion, batch, fed-batch, repeated batch, or continuous (e.g. a
continuous stirred-tank reactor models), for example. Typically,
the bioreactors comprise at least one vessel designed and are
configured to house medium (e.g., a chemically defined nutrient
medium). The vessel also typically comprises at least one inlet
designed and configured to flow fresh nutrient medium into the
vessel. The vessel also typically comprises at least one outlet
designed and configured to flow waste medium out of the vessel. In
some embodiments, the vessel may further comprise at least one
filter designed and configured to minimize the extent to which
isolated cells in the vessel are passed out through the at least
one outlet with waste medium. The bioreactor may also be fitted
with one or more other components designed to maintain conditions
suitable for cell growth. For example, the bioreactor may be fitted
with one or more circulation or mixing devices designed and
configured to circulate or mix the nutrient medium within the
vessel. Typically, the isolated cells that are engineered to
express recombinant therapeutic glycoproteins are suspended in the
nutrient medium. Therefore, in some cases, the circulation device
ensures that the isolated cells remain in suspension in the
nutrient medium. In some cases, the cells are attached to a
substrate. In some cases, the cells are attached to one or more
substrates (e.g., microbeads) that are suspended in the nutrient
medium. The bioreactor may comprise one or more ports for obtaining
a sample of the cell suspension from the vessel. The bioreactor may
be configured with one or more components for monitoring and/or
controlling conditions of the culture, including conditions such as
gas content (e.g., air, oxygen, nitrogen, carbon dioxide), flow
rates, temperature, pH and dissolved oxygen levels, and agitation
speed/circulation rate.
[0132] Vessels of any appropriate size may be used in the
bioreactors. Typically, the vessel size is suitable for satisfying
the production demands of manufacturing recombinant I2S. In some
embodiments, the vessel is designed and configured to contain up to
1 L, up to 10 L, up to 100 L, up to 500 L, up to 1000 L, up to 1500
L, up to 2000 L, or more of the nutrient medium. In some
embodiments, the volume of the production bioreactor is at least 10
L, at least 50 L, 100 L, at least 200 L, at least 250 L, at least
500 L, at least 1000 L, at least 1500 L, at least 2000 L, at least
2500 L, at least 5000 L, at least 8000 L, at least 10,000 L, or at
least 12,000 L, or more, or any volume in between. The production
bioreactor may be constructed of any material that is conducive to
cell growth and viability that does not interfere with expression
or stability or activity of the produced therapeutic glycoprotein.
Exemplary material may include, but not be limited to, glass,
plastic, or metal.
[0133] In some embodiments, cells may be cultured in a chemically
defined medium that is housed in a vessel of a bioreactor. The
culture methods often involve perfusing fresh nutrient medium into
the vessel through the at least one inlet and bleeding waste
nutrient medium out from vessel through the at least one outlet.
Bleeding is performed at a rate of up to about 0.1 vessel volume
per day, about 0.2 vessel volume per day, about 0.3 vessel volume
per day, about 0.4 vessel volume per day, about 0.5 vessel volume
per day, about 1 vessel volume per day, about 1.5 vessel volumes
per day or more. The methods also involve harvesting nutrient
medium that comprises recombinant the recombinant glycoprotein. In
some embodiments, the therapeutic glycoproteins may be a lysosomal
enzyme. In some embodiments, the therapeutic glycoprotein may be a
fusion protein. Harvesting may be performed at a rate of up to
about 0.1 vessel volume per day, about 0.2 vessel volume per day,
about 0.3 vessel volume per day, about 0.4 vessel volume per day,
about 0.5 vessel volume per day, about 1 vessel volume per day,
about 1.5 vessel volumes per day or more. Perfusing is also
performed, typically at a rate equivalent to the sum of the
bleeding rate and the harvesting rate. For example, perfusion rate
may be great than about 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8,
0.9, 1.0, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2.0 vessel
volume per day. In some embodiments, perfusion rate is less than
about 5.0, 4.5, 4.0, 3.5, 3.0, 2.5, 2.0, 1.5, 1.4, 1.3, 1.2, 1.1,
1.0, 0.9, 0.8, 0.7, 0.6, 0.5 vessel volume per day.
Monitoring Culture Conditions
[0134] In certain embodiments of the present invention, the
practitioner may find it beneficial or necessary to periodically
monitor particular conditions of the growing cell culture.
Monitoring cell culture conditions allows the practitioner to
determine whether the cell culture is producing recombinant
polypeptide or protein at suboptimal levels or whether the culture
is about to enter into a suboptimal production phase. In order to
monitor certain cell culture conditions, it will be necessary to
remove small aliquots of the culture for analysis.
[0135] As non-limiting example, it may be beneficial or necessary
to monitor temperature, pH, cell density, cell viability,
integrated viable cell density, osmolarity, or titer or activity of
the expressed therapeutic glycoprotein protein. Numerous techniques
are well known in the art that will allow one of ordinary skill in
the art to measure these conditions. For example, cell density may
be measured using a hemacytometer, a Coulter counter, or Cell
density examination (CEDEX). Viable cell density may be determined
by staining a culture sample with Trypan blue. Since only dead
cells take up the Trypan blue, viable cell density can be
determined by counting the total number of cells, dividing the
number of cells that take up the dye by the total number of cells,
and taking the reciprocal. Alternatively, the level of the
expressed therapeutic glycoprotein can be determined by standard
molecular biology techniques such as coomassie staining of SDS-PAGE
gels, Western blotting, Bradford assays, Lowry assays, Biuret
assays, and UV absorbance.
[0136] It may also be beneficial or necessary to monitor the level
of M6P phosphorylation and/or glycan pattern to generate a
glycosylation profile, as demonstrated in FIGS. 9-10 of the
application. For example, in some embodiments, analysis can be
performed to monitor glycan critical quality attributes such as
antennary fucosylation, glactosylation, sialyation, high mannose
structure and phosphorylation structures. In some embodiments,
analysis can be performed to characterize the recombinantly
produced glycoprotein in relation to a reference sample. In some
embodiments, analysis can be performed to characterize the
recombinantly produced glycoprotein in relation to a previous lot
or batch of the sample protein produced under identical conditions.
In some embodiments, analysis can be performed to characterize the
recombinantly produced glycoprotein in relation to a previous lot
or batch of the sample protein produced under different conditions.
In some embodiments, analysis can be performed to characterize the
recombinantly produced glycoprotein in relation to a previous lot
or batch of the sample protein produced at a different stage of
production.
[0137] The GNPT cell line can be used to recombinantly produce a
therapeutic glycoprotein with increased levels of M6P
phosphorylation. In some embodiments, the recombinant therapeutic
glycoprotein produced using the GNPT cell line has M6P levels
higher than the naturally occurring therapeutic glycoprotein. In
some embodiments, the recombinant therapeutic glycoprotein has M6P
phosphorylation levels higher than the same therapeutic
glycoprotein recombinantly produced in the non-GNPT overexpressing
cell line. In some embodiments, the increase in M6P phosphorylation
compared to production in a non-GNPT overexpressing cell line is at
least 1 fold. In some embodiments, the increase in M6P
phosphorylation is at least a 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20,
25, 30, 35, 40, 45, 50, 60, 70, 80, or 90 fold increase. In some
embodiments, the increase in M6P phosphorylation at least a 1%, 2%,
3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 15%, 20%, 30%, 40%, 50%, 60%, 70%,
80% or 90% increase. In some embodiments, the recombinant
therapeutic glycoprotein produced using the GNPT cell line has M6P
levels at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, or 90%
higher than the wild-type therapeutic glycoprotein.
[0138] In some embodiments, a recombinant therapeutic glycoprotein
produced using the GNPT cell line may contain at least one M6P
residue. In some embodiments, a recombinant therapeutic
glycoprotein may contain a plurality of M6P residues. In some
embodiments, a recombinant therapeutic glycoprotein may be a
bis-phosphorylated glycoprotein with two M6P residues. In some
embodiments, a recombinant therapeutic glycoprotein produced using
the GNPT cell line may contain at least 1, 2, 3, 4, 5, 6, 7, 8, 9
or 10 M6P residues compared to the wild-type protein.
[0139] In some embodiments, a recombinant therapeutic glycoprotein
may contain an increase in the number and/or level of high mannose
structures, such as, M3, M4, M5, M6, M7 or M8 structures, compared
to the wild-type protein. In some embodiments, the increase in M3
structures is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15,
20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, or 90
percent compared to the wild-type protein. In some embodiments, the
increase in M4 structures is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12,
13, 14, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85,
or 90 percent compared to the wild-type protein. In some
embodiments, the increase in M5 structures is 1, 2, 3, 4, 5, 6, 7,
8, 9, 10, 11, 12, 13, 14, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60,
65, 70, 75, 80, 85, or 90 percent compared to the wild-type
protein. In some embodiments, the increase in M6 structures is 1,
2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 20, 25, 30, 35, 40,
45, 50, 55, 60, 65, 70, 75, 80, 85, or 90 percent compared to the
wild-type protein. In some embodiments, the increase in M7
structures is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15,
20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, or 90
percent compared to the wild-type protein. In some embodiments, the
increase in M8 structures is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12,
13, 14, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85,
or 90 percent compared to the wild-type protein.
[0140] In some embodiments, the recombinant therapeutic
glycoprotein may contain and increase in the number and/or level of
total phosphorylated structures, compared to the wild-type protein.
In some embodiments, the increase in total phosphorylated
structures compared to the wild-type protein is 1, 2, 3, 4, 5, 6,
7, 8, 9, 10, 11, 12, 13, 14, 15, 20, 25, 30, 35, 40, 45, 50, 55,
60, 65, 70, 75, 80, 85, or 90 percent.
[0141] In some embodiments, a recombinant therapeutic glycoprotein
produced using the GNPT cell line may contain a higher level of
antennary structures compared to the wild type protein. In
particular, therapeutic glycoproteins produced with the GNPT cell
line may have a higher level of A0 antennarity, which is indicative
of M6P phosphorylation. In some embodiments, the increase in A0
level compared to the wild-type protein is at least 1, 2, 3, 4, 5,
6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 20, 25, 30, 35, 40, 45, 50, 55,
60, 65, 70, 75, 80, 85, or 90 percent.
[0142] In some embodiments, a therapeutic protein suitable for the
present invention may contain a bis-phosphorylated oligosaccharides
which have higher binding affinity to the CI-M6PR. In some
embodiments, a suitable enzyme contains up to about an average of
about at least 20% bis-phosphorylated oligosaccharides per enzyme.
In other embodiments, a suitable enzyme may contain about 10%, 15%,
18%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60% bis-phosphorylated
oligosaccharides per enzyme.
[0143] In some embodiments, the increased level in M6P
phosphorylation of the therapeutic glycoprotein, as a result of
producing using the GNPT cell line, may be tested using any of a
variety of well-known binding assays to evaluate the level of
CI-M6PR binding. In some embodiments, therapeutic glycoproteins
produced using the GNPT cell line have an increase in CI-M6PR of 1,
2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20,
25, 30, 35, 40, 45, 50, 60, 70, 80, or 90 fold compared to the
native protein.
[0144] In some embodiments, the level of M6P phosphorylation may be
tested by monitoring binding of a therapeutic glycoprotein to the
CI-M6PR using any of a variety of well-known in vitro binding assay
such as, but not limited to, radiolabeled run on assay,
radiolabeled binding assay, ELISA, Surface Plasmone Resonance and
Isothermal Titration calorimetry. In some embodiments, the level of
M6P phosphorylation may be tested by monitoring binding of a
therapeutic glycoprotein to the CI-M6PR by evaluating cellular
uptake using methods such as, but not limited to,
immunohistochemistry staining, fluorescent immunohistochemistry
staining and confocal microscopy. In some embodiments, the level of
M6P phosphorylation may be assays using analytical techniques such
as, but not limited to, glycosidase treatment, mass spectrometry
and HPLC.
Purification of Therapeutic Glycoproteins
[0145] Various methods may be used to purify or isolate therapeutic
glycoproteins produced according to various methods described
herein. In some embodiments, the expressed therapeutic glycoprotein
protein (e.g., lysosomal enzymes) may be secreted into the medium
and thus cells and other solids may be removed, as by
centrifugation or filtering for example, as a first step in the
purification process. Alternatively or additionally, the expressed
therapeutic glycoprotein may be bound to the surface of the host
cell. In this embodiment, the host cells (for example, yeast cells)
expressing the polypeptide or protein are lysed for purification.
Lysis of host cells (e.g., yeast cells) can be achieved by any
number of means well known to those of ordinary skill in the art,
including physical disruption by glass beads and exposure to high
pH conditions.
[0146] The therapeutic glycoprotein may be isolated and purified by
standard methods including, but not limited to, chromatography
(e.g., ion exchange, affinity, size exclusion, and hydroxyapatite
chromatography), gel filtration, centrifugation, or differential
solubility, ethanol precipitation or by any other available
technique for the purification of proteins (See, e.g., Scopes,
Protein Purification Principles and Practice 2nd Edition,
Springer-Verlag, New York, 1987; Higgins, S. J. and Hames, B. D.
(eds.), Protein Expression: A Practical Approach, Oxford Univ
Press, 1999; and Deutscher, M. P., Simon, M. I., Abelson, J. N.
(eds.), Guide to Protein Purification: Methods in Enzymology
(Methods in Enzymology Series, Vol 182), Academic Press, 1997, all
incorporated herein by reference). For immunoaffinity
chromatography in particular, the protein may be isolated by
binding it to an affinity column comprising antibodies that were
raised against that protein and were affixed to a stationary
support. Alternatively, affinity tags such as an influenza coat
sequence, poly-histidine, or glutathione-S-transferase can be
attached to the protein by standard recombinant techniques to allow
for easy purification by passage over the appropriate affinity
column. Protease inhibitors such as phenyl methyl sulfonyl fluoride
(PMSF), leupeptin, pepstatin or aprotinin may be added at any or
all stages in order to reduce or eliminate degradation of the
polypeptide or protein during the purification process. Protease
inhibitors are particularly desired when cells must be lysed in
order to isolate and purify the expressed polypeptide or protein.
Exemplary purification methods are described in the Examples
sections below.
Pharmaceutical Compositions and Administration
[0147] The present invention further provides pharmaceutical
compositions containing targeted therapeutics according to the
present invention. Typically, suitable pharmaceutical compositions
contain at least one pharmaceutically acceptable excipient and are
formulated for administration to humans.
[0148] For example, pharmaceutical compositions provided herein may
be provided in a sterile injectable form (e.g., a form that is
suitable for subcutaneous, intravenous, or intrathecal injection).
For example, in some embodiments, pharmaceutical compositions are
provided in a liquid dosage form that is suitable for injection. In
some embodiments, pharmaceutical compositions are provided as
powders (e.g., lyophilized and/or sterilized), optionally under
vacuum, which are reconstituted with an aqueous diluent (e.g.,
water, buffer, salt solution, etc.) prior to injection. In some
embodiments, pharmaceutical compositions are diluted and/or
reconstituted in water, sodium chloride solution, sodium acetate
solution, benzyl alcohol solution, phosphate buffered saline, etc.
In some embodiments, powder should be mixed gently with the aqueous
diluent (e.g., not shaken).
[0149] In some embodiments, provided pharmaceutical compositions
comprise one or more pharmaceutically acceptable excipients (e.g.,
preservative, inert diluent, dispersing agent, surface active agent
and/or emulsifier, buffering agent, etc.). In some embodiments,
pharmaceutical compositions comprise one or more preservatives. In
some embodiments, pharmaceutical compositions comprise no
preservative.
[0150] Compositions of the pharmaceutical compositions described
herein may be prepared by any method known or hereafter developed
in the art of pharmacology. In some embodiments, such preparatory
methods include the step of bringing active ingredient into
association with one or more excipients and/or one or more other
accessory ingredients, and then, if necessary and/or desirable,
shaping and/or packaging the product into a desired single- or
multi-dose unit.
[0151] A pharmaceutical composition in accordance with the
invention may be prepared, packaged, and/or sold in bulk, as a
single unit dose, and/or as a plurality of single unit doses. As
used herein, a "unit dose" is discrete amount of the pharmaceutical
composition comprising a predetermined amount of the active
ingredient. The amount of the active ingredient is generally equal
to a dose which would be administered to a subject and/or a
convenient fraction of such a dose such as, for example, one-half
or one-third of such a dose.
[0152] Relative amounts of active ingredient, pharmaceutically
acceptable excipient, and/or any additional ingredients in a
pharmaceutical composition in accordance with the invention may
vary, depending upon the identity, size, and/or condition of the
subject treated and/or depending upon the route by which the
composition is to be administered. By way of example, the
composition may comprise between 0.1% and 100% (w/w) active
ingredient.
[0153] Pharmaceutical compositions of the present invention may
additionally comprise a pharmaceutically acceptable excipient,
which, as used herein, may be or comprise solvents, dispersion
media, diluents, or other liquid vehicles, dispersion or suspension
aids, surface active agents, isotonic agents, thickening or
emulsifying agents, preservatives, solid binders, lubricants and
the like, as suited to the particular dosage form desired.
Remington's The Science and Practice of Pharmacy, 21st Edition, A.
R. Gennaro, (Lippincott, Williams & Wilkins, Baltimore, Md.,
2006) discloses various excipients used in formulating
pharmaceutical compositions and known techniques for the
preparation thereof. Except insofar as any conventional excipient
medium is incompatible with a substance or its derivatives, such as
by producing any undesirable biological effect or otherwise
interacting in a deleterious manner with any other component(s) of
the pharmaceutical composition, its use is contemplated to be
within the scope of this invention.
[0154] In some embodiments, pharmaceutical compositions according
to the present invention can be used for CNS delivery via various
techniques and routes including, but not limited to,
intraparenchymal, intracerebral, intravetricular cerebral (ICV),
intrathecal (e.g., IT-Lumbar, IT-cistema magna) administrations and
any other techniques and routes for injection directly or
indirectly to the CNS and/or CSF.
Intrathecal Delivery
[0155] In some embodiments, pharmaceutical compositions according
to the present invention can be used for intrathecal
administration. As used herein, intrathecal administration (also
referred to as intrathecal injection or intrathecal delivery)
refers to an injection into the spinal canal (intrathecal space
surrounding the spinal cord). Various formulations for intrathecal
administration are described in WO/2011/163652, the contents of
which are incorporated herein by reference.
[0156] According to the present invention, a pharmaceutical
composition containing a targeted therapeutics may be injected at
any region surrounding the spinal canal. In some embodiments, a
pharmaceutical composition containing a targeted therapeutics is
injected into the lumbar area or the cisterna magna or
intraventricularly into a cerebral ventricle space. As used herein,
the term "lumbar region" or "lumbar area" refers to the area
between the third and fourth lumbar (lower back) vertebrae and,
more inclusively, the L2-S1 region of the spine. Typically,
intrathecal injection via the lumbar region or lumber area is also
referred to as "lumbar IT delivery" or "lumbar IT
administration."
[0157] Various devices may be used for intrathecal delivery
according to the present invention. In some embodiments, a device
for intrathecal administration contains a fluid access port (e.g.,
injectable port); a hollow body (e.g., catheter) having a first
flow orifice in fluid communication with the fluid access port and
a second flow orifice configured for insertion into spinal cord;
and a securing mechanism for securing the insertion of the hollow
body in the spinal cord. As a non-limiting example, a suitable
securing mechanism contains one or more nobs mounted on the surface
of the hollow body and a sutured ring adjustable over the one or
more nobs to prevent the hollow body (e.g., catheter) from slipping
out of the spinal cord. In various embodiments, the fluid access
port comprises a reservoir. In some embodiments, the fluid access
port comprises a mechanical pump (e.g., an infusion pump). In some
embodiments, an implanted catheter is connected to either a
reservoir (e.g., for bolus delivery), or an infusion pump. The
fluid access port may be implanted or external
[0158] In some embodiments, intrathecal administration may be
performed by either lumbar puncture (i.e., slow bolus) or via a
port-catheter delivery system (i.e., infusion or bolus). In some
embodiments, the catheter is inserted between the laminae of the
lumbar vertebrae and the tip is threaded up the thecal space to the
desired level (generally L3-L4).
[0159] For injection, formulations of the invention can be
formulated in liquid solutions. In addition, the enzyme may be
formulated in solid form and re-dissolved or suspended immediately
prior to use. Lyophilized forms are also included. The injection
can be, for example, in the form of a bolus injection or continuous
infusion (e.g., using infusion pumps) of the enzyme.
Treatment of San B and Other Lysosomal Storage Diseases
[0160] The present invention may be used to effectively treat
Sanfilippo Syndrome Type B and other lysosomal storage diseases.
Sanfilippo Syndrome Type B, or Mucopolysaccharidosis III B (MPS III
B), is a heritable metabolic disorder resulting from a deficiency
of the enzyme Naglu. Naglu is localized to lysosomes and plays an
important role in the catabolism of glycosaminoglycans (GAGs)
heparan- and dermatan-sulfate. In the absence of enzyme, these
substrates accumulate within cells, ultimately causing engorgement,
followed by cellular death and tissue destruction. Due to the
widespread expression of enzyme, multiple cell types and organ
systems are affected in MPS III B patients.
[0161] A defining clinical feature of this disorder is central
nervous system (CNS) degeneration, which results in cognitive
impairment (e.g., decrease in IQ). Additionally, MRI scans of
affected individuals have revealed white matter lesions, dilated
perivascular spaces in the brain parenchyma, ganglia, corpus
callosum, and brainstem; atrophy; and ventriculomegaly (Wang et al.
Molecular Genetics and Metabolism, 2009). The disease typically
manifests itself in the first years of life with organomegaly and
skeletal abnormalities. Some affected individuals experience a
progressive loss of cognitive function, with most affected
individuals dying of disease-associated complications in their
first or second decade.
[0162] Compositions and methods of the present invention may be
used to effectively treat individuals suffering from or susceptible
to Sanfilippo Syndrome Type B. The terms, "treat" or "treatment,"
as used herein, refers to amelioration of one or more symptoms
associated with the disease, prevention or delay of the onset of
one or more symptoms of the disease, and/or lessening of the
severity or frequency of one or more symptoms of the disease.
[0163] In some embodiments, treatment refers to partially or
complete alleviation, amelioration, relief, inhibition, delaying
onset, reducing severity and/or incidence of neurological
impairment in a Sanfilippo Syndrome Type B patient. As used herein,
the term "neurological impairment" includes various symptoms
associated with impairment of the central nervous system (e.g., the
brain and spinal cord). Symptoms of neurological impairment may
include, for example, e.g., cognitive impairment; white matter
lesions; dilated perivascular spaces in the brain parenchyma,
ganglia, corpus callosum, and/or brainstem; atrophy; and/or
ventriculomegaly, among others.
[0164] The terms, "improve," "increase" or "reduce," as used
herein, indicate values that are relative to a control. In some
embodiments, a suitable control is a baseline measurement, such as
a measurement in the same individual prior to initiation of the
treatment described herein, or a measurement in a control
individual (or multiple control individuals) in the absence of the
treatment described herein. A "control individual" is an individual
afflicted with a lysosomal storage disease (e.g., Sanfillipo
Syndrome Type B), who is about the same age and/or gender as the
individual suffering from the same lysosmal storage disease, who is
being treated (to ensure that the stages of the disease in the
treated individual and the control individual(s) are
comparable).
[0165] The individual (also referred to as "patient" or "subject")
being treated is an individual (fetus, infant, child, adolescent,
or adult human) having a lysosomal storage disease or having the
potential to develop a lysosmal storage disease. In some
embodiments, the lysosmal storage disease is Sanfilippo Syndrome.
In some specific embodiments the lysosomal storage disease is
Sanfilippo Syndrome Type B. The individual can have residual
endogenous Naglu expression and/or activity, or no measurable
activity. For example, the individual having Sanfilipo Syndrome
Type B may have Naglu expression levels that are less than about
30-50%, less than about 25-30%, less than about 20-25%, less than
about 15-20%, less than about 10-15%, less than about 5-10%, less
than about 0.1-5% of normal Naglu expression levels.
[0166] In some embodiments, the individual is an individual who has
been recently diagnosed with the disease. Typically, early
treatment (treatment commencing as soon as possible after
diagnosis) is important to minimize the effects of the disease and
to maximize the benefits of treatment.
[0167] The invention will be more fully understood by reference to
the following examples. They should not, however, be construed as
limiting the scope of the invention. All references cited herein
are incorporated herein by reference in their entirety.
EXAMPLES
Example 1: Creation of Naglu Fusion Proteins
[0168] Several proteins have been implicated in binding to
different extracellular regions/domains within the CI-M6P receptor
via a M6P-dependant manner, such as Leukemia Inhibitory Factor,
Cellular Repressor of E1A-stimulated Genes and Saposin, or via a
M6P-independent manner, such as Insulin-like Growth Factor II.
Therefore, a series of Naglu-fusion proteins were designed that
might bind to CI-M6PR and therefore might facilitate
CI-M6PR-mediated delivery of rhNaglu to the lysosome, as described
below.
Naglu-SapDC
[0169] The Naglu-SapDC protein is a fusion of Naglu and portions of
the saposin protein as described below. To create the Naglu-SapDC
fusion protein, a nucleic acid construct was created by coupling
the human Naglu gene to the sequence encoding the conserved C
terminal region and D functional domain of Saposin (SapDC). A GAG3
linker was inserted between the two sequences to allow for rotation
between the two peptides following translation. In the resulting
Full-Length Naglu-SapDC fusion protein (SEQ ID NO:18), Full-Length
Naglu constitutes the N-terminus and the SapDC peptide sequences
the C-terminus.
TABLE-US-00007 (SEQ ID NO: 18)
MEAVAVAAAVGVLLLAGAGGAAGDEAREAAAVRALVARLLGPGPAADFSV
SVERALAAKPGLDTYSLGGGGAARVRVRGSTGVAAAAGLHRYLRDFCGCH
VAWSGSQLRLPRPLPAVPGELTEATPNRYRYYQNVCTQSYSFVWWDWARW
EREIDWMALNGINLALAWSGQEAIWQRVYLALGLTQAEINEFFTGPAFLA
WGRMGNLHTWDGPLPPSWHIKQLYLQHRVLDQMRSFGMTPVLPAFAGHVP
EAVTRVFPQVNVTKMGSWGHFNCSYSCSFLLAPEDPIFPIIGSLFLRELI
KEFGTDHIYGADTFNEMQPPSSEPSYLAAATTAVYEAMTAVDTEAVWLLQ
GWLFQHQPQFWGPAQIRAVLGAVPRGRLLVLDLFAESQPVYTRTASFQGQ
PFIWCMLHNFGGNHGLFGALEAVNGGPEAARLFPNSTMVGTGMAPEGISQ
NEVVYSLMAELGWRKDPVPDLAAWVTSFAARRYGVSHPDAGAAWRLLLRS
VYNCSGEACRGHNRSPLVRRPSLQMNTSIWYNRSDVFEAWRLLLTSAPSL
ATSPAFRYDLLDLTRQAVQELVSLYYEEARSAYLSKELASLLRAGGVLAY
ELLPALDEVLASDSRFLLGSWLEQARAAAVSEAEADFYEQNSRYQLTLWG
PEGNILDYANKQLAGLVANYYTPRWRLFLEALVDSVAQGIPFQQHQFDKN
VFQLEQAFVLSKQRYPSQPRGDTVDLAKKIFLKYYPRWVAGSWGAPGGGG
GAAAAAGGGGGGAPGGGGGAAAAAGGGGGGAPGGGGGAAAAAGGGGGGAP
DGGFCEVCKKLVGYLDRNLEK(N)STKQEILAALEKGCSFLPDPYQKQCD
QFVAEYEPVLIEILVEVMDPSFVCLKIGACPSAHKPLLGTEKCIWGPSYW
CQNTETAAQCNAVEHCKRHVWN Human Full length Naglu-in italics GAG3
Linker-in bold SapDC-underlined Sites within SapDC sequence
(N)-denotes glycosylation sites
Naglu-LIF
[0170] The Naglu-LIF protein is a fusion of Naglu and portions of
the Leukemia Inhibitory Factor protein as described below. To
create the Naglu-LIF fusion protein, a nucleic acid construct was
created by coupling the human Naglu gene to the sequence encoding
the 102 amino acid sequence comprising the glycosylation sites, and
helicies A, B, C and D of the 4 helix bundle of Leukcmia Inhibitory
Factor (LIF). A GAG3 linker was inserted between the two sequences
to allow for rotation between the two peptides following
translation. In the resulting Full-Length Naglu-LIF fusion protein
(SEQ ID NO:19), Full-Length Naglu constitutes the N-terminus and
the LIF peptide sequences the C-terminus.
TABLE-US-00008 (SEQ ID NO: 19)
MEAVAVAAAVGVLLLAGAGGAAGDEAREAAAVRALVARLLGPGPAADFSV
SVERALAAKPGLDTYSLGGGGAARVRVRGSTGVAAAAGLHRYLRDFCGCH
VAWSGSQLRLPRPLPAVPGELTEATPNRYRYYQNVCTQSYSFVWWDWARW
EREIDWMALNGINLALAWSGQEAIWQRVYLALGLTQAEINEFFTGPAFLA
WGRMGNLHTWDGPLPPSWHIKQLYLQHRVLDQMRSFGMTPVLPAFAGHVP
EAVTRVFPQVNVTKMGSWGHFNCSYSCSFLLAPEDPIFPIIGSLFLRELI
KEFGTDHIYGADTFNEMQPPSSEPSYLAAATTAVYEAMTAVDTEAVWLLQ
GWLFQHQPQFWGPAQTRAVLGAVPRGRLLVLDLFAESQPVYTRTASFQGQ
PFIWCMLHNFGGNHGLFGALEAVNGGPEAARLFPNSTMVGTGMAPEGISQ
NEVVYSLMAELGWRKDPVPDLAAWVTSFAARRYGVSHPDAGAAWRLLLRS
VYNCSGEACRGHNRSPLVRRPSLQMNTSIWYNRSDVFEAWRLLLTSAPSL
ATSPAFRYDLLDLTRQAVQELVSLYYEEARSAYLSKELASLLRAGGVLAY
ELLPALDEVLASDSRFLLGSWLEQARAAAVSEAEADFYEQNSRYQLTLWG
PEGNILDYANKQLAGLVANYYTPRWRLFLEALVDSVAQGIPFQQHQFDKN
VFQLEQAFVLSKQRYPSQPRGDTVDLAKKIFLKYYPRWVAGSWGAPGGGG
GAAAAAGGGGGGAPGGGGGAAAAAGGGGGGAPGGGGGAAAAAGGGGGGAP
SPLPITPVNATCAIRHPCHNNLMN{A}IR{A}QLA{A}L(N)GSANALFI
LYYTAQGEPFPNNLDKLCGP(N)VTDFPPFHA(N)GTEKAKLVELYRIVV
YLGTSLG(N)ITRDQKILNPSALSLHSKL(N)ATADILRGLLSNVLCRLC
SKYHVGHVDVTYGPDTSGKDV[[A]]QK[[A]]KLGCQLLGKYKQIIAVL AQAF Human
Full-Length Naglu-in italics GAG3-in bold LIF-underlined Sites
within LIF sequence (N)-denotes glycosylation sites { }-denoates
mutations made to alter recognition of the receptor gp130; [[
]]-denotesmutations made to alter recognition of the LIF
receptor
Naglu-CREG
[0171] The Naglu-CREG protein is a fusion of Naglu and portions of
the Cellular Repressor of E1A-stimulated Genes protein as described
below. To create the Naglu-CREG fusion protein, a nucleic acid
construct was created by coupling the human Naglu gene to the
sequence encoding the CREG amino acid sequence. A GAG3 linker was
inserted between the two sequences to allow for rotation between
the two peptides following translation. In the resulting
Full-Length Naglu-CREG fusion protein (SEQ ID NO:20), Full-Length
Naglu constitutes the N-terminus and the CREG peptide sequences the
C-terminus.
TABLE-US-00009 (SEQ ID NO: 20)
MEAVAVAAAVGVLLLAGAGGAAGDEAREAAAVRALVARLLGPGPAADFSV
SVERALAAKPGLDTYSLGGGGAARVRVRGSTGVAAAAGLHRYLRDFCGCH
VAWSGSQLRLPRPLPAVPGELTEATPNRYRYYQNVCTQSYSFVWWDWARW
EREIDWMALNGINLALAWSGQEAIWQRVYLALGLTQAEINEFFTGPAFLA
WGRMGNLHTWDGPLPPSWHIKQLYLQHRVLDQMRSFGNITPVLPAFAGHV
PEAVTRVFPQVNVTKAIGSWGHFNCSYSCSFLLAPEDPIFPIIGSLFLRE
LIKEFGTDHIYGADTFNEMQPPSSEPSYLAAATTAVYEAMTAVDTEAVWL
LQGWLFQHQPQFWGPAQIRAVLGAVPRGRLLVLDLFAESQPVYTRTASFQ
GQPFIWCMLHNFGGNHGLFGALEAVNGGPEAARLFPNSTMVGTGMAPEGI
SQNEVVYSLMAELGWRKDPVPDLAAWVTSFAARRYGVSHPDAGAAWRLLL
RSVYNCSGEACRGHNRSPLVRRPSLQMNTSIWYNRSDVFEAWRLLLTSAP
SLATSPAFRYDLLDLTRQAVQELVSLYYEEARSAYLSKELASLLRAGGVL
AYELLPALDEVLASDSRFLLGSWLEQARAAAVSEAEADFYEQNSRYQLTL
WGPEGNILDYANKQLAGLVANYYTPRWRLFLEALVDSVAQGIPFQQHQFD
KNVFQLEQAFVLSKQRYPSQPRGDTVDLAKKIFLKYYPRWVAGSWGAPGG
GGGAAAAAGGGGGGAPGGGGGAAAAAGGGGGGAPGGGGGAAAAAGGGGGG
APRGGRDHGDWDEASRLPPLPPREDAARVARFVTHVSDWGALATISTLEA
VRGRPFADVLSLSDGPPGAGSGVPYFYLSPLQLSVSNLQENPYATLTMTL
AQTNFCKKHGFPLCVHIMLSGTVTKV(N)ETEMDIAKHSLFIRHPEMKTW
PSSHNWFFAKL(N)ITNIWVLDYFGGPKIVTPEEYY(N)VTVQ Human Full-Length
Naglu-in italics GAG3 Linke-in bold CREG-underlined Sites within
CREG sequence (N)-denotes glycosylation sites
Naglu-IGFII
[0172] The Naglu-IGFII protein is a fusion of Naglu and portions of
the Insulin-like Growth Factor II peptide as described below. To
create the Naglu-IGFII fusion protein, a nucleic acid construct was
created by coupling the human Naglu gene to the sequence encoding
the amino acid residues 8-67 (SEQ ID NO:22) of the Insulin-like
Growth Factor II peptide IGFII). A GAG3 linker was inserted between
the two sequences to allow for rotation between the two peptides
following translation. In the resulting Full-Length Naglu-IGFII
fusion protein (SEQ ID NO:21), Full-Length Naglu constitutes the
N-terminus and the IGFII peptide sequences the C-terminus. Compared
to the full-length IGFII molecule, 8-67 IGFII is reported to bind
to M6P/IGF II receptor with a 2-10 fold higher affinity while its
ability to bind to the IGF I receptor is decreased 30 fold
(Hashimoto R, JBC 1995 270(30):18013-18018).
TABLE-US-00010 (SEQ ID NO: 21)
MEAVAVAAAVGVLLLAGAGGAAGDEAREAAAVRALVARLLGPGPAADFSV
SVERALAAKPGLDTYSLGGGGAARVRVRGSTGVAAAAGLHRYLRDFCGCH
VAWSGSQLRLPRPLPAVPGELTEATPNRYRYYQNVCTQSYSFVWWDWARW
EREIDWMALNGINLALAWSGQEAIWQRVYLALGLTQAEINEFFTGPAFLA
WGRMGNLHTWDGPLPPSWHIKQLYLQHRVLDQMRSFGMTPVLPAFAGHVP
EAVTRVFPQVNVTKMGSWGHFNCSYSCSFLLAPEDPIFPIIGSLFLRELI
KEFGTDHIYGADTFNEMQPPSSEPSYLAAATTAVYEAMTAVDTEAVWLLQ
GWLFQHQPQFWGPAQIRAVLGAVPRGRLLVLDLFAESQPVYTRTASFQGQ
PFIWCMLHNFGGNHGLFGALEAVNGGPEAARLFPNSTMVGTGMAPEGISQ
NEVVYSLMAELGWRKDPVPDLAAWVTSFAARRYGVSHPDAGAAWRLLLRS
VYNCSGEACRGHNRSPLVRRPSLQMNTSIWYNRSDVFEAWRLLLTSAPSL
ATSPAFRYDLLDLTRQAVQELVSLYYEEARSAYLSKELASLLRAGGVLAY
ELLPALDEVLASDSRFLLGSWLEQARAAAVSEAEADFYEQNSRYQLTLWG
PEGNILDYANKQLAGLVANYYTPRWRLFLEALVDSVAQGIPFQQHQFDKN
VFQLEQAFVLSKQRYPSQPRGDTVDLAKKIFLKYYPRWVAGSWGAPGGGG
GAAAAAGGGGGGAPGGGGGAAAAAGGGGGGAPGGGGGAAAAAGGGGGGAP
LCGGELVDTLQFVCGDRGFYFSRPASRVSRRSRGIVEECCFRSCDLALLE TYCATPAKSE Human
Full-Length Naglu-in italics GAG3 Linker-in bold IGFII-underlined
(SEQ ID NO: 22) LCGGELVDTLQFVCGDRGFYFSRPASRVSRRSRGIVEECCFRSCDLALLE
TYCATPAKSE
[0173] SEQ ID NO:18-21 are the amino acid sequences of Full-Length
Naglu fusion proteins, which still contain those 23 N-terminal
amino acids of the Full-Length Naglu protein that are removed
during intracellular processing. The amino acid sequences provided
below (SEQ ID NO:23-26) are the corresponding sequences that do not
include these 23 N-terminal amino acids.
TABLE-US-00011 Naglu-SapDC: (SEQ ID NO: 23)
DEAREAAAVRALVARLLGPGPAADFSVSVERALAAKPGLDTYSLGGGGAARVRVRGSTGVAAA
AGLHRYLRDFCGCHVAWSGSQLRLPRPLPAVPGELTEATPNRYRYYQNVCTQSYSFVWWDWA
RWEREIDWMALNGINLALAWSGQEAIWQRVYLALGLTQAEINEFFTGPAFLAWGRMGNLHTW
DGPLPPSWHIKQLYLQHRVLDQMRSFGMTPVLPAFAGHVPEAVTRVFPQVNVTTCLIGSWGHF
NCSYSCSFLLAPEDPIFPIIGSLFLRELIKEFGTDHIYGADTFNEMQPPSSEPSYLAAATTAVYEA
MTAVDTEAVWLLQGWLFQHQPQFWGPAQTRAVLGAVPRGRLLVLDLFAESQPVYTRTASFQG
QPFIWCMLHNFGGNHGLFGALEAVNGGPEAARLFPNSTMVGTGMAPEGISQNEVVYSLMAE
LGWRKDPVPDLAAWVTSFAARRYGVSHPDAGAAWRLLLRSVYNCSGEACRGHNRSPLVRRPS
LQMNTSIWYNRSDVFEAWRLLLTSAPSLATSPAFRYDLLDLTRQAVQELVSLYYEEARSAYLSKE
LASLLRAGGVLAYELLPALDEVLASDSRFLLGSWLEQARAAAVSEAEADFYEQNSRYQLTLWGP
EGNILDYANKQLAGLVANYYTPRWRLFLEALVDSVAQGIPFQQHQFDKNVFQLEQAFVLSKQ
RYPSQPRGDTVDLAKKIFLKYYPRWVAGSWGAPGGGGGAAAAAGGGGGGAPGGGGGA
AAAAGGGGGGAPGGGGGAAAAAGGGGGGAPDGGFCEVCKKLVGYLDRNLEK(N)S
TKQEILAALEKGCSFLPDPYQKQCDQFVAEYEPVLIEILVEVMDPSFVCLKIGACPSAHKP
LLGTEKCIWGPSYWCQNTETAAQCNAVEHCKRHVWN Human Full length Naglu-in
italics GAG3 Linker-in bold SapDC-underlined Sites within SapDC
sequence (N)-denotes glycosylation sites Naglu-LIF: (SEQ ID NO: 24)
DEAREAAAVRALVARLLGPGPAADFSVSVERALAAKPGLDTYSLGGGGAARVRVRGSTGVAAA
AGLHRYLRDFCGCHVAWSGSQLRLPRPLPAVPGELTEATPNRYRYYQNVCTQSYSFVWWDW
ARWEREIDWMALNGINLALAWSGQEAIWQRVYLALGLTQAEINEFFTGPAFLAWGRMGNLHT
WDGPLPPSWHIKQLYLQHRVLDQMRSFGMTPVLPAFAGHVPEAVTRVFPQVNVTKMGSWG
HFNCSYSCSFLLAPEDPIFPHGSLFLRELIKEFGTDHIYGADTFNEMQPPSSEPSYLAAATTAVY
EAMTAVDTEAVTYLLQGWLFQHQPQFWGPAQIRAVLGAVPRGRLLVLDLFAESQPVYTRTASF
QGQPFIWCMLHNFGGNHGLFGALEAVNGGPEAARLFPNSTMVGTGMAPEGISQNEVVYSL
MAELGWRKDPVPDLAAWVTSFAARRYGVSHPDAGAAWRLLLRSVYNCSGEACRGHNRSPLV
RRPSLQMNTSIWYNRSDVFEAWRLLLTSAPSLATSPAFRYDLLDLTRQAVQELVSLYYEEARSAY
LSKELASLLRAGGVLAYELLPALDEVLASDSRFLLGSWLEQARAAAVSEAEADFYEQNSRYQLT
LWGPEGNILDYANKQLAGLVANYYTPRWRLFLEALVDSVAQGIPFQQHQFDKNVFQLEQAF
VLSKQRYPSQPRGDTVDLAKKIFLKYYPRWVAGSWGAPGGGGGAAAAAGGGGGGAPGG
GGGAAAAAGGGGGGAPGGGGGAAAAAGGGGGGAPSPLPITPVNATCAIRHPCHNN
LMN{A}IR{A}QLA{A}L(N)GSANALFILYYTAQGEPFPNNLDKLCGP(N)VTDFPPFHA(N)
GTEKAKL VELYRIV VYLGTSLG(N)ITRDQKILNPSALSLHSKL(N)ATADILRGLLSNVLC
RLCSKYHVGHVDVTYGPDTSGKDV[[A]]QK[[A]]KLGCQLLGKYKQIIAVLAQAF Human
Full-Length Naglu-in italics GAG3 Linker-in bold LIF-underlined
Sites within LIF sequence (N)-denotes glycosylation sites {
}-denoates mutations made to alter recognition of the receptor
gp130; [[ ]]-denotes mutations made to alter recognition of the LIF
receptor Naglu-CREG: (SEQ ID NO: 25)
DEAREAAAVRALVARLLGPGPAADFSVSVERALAAKPGLDTYSLGGGGAARVRVRGSTGVAAA
AGLHRYLRDFCGCHVAWSGSQLRLPRPLPAVPGELTEATPNRYRYYQNVCTQSYSFVWWDWA
RWEREIDWMALNGINLALAWSGQEAIWQRVYLALGLTQAEINEFFTGPAFLAWGRMGNLHTW
DGPLPPSWHIKQLYLQHRVLDQMRSFGMTPVLPAFAGHVPEAVTRVFPQVNVTKMGSWGHF
NCSYSCSFLLAPEDPIFPHGSLFLRELIKEFGTDHIYGADTFNEMQPPSSEPSYLAAATTAVYEA
MTAVDTEAVWLLQGWLFQHQPQFWGPAQIRAVLGAVPRGRLLVLDLFAESQPVYTRTASFQG
QPFIWCMLHNFGGNHGLFGALEAVNGGPEAARLFPNSTM-VGTGMAEPEGISQNEVVYSLMA
LGWRKDPVPDLAAWVTSFAARRYGVSHPDAGAAWRLLLRSVYNCSGEACRGHNRSPLVRRPS
LQMNTSIWYNRSDVFEAWRLLLTSAPSLATSPAFRYDLLDLTRQAVQELVSLYYEEARSAYLSKE
LASLLRAGGVLAYELLPALDEVLASDSRFLLGSWLEQARAAAVSEAEADFYEQNSRYQLTLWGP
EGNILDYANKQLAGLVANYYTPRWRLFLEALVDSVAQGIPFQQHQFDKNVFQLEQAFVLSKQ
RYPSQPRGDTVDLAKKIFLKYYPRWVAGSWGAPGGGGGAAAAAGGGGGGAPGGGGGA
AAAAGGGGGGAPGGGGGAAAAAGGGGGGAPRGGRDHGDWDEASRLPPLPPREDA
ARVARFVTHVSDWGALATISTLEAVRGRPFADVLSLSDGPPGAGSGVPYFYLSPLQLSV
SNLQENPYATLTMTLAQTNFCKKHGFPLCVHIMLSGTVTKV(N)ETEMDIAKHSLFIRHP
EMKTWPSSHNWFFAKL(N)ITNIWVLDYFGGPKIVTPEEYY(N)VTVQ Human Full-Length
Naglu-in italics GAG3 Linker-in bold CREG-underlined Sites within
CREG sequence (N)-denotes glycosylation sites Naglu-IGFII: (SEQ ID
NO: 26)
DEAREAAAVRALVARLLGPGPAADFSVSVERALAAKPGLDTYSLGGGGAARVRVRGSTGVAAA
AGLHRYLRDFCGCHVAWSGSQLRLPRPLPAVPGELTEATPNRYRYYQNVCTQSYSFVWWDWA
RWEREIDWMALNGINLALAWSGQEAIWQRVYLALGLTQAEINEFFTGPAFLAWGRMGNLHTW
DGPLPPSWHIKQLYLQHRVLDQMRSFGMTPVLPAFAGHVPEAVTRVFPQVNVTKMGSWGHF
NCSYSCSFLLAPEDPIFPIIGSLFLRELIKEFGTDHIYGADTFNEMQPPSSEPSYLAAATTAVYEA
MTAVDTEAVWLLQGWLFQHQPQFWGPAQIRAVLGAVPRGRLLVLDLFAESQPVYTRTASFQG
QPFIWCAILHNFGGNHGLFGALEAVNGGPEAARLFPNSTMVGTGMAPEGISQNEVVYSLMAE
LGWRKDPVPDLAAWVTSFAARRYGVSHPDAGAAWRLLLRSVYNCSGEACRGHNRSPLVRRPS
LQMNTSIWYNRSDVFEAWRLLLTSAPSLATSPAFRYDLLDLTRQAVQELVSLYYEEARSAYLSKE
LASLLRAGGVLAYELLPALDEVLASDSRFLLGSWLEQARAAAVSEAEADFYEQNSRYQLTLWGP
EGNILDYANKQLAGLVANYYTPRWRLFLEALVDSVAQGIPFQQHQFDKNVFQLEQAFVLSKQ
RYPSQPRGDTVDLAKKIFLKYYPRWVAGSWGAPGGGGGAAAAAGGGGGGAPGGGGGA
AAAAGGGGGGAPGGGGGAAAAAGGGGGGAPLCGGELVDTLQFVCGDRGFYFSRPA
SRVSRRSRGIVEECCFRSCDLALLETYCATPAKSE Human Full-Length Naglu-in
italics GAG3 Linker-in bold IGFII-underlined
Example 2: In Vitro M6P Receptor Binding and Cellular Uptake of
Naglu-Fusion Proteins
[0174] The nucleic acid encoding each of the different Naglu fusion
proteins was subcloned into a mammalian expression vector and
transfected into HT-1080 cells using standard technologies.
Transfectants were then screened to generate a HT-1080
overexpressing cell line, using technologies generally known in the
art. For all in vitro protein based assays and receptor binding
experiments, recombinant protein was produced in a wave bioreactor,
using a mammalian cell culture expressing system. Following
expression, each fusion protein was subjected to a three step
purification process. First, the conditioned media was concentrated
using an ultra-filtration (UF) device (Pall Corporation, Port
Washington, N.Y. 11050). The concentrated media was then applied to
a butyl sepharose chromatography column (Butyl), and the eluate of
this purification step was then further purified using a Q
sepharose chromatography column (Q). The purified protein was
buffer exchanged into PBS (11.9 mM sodium phosphate, 2.7 mM
potassium phosphate, 137 mM sodium chloride at pH 7.4) for storage.
The resulting fusion proteins were purified to greater than 90%
purity.
[0175] Following purification, ELISAs was performed with various
concentrations of fusion proteins and with an antibody directed to
human Naglu to evaluate the binding of the Naglu-fusion proteins to
CI-M6PR. The data demonstrate that the Naglu-IGFII, Naglu-CREG,
Naglu-SapDC and Naglu-LIF fusion proteins were able to bind to
CI-M6PR (FIGS. 1 and 2). Of the four fusion proteins, Naglu-IGFII
showed the strongest binding with a K.sub.D of approximately 0.21
nM. Naglu-CREG, Naglu-SapDC and Naglu LIF also showed strong
binding to CI-M6PR (K.sub.D of 1.25, 5.5 and 32.4 nM,
respectively), which was not as strong as the the binding by
Naglu-IGFII however (FIGS. 1 and 2).
[0176] In vitro CI-M6PR mediated targeting and cellular uptake of
each Naglu-fusion protein was also evaluated. Briefly, cells
overexpressing CI-M6PR were grown to confluence and treated with a
solution of recombinant Naglu-IGFII, Naglu-CREG, Naglu-SapDC or
Naglu-LIF at various concentrations, which contained additional
mannose-6-phosphate or not (FIG. 3). After a specified period of
time, the supernatant was removed and the cells were washed
repeatedly. Following lysis of the cells, each sample was assayed
for intracellular Naglu enzyme activity. The data indicate that,
following treatment with each fusion protein, there was an increase
in the level of intracellular Naglu activity, suggesting that all
four fusion proteins were effectively internalized by the cells
(FIG. 3). This was in stark contrast to incubation of the
Naglu-fusion protein in the presence of mannose-6-phosphate, which
completely blocked cellular internalization (FIG. 3). The ability
of mannose-6-phosphate to prevent CI-M6PR targeting and lysosomal
entry of each Naglu-fusion protein (Naglu-CREG, Naglu-SapDC and
Naglu-LIF) suggests that mannose-6-phosphate is able to effectively
outcompete Naglu-fusion protein binding to CI-M6PR. This
demonstrates that the observed cellular internalization and
lysosomal entry of each Naglu-fusion protein was the result of
CI-M6PR. The data also indicate that cellular uptake of the
Naglu-fusion proteins occurs in a dose dependent fashion since
intracellular Naglu activity increased in proportion to increasing
concentrations of Naglu-fusion protein added to the assay (FIG. 3)
Binding of Naglu-IGFII to CI-M6PR is mediated through a
protein-protein interaction between the IGF-II domain and CI-M6PR.
Cellular uptake of Naglu-IGFII is therefore not dependent on
mannose-6-phosphate and not outcompeted by it.
Example 3: Creation of GNPT Cell Lines
[0177] Given the critical role GNPT plays in the post-translation
modification of N-linked glycans, specifically the
M6P-phosphorylation of soluble lysosomal enzymes, experiments were
performed to evaluate if overexpression of the GNPT enzyme could
result in increased M6P-phosphorylation levels on lysosomal
emzymes.
[0178] N-acetylglucosamine-1-phosphotransferase (GNTP) protein is a
hexameric complex containing two copies each of three distinct
subunits (.alpha., .beta., .gamma.). The nucleic acid encoding the
alpha/beta subunits and the gamma subunit of the GNTP protein were
cloned into one mammalian expression vector containing an
expression cassette for the alpha/beta subunit and one expression
cassette for the gamma subunit. The vector was transfected into
human HT-1080 cells, which were then grown under selection pressure
to establish cell lines overexpressing recombinant GNTP. A cell
line overexpressing GNPT was selected and used for secondary
transfection with a mammalian expression vector expressing
Naglu-LTF, Naglu-CREG, Naglu-SapDC or Naglu-IGFII, or the native
form of one of various lysosomal storage disease enzymes (ARSA,
I2S, FINS, GAA, GCB). Overexpression of GNTP and of each
co-expression product was confirmed by qPCR, ELISA, lysosomal
enzyme activity assay and/or Western blot analysis.
[0179] The GNPT enzyme exists as a hexameric protein complex
containing two copies each of three distinct subunits (.alpha.,
.beta., .gamma.). One construct was generated containing two
expression cassettes, one encoding the alpha/beta subunits and a
second one encoding the gamma subunit.
GNTP Alpha/Beta
[0180] To facilitate expression of the alpha and beta subunits of
GNPT a nucleic acid construct was created using the nucleic acid
sequence of SEQ ID NO:27.
TABLE-US-00012 (SEQ ID NO: 27)
ATGCTGTTCAAGCTCCTGCAGAGACAGACCTATACCTGCCTGTCCCACAGGTATGGGCTCTACG
TGTGCTTCTTGGGCGTCGTTGTCACCATCGTCTCCGCCTTCCAGTTCGGAGAGGTGGTTCTGGA
ATGGAGCCGAGATCAATACCATGTTTTGTTTGATTCCTATAGAGACAATATTGCTGGAAAGTCC
TTTCAGAATCGGCTTTGTCTGCCCATGCCGATTGACGTTGTTTACACCTGGGTGAATGGCACAG
ATCTTGAACTACTGAAGGAACTACAGCAGGTCAGAGAACAGATGGAGGAGGAGCAGAAAGCAAT
GAGAGAAATCCTTGGGAAAAACACAACGGAACCTACTAAGAAGAGTGAGAAGCAGTTAGAGTGT
TTGCTAACACACTGCATTAAGGTGCCAATGCTTGTCCTGGACCCAGCCCTGCCAGCCAACATCA
CCCTGAAGGACCTGCCATCTCTTTATCCTTCTTTTCATTCTGCCAGTGACATTTTCAATGITGC
AAAACCAAAAAACCCTTCTACCAATGTCTCAGTTGTTGTTTTTGACAGTACTAAGGATGTTGAA
GATGCCCACTCTGGACTGCTTAAAGGAAATAGCAGACAGACAGTATGGAGGGGCTACTTGACAA
CAGATAAAGAAGTCCCTGGATTAGIGCTAATGCAAGATTTGGCTTTCCTGAGTGGATTTCCACC
AACATTCAAGGAAACAAATCAACTAAAAACAAAATTGCCAGAAAATCTTTCCTCTAAAGTCAAA
CTGTTGCAGTTGTATTCAGAGGCCAGTGTAGCGCTTCTAAAACTGAATAACCCCAAGGATTTTC
AAGAATTGAATAAGCAAACTAAGAAGAACATGACCATTGATGGAAAAGAACTGACCATAAGTCC
TGCATATTTATTATGGGATCTGAGCGCCATCAGCCAGTCTAAGCAGGATGAAGACATCTCTGCC
AGTCGTTTTGAAGATAACGAAGAACTGAGGTACTCATTGCGATCTATCGAGAGGCATGCACCAT
GGGTTCGGAATATTTTCATTGTCACCAACGGGCAGATTCCATCCTGGCTGAACCTTGACAATCC
TCGAGTGACAATAGTAACACACCAGGATGTTTTTCGAAATTTGAGCCACTTGCCTACCTTTAGT
TCACCTGCTATTGAAAGTCACATTCATCGCATCGAAGGGCTGTCCCAGAAGTTTATTTACCTAA
ATGATGATGTCATGTTTGGGAAGGATGTCTGGCCAGATGATTTTTACAGTCACTCCAAAGGCCA
GAAGGTTTATTTGACATGGCCTGTGCCAAACTGTGCCGAGGGCTGCCCAGGTTCCTGGATTAAG
GATGGCTATTGTGACAAGGCTTGTAATAATTCAGCCTGCGATTGGGATGGTGGGGATTGCTCTG
GAAACAGTGGAGGGAGTCGCTATATTGCAGGAGGTGGAGGTACTGGGAGTATTGGAGTTGGACA
GCCCTGGCAGTTTGGTGGAGGAATAAACAGTGTCTCTTACTGTAATCAGGGATGTGCGAATTCC
TGGCTCGCTGATAAGTTCTGTGACCAAGCATGCAATGTCTTGTCCTGTGGGTTTGATGCTGGCG
ACTGTGGGCAAGATCATTTTCATGAATTGTATAAAGTGATCCTTCTCCCAAACCAGACTCACTA
TATTATTCCAAAAGGTGAATGCCTGCCTTATTTCAGCTTTGCAGAAGTAGCCAAAAGAGGAGTT
GAAGGTGCCTATAGTGACAATCCAATAATTCGACATGCTTCTATTGCCAACAAGTGGAAAACCA
TCCACCTCATAATGCACAGTGGAATGAATGCCACCACAATACATTTTAATCTCACGTTTCAAAA
TACAAACGATGAAGAGTTCAAAATGCAGATAACAGTGGAGGTGGACACAAGGGAGGGACCAAAA
CTGAATTCTACAGCCCAGAAGGGTTACGAAAATTTAGTTAGTCCCATAACACTTCTTCCAGAGG
CGGAAATCCTTTTTGAGGATATTCCCAAAGAAAAACGCTTCCCGAAGTTTAAGAGACATGATGT
TAACTCAACAAGGAGAGCCCAGGAAGAGGTGAAAATTCCCCTGGTAAATATTTCACTCCTTCCA
AAAGACGCCCAGTTGAGTCTCAATACCTTGGATTTGCAACtGGAACAtGGAGACATCACTtTGA
AAGGATACAATTTGTCCAAGTCAGCCTTGCTGAGATCATTTCTGATGAACTCACAGCATGCTAA
AATAAAAAATCAAGCTATAATAACAGATGAAACAAATGACAGTTTGGTGGCTCCACAGGAAAAA
CAGGTTCATAAAAGCATCTTGCCAAACAGCTTAGGAGTGTCTGAAAGATTGCAGAGGTTGACTT
TTCCTGCAGTGAGTGTAAAAGTGAATGGTCATGACCAGGGTCAGAATCCACCCCTGGACTTGGA
GACCACAGCAAGATTTAGAGTGGAAACTCACACCCAAAAAACCATAGGCGGAAATGTGACAAAA
GAAAAGCCCCCATCTCTGATTGTTCCACTGGAAAGCCAGATGACAAAAGAAAAGAAAATCACAG
GGAAAGAAAAAGAGAACAGTAGAATGGAGGAAAATGCTGAAAATCACATAGGCGTTACTGAAGT
GTTACTTGGAAGAAAGCTGCAGCATTACACAGATAGTTACTTGGGCTTTTTGCCATGGGAGAAA
AAAAAGTATTTCCAAGATCTTCTCGACGAAGAAGAGTCATTGAAGACACAATTGGCATACTTCA
CTGATAGCAAAAATACTGGGAGGCAACTAAAAGATACATTTGCAGATTCCCTCAGATATGTAAA
TAAAATTCTAAATAGCAAGTTTGGATTCACATCGCGGAAAGTCCCTGCTCACATGCCTCACATG
ATTGACCGGATTGTTATGCAAGAACTGCAAGATATGTTCCCTGAAGAATTTGACAAGACGTCAT
TTCACAAAGTGCGCCATTCTGAGGATATGCAGTTTGCCTTCTCTTATTTTTATTATCTCATGAG
TGCAGTGCAGCCACTGAATATATCTCAAGTCTTTGATGAAGTTGATACAGATCAATCTGGTGTC
TTGTCTGACAGAGAAATCCGAACACTGGCTACCAGAATTCACGAACTGCCGTTAAGTTTGCAGG
ATTTGACAGGTCTGGAACACATGCTAATAAATTGCTCAAAAATGCTTCCTGCTGATATCACGCA
GCTAAATAATATTCCACCAACTCAGGAATCCTACTATGATCCCAACCTGCCACCGGTCACTAAA
AGTCTAGTAACAAACTGTAAACCAGTAACTGACAAAATCCACAAAGCATATAAGGACAAAAACA
AATATAGGTTTGAAATCATGGGAGAAGAAGAAATCGCTTTTAAAATGATTCGTACCAACGTTTC
TCATGTGGTTGGCCAGTTGGATGACATAAGAAAAAACCCTAGGAAGTTTGTTTGCCTGAATGAC
AACATTGACCACAATCATAAAGATGCTCAGACAGTGAAGGCTGTTCTCAGGGACTTCTATGAAT
CCATGTTCCCCATACCTTCCCAATTTGAACTGCCAAGAGAGTATCGAAACCGTTTCCTTCATAT
GCATGAGCTGCAGGAATGGAGGGCTTATCGAGACAAATTGAAGTTTTGGACCCATTGTGTACTA
GCAACATTGATTATGTTTACTATATTCTCATTTTTTGCTGAGCAGTTAATTGCACTTAAGCGGA
AGATATTTCCCAGAAGGAGGATACACAAAGAAGCTAGTCCCAATCGAATCAGAGTATAG
[0181] The resulting amino acid sequence (SEQ ID NO:28) included
the alpha and beta subunits of GNPT along with the required
cleavage recognition site.
TABLE-US-00013 (SEQ ID NO: 28)
MLFKLLQRQTYTCLSHRYGLYVCFLGVVVTIVSAFQFGEVVLEWSRDQYHV
LFDSYRDNIAGKSFQNRLCLPMPIDVVYTWVNGTDLELLKELQQVREQMEE
EQKAMREILGKNTTEPTKKSEKQLECLLTHCIKVPMLVLDPALPANITLKD
LPSLYPSFHSASDIFNVAKPKNPSTNVSVVVFDSTKDVEDAHSGLLKGNSR
QTVWRGYLTTDKEVPGLVLMQDLAFLSGFPPTFKETNQLKTKLPENLSSKV
KLLQLYSEASVALLKLNNPKDFQELNKQTKKNMTIDGKELTISPAYLLWDL
SAISQSKQDEDISASRFEDNEELRYSLRSIERHAPWVRNIFIVTNGQIPSW
LNLDNPRVTIVTHQDVFRNLSHLPTFSSPATESHIHRIEGLSQKFIYLNDD
VMFGKDVWPDDFYSHSKGQKVYLTWPVPNCAEGCPGSWIKDGYCDKACNNS
ACDWDGGDCSGNSGGSRYIAGGGGTGSIGVGQPWQFGGGINSVSYCNQGCA
NSWLADKFCDQACNVLSCGFDAGDCGQDHFHELYKVILLPNQTHYIIPKGE
CLPYFSFAEVAKRGVEGAYSDNPIIRHASIANKWKTIHLIMHSGMNATTIH
FNLTFQNTNDEEFKMQITVEVDTREGPKLNSTAQKGYENLVSPITLLPEAE
ILFEDIPKEKRFPKFKRHDVNSTRRAQEEVKIPLVNISLLPKDAQLSLNTL
DLQLEHGDITLKGYNLSKSALLRSFLMNSQHAKIKNQAIITDETNDSLVAP
QEKQVHKSILPNSLGVSERLQRLTFPAVSVKVNGHDQGQNPPLDLETTARF
RVETHTQKTIGGNVTKEKPPSLIVPLESQMTKEKKITGKEKENSRMEENAE
NHIGVTEVLLGRKLQHYTDSYLGFLPWEKKKYFQDLLDEEESLKTQLAYFT
DSKNTGRQLKDTFADSLRYVNKILNSKFGFTSRKVPAHMPHMIDRIVMQEL
QDMFPEEFDKTSFHKVRHSEDMQFAFSYFYYLMSAVQPLNISQVFDEVDTD
QSGVLSDREIRTLATRIHELPLSLQDLTGLEHMLINCSKMLPADITQLNNI
PPTQESYYDPNLPPVTKSLVTNCKPVTDKIHKAYKDKNKYRFEIMGEEEIA
FKMIRTNVSHVVGQLDDIRKNPRKFVCLNDNIDHNHKDAQTVKAVLRDFYE
IMFTIFSFFAEQLIALKRKIFPRRRIHKEASPNRIRVSMFPIPSQFELPRE
YRNRFLHMHELQEWRAYRDKLKFWTHCVLATL *Alpha/Beta cleavage recognition
site-in bold and underlined
GNPT Gamma
[0182] To facilitate expression of the gamma subunit of GNPT a
nucleic acid construct was created using the nucleic acid sequence
of SEQ ID NO:29.
TABLE-US-00014 (SEQ ID NO: 29)
ATGGCGGCGGGGCTGGCGCGGCTCCTGTTGCTCCTCGGGCTCTCGGCCGG
CGGGCCCGCGCCGGCAGGTGCAGCGAAGATGAAGGTGGTGGAGGAGCCCA
ACGCGTTTGGGGTGAACAACCCGTTCTTGCCTCAGGCCAGTCGCCTCCAG
GCCAAGAGGGATCCTTCACCCGTGTCTGGACCCGTGCATCTCTTCCGACT
CTCGGGCAAGTGCTTCAGCCTGGTGGAGTCCACGTACAAGTATGAGTTCT
GCCCGTTCCACAACGTGACCCAGCACGAGCAGACCTTCCGCTGGAACGCC
TACAGTGGGATCCTCGGCATCTGGCACGAGTGGGAGATCGCCAACAACAC
CTTCACGGGCATGTGGATGAGGGACGGTGACGCCTGCCGTTCCCGGAGCC
GGCAGAGCAAGGTGGAGCTGGCGTGTGGAAAAAGCAACCGGCTGGCCCAT
GTGTCCGAGCCGAGCACCTGCGTCTACGCGCTGACGTTCGAGACCCCCCT
CGTCTGCCACCCCCACGCCTTGCTAGTGTACCCAACCCTGCCAGAGGCCC
TGCAGCGGCAGTGGGACCAGGTAGAGCAGGACCTGGCCGATGAGCTGATC
ACCCCCCAGGGCCATGAGAAGTTGCTGAGGACACTTTTTGAGGATGCTGG
CTACTTAAAGACCCCAGAAGAAAATGAACCCACCCAGCTGGAGGGAGGTC
CTGACAGCTTGGGGTTTGAGACCCTGGAAAACTGCAGGAAGGCTCATAAA
GAACTCTCAAAGGAGATCAAAAGGCTGAAAGGTTTGCTCACCCAGCACGG
CATCCCCTACACGAGGCCCACAGAAACTTCCAACTTGGAGCACTTGGGCC
ACGAGACGCCCAGAGCCAAGTCTCCAGAGCAGCTGCGGGGTGACCCAGGA
CTGCGTGGGAGTTTGTGA
[0183] The resulting amino acid sequence (SEQ ID NO:30) included
the gamma subunit of GNPT.
TABLE-US-00015 (SEQ ID NO: 30)
MAAGLARLLLLLGLSAGGPAPAGAAKMKVVEEPNAFGVNNPFLPQASRLQ
AKRDPSPVSGPVHLFRLSGKCFSLVESTYKYEFCPFHNVTQHEQTFRWNA
YSGILGIWHEWEIANNTFTGMWMRDGDACRSRSRQSKVELACGKSNRLAH
VSEPSTCVYALTFETPLVCHPHALLVYPTLPEALQRQWDQVEQDLADELI
TPQGHEKLLRTLFEDAGYLKTPEENEPTQLEGGPDSLGFETLENCRKAHK
ELSKEIKRLKGLLTQHGIPYTRPTETSNLEHLGHETPRAKSPEQLRGDPG LRGSL
Example 4: Evaluation of Lysosomal Enzymes Produced in a GNPT Cell
Line
[0184] Given the critical role GNPT plays in the post-translation
modification of N-linked glycans, specifically the
M6P-phosphorylation of soluble lysosomal enzymes, experiments were
performed to evaluate if overexpression of the GNPT enzyme could
result in increased M6P-phosphorylation levels on lysosomal
emzymes. Briefly, recombinant HT-1080 cell lines, overexpressing a
single lysosomal enzyme (GAA, LAL, ARSA, I2S, HNS or GCB) or
co-overexpressing a lysosomal enzyme and GNPT (GAA-GNPT, ARSA-GNPT,
I2S-GNPT, HNS-GNPT or GCB-GNPT) were generated using standard
methodologies. Similar cell lines were generated overexpressing
only Naglu-LIF (Naglu-LIF) or Naglu-LIF and GNPT (Naglu-LIF-GNPT).
Each of the cell lines was grown to confluence and conditioned cell
medium was collected and concentrated as described. For control
purposes, fractions of the concentrated sample were then treated
with Endoglycosidase H, and treated and untreated samples were
separated by SDS-PAGE (proteins were quantified by ELISA or an
enzymatic activity assay and 250 ng of each lysosomal enzyme were
loaded) and analyzed by Western blotting using an anti-M6P
antibody. As shown in FIG. 4, co-expression of each lysosomal
enzyme with GNPT resulted in a dramatic increase in M6P
phosphorylation signal, compared to expression of lysosomal enzymes
alone. Surprisingly, while Naglu-LIF protein overexpressed alone is
not M6P-phosphorylated in any detectable manner, co-expression of
Naglu-LIF with GNPT resulted in increased M6P-phosporylation (FIG.
4).
[0185] These data indicate that it is possible to use fusion
proteins and/or co-expression of GNPT to increase
M6P-phosporylation of lysosomal enzymes (e.g., Naglu).
Example 5: Increased M6P Receptor Binding and Cellular Uptake of
Recombinant I2S Produced Using a GNPT Cell Line
[0186] To further evaluate if expression of recombinant lysosomal
proteins in a GNPT overexpressing cell line results in increased
M6P-phosphorylation, additional studies were performed regarding
the lysosomal enzyme I2S. Recombinant HT-1080 cell lines
overexpressing I2S alone or co-overexpressing I2S and GNPT were
generated using standard methodologies. Each of the cell lines was
grown to confluence and conditioned cell medium was collected and
concentrated as described. Each protein was tested for any change
in receptor affinity using the CI-M6PR binding assay described in
Example 4. This analysis revealed that I2S from cells
overexpressing this protein alone bound to CI-M6PR more weakly than
I2S from cells also overexpressing GNPT (FIG. 5).
[0187] In vitro M6P receptor mediated targeting and cellular uptake
of recombinantly produced I2S proteins was also evaluated. Briefly,
cells overexpressing CI-M6PR were grown to confluence and treated
with a solution of recombinant I2S produced in HT-1080 cells only
overexpressing this protein, or recombinant I2S produced in HT-1080
cells also overexpressing GNPT. After a specified period of time,
the supernatant was removed and the cells were washed repeatedly.
Following lysis, each cell sample was assayed for intracellular I2S
enzyme activity. The data demonstrate that I2S produced in GNPT
overexpressing cell line was internalized more quickly and
displayed a higher affinity (K.sub.D=1.65 nM) for CI-M6PR than I2S
produced in cells overexpressing I2S alone (K.sub.D=27.89 nM) (FIG.
6). Taken together, this increase in cellular uptake is consistent
with both the observed increased M6P-phosphorylation (FIG. 4) and
tighter receptor binding (FIG. 5), suggesting that enhanced
CI-M6PR-mediated cellular uptake by the cell is the result of
increased M6P-phosphorylation and CI-M6PR binding affinity.
Example 6: Increased M6P Receptor Binding of Native and
Naglu-Fusion Proteins Produced Using a GNPT Cell Line
[0188] As described above, the level of M6P-phosphorylation of
recombinantly produced therapeutic glycoproteins could be increased
by using GNPT overexpressing cell lines (FIG. 4). In particular, an
increase in M6P-phosphorylation was observed for the therapeutic
fusion glycoprotein Naglu-LIF (FIG. 4). Surprisingly, this increase
in M6P-phosphorylation of the Naglu protein when fused to LIF,
produced a level of M6P-phosphorylation which was not observed for
the native Naglu protein, or with respect to non-fusion rhNaglu
generated in cells not co-overexpressing GNPT. To further evaluate
this phenomenon, additional studies were conducted to evaluate
M6P-phosphorylation levels for rhNaglu and various fusion proteins
recombinantly produced using GNPT overexpressing cell lines.
Briefly, clonal HT-1080 cell lines expressing a single recombinant
protein (Naglu-IGFII, rhNaglu, Naglu-GREG or Naglu-SapDC) or
co-expressing a lysosomal enzyme and GNPT (GNPT/Naglu-SapDC,
GNPT/Naglu-CREG, GNPT/rhNaglu) were generated using standard
procedures. Each cell line was grown to confluence, conditioned
medium was collected and the respective lysosomal protein and/or
fusion protein was purified to greater than 90% purity.
[0189] For the initial study, an in vitro assay was used to
evaluate CI-M6PR binding. This analysis revealed that
overexpression of rhNaglu alone resulted in an extremely low
binding affinity for CI-M6PR, with a K.sub.d below the assay's
detection threshold (FIG. 7). This result is in accordance with our
previous findings, which demonstrated little to no
M6P-phosphorylation of the rhNaglu enzyme when generated by
overexpression in HT-1080 cells. By contrast, rhNaglu produced
using a GNPT overexpressing cell line demonstrated, for the first
time, a weak binding affinity for CI-M6PR with a K.sub.d value of
approximately 65 nM (FIG. 7). While this binding affinity is much
lower than the binding affinity of Naglu-IGFII (used as a positive
control) (FIG. 7), binding of the latter is mediated through a
protein-protein interaction between the IGF-II domain and CI-M6PR.
Thus, our data indicate that the low M6P-phosphorylation levels of
certain therapeutic glycoproteins, such as Naglu, can be enhanced
by the manufacture of these proteins in GNPT overexpressing cell
lines. Furthermore, our data suggest that therapeutic glycoproteins
which have reduced M6P-phosphorylation levels can be targeted for
CI-M6PR-mediated lysosomal delivery by creating fusion proteins
where the fused region is capable of N-linked glycosylationand and
M6P-phosphorylation.
[0190] In the second phase of our study, CI-M6PR binding of the
remaining Naglu fusion proteins was evaluated, using the same in
vitro binding assay as described above. Consistent with our
previous findings, the data indicate that both Naglu-CREG and
Naglu-SapDC fusion proteins were able to bind CI-M6PR. This
demonstrated that therapeutic glycoproteins can be engineered as
fusion proteins with lysosomal targeting moieties to facilitate
binding to CI-M6PR. Naglu-SapDC and Naglu-CREG fusion proteins had
a binding affinity for CI-M6PR of 2.90 nM and 2.50 nM, respectively
(FIG. 8), which was greater than the binding affinity of rhNaglu
(none detected) and rhNaglu produced in cells overexpressing GNPT
(65 nM) (FIG. 7). The production of fusion proteins in GNPT
overexpressing cells lead to a further increase of their
M6P-phosphorylation, as evident from the dramatic increase of their
binding affinity for CI-M6PR, (0.23 nM for GNPT/Naglu-SapDC and
0.41 nM for GNPT/Naglu-CREG) that was similar to the binding
affinity observed regarding Naglu-IGF-II (0.24 nM) (FIG. 8).
Example 7: Increased M6P-Modification of Naglu-Fusion Proteins
Produced Using a GNPT Cell Line
[0191] To further confirm the increases of M6P on Naglu-LIF and
Naglu-SapDC after coexpression with GNPT, a monosaccharide M6P
quantitation assay was performed. This method used acid hydrolysis
to break down all the glycans on the analyzed glycoproteins; the
released monosaccharide, such as M6P, was then detected and
quantified by PAD-HPLC (Pulsed Amperometric Detection High
Performance Liquid Chromatography). Briefly, M6P disodium salt was
used to identify the peak of the M6P liberated from the analyzed
samples. The area under the curve for the M6P standard was used to
generate a standard curve, allowing the quantification of the
liberated M6P from the samples. The M6P content of Naglu-SapDC
increased from 0.75 to 1.84 mol/mol (M6P/Protein) after
co-expression with GNPT, and the M6P content of Naglu-LIF increased
to 1.07 after co-expression with GNPT (FIG. 9). Both, the GNPT
co-expressed Naglu-SapDC and the GNPT co-expressed Naglu-LIF had
M6P a content that was very similar to the M6P a content of the
control protein I2S (FIG. 9). This result indicates that GNPT
co-expression increased the M6P content of Naglu fusion proteins to
a level that is therapeutically meaningful. The method used to
quantify M6P on Naglu-LIF and Naglu-SapDC is set forth in more
detail below.
[0192] The procedure for mannose-6-phosphate quantitation by
PAD-HPLC was adapted from Zhou et al. 2002, Anal Biochem. 15;
306(2):163-170). Protein samples were mixed with 13 M
trifluoroacctic acid (TFA) to a final concentration of 6.75 M TFA.
The mixture was then transferred to a 0.5 ml glass vial with a
screw cap. Standards were prepared using mannose-6-phosphate
disodium purchased from Sigma-Aldrich Corporation (St. Louis. Mo.).
The standards were prepared in H.sub.2O/TFA in concentrations
ranging from 50 to 1000 pM, and were then transferred to glass
vials with screw caps. All samples were heated at 100.degree. C.
for 1.5 hours in a heating block. After heating, the glass vials
were placed on ice for 5 minutes, and the samples were transferred
to Eppendorf tubes. The samples were centrifuged at 14,000 rpm for
2 minutes and then dried in a SpeedVac.RTM. concentrator for
.about.1.5 hours. 200 .mu.l of water were then added to each sample
and the samples were again dried as before. Then, 100 .mu.l of
water were added to each sample and the samples were filtered
through a 0.45 .mu.m Durapore.RTM. centrifugal filter unit
(Millipore, Billerica, Mass.) at 14,000 rpm for 5 minutes. 20
microliters of each sample and of the standards were injected in a
chromatography system with a borate trap column in line with a
CarboPac PA 10 analytical column (Dionex ICS-3000 Ion
Chromatography System (Thermo Fisher Scientific, Inc., Sunnyvale,
Calif.)). An isocratic gradient was run with at 80% Eluent A (100
mM NaOH) and 20% Eluent B (1 M NaAc in 100 mM NaOH) for 15 minutes
with a step up to 70% Eluent B for 5 minutes, then back to 20%
Eluent B.
Example 8: Increased Cellular Uptake of Naglu-Fusion Proteins
Produced Using a GNPT Cell Line
[0193] In vitro CI-M6PR mediated targeting and cellular uptake of
Naglu-SapDC and Naglu-LIF produced in GNPT overexpression cell
lines was also evaluated (FIGS. 10-11). Cellular uptake of
Naglu-IGFII was determined for comparison. All samples were run in
parallel, under identical conditions, to make the resulting data
comparable.
[0194] Briefly, cells overexpressing CI-M6PR were grown to
confluence and treated with a solution of either recombinant
Naglu-SapDC and Naglu-Lif produced in HT-1080 cells alone, or
recombinant Naglu-SapDC and Naglu-Lif produced in HT-1080 cells
also overexpressing GNPT. After a specified period of time, the
supernatant was removed and the cells were washed repeatedly.
Following lysis, each cell sample was assayed for intracellular
Naglu enzyme activity, and the total protein was measured by a BCA
protein assay. The x-axis of the graphs in FIGS. 10-11 represented
the amount of Naglu-SapDC or Naglu-LIF applied to the cells. The
y-axis represented the intracellular Naglu activity, or the
equivalent amount of Naglu enzyme in nanogram (ng) (see FIG. 10 top
panel) normalized by the total protein amount.
[0195] The data show that Naglu-SapDC produced in GNPT
overexpressing cells has an affinity for CI-M6PR that is 14 times
higher than the affinity of Naglu-SapDC produced in cells
expressing Naglu-SapDC alone (K.sub.D=14.88 vs. K.sub.D=208.78 nM,
respectively) (FIG. 10). The affinity of Naglu-IGFII for CI-M6PR is
only 3.6 times higher than the affinity of Naglu-SapDC produced in
GNPT overexpressing cells (K.sub.D of 4.07 vs. 14.88 nM,
respectively) (FIG. 10). No cellular uptake of Naglu-SapDC was
observed when exogenous M6P was added to Naglu-SapDC samples,
further confirming that cellular uptake of Naglu-SapDC is mediated
by CI-M6PR via the M6P binding domain (FIG. 10).
[0196] Similarly, our data show that Naglu-LIF produced in GNPT
overexpressing cells has an affinity for CI-M6PR that is higher
than the affinity of Naglu-LIF produced in cells expressing
Naglu-LIF alone (FIG. 11). The affinity of Naglu-IGFII for CI-M6PR
is similar to the affinity of Naglu-LIF produced in GNPT
overexpressing cells (K.sub.D of 18 vs. 15 nM, respectively), while
the affinity of Naglu-LIF expressed in HT1080 cells alone is
significantly lower than the affinity of Naglu-IGFII (K.sub.D of
178 nM vs. 2 nM, respectively). No cellular uptake of Naglu-LIF was
observed when exogenous M6P was added to Naglu-LIF samples, further
confirming that cellular uptake of Naglu-LIF is mediated by CI-M6PR
via the M6P binding domain.
Example 9: In Vivo Delivery of Naglu-SapDC Fusion Protein
Intracellular Accumulation of Naglu-SapDC
[0197] For in vivo experiments described herein, Naglu-SapDC
prepared in GNPT overexpressing cells was utilized.
[0198] To further evaluate intracellular accumulation of the
Naglu-SapDC fusion protein, an in vivo study was performed using
Naglu KO mice subjected to intrathecal administration of vehicle
control (PBS) or Naglu-SapDC; in accordance with the experimental
conditions described in Table 7.
TABLE-US-00016 TABLE 7 Experimental Design to Assay Efficacy of
Naglu-SapDC Dose (mg/kg Group No. Treatment brain) Route Frequency
Sacrifice A 3 Vehicle N/A Intra- 2.times. Weekly 24 hrs post B 6
Naglu- 520 thecal final dose SapDC C 3 Vehicle N/A Intra- 3.times.
Weekly 24 hrs post D 6 Naglu- 520 thecal final dose SapDC
[0199] Following each respective treatment, Naglu KO mice were
sacrificed 24 hours after their final injection and assayed for
Naglu enzyme activity in various tissues. Total Naglu activity was
assayed using a well-established enzyme activity assay. Briefly,
tissue homogenate was incubated in the presence of the Naglu
specific substrate
methylumbelliferyl-N-acetyl-.alpha.-D-glucosainide for a specified
period of time, after which accumulation of cleavage product was
measured by examining fluorescence intensity at 360/460
(excitation/emission) using a fluorescent plate reader. The data
demonstrate that treatment with Naglu-SapDC resulted in a dramatic
increase in Naglu activity in both liver (FIG. 12A) and brain (FIG.
12B) tissue, when compared to vehicle control. This increase in
enzyme activity was observed over the duration of the 3 week
treatment period.
Biodistribution of Naglu-SapDC Fusion Protein
[0200] Tissue samples were also collected from the treated mice,
fixed in 10% NBF and processed for paraffin embedding. For each
tissue assayed, 5 .mu.m paraffin sections were subjected to
immunostaining using an antibody specific for human Naglu. The data
clearly demonstrate lysosomal delivery of Naglu-SapDC to neurons of
the cerebral cortex (FIG. 13 A, B). Most strikingly, the data also
demonstrate lysosomal delivery of Naglu-SapDC in hepatic cells of
the liver (FIG. 13E). This is surprising insofar as Naglu-SapDC was
administered via intrathecal delivery, indicating Naglu-SapDC can
reach targets and enter cells far away from its site of
administration.
Efficacy of Naglu-SapDC Fusion Protein
[0201] The in vivo activity of Naglu enzyme in Naglu knockout mice
treated by intrathecal administration of Naglu-SapDC was evaluated
by examining the intracellular accumulation of different
glycoproteins and their respective cleavage products. One such
assay was designed to evaluate the amount of glucosoaminoglycan
(GAG) in liver (FIG. 14) and brain tissue (FIG. 15).
[0202] Briefly, liver tissues were homogenized and then assayed for
GAG according to Jong et al. (Clin Chem 38(6):803-807, 1992). In
the case of brain tissue, the homnogenized samples were further
digested using pronase and benzonase to break down protein and
nucleic acid. GAG was then extracted by passing the sample through
a DEAE column. The buffer of the eluate was then exchanged by means
of a desalting column, and the total amount of GAG was determined
using dimethylmethylene blue staining (Lawrence et al., Nat Chem
Biol. 2012 Jan. 8; 8(2):197-204). As shown in FIGS. 14 and 15,
intreathecal delivery of Naglu-SapDC resulted in a significant
reduction in total GAG concentration within the liver and brain of
Naglu knockout mice, when compared to the vehicle control. The
reduction of the total GAG level within the brain was maintained
over the entire 3 week treatment period (FIG. 15).
[0203] A second assay was performed to evaluate the amount of
heparan sulfate in brain tissue following intrathecal delivery of
Naglu-SapDC fusion protein. Heparin sulfate is a specific type of
GAG which has been shown to accumulate in the brain of San B
patients. To measure heparan sulfate in brain tissue, a highly
sensitive LC/MS method was used, as described by Lawrence et al.
(Nat Chem Biol. 2012 Jan. 8; 8(2):197-204). Briefly, total GAG from
brain tissue was extracted using a DEAE column and a desalting
column, ad was then dried and weighted. The weighted GAG was then
treated with heparin lyases that specifically release unique mono,
di and tri saccharides from heparan sulfate. Released disaccharides
were then analyzed, identified and quantified by LC/MS and
comparison to commercially available disaccharide standards. As in
the case of the GAG study described above, the data illustrate
that, when compared to vehicle control, treatment with Naglu-SapDC
leads to a strong reduction in the total amount of heparan sulfate
accumulation in the brain over the duration of the three week
treatment period (FIG. 16).
[0204] Lastly, intracellular delivery of Naglu-SapDC was evaluated
by assaying the presence of different biomarkers for San B, using
the same LC/MS methodology used for the determination of heparin
sulfate tissue content, described above. Deficiency of Naglu causes
the accumulation of aberrant heparan sulfate cleavage products in
tissues (presented in FIGS. 17-19) which are natural substrates of
Naglu. The data show that intrathecal delivery of Naglu-SapDC to
Naglu knockout mice results in a dramatic decrease in the brain of
the accumulation of all three aberrant heparan sulfate cleavage
products analyzed (FIGS. 17-19). Taken together, all these three
experimental approaches demonstrate an overall reduction in GAG
levels and cleavage products, suggesting Naglu-SapDC is efficiently
internalized into the lysosome of the cell, where it maintains
enzyme activity.
[0205] To further elucidate and confirm the intracellular delivery
of Naglu-SapDC to lysosomes, the inventors examined the
distribution of the lysosomal marker Lamp-1 in each experimental
treatment group. Vehicle control mice, when compared to Naglu-SapDC
treated mice, showed an increased immunohistochemical staining for
Lamp-1 over the entire 3 week period in the apparently enlarged
lysosomes of neuronal and glia cells of the cerebral cortex (FIG.
20), cerebellum (FIG. 21), thalamus (FIG. 22), striatum (FIG. 23),
white matter (FIG. 24) and liver (FIG. 25).
[0206] In addition to Lamp-1, two additional cellular biomarkers
were used to further evaluate lysosomal targeting and intracellular
delivery of Naglu-SapDC. For this study, the ionized
calcium-binding adapter molecule 1 (Iba-1) and glial fibrillary
acidic protein (GFAP) were chosen as markers. Each protein is a
well-established indicator of cellular inflammatory response and
can be used to gauge the level and intensity of inflammation in
specific cell types. In particular, GFAP staining has been used
extensively to demonstrate the size and number of processes in
astrocytes, while Iba-1 staining is predominantly used for
evaluating microglial cells. As demonstrated in FIG. 26, mice
subjected to intrathecal delivery of Naglu-SapDC had a reduced
amount of GFAP staining in each neuronal tissue examined,
indicating reduced lysosome size and a low number of processes. A
similar trend was also observed for Iba-1 staining, for each
neuronal sample analyzed (FIG. 27).
[0207] These data provide strong in vivo support that Naglu-SapDC
is able to bind the CI-M6P receptor, which facilitates lysosomal
targeting and entry in various tissues types. Surprisingly,
intrathecal delivery of Naglu-SapDC resulted in extensive
bioavailability throughout the body, demonstrating localization in
neuronal tissue as well as various organ systems of the body, such
as the liver. The data further suggest, that upon entry into the
cell, the Naglu-SapDC fusion protein maintains enzyme activity and
proper function, as demonstrated by the overall reduction in excess
accumulation of glucosaminoglycans, heparin sulfate and San B
specific biomarkers in Naglu knockout mice, which is a hallmark of
Sanfilippo type B disease. Given the fusion protein's excellent
efficacy, biodistribution and ability to target lysosomal delivery
using the CI-M6P receptor meditated pathway, the data strongly
suggest that such an approach could be extremely effective for
enzyme replacement therapy. Despite the d little to no
M6P-phosphorylation of rhNaglu, Naglu-SapDC fusion proteins can be
M6P-phosphorylated and thus targeted for CI-M6P receptor mediated
lysosomal delivery. Our data clearly suggest that the described
lysosomal targeting approach is applicable not only to the
treatment of Sanfillipo type B syndrome, but also to the treatment
of any other lysosomal storage diseases in which, for example, the
native protein lacks a proper M6P-phosphorylation site, or is
poorly M6P-phosphorylated due competitive inhibition or steric
hindrance of the GNPT enzymatic pocket.
Example 10: In Vivo Delivery of Naglu-LIF Fusion Protein
Intracellular Accumulation of Naglu-LIF
[0208] For in vivo experiments described herein, Naglu-LIF prepared
in GNPT overexpressing cells was utilized.
[0209] To further evaluate intracellular accumulation of the
Naglu-LIF fusion protein, an in vivo study was performed using
Naglu KO mice subjected to intrathecal administration of vehicle
control (PBS) or Naglu-LIF; in accordance with the experimental
conditions described in Table 8.
TABLE-US-00017 TABLE 8 Experimental Design to Assay Efficacy of
Naglu-LIF Dose (mg/kg Group No. Treatment brain) Route Frequency
Sacrifice A 3 Vehicle N/A Intra- 2.times. Weekly 24 hrs post B 6
Naglu-LIF 520 thecal final dose C 3 Vehicle N/A Intra- 3.times.
Weekly 24 hrs post D 6 Naglu-LIF 520 thecal final dose
[0210] Following each respective treatment, Naglu KO mice were
sacrificed 24 hours after their final injection and assayed for
Naglu enzyme activity in various tissues. Total Naglu activity was
assayed using a well-established enzyme activity assay. Briefly,
total cell lysate was incubated in the presence of the Naglu
specific substrate
methylumbelliferyl-N-acetyl-.alpha.-D-glucosainide for a specified
period of time, after which accumulation of cleavage product was
measured by examining fluorescence intensity at 360/460
(excitation/emission) using a fluorescent plate reader. The data
demonstrate that treatment with Naglu-LIF resulted in a dramatic
increase in Naglu activity in both liver and brain tissue, when
compared to vehicle control. This increase in enzyme activity was
observed over the duration of the 3 week treatment period.
Biodistribution of Naglu-LIF Fusion Protein
[0211] Tissue samples were also collected from the treated mice,
fixed in 10% NBF and processed for paraffin embedding. For each
tissue assayed, 5 .mu.m paraffin sections was subjected to
immunostaining using an antibody specific for human Naglu. The data
clearly demonstrate lysosomal delivery of Naglu-LIF to neurons and
glia cells of the spinal cord, meninges of the cerebellum, meninges
of the cerebral cortex, and neurons and glia cells of limited areas
in the cerebral cortex (FIGS. 13C and D). Most strikingly, the data
also demonstrate lysosomal delivery of Naglu-LIF in hepatic cells
of the liver (FIG. 13F). This is surprising insofar as Naglu-LIF
was administered via intrathecal delivery, indicating Naglu-LIF can
reach targets and enter cells far away from its site of
administration.
Efficacy of Naglu-LIF Fusion Protein
[0212] The in vivo activity of Naglu was evaluated by examining the
intracellular accumulation of GAG and heparin sulfate, as described
above in connection with the analysis of Naglu-SapDC treated
knockout mice. As shown in FIG. 14 and FIG. 15, intreathecal
delivery of Naglu-LIF resulted in a significant reduction in total
GAG concentration within the liver and brain, respectively, of
Naglu knockout mice, when compared to the vehicle control. The
reduction of the total GAG level within the brain was maintained
over the entire 3 week treatment period (FIG. 15).
[0213] The data also illustrate that, when compared to vehicle
control, treatment with Naglu-LIF leads to a strong reduction in
the total amount of heparan sulfate accumulation in the brain over
the duration of the three week treatment period (FIG. 16). Taken
together, all data demonstrate an overall reduction in GAG levels,
suggesting Naglu-LIF is able to be efficiently internalized into
the lysosome of the cell, where it maintains enzyme activity.
[0214] To further elucidate and confirm the intracellular delivery
of Naglu-LIF to lysosomes, the inventors examined the distribution
of the lysosomal marker Lamp-1 in each each experimental treatment
group. Vehicle control mice, when compared to Naglu-LIF treated
mice, showed an increased immunohistochemical staining for Lamp-1
over the entire 3 week period in the apparently enlarged lysosomes
of neuronal and glia cells of the cerebral cortex (FIG. 28)
cerebellum (FIG. 29), thalamus (FIG. 30), striatum (FIG. 31), white
matter (FIG. 32) and liver (FIG. 33).
[0215] In addition to Lamp-1, two additional cellular biomarkers
were used to further evaluate lysosomal targeting and intracellular
delivery of Naglu-LIF. For this study, the ionized calcium-binding
adapter molecule 1 (Iba-1) and glial fibrillary acidic protein
(GFAP) were chosen as markers. Each protein is a well-established
indicator of cellular inflammatory response and can be used to
gauge the level and intensity of inflammation in specific cell
types. In particular, GFAP staining has been used extensively to
demonstrate the size and number of processes in astrocytes, while
Iba-1 staining is predominantly used for evaluating microglial
cells. As demonstrated in FIG. 34, mice subjected to intrathecal
delivery of Naglu-LIF had a reduced amount of GFAP staining for
each neuronal tissue examined, indicating reduced lysosome size and
a low number of processes. A similar trend was also observed for
Iba-1 staining, for each neuronal sample analyzed (FIG. 35).
[0216] These data provide strong in vivo support that Naglu-LIF is
able to bind the CI-M6P receptor, which facilitates lysosomal
targeting and entry in various tissues types. Surprisingly,
intrathecal delivery of Naglu-LIF resulted in extensive
bioavailability throughout the body, demonstrating localization in
neuronal tissue as well as various organ systems of the body, such
as the liver. The data further suggest that upon entry into the
cell the Naglu-LIF fusion protein maintains enzyme activity and
proper function, as demonstrated by the overall reduction in excess
accumulation of glucosaminoglycans and heparan sulfate in Naglu
knockout mice, which is a hallmark of Sanfilippo type B disease.
Given the fusion protein's excellent efficacy, biodistribution and
ability to target lysosomal delivery using the CI-M6P receptor
meditated pathway, the data strongly suggest that such an approach
could be extremely effective for enzyme replacement therapy.
Despite the poor M6P-phosphorylation of native Naglu and little to
no M6P-phosphorylation of rhNaglu, Naglu-LIF fusion proteins can be
M6P-phosphorylated and thus targeted for CI-M6P receptor mediated
lysosomal delivery. Our data clearly suggest that that the
described lysosomal targeting approach is applicable not only to
the treatment of Sanfillipo type B syndrome, but also to the
treatment of any other lysosomal storage diseases in which, for
example, the native protein lacks a proper M6P-phosphorylation
site, or is poorly M6P-phosphorylated due competitive inhibition or
steric hindrance of the GNPT enzymatic pocket.
Sequence CWU 1
1
301720PRTHomo sapiens 1Asp Glu Ala Arg Glu Ala Ala Ala Val Arg Ala
Leu Val Ala Arg Leu1 5 10 15Leu Gly Pro Gly Pro Ala Ala Asp Phe Ser
Val Ser Val Glu Arg Ala 20 25 30Leu Ala Ala Lys Pro Gly Leu Asp Thr
Tyr Ser Leu Gly Gly Gly Gly 35 40 45Ala Ala Arg Val Arg Val Arg Gly
Ser Thr Gly Val Ala Ala Ala Ala 50 55 60Gly Leu His Arg Tyr Leu Arg
Asp Phe Cys Gly Cys His Val Ala Trp65 70 75 80Ser Gly Ser Gln Leu
Arg Leu Pro Arg Pro Leu Pro Ala Val Pro Gly 85 90 95Glu Leu Thr Glu
Ala Thr Pro Asn Arg Tyr Arg Tyr Tyr Gln Asn Val 100 105 110Cys Thr
Gln Ser Tyr Ser Phe Val Trp Trp Asp Trp Ala Arg Trp Glu 115 120
125Arg Glu Ile Asp Trp Met Ala Leu Asn Gly Ile Asn Leu Ala Leu Ala
130 135 140Trp Ser Gly Gln Glu Ala Ile Trp Gln Arg Val Tyr Leu Ala
Leu Gly145 150 155 160Leu Thr Gln Ala Glu Ile Asn Glu Phe Phe Thr
Gly Pro Ala Phe Leu 165 170 175Ala Trp Gly Arg Met Gly Asn Leu His
Thr Trp Asp Gly Pro Leu Pro 180 185 190Pro Ser Trp His Ile Lys Gln
Leu Tyr Leu Gln His Arg Val Leu Asp 195 200 205Gln Met Arg Ser Phe
Gly Met Thr Pro Val Leu Pro Ala Phe Ala Gly 210 215 220His Val Pro
Glu Ala Val Thr Arg Val Phe Pro Gln Val Asn Val Thr225 230 235
240Lys Met Gly Ser Trp Gly His Phe Asn Cys Ser Tyr Ser Cys Ser Phe
245 250 255Leu Leu Ala Pro Glu Asp Pro Ile Phe Pro Ile Ile Gly Ser
Leu Phe 260 265 270Leu Arg Glu Leu Ile Lys Glu Phe Gly Thr Asp His
Ile Tyr Gly Ala 275 280 285Asp Thr Phe Asn Glu Met Gln Pro Pro Ser
Ser Glu Pro Ser Tyr Leu 290 295 300Ala Ala Ala Thr Thr Ala Val Tyr
Glu Ala Met Thr Ala Val Asp Thr305 310 315 320Glu Ala Val Trp Leu
Leu Gln Gly Trp Leu Phe Gln His Gln Pro Gln 325 330 335Phe Trp Gly
Pro Ala Gln Ile Arg Ala Val Leu Gly Ala Val Pro Arg 340 345 350Gly
Arg Leu Leu Val Leu Asp Leu Phe Ala Glu Ser Gln Pro Val Tyr 355 360
365Thr Arg Thr Ala Ser Phe Gln Gly Gln Pro Phe Ile Trp Cys Met Leu
370 375 380His Asn Phe Gly Gly Asn His Gly Leu Phe Gly Ala Leu Glu
Ala Val385 390 395 400Asn Gly Gly Pro Glu Ala Ala Arg Leu Phe Pro
Asn Ser Thr Met Val 405 410 415Gly Thr Gly Met Ala Pro Glu Gly Ile
Ser Gln Asn Glu Val Val Tyr 420 425 430Ser Leu Met Ala Glu Leu Gly
Trp Arg Lys Asp Pro Val Pro Asp Leu 435 440 445Ala Ala Trp Val Thr
Ser Phe Ala Ala Arg Arg Tyr Gly Val Ser His 450 455 460Pro Asp Ala
Gly Ala Ala Trp Arg Leu Leu Leu Arg Ser Val Tyr Asn465 470 475
480Cys Ser Gly Glu Ala Cys Arg Gly His Asn Arg Ser Pro Leu Val Arg
485 490 495Arg Pro Ser Leu Gln Met Asn Thr Ser Ile Trp Tyr Asn Arg
Ser Asp 500 505 510Val Phe Glu Ala Trp Arg Leu Leu Leu Thr Ser Ala
Pro Ser Leu Ala 515 520 525Thr Ser Pro Ala Phe Arg Tyr Asp Leu Leu
Asp Leu Thr Arg Gln Ala 530 535 540Val Gln Glu Leu Val Ser Leu Tyr
Tyr Glu Glu Ala Arg Ser Ala Tyr545 550 555 560Leu Ser Lys Glu Leu
Ala Ser Leu Leu Arg Ala Gly Gly Val Leu Ala 565 570 575Tyr Glu Leu
Leu Pro Ala Leu Asp Glu Val Leu Ala Ser Asp Ser Arg 580 585 590Phe
Leu Leu Gly Ser Trp Leu Glu Gln Ala Arg Ala Ala Ala Val Ser 595 600
605Glu Ala Glu Ala Asp Phe Tyr Glu Gln Asn Ser Arg Tyr Gln Leu Thr
610 615 620Leu Trp Gly Pro Glu Gly Asn Ile Leu Asp Tyr Ala Asn Lys
Gln Leu625 630 635 640Ala Gly Leu Val Ala Asn Tyr Tyr Thr Pro Arg
Trp Arg Leu Phe Leu 645 650 655Glu Ala Leu Val Asp Ser Val Ala Gln
Gly Ile Pro Phe Gln Gln His 660 665 670Gln Phe Asp Lys Asn Val Phe
Gln Leu Glu Gln Ala Phe Val Leu Ser 675 680 685Lys Gln Arg Tyr Pro
Ser Gln Pro Arg Gly Asp Thr Val Asp Leu Ala 690 695 700Lys Lys Ile
Phe Leu Lys Tyr Tyr Pro Arg Trp Val Ala Gly Ser Trp705 710 715
7202743PRTHomo sapiens 2Met Glu Ala Val Ala Val Ala Ala Ala Val Gly
Val Leu Leu Leu Ala1 5 10 15Gly Ala Gly Gly Ala Ala Gly Asp Glu Ala
Arg Glu Ala Ala Ala Val 20 25 30Arg Ala Leu Val Ala Arg Leu Leu Gly
Pro Gly Pro Ala Ala Asp Phe 35 40 45Ser Val Ser Val Glu Arg Ala Leu
Ala Ala Lys Pro Gly Leu Asp Thr 50 55 60Tyr Ser Leu Gly Gly Gly Gly
Ala Ala Arg Val Arg Val Arg Gly Ser65 70 75 80Thr Gly Val Ala Ala
Ala Ala Gly Leu His Arg Tyr Leu Arg Asp Phe 85 90 95Cys Gly Cys His
Val Ala Trp Ser Gly Ser Gln Leu Arg Leu Pro Arg 100 105 110Pro Leu
Pro Ala Val Pro Gly Glu Leu Thr Glu Ala Thr Pro Asn Arg 115 120
125Tyr Arg Tyr Tyr Gln Asn Val Cys Thr Gln Ser Tyr Ser Phe Val Trp
130 135 140Trp Asp Trp Ala Arg Trp Glu Arg Glu Ile Asp Trp Met Ala
Leu Asn145 150 155 160Gly Ile Asn Leu Ala Leu Ala Trp Ser Gly Gln
Glu Ala Ile Trp Gln 165 170 175Arg Val Tyr Leu Ala Leu Gly Leu Thr
Gln Ala Glu Ile Asn Glu Phe 180 185 190Phe Thr Gly Pro Ala Phe Leu
Ala Trp Gly Arg Met Gly Asn Leu His 195 200 205Thr Trp Asp Gly Pro
Leu Pro Pro Ser Trp His Ile Lys Gln Leu Tyr 210 215 220Leu Gln His
Arg Val Leu Asp Gln Met Arg Ser Phe Gly Met Thr Pro225 230 235
240Val Leu Pro Ala Phe Ala Gly His Val Pro Glu Ala Val Thr Arg Val
245 250 255Phe Pro Gln Val Asn Val Thr Lys Met Gly Ser Trp Gly His
Phe Asn 260 265 270Cys Ser Tyr Ser Cys Ser Phe Leu Leu Ala Pro Glu
Asp Pro Ile Phe 275 280 285Pro Ile Ile Gly Ser Leu Phe Leu Arg Glu
Leu Ile Lys Glu Phe Gly 290 295 300Thr Asp His Ile Tyr Gly Ala Asp
Thr Phe Asn Glu Met Gln Pro Pro305 310 315 320Ser Ser Glu Pro Ser
Tyr Leu Ala Ala Ala Thr Thr Ala Val Tyr Glu 325 330 335Ala Met Thr
Ala Val Asp Thr Glu Ala Val Trp Leu Leu Gln Gly Trp 340 345 350Leu
Phe Gln His Gln Pro Gln Phe Trp Gly Pro Ala Gln Ile Arg Ala 355 360
365Val Leu Gly Ala Val Pro Arg Gly Arg Leu Leu Val Leu Asp Leu Phe
370 375 380Ala Glu Ser Gln Pro Val Tyr Thr Arg Thr Ala Ser Phe Gln
Gly Gln385 390 395 400Pro Phe Ile Trp Cys Met Leu His Asn Phe Gly
Gly Asn His Gly Leu 405 410 415Phe Gly Ala Leu Glu Ala Val Asn Gly
Gly Pro Glu Ala Ala Arg Leu 420 425 430Phe Pro Asn Ser Thr Met Val
Gly Thr Gly Met Ala Pro Glu Gly Ile 435 440 445Ser Gln Asn Glu Val
Val Tyr Ser Leu Met Ala Glu Leu Gly Trp Arg 450 455 460Lys Asp Pro
Val Pro Asp Leu Ala Ala Trp Val Thr Ser Phe Ala Ala465 470 475
480Arg Arg Tyr Gly Val Ser His Pro Asp Ala Gly Ala Ala Trp Arg Leu
485 490 495Leu Leu Arg Ser Val Tyr Asn Cys Ser Gly Glu Ala Cys Arg
Gly His 500 505 510Asn Arg Ser Pro Leu Val Arg Arg Pro Ser Leu Gln
Met Asn Thr Ser 515 520 525Ile Trp Tyr Asn Arg Ser Asp Val Phe Glu
Ala Trp Arg Leu Leu Leu 530 535 540Thr Ser Ala Pro Ser Leu Ala Thr
Ser Pro Ala Phe Arg Tyr Asp Leu545 550 555 560Leu Asp Leu Thr Arg
Gln Ala Val Gln Glu Leu Val Ser Leu Tyr Tyr 565 570 575Glu Glu Ala
Arg Ser Ala Tyr Leu Ser Lys Glu Leu Ala Ser Leu Leu 580 585 590Arg
Ala Gly Gly Val Leu Ala Tyr Glu Leu Leu Pro Ala Leu Asp Glu 595 600
605Val Leu Ala Ser Asp Ser Arg Phe Leu Leu Gly Ser Trp Leu Glu Gln
610 615 620Ala Arg Ala Ala Ala Val Ser Glu Ala Glu Ala Asp Phe Tyr
Glu Gln625 630 635 640Asn Ser Arg Tyr Gln Leu Thr Leu Trp Gly Pro
Glu Gly Asn Ile Leu 645 650 655Asp Tyr Ala Asn Lys Gln Leu Ala Gly
Leu Val Ala Asn Tyr Tyr Thr 660 665 670Pro Arg Trp Arg Leu Phe Leu
Glu Ala Leu Val Asp Ser Val Ala Gln 675 680 685Gly Ile Pro Phe Gln
Gln His Gln Phe Asp Lys Asn Val Phe Gln Leu 690 695 700Glu Gln Ala
Phe Val Leu Ser Lys Gln Arg Tyr Pro Ser Gln Pro Arg705 710 715
720Gly Asp Thr Val Asp Leu Ala Lys Lys Ile Phe Leu Lys Tyr Tyr Pro
725 730 735Arg Trp Val Ala Gly Ser Trp 7403524PRTHomo sapiens 3Met
Tyr Ala Leu Phe Leu Leu Ala Ser Leu Leu Gly Ala Ala Leu Ala1 5 10
15Gly Pro Val Leu Gly Leu Lys Glu Cys Thr Arg Gly Ser Ala Val Trp
20 25 30Cys Gln Asn Val Lys Thr Ala Ser Asp Cys Gly Ala Val Lys His
Cys 35 40 45Leu Gln Thr Val Trp Asn Lys Pro Thr Val Lys Ser Leu Pro
Cys Asp 50 55 60Ile Cys Lys Asp Val Val Thr Ala Ala Gly Asp Met Leu
Lys Asp Asn65 70 75 80Ala Thr Glu Glu Glu Ile Leu Val Tyr Leu Glu
Lys Thr Cys Asp Trp 85 90 95Leu Pro Lys Pro Asn Met Ser Ala Ser Cys
Lys Glu Ile Val Asp Ser 100 105 110Tyr Leu Pro Val Ile Leu Asp Ile
Ile Lys Gly Glu Met Ser Arg Pro 115 120 125Gly Glu Val Cys Ser Ala
Leu Asn Leu Cys Glu Ser Leu Gln Lys His 130 135 140Leu Ala Glu Leu
Asn His Gln Lys Gln Leu Glu Ser Asn Lys Ile Pro145 150 155 160Glu
Leu Asp Met Thr Glu Val Val Ala Pro Phe Met Ala Asn Ile Pro 165 170
175Leu Leu Leu Tyr Pro Gln Asp Gly Pro Arg Ser Lys Pro Gln Pro Lys
180 185 190Asp Asn Gly Asp Val Cys Gln Asp Cys Ile Gln Met Val Thr
Asp Ile 195 200 205Gln Thr Ala Val Arg Thr Asn Ser Thr Phe Val Gln
Ala Leu Val Glu 210 215 220His Val Lys Glu Glu Cys Asp Arg Leu Gly
Pro Gly Met Ala Asp Ile225 230 235 240Cys Lys Asn Tyr Ile Ser Gln
Tyr Ser Glu Ile Ala Ile Gln Met Met 245 250 255Met His Met Gln Pro
Lys Glu Ile Cys Ala Leu Val Gly Phe Cys Asp 260 265 270Glu Val Lys
Glu Met Pro Met Gln Thr Leu Val Pro Ala Lys Val Ala 275 280 285Ser
Lys Asn Val Ile Pro Ala Leu Glu Leu Val Glu Pro Ile Lys Lys 290 295
300His Glu Val Pro Ala Lys Ser Asp Val Tyr Cys Glu Val Cys Glu
Phe305 310 315 320Leu Val Lys Glu Val Thr Lys Leu Ile Asp Asn Asn
Lys Thr Glu Lys 325 330 335Glu Ile Leu Asp Ala Phe Asp Lys Met Cys
Ser Lys Leu Pro Lys Ser 340 345 350Leu Ser Glu Glu Cys Gln Glu Val
Val Asp Thr Tyr Gly Ser Ser Ile 355 360 365Leu Ser Ile Leu Leu Glu
Glu Val Ser Pro Glu Leu Val Cys Ser Met 370 375 380Leu His Leu Cys
Ser Gly Thr Arg Leu Pro Ala Leu Thr Val His Val385 390 395 400Thr
Gln Pro Lys Asp Gly Gly Phe Cys Glu Val Cys Lys Lys Leu Val 405 410
415Gly Tyr Leu Asp Arg Asn Leu Glu Lys Asn Ser Thr Lys Gln Glu Ile
420 425 430Leu Ala Ala Leu Glu Lys Gly Cys Ser Phe Leu Pro Asp Pro
Tyr Gln 435 440 445Lys Gln Cys Asp Gln Phe Val Ala Glu Tyr Glu Pro
Val Leu Ile Glu 450 455 460Ile Leu Val Glu Val Met Asp Pro Ser Phe
Val Cys Leu Lys Ile Gly465 470 475 480Ala Cys Pro Ser Ala His Lys
Pro Leu Leu Gly Thr Glu Lys Cys Ile 485 490 495Trp Gly Pro Ser Tyr
Trp Cys Gln Asn Thr Glu Thr Ala Ala Gln Cys 500 505 510Asn Ala Val
Glu His Cys Lys Arg His Val Trp Asn 515 5204120PRTHomo sapiens 4Asp
Gly Gly Phe Cys Glu Val Cys Lys Lys Leu Val Gly Tyr Leu Asp1 5 10
15Arg Asn Leu Glu Lys Asn Ser Thr Lys Gln Glu Ile Leu Ala Ala Leu
20 25 30Glu Lys Gly Cys Ser Phe Leu Pro Asp Pro Tyr Gln Lys Gln Cys
Asp 35 40 45Gln Phe Val Ala Glu Tyr Glu Pro Val Leu Ile Glu Ile Leu
Val Glu 50 55 60Val Met Asp Pro Ser Phe Val Cys Leu Lys Ile Gly Ala
Cys Pro Ser65 70 75 80Ala His Lys Pro Leu Leu Gly Thr Glu Lys Cys
Ile Trp Gly Pro Ser 85 90 95Tyr Trp Cys Gln Asn Thr Glu Thr Ala Ala
Gln Cys Asn Ala Val Glu 100 105 110His Cys Lys Arg His Val Trp Asn
115 1205202PRTHomo sapiens 5Met Lys Val Leu Ala Ala Gly Val Val Pro
Leu Leu Leu Val Leu His1 5 10 15Trp Lys His Gly Ala Gly Ser Pro Leu
Pro Ile Thr Pro Val Asn Ala 20 25 30Thr Cys Ala Ile Arg His Pro Cys
His Asn Asn Leu Met Asn Gln Ile 35 40 45Arg Ser Gln Leu Ala Gln Leu
Asn Gly Ser Ala Asn Ala Leu Phe Ile 50 55 60Leu Tyr Tyr Thr Ala Gln
Gly Glu Pro Phe Pro Asn Asn Leu Asp Lys65 70 75 80Leu Cys Gly Pro
Asn Val Thr Asp Phe Pro Pro Phe His Ala Asn Gly 85 90 95Thr Glu Lys
Ala Lys Leu Val Glu Leu Tyr Arg Ile Val Val Tyr Leu 100 105 110Gly
Thr Ser Leu Gly Asn Ile Thr Arg Asp Gln Lys Ile Leu Asn Pro 115 120
125Ser Ala Leu Ser Leu His Ser Lys Leu Asn Ala Thr Ala Asp Ile Leu
130 135 140Arg Gly Leu Leu Ser Asn Val Leu Cys Arg Leu Cys Ser Lys
Tyr His145 150 155 160Val Gly His Val Asp Val Thr Tyr Gly Pro Asp
Thr Ser Gly Lys Asp 165 170 175Val Phe Gln Lys Lys Lys Leu Gly Cys
Gln Leu Leu Gly Lys Tyr Lys 180 185 190Gln Ile Ile Ala Val Leu Ala
Gln Ala Phe 195 2006180PRTHomo sapiens 6Ser Pro Leu Pro Ile Thr Pro
Val Asn Ala Thr Cys Ala Ile Arg His1 5 10 15Pro Cys His Asn Asn Leu
Met Asn Ala Ile Arg Ala Gln Leu Ala Ala 20 25 30Leu Asn Gly Ser Ala
Asn Ala Leu Phe Ile Leu Tyr Tyr Thr Ala Gln 35 40 45Gly Glu Pro Phe
Pro Asn Asn Leu Asp Lys Leu Cys Gly Pro Asn Val 50 55 60Thr Asp Phe
Pro Pro Phe His Ala Asn Gly Thr Glu Lys Ala Lys Leu65 70 75 80Val
Glu Leu Tyr Arg Ile Val Val Tyr Leu Gly Thr Ser Leu Gly Asn 85 90
95Ile Thr Arg Asp Gln Lys Ile Leu Asn Pro Ser Ala Leu Ser Leu His
100 105 110Ser Lys Leu Asn Ala Thr Ala Asp Ile Leu Arg Gly Leu Leu
Ser Asn 115 120 125Val Leu Cys Arg Leu Cys Ser Lys Tyr His Val Gly
His Val Asp Val 130 135 140Thr Tyr Gly Pro Asp Thr Ser Gly Lys Asp
Val Ala Gln Lys Ala Lys145
150 155 160Leu Gly Cys Gln Leu Leu Gly Lys Tyr Lys Gln Ile Ile Ala
Val Leu 165 170 175Ala Gln Ala Phe 1807220PRTHomo sapiens 7Met Ala
Gly Leu Ser Arg Gly Ser Ala Arg Ala Leu Leu Ala Ala Leu1 5 10 15Leu
Ala Ser Thr Leu Leu Ala Leu Leu Val Ser Pro Ala Arg Gly Arg 20 25
30Gly Gly Arg Asp His Gly Asp Trp Asp Glu Ala Ser Arg Leu Pro Pro
35 40 45Leu Pro Pro Arg Glu Asp Ala Ala Arg Val Ala Arg Phe Val Thr
His 50 55 60Val Ser Asp Trp Gly Ala Leu Ala Thr Ile Ser Thr Leu Glu
Ala Val65 70 75 80Arg Gly Arg Pro Phe Ala Asp Val Leu Ser Leu Ser
Asp Gly Pro Pro 85 90 95Gly Ala Gly Ser Gly Val Pro Tyr Phe Tyr Leu
Ser Pro Leu Gln Leu 100 105 110Ser Val Ser Asn Leu Gln Glu Asn Pro
Tyr Ala Thr Leu Thr Met Thr 115 120 125Leu Ala Gln Thr Asn Phe Cys
Lys Lys His Gly Phe Asp Pro Gln Ser 130 135 140Pro Leu Cys Val His
Ile Met Leu Ser Gly Thr Val Thr Lys Val Asn145 150 155 160Glu Thr
Glu Met Asp Ile Ala Lys His Ser Leu Phe Ile Arg His Pro 165 170
175Glu Met Lys Thr Trp Pro Ser Ser His Asn Trp Phe Phe Ala Lys Leu
180 185 190Asn Ile Thr Asn Ile Trp Val Leu Asp Tyr Phe Gly Gly Pro
Lys Ile 195 200 205Val Thr Pro Glu Glu Tyr Tyr Asn Val Thr Val Gln
210 215 22084PRTHomo sapiens 8Asp Pro Gln Ser19185PRTHomo sapiens
9Arg Gly Gly Arg Asp His Gly Asp Trp Asp Glu Ala Ser Arg Leu Pro1 5
10 15Pro Leu Pro Pro Arg Glu Asp Ala Ala Arg Val Ala Arg Phe Val
Thr 20 25 30His Val Ser Asp Trp Gly Ala Leu Ala Thr Ile Ser Thr Leu
Glu Ala 35 40 45Val Arg Gly Arg Pro Phe Ala Asp Val Leu Ser Leu Ser
Asp Gly Pro 50 55 60Pro Gly Ala Gly Ser Gly Val Pro Tyr Phe Tyr Leu
Ser Pro Leu Gln65 70 75 80Leu Ser Val Ser Asn Leu Gln Glu Asn Pro
Tyr Ala Thr Leu Thr Met 85 90 95Thr Leu Ala Gln Thr Asn Phe Cys Lys
Lys His Gly Phe Pro Leu Cys 100 105 110Val His Ile Met Leu Ser Gly
Thr Val Thr Lys Val Asn Glu Thr Glu 115 120 125Met Asp Ile Ala Lys
His Ser Leu Phe Ile Arg His Pro Glu Met Lys 130 135 140Thr Trp Pro
Ser Ser His Asn Trp Phe Phe Ala Lys Leu Asn Ile Thr145 150 155
160Asn Ile Trp Val Leu Asp Tyr Phe Gly Gly Pro Lys Ile Val Thr Pro
165 170 175Glu Glu Tyr Tyr Asn Val Thr Val Gln 180
1851013PRTArtificial Sequencesource/note="Description of Artificial
Sequence Synthetic peptide" 10Gly Gly Gly Gly Gly Ala Ala Ala Ala
Gly Gly Gly Gly1 5 10113PRTArtificial
Sequencesource/note="Description of Artificial Sequence Synthetic
peptide" 11Gly Ala Pro1126PRTArtificial
Sequencesource/note="Description of Artificial Sequence Synthetic
peptide" 12Gly Gly Gly Gly Gly Pro1 51335PRTArtificial
Sequencesource/note="Description of Artificial Sequence Synthetic
polypeptide" 13Gly Ala Pro Gly Gly Gly Gly Gly Ala Ala Ala Ala Gly
Gly Gly Gly1 5 10 15Gly Ala Pro Gly Gly Gly Gly Gly Ala Ala Ala Ala
Gly Gly Gly Gly 20 25 30Gly Ala Pro 351457PRTArtificial
Sequencesource/note="Description of Artificial Sequence Synthetic
polypeptide" 14Gly Ala Pro Gly Gly Gly Gly Gly Ala Ala Ala Ala Ala
Gly Gly Gly1 5 10 15Gly Gly Gly Ala Pro Gly Gly Gly Gly Gly Ala Ala
Ala Ala Ala Gly 20 25 30Gly Gly Gly Gly Gly Ala Pro Gly Gly Gly Gly
Gly Ala Ala Ala Ala 35 40 45Ala Gly Gly Gly Gly Gly Gly Ala Pro 50
551557PRTArtificial Sequencesource/note="Description of Artificial
Sequence Synthetic polypeptide" 15Gly Ala Pro Gly Gly Gly Gly Gly
Ala Ala Ala Ala Ala Gly Gly Gly1 5 10 15Gly Gly Gly Ala Pro Gly Gly
Gly Gly Gly Ala Ala Ala Ala Ala Gly 20 25 30Gly Gly Gly Gly Gly Ala
Pro Gly Gly Gly Gly Gly Ala Ala Ala Ala 35 40 45Ala Gly Gly Gly Gly
Gly Gly Ala Pro 50 55161256PRTHomo sapiens 16Met Leu Phe Lys Leu
Leu Gln Arg Gln Thr Tyr Thr Cys Leu Ser His1 5 10 15Arg Tyr Gly Leu
Tyr Val Cys Phe Leu Gly Val Val Val Thr Ile Val 20 25 30Ser Ala Phe
Gln Phe Gly Glu Val Val Leu Glu Trp Ser Arg Asp Gln 35 40 45Tyr His
Val Leu Phe Asp Ser Tyr Arg Asp Asn Ile Ala Gly Lys Ser 50 55 60Phe
Gln Asn Arg Leu Cys Leu Pro Met Pro Ile Asp Val Val Tyr Thr65 70 75
80Trp Val Asn Gly Thr Asp Leu Glu Leu Leu Lys Glu Leu Gln Gln Val
85 90 95Arg Glu Gln Met Glu Glu Glu Gln Lys Ala Met Arg Glu Ile Leu
Gly 100 105 110Lys Asn Thr Thr Glu Pro Thr Lys Lys Ser Glu Lys Gln
Leu Glu Cys 115 120 125Leu Leu Thr His Cys Ile Lys Val Pro Met Leu
Val Leu Asp Pro Ala 130 135 140Leu Pro Ala Asn Ile Thr Leu Lys Asp
Leu Pro Ser Leu Tyr Pro Ser145 150 155 160Phe His Ser Ala Ser Asp
Ile Phe Asn Val Ala Lys Pro Lys Asn Pro 165 170 175Ser Thr Asn Val
Ser Val Val Val Phe Asp Ser Thr Lys Asp Val Glu 180 185 190Asp Ala
His Ser Gly Leu Leu Lys Gly Asn Ser Arg Gln Thr Val Trp 195 200
205Arg Gly Tyr Leu Thr Thr Asp Lys Glu Val Pro Gly Leu Val Leu Met
210 215 220Gln Asp Leu Ala Phe Leu Ser Gly Phe Pro Pro Thr Phe Lys
Glu Thr225 230 235 240Asn Gln Leu Lys Thr Lys Leu Pro Glu Asn Leu
Ser Ser Lys Val Lys 245 250 255Leu Leu Gln Leu Tyr Ser Glu Ala Ser
Val Ala Leu Leu Lys Leu Asn 260 265 270Asn Pro Lys Asp Phe Gln Glu
Leu Asn Lys Gln Thr Lys Lys Asn Met 275 280 285Thr Ile Asp Gly Lys
Glu Leu Thr Ile Ser Pro Ala Tyr Leu Leu Trp 290 295 300Asp Leu Ser
Ala Ile Ser Gln Ser Lys Gln Asp Glu Asp Ile Ser Ala305 310 315
320Ser Arg Phe Glu Asp Asn Glu Glu Leu Arg Tyr Ser Leu Arg Ser Ile
325 330 335Glu Arg His Ala Pro Trp Val Arg Asn Ile Phe Ile Val Thr
Asn Gly 340 345 350Gln Ile Pro Ser Trp Leu Asn Leu Asp Asn Pro Arg
Val Thr Ile Val 355 360 365Thr His Gln Asp Val Phe Arg Asn Leu Ser
His Leu Pro Thr Phe Ser 370 375 380Ser Pro Ala Ile Glu Ser His Ile
His Arg Ile Glu Gly Leu Ser Gln385 390 395 400Lys Phe Ile Tyr Leu
Asn Asp Asp Val Met Phe Gly Lys Asp Val Trp 405 410 415Pro Asp Asp
Phe Tyr Ser His Ser Lys Gly Gln Lys Val Tyr Leu Thr 420 425 430Trp
Pro Val Pro Asn Cys Ala Glu Gly Cys Pro Gly Ser Trp Ile Lys 435 440
445Asp Gly Tyr Cys Asp Lys Ala Cys Asn Asn Ser Ala Cys Asp Trp Asp
450 455 460Gly Gly Asp Cys Ser Gly Asn Ser Gly Gly Ser Arg Tyr Ile
Ala Gly465 470 475 480Gly Gly Gly Thr Gly Ser Ile Gly Val Gly Gln
Pro Trp Gln Phe Gly 485 490 495Gly Gly Ile Asn Ser Val Ser Tyr Cys
Asn Gln Gly Cys Ala Asn Ser 500 505 510Trp Leu Ala Asp Lys Phe Cys
Asp Gln Ala Cys Asn Val Leu Ser Cys 515 520 525Gly Phe Asp Ala Gly
Asp Cys Gly Gln Asp His Phe His Glu Leu Tyr 530 535 540Lys Val Ile
Leu Leu Pro Asn Gln Thr His Tyr Ile Ile Pro Lys Gly545 550 555
560Glu Cys Leu Pro Tyr Phe Ser Phe Ala Glu Val Ala Lys Arg Gly Val
565 570 575Glu Gly Ala Tyr Ser Asp Asn Pro Ile Ile Arg His Ala Ser
Ile Ala 580 585 590Asn Lys Trp Lys Thr Ile His Leu Ile Met His Ser
Gly Met Asn Ala 595 600 605Thr Thr Ile His Phe Asn Leu Thr Phe Gln
Asn Thr Asn Asp Glu Glu 610 615 620Phe Lys Met Gln Ile Thr Val Glu
Val Asp Thr Arg Glu Gly Pro Lys625 630 635 640Leu Asn Ser Thr Ala
Gln Lys Gly Tyr Glu Asn Leu Val Ser Pro Ile 645 650 655Thr Leu Leu
Pro Glu Ala Glu Ile Leu Phe Glu Asp Ile Pro Lys Glu 660 665 670Lys
Arg Phe Pro Lys Phe Lys Arg His Asp Val Asn Ser Thr Arg Arg 675 680
685Ala Gln Glu Glu Val Lys Ile Pro Leu Val Asn Ile Ser Leu Leu Pro
690 695 700Lys Asp Ala Gln Leu Ser Leu Asn Thr Leu Asp Leu Gln Leu
Glu His705 710 715 720Gly Asp Ile Thr Leu Lys Gly Tyr Asn Leu Ser
Lys Ser Ala Leu Leu 725 730 735Arg Ser Phe Leu Met Asn Ser Gln His
Ala Lys Ile Lys Asn Gln Ala 740 745 750Ile Ile Thr Asp Glu Thr Asn
Asp Ser Leu Val Ala Pro Gln Glu Lys 755 760 765Gln Val His Lys Ser
Ile Leu Pro Asn Ser Leu Gly Val Ser Glu Arg 770 775 780Leu Gln Arg
Leu Thr Phe Pro Ala Val Ser Val Lys Val Asn Gly His785 790 795
800Asp Gln Gly Gln Asn Pro Pro Leu Asp Leu Glu Thr Thr Ala Arg Phe
805 810 815Arg Val Glu Thr His Thr Gln Lys Thr Ile Gly Gly Asn Val
Thr Lys 820 825 830Glu Lys Pro Pro Ser Leu Ile Val Pro Leu Glu Ser
Gln Met Thr Lys 835 840 845Glu Lys Lys Ile Thr Gly Lys Glu Lys Glu
Asn Ser Arg Met Glu Glu 850 855 860Asn Ala Glu Asn His Ile Gly Val
Thr Glu Val Leu Leu Gly Arg Lys865 870 875 880Leu Gln His Tyr Thr
Asp Ser Tyr Leu Gly Phe Leu Pro Trp Glu Lys 885 890 895Lys Lys Tyr
Phe Gln Asp Leu Leu Asp Glu Glu Glu Ser Leu Lys Thr 900 905 910Gln
Leu Ala Tyr Phe Thr Asp Ser Lys Asn Thr Gly Arg Gln Leu Lys 915 920
925Asp Thr Phe Ala Asp Ser Leu Arg Tyr Val Asn Lys Ile Leu Asn Ser
930 935 940Lys Phe Gly Phe Thr Ser Arg Lys Val Pro Ala His Met Pro
His Met945 950 955 960Ile Asp Arg Ile Val Met Gln Glu Leu Gln Asp
Met Phe Pro Glu Glu 965 970 975Phe Asp Lys Thr Ser Phe His Lys Val
Arg His Ser Glu Asp Met Gln 980 985 990Phe Ala Phe Ser Tyr Phe Tyr
Tyr Leu Met Ser Ala Val Gln Pro Leu 995 1000 1005Asn Ile Ser Gln
Val Phe Asp Glu Val Asp Thr Asp Gln Ser Gly 1010 1015 1020Val Leu
Ser Asp Arg Glu Ile Arg Thr Leu Ala Thr Arg Ile His 1025 1030
1035Glu Leu Pro Leu Ser Leu Gln Asp Leu Thr Gly Leu Glu His Met
1040 1045 1050Leu Ile Asn Cys Ser Lys Met Leu Pro Ala Asp Ile Thr
Gln Leu 1055 1060 1065Asn Asn Ile Pro Pro Thr Gln Glu Ser Tyr Tyr
Asp Pro Asn Leu 1070 1075 1080Pro Pro Val Thr Lys Ser Leu Val Thr
Asn Cys Lys Pro Val Thr 1085 1090 1095Asp Lys Ile His Lys Ala Tyr
Lys Asp Lys Asn Lys Tyr Arg Phe 1100 1105 1110Glu Ile Met Gly Glu
Glu Glu Ile Ala Phe Lys Met Ile Arg Thr 1115 1120 1125Asn Val Ser
His Val Val Gly Gln Leu Asp Asp Ile Arg Lys Asn 1130 1135 1140Pro
Arg Lys Phe Val Cys Leu Asn Asp Asn Ile Asp His Asn His 1145 1150
1155Lys Asp Ala Gln Thr Val Lys Ala Val Leu Arg Asp Phe Tyr Glu
1160 1165 1170Ser Met Phe Pro Ile Pro Ser Gln Phe Glu Leu Pro Arg
Glu Tyr 1175 1180 1185Arg Asn Arg Phe Leu His Met His Glu Leu Gln
Glu Trp Arg Ala 1190 1195 1200Tyr Arg Asp Lys Leu Lys Phe Trp Thr
His Cys Val Leu Ala Thr 1205 1210 1215Leu Ile Met Phe Thr Ile Phe
Ser Phe Phe Ala Glu Gln Leu Ile 1220 1225 1230Ala Leu Lys Arg Lys
Ile Phe Pro Arg Arg Arg Ile His Lys Glu 1235 1240 1245Ala Ser Pro
Asn Arg Ile Arg Val 1250 125517305PRTHomo sapiens 17Met Ala Ala Gly
Leu Ala Arg Leu Leu Leu Leu Leu Gly Leu Ser Ala1 5 10 15Gly Gly Pro
Ala Pro Ala Gly Ala Ala Lys Met Lys Val Val Glu Glu 20 25 30Pro Asn
Ala Phe Gly Val Asn Asn Pro Phe Leu Pro Gln Ala Ser Arg 35 40 45Leu
Gln Ala Lys Arg Asp Pro Ser Pro Val Ser Gly Pro Val His Leu 50 55
60Phe Arg Leu Ser Gly Lys Cys Phe Ser Leu Val Glu Ser Thr Tyr Lys65
70 75 80Tyr Glu Phe Cys Pro Phe His Asn Val Thr Gln His Glu Gln Thr
Phe 85 90 95Arg Trp Asn Ala Tyr Ser Gly Ile Leu Gly Ile Trp His Glu
Trp Glu 100 105 110Ile Ala Asn Asn Thr Phe Thr Gly Met Trp Met Arg
Asp Gly Asp Ala 115 120 125Cys Arg Ser Arg Ser Arg Gln Ser Lys Val
Glu Leu Ala Cys Gly Lys 130 135 140Ser Asn Arg Leu Ala His Val Ser
Glu Pro Ser Thr Cys Val Tyr Ala145 150 155 160Leu Thr Phe Glu Thr
Pro Leu Val Cys His Pro His Ala Leu Leu Val 165 170 175Tyr Pro Thr
Leu Pro Glu Ala Leu Gln Arg Gln Trp Asp Gln Val Glu 180 185 190Gln
Asp Leu Ala Asp Glu Leu Ile Thr Pro Gln Gly His Glu Lys Leu 195 200
205Leu Arg Thr Leu Phe Glu Asp Ala Gly Tyr Leu Lys Thr Pro Glu Glu
210 215 220Asn Glu Pro Thr Gln Leu Glu Gly Gly Pro Asp Ser Leu Gly
Phe Glu225 230 235 240Thr Leu Glu Asn Cys Arg Lys Ala His Lys Glu
Leu Ser Lys Glu Ile 245 250 255Lys Arg Leu Lys Gly Leu Leu Thr Gln
His Gly Ile Pro Tyr Thr Arg 260 265 270Pro Thr Glu Thr Ser Asn Leu
Glu His Leu Gly His Glu Thr Pro Arg 275 280 285Ala Lys Ser Pro Glu
Gln Leu Arg Gly Asp Pro Gly Leu Arg Gly Ser 290 295
300Leu30518920PRTArtificial Sequencesource/note="Description of
Artificial Sequence Synthetic polypeptide" 18Met Glu Ala Val Ala
Val Ala Ala Ala Val Gly Val Leu Leu Leu Ala1 5 10 15Gly Ala Gly Gly
Ala Ala Gly Asp Glu Ala Arg Glu Ala Ala Ala Val 20 25 30Arg Ala Leu
Val Ala Arg Leu Leu Gly Pro Gly Pro Ala Ala Asp Phe 35 40 45Ser Val
Ser Val Glu Arg Ala Leu Ala Ala Lys Pro Gly Leu Asp Thr 50 55 60Tyr
Ser Leu Gly Gly Gly Gly Ala Ala Arg Val Arg Val Arg Gly Ser65 70 75
80Thr Gly Val Ala Ala Ala Ala Gly Leu His Arg Tyr Leu Arg Asp Phe
85 90 95Cys Gly Cys His Val Ala Trp Ser Gly Ser Gln Leu Arg Leu Pro
Arg 100 105 110Pro Leu Pro Ala Val Pro Gly Glu Leu Thr Glu Ala Thr
Pro Asn Arg 115 120 125Tyr Arg Tyr Tyr Gln Asn Val Cys Thr Gln Ser
Tyr Ser Phe Val Trp 130 135 140Trp Asp Trp Ala Arg Trp Glu Arg Glu
Ile Asp Trp Met Ala Leu Asn145 150 155 160Gly Ile Asn Leu Ala Leu
Ala Trp Ser Gly Gln Glu Ala Ile Trp Gln 165 170 175Arg Val Tyr Leu
Ala Leu Gly Leu Thr Gln Ala Glu Ile Asn Glu Phe 180 185 190Phe Thr
Gly Pro Ala Phe
Leu Ala Trp Gly Arg Met Gly Asn Leu His 195 200 205Thr Trp Asp Gly
Pro Leu Pro Pro Ser Trp His Ile Lys Gln Leu Tyr 210 215 220Leu Gln
His Arg Val Leu Asp Gln Met Arg Ser Phe Gly Met Thr Pro225 230 235
240Val Leu Pro Ala Phe Ala Gly His Val Pro Glu Ala Val Thr Arg Val
245 250 255Phe Pro Gln Val Asn Val Thr Lys Met Gly Ser Trp Gly His
Phe Asn 260 265 270Cys Ser Tyr Ser Cys Ser Phe Leu Leu Ala Pro Glu
Asp Pro Ile Phe 275 280 285Pro Ile Ile Gly Ser Leu Phe Leu Arg Glu
Leu Ile Lys Glu Phe Gly 290 295 300Thr Asp His Ile Tyr Gly Ala Asp
Thr Phe Asn Glu Met Gln Pro Pro305 310 315 320Ser Ser Glu Pro Ser
Tyr Leu Ala Ala Ala Thr Thr Ala Val Tyr Glu 325 330 335Ala Met Thr
Ala Val Asp Thr Glu Ala Val Trp Leu Leu Gln Gly Trp 340 345 350Leu
Phe Gln His Gln Pro Gln Phe Trp Gly Pro Ala Gln Ile Arg Ala 355 360
365Val Leu Gly Ala Val Pro Arg Gly Arg Leu Leu Val Leu Asp Leu Phe
370 375 380Ala Glu Ser Gln Pro Val Tyr Thr Arg Thr Ala Ser Phe Gln
Gly Gln385 390 395 400Pro Phe Ile Trp Cys Met Leu His Asn Phe Gly
Gly Asn His Gly Leu 405 410 415Phe Gly Ala Leu Glu Ala Val Asn Gly
Gly Pro Glu Ala Ala Arg Leu 420 425 430Phe Pro Asn Ser Thr Met Val
Gly Thr Gly Met Ala Pro Glu Gly Ile 435 440 445Ser Gln Asn Glu Val
Val Tyr Ser Leu Met Ala Glu Leu Gly Trp Arg 450 455 460Lys Asp Pro
Val Pro Asp Leu Ala Ala Trp Val Thr Ser Phe Ala Ala465 470 475
480Arg Arg Tyr Gly Val Ser His Pro Asp Ala Gly Ala Ala Trp Arg Leu
485 490 495Leu Leu Arg Ser Val Tyr Asn Cys Ser Gly Glu Ala Cys Arg
Gly His 500 505 510Asn Arg Ser Pro Leu Val Arg Arg Pro Ser Leu Gln
Met Asn Thr Ser 515 520 525Ile Trp Tyr Asn Arg Ser Asp Val Phe Glu
Ala Trp Arg Leu Leu Leu 530 535 540Thr Ser Ala Pro Ser Leu Ala Thr
Ser Pro Ala Phe Arg Tyr Asp Leu545 550 555 560Leu Asp Leu Thr Arg
Gln Ala Val Gln Glu Leu Val Ser Leu Tyr Tyr 565 570 575Glu Glu Ala
Arg Ser Ala Tyr Leu Ser Lys Glu Leu Ala Ser Leu Leu 580 585 590Arg
Ala Gly Gly Val Leu Ala Tyr Glu Leu Leu Pro Ala Leu Asp Glu 595 600
605Val Leu Ala Ser Asp Ser Arg Phe Leu Leu Gly Ser Trp Leu Glu Gln
610 615 620Ala Arg Ala Ala Ala Val Ser Glu Ala Glu Ala Asp Phe Tyr
Glu Gln625 630 635 640Asn Ser Arg Tyr Gln Leu Thr Leu Trp Gly Pro
Glu Gly Asn Ile Leu 645 650 655Asp Tyr Ala Asn Lys Gln Leu Ala Gly
Leu Val Ala Asn Tyr Tyr Thr 660 665 670Pro Arg Trp Arg Leu Phe Leu
Glu Ala Leu Val Asp Ser Val Ala Gln 675 680 685Gly Ile Pro Phe Gln
Gln His Gln Phe Asp Lys Asn Val Phe Gln Leu 690 695 700Glu Gln Ala
Phe Val Leu Ser Lys Gln Arg Tyr Pro Ser Gln Pro Arg705 710 715
720Gly Asp Thr Val Asp Leu Ala Lys Lys Ile Phe Leu Lys Tyr Tyr Pro
725 730 735Arg Trp Val Ala Gly Ser Trp Gly Ala Pro Gly Gly Gly Gly
Gly Ala 740 745 750Ala Ala Ala Ala Gly Gly Gly Gly Gly Gly Ala Pro
Gly Gly Gly Gly 755 760 765Gly Ala Ala Ala Ala Ala Gly Gly Gly Gly
Gly Gly Ala Pro Gly Gly 770 775 780Gly Gly Gly Ala Ala Ala Ala Ala
Gly Gly Gly Gly Gly Gly Ala Pro785 790 795 800Asp Gly Gly Phe Cys
Glu Val Cys Lys Lys Leu Val Gly Tyr Leu Asp 805 810 815Arg Asn Leu
Glu Lys Asn Ser Thr Lys Gln Glu Ile Leu Ala Ala Leu 820 825 830Glu
Lys Gly Cys Ser Phe Leu Pro Asp Pro Tyr Gln Lys Gln Cys Asp 835 840
845Gln Phe Val Ala Glu Tyr Glu Pro Val Leu Ile Glu Ile Leu Val Glu
850 855 860Val Met Asp Pro Ser Phe Val Cys Leu Lys Ile Gly Ala Cys
Pro Ser865 870 875 880Ala His Lys Pro Leu Leu Gly Thr Glu Lys Cys
Ile Trp Gly Pro Ser 885 890 895Tyr Trp Cys Gln Asn Thr Glu Thr Ala
Ala Gln Cys Asn Ala Val Glu 900 905 910His Cys Lys Arg His Val Trp
Asn 915 92019980PRTArtificial Sequencesource/note="Description of
Artificial Sequence Synthetic polypeptide" 19Met Glu Ala Val Ala
Val Ala Ala Ala Val Gly Val Leu Leu Leu Ala1 5 10 15Gly Ala Gly Gly
Ala Ala Gly Asp Glu Ala Arg Glu Ala Ala Ala Val 20 25 30Arg Ala Leu
Val Ala Arg Leu Leu Gly Pro Gly Pro Ala Ala Asp Phe 35 40 45Ser Val
Ser Val Glu Arg Ala Leu Ala Ala Lys Pro Gly Leu Asp Thr 50 55 60Tyr
Ser Leu Gly Gly Gly Gly Ala Ala Arg Val Arg Val Arg Gly Ser65 70 75
80Thr Gly Val Ala Ala Ala Ala Gly Leu His Arg Tyr Leu Arg Asp Phe
85 90 95Cys Gly Cys His Val Ala Trp Ser Gly Ser Gln Leu Arg Leu Pro
Arg 100 105 110Pro Leu Pro Ala Val Pro Gly Glu Leu Thr Glu Ala Thr
Pro Asn Arg 115 120 125Tyr Arg Tyr Tyr Gln Asn Val Cys Thr Gln Ser
Tyr Ser Phe Val Trp 130 135 140Trp Asp Trp Ala Arg Trp Glu Arg Glu
Ile Asp Trp Met Ala Leu Asn145 150 155 160Gly Ile Asn Leu Ala Leu
Ala Trp Ser Gly Gln Glu Ala Ile Trp Gln 165 170 175Arg Val Tyr Leu
Ala Leu Gly Leu Thr Gln Ala Glu Ile Asn Glu Phe 180 185 190Phe Thr
Gly Pro Ala Phe Leu Ala Trp Gly Arg Met Gly Asn Leu His 195 200
205Thr Trp Asp Gly Pro Leu Pro Pro Ser Trp His Ile Lys Gln Leu Tyr
210 215 220Leu Gln His Arg Val Leu Asp Gln Met Arg Ser Phe Gly Met
Thr Pro225 230 235 240Val Leu Pro Ala Phe Ala Gly His Val Pro Glu
Ala Val Thr Arg Val 245 250 255Phe Pro Gln Val Asn Val Thr Lys Met
Gly Ser Trp Gly His Phe Asn 260 265 270Cys Ser Tyr Ser Cys Ser Phe
Leu Leu Ala Pro Glu Asp Pro Ile Phe 275 280 285Pro Ile Ile Gly Ser
Leu Phe Leu Arg Glu Leu Ile Lys Glu Phe Gly 290 295 300Thr Asp His
Ile Tyr Gly Ala Asp Thr Phe Asn Glu Met Gln Pro Pro305 310 315
320Ser Ser Glu Pro Ser Tyr Leu Ala Ala Ala Thr Thr Ala Val Tyr Glu
325 330 335Ala Met Thr Ala Val Asp Thr Glu Ala Val Trp Leu Leu Gln
Gly Trp 340 345 350Leu Phe Gln His Gln Pro Gln Phe Trp Gly Pro Ala
Gln Ile Arg Ala 355 360 365Val Leu Gly Ala Val Pro Arg Gly Arg Leu
Leu Val Leu Asp Leu Phe 370 375 380Ala Glu Ser Gln Pro Val Tyr Thr
Arg Thr Ala Ser Phe Gln Gly Gln385 390 395 400Pro Phe Ile Trp Cys
Met Leu His Asn Phe Gly Gly Asn His Gly Leu 405 410 415Phe Gly Ala
Leu Glu Ala Val Asn Gly Gly Pro Glu Ala Ala Arg Leu 420 425 430Phe
Pro Asn Ser Thr Met Val Gly Thr Gly Met Ala Pro Glu Gly Ile 435 440
445Ser Gln Asn Glu Val Val Tyr Ser Leu Met Ala Glu Leu Gly Trp Arg
450 455 460Lys Asp Pro Val Pro Asp Leu Ala Ala Trp Val Thr Ser Phe
Ala Ala465 470 475 480Arg Arg Tyr Gly Val Ser His Pro Asp Ala Gly
Ala Ala Trp Arg Leu 485 490 495Leu Leu Arg Ser Val Tyr Asn Cys Ser
Gly Glu Ala Cys Arg Gly His 500 505 510Asn Arg Ser Pro Leu Val Arg
Arg Pro Ser Leu Gln Met Asn Thr Ser 515 520 525Ile Trp Tyr Asn Arg
Ser Asp Val Phe Glu Ala Trp Arg Leu Leu Leu 530 535 540Thr Ser Ala
Pro Ser Leu Ala Thr Ser Pro Ala Phe Arg Tyr Asp Leu545 550 555
560Leu Asp Leu Thr Arg Gln Ala Val Gln Glu Leu Val Ser Leu Tyr Tyr
565 570 575Glu Glu Ala Arg Ser Ala Tyr Leu Ser Lys Glu Leu Ala Ser
Leu Leu 580 585 590Arg Ala Gly Gly Val Leu Ala Tyr Glu Leu Leu Pro
Ala Leu Asp Glu 595 600 605Val Leu Ala Ser Asp Ser Arg Phe Leu Leu
Gly Ser Trp Leu Glu Gln 610 615 620Ala Arg Ala Ala Ala Val Ser Glu
Ala Glu Ala Asp Phe Tyr Glu Gln625 630 635 640Asn Ser Arg Tyr Gln
Leu Thr Leu Trp Gly Pro Glu Gly Asn Ile Leu 645 650 655Asp Tyr Ala
Asn Lys Gln Leu Ala Gly Leu Val Ala Asn Tyr Tyr Thr 660 665 670Pro
Arg Trp Arg Leu Phe Leu Glu Ala Leu Val Asp Ser Val Ala Gln 675 680
685Gly Ile Pro Phe Gln Gln His Gln Phe Asp Lys Asn Val Phe Gln Leu
690 695 700Glu Gln Ala Phe Val Leu Ser Lys Gln Arg Tyr Pro Ser Gln
Pro Arg705 710 715 720Gly Asp Thr Val Asp Leu Ala Lys Lys Ile Phe
Leu Lys Tyr Tyr Pro 725 730 735Arg Trp Val Ala Gly Ser Trp Gly Ala
Pro Gly Gly Gly Gly Gly Ala 740 745 750Ala Ala Ala Ala Gly Gly Gly
Gly Gly Gly Ala Pro Gly Gly Gly Gly 755 760 765Gly Ala Ala Ala Ala
Ala Gly Gly Gly Gly Gly Gly Ala Pro Gly Gly 770 775 780Gly Gly Gly
Ala Ala Ala Ala Ala Gly Gly Gly Gly Gly Gly Ala Pro785 790 795
800Ser Pro Leu Pro Ile Thr Pro Val Asn Ala Thr Cys Ala Ile Arg His
805 810 815Pro Cys His Asn Asn Leu Met Asn Ala Ile Arg Ala Gln Leu
Ala Ala 820 825 830Leu Asn Gly Ser Ala Asn Ala Leu Phe Ile Leu Tyr
Tyr Thr Ala Gln 835 840 845Gly Glu Pro Phe Pro Asn Asn Leu Asp Lys
Leu Cys Gly Pro Asn Val 850 855 860Thr Asp Phe Pro Pro Phe His Ala
Asn Gly Thr Glu Lys Ala Lys Leu865 870 875 880Val Glu Leu Tyr Arg
Ile Val Val Tyr Leu Gly Thr Ser Leu Gly Asn 885 890 895Ile Thr Arg
Asp Gln Lys Ile Leu Asn Pro Ser Ala Leu Ser Leu His 900 905 910Ser
Lys Leu Asn Ala Thr Ala Asp Ile Leu Arg Gly Leu Leu Ser Asn 915 920
925Val Leu Cys Arg Leu Cys Ser Lys Tyr His Val Gly His Val Asp Val
930 935 940Thr Tyr Gly Pro Asp Thr Ser Gly Lys Asp Val Ala Gln Lys
Ala Lys945 950 955 960Leu Gly Cys Gln Leu Leu Gly Lys Tyr Lys Gln
Ile Ile Ala Val Leu 965 970 975Ala Gln Ala Phe
98020985PRTArtificial Sequencesource/note="Description of
Artificial Sequence Synthetic polypeptide" 20Met Glu Ala Val Ala
Val Ala Ala Ala Val Gly Val Leu Leu Leu Ala1 5 10 15Gly Ala Gly Gly
Ala Ala Gly Asp Glu Ala Arg Glu Ala Ala Ala Val 20 25 30Arg Ala Leu
Val Ala Arg Leu Leu Gly Pro Gly Pro Ala Ala Asp Phe 35 40 45Ser Val
Ser Val Glu Arg Ala Leu Ala Ala Lys Pro Gly Leu Asp Thr 50 55 60Tyr
Ser Leu Gly Gly Gly Gly Ala Ala Arg Val Arg Val Arg Gly Ser65 70 75
80Thr Gly Val Ala Ala Ala Ala Gly Leu His Arg Tyr Leu Arg Asp Phe
85 90 95Cys Gly Cys His Val Ala Trp Ser Gly Ser Gln Leu Arg Leu Pro
Arg 100 105 110Pro Leu Pro Ala Val Pro Gly Glu Leu Thr Glu Ala Thr
Pro Asn Arg 115 120 125Tyr Arg Tyr Tyr Gln Asn Val Cys Thr Gln Ser
Tyr Ser Phe Val Trp 130 135 140Trp Asp Trp Ala Arg Trp Glu Arg Glu
Ile Asp Trp Met Ala Leu Asn145 150 155 160Gly Ile Asn Leu Ala Leu
Ala Trp Ser Gly Gln Glu Ala Ile Trp Gln 165 170 175Arg Val Tyr Leu
Ala Leu Gly Leu Thr Gln Ala Glu Ile Asn Glu Phe 180 185 190Phe Thr
Gly Pro Ala Phe Leu Ala Trp Gly Arg Met Gly Asn Leu His 195 200
205Thr Trp Asp Gly Pro Leu Pro Pro Ser Trp His Ile Lys Gln Leu Tyr
210 215 220Leu Gln His Arg Val Leu Asp Gln Met Arg Ser Phe Gly Met
Thr Pro225 230 235 240Val Leu Pro Ala Phe Ala Gly His Val Pro Glu
Ala Val Thr Arg Val 245 250 255Phe Pro Gln Val Asn Val Thr Lys Met
Gly Ser Trp Gly His Phe Asn 260 265 270Cys Ser Tyr Ser Cys Ser Phe
Leu Leu Ala Pro Glu Asp Pro Ile Phe 275 280 285Pro Ile Ile Gly Ser
Leu Phe Leu Arg Glu Leu Ile Lys Glu Phe Gly 290 295 300Thr Asp His
Ile Tyr Gly Ala Asp Thr Phe Asn Glu Met Gln Pro Pro305 310 315
320Ser Ser Glu Pro Ser Tyr Leu Ala Ala Ala Thr Thr Ala Val Tyr Glu
325 330 335Ala Met Thr Ala Val Asp Thr Glu Ala Val Trp Leu Leu Gln
Gly Trp 340 345 350Leu Phe Gln His Gln Pro Gln Phe Trp Gly Pro Ala
Gln Ile Arg Ala 355 360 365Val Leu Gly Ala Val Pro Arg Gly Arg Leu
Leu Val Leu Asp Leu Phe 370 375 380Ala Glu Ser Gln Pro Val Tyr Thr
Arg Thr Ala Ser Phe Gln Gly Gln385 390 395 400Pro Phe Ile Trp Cys
Met Leu His Asn Phe Gly Gly Asn His Gly Leu 405 410 415Phe Gly Ala
Leu Glu Ala Val Asn Gly Gly Pro Glu Ala Ala Arg Leu 420 425 430Phe
Pro Asn Ser Thr Met Val Gly Thr Gly Met Ala Pro Glu Gly Ile 435 440
445Ser Gln Asn Glu Val Val Tyr Ser Leu Met Ala Glu Leu Gly Trp Arg
450 455 460Lys Asp Pro Val Pro Asp Leu Ala Ala Trp Val Thr Ser Phe
Ala Ala465 470 475 480Arg Arg Tyr Gly Val Ser His Pro Asp Ala Gly
Ala Ala Trp Arg Leu 485 490 495Leu Leu Arg Ser Val Tyr Asn Cys Ser
Gly Glu Ala Cys Arg Gly His 500 505 510Asn Arg Ser Pro Leu Val Arg
Arg Pro Ser Leu Gln Met Asn Thr Ser 515 520 525Ile Trp Tyr Asn Arg
Ser Asp Val Phe Glu Ala Trp Arg Leu Leu Leu 530 535 540Thr Ser Ala
Pro Ser Leu Ala Thr Ser Pro Ala Phe Arg Tyr Asp Leu545 550 555
560Leu Asp Leu Thr Arg Gln Ala Val Gln Glu Leu Val Ser Leu Tyr Tyr
565 570 575Glu Glu Ala Arg Ser Ala Tyr Leu Ser Lys Glu Leu Ala Ser
Leu Leu 580 585 590Arg Ala Gly Gly Val Leu Ala Tyr Glu Leu Leu Pro
Ala Leu Asp Glu 595 600 605Val Leu Ala Ser Asp Ser Arg Phe Leu Leu
Gly Ser Trp Leu Glu Gln 610 615 620Ala Arg Ala Ala Ala Val Ser Glu
Ala Glu Ala Asp Phe Tyr Glu Gln625 630 635 640Asn Ser Arg Tyr Gln
Leu Thr Leu Trp Gly Pro Glu Gly Asn Ile Leu 645 650 655Asp Tyr Ala
Asn Lys Gln Leu Ala Gly Leu Val Ala Asn Tyr Tyr Thr 660 665 670Pro
Arg Trp Arg Leu Phe Leu Glu Ala Leu Val Asp Ser Val Ala Gln 675 680
685Gly Ile Pro Phe Gln Gln His Gln Phe Asp Lys Asn Val Phe Gln Leu
690 695 700Glu Gln Ala Phe Val Leu Ser Lys Gln Arg Tyr Pro Ser Gln
Pro Arg705 710 715 720Gly Asp Thr Val Asp Leu Ala Lys Lys Ile Phe
Leu Lys Tyr Tyr Pro 725 730 735Arg Trp Val
Ala Gly Ser Trp Gly Ala Pro Gly Gly Gly Gly Gly Ala 740 745 750Ala
Ala Ala Ala Gly Gly Gly Gly Gly Gly Ala Pro Gly Gly Gly Gly 755 760
765Gly Ala Ala Ala Ala Ala Gly Gly Gly Gly Gly Gly Ala Pro Gly Gly
770 775 780Gly Gly Gly Ala Ala Ala Ala Ala Gly Gly Gly Gly Gly Gly
Ala Pro785 790 795 800Arg Gly Gly Arg Asp His Gly Asp Trp Asp Glu
Ala Ser Arg Leu Pro 805 810 815Pro Leu Pro Pro Arg Glu Asp Ala Ala
Arg Val Ala Arg Phe Val Thr 820 825 830His Val Ser Asp Trp Gly Ala
Leu Ala Thr Ile Ser Thr Leu Glu Ala 835 840 845Val Arg Gly Arg Pro
Phe Ala Asp Val Leu Ser Leu Ser Asp Gly Pro 850 855 860Pro Gly Ala
Gly Ser Gly Val Pro Tyr Phe Tyr Leu Ser Pro Leu Gln865 870 875
880Leu Ser Val Ser Asn Leu Gln Glu Asn Pro Tyr Ala Thr Leu Thr Met
885 890 895Thr Leu Ala Gln Thr Asn Phe Cys Lys Lys His Gly Phe Pro
Leu Cys 900 905 910Val His Ile Met Leu Ser Gly Thr Val Thr Lys Val
Asn Glu Thr Glu 915 920 925Met Asp Ile Ala Lys His Ser Leu Phe Ile
Arg His Pro Glu Met Lys 930 935 940Thr Trp Pro Ser Ser His Asn Trp
Phe Phe Ala Lys Leu Asn Ile Thr945 950 955 960Asn Ile Trp Val Leu
Asp Tyr Phe Gly Gly Pro Lys Ile Val Thr Pro 965 970 975Glu Glu Tyr
Tyr Asn Val Thr Val Gln 980 98521860PRTArtificial
Sequencesource/note="Description of Artificial Sequence Synthetic
polypeptide" 21Met Glu Ala Val Ala Val Ala Ala Ala Val Gly Val Leu
Leu Leu Ala1 5 10 15Gly Ala Gly Gly Ala Ala Gly Asp Glu Ala Arg Glu
Ala Ala Ala Val 20 25 30Arg Ala Leu Val Ala Arg Leu Leu Gly Pro Gly
Pro Ala Ala Asp Phe 35 40 45Ser Val Ser Val Glu Arg Ala Leu Ala Ala
Lys Pro Gly Leu Asp Thr 50 55 60Tyr Ser Leu Gly Gly Gly Gly Ala Ala
Arg Val Arg Val Arg Gly Ser65 70 75 80Thr Gly Val Ala Ala Ala Ala
Gly Leu His Arg Tyr Leu Arg Asp Phe 85 90 95Cys Gly Cys His Val Ala
Trp Ser Gly Ser Gln Leu Arg Leu Pro Arg 100 105 110Pro Leu Pro Ala
Val Pro Gly Glu Leu Thr Glu Ala Thr Pro Asn Arg 115 120 125Tyr Arg
Tyr Tyr Gln Asn Val Cys Thr Gln Ser Tyr Ser Phe Val Trp 130 135
140Trp Asp Trp Ala Arg Trp Glu Arg Glu Ile Asp Trp Met Ala Leu
Asn145 150 155 160Gly Ile Asn Leu Ala Leu Ala Trp Ser Gly Gln Glu
Ala Ile Trp Gln 165 170 175Arg Val Tyr Leu Ala Leu Gly Leu Thr Gln
Ala Glu Ile Asn Glu Phe 180 185 190Phe Thr Gly Pro Ala Phe Leu Ala
Trp Gly Arg Met Gly Asn Leu His 195 200 205Thr Trp Asp Gly Pro Leu
Pro Pro Ser Trp His Ile Lys Gln Leu Tyr 210 215 220Leu Gln His Arg
Val Leu Asp Gln Met Arg Ser Phe Gly Met Thr Pro225 230 235 240Val
Leu Pro Ala Phe Ala Gly His Val Pro Glu Ala Val Thr Arg Val 245 250
255Phe Pro Gln Val Asn Val Thr Lys Met Gly Ser Trp Gly His Phe Asn
260 265 270Cys Ser Tyr Ser Cys Ser Phe Leu Leu Ala Pro Glu Asp Pro
Ile Phe 275 280 285Pro Ile Ile Gly Ser Leu Phe Leu Arg Glu Leu Ile
Lys Glu Phe Gly 290 295 300Thr Asp His Ile Tyr Gly Ala Asp Thr Phe
Asn Glu Met Gln Pro Pro305 310 315 320Ser Ser Glu Pro Ser Tyr Leu
Ala Ala Ala Thr Thr Ala Val Tyr Glu 325 330 335Ala Met Thr Ala Val
Asp Thr Glu Ala Val Trp Leu Leu Gln Gly Trp 340 345 350Leu Phe Gln
His Gln Pro Gln Phe Trp Gly Pro Ala Gln Ile Arg Ala 355 360 365Val
Leu Gly Ala Val Pro Arg Gly Arg Leu Leu Val Leu Asp Leu Phe 370 375
380Ala Glu Ser Gln Pro Val Tyr Thr Arg Thr Ala Ser Phe Gln Gly
Gln385 390 395 400Pro Phe Ile Trp Cys Met Leu His Asn Phe Gly Gly
Asn His Gly Leu 405 410 415Phe Gly Ala Leu Glu Ala Val Asn Gly Gly
Pro Glu Ala Ala Arg Leu 420 425 430Phe Pro Asn Ser Thr Met Val Gly
Thr Gly Met Ala Pro Glu Gly Ile 435 440 445Ser Gln Asn Glu Val Val
Tyr Ser Leu Met Ala Glu Leu Gly Trp Arg 450 455 460Lys Asp Pro Val
Pro Asp Leu Ala Ala Trp Val Thr Ser Phe Ala Ala465 470 475 480Arg
Arg Tyr Gly Val Ser His Pro Asp Ala Gly Ala Ala Trp Arg Leu 485 490
495Leu Leu Arg Ser Val Tyr Asn Cys Ser Gly Glu Ala Cys Arg Gly His
500 505 510Asn Arg Ser Pro Leu Val Arg Arg Pro Ser Leu Gln Met Asn
Thr Ser 515 520 525Ile Trp Tyr Asn Arg Ser Asp Val Phe Glu Ala Trp
Arg Leu Leu Leu 530 535 540Thr Ser Ala Pro Ser Leu Ala Thr Ser Pro
Ala Phe Arg Tyr Asp Leu545 550 555 560Leu Asp Leu Thr Arg Gln Ala
Val Gln Glu Leu Val Ser Leu Tyr Tyr 565 570 575Glu Glu Ala Arg Ser
Ala Tyr Leu Ser Lys Glu Leu Ala Ser Leu Leu 580 585 590Arg Ala Gly
Gly Val Leu Ala Tyr Glu Leu Leu Pro Ala Leu Asp Glu 595 600 605Val
Leu Ala Ser Asp Ser Arg Phe Leu Leu Gly Ser Trp Leu Glu Gln 610 615
620Ala Arg Ala Ala Ala Val Ser Glu Ala Glu Ala Asp Phe Tyr Glu
Gln625 630 635 640Asn Ser Arg Tyr Gln Leu Thr Leu Trp Gly Pro Glu
Gly Asn Ile Leu 645 650 655Asp Tyr Ala Asn Lys Gln Leu Ala Gly Leu
Val Ala Asn Tyr Tyr Thr 660 665 670Pro Arg Trp Arg Leu Phe Leu Glu
Ala Leu Val Asp Ser Val Ala Gln 675 680 685Gly Ile Pro Phe Gln Gln
His Gln Phe Asp Lys Asn Val Phe Gln Leu 690 695 700Glu Gln Ala Phe
Val Leu Ser Lys Gln Arg Tyr Pro Ser Gln Pro Arg705 710 715 720Gly
Asp Thr Val Asp Leu Ala Lys Lys Ile Phe Leu Lys Tyr Tyr Pro 725 730
735Arg Trp Val Ala Gly Ser Trp Gly Ala Pro Gly Gly Gly Gly Gly Ala
740 745 750Ala Ala Ala Ala Gly Gly Gly Gly Gly Gly Ala Pro Gly Gly
Gly Gly 755 760 765Gly Ala Ala Ala Ala Ala Gly Gly Gly Gly Gly Gly
Ala Pro Gly Gly 770 775 780Gly Gly Gly Ala Ala Ala Ala Ala Gly Gly
Gly Gly Gly Gly Ala Pro785 790 795 800Leu Cys Gly Gly Glu Leu Val
Asp Thr Leu Gln Phe Val Cys Gly Asp 805 810 815Arg Gly Phe Tyr Phe
Ser Arg Pro Ala Ser Arg Val Ser Arg Arg Ser 820 825 830Arg Gly Ile
Val Glu Glu Cys Cys Phe Arg Ser Cys Asp Leu Ala Leu 835 840 845Leu
Glu Thr Tyr Cys Ala Thr Pro Ala Lys Ser Glu 850 855 8602260PRTHomo
sapiens 22Leu Cys Gly Gly Glu Leu Val Asp Thr Leu Gln Phe Val Cys
Gly Asp1 5 10 15Arg Gly Phe Tyr Phe Ser Arg Pro Ala Ser Arg Val Ser
Arg Arg Ser 20 25 30Arg Gly Ile Val Glu Glu Cys Cys Phe Arg Ser Cys
Asp Leu Ala Leu 35 40 45Leu Glu Thr Tyr Cys Ala Thr Pro Ala Lys Ser
Glu 50 55 6023897PRTArtificial Sequencesource/note="Description of
Artificial Sequence Synthetic polypeptide" 23Asp Glu Ala Arg Glu
Ala Ala Ala Val Arg Ala Leu Val Ala Arg Leu1 5 10 15Leu Gly Pro Gly
Pro Ala Ala Asp Phe Ser Val Ser Val Glu Arg Ala 20 25 30Leu Ala Ala
Lys Pro Gly Leu Asp Thr Tyr Ser Leu Gly Gly Gly Gly 35 40 45Ala Ala
Arg Val Arg Val Arg Gly Ser Thr Gly Val Ala Ala Ala Ala 50 55 60Gly
Leu His Arg Tyr Leu Arg Asp Phe Cys Gly Cys His Val Ala Trp65 70 75
80Ser Gly Ser Gln Leu Arg Leu Pro Arg Pro Leu Pro Ala Val Pro Gly
85 90 95Glu Leu Thr Glu Ala Thr Pro Asn Arg Tyr Arg Tyr Tyr Gln Asn
Val 100 105 110Cys Thr Gln Ser Tyr Ser Phe Val Trp Trp Asp Trp Ala
Arg Trp Glu 115 120 125Arg Glu Ile Asp Trp Met Ala Leu Asn Gly Ile
Asn Leu Ala Leu Ala 130 135 140Trp Ser Gly Gln Glu Ala Ile Trp Gln
Arg Val Tyr Leu Ala Leu Gly145 150 155 160Leu Thr Gln Ala Glu Ile
Asn Glu Phe Phe Thr Gly Pro Ala Phe Leu 165 170 175Ala Trp Gly Arg
Met Gly Asn Leu His Thr Trp Asp Gly Pro Leu Pro 180 185 190Pro Ser
Trp His Ile Lys Gln Leu Tyr Leu Gln His Arg Val Leu Asp 195 200
205Gln Met Arg Ser Phe Gly Met Thr Pro Val Leu Pro Ala Phe Ala Gly
210 215 220His Val Pro Glu Ala Val Thr Arg Val Phe Pro Gln Val Asn
Val Thr225 230 235 240Lys Met Gly Ser Trp Gly His Phe Asn Cys Ser
Tyr Ser Cys Ser Phe 245 250 255Leu Leu Ala Pro Glu Asp Pro Ile Phe
Pro Ile Ile Gly Ser Leu Phe 260 265 270Leu Arg Glu Leu Ile Lys Glu
Phe Gly Thr Asp His Ile Tyr Gly Ala 275 280 285Asp Thr Phe Asn Glu
Met Gln Pro Pro Ser Ser Glu Pro Ser Tyr Leu 290 295 300Ala Ala Ala
Thr Thr Ala Val Tyr Glu Ala Met Thr Ala Val Asp Thr305 310 315
320Glu Ala Val Trp Leu Leu Gln Gly Trp Leu Phe Gln His Gln Pro Gln
325 330 335Phe Trp Gly Pro Ala Gln Ile Arg Ala Val Leu Gly Ala Val
Pro Arg 340 345 350Gly Arg Leu Leu Val Leu Asp Leu Phe Ala Glu Ser
Gln Pro Val Tyr 355 360 365Thr Arg Thr Ala Ser Phe Gln Gly Gln Pro
Phe Ile Trp Cys Met Leu 370 375 380His Asn Phe Gly Gly Asn His Gly
Leu Phe Gly Ala Leu Glu Ala Val385 390 395 400Asn Gly Gly Pro Glu
Ala Ala Arg Leu Phe Pro Asn Ser Thr Met Val 405 410 415Gly Thr Gly
Met Ala Pro Glu Gly Ile Ser Gln Asn Glu Val Val Tyr 420 425 430Ser
Leu Met Ala Glu Leu Gly Trp Arg Lys Asp Pro Val Pro Asp Leu 435 440
445Ala Ala Trp Val Thr Ser Phe Ala Ala Arg Arg Tyr Gly Val Ser His
450 455 460Pro Asp Ala Gly Ala Ala Trp Arg Leu Leu Leu Arg Ser Val
Tyr Asn465 470 475 480Cys Ser Gly Glu Ala Cys Arg Gly His Asn Arg
Ser Pro Leu Val Arg 485 490 495Arg Pro Ser Leu Gln Met Asn Thr Ser
Ile Trp Tyr Asn Arg Ser Asp 500 505 510Val Phe Glu Ala Trp Arg Leu
Leu Leu Thr Ser Ala Pro Ser Leu Ala 515 520 525Thr Ser Pro Ala Phe
Arg Tyr Asp Leu Leu Asp Leu Thr Arg Gln Ala 530 535 540Val Gln Glu
Leu Val Ser Leu Tyr Tyr Glu Glu Ala Arg Ser Ala Tyr545 550 555
560Leu Ser Lys Glu Leu Ala Ser Leu Leu Arg Ala Gly Gly Val Leu Ala
565 570 575Tyr Glu Leu Leu Pro Ala Leu Asp Glu Val Leu Ala Ser Asp
Ser Arg 580 585 590Phe Leu Leu Gly Ser Trp Leu Glu Gln Ala Arg Ala
Ala Ala Val Ser 595 600 605Glu Ala Glu Ala Asp Phe Tyr Glu Gln Asn
Ser Arg Tyr Gln Leu Thr 610 615 620Leu Trp Gly Pro Glu Gly Asn Ile
Leu Asp Tyr Ala Asn Lys Gln Leu625 630 635 640Ala Gly Leu Val Ala
Asn Tyr Tyr Thr Pro Arg Trp Arg Leu Phe Leu 645 650 655Glu Ala Leu
Val Asp Ser Val Ala Gln Gly Ile Pro Phe Gln Gln His 660 665 670Gln
Phe Asp Lys Asn Val Phe Gln Leu Glu Gln Ala Phe Val Leu Ser 675 680
685Lys Gln Arg Tyr Pro Ser Gln Pro Arg Gly Asp Thr Val Asp Leu Ala
690 695 700Lys Lys Ile Phe Leu Lys Tyr Tyr Pro Arg Trp Val Ala Gly
Ser Trp705 710 715 720Gly Ala Pro Gly Gly Gly Gly Gly Ala Ala Ala
Ala Ala Gly Gly Gly 725 730 735Gly Gly Gly Ala Pro Gly Gly Gly Gly
Gly Ala Ala Ala Ala Ala Gly 740 745 750Gly Gly Gly Gly Gly Ala Pro
Gly Gly Gly Gly Gly Ala Ala Ala Ala 755 760 765Ala Gly Gly Gly Gly
Gly Gly Ala Pro Asp Gly Gly Phe Cys Glu Val 770 775 780Cys Lys Lys
Leu Val Gly Tyr Leu Asp Arg Asn Leu Glu Lys Asn Ser785 790 795
800Thr Lys Gln Glu Ile Leu Ala Ala Leu Glu Lys Gly Cys Ser Phe Leu
805 810 815Pro Asp Pro Tyr Gln Lys Gln Cys Asp Gln Phe Val Ala Glu
Tyr Glu 820 825 830Pro Val Leu Ile Glu Ile Leu Val Glu Val Met Asp
Pro Ser Phe Val 835 840 845Cys Leu Lys Ile Gly Ala Cys Pro Ser Ala
His Lys Pro Leu Leu Gly 850 855 860Thr Glu Lys Cys Ile Trp Gly Pro
Ser Tyr Trp Cys Gln Asn Thr Glu865 870 875 880Thr Ala Ala Gln Cys
Asn Ala Val Glu His Cys Lys Arg His Val Trp 885 890
895Asn24957PRTArtificial Sequencesource/note="Description of
Artificial Sequence Synthetic polypeptide" 24Asp Glu Ala Arg Glu
Ala Ala Ala Val Arg Ala Leu Val Ala Arg Leu1 5 10 15Leu Gly Pro Gly
Pro Ala Ala Asp Phe Ser Val Ser Val Glu Arg Ala 20 25 30Leu Ala Ala
Lys Pro Gly Leu Asp Thr Tyr Ser Leu Gly Gly Gly Gly 35 40 45Ala Ala
Arg Val Arg Val Arg Gly Ser Thr Gly Val Ala Ala Ala Ala 50 55 60Gly
Leu His Arg Tyr Leu Arg Asp Phe Cys Gly Cys His Val Ala Trp65 70 75
80Ser Gly Ser Gln Leu Arg Leu Pro Arg Pro Leu Pro Ala Val Pro Gly
85 90 95Glu Leu Thr Glu Ala Thr Pro Asn Arg Tyr Arg Tyr Tyr Gln Asn
Val 100 105 110Cys Thr Gln Ser Tyr Ser Phe Val Trp Trp Asp Trp Ala
Arg Trp Glu 115 120 125Arg Glu Ile Asp Trp Met Ala Leu Asn Gly Ile
Asn Leu Ala Leu Ala 130 135 140Trp Ser Gly Gln Glu Ala Ile Trp Gln
Arg Val Tyr Leu Ala Leu Gly145 150 155 160Leu Thr Gln Ala Glu Ile
Asn Glu Phe Phe Thr Gly Pro Ala Phe Leu 165 170 175Ala Trp Gly Arg
Met Gly Asn Leu His Thr Trp Asp Gly Pro Leu Pro 180 185 190Pro Ser
Trp His Ile Lys Gln Leu Tyr Leu Gln His Arg Val Leu Asp 195 200
205Gln Met Arg Ser Phe Gly Met Thr Pro Val Leu Pro Ala Phe Ala Gly
210 215 220His Val Pro Glu Ala Val Thr Arg Val Phe Pro Gln Val Asn
Val Thr225 230 235 240Lys Met Gly Ser Trp Gly His Phe Asn Cys Ser
Tyr Ser Cys Ser Phe 245 250 255Leu Leu Ala Pro Glu Asp Pro Ile Phe
Pro Ile Ile Gly Ser Leu Phe 260 265 270Leu Arg Glu Leu Ile Lys Glu
Phe Gly Thr Asp His Ile Tyr Gly Ala 275 280 285Asp Thr Phe Asn Glu
Met Gln Pro Pro Ser Ser Glu Pro Ser Tyr Leu 290 295 300Ala Ala Ala
Thr Thr Ala Val Tyr Glu Ala Met Thr Ala Val Asp Thr305 310 315
320Glu Ala Val Trp Leu Leu Gln Gly Trp Leu Phe Gln His Gln Pro Gln
325 330 335Phe Trp Gly Pro Ala Gln Ile Arg Ala Val Leu Gly Ala Val
Pro Arg 340 345 350Gly Arg Leu Leu Val Leu Asp Leu Phe Ala Glu
Ser Gln Pro Val Tyr 355 360 365Thr Arg Thr Ala Ser Phe Gln Gly Gln
Pro Phe Ile Trp Cys Met Leu 370 375 380His Asn Phe Gly Gly Asn His
Gly Leu Phe Gly Ala Leu Glu Ala Val385 390 395 400Asn Gly Gly Pro
Glu Ala Ala Arg Leu Phe Pro Asn Ser Thr Met Val 405 410 415Gly Thr
Gly Met Ala Pro Glu Gly Ile Ser Gln Asn Glu Val Val Tyr 420 425
430Ser Leu Met Ala Glu Leu Gly Trp Arg Lys Asp Pro Val Pro Asp Leu
435 440 445Ala Ala Trp Val Thr Ser Phe Ala Ala Arg Arg Tyr Gly Val
Ser His 450 455 460Pro Asp Ala Gly Ala Ala Trp Arg Leu Leu Leu Arg
Ser Val Tyr Asn465 470 475 480Cys Ser Gly Glu Ala Cys Arg Gly His
Asn Arg Ser Pro Leu Val Arg 485 490 495Arg Pro Ser Leu Gln Met Asn
Thr Ser Ile Trp Tyr Asn Arg Ser Asp 500 505 510Val Phe Glu Ala Trp
Arg Leu Leu Leu Thr Ser Ala Pro Ser Leu Ala 515 520 525Thr Ser Pro
Ala Phe Arg Tyr Asp Leu Leu Asp Leu Thr Arg Gln Ala 530 535 540Val
Gln Glu Leu Val Ser Leu Tyr Tyr Glu Glu Ala Arg Ser Ala Tyr545 550
555 560Leu Ser Lys Glu Leu Ala Ser Leu Leu Arg Ala Gly Gly Val Leu
Ala 565 570 575Tyr Glu Leu Leu Pro Ala Leu Asp Glu Val Leu Ala Ser
Asp Ser Arg 580 585 590Phe Leu Leu Gly Ser Trp Leu Glu Gln Ala Arg
Ala Ala Ala Val Ser 595 600 605Glu Ala Glu Ala Asp Phe Tyr Glu Gln
Asn Ser Arg Tyr Gln Leu Thr 610 615 620Leu Trp Gly Pro Glu Gly Asn
Ile Leu Asp Tyr Ala Asn Lys Gln Leu625 630 635 640Ala Gly Leu Val
Ala Asn Tyr Tyr Thr Pro Arg Trp Arg Leu Phe Leu 645 650 655Glu Ala
Leu Val Asp Ser Val Ala Gln Gly Ile Pro Phe Gln Gln His 660 665
670Gln Phe Asp Lys Asn Val Phe Gln Leu Glu Gln Ala Phe Val Leu Ser
675 680 685Lys Gln Arg Tyr Pro Ser Gln Pro Arg Gly Asp Thr Val Asp
Leu Ala 690 695 700Lys Lys Ile Phe Leu Lys Tyr Tyr Pro Arg Trp Val
Ala Gly Ser Trp705 710 715 720Gly Ala Pro Gly Gly Gly Gly Gly Ala
Ala Ala Ala Ala Gly Gly Gly 725 730 735Gly Gly Gly Ala Pro Gly Gly
Gly Gly Gly Ala Ala Ala Ala Ala Gly 740 745 750Gly Gly Gly Gly Gly
Ala Pro Gly Gly Gly Gly Gly Ala Ala Ala Ala 755 760 765Ala Gly Gly
Gly Gly Gly Gly Ala Pro Ser Pro Leu Pro Ile Thr Pro 770 775 780Val
Asn Ala Thr Cys Ala Ile Arg His Pro Cys His Asn Asn Leu Met785 790
795 800Asn Ala Ile Arg Ala Gln Leu Ala Ala Leu Asn Gly Ser Ala Asn
Ala 805 810 815Leu Phe Ile Leu Tyr Tyr Thr Ala Gln Gly Glu Pro Phe
Pro Asn Asn 820 825 830Leu Asp Lys Leu Cys Gly Pro Asn Val Thr Asp
Phe Pro Pro Phe His 835 840 845Ala Asn Gly Thr Glu Lys Ala Lys Leu
Val Glu Leu Tyr Arg Ile Val 850 855 860Val Tyr Leu Gly Thr Ser Leu
Gly Asn Ile Thr Arg Asp Gln Lys Ile865 870 875 880Leu Asn Pro Ser
Ala Leu Ser Leu His Ser Lys Leu Asn Ala Thr Ala 885 890 895Asp Ile
Leu Arg Gly Leu Leu Ser Asn Val Leu Cys Arg Leu Cys Ser 900 905
910Lys Tyr His Val Gly His Val Asp Val Thr Tyr Gly Pro Asp Thr Ser
915 920 925Gly Lys Asp Val Ala Gln Lys Ala Lys Leu Gly Cys Gln Leu
Leu Gly 930 935 940Lys Tyr Lys Gln Ile Ile Ala Val Leu Ala Gln Ala
Phe945 950 95525962PRTArtificial Sequencesource/note="Description
of Artificial Sequence Synthetic polypeptide" 25Asp Glu Ala Arg Glu
Ala Ala Ala Val Arg Ala Leu Val Ala Arg Leu1 5 10 15Leu Gly Pro Gly
Pro Ala Ala Asp Phe Ser Val Ser Val Glu Arg Ala 20 25 30Leu Ala Ala
Lys Pro Gly Leu Asp Thr Tyr Ser Leu Gly Gly Gly Gly 35 40 45Ala Ala
Arg Val Arg Val Arg Gly Ser Thr Gly Val Ala Ala Ala Ala 50 55 60Gly
Leu His Arg Tyr Leu Arg Asp Phe Cys Gly Cys His Val Ala Trp65 70 75
80Ser Gly Ser Gln Leu Arg Leu Pro Arg Pro Leu Pro Ala Val Pro Gly
85 90 95Glu Leu Thr Glu Ala Thr Pro Asn Arg Tyr Arg Tyr Tyr Gln Asn
Val 100 105 110Cys Thr Gln Ser Tyr Ser Phe Val Trp Trp Asp Trp Ala
Arg Trp Glu 115 120 125Arg Glu Ile Asp Trp Met Ala Leu Asn Gly Ile
Asn Leu Ala Leu Ala 130 135 140Trp Ser Gly Gln Glu Ala Ile Trp Gln
Arg Val Tyr Leu Ala Leu Gly145 150 155 160Leu Thr Gln Ala Glu Ile
Asn Glu Phe Phe Thr Gly Pro Ala Phe Leu 165 170 175Ala Trp Gly Arg
Met Gly Asn Leu His Thr Trp Asp Gly Pro Leu Pro 180 185 190Pro Ser
Trp His Ile Lys Gln Leu Tyr Leu Gln His Arg Val Leu Asp 195 200
205Gln Met Arg Ser Phe Gly Met Thr Pro Val Leu Pro Ala Phe Ala Gly
210 215 220His Val Pro Glu Ala Val Thr Arg Val Phe Pro Gln Val Asn
Val Thr225 230 235 240Lys Met Gly Ser Trp Gly His Phe Asn Cys Ser
Tyr Ser Cys Ser Phe 245 250 255Leu Leu Ala Pro Glu Asp Pro Ile Phe
Pro Ile Ile Gly Ser Leu Phe 260 265 270Leu Arg Glu Leu Ile Lys Glu
Phe Gly Thr Asp His Ile Tyr Gly Ala 275 280 285Asp Thr Phe Asn Glu
Met Gln Pro Pro Ser Ser Glu Pro Ser Tyr Leu 290 295 300Ala Ala Ala
Thr Thr Ala Val Tyr Glu Ala Met Thr Ala Val Asp Thr305 310 315
320Glu Ala Val Trp Leu Leu Gln Gly Trp Leu Phe Gln His Gln Pro Gln
325 330 335Phe Trp Gly Pro Ala Gln Ile Arg Ala Val Leu Gly Ala Val
Pro Arg 340 345 350Gly Arg Leu Leu Val Leu Asp Leu Phe Ala Glu Ser
Gln Pro Val Tyr 355 360 365Thr Arg Thr Ala Ser Phe Gln Gly Gln Pro
Phe Ile Trp Cys Met Leu 370 375 380His Asn Phe Gly Gly Asn His Gly
Leu Phe Gly Ala Leu Glu Ala Val385 390 395 400Asn Gly Gly Pro Glu
Ala Ala Arg Leu Phe Pro Asn Ser Thr Met Val 405 410 415Gly Thr Gly
Met Ala Pro Glu Gly Ile Ser Gln Asn Glu Val Val Tyr 420 425 430Ser
Leu Met Ala Glu Leu Gly Trp Arg Lys Asp Pro Val Pro Asp Leu 435 440
445Ala Ala Trp Val Thr Ser Phe Ala Ala Arg Arg Tyr Gly Val Ser His
450 455 460Pro Asp Ala Gly Ala Ala Trp Arg Leu Leu Leu Arg Ser Val
Tyr Asn465 470 475 480Cys Ser Gly Glu Ala Cys Arg Gly His Asn Arg
Ser Pro Leu Val Arg 485 490 495Arg Pro Ser Leu Gln Met Asn Thr Ser
Ile Trp Tyr Asn Arg Ser Asp 500 505 510Val Phe Glu Ala Trp Arg Leu
Leu Leu Thr Ser Ala Pro Ser Leu Ala 515 520 525Thr Ser Pro Ala Phe
Arg Tyr Asp Leu Leu Asp Leu Thr Arg Gln Ala 530 535 540Val Gln Glu
Leu Val Ser Leu Tyr Tyr Glu Glu Ala Arg Ser Ala Tyr545 550 555
560Leu Ser Lys Glu Leu Ala Ser Leu Leu Arg Ala Gly Gly Val Leu Ala
565 570 575Tyr Glu Leu Leu Pro Ala Leu Asp Glu Val Leu Ala Ser Asp
Ser Arg 580 585 590Phe Leu Leu Gly Ser Trp Leu Glu Gln Ala Arg Ala
Ala Ala Val Ser 595 600 605Glu Ala Glu Ala Asp Phe Tyr Glu Gln Asn
Ser Arg Tyr Gln Leu Thr 610 615 620Leu Trp Gly Pro Glu Gly Asn Ile
Leu Asp Tyr Ala Asn Lys Gln Leu625 630 635 640Ala Gly Leu Val Ala
Asn Tyr Tyr Thr Pro Arg Trp Arg Leu Phe Leu 645 650 655Glu Ala Leu
Val Asp Ser Val Ala Gln Gly Ile Pro Phe Gln Gln His 660 665 670Gln
Phe Asp Lys Asn Val Phe Gln Leu Glu Gln Ala Phe Val Leu Ser 675 680
685Lys Gln Arg Tyr Pro Ser Gln Pro Arg Gly Asp Thr Val Asp Leu Ala
690 695 700Lys Lys Ile Phe Leu Lys Tyr Tyr Pro Arg Trp Val Ala Gly
Ser Trp705 710 715 720Gly Ala Pro Gly Gly Gly Gly Gly Ala Ala Ala
Ala Ala Gly Gly Gly 725 730 735Gly Gly Gly Ala Pro Gly Gly Gly Gly
Gly Ala Ala Ala Ala Ala Gly 740 745 750Gly Gly Gly Gly Gly Ala Pro
Gly Gly Gly Gly Gly Ala Ala Ala Ala 755 760 765Ala Gly Gly Gly Gly
Gly Gly Ala Pro Arg Gly Gly Arg Asp His Gly 770 775 780Asp Trp Asp
Glu Ala Ser Arg Leu Pro Pro Leu Pro Pro Arg Glu Asp785 790 795
800Ala Ala Arg Val Ala Arg Phe Val Thr His Val Ser Asp Trp Gly Ala
805 810 815Leu Ala Thr Ile Ser Thr Leu Glu Ala Val Arg Gly Arg Pro
Phe Ala 820 825 830Asp Val Leu Ser Leu Ser Asp Gly Pro Pro Gly Ala
Gly Ser Gly Val 835 840 845Pro Tyr Phe Tyr Leu Ser Pro Leu Gln Leu
Ser Val Ser Asn Leu Gln 850 855 860Glu Asn Pro Tyr Ala Thr Leu Thr
Met Thr Leu Ala Gln Thr Asn Phe865 870 875 880Cys Lys Lys His Gly
Phe Pro Leu Cys Val His Ile Met Leu Ser Gly 885 890 895Thr Val Thr
Lys Val Asn Glu Thr Glu Met Asp Ile Ala Lys His Ser 900 905 910Leu
Phe Ile Arg His Pro Glu Met Lys Thr Trp Pro Ser Ser His Asn 915 920
925Trp Phe Phe Ala Lys Leu Asn Ile Thr Asn Ile Trp Val Leu Asp Tyr
930 935 940Phe Gly Gly Pro Lys Ile Val Thr Pro Glu Glu Tyr Tyr Asn
Val Thr945 950 955 960Val Gln26837PRTArtificial
Sequencesource/note="Description of Artificial Sequence Synthetic
polypeptide" 26Asp Glu Ala Arg Glu Ala Ala Ala Val Arg Ala Leu Val
Ala Arg Leu1 5 10 15Leu Gly Pro Gly Pro Ala Ala Asp Phe Ser Val Ser
Val Glu Arg Ala 20 25 30Leu Ala Ala Lys Pro Gly Leu Asp Thr Tyr Ser
Leu Gly Gly Gly Gly 35 40 45Ala Ala Arg Val Arg Val Arg Gly Ser Thr
Gly Val Ala Ala Ala Ala 50 55 60Gly Leu His Arg Tyr Leu Arg Asp Phe
Cys Gly Cys His Val Ala Trp65 70 75 80Ser Gly Ser Gln Leu Arg Leu
Pro Arg Pro Leu Pro Ala Val Pro Gly 85 90 95Glu Leu Thr Glu Ala Thr
Pro Asn Arg Tyr Arg Tyr Tyr Gln Asn Val 100 105 110Cys Thr Gln Ser
Tyr Ser Phe Val Trp Trp Asp Trp Ala Arg Trp Glu 115 120 125Arg Glu
Ile Asp Trp Met Ala Leu Asn Gly Ile Asn Leu Ala Leu Ala 130 135
140Trp Ser Gly Gln Glu Ala Ile Trp Gln Arg Val Tyr Leu Ala Leu
Gly145 150 155 160Leu Thr Gln Ala Glu Ile Asn Glu Phe Phe Thr Gly
Pro Ala Phe Leu 165 170 175Ala Trp Gly Arg Met Gly Asn Leu His Thr
Trp Asp Gly Pro Leu Pro 180 185 190Pro Ser Trp His Ile Lys Gln Leu
Tyr Leu Gln His Arg Val Leu Asp 195 200 205Gln Met Arg Ser Phe Gly
Met Thr Pro Val Leu Pro Ala Phe Ala Gly 210 215 220His Val Pro Glu
Ala Val Thr Arg Val Phe Pro Gln Val Asn Val Thr225 230 235 240Lys
Met Gly Ser Trp Gly His Phe Asn Cys Ser Tyr Ser Cys Ser Phe 245 250
255Leu Leu Ala Pro Glu Asp Pro Ile Phe Pro Ile Ile Gly Ser Leu Phe
260 265 270Leu Arg Glu Leu Ile Lys Glu Phe Gly Thr Asp His Ile Tyr
Gly Ala 275 280 285Asp Thr Phe Asn Glu Met Gln Pro Pro Ser Ser Glu
Pro Ser Tyr Leu 290 295 300Ala Ala Ala Thr Thr Ala Val Tyr Glu Ala
Met Thr Ala Val Asp Thr305 310 315 320Glu Ala Val Trp Leu Leu Gln
Gly Trp Leu Phe Gln His Gln Pro Gln 325 330 335Phe Trp Gly Pro Ala
Gln Ile Arg Ala Val Leu Gly Ala Val Pro Arg 340 345 350Gly Arg Leu
Leu Val Leu Asp Leu Phe Ala Glu Ser Gln Pro Val Tyr 355 360 365Thr
Arg Thr Ala Ser Phe Gln Gly Gln Pro Phe Ile Trp Cys Met Leu 370 375
380His Asn Phe Gly Gly Asn His Gly Leu Phe Gly Ala Leu Glu Ala
Val385 390 395 400Asn Gly Gly Pro Glu Ala Ala Arg Leu Phe Pro Asn
Ser Thr Met Val 405 410 415Gly Thr Gly Met Ala Pro Glu Gly Ile Ser
Gln Asn Glu Val Val Tyr 420 425 430Ser Leu Met Ala Glu Leu Gly Trp
Arg Lys Asp Pro Val Pro Asp Leu 435 440 445Ala Ala Trp Val Thr Ser
Phe Ala Ala Arg Arg Tyr Gly Val Ser His 450 455 460Pro Asp Ala Gly
Ala Ala Trp Arg Leu Leu Leu Arg Ser Val Tyr Asn465 470 475 480Cys
Ser Gly Glu Ala Cys Arg Gly His Asn Arg Ser Pro Leu Val Arg 485 490
495Arg Pro Ser Leu Gln Met Asn Thr Ser Ile Trp Tyr Asn Arg Ser Asp
500 505 510Val Phe Glu Ala Trp Arg Leu Leu Leu Thr Ser Ala Pro Ser
Leu Ala 515 520 525Thr Ser Pro Ala Phe Arg Tyr Asp Leu Leu Asp Leu
Thr Arg Gln Ala 530 535 540Val Gln Glu Leu Val Ser Leu Tyr Tyr Glu
Glu Ala Arg Ser Ala Tyr545 550 555 560Leu Ser Lys Glu Leu Ala Ser
Leu Leu Arg Ala Gly Gly Val Leu Ala 565 570 575Tyr Glu Leu Leu Pro
Ala Leu Asp Glu Val Leu Ala Ser Asp Ser Arg 580 585 590Phe Leu Leu
Gly Ser Trp Leu Glu Gln Ala Arg Ala Ala Ala Val Ser 595 600 605Glu
Ala Glu Ala Asp Phe Tyr Glu Gln Asn Ser Arg Tyr Gln Leu Thr 610 615
620Leu Trp Gly Pro Glu Gly Asn Ile Leu Asp Tyr Ala Asn Lys Gln
Leu625 630 635 640Ala Gly Leu Val Ala Asn Tyr Tyr Thr Pro Arg Trp
Arg Leu Phe Leu 645 650 655Glu Ala Leu Val Asp Ser Val Ala Gln Gly
Ile Pro Phe Gln Gln His 660 665 670Gln Phe Asp Lys Asn Val Phe Gln
Leu Glu Gln Ala Phe Val Leu Ser 675 680 685Lys Gln Arg Tyr Pro Ser
Gln Pro Arg Gly Asp Thr Val Asp Leu Ala 690 695 700Lys Lys Ile Phe
Leu Lys Tyr Tyr Pro Arg Trp Val Ala Gly Ser Trp705 710 715 720Gly
Ala Pro Gly Gly Gly Gly Gly Ala Ala Ala Ala Ala Gly Gly Gly 725 730
735Gly Gly Gly Ala Pro Gly Gly Gly Gly Gly Ala Ala Ala Ala Ala Gly
740 745 750Gly Gly Gly Gly Gly Ala Pro Gly Gly Gly Gly Gly Ala Ala
Ala Ala 755 760 765Ala Gly Gly Gly Gly Gly Gly Ala Pro Leu Cys Gly
Gly Glu Leu Val 770 775 780Asp Thr Leu Gln Phe Val Cys Gly Asp Arg
Gly Phe Tyr Phe Ser Arg785 790 795 800Pro Ala Ser Arg Val Ser Arg
Arg Ser Arg Gly Ile Val Glu Glu Cys 805 810 815Cys Phe Arg Ser Cys
Asp Leu Ala Leu Leu Glu Thr Tyr Cys Ala Thr 820 825 830Pro Ala Lys
Ser Glu 835273771DNAArtificial Sequencesource/note="Description of
Artificial Sequence Synthetic polynucleotide" 27atgctgttca
agctcctgca gagacagacc tatacctgcc tgtcccacag gtatgggctc 60tacgtgtgct
tcttgggcgt cgttgtcacc atcgtctccg ccttccagtt cggagaggtg
120gttctggaat ggagccgaga tcaataccat gttttgtttg attcctatag
agacaatatt 180gctggaaagt cctttcagaa
tcggctttgt ctgcccatgc cgattgacgt tgtttacacc 240tgggtgaatg
gcacagatct tgaactactg aaggaactac agcaggtcag agaacagatg
300gaggaggagc agaaagcaat gagagaaatc cttgggaaaa acacaacgga
acctactaag 360aagagtgaga agcagttaga gtgtttgcta acacactgca
ttaaggtgcc aatgcttgtc 420ctggacccag ccctgccagc caacatcacc
ctgaaggacc tgccatctct ttatccttct 480tttcattctg ccagtgacat
tttcaatgtt gcaaaaccaa aaaacccttc taccaatgtc 540tcagttgttg
tttttgacag tactaaggat gttgaagatg cccactctgg actgcttaaa
600ggaaatagca gacagacagt atggaggggc tacttgacaa cagataaaga
agtccctgga 660ttagtgctaa tgcaagattt ggctttcctg agtggatttc
caccaacatt caaggaaaca 720aatcaactaa aaacaaaatt gccagaaaat
ctttcctcta aagtcaaact gttgcagttg 780tattcagagg ccagtgtagc
gcttctaaaa ctgaataacc ccaaggattt tcaagaattg 840aataagcaaa
ctaagaagaa catgaccatt gatggaaaag aactgaccat aagtcctgca
900tatttattat gggatctgag cgccatcagc cagtctaagc aggatgaaga
catctctgcc 960agtcgttttg aagataacga agaactgagg tactcattgc
gatctatcga gaggcatgca 1020ccatgggttc ggaatatttt cattgtcacc
aacgggcaga ttccatcctg gctgaacctt 1080gacaatcctc gagtgacaat
agtaacacac caggatgttt ttcgaaattt gagccacttg 1140cctaccttta
gttcacctgc tattgaaagt cacattcatc gcatcgaagg gctgtcccag
1200aagtttattt acctaaatga tgatgtcatg tttgggaagg atgtctggcc
agatgatttt 1260tacagtcact ccaaaggcca gaaggtttat ttgacatggc
ctgtgccaaa ctgtgccgag 1320ggctgcccag gttcctggat taaggatggc
tattgtgaca aggcttgtaa taattcagcc 1380tgcgattggg atggtgggga
ttgctctgga aacagtggag ggagtcgcta tattgcagga 1440ggtggaggta
ctgggagtat tggagttgga cagccctggc agtttggtgg aggaataaac
1500agtgtctctt actgtaatca gggatgtgcg aattcctggc tcgctgataa
gttctgtgac 1560caagcatgca atgtcttgtc ctgtgggttt gatgctggcg
actgtgggca agatcatttt 1620catgaattgt ataaagtgat ccttctccca
aaccagactc actatattat tccaaaaggt 1680gaatgcctgc cttatttcag
ctttgcagaa gtagccaaaa gaggagttga aggtgcctat 1740agtgacaatc
caataattcg acatgcttct attgccaaca agtggaaaac catccacctc
1800ataatgcaca gtggaatgaa tgccaccaca atacatttta atctcacgtt
tcaaaataca 1860aacgatgaag agttcaaaat gcagataaca gtggaggtgg
acacaaggga gggaccaaaa 1920ctgaattcta cagcccagaa gggttacgaa
aatttagtta gtcccataac acttcttcca 1980gaggcggaaa tcctttttga
ggatattccc aaagaaaaac gcttcccgaa gtttaagaga 2040catgatgtta
actcaacaag gagagcccag gaagaggtga aaattcccct ggtaaatatt
2100tcactccttc caaaagacgc ccagttgagt ctcaatacct tggatttgca
actggaacat 2160ggagacatca ctttgaaagg atacaatttg tccaagtcag
ccttgctgag atcatttctg 2220atgaactcac agcatgctaa aataaaaaat
caagctataa taacagatga aacaaatgac 2280agtttggtgg ctccacagga
aaaacaggtt cataaaagca tcttgccaaa cagcttagga 2340gtgtctgaaa
gattgcagag gttgactttt cctgcagtga gtgtaaaagt gaatggtcat
2400gaccagggtc agaatccacc cctggacttg gagaccacag caagatttag
agtggaaact 2460cacacccaaa aaaccatagg cggaaatgtg acaaaagaaa
agcccccatc tctgattgtt 2520ccactggaaa gccagatgac aaaagaaaag
aaaatcacag ggaaagaaaa agagaacagt 2580agaatggagg aaaatgctga
aaatcacata ggcgttactg aagtgttact tggaagaaag 2640ctgcagcatt
acacagatag ttacttgggc tttttgccat gggagaaaaa aaagtatttc
2700caagatcttc tcgacgaaga agagtcattg aagacacaat tggcatactt
cactgatagc 2760aaaaatactg ggaggcaact aaaagataca tttgcagatt
ccctcagata tgtaaataaa 2820attctaaata gcaagtttgg attcacatcg
cggaaagtcc ctgctcacat gcctcacatg 2880attgaccgga ttgttatgca
agaactgcaa gatatgttcc ctgaagaatt tgacaagacg 2940tcatttcaca
aagtgcgcca ttctgaggat atgcagtttg ccttctctta tttttattat
3000ctcatgagtg cagtgcagcc actgaatata tctcaagtct ttgatgaagt
tgatacagat 3060caatctggtg tcttgtctga cagagaaatc cgaacactgg
ctaccagaat tcacgaactg 3120ccgttaagtt tgcaggattt gacaggtctg
gaacacatgc taataaattg ctcaaaaatg 3180cttcctgctg atatcacgca
gctaaataat attccaccaa ctcaggaatc ctactatgat 3240cccaacctgc
caccggtcac taaaagtcta gtaacaaact gtaaaccagt aactgacaaa
3300atccacaaag catataagga caaaaacaaa tataggtttg aaatcatggg
agaagaagaa 3360atcgctttta aaatgattcg taccaacgtt tctcatgtgg
ttggccagtt ggatgacata 3420agaaaaaacc ctaggaagtt tgtttgcctg
aatgacaaca ttgaccacaa tcataaagat 3480gctcagacag tgaaggctgt
tctcagggac ttctatgaat ccatgttccc cataccttcc 3540caatttgaac
tgccaagaga gtatcgaaac cgtttccttc atatgcatga gctgcaggaa
3600tggagggctt atcgagacaa attgaagttt tggacccatt gtgtactagc
aacattgatt 3660atgtttacta tattctcatt ttttgctgag cagttaattg
cacttaagcg gaagatattt 3720cccagaagga ggatacacaa agaagctagt
cccaatcgaa tcagagtata g 3771281256PRTArtificial
Sequencesource/note="Description of Artificial Sequence Synthetic
polypeptide" 28Met Leu Phe Lys Leu Leu Gln Arg Gln Thr Tyr Thr Cys
Leu Ser His1 5 10 15Arg Tyr Gly Leu Tyr Val Cys Phe Leu Gly Val Val
Val Thr Ile Val 20 25 30Ser Ala Phe Gln Phe Gly Glu Val Val Leu Glu
Trp Ser Arg Asp Gln 35 40 45Tyr His Val Leu Phe Asp Ser Tyr Arg Asp
Asn Ile Ala Gly Lys Ser 50 55 60Phe Gln Asn Arg Leu Cys Leu Pro Met
Pro Ile Asp Val Val Tyr Thr65 70 75 80Trp Val Asn Gly Thr Asp Leu
Glu Leu Leu Lys Glu Leu Gln Gln Val 85 90 95Arg Glu Gln Met Glu Glu
Glu Gln Lys Ala Met Arg Glu Ile Leu Gly 100 105 110Lys Asn Thr Thr
Glu Pro Thr Lys Lys Ser Glu Lys Gln Leu Glu Cys 115 120 125Leu Leu
Thr His Cys Ile Lys Val Pro Met Leu Val Leu Asp Pro Ala 130 135
140Leu Pro Ala Asn Ile Thr Leu Lys Asp Leu Pro Ser Leu Tyr Pro
Ser145 150 155 160Phe His Ser Ala Ser Asp Ile Phe Asn Val Ala Lys
Pro Lys Asn Pro 165 170 175Ser Thr Asn Val Ser Val Val Val Phe Asp
Ser Thr Lys Asp Val Glu 180 185 190Asp Ala His Ser Gly Leu Leu Lys
Gly Asn Ser Arg Gln Thr Val Trp 195 200 205Arg Gly Tyr Leu Thr Thr
Asp Lys Glu Val Pro Gly Leu Val Leu Met 210 215 220Gln Asp Leu Ala
Phe Leu Ser Gly Phe Pro Pro Thr Phe Lys Glu Thr225 230 235 240Asn
Gln Leu Lys Thr Lys Leu Pro Glu Asn Leu Ser Ser Lys Val Lys 245 250
255Leu Leu Gln Leu Tyr Ser Glu Ala Ser Val Ala Leu Leu Lys Leu Asn
260 265 270Asn Pro Lys Asp Phe Gln Glu Leu Asn Lys Gln Thr Lys Lys
Asn Met 275 280 285Thr Ile Asp Gly Lys Glu Leu Thr Ile Ser Pro Ala
Tyr Leu Leu Trp 290 295 300Asp Leu Ser Ala Ile Ser Gln Ser Lys Gln
Asp Glu Asp Ile Ser Ala305 310 315 320Ser Arg Phe Glu Asp Asn Glu
Glu Leu Arg Tyr Ser Leu Arg Ser Ile 325 330 335Glu Arg His Ala Pro
Trp Val Arg Asn Ile Phe Ile Val Thr Asn Gly 340 345 350Gln Ile Pro
Ser Trp Leu Asn Leu Asp Asn Pro Arg Val Thr Ile Val 355 360 365Thr
His Gln Asp Val Phe Arg Asn Leu Ser His Leu Pro Thr Phe Ser 370 375
380Ser Pro Ala Ile Glu Ser His Ile His Arg Ile Glu Gly Leu Ser
Gln385 390 395 400Lys Phe Ile Tyr Leu Asn Asp Asp Val Met Phe Gly
Lys Asp Val Trp 405 410 415Pro Asp Asp Phe Tyr Ser His Ser Lys Gly
Gln Lys Val Tyr Leu Thr 420 425 430Trp Pro Val Pro Asn Cys Ala Glu
Gly Cys Pro Gly Ser Trp Ile Lys 435 440 445Asp Gly Tyr Cys Asp Lys
Ala Cys Asn Asn Ser Ala Cys Asp Trp Asp 450 455 460Gly Gly Asp Cys
Ser Gly Asn Ser Gly Gly Ser Arg Tyr Ile Ala Gly465 470 475 480Gly
Gly Gly Thr Gly Ser Ile Gly Val Gly Gln Pro Trp Gln Phe Gly 485 490
495Gly Gly Ile Asn Ser Val Ser Tyr Cys Asn Gln Gly Cys Ala Asn Ser
500 505 510Trp Leu Ala Asp Lys Phe Cys Asp Gln Ala Cys Asn Val Leu
Ser Cys 515 520 525Gly Phe Asp Ala Gly Asp Cys Gly Gln Asp His Phe
His Glu Leu Tyr 530 535 540Lys Val Ile Leu Leu Pro Asn Gln Thr His
Tyr Ile Ile Pro Lys Gly545 550 555 560Glu Cys Leu Pro Tyr Phe Ser
Phe Ala Glu Val Ala Lys Arg Gly Val 565 570 575Glu Gly Ala Tyr Ser
Asp Asn Pro Ile Ile Arg His Ala Ser Ile Ala 580 585 590Asn Lys Trp
Lys Thr Ile His Leu Ile Met His Ser Gly Met Asn Ala 595 600 605Thr
Thr Ile His Phe Asn Leu Thr Phe Gln Asn Thr Asn Asp Glu Glu 610 615
620Phe Lys Met Gln Ile Thr Val Glu Val Asp Thr Arg Glu Gly Pro
Lys625 630 635 640Leu Asn Ser Thr Ala Gln Lys Gly Tyr Glu Asn Leu
Val Ser Pro Ile 645 650 655Thr Leu Leu Pro Glu Ala Glu Ile Leu Phe
Glu Asp Ile Pro Lys Glu 660 665 670Lys Arg Phe Pro Lys Phe Lys Arg
His Asp Val Asn Ser Thr Arg Arg 675 680 685Ala Gln Glu Glu Val Lys
Ile Pro Leu Val Asn Ile Ser Leu Leu Pro 690 695 700Lys Asp Ala Gln
Leu Ser Leu Asn Thr Leu Asp Leu Gln Leu Glu His705 710 715 720Gly
Asp Ile Thr Leu Lys Gly Tyr Asn Leu Ser Lys Ser Ala Leu Leu 725 730
735Arg Ser Phe Leu Met Asn Ser Gln His Ala Lys Ile Lys Asn Gln Ala
740 745 750Ile Ile Thr Asp Glu Thr Asn Asp Ser Leu Val Ala Pro Gln
Glu Lys 755 760 765Gln Val His Lys Ser Ile Leu Pro Asn Ser Leu Gly
Val Ser Glu Arg 770 775 780Leu Gln Arg Leu Thr Phe Pro Ala Val Ser
Val Lys Val Asn Gly His785 790 795 800Asp Gln Gly Gln Asn Pro Pro
Leu Asp Leu Glu Thr Thr Ala Arg Phe 805 810 815Arg Val Glu Thr His
Thr Gln Lys Thr Ile Gly Gly Asn Val Thr Lys 820 825 830Glu Lys Pro
Pro Ser Leu Ile Val Pro Leu Glu Ser Gln Met Thr Lys 835 840 845Glu
Lys Lys Ile Thr Gly Lys Glu Lys Glu Asn Ser Arg Met Glu Glu 850 855
860Asn Ala Glu Asn His Ile Gly Val Thr Glu Val Leu Leu Gly Arg
Lys865 870 875 880Leu Gln His Tyr Thr Asp Ser Tyr Leu Gly Phe Leu
Pro Trp Glu Lys 885 890 895Lys Lys Tyr Phe Gln Asp Leu Leu Asp Glu
Glu Glu Ser Leu Lys Thr 900 905 910Gln Leu Ala Tyr Phe Thr Asp Ser
Lys Asn Thr Gly Arg Gln Leu Lys 915 920 925Asp Thr Phe Ala Asp Ser
Leu Arg Tyr Val Asn Lys Ile Leu Asn Ser 930 935 940Lys Phe Gly Phe
Thr Ser Arg Lys Val Pro Ala His Met Pro His Met945 950 955 960Ile
Asp Arg Ile Val Met Gln Glu Leu Gln Asp Met Phe Pro Glu Glu 965 970
975Phe Asp Lys Thr Ser Phe His Lys Val Arg His Ser Glu Asp Met Gln
980 985 990Phe Ala Phe Ser Tyr Phe Tyr Tyr Leu Met Ser Ala Val Gln
Pro Leu 995 1000 1005Asn Ile Ser Gln Val Phe Asp Glu Val Asp Thr
Asp Gln Ser Gly 1010 1015 1020Val Leu Ser Asp Arg Glu Ile Arg Thr
Leu Ala Thr Arg Ile His 1025 1030 1035Glu Leu Pro Leu Ser Leu Gln
Asp Leu Thr Gly Leu Glu His Met 1040 1045 1050Leu Ile Asn Cys Ser
Lys Met Leu Pro Ala Asp Ile Thr Gln Leu 1055 1060 1065Asn Asn Ile
Pro Pro Thr Gln Glu Ser Tyr Tyr Asp Pro Asn Leu 1070 1075 1080Pro
Pro Val Thr Lys Ser Leu Val Thr Asn Cys Lys Pro Val Thr 1085 1090
1095Asp Lys Ile His Lys Ala Tyr Lys Asp Lys Asn Lys Tyr Arg Phe
1100 1105 1110Glu Ile Met Gly Glu Glu Glu Ile Ala Phe Lys Met Ile
Arg Thr 1115 1120 1125Asn Val Ser His Val Val Gly Gln Leu Asp Asp
Ile Arg Lys Asn 1130 1135 1140Pro Arg Lys Phe Val Cys Leu Asn Asp
Asn Ile Asp His Asn His 1145 1150 1155Lys Asp Ala Gln Thr Val Lys
Ala Val Leu Arg Asp Phe Tyr Glu 1160 1165 1170Ser Met Phe Pro Ile
Pro Ser Gln Phe Glu Leu Pro Arg Glu Tyr 1175 1180 1185Arg Asn Arg
Phe Leu His Met His Glu Leu Gln Glu Trp Arg Ala 1190 1195 1200Tyr
Arg Asp Lys Leu Lys Phe Trp Thr His Cys Val Leu Ala Thr 1205 1210
1215Leu Ile Met Phe Thr Ile Phe Ser Phe Phe Ala Glu Gln Leu Ile
1220 1225 1230Ala Leu Lys Arg Lys Ile Phe Pro Arg Arg Arg Ile His
Lys Glu 1235 1240 1245Ala Ser Pro Asn Arg Ile Arg Val 1250
125529918DNAArtificial Sequencesource/note="Description of
Artificial Sequence Synthetic polynucleotide" 29atggcggcgg
ggctggcgcg gctcctgttg ctcctcgggc tctcggccgg cgggcccgcg 60ccggcaggtg
cagcgaagat gaaggtggtg gaggagccca acgcgtttgg ggtgaacaac
120ccgttcttgc ctcaggccag tcgcctccag gccaagaggg atccttcacc
cgtgtctgga 180cccgtgcatc tcttccgact ctcgggcaag tgcttcagcc
tggtggagtc cacgtacaag 240tatgagttct gcccgttcca caacgtgacc
cagcacgagc agaccttccg ctggaacgcc 300tacagtggga tcctcggcat
ctggcacgag tgggagatcg ccaacaacac cttcacgggc 360atgtggatga
gggacggtga cgcctgccgt tcccggagcc ggcagagcaa ggtggagctg
420gcgtgtggaa aaagcaaccg gctggcccat gtgtccgagc cgagcacctg
cgtctacgcg 480ctgacgttcg agacccccct cgtctgccac ccccacgcct
tgctagtgta cccaaccctg 540ccagaggccc tgcagcggca gtgggaccag
gtagagcagg acctggccga tgagctgatc 600accccccagg gccatgagaa
gttgctgagg acactttttg aggatgctgg ctacttaaag 660accccagaag
aaaatgaacc cacccagctg gagggaggtc ctgacagctt ggggtttgag
720accctggaaa actgcaggaa ggctcataaa gaactctcaa aggagatcaa
aaggctgaaa 780ggtttgctca cccagcacgg catcccctac acgaggccca
cagaaacttc caacttggag 840cacttgggcc acgagacgcc cagagccaag
tctccagagc agctgcgggg tgacccagga 900ctgcgtggga gtttgtga
91830305PRTArtificial Sequencesource/note="Description of
Artificial Sequence Synthetic polypeptide" 30Met Ala Ala Gly Leu
Ala Arg Leu Leu Leu Leu Leu Gly Leu Ser Ala1 5 10 15Gly Gly Pro Ala
Pro Ala Gly Ala Ala Lys Met Lys Val Val Glu Glu 20 25 30Pro Asn Ala
Phe Gly Val Asn Asn Pro Phe Leu Pro Gln Ala Ser Arg 35 40 45Leu Gln
Ala Lys Arg Asp Pro Ser Pro Val Ser Gly Pro Val His Leu 50 55 60Phe
Arg Leu Ser Gly Lys Cys Phe Ser Leu Val Glu Ser Thr Tyr Lys65 70 75
80Tyr Glu Phe Cys Pro Phe His Asn Val Thr Gln His Glu Gln Thr Phe
85 90 95Arg Trp Asn Ala Tyr Ser Gly Ile Leu Gly Ile Trp His Glu Trp
Glu 100 105 110Ile Ala Asn Asn Thr Phe Thr Gly Met Trp Met Arg Asp
Gly Asp Ala 115 120 125Cys Arg Ser Arg Ser Arg Gln Ser Lys Val Glu
Leu Ala Cys Gly Lys 130 135 140Ser Asn Arg Leu Ala His Val Ser Glu
Pro Ser Thr Cys Val Tyr Ala145 150 155 160Leu Thr Phe Glu Thr Pro
Leu Val Cys His Pro His Ala Leu Leu Val 165 170 175Tyr Pro Thr Leu
Pro Glu Ala Leu Gln Arg Gln Trp Asp Gln Val Glu 180 185 190Gln Asp
Leu Ala Asp Glu Leu Ile Thr Pro Gln Gly His Glu Lys Leu 195 200
205Leu Arg Thr Leu Phe Glu Asp Ala Gly Tyr Leu Lys Thr Pro Glu Glu
210 215 220Asn Glu Pro Thr Gln Leu Glu Gly Gly Pro Asp Ser Leu Gly
Phe Glu225 230 235 240Thr Leu Glu Asn Cys Arg Lys Ala His Lys Glu
Leu Ser Lys Glu Ile 245 250 255Lys Arg Leu Lys Gly Leu Leu Thr Gln
His Gly Ile Pro Tyr Thr Arg 260 265 270Pro Thr Glu Thr Ser Asn Leu
Glu His Leu Gly His Glu Thr Pro Arg 275 280 285Ala Lys Ser Pro Glu
Gln Leu Arg Gly Asp Pro Gly Leu Arg Gly Ser 290 295 300Leu305
* * * * *