U.S. patent application number 15/978075 was filed with the patent office on 2018-09-06 for methods and compositions for treatment of pompe disease.
The applicant listed for this patent is Valerion Therapeutics, LLC. Invention is credited to Dustin D. Armstrong, Jeffrey C. Way.
Application Number | 20180251571 15/978075 |
Document ID | / |
Family ID | 51391819 |
Filed Date | 2018-09-06 |
United States Patent
Application |
20180251571 |
Kind Code |
A1 |
Armstrong; Dustin D. ; et
al. |
September 6, 2018 |
METHODS AND COMPOSITIONS FOR TREATMENT OF POMPE DISEASE
Abstract
In certain embodiments, the present disclosure provides
compositions and methods for treating Pompe disease.
Inventors: |
Armstrong; Dustin D.;
(Quincy, MA) ; Way; Jeffrey C.; (Cambridge,
MA) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
Valerion Therapeutics, LLC |
Concord |
MA |
US |
|
|
Family ID: |
51391819 |
Appl. No.: |
15/978075 |
Filed: |
May 11, 2018 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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14769270 |
Aug 20, 2015 |
10017581 |
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PCT/US2014/017483 |
Feb 20, 2014 |
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15978075 |
|
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61926874 |
Jan 13, 2014 |
|
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61767016 |
Feb 20, 2013 |
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Current U.S.
Class: |
1/1 |
Current CPC
Class: |
C07K 2319/33 20130101;
A61K 38/00 20130101; C07K 2319/70 20130101; C12Y 302/0102 20130101;
C07K 2317/56 20130101; C07K 2317/622 20130101; A61K 9/0019
20130101; A61K 38/47 20130101; C07K 2319/10 20130101; C07K 2317/14
20130101; A61P 3/08 20180101; C07K 2317/24 20130101; C07K 2317/92
20130101; C07K 2317/94 20130101; C12N 9/2408 20130101; A61P 9/00
20180101; C07K 2317/55 20130101; C07K 2317/565 20130101; A61P 21/00
20180101; A61K 2039/505 20130101; C07K 2317/77 20130101; C07K 16/40
20130101; C07K 16/44 20130101; A61P 43/00 20180101; C07K 2319/30
20130101; A61K 47/6871 20170801 |
International
Class: |
C07K 16/40 20060101
C07K016/40; C12N 9/26 20060101 C12N009/26; A61K 9/00 20060101
A61K009/00; A61K 38/47 20060101 A61K038/47; C07K 16/44 20060101
C07K016/44; A61K 47/68 20170101 A61K047/68 |
Claims
1. A chimeric polypeptide comprising: (i) a GAA polypeptide
comprising the amino acid sequence set forth in SEQ ID NO: 22 and
(ii) an internalizing moiety that binds DNA with a K.sub.D of less
than 100 nM and/or promotes transit across cellular membranes via
an equilibrative nucleoside transporter 2 (ENT2) transporter;
wherein the internalizing moiety is an antibody comprising a heavy
chain variable domain comprising the amino acid sequence of SEQ ID
NO: 9, or a humanized variant thereof, and a light chain variable
domain comprising the amino acid sequence of SEQ ID NO: 10, or a
humanized variant thereof.
2. The chimeric polypeptide of claim 1, further comprises a linker
sequence comprising the amino acid sequence set forth in SEQ ID NO:
30.
3. The chimeric polypeptide of claim 1, wherein the chimeric
polypeptide does not comprise the full length, GAA precursor
polypeptide set forth in SEQ ID NO: 1.
4. The chimeric polypeptide of claim 1, wherein the chimeric
polypeptide does not comprise the portion of GAA polypeptide set
forth in residues 1-57 of SEQ ID NO: 1 or 2.
5. The chimeric polypeptide of claim 1, wherein the chimeric
polypeptide has acid alpha-glucosidase activity.
6. The chimeric polypeptide of claim 1, wherein neither the GAA
polypeptide nor the chimeric polypeptide comprise a contiguous
amino acid sequence corresponding to the amino acids 1-60 of SEQ ID
NO: 1 or 2.
7. The chimeric polypeptide of claim 1, wherein neither the GAA
polypeptide nor the chimeric polypeptide comprise a contiguous
amino acid sequence corresponding to the amino acids 1-66 of SEQ ID
NO: 1 or 2.
8. The chimeric polypeptide of claim 1, wherein the mature GAA
polypeptide has a glycosylation pattern that differs from that of
naturally occurring human GAA.
9. The chimeric polypeptide of claim 1, wherein the internalizing
moiety promotes delivery of the chimeric polypeptide into cytoplasm
of cells.
10. The chimeric polypeptide of claim 1, wherein the chimeric
polypeptide is capable of being taken up by an autophagic
vacuole.
11. The chimeric polypeptide of claim 1, wherein the internalizing
moiety promotes transport of said chimeric polypeptide into muscle
cells or hepatocytes.
12. The chimeric polypeptide of claim 1, wherein the chimeric
polypeptide further comprises one or more polypeptide portions that
enhance one or more of in vivo stability, in vivo half life,
uptake/administration, production, or purification.
13. The chimeric polypeptide of claim 1, wherein the antibody is:
a) a monoclonal antibody 3E10, or a variant thereof that retains
cell penetrating activity, or a variant thereof that binds the same
epitope as 3E10, or an antibody that has substantially the same
cell penetrating activity as 3E10 and binds the same epitope as
3E10; or b) a monoclonal antibody 3E10, or a variant thereof that
retains the cell penetrating activity of 3E10.
14. The chimeric polypeptide of claim 1, wherein the antibody is a
chimeric, humanized, or fully human antibody.
15. The chimeric polypeptide of claim 1, wherein the antibody
comprises a VH CDR1 having the amino acid sequence of SEQ ID NO 24;
a VH CDR2 having the amino acid sequence of SEQ ID NO: 25; a VH
CDR3 having the amino acid sequence of SEQ ID NO: 26; a VL CDR1
having the amino acid sequence of SEQ ID NO: 27; a VL CDR2 having
the amino acid sequence of SEQ ID NO: 28; and a VL CDR3 having the
amino acid sequence of SEQ ID NO: 29; which CDRs are according to
the IMGT system.
16. A nucleic acid construct, comprising a nucleotide sequence that
encodes the chimeric polypeptide of claim 1.
17. A nucleic acid construct, comprising a nucleotide sequence that
encodes a GAA polypeptide, operably linked to a nucleotide sequence
that encodes an internalizing moiety, wherein the nucleic acid
construct encodes a chimeric polypeptide comprising: (i) the amino
acid sequence set forth in SEQ ID NO: 22 and (ii) an internalizing
moiety that promotes transit across cellular membranes via an
equilibrative nucleoside transporter 2 (ENT2) transporter and/or
that binds DNA with a K.sub.D of less than 100 nM; wherein the
internalizing moiety is an antibody comprising a heavy chain
variable domain comprising the amino acid sequence of SEQ ID NO: 9,
or a humanized variant thereof, and a light chain variable domain
comprising the amino acid sequence of SEQ ID NO: 10, or a humanized
variant thereof.
18. A vector comprising the nucleic acid construct of claim
1760.
19. A host cell comprising the vector of claim 18.
20. A method of producing a chimeric polypeptide comprising
culturing the host cell of claim 19 under appropriate conditions to
allow expression of the polypeptide to occur.
21. A method of treating Pompe disease in a subject in need
thereof, comprising administering to the subject an effective
amount of the chimeric polypeptide of claim 1.
22. The method of claim 21, wherein said subject in need thereof is
a subject: a) whose disease has been refractory to one or more
previous enzyme replacement therapies; b) having pathologic
cytoplasmic glycogen accumulation prior to initiation of treatment
with said chimeric polypeptide; c) diagnosed with Pompe disease
greater than six months prior to initiation of treatment with said
chimeric polypeptide; d) diagnosed with Pompe disease at least one
year prior to initiation of treatment with said chimeric
polypeptide; e) in whom the onset of symptoms of Pompe disease
occurred greater than six months prior to initiation of treatment
with said chimeric polypeptide; and/or f) in whom the onset of
symptoms of Pompe disease occurred at least one year prior to
initiation of treatment with said chimeric polypeptide.
23. A method of decreasing glycogen accumulation in cytoplasm,
lysosomes, and/or autophagic vacuoles of muscle cells, comprising
contacting muscle cells with a chimeric polypeptide, which chimeric
polypeptide comprising: (i) the amino acid sequence set forth in
SEQ ID NO: 22, and (ii) an internalizing moiety that promotes
transit across cellular membranes via an equilibrative nucleoside
transporter 2 (ENT2) transporter; wherein the internalizing moiety
is an antibody comprising a heavy chain variable domain comprising
the amino acid sequence of SEQ ID NO: 9, or a humanized variant
thereof, and a light chain variable domain comprising the amino
acid sequence of SEQ ID NO: 10, or a humanized variant thereof.
Description
CROSS-REFERENCE TO RELATED APPLICATIONS
[0001] This application is a continuation of U.S. application Ser.
No. 14/769,270, filed on Aug. 20, 2015, which is a national stage
filing under 35 U.S.C. .sctn. 371 of International Application No.
PCT/US2014/017483, filed Feb. 20, 2014, which claims the benefit of
the filing date under 35 U.S.C. .sctn. 119(e) to U.S. provisional
application Ser. No. 61/767,016, filed Feb. 20, 2013, and U.S.
provisional application Ser. No. 61/926,874, filed Jan. 13, 2014,
the entire contents of which are hereby incorporated by reference.
International Application No. PCT/US2014/017483 was published under
PCT Article 21(2) in English.
BACKGROUND OF THE DISCLOSURE
[0002] Glycogen storage disease type II (GSDII or Pompe disease) is
an autosomal recessive metabolic disorder characterized by a
deficiency in the lysosomal enzyme acid .alpha.-glucosidase (GAA).
Patients suffering from the disorder are unable to convert
lysosomal stores of glycogen into glucose, which leads initially to
accumulation of glycogen in the lysosome, and later to accumulation
of glycogen in the cytoplasm and autophagic vesicles of cells.
Eventually, the buildup of toxic levels of glycogen damages the
cells and impairs proper function. In particular, muscle cell
dysfunction is a hallmark of Pompe disease, with symptoms ranging
from hypertrophic cardiomyopathy, weakness, skeletal muscle
dysfunction and early infant death in infantile onset forms of the
disease, to progressive degeneration of skeletal muscle function
and respiratory muscle dysfunction in juvenile and adult onset
forms of the disease.
[0003] Treatment of Pompe disease with enzyme replacement therapy
(ERT) has provided partial restoration of muscle function and
prolonged survival in some patients. However, prior therapies based
on delivery of the 110 kDa precursor GAA protein have achieved
delivery of protein only to the lysosome. Delivery of protein
exclusively to the lysosome has proven ineffective to clear
glycogen build-up in the cytoplasm or other extra-lysosomal spaces.
Additionally, approaches based on delivering protein to the
lysosome have relied on uptake through mannose-6-phosphate
receptors in the lysosome, and high dosages appear to be
required.
SUMMARY OF THE DISCLOSURE
[0004] There is a need in the art for methods and compositions for
clearing cytoplasmic glycogen build-up in patients with Pompe
disease, as well as a need for alternative therapies for treating
Pompe disease. Such methods and compositions would improve
treatment of Pompe disease, particularly in patients whose disease
is severe enough and/or advanced enough to have significant
cytoplasmic glycogen accumulation. The present disclosure provides
such methods and compositions. In certain embodiments, the methods
and compositions provided herein decrease glycogen build-up in, at
least, the cytoplasm. In certain embodiments, the methods and
compositions also decrease glycogen build-up in lysosomes and
autophagic vesicles. Similarly, the methods and compositions
provided herein can be used to improve deleterious symptoms of
Pompe Disease, for example, to decrease levels of one or more of
alanine transaminase, aspartate transaminase, alkaline phosphatase,
and creatine phosphokinase (e.g., to decrease abnormally elevated
levels of one or more such enzymes, such as in serum).
[0005] In a first aspect, the disclosure provides a chimeric
polypeptide comprising: (i) a mature acid alpha-glucosidase (GAA)
polypeptide and (ii) an internalizing moiety that promotes delivery
into cells. In other words, the disclosure provides chimeric
polypeptides having two portions: a portion comprising a mature GAA
polypeptide (a GAA polypeptide comprising mature GAA; a GAA portion
comprising a GAA polypeptide comprising a mature GAA) and a portion
comprising an internalizing moiety that promotes delivery into
cells. In certain embodiments, the internalizing moiety promotes
transport into cytoplasm of cells. In certain embodiments, the
chimeric polypeptide has acid alpha-glucosidase activity, and does
not comprise a GAA precursor polypeptide of approximately 110
kilodaltons, particularly does not comprise the precursor
polypeptide of 110 kilodaltons generated endogenously by the
cleavage of amino acids 1-56 of SEQ ID NO: 1 by signal peptidase
and protease 1. In certain embodiments, the chimeric polypeptide
does not comprise the signal sequence of a GAA precursor
polypeptide (e.g., see the signal sequence depicted for SEQ ID NO:
1 or 2). In other words, the chimeric polypeptide comprises a
mature GAA portion and an internalizing moiety portion but does not
include amino acids 1-56 of SEQ ID NO: 1 (e.g., the GAA portion
comprises a GAA polypeptide but does not include amino acids 1-56
of SEQ ID NO: 1). The chimeric polypeptide may further comprise
additional portions, such as linker moieties and/or tags but, in
certain embodiments, does not include the GAA precursor polypeptide
of approximately 110 kilodaltons (e.g., the GAA precursor as
defined in Moreland et al, 2005, J. Biol. Chem. 280: 6780) and/or
the full length GAA polypeptide set forth in SEQ ID NO: 1 or 2.
[0006] In certain embodiments, the mature GAA polypeptide has a
molecular weight of approximately 70-76 kilodaltons. In certain
embodiments, the mature GAA polypeptide has a molecular weight of
about 70 kDa or about 76 kDa. In certain embodiments, the mature
GAA polypeptide comprises an amino acid sequence selected from
about: residues 122-782 of SEQ ID NOs: 1 or 2; residues 123-782 of
SEQ ID NOs: 1 or 2; residues 204-782 of SEQ ID NOs: 1 or 2;
residues 206-782 of SEQ ID NOs: 1 or 2; or residues 288-782 of SEQ
ID NOs: 1 or 2. In certain embodiments, the mature GAA polypeptide
consists of an amino acid sequence selected from about residues:
residues 122-782 of SEQ ID NOs: 1 or 2; residues 123-782 of SEQ ID
NOs: 1 or 2; residues 204-782 of SEQ ID NOs: 1 or 2; residues
206-782 of SEQ ID NOs: 1 or 2; or residues 288-782 of SEQ ID NOs: 1
or 2. In other embodiments, the C-terminal amino acid residues of
the mature GAA polypeptide varies, such that the C-terminal amino
acid residues is any of residues 816-881, as set forth in SEQ ID
NOs: 1 or 2. In certain embodiments, the mature GAA polypeptide
comprises or consists of the amino acid sequence of SEQ ID NO: 3 or
SEQ ID NO: 4. In certain embodiments, the chimeric polypeptide
comprises any of the foregoing. In certain embodiments, the GAA
polypeptide portion of the chimeric polypeptide consists of any of
the foregoing examples of mature GAA. Although the chimeric
polypeptide comprises additional amino acid sequence, in certain
embodiments, the chimeric polypeptide does include additional GAA
amino acid sequence contiguous with the mature GAA polypeptide
portion. In other embodiments, the chimeric polypeptide comprises
mature GAA and also comprises additional N- and or C-terminal
contiguous GAA polypeptide sequence, such as the longer active GAA
polypeptides described herein (e.g., the GAA portion comprises a
GAA polypeptide comprising mature GAA). As used herein, "GAA
polypeptide" refers to a polypeptide that comprises a portion
corresponding to mature GAA but may also include additional N-
and/or C-terminal portions naturally present in a GAA polypeptide
(e.g., a native GAA polypeptide). In certain embodiments, a GAA
polypeptide for use in the chimeric polypeptides and methods of the
disclosure does not include residues 1-56 of SEQ ID NO: 1. In
certain embodiments, the GAA polypeptide does not correspond to the
110 kilodalton, GAA precursor polypeptide.
[0007] In some embodiments, the chimeric polypeptide has acid
alpha-glucosidase activity.
[0008] In some embodiments, the chimeric polypeptide does not
comprise the full length, GAA translation product set forth in SEQ
ID NO: 1 (e.g., the GAA polypeptide portion does not include the
full length, GAA translation product set forth in SEQ ID NO: 1). In
some embodiments, neither the GAA polypeptide nor the chimeric
polypeptide comprise a contiguous amino acid sequence corresponding
to the amino acids 1-56 of SEQ ID NO: 1 or 2 (e.g., the GAA
polypeptide lacks the portion corresponding to amino acids 1-56,
preferably 1-57 of SEQ ID NO: 1 or 2, and this region is not
present in the chimeric polypeptide). In some embodiments, the
chimeric polypeptide comprises a GAA polypeptide that lacks at
least a portion of the GAA full linker region, wherein the full
linker region (SEQ ID NO: 31) corresponds to the amino acids 57-78
of SEQ ID NOs: 1 or 2 (e.g., the chimeric polypeptide may include
some of the full linker region but does not include all of the full
linker region). In some embodiments, neither the GAA polypeptide
nor the chimeric polypeptide comprise a contiguous amino acid
sequence corresponding to the amino acids 1-60 of SEQ ID NO: 1 or 2
(e.g., the GAA polypeptide lacks the portion corresponding to amino
acids 1-60 of SEQ ID NO: 1 or 2, and this region is not present in
the chimeric polypeptide). In some embodiments, the chimeric
polypeptide or GAA polypeptide comprises the amino acid sequence of
SEQ ID NO: 21. In some embodiments, the GAA polypeptide comprises
amino acids 61-952 of SEQ ID NO: 1. In some embodiments, neither
the GAA polypeptide nor the chimeric polypeptide comprise a
contiguous amino acid sequence corresponding to the amino acids
1-66 of SEQ ID NO: 1 or 2 (e.g., the GAA polypeptide lacks the
portion corresponding to amino acids 1-66 of SEQ ID NO: 1 or 2, and
this region is not present in the chimeric polypeptide). In some
embodiments, the chimeric polypeptide or GAA polypeptide comprises
the amino acid sequence of SEQ ID NO: 22. In some embodiments, the
GAA polypeptide comprises amino acids 67-952 of SEQ ID NO: 1. In
some embodiments, neither the GAA polypeptide nor the chimeric
polypeptide comprise a contiguous amino acid sequence corresponding
to the amino acids 1-69 of SEQ ID NO: 1 or 2 (e.g., the GAA
polypeptide lacks the portion corresponding to amino acids 1-69 of
SEQ ID NO: 1 or 2, and this region is not present in the chimeric
polypeptide). In some embodiments, the chimeric polypeptide or GAA
polypeptide comprises the sequence of SEQ ID NO: 23. In some
embodiments, the GAA polypeptide comprises amino acids 70-952 of
SEQ ID NO: 1. The disclosure contemplates combinations of any one
or more of these features.
[0009] In certain embodiments, the chimeric polypeptide comprises
or consists of the amino acid sequence set forth in SEQ ID NO: 11
or 12, or set forth in either of these sequence identifiers but in
the absence of one or more epitope tags (e.g., in the absence of,
for example, a His and/or Myc epitope tag).
[0010] In certain embodiments, the chimeric polypeptide and/or the
mature GAA is glycosylated. In certain embodiments, the chimeric
polypeptide and/or mature GAA is not glycosylated. In certain
embodiments, the mature GAA has a glycosylation pattern that
differs from that of naturally occurring human GAA.
[0011] In certain embodiments, the internalizing moiety promotes
delivery of the chimeric polypeptide into cytoplasm of cells. In
certain embodiments, the internalizing moiety promotes delivery of
said chimeric polypeptide into muscle cells, such as skeletal or
cardiac muscle cells (e.g., promotes delivery into cytoplasm of
such cells). In certain embodiments, the internalizing moiety
promotes delivery of said chimeric polypeptide into neurons or
hepatocytes (e.g., promotes delivery into cytoplasm of such
cells).
[0012] In certain embodiments, the chimeric polypeptide comprises
N-linked oligosaccharide chains modified with M6P residues. In
certain embodiments, the chimeric polypeptide comprises a
KFERQ-like sequence (SEQ ID NO: 33).
[0013] In certain embodiments, the chimeric polypeptide further
comprises one or more polypeptide portions that enhance one or more
of in vivo stability, in vivo half life, uptake/administration,
production, or purification. Exemplary polypeptide portions include
epitope tags, such as HA and myc tags, as well as the Fc region of
an immunoglobulin or all or a portion of HSA.
[0014] In certain embodiments, the internalizing moiety comprises
an antibody or antigen binding fragment. In certain embodiments,
the antibody or antigen binding fragment is a monoclonal antibody
or fragment. In certain embodiments, the antibody or antigen
binding fragment is human or humanized. In other embodiments, the
antibody or antigen binding fragment is murine. Exemplary antigen
binding fragments include, scFv, Fv, Fab, and the like. Further
exemplary antigen binding fragments comprise a heavy chain variable
region (VH) comprising CDR1, CDR2, and CDR3, and a light chain
variable region (VL) comprising CDR1, CDR2, and CDR3. When
referring to suitable internalizing moieties, they preferably
retain the antigen/target binding characteristics and cell
penetrating characteristics when present in the context of the
chimeric polypeptide.
[0015] In certain embodiments, the chimeric polypeptides comprise
as an internalizing moiety, an antibody or antigen binding fragment
thereof select from: monoclonal antibody 3E10, or a variant thereof
that retains cell penetrating activity, or a variant thereof that
binds the same epitope as 3E10, or an antibody that has
substantially the same cell penetrating activity as 3E10 and binds
the same epitope as 3E10, or an antigen binding fragment of any of
the foregoing. In certain embodiments, the antibody or antigen
binding fragment thereof is a monoclonal antibody 3E10, or a
variant thereof that retains cell penetrating activity, or an
antigen binding fragment of 3E10 or said 3E10 variant. In some
embodiments, the antibody or antigen binding fragment is a
chimeric, humanized, or fully human antibody or antigen binding
fragment. In some embodiments, the antibody or antigen binding
fragment comprises a heavy chain variable domain comprising an
amino acid sequence at least 95% identical to SEQ ID NO: 9, or a
humanized variant thereof. In some embodiments, the antibody or
antigen binding fragment comprises a light chain variable domain
comprising an amino acid sequence at least 95% identical to SEQ ID
NO: 10, or a humanized variant thereof. In some embodiments, the
antibody or antigen binding fragment comprises a heavy chain
variable domain comprising the amino acid sequence of SEQ ID NO: 9
and a light chain variable domain comprising the amino acid
sequence of SEQ ID NO: 10, or a humanized variant thereof. For any
description of an antibody or antigen binding fragment, the
disclosure contemplates that the antibody or antigen binding
fragment may comprise a heavy chain and a light chain, such as a
heavy chain comprising a heavy chain variable domain and a light
chain comprising a light chain variable domain. In some
embodiments, the antibody or antigen binding fragment
comprises:
[0016] a VH CDR1 having the amino acid sequence of SEQ ID NO
13;
[0017] a VH CDR2 having the amino acid sequence of SEQ ID NO:
14;
[0018] a VH CDR3 having the amino acid sequence of SEQ ID NO:
15;
[0019] a VL CDR1 having the amino acid sequence of SEQ ID NO:
16;
[0020] a VL CDR2 having the amino acid sequence of SEQ ID NO: 17;
and
[0021] a VL CDR3 having the amino acid sequence of SEQ ID NO: 18,
which CDRs are according to the Kabat system.
In some embodiments, the antibody or antigen binding fragment
comprises:
[0022] a VH CDR1 having the amino acid sequence of SEQ ID NO
24;
[0023] a VH CDR2 having the amino acid sequence of SEQ ID NO:
25;
[0024] a VH CDR3 having the amino acid sequence of SEQ ID NO:
26;
[0025] a VL CDR1 having the amino acid sequence of SEQ ID NO:
27;
[0026] a VL CDR2 having the amino acid sequence of SEQ ID NO: 28;
and
[0027] a VL CDR3 having the amino acid sequence of SEQ ID NO: 29,
which CDRs are according to the IMGT system.
In other words, in certain embodiments, the antibody comprises a
heavy chain comprising VH CDR1, VH CDR2, and VH CDR3 of the 3E10
antibody, as determined by Kabat and set forth above, and a light
chain comprising a VL CDR1, a VL CD2, and a VL CD3 of the 3E10
antibody, as determined by Kabat and set forth above; or is an
antigen binding fragment thereof. In certain other embodiments, the
antibody comprises a heavy chain comprising VH CDR1, VH CDR2, and
VH CDR3 of the 3E10 antibody, as determined by the IGMT system and
set forth above, and a light chain comprising a VL CDR1, a VL CD2,
and a VL CD3 of the 3E10 antibody, as determined by the IGMT system
and set forth above; or is an antigen binding fragment thereof.
[0028] In certain embodiments, the 3E10 antibody, fragment, or
variant comprises a heavy chain comprising an amino acid sequence
at least 80%, 85%, 90%, 92%, 95%, 96%, 97%, 98%, 99%, or even 100%
identical to SEQ ID NO: 9. In certain embodiments, the 3E10
antibody, fragment, or variant comprises a light chain comprising
an amino acid sequence at least 80%, 85%, 90%, 92%, 95%, 96%, 97%,
98%, 99%, or even 100% identical to SEQ ID NO: 10. In certain
embodiments, the 3E10 antibody, fragment, or variant comprises a
heavy chain comprising an amino acid sequence at least 80%, 85%,
90%, 92%, 95%, 96%, 97%, 98%, 99%, or even 100% identical to SEQ ID
NO: 9 and a light chain comprising an amino acid sequence at least
80%, 85%, 90%, 92%, 95%, 96%, 97%, 98%, 99%, or even 100% identical
to SEQ ID NO: 10. It is understood, that these heavy and light
chain regions may, in certain embodiments, be connected by a linker
(e.g., such as in the context of an scFv--see SEQ ID NO: 11 and
12). Chimeric polypeptides having any combination of the foregoing
or following internalizing moieties and mature GAA portions are
contemplated. Moreover, chimeric polypeptides having any
combination of the foregoing or following internalizing moieties
and GAA portions (e.g., GAA polypeptides) described herein are
contemplated. Any such chimeric polypeptides are suitable for use
in any of the methods of the disclosure described herein.
[0029] In some embodiments, the internalizing moiety is an antibody
or antigen-binding fragment (e.g., an antibody fragment). In some
embodiments, the internalizing moiety is an antibody fragment,
particularly an scFv. In other embodiments, the antibody fragment
is a Fab. In some embodiments, the N-terminus of the GAA
polypeptide portion is fused to the C-terminus of the heavy chain
constant region portion of the Fab. In some embodiments, the
N-terminus of the GAA polypeptide portion is fused to the
C-terminus of the heavy chain constant region portion of the Fab by
means of a linker. In some embodiments, the linker comprises the
amino acid sequence of SEQ ID NO: 30. In some embodiments, the
antibody or antigen binding fragment is an antibody. In some
embodiments, the N-terminus of the GAA polypeptide portion is fused
to the C-terminus of the heavy chain Fc portion of the antibody. In
some embodiments, the N-terminus of the GAA polypeptide portion is
fused to the C-terminus of the heavy chain Fc portion of the
antibody by means of a linker. In some embodiments, the linker
comprises the amino acid sequence of SEQ ID NO: 30.
[0030] In certain embodiments, the internalizing moiety comprises a
homing peptide. In certain embodiments, the internalizing moiety
transits cellular membranes via an equilibrative nucleoside
transporter 1 (ENT1), ENT2, ENT3 or ENT4 transporter. In certain
embodiments, the internalizing moiety transits cellular membranes
via an ENT2 transporter. By way of example, 3E10 antibody or
antigen binding fragment thereof transits cellular membranes via
ENT2.
[0031] In certain embodiments, the internalizing moiety is capable
of binding polynucleotides. In certain embodiments, the
internalizing moiety is capable of binding DNA. For example, 3E10
and the particular 3E10 variant described herein are known to bind
DNA (e.g., their target--or antigen is DNA). Although these and
other DNA binding antibodies are typically not specifically
reactive with a single antigen, they do bind DNA with relatively
strong affinity. In certain embodiments, the internalizing moiety
is capable of binding DNA with a K.sub.D of less than 1 .mu.M. In
certain embodiments, the internalizing moiety is capable of binding
DNA with a K.sub.D of less than 100 nM, less than 75 nM, less than
50 nM, or even less than 30 nM. For the foregoing, K.sub.D may be
determined using, for example, SPR or QCM according to standard
protocols.
[0032] In certain embodiments, the chimeric polypeptide is a
chemical conjugate of mature GAA polypeptide to the internalizing
moiety, such as a chemical conjugate comprising a GAA polypeptide
portion and an internalizing moiety portion. In other words, the
mature GAA portion and the internalizing moiety portion are
interconnected, directly or indirectly, via chemical conjugation.
In certain embodiments, the chimeric polypeptide is a recombinant,
co-translational fusion protein comprising the mature GAA
polypeptide and the internalizing moiety. When the chimeric
polypeptide is a fusion protein, the mature GAA portion and the
internalizing moiety portion may be interconnected directly or
indirectly. In certain embodiments, the chimeric polypeptide is
produced recombinantly to recombinantly conjugate the mature GAA
polypeptide to the internalizing moiety. In certain embodiments,
the chimeric polypeptide is produced in a prokaryotic or eukaryotic
cell, such as a yeast cell, an avian cell, an insect cell, or a
mammalian cell. In certain embodiments, the chimeric polypeptide is
produced in a prokaryotic cell, such as a bacterial cell. In
certain embodiments, the internalizing moiety is an antibody or an
antibody fragment. When the internalizing moiety is an antibody or
antibody fragment, the GAA polypeptide may be produced as a fusion
to any portion of the antibody or antibody fragment. For example,
if the internalizing moiety is a full length antibody or an Fab,
the GAA polypeptide may be fused recombinantly to (or chemically
conjugated to), for example, the C-terminus of the heavy chain of
the antibody or Fab.
[0033] Whether chemically or genetically conjugated, in certain
embodiments, the conjugate comprises a linker that conjugates or
joins, directly or indirectly, the mature GAA polypeptide to the
internalizing moiety. In certain embodiments, the conjugate does
not include a linker, and the mature GAA polypeptide is conjugated
or joined directly to the internalizing moiety. Regardless of
whether a linker joins the mature GAA and the internalizing moiety,
portions of the internalizing moiety may be joined via a linker
(e.g., an scFv has a linker joining VH and VL domains). In certain
embodiments, the chimeric polypeptide has a total of 0, 1, or 2
linkers. In other embodiments, the chimeric polypeptide has more
than two linkers. Any linkers may be cleavable. Chimeric
polypeptides of the disclosure comprise a GAA portion (e.g., a GAA
polypeptide) and an internalizing moiety portion. The disclosure
contemplates that any of the GAA portions (e.g., a GAA polypeptide
comprising mature GAA) and internalizing moiety portions can be
joined, as described above,
[0034] In certain embodiments, the internalizing moiety is
conjugated or joined, directly or indirectly, to the N-terminal or
C-terminal amino acid of the mature GAA polypeptide or to a longer
GAA polypeptide comprising a mature GAA polypeptide. In other
words, regardless of whether the mature GAA portion and the
internalizing portion are contiguous or separated by one or more
amino acid residues, the disclosure contemplates embodiments in
which the mature GAA portion is located N-terminal to the
internalizing moiety portion and embodiments in which the mature
GAA portion is located C-terminal to the internalizing moiety
portion. In certain embodiments, the internalizing moiety is
conjugated or joined to an internal amino acid of the mature GAA
polypeptide.
[0035] In a related aspect, the disclosure provides compositions
comprising one or more chimeric polypeptides of the disclosure.
Chimeric polypeptides for use in such compositions may have any
combination of features, as set forth above. In one embodiment, the
disclosure provides a composition comprising (a) a first chimeric
polypeptide comprising: (i) a mature acid alpha-glucosidase (GAA)
polypeptide having a molecular weight of approximately 76 kDa and
(ii) an internalizing moiety; and (b) a second chimeric polypeptide
comprising: (i) a mature acid alpha-glucosidase (GAA) polypeptide
having a molecular weight of approximately 70 kDa and (ii) an
internalizing moiety; wherein the first chimeric polypeptide and
the second chimeric polypeptide each have acid alpha-glucosidase
activity, and wherein neither the first chimeric polypeptide nor
the second chimeric polypeptide comprise a GAA precursor
polypeptide of approximately 110 kilodaltons. The foregoing is
merely exemplary of suitable compositions. The internalizing
moities for the first and second chimeric polypeptide may be the
same or different.
[0036] In certain embodiments, the composition of two or more
chimeric polypeptides further comprises a polypeptide comprising a
precursor GAA polypeptide having a molecular weight of about 110
kDa. Also contemplated are compositions in which one or more
chimeric polypeptides of the disclosure that do not include a GAA
precursor polypeptide of approximately 110 kDa is combined with a
polypeptide comprising a precursor GAA polypeptide having a
molecular weight of about 110 kDa.
[0037] In a related embodiment, the disclosure provides a
composition comprising (a) a chimeric polypeptide comprising: (i) a
mature acid alpha-glucosidase (GAA) polypeptide and (ii) a
internalizing moiety that promotes transport into cytoplasm of
cells; wherein the chimeric polypeptide has acid alpha-glucosidase
activity, and wherein the chimeric polypeptide does not comprise a
GAA precursor polypeptide of approximately 110 kilodaltons; and (b)
a polypeptide comprising a precursor GAA polypeptide having a
molecular weight of about 110 kDa. The foregoing is merely
exemplary of suitable compositions.
[0038] In another aspect, the disclosure provides a nucleic acid
construct comprising a nucleotide sequence that encodes any of the
chimeric polypeptides of the disclosure. For example, the
disclosure provides a nucleic acid construct, comprising a
nucleotide sequence that encodes a mature GAA polypeptide (or a GAA
polypeptide portion), operably linked to a nucleotide sequence that
encodes an internalizing moiety, wherein the nucleic acid construct
encodes a chimeric polypeptide having acid alpha-glucosidase
enzymatic activity and having the internalizing activity of the
internalizing moiety, and wherein the nucleic acid construct does
not encode a chimeric polypeptide comprising a GAA precursor
polypeptide of approximately 110 kilodaltons or does not encode a
polypeptide comprising a portion corresponding to residues 1-56 of
SEQ ID NO: 1.
[0039] In some embodiments, the disclosure provides a vector
comprising any of the nucleic acid constructs of the disclosure. In
some embodiments, the disclosure provides a host cell comprising
any of the vectors of the disclosure. In some embodiments, the host
cell comprises and is capable of expressing the vector. In some
embodiments, the disclosure provides method of producing a chimeric
polypeptide comprising culturing any of the host cells of the
disclosure under appropriate conditions to allow expression of the
polypeptide to occur. The disclosure contemplates embodiments in
which a chimeric polypeptide comprises a single polypeptide chain,
as well as embodiments in which a chimeric polypeptide comprises
more than one polypeptide chain. When a chimeric polypeptide
comprises more than one polypeptide chain that associates in the
active polypeptide, the disclosure contemplates methods and
compositions in which both polypeptide chains are present in and
expressed from the same vector, as well as methods and compositions
in which each chain is present in and expressed from a different
vector which may be co-expressed in the same host cell.
[0040] In another aspect, the disclosure provides a composition
comprising any of the chimeric polypeptides of the disclosure,
including any chimeric polypeptide having any combination of the
foregoing aspects and embodiments, and a pharmaceutically
acceptable carrier. In certain embodiments, the composition is a
sterile composition. In certain embodiments, the composition is
substantially pyrogen-free.
[0041] In another aspect, the disclosure provides a variety of in
vitro and in vivo methods. In one aspect, the disclosure provides a
method of treating Pompe disease in a subject in need thereof,
comprising administering to the subject an effective amount of any
one or more of the chimeric polypeptides or compositions of the
disclosure.
[0042] In another aspect, the disclosure provides a method of
treating Pompe disease in a subject in need thereof, comprising
administering to the subject an effective amount of a chimeric
polypeptide comprising: (i) a mature acid alpha-glucosidase (GAA)
polypeptide and (ii) an internalizing moiety; wherein the chimeric
polypeptide has acid alpha-glucosidase activity, and wherein the
chimeric polypeptide does not comprise a GAA precursor polypeptide
of approximately 110 kilodaltons.
[0043] In another aspect, the disclosure provides a method of
increasing acid alpha-glucosidase enzyme activity in a cell,
comprising contacting the cell with a chimeric polypeptide
comprising: (i) a mature acid alpha-glucosidase (GAA) polypeptide
and (ii) an internalizing moiety; wherein the chimeric polypeptide
has acid alpha-glucosidase activity, and wherein the chimeric
polypeptide does not comprise a GAA precursor polypeptide of
approximately 110 kilodaltons.
[0044] In another aspect, the disclosure provides a method of
decreasing glycogen accumulation in cytoplasm of muscle cells,
comprising contacting muscle cells with a chimeric polypeptide,
which chimeric polypeptide comprises (i) a mature acid
alpha-glucosidase (GAA) polypeptide and (ii) a internalizing moiety
that promotes delivery into cytoplasm of cells; wherein the
chimeric polypeptide has acid alpha-glucosidase activity, and
wherein the chimeric polypeptide does not comprise a GAA precursor
polypeptide of approximately 110 kilodaltons.
[0045] In another aspect, the disclosure provides a method of
decreasing glycogen accumulation in cytoplasm and lysosomes of
muscle cells, comprising contacting muscle cells with a chimeric
polypeptide, which chimeric polypeptide comprises (i) a mature acid
alpha-glucosidase (GAA) polypeptide and (ii) a internalizing
moiety; wherein the chimeric polypeptide has acid alpha-glucosidase
activity, and wherein the chimeric polypeptide does not comprise a
GAA precursor polypeptide of approximately 110 kilodaltons.
[0046] In another aspect, the disclosure provides a method of
decreasing glycogen accumulation in cytoplasm, lysosomes, and
autophagic vacuoles of muscle cells, comprising contacting muscle
cells with a chimeric polypeptide, which chimeric polypeptide
comprises (i) a mature acid alpha-glucosidase (GAA) polypeptide and
(ii) a internalizing moiety; wherein the chimeric polypeptide has
acid alpha-glucosidase activity, and wherein the chimeric
polypeptide does not comprise a GAA precursor polypeptide of
approximately 110 kilodaltons.
[0047] The following exemplary embodiments are applicable to any of
the foregoing methods of the disclosure. Moreover, the disclosure
contemplates combinations of these features with each other, as
well as with the aspects and embodiments of the disclosure detailed
above and throughout the specification. For example, any of the
chimeric polypeptides described herein, such as a chimeric
polypeptide comprising a GAA polypeptide portion comprising a
mature GAA polypeptide (e.g., a GAA polypeptide), as described
herein, and an internalizing moiety portion, as described herein
may be used in any of the in vivo or in vitro methods of the
disclosure.
[0048] In certain embodiments, the method comprises use of a
chimeric polypeptide, wherein the mature GAA polypeptide has a
molecular weight of approximately 70-76 kilodaltons. In certain
embodiments, the method comprises the use of a chimeric
polypeptide, wherein the mature GAA polypeptide has a molecular
weight of approximately 70 kilodaltons or approximately 76 kDa. In
certain embodiments, the mature GAA polypeptide has a molecular
weight of approximately 70-76 kilodaltons. In certain embodiments,
the mature GAA polypeptide has a molecular weight of about 70 kDa
or about 76 kDa. In certain embodiments, the mature GAA polypeptide
comprises an amino acid sequence selected from: residues 122-782 of
SEQ ID NOs: 1 or 2; residues 123-782 of SEQ ID NOs: 1 or 2;
residues 204-782 of SEQ ID NOs: 1 or 2; residues 206-782 of SEQ ID
NOs: 1 or 2; or residues 288-782 of SEQ ID NOs: 1 or 2. In certain
embodiments, the mature GAA polypeptide consists of an amino acid
sequence selected from residues: residues 122-782 of SEQ ID NOs: 1
or 2; residues 123-782 of SEQ ID NOs: 1 or 2; residues 204-782 of
SEQ ID NOs: 1 or 2; residues 206-782 of SEQ ID NOs: 1 or 2; or
residues 288-782 of SEQ ID NOs: 1 or 2. In other embodiments, the
C-terminal amino acid residues of the mature GAA polypeptide
varies, such that the C-terminal amino acid residues is any of
residues 816-881, as set forth in SEQ ID NOs: 1 or 2. In certain
embodiments, the mature GAA polypeptide comprises or consists of
the amino acid sequence of SEQ ID NO: 3 or SEQ ID NO: 4.
[0049] In some embodiments, the method comprises use of a chimeric
polypeptide that has acid alpha-glucosidase activity. The
disclosure contemplates chimeric polypeptides comprising a GAA
polypeptide and an internalizing moiety, as described herein, and
methods of using any such chimeric polypeptides. In some
embodiments, the method comprises use of a chimeric polypeptide
that does not comprise the full length, GAA polypeptide set forth
in SEQ ID NO: 1. In some embodiments, neither the GAA polypeptide
nor the chimeric polypeptide comprise a contiguous amino acid
sequence corresponding to the amino acids 1-56 of SEQ ID NO: 1 or 2
(e.g., the GAA polypeptide portion lacks the portion corresponding
to amino acids 1-56, preferably 1-57 of SEQ ID NO: 1 or 2, and this
region is not present in the chimeric polypeptide). In some
embodiments, the chimeric polypeptide comprises a GAA polypeptide
that lacks at least a portion of the GAA full linker region,
wherein the full linker region corresponds to the amino acids 57-78
of SEQ ID NOs: 1 or 2 (i.e., SEQ ID NO: 31). In some embodiments,
neither the GAA polypeptide nor the chimeric polypeptide comprise a
contiguous amino acid sequence corresponding to the amino acids
1-60 of SEQ ID NO: 1 or 2 (e.g., the GAA polypeptide portion lacks
the portion corresponding to amino acids 1-60 of SEQ ID NO: 1 or 2,
and this region is not present in the chimeric polypeptide). In
some embodiments, the chimeric polypeptide or GAA polypeptide
comprises the amino acid sequence of SEQ ID NO: 21. In some
embodiments, the GAA polypeptide, for use in a chimeric polypeptide
of the disclosure, comprises amino acids 61-952 of SEQ ID NO:1. In
some embodiments, neither the GAA polypeptide nor the chimeric
polypeptide comprise a contiguous amino acid sequence corresponding
to the amino acids 1-66 of SEQ ID NO: 1 or 2 (e.g., the GAA
polypeptide portion lacks the portion corresponding to amino acids
1-66, of SEQ ID NO: 1 or 2, and this region is not present in the
chimeric polypeptide). In some embodiments, the chimeric
polypeptide or GAA polypeptide comprises the amino acid sequence of
SEQ ID NO: 22. In some embodiments, the GAA polypeptide, for use in
a chimeric polypeptide of the disclosure, comprises amino acids
67-952 of SEQ ID NO: 1. In some embodiments, neither the GAA
polypeptide nor the chimeric polypeptide comprise a contiguous
amino acid sequence corresponding to the amino acids 1-69 of SEQ ID
NO: 1 or 2 (e.g., the GAA polypeptide portion lacks the portion
corresponding to amino acids 1-69 of SEQ ID NO: 1 or 2, and this
region is not present in the chimeric polypeptide). In some
embodiments, the chimeric polypeptide or GAA polypeptide comprises
the sequence of SEQ ID NO: 23. In some embodiments, the GAA
polypeptide, for use in a chimeric polypeptide of the disclosure,
comprises amino acids 70-952 of SEQ ID NO: 1.
[0050] In certain embodiments, the disclosure contemplates chimeric
polypeptides comprising the amino acid sequence set forth in SEQ ID
NO: 21, 22, or 23, and methods of using such chimeric polypeptides.
In other words, the GAA polypeptide portion of such chimeric
polypeptides comprises (or consists of) the amino acid sequence set
forth in SEQ ID NO: 21, 22, or 23. Also contemplated are variants
for use in a chimeric polypeptide of the disclosure, such as
variants at least 80%, 85%, 90%, 92%, 93%, 94%, 95%, 96%, 97%, 98%,
or 99% identical to SEQ ID NO: 21, 22, or 23.
[0051] In certain embodiments, the method comprises use of a
chimeric polypeptide comprising or consisting of the amino acid
sequence set forth in SEQ ID NO: 11 or 12, or set forth in either
of these sequence identifiers but in the absence of one or more
epitope tags.
[0052] In certain embodiments, the chimeric polypeptide and/or the
mature GAA is glycosylated. In certain embodiments, the chimeric
polypeptide and/or mature GAA is not glycosylated. In certain
embodiments, the mature GAA has a glycosylation pattern that
differs from that of naturally occurring human GAA. In certain
embodiments, the mature GAA comprises a KFERQ-like motif (SEQ ID
NO: 33).
[0053] In certain embodiments of any of the foregoing, the
internalizing moiety promotes delivery of the chimeric polypeptide
into cytoplasm of cells. In certain embodiments, the internalizing
moiety promotes delivery of said chimeric polypeptide into muscle
cells, such as skeletal or cardiac muscle cells (e.g., promotes
delivery into, at least, cytoplasm of muscle cells). In certain
embodiments, the internalizing moiety promotes delivery of said
chimeric polypeptide into hepatocytes or neurons (e.g., promotes
delivery into, at least, cytoplasm of hepatocytes or neurons). It
should be noted that when an internalizing moiety is described as
promoting delivery into muscle cells, that does not imply that
delivery is exclusive to muscle cells. All that is implied is that
delivery is somewhat enriched to muscle cells versus one or more
other cell types and that transit into cells is not ubiquitous
across all cell types.
[0054] In certain embodiments, the chimeric polypeptide comprises
N-linked oligosaccharide chains modified with M6P residues.
[0055] In certain embodiments, the method comprises use of chimeric
polypeptides further comprising one or more polypeptide portions
that enhance one or more of in vivo stability, in vivo half life,
uptake/administration, production, or purification. Exemplary
polypeptide portions include epitope tags, such as HA and myc tags,
as well as the Fc region of an immunoglobulin or all or a portion
of HSA.
[0056] In certain embodiments, the method comprises use of a
chimeric polypeptide comprising an internalizing moiety comprising
an antibody or antigen binding fragment. In certain embodiments,
the antibody or antigen binding fragment is a monoclonal antibody
or fragment. In certain embodiments, the antibody or antigen
binding fragment is human or humanized. In other embodiments, the
antibody or antigen binding fragment is murine.
[0057] Exemplary antigen binding fragments include, scFv, Fv, Fab,
and the like. Further exemplary antigen binding fragments comprise
a heavy chain variable region (VH) comprising CDR1, CDR2, and CDR3,
and a light chain variable region (VL) comprising CDR1, CDR2, and
CDR3. When referring to suitable internalizing moieties, they
preferably retain the antigen binding characteristics and cell
penetrating characteristics when present in the context of the
chimeric polypeptide. In some embodiments, the antigen or
antigen-binding fragment is a scFv. In other embodiments, the
antibody or antigen binding fragment is a Fab. In some embodiments,
the GAA polypeptide is fused to the C-terminus of the heavy chain
segment of the Fab. In some embodiments, the GAA polypeptide is
fused to the C-terminus of the heavy chain segment of the Fab by
means of a linker. In some embodiments, the linker comprises the
amino acid sequence of SEQ ID NO: 30. In some embodiments, the
antibody or antigen binding fragment is an antibody. In some
embodiments, the GAA polypeptide portion is fused to the C-terminus
of the heavy chain portion of the antibody. In some embodiments,
the GAA polypeptide portion is fused to the C-terminus of the heavy
chain portion of the antibody by means of a linker. In some
embodiments, the linker comprises the amino acid sequence of SEQ ID
NO: 30.
[0058] In certain embodiments, the method comprises the use of a
chimeric polypeptide comprising an antibody or antigen binding
fragment thereof that is a monoclonal antibody 3E10, or a variant
thereof that retains cell penetrating activity, or a variant
thereof that binds the same epitope as 3E10, or an antibody that
has substantially the same cell penetrating activity as 3E10 and
binds the same epitope as 3E10, or an antigen binding fragment of
any of the foregoing. In certain embodiments, the antibody or
antigen binding fragment thereof is monoclonal antibody 3E10, or a
variant thereof that retains cell penetrating activity, or an
antigen binding fragment of 3E10 or said 3E10 variant. In some
embodiments, the antibody or antigen binding fragment is a
chimeric, humanized, or fully human antibody or antigen binding
fragment. In some embodiments, the antibody or antigen binding
fragment comprises a heavy chain variable domain comprising an
amino acid sequence at least 95% identical to SEQ ID NO: 9, or a
humanized variant thereof. In some embodiment, the antibody or
antigen binding fragment comprises a light chain variable domain
comprising an amino acid sequence at least 95% identical to SEQ ID
NO: 10, or a humanized variant thereof. In some embodiments, the
antibody or antigen binding fragment comprises a heavy chain
variable domain comprising the amino acid sequence of SEQ ID NO: 9
and a light chain variable domain comprising the amino acid
sequence of SEQ ID NO: 10, or a humanized variant thereof. For any
description of an antibody or antigen binding fragment, the
disclosure contemplates that the antibody or antigen binding
fragment may comprise a heavy chain and a light chain, such as a
heavy chain comprising a heavy chain variable domain and a light
chain comprising a light chain variable domain. In some
embodiments, the antibody or antigen binding fragment comprises
[0059] a VH CDR1 having the amino acid sequence of SEQ ID NO
13;
[0060] a VH CDR2 having the amino acid sequence of SEQ ID NO:
14;
[0061] a VH CDR3 having the amino acid sequence of SEQ ID NO:
15;
[0062] a VL CDR1 having the amino acid sequence of SEQ ID NO:
16;
[0063] a VL CDR2 having the amino acid sequence of SEQ ID NO: 17;
and
[0064] a VL CDR3 having the amino acid sequence of SEQ ID NO: 18,
which CDRs are according to Kabat. In some embodiments, the
antibody or antigen binding fragment comprises:
[0065] a VH CDR1 having the amino acid sequence of SEQ ID NO
24;
[0066] a VH CDR2 having the amino acid sequence of SEQ ID NO:
25;
[0067] a VH CDR3 having the amino acid sequence of SEQ ID NO:
26;
[0068] a VL CDR1 having the amino acid sequence of SEQ ID NO:
27;
[0069] a VL CDR2 having the amino acid sequence of SEQ ID NO: 28;
and
[0070] a VL CDR3 having the amino acid sequence of SEQ ID NO: 29,
which CDRs are according to the IMGT system.
[0071] In certain embodiments, the 3E10 antibody, fragment, or
variant comprises a heavy chain comprising an amino acid sequence
at least 80%, 85%, 90%, 92%, 95%, 96%, 97%, 98%, 99%, or even 100%
identical to SEQ ID NO: 9. In certain embodiments, the 3E10
antibody, fragment, or variant comprises a light chain comprising
an amino acid sequence at least 80%, 85%, 90%, 92%, 95%, 96%, 97%,
98%, 99%, or even 100% identical to SEQ ID NO: 10. In certain
embodiments, the 3E10 antibody, fragment, or variant comprises a
heavy chain comprising an amino acid sequence at least 80%, 85%,
90%, 92%, 95%, 96%, 97%, 98%, 99%, or even 100% identical to SEQ ID
NO: 9 and a light chain comprising an amino acid sequence at least
80%, 85%, 90%, 92%, 95%, 96%, 97%, 98%, 99%, or even 100% identical
to SEQ ID NO: 10. Is in understood, that these heavy and light
chain regions may, in certain embodiments, be connected by a linker
(e.g., such as in the context of an scFv--see SEQ ID NO: 11 and
12). Chimeric polypeptides having any combination of the foregoing
or following internalizing moieties and mature GAA portions are
contemplated. In certain embodiments, the 3E10 antibody, fragment,
or variant comprises:
[0072] a heavy chain CDR1 having the amino acid sequence of SEQ ID
NO: 13, a heavy chain CDR2 having the amino acid sequence of SEQ ID
NO: 14, a heavy chain CDR3 having the amino acid sequence of SEQ ID
NO: 15, a light chain CDR1 having the amino acid sequence of SEQ ID
NO: 16, a light chain CDR2 having the amino acid sequence of SEQ ID
NO: 17, and/or a light chain CDR3 having the amino acid sequence of
SEQ ID NO: 18, which CDRs are according to Kabat. In some
embodiments, the antibody or antigen binding fragment
comprises:
[0073] a VH CDR1 having the amino acid sequence of SEQ ID NO
24;
[0074] a VH CDR2 having the amino acid sequence of SEQ ID NO:
25;
[0075] a VH CDR3 having the amino acid sequence of SEQ ID NO:
26;
[0076] a VL CDR1 having the amino acid sequence of SEQ ID NO:
27;
[0077] a VL CDR2 having the amino acid sequence of SEQ ID NO: 28;
and
[0078] a VL CDR3 having the amino acid sequence of SEQ ID NO: 29,
which CDRs are according to the IMGT system.
[0079] In certain embodiments, the internalizing moiety comprises a
homing peptide. In certain embodiments, the internalizing moiety
transits cellular membranes via an equilibrative nucleoside
transporter 1 (ENT1), ENT2, ENT3 or ENT4 transporter. In certain
embodiments, the internalizing moiety transits cellular membranes
via an equilibrative nucleoside transporter 2 (ENT2)
transporter.
[0080] In certain embodiments, the internalizing moiety is capable
of binding polynucleotides. In certain embodiments, the
internalizing moiety is capable of binding DNA. In certain
embodiments, the internalizing moiety is capable of binding DNA
with a K.sub.D of less than 1 .mu.M. In certain embodiments, the
internalizing moiety is capable of binding DNA with a K.sub.D of
less than 100 nM.
[0081] In certain embodiments, the subject in need of treatment is
a subject whose Pompe disease has been refractory to one or more
previous enzyme replacement therapies.
[0082] In certain embodiments, the subject in need of treatment is
a human patient with infantile Pompe disease. In certain
embodiments, the subject in need of treatment is a human patient
with juvenile onset or adult onset Pompe disease. In certain
embodiments, the subject in need of treatment is a human
patient.
[0083] In certain embodiments, an administered chimeric polypeptide
is transported to one or more of cytoplasm, lysosomes, and
autophagic vesicles of a cell. In certain embodiments,
administering the chimeric polypeptide reduces cytoplasmic glycogen
accumulation. In certain embodiments, administering the chimeric
polypeptide reduces lysosomal glycogen accumulation. In certain
embodiments, administering the chimeric polypeptide reduces
autophagic vacuole glycogen accumulation.
[0084] In certain embodiments, the subject in need thereof is a
subject diagnosed with or expected of having Pompe disease. In
certain embodiments, the subject in need thereof is a subject whose
disease has been refractory to one or more previous enzyme
replacement therapies. In certain embodiments, the subject in need
thereof is a subject having pathologic cytoplasmic glycogen
accumulation prior to initiation of treatment with said chimeric
polypeptide. In certain embodiments, the subject in need thereof is
a subject diagnosed with Pompe disease greater than six months
prior to initiation of treatment with said chimeric polypeptide. In
certain embodiments, the subject in need thereof is a subject
diagnosed with Pompe disease at least one year prior to initiation
of treatment with said chimeric polypeptide. In certain
embodiments, the subject in need thereof is a subject in whom the
onset of symptoms of Pompe disease occurred greater than six months
prior to initiation of treatment with said chimeric polypeptide. In
certain embodiments, the subject in need thereof is a subject in
whom the onset of symptoms of Pompe disease occurred at least one
year prior to initiation of treatment with said chimeric
polypeptide. In certain embodiments, the subject in need thereof is
a subject with Pompe disease in which pathologic cytoplasmic
glycogen accumulation has not yet occurred. In such cases,
administration of chimeric polypeptides of the disclosure (e.g.,
polypeptides that can be delivered into the cytoplasm) is used to
prevent pathologic cytoplasmic glycogen accumulation from
occurring.
[0085] In certain embodiments, the method comprises systemically
administering the chimeric polypeptide. In certain embodiments, the
method comprises intravenously administering the chimeric
polypeptides.
[0086] The disclosure contemplates all combinations of any of the
foregoing aspects and embodiments, as well as combinations with any
of the embodiments set forth in the detailed description and
examples. For example, any of the chimeric polypeptides and
compositions, including chimeric polypeptides and compositions
having any combination of mature GAA portions and an internalizing
moiety portions, can be used in any of the methods described
herein. Moreover, the disclosure contemplates that, in certain
embodiments, while comprising mature GAA, chimeric polypeptides of
the disclosure may also include additional contiguous GAA sequence
(e.g., the GAA portion comprises a GAA polypeptide that is larger
than just mature GAA, but does not include the complete amino acid
sequence set forth in SEQ ID NO: 1 and, preferably, does not
include a portion corresponding to residues 1-56 or 1-57 of SEQ ID
NO: 1 or 2).
BRIEF DESCRIPTION OF THE FIGURES
[0087] The patent or application file contains at least one drawing
executed in color.
[0088] Copies of this patent or patent application publication with
color drawings will be provided by the Office upon request and
payment of the necessary fee.
[0089] FIGS. 1A-1C is a diagram schematically depicting two
different fusion constructs generated. FIG. 1A is a diagram
schematically depicting the full-length GAA protein and its
different regions, as well as the murine heavy and light chains.
Amino acid residues 1-28 correspond to the signal sequence
("SigSeq") region, amino acids 29-56 correspond to the prepro
region, and amino acids 57-78 corresponds to the full linker
region. Residues 1-56 are highlighted in SEQ ID NO: 1 because, in
accordance with Moreland et al., this is the portion of the GAA
translation product that is cleaved by a signal peptidase and
protease to produce the precursor GAA polypeptide of approximately
110 kilodaltons. FIG. 1B is a diagram schematically depicting the
murine 3E10 Fab-GAA fusion construct, while FIG. 1C is a diagram
schematically depicting the murine 3E10 mAb-GAA fusion
construct.
DETAILED DESCRIPTION OF THE DISCLOSURE
[0090] Glycogen is a complex polysaccharide that provides a ready
store of glucose to cells in the human body. Glycogen is found
principally in the liver, where it is hydrolyzed and released into
the bloodstream to provide glucose to other cells, and in muscle,
where the glucose resulting from glycogen hydrolysis provides
energy for muscle cells. The lysosomal enzyme acid
.alpha.-glucosidase (GAA) is one of the enzymes that mediates
glycogen hydrolysis.
[0091] Disruption of glycogen hydrolysis, typically resulting from
genetic mutations in genes associated with the process, can lead to
glycogen storage disorders. In many cases, the severity of the
disease symptoms correlates directly with the extent of the
mutation. A debilitating glycogen storage disorder is Glycogen
storage disease type II (GSDII or Pompe disease), an autosomal
recessive metabolic disorder characterized by a deficiency in the
lysosomal enzyme acid .alpha.-glucosidase (GAA). Over 300 variants
in GAA are known, and disease phenotype depends largely on the
amount of residual enzyme activity (Schoser et al., Therapeutic
approaches in Glycogen Storage Disease type II (GSDII)/Pompe
disease, Neurotherapeutics, 5(4): 569-578, 2008). The most severe
form of Pompe disease is an infantile onset form, in which the
initial diagnosis occurs between 0-7 months of age. Infantile onset
Pompe disease is characterized by hypertrophic cardiomyopathy and
profound generalized weakness, and death typically occurs by 1 year
of age. Late onset Pompe disease (juvenile and adult onset) is less
severe, as residual GAA activity is present. Symptoms appear after
1 year of age or in adulthood, and dysfunction occurs primarily in
skeletal and respiratory muscles (Case and Kishnani, Physical
therapy management of Pompe disease, Genet Med 8(5): 318-327,
2006). Although juvenile and adult onset Pompe disease are less
severe, muscle dysfunction leads to increased and significant
disability over time. In some cases, patients become wheelchair
and/or ventilator dependent.
[0092] In all forms of Pompe disease, dysfunction of GAA impairs
the hydrolysis of glycogen in the lysosomes and causes toxic levels
of glycogen to accumulate (Geel et al., Pompe disease: Current
state of treatment modalities and animal models, Molecular Genetics
and Metabolism, 92:299-307, 2007). Initially, lysosomes in affected
cells increase in size and number. Subsequently, the lysosomes
rupture and leak glycogen into the cytoplasm. In muscle fibers,
high levels of glycogen accompanied by a lack of glycogen
hydrolysis may lead to local starvation and to an increased
autophagic response. However, the autophagic vesicles cannot fuse
properly with the lysosomes. In addition, cells uptake water to
counteract the high concentrations of glycogen, which leads to cell
swelling. Over a relatively brief period of time, glycogen
accumulates not only in lysosomes but also in the cytoplasm.
[0093] Endogenous human GAA is a 952 amino acid protein, encoded by
a gene of approximately 28 kb in length. In humans, 3 transcript
variants are known (NM_000152.3 which encodes NP000143.2;
NM_001079803.1 which encodes NP_001073271.1; and NM_001079804.1
which encodes NP_001073272.1). However, all three transcript
variants encode a protein having substantially the same amino acid
sequence. Endogenously, the GAA gene encodes a 952 or 957 amino
acid polypeptide which includes a signal sequence. This polypeptide
is glycosylated in the endoplasmic reticulum and the Golgi
apparatus, resulting in a glycosylated precursor with an apparent
molecular mass of 110 kDa. There are 7 potential glycosylation
sites on the immature precursor, located at residues 140, 233, 390,
470, 652, 882, and 925 of SEQ ID NOs: 1 or 2. The immature
precursor is targeted to the lysosomes through mannose-6-phosphate
receptors (MPRs) and a mannose-6-phosphate (M6P)-independent
pathway. The 110 kDa precursor protein is cleaved to give rise to
an endosomal intermediate form of GAA having a molecular weight of
about 95 kDa. Subsequent N-terminal and C-terminal proteolytic
cleavages generate, in the lysosome, mature, active forms of GAA
having molecular weights of about 76 kDa and about 70 kDa (Moreland
et al., Lysosomal Acid .alpha.-Glucosidase Consists of Four
Different Peptides Processed from a Single Chain Precursor, Journal
of Biological Chemistry, 280(8): 6780-6791, 2005; which is
incorporated by reference in its entirety). Owing to heterogeneity
in the cleavage sites, alternative starting residues and/or ending
residues may define the N and C terminal boundaries of mature GAA
polypeptides, such as mature GAA polypeptides for use in the
claimed disclosure. For example, the N-terminal residue of a mature
GAA polypeptide of about 76 kDa may, in certain embodiments,
correspond to residue 122 (Met) or 123 (Gly) of SEQ ID NOs: 1 or 2,
while the N-terminal residue of a mature GAA polypeptide of about
70 kDa may, in certain embodiments, correspond to any of residues
204 (Ala), 206 (Ser), or 288 (Gly) of SEQ ID NOs: 1 or 2.
Polypeptides having any of the foregoing N-terminal residues may
have, for example, a C-terminal residue corresponding to any of
residues 816 through 881 of SEQ ID NO:1 or 2, and may be residue
782 of SEQ ID NOs: 1 or 2. Additionally, the C-terminal residue may
be any of residues 782 through 816, or residues 782 through 881,
inclusive. The molecular weight of the mature GAA polypeptides may
be about 76 kDa or about 70 kDa, or may vary according to the
foregoing alternative starting and/or ending N and C terminal
residues (e.g., corresponding to portions generated due to
alternative cleavage).
[0094] The FDA approved a version of GAA referred to as
alglucosidase alfa (Myozyme, Genzyme Corporation), a recombinant
human GAA (rhGAA) analog of the 110 kDa precursor form of GAA,
produced in CHO cells. Myozyme is believed to be targeted to the
endocytic/lysosomal pathway, and is thought to exert its effects in
the lysosome. Myozyme does not appear to treat glycogen
accumulation in cytoplasm (Schoser et al., Therapeutic approaches
in Glycogen Storage Disease type II (GSDII)/Pompe disease,
Neurotherapeutics, 5(4): 569-578, 2008). As noted above, this
therapy is believed to target the lysosome and is based on delivery
of the immature precursor form of the protein. However, the
precursor form of the protein is less active than the 76 kDa mature
form of the GAA (Human Molecular Genetics, 7(11): 1815-1824, 1998).
Thus, in certain aspects, it may be beneficial to either (i)
deliver a mature form of GAA as a chimeric polypeptide, (ii)
deliver a GAA polypeptide that, although longer than the mature
form is shorter than the 110 kDa precursor form as a chimeric
polypeptide, and/or (iii) to deliver a GAA polypeptide with
activity of any size as a chimeric polypeptide connected to an
internalizing moiety to facilitate delivery of polypeptide into
cells, and even into the appropriate subcellular compartment.
Without being bound by theory, even if a polypeptide of the
disclosure has substantially the same activity as a precursor GAA
polypeptide, delivery to the proper cellular location, optionally
facilitated by an internalizing moiety that promotes delivery to
the cytoplasm, would increase the effective GAA activity delivered
to cells. In certain embodiments, the disclosure provides a
chimeric polypeptide comprising a GAA polypeptide comprising mature
GAA (a GAA portion comprising mature GAA) and an internalizing
moiety portion that facilitate deliver into cells. In other words,
the disclosure contemplates chimeric polypeptides comprising a GAA
polypeptide and an internalizing moiety.
[0095] In one aspect, the disclosure provides compositions and
methods for cytoplasmic delivery of a mature GAA molecule to
affected cells, for example, skeletal muscle cells. Pompe patients
exhibit a buildup of glycogen not only in lysosomes but also in
cytoplasm and autophagic vesicles. GAA that is targeted to the
lysosomes or to endocytic vesicles may not hydrolyze cytoplasmic
glycogen. For those patients who begin treatment after the disease
has progressed (in some cases, this may be patients who begin
treatment after 6 months with GAA dysfunction), it may be too late
for lysosome-targeted forms of GAA to clear glycogen effectively
from the cells. In contrast, cytoplasm-targeted mature GAA can
clear glycogen from the cytoplasm. Not only is mature GAA more
active than immature GAA, but mature GAA also remains active at
neutral pH, showing approximately 40% activity at neutral pH
relative to the acidic environment of the lysosome (Human Molecular
Genetics, 11(14), 2002). In fact, although the activity of mature
GAA is reduced at neutral pH relative to its activity at acidic pH,
even this reduced activity is greater than that of immature
GAA--even when assessed under the endogenous acidic conditions of
the lysosome. In addition, not only can cytoplasmically delivered
mature GAA decrease glycogen accumulation in the cytoplasm, but
such GAA may also be incorporated into autophagic vesicles and
lysosomes. Without being bound by theory, autophagic vesicles that
ultimately fuse with lysosomes may be one of the mechanisms to help
promote delivery of cytoplasmic GAA to lysosomes as well.
Accordingly, chimeric polypeptides of the disclosure delivered to
the cytoplasm are, at least, useful for decreasing glycogen
accumulation in cytoplasm and may also help decrease glycogen
accumulation in lysosomes and autophagic vesicles.
[0096] In certain embodiments, the disclosure provides a chimeric
polypeptide comprising a GAA polypeptide and an internalizing
moiety, as described herein. Any such chimeric polypeptide of the
disclosure can comprise any of the GAA polypeptides described
herein associated with any of the internalizing moiety portions
described herein, and these chimeric polypeptides can be used in
any of the methods of the disclosure.
[0097] In certain embodiments, chimeric polypeptides of the
disclosure comprise a mature GAA polypeptide and may also contain
some additional contiguous amino acid sequence from a GAA
polypeptide (but not including the 110 kD precursor polypeptide or
the signal sequence of the GAA precursor polypeptide). In other
embodiments, the chimeric polypeptides of the disclosure comprise a
mature GAA polypeptide but do not include additional contiguous
amino acid sequence from a GAA polypeptide other than the mature
GAA polypeptide. Thus, the disclosure contemplates chimeric
polypeptides in which the GAA portion comprises or consists of a
mature GAA polypeptide. Exemplary mature GAA polypeptides having a
molecular weight of 70-76 kD are described herein. In certain
embodiments, the chimeric polypeptide does not include the signal
sequence of the precursor GAA polypeptide. In certain embodiments,
the chimeric polypeptide does not include a portion corresponding
to residues 1-56 of SEQ ID NO: 1 or 2.
[0098] In certain embodiments, the disclosure provides a chimeric
polypeptide comprising a GAA polypeptide and an internalizing
moiety, as described herein. Any such chimeric polypeptide of the
disclosure can comprise any of the GAA polypeptides described
herein associated with any of the internalizing moiety portions
described herein, and these chimeric polypeptides can be used in
any of the methods of the disclosure.
[0099] In certain embodiments, the GAA polypeptide portion
comprises the amino acid sequence of SEQ ID NO: 21 (e.g., the GAA
polypeptide comprises SEQ ID NO: 21), and thus, the chimeric
polypeptide comprises a mature GAA having the amino acid sequence
of SEQ ID NO: 3 or 4. In certain embodiments, the chimeric
polypeptide does not include additional contiguous amino acid
sequence from human GAA--other than SEQ ID NO: 21. In certain
embodiments, the GAA polypeptide or chimeric polypeptide does not
include residues 1-56 of SEQ ID NO: 1. In certain embodiments, the
GAA polypeptide or chimeric polypeptide does not include residues
1-60 of SEQ ID NO: 1. In certain embodiments, the GAA polypeptide
portion comprises the amino acid sequence of SEQ ID NO: 22 (e.g.,
the GAA polypeptide comprises SEQ ID NO: 22), and thus, the
chimeric polypeptide comprises a mature GAA having the amino acid
sequence of SEQ ID NO: 3 or 4. In certain embodiments, the chimeric
polypeptide does not include additional contiguous amino acid
sequence from human GAA--other than SEQ ID NO: 22. In certain
embodiments, the GAA polypeptide or chimeric polypeptide does not
include residues 1-66 of SEQ ID NO: 1. In certain embodiments, the
GAA polypeptide portion comprises the amino acid sequence of SEQ ID
NO: 23 (e.g., the GAA polypeptide comprises SEQ ID NO: 23), and
thus, the chimeric polypeptide comprises a mature GAA having the
amino acid sequence of SEQ ID NO: 3 or 4. In certain embodiments,
the chimeric polypeptide does not include additional contiguous
amino acid sequence from human GAA--other than SEQ ID NO: 23. In
certain embodiments, the GAA polypeptide or chimeric polypeptide
does not include residues 1-69 of SEQ ID NO: 1.
[0100] Thus, in certain aspects, the disclosure provides chimeric
polypeptides comprising a mature acid alpha-glucosidase (GAA)
polypeptide that may be used to treat symptoms associated with
Pompe disease.
[0101] In certain embodiments, the disclosure provides a chimeric
polypeptide comprising (i) a mature GAA polypeptide; and (ii) an
internalizing moiety that promotes delivery into cells, such as
into cytoplasm of cells. In a particular embodiment, the
internalizing moiety helps target delivery of the chimeric
polypeptide to muscle cells, such as skeletal muscle cells.
I. GAA Polypeptides
[0102] It has been demonstrated that mature GAA polypeptides have
enhanced glycogen clearance as compared to the full length,
precursor GAA (Bijvoet, et al., 1998, Hum Mol Genet, 7(11):
1815-24), whether at low pH (i.e., the pH of the lysosome or
autophagic vacuole) or neutral pH (i.e., the pH of the cytoplasm)
conditions. In addition, while mature GAA is a lysosomal protein
that has optimal activity at lower pHs, mature GAA retains
approximately 40% activity at neutral pH (i.e., the pH of the
cytoplasm) (Martin-Touaux et al., 2002, Hum Mol Genet, 11(14):
1637-45). Accordingly, a GAA polypeptide comprising mature GAA is
suitable for cytoplasmic delivery, and thus, suitable to address an
unaddressed issue of Pompe disease: cytoplasmic glycogen
accumulation. However, regardless of whether the GAA portion of a
chimeric polypeptide comprises or consists of mature HAA, providing
the GAA polypeptide in association with an internalizing moiety of
the disclosure facilitates delivery into cells and, in certain
embodiments, delivery to cytoplasm.
[0103] As used herein, the mature GAA polypeptides include
variants, and in particular the mature, active forms of the protein
(the active about 76 kDa or about 70 kDa forms or similar forms
having an alternative starting and/or ending residue, collectively
termed "mature GAA"). The term "mature GAA" refers to a polypeptide
having an amino acid sequence corresponding to that portion of the
immature GAA protein that, when processed endogenously, has an
apparent molecular weight by SDS-PAGE of about 70 kDa to about 76
kDa, as well as similar polypeptides having alternative starting
and/or ending residues, as described above. In some embodiments,
the GAA polypeptide lacks the signal sequence (amino acids 1-27 of
SEQ ID NOs: 1 or 2 or the sequence designated by amino acids 1-56
of SEQ ID NO: 1-56). Exemplary mature GAA polypeptides include
polypeptides having residues 122-782 of SEQ ID NOs: 1 or 2;
residues 123-782 of SEQ ID NOs: 1 or 2; or residues 204-782 of SEQ
ID NOs: 1 or 2. The term "mature GAA" includes polypeptides that
are glycosylated in the same or substantially the same way as the
endogenous, mature proteins, and thus have a molecular weight that
is the same or similar to the predicted molecular weight. The term
also includes polypeptides that are not glycosylated or are
hyper-glycosylated, such that their apparent molecular weight
differ despite including the same primary amino acid sequence. Any
such variants or isoforms, functional fragments or variants, fusion
proteins, and modified forms of the mature GAA polypeptides have at
least a portion of the amino acid sequence of substantial sequence
identity to the native mature GAA protein, and retain enzymatic
activity. In certain embodiments, a functional fragment, variant,
or fusion protein of a mature GAA polypeptide comprises an amino
acid sequence that is at least 80%, 85%, 90%, 95%, 97%, 98%, 99% or
100% identical to mature GAA polypeptides set forth in one or both
of SEQ ID NOs: 3 and 4, or is at least 80%, 85%, 90%, 95%, 97%,
98%, 99% or 100% identical to mature GAA polypeptides corresponding
to one or more of: residues 122-782 of SEQ ID NOs: 1 or 2; residues
123-782 of SEQ ID NOs: 1 or 2; or residues 204-782 of SEQ ID NOs: 1
or 2. In some embodiments, the GAA polypeptide is a GAA polypeptide
from a non-human species, e.g., mouse, rat, dog, zebrafish, pig,
goat, cow, horse, monkey or ape. In some embodiments, the GAA
protein comprises the mature form, but not the full-length form, of
a bovine GAA protein having the amino acid sequence of SEQ ID NO:
32.
[0104] Here and elsewhere in the specification, sequence identity
refers to the percentage of residues in the candidate sequence that
are identical with the residue of a corresponding sequence to which
it is compared, after aligning the sequences and introducing gaps,
if necessary to achieve the maximum percent identity for the entire
sequence, and not considering any conservative substitutions as
part of the sequence identity. Neither N- or C-terminal extensions
nor insertions shall be construed as reducing identity or
homology.
[0105] Methods and computer programs for the alignment of sequences
and the calculation of percent identity are well known in the art
and readily available. Sequence identity may be measured using
sequence analysis software. For example, alignment and analysis
tools available through the ExPasy bioinformatics resource portal,
such as ClustalW algorithm, set to default parameters. Suitable
sequence alignments and comparisons based on pair-wise or global
alignment can be readily selected. One example of an algorithm that
is suitable for determining percent sequence identity and sequence
similarity is the BLAST algorithm, which is described in Altschul
et al., J Mol Biol 215:403-410 (1990). Software for performing
BLAST analyses is publicly available through the National Center
for Biotechnology Information (www.ncbi.nlm.nih.gov/). In certain
embodiments, the now current default settings for a particular
program are used for aligning sequences and calculating percent
identity.
[0106] In certain specific embodiments, the chimeric polypeptide
comprises a mature GAA polypeptide, such as a GAA polypeptide
comprising mature GAA. The mature GAA has an activity that is
similar to or substantially equivalent to the activity of
endogenous forms of human GAA that are about 76 kDa or about 70
kDa. For example, the mature GAA may be 7-10 fold more active for
glycogen hydrolysis than the 110 kDa precursor form, with the
comparison being made under the same or similar conditions (e.g.
the mature GAA-chimeric polypeptides disclosed herein as compared
with endogenous human immature precursor GAA under acidic or
neutral pH conditions) The mature GAA polypeptide may be the 76 kDa
or the 70 kDa form of GAA, or similar forms that use alternative
starting and/or ending residues. As noted in Moreland et al.
(Lysosomal Acid .alpha.-Glucosidase Consists of Four Different
Peptides Processed from a Single Chain Precursor, Journal of
Biological Chemistry, 280(8): 6780-6791, 2005), the nomenclature
used for the processed forms of GAA is based on an apparent
molecular mass as determined by SDS-PAGE. In some embodiments,
mature GAA may lack the N-terminal sites that are normally
glycosylated in the endoplasmic reticulum. An exemplary mature GAA
polypeptide comprises SEQ ID NO: 3 or SEQ ID NO:4. Further
exemplary mature GAA polypeptide may comprise or consist of an
amino acid sequence corresponding to about: residues 122-782 of SEQ
ID NOs: 1 or 2; residues 123-782 of SEQ ID NOs: 1 or 2, such as
shown in SEQ ID NO: 3; residues 204-782 of SEQ ID NOs: 1 or 2;
residues 206-782 of SEQ ID NOs: 1 or 2; residues 288-782 of SEQ ID
NOs: 1 or 2, as shown in SEQ ID NO: 4. Mature GAA polypeptides may
also have the N-terminal and or C-terminal residues described
above.
[0107] In certain embodiments, the chimeric polypeptide does not
comprise a full-length GAA polypeptide, but comprises a mature GAA
polypeptide and at least a portion of the full-length GAA
polypeptide. In other words, in certain embodiments, the chimeric
polypeptide comprises a GAA polypeptide and an internalizing
moiety. In some embodiments, the chimeric polypeptide does not
comprise a full-length GAA polypeptide comprising the amino acid
sequence of SEQ ID NO: 1 or 2, but comprises a mature GAA
polypeptide sequence comprising the amino acid sequences of SEQ ID
NOs: 3 or 4 and at least a portion of the amino acids corresponding
to amino acids 1-121 of SEQ ID NOs: 1-2 and/or at least a portion
of the amino acids corresponding to amino acids 783-952 of SEQ ID
NO: 1. In some embodiments, the chimeric polypeptide does not
comprise a full-length GAA polypeptide comprising the amino acid
sequence of SEQ ID NO: 1 or 2, but comprises a mature GAA
polypeptide sequence comprising the amino acid sequences of SEQ ID
NOs: 3 or 4 and at least a portion of the amino acids corresponding
to amino acids 783-952 of SEQ ID NO: 1. In some embodiments, the
chimeric polypeptide does not comprise a full-length GAA
polypeptide comprising the amino acid sequence of SEQ ID NO: 1 or
2, but comprises a mature GAA polypeptide sequence comprising the
amino acid sequences of SEQ ID NOs: 3 or 4 and at least a portion
of the amino acids corresponding to amino acids 783-957 of SEQ ID
NO: 2. These are exemplary of GAA polypeptides.
[0108] In certain embodiments, the GAA polypeptide portion (e.g.,
the portion comprising a GAA polypeptide comprising mature GAA;
e.g., a GAA polypeptide) of the chimeric proteins described herein
comprise a mature form of GAA but does not comprise a GAA
translation product set forth in SEQ ID NO: 1. In some embodiments,
neither the GAA polypeptide nor the chimeric polypeptide comprise a
contiguous amino acid sequence corresponding to the amino acids
1-27 or 1-56 of SEQ ID NO: 1 or 2. In some embodiments, the GAA
polypeptide lacks at least a portion of the GAA full linker region,
wherein the full linker region corresponds to amino acids 57-78 of
SEQ ID NOs: 1 or 2 (i.e., SEQ ID NO: 31). In some embodiments, the
GAA polypeptide does not comprise a contiguous amino acid sequence
corresponding to the amino acids 1-60, 1-61, 1-62, 1-63, 1-64,
1-65, 1-66, 1-67, 1-68, 1-69, 1-70, 1-71, 1-72, 1-73, 1-74, 1-75,
1-80, 1-85, 1-90, 1-95, 1-100, 1-105, 1-110, 1-115, 1-120 or 1-121
of SEQ ID NOs: 1 or 2. In particular embodiments, the GAA
polypeptide does not comprise a contiguous amino acid sequence
corresponding to the amino acids 1-60 of SEQ ID NOs: 1 or 2 (e.g.,
the chimeric polypeptide comprises a GAA polypeptide portion
comprising a GAA polypeptide comprising the amino acid sequence of
SEQ ID NO: 21). In other embodiments, the GAA portion does not
comprise a contiguous amino acid sequence corresponding to the
amino acids 1-66 of SEQ ID NO: 1 or 2 (e.g., the chimeric
polypeptide comprises a GAA polypeptide comprising the amino acid
sequence of SEQ ID NO: 22). In some embodiments, the GAA portion
does not comprise a contiguous amino acid sequence corresponding to
the amino acids 1-69 of SEQ ID NO: 1 or 2 (e.g., the chimeric
polypeptide comprises a GAA polypeptide portion comprising a GAA
polypeptide comprising the amino acid sequence of SEQ ID NO:
23).
[0109] In other embodiments, the mature GAA polypeptides may be
glycosylated, or may be not glycosylated. For those mature GAA
polypeptides that are glycosylated, the glycosylation pattern may
be the same as that of naturally-occurring human GAA or may be
different. One or more of the glycosylation sites on the precursor
GAA protein may be removed in the final mature GAA construct.
[0110] Mature GAA has been isolated from tissues such as bovine
testes, rat liver, pig liver, human liver, rabbit muscle, human
heart, human urine, and human placenta. Mature GAA may also be
produced using recombinant techniques, for example by transfecting
Chinese hamster ovary (CHO) cells with a vector that expresses
full-length human GAA or a vector that expresses mature GAA.
Recombinant human GAA (rhGAA) or mature GAA is then purified from
CHO-conditioned medium, using a series of ultrafiltration,
diafiltration, washing, and eluting steps, as described by Moreland
et al. (Lysosomal Acid .alpha.-Glucosidase Consists of Four
Different Peptides Processsed from a Single Chain Precursor,
Journal of Biological Chemistry, 280(8): 6780-6791, 2005). Mature
GAA fragments may be separated according to methods known in the
art, such as affinity chromatography and SDS page.
[0111] In certain embodiments, mature GAA, or fragments or variants
are human mature GAA.
[0112] In certain embodiments, fragments or variants of the mature
GAA polypeptides can be obtained by screening polypeptides
recombinantly produced from the corresponding fragment of the
nucleic acid encoding a mature GAA polypeptide. In addition,
fragments or variants can be chemically synthesized using
techniques known in the art such as conventional Merrifield solid
phase f-Moc or t-Boc chemistry. The fragments or variants can be
produced (recombinantly or by chemical synthesis) and tested to
identify those fragments or variants that can function as a native
GAA protein, for example, by testing their ability hydrolyze
glycogen and/or treat symptoms of Pompe disease.
[0113] In certain embodiments, the present disclosure contemplates
modifying the structure of a mature GAA polypeptide for such
purposes as enhancing therapeutic or prophylactic efficacy, or
stability (e.g., ex vivo shelf life and resistance to proteolytic
degradation in vivo). Such modified mature GAA polypeptides are
considered functional equivalents of the naturally-occurring GAA
polypeptide. Modified polypeptides can be produced, for instance,
by amino acid substitution, deletion, or addition. For instance, it
is reasonable to expect, for example, that an isolated replacement
of a leucine with an isoleucine or valine, an aspartate with a
glutamate, a threonine with a serine, or a similar replacement of
an amino acid with a structurally related amino acid (e.g.,
conservative mutations) will not have a major effect on the GAA
biological activity of the resulting molecule. Conservative
replacements are those that take place within a family of amino
acids that are related in their side chains.
[0114] This disclosure further contemplates generating sets of
combinatorial mutants of an mature GAA polypeptide, as well as
truncation mutants, and is especially useful for identifying
functional variant sequences. Combinatorially-derived variants can
be generated which have a selective potency relative to a naturally
occurring GAA polypeptide. Likewise, mutagenesis can give rise to
variants which have intracellular half-lives dramatically different
than the corresponding wild-type GAA polypeptide. For example, the
altered protein can be rendered either more stable or less stable
to proteolytic degradation or other cellular process which result
in destruction of, or otherwise inactivation of GAA function. Such
variants can be utilized to alter the mature GAA polypeptide level
by modulating their half-life. There are many ways by which the
library of potential mature GAA variants sequences can be
generated, for example, from a degenerate oligonucleotide sequence.
Chemical synthesis of a degenerate gene sequence can be carried out
in an automatic DNA synthesizer, and the synthetic genes then be
ligated into an appropriate gene for expression. The purpose of a
degenerate set of genes is to provide, in one mixture, all of the
sequences encoding the desired set of potential polypeptide
sequences. The synthesis of degenerate oligonucleotides is well
known in the art (see for example, Narang, S A (1983) Tetrahedron
39:3; Itakura et al., (1981) Recombinant DNA, Proc. 3rd Cleveland
Sympos. Macromolecules, ed. AG Walton, Amsterdam: Elsevier pp
273-289; Itakura et al., (1984) Annu. Rev. Biochem. 53:323; Itakura
et al., (1984) Science 198:1056; Ike et al., (1983) Nucleic Acid
Res. 11:477). Such techniques have been employed in the directed
evolution of other proteins (see, for example, Scott et al., (1990)
Science 249:386-390; Roberts et al., (1992) PNAS USA 89:2429-2433;
Devlin et al., (1990) Science 249: 404-406; Cwirla et al., (1990)
PNAS USA 87: 6378-6382; as well as U.S. Pat. Nos. 5,223,409,
5,198,346, and 5,096,815).
[0115] Alternatively, other forms of mutagenesis can be utilized to
generate a combinatorial library. For example, mature GAA
polypeptide variants can be generated and isolated from a library
by screening using, for example, alanine scanning mutagenesis and
the like (Ruf et al., (1994) Biochemistry 33:1565-1572; Wang et
al., (1994) J. Biol. Chem. 269:3095-3099; Balint et al., (1993)
Gene 137:109-118; Grodberg et al., (1993) Eur. J. Biochem.
218:597-601; Nagashima et al., (1993) J. Biol. Chem. 268:2888-2892;
Lowman et al., (1991) Biochemistry 30:10832-10838; and Cunningham
et al., (1989) Science 244:1081-1085), by linker scanning
mutagenesis (Gustin et al., (1993) Virology 193:653-660; Brown et
al., (1992) Mol. Cell Biol. 12:2644-2652; McKnight et al., (1982)
Science 232:316); by saturation mutagenesis (Meyers et al., (1986)
Science 232:613); by PCR mutagenesis (Leung et al., (1989) Method
Cell Mol Biol 1:11-19); or by random mutagenesis, including
chemical mutagenesis, etc. (Miller et al., (1992) A Short Course in
Bacterial Genetics, CSHL Press, Cold Spring Harbor, N.Y.; and
Greener et al., (1994) Strategies in Mol Biol 7:32-34). Linker
scanning mutagenesis, particularly in a combinatorial setting, is
an attractive method for identifying truncated (bioactive) forms of
mature GAA.
[0116] A wide range of techniques are known in the art for
screening gene products of combinatorial libraries made by point
mutations and truncations, and, for that matter, for screening cDNA
libraries for gene products having a certain property. Such
techniques will be generally adaptable for rapid screening of the
gene libraries generated by the combinatorial mutagenesis of the
mature GAA polypeptides. The most widely used techniques for
screening large gene libraries typically comprises cloning the gene
library into replicable expression vectors, transforming
appropriate cells with the resulting library of vectors, and
expressing the combinatorial genes under conditions in which
detection of a desired activity facilitates relatively easy
isolation of the vector encoding the gene whose product was
detected. Each of the illustrative assays described below are
amenable to high through-put analysis as necessary to screen large
numbers of degenerate sequences created by combinatorial
mutagenesis techniques.
[0117] In certain embodiments, a mature GAA polypeptide may include
a peptide and a peptidomimetic. As used herein, the term
"peptidomimetic" includes chemically modified peptides and
peptide-like molecules that contain non-naturally occurring amino
acids, peptoids, and the like. Peptidomimetics provide various
advantages over a peptide, including enhanced stability when
administered to a subject. Methods for identifying a peptidomimetic
are well known in the art and include the screening of databases
that contain libraries of potential peptidomimetics. For example,
the Cambridge Structural Database contains a collection of greater
than 300,000 compounds that have known crystal structures (Allen et
al., Acta Crystallogr. Section B, 35:2331 (1979)). Where no crystal
structure of a target molecule is available, a structure can be
generated using, for example, the program CONCORD (Rusinko et al.,
J. Chem. Inf. Comput. Sci. 29:251 (1989)). Another database, the
Available Chemicals Directory (Molecular Design Limited,
Informations Systems; San Leandro Calif.), contains about 100,000
compounds that are commercially available and also can be searched
to identify potential peptidomimetics of the mature GAA
polypeptides.
[0118] In certain embodiments, a mature GAA polypeptide may further
comprise post-translational modifications. Exemplary
post-translational protein modification include phosphorylation,
acetylation, methylation, ADP-ribosylation, ubiquitination,
glycosylation, carbonylation, sumoylation, biotinylation or
addition of a polypeptide side chain or of a hydrophobic group. As
a result, the modified mature GAA polypeptides may contain
non-amino acid elements, such as lipids, poly- or mono-saccharide,
and phosphates. Effects of such non-amino acid elements on the
functionality of a mature GAA polypeptide may be tested for its
biological activity, for example, its ability to treat Pompe
disease. In certain embodiments, the mature GAA polypeptide may
further comprise one or more polypeptide portions that enhance one
or more of in vivo stability, in vivo half life,
uptake/administration, and/or purification. In other embodiments,
the internalizing moiety comprises an antibody or an
antigen-binding fragment thereof.
[0119] In one specific embodiment of the present disclosure, a
mature GAA polypeptide may be modified with nonproteinaceous
polymers. In one specific embodiment, the polymer is polyethylene
glycol ("PEG"), polypropylene glycol, or polyoxyalkylenes, in the
manner as set forth in U.S. Pat. Nos. 4,640,835; 4,496,689;
4,301,144; 4,670,417; 4,791,192 or 4,179,337. PEG is a well-known,
water soluble polymer that is commercially available or can be
prepared by ring-opening polymerization of ethylene glycol
according to methods well known in the art (Sandler and Karo,
Polymer Synthesis, Academic Press, New York, Vol. 3, pages
138-161).
[0120] By the terms "biological activity", "bioactivity" or
"functional" is meant the ability of the mature GAA protein to
carry out the functions associated with wildtype GAA proteins, for
example, the hydrolysis of .alpha.-1,4- and .alpha.-1,6-glycosidic
linkages of glycogen, for example lysosomal glycogen. The terms
"biological activity", "bioactivity", and "functional" are used
interchangeably herein. In certain embodiments, and as described
herein, a mature GAA protein or chimeric polypeptide having
biological activity has the ability to hydrolyze glycogen. In other
embodiments, a mature GAA protein or chimeric polypeptide having
biological activity has the ability to lower the concentration of
lysosomal and/or cytoplasmic glycogen. In still other embodiments,
a mature GAA protein or chimeric polypeptide has the ability to
treat symptoms associated with Pompe disease. As used herein,
"fragments" are understood to include bioactive fragments (also
referred to as functional fragments) or bioactive variants that
exhibit "bioactivity" as described herein. That is, bioactive
fragments or variants of mature GAA exhibit bioactivity that can be
measured and tested. For example, bioactive fragments/functional
fragments or variants exhibit the same or substantially the same
bioactivity as native (i.e., wild-type, or normal) GAA protein, and
such bioactivity can be assessed by the ability of the fragment or
variant to, e.g., hydrolyze glycogen in vitro or in vivo. As used
herein, "substantially the same" refers to any parameter (e.g.,
activity) that is at least 70% of a control against which the
parameter is measured. In certain embodiments, "substantially the
same" also refers to any parameter (e.g., activity) that is at
least 75%, 80%, 85%, 90%, 92%, 95%, 97%, 98%, 99%, 100%, 102%,
105%, or 110% of a control against which the parameter is measured,
when assessed under the same or substantially the same conditions.
In certain embodiments, fragments or variants of the mature GAA
polypeptide will preferably retain at least 50%, 60%, 70%, 80%,
85%, 90%, 95% or 100% of the GAA biological activity associated
with the native GAA polypeptide, when assessed under the same or
substantially the same conditions. In certain embodiments,
fragments or variants of the mature GAA polypeptide have a
half-life (t.sub.1/2) which is enhanced relative to the half-life
of the native protein. Preferably, the half-life of mature GAA
fragments or variants is enhanced by at least 10%, 20%, 30%, 40%,
50%, 60%, 70%, 80%, 90%, 100%, 125%, 150%, 175%, 200%, 250%, 300%,
400% or 500%, or even by 1000% relative to the half-life of the
native GAA protein, when assessed under the same or substantially
the same conditions. In some embodiments, the protein half-life is
determined in vitro, such as in a buffered saline solution or in
serum. In other embodiments, the protein half-life is an in vivo
half life, such as the half-life of the protein in the serum or
other bodily fluid of an animal. In addition, fragments or variants
can be chemically synthesized using techniques known in the art
such as conventional Merrifield solid phase f-Moc or t-Boc
chemistry. The fragments or variants can be produced (recombinantly
or by chemical synthesis) and tested to identify those fragments or
variants that can function as well as or substantially similarly to
a native GAA protein.
[0121] With respect to methods of increasing GAA bioactivity in
cells, the disclosure contemplates all combinations of any of the
foregoing aspects and embodiments, as well as combinations with any
of the embodiments set forth in the detailed description and
examples. The described methods based on administering chimeric
polypeptides or contacting cells with chimeric polypeptides can be
performed in vitro (e.g., in cells or culture) or in vivo (e.g., in
a patient or animal model). In certain embodiments, the method is
an in vitro method. In certain embodiments, the method is an in
vivo method.
[0122] In some aspects, the present disclosure also provides a
method of producing any of the foregoing chimeric polypeptides as
described herein. Further, the present disclosure contemplates any
number of combinations of the foregoing methods and
compositions.
[0123] In certain aspects, a mature GAA polypeptide may be a fusion
protein which further comprises one or more fusion domains.
Well-known examples of such fusion domains include, but are not
limited to, polyhistidine, Glu-Glu, glutathione S transferase
(GST), thioredoxin, protein A, protein G, and an immunoglobulin
heavy chain constant region (Fc), maltose binding protein (MBP),
which are particularly useful for isolation of the fusion proteins
by affinity chromatography. For the purpose of affinity
purification, relevant matrices for affinity chromatography, such
as glutathione-, amylase-, and nickel- or cobalt-conjugated resins
are used. Fusion domains also include "epitope tags," which are
usually short peptide sequences for which a specific antibody is
available. Well known epitope tags for which specific monoclonal
antibodies are readily available include FLAG, influenza virus
haemagglutinin (HA), His, and c-myc tags. An exemplary His tag has
the sequence HHHHHH (SEQ ID NO: 7), and an exemplary c-myc tag has
the sequence EQKLISEEDL (SEQ ID NO: 8). It is recognized that any
such tags or fusions may be appended to the mature GAA portion of
the chimeric polypeptide or may be appended to the internalizing
moiety portion of the chimeric polypeptide, or both. In certain
embodiments, the chimeric polypeptides comprise a "AGIH" portion
(SEQ ID NO: 19) on the N-terminus (or within 10 amino acid residues
of the N-terminus) of the chimeric polypeptide, and such chimeric
polypeptides may be provided in the presence or absence of one or
more epitope tags. In further embodiments, the chimeric polypeptide
comprises a serine at the N-terminal most position of the
polypeptide. In some embodiments, the chimeric polypeptides
comprise an "SAGIH" (SEQ ID NO: 20) portion at the N-terminus (or
within 10 amino acid residues of the N-terminus) of the
polypeptide, and such chimeric polypeptides may be provided in the
presence or absence of one or more epitope tags.
[0124] In some cases, the fusion domains have a protease cleavage
site, such as for Factor Xa or Thrombin, which allows the relevant
protease to partially digest the fusion proteins and thereby
liberate the recombinant proteins therefrom. The liberated proteins
can then be isolated from the fusion domain by subsequent
chromatographic separation. In certain embodiments, the mature GAA
polypeptides may contain one or more modifications that are capable
of stabilizing the polypeptides. For example, such modifications
enhance the in vitro half life of the polypeptides, enhance
circulatory half life of the polypeptides or reducing proteolytic
degradation of the polypeptides.
[0125] In some embodiments, a mature GAA polypeptide may be a
fusion protein with an Fc region of an immunoglobulin. As is known,
each immunoglobulin heavy chain constant region comprises four or
five domains. The domains are named sequentially as follows:
CH1-hinge-CH2-CH3(-CH4). The DNA sequences of the heavy chain
domains have cross-homology among the immunoglobulin classes, e.g.,
the CH2 domain of IgG is homologous to the CH2 domain of IgA and
IgD, and to the CH3 domain of IgM and IgE. As used herein, the
term, "immunoglobulin Fc region" is understood to mean the
carboxyl-terminal portion of an immunoglobulin chain constant
region, preferably an immunoglobulin heavy chain constant region,
or a portion thereof. For example, an immunoglobulin Fc region may
comprise 1) a CH1 domain, a CH2 domain, and a CH3 domain, 2) a CH1
domain and a CH2 domain, 3) a CH1 domain and a CH3 domain, 4) a CH2
domain and a CH3 domain, or 5) a combination of two or more domains
and an immunoglobulin hinge region. In a preferred embodiment the
immunoglobulin Fc region comprises at least an immunoglobulin hinge
region a CH2 domain and a CH3 domain, and preferably lacks the CH1
domain. In one embodiment, the class of immunoglobulin from which
the heavy chain constant region is derived is IgG (Ig.gamma.)
(.gamma. subclasses 1, 2, 3, or 4). Other classes of
immunoglobulin, IgA (Ig.alpha.), IgD (Ig.delta.), IgE (Ig.epsilon.)
and IgM (Ig.mu.), may be used. The choice of appropriate
immunoglobulin heavy chain constant regions is discussed in detail
in U.S. Pat. Nos. 5,541,087, and 5,726,044. The choice of
particular immunoglobulin heavy chain constant region sequences
from certain immunoglobulin classes and subclasses to achieve a
particular result is considered to be within the level of skill in
the art. The portion of the DNA construct encoding the
immunoglobulin Fc region preferably comprises at least a portion of
a hinge domain, and preferably at least a portion of a CH.sub.3
domain of Fc .gamma. or the homologous domains in any of IgA, IgD,
IgE, or IgM. Furthermore, it is contemplated that substitution or
deletion of amino acids within the immunoglobulin heavy chain
constant regions may be useful in the practice of the disclosure.
One example would be to introduce amino acid substitutions in the
upper CH2 region to create a Fc variant with reduced affinity for
Fc receptors (Cole et al. (1997) J. IMMUNOL. 159:3613). One of
ordinary skill in the art can prepare such constructs using well
known molecular biology techniques.
[0126] In certain embodiments of any of the foregoing, the GAA
portion of the chimeric protein comprises one of the mature forms
of GAA, e.g., the 76 kDa fragment, the 70 kDa fragment, similar
forms that use an alternative start and/or stop site, or a
functional fragment thereof. In certain embodiments, such mature
GAA polypeptide or functional fragment thereof retains the ability
of to hydrolyze glycogen, as evaluated in vitro or in vivo.
Further, in certain embodiments, the chimeric polypeptide that
comprises such a mature GAA polypeptide or functional fragment
thereof can hydrolyze glycogen. Exemplary bioactive fragments
comprise at least 50, at least 60, at least 75, at least 100, at
least 125, at least 150, at least 175, at least 200, at least 225,
at least 230, at least 250, at least 260, at least 275, or at least
300 consecutive amino acid residues of a full length mature GAA
polypeptide.
[0127] In certain embodiments, the GAA polypeptide portion of the
chimeric proteins described herein comprise a mature form of GAA
but does not comprise a GAA polypeptide set forth in SEQ ID NO: 1.
In some embodiments, the GAA polypeptide lacks at least a portion
of the GAA full linker region, wherein the full linker region
corresponds to amino acids 57-78 of SEQ ID NOs: 1 or 2 (i.e., SEQ
ID NO: 31). In some embodiments, the GAA polypeptide does not
comprise a contiguous amino acid sequence corresponding to the
amino acids 1-60, 1-61, 1-62, 1-63, 1-64, 1-65, 1-66, 1-67, 1-68,
1-69, 1-70, 1-71, 1-72, 1-73, 1-74, 1-75, 1-80, 1-85, 1-90, 1-95,
1-100, 1-105, 1-110, 1-115, 1-120 or 1-121 of SEQ ID NOs: 1 or 2.
In particular embodiments, the GAA polypeptide does not comprise a
contiguous amino acid sequence corresponding to the amino acids
1-60 of SEQ ID NOs: 1 or 2 (e.g., the chimeric polypeptide
comprises a GAA polypeptide portion comprising a GAA polypeptide
comprising the amino acid sequence of SEQ ID NO: 21). In other
embodiments, the GAA portion does not comprise a contiguous amino
acid sequence corresponding to the amino acids 1-66 of SEQ ID NO: 1
or 2 (e.g., the chimeric polypeptide comprises a GAA polypeptide
portion comprising a GAA polypeptide comprising the amino acid
sequence of SEQ ID NO: 22). In some embodiments, the GAA portion
does not comprise a contiguous amino acid sequence corresponding to
the amino acids 1-69 of SEQ ID NO: 1 or 2 (e.g., the chimeric
polypeptide comprises a GAA polypeptide portion comprising a GAA
polypeptide comprising the amino acid sequence of SEQ ID NO:
23).
[0128] In certain embodiments, the disclosure contemplates chimeric
proteins where the mature GAA portion is a variant of any of the
foregoing mature GAA polypeptides or functional fragments.
Exemplary variants have an amino acid sequence at least 90%, 92%,
95%, 96%, 97%, 98%, or at least 99% identical to the amino acid
sequence of a native GAA polypeptide or bioactive fragment thereof,
and such variants retain the ability of native GAA to hydrolyze
glycogen, as evaluated in vitro or in vivo. The disclosure
contemplates chimeric proteins and the use of such proteins wherein
the GAA portion comprises any of the mature GAA polypeptides,
forms, or variants described herein in combination with any
internalizing moiety described herein. Exemplary mature GAA
polypeptides are set forth in SEQ ID NOs: 3 and 4. Moreover, in
certain embodiments, the mature GAA portion of any of the foregoing
chimeric polypeptides may, in certain embodiments, by a fusion
protein. Any such chimeric polypeptides comprising any combination
of GAA portions and internalizing moiety portions, and optionally
including one or more linkers, one or more tags, etc., may be used
in any of the methods of the disclosure.
II. Internalizing Moieties
[0129] As used herein, the term "internalizing moiety" refers to a
moiety capable of interacting with a target tissue or a cell type
to effect delivery of the attached molecule into the cell (i.e.,
penetrate desired cell; transport across a cellular membrane;
deliver across cellular membranes to, at least, the cytoplasm).
Preferably, this disclosure relates to an internalizing moiety
which promotes delivery to, for example, muscle cells and liver
cells. Internalizing moieties having limited cross-reactivity are
generally preferred. In certain embodiments, this disclosure
relates to an internalizing moiety which selectively, although not
necessarily exclusively, targets and penetrates muscle cells. In
certain embodiments, the internalizing moiety has limited
cross-reactivity, and thus preferentially targets a particular cell
or tissue type. However, it should be understood that internalizing
moieties of the subject disclosure do not exclusively target
specific cell types. Rather, the internalizing moieties promote
delivery to one or more particular cell types, preferentially over
other cell types, and thus provide for delivery that is not
ubiquitous. In certain embodiments, suitable internalizing moieties
include, for example, antibodies, monoclonal antibodies, or
derivatives or analogs thereof. Other internalizing moieties
include for example, homing peptides, fusion proteins, receptors,
ligands, aptamers, peptidomimetics, and any member of a specific
binding pair. In certain embodiments, the internalizing moiety
mediates transit across cellular membranes via an ENT2 transporter.
In some embodiments, the internalizing moiety helps the chimeric
polypeptide effectively and efficiently transit cellular membranes.
In some embodiments, the internalizing moiety transits cellular
membranes via an equilibrative nucleoside (ENT) transporter. In
some embodiments, the internalizing moiety transits cellular
membranes via an ENT1, ENT2, ENT3 or ENT4 transporter. In some
embodiments, the internalizing moiety transits cellular membranes
via an equilibrative nucleoside transporter 2 (ENT2) transporter.
In some embodiments, the internalizing moiety promotes delivery
into muscle cells (e.g., skeletal or cardiac muscle). In other
embodiments, the internalizing moiety promotes delivery into cells
other than muscle cells, e.g., neurons, epithelial cells, liver
cells, kidney cells or Leydig cells. For any of the foregoing, in
certain embodiments, the internalizing moiety promotes delivery of
a chimeric polypeptide into the cytoplasm.
[0130] In certain embodiments, the internalizing moiety promotes
delivery of a chimeric polypeptide into the cytoplasm. Without
being bound by theory, regardless of whether the GAA polypeptide
portion of the chimeric polypeptide comprises or consists of mature
GAA, this facilitates delivery to the cytoplasm and, optionally, to
the lysosome and/or autophagic vesicles.
[0131] In certain embodiments, the internalizing moiety is capable
of binding polynucleotides. In certain embodiments, the
internalizing moiety is capable of binding DNA. In certain
embodiments, the internalizing moiety is capable of binding DNA
with a K.sub.D of less than 1 .mu.M. In certain embodiments, the
internalizing moiety is capable of binding DNA with a K.sub.D of
less than 100 nM, less than 75 nM, less than 50 nM, or even less
than 30 nM. K.sub.D can be measured using Surface Plasmon Resonance
(SPR) or Quartz Crystal Microbalance (QCM), in accordance with
currently standard methods. By way of example, a 3E10 antibody or
antibody fragment, including an antibody or antibody fragment
comprising a VH having the amino acid sequence set forth in SEQ ID
NO: 9 and a VL having an amino acid sequence set forth in SEQ ID
NO: 10) is know to bind DNA with a K.sub.D of less than 100 nM.
[0132] In some embodiments, the internalizing moiety targets a
mature GAA polypeptide to muscle cells, and mediates transit of the
polypeptide across the cellular membrane into the cytoplasm of the
muscle cells.
[0133] As used herein, the term "internalizing moiety" refers to a
moiety capable of interacting with a target tissue or a cell type.
Preferably, this disclosure relates to an internalizing moiety
which promotes delivery to, for example, muscle cells and liver
cells. Internalizing moieties having limited cross-reactivity are
generally preferred. However, it should be understood that
internalizing moieties of the subject disclosure do not exclusively
target specific cell types. Rather, the internalizing moieties
promote delivery to one or more particular cell types,
preferentially over other cell types, and thus provide for delivery
that is not ubiquitous. In certain embodiments, suitable
internalizing moieties include, for example, antibodies, monoclonal
antibodies, or derivatives or analogs thereof; and other
internalizing moieties include for example, homing peptides, fusion
proteins, receptors, ligands, aptamers, peptidomimetics, and any
member of a specific binding pair. In some embodiments, the
internalizing moiety helps the chimeric polypeptide effectively and
efficiently transit cellular membranes. In some embodiments, the
internalizing moiety transits cellular membranes via an
equilibrative nucleoside (ENT) transporter. In some embodiments,
the internalizing moiety transits cellular membranes via an ENT1,
ENT2, ENT3 or ENT4 transporter. In some embodiments, the
internalizing moiety transits cellular membranes via an
equilibrative nucleoside transporter 2 (ENT2) transporter. In some
embodiments, the internalizing moiety promotes delivery into muscle
cells (e.g., skeletal or cardiac muscle). In other embodiments, the
internalizing moiety promotes delivery into cells other than muscle
cells, e.g., neurons, epithelial cells, liver cells, kidney cells
or Leydig cells.
[0134] (a) Antibodies
[0135] In certain aspects, an internalizing moiety may comprise an
antibody, including a monoclonal antibody, a polyclonal antibody,
and a humanized antibody. Without being bound by theory, such
antibody may bind to an antigen of a target tissue and thus mediate
the delivery of the subject chimeric polypeptide to the target
tissue (e.g., muscle). In some embodiments, internalizing moieties
may comprise antibody fragments, derivatives or analogs thereof,
including without limitation: Fv fragments, single chain Fv (scFv)
fragments, Fab' fragments, F(ab')2 fragments, single domain
antibodies, camelized antibodies and antibody fragments, humanized
antibodies and antibody fragments, human antibodies and antibody
fragments, and multivalent versions of the foregoing; multivalent
internalizing moieties including without limitation: Fv fragments,
single chain Fv (scFv) fragments, Fab' fragments, F(ab')2
fragments, single domain antibodies, camelized antibodies and
antibody fragments, humanized antibodies and antibody fragments,
human antibodies and antibody fragments, and multivalent versions
of the foregoing; multivalent internalizing moieties including
without limitation: monospecific or bispecific antibodies, such as
disulfide stabilized Fv fragments, scFv tandems ((scFv).sub.2
fragments), diabodies, tribodies or tetrabodies, which typically
are covalently linked or otherwise stabilized (i.e., leucine zipper
or helix stabilized) scFv fragments; receptor molecules which
naturally interact with a desired target molecule. In some
embodiments, the antibodies or variants thereof may be chimeric,
e.g., they may include variable heavy or light regions from the
murine 3E10 antibody, but may include constant regions from an
antibody of another species (e.g., a human). In some embodiments,
the antibodies or variants thereof may comprise a constant region
that is a hybrid of several different antibody subclass constant
domains (e.g., any combination of IgG1, IgG2a, IgG2b, IgG3 and
IgG4).
[0136] In certain embodiments, the antibodies or variants thereof,
may be modified to make them less immunogenic when administered to
a subject. For example, if the subject is human, the antibody may
be "humanized"; where the complementarity determining region(s) of
the hybridoma-derived antibody has been transplanted into a human
monoclonal antibody, for example as described in Jones, P. et al.
(1986), Nature, 321, 522-525 or Tempest et al. (1991),
Biotechnology, 9, 266-273. The term humanization and humanized is
well understood in the art when referring to antibodies. In some
embodiments, the internalizing moiety is any peptide or
antibody-like protein having the complementarity determining
regions (CDRs) of the 3E10 antibody sequence, or of an antibody
that binds the same epitope (e.g., the same target, such as DNA) as
3E10. Also, transgenic mice, or other mammals, may be used to
express humanized or human antibodies. Such humanization may be
partial or complete.
[0137] In certain embodiments, the internalizing moiety comprises
the monoclonal antibody 3E10 or an antigen binding fragment
thereof. For example, the antibody or antigen binding fragment
thereof may be monoclonal antibody 3E10, or a variant thereof that
retains cell penetrating activity, or an antigen binding fragment
of 3E10 or said 3E10 variant. Additionally, the antibody or antigen
binding fragment thereof may be an antibody that binds to the same
epitope (e.g., target, such as DNA) as 3E10, or an antibody that
has substantially the same cell penetrating activity as 3E10, or an
antigen binding fragment thereof. These are exemplary of agents
that target ENT2. In certain embodiments, the internalizing moiety
is capable of binding polynucleotides. In certain embodiments, the
internalizing moiety is capable of binding DNA. In certain
embodiments, the internalizing moiety is capable of binding DNA
with a K.sub.D of less than 1 .mu.M. In certain embodiments, the
internalizing moiety is capable of binding DNA with a K.sub.D of
less than 100 nM, less than 75 nM, less than 50 nM, or even less
than 30 nM. K.sub.D is determined using SPR or QCM, according to
manufacturer's instructions and current practice.
[0138] In certain embodiments, the antigen binding fragment is an
Fv or scFv fragment thereof. Monoclonal antibody 3E10 can be
produced by a hybridoma 3E10 placed permanently on deposit with the
American Type Culture Collection (ATCC) under ATCC accession number
PTA-2439 and is disclosed in U.S. Pat. No. 7,189,396. Additionally
or alternatively, the 3E10 antibody can be produced by expressing
in a host cell nucleotide sequences encoding the heavy and light
chains of the 3E10 antibody. The term "3E10 antibody" or
"monoclonal antibody 3E10" are used to refer to the antibody,
regardless of the method used to produce the antibody. Similarly,
when referring to variants or antigen-binding fragments of 3E10,
such terms are used without reference to the manner in which the
antibody was produced. At this point, 3E10 is generally not
produced by the hybridoma but is produced recombinantly. Thus, in
the context of the present application, 3E10 antibody will refer to
an antibody having the sequence of the hybridoma or comprising a
variable heavy chain domain comprising the amino acid sequence set
forth in SEQ ID NO: 9 (which has a one amino acid substitution
relative to that of the 3E10 antibody deposited with the ATCC, as
described herein) and the variable light chain domain comprising
the amino acid sequence set forth in SEQ ID NO: 10.
[0139] The internalizing moiety may also comprise variants of mAb
3E10, such as variants of 3E10 which retain the same cell
penetration characteristics as mAb 3E10, as well as variants
modified by mutation to improve the utility thereof (e.g., improved
ability to target specific cell types, improved ability to
penetrate the cell membrane, improved ability to localize to the
cellular DNA, convenient site for conjugation, and the like). Such
variants include variants wherein one or more conservative
substitutions are introduced into the heavy chain, the light chain
and/or the constant region(s) of the antibody. Such variants
include humanized versions of 3E10 or a 3E10 variant. In some
embodiments, the light chain or heavy chain may be modified at the
N-terminus or C-terminus. Similarly, the foregoing description of
variants applies to antigen binding fragments. Any of these
antibodies, variants, or fragments may be made recombinantly by
expression of the nucleotide sequence(s) in a host cell.
[0140] Monoclonal antibody 3E10 has been shown to penetrate cells
to deliver proteins and nucleic acids into the cytoplasmic or
nuclear spaces of target tissues (Weisbart R H et al., J Autoimmun.
1998 October; 11(5):539-46; Weisbart R H, et al. Mol Immunol. 2003
March; 39(13):783-9; Zack D J et al., J Immunol. 1996 Sep. 1;
157(5):2082-8.). Further, the VH and Vk sequences of 3E10 are
highly homologous to human antibodies, with respective humanness
z-scores of 0.943 and -0.880. Thus, Fv3E10 is expected to induce
less of an anti-antibody response than many other approved
humanized antibodies (Abhinandan K R et al., Mol. Biol. 2007 369,
852-862). A single chain Fv fragment of 3E10 possesses all the cell
penetrating capabilities of the original monoclonal antibody, and
proteins such as catalase, dystrophin, HSP70 and p53 retain their
activity following conjugation to Fv3E10 (Hansen J E et al., Brain
Res. 2006 May 9; 1088(1):187-96; Weisbart R H et al., Cancer Lett.
2003 Jun. 10; 195(2):211-9; Weisbart R H et al., J Drug Target.
2005 February; 13(2):81-7; Weisbart R H et al., J Immunol. 2000
Jun. 1; 164(11):6020-6; Hansen J E et al., J Biol Chem. 2007 Jul.
20; 282(29):20790-3). The 3E10 is built on the antibody scaffold
present in all mammals; a mouse variable heavy chain and variable
kappa light chain. 3E10 gains entry to cells via the ENT2
nucleotide transporter that is particularly enriched in skeletal
muscle and cancer cells, and in vitro studies have shown that 3E10
is nontoxic. (Weisbart R H et al., Mol Immunol. 2003 March;
39(13):783-9; Pennycooke M et al., Biochem Biophys Res Commun. 2001
Jan. 26; 280(3):951-9).
[0141] The internalizing moiety may also include mutants of mAb
3E10, such as variants of 3E10 which retain the same or
substantially the same cell penetration characteristics as mAb
3E10, as well as variants modified by mutation to improve the
utility thereof (e.g., improved ability to target specific cell
types, improved ability to penetrate the cell membrane, improved
ability to localize to the cellular DNA, improved binding affinity,
and the like). Such mutants include variants wherein one or more
conservative substitutions are introduced into the heavy chain, the
light chain and/or the constant region(s) of the antibody. Numerous
variants of mAb 3E10 have been characterized in, e.g., U.S. Pat.
No. 7,189,396 and WO 2008/091911, the teachings of which are
incorporated by reference herein in their entirety.
[0142] In certain embodiments, the internalizing moiety comprises
an antibody or antigen binding fragment comprising an VH domain
comprising an amino acid sequence at least 80%, 85%, 90%, 95%, 96%,
97%, 99%, or 100% identical to SEQ ID NO: 9 and/or a VL domain
comprising an amino acid sequence at least 85%, 90%, 95%, 96%, 97%,
99%, or 100% identical to SEQ ID NO: 10, or a humanized variant
thereof. Of course, such internalizing moieties transit cells via
ENT2 and/or bind the same epitope (e.g., target, such as DNA) as
3E10.
[0143] In certain embodiments, the internalizing moiety is capable
of binding polynucleotides. In certain embodiments, the
internalizing moiety is capable of binding DNA. In certain
embodiments, the internalizing moiety is capable of binding DNA
with a K.sub.D of less than 1 .mu.M. In certain embodiments, the
internalizing moiety is capable of binding DNA with a K.sub.D of
less than 100 nM.
[0144] In certain embodiments, the internalizing moiety is an
antigen binding fragment, such as a single chain Fv of 3E10 (scFv)
comprising SEQ ID NOs: 9 and 10. In certain embodiments, the
internalizing moiety comprises a single chain Fv of 3E10 (or
another antigen binding fragment), and the amino acid sequence of
the V.sub.H domain is at least 90%, 95%, 96%, 97%, 98%, 99%, or
100% identical to SEQ ID NO: 9, and amino acid sequence of the
V.sub.L domain is at least 90%, 95%, 96%, 97%, 98%, 99%, or 100%
identical to SEQ ID NO: 10. The variant 3E10 or fragment thereof
retains the function of an internalizing moiety. When the
internalizing moiety is an scFv, the VH and VL domains are
typically connected via a linker, such as a gly/ser linker. The VH
domain may be N-terminal to the VL domain or vice versa.
[0145] In some embodiments, the internalizing moiety comprises one
or more of the CDRs of the 3E10 antibody. In certain embodiments,
the internalizing moiety comprises one or more of the CDRs of a
3E10 antibody comprising the amino acid sequence of a V.sub.H
domain that is identical to SEQ ID NO: 9 and the amino acid
sequence of a V.sub.L domain that is identical to SEQ ID NO: 10.
The CDRs of the 3E10 antibody may be determined using any of the
CDR identification schemes available in the art. For example, in
some embodiments, the CDRs of the 3E10 antibody are defined
according to the Kabat definition as set forth in Kabat et al.
Sequences of Proteins of Immunological Interest, 5th Ed. Public
Health Service, National Institutes of Health, Bethesda, Md.
(1991). In other embodiments, the CDRs of the 3E10 antibody are
defined according to Chothia et al., 1987, J Mol Biol. 196: 901-917
and Chothia et al., 1989, Nature. 342:877-883. In other
embodiments, the CDRs of the 3E10 antibody are defined according to
the international ImMunoGeneTics database (IMGT) as set forth in
LeFranc et al., 2003, Development and Comparative Immunology, 27:
55-77. In other embodiments, the CDRs of the 3E10 antibody are
defined according to Honegger A, Pluckthun A., 2001, J Mol Biol.,
309:657-670. In some embodiments, the CDRs of the 3E10 antibody are
defined according to any of the CDR identification schemes
discussed in Kunik et al., 2012, PLoS Comput Biol. 8(2): e1002388.
In order to number residues of a 3E10 antibody for the purpose of
identifying CDRs according to any of the CDR identification schemes
known in the art, one may align the 3E10 antibody at regions of
homology of the sequence of the antibody with a "standard" numbered
sequence known in the art for the elected CDR identification
scheme. Maximal alignment of framework residues frequently requires
the insertion of "spacer" residues in the numbering system, to be
used for the Fv region. In addition, the identity of certain
individual residues at any given site number may vary from antibody
chain to antibody chain due to interspecies or allelic
divergence.
[0146] In certain embodiments, the internalizing moiety comprises
at least 1, 2, 3, 4, or 5 of the CDRs of 3E10 as determined using
the Kabat CDR identification scheme (e.g., the CDRs set forth in
SEQ ID NOs: 13-18). In other embodiments, the internalizing moiety
comprises at least 1, 2, 3, 4 or 5 of the CDRs of 3E10 as
determined using the IMGT identification scheme (e.g., the CDRs set
forth in SEQ ID NOs: 24-29). In certain embodiments, the
internalizing moiety comprises all six CDRs of 3E10 as determined
using the Kabat CDR identification scheme (e.g., comprises SEQ ID
NOs 13-18). In other embodiments, the internalizing moiety
comprises all six CDRS of 3E10 as determined using the IMGT
identification scheme (e.g., which are set forth as SEQ ID NOs:
24-29). For any of the foregoing, in certain embodiments, the
internalizing moiety is an antibody that binds the same epitope
(e.g., the same target, such as DNA) as 3E10 and/or the
internalizing moiety competes with 3E10 for binding to antigen.
Exemplary internalizing moieties target and transit cells via
ENT2.
[0147] The present disclosure utilizes the cell penetrating ability
of 3E10 or 3E10 fragments or variants to promote delivery of mature
GAA and GAA polypeptides comprising mature GAA in vivo or into
cells in vitro, such as into cytoplasm of cells. 3E10 and 3E10
variants and fragments are particularly well suited for this
because of their demonstrated ability to effectively promote
delivery to muscle cells, including skeletal and cardiac muscle, as
well as diaphragm. Thus, in certain embodiments, 3E10 and 3E10
variants and fragments (or antibodies or antibody fragments that
bind the same epitope and/or transit cells via ENT2) are useful for
promoting effective delivery into cells in subjects, such as human
patients or model organisms, having Pompe Disease or symptoms that
recapitulate Pompe Disease. In certain embodiments, chimeric
polypeptides in which the internalizing moiety is related to 3E10
are suitable to facilitate delivery of a GAA polypeptide comprising
mature GAA to the cytoplasm of cells.
[0148] As described further below, a recombinant 3E10 or 3E10-like
variant or fragment can be conjugated, linked or otherwise joined
to a mature GAA polypeptide, such as to a GAA polypeptide
comprising a mature GAA polypeptide. In the context of making
chimeric polypeptides to mature GAA, chemical conjugation, as well
as making the chimeric polypeptide as a fusion protein is available
and known in the art.
[0149] Preparation of antibodies or fragments thereof (e.g., a
single chain Fv fragment encoded by V.sub.H-linker-V.sub.L or
V.sub.L-linker-V.sub.H or a Fab) is well known in the art. In
particular, methods of recombinant production of mAb 3E10 antibody
fragments have been described in WO 2008/091911. Further, methods
of generating scFv fragments of antibodies or Fabs are well known
in the art. When recombinantly producing an antibody or antibody
fragment, a linker may be used. For example, typical surface amino
acids in flexible protein regions include Gly, Asn and Ser. One
exemplary linker is provided in SEQ ID NO: 5 or 6. Permutations of
amino acid sequences containing Gly, Asn and Ser would be expected
to satisfy the criteria (e.g., flexible with minimal hydrophobic or
charged character) for a linker sequence. Another exemplary linker
is of the formula (G.sub.4S)n, wherein n is an integer from 1-10,
such as 2, 3, or 4 (SEQ ID NO: 34). Other near neutral amino acids,
such as Thr and Ala, can also be used in the linker sequence.
[0150] In addition to linkers interconnecting portions of, for
example, an scFv, the disclosure contemplates the use of additional
linkers to, for example, interconnect the mature GAA portion to the
antibody portion of the chimeric polypeptide.
[0151] Preparation of antibodies may be accomplished by any number
of well-known methods for generating monoclonal antibodies. These
methods typically include the step of immunization of animals,
typically mice, with a desired immunogen (e.g., a desired target
molecule or fragment thereof). Once the mice have been immunized,
and preferably boosted one or more times with the desired
immunogen(s), monoclonal antibody-producing hybridomas may be
prepared and screened according to well known methods (see, for
example, Kuby, Janis, Immunology, Third Edition, pp. 131-139, W.H.
Freeman & Co. (1997), for a general overview of monoclonal
antibody production, that portion of which is incorporated herein
by reference). Over the past several decades, antibody production
has become extremely robust. In vitro methods that combine antibody
recognition and phage display techniques allow one to amplify and
select antibodies with very specific binding capabilities. See, for
example, Holt, L. J. et al., "The Use of Recombinant Antibodies in
Proteomics," Current Opinion in Biotechnology, 2000, 11:445-449,
incorporated herein by reference. These methods typically are much
less cumbersome than preparation of hybridomas by traditional
monoclonal antibody preparation methods. In one embodiment, phage
display technology may be used to generate an internalizing moiety
specific for a desired target molecule. An immune response to a
selected immunogen is elicited in an animal (such as a mouse,
rabbit, goat or other animal) and the response is boosted to expand
the immunogen-specific B-cell population. Messenger RNA is isolated
from those B-cells, or optionally a monoclonal or polyclonal
hybridoma population. The mRNA is reverse-transcribed by known
methods using either a poly-A primer or murine
immunoglobulin-specific primer(s), typically specific to sequences
adjacent to the desired V.sub.H and V.sub.L chains, to yield cDNA.
The desired V.sub.H and V.sub.L chains are amplified by polymerase
chain reaction (PCR) typically using V.sub.H and V.sub.L specific
primer sets, and are ligated together, separated by a linker.
V.sub.H and V.sub.L specific primer sets are commercially
available, for instance from Stratagene, Inc. of La Jolla, Calif.
Assembled V.sub.H-linker-V.sub.L product (encoding an scFv
fragment) is selected for and amplified by PCR. Restriction sites
are introduced into the ends of the V.sub.H-linker-V.sub.L product
by PCR with primers including restriction sites and the scFv
fragment is inserted into a suitable expression vector (typically a
plasmid) for phage display. Other fragments, such as an Fab'
fragment, may be cloned into phage display vectors for surface
expression on phage particles. The phage may be any phage, such as
lambda, but typically is a filamentous phage, such as fd and M13,
typically M13.
[0152] In certain embodiments, an antibody or antibody fragment is
made recombinantly in a host cell. In other words, once the
sequence of the antibody is known (for example, using the methods
described above), the antibody can be made recombinantly using
standard techniques.
[0153] In certain embodiments, the internalizing moieties may be
modified to make them more resistant to cleavage by proteases. For
example, the stability of an internalizing moiety comprising a
polypeptide may be increased by substituting one or more of the
naturally occurring amino acids in the (L) configuration with
D-amino acids. In various embodiments, at least 1%, 5%, 10%, 20%,
50%, 80%, 90% or 100% of the amino acid residues of internalizing
moiety may be of the D configuration. The switch from L to D amino
acids neutralizes the digestion capabilities of many of the
ubiquitous peptidases found in the digestive tract. Alternatively,
enhanced stability of an internalizing moiety comprising an peptide
bond may be achieved by the introduction of modifications of the
traditional peptide linkages. For example, the introduction of a
cyclic ring within the polypeptide backbone may confer enhanced
stability in order to circumvent the effect of many proteolytic
enzymes known to digest polypeptides in the stomach or other
digestive organs and in serum. In still other embodiments, enhanced
stability of an internalizing moiety may be achieved by
intercalating one or more dextrorotatory amino acids (such as,
dextrorotatory phenylalanine or dextrorotatory tryptophan) between
the amino acids of internalizing moiety. In exemplary embodiments,
such modifications increase the protease resistance of an
internalizing moiety without affecting the activity or specificity
of the interaction with a desired target molecule.
[0154] (b) Homing Peptides
[0155] In certain aspects, an internalizing moiety may comprise a
homing peptide which selectively directs the subject chimeric
mature GAA polypeptide to a target tissue (e.g., muscle). For
example, delivering a chimeric polypeptide to the muscle can be
mediated by a homing peptide comprising an amino acid sequence of
ASSLNIA (SEQ ID NO: 35). Further exemplary homing peptides are
disclosed in WO 98/53804. Homing peptides for a target tissue (or
organ) can be identified using various methods well known in the
art. Additional examples of homing peptides include the HIV
transactivator of transcription (TAT) which comprises the nuclear
localization sequence Tat48-60; Drosophila antennapedia
transcription factor homeodomain (e.g., Penetratin which comprises
Antp43-58 homeodomain 3rd helix); Homo-arginine peptides (e.g.,
Arg7 peptide-PKC-.epsilon. agonist protection of ischemic rat heart
("Arg7" is disclosed as SEQ ID NO: 36)); alpha-helical peptides;
cationic peptides ("superpositively" charged proteins). In some
embodiments, the homing peptide transits cellular membranes via an
equilibrative nucleoside (ENT) transporter. In some embodiments,
the homing peptide transits cellular membranes via an ENT1, ENT2,
ENT3 or ENT4 transporter. In some embodiments, the homing peptide
targets ENT2. In other embodiments, the homing peptide targets
muscle cells. The muscle cells targeted by the homing peptide may
include skeletal, cardiac or smooth muscle cells. In other
embodiments, the homing peptide targets neurons, epithelial cells,
liver cells, kidney cells or Leydig cells.
[0156] In certain embodiments, the homing peptide is capable of
binding polynucleotides. In certain embodiments, the homing peptide
is capable of binding DNA. In certain embodiments, the homing
peptide is capable of binding DNA with a K.sub.D of less than 1
.mu.M. In certain embodiments, the homing peptide is capable of
binding DNA with a K.sub.D of less than 100 nM.
[0157] Additionally, homing peptides for a target tissue (or organ)
can be identified using various methods well known in the art. Once
identified, a homing peptide that is selective for a particular
target tissue can be used, in certain embodiments.
[0158] An exemplary method is the in vivo phage display method.
Specifically, random peptide sequences are expressed as fusion
peptides with the surface proteins of phage, and this library of
random peptides are infused into the systemic circulation. After
infusion into host mice, target tissues or organs are harvested,
the phage is then isolated and expanded, and the injection
procedure repeated two more times. Each round of injection
includes, by default, a negative selection component, as the
injected virus has the opportunity to either randomly bind to
tissues, or to specifically bind to non-target tissues. Virus
sequences that specifically bind to non-target tissues will be
quickly eliminated by the selection process, while the number of
non-specific binding phage diminishes with each round of selection.
Many laboratories have identified the homing peptides that are
selective for vasculature of brain, kidney, lung, skin, pancreas,
intestine, uterus, adrenal gland, retina, muscle, prostate, or
tumors. See, for example, Samoylova et al., 1999, Muscle Nerve,
22:460; Pasqualini et al., 1996, Nature, 380:364; Koivunen et al.,
1995, Biotechnology, 13:265; Pasqualini et al., 1995, J. Cell
Biol., 130:1189; Pasqualini et al., 1996, Mole. Psych., 1:421, 423;
Rajotte et al., 1998, J. Clin. Invest., 102:430; Rajotte et al.,
1999, J. Biol. Chem., 274:11593. See, also, U.S. Pat. Nos.
5,622,699; 6,068,829; 6,174,687; 6,180,084; 6,232,287; 6,296,832;
6,303,573; 6,306,365. Homing peptides that target any of the above
tissues may be used for targeting a mature GAA protein to that
tissue.
[0159] (c) Additional Targeting to Lysosomes and Autophagic
Vesicles
[0160] A traditional method of targeting a protein to lysosomes is
modification of the protein with M6P residues, which directs their
transport to lysosomes through interaction of M6P residues and M6PR
molecules on the inner surface of structures such as the Golgi
apparatus or late endosome. Transport of endogenous GAA to the
lysosome depends on M6P and M6PR interaction. There are also forms
of M6P independent transport of GAA, as evidenced by normal
activity of GAA even in patients with I-cell disease, which
manifests with severe deficiencies in other lysosomal enzymes
(Wisselar et al., J. Biological Chemistry, 268(3): 2223-2231,
1993). Further evidence of M6P independent transport of GAA is
evidenced by a study showing no disruption in lysosomal GAA in
muscle-specific M6PR-knockout mice targeting (Wylie et al., 2003,
Am J Pathol, 162(1): 321-28). In certain embodiments, chimeric
polypeptides of the present disclosure (e.g., polypeptides
comprising mature GAA and an internalizing moiety) may further
include modification to facilitate additional targeting to the
lysosome through M6PRs or in pathways independent of M6PRs. Such
targeting moieties may be added, for example, at the N-terminus or
C-terminus of a chimeric polypeptide, and via conjugation to 3E10
or mature GAA. In other embodiments, the GAA portion of a chimeric
polypeptide comprises all or some of the endogenous sequences to
facilitate M6P transport.
[0161] In some embodiments, the chimeric polypeptides of the
present disclosure are transported to lysosomes via the cellular
process of autophagy. Autophagy is a catabolic mechanism that
involves cell degradation of unnecessary or dysfunctional cellular
components through the lysosomal machinery. During this process,
targeted cytoplasmic constituents are isolated from the rest of the
cell within vesicles called autophagosomes, which are then fused
with lysosomes and degraded or recycled. Uptake of proteins into
autophagic vesicles is mediated by the formation of a membrane
around the targeted region of a cell and subsequent fusion of the
vesicle with a lysosome. Several mechanisms for autophagy are
known, including macroautophagy in which organelles and proteins
are sequestered within the cell in a vesicle called an autophagic
vacuole. Upon fusion with the lysosome, the contents of the
autophagic vacuole are degraded by acidic lysosomal hydrolases. In
microautophagy, lysosomes engulf cytoplasm directly, and in
chaperone-mediated autophagy, proteins with a consensus peptide
sequence are bound by a hsc70-containing chaperone-cochaperone
complex, which is recognized by a lysosomal protein and
translocated across the lysosomal membrane. Autophagic vacuoles
have a lysosomal environment (low pH), which is conducive for
activity of enzymes such as mature GAA.
[0162] Autophagy naturally occurs in muscle cells of mammals
(Masiero et al, 2009, Cell Metabolism, 10(6): 507-15). It also has
been demonstrated that autophagic degradation is enhanced in Pompe
Disease (Malicdan et al., 2008, Neuromuscular Disorders, 18:
521-29; Fukuda et al, 2006, Mol Ther, 14(6): 831-39; Takikita et
al, 2009, Autophagy, 5(5): 729-31; Raben et al., 2008, 17(24):
3897-3908). Moreover, the autophagic vacuoles present in Pompe
Disease contain glycogen (Malicdan et al., 2008, Neuromuscular
Disorders, 18: 521-29). As the autophagic vacuoles take up proteins
from the cytoplasm, the chimeric polypeptides of the present
disclosure are expected to be taken up by glycogen-containing
autophagic vesicles, where the chimeric polypeptides would be free
to degrade the glycogen present within those vacuoles. As such, in
some embodiments, the chimeric polypeptides are capable of taken up
by autophagic vacuoles without addition of any autophagic
vacuole-specific targeting motif.
[0163] In certain embodiments, the chimeric polypeptides of the
present disclosure may further include modification to facilitate
additional targeting to autophagic vesicles. One known
chaperone-targeting motif is KFERQ-like motif (SEQ ID NO: 33).
Accordingly, this motif can be added to chimeric polypeptides as
described herein, in order to target the polypeptides for
autophagy. Such targeting moieties may be added, for example, at
the N-terminus or C-terminus of a chimeric polypeptide, and via
conjugation to 3E10 or mature GAA.
[0164] M6P residues or chaperone-targeting motifs may be added to
the mature GAA polypeptides. Mature GAA polypeptides of the present
disclosure may comprise, for example, the 76 kDa form of GAA or the
70 kDa form of GAA or similar forms that use an alternative
starting and/or ending site, administered either separately or in
combination. For combinations of 70 kDa and 76 kDa forms of GAA, or
similar forms of GAA as described herein, the internalizing motifs
may be added to either or both of the mature GAA polypeptides.
III. Chimeric Polypeptides
[0165] Chimeric polypeptides of the present disclosure can be made
in various manners. The chimeric polypeptides may comprise any of
the internalizing moiety portions and the mature GAA polypeptide
portions disclosed herein (e.g., a GAA polypeptide comprising
mature GAA, as described herein). As used herein, chimeric
polypeptides of the disclosure comprising (i) a GAA polypeptide
portion and (ii) an internalizing moiety portion, such as a GAA
polypeptide portion comprising a GAA polypeptide comprising a
mature GAA (e.g., the GAA polypeptide portion comprises a GAA
polypeptide which includes mature GAA but is longer than the mature
GAA generated in vivo by endogenous processing of a GAA precursor.
In addition, any of the chimeric polypeptides disclosed herein may
be utilized in any of the methods or compositions disclosed herein.
In some embodiments, an internalizing moiety (e.g. an antibody or a
homing peptide) is linked, directly or indirectly, to any one of
the mature GAA polypeptides, fragments or variants disclosed
herein. In some embodiments, the chimeric polypeptide does not
comprise an: i) immature GAA polypeptide of approximately 110 kDa
and/or, ii) immature GAA possessing the signal sequence, i.e.,
amino acid residues 1-27 of SEQ ID NO: 1 or 2. In other words, the
disclosure contemplates chimeric polypeptides in which the chimeric
polypeptide comprises a mature GAA polypeptide, but may also
include additional polypeptide sequence from a GAA polypeptide,
including sequence contiguous with the mature GAA polypeptide
(e.g., the GAA polypeptide portion comprises a GAA polypeptide
comprising a mature GAA polypeptide sequence). For example, in some
embodiments, the chimeric polypeptides comprise a GAA polypeptide
comprising the amino acid sequence of any of SEQ ID NOs: 21-23
(e.g., SEQ ID NOs 21-23 are exemplary of GAA polypeptides
comprising mature GAA but which also include additional contiguous
amino acids of a GAA polypeptide). The disclosure also contemplates
embodiments in which the chimeric polypeptide comprises a mature
GAA polypeptide but does not include additional GAA polypeptide
sequence contiguous with the mature GAA polypeptide portion.
Finally, the disclosure contemplates embodiments in which the
chimeric polypeptide does not include additional GAA polypeptide
portions in addition to the mature GAA polypeptide.
[0166] In certain embodiments, it may be desirable to conjugate any
of the internalizing moieties described herein with a mature GAA
polypeptide (e.g., a GAA polypeptide having the amino acid sequence
of SEQ ID NO: 3 or 4) in order to reduce the likelihood that a
chimeric polypeptide comprising a larger GAA polypeptide (e.g., a
GAA polypeptide having the amino acid sequence of any of SEQ ID
NOs: 21-24) is inadvertently cleaved at any of the cleavage sites
present in the full-length GAA polypeptide (e.g., cleaving between
any of the amino acids corresponding to amino acids 56-57, 77-78,
113-114, 121-122, 200-201, 203-204, 781-782, or 791-792 of SEQ ID
NO: 1) by a subject's proteases prior to uptake of the chimeric
polypeptide by a targeted cell in the subject.
[0167] In certain embodiments, the C-terminus of a mature GAA
polypeptide can be linked, directly or indirectly, to the
N-terminus of an internalizing moiety (e.g., an antibody, an
antibody fragment, or a homing peptide). Alternatively, the
C-terminus of an internalizing moiety (e.g., an antibody, an
antibody fragment, or a homing peptide) can be linked, directly or
indirectly, to the N-terminus of a mature GAA polypeptide. For
example, chimeric polypeptides can be designed to place the mature
GAA polypeptide at the amino or carboxy terminus of either the
antibody heavy or light chain of mAb 3E10. In some embodiments, the
GAA polypeptide comprises the amino acid sequence of SEQ ID NO: 22
or 23 fused to the C-terminus of an internalizing moiety. In some
embodiments, the GAA polypeptide comprises the amino acid sequence
of SEQ ID NO: 22 or 23 fused to the C-terminus of the heavy chain
segment of a Fab internalizing moiety. In some embodiments, the GAA
polypeptide comprises the amino acid sequence of SEQ ID NO: 22 or
23 fused to the C-terminus of the heavy chain segment of a
full-length antibody internalizing moiety.
[0168] In certain embodiments, potential configurations include the
use of truncated portions of an antibody's heavy and light chain
sequences (e.g., mAB 3E10) as needed to maintain the functional
integrity of the attached mature GAA polypeptide. Further still,
the internalizing moiety can be linked to an exposed internal
(non-terminus) residue of mature GAA or a variant thereof. In
further embodiments, any combination of the mature
GAA-internalizing moiety configurations can be employed, thereby
resulting in a mature GAA:internalizing moiety ratio that is
greater than 1:1 (e.g., two mature GAA molecules to one
internalizing moiety).
[0169] The mature GAA polypeptide and the internalizing moiety may
be linked directly to each other. Alternatively, they may be linked
to each other via a linker sequence, which separates the mature GAA
polypeptide and the internalizing moiety by a distance sufficient
to ensure that each domain properly folds into its secondary and
tertiary structures. Preferred linker sequences (1) should adopt a
flexible extended conformation, (2) should not exhibit a propensity
for developing an ordered secondary structure which could interact
with the functional domains of the mature GAA polypeptide or the
internalizing moiety, and (3) should have minimal hydrophobic or
charged character, which could promote interaction with the
functional protein domains. Typical surface amino acids in flexible
protein regions include Gly, Asn and Ser. Permutations of amino
acid sequences containing Gly, Asn and Ser would be expected to
satisfy the above criteria for a linker sequence. Other near
neutral amino acids, such as Thr and Ala, can also be used in the
linker sequence. In a specific embodiment, a linker sequence length
of about 20 amino acids can be used to provide a suitable
separation of functional protein domains, although longer or
shorter linker sequences may also be used. The length of the linker
sequence separating the mature GAA polypeptide and the
internalizing moiety can be from 5 to 500 amino acids in length, or
more preferably from 5 to 100 amino acids in length. Preferably,
the linker sequence is from about 5-30 amino acids in length. In
preferred embodiments, the linker sequence is from about 5 to about
20 amino acids, and is advantageously from about 10 to about 20
amino acids. In other embodiments, the linker joining the mature
GAA polypeptide to an internalizing moiety can be a constant domain
of an antibody (e.g., constant domain of mAb 3E10 or all or a
portion of an Fc region of another antibody). In certain
embodiments, the linker is a cleavable linker. In certain
embodiments, the linker sequence comprises the linker sequence of
SEQ ID NO: 30.
[0170] In other embodiments, the mature GAA polypeptide or
functional fragment thereof may be conjugated or joined directly to
the internalizing moiety. For example, a recombinantly conjugated
chimeric polypeptide can be produced as an in-frame fusion of the
mature GAA portion and the internalizing moiety portion. In certain
embodiments, the linker may be a cleavable linker. In any of the
foregoing embodiments, the internalizing moiety may be conjugated
(directly or via a linker) to the N-terminal or C-terminal amino
acid of the mature GAA polypeptide, such as to the N-terminal or
C-terminal amino acid of a GAA polypeptide comprising a mature GAA.
In other embodiments, the internalizing moiety may be conjugated
(directly or indirectly) to an internal amino acid of the mature
GAA polypeptide. Note that the two portions of the construct are
conjugated/joined to each other. Unless otherwise specified,
describing the chimeric polypeptide as a conjugation of the mature
GAA portion to the internalizing moiety is used equivalently as a
conjugation of the internalizing moiety to the mature GAA portion.
Further, unless otherwise specified, conjugation and/or joining
refers to either chemical or genetic conjugation.
[0171] In certain embodiments, the chimeric polypeptides of the
present disclosure can be generated using well-known cross-linking
reagents and protocols. For example, there are a large number of
chemical cross-linking agents that are known to those skilled in
the art and useful for cross-linking the mature GAA polypeptide
with an internalizing moiety (e.g., an antibody). For example, the
cross-linking agents are heterobifunctional cross-linkers, which
can be used to link molecules in a stepwise manner.
Heterobifunctional cross-linkers provide the ability to design more
specific coupling methods for conjugating proteins, thereby
reducing the occurrences of unwanted side reactions such as
homo-protein polymers. A wide variety of heterobifunctional
cross-linkers are known in the art, including succinimidyl
4-(N-maleimidomethyl) cyclohexane-1-carboxylate (SMCC),
m-Maleimidobenzoyl-N-hydroxysuccinimide ester (MBS); N-succinimidyl
(4-iodoacetyl) aminobenzoate (STAB), succinimidyl
4-(p-maleimidophenyl) butyrate (SMPB),
1-ethyl-3-(3-dimethylaminopropyl) carbodiimide hydrochloride (EDC);
4-succinimidyloxycarbonyl-a-methyl-a-(2-pyridyldithio)-tolune
(SMPT), N-succinimidyl 3-(2-pyridyldithio) propionate (SPDP),
succinimidyl 6-[3-(2-pyridyldithio) propionate] hexanoate
(LC-SPDP). Those cross-linking agents having N-hydroxysuccinimide
moieties can be obtained as the N-hydroxysulfosuccinimide analogs,
which generally have greater water solubility. In addition, those
cross-linking agents having disulfide bridges within the linking
chain can be synthesized instead as the alkyl derivatives so as to
reduce the amount of linker cleavage in vivo. In addition to the
heterobifunctional cross-linkers, there exists a number of other
cross-linking agents including homobifunctional and photoreactive
cross-linkers. Disuccinimidyl subcrate (DSS), bismaleimidohexane
(BMI-1) and dimethylpimelimidate.2 HCl (DMP) are examples of useful
homobifunctional cross-linking agents, and
bis-[B-(4-azidosalicylamido)ethyl]disulfide (BASED) and
N-succinimidyl-6(4'-azido-2'-nitrophenylamino)hexanoate (SANPAH)
are examples of useful photoreactive cross-linkers for use in this
disclosure. For a recent review of protein coupling techniques, see
Means et al. (1990) Bioconjugate Chemistry. 1:2-12, incorporated by
reference herein.
[0172] One particularly useful class of heterobifunctional
cross-linkers, included above, contain the primary amine reactive
group, N-hydroxysuccinimide (NETS), or its water soluble analog
N-hydroxysulfosuccinimide (sulfo-NHS). Primary amines (lysine
epsilon groups) at alkaline pH's are unprotonated and react by
nucleophilic attack on NHS or sulfo-NETS esters. This reaction
results in the formation of an amide bond, and release of NETS or
sulfo-NHS as a by-product. Another reactive group useful as part of
a heterobifunctional cross-linker is a thiol reactive group. Common
thiol reactive groups include maleimides, halogens, and pyridyl
disulfides. Maleimides react specifically with free sulfhydryls
(cysteine residues) in minutes, under slightly acidic to neutral
(pH 6.5-7.5) conditions. Halogens (iodoacetyl functions) react with
--SH groups at physiological pH's. Both of these reactive groups
result in the formation of stable thioether bonds. The third
component of the heterobifunctional cross-linker is the spacer arm
or bridge. The bridge is the structure that connects the two
reactive ends. The most apparent attribute of the bridge is its
effect on steric hindrance. In some instances, a longer bridge can
more easily span the distance necessary to link two complex
biomolecules.
[0173] In some embodiments, the chimeric polypeptide comprises
multiple linkers. For example, if the chimeric polypeptide
comprises an scFv internalizing moiety, the chimeric polypeptide
may comprise a first linker conjugating the GAA to the
internalizing moiety, and a second linker in the scFv conjugating
the V.sub.H domain (e.g., SEQ ID NO: 9) to the V.sub.L domain
(e.g., SEQ ID NO: 10).
[0174] Preparing protein-conjugates using heterobifunctional
reagents is a two-step process involving the amine reaction and the
sulfhydryl reaction. For the first step, the amine reaction, the
protein chosen should contain a primary amine. This can be lysine
epsilon amines or a primary alpha amine found at the N-terminus of
most proteins. The protein should not contain free sulfhydryl
groups. In cases where both proteins to be conjugated contain free
sulfhydryl groups, one protein can be modified so that all
sulfhydryls are blocked using for instance, N-ethylmaleimide (see
Partis et al. (1983) J. Pro. Chem. 2:263, incorporated by reference
herein). Ellman's Reagent can be used to calculate the quantity of
sulfhydryls in a particular protein (see for example Ellman et al.
(1958) Arch. Biochem. Biophys. 74:443 and Riddles et al. (1979)
Anal. Biochem. 94:75, incorporated by reference herein).
[0175] In certain specific embodiments, chimeric polypeptides of
the disclosure can be produced by using a universal carrier system.
For example, a mature GAA polypeptide can be conjugated to a common
carrier such as protein A, poly-L-lysine, hex-histidine, and the
like. The conjugated carrier will then form a complex with an
antibody which acts as an internalizing moiety. A small portion of
the carrier molecule that is responsible for binding immunoglobulin
could be used as the carrier.
[0176] In certain embodiments, chimeric polypeptides of the
disclosure can be produced by using standard protein chemistry
techniques such as those described in Bodansky, M. Principles of
Peptide Synthesis, Springer Verlag, Berlin (1993) and Grant G. A.
(ed.), Synthetic Peptides: A User's Guide, W. H. Freeman and
Company, New York (1992). In addition, automated peptide
synthesizers are commercially available (e.g., Advanced ChemTech
Model 396; Milligen/Biosearch 9600). In any of the foregoing
methods of cross-linking for chemical conjugation of mature GAA to
an internalizing moiety, a cleavable domain or cleavable linker can
be used. Cleavage will allow separation of the internalizing moiety
and the mature GAA polypeptide. For example, following penetration
of a cell by a chimeric polypeptide, cleavage of the cleavable
linker would allow separation of mature GAA from the internalizing
moiety.
[0177] In certain embodiments, the chimeric polypeptides comprising
a GAA polypeptide portion (e.g., a GAA polypeptide comprising a
mature GA polypeptide sequence) and an internalizing moiety portion
can be generated as a fusion protein containing the GAA polypeptide
and the internalizing moiety. In certain embodiments, the chimeric
polypeptides of the present disclosure can be generated as a fusion
protein containing a mature GAA polypeptide and an internalizing
moiety (e.g., an antibody or a homing peptide), expressed as one
contiguous polypeptide chain. In certain embodiments, the chimeric
polypeptide is generated as a fusion protein that comprises a GAA
polypeptide portion and internalizing moiety portion, wherein the
GAA polypeptide portion comprises a mature GAA polypeptide and also
includes additional polypeptide sequence from a GAA polypeptide,
including sequence contiguous with the mature GAA polypeptide. In
preparing such fusion protein, a fusion gene is constructed
comprising nucleic acids which encode a mature GAA polypeptide and
an internalizing moiety, and optionally, a peptide linker sequence
to span the mature GAA polypeptide and the internalizing moiety.
The use of recombinant DNA techniques to create a fusion gene, with
the translational product being the desired fusion protein, is well
known in the art. Both the coding sequence of a gene and its
regulatory regions can be redesigned to change the functional
properties of the protein product, the amount of protein made, or
the cell type in which the protein is produced. The coding sequence
of a gene can be extensively altered--for example, by fusing part
of it to the coding sequence of a different gene to produce a novel
hybrid gene that encodes a fusion protein. Examples of methods for
producing fusion proteins are described in PCT applications
PCT/US87/02968, PCT/US89/03587 and PCT/US90/07335, as well as
Traunecker et al. (1989) Nature 339:68, incorporated by reference
herein. Essentially, the joining of various DNA fragments coding
for different polypeptide sequences is performed in accordance with
conventional techniques, employing blunt-ended or stagger-ended
termini for ligation, restriction enzyme digestion to provide for
appropriate termini, filling in of cohesive ends as appropriate,
alkaline phosphatase treatment to avoid undesirable joining, and
enzymatic ligation. Alternatively, the fusion gene can be
synthesized by conventional techniques including automated DNA
synthesizers. In another method, PCR amplification of gene
fragments can be carried out using anchor primers which give rise
to complementary overhangs between two consecutive gene fragments
which can subsequently be annealed to generate a chimeric gene
sequence (see, for example, Current Protocols in Molecular Biology,
Eds. Ausubel et al. John Wiley & Sons: 1992). The chimeric
polypeptides encoded by the fusion gene may be recombinantly
produced using various expression systems as is well known in the
art (also see below).
[0178] Recombinantly conjugated chimeric polypeptides include
embodiments in which the mature GAA polypeptide is conjugated to
the N-terminus or C-terminus of the internalizing moiety. Exemplary
chimeric polypeptides in which mature GAA polypeptides are
conjugated to variant light and heavy chains of Fv3E10 are
indicated in SEQ ID NOS: 11 and 12. In certain embodiments, a
chimeric polypeptide of the disclosure further comprises, at the
N-terminus (at or within 10 amino acid residues of the N-terminus),
an amino acid sequence set forth in SEQ ID NO: 19 or 20.
[0179] Recombinantly conjugated chimeric polypeptides include
embodiments in which the internalizing moiety is N-terminal to the
GAA polypeptide and embodiments in which the internalizing moiety
is C-terminal to the GAA polypeptide portion.
[0180] We note that methods of making fusion proteins recombinantly
are well known in the art. Any of the chimeric proteins described
herein can readily be made recombinantly. This includes proteins
having one or more tags and/or one or more linkers. For example, if
the chimeric polypeptide comprises an scFv internalizing moiety,
the chimeric polypeptide may comprise a first linker
interconnection the internalizing moiety to the mature GAA
polypeptide portion, and a second linker in the scFv conjugating
the V.sub.H domain. Moreover, in certain embodiments, the chimeric
polypeptides comprise a "AGIH" portion (SEQ ID NO: 19) on the
N-terminus of the chimeric polypeptide (or within 10 amino acid
residues of the N-terminus), and such chimeric polypeptides may be
provided in the presence or absence of one or more epitope tags. In
further embodiments, the chimeric polypeptide comprises a serine at
the N-terminal most position of the polypeptide. In some
embodiments, the chimeric polypeptides comprise an "SAGIH" (SEQ ID
NO: 20) portion at the N-terminus of the polypeptide (or within 10
amino acid residues of the N-terminus), and such chimeric
polypeptides may be provided in the presence or absence of one or
more epitope tags.
[0181] In some embodiments, the immunogenicity of the chimeric
polypeptide may be reduced by identifying a candidate T-cell
epitope within a junction region spanning the chimeric polypeptide
and changing an amino acid within the junction region as described
in U.S. Patent Publication No. 2003/0166877.
[0182] Chimeric polypeptides according to the disclosure can be
used for numerous purposes. We note that any of the chimeric
polypeptides described herein can be used in any of the methods
described herein, and such suitable combinations are specifically
contemplated.
[0183] Chimeric polypeptides described herein can be used to
deliver mature GAA polypeptide to cells, particular to a muscle
cell. In certain embodiments, chimeric polypeptides deliver mature
GAA to liver cells. Thus, the chimeric polypeptides can be used to
facilitate transport of mature GAA to cells in vitro or in vivo. By
facilitating transport to cells, the chimeric polypeptides improve
delivery efficiency, thus facilitating working with mature GAA
polypeptide in vitro or in vivo. Further, by increasing the
efficiency of transport, the chimeric polypeptides may help
decrease the amount of mature GAA needed for in vitro or in vivo
experimentation. Moreover, by facilitating delivery to the
cytoplasm, the chimeric polypeptides and methods of the disclosure
can address the problems associated with cytoplasmic accumulation
of glycogen in, for example, Pompe disease.
[0184] The chimeric polypeptides can be used to study the function
of mature GAA in cells in culture, as well as to study transport of
mature GAA. The chimeric polypeptides can be used to identify
binding partners for mature GAA in cells, such as transport between
cytoplasm and lysosome. The chimeric polypeptides can be used in
screens to identify modifiers (e.g., small organic molecules or
polypeptide modifiers) of mature GAA activity in a cell. The
chimeric polypeptides can be used to help treat or alleviate the
symptoms of Pompe disease in humans or in an animal model. The
foregoing are merely exemplary of the uses for the subject chimeric
polypeptides.
[0185] Any of the chimeric polypeptides described herein, including
chimeric polypeptides combining any of the features of the GAA
polypeptides, internalizing moieties, and linkers, may be used in
any of the methods of the disclosure.
IV. GAA Related Nucleic Acids and Expression
[0186] In certain embodiments, the present disclosure makes use of
nucleic acids for producing a mature GAA polypeptide (including
functional fragments, variants, and fusions thereof), such as for
producing GAA polypeptides comprising a mature GAA polypeptide. In
certain specific embodiments, the nucleic acids may further
comprise DNA which encodes an internalizing moiety (e.g., an
antibody or a homing peptide) for making a recombinant chimeric
protein of the disclosure. In certain embodiments, the nucleic acid
construct does not encode a chimeric polypeptide comprising a GAA
precursor polypeptide of approximately 110 kDa. In certain
embodiments, the nucleic acid construct encodes a GAA polypeptide
comprising mature GAA but does not encode a GAA polypeptide
comprising (i) the amino acid sequence set forth in SEQ ID NO: 1 or
2 or (ii) a portion corresponding to residues 1-27 and/or 1-56 of
SEQ ID NO: 1 or 2. All these nucleic acids are collectively
referred to as mature GAA nucleic acids because they encode a
polypeptide comprising a mature GAA polypeptide and, optionally,
additional contiguous portions of a GAA polypeptide.
[0187] The nucleic acids may be single-stranded or double-stranded,
DNA or RNA molecules. In certain embodiments, the disclosure
relates to isolated or recombinant nucleic acid sequences that are
at least 80%, 85%, 90%, 95%, 97%, 98%, 99% or 100% identical to a
region of a GAA nucleotide sequence (e.g., GenBank Accession No.:
NM_000152.3 which encodes NP000143.2; NM_001079803.1 which encodes
NP_001073271.1; and NM_001079804.1 which encodes NP_001073272.1)
that encodes mature GAA (e.g., mature GAA nucleotide sequence). The
nucleotide sequences for GAA are hereby incorporated by reference
in their entirety. In further embodiments, the GAA nucleic acid
sequences can be isolated, recombinant, and/or fused with a
heterologous nucleotide sequence, or in a DNA library.
[0188] In certain embodiments, mature GAA nucleic acids also
include nucleotide sequences that hybridize under highly stringent
conditions to any of the above-mentioned native GAA nucleotide
sequences (e.g., GenBank Accession No.: NM.sub.-- 000152.3;
NM_001079803.1; and NM_001079804.1), or complement sequences
thereof. One of ordinary skill in the art will understand readily
that appropriate stringency conditions which promote DNA
hybridization can be varied. For example, one could perform the
hybridization at 6.0.times. sodium chloride/sodium citrate (SSC) at
about 45.degree. C., followed by a wash of 2.0.times.SSC at
50.degree. C. For example, the salt concentration in the wash step
can be selected from a low stringency of about 2.0.times.SSC at
50.degree. C. to a high stringency of about 0.2.times.SSC at
50.degree. C. In addition, the temperature in the wash step can be
increased from low stringency conditions at room temperature, about
22.degree. C., to high stringency conditions at about 65.degree. C.
Both temperature and salt may be varied, or temperature or salt
concentration may be held constant while the other variable is
changed. In one embodiment, the disclosure provides nucleic acids
which hybridize under low stringency conditions of 6.times. SSC at
room temperature followed by a wash at 2.times.SSC at room
temperature.
[0189] Isolated nucleic acids which differ from the native mature
GAA nucleic acids due to degeneracy in the genetic code are also
within the scope of the disclosure. For example, a number of amino
acids are designated by more than one triplet. Codons that specify
the same amino acid, or synonyms (for example, CAU and CAC are
synonyms for histidine) may result in "silent" mutations which do
not affect the amino acid sequence of the protein. However, it is
expected that DNA sequence polymorphisms that do lead to changes in
the amino acid sequences of the subject proteins will exist among
mammalian cells. One skilled in the art will appreciate that these
variations in one or more nucleotides (up to about 3-5% of the
nucleotides) of the nucleic acids encoding a particular protein may
exist among individuals of a given species due to natural allelic
variation. Any and all such nucleotide variations and resulting
amino acid polymorphisms are within the scope of this
disclosure.
[0190] In certain embodiments, the recombinant mature GAA nucleic
acids may be operably linked to one or more regulatory nucleotide
sequences in an expression construct. Regulatory nucleotide
sequences will generally be appropriate for a host cell used for
expression. Numerous types of appropriate expression vectors and
suitable regulatory sequences are known in the art for a variety of
host cells. Typically, said one or more regulatory nucleotide
sequences may include, but are not limited to, promoter sequences,
leader or signal sequences, ribosomal binding sites,
transcriptional start and termination sequences, translational
start and termination sequences, and enhancer or activator
sequences. Constitutive or inducible promoters as known in the art
are contemplated by the disclosure. The promoters may be either
naturally occurring promoters, or hybrid promoters that combine
elements of more than one promoter. An expression construct may be
present in a cell on an episome, such as a plasmid, or the
expression construct may be inserted in a chromosome. In a
preferred embodiment, the expression vector contains a selectable
marker gene to allow the selection of transformed host cells.
Selectable marker genes are well known in the art and will vary
with the host cell used. In certain aspects, this disclosure
relates to an expression vector comprising a nucleotide sequence
encoding a mature GAA polypeptide and operably linked to at least
one regulatory sequence. Regulatory sequences are art-recognized
and are selected to direct expression of the encoded polypeptide.
Accordingly, the term regulatory sequence includes promoters,
enhancers, and other expression control elements. Exemplary
regulatory sequences are described in Goeddel; Gene Expression
Technology: Methods in Enzymology, Academic Press, San Diego,
Calif. (1990). It should be understood that the design of the
expression vector may depend on such factors as the choice of the
host cell (e.g., Chines Hamster Ovary cells) to be transformed
and/or the type of protein desired to be expressed. Moreover, the
vector's copy number, the ability to control that copy number and
the expression of any other protein encoded by the vector, such as
antibiotic markers, should also be considered.
[0191] In some embodiments, a nucleic acid construct, comprising a
nucleotide sequence that encodes a mature GAA polypeptide or a
bioactive fragment thereof, is operably linked to a nucleotide
sequence that encodes an internalizing moiety, wherein the nucleic
acid construct encodes a chimeric polypeptide having mature GAA
biological activity. In certain embodiments, the nucleic acid
constructs may further comprise a nucleotide sequence that encodes
a linker.
[0192] This disclosure also pertains to a host cell transfected
with a recombinant gene which encodes a mature GAA polypeptide or a
chimeric polypeptide of the disclosure. The host cell may be any
prokaryotic or eukaryotic cell. For example, a mature GAA
polypeptide or a chimeric polypeptide may be expressed in bacterial
cells such as E. coli, insect cells (e.g., using a baculovirus
expression system), yeast, or mammalian cells. Other suitable host
cells are known to those skilled in the art.
[0193] The present disclosure further pertains to methods of
producing a mature GAA polypeptide or a chimeric polypeptide of the
disclosure. For example, a host cell transfected with an expression
vector encoding a mature GAA polypeptide or a chimeric polypeptide
can be cultured under appropriate conditions to allow expression of
the polypeptide to occur. The polypeptide may be secreted and
isolated from a mixture of cells and medium containing the
polypeptides. Alternatively, the polypeptides may be retained
cytoplasmically or in a membrane fraction and the cells harvested,
lysed and the protein isolated. A cell culture includes host cells,
media and other byproducts. Suitable media for cell culture are
well known in the art. The polypeptides can be isolated from cell
culture medium, host cells, or both using techniques known in the
art for purifying proteins, including ion-exchange chromatography,
gel filtration chromatography, ultrafiltration, electrophoresis,
and immunoaffinity purification with antibodies specific for
particular epitopes of the polypeptides (e.g., a GAA polypeptide).
In a preferred embodiment, the polypeptide is a fusion protein
containing a domain which facilitates its purification.
[0194] A recombinant mature GAA nucleic acid can be produced by
ligating the cloned gene, or a portion thereof, into a vector
suitable for expression in either prokaryotic cells, eukaryotic
cells (yeast, avian, insect or mammalian), or both. Expression
vehicles for production of a recombinant polypeptide include
plasmids and other vectors. For instance, suitable vectors include
plasmids of the types: pBR322-derived plasmids, pEMBL-derived
plasmids, pEX-derived plasmids, pBTac-derived plasmids and
pUC-derived plasmids for expression in prokaryotic cells, such as
E. coli. The preferred mammalian expression vectors contain both
prokaryotic sequences to facilitate the propagation of the vector
in bacteria, and one or more eukaryotic transcription units that
are expressed in eukaryotic cells. The pcDNAI/amp, pcDNAI/neo,
pRc/CMV, pSV2gpt, pSV2neo, pSV2-dhfr, pTk2, pRSVneo, pMSG, pSVT7,
pko-neo and pHyg derived vectors are examples of mammalian
expression vectors suitable for transfection of eukaryotic cells.
Some of these vectors are modified with sequences from bacterial
plasmids, such as pBR322, to facilitate replication and drug
resistance selection in both prokaryotic and eukaryotic cells.
Alternatively, derivatives of viruses such as the bovine papilloma
virus (BPV-1), or Epstein-Barr virus (pHEBo, pREP-derived and p205)
can be used for transient expression of proteins in eukaryotic
cells. The various methods employed in the preparation of the
plasmids and transformation of host organisms are well known in the
art. For other suitable expression systems for both prokaryotic and
eukaryotic cells, as well as general recombinant procedures, see
Molecular Cloning A Laboratory Manual, 2nd Ed., ed. by Sambrook,
Fritsch and Maniatis (Cold Spring Harbor Laboratory Press, 1989)
Chapters 16 and 17. In some instances, it may be desirable to
express the recombinant polypeptide by the use of a baculovirus
expression system. Examples of such baculovirus expression systems
include pVL-derived vectors (such as pVL1392, pVL1393 and pVL941),
pAcUW-derived vectors (such as pAcUW1), and pBlueBac-derived
vectors (such as the -gal containing pBlueBac III).
[0195] Techniques for making fusion genes are well known.
Essentially, the joining of various DNA fragments coding for
different polypeptide sequences is performed in accordance with
conventional techniques, employing blunt-ended or stagger-ended
termini for ligation, restriction enzyme digestion to provide for
appropriate termini, filling-in of cohesive ends as appropriate,
alkaline phosphatase treatment to avoid undesirable joining, and
enzymatic ligation. In another embodiment, the fusion gene can be
synthesized by conventional techniques including automated DNA
synthesizers. Alternatively, PCR amplification of gene fragments
can be carried out using anchor primers which give rise to
complementary overhangs between two consecutive gene fragments
which can subsequently be annealed to generate a chimeric gene
sequence (see, for example, Current Protocols in Molecular Biology,
eds. Ausubel et al., John Wiley & Sons: 1992).
[0196] The disclosure contemplates methods of producing chimeric
proteins recombinantly, such as described above. Suitable vectors
and host cells may be readily selected for expression of proteins
in, for example, yeast or mammalian cells. Host cells may express a
vector encoding a chimeric polypeptide stably or transiently. Such
host cells may be cultured under suitable conditions to express
chimeric polypeptide which can be readily isolated from the cell
culture medium.
[0197] Chimeric polypeptides of the disclosure (e.g., polypeptides
comprising a GAA portion comprising mature GAA and an internalizing
moiety portion) may be expressed as a single polypeptide chain or
as more than one polypeptide chains. An example of a single
polypeptide chain is when a GAA portion is fused inframe to an
internalizing moiety, which internalizing moiety is an scFv. In
certain embodiments, this single polypeptide chain is expressed
from a single vectors as a fusion protein.
[0198] An example of more than one polypeptide chains is when the
internalizing moiety is an antibody or Fab. In certain embodiments,
the heavy and light chains of the antibody or Fab may be expressed
in a host cell expressing a single vector or two vectors (one
expressing the heavy chain and one expressing the light chain). In
either case, the GAA polypeptide may be expressed as an inframe
fusion to, for example, the C-terminus of the heavy chain such that
the GAA polypeptide is appended to the internalizing moiety but at
a distance to the antigen binding region of the internalizing
moiety.
[0199] As noted above, methods for recombinantly expressing
polypeptides, including chimeric polypeptides, are well known in
the art. Nucleotide sequences expressing a GAA polypeptide, such as
a human GAA polypeptide, having a particular amino acid sequence
are available and can be used. Moreover, nucleotide sequences
expressing an internalizing moiety portion, such as expressing a
3E10 antibody, scFv, or Fab comprising the VH and VL set forth in
SEQ ID NO: 9 and 10) are publicly available and can be combined
with nucleotide sequence encoding suitable heavy and light chain
constant regions. The disclosure contemplates nucleotide sequences
encoding any of the chimeric polypeptides of the disclosure,
vectors (single vector or set of vectors) comprising such
nucleotide sequences, host cells comprising such vectors, and
methods of culturing such host cells to express chimeric
polypeptides of the disclosure.
V. Methods of Treatment and Other Methods of Use
[0200] For any of the methods described herein, the disclosure
contemplates the use of any of the chimeric polypeptides and/or
compositions described throughout the application. In addition, for
any of the methods described herein, the disclosure contemplates
the combination of any step or steps of one method with any step or
steps from another method.
[0201] For example, a chimeric polypeptide of the disclosure
comprising a GAA polypeptide portion and an internalizing moiety
portion can be used in any of the methods of the disclosure.
[0202] In certain embodiments, GAA polypeptides may comprise one of
the mature, active forms of the GAA protein, such as the 70 kDa
form or the mature 76 kDa form, or a combination of the two. Mature
GAA polypeptides may also be administered in combination with the
immature 110 kDa form of GAA, in order to target as many organelles
and cellular regions/compartments as possible. In addition, mature
GAA polypeptides may be administered in combination with and/or
following administration of immunotolerizing fragments of GAA, such
as small fragments of GAA, and/or immunosuppressive compounds. In
some embodiments, the GAA polypeptides comprise a mature GAA
polypeptide as well as additional polypeptide sequence from a GAA
polypeptide, such as sequence contiguous with the mature GAA
polypeptide.
[0203] In certain embodiments, the present disclosure provides
methods of delivering chimeric polypeptides to cells, including
cells in culture (in vitro or ex vivo) and cells in a subject.
Delivery to cells in culture, such as healthy cells or cells from a
model of disease, have numerous uses. These uses include to
identify GAA substrates or binding partners, to evaluate
localization and/or trafficking (e.g., to cytoplasm, lysosome,
and/or autophagic vesicles), to evaluate enzymatic activity under a
variety of conditions (e.g., pH), to assess glycogen accumulation,
and the like. In certain embodiments, chimeric polypeptides of the
disclosure can be used as reagents to understand GAA activity,
localization, and trafficking in healthy or disease contexts.
[0204] Delivery to subjects, such as to cells in a subject, have
numerous uses. Exemplary therapeutic uses are described below.
Moreover, the chimeric polypeptides may be used for diagnostic or
research purposes. For example, a chimeric polypeptide of the
disclosure may be detectably labeled and administered to a subject,
such as an animal model of disease or a patient, and used to image
the chimeric polypeptide in the subject's tissues (e.g.,
localization to muscle and/or liver). Additionally exemplary uses
include delivery to cells in a subject, such as to an animal model
of disease (e.g., Pompe disease). By way of example, chimeric
polypeptides of the disclosure may be used as reagents and
delivered to animals to understand GAA bioactivity, localization
and trafficking, protein-protein interactions, enzymatic activity,
and impacts on animal physiology in healthy or diseased
animals.
[0205] In certain embodiments, the present disclosure provides
methods of treating conditions associated with dysfunction of GAA
and Pompe disease. Such conditions include, but are not limited to,
aberrant accumulation of glycogen in the lysosomes, cytoplasm,
and/or autophagic vesicles of affected cells, for example heart and
skeletal muscle cells; cell starvation; disorganization of
microtubule structure; increase in number and/or size of lysosomes,
rupture of lysosomes; accumulation of cellular debris including
autophagic components; disruption of mitochondrial structure; cell
swelling; motorneuron disease; muscle weakness; progressive muscle
decline; damage to skeletal, respiratory, and/or cardiac muscles;
and premature death. Some symptoms of Pompe disease do not manifest
until the patients have lived without functional GAA or with
diminished levels of GAA for extended periods of time, such as 6
months or longer. In these cases, there has been additional time
for glycogen to accumulate not only in the lysosomes but also in
cytoplasm and autophagic vacuoles of the patient's cells,
triggering disruption of cell and organelle function as described
above. When the disease progresses to this stage, traditional
enzyme replacement therapies that target GAA to the lysosome may no
longer be effective or may be inadequate to treat the condition.
Thus, in some embodiments, administration of the mature GAA
polypeptides of the present disclosure targets polypeptides
comprising the mature GAA to the cytoplasm of affected cells and
treats symptoms associated with accumulation of glycogen in the
cytoplasm and/or autophagic vacuoles.
[0206] These methods involve, in certain embodiments, administering
to the individual a therapeutically effective amount of a chimeric
polypeptide as described above (e.g., a chimeric polypeptide
comprising (i) a GAA portion comprising a GAA polypeptide and (ii)
an internalizing moiety portion). These methods are particularly
aimed at therapeutic and prophylactic treatments of animals, and
more particularly, humans. With respect to methods for Pompe
disease, the disclosure contemplates all combinations of any of the
foregoing aspects and embodiments, as well as combinations with any
of the embodiments set forth in the detailed description and
examples.
[0207] The present disclosure provides a method of delivering a
chimeric polypeptide or nucleic acid construct into a cell via an
equilibrative nucleoside transporter (ENT2) pathway, comprising
contacting a cell with a chimeric polypeptide or nucleic acid
construct. In certain embodiments, the method comprises contacting
a cell with a chimeric polypeptide, which chimeric polypeptide
comprises a mature GAA polypeptide or bioactive fragment thereof
and an internalizing moiety which mediates transport across a
cellular membrane via an ENT2 pathway, thereby delivering the
chimeric polypeptide into the cell. In certain embodiments, the
cell is a muscle cell. The muscle cells targeted using the claimed
method may include skeletal, cardiac or smooth muscle cells. In
other embodiments, the chimeric polypeptides are delivered to
liver.
[0208] The present disclosure also provides a method of delivering
a chimeric polypeptide or nucleic acid construct into a cell via a
pathway that allows access to cells other than muscle cells. Other
cell types that could be targeted using the claimed method include,
for example, liver cells, neurons, epithelial cells, uterine cells,
and kidney cells.
[0209] Conditions associated with GAA dysfunction and Pompe disease
are manifold. Pompe disease is characterized by massive
accumulation of glycogen in many tissues, but predominantly
skeletal and cardiac muscles, leading to severe dysfunction of the
affected tissues. The disease symptoms are progressive. Early onset
form of the disease manifests clinically in infants as a
combination of hypotonia and generalized muscle weakness, such as a
head lag or a "floppy baby" appearance. Cardiomegaly appears in an
estimated 92-95% of all infant patients, and heart failure often
occurs. Respiratory failure due to weakness of the diaphragm is
common, and infants may present with difficulties feeding. Without
treatment, infants usually die within the first two years of
life.
[0210] In juvenile and adult onset forms of the disease, symptoms
include musculoskeletal dysfunction, such as muscle weakness, gait
abnormalities, muscle pain, frequent falls, difficulty chewing;
respiratory complications due to weakening of the diaphragm and
other respiratory muscles; cardiac abnormalities such as
arrhythmias; and gastrointestinal problems such as difficulty
swallowing or feeding. Patients may become dependent on ventilators
as the respiratory complications progress, or on wheelchairs as
motor function declines.
[0211] The terms "treatment", "treating", and the like are used
herein to generally mean obtaining a desired pharmacologic and/or
physiologic effect. "Treating" a condition or disease refers to
curing as well as ameliorating at least one symptom of the
condition or disease, and includes administration of a composition
which reduces the frequency of, or delays the onset of, symptoms of
a medical condition in a subject in need relative to a subject
which does not receive the composition. "Treatment" as used herein
covers any treatment of a disease or condition of a mammal,
particularly a human, and includes: (a) preventing symptoms of the
disease or condition from occurring in a subject which may be
predisposed to the disease or condition but has not yet begun
experiencing symptoms; (b) inhibiting the disease or condition
(e.g., arresting its development); or (c) relieving the disease or
condition (e.g., causing regression of the disease or condition,
providing improvement in one or more symptoms). For example,
"treatment" of Pompe disease encompasses a complete reversal or
cure of the disease, or any range of improvement in symptoms and/or
adverse effects attributable to Pompe disease. Merely to
illustrate, "treatment" of Pompe disease includes an improvement in
any of the following effects associated with dysfunction of GAA (or
combination thereof): decreased GAA activity (e.g., treatment
increases GAA activity), glycogen accumulation in cells (e.g.,
treatment decreases glycogen accumulation), increased creatine
kinase levels, elevation of urinary glucose tetrasaccharide,
reduction in heart size, hypertrophic cardiomyopathy, respiratory
complications, dependence on a ventilator, muscle dysfunction
and/or weakening, loss of motor function, dependence on a
wheelchair or other form of mobility assistance, dependence on neck
or abdominal support for sitting upright, ultrastructural damage of
muscle fibers, loss of muscle tone and function. Improvements in
any of these symptoms can be readily assessed according to standard
methods and techniques known in the art.
[0212] Other symptoms not listed above may also be monitored in
order to determine the effectiveness of treating Pompe disease. The
population of subjects treated by the method of the disclosure
includes subjects suffering from the undesirable condition or
disease, as well as subjects at risk for development of the
condition or disease. In certain embodiments, administering a
mature GAA chimeric polypeptide may have any one or more of the
following affects: decrease accumulation of glycogen in cytoplasm
of cells, decrease accumulation of glycogen in cytoplasm of muscle
cells, decrease accumulation of glycogen in cytoplasm of liver
cells, decrease accumulation of glycogen in cytoplasm of neurons,
decrease accumulation of glycogen in lysosomes of cells, decrease
accumulation of glycogen in lysosomes of muscle cells, decrease
accumulation of glycogen in lysosomes of liver cells, decrease
accumulation of glycogen in lysosomes of neurons, decrease
accumulation of glycogen in autophagic vacuoles of cells, decrease
accumulation of glycogen in autophagic vacuoles of muscle cells,
decrease accumulation of autophagic vacuoles in cytoplasm of liver
cells, decrease accumulation of glycogen in autophagic vacuoles of
neurons, decrease elevated levels of alanine transaminase (such as
elevated levels in serum), decrease elevated levels of aspartate
transaminase (such as elevated levels in serum), decrease elevated
levels of alkaline phosphatase (such as elevated levels in serum),
and/or decrease elevated levels of creatine phosphokinase (such as
elevated levels in serum). It should be noted that any of the GAA
chimeric polypeptides described above or herein may be used in any
of the methods described herein.
[0213] In certain embodiments, the subjects in need of treatment
are subjects having infantile form of Pompe disease. In other
embodiments, the subjects in need of treatment are subjects having
juvenile onset
[0214] By the term "therapeutically effective dose" is meant a dose
that produces the desired effect for which it is administered. The
exact dose will depend on the purpose of the treatment, and will be
ascertainable by one skilled in the art using known techniques
(see, e.g., Lloyd (1999) The Art, Science and Technology of
Pharmaceutical Compounding).
[0215] In certain embodiments, one or more chimeric polypeptides of
the present disclosure can be administered, together
(simultaneously) or at different times (sequentially). In addition,
chimeric polypeptides of the present disclosure can be administered
alone or in combination with one or more additional compounds or
therapies for treating Pompe disease. For example, one or more
chimeric polypeptides can be co-administered in conjunction with
one or more other therapeutic compounds. In some embodiments, the
one or more chimeric polypeptides can be co-administered in
conjunction with alglucosidase alfa (Myozyme, Genzyme Corporation).
When co-administration is indicated, the combination therapy may
encompass simultaneous or alternating administration. In addition,
the combination may encompass acute or chronic administration.
Optionally, the chimeric polypeptide of the present disclosure and
additional compounds act in an additive or synergistic manner for
treating Pompe disease. Additional compounds to be used in
combination therapies include, but are not limited to, small
molecules, polypeptides, antibodies, antisense oligonucleotides,
and siRNA molecules. Depending on the nature of the combinatory
therapy, administration of the chimeric polypeptides of the
disclosure may be continued while the other therapy is being
administered and/or thereafter. Administration of the chimeric
polypeptides may be made in a single dose, or in multiple doses. In
some instances, administration of the chimeric polypeptides is
commenced at least several days prior to the other therapy, while
in other instances, administration is begun either immediately
before or at the time of the administration of the other
therapy.
[0216] One type of combination therapy makes use of molecules that
promote muscle synthesis and/or fat reduction. Molecules such as
IGF-1, growth hormones, steroids, (.beta.-2 agonists (for example
Clenbuterol), and myostatin inhibitors may be administered to
patients in order to build muscle tissue and reduce fat
infiltration. These molecules may also increase ENT2 levels.
Accordingly, the molecules may be administered before treatment
with mature GAA polypeptides begins, in between treatments with
mature GAA polypeptides, or after treatment with mature GAA
polypeptides.
[0217] In another example of combination therapy, one or more
chimeric polypeptides of the disclosure can be used as part of a
therapeutic regimen combined with one or more additional treatment
modalities. By way of example, such other treatment modalities
include, but are not limited to, dietary therapy, occupational
therapy, physical therapy, ventilator supportive therapy, massage,
acupuncture, acupressure, mobility aids, assistance animals, and
the like.
[0218] Note that although the chimeric polypeptides described
herein can be used in combination with other therapies, in certain
embodiments, a chimeric polypeptide is provided as the sole form of
therapy. Regardless of whether administrated alone or in
combination with other medications or therapeutic regiments, the
dosage, frequency, route of administration, and timing of
administration of the chimeric polypeptides is determined by a
physician based on the condition and needs of the patient.
[0219] Chimeric polypeptides of the disclosure have numerous uses,
including in vitro and in vivo uses. In vivo uses include not only
therapeutic uses but also diagnostic and research uses in, for
example, any of the foregoing animal models. By way of example,
chimeric polypeptides of the disclosure may be used as research
reagents and delivered to animals to understand GAA bioactivity,
localization and trafficking, protein-protein interactions,
enzymatic activity, and impacts on animal physiology in healthy or
diseases animals.
[0220] Chimeric polypeptides may also be used in vitro to evaluate,
for example, GAA bioactivity, localization and trafficking,
protein-protein interactions, and enzymatic activity in cells in
culture, including healthy and GAA deficient cells in culture. The
disclosure contemplates that chimeric polypeptides of the
disclosure may be used to deliver GAA to cytoplasm, lysosome,
and/or autophagic vesicles of cells, including cells in
culture.
VI. Gene Therapy
[0221] Conventional viral and non-viral based gene transfer methods
can be used to introduce nucleic acids encoding polypeptides of
mature GAA and or chimeric polypeptides comprising mature GAA in
mammalian cells or target tissues. In certain embodiments, the
chimeric polypeptides for use in the methods described herein
comprise a mature GAA polypeptide, but also include additional
polypeptide sequence from a GAA polypeptide, including sequence
contiguous with the mature GAA polypeptide. Such methods can be
used to administer nucleic acids encoding polypeptides of the
disclosure (e.g., mature GAA, including variants thereof, and
include chimeric polypeptides) to cells in vitro. The disclosure
contemplates that gene transfer methods may be used to deliver
nucleic acid encoding any of the chimeric polypeptides of the
disclosure or GAA polypeptides. In some embodiments, the nucleic
acids encoding mature GAA are administered for in vivo or ex vivo
gene therapy uses. In other embodiments, gene delivery techniques
are used to study the activity of chimeric polypeptides or GAA
polypeptide or to study Pompe disease in cell based or animal
models, such as to evaluate cell trafficking, enzyme activity, and
protein-protein interactions following delivery to healthy or
diseased cells and tissues. Non-viral vector delivery systems
include DNA plasmids, naked nucleic acid, and nucleic acid
complexed with a delivery vehicle such as a liposome. Viral vector
delivery systems include DNA and RNA viruses, which have either
episomal or integrated genomes after delivery to the cell. Such
methods are well known in the art.
[0222] Methods of non-viral delivery of nucleic acids encoding
engineered polypeptides of the disclosure include lipofection,
microinjection, biolistics, virosomes, liposomes, immunoliposomes,
polycation or lipid:nucleic acid conjugates, naked DNA, artificial
virions, and agent-enhanced uptake of DNA. Lipofection methods and
lipofection reagents are well known in the art (e.g.,
Transfectam.TM. and Lipofectin.TM.). Cationic and neutral lipids
that are suitable for efficient receptor-recognition lipofection of
polynucleotides include those of Felgner, WO 91/17424, WO 91/16024.
Delivery can be to cells (ex vivo administration) or target tissues
(in vivo administration). The preparation of lipid:nucleic acid
complexes, including targeted liposomes such as immunolipid
complexes, is well known to one of skill in the art.
[0223] The use of RNA or DNA viral based systems for the delivery
of nucleic acids encoding mature GAA or its variants take advantage
of highly evolved processes for targeting a virus to specific cells
in the body and trafficking the viral payload to the nucleus. Viral
vectors can be administered directly to patients (in vivo) or they
can be used to treat cells in vitro and the modified cells are
administered to patients (ex vivo). Conventional viral based
systems for the delivery of polypeptides of the disclosure could
include retroviral, lentivirus, adenoviral, adeno-associated and
herpes simplex virus vectors for gene transfer. Viral vectors are
currently the most efficient and versatile method of gene transfer
in target cells and tissues. Integration in the host genome is
possible with the retrovirus, lentivirus, and adeno-associated
virus gene transfer methods, often resulting in long term
expression of the inserted transgene. Additionally, high
transduction efficiencies have been observed in many different cell
types and target tissues.
[0224] The tropism of a retrovirus can be altered by incorporating
foreign envelope proteins, expanding the potential target
population of target cells. Lentiviral vectors are retroviral
vectors that are able to transduce or infect non-dividing cells and
typically produce high viral titers. Selection of a retroviral gene
transfer system would therefore depend on the target tissue.
Retroviral vectors are comprised of cis-acting long terminal
repeats with packaging capacity for up to 6-10 kb of foreign
sequence. The minimum cis-acting LTRs are sufficient for
replication and packaging of the vectors, which are then used to
integrate the therapeutic gene into the target cell to provide
permanent transgene expression. Widely used retroviral vectors
include those based upon murine leukemia virus (MuLV), gibbon ape
leukemia virus (GaLV), Simian Immuno deficiency virus (SW), human
immuno deficiency virus (HIV), and combinations thereof, all of
which are well known in the art.
[0225] In applications where transient expression of the
polypeptides of the disclosure is preferred, adenoviral based
systems are typically used. Adenoviral based vectors are capable of
very high transduction efficiency in many cell types and do not
require cell division. With such vectors, high titer and levels of
expression have been obtained. This vector can be produced in large
quantities in a relatively simple system. Adeno-associated virus
("AAV") vectors are also used to transduce cells with target
nucleic acids, e.g., in the in vitro production of nucleic acids
and peptides, and for in vivo and ex vivo gene therapy procedures.
Construction of recombinant AAV vectors are described in a number
of publications, including U.S. Pat. No. 5,173,414; Tratschin et
al., Mol. Cell. Biol. 5:3251-3260 (1985); Tratschin, et al.; Mol.
Cell. Biol. 4:2072-2081 (1984); Hermonat & Muzyczka, PNAS
81:6466-6470 (1984); and Samulski et al., J. Virol. 63:03822-3828
(1989).
[0226] Recombinant adeno-associated virus vectors (rAAV) are a
promising alternative gene delivery systems based on the defective
and nonpathogenic parvovirus adeno-associated type 2 virus. All
vectors are derived from a plasmid that retains only the AAV 145 bp
inverted terminal repeats flanking the transgene expression
cassette. Efficient gene transfer and stable transgene delivery due
to integration into the genomes of the transduced cell are key
features for this vector system.
[0227] Replication-deficient recombinant adenoviral vectors (Ad)
can be engineered such that a transgene replaces the Ad E1a, E1b,
and E3 genes; subsequently the replication defector vector is
propagated in human 293 cells that supply deleted gene function in
trans. Ad vectors can transduce multiple types of tissues in vivo,
including nondividing, differentiated cells such as those found in
the liver, kidney and muscle system tissues. Conventional Ad
vectors have a large carrying capacity.
[0228] Packaging cells are used to form virus particles that are
capable of infecting a host cell. Such cells include 293 cells,
which package adenovirus, and 42 cells or PA317 cells, which
package retrovirus. Viral vectors used in gene therapy are usually
generated by producer cell line that packages a nucleic acid vector
into a viral particle. The vectors typically contain the minimal
viral sequences required for packaging and subsequent integration
into a host, other viral sequences being replaced by an expression
cassette for the protein to be expressed. The missing viral
functions are supplied in trans by the packaging cell line. For
example, AAV vectors used in gene therapy typically only possess
ITR sequences from the AAV genome which are required for packaging
and integration into the host genome. Viral DNA is packaged in a
cell line, which contains a helper plasmid encoding the other AAV
genes, namely rep and cap, but lacking ITR sequences. The cell line
is also infected with adenovirus as a helper. The helper virus
promotes replication of the AAV vector and expression of AAV genes
from the helper plasmid. The helper plasmid is not packaged in
significant amounts due to a lack of ITR sequences. Contamination
with adenovirus can be reduced by, e.g., heat treatment to which
adenovirus is more sensitive than AAV.
[0229] In many gene therapy applications, it is desirable that the
gene therapy vector be delivered with a high degree of specificity
to a particular tissue type. A viral vector is typically modified
to have specificity for a given cell type by expressing a ligand as
a fusion protein with a viral coat protein on the viruses outer
surface. The ligand is chosen to have affinity for a receptor known
to be present on the cell type of interest. This principle can be
extended to other pairs of virus expressing a ligand fusion protein
and target cell expressing a receptor. For example, filamentous
phage can be engineered to display antibody fragments (e.g., FAB or
Fv) having specific binding affinity for virtually any chosen
cellular receptor. Although the above description applies primarily
to viral vectors, the same principles can be applied to nonviral
vectors. Such vectors can be engineered to contain specific uptake
sequences thought to favor uptake by specific target cells, such as
muscle cells.
[0230] Gene therapy vectors can be delivered in vivo by
administration to an individual patient, by systemic administration
(e.g., intravenous, intraperitoneal, intramuscular, subdermal, or
intracranial infusion) or topical application. Alternatively,
vectors can be delivered to cells ex vivo, such as cells explanted
from an individual patient (e.g., lymphocytes, bone marrow
aspirates, tissue biopsy) or universal donor hematopoietic stem
cells, followed by reimplantation of the cells into a patient,
usually after selection for cells which have incorporated the
vector.
[0231] Ex vivo cell transfection for diagnostics, research, or for
gene therapy (e.g., via re-infusion of the transfected cells into
the host organism) is well known to those of skill in the art. For
example, cells are isolated from the subject organism, transfected
with a nucleic acid (gene or cDNA) encoding, e.g., mature GAA or
its variants, and re-infused back into the subject organism (e.g.,
patient). Various cell types suitable for ex vivo transfection are
well known to those of skill in the art.
[0232] In certain embodiments, stem cells are used in ex vivo
procedures for cell transfection and gene therapy. The advantage to
using stem cells is that they can be differentiated into other cell
types in vitro, or can be introduced into a mammal (such as the
donor of the cells) where they will engraft in the bone marrow.
Stem cells are isolated for transduction and differentiation using
known methods.
[0233] Vectors (e.g., retroviruses, adenoviruses, liposomes, etc.)
containing therapeutic nucleic acids can be also administered
directly to the organism for transduction of cells in vivo.
Alternatively, naked DNA can be administered. Administration is by
any of the routes normally used for introducing a molecule into
ultimate contact with blood or tissue cells. Suitable methods of
administering such nucleic acids are available and well known to
those of skill in the art, and, although more than one route can be
used to administer a particular composition, a particular route can
often provide a more immediate and more effective reaction than
another route.
[0234] Pharmaceutically acceptable carriers are determined in part
by the particular composition being administered, as well as by the
particular method used to administer the composition. Accordingly,
there is a wide variety of suitable formulations of pharmaceutical
compositions of the present disclosure, as described herein.
VII. Methods of Administration
[0235] Various delivery systems are known and can be used to
administer the chimeric polypeptides of the disclosure. Any such
methods may be used to administer any of the chimeric polypeptides
described herein. Methods of introduction can be enteral or
parenteral, including but not limited to, intradermal,
intramuscular, intraperitoneal, intramyocardial, intravenous,
subcutaneous, pulmonary, intranasal, intraocular, epidural, and
oral routes. The chimeric polypeptides may be administered by any
convenient route, for example, by infusion or bolus injection, by
absorption through epithelial or mucocutaneous linings (e.g., oral
mucosa, rectal and intestinal mucosa, etc.) and may be administered
together with other biologically active agents. Administration can
be systemic or local.
[0236] In certain embodiments, the chimeric polypeptide is
administered intravenously.
[0237] In certain embodiments, it may be desirable to administer
the chimeric polypeptides of the disclosure locally to the area in
need of treatment (e.g., muscle); this may be achieved, for
example, and not by way of limitation, by local infusion during
surgery, by means of a catheter, or by means of an implant, the
implant being of a porous, non-porous, or gelatinous material,
including membranes, such as sialastic membranes, fibers, or
commercial skin substitutes.
[0238] In another embodiment, such local administration can be to
all or a portion of the heart. For example, administration can be
by intrapericardial or intramyocardial administration. Similarly,
administration to cardiac tissue can be achieved using a catheter,
wire, and the like intended for delivery of agents to various
regions of the heart.
[0239] In another embodiment, local administration is directed to
the liver. Glycogen storage and glycogenolysis in the liver affect
the availability of glycogen for many other tissues in the body.
For example, a venous catheter may be placed in the hepatic portal
vein to deliver chimeric polypeptides directly to the liver. In
addition, in some embodiments where the internalizing moieties of
the chimeric polypeptides show a lower affinity for liver cells
than for other cell types, delivery through the hepatic portal vein
ensures that adequate concentrations of mature GAA reach the liver
cells.
[0240] Note that the disclosure contemplates methods in which
chimeric polypeptides are administered, at the same or different
times, via one than one route of administration. For example, the
disclosure contemplates a regimen in which chimeric polypeptides
are administered systemically, such as by intravenous infusion, in
combination with local administration via the hepatic portal
vein.
[0241] In other embodiments, the chimeric polypeptides of the
disclosure can be delivered in a vesicle, in particular, a liposome
(see Langer, 1990, Science 249:1527-1533). In yet another
embodiment, the chimeric polypeptides of the disclosure can be
delivered in a controlled release system. In another embodiment, a
pump may be used (see Langer, 1990, supra). In another embodiment,
polymeric materials can be used (see Howard et al., 1989, J.
Neurosurg. 71:105). In certain specific embodiments, the chimeric
polypeptides of the disclosure can be delivered intravenously.
[0242] In certain embodiments, the chimeric polypeptides are
administered by intravenous infusion. In certain embodiments, the
chimeric polypeptides are infused over a period of at least 10, at
least 15, at least 20, or at least 30 minutes. In other
embodiments, the chimeric polypeptides are infused over a period of
at least 60, 90, or 120 minutes. Regardless of the infusion period,
the disclosure contemplates that each infusion is part of an
overall treatment plan where chimeric polypeptide is administered
according to a regular schedule (e.g., weekly, monthly, etc.).
[0243] The foregoing applies to any of the chimeric polypeptides,
compositions, and methods described herein. The disclosure
specifically contemplates any combination of the features of such
chimeric polypeptides, compositions, and methods (alone or in
combination) with the features described for the various
pharmaceutical compositions and route of administration described
in this section.
VIII. Pharmaceutical Compositions
[0244] In certain embodiments, the subject chimeric polypeptides of
the present disclosure are formulated with a pharmaceutically
acceptable carrier. One or more chimeric polypeptides can be
administered alone or as a component of a pharmaceutical
formulation (composition). Any of the chimeric polypeptides
described herein may be formulated, as described herein. In certain
embodiments, the composition includes two or more chimeric
polypeptides of the disclosure, such as a chimeric polypeptide
comprising a mature GAA of approximately 70 kDa and a chimeric
polypeptide comprising a mature GAA of approximately 76 kDa. The
chimeric polypeptides may be formulated for administration in any
convenient way for use in human or veterinary medicine. Wetting
agents, emulsifiers and lubricants, such as sodium lauryl sulfate
and magnesium stearate, as well as coloring agents, release agents,
coating agents, sweetening, flavoring and perfuming agents,
preservatives and antioxidants can also be present in the
compositions.
[0245] Formulations of the subject chimeric polypeptides include,
for example, those suitable for oral, nasal, topical, parenteral,
rectal, and/or intravaginal administration. The formulations may
conveniently be presented in unit dosage form and may be prepared
by any methods well known in the art of pharmacy. The amount of
active ingredient which can be combined with a carrier material to
produce a single dosage form will vary depending upon the host
being treated and the particular mode of administration. The amount
of active ingredient which can be combined with a carrier material
to produce a single dosage form will generally be that amount of
the compound which produces a therapeutic effect.
[0246] In certain embodiments, methods of preparing these
formulations or compositions include combining another type of
therapeutic agents and a carrier and, optionally, one or more
accessory ingredients. In general, the formulations can be prepared
with a liquid carrier, or a finely divided solid carrier, or both,
and then, if necessary, shaping the product.
[0247] Formulations for oral administration may be in the form of
capsules, cachets, pills, tablets, lozenges (using a flavored
basis, usually sucrose and acacia or tragacanth), powders,
granules, or as a solution or a suspension in an aqueous or
non-aqueous liquid, or as an oil-in-water or water-in-oil liquid
emulsion, or as an elixir or syrup, or as pastilles (using an inert
base, such as gelatin and glycerin, or sucrose and acacia) and/or
as mouth washes and the like, each containing a predetermined
amount of a subject chimeric polypeptide therapeutic agent as an
active ingredient. Suspensions, in addition to the active
compounds, may contain suspending agents such as ethoxylated
isostearyl alcohols, polyoxyethylene sorbitol, and sorbitan esters,
microcrystalline cellulose, aluminum metahydroxide, bentonite,
agar-agar and tragacanth, and mixtures thereof.
[0248] In solid dosage forms for oral administration (capsules,
tablets, pills, dragees, powders, granules, and the like), one or
more chimeric polypeptide therapeutic agents of the present
disclosure may be mixed with one or more pharmaceutically
acceptable carriers, such as sodium citrate or dicalcium phosphate,
and/or any of the following: (1) fillers or extenders, such as
starches, lactose, sucrose, glucose, mannitol, and/or silicic acid;
(2) binders, such as, for example, carboxymethylcellulose,
alginates, gelatin, polyvinyl pyrrolidone, sucrose, and/or acacia;
(3) humectants, such as glycerol; (4) disintegrating agents, such
as agar-agar, calcium carbonate, potato or tapioca starch, alginic
acid, certain silicates, and sodium carbonate; (5) solution
retarding agents, such as paraffin; (6) absorption accelerators,
such as quaternary ammonium compounds; (7) wetting agents, such as,
for example, cetyl alcohol and glycerol monostearate; (8)
absorbents, such as kaolin and bentonite clay; (9) lubricants, such
a talc, calcium stearate, magnesium stearate, solid polyethylene
glycols, sodium lauryl sulfate, and mixtures thereof; and (10)
coloring agents. In the case of capsules, tablets and pills, the
pharmaceutical compositions may also comprise buffering agents.
Solid compositions of a similar type may also be employed as
fillers in soft and hard-filled gelatin capsules using such
excipients as lactose or milk sugars, as well as high molecular
weight polyethylene glycols and the like. Liquid dosage forms for
oral administration include pharmaceutically acceptable emulsions,
microemulsions, solutions, suspensions, syrups, and elixirs. In
addition to the active ingredient, the liquid dosage forms may
contain inert diluents commonly used in the art, such as water or
other solvents, solubilizing agents and emulsifiers, such as ethyl
alcohol, isopropyl alcohol, ethyl carbonate, ethyl acetate, benzyl
alcohol, benzyl benzoate, propylene glycol, 1,3-butylene glycol,
oils (in particular, cottonseed, groundnut, corn, germ, olive,
castor, and sesame oils), glycerol, tetrahydrofuryl alcohol,
polyethylene glycols and fatty acid esters of sorbitan, and
mixtures thereof. Besides inert diluents, the oral compositions can
also include adjuvants such as wetting agents, emulsifying and
suspending agents, sweetening, flavoring, coloring, perfuming, and
preservative agents.
[0249] In certain embodiments, methods of the disclosure include
topical administration, either to skin or to mucosal membranes such
as those on the cervix and vagina. The topical formulations may
further include one or more of the wide variety of agents known to
be effective as skin or stratum corneum penetration enhancers.
Examples of these are 2-pyrrolidone, N-methyl-2-pyrrolidone,
dimethylacetamide, dimethylformamide, propylene glycol, methyl or
isopropyl alcohol, dimethyl sulfoxide, and azone. Additional agents
may further be included to make the formulation cosmetically
acceptable. Examples of these are fats, waxes, oils, dyes,
fragrances, preservatives, stabilizers, and surface active agents.
Keratolytic agents such as those known in the art may also be
included. Examples are salicylic acid and sulfur. Dosage forms for
the topical or transdermal administration include powders, sprays,
ointments, pastes, creams, lotions, gels, solutions, patches, and
inhalants. The subject polypeptide therapeutic agents may be mixed
under sterile conditions with a pharmaceutically acceptable
carrier, and with any preservatives, buffers, or propellants which
may be required. The ointments, pastes, creams and gels may
contain, in addition to a subject chimeric polypeptide agent,
excipients, such as animal and vegetable fats, oils, waxes,
paraffins, starch, tragacanth, cellulose derivatives, polyethylene
glycols, silicones, bentonites, silicic acid, talc and zinc oxide,
or mixtures thereof. Powders and sprays can contain, in addition to
a subject chimeric polypeptides, excipients such as lactose, talc,
silicic acid, aluminum hydroxide, calcium silicates, and polyamide
powder, or mixtures of these substances. Sprays can additionally
contain customary propellants, such as chlorofluorohydrocarbons and
volatile unsubstituted hydrocarbons, such as butane and
propane.
[0250] Pharmaceutical compositions suitable for parenteral
administration may comprise one or more chimeric polypeptides in
combination with one or more pharmaceutically acceptable sterile
isotonic aqueous or nonaqueous solutions, dispersions, suspensions
or emulsions, or sterile powders which may be reconstituted into
sterile injectable solutions or dispersions just prior to use,
which may contain antioxidants, buffers, bacteriostats, solutes
which render the formulation isotonic with the blood of the
intended recipient or suspending or thickening agents. Examples of
suitable aqueous and nonaqueous carriers which may be employed in
the pharmaceutical compositions of the disclosure include water,
ethanol, polyols (such as glycerol, propylene glycol, polyethylene
glycol, and the like), and suitable mixtures thereof, vegetable
oils, such as olive oil, and injectable organic esters, such as
ethyl oleate. Proper fluidity can be maintained, for example, by
the use of coating materials, such as lecithin, by the maintenance
of the required particle size in the case of dispersions, and by
the use of surfactants.
[0251] These compositions may also contain adjuvants, such as
preservatives, wetting agents, emulsifying agents and dispersing
agents. Prevention of the action of microorganisms may be ensured
by the inclusion of various antibacterial and antifungal agents,
for example, paraben, chlorobutanol, phenol sorbic acid, and the
like. It may also be desirable to include isotonic agents, such as
sugars, sodium chloride, and the like into the compositions. In
addition, prolonged absorption of the injectable pharmaceutical
form may be brought about by the inclusion of agents which delay
absorption, such as aluminum monostearate and gelatin.
[0252] Injectable depot forms are made by forming microencapsule
matrices of one or more polypeptide therapeutic agents in
biodegradable polymers such as polylactide-polyglycolide. Depending
on the ratio of drug to polymer, and the nature of the particular
polymer employed, the rate of drug release can be controlled.
Examples of other biodegradable polymers include poly(orthoesters)
and poly(anhydrides). Depot injectable formulations are also
prepared by entrapping the drug in liposomes or microemulsions
which are compatible with body tissue.
[0253] In a preferred embodiment, the chimeric polypeptides of the
present disclosure are formulated in accordance with routine
procedures as a pharmaceutical composition adapted for intravenous
administration to human beings. Where necessary, the composition
may also include a solubilizing agent and a local anesthetic such
as lidocaine to ease pain at the site of the injection. Where the
composition is to be administered by infusion, it can be dispensed
with an infusion bottle containing sterile pharmaceutical grade
water or saline. Where the composition is administered by
injection, an ampoule of sterile water for injection or saline can
be provided so that the ingredients may be mixed prior to
administration.
[0254] In another embodiment, the chimeric polypeptides of the
present disclosure are formulated for subcutaneous administration
to human beings.
[0255] In certain embodiments, the chimeric polypeptides of the
present disclosure are formulated for deliver to the heart, such as
for intramyocardial or intrapericaridal delivery.
[0256] In certain embodiments, the composition is intended for
local administration to the liver via the hepatic portal vein, and
the chimeric polypeptides are formulated accordingly.
[0257] Note that, in certain embodiments, a particular formulation
is suitable for use in the context of deliver via more than one
route. Thus, for example, a formulation suitable for intravenous
infusion may also be suitable for delivery via the hepatic portal
vein. However, in other embodiments, a formulation is suitable for
use in the context of one route of delivery, but is not suitable
for use in the context of a second route of delivery.
[0258] The amount of the chimeric polypeptides of the disclosure
which will be effective in the treatment of a tissue-related
condition or disease (e.g., Pompe disease) can be determined by
standard clinical techniques. In addition, in vitro assays may
optionally be employed to help identify optimal dosage ranges. The
precise dose to be employed in the formulation will also depend on
the route of administration, and the seriousness of the condition,
and should be decided according to the judgment of the practitioner
and each subject's circumstances. However, suitable dosage ranges
for intravenous administration are generally about 20-5000
micrograms of the active chimeric polypeptide per kilogram body
weight. Suitable dosage ranges for intranasal administration are
generally about 0.01 pg/kg body weight to 1 mg/kg body weight.
Effective doses may be extrapolated from dose-response curves
derived from in vitro or animal model test systems.
[0259] In certain embodiments, compositions of the disclosure,
including pharmaceutical preparations, are non-pyrogenic. In other
words, in certain embodiments, the compositions are substantially
pyrogen free. In one embodiment the formulations of the disclosure
are pyrogen-free formulations which are substantially free of
endotoxins and/or related pyrogenic substances. Endotoxins include
toxins that are confined inside a microorganism and are released
only when the microorganisms are broken down or die. Pyrogenic
substances also include fever-inducing, thermostable substances
(glycoproteins) from the outer membrane of bacteria and other
microorganisms. Both of these substances can cause fever,
hypotension and shock if administered to humans. Due to the
potential harmful effects, even low amounts of endotoxins must be
removed from intravenously administered pharmaceutical drug
solutions. The Food & Drug Administration ("FDA") has set an
upper limit of 5 endotoxin units (EU) per dose per kilogram body
weight in a single one hour period for intravenous drug
applications (The United States Pharmacopeial Convention,
Pharmacopeial Forum 26 (1):223 (2000)). When therapeutic proteins
are administered in relatively large dosages and/or over an
extended period of time (e.g., such as for the patient's entire
life), even small amounts of harmful and dangerous endotoxin could
be dangerous. In certain specific embodiments, the endotoxin and
pyrogen levels in the composition are less then 10 EU/mg, or less
then 5 EU/mg, or less then 1 EU/mg, or less then 0.1 EU/mg, or less
then 0.01 EU/mg, or less then 0.001 EU/mg.
[0260] The foregoing applies to any of the chimeric polypeptides,
compositions, and methods described herein. The disclosure
specifically contemplates any combination of the features of such
chimeric polypeptides, compositions, and methods (alone or in
combination) with the features described for the various
pharmaceutical compositions and route of administration described
in this section.
IX. Animal Models
[0261] Pompe disease has been modeled in animals such as Brahman
and Shorthorn cattle, Lapland dog, cats, sheep, and a strain of
Japanese quail (Kikuchi et al., Clinical and Metabolic Correction
of Pompe Disease by Enzyme Therapy in Acid Maltase-deficient Quail,
J. Clin. Invest., 101(4): 827-833, 1998). In addition, mouse models
have been developed by targeted disruption of the GAA gene
(summarized in Geel et al., Pompe disease: Current state of
treatment modalities and animal models, Molecular Genetics and
Metabolism, 92:299-307, 2007). Briefly, mice possessing a knockout
in exon 13 of the GAA gene exhibit glycogen accumulation in
lysosomes of liver, heart, and skeletal muscle cells, but remain
phenotypically normal (Bijvoet et al., Generalized glycogen storage
and cardiomegaly in a knockout mouse model of Pompe disease, Human
Molecular Genetics, 7(1): 53-62, 1998). Mice in which exon 6 of the
GAA gene was replaced by a neomycin resistance gene flanked by LoxP
sites was developed, and lacked GAA function in several tissues.
This mouse has also been crossed with Cre-producing mice, and the
resultant progeny have abnormal lysosomal glycogen storage in heart
and skeletal muscle (Raben et al., Targeted Disruption of the Acid
.alpha.-Glucosidase Gene in Mice Causes an Illness with Critical
Features of Both Infantile and Adult Human Glycogen Storage Disease
Type II, J. Biological Chemistry, 272(30): 19086-19092, 1998). A
similar mouse model has targeted replacement of exon 14 with a
neomycin cassette and is comparable to the neomycin-exon 6 mouse
(Raben et al., Modulation of disease severity in mice with targeted
disruption of the acid alpha-glucosidase gene, Neuromuscl. Disord.
10: 283-291, 2000). Two additional mouse models have been developed
to address issues of immune response: one mouse model in which the
exon 6 deletion was targeted to maintain GAA function in the liver
while keeping the disease phenotype in other tissues, and one GAA
knockout mouse model in SCID mice, which do not produce anti-hGAA
antibodies upon administration of hGAA (Raben et al., Induction of
tolerance to a recombinant human enzyme, acid alpha-glucosidase, in
enzyme deficient knockout mice, Transgenic Research, 12:171-178,
2003; Xu et al., Improved efficacy of gene therapy approaches for
Pompe disease using a new, immune-deficient GSD-II mouse model,
Gene Therapy, 11:15890-1598, 2004). More recently, a double KO
mouse has been developed that pairs deletion of GAA and deletion of
glycogen synthase 1 to help determine the effects of decreased
glycogen production (Xu et al., Impaired organization and function
of myofilaments in single muscle fibers from a mouse model of Pompe
disease, J Appl Physiol 108: 1383-1388, 2010).
[0262] Accordingly, in certain embodiments, the present disclosure
contemplates methods of surveying improvements in disease
phenotypes using the mature GAA constructs (e.g., the chimeric
polypeptides comprising mature GAA) disclosed herein in any one or
more animal models, such as the mouse models described herein. By
way of example, various parameters can be examined in experimental
animals treated with a subject chimeric polypeptide, and such
animals can be compared to controls. Exemplary parameters that can
be assessed to evaluate potential efficacy include, but are not
limited to: increase in lifespan; increase in glycogen clearance,
decrease in glycogen accumulation, decrease in alanine transaminase
serum levels, decrease in aspartate transaminase serum levels,
decrease in alkaline phosphatase serum levels, decrease in creatine
phosphokinase serum levels, improved muscle strength, for example
in open field and open wire hang paradigms, restoration of function
of GAA in lysosomes in liver, skeletal muscle, smooth muscle and/or
cardiac muscle. Increase in glycogen clearance and decrease in
glycogen accumulation may be assessed, for example, by periodic
acid Schiff staining in a biopsy (e.g., muscle, liver or neuronal)
from a treated or untreated animal model.
[0263] Moreover, once it is established that, for example,
3E10*mature GAA results in an improvement in any one or more of
these phenotypes, a complete pharmacokinetic study to determine the
effective dose, clearance rate, volume of distribution, and
half-life of 3E10-mature GAA can be determined. The PK/PD/TK of the
final product can be examined in larger animals such as rats, dogs,
and primates.
[0264] The above models are exemplary of suitable animal model
systems for assessing the activity and effectiveness of the subject
chimeric polypeptides and/or formulations. These models have
correlations with symptoms of GAA deficiency, and thus provide
appropriate models for studying Pompe disease. Activity of the
subject chimeric polypeptides and/or formulations can be assessed
in any one or more of these models, and the results compared to
that observed in wildtype control animals and animals not treated
with the chimeric polypeptides. Similarly, the subject chimeric
polypeptides can be evaluated using cells in culture, for example,
cells prepared from any of the foregoing mutant mice or other
animals, as well as wild type cells, such as fibroblasts
Additionally, cell free systems may be used to assess, for example,
enzymatic activity of the subject chimeric polypeptides. An example
of an in vitro assay for testing activity of the chimeric
polypeptides disclosed herein would be to treat Pompe cells with or
without the chimeric polypeptides and then, after a period of
incubation, stain the cells for the presence of glycogen, e.g., by
using a periodic acid Schiff (PAS) stain. Another example of an in
vitro assay for testing activity of the chimeric polypeptides
disclosed herein would be a cell or cell-free assay in which
whether the ability of the chimeric polypeptides to hydrolyze
4-methylumbelliferyl-.alpha.-D-glucoside as a substrate is
assessed.
[0265] Chimeric polypeptides of the disclosure have numerous uses,
including in vitro and in vivo uses. In vivo uses include not only
therapeutic uses but also diagnostic and research uses in, for
example, any of the foregoing animal models. By way of example,
chimeric polypeptides of the disclosure may be used as research
reagents and delivered to animals to understand GAA bioactivity,
localization and trafficking, protein-protein interactions,
enzymatic activity, and impacts on animal physiology in healthy or
diseases animals.
[0266] Chimeric polypeptides may also be used in vitro to evaluate,
for example, GAA bioactivity, localization and trafficking,
protein-protein interactions, and enzymatic activity in cells in
culture, including healthy and GAA deficient cells in culture. The
disclosure contemplates that chimeric polypeptides of the
disclosure may be used to deliver GAA to cytoplasm, lysosome,
and/or autophagic vesicles of cells, including cells in
culture.
X. Kits
[0267] In certain embodiments, the disclosure also provides a
pharmaceutical package or kit comprising one or more containers
filled with at least one chimeric polypeptide of the disclosure.
Optionally associated with such container(s) can be a notice in the
form prescribed by a governmental agency regulating the
manufacture, use or sale of pharmaceuticals or biological products,
which notice reflects (a) approval by the agency of manufacture,
use or sale for human administration, (b) directions for use, or
both.
[0268] In certain embodiments, the kit includes additional
materials to facilitate delivery of the subject chimeric
polypeptides. For example, the kit may include one or more of a
catheter, tubing, infusion bag, syringe, and the like. In certain
embodiments, the chimeric polypeptide is packaged in a lyophilized
form, and the kit includes at least two containers: a container
comprising the lyophilized chimeric polypeptide and a container
comprising a suitable amount of water, buffer, or other liquid
suitable for reconstituting the lyophilized material.
[0269] The foregoing applies to any of the chimeric polypeptides,
compositions, and methods described herein. The disclosure
specifically contemplates any combination of the features of such
chimeric polypeptides, compositions, and methods (alone or in
combination) with the features described for the various kits
described in this section.
EXEMPLIFICATION
[0270] The disclosure now being generally described, it will be
more readily understood by reference to the following examples,
which are included merely for purposes of illustration of certain
aspects and embodiments of the present disclosure, and are not
intended to limit the disclosure. For example, the particular
constructs and experimental design disclosed herein represent
exemplary tools and methods for validating proper function. As
such, it will be readily apparent that any of the disclosed
specific constructs and experimental plan can be substituted within
the scope of the present disclosure.
Example 1: Chemical Conjugation of 3E10 and Human Mature GAA
(mAb3E10*Mature GAA)
Chemical Conjugation
[0271] In this example, ten milligrams (10 mg) of an exemplary 3E10
antigen binding fragment comprising a heavy chain variable domain
comprising the amino acid sequence of SEQ ID NO: 9 and a light
chain variable domain comprising the amino acid sequence of SEQ ID
NO: 10 (e.g., such as an scFv in which the VH and VL domains are
interconnected via a linker) are conjugated covalently, directly or
indirectly, to 76 kDa or 70 kDa mature human GAA, or to a GAA
polypeptide comprising a mature human GAA, in a 1/1 molar ratio
with the use of two different heterobifunctional reagents,
succinimidyl 3-(2-pyridyldithio) propionate and succinimidyl
trans-4-(maleimidylmethyl) cyclo-hexane-1-carboxylate. This
reaction modifies the lysine residues of 3E10 into thiols and adds
thiolreactive maleimide groups to mature GAA (Weisbart R H, et al.,
J Immunol. 2000 Jun. 1; 164(11): 6020-6). After deprotection, each
modified protein is reacted to each other to create a stable
thioether bond. Chemical conjugation is performed, and the products
are fractionated by gel filtration chromatography. The composition
of the fractions is assessed by native and SDS-PAGE in reducing and
nonreducing environments. Fractions containing the greatest ratio
of 3E10-mature GAA conjugate to free 3E10 and free mature GAA are
pooled and selected for use in later studies.
[0272] Similarly, conjugates are made in which an antigen binding
portion of 3E10 (such as a single chain Fv fragment) or a full
length 3E10 or a 3E10 Fab is conjugated to a mature GAA
polypeptide, such as mature GAA polypeptide having a molecular
weight of approximately 70-76 kDa (e.g., mature, active forms of
GAA), or similar forms that use an alternative starting and/or
ending residue, or to a GAA polypeptide comprising a mature GAA
polypeptide. Other exemplary conjugates include conjugates in which
the internalizing moiety is either a full length 3E10 mAb, or
variant thereof, or an antigen binding fragment of the foregoing.
The foregoing methods can be used to make chemical conjugates that
include any combination of GAA portions and internalizing moiety
portions, and the foregoing are merely exemplary. Both N-terminal
and C-terminal conjugates are made (e.g., conjugates in which the
3E10 portion is N-terminal to the mature GAA portion and conjugates
in which the 3E10 portion is C-terminal to the mature GAA portion).
Moreover, the experimental approaches detailed herein can be used
to evaluate any such chimeric polypeptide or to compare activity
amongst chimeric polypeptides.
In Vitro Assessment of Chemically Conjugated 3E10 and Mature
GAA
[0273] Preparations of conjugated Fv3E10-mature GAA chimeric
polypeptides and suitable control polypeptide are summarized in
Table 1. The listed chimeric polypeptides are solely for
illustrative purposes, and any chimeric polypeptides of the
disclosure comprising a GAA polypeptide portion comprising a mature
GAA and an internalizing moiety portion are similarly contemplated.
Subject chimeric polypeptides are added, for example, to cell
cultures and the extent of protein uptake, protein localization
and/or GAA enzymatic activity are determined and compared to
controls. Similarly, GAA enzymatic activity can be assessed in cell
free systems. We note that although, in this example, the
internalizing moiety portion and GAA portion are chemically
conjugated, each individual portion may be made recombinantly
(e.g., by expressing nucleotide sequence encoding the polypeptide
in a cell in culture and purifying the expressed polypeptide).
TABLE-US-00001 TABLE 1 Exemplary Chemically Conjugated Fv3E10 to
human mature GAA Group Polypeptides 1 Fv3E10*mature GAA
Fv3E10*mature GAA (76 kDa) Chemically (70 kDa) Chemically
conjugated conjugated 2 Fv3E10 & mature GAA Fv3E10 & mature
GAA (76 kDa) Mixed (70 kDa) Mixed unconjugated unconjugated 3
Fv3E10 alone Fv3E10 alone 4 mature GAA(76 kDa) mature GAA(70 kDa)
alone alone Note: Fv3E10 refers to an scFv antigen binding fragment
of 3E10, as described above
[0274] i) Enzymatic Activity of 3E10-Mature GAA
[0275] GAA enzymatic activity is measured by determining the rate
of 3E10-mature GAA catalyzed hydrolysis of a synthetic substrate,
p-nitrophenyl-D-.alpha.-glucopyranoside, in 50 mM sodium acetate,
0.1% BSA, pH 4.3, as described in McVie et al. (Biochemical and
Pharmacological Characterization of Different Recombinant Acid
.alpha.-Glucosidase Preparations Evaluated for the Treatment of
Pompe Disease, Mol Genet Metab., 94(4): 448-455, 2008). The
released chromophore, p-nitrophenol, is quantified
spectrophotometrically at an alkaline pH (>10.2) at 400 nm. One
unit of mature GAA is defined as that amount of activity which
resulted in the hydrolysis of 1 .mu.mol of substrate per minute at
37.degree. C. under the assay conditions. Duplicate experiments are
performed for Fv3E10 and mature GAA, Fv3E10 alone, or mature GAA
alone.
[0276] ii) Uptake of 3E10-Mature GAA
[0277] Uptake of 3E10-mature GAA is first assessed in COS-7 cells.
Previous studies indicate that ENT2 is involved in 3E10 transport
across the membrane of COS-7 cells (Hansen et al., J. Biol. Chem.,
282: 20790-20793, 2007), and a similar strategy can be used to
determine transport of the chimeric 3E10-mature GAA across the
membrane. Briefly, purified chimeric polypeptides are prepared in
PBS with 10% fetal calf serum; control buffer is PBS with 10% fetal
calf serum. 50 pt of control buffer or 3E10-mature GAA is added to
COS-7 cells and incubated for 1 hour. The buffer is aspirated,
cells are washed, fixed in chilled 100% ethanol, and stained with
either an antibody to 3E10 or to GAA.
[0278] To demonstrate that muscle cells also uptake Fv3E10-mature
GAA polypeptides, the same experiment is conducted in muscle cells.
The murine cardiomyocte HL-1 cell line expresses ENT2 (Naydenova et
al., Inosine and equilibrative nucleoside transporter 2 contribute
to hypoxic preconditioning in the murine cardiomyocyte HL-1 cell
line, Am J Physiol. Heart Circ. Physiol., 294(6):H2687-2692, 2008),
and this cell line can be used in place of COS-7 cells in the above
experiment.
[0279] Human Pompe fibroblasts (TR4192) are grown to confluence in
a T-75 flask using MEM/FBS media. The cells are washed with PBS,
trypsinized and plated at 1.times.10.sup.6 cells/mL in a 96-well
plate (100 .mu.L/well). Plates are incubated overnight at
37.degree. C. Following incubation, samples of Fv3E10 and mature
GAA, Fv3E10 alone, or mature GAA alone (each assayed in triplicate)
are diluted in reduced serum media (MEM/1% FBS) and added to the
cells. Following a 24 hour incubation at 37.degree. C., cells are
washed and lysed with the addition of PBS/1% Triton X-100 and
frozen at -80.degree. C. A protein determination assay using
bicinchoninic acid (BCA) and an activity analysis (using
4-methylumbelliferyl-.alpha.-D-glucoside as the substrate for GAA)
are performed on the cell lysates in order to determine the extent
of mature GAA uptake by cells McVie et al. (Biochemical and
Pharmacological Characterization of Different Recombinant Acid
.alpha.-Glucosidease Preparations Evaluated for the Treatment of
Pompe Disease, Mol Genet Metab., 94(4): 448-455, 2008).
[0280] iii) Immunoblot Detection of Cell-Penetrating 3E10-Mature
GAA
[0281] Additional tests are performed to determine the uptake of
3E10-mature GAA in muscle fibers isolated from either wildtype or
GAA KO mice (Bijvoet et al., Generalized glycogen storage and
cardiomegaly in a knockout mouse model of Pompe disease, Human
Molecular Genetics, 7(1): 53-62, 1998). White gastrocnemius (G),
tibialis anterior (TA) and extensor digitorum longus (EDL) muscles
are removed immediately after sacrifice from WT and GAA-KO mice and
pinned to Sylgaard coated dishes for fixation with 2%
paraformaldehyde in 0.1M phosphate buffer for 1 h, followed by
fixation in methanol (-20.degree. C.) for 6 min (Fukuda et al.,
Autophagy and Mis-targeting of Therapeutic Enzyme in Skeletal
Muscle in Pompe Disease, Mol Ther., 14(6): 831-839, 2006). Single
fibers are obtained by manual teasing.
[0282] Ten to 100 .mu.M of chemically conjugated 3E10-mature GAA,
an unconjugated mixture of Fv3E10 and mature GAA, Fv3E10 alone, or
mature GAA alone are cultured with the isolated myofibers. The
specificity of 3E10-mature GAA for the ENT2 transporter is
validated by addition of nitrobenzylmercaptopurine riboside
(NBMPR), an ENT2 specific inhibitor (Hansen et al., 2007, J. Biol.
Chem., 282(29): 20790-3) to ENT2 transfected cells just prior to
addition of 3E10-mature GAA. Eight to 24 hours later the myofibers
are collected for immunoblot and Periodic acid-Schiff (PAS) stain
for glycogen.
[0283] Myofibers are collected and resuspended in 500 ul PBS,
lysed, and the supernatants are collected for immunoblot analysis
of 3E10 and mature GAA. Epitope tagging will not, in certain
embodiments, be employed, therefore the presence of a coincident
anti-3E10 and anti-GAA immunoreactive band of .about.150-156 kDa
(for the full length 3E10+76 kDa GAA or full length 3E10+70 kDa
GAA) in 3E10*mature GAA treated cells versus 3E10-alone and
GAA-alone controls will constitute successful penetration of
chemically conjugated 3E10*mature GAA. Tubulin detection is used as
a loading control.
[0284] In addition, coverslips of treated cells are washed, fixed
in 100% ethanol, rehydrated, and 3E10 and mature GAA are detected
with previously described antibodies (Fukuda et al., Autophagy and
Mis-targeting of Therapeutic Enzyme in Skeletal Muscle in Pompe
Disease, Mol Ther., 14(6): 831-839, 2006).
[0285] Respective tissues are frozen, homogenized, and centrifuged
to remove insoluble proteins, and protein content of the
supernatants are measured by the Bradford assay. Equivalent amounts
of protein are electrophoretically separated in a 10%
polyacrylamide-SDS gel, are transferred to a nylon membrane, and
are probed with a rabbit anti-human GAA polyclonal antibody.
Detection of the bound anti-GAA antibody is visualized via the ECL
detection system (Amersham Pharmacia).
[0286] iv) Tissue Glycogen Content
[0287] Myofibers from treated wildtype mice and GAA KO mice are
fixed with 5% formaldehyde in 95% ethanol for 5 minutes at room
temperature. Glycogen content is evaluated using high resolution
light microscopy and computer assisted histomorphometry as
described in McVie et al. (Biochemical and Pharmacological
Characterization of Different Recombinant Acid .alpha.-Glucosidease
Preparations Evaluated for the Treatment of Pompe Disease, Mol
Genet Metab., 94(4): 448-455, 2008). Briefly, representative
samples of muscle tissue are fixed in 3% gluteraldehyde in 0.2
mol/L sodium cacodylate buffer (Electron Microscopy Sciences, Fort
Washington, Pa.) and embedded in epon-araldite. One micron sections
are stained with Periodic acid-Schiff (PAS) reaction and
counterstained with Richardson's solution. This results in high
quality tissue preservation in which glycogen is fully retained and
appears purple against a blue counterstain of myocyte cytoplasm.
One representative field from each slide will be photographed with
a Nikon DXM1200 digital camera (Nikon Inc, Instrument Group,
Meville, N.Y.) and analyzed using Metamorph Imaging Processing and
Analysis software (version 4.6; Universal Imaging Corporation). For
each image, glycogen load will be expressed as a percentage of
total tissue area.
Example 2 Genetic Construct of 3E10 and Human Mature GAA
(Fv3E10-GS3-Mature GAA)
[0288] Mammalian expression vectors encoding a genetic fusion of
Fv3E10 and one of the two mature forms of human GAA (e.g., such as
a GAA polypeptide comprising a mature GAA) (fv3E10-GS3-mature GAA,
comprising the scFv of mAb 3E10 fused to mature GAA by, for
example, the GS3 linker) will be generated. Note that in the
examples, we have used "Fv3E10" to refer to an scFv of 3E10. Note
that these genetic fusions are also referred to as recombinant
conjugates or recombinantly produced conjugates. Other linkers may
similarly be used. Further, linkerless fusions where the 3E10
moiety and the mature GAA moiety are directly fused may also be
used. Similarly fusions to a portion of a full length antibody or
Fab may be made. As with the chemical conjugates, recombinant
fusions comprising any of the chimeric polypeptides of the
disclosure are contemplated. Recombinantly produced chimeric
polypeptides may comprises a GAA polypeptide portion, according to
the disclosure (e.g., a GAA polypeptide comprising a mature GAA)
and an internalizing moiety portion, according to the
disclosure.
[0289] Additional recombinantly produced conjugates will similarly
be made for later testing. By way of non-limiting example: (a)
mature GAA-GS3-3E10, (b) 3E10-GS3-mature GAA, (c) mature
GAA-GS3-Fv3E10, (d) mature GAA-3E10, (e) 3E10-mature GAA, (f)
mature GAA-Fv3E10. Note that throughout the examples, the
abbreviation Fv is used to refer to a single chain Fv of 3E10.
Similarly, mAb 3E10 and 3E10 are used interchangeably. Similarly,
mature GAA refers to a mature GAA protein having a molecular weight
of from about 70-76 kDa, such as a mature GAA protein having a
molecular weight of about 76 kDa or about 70 kDa. These and other
chimeric polypeptides can be tested using, for example, the assays
detailed herein. Further polypeptides in which the chimeric
polypeptides comprise a mature GAA polypeptide, but which
polypeptides also include additional contiguous GAA polypeptide
sequence (but not the entire 110 kD precursor polypeptide and/or
not the signal sequence of the set forth in residues 1-56 or SEQ ID
NO: 1 or 2) are also contemplated and can similarly be made and
tested.
Create the cDNA for Human GAA and Confirm Activity In Vitro
[0290] i) Synthesis of the cDNA for GAA
[0291] The full-length, 3.6 kb human GAA cDNA that encodes a full
length, precursor form of human GAA (hGAA cDNA) may be found at
http://www.ncbi.nlm.nih.gov/sites/entrez, for example, under
GenBank Accession No. NM_000152.3. This cDNA sequences and other
transcript variants are hereby incorporated in their entirety. A
portion of such a human cDNA sequence corresponding approximately
to the region that encodes mature GAA is used herein to generate a
recombinant construct. However, it is also contemplated that the
full length cDNA can be used.
[0292] The mature GAA cDNA along with flanking restriction sites
that facilitate cloning into appropriate expression vectors will be
synthesized and sequenced by Genscript or other qualified
manufacturer of gene sequences. To maximize expression, the mature
GAA cDNA will be codon optimized for mammalian and pichia
expression. In the event that mammals or pichia prefer a different
codon for a given amino acid, we will use the next best candidate
to unify the preference. The resulting cDNA is cloned into a
CMV-based mammalian expression cassette and large scale preps of
the plasmid pCMV-mature GAA will be made using the Qiagen Mega
Endo-free plasmid purification kit. To avoid complicating immune
responses to the 3E10-GAA protein, epitopes or purification tags
are not, in certain embodiments, included. However, conjugates that
do include such tags may also be made and tested.
[0293] ii) Transfection of Cells In Vitro
[0294] A strategy to assess the function of GAA in transfected
cells is described above. Ten micrograms of the plasmid pCMV (mock)
or pCMV-mature GAA is transfected into 1) COS-7 cells, 2) HL-1
cells, 3) myofibers from wildtype mice, and 4) myofibers from GAA
KO mice using commercially available transfection reagents (Table
2). To track the efficiency of transfection, duplicate
transfections with plasmids encoding a suitable reporter such as
beta-galactosidase or GFP is performed. Forty-eight hours later
transfected cells are pelleted by centrifugation resuspended in 500
.mu.l PBS for protein and immunoblot analysis.
TABLE-US-00002 TABLE 2 Transfection strategy for pCMV and
pCMV-mature GAA Group Cells Transfected plasmid 1 COS-7 pCMV (mock)
2 COS-7 pCMV-mature GAA (76 kDa or 70 kDa) 3 HL-1 pCMV (mock) 4
HL-1 pCMV-mature GAA (76 kDa or 70 kDa) 5 Myofibers from pCMV
(mock) GAA KO mice 6.sup.neo/6.sup.neo 6 Myofibers from pCMV-mature
GAA GAA KO mice (76 kDa or 70 kDa) 6.sup.neo/6.sup.neo 7 Myofibers
from pCMV (mock) WT mice 8 Myofibers from pCMV-mature GAA WT mice
(76 kDa or 70 kDa)
[0295] iii) Viral Infection with AAV cDNA Construct
[0296] Constructs described above are cloned into an adenovirus
vector plasmid, according to methods described in Sun et al.,
(Enhanced Efficacy of an AAV Vector Encoding Chimeric,
Highly-Secreted Acid .alpha.-glucosidase in Glycogen Storage
Disease Type II, Mol Ther., 14(6): 822-830, 2006). These constructs
provide a means to test the cDNA constructs in cells, and/or use
constructs in vivo for gene therapy.
[0297] Briefly, 293 cells are transfected with an AAV vector
plasmid, the AAV packaging plasmid p5E18-VD 2/8, and pAdHelper
(Stratagene, La Jolla, Calif.). Cell lysate is harvested 48 hours
following infection, freeze-thawed 3 times, and isolated by sucrose
cushion pelleting followed by 2 cesium chloride gradient
centrifugation steps. AAV stocks are dialyzed against 3 changes of
Hanks buffer, and aliquots are stored at -80.degree. C. The number
of vector DNA containing-particles is determined by DNase I
digestion, DNA extraction, and Southern blot analysis. All viral
vector stocks are handled according to Biohazard Safety Level 2
guidelines published by the NIH.
[0298] The uptake of chimeric mature GAA is analyzed in (1)COS-7
cells, (2) HL-1 cells, and (3) Pompe disease patient cells as
described in Example 1 above. COS-7 cells, HL-1 cells, or
fibroblasts from a GSD-II patient are grown in medium containing
10% FBS and incubated for 40 hours with the medium of transfected
293 cells producing chimeric hGAA with activity of 300 nmolhrml.
GAA activity and glycogen in cultured patient fibroblasts is
analyzed as described above.
[0299] iii) Immunoblot Detection of Transfected Human GAA, and
Assay of GAA Mediated Hydrolysis of Glycogen.
[0300] The same procedures described in Example 1 are utilized.
Create and Validate cDNA Fv3E10 Genetically Conjugated to 76 kDa or
70 kDa Forms of GAA
[0301] i) Synthesis of the cDNA for Fv3E10
[0302] The cDNA encoding the mouse Fv3E10 variable light chain
linked to the 3E10 heavy chain (SEQ ID NOs: 9-10) contains a
mutation in the VH CDR1 that enhances the cell penetrating capacity
of the Fv fragment (Zack et al., 1996, J Immunol, 157(5): 2082-8).
The 3E10 cDNA is flanked by restriction sites that facilitate
cloning in frame with the cDNA coding sequence that corresponds to
the amino acid sequences in the mature forms of GAA (SEQ ID NOS:
3-4) or with a GAA polypeptide comprising mature GAA. The
constructs are synthesized and sequenced by Genscript or other
qualified manufacturer of gene sequences. To maximize expression
the 3E10 cDNA will be codon optimized for mammalian and pichia
expression. In the event that mammals or pichia prefer a different
codon for a given amino acid, the next best candidate to unify the
preference will be used. The resulting cDNA will be cloned into a
mammalian expression cassette and large scale preps of the plasmid
pCMV-3E10-mature GAA will be made using the Qiagen Mega Endo-free
plasmid purification kit. The constructs will be tested in 1)COS-7
cells, 2) HL-1 cells, 3) myofibers from wildtype mice, and 4)
myofibers from GAA KO mice. A transfection strategy is outlined in
Table 3.
TABLE-US-00003 TABLE 3 Transfection strategy for pCMV 3E10-GS3-GAA
Group Cells Transfected plasmid 1 COS-7 pCMV 2 COS-7 pCMV-GAA (76
kDa or 70 kDa) 3 COS-7 pCMV Fv3E10-GS3-GAA (76 kDa or 70 kDa) 4
HL-1 pCMV 5 HL-1 pCMV-GAA (76 kDa or 70 kDa) 6 HL-1 pCMV
Fv3E10-GS3-GAA (76 kDa or 70 kDa) 7 Myofibers from pCMV GAA KO mice
6.sup.neo/6.sup.neo 8 Myofibers from pCMV-GAA (76 kDa or 70 kDa)
GAA KO mice 6.sup.neo/6.sup.neo 9 Myofibers from pCMV
Fv3E10-GS3-GAA GAA KO mice (76 kDa or 70 kDa) 6.sup.neo/6.sup.neo
10 Myofibers from pCMV (mock) WT mice 11 Myofibers from pCMV-GAA
(76 kDa or 70 kDa) WT mice 12 Myofibers from pCMV Fv3E10-GS3-GAA WT
mice (76 kDa or 70 kDa)
[0303] ii) Transfection of Cells
[0304] The strategy to test the expression and glycogen hydrolysis
of the 3E10-GS3-mature GAA genetic fusion is described above. The
transfection procedure will be the same as described above for
transfection of the human GAA cDNA. Transfected cells will be
assayed for expression of hGAA and hydrolysis of glycogen.
Production of Recombinant 3E10 Genetically Conjugated to Mature
GAA
[0305] i) Construction of protein expression vectors for pichia.
Plasmid construction, transfection, colony selection and culture of
Pichia will use kits and manuals per the manufacturer's
instructions (Invitrogen). The cDNAs for genetically conjugated
3E10-GS3-mature GAA created and validated as described above will
be cloned into two alternative plasmids; PICZ for intracellular
expression and PICZalpha for secreted expression. Protein
expression from each plasmid is driven by the AOX1 promoter.
Transfected pichia will be selected with Zeocin and colonies will
be tested for expression of recombinant 3E10-GS3-mature GAA. High
expressers will be selected and scaled for purification.
[0306] ii) Purification of Recombinant 3E10-GS3-Mature GAA
[0307] cDNA fusions with mAb 3E10 Fv are ligated into the yeast
expression vector pPICZA which is subsequently electroporated into
the Pichia pastoris X-33 strain. Colonies are selected with Zeocin
(Invitrogen, Carlsbad, Calif.) and identified with anti-his6
antibodies (Qiagen Inc, Valencia, Calif. X-33 cells are grown in
baffled shaker flasks with buffered glycerol/methanol medium, and
protein synthesis is induced with 0.5% methanol according to the
manufacturer's protocol (EasySelect Pichia Expression Kit,
Invitrogen, Carlsbad, Calif.). The cells are lysed by two passages
through a French Cell Press at 20,000 lbs/in2, and recombinant
protein is purified from cell pellets solubilized in 9M guanidine
HCl and 2% NP40 by immobilized metal ion affinity chromatography
(IMAC) on Ni-NTAAgarose (Qiagen, Valencia, Calif.). Bound protein
is eluted in 50 mM NaH2PO4 containing 300 mM NaCl, 500 mM
imidazole, and 25% glycerol. Samples of eluted fractions are
electrophoresed in 4-20% gradient SDSPAGE (NuSep Ltd, Frenchs
Forest, Australia), and recombinant proteins is identified by
Western blotting to nitrocellulose membranes developed with
cargo-specific mouse antibodies followed by
alkalinephosphatase-conjugated goat antibodies to mouse IgG.
Alkaline phosphatase activity is measured by the chromogenic
substrate, nitroblue tetrazolium
chloride/5-bromo-4-chloro-3-indolylphosphate p-toluidine salt.
Proteins are identified in SDS-PAGE gels with GelCode Blue Stain
Reagent (Pierce Chemical Co., Rockford, Ill.). Eluted protein is
concentrated, reconstituted with fetal calf serum to 5%, and
exchange dialyzed 100-fold in 30,000 MWCO spin filters (Millipore
Corp., Billerica, Mass.) against McCoy's medium (Mediatech, Inc.,
Herndon, Va.) containing 5% glycerol. Although in this example a
Pichia expression system is illustrated, protein may also be
produced in other expression systems, including mammalian
expressions systems such as CHO cells. Vectors and methodologies,
including contract manufacturing services, for expressing proteins
in CHO cells are available, for example, from Lonza.
[0308] iii) Quality Assessment and Formulation
[0309] Immunoblot against 3E10 and mature GAA is used to verify the
size and identity of recombinant proteins, followed by silver
staining to identify the relative purity among preparations of
3E10, GAA and 3E10-GS3-mature GAA. Recombinant material is
formulated in a buffer and concentration (.about.0.5 mg/ml).
[0310] iv) In Vitro Assessment of Recombinant Material
[0311] The activity of 3E10-GS3-mature GAA protein is evaluated
using any one or more of the assays detailed in Example 1. Cell
penetration and/or enzymatic activity is compared to suitable
controls. Moreover, the amount of 3E10-GS3-mature GAA protein
needed to alleviate the GAA deficiency is determined using the
methods described above. The amounts of GAA activity in mammalian
cell-derived and pichia-derived recombinant 3E10-GS3-mature GAA can
be tested, for example, on (1) fibroblasts from Pompe disease
patients and control patients and (2) myofibers isolated from
wildtype and GAA KO mice.
Example 3 In Vivo Assessment of Muscle Targeted GAA in GAA KO
Mice
GAA Mouse Models for Evaluation
[0312] Several GAA KO mice have been characterized previously: a
GAA KO line (Bijvoet et al., Generalized glycogen storage and
cardiomegaly in a knockout mouse model of Pompe disease, Human
Molecular Genetics, 7(1): 53-62, 1998); a line in which exon 6 has
been replaced with loxP-flanked neo gene (6.sup.neo/76.sup.neo) or
deleted (.DELTA.6/.DELTA.6) (Raben et al., Targeted Disruption of
the Acid .alpha.-Glucosidase Gene in Mice Causes an Illness with
Critical Features of Both Infantile and Adult Human Glycogen
Storage Disease Type II, J. Biological Chemistry, 272(30):
19086-19092, 1998); a mouse line in which exon 14 has been deleted
(.DELTA.14.sup.neo/.DELTA.14.sup.neo) (Raben et al., Modulation of
disease severity in mice with targeted disruption of the acid
alpha-glucosiase gene, Neuromuscl. Disord. 10: 283-291, 2000); a
GAA KO crossed with an inducible hGAA that restores GAA
specifically to the liver or muscles (Raben et al., Conditional
tissue-specific expression of the acid alpha-glucosidase (GAA) gene
in the GAA knockout mouse: implications for therapy, Hum. Molec.
Genet. 10: 2039-2047, 2001); a GAA KO crossed with an inducible
hGAA mouse line in which expression of GAA in the liver induces
immunological tolerance to GAA; (Raben et al., Induction of
tolerance to a recombinant human enzyme, acid alpha-glucosidase, in
enzyme deficient knockout mice, Transgenic Research, 12:171-178,
2003); a GAA KO/SCID mouse developed to avoid an immune response to
GAA in GAA KO mice (Xu et al., Improved efficacy of gene therapy
approaches for Pompe disease using a new, immune-deficient GSD-II
mouse model, Gene Therapy, 11:15890-1598, 2004); and a double KO
mouse of GAA and glycogen synthase 1, in which the effects of
decreased glycogen production are studied (Xu et al., Impaired
organization and function of myofilaments in single muscle fibers
from a mouse model of Pompe disease, J Appl Physiol 108: 1383-1388,
2010).
[0313] Of these mice, the 6.sup.neo/6.sup.neo and
.DELTA.14.sup.neo/.DELTA.14.sup.neo mice show features of the
infantile and adult phenotypes of Pompe disease, while
.DELTA.6/.DELTA.6 shows muscle weakness at a later age. All mice
show abnormal glycogen storage in heart and skeletal muscle.
Accordingly, all of these models serve as appropriate animal models
to test the efficacy of 3E10-mature GAA therapy. If the mice
develop an immunological response to high doses of 3E10-mature GAA,
then one of the immune tolerant mouse models is used.
Selection of Dose of Mature GAA
[0314] To determine a dosage that treats glycogen accumulation in
skeletal muscle without inducing a harmful immune response, weekly
doses of 1 mg/kg, 20 mg/kg, or 100 mg/kg of fv3E10*mature GAA or
fv3E10-GS3-mature GAA are injected intravenously to GAA KO mice,
followed by assessment of changes in glycogen accumulation. The
development of anti-3E10-GAA antibodies are also monitored.
Assessment of changes in muscle morphology, muscle fiber strength,
and decrease in autophagic vacuoles are also evaluated. The below
table illustrates exemplary chimeric polypeptides that can be
evaluated. As noted above, any of the chimeric polypeptides of the
disclosure are similarly made and tested.
TABLE-US-00004 TABLE 4 In vivo dosing plan for chemically and
genetically conjugated Fv3E10-GAA *Age # of Dose *Months of Group
Mouse Genotype (months) mice Treatment (mg/kg) treatment 1 GAA KO
(or 1.5 5 Fv3E10*mature GAA 1, 20, 3 immune tolerant (76 kDa or 70
kDa) or 100 GAA KO line) Chemically conjugated 2 GAA KO (or 1.5 5
Fv3E10 & mature GAA 1, 20, 3 immune tolerant (76 kDa or 70 kDa)
or 100 GAA KO line) Mixed unconjugated 3 GAA KO (or 1.5 5
Fv3E10-GS3-mature GAA 1, 20, 3 immune tolerant (76 kDa or 70 kDa)
or 100 GAA KO line) Genetic conjugate 4 GAA KO (or 1.5 5 Vehicle
N/A 3 immune tolerant GAA KO line) 5 WT 1.5 5 Fv3E10*mature GAA 1,
20, 3 (76 kDa or 70 kDa) or 100 Chemically conjugated 6 WT 1.5 5
Fv3E10 & mature GAA 1, 20, 3 (76 kDa or 70 kDa) or 100 Mixed
unconjugated 7 WT 1.5 5 Fv3E10-GS3-mature GAA 1, 20, 3 (76 kDa or
70 kDa) or 100 Genetic conjugate 8 WT 1.5 5 Vehicle N/A 3 *Animals
at 3.5 months or 6 months will also be tested. The months of
treatment for these two groups will be 5 months and 1-2 months,
respectively. Note that chemical or genetic conjugates with 3E10 or
Fv3E10 are tested.
Materials and Methods
[0315] i) Injection of Chemically and Genetically Conjugated
3E10-Mature GAA
[0316] Fv3E10*mature GAA or Fv3E10-GS3-mature GAA is formulated and
diluted in a buffer that is consistent with intravenous injection
(e.g. sterile saline solution or a buffered solution of 50 mM
Tris-HCl, pH 7.4, 0.15 M NaCl). The amount of 3E10*mature GAA or
3E10-GS3-mature GAA given to each mouse is calculated as follows:
dose (mg/kg).times.mouse weight (kg).times.stock concentration
(mg/ml)=volume (ml) of stock per mouse, q.s. to 100 .mu.l with
vehicle. Exemplary Fv3E10-GS3-mature GAA chimeric polypeptides for
use in these experiments are set forth in SEQ ID NO: 11 or 12.
[0317] ii) Measurement of Anti-GAA Antibodies
[0318] The immune response to 3E10*mature GAA is determined as
described in Raben et al., (Enzyme replacement therapy in the mouse
model of Pompe disease, Molecular Genetics and Metabolism, 80:
159-166, 2003). Briefly, blood is drawn from the tail vein at
various time points to test the sera for the presence of antibodies
to the enzyme by ELISA using 96-well plates. Plates are coated
overnight at 4.degree. C. with 5 .mu.g/ml of rhGAA in PBS,
incubated with blocking solution (0.1% BSA in PBS) for 2 h at
37.degree. C., and washed with TPBS (0.1% Tween-20 in PBS). Next,
the plates are incubated with serial dilutions of test sera
(1:100-1:12 800) in triplicates for 1 h at 37.degree. C., washed
extensively, incubated for 1 h at 37.degree. C. with 1:12000
goat-anti-mouse IgG-horseradish peroxidase (Southern Biotechnology,
Birmingham, Ala.), washed again, and developed with
trimethylbenzidine (TMB) substrate for 15 min at room temperature
(KPL, Gaithersburg, Md.). The reaction will be stopped by adding 1N
HCL, and the plates will be read in a microplate reader at 450/650
nm.
[0319] iii) Tissue Collection and Preparation
[0320] Sampled tissues are obtained from the liver, heart,
diaphragm, and lower leg skeletal muscles of 3E10*mature GAA,
3E10-G53-mature GAA, or control untreated mice at 48-72 hour after
the final dose of mature enzyme. Tissues are homogenized for
measuring GAA activity, extracts are prepared for glycogen
measurements and/or tissues are fixed, embedded, and sectioned into
1 .mu.m sections for staining. For electron microscopy of muscle
tissues, clamped muscle specimens are fixed in 2.5% buffered
glutaraldehyde in 0.1 M sodium cacodylate buffer for 2-4 h at
4.degree. C., stored in 0.1M cacodylate buffer (pH 7.4) and further
processed.
[0321] iv) Histological Evaluation
[0322] For periodic acid Schiff analysis, tissues are fixed in 10%
formalin, embedded in paraffin, sectioned, and stained with
periodic acid-Schiff (PAS) by standard methods. Additionally,
PAS-staining and histochemistry are performed on snap-frozen muscle
biopsies. Serial transverse sections (7 .mu.m) are stained with
alkaline ATPase (pH 10.4) or NADH-TR by standard methods.
[0323] v) Immunohistochemistry
[0324] To assess proliferation of lysosomes, as described by Raben
et al., (Enzyme replacement therapy in the mouse model of Pompe
disease, Molecular Genetics and Metabolism, 80: 159-166, 2003),
antibodies to the lysosomal membrane protein LAMP are used. In
addition, MPR antibodies are used to determine which histochemical
muscle fiber types (as assessed by staining for alkaline ATPase or
NADH) are able to clear glycogen most effectively. The following
primary antibodies may be used: (FITC)-conjugated rat anti-mouse
CD107b (LAMP-2; 1:20) (BD Biosciences, San Diego, Calif.);
(FITC)-conjugated rat antimouse CD107a (LAMP-1; 1:20); and rabbit
anti-bovine CI-MPR (1:500). Seven micrometer sections of muscle
biopsies, snap-frozen in liquid nitrogen cooled isopentane, are
collected on cover slips coated with poly-L-lysine and fixed in
cold acetone for 10 min followed by re-hydration by immersing in
Tris-buffered saline (TB S) for 10 min. Non-specific binding sites
are blocked with 10% horse serum in phosphate-buffered saline (PBS)
for 1 h in a humidified chamber at room temperature followed by
incubation with primary antibodies for 24 h at 4.degree. C. in a
humid chamber. After several washes in PBS, the sections are either
mounted with antifade mounting reagent mixture (ProLong Antifade
kit, Molecular Probes, Inc., Eugene, Oreg.) or developed with
phycoerythrin (PE) conjugated goat F (ab')2 anti-rabbit IgG (H+L)
(1:200) secondary antibody (Caltag laboratories, Burlingame,
Calif.) (for CI-MPR) prior to mounting.
[0325] vi) Tests of Muscle Function
[0326] Assessment of motor ability: locomotor activity in an open
field is measured in a Digiscan apparatus (Omnitech Electronics),
as described in Raben et al. (Enzyme replacement therapy in the
mouse model of Pompe disease, Molecular Genetics and Metabolism,
80: 159-166, 2003). Total distance, horizontal activity, and
vertical activity is measured by the total number of photocell beam
breaks in 10-min intervals during 1-h sessions. Three independent
sessions are conducted for each animal over a period of 1 week.
Animals are starved overnight before sacrifice. The student's t
test will be used for comparisons between the groups. Differences
are considered significant at p<0:05.
[0327] In addition, all mice are subjected to a battery of motor
tests to determine muscle function, as described in Sidman et al.,
Temporal Neuropathological and Behavioral Phenotype of
6.sup.neo/6.sup.neo Pompe Disease Mice, J Neuropathol Exp Neurol.
67(8): 803-818, 2008). Briefly, all mice are evaluated once a week
from 3 months of age. Motor coordination and balance are measured
with a rotating rod apparatus (SmartRod, Accuscan Instruments,
Columbus, Ohio). For the rocking rotorod test, the rod is
programmed to rock backwards and forwards for up to 2.5 sec
duration with the overall acceleration in either direction
increasing to 25 rpm. Cutoff times are 60 seconds for the
accelerating test and 54 seconds for the rocking test. Animals are
tested three times on each version of the test with a rest period
of at least 5 minutes between measurements. Average fall latency
from the rod (or cutoff time) is recorded for each animal and used
for statistical analysis. Analyses of locomotor function is
performed with the Student t test (Prism GraphPad, San Diego,
Calif.). Data are mean.+-.SEM. p<0.05 is considered as a
statistically significant difference. For the Foot Fault Test, each
animal is placed on a wire rack with square holes for 60 seconds
and the number of times the paws slipped into the holes is
recorded. Each animal is tested twice. Mean values are used for
statistical analysis. Strength is measured with a Wire Hang Test.
The ability to hang upside down from a wire screen placed 60 cm
above a large housing cage is measured as a latency to fall into
the cage. A score of zero is assigned to animals that fall
immediately and a score of 60 seconds is assigned to animals that
did not fall. Cut-off time is 60 seconds. Each animal is tested
twice and means are used for statistical analyses. Data will be
expressed as mean.+-.SEM. For two groups the Student t test is
used; for more than two group comparisons, one-way ANOVA is used
followed by the post hoc Bonferroni multiple comparison test.
[0328] vii) Assessing Serum Enzyme Levels
[0329] Blood is collected from tail veins or from the venous sinus
from each mouse every three to four days for the duration of the
study. Samples are tested for levels of alanine transaminase,
aspartate transaminase, alkaline phosphatase, and/or creatine
phosphokinase. Decrease in the elevated levels of one or more of
these enzymes is indicative of reduction of some of the
pathological effects of cytoplasmic glycogen accumulation.
[0330] viii) Survival Assessment
[0331] Those treated and untreated diseased and control mice that
are not sacrificed in the experiments described above will be
monitored in a survival study. Specifically, the disease state,
treatment conditions and date of death of the animals are recorded.
A survival curve will be prepared based on the results of this
study.
Example 4: Trials of 3E10*Mature GAA and 3E10-GS3-Mature GAA in
Human Pompe Disease Patients
[0332] Following the examples of clinical trials in infantile-onset
Pompe disease patients and late-onset Pompe patients (summarized by
Schoser et al., Therapeutic approaches in Glycogen Storage Disease
type II (GSDII)/Pompe disease, Neurotherapeutics, 5(4): 569-578,
2008), 3E10*mature GAA and/or 3E10-GS3-mature GAA is administered
intravenously to patients in, for example, dosages of up to 40
mg/kg weekly (such as chimeric polypeptides comprising a GAA
portion comprising mature GAA and an internalizing moiety portion).
Patients may receive 10 mg/kg weekly for 52 weeks, or may receive
5-20 mg/kg weekly or biweekly for 153 weeks. Patients are monitored
for tolerance of the therapeutic and for improvements in glycogen
clearance, tissue morphology, motor function, and/or cardiac
function. Muscle biopsies are taken, and analyzed by
high-resolution light microscopy, digital histomorphometry,
electron microscopy, capillary density, fiber type analysis, and/or
confocal microscopy. The left ventricular mass index (LVMI) of
infants is monitored. Motor milestones, for example walking or
sitting upright, of infants and toddlers undergoing treatment is
compared with age-matched subjects not suffering from Pompe
disease. Dependence on ventilator support for breathing is also be
monitored.
[0333] The foregoing experimental schemes will similarly be used to
evaluate other chimeric polypeptides. By way of non-limiting
example, this scheme will be used to evaluate chemical conjugates
and fusion proteins having a GAA portion (or a fragment thereof)
and an internalizing moiety portion. By way of further example, the
foregoing methods may also be used to evaluate use of compositions
comprising a mixture of two or more conjugates, such as a mixture
of 3E10*mature GAA (70 kDa) and 3E10*mature GAA (76 kDa).
[0334] The particular chimeric polypeptides described above for
evaluation in examples 1-4 are exemplary of the chimeric
polypeptides of the disclosure--any of which can be made and
evaluated using, for example, the methods described in examples
1-4. By way of example, chemical and genetic conjugates in the
presence or absence of a linker are made and tested. Conjugates in
which the mature GAA moiety is located N-terminal to the
internalizing moiety, as well as conjugates in which the mature GAA
moiety is located C-terminal to the internalizing moiety are made
and test. Any of a range of internalizing moieties and linker
moieties are used.
[0335] By way of non-limiting example the following chimeric
polypeptides are made and tested: (a) mature GAA-GS3-3E10, (b)
3E10-GS3-mature GAA, (c) mature GAA-GS3-Fv3E10, (d) mature
GAA-3E10, (e) 3E10-mature GAA, (f) mature GAA-Fv3E10, (g)
3E10*mature GAA, (h) mature GAA*3E10, (i) internalizing
moiety*mature GAA, (j) mature GAA*internalizing moiety, (k)
internalizing moiety-GS3-mature GAA, (1) mature
GAA-GS3-internalizing moiety. Note that throughout the examples,
the abbreviation Fv is used to refer to a single chain Fv of 3E10.
Similarly, mAb 3E10 and 3E10 are used interchangeably. Similarly,
mature GAA refers to a mature GAA protein having a molecular weight
of from about 70-76 kDa, such as a mature GAA protein having a
molecular weight of about 76 kDa or about 70 kDa. These and other
chimeric polypeptides can be tested using, for example, the assays
detailed herein.
[0336] By way of further example, the foregoing methods may also be
used to evaluate use of compositions comprising a mixture of two or
more conjugates, such as a mixture of 3E10*mature GAA (70 kDa) and
3E10*mature GAA (76 kDa).
Example 5: Generation and Characterization of 3E10 mAb-GAA and 3E10
Fab-GAA Fusion Constructs
[0337] We expressed representative chimeric polypeptides according
to the protocol described in Hacker et al., 2013, Protein Expr
Purif. 92: 67. Specifically, chimeric polypeptides comprising a GAA
polypeptide portion and an internalizing moiety portion were made
recombinantly. In this experiment, a GAA polypeptide comprising
mature GAA (the GAA polypeptide portion of the chimeric
polypeptide) was fused to either a full-length murine monoclonal
3E10 antibody comprising the light chain variable domain set forth
in SEQ ID NO: 10 and the heavy chain variable domain set forth in
SEQ ID NO: 9 (the internalizing moiety portion), or to a Fab of
this 3E10 antibody (see FIG. 1). Specifically, in this example, the
N-terminus of a GAA polypeptide comprising a mature GAA and having
the amino acid sequence of SEQ ID NO: 22 was fused to the
C-terminus of either the heavy chain constant region of a murine
3E10 Fab fragment or to the C-terminus of the heavy chain constant
region of a full-length murine 3E10 monoclonal antibody (mAb). In
this example, the heavy chain of the internalizing moiety comprises
murine 3E10 antibody comprises the foregoing VH and a murine heavy
chain constant domain comprising CH1, hinge, CH2, and CH3 regions,
such as constant domain regions from an IgG1, IgG2a, IgG2b, or IgG4
antibody. In either case, a nucleotide sequence expressing the
recombinant heavy chain and 3E10 light chain comprising the
foregoing 3E10 VL were inserted into separate vectors and
transiently transfected into CHO-DG44 cells in order to produce to
produce the recombinant, chimeric protein. Similarly, the
nucleotide sequence encoding the heavy and light chains could be
expressed from a single vector. The chimeric constructs are shown
schematically in FIG. 1.
[0338] In this example, a linker sequence was used to fuse the GAA
polypeptide to the Fab or mAb heavy chains, and that linker had the
amino acid sequence of SEQ ID NO: 30.
Exemplary Sequences
TABLE-US-00005 [0339] SEQ ID NO: 1 = full-length, immature GAA
amino acid sequence (952 amino acids; signal sequence indicated in
bold/underline)
MGVRHPPCSHRLLAVCALVSLATAALLGHILLHDFLLVPRELSGSSPVLEETHPAHQQGASRPGPRDAQAHPGR-
PRAVPTQCDVP
PNSRFDCAPDKAITQEQCEARGCCYIPAKQGLQGAQMGQPWCFFPPSYPSYKLENLSSSEMGYTATLTRTTPTF-
FPKDILTLRLD
VMMETENRLHFTIKDPANRRYEVPLETPHVHSRAPSPLYSVEFSEEPFGVIVRRQLDGRVLLNTTVAPLFFADQ-
FLQLSTSLPSQ
YITGLAEHLSPLMLSTSWTRITLWNRDLAPTPGANLYGSHPFYLALEDGGSAHGVFLLNSNAMDVVLQPSPALS-
WRSTGGILDVY
IFLGPEPKSVVQQYLDVVGYPFMPPYWGLGFHLCRWGYSSTAITRQVVENMTRAHFPLDVQWNDLDYMDSRRDF-
TFNKDGFRDFP
AMVQELHQGGRRYMMIVDPAISSSGPAGSYRPYDEGLRRGVFITNETGQPLIGKVWPGSTAFPDFTNPTALAWW-
EDMVAEFHDQV
PFDGMWIDMNEPSNFIRGSEDGCPNNELENPPYVPGVVGGTLQAATICASSHQFLSTHYNLHNLYGLTEAIASH-
RALVKARGTRP
FVISRSTFAGHGRYAGHWTGDVWSSWEQLASSVPEILQFNLLGVPLVGADVCGFLGNTSEELCVRWTQLGAFYP-
FMRNHNSLLSL
PQEPYSFSEPAQQAMRKALTLRYALLPHLYTLFHQAHVAGETVARPLFLEFPKDSSTWTVDHQLLWGEALLITP-
VLQAGKAEVTG
YFPLGTWYDLQTVPVEALGSLPPPPAAPREPAIHSEGQWVTLPAPLDTINVHLRAGYIIPLQGPGLTTTESRQQ-
PMALAVALTKG
GEARGELFWDDGESLEVLERGAYTQVIFLARNNTIVNELVRVTSEGAGLQLQKVTVLGVATAPQQVLSNGVPVS-
NFTYSPDTKVL DICVSLLMGEQFLVSWC SEQ ID NO: 2 = full-length, immature
GAA amino acid sequence (957 amino acids; signal sequence indicated
in bold/underline) (GenBank Accession No. EAW89583.1)
MGVRHPPCSHRLLAVCALVSLATAALLGHILLHDFLLVPRELSGSSPVLEETHPAHQQGASRPGPRDAQAHPGR-
PRAVPTQCDVP
PNSRFDCAPDKAITQEQCEARGCCYIPAKQGLQGAQMGQPWCFFPPSYPSYKLENLSSSEMGYTATLTRTTPTF-
FPKDILTLRLD
VMMETENRLHFTIKDPANRRYEVPLETPHVHSRAPSPLYSVEFSEEPFGVIVRRQLDGRVLLNTTVAPLFFADQ-
FLQLSTSLPSQ
YITGLAEHLSPLMLSTSWTRITLWNRDLAPTPGANLYGSHPFYLALEDGGSAHGVFLLNSNAMDVVLQPSPALS-
WRSTGGILDVY
IFLGPEPKSVVQQYLDVVGYPFMPPYWGLGFHLCRWGYSSTAITRQVVENMTRAHFPLDVQWNDLDYMDSRRDF-
TFNKDGFRDFP
AMVQELHQGGRRYMMIVDPAISSSGPAGSYRPYDEGLRRGVFITNETGQPLIGKVWPGSTAFPDFTNPTALAWW-
EDMVAEFHDQV
PFDGMWIDMNEPSNFIRGSEDGCPNNELENPPYVPGVVGGTLQAATICASSHQFLSTHYNLHNLYGLTEAIASH-
RALVKARGTRP
FVISRSTFAGHGRYAGHWTGDVWSSWEQLASSVPEILQFNLLGVPLVGADVCGFLGNTSEELCVRWTQLGAFYP-
FMRNHNSLLSL
PQEPYSFSEPAQQAMRKALTLRYALLPHLYTLFHQAHVAGETVARPLFLEFPKDSSTWTVDHQLLWGEALLITP-
VLQAGKAEVTG
YFPLGTWYDLQTVPIEALGSLPPPPAAPREPAIHSEGQWVTLPAPLDTINVHLRAGYIIPLQGPGLTTTESRQQ-
PMALAVALTKG
GEARGELFWDDGESLEVLERGAYTQVIFLARNNTIVNELVRVTSEGAGLQLQKVTVLGVATAPQQVLSNGVPVS-
NFTYSPDTKAR GPRVLDICVSLLMGEQFLVSWC SEQ ID NO: 3 = exemplary mature
GAA amino acid sequence (corresponding to residues 123-782 of SEQ
ID NO: 1; one embodiment of a mature GAA polypeptide)
GQPWCFFPPSYPSYKLENLSSSEMGYTATLTRTTPTFFPKDILTLRLDVMMETENRLHFTIKDPANRRYEVPLE-
TPHVHSRAPSP
LYSVEFSEEPFGVIVRRQLDGRVLLNTTVAPLFFADQFLQLSTSLPSQYITGLAEHLSPLMLSTSWTRITLWNR-
DLAPTPGANLY
GSHPFYLALEDGGSAHGVFLLNSNAMDVVLQPSPALSWRSTGGILDVYIFLGPEPKSVVQQYLDVVGYPFMPPY-
WGLGFHLCRWG
YSSTAITRQVVENMTRAHFPLDVQWNDLDYMDSRRDFTFNKDGFRDFPAMVQELHQGGRRYMMIVDPAISSSGP-
AGSYRPYDEGL
RRGVFITNETGQPLIGKVWPGSTAFPDFTNPTALAWWEDMVAEFHDQVPFDGMWIDMNEPSNFIRGSEDGCPNN-
ELENPPYVPGV
VGGTLQAATICASSHQFLSTHYNLHNLYGLTEAIASHRALVKARGTRPFVISRSTFAGHGRYAGHWTGDVWSSW-
EQLASSVPEIL
QFNLLGVPLVGADVCGFLGNTSEELCVRWTQLGAFYPFMRNHNSLLSLPQEPYSFSEPAQQAMRKALTLRYALL-
PHLYTLFHQAH
VAGETVARPLFLEFPKDSSTWTVDHQLLWGEALLITPVLQAGKAEVTGYFPLGTWYDLQTVPVEA
SEQ ID NO: 4 = exemplary mature GAA amino acid sequence
(corresponding to residues 288-782 of SEQ ID NO: 1; one embodiment
of a mature GAA polypeptide)
GANLYGSHPFYLALEDGGSAHGVFLLNSNAMDVVLQPSPALSWRSTGGILDVYIFLGPEPKSVVQQYLDVVGYP-
FMPPYWGLGFH
LCRWGYSSTAITRQVVENMTRAHFPLDVQWNDLDYMDSRRDFTFNKDGFRDFPAMVQELHQGGRRYMMIVDPAI-
SSSGPAGSYRP
YDEGLRRGVFITNETGQPLIGKVWPGSTAFPDFTNPTALAWWEDMVAEFHDQVPFDGMWIDMNEPSNFIRGSED-
GCPNNELENPP
YVPGVVGGTLQAATICASSHQFLSTHYNLHNLYGLTEAIASHRALVKARGTRPFVISRSTFAGHGRYAGHWTGD-
VWSSWEQLASS
VPEILQFNLLGVPLVGADVCGFLGNTSEELCVRWTQLGAFYPFMRNHNSLLSLPQEPYSFSEPAQQAMRKALTL-
RYALLPHLYTL
FHQAHVAGETVARPLFLEFPKDSSTWTVDHQLLWGEALLITPVLQAGKAEVTGYFPLGTWYDLQTVPVEA
SEQ ID NO: 5 = GS3 linker GGGGSGGGGSGGGGS SEQ ID NO: 6 = Linker
GSTSGSGKSSEGKG SEQ ID NO: 7 = His tag HHHHHH SEQ ID NO: 8 = c-Myc
tag EQKLISEEDL SEQ ID NO: 9 = exemplary 3E10 Variable Heavy Chain
(V.sub.H having D31N substitution; see examples)
EVQLVESGGGLVKPGGSRKLSCAASGFTFSNYGMHWVRQAPEKGLEWVAYISSGSSTIYYADTVKGRFTISRDN-
AKNTLFLQMTS LRSEDTAMYYCARRGLLLDYWGQGTTLTVSS SEQ ID NO: 10 = 3E10
Variable Light Chain (V.sub.L)
DIVLTQSPASLAVSLGQRATISCRASKSVSTSSYSYMHWYQQKPGQPPKLLIKYASYLESGVPARFSGSGSGTD-
FHLNIHPVEEE DAATYYCQHSREFPWTFGGGTKLELK SEQ ID NO: 11 = Exemplary
chimeric polypeptide, Fv3E10-GAA (123-782)
DIVLTQSPASLAVSLGQRATISCRASKSVSTSSYSYMHWYQQKPGQPPKLLIKYASYLESGVPARFSGSGSGTD-
FHLNIHPVEEE
DAATYYCQHSREFPWTFGGGTKLELKGGGGSGGGGSGGGGSEVQLVESGGGLVKPGGSRKLSCAASGFTFSNYG-
MHWVRQAPEKG
LEWVAYISSGSSTIYYADTVKGRFTISRDNAKNTLFLQMTSLRSEDTAMYYCARRGLLLDYWGQGTTLTVSSEQ-
KLSEEDLGSTS
GSGKSSEGKGGQPWCFFPPSYPSYKLENLSSSEMGYTATLTRTTPTFFPKDILTLRLDVMMETENRLHFTIKDP-
ANRRYEVPLET
PRVHSRAPSPLYSVEFSEEPFGVIVHRQLDGRVLLNTTVAPLFFADQFLQLSTSLPSQYITGLAEHLSPLMLST-
SWTRITLWNRD
LAPTPGANLYGSHPFYLALEDGGSAHGVFLLNSNAMDVVLQPSPALSWRSTGGILDVYIFLGPEPKSVVQQYLD-
VVGYPFMPPYW
GLGFHLCRWGYSSTAITRQVVENMTRAHFPLDVQWNDLDYMDSRRDFTFNKDGFRDFPAMVQELHQGGRRYMMI-
VDPAISSSGPA
GSYRLYDEGLRRGVFITNETGQPLIGKVWPGSTAFPDFTNPTALAWWEDMVAEFHDQVPFDGMWIDMNEPSNFI-
RGSEDGCPNNE
LENPPYVPGVVGGTLQAATICASSHQFLSTHYNLHNLYGLTEAIASHRALVKARGTRPFVISRSTFAGHGRYAG-
HWTGDVWSSWE
QLASSVPEILQFNLLGVPLVGADVCGFLGNTSEELCVRWTQLGAFYPFMRNHNSLLSLPQEPYSFSEPAQQAMR-
KALTLRYALLP
HLYTLFHQAHVAGETVARPLFLEFPKDSSTWTVDHQLLWGEALLITPVLQAGKAEVTGYFPLGTWYDLQTVPIE-
AHRHHHH SEQ ID NO: 12 = Exemplary chimeric polypeptide, Fv3E10-GAA
(288-782)
DIVLTQSPASLAVSLGQRATISCRASKSVSTSSYSYMHWYQQKPGQPPKLLIKYASYLESGVPARFSGSGSGTD-
FHLNIHPVEEE
DAATYYCQHSREFPWTFGGGTKLELKGGGGSGGGGSGGGGSEVQLVESGGGLVKPGGSRKLSCAASGFTFSNYG-
MHWVRQAPEKG
LEWVAYISSGSSTIYYADTVKGRFTISRDNAKNTLFLQMTSLRSEDTAMYYCARRGLLLDYWGQGTTLTVSSEQ-
KLSEEDLGSTS
GSGKSSEGKGGANLYGSHPFYLALEDGGSAHGVFLLNSNAMDVVLQPSPALSWRSTGGILDVYIFLGPEPKSVV-
QQYLDVVGYPF
MPPYWGLGFHLCRWGYSSTAITRQVVENMTRAHFPLDVQWNDLDYMDSRRDFTFNKDGFRDFPAMVQELHQGGR-
RYMMIVDPAIS
SSGPAGSYRLYDEGLRRGVFITNETGQPLIGKVWPGSTAFPDFTNPTALAWWEDMVAEFHDQVPFDGMWIDMNE-
PSNFIRGSEDG
CPNNELENPPYVPGVVGGTLQAATICASSHQFLSTHYNLHNLYGLTEAIASHRALVKARGTRPFVISRSTFAGH-
GRYAGHWTGDV
WSSWEQLASSVPEILQFNLLGVPLVGADVCGFLGNTSEELCVRWTQLGAFYPFMRNHNSLLSLPQEPYSFSEPA-
QQAMRKALTLR
YALLPHLYTLFHQAHVAGETVARPLFLEFPKDSSTWTVDHQLLWGEALLITPVLQAGKAEVTGYFPLGTWYDLQ-
TVPIEAHHHHH H SEQ ID NO: 13-heavy chain variable domain CDR1 of
3E10 VH (as that VH is defined with reference to SEQ ID NO: 9), in
accordance with Kabat system NYGMH SEQ ID NO: 14-heavy chain
variable domain CDR2 of 3E10 VH (as that VH is defined with
reference to SEQ ID NO: 9), in accordance with Kabat system
YISSGSSTIYYADTVKG SEQ ID NO: 15-heavy chain variable domain CDR3 of
3E10 VH (as that VH is defined with reference to SEQ ID NO: 9), in
accordance with Kabat system RGLLLDY SEQ ID NO: 16-light chain
variable domain CDR1 of 3E10 VL (as that VL is defined with
reference to SEQ ID NO: 10), in accordance with Kabat system
RASKSVSTSSYSYMH SEQ ID NO: 17-light chain variable domain CDR2 of
3E10 VL (as that VL is defined with reference to SEQ ID NO: 10), in
accordance with Kabat system YASYLES SEQ ID NO: 18-light chain
variable domain CDR3 of 3E10 VL (as that VL is defined with
reference to SEQ ID NO: 10), in accordance with Kabat system
QHSREFPWT SEQ ID NO: 19 AGIH SEQ ID NO: 20 SAGIH SEQ ID NO:
21-Exemplary GAA polypeptide comprising mature GAA (residues
61-952; one embodiment of a GAA polypeptide)
SRPGPRDAQAHPGRPRAVPTQCDVPPNSRFDCAPDKAITQEQCEARGCCYIPAKQGLQGAQMGQPWCFFPPSYP-
SYKLENLSSSE
MGYTATLTRTTPTFFPKDILTLRLDVMMETENRLHFTIKDPANRRYEVPLETPHVHSRAPSPLYSVEFSEEPFG-
VIVRRQLDGRV
LLNTTVAPLFFADQFLQLSTSLPSQYITGLAEHLSPLMLSTSWTRITLWNRDLAPTPGANLYGSHPFYLALEDG-
GSAHGVFLLNS
NAMDVVLQPSPALSWRSTGGILDVYIFLGPEPKSVVQQYLDVVGYPFMPPYWGLGFHLCRWGYSSTAITRQVVE-
NIVITRAHFPL
DVQWNDLDYMDSRRDFTFNKDGFRDFPAMVQELHQGGRRYMMIVDPAISSSGPAGSYRPYDEGLRRGVFITNET-
GQPLIGKVWPG
STAFPDFTNPTALAWWEDMVAEFHDQVPFDGMWIDMNEPSNFIRGSEDGCPNNELENPPYVPGVVGGTLQAATI-
CASSHQFLSTH
YNLHNLYGLTEAIASHRALVKARGTRPFVISRSTFAGHGRYAGHWTGDVWSSWEQLASSVPEILQFNLLGVPLV-
GADVCGFLGNT
SEELCVRWTQLGAFYPFMRNHNSLLSLPQEPYSFSEPAQQAMRKALTLRYALLPHLYTLFHQAHVAGETVARPL-
FLEFPKDSSTW
TVDHQLLWGEALLITPVLQAGKAEVTGYFPLGTWYDLQTVPVEALGSLPPPPAAPREPAIHSEGQWVTLPAPLD-
TINVHLRAGYI
IPLQGPGLTTTESRQQPMALAVALTKGGEARGELFWDDGESLEVLERGAYTQVIFLARNNTIVNELVRVTSEGA-
GLQLQKVTVLG VATAPQQVLSNGVPVSNFTYSPDTKVLDICVSLLMGEQFLVSWC SEQ ID NO:
22-Exemplary GAA polypeptide comprising mature GAA (residues
67-952; one embodiment of a GAA polypeptide)
DAQAHPGRPRAVPTQCDVPPNSRFDCAPDKAITQEQCEARGCCYIPAKQGLQGAQMGQPWCFFPPSYPSYKLEN-
LSSSEMGYTAT
LTRTTPTFFPKDILTLRLDVMMETENRLHFTIKDPANRRYEVPLETPHVHSRAPSPLYSVEFSEEPFGVIVRRQ-
LDGRVLLNTTV
APLFFADQFLQLSTSLPSQYITGLAEHLSPLMLSTSWTRITLWNRDLAPTPGANLYGSHPFYLALEDGGSAHGV-
FLLNSNAMDVV
LQPSPALSWRSTGGILDVYIFLGPEPKSVVQQYLDVVGYPFMPPYWGLGFHLCRWGYSSTAITRQVVENMTRAH-
FPLDVQWNDLD
YMDSRRDFTFNKDGFRDFPAMVQELHQGGRRYMMIVDPAISSSGPAGSYRPYDEGLRRGVFITNETGQPLIGKV-
WPGSTAFPDFT
NPTALAWWEDMVAEFHDQVPFDGMWIDMNEPSNFIRGSEDGCPNNELENPPYVPGVVGGTLQAATICASSHQFL-
STHYNLHNLYG
LTEAIASHRALVKARGTRPFVISRSTFAGHGRYAGHWTGDVWSSWEQLASSVPEILQFNLLGVPLVGADVCGFL-
GNTSEELCVRW
TQLGAFYPFMRNHNSLLSLPQEPYSFSEPAQQAMRKALTLRYALLPHLYTLFHQAHVAGETVARPLFLEFPKDS-
STWTVDHQLLW
GEALLITPVLQAGKAEVTGYFPLGTWYDLQTVPVEALGSLPPPPAAPREPAIHSEGQWVTLPAPLDTINVHLRA-
GYIIPLQGPGL
TTTESRQQPMALAVALTKGGEARGELFWDDGESLEVLERGAYTQVIFLARNNTIVNELVRVTSEGAGLQLQKVT-
VLGVATAPQQV LSNGVPVSNFTYSPDTKVLDICVSLLMGEQFLVSWC SEQ ID NO:
23-Exemplary GAA polypeptide comprising mature GAA (residues
70-952; one embodiment of a GAA polypeptide)
AHPGRPRAVPTQCDVPPNSRFDCAPDKAITQEQCEARGCCYIPAKQGLQGAQMGQPWCFFPPSYPSYKLENLSS-
SEMGYTATLTR
TTPTFFPKDILTLRLDVMMETENRLHFTIKDPANRRYEVPLETPHVHSRAPSPLYSVEFSEEPFGVIVRRQLDG-
RVLLNTTVAPL
FFADQFLQLSTSLPSQYITGLAEHLSPLMLSTSWTRITLWNRDLAPTPGANLYGSHPFYLALEDGGSAHGVFLL-
NSNAMDVVLQP
SPALSWRSTGGILDVYIFLGPEPKSVVQQYLDVVGYPFMPPYWGLGFHLCRWGYSSTAITRQVVENMTRAHFPL-
DVQWNDLDYMD
SRRDFTFNKDGFRDFPAMVQELHQGGRRYMMIVDPAISSSGPAGSYRPYDEGLRRGVFITNETGQPLIGKVWPG-
STAFPDFTNPT
ALAWWEDMVAEFHDQVPFDGMWIDMNEPSNFIRGSEDGCPNNELENPPYVPGVVGGTLQAATICASSHQFLSTH-
YNLHNLYGLTE
AIASHRALVKARGTRPFVISRSTFAGHGRYAGHWTGDVWSSWEQLASSVPEILQFNLLGVPLVGADVCGFLGNT-
SEELCVRWTQL
GAFYPFMRNHNSLLSLPQEPYSFSEPAQQAMRKALTLRYALLPHLYTLFHQAHVAGETVARPLFLEFPKDSSTW-
TVDHQLLWGEA
LLITPVLQAGKAEVTGYFPLGTWYDLQTVPVEALGSLPPPPAAPREPAIHSEGQWVTLPAPLDTINVHLRAGYI-
IPLQGPGLTTT
ESRQQPMALAVALTKGGEARGELFWDDGESLEVLERGAYTQVIFLARNNTIVNELVRVTSEGAGLQLQKVTVLG-
VATAPQQVLSN GVPVSNFTYSPDTKVLDICVSLLMGEQFLVSWC SEQ ID NO: 24-heavy
chain variable (VH) domain CDR1 of exemplary 3E10 V.sub.H (as that
VH is defined with reference to SEQ ID NO: 9), in accordance with
the IMGT system GFTFSNYG SEQ ID NO: 25-heavy chain variable (VH)
domain CDR2 of exemplary 3E10 V.sub.H (as that VH is defined with
reference to SEQ ID NO: 9), in accordance with the IMGT system
ISSGSSTI SEQ ID NO: 26-heavy chain variable (VH) domain CDR3 of
exemplary 3E10 V.sub.H (as that VH is defined with reference to SEQ
ID NO: 9), in accordance with the IMGT system ARRGLLLDY SEQ ID NO:
27-light chain variable (VL) domain CDR1 of exemplary 3E10 V.sub.L
(as that VL is defined with reference to SEQ ID NO: 10), in
accordance with the IMGT system KSVSTSSYSY SEQ ID NO: 28-light
chain variable (VL) domain CR2 of exemplary 3E10 V.sub.L (as that
VL is defined with reference to SEQ ID NO: 10), in accordance with
the IMGT system YAS SEQ ID NO: 29-light chain variable (VL) domain
CDR3 of exemplary 3E10 V.sub.L (as that VL is defined with
reference to SEQ ID NO: 10), in accordance with the IMGT system
QHSREFPWT SEQ ID NO: 30-linker sequence GGSGGGSGGGSGG SEQ ID NO:
31-full linker region (residues 57-78 of GAA)
HILLHDFLLVPRELSGSSPVLEETHPAH SEQ ID NO: 32-bovine GAA precursor
protein (GenBank Accession No. NP776338.1)
MMRWPPCSRPLLGVCTLLSLALLGHILLHDLEVVPRELRGFSQDEIHQACQPGASSPECRGSPRAAPTQCDLPP-
NSRFDCAPDKG
ITPQQCEARGCCYMPAEWPPDAQMGQPWCFFPPSYPSYRLENLTTTETGYTATLTRAVPTFFPKDIMTLRLDML-
METESRLHFTI
KDPANRRYEVPLETPRVYSQAPFTLYSVEFSEEPFGVVVRRKLDGRVLLNTTVAPLFFADQFLQLSTSLPSQHI-
TGLAEHLGSLM
LSTNWTKITLWNRDIAPEPNVNLYGSHPFYLVLEDGGLAHGVFLLNSNAMDVVLQPSPALSWRSTGGILDVYIF-
LGPEPKSVVQQ
YLDVVGYPFMPPYWGLGFHLCRWGYSTSAITRQVVENMTRAYFPLDVQWNDLDYMDARRDFTFNKDHFGDFPAM-
VQELHQGGRRY
IMIVDPAISSSGPAGTYRPYDEGLRRGVFITNETGQPLIGQVWPGLTAFPDFTNPETLDWWQDMVTEFHAQVPF-
DGMWIDMNEPS
NFVRGSVDGCPDNSLENPPYLPGVVGGTLRAATICASSHQFLSTHYDLHNLYGLTEALASHRALVKARGMRPFV-
ISRSTFAGHGR
YSGHWTGDVWSNWEQLSYSVPEILLFNLLGVPLVGADICGFLGNTSEELCVRWTQLGAFYPFMRNHNALNSQPQ-
EPYRFSETAQQ
AMRKAFTLRYVLLPYLYTLFHRAHVRGETVARPLFLEFPEDPSTWTVDRQLLWGEALLITPVLEAEKVEVTGYF-
PQGTWYDLQTV
PMEAFGSLPPPAPLTSVIHSKGQWVTLSAPLDTINVHLRAGHIIPMQGPALTTTESRKQHMALAVALTASGEAQ-
GELFWDDGESL
GVLDGGDYTQLIFLAKNNTFVNKLVHVSSEGASLQLRNVTVLGVATAPQQVLCNSVPVSNFTFSPDTETLAIPV-
SLTMGEQFVIS WS SEQ ID NO: 33-KFERQ KFERQ SEQ ID NO:
34-(G.sub.4S).sub.n, wherein n is an integer from 1-10
(GGGGS).sub.n SEQ ID NO: 35-ASSLNIA homing peptide ASSLNIA SEQ ID
NO: 36-Arg7 Peptide RRRRRRR
INCORPORATION BY REFERENCE
[0340] All publications and patents mentioned herein are hereby
incorporated by reference in their entirety as if each individual
publication or patent was specifically and individually indicated
to be incorporated by reference.
[0341] While specific embodiments of the subject disclosure have
been discussed, the above specification is illustrative and not
restrictive. Many variations of the disclosure will become apparent
to those skilled in the art upon review of this specification and
the claims below. The full scope of the disclosure should be
determined by reference to the claims, along with their full scope
of equivalents, and the specification, along with such variations.
Sequence CWU 1
1
361952PRTHomo sapiens 1Met Gly Val Arg His Pro Pro Cys Ser His Arg
Leu Leu Ala Val Cys 1 5 10 15 Ala Leu Val Ser Leu Ala Thr Ala Ala
Leu Leu Gly His Ile Leu Leu 20 25 30 His Asp Phe Leu Leu Val Pro
Arg Glu Leu Ser Gly Ser Ser Pro Val 35 40 45 Leu Glu Glu Thr His
Pro Ala His Gln Gln Gly Ala Ser Arg Pro Gly 50 55 60 Pro Arg Asp
Ala Gln Ala His Pro Gly Arg Pro Arg Ala Val Pro Thr 65 70 75 80 Gln
Cys Asp Val Pro Pro Asn Ser Arg Phe Asp Cys Ala Pro Asp Lys 85 90
95 Ala Ile Thr Gln Glu Gln Cys Glu Ala Arg Gly Cys Cys Tyr Ile Pro
100 105 110 Ala Lys Gln Gly Leu Gln Gly Ala Gln Met Gly Gln Pro Trp
Cys Phe 115 120 125 Phe Pro Pro Ser Tyr Pro Ser Tyr Lys Leu Glu Asn
Leu Ser Ser Ser 130 135 140 Glu Met Gly Tyr Thr Ala Thr Leu Thr Arg
Thr Thr Pro Thr Phe Phe 145 150 155 160 Pro Lys Asp Ile Leu Thr Leu
Arg Leu Asp Val Met Met Glu Thr Glu 165 170 175 Asn Arg Leu His Phe
Thr Ile Lys Asp Pro Ala Asn Arg Arg Tyr Glu 180 185 190 Val Pro Leu
Glu Thr Pro His Val His Ser Arg Ala Pro Ser Pro Leu 195 200 205 Tyr
Ser Val Glu Phe Ser Glu Glu Pro Phe Gly Val Ile Val Arg Arg 210 215
220 Gln Leu Asp Gly Arg Val Leu Leu Asn Thr Thr Val Ala Pro Leu Phe
225 230 235 240 Phe Ala Asp Gln Phe Leu Gln Leu Ser Thr Ser Leu Pro
Ser Gln Tyr 245 250 255 Ile Thr Gly Leu Ala Glu His Leu Ser Pro Leu
Met Leu Ser Thr Ser 260 265 270 Trp Thr Arg Ile Thr Leu Trp Asn Arg
Asp Leu Ala Pro Thr Pro Gly 275 280 285 Ala Asn Leu Tyr Gly Ser His
Pro Phe Tyr Leu Ala Leu Glu Asp Gly 290 295 300 Gly Ser Ala His Gly
Val Phe Leu Leu Asn Ser Asn Ala Met Asp Val 305 310 315 320 Val Leu
Gln Pro Ser Pro Ala Leu Ser Trp Arg Ser Thr Gly Gly Ile 325 330 335
Leu Asp Val Tyr Ile Phe Leu Gly Pro Glu Pro Lys Ser Val Val Gln 340
345 350 Gln Tyr Leu Asp Val Val Gly Tyr Pro Phe Met Pro Pro Tyr Trp
Gly 355 360 365 Leu Gly Phe His Leu Cys Arg Trp Gly Tyr Ser Ser Thr
Ala Ile Thr 370 375 380 Arg Gln Val Val Glu Asn Met Thr Arg Ala His
Phe Pro Leu Asp Val 385 390 395 400 Gln Trp Asn Asp Leu Asp Tyr Met
Asp Ser Arg Arg Asp Phe Thr Phe 405 410 415 Asn Lys Asp Gly Phe Arg
Asp Phe Pro Ala Met Val Gln Glu Leu His 420 425 430 Gln Gly Gly Arg
Arg Tyr Met Met Ile Val Asp Pro Ala Ile Ser Ser 435 440 445 Ser Gly
Pro Ala Gly Ser Tyr Arg Pro Tyr Asp Glu Gly Leu Arg Arg 450 455 460
Gly Val Phe Ile Thr Asn Glu Thr Gly Gln Pro Leu Ile Gly Lys Val 465
470 475 480 Trp Pro Gly Ser Thr Ala Phe Pro Asp Phe Thr Asn Pro Thr
Ala Leu 485 490 495 Ala Trp Trp Glu Asp Met Val Ala Glu Phe His Asp
Gln Val Pro Phe 500 505 510 Asp Gly Met Trp Ile Asp Met Asn Glu Pro
Ser Asn Phe Ile Arg Gly 515 520 525 Ser Glu Asp Gly Cys Pro Asn Asn
Glu Leu Glu Asn Pro Pro Tyr Val 530 535 540 Pro Gly Val Val Gly Gly
Thr Leu Gln Ala Ala Thr Ile Cys Ala Ser 545 550 555 560 Ser His Gln
Phe Leu Ser Thr His Tyr Asn Leu His Asn Leu Tyr Gly 565 570 575 Leu
Thr Glu Ala Ile Ala Ser His Arg Ala Leu Val Lys Ala Arg Gly 580 585
590 Thr Arg Pro Phe Val Ile Ser Arg Ser Thr Phe Ala Gly His Gly Arg
595 600 605 Tyr Ala Gly His Trp Thr Gly Asp Val Trp Ser Ser Trp Glu
Gln Leu 610 615 620 Ala Ser Ser Val Pro Glu Ile Leu Gln Phe Asn Leu
Leu Gly Val Pro 625 630 635 640 Leu Val Gly Ala Asp Val Cys Gly Phe
Leu Gly Asn Thr Ser Glu Glu 645 650 655 Leu Cys Val Arg Trp Thr Gln
Leu Gly Ala Phe Tyr Pro Phe Met Arg 660 665 670 Asn His Asn Ser Leu
Leu Ser Leu Pro Gln Glu Pro Tyr Ser Phe Ser 675 680 685 Glu Pro Ala
Gln Gln Ala Met Arg Lys Ala Leu Thr Leu Arg Tyr Ala 690 695 700 Leu
Leu Pro His Leu Tyr Thr Leu Phe His Gln Ala His Val Ala Gly 705 710
715 720 Glu Thr Val Ala Arg Pro Leu Phe Leu Glu Phe Pro Lys Asp Ser
Ser 725 730 735 Thr Trp Thr Val Asp His Gln Leu Leu Trp Gly Glu Ala
Leu Leu Ile 740 745 750 Thr Pro Val Leu Gln Ala Gly Lys Ala Glu Val
Thr Gly Tyr Phe Pro 755 760 765 Leu Gly Thr Trp Tyr Asp Leu Gln Thr
Val Pro Val Glu Ala Leu Gly 770 775 780 Ser Leu Pro Pro Pro Pro Ala
Ala Pro Arg Glu Pro Ala Ile His Ser 785 790 795 800 Glu Gly Gln Trp
Val Thr Leu Pro Ala Pro Leu Asp Thr Ile Asn Val 805 810 815 His Leu
Arg Ala Gly Tyr Ile Ile Pro Leu Gln Gly Pro Gly Leu Thr 820 825 830
Thr Thr Glu Ser Arg Gln Gln Pro Met Ala Leu Ala Val Ala Leu Thr 835
840 845 Lys Gly Gly Glu Ala Arg Gly Glu Leu Phe Trp Asp Asp Gly Glu
Ser 850 855 860 Leu Glu Val Leu Glu Arg Gly Ala Tyr Thr Gln Val Ile
Phe Leu Ala 865 870 875 880 Arg Asn Asn Thr Ile Val Asn Glu Leu Val
Arg Val Thr Ser Glu Gly 885 890 895 Ala Gly Leu Gln Leu Gln Lys Val
Thr Val Leu Gly Val Ala Thr Ala 900 905 910 Pro Gln Gln Val Leu Ser
Asn Gly Val Pro Val Ser Asn Phe Thr Tyr 915 920 925 Ser Pro Asp Thr
Lys Val Leu Asp Ile Cys Val Ser Leu Leu Met Gly 930 935 940 Glu Gln
Phe Leu Val Ser Trp Cys 945 950 2957PRTHomo sapiens 2Met Gly Val
Arg His Pro Pro Cys Ser His Arg Leu Leu Ala Val Cys 1 5 10 15 Ala
Leu Val Ser Leu Ala Thr Ala Ala Leu Leu Gly His Ile Leu Leu 20 25
30 His Asp Phe Leu Leu Val Pro Arg Glu Leu Ser Gly Ser Ser Pro Val
35 40 45 Leu Glu Glu Thr His Pro Ala His Gln Gln Gly Ala Ser Arg
Pro Gly 50 55 60 Pro Arg Asp Ala Gln Ala His Pro Gly Arg Pro Arg
Ala Val Pro Thr 65 70 75 80 Gln Cys Asp Val Pro Pro Asn Ser Arg Phe
Asp Cys Ala Pro Asp Lys 85 90 95 Ala Ile Thr Gln Glu Gln Cys Glu
Ala Arg Gly Cys Cys Tyr Ile Pro 100 105 110 Ala Lys Gln Gly Leu Gln
Gly Ala Gln Met Gly Gln Pro Trp Cys Phe 115 120 125 Phe Pro Pro Ser
Tyr Pro Ser Tyr Lys Leu Glu Asn Leu Ser Ser Ser 130 135 140 Glu Met
Gly Tyr Thr Ala Thr Leu Thr Arg Thr Thr Pro Thr Phe Phe 145 150 155
160 Pro Lys Asp Ile Leu Thr Leu Arg Leu Asp Val Met Met Glu Thr Glu
165 170 175 Asn Arg Leu His Phe Thr Ile Lys Asp Pro Ala Asn Arg Arg
Tyr Glu 180 185 190 Val Pro Leu Glu Thr Pro His Val His Ser Arg Ala
Pro Ser Pro Leu 195 200 205 Tyr Ser Val Glu Phe Ser Glu Glu Pro Phe
Gly Val Ile Val Arg Arg 210 215 220 Gln Leu Asp Gly Arg Val Leu Leu
Asn Thr Thr Val Ala Pro Leu Phe 225 230 235 240 Phe Ala Asp Gln Phe
Leu Gln Leu Ser Thr Ser Leu Pro Ser Gln Tyr 245 250 255 Ile Thr Gly
Leu Ala Glu His Leu Ser Pro Leu Met Leu Ser Thr Ser 260 265 270 Trp
Thr Arg Ile Thr Leu Trp Asn Arg Asp Leu Ala Pro Thr Pro Gly 275 280
285 Ala Asn Leu Tyr Gly Ser His Pro Phe Tyr Leu Ala Leu Glu Asp Gly
290 295 300 Gly Ser Ala His Gly Val Phe Leu Leu Asn Ser Asn Ala Met
Asp Val 305 310 315 320 Val Leu Gln Pro Ser Pro Ala Leu Ser Trp Arg
Ser Thr Gly Gly Ile 325 330 335 Leu Asp Val Tyr Ile Phe Leu Gly Pro
Glu Pro Lys Ser Val Val Gln 340 345 350 Gln Tyr Leu Asp Val Val Gly
Tyr Pro Phe Met Pro Pro Tyr Trp Gly 355 360 365 Leu Gly Phe His Leu
Cys Arg Trp Gly Tyr Ser Ser Thr Ala Ile Thr 370 375 380 Arg Gln Val
Val Glu Asn Met Thr Arg Ala His Phe Pro Leu Asp Val 385 390 395 400
Gln Trp Asn Asp Leu Asp Tyr Met Asp Ser Arg Arg Asp Phe Thr Phe 405
410 415 Asn Lys Asp Gly Phe Arg Asp Phe Pro Ala Met Val Gln Glu Leu
His 420 425 430 Gln Gly Gly Arg Arg Tyr Met Met Ile Val Asp Pro Ala
Ile Ser Ser 435 440 445 Ser Gly Pro Ala Gly Ser Tyr Arg Pro Tyr Asp
Glu Gly Leu Arg Arg 450 455 460 Gly Val Phe Ile Thr Asn Glu Thr Gly
Gln Pro Leu Ile Gly Lys Val 465 470 475 480 Trp Pro Gly Ser Thr Ala
Phe Pro Asp Phe Thr Asn Pro Thr Ala Leu 485 490 495 Ala Trp Trp Glu
Asp Met Val Ala Glu Phe His Asp Gln Val Pro Phe 500 505 510 Asp Gly
Met Trp Ile Asp Met Asn Glu Pro Ser Asn Phe Ile Arg Gly 515 520 525
Ser Glu Asp Gly Cys Pro Asn Asn Glu Leu Glu Asn Pro Pro Tyr Val 530
535 540 Pro Gly Val Val Gly Gly Thr Leu Gln Ala Ala Thr Ile Cys Ala
Ser 545 550 555 560 Ser His Gln Phe Leu Ser Thr His Tyr Asn Leu His
Asn Leu Tyr Gly 565 570 575 Leu Thr Glu Ala Ile Ala Ser His Arg Ala
Leu Val Lys Ala Arg Gly 580 585 590 Thr Arg Pro Phe Val Ile Ser Arg
Ser Thr Phe Ala Gly His Gly Arg 595 600 605 Tyr Ala Gly His Trp Thr
Gly Asp Val Trp Ser Ser Trp Glu Gln Leu 610 615 620 Ala Ser Ser Val
Pro Glu Ile Leu Gln Phe Asn Leu Leu Gly Val Pro 625 630 635 640 Leu
Val Gly Ala Asp Val Cys Gly Phe Leu Gly Asn Thr Ser Glu Glu 645 650
655 Leu Cys Val Arg Trp Thr Gln Leu Gly Ala Phe Tyr Pro Phe Met Arg
660 665 670 Asn His Asn Ser Leu Leu Ser Leu Pro Gln Glu Pro Tyr Ser
Phe Ser 675 680 685 Glu Pro Ala Gln Gln Ala Met Arg Lys Ala Leu Thr
Leu Arg Tyr Ala 690 695 700 Leu Leu Pro His Leu Tyr Thr Leu Phe His
Gln Ala His Val Ala Gly 705 710 715 720 Glu Thr Val Ala Arg Pro Leu
Phe Leu Glu Phe Pro Lys Asp Ser Ser 725 730 735 Thr Trp Thr Val Asp
His Gln Leu Leu Trp Gly Glu Ala Leu Leu Ile 740 745 750 Thr Pro Val
Leu Gln Ala Gly Lys Ala Glu Val Thr Gly Tyr Phe Pro 755 760 765 Leu
Gly Thr Trp Tyr Asp Leu Gln Thr Val Pro Ile Glu Ala Leu Gly 770 775
780 Ser Leu Pro Pro Pro Pro Ala Ala Pro Arg Glu Pro Ala Ile His Ser
785 790 795 800 Glu Gly Gln Trp Val Thr Leu Pro Ala Pro Leu Asp Thr
Ile Asn Val 805 810 815 His Leu Arg Ala Gly Tyr Ile Ile Pro Leu Gln
Gly Pro Gly Leu Thr 820 825 830 Thr Thr Glu Ser Arg Gln Gln Pro Met
Ala Leu Ala Val Ala Leu Thr 835 840 845 Lys Gly Gly Glu Ala Arg Gly
Glu Leu Phe Trp Asp Asp Gly Glu Ser 850 855 860 Leu Glu Val Leu Glu
Arg Gly Ala Tyr Thr Gln Val Ile Phe Leu Ala 865 870 875 880 Arg Asn
Asn Thr Ile Val Asn Glu Leu Val Arg Val Thr Ser Glu Gly 885 890 895
Ala Gly Leu Gln Leu Gln Lys Val Thr Val Leu Gly Val Ala Thr Ala 900
905 910 Pro Gln Gln Val Leu Ser Asn Gly Val Pro Val Ser Asn Phe Thr
Tyr 915 920 925 Ser Pro Asp Thr Lys Ala Arg Gly Pro Arg Val Leu Asp
Ile Cys Val 930 935 940 Ser Leu Leu Met Gly Glu Gln Phe Leu Val Ser
Trp Cys 945 950 955 3660PRTHomo sapiens 3Gly Gln Pro Trp Cys Phe
Phe Pro Pro Ser Tyr Pro Ser Tyr Lys Leu 1 5 10 15 Glu Asn Leu Ser
Ser Ser Glu Met Gly Tyr Thr Ala Thr Leu Thr Arg 20 25 30 Thr Thr
Pro Thr Phe Phe Pro Lys Asp Ile Leu Thr Leu Arg Leu Asp 35 40 45
Val Met Met Glu Thr Glu Asn Arg Leu His Phe Thr Ile Lys Asp Pro 50
55 60 Ala Asn Arg Arg Tyr Glu Val Pro Leu Glu Thr Pro His Val His
Ser 65 70 75 80 Arg Ala Pro Ser Pro Leu Tyr Ser Val Glu Phe Ser Glu
Glu Pro Phe 85 90 95 Gly Val Ile Val Arg Arg Gln Leu Asp Gly Arg
Val Leu Leu Asn Thr 100 105 110 Thr Val Ala Pro Leu Phe Phe Ala Asp
Gln Phe Leu Gln Leu Ser Thr 115 120 125 Ser Leu Pro Ser Gln Tyr Ile
Thr Gly Leu Ala Glu His Leu Ser Pro 130 135 140 Leu Met Leu Ser Thr
Ser Trp Thr Arg Ile Thr Leu Trp Asn Arg Asp 145 150 155 160 Leu Ala
Pro Thr Pro Gly Ala Asn Leu Tyr Gly Ser His Pro Phe Tyr 165 170 175
Leu Ala Leu Glu Asp Gly Gly Ser Ala His Gly Val Phe Leu Leu Asn 180
185 190 Ser Asn Ala Met Asp Val Val Leu Gln Pro Ser Pro Ala Leu Ser
Trp 195 200 205 Arg Ser Thr Gly Gly Ile Leu Asp Val Tyr Ile Phe Leu
Gly Pro Glu 210 215 220 Pro Lys Ser Val Val Gln Gln Tyr Leu Asp Val
Val Gly Tyr Pro Phe 225 230 235 240 Met Pro Pro Tyr Trp Gly Leu Gly
Phe His Leu Cys Arg Trp Gly Tyr 245 250 255 Ser Ser Thr Ala Ile Thr
Arg Gln Val Val Glu Asn Met Thr Arg Ala 260 265 270 His Phe Pro Leu
Asp Val Gln Trp Asn Asp Leu Asp Tyr Met Asp Ser 275 280 285 Arg Arg
Asp Phe Thr Phe Asn Lys Asp Gly Phe Arg Asp Phe Pro Ala 290 295 300
Met Val Gln Glu Leu His Gln Gly Gly Arg Arg Tyr Met Met Ile Val 305
310 315 320 Asp Pro Ala Ile Ser Ser Ser Gly Pro Ala Gly Ser Tyr Arg
Pro Tyr 325 330 335 Asp Glu Gly Leu Arg Arg Gly Val Phe Ile Thr Asn
Glu Thr Gly Gln 340 345 350 Pro Leu Ile Gly Lys Val Trp Pro Gly Ser
Thr Ala Phe Pro Asp Phe 355 360 365 Thr Asn Pro Thr Ala Leu Ala Trp
Trp Glu Asp Met Val Ala Glu Phe 370 375 380 His Asp Gln Val Pro Phe
Asp Gly Met Trp Ile
Asp Met Asn Glu Pro 385 390 395 400 Ser Asn Phe Ile Arg Gly Ser Glu
Asp Gly Cys Pro Asn Asn Glu Leu 405 410 415 Glu Asn Pro Pro Tyr Val
Pro Gly Val Val Gly Gly Thr Leu Gln Ala 420 425 430 Ala Thr Ile Cys
Ala Ser Ser His Gln Phe Leu Ser Thr His Tyr Asn 435 440 445 Leu His
Asn Leu Tyr Gly Leu Thr Glu Ala Ile Ala Ser His Arg Ala 450 455 460
Leu Val Lys Ala Arg Gly Thr Arg Pro Phe Val Ile Ser Arg Ser Thr 465
470 475 480 Phe Ala Gly His Gly Arg Tyr Ala Gly His Trp Thr Gly Asp
Val Trp 485 490 495 Ser Ser Trp Glu Gln Leu Ala Ser Ser Val Pro Glu
Ile Leu Gln Phe 500 505 510 Asn Leu Leu Gly Val Pro Leu Val Gly Ala
Asp Val Cys Gly Phe Leu 515 520 525 Gly Asn Thr Ser Glu Glu Leu Cys
Val Arg Trp Thr Gln Leu Gly Ala 530 535 540 Phe Tyr Pro Phe Met Arg
Asn His Asn Ser Leu Leu Ser Leu Pro Gln 545 550 555 560 Glu Pro Tyr
Ser Phe Ser Glu Pro Ala Gln Gln Ala Met Arg Lys Ala 565 570 575 Leu
Thr Leu Arg Tyr Ala Leu Leu Pro His Leu Tyr Thr Leu Phe His 580 585
590 Gln Ala His Val Ala Gly Glu Thr Val Ala Arg Pro Leu Phe Leu Glu
595 600 605 Phe Pro Lys Asp Ser Ser Thr Trp Thr Val Asp His Gln Leu
Leu Trp 610 615 620 Gly Glu Ala Leu Leu Ile Thr Pro Val Leu Gln Ala
Gly Lys Ala Glu 625 630 635 640 Val Thr Gly Tyr Phe Pro Leu Gly Thr
Trp Tyr Asp Leu Gln Thr Val 645 650 655 Pro Val Glu Ala 660
4495PRTHomo sapiens 4Gly Ala Asn Leu Tyr Gly Ser His Pro Phe Tyr
Leu Ala Leu Glu Asp 1 5 10 15 Gly Gly Ser Ala His Gly Val Phe Leu
Leu Asn Ser Asn Ala Met Asp 20 25 30 Val Val Leu Gln Pro Ser Pro
Ala Leu Ser Trp Arg Ser Thr Gly Gly 35 40 45 Ile Leu Asp Val Tyr
Ile Phe Leu Gly Pro Glu Pro Lys Ser Val Val 50 55 60 Gln Gln Tyr
Leu Asp Val Val Gly Tyr Pro Phe Met Pro Pro Tyr Trp 65 70 75 80 Gly
Leu Gly Phe His Leu Cys Arg Trp Gly Tyr Ser Ser Thr Ala Ile 85 90
95 Thr Arg Gln Val Val Glu Asn Met Thr Arg Ala His Phe Pro Leu Asp
100 105 110 Val Gln Trp Asn Asp Leu Asp Tyr Met Asp Ser Arg Arg Asp
Phe Thr 115 120 125 Phe Asn Lys Asp Gly Phe Arg Asp Phe Pro Ala Met
Val Gln Glu Leu 130 135 140 His Gln Gly Gly Arg Arg Tyr Met Met Ile
Val Asp Pro Ala Ile Ser 145 150 155 160 Ser Ser Gly Pro Ala Gly Ser
Tyr Arg Pro Tyr Asp Glu Gly Leu Arg 165 170 175 Arg Gly Val Phe Ile
Thr Asn Glu Thr Gly Gln Pro Leu Ile Gly Lys 180 185 190 Val Trp Pro
Gly Ser Thr Ala Phe Pro Asp Phe Thr Asn Pro Thr Ala 195 200 205 Leu
Ala Trp Trp Glu Asp Met Val Ala Glu Phe His Asp Gln Val Pro 210 215
220 Phe Asp Gly Met Trp Ile Asp Met Asn Glu Pro Ser Asn Phe Ile Arg
225 230 235 240 Gly Ser Glu Asp Gly Cys Pro Asn Asn Glu Leu Glu Asn
Pro Pro Tyr 245 250 255 Val Pro Gly Val Val Gly Gly Thr Leu Gln Ala
Ala Thr Ile Cys Ala 260 265 270 Ser Ser His Gln Phe Leu Ser Thr His
Tyr Asn Leu His Asn Leu Tyr 275 280 285 Gly Leu Thr Glu Ala Ile Ala
Ser His Arg Ala Leu Val Lys Ala Arg 290 295 300 Gly Thr Arg Pro Phe
Val Ile Ser Arg Ser Thr Phe Ala Gly His Gly 305 310 315 320 Arg Tyr
Ala Gly His Trp Thr Gly Asp Val Trp Ser Ser Trp Glu Gln 325 330 335
Leu Ala Ser Ser Val Pro Glu Ile Leu Gln Phe Asn Leu Leu Gly Val 340
345 350 Pro Leu Val Gly Ala Asp Val Cys Gly Phe Leu Gly Asn Thr Ser
Glu 355 360 365 Glu Leu Cys Val Arg Trp Thr Gln Leu Gly Ala Phe Tyr
Pro Phe Met 370 375 380 Arg Asn His Asn Ser Leu Leu Ser Leu Pro Gln
Glu Pro Tyr Ser Phe 385 390 395 400 Ser Glu Pro Ala Gln Gln Ala Met
Arg Lys Ala Leu Thr Leu Arg Tyr 405 410 415 Ala Leu Leu Pro His Leu
Tyr Thr Leu Phe His Gln Ala His Val Ala 420 425 430 Gly Glu Thr Val
Ala Arg Pro Leu Phe Leu Glu Phe Pro Lys Asp Ser 435 440 445 Ser Thr
Trp Thr Val Asp His Gln Leu Leu Trp Gly Glu Ala Leu Leu 450 455 460
Ile Thr Pro Val Leu Gln Ala Gly Lys Ala Glu Val Thr Gly Tyr Phe 465
470 475 480 Pro Leu Gly Thr Trp Tyr Asp Leu Gln Thr Val Pro Val Glu
Ala 485 490 495 515PRTArtificial SequenceDescription of Artificial
Sequence Synthetic peptide 5Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser
Gly Gly Gly Gly Ser 1 5 10 15 614PRTArtificial SequenceDescription
of Artificial Sequence Synthetic peptide 6Gly Ser Thr Ser Gly Ser
Gly Lys Ser Ser Glu Gly Lys Gly 1 5 10 76PRTArtificial
SequenceDescription of Artificial Sequence Synthetic 6xHis tag 7His
His His His His His 1 5 810PRTArtificial SequenceDescription of
Artificial Sequence Synthetic peptide 8Glu Gln Lys Leu Ile Ser Glu
Glu Asp Leu 1 5 10 9116PRTArtificial SequenceDescription of
Artificial Sequence Synthetic polypeptide 9Glu Val Gln Leu Val Glu
Ser Gly Gly Gly Leu Val Lys Pro Gly Gly 1 5 10 15 Ser Arg Lys Leu
Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Asn Tyr 20 25 30 Gly Met
His Trp Val Arg Gln Ala Pro Glu Lys Gly Leu Glu Trp Val 35 40 45
Ala Tyr Ile Ser Ser Gly Ser Ser Thr Ile Tyr Tyr Ala Asp Thr Val 50
55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Thr Leu
Phe 65 70 75 80 Leu Gln Met Thr Ser Leu Arg Ser Glu Asp Thr Ala Met
Tyr Tyr Cys 85 90 95 Ala Arg Arg Gly Leu Leu Leu Asp Tyr Trp Gly
Gln Gly Thr Thr Leu 100 105 110 Thr Val Ser Ser 115
10111PRTArtificial SequenceDescription of Artificial Sequence
Synthetic polypeptide 10Asp Ile Val Leu Thr Gln Ser Pro Ala Ser Leu
Ala Val Ser Leu Gly 1 5 10 15 Gln Arg Ala Thr Ile Ser Cys Arg Ala
Ser Lys Ser Val Ser Thr Ser 20 25 30 Ser Tyr Ser Tyr Met His Trp
Tyr Gln Gln Lys Pro Gly Gln Pro Pro 35 40 45 Lys Leu Leu Ile Lys
Tyr Ala Ser Tyr Leu Glu Ser Gly Val Pro Ala 50 55 60 Arg Phe Ser
Gly Ser Gly Ser Gly Thr Asp Phe His Leu Asn Ile His 65 70 75 80 Pro
Val Glu Glu Glu Asp Ala Ala Thr Tyr Tyr Cys Gln His Ser Arg 85 90
95 Glu Phe Pro Trp Thr Phe Gly Gly Gly Thr Lys Leu Glu Leu Lys 100
105 110 11931PRTArtificial SequenceDescription of Artificial
Sequence Synthetic polypeptide 11Asp Ile Val Leu Thr Gln Ser Pro
Ala Ser Leu Ala Val Ser Leu Gly 1 5 10 15 Gln Arg Ala Thr Ile Ser
Cys Arg Ala Ser Lys Ser Val Ser Thr Ser 20 25 30 Ser Tyr Ser Tyr
Met His Trp Tyr Gln Gln Lys Pro Gly Gln Pro Pro 35 40 45 Lys Leu
Leu Ile Lys Tyr Ala Ser Tyr Leu Glu Ser Gly Val Pro Ala 50 55 60
Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe His Leu Asn Ile His 65
70 75 80 Pro Val Glu Glu Glu Asp Ala Ala Thr Tyr Tyr Cys Gln His
Ser Arg 85 90 95 Glu Phe Pro Trp Thr Phe Gly Gly Gly Thr Lys Leu
Glu Leu Lys Gly 100 105 110 Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly
Gly Gly Gly Ser Glu Val 115 120 125 Gln Leu Val Glu Ser Gly Gly Gly
Leu Val Lys Pro Gly Gly Ser Arg 130 135 140 Lys Leu Ser Cys Ala Ala
Ser Gly Phe Thr Phe Ser Asn Tyr Gly Met 145 150 155 160 His Trp Val
Arg Gln Ala Pro Glu Lys Gly Leu Glu Trp Val Ala Tyr 165 170 175 Ile
Ser Ser Gly Ser Ser Thr Ile Tyr Tyr Ala Asp Thr Val Lys Gly 180 185
190 Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Thr Leu Phe Leu Gln
195 200 205 Met Thr Ser Leu Arg Ser Glu Asp Thr Ala Met Tyr Tyr Cys
Ala Arg 210 215 220 Arg Gly Leu Leu Leu Asp Tyr Trp Gly Gln Gly Thr
Thr Leu Thr Val 225 230 235 240 Ser Ser Glu Gln Lys Leu Ser Glu Glu
Asp Leu Gly Ser Thr Ser Gly 245 250 255 Ser Gly Lys Ser Ser Glu Gly
Lys Gly Gly Gln Pro Trp Cys Phe Phe 260 265 270 Pro Pro Ser Tyr Pro
Ser Tyr Lys Leu Glu Asn Leu Ser Ser Ser Glu 275 280 285 Met Gly Tyr
Thr Ala Thr Leu Thr Arg Thr Thr Pro Thr Phe Phe Pro 290 295 300 Lys
Asp Ile Leu Thr Leu Arg Leu Asp Val Met Met Glu Thr Glu Asn 305 310
315 320 Arg Leu His Phe Thr Ile Lys Asp Pro Ala Asn Arg Arg Tyr Glu
Val 325 330 335 Pro Leu Glu Thr Pro Arg Val His Ser Arg Ala Pro Ser
Pro Leu Tyr 340 345 350 Ser Val Glu Phe Ser Glu Glu Pro Phe Gly Val
Ile Val His Arg Gln 355 360 365 Leu Asp Gly Arg Val Leu Leu Asn Thr
Thr Val Ala Pro Leu Phe Phe 370 375 380 Ala Asp Gln Phe Leu Gln Leu
Ser Thr Ser Leu Pro Ser Gln Tyr Ile 385 390 395 400 Thr Gly Leu Ala
Glu His Leu Ser Pro Leu Met Leu Ser Thr Ser Trp 405 410 415 Thr Arg
Ile Thr Leu Trp Asn Arg Asp Leu Ala Pro Thr Pro Gly Ala 420 425 430
Asn Leu Tyr Gly Ser His Pro Phe Tyr Leu Ala Leu Glu Asp Gly Gly 435
440 445 Ser Ala His Gly Val Phe Leu Leu Asn Ser Asn Ala Met Asp Val
Val 450 455 460 Leu Gln Pro Ser Pro Ala Leu Ser Trp Arg Ser Thr Gly
Gly Ile Leu 465 470 475 480 Asp Val Tyr Ile Phe Leu Gly Pro Glu Pro
Lys Ser Val Val Gln Gln 485 490 495 Tyr Leu Asp Val Val Gly Tyr Pro
Phe Met Pro Pro Tyr Trp Gly Leu 500 505 510 Gly Phe His Leu Cys Arg
Trp Gly Tyr Ser Ser Thr Ala Ile Thr Arg 515 520 525 Gln Val Val Glu
Asn Met Thr Arg Ala His Phe Pro Leu Asp Val Gln 530 535 540 Trp Asn
Asp Leu Asp Tyr Met Asp Ser Arg Arg Asp Phe Thr Phe Asn 545 550 555
560 Lys Asp Gly Phe Arg Asp Phe Pro Ala Met Val Gln Glu Leu His Gln
565 570 575 Gly Gly Arg Arg Tyr Met Met Ile Val Asp Pro Ala Ile Ser
Ser Ser 580 585 590 Gly Pro Ala Gly Ser Tyr Arg Leu Tyr Asp Glu Gly
Leu Arg Arg Gly 595 600 605 Val Phe Ile Thr Asn Glu Thr Gly Gln Pro
Leu Ile Gly Lys Val Trp 610 615 620 Pro Gly Ser Thr Ala Phe Pro Asp
Phe Thr Asn Pro Thr Ala Leu Ala 625 630 635 640 Trp Trp Glu Asp Met
Val Ala Glu Phe His Asp Gln Val Pro Phe Asp 645 650 655 Gly Met Trp
Ile Asp Met Asn Glu Pro Ser Asn Phe Ile Arg Gly Ser 660 665 670 Glu
Asp Gly Cys Pro Asn Asn Glu Leu Glu Asn Pro Pro Tyr Val Pro 675 680
685 Gly Val Val Gly Gly Thr Leu Gln Ala Ala Thr Ile Cys Ala Ser Ser
690 695 700 His Gln Phe Leu Ser Thr His Tyr Asn Leu His Asn Leu Tyr
Gly Leu 705 710 715 720 Thr Glu Ala Ile Ala Ser His Arg Ala Leu Val
Lys Ala Arg Gly Thr 725 730 735 Arg Pro Phe Val Ile Ser Arg Ser Thr
Phe Ala Gly His Gly Arg Tyr 740 745 750 Ala Gly His Trp Thr Gly Asp
Val Trp Ser Ser Trp Glu Gln Leu Ala 755 760 765 Ser Ser Val Pro Glu
Ile Leu Gln Phe Asn Leu Leu Gly Val Pro Leu 770 775 780 Val Gly Ala
Asp Val Cys Gly Phe Leu Gly Asn Thr Ser Glu Glu Leu 785 790 795 800
Cys Val Arg Trp Thr Gln Leu Gly Ala Phe Tyr Pro Phe Met Arg Asn 805
810 815 His Asn Ser Leu Leu Ser Leu Pro Gln Glu Pro Tyr Ser Phe Ser
Glu 820 825 830 Pro Ala Gln Gln Ala Met Arg Lys Ala Leu Thr Leu Arg
Tyr Ala Leu 835 840 845 Leu Pro His Leu Tyr Thr Leu Phe His Gln Ala
His Val Ala Gly Glu 850 855 860 Thr Val Ala Arg Pro Leu Phe Leu Glu
Phe Pro Lys Asp Ser Ser Thr 865 870 875 880 Trp Thr Val Asp His Gln
Leu Leu Trp Gly Glu Ala Leu Leu Ile Thr 885 890 895 Pro Val Leu Gln
Ala Gly Lys Ala Glu Val Thr Gly Tyr Phe Pro Leu 900 905 910 Gly Thr
Trp Tyr Asp Leu Gln Thr Val Pro Ile Glu Ala His His His 915 920 925
His His His 930 12766PRTArtificial SequenceDescription of
Artificial Sequence Synthetic polypeptide 12Asp Ile Val Leu Thr Gln
Ser Pro Ala Ser Leu Ala Val Ser Leu Gly 1 5 10 15 Gln Arg Ala Thr
Ile Ser Cys Arg Ala Ser Lys Ser Val Ser Thr Ser 20 25 30 Ser Tyr
Ser Tyr Met His Trp Tyr Gln Gln Lys Pro Gly Gln Pro Pro 35 40 45
Lys Leu Leu Ile Lys Tyr Ala Ser Tyr Leu Glu Ser Gly Val Pro Ala 50
55 60 Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe His Leu Asn Ile
His 65 70 75 80 Pro Val Glu Glu Glu Asp Ala Ala Thr Tyr Tyr Cys Gln
His Ser Arg 85 90 95 Glu Phe Pro Trp Thr Phe Gly Gly Gly Thr Lys
Leu Glu Leu Lys Gly 100 105 110 Gly Gly Gly Ser Gly Gly Gly Gly Ser
Gly Gly Gly Gly Ser Glu Val 115 120 125 Gln Leu Val Glu Ser Gly Gly
Gly Leu Val Lys Pro Gly Gly Ser Arg 130 135 140 Lys Leu Ser Cys Ala
Ala Ser Gly Phe Thr Phe Ser Asn Tyr Gly Met 145 150 155 160 His Trp
Val Arg Gln Ala Pro Glu Lys Gly Leu Glu Trp Val Ala Tyr 165 170 175
Ile Ser Ser Gly Ser Ser Thr Ile Tyr Tyr Ala Asp Thr Val Lys Gly 180
185 190 Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Thr Leu Phe Leu
Gln 195 200 205 Met Thr Ser Leu Arg Ser Glu Asp Thr Ala Met Tyr Tyr
Cys Ala Arg 210 215 220 Arg Gly Leu Leu Leu Asp Tyr Trp Gly Gln Gly
Thr Thr Leu Thr Val 225 230 235
240 Ser Ser Glu Gln Lys Leu Ser Glu Glu Asp Leu Gly Ser Thr Ser Gly
245 250 255 Ser Gly Lys Ser Ser Glu Gly Lys Gly Gly Ala Asn Leu Tyr
Gly Ser 260 265 270 His Pro Phe Tyr Leu Ala Leu Glu Asp Gly Gly Ser
Ala His Gly Val 275 280 285 Phe Leu Leu Asn Ser Asn Ala Met Asp Val
Val Leu Gln Pro Ser Pro 290 295 300 Ala Leu Ser Trp Arg Ser Thr Gly
Gly Ile Leu Asp Val Tyr Ile Phe 305 310 315 320 Leu Gly Pro Glu Pro
Lys Ser Val Val Gln Gln Tyr Leu Asp Val Val 325 330 335 Gly Tyr Pro
Phe Met Pro Pro Tyr Trp Gly Leu Gly Phe His Leu Cys 340 345 350 Arg
Trp Gly Tyr Ser Ser Thr Ala Ile Thr Arg Gln Val Val Glu Asn 355 360
365 Met Thr Arg Ala His Phe Pro Leu Asp Val Gln Trp Asn Asp Leu Asp
370 375 380 Tyr Met Asp Ser Arg Arg Asp Phe Thr Phe Asn Lys Asp Gly
Phe Arg 385 390 395 400 Asp Phe Pro Ala Met Val Gln Glu Leu His Gln
Gly Gly Arg Arg Tyr 405 410 415 Met Met Ile Val Asp Pro Ala Ile Ser
Ser Ser Gly Pro Ala Gly Ser 420 425 430 Tyr Arg Leu Tyr Asp Glu Gly
Leu Arg Arg Gly Val Phe Ile Thr Asn 435 440 445 Glu Thr Gly Gln Pro
Leu Ile Gly Lys Val Trp Pro Gly Ser Thr Ala 450 455 460 Phe Pro Asp
Phe Thr Asn Pro Thr Ala Leu Ala Trp Trp Glu Asp Met 465 470 475 480
Val Ala Glu Phe His Asp Gln Val Pro Phe Asp Gly Met Trp Ile Asp 485
490 495 Met Asn Glu Pro Ser Asn Phe Ile Arg Gly Ser Glu Asp Gly Cys
Pro 500 505 510 Asn Asn Glu Leu Glu Asn Pro Pro Tyr Val Pro Gly Val
Val Gly Gly 515 520 525 Thr Leu Gln Ala Ala Thr Ile Cys Ala Ser Ser
His Gln Phe Leu Ser 530 535 540 Thr His Tyr Asn Leu His Asn Leu Tyr
Gly Leu Thr Glu Ala Ile Ala 545 550 555 560 Ser His Arg Ala Leu Val
Lys Ala Arg Gly Thr Arg Pro Phe Val Ile 565 570 575 Ser Arg Ser Thr
Phe Ala Gly His Gly Arg Tyr Ala Gly His Trp Thr 580 585 590 Gly Asp
Val Trp Ser Ser Trp Glu Gln Leu Ala Ser Ser Val Pro Glu 595 600 605
Ile Leu Gln Phe Asn Leu Leu Gly Val Pro Leu Val Gly Ala Asp Val 610
615 620 Cys Gly Phe Leu Gly Asn Thr Ser Glu Glu Leu Cys Val Arg Trp
Thr 625 630 635 640 Gln Leu Gly Ala Phe Tyr Pro Phe Met Arg Asn His
Asn Ser Leu Leu 645 650 655 Ser Leu Pro Gln Glu Pro Tyr Ser Phe Ser
Glu Pro Ala Gln Gln Ala 660 665 670 Met Arg Lys Ala Leu Thr Leu Arg
Tyr Ala Leu Leu Pro His Leu Tyr 675 680 685 Thr Leu Phe His Gln Ala
His Val Ala Gly Glu Thr Val Ala Arg Pro 690 695 700 Leu Phe Leu Glu
Phe Pro Lys Asp Ser Ser Thr Trp Thr Val Asp His 705 710 715 720 Gln
Leu Leu Trp Gly Glu Ala Leu Leu Ile Thr Pro Val Leu Gln Ala 725 730
735 Gly Lys Ala Glu Val Thr Gly Tyr Phe Pro Leu Gly Thr Trp Tyr Asp
740 745 750 Leu Gln Thr Val Pro Ile Glu Ala His His His His His His
755 760 765 135PRTArtificial SequenceDescription of Artificial
Sequence Synthetic peptide 13Asn Tyr Gly Met His 1 5
1417PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 14Tyr Ile Ser Ser Gly Ser Ser Thr Ile Tyr Tyr Ala
Asp Thr Val Lys 1 5 10 15 Gly 157PRTArtificial SequenceDescription
of Artificial Sequence Synthetic peptide 15Arg Gly Leu Leu Leu Asp
Tyr 1 5 1615PRTArtificial SequenceDescription of Artificial
Sequence Synthetic peptide 16Arg Ala Ser Lys Ser Val Ser Thr Ser
Ser Tyr Ser Tyr Met His 1 5 10 15 177PRTArtificial
SequenceDescription of Artificial Sequence Synthetic peptide 17Tyr
Ala Ser Tyr Leu Glu Ser 1 5 189PRTArtificial SequenceDescription of
Artificial Sequence Synthetic peptide 18Gln His Ser Arg Glu Phe Pro
Trp Thr 1 5 194PRTArtificial SequenceDescription of Artificial
Sequence Synthetic peptide 19Ala Gly Ile His 1 205PRTArtificial
SequenceDescription of Artificial Sequence Synthetic peptide 20Ser
Ala Gly Ile His 1 5 21892PRTArtificial SequenceDescription of
Artificial Sequence Synthetic polypeptide 21Ser Arg Pro Gly Pro Arg
Asp Ala Gln Ala His Pro Gly Arg Pro Arg 1 5 10 15 Ala Val Pro Thr
Gln Cys Asp Val Pro Pro Asn Ser Arg Phe Asp Cys 20 25 30 Ala Pro
Asp Lys Ala Ile Thr Gln Glu Gln Cys Glu Ala Arg Gly Cys 35 40 45
Cys Tyr Ile Pro Ala Lys Gln Gly Leu Gln Gly Ala Gln Met Gly Gln 50
55 60 Pro Trp Cys Phe Phe Pro Pro Ser Tyr Pro Ser Tyr Lys Leu Glu
Asn 65 70 75 80 Leu Ser Ser Ser Glu Met Gly Tyr Thr Ala Thr Leu Thr
Arg Thr Thr 85 90 95 Pro Thr Phe Phe Pro Lys Asp Ile Leu Thr Leu
Arg Leu Asp Val Met 100 105 110 Met Glu Thr Glu Asn Arg Leu His Phe
Thr Ile Lys Asp Pro Ala Asn 115 120 125 Arg Arg Tyr Glu Val Pro Leu
Glu Thr Pro His Val His Ser Arg Ala 130 135 140 Pro Ser Pro Leu Tyr
Ser Val Glu Phe Ser Glu Glu Pro Phe Gly Val 145 150 155 160 Ile Val
Arg Arg Gln Leu Asp Gly Arg Val Leu Leu Asn Thr Thr Val 165 170 175
Ala Pro Leu Phe Phe Ala Asp Gln Phe Leu Gln Leu Ser Thr Ser Leu 180
185 190 Pro Ser Gln Tyr Ile Thr Gly Leu Ala Glu His Leu Ser Pro Leu
Met 195 200 205 Leu Ser Thr Ser Trp Thr Arg Ile Thr Leu Trp Asn Arg
Asp Leu Ala 210 215 220 Pro Thr Pro Gly Ala Asn Leu Tyr Gly Ser His
Pro Phe Tyr Leu Ala 225 230 235 240 Leu Glu Asp Gly Gly Ser Ala His
Gly Val Phe Leu Leu Asn Ser Asn 245 250 255 Ala Met Asp Val Val Leu
Gln Pro Ser Pro Ala Leu Ser Trp Arg Ser 260 265 270 Thr Gly Gly Ile
Leu Asp Val Tyr Ile Phe Leu Gly Pro Glu Pro Lys 275 280 285 Ser Val
Val Gln Gln Tyr Leu Asp Val Val Gly Tyr Pro Phe Met Pro 290 295 300
Pro Tyr Trp Gly Leu Gly Phe His Leu Cys Arg Trp Gly Tyr Ser Ser 305
310 315 320 Thr Ala Ile Thr Arg Gln Val Val Glu Asn Met Thr Arg Ala
His Phe 325 330 335 Pro Leu Asp Val Gln Trp Asn Asp Leu Asp Tyr Met
Asp Ser Arg Arg 340 345 350 Asp Phe Thr Phe Asn Lys Asp Gly Phe Arg
Asp Phe Pro Ala Met Val 355 360 365 Gln Glu Leu His Gln Gly Gly Arg
Arg Tyr Met Met Ile Val Asp Pro 370 375 380 Ala Ile Ser Ser Ser Gly
Pro Ala Gly Ser Tyr Arg Pro Tyr Asp Glu 385 390 395 400 Gly Leu Arg
Arg Gly Val Phe Ile Thr Asn Glu Thr Gly Gln Pro Leu 405 410 415 Ile
Gly Lys Val Trp Pro Gly Ser Thr Ala Phe Pro Asp Phe Thr Asn 420 425
430 Pro Thr Ala Leu Ala Trp Trp Glu Asp Met Val Ala Glu Phe His Asp
435 440 445 Gln Val Pro Phe Asp Gly Met Trp Ile Asp Met Asn Glu Pro
Ser Asn 450 455 460 Phe Ile Arg Gly Ser Glu Asp Gly Cys Pro Asn Asn
Glu Leu Glu Asn 465 470 475 480 Pro Pro Tyr Val Pro Gly Val Val Gly
Gly Thr Leu Gln Ala Ala Thr 485 490 495 Ile Cys Ala Ser Ser His Gln
Phe Leu Ser Thr His Tyr Asn Leu His 500 505 510 Asn Leu Tyr Gly Leu
Thr Glu Ala Ile Ala Ser His Arg Ala Leu Val 515 520 525 Lys Ala Arg
Gly Thr Arg Pro Phe Val Ile Ser Arg Ser Thr Phe Ala 530 535 540 Gly
His Gly Arg Tyr Ala Gly His Trp Thr Gly Asp Val Trp Ser Ser 545 550
555 560 Trp Glu Gln Leu Ala Ser Ser Val Pro Glu Ile Leu Gln Phe Asn
Leu 565 570 575 Leu Gly Val Pro Leu Val Gly Ala Asp Val Cys Gly Phe
Leu Gly Asn 580 585 590 Thr Ser Glu Glu Leu Cys Val Arg Trp Thr Gln
Leu Gly Ala Phe Tyr 595 600 605 Pro Phe Met Arg Asn His Asn Ser Leu
Leu Ser Leu Pro Gln Glu Pro 610 615 620 Tyr Ser Phe Ser Glu Pro Ala
Gln Gln Ala Met Arg Lys Ala Leu Thr 625 630 635 640 Leu Arg Tyr Ala
Leu Leu Pro His Leu Tyr Thr Leu Phe His Gln Ala 645 650 655 His Val
Ala Gly Glu Thr Val Ala Arg Pro Leu Phe Leu Glu Phe Pro 660 665 670
Lys Asp Ser Ser Thr Trp Thr Val Asp His Gln Leu Leu Trp Gly Glu 675
680 685 Ala Leu Leu Ile Thr Pro Val Leu Gln Ala Gly Lys Ala Glu Val
Thr 690 695 700 Gly Tyr Phe Pro Leu Gly Thr Trp Tyr Asp Leu Gln Thr
Val Pro Val 705 710 715 720 Glu Ala Leu Gly Ser Leu Pro Pro Pro Pro
Ala Ala Pro Arg Glu Pro 725 730 735 Ala Ile His Ser Glu Gly Gln Trp
Val Thr Leu Pro Ala Pro Leu Asp 740 745 750 Thr Ile Asn Val His Leu
Arg Ala Gly Tyr Ile Ile Pro Leu Gln Gly 755 760 765 Pro Gly Leu Thr
Thr Thr Glu Ser Arg Gln Gln Pro Met Ala Leu Ala 770 775 780 Val Ala
Leu Thr Lys Gly Gly Glu Ala Arg Gly Glu Leu Phe Trp Asp 785 790 795
800 Asp Gly Glu Ser Leu Glu Val Leu Glu Arg Gly Ala Tyr Thr Gln Val
805 810 815 Ile Phe Leu Ala Arg Asn Asn Thr Ile Val Asn Glu Leu Val
Arg Val 820 825 830 Thr Ser Glu Gly Ala Gly Leu Gln Leu Gln Lys Val
Thr Val Leu Gly 835 840 845 Val Ala Thr Ala Pro Gln Gln Val Leu Ser
Asn Gly Val Pro Val Ser 850 855 860 Asn Phe Thr Tyr Ser Pro Asp Thr
Lys Val Leu Asp Ile Cys Val Ser 865 870 875 880 Leu Leu Met Gly Glu
Gln Phe Leu Val Ser Trp Cys 885 890 22886PRTArtificial
SequenceDescription of Artificial Sequence Synthetic polypeptide
22Asp Ala Gln Ala His Pro Gly Arg Pro Arg Ala Val Pro Thr Gln Cys 1
5 10 15 Asp Val Pro Pro Asn Ser Arg Phe Asp Cys Ala Pro Asp Lys Ala
Ile 20 25 30 Thr Gln Glu Gln Cys Glu Ala Arg Gly Cys Cys Tyr Ile
Pro Ala Lys 35 40 45 Gln Gly Leu Gln Gly Ala Gln Met Gly Gln Pro
Trp Cys Phe Phe Pro 50 55 60 Pro Ser Tyr Pro Ser Tyr Lys Leu Glu
Asn Leu Ser Ser Ser Glu Met 65 70 75 80 Gly Tyr Thr Ala Thr Leu Thr
Arg Thr Thr Pro Thr Phe Phe Pro Lys 85 90 95 Asp Ile Leu Thr Leu
Arg Leu Asp Val Met Met Glu Thr Glu Asn Arg 100 105 110 Leu His Phe
Thr Ile Lys Asp Pro Ala Asn Arg Arg Tyr Glu Val Pro 115 120 125 Leu
Glu Thr Pro His Val His Ser Arg Ala Pro Ser Pro Leu Tyr Ser 130 135
140 Val Glu Phe Ser Glu Glu Pro Phe Gly Val Ile Val Arg Arg Gln Leu
145 150 155 160 Asp Gly Arg Val Leu Leu Asn Thr Thr Val Ala Pro Leu
Phe Phe Ala 165 170 175 Asp Gln Phe Leu Gln Leu Ser Thr Ser Leu Pro
Ser Gln Tyr Ile Thr 180 185 190 Gly Leu Ala Glu His Leu Ser Pro Leu
Met Leu Ser Thr Ser Trp Thr 195 200 205 Arg Ile Thr Leu Trp Asn Arg
Asp Leu Ala Pro Thr Pro Gly Ala Asn 210 215 220 Leu Tyr Gly Ser His
Pro Phe Tyr Leu Ala Leu Glu Asp Gly Gly Ser 225 230 235 240 Ala His
Gly Val Phe Leu Leu Asn Ser Asn Ala Met Asp Val Val Leu 245 250 255
Gln Pro Ser Pro Ala Leu Ser Trp Arg Ser Thr Gly Gly Ile Leu Asp 260
265 270 Val Tyr Ile Phe Leu Gly Pro Glu Pro Lys Ser Val Val Gln Gln
Tyr 275 280 285 Leu Asp Val Val Gly Tyr Pro Phe Met Pro Pro Tyr Trp
Gly Leu Gly 290 295 300 Phe His Leu Cys Arg Trp Gly Tyr Ser Ser Thr
Ala Ile Thr Arg Gln 305 310 315 320 Val Val Glu Asn Met Thr Arg Ala
His Phe Pro Leu Asp Val Gln Trp 325 330 335 Asn Asp Leu Asp Tyr Met
Asp Ser Arg Arg Asp Phe Thr Phe Asn Lys 340 345 350 Asp Gly Phe Arg
Asp Phe Pro Ala Met Val Gln Glu Leu His Gln Gly 355 360 365 Gly Arg
Arg Tyr Met Met Ile Val Asp Pro Ala Ile Ser Ser Ser Gly 370 375 380
Pro Ala Gly Ser Tyr Arg Pro Tyr Asp Glu Gly Leu Arg Arg Gly Val 385
390 395 400 Phe Ile Thr Asn Glu Thr Gly Gln Pro Leu Ile Gly Lys Val
Trp Pro 405 410 415 Gly Ser Thr Ala Phe Pro Asp Phe Thr Asn Pro Thr
Ala Leu Ala Trp 420 425 430 Trp Glu Asp Met Val Ala Glu Phe His Asp
Gln Val Pro Phe Asp Gly 435 440 445 Met Trp Ile Asp Met Asn Glu Pro
Ser Asn Phe Ile Arg Gly Ser Glu 450 455 460 Asp Gly Cys Pro Asn Asn
Glu Leu Glu Asn Pro Pro Tyr Val Pro Gly 465 470 475 480 Val Val Gly
Gly Thr Leu Gln Ala Ala Thr Ile Cys Ala Ser Ser His 485 490 495 Gln
Phe Leu Ser Thr His Tyr Asn Leu His Asn Leu Tyr Gly Leu Thr 500 505
510 Glu Ala Ile Ala Ser His Arg Ala Leu Val Lys Ala Arg Gly Thr Arg
515 520 525 Pro Phe Val Ile Ser Arg Ser Thr Phe Ala Gly His Gly Arg
Tyr Ala 530 535 540 Gly His Trp Thr Gly Asp Val Trp Ser Ser Trp Glu
Gln Leu Ala Ser 545 550 555 560 Ser Val Pro Glu Ile Leu Gln Phe Asn
Leu Leu Gly Val Pro Leu Val 565 570 575 Gly Ala Asp Val Cys Gly Phe
Leu Gly Asn Thr Ser Glu Glu Leu Cys 580 585 590 Val Arg Trp Thr Gln
Leu Gly Ala Phe Tyr Pro Phe Met Arg Asn His 595 600 605 Asn Ser Leu
Leu Ser Leu Pro Gln Glu Pro Tyr Ser Phe Ser Glu Pro 610 615 620 Ala
Gln Gln Ala Met Arg Lys Ala Leu Thr Leu Arg Tyr Ala Leu Leu 625 630
635 640 Pro His Leu Tyr Thr Leu Phe His Gln Ala His Val Ala Gly Glu
Thr 645 650 655 Val Ala Arg Pro Leu Phe Leu Glu Phe Pro Lys Asp Ser
Ser Thr Trp 660 665 670 Thr Val Asp His Gln Leu Leu Trp Gly Glu Ala
Leu Leu Ile Thr Pro 675 680 685 Val Leu Gln Ala Gly Lys Ala Glu Val
Thr Gly Tyr Phe Pro Leu Gly 690 695 700 Thr Trp Tyr Asp
Leu Gln Thr Val Pro Val Glu Ala Leu Gly Ser Leu 705 710 715 720 Pro
Pro Pro Pro Ala Ala Pro Arg Glu Pro Ala Ile His Ser Glu Gly 725 730
735 Gln Trp Val Thr Leu Pro Ala Pro Leu Asp Thr Ile Asn Val His Leu
740 745 750 Arg Ala Gly Tyr Ile Ile Pro Leu Gln Gly Pro Gly Leu Thr
Thr Thr 755 760 765 Glu Ser Arg Gln Gln Pro Met Ala Leu Ala Val Ala
Leu Thr Lys Gly 770 775 780 Gly Glu Ala Arg Gly Glu Leu Phe Trp Asp
Asp Gly Glu Ser Leu Glu 785 790 795 800 Val Leu Glu Arg Gly Ala Tyr
Thr Gln Val Ile Phe Leu Ala Arg Asn 805 810 815 Asn Thr Ile Val Asn
Glu Leu Val Arg Val Thr Ser Glu Gly Ala Gly 820 825 830 Leu Gln Leu
Gln Lys Val Thr Val Leu Gly Val Ala Thr Ala Pro Gln 835 840 845 Gln
Val Leu Ser Asn Gly Val Pro Val Ser Asn Phe Thr Tyr Ser Pro 850 855
860 Asp Thr Lys Val Leu Asp Ile Cys Val Ser Leu Leu Met Gly Glu Gln
865 870 875 880 Phe Leu Val Ser Trp Cys 885 23883PRTArtificial
SequenceDescription of Artificial Sequence Synthetic polypeptide
23Ala His Pro Gly Arg Pro Arg Ala Val Pro Thr Gln Cys Asp Val Pro 1
5 10 15 Pro Asn Ser Arg Phe Asp Cys Ala Pro Asp Lys Ala Ile Thr Gln
Glu 20 25 30 Gln Cys Glu Ala Arg Gly Cys Cys Tyr Ile Pro Ala Lys
Gln Gly Leu 35 40 45 Gln Gly Ala Gln Met Gly Gln Pro Trp Cys Phe
Phe Pro Pro Ser Tyr 50 55 60 Pro Ser Tyr Lys Leu Glu Asn Leu Ser
Ser Ser Glu Met Gly Tyr Thr 65 70 75 80 Ala Thr Leu Thr Arg Thr Thr
Pro Thr Phe Phe Pro Lys Asp Ile Leu 85 90 95 Thr Leu Arg Leu Asp
Val Met Met Glu Thr Glu Asn Arg Leu His Phe 100 105 110 Thr Ile Lys
Asp Pro Ala Asn Arg Arg Tyr Glu Val Pro Leu Glu Thr 115 120 125 Pro
His Val His Ser Arg Ala Pro Ser Pro Leu Tyr Ser Val Glu Phe 130 135
140 Ser Glu Glu Pro Phe Gly Val Ile Val Arg Arg Gln Leu Asp Gly Arg
145 150 155 160 Val Leu Leu Asn Thr Thr Val Ala Pro Leu Phe Phe Ala
Asp Gln Phe 165 170 175 Leu Gln Leu Ser Thr Ser Leu Pro Ser Gln Tyr
Ile Thr Gly Leu Ala 180 185 190 Glu His Leu Ser Pro Leu Met Leu Ser
Thr Ser Trp Thr Arg Ile Thr 195 200 205 Leu Trp Asn Arg Asp Leu Ala
Pro Thr Pro Gly Ala Asn Leu Tyr Gly 210 215 220 Ser His Pro Phe Tyr
Leu Ala Leu Glu Asp Gly Gly Ser Ala His Gly 225 230 235 240 Val Phe
Leu Leu Asn Ser Asn Ala Met Asp Val Val Leu Gln Pro Ser 245 250 255
Pro Ala Leu Ser Trp Arg Ser Thr Gly Gly Ile Leu Asp Val Tyr Ile 260
265 270 Phe Leu Gly Pro Glu Pro Lys Ser Val Val Gln Gln Tyr Leu Asp
Val 275 280 285 Val Gly Tyr Pro Phe Met Pro Pro Tyr Trp Gly Leu Gly
Phe His Leu 290 295 300 Cys Arg Trp Gly Tyr Ser Ser Thr Ala Ile Thr
Arg Gln Val Val Glu 305 310 315 320 Asn Met Thr Arg Ala His Phe Pro
Leu Asp Val Gln Trp Asn Asp Leu 325 330 335 Asp Tyr Met Asp Ser Arg
Arg Asp Phe Thr Phe Asn Lys Asp Gly Phe 340 345 350 Arg Asp Phe Pro
Ala Met Val Gln Glu Leu His Gln Gly Gly Arg Arg 355 360 365 Tyr Met
Met Ile Val Asp Pro Ala Ile Ser Ser Ser Gly Pro Ala Gly 370 375 380
Ser Tyr Arg Pro Tyr Asp Glu Gly Leu Arg Arg Gly Val Phe Ile Thr 385
390 395 400 Asn Glu Thr Gly Gln Pro Leu Ile Gly Lys Val Trp Pro Gly
Ser Thr 405 410 415 Ala Phe Pro Asp Phe Thr Asn Pro Thr Ala Leu Ala
Trp Trp Glu Asp 420 425 430 Met Val Ala Glu Phe His Asp Gln Val Pro
Phe Asp Gly Met Trp Ile 435 440 445 Asp Met Asn Glu Pro Ser Asn Phe
Ile Arg Gly Ser Glu Asp Gly Cys 450 455 460 Pro Asn Asn Glu Leu Glu
Asn Pro Pro Tyr Val Pro Gly Val Val Gly 465 470 475 480 Gly Thr Leu
Gln Ala Ala Thr Ile Cys Ala Ser Ser His Gln Phe Leu 485 490 495 Ser
Thr His Tyr Asn Leu His Asn Leu Tyr Gly Leu Thr Glu Ala Ile 500 505
510 Ala Ser His Arg Ala Leu Val Lys Ala Arg Gly Thr Arg Pro Phe Val
515 520 525 Ile Ser Arg Ser Thr Phe Ala Gly His Gly Arg Tyr Ala Gly
His Trp 530 535 540 Thr Gly Asp Val Trp Ser Ser Trp Glu Gln Leu Ala
Ser Ser Val Pro 545 550 555 560 Glu Ile Leu Gln Phe Asn Leu Leu Gly
Val Pro Leu Val Gly Ala Asp 565 570 575 Val Cys Gly Phe Leu Gly Asn
Thr Ser Glu Glu Leu Cys Val Arg Trp 580 585 590 Thr Gln Leu Gly Ala
Phe Tyr Pro Phe Met Arg Asn His Asn Ser Leu 595 600 605 Leu Ser Leu
Pro Gln Glu Pro Tyr Ser Phe Ser Glu Pro Ala Gln Gln 610 615 620 Ala
Met Arg Lys Ala Leu Thr Leu Arg Tyr Ala Leu Leu Pro His Leu 625 630
635 640 Tyr Thr Leu Phe His Gln Ala His Val Ala Gly Glu Thr Val Ala
Arg 645 650 655 Pro Leu Phe Leu Glu Phe Pro Lys Asp Ser Ser Thr Trp
Thr Val Asp 660 665 670 His Gln Leu Leu Trp Gly Glu Ala Leu Leu Ile
Thr Pro Val Leu Gln 675 680 685 Ala Gly Lys Ala Glu Val Thr Gly Tyr
Phe Pro Leu Gly Thr Trp Tyr 690 695 700 Asp Leu Gln Thr Val Pro Val
Glu Ala Leu Gly Ser Leu Pro Pro Pro 705 710 715 720 Pro Ala Ala Pro
Arg Glu Pro Ala Ile His Ser Glu Gly Gln Trp Val 725 730 735 Thr Leu
Pro Ala Pro Leu Asp Thr Ile Asn Val His Leu Arg Ala Gly 740 745 750
Tyr Ile Ile Pro Leu Gln Gly Pro Gly Leu Thr Thr Thr Glu Ser Arg 755
760 765 Gln Gln Pro Met Ala Leu Ala Val Ala Leu Thr Lys Gly Gly Glu
Ala 770 775 780 Arg Gly Glu Leu Phe Trp Asp Asp Gly Glu Ser Leu Glu
Val Leu Glu 785 790 795 800 Arg Gly Ala Tyr Thr Gln Val Ile Phe Leu
Ala Arg Asn Asn Thr Ile 805 810 815 Val Asn Glu Leu Val Arg Val Thr
Ser Glu Gly Ala Gly Leu Gln Leu 820 825 830 Gln Lys Val Thr Val Leu
Gly Val Ala Thr Ala Pro Gln Gln Val Leu 835 840 845 Ser Asn Gly Val
Pro Val Ser Asn Phe Thr Tyr Ser Pro Asp Thr Lys 850 855 860 Val Leu
Asp Ile Cys Val Ser Leu Leu Met Gly Glu Gln Phe Leu Val 865 870 875
880 Ser Trp Cys 248PRTArtificial SequenceDescription of Artificial
Sequence Synthetic peptide 24Gly Phe Thr Phe Ser Asn Tyr Gly 1 5
258PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 25Ile Ser Ser Gly Ser Ser Thr Ile 1 5
269PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 26Ala Arg Arg Gly Leu Leu Leu Asp Tyr 1 5
2710PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 27Lys Ser Val Ser Thr Ser Ser Tyr Ser Tyr 1 5 10
283PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 28Tyr Ala Ser 1 299PRTArtificial
SequenceDescription of Artificial Sequence Synthetic peptide 29Gln
His Ser Arg Glu Phe Pro Trp Thr 1 5 3013PRTArtificial
SequenceDescription of Artificial Sequence Synthetic peptide 30Gly
Gly Ser Gly Gly Gly Ser Gly Gly Gly Ser Gly Gly 1 5 10
3128PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 31His Ile Leu Leu His Asp Phe Leu Leu Val Pro Arg
Glu Leu Ser Gly 1 5 10 15 Ser Ser Pro Val Leu Glu Glu Thr His Pro
Ala His 20 25 32937PRTBos taurus 32Met Met Arg Trp Pro Pro Cys Ser
Arg Pro Leu Leu Gly Val Cys Thr 1 5 10 15 Leu Leu Ser Leu Ala Leu
Leu Gly His Ile Leu Leu His Asp Leu Glu 20 25 30 Val Val Pro Arg
Glu Leu Arg Gly Phe Ser Gln Asp Glu Ile His Gln 35 40 45 Ala Cys
Gln Pro Gly Ala Ser Ser Pro Glu Cys Arg Gly Ser Pro Arg 50 55 60
Ala Ala Pro Thr Gln Cys Asp Leu Pro Pro Asn Ser Arg Phe Asp Cys 65
70 75 80 Ala Pro Asp Lys Gly Ile Thr Pro Gln Gln Cys Glu Ala Arg
Gly Cys 85 90 95 Cys Tyr Met Pro Ala Glu Trp Pro Pro Asp Ala Gln
Met Gly Gln Pro 100 105 110 Trp Cys Phe Phe Pro Pro Ser Tyr Pro Ser
Tyr Arg Leu Glu Asn Leu 115 120 125 Thr Thr Thr Glu Thr Gly Tyr Thr
Ala Thr Leu Thr Arg Ala Val Pro 130 135 140 Thr Phe Phe Pro Lys Asp
Ile Met Thr Leu Arg Leu Asp Met Leu Met 145 150 155 160 Glu Thr Glu
Ser Arg Leu His Phe Thr Ile Lys Asp Pro Ala Asn Arg 165 170 175 Arg
Tyr Glu Val Pro Leu Glu Thr Pro Arg Val Tyr Ser Gln Ala Pro 180 185
190 Phe Thr Leu Tyr Ser Val Glu Phe Ser Glu Glu Pro Phe Gly Val Val
195 200 205 Val Arg Arg Lys Leu Asp Gly Arg Val Leu Leu Asn Thr Thr
Val Ala 210 215 220 Pro Leu Phe Phe Ala Asp Gln Phe Leu Gln Leu Ser
Thr Ser Leu Pro 225 230 235 240 Ser Gln His Ile Thr Gly Leu Ala Glu
His Leu Gly Ser Leu Met Leu 245 250 255 Ser Thr Asn Trp Thr Lys Ile
Thr Leu Trp Asn Arg Asp Ile Ala Pro 260 265 270 Glu Pro Asn Val Asn
Leu Tyr Gly Ser His Pro Phe Tyr Leu Val Leu 275 280 285 Glu Asp Gly
Gly Leu Ala His Gly Val Phe Leu Leu Asn Ser Asn Ala 290 295 300 Met
Asp Val Val Leu Gln Pro Ser Pro Ala Leu Ser Trp Arg Ser Thr 305 310
315 320 Gly Gly Ile Leu Asp Val Tyr Ile Phe Leu Gly Pro Glu Pro Lys
Ser 325 330 335 Val Val Gln Gln Tyr Leu Asp Val Val Gly Tyr Pro Phe
Met Pro Pro 340 345 350 Tyr Trp Gly Leu Gly Phe His Leu Cys Arg Trp
Gly Tyr Ser Thr Ser 355 360 365 Ala Ile Thr Arg Gln Val Val Glu Asn
Met Thr Arg Ala Tyr Phe Pro 370 375 380 Leu Asp Val Gln Trp Asn Asp
Leu Asp Tyr Met Asp Ala Arg Arg Asp 385 390 395 400 Phe Thr Phe Asn
Lys Asp His Phe Gly Asp Phe Pro Ala Met Val Gln 405 410 415 Glu Leu
His Gln Gly Gly Arg Arg Tyr Ile Met Ile Val Asp Pro Ala 420 425 430
Ile Ser Ser Ser Gly Pro Ala Gly Thr Tyr Arg Pro Tyr Asp Glu Gly 435
440 445 Leu Arg Arg Gly Val Phe Ile Thr Asn Glu Thr Gly Gln Pro Leu
Ile 450 455 460 Gly Gln Val Trp Pro Gly Leu Thr Ala Phe Pro Asp Phe
Thr Asn Pro 465 470 475 480 Glu Thr Leu Asp Trp Trp Gln Asp Met Val
Thr Glu Phe His Ala Gln 485 490 495 Val Pro Phe Asp Gly Met Trp Ile
Asp Met Asn Glu Pro Ser Asn Phe 500 505 510 Val Arg Gly Ser Val Asp
Gly Cys Pro Asp Asn Ser Leu Glu Asn Pro 515 520 525 Pro Tyr Leu Pro
Gly Val Val Gly Gly Thr Leu Arg Ala Ala Thr Ile 530 535 540 Cys Ala
Ser Ser His Gln Phe Leu Ser Thr His Tyr Asp Leu His Asn 545 550 555
560 Leu Tyr Gly Leu Thr Glu Ala Leu Ala Ser His Arg Ala Leu Val Lys
565 570 575 Ala Arg Gly Met Arg Pro Phe Val Ile Ser Arg Ser Thr Phe
Ala Gly 580 585 590 His Gly Arg Tyr Ser Gly His Trp Thr Gly Asp Val
Trp Ser Asn Trp 595 600 605 Glu Gln Leu Ser Tyr Ser Val Pro Glu Ile
Leu Leu Phe Asn Leu Leu 610 615 620 Gly Val Pro Leu Val Gly Ala Asp
Ile Cys Gly Phe Leu Gly Asn Thr 625 630 635 640 Ser Glu Glu Leu Cys
Val Arg Trp Thr Gln Leu Gly Ala Phe Tyr Pro 645 650 655 Phe Met Arg
Asn His Asn Ala Leu Asn Ser Gln Pro Gln Glu Pro Tyr 660 665 670 Arg
Phe Ser Glu Thr Ala Gln Gln Ala Met Arg Lys Ala Phe Thr Leu 675 680
685 Arg Tyr Val Leu Leu Pro Tyr Leu Tyr Thr Leu Phe His Arg Ala His
690 695 700 Val Arg Gly Glu Thr Val Ala Arg Pro Leu Phe Leu Glu Phe
Pro Glu 705 710 715 720 Asp Pro Ser Thr Trp Thr Val Asp Arg Gln Leu
Leu Trp Gly Glu Ala 725 730 735 Leu Leu Ile Thr Pro Val Leu Glu Ala
Glu Lys Val Glu Val Thr Gly 740 745 750 Tyr Phe Pro Gln Gly Thr Trp
Tyr Asp Leu Gln Thr Val Pro Met Glu 755 760 765 Ala Phe Gly Ser Leu
Pro Pro Pro Ala Pro Leu Thr Ser Val Ile His 770 775 780 Ser Lys Gly
Gln Trp Val Thr Leu Ser Ala Pro Leu Asp Thr Ile Asn 785 790 795 800
Val His Leu Arg Ala Gly His Ile Ile Pro Met Gln Gly Pro Ala Leu 805
810 815 Thr Thr Thr Glu Ser Arg Lys Gln His Met Ala Leu Ala Val Ala
Leu 820 825 830 Thr Ala Ser Gly Glu Ala Gln Gly Glu Leu Phe Trp Asp
Asp Gly Glu 835 840 845 Ser Leu Gly Val Leu Asp Gly Gly Asp Tyr Thr
Gln Leu Ile Phe Leu 850 855 860 Ala Lys Asn Asn Thr Phe Val Asn Lys
Leu Val His Val Ser Ser Glu 865 870 875 880 Gly Ala Ser Leu Gln Leu
Arg Asn Val Thr Val Leu Gly Val Ala Thr 885 890 895 Ala Pro Gln Gln
Val Leu Cys Asn Ser Val Pro Val Ser Asn Phe Thr 900 905 910 Phe Ser
Pro Asp Thr Glu Thr Leu Ala Ile Pro Val Ser Leu Thr Met 915 920 925
Gly Glu Gln Phe Val Ile Ser Trp Ser 930 935 335PRTArtificial
SequenceDescription of Artificial Sequence Synthetic peptide 33Lys
Phe Glu Arg Gln 1 5 3450PRTArtificial SequenceDescription of
Artificial Sequence Synthetic polypeptidemisc_feature(1)..(50)This
sequence may encompass 1-10 repeating "Gly Gly Gly Gly Ser"
repeating units 34Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly
Gly Gly Ser Gly 1 5 10 15 Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly
Gly Gly Gly Ser Gly Gly 20 25 30 Gly Gly Ser Gly Gly Gly Gly Ser
Gly Gly Gly Gly Ser Gly Gly Gly 35 40 45 Gly Ser 50
357PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 35Ala Ser Ser Leu Asn Ile Ala 1 5
367PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 36Arg Arg Arg Arg Arg Arg Arg 1 5
* * * * *
References