U.S. patent application number 16/893265 was filed with the patent office on 2020-09-24 for methods, compounds, and compositions for treatment and prophylaxis in the respiratory tract.
This patent application is currently assigned to Ansun Biopharma, Inc.. The applicant listed for this patent is Ansun Biopharma, Inc.. Invention is credited to Fang Fang, Michael P. Malakhov, Ron Moss, David Wurtman.
Application Number | 20200297822 16/893265 |
Document ID | / |
Family ID | 1000004870474 |
Filed Date | 2020-09-24 |
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United States Patent
Application |
20200297822 |
Kind Code |
A1 |
Fang; Fang ; et al. |
September 24, 2020 |
Methods, Compounds, and Compositions For Treatment and Prophylaxis
in the Respiratory Tract
Abstract
The present invention provides a method of reducing the quantity
of mucus in the respiratory tract of a subject with elevated levels
of mucus in said respiratory tract. The method includes
administering to the subject a compound or composition containing a
therapeutically effective amount of a fusion protein comprising a
sialidase or an active portion thereof and an anchoring domain. The
therapeutically effective amount comprises an amount of the fusion
protein that results in a reduction of the quantity of mucus in the
respiratory tract after administration of the compound or
composition when compared to the quantity of mucus present prior to
administration of the compound or composition.
Inventors: |
Fang; Fang; (Rancho Santa
Fe, CA) ; Wurtman; David; (San Diego, CA) ;
Moss; Ron; (Encinitas, CA) ; Malakhov; Michael
P.; (San Francisco, CA) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
Ansun Biopharma, Inc. |
San Diego |
CA |
US |
|
|
Assignee: |
Ansun Biopharma, Inc.
San Diego
CA
|
Family ID: |
1000004870474 |
Appl. No.: |
16/893265 |
Filed: |
June 4, 2020 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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15865217 |
Jan 8, 2018 |
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16893265 |
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15136751 |
Apr 22, 2016 |
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15865217 |
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14489428 |
Sep 17, 2014 |
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15136751 |
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13769095 |
Feb 15, 2013 |
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14489428 |
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12940742 |
Nov 5, 2010 |
8398971 |
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13769095 |
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61259033 |
Nov 6, 2009 |
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61259055 |
Nov 6, 2009 |
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61322813 |
Apr 9, 2010 |
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61332063 |
May 6, 2010 |
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61381420 |
Sep 9, 2010 |
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Current U.S.
Class: |
1/1 |
Current CPC
Class: |
A61K 49/0008 20130101;
C07K 14/485 20130101; A61K 33/14 20130101; C12N 9/2477 20130101;
A61K 9/06 20130101; A61K 31/198 20130101; A61K 9/0014 20130101;
A61K 45/06 20130101; A61K 38/1808 20130101; A61K 31/05 20130101;
C12Y 302/01018 20130101; A61K 38/47 20130101; A61K 9/0073 20130101;
G01N 2800/122 20130101; A61K 9/0043 20130101; G01N 2500/00
20130101; A61K 47/26 20130101; A61K 31/4172 20130101; A61K 31/7016
20130101; A61K 33/06 20130101; A61K 47/32 20130101; C07K 2319/00
20130101; A61K 9/0019 20130101; A61K 47/22 20130101; C12Q 1/34
20130101; A61K 47/34 20130101; A61K 9/0053 20130101; A61K 47/02
20130101; C12N 9/2402 20130101 |
International
Class: |
A61K 38/47 20060101
A61K038/47; A61K 9/00 20060101 A61K009/00; A61K 9/06 20060101
A61K009/06; A61K 31/05 20060101 A61K031/05; A61K 31/198 20060101
A61K031/198; A61K 33/06 20060101 A61K033/06; A61K 33/14 20060101
A61K033/14; A61K 47/32 20060101 A61K047/32; A61K 47/34 20060101
A61K047/34; C12N 9/24 20060101 C12N009/24; A61K 31/4172 20060101
A61K031/4172; A61K 31/7016 20060101 A61K031/7016; A61K 45/06
20060101 A61K045/06; C12Q 1/34 20060101 C12Q001/34; A61K 38/18
20060101 A61K038/18; C07K 14/485 20060101 C07K014/485; A61K 47/02
20060101 A61K047/02; A61K 47/22 20060101 A61K047/22; A61K 47/26
20060101 A61K047/26; A61K 49/00 20060101 A61K049/00 |
Goverment Interests
FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT
[0002] This invention was made with Government support under
contract number HHSN266200600015C awarded by the United States
Department of Health and Human Services, National Institutes of
Health. The Government has certain rights in the invention.
Claims
1. A method of reducing the quantity or level of mucus or
preventing an increase in the quantity or level of mucus in a
respiratory tract of a subject, the method comprising:
administering to the subject a compound or composition comprising a
therapeutically effective amount of a fusion protein, wherein the
fusion protein comprises at least one catalytic domain of a
sialidase, wherein the catalytic domain of the sialidase comprises
the sequence of amino acids extending from amino acid 274 to amino
acid 666 of SEQ ID NO:12, inclusive, and at least one anchoring
domain, wherein the anchoring domain is a glycosaminoglycan (GAG)
binding domain of human amphiregulin comprising the amino acid
sequence of SEQ ID NO:7; and the therapeutically effective amount
comprises an amount of the fusion protein that results in a
reduction of the quantity of mucus in the respiratory tract after
administration of the compound or composition when compared to the
quantity of mucus present prior to administration of the compound
or composition.
Description
CLAIM OF PRIORITY
[0001] This application claims the benefit of U.S. patent
application Ser. No. 15/865,217, filed on Jan. 8, 2018, which
claims the benefit of U.S. patent application Ser. No. 15/136,751,
filed on Apr. 22, 2016, which claims the benefit of U.S. patent
application Ser. No. 14/489,428, filed on Sep. 17, 2014, which
claims the benefit of U.S. patent application Ser. No. 13/769,095,
filed on Feb. 15, 2013, which claims the benefit of U.S. patent
application Ser. No. 12/940,742, filed on Nov. 5, 2010, (U.S. Pat.
No. 8,398,971) which claims the benefit of U.S. Provisional Patent
Application Ser. No. 61/259,033, filed on Nov. 6, 2009, and U.S.
Provisional Patent Application Ser. No. 61/259,055, filed on Nov.
6, 2009, U.S. Provisional Patent Application Ser. No. 61/322,813,
filed on Apr. 9, 2010, U.S. Provisional Patent Application Ser. No.
61/332,063, filed on May 6, 2010, and U.S. Provisional Patent
Application Ser. No. 61/381,420 filed on Sep. 9, 2010, the entire
contents of each of which are hereby incorporated by reference.
BACKGROUND
[0003] Respiratory tract infections (RTIs) are among the most
common, and potentially most severe, types of infectious diseases.
Examples of RTIs include influenza, parainfluenza, RSV, sinusitis,
otitis, laryngitis, bronchitis and pneumonia.
[0004] One common feature of agents that cause RTIs, such as
respiratory pathogenic bacteria, is that they establish commensal
colonization on the mucosal surface of the upper airway; such
colonization precedes an infection and generally is prerequisite
for infections. Bacterial colonization in a neonate occurs shortly
after birth. During one's lifetime, the upper airway, specifically
the nasopharynx and oropharynx, remains a dynamic ecological
reservoir of microbial species with bacteria being acquired,
eliminated and re-acquired continually. In most cases, the
bacterial flora in the pharynx are harmless. However, when the
condition of the host is altered, some microorganisms may invade
adjacent tissues or bloodstream to cause diseases.
[0005] In addition to serving as the port of entry for mucosal and
invasive infections by both bacteria and viruses, the nasopharynx
and oropharynx are also the major source of spreading the
pathogenic microorganisms between individuals, as well as the
reservoir where antibiotic-resistant bacteria are selected
(Garcia-Rodriguez and Martinez, J Antimicrob Chemother, (2002)
50(Suppl S2), 59-73; Soriano and Rodriguez-Cerrato, J Antimicrob
Chemother, (2002) 50 Suppl S2, 51-58). It is well established
clinically that individuals who are prone to RTIs tend to be
persistent and recurrent carriers of pathogenic bacteria
(Garcia-Rodriguez and Martinez, J Antimicrob Chemother, (2002)
50(Suppl S2), 59-73; Mbaki et al., Tohoku J Exp. Med., (1987)
153(2), 111-121). For example, Helicobacter pylori is a human
pathogen implicated in gastritis and peptic ulcer. The bacterium
resides in the human stomach and binds to epithelial cells of the
gastric antrum.
[0006] Other disorders of the respiratory tract (more broadly
termed, RTDs) may not be caused by infectious agents, although they
could arise as a consequence of infection. Examples of RTDs include
a variety of obstructive lung diseases such as allergic and
non-allergic asthma, COPD, bronchiectasis, vasculitis, mucous
plugging, Wegener's granulomatosis and cystic fibrosis (CF). RTDs
can have a genetic basis (for example, CF), can arise due to
immunodeficiencies, can arise due to other deficiencies (for
example, alpha-1-antitrypsin deficiency can make people more
susceptible to bronchiectasis), can be caused by allergens and/or
chemical pollutants, or can present as complications of other
infectious diseases such as the RTIs described above or
inflammatory diseases such as inflammatory bowel syndrome or
Crohn's disease.
[0007] Common indications of RTIs and RTDs include inflammation and
elevated levels of mucous in the respiratory tract. However,
currently available drugs that are used to treat RTIs and RTDs
often are unable to ameliorate these associated conditions. For
example, Relenza.RTM. is a well-known treatment for influenza, but
it is not recommended for patients who suffer from underlying
airway disease, such as asthma and COPD. Thus, in addition to the
need for drugs that reduce inflammation and/or reduce mucus in the
respiratory tract or limit its increase are drugs that are capable
of treating respiratory infectious diseases, such as influenza,
parainfluenza and RSV, without aggravating underlying respiratory
conditions, such as asthma, bronchitis, bronchiectasis, and COPD,
of patients.
[0008] The present invention recognizes that drugs currently
available for medical use have limited efficacy with respect to
reducing inflammation, and/or reducing mucus in the respiratory
tract or limiting its increase in the respiratory tract, and those
that are available are associated with side effects. The present
invention also recognizes that there is a need for drugs for
treating respiratory infectious diseases in patients with
underlying airway disease, such as asthma, bronchitis,
bronchiectasis and COPD. Thus, there is a need for new drugs that
are able to reduce inflammation, and/or drugs that reduce mucus in
the respiratory tract or limit its increase in the respiratory
tract. There is also a need for drugs that can treat respiratory
infectious diseases while reducing inflammation, and/or while
reducing mucus in the respiratory tract or limiting its increase in
the respiratory tract.
SUMMARY
[0009] The compositions, components of compostions and methods
provided below are characterized by a variety of component
ingredients, steps of preparation, and biophysical, physical,
biochemical or chemical parameters. As would be apparent to those
of skill in the art, the compositions and methods provided herein
include any and all permutations and combinations of the
ingredients, steps and/or parameters described below.
[0010] The invention relates to the use of therapeutic compounds
and compositions that have anti-inflammatory effects in the
respiratory tract and to methods of treating respiratory
inflammation and prophylaxis against respiratory inflammation. The
invention also relates to therapeutic compounds and compositions
that can be used to prevent or treat diseases that are caused by,
cause, or are exacerbated by respiratory inflammation, including,
but not limited to, inflammation not caused by allergies or
allergic reactions.
[0011] The invention also relates to the use of therapeutic
compounds and compositions to reduce the quantity of mucus in the
respiratory tract of subjects with elevated levels of mucus in
their respiratory tracts, and to corresponding methods of
treatment. The invention also relates to the use of therapeutic
compounds and compositions to limit an increase in the quantity of
mucus in the respiratory tract of subjects above a baseline level
of mucus in their respiratory tract and to corresponding methods of
treatment. The invention also relates to therapeutic compounds and
compositions that can be used to prevent or treat conditions and/or
diseases that are caused by, cause, or are exacerbated by increased
mucus in the respiratory tract, such as, both allergic and
non-allergic asthma, chronic obstructive pulmonary disease (COPD),
bronchitis (both acute and non-acute), bronchiectasis, cystic
fibrosis (CF), vasculitis, mucus plugging, Wegener's
granulomatosis, pneumonia, tuberculosis, cancers involving the
lungs or the respiratory tract, Kartagener syndrome, Young's
syndrome, chronic sinopulmonary infections, alpha 1-antitrypsin
deficiency, primary immunodeficiencies, acquired immune deficiency
syndrome, opportunistic infections, infectious and post infectious
states, common cold, exercise-induced asthma, exercise induced
hypersecretion of mucus, inflammatory bowel disease, ulcerative
colitis, Crohn's disease, allergic reactions to inhaled fungus
spores, respiratory infections, respiratory obstructions,
inhalation or aspiration of ammonia and other toxic gases,
pulmonary aspiration, alcoholism, various allergies, and any other
disorder that causes increased mucus production in the respiratory
tract or is caused by or exacerbated by increased mucus production
in the respiratory tract. In some embodiments, the subject has more
than one of the aforementioned conditions and/or diseases. In other
embodiments, the subject having one or more of the aforementioned
conditions and/or diseases does not have an accompanying infectious
disease (RTI), such as influenza, parainfluenza or RSV. In other
embodiments, the subject having one or more of the aforementioned
conditions and/or diseases has one or more accompanying infectious
diseases, such as influenza, parainfluenza or RSV. Thus, provided
herein are methods, compounds and compositions for treating
inflammatory and/or allergic responses associated with an RTI, an
RTD, or combinations thereof.
[0012] The compounds and compositions provided herein can reduce
mucus production in the respiratory tract and/or reduce the levels
of inflammatory cells that cause allergic or non-allergic types of
inflammation, including, without limitation, monocytes,
macrophages, dendritic cells, histiocytes, Kuppfer cells,
mastocytes and neutrophiles. The compounds and compositions
provided herein include a sialidase or active portion thereof.
Without being bound by any theory, sialic acids have been
implicated in allergic and/or inflammatory responses associated
with RTIs and RTDs. For example, siglecs (sialic acid binding
Ig-like lectins) are members of the immunoglobulin (Ig) superfamily
that bind to sialic acid and are mainly expressed by cells of the
hematopoietic system. At least 11 siglecs have been discovered and
they seem to exclusively recognize cell surface sialic acid as the
ligand. It is believed that the binding of siglecs to sialic acid
mediates cell-cell adhesion and interactions (Crocker and Varki,
Trends Immunol., (2001) 22(6), 337-342; Angata and Brinkman-Van der
Linden, Biochim. Biophys. Acta, (2002) 1572(2-3), 294-316).
Siglec-8 (SAF-2) is an adhesion molecule that is highly restricted
to the surface of eosinophils, basophils, and mast cells, which are
the central effector cells in allergic conditions including
allergic rhinitis, asthma and eczema. Siglec-8 (homologous to
Siglec-F in mice) is considered to be responsible for mediating the
recruitment of the three allergic cell types to the airway, the
lungs and other sites of allergy. Siglec-1 (sialoadhesion) and
siglec-2 (CD22) are the adhesion molecules on macrophages and B
cells, both types of cells play central roles in immune reactions
that lead to inflammation. Siglec-9 is predominantly expressed on
neutrophils, which are known to be important effector cells in
inflammation (von Gunten, Yousefi, Seitz, Jakob, Schaffner, Seger,
Takala, Villiger, and Simon (2005) Blood 106:1423-1431). Further,
without being bound by any particular theory, sialic acid residues
have been implicated in the interaction of muscaranic receptors
with agonists; thus, sialidases can affect the interecation of
muscarinic receptors with their agonists.
[0013] The present invention provides a method of reducing the
quantity of mucus in the respiratory tract of a subject with
elevated levels of mucus in said respiratory tract. The method
includes administering to the subject a compound or composition
containing a therapeutically effective amount of a fusion protein
having a sialidase or an active portion thereof and an anchoring
domain. The therapeutically effective amount includes an amount of
the fusion protein that results in a reduction of the quantity of
mucus in the respiratory tract after administration of the compound
or composition when compared to the quantity of mucus present prior
to administration of the composition.
[0014] In another embodiment, another method of reducing the
quantity of mucus in the respiratory tract of a subject with
elevated levels of mucus in said respiratory tract is provided. The
method includes administering to the subject a compound or
composition containing a therapeutically effective amount of a
fusion protein. The fusion protein has at least one catalytic
domain of a sialidase, wherein the catalytic domain of the
sialidase includes the sequence of amino acids extending from amino
acid 274 to amino acid 666 of SEQ ID NO:12, inclusive, and at least
one anchoring domain. The anchoring domain can be a
glycosaminoglycan (GAG) binding domain of human amphiregulin
including the amino acid sequence of SEQ ID NO:7. The
therapeutically effective amount includes an amount of the fusion
protein that results in a reduction of the quantity of mucus in the
respiratory tract after administration of the compound or
composition when compared to the quantity of mucus present prior to
administration of the composition.
[0015] In another embodiment, another method of reducing the
quantity of mucus in the respiratory tract of a subject with
elevated levels of mucus in said respiratory tract is provided. The
method includes administering to the subject a compound or
composition containing a therapeutically effective amount of a
protein or peptide having a sialidase or an active portion thereof.
The therapeutically effective amount includes an amount of the
protein or peptide that results in a reduction of the quantity of
mucus in the respiratory tract after administration of the compound
or composition when compared to the quantity of mucus present prior
to administration of the compound or composition.
[0016] In another embodiment, a method of treating or ameliorating
the effects of chronic obstructive pulmonary disease (COPD),
bronchitis, bronchiectasis, cystic fibrosis (CF), vasculitis, mucus
plugging, Wegener's granulomatosis, pneumonia, tuberculosis, cancer
involving the lungs or the respiratory tract, Kartagener syndrome,
Young's syndrome, chronic sinopulmonary infection, alpha
1-antitrypsin deficiency, primary immunodeficiency, acquired immune
deficiency syndrome, opportunistic infection, an infectious state,
a post infectious state, common cold, exercise induced
hypersecretion of mucus, inflammatory bowel disease, ulcerative
colitis, Crohn's disease, respiratory infection, respiratory
obstruction, inhalation or aspiration of a toxic gas, pulmonary
aspiration, or alcoholism in a subject with an elevated level of
mucus in his or her respiratory tract is provided. The method
includes administering to the subject a compound or composition
containing a therapeutically effective amount of a fusion protein.
The fusion protein has at least one catalytic domain of a
sialidase, wherein the catalytic domain of the sialidase includes
the sequence of amino acids extending from amino acid 274 to amino
acid 666 of SEQ ID NO:12, inclusive, and at least one anchoring
domain. The anchoring domain can be a glycosaminoglycan (GAG)
binding domain of human amphiregulin including the amino acid
sequence of SEQ ID NO:7. The therapeutically effective amount
includes an amount of the fusion protein that results in a
reduction of the quantity of mucus in the respiratory tract after
administration of the compound or composition when compared to the
quantity of mucus present prior to administration of the compound
or composition.
[0017] In another embodiment, another method of treating or
ameliorating the effects of chronic obstructive pulmonary disease
(COPD), bronchitis, bronchiectasis, cystic fibrosis (CF),
vasculitis, mucus plugging, Wegener's granulomatosis, pneumonia,
tuberculosis, cancer involving the lungs or the respiratory tract,
Kartagener syndrome, Young's syndrome, chronic sinopulmonary
infection, alpha 1-antitrypsin deficiency, primary
immunodeficiency, acquired immune deficiency syndrome,
opportunistic infection, an infectious state, a post infectious
state, common cold, exercise induced hypersecretion of mucus,
inflammatory bowel disease, ulcerative colitis, Crohn's disease,
respiratory infection, respiratory obstruction, inhalation or
aspiration of a toxic gas, pulmonary aspiration, or alcoholism in a
subject with an elevated level of mucus in his or her respiratory
tract is provided. The method includes administering to the subject
a compound or composition containing a therapeutically effective
amount of a fusion protein. The fusion protein has a sialidase or
an active portion thereof and an anchoring domain. The
therapeutically effective amount includes an amount of the fusion
protein that results in a reduction of the quantity of mucus in the
respiratory tract after administration of the compound or
composition when compared to the quantity of mucus present prior to
administration of the compound or composition.
[0018] In another embodiment, another method of treating or
ameliorating the effects of chronic obstructive pulmonary disease
(COPD), bronchitis, bronchiectasis, cystic fibrosis (CF),
vasculitis, mucus plugging, Wegener's granulomatosis, pneumonia,
tuberculosis, cancer involving the lungs or the respiratory tract,
Kartagener syndrome, Young's syndrome, chronic sinopulmonary
infection, alpha 1-antitrypsin deficiency, primary
immunodeficiency, acquired immune deficiency syndrome,
opportunistic infection, an infectious state, a post infectious
state, common cold, exercise induced hypersecretion of mucus,
inflammatory bowel disease, ulcerative colitis, Crohn's disease,
respiratory infection, respiratory obstruction, inhalation or
aspiration of a toxic gas, pulmonary aspiration, or alcoholism in a
subject with an elevated level of mucus in his or her respiratory
tract is provided. The method includes administering to the subject
a compound or composition containing a therapeutically effective
amount of a protein or peptide having a sialidase or an active
portion thereof. The therapeutically effective amount includes an
amount of the protein or peptide that results in a reduction of the
quantity of mucus in the respiratory tract after administration of
the compound or composition when compared to the quantity of mucus
present prior to administration of the compound or composition.
[0019] In another embodiment, a method of limiting an increase in
the quantity of mucus in the respiratory tract of a subject above a
baseline level of mucus in said subject's respiratory tract is
provided. The method includes administering to the subject a
compound or composition containing a therapeutically effective
amount of a fusion protein. The fusion protein has at least one
catalytic domain of a sialidase, wherein the catalytic domain of
the sialidase includes the sequence of amino acids extending from
amino acid 274 to amino acid 666 of SEQ ID NO:12, inclusive, and at
least one anchoring domain, wherein the anchoring domain is a
glycosaminoglycan (GAG) binding domain of human amphiregulin
comprising the amino acid sequence of SEQ ID NO:7. The
therapeutically effective amount includes an amount of the fusion
protein that limits an increase in the quantity of mucus in the
respiratory tract of said subject above a baseline level after
administration of the compound or composition.
[0020] In another embodiment, another method of limiting an
increase in the quantity of mucus in the respiratory tract of a
subject above a baseline level of mucus in said subject's
respiratory tract is provided. The method includes administering to
the subject a compound or composition containing a therapeutically
effective amount of a fusion protein having a sialidase or an
active portion thereof and an anchoring domain. The therapeutically
effective amount includes an amount of the fusion protein that
limits an increase in the quantity of mucus in the respiratory
tract of said subject above a baseline level after administration
of the compound or composition.
[0021] In yet another embodiment, another method of limiting an
increase in the quantity of mucus in the respiratory tract of a
subject above a baseline level of mucus in said subject's
respiratory tract is provided. The method includes administering to
the subject a compound or composition containing a therapeutically
effective amount of a protein or peptide having a sialidase or an
active portion thereof. The therapeutically effective amount
includes an amount of the protein or peptide that limits an
increase in the quantity of mucus in the respiratory tract of the
subject above a baseline level after administration of the compound
or composition.
[0022] Also contemplated herein are methods of identifying
sialidases or active portions thereof according to the compounds or
compositions provided herein, where the sialidases or active
portions thereof are effective at reducing the quantity of mucus in
the respiratory tract of subjects. The reduction in mucus can be
measured directly in standard assays known to those of skill in the
art. For example, in some embodiments, a single compound or a
library or collection of compounds or compositions comprising
sialidase(s) and/or catalytically active portion(s) thereof are
administered to an animal model of asthma having an associated
inflammatory response, such as the guinea pig and the mouse as
described in Example 1 and Example 2, respectively. An asthmatic or
other inflammatory condition is created in the animal whereby the
accumulation of mucus in the lung or respitatory tract is
increased. The level of mucus is then quantitated and compared to
the level after treatment with a sialidase or active portion
thereof. If there is a reduction of the mucus level in the presence
of the sialidase or active portion thereof, the sialidase or active
portion thereof is identified or selected as one that can be used
in the methods provided herein for treating inflammation, allergies
and/or associated inflammatory/allergic responses, such as the
overproduction of mucus.
[0023] In some embodiments, a sialidase or active portion thereof
according to the compounds and compositions provided herein is
identified as being suitable for treating inflammation, allergies
or associated responses by measuring its ability to disrupt
muscarinic receptor-agonist interactions according to standard
methods known to those of skill in the art. For example, provided
herein is a method of assessing whether a compound or composition
comprising a sialidase and/or catalytically active portion thereof
reduces the quantity of mucus in the respiratory tract of a
subject, by
[0024] (a) contacting the muscarinic receptors of an animal subject
with a compound or composition that includes a sialidase and/or a
catalytically active portion thereof;
[0025] (b) administering a muscarinic receptor agonist to the
subject;
[0026] (c) quantitating the airway resistance in the subject;
[0027] (d) comparing the airway resistance level measured in (c)
with the airway resistance in the absence of contact with the
compound or composition;
[0028] (e) identifying whether the compound or composition reduces
the airway resistance relative to the airway resistance in the
absence of contact with the compound or composition; and
[0029] (f) if the compound or composition reduces the airway
resistance as determined in (e), assessing the compound or
composition as one that reduces the quantity of mucus in the
respiratory tract of the subject. Such a method is exemplified in
Example 3.
[0030] Unless otherwise defined, all technical and scientific terms
used herein have the same meaning as commonly understood by one of
ordinary skill in the art to which this invention belongs. Methods
and materials are described herein for use in the present
invention; other, suitable methods and materials known in the art
can also be used. The materials, methods, and examples are
illustrative only and not intended to be limiting. All
publications, patent applications, patents, sequences, database
entries, and other references mentioned herein are incorporated by
reference in their entirety. In case of conflict, the present
specification, including definitions, will control.
[0031] Other features and advantages of the invention will be
apparent from the following detailed description and figures, and
from the claims.
BRIEF DESCRIPTION OF THE DRAWINGS
[0032] FIG. 1 shows GAG-binding sequences of four human genes: PF4,
human platelet factor 4; IL8, human interleukin 8; AT III, human
antithrombin III; ApoE, human apolipoprotein E; AAMP, human
angio-associated migratory cell protein; human amphiregulin.
[0033] FIG. 2 is a sequence comparison between human sialidases
NEU2 and NEU4 (SEQ ID NOs: 8 & 9).
[0034] FIG. 3 is a table comparing substrate specificity of
bacterial and fungal sialidases.
[0035] FIG. 4 depicts the nucleotide and amino acid sequence (SEQ
ID NOs: 28 & 29) of a construct of the present invention
encoding His6-AvCD. NcoI and HindIII sites used for cloning into
pTrc99a are shown in bold.
[0036] FIG. 5 depicts the nucleotide and amino acid sequences (SEQ
ID NOs: 18 & 19) of another construct of the present invention
encoding AR-AvCD. NcoI and HindIII sites used for cloning into
pTrc99a are shown in bold.
[0037] FIG. 6 depicts the nucleotide and amino acid sequences (SEQ
ID NO: 36 & 37) of another construct of the present invention
encoding AR-G.sub.4S-AvCD. NcoI and HindIII sites used for cloning
into pTrc99a are shown in bold.
[0038] FIGS. 7A-B are graphs showing that topical administration of
recombinant AR-AvCD sialidase fusion protein reduces the
inflammatory responses of ferrets infected with an influenza A
(H1N1) virus. FIG. 7A shows the total number of inflammatory cells
from nasal wash samples obtained from infected animals at the
indicated times after infection. he protein concentration was
determined in cell-free nasal wash samples of infected ferrets.
Infected ferrets were vehicle-treated (squares) or were treated
with recombinant AR-AvCD sialidase fusion protein made from
Construct #2 (triangles). Uninfected animals were also treated with
recombinant AR-AvCD sialidase fusion protein (diamonds).
Statistically significant values are labeled with * (p<0.05) and
** (p<0.01).
[0039] FIG. 8 provides graphs showing formula and explanation of
the Enhanced Pause (PENH), the parameter used for measuring
bronchoconstriction in conscious unrestrained animals.
[0040] FIG. 9 provides a graph showing early asthmatic reaction in
response to an OVA-aerosol. Results are expressed as arithmetic
average .+-.SEM. *p<0.05, ***p<0.001 using student's
t-test.
[0041] FIG. 10 provides a graph showing the total number of cells
in guinea pigs on the day of section. Results are expressed as
arithmetic average .+-.SEM. **p<0.01, ***p<0.001 using
student's t-test.
[0042] FIG. 11 provides a graph showing the total number of
macrophages recovered in guinea pig BAL fluid on the day of
section. Results are expressed as arithmetic average .+-.SEM.
**p<0.01.
[0043] FIG. 12 provides a graph showing the total number of
lymphocytes recovered in guinea pig BAL fluid on the day of
section. Results are expressed as arithmetic average .+-.SEM.
*p<0.05.
[0044] FIG. 13 provides a graph showing the total number of
neutrophils recovered in guinea pig BAL fluid on the day of
section. Results are expressed as arithmetic average .+-.SEM.
*p<0.05, ***p<0.001.
[0045] FIG. 14 provides a graph showing the total number of
eosinophils recovered in guinea pig BAL fluid on the day of
section. Results are expressed as arithmetic average .+-.SEM.
*p<0.05, ***p<0.001.
[0046] FIG. 15 provides a graph showing the percent change in Penh
at Mch 48 mg/mL in the effect of sialidase treatment on the early
and late asthmatic reaction in guinea pigs.
[0047] FIG. 16 provides a graph showing the percent change in Pehn
at a range of Mch concentrations in the effect of sialidase
treatment on the early and late asthmatic reaction in guinea
pigs.
[0048] FIG. 17 provides a graph showing blood Eosinophils in the
effect of sialidase treatment on the early and late asthmatic
reaction in guinea pigs.
[0049] FIG. 18 provides a graph showing PAS staining for lung mucus
in the effect of sialidase treatment on the early and late
asthmatic reaction in guinea pigs.
[0050] FIGS. 19A-F provide a PAS staining for lung mucus.
[0051] FIG. 20 provides a graph showing MBP immunostaining for
eosinophils in the effect of sialidase treatment on the early and
late asthmatic reaction in guinea pigs.
[0052] FIGS. 21A-B provide graphs showing reduced airway resistance
in naive mice treated intranasally with low doses of DAS181
(methacholine challenged).
[0053] FIG. 22 provides a graph showing reduced airway resistance
in naive mice treated intranasally with a low dose of DAS181
(methacholine challenged).
[0054] FIG. 23 provides a graph showing reduced airway resistance
in naive mice treated intranasally with DAS181 (carbachol
challenged).
[0055] FIG. 24 provides a graph showing airway resistance in naive
mice treated intranasally with a low dose of DAS185 (methacholine
challenged).
[0056] FIG. 25 provides graphsshowing time-course of DAS185
mediated reduction of airway resistance (methacholine
challenged).
[0057] FIG. 26 provides a graph showing reduced airway resistance
in naive mice treated intranasally with very lose doses of DAS181
(methacholine challenged).
[0058] Like reference symbols in the various drawings indicate like
elements.
DETAILED DESCRIPTION
[0059] The present disclosure provides, inter alia, novel methods
of use for compounds described in U.S. patent application Ser. Nos.
10/718,986 and 10/939,262 (both of which are hereby incorporated by
reference in their entirety) to reduce mucus, e.g., in the
respiratory tract of subjects with elevated levels of mucus in
their respiratory tract. In some embodiments, the present
disclosure provides compositions and methods for reducing mucus
(e.g., mucus levels) in a subject in need of reduced mucus levels
and that does not have influenza (e.g., is not infected with
influenza at the time of treatment) or ashma.
[0060] In some embodiments, the compounds can include compounds
made by NexBio, Inc. under the compound name DAS181 and under the
trademark Fludase.RTM. (provided herein as SEQ ID NO:21). DAS181 is
a fusion protein comprising a catalytic domain of a sialidase, and
an anchoring domain. Several of the examples described herein use
DAS181 or compositions containing DAS181.
Definitions
[0061] Unless defined otherwise, all technical and scientific terms
used herein have the same meaning as commonly understood by one of
ordinary skill in the art to which this invention belongs.
Generally, the nomenclature used herein and the manufacture or
laboratory procedures described below are well known and commonly
employed in the art. Conventional methods are used for these
procedures, such as those provided in the art and various general
references. Where a term is provided in the singular, the inventors
also contemplate the plural of that term. Where there are
discrepancies in terms and definitions used in references that are
incorporated by reference, the terms used in this application shall
have the definitions given herein. As employed throughout the
disclosure, the following terms, unless otherwise indicated, shall
be understood to have the following meanings:
[0062] As used herein, a "subject" includes any animal for whom
diagnosis, screening, monitoring or treatment is contemplated.
Animals include mammals such as primates and domesticated animals.
An exemplary primate is human. A patient refers to a subject such
as a mammal, primate, human or livestock subject afflicted with a
disease condition or for which a disease condition is to be
determined or risk of a disease condition is to be determined.
[0063] In some embodiments, the methods disclosed herein can
include selecting a a subject in need of reduced mucus levels and
that is not infected with one or more of influenza, parainfluenza,
and/or respiratory syncytial virus (RSV). In some instances, the
terms infected or infection can include the presence of a influenza
and/or parainfluenza virus and/or RSV in a subject. In some
instances, the terms infected or infection can include the presence
of active or replicating influenza and/or parainfluenza virus
and/or RSV in a subject. In some embodiments, a subject with an
active or replicating influenza and/or parainfluenza virus and/or
RSV infection can be selected based on the presence or detection of
influenza and/or parainfluenza virus shedding and/or RSV shedding
in the subject (e.g., in a sample from the subject). In some
embodiments, the methods disclosed herein can include selecting a
subject in need of reduced mucus levels, wherein the subject has a
latent influenza, parainfluenza, and/or RSV infection.
[0064] An "animal model" as used herein means an animal that
sufficiently mimics, resembles or reproduces a disease or condition
of interest in its anatomy, physiology, or response (to a pathogen
or allergen, e.g.) so as to be useful in medical research that can
be extrapolated to the disease or condition of interest (e.g., to
screen for diagnostic or therapeutic agents; to measure therapeutic
efficacy of a compound or composition, etc.). For example, the
guinea pig and the mouse can be animal models to mimic inflammatory
and/or allergic responses associated with asthma, as demonstrated
in Examples 1 and 2, respectively. The mouse also can be an animal
model to study the interaction of muscarinic receptors with their
agonists, and the disruption thereof by agents such as the
compounds and compositions provided herein (see Example 3).
[0065] A "pathogen" can be any virus or microorganism that can
infect a cell, a tissue or an organism. A pathogen can be a virus,
bacterium, or protozoan.
[0066] A "target cell" is any cell that can be infected by a
pathogen or any cell that can interact with inflammatory cells, or
a host cell that is the intended destination for an exogenous gene
transferred by a recombinant virus.
[0067] "Inflammatory cells" are the cells that carry out or
participate in inflammatory responses of the immune system.
Inflammatory cells include include B lymphocytes, T lymphocytes,
macrophages, basophils, eosinophils, mast cells, NK cells,
monocytes, and neutrophils.
[0068] An "extracellular activity that can inhibit adhesion or
function of inflammatory cells" is any activity that can prevent
inflammatory cells from contacting the target cell and affecting
the normal physiological status of the target cell.
[0069] A "domain that can anchor said at least one therapeutic
domain to the membrane of a target cell", also called an
"extracellular anchoring domain" or simply, "anchoring domain"
refers to a chemical entity can that can stably bind a moiety that
is at or on the exterior of a cell surface or is in close proximity
to the surface of a cell. An extracellular anchoring domain can be
reversibly or irreversibly linked to one or more moieties, such as
one or more therapeutic domains, and thereby cause the one or more
attached therapeutic moieties to be retained at or in close
proximity to the exterior surface of a eukaryotic cell. An
extracellular anchoring domain can bind at least one molecule on
the surface of a target cell or at least one molecule found in
close association with the surface of a target cell. For example,
an extracellular anchoring domain can bind a molecule covalently or
noncovalently associated with the cell membrane of a target cell,
or can bind a molecule present in the extracellular matrix
surrounding a target cell. An extracellular anchoring domain can be
a peptide, polypeptide, or protein, and can also comprise any
additional type of chemical entity, including one or more
additional proteins, polypeptides, or peptides, a nucleic acid,
peptide nucleic acid, nucleic acid analogue, nucleotide, nucleotide
analogue, small organic molecule, polymer, lipids, steroid, fatty
acid, carbohydrate, or a combination of any of these.
[0070] As used herein, a protein or peptide sequences is
"substantially homologous" to a reference sequence when it is
either identical to a reference sequence, or comprises one or more
amino acid deletions, one or more additional amino acids, or more
one or more conservative amino acid substitutions, and retains the
same or essentially the same activity as the reference sequence.
Conservative substitutions may be defined as exchanges within one
of the following five groups: [0071] I. Small, aliphatic, nonpolar
or slightly polar residues: Ala, Ser, Thr, Pro, Gly [0072] II.
Polar, negatively charged residues and their amides: Asp, Asn, Glu,
Gln [0073] III. Polar, positively charged residues: His, Arg, Lys
[0074] IV. Large, aliphatic nonpolar residues: Met, Leu, Ile, Val,
Cys [0075] V. Large aromatic residues: Phe, Try, Trp
[0076] Within the foregoing groups, the following substitutions are
considered to be "highly conservative": Asp/Glu, His/Arg/Lys,
Phe/Tyr/Trp, and Met/Leu/Ile/Val. Semi-conservative substitutions
are defined to be exchanges between two of groups (I)-(IV) above
which are limited to supergroup (A), comprising (I), (II), and
(III) above, or to supergroup (B), comprising (IV) and (V) above.
In addition, where hydrophobic amino acids are specified in the
application, they refer to the amino acids Ala, Gly, Pro, Met, Leu,
Ile, Val, Cys, Phe, and Trp, whereas hydrophilic amino acids refer
to Ser, Thr, Asp, Asn, Glu, Gln, His, Arg, Lys, and Tyr.
[0077] A "sialidase" is an enzyme that can remove a sialic acid
residue from a substrate molecule. The sialidases
(N-acylneuraminosylglycohydrolases, EC 3.2.1.18) are a group of
enzymes that hydrolytically remove sialic acid residues from
sialo-glycoconjugates. Sialic acids are alpha-keto acids with
9-carbon backbones that are usually found at the outermost
positions of the oligosaccharide chains that are attached to
glycoproteins and glycolipids. One of the major types of sialic
acids is N-acetylneuraminic acid (Neu5Ac), which is the
biosynthetic precursor for most of the other types. The substrate
molecule can be, as nonlimiting examples, an oligosaccharide, a
polysaccharide, a glycoprotein, a ganglioside, or a synthetic
molecule. For example, a sialidase can cleave bonds having
alpha(2,3)-Gal, alpha(2,6)-Gal, or alpha(2,8)-Gal linkages between
a sialic acid residue and the remainder of a substrate molecule. A
sialidase can also cleave any or all of the linkages between the
sialic acid residue and the remainder of the substrate molecule.
Two major linkages between Neu5Ac and the penultimate galactose
residues of carbohydrate side chains are found in nature, Neu5Ac
alpha (2,3)-Gal and Neu5Ac alpha (2,6)-Gal. Both Neu5Ac alpha
(2,3)-Gal and Neu5Ac alpha (2,6)-Gal molecules can be recognized by
influenza viruses as the receptor, although human viruses seem to
prefer Neu5Ac alpha (2,6)-Gal, avian and equine viruses
predominantly recognize Neu5Ac alpha (2,3)-Gal. A sialidase can be
a naturally-occurring sialidase, an engineered sialidase (such as,
but not limited to a sialidase whose amino acid sequence is based
on the sequence of a naturally-occurring sialidase, including a
sequence that is substantially homologous to the sequence of a
naturally-occurring sialidase). As used herein, "sialidase" can
also mean the active portion of a naturally-occurring sialidase, or
a peptide or protein that comprises sequences based on the active
portion of a naturally-occurring sialidase.
[0078] A "fusion protein" is a protein comprising amino acid
sequences from at least two different sources. A fusion protein can
comprise amino acid sequence that is derived from a naturally
occurring protein or is substantially homologous to all or a
portion of a naturally occurring protein, and in addition can
comprise from one to a very large number of amino acids that are
derived from or substantially homologous to all or a portion of a
different naturally occurring protein. In the alternative, a fusion
protein can comprise amino acid sequence that is derived from a
naturally occurring protein or is substantially homologous to all
or a portion of a naturally occurring protein, and in addition can
comprise from one to a very large number of amino acids that are
synthetic sequences.
[0079] A "sialidase catalytic domain protein" is a protein that
comprises the catalytic domain of a sialidase, or an amino acid
sequence that is substantially homologous to the catalytic domain
of a sialidase, but does not comprises the entire amino acid
sequence of the sialidase the catalytic domain is derived from,
wherein the sialidase catalytic domain protein retains
substantially the same activity as the intact sialidase the
catalytic domain is derived from. A sialidase catalytic domain
protein can comprise amino acid sequences that are not derived from
a sialidase, but this is not required. A sialidase catalytic domain
protein can comprise amino acid sequences that are derived from or
substantially homologous to amino acid sequences of one or more
other known proteins, or can comprise one or more amino acids that
are not derived from or substantially homologous to amino acid
sequences of other known proteins.
[0080] "Therapeutically effective amount" means an amount of a
composition or compound that is needed for a desired therapeutic,
prophylactic, or other biological effect or response when a
composition or compound is administered to a subject in a single
dosage form. The particular amount of the composition or compound
will vary widely according to conditions such as the nature of the
composition or compound, the nature of the condition being treated,
the age and size of the subject.
[0081] "Treatment" means any manner in which one or more of the
symptoms of a condition, disorder or disease are ameliorated or
otherwise beneficially altered. Treatment also encompasses any
pharmaceutical use of the composition or compound herein, such as
for reducing mucus in the respiratory tract.
[0082] "Respiratory tract" means the air passages from the nose to
the pulmonary alveoli, including the nose, throat, pharynx, larynx,
trachea, and bronchi, and it also includes the lungs, and is
sometimes referred to by medical practitioners as the respiratory
system.
[0083] "Inhaler" means a device for giving medicines in the form of
a spray or dry powder that is inhaled (breathed in either naturally
or mechanically forced in to the lungs) through the nose or mouth,
and includes without limitation, a passive or active ventilator
(mechanical with or with an endotracheal tube), nebulizer, dry
powder inhaler, metered dose inhaler, and pressureized metered dose
inhaler.
[0084] "Inhalant" is any substance that is inhaled through the nose
or mouth.
[0085] "Reducing the quantity of mucus" means diminishing all or
some, generally more than by 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%,
80%, 85%, 90% 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% or more
of the amount of mucus in the respiratory tract when compared with
the amount prior to administration of the compositions or compounds
described herein. "Reducing the quantity of mucus" can also mean
reducing the amount of mucus in an amount that is observable by a
healthcare practitioner using whatever medical implements are
available for such observation, such as, e.g., by auscultation, by
MM or other radiographic study, by direct visualization with a
bronchoscope or other visualization device, or by measuring patient
mucus over time. "Reducing the quantity of mucus" can also mean
reducing the amount of mucus in an amount that is observable by the
patient or subject himself or herself with self-reporting or
self-observation, such as, e.g., monitoring the amount of
expectorated or swallowed mucus over time, or by subjectively
observing the sense of congestion in his or her lungs over
time.
[0086] "Limiting an increase in the quantity of mucus" means that
the amount of mucus in the respiratory tract after administration
of the compositions and compounds described herein does not
increase more than if they had not been administered. "Limiting an
increase in the quantity of mucus" also means that the amount of
mucus in the respiratory tract after administration of the
compositions and compounds described herein does not increase after
their administration of the compositions and compounds. "Limiting
an increase in the quantity of mucus" can also mean limiting an
increase over the patient's baseline at the time of administration
of the compounds or compositions in an amount that is observable or
ascertainable by a healthcare practitioner using whatever medical
implements and analytical systems are available for such
observations, such as, e.g., by ausculation, by MM or other
radiographic study, by direct visualization with a bronchoscope or
other visualization device, or by measuring patient sputum over
time. "Limiting an increase in the quantity of mucus" can also mean
limiting an increase over the patient's baseline at the time of
administration of the compounds or compositions in an amount that
is observable by the patient or subject himself or herself with
self-reporting or self-observation, such as, e.g., monitoring the
amount of expectorated or swallowed mucus over time, or by
subjectively observing the sense of congestion in his or her lungs
over time.
[0087] "Excipient" as used herein means one or more inactive
substances or compounds that either alone or in combination are
used as a carrier for the active ingredients of a medication. As
used herein "excipient" can also mean one or more substances or
compounds that are included in a pharmaceutical composition to
improve its beneficial effects or that have a synergistic effect
with the active ingredient.
Peptide or Protein Based Compounds
[0088] The present invention includes peptide or protein-based
compounds that comprise at least one domain that can anchor the
compound to the membrane of a eukaryotic cell and at least one
additional domain that is a therapeutic domain. By "peptide or
protein-based" compounds, it is meant that the two major domains of
the compound have an amino acid framework, in which the amino acids
are joined by peptide bonds. A peptide or protein-based compound
can also have other chemical compounds or groups attached to the
amino acid framework or backbone, including moieties that
contribute to the anchoring activity of the anchoring domain, or
moieties that contribute to the therapeutic activity of the
therapeutic domain. For example, the protein-based therapeutics
used in the present invention can comprise compounds and molecules
such as but not limited to: carbohydrates, fatty acids, lipids,
steroids, nucleotides, nucleotide analogues, nucleic acid
molecules, nucleic acid analogues, peptide nucleic acid molecules,
small organic molecules, or even polymers. The protein-based
therapeutics of the present invention can also comprise modified or
non-naturally occurring amino acids. Non-amino acid portions of the
compounds can serve any purpose, including but not limited to:
facilitating the purification of the compound, improving the
solubility or distribution or the compound (such as in a
therapeutic formulation), linking domains of the compound or
linking chemical moieties to the compound, contributing to the
two-dimensional or three-dimensional structure of the compound,
increasing the overall size of the compound, increasing the
stability of the compound, and contributing to the anchoring
activity or therapeutic activity of the compound.
[0089] The peptide or protein-based compounds of the present
invention can also include protein or peptide sequences in addition
to those that comprise anchoring domains or therapeutic domains.
The additional protein sequences can serve any purpose, including
but not limited to any of the purposes outlined above (facilitating
the purification of the compound, improving the solubility or
distribution or the compound, linking domains of the compound or
linking chemical moieties to the compound, contributing to the
two-dimensional or three-dimensional structure of the compound,
increasing the overall size of the compound, increasing the
stability of the compound, or contributing to the anchoring
activity or therapeutic activity of the compound). Any additional
protein or amino acid sequences can be part of a single polypeptide
or protein chain that includes the anchoring domain or domains and
therapeutic domain or domains, but any feasible arrangement of
protein sequences is within the scope of the present invention.
[0090] The anchoring domain and therapeutic domain can be arranged
in any appropriate way that allows the compound to bind at or near
a target cell membrane. The compound can have at least one protein
or peptide-based anchoring domain and at least one peptide or
protein-based therapeutic domain. In this case, the domains can be
arranged linearly along the peptide backbone in any order. The
anchoring domain can be N-terminal to the therapeutic domain, or
can be C-terminal to the therapeutic domain. It is also possible to
have one or more therapeutic domains flanked by at least one
anchoring domain on each end. Alternatively, one or more anchoring
domains can be flanked by at least one therapeutic domain on each
end. Chemical or peptide linkers can optionally be used to join
some or all of the domains of a compound.
[0091] It is also possible to have the domains in a nonlinear,
branched arrangement. For example, the therapeutic domain can be
attached to a derivatized side chain of an amino acid that is part
of a polypeptide chain that also includes, or is linked to, the
anchoring domain.
[0092] A compound of the present invention can have more than one
anchoring domain. In cases in which a compound has more than one
anchoring domain, the anchoring domains can be the same or
different. A compound used in the present invention can have more
than one therapeutic domain. In cases in which a compound has more
than one therapeutic domain, the therapeutic domains can be the
same or different. Where a compound comprises multiple anchoring
domains, the anchoring domains can be arranged in tandem (with or
without linkers) or on alternate sides of other domains, such as
therapeutic domains. Where a compound comprises multiple
therapeutic domains, the therapeutic domains can be arranged in
tandem (with or without linkers) or on alternate sides of other
domains, such as, but not limited to, anchoring domains.
[0093] A peptide or protein-based compound of the present invention
can be made by any appropriate way, including purifying naturally
occurring proteins, optionally proteolytically cleaving the
proteins to obtain the desired functional domains, and conjugating
the functional domains to other functional domains. Peptides can
also be chemically synthesized, and optionally chemically
conjugated to other peptides or chemical moieties. A peptide or
protein-based compound of the present invention can be made by
engineering a nucleic acid construct to encode at least one
anchoring domain and at least one therapeutic domain together (with
or without nucleic acid linkers) in a continuous polypeptide. The
nucleic acid constructs, in some embodiments having appropriate
expression sequences, can be transfected into prokaryotic or
eukaryotic cells, and the therapeutic protein-based compound can be
expressed by the cells and purified. Any desired chemical moieties
can optionally be conjugated to the peptide or protein-based
compound after purification. In some cases, cell lines can be
chosen for expressing the protein-based therapeutic for their
ability to perform desirable post-translational modifications (such
as, but not limited to glycosylation).
[0094] A great variety of constructs can be designed and their
protein products tested for desirable activities (such as, for
example, binding activity of an anchoring domain, or a binding,
catalytic, or inhibitory activity of a therapeutic domain).
Anchoring Domain
[0095] As used herein, an "extracellular anchoring domain" or
"anchoring domain" is any moiety that can stably bind an entity
that is at or on the exterior surface of a target cell or is in
close proximity to the exterior surface of a target cell. An
anchoring domain serves to retain a compound used in the present
invention at or near the external surface of a target cell.
[0096] An extracellular anchoring domain can bind 1) a molecule
expressed on the surface of a target cell, or a moiety, domain, or
epitope of a molecule expressed on the surface of a target cell, 2)
a chemical entity attached to a molecule expressed on the surface
of a target cell, or 3) a molecule of the extracellular matrix
surrounding a target cell.
[0097] An anchoring domain can be a peptide or protein domain
(including a modified or derivatized peptide or protein domain), or
comprises a moiety coupled to a peptide or protein. A moiety
coupled to a peptide or protein can be any type of molecule that
can contribute to the binding of the anchoring domain to an entity
at or near the target cell surface, and in some embodiments is an
organic molecule, such as, for example, nucleic acid, peptide
nucleic acid, nucleic acid analogue, nucleotide, nucleotide
analogue, small organic molecule, polymer, lipids, steroid, fatty
acid, carbohydrate, or any combination of any of these.
[0098] A molecule, complex, domain, or epitope that is bound by an
anchoring domain may or may not be specific for the target cell.
For example, an anchoring domain may bind an epitope present on
molecules on or in close proximity to the target cell and that
occur at sites other than the vicinity of the target cell as well.
In many cases, however, localized delivery of a therapeutic
compound of the present invention will restrict its occurrence
primarily to the surface of target cells. In other cases, a
molecule, complex, moiety, domain, or epitope bound by an anchoring
domain may be specific to a target tissue or target cell type.
[0099] Target tissue or target cell type includes the sites in an
animal or human body where a pathogen invades or amplifies. For
example, a target cell can be an endothelial cell that can be
infected by a pathogen. A composition used in the present invention
can comprise an anchoring domain that can bind a cell surface
epitope, for example, that is specific for the endothelial cell
type. In another example, a target cell can be an epithelial cell
and a composition of the present invention can bind an epitope
present on the cell surface of many epithelial cell types, or
present in the extracellular matrix of different types of
epithelial cells. In this case localized delivery of the
composition can restrict its localization to the site of the
epithelial cells that are targets of the pathogen.
[0100] Compounds used in the present invention can have one or more
anchoring domains that can bind at or near the surface of
epithelial cells. For example, heparan sulfate, closely related to
heparin, is a type of glycosaminoglycan (GAG) that is ubiquitously
present on cell membranes, including the surface of respiratory
epithelium. Many proteins specifically bind to heparin/heparan
sulfate, and the GAG-binding sequences in these proteins have been
identified (Meyer, F A, King, M and Gelman, R A. (1975) Biochimica
et Biophysica Acta 392: 223-232; Schauer, S. ed., pp 233. Sialic
Acids Chemistry, Metabolism and Function. Springer-Verlag, 1982).
For example, the GAG-binding sequences of human platelet factor 4
(PF4) (SEQ ID NO:2), human interleukin 8 (IL8) (SEQ ID NO:3), human
antithrombin III (AT III) (SEQ ID NO:4), human apoprotein E (ApoE)
(SEQ ID NO:5), human angio-associated migratory cell protein (AAMP)
(SEQ ID NO:6), or human amphiregulin (SEQ ID NO:7) (FIG. 1) have
been shown to have very high affinity (in the nanomolar range)
towards heparin (Lee, M K and Lander, A D. (1991) Pro Natl Acad Sci
USA 88:2768-2772; Goger, B, Halden, Y, Rek, A, Mosl, R, Pye, D.
Gallagher, J and Kungl, A J. (2002) Biochem. 41:1640-1646; Witt, D
P and Lander A D (1994) Curr Bio 4:394-400; Weisgraber, K H, Rall,
S C, Mahley, R W, Milne, R W and Marcel, Y. (1986) J Bio Chem
261:2068-2076). The GAG-binding sequences of these proteins are
distinct from their receptor-binding sequences, so they will not
induce the biological activities associated with the full-length
proteins or the receptor-binding domains. These sequences, or other
sequences that have been identified or are identified in the future
as heparin/heparan sulfate binding sequences, or sequences
substantially homologous to identified heparin/heparan sulfate
binding sequences that have heparin/heparan sulfate binding
activity, can be used as epithelium-anchoring-domains in compounds
used in the present invention.
[0101] An anchoring domain can bind a moiety that is specific to
the target cell type of a particular species or can bind a moiety
that is found in the target cell type of more than one species.
Therapeutic Domain
[0102] A compound used in the present invention includes at least
one therapeutic domain or active portion, those terms being used
interchangeable herein. The therapeutic activity can be, as
nonlimiting examples, a binding activity, a catalytic activity, or
an inhibitory activity. A therapeutic domain can modify or inhibit
a function of the target cell or target organism. An active portion
of a compound has therapeutic activity. For example, the catalytic
domain or active portion of a sialidase can be its therapeutic
domain.
[0103] The therapeutic domain can act extracellularly, meaning that
its infection-preventing, inflammatory response-modulating, or
transduction-enhancing activity takes place at the target cell
surface or in the immediate area surrounding the target cell,
including sites within the extracellular matrix, intracellular
spaces, or luminal spaces of tissues.
[0104] A therapeutic domain can be a peptide or protein domain
(including a modified or derivatized peptide or protein domain), or
comprises a moiety coupled to a peptide or protein. A moiety
coupled to a peptide or protein can be any type of molecule, and is
in some embodiments an organic molecule, such as, for example,
nucleic acid, peptide nucleic acid, nucleic acid analogue,
nucleotide, nucleotide analogue, small organic molecule, polymer,
lipids, steroid, fatty acid, carbohydrate, or any combination of
any of these.
[0105] A therapeutic domain can be a synthetic peptide or
polypeptide, or can comprise a synthetic molecule that can be
conjugated to a peptide or polypeptide, can be a
naturally-occurring peptide or protein, or a domain of
naturally-occurring protein. A therapeutic domain can also be a
peptide or protein that is substantially homologous to a
naturally-occurring peptide or protein.
Linkers
[0106] A compound used in the present invention can optionally
include one or more linkers that can join domains of the compound.
Linkers can be used to provide optimal spacing or folding of the
domains of a compound. The domains of a compound joined by linkers
can be therapeutic domains, anchoring domains, or any other domains
or moieties of the compound that provide additional functions such
as enhancing compound stability, facilitating purification, etc. A
linker used to join domains of compounds of the present invention
can be a chemical linker or an amino acid or peptide linker. Where
a compound comprises more than one linker, the linkers can be the
same or different. Where a compound comprises more than one linker,
the linkers can be of the same or different lengths.
[0107] Many chemical linkers of various compositions, polarity,
reactivity, length, flexibility, and cleavability are known in the
art of organic chemistry. Preferred linkers include amino acid or
peptide linkers. Peptide linkers are well known in the art. Some
embodiments of linkers are between one and about one hundred amino
acids in length, and between one and about thirty amino acids in
length, although length is not a limitation in the linkers of the
compounds of the present invention. The linkder amino acid
sequences can be selected such that they do not interfere with the
mucus-reducing and/or anti-inflammatory activity of the compounds
and compositions used in the present invention. Some embodiments of
linkers are those that include the amino acid glycine. For example,
linkers having the sequence:
(GGGGS (SEQ ID NO:10))n, where n is a whole number between 1 and
20, or between 1 and 12, can be used to link domains of therapeutic
compounds used in the present invention.
Composition Comprising at Least One Anchoring Domain and at Least
One Catalytic Activity
[0108] In some aspects, the present invention can use compounds
that have a therapeutic domain that has an enzymatic activity. The
enzymatic activity can be a catalytic activity that removes,
degrades or modifies a host molecule or complex. In some
embodiments the host molecule or complex can be removed, degraded,
or modified by the enzymatic activity of a compound of the present
invention is on, at, or near the surface of a target cell.
[0109] Compounds used in the present invention can have, for
example, one of the following structures: [0110] (Anchoring
Domain)n-[linker]-(Enzymatic Activity)n (n=1,2, 3 or more) or:
[0111] (Enzymatic Activity)n (n=1,2, 3 or more)-[linker]-(Anchoring
Domain)n, [0112] where the linkers are optional.
[0113] The enzymatic activity can be a monomeric form of a peptide
or polypeptide or can be multiple copies of the same polypeptide
that are either linked directly or with spacing sequence in
between. The polypeptides or peptides can be linked directly or via
a spacer composed of peptide linker sequence. The anchoring domain
can be any peptide or polypeptide that can bind to or near the
surface of target cells.
[0114] In one embodiment, a therapeutic domain comprises a
sialidase that can eliminate or greatly reduce the level of sialic
acid on the surface of epithelial cells. The therapeutic domain can
comprise a complete sialidase protein, or an active portion
thereof, wherein the active portion thereof retains the ability to
perform the catalytic function(s) of the sialidase protein (e.g.,
cleaving sialic acid residues).
[0115] Sialic acid mediates cell adhesion and interactions between
inflammatory cells and target cells. Therefore, treating the
surface of respiratory epithelial cells with a sialidase can
prevent the recruitment of inflammatory cells to the airway
surface, and therefore can treat allergic reactions including
asthma and allergic rhinitis. It also unexpectedly results in
reducing the quantity of mucus in the respiratory tract of subjects
with elevated levels of mucus in their respiratory tract, and
limiting increase in the quantity of mucus in the respiratory tract
of subjects above a baseline of mucus in the respiratory tract of
those subjects.
[0116] Among the sialidases contemplated for use in the methods
described herein are the large bacterial sialidases that can
degrade the receptor sialic acids Neu5Ac alpha(2,6)-Gal and Neu5Ac
alpha(2,3)-Gal. For example, the bacterial sialidase enzymes from
Clostridium perfringens (Genbank Accession Number X87369),
Actinomyces viscosus, Arthrobacter ureafaciens, or Micromonospora
viridifaciens (Genbank Accession Number D01045) can be used.
Therapeutic domains of compounds of the present invention can
comprise all or a portion of the amino acid sequence of a large
bacterial sialidase or can comprise amino acid sequences that are
substantially homologous to all or a portion of the amino acid
sequence of a large bacterial sialidase. In one preferred
embodiment, a therapeutic domain comprises a sialidase encoded by
Actinomyces viscosus, such as that of SEQ ID NO:12, or such as
sialidase sequence substantially homologous to SEQ ID NO:12. In yet
another preferred embodiment, a therapeutic domain comprises the
catalytic domain of the Actinomyces viscosus sialidase extending
from amino acids 274-667 of SEQ ID NO:12, or a substantially
homologous sequence.
[0117] Other sialidases contemplated for use in the methods
described herein are the human sialidases such as those encoded by
the genes NEU2 (SEQ ID NO:8; Genbank Accession Number Y16535;
Monti, E, Preti, Rossi, E., Ballabio, A and Borsani G. (1999)
Genomics 57:137-143) and NEU4 (SEQ ID NO:9; Genbank Accession
Number NM080741; Monti, E, Preti, A, Venerando, B and Borsani, G.
(2002) Neurochem Res 27:646-663) (FIG. 2). Therapeutic domains of
compounds used in the present invention can comprise all or a
portion of the amino acid sequences of a human sialidase or can
comprise amino acid sequences that are substantially homologous to
all or a portion of the amino acid sequences of a human sialidase.
Where a therapeutic domain comprises a portion of the amino acid
sequences of a naturally occurring sialidase, or sequences
substantially homologous to a portion of the amino acid sequences
of a naturally occurring sialidase, the portion can have
essentially the same activity as the human sialidase.
[0118] A compound for reducing elevated levels of mucus in the
respiratory tract can in some embodiments have one or anchoring
domains that can bind at or near the surface of epithelial cells.
In some embodiments, the epithelium-anchoring domain is a
GAG-binding sequence from a human protein, such as, for example,
the GAG-binding amino acid sequences of human platelet factor 4
(PF4) (SEQ ID NO:2), human interleukin 8 (IL8) (SEQ ID NO:3), human
antithrombin III (AT III) (SEQ ID NO:4), human apoprotein E (ApoE)
(SEQ ID NO:5), human angio-associated migratory cell protein (AAMP)
(SEQ ID NO:6), and human amphiregulin (SEQ ID NO:7) (FIG. 1). An
epithelial anchoring domain can also be substantially homologous to
a naturally occurring GAG-binding sequence, such as those listed in
FIG. 1. Such compounds can be formulated for nasal, tracheal,
bronchial, oral, or topical administration, or can be formulated as
an injectable solution or as eyedrops, or formulated into a
solution or dry powder and inhaled with inhalers.
[0119] A pharmaceutical composition comprising such compounds can
be used to treat or prevent allergy or inflammatory response. In
addition, such compounds have been shown herein to reduce the
quantity of mucus in the respiratory tract of subjects with
elevated levels of mucus in their respiratory tracts, and to limit
increases in the quantity of mucus in the respiratory tract of
subjects above a baseline of mucus in their respiratory tracts.
Therefore, such compounds can be used to as therapeutic treatments
to reduce the quantity of mucus in the respiratory tract of
subjects with elevated levels of mucus in their respiratory tracts,
or as prophylactic treatments to limit increases in the quantity of
mucus in the respiratory tract of subjects above a baseline of
mucus in their respiratory tracts. Due to their effect on mucus in
the respiratory tract, these compounds can also be used to prevent,
treat, or ameliorate the effects of chronic obstructive pulmonary
disease (COPD), bronchitis, bronchiectasis, cystic fibrosis (CF),
vasculitis, mucus plugging, Wegener's granulomatosis, pneumonia,
tuberculosis, cancer involving the lungs or the respiratory tract,
Kartagener syndrome, Young's syndrome, chronic sinopulmonary
infection, alpha 1-antitrypsin deficiency, primary
immunodeficiency, acquired immune deficiency syndrome,
opportunistic infection, an infectious state, a post infectious
state, common cold, exercise induced hypersecretion of mucus,
inflammatory bowel disease, ulcerative colitis, Crohn's disease,
respiratory infection, respiratory obstruction, inhalation or
aspiration of a toxic gas, pulmonary aspiration, or alcoholism in
subjects with elevated levels of mucus in their respiratory tract
or who are at risk of having increased levels of mucus in their
respiratory tract.
[0120] It is also within the scope of the present invention to use
compounds or compositions comprising a human sialidase, such as any
of those described herein, or an active portion thereof, or a
compound with substantial homology to a sialidase, in the absence
of an anchoring domain (a) to treat or prevent allergic and
inflammatory responses in the respiratory tract, (b) to reduce the
quantity of mucus in the respiratory tract of subjects with
elevated levels of mucus in their respiratory tracts, (c) to limit
increases in the quantity of mucus in the respiratory tract of
subjects above a baseline of mucus in their respiratory tracts,
and/or (d) to prevent, treat, or ameliorate the effects of chronic
obstructive pulmonary disease (COPD), bronchitis, bronchiectasis,
cystic fibrosis (CF), vasculitis, mucus plugging, Wegener's
granulomatosis, pneumonia, tuberculosis, cancer involving the lungs
or the respiratory tract, Kartagener syndrome, Young's syndrome,
chronic sinopulmonary infection, alpha 1-antitrypsin deficiency,
primary immunodeficiency, acquired immune deficiency syndrome,
opportunistic infection, an infectious state, a post infectious
state, common cold, exercise induced hypersecretion of mucus,
inflammatory bowel disease, ulcerative colitis, Crohn's disease,
respiratory infection, respiratory obstruction, inhalation or
aspiration of a toxic gas, pulmonary aspiration, or alcoholism in
subjects with elevated levels of mucus in their respiratory tract
or who are at risk of having increased levels of mucus in their
respiratory tract. The present invention recognizes that elevated
levels of mucus in the respiratory tract can be reduced by the use
of a sialidase or an active portion of a sialidase, and that such
sialidases or active portions thereof can optionally be adapted, by
genetic or chemical engineering, or by pharmaceutical formulation,
to improve their half life or retention at the respiratory
epithelium.
[0121] These compounds and pharmaceutical compositions can be
delivered to the upper respiratory tract as a nasal spray, or
delivered to the respiratory tract as an inhalant with
inhalers.
[0122] The compounds described herein can be formulated into
pharmaceutical compositions that include various additional
compounds either alone or in various combinations, such as,
Na.sub.2SO.sub.4, MgSO.sub.4, CaCl.sub.2), Histidine, Histine-HCl,
and Trehalose or their analogs. These additional compounds can be
included in the pharmaceutical compositions to act as excipients or
as active ingredients that provide additional beneficial
effects.
Therapeutic Composition Comprising at Least One Sialidase
Activity
[0123] The present invention includes methods that use therapeutic
compounds and compositions that comprise at least one sialidase
activity. The sialidase activity can be a sialidase isolated from
any source, such as, for example, a bacterial or mammalian source,
or can be a recombinant protein that is substantially homologous to
at least a portion of a naturally occurring sialidase. In some
embodiments sialidases are the large bacterial sialidases that can
degrade the receptor sialic acids Neu5Ac alpha(2,6)-Gal and Neu5Ac
alpha(2,3)-Gal. For example, the bacterial sialidase enzymes from
Clostridium perfringens (Genbank Accession Number X87369),
Actinomyces viscosus (Genbank Accession Number L06898),
Arthrobacter ureafaciens, or Micromonospora viridifaciens (Genbank
Accession Number D01045) or substantially homologous proteins can
be used.
[0124] For example, therapeutic compounds and compositions used in
the present invention can comprise a large bacterial sialidase or
can comprise a protein with the amino acid sequence of a large
bacterial sialidase or can comprise amino acid sequences that are
substantially homologous to the amino acid sequence of a large
bacterial sialidase. A pharmaceutical composition that can be used
in the present invention comprises the A. viscosus sialidase (SEQ
ID NO:12), or comprises a protein substantially homologous to the
A. viscosus sialidase.
[0125] Other sialidases that can be used in the compositions,
compounds and methods described herein are the human sialidases
such as those encoded by the genes NEU2 (SEQ ID NO:8; Genbank
Accession Number Y16535; Monti, E, Preti, Rossi, E., Ballabio, A
and Borsani G. (1999) Genomics 57:137-143) and NEU4 (SEQ ID NO:9;
Genbank Accession Number NM080741; Monti, E, Preti, A, Venerando, B
and Borsani, G. (2002) Neurochem Res 27:646-663) (FIG. 2).
Therapeutic domains of compounds of the present invention can
comprise a human sialidase protein that is substantially homologous
to the amino acid sequences of a human sialidase or can comprise
amino acid sequences that are substantially homologous to all or a
portion of the amino acid sequences of a human sialidase. Where a
therapeutic domain comprises a portion of the amino acid sequences
of a naturally occurring sialidase, or sequences substantially
homologous to a portion of the amino acid sequences of a naturally
occurring sialidase, the portion can have essentially the same
activity as the human sialidase, e.g., an active portion of the
sialidase.
[0126] Generally, sialidases that can effectively degrade on
respiratory epithelial cells both receptor sialic acids Neu5Ac
a(2,6)-Gal and Neu5Ac a(2,3)-Gal, can be used. Sialidases are found
in higher eukaryotes, as well as in some mostly pathogenic
microbes, including viruses, bacteria and protozoans. Viral and
bacterial sialidases have been well characterized, and the
three-dimensional structures of some of them have been determined
(Crennell, S J, Garman, E, Laver, G, Vimr, E and Taylor, G. (1994)
Structure 2:535-544; Janakiraman, M N, White, C L, Laver, W G, Air,
G M and Luo, M. (1994) Biochemistry 33:8172-8179; Pshezhetsky, A,
Richard, C, Michaud, L, Igdoura, S, Wang, S, Elsliger, M, Qu, J,
Leclerc, D, Gravel, R, Dallaire, L and Potier, M. (1997) Nature
Genet 15: 316-320). Several human sialidases have also been cloned
in the recent years (Milner, C M, Smith, S V, Carrillo M B, Taylor,
G L, Hollinshead, M and Campbell, R D. (1997) J Bio Chem
272:4549-4558; Monti, E, Preti, A, Nesti, C, Ballabio, A and
Borsani G. 1999. Glycobiol 9:1313-1321; Wada, T, Yoshikawa, Y,
Tokuyama, S, Kuwabara, M, Akita, H and Miyagi, T. (1999) Biochem
Biophy Res Communi 261:21-27; Monti, E, Bassi, M T, Papini, N,
Riboni, M, Manzoni, M, Veneranodo, B, Croci, G, Preti, A, Ballabio,
A, Tettamanti, G and Borsani, G. (2000) Bichem J 349:343-351).
DAS181, which contains an active portion of a sialidase, has also
been cloned.
[0127] All the sialidases characterized share a four amino acid
motif in the amino terminal portion followed by the Asp box motif
which is repeated three to five times depending on the protein.
(Monti, E, Bassi, M T, Papini, N, Riboni, M, Manzoni, M,
Veneranodo, B, Croci, G, Preti, A, Ballabio, A, Tettamanti, G and
Borsani, G. (2000) Bichem J349:343-351; Copley, R R, Russell, R B
and Ponting, C P. (2001) Protein Sci 10:285-292). While the overall
amino acid identity of the sialidase superfamily is relatively low
at about 20-30%, the overall fold of the molecules, especially the
catalytic amino acids, are remarkably similar (Wada, T, Yoshikawa,
Y, Tokuyama, S, Kuwabara, M, Akita, H and Miyagi, T. (1999) Biochem
Biophy Res Communi 261:21-27; Monti, E, Bassi, M T, Papini, N,
Riboni, M, Manzoni, M, Veneranodo, B, Croci, G, Preti, A, Ballabio,
A, Tettamanti, G and Borsani, G. (2000) Bichem J 349:343-351;
Copley, R R, Russell, R B and Ponting, C P. (2001) Protein Sci
10:285-292).
[0128] The sialidases are generally divided into two families:
"small" sialidases have molecular weight of about 42 kDa and do not
require divalent metal ion for maximal activity; "large" sialidases
have molecular weight above 65 kDa and may require divalent metal
ion for activity (Wada, T, Yoshikawa, Y, Tokuyama, S, Kuwabara, M,
Akita, H and Miyagi, T. (1999) Biochem Biophy Res Communi
261:21-27; Monti, E, Bassi, M T, Papini, N, Riboni, M, Manzoni, M,
Veneranodo, B, Croci, G, Preti, A, Ballabio, A, Tettamanti, G and
Borsani, G. (2000) Bichem J 349:343-351; Copley, R R, Russell, R B
and Ponting, C P. (2001) Protein Sci 10:285-292).
[0129] Over fifteen sialidase proteins have been purified and they
vary greatly from one another in substrate specificities and
enzymatic kinetics. Large bacterial sialidases can effectively
cleave sialic acid in both (.alpha.,2-6) linkage and (.alpha.,2-3)
linkage in the context of most natural substrates (FIG. 4; Vimr, D
R. (1994) Trends Microbiol 2: 271-277; Drzeniek, R. (1973)
Histochem J5:271-290; Roggentin, P, Kleineidam, R G and Schauer, R.
(1995) Biol Chem Hoppe-Seyler 376:569-575; Roggentin, P, Schauer,
R, Hoyer, L L and Vimr, E R. (1993) Mol Microb 9:915-921). Because
of their broad substrate specificities, large bacterial sialidases
make good candidates.
[0130] FIG. 4 shows several of the large bacterial sialidases with
known substrate specificity. These enzymes have high specific
activity (600 U/mg protein for C. perfringens (Corfield, A P, Veh,
R W, Wember, M, Michalski, J C and Schauer, R. (1981) Bichem J
197:293-299) and 680 U/mg protein for A. viscosus (Teufel, M,
Roggentin, P. and Schauer, R. (1989) Biol Chem Hoppe Seyler
370:435-443)), are fully active without divalent metal iron, and
have been cloned and purified as recombinant proteins from E. coli
(Roggentin, P, Kleineidam, R G and Schauer, R. (1995) Biol Chem
Hoppe-Seyler 376:569-575, Teufel, M, Roggentin, P. and Schauer, R.
(1989) Biol Chem Hoppe Seyler 370:435-443, Sakurada, K, Ohta, T and
Hasegawa, M. (1992) J Bacteriol 174: 6896-6903). In addition, C.
perfringens is stable in solution at 2-8.degree. C. for several
weeks, and at 4.degree. C. in the presence of albumin for more than
two years (Wang, F Z, Akula, S M, Pramod, N P, Zeng, L and
Chandran, B. (2001) J Virol 75:7517-27). A. viscosus is labile
towards freezing and thawing, but is stable at 4.degree. C. in 0.1
M acetate buffer, pH 5 (Teufel, M, Roggentin, P. and Schauer, R.
(1989) Biol Chem Hoppe Seyler 370:435-443).
[0131] A pharmaceutical composition comprising a sialidase can
include other compounds, including but not limited to other
proteins, that can also have therapeutic activity. A pharmaceutical
composition comprising a sialidase can include other compounds that
can enhance the stability, solubility, packaging, delivery,
consistency, taste, or fragrance of the composition.
[0132] Compounds comprising a sialidase can be formulated for
nasal, tracheal, bronchial, oral, or topical administration, or can
be formulated as an injectable solution or as eyedrops, or
formulated into a solution or dry powder and inhaled with inhalers.
The sialidases described herein can be formulated into
pharmaceutical compositions that include various additional
compounds such as, MgSO.sub.4, CaCl.sub.2), Histidine, Histine-HCl,
and Trehalose or their analogs.
[0133] These sialidases or pharmaceutical compositions containing
them can be used (a) to treat or prevent allergic and inflammatory
responses in the respiratory tract, (b) to reduce the quantity of
mucus in the respiratory tract of subjects with elevated levels of
mucus in their respiratory tracts, (c) to limit increases in the
quantity of mucus in the respiratory tract of subjects above a
baseline of mucus in their respiratory tracts, and/or (d) to
prevent, treat, or ameliorate the effects of chronic obstructive
pulmonary disease (COPD), bronchitis, bronchiectasis, cystic
fibrosis (CF), vasculitis, mucus plugging, Wegener's
granulomatosis, pneumonia, tuberculosis, cancer involving the lungs
or the respiratory tract, Kartagener syndrome, Young's syndrome,
chronic sinopulmonary infection, alpha 1-antitrypsin deficiency,
primary immunodeficiency, acquired immune deficiency syndrome,
opportunistic infection, an infectious state, a post infectious
state, common cold, exercise induced hypersecretion of mucus,
inflammatory bowel disease, ulcerative colitis, Crohn's disease,
respiratory infection, respiratory obstruction, inhalation or
aspiration of a toxic gas, pulmonary aspiration, or alcoholism in
subjects with elevated levels of mucus in their respiratory tract
or who are at risk of having increased levels of mucus in their
respiratory tract. In some embodiments, subjects with elevated
levels of mucus in their respiratory tract do not include subjects
with one or more of influenza, parainfluenza, and/or respiratory
syncytial virus (RSV).
Sialidase Catalytic Domain Proteins or Peptides
[0134] As used herein a "sialidase catalytic domain protein or
peptide" comprises a catalytic domain of a sialidase but does not
comprise the entire amino acid sequence of the sialidase from which
the catalytic domain is derived. A sialidase catalytic domain
protein or peptide has sialidase activity. A sialidase catalytic
domain protein or peptide can have at least 10%, at least 20%, at
least 50%, at least 70% of the activity of the sialidase from which
the catalytic domain sequence is derived. A sialidase catalytic
domain protein or peptide can have at least 90% of the activity of
the sialidase from which the catalytic domain sequence is
derived.
[0135] A sialidase catalytic domain protein or peptide can include
other amino acid sequences, such as but not limited to additional
sialidase sequences, sequences derived from other proteins, or
sequences that are not derived from sequences of
naturally-occurring proteins. Additional amino acid sequences can
perform any of a number of functions, including contributing other
activities to the catalytic domain protein, enhancing the
expression, processing, folding, or stability of the sialidase
catalytic domain protein, or even providing a desirable size or
spacing of the protein or peptide.
[0136] A preferred sialidase catalytic domain protein or peptide is
a protein that comprises the catalytic domain of the A. viscosus
sialidase. An A. viscosus sialidase catalytic domain protein or
peptide can include amino acids 270-667 of the A. viscosus
sialidase sequence (SEQ ID NO:12). An A. viscosus sialidase
catalytic domain protein or peptide can include amino acid sequence
that begins at any of the amino acids from amino acid 270 to amino
acid 290 of the A. viscosus sialidase sequence (SEQ ID NO:12) and
ends at any of the amino acids from amino acid 665 to amino acid
901 of said A. viscosus sialidase sequence (SEQ ID NO:12), and
lacks any A. viscosus sialidase protein sequence extending from
amino acid 1 to amino acid 269. (As used herein "lacks any A.
viscosus sialidase protein sequence extending from amino acid 1 to
amino acid 269" means lacks any stretch of four or more consecutive
amino acids as they appear in the designated protein or amino acid
sequence.)
[0137] In some embodiments, an A. viscosus sialidase catalytic
domain protein or peptide comprises amino acids 274-681 of the A.
viscosus sialidase sequence (SEQ ID NO:12) and lacks other A.
viscosus sialidase sequence. In other embodiments, an A. viscosus
sialidase catalytic domain protein comprises amino acids 290-666 or
290-667 of the A. viscosus sialidase sequence (SEQ ID NO:12) and
lacks any other A. viscosus sialidase sequence. In yet other
embodiments, an A. viscosus sialidase catalytic domain protein or
peptide comprises amino acids 274-666 of the A. viscosus sialidase
sequence (SEQ ID NO:12) and lacks any other A. viscosus sialidase
sequence. In yet other embodiments, an A. viscosus sialidase
catalytic domain protein or peptide comprises amino acids 290-666
or 290-667 of the A. viscosus sialidase sequence (SEQ ID NO:12) and
lacks any other A. viscosus sialidase sequence. In yet other
embodiments, an A. viscosus sialidase catalytic domain protein or
peptide comprises amino acids 290-681 of the A. viscosus sialidase
sequence (SEQ ID NO:12) and lacks any other A. viscosus sialidase
sequence.
[0138] Such sialidase catalytic domain proteins or peptides can be
formulated for nasal, tracheal, bronchial, oral, or topical
administration, or can be formulated as an injectable solution or
as eyedrops, or formulated into a solution or dry powder and
inhaled with an inhaler. The sialidase catalytic domain proteins or
peptides described herein can be formulated into pharmaceutical
compositions that include various additional compounds, such as,
MgSO.sub.4, CaCl.sub.2), Histidine, Histine-HCl, and Trehalose or
their analogs. These additional compounds can be included in the
pharmaceutical compositions either alone or in various
combinations, such as, Na.sub.2SO.sub.4, MgSO.sub.4, CaCl.sub.2),
Histidine, Histine-HCl, and Trehalose or their analogs. These
additional compounds can be included in the pharmaceutical
compositions to act as excipients or as active ingredients that
provide additional beneficial effects.
[0139] Such sialidase catalytic domain proteins or peptides or
pharmaceutical compositions containing them can be used (a) to
treat or prevent allergic and inflammatory responses in the
respiratory tract, (b) to reduce the quantity of mucus in the
respiratory tract of subjects with elevated levels of mucus in
their respiratory tracts, (c) to limit increases in the quantity of
mucus in the respiratory tract of subjects above a baseline of
mucus in their respiratory tracts, and/or (d) to prevent, treat, or
ameliorate the effects of chronic obstructive pulmonary disease
(COPD), bronchitis, bronchiectasis, cystic fibrosis (CF),
vasculitis, mucus plugging, Wegener's granulomatosis, pneumonia,
tuberculosis, cancer involving the lungs or the respiratory tract,
Kartagener syndrome, Young's syndrome, chronic sinopulmonary
infection, alpha 1-antitrypsin deficiency, primary
immunodeficiency, acquired immune deficiency syndrome,
opportunistic infection, an infectious state, a post infectious
state, common cold, exercise induced hypersecretion of mucus,
inflammatory bowel disease, ulcerative colitis, Crohn's disease,
respiratory infection, respiratory obstruction, inhalation or
aspiration of a toxic gas, pulmonary aspiration, or alcoholism in
subjects with elevated levels of mucus in their respiratory tract
or who are at risk of having increased levels of mucus in their
respiratory tract.
Fusion Proteins
[0140] Sialidase catalytic domain proteins can be fusion proteins,
in which the fusion protein comprises at least one sialidase
catalytic domain and at least one other protein domain, including
but not limited to: a purification domain, a protein tag, a protein
stability domain, a solubility domain, a protein size-increasing
domain, a protein folding domain, a protein localization domain, an
anchoring domain, an N-terminal domain, a C-terminal domain, a
catalytic activity domain, a binding domain, or a catalytic
activity-enhancing domain. The at least one other protein domain
can be derived from another source, such as, but not limited to,
sequences from another protein. The at least one other protein
domain need not be based on any known protein sequence, but can be
engineered and empirically tested to perform any function in the
fusion protein.
[0141] Purification domains can include, as nonlimiting examples,
one or more of a his tag, a calmodulin binding domain, a maltose
binding protein domain, a streptavidin domain, a streptavidin
binding domain, an intein domain, or a chitin binding domain.
Protein tags can comprise sequences that can be used for antibody
detection of proteins, such as, for example, the myc tag, the
hemagglutinin tag, or the FLAG tag. Protein domains that enhance
protein expression, modification, folding, stability, size, or
localization can be based on sequences of know proteins or
engineered. Other protein domains can have binding or catalytic
activity or enhance the catalytic activity of the sialidase
catalytic domain.
[0142] Fusion proteins used in the compositions, compounds and
methods of the present invention comprise at least one sialidase
catalytic domain and at least one anchoring domain. In some
embodiments, anchoring domains include GAG-binding domains, such as
the GAG-binding domain or human amphiregulin (SEQ ID NO:7).
[0143] Sialidase catalytic domains and other domains of a fusion
protein used in the present invention can optionally be joined by
linkers, such as but not limited to peptide linkers. A variety of
peptide linkers are known in the art. In one embodiment a linker
can be a peptide linker comprising glycine, such as G-G-G-G-S(SEQ
ID NO:10).
[0144] Such fusion proteins can be formulated for nasal, tracheal,
bronchial, oral, or topical administration, or can be formulated as
an injectable solution or as eyedrops or formulated into a solution
or dry powder and inhaled with an inhaler. These fusion proteins
can be formulated into pharmaceutical compositions that include
various additional compounds either alone or in various
combinations, such as, Na.sub.2SO.sub.4, MgSO.sub.4, CaCl.sub.2),
Histidine, Histine-HCl, and Trehalose or their analogs. These
additional compounds can be included in the pharmaceutical
compositions to act as excipients or as active ingredients that
provide additional beneficial effects.
[0145] Such fusion proteins or pharmaceutical compositions
containing them can be used (a) to treat or prevent allergic and
inflammatory responses in the respiratory tract, (b) to reduce the
quantity of mucus in the respiratory tract of subjects with
elevated levels of mucus in their respiratory tracts, (c) to limit
increases in the quantity of mucus in the respiratory tract of
subjects above a baseline of mucus in their respiratory tracts,
and/or (d) to prevent, treat, or ameliorate the effects of chronic
obstructive pulmonary disease (COPD), bronchitis, bronchiectasis,
cystic fibrosis (CF), vasculitis, mucus plugging, Wegener's
granulomatosis, pneumonia, tuberculosis, cancer involving the lungs
or the respiratory tract, Kartagener syndrome, Young's syndrome,
chronic sinopulmonary infection, alpha 1-antitrypsin deficiency,
primary immunodeficiency, acquired immune deficiency syndrome,
opportunistic infection, an infectious state, a post infectious
state, common cold, exercise induced hypersecretion of mucus,
inflammatory bowel disease, ulcerative colitis, Crohn's disease,
respiratory infection, respiratory obstruction, inhalation or
aspiration of a toxic gas, pulmonary aspiration, or alcoholism in
subjects with elevated levels of mucus in their respiratory tract
or who are at risk of having increased levels of mucus in their
respiratory tract.
[0146] Various constructs of fusion proteins are shown in FIGS.
4-6, as well as in the sequences provided in the sequence listing
provided herein.
Methods for Testing the Compounds and Compositions and/or for
Screening to Identify Sialidases and/or Active Portions Thereof to
Treat Diseases Accompanied by Inflammation
[0147] The compounds and compositions provided herein can be tested
for their activity in reducing inflammation, allergies or
associated responses, such as mucus overproduction, using standard
assays known to those of skill in the art. Several cell-based
(e.g., tracheal cell cultures) and animal-based assays (mouse
models, guinea pig models) for measuring inflammation or mucus
overproduction are known (see, e.g., Nakao et al., J. Immunol.,
180:6262-6269 (2008); Westerhof et al., Mediators Inflamm.,
10(3):143-154 (2001); Miller et al., J. Immunol., 170:3348-3356
(2003); Nakanishi et al., Proc. Natl. Acad. Sci. USA,
98(9):5175-5180 (2001); and DuBuske, Allergy Proc., 16(2):55-58
(1995), the contents of each of which are incorporated in their
entirety by reference herein). The compounds and compositions
provided herein can be tested for their ability to reduce
inflammation or mucus overproduction in any of these assays or
other standard assays known to those of skill in the art. In
addition, sialidases or active portions thereof can be identified
and/or selected for their anti-inflammatory activity and/or ability
to reduce associated responses, such as mucus overproduction, using
such assays. Exemplary assays and protocols are described herein in
Example 1 and Example 2.
[0148] In addition to assays that measure inflammation or
associated responses, such as mucus overproduction, the compounds
and compositions provided herein can be tested for their activity
by assessing their ability to disrupt muscarinic receptor--mediated
signaling in the presence of an agonist. Muscarinic receptors, or
mAChRs, are G protein-coupled acetylcholine receptors found in the
plasma membranes of certain neurons- and other cells. They play
several roles, including acting as the main end-receptor stimulated
by acetylcholine released from postganglionic fibers in the
parasympathetic nervous system.
[0149] Muscarinic receptor-agonist interactions, and the resulting
signaling, is believed to play a role in diseases that have
associated inflammatory and/or allergic responses, such as asthma
and COPD (see, e.g., "Muscarinic Receptors in Airways Diseases,"
Birkhauser-Verlag publ., Zangsma et al., Eds.).
[0150] More specifically, acetylcholinergic mechanisms are
recognized to influence the following normal and pathogenic
respiratory functions: [0151] 1. secretion of mucus, [0152] 2.
active transport of ions across the respiratory epithelium and
during mucociliary transport, [0153] 3. smooth muscle tone of the
airways, [0154] 4. immunologic and inflammatory response of the
airways, [0155] 5. reflex regulation of the airways, [0156] 6.
respiratory responses of the airways in asthma and in other
hypersensitivity states of the respiratory tract.
[0157] Consequently, certain anti-muscarinic agents have been
effective against: (a) acetylcholinergically induced
bronchoconstriction; (b) iatrogenic airway spasms induced by beta
blockers; and (c) psychogenic bronchospasm. The two main pulmonary
applications of anti-muscarinic agents has been chronic bronchitis
and bronchial asthma (Pharmacology of Anti-Muscarinic Agents,
Laszlo Gyermek (1998)).
[0158] There are five broad classes of muscarinic receptors, based
on their physiological roles, and agonists for each of these
receptors are known to those of skill in the art:
[0159] M1 receptor--exemplary agonists include acetylcholine,
oxotremorine, muscarine, carbachol and McNA343
[0160] M2 receptor--exemplary agonists include acetylcholine,
methacholine, carbachol, oxotremorine and muscarine
[0161] M3 receptor--exemplary agonists include acetylcholine,
bethanechol, carbachol, oxotremorine and pilocarpine
[0162] M4 receptor--exemplary agonists include acetylcholine,
carbachol and oxotremorine
[0163] M5 receptor--exemplary agonists include acetylcholine,
carbachol and oxotremorine
[0164] In some embodiments, the compounds and compositions provided
herein can be tested for the ability to reduce inflammation and/or
allergic responses, including mucus overproduction, associated with
RTIs or RTDs by assessing their ability to disrupt muscarinic
receptor-agonist interactions. Further, sialidases and/or active
portions thereof can be screened, identified and selected for their
ability to reduce inflammation, allergies, and/or associated
responses such as mucus overproduction by assessing their ability
to disrupt muscarinic receptor--agonist interactions. These tests
and screens can be performed using standard assays known to those
of skill in the art (see, e.g, Armstrong et al., Curr. Protocols in
Pharmacol., UNIT 12-13 (2010), the contents of which are
incorporated in their entirety by reference herein). An exemplary
assay and protocol is provided herein in Example 3.
Pharmaceutical Compositions
[0165] The present invention includes compounds of the present
invention formulated as pharmaceutical compositions. The
pharmaceutical compositions comprise a pharmaceutically acceptable
carrier prepared for storage and subsequent administration, which
have a pharmaceutically effective amount of the compound in a
pharmaceutically acceptable carrier or diluent. Acceptable carriers
or diluents for therapeutic use are well known in the
pharmaceutical art, and are described, for example, in Remington's
Pharmaceutical Sciences, 18th Ed., Mack Publishing Co., Easton, Pa.
(1990)). Preservatives, stabilizers, dyes and even flavoring agents
can be provided in the pharmaceutical composition. For example,
sodium benzoate, sorbic acid and esters of p-hydroxybenzoic acid
can be added as preservatives. In addition, antioxidants and
suspending agents can be used.
[0166] Depending on the target cell, the compounds of the present
invention can be formulated and used as tablets, capsules or
elixirs for oral or inhaled administration; salves or ointments for
topical application; suppositories for rectal administration;
sterile solutions, suspensions, and encapsulated powders and the
like for use as inhalants or nasal sprays. Injectables can also be
prepared in conventional forms either as liquid solutions or
suspensions, solid forms suitable for solution or suspension in
liquid prior to injection, or as emulsions.
[0167] Suitable excipients are, for example, water, saline,
dextrose, mannitol, lactose, lecithin, albumin, sodium glutamate,
cysteine hydrochloride and the like. In addition to those
excipients, additional compounds that can be included in the
pharmaceutical compositions described herein either alone or in
various combinations include Na.sub.2SO.sub.4, MgSO.sub.4,
CaCl.sub.2), Histidine, Histine-HCl, and Trehalose or their analogs
or Mg salts and/or Ca salts. These additional compounds can be
included in the pharmaceutical compositions to act as excipients or
as active ingredients that provide additional beneficial effects.
In addition, if desired, the injectable pharmaceutical compositions
can contain minor amounts of nontoxic auxiliary substances, such as
wetting agents, pH buffering agents and the like.
[0168] The pharmaceutically effective amount of a test compound
required as a dose will depend on the route of administration, the
type of animal or patient being treated, and the physical
characteristics of the specific animal under consideration. The
dose can be tailored to achieve a desired effect, such as reduction
of elevated levels of mucus in the respiratory tract, but will
depend on such factors as weight, diet, concurrent medication and
other factors which those skilled in the medical arts will
recognize. In practicing the methods of the present invention, the
pharmaceutical compositions can be used alone or in combination
with one another, or in combination with other therapeutic or
diagnostic agents. These products can be utilized in vivo in a
non-human animal subject, in a mammalian subject, in a human
subject, or in vitro. In employing them in vivo, the pharmaceutical
compositions can be administered to the patient or subject in a
variety of ways, including topically, parenterally, intravenously,
subcutaneously, intramuscularly, colonically, rectally, nasally or
intraperitoneally, employing a variety of dosage forms. Such
methods can also be used in testing the activity of test compounds
in vivo.
[0169] In some embodiments, these pharmaceutical compositions may
be in the form of orally-administrable suspensions, solutions,
tablets or lozenges; nasal sprays; inhalants; injectables, topical
sprays, ointments, powders, or gels, or formulated into a solutions
or dry powders and inhaled with an inhaler.
[0170] When administered orally as a suspension, compositions of
the present invention are prepared according to techniques
well-known in the art of pharmaceutical formulation and may contain
microcrystalline cellulose for imparting bulk, alginic acid or
sodium alginate as a suspending agent, methylcellulose as a
viscosity enhancer, and sweeteners/flavoring agents known in the
art. As immediate release tablets, these compositions may contain
microcrystalline cellulose, dicalcium phosphate, starch, magnesium
stearate and lactose and/or other excipients, binders, extenders,
disintegrants, diluents and lubricants known in the art. Components
in the formulation of a mouthwash or rinse include antimicrobials,
surfactants, cosurfactants, oils, water and other additives such as
sweeteners/flavoring agents known in the art.
[0171] When administered by a drinking solution, the composition
comprises one or more of the compounds of the present invention,
dissolved in water, with appropriate pH adjustment, and with
carrier. The compound can be dissolved in distilled water, tap
water, spring water, and the like. The pH can in some embodiments
be adjusted to between about 3.5 and about 8.5. Sweeteners can be
added, e.g., 1% (w/v) sucrose.
[0172] Lozenges can be prepared according to U.S. Pat. No.
3,439,089, herein incorporated by reference for these purposes.
[0173] When administered by nasal aerosol or inhalation, the
pharmaceutical compositions are prepared according to techniques
well-known in the art of pharmaceutical formulation and can be
prepared as solutions in saline, employing benzyl alcohol or other
suitable preservatives, absorption promoters to enhance
bioavailability, fluorocarbons, and/or other solubilizing or
dispersing agents known in the art. See, for example, Ansel, H. C.
et al., Pharmaceutical Dosage Forms and Drug Delivery Systems,
Sixth Ed. (1995). Inhaled powders can also be prepared using
techniques described in U.S. patent application Ser. Nos.
11/657,813 and 12/179,520, both of which are incorporated herein by
reference in their entirety. These compositions and formulations
can generally be prepared with suitable nontoxic pharmaceutically
acceptable ingredients. These ingredients are known to those
skilled in the preparation of nasal dosage forms and some of these
can be found in Remington's Pharmaceutical Sciences, 18th Ed., Mack
Publishing Co., Easton, Pa. (1990, a standard reference in the
field. The choice of suitable carriers is highly dependent upon the
exact nature of the nasal dosage form desired, e.g., solutions,
suspensions, ointments, or gels. Nasal dosage forms generally
contain large amounts of water in addition to the active
ingredient. Minor amounts of other ingredients such as pH
adjusters, emulsifiers or dispersing agents, preservatives,
surfactants, jelling agents, or buffering and other stabilizing and
solubilizing agents can also be present. Generally, the nasal
dosage form can be isotonic with nasal secretions.
[0174] Nasal formulations can be administers as drops, sprays,
aerosols or by any other intranasal dosage form. Optionally, the
delivery system can be a unit dose delivery system. The volume of
solution or suspension delivered per dose can be anywhere from
about 5 to about 2000 microliters, from about 10 to about 1000
microliters, or from about 50 to about 500 microliters. Delivery
systems for these various dosage forms can be dropper bottles,
plastic squeeze units, atomizers, nebulizers or pharmaceutical
aerosols in either unit dose or multiple dose packages.
[0175] The formulations of this invention can be varied to include;
(1) other acids and bases to adjust the pH; (2) other tonicity
imparting agents such as sorbitol, glycerin and dextrose; (3) other
antimicrobial preservatives such as other parahydroxy benzoic acid
esters, sorbate, benzoate, propionate, chlorbutanol, phenylethyl
alcohol, benzalkonium chloride, and mercurials; (4) other viscosity
imparting agents such as sodium carboxymethylcellulose,
microcrystalline cellulose, polyvinylpyrrolidone, polyvinyl alcohol
and other gums; (5) suitable absorption enhancers; (6) stabilizing
agents such as antioxidants, like bisulfite and ascorbate, metal
chelating agents such as sodium edetate and drug solubility
enhancers such as polyethylene glycols.
[0176] One embodiment of the invention includes pharmaceutical
compositions that at various dosage levels, such as dosage levels
between about 0.01 mg and about 100 mg, reduce the quantity of
mucus in the respiratory tract of subjects with elevated levels of
mucus in their respiratory tracts, and/or that limit increases in
the quantity of mucus in the respiratory tract of subjects above a
baseline of mucus in their respiratory tracts. Examples of such
dosage levels include doses of about 0.05 mg, 0.06 mg, 0.1 mg, 0.5
mg, 1 mg, 5 mg, 10 mg, 20 mg, 50 mg, or 100 mg. Another embodiment
of the invention includes pharameutical compositions that at
various dosage levels, such as dosage levels between about 0.01 mg
and about 100 mg, reduce inflammation in the respiratory tract or
prevent worsening of inflammation in the respiratory tract.
Examples of such dosage levels include doses of about 0.05 mg, 0.06
mg, 0.1 mg, 0.5 mg, 1 mg, 5 mg, 10 mg, 20 mg, 50 mg, or 100 mg. The
foregoing doses can be administered one or more times per day, for
one day, two days, three days, four days, five days, six days,
seven days, eight days, nine days, ten days, eleven days, twelve
days, thirteen days, or fourteen or more days. Higher doses or
lower doses can also be administered. Typically, dosages can be
between about 1 ng/kg and about 10 mg/kg, between about 10 ng/kg
and about 1 mg/kg, and between about 100 ng/kg and about 100
micrograms/kg. In various examples described herein, mice were
treated with various dosages of the compositions described herein,
including dosages of 0.0008 mg/kg, 0.004 mg/kg, 0.02 mg/kg, 0.06
mg/kg, 0.1 mg/kg, 0.3 mg/kg, 0.6 mg/kg, and 1.0 gm/kg.
[0177] In one embodiment a pharmaceutical composition includes
DAS181, MgSO.sub.4 1.446 mg/ml, CaCl.sub.2) 0.059 mg/ml, Histidine
1.427 mg/ml, Histidine-HCl 1.943 mg/ml, and Trehalose 3.000
mg/ml.
[0178] In another embodiment a pharameutical composition includes
DAS181, MgSO.sub.4, CaCl.sub.2), Histidine, Histidine-HCl, and
Trehalose.
[0179] In another embodiment a pharameutical composition includes
DAS181, Na.sub.2SO.sub.4, and CaCl.sub.2).
[0180] In another embodiment a pharmaceutical composition includes
DAS181 and any combination of one or more of the following:
Na.sub.2SO.sub.4, MgSO.sub.4, CaCl.sub.2), Histidine,
Histidine-HCl, and Trehalose.
[0181] In another embodiment a pharmaceutical composition includes
(a) a naturally occurring sialidase protein or peptide or an active
portion thereof, or a recombinant protein substantially homologous
to at least a portion of a naturally occurring sialidase, (b)
MgSO.sub.4 1.446 mg/ml, (c) CaCl.sub.2) 0.059 mg/ml, (d) Histidine
1.427 mg/ml, (e) Histidine-HCl 1.943 mg/ml, and (f) Trehalose 3.000
mg/ml. In one embodiment, the protein or peptide is a sialidase
with substantial homology to the A. viscosus sialidase (SEQ ID
NO:12) or substantial homology to an active portion thereof, such
as amino acids 274-666, 274-667, 270-667, 274-681, or 290-681 of
SEQ ID NO:12, or any other catalytic domain of Actinomyces viscosis
sialidase. In other embodiments, the protein or peptide is from one
of the large bacterial sialidases that can degrade the receptor
sialic acids Neu5Ac alpha(2,6)-Gal and Neu5Ac alpha(2,3)-Gal. For
example, the bacterial sialidase enzymes from Clostridium
perfringens (Genbank Accession Number X87369), Arthrobacter
ureafaciens, or Micromonospora viridifaciens (Genbank Accession
Number D01045) or proteins or peptides that are substantially
homologous to those sialidases or their active portions. In other
embodiments, the protein or peptide is from other sialidases, such
as those encoded by the genes NEU2 (SEQ ID NO:8; Genbank Accession
Number Y16535; Monti, E, Preti, Rossi, E., Ballabio, A and Borsani
G. (1999) Genomics 57:137-143) and NEU4 (SEQ ID NO:9; Genbank
Accession Number NM080741; Monti, E, Preti, A, Venerando, B and
Borsani, G. (2002) Neurochem Res 27:646-663) (FIG. 2), or active
portions of those sialidases.
[0182] In another embodiment a pharmaceutical composition includes
(a) a naturally occurring sialidase protein or peptide or an active
portion thereof, or a recombinant protein substantially homologous
to at least a portion of a naturally occurring sialidase, (b)
MgSO.sub.4, (c) CaCl.sub.2), (d) Histidine, (e) Histidine-HCl, and
(f) Trehalose. In one embodiment, the protein or peptide is a
sialidase with substantial homology to the A. viscosus sialidase
(SEQ ID NO:12) or substantial homology to an active portion
thereof, such as amino acids 274-666, 274-667, 270-667, 274-681, or
290-681 of SEQ ID NO:12, or any other catalytic domain of
Actinomyces viscosis sialidase. In other embodiments, the protein
or peptide is from one of the large bacterial sialidases that can
degrade the receptor sialic acids Neu5Ac alpha(2,6)-Gal and Neu5Ac
alpha(2,3)-Gal. For example, the bacterial sialidase enzymes from
Clostridium perfringens (Genbank Accession Number X87369),
Arthrobacter ureafaciens, or Micromonospora viridifaciens (Genbank
Accession Number D01045) or proteins or peptides that are
substantially homologous to those sialidases or their active
portions. In other embodiments, the protein or peptide is from
other sialidases, such as those encoded by the genes NEU2 (SEQ ID
NO:8; Genbank Accession Number Y16535; Monti, E, Preti, Rossi, E.,
Ballabio, A and Borsani G. (1999) Genomics 57:137-143) and NEU4
(SEQ ID NO:9; Genbank Accession Number NM080741; Monti, E, Preti,
A, Venerando, B and Borsani, G. (2002) Neurochem Res 27:646-663)
(FIG. 2), or active portions of those sialidases.
[0183] In another embodiment a pharmaceutical composition includes
(a) a naturally occurring sialidase protein or peptide or an active
portion thereof, or a recombinant protein substantially homologous
to at least a portion of a naturally occurring sialidase, (b)
Na.sub.2SO.sub.4, and (c) CaCl.sub.2). In one embodiment, the
protein or peptide is a sialidase with substantial homology to the
A. viscosus sialidase (SEQ ID NO:12) or substantial homology to an
active portion thereof, such as amino acids 274-666, 274-667,
270-667, 274-681, or 290-681 of SEQ ID NO:12, or any other
catalytic domain of Actinomyces viscosis sialidase. In one
embodiment, the protein or peptide is a sialidase with substantial
homology to the A. viscosus sialidase (SEQ ID NO:12) or substantial
homology to an active portion thereof, such as amino acids 274-666,
274-667, 270-667, 274-681, or 290-681 of SEQ ID NO:12, or any other
catalytic domain of Actinomyces viscosis sialidase. In other
embodiments, the protein or peptide is from one of the large
bacterial sialidases that can degrade the receptor sialic acids
Neu5Ac alpha(2,6)-Gal and Neu5Ac alpha(2,3)-Gal. For example, the
bacterial sialidase enzymes from Clostridium perfringens (Genbank
Accession Number X87369), Arthrobacter ureafaciens, or
Micromonospora viridifaciens (Genbank Accession Number D01045) or
proteins or peptides that are substantially homologous to those
sialidases or their active portions. In other embodiments, the
protein or peptide is from other sialidases, such as those encoded
by the genes NEU2 (SEQ ID NO:8; Genbank Accession Number Y16535;
Monti, E, Preti, Rossi, E., Ballabio, A and Borsani G. (1999)
Genomics 57:137-143) and NEU4 (SEQ ID NO:9; Genbank Accession
Number NM080741; Monti, E, Preti, A, Venerando, B and Borsani, G.
(2002) Neurochem Res 27:646-663) (FIG. 2), or active portions of
those sialidases.
[0184] In another embodiment a pharmaceutical composition includes
(a) a naturally occurring sialidase protein or peptide or an active
portion thereof, or a recombinant protein substantially homologous
to at least a portion of a naturally occurring sialidase, and any
combination of one or more of the following: Na.sub.2SO.sub.4,
MgSO.sub.4, CaCl.sub.2), Histidine, Histidine-HCl, and Trehalose.
In one embodiment, the protein or peptide is a sialidase with
substantial homology to the A. viscosus sialidase (SEQ ID NO:12) or
substantial homology to an active portion thereof, such as amino
acids 274-666, 274-667, 270-667, 274-681, or 290-681 of SEQ ID
NO:12, or any other catalytic domain of Actinomyces viscosis
sialidase. In other embodiments, the protein or peptide is from one
of the large bacterial sialidases that can degrade the receptor
sialic acids Neu5Ac alpha(2,6)-Gal and Neu5Ac alpha(2,3)-Gal. For
example, the bacterial sialidase enzymes from Clostridium
perfringens (Genbank Accession Number X87369), Arthrobacter
ureafaciens, or Micromonospora viridifaciens (Genbank Accession
Number D01045) or proteins or peptides that are substantially
homologous to those sialidases or their active portions. In other
embodiments, the protein or peptide is from other sialidases, such
as those encoded by the genes NEU2 (SEQ ID NO:8; Genbank Accession
Number Y16535; Monti, E,
[0185] Preti, Rossi, E., Ballabio, A and Borsani G. (1999) Genomics
57:137-143) and NEU4 (SEQ ID NO:9; Genbank Accession Number
NM080741; Monti, E, Preti, A, Venerando, B and Borsani, G. (2002)
Neurochem Res 27:646-663) (FIG. 2), or active portions of those
sialidases.
[0186] In another embodiment a pharmaceutical composition includes
(a) a fusion protein that has at least one catalytic domain of a
sialidase, wherein the catalytic domain of the sialidase includes
the sequence of amino acids extending from amino acid 274 to amino
acid 666 of SEQ ID NO:12 (alternatively, 274 to 666, 270-667,
274-681, 290-681 of SEQ ID NO:12, or any other catalytic domain of
Actinomyces viscosis), inclusive, and at least one anchoring
domain, wherein the anchoring domain is a glycosaminoglycan (GAG)
binding domain of human amphiregulin including the amino acid
sequence of SEQ ID NO:7, (b) MgSO.sub.4 1.446 mg/ml, (c)
CaCl.sub.2) 0.059 mg/ml, (d) Histidine 1.427 mg/ml, (e)
Histidine-HCl 1.943 mg/ml, and (f) Trehalose 3.000 mg/ml.
[0187] In another embodiment a pharmaceutical composition includes
(a) a fusion protein that has at least one catalytic domain of a
sialidase, wherein the catalytic domain of the sialidase includes
the sequence of amino acids extending from amino acid 274 to amino
acid 666 of SEQ ID NO:12 (alternatively, 274 to 666, 270-667,
274-681, 290-681 of SEQ ID NO:12, or any other catalytic domain of
Actinomyces viscosis), inclusive, and at least one anchoring
domain, wherein the anchoring domain is a glycosaminoglycan (GAG)
binding domain of human amphiregulin including the amino acid
sequence of SEQ ID NO:7, (b) MgSO.sub.4, (c) CaCl.sub.2), (d)
Histidine, (e) Histidine-HCl, and (f) Trehalose.
[0188] In another embodiment a pharmaceutical composition includes
(a) a fusion protein that has at least one catalytic domain of a
sialidase, wherein the catalytic domain of the sialidase includes
the sequence of amino acids extending from amino acid 274 to amino
acid 666 of SEQ ID NO:12 (alternatively, 274 to 666, 270-667,
274-681, 290-681 of SEQ ID NO:12, or any other catalytic domain of
Actinomyces viscosis), inclusive, and at least one anchoring
domain, wherein the anchoring domain is a glycosaminoglycan (GAG)
binding domain of human amphiregulin including the amino acid
sequence of SEQ ID NO:7, (b) Na.sub.2SO.sub.4, and (c)
CaCl.sub.2).
[0189] In another embodiment a pharmaceutical composition includes
(a) a fusion protein that has at least one catalytic domain of a
sialidase, wherein the catalytic domain of the sialidase includes
the sequence of amino acids extending from amino acid 274 to amino
acid 666 of SEQ ID NO:12 (alternatively, 274 to 666, 270-667,
274-681, 290-681 of SEQ ID NO:12, or any other catalytic domain of
Actinomyces viscosis), inclusive, and at least one anchoring
domain, wherein the anchoring domain is a glycosaminoglycan (GAG)
binding domain of human amphiregulin including the amino acid
sequence of SEQ ID NO:7, and (b) any combination of one or more of
the following: Na.sub.2SO.sub.4, MgSO.sub.4, CaCl.sub.2),
Histidine, Histidine-HCl, and Trehalose.
[0190] In another embodiment a pharmaceutical composition includes
(a) a fusion protein having a sialidase or an active portion
thereof and an anchoring domain, (b) MgSO.sub.4 1.446 mg/ml, (c)
CaCl.sub.2) 0.059 mg/ml, (d) Histidine 1.427 mg/ml, (e)
Histidine-HCl 1.943 mg/ml, and (f) Trehalose 3.000 mg/ml.
[0191] In another embodiment a pharmaceutical composition includes
(a) a fusion protein having a sialidase or an active portion
thereof and an anchoring domain, (b) MgSO.sub.4, (c) CaCl.sub.2),
(d) Histidine, (e) Histidine-HCl, and (f) Trehalose.
[0192] In another embodiment a pharmaceutical composition includes
(a) a fusion protein having a sialidase or an active portion
thereof and an anchoring domain, (b) Na.sub.4SO.sub.4, and (c)
CaCl.sub.2).
[0193] In another embodiment a pharmaceutical composition includes
(a) a fusion protein having a sialidase or an active portion
thereof and an anchoring domain, and (b) any combination of one or
more of the following: Na.sub.2SO.sub.4, MgSO.sub.4, CaCl.sub.2),
Histidine, Histidine-HCl, and Trehalose.
[0194] Another representative example of a pharmaceutical
composition of the present invention and that can be used in the
methods described herein includes the following: DAS181, histidine,
magnesium sulfate (or citrate salt), calcium chloride, trehalose,
water, Na-Acetate, and acetic acid.
[0195] Yet another representative example of a pharmaceutical
composition of the present invention and that can be used in the
methods described herein includes DAS181 (in any concentration
between about 0.01% and about 100% w/w, between about 1.00% and
about 90.0% w/w, between about 5.00% and about 80.0% w/w, between
about 10.0% and about 70.0% w/w, between about 20.0% and about 70%
w/w, between about 30.0% and about 70.0% w/w, between about 40.0%
and about 70.0% w/w, between about 50.0% and about 70% w/w, between
about 60.0% and about 70.0% w/w) in combination with any of the
following: histidine or histidine-HCl (in any concentration between
about 0.00% and about 90.0% w/w, between about 0.01% and about
80.0% w/w, between about 1.00% and about 75.0% w/w, between about
2.00% and about 70.0% w/w, between about 3.00% and about 60% w/w,
between about 4.00% and about 50.0% w/w, between about 5.00% and
about 40.0% w/w, between about 6.00% and about 30% w/w, between
about 7.00% and about 20.0% w/w), magnesium sulfate (or citrate
salt or sodium sulfate)(in any concentration between about 0.00%
and about 90.0% w/w, between about 0.01% and about 80.0% w/w,
between about 1.00% and about 75.0% w/w, between about 2.00% and
about 70.0% w/w, between about 3.00% and about 60% w/w, between
about 4.00% and about 50.0% w/w, between about 5.00% and about
40.0% w/w, between about 6.00% and about 30% w/w, between about
7.00% and about 20.0% w/w), calcium chloride (in any concentration
between about 0.00% and about 90.0% w/w, between about 0.01% and
about 80.0% w/w, between about 0.01% and about 75.0% w/w, between
about 0.01% and about 70.0% w/w, between about 0.01% and about 60%
w/w, between about 0.01% and about 50.0% w/w, between about 0.01%
and about 40.0% w/w, between about 0.01% and about 30% w/w, between
about 0.10% and about 20.0% w/w), trehalose (in any concentration
between about 0.00% and about 90.0% w/w, between about 0.01% and
about 80.0% w/w, between about 1.00% and about 75.0% w/w, between
about 2.00% and about 70.0% w/w, between about 3.00% and about 60%
w/w, between about 4.00% and about 50.0% w/w, between about 5.00%
and about 40.0% w/w, between about 6.00% and about 30% w/w, between
about 7.00% and about 20.0% w/w), water (in any concentration
between about 0.00% and about 90.0% w/w, between about 0.01% and
about 80.0% w/w, between about 1.00% and about 75.0% w/w, between
about 2.00% and about 70.0% w/w, between about 3.00% and about 60%
w/w, between about 4.00% and about 50.0% w/w, between about 5.00%
and about 40.0% w/w, between about 6.00% and about 30% w/w, between
about 7.00% and about 20.0% w/w), Na-Acetate (in any concentration
between about 0.00% and about 90.0% w/w, between about 0.01% and
about 80.0% w/w, between about 0.01% and about 75.0% w/w, between
about 0.01% and about 70.0% w/w, between about 0.01% and about 60%
w/w, between about 0.01% and about 50.0% w/w, between about 0.01%
and about 40.0% w/w, between about 0.01% and about 30% w/w, between
about 0.10% and about 20.0% w/w), and acetic acid (in any
concentration between about 0.00% and about 90.0% w/w, between
about 0.01% and about 80.0% w/w, between about 0.01% and about
75.0% w/w, between about 0.01% and about 70.0% w/w, between about
0.01% and about 60% w/w, between about 0.01% and about 50.0% w/w,
between about 0.01% and about 40.0% w/w, between about 0.01% and
about 30% w/w, between about 0.10% and about 20.0% w/w).
[0196] Any of the above pharmaceutical compositions may in addition
include MgCl.sub.2 in various concentrations ranging from about 0%
to about 75% w/w.
Reducing mucus in the respiratory tract and limiting its
increase
[0197] Accumulation or elevated levels of mucus in the respiratory
airway tree can be caused by an increased volume of mucus produced,
and also by decreased clearance due to defects in the ciliary
clearance apparatus in the respiratory tract. Hypersecretion of
mucus can be chronic, but increased volumes are produced in
exacerbations of COPD, during attacks of asthma, and in
bronchiectatic and cystic fibrosis patients (W. D. Kim, Eur Respir.
J. 1997, 10:1914-1917). Intraluminal mucus accumulation (i.e.,
elevated levels of mucus) in the airways associated with
hypersecretion of mucus or decreased clearance thereof creates a
clinical problem in almost all pulmonary diseases and diseases that
have an affect on the respiratory tract, including without
limitation chronic obstructive pulmonary disease (COPD),
bronchitis, bronchiectasis, cystic fibrosis (CF), vasculitis, mucus
plugging, Wegener's granulomatosis, pneumonia, tuberculosis, cancer
involving the lungs or the respiratory tract, Kartagener syndrome,
Young's syndrome, chronic sinopulmonary infection, alpha
1-antitrypsin deficiency, primary immunodeficiency, acquired immune
deficiency syndrome, opportunistic infection, an infectious state,
a post infectious state, common cold, exercise induced
hypersecretion of mucus, inflammatory bowel disease, ulcerative
colitis, Crohn's disease, respiratory infection, respiratory
obstruction, inhalation or aspiration of a toxic gas, pulmonary
aspiration, or alcoholism. Elevated levels of mucus in the
respiratory tract are an important determinant in the prognosis and
clinical feastures of various pulmonary diseases, such as chronic
bronchitis, bronchiectasis and bronchial asthma, in addition to
cystic fibrosis and COPD (W. D. Kim, Eur Respir. J. 1997,
10:1914-1917). Accordingly, in some embodiments, the present
disclosure include methods in which a subject with one or more of
these conditions or diseases is selected for treatment. In some
embodiments, the methods can include selecting a subject with one
or more of the conditions or diseases provided herein and that is
not infected with one or more of influenza, parainfluenza, and/or
respiratory syncytial virus (RSV). Following selection, the subject
can be treated by administration of one or more of the compositions
disclosed herein.
[0198] Provided herein are methods that include the administration
of the compounds described herein and in U.S. application Ser. Nos.
10/718,986 and 10/939,262, or compositions containing them, to
reduce the quantity of mucus in the respiratory tract of subjects
with elevated levels of mucus in their respiratory tracts and to
limit increases in the quantity of mucus in the respiratory tract
of subjects above a baseline of mucus in their respiratory tracts.
Thus, the invention relates to method of using the therapeutic
compounds and/or compositions described herein to prevent or treat
diseases that are caused by, cause, or are exacerbated by
respiratory inflammation or increased mucus production, such as,
both allergic and non-allergic asthma, chronic obstructive
pulmonary disease (COPD), bronchitis (both acute and non-acute),
bronchiectasis, cystic fibrosis (CF), vasculitis, mucuous plugging,
Wegener's granulomatosis, and any other disorder that causes
inflammation or increased mucus production in the respiratory tract
or is caused by or exacerbated by inflammation or increased mucus
production in the respiratory tract. The invention also includes
methods of using the therapeutic compounds and/or compositions
described herein to reduce the quantity of mucus in the respiratory
tract of subjects with elevated levels of mucus in their
respiratory tracts and limit increases in the quantity of mucus in
the respiratory tract of subjects above a baseline of mucus in
their respiratory tracts.
[0199] In some embodiments, the methods include administering a
composition or compound containing a therapeutically effective
amount of a protein or peptide having a sialidase or an active
portion thereof to a subject. The protein or peptide can be an
isolated naturally occurring sialidase protein, or a recombinant
protein substantially homologous to at least a portion of a
naturally occurring sialidase. In one embodiment, a pharmaceutical
composition or compound contains a sialidase with substantial
homology to the A. viscosus sialidase (SEQ ID NO:12) or substantial
homology to an active portion thereof, such as amino acids 274-666,
274-667, 270-667, 274-681, or 290-681 of SEQ ID NO:12, or any other
catalytic domain of Actinomyces viscosis sialidase. The
therapeutically effective amount includes an amount of the protein
or peptide that results in a reduction of the quantity of mucus in
the respiratory tract after administration of the composition or
compound when compared to the quantity of mucus present prior to
administration of the composition.
[0200] In other embodiments, the methods include administering a
composition or compound containing a therapeutically effective
amount of a fusion protein, wherein the fusion protein has at least
one catalytic domain of a sialidase, wherein the catalytic domain
of the sialidase includes the sequence of amino acids extending
from amino acid 274 to amino acid 666 of SEQ ID NO:12
(alternatively, 274 to 666, 270-667, 274-681, 290-681 of SEQ ID
NO:12, or any other catalytic domain of Actinomyces viscosis),
inclusive, and at least one anchoring domain, wherein the anchoring
domain is a glycosaminoglycan (GAG) binding domain of human
amphiregulin including the amino acid sequence of SEQ ID NO:7. The
therapeutically effective amount includes an amount of the fusion
protein that results in a reduction of the quantity of mucus in the
respiratory tract after administration of the composition or
compound when compared to the quantity of mucus present prior to
administration of the composition.
[0201] In yet other embodiments, the methods include administering
a composition containing a therapeutically effective amount of a
fusion protein having a sialidase or an active portion thereof and
an anchoring domain. The therapeutically effective amount includes
an amount of the fusion protein that results in a reduction of the
quantity of mucus in the respiratory tract after administration of
the composition or compound when compared to the quantity of mucus
present prior to administration of the composition.
[0202] Other embodiments include methods of preventing, treating or
ameliorating the effects of chronic obstructive pulmonary disease
(COPD), bronchitis, bronchiectasis, cystic fibrosis (CF),
vasculitis, mucus plugging, Wegener's granulomatosis, pneumonia,
tuberculosis, cancer involving the lungs or the respiratory tract,
Kartagener syndrome, Young's syndrome, chronic sinopulmonary
infection, alpha 1-antitrypsin deficiency, primary
immunodeficiency, acquired immune deficiency syndrome,
opportunistic infection, an infectious state, a post infectious
state, common cold, exercise induced hypersecretion of mucus,
inflammatory bowel disease, ulcerative colitis, Crohn's disease,
respiratory infection, respiratory obstruction, inhalation or
aspiration of a toxic gas, pulmonary aspiration, or alcoholism in a
subject with an elevated level of mucus in his or her respiratory
tract. The methods include administering (a) a composition
containing a therapeutically effective amount of a protein or
peptide having a sialidase or an active portion thereof to a
subject, (b) a composition containing a therapeutically effective
amount of a fusion protein, wherein the fusion protein has at least
one catalytic domain of a sialidase, wherein the catalytic domain
of the sialidase includes the sequence of amino acids extending
from amino acid 274 to amino acid 666 of SEQ ID NO:12
(alternatively, 274 to 666, 270-667, 274-681, 290-681 of SEQ ID
NO:12, or any other catalytic domain of Actinomyces viscosis),
inclusive, and at least one anchoring domain, wherein the anchoring
domain is a glycosaminoglycan (GAG) binding domain of human
amphiregulin including the amino acid sequence of SEQ ID NO:7, or
(c) a composition or compound containing a therapeutically
effective amount of a fusion protein having a sialidase or an
active portion thereof and an anchoring domain. The therapeutically
effective amount of these compositions or compounds includes an
amount that results in a reduction of the quantity of mucus in the
respiratory tract after administration of the composition when
compared to the quantity of mucus present prior to administration
of the composition or compound.
[0203] Yet other embodiments include methods of limiting an
increase in the quantity of mucus in the respiratory tract of a
subject above a baseline level of mucus in said subject's
respiratory tract. The methods include administering (a) a
composition or compound containing a therapeutically effective
amount of a protein or peptide having a sialidase or an active
portion thereof to a subject, (b) a composition or compound
containing a therapeutically effective amount of a fusion protein,
wherein the fusion protein has at least one catalytic domain of a
sialidase, wherein the catalytic domain of the sialidase includes
the sequence of amino acids extending from amino acid 274 to amino
acid 666 of SEQ ID NO:12 (alternatively, 274 to 666, 270-667,
274-681, 290-681 of SEQ ID NO:12, or any other catalytic domain of
Actinomyces viscosis), inclusive, and at least one anchoring
domain, wherein the anchoring domain is a glycosaminoglycan (GAG)
binding domain of human amphiregulin including the amino acid
sequence of
[0204] SEQ ID NO:7, or (c) a composition or compound containing a
therapeutically effective amount of a fusion protein having a
sialidase or an active portion thereof and an anchoring domain. The
therapeutically effective amount of these compositions or compounds
includes an amount that limits an increase in the quantity of mucus
in the respiratory tract of the subject above a baseline level
after administration of the composition.
[0205] In some embodiments, the compositions or compounds used can
include additional compounds, including, without limitation, any
one or more of the following either alone or in various
combinations: Na.sub.2SO.sub.4, MgSO.sub.4, CaCl.sub.2), Histidine,
Histine-HCl, and Trehalose or their analogs, Mg salts and/or Ca
salts. These additional compounds can be included in the
pharmaceutical compositions to act as excipients or as active
ingredients that provide additional beneficial effects.
[0206] The subjects to be treated with the foregoing methods can be
human subjects or non-human animal subjects. The compounds and
compositions described herein can be administered to epithelial
cells of the subject through various routes of administration,
including, without limitation, by using inhalers to introduce the
compounds or compositions into the respiratory tract of the
subject.
[0207] In some preferred embodiments, compounds described herein
can be delivered as an inhalant with an inhaler or as a nasal
spray. They can also be administered as eye drops, ear drops, or
sprays, ointments, lotions, or gels to be applied to the skin. They
can also be administered intravenously or as a local injection.
Reducing or Preventing Inflammation in the Respiratory Tract
[0208] The present invention involves the unexpected discovery that
administration of the compounds described in U.S. application Ser.
Nos. 10/718,986 and 10/939,262, or compositions containing them, to
reduce the amount of inflammatory cells in the respiratory tract.
Thus, the invention relates to therapeutic compositions or
compounds that can be used to reduce inflammation in the
respiratory tract or prevent worsening of inflammation in the
respiratory tract. The invention also includes methods of reducing
inflammation in the respiratory tract or preventing worsening of
inflammation in the respiratory tract. In addition, the invention
relates to therapeutic compositions or compounds that can be used
to prevent or treat diseases that are caused by, cause, or are
exacerbated by respiratory inflammation, such as, both allergic and
non-allergic asthma, allergic rhinitis, eczema, psoriasis,
reactions to plant or animal toxins, autoimmune conditions, and any
other disorder, disease or condition that causes inflammation in
the respiratory tract or is caused by or exacerbated by
inflammation in the respiratory tract.
[0209] In some preferred embodiments, the methods include
administering a composition or compound containing a
therapeutically effective amount of a protein or peptide having a
sialidase or an active portion thereof to a subject. The protein or
peptide can be an isolated naturally occurring sialidase protein,
or a recombinant protein substantially homologous to at least a
portion of a naturally occurring sialidase. A preferred
pharmaceutical composition contains a sialidase with substantial
homology to the A. viscosus sialidase (SEQ ID NO:12) or substantial
homology to an active portion thereof, such as amino acids 274-666,
274-667, 270-667, 274-681, or 290-681 of SEQ ID NO:12, or any other
catalytic domain of Actinomyces viscosis sialidase. The
therapeutically effective amount includes an amount of the protein
or peptide that prevents or reduces an allergic or inflammatory
response in the respiratory tract the respiratory tract after
administration of the composition or compound.
[0210] In other embodiments, the methods include administering a
composition or compound containing a therapeutically effective
amount of a fusion protein, wherein the fusion protein has at least
one catalytic domain of a sialidase, wherein the catalytic domain
of the sialidase includes the sequence of amino acids extending
from amino acid 274 to amino acid 666 of SEQ ID NO:12
(alternatively, 274 to 666, 270-667, 274-681, 290-681 of SEQ ID
NO:12, or any other catalytic domain of Actinomyces viscosis),
inclusive, and at least one anchoring domain, wherein the anchoring
domain is a glycosaminoglycan (GAG) binding domain of human
amphiregulin including the amino acid sequence of SEQ ID NO:7. The
therapeutically effective amount includes an amount of the fusion
protein that prevents or reduces an allergic or inflammatory
response in the respiratory tract the respiratory tract after
administration of the composition or compound.
[0211] In yet other embodiments, the methods include administering
a composition or compound containing a therapeutically effective
amount of a fusion protein having a sialidase or an active portion
thereof and an anchoring domain. The therapeutically effective
amount includes an amount of the fusion protein that prevents or
reduces an allergic or inflammatory response in the respiratory
tract the respiratory tract after administration of the composition
or compound.
[0212] In some embodiments, the compositions or compounds used can
include additional compounds, including, without limitation, any
one or more of the following either alone or in various
combinations: Na.sub.2SO.sub.4, MgSO.sub.4, CaCl.sub.2), Histidine,
Histine-HCl, and Trehalose or their analogs. These additional
compounds can be included in the pharmaceutical compositions to act
as excipients or as active ingredients that provide additional
beneficial effects.
[0213] The subjects to be treated with the foregoing methods can be
human subjects or non-human animal subjects. The compositions and
compounds described herein can be administered to epithelial cells
of the subject through various routes of administration, including,
without limitation, by using inhalers to introduce the compounds or
compositions into the respiratory tract of the subject.
[0214] In some preferred embodiments, compositions or compounds
described herein can be delivered as an inhalant with an inhaler or
as a nasal spray. They can also be administered as eye drops, ear
drops, or sprays, ointments, lotions, or gels to be applied to the
skin. They can also be administered intravenously or as a local
injection. FIGS. 7A-B show the results of the effect of the use of
one of the fusion protein construct depicted in FIG. 5 on
inflammatory cells of ferrets infected with human unadapted
influenza. In ferrets that shed the virus despite treatment with
fusion protein (n=8), the inflammatory response was reduced and
animals appeared to be more alert and active compared to the
untreated ferrets that were invariably lethargic and feverish. For
this group of 8 infected, fusion-protein treated animals, the mean
AUC (area under the curve) value calculated for the nasal protein
concentrations was reduced by approximately 40% (2.68 vs. 4.48,
arbitrary units) compared to the vehicle-treated (phosphate buffer
saline) infected animals (FIG. 7B). In vehicle-treated infected
animals, the number of inflammatory cells in nasal washes was
increased to approximately 100-fold above those in uninfected
animals on day 2 post challenge. These levels were sustained for 4
additional days. The fusion protein-treated animals exhibited a
significant reduction in the number of inflammatory cells in the
nasal washes. Specifically, the AUC value for cell counts was
reduced by approximately 3-fold in the fusion protein-treated
animals compared to the vehicle-treated infected animals (1965 vs.
674, arbitrary units, (FIG. 7B). The observed reduction in the
inflammatory response indicates the importance of inhibiting viral
replication at the early stage of infection.
Dosage
[0215] As will be readily apparent to one skilled in the art, the
useful in vivo dosage to be administered and the particular mode of
administration will vary depending upon the age, weight and type of
patient being treated, the particular pharmaceutical composition
employed, and the specific use for which the pharmaceutical
composition is employed. The determination of effective dosage
levels, that is the dose levels necessary to achieve the desired
result, can be accomplished by one skilled in the art using routine
methods as discussed above. In non-human animal studies,
applications of the pharmaceutical compositions are commenced at
higher dose levels, with the dosage being decreased until the
desired effect is no longer achieved or adverse side effects are
reduced or disappear. The dosage for a compound of the present
invention can range broadly depending upon the desired affects, the
therapeutic indication, route of administration and purity and
activity of the compound. Typically, human clinical applications of
products are commenced at lower dosage levels, with dosage level
being increased until the desired effect is achieved.
Alternatively, acceptable in vitro studies can be used to establish
useful doses and routes of administration of the test compound.
Typically, dosages can be between about 1 ng/kg and about 10 mg/kg,
between about 10 ng/kg and about 1 mg/kg, and between about 100
ng/kg and about 100 micrograms/kg. In various examples described
herein, mice were treated with various dosages of the compositions
described herein, including dosages of 0.0008 mg/kg, 0.004 mg/kg,
0.02 mg/kg, 0.06 mg/kg, 0.1 mg/kg, 0.3 mg/kg, 0.6 mg/kg, and 1.0
gm/kg. As nonlimiting examples, the compositions described herein
can be administered to humans in doses of between about 0.01 mg and
about 100 mg, such as about 0.05 mg, 0.06 mg, 0.1 mg, 0.5 mg, 1 mg,
5 mg, 10 mg, 20 mg, 50 mg, or 100 mg, and can be administered one
or more times per day, for one day, two days, three days, four
days, five days, six days, seven days, eight days, nine days, ten
days, eleven days, twelve days, thirteen days, or fourteen or more
days. Higher doses or lower doses can also be administered. In one
embodiment, as shown in Example 3 below, a dose of 0.06 mg/kg of a
sialidase compound is sufficient to desialylate muscarinic
receptors resulting in reduced airway responsiveness to muscarinic
receptor agonists, and thus potentially resulting in reducing
airway constriction, airway hypersensitivity, inflammation,
allergies or associated responses, such as bronchoconstriction,
asthma, and mucus overproduction. Efficacy in low doses, such as
0.06 mg/kg (translating in adult humans into a dose of about 4 or 5
mg), or 0.02 mg/kg (translating in adult humans into a dose of
about 1 or 2 mg), makes the sialidase-based compounds described
herein good candidates for use in chronic diseases that require
repeated long-term administration.
[0216] A treatment regimen can include administration of the
compounds and compositions described herein from once per day to
ten times per day, from once per day to six times per day, from
once per day to five times per day, from once per day to four times
per day, from once per day to three times per day, from once per
day to twice per day, and just once per day. The treatment can last
from just one day to daily, weekly, monthly, or other periodic use
for a predetermined period of time or for the remainder of the
subject's life.
[0217] The exact formulation, route of administration and dosage
can be chosen by the individual physician in view of the patient's
condition (see, Fingle et al., in The Pharmacological Basis of
Therapeutics (1975)). It should be noted that the attending
physician would know how to and when to terminate, interrupt or
adjust administration due to toxicity, organ dysfunction or other
adverse effects. Conversely, the attending physician would also
know to adjust treatment to higher levels if the clinical response
were not adequate. The magnitude of an administrated does in the
management of the disorder of interest will vary with the severity
of the condition to be treated and to the route of administration.
The severity of the condition may, for example, be evaluated, in
part, by standard prognostic evaluation methods. Further, the dose
and perhaps dose frequency, will also vary according to the age,
body weight and response of the individual patient, including those
for veterinary applications.
[0218] In some preferred regimens, appropriate dosages are
administered to each patient by either inhaler, nasal spray, or by
topical application. It will be understood, however, that the
specific dose level and frequency of dosage for any particular
patient may be varied and will depend upon a variety of factors
including the activity of the specific salt or other form employed,
the metabolic stability and length of action of that compound, the
age, body weight, general health, sex, diet, mode and time of
administration, rate of excretion, drug combination, the severity
of the particular condition, and the host undergoing therapy.
[0219] In some embodiments, the present disclosure provides methods
for using any one or more of the compositions (indicated below as
`X`) disclosed herein in the following methods:
[0220] Substance X for use as a medicament in the treatment of
excess mucus or abnormal (e.g., above normal mucus levels as
compared to one or more healthy subjects (e.g., of the same
ethnicity and/or in the same or similar geographical location)
and/or as indicated by a health care practitioner), elevated mucus
production, and/or any one or more of the diseases/conditions
disclosed herein; (each of which is collectively referred to in the
following examples as `Y.`
[0221] Use of substance X for the manufacture of a medicament for
the treatment of Y; and
[0222] Substance X for use in the treatment of Y.
EXAMPLES
[0223] The invention is further described in the following
examples, which do not limit the scope of the invention described
in the claims.
Example 1. Effect of Sialidase Treatment on the Early and Late
Asthmatic Reaction in Guinea Pigs
[0224] 1. Overview
[0225] In this study Fludase.RTM. was tested in a guinea pig model
of allergic asthma. Guinea pigs were sensitised with ovabumin (OVA)
or saline and after 15 or 20 days they were treated with
Fludase.RTM. or sodium sulfate. On day 21, all the animals were
challenged with OVA to measure the early asthmatic reaction. Airway
compliance/resistance were determined and broncho-alveolar lavage
(BAL) fluid was taken from the left lung to count the total number
of cells and to differentiate them.
[0226] 2. Introduction
[0227] The main purpose of this study was to achieve a
characterization of the effect of Fludase.RTM. on the early and
late reactions in a guinea pig model for asthma. The guinea pigs
involved in the study were naive, and thus not infected with
influenza or other infectious agent as part of the experiment.
Asthma was induced by sensitising the guinea pigs on day 0 with
OVA. After 15 or 20 days the guinea pigs were treated intatracheal
with Fludase.RTM. (0.3 mg/kg) or sodium sulfate (0.143 mM, pH 5.0).
On day 21 the guinea pigs received an OVA aerosol and the airway
responsiveness (PenH) was measured. On day 22 the pulmonary
resistance and compliance were determined. At time intervals of 2
minutes doses of histamine from 0.2-2 .mu.g/kg were administered by
intravenously injection. At the end of the experiment guinea pigs
were sacrificed and the left lung was lavaged and the isolated BAL
cells were washed, counted and differentiated into macrophages,
lymphocytes, neutrophils and eosinophils.
[0228] 3. Materials & Methods
[0229] Animals
[0230] Male Hartley-strain guinea pigs (HSD Poc: DH, weighing
400-500) of specific pathogen free quality were obtained from
Harlan-CPB (Zeist, The Netherlands). They were used after 1 week of
acclimatisation to their housing conditions. Water and commercial
chow were allowed ad libitum. The experiments were approved by the
Animal Ethics Committee of the Utrecht University (Utrecht, The
Netherlands).
[0231] Sensitisation, Pre-Treatment & Challenge
[0232] Guinea pigs were sensitised with saline (solutions contains
100 mg/ml Al(OH).sub.3) or OVA (solution contains 20 .mu.g/ml OVA
and 100 mg/ml Al(OH).sub.3), administered intraperitoneally 0.5 ml
and subcutanously 5.times.0.1 ml, total injection volume 1 ml.
After 15 or 20 days animals were treated once with 0.3 mg/kg
Fludase.RTM. and the control animals were treated with sodium
sulfate (0.143 mM, pH 5.0) on day 20 by tracheal instillation. A
laryngoscope was used to facilitate the location of the epiglottis.
Then the Fludase.RTM. or sodium sulfate was given with a liquid
aerosol using the IA-1C MicroSprayer (Penn Century, Inc,
Philadelphia, USA). The guinea pig was in an upright position
during the tracheal instillation.
[0233] During this tracheal instillation the guinea pigs were
anaesthetized with 150 .mu.l of a mixture of Ketamine.RTM.,
Xylazin.RTM., Atropin and saline (3.5:3:1:3), injected intra
muscular in the hind paw.
[0234] On day 21 the guinea pigs were challenged by exposure to an
aerosol OVA (0.1% wt/vol in sterile saline). The aerosol was
generated into a 3 liter perspex chamber in which the guinea pigs
were placed. First the basal bronchoconstriction (PenH) was
measured. The guinea pigs were provoked with OVA aerosol for 10
seconds. Directly after the challenge the early asthmatic reaction
was (PenH) was measured.
[0235] Allergen-Induced Early Asthmatic Reaction in Conscious
Unrestrained Guinea Pigs
[0236] Airway function of the animals was measured directly after
exposure to aerosolised OVA in a ventilated bias flow whole body
plethysmograph (Buxco Electronics, Sharon Conn., USA). The
plethysmograph consists of a reference chamber and an animal
chamber. The animal chamber is attached to the outside via a
pneumotachograph in the top of the plethysmograph. An aerosol inlet
to the animal chamber is centrically located in the roof of the
animal chamber. When an animal is placed in the animal chamber and
is breathing quietly, it creates pressure between tidal volume and
thoracic movement during respiration. The differential pressure
transducer measures the changes in pressure between animal chamber
and the reference chamber and brings these data to a preamplifier.
Thereafter, data is sent to a computer where several parameters are
calculated, which represents animal's lung function. All guinea
pigs used were measured basal for 5 minutes and after the aerosol
for 15 minutes in the whole body plethysmograph. Besides known lung
function parameters as peak expiratory flow (PEF) and tidal volume
(TV), the enhanced pause (PenH) was also measured. The formula and
explanation of the PenH is shown in FIG. 8. During
bronchoconstriction peak expiratory flow and peak inspiratory flow
are increased, while relaxation time and expiratory time are
decreased. This results in an increased PenH. Data from
bronchoconstriction in conscious unrestrained guinea pigs are
presented in PenH (FIG. 8).
Airway Responsiveness In Vivo
[0237] On day 22 the guinea pigs were anaesthetized with urethane 2
g/kg intra peritoneally. The animals were allowed to breathe
spontaneously. An anaesthesia-induced fall in body temperature was
avoided by placing the animals in a heated chamber, which kept the
body temperature at 37.degree. C. The guinea pigs were prepared for
the measurement of pulmonary resistance (R.sub.L) and compliance
(C) as follows. A small polyethylene catheter (PE-50) was placed in
the right jugular vein for intra venous administration of
increasing doses of histamine (0.2-2 .mu.g/kg). First the basal
R.sub.L and C were measured for 5 minutes. Thereafter an increasing
dose of histamine was injected and R.sub.L and C were measured for
2 minutes. Airflow and tidal volume were determined by cannulating
and connecting the trachea with Fleisch flow head (nr 000;
Meijnhart, Bunnik, The Netherlands) to a pneumotachograph. A
pressure transducer (model MP45-2; Validyne Engineering Corp.,
Northridge, Calif.) measured the transpulmonary pressure by
determing pressure differences between the tracheal cannula and a
cannula filled with saline inserted in the oesophagus. R.sub.L and
C were determined breath by breath with a respiratory analyser.
R.sub.L was yielded by dividing transpulmonary pressure by airflow
at isovolume points. C was determined by dividing volume by
transpulmonary pressure at isoflow points. Data are presented as
maximal R.sub.L and minimal C in cm H.sub.2O/ml*sec.sup.-1 an ml/cm
H.sub.2O, respectively.
[0238] Collection of Broncho-Alveolar Lung Lavage Cells
[0239] Broncho-alveolar lavage cells were obtained as follows. The
trachea was trimmed free of connective tissue and blood vessels and
a small incision was made for insertion of a cannula into the
trachea. The right lung was tied up so only the left lung was
lavaged. The left lung was filled with 5 ml saline (0.9% NaCl) of
37.degree. C. in situ. Fluid was withdrawn from the lung after
gentle massage and collected in a plastic tube on ice (4.degree.
C.). This procedure was repeated 3 times (total 15 ml) and the cell
suspensions recovered from each animal were pooled. Thereafter,
cells were sedimented by centrifugation at 1500 rpm for 5 minutes
at 4.degree. C. The supernatant solution was thrown away and the
pellet was resuspended in 1 ml saline. Only plastic tubes were used
throughout the isolation procedure in order to minimize adherence
of the cells to the walls of the tubes.
[0240] Cell Count and Differentiation
[0241] The cells were stained with Turk solution and counted in a
Burker-Turk bright-line counting chamber (microscope, magnification
100.times.). For differential BAL cell counts cytospin preparations
were made and stained with Diff-Quick (Merz & Dade A. G.,
Dudingen, Switzerland). After coding all cytospin preparations were
evaluated by one observer using oil immersion microscopy
(magnification 1000.times.). Cells were differentiated into
macrophages, lymphocytes, neutrophils and eosinophils by standard
morphology. At least 200 cells per cytospin preparation were
counted and the absolute number of each cell type was
calculated.
[0242] Statistical Analysis
[0243] Unless stated otherwise, data are expressed as arithmetic
average .+-.standard error of mean and comparisons between groups
were made using Student's t-test. A probability value p<0.05 was
considered significant.
[0244] 4. Results
[0245] Airway Responsiveness in Conscious Unrestrained Guinea
Pigs
[0246] As shown in FIG. 9, basal airway resistance was not
different between the saline guinea pigs treated with sodium
sulfate or DAS181 on day 15 or day 20 (PenH=0.24.+-.0.009
sal-sodium sulfate, 0.25.+-.0.01 saline-Flu day 15, 0.28.+-.0.01
sal-Flu day 20 treated guinea pigs). There was also no difference
between the OVA guinea pigs treated with sodium sulfate or
Fludase.RTM. on day 15 or day 20 at basal level (PenH=0.25.+-.0.01
OVA-sodium sulfate, 0.27.+-.0.02 OVA-Flu day 15, 0.31.+-.0.03
OVA-Flu day 20 treated guinea pigs).
[0247] Ova challenge slightly increased the basal airway resistance
in saline sensitized animals (SOD, Flu 15 and 20, FIG. 2). However,
the early asthmatic reaction in response to the OVA aerosol was
strongly increased in the OVA-sodium sulfate treated guinea pigs
(PenH=1.57.+-.0.45). After treatment with Fludase.RTM. the early
asthmatic reaction was decreased by nearly 30% on day 15, but not
on day 20.
[0248] Cell Count in Broncho-Alveolar Lavage Fluid
[0249] As shown in FIG. 10, Fludase.RTM. decreases the total number
of cells both in saline and OVA guinea pigs.
[0250] Differential Cell Count in the Broncho-Alveolar Lavage
Fluid
[0251] As shown in FIG. 11, the number of macrophages was enhanced
by (30%) in the OVA guinea pigs compared to the saline treated
group. Total number of macrophages was strongly decreased after
treatment with Fludase.RTM. on day 15 and 20, both in saline and
OVA treated guinea pigs.
[0252] As shown in FIG. 12, a similar pattern was observed with the
number of lymphocytes. The number of lymphocytes is decreased after
Fludase.RTM. treatment.
[0253] As shown in FIG. 13, compared to historical controls, SOD
induces a strong increase in the number of neutrophils into the
lungs. Interestingly, Fludase.RTM. completely prevented this influx
in both the saline and OVA groups.
[0254] As shown in FIG. 14, compared to historical controls SOD
induces also an eosinophil influx into the airways, which is
further increased by OVA challenge. Interestingly, Fludase.RTM.
treatment can restore the number of eosinophils up to historical
control levels.
[0255] 5. Discussion/Conclusion
[0256] OVA-sensitised and challenged animals demonstrate an early
and late asthmatic reaction as measured by an increase in PENH up
on OVA-challenge, and an increase in the number of inflammatory
cells in the BAL-fluid.
[0257] Interestingly, Fludase.RTM. (Flu15) reduced the early
asthmatic response by nearly 30% (FIG. 9), suggesting an effect of
this compound on mast cell stimulation. Moreover, Fludase.RTM. had
a tremendous effect on the inflammation caused by SOD or OVA. The
number of all inflammatory cells (macrophages, lymphocytes,
neutrophils and eosinophils) was significantly decreased by this
compound.
[0258] In conclusion, Fludase.RTM. demonstrated to be effective in
both the SOD-induced inflammation and the OVA-induced
inflammation.
Example 2. The Effect of Fludase in a Mouse Model of Acute OVA
Induced Asthma
[0259] 1. Introduction
[0260] The aim of the study was to investigate whether Fludase.RTM.
(also referred to as DAS181) (1) inhibits allergen induced airway
inflammation and airway hyperreactivity in an acute OVA challenge
mouse model of asthma. In addition to studying the effect of DAS
181 alone as an intervention, three other interventions in the
mouse asthma model were studied including a) DAS181+an excipient
(used as the dry powder formulation to deliver DAS181 in vivo), b)
the excipient alone, and c) dexamethasone (as a comparator).
[0261] 2. Materials and Methods
[0262] Mice
[0263] Female BALB/c mice age 12 weeks were purchased from Charles
River and housed in the UCSD vivarium. They were used after
approximately one week of acclimatization in the UCSD vivarium.
[0264] OVA Sensitization and OVA Challenge
[0265] Mice were immunized s.c. on days 0, 7, 14, and 21 with 25 ug
of OVA adsorbed to 1 mg of alum in 200 ul normal saline to induce a
predominant Th2 immune response. Intranasal OVA challenges (20
ug/50 ul) were started on day 26 and then repeated on day 28 and
day 30. In the no OVA group, mice were sensitized to OVA but not
challenged intranasally with OVA. Mice had airway responsiveness to
methacholine (Mch) measurements performed 24 hours after the final
OVA challenge by Penh on day 31. The mice involved in the study
were naive and thus not infected with influenza or other infectious
agent as part of the experiment. Mice were then immediately
sacrificed. Bronchoalveolar lavage fluid (BAL), blood, and lung
tissue were processed for outcomes detailed below.
[0266] Administration of Test Compounds to Mice
[0267] 1. Compounds Tested
[0268] The following compounds were studied in the mouse model of
asthma [0269] a) DAS181 with excipient (0.6 mg/kg intranasal):
DAS181-F02 (NexBio, Inc. part #43-071, lot #47-034) was prepared in
PBS to 20 mgDAS/ml. Before each dosing, it was freshly diluted in
PBS to 0.6 mgDAS181/kg with dosing volume of 50 ul, for mice with
an average body weight of 21 g. [0270] b) DAS181 (0.6 mg/kg
intranasal) [0271] c) Excipient (50 .mu.L/mouse): Excipient
(416TL022A) was supplied by NexBio, Inc. as a solution, and the
concentration of each of the excipient component is equivalent to
that in 20 mgDAS181/ml (MgSO.sub.4 1.446 mg/ml, CaCl.sub.2) 0.059
mg/ml, Histidine 1.427 mg/ml, Histidine-HCl 1.943 mg/ml, and
Trehalose 3.000 mg/ml). It was freshly diluted in PBS for dosing as
in a). The final concentration of each excipient component is
equivalent to that in 0.6 mgDAS181/kg, in 50 ul, for mice with
average body weight of 21 g. [0272] d) Dexamethasone (1.0 mg/kg
intraperitoneally)
[0273] Excipient solution was prepared in the following manner. The
target final concentration for each excipient was calculated to
reach equivalent concentration when DAS181-F02 bulk dry powder is
reconstituted at a 20 mg protein/mL. The 10.times. stock solutions
(100.times. for Calcium Chloride) for each excipient (10 mL each)
were then prepared. Materials used to prepare the stock solutions
are listed in Table 1. All materials are USP grade, or equivalent.
Appropriate amounts of each excipient were weighed into a 15 mL
conical tube according to Table 2, and water was added to bring the
total weight to 10 grams and vortex to dissolve the material
completely. The final 1.times. excipient solution was prepared by
adding 1 mL of each stock solution, and then bringing the volume to
10 mL using water. All sample preparation was preformed
gravimetrically, assuming solution density of 1 gram/mL.
[0274] Although referred to as "excipient" or "excipient" solution
in these examples, these additional compounds can have additional
beneficial effects with respect to reduction of mucus and reduction
of inflammation and inflammatory cells. These excpients can also
have a synergistic effect with DAS181.
TABLE-US-00001 TABLE 1 Material Information Description
Manufacturer Mfg. Part # Mfg. Lot # Expiry L-Histidine Sigma
H6034-100g 078K0179 September 2012 L-Histidine Sigma H4036-1kg
068K8310 January 2012 monohydrochloride monohydrate a,a -
Trehalose, Dihydrate J. T. Baker 4226-04 G47596 November 2010
Magnesium Sulfate EMD 1.05882.0500 K38528682R February 2013
Heptahydrate Calcium Chloride, Dihydrate Mallinckrodt 4616-04
G24475 September 2009 WFI B. Braun S9200-SS J8K015 August 2010
TABLE-US-00002 TABLE 2 Stock Solution Preparation Sheet
(Theoretical) Final conc. Dilution Stock conc. Wt. of Salt
Composition (mg/mL) Factor (mg/mL) for 10 mL (mg) MgSO.sub.4 1.446
10 14.457 296 CaCl.sub.2 0.059 100 5.943 79 Histidine 1.427 10
14.266 143 Histidine.cndot.HCl 1.943 10 19.431 213 Trehalose 3.000
10 30.000 332
[0275] 2. Mouse Model of Asthma
[0276] The following groups of Balb/c mice (n=10 female mice/group)
were studied.
[0277] a) No OVA
[0278] b) OVA
[0279] c) OVA+DAS181 with excipient
[0280] d) OVA+DAS181
[0281] e) OVA+excipient
[0282] f) OVA+dexamethasone
[0283] 3. Timing of Administration of Test Compounds
[0284] The test compounds were administered one hour prior to each
of the three intranasal OVA challenges on days 26, 28, and 30.
[0285] Timing of End-Points Studied
[0286] Mice were sacrificed 24 hours after the final OVA challenge
and blood, BAL, and lungs were analyzed (27).
[0287] 4. End-Points Studied
[0288] a) Penh
[0289] Airway responsiveness was assessed on day 31, twenty four
hours after the final OVA inhalation, using a single chamber whole
body plethysmograph obtained from Buxco (Troy, N.Y.). In this
system, an unrestrained, spontaneously breathing mouse is placed
into the main chamber of the plethysmograph, and pressure
differences between this chamber and a reference chamber are
recorded. The resulting box pressure signal is caused by volume and
resultant pressure changes during the respiratory cycle of the
mouse. A low pass filter in the wall of the main chamber allows
thermal compensation. From these box pressure signals, the phases
of the respiratory cycle, tidal volumes, and the enhanced pause
(Penh) can be calculated. Penh is a dimensionless value that
represents a function of the proportion of maximal expiratory to
maximal inspiratory box pressure signals and of the timing of
expiration. It correlates closely with pulmonary resistance
measured by conventional two-chamber plethysmography in ventilated
mice. In the plethysmograph, mice were exposed for 3 min to
nebulized PBS and subsequently to increasing concentrations of
nebulized metacholine (MCh)(3, 6, 12, 24, 48 mg/ml Mch) (Sigma, St.
Louis, Mo.) in PBS using an Aerosonic ultrasonic nebulizer
(DeVilbiss). After each nebulization, recordings were taken for 3
min. The Penh values measured during each 3-min sequence were
averaged and are expressed for each MCh concentration as the
percentage of baseline Penh values following PBS exposure.
[0290] b) Blood Eosinophil Counts
[0291] Peripheral blood was collected from mice by cardiac puncture
into EDTA-containing tubes. Erythrocytes were lysed using a 1:10
solution of 100 mM potassium carbonate-1.5 M ammonium chloride. The
remaining cells were resuspended in 1 mL PBS. To perform
differential cell counts, 200 .mu.L resuspended peripheral-blood
leukocyte suspensions were cytospun onto microscope slides and
air-dried. Slides were stained with Wright-Giemsa and the % of
eosinophils in the total number of white blood cells were assessed
under a light microscope.
[0292] c) PAS Staining for Lung Mucus
[0293] To quantitate the level of mucus expression in the airway,
the number of periodic acid Schiff (PAS)-positive and PAS-negative
epithelial cells in individual bronchioles were counted as
previously described (Zhang, M., T. Angata, J. Y. Cho, M. Miller,
D. H. Broide, A. Varki. 2007 Blood. 109:4280-4287). At least ten
bronchioles were counted in each slide. Results are expressed as
the percentage of PAS-positive cells per bronchiole, which is
calculated from the number of PAS-positive epithelial cells per
bronchus divided by the total number of epithelial cells of each
bronchiole. Slides of lung tissue with no OVA, OVA and OVA+DAS181
were also taken and observed.
[0294] d) MBP Staining of Lungs for Peribronchial Eosinophils
[0295] Lungs from the different experimental groups were processed
as a batch for either histologic staining or immunostaining under
identical conditions as described in Zhang et al. Stained and
immunostained slides were all quantified under identical light
microscope conditions, including magnification (20 X), gain, camera
position, and background illumination. Lung sections were processed
for MBP immunohistochemistry as described above, using an
anti-mouse MBP (Major Basic Protein) Ab (kindly provided by James
Lee PhD, Mayo Clinic, Scottsdale, Ariz.) and the immunoperoxidase
method as previously described in Zhang et al. Major Basic Protein
is an eosinophil cytoplasmic granule protein which serves as a
marker of eosinophils in tissues. The number of individual cells
staining positive for MBP in the peribronchial space were counted
using a light microscope. Results are expressed as the number of
peribronchial cells staining positive for MBP per bronchiole with
150-200 .mu.m of internal diameter. At least ten bronchioles were
counted in each slide.
[0296] 5. Statistical Analysis
[0297] Results in the different groups of mice were compared by
Mann Whitney non-parametric T test. All results are presented as
mean+SEM. A statistical software package (Graph Pad Prism, San
Diego, Calif.) was used for the analysis. P values of <0.05 were
considered statistically significant.
[0298] 6. Results
[0299] a) Penh
[0300] As shown in FIG. 15, OVA challenge induced a significant
increase in airway responsiveness as assessed by changes in Penh
(OVA vs no OVA; p<0.0001).
[0301] OVA challenged mice pre-treated with DAS181+excipient had a
significant reduction in Penh compared to OVA challenged mice (OVA
vs OVA+DAS181+excipient; p<0.01).
[0302] OVA challenged mice pre-treated with DAS181 resulted in a
reduction in Penh compared to OVA challenged mice (OVA vs
OVA+DAS181; p<0.005).
[0303] As shown in FIG. 16, measurement of Penh at 48 mg/ml Mch
provides the largest difference between positive and negative
controls (no OVA vs OVA) and is why this dose of Mch is used to
assess the effect of an intervention such as DAS181.
[0304] b) Blood Eosinophils
[0305] As shown in FIG. 17, OVA challenge induced a significant
increase in blood eosinophils (OVA vs no OVA; p<0.0005).
[0306] DAS 181 significantly reduced blood eosinophils (OVA vs
OVA+DAS, p=0.04)
[0307] c) PAS Staining for Lung Mucus
[0308] As shown in FIGS. 18-19A-F, OVA challenge induced a
significant increase in the % of airway epithelium staining
positive for PAS (OVA vs no OVA; p<0.0001).
[0309] OVA challenged mice pre-treated with DAS181+excipient had a
significant reduction in PAS staining compared to OVA challenged
mice (OVA vs OVA+DAS181+excipient; p<0.0001).
[0310] OVA challenged mice pre-treated with DAS181 had a
statistically significant reduction in PAS staining compared to OVA
challenged mice (OVA vs OVA+DAS181; p<0.0001).
[0311] Effect on Mucus
[0312] DAS181 with excipient as well as DAS181 alone significantly
reduced PAS staining showing that there is an inhibitory effect of
DAS 181 on PAS staining. This shows that DAS181 with excipient or
DAS181 alone reduces mucus in the respiratory tract.
[0313] d) MBP Immunostaining of Lungs
[0314] As shown in FIG. 20, OVA challenge induced a significant
increase in the number of peribronchial MBP+eosinophils (OVA vs no
OVA; p<0.0001).
[0315] OVA challenged mice pre-treated with DAS181+excipient had a
significant reduction in the number of peribronchial
MBP+eosinophils compared to OVA challenged mice (OVA vs
OVA+DAS181+excipient; p=0.02).
Example 3. Reduced Airway Resistance in Naive Mice Treated
Intranasally with Low Doses of DAS181 (Methacholine Challenged)
[0316] The objective of the study was to test the effect of
different dose levels of DAS181 on muscarinic receptor mediated
airway resistance in naive mice. BALB/c mice (N=4) were treated
intranasally with PBS or DAS181 at 0.06, 0.1 or 0.6 mg/kg once
daily for three days. Eight hours post the final treatment animals
were challenged with increasing doses of muscarinic receptor
agonist Methacholine (Mch). Airway responsiveness was assessed
using whole body plethysmography. Changes in airway resistance were
expressed as the enhanced pause (Penh), a dimensionless value that
represents a function of the proportion of maximal expiratory to
maximal inspiratory box pressure signals and of the timing of
expiration. Mice were exposed to nebulized PBS and subsequently to
increasing concentrations of nebulized MCh (12, 24, 48 mg/ml Mch)
for 2 min in PBS. Recordings were performed for 3 min following
each exposure. The obtained Penh values were averaged and expressed
as the percentage of baseline following PBS exposure.
[0317] Results: 24 and 48 mg/ml of the muscarinic receptor agonist
methacholine increased airway resistance above baseline. All
animals treated with DAS181 had significantly reduced airway
resistance at 48 mg/ml of Mch (Fig). No difference was observed
between the different dose groups.
[0318] Conclusions: Consistent with previous data, intranasal
treatment with DAS181 reduced bronchoconstriction in response to
the muscarinic receptor agonist Mch, further supporting the
hypothesis that DAS181 dependent desialylation causes a reduction
in muscarinic receptor signaling. Surprisingly, the two higher dose
levels did not exert any greater effect than the lowest dose,
suggesting that a dosage level as low as 0.06 mg/kg of intranasal
DAS181 is sufficient to desialylate muscarinic receptors resulting
in reduced airway responsiveness to muscarinic receptor agonists,
and thus potentially resulting in reducing inflammation, allergies
or acetylcholine-associated responses, such as bronchoconstriction,
asthma, and mucus overproduction. These results are depicted in
FIGS. 21A and 21B.
Example 4. Reduced Airway Resistance in Naive Mice Treated
Intranasally with a Low Dose of DAS181 (Methacholine
Challenged)
[0319] The objective of the study was to test the effect of a 0.6
mg/kg once daily dose of DAS181 on muscarinic receptor mediated
airway resistance in naive mice. BALB/c mice (N=4) were treated
intranasally with PBS or DAS181 at 0.6 mg/kg once daily for three
days. Eight hours post the final treatment animals were challenged
with increasing doses of muscarinic receptor agonist Methacholine
(Mch). Airway responsiveness was assessed using whole body
plethysmography. Changes in airway resistance were expressed as the
enhanced pause (Penh), a dimensionless value that represents a
function of the proportion of maximal expiratory to maximal
inspiratory box pressure signals and of the timing of expiration.
Mice were exposed to nebulized PBS and subsequently to increasing
concentrations of nebulized MCh (3, 6, 12, 24, 48 mg/ml Mch) for 2
min in PBS. Recordings were performed for 3 min following each
exposure. The obtained Penh values were averaged and expressed as
the percentage of baseline following PBS exposure.
[0320] Results: 3, 6, 12, 24 and 48 mg/ml of the muscarinic
receptor agonist methacholine increased airway resistance above
baseline in PBS treated animals, and 12, 24 and 48 mg/ml of
methacholine increased airway resistance above baseline in DAS181
treated animals, while 3 and 6 mg/ml of methacholine did not
increase airway resistance above baseline in DAS181 treated
animals. All animals treated with DAS181 had significantly reduced
airway resistance at 6, 12, 24 and 48 mg/ml of Mch compared to the
control.
[0321] Conclusions: Consistent with previous data, intranasal
treatment with DAS181 reduced bronchoconstriction in response to
the muscarinic receptor agonist Mch, further supporting the
hypothesis that DAS181 dependent desialylation causes a reduction
in muscarinic receptor signaling. These results are depicted in
FIG. 22.
Example 5. Reduced Airway Resistance in Naive Mice Treated
Intranasally with DAS181 (Carbachol Challenged)
[0322] The objective of the study was to test the effect of a 0.6
mg/kg once daily dose of DAS181 on muscarinic receptor mediated
airway resistance in naive mice. BALB/c mice (N=4) were treated
intranasally with PBS or DAS181 at 0.6 mg/kg once daily for three
days. Eight hours post the final treatment animals were challenged
with increasing doses of muscarinic receptor agonist carbachol.
Airway responsiveness was assessed using whole body
plethysmography. Changes in airway resistance were expressed as the
enhanced pause (Penh), a dimensionless value that represents a
function of the proportion of maximal expiratory to maximal
inspiratory box pressure signals and of the timing of expiration.
Mice were exposed to nebulized PBS and subsequently to increasing
concentrations of nebulized Carbachol (1.25, 2.5, 5, 10, 20 mg/ml
carbachol) for 2 min in PBS. Recordings were performed for 3 min
following each exposure. The obtained Penh values were averaged and
expressed as the percentage of baseline following PBS exposure.
[0323] Results: 5, 10, and 20 mg/ml of the muscarinic receptor
agonist carbachol increased airway resistance above baseline in
both PBS treated and DAS181 animals. All animals treated with
DAS181 had significantly reduced airway resistance at 5, 10, and 20
mg/ml of carbachol.
[0324] Conclusions: Consistent with previous data, intranasal
treatment with DAS181 reduced bronchoconstriction in response to
the muscarinic receptor agonist carbachol, further supporting the
hypothesis that DAS181 dependent desialylation causes a reduction
in muscarinic receptor signaling. These results are depicted in
FIG. 23.
Example 6. Airway Resistance in Naive Mice Treated Intranasally
with a Low Dose of DAS185 (Methacholine Challenged)
[0325] The objective of the study was to test the effect of a 0.6
mg/kg once daily dose of DAS185 on muscarinic receptor mediated
airway resistance in naive mice. DAS185 is an enzymatically
inactive version of DAS181, in which a mutation in the sialidase
portion renders the sialidase inactive. BALB/c mice (N=4) were
treated intranasally with PBS or DAS185 at 0.6 mg/kg once daily for
three days. Eight hours post the final treatment animals were
challenged with increasing doses of muscarinic receptor agonist
Methacholine (Mch). Airway responsiveness was assessed using whole
body plethysmography. Changes in airway resistance were expressed
as the enhanced pause (Penh), a dimensionless value that represents
a function of the proportion of maximal expiratory to maximal
inspiratory box pressure signals and of the timing of expiration.
Mice were exposed to nebulized PBS and subsequently to increasing
concentrations of nebulized MCh (3, 6, 12, 24, 48 mg/ml Mch) for 2
min in PBS. Recordings were performed for 3 min following each
exposure. The obtained Penh values were averaged and expressed as
the percentage of baseline following PBS exposure.
[0326] Results: There was no difference in airway resistance in
response to the Mch challenge between the DAS185 treated and PBS
treated animals.
[0327] Conclusions: Whereas there was a difference in airway
resistance between DAS181 and PBS treated animals in example 4
above, there was no difference when DAS181 was replaced with
enzymatically inactive DAS185. This experiment shows that reduction
in airway resistance in response to DAS181 is sialidase dependent.
These results are depicted in FIG. 24.
Example 7. Time-Course of DAS181 Mediated Reduction of Airway
Resistance (Methacholine Challenged)
[0328] The objective of the study was to test the effect of a 0.6
mg/kg once daily dose of DAS181 for one, two or three days on
muscarinic receptor mediated airway resistance in naive mice.
BALB/c mice (N=4) were treated intranasally with PBS or DAS181 at
0.6 mg/kg once daily for one, two or three days. Eight hours post
the final treatment animals were challenged with increasing doses
of muscarinic receptor agonist Methacholine (Mch). Airway
responsiveness was assessed using whole body plethysmography.
Changes in airway resistance were expressed as the enhanced pause
(Penh), a dimensionless value that represents a function of the
proportion of maximal expiratory to maximal inspiratory box
pressure signals and of the timing of expiration. Mice were exposed
to nebulized PBS and subsequently to increasing concentrations of
nebulized MCh (3, 6, 12, 24, 48 mg/ml Mch) for 2 min in PBS.
Recordings were performed for 3 min following each exposure. The
obtained Penh values were averaged and expressed as the percentage
of baseline following PBS exposure.
[0329] Results: For one day of treatment, there was no difference
in airway resistance in response to the Mch challenge between the
DAS181 treated and PBS treated animals. For two days of treatment,
DAS181 appears to reduce airway resistance relative to PBS at 12
and 24 mg/ml methacholine, but not at 3, 6 and 48 mg/ml of
methacholine. At three days of treatment, DAS181 had significantly
reduced airway resistance at 24 and 48 mg/ml of Mch.
[0330] Conclusions: Consistent with previous data, there was a
difference in airway resistance between DAS181 and PBS treated
animals on day 3. There was no difference when following one
treatment dose, and partial reduction following two days of
treatment with DAS181. This experiment shows that 2-3 days of
treatment is optimal to achieve a reduction in airway resistance.
These results are depicted in FIG. 25.
Example 8. Reduced Airway Resistance in Naive Mice Treated
Intranasally with Very Low Doses of DAS181 (Methacholine
Challenged)
[0331] The objective of the study was to test the effect of
different low-dose levels of DAS181 on muscarinic receptor mediated
airway resistance in naive mice. BALB/c mice (N=4) were treated
intranasally with PBS or DAS181 at 0.0008, 0.004, 0.02, or 0.1
mg/kg once daily for three days. Eight hours post the final
treatment animals were challenged with increasing doses of
muscarinic receptor agonist Methacholine (Mch). Airway
responsiveness was assessed using whole body plethysmography.
Changes in airway resistance were expressed as the enhanced pause
(Penh), a dimensionless value that represents a function of the
proportion of maximal expiratory to maximal inspiratory box
pressure signals and of the timing of expiration. Mice were exposed
to nebulized PBS and subsequently to increasing concentrations of
nebulized MCh (12, 24, 48 mg/ml Mch) for 2 min in PBS. Recordings
were performed for 3 min following each exposure. The obtained Penh
values were averaged and expressed as the percentage of baseline
following PBS exposure.
[0332] Results: 24 and 48 mg/ml of the muscarinic receptor agonist
methacholine increased airway resistance above baseline. All
animals treated with DAS181 had significantly reduced airway
resistance at 24 and 48 mg/ml of Mch. No difference was observed
between the different dose groups of DAS181.
[0333] Conclusions: Intranasal treatment with very low doses of
DAS181 reduced bronchoconstriction in response to the muscarinic
receptor agonist Mch, further supporting the hypothesis that DAS181
dependent desialylation causes a reduction in muscarinic receptor
signaling even at very low doses of DAS181. This experiment shows
that dosage levels as low as 0.0008 mg/kg of intranasal DAS181 is
sufficient to desialylate muscarinic receptors resulting in reduced
airway responsiveness to muscarinic receptor agonists, and thus
potentially resulting in reducing inflammation, allergies or
acetylcholine-associated responses, such as bronchoconstriction,
asthma, and mucus overproduction. These results are depicted in
FIG. 26.
Example 9. The Reduced Airway Resistance in Naive Mice Treated
Intranasally with DAS181 is Dose-Dependent (Methacholine
Challenged)
[0334] The objective of the study was to test the effect of
different dose levels of DAS181 on muscarinic receptor mediated
airway resistance in naive mice. BALB/c mice (N=4) were treated
intranasally with PBS or DAS181 at 10 ng/kg, 0.1 .mu.g/kg, 1
.mu.g/kg, 10 .mu.g/kg or 0.1 mg/kg once daily for three days
followed by increasing doses of muscarinic receptor agonist
methacholine (Mch). Airway responsiveness was assessed using whole
body plethysmography. Changes in airway resistance were expressed
as the enhanced pause (Penh), a dimensionless value that represents
a function of the proportion of maximal expiratory to maximal
inspiratory box pressure signals and of the timing of expiration.
Mice were exposed to nebulized PBS and subsequently to increasing
concentrations of nebulized MCh (12, 24, 48 mg/ml Mch) for 2 min in
PBS. Recordings were performed for 3 min following each exposure.
The obtained Penh values were averaged and expressed as the
percentage of baseline following PBS exposure.
[0335] Results: 24 mg/ml and higher concentrations of the
muscarinic receptor agonist methacholine increased airway
resistance above baseline. The airway responsiveness was assessed 8
hours post-treatment on Day 3. All animals treated with DAS181
showed reduced airway resistance at 24 mg/ml of Mch, with average
percentage change Penh values (representing increasein airway
resistance over baseline) as follows:
[0336] Control (PBS): 550-560%
[0337] 10 ng/kg DAS181: 525-530%
[0338] 0.1 .mu.g/kg DAS181: 450%
[0339] 1 .mu.g/kg DAS181: 525%
[0340] 10 .mu.g/kg DAS181: 225%
[0341] 0.1 mg/kg DAS181: 200%
[0342] As seen from the above results, dose levels of 0.01 or 0.1
mg/kg of DAS181 significantly reduced airway resistance in response
to Mch.
[0343] Conclusion: The results demonstrate that reduced airway
resistance in response to intranasal treatment with DAS181 is
dose-dependent.
Example 10. Effect of DAS181 on M2 and M3 Muscarinic Receptor
Signaling In Vitro
[0344] The objective of the study was to assess muscarinic receptor
desialylation as a potential mechanism for airway protection, using
an in vitro model. Human chem-1 cells (Millipore) stably
transfected with M2 or M3 muscarinic receptor were treated with
0.4, 2 or 10 .mu.M of DAS181 for 30 min at 37.degree. C. prior to
the addition of a receptor agonist (acetylcholine; ACh). Receptor
signaling was determined using a fluorescence reporter for
intracellular calcium.
[0345] Results:
[0346] At all doses of DAS181 tested, treatment of human chem-1
cells with DAS181 increased the potency of agonist-mediated M2
receptor signaling and decreased the potency of agonist-mediated M3
receptor signaling. The results are shown below:
[0347] A. M2 Receptor
TABLE-US-00003 Treatment Predicted EC50 Potency Value (M) ACh alone
(no DAS181) 390 nM ACh + 10 .mu.M DAS181 140 nM ACh + 2 .mu.M
DAS181 130 nM ACh + 0.4 .mu.M DAS181 140 nM
[0348] B. M3 Receptor
TABLE-US-00004 Treatment Predicted EC50 Potency Value (M) ACh alone
(no DAS181) 6.3 nM ACh + 10 .mu.M DAS181 270 nM ACh + 2 .mu.M
DAS181 51 nM ACh + 0.4 .mu.M DAS181 110 nM
[0349] Conclusion:
[0350] The results demonstrate that DAS181 increases signaling
through the M2 muscarinic receptor and decreases signaling through
the M3 muscarinic receptor. Thus, the DAS181-mediated responses
could be indicative of positive allosteric modulation of the M2,
and either antagonist or negative modulation M3. Desialylation may
offer airway protection by reducing the stimulatory signal as well
as enhancing the inhibitory signal mediated by muscarinic
receptors.
Example 11. Therapeutic Efficacy of DAS181 Microparticle
Formulations Against Parainfluenza
[0351] The efficacy of DAS181 was tested against parainfluenza
virus (NV), which is an acute respiratory infection. A 63 year old
female patient tested positive for PIV on Jul. 14, 2010 and Jul.
21, 2010; shedding of the PIV antigen in nasal swabs and sputum
samples collected from the patient on those days was detected by
PCR.
[0352] The patient was treated on Jul. 23, 24 and 25, 2010 with one
capsule (10 mg delivered dose) a day of a dry powder formulation of
DAS181 whose components and wt/wt % in the composition are as
follows: DAS181: 64.54-64.69%; Histidine free base: 4.32-4.60%;
Histidine HCl: 5.85-6.27%; Trehalose: 9.06-9.68%; Magnesium
sulfate: 4.66-5.84%; Calcium chloride: 0.19%; Sodium acetate:
0.04-0.05%; Acetic acid: 0.02%; Water: 10%; Isopropanol: trace
amounts. Each capsule contained 13 mg of the dry powder in a type 3
clear HPMC capsule (Capsugel), giving a delivered dose of 10 mg.
The patient was administered one capsule a day for three days (Jul.
23, Jul. 24 and Jul. 25, 2010) by inhalation. The patient tested
positive for PIV on the day after completion of the treatment (July
26), and tested negative for PIV on the fifth day following
treatment (Jul. 30, 2010). The results demonstrate the
effectiveness of DAS181 against parainfluenza, i.e., a respiratory
infection of the upper respiratory tract.
Example 12. Therapeutic Efficacy of DAS181 Microparticle
Formulations Against Asthma
[0353] The efficacy of the DAS181 microparticle formulation used in
Example 9 above was tested against asthma. A 20 year old male
Caucasian asthma patient was tested for changes in airflow prior to
and 1 hour after oral administration of a 10 mg delivered dose (13
mg capsule) of Formulation A, as measured by FEV1 (forced
expiratory volume of air in 1 second). Prior to administration of
the drug, the FEV1 of the patient was at 82% of the predicted
normal lung function. One hour after administration of the drug,
the FEV1 of the patient indicated a clinically significant
improvement in lung function, with a 10% increase in value to 92%.
The results demonstrate that a DAS181 formulation can be effective
against asthma, i.e., a non-infectious respiratory disorder
affecting the central to upper respiratory tract.
OTHER EMBODIMENTS
[0354] It is to be understood that while the invention has been
described in conjunction with the detailed description thereof, the
foregoing description is intended to illustrate and not limit the
scope of the invention, which is defined by the scope of the
appended claims. Other aspects, advantages, and modifications are
within the scope of the following claims.
Sequence CWU 1
1
39158PRTBos taurus 1Arg Pro Asp Phe Cys Leu Glu Pro Pro Tyr Thr Gly
Pro Cys Lys Ala1 5 10 15Arg Ile Ile Arg Tyr Phe Tyr Asn Ala Lys Ala
Gly Leu Cys Gln Thr 20 25 30Phe Val Tyr Gly Gly Cys Arg Ala Lys Arg
Asn Asn Phe Lys Ser Ala 35 40 45Glu Asp Cys Met Arg Thr Cys Gly Gly
Ala 50 55224PRTHomo sapiens 2Asn Gly Arg Arg Ile Cys Leu Asp Leu
Gln Ala Pro Leu Tyr Lys Lys1 5 10 15Ile Ile Lys Lys Leu Leu Glu Ser
20327PRTHomo sapiens 3Gly Arg Glu Leu Cys Leu Asp Pro Lys Glu Asn
Trp Val Gln Arg Val1 5 10 15Val Glu Lys Phe Leu Lys Arg Ala Glu Asn
Ser 20 25434PRTHomo sapiens 4Gln Ile His Phe Phe Phe Ala Lys Leu
Asn Cys Arg Leu Tyr Arg Lys1 5 10 15Ala Asn Lys Ser Ser Lys Leu Val
Ser Ala Asn Arg Leu Phe Gly Asp 20 25 30Lys Ser534PRTHomo sapiens
5Glu Leu Arg Val Arg Leu Ala Ser His Leu Arg Lys Leu Arg Lys Arg1 5
10 15Leu Leu Arg Asp Ala Asp Asp Leu Gln Lys Arg Leu Ala Val Tyr
Gln 20 25 30Ala Gly612PRTHomo sapiens 6Arg Arg Leu Arg Arg Met Glu
Ser Glu Ser Glu Ser1 5 10721PRTHomo sapiens 7Lys Arg Lys Lys Lys
Gly Gly Lys Asn Gly Lys Asn Arg Arg Asn Arg1 5 10 15Lys Lys Lys Asn
Pro 208379PRTHomo sapiens 8Met Ala Ser Leu Pro Val Leu Gln Lys Glu
Ser Val Phe Gln Ser Gly1 5 10 15Ala His Ala Tyr Arg Ile Pro Ala Leu
Leu Tyr Leu Pro Gly Gln Gln 20 25 30Ser Leu Leu Ala Phe Ala Glu Gln
Arg Ala Ser Lys Lys Asp Glu His 35 40 45Ala Glu Leu Ile Val Leu Arg
Arg Gly Asp Tyr Asp Ala Pro Thr His 50 55 60Gln Val Gln Trp Gln Ala
Gln Glu Val Val Ala Gln Ala Arg Leu Asp65 70 75 80Gly His Arg Ser
Met Asn Pro Cys Pro Leu Tyr Asp Ala Gln Thr Gly 85 90 95Thr Leu Phe
Leu Phe Phe Ile Ala Ile Pro Gly Gln Val Thr Glu Gln 100 105 110Gln
Gln Leu Gln Thr Arg Ala Asn Val Thr Arg Leu Cys Gln Val Thr 115 120
125Ser Thr Asp His Gly Arg Thr Trp Ser Ser Pro Arg Asp Leu Thr Asp
130 135 140Ala Ala Ile Gly Pro Ala Tyr Arg Glu Trp Ser Thr Phe Ala
Val Gly145 150 155 160Pro Gly His Cys Leu Gln Leu Asn Asp Arg Ala
Arg Ser Leu Val Val 165 170 175Pro Ala Tyr Ala Tyr Arg Lys Leu His
Pro Ile Gln Arg Pro Ile Pro 180 185 190Ser Ala Phe Cys Phe Leu Ser
His Asp His Gly Arg Thr Trp Ala Arg 195 200 205Gly His Phe Val Ala
Gln Asp Thr Leu Glu Cys Gln Val Ala Glu Val 210 215 220Glu Thr Gly
Glu Gln Arg Val Val Thr Leu Asn Ala Arg Ser His Leu225 230 235
240Arg Ala Arg Val Gln Ala Gln Ser Thr Asn Asp Gly Leu Asp Phe Gln
245 250 255Glu Ser Gln Leu Val Lys Lys Leu Val Glu Pro Pro Pro Gln
Gly Cys 260 265 270Gln Gly Ser Val Ile Ser Phe Pro Ser Pro Arg Ser
Gly Pro Gly Ser 275 280 285Pro Gln Trp Leu Leu Tyr Thr His Pro Thr
His Ser Trp Gln Arg Ala 290 295 300Asp Leu Gly Ala Tyr Leu Asn Pro
Arg Pro Pro Ala Pro Glu Ala Trp305 310 315 320Ser Glu Pro Val Leu
Leu Ala Lys Gly Ser Cys Ala Tyr Ser Asp Leu 325 330 335Gln Ser Met
Gly Thr Gly Pro Asp Gly Ser Pro Leu Phe Gly Cys Leu 340 345 350Tyr
Glu Ala Asn Asp Tyr Glu Glu Ile Val Phe Leu Met Phe Thr Leu 355 360
365Lys Gln Ala Phe Pro Ala Glu Tyr Leu Pro Gln 370 3759424PRTHomo
sapiens 9Leu Ala Gly Gly Ser Val Arg Trp Gly Ala Leu His Val Leu
Gly Thr1 5 10 15Ala Ala Leu Ala Glu His Arg Ser Met Asn Pro Cys Pro
Val His Asp 20 25 30Ala Gly Thr Gly Thr Val Phe Leu Phe Phe Ile Ala
Val Leu Gly His 35 40 45Thr Pro Glu Ala Val Gln Ile Ala Thr Gly Arg
Asn Ala Ala Arg Leu 50 55 60Cys Cys Val Ala Ser Arg Asp Ala Gly Leu
Ser Trp Gly Ser Ala Arg65 70 75 80Asp Leu Thr Glu Glu Ala Ile Gly
Gly Ala Val Gln Asp Trp Ala Thr 85 90 95Phe Ala Val Gly Pro Gly His
Gly Val Gln Leu Pro Ser Gly Arg Leu 100 105 110Leu Val Pro Ala Tyr
Thr Tyr Arg Val Asp Arg Leu Glu Cys Phe Gly 115 120 125Lys Ile Cys
Arg Thr Ser Pro His Ser Phe Ala Phe Tyr Ser Asp Asp 130 135 140His
Gly Arg Thr Trp Arg Cys Gly Gly Leu Val Pro Asn Leu Arg Ser145 150
155 160Gly Glu Cys Gln Leu Ala Ala Val Asp Gly Gly Gln Ala Gly Ser
Phe 165 170 175Leu Tyr Cys Asn Ala Arg Ser Pro Leu Gly Ser Arg Val
Gln Ala Leu 180 185 190Ser Thr Asp Glu Gly Thr Ser Phe Leu Pro Ala
Glu Arg Val Ala Ser 195 200 205Leu Pro Glu Thr Ala Trp Gly Cys Gln
Gly Ser Ile Val Gly Phe Pro 210 215 220Ala Pro Ala Pro Asn Arg Pro
Arg Asp Asp Ser Trp Ser Val Gly Pro225 230 235 240Arg Ser Pro Leu
Gln Pro Pro Leu Leu Gly Pro Gly Val His Glu Pro 245 250 255Pro Glu
Glu Ala Ala Val Asp Pro Arg Gly Gly Gln Val Pro Gly Gly 260 265
270Pro Phe Ser Arg Leu Gln Pro Arg Gly Asp Gly Pro Arg Gln Pro Gly
275 280 285Pro Arg Pro Gly Val Ser Gly Asp Val Gly Ser Trp Thr Leu
Ala Leu 290 295 300Pro Met Pro Phe Ala Ala Pro Pro Gln Ser Pro Thr
Trp Leu Leu Tyr305 310 315 320Ser His Pro Val Gly Arg Arg Ala Arg
Leu His Met Gly Ile Arg Leu 325 330 335Ser Gln Ser Pro Leu Asp Pro
Arg Ser Trp Thr Glu Pro Trp Val Ile 340 345 350Tyr Glu Gly Pro Ser
Gly Tyr Ser Asp Leu Ala Ser Ile Gly Pro Ala 355 360 365Pro Glu Gly
Gly Leu Val Phe Ala Cys Leu Tyr Glu Ser Gly Ala Arg 370 375 380Thr
Ser Tyr Asp Glu Ile Ser Phe Cys Thr Phe Ser Leu Arg Glu Val385 390
395 400Leu Glu Asn Val Pro Ala Ser Pro Lys Pro Pro Asn Leu Gly Asp
Lys 405 410 415Pro Arg Gly Cys Cys Trp Pro Ser 420105PRTArtificial
SequenceSynthetic Construct 10Gly Gly Gly Gly Ser1
5112706DNAActinomyces viscosus 11atgacatcgc atagtccttt ctcccggagg
cgcctgccgg ccctcctggg ctccctgcca 60ctggccgcca ccggcctgat cgccgccgca
cccccggcgc acgccgtccc cacgtctgac 120ggcctggccg acgtcaccat
cacgcaggtg aacgcgcccg cggacggcct ctactccgtc 180ggcgatgtca
tgaccttcaa catcaccctg accaacacca gcggcgaggc ccactcctac
240gccccggcct cgacgaacct gtccgggaac gtctccaagt gccggtggcg
caacgtcccg 300gccgggacga ccaagaccga ctgcaccggc ctggccacgc
acacggtgac cgccgaggac 360ctcaaggccg gtggcttcac cccgcagatc
gcctacgagg tcaaggccgt ggagtacgcc 420gggaaggccc tgagcacccc
ggagacgatc aagggcgcga cgagcccagt caaggccaac 480tcgctgcggg
tcgagtcgat cacgccgtcg tcgagccagg agaactacaa gctgggcgac
540accgtcagct acacggtgcg cgtgcgctcg gtgtcggaca agacgatcaa
cgtcgccgcc 600accgaatcct ccttcgacga cctgggccgc cagtgccact
ggggcggcct caagccgggc 660aagggcgccg tctacaactg caagccgctc
acccacacga tcacgcaagc cgacgtcgac 720gccggccgct ggacgccatc
gatcaccctg acggccaccg gaaccgacgg cgccaccctc 780cagacgctca
ccgccaccgg caacccgatc aacgtcgtcg gcgaccaccc gcaggccacg
840cccgcaccgg cgcccgacgc gagcacggag ctgccggcct caatgagcca
ggcccagcac 900ctggccgcca acacggccac cgacaactac cgcatcccgg
cgatcaccac cgcccccaat 960ggggacctgc tcatctccta cgacgagcgc
ccgaaggaca acggcaacgg cggcagcgac 1020gcccccaacc cgaaccacat
cgtccagcgc cgctccaccg acggcggcaa gacctggtcg 1080gcgcccacct
acatccacca gggcacggag accggcaaga aggtcggcta ctccgacccg
1140agctacgtcg tcgatcacca gacgggcacg atcttcaact tccacgtcaa
gtcctacgac 1200cagggctggg gcggctcgcg cggcggcacc gacccggaga
accggggcat catccaggcc 1260gaggtgtcga cctccacgga caacggctgg
acctggacgc accgcacgat caccgcggac 1320atcacgaagg acaagccgtg
gaccgcgcgt ttcgcggcct cgggccaggg catccagatt 1380cagcacgggc
cccacgccgg gcgcctggtg cagcagtaca cgatcaggac cgccggcggc
1440gcggtgcagg ccgtctcggt ctactccgac gaccacggga agacgtggca
ggccggcacg 1500ccgatcggga ccggcatgga tgagaacaag gtcgttgagc
tctccgacgg ctccctcatg 1560ctcaactcgc gcgcctcgga tggctccggc
ttccgcaagg tggcccactc caccgacggt 1620gggcagacct ggagcgagcc
ggtgtccgac aagaacctgc ccgactcggt ggacaacgcc 1680cagatcatcc
gagccttccc gaacgccgcg ccggacgacc cgcgcgccaa ggtgctgctg
1740ctgagccact caccgaaccc gcggccgtgg tcgcgtgacc gcggcaccat
ctcgatgtcc 1800tgcgacgacg gcgcctcctg gacgaccagc aaggtcttcc
acgagccctt cgtcggatac 1860acgacgatcg cggtgcagtc cgacggcagc
atcgggctgc tcagcgagga cgcccacaac 1920ggcgccgact acggcggcat
ctggtaccgc aacttcacga tgaactggct cggcgagcag 1980tgcggccaga
agccggcgga gccgagcccg gcgccgtcgc cgacggcggc accctcagcg
2040gcaccgacgg agaagccggc cccgtcggcc gcgccgagcg ctgagcccac
gcaggcaccg 2100gcaccatcct ccgcgcccga gccgagcgct gcgcccgagc
cgagcagcgc cccggcgccg 2160gagcccacga ccgctccgag cacggagccc
acaccggctc ctgcgcccag ctccgcacct 2220gagcagaccg atgggccgac
cgctgcgccc gcaccggaga cgtcctctgc accggccgcc 2280gaaccgacgc
aggccccgac ggtggcgcct tctgttgagc ccacgcaggc tccgggtgcg
2340cagccgagct cagcacccaa gccgggggcg acgggtcggg ccccgtcggt
ggtgaacccg 2400aaggcgaccg gggcggcgac ggagcctggg acgccgtcat
cgagcgcgag cccggcaccg 2460agccggaacg cggcgccgac gccgaagccg
ggcatggagc ccgatgagat tgatcggccg 2520tctgacggca ccatggcgca
gccgaccggt ggcgccagcg cgccgagtgc cgcgccgacg 2580caggcggcga
aggccggcag caggctgtct cgcacgggga ccaacgcgct gctgatcctg
2640ggccttgcgg gtgtcgcggt tgtcggcggg tacctgctgc tgcgggctcg
ccgttcgaag 2700aactga 270612901PRTActinomyces viscosus 12Met Thr
Ser His Ser Pro Phe Ser Arg Arg Arg Leu Pro Ala Leu Leu1 5 10 15Gly
Ser Leu Pro Leu Ala Ala Thr Gly Leu Ile Ala Ala Ala Pro Pro 20 25
30Ala His Ala Val Pro Thr Ser Asp Gly Leu Ala Asp Val Thr Ile Thr
35 40 45Gln Val Asn Ala Pro Ala Asp Gly Leu Tyr Ser Val Gly Asp Val
Met 50 55 60Thr Phe Asn Ile Thr Leu Thr Asn Thr Ser Gly Glu Ala His
Ser Tyr65 70 75 80Ala Pro Ala Ser Thr Asn Leu Ser Gly Asn Val Ser
Lys Cys Arg Trp 85 90 95Arg Asn Val Pro Ala Gly Thr Thr Lys Thr Asp
Cys Thr Gly Leu Ala 100 105 110Thr His Thr Val Thr Ala Glu Asp Leu
Lys Ala Gly Gly Phe Thr Pro 115 120 125Gln Ile Ala Tyr Glu Val Lys
Ala Val Glu Tyr Ala Gly Lys Ala Leu 130 135 140Ser Thr Pro Glu Thr
Ile Lys Gly Ala Thr Ser Pro Val Lys Ala Asn145 150 155 160Ser Leu
Arg Val Glu Ser Ile Thr Pro Ser Ser Ser Gln Glu Asn Tyr 165 170
175Lys Leu Gly Asp Thr Val Ser Tyr Thr Val Arg Val Arg Ser Val Ser
180 185 190Asp Lys Thr Ile Asn Val Ala Ala Thr Glu Ser Ser Phe Asp
Asp Leu 195 200 205Gly Arg Gln Cys His Trp Gly Gly Leu Lys Pro Gly
Lys Gly Ala Val 210 215 220Tyr Asn Cys Lys Pro Leu Thr His Thr Ile
Thr Gln Ala Asp Val Asp225 230 235 240Ala Gly Arg Trp Thr Pro Ser
Ile Thr Leu Thr Ala Thr Gly Thr Asp 245 250 255Gly Ala Thr Leu Gln
Thr Leu Thr Ala Thr Gly Asn Pro Ile Asn Val 260 265 270Val Gly Asp
His Pro Gln Ala Thr Pro Ala Pro Ala Pro Asp Ala Ser 275 280 285Thr
Glu Leu Pro Ala Ser Met Ser Gln Ala Gln His Leu Ala Ala Asn 290 295
300Thr Ala Thr Asp Asn Tyr Arg Ile Pro Ala Ile Thr Thr Ala Pro
Asn305 310 315 320Gly Asp Leu Leu Ile Ser Tyr Asp Glu Arg Pro Lys
Asp Asn Gly Asn 325 330 335Gly Gly Ser Asp Ala Pro Asn Pro Asn His
Ile Val Gln Arg Arg Ser 340 345 350Thr Asp Gly Gly Lys Thr Trp Ser
Ala Pro Thr Tyr Ile His Gln Gly 355 360 365Thr Glu Thr Gly Lys Lys
Val Gly Tyr Ser Asp Pro Ser Tyr Val Val 370 375 380Asp His Gln Thr
Gly Thr Ile Phe Asn Phe His Val Lys Ser Tyr Asp385 390 395 400Gln
Gly Trp Gly Gly Ser Arg Gly Gly Thr Asp Pro Glu Asn Arg Gly 405 410
415Ile Ile Gln Ala Glu Val Ser Thr Ser Thr Asp Asn Gly Trp Thr Trp
420 425 430Thr His Arg Thr Ile Thr Ala Asp Ile Thr Lys Asp Lys Pro
Trp Thr 435 440 445Ala Arg Phe Ala Ala Ser Gly Gln Gly Ile Gln Ile
Gln His Gly Pro 450 455 460His Ala Gly Arg Leu Val Gln Gln Tyr Thr
Ile Arg Thr Ala Gly Gly465 470 475 480Ala Val Gln Ala Val Ser Val
Tyr Ser Asp Asp His Gly Lys Thr Trp 485 490 495Gln Ala Gly Thr Pro
Ile Gly Thr Gly Met Asp Glu Asn Lys Val Val 500 505 510Glu Leu Ser
Asp Gly Ser Leu Met Leu Asn Ser Arg Ala Ser Asp Gly 515 520 525Ser
Gly Phe Arg Lys Val Ala His Ser Thr Asp Gly Gly Gln Thr Trp 530 535
540Ser Glu Pro Val Ser Asp Lys Asn Leu Pro Asp Ser Val Asp Asn
Ala545 550 555 560Gln Ile Ile Arg Ala Phe Pro Asn Ala Ala Pro Asp
Asp Pro Arg Ala 565 570 575Lys Val Leu Leu Leu Ser His Ser Pro Asn
Pro Arg Pro Trp Ser Arg 580 585 590Asp Arg Gly Thr Ile Ser Met Ser
Cys Asp Asp Gly Ala Ser Trp Thr 595 600 605Thr Ser Lys Val Phe His
Glu Pro Phe Val Gly Tyr Thr Thr Ile Ala 610 615 620Val Gln Ser Asp
Gly Ser Ile Gly Leu Leu Ser Glu Asp Ala His Asn625 630 635 640Gly
Ala Asp Tyr Gly Gly Ile Trp Tyr Arg Asn Phe Thr Met Asn Trp 645 650
655Leu Gly Glu Gln Cys Gly Gln Lys Pro Ala Glu Pro Ser Pro Ala Pro
660 665 670Ser Pro Thr Ala Ala Pro Ser Ala Ala Pro Thr Glu Lys Pro
Ala Pro 675 680 685Ser Ala Ala Pro Ser Ala Glu Pro Thr Gln Ala Pro
Ala Pro Ser Ser 690 695 700Ala Pro Glu Pro Ser Ala Ala Pro Glu Pro
Ser Ser Ala Pro Ala Pro705 710 715 720Glu Pro Thr Thr Ala Pro Ser
Thr Glu Pro Thr Pro Ala Pro Ala Pro 725 730 735Ser Ser Ala Pro Glu
Gln Thr Asp Gly Pro Thr Ala Ala Pro Ala Pro 740 745 750Glu Thr Ser
Ser Ala Pro Ala Ala Glu Pro Thr Gln Ala Pro Thr Val 755 760 765Ala
Pro Ser Val Glu Pro Thr Gln Ala Pro Gly Ala Gln Pro Ser Ser 770 775
780Ala Pro Lys Pro Gly Ala Thr Gly Arg Ala Pro Ser Val Val Asn
Pro785 790 795 800Lys Ala Thr Gly Ala Ala Thr Glu Pro Gly Thr Pro
Ser Ser Ser Ala 805 810 815Ser Pro Ala Pro Ser Arg Asn Ala Ala Pro
Thr Pro Lys Pro Gly Met 820 825 830Glu Pro Asp Glu Ile Asp Arg Pro
Ser Asp Gly Thr Met Ala Gln Pro 835 840 845Thr Gly Gly Ala Ser Ala
Pro Ser Ala Ala Pro Thr Gln Ala Ala Lys 850 855 860Ala Gly Ser Arg
Leu Ser Arg Thr Gly Thr Asn Ala Leu Leu Ile Leu865 870 875 880Gly
Leu Ala Gly Val Ala Val Val Gly Gly Tyr Leu Leu Leu Arg Ala 885 890
895Arg Arg Ser Lys Asn 900131887DNAActinomyces viscosus
13ggcgaccacc cgcaggccac gcccgcaccg gcgcccgacg cgagcacgga gctgccggcc
60tcaatgagcc aggcccagca cctggccgcc aacacggcca ccgacaacta ccgcatcccg
120gcgatcacca ccgcccccaa tggggacctg ctcatctcct acgacgagcg
cccgaaggac 180aacggcaacg gcggcagcga cgcccccaac ccgaaccaca
tcgtccagcg ccgctccacc 240gacggcggca agacctggtc ggcgcccacc
tacatccacc agggcacgga gaccggcaag 300aaggtcggct actccgaccc
gagctacgtc gtcgatcacc agacgggcac gatcttcaac 360ttccacgtca
agtcctacga ccagggctgg
ggcggctcgc gcggcggcac cgacccggag 420aaccggggca tcatccaggc
cgaggtgtcg acctccacgg acaacggctg gacctggacg 480caccgcacga
tcaccgcgga catcacgaag gacaagccgt ggaccgcgcg tttcgcggcc
540tcgggccagg gcatccagat tcagcacggg ccccacgccg ggcgcctggt
gcagcagtac 600acgatcagga ccgccggcgg cgcggtgcag gccgtctcgg
tctactccga cgaccacggg 660aagacgtggc aggccggcac gccgatcggg
accggcatgg atgagaacaa ggtcgttgag 720ctctccgacg gctccctcat
gctcaactcg cgcgcctcgg atggctccgg cttccgcaag 780gtggcccact
ccaccgacgg tgggcagacc tggagcgagc cggtgtccga caagaacctg
840cccgactcgg tggacaacgc ccagatcatc cgagccttcc cgaacgccgc
gccggacgac 900ccgcgcgcca aggtgctgct gctgagccac tcaccgaacc
cgcggccgtg gtcgcgtgac 960cgcggcacca tctcgatgtc ctgcgacgac
ggcgcctcct ggacgaccag caaggtcttc 1020cacgagccct tcgtcggata
cacgacgatc gcggtgcagt ccgacggcag catcgggctg 1080ctcagcgagg
acgcccacaa cggcgccgac tacggcggca tctggtaccg caacttcacg
1140atgaactggc tcggcgagca gtgcggccag aagccggcgg agccgagccc
ggcgccgtcg 1200ccgacggcgg caccctcagc ggcaccgacg gagaagccgg
ccccgtcggc cgcgccgagc 1260gctgagccca cgcaggcacc ggcaccatcc
tccgcgcccg agccgagcgc tgcgcccgag 1320ccgagcagcg ccccggcgcc
ggagcccacg accgctccga gcacggagcc cacaccggct 1380cctgcgccca
gctccgcacc tgagcagacc gatgggccga ccgctgcgcc cgcaccggag
1440acgtcctctg caccggccgc cgaaccgacg caggccccga cggtggcgcc
ttctgttgag 1500cccacgcagg ctccgggtgc gcagccgagc tcagcaccca
agccgggggc gacgggtcgg 1560gccccgtcgg tggtgaaccc gaaggcgacc
ggggcggcga cggagcctgg gacgccgtca 1620tcgagcgcga gcccggcacc
gagccggaac gcggcgccga cgccgaagcc gggcatggag 1680cccgatgaga
ttgatcggcc gtctgacggc accatggcgc agccgaccgg tggcgccagc
1740gcgccgagtg ccgcgccgac gcaggcggcg aaggccggca gcaggctgtc
tcgcacgggg 1800accaacgcgc tgctgatcct gggccttgcg ggtgtcgcgg
ttgtcggcgg gtacctgctg 1860ctgcgggctc gccgttcgaa gaactga
188714628PRTActinomyces viscosus 14Gly Asp His Pro Gln Ala Thr Pro
Ala Pro Ala Pro Asp Ala Ser Thr1 5 10 15Glu Leu Pro Ala Ser Met Ser
Gln Ala Gln His Leu Ala Ala Asn Thr 20 25 30Ala Thr Asp Asn Tyr Arg
Ile Pro Ala Ile Thr Thr Ala Pro Asn Gly 35 40 45Asp Leu Leu Ile Ser
Tyr Asp Glu Arg Pro Lys Asp Asn Gly Asn Gly 50 55 60Gly Ser Asp Ala
Pro Asn Pro Asn His Ile Val Gln Arg Arg Ser Thr65 70 75 80Asp Gly
Gly Lys Thr Trp Ser Ala Pro Thr Tyr Ile His Gln Gly Thr 85 90 95Glu
Thr Gly Lys Lys Val Gly Tyr Ser Asp Pro Ser Tyr Val Val Asp 100 105
110His Gln Thr Gly Thr Ile Phe Asn Phe His Val Lys Ser Tyr Asp Gln
115 120 125Gly Trp Gly Gly Ser Arg Gly Gly Thr Asp Pro Glu Asn Arg
Gly Ile 130 135 140Ile Gln Ala Glu Val Ser Thr Ser Thr Asp Asn Gly
Trp Thr Trp Thr145 150 155 160His Arg Thr Ile Thr Ala Asp Ile Thr
Lys Asp Lys Pro Trp Thr Ala 165 170 175Arg Phe Ala Ala Ser Gly Gln
Gly Ile Gln Ile Gln His Gly Pro His 180 185 190Ala Gly Arg Leu Val
Gln Gln Tyr Thr Ile Arg Thr Ala Gly Gly Ala 195 200 205Val Gln Ala
Val Ser Val Tyr Ser Asp Asp His Gly Lys Thr Trp Gln 210 215 220Ala
Gly Thr Pro Ile Gly Thr Gly Met Asp Glu Asn Lys Val Val Glu225 230
235 240Leu Ser Asp Gly Ser Leu Met Leu Asn Ser Arg Ala Ser Asp Gly
Ser 245 250 255Gly Phe Arg Lys Val Ala His Ser Thr Asp Gly Gly Gln
Thr Trp Ser 260 265 270Glu Pro Val Ser Asp Lys Asn Leu Pro Asp Ser
Val Asp Asn Ala Gln 275 280 285Ile Ile Arg Ala Phe Pro Asn Ala Ala
Pro Asp Asp Pro Arg Ala Lys 290 295 300Val Leu Leu Leu Ser His Ser
Pro Asn Pro Arg Pro Trp Ser Arg Asp305 310 315 320Arg Gly Thr Ile
Ser Met Ser Cys Asp Asp Gly Ala Ser Trp Thr Thr 325 330 335Ser Lys
Val Phe His Glu Pro Phe Val Gly Tyr Thr Thr Ile Ala Val 340 345
350Gln Ser Asp Gly Ser Ile Gly Leu Leu Ser Glu Asp Ala His Asn Gly
355 360 365Ala Asp Tyr Gly Gly Ile Trp Tyr Arg Asn Phe Thr Met Asn
Trp Leu 370 375 380Gly Glu Gln Cys Gly Gln Lys Pro Ala Glu Pro Ser
Pro Ala Pro Ser385 390 395 400Pro Thr Ala Ala Pro Ser Ala Ala Pro
Thr Glu Lys Pro Ala Pro Ser 405 410 415Ala Ala Pro Ser Ala Glu Pro
Thr Gln Ala Pro Ala Pro Ser Ser Ala 420 425 430Pro Glu Pro Ser Ala
Ala Pro Glu Pro Ser Ser Ala Pro Ala Pro Glu 435 440 445Pro Thr Thr
Ala Pro Ser Thr Glu Pro Thr Pro Ala Pro Ala Pro Ser 450 455 460Ser
Ala Pro Glu Gln Thr Asp Gly Pro Thr Ala Ala Pro Ala Pro Glu465 470
475 480Thr Ser Ser Ala Pro Ala Ala Glu Pro Thr Gln Ala Pro Thr Val
Ala 485 490 495Pro Ser Val Glu Pro Thr Gln Ala Pro Gly Ala Gln Pro
Ser Ser Ala 500 505 510Pro Lys Pro Gly Ala Thr Gly Arg Ala Pro Ser
Val Val Asn Pro Lys 515 520 525Ala Thr Gly Ala Ala Thr Glu Pro Gly
Thr Pro Ser Ser Ser Ala Ser 530 535 540Pro Ala Pro Ser Arg Asn Ala
Ala Pro Thr Pro Lys Pro Gly Met Glu545 550 555 560Pro Asp Glu Ile
Asp Arg Pro Ser Asp Gly Thr Met Ala Gln Pro Thr 565 570 575Gly Gly
Ala Ser Ala Pro Ser Ala Ala Pro Thr Gln Ala Ala Lys Ala 580 585
590Gly Ser Arg Leu Ser Arg Thr Gly Thr Asn Ala Leu Leu Ile Leu Gly
595 600 605Leu Ala Gly Val Ala Val Val Gly Gly Tyr Leu Leu Leu Arg
Ala Arg 610 615 620Arg Ser Lys Asn625151182DNAArtificial
SequenceSynthetic Construct 15ggagatcatc cacaagctac accagcacct
gcaccagatg ctagcactga gctgccagca 60agcatgtctc aggctcagca tcttgcagca
aatacggcta ctgataatta tcgcattcca 120gcgattacaa ccgctccgaa
tggtgattta ctgattagct atgatgaacg gccgaaggac 180aatggaaatg
gtggttccga tgcccctaac ccgaatcata ttgttcagcg tcgctccaca
240gatggcggta aaacttggag cgcgccaacc tatattcatc agggtacgga
gactggcaag 300aaagtgggat attccgaccc ctcttatgtg gtggatcatc
aaaccggtac aatcttcaat 360tttcatgtga aatcatacga tcagggctgg
ggaggtagcc gtgggggaac agacccggaa 420aaccgcggga ttattcaggc
agaggtgtct acgagcacgg ataatggatg gacgtggaca 480catcgcacca
tcaccgcgga tattacgaaa gataaaccgt ggaccgcgcg ttttgcggcg
540tccggccaag gcattcagat ccagcatggg ccgcatgccg gccgtctggt
gcaacagtat 600accattcgta cggccggtgg agcggtgcag gctgtatcgg
tttattccga tgatcatggg 660aaaacgtggc aggctggcac cccgattggg
acgggtatgg atgaaaacaa agttgtagag 720ctgtctgacg gctctctgat
gctgaacagt cgtgcgtcgg acgggagcgg ctttcgtaag 780gttgcgcata
gcactgatgg tgggcagacc tggtccgaac cggtttcgga caaaaatttg
840ccggattcgg ttgataatgc ccagataatt cgtgcgtttc ctaatgctgc
ccccgatgac 900ccgcgcgcga aagtacttct tctgagtcat tccccaaatc
cacgtccgtg gtcccgggat 960cgtggtacga taagcatgtc atgtgatgac
ggggcctcat ggaccacttc caaagttttt 1020cacgaaccgt ttgtgggcta
cacgactatt gcagttcaga gtgatggaag catcggtctg 1080ctgtcggagg
acgcgcacaa tggcgctgat tatggtggca tctggtatcg taattttacg
1140atgaactggc tgggagaaca atgtggacaa aaacccgcgg aa
118216394PRTActinomyces viscosus 16Gly Asp His Pro Gln Ala Thr Pro
Ala Pro Ala Pro Asp Ala Ser Thr1 5 10 15Glu Leu Pro Ala Ser Met Ser
Gln Ala Gln His Leu Ala Ala Asn Thr 20 25 30Ala Thr Asp Asn Tyr Arg
Ile Pro Ala Ile Thr Thr Ala Pro Asn Gly 35 40 45Asp Leu Leu Ile Ser
Tyr Asp Glu Arg Pro Lys Asp Asn Gly Asn Gly 50 55 60Gly Ser Asp Ala
Pro Asn Pro Asn His Ile Val Gln Arg Arg Ser Thr65 70 75 80Asp Gly
Gly Lys Thr Trp Ser Ala Pro Thr Tyr Ile His Gln Gly Thr 85 90 95Glu
Thr Gly Lys Lys Val Gly Tyr Ser Asp Pro Ser Tyr Val Val Asp 100 105
110His Gln Thr Gly Thr Ile Phe Asn Phe His Val Lys Ser Tyr Asp Gln
115 120 125Gly Trp Gly Gly Ser Arg Gly Gly Thr Asp Pro Glu Asn Arg
Gly Ile 130 135 140Ile Gln Ala Glu Val Ser Thr Ser Thr Asp Asn Gly
Trp Thr Trp Thr145 150 155 160His Arg Thr Ile Thr Ala Asp Ile Thr
Lys Asp Lys Pro Trp Thr Ala 165 170 175Arg Phe Ala Ala Ser Gly Gln
Gly Ile Gln Ile Gln His Gly Pro His 180 185 190Ala Gly Arg Leu Val
Gln Gln Tyr Thr Ile Arg Thr Ala Gly Gly Ala 195 200 205Val Gln Ala
Val Ser Val Tyr Ser Asp Asp His Gly Lys Thr Trp Gln 210 215 220Ala
Gly Thr Pro Ile Gly Thr Gly Met Asp Glu Asn Lys Val Val Glu225 230
235 240Leu Ser Asp Gly Ser Leu Met Leu Asn Ser Arg Ala Ser Asp Gly
Ser 245 250 255Gly Phe Arg Lys Val Ala His Ser Thr Asp Gly Gly Gln
Thr Trp Ser 260 265 270Glu Pro Val Ser Asp Lys Asn Leu Pro Asp Ser
Val Asp Asn Ala Gln 275 280 285Ile Ile Arg Ala Phe Pro Asn Ala Ala
Pro Asp Asp Pro Arg Ala Lys 290 295 300Val Leu Leu Leu Ser His Ser
Pro Asn Pro Arg Pro Trp Ser Arg Asp305 310 315 320Arg Gly Thr Ile
Ser Met Ser Cys Asp Asp Gly Ala Ser Trp Thr Thr 325 330 335Ser Lys
Val Phe His Glu Pro Phe Val Gly Tyr Thr Thr Ile Ala Val 340 345
350Gln Ser Asp Gly Ser Ile Gly Leu Leu Ser Glu Asp Ala His Asn Gly
355 360 365Ala Asp Tyr Gly Gly Ile Trp Tyr Arg Asn Phe Thr Met Asn
Trp Leu 370 375 380Gly Glu Gln Cys Gly Gln Lys Pro Ala Glu385
390176PRTArtificial SequenceSynthetic Construct 17Met Val Lys Arg
Lys Lys1 5181281DNAArtificial SequenceSynthetic Construct
18ccatggttaa gcgcaaaaaa aaaggcggca aaaacggtaa aaatcgtcgt aaccgtaaga
60aaaaaaatcc tggagatcat ccacaagcta caccagcacc tgcaccagat gctagcactg
120agctgccagc aagcatgtct caggctcagc atcttgcagc aaatacggct
actgataatt 180atcgcattcc agcgattaca accgctccga atggtgattt
actgattagc tatgatgaac 240ggccgaagga caatggaaat ggtggttccg
atgcccctaa cccgaatcat attgttcagc 300gtcgctccac agatggcggt
aaaacttgga gcgcgccaac ctatattcat cagggtacgg 360agactggcaa
gaaagtggga tattccgacc cctcttatgt ggtggatcat caaaccggta
420caatcttcaa ttttcatgtg aaatcatacg atcagggctg gggaggtagc
cgtgggggaa 480cagacccgga aaaccgcggg attattcagg cagaggtgtc
tacgagcacg gataatggat 540ggacgtggac acatcgcacc atcaccgcgg
atattacgaa agataaaccg tggaccgcgc 600gttttgcggc gtccggccaa
ggcattcaga tccagcatgg gccgcatgcc ggccgtctgg 660tgcaacagta
taccattcgt acggccggtg gagcggtgca ggctgtatcg gtttattccg
720atgatcatgg gaaaacgtgg caggctggca ccccgattgg gacgggtatg
gatgaaaaca 780aagttgtaga gctgtctgac ggctctctga tgctgaacag
tcgtgcgtcg gacgggagcg 840gctttcgtaa ggttgcgcat agcactgatg
gtgggcagac ctggtccgaa ccggtttcgg 900acaaaaattt gccggattcg
gttgataatg cccagataat tcgtgcgttt cctaatgctg 960cccccgatga
cccgcgcgcg aaagtacttc ttctgagtca ttccccaaat ccacgtccgt
1020ggtcccggga tcgtggtacg ataagcatgt catgtgatga cggggcctca
tggaccactt 1080ccaaagtttt tcacgaaccg tttgtgggct acacgactat
tgcagttcag agtgatggaa 1140gcatcggtct gctgtcggag gacgcgcaca
atggcgctga ttatggtggc atctggtatc 1200gtaattttac gatgaactgg
ctgggagaac aatgtggaca aaaacccgcg gaataagctt 1260aaaaacccgc
ggaataagct t 128119444PRTArtificial SequenceSynthetic Construct
19Met Val Lys Arg Lys Lys Lys Gly Gly Lys Asn Gly Lys Asn Arg Arg1
5 10 15Asn Arg Lys Lys Lys Asn Pro Gly Asp His Pro Gln Ala Thr Pro
Ala 20 25 30Pro Ala Pro Asp Ala Ser Thr Glu Leu Pro Ala Ser Met Ser
Gln Ala 35 40 45Gln His Leu Ala Ala Asn Thr Ala Thr Asp Asn Tyr Arg
Ile Pro Ala 50 55 60Ile Thr Thr Ala Pro Asn Gly Asp Leu Leu Ile Ser
Tyr Asp Glu Arg65 70 75 80Pro Lys Asp Asn Gly Asn Gly Gly Ser Asp
Ala Pro Asn Pro Asn His 85 90 95Ile Val Gln Arg Arg Ser Thr Asp Gly
Gly Lys Thr Trp Ser Ala Pro 100 105 110Thr Tyr Ile His Gln Gly Thr
Glu Thr Gly Lys Lys Val Gly Tyr Ser 115 120 125Asp Pro Ser Tyr Val
Val Asp His Gln Thr Gly Thr Ile Phe Asn Phe 130 135 140His Val Lys
Ser Tyr Asp Gln Gly Trp Gly Gly Ser Arg Gly Gly Thr145 150 155
160Asp Pro Glu Asn Arg Gly Ile Ile Gln Ala Glu Val Ser Thr Ser Thr
165 170 175Asp Asn Gly Trp Thr Trp Thr His Arg Thr Ile Thr Ala Asp
Ile Thr 180 185 190Lys Asp Lys Pro Trp Thr Ala Arg Phe Ala Ala Ser
Gly Gln Gly Ile 195 200 205Gln Ile Gln His Gly Pro His Ala Gly Arg
Leu Val Gln Gln Tyr Thr 210 215 220Ile Arg Thr Ala Gly Gly Ala Val
Gln Ala Val Ser Val Tyr Ser Asp225 230 235 240Asp His Gly Lys Thr
Trp Gln Ala Gly Thr Pro Ile Gly Thr Gly Met 245 250 255Asp Glu Asn
Lys Val Val Glu Leu Ser Asp Gly Ser Leu Met Leu Asn 260 265 270Ser
Arg Ala Ser Asp Gly Ser Gly Phe Arg Lys Val Ala His Ser Thr 275 280
285Asp Gly Gly Gln Thr Trp Ser Glu Pro Val Ser Asp Lys Asn Leu Pro
290 295 300Asp Ser Val Asp Asn Ala Gln Ile Ile Arg Ala Phe Pro Asn
Ala Ala305 310 315 320Pro Asp Asp Pro Arg Ala Lys Val Leu Leu Leu
Ser His Ser Pro Asn 325 330 335Pro Arg Pro Trp Ser Arg Asp Arg Gly
Thr Ile Ser Met Ser Cys Asp 340 345 350Asp Gly Ala Ser Trp Thr Thr
Ser Lys Val Phe His Glu Pro Phe Val 355 360 365Gly Tyr Thr Thr Ile
Ala Val Gln Ser Asp Gly Ser Ile Gly Leu Leu 370 375 380Ser Glu Asp
Ala His Asn Gly Ala Asp Tyr Gly Gly Ile Trp Tyr Arg385 390 395
400Asn Phe Thr Met Asn Trp Leu Gly Glu Gln Cys Gly Gln Lys Pro Ala
405 410 415Glu Gly Ala Asp Tyr Gly Gly Ile Trp Tyr Arg Asn Phe Thr
Met Asn 420 425 430Trp Leu Gly Glu Gln Cys Gly Gln Lys Pro Ala Glu
435 440201248DNAArtificial SequenceSynthetic Construct 20atgggagatc
atccacaagc tacaccagca cctgcaccag atgctagcac tgagctgcca 60gcaagcatgt
ctcaggctca gcatcttgca gcaaatacgg ctactgataa ttatcgcatt
120ccagcgatta caaccgctcc gaatggtgat ttactgatta gctatgatga
acggccgaag 180gacaatggaa atggtggttc cgatgcccct aacccgaatc
atattgttca gcgtcgctcc 240acagatggcg gtaaaacttg gagcgcgcca
acctatattc atcagggtac ggagactggc 300aagaaagtgg gatattccga
cccctcttat gtggtggatc atcaaaccgg tacaatcttc 360aattttcatg
tgaaatcata cgatcagggc tggggaggta gccgtggggg aacagacccg
420gaaaaccgcg ggattattca ggcagaggtg tctacgagca cggataatgg
atggacgtgg 480acacatcgca ccatcaccgc ggatattacg aaagataaac
cgtggaccgc gcgttttgcg 540gcgtccggcc aaggcattca gatccagcat
gggccgcatg ccggccgtct ggtgcaacag 600tataccattc gtacggccgg
tggagcggtg caggctgtat cggtttattc cgatgatcat 660gggaaaacgt
ggcaggctgg caccccgatt gggacgggta tggatgaaaa caaagttgta
720gagctgtctg acggctctct gatgctgaac agtcgtgcgt cggacgggag
cggctttcgt 780aaggttgcgc atagcactga tggtgggcag acctggtccg
aaccggtttc ggacaaaaat 840ttgccggatt cggttgataa tgcccagata
attcgtgcgt ttcctaatgc tgcccccgat 900gacccgcgcg cgaaagtact
tcttctgagt cattccccaa atccacgtcc gtggtcccgg 960gatcgtggta
cgataagcat gtcatgtgat gacggggcct catggaccac ttccaaagtt
1020tttcacgaac cgtttgtggg ctacacgact attgcagttc agagtgatgg
aagcatcggt 1080ctgctgtcgg aggacgcgca caatggcgct gattatggtg
gcatctggta tcgtaatttt 1140acgatgaact ggctgggaga acaatgtgga
caaaaacccg cgaagcgcaa aaaaaaaggc 1200ggcaaaaacg gtaaaaatcg
tcgtaaccgt aagaaaaaaa atccttga 124821415PRTArtificial
SequenceSynthetic Construct 21Met Gly Asp His Pro Gln Ala Thr Pro
Ala Pro Ala Pro Asp Ala Ser1 5 10 15Thr Glu Leu Pro Ala Ser Met Ser
Gln Ala Gln His Leu Ala Ala Asn 20 25 30Thr Ala Thr Asp Asn Tyr Arg
Ile Pro Ala Ile Thr Thr Ala Pro Asn 35 40 45Gly Asp Leu Leu Ile Ser
Tyr Asp Glu Arg Pro Lys Asp Asn Gly Asn 50 55 60Gly Gly Ser Asp Ala
Pro Asn Pro Asn His Ile Val Gln Arg Arg Ser65 70 75 80Thr Asp Gly
Gly Lys Thr Trp Ser Ala Pro Thr Tyr Ile His
Gln Gly 85 90 95Thr Glu Thr Gly Lys Lys Val Gly Tyr Ser Asp Pro Ser
Tyr Val Val 100 105 110Asp His Gln Thr Gly Thr Ile Phe Asn Phe His
Val Lys Ser Tyr Asp 115 120 125Gln Gly Trp Gly Gly Ser Arg Gly Gly
Thr Asp Pro Glu Asn Arg Gly 130 135 140Ile Ile Gln Ala Glu Val Ser
Thr Ser Thr Asp Asn Gly Trp Thr Trp145 150 155 160Thr His Arg Thr
Ile Thr Ala Asp Ile Thr Lys Asp Lys Pro Trp Thr 165 170 175Ala Arg
Phe Ala Ala Ser Gly Gln Gly Ile Gln Ile Gln His Gly Pro 180 185
190His Ala Gly Arg Leu Val Gln Gln Tyr Thr Ile Arg Thr Ala Gly Gly
195 200 205Ala Val Gln Ala Val Ser Val Tyr Ser Asp Asp His Gly Lys
Thr Trp 210 215 220Gln Ala Gly Thr Pro Ile Gly Thr Gly Met Asp Glu
Asn Lys Val Val225 230 235 240Glu Leu Ser Asp Gly Ser Leu Met Leu
Asn Ser Arg Ala Ser Asp Gly 245 250 255Ser Gly Phe Arg Lys Val Ala
His Ser Thr Asp Gly Gly Gln Thr Trp 260 265 270Ser Glu Pro Val Ser
Asp Lys Asn Leu Pro Asp Ser Val Asp Asn Ala 275 280 285Gln Ile Ile
Arg Ala Phe Pro Asn Ala Ala Pro Asp Asp Pro Arg Ala 290 295 300Lys
Val Leu Leu Leu Ser His Ser Pro Asn Pro Arg Pro Trp Ser Arg305 310
315 320Asp Arg Gly Thr Ile Ser Met Ser Cys Asp Asp Gly Ala Ser Trp
Thr 325 330 335Thr Ser Lys Val Phe His Glu Pro Phe Val Gly Tyr Thr
Thr Ile Ala 340 345 350Val Gln Ser Asp Gly Ser Ile Gly Leu Leu Ser
Glu Asp Ala His Asn 355 360 365Gly Ala Asp Tyr Gly Gly Ile Trp Tyr
Arg Asn Phe Thr Met Asn Trp 370 375 380Leu Gly Glu Gln Cys Gly Gln
Lys Pro Ala Lys Arg Lys Lys Lys Gly385 390 395 400Gly Lys Asn Gly
Lys Asn Arg Arg Asn Arg Lys Lys Lys Asn Pro 405 410
415221293DNAArtificial SequenceSynthetic Construct 22atgggagatc
atccacaagc tacaccagca cctgcaccag atgctagcac tgagctgcca 60gcaagcatgt
ctcaggctca gcatcttgca gcaaatacgg ctactgataa ttatcgcatt
120ccagcgatta caaccgctcc gaatggtgat ttactgatta gctatgatga
acggccgaag 180gacaatggaa atggtggttc cgatgcccct aacccgaatc
atattgttca gcgtcgctcc 240acagatggcg gtaaaacttg gagcgcgcca
acctatattc atcagggtac ggagactggc 300aagaaagtgg gatattccga
cccctcttat gtggtggatc atcaaaccgg tacaatcttc 360aattttcatg
tgaaatcata cgatcagggc tggggaggta gccgtggggg aacagacccg
420gaaaaccgcg ggattattca ggcagaggtg tctacgagca cggataatgg
atggacgtgg 480acacatcgca ccatcaccgc ggatattacg aaagataaac
cgtggaccgc gcgttttgcg 540gcgtccggcc aaggcattca gatccagcat
gggccgcatg ccggccgtct ggtgcaacag 600tataccattc gtacggccgg
tggagcggtg caggctgtat cggtttattc cgatgatcat 660gggaaaacgt
ggcaggctgg caccccgatt gggacgggta tggatgaaaa caaagttgta
720gagctgtctg acggctctct gatgctgaac agtcgtgcgt cggacgggag
cggctttcgt 780aaggttgcgc atagcactga tggtgggcag acctggtccg
aaccggtttc ggacaaaaat 840ttgccggatt cggttgataa tgcccagata
attcgtgcgt ttcctaatgc tgcccccgat 900gacccgcgcg cgaaagtact
tcttctgagt cattccccaa atccacgtcc gtggtcccgg 960gatcgtggta
cgataagcat gtcatgtgat gacggggcct catggaccac ttccaaagtt
1020tttcacgaac cgtttgtggg ctacacgact attgcagttc agagtgatgg
aagcatcggt 1080ctgctgtcgg aggacgcgca caatggcgct gattatggtg
gcatctggta tcgtaatttt 1140acgatgaact ggctgggaga acaatgtgga
caaaaacccg cggaaccgag cccagcccct 1200agccctactg cagcaccgtc
cgctgcaaag cgcaaaaaaa aaggcggcaa aaacggtaaa 1260aatcgtcgta
accgtaagaa aaaaaatcct tga 129323430PRTArtificial SequenceSynthetic
Construct 23Met Gly Asp His Pro Gln Ala Thr Pro Ala Pro Ala Pro Asp
Ala Ser1 5 10 15Thr Glu Leu Pro Ala Ser Met Ser Gln Ala Gln His Leu
Ala Ala Asn 20 25 30Thr Ala Thr Asp Asn Tyr Arg Ile Pro Ala Ile Thr
Thr Ala Pro Asn 35 40 45Gly Asp Leu Leu Ile Ser Tyr Asp Glu Arg Pro
Lys Asp Asn Gly Asn 50 55 60Gly Gly Ser Asp Ala Pro Asn Pro Asn His
Ile Val Gln Arg Arg Ser65 70 75 80Thr Asp Gly Gly Lys Thr Trp Ser
Ala Pro Thr Tyr Ile His Gln Gly 85 90 95Thr Glu Thr Gly Lys Lys Val
Gly Tyr Ser Asp Pro Ser Tyr Val Val 100 105 110Asp His Gln Thr Gly
Thr Ile Phe Asn Phe His Val Lys Ser Tyr Asp 115 120 125Gln Gly Trp
Gly Gly Ser Arg Gly Gly Thr Asp Pro Glu Asn Arg Gly 130 135 140Ile
Ile Gln Ala Glu Val Ser Thr Ser Thr Asp Asn Gly Trp Thr Trp145 150
155 160Thr His Arg Thr Ile Thr Ala Asp Ile Thr Lys Asp Lys Pro Trp
Thr 165 170 175Ala Arg Phe Ala Ala Ser Gly Gln Gly Ile Gln Ile Gln
His Gly Pro 180 185 190His Ala Gly Arg Leu Val Gln Gln Tyr Thr Ile
Arg Thr Ala Gly Gly 195 200 205Ala Val Gln Ala Val Ser Val Tyr Ser
Asp Asp His Gly Lys Thr Trp 210 215 220Gln Ala Gly Thr Pro Ile Gly
Thr Gly Met Asp Glu Asn Lys Val Val225 230 235 240Glu Leu Ser Asp
Gly Ser Leu Met Leu Asn Ser Arg Ala Ser Asp Gly 245 250 255Ser Gly
Phe Arg Lys Val Ala His Ser Thr Asp Gly Gly Gln Thr Trp 260 265
270Ser Glu Pro Val Ser Asp Lys Asn Leu Pro Asp Ser Val Asp Asn Ala
275 280 285Gln Ile Ile Arg Ala Phe Pro Asn Ala Ala Pro Asp Asp Pro
Arg Ala 290 295 300Lys Val Leu Leu Leu Ser His Ser Pro Asn Pro Arg
Pro Trp Ser Arg305 310 315 320Asp Arg Gly Thr Ile Ser Met Ser Cys
Asp Asp Gly Ala Ser Trp Thr 325 330 335Thr Ser Lys Val Phe His Glu
Pro Phe Val Gly Tyr Thr Thr Ile Ala 340 345 350Val Gln Ser Asp Gly
Ser Ile Gly Leu Leu Ser Glu Asp Ala His Asn 355 360 365Gly Ala Asp
Tyr Gly Gly Ile Trp Tyr Arg Asn Phe Thr Met Asn Trp 370 375 380Leu
Gly Glu Gln Cys Gly Gln Lys Pro Ala Glu Pro Ser Pro Ala Pro385 390
395 400Ser Pro Thr Ala Ala Pro Ser Ala Ala Lys Arg Lys Lys Lys Gly
Gly 405 410 415Lys Asn Gly Lys Asn Arg Arg Asn Arg Lys Lys Lys Asn
Pro 420 425 430241203DNAArtificial SequenceSynthetic Construct
24atgggagagc tgccagcaag catgtctcag gctcagcatc ttgcagcaaa tacggctact
60gataattatc gcattccagc gattacaacc gctccgaatg gtgatttact gattagctat
120gatgaacggc cgaaggacaa tggaaatggt ggttccgatg cccctaaccc
gaatcatatt 180gttcagcgtc gctccacaga tggcggtaaa acttggagcg
cgccaaccta tattcatcag 240ggtacggaga ctggcaagaa agtgggatat
tccgacccct cttatgtggt ggatcatcaa 300accggtacaa tcttcaattt
tcatgtgaaa tcatacgatc agggctgggg aggtagccgt 360gggggaacag
acccggaaaa ccgcgggatt attcaggcag aggtgtctac gagcacggat
420aatggatgga cgtggacaca tcgcaccatc accgcggata ttacgaaaga
taaaccgtgg 480accgcgcgtt ttgcggcgtc cggccaaggc attcagatcc
agcatgggcc gcatgccggc 540cgtctggtgc aacagtatac cattcgtacg
gccggtggag cggtgcaggc tgtatcggtt 600tattccgatg atcatgggaa
aacgtggcag gctggcaccc cgattgggac gggtatggat 660gaaaacaaag
ttgtagagct gtctgacggc tctctgatgc tgaacagtcg tgcgtcggac
720gggagcggct ttcgtaaggt tgcgcatagc actgatggtg ggcagacctg
gtccgaaccg 780gtttcggaca aaaatttgcc ggattcggtt gataatgccc
agataattcg tgcgtttcct 840aatgctgccc ccgatgaccc gcgcgcgaaa
gtacttcttc tgagtcattc cccaaatcca 900cgtccgtggt cccgggatcg
tggtacgata agcatgtcat gtgatgacgg ggcctcatgg 960accacttcca
aagtttttca cgaaccgttt gtgggctaca cgactattgc agttcagagt
1020gatggaagca tcggtctgct gtcggaggac gcgcacaatg gcgctgatta
tggtggcatc 1080tggtatcgta attttacgat gaactggctg ggagaacaat
gtggacaaaa acccgcgaag 1140cgcaaaaaaa aaggcggcaa aaacggtaaa
aatcgtcgta accgtaagaa aaaaaatcct 1200tga 120325400PRTArtificial
SequenceSynthetic Construct 25Met Gly Glu Leu Pro Ala Ser Met Ser
Gln Ala Gln His Leu Ala Ala1 5 10 15Asn Thr Ala Thr Asp Asn Tyr Arg
Ile Pro Ala Ile Thr Thr Ala Pro 20 25 30Asn Gly Asp Leu Leu Ile Ser
Tyr Asp Glu Arg Pro Lys Asp Asn Gly 35 40 45Asn Gly Gly Ser Asp Ala
Pro Asn Pro Asn His Ile Val Gln Arg Arg 50 55 60Ser Thr Asp Gly Gly
Lys Thr Trp Ser Ala Pro Thr Tyr Ile His Gln65 70 75 80Gly Thr Glu
Thr Gly Lys Lys Val Gly Tyr Ser Asp Pro Ser Tyr Val 85 90 95Val Asp
His Gln Thr Gly Thr Ile Phe Asn Phe His Val Lys Ser Tyr 100 105
110Asp Gln Gly Trp Gly Gly Ser Arg Gly Gly Thr Asp Pro Glu Asn Arg
115 120 125Gly Ile Ile Gln Ala Glu Val Ser Thr Ser Thr Asp Asn Gly
Trp Thr 130 135 140Trp Thr His Arg Thr Ile Thr Ala Asp Ile Thr Lys
Asp Lys Pro Trp145 150 155 160Thr Ala Arg Phe Ala Ala Ser Gly Gln
Gly Ile Gln Ile Gln His Gly 165 170 175Pro His Ala Gly Arg Leu Val
Gln Gln Tyr Thr Ile Arg Thr Ala Gly 180 185 190Gly Ala Val Gln Ala
Val Ser Val Tyr Ser Asp Asp His Gly Lys Thr 195 200 205Trp Gln Ala
Gly Thr Pro Ile Gly Thr Gly Met Asp Glu Asn Lys Val 210 215 220Val
Glu Leu Ser Asp Gly Ser Leu Met Leu Asn Ser Arg Ala Ser Asp225 230
235 240Gly Ser Gly Phe Arg Lys Val Ala His Ser Thr Asp Gly Gly Gln
Thr 245 250 255Trp Ser Glu Pro Val Ser Asp Lys Asn Leu Pro Asp Ser
Val Asp Asn 260 265 270Ala Gln Ile Ile Arg Ala Phe Pro Asn Ala Ala
Pro Asp Asp Pro Arg 275 280 285Ala Lys Val Leu Leu Leu Ser His Ser
Pro Asn Pro Arg Pro Trp Ser 290 295 300Arg Asp Arg Gly Thr Ile Ser
Met Ser Cys Asp Asp Gly Ala Ser Trp305 310 315 320Thr Thr Ser Lys
Val Phe His Glu Pro Phe Val Gly Tyr Thr Thr Ile 325 330 335Ala Val
Gln Ser Asp Gly Ser Ile Gly Leu Leu Ser Glu Asp Ala His 340 345
350Asn Gly Ala Asp Tyr Gly Gly Ile Trp Tyr Arg Asn Phe Thr Met Asn
355 360 365Trp Leu Gly Glu Gln Cys Gly Gln Lys Pro Ala Lys Arg Lys
Lys Lys 370 375 380Gly Gly Lys Asn Gly Lys Asn Arg Arg Asn Arg Lys
Lys Lys Asn Pro385 390 395 400261248DNAArtificial SequenceSynthetic
Construct 26atgggagagc tgccagcaag catgtctcag gctcagcatc ttgcagcaaa
tacggctact 60gataattatc gcattccagc gattacaacc gctccgaatg gtgatttact
gattagctat 120gatgaacggc cgaaggacaa tggaaatggt ggttccgatg
cccctaaccc gaatcatatt 180gttcagcgtc gctccacaga tggcggtaaa
acttggagcg cgccaaccta tattcatcag 240ggtacggaga ctggcaagaa
agtgggatat tccgacccct cttatgtggt ggatcatcaa 300accggtacaa
tcttcaattt tcatgtgaaa tcatacgatc agggctgggg aggtagccgt
360gggggaacag acccggaaaa ccgcgggatt attcaggcag aggtgtctac
gagcacggat 420aatggatgga cgtggacaca tcgcaccatc accgcggata
ttacgaaaga taaaccgtgg 480accgcgcgtt ttgcggcgtc cggccaaggc
attcagatcc agcatgggcc gcatgccggc 540cgtctggtgc aacagtatac
cattcgtacg gccggtggag cggtgcaggc tgtatcggtt 600tattccgatg
atcatgggaa aacgtggcag gctggcaccc cgattgggac gggtatggat
660gaaaacaaag ttgtagagct gtctgacggc tctctgatgc tgaacagtcg
tgcgtcggac 720gggagcggct ttcgtaaggt tgcgcatagc actgatggtg
ggcagacctg gtccgaaccg 780gtttcggaca aaaatttgcc ggattcggtt
gataatgccc agataattcg tgcgtttcct 840aatgctgccc ccgatgaccc
gcgcgcgaaa gtacttcttc tgagtcattc cccaaatcca 900cgtccgtggt
cccgggatcg tggtacgata agcatgtcat gtgatgacgg ggcctcatgg
960accacttcca aagtttttca cgaaccgttt gtgggctaca cgactattgc
agttcagagt 1020gatggaagca tcggtctgct gtcggaggac gcgcacaatg
gcgctgatta tggtggcatc 1080tggtatcgta attttacgat gaactggctg
ggagaacaat gtggacaaaa acccgcggaa 1140ccgagcccag cccctagccc
tactgcagca ccgtccgctg caaagcgcaa aaaaaaaggc 1200ggcaaaaacg
gtaaaaatcg tcgtaaccgt aagaaaaaaa atccttga 124827415PRTArtificial
SequenceSynthetic Construct 27Met Gly Glu Leu Pro Ala Ser Met Ser
Gln Ala Gln His Leu Ala Ala1 5 10 15Asn Thr Ala Thr Asp Asn Tyr Arg
Ile Pro Ala Ile Thr Thr Ala Pro 20 25 30Asn Gly Asp Leu Leu Ile Ser
Tyr Asp Glu Arg Pro Lys Asp Asn Gly 35 40 45Asn Gly Gly Ser Asp Ala
Pro Asn Pro Asn His Ile Val Gln Arg Arg 50 55 60Ser Thr Asp Gly Gly
Lys Thr Trp Ser Ala Pro Thr Tyr Ile His Gln65 70 75 80Gly Thr Glu
Thr Gly Lys Lys Val Gly Tyr Ser Asp Pro Ser Tyr Val 85 90 95Val Asp
His Gln Thr Gly Thr Ile Phe Asn Phe His Val Lys Ser Tyr 100 105
110Asp Gln Gly Trp Gly Gly Ser Arg Gly Gly Thr Asp Pro Glu Asn Arg
115 120 125Gly Ile Ile Gln Ala Glu Val Ser Thr Ser Thr Asp Asn Gly
Trp Thr 130 135 140Trp Thr His Arg Thr Ile Thr Ala Asp Ile Thr Lys
Asp Lys Pro Trp145 150 155 160Thr Ala Arg Phe Ala Ala Ser Gly Gln
Gly Ile Gln Ile Gln His Gly 165 170 175Pro His Ala Gly Arg Leu Val
Gln Gln Tyr Thr Ile Arg Thr Ala Gly 180 185 190Gly Ala Val Gln Ala
Val Ser Val Tyr Ser Asp Asp His Gly Lys Thr 195 200 205Trp Gln Ala
Gly Thr Pro Ile Gly Thr Gly Met Asp Glu Asn Lys Val 210 215 220Val
Glu Leu Ser Asp Gly Ser Leu Met Leu Asn Ser Arg Ala Ser Asp225 230
235 240Gly Ser Gly Phe Arg Lys Val Ala His Ser Thr Asp Gly Gly Gln
Thr 245 250 255Trp Ser Glu Pro Val Ser Asp Lys Asn Leu Pro Asp Ser
Val Asp Asn 260 265 270Ala Gln Ile Ile Arg Ala Phe Pro Asn Ala Ala
Pro Asp Asp Pro Arg 275 280 285Ala Lys Val Leu Leu Leu Ser His Ser
Pro Asn Pro Arg Pro Trp Ser 290 295 300Arg Asp Arg Gly Thr Ile Ser
Met Ser Cys Asp Asp Gly Ala Ser Trp305 310 315 320Thr Thr Ser Lys
Val Phe His Glu Pro Phe Val Gly Tyr Thr Thr Ile 325 330 335Ala Val
Gln Ser Asp Gly Ser Ile Gly Leu Leu Ser Glu Asp Ala His 340 345
350Asn Gly Ala Asp Tyr Gly Gly Ile Trp Tyr Arg Asn Phe Thr Met Asn
355 360 365Trp Leu Gly Glu Gln Cys Gly Gln Lys Pro Ala Glu Pro Ser
Pro Ala 370 375 380Pro Ser Pro Thr Ala Ala Pro Ser Ala Ala Lys Arg
Lys Lys Lys Gly385 390 395 400Gly Lys Asn Gly Lys Asn Arg Arg Asn
Arg Lys Lys Lys Asn Pro 405 410 415281221DNAArtificial
SequenceSynthetic Construct 28ccatggggca tcaccatcac catcatctag
agggagatca tccacaagct acaccagcac 60ctgcaccaga tgctagcact gagctgccag
caagcatgtc tcaggctcag catcttgcag 120caaatacggc tactgataat
tatcgcattc cagcgattac aaccgctccg aatggtgatt 180tactgattag
ctatgatgaa cggccgaagg acaatggaaa tggtggttcc gatgccccta
240acccgaatca tattgttcag cgtcgctcca cagatggcgg taaaacttgg
agcgcgccaa 300cctatattca tcagggtacg gagactggca agaaagtggg
atattccgac ccctcttatg 360tggtggatca tcaaaccggt acaatcttca
attttcatgt gaaatcatac gatcagggct 420ggggaggtag ccgtggggga
acagacccgg aaaaccgcgg gattattcag gcagaggtgt 480ctacgagcac
ggataatgga tggacgtgga cacatcgcac catcaccgcg gatattacga
540aagataaacc gtggaccgcg cgttttgcgg cgtccggcca aggcattcag
atccagcatg 600ggccgcatgc cggccgtctg gtgcaacagt ataccattcg
tacggccggt ggagcggtgc 660aggctgtatc ggtttattcc gatgatcatg
ggaaaacgtg gcaggctggc accccgattg 720ggacgggtat ggatgaaaac
aaagttgtag agctgtctga cggctctctg atgctgaaca 780gtcgtgcgtc
ggacgggagc ggctttcgta aggttgcgca tagcactgat ggtgggcaga
840cctggtccga accggtttcg gacaaaaatt tgccggattc ggttgataat
gcccagataa 900ttcgtgcgtt tcctaatgct gcccccgatg acccgcgcgc
gaaagtactt cttctgagtc 960attccccaaa tccacgtccg tggtcccggg
atcgtggtac gataagcatg tcatgtgatg 1020acggggcctc atggaccact
tccaaagttt ttcacgaacc gtttgtgggc tacacgacta 1080ttgcagttca
gagtgatgga agcatcggtc tgctgtcgga ggacgcgcac aatggcgctg
1140attatggtgg catctggtat cgtaatttta cgatgaactg gctgggagaa
caatgtggac 1200aaaaacccgc ggaataagct t 122129404PRTArtificial
SequenceSynthetic Construct 29Met Gly His His His
His His His Leu Glu Gly Asp His Pro Gln Ala1 5 10 15Thr Pro Ala Pro
Ala Pro Asp Ala Ser Thr Glu Leu Pro Ala Ser Met 20 25 30Ser Gln Ala
Gln His Leu Ala Ala Asn Thr Ala Thr Asp Asn Tyr Arg 35 40 45Ile Pro
Ala Ile Thr Thr Ala Pro Asn Gly Asp Leu Leu Ile Ser Tyr 50 55 60Asp
Glu Arg Pro Lys Asp Asn Gly Asn Gly Gly Ser Asp Ala Pro Asn65 70 75
80Pro Asn His Ile Val Gln Arg Arg Ser Thr Asp Gly Gly Lys Thr Trp
85 90 95Ser Ala Pro Thr Tyr Ile His Gln Gly Thr Glu Thr Gly Lys Lys
Val 100 105 110Gly Tyr Ser Asp Pro Ser Tyr Val Val Asp His Gln Thr
Gly Thr Ile 115 120 125Phe Asn Phe His Val Lys Ser Tyr Asp Gln Gly
Trp Gly Gly Ser Arg 130 135 140Gly Gly Thr Asp Pro Glu Asn Arg Gly
Ile Ile Gln Ala Glu Val Ser145 150 155 160Thr Ser Thr Asp Asn Gly
Trp Thr Trp Thr His Arg Thr Ile Thr Ala 165 170 175Asp Ile Thr Lys
Asp Lys Pro Trp Thr Ala Arg Phe Ala Ala Ser Gly 180 185 190Gln Gly
Ile Gln Ile Gln His Gly Pro His Ala Gly Arg Leu Val Gln 195 200
205Gln Tyr Thr Ile Arg Thr Ala Gly Gly Ala Val Gln Ala Val Ser Val
210 215 220Tyr Ser Asp Asp His Gly Lys Thr Trp Gln Ala Gly Thr Pro
Ile Gly225 230 235 240Thr Gly Met Asp Glu Asn Lys Val Val Glu Leu
Ser Asp Gly Ser Leu 245 250 255Met Leu Asn Ser Arg Ala Ser Asp Gly
Ser Gly Phe Arg Lys Val Ala 260 265 270His Ser Thr Asp Gly Gly Gln
Thr Trp Ser Glu Pro Val Ser Asp Lys 275 280 285Asn Leu Pro Asp Ser
Val Asp Asn Ala Gln Ile Ile Arg Ala Phe Pro 290 295 300Asn Ala Ala
Pro Asp Asp Pro Arg Ala Lys Val Leu Leu Leu Ser His305 310 315
320Ser Pro Asn Pro Arg Pro Trp Ser Arg Asp Arg Gly Thr Ile Ser Met
325 330 335Ser Cys Asp Asp Gly Ala Ser Trp Thr Thr Ser Lys Val Phe
His Glu 340 345 350Pro Phe Val Gly Tyr Thr Thr Ile Ala Val Gln Ser
Asp Gly Ser Ile 355 360 365Gly Leu Leu Ser Glu Asp Ala His Asn Gly
Ala Asp Tyr Gly Gly Ile 370 375 380Trp Tyr Arg Asn Phe Thr Met Asn
Trp Leu Gly Glu Gln Cys Gly Gln385 390 395 400Lys Pro Ala
Glu301257DNAArtificial SequenceSynthetic Construct 30ccatgaagcg
caaaaaaaaa ggcggcaaaa acggtaaaaa tcgtcgtaac cgtaagaaaa 60aaaatcctgg
agatcatcca caagctacac cagcacctgc accagatgct agcactgagc
120tgccagcaag catgtctcag gctcagcatc ttgcagcaaa tacggctact
gataattatc 180gcattccagc gattacaacc gctccgaatg gtgatttact
gattagctat gatgaacggc 240cgaaggacaa tggaaatggt ggttccgatg
cccctaaccc gaatcatatt gttcagcgtc 300gctccacaga tggcggtaaa
acttggagcg cgccaaccta tattcatcag ggtacggaga 360ctggcaagaa
agtgggatat tccgacccct cttatgtggt ggatcatcaa accggtacaa
420tcttcaattt tcatgtgaaa tcatacgatc agggctgggg aggtagccgt
gggggaacag 480acccggaaaa ccgcgggatt attcaggcag aggtgtctac
gagcacggat aatggatgga 540cgtggacaca tcgcaccatc accgcggata
ttacgaaaga taaaccgtgg accgcgcgtt 600ttgcggcgtc cggccaaggc
attcagatcc agcatgggcc gcatgccggc cgtctggtgc 660aacagtatac
cattcgtacg gccggtggag cggtgcaggc tgtatcggtt tattccgatg
720atcatgggaa aacgtggcag gctggcaccc cgattgggac gggtatggat
gaaaacaaag 780ttgtagagct gtctgacggc tctctgatgc tgaacagtcg
tgcgtcggac gggagcggct 840ttcgtaaggt tgcgcatagc actgatggtg
ggcagacctg gtccgaaccg gtttcggaca 900aaaatttgcc ggattcggtt
gataatgccc agataattcg tgcgtttcct aatgctgccc 960ccgatgaccc
gcgcgcgaaa gtacttcttc tgagtcattc cccaaatcca cgtccgtggt
1020cccgggatcg tggtacgata agcatgtcat gtgatgacgg ggcctcatgg
accacttcca 1080aagtttttca cgaaccgttt gtgggctaca cgactattgc
agttcagagt gatggaagca 1140tcggtctgct gtcggaggac gcgcacaatg
gcgctgatta tggtggcatc tggtatcgta 1200attttacgat gaactggctg
ggagaacaat gtggacaaaa acccgcggaa taagctt 125731416PRTArtificial
SequenceSynthetic Construct 31Met Lys Arg Lys Lys Lys Gly Gly Lys
Asn Gly Lys Asn Arg Arg Asn1 5 10 15Arg Lys Lys Lys Asn Pro Gly Asp
His Pro Gln Ala Thr Pro Ala Pro 20 25 30Ala Pro Asp Ala Ser Thr Glu
Leu Pro Ala Ser Met Ser Gln Ala Gln 35 40 45His Leu Ala Ala Asn Thr
Ala Thr Asp Asn Tyr Arg Ile Pro Ala Ile 50 55 60Thr Thr Ala Pro Asn
Gly Asp Leu Leu Ile Ser Tyr Asp Glu Arg Pro65 70 75 80Lys Asp Asn
Gly Asn Gly Gly Ser Asp Ala Pro Asn Pro Asn His Ile 85 90 95Val Gln
Arg Arg Ser Thr Asp Gly Gly Lys Thr Trp Ser Ala Pro Thr 100 105
110Tyr Ile His Gln Gly Thr Glu Thr Gly Lys Lys Val Gly Tyr Ser Asp
115 120 125Pro Ser Tyr Val Val Asp His Gln Thr Gly Thr Ile Phe Asn
Phe His 130 135 140Val Lys Ser Tyr Asp Gln Gly Trp Gly Gly Ser Arg
Gly Gly Thr Asp145 150 155 160Pro Glu Asn Arg Gly Ile Ile Gln Ala
Glu Val Ser Thr Ser Thr Asp 165 170 175Asn Gly Trp Thr Trp Thr His
Arg Thr Ile Thr Ala Asp Ile Thr Lys 180 185 190Asp Lys Pro Trp Thr
Ala Arg Phe Ala Ala Ser Gly Gln Gly Ile Gln 195 200 205Ile Gln His
Gly Pro His Ala Gly Arg Leu Val Gln Gln Tyr Thr Ile 210 215 220Arg
Thr Ala Gly Gly Ala Val Gln Ala Val Ser Val Tyr Ser Asp Asp225 230
235 240His Gly Lys Thr Trp Gln Ala Gly Thr Pro Ile Gly Thr Gly Met
Asp 245 250 255Glu Asn Lys Val Val Glu Leu Ser Asp Gly Ser Leu Met
Leu Asn Ser 260 265 270Arg Ala Ser Asp Gly Ser Gly Phe Arg Lys Val
Ala His Ser Thr Asp 275 280 285Gly Gly Gln Thr Trp Ser Glu Pro Val
Ser Asp Lys Asn Leu Pro Asp 290 295 300Ser Val Asp Asn Ala Gln Ile
Ile Arg Ala Phe Pro Asn Ala Ala Pro305 310 315 320Asp Asp Pro Arg
Ala Lys Val Leu Leu Leu Ser His Ser Pro Asn Pro 325 330 335Arg Pro
Trp Ser Arg Asp Arg Gly Thr Ile Ser Met Ser Cys Asp Asp 340 345
350Gly Ala Ser Trp Thr Thr Ser Lys Val Phe His Glu Pro Phe Val Gly
355 360 365Tyr Thr Thr Ile Ala Val Gln Ser Asp Gly Ser Ile Gly Leu
Leu Ser 370 375 380Glu Asp Ala His Asn Gly Ala Asp Tyr Gly Gly Ile
Trp Tyr Arg Asn385 390 395 400Phe Thr Met Asn Trp Leu Gly Glu Gln
Cys Gly Gln Lys Pro Ala Glu 405 410 4153243DNAArtificial
SequenceSynthetic Constructmisc_feature17, 18, 20, 21n = A,T,C or G
32ttttcgtctc ccatgvnnvn naagcgcaaa aaaaaaggcg gca
433310PRTArtificial SequenceSynthetic ConstructVARIANT2, 3Xaa = Any
Amino Acid 33Met Xaa Xaa Lys Arg Lys Lys Lys Gly Gly1 5
10341272DNAArtificial SequenceSynthetic Construct 34ccatgaagcg
caaaaaaaaa ggcggcaaaa acggtaaaaa tcgtcgtaac cgtaagaaaa 60aaaatcctgg
tggtggtggt tctggagatc atccacaagc tacaccagca cctgcaccag
120atgctagcac tgagctgcca gcaagcatgt ctcaggctca gcatcttgca
gcaaatacgg 180ctactgataa ttatcgcatt ccagcgatta caaccgctcc
gaatggtgat ttactgatta 240gctatgatga acggccgaag gacaatggaa
atggtggttc cgatgcccct aacccgaatc 300atattgttca gcgtcgctcc
acagatggcg gtaaaacttg gagcgcgcca acctatattc 360atcagggtac
ggagactggc aagaaagtgg gatattccga cccctcttat gtggtggatc
420atcaaaccgg tacaatcttc aattttcatg tgaaatcata cgatcagggc
tggggaggta 480gccgtggggg aacagacccg gaaaaccgcg ggattattca
ggcagaggtg tctacgagca 540cggataatgg atggacgtgg acacatcgca
ccatcaccgc ggatattacg aaagataaac 600cgtggaccgc gcgttttgcg
gcgtccggcc aaggcattca gatccagcat gggccgcatg 660ccggccgtct
ggtgcaacag tataccattc gtacggccgg tggagcggtg caggctgtat
720cggtttattc cgatgatcat gggaaaacgt ggcaggctgg caccccgatt
gggacgggta 780tggatgaaaa caaagttgta gagctgtctg acggctctct
gatgctgaac agtcgtgcgt 840cggacgggag cggctttcgt aaggttgcgc
atagcactga tggtgggcag acctggtccg 900aaccggtttc ggacaaaaat
ttgccggatt cggttgataa tgcccagata attcgtgcgt 960ttcctaatgc
tgcccccgat gacccgcgcg cgaaagtact tcttctgagt cattccccaa
1020atccacgtcc gtggtcccgg gatcgtggta cgataagcat gtcatgtgat
gacggggcct 1080catggaccac ttccaaagtt tttcacgaac cgtttgtggg
ctacacgact attgcagttc 1140agagtgatgg aagcatcggt ctgctgtcgg
aggacgcgca caatggcgct gattatggtg 1200gcatctggta tcgtaatttt
acgatgaact ggctgggaga acaatgtgga caaaaacccg 1260cggaataagc tt
127235421PRTArtificial SequenceSynthetic Construct 35Met Lys Arg
Lys Lys Lys Gly Gly Lys Asn Gly Lys Asn Arg Arg Asn1 5 10 15Arg Lys
Lys Lys Asn Pro Gly Gly Gly Gly Ser Gly Asp His Pro Gln 20 25 30Ala
Thr Pro Ala Pro Ala Pro Asp Ala Ser Thr Glu Leu Pro Ala Ser 35 40
45Met Ser Gln Ala Gln His Leu Ala Ala Asn Thr Ala Thr Asp Asn Tyr
50 55 60Arg Ile Pro Ala Ile Thr Thr Ala Pro Asn Gly Asp Leu Leu Ile
Ser65 70 75 80Tyr Asp Glu Arg Pro Lys Asp Asn Gly Asn Gly Gly Ser
Asp Ala Pro 85 90 95Asn Pro Asn His Ile Val Gln Arg Arg Ser Thr Asp
Gly Gly Lys Thr 100 105 110Trp Ser Ala Pro Thr Tyr Ile His Gln Gly
Thr Glu Thr Gly Lys Lys 115 120 125Val Gly Tyr Ser Asp Pro Ser Tyr
Val Val Asp His Gln Thr Gly Thr 130 135 140Ile Phe Asn Phe His Val
Lys Ser Tyr Asp Gln Gly Trp Gly Gly Ser145 150 155 160Arg Gly Gly
Thr Asp Pro Glu Asn Arg Gly Ile Ile Gln Ala Glu Val 165 170 175Ser
Thr Ser Thr Asp Asn Gly Trp Thr Trp Thr His Arg Thr Ile Thr 180 185
190Ala Asp Ile Thr Lys Asp Lys Pro Trp Thr Ala Arg Phe Ala Ala Ser
195 200 205Gly Gln Gly Ile Gln Ile Gln His Gly Pro His Ala Gly Arg
Leu Val 210 215 220Gln Gln Tyr Thr Ile Arg Thr Ala Gly Gly Ala Val
Gln Ala Val Ser225 230 235 240Val Tyr Ser Asp Asp His Gly Lys Thr
Trp Gln Ala Gly Thr Pro Ile 245 250 255Gly Thr Gly Met Asp Glu Asn
Lys Val Val Glu Leu Ser Asp Gly Ser 260 265 270Leu Met Leu Asn Ser
Arg Ala Ser Asp Gly Ser Gly Phe Arg Lys Val 275 280 285Ala His Ser
Thr Asp Gly Gly Gln Thr Trp Ser Glu Pro Val Ser Asp 290 295 300Lys
Asn Leu Pro Asp Ser Val Asp Asn Ala Gln Ile Ile Arg Ala Phe305 310
315 320Pro Asn Ala Ala Pro Asp Asp Pro Arg Ala Lys Val Leu Leu Leu
Ser 325 330 335His Ser Pro Asn Pro Arg Pro Trp Ser Arg Asp Arg Gly
Thr Ile Ser 340 345 350Met Ser Cys Asp Asp Gly Ala Ser Trp Thr Thr
Ser Lys Val Phe His 355 360 365Glu Pro Phe Val Gly Tyr Thr Thr Ile
Ala Val Gln Ser Asp Gly Ser 370 375 380Ile Gly Leu Leu Ser Glu Asp
Ala His Asn Gly Ala Asp Tyr Gly Gly385 390 395 400Ile Trp Tyr Arg
Asn Phe Thr Met Asn Trp Leu Gly Glu Gln Cys Gly 405 410 415Gln Lys
Pro Ala Glu 420361275DNAArtificial SequenceSynthetic Construct
36ccatggttaa gcgcaaaaaa aaaggcggca aaaacggtaa aaatcgtcgt aaccgtaaga
60aaaaaaatcc tggtggtggt ggttctggag atcatccaca agctacacca gcacctgcac
120cagatgctag cactgagctg ccagcaagca tgtctcaggc tcagcatctt
gcagcaaata 180cggctactga taattatcgc attccagcga ttacaaccgc
tccgaatggt gatttactga 240ttagctatga tgaacggccg aaggacaatg
gaaatggtgg ttccgatgcc cctaacccga 300atcatattgt tcagcgtcgc
tccacagatg gcggtaaaac ttggagcgcg ccaacctata 360ttcatcaggg
tacggagact ggcaagaaag tgggatattc cgacccctct tatgtggtgg
420atcatcaaac cggtacaatc ttcaattttc atgtgaaatc atacgatcag
ggctggggag 480gtagccgtgg gggaacagac ccggaaaacc gcgggattat
tcaggcagag gtgtctacga 540gcacggataa tggatggacg tggacacatc
gcaccatcac cgcggatatt acgaaagata 600aaccgtggac cgcgcgtttt
gcggcgtccg gccaaggcat tcagatccag catgggccgc 660atgccggccg
tctggtgcaa cagtatacca ttcgtacggc cggtggagcg gtgcaggctg
720tatcggttta ttccgatgat catgggaaaa cgtggcaggc tggcaccccg
attgggacgg 780gtatggatga aaacaaagtt gtagagctgt ctgacggctc
tctgatgctg aacagtcgtg 840cgtcggacgg gagcggcttt cgtaaggttg
cgcatagcac tgatggtggg cagacctggt 900ccgaaccggt ttcggacaaa
aatttgccgg attcggttga taatgcccag ataattcgtg 960cgtttcctaa
tgctgccccc gatgacccgc gcgcgaaagt acttcttctg agtcattccc
1020caaatccacg tccgtggtcc cgggatcgtg gtacgataag catgtcatgt
gatgacgggg 1080cctcatggac cacttccaaa gtttttcacg aaccgtttgt
gggctacacg actattgcag 1140ttcagagtga tggaagcatc ggtctgctgt
cggaggacgc gcacaatggc gctgattatg 1200gtggcatctg gtatcgtaat
tttacgatga actggctggg agaacaatgt ggacaaaaac 1260ccgcggaata agctt
127537422PRTArtificial SequenceSynthetic Construct 37Met Val Lys
Arg Lys Lys Lys Gly Gly Lys Asn Gly Lys Asn Arg Arg1 5 10 15Asn Arg
Lys Lys Lys Asn Pro Gly Gly Gly Gly Ser Gly Asp His Pro 20 25 30Gln
Ala Thr Pro Ala Pro Ala Pro Asp Ala Ser Thr Glu Leu Pro Ala 35 40
45Ser Met Ser Gln Ala Gln His Leu Ala Ala Asn Thr Ala Thr Asp Asn
50 55 60Tyr Arg Ile Pro Ala Ile Thr Thr Ala Pro Asn Gly Asp Leu Leu
Ile65 70 75 80Ser Tyr Asp Glu Arg Pro Lys Asp Asn Gly Asn Gly Gly
Ser Asp Ala 85 90 95Pro Asn Pro Asn His Ile Val Gln Arg Arg Ser Thr
Asp Gly Gly Lys 100 105 110Thr Trp Ser Ala Pro Thr Tyr Ile His Gln
Gly Thr Glu Thr Gly Lys 115 120 125Lys Val Gly Tyr Ser Asp Pro Ser
Tyr Val Val Asp His Gln Thr Gly 130 135 140Thr Ile Phe Asn Phe His
Val Lys Ser Tyr Asp Gln Gly Trp Gly Gly145 150 155 160Ser Arg Gly
Gly Thr Asp Pro Glu Asn Arg Gly Ile Ile Gln Ala Glu 165 170 175Val
Ser Thr Ser Thr Asp Asn Gly Trp Thr Trp Thr His Arg Thr Ile 180 185
190Thr Ala Asp Ile Thr Lys Asp Lys Pro Trp Thr Ala Arg Phe Ala Ala
195 200 205Ser Gly Gln Gly Ile Gln Ile Gln His Gly Pro His Ala Gly
Arg Leu 210 215 220Val Gln Gln Tyr Thr Ile Arg Thr Ala Gly Gly Ala
Val Gln Ala Val225 230 235 240Ser Val Tyr Ser Asp Asp His Gly Lys
Thr Trp Gln Ala Gly Thr Pro 245 250 255Ile Gly Thr Gly Met Asp Glu
Asn Lys Val Val Glu Leu Ser Asp Gly 260 265 270Ser Leu Met Leu Asn
Ser Arg Ala Ser Asp Gly Ser Gly Phe Arg Lys 275 280 285Val Ala His
Ser Thr Asp Gly Gly Gln Thr Trp Ser Glu Pro Val Ser 290 295 300Asp
Lys Asn Leu Pro Asp Ser Val Asp Asn Ala Gln Ile Ile Arg Ala305 310
315 320Phe Pro Asn Ala Ala Pro Asp Asp Pro Arg Ala Lys Val Leu Leu
Leu 325 330 335Ser His Ser Pro Asn Pro Arg Pro Trp Ser Arg Asp Arg
Gly Thr Ile 340 345 350Ser Met Ser Cys Asp Asp Gly Ala Ser Trp Thr
Thr Ser Lys Val Phe 355 360 365His Glu Pro Phe Val Gly Tyr Thr Thr
Ile Ala Val Gln Ser Asp Gly 370 375 380Ser Ile Gly Leu Leu Ser Glu
Asp Ala His Asn Gly Ala Asp Tyr Gly385 390 395 400Gly Ile Trp Tyr
Arg Asn Phe Thr Met Asn Trp Leu Gly Glu Gln Cys 405 410 415Gly Gln
Lys Pro Ala Glu 42038416PRTArtificial SequenceSynthetic Construct
38Met Lys Arg Lys Lys Lys Gly Gly Lys Asn Gly Lys Asn Arg Arg Asn1
5 10 15Arg Lys Lys Lys Asn Pro Gly Asp His Pro Gln Ala Thr Pro Ala
Pro 20 25 30Ala Pro Asp Ala Ser Thr Glu Leu Pro Ala Ser Met Ser Gln
Ala Gln 35 40 45His Leu Ala Ala Asn Thr Ala Thr Asp Asn Tyr Arg Ile
Pro Ala Ile 50 55 60Thr Thr Ala Pro Asn Gly Asp Leu Leu Ile Ser Tyr
Asp Glu Arg Pro65 70 75 80Lys Asp Asn Gly Asn Gly Gly Ser Asp Ala
Pro
Asn Pro Asn His Ile 85 90 95Val Gln Arg Arg Ser Thr Asp Gly Gly Lys
Thr Trp Ser Ala Pro Thr 100 105 110Tyr Ile His Gln Gly Thr Glu Thr
Gly Lys Lys Val Gly Tyr Ser Asp 115 120 125Pro Ser Tyr Val Val Asp
His Gln Thr Gly Thr Ile Phe Asn Phe His 130 135 140Val Lys Ser Tyr
Asp Gln Gly Trp Gly Gly Ser Arg Gly Gly Thr Asp145 150 155 160Pro
Glu Asn Arg Gly Ile Ile Gln Ala Glu Val Ser Thr Ser Thr Asp 165 170
175Asn Gly Trp Thr Trp Thr His Arg Thr Ile Thr Ala Asp Ile Thr Lys
180 185 190Asp Lys Pro Trp Thr Ala Arg Phe Ala Ala Ser Gly Gln Gly
Ile Gln 195 200 205Ile Gln His Gly Pro His Ala Gly Arg Leu Val Gln
Gln Tyr Thr Ile 210 215 220Arg Thr Ala Gly Gly Ala Val Gln Ala Val
Ser Val Tyr Ser Asp Asp225 230 235 240His Gly Lys Thr Trp Gln Ala
Gly Thr Pro Ile Gly Thr Gly Met Asp 245 250 255Glu Asn Lys Val Val
Glu Leu Ser Asp Gly Ser Leu Met Leu Asn Ser 260 265 270Arg Ala Ser
Asp Gly Ser Gly Phe Arg Lys Val Ala His Ser Thr Asp 275 280 285Gly
Gly Gln Thr Trp Ser Glu Pro Val Ser Asp Lys Asn Leu Pro Asp 290 295
300Ser Val Asp Asn Ala Gln Ile Ile Arg Ala Phe Pro Asn Ala Ala
Pro305 310 315 320Asp Asp Pro Arg Ala Lys Val Leu Leu Leu Ser His
Ser Pro Asn Pro 325 330 335Arg Pro Trp Ser Arg Asp Arg Gly Thr Ile
Ser Met Ser Cys Asp Asp 340 345 350Gly Ala Ser Trp Thr Thr Ser Lys
Val Phe His Glu Pro Phe Val Gly 355 360 365Tyr Thr Thr Ile Ala Val
Gln Ser Asp Gly Ser Ile Gly Leu Leu Ser 370 375 380Glu Asp Ala His
Asn Gly Ala Asp Tyr Gly Gly Ile Trp Tyr Arg Asn385 390 395 400Phe
Thr Met Asn Trp Leu Gly Glu Gln Cys Gly Gln Lys Pro Ala Glu 405 410
41539421PRTArtificial SequenceSynthetic Construct 39Val Lys Arg Lys
Lys Lys Gly Gly Lys Asn Gly Lys Asn Arg Arg Asn1 5 10 15Arg Lys Lys
Lys Asn Pro Gly Gly Gly Gly Ser Gly Asp His Pro Gln 20 25 30Ala Thr
Pro Ala Pro Ala Pro Asp Ala Ser Thr Glu Leu Pro Ala Ser 35 40 45Met
Ser Gln Ala Gln His Leu Ala Ala Asn Thr Ala Thr Asp Asn Tyr 50 55
60Arg Ile Pro Ala Ile Thr Thr Ala Pro Asn Gly Asp Leu Leu Ile Ser65
70 75 80Tyr Asp Glu Arg Pro Lys Asp Asn Gly Asn Gly Gly Ser Asp Ala
Pro 85 90 95Asn Pro Asn His Ile Val Gln Arg Arg Ser Thr Asp Gly Gly
Lys Thr 100 105 110Trp Ser Ala Pro Thr Tyr Ile His Gln Gly Thr Glu
Thr Gly Lys Lys 115 120 125Val Gly Tyr Ser Asp Pro Ser Tyr Val Val
Asp His Gln Thr Gly Thr 130 135 140Ile Phe Asn Phe His Val Lys Ser
Tyr Asp Gln Gly Trp Gly Gly Ser145 150 155 160Arg Gly Gly Thr Asp
Pro Glu Asn Arg Gly Ile Ile Gln Ala Glu Val 165 170 175Ser Thr Ser
Thr Asp Asn Gly Trp Thr Trp Thr His Arg Thr Ile Thr 180 185 190Ala
Asp Ile Thr Lys Asp Lys Pro Trp Thr Ala Arg Phe Ala Ala Ser 195 200
205Gly Gln Gly Ile Gln Ile Gln His Gly Pro His Ala Gly Arg Leu Val
210 215 220Gln Gln Tyr Thr Ile Arg Thr Ala Gly Gly Ala Val Gln Ala
Val Ser225 230 235 240Val Tyr Ser Asp Asp His Gly Lys Thr Trp Gln
Ala Gly Thr Pro Ile 245 250 255Gly Thr Gly Met Asp Glu Asn Lys Val
Val Glu Leu Ser Asp Gly Ser 260 265 270Leu Met Leu Asn Ser Arg Ala
Ser Asp Gly Ser Gly Phe Arg Lys Val 275 280 285Ala His Ser Thr Asp
Gly Gly Gln Thr Trp Ser Glu Pro Val Ser Asp 290 295 300Lys Asn Leu
Pro Asp Ser Val Asp Asn Ala Gln Ile Ile Arg Ala Phe305 310 315
320Pro Asn Ala Ala Pro Asp Asp Pro Arg Ala Lys Val Leu Leu Leu Ser
325 330 335His Ser Pro Asn Pro Arg Pro Trp Ser Arg Asp Arg Gly Thr
Ile Ser 340 345 350Met Ser Cys Asp Asp Gly Ala Ser Trp Thr Thr Ser
Lys Val Phe His 355 360 365Glu Pro Phe Val Gly Tyr Thr Thr Ile Ala
Val Gln Ser Asp Gly Ser 370 375 380Ile Gly Leu Leu Ser Glu Asp Ala
His Asn Gly Ala Asp Tyr Gly Gly385 390 395 400Ile Trp Tyr Arg Asn
Phe Thr Met Asn Trp Leu Gly Glu Gln Cys Gly 405 410 415Gln Lys Pro
Ala Glu 420
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