U.S. patent application number 13/616694 was filed with the patent office on 2013-05-23 for methods for identifying polypeptide targets and uses thereof for treating immunological diseases.
This patent application is currently assigned to Viral Logic Systems Technology Corp.. The applicant listed for this patent is Ajamete Kaykas, Craig A. Smith, Steven Wiley. Invention is credited to Ajamete Kaykas, Craig A. Smith, Steven Wiley.
Application Number | 20130129748 13/616694 |
Document ID | / |
Family ID | 38229867 |
Filed Date | 2013-05-23 |
United States Patent
Application |
20130129748 |
Kind Code |
A1 |
Wiley; Steven ; et
al. |
May 23, 2013 |
METHODS FOR IDENTIFYING POLYPEPTIDE TARGETS AND USES THEREOF FOR
TREATING IMMUNOLOGICAL DISEASES
Abstract
The present invention provides methods for identifying viral
virulence factors and for identifying cellular polypeptides to
which the viral polypeptides bind. The cellular polypeptide is
useful as a therapeutic target or as a therapeutic agent for
treating diseases and disorders, including immunological diseases
or disorders.
Inventors: |
Wiley; Steven; (Seattle,
WA) ; Smith; Craig A.; (Seattle, WA) ; Kaykas;
Ajamete; (Seattle, WA) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
Wiley; Steven
Smith; Craig A.
Kaykas; Ajamete |
Seattle
Seattle
Seattle |
WA
WA
WA |
US
US
US |
|
|
Assignee: |
Viral Logic Systems Technology
Corp.
Seattle
WA
|
Family ID: |
38229867 |
Appl. No.: |
13/616694 |
Filed: |
September 14, 2012 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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|
11728329 |
Mar 22, 2007 |
8293500 |
|
|
13616694 |
|
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|
|
60784620 |
Mar 22, 2006 |
|
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Current U.S.
Class: |
424/172.1 ;
435/5; 435/6.1; 435/6.13; 435/7.21; 435/7.23; 435/7.24 |
Current CPC
Class: |
A61P 1/04 20180101; A61P
3/10 20180101; A61P 19/02 20180101; G01N 33/56983 20130101; A61P
25/00 20180101; A61P 9/12 20180101; A61P 37/02 20180101; A61K
39/3955 20130101; A61P 13/12 20180101; G01N 33/6845 20130101; A61P
3/00 20180101; A61P 9/10 20180101; C12Q 1/70 20130101; A61P 31/04
20180101; A61P 7/00 20180101; A61P 11/00 20180101; G01N 2500/02
20130101; A61P 17/06 20180101; G01N 33/5008 20130101; A61P 15/00
20180101; A61P 21/04 20180101; C12Q 1/18 20130101; A61P 37/00
20180101; A61P 11/06 20180101; A61P 29/00 20180101; A61P 37/06
20180101 |
Class at
Publication: |
424/172.1 ;
435/7.24; 435/7.21; 435/7.23; 435/5; 435/6.13; 435/6.1 |
International
Class: |
G01N 33/68 20060101
G01N033/68; A61K 39/395 20060101 A61K039/395; C12Q 1/70 20060101
C12Q001/70 |
Goverment Interests
STATEMENT REGARDING SEQUENCE LISTING
[0002] The Sequence Listing associated with this application is
provided in text format in lieu of a paper copy, and is hereby
incorporated by reference into the specification. The name of the
text file containing the sequence listing
930118.402C1_SEQUENCE_LISTING_TXT. The text file is 114 kb, was
created on Sep. 14, 2012, and is being submitted electronically via
efs-web.
Claims
1. A method of identifying a cellular polypeptide to which a viral
polypeptide binds comprising: (a) contacting a cell, or a fraction
or a supernatant of the cell, and a fusion protein comprising a
viral polypeptide fused to an affinity tag comprising a polypeptide
tag, under conditions and for a time sufficient that permit a viral
polypeptide moiety of the fusion protein to interact with a
polypeptide associated with the cell, or the fraction or the
supernatant of the cell, to provide a fusion protein:cellular
polypeptide complex, wherein the viral polypeptide exhibits at
least one virulence trait; (b) isolating the fusion
protein:cellular polypeptide complex; and (c) determining the amino
acid sequence of the cellular polypeptide or of at least one
cellular polypeptide fragment comprising at least eight amino
acids, and thereby identifying a cellular polypeptide to which a
viral polypeptide binds.
2. The method of claim 1 further comprising prior to step (a), (i)
identifying in the genome of a virus, a polynucleotide sequence
that encodes a viral polypeptide, which viral polypeptide comprises
at least 40 amino acids; and (ii) producing a fusion protein
comprising the viral polypeptide fused to an affinity tag
sequence.
3. The method of claim 2, wherein the at least one virulence trait
is selected from (a) the trait that expression of a mutant viral
polypeptide in a cell infected by the virus correlates with a
decrease in virulence of the virus; (b) the trait that absence of
expression of the viral polypeptide in a cell infected by the virus
correlates with a decrease in virulence of the virus; (c) the trait
that the viral polypeptide is secreted by a cell infected with the
virus or the viral polypeptide is associated with a cellular
membrane of a cell infected by the virus; and (d) the trait that
the polynucleotide sequence in the virus genome is located in a
genomic region that encodes at least one other viral polypeptide
that is a viral virulence factor.
4. (canceled)
5. The method of claim 1 wherein the viral polypeptide (A) is
secreted by a cell infected with the virus, (B) is associated with
a cellular membrane, or (C) is intracellular.
6.-11. (canceled)
12. The method according to claim 1 wherein prior to step (b) the
cell is subjected to at least one stimulus, wherein the at least
one stimulus is selected from (a) an antibody that specifically
binds to a cognate antigen expressed by the cell; (b) a phorbol
ester; (c) concanavalin A; (d) a cytokine; (e) a chemokine; and (f)
ionomycin.
13. (canceled)
14. The method according to claim 1 wherein the fraction of the
cell is selected from a cell lysate, a cell extract, or at least
one isolated cell organelle.
15. The method according to claim 1 wherein the affinity tag
further comprises a detectable moiety.
16.-18. (canceled)
19. The method according to claim 1 wherein the affinity tag
further comprises a protease recognition sequence.
20. (canceled)
21. The method according claim 1, wherein the polypeptide tag is
selected from a hemagglutinin peptide; a calmodulin binding
polypeptide, a streptavidin binding peptide, an immunoglobulin Fc
polypeptide, an immunoglobulin mutein Fc polypeptide, a protein
C-tag, an at least one immunoglobulin binding staphylococcal
protein A domain, and Softag.TM..
22. (canceled)
23. The method according to claim 21 wherein the immunoglobulin Fc
polypeptide is a human IgG immunoglobulin Fc polypeptide or an
immunoglobulin mutein Fc polypeptide wherein the immunoglobulin
mutein Fc polypeptide is a human IgG1 immunoglobulin mutein Fc
polypeptide.
24.-25. (canceled)
26. The method according to claim 1 wherein the affinity tag
comprises a first polypeptide tag and a second polypeptide tag.
27. The method according to claim 26 wherein the affinity tag
further comprises a protease recognition sequence.
28. The method according to claim 27 wherein the protease
recognition sequence is located between the first polypeptide tag
and the second polypeptide tag.
29. The method according to claim 28 wherein the first polypeptide
tag and the second polypeptide tag are selected from a
hemagglutinin peptide; a calmodulin binding polypeptide, a
streptavidin binding peptide, an immunoglobulin Fc polypeptide, an
immunoglobulin mutein Fc polypeptide, a protein C-tag, an at least
one immunoglobulin binding staphylococcal protein A domain, and
Softag.TM..
30. The method according to claim 26 wherein the affinity tag
further comprises a third polypeptide tag.
31. The method according to claim 30 wherein the first polypeptide
tag, the second polypeptide tag, and the third polypeptide tag are
selected from a hemagglutinin peptide; a calmodulin binding
polypeptide, a streptavidin binding peptide, an immunoglobulin Fc
polypeptide, an immunoglobulin mutein Fc polypeptide, a protein
C-tag, an at least one immunoglobulin binding staphylococcal
protein A domain, and Softag.TM..
32. (canceled)
33. The method according to claim 30 wherein the affinity tag
comprises at least one protease recognition sequence.
34.-36. (canceled)
37. The method according to claim 30 wherein the affinity tag
further comprises a fourth polypeptide tag.
38. The method according to claim 37 wherein the first, second,
third, and fourth polypeptide are selected from a hemagglutinin
peptide; a calmodulin binding polypeptide, a streptavidin binding
peptide, an immunoglobulin Fc polypeptide, an immunoglobulin mutein
Fc polypeptide, a protein C-tag, an at least one immunoglobulin
binding staphylococcal protein A domain, and Softag.TM..
39. (canceled)
40. The method according to claim 37 wherein the fourth polypeptide
tag is the same as the first, second, or third polypeptide tag.
41.-43. (canceled)
44. The method according to claim 33 wherein the affinity tag
further comprises a second protease recognition sequence.
45.-46. (canceled)
47. The method according to claim 1 wherein step (c) comprises (i)
cleaving the isolated cellular polypeptide with a protease to
generate a plurality of polypeptide fragments of the cellular
polypeptide; (ii) determining the amino acid sequence of at least
one polypeptide fragment, wherein the fragment comprises at least
eight amino acids; and (iii) comparing the amino acid sequence of
the at least one polypeptide fragment with the amino acid sequence
of a known cellular polypeptide, thereby identifying the cellular
polypeptide to which the viral polypeptide binds.
48. The method of claim 47 wherein the amino acid sequence is
determined by a method comprising liquid chromatography and mass
spectrometry.
49. The method of claim 1 wherein step (b) comprises (i) contacting
the fusion protein:cellular polypeptide complex and a cognate
ligand of the affinity tag under conditions and for a time
sufficient to permit formation of a cognate ligand:fusion
protein:cellular polypeptide complex; and (ii) isolating the fusion
polypeptide:cellular polypeptide from the cognate ligand:fusion
protein:cellular polypeptide complex.
50. The method of claim 1 wherein the fusion protein is
recombinantly expressed.
51. The method of claim 1 wherein the fusion protein is
recombinantly expressed by the cell of step (a).
52. The method of claim 1 further comprising identifying a cell
type that comprises a cellular polypeptide to which the viral
polypeptide binds comprising: (i) contacting the fusion protein and
a biological sample comprising at least one cell, or a fraction of
the cell or a supernatant of the cell, under conditions and for a
time sufficient to permit the viral polypeptide moiety of the
fusion protein to interact with the at least one cell, or the cell
fraction or the cell supernatant; (ii) determining the presence or
absence of binding of the fusion protein to the at least one cell,
or the fraction or the supernatant thereof; (iii) isolating the
cell to which the fusion protein binds; and (iv) characterizing the
cell, and therefrom determining the cell type that comprises a
cellular polypeptide to which the viral polypeptide binds.
53. A method for identifying a cellular polypeptide to which a
viral polypeptide binds comprising: (a) identifying in the genome
of a virus, a polynucleotide sequence that encodes a viral
polypeptide, which viral polypeptide comprises at least 40 amino
acids; (b) introducing into a cell a recombinant expression
construct comprising a promoter operatively linked to a
polynucleotide encoding the viral polypeptide fused in frame with
an affinity tag; (c) isolating from the cell, or from a fraction of
the cell, or from a supernatant of the cell, a fusion
protein:cellular polypeptide complex; (d) isolating the fusion
protein:cellular polypeptide complex; and (e) determining the amino
acid sequence of the cellular polypeptide or of at least one
cellular polypeptide fragment comprising at least eight amino
acids, and thereby identifying a cellular polypeptide to which a
viral polypeptide binds.
54. A method for identifying a cellular polypeptide to which a
viral polypeptide binds comprising: (a) contacting a cell, or a
fraction or a supernatant of the cell, and a fusion protein
comprising a viral polypeptide moiety fused to an affinity tag
moiety, under conditions and for a time sufficient that permit the
viral polypeptide moiety of the fusion protein to interact with a
polypeptide associated with the cell, or the fraction or the
supernatant of the cell, to provide a fusion protein:cellular
polypeptide complex, wherein the viral polypeptide has at least one
virulence trait, and wherein the affinity tag comprises at least a
first polypeptide tag, a second polypeptide tag, and at least one
protease recognition sequence; (b) isolating the fusion
protein:cellular polypeptide complex, wherein said step of
isolating comprises: (i) contacting the fusion protein:cellular
polypeptide complex with a first cognate ligand of the first
polypeptide tag under conditions and for a time sufficient to
permit the affinity tag moiety of the fusion protein to interact
with the first cognate ligand to provide a first cognate
ligand:fusion protein:cellular polypeptide complex; (ii) contacting
the first cognate ligand:fusion protein:cellular polypeptide
complex with a protease capable of cleaving the fusion protein at
or near the protease recognition sequence to provide a cleaved
fusion protein:cellular polypeptide complex; (iii) contacting the
cleaved fusion protein:cellular polypeptide complex with a second
cognate ligand that specifically binds to the second polypeptide
tag, under conditions and for a time sufficient that permit the
second cognate ligand and the cleaved fusion protein:cellular
polypeptide complex to interact to form a second cognate
ligand:cleaved fusion protein:cellular polypeptide complex; and
(iv) isolating the cleaved fusion protein:cellular polypeptide
complex from the second cognate ligand:cleaved fusion
protein:cellular polypeptide complex; and (c) determining the amino
acid sequence of the cellular polypeptide or of at least one
polypeptide fragment of the cellular polypeptide, wherein the at
least one polypeptide fragment comprises at least eight amino
acids, and thereby identifying a cellular polypeptide to which a
viral polypeptide binds.
55. The method of claim 54 further comprising prior to the step of
contacting the cell, or a fraction or a supernatant of the cell,
and a fusion protein the steps of (a) identifying in the genome of
a virus, a polynucleotide sequence that encodes a viral
polypeptide, which viral polypeptide comprises at least 40 amino
acids; and (b) producing a fusion protein comprising the viral
polypeptide fused to an affinity tag sequence.
56. A method of identifying a cellular polypeptide to which a viral
polypeptide binds comprising: (a) identifying in the genome of a
virus, a polynucleotide sequence that encodes a viral polypeptide,
wherein the viral polypeptide comprises at least 40 amino acids;
(b) producing a fusion protein comprising the viral polypeptide
fused to an affinity tag sequence, wherein the affinity tag
sequence comprises a first polypeptide tag sequence, a second
polypeptide tag sequence, and a protease recognition sequence
located between the first and second polypeptide tag sequences; (c)
contacting the fusion protein and a cell, or a fraction or a
supernatant thereof, under conditions and for a time sufficient
that permit the viral polypeptide moiety of the fusion protein to
interact with a polypeptide associated with the cell, or the
fraction or the supernatant thereof, to provide a fusion
protein:cellular polypeptide complex; (d) isolating the fusion
protein:cellular polypeptide complex, wherein said step of
isolating comprises: (i) contacting the fusion protein:cellular
polypeptide complex with a first cognate ligand of the first
polypeptide tag sequence under conditions and for a time sufficient
to permit the affinity tag moiety of the fusion protein to interact
with the first cognate ligand to provide a first cognate ligand:
fusion protein:cellular polypeptide complex; (ii) contacting the
first cognate ligand:fusion protein:cellular polypeptide complex
with a protease capable of cleaving the fusion protein at or near
the protease recognition sequence to provide a cleaved fusion
protein:cellular polypeptide complex; (iii) contacting the cleaved
fusion protein:cellular polypeptide complex with a second cognate
ligand that specifically binds to the second polypeptide tag, under
conditions and for a time sufficient that permit the second cognate
ligand and the cleaved fusion protein:cellular polypeptide complex
to interact to form a second cognate ligand:cleaved fusion
protein:cellular polypeptide complex; and (iv) isolating the
cleaved fusion protein:cellular polypeptide complex; and (e)
determining the amino acid sequence of the cellular polypeptide or
of at least one cellular polypeptide fragment comprising at least
eight amino acids, and therefrom identifying a cellular polypeptide
to which a viral polypeptide binds.
57. A method of identifying an agent for treating an immunological
disease or disorder comprising: (a) identifying a cellular
polypeptide to which a viral polypeptide binds according to the
method of claim 1, wherein interaction between the cellular
polypeptide and the viral polypeptide alters immunoresponsiveness
of an immune cell; (b) contacting (i) the cellular polypeptide, or
a cell comprising the cellular polypeptide; (ii) the viral
polypeptide; (iii) and a candidate agent, under conditions and for
a time sufficient that permit the cellular polypeptide and the
viral polypeptide to interact; (c) determining the level of binding
of the viral polypeptide to the cellular polypeptide in the
presence of the candidate agent to the level of binding of the
viral polypeptide to the cellular polypeptide in the absence of the
candidate agent, wherein a decrease in the level of binding of the
viral polypeptide to the cellular polypeptide in the presence of
the candidate agent compared with the level of binding of the viral
polypeptide to the cellular polypeptide in the absence of the
candidate agent thereby identifies an agent for treating an
immunological disease or disorder.
58. The method according to claim 57 wherein the agent is selected
from (a) an antibody, or antigen-binding fragment thereof, (b) a
viral polypeptide/Fc polypeptide fusion protein; (c) a peptide/Fc
polypeptide fusion protein; (d) a small molecule; (e) a small
interfering RNA (siRNA); (f) an antisense polynucleotide; and (g)
an aptamer.
59.-61. (canceled)
62. A method of treating a disease or disorder comprising
administering to a subject in need thereof (a) a pharmaceutically
suitable carrier; and (b) an agent identified according to the
method of claim 57, wherein the disease or disorder is an
immunological disease or disorder, a cardiovascular disease or
disorder, a metabolic disease or disorder, or a proliferative
disease or disorder.
63. The method according to claim 62 wherein the agent is an
antibody, or antigen-binding fragment thereof.
64. A method for guiding the selection of a therapeutic agent for
treating a disease or medical disorder, comprising: (a) identifying
a viral polypeptide that increases the virulence of a virus in a
host infected with the virus; (b) identifying a cellular
polypeptide to which the viral polypeptide binds, wherein binding
of the viral polypeptide to the cellular polypeptide alters at
least one biological activity of a cell; (c) identifying one or
more agents that inhibit binding of the viral polypeptide to the
cellular polypeptide; (d) categorizing the capability of the one or
more agents identified in step (c) to alter at least one biological
effect of a cell, wherein altering the at least one biological
effect reduces the risk of developing a disease or medical disorder
or reduces at least one symptom of a disease or medical disorder in
a host; and (e) selecting at least one agent from step (d) for
testing in preclinical and clinical methods, and therefrom guiding
the selection of a therapeutic agent for treating a disease or
disorder.
65. The method according to claim 64 wherein the at least one
biological activity of the cell is immunoresponsiveness and the
cell is an immune cell.
66.-68. (canceled)
Description
CROSS-REFERENCES TO RELATED APPLICATIONS
[0001] This application is a Continuation of U.S. patent
application Ser. No. 11/728,329, filed Mar. 22, 2007, now allowed,
which claims the benefit U.S. Provisional Patent Application No.
60/784,620 filed Mar. 22, 2006, which applications are incorporated
herein by reference in their entireties.
BACKGROUND OF THE INVENTION
[0003] 1. Field of the Invention
[0004] The present invention provides a method for identifying
cellular polypeptides, which when a biological activity of the
cellular polypeptide is altered, a disease or disorder, in
particular, an immunological disease or disorder may be treated.
The method comprises identifying a viral virulence factor and its
cellular target(s). Also provided herein are agents and methods for
identifying such agents that affect a biological activity of the
cellular target and that may be used as therapeutic molecules for
treating a disease or disorder. Such agents are useful for altering
immunoresponsiveness of the immune system and for treating
immunological disorders in a subject.
[0005] 2. Description of the Related Art
[0006] Immunological diseases and disorders, including autoimmune
diseases and inflammatory diseases, afflict more than twenty
million people in the United States. Many immunological diseases
are debilitating and chronic, and thus affect a patient's
productivity, well-being, as well as general health.
[0007] A need exists to identify cellular polypeptides that are
effectors or modulators of an immune response and also to identify
agents that modulate the immune response by interacting with the
cellular polypeptides. Such agents are useful for treating and/or
preventing immunological diseases and disorders and other related
diseases and disorders. Provided herein are methods for identifying
cellular polypeptides that are useful as therapeutic targets.
BRIEF SUMMARY OF THE INVENTION
[0008] The present invention relates to the discovery of a method
for rapidly identifying cellular targets that are important in
modulating the human immune system, and then identifying the
counterstructures that will bind and modulate those targets.
[0009] In one embodiment, a method is provided for identifying a
cellular polypeptide to which a viral polypeptide binds comprising
(a) contacting a cell, or a fraction or a supernatant of the cell,
and a fusion protein comprising a viral polypeptide fused to an
affinity tag, under conditions and for a time sufficient that
permit a viral polypeptide moiety of the fusion protein to interact
with a polypeptide associated with the cell, or the fraction or the
supernatant of the cell, to provide a fusion protein:cellular
polypeptide complex, wherein the viral polypeptide exhibits at
least one virulence trait; (b) isolating the fusion
protein:cellular polypeptide complex; and (c) determining the amino
acid sequence of the cellular polypeptide or of at least one
cellular polypeptide fragment comprising at least eight amino
acids, and thereby identifying a cellular polypeptide to which a
viral polypeptide binds. In a certain embodiment, further
comprising prior to step (a), (i) identifying in the genome of a
virus, a polynucleotide sequence that encodes a viral polypeptide,
which viral polypeptide comprises at least 40 amino acids; and (ii)
producing a fusion protein comprising the viral polypeptide fused
to an affinity tag sequence. In a specific embodiment at least one
virulence trait comprises the trait that expression of a mutant
viral polypeptide in a cell infected by the virus correlates with a
decrease in virulence of the virus. In another specific embodiment,
at least one virulence trait comprises the trait that absence of
expression of the viral polypeptide in a cell infected by the virus
correlates with a decrease in virulence of the virus. In yet
another specific embodiment, the viral polypeptide (a) is secreted
by a cell infected with the virus, (b) is associated with a
cellular membrane, or (c) is intracellular. In a particular
embodiment, the viral polypeptide is secreted by the infected cell.
In another specific embodiment, the at least one virulence trait
comprises the trait that the viral polypeptide is secreted by a
cell infected with the virus or the viral polypeptide is associated
with a cellular membrane of a cell that is infected by the
virus.
[0010] In another specific embodiment of the aforementioned method,
the at least one virulence trait comprises the trait that the
polynucleotide sequence in the virus genome is located in a genomic
region that encodes at least one other viral polypeptide (i.e., a
second viral polypeptide) that is a viral virulence factor, wherein
the region is at the 5' terminal end or the 3' terminal end of the
virus genome. In one certain embodiment, the virus is a poxvirus.
In certain embodiments, the virus comprises a DNA genome, a
double-stranded RNA genome, or a single-stranded RNA genome. In a
particular embodiment, the virus comprises a DNA genome, and the
virus is a poxvirus, adenovirus, herpesvirus, or a hepatitis B
virus. In another specific embodiment, the virus comprises an RNA
genome and the virus is selected from a picornavirus, a retrovirus,
a hemorrhagic fever virus, or a hepatitis C virus.
[0011] In certain embodiments, prior to step (b) of the
aforementioned method, the cell is subjected to at least one
stimulus, and in certain embodiments, the cell is an immune cell
and the at least one stimulus is selected from (a) an antibody that
specifically binds to a cognate antigen expressed by the cell; (b)
a phorbol ester; (c) concanavalin A; (d) a cytokine; (e) a
chemokine; and (f) ionomycin. In other certain embodiments, the
immune cell is subjected to at least two or to at least three of
the aforementioned stimuli. In another embodiment, the fraction of
the cell is selected from a cell lysate, a cell extract, or at
least one isolated cell organelle. In a particular embodiment, the
affinity tag comprises a detectable moiety, and in other particular
embodiments, the affinity tag comprises a polypeptide tag and a
detectable moiety. In a particular embodiment, the detectable
moiety is selected from a fluorophore, a radionuclide, an enzyme,
and biotin.
[0012] In yet another embodiment of the aforementioned methods, the
affinity tag comprises a polypeptide tag. In a specific embodiment,
the affinity tag further comprises a protease recognition sequence.
In yet another specific embodiment, the protease recognition
sequence is located between the viral polypeptide and the
polypeptide tag. In particular embodiments, the polypeptide tag is
selected from a hemagglutinin peptide; a calmodulin binding
polypeptide, a streptavidin binding peptide, an immunoglobulin Fc
polypeptide, an immunoglobulin mutein Fc polypeptide, a protein
C-tag, an at least one immunoglobulin binding staphylococcal
protein A domain, and Softag.TM.. In a particular embodiment, the
affinity tag comprises the hemagglutinin peptide, which comprises
the amino acid sequence YPYDVDYA (SEQ ID NO:1). In another
particular embodiment, the affinity tag comprises the calmodulin
binding polypeptide, which comprises the amino acid sequence
KRRWKKNFIAVSAANRFKKISSSGAL (SEQ ID NO:3). In still another
particular embodiment, the affinity tag comprises an immunoglobulin
Fc polypeptide wherein the Fc polypeptide is a human IgG
immunoglobulin Fc polypeptide, and in certain particular
embodiments, the human IgG immunoglobulin Fc polypeptide is a human
IgG1 immunoglobulin Fc polypeptide. In certain other particular
embodiments, the polypeptide tag is an immunoglobulin mutein Fc
polypeptide, wherein the immunoglobulin mutein Fc polypeptide is a
human IgG1 immunoglobulin mutein Fc polypeptide. In still another
particular embodiment, the affinity tag comprises the protein
C-tag, which comprises the amino acid sequence EDQVDPRLIDGK (SEQ ID
NO:4). In yet still another particular embodiment, the affinity tag
comprises the streptavidin binding peptide comprises the amino acid
sequence selected from MDEKTTGWRGGHVVEGLAGELEQLRARLEHHPQGQREP (SEQ
ID NO:6) or DVEAWLDERVPLVET; SEQ ID NO:7). In still another
particular embodiment, the affinity tag comprises the at least one
immunoglobulin binding staphylococcal protein A domain, which
comprises an IgG-binding protein ZZ. In yet another specific
embodiment, the Softag.TM. comprises the amino sequence
SLAELLNAGLGGS (SEQ ID NO:11).
[0013] In another embodiment of the aforementioned methods, the
affinity tag comprises a first polypeptide tag and a second
polypeptide tag. In a specific embodiment, the affinity tag further
comprises a protease recognition sequence, and in a particular
embodiment, the protease recognition sequence is located between
the first polypeptide tag and the second polypeptide tag. In
certain embodiments, the first polypeptide tag and the second
polypeptide tag are selected from a hemagglutinin peptide; a
calmodulin binding polypeptide, a streptavidin binding peptide, an
immunoglobulin Fc polypeptide, an immunoglobulin mutein Fc
polypeptide, a protein C-tag, an at least one immunoglobulin
binding staphylococcal protein A domain, and Softag.TM..
[0014] In still other embodiments, the affinity tag further
comprises a third polypeptide tag, wherein the first polypeptide
tag, the second polypeptide tag, and the third polypeptide tag are
each selected from a hemagglutinin peptide; a calmodulin binding
polypeptide, a streptavidin binding peptide, an immunoglobulin Fc
polypeptide, an immunoglobulin mutein Fc polypeptide, a protein
C-tag, an at least one immunoglobulin binding staphylococcal
protein A domain, and Softag.TM.. In a specific embodiment, the
first polypeptide tag is a hemagglutinin peptide; the second
polypeptide tag is a protein C-tag; and the third polypeptide tag
is Softag.TM.. In other certain embodiments, the affinity tag
comprises at least one protease recognition sequence. In a specific
embodiment, the first polypeptide tag is a hemagglutinin peptide;
the second polypeptide tag is a protein C-tag; and the third
polypeptide tag is an at least one immunoglobulin binding
staphylococcal protein A domain; and the at least one protease
recognition sequence is a tobacco etch virus protease recognition
sequence or a Human Rhinovirus HRV3C protease recognition sequence.
In still yet another specific embodiment, the first polypeptide tag
is a hemagglutinin peptide; the second polypeptide tag is a protein
C-tag; and the third polypeptide tag is an immunoglobulin mutein Fc
polypeptide; and the at least one protease recognition sequence is
a tobacco etch virus protease recognition sequence or a Human
Rhinovirus HRV3C protease recognition sequence. In yet another
specific embodiment, the affinity tag comprises a first polypeptide
tag, a second polypeptide tag, a third polypeptide tag, and at
least one protease recognition sequence, wherein the at least one
protease recognition sequence is located between the first
polypeptide tag and the second polypeptide tag, or wherein the
protease recognition sequence is located between the second
polypeptide tag and the third polypeptide tag. In another specific
embodiment, the affinity tag further comprises a second protease
recognition sequence.
[0015] In yet another embodiment of the aforementioned methods, the
affinity tag further comprises a fourth polypeptide tag. In a
particular embodiment, each of the first, second, third, and fourth
polypeptide tags is selected from a hemagglutinin peptide, a
calmodulin binding polypeptide, a streptavidin binding peptide, an
immunoglobulin Fc polypeptide, an immunoglobulin mutein Fc
polypeptide, a protein C-tag, an at least one immunoglobulin
binding staphylococcal protein A domain, and Softag.TM.. In yet
another particular embodiment, the first polypeptide tag is a
hemagglutinin tag; the second polypeptide tag is a calmodulin
binding polypeptide; the third polypeptide tag is a streptavidin
binding peptide; the fourth polypeptide tag is an immunoglobulin
mutein Fc polypeptide; and the at least one protease recognition
sequence is a tobacco etch virus protease recognition sequence. In
still other embodiments, the fourth polypeptide tag is the same as
the first, second, or third polypeptide tag. In a specific
embodiment, the first polypeptide tag is a hemagglutinin
polypeptide; the second polypeptide tag is a protein C-tag; the
third polypeptide tag and is a streptavidin binding peptide; and
the fourth polypeptide tag is a repeat of the third polypeptide
tag. In a particular embodiment, the affinity tag further comprises
a protease recognition sequence between the second polypeptide tag
and the third polypeptide tag, wherein in a specific embodiment,
the protease recognition sequence is a Human Rhinovirus HRV3C
protease recognition sequence. In another specific embodiment, the
affinity tag further comprises a second protease recognition
sequence.
[0016] According to any of the aforementioned methods, the fusion
protein further comprises a signal peptide sequence a signal
peptide sequence. In certain embodiments, the signal peptide
sequence comprises the amino acid sequence of a human growth
hormone signal peptide sequence, which in certain embodiments
comprises the amino acid sequence MATGSRTSLLLAFGLLCLPWLQEGSA (SEQ
ID NO:12).
[0017] In another embodiment of the aforementioned methods, step
(c) comprises (i) cleaving the isolated cellular polypeptide with a
protease to generate at least one polypeptide fragment or a
plurality of polypeptide fragments of the cellular polypeptide;
(ii) determining the amino acid sequence of at least one
polypeptide fragment, wherein the fragment comprises at least eight
amino acids; and (iii) comparing the amino acid sequence of the at
least one polypeptide fragment with the amino acid sequence of a
known cellular polypeptide, thereby identifying the cellular
polypeptide to which the viral polypeptide binds. In a particular
embodiment, the amino acid sequence is determined by a method
comprising liquid chromatography and mass spectrometry, wherein in
certain embodiments, the method comprises liquid chromatography and
tandem mass spectrometry.
[0018] In still another embodiment of the aforementioned methods,
step (b) comprises (i) contacting the fusion protein:cellular
polypeptide complex and a cognate ligand of the affinity tag under
conditions and for a time sufficient to permit formation of a
cognate ligand:fusion protein:cellular polypeptide complex; and
(ii) isolating the fusion polypeptide: cellular polypeptide from
the cognate ligand: fusion protein:cellular polypeptide complex. In
certain embodiments, the fusion protein is recombinantly expressed.
In a particular embodiment, the fusion protein is recombinantly
expressed by the cell of step (a).
[0019] In yet another embodiment of the aforementioned methods, the
method further comprises identifying a cell type that comprises a
cellular polypeptide to which a viral polypeptide that exhibits at
least one virulence trait binds comprising (i) contacting the
fusion protein and a biological sample comprising at least one
cell, or a fraction of the cell or a supernatant of the cell, under
conditions and for a time sufficient to permit the viral
polypeptide moiety of the fusion protein to interact with the at
least one cell, or the cell fraction or the cell supernatant; (ii)
determining the presence or absence of binding of the fusion
protein to the at least one cell, or the fraction or the
supernatant thereof; (iii) isolating the cell to which the fusion
protein binds; and (iv) characterizing the cell, and therefrom
determining the cell type that comprises a cellular polypeptide to
which the viral polypeptide binds.
[0020] In another embodiment, a method is provided for identifying
a cellular polypeptide to which a viral polypeptide binds
comprising (a) identifying in the genome of a virus, a
polynucleotide sequence that encodes a viral polypeptide, which
viral polypeptide comprises at least 40 amino acids; (b) producing
a fusion protein comprising the viral polypeptide fused to an
affinity tag sequence; (c) contacting the fusion protein and a
cell, or a fraction or a supernatant of the cell, under conditions
and for a time sufficient that permit the viral polypeptide moiety
of the fusion protein to interact with a polypeptide associated
with the cell, or the fraction or the supernatant thereof, to
provide a fusion protein:cellular polypeptide complex; (d)
isolating the fusion protein:cellular polypeptide complex; and (e)
determining the amino acid sequence of all or a portion of the
cellular polypeptide, and thereby identifying a cellular
polypeptide to which a viral polypeptide binds. In a particular
embodiment, the fusion protein is produced by the cell of step (c).
In a particular embodiment, the viral polypeptide exhibits at least
one virulence trait that comprises the trait that expression of a
mutant viral polypeptide in a cell infected by the virus correlates
with a decrease in virulence of the virus. In another particular
embodiment, the at least one virulence trait comprises the trait
that absence of expression of the viral polypeptide in a cell
infected by the virus correlates with a decrease in virulence of
the virus. In yet another particular embodiment, the viral
polypeptide (i) is secreted by a cell infected with the virus, (ii)
is associated with a cellular membrane, or (iii) is intracellular.
In certain particular embodiments, the at least one virulence trait
of the viral polypeptide comprises the trait that the viral
polypeptide is secreted by a cell infected with the virus; in
another certain embodiment, the at least one virulence trait
comprises the trait that the viral polypeptide is associated with a
cellular membrane of the cell infected by the virus. In another
embodiment of the aforementioned method, the at least one virulence
trait comprises the trait that the polynucleotide sequence in the
virus genome is located in a genomic region that encodes at least
one other viral polypeptide (i.e., a second viral polypeptide) that
is a viral virulence factor, wherein the region is at the 5'
terminal end or the 3' terminal end of the virus genome. In certain
embodiments, the virus is a poxvirus. In certain embodiments, the
virus comprises a DNA genome, a double-stranded RNA genome, or a
single-stranded RNA genome. In a particular embodiment, the virus
comprises a DNA genome, and the virus is a poxvirus, adenovirus,
herpesvirus, or a hepatitis B virus. In another specific
embodiment, the virus comprises an RNA genome and the virus is
selected from a picornavirus, a retrovirus, a hemorrhagic fever
virus, or a hepatitis C virus. In certain embodiments, prior to the
step of contacting the fusion protein and a cell, or a fraction or
a supernatant of the cell, the cell is subjected to at least one
stimulus, and in certain embodiments, the cell is an immune cell
and the at least one stimulus is selected from (a) an antibody that
specifically binds to a cognate antigen expressed by the cell; (b)
a phorbol ester; (c) concanavalin A; (d) a cytokine; (e) a
chemokine; and (f) ionomycin. In other certain embodiments, the
immune cell is subjected to at least two or to at least three of
the aforementioned stimuli. In another embodiment, the fraction of
the cell is selected from a cell lysate, a cell extract, or at
least one isolated cell organelle. In a particular embodiment, the
affinity tag comprises a detectable moiety, and in other particular
embodiments, the affinity tag comprises a polypeptide tag and a
detectable moiety. In a particular embodiment, the detectable
moiety is selected from a fluorophore, a radionuclide, an enzyme,
and biotin. In certain embodiments, the affinity tag comprises at
least one polypeptide tag, and the polypeptide tag is selected from
a hemagglutinin peptide, a calmodulin binding polypeptide, a
streptavidin binding peptide, an immunoglobulin Fc polypeptide, an
immunoglobulin mutein Fc polypeptide, a protein C-tag, an at least
one immunoglobulin binding staphylococcal protein A domain, and
Softag.TM.. In another embodiment, the affinity tag comprises at
least two, three, or four polypeptide tags, and each of the at
least two, three, or four polypeptide tags is selected from
hemagglutinin peptide, a calmodulin binding polypeptide, a
streptavidin binding peptide, an immunoglobulin Fc polypeptide, an
immunoglobulin mutein Fc polypeptide, a protein C-tag, an at least
one immunoglobulin binding staphylococcal protein A domain, and
Softag.TM.. In a specific embodiment, the affinity tag further
comprises at least one protease recognition sequence, wherein the
at least one protease recognition sequence is either a tobacco etch
virus protease recognition sequence or a Human Rhinovirus HRV3C
protease recognition sequence. In yet another specific embodiment,
the affinity tag further comprises at least two protease
recognition sequences, wherein at least one protease recognition
sequence is either a tobacco etch virus protease recognition
sequence or a Human Rhinovirus HRV3C protease recognition
sequence.
[0021] In one embodiment, a method is provided for identifying a
cellular polypeptide to which a viral polypeptide binds, comprising
(a) identifying in the genome of a virus, a polynucleotide sequence
that encodes a viral polypeptide, which viral polypeptide comprises
at least 40 amino acids; (b) introducing into a cell a recombinant
expression construct comprising a promoter operatively linked to a
polynucleotide encoding the viral polypeptide fused in frame with
an affinity tag; (c) isolating from the cell, or from a fraction of
the cell, or from a supernatant of the cell, a fusion
protein:cellular polypeptide complex; (d) isolating the fusion
protein:cellular polypeptide complex; and (e) determining the amino
acid sequence of the cellular polypeptide or of at least one
cellular polypeptide fragment comprising at least eight amino
acids, and thereby identifying a cellular polypeptide to which a
viral polypeptide binds. In a particular embodiment, the viral
polypeptide comprises at least one virulence trait. In a specific
embodiment, the at least one virulence trait comprises the trait
that expression of a mutant viral polypeptide in a cell infected by
the virus correlates with a decrease in virulence of the virus. In
another particular embodiment, the at least one virulence trait
comprises the trait that absence of expression of the viral
polypeptide in a cell infected by the virus correlates with a
decrease in virulence of the virus. In yet another particular
embodiment, the viral polypeptide (i) is secreted by a cell
infected with the virus, (ii) is associated with a cellular
membrane, or (iii) is intracellular. In certain particular
embodiments, the at least one virulence trait of the viral
polypeptide comprises the trait that the viral polypeptide is
secreted by a cell infected with the virus; in another certain
embodiment, the at least one virulence trait comprises the trait
that the viral polypeptide is associated with a cellular membrane
of the cell infected by the virus. In another embodiment of the
aforementioned method, the at least one virulence trait comprises
the trait that the polynucleotide sequence in the virus genome is
located in a genomic region that encodes at least one other viral
polypeptide (i.e., a second viral polypeptide) that is a viral
virulence factor, wherein the region is at the 5' terminal end or
the 3' terminal end of the virus genome. In certain embodiments,
the virus is a poxvirus. In certain embodiments, the virus
comprises a DNA genome, a double-stranded RNA genome, or a
single-stranded RNA genome. In a particular embodiment, the virus
comprises a DNA genome, and the virus is a poxvirus, adenovirus,
herpesvirus, or a hepatitis B virus. In another specific
embodiment, the virus comprises an RNA genome and the virus is
selected from a picornavirus, a retrovirus, a hemorrhagic fever
virus, or a hepatitis C virus. In certain embodiments, the method
further comprises prior to step (c) the cell is subjected to at
least one stimulus, and in certain embodiments, the cell is an
immune cell and the at least one stimulus is selected from (a) an
antibody that specifically binds to a cognate antigen expressed by
the cell; (b) a phorbol ester; (c) concanavalin A; (d) a cytokine;
(e) a chemokine; and (f) ionomycin. In other certain embodiments,
the immune cell is subjected to at least two or to at least three
of the aforementioned stimuli. In another embodiment, the fraction
of the cell is selected from a cell lysate, a cell extract, or at
least one isolated cell organelle. In a particular embodiment, the
affinity tag comprises a detectable moiety, and in other particular
embodiments, the affinity tag comprises a polypeptide tag and a
detectable moiety. In a particular embodiment, the detectable
moiety is selected from a fluorophore, a radionuclide, an enzyme,
and biotin. In certain embodiments, the affinity tag comprises at
least one polypeptide tag, and the polypeptide tag is selected from
a hemagglutinin peptide; a calmodulin binding polypeptide, a
streptavidin binding peptide, an immunoglobulin Fc polypeptide, an
immunoglobulin mutein Fc polypeptide, a protein C-tag, an at least
one immunoglobulin binding staphylococcal protein A domain, and
Softag.TM.. In another embodiment, the affinity tag comprises at
least two, three, or four polypeptide tags, and each of the at
least two, three, or four polypeptide tags is selected from a
hemagglutinin peptide; a calmodulin binding polypeptide, a
streptavidin binding peptide, an immunoglobulin Fc polypeptide, an
immunoglobulin mutein Fc polypeptide, a protein C-tag, an at least
one immunoglobulin binding staphylococcal protein A domain, and
Softag.TM.. In a specific embodiment, the affinity tag further
comprises at least one protease recognition sequence, wherein the
at least one protease recognition sequence is either a tobacco etch
virus protease recognition sequence or a Human Rhinovirus HRV3C
protease recognition sequence. In yet another specific embodiment,
the affinity tag further comprises at least two protease
recognition sequences, wherein at least one protease recognition
sequence is either a tobacco etch virus protease recognition
sequence or a Human Rhinovirus HRV3C protease recognition
sequence.
[0022] In yet another embodiment, a method is provided for
identifying a cellular polypeptide to which a viral polypeptide
binds comprising (a) contacting a cell, or a fraction or a
supernatant of the cell, and a fusion protein comprising a viral
polypeptide moiety fused to an affinity tag moiety, under
conditions and for a time sufficient that permit the viral
polypeptide moiety of the fusion protein to interact with a
polypeptide associated with the cell, or the fraction or the
supernatant of the cell, to provide a fusion protein:cellular
polypeptide complex, wherein the viral polypeptide has at least one
virulence trait, and wherein the affinity tag comprises at least a
first polypeptide tag, a second polypeptide tag, and at least one
protease recognition sequence; (b) isolating the fusion
protein:cellular polypeptide complex, wherein said step of
isolating comprises (i) contacting the fusion protein:cellular
polypeptide complex with a first cognate ligand of the first
polypeptide tag under conditions and for a time sufficient to
permit the affinity tag moiety of the fusion protein to interact
with the first cognate ligand to provide a first cognate
ligand:fusion protein:cellular polypeptide complex; (ii) contacting
the first cognate ligand:fusion protein:cellular polypeptide
complex with a protease capable of cleaving the fusion protein at
or near the protease recognition sequence to provide a cleaved
fusion protein:cellular polypeptide complex; (iii) contacting the
cleaved fusion protein:cellular polypeptide complex with a second
cognate ligand that specifically binds to the second polypeptide
tag, under conditions and for a time sufficient that permit the
second cognate ligand and the cleaved fusion protein:cellular
polypeptide complex to interact to form a second cognate
ligand:cleaved fusion protein:cellular polypeptide complex; and
(iv) isolating the cleaved fusion protein:cellular polypeptide
complex from the second cognate ligand:cleaved fusion
protein:cellular polypeptide complex; and (c) determining the amino
acid sequence of the cellular polypeptide or of at least one
polypeptide fragment of the cellular polypeptide, wherein the at
least one polypeptide fragment comprises at least eight amino
acids, and thereby identifying a cellular polypeptide to which a
viral polypeptide binds. In a specific embodiment, the method
further comprises prior to the step of contacting the cell, or a
fraction or a supernatant of the cell, and a fusion protein the
steps of (a) identifying in the genome of a virus, a polynucleotide
sequence that encodes a viral polypeptide, which viral polypeptide
comprises at least 40 amino acids; and (b) producing a fusion
protein comprising the viral polypeptide fused to an affinity tag
sequence. In a certain embodiment, prior to the step of contacting
the cell, or a fraction or a supernatant of the cell, and a fusion
protein further comprises (i) identifying in the genome of a virus,
a polynucleotide sequence that encodes a viral polypeptide, which
viral polypeptide comprises at least 40 amino acids; and (ii)
producing a fusion protein comprising the viral polypeptide fused
to an affinity tag sequence. In a particular embodiment, the viral
polypeptide comprises at least one virulence trait. In a specific
embodiment, the at least one virulence trait comprises the trait
that expression of a mutant viral polypeptide in a cell infected by
the virus correlates with a decrease in virulence of the virus. In
another particular embodiment, the at least one virulence trait
comprises the trait that absence of expression of the viral
polypeptide in a cell infected by the virus correlates with a
decrease in virulence of the virus. In yet another particular
embodiment, the viral polypeptide (i) is secreted by a cell
infected with the virus, (ii) is associated with a cellular
membrane, or (iii) is intracellular. In certain particular
embodiments, the at least one virulence trait of the viral
polypeptide comprises the trait that the viral polypeptide is
secreted by a cell infected with the virus; in another certain
embodiment, the at least one virulence trait comprises the trait
that the viral polypeptide is associated with a cellular membrane
of the cell infected by the virus. In another embodiment of the
aforementioned method, the at least one virulence trait comprises
the trait that the polynucleotide sequence in the virus genome is
located in a genomic region that encodes at least one other viral
polypeptide (i.e., a second viral polypeptide) that is a viral
virulence factor, wherein the region is at the 5' terminal end or
the 3' terminal end of the virus genome. In certain embodiments,
the virus is a poxvirus. In certain embodiments, the virus
comprises a DNA genome, a double-stranded RNA genome, or a
single-stranded RNA genome. In a particular embodiment, the virus
comprises a DNA genome, and the virus is a poxvirus, adenovirus,
herpesvirus, or a hepatitis B virus. In another specific
embodiment, the virus comprises an RNA genome and the virus is
selected from a picornavirus, a retrovirus, a hemorrhagic fever
virus, or a hepatitis C virus. In certain embodiments, prior to
step (a) the cell is subjected to at least one stimulus, and in
certain embodiments, the cell is an immune cell and the at least
one stimulus is selected from (a) an antibody that specifically
binds to a cognate antigen expressed by the cell; (b) a phorbol
ester; (c) concanavalin A; (d) a cytokine; (e) a chemokine; and (f)
ionomycin. In other certain embodiments, the immune cell is
subjected to at least two or to at least three of the
aforementioned stimuli. In another embodiment, the fraction of the
cell is selected from a cell lysate, a cell extract, or at least
one isolated cell organelle. In a particular embodiment, the
affinity tag comprises a detectable moiety, and in other particular
embodiments, the affinity tag comprises a polypeptide tag and a
detectable moiety. In a particular embodiment, the detectable
moiety is selected from a fluorophore, a radionuclide, an enzyme,
and biotin. In certain embodiments, the affinity tag comprises at
least one polypeptide tag, and wherein the polypeptide tag is
selected from a hemagglutinin peptide; a calmodulin binding
polypeptide, a streptavidin binding peptide, an immunoglobulin Fc
polypeptide, an immunoglobulin mutein Fc polypeptide, a protein
C-tag, an at least one immunoglobulin binding staphylococcal
protein A domain, and Softag.TM.. In another embodiment, the
affinity tag comprises at least two, three, or four polypeptide
tags, and wherein each of the at least two, three, or four
polypeptide tags is selected from hemagglutinin peptide, a
calmodulin binding polypeptide, a streptavidin binding peptide, an
immunoglobulin Fc polypeptide, an immunoglobulin mutein Fc
polypeptide, a protein C-tag, an at least one immunoglobulin
binding staphylococcal protein A domain, and Softag.TM.. In a
specific embodiment, the affinity tag further comprises at least
one protease recognition sequence, wherein the at least one
protease recognition sequence is either a tobacco etch virus
protease recognition sequence or a Human Rhinovirus HRV3C protease
recognition sequence. In yet another specific embodiment, the
affinity tag further comprises at least two protease recognition
sequences, wherein at least one protease recognition sequence is
either a tobacco etch virus protease recognition sequence or a
Human Rhinovirus HRV3C protease recognition sequence.
[0023] In another embodiment, a method for identifying a cellular
polypeptide to which a viral polypeptide binds comprises (a)
contacting an isolated viral polypeptide with a cell, or a fraction
or supernatant of the cell, under conditions and for a time
sufficient that permit the viral polypeptide moiety of the fusion
protein to interact with a polypeptide associated with the cell,
providing a viral polypeptide:cellular polypeptide complex, wherein
the viral polypeptide comprises at least one virulence trait
selected from (i) expression of a mutant of the viral polypeptide
in a cell infected by a virus correlates with a decrease in
virulence of the virus, wherein the virus comprises a genome that
encodes the viral polypeptide; (ii) absence of expression of the
viral polypeptide in a cell infected by a virus correlates with a
decrease in virulence of the virus, wherein the virus comprises a
genome that encodes the viral polypeptide; (iii) the viral
polypeptide is secreted by a cell infected with a virus wherein the
virus comprises a genome that encodes the viral polypeptide; (iv)
the viral polypeptide is encoded by a polynucleotide sequence
present in the genome of a virus, wherein the polynucleotide
sequence is located at a genomic region that encodes at least one
other viral polypeptide (i.e., a second viral polypeptide) that is
a viral virulence factor; (v) the viral polypeptide is encoded by a
polynucleotide sequence present in the genome of a virus, wherein
the polynucleotide sequence is located at the 5' terminal end or
the 3' terminal end of the viral genome; and (vi) the viral
polypeptide comprises at least 40 amino acids; and (b) isolating
the viral polypeptide:cellular polypeptide complex; and (c)
determining the amino acid sequence of the cellular polypeptide or
of at least one cellular polypeptide fragment comprising at least
eight amino acids, and thereby identifying a cellular polypeptide
to which a viral polypeptide binds. In another embodiment of the
aforementioned method, the polynucleotide sequence in the virus
genome is located in a genomic region that encodes at least one
viral virulence factor, wherein the region is at the 5' terminal
end or the 3' terminal end of the virus genome, and wherein in
certain embodiments, the virus is a poxvirus. In certain
embodiments, the virus comprises a DNA genome, a double-stranded
RNA genome, or a single-stranded RNA genome. In a particular
embodiment, the virus comprises a DNA genome, and the virus is a
poxvirus, adenovirus, herpesvirus, or a hepatitis B virus. In
another specific embodiment, the virus comprises an RNA genome and
the virus is selected from a picornavirus, a retrovirus, a
hemorrhagic fever virus, or a hepatitis C virus. In certain
embodiments, prior to step (a) the cell is subjected to at least
one stimulus, and in certain embodiments, the cell is an immune
cell and the at least one stimulus is selected from (a) an antibody
that specifically binds to a cognate antigen expressed by the cell;
(b) a phorbol ester; (c) concanavalin A; (d) a cytokine; (e) a
chemokine; and (f) ionomycin. In other certain embodiments, the
immune cell is subjected to at least two or to at least three of
the aforementioned stimuli. In another embodiment, the fraction of
the cell is selected from a cell lysate, a cell extract, or at
least one isolated cell organelle. In a particular embodiment, the
affinity tag comprises a detectable moiety, and in other particular
embodiments, the affinity tag comprises a polypeptide tag and a
detectable moiety. In a particular embodiment, the detectable
moiety is selected from a fluorophore, a radionuclide, an enzyme,
and biotin. In certain embodiments, the affinity tag comprises at
least one polypeptide tag, and the polypeptide tag is selected from
a hemagglutinin peptide, a calmodulin binding polypeptide, a
streptavidin binding peptide, an immunoglobulin Fc polypeptide, an
immunoglobulin mutein Fc polypeptide, a protein C-tag, an at least
one immunoglobulin binding staphylococcal protein A domain, and
Softag.TM.. In another embodiment, the affinity tag comprises at
least two, three, or four polypeptide tags, and each of the at
least two, three, or four polypeptide tags is selected from
hemagglutinin peptide, a calmodulin binding polypeptide, a
streptavidin binding peptide, an immunoglobulin Fc polypeptide, an
immunoglobulin mutein Fc polypeptide, a protein C-tag, an at least
one immunoglobulin binding staphylococcal protein A domain, and
Softag.TM.. In a specific embodiment, the affinity tag further
comprises at least one protease recognition sequence, wherein the
at least one protease recognition is either a tobacco etch virus
protease recognition sequence or a Human Rhinovirus HRV3C protease
recognition sequence. In yet another specific embodiment, the
affinity tag further comprises at least one protease recognition
sequence, wherein at least one protease recognition sequence is
either a tobacco etch virus protease recognition sequence or a
Human Rhinovirus HRV3C protease recognition sequence.
[0024] In another embodiment, a method of identifying a cellular
polypeptide to which a viral polypeptide binds, comprises (a)
identifying in the genome of a virus, a polynucleotide sequence
that encodes a viral polypeptide, wherein the viral polypeptide
comprises at least 40 amino acids; (b) producing a fusion protein
comprising the viral polypeptide fused to an affinity tag sequence,
wherein the affinity tag sequence comprises a first polypeptide tag
sequence, a second polypeptide tag sequence, and a protease
recognition sequence located between the first and second
polypeptide tag sequences; (c) contacting the fusion protein and a
cell, or a fraction or a supernatant of the cell, under conditions
and for a time sufficient that permit the viral polypeptide moiety
of the fusion protein to interact with a polypeptide associated
with the cell, or the fraction or the supernatant thereof, to
provide a fusion protein:cellular polypeptide complex; (d)
isolating the fusion protein:cellular polypeptide complex, wherein
said step of isolating comprises (i) contacting the fusion
protein:cellular polypeptide complex with a first cognate ligand of
the first polypeptide tag under conditions and for a time
sufficient to permit the affinity tag moiety of the fusion protein
to interact with the first cognate ligand to provide a first
cognate ligand:fusion protein:cellular polypeptide complex; (ii)
contacting the first cognate ligand:fusion protein:cellular
polypeptide complex with a protease capable of cleaving the fusion
protein at or near the protease recognition sequence to provide a
cleaved fusion protein:cellular polypeptide complex; (iii)
contacting the cleaved fusion protein:cellular polypeptide complex
with a second cognate ligand that specifically binds to the second
polypeptide tag, under conditions and for a time sufficient that
permit the second cognate ligand and the cleaved fusion
protein:cellular polypeptide complex to interact to form a second
cognate ligand:cleaved fusion protein:cellular polypeptide complex;
and (iv) isolating the cleaved fusion protein:cellular polypeptide
complex; and (e) determining the amino acid sequence of the
cellular polypeptide or of at least one cellular polypeptide
fragment comprising at least eight amino acids, and therefrom
identifying a cellular polypeptide to which a viral polypeptide
binds. In a particular embodiment, the viral polypeptide comprises
at least one virulence trait. In a specific embodiment, the at
least one virulence trait comprises the trait that expression of a
mutant viral polypeptide in a cell infected by the virus correlates
with a decrease in virulence of the virus. In another particular
embodiment, the at least one virulence trait comprises the trait
that absence of expression of the viral polypeptide in a cell
infected by the virus correlates with a decrease in virulence of
the virus. In yet another particular embodiment, the viral
polypeptide (i) is secreted by a cell infected with the virus, (ii)
is associated with a cellular membrane, or (iii) is intracellular.
In certain particular embodiments, the at least one virulence trait
of the viral polypeptide comprises the trait that the viral
polypeptide is secreted by a cell infected with the virus; in
another certain embodiment, the at least one virulence trait
comprises the trait that the viral polypeptide is associated with a
cellular membrane of the cell infected by the virus. In another
embodiment of the aforementioned method, the at least one virulence
trait comprises the trait that the polynucleotide sequence in the
virus genome is located in a genomic region that encodes at least
one other viral polypeptide (i.e., a second viral polypeptide) that
is a viral virulence factor, wherein the region is at the 5'
terminal end or the 3' terminal end of the virus genome. In certain
embodiments, the virus is a poxvirus. In certain embodiments, the
virus comprises a DNA genome, a double-stranded RNA genome, or a
single-stranded RNA genome. In a particular embodiment, the virus
comprises a DNA genome, and the virus is a poxvirus, adenovirus,
herpesvirus, or a hepatitis B virus. In another specific
embodiment, the virus comprises an RNA genome and the virus is
selected from a picornavirus, a retrovirus, a hemorrhagic fever
virus, or a hepatitis C virus. In certain embodiments, prior to
step (c) the cell is subjected to at least one stimulus, and in
certain embodiments, the cell is an immune cell and the at least
one stimulus is selected from (a) an antibody that specifically
binds to a cognate antigen expressed by the cell; (b) a phorbol
ester; (c) concanavalin A; (d) a cytokine; (e) a chemokine; and (f)
ionomycin. In other certain embodiments, the immune cell is
subjected to at least two or to at least three of the
aforementioned stimuli. In another embodiment, the fraction of the
cell is selected from a cell lysate, a cell extract, or at least
one isolated cell organelle. In a particular embodiment, the
affinity tag further comprises a detectable moiety. In a particular
embodiment, the detectable moiety is selected from a fluorophore, a
radionuclide, an enzyme, and biotin. In certain embodiments, the at
least two polypeptide tags that comprise the affinity tag, are each
selected from a hemagglutinin peptide; a calmodulin binding
polypeptide, a streptavidin binding peptide, an immunoglobulin Fc
polypeptide, an immunoglobulin mutein Fc polypeptide, a protein
C-tag, an at least one immunoglobulin binding staphylococcal
protein A domain, and Softag.TM.. In another embodiment, the
affinity tag comprises at least three or four polypeptide tags, and
each of the at least three or four polypeptide tags is selected
from hemagglutinin peptide; a calmodulin binding polypeptide, a
streptavidin binding peptide, an immunoglobulin Fc polypeptide, an
immunoglobulin mutein Fc polypeptide, a protein C-tag, an at least
one immunoglobulin binding staphylococcal protein A domain, and
Softag.TM.. In a specific embodiment, the affinity tag further
comprises at least one second protease recognition sequence,
wherein the at least one second protease recognition sequence is
either a tobacco etch virus protease recognition sequence or a
Human Rhinovirus HRV3C protease recognition sequence.
[0025] Also provided herein is a viral virulence factor comprising
a viral polypeptide that binds to a host cell wherein the viral
polypeptide comprises at least one trait selected from (a)
expression of a mutant of the viral polypeptide in a cell infected
by a virus correlates with a decrease in virulence of the virus,
wherein the virus comprises a genome that encodes the viral
polypeptide; (b) absence of expression of the viral polypeptide in
a cell infected by a virus correlates with a decrease in virulence
of the virus, wherein the virus comprises a genome encodes the
viral polypeptide; (c) the viral polypeptide is secreted by a cell
infected with a virus wherein the virus comprises a genome that
encodes the viral polypeptide; (d) the viral polypeptide is encoded
by a polynucleotide sequence present in the genome of a virus,
wherein the polynucleotide sequence is located at a genomic region
that encodes a second viral polypeptide that is a viral virulence
factor; (e) the viral polypeptide is encoded by a polynucleotide
sequence present in the genome of a virus, wherein the
polynucleotide sequence is located at the 5' terminal end or the 3'
terminal end of the viral genome; and (f) the viral polypeptide
comprises at least 40 amino acids; and wherein binding of the viral
polypeptide to a host cell alters at least one biological activity
of the host cell such that the host exhibits an increased
susceptibility to infection by a virus that comprises a genome
encoding the viral polypeptide. In certain embodiments, the virus
comprises a DNA genome, a double-stranded RNA genome, or a
single-stranded RNA genome. In a particular embodiment the virus
comprises a DNA genome, wherein the virus is a poxvirus, an
adenovirus, a hepatitis B virus, or a herpesvirus. In another
particular embodiment, the virus comprises an RNA genome and the
virus is selected from a picornavirus, a retrovirus, a hemorrhagic
fever virus, or a hepatitis C virus.
[0026] In another embodiment, provided herein is an isolated
polypeptide comprising a cellular polypeptide that binds to a viral
polypeptide, wherein the cellular polypeptide is isolated according
to a method comprising (a) identifying in the genome of a virus, a
polynucleotide sequence that encodes a viral polypeptide, which
viral polypeptide comprises at least 40 amino acids; (b) producing
a fusion protein comprising the viral polypeptide fused to an
affinity tag sequence; (c) contacting the fusion protein and a
cell, or a fraction of the cell or a supernatant of the cell, under
conditions and for a time sufficient that permit the viral
polypeptide moiety of the fusion protein to interact with a
polypeptide present in the cell, or the fraction of the cell or the
supernatant of the cell, to provide a fusion protein:cellular
polypeptide complex; (d) isolating the fusion protein:cellular
polypeptide complex; and (e) determining the amino acid sequence of
the cellular polypeptide or of at least one cellular polypeptide
fragment comprising at least eight amino acids, and thereby
identifying a cellular polypeptide to which a viral polypeptide
binds. In certain embodiments, the virus comprises a DNA genome, a
double-stranded RNA genome, or a single-stranded RNA genome. In a
particular embodiment the virus comprises a DNA genome, wherein the
virus is a poxvirus, an adenovirus, a hepatitis B virus, or a
herpesvirus. In another particular embodiment, the virus comprises
an RNA genome and the virus is selected from a picornavirus, a
retrovirus, a hemorrhagic fever virus, or a hepatitis C virus. In
certain embodiments, the viral polypeptide is a viral virulence
factor comprising a viral polypeptide that binds to a host cell
wherein the viral polypeptide comprises at least one virulence
trait as described above.
[0027] Also provided herein is a method of identifying an agent for
treating an immunological disease or disorder comprising (a)
identifying a cellular polypeptide to which a viral polypeptide
binds according to any of the aforementioned methods for
identifying a cellular polypeptide, wherein interaction between the
cellular polypeptide and the viral polypeptide alters
immunoresponsiveness of an immune cell; (b) contacting (i) the
cellular polypeptide, or a cell comprising the cellular
polypeptide; (ii) the viral polypeptide; (iii) and a candidate
agent, under conditions and for a time sufficient that permit the
cellular polypeptide and the viral polypeptide to interact; (c)
determining the level of binding of the viral polypeptide to the
cellular polypeptide in the presence of the candidate agent to the
level of binding of the viral polypeptide to the cellular
polypeptide in the absence of the candidate agent, wherein a
decrease in the level of binding of the viral polypeptide to the
cellular polypeptide in the presence of the candidate agent
compared with the level of binding of the viral polypeptide to the
cellular polypeptide in the absence of the candidate agent thereby
identifies an agent for treating an immunological disease or
disorder. In certain embodiments, the agent is selected from (a) an
antibody, or antigen-binding fragment thereof, (b) a viral
polypeptide/Fc polypeptide fusion protein; (c) a peptide/Fc
polypeptide fusion protein; (d) a small molecule; (e) a small
interfering RNA (siRNA); (f) an antisense polynucleotide; and (g)
an aptamer.
[0028] In another embodiment, antibody, or antigen-binding fragment
thereof, is provided that specifically binds to the cellular
polypeptide, which cellular polypeptide binds to a viral
polypeptide as described above and herein, and which cellular
polypeptide is identified according to any one of the methods
described above or herein. In certain embodiments, the antibody is
a polyclonal antibody. In certain other embodiments, the antibody
is a monoclonal antibody, wherein the monoclonal antibody is
selected from a mouse monoclonal antibody, a human monoclonal
antibody, a rat monoclonal antibody, and a hamster monoclonal
antibody. In other embodiments, the antibody is a humanized
antibody or a chimeric antibody. Also provided herein is a host
cell that expresses the antibody, and in certain embodiments, the
host cell is a hybridoma cell. In a specific embodiment, the
antigen-binding fragment is selected from F(ab).sub.2, Fab', Fab,
Fd, and Fv; and in other specific embodiments, the antigen-binding
fragment is of human, mouse, chicken, or rabbit origin. In another
specific embodiment, the antigen-binding fragment is a single chain
Fv (scFv). In another embodiment, a method is provided for treating
a disease or disorder comprising administering to a subject in need
thereof (a) a pharmaceutically suitable carrier; and (b) the
antibody, or an antigen binding fragment thereof. In particular
embodiments, the disease or disorder is an immunological disease or
disorder, a cardiovascular disease or disorder, a metabolic disease
or disorder, or a proliferative disease or disorder. In a specific
embodiment, the immunological disease or disorder is an autoimmune
disease or an inflammatory disease. In yet other specific
embodiments, the immunological disease or disorder is multiple
sclerosis, rheumatoid arthritis, systemic lupus erythematosus,
graft versus host disease, sepsis, diabetes, psoriasis,
atherosclerosis, Sjogren's syndrome, progressive systemic
sclerosis, scleroderma, acute coronary syndrome, ischemic
reperfusion, Crohn's Disease, endometriosis, glomerulonephritis,
myasthenia gravis, idiopathic pulmonary fibrosis, asthma, acute
respiratory distress syndrome (ARDS), vasculitis, or inflammatory
autoimmune myositis. In another specific embodiment, the disease or
disorder is a cardiovascular disease or disorder, wherein the
cardiovascular disease or disorder is atherosclerosis,
endocarditis, hypertension, or peripheral ischemic disease.
[0029] In another embodiment, a method is provided for treating an
immunological disease or disorder in a subject, which method
comprises administering to the subject an antibody, or
antigen-binding fragment thereof, that inhibits binding of a
cellular polypeptide with a viral polypeptide that exhibits at
least one virulence trait, as described above and herein. In yet
another embodiment, a method is provided for treating a disease or
disorder comprising administering to a subject in need thereof (a)
a pharmaceutically suitable carrier; and (b) a cellular polypeptide
that binds to a viral polypeptide that exhibits at least one
virulence trait as described above and herein, or that binds to the
cellular polypeptide identified according to any one of the methods
described above or herein, or an extracellular domain thereof. In
certain embodiments, the cellular polypeptide or at least one
extracellular domain of the cellular polypeptide is fused to a
second polypeptide sequence. In a specific embodiment, the second
polypeptide sequence is an immunoglobulin Fc polypeptide or an
immunoglobulin mutein Fc polypeptide. In particular embodiments,
the disease or disorder is an immunological disease or disorder, a
cardiovascular disease or disorder, a metabolic disease or
disorder, or a proliferative disease or disorder. In a specific
embodiment, the immunological disease or disorder is an autoimmune
disease or an inflammatory disease. In yet other specific
embodiments, the immunological disease or disorder is multiple
sclerosis, rheumatoid arthritis, systemic lupus erythematosus,
graft versus host disease, sepsis, diabetes, psoriasis,
atherosclerosis, Sjogren's syndrome, progressive systemic
sclerosis, scleroderma, acute coronary syndrome, ischemic
reperfusion, Crohn's Disease, endometriosis, glomerulonephritis,
myasthenia gravis, idiopathic pulmonary fibrosis, asthma, acute
respiratory distress syndrome (ARDS), vasculitis, or inflammatory
autoimmune myositis. In another specific embodiment, the disease or
disorder is a cardiovascular disease or disorder, wherein the
cardiovascular disease or disorder is atherosclerosis,
endocarditis, hypertension, or peripheral ischemic disease.
[0030] Also provided is a method of treating a disease or disorder
comprising administering to a subject in need thereof (a) a
pharmaceutically suitable carrier; and (b) an agent identified
according to method comprising (a) identifying a cellular
polypeptide to which a viral polypeptide binds according to any of
the aforementioned methods for identifying a cellular polypeptide,
wherein interaction between the cellular polypeptide and the viral
polypeptide alters immunoresponsiveness of an immune cell; (b)
contacting (i) the cellular polypeptide, or a cell comprising the
cellular polypeptide; (ii) the viral polypeptide; (iii) and a
candidate agent, under conditions and for a time sufficient that
permit the cellular polypeptide and the viral polypeptide to
interact; (c) determining a level of binding of the viral
polypeptide to the cellular polypeptide in the presence of the
candidate agent to a level of binding of the viral polypeptide to
the cellular polypeptide in the absence of the candidate agent,
wherein a decrease in the level of binding of the viral polypeptide
to the cellular polypeptide in the presence of the candidate agent
compared with the level of binding of the viral polypeptide to the
cellular polypeptide in the absence of the candidate agent thereby
identifies an agent for treating an immunological disease or
disorder. In particular embodiments, the disease or disorder is an
immunological disease or disorder, a cardiovascular disease or
disorder, a metabolic disease or disorder, or a proliferative
disease or disorder. In a specific embodiment, the immunological
disease or disorder is an autoimmune disease or an inflammatory
disease. In yet other specific embodiments, the immunological
disease or disorder is multiple sclerosis, rheumatoid arthritis,
systemic lupus erythematosus, graft versus host disease, sepsis,
diabetes, psoriasis, atherosclerosis, Sjogren's syndrome,
progressive systemic sclerosis, scleroderma, acute coronary
syndrome, ischemic reperfusion, Crohn's Disease, endometriosis,
glomerulonephritis, myasthenia gravis, idiopathic pulmonary
fibrosis, asthma, acute respiratory distress syndrome (ARDS),
vasculitis, or inflammatory autoimmune myositis. In another
specific embodiment, the disease or disorder is a cardiovascular
disease or disorder, wherein the cardiovascular disease or disorder
is atherosclerosis, endocarditis, hypertension, or peripheral
ischemic disease.
[0031] The invention further provides a business method comprising
(a) identifying a viral polypeptide that is a viral virulence
factor; (b) identifying a cellular polypeptide to which the viral
virulence factor binds, wherein binding of the viral virulence
factor to the cellular polypeptide alters at least one biological
activity of a cell; (c) identifying an agent that inhibits binding
of the viral virulence factor to the cellular polypeptide, thereby
identifying an agent that alters the at least one biological
activity of the cell; and (d) designing and executing at least one
pre-clinical study to determine whether altering the at least one
biological activity of the cell by the agent indicates that the
agent is useful for treating a disease or medical disorder in a
human subject. In a particular embodiment, the business method
further comprises designing and executing at least one clinical
study to evaluate the safety of the agent in a human subject, which
in certain embodiments further comprises designing and executing at
least one clinical study to evaluate the efficacy of the agent in a
human subject in need of the agent, and in still certain other
embodiments, further comprises selling the agent. In specific
embodiments, the business method comprises a step of licensing of
the viral polypeptide from a licensing organization to an acquiring
company. In another specific embodiment, comprises a step of
licensing of the cellular polypeptide from a licensing organization
to an acquiring company. In yet another specific embodiment, the
method comprises a step of licensing of the agent from a licensing
organization to an acquiring company, wherein, in certain
embodiments, the licensing organization is a biopharmaceutical
company. In other certain embodiments, the acquiring company is a
biopharmaceutical company. In one particular embodiment, the
biopharmaceutical company performs experiments to identify the
cellular polypeptide. In other particular embodiments, a
biopharmaceutical company performs experiments to identify the
agent. In still another embodiment, the business method further
comprises licensing the right from a biopharmaceutical company to a
selling company to sell the agent. In another specific embodiment,
the business method further comprises collecting a royalty fee from
the selling company by a biopharmaceutical company. In one
embodiment, the agent is selected from (a) an antibody, or
antigen-binding fragment thereof, (b) a viral polypeptide/Fc
polypeptide fusion protein; (c) a peptide/Fc polypeptide fusion
protein; (d) a domain of the cellular polypeptide, or a fragment
thereof comprising at least eight amino acids, fused to an Fc
polypeptide; (e) a small molecule; (f) a small interfering RNA
(siRNA); (g) an antisense polynucleotide; and (h) an aptamer. In
another certain embodiment, the at least one biological activity of
the cell is immunoresponsiveness and the cell is an immune cell. In
specific embodiments, the disease or disorder is an immunological
disease or disorder, a cardiovascular disease or disorder, a
metabolic disease or disorder, or a proliferative disease or
disorder. In a specific embodiment, the immunological disease or
disorder is an autoimmune disease or an inflammatory disease. In
yet other specific embodiments, the immunological disease or
disorder is multiple sclerosis, rheumatoid arthritis, systemic
lupus erythematosus, graft versus host disease, sepsis, diabetes,
psoriasis, atherosclerosis, Sjogren's syndrome, progressive
systemic sclerosis, scleroderma, acute coronary syndrome, ischemic
reperfusion, Crohn's Disease, endometriosis, glomerulonephritis,
myasthenia gravis, idiopathic pulmonary fibrosis, asthma, acute
respiratory distress syndrome (ARDS), vasculitis, or inflammatory
autoimmune myositis. In another specific embodiment, the disease or
disorder is a cardiovascular disease or disorder, wherein the
cardiovascular disease or disorder is atherosclerosis,
endocarditis, hypertension, or peripheral ischemic disease.
[0032] In another embodiment, a method is provided for guiding the
selection of a therapeutic agent for treating a disease or medical
disorder, comprising (a) identifying a viral polypeptide that
increases the virulence of a virus in a host infected with the
virus; (b) identifying a cellular polypeptide to which the viral
polypeptide binds, wherein binding of the viral polypeptide to the
cellular polypeptide alters at least one biological activity of a
cell; (c) identifying one or more agents that inhibit binding of
the viral polypeptide to the cellular polypeptide; (d) categorizing
the capability of the one or more agents identified in step (c) to
alter at least one biological effect of a cell, wherein altering
the at least one biological effect reduces the risk of developing a
disease or medical disorder or reduces at least one symptom of a
disease or medical disorder in a host; and (e) selected at least
one agent for testing in a preclinical and/or a clinical method,
and therefrom guiding the selection of a therapeutic agent for
treating a disease or disorder. In another certain embodiment, the
at least one biological activity of the cell is
immunoresponsiveness and the cell is an immune cell. In specific
embodiments, the disease or disorder is an immunological disease or
disorder, a cardiovascular disease or disorder, a metabolic disease
or disorder, or a proliferative disease or disorder. In a specific
embodiment, the immunological disease or disorder is an autoimmune
disease or an inflammatory disease. In yet other specific
embodiments, the immunological disease or disorder is multiple
sclerosis, rheumatoid arthritis, systemic lupus erythematosus,
graft versus host disease, sepsis, diabetes, psoriasis,
atherosclerosis, Sjogren's syndrome, progressive systemic
sclerosis, scleroderma, acute coronary syndrome, ischemic
reperfusion, Crohn's Disease, endometriosis, glomerulonephritis,
myasthenia gravis, idiopathic pulmonary fibrosis, asthma, acute
respiratory distress syndrome (ARDS), vasculitis, or inflammatory
autoimmune myositis. In another specific embodiment, the disease or
disorder is a cardiovascular disease or disorder, wherein the
cardiovascular disease or disorder is atherosclerosis,
endocarditis, hypertension, or peripheral ischemic disease. In
another specific embodiment, step (a) is performed using a computer
device comprising (i) a first knowledge base comprising a plurality
of different polynucleotide sequences encoding a plurality of viral
polypeptides; and (ii) a second knowledge base comprising a
plurality of rules for evaluating and selecting a viral polypeptide
that is a viral virulence factor, wherein the viral polypeptide is
identified from information received in step (i).
[0033] In another embodiment, provided herein is a business method
for selling a therapeutic agent to treat a disease or disorder,
comprising (a) receiving information regarding a viral polypeptide
that increases the virulence of a virus in a host infected with the
virus; (b) identifying a cellular polypeptide to which the viral
polypeptide binds, wherein binding of the viral polypeptide to the
cellular polypeptide alters at least one biological activity of a
cell; (c) identifying one or more agents that inhibit binding of
the viral polypeptide to the cellular polypeptide and that alter
the at least one biological activity of the cell, wherein altering
the at least one biological effect reduces the risk of developing
an disease or disorder or reduces at least one symptom of an
disease or medical disorder in a host; and (d) selling an agent
identified in step (c) to a medical professional or patient for
treatment of the disease or medical disorder. In a specific
embodiment, the immunological disease or disorder is an autoimmune
disease or an inflammatory disease. In yet other specific
embodiments, the immunological disease or disorder is multiple
sclerosis, rheumatoid arthritis, systemic lupus erythematosus,
graft versus host disease, sepsis, diabetes, psoriasis,
atherosclerosis, Sjogren's syndrome, progressive systemic
sclerosis, scleroderma, acute coronary syndrome, ischemic
reperfusion, Crohn's Disease, endometriosis, glomerulonephritis,
myasthenia gravis, idiopathic pulmonary fibrosis, asthma, acute
respiratory distress syndrome (ARDS), vasculitis, or inflammatory
autoimmune myositis. In another specific embodiment, the disease or
disorder is a cardiovascular disease or disorder, wherein the
cardiovascular disease or disorder is atherosclerosis,
endocarditis, hypertension, or peripheral ischemic disease. In
another specific embodiment, step (a) is performed using a computer
device comprising (i) a first knowledge base comprising a plurality
of different polynucleotide sequences encoding a plurality of viral
polypeptides; and (ii) a second knowledge base comprising a
plurality of rules for evaluating and selecting a viral polypeptide
that is a viral virulence factor, wherein the viral polypeptide is
identified from information received in step (i).
[0034] Also provided herein is a system for guiding the selection
of a viral polypeptide to achieve a desired result, comprising (a)
a computing device comprising (i) a first knowledge base comprising
a plurality of polynucleotide sequences encoding a plurality of
viral polypeptides; and (ii) a second knowledge base comprising a
plurality of rules for evaluating and selecting a viral polypeptide
that is a viral virulence factor based upon information received in
step (i); (b) means for providing information regarding a target
viral virulence factor and a desired result to said computing
device; and (c) means in said computing device for identifying and
categorizing or ranking at least one polynucleotide sequence
encoding a viral polypeptide that may be used to identify a
cellular polypeptide with which the viral polypeptide binds,
wherein binding of the viral polypeptide to the cellular
polypeptide alters at least one biological activity of a cell.
[0035] In another embodiment, a computer program product is
provided for guiding the selection of a viral polypeptide to
achieve a desired result, said computer program product comprising
a computer usable storage medium having computer readable program
code means embodied in the medium, the computer readable program
code means comprising (a) computer readable program code means for
generating (i) a first knowledge base comprising a plurality of
polynucleotide sequences encoding a plurality of viral
polypeptides; and (ii) a second knowledge base comprising a
plurality of rules for evaluating and selecting a viral polypeptide
that is a viral virulence factor based upon information received in
step (i); (b) a computer readable program code means for providing
information regarding a target viral virulence factor and a desired
result to said computing device; and (c) computer readable program
code means for identifying and categorizing or ranking a target
viral virulence factor that may be used to identify a cellular
polypeptide to which the viral polypeptide binds, wherein binding
of the viral polypeptide to the cellular polypeptide alters at
least one biological activity of a cell.
[0036] In another embodiment is provided a method of manufacture
for producing a cellular polypeptide that binds to a viral
polypeptide comprising (a) identifying a cellular polypeptide to
which a viral polypeptide binds according to the methods described
above and herein; (b) determining a nucleotide sequence that
encodes the cellular polypeptide; (c) preparing a recombinant
expression vector comprising a promoter operatively linked to the
nucleotide sequence that encodes the cellular polypeptide; (d)
transfecting or transforming a host cell with the recombinant
expression vector prepared in step (c); (e) culturing the host cell
of step (d) under conditions that permit expression of the cellular
polypeptide; and (f) isolating the cellular polypeptide from the
host cell culture.
[0037] In another embodiment, method of manufacture is provided for
producing an agent for treating an immunological disease or
disorder comprising (a) identifying an agent for treating an
immunological disease or disorder, wherein the step of identifying
comprises (i) identifying a cellular polypeptide to which a viral
polypeptide binds according to any of the methods described above
or herein, wherein interaction between the cellular polypeptide and
the viral polypeptide alters immunoresponsiveness of an immune
cell; (ii) contacting (A) the cellular polypeptide, or a cell
comprising the cellular polypeptide; (B) the viral polypeptide; (C)
and a candidate agent, under conditions and for a time sufficient
that permit the cellular polypeptide and the viral polypeptide to
interact; (iii) determining a level of binding of the viral
polypeptide to the cellular polypeptide in the presence of the
candidate agent to a level of binding of the viral polypeptide to
the cellular polypeptide in the absence of the candidate agent,
thereby identifying an agent for treating an immunological disease
or disorder; and (b) producing the agent identified in step (a). In
a specific embodiment, the agent is selected from (a) an antibody,
or antigen-binding fragment thereof, (b) a viral polypeptide/Fc
polypeptide fusion protein; (c) a peptide/Fc polypeptide fusion
protein; (d) a domain of the cellular polypeptide, or a fragment
thereof comprising at least eight amino acids, fused to an Fc
polypeptide; (e) a small molecule; (f) a small interfering RNA
(siRNA); (g) an antisense polynucleotide; and (h) an aptamer.
[0038] As used herein and in the appended claims, the singular
forms "a," "and," and "the" include plural referents unless the
context clearly dictates otherwise. Thus, for example, reference to
"an agent" includes a plurality of such agents, and reference to
"the cell" includes reference to one or more cells and equivalents
thereof known to those skilled in the art, and so forth. The term
"comprising" (and related terms such as "comprise" or "comprises"
or "having" or "including") is not intended to exclude that, for
example, any composition of matter, composition, method, or
process, or the like, described herein may "consist of" or "consist
essentially of" the described features.
[0039] All U.S. patents, U.S. patent application publications, U.S.
patent applications, foreign patents, foreign patent applications,
and non-patent publications referred to in this application and/or
listed in the Application Data Sheet, are incorporated herein by
reference, in their entireties.
BRIEF DESCRIPTION OF THE DRAWINGS
[0040] FIG. 1 presents a schematic of an A41L fusion polypeptide
encoded by a recombinant expression construct (A41LCRFC) for
expression of the fusion polypeptide used for tandem affinity
purification (TAP). The encoded fusion polypeptide includes mature
A41L from Cowpox virus that was fused at its amino terminal end to
the carboxy terminus of the human growth hormone leader peptide (GH
Leader). The tandem affinity tag (CRFC) was fused to the carboxy
terminus of A41L and included a human influenza virus hemagglutinin
(HA) epitope (YPYDVDYA, SEQ ID NO:1) in frame with a Protein C-TAG
(EDQVDPRLIDGK (SEQ ID NO:4), derived from the heavy chain of human
Protein C); human rhinovirus HRV3C protease site (HRV3C cleavage
site) (LEVLFQGP (SEQ ID NO:16); and a mutein derivative of the Fc
portion of a human IgG immunoglobulin (Mutein FC).
[0041] FIG. 2 presents a schematic of the TAP procedure for
identifying cellular polypeptides that bind to A41L.
[0042] FIG. 3 illustrates peptides of LAR, RPTP-.delta., and
RPTP-.sigma. identified by tandem affinity purification (TAP) and
LC/MS/MS analysis. FIG. 3A illustrates the sequences of peptides
(bold typeface) within LAR (SEQ ID NO:43) that were identified by
LC/MS/MS after TAP. FIG. 3B illustrates the sequences of peptides
(bold typeface) within RPTP-.sigma. (SEQ ID NO:18) that were
identified by LC/MS/MS after TAP. FIG. 3C illustrates the sequences
of peptides (bold typeface) within RPTP-.delta. (SEQ ID NO:19) that
were identified by LC/MS/MS after TAP.
[0043] FIG. 4 provides a schematic of various fusion protein
constructs that may be used for tandem affinity purification
procedures. A human growth hormone (GH) signal peptide is shown at
the amino terminal end of each construct. ORF (open reading frame)
refers to an encoded viral virulence factor, which may be the
full-length polypeptide or a portion thereof. The affinity tag
polypeptides include HA (human influenza virus hemagglutinin
peptide (also called hemagglutinin epitope)); CBP, calmodulin
binding protein; SBP, streptavidin binding protein; 2.times.SBP,
tandem repeat of streptavidin binding protein; mutein Fc, mutein
immunoglobulin Fc polypeptide; C-TAG, protein C-tag derived from
the heavy chain of human protein C; Softag.TM. peptide; and ZZ, an
IgG-binding ZZ polypeptide. Protease sites include Rhino, human
rhinovirus 3C(HRV3C) protease site; and TEV, tobacco etch virus
protease site.
[0044] FIG. 5 presents a schematic of tandem affinity purification
using a CRFC affinity tag fused in frame to a viral virulence
factor open reading frame (Viral ORF). The CRFC Tag includes from
the amino terminal end, HA (hemagglutinin peptide (also called
hemagglutinin epitope); C-TAG; HRV3C protease site; and
immunoglobulin Fc polypeptide, which can be either a wildtype Fc
polypeptide or a mutein Fc polypeptide.
[0045] FIG. 6 presents an amino acid sequence alignment between (i)
an A41L/Fc fusion polypeptide comprising an A41L signal peptide
sequence, an A41L polypeptide, and a human IgG1 Fc polypeptide
(A41L/Fc) (SEQ ID NO:32) and (ii) an A41L/mutein Fc fusion
polypeptide comprising a human growth hormone signal peptide
sequence, an A41L polypeptide variant, and a mutein Fc polypeptide
(A41L/mutein Fc) (SEQ ID NO:31). The consensus sequence (SEQ ID
NO:44) is also shown. The vertical dotted lines indicate the amino
terminal and carboxy terminal ends of the A41L polypeptide.
DETAILED DESCRIPTION OF THE INVENTION
[0046] The present invention relates to methods for rapidly
identifying cellular targets that are important in modulating the
human immune system, and then identifying the counterstructures
that will bind and modulate those targets. The method described
herein comprises identifying viral virulence genes using
bioinformatics and expressing the virulence genes, or portions (or
fragments) thereof, that encode a viral virulence polypeptide, or a
fragment thereof (such as a fragment that binds to a cellular
polypeptide). The expressed viral virulence polypeptides may
further comprise one or more sequence tags for detection and
isolation. The expressed, tagged virulence gene products are used
to screen cells (for example by fluorescence-activated cell
sorting) or to screen isolated cell membranes (such as by BIAcore)
to identify a cellular target molecule that interacts with the
virulence gene product. The cellular target can then be isolated
and identified, for example by, determining its amino acid
sequence.
[0047] Methods described herein comprise contacting a cell, a
fraction of a cell, or a supernatant of a cell, with a viral
polypeptide that is a viral virulence factor (also called a viral
virulence polypeptide) and then identifying the cellular target to
which the viral virulence polypeptide binds. Viral virulence genes
and the polypeptides encoded by the virulence genes are identified,
for example, by bioinformatics methods. Fusion proteins may be
prepared that comprise the viral virulence polypeptide (or a
fragment or portion thereof that interacts with a cellular
polypeptide) and an affinity tag (also called an epitope tag). In
certain embodiments, the methods comprise a modification of a
tandem affinity purification (TAP) tag procedure for isolating the
cellular polypeptide target, including a cellular polypeptide
target that is secreted by a cell.
[0048] Viruses have evolved numerous mechanisms to evade detection
and elimination by the immune system of an infected host by
encoding proteins that are viral homologues of cell cytokines, cell
chemokines or the receptor(s) of a cytokine or chemokine For
example, the genomes of poxviruses encode a soluble viral tumor
necrosis factor (TNF) receptor, which binds to and inhibits the
inflammation-inducing cytokine, TNF. Other cellular polypeptides
targeted by viral polypeptides include interleukin 1, various
chemokines, and CD30.
[0049] Viruses have evolved to withstand mechanisms that an
infected host has developed to limit infection and to adversely
affect the replication cycle of the virus. Viruses comprise genes
encoding proteins that have properties, characteristics, and/or
functions that enable the virus to evade or modulate the immune
response of the host. In addition, viruses have the ability to
acquire genes from the host and/or to evolve viral homologues of
host genes and/or to evolve viral modulators of host genes.
Accordingly, a virus, which has the capability to evolve and/or the
capability to acquire genes from the host, comprises a genome that
encodes proteins called virulence factors (also called herein a
viral virulence polypeptide) that modulate an immune response of
the host to the virus. A cellular component in the host that is a
ligand for a viral virulence factor may be, therefore, an important
immunomodulatory target.
[0050] In particular, identification of cellular polypeptides,
including cell surface polypeptides, secreted polypeptides, and
intracellular polypeptides that are expressed by immune cells and
that interact with a viral virulence factor (i.e., a viral
virulence polypeptide or a viral polypeptide that exhibits at least
one viral virulence trait) will be useful and beneficial for
identifying agents that may be used for treating immunological
disorders, such as, for example, inflammatory diseases and
autoimmune diseases, including multiple sclerosis, rheumatoid
arthritis, and systemic lupus erythematosus (SLE). In another
embodiment, identification of a viral virulence factor is useful
for identifying agents that are used to treat and/or prevent a
viral infection that is caused by the virus (or a related virus)
that encodes the virulence factor. A need exists to identify and
develop compositions that can be used for treatment and prophylaxis
of such immunological diseases and disorders and viral
infections.
Methods for Identifying a Cellular Polypeptide Therapeutic
Target
[0051] Methods described herein for identifying cellular
polypeptides that are suitable targets for altering a cellular
activity or function include methods for identifying a cellular
polypeptide to which a viral polypeptide (e.g., a viral virulence
polypeptide or virulence factor) binds. Such methods comprise
contacting a cell, or a fraction or a supernatant of the cell, with
a fusion protein. The fusion protein comprises the viral virulence
polypeptide that is fused to an affinity tag. The fusion protein
and cell (or a cell fraction, cell culture supernatant, cell
lysate, cell extract, or extracellular supernatant comprising the
cognate cellular polypeptide(s)) are permitted to interact under
conditions and for a time sufficient to permit the viral
polypeptide moiety of the fusion protein to interact with a
polypeptide associated with the cell, a fraction of the cell, or a
supernatant of the cell, and form a fusion protein/cellular
polypeptide complex. The complex may be isolated via the affinity
tag, which is permitted to bind to a cognate ligand of the tag. The
identity of the cellular polypeptide may be determined according to
methods described herein and practiced in the art, including but
not limited to LC-MS/MS, MALDI-TOF, immunoassays, peptide mapping,
and amino acid analyses, including amino terminal end analysis
(e.g., Edman degradation).
[0052] An exemplary method for affinity isolation of a target
cellular polypeptide is tandem affinity purification (TAP) (also
called TAP tag) (see, e.g., Rigaut et al. Nat. Biotech. 17:1030-32
(1999); Puig et al., Methods 24:218-29 (2001); Knuesel et al. Mol.
Cell. Proteomics 2:1225-33 (2003)). The TAP method permits
identification of components present in biological complexes.
Purification may be rapid and is performed under conditions that in
general do not require denaturation of any components or of the
biological complex. Typically, a TAP tag (or affinity tag) is fused
to the amino terminal or carboxy terminal end of a polypeptide of
interest (in this instance, a viral virulence polypeptide, or a
portion thereof encoded by an open reading frame of the viral
genome), which is contacted with a cell or a cell fraction to
permit interaction between the polypeptide of interest and the
cellular polypeptide such that a complex is formed between the
polypeptide of interest and the cellular polypeptide. The complex
is then isolated by exploiting the binding properties of the
affinity tag. Subsequent to TAP procedures, in certain embodiments
the identity of the target cellular polypeptide is determined by
liquid chromatograph tandem mass spectrometry, referred to as
LC-MS/MS, which is described in greater detail herein.
Viral Polypeptides
[0053] Viral polypeptides that are useful in methods for
identifying a cellular polypeptide that is a therapeutic target
include viral polypeptides that maintain or increase the ability of
a virus to cause disease in a host, that is, that affect the
virulence of the virus. Such viral polypeptides are referred to
herein as a viral virulence polypeptide, which is a viral
polypeptide that exhibits at least one virulence trait. In a
certain embodiment, a method for identifying a cellular polypeptide
to which a viral virulence polypeptide binds and to which
therapeutic agents may be targeted, further comprises identifying
in the genome of a virus, a polynucleotide sequence that encodes a
viral polypeptide (e.g., for example, identifying a polynucleotide
sequence that encodes for at least 20, 30, or 40 contiguous amino
acids) that has one or more virulence traits. The identified viral
polypeptide is then fused to an affinity tag, which is described in
further detail herein.
[0054] Virulence or the ability of a virus to cause disease
includes the extent to which the virus has the capability to
overcome or minimize one or more host defense mechanisms.
Components of microorganisms, including viruses, that maintain or
increase the virulence of the microorganism are also called
virulence factors. A viral polypeptide that is a viral virulence
factor (or viral virulence polypeptide) has the capability to evade
or modulate the immune response of the host. As described herein,
virulence of a virus is maintained in part by evolution of the
genetic information contained within a virus and by the ability of
a virus to acquire genes from the host and/or to evolve viral
homologues of host genes and/or to evolve viral modulators of host
genes. Viral polypeptides that are viral virulence factors include
polypeptides that when bound to cellular polypeptides affect (i.e.,
modulate or alter) a biological function or activity of the
cellular polypeptide, which alters the ability of the host to
effect an immune response that will prevent, minimize, reduce,
suppress, or inhibit infection by the virus and that will prevent,
minimize, reduce, suppress, or inhibit the sequelae of the disease
associated therewith.
[0055] The genome of a virus that encodes one or more viral
polypeptides that are viral virulence factors may be a
single-stranded DNA genome, double-stranded DNA genome,
double-stranded RNA genome, or single-stranded RNA genome (sense or
anti-sense). Exemplary DNA viruses include, but are not limited to,
large DNA genome (double-stranded DNA) viruses, such as
herpesviruses, adenoviruses, and poxviruses. Other viruses that
encode polypeptides that contribute to the virulence of a virus
include but are not limited to picornaviruses (RNA containing
viruses e.g., an enterovirus, rhinoviruses, hepatovirus (Hepatitis
A virus), cardiovirus, aphthovirus, parechovirus, erbovirus,
kobuvirus, and teschovirus); hemorrhagic fever viruses (RNA
containing viruses e.g., arenaviruses, filoviruses, bunyviruses,
flaviviruses); influenza viruses (single stranded RNA viruses);
retroviruses (e.g., oncoviruses and lentiviruses (RNA containing
viruses, for example, HIV-1, HIV-2, HTLV-1, HTLV-2)); hepatitis B
virus (DNA containing virus); hepatitis C virus (RNA containing
virus); and coronaviruses. An antisense RNA virus is also referred
to in the art as negative RNA-stranded virus and includes, for
example, measles virus, mumps virus, influenza virus, Ebola virus,
and respiratory syncytial virus. Positive-stranded RNA viruses,
also referred to as sense RNA viruses include, for example,
polioviruses, rhinoviruses, coronaviruses, rubella, yellow fever
virus, West Nile virus, dengue fever viruses, hepatitis A and
hepatitis C viruses.
[0056] Poxviruses form a group of double-stranded DNA viruses that
replicate in the cytoplasm of a cell and have adapted to replicate
in numerous different hosts. An adaptive mechanism of many
poxviruses involves the acquisition of host genes that allow the
viruses to evade the host's immune system and/or facilitate viral
replication (Smith et al., Science 248:1019 (1990); Bugert and
Darai, Virus Genes 21:111 (2000); Alcami et al., Semin. Virol.
8:419 (1998); McFadden and Barry, Semin. Virol. 8:429 (1998)). This
process is facilitated by the relatively large size and complexity
of the poxvirus genome. Poxviruses include, for example, orthopox
viruses such as vaccinia, monkeypox, cowpox, and variola viruses
(e.g., smallpox virus), leporipoxviruses, such as myxoma and Shope
fibroma virus, molluscipox (e.g., Molluscum contagiosum),
yatapoxvirus (such as Yaba-like disease virus), parapoxvirus (e.g.,
ORF virus). By way of example, vaccinia virus, a prototype poxvirus
widely used as a smallpox vaccine, has a genome of approximately
190 kilobases, which could potentially encode more than 200
proteins (Goebel et al., Virology 179:247 (1990)). Even though the
entire genome of Vaccinia virus and other poxviruses have been
sequenced, the function of many of the potential open reading
frames (ORFs), and the existence of polypeptides encoded by the
ORFs, remains unknown.
[0057] Expeditious inspection of polynucleotide sequences within a
viral genome to identify those sequences that encode a viral
virulence factor among the many ORFs of a viral genome, for
example, a large DNA genome-containing virus such as a poxvirus, is
useful for the methods described herein for identifying a cellular
polypeptide that is a therapeutic target. In certain embodiments, a
genome of a virus, such as a poxvirus, is analyzed to identify
polynucleotide sequences within the viral genome that encode viral
polypeptides that contribute to the virulence of the virus. Such
viral polypeptides exhibit at least one (i.e., one or more)
virulence characteristics or virulence traits.
[0058] Exemplary virulence traits of a viral virulence factor
include traits that may be observed in a host infected with the
virus that expresses the viral virulence factor or may be observed
in cells propagated in tissue culture. A viral polypeptide that
contributes to virulence of the virus includes a polypeptide that
when it is altered such as when it comprises a mutation (at least
one substitution, insertion, or deletion of an amino acid either as
a consequence of natural selection or by any number of different
mutagenesis techniques practiced by persons skilled in the
molecular biology art) that alters (such as decreases in a
statistically significant or biologically significant manner) a
biological activity of the viral polypeptide. When such a viral
polypeptide mutant (or altered viral polypeptide) is expressed in a
cell infected by the virus comprising the genome that encodes the
mutant (or altered) viral polypeptide, the expression of the mutant
(or altered) viral polypeptide correlates with a decrease in
virulence of the virus.
[0059] Also, a virulence trait of a viral polypeptide that is a
viral virulence factor is indicated by the correlation between the
absence of expression of the viral polypeptide in a cell infected
by a virus with a decrease in virulence of the virus. The
correlation between lack of expression of a viral polypeptide and
decreased virulence may be observed by infecting cells with a
recombinant virus in which the gene encoding the viral polypeptide
is deleted, silenced (e.g., by treating a cell infected with the
virus with an antisense polynucleotide or by RNA interference using
a small interfering RNA (siRNA)), or knocked out. In tissue
culture, passage of cells may not be adversely affected when the
cells are infected with a virus that expresses a mutant of the
particular viral polypeptide of interest or that fails to express
the viral polypeptide. In a host, a viral polypeptide that exhibits
at least one virulence trait includes a viral polypeptide that when
its expression is altered (for example, by introduction of at least
one mutation that alters a biological activity of the viral
polypeptide or by reduced expression or lack of expression), the
altered expression correlates with decreased virulence, that is, a
decreased ability of the virus to cause disease, and/or an increase
in inflammation, and/or an increase in other types of immune
responses. That is, the host has increased immunoresponsiveness to
the virus comprising a genome that encodes a mutant viral
polypeptide or that does not contain a polynucleotide sequence that
encodes the polypeptide.
[0060] A virulence trait also relates to the cellular or
extracellular location of the viral polypeptide after it is
expressed in a cell infected with a virus that comprises the genome
encoding the viral polypeptide. After expression in an infected
cell, the viral polypeptide may remain at an intracellular
location, may be a membrane spanning polypeptide having
extracellular domains, or may be secreted by the infected cell. A
viral polypeptide that is secreted by an infected cell or that has
extracellular domains contributes to the virulence of the virus by
interacting with other cells of the host or with other molecules
associated with or secreted by other cells of the host. Such
molecules include, but are not limited to, cell surface antigens,
cytokines, chemokines, hormones, and other molecules that
contribute to host defense.
[0061] Viral polypeptides that contribute to viral virulence are
typically encoded by polynucleotide sequences that are located
within proximity of each other in the viral genome. Accordingly, a
virulence trait of a viral polypeptide includes that the
polypeptide is encoded by a polynucleotide sequence that is located
at a genomic region that encodes at least one other polypeptide
that is either known in the art to be a viral virulence polypeptide
or is determined to be a viral virulence polypeptide according to
methods described herein and practiced in the art. For example in
poxviruses, a polynucleotide sequence present in the viral genome
that encodes a viral polypeptide that is a virulence viral
polypeptide is located toward the 5' terminal end or toward the 3'
terminal end of the viral genome. In certain instances a
polynucleotide sequence encoding a viral polypeptide that is a
virulence factor may be found within about one-third or within
about one-quarter of the 5' terminal end or the 3' terminal end of
the genome (i.e., such a polynucleotide sequence comprises
nucleotides that are located within at least 20%, 25%, 30%, 33%, or
at least 35% of either the 5' terminal end or the 3' terminal end
of the viral genome).
[0062] Accordingly, also provided herein are viral virulence
factors that are viral virulence polypeptides that bind to a host
cell. Such a virus virulence factor is a viral polypeptide that
comprises at least one trait (virulence trait) such as (a)
expression of a mutant of the viral polypeptide (or an altered
viral polypeptide) in a cell infected by a virus, which correlates
with a decrease in virulence of the virus, wherein the virus
comprises a genome that encodes the viral polypeptide; (b) absence
of expression of the viral polypeptide in a cell infected by a
virus correlates with a decrease in virulence of the virus, wherein
the virus comprises a genome that encodes the viral polypeptide;
(c) the viral polypeptide is secreted by a cell infected with a
virus wherein the virus comprises a genome that encodes the viral
polypeptide; (d) the viral polypeptide is encoded by a
polynucleotide sequence present in the genome of a virus, wherein
the polynucleotide sequence is located at a genomic region that
encodes at least one viral virulence factor; (e) the viral
polypeptide is encoded by a polynucleotide sequence present in the
genome of a virus, wherein the polynucleotide sequence is located
at the 5' terminal end or the 3' terminal end of the viral genome;
and (f) the viral polypeptide comprises at least 40 amino acids
(which may include a signal peptide sequence). When a viral
polypeptide that is a virulence factor interacts with a cell
(including specifically binding to a cellular polypeptide that is
intracellular, located in a cellular membrane, or is secreted), the
viral polypeptide alters at least one biological activity of the
host cell such that the host exhibits an increased susceptibility
to infection (or a decreased capability to resist infection) by the
virus that comprises a genome encoding the viral polypeptide.
[0063] A viral virulence polypeptide (or a variant, derivative, or
fragment thereof) may also be used as a therapeutic agent. In one
embodiment, a viral virulence polypeptide or a fusion polypeptide
comprising the virulence polypeptide may be used for treating a
patient or subject in need thereof. In a specific embodiment, the
subject presents an acute immune response, such as, by way of
nonlimiting example, a subject who presents an acute respiratory
distress syndrome (ARDS). To reduce or minimize the possibility or
the extent of an immune response by the subject that is specific
for a viral virulence polypeptide (or a variant, derivative, or
fragment thereof) the viral polypeptide may be administered in a
limited number of doses, may be produced or derived in a manner
that alters a post-translational modification of the viral
polypeptide and thus decreases the immunogenicity of the viral
polypeptide, and may be administered under conditions that reduce
or minimize antigenicity of the viral polypeptide. For example, a
viral virulence polypeptide may be administered prior to,
concurrently with, or subsequent to the administration in the
subject of a second composition that suppresses an immune response,
particularly a response that is specific for the viral polypeptide.
In addition, persons skilled in the art are familiar with methods
for increasing the half-life and/or improving the pharmacokinetic
properties of a polypeptide, such as by pegylating the polypeptide,
which may increase the effectiveness of a therapeutic agent,
including agents that are not administered multiple times.
[0064] Viral polypeptides that are known to contribute to virulence
of the virus are typically at least 20, 30, or 40 amino acids in
length or at least 50, 60, 70, 80, 90, 100, 120, 140, 160, 180,
200, 250, 300, or 400 or more amino acids in length. To identify a
polynucleotide sequences in a viral genome that may encode a
polypeptide that is a virulence factor, the sequence of the genome
is inspected to identify regions containing open reading frames for
at least 20, 30, 40, 50 or more amino acids (i.e., 20, 30, 40, or
50 contiguous amino acids). In certain embodiments, the
polynucleotide sequence comprises an open reading frame that
encodes a polypeptide comprising at least 40 amino acids. The viral
polypeptide encoded by the open reading frame identified in the
methods described herein may be a full-length viral polypeptide or
may be a portion or fragment of a full-length polypeptide. In
particular embodiments, the fragment of a viral virulence
polypeptide is a fragment that comprises at least 10, 15, 20, 25,
30, 40, 50 or more, consecutive (or contiguous) amino acids of the
full-length polypeptide and that is capable of binding to a
cellular polypeptide.
[0065] The polynucleotide sequence may include nucleotides that
encode a signal peptide sequence and the mature polypeptide or a
portion of a full-length, mature polypeptide. Signal peptides,
which facilitate translocation of secretory proteins and
cell-surface proteins across intracellular membranes and to final
localization, are located at the N-terminus of such proteins and
are typically 13-40 amino acids in length. A polypeptide comprising
a signal peptide sequence is also called a pre-protein. The signal
peptide is normally cleaved from the pre-protein to provide the
mature protein. Signal peptides exhibit certain sequence
characteristics and other properties such that the signal peptide
sequence can be distinguished from the mature polypeptide sequence;
computer programs may be used to aid determining the amino acid
sequence of the signal polypeptide (Zhang et al., Protein Sci.
13:2819-24 (2004)).
[0066] Inspection of polynucleotide sequences that encode viral
polypeptides may be accomplished by scanning the polynucleotide
sequence of a viral genome or putative viral genome, or any portion
or fraction thereof, for example, by using a computer and
applicable software programs. The polynucleotide sequence of a
viral genome or a portion of the viral genome can be translated
into six open reading frames (three forward and three reverse)
using readily available computer programs. Software programs
include programs that are available commercially and include
programs that may be custom designed and prepared. The six open
reading frames may then be inspected, with or without the aid of
additional computer software programs, to identify locations in the
viral genome that contain open reading frames that encode for at
least 20, 30, or 40 amino acids.
[0067] A viral polypeptide encoded by the open reading frame may be
a full-length viral virulence polypeptide, or a fragment or portion
of the viral virulence polypeptide, as described herein, and may be
used for identifying a cellular polypeptide, which is a therapeutic
target. A fragment or portion of a viral polypeptide that comprises
at least 20 consecutive (or contiguous) amino acids, at least 30 or
at least 40 amino acids or at least 50, 60, 70, 80, 90, 100, 120,
140, 160, 180, 200 amino acids, or more (or any integer between the
ranges described) may be attached to or fused to and affinity tag.
A fragment or a portion of a viral polypeptide useful for the
methods described herein includes an extracellular portion of a
viral polypeptide that is a membrane polypeptide or includes a
portion of the viral polypeptide that comprises at least one
binding domain.
[0068] A protein (or polypeptide) domain for any of the viral
virulence polypeptides or cellular polypeptides or other
polypeptides described herein refers to a region of a polypeptide
or protein that can be defined or described structurally and/or
functionally. For example, a domain of a polypeptide may represent
an enzymatic motif or region, a binding domain (for a ligand or a
specifically-binding antibody), a location (e.g., an extracellular
domain or intracellular domain), and independently folding
structural unit, and other definitions understood in the art. The
presence of a domain in a polypeptide may be determined by
inspection of the primary sequence to determine sequence homology
with a known or similar domain or may be determined (or the
likelihood of the presence of domain determined) with computer
programs such as PROSITE, BLOCKS, PRINTS, DOMAK, and PFAM that are
readily available (see also, e.g., Siddiquiet al., Protein Science
4:872-884 (1995)).
[0069] A viral polypeptide described herein also includes a viral
polypeptide variant. A viral polypeptide variant includes a viral
strain variant or other variant. Variants may result from natural
polymorphisms or may be synthesized by recombinant methodology
(e.g., to obtain codon optimization for expression in a particular
host or to introduce an amino acid mutation) or chemical synthesis,
and may differ from wild-type polypeptides by one or more amino
acid substitutions, insertions, deletions. A variant of a viral
polypeptide identified as described herein has at least 70% to 100%
amino acid identity (that is, at least 70%, 75%, 80%, 85%, 90%,
95%, or 99% identity) to the amino acid sequence encoded by the
viral genome. Preferably a variant viral polypeptide that comprises
at least one substitution, deletion, or insertion of an amino acid
retains the same biological activity, including the capability to
bind to at least one cellular polypeptide. A viral polypeptide
variant that comprises one or more substitutions preferably
comprises conservative substitution(s) compared with the wildtype
polypeptide sequence.
[0070] A conservative substitution is one in which an amino acid is
substituted for another amino acid that has similar properties,
such that one skilled in the art of peptide chemistry would expect
the secondary structure and hydropathic nature of the polypeptide
to be substantially unchanged. Amino acid substitutions may
generally be made on the basis of similarity in polarity, charge,
solubility, hydrophobicity, hydrophilicity and/or the amphipathic
nature of the residues. For example, negatively charged amino acids
include aspartic acid and glutamic acid; positively charged amino
acids include lysine and arginine; and amino acids with uncharged
polar head groups having similar hydrophilicity values include
leucine, isoleucine and valine; glycine and alanine; asparagine and
glutamine; and serine, threonine, phenylalanine and tyrosine.
Examples of conservative substitutions include substituting one
aliphatic amino acid for another, such as isoleucine, valine,
leucine, or alanine, or substituting one polar residue for another,
such as between lysine and arginine, glutamic acid and aspartic
acid, or glutamine and asparagine. A similar amino acid or a
conservative amino acid substitution is also one in which an amino
acid residue is replaced with an amino acid residue having a
similar side chain, which include amino acids with basic side
chains (e.g., lysine, arginine, histidine); acidic side chains
(e.g., aspartic acid, glutamic acid); uncharged polar side chains
(e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine,
cysteine, histidine); nonpolar side chains (e.g., alanine, valine,
leucine, isoleucine, proline, phenylalanine, methionine,
tryptophan); beta-branched side chains (e.g., threonine, valine,
isoleucine), and aromatic side chains (e.g., tyrosine,
phenylalanine, tryptophan). Proline, which is considered more
difficult to classify, shares properties with amino acids that have
aliphatic side chains (e.g., Leu, Val, Ile, and Ala). In certain
circumstances, substitution of glutamine for glutamic acid or
asparagine for aspartic acid may be considered a similar
substitution in that glutamine and asparagine are amide derivatives
of glutamic acid and aspartic acid, respectively. A variant may
also, or alternatively, contain nonconservative changes that do not
adversely alter the properties, including the binding properties,
of the viral polypeptide.
[0071] Variants may also (or alternatively) be modified by, for
example, the deletion or addition of amino acids that have minimal
influence on the activity of the polypeptide. In particular,
variants may contain additional amino acid sequences at the amino
and/or carboxy termini. Such sequences may be used, for example, to
facilitate purification or detection of the polypeptide.
[0072] A polynucleotide sequence that encodes the viral virulence
polypeptide, fragment, portion, or variant thereof, includes the
polynucleotide sequence identified in the viral genome and also
includes a polynucleotide variant that differs from the genomic
sequence due to degeneracy of the genetic code. A polynucleotide
variant also includes a polynucleotide sequence that encodes a
viral virulence polypeptide variant as described herein.
[0073] Persons skilled in the art may readily introduce mutations
into a polynucleotide sequence for preparing a polypeptide variant
using any one of a variety of mutagenesis techniques routinely used
by a person skilled in the art. Mutations may be introduced at
particular loci by synthesizing oligonucleotides that contain a
mutant sequence that are flanked by restriction sites, enabling
ligation to fragments of the native sequence. Following ligation,
the resulting reconstructed sequence encodes a derivative or
variant having the desired amino acid insertion, substitution, or
deletion.
[0074] Alternatively, oligonucleotide-directed site-specific (or
segment specific) mutagenesis procedures may be employed to provide
an altered polynucleotide having particular codons altered
according to the substitution, deletion, or insertion. Exemplary
methods of making the alterations set forth above are disclosed by
Walder et al. (Gene 42:133, 1986); Bauer et al. (Gene 37:73, 1985);
Craik (BioTechniques, January 1985, 12-19); Smith et al. (Genetic
Engineering: Principles and Methods, Plenum Press, 1981); and
Sambrook et al. (supra). Deletion or truncation derivatives of a
viral virulence polypeptide (e.g., a soluble extracellular portion)
may also be constructed by using convenient restriction
endonuclease sites adjacent to the desired deletion. Subsequent to
restriction, overhangs may be filled in and the DNA religated.
Exemplary methods of making the alterations set forth above are
disclosed by Sambrook et al. (Molecular Cloning: A Laboratory
Manual, 3d Ed., Cold Spring Harbor Laboratory Press (2001)).
[0075] Mutations that are made in a polynucleotide preferably
preserve the reading frame of the coding sequences. Furthermore,
the mutations will preferably not create complementary regions that
when transcribed could hybridize to produce secondary mRNA
structures, such as loops or hairpins, which would adversely affect
translation of the mRNA. Although a mutation site may be
predetermined, the nature of the mutation need not per se be
predetermined. For example, random mutagenesis may be conducted at
the target codon and the expressed mutants screened for gain, loss,
or retention of biological activity. Alternatively, mutations may
be introduced at particular loci by synthesizing oligonucleotides
containing a mutant sequence, flanked by restriction sites enabling
ligation to fragments of the native sequence. Following ligation,
the resulting reconstructed sequence encodes a derivative having
the desired amino acid insertion, substitution, or deletion.
Nucleic acid molecules that encode viral virulence polypeptides or
fusion proteins as described herein may also be constructed using
techniques such as polymerase chain reaction (PCR) mutagenesis,
chemical mutagenesis (Drinkwater and Klinedinst, Proc. Natl. Acad.
Sci. USA 83:3402-3406, 1986); forced nucleotide misincorporation
(e.g., Liao and Wise Gene 88:107-111, 1990); or use of randomly
mutagenized oligonucleotides (Horwitz et al., Genome 3:112-117,
1989).
[0076] Nucleotide sequences and amino acid sequences of two or more
viral polynucleotides and the encoded polypeptides and variants
thereof, respectively, can be compared using any standard software
program, such as BLAST, tBLAST, pBLAST, or MegAlign. Still others
include those provided in the Lasergene bioinformatics computing
suite, which is produced by DNASTAR.RTM. (Madison, Wis.); CLUSTALW
program (Thompson et al., Nucleic Acids Res. 22:4673-80 (1991));
and "GeneDoc" (Nicholas et al., EMBNEW News 4:14 (1991)).
References for algorithms such as ALIGN or BLAST may be found in,
for example, Altschul, J. Mol. Biol. 219:555-565, 1991; or Henikoff
and Henikoff, Proc. Natl. Acad. Sci. USA 89:10915-10919, 1992.
BLAST is available at the NCBI website. Such algorithms include
Align or the BLAST algorithm (see, e.g., Altschul, J. Mol. Biol.
219:555-565, 1991; Henikoff and Henikoff, Proc. Natl. Acad. Sci.
USA 89:10915-10919, 1992), which are available at the NCBI website
(see [online] Internet at ncbi.nlm.nih.gov/cgi-bin/BLAST). Default
parameters may be used. Other methods for comparing two nucleotide
or amino acid sequences by determining optimal alignment are
practiced by those having skill in the art (see, for example,
Peruski and Peruski, The Internet and the New Biology: Tools for
Genomic and Molecular Research (ASM Press, Inc. 1997); Wu et al.
(eds.), "Information Superhighway and Computer Databases of Nucleic
Acids and Proteins," in Methods in Gene Biotechnology, pages
123-151 (CRC Press, Inc. 1997); and Bishop (ed.), Guide to Human
Genome Computing, 2nd Ed. (Academic Press, Inc. 1998)).
[0077] As used herein, "percent identity" is the percent value
returned by comparing a viral polypeptide (i.e., a viral virulence
polypeptide that is a viral polypeptide that exhibits at least one
virulence trait), fragment, or variant thereof, sequence to a test
sequence using a computer implemented algorithm, typically with
default parameters. A variant polypeptide could be made to include
one or more of a variety of mutations, such as point mutations,
frameshift mutations, missense mutations, additions, deletions, and
the like, or the variants can be a result of modifications, such as
by certain chemical substituents, including glycosylation,
alkylation, etc. As used herein, "similarity" between two peptides
or polypeptides is generally determined by comparing the amino acid
sequence of one peptide or polypeptide to the amino acid sequence
and conserved amino acid substitutes thereto of a second peptide or
polypeptide.
[0078] A viral polypeptide, including a viral virulence
polypeptide, may be prepared by chemically synthesizing the
polypeptide according to chemical synthesis methods practiced in
the art, including synthesis by automated procedure. Equipment for
automated synthesis of polypeptides is commercially available from
suppliers such as Perkin-Elmer, Inc., Applied BioSystems Division
(Foster City, Calif.), and may be operated according to the
manufacturer's instructions. Such polypeptides may be synthesized
using any of the commercially available solid-phase techniques,
such as the Merrifield solid-phase synthesis method, by which amino
acids are sequentially added to a growing amino acid chain (see,
e.g., Merrifield, J. Am. Chem. Soc. 85:2149 (1963)). For example,
polypeptides may be synthesized using
N-alpha-(9-fluorenylmethyloxycarbonyl (Fmoc) or tert-butoxycarbonyl
(tBoc)-protection strategies with
2-(1H-7-Azabenzotriazol-1-yl)-1,1,3,3-tetramethyl uronium
hexafluorophosphate (HATU) or
2-(1H-Benzotriazol-1-yl)-1,1,3,3-tetramethyluronium
hexafluorophosphate (HBTU) as the coupling agent (see, e.g.,
Schnolzer, et al. Int. J. Pept. Protein Res. 40, 180-193 (1992);
Hackeng et al., Proc. Natl. Acad. Sci. USA 94:7845-50 (1997)). The
crude polypeptide may be further purified using preparative reverse
phase chromatography. Other purification methods, such as partition
chromatography, gel filtration, gel electrophoresis, or
ion-exchange chromatography may be used. In addition, any naturally
occurring amino acid or derivative thereof may be used, including
D-amino acids or L-amino acids, and combinations thereof. In
certain embodiments, a synthetic viral polypeptide has an amino
acid sequence that is identical to, or at least 80% identical
(which includes at least 85%, 90%, or 95% or any percent in between
80% and 100%) to the amino acid sequence encoded by the viral
genome.
[0079] Alternatively, the viral virulence polypeptide may be
prepared by recombinant expression methods described herein and/or
practiced routinely in the art, wherein the viral polypeptide, or
fusion protein comprising the viral polypeptide, is expressed from
a polynucleotide that is operatively linked to an expression
control sequence (e.g., a promoter, enhancer, transcription
initiation site) in a nucleic acid expression construct. A viral
polypeptide and a fusion polypeptide comprising a viral polypeptide
as described in greater detail herein may be expressed using
vectors and constructs, particularly recombinant expression
constructs, that include any polynucleotide encoding such
polypeptides. Host cells are genetically engineered with vectors
and/or constructs to produce these polypeptides and fusion
proteins, or fragments or variants thereof, by recombinant
techniques. Each of the polypeptides and fusion polypeptides
described herein can be expressed in mammalian cells, yeast,
bacteria, or other cells under the control of appropriate
promoters. Cell-free translation systems can also be employed to
produce such proteins using RNAs derived from DNA constructs.
Appropriate cloning and expression vectors for use with prokaryotic
and eukaryotic hosts are described, for example, by Sambrook, et
al., Molecular Cloning: A Laboratory Manual, Third Edition, Cold
Spring Harbor, N.Y., (2001).
[0080] Cells And Cellular Polypeptides
[0081] Described herein are methods for identifying cellular
polypeptides that in some manner are affected by or are effectors
of a disease process. By altering (increasing or decreasing in a
statistically significant or biologically significant manner) at
least one (i.e., one or more) biological activity of a cellular
polypeptide, the disease process may be inhibited, abrogated,
slowed, or interfered with such that the disease or disorder in an
affected host is treated and/or prevented, and/or at least one
symptom of the disease or disorder is inhibited, reduced, or
abrogated.
[0082] As described herein viruses, such as poxviruses, encode
proteins that are expressed during infection that contribute to the
capability of the virus to evade a host's immune system and/or to
suppress the host's immune system by interacting with one or more
cellular molecules. These viral polypeptides interact with cellular
polypeptides that include, but are not limited to, cell surface
antigens, cell surface receptors, cytokines, chemokines, cytokine
or chemokine binding proteins, intracellular signaling
polypeptides, or substrates of cell surface receptors or signaling
molecules, and other immunoregulatory molecules that affect and
regulate an immune response (see, e.g., U.S. Pat. No. 5,359,039;
U.S. Pat. No. 6,852,486; U.S. Pat. No. 5,871,740; U.S. Pat. No.
6,843,991; U.S. Pat. No. 6,355,252).
[0083] Cellular polypeptides may be identified according to the
methods described herein using a biological sample that comprises
an intact cell, a cell fraction, and/or a cell supernatant. A
"biological sample" as used herein refers in certain embodiments to
a sample containing at least one cell or a fraction of a cell or a
supernatant of a cell. A biological sample may be a blood sample
(from which serum or plasma may be prepared), biopsy specimen, body
fluids (e.g., lung lavage, ascites, mucosal washings, synovial
fluid), bone marrow, lymph nodes, tissue explant, organ culture, or
any other tissue or cell preparation from a subject or a biological
source.
[0084] The subject or biological source may be a human or non-human
animal, a primary cell culture (e.g., immune cells, virus infected
cells), or culture adapted cell line, including but not limited to,
genetically engineered cell lines that may contain chromosomally
integrated or episomal recombinant nucleic acid sequences,
immortalized or immortalizable cell lines, somatic cell hybrid cell
lines, differentiated or differentiatable cell lines, transformed
cell lines, and the like. A variety of normal cells and tumor cell
types may be used to identify cellular polypeptides that bind to or
interact with a viral virulence polypeptide, including B cells and
T cells (activated or non-activated), macrophages, epithelial
cells, fibroblasts, and cell lines such as Raji (B cell lymphoma),
THP-1 (acute monocytic leukemia), and Jurkat (T cell leukemia).
Cells useful for the methods described herein are immune cells,
including T cells, B cells, natural killer cells, macrophages, etc.
The immune cell may be present in or isolated from a biological
sample as described herein. For example, the immune cell or any
other cell may be obtained from a primary or long-term cell culture
or may be present in or isolated from a biological sample obtained
from a subject (human or non-human animal).
[0085] A sample may further refer to a tissue or cell preparation
in which the morphological integrity or physical state has been
disrupted, for example, by dissection, dissociation,
solubilization, fractionation, homogenization, biochemical or
chemical extraction, pulverization, lyophilization, sonication, or
combination thereof, or any other means for processing a sample
derived from a subject or biological source. Such a cell
preparation includes a cell fraction such as a cell lysate or cell
extract that may be used in the methods described herein. A cell
fraction also includes a preparation of one or more isolated
organelles from a cell. A cell organelle includes but is not
limited to nucleus, mitochondrion, nucleolus, centriole,
centrosome, Golgi, cytoskeleton, cytosol, secretory vesicle,
lysosome, peroxisome, vacuole, cell membrane, and endoplasmic
reticulum. A cell fraction also includes complex multi-molecular
structures such as lipid rafts and other trafficking and transport
complexes. A cell fraction and an isolated cell organelle may be
prepared according to methods routinely practiced in the art.
[0086] As described herein, a cellular polypeptide to which a viral
virulence polypeptide binds includes a cellular polypeptide that is
secreted by a cell. Accordingly, a cell supernatant, which
includes, for example, cellular washes, cell culture media, or
conditioned media (i.e., media from cells in culture that have been
propagated for a period of time sufficient for the cells to secrete
such cellular polypeptides), or any other extracellular preparation
may be used in the methods described herein. A biological sample,
such as blood, serum, or plasma, cerebral spinal fluid, or other
body fluids described herein, may also contain one or more cellular
polypeptides to which a viral polypeptide binds and be used as a
source for detecting the presence of the cellular polypeptide that
is secreted by a cell or that is released by a cell via other
processes including normal or abnormal cell death.
[0087] In certain embodiments, a cell is stimulated prior to, at
the same time, or after, a cell, or a fraction or a supernatant of
the cell, is contacted with a fusion protein comprising a viral
polypeptide fused to an affinity tag, as described in detail
herein. A cell may be stimulated with at least one stimulus, at
least two stimuli, or more than two different stimuli. Exemplary
stimuli include an antibody that specifically binds to a cognate
antigen (e.g., a cell surface marker antigen or cell surface
receptor) expressed by the cell; a phorbol ester (which modulate
gene expression, reorganize the cytoskeleton, and/or stimulate bulk
protein synthesis) (e.g., Phorbol 12-myristate 13-acetate (PMA)),
and other mitogens (e.g., concanavalin A and other lectins;
lipospolysaccharide, phytohemagglutinin (PHA), pokeweed mitogen
(PWM), insulin, polypeptide growth factors); a cytokine; a
chemokine; and ionomycin. In certain embodiments, a cell may be
exposed to a combination of at least two agents, for example, PMA
and ionomycin or PWM and insulin.
[0088] Detection of a Viral Polypeptide/Cellular Polypeptide
Complex
[0089] As described herein, methods are provided for identifying a
cellular polypeptide to which a viral virulence polypeptide binds
by contacting (mixing, combining, or in some manner permitting
interaction) a source of the cellular polypeptide (e.g., a cell,
cell fraction, or cell supernatant) and a viral polypeptide under
conditions and for a time sufficient for the viral polypeptide and
the cellular polypeptide to form a complex. The viral
polypeptide/cellular polypeptide complex may then be detected
and/or isolated. In certain embodiments, the viral polypeptide is
fused to an affinity tag to form a fusion protein. Such fusion
proteins may be used in methods such as tandem affinity
purification for purification and/or isolation of the cellular
polypeptide. The identity of the cellular polypeptide may be
determined according to a variety of methods practiced in the art
and described herein, such as LC-MS/MS.
[0090] Fusion Proteins: Viral Polypeptide Fused to an Affinity
Tag
[0091] In one embodiment, the viral virulence polypeptide (or
fragment thereof) is fused to an affinity tag, which may be used in
methods described herein for identifying a cellular polypeptide.
The affinity tag may comprise at least one polypeptide tag and/or
at least one detectable moiety (or label or reporter molecule) such
as an enzyme, cytotoxicity agent, or other reporter molecule,
including a dye, radionuclide, luminescent group, fluorescent
group, or biotin, or the like according to methods practiced in the
art. Techniques for radiolabeling of polypeptides are known in the
art (see, e.g., Adams, In Vivo 12:11-21 (1998); Hiltunen, Acta
Oncol. 32:831-9 (1993)). The detectable moiety may be attached to
the viral polypeptide or the polypeptide tag, such as through any
available amino acid side-chain, terminal amino acid, or
carbohydrate functional group located in the polypeptide, provided
that the attachment or attachment process does not adversely affect
the binding properties such that the usefulness of the molecule is
abrogated. Particular functional groups include, for example, any
free amino, imino, thiol, hydroxyl, carboxyl, or aldehyde group.
Attachment of the polypeptide (either the viral polypeptide or a
polypeptide tag portion of the affinity tag) and the detectable
moiety may be achieved via such groups and an appropriate
functional group in the detectable moiety. The linkage may be
direct or indirect through spacing or bridging groups (see, e.g.,
International Patent Application Publication Nos. WO 93/06231, WO
92/22583, WO 90/091195, and WO 89/01476; see also, e.g., commercial
vendors such as Pierce Biotechnology, Rockford, Ill.).
[0092] An affinity tag comprising a polypeptide tag may be attached
to the viral polypeptide by any of a variety of techniques with
which those skilled in the art will be familiar. A fusion protein
comprising a viral polypeptide and an affinity tag may be detected,
identified, or isolated when bound to a cellular polypeptide
according to methods and techniques including, for example,
interaction of the polypeptide tag to a detectable cognate binding
molecule (i.e., cognate ligand), direct covalent modification of a
fusion protein with a detectable moiety (e.g., a labeling moiety),
non-covalent binding of the fusion protein to a specific labeled
reporter molecule, enzymatic modification of a detectable substrate
by a fusion protein that includes a portion having enzyme activity,
or immobilization (covalent or non-covalent) of the fusion protein
on a solid-phase support. A cognate ligand of an affinity tag,
which includes a cognate ligand of a polypeptide tag is a molecule
with which a polypeptide tag is capable of interacting to form a
complex. Examples of cognate ligands include but are not limited to
an antibody (or a fragment or derivative thereof) that specifically
binds to the polypeptide tag, a small molecule, a polypeptide,
peptide, carbohydrate, hormone, cell receptor polypeptide (or
fragment or domain thereof), cell surface antigen, or other
cellular molecule to which the polypeptide tag binds. A viral
polypeptide may be fused to another polypeptide such as a peptide
tag having desirable affinity properties according to methods
described in the art and routinely practiced by skilled artisans
(see, e.g., U.S. Pat. No. 5,100,788; WO 89/03422; U.S. Pat. No.
5,489,528; U.S. Pat. No. 5,672,691; WO 93/24631; U.S. Pat. No.
5,168,049; U.S. Pat. No. 5,272,254; EP 511,747).
[0093] In certain embodiments, the affinity tag that is attached to
a viral virulence polypeptide comprises at least one polypeptide
tag; in certain other embodiments, the affinity tag comprises at
least two, three, or four, or more polypeptide tags. Examples of
polypeptide tags include but are not limited to an immunoglobulin
Fc polypeptide, an immunoglobulin mutein Fc polypeptide, a
hemagglutinin peptide, a calmodulin binding polypeptide (or a
domain or peptide thereof), a protein C-tag, a streptavidin binding
peptide (or fragments thereof), a protein A fragment (e.g., an
IgG-binding ZZ polypeptide), and a Softag.TM. peptide. Additional
affinity tags include polyhistidine tag (his tag) or FLAG.RTM.
epitope tag (DYKDDDDK, SEQ ID NO:20), beta-galactosidase, alkaline
phosphatase, GST, or the XPRESS.TM. epitope tag (DLYDDDDK, SEQ ID
NO:21; (Invitrogen Corp., Carlsbad, Calif.) and the like (see,
e.g., U.S. Pat. No. 5,011,912; Hopp et al., Bio/Technology 6:1204
(1988)). The affinity sequence may be supplied by a vector, such
as, for example, a hexa-histidine tag that is provided in pBAD/His
(Invitrogen). Alternatively, the affinity sequence may be added
either synthetically or engineered into the primers used to
recombinantly generate the nucleic acid coding sequence (e.g.,
using the polymerase chain reaction).
[0094] In one embodiment, at least one polypeptide tag is an Fc
polypeptide. In a particular embodiment, the Fc polypeptide is of
human origin and may be from any of the immunoglobulin classes,
such as human IgG1, IgG2, IgG3, IgG4, or IgA. In a certain
embodiment, the Fc polypeptide is derived from a human IgG1
immunoglobulin, (see Kabat et al., in Sequences of Proteins of
Immunological Interest, 4th ed., (U.S. Dept. of Health and Human
Services, U.S. Government Printing Office, 1991)). In another
embodiment, the Fc polypeptide comprises an amino acid sequence of
an Fc polypeptide from a non-human animal, for example, but not
limited to, a mouse, rat, rabbit, or hamster. Binding partners (or
cognate ligands) of an Fc polypeptide include, for example, protein
A (or at least one domain of protein A that binds to an
immunoglobulin Fc polypeptide); protein G; an antibody (or fragment
or derivative thereof) that specifically binds to the Fc
polypeptide from a particular species (e.g., an antibody that
specifically binds to an Fc polypeptide of a particular class or
isotype and from a particular species, such as an antibody that
specifically binds to a human IgG1 Fc polypeptide). A fusion
protein that comprises an affinity tag comprising an Fc polypeptide
may be identified, detected, or isolated, for example, when the
fusion protein has formed a complex with a cellular polypeptide by
contacting the complex with any one of the Fc polypeptide binding
partners or ligands described herein or known in the art.
[0095] The amino acid sequences of Fc polypeptides of human origin
and of a variety of species are available in the art, for example,
in Kabat et al. (in Sequences of Proteins of Immunological
Interest, 4th ed. (U.S. Dept. of Health and Human Services, U.S.
Government Printing Office, 1991)). By way of example, the Fc
polypeptide tag of a fusion protein described herein may comprise
the amino acid sequence of all or a portion of the hinge region,
CH2 domain, and CH3 domain of a human immunoglobulin, for example,
an IgG1. An Fc polypeptide is capable of interacting with a second
Fc polypeptide to form a dimer via covalent, such as disulfide bond
formation, and noncovalent interactions.
[0096] An Fc polypeptide as described herein also includes Fc
polypeptide variants. One such Fc polypeptide variant has one or
more cysteine residues (such as one or more cysteine residues in
the hinge region) that forms an interchain disulfide bond
substituted with another amino acid, such as serine, to reduce the
number of interchain disulfide bonds that can form between the two
heavy chain constant region polypeptides that form an Fc
polypeptide. In addition, or alternatively, the most amino terminal
cysteine residue of the hinge region that typically forms a
disulfide bond with a light chain constant region in an
immunoglobulin molecule may be deleted or substituted, for example,
substituted with a serine residue. Another example of an Fc
polypeptide variant is a variant that has one or more amino acids
involved in an effector function substituted or deleted such that
the Fc polypeptide has a reduced level of an effector function. For
example, amino acids in the Fc region may be substituted to reduce
or abrogate binding of a component of the complement cascade (see,
e.g., Duncan et al., Nature 332:563-64 (1988); Morgan et al.,
Immunology 86:319-24 (1995)) or to reduce or abrogate the ability
of the Fc fragment to bind to an IgG Fc receptor expressed by an
immune cell (Wines et al., J. Immunol. 164:5313-18 (2000); Chappel
et al., Proc. Natl. Acad. Sci. USA 88:9036 (1991); Canfield et al.,
J. Exp. Med. 173:1483 (1991); Duncan et al., supra); or to alter
antibody-dependent cellular cytotoxicity. Such an Fc polypeptide
variant that differs from the wildtype Fc polypeptide is also
called herein a mutein Fc polypeptide.
[0097] In one embodiment, a viral virulence polypeptide (or
fragment or variant thereof) is fused in frame with an Fc
polypeptide that comprises at least one substitution of a residue
that in the wildtype Fc region polypeptide contributes to binding
of an Fc polypeptide or immunoglobulin to one or more IgG Fc
receptors expressed on certain immune cells. Such a mutein Fc
polypeptide comprises at least one substitution of an amino acid
residue in the CH2 domain of the mutein Fc polypeptide, such that
the capability of the fusion polypeptide to bind to an IgG Fc
receptor, such as an IgG Fc receptor present on the surface of an
immune cell, is reduced.
[0098] By way of background, on human leukocytes three distinct
types of Fc IgG-receptors are expressed that are distinguishable by
structural and functional properties, as well as by antigenic
structures, which differences are detected by CD specific
monoclonal antibodies. The IgG Fc receptors are designated
Fc.gamma.RI (CD64), Fc.gamma.RII (CD32), and Fc.gamma.RIII (CD16)
and are differentially expressed on overlapping subsets of
leukocytes.
[0099] Fc.gamma.RI (CD64), a high-affinity receptor expressed on
monocytes, macrophages, neutrophils, myeloid precursors, and
dendritic cells, comprises isoforms 1a and 1b. Fc.gamma.RII (CD32),
comprised of isoforms IIa, IIb1, IIb2, IIb3, and IIc, is a
low-affinity receptor that is the most widely distributed human
Fc.gamma.R type; it is expressed on most types of blood leukocytes,
as well as on Langerhans cells, dendritic cells, and platelets.
Fc.gamma.RIII (CD16) has two isoforms, both of which are capable of
binding to human IgG1 and IgG3. The Fc.gamma.RIIIa isoform has an
intermediate affinity for IgG and is expressed on macrophages,
monocytes, natural killer (NK) cells, and subsets of T cells.
Fc.gamma.RIIIb is a low-affinity receptor for IgG and is
selectively expressed on neutrophils.
[0100] Residues in the amino terminal portion of the CH2 domain
that contribute to IgG Fc receptor binding include residues at
positions Leu234-Ser239 (Leu-Leu-Gly-Gly-Pro-Ser (SEQ ID NO:22) (EU
numbering system, Kabat et al., supra) (see, e.g., Morgan et al.,
Immunology 86:319-24 (1995), and references cited therein). These
positions correspond to positions 15-20 of the amino acid sequence
of an exemplary human IgG1 Fc polypeptide (SEQ ID NO:25).
Substitution of the amino acid at one or more of these six
positions (i.e., one, two, three, four, five, or all six) in the
CH2 domain results in a reduction of the capability of the Fc
polypeptide to bind to one or more of the IgG Fc receptors (or
isoforms thereof) (see, e.g., Burton et al., Adv. Immunol. 51:1
(1992); Hulett et al., Adv. Immunol. 57:1 (1994); Jefferis et al.,
Immunol. Rev. 163:59 (1998); Lund et al., J. Immunol. 147:2657
(1991); Sarmay et al., Mol. Immunol. 29:633 (1992); Lund et al.,
Mol. Immunol. 29:53 (1992); Morgan et al., supra). In addition to
substitution of one or more amino acids at EU positions 234-239,
one, two, or three or more amino acids adjacent to this region
(either to the carboxy terminal side of position 239 or to the
amino terminal side of position 234) may also be substituted.
[0101] By way of example, substitution of the leucine residue at
position 235 (which corresponds to position 16 of SEQ ID NO:25)
with a glutamic acid residue or an alanine residue abolishes or
reduces, respectively, the affinity of an immunoglobulin (such as
human IgG3) for Fc.gamma.RI (Lund et al., 1991, supra; Canfield et
al., supra; Morgan et al., supra). As another example, replacement
of the leucine residues at positions 234 and 235 (which correspond
to positions 15 and 16 of SEQ ID NO:25), for example, with alanine
residues, abrogates binding of an immunoglobulin to Fc.gamma.RIIa
(see, e.g., Wines et al., supra). Alternatively, leucine at
position 234 (corresponding to position 15 of SEQ ID NO:25),
leucine at position 235 (corresponding to position 16 of SEQ ID
NO:25), and glycine at position 237 (corresponding to position 18
of SEQ ID NO:25), each may be substituted with a different amino
acid, such as leucine at position 234 may be substituted with an
alanine residue (L234A), leucine at 235 may be substituted with an
alanine residue (L235A) or with a glutamic acid residue (L235E),
and the glycine residue at position 237 may be substituted with
another amino acid, for example an alanine residue (G237A).
[0102] In one embodiment, a mutein Fc polypeptide that is fused in
frame to a viral virulence polypeptide (or variant or fragment
thereof) comprises one, two, three, four, five, or six mutations at
positions 15-20 of SEQ ID NO:25 that correspond to positions
234-239 of a human IgG1 CH2 domain (EU numbering system) as
described herein. An exemplary mutein Fc polypeptide comprises the
amino acid sequence set forth in SEQ ID NO:23 in which
substitutions corresponding to (L234A), (L235E), and (G237A) may be
found at positions 13, 14, and 16 of SEQ ID NO:23.
[0103] In another embodiment, a mutein Fc polypeptide comprises a
mutation of a cysteine residue in the hinge region of an Fc
polypeptide. In one embodiment, the cysteine residue most proximal
to the amino terminus of the hinge region of an Fc polypeptide that
in a whole immunoglobulin molecule forms a disulfide bond with a
cysteine in the constant region of the light chain (e.g., for
example, the cysteine residue most proximal to the amino terminus
of the hinge region of the Fc portion of a wildtype IgG1
immunoglobulin) is deleted or substituted with another amino acid.
That is, by way of illustration, the cysteine residue that
corresponds to the cysteine at position 1 of the exemplary Fc
polypeptide having the sequence set forth in SEQ ID NO:25, is
deleted, or the cysteine residue at this position is substituted
with another amino acid that is incapable of forming a disulfide
bond, for example, a serine residue. In another embodiment, a
mutein Fc polypeptide comprises a deletion or substitution of the
cysteine residue most proximal to the amino terminus of the hinge
region of an Fc polypeptide further comprises deletion or
substitution of the adjacent C-terminal amino acid. In a certain
embodiment, this cysteine residue and the adjacent C-terminal
residue are both deleted from the hinge region of a mutein Fc
polypeptide. In a specific embodiment, a mutein Fc polypeptide
comprises an amino acid sequence wherein the cysteine residue at a
position corresponding to position 1 of SEQ ID NO:25 and the
aspartic acid at a position corresponding to position 2 of SEQ ID
NO:25 are deleted. Fc polypeptides that comprise deletion of these
cysteine and aspartic acid residues in the hinge region may be
efficiently expressed in a host cell, and in certain instances, may
be more efficiently expressed in a cell than an Fc polypeptide that
retains the wildtype cysteine and aspartate residues.
[0104] In a specific embodiment, a mutein Fc polypeptide comprises
the amino acid sequence set forth in SEQ ID NO:23, which differs
from the wildtype Fc polypeptide (SEQ ID NO:25) wherein the
cysteine residue at position 1 of SEQ ID NO:25 is deleted and the
aspartic acid at position 2 of SEQ ID NO:25 is deleted and the
leucine reside at position 15 of SEQ ID NO:25 is substituted with
an alanine residue (i.e., position 13 of SEQ ID NO:23), the leucine
residue at position 16 is substituted with a glutamic acid residue
(i.e., position 14 of SEQ ID NO:23), and the glycine at position 18
is substituted with an alanine residue (i.e., position 16 of SEQ ID
NO:23) (see FIG. 6; SEQ ID NO:23). Thus, an exemplary mutein Fc
polypeptide comprises an amino acid sequence at its amino terminal
portion of KTHTCPPCPAPEAEGAPS (SEQ ID NO:26) (see SEQ ID NO:23, an
exemplary Fc mutein sequence).
[0105] Other Fc variants encompass similar amino acid sequences of
known Fc polypeptide sequences that have only minor changes, for
example by way of illustration and not limitation, covalent
chemical modifications, insertions, deletions and/or substitutions,
which may further include conservative substitutions. Amino acid
sequences that are similar to one another may share substantial
regions of sequence homology. Similarly, nucleotide sequences that
encode the Fc variants may encompass substantially similar
nucleotide sequences and have only minor changes, for example by
way of illustration and not limitation, covalent chemical
modifications, insertions, deletions, and/or substitutions, which
may further include silent mutations owing to degeneracy of the
genetic code. Nucleotide sequences that are similar to one another
may share substantial regions of sequence homology.
[0106] Another polypeptide tag that may be used as an affinity tag,
either alone or with at least one additional polypeptide tag,
includes a hemagglutinin peptide, which in certain embodiments is a
human influenza hemagglutinin peptide. The amino acid sequence of
an exemplary hemagglutinin peptide comprises YPYDVDYA (SEQ ID
NO:1). Antibodies that specifically bind to the hemagglutinin
peptide are examples of cognate ligands for a hemagglutinin peptide
and are available commercially (e.g., Roche Diagnostics Corp.,
Roche Applied Science, Indianapolis, Ind.; Vector Laboratories,
Burlingame, Calif.).
[0107] In another embodiment, a calmodulin binding polypeptide
(CBP) derived from cAMP kinase or a CBP domain or CBP peptide may
be used as at least one polypeptide tag (see, e.g., Puig et al.,
Methods 24:218-29 (2001)). The CBP moiety, which may be a peptide
or domain of the CBP full-length polypeptide is capable of binding
to calmodulin (i.e., a cognate ligand) in the presence of calcium
(Ca.sup.2+). An exemplary CBP peptide comprises the sequence set
forth in SEQ ID NO:3 (KRRWKKNFIAVSAANRFKKISSSGAL), which may have
at least one, two, three, four, five, six, seven, or more amino
acids at either the amino terminus or carboxy terminus that are the
adjacent amino acids of the calmodulin binding protein or that
represent a spacer peptide. The interaction between the CBP moiety
and calmodulin may be disrupted by the addition of a chelating
agent, such as EGTA or EDTA. In certain instances, endogenous
calmodulin is present in the cell, cell fraction, or cell
supernatant that is used in the methods described herein for
identifying a cellular polypeptide to which a viral virulence
polypeptide may bind. Binding of the fusion protein that comprises
a CBP moiety to endogenous calmodulin may be prevented or
significantly reduced by adding a chelating agent prior to exposure
of a fusion protein:cellular polypeptide complex (or other complex
formed according to the methods described herein) to an exogenous
source of calmodulin.
[0108] An affinity tag may also comprise a protein C-tag as a
polypeptide tag. A protein C-tag comprises the amino acid sequence
EDQVDPRLIDGK (SEQ ID NO:4), derived from the heavy chain of human
protein C (vitamin K-dependent serine protease). Antibodies are
commercially available that may be used to detect the protein C-tag
polypeptide tag or to isolate a complex or fusion protein that
comprises the protein C-tag polypeptide tag (see, e.g., Roche
Applied Science; Delta BioLabs, Gilroy, Calif.; Abcam Inc.,
Cambridge, Mass.; Immunology Consultants Laboratory, Inc., Newburg,
Oreg.). Binding of the C-tag peptide to an antibody called HPC4
(Roche Applied Science) is calcium dependent, wherein the calcium
binding domain resides on the antibody. A fusion polypeptide
comprising the protein C tag as a polypeptide tag that is bound to
a calcium-dependent specific antibody may be eluted (i.e., the
binding interaction between the protein C tag and the antibody is
disrupted) by using a chelating agent such as EDTA.
[0109] An affinity tag may also comprise a streptavidin binding
peptide (SBP) (or fragments thereof). The amino acid sequence of
SBP comprises, for example, MDEKTTGWRGGHVVEGLAGELEQLRARLEHHPQGQREP
(SEQ ID NO:6; encoded by the nucleotide sequence set forth in SEQ
ID NO:9). An affinity tag may comprise one SBP moiety or may
comprise two SBP moieties (i.e., in tandem) (e.g., SEQ ID NO:8,
encoded by the nucleotide sequence set forth in SEQ ID NO:10),
which increases the affinity of the interaction between
streptavidin binding peptide and streptavidin, its cognate ligand.
In certain embodiments, a lower affinity interaction between
streptavidin and a SBP peptide tag may be desirable. Accordingly, a
SBP peptide comprising 15 amino acids (DVEAWLDERVPLVET; SEQ ID
NO:7), may be used (see also, e.g., Lamla, Protein Expr. Purif.
33:39-47 (2004)). Binding of an SBP polypeptide to streptavidin can
be disrupted by addition of biotin, which will compete with SBP for
binding to streptavidin.
[0110] Another polypeptide tag that may be incorporated into the
affinity tag portion of the viral polypeptide fusion protein is a
staphylococcal protein A binding domain that binds to the Fc
portion of certain classes and isotypes of immunoglobulins, which
accordingly serve as cognate ligands for a staphylococcal protein A
binding domain tag. The affinity tag may comprise at least two
staphylococcal protein A binding domains. In one embodiment, the
staphylococcal protein A binding domain comprises an IgG-binding
protein ZZ, which binds to the Fc portion of immunoglobulins (see,
e.g., Nizard et al., Protein Eng. 14:439-446 (2001); Nizard et al.,
FEBS Lett. 433:83-88 (1998)). The ZZ polypeptide is prepared by
duplicating a mutated B domain of staphylococcal protein A, the
polypeptide and encoding nucleotide sequences of which are long
known in the art (see, e.g., Nizard et al., (2001), supra; Nilsson
et al., Protein Eng. 1:107-13 (1987); Ljungberg et al., Mol.
Immunol. 30:1279-85 (1993); Jansson et al., FEMS Immunol. Med.
Microbiol. 20:69-78 (1998); see also, e.g., GenBank Accession Nos.
M74186 (Jun. 21, 1993) and M74187 (May 23, 1996)).
[0111] An affinity tag may comprise a polypeptide tag that is a
Softag.TM. peptide, which comprises the amino acid sequence
SLAELLNAGLGGS (SEQ ID NO:11) (an epitope of Escherichia coli RNA
polymerase) (NeoClone, Madison, Wis.). This peptide tag
specifically binds to an antibody called NT73, which is an example
of a cognate ligand for Softag.TM. (Thompson et al., Biochemistry
31:7003-7008 (1992); Anthony et al., J. Biol. Chem. 277:46433-41
(2002)). A binding interaction between Softag.TM. and NT73 may be
disrupted in the presence of a low molecular weight
polyhydroxylated compound (polyol) and a non-chaotropic salt (see
Thompson et al., supra).
[0112] As described herein, in certain embodiments, a fusion
protein comprising a viral virulence polypeptide and an affinity
tag is used in methods described herein for identifying a cellular
polypeptide that may be a suitable target for a therapeutic agent
and may be used in methods for identifying a cell type that
expresses such a cellular polypeptide. The affinity tag, in certain
embodiments, comprises at least one polypeptide tag, and in certain
other embodiments, the affinity tag may comprise at least two,
three, or four polypeptide tags. The fusion protein may be
constructed using synthetic biochemical and organic chemical
methods that are described herein and routinely practiced in the
art. The fusion protein may also be constructed recombinantly using
molecular biology techniques and procedures, also described herein
and routinely practiced in the molecular biology art, and then
expressed in eukaryotic or prokaryotic cells as a recombinant
protein.
[0113] As used herein, a fusion protein that comprises a viral
polypeptide and an affinity tag may further comprise spacer peptide
sequences between moieties of the fusion polypeptide. For example,
a spacer peptide may be located between the viral polypeptide and
the affinity tag, or when the affinity tag comprises at least two
polypeptide tags, an amino acid spacer sequence may also be between
the polypeptide tags. The spacer peptide may be at least one, two,
three, four, five, six, or seven or more amino acids. Spacer
peptides may be incorporated into a fusion protein to enable or
ensure or facilitate proper folding of each moiety of the fusion
protein. In addition, or alternatively, when the fusion protein is
prepared recombinantly, the spacer peptide may be the translational
product (i.e., encoded amino acid sequence) of a polynucleotide
restriction site that is incorporated into the nucleotide sequence
of a recombinant construct and that is useful for cloning purposes.
In certain other embodiments one, two, or three amino acids at the
amino terminal end or carboxy terminal end of a polypeptide tag
(such as those described herein) may be deleted or substituted,
which may be useful for accommodating a restriction site sequence
or a spacer sequence.
[0114] An affinity tag may further comprise at least one protease
recognition sequence. A protease recognition sequence refers to a
consecutive amino acid sequence that is recognized and required for
proteolytic cleavage by a particular protease. A protease
recognition sequence may be coincident with the protease cleavage
site, that is, cleavage occurs at the protease recognition
sequence. The protease recognition sequence may include one or more
amino acids on either side of the peptide bond to be hydrolyzed by
the protease. Alternatively, the protease recognition sequence may
be one, two, or more amino acids distal, toward the amino or
carboxy terminus, to the cleavage site of the protease.
Accordingly, the protease cleaves the polypeptide comprising the
protease recognition sequence at or near the protease recognition
sequence.
[0115] In one embodiment, a protease recognition sequence comprises
the protease cleavage site of tobacco etch virus (TEV) protease.
TEV protease recognizes a linear epitope of the general formula
E-X-X-Y-X-Q-(G/S) (SEQ ID NO:28), wherein X refers to any amino
acid. In a particular embodiment, a fusion protein comprises a TEV
protease recognition sequence having the amino acid sequence
ENLYFQS (SEQ ID NO:29). Another commonly used TEV protease
recognition sequence comprises the amino acids ENLYFQG (SEQ ID
NO:30). Incorporation of certain other amino acids at the variable
amino acid positions results in a peptide sequence that is less
efficiently cleaved by the TEV protease, which in certain specific
embodiments, may be desirable. The protease cleaves between the
glutamine and glycine or serine residues (-Q-(G/S)).
[0116] In certain embodiments, the affinity tag may comprise a
human rhinovirus 3C (HRV3C) protease site. The protease recognition
sequence comprises the amino acids LEVLFQGP (SEQ ID NO:16). In
certain other embodiments, the affinity tag may comprise at least
two protease recognition sequences, such as the TEV protease
recognition sequence and the human rhinovirus HRV3C protease
recognition sequence.
[0117] In certain embodiments, the affinity tag comprises at least
two polypeptide tags. In other certain embodiments, the affinity
tag may comprise at least two protease recognition sequences. For
example, the affinity tag may comprise at least three polypeptide
tags, and one protease recognition sequence may be between a first
and a second polypeptide tag and a second protease recognition
sequence may be located between the second polypeptide tag and a
third polypeptide tag. Alternatively, a fusion protein that
comprises at least two protease recognition sequences may comprise
a first protease recognition sequence between the viral polypeptide
and the affinity tag and may comprise a second protease recognition
sequence between any of two polypeptide tags present in the
affinity tag. Persons skilled in the art will appreciate that a
protease recognition sequence in the fusion proteins and affinity
tags described herein may be located between any two polypeptide
tags of the affinity tag or may be located between the viral
polypeptide and the affinity tag.
[0118] An affinity tag may be located at the amino terminal end of
the viral polypeptide or may be located at the carboxy terminal end
of the viral polypeptide of a fusion protein. When a fusion protein
is expressed recombinantly, a person skilled in the art using
standard molecular biology and recombinant expression methods and
procedures will be able to determine readily if locating an
affinity tag at either terminal end of the viral polypeptide
adversely affects expression, that is, any one of translation,
folding, and/or transport of the fusion protein. The recombinant
vector can then be constructed accordingly so that expression of
the fusion protein is not significantly adversely affected.
[0119] In one embodiment, the affinity tag comprises one
polypeptide tag, such as a mutein Fc polypeptide. In another
embodiment, an affinity tag comprises at least two, at least three,
at least four, or at least five or six polypeptide tags described
herein, including but not limited to an immunoglobulin Fc
polypeptide, an immunoglobulin mutein Fc polypeptide, a
hemagglutinin peptide, a calmodulin binding peptide or a calmodulin
domain, a protein C-tag, a streptavidin binding peptide (or
fragments thereof), a His tag, a protein A fragment (e.g., an
IgG-binding ZZ polypeptide), and a Softag.TM. peptide. In certain
embodiments, one or more of the polypeptide tags is repeated, that
is, at least two amino acid sequences of the same polypeptide tag
are repeated in the affinity tag. The repeated polypeptide tags may
be immediately adjacent to each other or separated by at least one
different polypeptide tag.
[0120] In a specific embodiment, the affinity tag comprises a
hemagglutinin peptide, a C-tag peptide, and a Softag.TM. peptide.
In another certain embodiment, the affinity tag comprises a
hemagglutinin peptide, a C-tag peptide, and a mutein Fc polypeptide
tag. In another specific embodiment, the affinity tag comprises a
hemagglutinin peptide, a C-tag peptide, and a protein A fragment,
such as an IgG-binding ZZ polypeptide. In another embodiment, the
affinity tag comprises a hemagglutinin peptide, a calmodulin
binding peptide or domain, a streptavidin binding peptide (SBP) (or
a fragment thereof). In another embodiment, the affinity tag
comprises a hemagglutinin peptide, a C-tag peptide, and a SBP or
fragment thereof. In a particular embodiment, the SBP peptide, or
fragment thereof, is repeated at least two times. In another
embodiment, the affinity tag comprises a hemagglutinin peptide, a
calmodulin binding peptide or domain, a SBP (or a fragment
thereof), and a mutein Fc polypeptide. In still other embodiments
as described herein, an affinity tag, including the specific
embodiments described herein, further comprises at least one
protease recognition sequence. By way of non-limiting example, an
affinity tag that comprises a hemagglutinin peptide, a calmodulin
binding peptide or domain, a SBP (or a fragment thereof), and a
mutein Fc polypeptide may further comprise a protease recognition
sequence, for example, a TEV protease recognition sequence or a
HRV3C protease sequence, between any two polypeptide tags. Affinity
tags comprising one polypeptide tag, such as mutein Fc polypeptide,
may also further comprise a protease recognition sequence between
the viral polypeptide sequence and the mutein Fc polypeptide.
[0121] Recombinant Expression Constructs
[0122] In certain embodiments, a fusion protein comprising a viral
virulence polypeptide and an affinity tag is recombinantly
expressed. According to the methods described herein, a fusion
protein may be used as a probe to identify a cellular polypeptide
that binds to a viral polypeptide or to identify a cell type with
which the cellular polypeptide is associated. In other embodiments,
the fusion protein is used to identify and to isolate a cellular
polypeptide to which a viral virulence polypeptide binds, using
methods described in further detail herein, which may include steps
similar to a tandem affinity purification (TAP) tag method, wherein
the fusion protein is expressed recombinantly in a cell with which
a target cellular polypeptide is associated (see, e.g., Rigaut et
al., Nat. Biotech. 17:1030-32 (1999); Puig et al., supra; Tasto et
al., Yeast 18:657-62 (2001); Gould et al., Methods 33:239-44
(2004)). Fusion proteins comprising the affinity tags described
herein are particularly useful for isolating and purifying a
cellular polypeptide to which a viral polypeptide binds.
[0123] A fusion protein comprising a viral virulence polypeptide
and an affinity tag may be prepared by recombinant expression
methods described herein and/or described in the art, wherein the
fusion protein comprising the viral polypeptide, is expressed from
a polynucleotide that is operatively linked to an expression
control sequence (e.g., a promoter, enhancer, transcription
initiation site) in a nucleic acid expression construct. The fusion
protein as described in greater detail herein may be expressed
using vectors and constructs, particularly recombinant expression
constructs, that include any polynucleotide encoding such
polypeptides. The nucleotide sequence of such polynucleotides that
encode the viral polypeptides and polypeptide tags can be readily
determined by a person skilled in the molecular biology art on the
basis of the amino acid sequence of the viral polypeptides and
polypeptide tags disclosed herein and known in the art, given the
art accepted and well characterized genetic code. Host cells may be
transfected, transformed, or transduced with vectors and/or
constructs to produce these polypeptides and fusion proteins, or
fragments or variants thereof, by recombinant techniques. Each of
the polypeptides and fusion polypeptides described herein can be
expressed in mammalian cells, yeast, bacteria, or other cells under
the control of appropriate promoters. Appropriate cloning and
expression vectors for use with prokaryotic and eukaryotic hosts
are described, for example, by Sambrook, et al., Molecular Cloning:
A Laboratory Manual, Third Edition, Cold Spring Harbor, N.Y.,
(2001). In particular embodiments, the host cell is a eukaryotic
cell, which is a mammalian cell, including, for example, a CV1/EBNA
cell, HEK293 cell, HEK293T cell, COS-7 cell, a CHO cell, and the
like.
[0124] A polynucleotide, nucleic acid, or nucleic acid molecule
refers to any of single-stranded or double-stranded
deoxyribonucleic acid (DNA) or ribonucleic acid (RNA)
polynucleotide, oligonucleotide, or fragment thereof.
Polynucleotides may be isolated from a biological source and/or may
be generated and amplified by standard molecular biology methods
practiced in the art, for cloning and amplification (such as the
polymerase chain reaction (PCR)). Polynucleotide fragments may be
obtained from a PCR product or from an isolated polynucleotide by
any of ligation, scission, endonuclease, and/or exonuclease
activity. Nucleic acids may be composed of monomers that are
naturally occurring nucleotides (such as deoxyribonucleotides and
ribonucleotides), analogs of naturally occurring nucleotides (e.g.,
.alpha.-enantiomeric forms of naturally-occurring nucleotides), or
a combination of both. Modified nucleotides can have modifications
in sugar moieties and/or in pyrimidine or purine base moieties.
[0125] In one embodiment, recombinant expression constructs
comprise a polynucleotide sequence that encodes a fusion protein
comprising a viral polypeptide that is fused in frame with an
affinity tag. As described herein the affinity tag may comprise at
least one, two, three, four, or more polypeptide tags and may
further comprise at least one or at least two protease recognition
sequences. As described herein, the recombinant expression
constructs also contain nucleotide sequences that encode spacer
peptides. When the amino acid sequence of a polypeptide is known,
such as the polypeptide tag sequences disclosed herein and used in
the art, a polynucleotide sequence that encodes such a polypeptide
may readily be designed and prepared according to standard
molecular biology knowledge (e.g., sequences of codons for each
amino acid) and methods routinely practiced by a person skilled in
the art.
[0126] A recombinant construct may further comprise a signal
peptide sequence operatively linked and fused in frame with the
fusion protein. A signal peptide may be incorporated into the
recombinant expression construct to facilitate translocation of the
fusion protein as a secretory protein or a cell-surface protein
across intracellular membranes and to final localization. The
signal peptide sequence is located at the N-terminus of a fusion
protein and is typically 13-40 amino acids in length. Accordingly,
a signal peptide may be located at the amino terminus of the viral
polypeptide when the affinity tag is attached or fused to the
carboxy terminal end of the viral polypeptide, or the signal
peptide may be located at the amino terminus of the affinity tag of
the fusion polypeptide when the affinity tag is attached or fused
to the amino terminal end of the viral polypeptide.
[0127] An example of a signal peptide sequence that is fused in
frame to the fusion protein is a human growth hormone signal
peptide sequence. The recombinant construct therefore comprises a
nucleotide sequence that encodes the amino acid sequence
MATGSRTSLLLAFGLLCLPWLQEGSA (SEQ ID NO:12). The signal peptide
sequence may further comprise amino acids that are encoded by the
nucleotide sequences of restriction sites. The restriction sites
may be useful for cloning and subcloning of the different
polynucleotide sequences to construct a polynucleotide sequence
that encodes the fusion polypeptide. Depending on the site at which
a cellular enzyme cleaves the signal peptide from the mature fusion
protein, the amino acids encoded by the restriction site nucleotide
sequences may in whole or in part be attached at the amino terminal
end of the fusion protein. In certain embodiments, the recombinant
construct comprises a polynucleotide that has a nucleotide sequence
that corresponds to the restriction site of the restriction enzyme
Spe1 or Asp718, which nucleotide sequences encode the amino acids
Thr-Ser and Gly-Thr, respectively. Accordingly, a human growth
hormone signal peptide sequence further comprising nucleotides
corresponding to a Spe1 and an Asp718 restriction site comprises
the amino acid sequence MATGSRTSLLLAFGLLCLPWLQEGSATSGT (SEQ ID
NO:13). A person skilled in the art can readily determine which
restriction site nucleotide sequences encode amino acids and may
incorporate additional restriction sites or alternative restriction
sites at the carboxy terminal end of a signal sequence. Restriction
site nucleotide sequences may also be incorporated between the
viral polypeptide and the affinity tag and/or between polypeptide
tags of the affinity tag.
[0128] Generally, recombinant expression vectors include origins of
replication, selectable markers permitting transformation of the
host cell, for example, the ampicillin resistance gene of E. coli
and S. cerevisiae TRP1 gene, and a promoter derived from a highly
expressed gene to direct transcription of a downstream structural
sequence. Promoters can be derived from operons encoding glycolytic
enzymes such as 3-phosphoglycerate kinase (PGK), .alpha.-factor,
acid phosphatase, or heat shock proteins, among others. The
heterologous structural sequence is assembled in appropriate phase
with translation initiation and termination sequences. Vectors that
may be used and modified for expression of the fusion polypeptides
described herein that comprises a viral virulence polypeptide and
an affinity tag are available from commercial sources and include,
for example, pcDNA.TM.3.1 and related vectors (Invitrogen),
adenovirus vectors and adeno associated virus vectors (e.g., pAAV
vectors, Stratagene, La Jolla, Calif.) and retroviral vectors
including a Lentiviral vector system (e.g., pSL9).
[0129] Host cells containing the described recombinant expression
constructs may be genetically engineered either by stably
introducing or transiently introducing (transducing, transforming,
or transfecting) the vectors and/or expression constructs (for
example, a cloning vector, a shuttle vector, or an expression
construct). Vector constructs comprising cloned polynucleotide
sequences encoding a fusion protein described herein can be
introduced into cultured mammalian cells by, for example,
liposome-mediated transfection, calcium phosphate-mediated
transfection (Wigler et al., Cell 14:725, 1978; Corsaro and
Pearson, Somatic Cell Genetics 7:603, 1981; Graham and Van der Eb,
Virology 52:456, 1973), electroporation (Neumann et al., EMBO J.
1:841-845, 1982), or DEAE-dextran mediated transfection (Ausubel et
al. (eds.), Current Protocols in Molecular Biology, John Wiley and
Sons, Inc., NY, 1987); retroviral, adenoviral and protoplast
fusion-mediated transfection (see Sambrook et al., supra). To
identify cells that have been stably transfected with the vector
containing the cloned DNA, a selectable marker is generally
introduced into the cells along with the polynucleotide of
interest. Preferred selectable markers for use in cultured
mammalian cells include genes that confer resistance to drugs, such
as neomycin, hygromycin, and methotrexate. The selectable marker
may be an amplifiable selectable marker. Preferred amplifiable
selectable markers are the DHFR gene and the neomycin resistance
gene. Selectable markers are reviewed by Thilly (Mammalian Cell
Technology, Butterworth Publishers, Stoneham, Mass.).
[0130] The vector or construct may be in the form of a plasmid, a
viral particle, a phage, etc. The engineered host cells can be
cultured in conventional nutrient media modified as appropriate for
activating promoters, selecting transformants, or amplifying
particular genes or encoding-nucleotide sequences. Selection and
maintenance of culture conditions for particular host cells, such
as temperature, pH and the like, will be readily apparent to the
ordinarily skilled artisan. Preferably the host cell can be adapted
to sustained propagation in culture to yield a cell line according
to art-established methodologies. In certain embodiments, the cell
line is an immortal cell line, which refers to a cell line that can
be repeatedly (at least ten times while remaining viable) passaged
in culture following log-phase growth. In other embodiments the
host cell used to generate a cell line is a cell that is capable of
unregulated growth, such as a cancer cell, or a transformed cell,
or a malignant cell.
[0131] Useful bacterial expression constructs for expressing a
viral virulence polypeptide or fusion protein comprising the viral
polypeptide are constructed by inserting into an expression vector
a structural DNA sequence encoding a desired protein together with
suitable translation initiation and termination signals in operable
reading phase with a functional promoter. The construct may
comprise one or more phenotypic selectable markers and an origin of
replication to ensure maintenance of the vector construct and, if
desirable, to provide amplification within the host. Suitable
prokaryotic hosts for transformation include E. coli, Bacillus
subtilis, Salmonella typhimurium and various species within the
genera Pseudomonas, Streptomyces, and Staphylococcus, although
others may also be employed as a matter of choice. Any other
plasmid or vector may be used as long as they are replicable and
viable in the host. Thus, for example, the polynucleotides as
described herein may be included in any one of a variety of
expression vector constructs as a recombinant expression construct
for expressing a polypeptide. Such vectors and constructs include
chromosomal, nonchromosomal, and synthetic DNA sequences, e.g.,
bacterial plasmids; phage DNA; baculovirus; yeast plasmids; vectors
derived from combinations of plasmids and phage DNA; viral DNA,
such as vaccinia, adenovirus, fowl pox virus, and pseudorabies.
However, any other vector may be used for preparation of a
recombinant expression construct as long as it is replicable and
viable in the host.
[0132] The appropriate DNA sequence(s) may be inserted into the
vector by a variety of procedures. In general, the DNA sequence is
inserted into an appropriate restriction endonuclease site(s) by
procedures known in the art. Standard techniques for cloning, DNA
isolation, amplification and purification, for enzymatic reactions
involving DNA ligase, DNA polymerase, restriction endonucleases and
the like, and various separation techniques are those known and
commonly employed by those skilled in the art. Numerous standard
techniques are described, for example, in Ausubel et al. (Current
Protocols in Molecular Biology (Greene Publ. Assoc. Inc. & John
Wiley & Sons, Inc., 1993)); Sambrook et al. (Molecular Cloning:
A Laboratory Manual, 3rd Ed., (Cold Spring Harbor Laboratory
2001)); Maniatis et al. (Molecular Cloning, (Cold Spring Harbor
Laboratory 1982)), and elsewhere.
[0133] The polynucleotide sequence encoding a polypeptide in the
expression vector is operatively linked to at least one appropriate
expression control sequences (e.g., a promoter or a regulated
promoter) to direct mRNA synthesis. Representative examples of such
expression control sequences include LTR or SV40 promoter, the E.
coli lac or trp, the phage lambda P.sub.L promoter, and other
promoters known to control expression of genes in prokaryotic or
eukaryotic cells or their viruses. Promoter regions can be selected
from any desired gene using CAT (chloramphenicol transferase)
vectors or other vectors with selectable markers. Particular
bacterial promoters include lad, lacZ, T3, T5, T7, gpt, lambda
P.sub.R, P.sub.L, and trp. Eukaryotic promoters include CMV
immediate early, HSV thymidine kinase, early and late SV40, LTRs
from retroviruses, and mouse metallothionein-I. Selection of the
appropriate vector and promoter and preparation of certain
recombinant expression constructs comprising at least one promoter
or regulated promoter operatively linked to a polynucleotide
described herein is well within the level of ordinary skill in the
art.
[0134] Design and selection of inducible, regulated promoters
and/or tightly regulated promoters are known in the art and will
depend on the particular host cell and expression system. The pBAD
Expression System (Invitrogen Life Technologies, Carlsbad, Calif.)
is an example of a tightly regulated expression system that uses
the E. coli arabinose operon (P.sub.BAD or P.sub.ARA) (see Guzman
et al., J. Bacteriology 177:4121-30 (1995); Smith et al., J. Biol.
Chem. 253:6931-33 (1978); Hirsh et al., Cell 11:545-50 (1977)),
which controls the arabinose metabolic pathway. A variety of
vectors employing this system are commercially available. Other
examples of tightly regulated promoter-driven expression systems
include PET Expression Systems (see U.S. Pat. No. 4,952,496)
available from Stratagene (La Jolla, Calif.) or tet-regulated
expression systems (Gossen et al., Proc. Natl. Acad. Sci. USA
89:5547-51 (1992); Gossen et al., Science 268:1766-69 (1995)). The
pLP-TRE2 Acceptor Vector (BD Biosciences Clontech, Palo Alto,
Calif.) is designed for use with CLONTECH's Creator.TM. Cloning
Kits to rapidly generate a tetracycline-regulated expression
construct for tightly controlled, inducible expression of a gene of
interest using the site-specific Cre-lox recombination system (see,
e.g., Sauer, Methods 14:381-92 (1998); Furth, J. Mamm. Gland Biol.
Neoplas. 2:373 (1997)), which may also be employed for host cell
immortalization (see, e.g., Cascio, Artif. Organs 25:529
(2001)).
[0135] The vector may be a viral vector such as a retroviral
vector. For example, retroviruses from which the retroviral plasmid
vectors may be derived include, but are not limited to, Moloney
Murine Leukemia Virus, spleen necrosis virus, Rous Sarcoma Virus,
Harvey Sarcoma virus, avian leukosis virus, gibbon ape leukemia
virus, human immunodeficiency virus, adenovirus, Myeloproliferative
Sarcoma Virus, and mammary tumor virus. A viral vector also
includes one or more promoters. Suitable promoters that may be
employed include, but are not limited to, the retroviral LTR; the
SV40 promoter; and the human cytomegalovirus (CMV) promoter
described in Miller et al., Biotechniques 7:980-990 (1989), or any
other promoter (e.g., eukaryotic cellular promoters including, for
example, the histone, pol III, and .beta.-actin promoters). Other
viral promoters that may be employed include, but are not limited
to, adenovirus promoters, thymidine kinase (TK) promoters, and B19
parvovirus promoters.
[0136] The retroviral plasmid vector is employed to transduce
packaging cell lines (e.g., PE501, PA317, .psi.-2, .psi.-AM, PA12,
T19-14X, VT-19-17-H2, .psi.CRE, .psi.CRIP, GP+E-86, GP+envAm12,
DAN; see also, e.g., Miller, Human Gene Therapy, 1:5-14 (1990)) to
form producer cell lines. The vector may transduce the packaging
cells through any means known in the art, such as, for example,
electroporation, the use of liposomes, and calcium phosphate
precipitation. The producer cell line generates infectious
retroviral vector particles that include the nucleic acid
sequence(s) encoding the polypeptides or fusion proteins described
herein. Such retroviral vector particles then may be employed, to
transduce eukaryotic cells, either in vitro or in vivo. Eukaryotic
cells that may be transduced include, for example, embryonic stem
cells, embryonic carcinoma cells, hematopoietic stem cells,
hepatocytes, fibroblasts, myoblasts, keratinocytes, endothelial
cells, bronchial epithelial cells, and other culture-adapted cell
lines.
[0137] As another example, host cells transduced by a recombinant
viral construct directing the expression of polypeptides or fusion
proteins may produce viral particles containing expressed
polypeptides or fusion proteins that are derived from portions of a
host cell membrane incorporated by the viral particles during viral
budding. The polypeptide-encoding nucleic acid sequences may be
cloned into a baculovirus shuttle vector, which is then recombined
with a baculovirus to generate a recombinant baculovirus expression
construct that is used to infect, for example, Sf9 host cells (see,
e.g., Baculovirus Expression Protocols, Methods in Molecular
Biology Vol. 39, Richardson, Ed. (Human Press 1995); Piwnica-Worms,
"Expression of Proteins in Insect Cells Using Baculoviral Vectors,"
Section II, Chapter 16 in Short Protocols in Molecular Biology,
2.sup.nd Ed., Ausubel et al., eds., (John Wiley & Sons 1992),
pages 16-32 to 16-48).
[0138] Methods for Identifying a Target Cellular Polypeptide
[0139] In one embodiment, a method is provided for identifying a
cellular polypeptide to which a viral virulence polypeptide binds
comprising contacting a cell, or a fraction or a supernatant of the
cell, and a fusion protein comprising a viral polypeptide fused to
an affinity tag, under conditions and for a time sufficient that
permit a viral polypeptide moiety of the fusion protein to interact
with a polypeptide associated with the cell, or the fraction or the
supernatant of the cell, to provide a fusion protein:cellular
polypeptide complex, wherein the viral polypeptide has at least one
virulence trait. The step of contacting (i.e., mixing, combining,
or in some manner permitting interaction between the cell and
fusion protein) may be performed in any suitable reaction vessel
used in the art. The fusion protein:cellular polypeptide complex is
then isolated, and the amino acid sequence of the cellular
polypeptide determined by determining the amino acid sequence of
the entire polypeptide or of at least one cellular polypeptide
fragment (or a plurality of fragments), wherein each fragment
comprises at least eight amino acids (which includes any
polypeptide fragment having eight or more amino acids in any whole
integer amount), and thereby identifying a cellular polypeptide to
which a viral polypeptide binds. An exemplary technique or
combination of techniques and methods for identifying a cellular
polypeptide include tandem affinity purification (TAP) followed by
LC-MS/MS (liquid chromatography-tandem mass spectrometry), which
are practiced in the art and described herein.
[0140] As described herein, the viral virulence polypeptide is a
polypeptide from a virus that contributes to the virulence of the
virus in an infected host. At least one virulence trait of a viral
polypeptide may be known in the art. Alternatively, the methods
described herein may further comprise determining whether a viral
polypeptide has at least one trait that is a virulence trait as
described in detail herein. The polypeptide sequence of the viral
polypeptide may also be known in the art or may be determined, for
example, according to the methods described herein. Similarly, a
trait of a viral polypeptide that is described herein as a
virulence trait may be known in the art and that the trait relates
to the virulence of the virus, may or may not be apparent to a
person skilled in the art. Procedures and techniques for
determining whether a viral polypeptide has one or more virulence
traits may be performed according to methods described herein and
with which a skilled artisan will be familiar. For example, prior
to the step of contacting a cell, a cell supernatant, or a cell
fraction, the polynucleotide sequence of a viral genome may be
inspected or scanned as described herein to identify at least one
open reading frame encoding at least forty amino acids (which may
include a signal peptide sequence). Alternatively, or in addition,
the method may further comprise any one or more of the following:
determining that expression of a mutant viral polypeptide in a cell
infected by the virus correlates with a decrease in virulence of
the virus; determining that absence of expression of the viral
polypeptide in a cell infected by the virus correlates with a
decrease in virulence of the virus; determining that the viral
polypeptide is secreted by a cell infected with the virus, is
associated with a cellular membrane, or is intracellular; and
determining that the polynucleotide sequence in the virus genome is
located in a genomic region that encodes at least one other viral
polypeptide that is a viral virulence factor, wherein the region is
at the 5' terminal end or the 3' terminal end of the virus
genome.
[0141] A fusion protein comprising the viral polypeptide and an
affinity tag may be prepared synthetically or recombinantly
according to methods described herein. Contacting the fusion
protein with a cell, a fraction of the cell, or a supernatant of
the cell, includes permitting the cell or fraction or supernatant
to interact, such as mixing or combining together in some manner
the cell, cell fraction, or cell supernatant and the fusion
polypeptide. A cellular polypeptide of a cell, which may be
expressed on the cell surface of a cell or is secreted from the
cell, and thus can be obtained in a cell supernatant, may be
contacted with a fusion protein described herein. In a specific
embodiment, a cell supernatant, such as conditioned media (i.e.,
media collected from a plurality of cells that have been cultured
for a time sufficient such that a cellular polypeptide is secreted,
or in some manner released, by the cell), is contacted with a
fusion polypeptide.
[0142] Contacting the cell, cell fraction, or cell supernatant and
the fusion polypeptide also includes contact when the fusion
protein is introduced into the cell. For example, a recombinant
expression construct comprising a polynucleotide sequence that
encodes the fusion protein may be introduced into the cell by a
transfection, transformation, or other method described herein and
practiced in the art such that the fusion protein is expressed by
the cell. Thus, the fusion protein is permitted to interact with a
cellular polypeptide that is intracellular, associated with a
membrane (either a membrane of a cellular organelle or the cellular
membrane), or that is secreted by the cell (and which may be
located in the supernatant of the cell). A polynucleotide encoding
the fusion protein may also be introduced into a cell as a "naked"
polynucleotide as described, for example, in Ulmer et al., Science
259:1745-49 (1993) and reviewed by Cohen, Science 259:1691-92
(1993). The uptake of a naked polynucleotide may be increased by
coating the polynucleotide onto biodegradable beads, which are
efficiently transported into the cells.
[0143] In particular embodiments, the fusion protein comprises a
signal peptide sequence such as the human growth hormone signal
peptide sequence that facilitates secretion of the fusion protein,
which more readily permits interaction between the fusion protein
and a cellular polypeptide that is a secreted cellular polypeptide.
Accordingly, for identifying a cellular polypeptide including a
polypeptide that is a secreted cellular polypeptide, the fusion
protein may comprise, for example, a growth hormone signal peptide
sequence fused in frame with the viral polypeptide, which is in
turn fused in frame with the affinity tag. In a specific
embodiment, the affinity tag comprises from the amino terminal end
of the affinity tag toward the carboxy terminal end, a
hemagglutinin peptide (e.g., YPYDVDYA (SEQ ID NO:1)), a calmodulin
binding polypeptide (e.g., KRRWKKNFIAVSAANRFKKISSSGAL (SEQ ID
NO:3)), a TEV protease recognition sequence, a streptavidin binding
peptide, and an immunoglobulin mutein Fc polypeptide (e.g., a human
IgG mutein Fc polypeptide). In another specific embodiment, the
affinity tag of the fusion protein comprises a hemagglutinin
peptide (e.g., YPYDVDYA (SEQ ID NO:1)), a calmodulin binding
polypeptide (e.g., KRRWKKNFIAVSAANRFKKISSSGAL (SEQ ID NO:3)), a
human rhinovirus 3C protease recognition sequence, and a
streptavidin binding peptide, and a second repeat of the
streptavidin binding peptide.
[0144] In certain embodiments, a cell is stimulated prior to, at
the same time, or after, a cell, a cell fraction or a cell
supernatant, is contacted with a fusion protein. As described
herein, a stimulus includes, for example, an antibody that
specifically binds to a cognate antigen (e.g., a cell surface
marker antigen or cell surface receptor) expressed by the cell; a
phorbol ester (e.g., PMA), and other mitogens; a cytokine; a
chemokine; and ionomycin; or a combination of at least two agents
(for example, PMA and ionomycin; PWM (pokeweed mitogen) and
insulin).
[0145] Also as described herein, a cell fraction such as a cell
lysate or cell extract that may be used in the methods described
herein. A cell fraction also includes a preparation of one or more
isolated organelles from a cell, which are described herein, and
also includes complex multi-molecular structures such as lipid
rafts and other trafficking and transport complexes. Cell
fractions, cell lysates, cell extracts, and isolated cell
organelles may be prepared according to methods and techniques
appropriate for a particular cell and may include one or a
combination of mechanical, physical, and chemical techniques with
which a skilled artisan is familiar. A cell supernatant, which
includes, for example, cellular washes, cell culture media, or
conditioned media (i.e., media from cells in culture that have been
propagated for a period of time sufficient for the cells to secrete
a cellular polypeptide to which a viral polypeptide binds), or any
other extracellular preparation, including a biological sample, may
be used in the methods described herein. As described herein, a
cell fraction or cell supernatant of a cell may be contacted with a
fusion polypeptide that is expressed by the cell. Alternatively, a
cell supernatant or cell fraction may be obtained and then
contacted with a fusion polypeptide.
[0146] Interaction of the fusion protein and the cell, cell
supernatant, or cell fraction permits the viral polypeptide moiety
of the fusion protein to interact with or bind to a cellular
polypeptide, which is a cellular polypeptide that the viral
polypeptide interacts with during infection of a host by the virus
that encodes and expresses the viral polypeptide. The interaction
occurs under suitable conditions (e.g., temperature, atmosphere,
nutrients, buffers, pH, etc.) and for a time sufficient to permit
formation of a fusion protein:cellular polypeptide complex that can
be isolated. As described herein, the fusion protein comprises an
affinity tag, which comprises a detectable moiety and/or at least
one polypeptide tag.
[0147] In one embodiment, the fusion protein:cellular polypeptide
complex may be isolated from the cell, cell fraction, or from a
cell supernatant by contacting the complex with a cognate ligand of
at least one polypeptide tag of the affinity tag. Examples of
polypeptide tags and cognate ligands are described in detail
herein. The cognate ligand may be bound to a solid support, such as
a plastic, glass, or metal surface, including but not limited to a
slide, bead, plate (including a multi-well plate), nanoparticle, or
other matrix, including polymeric matrices, negatively charged
matrices, and positively charged matrices. For isolating the fusion
protein:cellular polypeptide complex from the cell, cell fraction,
or cell supernatant, the complex is permitted to interact with the
cognate ligand under suitable conditions and for a time sufficient
for a polypeptide tag and its cognate ligand to bind, thus forming
a cognate ligand:fusion protein:cellular polypeptide complex. The
fusion protein:cellular polypeptide complex may then be isolated by
disrupting the interaction between the cognate ligand and the
polypeptide tag and separating the complex from the cognate ligand.
Persons skilled in the art can readily determine the suitable
conditions for forming a complex formation and for disrupting a
complex (e.g., pH; buffer; absence or presence of ions, salts,
cations, or chelating agents; etc.) and time sufficient for
formation of a complex between the cognate ligand and the
polypeptide tag.
[0148] Alternatively, when the affinity tag comprises a protease
recognition sequence, the respective protease may be contacted with
the cognate ligand:fusion protein:cellular polypeptide complex
under conditions and for a time sufficient for the protease to
cleave the fusion polypeptide to release a cleaved fusion
protein:cellular polypeptide complex. Certain polypeptide tags
exhibit a high affinity for the respective cognate ligand (e.g.,
SBP peptide (SEQ ID NO:6) or tandem SBP tags (SEQ ID NO:8); Fc
polypeptide-cognate ligand interactions; or certain
antibody-polypeptide tag specific binding) such that disruption of
the interaction between the polypeptide tag and cognate ligand
requires conditions (e.g., low pH, chaotropic agents, high salt,
etc.) that could (but not necessarily) adversely affect the
structure of the cellular polypeptide or disrupt the interaction
between the viral polypeptide and cellular polypeptide.
Incorporation of at least one protease recognition sequence into
the affinity tag provides the capability to separate and isolate
the fusion protein (or a portion thereof):cellular polypeptide
complex, thus minimizing possible adverse alteration of the
cellular polypeptide and/or the viral polypeptide:cellular
polypeptide interaction. In addition, incorporation of at least one
protease recognition sequence into the affinity tag and subsequent
proteolytic cleavage of a portion of the affinity tag from the
fusion protein:cellular polypeptide complex increases the mass
ratio of the cellular polypeptide to fusion protein and thus
increases the sensitivity of detection and analysis of the cellular
polypeptide by methods described herein, such as LC-MS/MS (liquid
chromatography followed by tandem mass spectrometry). A cellular
polypeptide:fusion protein complex may be identified and detected
by methods described in further detail herein and known in the art
including, for example, surface plasmon resonance (SPR);
Fluorescence Activated Cell Sorting (FACS) (e.g., for identifying a
cell surface expressed polypeptide); and kinetic protein
interaction measuring devices such as a KinExA.TM. device (e.g.,
Sapidyne Instruments, Inc., Boise, Id.) (e.g., for identifying a
cellular polypeptide in a cell supernatant such as conditioned
media), which methods may further comprise use of radioactive
labeling (e.g., S.sup.35, P.sup.32, and the like).
[0149] Subsequent to isolation of a fusion protein:cellular
polypeptide complex from the cell, cell fraction, or cell
supernatant, the cellular polypeptide is identified. In certain
embodiments, the identity of the cellular polypeptide may be
determined by analyzing the full-length polypeptide, for example,
as described herein, by performing immunoassays using antibodies
that have been characterized by their ability to specifically bind
to a particular cellular polypeptide. The identity of the cellular
polypeptide (either in the complex or isolated from the complex)
can be determined by methods, for example, immunochemical methods,
such as immunoblotting, immunoassay (e.g., an ELISA),
radioimmunoassay, competition assays, and other assays practiced in
the art. An immunoassay may be performed in a matrix or multi-plex
or high throughput method in which the cellular polypeptide is
contacted with numerous antibodies with specificities for different
cellular polypeptides. Other exemplary procedures for identifying
longer cellular polypeptide fragments or full-length polypeptides
include MALDI-TOF, which is routinely practiced in the art and
described herein. As described herein, such methods may be used for
analyzing the full-length cellular polypeptide or fragments
thereof, which may be fragments at least 6 amino acids or at least
any number of amino acids between 6 amino acids and the number of
amino acids that comprises the full-length of the polypeptide.
[0150] In other embodiments, the amino acid sequence of the
cellular polypeptide or of at least one peptide thereof, can be
determined. In certain embodiments, the cellular polypeptide may be
isolated from the complex. The amino acid sequence of the cellular
polypeptide or a peptide thereof may be determined by any one of a
number of methods practiced by a person skilled in the art and
described herein. For example, partial hydrolysis of a cellular
polypeptide will generate peptides for which the amino acid
sequence can be determined. Partial hydrolysis may be performed by
chemical methods or by enzymatic methods. Compounds used for
partial chemical hydrolysis of proteins include mild acid (e.g.,
formic acid at 40.degree. C.) (specificity: Asp-Pro); hydroxylamine
(specificity: Asn:Gly); cyanogens bromide (specificity: carboxyl
side of Met); iodosobenzoic acid (specificity: carboxyl side of
Trp); and 2-nitro-5-thiocyanobenzoate followed by alkali treatment
(specificity: amine side of Cys). Proteolytic enzymes that are
useful for generating peptide fragments that can be sequenced
include for example, trypsin (specificity: carboxyl side of Arg and
Lys); chymotrypsin (specificity: carboxyl side of Tyr, Phe, and
Trp); elastase (specificity: carboxyl side of Ala and Gly); ficin
(specificity: uncharged, aromatic amino acids); papain (carboxyl
side of Arg, Lys, and Phe); pepsin (specificity: carboxyl side of
Phe and Leu; non-polar pairs); thermolysin (specificity: amine side
of Leu and Phe; non-polar residues); and thrombin (specificity:
carboxyl side of Arg). Proteases that are particularly useful to
generate peptides that will be subjected to mass spectrometry for
amino acid sequence analysis include trypsin, and also include
proteases called Asp-N (specificity: amine side of Asp and Cys),
Glu-C (specificity: carboxyl side of Glu and Asp), Lys-C
(specificity: carboxyl side of Lys), and Arg-C (specificity:
carboxyl side of Arg), which are available commercially (see, e.g.,
Sigma Aldrich, St. Louis, Mo.). The three-letter and single letter
nomenclature for amino acids that is used herein conforms to the
art accepted standard for such abbreviations.
[0151] In certain embodiments, a cellular polypeptide is identified
by contacting the cellular polypeptide (or cellular
polypeptide:fusion protein complex) with a protease to generate
peptide fragments of at least 8-20 (and any integer between the
specifically named length range, e.g., 8, 10, 12, 14, 16, 18, or 20
amino acids) in length or longer. The cellular peptide fragments
may then be separated or isolated so that individual fragments may
be subjected to amino acid analysis. Separation methods include,
for example, any of a variety of liquid chromatography (LC)
techniques (high performance liquid chromatography, fast protein
liquid chromatography), including two-dimensional LC, other
chromatography methods (e.g., affinity, ion exchange, thin layer,
gel chromatography, etc.), and various electrophoresis techniques
that persons skilled in the art routinely use. The amino acid
sequence of the peptides that are obtained, for example, from the
LC step, may then be determined by a variety of sequencing methods
practiced in the art, for example, mass spectrometry, which
includes tandem mass spectrometry (MS/MS) that provides ion spectra
identification, which may further comprise induced fragmentation of
the parental peptide ions. The mass spectrometry data may then be
analyzed by software programs (e.g., SEQUEST (Sequest Technologies,
Inc. Lisle, Ill.); MASCOT (Matrix Science Ltd., London, UK); or
X!TANDEM (open source software, Global Proteome Machine
Organization); see also, e.g., Clauser et al., Anal. Chem. 71:2871
(1999)) that compare the data with information in databases
regarding known polypeptides. Ion spectra may also be identified by
comparing to databases of spectra that are associated with high
confidence to specific peptides using software packages such as
X!HUNTER.
[0152] Mass spectrometry methods include electrospray ionization,
which provides high sensitivity and is directly applicable to the
analysis of peptides and proteins. Multiple charging through the
use of electrospray ionization is sample dependent, but can extend
the upper mass limit that can be analyzed using a triple quadrupole
instrument into the 50-80 kDa range for some proteins. For mass
spectral analysis of peptides and proteins, preferably the sample
is soluble in dilute acid. In many instances, the sample undergoes
at least one round of HPLC. A variety of information about the
polypeptide/peptides can be obtained through the use of
electrospray-mass spectrometry on a triple quadrupole instrument.
These include molecular mass measurement, assessment of chemical
modifications through mass increases, daughter ion scans (for
sequence analysis), precursor ion scans, constant mass difference
scans and selected ion monitoring.
[0153] For analysis of peptides, triple quadrupole technology is
used for performing collision-induced dissociation (CID) of the ion
for sequence/structural information. The maximum number of residues
that can be sequenced is usually about 20 amino acids. Sequence
analysis from larger peptides/proteins usually requires a protease
digestion step before analysis.
[0154] For example, LC-MS/MS may be performed using an ion trap
mass spectrometer (such as LC/MS/MS LCQ Deca XP (ThermoFinnigan,
Thermo Electron Corp., Waltham, Mass.); QSTAR.RTM. Elite LC/MS/MS
System, Applied Biosystems/MDS Sciex) to identify a target cellular
polypeptide on the basis of the amino acid sequence of fragments of
the polypeptide (MS/MS sequence). The mass spectrometer can be
linked to an LC system, such as an HPLC system. Subsequent to
protease digestion of the target cellular polypeptide, for example,
after digestion with trypsin, the peptides are injected onto a
reversed phase column and separated based on their hydrophobicity.
Peptides desorbed from the column are eluted directly into the mass
spectrometer. In the ion trap, the mass of the intact peptides is
measured. Each peptide is then in turn isolated in the trap, and
the collision energy is increased, which fragments the peptide,
providing an MS/MS spectrum that represents the sequence of the
peptide. The MS/MS spectra are subjected to search against a
database (protein, DNA, or EST) to identify a peptide with the
corresponding intact mass and fragment masses. Typically, protein
identification software assigns a score for the match between the
measured MS/MS mass spectrum and the theoretical peptide mass
spectrum calculated from proteins in the database as described
above. Other exemplary methods are described in U.S. Pat. No.
6,829,539 and U.S. Pat. No. 6,908,740. See also, for example, Lin
et al., J. Biomol. Techniques 14:149-55 (2003); Tomlinson et al.,
Rapid Commun. Mass Spectrom. 17:909-16 (2003); Yi et al., Rapid
Commun. Mass Spectrom. 17:2093-98 (2003)).
[0155] Other mass spectrometry methods that may be used for
determining the identity of a target cellular polypeptide include
matrix-assisted laser desorption ionization time-of-flight
(MALDI-TOF), MALDI-TOF-MS. Procedures are available in the art for
analyzing peptides from proteins that are subjected to protease
digest and sodium dodecyl sulfate (SDS) gel electrophoresis (see,
e.g., Egelhofer et al., Anal. Chem. 74:1760-71 (2002); Cohen et
al., Anal. Biochem. 247:257-67 (1997); Cottrell et al., Protein and
peptide analysis by mass spectrometry. J. R. Chapman. Totowa, N.J.,
Humana Press. 61:67-82 (1996); Fernandez et al., Electrophoresis
19:1036-45 (1998) Jensen et al., Proteins Suppl. 2: 74-79 (1998)).
See also, for example, Andersen et al., Nat. Biotechnol. 14: 449-57
(1996); Chapman, Protein and peptide analysis by mass spectrometry
C. J. R. Totowa, N.J., Humana Press. 61:9-28 (1996); Gillece-Castro
et al., Methods Enzymol. 271:427-48 (1996); Hillenkamp et al.,
Methods Enzymol. 193: 280-95 (1990); Katta et al., Anal. Chem.
70:4410-6 (1998); Patterson et al., Anal. Chem. 67:3971-78 (1995);
Wang et al., Protein and peptide analysis by mass spectrometry J.
R. Chapman. Totowa, N.J., Humana Press. 61:161-70 (1996); Oliver et
al., Methods Mol. Biol. 61:295-309 (1996); Covey, Methods Mol.
Biol. 61:83-99 (1996); Ducret et al., Protein Sci. 7:706-19 (1998);
Fearnley et al., Biochem. Soc. Trans. 24:912-7 (1996); Yates,
Methods Enzymol. 271:351-77 (1996)).
[0156] Additional methods known in the art for determining the
amino acids sequence of a polypeptide or a peptide thereof may be
used. Such methods include, for example, N-terminal group analysis
using Edmund degradation that may be used in conjunction with
aminopeptidase M cleavage; C-terminal analysis; and enzymatic
C-terminal amino acid cleavage using any one of several
carboxypeptidase enzymes (e.g., carboxypeptidase C,
carboxypeptidase Y).
[0157] In certain embodiments, methods for identifying a cellular
polypeptide to which a viral polypeptide (i.e., a viral virulence
polypeptide that is a viral polypeptide that exhibits at least one
virulence trait) interacts and which cellular polypeptide may be
useful as a therapeutic target further comprise identifying a cell
type that comprises the cellular polypeptide. A fusion protein
comprising a viral polypeptide and an affinity tag can be contacted
with a biological sample that comprises at least one cell, or a
fraction of the cell or a supernatant of a cell, under conditions
and for a time sufficient to permit the viral polypeptide moiety of
the fusion protein to interact with the at least one cell, or the
cell fraction or the cell supernatant. The level of binding of the
fusion protein to the cell (or cell fraction or cell supernatant)
can be determined, which indicates the presence or absence of
binding of the fusion protein to the biological sample. The cell
may then be isolated and characterized, thus identifying at least
one cell type that comprises a cellular polypeptide to which the
viral polypeptide binds. In certain embodiments, the affinity tag
comprises a detectable moiety, for example, a fluorophore, a
radionuclide, an enzyme, or biotin that is useful for isolating the
fusion protein:cell complex and/or characterizing a cell type.
[0158] A biological sample, such as for example, blood, bone
marrow, various tissue samples, may comprise different types of
cells. A type of cell (or cell type) as referred to herein includes
cells of different lineages, such as a hematopoietic cell and a
neuronal cell, for example, and also refers to different types of
cells that are more highly related, for example, the various types
of immune cells (T cells, B cells, natural killer cells,
macrophages, etc.). Types of cells may be distinguished and
characterized according to cell surface antigen expression,
morphology, response to stimuli and other features, which can be
readily accomplished using standard reagents and methods (e.g.,
immunoassays, microscopy, and bioactivity assays).
[0159] In one embodiment, a method is provided for identifying a
cellular polypeptide to which a viral virulence polypeptide binds
that comprises contacting (mixing, combining, or in some manner
permitting interaction, including expression of the fusion protein
in the cell) a cell, or a fraction or a supernatant of the cell,
and a fusion protein comprising a viral polypeptide (that has at
least one virulence trait) fused to an affinity tag, under
conditions and for a time sufficient that permit a viral
polypeptide moiety of the fusion protein to interact with a
polypeptide associated with the cell, or the fraction or the
supernatant of the cell, to provide a fusion protein:cellular
polypeptide complex. In certain embodiments, the affinity tag
comprises at least a first polypeptide tag and a second polypeptide
tag, and may further comprise at least one or two additional
polypeptide tags, and also comprises at least one protease
recognition sequence. The fusion protein:cellular polypeptide
complex may then be isolated by contacting the fusion
protein:cellular polypeptide complex and a first cognate ligand of
the first polypeptide tag under conditions and for a time
sufficient to permit the affinity tag moiety of the fusion protein
to interact with the first cognate ligand to provide a first
cognate ligand:fusion protein:cellular polypeptide complex. The
first cognate ligand:fusion protein:cellular polypeptide complex
may then be contacted, mixed, or combined with a protease capable
of cleaving the fusion protein at or near the protease recognition
sequence to provide a cleaved fusion protein:cellular polypeptide
complex. In a subsequent step, the cleaved fusion protein:cellular
polypeptide complex is contacted (mixed, combined, or in some
manner permitted to interact) with a second cognate ligand that
specifically binds to the second polypeptide tag, under conditions
and for a time sufficient that permit the second cognate ligand and
the cleaved fusion protein:cellular polypeptide complex to interact
to form a second cognate ligand:cleaved fusion protein:cellular
polypeptide complex. The cleaved fusion protein:cellular
polypeptide complex may then be isolated from the second cognate
ligand:cleaved fusion protein:cellular polypeptide complex, and the
amino acid sequence of the cellular polypeptide or of at least one
polypeptide fragment of the cellular polypeptide, wherein the at
least one polypeptide fragment comprises at least eight amino acids
(or at least 10, 12, 14, 16, 18, or 20 amino acids in length or
longer) is determined.
[0160] Cellular Polypeptides and Agents
[0161] Also provided herein are cellular polypeptides that bind to
viral polypeptides. As described herein a cellular polypeptide can
be isolated according to a method comprising identifying in the
genome of a virus, a polynucleotide sequence that encodes a viral
polypeptide, which viral polypeptide exhibits at least one
virulence trait as described herein, including comprising at least
40 amino acids (which may include a signal peptide sequence. In
certain embodiments, a fusion protein comprising the viral
polypeptide fused or in some manner attached to an affinity tag
sequence is produced and then contacted with a cell, or a fraction
of the cell or a supernatant of the cell, under conditions and for
a time sufficient that permit the viral polypeptide moiety of the
fusion protein to interact with a polypeptide present in the cell,
or the fraction of the cell or the supernatant of the cell, to
provide a fusion protein:cellular polypeptide complex. As described
in detail herein the fusion protein:cellular polypeptide complex is
isolated and the amino acid sequence of the cellular polypeptide or
of at least one fragment of the polypeptide, wherein the fragment
comprises at least eight amino acids (or at least 10, 12, 14, 16,
18, or 20 amino acids in length or longer) is determined according
to the methods described herein. As described herein, the viral
polypeptide may be encoded by the genome (RNA or DNA) of a virus,
including large DNA genome viruses, such as poxviruses,
adenoviruses, and herpesviruses, and including the genome of any
other virus described herein or known in the art or a genome that
becomes known or available.
[0162] Cellular polypeptides that are identified by the methods
described herein include but are not limited to cell surface
antigens, cell surface receptors, cytokines, chemokines, cytokine
or chemokine binding proteins, intracellular signaling
polypeptides, or substrates of cell surface receptors or signaling
molecules. Exemplary cellular polypeptides that bind to a viral
polypeptide include receptor-like protein tyrosine phosphatases
(RPTP) (e.g., leukocyte common antigen related protein (LAR),
RPTP-.sigma., and RPTP-.delta.) (see, e.g., U.S. Pat. No.
6,852,486; International Patent Application Publication WO
98/37217; Ng et al., J. Gen. Virol. 82:2095-105 (2001); U.S. Ser.
No. 60/721,876). The viral polypeptide A41L that binds to the RPTPs
is present in several different poxviruses, including Cowpox virus
(CPV), vaccinia virus (strains Copenhagen, Ankara, Tian Tan and WR)
and variola virus (including strains Harvey, India-1967 and
Garcia-1966). Binding of A41L to LAR, RPTP-.delta., and/or
RPTP-.sigma. alters at least one biological function of these
phosphatases, and as described herein the interaction between A41L
and LAR, RPTP-.delta., and/or RPTP-.sigma. expressed on the cell
surface of an immune cell alters (e.g., suppresses or enhances) the
immunoresponsiveness of the cell.
[0163] Alteration of a biological activity of a cell as a result of
the interaction between a viral polypeptide and a cellular
polypeptide (for example, interaction between the viral polypeptide
A41L and a cellular RPTP, which alters the immunoresponsiveness of
an immune cell) may also be effected by a bioactive agent (compound
or molecule) in a manner similar to the viral polypeptide.
Bioactive agents include, for example, small molecules, nucleic
acids (such as aptamers, siRNAs, antisense nucleic acids),
antibodies and fragments thereof, and fusion proteins (such as
peptide-Fc fusion proteins and cell polypeptide domains or
fragments (at least 8, 10, 12, 14, 16, 18, 20, 25, or 30 amino
acids) that are fused to other moieties such as an immunoglobulin
Fc polypeptide). An agent may interact with and bind to at least
one cellular polypeptide at a location on the cellular polypeptide
that is the same location or proximal to the same location as where
the viral polypeptide binds. Alternatively, alteration of at least
one biological function by an agent in a manner similar to the
effect of a viral polypeptide may result from binding or
interaction of the agent with the cellular polypeptide at a
location distal from that at which the viral polypeptide binds.
Binding studies, including competitive binding assays, and
functional assays, which indicate the level of immunoresponsiveness
of a cell, may be performed according to methods described herein
and practiced in the art to determine and compare the capability
and level with which an agent binds to and affects the
immunoresponsiveness of an immune cell.
[0164] Provided herein are methods for identifying an agent for
treating a disease or disorder, such as a cardiovascular disease or
disorder, metabolic disease or disorder, or proliferative disease
or disorder, or immunological disease or disorder. An agent that is
useful for treating such diseases or disorders is capable of
altering at least one biological function of a cellular
polypeptide. The method comprises contacting (i.e., mixing,
combining, or in some manner permitting interaction) among (i) the
cellular polypeptide, or a cell comprising the cellular
polypeptide, or a cell supernatant or cell fraction; (ii) the viral
polypeptide (i.e., a viral polypeptide that is a viral virulence
polypeptide and that exhibits at least one virulence trait as
described herein); (iii) and a candidate agent, under conditions
and for a time sufficient that permit the cellular polypeptide and
the viral polypeptide to interact. The cellular polypeptide, or a
cell supernatant or cell fraction comprising the cellular
polypeptide is contacted (i.e., combined etc.) in a reaction vessel
with and without the candidate agent. The level of binding of the
cellular polypeptide to the viral polypeptide in the presence of
the candidate agent (a first level of binding) is then determined
and compared with the level of binding of the cellular polypeptide
to the viral polypeptide in the absence of the candidate agent (a
second level of binding) according to methods routinely practiced
by persons skilled in the art and described herein. The level of
binding of the viral polypeptide and cellular polypeptide without a
candidate agent may be determined at the same time as the level of
binding in the presence of the agent, or the level of binding of
the viral polypeptide and cellular polypeptide in the absence of
the candidate agent may be historically determined. A decrease in
the level of binding of the viral polypeptide to the cellular
polypeptide in the presence of the candidate agent compared to the
level of binding of the viral polypeptide to the cellular
polypeptide in the absence of the candidate agent indicates that
the agent inhibits (partially or in total) binding of the viral
polypeptide to the cellular polypeptide, thus identifying an agent
that is useful for treating the disease or disorder. Binding of the
agent to the cellular polypeptide thus affects at least one or more
of the biological functions (e.g., the immunoresponsiveness of an
immune cell) of the cellular polypeptide that is affected by
binding of the viral polypeptide to the cellular polypeptide.
Methods for identifying such an agent include any additional
appropriate controls with which a person skilled in the art is
familiar.
[0165] In a specific embodiment, a method is provided for
identifying an agent for treating an immunological disease or
disorder, comprising identifying a cellular polypeptide to which a
viral polypeptide binds according to the methods described herein,
wherein interaction between the cellular polypeptide and the viral
polypeptide (i.e., a viral polypeptide that is a viral virulence
polypeptide and that exhibits at least one virulence trait as
described herein) alters (i.e., increases or decreases in a
statistically significant or biologically significant manner)
immunoresponsiveness of an immune cell. The method comprises
contacting (i.e., mixing, combining, or in some manner permitting
interaction) among (i) the cellular polypeptide, or a cell
comprising the cellular polypeptide, or a cell supernatant or cell
fraction; (ii) the viral polypeptide; (iii) and a candidate agent,
under conditions and for a time sufficient that permit the cellular
polypeptide and the viral polypeptide to interact. As described
above, the level of binding of the viral polypeptide to the
cellular polypeptide in the presence of the candidate agent is
compared to a level of binding of the viral polypeptide to the
cellular polypeptide in the absence of the candidate agent. If the
level of binding of the viral polypeptide to the cellular
polypeptide in the presence of the candidate agent is decreased
compared with the level of binding of the of the viral polypeptide
to the cellular polypeptide in the absence of the agent, the agent
may be useful for treating an immunological disease or disorder. A
candidate agent that inhibits (or prevents, reduces, minimizes, or
abrogates) binding of the viral polypeptide to the cellular
polypeptide may mimic or act in the same manner as the viral
polypeptide, and thus affect at least one biological activity of
the cellular polypeptide. Such a biological activity includes but
is not limited to, altering immunoresponsiveness of an immune cell
(e.g., in certain embodiments, to suppress the immunoresponsiveness
of the immune cell and in certain other embodiments, to enhance
immunoresponsiveness of the immune cell) that comprises the
cellular polypeptide. Accordingly, such an agent is useful for
treating an immunological disease or disorder.
[0166] Immunoresponsiveness may be determined according to methods
practiced by a person skilled in the art such as measuring levels
of cytokines, proliferation, and stimulation. Immunoresponsiveness
of an immune cell may also be determined by evaluating changes in
cell adhesion and cell migration and by examining the tyrosine
phosphorylation pattern of cellular proteins, including but not
limited to cytoskeletal proteins and other proteins that affect
cell adhesion and migration.
[0167] The agents described herein may be useful for treating or
preventing, inhibiting, slowing the progression of, or reducing the
symptoms associated with, an immunological disease or disorder, a
cardiovascular disease or disorder, a metabolic disease or
disorder, or a proliferative disease or disorder. An immunological
disorder includes an inflammatory disease or disorder and an
autoimmune disease or disorder. While inflammation or an
inflammatory response is a host's normal and protective response to
an injury, inflammation can cause undesired damage. For example,
atherosclerosis is, at least in part, a pathological response to
arterial injury and the consequent inflammatory cascade.
[0168] An agent or cellular polypeptide (or fragment thereof) is
useful for treating a subject who has or who is suspected of having
a disorder or disease as described herein, such as an immunological
disease or disorder. A subject in need of such treatment may be a
human or may be a non-human primate or other animal (i.e.,
veterinary use) who has developed symptoms of an immunological
disease or who is at risk for developing an immunological disease.
Examples of non-human primates and other animals include but are
not limited to farm animals, pets, and zoo animals (e.g., horses,
cows, buffalo, llamas, goats, rabbits, cats, dogs, chimpanzees,
orangutans, gorillas, monkeys, elephants, bears, large cats,
etc.).
[0169] Examples of immunological disorders that may be treated with
an antibody or antigen-binding fragment thereof described herein
include but are not limited to multiple sclerosis, rheumatoid
arthritis, systemic lupus erythematosus (SLE), graft versus host
disease (GVHD), sepsis, diabetes, psoriasis, atherosclerosis,
Sjogren's syndrome, progressive systemic sclerosis, scleroderma,
acute coronary syndrome, ischemic reperfusion, Crohn's Disease,
endometriosis, glomerulonephritis, myasthenia gravis, idiopathic
pulmonary fibrosis, asthma, acute respiratory distress syndrome
(ARDS), vasculitis, or inflammatory autoimmune myositis and other
inflammatory and muscle degenerative diseases (e.g.,
dermatomyositis, polymyositis, juvenile dermatomyositis, inclusion
body myositis). ARDS, which may develop in adults and in children,
often follows a direct pulmonary or systemic insult (for example,
sepsis, pneumonia, aspiration) that injures the alveolar-capillary
unit. Several cytokines are associated with development of the
syndrome, including, for example, tumor necrosis factor-alpha
(TNF-.alpha.), interleukin-beta (IL-.beta.), IL-10, and soluble
intercellular adhesion molecule 1 (sICAM-1). The increased or
decreased level of these factors and cytokines in a biological
sample may be readily determined by methods and assays described
herein and practiced routinely in the art to monitor the acute
state and to monitor the effect of treatment.
[0170] A cardiovascular disease or disorder that may be treated,
which may include a disease and disorder that is also considered an
immunological disease/disorder, includes for example,
atherosclerosis, endocarditis, hypertension, or peripheral ischemic
disease. A metabolic disease or disorder includes diabetes,
obesity, and diseases and disorders associated with abnormal or
altered mitochondrial function.
[0171] As described herein an agent may be a compound referred to
as a small molecule. A small molecule agent may be provided as a
member of a "library" or collection of compounds, compositions, or
molecules. Small molecules typically have molecular weights less
than 10.sup.5 daltons, less than 10.sup.4 daltons, or less than
10.sup.3 daltons. For example, members of a library of test
compounds can be administered to a plurality of samples, each
containing at least one cellular polypeptide or source of a
cellular polypeptide as provided herein, and then the samples are
assayed for their capability to enhance or inhibit a biological
activity of the cellular polypeptide or for the capability to
inhibit or enhance interaction between a viral polypeptide and a
cellular polypeptide.
[0172] A bioactive agent that is used for altering the biological
function of a cell, such as immunoresponsiveness of an immune cell,
and that may be used for treating a disease or disorder is a
peptide-immunoglobulin (Ig) constant region fusion polypeptide,
which includes a peptide-IgFc fusion polypeptide. The peptide may
be any naturally occurring or recombinantly prepared molecule. A
peptide-Ig constant region fusion polypeptide, such as a
peptide-IgFc fusion polypeptide (also referred to in the art as a
peptibody (see, e.g., U.S. Pat. No. 6,660,843)), comprises a
biologically active peptide or polypeptide capable of altering the
activity of a protein of interest. The Fc polypeptide may also be a
mutein Fc polypeptide as described herein. Peptides that alter a
biological function of a cell, such as the immunoresponsiveness of
an immune cell, may be identified and isolated from combinatorial
libraries (see, e.g., International Patent Application Nos.
PCT/US91/08694 and PCT/US91/04666) and from phage display peptide
libraries (see, e.g., Scott et al., Science 249:386 (1990); Devlin
et al., Science 249:404 (1990); Cwirla et al., Science 276: 1696-99
(1997); U.S. Pat. No. 5,223,409; U.S. Pat. No. 5,733,731; U.S. Pat.
No. 5,498,530; U.S. Pat. No. 5,432,018; U.S. Pat. No. 5,338,665;
1994; U.S. Pat. No. 5,922,545; International Application
Publication Nos. WO 96/40987 and WO 98/15833).
[0173] In certain embodiments, polynucleotides and oligonucleotides
are provided that are complementary to at least a portion of a
sequence encoding a cellular polypeptide of interest (e.g., a short
interfering nucleic acid, an antisense polynucleotide, a ribozyme,
or a peptide nucleic acid) and that may be used to alter gene
and/or protein expression. As described herein, these
polynucleotides that specifically bind to or hybridize to nucleic
acid molecules that encode a cellular polypeptide may be prepared
using the nucleotide sequences available in the art. In another
embodiment, nucleic acid molecules such as aptamers that are not
sequence-specific may also be used to alter gene and/or protein
expression.
[0174] Antisense polynucleotides bind in a sequence-specific manner
to nucleic acids such as mRNA or DNA. Identification of
oligonucleotides and ribozymes for use as antisense agents and
identification of DNA encoding the genes for targeted delivery
involve methods well known in the art. For example, the desirable
properties, lengths, and other characteristics of such
oligonucleotides are well known. Antisense technology can be used
to control gene expression through interference with binding of
polymerases, transcription factors, or other regulatory molecules
(see Gee et al., In Huber and Can, Molecular and Immunologic
Approaches, Futura Publishing Co. (Mt. Kisco, N.Y.; 1994)).
[0175] Short interfering RNAs may be used for modulating
(decreasing or inhibiting) the expression of a gene encoding a
cellular polypeptide of interest. The disclosure herein relates to
compounds, compositions, and methods useful for modulating the
expression and activity of genes by RNA interference using small
nucleic acid molecules. In particular, small nucleic acid
molecules, such as short interfering RNA (siRNA), micro-RNA
(miRNA), and short hairpin RNA (shRNA) molecules may be used
according to the methods described herein to modulate the
expression of a cellular polypeptide of interest. A siRNA
polynucleotide preferably comprises a double-stranded RNA (dsRNA)
but may comprise a single-stranded RNA (see, e.g., Martinez et al.
Cell 110:563-74 (2002)). A siRNA polynucleotide may comprise other
naturally occurring, recombinant, or synthetic single-stranded or
double-stranded polymers of nucleotides (ribonucleotides or
deoxyribonucleotides or a combination of both) and/or nucleotide
analogues as provided herein and known and used by persons skilled
in the art.
[0176] At least one strand of a double-stranded siRNA
polynucleotide has at least one, and preferably two nucleotides
that "overhang" (i.e., that do not base pair with a complementary
base in the opposing strand) at the 3' end of either strand, or
preferably both strands, of the siRNA polynucleotide. Typically,
each strand of the siRNA polynucleotide duplex has a two-nucleotide
overhang at the 3' end. The two-nucleotide overhang may be a
thymidine dinucleotide (TT) or may comprise other bases, for
example, a TC dinucleotide or a TG dinucleotide, or any other
dinucleotide (see, e.g., International Patent Application
Publication No. WO 01/75164). Alternatively, the siRNA
polynucleotide may have blunt ends, that is, each nucleotide in one
strand of the duplex is perfectly complementary (e.g., by
Watson-Crick base-pairing) with a nucleotide of the opposite
strand.
[0177] In another embodiment, peptide nucleic acids (PNAs) can be
prepared by modifying the deoxyribose phosphate backbone of a
polynucleotide (or a portion thereof) that encodes a cellular
polypeptide of interest (see, e.g., Hyrup B. et al., Bioorganic
& Medicinal Chemistry 4:5-23) (1996)). The terms "peptide
nucleic acid" or "PNA" refers to a nucleic acid mimic, for example,
a DNA mimic, in which the deoxyribose phosphate backbone is
replaced by a pseudopeptide backbone wherein only the four natural
nucleobases are retained. The neutral backbone of a PNA has been
shown to allow for specific hybridization to DNA and RNA under
conditions of low ionic strength. The synthesis of PNA oligomers
can be performed using standard solid phase peptide synthesis
protocols (see, e.g., Hyrup B., supra; Perry-O'Keefe et al., Proc.
Natl. Acad. Sci. USA 93:14670-75 (1996)). A PNA molecule that is
specific for a cellular polypeptide can be used as an antisense or
anti-gene agent for sequence-specific modulation of gene expression
for example, by inducing transcription or translation arrest or by
inhibiting replication.
[0178] Aptamers are DNA or RNA molecules, generally
single-stranded, that have been selected from random pools based on
their ability to bind other molecules, including nucleic acids,
proteins, lipids, etc. Unlike antisense polynucleotides, short
interfering RNA (siRNA), or ribozymes that bind to a polynucleotide
that comprises a sequence that encodes a polypeptide of interest
and that alter transcription or translation, aptamers can target
and bind to polypeptides. Aptamers may be selected from random or
unmodified oligonucleotide libraries by their ability to bind to
specific targets (see, e.g., U.S. Pat. No. 6,867,289; U.S. Pat. No.
5,567,588). Aptamers have capacity to form a variety of two- and
three-dimensional structures and have sufficient chemical
versatility available within their monomers to act as ligands
(i.e., to form specific binding pairs) with virtually any chemical
compound, whether monomeric or polymeric. Molecules of any size or
composition can serve as targets. An iterative process of in vitro
selection may be used to enrich the library for species with high
affinity to the target. This process involves repetitive cycles of
incubation of the library with a desired target, separation of free
oligonucleotides from those bound to the target, and amplification
of the bound oligonucleotide subset, such as by using the
polymerase chain reaction (PCR). From the selected sub-population
of sequences that have high affinity for the target, a
sub-population may be subcloned and particular aptamers examined in
further detail to identify aptamers that alter a biological
function of the target (see, e.g., U.S. Pat. No. 6,699,843).
[0179] Aptamers may comprise any deoxyribonucleotide or
ribonucleotide or modifications of these bases, such as
deoxythiophosphosphate (or phosphorothioate), which have sulfur in
place of oxygen as one of the non-bridging ligands bound to the
phosphorus. Monothiophosphates .alpha.S have one sulfur atom and
are thus chiral around the phosphorus center. Dithiophosphates are
substituted at both oxygens and are thus achiral. Phosphorothioate
nucleotides are commercially available or can be synthesized by
several different methods known in the art.
[0180] An agent includes an antibody, or antigen binding fragment
thereof, that specifically binds to a cellular polypeptide of
interest. These specific antibodies may be polyclonal or
monoclonal, prepared by immunization of animals and subsequent
isolation of the antibody, or the antibodies may be recombinant
antibodies.
[0181] As used herein, an antibody is said to be "immunospecific,"
"specific for" or to "specifically bind" to a cellular polypeptide
of interest if it reacts at a detectable level with the cellular
polypeptide, preferably with an affinity constant, K.sub.a, of
greater than or equal to about 10.sup.4 M.sup.-1, or greater than
or equal to about 10.sup.5 M.sup.-1, greater than or equal to about
10.sup.6 M.sup.-1, greater than or equal to about 10.sup.7
M.sup.-1, or greater than or equal to 10.sup.8 M.sup.-1. Affinity
of an antibody for its cognate antigen is also commonly expressed
as a dissociation constant K.sub.D, and an anti-cellular
polypeptide antibody specifically binds to a cellular polypeptide
if it binds with a K.sub.D of less than or equal to 10.sup.-4 M,
less than or equal to about 10.sup.-5 M, less than or equal to
about 10.sup.-6 M, less than or equal to 10.sup.-7 M, or less than
or equal to 10.sup.-8 M. These definitions are also applicable to
other antigen-antibody interactions described herein, for example,
polypeptide tags and their cognate ligands.
[0182] Affinities of binding partners or antibodies can be readily
determined using conventional techniques, for example, those
described by Scatchard et al. (Ann. N.Y. Acad. Sci. USA 51:660
(1949)) and by surface plasmon resonance (SPR; BIAcore.TM.,
Biosensor, Piscataway, N.J.). For surface plasmon resonance, target
molecules are immobilized on a solid phase and exposed to ligands
in a mobile phase running along a flow cell. If ligand binding to
the immobilized target occurs, the local refractive index changes,
leading to a change in SPR angle, which can be monitored in real
time by detecting changes in the intensity of the reflected light.
The rates of change of the surface plasmon resonance signal can be
analyzed to yield apparent rate constants for the association and
dissociation phases of the binding reaction. The ratio of these
values gives the apparent equilibrium constant (affinity) (see,
e.g., Wolff et al., Cancer Res. 53:2560-2565 (1993)).
[0183] Binding properties of an antibody to a cellular polypeptide
described herein may generally be determined and assessed using
immunodetection methods including, for example, an enzyme-linked
immunosorbent assay (ELISA), immunoprecipitation, immunoblotting,
countercurrent immunoelectrophoresis, radioimmunoassays, dot blot
assays, inhibition or competition assays, and the like, which may
be readily performed by those having ordinary skill in the art
(see, e.g., U.S. Pat. Nos. 4,376,110 and 4,486,530; Harlow et al.,
Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory
(1988)). Immunoassay methods may include controls and procedures to
determine whether antibodies bind specifically to the cellular
polypeptide and do not recognize or cross-react with other cellular
polypeptides.
[0184] An antibody according may belong to any immunoglobulin
class, for example IgG, IgE, IgM, IgD, or IgA. It may be obtained
from or derived from an animal, for example, fowl (e.g., chicken)
and mammals, which include but are not limited to a mouse, rat,
hamster, rabbit, or other rodent, a cow, horse, sheep, goat, camel,
human, or other primate. The antibody may be an internalising
antibody. In one such technique, an animal is immunized with a
cellular polypeptide or fragment thereof (at least 6 amino acids)
as described herein as an antigen to generate polyclonal antisera.
Suitable animals include, for example, rabbits, sheep, goats, pigs,
cattle, and may also include smaller mammalian species, such as
mice, rats, and hamsters, or other species.
[0185] Antibodies may generally be prepared by any of a variety of
techniques known to persons having ordinary skill in the art. See,
e.g., Harlow et al., Antibodies: A Laboratory Manual, Cold Spring
Harbor Laboratory (1988); Peterson, ILAR J. 46:314-19 (2005)).
Polyclonal antibodies that bind specifically to a cellular
polypeptide can be prepared using methods described and practiced
by persons skilled in the art (see, for example, Green et al.,
"Production of Polyclonal Antisera," in Immunochemical Protocols
(Manson, ed.), pages 1-5 (Humana Press 1992); Harlow et al.,
Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory
(1988); Williams et al., "Expression of foreign proteins in E. coli
using plasmid vectors and purification of specific polyclonal
antibodies," in DNA Cloning 2: Expression Systems, 2nd Edition,
Glover et al. (eds.), page 15 (Oxford University Press 1995)).
Although polyclonal antibodies are typically raised in animals such
as rats, mice, rabbits, goats, cattle, or sheep, an anti-cellular
polypeptide antibody may also be obtained from a subhuman primate.
General techniques for raising diagnostically and therapeutically
useful antibodies in baboons may be found, for example, in
International Patent Application Publication No. WO 91/11465 (1991)
and in Losman et al., Int. J. Cancer 46:310, 1990.
[0186] Monoclonal antibodies that specifically bind to a cellular
polypeptide of interest and hybridomas, which are examples of
immortal eukaryotic cell lines, that produce monoclonal antibodies
having the desired binding specificity, may also be prepared, for
example, using the technique of Kohler and Milstein (Nature,
256:495-97 (1976), Eur. J. Immunol. 6:511-19 (1975)) and
improvements thereto (see, e.g., Coligan et al. (eds.), Current
Protocols in Immunology, 1:2.5.1-2.6.7 (John Wiley & Sons
1991); U.S. Pat. Nos. 4,902,614, 4,543,439, and 4,411,993;
Monoclonal Antibodies, Hybridomas: A New Dimension in Biological
Analyses, Plenum Press, Kennett et al. (eds.) (1980); and
Antibodies: A Laboratory Manual, Harlow and Lane (eds.), Cold
Spring Harbor Laboratory Press (1988); see also, e.g., Brand et
al., Planta Med. 70:986-92 (2004); Pasqualini et al., Proc. Natl.
Acad. Sci. USA 101:257-59 (2004)). An animal, for example, a rat,
hamster, or more commonly, a mouse, is immunized with a cellular
polypeptide immunogen prepared according to methods practiced in
the art. The presence of specific antibody production may be
monitored after the initial injection (injections may be
administered by any one of several routes as described herein for
generation of polyclonal antibodies) and/or after a booster
injection by obtaining a serum sample and detecting the presence of
an antibody that binds to the cellular polypeptide using any one of
several immunodetection methods known in the art and described
herein.
[0187] An antibody that specifically binds to a cellular
polypeptide may be a human monoclonal antibody. Human monoclonal
antibodies may be generated by any number of techniques with which
those having ordinary skill in the art will be familiar. Such
methods include, but are not limited to, Epstein Barr Virus (EBV)
transformation of human peripheral blood cells (e.g., containing B
lymphocytes) (see, e.g., U.S. Pat. No. 4,464,456; see also, e.g.,
Glasky et al., Hybridoma 8:377-89 (1989)); in vitro immunization of
human B cells (see, e.g., Boerner et al., J. Immunol. 147:86-95
(1991)); fusion of spleen cells from immunized transgenic mice
carrying inserted human immunoglobulin genes (see, e.g., Green et
al., Nature Genet. 7:13 (1994); Lonberg et al., Nature 368:856
(1994); Taylor et al., Int. Immun. 6:579 (1994); U.S. Pat. No.
5,877,397; Bruggemann et al., Curr. Opin. Biotechnol. 8:455-58
(1997); Jakobovits et al., Ann. N.Y. Acad. Sci. 764:525-35 (1995));
isolation from human immunoglobulin V region phage libraries;
cloning the light chain and heavy chain variable regions from a B
cell that is producing an anti-cellular polypeptide antibody (WO
92/02551; U.S. Pat. No. 5,627,052; Babcook et al., Proc. Natl.
Acad. Sci. USA 93:7843-48 (1996)); or other procedures as known in
the art and based on the disclosure herein.
[0188] Chimeric antibodies, specific for a cellular polypeptide of
interest, including humanized antibodies, may also be generated
according to the present invention. A chimeric antibody has at
least one constant region domain derived from a first mammalian
species and at least one variable region domain derived from a
second, distinct mammalian species. See, e.g., Morrison et al.,
Proc. Natl. Acad. Sci. USA, 81:6851-55 (1984). In one embodiment, a
chimeric antibody may be constructed by cloning the polynucleotide
sequence that encodes at least one variable region domain derived
from a non-human monoclonal antibody, such as the variable region
derived from a murine, rat, or hamster monoclonal antibody, into a
vector containing a nucleic acid sequence that encodes at least one
human constant region (see, e.g., Shin et al., Methods Enzymol.
178:459-76 (1989); Walls et al., Nucleic Acids Res. 21:2921-29
(1993)).
[0189] A non-human/human chimeric antibody may be further
genetically engineered to create a "humanized" antibody. Such a
humanized antibody may comprise a plurality of CDRs derived from an
immunoglobulin of a non-human mammalian species, at least one human
variable framework region, and at least one human immunoglobulin
constant region. Useful strategies for designing humanized
antibodies may therefore include, for example by way of
illustration and not limitation, identification of human variable
framework regions that are most homologous to the non-human
framework regions of the chimeric antibody (see, e.g., Jones et
al., Nature 321:522-25 (1986); Riechmann et al., Nature 332:323-27
(1988)).
[0190] Designing a humanized antibody may therefore include
determining CDR loop conformations and structural determinants of
the non-human variable regions, for example, by computer modeling,
and then comparing the CDR loops and determinants to known human
CDR loop structures and determinants (see, e.g., Padlan et al.,
FASEB 9:133-39 (1995); Chothia et al., Nature, 342:377-83 (1989)).
Computer modeling may also be used to compare human structural
templates selected by sequence homology with the non-human variable
regions (see, e.g., Bajorath et al., Ther. Immunol. 2:95-103
(1995); EP-0578515-A3; Davies et al., Ann. Rev. Biochem. 59:439-73,
(1990)). If humanization of the non-human CDRs results in a
decrease in binding affinity, computer modeling may aid in
identifying specific amino acid residues that could be changed by
site-directed or other mutagenesis techniques to partially,
completely, or supra-optimally (i.e., increase to a level greater
than that of the non-humanized antibody) restore affinity. Those
having ordinary skill in the art are familiar with these techniques
and will readily appreciate numerous variations and modifications
to such design strategies.
[0191] For particular uses, antigen-binding fragments of antibodies
may be desired. Antibody fragments, F(ab').sub.2, Fab, Fab', Fv,
and Fd, can be obtained, for example, by proteolytic hydrolysis of
the antibody, for example, pepsin or papain digestion of whole
antibodies according to conventional methods. As an illustration,
antibody fragments can be produced by enzymatic cleavage of
antibodies with pepsin to provide a fragment denoted F(ab').sub.2.
This fragment can be further cleaved using a thiol reducing agent
to produce an Fab' monovalent fragment. Optionally, the cleavage
reaction can be performed using a blocking group for the sulfhydryl
groups that result from cleavage of disulfide linkages. As an
alternative, an enzymatic cleavage of an antibody using papain
produces two monovalent Fab fragments and an Fc fragment (see,
e.g., U.S. Pat. No. 4,331,647; Nisonoff et al., Arch. Biochem.
Biophys. 89:230 (1960); Porter, Biochem. J. 73:119 (1959); Edelman
et al., in Methods in Enzymology 1:422 (Academic Press 1967); Weir,
Handbook of Experimental Immunology, Blackwell Scientific, Boston
(1986)). The antigen binding fragments may be separated from the Fc
fragments by affinity chromatography, for example, using
immobilized protein A, protein G, an Fc specific antibody, or
immobilized cellular polypeptide or a fragment thereof. Other
methods for cleaving antibodies, such as separating heavy chains to
form monovalent light-heavy chain fragments (Fd), further cleaving
of fragments, or other enzymatic, chemical, or genetic techniques
may also be used, so long as the fragments bind to the cellular
polypeptide that is recognized by the intact antibody.
[0192] An antibody fragment may also be any synthetic or
genetically engineered protein that acts like an antibody in that
it binds to a specific antigen to form a complex. For example,
antibody fragments include isolated fragments consisting of the
light chain variable region, Fv fragments consisting of the
variable regions of the heavy and light chains, recombinant single
chain polypeptide molecules in which light and heavy variable
regions are connected by a peptide linker (scFv proteins), and
minimal recognition units consisting of the amino acid residues
that mimic the hypervariable region. The antibody of the present
invention preferably comprises at least one variable region domain.
The variable region domain may be of any size or amino acid
composition and will generally comprise at least one hypervariable
amino acid sequence responsible for antigen binding and which is
adjacent to or in frame with one or more framework sequences. In
general terms, the variable (V) region domain may be any suitable
arrangement of immunoglobulin heavy (V.sub.H) and/or light
(V.sub.L) chain variable domains. Thus, for example, the V region
domain may be monomeric and be a V.sub.H or V.sub.L domain, which
is capable of independently binding antigen with acceptable
affinity. Alternatively, the V region domain may be dimeric and
contain V.sub.H-V.sub.H, V.sub.H-V.sub.L, or V.sub.L-V.sub.L,
dimers. Preferably, the V region dimer comprises at least one
V.sub.H and at least one V.sub.L chain that are non-covalently
associated (hereinafter referred to as F.sub.v). If desired, the
chains may be covalently coupled either directly, for example via a
disulfide bond between the two variable domains, or through a
linker, for example a peptide linker, to form a single chain Fv
(scF.sub.v).
[0193] A minimal recognition unit is an antibody fragment
comprising a single complementarity-determining region (CDR). Such
CDR peptides can be obtained by constructing polynucleotides that
encode the CDR of an antibody of interest. The polynucleotides are
prepared, for example, by using the polymerase chain reaction to
synthesize the variable region using mRNA isolated from or
contained within antibody-producing cells as a template according
to methods practiced by persons skilled in the art (see, for
example, Larrick et al., Methods: A Companion to Methods in
Enzymology 2:106, (1991); Courtenay-Luck, "Genetic Manipulation of
Monoclonal Antibodies," in Monoclonal Antibodies: Production,
Engineering and Clinical Application, Ritter et al. (eds.), page
166 (Cambridge University Press 1995); and Ward et al., "Genetic
Manipulation and Expression of Antibodies," in Monoclonal
Antibodies: Principles and Applications, Birch et al., (eds.), page
137 (Wiley-Liss, Inc. 1995)). Alternatively, such CDR peptides and
other antibody fragment can be synthesized using an automated
peptide synthesizer.
[0194] According to certain embodiments, non-human, human, or
humanized heavy chain and light chain variable regions of any of
the Ig molecules described herein may be constructed as scFv
polypeptide fragments (single chain antibodies). See, e.g., Bird et
al., Science 242:423-426 (1988); Huston et al., Proc. Natl. Acad.
Sci. USA 85:5879-83 (1988)). Multi-functional scFv fusion proteins
may be generated by linking a polynucleotide sequence encoding an
scFv polypeptide in-frame with at least one polynucleotide sequence
encoding any of a variety of known effector proteins. These methods
are known in the art, and are disclosed, for example, in
EP-B1-0318554, U.S. Pat. No. 5,132,405, U.S. Pat. No. 5,091,513,
and U.S. Pat. No. 5,476,786. By way of example, effector proteins
may include immunoglobulin constant region sequences. See, e.g.,
Hollenbaugh et al., J. Immunol. Methods 188:1-7 (1995). Other
examples of effector proteins are enzymes. As a non-limiting
example, such an enzyme may provide a biological activity for
therapeutic purposes (see, e.g., Siemers et al., Bioconjug. Chem.
8:510-19 (1997)), or may provide a detectable activity, such as
horseradish peroxidase-catalyzed conversion of any of a number of
well-known substrates into a detectable product, for diagnostic
uses.
[0195] Antibodies may also be identified and isolated from human
immunoglobulin phage libraries, from rabbit immunoglobulin phage
libraries, from mouse immunoglobulin phage libraries, and/or from
chicken immunoglobulin phage libraries (see, e.g., Winter et al.,
Annu. Rev. Immunol. 12:433-55 (1994); Burton et al., Adv. Immunol.
57:191-280 (1994); U.S. Pat. No. 5,223,409; Huse et al., Science
246:1275-81 (1989); Schlebusch et al., Hybridoma 16:47-52 (1997)
and references cited therein; Rader et al., J. Biol. Chem.
275:13668-76 (2000); Popkov et al., J. Mol. Biol. 325:325-35
(2003); Andris-Widhopf et al., J. Immunol. Methods 242:159-31
(2000)). Antibodies isolated from non-human species or non-human
immunoglobulin libraries may be genetically engineered according to
methods described herein and known in the art to "humanize" the
antibody or fragment thereof. Immunoglobulin variable region gene
combinatorial libraries may be created in phage vectors that can be
screened to select Ig fragments (Fab, Fv, scFv, or multimers
thereof) that bind specifically to a cellular polypeptide of
interest (see, e.g., U.S. Pat. No. 5,223,409; Huse et al., Science
246:1275-81 (1989); Sastry et al., Proc. Natl. Acad. Sci. USA
86:5728-32 (1989); Alting-Mees et al., Strategies in Molecular
Biology 3:1-9 (1990); Kang et al., Proc. Natl. Acad. Sci. USA
88:4363-66 (1991); Hoogenboom et al., J. Molec. Biol. 227:381-388
(1992); Schlebusch et al., Hybridoma 16:47-52 (1997) and references
cited therein; U.S. Pat. No. 6,703,015).
[0196] In certain other embodiments, cellular polypeptide-specific
antibodies are multimeric antibody fragments. Useful methodologies
are described generally, for example in Hayden et al., Curr Opin.
Immunol. 9:201-12 (1997) and Coloma et al., Nat. Biotechnol.
15:159-63 (1997). For example, multimeric antibody fragments may be
created by phage techniques to form miniantibodies (U.S. Pat. No.
5,910,573) or diabodies (Holliger et al., Cancer Immunol.
Immunother. 45:128-30 (1997)). Multimeric fragments may be
generated that are multimers of a cellular polypeptide-specific Fv.
Multimeric antibodies include bispecific and bifunctional
antibodies comprising a first Fv specific for an antigen associated
with a second Fv having a different antigen specificity (see, e.g.,
Drakeman et al., Expert Opin. Investig. Drugs 6:1169-78 (1997);
Koelemij et al., J. Immunother. 22:514-24 (1999); Marvin et al.,
Acta Pharmacol. Sin. 26:649-58 (2005); Das et al., Methods Mol.
Med. 109:329-46 (2005)).
[0197] A minimal recognition unit is an antibody fragment
comprising a single complementarity-determining region (CDR). Such
CDR peptides can be obtained by constructing polynucleotides that
encode the CDR of an antibody of interest. The polynucleotides are
prepared, for example, by using the polymerase chain reaction to
synthesize the variable region using mRNA isolated from or
contained within antibody-producing cells as a template according
to methods practiced by persons skilled in the art (see, for
example, Larrick et al., Methods: A Companion to Methods in
Enzymology 2:106, (1991); Courtenay-Luck, "Genetic Manipulation of
Monoclonal Antibodies," in Monoclonal Antibodies: Production,
Engineering and Clinical Application, Ritter et al. (eds.), page
166 (Cambridge University Press 1995); and Ward et al., "Genetic
Manipulation and Expression of Antibodies," in Monoclonal
Antibodies: Principles and Applications, Birch et al., (eds.), page
137 (Wiley-Liss, Inc. 1995)). Alternatively, such CDR peptides and
other antibody fragment can be synthesized using an automated
peptide synthesizer.
[0198] In other embodiments, a minimal recognition unit may be
identified from a peptide library. Such peptides may be identified
and isolated from combinatorial libraries (see, e.g., International
Patent Application Nos. PCT/US91/08694 and PCT/US91/04666) and from
phage display peptide libraries (see, e.g., Scott et al., Science
249:386 (1990); Devlin et al., Science 249:404 (1990); Cwirla et
al., Science 276: 1696-99 (1997); U.S. Pat. No. 5,223,409; U.S.
Pat. No. 5,733,731; U.S. Pat. No. 5,498,530; U.S. Pat. No.
5,432,018; U.S. Pat. No. 5,338,665; 1994; U.S. Pat. No. 5,922,545;
International Application Publication Nos. WO 96/40987 and WO
98/15833). In phage display peptide libraries, random peptide
sequences are fused to a phage coat protein such that the peptides
are displayed on the external surface of a filamentous phage
particle.
[0199] A peptide that is a minimal recognition unit or a CDR (i.e.,
any one or more of three CDRs present in a heavy chain variable
region and/or one or more of three CDRs present in a light chain
variable region) may be identified by computer modeling techniques,
which can be used for comparing and predicting a peptide sequence
that will specifically bind to a cellular polypeptide as described
herein (see, e.g., Bradley et al., Science 309:1868 (2005);
Schueler-Furman et al., Science 310:638 (2005)). Such
computer-assisted predictive modeling techniques may also be useful
for altering the binding affinity of an antibody that binds to a
cellular polypeptide. By comparing the predicted three-dimensional
structure of a minimal recognition unit and/or of one or more CDRs
with the predicted three-dimensional structure of a viral
polypeptide that specifically binds the cellular polypeptide, the
modeling techniques provide a method to identify residues within
the minimal recognition unit and/or of one or more CDRs that can be
substituted and that will more closely approximate the binding
interaction between the cellular polypeptide and the viral
polypeptide. Amino acid substitutions may be readily accomplished
using any one of a number of mutagenesis techniques described
herein and used routinely in the art for making polynucleotide and
polypeptide variants.
[0200] In certain embodiments, anti-idiotype antibodies that
recognize and bind specifically to an antibody (or antigen-binding
fragment thereof) that specifically binds to a cellular polypeptide
of interest are provided. Anti-idiotype antibodies may be generated
as polyclonal antibodies or as monoclonal antibodies by the methods
described herein, using an antibody (or antigen-binding fragment
thereof) that specifically binds to the cellular polypeptide as
immunogen. Anti-idiotype antibodies or antigen-binding fragments
thereof may also be generated by any of the recombinant genetic
engineering methods described above or by phage display selection.
Anti-idiotype antibodies may be further engineered to provide a
chimeric or humanized anti-idiotype antibody, according to the
description provided in detail herein and according to methods
routinely practiced in the art. An anti-idiotype antibody may bind
specifically to the antigen-binding site of the anti-cellular
polypeptide antibody such that binding of the antibody to the
cellular polypeptide is competitively inhibited. Alternatively, an
anti-idiotype antibody as provided herein may not competitively
inhibit binding of an anti-cellular polypeptide antibody to the
cellular polypeptide.
[0201] An agent also includes a peptide-immunoglobulin (Ig)
constant region fusion polypeptide, which includes a peptide-IgFc
fusion polypeptide. The peptide may be any naturally occurring or
recombinantly prepared molecule. A peptide-Ig constant region
fusion polypeptide, such as a peptide-IgFc fusion polypeptide (also
referred to in the art as a peptibody (see, e.g., U.S. Pat. No.
6,660,843)), comprises a biologically active peptide or polypeptide
capable of altering the activity of a cellular polypeptide of
interest that is fused in-frame with a portion, at least one
constant region domain (e.g., CH1, CH2, CH3, and/or CH4), or the Fc
portion (CH2-CH3) of an immunoglobulin. The Fc portion is also
referred to herein as the Fc region.
[0202] In one embodiment, the peptide portion of the fusion
polypeptide is capable of interacting with or binding to the
cellular polypeptide to which the viral polypeptide binds and
effecting the same biological activity as the viral polypeptide
when it binds to the cellular polypeptide. In certain embodiments,
binding of the peptide-Fc fusion polypeptide suppresses (inhibits,
prevents, decreases, or abrogates) the immunoresponsiveness of an
immune cell that expresses the cellular polypeptide. For example,
such a peptide may be identified by determining its capability to
inhibit or block binding of the viral polypeptide to a cell that
expresses the cellular polypeptide. Alternatively, a candidate
peptide may be permitted to contact or interact with a cell that
expresses the cellular polypeptide, and the capability of the
candidate peptide to suppress or enhance at least one biological
function of the cell can be measured according to methods described
herein and practiced in the art.
[0203] Candidate peptides may be provided as members of a
combinatorial library, which includes synthetic peptides prepared
according to a plurality of predetermined chemical reactions
performed in a plurality of reaction vessels. For example, various
starting peptides may be prepared according to standard peptide
synthesis techniques with which a skilled artisan will be
familiar.
[0204] Peptides that alter at least one biological activity of a
cell may be identified and isolated from combinatorial libraries
(see, e.g., International Patent Application Nos. PCT/US91/08694
and PCT/US91/04666) and from phage display peptide libraries (see,
e.g., Scott et al., Science 249:386 (1990); Devlin et al., Science
249:404 (1990); Cwirla et al., Science 276: 1696-99 (1997); U.S.
Pat. No. 5,223,409; U.S. Pat. No. 5,733,731; U.S. Pat. No.
5,498,530; U.S. Pat. No. 5,432,018; U.S. Pat. No. 5,338,665; 1994;
U.S. Pat. No. 5,922,545; International Application Publication Nos.
WO 96/40987 and WO 98/15833). In phage display peptide libraries,
random peptide sequences are fused to a phage coat protein such
that the peptides are displayed on the external surface of a
filamentous phage particle. Typically, the displayed peptides are
contacted with a ligand or binding molecule of interest to permit
interaction between the peptide and the ligand or binding molecule,
unbound phage are removed, and the bound phage are eluted and
subsequently enriched by successive rounds of affinity purification
and repropagation. The peptides with the greatest affinity for the
ligand or binding molecule or target molecule of interest (e.g.,
the cellular polypeptides identified and described herein) may be
sequenced to identify key residues, which may identify peptides
within one or more structurally related families of peptides.
Comparison of sequences of peptides may also indicate which
residues in such peptides may be safely substituted or deleted by
mutagenesis. These peptides may then be incorporated into
additional peptide libraries that can be screened, and peptides
with optimized affinity can be identified.
[0205] Additional methods for identifying peptides that may alter
at least one biological activity of a cell and thus be useful for
treating and/or preventing an immunological disease or disorder or
a viral infection include, but are not limited to, (1) structural
analysis of protein-protein interaction such as analyzing the
crystal structure of the cellular polypeptide target (see, e.g.,
Jia, Biochem. Cell Biol. 75:17-26 (1997)) to identity and to
determine the orientation of critical residues of the cellular
polypeptide, which will be useful for designing a peptide (see,
e.g., Takasaki et al., Nature Biotech. 15: 1266-70 (1997)); (2) a
peptide library comprising peptides fused to a
peptidoglycan-associated lipoprotein and displayed on the outer
surface of bacteria such as E. coli; (3) generating a library of
peptides by disrupting translation of polypeptides to generate
RNA-associated peptides; and (4) generating peptides by digesting
polypeptides with one or more proteases. (See also, e.g., U.S. Pat.
Nos. 6,660,843; 5,773,569; 5,869,451; 5,932,946; 5,608,035;
5,786,331; 5,880,096). A peptide may comprise any number of amino
acids between 3 and 75 amino acids, 3 and 60 amino acids, 3 and 50
amino acids, 3 and 40 amino acids, 3 and 30 amino acids, 3 and 20
amino acids, or 3 and 10 amino acids. A peptide that has the
capability of alter a biological activity of a cell, such as for
example, alter immunoresponsiveness of an immune cell (e.g., in
certain embodiments, to suppress the immunoresponsiveness of the
immune cell and in certain other embodiments, to enhance
immunoresponsiveness of the immune cell) may also be further
derivatized to add or insert amino acids that are useful for
constructing a peptide-Ig constant region fusion protein (such as
amino acids that are linking sequences or that are spacer
sequences).
[0206] A peptide that may be used to construct a peptide-Ig
constant region fusion polypeptide (including a peptide-IgFc fusion
polypeptide) may be derived from the viral polypeptide that binds
to the cellular polypeptide. Peptides may be randomly generated
from the viral polypeptide by proteolytic digestion using any one
or more of various proteases, isolated, and then analyzed for their
capability to alter at least one biological activity of a cell.
Peptides of a viral polypeptide may also be generated using
recombinant methods described herein and practiced in the art.
Randomly generated peptides may also be used to prepare peptide
combinatorial libraries or phage libraries as described herein and
in the art. Alternatively, the amino acid sequences of portions of
a viral polypeptide that interact with the cellular polypeptide may
be determined by computer modeling of the cellular polypeptide, or
of a portion of the polypeptide, for example, the extracellular
portion, and/or x-ray crystallography (which may include
preparation and analysis of crystals of the cellular polypeptide
(or a fragment thereof) only or of the cellular polypeptide-viral
polypeptide (or fragments of the cellular polypeptide and/or the
viral polypeptide complex).
[0207] As described in detail herein, an Fc polypeptide (or portion
or region) of an immunoglobulin comprises the heavy chain CH2
domain and CH3 domain and a portion of or the entire hinge region
that is located between CH1 and CH2. Historically, an Fc fragment
was derived by papain digestion of an immunoglobulin and included
the hinge region of the immunoglobulin. An Fc region or Fc
polypeptide referred to herein is a monomeric polypeptide that may
be linked or associated to form dimeric or multimeric forms by
covalent (e.g., particularly disulfide bonds) and non-covalent
association. The number of intermolecular disulfide bonds between
monomeric subunits of Fc polypeptides varies depending on the
immunoglobulin class (e.g., IgG, IgA, IgE) or subclass (e.g., human
IgG1, IgG2, IgG3, IgG4, IgA1, IgA2). The Fc portion of the
immunoglobulin mediates certain effector functions of an
immunoglobulin. Three general categories of effector functions
associated with the Fc region include (1) activation of the
classical complement cascade, (2) interaction with effector cells,
and (3) compartmentalization of immunoglobulins. Presently, an Fc
polypeptide, and any one or more constant region domains, and
fusion proteins comprising at least one immunoglobulin constant
region domain can be readily prepared according to recombinant
molecular biology techniques with which a skilled artisan is quite
familiar.
[0208] The Fc polypeptide is preferably prepared using the
nucleotide and the encoded amino acid sequences derived from the
animal species for whose use the peptide-IgFc fusion polypeptide is
intended. In one embodiment, the Fc fragment is of human origin and
may be from any of the immunoglobulin classes, such as human IgG1
and IgG2.
[0209] An Fc polypeptide as described herein also includes Fc
polypeptide variants. One such Fc polypeptide variant has one or
more cysteine residues (such as one or more cysteine residues in
the hinge region) that forms a disulfide bond with another Fc
polypeptide substituted with another amino acid, such as serine, to
reduce the number of disulfide bonds formed between two Fc
polypeptides. Another example of an Fc polypeptide variant is a
variant that has one or more amino acids involved in an effector
function substituted such that the Fc polypeptide has a reduced
level of an effector function. For example, amino acids in the Fc
region may be substituted to reduce or abrogate binding of a
component of the complement cascade (see, e.g., Duncan et al.,
Nature 332:563-64 (1988); Morgan et al., Immunology 86:319-24
(1995)); to reduce or abrogate the ability of the Fc polypeptide to
bind to an Fc receptor expressed by an immune cell; or to alter
antibody-dependent cellular cytotoxicity.
[0210] Other Fc variants encompass similar amino acid sequences of
known Fc polypeptide sequences that have only minor changes, for
example by way of illustration and not limitation, covalent
chemical modifications, insertions, deletions and/or substitutions,
which may further include conservative substitutions. Amino acid
sequences that are similar to one another may share substantial
regions of sequence homology. Similarly, nucleotide sequences that
encode the Fc variants may encompass substantially similar
nucleotide sequences and have only minor changes, for example by
way of illustration and not limitation, covalent chemical
modifications, insertions, deletions, and/or substitutions, which
may further include silent mutations owing to degeneracy of the
genetic code. Nucleotide sequences that are similar to one another
may share substantial regions of sequence homology.
[0211] An Fc polypeptide or at least one immunoglobulin constant
region, or portion thereof, when fused to a peptide or polypeptide
of interest acts, at least in part, as a vehicle or carrier moiety
that prevents degradation and/or increases half-life, reduces
toxicity, reduces immunogenicity, and/or increases biological
activity of the peptide such as by forming dimers or other
multimers (see, e.g., U.S. Pat. Nos. 6,018,026; 6,291,646;
6,323,323; 6,300,099; 5,843,725). (See also, e.g., U.S. Pat. No.
5,428,130; U.S. Pat. No. 6,660,843; U.S. Patent Application
Publication Nos. 2003/064480; 2001/053539; 2004/087778;
2004/077022; 2004/071712; 2004/057953/ 2004/053845/ 2004/044188;
2004/001853; 2004/082039). Alternative moieties to an
immunoglobulin constant region such as an Fc polypeptide that may
be linked or fused to a peptide that binds to a viral polypeptide
and/or that alters at least one biological activity of a cell
include, for example, a linear polymer (e.g., polyethylene glycol,
polylysine, dextran, etc.; see, for example, U.S. Pat. No.
4,289,872; International Patent Application Publication No. WO
93/21259); a lipid; a cholesterol group (such as a steroid); a
carbohydrate or oligosaccharide.
[0212] Provided herein are methods of manufacture for producing a
cellular polypeptide to which a viral polypeptide (particularly a
viral polypeptide that has at least one virulence trait described
herein). For example a process (or method) for manufacturing a
cellular polypeptide comprises identifying a cellular polypeptide
to which a viral polypeptide binds according to any one of the
method described herein. After the amino acid sequence of the
cellular polypeptide is determined, a nucleotide sequence that
encodes the cellular polypeptide may be determining according to
principles based on the genetic code. Alternatively, the nucleotide
sequence of the genomic DNA or mRNA of a cell that encodes the
cellular polypeptide may be determined by using standard molecular
biology techniques, including primer design, hybridization, nucleic
acid isolation, cloning, and amplification, and sequencing. A
polynucleotide comprising a nucleotide sequence encoding the
cellular polypeptide may be incorporated into a recombinant
expression construct (i.e., vector) according to well known methods
and principles known in the molecular biology art and described
herein for preparing a recombinant expression vector. The vector
also includes a promoter operatively linked to the nucleotide
sequence that encodes the cellular polypeptide as well as other
regulatory elements (e.g., enhancer or transcription initiation
site) with which a skilled artisan is familiar. The vector may then
be introduced into a host cell (e.g., a prokaryotic, eukaryotic,
insect, yeast, or other suitable host cell) such as by transfecting
or transforming the host cell with the recombinant expression
vector. After culturing the host cell under conditions and for a
time sufficient that permit expression of the cellular polypeptide,
the cellular polypeptide may be isolated from the host cell culture
or from the host cells.
[0213] The nucleic acid molecules encoding the cellular polypeptide
that specifically binds to the viral polypeptide, as described
herein, may be propagated and expressed according to any of a
variety of well-known procedures for nucleic acid excision,
ligation, transformation, and transfection. Thus, in certain
embodiments expression of the cellular polypeptide may be preferred
in a prokaryotic host cell, such as Escherichia coli (see, e.g.,
Pluckthun et al., Methods Enzymol. 178:497-515 (1989)). In certain
other embodiments, expression of the cellular polypeptide may be
preferred in a eukaryotic host cell, including yeast (e.g.,
Saccharomyces cerevisiae, Schizosaccharomyces pombe, and Pichia
pastoris); animal cells (including mammalian cells); or plant
cells. Examples of suitable animal cells include, but are not
limited to, myeloma, HEK293, COS, or CHO cells. Examples of plant
cells include tobacco, corn, soybean, and rice cells. By methods
known to those having ordinary skill in the art and based on the
present disclosure, a nucleic acid vector may be designed for
expressing foreign sequences in a particular host system, and then
polynucleotide sequences encoding the cellular polypeptide may be
inserted. The regulatory elements will vary according to the
particular host.
[0214] Also provided herein are methods of manufacture for
producing an agent that is useful for treating a subject who has or
who is at risk of developing a disease or disorder as described
herein, including an immunological disease or disorder, or who is
has a viral infection or who is at risk for developing a viral
infection. In one embodiment, such a method of manufacture
comprises (a) identifying an agent for treating a disease or
disorder, such as an immunological disease or disorder or a viral
infection according to methods described herein and practiced in
the art. For example, identifying an agent comprises identifying a
cellular polypeptide to which a viral polypeptide binds according
to the methods described herein, wherein interaction between the
cellular polypeptide and the viral polypeptide alters
immunoresponsiveness of an immune cell. The method for identifying
an agent comprises contacting the cellular polypeptide, or a cell
comprising the cellular polypeptide; the viral polypeptide; and a
candidate agent, which agents are described herein in detail, under
conditions and for a time sufficient that permit the cellular
polypeptide and the viral polypeptide to interact. The level of
binding of the viral polypeptide to the cellular polypeptide in the
presence of the candidate agent is then determined and compared
with the level of binding of the viral polypeptide to the cellular
polypeptide in the absence of the candidate agent. A candidate
agent that inhibits (or prevents, reduces, minimizes, or abrogates)
binding of the viral polypeptide to the cellular polypeptide may
mimic or act in the same manner as the viral polypeptide, and thus
affect at least one biological activity of the cellular
polypeptide. Such a biological activity includes but is not limited
to, altering immunoresponsiveness of an immune cell (e.g., in
certain embodiments, to suppress the immunoresponsiveness of the
immune cell and in certain other embodiments, to enhance
immunoresponsiveness of the immune cell) that comprises the
cellular polypeptide. Accordingly, such an agent is useful for
treating an immunological disease or disorder. The agent is then
produced according to methods known in the art for producing the
agent.
[0215] The agent may be any agent described herein, such as, for
example, an antibody, or antigen-binding fragment thereof; a small
molecule; an aptamer; an antisense polynucleotide; a small
interfering RNA (siRNA); and a peptide-IgFc fusion polypeptide. In
a particular embodiment, the agent is an antibody, or
antigen-binding fragment thereof, which may be produced according
to methods described herein and that are adapted for large-scale
manufacture. For example, production methods include batch cell
culture, which is monitored and controlled to maintain appropriate
culture conditions. Purification of the antibody, or
antigen-binding fragment thereof, may be performed according to
methods described herein and known in the art and that comport with
guidelines of domestic and foreign regulatory agencies.
[0216] An agent (such as, but not limited to, an antibody, or
antigen-binding fragment thereof that binds to a target cellular
polypeptide) identified according to the methods described herein
that may be useful for treating or preventing a disease or
disorder, including a cardiovascular disease or disorder, a
metabolic disease or disorder, a proliferative disease or disorder,
or an immunological disease or disorder may be combined (i.e.,
formulated) with a pharmaceutically (i.e., physiologically)
suitable excipient for administration to a subject. A
pharmaceutical composition may be a sterile aqueous or non-aqueous
solution, suspension or emulsion, which additionally comprises a
physiologically acceptable excipient (pharmaceutically acceptable
or suitable excipient or carrier) (i.e., a non-toxic material that
does not interfere with the activity of the active ingredient).
Such compositions may be in the form of a solid, liquid, or gas
(aerosol). Alternatively, compositions described herein may be
formulated as a lyophilizate, or compounds may be encapsulated
within liposomes using technology known in the art. Pharmaceutical
compositions may also contain other components, which may be
biologically active or inactive. Such components include, but are
not limited to, buffers (e.g., neutral buffered saline or phosphate
buffered saline), carbohydrates (e.g., glucose, mannose, sucrose or
dextrans), mannitol, proteins, polypeptides or amino acids such as
glycine, antioxidants, chelating agents such as EDTA or
glutathione, stabilizers, dyes, flavoring agents, and suspending
agents and/or preservatives.
[0217] Any suitable excipient or carrier known to those of ordinary
skill in the art for use in pharmaceutical compositions may be
employed in the compositions described herein. Excipients for
therapeutic use are well known, and are described, for example, in
Remingtons Pharmaceutical Sciences, Mack Publishing Co. (A. R.
Gennaro ed. 1985). In general, the type of excipient is selected
based on the mode of administration. Pharmaceutical compositions
may be formulated for any appropriate manner of administration.
[0218] Pharmaceutical compositions may be administered in a manner
appropriate to the disease to be treated (or prevented) as
determined by persons skilled in the medical arts. An appropriate
dose and a suitable duration and frequency of administration will
be determined by such factors as the condition of the patient, the
type and severity of the patient's disease, the particular form of
the active ingredient, and the method of administration. In
general, an appropriate dose and treatment regimen provides the
composition(s) in an amount sufficient to provide therapeutic
and/or prophylactic benefit (e.g., an improved clinical outcome,
such as more frequent complete or partial remissions, or longer
disease-free and/or overall survival, or a lessening of symptom
severity). For prophylactic use, a dose should be sufficient to
prevent, delay the onset of, or diminish the severity of a disease
such as an immunological disease or disorder.
[0219] Optimal doses may generally be determined using experimental
models and/or clinical trials. The optimal dose may depend upon the
body mass, weight, or blood volume of the patient. In general, the
amount of a polypeptide, such as an antibody or antigen-binding
fragment thereof, as described herein, present in a dose, or
produced in situ by DNA present in a dose, ranges from about 0.01
.mu.g to about 1000 .mu.g per kg of subject. The use of the minimum
dosage that is sufficient to provide effective therapy is usually
preferred. Patients may generally be monitored for therapeutic or
prophylactic effectiveness using assays suitable for the condition
being treated or prevented, which assays will be familiar to those
having ordinary skill in the art. Suitable dose sizes will vary
with the size of the patient, but will typically range from about 1
ml to about 500 ml for a 10-60 kg subject.
[0220] Business Methods
[0221] Provided herein are methods and systems for identifying
agents that may be used as therapeutic bioactive agents for
treating diseases and disorders, including immunological diseases
and disorders, testing and evaluating the agents in pre-clinical
and clinical trials, and then selling the agents to health care
professionals. Systems and methods for selling a therapeutic agent
include, but are not limited to, (1) scientific methods, which may
include computer systems and methods, for identifying and analyzing
a viral polypeptide that is a viral virulence factor; (2)
scientific methods for identifying a cellular polypeptide that
binds to the viral virulence factor and which cellular polypeptide
is therefore a target for a therapeutic agent; (3) scientific
methods for identifying and analyzing an agent that inhibits
binding of the viral polypeptide to the cellular polypeptide, and
thus alters at least one biological activity of the cellular
polypeptide; (4) methods and systems for clinical development of
the agent; (5) selling the agent to health care professionals for
treatment of patients in need of the agent.
[0222] In one embodiment, a business method is provided that
comprises identifying a viral polypeptide that is a viral virulence
factor; identifying a cellular polypeptide to which a viral
virulence factor binds, wherein binding of the viral virulence
factor to the cellular polypeptide alters at least one biological
activity of the cell with which the cellular polypeptide is
associated; identifying an agent that inhibits binding of the
cellular polypeptide and the viral polypeptide, thereby identifying
an agent that alters at least one biological activity of the cell;
and designing and executing at least one pre-clinical study. In
certain other embodiments, the business method further comprises
designing and executing at least one clinical trial in human
subjects to determine the safety profile of the agent in humans.
The business method may also further comprises the design and
execution of at least one clinical trial for evaluating the
efficacy of the agent in human subjects who are in need of such an
agent for treatment (which includes prevention) of a disease or
medical disorder. The method may still further comprise selling the
agent to a health care professional or to a distributing entity
that provides the agent to a health care professional.
[0223] In a specific embodiment, the at least one biological effect
is immunoresponsiveness and the cell is an immune cell, and the
agent alters immunoresponsiveness of the immune cell. The agent
that inhibits binding between the viral and cellular polypeptides
is, thus, also capable of altering immunoresponsiveness of an
immune cell. As described in detail herein, the agent includes, but
is not limited to, (a) an antibody, or antigen-binding fragment
thereof, (b) a viral polypeptide/Fc polypeptide fusion protein; (c)
a peptide/Fc polypeptide fusion protein; (d) a domain of the
cellular polypeptide, or a fragment thereof comprising at least
eight amino acids, fused to an Fc polypeptide; (e) a small
molecule; (f) a small interfering RNA (siRNA); (g) an antisense
polynucleotide; and (h) an aptamer.
[0224] As described herein, the business method comprises designing
and executing at least one pre-clinical study to determine whether
altering the at least one biological activity of the cell by the
agent indicates that the agent is useful for treating a disease or
medical disorder in a human subject. Preclinical studies include
experiments that contribute to the determination of the therapeutic
window of the agent (i.e., the relationship between the efficacy
and safety of the agent). Procedures may be designed and performed
or executed in cell culture studies to determine the level of the
agent that induces at least one toxic effect in a cell. A toxic
effect is understood to mean an undesirable effect in a cell. For
example, if the agent is being tested for its capability to enhance
or prolong survival of a cell or to maintain a normal biological
activity or function, a toxic effect would include induction of
apoptosis, an abnormal change in structural cell morphology or
integrity, or induction and maintenance of any cellular pathway
that is not considered normal for that particular cell type or that
alters the capability of the cell to thrive. If the agent were
being tested for its capability to treat a cancer or malignancy
and/or act as an anti-proliferative agent, induction of apoptosis
and cell death are desirable effects and such analyses would
indicate potential efficacy of the agent. Preclinical studies that
may be designed and executed for toxicity analysis include studies
in animals. Examples of other preclinical studies that may be
conducted using cell culture methods and/or animal models include
determining teratotagenicity; effects on male and female
reproductive systems, including effects of the agent on the birth
of offspring; pharmacokinetic analyses; and stability studies.
[0225] Preclinical studies also include cell culture and animal
studies that indicate and evaluate the efficacy of the agent.
Agents that are useful for treating an immunological disease or
disorder, such as an autoimmune disease or inflammatory disease or
disorder, cardiovascular disease or disorder, a metabolic disease
or disorder, or a proliferative disease or disorder, may be
determined and evaluated in any one of a number of animal models
described herein and used by persons skilled in the art (see, e.g.,
reviews by Taneja et al., Nat. Immunol. 2:781-84 (2001); Lam-Tse et
al., Springer Semin. Immunopathol. 24:297-321 (2002)). For example,
mice that have three genes, Tyro3, Mer, and Axl that encode
receptor tyrosine kinases, knocked out exhibit several symptoms of
autoimmune diseases, including rheumatoid arthritis and SLE (Lu et
al., Science 293:228-29 (2001)). A murine model of spontaneous
lupus-like disease has been described using NZB/WF1 hybrid mice
(see, e.g., Drake et al., Immunol. Rev. 144:51-74 (1995)). An
animal model for type I diabetes that permits testing of agents and
molecules that affect onset, modulation, and/or protection of the
animal from disease uses MHC transgenic (Tg) mice. Mice that
express the HLA-DQ8 transgene (HLA-DQ8 is the predominant
predisposing gene in human type 1 diabetes) and the HLA-DQ6
transgene (which is diabetes protective) were crossed with RIP(rat
insulin promoter). B7-1-Tg mice to provide HLA-DQ8 RIP.B7-1
transgenic mice that develop spontaneous diabetes (see Wakeland et
al., Curr. Opin. Immunol. 11:701-707 (1999); Wen et al., J. Exp.
Med. 191:97-104 (2000)). (See also Brondum et al., Horm. Metab.
Res. 37 Suppl 1:56-60 (2005)).
[0226] Animal models that may be used for characterizing agents
that are useful for treating rheumatoid arthritis include a
collagen-induced arthritis model (see, e.g., Kakimoto, Chin. Med.
Sci. J. 6:78-83 (1991); Myers et al., Life Sci. 61:1861-78 (1997))
and an anti-collagen antibody-induced arthritis model (see, e.g.,
Kakimoto, supra). Other applicable animal models for immunological
diseases include an experimental autoimmune encephalomyelitis model
(also called experimental allergic encephalomyelitis model), an
animal model of multiple sclerosis; a psoriasis model that uses
AGR129 mice that are deficient in type I and type II interferon
receptors and deficient for the recombination activating gene 2
(Zenz et al., Nature 437:369-75 (2005); Boyman et al., J. Exp. Med.
199:731-36 (2004); published online Feb. 23, 2004); and a TNBS
(2,4,6-trinitrobenzene sulphonic acid) mouse model for inflammatory
bowel disease. Numerous animal models for cardiovascular disease
are available and include models described in van Vlijmen et al.,
J. Clin. Invest. 93:1403-10 (1994); Kiriazis et al., Annu. Rev.
Physiol. 62:321-51 (2000); Babu et al., Methods Mol. Med.
112:365-77 (2005).
[0227] As described herein, the business method comprises designing
and executing at least one clinical study to determine whether
altering the at least one biological activity of the cell by the
agent indicates that the agent is useful (i.e., safe and effective)
for treating a disease or medical disorder in a human subject.
Clinical studies are performed by persons skilled in the art and
according to statutes and rules (see 21 U.S.C. (The Federal Food,
Drug, and Cosmetics Act) and 21 C.F.R., particularly 21 C.F.R.
.sctn..sctn.312, 314, 316, 50, 54, 56, 58, and 201) and guidance
documents provided by the Food and Drug Administration (FDA) for
designing, executing, evaluating, and reporting the results of
clinical studies. In certain embodiments, the business method
comprises design and execution of at least one Phase I clinical
trial, at least one Phase II clinical trial, and at least one Phase
III clinical trial. In another embodiment, the method comprises
design and execution of at least one Phase I clinical trial and at
least one Phase II clinical trial, and in another embodiment the
business method comprises design and execution of at least one
Phase I clinical trial.
[0228] A Phase I clinical trial typically includes design and
execution of studies to evaluate the safety of the agent in human
subjects. A Phase I study or a second Phase I study may include
dose escalation studies. In certain instances, the Phase I trial
may be tested in a patient group that may gain some clinical
benefit from receiving the agent. A Phase I study is designed to
determine how an agent is absorbed, metabolized, and excreted in
the human body and also to assess adverse effects. Designing a
Phase I clinical trial including preparation of an Investigational
New Drug (IND) Application and submission to the FDA for evaluation
and approval. The content of an IND and the studies that are
performed that are included in the content of the IND are
determined, for example, by the agent, therapeutic indication, and
the appropriate pre-clinical in vivo (i.e., animal studies) and in
vitro (cell culture and other biological and chemical assays)
studies that inform a clinician or other health care professional
about the therapeutic agent.
[0229] A phase II study includes the early controlled clinical
studies conducted to obtain some preliminary data on the
effectiveness of the drug for a particular indication or
indications in patients with the disease or condition. This phase
of testing also helps determine the common short-term side effects
and risks associated with the drug. Phase II studies are typically
well-controlled, closely monitored, and conducted in a relatively
small number of patients, usually involving several hundred people.
Phase III studies are expanded controlled and uncontrolled trials.
These studies are performed after preliminary evidence suggesting
effectiveness of the drug has been obtained in Phase II, and are
intended to gather the additional information about effectiveness
and safety that is needed to evaluate the overall benefit-risk
relationship of the drug. Phase III studies also provide an
adequate basis for extrapolating the results to the general
population and transmitting that information in the physician
labeling. Phase III studies usually include several hundred to
several thousand people.
[0230] Design and execution of each clinical study includes
clinical study protocol design (including but not limited to
inclusion and exclusion criteria, statistical design and analyses
for primary and secondary endpoint determinations; adverse effect
analysis and protocols for reporting an adverse event; background
about the agent and the disease indication; patient information
(background and monitoring); clinical administration protocol;
etc.); study evaluation (including but not limited to statistical
analyses of all measured parameters outlined in the clinical
protocol; statistical analysis and categorization of adverse
events; narratives for serious adverse events; and other summaries
as required by the FDA); and conclusions. Design, results, and
conclusions may be discussed with the FDA informally as well as
formally, which is required by statute. Design and execution of
clinical studies may include additional methods and systems with
which persons skilled in the art of design and execution of
clinical studies will be very familiar.
[0231] The business methods described herein may further comprise
preparation of a New Drug Application (NDA) or Biological License
Application (BLA) as appropriate for the particular agent and as
provided in guidelines of FDA sub-agencies (CDER and CBER) and
presentation to the FDA to obtain marketing approval of the drug.
Accordingly, the business methods may further comprise selling the
agent. The agent may be sold directly to a health care
professional, including but not limited to, a physician or a
pharmacist, or may be sold to a distributor of marketed drugs and
biologics, which then sells the drug to the health care
professional. In certain circumstances, the drug may be sold
directly over the counter by retail and wholesale entities to the
consumer.
[0232] The business methods described herein may comprise licensing
by a pharmaceutical company to another biopharmaceutical company,
research or medical institution, large pharmaceutical company, or a
generic drug manufacturing company the rights to make and use the
viral polypeptide, the cellular polypeptide to which the viral
polypeptide binds, and/or the agent. The rights to make and use
each of the aforementioned viral polypeptide, cellular polypeptide,
and agent may include a sale or assignment of the rights or
licensing of the rights. The entity that sells or licenses the
viral polypeptide, cellular polypeptide, and/or agent to another
party is referred to as the selling company or licensing
organization, respectively, and the party to which the viral
polypeptide, cellular polypeptide, and/or agent is sold or licensed
to is referred to as the acquiring company. The term "company"
refers to any business entity or government institution or other
public institution that may be legally formed within a country or
within a state or province of the country, which may be a
for-profit or a non-profit entity.
[0233] In certain particular embodiments, the business arrangement
between the acquiring company and the licensing organization may
provide for the acquiring company to acquire intellectual property
to the viral polypeptide, cellular polypeptide, and/or agent, and
also to acquire certain associated technical information and
know-how. The acquiring company may pay the licensing organization
a combination of any one of upfront fees, ongoing research and
development costs, royalties, milestone payments (for example,
payment upon the acquiring company initiating and/or completing one
or more stages of clinical development, revenue creation, and
technical success milestones), in addition to other consideration
agreed to by the acquiring company and the licensing organization.
The payments may be in the form of cash, equity, and/or traded
assets (including rights to other agents, viral polypeptides,
and/or cellular polypeptides), or other agreed upon payment. In
return, the licensing organization may grant to the acquiring
company exclusive or non-exclusive licenses to the intellectual
property rights associated with the viral polypeptide, cellular
polypeptide, and/or agent, or assign the intellectual property
rights associated with the viral polypeptide, cellular polypeptide,
and/or agent. The rights may be granted in total or in specific
fields (e.g., use of the agent for a particular disease indication)
or in specific territories.
[0234] In a particular embodiment, the licensing organization is a
biopharmaceutical company and in other particular embodiments, the
acquiring organization is a biopharmaceutical company. In other
embodiments, the biopharmaceutical company performs experiments to
identify the viral polypeptide, the cellular polypeptide to which
the viral polypeptide binds, and/or to identify the agent that
alters at least one biological activity of a cell. In certain
embodiments, the agent alters immunoresponsiveness of an immune
cell.
[0235] Also provided herein is a method for guiding the selection
of a therapeutic agent for treating a disease or medical disorder.
The method comprises receiving information regarding a viral
polypeptide that increases the virulence of a virus in a host
infected with the virus; identifying a cellular polypeptide to
which the viral polypeptide binds, wherein binding of the viral
polypeptide to the cellular polypeptide alters at least one
biological activity of a cell; identifying one or more agents that
inhibit binding of the viral polypeptide to the cellular
polypeptide; categorizing the capability of the one or more agents
to alter at least one biological effect of a cell, wherein altering
the at least one biological effect reduces the risk of developing a
disease or medical disorder or reduces at least one symptom of a
disease or medical disorder in a host; and providing the
categorization of the capability of the agent to alter at least one
biological effect of a cell to a medical research professional to
assist in selecting the agent for testing in preclinical and
clinical methods, and therefrom guiding the selection of a
therapeutic agent for treating a disease or disorder. In a
particular embodiment the at least one biological effect is
immunoresponsiveness and the cell is an immune cell.
[0236] The capability of at least one agent to reduce the risk of
developing a disease include the capability of the at least one
agent (or more than one agent) to increase or prolong the time
between when a subject is suspected or having a disease or disorder
or determined to be at risk for developing a disease or disorder to
when the subject exhibits at least one symptom or sequelae of the
disease or disorder. Reducing at least one symptom includes the
capability of the agent to decrease, ameliorate, or otherwise
minimize the intensity, development, or exacerbation of a symptom
(or sequelae) of the disease or disorder. The agent may also
prevent development of a disease or disorder.
[0237] As described herein the capability of the one or more agents
to alter at least one biological effect of a cell, wherein altering
the at least one biological effect reduces the risk of developing a
disease or medical disorder or reduces at least one symptom of a
disease or medical disorder in a host may be determined using
experimental models and/or clinical trials. Pharmaceutical
compositions may be administered in a manner appropriate to the
disease to be treated (or prevented) as determined by persons
skilled in the medical arts. An appropriate dose and a suitable
duration and frequency of administration will be determined by such
factors as the condition of the subject or host, the type and
severity of the subject's disease, the particular form of the
active ingredient, and the method of administration. In general, an
appropriate dose and treatment regimen provides the composition(s)
in an amount sufficient to provide therapeutic and/or prophylactic
benefit (e.g., an improved clinical outcome, such as more frequent
complete or partial remissions, or longer disease-free and/or
overall survival, or a lessening of symptom severity). For
prophylactic use, a dose should be sufficient to prevent, delay the
onset of, or diminish the severity of a symptom associated with a
disease or medical disorder including an immunological disease or
disorder.
[0238] In another embodiment, business method is provided for
selling a therapeutic agent to treat a disease or disorder. The
business method comprises receiving information regarding a viral
polypeptide that increases the virulence of a virus in a host
infected with the virus, and identifying a cellular polypeptide to
which the viral polypeptide binds, wherein binding of the viral
polypeptide to the cellular polypeptide alters at least one
biological activity of a cell. In particular embodiments, the at
least one biological activity is immunoresponsiveness and the cell
is an immune cell. The method may further comprise identifying one
or more agents that inhibit binding of the viral polypeptide to the
cellular polypeptide and that alter the at least one biological
activity of the cell. As described herein when at least one
biological effect is altered, the risk of developing a disease or
medical disorder is reduced or at least one symptom of the disease
or medical disorder is reduced in a subject (or patient or host).
Such methods may further comprise selling the agent to a medical
professional or health caregiver, a distributor that sells the drug
to the medical professional, or to a patient in need of the
treatment for the disease or disorder.
[0239] Also provided herein is a system for guiding the selection
of a viral polypeptide to achieve a desired result. The system
comprises a computing device, which includes a knowledge base
comprising a plurality of polynucleotide sequences encoding a
plurality of viral polypeptides. The system also includes a second
knowledge base that comprises a plurality of rules for evaluating
and selecting a viral polypeptide that is a viral virulence factor
based upon information received regarding polynucleotide sequences
encoding viral polypeptides. The system further comprises means for
providing information regarding a target viral virulence factor and
a desired result to said computing device; and a means in the
computing device for identifying and categorizing or ranking at
least one polynucleotide sequence encoding a viral polypeptide that
may be used to identify a cellular polypeptide with which the viral
polypeptide binds.
[0240] Also contemplated is a computer program product for guiding
the selection of a viral polypeptide to achieve a desired result.
The computer program product includes a computer usable storage
medium having computer readable program code means embodied in the
medium. The computer readable program code means comprises computer
readable program code means for generating one knowledge base
comprising a plurality of polynucleotide sequences encoding a
plurality of viral polypeptides, and a second knowledge base that
comprises a plurality of rules for evaluating and selecting a viral
polypeptide that is a viral virulence factor based upon information
regarding polynucleotide sequences that encode viral polypeptides.
A computer program product may also comprises a computer readable
program code means for providing information regarding a target
viral virulence factor and a desired result to the computing
device; and computer readable program code means for identifying
and categorizing or ranking a target viral virulence factor that
may be used to identify a cellular polypeptide to which the viral
polypeptide binds, and wherein binding of the viral polypeptide to
the cellular polypeptide alters at least one biological activity of
a cell.
[0241] Categorizing the capability of at least one agent that
inhibits binding of a viral polypeptide that is a virulence factor
to a cellular polypeptide comprises analyzing the output from
scientific method analysis, chemical composition analysis, and/or
biological comparison analysis, for various techniques and assays
and sorting them according to their predicted effect (i.e., the
capability to alter at least one biological activity in a cell).
The results may include plotting of various assay results and data,
assigning numerical scores or values to the various results and
data, based upon one or more predicted effects, and ranking of
various assay results and data based upon the desired effect or
outcome. In addition, biological reaction analysis also includes
designing appropriate assay controls and methods of efficacy
measurement. A variety of charts, graphs, graphical models, and
other documents related to categorizing results and data from the
assays according to any of a variety of different factors,
including predicted potency and specificity, may be generated and
stored.
[0242] The methods and systems described herein may be practiced
without the aid of computers or related software. However, in
certain embodiments, the methods and systems described herein are
practiced using computers and software to accomplish one or more of
the analyses described. For example, computers and software may be
used for receiving information regarding a viral polypeptide that
increases the virulence of a virus in a host infected with the
virus may be performed using a computer device. In certain
embodiments, the device comprises at least one knowledge base. For
example, one knowledge base comprises a plurality of different
polynucleotide sequences that encode a plurality of viral
polypeptides. Another knowledge base useful for the methods
described herein comprises a plurality of rules for evaluating and
selecting a viral polypeptide that is a viral virulence factor. The
rules include whether a viral polypeptide identified from the
plurality of polynucleotide sequences comprises at least one of the
virulence traits of viral polypeptides, which are described in
detail herein.
[0243] Receiving information may be embodied in many different
forms, including, e.g., a method data processing system or computer
program product. Furthermore, the methods and systems described
herein may comprise an entirely hardware embodiment, an entirely
software embodiment, or an embodiment combining software and
hardware aspects. Furthermore, the methods may provide a computer
program product on a computer-usable storage medium that has
computer readable program code means embodied in the medium. Any
suitable computer readable medium may be used including, but not
limited to, hard disks, CD-ROMs, optical storage devices, and
magnetic storage devices.
[0244] The business methods and methods for selecting a therapeutic
agent described herein may further comprise cataloging and document
creation, which comprises sorting, serializing, and/or storing all
output from various sources and analysis for documentation and
retrieval. In certain embodiments, the output is organized and
rendered into a final document that is delivered to the medical
professional, medical research professional, or other health
caregiver. The documents may further comprise predictions,
prediction models, designs, and serialized custom products. Thus,
in certain embodiments, the methods and systems of the present
invention include organizing results of one or more of the analyses
described above into groups and serializing and cataloguing these
results for each customer or user (i.e., medical professional,
medical research professional, or caregiver or the like).
[0245] In particular embodiments, the computer or other
programmable data processing apparatus contain one or more
knowledge bases that include information and/or rules useful in
performing analyses. In certain embodiments, the computer or other
programmable data processing apparatus includes means for
determining or obtaining a gene or polynucleotide sequence that
encodes a viral polypeptide, based upon receiving information
regarding said sequence in any of a variety of formats, including
the entry of the sequence itself, the name of the gene and
organism, or a sequence identifier number from any one of a number
of available databases (either public or that may be
purchased).
[0246] In another embodiment, a knowledge base includes a variety
of different biological assays or information assigned for
achieving different results, such as a group of assays suitable for
determining levels of viral polypeptide expression, localization of
the viral polypeptide after it is expressed by an infected cell,
determining the effect on virulence of the virus by substituting,
deleting or inserting one or more amino acids in the viral
polypeptide, or information related to the location of the
polynucleotide sequence encoding the viral polypeptide in the viral
genome, and other assays and information. Such a knowledge base may
further comprise expert rules for determining possible biological
assays based upon user input regarding the desired result.
[0247] In other embodiments, a knowledge base comprises expert
rules for determining structural characteristics of a
polynucleotide sequence (e.g., a polynucleotide sequence that
encodes a viral polypeptide as described herein), such as rules
provided in the programs recited herein. In a related embodiment, a
knowledge base comprises expert rules for comparing a
polynucleotide sequence to another database, e.g., human genome
database, to identify and/or predict biological interactions of a
reagent in a cellular or genomic environment.
[0248] Thus, in one embodiment, information regarding a
polynucleotide that encodes a viral virulence factor, a target
cellular polypeptide, and an agent that alters the interaction
between the viral virulence factor and the cellular polypeptide and
desired result, e.g., altering a biological activity of a cell
(e.g., immunoresponsiveness of an immune cell), is inputted into a
computer comprising a knowledge base regarding agents, and the
computer selects appropriate agents based upon the desired result.
The computer then designs reagents agents suitable for treating a
disease or medical disorder, based upon the identified viral
polypeptide and cellular polypeptide target and agent, using expert
rules for analyzing the chemical composition of potential agents,
as well as analyzing potential interactions in a biological system,
such as a cell or genomic environment. The agents are then ranked
using several criteria, with related documentation produced.
[0249] As described herein a medical disease or disorder includes
an immunological disease or disorder, a cardiovascular disease or
disorder, a metabolic disease or disorder, or a proliferative
disease or disorder. An immunological disorder or disease includes
but is not limited to an autoimmune disease or an inflammatory
disease. Exemplary immunological diseases and disorders include but
are not limited to multiple sclerosis, rheumatoid arthritis,
systemic lupus erythematosus, graft versus host disease, sepsis,
diabetes, psoriasis, atherosclerosis, Sjogren's syndrome,
progressive systemic sclerosis, scleroderma, acute coronary
syndrome, ischemic reperfusion, Crohn's Disease, endometriosis,
glomerulonephritis, myasthenia gravis, idiopathic pulmonary
fibrosis, asthma, acute respiratory distress syndrome (ARDS),
vasculitis, and inflammatory autoimmune myositis. Examples of
cardiovascular diseases include atherosclerosis, endocarditis,
hypertension, and peripheral ischemic disease.
[0250] The following Examples are offered for the purpose of
illustrating the embodiments disclosed herein and are not to be
construed to limit the scope of this invention.
EXAMPLES
Example 1
Identification of RPTPs Expressed on Immune Cells that Bind
A41L
[0251] This Example describes a method for identifying cell surface
polypeptides that bind to A41L.
[0252] A recombinant expression vector comprising a polynucleotide
that encoded a Cowpox A41L fusion polypeptide was constructed for a
tandem affinity purification (TAP) procedure (also called TAP tag
procedure) (see also, e.g., Rigaut et al. Nat. Biotech. 17:1030-32
(1999); Puig et al., Methods 24:218-29 (2001); Knuesel et al. Mol.
Cell. Proteomics 2:1225-33 (2003)). The construct called A41LCRFC
was prepared and the fusion polypeptide expressed and isolated
according to standard molecular biology and affinity purification
techniques and methods. A schematic of the construct is provided in
FIG. 1. The A41LCRFC construct included a nucleotide sequence that
encoded a mature A41L coding sequence from Cowpox virus fused to
the C-terminus of the human growth hormone leader peptide. The CRFC
tandem affinity tag was fused to the C-terminus of A41L. The CRFC
tag included a human influenza virus hemagglutinin peptide, the HA
epitope, amino acids YPYDVDYA (SEQ ID NO:1, which is encoded by the
nucleotide sequence set forth in SEQ ID NO:2), for which antibodies
are commercially available, permitting detection of the expression
fusion polypeptide by immunochemistry methods, such as fluorescence
activated cell sorting (FACS) or immunoblotting. Fused to the
carboxyl terminal end of the HA epitope was a Protein C-tag, amino
acids EDQVDPRLIDGK (SEQ ID NO:4, which is encoded by the nucleotide
sequence set forth in SEQ ID NO:5), which is derived from the heavy
chain of human Protein C. To the carboxyl end of the Protein C-tag
was fused a Human Rhinovirus HRV3C protease site, amino acids
LEVLFQGP (SEQ ID NO:16, which is encoded by the nucleotide sequence
set forth in SEQ ID NO:17); and to the carboxyl end of the HRV3C
protease site was fused a mutein derivative of the Fc portion of a
human IgG.
[0253] A schematic illustrating the TAP tag procedure is presented
in FIG. 2. Ten .mu.g of the A41LCRFC fusion polypeptide that was
bound to Protein A was incubated with cell lysates prepared from
5.times.10.sup.6 monocytes. A variety of normal cells and tumor
cell types may be used to identify cellular polypeptides that bind
to or interact with A41L, including B cells and T cells (activated
or non-activated), macrophages, epithelial cells, fibroblasts, and
cell lines such as Raji (B cell lymphoma), THP-1 (acute monocytic
leukemia), and Jurkat (T cell leukemia).
[0254] The A41LCRFC/cell lysate complexes were washed and then
subjected to cleavage by the HRV3C protease, which released A41L
and associated proteins. Calcium chloride (1 M) was added to the
released A41L/cell lysate complexes, which were then applied to an
anti-protein C-Tag affinity resin. Calcium chloride is required for
the interaction of anti-C-tag and the C-tag epitope. The complexes
bound to the anti-protein C-Tag affinity resin were washed in a
buffer containing calcium chloride and then eluted by calcium
chelation using EGTA. The subsequent eluent was digested with
trypsin and the digested A41L complexes were subjected to direct
tandem mass spectrometry to identify A41L and its associated
proteins.
[0255] The sequences of the trypsin-generated peptides were
identified by mass spectrometry. The peptides were identified as
portions of the receptor-like protein tyrosine phosphatases, LAR,
RPTP-.sigma., and RPTP-.delta. as shown in FIGS. 3A, 3B, and 3C,
respectively.
Example 2
Preparation of A41L-Fc Fusion Polypeptides
[0256] This example describes preparation of recombinant expression
vectors for expression of an A41L-Fc fusion polypeptide and an
A41L-mutein Fc fusion polypeptide.
[0257] Recombinant expression vectors were prepared according to
methods routinely practiced by a person skilled in the molecular
biology art. A polynucleotide encoding A41L-Fc and a polynucleotide
encoding A41L-mutein Fc were cloned into the multiple cloning site
of the vector, pDC409 (SEQ ID NO:41) (see, e.g., U.S. Pat. No.
6,512,095 and U.S. Pat. No. 6,680,840, and references cited
therein). The amino acid sequence of the A41L-Fc polypeptide is set
forth in SEQ ID NO:32, and the amino acid sequence of the
A41L-mutein Fc polypeptide is set forth in SEQ ID NO: 31 (see FIG.
6). The nucleotide sequence that encodes the mutein Fc (human IgG1)
polypeptide (SEQ ID NO:23) is set forth in SEQ ID NO:24. Ten to
twenty micrograms of each expression plasmid were transfected into
a HEK293T cells or COS-7 cells (American Type Tissue Collection
(ATCC), Manassas, Va.) that were grown in 10 cm diameter standard
tissue culture plates to approximately 80% confluency. Transfection
was performed using Lipofectamine.TM. Plus.TM. (Invitrogen Corp.,
Carlsbad, Calif.). The transfected cells were cultured for 48
hours, and then supernatant from the cell cultures was harvested.
The A41L fusion proteins were purified by Protein A sepharose
affinity chromatography according to standard procedures.
Example 3
Preparation of Affinity Tags for Fusion Polypeptides
[0258] This example describes preparation of recombinant expression
vectors for expression of various affinity tags.
[0259] Fusion proteins, such as a fusion protein comprising a
virulence factor polypeptide, or portion thereof, encoded by a
viral virulence gene are fused in frame to an affinity tag for
detection and/or isolation, for example, by tandem affinity
purification (TAP). The fusion polypeptide may comprise more than
one affinity tag. Recombinant expression vectors that comprise
polynucleotide sequences encoding fusion proteins are prepared
according to methods and techniques well known and routinely used
by a person skilled in the molecular biology art (see also Example
2). As described herein, a fusion protein may further comprise at
least one protease site. The polynucleotides encoding the fusion
proteins may be inserted into any number of recombinant expression
vectors available from commercial vendors and manufacturers. The
vectors may be further adapted for insertion of polynucleotides
described herein, for example, to introduce or remove a restriction
site or to introduce or remove a regulatory element. Exemplary
vectors include but are not limited to pcDNA.TM.3.1 that contains
the CMV promoter (SEQ ID NO:39) (Invitrogen) (see, e.g., U.S. Pat.
Nos. 5,168,062 and 5,385,839); pSL9, a lentiviral expression
plasmid (SEQ ID NO:40); pDC409 (SEQ ID NO:41); and pAAV, an
adeno-associated virus expression plasmid (see, e.g., Stratagene,
La Jolla, Calif.) (for example, SEQ ID NO:42).
[0260] Examples of polypeptide and peptide sequences that may be
included in a fusion protein are presented in FIG. 4. The
expression constructs comprise a growth hormone (GH) signal peptide
sequence (SEQ ID NO:12) (encoded by the polynucleotide set forth in
SEQ ID NO:14). In certain embodiments, the sequence includes
restriction sites, for example, SpeI and Asp718 are added to the
C-terminus of the signal peptide (SEQ ID NO:13) to permit a
polypeptide moiety to be fused to the signal peptide sequence
(nucleotide sequence encoding GH with restriction sites: SEQ ID
NO:15).
[0261] As described herein, fusion polypeptides comprise one, two,
three, four, or more affinity tags and one or more protease sites.
Peptide spacer sequences may be included between any two
polypeptide moieties, such as between two affinity tags or between
and affinity tag and a protease site, or between a virulence factor
polypeptide open reading frame (ORF) and an affinity tag or
protease site. The peptide spacer sequences may be, but not
necessarily be, encoded by a nucleotide sequence that is a cleavage
site or recognition site for a restriction enzyme. An example of an
affinity tag includes an affinity tag combination that has more
than one polypeptide. For instance, an HAC tag comprises an
HA-epitope tag, C-TAG, and 2.times.SBP, which may be present in a
fusion protein in any order (see, e.g., SEQ ID NO:35 sets forth the
amino acid sequence of the HAC tag, wherein the HA epitope is
located at the amino terminal end of the affinity tag fused to a
C-TAG, which is fused to 2.times.SBP; SEQ ID NO:36 provides the
nucleotide sequence encoding this HAC tag).
[0262] Another affinity tag that is an affinity tag combination is
called herein a CRFC tag (see Example 1). A CRFC tag is a
combination of an HA-epitope tag, a C-TAG, a human Rhinovirus HRV3C
protease site, and an Fc polypeptide. An exemplary polypeptide
sequence is provided in SEQ ID NO:37 and the nucleotide sequence
encoding the CRFC tag is set forth in SEQ ID NO:38. The fusion
polypeptides comprising any of the affinity tags described herein,
which include affinity tag combinations, are used for tandem
affinity purification of target cellular polypeptides that interact
with a viral virulence factor, or a portion thereof.
[0263] From the foregoing it will be appreciated that, although
specific embodiments of the invention have been described herein
for purposes of illustration, various modifications may be made
without deviating from the spirit and scope of the invention. Those
skilled in the art will recognize, or be able to ascertain, using
no more than routine experimentation, many equivalents to the
specific embodiments of the invention described herein. Such
equivalents are intended to be encompassed by the following claims.
Sequence CWU 1
1
4418PRTArtificial Sequencehemagglutinin peptide sequence 1Tyr Pro
Tyr Asp Val Asp Tyr Ala 1 5 227DNAArtificial Sequencehemagglutinin
nucleotide sequence 2tacccctacg acgtgcccga ctacgcc
27326PRTArtificial Sequencecalmodulin binding polypeptide sequence
3Lys Arg Arg Trp Lys Lys Asn Phe Ile Ala Val Ser Ala Ala Asn Arg 1
5 10 15 Phe Lys Lys Ile Ser Ser Ser Gly Ala Leu 20 25
412PRTArtificial SequenceProtein C-tag peptide sequence 4Glu Asp
Gln Val Asp Pro Arg Leu Ile Asp Gly Lys 1 5 10 536DNAArtificial
SequenceProtein C-tag nucleotide sequence 5gaggaccagg tggacccccg
gctgatcgac ggcaag 36638PRTArtificial SequenceStreptavidin binding
protein sequence 6Met Asp Glu Lys Thr Thr Gly Trp Arg Gly Gly His
Val Val Glu Gly 1 5 10 15 Leu Ala Gly Glu Leu Glu Gln Leu Arg Ala
Arg Leu Glu His His Pro 20 25 30 Gln Gly Gln Arg Glu Pro 35
715PRTArtificial SequenceLow affinity streptavidin binding protein
sequence 7Asp Val Glu Ala Trp Leu Asp Glu Arg Val Pro Leu Val Glu
Thr 1 5 10 15 879PRTArtificial SequenceTandem streptavidin binding
protein sequence 8Met Asp Glu Lys Thr Thr Gly Trp Arg Gly Gly His
Val Val Glu Gly 1 5 10 15 Leu Ala Gly Glu Leu Glu Gln Leu Arg Ala
Arg Leu Glu His His Pro 20 25 30 Gln Gly Gln Arg Glu Pro Gly Ser
Gly Met Asp Glu Lys Thr Thr Gly 35 40 45 Trp Arg Gly Gly His Val
Val Glu Gly Leu Ala Gly Glu Leu Glu Gln 50 55 60 Leu Arg Ala Arg
Leu Glu His His Pro Gln Gly Gln Arg Glu Pro 65 70 75
9114DNAArtificial SequenceLow affinity streptavidin binding
nucleotide sequence 9atggacgaga agaccaccgg ctggcggggc ggccacgtgg
tggagggcct ggccggcgag 60ctggagcagc tgcgggcccg gctggagcac cacccccagg
gccagcggga gccc 11410240DNAArtificial SequenceTandem streptavidin
binding nucleotide sequence 10atggacgaga agaccaccgg ctggcggggc
ggccacgtgg tggagggcct ggccggcgag 60ctggagcagc tgcgggcccg gctggagcac
cacccccagg gccagcggga gcccggaagc 120ggtatggatg aaaaaactac
tggttggaga gggggacatg tagtcgaagg tctggccggc 180gagttagaac
aattaagagc tagattggaa catcatccac aaggtcaaag agaaccttag
2401113PRTArtificial SequenceSoftagTM peptide sequence 11Ser Leu
Ala Glu Leu Leu Asn Ala Gly Leu Gly Gly Ser 1 5 10 1226PRTHomo
sapiens 12Met Ala Thr Gly Ser Arg Thr Ser Leu Leu Leu Ala Phe Gly
Leu Leu 1 5 10 15 Cys Leu Pro Trp Leu Gln Glu Gly Ser Ala 20 25
1330PRTArtificial SequenceSynthesized human growth hormone peptide
with restriction sites 13Met Ala Thr Gly Ser Arg Thr Ser Leu Leu
Leu Ala Phe Gly Leu Leu 1 5 10 15 Cys Leu Pro Trp Leu Gln Glu Gly
Ser Ala Thr Ser Gly Thr 20 25 30 1478DNAHomo sapiens 14atggctacag
gctcccggac gtccctgctc ctggcttttg gcctgctctg cctgccctgg 60cttcaagagg
gcagtgca 781590DNAArtificial SequenceHuman growth hormone
nucleotide with restriction sites 15atggctacag gctcccggac
gtccctgctc ctggcttttg gcctgctctg cctgccctgg 60cttcaagagg gcagtgcaac
tagtggtacc 90168PRTHuman rhinovirus 16Leu Glu Val Leu Phe Gln Gly
Pro 1 5 1724DNAHuman rhinovirus 17ctggaggtgc tgttccaggg cccc
24181948PRTHomo sapiens 18Met Ala Pro Thr Trp Gly Pro Gly Met Val
Ser Val Val Gly Pro Met 1 5 10 15 Gly Leu Leu Val Val Leu Leu Val
Gly Gly Cys Ala Ala Glu Glu Pro 20 25 30 Pro Arg Phe Ile Lys Glu
Pro Lys Asp Gln Ile Gly Val Ser Gly Gly 35 40 45 Val Ala Ser Phe
Val Cys Gln Ala Thr Gly Asp Pro Lys Pro Arg Val 50 55 60 Thr Trp
Asn Lys Lys Gly Lys Lys Val Asn Ser Gln Arg Phe Glu Thr 65 70 75 80
Ile Glu Phe Asp Glu Ser Ala Gly Ala Val Leu Arg Ile Gln Pro Leu 85
90 95 Arg Thr Pro Arg Asp Glu Asn Val Tyr Glu Cys Val Ala Gln Asn
Ser 100 105 110 Val Gly Glu Ile Thr Val His Ala Lys Leu Thr Val Leu
Arg Glu Asp 115 120 125 Gln Leu Pro Ser Gly Phe Pro Asn Ile Asp Met
Gly Pro Gln Leu Lys 130 135 140 Val Val Glu Arg Thr Arg Thr Ala Thr
Met Leu Cys Ala Ala Ser Gly 145 150 155 160 Asn Pro Asp Pro Glu Ile
Thr Trp Phe Lys Asp Phe Leu Pro Val Asp 165 170 175 Pro Ser Ala Ser
Asn Gly Arg Ile Lys Gln Leu Arg Ser Asp Gln Ala 180 185 190 Phe Ser
His Leu Pro Thr Gly Ala Leu Gln Ile Glu Ser Ser Glu Glu 195 200 205
Thr Asp Gln Gly Lys Tyr Glu Cys Val Ala Thr Asn Ser Ala Gly Val 210
215 220 Arg Tyr Ser Ser Pro Ala Asn Leu Tyr Val Arg Ala Leu Leu Lys
Leu 225 230 235 240 Arg Arg Val Ala Pro Arg Phe Ser Ile Leu Pro Met
Ser His Glu Ile 245 250 255 Met Pro Gly Gly Asn Val Asn Ile Thr Cys
Val Ala Val Gly Ser Pro 260 265 270 Met Pro Tyr Val Lys Trp Met Gln
Gly Ala Glu Asp Leu Thr Pro Glu 275 280 285 Asp Asp Met Pro Val Gly
Arg Asn Val Leu Glu Leu Thr Asp Val Lys 290 295 300 Asp Ser Ala Asn
Tyr Thr Cys Val Ala Met Ser Ser Leu Gly Val Ile 305 310 315 320 Glu
Ala Val Ala Gln Ile Thr Val Lys Ser Leu Pro Lys Ala Pro Gly 325 330
335 Thr Pro Met Val Thr Glu Asn Thr Ala Thr Ser Ile Thr Ile Thr Trp
340 345 350 Asp Ser Gly Asn Pro Asp Pro Val Ser Tyr Tyr Val Ile Glu
Tyr Lys 355 360 365 Ser Lys Ser Gln Asp Gly Pro Tyr Gln Ile Lys Glu
Asp Ile Thr Thr 370 375 380 Thr Arg Tyr Ser Ile Gly Gly Leu Ser Pro
Asn Ser Glu Tyr Glu Ile 385 390 395 400 Trp Val Ser Ala Val Asn Ser
Ile Gly Gln Gly Pro Pro Ser Glu Ser 405 410 415 Val Val Thr Arg Thr
Gly Glu Gln Ala Pro Ala Ser Ala Pro Arg Asn 420 425 430 Val Gln Ala
Arg Met Leu Ser Ala Thr Thr Met Ile Val Gln Trp Glu 435 440 445 Glu
Pro Val Glu Pro Asn Gly Leu Ile Arg Gly Tyr Arg Val Tyr Tyr 450 455
460 Thr Met Glu Pro Glu His Pro Val Gly Asn Trp Gln Lys His Asn Val
465 470 475 480 Asp Asp Ser Leu Leu Thr Thr Val Gly Ser Leu Leu Glu
Asp Glu Thr 485 490 495 Tyr Thr Val Arg Val Leu Ala Phe Thr Ser Val
Gly Asp Gly Pro Leu 500 505 510 Ser Asp Pro Ile Gln Val Lys Thr Gln
Gln Gly Val Pro Gly Gln Pro 515 520 525 Met Asn Leu Arg Ala Glu Ala
Arg Ser Glu Thr Ser Ile Thr Leu Ser 530 535 540 Trp Ser Pro Pro Arg
Gln Glu Ser Ile Ile Lys Tyr Glu Leu Leu Phe 545 550 555 560 Arg Glu
Gly Asp His Gly Arg Glu Val Gly Arg Thr Phe Asp Pro Thr 565 570 575
Thr Ser Tyr Val Val Glu Asp Leu Lys Pro Asn Thr Glu Tyr Ala Phe 580
585 590 Arg Leu Ala Ala Arg Ser Pro Gln Gly Leu Gly Ala Phe Thr Pro
Val 595 600 605 Val Arg Gln Arg Thr Leu Gln Ser Lys Pro Ser Ala Pro
Pro Gln Asp 610 615 620 Val Lys Cys Val Ser Val Arg Ser Thr Ala Ile
Leu Val Ser Trp Arg 625 630 635 640 Pro Pro Pro Pro Glu Thr His Asn
Gly Ala Leu Val Gly Tyr Ser Val 645 650 655 Arg Tyr Arg Pro Leu Gly
Ser Glu Asp Pro Glu Pro Lys Glu Val Asn 660 665 670 Gly Ile Pro Pro
Thr Thr Thr Gln Ile Leu Leu Glu Ala Leu Glu Lys 675 680 685 Trp Thr
Gln Tyr Arg Ile Thr Thr Val Ala His Thr Glu Val Gly Pro 690 695 700
Gly Pro Glu Ser Ser Pro Val Val Val Arg Thr Asp Glu Asp Val Pro 705
710 715 720 Ser Ala Pro Pro Arg Lys Val Glu Ala Glu Ala Leu Asn Ala
Thr Ala 725 730 735 Ile Arg Val Leu Trp Arg Ser Pro Ala Pro Gly Arg
Gln His Gly Gln 740 745 750 Ile Arg Gly Tyr Gln Val His Tyr Val Arg
Met Glu Gly Ala Glu Ala 755 760 765 Arg Gly Pro Pro Arg Ile Lys Asp
Val Met Leu Ala Asp Ala Gln Trp 770 775 780 Glu Thr Asp Asp Thr Ala
Glu Tyr Glu Met Val Ile Thr Asn Leu Gln 785 790 795 800 Pro Glu Thr
Ala Tyr Ser Ile Thr Val Ala Ala Tyr Thr Met Lys Gly 805 810 815 Asp
Gly Ala Arg Ser Lys Pro Lys Val Val Val Thr Lys Gly Ala Val 820 825
830 Leu Gly Arg Pro Thr Leu Ser Val Gln Gln Thr Pro Glu Gly Ser Leu
835 840 845 Leu Ala Arg Trp Glu Pro Pro Ala Gly Thr Ala Glu Asp Gln
Val Leu 850 855 860 Gly Tyr Arg Leu Gln Phe Gly Arg Glu Asp Ser Thr
Pro Leu Ala Thr 865 870 875 880 Leu Glu Phe Pro Pro Ser Glu Asp Arg
Tyr Thr Ala Ser Gly Val His 885 890 895 Lys Gly Ala Thr Tyr Val Phe
Arg Leu Ala Ala Arg Ser Arg Gly Gly 900 905 910 Leu Gly Glu Glu Ala
Ala Glu Val Leu Ser Ile Pro Glu Asp Thr Pro 915 920 925 Arg Gly His
Pro Gln Ile Leu Glu Ala Ala Gly Asn Ala Ser Ala Gly 930 935 940 Thr
Val Leu Leu Arg Trp Leu Pro Pro Val Pro Ala Glu Arg Asn Gly 945 950
955 960 Ala Ile Val Lys Tyr Thr Val Ala Val Arg Glu Ala Gly Ala Leu
Gly 965 970 975 Pro Ala Arg Glu Thr Glu Leu Pro Ala Ala Ala Glu Pro
Gly Ala Glu 980 985 990 Asn Ala Leu Thr Leu Gln Gly Leu Lys Pro Asp
Thr Ala Tyr Asp Leu 995 1000 1005 Gln Val Arg Ala His Thr Arg Arg
Gly Pro Gly Pro Phe Ser Pro 1010 1015 1020 Pro Val Arg Tyr Arg Thr
Phe Leu Arg Asp Gln Val Ser Pro Lys 1025 1030 1035 Asn Phe Lys Val
Lys Met Ile Met Lys Thr Ser Val Leu Leu Ser 1040 1045 1050 Trp Glu
Phe Pro Asp Asn Tyr Asn Ser Pro Thr Pro Tyr Lys Ile 1055 1060 1065
Gln Tyr Asn Gly Leu Thr Leu Asp Val Asp Gly Arg Thr Thr Lys 1070
1075 1080 Lys Leu Ile Thr His Leu Lys Pro His Thr Phe Tyr Asn Phe
Val 1085 1090 1095 Leu Thr Asn Arg Gly Ser Ser Leu Gly Gly Leu Gln
Gln Thr Val 1100 1105 1110 Thr Ala Trp Thr Ala Phe Asn Leu Leu Asn
Gly Lys Pro Ser Val 1115 1120 1125 Ala Pro Lys Pro Asp Ala Asp Gly
Phe Ile Met Val Tyr Leu Pro 1130 1135 1140 Asp Gly Gln Ser Pro Val
Pro Val Gln Ser Tyr Phe Ile Val Met 1145 1150 1155 Val Pro Leu Arg
Lys Ser Arg Gly Gly Gln Phe Leu Thr Pro Leu 1160 1165 1170 Gly Ser
Pro Glu Asp Met Asp Leu Glu Glu Leu Ile Gln Asp Ile 1175 1180 1185
Ser Arg Leu Gln Arg Arg Ser Leu Arg His Ser Arg Gln Leu Glu 1190
1195 1200 Val Pro Arg Pro Tyr Ile Ala Ala Arg Phe Ser Val Leu Pro
Pro 1205 1210 1215 Thr Phe His Pro Gly Asp Gln Lys Gln Tyr Gly Gly
Phe Asp Asn 1220 1225 1230 Arg Gly Leu Glu Pro Gly His Arg Tyr Val
Leu Phe Val Leu Ala 1235 1240 1245 Val Leu Gln Lys Ser Glu Pro Thr
Phe Ala Ala Ser Pro Phe Ser 1250 1255 1260 Asp Pro Phe Gln Leu Asp
Asn Pro Asp Pro Gln Pro Ile Val Asp 1265 1270 1275 Gly Glu Glu Gly
Leu Ile Trp Val Ile Gly Pro Val Leu Ala Val 1280 1285 1290 Val Phe
Ile Ile Cys Ile Val Ile Ala Ile Leu Leu Tyr Lys Asn 1295 1300 1305
Lys Pro Asp Ser Lys Arg Lys Asp Ser Glu Pro Arg Thr Lys Cys 1310
1315 1320 Leu Leu Asn Asn Ala Asp Leu Ala Pro His His Pro Lys Asp
Pro 1325 1330 1335 Val Glu Met Arg Arg Ile Asn Phe Gln Thr Pro Asp
Ser Gly Leu 1340 1345 1350 Arg Ser Pro Leu Arg Glu Pro Gly Phe His
Phe Glu Ser Met Leu 1355 1360 1365 Ser His Pro Pro Ile Pro Ile Ala
Asp Met Ala Glu His Thr Glu 1370 1375 1380 Arg Leu Lys Ala Asn Asp
Ser Leu Lys Leu Ser Gln Glu Tyr Glu 1385 1390 1395 Ser Ile Asp Pro
Gly Gln Gln Phe Thr Trp Glu His Ser Asn Leu 1400 1405 1410 Glu Val
Asn Lys Pro Lys Asn Arg Tyr Ala Asn Val Ile Ala Tyr 1415 1420 1425
Asp His Ser Arg Val Ile Leu Gln Pro Ile Glu Gly Ile Met Gly 1430
1435 1440 Ser Asp Tyr Ile Asn Ala Asn Tyr Val Asp Gly Tyr Arg Cys
Gln 1445 1450 1455 Asn Ala Tyr Ile Ala Thr Gln Gly Pro Leu Pro Glu
Thr Phe Gly 1460 1465 1470 Asp Phe Trp Arg Met Val Trp Glu Gln Arg
Ser Ala Thr Ile Val 1475 1480 1485 Met Met Thr Arg Leu Glu Glu Lys
Ser Arg Ile Lys Cys Asp Gln 1490 1495 1500 Tyr Trp Pro Asn Arg Gly
Thr Glu Thr Tyr Gly Phe Ile Gln Val 1505 1510 1515 Thr Leu Leu Asp
Thr Ile Glu Leu Ala Thr Phe Cys Val Arg Thr 1520 1525 1530 Phe Ser
Leu His Lys Asn Gly Ser Ser Glu Lys Arg Glu Val Arg 1535 1540 1545
Gln Phe Gln Phe Thr Ala Trp Pro Asp His Gly Val Pro Glu Tyr 1550
1555 1560 Pro Thr Pro Phe Leu Ala Phe Leu Arg Arg Val Lys Thr Cys
Asn 1565 1570 1575 Pro Pro Asp Ala Gly Pro Ile Val Val His Cys Ser
Ala Gly Val 1580 1585 1590 Gly Arg Thr Gly Cys Phe Ile Val Ile Asp
Ala Met Leu Glu Arg 1595 1600 1605 Ile Lys Pro Glu Lys Thr Val Asp
Val Tyr Gly His Val Thr Leu 1610 1615 1620 Met Arg Ser Gln Arg Asn
Tyr Met Val Gln Thr Glu Asp Gln Tyr 1625 1630 1635 Ser Phe Ile His
Glu Ala Leu Leu Glu Ala Val Gly Cys Gly Asn 1640 1645 1650 Thr Glu
Val Pro Ala Arg Ser Leu Tyr Ala Tyr Ile Gln Lys Leu 1655 1660 1665
Ala Gln Val Glu Pro Gly Glu His Val Thr Gly Met Glu Leu Glu 1670
1675 1680 Phe Lys Arg Leu Ala Asn Ser Lys Ala His Thr Ser Arg Phe
Ile 1685 1690 1695 Ser Ala Asn Leu Pro Cys Asn Lys Phe Lys Asn Arg
Leu Val Asn 1700 1705 1710 Ile Met Pro Tyr Glu Ser Thr Arg Val Cys
Leu Gln Pro Ile Arg 1715 1720 1725 Gly Val Glu Gly Ser Asp Tyr Ile
Asn Ala Ser Phe Ile Asp Gly 1730 1735 1740 Tyr Arg Gln Gln Lys Ala
Tyr Ile Ala Thr Gln Gly Pro Leu Ala 1745
1750 1755 Glu Thr Thr Glu Asp Phe Trp Arg Met Leu Trp Glu Asn Asn
Ser 1760 1765 1770 Thr Ile Val Val Met Leu Thr Lys Leu Arg Glu Met
Gly Arg Glu 1775 1780 1785 Lys Cys His Gln Tyr Trp Pro Ala Glu Arg
Ser Ala Arg Tyr Gln 1790 1795 1800 Tyr Phe Val Val Asp Pro Met Ala
Glu Tyr Asn Met Pro Gln Tyr 1805 1810 1815 Ile Leu Arg Glu Phe Lys
Val Thr Asp Ala Arg Asp Gly Gln Ser 1820 1825 1830 Arg Thr Val Arg
Gln Phe Gln Phe Thr Asp Trp Pro Glu Gln Gly 1835 1840 1845 Val Pro
Lys Ser Gly Glu Gly Phe Ile Asp Phe Ile Gly Gln Val 1850 1855 1860
His Lys Thr Lys Glu Gln Phe Gly Gln Asp Gly Pro Ile Ser Val 1865
1870 1875 His Cys Ser Ala Gly Val Gly Arg Thr Gly Val Phe Ile Thr
Leu 1880 1885 1890 Ser Ile Val Leu Glu Arg Met Arg Tyr Glu Gly Val
Val Asp Ile 1895 1900 1905 Phe Gln Thr Val Lys Met Leu Arg Thr Gln
Arg Pro Ala Met Val 1910 1915 1920 Gln Thr Glu Asp Glu Tyr Gln Phe
Cys Tyr Gln Ala Ala Leu Glu 1925 1930 1935 Tyr Leu Gly Ser Phe Asp
His Tyr Ala Thr 1940 1945 191913PRTHomo sapiens 19Met Val His Val
Ala Arg Leu Leu Leu Leu Leu Leu Thr Phe Phe Leu 1 5 10 15 Arg Thr
Asp Ala Glu Thr Pro Pro Arg Phe Thr Arg Thr Pro Val Asp 20 25 30
Gln Thr Gly Val Ser Gly Gly Val Ala Ser Phe Ile Cys Gln Ala Thr 35
40 45 Gly Asp Pro Arg Pro Lys Ile Val Trp Asn Lys Lys Gly Lys Lys
Val 50 55 60 Ser Asn Gln Arg Phe Glu Val Ile Glu Phe Asp Asp Gly
Ser Gly Ser 65 70 75 80 Val Leu Arg Ile Gln Pro Leu Arg Thr Pro Arg
Asp Glu Ala Ile Tyr 85 90 95 Glu Cys Val Ala Ser Asn Asn Val Gly
Glu Ile Ser Val Ser Thr Arg 100 105 110 Leu Thr Val Leu Arg Glu Asp
Gln Ile Pro Arg Gly Phe Pro Thr Ile 115 120 125 Asp Met Gly Pro Gln
Leu Lys Val Val Glu Arg Thr Arg Thr Ala Thr 130 135 140 Met Leu Cys
Ala Ala Ser Gly Asn Pro Asp Pro Glu Ile Thr Trp Phe 145 150 155 160
Lys Asp Phe Leu Pro Val Asp Thr Ser Asn Asn Asn Gly Arg Ile Lys 165
170 175 Gln Leu Arg Ser Gly Arg Val Phe Lys Arg Leu Asn Arg Arg Ala
Leu 180 185 190 Gln Ile Glu Gln Ser Glu Glu Ser Asp Gln Gly Lys Tyr
Glu Cys Val 195 200 205 Ala Thr Asn Ser Ala Gly Thr Arg Tyr Ser Ala
Pro Ala Asn Leu Tyr 210 215 220 Val Arg Val Glu Thr Pro Gln Val Arg
Arg Val Pro Pro Arg Phe Ser 225 230 235 240 Ile Pro Pro Thr Asn His
Glu Ile Met Pro Gly Gly Ser Val Asn Ile 245 250 255 Thr Cys Val Ala
Val Gly Ser Pro Met Pro Tyr Val Lys Trp Met Leu 260 265 270 Gly Ala
Glu Asp Leu Thr Pro Glu Asp Asp Met Pro Ile Gly Arg Asn 275 280 285
Val Leu Glu Leu Asn Asp Val Arg Gln Ser Ala Asn Tyr Thr Cys Val 290
295 300 Ala Met Ser Thr Leu Gly Val Ile Glu Ala Ile Ala Gln Ile Thr
Val 305 310 315 320 Lys Ala Leu Pro Lys Pro Pro Gly Thr Pro Val Val
Thr Glu Ser Thr 325 330 335 Ala Thr Ser Ile Thr Leu Thr Trp Asp Ser
Gly Asn Pro Glu Pro Val 340 345 350 Ser Tyr Tyr Ile Ile Gln His Lys
Pro Lys Asn Ser Glu Glu Leu Tyr 355 360 365 Lys Glu Ile Asp Gly Val
Ala Thr Thr Arg Tyr Ser Val Ala Gly Leu 370 375 380 Ser Pro Tyr Ser
Asp Tyr Glu Phe Arg Val Val Ala Val Asn Asn Ile 385 390 395 400 Gly
Arg Gly Pro Pro Ser Glu Pro Val Leu Thr Gln Thr Ser Glu Gln 405 410
415 Ala Pro Ser Ser Ala Pro Arg Asp Val Gln Ala Arg Met Leu Ser Ser
420 425 430 Thr Thr Ile Leu Val Gln Trp Lys Glu Pro Glu Glu Pro Asn
Gly Gln 435 440 445 Ile Gln Gly Tyr Arg Val Tyr Tyr Thr Met Asp Pro
Thr Gln His Val 450 455 460 Asn Asn Trp Met Lys His Asn Val Ala Asp
Ser Gln Ile Thr Thr Ile 465 470 475 480 Gly Asn Leu Val Pro Gln Lys
Thr Tyr Ser Val Lys Val Leu Ala Phe 485 490 495 Thr Ser Ile Gly Asp
Gly Pro Leu Ser Ser Asp Ile Gln Val Ile Thr 500 505 510 Gln Thr Gly
Val Pro Gly Gln Pro Leu Asn Phe Lys Ala Glu Pro Glu 515 520 525 Ser
Glu Thr Ser Ile Leu Leu Ser Trp Thr Pro Pro Arg Ser Asp Thr 530 535
540 Ile Ala Asn Tyr Glu Leu Val Tyr Lys Asp Gly Glu His Gly Glu Glu
545 550 555 560 Gln Arg Ile Thr Ile Glu Pro Gly Thr Ser Tyr Arg Leu
Gln Gly Leu 565 570 575 Lys Pro Asn Ser Leu Tyr Tyr Phe Arg Leu Ala
Ala Arg Ser Pro Gln 580 585 590 Gly Leu Gly Ala Ser Thr Ala Glu Ile
Ser Ala Arg Thr Met Gln Ser 595 600 605 Lys Pro Ser Ala Pro Pro Gln
Asp Ile Ser Cys Thr Ser Pro Ser Ser 610 615 620 Thr Ser Ile Leu Val
Ser Trp Gln Pro Pro Pro Val Glu Lys Gln Asn 625 630 635 640 Gly Ile
Ile Thr Glu Tyr Ser Ile Lys Tyr Thr Ala Val Asp Gly Glu 645 650 655
Asp Asp Lys Pro His Glu Ile Leu Gly Ile Pro Ser Asp Thr Thr Lys 660
665 670 Tyr Leu Leu Glu Gln Leu Glu Lys Trp Thr Glu Tyr Arg Ile Thr
Val 675 680 685 Thr Ala His Thr Asp Val Gly Pro Gly Pro Glu Ser Leu
Ser Val Leu 690 695 700 Ile Arg Thr Asn Glu Asp Val Pro Ser Gly Pro
Pro Arg Lys Val Glu 705 710 715 720 Val Glu Ala Val Asn Ser Thr Ser
Val Lys Val Ser Trp Arg Ser Pro 725 730 735 Val Pro Asn Lys Gln His
Gly Gln Ile Arg Gly Tyr Gln Val His Tyr 740 745 750 Val Arg Met Glu
Asn Gly Glu Pro Lys Gly Gln Pro Met Leu Lys Asp 755 760 765 Val Met
Leu Ala Asp Ala Gln Trp Glu Phe Asp Asp Thr Thr Glu His 770 775 780
Asp Met Ile Ile Ser Gly Leu Gln Pro Glu Thr Ser Tyr Ser Leu Thr 785
790 795 800 Val Thr Ala Tyr Thr Thr Lys Gly Asp Gly Ala Arg Ser Lys
Pro Lys 805 810 815 Leu Val Ser Thr Thr Gly Ala Val Pro Gly Lys Pro
Arg Leu Val Ile 820 825 830 Asn His Thr Gln Met Asn Thr Ala Leu Ile
Gln Trp His Pro Pro Val 835 840 845 Asp Thr Phe Gly Pro Leu Gln Gly
Tyr Arg Leu Lys Phe Gly Arg Lys 850 855 860 Asp Met Glu Pro Leu Thr
Thr Leu Glu Phe Ser Glu Lys Glu Asp His 865 870 875 880 Phe Thr Ala
Thr Asp Ile His Lys Gly Ala Ser Tyr Val Phe Arg Leu 885 890 895 Ser
Ala Arg Asn Lys Val Gly Phe Gly Glu Glu Met Val Lys Glu Ile 900 905
910 Ser Ile Pro Glu Glu Val Pro Thr Gly Phe Pro Gln Asn Leu His Ser
915 920 925 Glu Gly Thr Thr Ser Thr Ser Val Gln Leu Ser Trp Gln Pro
Pro Val 930 935 940 Leu Ala Glu Arg Asn Gly Ile Ile Thr Lys Tyr Thr
Leu Leu Tyr Arg 945 950 955 960 Asp Ile Asn Ile Pro Leu Leu Pro Met
Glu Gln Leu Ile Val Pro Ala 965 970 975 Asp Thr Thr Met Thr Leu Thr
Gly Leu Lys Pro Asp Thr Thr Tyr Asp 980 985 990 Val Lys Val Arg Ala
His Thr Ser Lys Gly Pro Gly Pro Tyr Ser Pro 995 1000 1005 Ser Val
Gln Phe Arg Thr Leu Pro Val Asp Gln Val Phe Ala Lys 1010 1015 1020
Asn Phe His Val Lys Ala Val Met Lys Thr Ser Val Leu Leu Ser 1025
1030 1035 Trp Glu Ile Pro Glu Asn Tyr Asn Ser Ala Met Pro Phe Lys
Ile 1040 1045 1050 Leu Tyr Asp Asp Gly Lys Met Val Glu Glu Val Asp
Gly Arg Ala 1055 1060 1065 Thr Gln Lys Leu Ile Val Asn Leu Lys Pro
Glu Lys Ser Tyr Ser 1070 1075 1080 Phe Val Leu Thr Asn Arg Gly Asn
Ser Ala Gly Gly Leu Gln His 1085 1090 1095 Arg Val Thr Ala Lys Thr
Ala Pro Asp Val Leu Arg Thr Lys Pro 1100 1105 1110 Ala Phe Ile Gly
Lys Thr Asn Leu Asp Gly Met Ile Thr Val Gln 1115 1120 1125 Leu Pro
Glu Val Pro Ala Asn Glu Asn Ile Lys Gly Tyr Tyr Ile 1130 1135 1140
Ile Ile Val Pro Leu Lys Lys Ser Arg Gly Lys Phe Ile Lys Pro 1145
1150 1155 Trp Glu Ser Pro Asp Glu Met Glu Leu Asp Glu Leu Leu Lys
Glu 1160 1165 1170 Ile Ser Arg Lys Arg Arg Ser Ile Arg Tyr Gly Arg
Glu Val Glu 1175 1180 1185 Leu Lys Pro Tyr Ile Ala Ala His Phe Asp
Val Leu Pro Thr Glu 1190 1195 1200 Phe Thr Leu Gly Asp Asp Lys His
Tyr Gly Gly Phe Thr Asn Lys 1205 1210 1215 Gln Leu Gln Ser Gly Gln
Glu Tyr Val Phe Phe Val Leu Ala Val 1220 1225 1230 Met Glu His Ala
Glu Ser Lys Met Tyr Ala Thr Ser Pro Tyr Ser 1235 1240 1245 Asp Pro
Val Val Ser Met Asp Leu Asp Pro Gln Pro Ile Thr Asp 1250 1255 1260
Glu Glu Glu Gly Leu Ile Trp Val Val Gly Pro Val Leu Ala Val 1265
1270 1275 Val Phe Ile Ile Cys Ile Val Ile Ala Ile Leu Leu Tyr Lys
Arg 1280 1285 1290 Lys Arg Ala Glu Ser Asp Ser Arg Lys Ser Ser Ile
Pro Asn Asn 1295 1300 1305 Lys Glu Ile Pro Ser His His Pro Thr Asp
Pro Val Glu Leu Arg 1310 1315 1320 Arg Leu Asn Phe Gln Thr Pro Gly
Met Ala Ser His Pro Pro Ile 1325 1330 1335 Pro Ile Leu Glu Leu Ala
Asp His Ile Glu Arg Leu Lys Ala Asn 1340 1345 1350 Asp Asn Leu Lys
Phe Ser Gln Glu Tyr Glu Ser Ile Asp Pro Gly 1355 1360 1365 Gln Gln
Phe Thr Trp Glu His Ser Asn Leu Glu Val Asn Lys Pro 1370 1375 1380
Lys Asn Arg Tyr Ala Asn Val Ile Ala Tyr Asp His Ser Arg Val 1385
1390 1395 Leu Leu Ser Ala Ile Glu Gly Ile Pro Gly Ser Asp Tyr Val
Asn 1400 1405 1410 Ala Asn Tyr Ile Asp Gly Tyr Arg Lys Gln Asn Ala
Tyr Ile Ala 1415 1420 1425 Thr Gln Gly Ser Leu Pro Glu Thr Phe Gly
Asp Phe Trp Arg Met 1430 1435 1440 Ile Trp Glu Gln Arg Ser Ala Thr
Val Val Met Met Thr Lys Leu 1445 1450 1455 Glu Glu Arg Ser Arg Val
Lys Cys Asp Gln Tyr Trp Pro Ser Arg 1460 1465 1470 Gly Thr Glu Thr
His Gly Leu Val Gln Val Thr Leu Leu Asp Thr 1475 1480 1485 Val Glu
Leu Ala Thr Tyr Cys Val Arg Thr Phe Ala Leu Tyr Lys 1490 1495 1500
Asn Gly Ser Ser Glu Lys Arg Glu Val Arg Gln Phe Gln Phe Thr 1505
1510 1515 Ala Trp Pro Asp His Gly Val Pro Glu His Pro Thr Pro Phe
Leu 1520 1525 1530 Ala Phe Leu Arg Arg Val Lys Thr Cys Asn Pro Pro
Asp Ala Gly 1535 1540 1545 Pro Met Val Val His Cys Ser Ala Gly Val
Gly Arg Thr Gly Cys 1550 1555 1560 Phe Ile Val Ile Asp Ala Met Leu
Glu Arg Ile Lys His Glu Lys 1565 1570 1575 Thr Val Asp Ile Tyr Gly
His Val Thr Leu Met Arg Ala Gln Arg 1580 1585 1590 Asn Tyr Met Val
Gln Thr Glu Asp Gln Tyr Ile Phe Ile His Asp 1595 1600 1605 Ala Leu
Leu Glu Ala Val Thr Cys Gly Asn Thr Glu Val Pro Ala 1610 1615 1620
Arg Asn Leu Tyr Ala Tyr Ile Gln Lys Leu Thr Gln Ile Glu Thr 1625
1630 1635 Gly Glu Asn Val Thr Gly Met Glu Leu Glu Phe Lys Arg Leu
Ala 1640 1645 1650 Ser Ser Lys Ala His Thr Ser Arg Phe Ile Ser Ala
Asn Leu Pro 1655 1660 1665 Cys Asn Lys Phe Lys Asn Arg Leu Val Asn
Ile Met Pro Tyr Glu 1670 1675 1680 Ser Thr Arg Val Cys Leu Gln Pro
Ile Arg Gly Val Glu Gly Ser 1685 1690 1695 Asp Tyr Ile Asn Ala Ser
Phe Ile Asp Gly Tyr Arg Gln Gln Lys 1700 1705 1710 Ala Tyr Ile Ala
Thr Gln Gly Pro Leu Ala Glu Thr Thr Glu Asp 1715 1720 1725 Phe Trp
Arg Met Leu Trp Glu His Asn Ser Thr Ile Val Val Met 1730 1735 1740
Leu Thr Lys Leu Arg Glu Met Gly Arg Glu Lys Cys His Gln Tyr 1745
1750 1755 Trp Pro Ala Glu Arg Ser Ala Arg Tyr Gln Tyr Phe Val Val
Asp 1760 1765 1770 Pro Met Ala Glu Tyr Asn Met Pro Gln Tyr Ile Leu
Arg Glu Phe 1775 1780 1785 Lys Val Thr Asp Ala Arg Asp Gly Gln Ser
Arg Thr Val Arg Gln 1790 1795 1800 Phe Gln Phe Thr Asp Trp Pro Glu
Gln Gly Val Pro Lys Ser Gly 1805 1810 1815 Glu Gly Phe Ile Asp Phe
Ile Gly Gln Val His Lys Thr Lys Glu 1820 1825 1830 Gln Phe Gly Gln
Asp Gly Pro Ile Ser Val His Cys Ser Ala Gly 1835 1840 1845 Val Gly
Arg Thr Gly Val Phe Ile Thr Leu Ser Ile Val Leu Glu 1850 1855 1860
Arg Met Arg Tyr Glu Gly Val Val Asp Ile Phe Gln Thr Val Lys 1865
1870 1875 Met Leu Arg Thr Gln Arg Pro Ala Met Val Gln Thr Glu Asp
Gln 1880 1885 1890 Tyr Gln Phe Ser Tyr Arg Ala Ala Leu Glu Tyr Leu
Gly Ser Phe 1895 1900 1905 Asp His Tyr Ala Thr 1910
208PRTArtificial SequenceFLAG epitope tag peptide 20Asp Tyr Lys Asp
Asp Asp Asp Lys 1 5 218PRTArtificial SequenceXPRESS epitope tag
peptide 21Asp Leu Tyr Asp Asp Asp Asp Lys 1 5 226PRTHomo sapiens
22Leu Leu Gly Gly Pro Ser 1 5 23226PRTArtificial SequenceMutated
human amino acid sequence (mutein Fc) 23Lys Thr His Thr Cys Pro Pro
Cys Pro Ala Pro Glu Ala Glu Gly Ala 1 5 10 15 Pro Ser Val Phe Leu
Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile 20 25 30 Ser Arg Thr
Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu 35 40 45 Asp
Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His 50 55
60 Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg
65 70 75 80 Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn
Gly Lys
85 90 95 Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro
Ile Glu 100 105 110 Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu
Pro Gln Val Tyr 115 120 125 Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr
Lys Asn Gln Val Ser Leu 130 135 140 Thr Cys Leu Val Lys Gly Phe Tyr
Pro Ser Asp Ile Ala Val Glu Trp 145 150 155 160 Glu Ser Asn Gly Gln
Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val 165 170 175 Leu Asp Ser
Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp 180 185 190 Lys
Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His 195 200
205 Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro
210 215 220 Gly Lys 225 24678DNAArtificial SequenceMutated human
oligonucleotide sequence (mutein Fc) 24aaaactcaca catgcccacc
gtgcccagca cctgaagccg agggcgcgcc gtcagtcttc 60ctcttccccc caaaacccaa
ggacaccctc atgatctccc ggacccctga ggtcacatgc 120gtggtggtgg
acgtgagcca cgaagaccct gaggtcaagt tcaactggta cgtggacggc
180gtggaggtgc ataatgccaa gacaaagccg cgggaggagc agtacaacag
cacgtaccgt 240gtggtcagcg tcctcaccgt cctgcaccag gactggctga
atggcaagga gtacaagtgc 300aaggtctcca acaaagccct cccagccccc
atcgagaaaa ccatctccaa agccaaaggg 360cagccccgag aaccacaggt
gtacaccctg cccccatccc gggatgagct gaccaagaac 420caggtcagcc
tgacctgcct ggtcaaaggc ttctatccca gcgacatcgc cgtggagtgg
480gagagcaatg ggcagccgga gaacaactac aagaccacgc ctcccgtgct
ggactccgac 540ggctccttct tcctctatag caagctcacc gtggacaaga
gcaggtggca gcaggggaac 600gtcttctcat gctccgtgat gcatgaggct
ctgcacaacc actacacgca gaagagcctc 660tccctgtctc cgggtaaa
67825228PRTHomo sapiens 25Cys Asp Lys Thr His Thr Cys Pro Pro Cys
Pro Ala Pro Glu Leu Leu 1 5 10 15 Gly Gly Pro Ser Val Phe Leu Phe
Pro Pro Lys Pro Lys Asp Thr Leu 20 25 30 Met Ile Ser Arg Thr Pro
Glu Val Thr Cys Val Val Val Asp Val Ser 35 40 45 His Glu Asp Pro
Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu 50 55 60 Val His
Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr 65 70 75 80
Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn 85
90 95 Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala
Pro 100 105 110 Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg
Glu Pro Gln 115 120 125 Val Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu
Thr Lys Asn Gln Val 130 135 140 Ser Leu Thr Cys Leu Val Lys Gly Phe
Tyr Pro Ser Asp Ile Ala Val 145 150 155 160 Glu Trp Glu Ser Asn Gly
Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro 165 170 175 Pro Val Leu Asp
Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr 180 185 190 Val Asp
Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val 195 200 205
Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu 210
215 220 Ser Pro Gly Lys 225 2618PRTArtificial SequenceMutated human
peptide sequence (NH2 end of mutein Fc) 26Lys Thr His Thr Cys Pro
Pro Cys Pro Ala Pro Glu Ala Glu Gly Ala 1 5 10 15 Pro Ser
2720PRTArtificial SequenceMutated human peptide sequence (NH2 end
of mutein Fc with 2 amino acid spacer) 27Gly Ser Lys Thr His Thr
Cys Pro Pro Cys Pro Ala Pro Glu Ala Glu 1 5 10 15 Gly Ala Pro Ser
20 287PRTUnknownTEV motif 28Glu Xaa Xaa Tyr Xaa Gln Xaa 1 5
297PRTTobacco etch virus 29Glu Asn Leu Tyr Phe Gln Ser 1 5
307PRTTobacco etch virus 30Glu Asn Leu Tyr Phe Gln Gly 1 5
31468PRTArtificial SequenceMutated human fusion peptide
(A41L-Mutein Fc) 31Met Ala Thr Gly Ser Arg Thr Ser Leu Leu Leu Ala
Phe Gly Leu Leu 1 5 10 15 Cys Leu Pro Trp Leu Gln Glu Gly Ser Ala
Thr Ser Gly Thr Thr Ser 20 25 30 Glu Pro Ala Tyr Asp Lys Ser Val
Cys Asp Ser Asn Asn Lys Glu Tyr 35 40 45 Met Gly Ile Glu Val Tyr
Val Glu Ala Thr Leu Asp Glu Pro Leu Lys 50 55 60 Gln Thr Thr Cys
Glu Ser Glu Ile His Lys Tyr Gly Ala Ser Val Ser 65 70 75 80 Asn Gly
Gly Leu Asn Ile Ser Val Asp Leu Leu Asn Cys Phe Leu Asn 85 90 95
Phe His Thr Val Gly Val Tyr Thr Asn Arg Asp Thr Gly Val Tyr Thr 100
105 110 Asn Arg Asp Thr Val Tyr Ala Lys Phe Ala Ser Leu Asp Pro Ser
Thr 115 120 125 Glu Pro Ile Asn Ser Met Thr His Asp Asp Leu Val Lys
Leu Thr Glu 130 135 140 Glu Cys Ile Val Asp Ile Tyr Leu Lys Cys Glu
Val Asp Lys Thr Lys 145 150 155 160 Asp Phe Met Lys Asn Gly Asn Arg
Leu Lys Pro Arg Asp Phe Lys Thr 165 170 175 Val Pro Pro Ser Asn Val
Gly Ser Met Ile Glu Leu Gln Ser Asp Tyr 180 185 190 Cys Val Glu Asp
Val Thr Ala Tyr Val Lys Ile Tyr Asp Glu Cys Gly 195 200 205 Asn Ile
Lys Gln His Ser Ile Pro Thr Leu Arg Asp Tyr Phe Thr Thr 210 215 220
Lys Asn Gly Gln Pro Arg Lys Ile Leu Lys Lys Lys Phe Asp Ser Cys 225
230 235 240 Gly Ser Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu
Ala Glu 245 250 255 Gly Ala Pro Ser Val Phe Leu Phe Pro Pro Lys Pro
Lys Asp Thr Leu 260 265 270 Met Ile Ser Arg Thr Pro Glu Val Thr Cys
Val Val Val Asp Val Ser 275 280 285 His Glu Asp Pro Glu Val Lys Phe
Asn Trp Tyr Val Asp Gly Val Glu 290 295 300 Val His Asn Ala Lys Thr
Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr 305 310 315 320 Tyr Arg Val
Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn 325 330 335 Gly
Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro 340 345
350 Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln
355 360 365 Val Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn
Gln Val 370 375 380 Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser
Asp Ile Ala Val 385 390 395 400 Glu Trp Glu Ser Asn Gly Gln Pro Glu
Asn Asn Tyr Lys Thr Thr Pro 405 410 415 Pro Val Leu Asp Ser Asp Gly
Ser Phe Phe Leu Tyr Ser Lys Leu Thr 420 425 430 Val Asp Lys Ser Arg
Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val 435 440 445 Met His Glu
Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu 450 455 460 Ser
Pro Gly Lys 465 32456PRTArtificial SequenceMutated human fusion
peptide (A41L-wild type Fc) 32Met Tyr Ser Leu Phe Ile Ile Leu Met
Gly Leu Pro Phe Ser Phe Gln 1 5 10 15 Thr Ser Glu Pro Ala Tyr Asp
Lys Ser Val Cys Asp Ser Asn Asn Lys 20 25 30 Glu Tyr Met Gly Ile
Glu Val Tyr Val Glu Ala Thr Leu Asp Glu Pro 35 40 45 Leu Arg Gln
Thr Thr Cys Glu Ser Glu Ile His Lys Tyr Gly Ala Ser 50 55 60 Val
Ser Asn Gly Gly Leu Asn Ile Ser Val Asp Leu Leu Asn Cys Phe 65 70
75 80 Leu Asn Phe His Thr Val Gly Val Tyr Thr Asn Arg Asp Thr Gly
Val 85 90 95 Tyr Thr Asn Arg Asp Thr Val Tyr Ala Lys Phe Ala Ser
Leu Asp Pro 100 105 110 Ser Thr Glu Pro Ile Asn Ser Met Thr His Asp
Asp Leu Val Lys Leu 115 120 125 Thr Glu Glu Cys Ile Val Asp Ile Tyr
Leu Lys Cys Glu Val Asp Lys 130 135 140 Thr Lys Asp Phe Met Lys Asn
Gly Asn Arg Leu Lys Pro Arg Asp Phe 145 150 155 160 Lys Thr Val Pro
Pro Ser Asn Val Gly Ser Met Ile Glu Leu Gln Ser 165 170 175 Asp Tyr
Cys Val Glu Asp Val Thr Ala Tyr Val Lys Ile Tyr Asp Glu 180 185 190
Cys Gly Asn Ile Lys Gln His Ser Ile Pro Thr Leu Arg Asp Tyr Phe 195
200 205 Thr Thr Lys Asn Gly Gln Pro Arg Lys Ile Leu Lys Lys Lys Phe
Asp 210 215 220 Ser Cys Gly Ser Cys Asp Lys Thr His Thr Cys Pro Pro
Cys Pro Ala 225 230 235 240 Pro Glu Leu Leu Gly Gly Pro Ser Val Phe
Leu Phe Pro Pro Lys Pro 245 250 255 Lys Asp Thr Leu Met Ile Ser Arg
Thr Pro Glu Val Thr Cys Val Val 260 265 270 Val Asp Val Ser His Glu
Asp Pro Glu Val Lys Phe Asn Trp Tyr Val 275 280 285 Asp Gly Val Glu
Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln 290 295 300 Tyr Asn
Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln 305 310 315
320 Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala
325 330 335 Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly
Gln Pro 340 345 350 Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg
Asp Glu Leu Thr 355 360 365 Lys Asn Gln Val Ser Leu Thr Cys Leu Val
Lys Gly Phe Tyr Pro Ser 370 375 380 Asp Ile Ala Val Glu Trp Glu Ser
Asn Gly Gln Pro Glu Asn Asn Tyr 385 390 395 400 Lys Thr Thr Pro Pro
Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr 405 410 415 Ser Lys Leu
Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe 420 425 430 Ser
Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys 435 440
445 Ser Leu Ser Leu Ser Pro Gly Lys 450 455 33210PRTPoxvirus (A41L)
33Thr Ser Glu Pro Ala Tyr Asp Lys Ser Val Cys Asp Ser Asn Asn Lys 1
5 10 15 Glu Tyr Met Gly Ile Glu Val Tyr Val Glu Ala Thr Leu Asp Glu
Pro 20 25 30 Leu Lys Gln Thr Thr Cys Glu Ser Glu Ile His Lys Tyr
Gly Ala Ser 35 40 45 Val Ser Asn Gly Gly Leu Asn Ile Ser Val Asp
Leu Leu Asn Cys Phe 50 55 60 Leu Asn Phe His Thr Val Gly Val Tyr
Thr Asn Arg Asp Thr Gly Val 65 70 75 80 Tyr Thr Asn Arg Asp Thr Val
Tyr Ala Lys Phe Ala Ser Leu Asp Pro 85 90 95 Ser Thr Glu Pro Ile
Asn Ser Met Thr His Asp Asp Leu Val Lys Leu 100 105 110 Thr Glu Glu
Cys Ile Val Asp Ile Tyr Leu Lys Cys Glu Val Asp Lys 115 120 125 Thr
Lys Asp Phe Met Lys Asn Gly Asn Arg Leu Lys Pro Arg Asp Phe 130 135
140 Lys Thr Val Pro Pro Ser Asn Val Gly Ser Met Ile Glu Leu Gln Ser
145 150 155 160 Asp Tyr Cys Val Glu Asp Val Thr Ala Tyr Val Lys Ile
Tyr Asp Glu 165 170 175 Cys Gly Asn Ile Lys Gln His Ser Ile Pro Thr
Leu Arg Asp Tyr Phe 180 185 190 Thr Thr Lys Asn Gly Gln Pro Arg Lys
Ile Leu Lys Lys Lys Phe Asp 195 200 205 Ser Cys 210 34226PRTPox
virus (A41L) 34Met Tyr Ser Leu Phe Ile Ile Leu Met Gly Leu Pro Phe
Ser Phe Gln 1 5 10 15 Thr Ser Glu Pro Ala Tyr Asp Lys Ser Val Cys
Asp Ser Asn Asn Lys 20 25 30 Glu Tyr Met Gly Ile Glu Val Tyr Val
Glu Ala Thr Leu Asp Glu Pro 35 40 45 Leu Arg Gln Thr Thr Cys Glu
Ser Glu Ile His Lys Tyr Gly Ala Ser 50 55 60 Val Ser Asn Gly Gly
Leu Asn Ile Ser Val Asp Leu Leu Asn Cys Phe 65 70 75 80 Leu Asn Phe
His Thr Val Gly Val Tyr Thr Asn Arg Asp Thr Gly Val 85 90 95 Tyr
Thr Asn Arg Asp Thr Val Tyr Ala Lys Phe Ala Ser Leu Asp Pro 100 105
110 Ser Thr Glu Pro Ile Asn Ser Met Thr His Asp Asp Leu Val Lys Leu
115 120 125 Thr Glu Glu Cys Ile Val Asp Ile Tyr Leu Lys Cys Glu Val
Asp Lys 130 135 140 Thr Lys Asp Phe Met Lys Asn Gly Asn Arg Leu Lys
Pro Arg Asp Phe 145 150 155 160 Lys Thr Val Pro Pro Ser Asn Val Gly
Ser Met Ile Glu Leu Gln Ser 165 170 175 Asp Tyr Cys Val Glu Asp Val
Thr Ala Tyr Val Lys Ile Tyr Asp Glu 180 185 190 Cys Gly Asn Ile Lys
Gln His Ser Ile Pro Thr Leu Arg Asp Tyr Phe 195 200 205 Thr Thr Lys
Asn Gly Gln Pro Arg Lys Ile Leu Lys Lys Lys Phe Asp 210 215 220 Ser
Cys 225 35106PRTArtificial SequencePeptide sequence of combined
affinity tags 35Ala Gly Gly Pro Gly Gly Tyr Pro Tyr Asp Val Pro Asp
Tyr Ala Glu 1 5 10 15 Asp Gln Val Asp Pro Arg Leu Ile Asp Gly Lys
Met Asp Glu Lys Thr 20 25 30 Thr Gly Trp Arg Gly Gly His Val Val
Glu Gly Leu Ala Gly Glu Leu 35 40 45 Glu Gln Leu Arg Ala Arg Leu
Glu His His Pro Gln Gly Gln Arg Glu 50 55 60 Pro Gly Ser Gly Met
Asp Glu Lys Thr Thr Gly Trp Arg Gly Gly His 65 70 75 80 Val Val Glu
Gly Leu Ala Gly Glu Leu Glu Gln Leu Arg Ala Arg Leu 85 90 95 Glu
His His Pro Gln Gly Gln Arg Glu Pro 100 105 36327DNAArtificial
SequenceNucleotide sequence of combined affinity tags 36gcggccgctg
gcggccccgg cggctacccc tacgacgtgc ccgactacgc cgaggaccag 60gtggaccccc
ggctgatcga cggcaagatg gacgagaaga ccaccggctg gcggggcggc
120cacgtggtgg agggcctggc cggcgagctg gagcagctgc gggcccggct
ggagcaccac 180ccccagggcc agcgggagcc cggaagcggt atggatgaaa
aaactactgg ttggagaggg 240ggacatgtag tcgaaggtct ggccggcgag
ttagaacaat taagagctag attggaacat 300catccacaag gtcaaagaga accttag
32737270PRTArtificial SequencePeptide sequence of combined affinity
tags 37Gly Gly Ser Gly Pro Gly Gly Tyr Pro Tyr Asp Val Pro Asp Tyr
Ala 1 5 10 15 Glu Asp Gln Val Asp Pro Arg Leu Ile Asp Gly Lys Leu
Glu Val Leu 20 25 30 Phe Gln Gly Pro Leu Glu Val Leu Phe Gln Gly
Pro Lys Thr His Thr 35 40 45 Cys Pro Pro Cys Pro Ala Pro Glu Ala
Glu Gly Ala Pro Ser Val Phe 50 55 60 Leu Phe Pro Pro Lys Pro Lys
Asp Thr Leu Met Ile Ser Arg Thr Pro 65 70 75 80 Glu Val Thr Cys Val
Val Val Asp Val Ser His Glu Asp Pro Glu Val 85 90 95 Lys Phe Asn
Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr 100 105 110 Lys
Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val 115 120
125 Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys
130
135 140 Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile
Ser 145 150 155 160 Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr
Thr Leu Pro Pro 165 170 175 Ser Arg Asp Glu Leu Thr Lys Asn Gln Val
Ser Leu Thr Cys Leu Val 180 185 190 Lys Gly Phe Tyr Pro Ser Asp Ile
Ala Val Glu Trp Glu Ser Asn Gly 195 200 205 Gln Pro Glu Asn Asn Tyr
Lys Thr Thr Pro Pro Val Leu Asp Ser Asp 210 215 220 Gly Ser Phe Phe
Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp 225 230 235 240 Gln
Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His 245 250
255 Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys 260 265
270 38821DNAArtificial SequenceNucleotide sequence of combined
affinity tags 38gcggccgctg gcggcagcgg ccccggcggc tacccctaca
cgtgcccgac tacgccgagg 60accaggtgga cccccggctg atcgacggca agctggaggt
cctctttcaa gggccactgg 120aagtcctgtt ccaaggacct aaaactcaca
catgcccacc gtgcccagca cctgaagccg 180agggcgcgcc gtcagtcttc
ctcttccccc caaaacccaa ggacaccctc atgatctccc 240ggacccctga
ggtcacatgc gtggtggtgg acgtgagcca cgaagaccct gaggtcaagt
300tcaactggta cgtggacggc gtggaggtgc ataatgccaa gacaaagccg
cgggaggagc 360agtacaacag cacgtaccgt gtggtcagcg tcctcaccgt
cctgcaccag gactggctga 420atggcaagga gtacaagtgc aaggtctcca
acaaagccct cccagccccc atcgagaaaa 480ccatctccaa agccaaaggg
cagccccgag aaccacaggt gtacaccctg cccccatccc 540gggatgagct
gaccaagaac caggtcagcc tgacctgcct ggtcaaaggc ttctatccca
600gcgacatcgc cgtggagtgg gagagcaatg ggcagccgga gaacaactac
aagaccacgc 660ctcccgtgct ggactccgac ggctccttct tcctctatag
caagctcacc gtggacaaga 720gcaggtggca gcaggggaac gtcttctcat
gctccgtgat gcatgaggct ctgcacaacc 780actacacgca gaagagcctc
tccctgtctc cgggtaaatg a 821395428DNAArtificial SequenceVector
sequence (PCDNA3.1) 39gacggatcgg gagatctccc gatcccctat ggtgcactct
cagtacaatc tgctctgatg 60ccgcatagtt aagccagtat ctgctccctg cttgtgtgtt
ggaggtcgct gagtagtgcg 120cgagcaaaat ttaagctaca acaaggcaag
gcttgaccga caattgcatg aagaatctgc 180ttagggttag gcgttttgcg
ctgcttcgcg atgtacgggc cagatatacg cgttgacatt 240gattattgac
tagttattaa tagtaatcaa ttacggggtc attagttcat agcccatata
300tggagttccg cgttacataa cttacggtaa atggcccgcc tggctgaccg
cccaacgacc 360cccgcccatt gacgtcaata atgacgtatg ttcccatagt
aacgccaata gggactttcc 420attgacgtca atgggtggag tatttacggt
aaactgccca cttggcagta catcaagtgt 480atcatatgcc aagtacgccc
cctattgacg tcaatgacgg taaatggccc gcctggcatt 540atgcccagta
catgacctta tgggactttc ctacttggca gtacatctac gtattagtca
600tcgctattac catggtgatg cggttttggc agtacatcaa tgggcgtgga
tagcggtttg 660actcacgggg atttccaagt ctccacccca ttgacgtcaa
tgggagtttg ttttggcacc 720aaaatcaacg ggactttcca aaatgtcgta
acaactccgc cccattgacg caaatgggcg 780gtaggcgtgt acggtgggag
gtctatataa gcagagctct ctggctaact agagaaccca 840ctgcttactg
gcttatcgaa attaatacga ctcactatag ggagacccaa gctggctagc
900gtttaaactt aagcttggta ccgagctcgg atccactagt ccagtgtggt
ggaattctgc 960agatatccag cacagtggcg gccgctcgag tctagagggc
ccgtttaaac ccgctgatca 1020gcctcgactg tgccttctag ttgccagcca
tctgttgttt gcccctcccc cgtgccttcc 1080ttgaccctgg aaggtgccac
tcccactgtc ctttcctaat aaaatgagga aattgcatcg 1140cattgtctga
gtaggtgtca ttctattctg gggggtgggg tggggcagga cagcaagggg
1200gaggattggg aagacaatag caggcatgct ggggatgcgg tgggctctat
ggcttctgag 1260gcggaaagaa ccagctgggg ctctaggggg tatccccacg
cgccctgtag cggcgcatta 1320agcgcggcgg gtgtggtggt tacgcgcagc
gtgaccgcta cacttgccag cgccctagcg 1380cccgctcctt tcgctttctt
cccttccttt ctcgccacgt tcgccggctt tccccgtcaa 1440gctctaaatc
gggggctccc tttagggttc cgatttagtg ctttacggca cctcgacccc
1500aaaaaacttg attagggtga tggttcacgt agtgggccat cgccctgata
gacggttttt 1560cgccctttga cgttggagtc cacgttcttt aatagtggac
tcttgttcca aactggaaca 1620acactcaacc ctatctcggt ctattctttt
gatttataag ggattttgcc gatttcggcc 1680tattggttaa aaaatgagct
gatttaacaa aaatttaacg cgaattaatt ctgtggaatg 1740tgtgtcagtt
agggtgtgga aagtccccag gctccccagc aggcagaagt atgcaaagca
1800tgcatctcaa ttagtcagca accaggtgtg gaaagtcccc aggctcccca
gcaggcagaa 1860gtatgcaaag catgcatctc aattagtcag caaccatagt
cccgccccta actccgccca 1920tcccgcccct aactccgccc agttccgccc
attctccgcc ccatggctga ctaatttttt 1980ttatttatgc agaggccgag
gccgcctctg cctctgagct attccagaag tagtgaggag 2040gcttttttgg
aggcctaggc ttttgcaaaa agctcccggg agcttgtata tccattttcg
2100gatctgatca agagacagga tgaggatcgt ttcgcatgat tgaacaagat
ggattgcacg 2160caggttctcc ggccgcttgg gtggagaggc tattcggcta
tgactgggca caacagacaa 2220tcggctgctc tgatgccgcc gtgttccggc
tgtcagcgca ggggcgcccg gttctttttg 2280tcaagaccga cctgtccggt
gccctgaatg aactgcagga cgaggcagcg cggctatcgt 2340ggctggccac
gacgggcgtt ccttgcgcag ctgtgctcga cgttgtcact gaagcgggaa
2400gggactggct gctattgggc gaagtgccgg ggcaggatct cctgtcatct
caccttgctc 2460ctgccgagaa agtatccatc atggctgatg caatgcggcg
gctgcatacg cttgatccgg 2520ctacctgccc attcgaccac caagcgaaac
atcgcatcga gcgagcacgt actcggatgg 2580aagccggtct tgtcgatcag
gatgatctgg acgaagagca tcaggggctc gcgccagccg 2640aactgttcgc
caggctcaag gcgcgcatgc ccgacggcga ggatctcgtc gtgacccatg
2700gcgatgcctg cttgccgaat atcatggtgg aaaatggccg cttttctgga
ttcatcgact 2760gtggccggct gggtgtggcg gaccgctatc aggacatagc
gttggctacc cgtgatattg 2820ctgaagagct tggcggcgaa tgggctgacc
gcttcctcgt gctttacggt atcgccgctc 2880ccgattcgca gcgcatcgcc
ttctatcgcc ttcttgacga gttcttctga gcgggactct 2940ggggttcgaa
atgaccgacc aagcgacgcc caacctgcca tcacgagatt tcgattccac
3000cgccgccttc tatgaaaggt tgggcttcgg aatcgttttc cgggacgccg
gctggatgat 3060cctccagcgc ggggatctca tgctggagtt cttcgcccac
cccaacttgt ttattgcagc 3120ttataatggt tacaaataaa gcaatagcat
cacaaatttc acaaataaag catttttttc 3180actgcattct agttgtggtt
tgtccaaact catcaatgta tcttatcatg tctgtatacc 3240gtcgacctct
agctagagct tggcgtaatc atggtcatag ctgtttcctg tgtgaaattg
3300ttatccgctc acaattccac acaacatacg agccggaagc ataaagtgta
aagcctgggg 3360tgcctaatga gtgagctaac tcacattaat tgcgttgcgc
tcactgcccg ctttccagtc 3420gggaaacctg tcgtgccagc tgcattaatg
aatcggccaa cgcgcgggga gaggcggttt 3480gcgtattggg cgctcttccg
cttcctcgct cactgactcg ctgcgctcgg tcgttcggct 3540gcggcgagcg
gtatcagctc actcaaaggc ggtaatacgg ttatccacag aatcagggga
3600taacgcagga aagaacatgt gagcaaaagg ccagcaaaag gccaggaacc
gtaaaaaggc 3660cgcgttgctg gcgtttttcc ataggctccg cccccctgac
gagcatcaca aaaatcgacg 3720ctcaagtcag aggtggcgaa acccgacagg
actataaaga taccaggcgt ttccccctgg 3780aagctccctc gtgcgctctc
ctgttccgac cctgccgctt accggatacc tgtccgcctt 3840tctcccttcg
ggaagcgtgg cgctttctca tagctcacgc tgtaggtatc tcagttcggt
3900gtaggtcgtt cgctccaagc tgggctgtgt gcacgaaccc cccgttcagc
ccgaccgctg 3960cgccttatcc ggtaactatc gtcttgagtc caacccggta
agacacgact tatcgccact 4020ggcagcagcc actggtaaca ggattagcag
agcgaggtat gtaggcggtg ctacagagtt 4080cttgaagtgg tggcctaact
acggctacac tagaagaaca gtatttggta tctgcgctct 4140gctgaagcca
gttaccttcg gaaaaagagt tggtagctct tgatccggca aacaaaccac
4200cgctggtagc ggtttttttg tttgcaagca gcagattacg cgcagaaaaa
aaggatctca 4260agaagatcct ttgatctttt ctacggggtc tgacgctcag
tggaacgaaa actcacgtta 4320agggattttg gtcatgagat tatcaaaaag
gatcttcacc tagatccttt taaattaaaa 4380atgaagtttt aaatcaatct
aaagtatata tgagtaaact tggtctgaca gttaccaatg 4440cttaatcagt
gaggcaccta tctcagcgat ctgtctattt cgttcatcca tagttgcctg
4500actccccgtc gtgtagataa ctacgatacg ggagggctta ccatctggcc
ccagtgctgc 4560aatgataccg cgagacccac gctcaccggc tccagattta
tcagcaataa accagccagc 4620cggaagggcc gagcgcagaa gtggtcctgc
aactttatcc gcctccatcc agtctattaa 4680ttgttgccgg gaagctagag
taagtagttc gccagttaat agtttgcgca acgttgttgc 4740cattgctaca
ggcatcgtgg tgtcacgctc gtcgtttggt atggcttcat tcagctccgg
4800ttcccaacga tcaaggcgag ttacatgatc ccccatgttg tgcaaaaaag
cggttagctc 4860cttcggtcct ccgatcgttg tcagaagtaa gttggccgca
gtgttatcac tcatggttat 4920ggcagcactg cataattctc ttactgtcat
gccatccgta agatgctttt ctgtgactgg 4980tgagtactca accaagtcat
tctgagaata gtgtatgcgg cgaccgagtt gctcttgccc 5040ggcgtcaata
cgggataata ccgcgccaca tagcagaact ttaaaagtgc tcatcattgg
5100aaaacgttct tcggggcgaa aactctcaag gatcttaccg ctgttgagat
ccagttcgat 5160gtaacccact cgtgcaccca actgatcttc agcatctttt
actttcacca gcgtttctgg 5220gtgagcaaaa acaggaaggc aaaatgccgc
aaaaaaggga ataagggcga cacggaaatg 5280ttgaatactc atactcttcc
tttttcaata ttattgaagc atttatcagg gttattgtct 5340catgagcgga
tacatatttg aatgtattta gaaaaataaa caaatagggg ttccgcgcac
5400atttccccga aaagtgccac ctgacgtc 5428406706DNAArtificial
SequenceVector sequence pSL9 40caggtggcac ttttcgggga aatgtgcgcg
gaacccctat ttgtttattt ttctaaatac 60attcaaatat gtatccgctc atgagacaat
aaccctgata aatgcttcaa taatattgaa 120aaaggaagag tatgagtatt
caacatttcc gtgtcgccct tattcccttt tttgcggcat 180tttgccttcc
tgtttttgct cacccagaaa cgctggtgaa agtaaaagat gctgaagatc
240agttgggtgc acgagtgggt tacatcgaac tggatctcaa cagcggtaag
atccttgaga 300gttttcgccc cgaagaacgt tttccaatga tgagcacttt
taaagttctg ctatgtggcg 360cggtattatc ccgtattgac gccgggcaag
agcaactcgg tcgccgcata cactattctc 420agaatgactt ggttgagtac
tcaccagtca cagaaaagca tcttacggat ggcatgacag 480taagagaatt
atgcagtgct gccataacca tgagtgataa cactgcggcc aacttacttc
540tgacaacgat cggaggaccg aaggagctaa ccgctttttt gcacaacatg
ggggatcatg 600taactcgcct tgatcgttgg gaaccggagc tgaatgaagc
cataccaaac gacgagcgtg 660acaccacgat gcctgtagca atggcaacaa
cgttgcgcaa actattaact ggcgaactac 720ttactctagc ttcccggcaa
caattaatag actggatgga ggcggataaa gttgcaggac 780cacttctgcg
ctcggccctt ccggctggct ggtttattgc tgataaatct ggagccggtg
840agcgtgggtc tcgcggtatc attgcagcac tggggccaga tggtaagccc
tcccgtatcg 900tagttatcta cacgacgggg agtcaggcaa ctatggatga
acgaaataga cagatcgctg 960agataggtgc ctcactgatt aagcattggt
aactgtcaga ccaagtttac tcatatatac 1020tttagattga tttaaaactt
catttttaat ttaaaaggat ctaggtgaag atcctttttg 1080ataatctcat
gaccaaaatc ccttaacgtg agttttcgtt ccactgagcg tcagaccccg
1140tagaaaagat caaaggatct tcttgagatc ctttttttct gcgcgtaatc
tgctgcttgc 1200aaacaaaaaa accaccgcta ccagcggtgg tttgtttgcc
ggatcaagag ctaccaactc 1260tttttccgaa ggtaactggc ttcagcagag
cgcagatacc aaatactgtc cttctagtgt 1320agccgtagtt aggccaccac
ttcaagaact ctgtagcacc gcctacatac ctcgctctgc 1380taatcctgtt
accagtggct gctgccagtg gcgataagtc gtgtcttacc gggttggact
1440caagacgata gttaccggat aaggcgcagc ggtcgggctg aacggggggt
tcgtgcacac 1500agcccagctt ggagcgaacg acctacaccg aactgagata
cctacagcgt gagctatgag 1560aaagcgccac gcttcccgaa gggagaaagg
cggacaggta tccggtaagc ggcagggtcg 1620gaacaggaga gcgcacgagg
gagcttccag ggggaaacgc ctggtatctt tatagtcctg 1680tcgggtttcg
ccacctctga cttgagcgtc gatttttgtg atgctcgtca ggggggcgga
1740gcctatggaa aaacgccagc aacgcggcct ttttacggtt cctggccttt
tgctggcctt 1800ttgctcacat gttctttcct gcgttatccc ctgattctgt
ggataaccgt attaccgcct 1860ttgagtgagc tgataccgct cgccgcagcc
gaacgaccga gcgcagcgag tcagtgagcg 1920aggaagcgga agagcgccca
atacgcaaac cgcctctccc cgcgcgttgg ccgattcatt 1980aatgcagctg
gcacgacagg tttcccgact ggaaagcggg cagtgagcgc aacgcaatta
2040atgtgagtta gctcactcat taggcacccc aggctttaca ctttatgctt
ccggctcgta 2100tgttgtgtgg aattgtgagc ggataacaat ttcacacagg
aaacagctat gaccatgatt 2160acgccaagcg cgcaattaac cctcactaaa
gggaacaaaa gctggagctg caagcttaat 2220gtagtcttat gcaatactct
tgtagtcttg caacatggta acgatgagtt agcaacatgc 2280cttacaagga
gagaaaaagc accgtgcatg ccgattggtg gaagtaaggt ggtacgatcg
2340tgccttatta ggaaggcaac agacgggtct gacatggatt ggacgaacca
ctgaattgga 2400ggcgtggcct gggcgggact ggggagtggc gagccctcag
atcctgcata taagcagctg 2460ctttttgcct gtactgggtc tctctggtta
gaccagatct gagcctggga gctctctggc 2520taactaggga acccactgct
taagcctcaa taaagcttgc cttgagtgct tcaagtagtg 2580tgtgcccgtc
tgttgtgtga ctctggtaac tagagatccc tcagaccctt ttagtcagtg
2640tggaaaatct ctagcagtgg cgcccgaaca gggacctgaa agcgaaaggg
aaaccagagc 2700tctctcgacg caggactcgg cttgctgaag cgcgcacggc
aagaggcgag gggcggcgac 2760tggtgagtac gccaaaaatt ttgactagcg
gaggctagaa ggagagagat gggtgcgaga 2820gcgtcagtat taagcggggg
agaattagat cgcgatggga aaaaattcgg ttaaggccag 2880ggggaaagaa
aaaatataaa ttaaaacata tagtatgggc aagcagggag ctagaacgat
2940tcgcagttaa tcctggcctg ttagaaacat cagaaggctg tagacaaata
ctgggacagc 3000tacaaccatc ccttcagaca ggatcagaag aacttagatc
attatataat acagtagcaa 3060ccctctattg tgtgcatcaa aggatagaga
taaaagacac caaggaagct ttagacaaga 3120tagaggaaga gcaaaacaaa
agtaagacca ccgcacagca agcggccgct gatcttcaga 3180cctggaggag
gagatatgag ggacaattgg agaagtgaat tatataaata taaagtagta
3240aaaattgaac cattaggagt agcacccacc aaggcaaaga gaagagtggt
gcagagagaa 3300aaaagagcag tgggaatagg agctttgttc cttgggttct
tgggagcagc aggaagcact 3360atgggcgcag cctcaatgac gctgacggta
caggccagac aattattgtc tggtatagtg 3420cagcagcaga acaatttgct
gagggctatt gaggcgcaac agcatctgtt gcaactcaca 3480gtctggggca
tcaagcagct ccaggcaaga atcctggctg tggaaagata cctaaaggat
3540caacagctcc tggggatttg gggttgctct ggaaaactca tttgcaccac
tgctgtgcct 3600tggaatgcta gttggagtaa taaatctctg gaacagattg
gaatcacacg acctggatgg 3660agtgggacag agaaattaac aattacacaa
gcttaataca ctccttaatt gaagaatcgc 3720aaaaccagca agaaaagaat
gaacaagaat tattggaatt agataaatgg gcaagtttgt 3780ggaattggtt
taacataaca aattggctgt ggtatataaa attattcata atgatagtag
3840gaggcttggt aggtttaaga atagtttttg ctgtactttc tatagtgaat
agagttaggc 3900agggatattc accattatcg tttcagaccc acctcccaac
cccgagggga cccgacaggc 3960ccgaaggaat agaagaagaa ggtggagaga
gagacagaga cagatccatt cgattagtga 4020acggatctcg acggtatcgg
ttttaaaaga aaagggggga ttggggggta cagtgcaggg 4080gaaagaatag
tagacataat agcaacagac atacaaacta aagaattaca aaaacaaatt
4140acaaaaattc aaaattttat cggggctgca ggaattcggc gcgccacgcg
tccgcggact 4200agtctcgagt taattaagct agcctagtgc catttgttca
gtggttcgta gggctttccc 4260ccactgtttg gctttcagtt atatggatga
tgtggtattg ggggccaagt ctgtacagca 4320tcttgagtcc ctttttaccg
ctgttaccaa ttttcttttg tctttgggta tacatttaaa 4380ccctaacaaa
acaaagagat ggggttactc tctaaatttt atgggttatg tcattggatg
4440ttatgggtcc ttgccacaag aacacatcat acaaaaaatc aaagaatgtt
ttagaaaact 4500tcctattaac aggcctattg attggaaagt atgtcaacga
attgtgggtc ttttgggttt 4560tgctgcccct tttacacaat gtggttatcc
tgcgttgatg cctttgtatg catgtattca 4620atctaagcag gctttcactt
tctcgccaac ttacaaggcc tttctgtgta aacaatacct 4680gaacctttac
cccgttgccc ggcaacggcc acctctgtgc caagtgtttg ctgacgcaac
4740ccccactggc tggggcttgg tcatgggcca tcagcgcatg cgtggaacct
tttcggctcc 4800tctgccgatc catactgcgg aactcctagc cgcttgtttt
gctcgcagca ggtctggagc 4860aaacattatc gggactgata actctgttgt
cctatcccgc aaatatacat cgtttccatg 4920gctgctaggc tgtgctgcca
actggatcct gcgcgggacg tcctttgttt acgtcccgtc 4980ggcgctgaat
cctgcggacg acccttctcg gggtcgcttg ggactctctc gtccccttct
5040ccgtctgccg ttccgaccga ccacggggcg cacctctctt tacgcggact
ccccgtctgt 5100gccttctcat ctgccggacc gtgtgcactt cgcttcacct
ctgcacgtcg catggagacc 5160accgtgaacg cccaccaaat attgcccaag
gtcttacata agaggactct tggactctca 5220gcaatgtcaa cgaccgacct
tgaggcatac ttcaaagact gtttgtttaa agactgggag 5280gagttggggg
aggagattag gttaaaggtc tttgtactag gaggctgtag gcataaattg
5340gtctgcgcac cagcaccatg tatcactaga gcggggtacc tttaagacca
atgacttaca 5400aggcagctgt agatcttagc cactttttaa aagaaaaggg
gggacttgga agggctaatt 5460cactcccaac gaagacaaga tctgcttttt
gcttgtactg ggtctctctg gttagaccag 5520atctgagcct gggagctctc
tggctaacta gggaacccac tgcttaagcc tcaataaagc 5580ttgccttgag
tgcttcaagt agtgtgtgcc cgtctgttgt gtgactctgg taactagaga
5640tccctcagac ccttttagtc agtgtggaaa atctctagca gtagtagttc
atgtcatctt 5700attattcagt atttataact tgcaaagaaa tgaatatcag
agagtgagag gaacttgttt 5760attgcagctt ataatggtta caaataaagc
aatagcatca caaatttcac aaataaagca 5820tttttttcac tgcattctag
ttgtggtttg tccaaactca tcaatgtatc ttatcatgtc 5880tggctctagc
tatcccgccc ctaactccgc ccagttccgc ccattctccg ccccatggct
5940gactaatttt ttttatttat gcagaggccg aggccgcctc ggcctctgag
ctattccaga 6000agtagtgagg aggctttttt ggaggcctag gcttttgcgt
cgagacgtac ccaattcgcc 6060ctatagtgag tcgtattacg cgcgctcact
ggccgtcgtt ttacaacgtc gtgactggga 6120aaaccctggc gttacccaac
ttaatcgcct tgcagcacat ccccctttcg ccagctggcg 6180taatagcgaa
gaggcccgca ccgatcgccc ttcccaacag ttgcgcagcc tgaatggcga
6240atggcgcgac gcgccctgta gcggcgcatt aagcgcggcg ggtgtggtgg
ttacgcgcag 6300cgtgaccgct acacttgcca gcgccctagc gcccgctcct
ttcgctttct tcccttcctt 6360tctcgccacg ttcgccggct ttccccgtca
agctctaaat cgggggctcc ctttagggtt 6420ccgatttagt gctttacggc
acctcgaccc caaaaaactt gattagggtg atggttcacg 6480tagtgggcca
tcgccctgat agacggtttt tcgccctttg acgttggagt ccacgttctt
6540taatagtgga ctcttgttcc aaactggaac aacactcaac cctatctcgg
tctattcttt 6600tgatttataa gggattttgc cgatttcggc ctattggtta
aaaaatgagc tgatttaaca 6660aaaatttaac gcgaatttta acaaaatatt
aacgtttaca atttcc 6706417749DNAArtificial SequenceVector sequence
pDC409 41gggctgtgga atgtgtgtca gttagggtgt ggaaagtccc caggctcccc
agcaggcaga 60agtatgcaaa gcatgcatct caattagtca gcaaccaggt gtggaaagtc
cccaggctcc 120ccagcaggca gaagtatgca aagcatgcat ctcaattagt
cagcaaccat agtcccgccc 180ctaactccgc ccatcccgcc cctaactccg
cccagttccg cccattctcc gccccatggc 240tgactaattt tttttattta
tgcagaggcc gaggccgcct cggcctctga gctattccag 300aagtagtgag
gaggcttttt tggaggccta ggcttttgca aaaagctctc tagatcgatg
360aattctcgag ccaccatgga gccagtagat cctagactag agccctggaa
gcatccagga 420agtcagccta aaactgcttg taccaattgc tattgtaaaa
agtgttgctt tcattgccaa 480gtttgtttca taacaaaggc cttaggcatc
tcctacggcc gcaagaagcg gagacagcga 540cgaagacctc ctcaaggcag
tcagactcat caagtttctc tatcaaagca acccacctcc 600caatcccgag
gggacccgac aggcccgaag gaataggtac caagcttcta gaggatcttt
660gtgaaggaac cttacttctg tggtgtgaca taattggaca aactacctac
agagatttaa 720agctctaagg taaatataaa atttttaagt gtataatgtg
ttaaactact gattctaatt 780gtttgtgtat tttagattcc aacctatgga
actgatgaat gggagcagtg gtggaatgcc 840tttaatgagg aaaacctgtt
ttgctcagaa gaaatgccat ctagtgatga tgaggctact 900gctgactctc
aacattctac tcctccaaaa aagaagagaa aggtagaaga ccccaaggac
960tttccttcag
aattgctaag ttttttgagt catgctgtgt ttagtaatag aactcttgct
1020tgctttgcta tttacaccac aaaggaaaaa gctgcactgc tatacaagaa
aattatggaa 1080aaatattctg taacctttat aagtaggcat aacagttata
atcataacat actgtttttt 1140cttactccac acaggcatag agtgtctgct
attaataact atgctcaaaa attgtgtacc 1200tttagctttt taatttgtaa
aggggttaat aaggaatatt tgatgtatag tgccttgact 1260agagatcata
atcagccata ccacatttgt agaggtttta cttgctttaa aaaacctccc
1320acacctcccc ctgaacctga aacataaaat gaatgcaatt gttgttgtta
acttgtttat 1380tgcagcttat aatggttaca aataaagcaa tagcatcaca
aatttcacaa ataaagcatt 1440tttttcactg cattctagtt gtggtttgtc
caaactcatc aatgtatctt atcatgtctg 1500gatccgagct tatcgatggt
accgcatcgg gagtacttca agaactgctg atatcgagct 1560tgctacaagg
gactttccgc tggggacttt ccagggaggc gtggcctggg cgggactggg
1620gagtggcgag ccctcagatc ctgcatataa gcagctgctt tttgcctgta
ctgggtctct 1680gccattagag gtcatctgag cctgggagct ctgaccctag
tggcggaacc cactgcttaa 1740gcctcaatag gatcctcttc cgcatcgctg
tctgcgaggg ccagctgttg ggctcgcggt 1800tgaggacaaa ctcttcgcgg
tctttccagt actcttggat cggaaacccg tcggcctccg 1860aacggtactc
cgccaccgag ggacctgagc gagtccgcat cgaccggatc ggaaaacctc
1920tcgagggcca cgcgtttaaa cgtcgacggc ccgggcggcc gctacagatc
tgtttaaact 1980agttagctag gcgcacccta tagtgagtcg tattaggatc
tacgcgattt gatgtatagt 2040gccttgacta gagatcataa tcagccatac
cacatttgta gaggttttac ttgctttaaa 2100aaacctccca cacctccccc
tgaacctgaa acataaaatg aatgcaattg ttgttgttaa 2160cttgtttatt
gcagcttata atggttacaa ataaagcaat agcatcacaa atttcacaaa
2220taaagcattt ttttcactgc attctagttg tggtttgtcc aaactcatca
atgtatctta 2280tcatgtctgg atccgagctt atcgactcta gaggatcccc
catggtcggg acgctctggc 2340cggtgaggcg tgcgcagtcg ttgacgctct
agaccgtgca aaaggagagc ctgtaagcgg 2400gcactcttcc gtggtctggt
ggataaattc gcaagggtat catggcggac gaccggggtt 2460cgaaccccgg
atccggccgt ccgccgtgat ccatgcggtt accgcccgcg tgtcgaaccc
2520aggtgtgcga cgtcagacaa cgggggagcg ctccttttgg cttccttcca
ggcgcggcgg 2580ctgctgcgct agcttttttg gccactggcc gcgcgcggcg
taagcggtta ggctggaaag 2640cgaaagcatt aagtggctcg ctccctgtag
ccggagggtt attttccaag ggttgagtcg 2700caggaccccc ggttcgagtc
tcgggccggc cggactgcgg cgaacggggg tttgcctccc 2760cgtcatgcaa
gaccccgctt gcaaattcct ccggaaacag ggacgagccc cttttttgct
2820tttcccagat gcatccggtg ctgcggcaga tgcgcccccc tcctcagcag
cggcaagagc 2880aagagcagcg gcagacatgc agggcaccct ccccttctcc
taccgcgtca ggaggggcaa 2940catccgggta ccgagctcga attcttgaag
acgaaagggc ctcgtgatac gcctattttt 3000ataggttaat gtcatgataa
taatggtttc ttagacgtca ggtggcactt ttcggggaaa 3060tgtgcgcgga
acccctattt gtttattttt ctaaatacat tcaaatatgt atccgctcat
3120gagacaataa ccctgataaa tgcttcaata atattgaaaa aggaagagta
tgagtattca 3180acatttccgt gtcgccctta ttcccttttt tgcggcattt
tgccttcctg tttttgctca 3240cccagaaacg ctggtgaaag taaaagatgc
tgaagatcag ttgggtgcac gagtgggtta 3300catcgaactg gatctcaaca
gcggtaagat ccttgagagt tttcgccccg aagaacgttt 3360tccaatgatg
agcactttta aagttctgct atgtggcgcg gtattatccc gtgttgacgc
3420cgggcaagag caactcggtc gccgcataca ctattctcag aatgacttgg
ttgagtactc 3480accagtcaca gaaaagcatc ttacggatgg catgacagta
agagaattat gcagtgctgc 3540cataaccatg agtgataaca ctgcggccaa
cttacttctg acaacgatcg gaggaccgaa 3600ggagctaacc gcttttttgc
acaacatggg ggatcatgta actcgccttg atcgttggga 3660accggagctg
aatgaagcca taccaaacga cgagcgtgac accacgatgc ctgcagcaat
3720ggcaacaacg ttgcgcaaac tattaactgg cgaactactt actctagctt
cccggcaaca 3780attaatagac tggatggagg cggataaagt tgcaggacca
cttctgcgct cggcccttcc 3840ggctggctgg tttattgctg ataaatctgg
agccggtgag cgtgggtctc gcggtatcat 3900tgcagcactg gggccagatg
gtaagccctc ccgtatcgta gttatctaca cgacggggag 3960tcaggcaact
atggatgaac gaaatagaca gatcgctgag ataggtgcct cactgattaa
4020gcattggtaa ctgtcagacc aagtttactc atatatactt tagattgatt
taaaacttca 4080tttttaattt aaaaggatct aggtgaagat cctttttgat
aatctcatga ccaaaatccc 4140ttaacgtgag ttttcgttcc actgagcgtc
agaccccgta gaaaagatca aaggatcttc 4200ttgagatcct ttttttctgc
gcgtaatctg ctgcttgcaa acaaaaaaac caccgctacc 4260agcggtggtt
tgtttgccgg atcaagagct accaactctt tttccgaagg taactggctt
4320cagcagagcg cagataccaa atactgtcct tctagtgtag ccgtagttag
gccaccactt 4380caagaactct gtagcaccgc ctacatacct cgctctgcta
atcctgttac cagtggctgc 4440tgccagtggc gataagtcgt gtcttaccgg
gttggactca agacgatagt taccggataa 4500ggcgcagcgg tcgggctgaa
cggggggttc gtgcacacag cccagcttgg agcgaacgac 4560ctacaccgaa
ctgagatacc tacagcgtga gcattgagaa agcgccacgc ttcccgaagg
4620gagaaaggcg gacaggtatc cggtaagcgg cagggtcgga acaggagagc
gcacgaggga 4680gcttccaggg ggaaacgcct ggtatcttta tagtcctgtc
gggtttcgcc acctctgact 4740tgagcgtcga tttttgtgat gctcgtcagg
ggggcggagc ctatggaaaa acgccagcaa 4800cgcggccttt ttacggttcc
tggccttttg ctggcctttt tgaagctgtc cctgatggtc 4860gtcatctacc
tgcctggaca gcatggcctg caacgcgggc atcccgatgc cgccggaagc
4920gagaagaatc ataatgggga aggccatcca gcctcgcgtc gcgaacgcca
gcaagacgta 4980gcccagcgcg tcggccgcca tgccggcgat aatggcctgc
ttctcgccga aacgtttggt 5040ggcgggacca gtgacgaagg cttgagcgag
ggcgtgcaag attccgaata ccgcaagcga 5100caggccgatc atcgtcgcgc
tccagcgaaa gcggtcctcg ccgaaaatga cccagagcgc 5160tgccggcacc
tgtcctacga gttgcatgat aaagaagaca gtcataagtg cggcgacgat
5220agtcatgccc cgcgcccacc ggaaggagct gactgggttg aaggctctca
agggcatcgg 5280tcgatgcagg aaaaggacaa gcagcgaaaa ttcacgcccc
cttgggaggt ggcggcatat 5340gcaaaggata gcactcccac tctactactg
ggtatcatat gctgactgta tatgcatgag 5400gatagcatat gctacccgga
tacagattag gatagcatat actacccaga tatagattag 5460gatagcatat
gctacccaga tatagattag gatagcctat gctacccaga tataaattag
5520gatagcatat actacccaga tatagattag gatagcatat gctacccaga
tatagattag 5580gatagcctat gctacccaga tatagattag gatagcatat
gctacccaga tatagattag 5640gatagcatat gctatccaga tatttgggta
gtatatgcta cccagatata aattaggata 5700gcatatacta ccctaatctc
tattaggata gcatatgcta cccggataca gattaggata 5760gcatatacta
cccagatata gattaggata gcatatgcta cccagatata gattaggata
5820gcctatgcta cccagatata aattaggata gcatatacta cccagatata
gattaggata 5880gcatatgcta cccagatata gattaggata gcctatgcta
cccagatata gattaggata 5940gcatatgcta tccagatatt tgggtagtat
atgctaccca tggcaacatt agcccaccgt 6000gctctcagcg acctcgtgaa
tatgaggacc aacaaccctg tgcttggcgc tcaggcgcaa 6060gtgtgtgtaa
tttgtcctcc agatcgcagc aatcgcgccc ctatcttggc ccgcccacct
6120acttatgcag gtattccccg gggtgccatt agtggttttg tgggcaagtg
gtttgaccgc 6180agtggttagc ggggttacaa tcagccaagt tattacaccc
ttattttaca gtccaaaacc 6240gcagggcggc gtgtgggggc tgacgcgtgc
ccccactcca caatttcaaa aaaaagagtg 6300gccacttgtc tttgtttatg
ggccccattg gcgtggagcc ccgtttaatt ttcgggggtg 6360ttagagacaa
ccagtggagt ccgctgctgt cggcgtccac tctctttccc cttgttacaa
6420atagagtgta acaacatggt tcacctgtct tggtccctgc ctgggacaca
tcttaataac 6480cccagtatca tattgcacta ggattatgtg ttgcccatag
ccataaattc gtgtgagatg 6540gacatccagt ctttacggct tgtccccacc
ccatggattt ctattgttaa agatattcag 6600aatgtttcat tcctacacta
gtatttattg cccaaggggt ttgtgagggt tatattggtg 6660tcatagcaca
atgccaccac tgaacccccc gtccaaattt tattctgggg gcgtcacctg
6720aaaccttgtt ttcgagcacc tcacatacac cttactgttc acaactcagc
agttattcta 6780ttagctaaac gaaggagaat gaagaagcag gcgaagattc
aggagagttc actgcccgct 6840ccttgatctt cagccactgc ccttgtgact
aaaatggttc actaccctcg tggaatcctg 6900accccatgta aataaaaccg
tgacagctca tggggtggga gatatcgctg ttccttagga 6960cccttttact
aaccctaatt cgatagcata tgcttcccgt tgggtaacat atgctattga
7020attagggtta gtctggatag tatatactac tacccgggaa gcatatgcta
cccgtttagg 7080gttaacaagg gggccttata aacactattg ctaatgccct
cttgagggtc cgcttatcgg 7140tagctacaca ggcccctctg attgacgttg
gtgtagcctc ccgtagtctt cctgggcccc 7200tgggaggtac atgtccccca
gcattggtgt aagagcttca gccaagagtt acacataaag 7260gcaatgttgt
gttgcagtcc acagactgca aagtctgctc caggatgaaa gccactcagt
7320gttggcaaat gtgcacatcc atttataagg atgtcaacta cagtcagaga
acccctttgt 7380gtttggtccc cccccgtgtc acatgtggaa cagggcccag
ttggcaagtt gtaccaacca 7440actgaaggga ttacatgcac tgcccccctc
gacgctctcc cttatgcgac tcctgcatta 7500ggaagcagcc cagtagtagg
ttgaggccgt tgagcaccgc cgccgcaagg aatggtgcat 7560gcaaggagat
ggcgcccaac agtcccccgg ccacggggcc tgccaccata cccacgccga
7620aacaagcgct catgagcccg aagtggcgag cccgatcttc cccatcggtg
atgtcggcga 7680tataggcgcc agcaaccgca cctgtggcgc cggtgatgcc
ggccacgatg cgtccggcgt 7740agaggatcc 7749424650DNAArtificial
SequenceVector sequence pAAV 42cctgcaggca gctgcgcgct cgctcgctca
ctgaggccgc ccgggcaaag cccgggcgtc 60gggcgacctt tggtcgcccg gcctcagtga
gcgagcgagc gcgcagagag ggagtggcca 120actccatcac taggggttcc
tgcggccgca cgcgtggagc tagttattaa tagtaatcaa 180ttacggggtc
attagttcat agcccatata tggagttccg cgttacataa cttacggtaa
240atggcccgcc tggctgaccg cccaacgacc cccgcccatt gacgtcaata
atgacgtatg 300ttcccatagt aacgtcaata gggactttcc attgacgtca
atgggtggag tatttacggt 360aaactgccca cttggcagta catcaagtgt
atcatatgcc aagtacgccc cctattgacg 420tcaatgacgg taaatggccc
gcctggcatt atgcccagta catgacctta tgggactttc 480ctacttggca
gtacatctac gtattagtca tcgctattac catggtgatg cggttttggc
540agtacatcaa tgggcgtgga tagcggtttg actcacgggg atttccaagt
ctccacccca 600ttgacgtcaa tgggagtttg ttttgcacca aaatcaacgg
gactttccaa aatgtcgtaa 660caactccgcc ccattgacgc aaatgggcgg
taggcgtgta cggtgggagg tctatataag 720cagagctcgt ttagtgaacc
gtcagatcgc ctggagacgc catccacgct gttttgacct 780ccatagaaga
caccgggacc gatccagcct ccgcggattc gaatcccggc cgggaacggt
840gcattggaac gcggattccc cgtgccaaga gtgacgtaag taccgcctat
agagtctata 900ggcccacaaa aaatgctttc ttcttttaat atactttttt
gtttatctta tttctaatac 960tttccctaat ctctttcttt cagggcaata
atgatacaat gtatcatgcc tctttgcacc 1020attctaaaga ataacagtga
taatttctgg gttaaggcaa tagcaatatt tctgcatata 1080aatatttctg
catataaatt gtaactgatg taagaggttt catattgcta atagcagcta
1140caatccagct accattctgc ttttatttta tggttgggat aaggctggat
tattctgagt 1200ccaagctagg cccttttgct aatcatgttc atacctctta
tcttcctccc acagctcctg 1260ggcaacgtgc tggtctgtgt gctggcccat
cactttggca aagaattggg attcgaacat 1320cgattgaatt ccccggggat
cctctagagt cgacctgcag aagcttgcct cgagcagcgc 1380tgctcgagag
atctacgggt ggcatccctg tgacccctcc ccagtgcctc tcctggccct
1440ggaagttgcc actccagtgc ccaccagcct tgtcctaata aaattaagtt
gcatcatttt 1500gtctgactag gtgtccttct ataatattat ggggtggagg
ggggtggtat ggagcaaggg 1560gcaagttggg aagacaacct gtagggcctg
cggggtctat tgggaaccaa gctggagtgc 1620agtggcacaa tcttggctca
ctgcaatctc cgcctcctgg gttcaagcga ttctcctgcc 1680tcagcctccc
gagttgttgg gattccaggc atgcatgacc aggctcagct aatttttgtt
1740tttttggtag agacggggtt tcaccatatt ggccaggctg gtctccaact
cctaatctca 1800ggtgatctac ccaccttggc ctcccaaatt gctgggatta
caggcgtgaa ccactgctcc 1860cttccctgtc cttctgattt tgtaggtaac
cacgtgcgga ccgagcggcc gcaggaaccc 1920ctagtgatgg agttggccac
tccctctctg cgcgctcgct cgctcactga ggccgggcga 1980ccaaaggtcg
cccgacgccc gggctttgcc cgggcggcct cagtgagcga gcgagcgcgc
2040agctgcctgc aggggcgcct gatgcggtat tttctcctta cgcatctgtg
cggtatttca 2100caccgcatac gtcaaagcaa ccatagtacg cgccctgtag
cggcgcatta agcgcggcgg 2160gtgtggtggt tacgcgcagc gtgaccgcta
cacttgccag cgccctagcg cccgctcctt 2220tcgctttctt cccttccttt
ctcgccacgt tcgccggctt tccccgtcaa gctctaaatc 2280gggggctccc
tttagggttc cgatttagtg ctttacggca cctcgacccc aaaaaacttg
2340atttgggtga tggttcacgt agtgggccat cgccctgata gacggttttt
cgccctttga 2400cgttggagtc cacgttcttt aatagtggac tcttgttcca
aactggaaca acactcaacc 2460ctatctcggg ctattctttt gatttataag
ggattttgcc gatttcggcc tattggttaa 2520aaaatgagct gatttaacaa
aaatttaacg cgaattttaa caaaatatta acgtttacaa 2580ttttatggtg
cactctcagt acaatctgct ctgatgccgc atagttaagc cagccccgac
2640acccgccaac acccgctgac gcgccctgac gggcttgtct gctcccggca
tccgcttaca 2700gacaagctgt gaccgtctcc gggagctgca tgtgtcagag
gttttcaccg tcatcaccga 2760aacgcgcgag acgaaagggc ctcgtgatac
gcctattttt ataggttaat gtcatgataa 2820taatggtttc ttagacgtca
ggtggcactt ttcggggaaa tgtgcgcgga acccctattt 2880gtttattttt
ctaaatacat tcaaatatgt atccgctcat gagacaataa ccctgataaa
2940tgcttcaata atattgaaaa aggaagagta tgagtattca acatttccgt
gtcgccctta 3000ttcccttttt tgcggcattt tgccttcctg tttttgctca
cccagaaacg ctggtgaaag 3060taaaagatgc tgaagatcag ttgggtgcac
gagtgggtta catcgaactg gatctcaaca 3120gcggtaagat ccttgagagt
tttcgccccg aagaacgttt tccaatgatg agcactttta 3180aagttctgct
atgtggcgcg gtattatccc gtattgacgc cgggcaagag caactcggtc
3240gccgcataca ctattctcag aatgacttgg ttgagtactc accagtcaca
gaaaagcatc 3300ttacggatgg catgacagta agagaattat gcagtgctgc
cataaccatg agtgataaca 3360ctgcggccaa cttacttctg acaacgatcg
gaggaccgaa ggagctaacc gcttttttgc 3420acaacatggg ggatcatgta
actcgccttg atcgttggga accggagctg aatgaagcca 3480taccaaacga
cgagcgtgac accacgatgc ctgtagcaat ggcaacaacg ttgcgcaaac
3540tattaactgg cgaactactt actctagctt cccggcaaca attaatagac
tggatggagg 3600cggataaagt tgcaggacca cttctgcgct cggcccttcc
ggctggctgg tttattgctg 3660ataaatctgg agccggtgag cgtgggtctc
gcggtatcat tgcagcactg gggccagatg 3720gtaagccctc ccgtatcgta
gttatctaca cgacggggag tcaggcaact atggatgaac 3780gaaatagaca
gatcgctgag ataggtgcct cactgattaa gcattggtaa ctgtcagacc
3840aagtttactc atatatactt tagattgatt taaaacttca tttttaattt
aaaaggatct 3900aggtgaagat cctttttgat aatctcatga ccaaaatccc
ttaacgtgag ttttcgttcc 3960actgagcgtc agaccccgta gaaaagatca
aaggatcttc ttgagatcct ttttttctgc 4020gcgtaatctg ctgcttgcaa
acaaaaaaac caccgctacc agcggtggtt tgtttgccgg 4080atcaagagct
accaactctt tttccgaagg taactggctt cagcagagcg cagataccaa
4140atactgtcct tctagtgtag ccgtagttag gccaccactt caagaactct
gtagcaccgc 4200ctacatacct cgctctgcta atcctgttac cagtggctgc
tgccagtggc gataagtcgt 4260gtcttaccgg gttggactca agacgatagt
taccggataa ggcgcagcgg tcgggctgaa 4320cggggggttc gtgcacacag
cccagcttgg agcgaacgac ctacaccgaa ctgagatacc 4380tacagcgtga
gctatgagaa agcgccacgc ttcccgaagg gagaaaggcg gacaggtatc
4440cggtaagcgg cagggtcgga acaggagagc gcacgaggga gcttccaggg
ggaaacgcct 4500ggtatcttta tagtcctgtc gggtttcgcc acctctgact
tgagcgtcga tttttgtgat 4560gctcgtcagg ggggcggagc ctatggaaaa
acgccagcaa cgcggccttt ttacggttcc 4620tggccttttg ctggcctttt
gctcacatgt 4650431897PRTHomo sapiens 43Met Ala Pro Glu Pro Ala Pro
Gly Arg Thr Met Val Pro Leu Val Pro 1 5 10 15 Ala Leu Val Met Leu
Gly Leu Val Ala Gly Ala His Gly Asp Ser Lys 20 25 30 Pro Val Phe
Ile Lys Val Pro Glu Asp Gln Thr Gly Leu Ser Gly Gly 35 40 45 Val
Ala Ser Phe Val Cys Gln Ala Thr Gly Glu Pro Lys Pro Arg Ile 50 55
60 Thr Trp Met Lys Lys Gly Lys Lys Val Ser Ser Gln Arg Phe Glu Val
65 70 75 80 Ile Glu Phe Asp Asp Gly Ala Gly Ser Val Leu Arg Ile Gln
Pro Leu 85 90 95 Arg Val Gln Arg Asp Glu Ala Ile Tyr Glu Cys Thr
Ala Thr Asn Ser 100 105 110 Leu Gly Glu Ile Asn Thr Ser Ala Lys Leu
Ser Val Leu Glu Glu Glu 115 120 125 Gln Leu Pro Pro Gly Phe Pro Ser
Ile Asp Met Gly Pro Gln Leu Lys 130 135 140 Val Val Glu Lys Ala Arg
Thr Ala Thr Met Leu Cys Ala Ala Gly Gly 145 150 155 160 Asn Pro Asp
Pro Glu Ile Ser Trp Phe Lys Asp Phe Leu Pro Val Asp 165 170 175 Pro
Ala Thr Ser Asn Gly Arg Ile Lys Gln Leu Arg Ser Gly Ala Leu 180 185
190 Gln Ile Glu Ser Ser Glu Glu Ser Asp Gln Gly Lys Tyr Glu Cys Val
195 200 205 Ala Thr Asn Ser Ala Gly Thr Arg Tyr Ser Ala Pro Ala Asn
Leu Tyr 210 215 220 Val Arg Val Arg Arg Val Ala Pro Arg Phe Ser Ile
Pro Pro Ser Ser 225 230 235 240 Gln Glu Val Met Pro Gly Gly Ser Val
Asn Leu Thr Cys Val Ala Val 245 250 255 Gly Ala Pro Met Pro Tyr Val
Lys Trp Met Met Gly Ala Glu Glu Leu 260 265 270 Thr Lys Glu Asp Glu
Met Pro Val Gly Arg Asn Val Leu Glu Leu Ser 275 280 285 Asn Val Val
Arg Ser Ala Asn Tyr Thr Cys Val Ala Ile Ser Ser Leu 290 295 300 Gly
Met Ile Glu Ala Thr Ala Gln Val Thr Val Lys Ala Leu Pro Lys 305 310
315 320 Pro Pro Ile Asp Leu Val Val Thr Glu Thr Thr Ala Thr Ser Val
Thr 325 330 335 Leu Thr Trp Asp Ser Gly Asn Ser Glu Pro Val Thr Tyr
Tyr Gly Ile 340 345 350 Gln Tyr Arg Ala Ala Gly Thr Glu Gly Pro Phe
Gln Glu Val Asp Gly 355 360 365 Val Ala Thr Thr Arg Tyr Ser Ile Gly
Gly Leu Ser Pro Phe Ser Glu 370 375 380 Tyr Ala Phe Arg Val Leu Ala
Val Asn Ser Ile Gly Arg Gly Pro Pro 385 390 395 400 Ser Glu Ala Val
Arg Ala Arg Thr Gly Glu Gln Ala Pro Ser Ser Pro 405 410 415 Pro Arg
Arg Val Gln Ala Arg Met Leu Ser Ala Ser Thr Met Leu Val 420 425 430
Gln Trp Glu Pro Pro Glu Glu Pro Asn Gly Leu Val Arg Gly Tyr Arg 435
440 445 Val Tyr Tyr Thr Pro Asp Ser Arg Arg Pro Pro Asn Ala Trp His
Lys 450 455 460 His Asn Thr Asp Ala Gly Leu Leu Thr Thr Val Gly Ser
Leu Leu Pro 465 470 475 480 Gly Ile Thr Tyr Ser Leu Arg Val Leu Ala
Phe Thr Ala Val Gly Asp 485 490 495 Gly Pro Pro Ser Pro Thr Ile Gln
Val Lys Thr Gln Gln Gly Val Pro 500 505 510 Ala Gln Pro Ala Asp Phe
Gln Ala Glu Val Glu Ser Asp Thr Arg Ile 515 520 525 Gln Leu Ser
Trp
Leu Leu Pro Pro Gln Glu Arg Ile Ile Met Tyr Glu 530 535 540 Leu Val
Tyr Trp Ala Ala Glu Asp Glu Asp Gln Gln His Lys Val Thr 545 550 555
560 Phe Asp Pro Thr Ser Ser Tyr Thr Leu Glu Asp Leu Lys Pro Asp Thr
565 570 575 Leu Tyr Arg Phe Gln Leu Ala Ala Arg Ser Asp Met Gly Val
Gly Val 580 585 590 Phe Thr Pro Thr Ile Glu Ala Arg Thr Ala Gln Ser
Thr Pro Ser Ala 595 600 605 Pro Pro Gln Lys Val Met Cys Val Ser Met
Gly Ser Thr Thr Val Arg 610 615 620 Val Ser Trp Val Pro Pro Pro Ala
Asp Ser Arg Asn Gly Val Ile Thr 625 630 635 640 Gln Tyr Ser Val Ala
Tyr Glu Ala Val Asp Gly Glu Asp Arg Gly Arg 645 650 655 His Val Val
Asp Gly Ile Ser Arg Glu His Ser Ser Trp Asp Leu Val 660 665 670 Gly
Leu Glu Lys Trp Thr Glu Tyr Arg Val Trp Val Arg Ala His Thr 675 680
685 Asp Val Gly Pro Gly Pro Glu Ser Ser Pro Val Leu Val Arg Thr Asp
690 695 700 Glu Asp Val Pro Ser Gly Pro Pro Arg Lys Val Glu Val Glu
Pro Leu 705 710 715 720 Asn Ser Thr Ala Val His Val Tyr Trp Lys Leu
Pro Val Pro Ser Lys 725 730 735 Gln His Gly Gln Ile Arg Gly Tyr Gln
Val Thr Tyr Val Arg Leu Glu 740 745 750 Asn Gly Glu Pro Arg Gly Leu
Pro Ile Ile Gln Asp Val Met Leu Ala 755 760 765 Glu Ala Gln Glu Thr
Thr Ile Ser Gly Leu Thr Pro Glu Thr Thr Tyr 770 775 780 Ser Val Thr
Val Ala Ala Tyr Thr Thr Lys Gly Asp Gly Ala Arg Ser 785 790 795 800
Lys Pro Lys Ile Val Thr Thr Thr Gly Ala Val Pro Gly Arg Pro Thr 805
810 815 Met Met Ile Ser Thr Thr Ala Met Asn Thr Ala Leu Leu Gln Trp
His 820 825 830 Pro Pro Lys Glu Leu Pro Gly Glu Leu Leu Gly Tyr Arg
Leu Gln Tyr 835 840 845 Cys Arg Ala Asp Glu Ala Arg Pro Asn Thr Ile
Asp Phe Gly Lys Asp 850 855 860 Asp Gln His Phe Thr Val Thr Gly Leu
His Lys Gly Thr Thr Tyr Ile 865 870 875 880 Phe Arg Leu Ala Ala Lys
Asn Arg Ala Gly Leu Gly Glu Glu Phe Glu 885 890 895 Lys Glu Ile Arg
Thr Pro Glu Asp Leu Pro Ser Gly Phe Pro Gln Asn 900 905 910 Leu His
Val Thr Gly Leu Thr Thr Ser Thr Thr Glu Leu Ala Trp Asp 915 920 925
Pro Pro Val Leu Ala Glu Arg Asn Gly Arg Ile Ile Ser Tyr Thr Val 930
935 940 Val Phe Arg Asp Ile Asn Ser Gln Gln Glu Leu Gln Asn Ile Thr
Thr 945 950 955 960 Asp Thr Arg Phe Thr Leu Thr Gly Leu Lys Pro Asp
Thr Thr Tyr Asp 965 970 975 Ile Lys Val Arg Ala Trp Thr Ser Lys Gly
Ser Gly Pro Leu Ser Pro 980 985 990 Ser Ile Gln Ser Arg Thr Met Pro
Val Glu Gln Val Phe Ala Lys Asn 995 1000 1005 Phe Arg Val Ala Ala
Ala Met Lys Thr Ser Val Leu Leu Ser Trp 1010 1015 1020 Glu Val Pro
Asp Ser Tyr Lys Ser Ala Val Pro Phe Lys Ile Leu 1025 1030 1035 Tyr
Asn Gly Gln Ser Val Glu Val Asp Gly His Ser Met Arg Lys 1040 1045
1050 Leu Ile Ala Asp Leu Gln Pro Asn Thr Glu Tyr Ser Phe Val Leu
1055 1060 1065 Met Asn Arg Gly Ser Ser Ala Gly Gly Leu Gln His Leu
Val Ser 1070 1075 1080 Ile Arg Thr Ala Pro Asp Leu Leu Pro His Lys
Pro Leu Pro Ala 1085 1090 1095 Ser Ala Tyr Ile Glu Asp Gly Arg Phe
Asp Leu Ser Met Pro His 1100 1105 1110 Val Gln Asp Pro Ser Leu Val
Arg Trp Phe Tyr Ile Val Val Val 1115 1120 1125 Pro Ile Asp Arg Val
Gly Gly Ser Met Leu Thr Pro Arg Trp Ser 1130 1135 1140 Thr Pro Glu
Glu Leu Glu Leu Asp Glu Leu Leu Glu Ala Ile Glu 1145 1150 1155 Gln
Gly Gly Glu Glu Gln Arg Arg Arg Arg Arg Gln Ala Glu Arg 1160 1165
1170 Leu Lys Pro Tyr Val Ala Ala Gln Leu Asp Val Leu Pro Glu Thr
1175 1180 1185 Phe Thr Leu Gly Asp Lys Lys Asn Tyr Arg Gly Phe Tyr
Asn Arg 1190 1195 1200 Pro Leu Ser Pro Asp Leu Ser Tyr Gln Cys Phe
Val Leu Ala Ser 1205 1210 1215 Leu Lys Glu Pro Met Asp Gln Lys Arg
Tyr Ala Ser Ser Pro Tyr 1220 1225 1230 Ser Asp Glu Ile Val Val Gln
Val Thr Pro Ala Gln Gln Gln Glu 1235 1240 1245 Glu Pro Glu Met Leu
Trp Val Thr Gly Pro Val Leu Ala Val Ile 1250 1255 1260 Leu Ile Ile
Leu Ile Val Ile Ala Ile Leu Leu Phe Lys Arg Lys 1265 1270 1275 Arg
Thr His Ser Pro Ser Ser Lys Asp Glu Gln Ser Ile Gly Leu 1280 1285
1290 Lys Asp Ser Leu Leu Ala His Ser Ser Asp Pro Val Glu Met Arg
1295 1300 1305 Arg Leu Asn Tyr Gln Thr Pro Gly Met Arg Asp His Pro
Pro Ile 1310 1315 1320 Pro Ile Thr Asp Leu Ala Asp Asn Ile Glu Arg
Leu Lys Ala Asn 1325 1330 1335 Asp Gly Leu Lys Phe Ser Gln Glu Tyr
Glu Ser Ile Asp Pro Gly 1340 1345 1350 Gln Gln Phe Thr Trp Glu Asn
Ser Asn Leu Glu Val Asn Lys Pro 1355 1360 1365 Lys Asn Arg Tyr Ala
Asn Val Ile Ala Tyr Asp His Ser Arg Val 1370 1375 1380 Ile Leu Thr
Ser Ile Asp Gly Val Pro Gly Ser Asp Tyr Ile Asn 1385 1390 1395 Ala
Asn Tyr Ile Asp Gly Tyr Arg Lys Gln Asn Ala Tyr Ile Ala 1400 1405
1410 Thr Gln Gly Pro Leu Pro Glu Thr Met Gly Asp Phe Trp Arg Met
1415 1420 1425 Val Trp Glu Gln Arg Thr Ala Thr Val Val Met Met Thr
Arg Leu 1430 1435 1440 Glu Glu Lys Ser Arg Val Lys Cys Asp Gln Tyr
Trp Pro Ala Arg 1445 1450 1455 Gly Thr Glu Thr Cys Gly Leu Ile Gln
Val Thr Leu Leu Asp Thr 1460 1465 1470 Val Glu Leu Ala Thr Tyr Thr
Val Arg Thr Phe Ala Leu His Lys 1475 1480 1485 Ser Gly Ser Ser Glu
Lys Arg Glu Leu Arg Gln Phe Gln Phe Met 1490 1495 1500 Ala Trp Pro
Asp His Gly Val Pro Glu Tyr Pro Thr Pro Ile Leu 1505 1510 1515 Ala
Phe Leu Arg Arg Val Lys Ala Cys Asn Pro Leu Asp Ala Gly 1520 1525
1530 Pro Met Val Val His Cys Ser Ala Gly Val Gly Arg Thr Gly Cys
1535 1540 1545 Phe Ile Val Ile Asp Ala Met Leu Glu Arg Met Lys His
Glu Lys 1550 1555 1560 Thr Val Asp Ile Tyr Gly His Val Thr Cys Met
Arg Ser Gln Arg 1565 1570 1575 Asn Tyr Met Val Gln Thr Glu Asp Gln
Tyr Val Phe Ile His Glu 1580 1585 1590 Ala Leu Leu Glu Ala Ala Thr
Cys Gly His Thr Glu Val Pro Ala 1595 1600 1605 Arg Asn Leu Tyr Ala
His Ile Gln Lys Leu Gly Gln Val Pro Pro 1610 1615 1620 Gly Glu Ser
Val Thr Ala Met Glu Leu Glu Phe Lys Leu Leu Ala 1625 1630 1635 Ser
Ser Lys Ala His Thr Ser Arg Phe Ile Ser Ala Asn Leu Pro 1640 1645
1650 Cys Asn Lys Phe Lys Asn Arg Leu Val Asn Ile Met Pro Tyr Glu
1655 1660 1665 Leu Thr Arg Val Cys Leu Gln Pro Ile Arg Gly Val Glu
Gly Ser 1670 1675 1680 Asp Tyr Ile Asn Ala Ser Phe Leu Asp Gly Tyr
Arg Gln Gln Lys 1685 1690 1695 Ala Tyr Ile Ala Thr Gln Gly Pro Leu
Ala Glu Ser Thr Glu Asp 1700 1705 1710 Phe Trp Arg Met Leu Trp Glu
His Asn Ser Thr Ile Ile Val Met 1715 1720 1725 Leu Thr Lys Leu Arg
Glu Met Gly Arg Glu Lys Cys His Gln Tyr 1730 1735 1740 Trp Pro Ala
Glu Arg Ser Ala Arg Tyr Gln Tyr Phe Val Val Asp 1745 1750 1755 Pro
Met Ala Glu Tyr Asn Met Pro Gln Tyr Ile Leu Arg Glu Phe 1760 1765
1770 Lys Val Thr Asp Ala Arg Asp Gly Gln Ser Arg Thr Ile Arg Gln
1775 1780 1785 Phe Gln Phe Thr Asp Trp Pro Glu Gln Gly Val Pro Lys
Thr Gly 1790 1795 1800 Glu Gly Phe Ile Asp Phe Ile Gly Gln Val His
Lys Thr Lys Glu 1805 1810 1815 Gln Phe Gly Gln Asp Gly Pro Ile Thr
Val His Cys Ser Ala Gly 1820 1825 1830 Val Gly Arg Thr Gly Val Phe
Ile Thr Leu Ser Ile Val Leu Glu 1835 1840 1845 Arg Met Arg Tyr Glu
Gly Val Val Asp Met Phe Gln Thr Val Lys 1850 1855 1860 Thr Leu Arg
Thr Gln Arg Pro Ala Met Val Gln Thr Glu Asp Gln 1865 1870 1875 Tyr
Gln Cys Tyr Arg Ala Ala Leu Glu Tyr Leu Gly Ser Phe Asp 1880 1885
1890 His Tyr Ala Thr 1895 44440PRTunknownConsensus sequence 44Leu
Leu Ile Gly Ser Thr Ser Glu Pro Ala Tyr Asp Lys Ser Val Cys 1 5 10
15 Asp Ser Asn Asn Lys Glu Tyr Met Gly Ile Glu Val Tyr Val Glu Ala
20 25 30 Thr Leu Asp Glu Pro Leu Lys Gln Thr Thr Cys Glu Ser Glu
Ile His 35 40 45 Lys Tyr Gly Ala Ser Val Ser Asn Gly Gly Leu Asn
Ile Ser Val Asp 50 55 60 Leu Leu Asn Cys Phe Leu Asn Phe His Thr
Val Gly Val Tyr Thr Asn 65 70 75 80 Arg Asp Thr Gly Val Tyr Thr Asn
Arg Asp Thr Val Tyr Ala Lys Phe 85 90 95 Ala Ser Leu Asp Pro Ser
Thr Glu Pro Ile Asn Ser Met Thr His Asp 100 105 110 Asp Leu Val Lys
Leu Thr Glu Glu Cys Ile Val Asp Ile Tyr Leu Lys 115 120 125 Cys Glu
Val Asp Lys Thr Lys Asp Phe Met Lys Asn Gly Asn Arg Leu 130 135 140
Lys Pro Arg Asp Phe Lys Thr Val Pro Pro Ser Asn Val Gly Ser Met 145
150 155 160 Ile Glu Leu Gln Ser Asp Tyr Cys Val Glu Asp Val Thr Ala
Tyr Val 165 170 175 Lys Ile Tyr Asp Glu Cys Gly Asn Ile Lys Gln His
Ser Ile Pro Thr 180 185 190 Leu Arg Asp Tyr Phe Thr Thr Lys Asn Gly
Gln Pro Arg Lys Ile Leu 195 200 205 Lys Lys Lys Phe Asp Ser Cys Gly
Lys Thr His Thr Cys Pro Pro Cys 210 215 220 Pro Ala Pro Glu Gly Ala
Pro Ser Val Phe Leu Phe Pro Pro Lys Pro 225 230 235 240 Lys Asp Thr
Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val 245 250 255 Val
Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val 260 265
270 Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln
275 280 285 Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu
His Gln 290 295 300 Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val
Ser Asn Lys Ala 305 310 315 320 Leu Pro Ala Pro Ile Glu Lys Thr Ile
Ser Lys Ala Lys Gly Gln Pro 325 330 335 Arg Glu Pro Gln Val Tyr Thr
Leu Pro Pro Ser Arg Asp Glu Leu Thr 340 345 350 Lys Asn Gln Val Ser
Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser 355 360 365 Asp Ile Ala
Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr 370 375 380 Lys
Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr 385 390
395 400 Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val
Phe 405 410 415 Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr
Thr Gln Lys 420 425 430 Ser Leu Ser Leu Ser Pro Gly Lys 435 440
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