U.S. patent application number 12/413308 was filed with the patent office on 2010-02-18 for neutralizing antibodies to influenza viruses.
This patent application is currently assigned to Sea Lane Biotechnologies. Invention is credited to Ramesh Bhatt, Lawrence Horowitz, Arun Kashyap.
Application Number | 20100040635 12/413308 |
Document ID | / |
Family ID | 41021445 |
Filed Date | 2010-02-18 |
United States Patent
Application |
20100040635 |
Kind Code |
A1 |
Horowitz; Lawrence ; et
al. |
February 18, 2010 |
NEUTRALIZING ANTIBODIES TO INFLUENZA VIRUSES
Abstract
The present invention concerns methods and means for
identifying, producing, and engineering neutralizing molecules
against influenza A viruses, and to the neutralizing molecules
produced. In particular, the invention concerns neutralizing
molecules against various influenza A virus subtypes, including
neutralizing antibodies against H5 and/or H3 and/or H1, such as,
for example all of H1, H3, and H5 subtypes, and methods and means
for making such molecules.
Inventors: |
Horowitz; Lawrence;
(Atherton, CA) ; Bhatt; Ramesh; (Belmont, CA)
; Kashyap; Arun; (Newark, CA) |
Correspondence
Address: |
Goodwin Procter LLP;Attn: Patent Administrator
135 Commonwealth Drive
Menlo Park
CA
94025-1105
US
|
Assignee: |
Sea Lane Biotechnologies
Atherton
CA
|
Family ID: |
41021445 |
Appl. No.: |
12/413308 |
Filed: |
March 27, 2009 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
|
61040459 |
Mar 28, 2008 |
|
|
|
Current U.S.
Class: |
424/159.1 ;
435/5; 506/18; 530/389.1; 530/389.4 |
Current CPC
Class: |
C07K 2317/56 20130101;
C07K 2317/92 20130101; A61K 39/145 20130101; C07K 2317/567
20130101; C12N 7/00 20130101; A61P 31/16 20180101; C12N 2760/16111
20130101; A61K 2039/505 20130101; C07K 2317/21 20130101; A61K
2039/545 20130101; A61K 39/42 20130101; C07K 16/1018 20130101; C07K
2317/622 20130101; C12N 2760/16134 20130101; C07K 2317/76 20130101;
C07K 2317/55 20130101; C07K 2317/565 20130101; A61K 39/00
20130101 |
Class at
Publication: |
424/159.1 ;
530/389.4; 530/389.1; 435/5; 506/18 |
International
Class: |
A61K 39/395 20060101
A61K039/395; C07K 16/00 20060101 C07K016/00; A61P 31/16 20060101
A61P031/16; C12Q 1/70 20060101 C12Q001/70; C40B 40/10 20060101
C40B040/10 |
Claims
1. A molecule, which is an antibody or antibody-like molecule,
wherein said molecule (i) neutralizes more than one subtype and/or
more than one isolate of an influenza A virus, (ii) binds to a
hemagglutinin (HA) antigen of the virus, and (iii) does not inhibit
hemagglutination.
2. The molecule of claim 1, which is a polypeptide comprising a
VpreB sequence and/or a .lamda.5 sequence.
3. The molecule of claim 1, which is a polypeptide comprising a
VpreB sequence fused to a .lamda.5 sequence.
4. The molecule of claim 1, which is a .kappa.-like surrogate light
chain (SLC) construct comprising a V.kappa.-like and/or a JC.kappa.
sequence.
5. The molecule of claim 1, which is an antibody.
6. The molecule of claim 1 which is cross-reactive with at least
two HA antigens selected from the group consisting of H1, H2, H3,
H5, H6, H7, H8 and H9.
7. The molecule of claim 1 which is cross-reactive with at least
two HA antigens selected from the group consisting of H1, H2, H3,
H5, and H9.
8. The molecule of claim 1 which binds to an epitope of an H1
subtype of the HA antigen.
9. The molecule of claim 1 which binds to an epitope of an H5
subtype of the HA antigen.
10. The molecule of claim 1 which binds to an epitope of an H3
subtype of the HA antigen.
11. The molecule of any one of claims 8, 9, or 10 wherein the
epitope is displayed on the surface of an influenza A virus.
12. The molecule of any one of claims 8, 9, or 10 which neutralizes
at least one of the H5, H3, and H1 influenza A virus subtypes.
13. The molecule of any one of claims 8, 9, or 10 which neutralizes
more than one isolate of an H5 and/or H3 and/or H1 subtype of an
influenza A virus.
14. The molecule of claim 1, which does not prevent the globular
head region of the influenza A virus from binding the surface of a
cell.
15. The molecule of claim 1 wherein at least one of said viruses
has the ability to infect humans.
16. The molecule of claim 1 wherein at least one of said isolates
has been obtained from a human subject.
17. The molecule of claim 1 wherein at least one of said isolates
has been obtained from a non-human animal.
18. The molecule of claim 17 wherein said non-human animal is a
bird.
19. The molecule of claim 18 wherein said bird is a wild-fowl or a
chicken.
20. The molecule of claim 1 which binds to an H1 HA antigen.
21. The molecule of claim 20 which binds to at least one additional
HA antigen.
22. The molecule of claim 21 wherein said additional HA antigen is
selected from the group consisting of H2, H3, H5, H6, H7, H8 and
H9.
23. The molecule of claim 21 which additionally binds HA antigen
H5.
24. The molecule of claim 21 which additionally binds HA antigens
H3 and H9.
25. The molecule of claim 21 which additionally binds HA antigens
H3, H5, and H9.
26. An antibody or antibody-like molecule which binds essentially
the same epitope as the epitope for an antibody or antibody-like
molecule comprising a heavy chain polypeptide comprising an amino
acid sequence selected from the group consisting of SEQ ID NO:4,
SEQ ID NO:45, SEQ ID NO:9, and SEQ ID NO:61; or a consensus or
variant sequence based upon said amino acid sequences.
27. The antibody or antibody-like molecule of claim 26 which binds
essentially the same epitope as the epitope for an antibody or
antibody-like molecule comprising a light chain polypeptide
comprising an amino acid sequence selected from the group
consisting of SEQ ID NO:71, SEQ ID NO:140, SEQ ID NO:81, SEQ ID
NO:158, SEQ ID NO:159, and SEQ ID NO:160; or a consensus or variant
sequence based upon said amino acid sequences.
28. An antibody or antibody-like molecule which binds essentially
the same epitope as the epitope for an antibody or antibody-like
molecule comprising a heavy chain polypeptide comprising an amino
acid sequence having the formula:
X.sub.1-X.sub.2-Q-L-V-Q-S-G-X.sub.3-E-V-X.sub.4-K-P-G-X.sub.5-S-V-X.sub.6-
-X.sub.7-S-C-K-X.sub.8-S-G-G-X.sub.9-F-S-S-Y-A-X.sub.10-X.sub.11-W-V-R-Q-A-
-P-G-Q-G-L-E-W-M-G-X.sub.12-G-I-I-X.sub.13-X.sub.14-F-G-T-T-X.sub.15-N-Y-A-
-Q-K-F-Q-G-R-X.sub.16-T-X.sub.17-T-A-D-X.sub.18-X.sub.19-T-S-T-A-Y-M-E-L-S-
-S-L-R-S-X.sub.20-D-T-A-V-Y-Y-C-A-R-G-S-Y-Y-Y-E-X.sub.21-X.sub.22-L-D-Y-W--
G-X.sub.23-G-T-X.sub.24; or a consensus or variant sequence based
upon said amino acid sequences, or a fragment thereof; wherein
X.sub.1 is Q or E; X.sub.2 is V or M; X.sub.3 is A or T; X.sub.4 is
K or Q; X.sub.5 is S or A; X.sub.6 is K or R; X.sub.7 is V or L;
X.sub.8 is A, T or V; X.sub.9 is T, S or A; X.sub.10 is I or V;
X.sub.11 is S or T; X.sub.12 is G or A; X.sub.13 is P or G;
X.sub.14 is I or M; X.sub.15 is A or T; X.sub.16 is V or L;
X.sub.17 is I, L, or M; X.sub.18 is K or E; X.sub.19 is S, L or M;
X.sub.20 is E or D; X.sub.2 is S, T or N; X.sub.22 is S or T;
X.sub.23 is Q, K, G or R; and X.sub.24 is L, T or M.
29. The antibody or antibody-like molecule of claim 28 which binds
essentially the same epitope as the epitope for an antibody or
antibody-like molecule comprising a light chain polypeptide
comprising an amino acid sequence selected from the group
consisting of SEQ ID NO:71, SEQ ID NO:140, SEQ ID NO:81, SEQ ID
NO:158, SEQ ID NO:159, and SEQ ID NO:160; or a consensus or variant
sequence based upon said amino acid sequences.
30. An antibody or antibody-like molecule comprising a heavy chain
polypeptide comprising an amino acid sequence selected from the
group consisting of SEQ ID NO:4, SEQ ID NO:45, SEQ ID NO:9, and SEQ
ID NO:61; or a consensus or variant sequence based upon said amino
acid sequences.
31. The antibody or antibody-like molecule of claim 30 further
comprising a light chain polypeptide comprising an amino acid
sequence selected from the group consisting of SEQ ID NO:71, SEQ ID
NO:140, SEQ ID NO:81, SEQ ID NO:158, SEQ ID NO:159, and SEQ ID
NO:160; or a consensus or variant sequence based upon said amino
acid sequences.
32. An antibody or antibody-like molecule comprising a heavy chain
polypeptide comprising an amino acid sequence having the formula:
X.sub.1-X.sub.2-Q-L-V-Q-S-G-X.sub.3-E-V-X.sub.4-K-P-G-X.sub.5-S-V-X.sub.6-
-X.sub.7-S-C-K-X.sub.8-S-G-G-X.sub.9-F-S-S-Y-A-X.sub.10-X.sub.11-W-V-R-Q-A-
-P-G-Q-G-L-E-W-M-G-X.sub.12-G-I-I-X.sub.13-X.sub.14-F-G-T-T-X.sub.15-N-Y-A-
-Q-K-F-Q-G-R-X.sub.16-T-X.sub.17-T-A-D-X.sub.18-X.sub.19-T-S-T-A-Y-M-E-L-S-
-S-L-R-S-X.sub.20-D-T-A-V-Y-Y-C-A-R-G-S-Y-Y-Y-E-X.sub.21-X.sub.22-L-D-Y-W--
G-X.sub.23-G-T-X.sub.24; or a consensus or variant sequence based
upon said amino acid sequences, or a fragment thereof; wherein
X.sub.1 is Q or E; X.sub.2 is V or M; X.sub.3 is A or T; X.sub.4 is
K or Q; X.sub.5 is S or A; X.sub.6 is K or R; X.sub.7 is V or L;
X.sub.8 is A, T or V; X.sub.9 is T, S or A; X.sub.10 is I or V;
X.sub.11 is S or T; X.sub.12 is G or A; X.sub.13 is P or G;
X.sub.14 is I or M; X.sub.15 is A or T; X.sub.16 is V or L;
X.sub.17 is I, L, or M; X.sub.18 is K or E; X.sub.19 is S, L or M;
X.sub.20 is E or D; X.sub.21 is S, T or N; X.sub.22 is S or T;
X.sub.23 is Q, K, G or R; and X.sub.24 is L, T or M.
33. The antibody or antibody-like molecule of claim 32 further
comprising a light chain polypeptide comprising an amino acid
sequence selected from the group consisting of SEQ ID NO:71, SEQ ID
NO:140, SEQ ID NO:81, SEQ ID NO:158, SEQ ID NO:159, and SEQ ID
NO:160; or a consensus or variant sequence based upon said amino
acid sequences.
34. The antibody or antibody-like molecule of any one of claims 26
to 33 which (i) neutralizes more than one subtype and/or more than
one isolate of an influenza A virus, (ii) binds to a hemagglutinin
(HA) antigen of the virus, and (iii) does not inhibit hem
agglutination.
35. A composition comprising a molecule according to any one of
claims 1 to 25.
36. A composition comprising an antibody or antibody-like molecule
according to any one of claims 26 to 34.
37. A molecule comprising an antibody heavy chain variable domain
comprising at least one substitution in the surface exposed cluster
determined by amino acid positions 52A, 53, 73, and 74, following
Kabat amino acid numbering, wherein said molecule is capable of
binding to and neutralizing a viral antigen.
38. The molecule of claim 37, comprising a substitution at least
one of amino acid positions 52A, 53, 73, and 74.
39. The molecule of claim 37, comprising a substitution at all of
amino acid positions 52A, 53, 73, and 74.
40. The molecule of claim 39, further comprising a substitution at
amino acid position 57.
41. The molecule of claim 39 comprising P52G, 153M, L73E, and
S74L/M substitutions.
42. The molecule of claim 41 additionally comprising an A57T
substitution.
43. The molecule of claim 42 additionally comprising a substitution
at least one of amino acid positions 24, 34, 35 and 50.
44. The molecule of claim 43 comprising substitutions at all of
amino acid positions 24, 34, 35 and 50.
45. The molecule of claim 44 comprising V24T, W34V, G35T and S50A
substitutions.
46. The molecule of any one of claims 37 to 45 wherein the heavy
chain variable domain sequence is from the V.sub.H 1e germ-line
heavy chain.
47. The molecule of claim 46 wherein the rest of the heavy chain
variable domain sequence retains the sequence of the V.sub.H 1e
germ-line heavy chain.
48. The molecule of claim 46 wherein the V.sub.H 1e germ-line heavy
chain variable domain comprises at least one additional
conservative substitution.
49. The molecule of any one of claims 38 to 48, further comprising
a light chain sequence.
50. The molecule of claim 49 wherein the light chain sequence is an
antibody .lamda. or .kappa. light chain sequence.
51. The molecule of claim 49 wherein the light chain sequence is a
surrogate light chain sequence.
52. The molecule of claim 51 wherein the surrogate light chain
sequence comprises a VpreB sequence and/or a .lamda.5 sequence.
53. The molecule of claim 52, wherein the surrogate light chain
sequence comprises a VpreB sequence fused to a .lamda.5
sequence.
54. The molecule of claim 51, wherein the surrogate light chain
sequence is a .kappa.-like surrogate light chain (SLC) construct
comprising a V.kappa.-like and/or a JC.kappa. sequence.
55. The molecule of any one of claims 37 to 54, wherein the viral
antigen is selected from the group consisting of viral antigens
from influenza viruses, HIV-1, HIV-2, HTLV-I and -II viruses, SARS
coronavirus, herpes simplex virus, Epstein Barr virus,
cytomegalovirus, HCV, HAV, HBV, HDV, HEV, toxoplasma gondii virus,
treponema pallidium virus, human T-lymphotrophic virus,
encephalitis virus, West Nile virus, Dengue virus, Varicella Zoster
Virus, rubeola, mumps, and rubella.
56. The molecule of claim 55 wherein the viral antigen is from an
influenza virus or an HIV-1 or HIV-2 virus.
57. A vaccine effective against an influenza A virus, comprising a
peptide or polypeptide functionally mimicking a neutralization
epitope of a molecule according to any one of claims 1 to 36.
58. A vaccine effective against a viral antigen, comprising a
peptide or polypeptide functionally mimicking a neutralization
epitope of a molecule according to any one of claims 37 to 57.
59. A method for identifying an antibody capable of neutralizing an
isolate of an H5 influenza A virus and/or an isolate of an H1
influenza A virus; or a subtype of an H5 influenza A virus and/or a
subtype of an H1 influenza A virus, comprising identifying, in an
antibody library, antibodies that react with both an H5 isolate
and/or an H1 isolate; or an H5 subtype and/or an H1 subtype, and
subjecting the antibodies identified to successive alternating
rounds of selection, based on their ability to bind said H5 and/or
H1 isolates or HA proteins; or said H5 and/or H1 subtypes or HA
proteins, respectively.
60. A collection of sequences shared by the neutralizing antibodies
identified by the method of claim 59.
61. A collection of sequences comprising one or more of the unique
heavy and/or light chain sequences shown in Table 2 or a consensus
or variant sequence based on said sequences.
62. A neutralizing antibody identifiable by the method of claim 59,
or a fragment thereof.
Description
CROSS-REFERENCE TO RELATED APPLICATIONS
[0001] This application claims priority under Section .sctn.119(e)
and the benefit of U.S. Provisional Application Ser. No. 61/040,459
filed Mar. 28, 2008, the disclosure of which is incorporated herein
by reference in its entirety.
FIELD OF THE INVENTION
[0002] The present invention concerns methods and means for
identifying, producing, and engineering neutralizing molecules
against viral antigens, including influenza A viruses, and to the
neutralizing molecules produced. The invention further concerns
various uses of the molecules produced, including the design and
production of vaccines utilizing the binding sites of the
neutralizing molecules of the present invention on the target viral
antigen, such as influenza A virus.
[0003] Viruses are infectious pathogens that can cause serious
diseases including major threats for global public health, such as
the influenza, AIDS, and hepatitis. A number of cancers have also
been linked to viruses in conjunction with environmental factors. A
typical virus is a sub-micrometer particle that has DNA or RNA
packaged in a shell known as the capsid. Viral antigens protrude
from the capsid and often fulfill important function in docking to
the host cell, fusion, and injection of viral DNA/RNA.
Antibody-based immune responses form a first layer of protection of
the host from viral infection, however, in many cases a vigorous
cellular immune response mediated by T-cells and NK-cells is
required for effective viral clearance. When cellular immunity is
unable to clear the virus, the infection can become chronic, and
serum antibodies to the viral pathogen are used as first indicator
for the diagnosis of the disease. Antibodies and antibody-like
molecules would be valuable tools for passive immunization against,
or for the treatment of such viral diseases.
[0004] One viral disease, the flu, is a contagious respiratory
illness caused by influenza viruses. It causes mild to severe
illness, and at times can lead to death. Annually, in the United
States, influenza is contracted by 5-20% of the population,
hospitalizing about 200,000, and causing the deaths of about
36,000.
[0005] Influenza viruses spread in respiratory droplets caused by
coughing and sneezing, which are usually transmitted from person to
person. Immunity to influenza surface antigens, particularly
hemagglutinin, reduces the likelihood of infection and severity of
disease if infection occurs. Although influenza vaccines are
available, because a vaccine against one influenza virus type or
subtype confers limited or no protection against another type or
subtype of influenza, it is necessary to incorporate one or more
new strains in each year's influenza vaccine.
[0006] Influenza viruses are segmented negative-strand RNA viruses
and belong to the Orthomyxoviridae family. Influenza A virus
consists of 9 structural proteins and codes additionally for one
nonstructural NS1 protein with regulatory functions. The
non-structural NS1 protein is synthesized in large quantities
during the reproduction cycle and is localized in the cytosol and
nucleus of the infected cells. The segmented nature of the viral
genome allows the mechanism of genetic reassortment (exchange of
genome segments) to take place during mixed infection of a cell
with different viral strains. The influenza A virus may be further
classified into various subtypes depending on the different
hemagglutinin (HA) and neuraminidase (NA) viral proteins displayed
on their surface. Influenza A virus subtypes are identified by two
viral surface glycoproteins, hemagglutinin (HA or H) and
neuraminidase (NA or N). Each influenza virus subtype is identified
by its combination of H and N proteins. There are 16 known HA
subtypes and 9 known NA subtypes. Influenza type A viruses can
infect people, birds, pigs, horses, and other animals, but wild
birds are the natural hosts for these viruses. Only some influenza
A subtypes (i.e., H1N1, H1N2, and H3N2) are currently in
circulation among people, but all combinations of the 16H and 9 NA
subtypes have been identified in avian species, especially in wild
waterfowl and shorebirds. In addition, there is increasing evidence
that H5 and H7 influenza viruses can also cause human illness.
[0007] The HA of influenza A virus comprises two structurally
distinct regions, namely, a globular head region and a stem region.
The globular head region contains a receptor binding site which is
responsible for virus attachment to a target cell and participates
in the hemagglutination activity of HA. The stem region contains a
fusion peptide which is necessary for membrane fusion between the
viral envelope and an endosomal membrane of the cell and thus
relates to fusion activity (Wiley et al., Ann. Rev. Biochem.,
56:365-394 (1987)).
[0008] A pandemic is a global disease outbreak. An influenza
pandemic occurs when a new influenza A virus: (1) emerges for which
there is little or no immunity in the human population, (2) begins
to cause serious illness, and then (3) spreads easily
person-to-person worldwide. During the 20.sup.th century there have
been three such influenza pandemics. First, in 1918, the "Spanish
Flu" influenza pandemic caused at least 500,000 deaths in the
United States and up to 40 million deaths worldwide. This pandemic
was caused by influenza A H1N1 subtype. Second, in 1957, the "Asian
Flu" influenza pandemic, caused by the influenza A H2N2 subtype,
resulted in at least 70,000 deaths in the United States and 1-2
million deaths worldwide. Most recently in 1968 the "Hong Kong Flu"
influenza pandemic, caused by the influenza A H3N2 subtype,
resulted in about 34,000 U.S. deaths and 700,000 deaths
worldwide.
[0009] In 1997, the first influenza A H5N1 cases were reported in
Hong Kong. This was the first time that this type of avian virus
directly infected humans, but a pandemic did not result because
human to human transmission was not observed.
[0010] Lu et al., Resp. Res. 7:43 (2006) (doi:
10.1186/1465-992-7-43) report the preparation of anti-H5N1 IgGs
from horses vaccinated with inactivated H5N1 virus, and of
H5N1-specific F(ab').sub.2 fragments, which were described to
protect BALB/c mice infected with H5N1 virus.
[0011] Hanson et al., Resp. Res. 7:126 (doi:
10.1186/1465-9921-7-126) describe the use of a chimeric monoclonal
antibody specific for influenza A H5 virus hemagglutinin for
passive immunization of mice.
[0012] Neutralizing antibodies to influenza viruses are disclosed
in U.S. Application Publication No. 20080014205, published on Jan.
17, 2008.
[0013] In view of the severity of the respiratory illness caused by
certain influenza A viruses, and the threat of a potential
pandemic, there is a great need for effective preventative and
treatment methods. The present invention addresses this need by
providing influenza A neutralizing molecules against various H
subtypes of the virus, including, without limitation, the H1, and
H3 subtypes, and the H5 subtype of the influenza A virus. The
invention further provides molecules capable of neutralizing more
than one, and preferably all, isolates (strains) of a given subtype
of the influenza A virus, including, without limitation, isolates
obtained from various human and non-human species and isolates from
victims and/or survivors of various influenza epidemics and/or
pandemics.
[0014] Such neutralizing molecules can be used for the prevention
and/or treatment influenza virus infection, including passive
immunization of infected or at risk populations in cases of
epidemics or pandemics.
SUMMARY OF THE INVENTION
[0015] In one aspect, the present invention concerns a molecule
that can neutralize at least one subtype of an influenza virus
and/or at least one isolate of an influenza virus. In one
embodiment, the molecule is an antibody or an antibody-like
molecule, wherein the molecule (i) neutralizes more than one
subtype and/or more than one isolate of an influenza A virus, (ii)
binds to a hemagglutinin (HA) antigen of the virus, and (iii) does
not inhibit hemagglutination. In another embodiment, the molecule
is a polypeptide comprising a VpreB sequence and/or a .lamda.5
sequence. In one other embodiment, the molecule is a polypeptide
comprising a VpreB sequence fused to a .lamda.5 sequence. In
another embodiment, the molecule is a .kappa.-like surrogate light
chain (SLC) construct comprising a V.kappa.-like and/or a JC.kappa.
sequence. In one embodiment, the molecule is an antibody. In
another embodiment, the molecule is cross-reactive with at least
two HA antigens selected from the group consisting of H1, H2, H3,
H5, H6, H7, H8 and H9. In yet another embodiment, the molecule is
cross-reactive with at least two HA antigens selected from the
group consisting of H1, H2, H3, H5, and H9.
[0016] In all embodiments, the molecule is an antibody or an
antibody-like molecule.
[0017] In all embodiments, the molecule is an antibody
fragment.
[0018] In one aspect, the present invention concerns a neutralizing
molecule neutralizing an influenza A virus subtype. In one
embodiment, the molecule is an antibody or an antibody-like
molecule, wherein the molecule (i) neutralizes more than one
subtype and/or more than one isolate of an influenza A virus, (ii)
binds to a hemagglutinin (HA) antigen of the virus, and (iii) does
not inhibit hemagglutination. In other embodiments, the molecule
does not prevent the influenza A virus' globular head region from
binding the surface of a cell. In another embodiment, the cell is a
cell to be infected. In another embodiment, the molecule does not
prevent the influenza A virus from attaching to a cell
[0019] In one embodiment, the molecule binds to an epitope of an H5
subtype of the HA antigen; an H1 subtype of the HA antigen; or an
H3 subtype of the HA antigen. In one other embodiment, the H5, H3,
or H1 epitope is displayed on the surface of an influenza A virus.
In another embodiment, the H5 subtype is an H5N1 subtype. In one
other embodiment, the H1 subtype is an H1N1 subtype. In another
embodiment, the molecule neutralizes more than one isolate of the
H5 and/or H3 and/or H1 influenza A virus subtypes. In another
embodiment, the molecule neutralizes more than one isolate of the
H5N1 and/or H3N1 and/or H1N1 influenza A virus subtypes. In one
embodiment, the molecule neutralizes all isolates of the H5 and/or
H3 and/or H1 influenza A virus subtypes.
[0020] In all embodiments, the influenza A virus subtypes that are
neutralized may be further characterized by a neuraminidase (N)
glycoprotein, including without limitation N1 and N2.
[0021] In one other embodiment, the molecule neutralizes at least
an H5 and/or H3 and/or an H1 influenza A virus subtypes.
[0022] In another embodiment, the molecule neutralizes all H5
and/or H3 and/or H1 influenza A virus subtypes.
[0023] In another embodiment, the molecule neutralizes more than
one H5 and/or H3 and/or H1 isolate of an influenza A virus
subtype.
[0024] In yet another embodiment, the molecule neutralizes all H5
and/or H3 and/or H1 isolates of an influenza A virus subtype.
[0025] In another embodiment, the molecule neutralizes all H5
and/or H3 and/or H1 isolates of an influenza A virus subtype where
the isolates are capable of infecting humans.
[0026] In all embodiments, the H5 subtype may comprise an H5
antigen and/or the H1 subtype may comprise an H1 antigen.
[0027] In one other embodiment, the present invention provides a
molecule which binds essentially the same epitope as the epitope
for a molecule having a heavy chain polypeptide containing an amino
acid sequence shown as SEQ ID NO:4, SEQ ID NO:45, SEQ ID NO:9, or
SEQ ID NO:61; or a consensus or variant sequence based upon said
amino acid sequences, or a fragment thereof. In another embodiment,
the molecule binds essentially the same epitope as the epitope for
a molecule comprising a light chain polypeptide containing an amino
acid sequence shown as SEQ ID NO:71, SEQ ID NO:140, SEQ ID NO:81,
SEQ ID NO:158, SEQ ID NO:159, or SEQ ID NO:160; or a consensus or
variant sequence based upon said amino acid sequences. In some
embodiments, the present invention provides a molecule comprising a
heavy chain polypeptide containing SEQ ID NO:4, SEQ ID NO:45, SEQ
ID NO:9, or SEQ ID NO:61, or a consensus or variant sequence based
upon said amino acid sequences, or a fragment thereof. In other
embodiments, the molecule further contains a light chain
polypeptide containing SEQ ID NO:71, SEQ ID NO:140, SEQ ID NO:81,
SEQ ID NO:158, SEQ ID NO:159, or SEQ ID NO:160, or a consensus or
variant sequence based upon said amino acid sequences, or a
fragment thereof.
[0028] In one embodiment, the molecule binds essentially the same
epitope as a molecule that includes a heavy chain polypeptide
containing an amino acid sequence having the formula:
X.sub.1-X.sub.2-Q-L-V-Q-S-G-X.sub.3-E-V-X.sub.4-K-P-G-X.sub.5-S-V-X.sub.6-
-X.sub.7-S-C-K-X.sub.8-S-G-G-X.sub.9-F-S-S-Y-A-X.sub.10-X.sub.11-W-V-R-Q-A-
-P-G-Q-G-L-E-W-M-G-X.sub.12-G-I-I-X.sub.13-X.sub.14-F-G-T-T-X.sub.15-N-Y-A-
-Q-K-F-Q-G-R-X.sub.16-T-X.sub.17-T-A-D-X.sub.19-X.sub.19-T-S-T-A-Y-M-E-L-S-
-S-L-R-S-X.sub.20-D-T-A-V-Y-Y-C-A-R-G-S-Y-Y-Y-E-X.sub.21-X.sub.22-L-D-Y-W--
G-X.sub.23-G-T-X.sub.24 (SEQ ID NO:161), or a consensus or variant
sequence based upon said amino acid sequences; wherein X.sub.1 is Q
or E; X.sub.2 is V or M; X.sub.3 is A or T; X.sub.4 is K or Q;
X.sub.5 is S or A; X.sub.6 is K or R; X.sub.7 is V or L; X.sub.8 is
A, T or V; X.sub.9 is T, S or A; X.sub.10 is I or V; X.sub.11 is S
or T; X.sub.12 is G or A; X.sub.13 is P or G; X.sub.14 is I or M;
X.sub.15 is A or T; X.sub.16 is V or L; X.sub.17 is I, L, or M;
X.sub.18 is K or E; X.sub.19 is S, L or M; X.sub.20 is E or D;
X.sub.21 is S, T or N; X.sub.22 is S or T; X.sub.23 is Q, K, G or
R; and X.sub.24 is L, T or M. In one embodiment, the amino acid
sequence shown as SEQ ID NO:161 further comprises -V-T-V-S-S or
-V-R-V-S-S at the C-terminal end following X.sub.24.
[0029] In yet another embodiment, the molecule binds essentially
the same epitope as the epitope for a molecule containing a light
chain polypeptide containing an amino acid sequence shown as SEQ ID
NO:71, SEQ ID NO:140, SEQ ID NO:81, SEQ ID NO:158, SEQ ID NO:159,
or SEQ ID NO:160, or a consensus or variant sequence based upon
said amino acid sequences.
[0030] In some embodiments, the present invention provides a
molecule comprising a heavy chain polypeptide containing a heavy
chain polypeptide containing an amino acid sequence having the
formula:
X.sub.1-X.sub.2-Q-L-V-Q-S-G-X.sub.3-E-V-X.sub.4-K-P-G-X.sub.5-S-V-X.sub.6-
-X.sub.7-S-C-K-X.sub.8-S-G-G-X.sub.9-F-S-S-Y-A-X.sub.10-X.sub.11-W-V-R-Q-A-
-P-G-Q-G-L-E-W-M-G-X.sub.12-G-I-I-X.sub.13-X.sub.14-F-G-T-T-X.sub.15-N-Y-A-
-Q-K-F-Q-G-R-X.sub.16-T-X.sub.17-T-A-D-X.sub.18-X.sub.19-T-S-T-A-Y-M-E-L-S-
-S-L-R-S-X.sub.20-D-T-A-V-Y-Y-C-A-R-G-S-Y-Y-Y-E-X.sub.21-X.sub.22-L-D-Y-W--
G-X.sub.23-G-T-X.sub.24 (SEQ ID NO:161), or a consensus or variant
sequence based upon said amino acid sequences; wherein X.sub.1 is Q
or E; X.sub.2 is V or M; X.sub.3 is A or T; X.sub.4 is K or Q;
X.sub.5 is S or A; X.sub.6 is K or R; X.sub.7 is V or L; X.sub.8 is
A, T or V; X.sub.9 is T, S or A; X.sub.10 is I or V; X.sub.11 is S
or T; X.sub.12 is G or A; X.sub.13 is P or G; X.sub.14 is I or M;
X.sub.15 is A or T; X.sub.16 is V or L; X.sub.17 is I, L, or M;
X.sub.18 is K or E; X.sub.19 is S, L or M; X.sub.20 is E or D;
X.sub.21 is S, T or N; X.sub.22 is S or T; X.sub.23 is Q, K, G or
R; and X.sub.24 is L, T or M. In one embodiment, the amino acid
sequence shown as SEQ ID NO:161 further comprises -V-T-V-S-S or
-V-R-V-S-S at the C-terminal end following X.sub.24.
[0031] In other embodiments, the molecule further contains a light
chain polypeptide containing an amino acid sequence shown as SEQ ID
NO:71, SEQ ID NO:140, SEQ ID NO:81, SEQ ID NO:158, SEQ ID NO:159,
or SEQ ID NO:160.
[0032] In all embodiments, the molecule is an antibody or an
antibody-like molecule.
[0033] In one embodiment, the antibody or an antibody-like molecule
(i) neutralizes more than one subtype and/or more than one isolate
of an influenza A virus, (ii) binds to a hemagglutinin (HA) antigen
of the virus, and (iii) does not inhibit hemagglutination.
[0034] In another embodiment, at least one of the virus subtypes
and/or isolates neutralized by the molecules herein has the ability
to infect humans.
[0035] In other embodiments, at least one of said isolates has been
obtained from a human subject. In another embodiment, the human
subject is or was diseased with an influenza virus at the time of
obtaining the isolate. In other embodiments, the human subject
recovered from infection with the influenza virus A. In another
embodiment, the influenza virus A is an H5 subtype and/or an H1
subtype of influenza virus A.
[0036] In one embodiment, at least one of said isolates has been
obtained from a non-human animal. In another embodiment, at least
one of the isolates is from a bird, including, without limitation,
wild-fowls and chicken.
[0037] In one embodiment, the molecules neutralize an H5 subtype
and an H1 subtype.
[0038] In another embodiment, the neutralizing molecules of the
present invention bind the H5 and/or H1 protein. In one embodiment,
the H5 protein is an H5 HA protein. Preferably, the molecules bind
more than one variant of the H5 protein, or, even more preferably,
substantially all variants of the H5 protein.
[0039] In another embodiment, the molecule also binds to at least
one additional HA antigen. In one other embodiment, the additional
HA antigen is an H1 HA antigen. In one other embodiment, the
molecule binds to substantially all variants of the H1 HA protein.
In one embodiment, the at least one additional HA antigen is
selected from the group consisting of H2, H3, H5, H6, H7, H8 and
H9. In another embodiment, the at least one additional HA antigen
also binds to HA antigen H5; HA antigens H3 and H9; or HA antigens
H3, H5, and H9.
[0040] In other embodiments, the molecules described herein bind to
the H5 protein and to at least one additional H protein, such as an
H1 protein.
[0041] In a different aspect, the invention concerns compositions
comprising the neutralizing molecules described herein. In one
embodiment, the compositions comprise an antibody or antibody-like
molecule described herein.
[0042] In a further aspect, the invention concerns a method for
identifying a molecule capable of neutralizing more than one
isolate of a single influenza A virus subtype or multiple influenza
A virus subtypes. This method comprises identifying molecules,
e.g., antibodies, antibody fragments, or antibody-like molecules in
an antibody library, that react with both a first and a second
isolate of the influenza A virus subtype or with a first and a
second subtype of the influenza A virus, and subjecting the
molecules identified to successive, alternating rounds of
selection, based on their ability to bind the first and second
isolates, or the first and second subtypes, respectively. In one
embodiment, the method further comprises isolating the identified
antibody.
[0043] In another embodiment, molecules that react with both a
first and a second influenza A virus subtype isolate have been
identified by at least two rounds of separate enrichment of
molecules reacting with the first isolate and the second isolate,
respectively, and recombining the molecules identified.
[0044] In another aspect, the present invention provides a method
of identifying an antibody capable of neutralizing an isolate of an
H5 influenza A virus and/or an isolate of an H1 influenza A virus;
or a subtype of an H5 influenza A virus and/or a subtype of an H1
influenza A virus. In one embodiment, the method comprises
identifying, in an antibody library, antibodies that react with
both an H5 isolate and/or an H1 isolate; or an H5 subtype and/or an
H1 subtype, and subjecting the antibodies identified to successive
alternating rounds of selection, based on their ability to bind
said H5 and/or H1 isolates or HA proteins; or said H5 and/or H1
subtypes or HA proteins, respectively. In another embodiment, the
method comprises at least two rounds of selection. In one
embodiment, the method further comprises isolating the identified
antibody. In another embodiment, the H5 isolate is an H5 subtype of
said influenza A virus or HA and/or said H1 isolate is an H1
subtype of said influenza A virus or HA. In yet another embodiment,
the antibodies that react with both a first and a second influenza
A virus subtype isolate or HA have been identified by at least two
rounds of separate enrichment of antibodies reacting with the first
isolate or HA and the second isolate or HA, respectively, and
recombining the antibodies identified. In one other embodiment, the
antibody that can react with both said H5 and said H1 influenza A
subtype isolates or HAs is subjected to mutagenesis prior to being
subjected to said successive alternating rounds of selection, based
on their ability to bind said H5 and second H1 subtype isolates or
HAs, respectively. In one other embodiment, the influenza A virus
subtype is an H5 subtype or HA and said influenza A virus subtype
is an H1 subtype or HA. In another embodiment, the H5 subtype is,
or the HA is from, a 2006 Turkish isolate of the H5 virus; the H5
subtype is, or the HA is from, a 2003/2004 Vietnam isolate of the
H5 virus; the H5 subtype is, or the HA is from, a 1997 Hong Kong
isolate of the H5 virus; the H1 subtype is, or the HA is from, a
New Calcdonia/20/99 isolate of the H1 virus; the H5 and/or said H1
subtypes or HAs originate from different species; or any
combination thereof. In one other embodiment, at least one of said
species is human; or at least one of said species is a bird. In
another embodiment, the antibodies capable of binding said H5
and/or said H1 isolates are additionally selected based on their
ability to bind more than one influenza A subtype.
[0045] In another embodiment, the molecule library is a phage
display library. In one embodiment, the selection is performed by
biopanning.
[0046] In another embodiment, the molecule that can react with both
the first and the second influenza A subtype isolate is subjected
to mutagenesis prior to being subjected to successive alternating
rounds of selection, based on its ability to bind the first and
second isolate, respectively. If desired, the molecules capable of
binding the first and the second isolate are additionally selected
based on their ability to bind more than one influenza A
subtype.
[0047] The application of such enrichment techniques can be
similarly applied to molecules in general, regardless of the target
to which they bind. Such general enrichment/selection methods are
specifically included as part of the invention.
[0048] In another embodiment, the invention concerns a collection
of sequences shared by the neutralizing molecules of the present
invention and identified by the methods described herein. In one
other embodiment, the collection of sequences comprises one or more
of the unique heavy and/or light chain sequences shown in Table 2
or a consensus or variant sequence based on said sequences. In
another embodiment, the present invention provides a neutralizing
antibody or a fragment thereof, identified by the methods described
herein.
[0049] In a still further aspect, the invention concerns a method
for treating an influenza A infection in a subject comprising of
administering to the subject an effective amount of a neutralizing
molecule or molecule composition herein.
[0050] In another aspect, the invention concerns a method for
preventing influenza A infection comprising of administering to a
subject at risk of developing influenza A infection an effective
amount of a neutralizing molecule or molecule composition described
herein. In one embodiment, the neutralizing molecule is a
neutralizing antibody, antibody fragment, or antibody-like
molecule.
[0051] In all embodiments, the subject is a human patient. In all
embodiments, the subject is a subject at risk of developing an
influenza A infection.
[0052] In a different aspect, the invention concerns a method for
producing a diverse multifunctional molecule collection,
comprising: (a) aligning CDR sequences of at least two functionally
different molecules, e.g., antibodies, antibody fragments, or
antibody-like molecules, (b) identifying amino acid residues
conserved between the CDR sequences aligned, and (c) performing
mutagenesis of multiple non-conserved amino acid residues in at
least one of the CDR sequences aligned, using degenerate
oligonucleotide probes encoding at least the amino acid residues
present in the functionally different molecules at the
non-conserved positions mutagenized to produce multiple variants of
the aligned CDR sequences, and, if desired, repeating steps (b) and
(c) with one or more of the variants until the molecule collection
reaches a desired degree of diversity and/or size.
[0053] In a particular embodiment, the CDR sequences aligned have
the same lengths.
[0054] In another embodiment, the conserved amino acid residues are
retained in at least two of the CDR sequences aligned.
[0055] In a further aspect, the invention concerns a molecule
collection comprising a plurality of neutralizing molecules, e.g.,
antibodies, antibody fragments, or antibody-like molecules, which
differ from each other in at least one property.
[0056] The invention further concerns a method for uniquely
identifying nucleic acids in a collection comprising labeling the
nucleic acids with a unique barcode linked to or incorporated in
the sequences of the nucleic acid present in such collection.
[0057] The invention further concerns a vaccine effective against
influenza A virus containing a peptide or polypeptide that
functionally mimics a neutralization epitope bound by a molecule of
the present invention. In one embodiment, the vaccine is a
synthetic vaccine. In another embodiment, the vaccine contains an
attenuated influenza A virus, or a part thereof. In one other
embodiment, the vaccine contains a killed influenza A virus, or
part thereof. In another embodiment, the molecule that binds a
neutralization epitope is one of the following:
[0058] (a) a molecule which (i) neutralizes more than one subtype
and/or more than one isolate of an influenza A virus, (ii) binds to
a hemagglutinin (HA) antigen of the virus, and (iii) does not
prevent hemagglutination;
[0059] (b) a molecule which binds essentially the same epitope as
the epitope for a molecule comprising a heavy chain polypeptide
comprising an amino acid sequence selected from the group
consisting of SEQ ID NO:4, SEQ ID NO:45, SEQ ID NO:9, and SEQ ID
NO:61; or a consensus or variant sequence based upon said amino
acid sequences, or a fragment thereof;
[0060] (c) a molecule which binds essentially the same epitope as
the epitope for a molecule comprising a heavy chain polypeptide
comprising an amino acid sequence having the formula:
X.sub.1-X.sub.2-Q-L-V-Q-S-G-X.sub.3-E-V-X.sub.4-K-P-G-X.sub.5-S-V-X.sub.6-
-X.sub.7-S-C-K-X.sub.8-S-G-G-X.sub.9-F-S-S-Y-A-X.sub.10-X.sub.11-W-V-R-Q-A-
-P-G-Q-G-L-E-W-M-G-X.sub.12-G-I-I-X.sub.13-X.sub.14-F-G-T-T-X.sub.15-N-Y-A-
-Q-K-F-Q-G-R-X.sub.16-T-X.sub.17-T-A-D-X.sub.18-X.sub.19-T-S-T-A-Y-M-E-L-S-
-S-L-R-S-X.sub.20-D-T-A-V-Y-Y-C-A-R-G-S-Y-Y-Y-E-X.sub.21-X.sub.22-L-D-Y-W--
G-X.sub.23-G-T-X.sub.24 (SEQ ID NO:161); or a consensus or variant
sequence based upon said amino acid sequences, or a fragment
thereof; wherein X.sub.1 is Q or E; X.sub.2 is V or M; X.sub.3 is A
or T; X.sub.4 is K or Q; X.sub.5 is S or A; X.sub.6 is K or R;
X.sub.7 is V or L; X.sub.8 is A, T or V; X.sub.9 is T, S or A;
X.sub.10 is I or V; X.sub.11 is S or T; X.sub.12 is G or A;
X.sub.13 is P or G; X.sub.14 is I or M; X.sub.15 is A or T;
X.sub.16 is V or L; X.sub.17 is I, L, or M; X.sub.18 is K or E;
X.sub.19 is S, L or M; X.sub.20 is E or D; X.sub.21 is S, T or N;
X.sub.22 is S or T; X.sub.23 is Q, K, G or R; and X.sub.24 is L, T
or M;
[0061] (d) a molecule comprising a heavy chain polypeptide
comprising an amino acid sequence selected from the group
consisting of SEQ ID NO:4, SEQ ID NO:45, SEQ ID NO:9, and SEQ ID
NO:61; or a consensus or variant sequence based upon said amino
acid sequences, or a fragment thereof; or
[0062] (e) a molecule comprising a heavy chain polypeptide
comprising an amino acid sequence having the formula:
X.sub.1-X.sub.2-Q-L-V-Q-S-G-X.sub.3-E-V-X.sub.4-K-P-G-X.sub.5-S-V-X.sub.6-
-X.sub.7-S-C-K-X.sub.8-S-G-G-X.sub.9-F-S-S-Y-A-X.sub.10-X.sub.11-W-V-R-Q-A-
-P-G-Q-G-L-E-W-M-G-X.sub.12-G-I-I-X.sub.13-X.sub.14-F-G-T-T-X.sub.15-N-Y-A-
-Q-K-F-Q-G-R-X.sub.16-T-X.sub.17-T-A-D-X.sub.18-X.sub.19-T-S-T-A-Y-M-E-L-S-
-S-L-R-S-X.sub.20-D-T-A-V-Y-Y-C-A-R-G-S-Y-Y-Y-E-X.sub.21-X.sub.22-L-D-Y-W--
G-X.sub.23-G-T-X.sub.24 (SEQ ID NO:161); or a consensus or variant
sequence based upon said amino acid sequences, or a fragment
thereof; wherein X.sub.1 is Q or E; X.sub.2 is V or M; X.sub.3 is A
or T; X.sub.4 is K or Q; X.sub.5 is S or A; X.sub.6 is K or R;
X.sub.7 is V or L; X.sub.8 is A, T or V; X.sub.9 is T, S or A;
X.sub.10 is I or V; X.sub.11 is S or T; X.sub.12 is G or A;
X.sub.13 is P or G; X.sub.14 is I or M; X.sub.15 is A or T;
X.sub.16 is V or L; X.sub.17 is I, L, or M; X.sub.18 is K or E;
X.sub.19 is S, L or M; X.sub.20 is E or D; X.sub.21 is S, T or N;
X.sub.22 is S or T; X.sub.23 is Q, K, G or R; and X.sub.24 is L, T
or M.
[0063] In one embodiment, the amino acid sequence shown as SEQ ID
NO:161 further comprises -V-T-V-S-S or -V-R-V-S-S at the C-terminal
end following X.sub.24.
[0064] In another embodiment, the vaccine is based on a molecule
that binds an HA antigen. In some other embodiments, the HA antigen
is an H5 subtype or an H1 subtype. In one other embodiment, the
antigen is displayed on the surface of an influenza A virus. In yet
another embodiment, the peptide or polypeptide contains antigenic
determinants that raise neutralizing molecules, e.g.,
antibodies.
[0065] In all embodiments, the present invention provides
compositions that comprise a molecule described herein. In all
embodiments, the molecule is an antibody, antibody fragment, or
antibody-like molecule.
[0066] In one aspect, the present invention provides neutralizing
antibodies identified by the methods described herein. In one
embodiment, the neutralizing antibody is an antibody or an antibody
fragment. In another embodiment, the neutralizing antibody or
antibody fragment is capable of conferring passive immunity to an
avian or mammalian subject against an influenza A virus infection.
In another embodiment, the mammalian subject is a human. In one
other embodiment, the influenza A virus infection is caused by an
H5 subtype and/or an H1 subtype.
[0067] In another aspect, the present invention provides molecules
capable of binding to and neutralizing a viral antigen. In one
embodiment, the molecule comprises an antibody heavy chain variable
domain comprising at least one substitution in the surface exposed
cluster determined by amino acid positions 52A, 53, 73, and 74,
following Kabat amino acid numbering, wherein said molecule is
capable of binding to and neutralizing a viral antigen. In another
embodiment, the molecule comprises a substitution at least one of
amino acid positions 52A, 53, 73, and 74. In another embodiment,
the molecule comprises a substitution at all of amino acid
positions 52A, 53, 73, and 74. In another embodiment, the molecule
further comprises a substitution at amino acid position 57. In
another embodiment, the molecule further comprises P52G, I53M,
L73E, and S74L/M substitutions. In another embodiment, the molecule
additionally comprises an A57T substitution. In another embodiment,
the molecule also comprises a substitution at least one of amino
acid positions 24, 34, 35 and 50. In another embodiment, the
molecule comprises substitutions at all of amino acid positions 24,
34, 35 and 50. In another embodiment, the molecule comprises V24T,
W34V, G35T and S50A substitutions.
[0068] In one aspect, the molecules of the present invention
comprise a heavy chain variable domain sequence from a germ-line
heavy chain. In one embodiment, the germ-line heavy chain is a
V.sub.H1e or a V.sub.H1-69 germ-line heavy chain. In another
embodiment, the rest of the heavy chain variable domain sequence
retains the sequence of the germ-line heavy chain. In another
embodiment, the germ-line heavy chain variable domain comprises at
least one additional conservative substitution.
[0069] In one embodiment, the molecules further comprise a light
chain sequence. In another embodiment, the light chain sequence is
an antibody .lamda. or .kappa. light chain sequence. In one
embodiment, the light chain sequence is a surrogate light chain
sequence. In one other embodiment, the surrogate light chain
sequence comprises a VpreB sequence and/or a .lamda.5 sequence. In
yet another embodiment, the surrogate light chain sequence
comprises a VpreB sequence fused to a .lamda.5 sequence. In another
embodiment, the surrogate light chain sequence is a .kappa.-like
surrogate light chain (SLC) construct comprising a V.kappa.-like
and/or a JC.kappa. sequence.
[0070] In one embodiment, the viral antigen neutralized by the
molecule is selected from the group consisting of viral antigens
from influenza viruses, HIV-1, HIV-2, HTLV-I and -II viruses, SARS
coronavirus, herpes simplex virus, Epstein Barr virus,
cytomegalovirus, hepatitis virus (HCV, HAV, HBV, HDV, HEV),
toxoplasma gondii virus, treponema pallidium virus, human
T-lymphotrophic virus, encephalitis virus, West Nile virus, Dengue
virus, Varicella Zoster Virus, rubeola, mumps, and rubella.
[0071] In another embodiment, the viral antigen is from an
influenza virus or an HIV-1 or HIV-2 virus.
[0072] In one other aspect, the present invention provides vaccines
effective against influenza A virus. In one embodiment, the vaccine
comprises a peptide or polypeptide functionally mimicking a
neutralization epitope of a molecule described herein. In another
embodiment, the vaccine effective against a viral antigen comprises
a peptide or polypeptide functionally mimicking a neutralization
epitope of a molecule described herein. In one embodiment, the
viral antigen is from an influenza virus or an HIV-1 or HIV-2
virus.
[0073] In another embodiment, the vaccine is a vaccine effective
against an influenza A virus, comprising a peptide or polypeptide
functionally mimicking a neutralization epitope of a molecule
described herein. In one embodiment, the molecule is an antibody.
In another embodiment, the antibody binds an HA antigen. In one
other embodiment, the HA antigen is an H5 subtype. In one other
embodiment, the HA antigen is an H1 subtype. In one other
embodiment, the antigen is displayed on the surface of an influenza
A virus. In one other embodiment, the peptide or polypeptide
comprises antigenic determinants that raise neutralizing
antibodies.
[0074] In one embodiment, the vaccine is a synthetic vaccine. In
another embodiment, the vaccine comprises an attenuated influenza A
virus, or a part thereof. In one other embodiment, the vaccine
comprises a killed influenza A virus, or part thereof.
[0075] In other embodiments, the vaccine is suitable for oral
administration, parenteral administration, transdermal delivery, or
transmucosal delivery. In one embodiment, the transmucosal delivery
is intra-nasal administration. In one other embodiment, the vaccine
is for childhood immunization.
BRIEF DESCRIPTION OF THE DRAWINGS
[0076] FIG. 1 illustrates a typical panning enrichment scheme for
increasing the reactive strength towards two different targets, A
and B. Each round of enrichment increases the reactive strength of
the pool towards the individual target(s).
[0077] FIG. 2 illustrates a strategy for the selection of clones
cross-reactive with targets A and B, in which each successive round
reinforces the reactive strength of the resulting pool towards both
targets.
[0078] FIG. 3 illustrates a strategy for increasing the reactive
strengths towards two different targets (targets A and B), by
recombining parallel discovery pools to generate/increase
cross-reactivity. Each round of selection of the recombined
antibody library increases the reactive strength of the resulting
pool towards both targets.
[0079] FIG. 4 illustrates a strategy for increasing
cross-reactivity to a target B while maintaining reactivity to a
target A. First, a clone reactive with target A is selected, then a
mutagenic library of the clones reactive with target A is prepared,
and selection is performed as shown, yielding one or more antibody
clones that show strong reactivity with both target A and target
B.
[0080] FIG. 5 illustrates a representative mutagenesis method for
generating a diverse multifunctional antibody collection by the
"destinational mutagenesis" method.
[0081] FIG. 6 shows the analysis of antibody binding to
hemagglutinins from different influenza A subtypes.
[0082] FIG. 7 shows the positions of H5 hemagglutinin binding Group
1 required and dominant mutations on the crystal structure of Fab
47e.
[0083] FIG. 8 shows the cross-reactive titers of Turkish avian
influenza survivors to the H5N1 Vietnam 1203/04 hemagglutinin
protein.
[0084] FIG. 9 illustrates the cloning and barcoding of annotated
repertoires.
DETAILED DESCRIPTION
A. Definitions
[0085] Unless defined otherwise, technical and scientific terms
used herein have the same meaning as commonly understood by one of
ordinary skill in the art to which this invention belongs.
Singleton et al., Dictionary of Microbiology and Molecular Biology
2nd ed., J. Wiley & Sons (New York, N.Y. 1994), provides one
skilled in the art with a general guide to many of the terms used
in the present application.
[0086] One skilled in the art will recognize many methods and
materials similar or equivalent to those described herein, which
could be used in the practice of the present invention. Indeed, the
present invention is in no way limited to the methods and materials
described. For purposes of the present invention, the following
terms are defined below.
[0087] The terms "influenza A subtype" or "influenza A virus
subtype" are used interchangeably, and refer to influenza A virus
variants that are characterized by a hemagglutinin (H) viral
surface protein, and thus are labeled by an H number, such as, for
example, H1, H3, and H5. In addition, the subtypes may be further
characterized by a neuraminidase (N) viral surface protein,
indicated by an N number, such as, for example, N1 and N2. As such,
a subtype may be referred to by both H and N numbers, such as, for
example, H1N1, H5N1, and H5N2. The terms specifically include all
strains (including extinct strains) within each subtype, which
usually result from mutations and show different pathogenic
profiles. Such strains will also be referred to as various
"isolates" of a viral subtype, including all past, present and
future isolates. Accordingly, in this context, the terms "strain"
and "isolate" are used interchangeably. Subtypes contain antigens
based upon an influenza A virus. The antigens may be based upon a
hemagglutinin viral surface protein and can be designated as "HA
antigen". In some instances, such antigens are based on the protein
of a particular subtype, such as, for example, an H1 subtype and an
H5 subtype, which may be designated an H1 antigen and an H5
antigen, respectively.
[0088] The term "influenza" is used to refer to a contagious
disease caused by an influenza virus.
[0089] In the context of the present invention, the term "antibody"
(Ab) is used in the broadest sense and includes polypeptides which
exhibit binding specificity to a specific antigen.
[0090] "Native antibodies" are usually heterotetrameric
glycoproteins of about 150,000 daltons, composed of two identical
light (L) chains and two identical heavy (H) chains. Each light
chain is linked to a heavy chain by covalent disulfide bond(s),
while the number of disulfide linkages varies between the heavy
chains of different immunoglobulin isotypes. Each heavy and light
chain also has regularly spaced intrachain disulfide bridges. Each
heavy chain has, at one end, a variable domain (V.sub.H) followed
by a number of constant domains. Each light chain has a variable
domain at one end (V.sub.L) and a constant domain at its other end;
the constant domain of the light chain is aligned with the first
constant domain of the heavy chain, and the light chain variable
domain is aligned with the variable domain of the heavy chain.
Particular amino acid residues are believed to form an interface
between the light- and heavy-chain variable domains, Chothia et
al., J. Mol. Biol. 186:651 (1985); Novotny and Haber, Proc. Natl.
Acad. Sci. U.S.A. 82:4592 (1985).
[0091] The term "variable" with reference to antibody chains is
used to refer to portions of the antibody chains which differ
extensively in sequence among antibodies and participate in the
binding and specificity of each particular antibody for its
particular antigen. Such variability is concentrated in three
segments called hypervariable regions both in the light chain and
the heavy chain variable domains. The more highly conserved
portions of variable domains are called the framework region (FR).
The variable domains of native heavy and light chains each comprise
four FRs (FR1, FR2, FR3 and FR4, respectively), largely adopting a
.beta.-sheet configuration, connected by three hypervariable
regions, which form loops connecting, and in some cases forming
part of, the .beta.-sheet structure. The hypervariable regions in
each chain are held together in close proximity by the FRs and,
with the hypervariable regions from the other chain, contribute to
the formation of the antigen-binding site of antibodies (see Kabat
et al., Sequences of Proteins of Immunological Interest, 5th Ed.
Public Health Service, National Institutes of Health, Bethesda, Md.
(1991), pages 647-669). The constant domains are not involved
directly in binding an antibody to an antigen, but exhibit various
effector functions, such as participation of the antibody in
antibody-dependent cellular toxicity.
[0092] The term "hypervariable region" when used herein refers to
the amino acid residues of an antibody which are responsible for
antigen-binding. The hypervariable region comprises amino acid
residues from a "complementarity determining region" or "CDR"
(i.e., residues 30-36 (L1), 46-55 (L2) and 86-96 (L3) in the light
chain variable domain and 30-35 (H1), 47-58 (H2) and 93-101 (H3) in
the heavy chain variable domain; MacCallum et al., J Mol. Biol.
1996. "Framework" or "FR" residues are those variable domain
residues other than the hypervariable region residues as herein
defined.
[0093] Depending on the amino acid sequence of the constant domain
of their heavy chains, antibodies can be assigned to different
classes. There are five major classes of antibodies IgA, IgD, IgE,
IgG, and IgM, and several of these may be further divided into
subclasses (isotypes), e.g., IgG1, IgG2, IgG3, IgG4, IgA, and
IgA2.
[0094] The heavy-chain constant domains that correspond to the
different classes of immunoglobulins are called .alpha., .delta.,
.epsilon., .gamma., and .mu., respectively.
[0095] The "light chains" of antibodies from any vertebrate species
can be assigned to one of two clearly distinct types, called kappa
(.kappa.) and lambda (.lamda.), based on the amino acid sequences
of their constant domains.
[0096] The term "antibody fragment" is a portion of a full length
antibody, generally the antigen binding or variable domain thereof.
Examples of antibody fragments include, but are not limited to,
Fab, Fab', F(ab').sub.2, and Fv fragments, linear antibodies,
single-chain antibody molecules, diabodies, and multispecific
antibodies formed from antibody fragments. Further examples of
antibody fragments include, but are not limited to, scFv,
(scFv).sub.2, dAbs (single-domain antibodies), and complementarity
determining region (CDR) fragments, and minibodies, which are
minimized variable domains whose two loops are amenable to
combinatorial mutagenesis.
[0097] The term "monoclonal antibody" is used to refer to an
antibody molecule synthesized by a single clone of B cells. The
modifier "monoclonal" indicates the character of the antibody as
being obtained from a substantially homogeneous population of
antibodies, and is not to be construed as requiring production of
the antibody by any particular method. Thus, monoclonal antibodies
may be made by the hybridoma method first described by Kohler and
Milstein, Nature 256:495 (1975); Eur. J. Immunol. 6:511 (1976), by
recombinant DNA techniques, or may also be isolated from phage
antibody libraries.
[0098] The term "polyclonal antibody" is used to refer to a
population of antibody molecules synthesized by a population of B
cells.
[0099] "Single-chain Fv" or "sFv" antibody fragments comprise the
V.sub.H and V.sub.L domains of antibody, wherein these domains are
present in a single polypeptide chain. Generally, the Fv
polypeptide further comprises a polypeptide linker between the
V.sub.H and V.sub.L domains which enables the sFv to form the
desired structure for antigen binding. For a review of sFv see
Pluckthun in The Pharmacology of Monoclonal Antibodies, vol. 113,
Rosenburg and Moore eds. Springer-Verlag, New York, pp. 269-315
(1994). Single-chain antibodies are disclosed, for example in WO
88/06630 and WO 92/01047.
[0100] The term "diabody" refers to small antibody fragments with
two antigen-binding sites, which fragments comprise a heavy chain
variable domain (V.sub.H) connected to a light chain variable
domain (V.sub.L) in the same polypeptide chain (V.sub.H-V.sub.L).
By using a linker that is too short to allow pairing between the
two domains on the same chain, the domains are forced to pair with
the complementary domains of another chain and create two
antigen-binding sites. Diabodies are described more fully in, for
example, EP 404,097; WO 93/11161; and Hollinger et al., Proc. Natl.
Acad. Sci. USA 90:6444-6448 (1993).
[0101] The term "minibody" is used to refer to an scFv-CH3 fusion
protein that self-assembles into a bivalent dimer of 80 kDa
(scFv-CH3).sub.2.
[0102] The term "aptamer" is used herein to refer to synthetic
nucleic acid ligands that bind to protein targets with high
specificity and affinity. Aptamers are known as potent inhibitors
of protein function.
[0103] A dAb fragment (Ward et al., Nature 341:544 546 (1989))
consists of a V.sub.H domain or a V.sub.L domain.
[0104] As used herein the term "antibody binding regions" refers to
one or more portions of an immunoglobulin or antibody variable
region capable of binding an antigen(s). Typically, the antibody
binding region is, for example, an antibody light chain (V.sub.L)
(or variable region thereof), an antibody heavy chain (V.sub.H) (or
variable region thereof), a heavy chain Fd region, a combined
antibody light and heavy chain (or variable region thereof) such as
a Fab, F(ab').sub.2, single domain, or single chain antibody
(scFv), or a full length antibody, for example, an IgG (e.g., an
IgG1, IgG2, IgG3, or IgG4 subtype), IgA1, IgA2, IgD, IgE, or IgM
antibody.
[0105] The term "bispecific antibody" refers to an antibody that
shows specificities to two different types of antigens. The term as
used herein specifically includes, without limitation, antibodies
which show binding specificity for a target antigen and to another
target that facilitates delivery to a particular tissue. Similarly,
multi-specific antibodies have two or more binding
specificities.
[0106] The expression "linear antibody" is used to refer to
comprising a pair of tandem Fd segments
(V.sub.H-C.sub.H1-V.sub.H-C.sub.H1) which form a pair of antigen
binding regions. Linear antibodies can be bispecific or
monospecific and are described, for example, by Zapata et al.,
Protein Eng 8(10):1057-1062 (1995).
[0107] For the purposes of the present invention, the term
"antibody-like molecule" includes any molecule, other than an
antibody fragment as hereinabove defined, that is capable of
binding to and neutralizing a viral antigen. The term specifically
includes, without limitation, pre-B cell receptor (pre-BCR) like
structures, referred to as "surrobodies," including surrogate light
chain (SLC) elements, as described, for example, in PCT Publication
No. WO 2008/118970, published Oct. 2, 2008, and in Xu et al. Proc.
Natl. Acad. Sci. USA, 105(31): 10756-61 (2008). The SLC is a
nondiversified heterodimer composed of the noncovalently associated
Vpre-B and .lamda.5 proteins. The VpreB chain is homologous to a
V.lamda. Ig domain, and the .lamda.5 chain is homologous to the
C.lamda. domain of canonical antibodies, respectively. The
heterodimeric SLC is covalently associated with the heavy chain in
the pre-BCR complex by disulfide bonds between the C.lamda. domain
and the first constant domain of the pre-BCR HC. A unique feature
of the SLC is that the VpreB1 and the .lamda.5 domains each have
noncanonical peptide extensions. VpreB1 has an additional 21
residues on its C terminus, and .lamda.5 has a 50-aa-long tail on
its N terminus (see, e.g. Vettermann et al., Semin. Immunol.
18:44-55 (2006)). The surrobody structures specifically include,
without limitation, the native trimeric pre-BCR-like functional
unit of the pre-BCR, fusion of VpreB1 to .lamda.5, and trimers that
eliminated either the .lamda.5 N-terminal 50 aa or the VpreB1
C-terminal 21 aa or both peptide extensions. In addition, chimeric
constructs using the constant components of classical antibody
light chains are specifically included within the definition of
surrobodies.
[0108] Other representatives of "antibody-like molecules," as
defined herein, are similar structures comprising antibody
surrogate .kappa. light chain sequences, where .kappa. light chain
sequences are optionally partnered with another polypeptide, such
as, for example, antibody heavy and/or light chain domain
sequences. A .kappa.-like B cell receptor (.kappa.-like BCR) has
been identified, utilizing a .kappa.-like surrogate light chain
(.kappa.-like SLC) (Frances et al., EMBO J 13:5937-43 (1994);
Thompson et al., Immunogenetics 48:305-11 (1998); Rangel et al., J
Biol Chem 280:17807-14 (2005)). Rangel et al., J Biol Chem
280(18):17807-17814 (2005) report the identification and molecular
characterization of a Vs-like protein that is the product of an
unrearranged V.kappa. gene, which turned out to the be identical to
the cDNA sequence previously reported by Thompson et al.,
Immunogenetics 48:305-311 (1998). Whereas, Frances et al., EMBO J.
13:5937-43 (1994) reported the identification and characterization
of a rearranged germline JCk that has the capacity to associate
with .mu. heavy chains at the surface of B cell precursors, thereby
providing an alternative to the .lamda.5 pathway for B cell
development. It has been proposed that .kappa.-like and
.lamda.-like pre-BCRs work in concert to promote light chain
rearrangement and ensure the maturation of B cell progenitors. For
a review, see McKeller and Martinez-Valdez Seminars in Immunology
18:4043 (2006).
[0109] The term ".lamda.5" is used herein in the broadest sense and
refers to any native sequence or variant .lamda.5 polypeptide,
specifically including, without limitation, native sequence human
and other mammalian .lamda.5 polypeptides, and variants formed by
posttranslational modifications, as well a variants of such native
sequence polypeptides.
[0110] The terms "`variant VpreB polypeptide" and "a variant of a
VpreB polypeptide" are used interchangeably, and are defined herein
as a polypeptide differing from a native sequence VpreB polypeptide
at one or more amino acid positions as a result of an amino acid
modification. The "variant VpreB polypeptide," as defined herein,
will be different from a native antibody .lamda. or .kappa. light
chain sequence, or a fragment thereof. The "variant VpreB
polypeptide" will preferably retain at least about 65%, or at least
about 70%, or at least about 75%, or at least about 80%, or at
least about 85%, or at least about 90%, or at least about 95%, or
at least about 98% sequence identity with a native sequence VpreB
polypeptide. In another preferred embodiment, the "variant VpreB
polypeptide" will be less then 95%, or less than 90%, or less then
85%, or less than 80%, or less than 75%, or less then 70%, or less
than 65%, or less than 60% identical in its amino acid sequence to
a native antibody .lamda. or .kappa. light chain sequence. Variant
VpreB polypeptides specifically include, without limitation, VpreB
polypeptides in which the non-Ig-like unique tail at the C-terminus
of the VpreB sequence is partially or completely removed. The terms
"variant .lamda.5 polypeptide" and "a variant of a .lamda.5
polypeptide" are used interchangeably, and are defined herein as a
polypeptide differing from a native sequence .lamda.5 polypeptide
at one or more amino acid positions as a result of an amino acid
modification. The "variant .lamda.5 polypeptide," as defined
herein, will be different from a native antibody .lamda. or K light
chain sequence, or a fragment thereof. The "variant .lamda.5
polypeptide" will preferably retain at least about 65%, or at least
about 70%, or at least about 75%, or at least about 80%, or at
least about 85%, or at least about 90%, or at least about 95%, or
at least about 98% sequence identity with a native sequence
.lamda.5 polypeptide. In another preferred embodiment, the
"`variant .lamda.5 polypeptide" will be less then 95%, or less than
90%, or less then 85%, or less than 80%, or less than 75%, or less
then 70%, or less than 65%, or less than 60% identical in its amino
acid sequence to a native antibody .lamda. or .kappa. light chain
sequence. Variant .lamda.5 polypeptides specifically include,
without limitation. .lamda.5 polypeptides in which the unique tail
at the N-terminus of the .lamda.5 sequence is partially or
completely removed.
[0111] The term "VpreB sequence" is used herein to refer to the
sequence of "VpreB," as hereinabove defined, or a fragment
thereof.
[0112] The term ".lamda.5 sequence" is used herein to refers to the
sequence of ".lamda.5," as hereinabove defined, or a fragment
thereof.
[0113] The term "surrogate light chain sequence," as defined
herein, means any polypeptide sequence that comprises a "VpreB
sequence" and/or a ".lamda.5 sequence," as hereinabove defined.
[0114] The terms ".kappa.-like surrogate light chain variable
domain," "V.kappa.-like SLC," and "V.kappa.-like" are used
interchangeably, and refer to any native sequence polypeptide that
is the product of an unrearranged V.kappa.; gene, and variants
thereof. In one embodiment, variants of native sequence
V.kappa.-like polypeptides comprise a C-terminal extension (tail)
relative to antibody .kappa. light chain sequences. In a particular
embodiment, variants of native sequence V.kappa.-like polypeptides
retain at least part, and preferably all, of the unique C-terminal
extension (tail) that distinguishes the V.kappa.-like polypeptides
from the corresponding antibody .kappa. light chains. In another
embodiment, the C-terminal tail of the variant V.kappa.-like
polypeptide is a sequence not naturally associated with the rest of
the sequence. In the latter embodiment, the difference between the
C-terminal tail naturally present in the native V.kappa.-like
sequence and the variant sequence may result from one or more amino
acid alterations (substitutions, insertions, deletions, and/or
additions), or the C-terminal tail may be identical with a tail
present in nature in a different V.kappa.-like protein. The
V.kappa.-like polypeptides may contain amino acid alterations in
regions corresponding to one or more of antibody .kappa. light
chain CDR1, CDR2 and CDR3 sequences. In all instances, the variants
can, and preferably do, include a C-terminal extension of at least
four, or at least five, or at least six, or at least seven, or at
least eight, or at least nine, or at least ten amino acids,
preferably 4-100, or 4-90, or 4-80, or 4-70, or 4-60, or 4-50, or
4-45, or 4-40, or 4-35, or 4-30, or 4-25, or 4-20, or 4-15, or 4-10
amino acid residues relative to a native antibody .kappa. light
chain variable region sequence. As defined herein, V.kappa.-like
polypeptide variant will be different from a native antibody
.kappa. or .lamda. light chain sequence or a fragment thereof, and
will preferably retain at least about 65%, or at least about 70%,
or at least about 75%, or at least about 80%, or at least about
85%, or at least about 90%, or at least about 95%, or at least
about 98% sequence identity with a native sequence V.kappa.
polypeptide. In another preferred embodiment, the V.kappa.-like
polypeptide variant will be less then 95%, or less than 90%, or
less then 85%, or less than 80%, or less than 75%, or less then
70%, or less than 65%, or less than 60%, or less then 55%, or less
than 50%, or less than 45%, or less than 40% identical in its amino
acid sequence to a native antibody .lamda. or .kappa. light chain
sequence. In other embodiments, the sequence identity is between
about 40% and about 95%, or between about 45% and about 90%, or
between about 50% and about 85%, or between about 55% and about
80%, or between about 60% and about 75%, or between about 60% and
about 80%, or between about 65% and about 85%, or between about 65%
and about 90%, or between about 65% and about 95%. In all
embodiments, preferably the V.kappa.-like polypeptides are capable
of binding to a target.
[0115] The terms "JC.kappa." and "JC.kappa.-like" are used
interchangeably, and refer to native sequence polypeptides that
include a portion identical to a native sequence .kappa. J-constant
(C) region segment and a unique N-terminal extension (tail), and
variants thereof. In one embodiment, variants of native sequence
JC.kappa.-like polypeptides comprise an N-terminal extension (tail)
that distinguishes them from an antibody JC segment. In a
particular embodiment, variants of native sequence JC.kappa.-like
polypeptides retain at least part, and preferably all, of the
unique N-terminal extension (tail) that distinguishes the
JC.kappa.-like polypeptides from the corresponding antibody .kappa.
light chain JC segments. In another embodiment, the N-terminal tail
of the variant JC.kappa.-like polypeptide is a sequence not
naturally associated with the rest of the sequence. In the latter
embodiment, the difference between the N-terminal tail naturally
present in the native JC.kappa.-like sequence and the variant
sequence may result from one or more amino acid alterations
(substitutions, insertions, deletions, and/or additions), or the
N-terminal tail may be identical with a tail present in nature in a
different JC.kappa.-like protein. Variants of native sequence
JC.kappa.-like polypeptides may contain one or more amino acid
alterations in the part of the sequence that is identical to a
native antibody .kappa. variable domain JC sequence. In all
instances, the variants can, and preferably do, include an
N-terminal extension (unique N-terminus) of at least four, or at
least five, or at least six, or at least seven, or at least eight,
or at least nine, or at least ten amino acids, preferably 4-100, or
4-90, or 4-80, or 4-70, or 4-60, 4-50, or 4-45, or 4-40, or 4-35,
or 4-30, or 4-25, or 4-20, or 4-15, or 4-10 amino acid residues
relative to a native antibody .kappa. light chain JC sequence. The
JC.kappa.-like polypeptide variant, as defined herein, will be
different from a native antibody .lamda. or .kappa. light chain JC
sequence, or a fragment thereof, and will preferably retain at
least about 65%, or at least about 70%, or at least about 75%, or
at least about 80%, or at least about 85%, or at least about 90%,
or at least about 95%, or at least about 98% sequence identity with
a native sequence JC polypeptide. In another preferred embodiment,
the JC.kappa.-like polypeptide variant will be less then 95%, or
less than 90%, or less then 85%, or less than 80%, or less than
75%, or less then 70%, or less than 65%, or less than 60% identical
in its amino acid sequence to a native antibody .lamda. or .kappa.
light chain JC sequence. In other embodiments, the sequence
identity is between about 40% and about 95%, or between about 45%
and about 90%, or between about 50% and about 85%, or between about
55% and about 80%, or between about 60% and about 75%, or between
about 60% and about 80%, or between about 65% and about 85%, or
between about 65% and about 90%, or between about 65% and about
95%.
[0116] Percent amino acid sequence identity may be determined using
the sequence comparison program NCBI-BLAST2 (Altschul et al.,
Nucleic Acids Res. 25:3389-3402 (1997)). The NCBI-BLAST2 sequence
comparison program may be downloaded from
http://www.ncbi.nlm.nih.gov or otherwise obtained from the National
Institute of Health, Bethesda, Md. NCBI-BLAST2 uses several search
parameters, wherein all of those search parameters are set to
default values including, for example, unmask=yes, strand=all,
expected occurrences=10, minimum low complexity length=15/5,
multi-pass e-value=0.01, constant for multi-pass=25, dropoff for
final gapped alignment=25 and scoring matrix=BLOSUM62.
[0117] The terms "conjugate," "conjugated," and "conjugation" refer
to any and all forms of covalent or non-covalent linkage, and
include, without limitation, direct genetic or chemical fusion,
coupling through a linker or a cross-linking agent, and
non-covalent association, for example through Van der Waals forces,
or by using a leucine zipper.
[0118] The term "fusion" is used herein to refer to the combination
of amino acid sequences of different origin in one polypeptide
chain by in-frame combination of their coding nucleotide sequences.
The term "fusion" explicitly encompasses internal fusions, i.e.,
insertion of sequences of different origin within a polypeptide
chain, in addition to fusion to one of its termini.
[0119] As used herein, the terms "peptide," "polypeptide" and
"protein" all refer to a primary sequence of amino acids that are
joined by covalent "peptide linkages." In general, a peptide
consists of a few amino acids, typically from about 2 to about 50
amino acids, and is shorter than a protein. The term "polypeptide,"
as defined herein, encompasses peptides and proteins.
[0120] The term "amino acid" or "amino acid residue" typically
refers to an amino acid having its art recognized definition such
as an amino acid selected from the group consisting of: alanine
(Ala); arginine (Arg); asparagine (Asn); aspartic acid (Asp);
cysteine (Cys); glutamine (Gln); glutamic acid (Glu); glycine
(Gly); histidine (His); isoleucine (Ile): leucine (Leu); lysine
(Lys); methionine (Met); phenylalanine (Phe); proline (Pro); serine
(Ser); threonine (Thr); tryptophan (Trp); tyrosine (Tyr); and
valine (Val) although modified, synthetic, or rare amino acids may
be used as desired. Thus, modified and unusual amino acids listed
in 37 CFR 1.822(b)(4) are specifically included within this
definition and expressly incorporated herein by reference. Amino
acids can be subdivided into various sub-groups. Thus, amino acids
can be grouped as having a nonpolar side chain (e.g., Ala, Cys,
Ile, Leu, Met, Phe, Pro, Val); a negatively charged side chain
(e.g., Asp, Glu); a positively charged side chain (e.g., Arg, His,
Lys); or an uncharged polar side chain (e.g., Asn, Cys, Gln, Gly,
His, Met, Phe, Ser, Thr, Trp, and Tyr). Amino acids can also be
grouped as small amino acids (Gly, Ala), nucleophilic amino acids
(Ser, His, Thr, Cys), hydrophobic amino acids (Val, Leu, Ile, Met,
Pro), aromatic amino acids (Phe, Tyr, Trp, Asp, Glu), amides (Asp,
Glu), and basic amino acids (Lys, Arg).
[0121] The term "polynucleotide(s)" refers to nucleic acids such as
DNA molecules and RNA molecules and analogues thereof (e.g., DNA or
RNA generated using nucleotide analogues or using nucleic acid
chemistry). As desired, the polynucleotides may be made
synthetically, e.g., using art-recognized nucleic acid chemistry or
enzymatically using, e.g., a polymerase, and, if desired, be
modified. Typical modifications include methylation, biotinylation,
and other art-known modifications. In addition, the nucleic acid
molecule can be single-stranded or double-stranded and, where
desired, linked to a detectable moiety.
[0122] The term "variant" with respect to a reference polypeptide
refers to a polypeptide that possesses at least one amino acid
mutation or modification (i.e., alteration) as compared to a native
polypeptide. Variants generated by "amino acid modifications" can
be produced, for example, by substituting, deleting, inserting
and/or chemically modifying at least one amino acid in the native
amino acid sequence.
[0123] An "amino acid modification" refers to a change in the amino
acid sequence of a predetermined amino acid sequence. Exemplary
modifications include an amino acid substitution, insertion and/or
deletion.
[0124] An "amino acid modification at a specified position," refers
to the substitution or deletion of the specified residue, or the
insertion of at least one amino acid residue adjacent the specified
residue. By insertion "adjacent" a specified residue is meant
insertion within one to two residues thereof. The insertion may be
N-terminal or C-terminal to the specified residue.
[0125] An "amino acid substitution" refers to the replacement of at
least one existing amino acid residue in a predetermined amino acid
sequence with another different "replacement" amino acid residue.
The replacement residue or residues may be "naturally occurring
amino acid residues" (i.e. encoded by the genetic code) and
selected from the group consisting of: alanine (Ala); arginine
(Arg); asparagine (Asn); aspartic acid (Asp); cysteine (Cys);
glutamine (Gln); glutamic acid (Glu); glycine (Gly); histidine
(His); isoleucine (Ile): leucine (Leu); lysine (Lys); methionine
(Met); phenylalanine (Phe); proline (Pro); serine (Ser); threonine
(Thr); tryptophan (Trp); tyrosine (Tyr); and valine (Val).
Substitution with one or more non-naturally occurring amino acid
residues is also encompassed by the definition of an amino acid
substitution herein.
[0126] A "non-naturally occurring amino acid residue" refers to a
residue, other than those naturally occurring amino acid residues
listed above, which is able to covalently bind adjacent amino acid
residues(s) in a polypeptide chain. Examples of non-naturally
occurring amino acid residues include norleucine, ornithine,
norvaline, homoserine and other amino acid residue analogues such
as those described in Ellman et al. Meth. Enzym. 202:301 336
(1991). To generate such non-naturally occurring amino acid
residues, the procedures of Noren et al. Science 244:182 (1989) and
Ellman et al., supra, can be used. Briefly, these procedures
involve chemically activating a suppressor tRNA with a
non-naturally occurring amino acid residue followed by in vitro
transcription and translation of the RNA.
[0127] An "amino acid insertion" refers to the incorporation of at
least one amino acid into a predetermined amino acid sequence.
While the insertion will usually consist of the insertion of one or
two amino acid residues, the present application contemplates
larger "peptide insertions", e.g. insertion of about three to about
five or even up to about ten amino acid residues. The inserted
residue(s) may be naturally occurring or non-naturally occurring as
disclosed above.
[0128] An "amino acid deletion" refers to the removal of at least
one amino acid residue from a predetermined amino acid
sequence.
[0129] The term "mutagenesis" refers to, unless otherwise
specified, any art recognized technique for altering a
polynucleotide or polypeptide sequence. Preferred types of
mutagenesis include error prone PCR mutagenesis, saturation
mutagenesis, or other site directed mutagenesis.
[0130] "Site-directed mutagenesis" is a technique standard in the
art, and is conducted using a synthetic oligonucleotide primer
complementary to a single-stranded phage DNA to be mutagenized
except for limited mismatching, representing the desired mutation.
Briefly, the synthetic oligonucleotide is used as a primer to
direct synthesis of a strand complementary to the single-stranded
phage DNA, and the resulting double-stranded DNA is transformed
into a phage-supporting host bacterium. Cultures of the transformed
bacteria are plated in top agar, permitting plaque formation from
single cells that harbor the phage. Theoretically, 50% of the new
plaques will contain the phage having, as a single strand, the
mutated form; 50% will have the original sequence. Plaques of
interest are selected by hybridizing with kinased synthetic primer
at a temperature that permits hybridization of an exact match, but
at which the mismatches with the original strand are sufficient to
prevent hybridization. Plaques that hybridize with the probe are
then selected, sequenced and cultured, and the DNA is
recovered.
[0131] The term "neutralizing molecule" is used herein in the
broadest sense and refers to any molecule that inhibits a virus
from replicatively infecting a target cell, irrespective of the
mechanism by which neutralization is achieved, The neutralizing
molecule preferably an antibody or an antibody-like molecule, as
hereinabove defined, Neutralization can be achieved, for example,
by inhibiting the attachment or adhesion of the virus to the cell
surface, e.g., by engineering an molecule, such as an antibody or
antibody-like molecule, that binds directly to, or close by, the
site responsible for the attachment or adhesion of the virus.
Neutralization can also be achieved by a molecule, such as an
antibody or antibody-like molecule, directed to the virion surface,
which results in the aggregation of virions. Neutralization can
further occur by inhibition of the fusion of viral and cellular
membranes following attachment of the virus to the target cell, by
inhibition of endocytosis, inhibition of progeny virus from the
infected cell, and the like. The neutralizing molecules, such as
antibodies or antibody-like molecules, of the present invention are
not limited by the mechanism by which neutralization is
achieved.
[0132] The term "antibody repertoire" is used herein in the
broadest sense and refers to a collection of antibodies or antibody
fragments which can be used to screen for a particular property,
such as binding ability, binding specificity, ability of
gastrointestinal transport, stability, affinity, and the like. The
term specifically includes antibody libraries, including all forms
of combinatorial libraries, such as, for example, antibody phage
display libraries, including, without limitation, single-chain Fv
(scFv) and Fab antibody phage display libraries from any source,
including naive, synthetic and semi-synthetic libraries.
[0133] Similarly, a "repertoire of antibody-like molecules" (as
hereinabove defined) refers to a collection of such molecules which
can be used to screen for a particular property, such as binding
ability, binding specificity, ability of gastrointestinal
transport, stability, affinity, and the like. The term specifically
includes surrobody libraries and libraries of .kappa.-like light
chain constructs (as hereinabove defined), including all forms of
combinatorial libraries, such as, for example, phage display
libraries. Combinatorial surrobody libraries are disclosed, for
example, in Xu et al., (2008), supra.
[0134] A "phage display library" is a protein expression library
that expresses a collection of cloned protein sequences as fusions
with a phage coat protein. Thus, the phrase "phage display library"
refers herein to a collection of phage (e.g., filamentous phage)
wherein the phage express an external (typically heterologous)
protein. The external protein is free to interact with (bind to)
other moieties with which the phage are contacted. Each phage
displaying an external protein is a "member" of the phage display
library.
[0135] An "antibody phage display library" refers to a phage
display library that displays antibodies or antibody fragments. The
antibody library includes the population of phage or a collection
of vectors encoding such a population of phage, or cell(s)
harboring such a collection of phage or vectors. The library can be
monovalent, displaying on average one single-chain antibody or
antibody fragment per phage particle, or multi-valent, displaying,
on average, two or more antibodies or antibody fragments per viral
particle. The term "antibody fragment" includes, without
limitation, single-chain Fv (scFv) fragments and Fab fragments.
Preferred antibody libraries comprise on average more than
10.sup.6, or more than 10.sup.7, or more than 10.sup.8, or more
than 10.sup.9 different members.
[0136] The term "filamentous phage" refers to a viral particle
capable of displaying a heterogenous polypeptide on its surface,
and includes, without limitation, f1, fd, Pf1, and M13. The
filamentous phage may contain a selectable marker such as
tetracycline (e.g., "fd-tet"). Various filamentous phage display
systems are well known to those of skill in the art (see, e.g.,
Zacher et al., Gene 9:127-140 (1980), Smith et al., Science
228:1315-1317 (1985); and Parmley and Smith, Gene 73:305-318
(1988)).
[0137] The term "panning" is used to refer to the multiple rounds
of screening process in identification and isolation of phages
carrying compounds, such as antibodies, with high affinity and
specificity to a target.
[0138] The term "non-human animal" as used herein includes, but is
not limited to, mammals such as, for example, non-human primates,
rodents (e.g., mice and rats), and non-rodent animals, such as, for
example, rabbits, pigs, sheep, goats, cows, pigs, horses and
donkeys. It also includes birds (e.g., chickens, turkeys, ducks,
geese and the like). The term "non-primate animal" as used herein
refers to mammals other than primates, including but not limited to
the mammals specifically listed above.
[0139] The phrase "functionally different antibodies," and
grammatical variants thereof, are used to refer to antibodies that
differ from each other in at least one property, including, without
limitation, binding specificity, binding affinity, and any
immunological or biological function, such as, for example, ability
to neutralize a target, extent or quality of biological activity,
etc.
[0140] The phrase "conserved amino acid residues" is used to refer
to amino acid residues that are identical between two or more amino
acid sequences aligned with each other.
[0141] The term "epitope" as used herein, refers to a sequence of
at least about 3 to 5, preferably at least about 5 to 10, or at
least about 5 to 15 amino acids, and typically not more than about
500, or about 1,000 amino acids, which define a sequence that by
itself, or as part of a larger sequence, binds to an antibody
generated in response to such sequence. An epitope is not limited
to a polypeptide having a sequence identical to the portion of the
parent protein from which it is derived. Indeed, viral genomes are
in a state of constant change and exhibit relatively high degrees
of variability between isolates. Thus the term "epitope"
encompasses sequences identical to the native sequence, as well as
modifications, such as deletions, substitutions and/or insertions
to the native sequence. Generally, such modifications are
conservative in nature but non-conservative modifications are also
contemplated. The term specifically includes "mimotopes," i.e.
sequences that do not identify a continuous linear native sequence
or do not necessarily occur in a native protein, but functionally
mimic an epitope on a native protein. The term "epitope"
specifically includes linear and conformational epitopes.
B. General Techniques
[0142] Techniques for performing the methods of the present
invention are well known in the art and described in standard
laboratory textbooks, including, for example, Ausubel et al.,
Current Protocols of Molecular Biology, John Wiley and Sons (1997);
Molecular Cloning: A Laboratory Manual, Third Edition, J. Sambrook
and D. W. Russell, eds., Cold Spring Harbor, N.Y., USA, Cold Spring
Harbor Laboratory Press, 2001; Antibody Phage Display: Methods and
Protocols, P. M. O'Brian and R. Aitken, eds., Humana Press, In:
Methods in Molecular Biology, Vol. 178; Phage Display: A Laboratory
Manual, C. F. Barbas III et al. eds., Cold Spring Harbor, N.Y.,
USA, Cold Spring Harbor Laboratory Press, 2001; and Antibodies, G.
Subramanian, ed., Kluwer Academic, 2004. Mutagenesis can, for
example, be performed using site-directed mutagenesis (Kunkel et
al., Proc. Natl. Acad. Sci. USA 82:488-492 (1985)).
[0143] In one aspect, the viral antigen neutralizing molecules of
the present invention are antibodies, which are typically selected
using antibody libraries. In the following description, the
invention is illustrated with reference to certain types of
antibody libraries, but the invention is not limited to the use of
any particular type of antibody library. Recombinant monoclonal
antibody libraries can be based on immune fragments or naive
fragments. Antibodies from immune antibody libraries are typically
constructed with V.sub.H and V.sub.L gene pools that are cloned
from source B cells into an appropriate vector for expression to
produce a random combinatorial library, which can subsequently be
selected for and/or screened. Other types of libraries may be
comprised of antibody fragments from a source of genes that is not
explicitly biased for clones that bind to an antigen. Thus, naive
antibody libraries derive from natural, unimmunized, rearranged V
genes. Synthetic antibody libraries are constructed entirely by in
vitro methods, introducing areas of complete or tailored degeneracy
into the CDRs of one or more V genes. Semi-synthetic libraries
combine natural and synthetic diversity, and are often created to
increase natural diversity while maintaining a desired level of
functional diversity. Thus, such libraries can, for example, be
created by shuffling natural CDR regions (Soderlind et al., Nat.
Biotechnol. 18:852-856 (2000)), or by combining naturally
rearranged CDR sequences from human B cells with synthetic CDR1 and
CDR2 diversity (Hoet et al., Nat. Biotechnol. 23:455-38 (2005)).
The present invention encompasses the use of naive, synthetic and
semi-synthetic antibody libraries, or any combination thereof.
[0144] Similarly, the methods of the present invention are not
limited by any particular technology used for the display of
antibodies. Although the invention is illustrated with reference to
phage display, antibodies of the present invention can also be
identified by other display and enrichment technologies. Antibody
fragments have been displayed on the surface of filamentous phage
that encode the antibody genes (Hoogenboom and Winter J. Mol.
Biol., 222:381 388 (1992); McCafferty et al., Nature 348(6301):552
554 (1990); Griffiths et al. EMBO J, 13(14):3245-3260 (1994)). For
a review of techniques for selecting and screening antibody
libraries see, e.g., Hoogenboom, Nature Biotechnol. 23(9):1105-1116
(2005). In addition, there are systems known in the art for display
of heterologous proteins and fragments thereof on the surface of
Escherichia coli (Agterberg et al., Gene 88:37-45 (1990); Charbit
et al., Gene 70:181-189 (1988); Francisco et al., Proc. Natl. Acad.
Sci. USA 89:2713-2717 (1992)), and yeast, such as Saccharomyces
cerevisiae (Boder and Wittrup, Nat. Biotechnol. 15:553-557 (1997);
Kieke et al., Protein Eng. 10:1303-1310 (1997)). Other known
display techniques include ribosome or mRNA display (Mattheakis et
al., Proc. Natl. Acad. Sci. USA 91:9022-9026 (1994); Hanes and
Pluckthun, Proc. Natl. Acad. Sci. USA 94:4937-4942 (1997)), DNA
display (Yonezawa et al., Nucl. Acid Res. 31(19):e118 (2003));
microbial cell display, such as bacterial display (Georgiou et al.,
Nature Biotech. 15:29-34 (1997)), display on mammalian cells, spore
display (Isticato et al., J. Bacteriol. 183:6294-6301 (2001); Cheng
et al., Appl. Environ. Microbiol. 71:3337-3341 (2005) and
co-pending provisional application Ser. No. 60/865,574, filed Nov.
13, 2006), viral display, such as retroviral display (Urban et al.,
Nucleic Acids Res. 33:e35 (2005), display based on protein-DNA
linkage (Odegrip et al., Proc. Acad. Natl. Sci. USA 101:2806-2810
(2004); Reiersen et al., Nucleic Acids Res. 33:e10 (2005)), and
microbead display (Sepp et al., FEBS Lett. 532:455-458 (2002)).
C. Detailed Description of Preferred Embodiments
[0145] In one aspect, the present invention concerns the selection,
production and use of monoclonal antibodies and antibody-like
molecules neutralizing more than one subtype and/or more than one
isolate of an influenza A virus, binding to a hemagglutinin (HA)
antigen of the virus, but not inhibiting hemagglutination.
[0146] The virions of influenza A virus contain 8 segments of
linear negative-sense single stranded RNA. The total genome length
is 13600 nucleotides, and the eight segments are 2350 nucleotides;
2350 nucleotides; of 2250 nucleotides; 1780 nucleotides; 1575
nucleotides; 1420 nucleotides; 1050 nucleotides; and 900
nucleotides, respectively, in length. Host specificity and
attenuation of influenza A virus have been attributed to viral
hemagglutinin (H, HA), nucleoprotein (NP), matrix (M), and
non-structural (NS) genes individually or in combinations of viral
genes (see, e.g., Rogers et al., Virology 127:361-373 (1983);
Scholtissek et al., Virology 147:287-294 (1985); Snyder et al., J.
Clin. Microbiol. 24:467-469 (1986); Tian et al., J. Virol.
53:771-775 (1985); Treanor et al., Virology 171:1-9 (1989).
[0147] Nucleotide and amino acid sequences of influenza A viruses
and their surface proteins, including hemagglutinins and
neuraminidase proteins, are available from GenBank and other
sequence databases, such as, for example, the Influenza Sequence
Database maintained by the Theoretical Biology and Biophysics Group
of Los Alamos National Laboratory. The amino acid sequences of 15
known H subtypes of the influenza A virus hemagglutinin (H1-H15)
are shown in U.S. Application Publication No. 20080014205,
published on Jan. 17, 2008, incorporated herein by reference in its
entirety. An additional influenza A virus hemagglutinin subtype
(H16) was isolated recently from black-headed gulls in Sweden, and
reported by Fouchier et al., J. Virol. 79(5):2814-22 (2005). A
large variety of strains of each H subtype are also known. For
example, the sequence of the HA protein designated H5 A/Hong
Kong/156/97 was determined from an influenza A H5N1 virus isolated
from a human in Hong Kong in May 1997, and is shown in comparison
with sequences of several additional strains obtained from other
related H5N1 isolates in Suarez et al., J. Virol. 72:6678-6688
(1998).
[0148] The structure of the catalytic and antigenic sites of
influenza virus neuraminidase have been published by Colman et al.,
Nature 303:41-4 (1983), and neuraminidase sequences are available
from GenBank and other sequence databases.
[0149] It has been known that virus-specific antibodies resulting
from the immune response of infected individuals typically
neutralize the virus via interaction with the viral hemagglutinin
(Ada et al., Curr. Top. Microbiol. Immunol. 128:1-54 (1986); Couch
et al., Annu. Rev. Micobiol. 37:529-549 (1983)). The
three-dimensional structures of influenza virus hemagglutinins and
crystal structures of complexes between influenza virus
hemagglutinins and neutralizing antibodies have also been
determined and published, see, e.g., Wilson et al., Nature
289:366-73 (1981); Ruigrok et al., J. Gen. Virol. 69 (Pt
11):2785-95 (1988); Wrigley et al., Virology 131(2):308-14 (1983);
Daniels et al., EMBO J. 6:1459-1465 (1987); and Bizebard et al.,
Nature 376:92-94 (2002).
[0150] According to the present invention, antibodies with the
desired properties are identified from one or more antibody
libraries, which can come from a variety of sources and can be of
different types.
[0151] Comprehensive Human Influenza Antibody Libraries
[0152] Comprehensive human influenza antibody libraries can be
created from antibodies obtained from convalescent patients of
various prior influenza, seasonal outbreaks epidemics, and
pandemics, including the 1968 Hong Kong flu (H3N2), the 1957 Asian
flu (H2N2), the 1918 Spanish flu (H1N1), and the 2004/2005 Avian
flu (H5N1). For example, see U.S. Application Publication No.
20080014205, published on Jan. 17, 2008, incorporated herein by
reference in its entirety. In order to prepare such libraries,
blood or bone marrow samples are collected from individuals known
or suspected to have been infected with an influenza virus.
Peripheral blood samples, especially from geographically distant
sources, may need to be stabilized prior to transportation and use.
Kits for this purpose are well known and commercially available,
such as, for example, BD Vacutainer.RTM. CPT.TM. cell preparation
tubes can be used for centrifugal purification of lymphocytes, and
guanidium, Trizol, or RNAlater used to stabilize the samples. Upon
receipt of the stabilized lymphocytes or whole bone marrow, RT-PCR
is performed to rescue heavy and light chain repertoires, using
immunoglobulin oligo primers known in the art. The PCR repertoire
products are combined with linker oligos to generate scFv libraries
to clone directly in frame with m13 pIII protein, following
procedures known in the art.
[0153] In a typical protocol, antibodies in the human sera can be
detected by well known serological assays, including, for example,
by the well-known hemagglutinin inhibition (HAI) assay (Kendal, A.
P., M. S. Pereira, and J. J. Skehel. 1982. Concepts and procedures
for laboratory-based influenza surveillance. U.S. Department of
Health and Human Services, Public Health Service, Centers for
Disease Control, Atlanta, Ga.), or the microneutralization assay
(Harmon et al., J. Clin. Microbiol. 26:333-337 (1988)). This
detection step might not be necessary if the serum sample has
already been confirmed to contain influenza neutralizing
antibodies. Lymphocytes from whole blood or those present in bone
marrow are next processed by methods known in the art. Whole RNA is
extracted by Tri BD reagent (Sigma) from fresh or RNAlater
stabilized tissue. Subsequently, the isolated donor total RNA is
further purified to mRNA using Oligotex purification (Qiagen). Next
first strand cDNA synthesis, is generated by using random nonamer
oligonucleotides and or oligo (dT).sub.18 primers according to the
protocol of AccuScript reverse transcriptase (Stratagene). Briefly,
100 ng mRNA, 0.5 mM dNTPs and 300 ng random nonamers and or 500 ng
oligo (dT).sub.18 primers in Accuscript RT buffer (Stratagene) are
incubated at 65.degree. C. for 5 min, followed by rapid cooling to
4.degree. C. Then, 100 mM DTT, Accuscript RT, and RNAse Block are
added to each reaction and incubated at 42.degree. C. for 1 h, and
the reverse transcriptase is inactivated by heating at 70.degree.
C. for 15 minutes. The cDNA obtained can be used as a template for
RT-PCR amplification of the antibody heavy and light chain V genes,
which can then be cloned into a vector, or, if phage display
library is intended, into a phagemid vector. This procedure
generates a repertoire of antibody heavy and light chain variable
region clones (V.sub.H and V.sub.L libraries), which can be kept
separate or combined for screening purposes.
[0154] Immunoglobulin repertoires from peripheral lymphocytes of
survivors of earlier epidemics and pandemics, such as the 1918
Spanish Flu, can be retrieved, stabilized, and rescued in a manner
similar to that described above. For additional H1 and H3 libraries
repertoires can be recovered from properly timed vaccinated
locally-sourced donors. As an additional option commercially
available bone marrow total RNA or mRNA can be purchased from
commercial sources to produce libraries suitable for H1 and H3, and
depending upon the background of donor also suitable for H2
antibody screening.
[0155] Synthetic Human-Like Repertoire
[0156] In the methods of the present invention, the synthetic human
antibody repertoire can be represented by a synthetic antibody
library, which can be made by methods known in the art or obtained
from commercial sources. Thus, for example, a fully synthetic human
repertoire is described in U.S. patent application Ser. No.
11/864,525 filed on Sep. 28, 2007, the entire disclosure of which
is hereby expressly incorporated by reference. In brief, this
patent application describes libraries of immunoglobulins in which
predetermined amino acids have been combinatorially introduced into
one or more complementarity-determining regions of the
immunoglobulin of interest. Additionally, for example, a universal
immunoglobulin library, including subsets of such library, are
described in U.S. Patent Application Publication No. 20030228302
published on Dec. 11, 2003, the entire disclosure of which is
hereby expressly incorporated by reference.
[0157] Specific sublibraries of antibody heavy and light chains
with various mutations can be combined to provide the framework
constructs for the antibodies of the present invention, which is
followed by introducing diversity in the CDRs of both heavy and
light chains. This diversity can be achieved by methods known in
the art, such as, for example, by Kunkel mutagenesis, and can be
repeated several times in order to further increase diversity.
Thus, for example, diversity into the heavy and light chain CDR1
and CD2 regions, separately or simultaneously, can be introduced by
multiple rounds of Kunkel mutagenesis. If necessary, the various
Kunkel clones can be segregated by CDR lengths and/or clones
lacking diversity in a targeted CDR (e.g., CDR1 or CDR3) can be
removed, e.g., by digestion with template-specific restriction
enzymes. Upon completion of these steps, the size of the library
should exceed about 10.sup.9 members, but libraries with lesser
members are also useful.
[0158] In a specific embodiment, both immunized antibody libraries
and synthetic antibody libraries are used for identifying the
neutralizing antibodies of the present invention. The two types of
libraries are fundamentally different. The synthetic antibody
libraries are synthesized collections of human antibodies with the
predicted ability to bind antigens, while an immunized repertoire
will contain sequences to specifically recognize avian H5
hemagglutinin, and/or H1, H2, or H3 hemagglutinin, as the case may
be. Thus, the immunized repertoires are theoretically optimized to
recognize critical components of targeted influenza subtype(s). As
a result these differences the two methods produce a different set
of antibodies and thus provide a more efficient approach for
identifying the desired neutralizing antibodies.
[0159] Hyperimmunized Non-Human Primate Antibody Libraries
[0160] In this method, an antibody library is rescued from
hyperimmunized non-human primates, such as, for example, macaque or
baboons. Specifically, non-human primates are immunized with
various subtypes of the influenza A virus or with various
hemagglutinin (H) proteins. Animals developing titers of antibody
recognizing the influenza A virus subtype or hemagglutinin they
were immunized with are sacrificed and their spleens harvested.
Blood or bone marrow of the immunized animals is collected, and
antibodies produced are collected and amplified as described above
for the comprehensive influenza antibody libraries.
[0161] Strategies for Isolating Neutralizing Antibodies of the
Invention
[0162] Regardless of the type of antibody library or libraries
used, antibodies with dual specificities, such as, for example,
showing reactivity with two different influenza A subtypes and/or
with two strains (isolates) of the same subtype, and/or with human
and non-human isolates, can be discovered and optimized through
controlled cross-reactive selection and/or directed combinatorial
and/or mutagenic engineering.
[0163] In a typical enrichment scheme, illustrated in FIG. 1, a
library including antibodies showing cross-reactivity to two
targets, designated as targets A and B, are subjected to multiple
rounds of enrichment (see U.S. Application Publication No.
20080014205, published on Jan. 17, 2008, incorporated herein by
reference in its entirety). If enrichment is based on reactivity
with target A, each round of enrichment will increase the reactive
strength of the pool towards target A. Similarly, if enrichment is
based on reactivity with target B, each round of enrichment will
increase the reactive strength of the pool towards target B.
Although this approach refers to panning, which is the selection
method used when screening phage display libraries (see below), the
approach is equally applicable to any type of library discussed
above, other otherwise known in the art, and to any type of display
technique. Targets A and B include any targets to which antibodies
bind, including but not limited to various isolates, types and
sub-types of influenza viruses.
[0164] Since the goal of the present invention is to identify
neutralizing antibodies with multiple specificities, a
cross-reactive discovery selection scheme has been developed. In
the interest of simplicity, this scheme is illustrated in FIG. 2
showing the selection of antibodies with dual specificities. In
this case, an antibody library including antibodies showing
reactivity with two targets, targets A and B, is first selected for
reactivity with one of the targets, e.g., target A, followed by
selection for reactivity with the other target, e.g., target B.
Each successive selection round reinforces the reactive strength of
the resulting pool towards both targets. (see also U.S. Application
Publication No. 20080014205, published on Jan. 17, 2008,
incorporated herein by reference in its entirety). Accordingly,
this method is particularly useful for identifying antibodies with
dual specificity. Of course, the method can be extended to
identifying antibodies showing reactivity towards further targets,
by including additional rounds of enrichment towards the additional
target(s). Again, if the library screened is a phage display
library, selection is performed by cross-reactive panning, but
other libraries and other selection methods can also be used.
[0165] A combination of the two methods discussed above includes
two separate enrichment rounds for reactivity towards target A and
target B, respectively, recombining the two pools obtained, and
subsequent cross-reactive selection rounds, as described above (see
U.S. Application Publication No. 20080014205, published on Jan. 17,
2008, incorporated herein by reference in its entirety). This
approach is illustrated in FIG. 3. Just as in the pure
cross-reactive selection, each round of selection of the recombined
library increases the reactive strength of the resulting pool
towards both targets.
[0166] In a further embodiment, illustrated in FIG. 4, first a
clone showing strong reactivity with a target A, and having
detectable cross-reactivity with target B is identified. Based on
this clone, a mutagenic library is prepared, which is then
selected, in alternating rounds, for reactivity with target B and
target A respectively. This scheme will result in antibodies that
maintain strong reactivity with target A, and have increased
reactivity with target B (see U.S. Application Publication No.
20080014205, published on Jan. 17, 2008, incorporated herein by
reference in its entirety). Just as before, selection is performed
by panning, if the libraries screened are phage display libraries,
but other libraries, other display techniques, and other selection
methods can also be used, following the same strategy.
[0167] As discussed above, targets A and B can, for example, be two
different subtypes of the influenza A virus, two different strains
(isolates) of the same influenza A virus, subtypes or isolates from
two different species, where one species is preferably human. Thus,
for example, target A may be an isolate of the 2004 Vietnam isolate
of the H5N1 virus, and target B may be a 1997 Hong Kong isolate of
the H5N1 virus. It is emphasized that these examples are merely
illustrative, and antibodies with dual and multiple specificities
to any two or multiple targets can be identified, selected and
optimized in an analogous manner.
[0168] Alternatively, if an antibody library such as the UAL that
allows segregation of discrete frameworks and CDR lengths is used
to find an antibody to target A, then an antigen B could be
screened for and the library could be restricted to a diverse
collection of similar parameters. Once an antibody to antigen B is
found then chimeric or mutagenic antibodies based upon the
respective A and B antibodies could be used to engineer a dual
specific collection.
[0169] Phage Display
[0170] In a particular embodiment, the present invention utilizes
phage display antibody libraries to functionally discover
neutralizing monoclonal antibodies with multiple (including dual)
specificities. Such antibodies can, for example, be monoclonal
antibodies capable of neutralizing more than one influenza A virus
subtype, including the H5, H7 and/or H9 subtypes, such as the H5
and H1; H5 and H2; H5 and H3; H5, H1, and H2; H5, H1, and H3; H5,
H2 and H3; H1, H2 and H3, etc., subtypes, and/or more than one
strain (isolate) of the same subtype.
[0171] To generate a phage antibody library, a cDNA library
obtained from any source, including the libraries discussed above,
is cloned into a phagemid vector.
[0172] Thus, for example, the collection of antibody heavy and
light chain repertoires rescued from lymphocytes or bone marrow by
RT-PCR as described above, is reassembled as a scFv library fused
to m13 pIII protein. The combinatorial library will contain about
more than 10.sup.6, or more than 10.sup.7, or more than 10.sup.8,
or more than 10.sup.9 different members, more than 10.sup.7
different members or above being preferred. For quality control
random clones are sequenced to assess overall repertoire
complexity.
[0173] Similarly, following the initial PCR rescue of heavy and
light chain variable regions from a naive or immunized human, or
hyperimmunized nonhuman primate antibody library, the PCR products
are combined with linker oligos to generate scFv libraries to clone
directly in frame with M13 pIII coat protein. The library will
contain about more than 10.sup.6, or more than 10.sup.7, or more
than 10.sup.8, or more than 10.sup.9 different members, more than
10.sup.7 different members or above being preferred. As a quality
control step, random clones are sequenced in order to assess
overall repertoire size and complexity.
[0174] Antibody phage display libraries may contain antibodies in
various formats, such as in a single-chain Fv (scFv) or Fab format.
For review see, e.g., Hoogenboom, Methods Mol. Biol. 178:1-37
(2002).
[0175] Screening
[0176] Screening methods for identifying antibodies with the
desired neutralizing properties have been described above.
Reactivity can be assessed based on direct binding to the desired
hemagglutinin proteins.
[0177] Hemagglutinin (HA) Protein Production
[0178] Hemagglutinin (HA) proteins can be produced by recombinant
DNA technology. In this method, HA genes are cloned into an
appropriate vector, preferably a baculovirus expression vector for
expression in baculovirus-infected insect cells, such as Spodoptera
frugiperda (Sf9) cells.
[0179] The nucleic acid coding for the HA protein is inserted into
a baculovirus expression vector, such as Bac-to-Bac (Invitrogen),
with or without a C-terminal epitope tag, such as a poly-his
(hexahistidine tag). A poly-his tag provides for easy purification
by nickel chelate chromatography.
[0180] In general the cloning involves making reference cDNAs by
assembly PCR from individually synthesized oligos. Corresponding
isolate variant HA proteins are made by either substituting
appropriate mutant oligos into additional assembly PCRs or by
mutagenesis techniques, such as by Kunkel mutagenesis. Two clusters
of HA protein sequences exist for H5, the 1997 and 2004 subtype
isolates. Therefore, a single reference protein is made for each
cluster. Similarly, reference proteins are generated for 1918
Spanish flu (H1), 1958 Asian Flu (H2), 1968 Hong Kong Flu (H3), and
current H1, H2, H3 isolates.
[0181] Recombinant baculovirus is generated by transfecting the
above Bacmid into Sf9 cells (ATCC CRL 1711) using lipofectin
(commercially available from Gibco-BRL). After 4-5 days of
incubation at 28.degree. C., the released viruses are harvested and
used for further amplifications. Viral infection and protein
expression are performed as described by O'Reilley et al.,
Baculovirus Expression Vectors: A Laboratory Manual (Oxford: Oxford
University Press, 1994).
[0182] Expressed poly-His-tagged HA polypeptides can then be
purified, for example, by Ni.sup.2+-chelate affinity chromatography
as follows. Supernatants are collected from recombinant
virus-infected Sf9 cells as described by Rupert et al., Nature
362:175-179 (1993). A Ni.sup.2+-NTA agarose column (commercially
available from Qiagen) is prepared with a bed volume of 5 mL,
washed with 25 mL of water, and equilibrated with 25 mL of loading
buffer. The filtered cell extract is loaded onto the column at 0.5
mL per minute. The column is washed to baseline A.sub.280 with
loading buffer, at which point fraction collection is started.
Next, the column is washed with a secondary wash buffer (50 mM
phosphate; 300 mM NaCl, 10% glycerol, pH 6.0), which elutes
non-specifically bound protein. After reaching A.sub.280 baseline
again, the column is developed with a 0 to 500 mM imidazole
gradient in the secondary wash buffer. One-mL fractions are
collected and analyzed by SDS-PAGE and silver staining or Western
blot with Ni.sup.2+-NTA-conjugated to alkaline phosphatase
(Qiagen). Fractions containing the eluted His.sub.10-tagged HA
polypeptide are pooled and dialyzed against loading buffer.
[0183] Alternatively, purification of an IgG-tagged (or Fc-tagged)
HA polypeptide can be performed using known chromatography
techniques, including, for instance, Protein A or protein G column
chromatography.
[0184] As an alternative to using Sf9 cells HA proteins can also be
produced in other recombinant host cells, prokaryote, yeast, or
higher eukaryote cells. Suitable prokaryotes include but are not
limited to eubacteria, such as Gram-negative or Gram-positive
organisms, for example, Enterobacteriaceae such as Escherichia,
e.g., E. coli, Enterobacter, Erwinia, Klebsiella, Proteus,
Salmonella, e.g., Salmonella typhimurium, Serratia, e.g., Serratia
marcescans, and Shigella, as well as Bacilli such as B. subtilis
and B. licheniformis (e.g., B. licheniformis 41P disclosed in DD
266,710 published 12 Apr. 1989), Pseudomonas such as P. aeruginosa,
and Streptomyces. Various E. coli strains are publicly available,
such as E. coli K12 strain MM294 (ATCC 31,446); E. coli X1776 (ATCC
31,537); E. coli strain W3110 (ATCC 27,325); and K5 772 (ATCC
53,635).
[0185] In addition to prokaryotes, eukaryotic microbes such as
filamentous fungi or yeast are suitable cloning or expression hosts
for vectors containing nucleic acid encoding an HA polypeptide.
Saccharomyces cerevisiae is a commonly used lower eukaryotic host
microorganism. However, a number of other genera, species, and
strains are commonly available and useful herein, such as
Schizosaccharomyces pombe (Beach and Nurse, Nature 290: 140 (1981);
EP 139,383 published 2 May 1985); Kluyveromyces hosts (U.S. Pat.
No. 4,943,529; Fleer et al., Bio/Technology 9:968-975 (1991)) such
as, e.g., K. lactis (MW98-8C, CBS683, CBS4574; Louvencourt et al.,
J. Bacteriol. 737 (1983)), K. fragilis (ATCC 12,424), K. bulgaricus
(ATCC 16,045), K. wickeramii (ATCC 24,178), K. waltii (ATCC
56,500), K. drosophilarum (ATCC 36,906; Van den Berg et al.,
Bio/Technology 8:135 (1990)), K. thermotolerans, and K. marxianus,
yarrowia (EP 402,226); Pichia pastoris (EP 183,070; Sreekrishna et
al., J. Basic Microbiol. 28:265-278 (1988)); Candida, Trichoderma
reesia (EP 244,234); Neurospora crassa (Case et al., Proc. Natl.
Acad. Sci. USA 76:5259-5263 (1979)); Schwanniomyces such as
Schwanniomyces occidentalis (EP 394,538 published 31 Oct. 1990);
and filamentous fungi such as, e.g., Neurospora, Penicillium,
Tolypocladium (WO 91/00357 published 10 Jan. 1991), and Aspergillus
hosts such as A. nidulans (Ballance et al., Biochem. Biophys. Res.
Commun. 112:284-289 (1983); Tilburn et al., Gene 26:205-221 (1983);
Yelton et al., Proc. Natl. Acad. Sci. USA 81:1470-1474 (1984)) and
A. niger Kelly and Hynes, EMBO J. 4:475-479 (1985). Methylotropic
yeasts are suitable herein and include, but are not limited to,
yeast capable of growth on methanol selected from the genera
consisting of Hansenula, Candida, Kloeckera, Pichia, Saccharomyces,
Torulopsis, and Rhodotorula. A list of specific species that are
exemplary of this class of yeasts may be found in C. Anthony, The
Biochemistry of Methylotrophs 269 (1982).
[0186] Suitable host cells for the expression of HA proteins
include cells of multicellular organisms. Examples of invertebrate
cells include the above-mentioned insect cells such as Drosophila
S2 and Spodoptera Sf9, as well as plant cells. Examples of useful
mammalian host cell lines include Chinese hamster ovary (CHO) and
COS cells. More specific examples include monkey kidney CV1 line
transformed by SV40 (COS-7, ATCC CRL 1651); human embryonic kidney
line (HEK 293 or HEK 293 cells subcloned for growth in suspension
culture (Graham et al., J. Gen Virol. 36:59 (1977)); Chinese
hamster ovary cells/-DHFR (CHO, Urlaub and Chasin, Proc. Natl.
Acad. Sci. USA 77:4216 (1980)); mouse sertoli cells (TM4, Mather,
Biol. Reprod. 23:243-251 (1980)); human lung cells (WI 38, ATCC CCL
75); human liver cells (Hep G2, HB 8065); and mouse mammary tumor
(MMT 060562, ATCC CCL51). The selection of the appropriate host
cell is deemed to be within the skill in the art.
[0187] Hemagglutinin (HA) Protein Panning
[0188] HA protein is immobilized on to the surface of microtiter
wells or magnetic beads to pan the described above libraries. In a
particular embodiment, each library is allowed to bind the H5
protein at 4 degrees for two hours and then washed extensively with
cold PBS, before eluting HA specific binding clones with 0.2M
glycine-HC1 buffer (pH2.5). The recovered phage is pH neutralized
and amplified by infecting a susceptible host E. coli.
Subsequently, phagemid production can be induced to repeat the
enrichment of positive clones and subsequent clones isolation for
triage. Upon sufficient enrichment the entire pool is transferred
by infection into a non amber suppressor E. coli strain such as
HB2151 to express soluble scFv proteins. Alternatively the pool(s)
could be subcloned into a monomeric scFv expression vector, such as
pBAD, and recombinant soluble scFv proteins are expressed for in
vitro analysis and characterization, as described below.
[0189] Characterization
[0190] H5 clones are first tested for binding affinity to an H5
protein produced as described above. In a particular example,
binding is tested to a 2004 H5 protein (Refseq AAS65618, Isolate;
A/Thailand/2(SP-33)/2004(H5N1)), and in parallel test to a 1997 H5
protein (Refseq AAF74331, Isolate; A/Hong Kong/486/97(H5N1)), but
other isolates can also be used alone or in any combination. The
positive clones obtained with the 2004 and the 1997 H5 proteins
will fall into two broad categories: 2004 selective and 2004/1997
nonselective. The typical functional test for neutralization
involves hemagglutination inhibition assays using whole virus
binding to red blood cells. Due to safety concerns, alternative
hemagglutination assays with recombinant protein and red blood
cells are preferred. In order to eliminate the need for whole
blood, the hemagglutinin binding inhibition assay can be preformed
on airway epithelial cells. The binding assay can be performed in
any configuration, including, without limitation, any flow
cytometric or cell ELISA (cELISA) based assays. Using cELISA is
advantageous in that it obviates the use of expensive flow
cytometry equipment and can provide for more automated clonal
assessment and greater data collection. On the other hand, flow
cytometry may provide greater sensitivity, consistency, and
speed.
[0191] H1 clones can be tested for binding to any H1 proteins,
including binding to the current 2004 H1 and, in parallel, for
binding to 1918 and 1976 proteins. The positive clones will fall
into two broad categories: 2004 selective and 2004 nonselective.
Once again it is critical to test for neutralization, using
methodologies similar to those described above.
[0192] Other HA proteins, such as H2, H3, H5, H6, H7, H8 and H9,
can be characterized in an analogous manner.
[0193] In one aspect, the antibodies of the present invention have
a binding affinity for an H2, H3, H5, H6, H7, H8, or H9 HA
containing influenza virus or an H1 HA containing influenza virus,
such as, for example, H1/H3, H1/H5, etc. Binding affinities of the
antibodies of the present invention can be determined by methods
known to those of skill in the art, for example by the Scatchard
analysis of Munson et al., Anal. Biochem., 107:220 (1980). In one
embodiment, the binding affinity of the antibody is from about
1.times.10.sup.-7 to about 1.times.10.sup.-13 M, from about
1.times.10.sup.-8 to about 1.times.10.sup.-12 M, or from about
1.times.10.sup.-9 to about 1.times.10.sup.-11 M. In other
embodiments, the binding affinity of the antibody is about
1.times.10.sup.-7 M, about 1.times.10.sup.-8 M, about
1.times.10.sup.-9 M, about 1.times.10.sup.-10 M, about
1.times.10.sup.-11 M, about 1.times.10.sup.-12 M, or about
1.times.10.sup.-13 M. For example, an antibody of the present
invention demonstrated a binding affinity of 13 .mu.M for an H5 HA
(Vietnam/1203/04) containing influenza virus (see antibodies from
survivor 2 in the Example below). Another antibody demonstrated a
binding affinity of single digit nM for an H5 HA (Vietnam/1203/04)
containing influenza virus (see antibodies from survivor 5 in the
Example below).
[0194] Optimization
[0195] For the efficient management of influenza epidemics and
pandemics, including a potential pandemic associated with human
infections caused by an avian (H5) virus, antibodies that
effectively neutralize current isolates of the H proteins, such as
the H5 protein, as well as future mutations, are needed. In order
to achieve this goal, diverse H (e.g., H5) neutralizing clones need
to be identified that bind all known isolates of the targeted
hemagglutinin subtype(s).
[0196] If desired, cross-reactivity can be further improved by
methods known in the art, such as, for example, by Look Through
Mutagenesis (LTM), as described in US. Patent Application
Publication No. 20050136428, published Jun. 23, 2005, the entire
disclosure of which is hereby expressly incorporated by
reference.
[0197] Look-through mutagenesis (LTM) is a multidimensional
mutagenesis method that simultaneously assesses and optimizes
combinatorial mutations of selected amino acids. The process
focuses on a precise distribution within one or more
complementarity determining region (CDR) domains and explores the
synergistic contribution of amino acid side-chain chemistry. LTM
generates a positional series of single mutations within a CDR
where each wild type residue is systematically substituted by one
of a number of selected amino acids. Mutated CDRs are combined to
generate combinatorial single-chain variable fragment (scFv)
libraries of increasing complexity and size without becoming
prohibitive to the quantitative display of all variants. After
positive selection, clones with improved properties are sequenced,
and those beneficial mutations are mapped. To identify synergistic
mutations for improved HA binding properties, combinatorial
libraries (combinatorial beneficial mutations, CBMs) expressing all
beneficial permutations can be produced by mixed DNA probes,
positively selected, and analyzed to identify a panel of optimized
scFv candidates. The procedure can be performed in a similar manner
with Fv and other antibody libraries.
[0198] Mutagenesis can also be performed by walk-through
mutagenesis (WTM), as described above.
[0199] Another useful mutagenic method to intentionally design
cross-reactivity of the antibodies herein with more than one
influenza A subtype and/or more than one isolate of the same
subtype, is referred herein as "destinational" mutagenesis.
Destinational mutagenesis can be used to rationally engineer a
collection of antibodies based upon one or more antibody clones,
preferably of differing reactivities. In the context of the present
invention, destinational mutagenesis is used to encode single or
multiple residues defined by analogous positions on like sequences
such as those in the individual CDRs of antibodies. In this case,
these collections are generated using oligo degeneracy to capture
the range of residues found in the comparable positions. It is
expected that within this collection a continuum of specificities
will exist between or even beyond those of the parental clones. The
objective of destinational mutagenesis is to generate diverse
multifunctional antibody collections, or libraries, between two or
more discrete entities or collections. In the case of influenza
this method can be utilized to use two antibodies that recognize
two distinct epitopes, isolates, or subtypes and morph both
functional qualities into a single antibody. As an example, a first
influenza A antibody can be specific to a Vietnam isolate of the H5
subtype and a second antibody is specific to a Thailand or Turkish
isolate of the H5 subtype of the influenza A virus. To create a
destinational mutagenesis library, the CDR sequences for both
antibodies are first attained and aligned. Next all positions of
conserved identity are fixed with a single codon to the matched
residue. At non-conserved positions a degenerate codon is
incorporated to encode both residues. In some instances the
degenerate codon will only encode the two parental residues at this
position. However, in some instances additional co-products are
produced. The level of co-product production can be dialed in to
force co-product production or eliminate this production dependent
upon size limits or goals.
[0200] Thus, for example, if the first position of the two
antibodies respectively are threonine and alanine, the degenerate
codon with A/G-C- in the first two positions would only encode
threonine or alanine, irrespective of the base in the third
position. If, for example, the next position residues are lysine
and arginine the degenerate codon A-A/G-A/G will only encode lysine
or arginine. However, if the degenerate codon A/C-A/G-A/G/C/T were
used then asparagine, histidine, glutamine, and serine coproducts
will be generated as well.
[0201] As a convenience it is simpler to use only antibodies with
matched CDR lengths. One way to force this is to screen a size
restricted library for the second antigen, based on the CDR length
and potentially even framework restrictions imparted by the
initially discovered antibody. It is noted, however, that using
CDRs of equal length is only a convenience and not a requirement.
It is easy to see that, while this method will be useful to create
large functionally diverse libraries of influenza A virus
neutralizing antibodies, its applicability is much broader. This
mutagenesis technique can be used to produce functionally diverse
libraries or collections of any antibody (see U.S. Application
Publication No. 20080014205, published on Jan. 17, 2008 and
incorporated herein by reference in its entirety). Thus, FIG. 5 is
included herein to illustrate the use of the destinational
mutagenesis method using CDRs of a TNF-.alpha. antibody and a CD11a
antibody as the parental sequences mutagenized.
[0202] Other exemplary mutagenesis methods include targeted random
mutagenesis, saturation mutagenesis and error prone PCR.
[0203] Targeted random mutagenesis (Matteuchi and Heyneker, Nucleic
Acids Research 11: 3113-3121 (1983)) using ambiguously synthesized
oligonucleotides is a technique that generates an intended codon as
well as all possible codons at specific ratios, with respect to
each other, at designated positions. Ambiguously synthesized
oligonucleotides result in the reduced accuracy of nucleotide
addition by the specific addition of non "wild type" bases at
designated positions, or codons. This is typically performed by
fixing the ratios of wild type and non wild type bases in the
oligonucleotide synthesizer and designating the mixture of the two
reagents at the time of synthesis.
[0204] Saturation mutagenesis (Hayashi et al., Biotechniques
17:310-315 (1994)) is a technique in which all 20 amino acids are
substituted in a particular position in a protein and clones
corresponding to each variant are assayed for a particular
phenotype. (See, also U.S. Pat. Nos. 6,171,820; 6,358,709 and
6,361,974.)
[0205] Error prone PCR (Leung et al., Technique 1:11-15 (1989);
Cadwell and Joyce, PCR Method Applic. 2:28-33 (1992)) is a modified
polymerase chain reaction (PCR) technique introducing random point
mutations into cloned genes. The resulting PCR products can be
cloned to produce random mutant libraries or transcribed directly
if a T7 promoter is incorporated within the appropriate PCR
primer.
[0206] Other mutagenesis techniques are also well known and
described, for example, in In Vitro Mutagenesis Protocols, J.
Braman, Ed., Humana Press, 2001.
[0207] In the present case, one of the main goals is to engineer an
antibody (or antibodies) to effectively treat current H5 (or H7 or
H9) isolates as well as future mutations. To engineer an antibody
with tolerances capable of recognizing mutations in new isolates H5
neutralizing clones that bind a variety of H5 isolates, including,
for example, both recent 2004 isolates and previous 1997 isolates
are to be identified. It is expected that if a clone is selected on
a 2004 isolate it will bind/neutralize a 1997 isolate to a lesser
degree. In this case the goal is to improve 1997 recognition
dramatically within the context of improving (or at least
maintaining) 2004 isolate binding. Therefore, selection is first
done for improvements on 1997 reference protein followed by
selection on the 2004 protein. Doing so provides a greater
selective pressure on the new strain, while maintaining pressure on
the second parameter.
[0208] Optimization can be based on any of the libraries discussed
above, or any other types of libraries known in the art, alone or
in any combination. In a particular embodiment, optimization can
begin by screening three types of LTM libraries; triple mutagenized
light chain library, triple mutagenized heavy chain library, and
hextuple mutagenized (light+heavy chain) library. H5 is panned
essentially as described above, although minor modifications might
be desirable. For example, prior to glycine-HCl elution one can
select for improved binding by increasing washing stringencies at
each round by either or both of the following methods: extensive
washing at RT or 37 degrees, or prolonged incubation in presence of
excess soluble parent scFv. These selection modifications should
improve off-rate kinetics in the resulting clones. After 3-4 rounds
of selection we will sequence random clones and test for binding by
ELISA. Following sequence analysis of the improved clones, all the
allowable improved mutations are combined into a combinatorial
beneficial mutagenesis (CBM) library to select for synergistic
improvements to binding of both subtype H5 isolates. The CBM
library is made by synthesizing degenerate oligo nucleotides to
represent all improved and original parental residues at all
positions. The resulting library is selected under increasing
stringencies, similarly to LTM screening. Following sufficient
selection the pool is subcloned into a pBAD expression vector to
express and purify monomeric scFv protein from E. coli for binding
and neutralization assays, described above.
[0209] H1 neutralizing antibodies can be optimized in an analogous
manner. In this case one can select and optimize using any
reference protein sequences from 1918, 1976, and current as either
a starting point or destination.
[0210] In addition, intertype recognition is tested with the
neutralizing antibody clones. An example of intertype recognition
is coincidental or engineered H1 binding from an H5 sourced or
optimized clone.
[0211] The handling of antibody libraries, such as libraries from
various donors or characterized by reactivity to different isolates
of subtypes of a virus, including but not limited to influenza
viruses, can be greatly facilitated by applying unique barcodes
distinguishing the various antibody collections. The barcodes
preferably are selected such that they are capable of propagating
along with the clone(s) labeled.
[0212] Thus the barcodes can be non-coding DNA sequences of about
1-24 non-coding nucleotides in length that can be deconvoluted by
sequencing or specific PCR primers. This way, a collection of
nucleic acids, such as an antibody repertoire, can be linked at the
cloning step.
[0213] In another example, the barcodes are coding sequences of
silent mutations. If the libraries utilize restriction enzymes that
recognize interrupted palidromes (e.g. Sfi GGCCNNNNNGGCC), distinct
nucleotides can be incorporated in place of the "N's" to
distinguish various collections of clones, such as antibody
libraries. This barcoding approach has the advantage that the
repertoire is linked at the amplification step.
[0214] In a different example, the barcodes are coding sequences
that encode immunologically distinct peptide or protein sequences
fused to phage particles. Examples include, for example, epitope
(e.g. Myc, HA, FLAG) fusions to pIII, pVIII, pVII, or pIX phages.
The epitopes can be used singly or in various combinations, and can
be provided in cis (on the library-encoding plasmid) or in trans
(specifically modified helper phage) configuration.
[0215] Other examples of possible barcodes include, without
limitation, chemical and enzymatic phage modifications (for phage
libraries) with haptens or fluorescent chromophores. Such tags are
preferred for a single round of selection.
[0216] While barcoding is illustrated herein for distinguishing
antibody libraries, one of ordinary skill will appreciate that the
described approaches are broadly applicable for uniquely labeling
and distinguishing nucleic acid molecules and collections of
nucleic acids in general.
[0217] Epitope Mapping of Neutralizing Antibodies
[0218] Once neutralizing antibodies with the desired properties
have been identified, it might be desirable to identify the
dominant epitope or epitopes recognized by the majority of such
antibodies. Methods for epitope mapping are well known in the art
and are disclosed, for example, in Morris, Glenn E., Epitope
Mapping Protocols, Totowa, N. J. ed., Humana Press, 1996; and
Epitope Mapping: A Practical Approach, Westwood and Hay, eds.,
Oxford University Press, 2001.
[0219] Epitope mapping concerns the identification of the epitope
to which an antibody binds. There are many methods known to those
of skill in the art for determining the location of epitopes on
proteins, including crystallography analysis of the
antibody-antigen complex, competition assays, gene fragment
expression assays, and synthetic peptide-based assays (see for
example, in Chapter 11 of Harlow and Lane, Using Antibodies, a
Laboratory Manual, Cold Spring Harbor Laboratory Press, Cold Spring
Harbor, N.Y., 1999; U.S. Pat. No. 7,332,579, each of which is
incorporated herein by reference in its entirety). An antibody
binds "essentially the same epitope" as a reference antibody, when
the two antibodies recognize epitopes that are identical or
sterically overlapping epitopes. A commonly used method for
determining whether two antibodies bind to identical or sterically
overlapping epitopes is the competition assay, which can be
configured in all number of different formats, using either labeled
antigen or labeled antibody. Usually, an antigen is immobilized on
a 96-well plate, and the ability of unlabeled antibodies to block
the binding of labeled antibodies is measured using radioactive or
enzyme labels.
[0220] Production of Neutralizing Antibodies
[0221] Once antibodies with the desired neutralizing properties are
identified, such antibodies, including antibody fragments can be
produced by methods well known in the art, including, for example,
hybridoma techniques or recombinant DNA technology.
[0222] In the hybridoma method, a mouse or other appropriate host
animal, such as a hamster, is immunized to elicit lymphocytes that
produce or are capable of producing antibodies that will
specifically bind to the protein used for immunization.
Alternatively, lymphocytes may be immunized in vitro. Lymphocytes
then are fused with myeloma cells using a suitable fusing agent,
such as polyethylene glycol, to form a hybridoma cell (Goding,
Monoclonal Antibodies: Principles and Practice, pp. 59-103
(Academic Press, 1986)).
[0223] The hybridoma cells thus prepared are seeded and grown in a
suitable culture medium that preferably contains one or more
substances that inhibit the growth or survival of the unfused,
parental myeloma cells. For example, if the parental myeloma cells
lack the enzyme hypoxanthine guanine phosphoribosyl transferase
(HGPRT or HPRT), the culture medium for the hybridomas typically
will include hypoxanthine, aminopterin, and thymidine (HAT medium),
which substances prevent the growth of HGPRT-deficient cells.
[0224] Preferred myeloma cells are those that fuse efficiently,
support stable high-level production of antibody by the selected
antibody-producing cells, and are sensitive to a medium such as HAT
medium. Among these cell lines, preferred myeloma cell lines are
murine myeloma lines, such as those derived from MOPC-21 and MPC-11
mouse tumors available from the Salk Institute Cell Distribution
Center, San Diego, Calif. USA, and SP-2 or X63-Ag8-653 cells
available from the American Type Culture Collection, Rockville, Md.
USA. Human myeloma and mouse-human heteromyeloma cell lines also
have been described for the production of human monoclonal
antibodies (Kozbor, J. Immunol. 133:3001 (1984); and Brodeur et
al., Monoclonal Antibody Production Techniques and Applications,
pp. 51-63 (Marcel Dekker, Inc., New York, 1987)).
[0225] Culture medium in which hybridoma cells are growing is
assayed for production of monoclonal antibodies directed against
the antigen. Preferably, the binding specificity of monoclonal
antibodies produced by hybridoma cells is determined by
immunoprecipitation or by an in vitro binding assay, such as
radioimmunoassay, (RIA) or enzyme-linked immunoabsorbent assay
(ELISA).
[0226] Recombinant monoclonal antibodies can, for example, be
produced by isolating the DNA encoding the required antibody chains
and co-transfecting a recombinant host cell with the coding
sequences for co-expression, using well known recombinant
expression vectors. Recombinant host cells can be prokaryotic and
eukaryotic cells, such as those described above.
[0227] The choice of human variable domains, both light and heavy,
to be used in making the humanized antibodies is very important to
reduce antigenicity. According to the so-called "best-fit" method,
the sequence of the variable domain of a rodent antibody is
screened against the entire library of known human variable-domain
sequences. The human sequence which is closest to that of the
rodent is then accepted as the human framework region (FR) for the
humanized antibody (Sims et al., J. Immunol. 151:2296 (1993);
Chothia et al., J. Mol. Biol. 196:901 (1987)). It is important that
antibodies be humanized with retention of high affinity for the
antigen and other favorable biological properties. To achieve this
goal, according to a preferred method, humanized antibodies are
prepared by a process of analysis of the parental sequences and
various conceptual humanized products using three-dimensional
models of the parental and humanized sequences.
[0228] In addition, human antibodies can be generated following
methods known in the art. For example, transgenic animals (e.g.,
mice) can be made that are capable, upon immunization, of producing
a full repertoire of human antibodies in the absence of endogenous
immunoglobulin production. See, e.g., Jakobovits et al., Proc.
Natl. Acad. Sci. USA 90:2551 (1993); Jakobovits et al., Nature
362:255-258 (1993); Bruggermann et al., Year in Immuno. 7:33
(1993); and U.S. Pat. Nos. 5,591,669, 5,589,369 and 5,545,807.
[0229] Neutralizing Antibodies
[0230] A number of neutralizing antibodies have been identified
through the use of the techniques described herein, including those
described in the Examples below. In one aspect, the present
invention provides neutralizing antibodies that bind to a
hemagglutinin protein epitope. In one embodiment, the neutralizing
antibody binds to at least one epitope on the HA1 subunit of the
hemagglutinin protein. In another embodiment, the neutralizing
antibody binds to at least two, at least three, at least four, at
least five, or at least six epitopes on the HA1 subunit of the
hemagglutinin protein. In a preferred embodiment, the neutralizing
antibody of the present invention binds to an epitope that is
substantially the same as the epitope for (i) an antibody
comprising a heavy chain amino acid sequence shown as SEQ ID NO: 4
and a light chain amino acid sequence shown as SEQ ID NO:71
(antibody 1 in the Example below and as shown in Table 1); (ii) an
antibody comprising a heavy chain amino acid sequence shown as SEQ
ID NO:45 and a light chain amino acid sequence shown as SEQ ID
NO:140 (antibody 2 in the Example below and as shown in Table 1);
(iii) an antibody comprising a heavy chain amino acid sequence
shown as SEQ ID NO:9 and a light chain amino acid sequence shown as
SEQ ID NO:81 (antibody 3 in the Examples below and as shown in
Table 1); (iv) an antibody comprising a heavy chain amino acid
sequence shown as SEQ ID NO:61 and a light chain amino acid
sequence shown as SEQ ID NO:158 (antibody 4 in the Example below
and as shown in Table 1); or (v) an antibody comprising a heavy
chain amino acid sequence shown as SEQ ID NO:61 and a light chain
amino acid sequence shown as SEQ ID NO:159 (antibody 5 in Table 1);
(vi) an antibody comprising a heavy chain amino acid sequence shown
as SEQ ID NO:61 and a light chain amino acid sequence shown as SEQ
ID NO:160 (antibody 6 in Table 1). This is summarized in Table 1
below.
[0231] In some embodiments, the antibodies of the present invention
neutralize viruses containing H5 and/or H1. In other embodiments,
the antibodies neutralize both H5 and H1. In one embodiment, the
antibodies of the present invention do not prevent
hemagglutination. In other embodiments, the antibodies do not
prevent the binding of an influenza A virus to a target cell to be
infected. In another embodiment, the anti-hemagglutinin antibody
does not prevent the receptor binding site on the globular head
region of the HA of an influenza A virus from attaching to a target
cell to allow hemagglutinin activity of HA to occur.
TABLE-US-00001 TABLE 1 Neutralizing Abs Antibody Heavy chain SEQ ID
NO: Light chain SEQ ID NO: Neutralizes 1
QVQLVQSGAEVKKPGSSVRVSCKTSGGTFSSYAVTWV
ETTLTQSPGTLSLSPGERATLSCRVSQSVSSNYLAWYQQKP H5, H1
RQAPGQGLEWMGGIIGMFGTTNYAQKFQGRLTITADE
GQAPRLLIYGASSRATGIPDRFSGSGSGTDFTLTISRLEPE
MTSTAYMELSSLRSEDTAVYYCARGSYYYETTLDYW DFAVYYCQQYGTSPRAFGHGTKVEIKRTV
GRGTL (SEQ ID NO: 71) (SEQ ID NO: 4) 2
QVQLVQSGAEVKKPGSSVKVSCKTSGGTFSSYAVTWV
ETTLTQSPATLSVSPGERATLSCRASQSVSRNLAWYQQKPG H5, H1
RQAPGQGLEWMGGIIGMFGTRNYAQKFQGRVTITADE
QAPRLLIYGASSRATGIPDRFSGSGSGTDFTLTISRLEPED
LTSTAYMELSSLRSEDTAVYYCARGSYYYESSLDYWG FAVYYCQQYGSSSITFGQGTRLEIKRTV
RGTL (SEQ ID NO: 140) (SEQ ID NO: 45) 3
EVQLVQSGAEVKKPGSSVKVSCKTSGGTFSSYAVTWV
QSVLTQPPSVSGAPGQRVTISCGGSRSNIGAGYDVHWYQQ H5, H1
RQAPGQGLEWMGAIIGMFGTTNYAQKEQGRVTITADE
FPGTAPKVVIYGNNNRPSGVPDRFSGSKSGTSASLAITGL
LTSTAYMELSSLRSDDTAVYYCARGSYYYESSLDYWG
QAEDEANYYCQSYDTNLGGSIFGGGTQVTVL KGTL (SEQ ID NO: 81) (SEQ ID NO: 9)
4 QVQLQESGPGLVKPSETLSLTCTVSGYSFDSGYYWGW
NFMLTQPHSVSESPGKTVTISCTGSGGNIARNYVQWYQQR H5
LRQPPGKGLEWIGSIYHSRNTYYNPSLKSRVTISVDT
PGSAPVTVILEDDKRPSGIPDRFSGSIDRSSNSASLTISG
SKNQFSLQLSSVTAADTAVYYCARGTWYSSNLRYWFD LRTEDEALYYCQSYDDSDLVVFGGGTKLT
PWGKGTL (SEQ ID NO: 158) (SEQ ID NO: 61) 5
QVQLQESGPGLVKPSETLSLTCTVSGYSFDSGYYWGW
NFMLTQPHSVSESPGKTVTISCTGSSGSIASNYVQWYQQRP H5
LRQPPGKGLEWIGSIYHSRNTYYNPSLKSRVTISVDT
GSAPTTVIYEDYQRPSGVPDRFSGSIDSSSNSASLTISGLK
SKNQFSLQLSSVTAADTAVYYCARGTWYSSNLRYWFD TEDEADYYCQSYDDSDHLIFGGGTKLTVL
PWGKGTL (SEQ ID NO: 159) (SEQ ID NO: 61) 6
QVQLQESGPGLVKPSETLSLTCTVSGYSFDSGYYWGW
QSVLTQPPSASGTPGQRVTJSCSGSSSNIGSNTVNWYKQLP H5
LRQPPGKGLEWIGSIYHSRNTYYNPSLKSRVTISVDT
GTAPRLLIYSNDQRPSGVPDRFSGSKSGTSASLAISGLQSE
SKNQFSLQLSSVTAADTAVYYCARGTWYSSNLRYWFD DEANYYCAAWDDSLSGWVFGGGTKLTVL
PWGKGTL (SEQ ID NO: 160) (SEQ ID NO: 61)
[0232] Based on the experiments described in the Examples below, a
number of H5 anti-hemagglutinin antibody heavy chain/light chain
pairings were identified. As shown in Table 2, column 1 provides
the heavy chain amino acid sequence, column 2 provides the
corresponding SEQ ID NO: for the heavy chain sequence, column 3
provides the amino acid sequence for those light chains that pair
with the heavy chains in the same row, and column 4 provides the
corresponding SEQ ID NOS: for the light chain sequence. For
example, the heavy chain sequence shown as SEQ ID NO:1 pairs with
the light chain sequence shown as SEQ ID NO:68, SEQ ID NO:2 pairs
with SEQ ID NO:69, etc. In some embodiments, a heavy chain can pair
with more than one light chain. For example, the heavy chain
sequence shown as SEQ ID NO:6 pairs with either the light chain
sequence shown as SEQ ID NO:74 or the light chain sequence shown as
SEQ ID NO:75; or the heavy chain sequence shown as SEQ ID NO:7
pairs with one of (i) the light chain sequence shown as SEQ ID
NO:75, (ii) the light chain sequence shown as SEQ ID NO:76, or
(iii) the light chain sequence shown as SEQ ID NO:77.
[0233] In one embodiment, the neutralizing antibodies of the
present invention contain at least one heavy chain polypeptide
containing an amino acid sequence shown in Table 2, and/or at least
one light chain polypeptide containing an amino acid sequence shown
in Table 2.
TABLE-US-00002 TABLE 2 SEQ SEQ ID ID Heavy Chain Sequence NO Light
Chain Sequence NO EVQLVQSGTEVKKPGSSVKLS 1 QSALTQPASVSGSPGQSITISC 68
CKASGGTFSSYAVTWVRQAPG TGTSSDFGGSNHVSWYQQHPGK QGLEWMGGIIGMFGTTNYAQK
APKLIIYDVSDRPSGVSNRFSG FQGRLTITADEMTSTAYMELS SKSGNTASLTVSGLQAEDEAHY
SLRSEDTAVYYCARGSYYYET YCSSYAGSNNFVFGTGTKVTVL TLDYWGRGTM
EVQLVQSGAEVKKPGSSVRVS 2 ETTLTQSPGTLSLSPGERATLS 69
CKTSGGTFSSYAVTWVRQAPG CRASQTVSSSYLAWYRQKPGQA QGLEWMGGIIGMFGTTNYAQK
PRLLIYGTSSRATGIPDRFSGS FQGRLTITADEMTSTAYMELS GSGTDFTLTISRLEPEDFAVYY
SLRSEDTAVYYCARGSYYYET CQQYGNSRVTFGPGTKVDIKRT TLDYWGRGTM V
QVQLQQSGAEVKK2GSSVRVS 3 EIVMTQSPGTLSLSPGERATLS 70
CKTSGGTFSSYAVTWVRQAPG CRASQSLSGSNVAWYQQKFGQA QGLEWMGGIIGMFGTTNYAQK
PRLLIHGASKRAAGIPDRFSGS FQGRLTITADEMTSTAYMELS GSGTDFTLTISRLQPDDYAVYY
SLRSEDTAVYYCARGSYYYET CQQYGTKPFTFGQGSKLEIKRT TLDYWGQGTM V
QVQLVQSGAEVKKPGSSVRVS 4 ETTLTQSPGTLSLSPGERATLS 71
CKTSGGTFSSYAVTWVRQAPG CRVSQSVSSNYLAWYQQKPGQA QGLEWMGGIIGMFGTTNYAQK
PRLLIYGASSRATGIPDRFSGS FQGRLTITADEMTSTAYMELS GSGTDFTLTISRLEPEDFAVYY
SLRSEDTAVYYCARGSYYYET CQQYGTSPRAFGHGTKVEIKRT TLDYWGRGTL V
QVQLQQSGAEVKKPGSSVRVS 5 QSALTQPPSASGSPGQSVTISC 72
CKTSGGTFSSYAVTWVRQAPG TGASSDIGGYKSVSWYQQHPGK QGLEWMGGIIGMFGTTNYAQK
APKLIIYDVTERPSGVPDRFSA FQGRLTITADEMTSTAYMELS SKSGNTASLTVSGLQAEDEADY
SLRSEDTAVYYCARGSYYYET YCSSYGGSNNLVVFGGGTKVTV TLDYWGKGTL L
DIQMTQSPSSVSASVGDRVTIT 73 SWLAWYQQKPGKAPKLLIYAAS
TLQRGVPSRFSGSGSGTDFTLT INSLQPEDFATYYCQQYNSYPL TFGGGTKVEIKR
EVQLVQSGAEVKKPGSSVRVS 6 EIVLTQSPGTLSLSPGERATLS 74
CKTSGGTFSSYAVTWVRQAPG CRASQSVSNNYLAWYQQKPGQA QGLEWMGGIIGMFGTTNYAQK
PRLLIYGASSRATGIPDRFSGS FQGRLTITADEMTSTAYMELS GSGTDFTLTISRLEPEDFAVYY
SLRSEDTAVYYCARGSYYYET CQQYGRSPRTFGQGTKVEIKRT TLDYWGKGTL
ETTLTQSPGTLSLSPGERATLS 75 CRASQSLGGANLGWYQQKFGQP
PRLLIYGASSRATGVPDRESGS GSGTDFALTISRLEPEDFAVYY
CQQYGSKPYTFGQGTKLEIKRT V EVQLVQSGAEVKKPGSSVRVS 7
ETTLTQSPGTLSLSPGERATLS 75 CKTSGGTFSSYAVTWVRQAPG
CRASQSLGGANLGWYQQKFGQP QGLEWMGGIIGMFGTTNYAQK PRLLIYGASSRATGVPDRFSGS
FQGRLTITADEMTSTAYMELS GSGTDFALTISRLEPEDFAVYY SLRSEDTAVYYCARGSYYYET
CQQYGSKPYTFGQGTKLEIKRT TLDYWGQGTL V ETTLTQSPATLSVSPGERATLS 76
CRASQSVSTNLAWYQQKPGQAP RLLIHGASTRATGIPARESGSG
SGTEFTLTISSLQSEDSAVYYC QQHNNWPPVTFGRGTKVEIKRT V
ETTLTQSPATLSVSPGERATLS 77 CRASQSVSRNLAWYQQKPGQAP
RLLIYGASSRATGIPDRFSGSG SGTDFTLTISRLEPEDFAVYYC
QQYGSSSITFGQGTRLEIKRTV V EVQLVQSGAEVKKPGSSVKVS 8
QSVLTQPPSASGAPGQRVTISC 78 CKTSGGTFSSYAVTWVRQAPG
TGSSSNIGAGYDVHWYQQLPGR QGLEWMGAIIGMFGTTNYAQK APKLLIYGNSNRPSGVPARFSG
FQGRVTLTADELTSTAYMELS SKSATSASLAITGLQAEDEADY SLRSEDTAVYYCARGSYYYES
YCQSYDSSLSGVVFGGGTKLTV SLDYWGRGTL L EIVMTQSPATLSVSPGERAILS 79
CRASRSVSTNLAWYQQKPGQAP RLLIYGASTRATGIPDRFSGSG
SGTDFTLTISRLEPEDFAVYYC QQYGSSPNFGGGTKVEIKR EVQLVQSGAEVKKPGSSVKVS 9
EIVMTQSPGTLSLSPGERATLS 80 CKTSGGTFSSYAVTWVRQAPG
CRASQSVPNRYIAWYQQKPGQA QGLEWMGAIIGMFGTTNYAQK PRLLTYGASSRATGIPDRFSGS
FQGRVTITADELTSTAYMELS GSGPDFTLTISRLEPEDFAVYY SLRSDDTAVYYCARGSYYYES
CQQYGRSPQTFGQGTKLETKGT SLDYWGKGTL V QSVLTQPPSVSGAPGQRVTISC 81
GGSRSNIGAGYDVHWYQQFPGT APKVVIYGNNNRPSGVPDRESG
SKSGTSASLAITGLQAEDEANY YCQSYDTNLGGSIEGGGTQVTV L
EIVMTQSPGTLSVSPGDAATLS 82 CRASRNINNNLAWYQQTPGQAP
RLLIYGASTRATGLPARFTGSG SGTEFTLTISSLQSEDFAVYYC QQYNNWPRTPGQGTKVEIKR
EVQLVQSGTEVKKPGSSVKVS 10 ETTLTQSPGTLSLSPGERATLS 83
CKVSGGTFSSYAVTWVRQAPG CRASQIVDSSYLAWYQHRPGQA QGLEWMGAIIGMFGTTNYAQK
PRILIYGASSRAPGVPDRFSGS FQGRVTITADELTSTAYMELS GSGTDFTLTISRLEPEDFAVYY
SLRSDDTAVYYCARGSYYYES CQQYAVSPRTFGQGTKVEIKRT SLDYWGGGTT V
EVQLVQSGAEVKKPGSSVKVS 11 EIVLTQSPGTLSLSPGDRATLS 84
CKTSGGTFSSYAVTWVRQAPG CRASQSLGTNYLAWYQQKPGQA QGLEWMGAIIGMFGTTNYAQK
PRLLIYGASSRATGIPDRFSGS FQGRVTITADELTSTAYMELS GSGTDFTLIISRLEPEDFAVYY
SLRSDDTAVYYCARGSYYYES CQQYGRSPQKFGQGTKVEIKRT SLDYWGQGTL V
QSVLTQPPSASGTPGQRVTISC 85 SGSSSNIGSNYVYWYQQLPGTA
PKLLIYRINNQRPSGVPDRFSG SKSGTSASLATSGLRSEDEANY
YCAAWDDSLSGWVFGGGTKLTV L EIVMTQSPATLSVSPGERAILS 86
CRASRSVSTNLAWYQQKPGQAP RLLTYGASTRATGIPDRFSGSG
SGTDFTLTISRLEPEDFAVYYC QQYGSSPNFGGGTKVEIKR EVQLVQSGAEVKKPGSSVKVS 12
DIQMTQSPSSVSASVGDRVTTT 87 CKTSGGTESSYAVTWVRQAPG
CRASQGISSWLAWYQQKPGKAP QGLEWMGAIIGMFGTTNYAQK KLLIYAASSLQSGVPSRFSGSG
FQGRVTITADELTSTAYMELS SGTDFTLTISSLQPEDVATYYC SLRSDDTAVYYCARGSYYYES
QKYNSAPRTFGQGTKVEIKR SLDYWGQGTM QVQLVQSGAEVKKPGSSVKVS 13
DIQLTQSPSSLSASVGDRVTIT 88 CKTSGGTFSSYAVTWVRQAPG
CRASQSISNYLNWYQQKPGKAP QGLEWMGAIIGMFGTTNYAQK KLLIYAASSLQSGVPSRESGSG
FQGRVTITADELTSTAYMELS SGTDFTLTISSLQPEDSATYYC SLRSDDTAVYYCARGSYYYES
QQSHSTPRTFGQGTKLEIKRTV SLDYWGQGTM QAVLTQPPSASGTPGQRVTISC 89
SGSSSNIGTNTVNWYQQLPGTA PKLLIYRNIQRPSGVPDRFSGS
KSGTSASLAISGLQSEDEADYY CAAWDDSLNGYVFGTGTKLTVL EVQLVQSGAEVKKPGSSVKVS
14 EIVLTQSPGTLSLSPGEKATLS 90 CKTTGGTFSSYAVTWVRQAPG
CRASQSVSNTYLAWYQQKPGQA QGLEWMGGIIGMFGTTNYAQK PRLLLYGASSRAPGIPDRFSGS
FQGRVTITADEMTSTAYMELS GSGTDFTLTISRLEAEDFAVYF SLRSEDTAVYYCARGSYYYES
CQQYAGSPRTFGQGTKVEIKRT SLDYWGQGTL V EIVLTQSPGSLSLSPGERATLS 91
CRASQSVSHGYLAWYQQKPGQA PRFLIYGASSRPTGIPDRFRGS
GSGTDFTLTISSLEPEDSAVYY CQQYSTSPLTFGGGTKVETKRT V
PELTQPPSASGTPGQRVTISCS 92 GSSSNIGSNYVYWYQQLPGTAP
KLLIYRNNQRPSGVPDRFSGSK SGTSASLAISGLRSEDEADYYC AAWDDSLSGWVFGGGTKLTVL
EVQLVQSGAEVKKPGSSVKVS 15 ETTLTQSPGTLSLSPGERATLS 93
CKTTGGTFSSYAVTWVRQAPG CRASQIVDSSYLAWYQHRPGQA QGLEWMGGIIGMFGTTNYAQK
PRILIYGASSRAPGVPDRFSGS FQGRVTITADEMTSTAYMELS GSGTDFTLTISRLEPEDFAVYY
SLRSEDTAVYYCARGSYYYES CQQYGSPPRTFGQGTKVEIKRT SLDYWGQGTM V
EVQLVQSGAEVKKPGSSVKVS 16 QSVLTQPPSTSGTPGQRVTISC 94
CKTSGGTFSSYAVTWVRQAPG SGSSSNIGRKTVNWYQQLPGTA QGLEWMGAIIGMFGTTNYAQK
PKLLIYNDNQRPSGVPDRFSGS FQGRVTLTADELTSTAYMELS KAGTSASLAISGLQSEDEADYY
SLRSEDTAVYYCARGSYYYES CAVWDDSLNAWVFGGGTKLTVL SLDYWGKGTL
EVQLVQSGAEVKKPGSSVKVS 17 DVVMTQSPLSLPVTPGEPAAIS 95
CKTSGGTFSSYAVTWVRQAPG CRSSQSLLHSDGNNYLDWYLQK QGLEWMGAIIGMFGTTNYAQK
PGQSPHLLIYLGSNRASGVPDR FQGRVTLTADELTSTAYMELS FSGSGSGTDFELKISRVEAEDA
SLRSEDTAVYYCARGSYYYES GVYYCMQASQTPRTFGQGTKLE SLDYWGKGTM LKRTV
EVQLVQSGAEVKKPGASVKVS 18 HVILTQPPSVSVAPGMTARMTC 96
CKASGGAFSSYAVTWVRQAPG GGDNVGRRNVHWYQQKPGQAPV QGLEWMGGIIGMFGTTNYAQK
LVVYDDGGRPSAIPARFSGSKS FQGRVTITADELTSTAYMELS GNTATLIISRVEAGDEADYYCQ
SLRSEDTAVYYCARGSYYYES MWHSSGDQWVFGGGTKLTVL SLDYWGQGTM
EVQLVQSGAEVKKPGSSVKVS 19 ETTLTQSPGTLSLSPGERATLS 97
CKTSGGTFSSYAVTWVRQAPG CRASQSISPNYLAWYQQRPGQA QGLEWMGGIIGMFGTTNYAQK
PRLLIYGASKRATGIPDRFSGS FQGRVTMTADEMTSTAYMELS GSGTDFTLTISSLEPEDSAVYY
SLRSEDTAVYYCARGSYYYES CQHQGFGQGTKVEIKRTV SLDYWGKGTL
EVQLVQSGAEVKKPGSSVKVS 20 QSVLTQPPSASGTPDQRVTISC 98
CKTSGGTFSSYAVTWVRQAPG SGSGSNIGSNYVYWYQQLPGAA QGLEWMGGIIGMFGTTNYAQK
PKLLMSRNNQRPSGVPDRFSGS FQGRVTITADEMTSTAYMELS KSGTSASLAISGLQSEDEADYY
SLRSEDTAVYYCARGSYYYES CAAWDDSLTGYVFGTGTKLTVL SLDYWGRGTL
EVQLVQSGAEVKKPGSSVKVS 21 SYVLTQPPSVSVAPGKTARITC 99
CKTSGGTFSSYAVTWVRQAPG GGKNIGSKSVHWYQQKSGQAPV QGLEWMGGIIGMFGTTNYAQK
LVIYGDSDRPSGIPERFSGSNS FQGRVTITADEMTSTAYMELS GNTATLTISGVEAEDEADYYCQ
SLRSEDTAVYYCARGSYYYES VWDNTSDHAGVFGGGTKVTVL SLDYWGRGTM
EVQLVQSGAEVKKPGSSVKVS 22 QSVLTQPPSASGTPGQRVTISC 100
CKTSGGTFSSYAVTWVRQAPG SGSSSNIGSNYVYWYQQLPGTA QGLEWMGAIIGMFGTTNYAQK
PKLLIYRNNQRSSGVPDRFSGS FQGRVTLTADELTSTAYMELS KSGTSASLAISGLRSEDEADYY
SLRSEDTAVYYCARGSYYYES CAAWDDSLSGLVFGGGTKLTVL SLDYWGQGTL
QSVLTQPPSVSAAPRQSVTISC 101 SGTTSNIGNNPVSWYQQFPGRA
PNLLIYYNDVVPSGVSDRFSAS KAGTSASLAISRLQSEDEADYY
CATWDDSLSAWVFGGGTQLTVL QAVLTQPPSASGTPGQRVTISC 102
SGSSSNIGSNYVYWYQQLPGTA PKLLIYSNNQRPSGVPDRFSGS
KSGTSASLVISGLQSEDETDYY CAAWDDSLNGWVFGGGTKLTVL EVQLVQSGAEVQKPGSSVKVS
23 QSALTQPPSASGSPGQSVTITC 103 CKTSGGTFSSYAVTWVRQAPG
AGASSDLGDYKSVSWYQQHPGK QGLEWMGAIIGMFGTTNYAQK APKLIIYDVIKRPAGVPDRFSA
FQGRVTLTADELTSTAYMELS SKSGNTASLTVSGLQAEDEADY SLRSEDTAVYYCARGSYYYES
YCSSYAGSNNIVIEGGGTKLTV SLDYWGQGTL L EVQLVQSGAEVKKPGSSVKVS 24
ETTLTQSPGTLSLSPGERATLS 104 CKTSGGTFSSYAVTWVRQAPG
CRASQGIDRKYLAWYQRKHGQA QGLEWMGGIIGMFGTTNYAQK PRLLIYGASNRATGIPDRFSGS
FQGRVTITADELTSTAYMELS GSGTDFTLTISRLEPEDFAVYY SLRSEDTAVYYCARGSYYYES
CQQYADSFVSFGQGTKLEIKRT SLDYWGQGTL V EVQLVQSGAEVKKPGSSVKVS 25
DIQLTQSPSTLSASVGDRVTIT 105
CKASGGTFSSYAISWVRQAPG CRASQSISRWLAWYQQKPGKTP QGLEWMGAIIGMFGTTNYAQK
KLLIYEASNLQSGVPSRFSGSG FQGRVTITADELTSTAYMELS SGTEFTLTISSLQPDDFATYYC
SLRSDDTAVYYCARGSYYYES QQYKSDFLVTFGPGTKVDIKRT SLDYWGQGTL V
EVQLVQSGAEVKKPGSSVKVS 26 QSVLTQPPSVSGAPGQKITISC 106
CKTSGGTFSSYAVTWVRQAPG TGSSSNIGTGYDVHWYQQLPGR QGLEWMGAIIGMFGTTNYAQK
APRLLISADANRPSGVPDRFSA FQGRVTLTADQLTSTAYMELS SKSGTSASLAITGLQAEDEADY
SLRSEDTAVYYCARGSYYYES YCQSYDTRLGGSIFGGGTQLTV SLDYWGRGTL L
EVQLVQSGAEVKKPGSSVKVS 27 EIVMTQSPGTLSVSPGDAATLS 107
CKTSGGTFSSYAVTWVRQAPG CRASRNINNNLAWYQQTPGQAP QGLEWMGAIIGMFGTTNYAQK
RLLIYGASTRATGLPARFTGSG FQGRVTITADELTSTAYMELS SGTEFTLTISSLQSEDFAVYYC
SLRSDDTAVYYCARGSYYYES QQYNNWPRTFGQGTKVEIKR SLDYWGKGTL
EVQLVQSGAEVKKPGSSVKVS 28 QSVLTQPPSVSAAPGQEVTTTC 108
CKTSGGTFSSYAVTWVRQAPG SGSGANIGNNYVSWYQQVPGTA QGLEWMGAIIGMFGTTNYAQK
PKLVIYDNNRRPSGIPDRFSGS FQGRVTITADELTSTAYMELS KSGTSATLGITGLQTGDEADYY
SLRSDDTAVYYCARGSYYYES CGTWDSSLSAVVFGGGTKVTVL SLDYWGKGTT
EVQLVQSGAEVKKPGSSVKVS 29 QAVLTQPPSASGTPGQTVTISC 109
AVTWVRQAPGQGLEWMGGIIG SGVTSNIGNNYVYWYQQLPGTA MFGTTNYAQKFQGRVTITADE
PRLLIYSNNQRPSGVPDRFSGS LTSTAYMELSSLRSEDTAVYY KSGTSASLAISGLRSEDEADYY
CARGSYYYESSLDYWGKGTL CSAWDDSLRENLFGTGTQLTVL EVQLVQSGAEVKKPGSSVKVS
30 QAVLTQPPSASGTPGQRVTISC 110 CKTSGGTFSSYAVTWVRQAPG
SGSSSNFGMNAVNWYQQLPGTA QGLEWMGAIIGMFGTTNYAQK PKLLMYSNSKRPSGVPDRFSGS
FQGRVTLTADELTSTAYMELS KSGTSASLAISGLQSEDEADYY SLRSEDTAVYYCARGSYYYES
CSAWDDNLNGWVEGGGTKVTVL SLDYWGRGTM EVQLVQSGAEVKKPGSSVKVS 31
QAVLTQPPSASGTPGQRVTISC 111 CKTSGGTFSSYAVTWVRQAPG
SGSSSNIGSNTVNWYQQLPGTA QGLEWMGAIIGMFGTTNYAQK PKLLIYRNNQRPSGVPDRFSGS
FQGRVTITADELTSTAYMELS KSGTSASLAISGLQSEDEADYY SLRSDDTAVYYCARGSYYYES
CATWDDSLTSVVFGGGTKVTVL SLDYWGKGTMVTVS QMQLVQSGAEVKKPGSSVKVS 32
ETTLTQSPSSLSASIGDRITIA 112 CKTSGGTFSSYAVTWVRQAPG
CQASQDIRNRLNWYLQRPGKAP QGLEWMGAIIGMFGTTNYAQK QLLIYDASNLETGVPSKFAGRG
FQGRVTITADELTSTAYMELS SGTDFTLTISSLQPEDIGTYFC SLRSDDTAVYYCARGSYYYES
QQYGDLSPLTFGGGTKVDIRRT SLDYWGKGTM V QMQLVQSGAEVKKPGSSVKVS 33
ETTLTQSPSSLSASIGDRITIA 113 CKTSGGTFSSYAVTWVRQAPG
CQASQDIRNRLNWYLQRPGKAP QGLEWMGAITGMFGTTNYAQK QLLIYDASNLETGVPSKFAGRG
FQGRVTLTADELTSTAYMELS SGTDFTLTISSLQPEDIGTYFC SLRSEDTAVYYCARGSYYYES
QQYGDLSPLTFGGGTKVDIRRT SLDYWGQGTLVTVS V DIQMTQSPSSLSASVGDRVTIT 114
CRASQSISNYLNWYQQKPGKAP KLLIYAASSLQSGVPSRFSGSG
SGTDFTLTISSLQPEDSATYYC QQSHSTPRTFGQGTKLEIKRTV QVQLVQSGAEVKKPGSSVKVS
34 QSVLTQPPSVSGAPGQRVTISC 115 CKTTGGTFSSYAVTWVRQAPG
TGTSSNIGAGFDVHWYQQFPGT QGLEWMGGIIGMEGTTNYAQK APKLLIYDNVKRPSGVPDRFSG
FQGRVTITADEMTSTAYMELS SKSGTSASLAITGLRAEDEADY SLRSEDTAVYYCARGSYYYES
YCQSYDTSLSRYVEGTGTKVTV SLDYWGKGTM L QVQLVQSGAEVKKPGSSVKVS 35
ETTLTQSPGTLSLSPGERATLS 116 CKASGGTFSSYAVTWVRQAPG
CRASQSVSSIYLAWYQQKPGQA QGLEWMGAIIGMFGTTNYAQK PRLVIHGASSRATGIPDRFSGS
FQGRVTLTADELTSTAYMELS GSGTDFTLTISRLEPEDFAVYY SLRSEDTAVYYCARGSYYYES
CQQYGSSPLTFGGGTKVEIKRT SLDYWGQGTL V QVQLVQSGAEVKKPGSSVKVS 36
ETTLTQSPGILSLSPGESATLS 117 CKTSGGTFSSYAVTWVRQAPG
CGASQTISSRYLAWYQQRPGQA QGLEWMGAIIGMFGTTFNYAQ PRLLIFDASRRATGVPDRFSGG
KFQGRVTLTADELTSTAYMEL GSGTDFTLTISRLEPEDFGVFY SSLRSEDTAVYYCARGSYYYE
CQQYGISPYTFGQGTKLEIKRT SSLDYWGQGTL V EIVLTQSPGTLSLSPGERATLS 118
CRASQSVSNNLAWYQQKFGQAP RLLIYAASSRATDIPARFSGSG
SGTDFTLTISRLEPEDFAVYYC QQYVDSPRTFGQGTKVEIKRTV
EIVLTQSPGTLSLSPGERATLS 119 CRASQSVSSSYLAWYQQKPGQA
PRLLIYGASSRATGIPDRFSGS GSGTDFTLTISRLEPEDFAVYY
CQQYGSSPLTFGGGTKVEIKRT V EIVLTQSPGTLSLSPGERATLS 120
CRASQSVSSSYLAWYQQKPGQA PRLLIYGASSRAAGIPDRFSGS
GSGTDFTLTISRLEPEDFAVYY CQQYGSSPKTFGQGTKVEIKRT V
QSVLTQPPSVSGAPGQGVTISC 121 SGSSSNIGANYVVHWYRQLPGA
APKLLIYDDIHRPSGVPDRFSG SRSGTSASLAITGLQPEDEADY
YCQTYDTSLRGSVFGGGTKLTV L QSVLTQPPSASGTPGQRVTISC 122
SGSSSNIGSNYVYWYQQLPGTA PKLLIYRNNQRPSGVPDRFSGS
KSGTSASLAISGLRSEDEADYY CAAWDDSLSGWVFGGGTKVTVL
QAVLTQPPSVSVAPGKTATITC 123 GVNNLGRKSVHWYQQKPGQAPV
LVVYDSNDRPSGIPERFSGSNS GNTATLIISRVEAGDEADYSCQ VWDNNVDHPVFGGGTKLTVL
HVILTQPPSVSVAPGMTARMTC 97 GGDNVGRRNVHWYQQKPGQAPV
LVVYDDGGRPSAIPARFSGSKS GNTATLIISRVEAGDEADYYCQ MWHSSGDQWVFGGGTKLTVL
SSELTQDPAVSVALGQTVRITC 124 QGDSLRSYYASWYQQKPGQAPV
LVIYGKNNRPSGIPDRFSGSSS GNTASLTITGAQAEDEADYYCN SRDSSGNHWVFGGGTKVTVL
EIVMTQSPGTLSLSPGERATLS 125 CRASQSVSSSLAWYQQKPGQAP
RLLIYGASNRATGIPDRFSGSG SGTDFTLTISRLEPEDEAVYYC QQYGGSPRTFGQGTKLEIKR
EIVLTQSPGTLSLSPGERATLS 126 CRASQSVSSSYLAWYQQKPGQA
PRLLIYGASSRATGIPDRFSGS GSGTDFTLTISRLEPEDFAVYY CQQYGSSPNFGGGTKVDIKR
QAVLTQPPSASGTPGQRVTISC 127 SGSSSNIGSNSVYWYQQLPGTA
PKLLIYRINNQRPSGVPDRFSG SKSGTSASLAISGLQSEDEADY
YCAAWDDSLNGVVFGGGTKLTV L QSVVTQPPSVSGAPGQRVTISC 128
TGSSSNIGAGYDVHWYQQLPGT APKLLIYGDTNRPSGVPDRFSG
SKSGTSASLAITGLQAEDEADY YCQSYDGSRSGLVFGGGTKLTV L
QVQLVQSGAEVKKPGSSVKVS 37 EIVLTQSPGTLSLSPGERATLS 129
CKTTGGTFSSYAVTWVRQAPG CRASQSLSNAYLAWYQQKPGQA QGLEWMGGIIGMFGTTNYAQK
PRLLLYGGSTRATGIPDRFSGS FQGRVTITADEMTSTAYMELS GSGTDFTLTISSLEAEDFAVYY
SLRSEDTAVYYCARGSYYYES CQQYGSSPRTFGQGTKVEIKRT SLDYWGKGTL V
QVQLLQSGAEVKKPGSSVKVS 38 EIVLTQSPGTLSLSPGERATLS 130
CKTTGGTFSSYAVTWVRQAPG CRASQSVSSSYLAWYQQKPGQA QGLEWMGGIIGMFGTTNYAQK
PRLLIYGTSSRATDIPDRFSGS FQGRVTITADEMTSTAYMELS GSGTDFTLTISRLEPEDFAVYY
SLRSEDTAVYYCARGSYYYES CQQYGRSPFTFGGGTKVEIKRT SLDYWGKGTL V
QVQLQQSGAEVKKPGSSVKVS 39 DIQLTQSPSSLSASVGDRVTIT 131
CKTSGGTFSSYAVTWVRQAPG CRASQGISNYLAWYQQKPGKVP QGLEWMGAIIGMFGTTNYAQK
KLLIYAASTLQSGVPSRFSGSG FQGRVTLTADELTSTAYMELS SGTDFTLTISSLQPEDVATYYC
SLRSEDTAVYYCARGSYYYES QKYNSAPRTFGQGTKVEIKRTV SLDYWGQGTL
QVQLVQSGAEVKKPGSSVKVS 40 DIQLTQSPSSLSASVGDRVTIT 132
CKTTGGTFSSYAVTWVRQAPG CRASQGISNYLAWYQQKPGKVP QGLEWMGGIIGMFGTTNYAQK
KLLIYAASTLQSGVPSRFSGSG FQGRVTITADEMTSTAYMELS SGTDFTLTISSLQPEDVATYYC
SLRSEDTAVYYCARGSYYYES QKYNSAPLTFGGGTKVEIKRTV SLDYWGQGTL
EIVMTQSPGTLSLSPGERATLS 133 CRASQSVSSSYLAWYQQKPGQA
PRLLTYGASSRATGIPDRFSGS GSGTDFTLTISRLEPEDFAVYY CQQYGSSPYTFGQGTKVEIK
QVQLVQSGAEVKKPGSSVKVS 41 QSVLTQPPSASGTPGQRVTISC 134
CKTSGGTFSSYAVTWVRQAPG SGSSSNIGSNFVYWYQQLPGTA QGLEWMGAIIGMFGTTNYAQK
PKLLIYKSNQRPSGVPDRFSGS ) FQGRVTLTADELTSTAYMELS
KSGTSASLAISGLRSEDEADYY SLRSEDTAVYYCARGSYYYES CAAWDDSLSGYVFGTGTQLTVL
SLDYWGQGTM QVQLVQSGAEVKKPGSSVKVS 42 ETTLTQSPDTLSLSPGERATLS 135
CKTSGGSFSSYAVTWVRQAPG CRASQSVSSGSLAWYQQKPGQA QGLEWMGGTIGMFGTTNYAQK
PRLLIYAASSRAAGIPDRESGS FQGRVTITADELTSTAYMELS GSGTDFTLTISRLEPEDFAVYY
SLRSEDTAVYYCARGSYYYES CQQYGSSPGLTFGGGTQVEIKR SLDYWGQGTM TV
QVQLVQSGAEVKKPGSSVKVS 43 ETTLTQSPGTLSLSPGERATLS 136
CKTSGGTFSSYAVTWVRQAPG CRASQSVSSSYLAWYQQKPGQA QGLEWMGGIIGMFGTTNYAQK
PRLLIYGASSRATGIPDRFSGS FQGRVTITADELRSTAYMELS GSGTDFTLTISRLEPEDFAVYY
SLRSEDTAVYYCARGSYYYES CQQYGSSPYTEGQGTKLEIKRT SLDYWGQGTL V
QVQLVQSGAEVKKPGSSVKVS 44 ETVLTQSPGTLSLSPGERATLS 137
CKTSGGTFSSYAVTWVRQAPG CRASQNIGWYLAWYQHKPGQAP QGLEWMGGIIGMFGTTNYAQK
RLIMYDASTRATGIPDRFSGSG FQGRVTITADELTSTAYMELS SGTDFTLTISRLEPEDFAVYYC
SLRSEDTAVYYCARGSYYYES QQYDSVPSTEGQGTNLEIKRTV SLDYWGQGTL
QSVLTQPPSVSGAPGQRVTISC 138 TGSSSNIGAGYDVHWYQQLPGT
TPKLLIYDNTNRPSGVPDRFSA SKSGASASLAITGLRDEDEADY
YCQSYDSSLSASVFGGGTKLTV L QVQLVQSGAEVKKPGSSVKVS 45
QSALTQPRSVSGSPGQSVTISC 139 CKTSGGTFSSYAVTWVRQAPG
TGTTSDVGGYNYVSWYQQHPGE QGLEWMGGIIGMFGTTNYAQK APKLIIYDVSNRPSGVSNRFSG
FQGRVTITADELTSTAYMELS SKSGNTASLTVSGLQAEDEADY SLRSEDTAVYYCARGSYYYES
YCSSEAGSSNLIFGGGTKLTVL SLDYWGRGTL ETTLTQSPATLSVSPGERATLS 140
CRASQSVSRNLAWYQQKPGQAP RLLIYGASSRATGIPDRFSGSG
SGTDFTLTISRLEPEDFAVYYC QQYGSSSITFGQGTRLEIKRTV QVQLVQSGAEVKRPGSSVKVS
46 SYELTQPPSASGTPGQRVTISC 141 CKASGGTFSSYAVTWVRQAPG
SGSSSNIGSNTVNWYQQLPGTA QGLEWMGAIIGMFGTTNYAQK PKLLIYSNNHRPSGVPDRFSGS
FQGRVTLTADELTSTAYMELS KSGTSASLAISGLQSEDEADYY SLRSEDTAVYYCARGSYYYES
CATWDDSLNGWVFGGGTKVTVL SLDYWGQGTM QVQLVQSGAEVKKPGSSVKVS 47
QSVLTQPPSASGTPGQRVTISC 142 CKASGGTFSSYAVTWVRQAPG
SGSSSNIGGNTVNWYQQVPGTA QGLEWMGAIIGMFGTTNYAQK PKLLIHSNNQRPSGVPDRESGS
FQGRVTITADELTSTAYMELS KSGTSASLAISGLLSEDEADYY SLRSDDTAVYYCARGSYYYES
CEVWDDSLNGRVEGGGTKLTVL SLDYWGQGTL QVQLVQSGAEVKKPGSSVKVS 48
DIQLTQSPSSLSASVGDRVTIT 143 CKTSGGTFSSYAVTWVRQAPG
CRASQGISNYLAWYQQKPGKVP QGLEWMGAIIGMFGTTNYAQK KLLIYAASTLQSGVPSRFSGSG
FQGRVTITADELTSTAYMELS SGTDFTLTISSLQPEDFATYYC SLRSDDTAVYYCARGSYYYES
QQSYSTPRTFGQGTKLETKR SLDYWGQGTL HPELTQPPSASGTPGQRVTISC 144
SGSSSNIGSNTVNWYQQLPGTG PKLLIYSNNQRPSGVPDRFSGS
KSGTSASLAISGLQSEDEADYY
CAAWDDSLNGWVFGGGTKVTVL EIVLTQSPGTLSLSPGERATLS 145
CRASQSVSSSYLAWYQQKPGQA PRLLIYGASSRATGIPDRFSGS
GSGTDFTLTISRLEPEDFAVYY CQQFGSSQVTFGGGTKVEIKR GVQLVQSGAEVKKPGSSVKVS
49 HVILTQPPSVSVAPGMTARMTC 146 CKTSGGTFSSYAVTWVRQAPG
GGDNVGRRNHWYQQKPGQAPVL QGLEWMGAIIGMFGTTNYAQK VVYDDGGRPSAIPARFSGSKSG
FQGRVTLTADELTSTAYMELS NTATLIISRVEAGDEADYYCQM SLRSEDTAVYYCARGSYYYES
WHSSGDQWVFGGGTKLTVL SLDYWGKGTM GVQLVQSGAEVKKPGSSVKVS 50
QAVLTQPPSASGTPGQRVTISC 147 CKTSGGTFSSYAVTWVRQAPG
SGSSSNIGSNYVYWYQHLPGTA QGLEWMGAIIGMFGTTNYAQK PKLLIDRNDQRPSGVPDRFSGS
FQGRVTITADELTSTAYMELS KSGTSASLAISGLRSEDEADYY SLRSDDTAVYYCARGSYYYES
CAAWDDNLSGVVFGGGTKVTVL SLDYWGRGTL QVQLVQSGAEVKKPGSSVKVS 51
QSVVTQPPSVSGAPGQRVTISC 148 CKASGGTFSSYAISWVRQAPG
TGSSIGAGYDVHWYQQLPRTAP QGLEWMGAIIGMFGTTNYAQK KLLIFGNTNRPSGVPDRFSGSK
FQGRVTLTADELTSTAYMELS SGTSASLTITGLQAEDEANYYC SLRSEDTAVYYCARGSYYYES
QTYDSSLSGWVFGGGTKLTVL SLDYWGKGTM QVQLVQSGAEVKKPGSSVKVS 52
PELTQPPSASGTPGQRVTISCS 149 CKASGGTFSSYAISWVRQAPG
GSSSNIGSNHVYWYQQLPGTAP QGLEWMGAIIGMFGTTNYAQK KLLIYRNNQRPSGVPDRFSGSK
FQGRVTITADELTSTAYMELS SGTSASLAISGLRSEDEADYYC SLRSDDTAVYYCARGSYYYES
ATWDDNLSGRLVEGGGTKLTVL SLDYWGKGTM QVQLVQSGAEVKKPGSSVKVS 53
QSVLTQPPSVSGAPGQRVTISC 150 CKTTGGTFSSYAVTWVRQAPG
IGSNSNIGANFAVHWYQQLPGA QGLEWMGGIIGMFGTTNYAQK APKLLIYDNTNRPSGVPDRFSG
FQGRVTITADEMTSTAYMELS SKSGTSASLDITGLQADDEADY SLRSEDTAVYYCARGSYYYES
YCQSYDARLNGWVFGGGTKLTV SLDYWGQGTM L QVQLQQSGAEVKKPGSSVKVS 54
QSALTQPPSASGSPGQSVTISC 151 CKTSGGTFSSYAVTWVRQAPG
AGASSDIGTYNSVSWYQQHPGK QGLEWMGAIIGMFGTTNYAQK APKLIIYEVTKRPSGVPDRFSG
FQGRVTITADELTSTAYMELS SKSGNTASLTVSGLQAEDEADY SLRSDDTAVYYCARGSYYYES
YCNSYAGTKGYVFGSGTKVTVL SLDYWGKGTM QVQLQQSGAEVKKPGSSVKVS 55
SSELTQDPAVSVALGQTVRITC 152 CKTSGGTFSSYAVTWVRQAPG
QGDSLRNEYASWYQQKPGQAPV QGLEWMGGITGMFGTTNYAQK LVMKGNNNRPSVIPDRFSGSRS
FQGRVTITADELTSTAYMELS GNTASLTITGAQAEDEADYYCS SLRSEDTAVYYCARGSYYYEN
SRDSSGNRFFGSGTKVTVL SLDYWGKGTL QVQLVQSGAEVKKPGSSVKVS 56
HPELTQPPSLSVSPGQTATISC 153 CKTSGGTFSSYAVTWVRQAPG
SGERLTNKYTSWYQQRPGQSPA QGLEWMGAIIGMFGTTNYAQK LVIYQDDKRPSGIPERFSGSNS
FQGRVTLTADELTSTAYMELS GNTATLTISGTQPMDEAVYYCQ SLRSEDTAVYYCARGSYYYES
AWDTNTQMTFGGGTKLTVL SLDYWGKGTM QVQLVQSGAEVKKPGSSVKVS 57
QAVLTQPPSASGTPGQRVTISC 154 CKTSGGTFSSYAVTWVRQAPG
SGSSSNIGTNTVNWYQQLPGTA QGLEWMGAIIGMFGTTNYAQK PKLLIYRNIQRPSGVPDRFSGS
FQGRVTITADELTSTAYMELS KSGTSASLAISGLQSEDEADYY SLRSDDTAVYYCARGSYYYES
CAAWDDSLNGYVFGTGTKLTVL SLDYWGQGTM QVQLVQSGAEVKKPGSSVKVS 58
ETTLTQSPGTVSLSPGERATLS 155 CKTSGGTFSSYAVTWVRQAPG
CRASQSVGGSNLAWYQQKPGQA QGLEWMGAIIGMFGTTNYAQK PRLVIYATSRKANGIPDRFSGS
FQGRVTITADELTSTAYMELS GSGTDFTLTISRLEPEDFAVYF SLRSDDTAVYYCARGSYYYES
CQQYGTSPPSVTFGGGTKVEIR SLDYWGRGTM R QVQLVQSGAEVKKPGSSVKVS 59
QAVLTQPPSASGTPGQRVTISC 156 CKASGGTFSSYAVTWVRQAPG
SGSSSNIGSNPVNWYQQLPGTA QGLEWMGAIIGMFGTTNYAQK PKLLIYSNNQRPSGVPDRFSGS
FQGRVTLTADELTSTAYMELS KSGTSASLAISGLQSEDEADYY SLRSEDTAVYYCARGSYYYES
CAAWDDSLTGYVFGTGTQLTVL SLDYWGKGTM QVQLVQSGAEVKKPGSSVKVS 60
SYVLTQPPSASGTPDQRVTISC 157 CKASGGTFSSYAISWVRQAPG
SGSSSNIGSNYVYWYQQFPGAA QGLEWMGGIIGMFGTTNYAQK PKLLMSRNNQRPSGVPDRFSGS
FQGRVTITADELTSTAYMELS KSGTSASLAISGLRSEDEAYYS SLRSEDTAVYYCARGSYYYES
CAAWDDSLNGLVFGGGTKVTVL SLDYWGKGTT QVQLQESGPGLVKPSETLSLT 61
NFMLTQPHSVSESPGKTVTISC 158 CTVSGYSFDSGYYWGWLRQPP
TGSGGNIARNYVQWYQQRPGSA GKGLEWIGSIYHSRNTYYNPS PVTVILEDDKRPSGIPDRPSGS
LKSRVTISVDTSKNQFSLQLS IDRSSNSASLTISGLRTEDEAL SVTAADTAVYYCARGTWYSSN
YYCQSYDDSDLVVFGGGTKLT LRYWFDPWGKGTL NFMLTQPHSVSESPGKTVTISC 159
TGSSGSIASNYVQWYQQRPGSA PTTVIYEDYQRPSGVPDRFSGS
IDSSSNSASLTISGLKTEDEAD YYCQSYDDSDHLIFGGGTKLTV L
QSVLTQPPSASGTPGQRVTISC 160 SGSSSNIGSNTVNWYKQLPGTA
PRLLIYSNDQRPSGVPDRPSGS KSGTSASLAISGLQSEDEANYY
CAAWDDSLSGWVFGGGTKLTVL PELTQPHSVSESPGKTVTISCT 161
GSGGRIATNHVQWYQQRPGSAP TIVIYENNQRPSGVPNRFSGSI
DDSSNSASLTISALRTEDEADY YCQSADATNVFFGGGTKVTVL PELTQPPSASGTPGQRVTISCS
GSSSNIGSNTVNWYQQLPGTAP KLLIYSNNQRPSGVPDRFSGSK
SGTSASLAISGLQSEDEADYYC AAWDDSLNGWVFGGGTKLTVL DIQMTQSPSSLSAFVGDRVTIT
163 CQASQDISNYLNWYQQKPGKAP KLLIYDATNLETGVPSRFSGSG
SGTDFTFTISSLQPEDIATYYC QQYDNLPLTFGGGTKVDIKR QVQLQESGPGQVKYSETLSLT
62 QSVLTQPPSASGTPGQRVTLSC 164 CTVSGYSFDSGYYWGWLRQPP
SGSSSNIGGNSVNWYQHVPGTA GKGLEWIGSIYHSRNTYYNPS PKLLMHSDDQRPSGVPDRFSGS
LKSRVTISVDTSKNQFSLQLS KSGTSASLAISGLQSEDEADYY SVTAADTAVYYCARGTWYSSN
CAAWDDSLNAWVFGGGTKVTVL LRYWFDPWGKGTT EVQLVQSGAAVKKPGSSVKVS 63
ETTLTQSPGTLSLSPGERATLS 165 CKASGGRFSSYAINWVRQAPG
CRASQSVSSRYLAWYQQKPGQA QGLEWMGGIIGMFGTTDYAQK PRLLIYGASNRATGVPDRESGS
FQGRVTITADEVTSTGYMELR GSGTDFTLTINRLEPEDFAVYY SLTSEDTAVYYCARGSGYHLQ
CQHYSRSLTFGGGTKVEIKRT NPFDLWGRGTM QVQLQQSGAAVKKPGSSVKVS 64
QSVLTQPPSVSAAPGQMVTISC 166 CKASGGRFSSYAINWVRQAPG
SGSNSNIGNNYISWYQQLPGSA QGLEWMGGIIGMFGTTDYAQK PRLLIYNNYKRPSGIPDRFSAS
PQGRVTITADEVTSTGYMELR KSGTSATLGITGLQTGDEADYY SLTSEDTAVYYCARGSGYHLQ
CGTWDSSLSSVVFGGGTKVTVL NPFDLWGKGTL EVQLVESGGGLVQPGGSLRLS 65
QSVLTQPPSVSGAPGQRVTISC 167 CAASGFPFSSYVMIWVRQVPG
TGSSSNTGAGNHVHWYQQVAGA KGLEWVSAIGGSGGSTYYADS APKLLISNNNNRPSGVPDRFSA
VKGRFTISRDNSKNTLYLQMN SKSGTSASLDITGLQAEDEADY SLRADDTAVYYCVLSPKSYYD
YCQSYDNSLNDWVFGGGTQLTV NSGIYFDFWGRGTL L EVQLVETGGGLVQPGGSLRLS 66
QSVVTQPPSESAAPGQKVTISC 168 CAASGFPFSSYVMIWVRQVPG
SGSSSNIGNNYVSWYQQFPGAA KGLEWVSAIGGSGGSTYYADS PKLLIFENNKRHSGIPDRFSGS
VKGRFTISRDNSKNTLYLQMN KSGTSATLGIAELQTGDEADYY SLRADDTAVYYCVLSPKSYYD
CGVWDSSLSAWVFGGGTQLTVL NSGIYFDFWGKGTL EVQLVESGGGLVQPGGSLRLS 67
NFMLTQPHSVSESPGKTVTFSC 169 CAASGFPFSSYVMIWVRQVPG
TRSSGSIASKYVQWYQQRPGSA KGLEWVSAIGGSGGSTYYADS PTIVIEENTKRPYGVPDRFSGS
VKGRFTISRDNSKNTLYLQMN IDSSSNSASLTISGLKTEDEAD SLRADDTAVYYCVLSPKSYYD
YYCQSYDSSNHWVFGGGTQLTV NSGIYFDFWGRGTL LS
[0234] Use of Neutralizing Antibodies
[0235] The influenza neutralizing antibodies of the present
invention can be used for the prevention and/or treatment of
influenza type A infections. For therapeutic applications, the
antibodies or other molecules, the delivery of which is facilitated
by using the antibodies or antibody-based transport sequences, are
usually used in the form of pharmaceutical compositions. Techniques
and formulations generally may be found in Remington's
Pharmaceutical Sciences, 18th Edition, Mack Publishing Co. (Easton,
Pa. 1990). See also, Wang and Hanson "Parenteral Formulations of
Proteins and Peptides: Stability and Stabilizers," Journal of
Parenteral Science and Technology, Technical Report No. 10, Supp.
42-2S (1988).
[0236] Antibodies are typically formulated in the form of
lyophilized formulations or aqueous solutions. Acceptable carriers,
excipients, or stabilizers are nontoxic to recipients at the
dosages and concentrations employed, and include buffers such as
phosphate, citrate, and other organic acids; antioxidants including
ascorbic acid and methionine; preservatives (such as
octadecyldimethylbenzyl ammonium chloride; hexamethonium chloride;
benzalkonium chloride, benzethonium chloride; phenol, butyl or
benzyl alcohol; alkyl parabens such as methyl or propyl paraben;
catechol; resorcinol; cyclohexanol; 3-pentanol; and m-cresol); low
molecular weight (less than about 10 residues) polypeptides;
proteins, such as serum albumin, gelatin, or immunoglobulins;
hydrophilic polymers such as polyvinylpyrrolidone; amino acids such
as glycine, glutamine, asparagine, histidine, arginine, or lysine;
monosaccharides, disaccharides, and other carbohydrates including
glucose, mannose, or dextrins; chelating agents such as EDTA;
sugars such as sucrose, mannitol, trehalose or sorbitol;
salt-forming counter-ions such as sodium; metal complexes (e.g.,
Zn-protein complexes); and/or non-ionic surfactants such as
TWEEN.TM., PLURONICS.TM. or polyethylene glycol (PEG).
[0237] The antibodies also may be entrapped in microcapsules
prepared, for example, by coacervation techniques or by interfacial
polymerization (for example, hydroxymethylcellulose or
gelatin-microcapsules and poly-(methylmethacylate) microcapsules,
respectively), in colloidal drug delivery systems (for example,
liposomes, albumin microspheres, microemulsions, nano-particles and
nanocapsules), or in macroemulsions. Such techniques are disclosed
in Remington's Pharmaceutical Sciences, supra.
[0238] The neutralizing antibodies disclosed herein may also be
formulated as immunoliposomes. Liposomes containing the antibody
are prepared by methods known in the art, such as described in
Epstein et al., Proc. Natl. Acad. Sci. USA 82:3688 (1985); Hwang et
al., Proc. Natl. Acad. Sci. USA 77:4030 (1980); U.S. Pat. Nos.
4,485,045 and 4,544,545; and WO97/38731 published Oct. 23, 1997.
Liposomes with enhanced circulation time are disclosed in U.S. Pat.
No. 5,013,556.
[0239] Particularly useful liposomes can be generated by the
reverse phase evaporation method with a lipid composition
comprising phosphatidylcholine, cholesterol and PEG-derivatized
phosphatidylethanolamine (PEG-PE). Liposomes are extruded through
filters of defined pore size to yield liposomes with the desired
diameter. Fab' fragments of the antibody of the present invention
can be conjugated to the liposomes as described in Martin et al. J.
Biol. Chem. 257:286-288 (1982) via a disulfide interchange
reaction. A chemotherapeutic agent is optionally contained within
the liposome. See Gabizon et al. J National Cancer Inst. 81(19)1484
(1989).
[0240] For the prevention or treatment of disease, the appropriate
dosage of antibody will depend on the type of infection to be
treated the severity and course of the disease, and whether the
antibody is administered for preventive or therapeutic purposes.
The antibody is suitably administered to the patient at one time or
over a series of treatments. Depending on the type and severity of
the disease, about 1 .mu.g/kg to about 15 mg/kg of antibody is a
typical initial candidate dosage for administration to the patient,
whether, for example, by one or more separate administrations, or
by continuous infusion.
[0241] The neutralizing antibodies of the present invention can be
additionally used as a tool for epitope mapping of antigenic
determinants of an influenza A virus, and are useful in vaccine
development. Indeed, as shown in the Example below, the inventors
herein have identified several broadly reactive neutralizing
antibodies that can be used as guides for vaccine design.
[0242] Thus, the neutralizing antibodies of the present invention
can be used to select peptides or polypeptides that functionally
mimic the neutralization epitopes to which the antibodies bind,
which, in turn, can be developed into vaccines against influenza A
virus infection. In one embodiment, the present invention provides
a vaccine effective against an influenza A virus comprising a
peptide or polypeptide that functionally mimics a neutralization
epitope bound by an antibody described herein. In one embodiment,
the vaccine comprises a peptide or polypeptide functionally
mimicking a neutralization epitope bound by an antibody that binds
a hemagglutinin (HA) antigen. In another embodiments, the vaccine
may be synthetic. In other embodiments, the vaccine may comprise
(i) an attenuated influenza A virus, or a part thereof; or (ii) a
killed influenza A virus, or part thereof. In one other embodiment,
the vaccine comprises a peptide or polypeptide functionally
mimicking a neutralization epitope bound by an antibody that binds
a hemagglutinin (HA) antigen. The HA antigen may be an H5 subtype
or an H1 subtype. In another embodiment, the HA antigen is
displayed on the surface of an influenza A virus.
[0243] In another embodiment, the peptides or polypeptides of the
vaccine contain antigenic determinants that raise influenza A virus
neutralizing antibodies.
[0244] In a more general aspect, the neutralizing molecules,
including but not limited to antibodies, are useful to prevent or
treat viral infections. Thus, the neutralizing molecules of the
present invention are useful in both immunotherapy, such as passive
immunization using one or more such molecules, and in the
development of vaccines directed at the viral antigenic
target(s).
[0245] Identification of Residues Important for Neutralizing
Function
[0246] In a significant aspect of the present invention, a cluster
of antibody residues important for neutralizing properties have
been identified. In particular, it has been found that antibodies
comprising an antibody heavy chain variable domain comprising at
least one substitution in the surface exposed cluster determined by
amino acid positions 52A, 53, 73, and 74, following Kabat amino
acid numbering, have excellent neutralizing properties, including
but not limited to neutralization of influenza viruses. In
particular, it has been found that the following mutations: 52A
(Pro.fwdarw.Gly), 53 (Ile.fwdarw.Met), 73 (Lys.fwdarw.Glu), and 74
(Ser .fwdarw.Leu or Met), relative to germ line chemistry, create a
remarkably tight cluster on the exposed surface of the 4 heavy
chain variable domain, where they form a ridge that protrudes
prominently from the protein surface. An additional mutation
important for neutralizing properties, 57 (Ala.fwdarw.Thr), is
partially buried at the base of the CDR2 loop. The surface-exposed
changes in CDR 2 and framework 3 are believed to have a direct role
in antigen binding, where the less exposed mutation at position 57
and some additional mutations are likely have indirect effects
through stabilizing and/or positioning of the CDR2 loop. Such
additional mutations include conservative changes in CDR1 at
position 34 (Ile.fwdarw.Val) and 35 (Ser.fwdarw.Thr) and also in
CDR2 at position 50 (Gly.fwdarw.Ala). These mutations are believed
to be important broadly for viral neutralizing properties,
including, without limitation, neutralization of influenza A
viruses, such as H5 HA, as well as HIV viruses.
[0247] These results are very valuable not only for understanding
the immunochemical basis of neutralization but also for designing
antibodies and antibody-like molecules with broad and improved
viral neutralizing properties, as disclosed and claimed herein.
[0248] Vaccine development and the development of neutralizing
antibodies with improved properties, using information about the
residues important or beneficial for neutralizing properties
disclosed herein, can additionally benefit from the combinatorial
libraries of conformationally constrained polypeptide sequence
described in PCT Application Publication No. WO 2008/089073,
published on Jul. 24, 2008.
[0249] Non-Antibody Molecules with Neutralizing Properties
[0250] Although in the previous description the invention is
illustrated with reference to antibody libraries, libraries of
other, non-antibody molecules, such as surrobodies, can be
prepared, used, and optimized in a similar manner. Thus, the
construction of unique combinatorial protein libraries based on the
pre-B cell receptor (pre-BCR) ("surrobody libraries") are described
in Xu et al., 2008, supra. As discussed before, the pre-BCR is a
protein that is produced during normal development of the antibody
repertoire. Unlike that of canonical antibodies, the pre-BCR
subunit is a trimer that is composed of an antibody heavy chain
paired with two surrogate light chain (SLC) components.
Combinatorial libraries based on these pre-BCR proteins in which
diverse heavy chains are paired with a fixed SLC were expressed in
mammalian, Escherichia coli, and phagemid systems. These libraries
contain members that have nanomolar affinity for a target antigen.
A description of the library construction, selective enrichment,
and biophysical characterization of library members is detailed in
the Materials and Methods section of the Xu et al. paper.
[0251] Further details of the invention are illustrated by the
following non-limiting Examples.
Example 1
Antibody Libraries from Survivors of Prior Bird Flu Outbreaks and
Preparation of Neutralizing Antibodies
[0252] The widespread incidence of H5N1 influenza viruses in bird
populations poses risks to human health. Even though the virus has
not yet adapted for facile transmission between humans, it can
cause severe disease and often death. Here we report the generation
of combinatorial antibody libraries from the bone marrow of five
survivors of the recent H5N1 avian influenza outbreak in Turkey. To
date, these libraries have yielded >300 unique antibodies
against H5N1 viral antigens. Amongst these antibodies, we have
identified several broadly reactive neutralizing antibodies that
could be used for passive immunization against H5N1 virus or as
guides for vaccine design. The large number of antibodies obtained
from these survivors provides a detailed immunochemical analysis of
individual human solutions to virus neutralization in the setting
of an actual virulent influenza outbreak. Remarkably, two of these
antibodies neutralized both H1 and H5 subtype influenza
viruses.
[0253] Newly emergent highly pathogenic influenza virus strains
pose a profound threat to man. Three influenza pandemics have
occurred within the past 100 years, each with devastating
consequences (Palese, P. & Shaw, M. L. In Fields Virology, Vol.
11 (Eds. Knipe, D M and Howley, P. M.) Lippencott Williams and
Wilkins, Philadelphia, 2006, 1648-1689)). The recent emergence of
the H5N1 virus strain, though mainly confined at present to avian
hosts, has already demonstrated virulence in humans, causing the
death of more than 200 people ((2008) (World Health Organization,
Geneva)). Therefore, healthcare officials, researchers, and
governments are actively considering their options should a
pandemic occur. One widely considered approach concerns the use of
passive antibodies either for prevention of disease or treatment
after exposure to virus (Luke, T. C. et al. (2006) Ann Intern Med
145, 599-609). The potential for passive immunization against
influenza has been evident since the Spanish influenza nearly a
century ago, where the benefits of transfused of blood, sera, and
blood products reduced the risk of mortality by more than 50%
(Id.). Recently the benefits of treatment with convalescent plasma
have also been reported in instances of H5N1 influenza (Kong L K
& Zhou B P (2006) Hong Kong Med J 12, 489; Zhou, B. et al.
(2007) N Engl J Med 357, 1450-1451). Additionally, passive
immunization with human and mouse monoclonal antibodies has been
reported to protect animals from death, even when given after H5N1
infection (Hanson, B. J. et al. (2006) Respir Res 7, 126)).
[0254] The most logical source of human antibodies for passive
therapy would be patients that have survived infection. Through the
use of modern combinatorial antibody library technologies, it is
now possible to capture the entire immunological history of an
individual's response to an infection (Law, M. et al. (2007) Nat
Med. January; 14(1):25-7. Epub 2007 Dec. 6; Lerner R A (2006) Angew
Chem Int Ed Engl 45, 8106-8125). Because antibody libraries contain
the complete record of an individual's response to pathogens, one
can recover the repertoire specific to a given agent by using a
laboratory process of selective enrichment. Such libraries both
give archival information as to the nature of antibodies made
during the infection and allow recovery of potentially therapeutic
human monoclonal antibodies. Importantly, antibody recovery is
independent of whether an active antibody response is still
occurring at the time the sample is taken. Thus, depending on when
the libraries are constructed, one may obtain antibodies that are
currently being made and/or are part of the individual's
immunological history. For infections that may be lethal, such
analyses carried out on surviving patients may be particularly
important because they chart some of the immunological mechanisms
used during a successful host defense in the actual clinical
setting of an outbreak.
[0255] Typically, when libraries are prepared from individuals who
have been infected with a virus, hundreds to thousands of different
antibodies are obtained, as opposed to only a few when other
methods are used (Lerner R A (2006) Angew Chem Int Ed Engl 45,
8106-8125). This has several consequences. A comparative sequence
analysis of these antibodies allows a detailed map of the chemistry
of antibody binding. Similarly, a comparison of neutralizing and
non-neutralizing antibodies can give important information about
the nature of binding interactions that are critical to
neutralization.
[0256] Here we describe the creation of the first comprehensive
avian influenza antibody libraries made from survivors of infection
with an avian influenza virus during a confirmed outbreak. We have
used these libraries to obtain large numbers of monoclonal
antibodies to the H5N1 avian influenza virus, some of which have
broad reactivity and are neutralizing across viral sub-types.
Ultimately these combinatorial antibody libraries may hold the key
to immunotherapy, such as passive immunization, using one or more
member antibodies, or they may guide the development of vaccines
utilizing the antigenic target(s) of the neutralizing antibodies in
the library.
[0257] The outbreak and source of material. Between December 2005
and January 2006 an outbreak of avian influenza H5N1 occurred in
Turkey (A. F. Oner et al., N Engl J Med 355, 2179 (Nov. 23, 2006)).
In total, twelve individuals were infected and only eight survived.
Because bone marrow RNA contains the archived record of all
antibodies made by an individual, we selected it as our source
material. We obtained bone marrow and serum from six of the Turkish
survivors approximately 4 months following recovery and
successfully prepared antibody libraries from five of the six bone
marrow samples. In the sixth sample the RNA was degraded.
[0258] Serological analysis. The hemagglutinin protein is essential
for binding the influenza virus to the cell that is being infected
and is generally considered to be the main target of neutralizing
antibodies (Palese, P. & Shaw, M. L. In Fields Virology, Vol.
11 (Eds. Knipe, D M and Howley, P. M.) Lippencott Williams and
Wilkins, Philadelphia, 2006, 1648-1689) (2008) (World Health
Organization, Geneva)). Therefore, we tested by ELISA each of the
individual serum samples at high serum dilutions for the detection
of antibodies against H5 hemagglutinin proteins (FIG. 8) and intact
viruses (data not shown). This analysis showed that the patients
had readily detectable serum antibodies, even when diluted
10,000-fold. We selected the Vietnam/1203/04 hemagglutinin as a
target because it was readily available and is thought to be
related to the influenza virus strain that caused the outbreak of
disease in Turkey.
[0259] Library construction. Our primary objectives were to
understand the nature of the immunological response to infection
and to identify specific antibodies that might be used passively
for the prevention of spread and/or treatment of H5N1 influenza
virus infections. We wished to recover every possible solution to
H5N1 infections with minimal or no bias. Because gene expression
for the individual immunoglobulin families is not equal, making
them prone to bias and over-representation, we decided against
using the standard pooled approach to immunoglobulin recovery.
Instead, we individually rescued 20 of the 23 distinctly
amplifiable gene families during the construction of the libraries.
The remaining three gene families (V.sub.H 2, 5, and 6) were
recovered as a pool because they are infrequently used. We further
normalized gene content by creating equimolar pools of each
immunoglobulin family DNA for cloning into a phagemid display
vector.
[0260] A unique DNA bar code was embedded into a non-disruptive
portion of the phagemid vector to allow each clone to be tracked
back to the original patient source (FIG. 9). This bar coding
allows assignment of clones to individual patients even when phage
libraries from multiple survivors are screened simultaneously. As a
result of this tagging, every clone isolated from any library can
be confidently attributed to the cognate survivor.
[0261] As illustrated in FIG. 9, cloning and barcoding of annotated
repertoires allows tracking of all clones to their sources. Each
survivor is assigned a unique barcoded vector and immunoglobulin
repertoires are cloned via restriction sites indicated in the upper
panel. Plasmids from any clone can be assigned to designated
sub-libraries via their light chain class and their survivor
barcode.
[0262] Using this vector with its coding system, we successfully
cloned repertoires from the bone marrow of five of the six
survivors in both single chain (scFv) and Fab phagemid formats.
Each collection from an individual survivor has a diversity of
greater than 1.0.times.10.sup.8 members. Furthermore, we created
additional bar coded libraries comprised of mixed survivor light
and heavy chains with a final diversity of 1.1.times.10.sup.9.
Collectively the 5 donor-specific collections and the pooled
libraries from all donors have a total diversity of
1.0.times.10.sup.9 as a scFv collection and 4.2.times.10.sup.9 as a
Fab-displayed collection (Table 3).
[0263] Table 3 shows the light chain and full library total
transformants in both scFv and Fab formats. Total diversity
represented by all libraries is 5.6.times.10.sup.9.
TABLE-US-00003 TABLE 3 Light Chains Completed Libraries Kappa
Lambda Kappa Lambda scFv H5-1 3.00E+06 4.00E+06 1.50E+08 1.20E+08
H5-2 3.00E+06 3.00E+06 4.00E+07 1.60E+07 H5-3 8.20E+05 1.70E+06
5.30E+07 1.50E+08 H5-5 7.00E+06 5.60E+06 6.50E+08 5.60E+07 H5-6
1.50E+06 5.00E+06 ND 1.00E+07 H5Pool Not Determined 1.80E+08
5.70E+08 Totals 1.10E+09 9.20E+08 scFv Total 2.00E+09 Fab H5-1
1.50E+06 2.90E+06 2.90E+08 4.60E+08 H5-2 3.10E+06 9.40E+05 4.40E+08
4.30E+08 H5-3 2.80E+06 2.30E+06 3.90E+08 3.90E+08 H5-5 7.00E+06
5.60E+06 7.20E+08 1.70E+08 H5-6 1.50E+06 5.00E+06 ND ND H5Pool
1.90E+07 2.60E+08 Totals 2.10E+09 1.45E+09 Fab Total 3.60E+09
[0264] Selecting binding antibodies. As indicated above, one
interesting feature of these studies was that we initially selected
antibodies for binding against a virus strain and antigen that was
related to, but different from, the one that caused the infection.
This was because viral isolates from the patients were not
available. The necessity of using a related strain to select
antibodies could have proven fortuitous because it may have led to
the isolation of more broadly neutralizing antibodies (see
below).
[0265] The libraries were panned against inactivated virus
containing the Vietnam/1203/04 virus HA and NA proteins and
recombinant purified hemagglutinin (Barbas, C. et al. (2001) Phage
Display, A Laboratory Manual (Cold Spring Harbor Laboratory
Press)). Typically, following three to four rounds of phage
panning, individual clones from enriched phage pools were analyzed
by ELISA against H5N1 virus or purified hemagglutinin and the
positive clones were sequenced to determine their heavy and light
chain sequences and to read their survivor bar code (D. W. Coomber,
Methods Mol Biol 178, 133 (2002)). From these studies, we isolated
specific H5 hemagglutinin binding clones from all five of the
individual libraries from survivors. In total, we have so far
recovered more than 300 hundred different antiviral antibodies, of
which 146 specifically bind the H5 hemagglutinin protein.
[0266] General Features of the Selected Clones. Overall, the
individual patients use different germ lines for both heavy and
light chains, demonstrating that individual patients have found
different solutions to the same potentially lethal immunological
challenge. The major features of combinatorial antibody libraries
that can be used both to give confidence as to the quality of the
obtained repertoire and to provide information as to the chemistry
of antibody binding and/or neutralization are seen in these clones.
These clones contain all the hallmarks of the previously described
repeated clones ("jackpot solution") to antigen binding that is
found in the natural progression of affinity maturation, as well as
in selected synthetic antibody libraries (Lerner, R. A. Angew Chem
Int Ed Engl 45, 8106 (Dec. 11, 2006); A. Rajpal et al., Proc Natl
Acad Sci USA 102, 8466 (Jun. 14, 2005)). The presence of "jackpots"
in these large collections validates the screening procedure
because, unless the phage were selected on the basis of activity,
the chance of obtaining the same clone multiple times is highly
improbable. Moreover, when one analyzes the heavy chain differences
within groups, it was observed that many of the amino acid
substitutions were chemically and structurally conservative (Table
1). As with repeated clones, the appearance of multiple amino acid
substitutions that are chemically reasonable is unlikely to be a
random event.
[0267] Binding Specificity of recovered antibodies. Initial testing
of a set of Fabs using Bio-Layer Interferometry binding to the H5
Vietnam hemagglutinin protein indicated that we had identified at
least four distinct epitopes (data not shown). We selected six
clones from three survivors that recognized two different epitopes
for conversion into full IgG, proteins. The binding of three of
these antibodies was mapped to the HA1 subunit of the hemagglutinin
protein by western blot analysis (data not shown).
[0268] One goal of these studies was to recover those rare
antibodies that broadly neutralize divergent viral strains. There
was a suggestion that some of our antibodies might be broadly
reactive because the serum from the donors had high titer
antibodies against a divergent subfamily of H5N1 viruses that
extended beyond the virus with which they were infected. To
determine the degree of cross reactivity at the level of individual
antibodies, we analyzed binding of our clones to different
influenza hemagglutinin antigens (FIG. 6).
[0269] FIG. 6 shows the cross-reactivity of H5N1 antibodies from
two survivors with hemagglutinins from H1N1 viruses. (A) Bars are
H5N1 Vietnam 1203/04 (dark grey), H5N1 Turkey/65596/06 (white),
H5N1 Indonesia/5/05 (diagonal stripes), H1N1 New Calcdonia/20/99
(vertical stripes), H1N1 South Carolina/1/18 (crosshatch stripes),
and H3N2 Wisconsin/67/05 (light gray). (B) Relative ranking of
antibodies by their ELISA signal over background ("+" is above
background and less than 2 fold, "++" is between 2 and 9-fold,
"+++" is between 9 and 15-fold, "++++" is greater than 15-fold
above background, and "-" is not measurably above background), on
the various proteins. Not surprisingly, these antibodies recognize
hemagglutinin from the corresponding infecting Turkey/65596/06
strain, and in addition recognize the heterologous hemagglutinin
from the Vietnam/1203/04 strain used for selection. Furthermore,
they recognize the antigenically divergent Indonesian/5/05H5
hemagglutinin. We performed kinetic binding analyses on prototype
antibodies and found that the antibodies from survivor 5 bound
Vietnam/1203/04 hemagglutinin with single digit nanomolar
affinities, while the survivor 2 antibody bound more strongly with
a measured affinity of 13 .mu.M. The available binding affinities
for H5 and H1 and the clonal identities of the Fab fragments for
the antibodies are as follows.
TABLE-US-00004 Identity H5 H1 A 10-40 nM 40-80 nM B 130 pM C 10-40
nM D 13 pM E 4 nM 90 nM
[0270] To determine if our antibodies are even more broadly
reactive, we studied their binding to a larger collection of
hemagglutinins from different influenza A subtypes (FIG. 6). We
found that the four prototype antibodies bound hemagglutinin from
the closely related subtype H1N1 contemporary reference strain New
Calcdonia/20/99. Notably, the three neutralizing antibodies
belonging to survivor 5 also bound hemagglutinin from the H1N1
South Carolina/1/18 isolate that emerged during the 1918 Spanish
Flu pandemic. Conversely, none of these four antibodies bound
hemagglutinin from the contemporary H3N2 Wisconsin/67/05 reference
strain, indicating that even though the antibodies display broad
spectrum binding amongst and between Influenza subtypes, the
reactivity did not extend to all influenza subtypes.
[0271] To further explore the immunochemical basis of the H1/H5
cross-reactivity, we re-screened the libraries against the H1N1 New
Calcdonia/20/99 hemagglutinin protein. From this selection, we
found clones (Table 6) that bore significant similarity to the
sequences previously obtained from survivor 5 when the H5
hemagglutinin protein was used in the panning (Table 5).
[0272] Neutralization Studies. Initially the antibodies were
assayed for their ability to neutralize an H5 HA (Vietnam/1203/04)
containing influenza virus. One antibody derived from survivor 2
and 3 from survivor 5 that recognized a common epitope (epitope
"A") were all neutralizing whereas the two antibodies derived from
survivor 1 that recognized a second epitope (epitope "B") were
not.
[0273] Based on the striking sequence similarity of clones
separately isolated from survivor 5 against either H5N1 or H1N1
hemagglutinin, we predicted that their cross reactivity would
extend beyond simple binding and they would also have the highly
unusual property of neutralizing both H5N1 and H1N1 virus. To test
the cross neutralizing activity of the IgGs, we tested
representative antibodies from the H5N1 screen in a neutralization
assay to see if they would also neutralize H1N1 or H3N2 virus
(Table 4). We studied the H1 bearing virus A/New Cal/20/99 and the
H3 bearing virus A/Hong Kong/68. A collection of viruses bearing H5
subtype hemagglutinin was also tested (A/Vn/1203/04; A/Indo/5/05;
A/Turkey/65596/06; A/Egypt/06). The antibodies showed no activity
against H3 subtype influenza. However, three of the monoclonal
antibodies (1-3) that neutralized H5 containing viruses also
strongly neutralized all viruses bearing HA from subtypes H1 (Table
4).
TABLE-US-00005 TABLE 4 Virus* H5 H1 H3 A/Vietnam A/Vietnam
A/Indonesia A/Turkey A/Egypt A/New Cal A/Hong 1203/04 .dagger-dbl.
1203/04 .dagger-dbl. 5/05 65596/06 14725/06 20/99 Kong/68 Ab
1.dagger. 11-21 2.3-9.3 9.3 9.3 1.2-2.3 9 >333 Ab 2.dagger. 63
54-217 27 108 7-13 54-108 >333 Ab 3.dagger. 58 18 16 31 4-8 8-16
>333 Ab 4.dagger. 1.7-6.3 0.5-2.2 >333 Not done Not done
>333 >333 Mab #8.sctn. 2.7 Not done Not done Not done Not
done Not done Not done The results of Table 4 were obtained as
follows. MDCK cells were inoculated with 100 TCID50 of virus in the
presence of 2-fold serial dilutions of monoclonal antibodies.
.dagger.Minimum inhibitory concentrations required to neutralize
virus in duplicate samples are presented in ug/ml. .dagger-dbl. -
The viral neutralization results from two independent experiments
are both shown. .sctn.Mab#8 is a mouse monoclonal H5N1 neutralizing
antibody raised against A/Vietnam/1203/04.
[0274] The results of Table 4 were obtained as follows. MDCK cells
were inoculated with 100 TCID50 of virus in the presence of 2-fold
serial dilutions of monoclonal antibodies. .dagger.--Minimum
inhibitory concentrations required to neutralize virus in duplicate
samples are presented in ug/ml. .dagger-dbl.--The viral
neutralization results from two independent experiments are both
shown. .sctn.--Mab#8 is a mouse monoclonal H5N1 neutralizing
antibody raised against A/Vietnam/1203/04.
[0275] Immunochemical basis of neutralization. One advantage of
antibody libraries is that when one obtains large numbers of
antibodies, they can be grouped as to their relatedness. Thus, when
a function for a given antibody in the collection is observed one
can predict that other members of the group to which it belongs
will have similar activity.
[0276] Table 5 shows example sequences displaying the
immunochemical basis of neutralization discovered from Survivor 5
libraries following H5N1 Vietnam panning. The 61 unique heavy chain
sequences aligned with their germline variable regions (variable
(V) region gene V.sub.H1e/V.sub.H1-69) from the 114 unique heavy
and light chain combinations. Requisite mutations are shown in
bolded, underlined text (column 5--PI to GM and A to T; column
6--KS to EL or EM or XL) and predominant mutations are shown in
italicized, underlined text (column 2--A to T; column 3--IS to VT;
column 5--G to A; column 8--K to Q or R). Heavy chains sequences
also discovered in H1N1 New Calcdonia panning are highlighted in
gray. Antibody regions and Kabat numbering ranges are listed at the
top of each sequence column. The heavy chain/light chain pairing is
indicated in the first column as follows: *--paired with 2 unique
light chains, .dagger.--paired with 3 unique light chains,
.dagger-dbl.--paired with 4 unique light chains, .sctn.--paired
with 5 unique light chains, and --paired with 12 unique light
chains.
TABLE-US-00006 TABLE 5 Group 1 heavy FR1 CDR1 FR2 CDR2 chains 1-29
30-35 36-46 47-56 Vh1e QVQLVQSGAEVKKPGSSVKVSCKASGGTF SSYAIS
WVRQAPGQGLE WMGGIIPIFGTAN 1 ----------------------------- ------
----------- ------GM---T- 2 ----------------------------- ------
----------- ---A--GM---T- 3 ----------------------------- ------
----------- ---A--GM---T- 4* ----------------------------- ----VT
----------- ---A--GM---T- 5 ----------------------------- ----VT
----------- ---A--GM---T- 6 ----------------------------- ----VT
----------- ---A--GM---T- 7* -----------------------T----- ----VT
----------- ------GM---T- 8* -----------------------T----- ----VT
----------- ------GM---T- 9.dagger. -----------------------T-----
----VT ----------- ---A--GM---T- 10 -----------------------T-----
----VT ----------- ---A--GM---T- 11* -----------------------T-----
----VT ----------- ---A--GM---T- 12 -----------------------T-----
----VT ----------- ---A--GM---T- 13* -----------------------T-----
----VT ----------- ---A--GM---T- 14 -----------------------T-----
----VT ----------- ---A--GM---T- 15 -----------------------T-----
----VT ----------- ---A--GM---T- 16* -----------------------T-----
----VT ----------- ---A--GM---T- 17 -----------------R-----T-----
----VT ----------- ------GM---T- 18 -----------------------T---S-
----VT ----------- ------GM---T- 19 -----------------------TT----
----VT ----------- ------GM---T- 20.sctn.
-----------------------TT---- ----VT ----------- ------GM---T- 21
-----------------------TT---- ----VT ----------- ------GM---T- 22
-----------------------TT---- ----VT ----------- ------GM---T- 23
-----------------------X----- ----VT ----------- ---A--GM---T- 24
E---------------------------- ------ ----------- ---A--GM---T- 25
E-------T----------L--------- ----VT ----------- ------GM---T- 26
E-------T--------------V----- ----VT ----------- ---A--GM---T- 27
E----------Q-----------T----- ----VT ----------- ---A--GM---T- 28
E--------------A-----------A- ----VT ----------- ------GM---T-
29.dagger. E-----------------R----T----- ----VT -----------
------GM---T- 30.dagger. E-----------------R----T----- ----VT
----------- ------GM---T- 31 E-----------------R----T----- ----VT
----------- ------GM---T- 32 E----------------------T----- ----VT
----------- ---A--GM---T- 33* E----------------------T----- ----VT
----------- ---A--GM---T- 34.dagger-dbl.
E----------------------T----- ----VT ----------- ---A--GM---T- 35
E----------------------T----- ----VT ----------- ---A--GM---T- 36
E----------------------T----- ----VT ----------- ---A--GM---T- 37
E----------------------T----- ----VT ----------- ---A--GM---T- 38
E----------------------T----- ----VT ----------- ---A--GM---T-
39.sctn. E----------------------T----- ----VT -----------
---A--GM---T- 40.sctn. E----------------------T----- ----VT
----------- ---A--GM---T- 41* E----------------------T----- ----VT
----------- ---A--GM---T- 42.dagger. E----------------------T-----
----VT ----------- ---A--GM---T- 43 E----------------------T-----
----VT ----------- ---A--GM---T- 44 E----------------------T-----
----VT ----------- ---A--GM---T- 45 E----------------------T-----
----VT ----------- ------GM---T- 46 E----------------------T-----
----VT ----------- ------GM---T- 47 E----------------------T-----
----VT ----------- ------GM---T- 48.dagger.
E----------------------T----- ----VT ----------- ------GM---T- 49
E----------------------T----- ----VT ----------- ------GM---T-
50.dagger. E----------------------TT---- ----VT -----------
------GM---T- 51 E----------------------TT---- ----VT -----------
------GM---T- 52 G----------------------T----- ----VT -----------
---A--GM---T- 53 G----------------------T----- ----VT -----------
---A--GM---T- 54 -M---------------------T----- ----VT -----------
---A--GM---T- 55.dagger. -M---------------------T----- ----VT
----------- ---A--GM---T- 56 ----L------------------TT---- ----VT
----------- ------GM---T- 57* ----Q------------------T----- ----VT
----------- ---A--GM---T- 58 ----Q------------------T----- ----VT
----------- ---A--GM---T- 59 ----Q------------------T----- ----VT
----------- ------GM---T- 60* ----Q-------------R----T----- ----VT
----------- ------GM---T- 61* ----Q-------------R----T----- ----VT
----------- ------GM---T- Group 1 heavy FR3 CDR3 FR4 chains 59-92
93-101 102-113 Vh1e YAQKFQGRVTITADKSTSTAYMELSSLRSEDTAVYYC
ARGSYYYESSLD YWGQGTLVTVSS 1 --------------EL---------------------
------------ ---K-T----- 2 ----------L---EL---------------------
------------ ---K-M----- 3 --------------EL-------------D-------
------------ ---K-M----- 4* ----------L---EL---------------------
------------ ------------ 5 --------------EL-------------D-------
------------ ------------ 6 ----------L---EL---------------------
------------ ---K-M----- 7* --------------ELR--------------------
------------ ------------ 8* --------------EL---------------------
------------ ---R------- 9.dagger.
--------------EL-------------D------- ------------ ------------ 10
--------------EL-------------D------- ------------ ---R-M----- 11*
--------------EL-------------D------- ------------ ------M----- 12
----------L---EL--------------------- ------------ ------------ 13*
----------L---EL--------------------- ------------ ------M----- 14
----------L---EL--------------------- ------------ ---K-M----- 15
----------L---EL-------------X------- ------------ ------------ 16*
----------L---EL--------------------- ------------ ------------ 17
--------L-----EM--------------------- --------TT-- ---R------- 18
--------------EL--------------------- ------------ ------M----- 19
--------------EM--------------------- ------------ ---K-M-----
20.sctn. --------------EM--------------------- ------------
------------ 21 --------------EM--------------------- ------------
---K------- 22 --------------EM--------------------- ------------
------M----- 23 --------------EL--------------------- ------------
------------ 24 --------------EL--------------------- ------------
------------ 25 --------L-----EM--------------------- ------------
---R-M----- 26 --------------EL-------------D------- --------TT--
---G--T----- 27 ----------L---EL--------------------- ------------
------------ 28 --------------EL--------------------- ------------
------M----- 29.dagger. --------L-----EM---------------------
------------ ------------ 30.dagger.
--------L-----EM--------------------- --------TT-- ---K------- 31
--------L-----EM--------------------- --------TT-- ---R-M----- 32
----------L---EL--------------------- --------TT-- ---R------- 33*
--------------EL-------------D------- ------------ ---K-------
34.dagger-dbl. --------------EL-------------D------- ------------
------------ 35 --------------EL-------------D------- ------------
------M----- 36 --------------EL-------------D------- ------------
---K------- 37 --------------EL-------------D------- ------------
---K-T----- 38 --------------EL-------------D------- ------------
---K-M----- 39.sctn. ----------L---EL---------------------
------------ ------------ 40.sctn.
----------L---EL--------------------- ------------ ---K------- 41*
----------L---EL--------------------- ------------ ---K-M-----
42.dagger. ----------L---EL--------------------- ------------
---R------- 43 ----------L---XL--------------------- ------------
---R------- 44 ----------L---EL--------------------- ------------
---R-M----- 45 ----------M---EM--------------------- ------------
---K------- 46 --------------EL--------------------- ------------
------------ 47 --------------EL--------------------- ------------
---K------- 48.dagger. --------------EM---------------------
------------ ---R------- 49 --------------EM---------------------
------------ ---R-M----- 50.dagger.
--------------EM--------------------- ------------ ------------ 51
--------------EM--------------------- ------------ ------M----- 52
----------L---EL--------------------- ------------ ---K-M-----
53 --------------EL-------------D------- ------------ ---R-------
54 --------------EL-------------D------- ------------ ---K-M-----
55.dagger. ----------L---EL--------------------- ------------
------------ 56 --------------EM--------------------- ------------
---K------- 57* ----------L---EL--------------------- ------------
------------ 58 --------------EL-------------D------- ------------
---K-M----- 59 --------------EL--------------------- --------N---
---K------- 60* --------L-----EM--------------------- --------TT--
------M----- 61* --------L-----EM--------------------- ---------TT-
---K-------
[0277] All members of the group that contained the neutralizing
antibody collection against epitope "A" from survivor 5, analyzed
to date, are shown in Table 1. The group is comprised of 61 unique
members that most closely resemble the variable (V) region gene
V.sub.H1e/V.sub.H1-69 germ line heavy chain. Some heavy chains are
paired with more than one light chain. In total these heavy chains
have 114 unique pairings to both kappa and lambda light chains.
Comparing these heavy chains to the highly related
V.sub.H1e/V.sub.H1-69 germline, we observe three types of point
substitutions. Some changes appear to be required, others are
dominant, and some residues have only been changed sporadically.
The changes that are required occur in every clone in the group
within CDR2 at position 52A (Pro>Gly), 53 (Ile>Met), and 57
(Ala>Thr), as well as in the framework 3 region at position 73
(Lys>Glu) and 74 (Ser>Leu or Met), all of which vary from the
germline side chain chemistries, suggesting that these mutations
are critical to antigen binding and neutralization. The second set
of mutations is dominant and found in most clones. The first, in
framework 1 at position 24 (Ala>Thr), represents a significant
chemical change. The next three are conservative changes in CDR1 at
positions 34 (Ile>Val) and 35 (Ser>Thr) and also in CDR2 at
position 50 (Gly>Ala). All four of these dominant substitutions,
however, are dispensable, suggesting that, while beneficial, they
are not essential. The sporadic changes found throughout framework
regions 1, 3, and 4, as well as CDR3, are all conservative and
likely represent minor optimization events.
[0278] Table 6 shows examples of sequences displaying the
Immunochemical basis of neutralization discovered from Survivor 5
libraries following H1N1 New Calcdonia panning. The 35 unique heavy
chain sequences aligned with their germline variable regions from
the 82 unique heavy and light chain combinations. Requisite
mutations are highlighted in bolded, underlined text (column 2--A
to T; column 3--IS to VT; column 5--PI to GM and A to T; column
6--KS to EL or EM and K to E) and predominant mutations are shown
in italicized, underlined text (column 2--Q to E; column 5--G to A;
column 6--I to L or M; column 8--Q to K or R or E). Heavy chains
sequences also discovered in H5N1 Vietnam panning are highlighted
in gray.
TABLE-US-00007 TABLE 6 Group 1 heavy FR1 CDR1 FR2 CDR2 chains
1-2930-35 30-40 47-56 Vh1e QVQLVQSGAEVKKPGSSVKVSCKASGGTF SSYAIS
WVRQAPGQGLE WMGGTIPIFGTAN 1* VT A GM T 2.dagger-dbl. T VT GM T 3
-----------------------T----- ----VT ----------- ------GM---T- 4
-----------------------T----- ----VT ----------- ------GM---T- 5
-----------------------T----- ----VT ----------- ------GM---T-
6.dagger. T VT A GM T 7 -----------------------T----- ----VT
----------- ------GM---T- 8* T VT A GM T 9 T VT A GM T 10* T VT A
GM T 11 -----------------------T----- ----VT -----------
------GM---T- 12 -----------------------T----- ----VT -----------
------GM---T- 13 -----------------------T----- ----VT -----------
------GM---T- 14 -----------------------T----- ----VT -----------
------GM---T- 15 -----------------------T----- ----VT -----------
------GM---T- 16.sctn. TT VT GM T 17 -----------------------TT----
----VT ----------- ------GM---T- 18 E--------------A-------T-----
----VT ----------- ------GM---T- 19.dagger. E R T VT GM T
20.dagger-dbl. E T VT A GM T 21 E T VT A GM T 22 E T VT A GM T
23.sctn. E T VT A GM T 24.sctn. E T VT A GM T 25* E T VT A GM T 26*
E----------------------T----- ----VT ----------- ------GM---T-
27.dagger. E T VT A GM T 28 E----------------------T----- ----VT
----------- ------GM---T- 29.dagger. E T VT GM T 30
E----------------------TT---- ----VT ----------- ------GM---T- 31
E----------------------TT---- ----VT ----------- ------GM---T-
32.dagger. M T VT A GM T 33 ----E------------------T----- ----VT
----------- ------GM---T- 34* Q T VT A GM T 35* Q R T VT GM T Group
1 heavy FR3 CDR3 FR4 chains 59-92 93-101 102-113 Vh1e
YAQKFQGRVTITADKSTSTAYMELSSLRSEDTAVYYC ARGSYYYESSLD YWGQGTLVTVSS 1*
L EL 2.dagger-dbl. ELR 3 ----------M---EM---------------------
------------ ------------ 4 ----------M---EM---------------------
------------ ------M----- 5 --------------EM---------------------
------------ ------------ 6.dagger. EL D 7
--------------EL-------------D------- ------------ ---K-------- 8*
EL D M 9 L EL 10* L EL M 11 ----------L---EL---------------------
------------ ------P----- 12 ----------L---EL---------------------
------------ ---R-------- 13 ----------L---EL---------------------
------------ ---R--M----- 14 ----------L---EL---------------------
------------ ---K-------- 15 --------------E----------------------
--------T--- ---K-------- 16.sctn. EM 17
--------------EM--------------------- ------------ ---R--M----- 18
--------------EM--------------------- ------------ ------------
19.dagger. --------L-----EM--------------------- TT K
20.dagger-dbl. EL D 21 EL D M 22 EL D K 23.sctn. L EL 24.sctn. L EL
K 25* L EL K M 26* ----------L---EL---------------------
------------ ---K--T----- 27.dagger. L EL R 28
--------------EL--------------------- --------N--- ---R--------
29.dagger. EM R 30 --------------EM---------------------
------------ ---R--M----- 31 --------------EM---------------------
------------ ---K--M----- 32.dagger. L EL 33
----------L---EL--------------------- ------------ ---E--T----- 34*
L EL 35* L EM TT M
[0279] In Table 6, antibody regions and Kabat numbering ranges are
listed at the top of each sequence column. (*--paired with 2 unique
light chains, .sup..dagger.--paired with 3 unique light chains,
.sup..dagger-dbl.--paired with 4 unique light chains,
.sup..sctn.--paired with 5 unique light chains,
.sup..dagger-dbl.--paired with 13 unique light chains).
[0280] FIG. 7 shows the positions of the required mutations in the
structure of the antibody superimposed on the crystal structure of
a highly related anti-HIV Fab called 47e (1rzi.pdb) (Huang, C. C.
et al. (2004) Proc. Nat. Acad. Sci. 101, 2706-2711). FIG. 7 shows
the positions of H5 hemagglutinin binding Group 1 required and
dominant mutations on the crystal structure of Fab 47e. The
required mutations are shown as G52 (52A (Pro>Gly)), M53
(Ile>Met), T57 (Ala>Thr), E73 (Lys>Glu) and LM74
(Ser>Leu or Met). The dominant mutations are shown as T24
(Ala>Thr), V34 (Ile>Val), T35 (Ser>Thr), and A50
(Gly>Ala). The required and dominant Group1 heavy chain
sequences identified in H5 Vietnam/1203/2004 HA biopanning are
superimposed on the crystal structure of the highly related
anti-HIV Fab 47e. Mutations are shown in both backbone (top) and
space-filling (bottom) models. A tight cluster is formed by four of
the required mutations in and adjacent to CDR2. The required
mutations 52A (Pro>Gly), 53 (Ile>Met), 73 (Lys>Glu) and 74
(Ser>Leu or Met) form a remarkably tight cluster on the exposed
surface of the heavy chain variable domain where they form a ridge
that prominently protrudes from the protein surface (FIG. 7). The
remaining required mutation 57 (Ala>Thr) is partially buried at
the base of the CDR2 loop. The surface exposed changes in CDR 2 and
framework 3 are likely to have a direct role in antigen binding
while the less exposed required mutation and the non-essential
dominant mutations may have indirect effects through stabilizing
and/or positioning the CDR2 loop.
[0281] The antibodies from survivor 2 are comprised of 2 unique
heavy chains that most closely resemble the V.sub.H4-4b germ line
heavy chain (Table 7). The first heavy chain has been found paired
with 5 unique lambda light chains, four of which are from the
infrequently used lambda 6 light chain family and the other is
paired with a single kappa light chain. Antibody 4 whose
neutralization profile was more restricted came from this
group.
TABLE-US-00008 TABLE 7 FR1 CDR1 FR2 CDR2 Group 2 (1-29)
(30-35)(36-46) (47-58) Heavy QVQLQESGPGLVKPSETLSLTCTVSGYSF DSGYYWG
WLRQPPGKGLE WIGSIYHSRNTY chain Lambda FMLTQPHSVSESPGKTVTISCTGSGGN
IARNYVQWY QQRPGSAPV TVILEDDKRP light FMLTQPHSVSESPGKTVTISCTGSSGS
IASNYVQWY QQRPGSAPT TVIYEDYQRP chains SVLTQFPSASGTPGQRVTISCSGSSSN
IGSNTVNWY KQLPGTAPR LLIYSNDQRP SVLTQPPSASGTPGQRVTLSCSGSSSN
IGGNSVNWY QHVPGTAPK LLMHSDDQRP PELTQPHSVSESPGKTVTISCTGSGGR
IATNHVQWY QQRPGSAPT IVIYENNQRP PELTQPPSASGTPCQRVTISCSGSSSN
IGSNTVNWY QQLPGTAPK LLIYSNNQRP Kappa DIQMTQSPSSLSAFVGDRVTITCQASQDI
SNYLNWY QQKPGKAPK LLIYDATNLE light chain FR3 CDR3 FR4 Group 2
(59-92) (93-101) (102-113) Heavy
YNPSLKSRVTISVDTSKNQFSLQLSSVTAADTAVYYC ARGTWYSSNLRYWFD PWGKGTLVRVSS
chain Lambda SGIPDRFSGSIDRSSNSASLTISGLRTEDEALYYC QSYDDSDLV
VFGGCTKLT light SGVPDRFSGSIDSSSNSASLTISGLKTEDEADYYC QSYDDSDHL
IFGGGTKLTVL chains SGVPDRFSGSKSGTSASLAISGLQSEDEANYYC AAWDDSLSGW
VFGGGTKLTVL SGVPDRFSGSKSGTSXSLAISGLQSEDEADYYC AXWDDSLNAW
VFGGXTKVTVL SGVPNRFSGSIDDSSNSASLTTSALRTEDEADYYC QSADATNV
FFGGGTKVTVL SGVPDRFSGSKSGTSASLAISGLQSEDEADYYC AAWDDSLNGW
VFGGXTKLTVL Kappa TGVPSRFSGSGSGTDFTFTISSLQPEDIATYYC QQYDNLPL
TFGGGTKVDIKR light chain
[0282] The probability that a given mutation is important to the
activity of an antibody increases as a function of the number of
times it was independently selected. To determine if the required
mutations were selected during somatic mutation from independent
clones or were from the progeny of a single clone that further
mutated during subsequent replications, the codon usage of the
dominant mutations were analyzed (Table 8A-8B). The data reveal
that although different codons were used they resulted in the same
amino acid changes, demonstrating that these mutations arose
independently in different clones and were, thus, selected multiple
times. This convergent outcome for independently selected events is
strong evidence that these dominant mutations play a critical role
in the binding to the virus and/or its neutralization.
[0283] As illustrated in Table 8A-8B, codon usage of individual
clones shows independent origin of selected H5 HA binding clones.
DNA alignment and encoded amino acids for 6 representative Group 1
antibodies against the VH1-e germline. The use of different codons
for the same amino acids demonstrates that each unique sequence is
of a distinct origin. Table 8A corresponds to CDR2 and Table 8B
corresponds to Framework 3. Germ line codons are shown as bolded
codons. A change from a germ line codon to the same amino acid is
shown as a plain text codon. A first change from a germ line amino
acid is shown as a bolded, underlined codon. A second change from a
germ line amino acid is shown as an italicized, underlined codon. A
third change from a germ line amino acid is shown as an underlined,
grayed-out codon.
TABLE-US-00009 TABLE 8A Kabat Sequence CDR2 Clone Number 47 48 49
50 51 52 52A 52B 520 53 54 55 56 57 58 VH1-e germilne TGG ATG GGA
AGG ATC ATC CCT ATC CTT GGT ATA GCA AAC TAC GCA W M G R I I P I L G
I A N Y A 27 TGG ATG GGC GCG ATC ATC GGT ATG TTT GGT ACA ACA AAC
TAC GCA W M G A I I G M F G T T N Y A 30 TGG ATG GGA GGG ATC ATC
GGT ATG TTT GGA ACA ACC AAC TAT GCA W M G G I I G M F G T T N Y A
33 TGG ATG GGA GCG ATC ATC GGT ATG TTT GGT ACA ACA AAC TAC GCA W M
G A I I G M F G T T N Y A 41 TGG ATG GGC GCG ATC ATC GGT ATG TTT
GGT ACA ACA AAC TAC GCA W M G A I I G M F G T T N Y A 50 TGG ATG
GGA GGG ATC ATC GGT ATG TTT GGT ACA ACG AAC TAT GCA W M G G I I G M
F G T T N Y A 17 TGG ATG GGA GGG ATC ATC GGT ATG TTT GGT ACA ACA
AAC TAC GCA W M G G I I G M F G T T N Y A #Codons used 1 1 2 2 1 1
1 1 1 2 1 3 1 2 1
TABLE-US-00010 TABLE 8B Kabat Sequence Clone Framework 3 Number 67
68 69 70 71 72 73 74 75 76 77 78 VH1-e GTC ACG ATT ACC GCG GAC AAA
TCC ACG AGC ACA GCC germline V T I T A D K S T S T A 27 GTC ACG CTT
ACC GCG GAC GAA TTA ACG TCC ACA GCC V T L T A D E L T S T A 30 CTC
ACA ATC ACC GCG GAC GAG ATG ACG TCC ACA GCC L T I T A D E M T S T A
33 GTC ACA ATC ACC GCG GAC GAA TTA ACG TCC ACA GCC V T I T A D E L
T S T A 41 GTC ACG CTT ACC GCG GAC GAA TTA ACG TCC ACA GCC V T L T
A D E L T S T A 50 GTC ACG ATT ACC GCG GAC GAG ATG ACG TCC ACA GCC
V T I T A D E M T S T A 17 GTC ACG ATT ACC GCG GAC GAA TTA ACG TCC
ACA GCC V T I T A D E L T S T A #Codons used 2 2 3 1 1 1 2 2 1 1 1
1
[0284] The present report raises two central issues relative to the
prevention and treatment of infections caused by the avian
influenza neutralized virus. The first concerns the importance of
antibodies relative to other components of the immune system. While
it has been known for over 80 years that passive administration of
immune sera can prevent infection Luke, T. C. et al., Kilbane E M,
Jackson J L, & Hoffman S L (2006) Ann Intern Med 145, 599-609),
more recent studies with monoclonal antibodies also offer
encouragement (Hanson, B. J. et al. (2006) Respir Res 7, 126;
Huang, C. C. et al. (2004) Proc. Nat. Acad. Sci. 101, 2706-2711;
Simmons C. P. et al. (2007) PLoS Med 4, e178). For example, Hanson
et. al. showed that a monoclonal antibody to H5N1 virus was
completely protective against lethal infection, even when given
three days post inoculation in mice (Hanson, B. J. et al. (2006)
Respir Res 7, 126). Given the possibility of a catastrophic
epidemic, the way forward seems clear to many in the field. It has
been suggested that governments should maintain stocks of
neutralizing antibodies such as those reported here. The facts that
our antibodies are fully human and have been isolated from
individuals who successfully combated viral infection may offer
advantages. However, even if such antibodies are stockpiled,
hurdles remain. For instance, if the gene encoding the epitope to
which the antibody binds were to mutate, then the antibody might be
less effective. Also, there is some evidence that cellular immunity
enhances clearance of the virus. Nevertheless, if the only effect
of passive immunization was to lessen the severity of infection,
thereby giving the necessary time for other immune effectors to
operate, it could be of critical importance for lessening mortality
in patients with weakened immune, cardiovascular, and respiratory
systems and in the elderly. Passive immunization might prevent the
cytokine storm against rapidly proliferating virus, as occurred
even in healthy young adults during the 1918 influenza
outbreak.
[0285] The second important feature of this report relates to the
special advantages that antibodies from combinatorial libraries
bring to the problem (Lerner R A (2006) Angew Chem Int Ed Engl 45,
8106-8125). The most general aspect is that, because such libraries
are nucleic acid based, they are not are not dependant on whether
an important antibody is currently being produced. This obviates
any concern about when in the course of the disease the sample was
obtained. Indeed, as is the case here, when the source of antibody
genes is the bone marrow, the entire immunologic history of an
individual's antibody response may be obtained, irrespective of
whether an antibody is actively expressed or is stored in the
memory compartment. Thus, in the analysis of antibody ontogeny in
the individuals studied here, the time factor is eliminated and one
can get a clearer view of the precursor product relationships
between related antibodies. In this respect, one of the most
remarkable features of some of our antibody collections (i.e. group
1) is that the required somatic mutations are confined to framework
3 or CDRH2 rather than CDRH3 where they would be most expected to
occur. This may be because the extreme virulence of the virus
imposes time pressure on the evolution of the immune response. To
survive an H5N1 avian influenza virus infection, one must mount an
effective immune response rapidly. Because the framework regions
and CDR2 of the protein are structurally rather constrained, the
evolutionary search of sequence space for increased binding energy
through somatic mutation may be more efficient for these regions
than for a similar search through the more flexible and diverse
CDR3 region. Indeed, it is well known, mostly from attempts to
humanize antibodies, that framework mutations can directly or
indirectly affect binding energy and/or specificity (Foote J &
Winter G, (1992) J Mol Biol 224, 487-499; Holmes, M. A. et al.
(2001) J Immunol 167, 296-301). Alternatively, the immune system
may use frameworks and/or CDRs that have been previously optimized,
perhaps in response to an earlier exposure to a similar virus.
Regardless of the exact mechanism, our results are in broad
agreement with those of Zinkernagel and colleagues who studied the
immune response against lethal vesicular stomatitis virus
infections in mice (Kalinke U et al. (1996) Immunity 5, 639-652;
Kalinke U et al., (2000) Proc Natl Acad Sci USA 97, 10126-10131).
In their studies, only one V.sub.H germline gene was used and the
primary neutralizing immune response was devoid of somatic
mutations. Only later did somatic mutations in the CDRs appear. It
should be emphasized that while our analysis to date has revealed
many interesting antibodies, we have so far only analyzed a small
fraction of the library. As further analyses are carried out, we
expect to see many other immunochemical solutions to the problem of
virus infection.
[0286] From an antibody engineering point of view, the large
database unique to antibody libraries creates a roadmap for
improving the binding energy and/or specificity of the antibodies,
if necessary. For example, one understands immediately that there
are heavy chains (Tables 1, 2, and S14) that are highly promiscuous
with respect to their light chain partners. These heavy chains are
ideal for light chain shuffling experiments where very large
numbers of new light chains are paired with a single promiscuous
heavy chain (Lerner R A (2006) Angew Chem Int Ed Engl 45,
8106-8125; Kang, A. S. et al. (1991) Proc Natl Acad Sci USA 88,
11120-11123). Ultimately, the best features of different antibodies
can be amalgamated into a single antibody that can be highly
effective and even overcome viral escape by mutation. This is
especially likely when consensus sequences important to
neutralization occur either in the different antibody chains or in
different CDRs or frameworks within a chain or both. Thus, many
combinations can be tested and an amalgamated antibody could
contain the best elements of these various loops and frameworks.
Critically, when some of the features incorporated into the
amalgamated antibodies represent alternative binding modes to a
neutralization target on the virus, one would expect viral escape
to be more difficult.
[0287] There is another feature that derives from the large numbers
of antibodies obtained from libraries that may be of particular
importance to the influenza problem. Many, if not most, of the
antibodies that result from an infection have little to do with
prevention of further infectivity and are simply a response to the
foreign nature of the virus. Thus, if one has only a few antibodies
to choose from, one might miss the most important rare antibodies
because they are under-represented in the bulk immune response.
Indeed, this may be a feature of the most potent antibodies since
they need only be present in small concentrations and/or may occur
late in an infection only after many other "attempts" were tried
during the evolution of an immune response. We have seen this
phenomenon in human libraries from cancer patients where antibodies
that prevent metastasis are present at the very rare frequency of
about one out of 1.0.times.10.sup.8 library members
(Felding-Habermann B, et al. (2004) Proc Natl Acad Sci USA 101,
17210-17215). The features that one might screen for that would be
expected to be rare are, for example, antibodies that exhibit broad
neutralization or have unusual access to important tissue
compartments. Toward this end, it will be interesting to see if
there are any neutralizing antibodies in our collection of clones
that bind virus but are not directed to the hemagglutinin.
[0288] The analysis of the immune response from actual cases can
give guidance for both new passive antibody therapy and vaccine
design. For example, we already know that patients make antibodies
against the hemagglutinin that are broadly reactive between H5 and
H1 strains, but skip H3. We could not learn this from simple
serology because serum contains a collection of activities as seen
here for our patients and, thus, it is impossible to determine the
clonal basis of any reactivity from an analysis of sera. The
localization of the cross-reactive epitopes already found here as
well as others is now relatively straight forward using antibodies
from the library as a guide. Access to multiple antibodies from
several survivors of the viral infection also enables the mapping
of common epitopes, other than hemagglutinin, to which all
survivors have developed high affinity antibodies. The knowledge of
several previously unknown epitopes could provide the foundation
for the design of novel vaccines.
[0289] Characterized neutralizing antibodies can also give
information regarding the potential efficacy of candidate vaccines.
For instance, one can determine if particular traditional or
recombinant vaccine preparations generate antibody classes that
have proven to be neutralizing from analysis of survivors of actual
infections. Furthermore, these antibodies can be used as test
reagents to ensure that epitopes that are important to
neutralization are properly presented in the vaccine constructs.
While this later point might seem trivial, there has heretofore
been no simple way to learn whether critical epitopes are destroyed
during construction of subunit vaccines or even during formulation
of intact virus preparations.
[0290] Finally, we come to the often asked interesting question of
whether it matters that the libraries were prepared from patients
who successfully combated an infection as opposed to animals or
people that simply have been immunized with viral antigens. Because
a substantial fraction of patients in our cohort died, it is
tempting to speculate that the survivors made antibodies that were
related to their favorable clinical outcome. This is a difficult
argument because so many factors contribute to patient survival,
several of which have little to do with the robustness of the
immune response. It simply should be said that natural antibodies
obtained from survivors can reasonably be expected to be at least
as good as, and perhaps better, than those obtained after simple
immunization with inert antigens. At the very least, one can be
certain that the virus has been presented in a manner that allowed
an immune response appropriate to survival of the individual. Thus,
we gained insight from this analysis about how the immunological
repertoire searches sequence space when, because of the virulence
of the infectious agent, time is short.
[0291] Materials and Methods
[0292] H5N1 Turkish cases--bone marrow recovery. Six H5N1 survivors
provided bone marrow and serum for this study. All were diagnosed
between December 2005 and January 2006. Descriptions of their
diagnoses have been previously reported (Oner et al., (2006) N.
Engl. J. Med. 355:2179-2185). Briefly, most samples were
nasopharyngeal swabs tested by ELISA, rapid influenza test, and/or
real-time polymerase chain reaction in Turkey. Additionally, four
of the six survivors were further verified by WHO laboratory
testing in London. Following four and five months post recovery
bone marrow aspirates and serum from the six survivors were
collected, minimally processed in RNALater (Ambion) to preserve RNA
integrity, and shipped frozen on dry ice to our laboratories. This
study was reviewed and approved by both the Turkish Ministry of
Health and the Yuzuncu Yil University, Van, Turkey. Written
guardian consent was provided for all donors.
[0293] Antibodies Proteins and viruses. Hemagglutinin proteins were
either purchased from Protein Sciences (H5 Protein
A/Vietnam/1203/2004, H1 Protein A/New Calcdonia/20/99, H3 Protein
A/Wisconsin/67/05) or generated by de novo synthesis (H1 Protein
A/South Carolina/1/18) as eukaryotic codon optimized soluble
secreted HA genes (DNA 2.0) and then subcloned into pCI (Promega)
for mammalian protein expression, sequence verified, and then
transfected into 293 Freestyle cells (Invitrogen) according to
manufacturers guidelines. Briefly 20 .mu.g of light chain and 10
.mu.g heavy chain encoding plasmid were combined with 1.0 m1293
fectin and incubated for 60 minutes. Following this preincubation
the DNA mixture was combined with 3.times.10.sup.7 cells in 30 ml
media for then and the resulting cell suspension was grown
according to manufacturers suggestion for 7 days. After seven days
the secreted immunoglobulins were purified from the culture
supernatants using protein A chromatography (Calbiochem). The
resulting purified antibodies were buffer exchanged into sterile
PBS using centrifugal size filtration (Centricon Plus-20) and their
protein concentrations determined by calorimetric BCA assay
(Pierce).
[0294] Recombinant viruses were genetically engineered and produced
as described elsewhere (Fodor et al., (1999) J. Virol.
73:9679-9682). Additionally, Indonesia, Turkey, and Egypt were
similarly made except their HA genes were synthetically assembled
using eukaryotic codon optimized sequences (DNA 2.0). Inactivated
viruses were made as described elsewhere (Fodor et al., supra).
[0295] Serology: Hemagglutinin and viral ELISA. Recombinant HA
proteins: H5 Protein A/Vietnam/1203/2004 (Protein Sciences)--10
ng/well; Recombinant HA H1 Protein A/New Calcdonia/20/99 (Protein
Sciences)--10 ng/well; Recombinant HA H3 Protein A/Wisconsin/67/05
(Protein Sciences)--10 ng/well; H1N1 Virus A/New Calcdonia
(BioSource)--70 ng/well; H3N2 Virus A/Panama/2007/99
(BioSource)--10 ng/well; FDA Influenza Virus Vaccine for H5N1
rgA/Vietnam/1203/2004 (CBER)-- 10 ng/well.
[0296] ELISA plates were coated as indicated with either
recombinant hemagglutinin protein or inactivated virus overnight
incubation at room temperature. The next day plates were
appropriately blocked (1% bovine serum albumin in PBS/0.05%
Tween-20) and then 0.1 ml serum samples, diluted in blocking
buffer, were incubated, washed, and detected using a peroxidase
conjugated anti-human Fe antibody (Jackson Immuno) and TMB
detection (BioFX). Absorbance at 450 nm was read, data recorded,
and reported herein.
[0297] Donor specific repertoire recovery. Between 2-2.5 ml of
donor bone marrow previously stored in 25 ml RNAlater (Ambion) was
processed with TRI-BD (Sigma) according to manufacturers directions
and then further processed to extract purified total RNA as
described elsewhere (Barbas et al., (2001) Phage Display: A
Laboratory Manual (Cold Spring Harbor Lab Press, Cold Spring
Harbor, N.Y.)). Next mRNA was purified by Oligotex spin column
purification (Qiagen). Next random nonamer primed reactions and
oligo dT reverse transcription reactions performed using Accuscript
(Stratagene) according to manufacturers directions.
[0298] For each donor the following was performed. For each of the
11 lambda light chain families a single PCR amplification was
performed with family specific V.sub.L primers, a mixture of
J.sub.L primers, and primed with 75 ng Oligo dT cDNA. Kappa
recovery was similarly performed for each of the six kappa light
families, except that 75 ng of random nonamer primed cDNA was used.
For heavy chain recovery VH1/7, VH3, and VH4 were individually
recovered, and VH 2,5, and 6 were pool amplified with gene specific
primers, a mixtures of J.sub.H primers, and each was primed with
100 ng of random nonamer primed cDNA. Primers and amplification
conditions were essential as described elsewhere using Platinum Pfx
polymerase (Invitrogen) (ref). PCR products were minimally
processed by PCR Cleanup (Qiagen) quantitated by A.sub.260. Heavy
chain reactions were gel purified and then, if necessary, amplified
again to produce quantities sufficient for cloning.
[0299] Phage Library Construction.
[0300] Light chain cloning. Donor specific barcoded vectors and
equimolar pools of Kappa and lambda light chains were separately
digested with NotI and BamHI and gel purified using (Qiagen).
Library ligations were performed with 200 ng of gel purified Kappa
or Lambda inserts and 1 .mu.g of gel purified vector. Incubation is
for at least one hour at RT or overnight at 14.degree. C. Ligations
were desalted using Edge BioSystem Perfroma spin columns. 5
electroporations per library were done in 80 .mu.l TG-1 aliquots,
each recovered in 1 ml SOC, pooled and outgrown for one hour at
37.degree. C. A sample of each was taken for plating and used to
determine the total number of transformants. The remainder was
transferred to 200 ml 2YT+100 .mu.g/ml Ampicillin+2% glucose and
grown overnight at 37.degree. C. Target number of
transformants/library was at least 1.times.10.sup.6/.mu.g vector
DNA. Light chain library plasmids were then pelleted and the
plasmids purified using a Qiagen High Speed Maxiprep Kit.
[0301] Heavy chain cloning and phage production. Donor specific
heavy chains (V.sub.H1, V.sub.H 3, V.sub.H 4, and V.sub.H 2, 5, 6
pool) and light chain library collections were separately digested
with a 40 Unit/.mu.g DNA with SfiI and XhoI and gel purified
(Qiagen). 5 .mu.g of kappa and lambda light chain libraries were
separately ligated, overnight, with 1.2 .mu.g of an equimolar mix
of the four donor specific heavy chain preparations. The library
ligations were spin column desalted (Edge BioSystem) and then
transformed in 16-20 electroporations per library. Processing to
determine the number of transformants is as described above. Phage
production proceeded as described elsewhere. Following phage
production the phage was harvested by PEG/NaCl precipitation and
resuspended and stored in PBS containing 50% glycerol.
[0302] Panning and Clonal ELISAs. Panning and clonal ELISAs were
performed as described previously (Fodor, E. et al. J Virol. 1999;
73(11): 9679-82).
[0303] Microneutralization. Cross sub-type neutralization by
antibodies recovered from survivors of avian influenza. Indonesia
and Turkey hemagglutinin genes were synthetically assembled using
human codon optimized sequences (DNA 2.0) and then used to generate
recombinant engineered viruses. Recombinant influenza viruses were
generated using reverse genetics as previously described (Fodor, E.
et al. J Virol. 1999; 73(11): 9679-82). Briefly, 1 ug each of 10
plasmids was transfected into 293 T cells in monolayer. Each
transfection contained ambisense plasmids (for the expression of
both vRNAs and mRNAs) for the A/Puerto Rico/8/34/PA, PB1, PB2, NP,
M, and NS segments, in addition to vRNA (pPOL1 type) and protein
expression plasmids (pCAGGS type) for A/Vietnam/1203/04 HA and NA
(pCAGGS expression plasmid was kindly provided by J. Miyazaki,
Osaka University, Osaka, Japan) (Miyazaki, J. et al. Gene 1989;
79(2):269-77). Twenty hours following transfection, 293T cells were
resuspended in cell culture supernatant, and used to inoculate
10-day-old embryonated eggs.
[0304] Antibodies were screened for neutralizing activity against
viruses as follows. Two fold serial dilutions of each Mab were
incubated with 100 TCID.sub.50 of virus in PBS at 370 for 1 h.
Madin-Darby Canine Kidney cell monolayers in 24 well plates were
washed once with PBS and inoculated with virus-antibody mixtures.
Following incubation for 1 h at 37.degree. C. in 5% CO.sub.2, the
inoculum was removed and monolayers were again washed once with
PBS. Opti-MEM supplemented with 0.3% BSA, 0.01% FBS and 1 ug/ml
TPCK-treated trypsin was added and cells were incubated for 72 h at
37.degree. C. The presence of virus in cell culture supernatants
was assessed by HA assays using 0.5% chicken red blood cells.
[0305] Cross-reaction IgG ELISA. Microtiter plates were coated with
0.1 ml of the following antigens diluted in coating buffer and
incubated overnight at room temperature: 100 ng/ml H5N1 Vietnam
1203/04, 250 ng/ml H5N1 Turkey/65596/06, 1 .mu.g/ml H5N1
[0306] Indonesia/5/05, 700 ng/ml H1N1 New Calcdonia/20/99, 1
.mu.g/ml H1N1 North Carolina/1/18, 100 ng/ml and H3N2
Wisconsin/67/05. Blocking was done with 0.3 ml of blocking buffer
(4% Non-fat dry milk in PBS/0.05% Tween-20). Following blocking
antibodies diluted to 0.5 .mu.g/ml in 2% non-fat dry milk blocking
buffer were incubated for two hours at 4 C, washed, and later
detected using a 1:3000 dilution of peroxidase conjugated
anti-human F.sub.c antibody (Jackson ImmunoResearch) in 2% non-fat
dry milk blocking buffer and standard TMB substrate detection
(BioFX). Absorbance at 450 nm was read, data recorded, and reported
herein. (B) Relative ranking of antibodies by their ELISA signal to
noise ratios (-<2, +=2-<9, ++=9-<15, +++=.gtoreq.15), on
various proteins and minimal inhibitory concentration (MIC) in
microneutralization assay. Suitable protocols can be found in
Barbas C. et al. (2001) Phage Display, A Laboratory Manual (Cold
Spring Harbor Laboratory Press).
[0307] Epitope Analysis of Hemagglutinin-Binding Fabs.
[0308] Biotinylation of HA Proteins: 100 ug of purified
Hemagglutinin protein is biotinylated at a 20:1 molar excess using
Pierce No-Weigh PEO4 biotin (cat #21329) according to manufacturers
instructions, incubated at room temperature for 1-3 hours with
intermittent mixing and then incubated overnight at 4 C. The excess
biotin is removed by size exclusion spin column and exchanged into
PBS.
[0309] Quantitation of Fabs: HA binding Fabs are purified by FPLC
using Ni.sup.2+ affinity chromatography, desalted to remove excess
imidazole, concentrated, and quantitated by quantitative light
chain ELISAs (Bethel Labs, cat # E80-115-.kappa., and
E80-116-.lamda.) are performed according to the manufacturers
instructions.
[0310] Sample set up: HA protein is bound to sensors and allowed to
reach new baseline. Next, sample and epitope binding standards are
tested for HA saturation using the conditions determined from
kinetic analysis. Desalted, concentrated Fabs were evaluated for HA
binding in a typical range of 0.5-16 ul in 200 ul sample diluent.
Using the conditions identified in saturation testing, standard
epitope binding antibodies are first loaded on to HA coated
biosensors. A new baseline is established and then the test samples
at half saturation concentrations are loaded on to the epitope
saturated sensors. Antibodies are tested against all possible
epitope recognition standards in this way. The following is a
summary of the sample type and time the sensors are held in each
column of solution:
TABLE-US-00011 Column 1 Baseline Sample Diluent 1-2 minutes Column
2 HA Binding Biotinylated HA 5-15 minutes Column 3 Baseline Sample
Diluent 1-2 minutes Column 4 Saturation Diluted antibodies 5-15
minutes Column 5 Baseline Sample Diluent 1-2 minutes Column 6
Sample binding Diluted test antibody 5-15 minutes Column 7 Baseline
Sample Diluent 1-2 minutes
[0311] Increased interference shift above saturation levels
indicates novel epitope recognition. Three possible results from
this type of analysis are:
[0312] 1) Complete blocking--No interference shift
[0313] 2) Restoration of saturation--If dissociation of the
standard occurs during the baseline after binding, sample binding
that restores the signal to saturation levels indicates binding to
the same epitope
[0314] 3) New epitope binding--Increased interference shift above
saturation levels
[0315] Kinetic Analysis of Hemagglutinin-Binding Fabs.
[0316] Biotinylation of HA Proteins: 100 ug of purified
Hemagglutinin protein is biotinylated at a 20:1 molar excess using
Pierce No-Weigh PEO4 biotin (cat #21329) according to manufacturers
instructions, incubated at room temperature for 1-3 hours with
intermittent mixing and then incubated overnight at 4 C. The excess
biotin is removed by size exclusion spin column and exchanged into
PBS.
[0317] Quantitation of Fabs: HA binding Fabs are purified by FPLC
using Ni.sup.2+ affinity chromatography, desalted to remove excess
imidazole, concentrated, and quantitated by quantitative light
chain ELISAs (Bethel Labs, cat # E80-115-.kappa., and
E80-116-.lamda.) are performed according to the manufacturers
instructions.
[0318] Kinetic Analysis: Kinetic analysis is performed on a range
of sample concentrations that are empirically determined. The first
range is typically 15 nM-500 nM in serial 2 fold dilutions and the
samples are incubated with biosensors coated with HA protein for up
to 15 minutes, then incubated in sample diluent for up to 1 hour.
All of these steps are done with sample plate rotation at 1500 RPM.
Association is measured during the Fab incubation with the
HA-coated biosensors and dissociation is measured in the sample
diluent incubation following binding. The following is a summary of
the sample type and time the sensors are held in each column of
solution:
TABLE-US-00012 Column 1 Baseline Sample Diluent 1-2 minutes Column
2 HA Binding Biotinylated HA 5-15 minutes Column 3 Baseline Sample
Diluent 1-2 minutes Column 4 Association Diluted antibodies 5-15
minutes Column 5 Dissociation Sample Diluent 15-180 minutes
[0319] Data analysis using the Forte Bio Kinetic Analysis software
provides estimates of on and off rates with r.sup.2 values. A value
is deemed to be reportable if of high confidence with r.sup.2
values >0.95. The k.sub.D is then accepted as the affinity of
the molecule.
[0320] Viral microneutralization. The VN activity of MAbs was
measured as follows. MAb dilutions (50 ml) in ISC-CM--0.1% BSA,
eight replicates per dilution, were dispensed into 96-well
flat-bottom tissue culture plates. PR8 (50 ml) in ISC-CM--0.1% BSA;
(100 TCID50) were added to each well, and the plates were incubated
for 1 h at 37.degree. C. MDCK cells were then added to each well
(25 ml ISC-CM--0.1% BSA containing 2 3 106 cells/ml), and the
plates were incubated for 8 to 14 h to permit MDCK cells to adhere.
The medium was then flicked out and replaced with 200 ml of
antibody-free ISC-CM--0.1% BSA supplemented with trypsin (2.5%
trypsin [Whittaker Bioproducts Inc.]) at a final dilution of
1/3,000; (8 mg/ml). After another 2.5 days of incubation, culture
supernatants were tested for the presence of virus by HA titer
determination. The MAb concentration at which 50% of the cultures
were protected from infection was computed by interpolation and
taken as the MAb VN activity. Note that low concentrations indicate
high VN activity (Mozdzanowska, K. et al. (1997) J. Virol. 71,
4347-4355).
Example 2
Generating Universal Influenza Vaccines
[0321] The goal of vaccine design against heterogeneous pathogens
is to identify and design effective and broadly protective
antigens. In the case of influenza, considerable historical efforts
have gone into the empirical testing of conserved linear sequences
and regions with little success. A plausible reason for these
failures is a lack of knowledge that focused responses against
antigenic test articles are actual bona fide productive sites for
neutralization of an antigen on the pathogen in the setting of an
actual infection. For influenza one would be expect to find these
bona fide solutions within the repertoires of survivors of an
influenza infection. In our case we have demonstrated that several
related antibodies amongst a large collection of antibodies,
derived from an H5N1 influenza survivor, (see Table 4 above) are
capable of broadly neutralizing several subtypes of Influenza.
These antibodies neutralize influenza through a novel mechanism
that does not involve classical inhibition of hemagglutination,
which has now been confirmed and delineated at a structural level
by two additional and independent groups. Collectively, we expect
that the design and assessment of vaccines utilizing such cross
neutralizing antibodies derived from bona fide survivors would aid
in the design and validity of cross reactive or "universal"
influenza vaccines.
[0322] Specifically cross neutralizing monoclonal antibodies can be
used in the design and validation of vaccine production processes
that maintain or enhance the quality and antigenicity of cross
neutralizing epitopes in current and future manufactured vaccines.
Assuming that antibody binding to vaccine is reflective of
structural integrity and antigenic potential, one would assess
binding of cross neutralizing antibodies, such as Ab-1 (see Table 4
above) to such vaccine process derivatives to quantitatively assess
their cross neutralizing potential.
[0323] To maximize the responses toward these universal epitopes
one would create derivatives to increase immunogenicity towards
these universal epitopes. In this case the resulting antigen would
again be tested to insure that not only the efficiency of binding
to target was maintained, but that a directed immunogenicity was
accomplished. This would either involve determining the specific
universal neutralizing titers contained in the serum from immunized
individuals or test animals, likely by competitive ELISA against
Ab-1 (or related antibody) from Table 4. As an in vitro surrogate,
one would combine the antigen-antibody binding data with that of an
in vitro or in silico predictive model for immunogenicity. To
further direct responses to the universal epitope one may
deimmunize known non-neutralizing hemagglutinin epitopes
[0324] It reasonable to extend this antibody for the design and
validation of engineered recombinant hemagglutinin chimeras,
fragments, and conformational mimics. For instance, it is well
established that influenza contains many immunodominant epitopes
that give rise to non-neutralizing responses. Utilizing the cross
protective antibodies it is possible to assess whether antigen
variants of vaccines that have been partially or fully deimmunized
for these immunodominant non-neutralizing epitopes have maintained
or created enhanced recognition of the universally protective
epitopes.
[0325] Additional ways to guide a specific response to a distinct
epitope is to simply remove non-neutralizing and non-conserved
regions from the recombinant vaccine design. As an example we would
remove the HA1, or HA0 globular sialic binding domain of
hemagglutinin to leave the more conserved stem region of
hemagglutinin as the principal target for an immune response. As
sequence space does not strictly correlate to physical space, this
will require the removal of middle coding regions for proteins to
create such aglobular constructs. Further as more of the globular
domain is removed this will cause residues that are normally
embedded within the protein structure to be exposed. These residues
that are not normally solvent exposed may need to be mutagenized
and deimmunized to residues that are better suited structurally or
more compatible to solvent exposure. Similar to efforts described
above, we would use the antibodies identified previously to insure
the integrity of these cross protective epitopes.
[0326] From these aglobular vaccine designs, one could minimize the
antigen epitopes and even remove them from the context of
hemagglutinin to create a conformational cross specific
antigen.
[0327] The strategies outlined above detail methods to guide a
response to a minimized neutralizing epitope or element. From the
knowledge of such minimized elements, which are likely be
conformationally dependent and exist within discontinuous sequence
space, it would be possible to recreate the conformational
neutralizing epitope in a combinatorial fashion within a smaller
polypeptide, as described previously (see Horowitz et al.,
Combinatorial Libraries of Conformationally Constrained Polypeptide
Sequences, PCT/US2008/050877) where the proximal placement of
discontinuous epitopes alone, or in the context of designed
structural support, can regenerate the essential properties of
conformational epitopes.
[0328] In such a design we would take the conformation epitope and
express them on hemagglutinin related and unrelated structural
scaffolds, or even as a collection of conformational epitopes
within a library that could be selected by conformationally
dependent antibodies such as Ab-1.
[0329] The reduction of discontinuous epitopes to a conformational
epitope would result in an even smaller sized peptide immunogen
than that possible with traditional protein engineering.
Furthermore these structural epitopes may be further enhanced,
reduced in size, or substituted through the use of nonpeptide
mimetics. In any event, any of these conformational derivatives or
mimics would be validated by the Ab-1, Ab-1 related antibody, or
corresponding antibody to the influenza virus of choice.
[0330] Methods and materials. Influenza fusion epitope spore
vaccine targets. [0331] 1. Mammalian expression of target as
secreted protein or on mammalian cell. [0332] a. Express stem (HA2
only) [0333] b. aglobular HA0 [0334] c. aglobular HA1/HA2 [0335] d.
aglobular HA1/topless HA2 [0336] 2. Detect conformational epitope
with A6-related antibody of secreted protein or on mammalian cell
[0337] 3. Transfer successful stem or aglobular antigen to spore
expression [0338] 4. Test for spore binding with A6-related
antibody [0339] 5. Immunize mice
Example 3
Increasing the Potency and Spectrum of Cross Subtype Neutralizing
Antibodies
[0340] As mentioned previously, the group of cross subtype
neutralizing antibodies that are partially represented by Ab-1, 2,
& 3 contain very distinct and seemingly requisite heavy chain
mutations within CDR2 and framework 3 (FR3), yet remarkably little
to no diversity within CDR3. Considering the shear number of clones
that were identified with these hallmark sequences, all of which
were restricted to a 1-e, or 1-e like frameworks, leads one to
suspect that this broad spectrum activity is principally driven by
the this specific heavy chain framework and the CDR2 and Framework
3 (FR3) mutations. Recently, two groups have confirmed this at a
structural level by analyzing co-crystals of hemagglutinin and
other broad spectrum antibodies that utilize the 1-e like, 1-69
germline framework (Kashyap et al. supra; Throsby et al., PLoS ONE
3(12): e3942). In each instance the predominant binding was driven
by CDR2 and FR3 sequence corresponding to the areas described by
Kashyap et al. (supra). To identify minimal binding elements for
this broad specificity one would begin by serially reverting back
each of the CDR2 and FR3 mutations to germline and assess broad
subtype influenza binding. In the case of CDR3, alanine scanning
would be used to further define the crossreactive minimal essential
elements.
[0341] Upon learning the range of sequence involved in broad
specificity binding we would use methods of mutagenesis to create
improved mutants for testing either individually or amongst a
collection in a library. Methods commonly used to introduce
mutations could be saturation mutagenesis at sites responsible for
binding or error-prone PCR mutagenesis throughout the regions known
to be responsible for binding. Similarly, the previously mentioned
mutagenesis methods could be applied to other areas of the heavy
chain that may influence recognition in a more global manner.
[0342] Once these 1-e or 1-e like optimized clones were identified
we next utilize recombinant methods to graft these defined minimal
elements onto other related and unrelated heavy chain frameworks.
This gives us the ability to explore additional optimized solutions
under different contexts that may be superior to the original. As a
next step these minimal elements would be modeled and/or grafted
onto other related and unrelated proteins. The success of these
efforts could provide superior pharmacological agents and even
avenues leading to minimized or constrained peptides that either
present or mimic the crossreactive binding motif mimetic. Success
at this stage would then be extended into the area of
nonpeptide-mimetics.
[0343] Finally, searching the sequence databases for other related
antibodies revealed numerous anti-infectious antibodies, suggesting
the 1-e or 1-e like framework may function as a first line defense
against infectious organisms and viruses. As such it is presumable
that 1-e or 1-e like repertoires would be ideal sources for de novo
identification of anti-infectious antibodies that could be
developed similar to the outlined steps for the Ab-1 and related
antibodies for influenza.
[0344] Materials and methods. 1-69/1-e anti-idiotype antibodies and
vaccines. [0345] 1. Pan minimized framework element antibody for
specific reagent. [0346] 2. Administer anti-idiotype antibody in
presence or absence of B cell stimulating agent to expand
anti-influenza repertoire. [0347] 3. Measure anti-influenza titer
(ex vivo from PBMC or bone marrow).
Example 4
Inducing 1-e and 1-e-Like Anti-Influenza Antibodies
[0348] Inducing the proliferation of memory B cells causes the
proliferation and secretion of the specific antibodies the
stimulated B cells. Presumably upon learning the minimal binding
elements required for cross subtype hemagglutinin binding one could
use this element as a selection tool to identify anti-idiotype
antibodies. The administration of such a framework and mutation
specific anti-idiotype antibodies would result in the expansion of
these broad specific memory B cells and the serological increase of
these anti-influenza antibodies in the setting of prophylaxis or
treatment of disease.
[0349] Again, as searching the sequence databases for other related
antibodies has revealed numerous anti-infectious antibodies,
suggesting the 1-e or 1-e like framework may function as a first
line defense against infectious organisms and viruses. It is
presumable that expansion of 1-e or 1-e like anti-idiotype
repertoires would be ideally suited for protection or treatment of
infectious disease.
[0350] Agents to induce or produce broadly specific antibodies
(1-69/1-e and related frameworks). Agents include rearranged Vh for
delivered as gene therapy (in vivo & ex vivo), engineered
transcriptional activators of Vh specific genes. Such agents would
be useful for influenza (antiviral) treatment/prophylaxis; as an
adjuvant for (antiviral) prophylaxis; ex vivo selection (and
possible expansion) of Vh specific B cells for treatment and
prophylaxis; influenza epitopes for Vh specific
induction/production; 1-69/1-e and related anti-idiotype
antibodies/surrobodies to expand Vh specific memory response (may
include costimulatory agent on a surrobody or separate
administration); vaccines directed to 1-69/1-e frameworks to induce
proliferation and production of 1-69/1-e and related antibodies;
and any combination of the above.
Example 5
Co-Administration of Vaccine and Antibody to Increase Potency and
Spectrum of Protection
[0351] Complexes of antibody and antigen are known to potently
induce responses against numerous microbial proteins and other
proteins in animals. One possible explanation is that a forced
uptake of the vaccine antibody complex occurs by Fc receptors on
antigen presenting cells. Complexes of cross reactive antibodies,
such as Ab-1 with seasonal vaccines would allow for increases in
potency from year to year and because Ab-1 and the related
antibodies recognize numerous hemagglutinin antigens, obviates the
need to recreate new antibodies when new viral isolates are
selected for each seasons Influenza vaccine. Furthermore, as these
antibodies are directed to conserved neutralizing regions they may
actually direct a more effective protective response towards these
critically conserved susceptible regions when complexed with
antigen. As described previously, the vaccine may be a traditional
live or killed virus, recombinant protein or protein fragment, or
even minimized peptide or non-peptidic conformationally epitope
complexed with an antibody, antibody fragment or derivative, or
surrobody.
[0352] Although in the foregoing description the invention is
illustrated with reference to certain embodiments, it is not so
limited. Indeed, various modifications of the invention in addition
to those shown and described herein will become apparent to those
skilled in the art from the foregoing description and fall within
the scope of the appended claims.
[0353] All references cited throughout the specification are hereby
expressly incorporated by reference in their entirety.
Sequence CWU 1
1
2961115PRTArtificial SequenceDescription of Artificial Sequence
Synthetic polypeptide 1Glu Val Gln Leu Val Gln Ser Gly Thr Glu Val
Lys Lys Pro Gly Ser1 5 10 15Ser Val Lys Leu Ser Cys Lys Ala Ser Gly
Gly Thr Phe Ser Ser Tyr 20 25 30Ala Val Thr Trp Val Arg Gln Ala Pro
Gly Gln Gly Leu Glu Trp Met 35 40 45Gly Gly Ile Ile Gly Met Phe Gly
Thr Thr Asn Tyr Ala Gln Lys Phe 50 55 60Gln Gly Arg Leu Thr Ile Thr
Ala Asp Glu Met Thr Ser Thr Ala Tyr65 70 75 80Met Glu Leu Ser Ser
Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95Ala Arg Gly Ser
Tyr Tyr Tyr Glu Thr Thr Leu Asp Tyr Trp Gly Arg 100 105 110Gly Thr
Met 1152115PRTArtificial SequenceDescription of Artificial Sequence
Synthetic polypeptide 2Glu Val Gln Leu Val Gln Ser Gly Ala Glu Val
Lys Lys Pro Gly Ser1 5 10 15Ser Val Arg Val Ser Cys Lys Thr Ser Gly
Gly Thr Phe Ser Ser Tyr 20 25 30Ala Val Thr Trp Val Arg Gln Ala Pro
Gly Gln Gly Leu Glu Trp Met 35 40 45Gly Gly Ile Ile Gly Met Phe Gly
Thr Thr Asn Tyr Ala Gln Lys Phe 50 55 60Gln Gly Arg Leu Thr Ile Thr
Ala Asp Glu Met Thr Ser Thr Ala Tyr65 70 75 80Met Glu Leu Ser Ser
Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95Ala Arg Gly Ser
Tyr Tyr Tyr Glu Thr Thr Leu Asp Tyr Trp Gly Arg 100 105 110Gly Thr
Met 1153115PRTArtificial SequenceDescription of Artificial Sequence
Synthetic polypeptide 3Gln Val Gln Leu Gln Gln Ser Gly Ala Glu Val
Lys Lys Pro Gly Ser1 5 10 15Ser Val Arg Val Ser Cys Lys Thr Ser Gly
Gly Thr Phe Ser Ser Tyr 20 25 30Ala Val Thr Trp Val Arg Gln Ala Pro
Gly Gln Gly Leu Glu Trp Met 35 40 45Gly Gly Ile Ile Gly Met Phe Gly
Thr Thr Asn Tyr Ala Gln Lys Phe 50 55 60Gln Gly Arg Leu Thr Ile Thr
Ala Asp Glu Met Thr Ser Thr Ala Tyr65 70 75 80Met Glu Leu Ser Ser
Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95Ala Arg Gly Ser
Tyr Tyr Tyr Glu Thr Thr Leu Asp Tyr Trp Gly Gln 100 105 110Gly Thr
Met 1154115PRTArtificial SequenceDescription of Artificial Sequence
Synthetic polypeptide 4Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val
Lys Lys Pro Gly Ser1 5 10 15Ser Val Arg Val Ser Cys Lys Thr Ser Gly
Gly Thr Phe Ser Ser Tyr 20 25 30Ala Val Thr Trp Val Arg Gln Ala Pro
Gly Gln Gly Leu Glu Trp Met 35 40 45Gly Gly Ile Ile Gly Met Phe Gly
Thr Thr Asn Tyr Ala Gln Lys Phe 50 55 60Gln Gly Arg Leu Thr Ile Thr
Ala Asp Glu Met Thr Ser Thr Ala Tyr65 70 75 80Met Glu Leu Ser Ser
Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95Ala Arg Gly Ser
Tyr Tyr Tyr Glu Thr Thr Leu Asp Tyr Trp Gly Arg 100 105 110Gly Thr
Leu 1155115PRTArtificial SequenceDescription of Artificial Sequence
Synthetic polypeptide 5Gln Val Gln Leu Gln Gln Ser Gly Ala Glu Val
Lys Lys Pro Gly Ser1 5 10 15Ser Val Arg Val Ser Cys Lys Thr Ser Gly
Gly Thr Phe Ser Ser Tyr 20 25 30Ala Val Thr Trp Val Arg Gln Ala Pro
Gly Gln Gly Leu Glu Trp Met 35 40 45Gly Gly Ile Ile Gly Met Phe Gly
Thr Thr Asn Tyr Ala Gln Lys Phe 50 55 60Gln Gly Arg Leu Thr Ile Thr
Ala Asp Glu Met Thr Ser Thr Ala Tyr65 70 75 80Met Glu Leu Ser Ser
Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95Ala Arg Gly Ser
Tyr Tyr Tyr Glu Thr Thr Leu Asp Tyr Trp Gly Lys 100 105 110Gly Thr
Leu 1156115PRTArtificial SequenceDescription of Artificial Sequence
Synthetic polypeptide 6Glu Val Gln Leu Val Gln Ser Gly Ala Glu Val
Lys Lys Pro Gly Ser1 5 10 15Ser Val Arg Val Ser Cys Lys Thr Ser Gly
Gly Thr Phe Ser Ser Tyr 20 25 30Ala Val Thr Trp Val Arg Gln Ala Pro
Gly Gln Gly Leu Glu Trp Met 35 40 45Gly Gly Ile Ile Gly Met Phe Gly
Thr Thr Asn Tyr Ala Gln Lys Phe 50 55 60Gln Gly Arg Leu Thr Ile Thr
Ala Asp Glu Met Thr Ser Thr Ala Tyr65 70 75 80Met Glu Leu Ser Ser
Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95Ala Arg Gly Ser
Tyr Tyr Tyr Glu Thr Thr Leu Asp Tyr Trp Gly Lys 100 105 110Gly Thr
Leu 1157115PRTArtificial SequenceDescription of Artificial Sequence
Synthetic polypeptide 7Glu Val Gln Leu Val Gln Ser Gly Ala Glu Val
Lys Lys Pro Gly Ser1 5 10 15Ser Val Arg Val Ser Cys Lys Thr Ser Gly
Gly Thr Phe Ser Ser Tyr 20 25 30Ala Val Thr Trp Val Arg Gln Ala Pro
Gly Gln Gly Leu Glu Trp Met 35 40 45Gly Gly Ile Ile Gly Met Phe Gly
Thr Thr Asn Tyr Ala Gln Lys Phe 50 55 60Gln Gly Arg Leu Thr Ile Thr
Ala Asp Glu Met Thr Ser Thr Ala Tyr65 70 75 80Met Glu Leu Ser Ser
Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95Ala Arg Gly Ser
Tyr Tyr Tyr Glu Thr Thr Leu Asp Tyr Trp Gly Gln 100 105 110Gly Thr
Leu 1158115PRTArtificial SequenceDescription of Artificial Sequence
Synthetic polypeptide 8Glu Val Gln Leu Val Gln Ser Gly Ala Glu Val
Lys Lys Pro Gly Ser1 5 10 15Ser Val Lys Val Ser Cys Lys Thr Ser Gly
Gly Thr Phe Ser Ser Tyr 20 25 30Ala Val Thr Trp Val Arg Gln Ala Pro
Gly Gln Gly Leu Glu Trp Met 35 40 45Gly Ala Ile Ile Gly Met Phe Gly
Thr Thr Asn Tyr Ala Gln Lys Phe 50 55 60Gln Gly Arg Val Thr Leu Thr
Ala Asp Glu Leu Thr Ser Thr Ala Tyr65 70 75 80Met Glu Leu Ser Ser
Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95Ala Arg Gly Ser
Tyr Tyr Tyr Glu Ser Ser Leu Asp Tyr Trp Gly Arg 100 105 110Gly Thr
Leu 1159115PRTArtificial SequenceDescription of Artificial Sequence
Synthetic polypeptide 9Glu Val Gln Leu Val Gln Ser Gly Ala Glu Val
Lys Lys Pro Gly Ser1 5 10 15Ser Val Lys Val Ser Cys Lys Thr Ser Gly
Gly Thr Phe Ser Ser Tyr 20 25 30Ala Val Thr Trp Val Arg Gln Ala Pro
Gly Gln Gly Leu Glu Trp Met 35 40 45Gly Ala Ile Ile Gly Met Phe Gly
Thr Thr Asn Tyr Ala Gln Lys Phe 50 55 60Gln Gly Arg Val Thr Ile Thr
Ala Asp Glu Leu Thr Ser Thr Ala Tyr65 70 75 80Met Glu Leu Ser Ser
Leu Arg Ser Asp Asp Thr Ala Val Tyr Tyr Cys 85 90 95Ala Arg Gly Ser
Tyr Tyr Tyr Glu Ser Ser Leu Asp Tyr Trp Gly Lys 100 105 110Gly Thr
Leu 11510115PRTArtificial SequenceDescription of Artificial
Sequence Synthetic polypeptide 10Glu Val Gln Leu Val Gln Ser Gly
Thr Glu Val Lys Lys Pro Gly Ser1 5 10 15Ser Val Lys Val Ser Cys Lys
Val Ser Gly Gly Thr Phe Ser Ser Tyr 20 25 30Ala Val Thr Trp Val Arg
Gln Ala Pro Gly Gln Gly Leu Glu Trp Met 35 40 45Gly Ala Ile Ile Gly
Met Phe Gly Thr Thr Asn Tyr Ala Gln Lys Phe 50 55 60Gln Gly Arg Val
Thr Ile Thr Ala Asp Glu Leu Thr Ser Thr Ala Tyr65 70 75 80Met Glu
Leu Ser Ser Leu Arg Ser Asp Asp Thr Ala Val Tyr Tyr Cys 85 90 95Ala
Arg Gly Ser Tyr Tyr Tyr Glu Ser Ser Leu Asp Tyr Trp Gly Gly 100 105
110Gly Thr Thr 11511115PRTArtificial SequenceDescription of
Artificial Sequence Synthetic polypeptide 11Glu Val Gln Leu Val Gln
Ser Gly Ala Glu Val Lys Lys Pro Gly Ser1 5 10 15Ser Val Lys Val Ser
Cys Lys Thr Ser Gly Gly Thr Phe Ser Ser Tyr 20 25 30Ala Val Thr Trp
Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met 35 40 45Gly Ala Ile
Ile Gly Met Phe Gly Thr Thr Asn Tyr Ala Gln Lys Phe 50 55 60Gln Gly
Arg Val Thr Ile Thr Ala Asp Glu Leu Thr Ser Thr Ala Tyr65 70 75
80Met Glu Leu Ser Ser Leu Arg Ser Asp Asp Thr Ala Val Tyr Tyr Cys
85 90 95Ala Arg Gly Ser Tyr Tyr Tyr Glu Ser Ser Leu Asp Tyr Trp Gly
Gln 100 105 110Gly Thr Leu 11512115PRTArtificial
SequenceDescription of Artificial Sequence Synthetic polypeptide
12Glu Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ser1
5 10 15Ser Val Lys Val Ser Cys Lys Thr Ser Gly Gly Thr Phe Ser Ser
Tyr 20 25 30Ala Val Thr Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu
Trp Met 35 40 45Gly Ala Ile Ile Gly Met Phe Gly Thr Thr Asn Tyr Ala
Gln Lys Phe 50 55 60Gln Gly Arg Val Thr Ile Thr Ala Asp Glu Leu Thr
Ser Thr Ala Tyr65 70 75 80Met Glu Leu Ser Ser Leu Arg Ser Asp Asp
Thr Ala Val Tyr Tyr Cys 85 90 95Ala Arg Gly Ser Tyr Tyr Tyr Glu Ser
Ser Leu Asp Tyr Trp Gly Gln 100 105 110Gly Thr Met
11513115PRTArtificial SequenceDescription of Artificial Sequence
Synthetic polypeptide 13Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val
Lys Lys Pro Gly Ser1 5 10 15Ser Val Lys Val Ser Cys Lys Thr Ser Gly
Gly Thr Phe Ser Ser Tyr 20 25 30Ala Val Thr Trp Val Arg Gln Ala Pro
Gly Gln Gly Leu Glu Trp Met 35 40 45Gly Ala Ile Ile Gly Met Phe Gly
Thr Thr Asn Tyr Ala Gln Lys Phe 50 55 60Gln Gly Arg Val Thr Ile Thr
Ala Asp Glu Leu Thr Ser Thr Ala Tyr65 70 75 80Met Glu Leu Ser Ser
Leu Arg Ser Asp Asp Thr Ala Val Tyr Tyr Cys 85 90 95Ala Arg Gly Ser
Tyr Tyr Tyr Glu Ser Ser Leu Asp Tyr Trp Gly Gln 100 105 110Gly Thr
Met 11514115PRTArtificial SequenceDescription of Artificial
Sequence Synthetic polypeptide 14Glu Val Gln Leu Val Gln Ser Gly
Ala Glu Val Lys Lys Pro Gly Ser1 5 10 15Ser Val Lys Val Ser Cys Lys
Thr Thr Gly Gly Thr Phe Ser Ser Tyr 20 25 30Ala Val Thr Trp Val Arg
Gln Ala Pro Gly Gln Gly Leu Glu Trp Met 35 40 45Gly Gly Ile Ile Gly
Met Phe Gly Thr Thr Asn Tyr Ala Gln Lys Phe 50 55 60Gln Gly Arg Val
Thr Ile Thr Ala Asp Glu Met Thr Ser Thr Ala Tyr65 70 75 80Met Glu
Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95Ala
Arg Gly Ser Tyr Tyr Tyr Glu Ser Ser Leu Asp Tyr Trp Gly Gln 100 105
110Gly Thr Leu 11515115PRTArtificial SequenceDescription of
Artificial Sequence Synthetic polypeptide 15Glu Val Gln Leu Val Gln
Ser Gly Ala Glu Val Lys Lys Pro Gly Ser1 5 10 15Ser Val Lys Val Ser
Cys Lys Thr Thr Gly Gly Thr Phe Ser Ser Tyr 20 25 30Ala Val Thr Trp
Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met 35 40 45Gly Gly Ile
Ile Gly Met Phe Gly Thr Thr Asn Tyr Ala Gln Lys Phe 50 55 60Gln Gly
Arg Val Thr Ile Thr Ala Asp Glu Met Thr Ser Thr Ala Tyr65 70 75
80Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys
85 90 95Ala Arg Gly Ser Tyr Tyr Tyr Glu Ser Ser Leu Asp Tyr Trp Gly
Gln 100 105 110Gly Thr Met 11516115PRTArtificial
SequenceDescription of Artificial Sequence Synthetic polypeptide
16Glu Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ser1
5 10 15Ser Val Lys Val Ser Cys Lys Thr Ser Gly Gly Thr Phe Ser Ser
Tyr 20 25 30Ala Val Thr Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu
Trp Met 35 40 45Gly Ala Ile Ile Gly Met Phe Gly Thr Thr Asn Tyr Ala
Gln Lys Phe 50 55 60Gln Gly Arg Val Thr Leu Thr Ala Asp Glu Leu Thr
Ser Thr Ala Tyr65 70 75 80Met Glu Leu Ser Ser Leu Arg Ser Glu Asp
Thr Ala Val Tyr Tyr Cys 85 90 95Ala Arg Gly Ser Tyr Tyr Tyr Glu Ser
Ser Leu Asp Tyr Trp Gly Lys 100 105 110Gly Thr Leu
11517115PRTArtificial SequenceDescription of Artificial Sequence
Synthetic polypeptide 17Glu Val Gln Leu Val Gln Ser Gly Ala Glu Val
Lys Lys Pro Gly Ser1 5 10 15Ser Val Lys Val Ser Cys Lys Thr Ser Gly
Gly Thr Phe Ser Ser Tyr 20 25 30Ala Val Thr Trp Val Arg Gln Ala Pro
Gly Gln Gly Leu Glu Trp Met 35 40 45Gly Ala Ile Ile Gly Met Phe Gly
Thr Thr Asn Tyr Ala Gln Lys Phe 50 55 60Gln Gly Arg Val Thr Leu Thr
Ala Asp Glu Leu Thr Ser Thr Ala Tyr65 70 75 80Met Glu Leu Ser Ser
Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95Ala Arg Gly Ser
Tyr Tyr Tyr Glu Ser Ser Leu Asp Tyr Trp Gly Lys 100 105 110Gly Thr
Met 11518115PRTArtificial SequenceDescription of Artificial
Sequence Synthetic polypeptide 18Glu Val Gln Leu Val Gln Ser Gly
Ala Glu Val Lys Lys Pro Gly Ala1 5 10 15Ser Val Lys Val Ser Cys Lys
Ala Ser Gly Gly Ala Phe Ser Ser Tyr 20 25 30Ala Val Thr Trp Val Arg
Gln Ala Pro Gly Gln Gly Leu Glu Trp Met 35 40 45Gly Gly Ile Ile Gly
Met Phe Gly Thr Thr Asn Tyr Ala Gln Lys Phe 50 55 60Gln Gly Arg Val
Thr Ile Thr Ala Asp Glu Leu Thr Ser Thr Ala Tyr65 70 75 80Met Glu
Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95Ala
Arg Gly Ser Tyr Tyr Tyr Glu Ser Ser Leu Asp Tyr Trp Gly Gln 100 105
110Gly Thr Met 11519115PRTArtificial SequenceDescription of
Artificial Sequence Synthetic polypeptide 19Glu Val Gln Leu Val Gln
Ser Gly Ala Glu Val Lys Lys Pro Gly Ser1 5 10 15Ser Val Lys Val Ser
Cys Lys Thr Ser Gly Gly Thr Phe Ser Ser Tyr 20 25 30Ala Val Thr Trp
Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met 35 40 45Gly Gly Ile
Ile Gly Met Phe Gly Thr Thr Asn Tyr Ala Gln Lys Phe 50 55 60Gln Gly
Arg Val Thr Met Thr Ala Asp Glu Met Thr Ser Thr Ala Tyr65 70 75
80Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys
85 90 95Ala Arg Gly Ser Tyr Tyr Tyr Glu Ser Ser Leu Asp Tyr Trp Gly
Lys 100 105 110Gly Thr Leu 11520115PRTArtificial
SequenceDescription of Artificial Sequence Synthetic polypeptide
20Glu Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ser1
5 10 15Ser Val Lys Val Ser Cys Lys Thr Ser Gly Gly Thr Phe Ser Ser
Tyr
20 25 30Ala Val Thr Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp
Met 35 40 45Gly Gly Ile Ile Gly Met Phe Gly Thr Thr Asn Tyr Ala Gln
Lys Phe 50 55 60Gln Gly Arg Val Thr Ile Thr Ala Asp Glu Met Thr Ser
Thr Ala Tyr65 70 75 80Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr
Ala Val Tyr Tyr Cys 85 90 95Ala Arg Gly Ser Tyr Tyr Tyr Glu Ser Ser
Leu Asp Tyr Trp Gly Arg 100 105 110Gly Thr Leu
11521115PRTArtificial SequenceDescription of Artificial Sequence
Synthetic polypeptide 21Glu Val Gln Leu Val Gln Ser Gly Ala Glu Val
Lys Lys Pro Gly Ser1 5 10 15Ser Val Lys Val Ser Cys Lys Thr Ser Gly
Gly Thr Phe Ser Ser Tyr 20 25 30Ala Val Thr Trp Val Arg Gln Ala Pro
Gly Gln Gly Leu Glu Trp Met 35 40 45Gly Gly Ile Ile Gly Met Phe Gly
Thr Thr Asn Tyr Ala Gln Lys Phe 50 55 60Gln Gly Arg Val Thr Ile Thr
Ala Asp Glu Met Thr Ser Thr Ala Tyr65 70 75 80Met Glu Leu Ser Ser
Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95Ala Arg Gly Ser
Tyr Tyr Tyr Glu Ser Ser Leu Asp Tyr Trp Gly Arg 100 105 110Gly Thr
Met 11522115PRTArtificial SequenceDescription of Artificial
Sequence Synthetic polypeptide 22Glu Val Gln Leu Val Gln Ser Gly
Ala Glu Val Lys Lys Pro Gly Ser1 5 10 15Ser Val Lys Val Ser Cys Lys
Thr Ser Gly Gly Thr Phe Ser Ser Tyr 20 25 30Ala Val Thr Trp Val Arg
Gln Ala Pro Gly Gln Gly Leu Glu Trp Met 35 40 45Gly Ala Ile Ile Gly
Met Phe Gly Thr Thr Asn Tyr Ala Gln Lys Phe 50 55 60Gln Gly Arg Val
Thr Leu Thr Ala Asp Glu Leu Thr Ser Thr Ala Tyr65 70 75 80Met Glu
Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95Ala
Arg Gly Ser Tyr Tyr Tyr Glu Ser Ser Leu Asp Tyr Trp Gly Gln 100 105
110Gly Thr Leu 11523115PRTArtificial SequenceDescription of
Artificial Sequence Synthetic polypeptide 23Glu Val Gln Leu Val Gln
Ser Gly Ala Glu Val Gln Lys Pro Gly Ser1 5 10 15Ser Val Lys Val Ser
Cys Lys Thr Ser Gly Gly Thr Phe Ser Ser Tyr 20 25 30Ala Val Thr Trp
Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met 35 40 45Gly Ala Ile
Ile Gly Met Phe Gly Thr Thr Asn Tyr Ala Gln Lys Phe 50 55 60Gln Gly
Arg Val Thr Leu Thr Ala Asp Glu Leu Thr Ser Thr Ala Tyr65 70 75
80Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys
85 90 95Ala Arg Gly Ser Tyr Tyr Tyr Glu Ser Ser Leu Asp Tyr Trp Gly
Gln 100 105 110Gly Thr Leu 11524115PRTArtificial
SequenceDescription of Artificial Sequence Synthetic polypeptide
24Glu Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ser1
5 10 15Ser Val Lys Val Ser Cys Lys Thr Ser Gly Gly Thr Phe Ser Ser
Tyr 20 25 30Ala Val Thr Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu
Trp Met 35 40 45Gly Gly Ile Ile Gly Met Phe Gly Thr Thr Asn Tyr Ala
Gln Lys Phe 50 55 60Gln Gly Arg Val Thr Ile Thr Ala Asp Glu Leu Thr
Ser Thr Ala Tyr65 70 75 80Met Glu Leu Ser Ser Leu Arg Ser Glu Asp
Thr Ala Val Tyr Tyr Cys 85 90 95Ala Arg Gly Ser Tyr Tyr Tyr Glu Ser
Ser Leu Asp Tyr Trp Gly Gln 100 105 110Gly Thr Leu
11525115PRTArtificial SequenceDescription of Artificial Sequence
Synthetic polypeptide 25Glu Val Gln Leu Val Gln Ser Gly Ala Glu Val
Lys Lys Pro Gly Ser1 5 10 15Ser Val Lys Val Ser Cys Lys Ala Ser Gly
Gly Thr Phe Ser Ser Tyr 20 25 30Ala Ile Ser Trp Val Arg Gln Ala Pro
Gly Gln Gly Leu Glu Trp Met 35 40 45Gly Ala Ile Ile Gly Met Phe Gly
Thr Thr Asn Tyr Ala Gln Lys Phe 50 55 60Gln Gly Arg Val Thr Ile Thr
Ala Asp Glu Leu Thr Ser Thr Ala Tyr65 70 75 80Met Glu Leu Ser Ser
Leu Arg Ser Asp Asp Thr Ala Val Tyr Tyr Cys 85 90 95Ala Arg Gly Ser
Tyr Tyr Tyr Glu Ser Ser Leu Asp Tyr Trp Gly Gln 100 105 110Gly Thr
Leu 11526115PRTArtificial SequenceDescription of Artificial
Sequence Synthetic polypeptide 26Glu Val Gln Leu Val Gln Ser Gly
Ala Glu Val Lys Lys Pro Gly Ser1 5 10 15Ser Val Lys Val Ser Cys Lys
Thr Ser Gly Gly Thr Phe Ser Ser Tyr 20 25 30Ala Val Thr Trp Val Arg
Gln Ala Pro Gly Gln Gly Leu Glu Trp Met 35 40 45Gly Ala Ile Ile Gly
Met Phe Gly Thr Thr Asn Tyr Ala Gln Lys Phe 50 55 60Gln Gly Arg Val
Thr Leu Thr Ala Asp Gln Leu Thr Ser Thr Ala Tyr65 70 75 80Met Glu
Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95Ala
Arg Gly Ser Tyr Tyr Tyr Glu Ser Ser Leu Asp Tyr Trp Gly Arg 100 105
110Gly Thr Leu 11527115PRTArtificial SequenceDescription of
Artificial Sequence Synthetic polypeptide 27Glu Val Gln Leu Val Gln
Ser Gly Ala Glu Val Lys Lys Pro Gly Ser1 5 10 15Ser Val Lys Val Ser
Cys Lys Thr Ser Gly Gly Thr Phe Ser Ser Tyr 20 25 30Ala Val Thr Trp
Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met 35 40 45Gly Ala Ile
Ile Gly Met Phe Gly Thr Thr Asn Tyr Ala Gln Lys Phe 50 55 60Gln Gly
Arg Val Thr Ile Thr Ala Asp Glu Leu Thr Ser Thr Ala Tyr65 70 75
80Met Glu Leu Ser Ser Leu Arg Ser Asp Asp Thr Ala Val Tyr Tyr Cys
85 90 95Ala Arg Gly Ser Tyr Tyr Tyr Glu Ser Ser Leu Asp Tyr Trp Gly
Lys 100 105 110Gly Thr Leu 11528115PRTArtificial
SequenceDescription of Artificial Sequence Synthetic polypeptide
28Glu Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ser1
5 10 15Ser Val Lys Val Ser Cys Lys Thr Ser Gly Gly Thr Phe Ser Ser
Tyr 20 25 30Ala Val Thr Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu
Trp Met 35 40 45Gly Ala Ile Ile Gly Met Phe Gly Thr Thr Asn Tyr Ala
Gln Lys Phe 50 55 60Gln Gly Arg Val Thr Ile Thr Ala Asp Glu Leu Thr
Ser Thr Ala Tyr65 70 75 80Met Glu Leu Ser Ser Leu Arg Ser Asp Asp
Thr Ala Val Tyr Tyr Cys 85 90 95Ala Arg Gly Ser Tyr Tyr Tyr Glu Ser
Ser Leu Asp Tyr Trp Gly Lys 100 105 110Gly Thr Thr
11529115PRTArtificial SequenceDescription of Artificial Sequence
Synthetic polypeptide 29Glu Val Gln Leu Val Gln Ser Gly Ala Glu Val
Lys Lys Pro Gly Ser1 5 10 15Ser Val Lys Val Ser Cys Lys Thr Ser Gly
Gly Thr Phe Ser Ser Tyr 20 25 30Ala Val Thr Trp Val Arg Gln Ala Pro
Gly Gln Gly Leu Glu Trp Met 35 40 45Gly Gly Ile Ile Gly Met Phe Gly
Thr Thr Asn Tyr Ala Gln Lys Phe 50 55 60Gln Gly Arg Val Thr Ile Thr
Ala Asp Glu Leu Thr Ser Thr Ala Tyr65 70 75 80Met Glu Leu Ser Ser
Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95Ala Arg Gly Ser
Tyr Tyr Tyr Glu Ser Ser Leu Asp Tyr Trp Gly Lys 100 105 110Gly Thr
Leu 11530115PRTArtificial SequenceDescription of Artificial
Sequence Synthetic polypeptide 30Glu Val Gln Leu Val Gln Ser Gly
Ala Glu Val Lys Lys Pro Gly Ser1 5 10 15Ser Val Lys Val Ser Cys Lys
Thr Ser Gly Gly Thr Phe Ser Ser Tyr 20 25 30Ala Val Thr Trp Val Arg
Gln Ala Pro Gly Gln Gly Leu Glu Trp Met 35 40 45Gly Ala Ile Ile Gly
Met Phe Gly Thr Thr Asn Tyr Ala Gln Lys Phe 50 55 60Gln Gly Arg Val
Thr Leu Thr Ala Asp Glu Leu Thr Ser Thr Ala Tyr65 70 75 80Met Glu
Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95Ala
Arg Gly Ser Tyr Tyr Tyr Glu Ser Ser Leu Asp Tyr Trp Gly Arg 100 105
110Gly Thr Met 11531119PRTArtificial SequenceDescription of
Artificial Sequence Synthetic polypeptide 31Glu Val Gln Leu Val Gln
Ser Gly Ala Glu Val Lys Lys Pro Gly Ser1 5 10 15Ser Val Lys Val Ser
Cys Lys Thr Ser Gly Gly Thr Phe Ser Ser Tyr 20 25 30Ala Val Thr Trp
Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met 35 40 45Gly Ala Ile
Ile Gly Met Phe Gly Thr Thr Asn Tyr Ala Gln Lys Phe 50 55 60Gln Gly
Arg Val Thr Ile Thr Ala Asp Glu Leu Thr Ser Thr Ala Tyr65 70 75
80Met Glu Leu Ser Ser Leu Arg Ser Asp Asp Thr Ala Val Tyr Tyr Cys
85 90 95Ala Arg Gly Ser Tyr Tyr Tyr Glu Ser Ser Leu Asp Tyr Trp Gly
Lys 100 105 110Gly Thr Met Val Thr Val Ser 11532115PRTArtificial
SequenceDescription of Artificial Sequence Synthetic polypeptide
32Gln Met Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ser1
5 10 15Ser Val Lys Val Ser Cys Lys Thr Ser Gly Gly Thr Phe Ser Ser
Tyr 20 25 30Ala Val Thr Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu
Trp Met 35 40 45Gly Ala Ile Ile Gly Met Phe Gly Thr Thr Asn Tyr Ala
Gln Lys Phe 50 55 60Gln Gly Arg Val Thr Ile Thr Ala Asp Glu Leu Thr
Ser Thr Ala Tyr65 70 75 80Met Glu Leu Ser Ser Leu Arg Ser Asp Asp
Thr Ala Val Tyr Tyr Cys 85 90 95Ala Arg Gly Ser Tyr Tyr Tyr Glu Ser
Ser Leu Asp Tyr Trp Gly Lys 100 105 110Gly Thr Met
11533119PRTArtificial SequenceDescription of Artificial Sequence
Synthetic polypeptide 33Gln Met Gln Leu Val Gln Ser Gly Ala Glu Val
Lys Lys Pro Gly Ser1 5 10 15Ser Val Lys Val Ser Cys Lys Thr Ser Gly
Gly Thr Phe Ser Ser Tyr 20 25 30Ala Val Thr Trp Val Arg Gln Ala Pro
Gly Gln Gly Leu Glu Trp Met 35 40 45Gly Ala Ile Ile Gly Met Phe Gly
Thr Thr Asn Tyr Ala Gln Lys Phe 50 55 60Gln Gly Arg Val Thr Leu Thr
Ala Asp Glu Leu Thr Ser Thr Ala Tyr65 70 75 80Met Glu Leu Ser Ser
Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95Ala Arg Gly Ser
Tyr Tyr Tyr Glu Ser Ser Leu Asp Tyr Trp Gly Gln 100 105 110Gly Thr
Leu Val Thr Val Ser 11534115PRTArtificial SequenceDescription of
Artificial Sequence Synthetic polypeptide 34Gln Val Gln Leu Val Gln
Ser Gly Ala Glu Val Lys Lys Pro Gly Ser1 5 10 15Ser Val Lys Val Ser
Cys Lys Thr Thr Gly Gly Thr Phe Ser Ser Tyr 20 25 30Ala Val Thr Trp
Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met 35 40 45Gly Gly Ile
Ile Gly Met Phe Gly Thr Thr Asn Tyr Ala Gln Lys Phe 50 55 60Gln Gly
Arg Val Thr Ile Thr Ala Asp Glu Met Thr Ser Thr Ala Tyr65 70 75
80Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys
85 90 95Ala Arg Gly Ser Tyr Tyr Tyr Glu Ser Ser Leu Asp Tyr Trp Gly
Lys 100 105 110Gly Thr Met 11535115PRTArtificial
SequenceDescription of Artificial Sequence Synthetic polypeptide
35Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ser1
5 10 15Ser Val Lys Val Ser Cys Lys Ala Ser Gly Gly Thr Phe Ser Ser
Tyr 20 25 30Ala Val Thr Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu
Trp Met 35 40 45Gly Ala Ile Ile Gly Met Phe Gly Thr Thr Asn Tyr Ala
Gln Lys Phe 50 55 60Gln Gly Arg Val Thr Leu Thr Ala Asp Glu Leu Thr
Ser Thr Ala Tyr65 70 75 80Met Glu Leu Ser Ser Leu Arg Ser Glu Asp
Thr Ala Val Tyr Tyr Cys 85 90 95Ala Arg Gly Ser Tyr Tyr Tyr Glu Ser
Ser Leu Asp Tyr Trp Gly Gln 100 105 110Gly Thr Leu
11536115PRTArtificial SequenceDescription of Artificial Sequence
Synthetic polypeptide 36Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val
Lys Lys Pro Gly Ser1 5 10 15Ser Val Lys Val Ser Cys Lys Thr Ser Gly
Gly Thr Phe Ser Ser Tyr 20 25 30Ala Val Thr Trp Val Arg Gln Ala Pro
Gly Gln Gly Leu Glu Trp Met 35 40 45Gly Ala Ile Ile Gly Met Phe Gly
Thr Thr Asn Tyr Ala Gln Lys Phe 50 55 60Gln Gly Arg Val Thr Leu Thr
Ala Asp Glu Leu Thr Ser Thr Ala Tyr65 70 75 80Met Glu Leu Ser Ser
Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95Ala Arg Gly Ser
Tyr Tyr Tyr Glu Ser Ser Leu Asp Tyr Trp Gly Gln 100 105 110Gly Thr
Leu 11537115PRTArtificial SequenceDescription of Artificial
Sequence Synthetic polypeptide 37Gln Val Gln Leu Val Gln Ser Gly
Ala Glu Val Lys Lys Pro Gly Ser1 5 10 15Ser Val Lys Val Ser Cys Lys
Thr Thr Gly Gly Thr Phe Ser Ser Tyr 20 25 30Ala Val Thr Trp Val Arg
Gln Ala Pro Gly Gln Gly Leu Glu Trp Met 35 40 45Gly Gly Ile Ile Gly
Met Phe Gly Thr Thr Asn Tyr Ala Gln Lys Phe 50 55 60Gln Gly Arg Val
Thr Ile Thr Ala Asp Glu Met Thr Ser Thr Ala Tyr65 70 75 80Met Glu
Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95Ala
Arg Gly Ser Tyr Tyr Tyr Glu Ser Ser Leu Asp Tyr Trp Gly Lys 100 105
110Gly Thr Leu 11538115PRTArtificial SequenceDescription of
Artificial Sequence Synthetic polypeptide 38Gln Val Gln Leu Leu Gln
Ser Gly Ala Glu Val Lys Lys Pro Gly Ser1 5 10 15Ser Val Lys Val Ser
Cys Lys Thr Thr Gly Gly Thr Phe Ser Ser Tyr 20 25 30Ala Val Thr Trp
Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met 35 40 45Gly Gly Ile
Ile Gly Met Phe Gly Thr Thr Asn Tyr Ala Gln Lys Phe 50 55 60Gln Gly
Arg Val Thr Ile Thr Ala Asp Glu Met Thr Ser Thr Ala Tyr65 70 75
80Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys
85 90 95Ala Arg Gly Ser Tyr Tyr Tyr Glu Ser Ser Leu Asp Tyr Trp Gly
Lys 100 105 110Gly Thr Leu 11539115PRTArtificial
SequenceDescription of Artificial Sequence Synthetic polypeptide
39Gln Val Gln Leu Gln Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ser1
5 10 15Ser Val Lys Val Ser Cys Lys Thr Ser Gly Gly Thr Phe Ser Ser
Tyr 20 25 30Ala Val Thr Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu
Trp Met 35 40 45Gly Ala Ile Ile Gly Met Phe Gly Thr Thr Asn Tyr Ala
Gln Lys Phe 50 55 60Gln Gly Arg Val Thr Leu
Thr Ala Asp Glu Leu Thr Ser Thr Ala Tyr65 70 75 80Met Glu Leu Ser
Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95Ala Arg Gly
Ser Tyr Tyr Tyr Glu Ser Ser Leu Asp Tyr Trp Gly Gln 100 105 110Gly
Thr Leu 11540115PRTArtificial SequenceDescription of Artificial
Sequence Synthetic polypeptide 40Gln Val Gln Leu Val Gln Ser Gly
Ala Glu Val Lys Lys Pro Gly Ser1 5 10 15Ser Val Lys Val Ser Cys Lys
Thr Thr Gly Gly Thr Phe Ser Ser Tyr 20 25 30Ala Val Thr Trp Val Arg
Gln Ala Pro Gly Gln Gly Leu Glu Trp Met 35 40 45Gly Gly Ile Ile Gly
Met Phe Gly Thr Thr Asn Tyr Ala Gln Lys Phe 50 55 60Gln Gly Arg Val
Thr Ile Thr Ala Asp Glu Met Thr Ser Thr Ala Tyr65 70 75 80Met Glu
Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95Ala
Arg Gly Ser Tyr Tyr Tyr Glu Ser Ser Leu Asp Tyr Trp Gly Gln 100 105
110Gly Thr Leu 11541115PRTArtificial SequenceDescription of
Artificial Sequence Synthetic polypeptide 41Gln Val Gln Leu Val Gln
Ser Gly Ala Glu Val Lys Lys Pro Gly Ser1 5 10 15Ser Val Lys Val Ser
Cys Lys Thr Ser Gly Gly Thr Phe Ser Ser Tyr 20 25 30Ala Val Thr Trp
Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met 35 40 45Gly Ala Ile
Ile Gly Met Phe Gly Thr Thr Asn Tyr Ala Gln Lys Phe 50 55 60Gln Gly
Arg Val Thr Leu Thr Ala Asp Glu Leu Thr Ser Thr Ala Tyr65 70 75
80Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys
85 90 95Ala Arg Gly Ser Tyr Tyr Tyr Glu Ser Ser Leu Asp Tyr Trp Gly
Gln 100 105 110Gly Thr Met 11542115PRTArtificial
SequenceDescription of Artificial Sequence Synthetic polypeptide
42Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ser1
5 10 15Ser Val Lys Val Ser Cys Lys Thr Ser Gly Gly Ser Phe Ser Ser
Tyr 20 25 30Ala Val Thr Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu
Trp Met 35 40 45Gly Gly Ile Ile Gly Met Phe Gly Thr Thr Asn Tyr Ala
Gln Lys Phe 50 55 60Gln Gly Arg Val Thr Ile Thr Ala Asp Glu Leu Thr
Ser Thr Ala Tyr65 70 75 80Met Glu Leu Ser Ser Leu Arg Ser Glu Asp
Thr Ala Val Tyr Tyr Cys 85 90 95Ala Arg Gly Ser Tyr Tyr Tyr Glu Ser
Ser Leu Asp Tyr Trp Gly Gln 100 105 110Gly Thr Met
11543115PRTArtificial SequenceDescription of Artificial Sequence
Synthetic polypeptide 43Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val
Lys Lys Pro Gly Ser1 5 10 15Ser Val Lys Val Ser Cys Lys Thr Ser Gly
Gly Thr Phe Ser Ser Tyr 20 25 30Ala Val Thr Trp Val Arg Gln Ala Pro
Gly Gln Gly Leu Glu Trp Met 35 40 45Gly Gly Ile Ile Gly Met Phe Gly
Thr Thr Asn Tyr Ala Gln Lys Phe 50 55 60Gln Gly Arg Val Thr Ile Thr
Ala Asp Glu Leu Arg Ser Thr Ala Tyr65 70 75 80Met Glu Leu Ser Ser
Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95Ala Arg Gly Ser
Tyr Tyr Tyr Glu Ser Ser Leu Asp Tyr Trp Gly Gln 100 105 110Gly Thr
Leu 11544115PRTArtificial SequenceDescription of Artificial
Sequence Synthetic polypeptide 44Gln Val Gln Leu Val Gln Ser Gly
Ala Glu Val Lys Lys Pro Gly Ser1 5 10 15Ser Val Lys Val Ser Cys Lys
Thr Ser Gly Gly Thr Phe Ser Ser Tyr 20 25 30Ala Val Thr Trp Val Arg
Gln Ala Pro Gly Gln Gly Leu Glu Trp Met 35 40 45Gly Gly Ile Ile Gly
Met Phe Gly Thr Thr Asn Tyr Ala Gln Lys Phe 50 55 60Gln Gly Arg Val
Thr Ile Thr Ala Asp Glu Leu Thr Ser Thr Ala Tyr65 70 75 80Met Glu
Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95Ala
Arg Gly Ser Tyr Tyr Tyr Glu Ser Ser Leu Asp Tyr Trp Gly Gln 100 105
110Gly Thr Leu 11545115PRTArtificial SequenceDescription of
Artificial Sequence Synthetic polypeptide 45Gln Val Gln Leu Val Gln
Ser Gly Ala Glu Val Lys Lys Pro Gly Ser1 5 10 15Ser Val Lys Val Ser
Cys Lys Thr Ser Gly Gly Thr Phe Ser Ser Tyr 20 25 30Ala Val Thr Trp
Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met 35 40 45Gly Gly Ile
Ile Gly Met Phe Gly Thr Thr Asn Tyr Ala Gln Lys Phe 50 55 60Gln Gly
Arg Val Thr Ile Thr Ala Asp Glu Leu Thr Ser Thr Ala Tyr65 70 75
80Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys
85 90 95Ala Arg Gly Ser Tyr Tyr Tyr Glu Ser Ser Leu Asp Tyr Trp Gly
Arg 100 105 110Gly Thr Leu 11546115PRTArtificial
SequenceDescription of Artificial Sequence Synthetic polypeptide
46Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ser1
5 10 15Ser Val Lys Val Ser Cys Lys Ala Ser Gly Gly Thr Phe Ser Ser
Tyr 20 25 30Ala Val Thr Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu
Trp Met 35 40 45Gly Ala Ile Ile Gly Met Phe Gly Thr Thr Asn Tyr Ala
Gln Lys Phe 50 55 60Gln Gly Arg Val Thr Leu Thr Ala Asp Glu Leu Thr
Ser Thr Ala Tyr65 70 75 80Met Glu Leu Ser Ser Leu Arg Ser Glu Asp
Thr Ala Val Tyr Tyr Cys 85 90 95Ala Arg Gly Ser Tyr Tyr Tyr Glu Ser
Ser Leu Asp Tyr Trp Gly Gln 100 105 110Gly Thr Met
11547115PRTArtificial SequenceDescription of Artificial Sequence
Synthetic polypeptide 47Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val
Lys Lys Pro Gly Ser1 5 10 15Ser Val Lys Val Ser Cys Lys Ala Ser Gly
Gly Thr Phe Ser Ser Tyr 20 25 30Ala Val Thr Trp Val Arg Gln Ala Pro
Gly Gln Gly Leu Glu Trp Met 35 40 45Gly Ala Ile Ile Gly Met Phe Gly
Thr Thr Asn Tyr Ala Gln Lys Phe 50 55 60Gln Gly Arg Val Thr Ile Thr
Ala Asp Glu Leu Thr Ser Thr Ala Tyr65 70 75 80Met Glu Leu Ser Ser
Leu Arg Ser Asp Asp Thr Ala Val Tyr Tyr Cys 85 90 95Ala Arg Gly Ser
Tyr Tyr Tyr Glu Ser Ser Leu Asp Tyr Trp Gly Gln 100 105 110Gly Thr
Leu 11548115PRTArtificial SequenceDescription of Artificial
Sequence Synthetic polypeptide 48Gln Val Gln Leu Val Gln Ser Gly
Ala Glu Val Lys Lys Pro Gly Ser1 5 10 15Ser Val Lys Val Ser Cys Lys
Thr Ser Gly Gly Thr Phe Ser Ser Tyr 20 25 30Ala Val Thr Trp Val Arg
Gln Ala Pro Gly Gln Gly Leu Glu Trp Met 35 40 45Gly Ala Ile Ile Gly
Met Phe Gly Thr Thr Asn Tyr Ala Gln Lys Phe 50 55 60Gln Gly Arg Val
Thr Ile Thr Ala Asp Glu Leu Thr Ser Thr Ala Tyr65 70 75 80Met Glu
Leu Ser Ser Leu Arg Ser Asp Asp Thr Ala Val Tyr Tyr Cys 85 90 95Ala
Arg Gly Ser Tyr Tyr Tyr Glu Ser Ser Leu Asp Tyr Trp Gly Gln 100 105
110Gly Thr Leu 11549115PRTArtificial SequenceDescription of
Artificial Sequence Synthetic polypeptide 49Gly Val Gln Leu Val Gln
Ser Gly Ala Glu Val Lys Lys Pro Gly Ser1 5 10 15Ser Val Lys Val Ser
Cys Lys Thr Ser Gly Gly Thr Phe Ser Ser Tyr 20 25 30Ala Val Thr Trp
Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met 35 40 45Gly Ala Ile
Ile Gly Met Phe Gly Thr Thr Asn Tyr Ala Gln Lys Phe 50 55 60Gln Gly
Arg Val Thr Leu Thr Ala Asp Glu Leu Thr Ser Thr Ala Tyr65 70 75
80Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys
85 90 95Ala Arg Gly Ser Tyr Tyr Tyr Glu Ser Ser Leu Asp Tyr Trp Gly
Lys 100 105 110Gly Thr Met 11550115PRTArtificial
SequenceDescription of Artificial Sequence Synthetic polypeptide
50Gly Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ser1
5 10 15Ser Val Lys Val Ser Cys Lys Thr Ser Gly Gly Thr Phe Ser Ser
Tyr 20 25 30Ala Val Thr Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu
Trp Met 35 40 45Gly Ala Ile Ile Gly Met Phe Gly Thr Thr Asn Tyr Ala
Gln Lys Phe 50 55 60Gln Gly Arg Val Thr Ile Thr Ala Asp Glu Leu Thr
Ser Thr Ala Tyr65 70 75 80Met Glu Leu Ser Ser Leu Arg Ser Asp Asp
Thr Ala Val Tyr Tyr Cys 85 90 95Ala Arg Gly Ser Tyr Tyr Tyr Glu Ser
Ser Leu Asp Tyr Trp Gly Arg 100 105 110Gly Thr Leu
11551115PRTArtificial SequenceDescription of Artificial Sequence
Synthetic polypeptide 51Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val
Lys Lys Pro Gly Ser1 5 10 15Ser Val Lys Val Ser Cys Lys Ala Ser Gly
Gly Thr Phe Ser Ser Tyr 20 25 30Ala Ile Ser Trp Val Arg Gln Ala Pro
Gly Gln Gly Leu Glu Trp Met 35 40 45Gly Ala Ile Ile Gly Met Phe Gly
Thr Thr Asn Tyr Ala Gln Lys Phe 50 55 60Gln Gly Arg Val Thr Leu Thr
Ala Asp Glu Leu Thr Ser Thr Ala Tyr65 70 75 80Met Glu Leu Ser Ser
Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95Ala Arg Gly Ser
Tyr Tyr Tyr Glu Ser Ser Leu Asp Tyr Trp Gly Lys 100 105 110Gly Thr
Met 11552115PRTArtificial SequenceDescription of Artificial
Sequence Synthetic polypeptide 52Gln Val Gln Leu Val Gln Ser Gly
Ala Glu Val Lys Lys Pro Gly Ser1 5 10 15Ser Val Lys Val Ser Cys Lys
Ala Ser Gly Gly Thr Phe Ser Ser Tyr 20 25 30Ala Ile Ser Trp Val Arg
Gln Ala Pro Gly Gln Gly Leu Glu Trp Met 35 40 45Gly Ala Ile Ile Gly
Met Phe Gly Thr Thr Asn Tyr Ala Gln Lys Phe 50 55 60Gln Gly Arg Val
Thr Ile Thr Ala Asp Glu Leu Thr Ser Thr Ala Tyr65 70 75 80Met Glu
Leu Ser Ser Leu Arg Ser Asp Asp Thr Ala Val Tyr Tyr Cys 85 90 95Ala
Arg Gly Ser Tyr Tyr Tyr Glu Ser Ser Leu Asp Tyr Trp Gly Lys 100 105
110Gly Thr Met 11553115PRTArtificial SequenceDescription of
Artificial Sequence Synthetic polypeptide 53Gln Val Gln Leu Val Gln
Ser Gly Ala Glu Val Lys Lys Pro Gly Ser1 5 10 15Ser Val Lys Val Ser
Cys Lys Thr Thr Gly Gly Thr Phe Ser Ser Tyr 20 25 30Ala Val Thr Trp
Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met 35 40 45Gly Gly Ile
Ile Gly Met Phe Gly Thr Thr Asn Tyr Ala Gln Lys Phe 50 55 60Gln Gly
Arg Val Thr Ile Thr Ala Asp Glu Met Thr Ser Thr Ala Tyr65 70 75
80Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys
85 90 95Ala Arg Gly Ser Tyr Tyr Tyr Glu Ser Ser Leu Asp Tyr Trp Gly
Gln 100 105 110Gly Thr Met 11554115PRTArtificial
SequenceDescription of Artificial Sequence Synthetic polypeptide
54Gln Val Gln Leu Gln Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ser1
5 10 15Ser Val Lys Val Ser Cys Lys Thr Ser Gly Gly Thr Phe Ser Ser
Tyr 20 25 30Ala Val Thr Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu
Trp Met 35 40 45Gly Ala Ile Ile Gly Met Phe Gly Thr Thr Asn Tyr Ala
Gln Lys Phe 50 55 60Gln Gly Arg Val Thr Ile Thr Ala Asp Glu Leu Thr
Ser Thr Ala Tyr65 70 75 80Met Glu Leu Ser Ser Leu Arg Ser Asp Asp
Thr Ala Val Tyr Tyr Cys 85 90 95Ala Arg Gly Ser Tyr Tyr Tyr Glu Ser
Ser Leu Asp Tyr Trp Gly Lys 100 105 110Gly Thr Met
11555115PRTArtificial SequenceDescription of Artificial Sequence
Synthetic polypeptide 55Gln Val Gln Leu Gln Gln Ser Gly Ala Glu Val
Lys Lys Pro Gly Ser1 5 10 15Ser Val Lys Val Ser Cys Lys Thr Ser Gly
Gly Thr Phe Ser Ser Tyr 20 25 30Ala Val Thr Trp Val Arg Gln Ala Pro
Gly Gln Gly Leu Glu Trp Met 35 40 45Gly Gly Ile Ile Gly Met Phe Gly
Thr Thr Asn Tyr Ala Gln Lys Phe 50 55 60Gln Gly Arg Val Thr Ile Thr
Ala Asp Glu Leu Thr Ser Thr Ala Tyr65 70 75 80Met Glu Leu Ser Ser
Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95Ala Arg Gly Ser
Tyr Tyr Tyr Glu Asn Ser Leu Asp Tyr Trp Gly Lys 100 105 110Gly Thr
Leu 11556115PRTArtificial SequenceDescription of Artificial
Sequence Synthetic polypeptide 56Gln Val Gln Leu Val Gln Ser Gly
Ala Glu Val Lys Lys Pro Gly Ser1 5 10 15Ser Val Lys Val Ser Cys Lys
Thr Ser Gly Gly Thr Phe Ser Ser Tyr 20 25 30Ala Val Thr Trp Val Arg
Gln Ala Pro Gly Gln Gly Leu Glu Trp Met 35 40 45Gly Ala Ile Ile Gly
Met Phe Gly Thr Thr Asn Tyr Ala Gln Lys Phe 50 55 60Gln Gly Arg Val
Thr Leu Thr Ala Asp Glu Leu Thr Ser Thr Ala Tyr65 70 75 80Met Glu
Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95Ala
Arg Gly Ser Tyr Tyr Tyr Glu Ser Ser Leu Asp Tyr Trp Gly Lys 100 105
110Gly Thr Met 11557115PRTArtificial SequenceDescription of
Artificial Sequence Synthetic polypeptide 57Gln Val Gln Leu Val Gln
Ser Gly Ala Glu Val Lys Lys Pro Gly Ser1 5 10 15Ser Val Lys Val Ser
Cys Lys Thr Ser Gly Gly Thr Phe Ser Ser Tyr 20 25 30Ala Val Thr Trp
Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met 35 40 45Gly Ala Ile
Ile Gly Met Phe Gly Thr Thr Asn Tyr Ala Gln Lys Phe 50 55 60Gln Gly
Arg Val Thr Ile Thr Ala Asp Glu Leu Thr Ser Thr Ala Tyr65 70 75
80Met Glu Leu Ser Ser Leu Arg Ser Asp Asp Thr Ala Val Tyr Tyr Cys
85 90 95Ala Arg Gly Ser Tyr Tyr Tyr Glu Ser Ser Leu Asp Tyr Trp Gly
Gln 100 105 110Gly Thr Met 11558115PRTArtificial
SequenceDescription of Artificial Sequence Synthetic polypeptide
58Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ser1
5 10 15Ser Val Lys Val Ser Cys Lys Thr Ser Gly Gly Thr Phe Ser Ser
Tyr 20 25 30Ala Val Thr Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu
Trp Met 35 40 45Gly Ala Ile Ile Gly Met Phe Gly Thr Thr Asn Tyr Ala
Gln Lys Phe 50 55 60Gln Gly Arg Val Thr Ile Thr Ala Asp Glu Leu Thr
Ser Thr Ala Tyr65 70 75 80Met Glu Leu Ser Ser Leu Arg Ser Asp Asp
Thr Ala Val Tyr Tyr Cys 85 90 95Ala Arg Gly Ser Tyr Tyr Tyr Glu Ser
Ser Leu Asp Tyr Trp Gly Arg
100 105 110Gly Thr Met 11559115PRTArtificial SequenceDescription of
Artificial Sequence Synthetic polypeptide 59Gln Val Gln Leu Val Gln
Ser Gly Ala Glu Val Lys Lys Pro Gly Ser1 5 10 15Ser Val Lys Val Ser
Cys Lys Ala Ser Gly Gly Thr Phe Ser Ser Tyr 20 25 30Ala Val Thr Trp
Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met 35 40 45Gly Ala Ile
Ile Gly Met Phe Gly Thr Thr Asn Tyr Ala Gln Lys Phe 50 55 60Gln Gly
Arg Val Thr Leu Thr Ala Asp Glu Leu Thr Ser Thr Ala Tyr65 70 75
80Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys
85 90 95Ala Arg Gly Ser Tyr Tyr Tyr Glu Ser Ser Leu Asp Tyr Trp Gly
Lys 100 105 110Gly Thr Met 11560115PRTArtificial
SequenceDescription of Artificial Sequence Synthetic polypeptide
60Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ser1
5 10 15Ser Val Lys Val Ser Cys Lys Ala Ser Gly Gly Thr Phe Ser Ser
Tyr 20 25 30Ala Ile Ser Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu
Trp Met 35 40 45Gly Gly Ile Ile Gly Met Phe Gly Thr Thr Asn Tyr Ala
Gln Lys Phe 50 55 60Gln Gly Arg Val Thr Ile Thr Ala Asp Glu Leu Thr
Ser Thr Ala Tyr65 70 75 80Met Glu Leu Ser Ser Leu Arg Ser Glu Asp
Thr Ala Val Tyr Tyr Cys 85 90 95Ala Arg Gly Ser Tyr Tyr Tyr Glu Ser
Ser Leu Asp Tyr Trp Gly Lys 100 105 110Gly Thr Thr
11561118PRTArtificial SequenceDescription of Artificial Sequence
Synthetic polypeptide 61Gln Val Gln Leu Gln Glu Ser Gly Pro Gly Leu
Val Lys Pro Ser Glu1 5 10 15Thr Leu Ser Leu Thr Cys Thr Val Ser Gly
Tyr Ser Phe Asp Ser Gly 20 25 30Tyr Tyr Trp Gly Trp Leu Arg Gln Pro
Pro Gly Lys Gly Leu Glu Trp 35 40 45Ile Gly Ser Ile Tyr His Ser Arg
Asn Thr Tyr Tyr Asn Pro Ser Leu 50 55 60Lys Ser Arg Val Thr Ile Ser
Val Asp Thr Ser Lys Asn Gln Phe Ser65 70 75 80Leu Gln Leu Ser Ser
Val Thr Ala Ala Asp Thr Ala Val Tyr Tyr Cys 85 90 95Ala Arg Gly Thr
Trp Tyr Ser Ser Asn Leu Arg Tyr Trp Phe Asp Pro 100 105 110Trp Gly
Lys Gly Thr Leu 11562118PRTArtificial SequenceDescription of
Artificial Sequence Synthetic polypeptide 62Gln Val Gln Leu Gln Glu
Ser Gly Pro Gly Gln Val Lys Pro Ser Glu1 5 10 15Thr Leu Ser Leu Thr
Cys Thr Val Ser Gly Tyr Ser Phe Asp Ser Gly 20 25 30Tyr Tyr Trp Gly
Trp Leu Arg Gln Pro Pro Gly Lys Gly Leu Glu Trp 35 40 45Ile Gly Ser
Ile Tyr His Ser Arg Asn Thr Tyr Tyr Asn Pro Ser Leu 50 55 60Lys Ser
Arg Val Thr Ile Ser Val Asp Thr Ser Lys Asn Gln Phe Ser65 70 75
80Leu Gln Leu Ser Ser Val Thr Ala Ala Asp Thr Ala Val Tyr Tyr Cys
85 90 95Ala Arg Gly Thr Trp Tyr Ser Ser Asn Leu Arg Tyr Trp Phe Asp
Pro 100 105 110Trp Gly Lys Gly Thr Thr 11563116PRTArtificial
SequenceDescription of Artificial Sequence Synthetic polypeptide
63Glu Val Gln Leu Val Gln Ser Gly Ala Ala Val Lys Lys Pro Gly Ser1
5 10 15Ser Val Lys Val Ser Cys Lys Ala Ser Gly Gly Arg Phe Ser Ser
Tyr 20 25 30Ala Ile Asn Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu
Trp Met 35 40 45Gly Gly Ile Ile Gly Met Phe Gly Thr Thr Asp Tyr Ala
Gln Lys Phe 50 55 60Gln Gly Arg Val Thr Ile Thr Ala Asp Glu Val Thr
Ser Thr Gly Tyr65 70 75 80Met Glu Leu Arg Ser Leu Thr Ser Glu Asp
Thr Ala Val Tyr Tyr Cys 85 90 95Ala Arg Gly Ser Gly Tyr His Leu Gln
Asn Pro Phe Asp Leu Trp Gly 100 105 110Arg Gly Thr Met
11564116PRTArtificial SequenceDescription of Artificial Sequence
Synthetic polypeptide 64Gln Val Gln Leu Gln Gln Ser Gly Ala Ala Val
Lys Lys Pro Gly Ser1 5 10 15Ser Val Lys Val Ser Cys Lys Ala Ser Gly
Gly Arg Phe Ser Ser Tyr 20 25 30Ala Ile Asn Trp Val Arg Gln Ala Pro
Gly Gln Gly Leu Glu Trp Met 35 40 45Gly Gly Ile Ile Gly Met Phe Gly
Thr Thr Asp Tyr Ala Gln Lys Phe 50 55 60Gln Gly Arg Val Thr Ile Thr
Ala Asp Glu Val Thr Ser Thr Gly Tyr65 70 75 80Met Glu Leu Arg Ser
Leu Thr Ser Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95Ala Arg Gly Ser
Gly Tyr His Leu Gln Asn Pro Phe Asp Leu Trp Gly 100 105 110Lys Gly
Thr Leu 11565119PRTArtificial SequenceDescription of Artificial
Sequence Synthetic polypeptide 65Glu Val Gln Leu Val Glu Ser Gly
Gly Gly Leu Val Gln Pro Gly Gly1 5 10 15Ser Leu Arg Leu Ser Cys Ala
Ala Ser Gly Phe Pro Phe Ser Ser Tyr 20 25 30Val Met Ile Trp Val Arg
Gln Val Pro Gly Lys Gly Leu Glu Trp Val 35 40 45Ser Ala Ile Gly Gly
Ser Gly Gly Ser Thr Tyr Tyr Ala Asp Ser Val 50 55 60Lys Gly Arg Phe
Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr65 70 75 80Leu Gln
Met Asn Ser Leu Arg Ala Asp Asp Thr Ala Val Tyr Tyr Cys 85 90 95Val
Leu Ser Pro Lys Ser Tyr Tyr Asp Asn Ser Gly Ile Tyr Phe Asp 100 105
110Phe Trp Gly Arg Gly Thr Leu 11566119PRTArtificial
SequenceDescription of Artificial Sequence Synthetic polypeptide
66Glu Val Gln Leu Val Glu Thr Gly Gly Gly Leu Val Gln Pro Gly Gly1
5 10 15Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Pro Phe Ser Ser
Tyr 20 25 30Val Met Ile Trp Val Arg Gln Val Pro Gly Lys Gly Leu Glu
Trp Val 35 40 45Ser Ala Ile Gly Gly Ser Gly Gly Ser Thr Tyr Tyr Ala
Asp Ser Val 50 55 60Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys
Asn Thr Leu Tyr65 70 75 80Leu Gln Met Asn Ser Leu Arg Ala Asp Asp
Thr Ala Val Tyr Tyr Cys 85 90 95Val Leu Ser Pro Lys Ser Tyr Tyr Asp
Asn Ser Gly Ile Tyr Phe Asp 100 105 110Phe Trp Gly Lys Gly Thr Leu
11567119PRTArtificial SequenceDescription of Artificial Sequence
Synthetic polypeptide 67Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu
Val Gln Pro Gly Gly1 5 10 15Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly
Phe Pro Phe Ser Ser Tyr 20 25 30Val Met Ile Trp Val Arg Gln Val Pro
Gly Lys Gly Leu Glu Trp Val 35 40 45Ser Ala Ile Gly Gly Ser Gly Gly
Ser Thr Tyr Tyr Ala Asp Ser Val 50 55 60Lys Gly Arg Phe Thr Ile Ser
Arg Asp Asn Ser Lys Asn Thr Leu Tyr65 70 75 80Leu Gln Met Asn Ser
Leu Arg Ala Asp Asp Thr Ala Val Tyr Tyr Cys 85 90 95Val Leu Ser Pro
Lys Ser Tyr Tyr Asp Asn Ser Gly Ile Tyr Phe Asp 100 105 110Phe Trp
Gly Arg Gly Thr Leu 11568110PRTArtificial SequenceDescription of
Artificial Sequence Synthetic polypeptide 68Gln Ser Ala Leu Thr Gln
Pro Ala Ser Val Ser Gly Ser Pro Gly Gln1 5 10 15Ser Ile Thr Ile Ser
Cys Thr Gly Thr Ser Ser Asp Phe Gly Gly Ser 20 25 30Asn His Val Ser
Trp Tyr Gln Gln His Pro Gly Lys Ala Pro Lys Leu 35 40 45Ile Ile Tyr
Asp Val Ser Asp Arg Pro Ser Gly Val Ser Asn Arg Phe 50 55 60Ser Gly
Ser Lys Ser Gly Asn Thr Ala Ser Leu Thr Val Ser Gly Leu65 70 75
80Gln Ala Glu Asp Glu Ala His Tyr Tyr Cys Ser Ser Tyr Ala Gly Ser
85 90 95Asn Asn Phe Val Phe Gly Thr Gly Thr Lys Val Thr Val Leu 100
105 11069111PRTArtificial SequenceDescription of Artificial
Sequence Synthetic polypeptide 69Glu Thr Thr Leu Thr Gln Ser Pro
Gly Thr Leu Ser Leu Ser Pro Gly1 5 10 15Glu Arg Ala Thr Leu Ser Cys
Arg Ala Ser Gln Thr Val Ser Ser Ser 20 25 30Tyr Leu Ala Trp Tyr Arg
Gln Lys Pro Gly Gln Ala Pro Arg Leu Leu 35 40 45Ile Tyr Gly Thr Ser
Ser Arg Ala Thr Gly Ile Pro Asp Arg Phe Ser 50 55 60Gly Ser Gly Ser
Gly Thr Asp Phe Thr Leu Thr Ile Ser Arg Leu Glu65 70 75 80Pro Glu
Asp Phe Ala Val Tyr Tyr Cys Gln Gln Tyr Gly Asn Ser Arg 85 90 95Val
Thr Phe Gly Pro Gly Thr Lys Val Asp Ile Lys Arg Thr Val 100 105
11070111PRTArtificial SequenceDescription of Artificial Sequence
Synthetic polypeptide 70Glu Ile Val Met Thr Gln Ser Pro Gly Thr Leu
Ser Leu Ser Pro Gly1 5 10 15Glu Arg Ala Thr Leu Ser Cys Arg Ala Ser
Gln Ser Leu Ser Gly Ser 20 25 30Asn Val Ala Trp Tyr Gln Gln Lys Phe
Gly Gln Ala Pro Arg Leu Leu 35 40 45Ile His Gly Ala Ser Lys Arg Ala
Ala Gly Ile Pro Asp Arg Phe Ser 50 55 60Gly Ser Gly Ser Gly Thr Asp
Phe Thr Leu Thr Ile Ser Arg Leu Gln65 70 75 80Pro Asp Asp Tyr Ala
Val Tyr Tyr Cys Gln Gln Tyr Gly Thr Lys Pro 85 90 95Phe Thr Phe Gly
Gln Gly Ser Lys Leu Glu Ile Lys Arg Thr Val 100 105
11071111PRTArtificial SequenceDescription of Artificial Sequence
Synthetic polypeptide 71Glu Thr Thr Leu Thr Gln Ser Pro Gly Thr Leu
Ser Leu Ser Pro Gly1 5 10 15Glu Arg Ala Thr Leu Ser Cys Arg Val Ser
Gln Ser Val Ser Ser Asn 20 25 30Tyr Leu Ala Trp Tyr Gln Gln Lys Pro
Gly Gln Ala Pro Arg Leu Leu 35 40 45Ile Tyr Gly Ala Ser Ser Arg Ala
Thr Gly Ile Pro Asp Arg Phe Ser 50 55 60Gly Ser Gly Ser Gly Thr Asp
Phe Thr Leu Thr Ile Ser Arg Leu Glu65 70 75 80Pro Glu Asp Phe Ala
Val Tyr Tyr Cys Gln Gln Tyr Gly Thr Ser Pro 85 90 95Arg Ala Phe Gly
His Gly Thr Lys Val Glu Ile Lys Arg Thr Val 100 105
11072111PRTArtificial SequenceDescription of Artificial Sequence
Synthetic polypeptide 72Gln Ser Ala Leu Thr Gln Pro Pro Ser Ala Ser
Gly Ser Pro Gly Gln1 5 10 15Ser Val Thr Ile Ser Cys Thr Gly Ala Ser
Ser Asp Ile Gly Gly Tyr 20 25 30Lys Ser Val Ser Trp Tyr Gln Gln His
Pro Gly Lys Ala Pro Lys Leu 35 40 45Ile Ile Tyr Asp Val Thr Glu Arg
Pro Ser Gly Val Pro Asp Arg Phe 50 55 60Ser Ala Ser Lys Ser Gly Asn
Thr Ala Ser Leu Thr Val Ser Gly Leu65 70 75 80Gln Ala Glu Asp Glu
Ala Asp Tyr Tyr Cys Ser Ser Tyr Gly Gly Ser 85 90 95Asn Asn Leu Val
Val Phe Gly Gly Gly Thr Lys Val Thr Val Leu 100 105
11073108PRTArtificial SequenceDescription of Artificial Sequence
Synthetic polypeptide 73Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Val
Ser Ala Ser Val Gly1 5 10 15Asp Arg Val Thr Ile Thr Cys Arg Ala Ser
Gln Gly Ile Ser Ser Trp 20 25 30Leu Ala Trp Tyr Gln Gln Lys Pro Gly
Lys Ala Pro Lys Leu Leu Ile 35 40 45Tyr Ala Ala Ser Thr Leu Gln Arg
Gly Val Pro Ser Arg Phe Ser Gly 50 55 60Ser Gly Ser Gly Thr Asp Phe
Thr Leu Thr Ile Asn Ser Leu Gln Pro65 70 75 80Glu Asp Phe Ala Thr
Tyr Tyr Cys Gln Gln Tyr Asn Ser Tyr Pro Leu 85 90 95Thr Phe Gly Gly
Gly Thr Lys Val Glu Ile Lys Arg 100 10574110PRTArtificial
SequenceDescription of Artificial Sequence Synthetic polypeptide
74Glu Ile Val Leu Thr Gln Ser Pro Gly Thr Leu Ser Leu Ser Pro Gly1
5 10 15Glu Arg Ala Thr Leu Ser Cys Arg Ala Ser Gln Ser Val Ser Asn
Asn 20 25 30Tyr Leu Ala Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Arg
Leu Leu 35 40 45Ile Tyr Gly Ala Ser Ser Arg Ala Thr Gly Ile Pro Asp
Arg Phe Ser 50 55 60Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile
Ser Arg Leu Glu65 70 75 80Pro Glu Asp Phe Ala Val Tyr Tyr Cys Gln
Gln Tyr Gly Arg Ser Pro 85 90 95Arg Thr Phe Gly Gln Gly Thr Lys Val
Glu Ile Lys Arg Thr 100 105 11075111PRTArtificial
SequenceDescription of Artificial Sequence Synthetic polypeptide
75Glu Thr Thr Leu Thr Gln Ser Pro Gly Thr Leu Ser Leu Ser Pro Gly1
5 10 15Glu Arg Ala Thr Leu Ser Cys Arg Ala Ser Gln Ser Leu Gly Gly
Ala 20 25 30Asn Leu Gly Trp Tyr Gln Gln Lys Phe Gly Gln Pro Pro Arg
Leu Leu 35 40 45Ile Tyr Gly Ala Ser Ser Arg Ala Thr Gly Val Pro Asp
Arg Phe Ser 50 55 60Gly Ser Gly Ser Gly Thr Asp Phe Ala Leu Thr Ile
Ser Arg Leu Glu65 70 75 80Pro Glu Asp Phe Ala Val Tyr Tyr Cys Gln
Gln Tyr Gly Ser Lys Pro 85 90 95Tyr Thr Phe Gly Gln Gly Thr Lys Leu
Glu Ile Lys Arg Thr Val 100 105 11076111PRTArtificial
SequenceDescription of Artificial Sequence Synthetic polypeptide
76Glu Thr Thr Leu Thr Gln Ser Pro Ala Thr Leu Ser Val Ser Pro Gly1
5 10 15Glu Arg Ala Thr Leu Ser Cys Arg Ala Ser Gln Ser Val Ser Thr
Asn 20 25 30Leu Ala Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Arg Leu
Leu Ile 35 40 45His Gly Ala Ser Thr Arg Ala Thr Gly Ile Pro Ala Arg
Phe Ser Gly 50 55 60Ser Gly Ser Gly Thr Glu Phe Thr Leu Thr Ile Ser
Ser Leu Gln Ser65 70 75 80Glu Asp Ser Ala Val Tyr Tyr Cys Gln Gln
His Asn Asn Trp Pro Pro 85 90 95Val Thr Phe Gly Arg Gly Thr Lys Val
Glu Ile Lys Arg Thr Val 100 105 11077111PRTArtificial
SequenceDescription of Artificial Sequence Synthetic polypeptide
77Glu Thr Thr Leu Thr Gln Ser Pro Ala Thr Leu Ser Val Ser Pro Gly1
5 10 15Glu Arg Ala Thr Leu Ser Cys Arg Ala Ser Gln Ser Val Ser Arg
Asn 20 25 30Leu Ala Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Arg Leu
Leu Ile 35 40 45Tyr Gly Ala Ser Ser Arg Ala Thr Gly Ile Pro Asp Arg
Phe Ser Gly 50 55 60Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser
Arg Leu Glu Pro65 70 75 80Glu Asp Phe Ala Val Tyr Tyr Cys Gln Gln
Tyr Gly Ser Ser Ser Ile 85 90 95Thr Phe Gly Gln Gly Thr Arg Leu Glu
Ile Lys Arg Thr Val Val 100 105 11078111PRTArtificial
SequenceDescription of Artificial Sequence Synthetic polypeptide
78Gln Ser Val Leu Thr Gln Pro Pro Ser Ala Ser Gly Ala Pro Gly Gln1
5 10 15Arg Val Thr Ile Ser Cys Thr Gly Ser Ser Ser Asn Ile Gly Ala
Gly 20 25 30Tyr Asp Val His Trp Tyr Gln Gln Leu Pro Gly Arg Ala Pro
Lys Leu 35 40 45Leu Ile Tyr
Gly Asn Ser Asn Arg Pro Ser Gly Val Pro Ala Arg Phe 50 55 60Ser Gly
Ser Lys Ser Ala Thr Ser Ala Ser Leu Ala Ile Thr Gly Leu65 70 75
80Gln Ala Glu Asp Glu Ala Asp Tyr Tyr Cys Gln Ser Tyr Asp Ser Ser
85 90 95Leu Ser Gly Val Val Phe Gly Gly Gly Thr Lys Leu Thr Val Leu
100 105 11079107PRTArtificial SequenceDescription of Artificial
Sequence Synthetic polypeptide 79Glu Ile Val Met Thr Gln Ser Pro
Ala Thr Leu Ser Val Ser Pro Gly1 5 10 15Glu Arg Ala Ile Leu Ser Cys
Arg Ala Ser Arg Ser Val Ser Thr Asn 20 25 30Leu Ala Trp Tyr Gln Gln
Lys Pro Gly Gln Ala Pro Arg Leu Leu Ile 35 40 45Tyr Gly Ala Ser Thr
Arg Ala Thr Gly Ile Pro Asp Arg Phe Ser Gly 50 55 60Ser Gly Ser Gly
Thr Asp Phe Thr Leu Thr Ile Ser Arg Leu Glu Pro65 70 75 80Glu Asp
Phe Ala Val Tyr Tyr Cys Gln Gln Tyr Gly Ser Ser Pro Asn 85 90 95Phe
Gly Gly Gly Thr Lys Val Glu Ile Lys Arg 100 10580111PRTArtificial
SequenceDescription of Artificial Sequence Synthetic polypeptide
80Glu Ile Val Met Thr Gln Ser Pro Gly Thr Leu Ser Leu Ser Pro Gly1
5 10 15Glu Arg Ala Thr Leu Ser Cys Arg Ala Ser Gln Ser Val Pro Asn
Arg 20 25 30Tyr Ile Ala Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Arg
Leu Leu 35 40 45Ile Tyr Gly Ala Ser Ser Arg Ala Thr Gly Ile Pro Asp
Arg Phe Ser 50 55 60Gly Ser Gly Ser Gly Pro Asp Phe Thr Leu Thr Ile
Ser Arg Leu Glu65 70 75 80Pro Glu Asp Phe Ala Val Tyr Tyr Cys Gln
Gln Tyr Gly Arg Ser Pro 85 90 95Gln Thr Phe Gly Gln Gly Thr Lys Leu
Glu Ile Lys Gly Thr Val 100 105 11081111PRTArtificial
SequenceDescription of Artificial Sequence Synthetic polypeptide
81Gln Ser Val Leu Thr Gln Pro Pro Ser Val Ser Gly Ala Pro Gly Gln1
5 10 15Arg Val Thr Ile Ser Cys Gly Gly Ser Arg Ser Asn Ile Gly Ala
Gly 20 25 30Tyr Asp Val His Trp Tyr Gln Gln Phe Pro Gly Thr Ala Pro
Lys Val 35 40 45Val Ile Tyr Gly Asn Asn Asn Arg Pro Ser Gly Val Pro
Asp Arg Phe 50 55 60Ser Gly Ser Lys Ser Gly Thr Ser Ala Ser Leu Ala
Ile Thr Gly Leu65 70 75 80Gln Ala Glu Asp Glu Ala Asn Tyr Tyr Cys
Gln Ser Tyr Asp Thr Asn 85 90 95Leu Gly Gly Ser Ile Phe Gly Gly Gly
Thr Gln Val Thr Val Leu 100 105 11082108PRTArtificial
SequenceDescription of Artificial Sequence Synthetic polypeptide
82Glu Ile Val Met Thr Gln Ser Pro Gly Thr Leu Ser Val Ser Pro Gly1
5 10 15Asp Ala Ala Thr Leu Ser Cys Arg Ala Ser Arg Asn Ile Asn Asn
Asn 20 25 30Leu Ala Trp Tyr Gln Gln Thr Pro Gly Gln Ala Pro Arg Leu
Leu Ile 35 40 45Tyr Gly Ala Ser Thr Arg Ala Thr Gly Leu Pro Ala Arg
Phe Thr Gly 50 55 60Ser Gly Ser Gly Thr Glu Phe Thr Leu Thr Ile Ser
Ser Leu Gln Ser65 70 75 80Glu Asp Phe Ala Val Tyr Tyr Cys Gln Gln
Tyr Asn Asn Trp Pro Arg 85 90 95Thr Phe Gly Gln Gly Thr Lys Val Glu
Ile Lys Arg 100 10583111PRTArtificial SequenceDescription of
Artificial Sequence Synthetic polypeptide 83Glu Thr Thr Leu Thr Gln
Ser Pro Gly Thr Leu Ser Leu Ser Pro Gly1 5 10 15Glu Arg Ala Thr Leu
Ser Cys Arg Ala Ser Gln Ile Val Asp Ser Ser 20 25 30Tyr Leu Ala Trp
Tyr Gln His Arg Pro Gly Gln Ala Pro Arg Ile Leu 35 40 45Ile Tyr Gly
Ala Ser Ser Arg Ala Pro Gly Val Pro Asp Arg Phe Ser 50 55 60Gly Ser
Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Arg Leu Glu65 70 75
80Pro Glu Asp Phe Ala Val Tyr Tyr Cys Gln Gln Tyr Ala Val Ser Pro
85 90 95Arg Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys Arg Thr Val
100 105 11084111PRTArtificial SequenceDescription of Artificial
Sequence Synthetic polypeptide 84Glu Ile Val Leu Thr Gln Ser Pro
Gly Thr Leu Ser Leu Ser Pro Gly1 5 10 15Asp Arg Ala Thr Leu Ser Cys
Arg Ala Ser Gln Ser Leu Gly Thr Asn 20 25 30Tyr Leu Ala Trp Tyr Gln
Gln Lys Pro Gly Gln Ala Pro Arg Leu Leu 35 40 45Ile Tyr Gly Ala Ser
Ser Arg Ala Thr Gly Ile Pro Asp Arg Phe Ser 50 55 60Gly Ser Gly Ser
Gly Thr Asp Phe Thr Leu Ile Ile Ser Arg Leu Glu65 70 75 80Pro Glu
Asp Phe Ala Val Tyr Tyr Cys Gln Gln Tyr Gly Arg Ser Pro 85 90 95Gln
Lys Phe Gly Gln Gly Thr Lys Val Glu Ile Lys Arg Thr Val 100 105
11085110PRTArtificial SequenceDescription of Artificial Sequence
Synthetic polypeptide 85Gln Ser Val Leu Thr Gln Pro Pro Ser Ala Ser
Gly Thr Pro Gly Gln1 5 10 15Arg Val Thr Ile Ser Cys Ser Gly Ser Ser
Ser Asn Ile Gly Ser Asn 20 25 30Tyr Val Tyr Trp Tyr Gln Gln Leu Pro
Gly Thr Ala Pro Lys Leu Leu 35 40 45Ile Tyr Arg Asn Asn Gln Arg Pro
Ser Gly Val Pro Asp Arg Phe Ser 50 55 60Gly Ser Lys Ser Gly Thr Ser
Ala Ser Leu Ala Thr Ser Gly Leu Arg65 70 75 80Ser Glu Asp Glu Ala
Asn Tyr Tyr Cys Ala Ala Trp Asp Asp Ser Leu 85 90 95Ser Gly Trp Val
Phe Gly Gly Gly Thr Lys Leu Thr Val Leu 100 105
11086107PRTArtificial SequenceDescription of Artificial Sequence
Synthetic polypeptide 86Glu Ile Val Met Thr Gln Ser Pro Ala Thr Leu
Ser Val Ser Pro Gly1 5 10 15Glu Arg Ala Ile Leu Ser Cys Arg Ala Ser
Arg Ser Val Ser Thr Asn 20 25 30Leu Ala Trp Tyr Gln Gln Lys Pro Gly
Gln Ala Pro Arg Leu Leu Ile 35 40 45Tyr Gly Ala Ser Thr Arg Ala Thr
Gly Ile Pro Asp Arg Phe Ser Gly 50 55 60Ser Gly Ser Gly Thr Asp Phe
Thr Leu Thr Ile Ser Arg Leu Glu Pro65 70 75 80Glu Asp Phe Ala Val
Tyr Tyr Cys Gln Gln Tyr Gly Ser Ser Pro Asn 85 90 95Phe Gly Gly Gly
Thr Lys Val Glu Ile Lys Arg 100 10587108PRTArtificial
SequenceDescription of Artificial Sequence Synthetic polypeptide
87Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Val Ser Ala Ser Val Gly1
5 10 15Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Gly Ile Ser Ser
Trp 20 25 30Leu Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu
Leu Ile 35 40 45Tyr Ala Ala Ser Ser Leu Gln Ser Gly Val Pro Ser Arg
Phe Ser Gly 50 55 60Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser
Ser Leu Gln Pro65 70 75 80Glu Asp Val Ala Thr Tyr Tyr Cys Gln Lys
Tyr Asn Ser Ala Pro Arg 85 90 95Thr Phe Gly Gln Gly Thr Lys Val Glu
Ile Lys Arg 100 10588110PRTArtificial SequenceDescription of
Artificial Sequence Synthetic polypeptide 88Asp Ile Gln Leu Thr Gln
Ser Pro Ser Ser Leu Ser Ala Ser Val Gly1 5 10 15Asp Arg Val Thr Ile
Thr Cys Arg Ala Ser Gln Ser Ile Ser Asn Tyr 20 25 30Leu Asn Trp Tyr
Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile 35 40 45Tyr Ala Ala
Ser Ser Leu Gln Ser Gly Val Pro Ser Arg Phe Ser Gly 50 55 60Ser Gly
Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro65 70 75
80Glu Asp Ser Ala Thr Tyr Tyr Cys Gln Gln Ser His Ser Thr Pro Arg
85 90 95Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys Arg Thr Val 100
105 11089110PRTArtificial SequenceDescription of Artificial
Sequence Synthetic polypeptide 89Gln Ala Val Leu Thr Gln Pro Pro
Ser Ala Ser Gly Thr Pro Gly Gln1 5 10 15Arg Val Thr Ile Ser Cys Ser
Gly Ser Ser Ser Asn Ile Gly Thr Asn 20 25 30Thr Val Asn Trp Tyr Gln
Gln Leu Pro Gly Thr Ala Pro Lys Leu Leu 35 40 45Ile Tyr Arg Asn Ile
Gln Arg Pro Ser Gly Val Pro Asp Arg Phe Ser 50 55 60Gly Ser Lys Ser
Gly Thr Ser Ala Ser Leu Ala Ile Ser Gly Leu Gln65 70 75 80Ser Glu
Asp Glu Ala Asp Tyr Tyr Cys Ala Ala Trp Asp Asp Ser Leu 85 90 95Asn
Gly Tyr Val Phe Gly Thr Gly Thr Lys Leu Thr Val Leu 100 105
11090111PRTArtificial SequenceDescription of Artificial Sequence
Synthetic polypeptide 90Glu Ile Val Leu Thr Gln Ser Pro Gly Thr Leu
Ser Leu Ser Pro Gly1 5 10 15Glu Lys Ala Thr Leu Ser Cys Arg Ala Ser
Gln Ser Val Ser Asn Thr 20 25 30Tyr Leu Ala Trp Tyr Gln Gln Lys Pro
Gly Gln Ala Pro Arg Leu Leu 35 40 45Leu Tyr Gly Ala Ser Ser Arg Ala
Pro Gly Ile Pro Asp Arg Phe Ser 50 55 60Gly Ser Gly Ser Gly Thr Asp
Phe Thr Leu Thr Ile Ser Arg Leu Glu65 70 75 80Ala Glu Asp Phe Ala
Val Tyr Phe Cys Gln Gln Tyr Ala Gly Ser Pro 85 90 95Arg Thr Phe Gly
Gln Gly Thr Lys Val Glu Ile Lys Arg Thr Val 100 105
11091111PRTArtificial SequenceDescription of Artificial Sequence
Synthetic polypeptide 91Glu Ile Val Leu Thr Gln Ser Pro Gly Ser Leu
Ser Leu Ser Pro Gly1 5 10 15Glu Arg Ala Thr Leu Ser Cys Arg Ala Ser
Gln Ser Val Ser His Gly 20 25 30Tyr Leu Ala Trp Tyr Gln Gln Lys Pro
Gly Gln Ala Pro Arg Phe Leu 35 40 45Ile Tyr Gly Ala Ser Ser Arg Pro
Thr Gly Ile Pro Asp Arg Phe Arg 50 55 60Gly Ser Gly Ser Gly Thr Asp
Phe Thr Leu Thr Ile Ser Ser Leu Glu65 70 75 80Pro Glu Asp Ser Ala
Val Tyr Tyr Cys Gln Gln Tyr Ser Thr Ser Pro 85 90 95Leu Thr Phe Gly
Gly Gly Thr Lys Val Glu Ile Lys Arg Thr Val 100 105
11092109PRTArtificial SequenceDescription of Artificial Sequence
Synthetic polypeptide 92Pro Glu Leu Thr Gln Pro Pro Ser Ala Ser Gly
Thr Pro Gly Gln Arg1 5 10 15Val Thr Ile Ser Cys Ser Gly Ser Ser Ser
Asn Ile Gly Ser Asn Tyr 20 25 30Val Tyr Trp Tyr Gln Gln Leu Pro Gly
Thr Ala Pro Lys Leu Leu Ile 35 40 45Tyr Arg Asn Asn Gln Arg Pro Ser
Gly Val Pro Asp Arg Phe Ser Gly 50 55 60Ser Lys Ser Gly Thr Ser Ala
Ser Leu Ala Ile Ser Gly Leu Arg Ser65 70 75 80Glu Asp Glu Ala Asp
Tyr Tyr Cys Ala Ala Trp Asp Asp Ser Leu Ser 85 90 95Gly Trp Val Phe
Gly Gly Gly Thr Lys Leu Thr Val Leu 100 10593111PRTArtificial
SequenceDescription of Artificial Sequence Synthetic polypeptide
93Glu Thr Thr Leu Thr Gln Ser Pro Gly Thr Leu Ser Leu Ser Pro Gly1
5 10 15Glu Arg Ala Thr Leu Ser Cys Arg Ala Ser Gln Ile Val Asp Ser
Ser 20 25 30Tyr Leu Ala Trp Tyr Gln His Arg Pro Gly Gln Ala Pro Arg
Ile Leu 35 40 45Ile Tyr Gly Ala Ser Ser Arg Ala Pro Gly Val Pro Asp
Arg Phe Ser 50 55 60Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile
Ser Arg Leu Glu65 70 75 80Pro Glu Asp Phe Ala Val Tyr Tyr Cys Gln
Gln Tyr Gly Ser Pro Pro 85 90 95Arg Thr Phe Gly Gln Gly Thr Lys Val
Glu Ile Lys Arg Thr Val 100 105 11094110PRTArtificial
SequenceDescription of Artificial Sequence Synthetic polypeptide
94Gln Ser Val Leu Thr Gln Pro Pro Ser Thr Ser Gly Thr Pro Gly Gln1
5 10 15Arg Val Thr Ile Ser Cys Ser Gly Ser Ser Ser Asn Ile Gly Arg
Lys 20 25 30Thr Val Asn Trp Tyr Gln Gln Leu Pro Gly Thr Ala Pro Lys
Leu Leu 35 40 45Ile Tyr Asn Asp Asn Gln Arg Pro Ser Gly Val Pro Asp
Arg Phe Ser 50 55 60Gly Ser Lys Ala Gly Thr Ser Ala Ser Leu Ala Ile
Ser Gly Leu Gln65 70 75 80Ser Glu Asp Glu Ala Asp Tyr Tyr Cys Ala
Val Trp Asp Asp Ser Leu 85 90 95Asn Ala Trp Val Phe Gly Gly Gly Thr
Lys Leu Thr Val Leu 100 105 11095115PRTArtificial
SequenceDescription of Artificial Sequence Synthetic polypeptide
95Asp Val Val Met Thr Gln Ser Pro Leu Ser Leu Pro Val Thr Pro Gly1
5 10 15Glu Pro Ala Ala Ile Ser Cys Arg Ser Ser Gln Ser Leu Leu His
Ser 20 25 30Asp Gly Asn Asn Tyr Leu Asp Trp Tyr Leu Gln Lys Pro Gly
Gln Ser 35 40 45Pro His Leu Leu Ile Tyr Leu Gly Ser Asn Arg Ala Ser
Gly Val Pro 50 55 60Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe
Glu Leu Lys Ile65 70 75 80Ser Arg Val Glu Ala Glu Asp Ala Gly Val
Tyr Tyr Cys Met Gln Ala 85 90 95Ser Gln Thr Pro Arg Thr Phe Gly Gln
Gly Thr Lys Leu Glu Leu Lys 100 105 110Arg Thr Val
11596108PRTArtificial SequenceDescription of Artificial Sequence
Synthetic polypeptide 96His Val Ile Leu Thr Gln Pro Pro Ser Val Ser
Val Ala Pro Gly Met1 5 10 15Thr Ala Arg Met Thr Cys Gly Gly Asp Asn
Val Gly Arg Arg Asn Val 20 25 30His Trp Tyr Gln Gln Lys Pro Gly Gln
Ala Pro Val Leu Val Val Tyr 35 40 45Asp Asp Gly Gly Arg Pro Ser Ala
Ile Pro Ala Arg Phe Ser Gly Ser 50 55 60Lys Ser Gly Asn Thr Ala Thr
Leu Ile Ile Ser Arg Val Glu Ala Gly65 70 75 80Asp Glu Ala Asp Tyr
Tyr Cys Gln Met Trp His Ser Ser Gly Asp Gln 85 90 95Trp Val Phe Gly
Gly Gly Thr Lys Leu Thr Val Leu 100 10597106PRTArtificial
SequenceDescription of Artificial Sequence Synthetic polypeptide
97Glu Thr Thr Leu Thr Gln Ser Pro Gly Thr Leu Ser Leu Ser Pro Gly1
5 10 15Glu Arg Ala Thr Leu Ser Cys Arg Ala Ser Gln Ser Ile Ser Pro
Asn 20 25 30Tyr Leu Ala Trp Tyr Gln Gln Arg Pro Gly Gln Ala Pro Arg
Leu Leu 35 40 45Ile Tyr Gly Ala Ser Lys Arg Ala Thr Gly Ile Pro Asp
Arg Phe Ser 50 55 60Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile
Ser Ser Leu Glu65 70 75 80Pro Glu Asp Ser Ala Val Tyr Tyr Cys Gln
His Gln Gly Phe Gly Gln 85 90 95Gly Thr Lys Val Glu Ile Lys Arg Thr
Val 100 10598110PRTArtificial SequenceDescription of Artificial
Sequence Synthetic polypeptide 98Gln Ser Val Leu Thr Gln Pro Pro
Ser Ala Ser Gly Thr Pro Asp Gln1 5 10 15Arg Val Thr Ile Ser Cys Ser
Gly Ser Gly Ser Asn Ile Gly Ser Asn 20 25 30Tyr Val Tyr Trp Tyr Gln
Gln Leu Pro Gly Ala Ala Pro Lys Leu Leu 35 40 45Met Ser Arg Asn Asn
Gln Arg Pro Ser Gly Val Pro Asp Arg Phe Ser 50 55 60Gly Ser Lys Ser
Gly Thr
Ser Ala Ser Leu Ala Ile Ser Gly Leu Gln65 70 75 80Ser Glu Asp Glu
Ala Asp Tyr Tyr Cys Ala Ala Trp Asp Asp Ser Leu 85 90 95Thr Gly Tyr
Val Phe Gly Thr Gly Thr Lys Leu Thr Val Leu 100 105
11099109PRTArtificial SequenceDescription of Artificial Sequence
Synthetic polypeptide 99Ser Tyr Val Leu Thr Gln Pro Pro Ser Val Ser
Val Ala Pro Gly Lys1 5 10 15Thr Ala Arg Ile Thr Cys Gly Gly Lys Asn
Ile Gly Ser Lys Ser Val 20 25 30His Trp Tyr Gln Gln Lys Ser Gly Gln
Ala Pro Val Leu Val Ile Tyr 35 40 45Gly Asp Ser Asp Arg Pro Ser Gly
Ile Pro Glu Arg Phe Ser Gly Ser 50 55 60Asn Ser Gly Asn Thr Ala Thr
Leu Thr Ile Ser Gly Val Glu Ala Glu65 70 75 80Asp Glu Ala Asp Tyr
Tyr Cys Gln Val Trp Asp Asn Thr Ser Asp His 85 90 95Ala Gly Val Phe
Gly Gly Gly Thr Lys Val Thr Val Leu 100 105100110PRTArtificial
SequenceDescription of Artificial Sequence Synthetic polypeptide
100Gln Ser Val Leu Thr Gln Pro Pro Ser Ala Ser Gly Thr Pro Gly Gln1
5 10 15Arg Val Thr Ile Ser Cys Ser Gly Ser Ser Ser Asn Ile Gly Ser
Asn 20 25 30Tyr Val Tyr Trp Tyr Gln Gln Leu Pro Gly Thr Ala Pro Lys
Leu Leu 35 40 45Ile Tyr Arg Asn Asn Gln Arg Ser Ser Gly Val Pro Asp
Arg Phe Ser 50 55 60Gly Ser Lys Ser Gly Thr Ser Ala Ser Leu Ala Ile
Ser Gly Leu Arg65 70 75 80Ser Glu Asp Glu Ala Asp Tyr Tyr Cys Ala
Ala Trp Asp Asp Ser Leu 85 90 95Ser Gly Leu Val Phe Gly Gly Gly Thr
Lys Leu Thr Val Leu 100 105 110101110PRTArtificial
SequenceDescription of Artificial Sequence Synthetic polypeptide
101Gln Ser Val Leu Thr Gln Pro Pro Ser Val Ser Ala Ala Pro Arg Gln1
5 10 15Ser Val Thr Ile Ser Cys Ser Gly Thr Thr Ser Asn Ile Gly Asn
Asn 20 25 30Pro Val Ser Trp Tyr Gln Gln Phe Pro Gly Arg Ala Pro Asn
Leu Leu 35 40 45Ile Tyr Tyr Asn Asp Val Val Pro Ser Gly Val Ser Asp
Arg Phe Ser 50 55 60Ala Ser Lys Ala Gly Thr Ser Ala Ser Leu Ala Ile
Ser Arg Leu Gln65 70 75 80Ser Glu Asp Glu Ala Asp Tyr Tyr Cys Ala
Thr Trp Asp Asp Ser Leu 85 90 95Ser Ala Trp Val Phe Gly Gly Gly Thr
Gln Leu Thr Val Leu 100 105 110102110PRTArtificial
SequenceDescription of Artificial Sequence Synthetic polypeptide
102Gln Ala Val Leu Thr Gln Pro Pro Ser Ala Ser Gly Thr Pro Gly Gln1
5 10 15Arg Val Thr Ile Ser Cys Ser Gly Ser Ser Ser Asn Ile Gly Ser
Asn 20 25 30Tyr Val Tyr Trp Tyr Gln Gln Leu Pro Gly Thr Ala Pro Lys
Leu Leu 35 40 45Ile Tyr Ser Asn Asn Gln Arg Pro Ser Gly Val Pro Asp
Arg Phe Ser 50 55 60Gly Ser Lys Ser Gly Thr Ser Ala Ser Leu Val Ile
Ser Gly Leu Gln65 70 75 80Ser Glu Asp Glu Thr Asp Tyr Tyr Cys Ala
Ala Trp Asp Asp Ser Leu 85 90 95Asn Gly Trp Val Phe Gly Gly Gly Thr
Lys Leu Thr Val Leu 100 105 110103111PRTArtificial
SequenceDescription of Artificial Sequence Synthetic polypeptide
103Gln Ser Ala Leu Thr Gln Pro Pro Ser Ala Ser Gly Ser Pro Gly Gln1
5 10 15Ser Val Thr Ile Thr Cys Ala Gly Ala Ser Ser Asp Leu Gly Asp
Tyr 20 25 30Lys Ser Val Ser Trp Tyr Gln Gln His Pro Gly Lys Ala Pro
Lys Leu 35 40 45Ile Ile Tyr Asp Val Ile Lys Arg Pro Ala Gly Val Pro
Asp Arg Phe 50 55 60Ser Ala Ser Lys Ser Gly Asn Thr Ala Ser Leu Thr
Val Ser Gly Leu65 70 75 80Gln Ala Glu Asp Glu Ala Asp Tyr Tyr Cys
Ser Ser Tyr Ala Gly Ser 85 90 95Asn Asn Ile Val Ile Phe Gly Gly Gly
Thr Lys Leu Thr Val Leu 100 105 110104111PRTArtificial
SequenceDescription of Artificial Sequence Synthetic polypeptide
104Glu Thr Thr Leu Thr Gln Ser Pro Gly Thr Leu Ser Leu Ser Pro Gly1
5 10 15Glu Arg Ala Thr Leu Ser Cys Arg Ala Ser Gln Gly Ile Asp Arg
Lys 20 25 30Tyr Leu Ala Trp Tyr Gln Arg Lys His Gly Gln Ala Pro Arg
Leu Leu 35 40 45Ile Tyr Gly Ala Ser Asn Arg Ala Thr Gly Ile Pro Asp
Arg Phe Ser 50 55 60Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile
Ser Arg Leu Glu65 70 75 80Pro Glu Asp Phe Ala Val Tyr Tyr Cys Gln
Gln Tyr Ala Asp Ser Phe 85 90 95Val Ser Phe Gly Gln Gly Thr Lys Leu
Glu Ile Lys Arg Thr Val 100 105 110105111PRTArtificial
SequenceDescription of Artificial Sequence Synthetic polypeptide
105Asp Ile Gln Leu Thr Gln Ser Pro Ser Thr Leu Ser Ala Ser Val Gly1
5 10 15Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Ser Ile Ser Arg
Trp 20 25 30Leu Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ile Pro Lys Leu
Leu Ile 35 40 45Tyr Glu Ala Ser Asn Leu Gln Ser Gly Val Pro Ser Arg
Phe Ser Gly 50 55 60Ser Gly Ser Gly Thr Glu Phe Thr Leu Thr Ile Ser
Ser Leu Gln Pro65 70 75 80Asp Asp Phe Ala Thr Tyr Tyr Cys Gln Gln
Tyr Lys Ser Asp Phe Leu 85 90 95Val Thr Phe Gly Pro Gly Thr Lys Val
Asp Ile Lys Arg Thr Val 100 105 110106111PRTArtificial
SequenceDescription of Artificial Sequence Synthetic polypeptide
106Gln Ser Val Leu Thr Gln Pro Pro Ser Val Ser Gly Ala Pro Gly Gln1
5 10 15Lys Ile Thr Ile Ser Cys Thr Gly Ser Ser Ser Asn Ile Gly Thr
Gly 20 25 30Tyr Asp Val His Trp Tyr Gln Gln Leu Pro Gly Arg Ala Pro
Arg Leu 35 40 45Leu Ile Ser Ala Asp Ala Asn Arg Pro Ser Gly Val Pro
Asp Arg Phe 50 55 60Ser Ala Ser Lys Ser Gly Thr Ser Ala Ser Leu Ala
Ile Thr Gly Leu65 70 75 80Gln Ala Glu Asp Glu Ala Asp Tyr Tyr Cys
Gln Ser Tyr Asp Thr Arg 85 90 95Leu Gly Gly Ser Ile Phe Gly Gly Gly
Thr Gln Leu Thr Val Leu 100 105 110107108PRTArtificial
SequenceDescription of Artificial Sequence Synthetic polypeptide
107Glu Ile Val Met Thr Gln Ser Pro Gly Thr Leu Ser Val Ser Pro Gly1
5 10 15Asp Ala Ala Thr Leu Ser Cys Arg Ala Ser Arg Asn Ile Asn Asn
Asn 20 25 30Leu Ala Trp Tyr Gln Gln Thr Pro Gly Gln Ala Pro Arg Leu
Leu Ile 35 40 45Tyr Gly Ala Ser Thr Arg Ala Thr Gly Leu Pro Ala Arg
Phe Thr Gly 50 55 60Ser Gly Ser Gly Thr Glu Phe Thr Leu Thr Ile Ser
Ser Leu Gln Ser65 70 75 80Glu Asp Phe Ala Val Tyr Tyr Cys Gln Gln
Tyr Asn Asn Trp Pro Arg 85 90 95Thr Phe Gly Gln Gly Thr Lys Val Glu
Ile Lys Arg 100 105108110PRTArtificial SequenceDescription of
Artificial Sequence Synthetic polypeptide 108Gln Ser Val Leu Thr
Gln Pro Pro Ser Val Ser Ala Ala Pro Gly Gln1 5 10 15Glu Val Thr Ile
Thr Cys Ser Gly Ser Gly Ala Asn Ile Gly Asn Asn 20 25 30Tyr Val Ser
Trp Tyr Gln Gln Val Pro Gly Thr Ala Pro Lys Leu Val 35 40 45Ile Tyr
Asp Asn Asn Arg Arg Pro Ser Gly Ile Pro Asp Arg Phe Ser 50 55 60Gly
Ser Lys Ser Gly Thr Ser Ala Thr Leu Gly Ile Thr Gly Leu Gln65 70 75
80Thr Gly Asp Glu Ala Asp Tyr Tyr Cys Gly Thr Trp Asp Ser Ser Leu
85 90 95Ser Ala Val Val Phe Gly Gly Gly Thr Lys Val Thr Val Leu 100
105 110109110PRTArtificial SequenceDescription of Artificial
Sequence Synthetic polypeptide 109Gln Ala Val Leu Thr Gln Pro Pro
Ser Ala Ser Gly Thr Pro Gly Gln1 5 10 15Thr Val Thr Ile Ser Cys Ser
Gly Val Thr Ser Asn Ile Gly Asn Asn 20 25 30Tyr Val Tyr Trp Tyr Gln
Gln Leu Pro Gly Thr Ala Pro Arg Leu Leu 35 40 45Ile Tyr Ser Asn Asn
Gln Arg Pro Ser Gly Val Pro Asp Arg Phe Ser 50 55 60Gly Ser Lys Ser
Gly Thr Ser Ala Ser Leu Ala Ile Ser Gly Leu Arg65 70 75 80Ser Glu
Asp Glu Ala Asp Tyr Tyr Cys Ser Ala Trp Asp Asp Ser Leu 85 90 95Arg
Glu Asn Leu Phe Gly Thr Gly Thr Gln Leu Thr Val Leu 100 105
110110110PRTArtificial SequenceDescription of Artificial Sequence
Synthetic polypeptide 110Gln Ala Val Leu Thr Gln Pro Pro Ser Ala
Ser Gly Thr Pro Gly Gln1 5 10 15Arg Val Thr Ile Ser Cys Ser Gly Ser
Ser Ser Asn Phe Gly Met Asn 20 25 30Ala Val Asn Trp Tyr Gln Gln Leu
Pro Gly Thr Ala Pro Lys Leu Leu 35 40 45Met Tyr Ser Asn Ser Lys Arg
Pro Ser Gly Val Pro Asp Arg Phe Ser 50 55 60Gly Ser Lys Ser Gly Thr
Ser Ala Ser Leu Ala Ile Ser Gly Leu Gln65 70 75 80Ser Glu Asp Glu
Ala Asp Tyr Tyr Cys Ser Ala Trp Asp Asp Asn Leu 85 90 95Asn Gly Trp
Val Phe Gly Gly Gly Thr Lys Val Thr Val Leu 100 105
110111110PRTArtificial SequenceDescription of Artificial Sequence
Synthetic polypeptide 111Gln Ala Val Leu Thr Gln Pro Pro Ser Ala
Ser Gly Thr Pro Gly Gln1 5 10 15Arg Val Thr Ile Ser Cys Ser Gly Ser
Ser Ser Asn Ile Gly Ser Asn 20 25 30Thr Val Asn Trp Tyr Gln Gln Leu
Pro Gly Thr Ala Pro Lys Leu Leu 35 40 45Ile Tyr Arg Asn Asn Gln Arg
Pro Ser Gly Val Pro Asp Arg Phe Ser 50 55 60Gly Ser Lys Ser Gly Thr
Ser Ala Ser Leu Ala Ile Ser Gly Leu Gln65 70 75 80Ser Glu Asp Glu
Ala Asp Tyr Tyr Cys Ala Thr Trp Asp Asp Ser Leu 85 90 95Thr Ser Val
Val Phe Gly Gly Gly Thr Lys Val Thr Val Leu 100 105
110112111PRTArtificial SequenceDescription of Artificial Sequence
Synthetic polypeptide 112Glu Thr Thr Leu Thr Gln Ser Pro Ser Ser
Leu Ser Ala Ser Ile Gly1 5 10 15Asp Arg Ile Thr Ile Ala Cys Gln Ala
Ser Gln Asp Ile Arg Asn Arg 20 25 30Leu Asn Trp Tyr Leu Gln Arg Pro
Gly Lys Ala Pro Gln Leu Leu Ile 35 40 45Tyr Asp Ala Ser Asn Leu Glu
Thr Gly Val Pro Ser Lys Phe Ala Gly 50 55 60Arg Gly Ser Gly Thr Asp
Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro65 70 75 80Glu Asp Ile Gly
Thr Tyr Phe Cys Gln Gln Tyr Gly Asp Leu Ser Pro 85 90 95Leu Thr Phe
Gly Gly Gly Thr Lys Val Asp Ile Arg Arg Thr Val 100 105
110113111PRTArtificial SequenceDescription of Artificial Sequence
Synthetic polypeptide 113Glu Thr Thr Leu Thr Gln Ser Pro Ser Ser
Leu Ser Ala Ser Ile Gly1 5 10 15Asp Arg Ile Thr Ile Ala Cys Gln Ala
Ser Gln Asp Ile Arg Asn Arg 20 25 30Leu Asn Trp Tyr Leu Gln Arg Pro
Gly Lys Ala Pro Gln Leu Leu Ile 35 40 45Tyr Asp Ala Ser Asn Leu Glu
Thr Gly Val Pro Ser Lys Phe Ala Gly 50 55 60Arg Gly Ser Gly Thr Asp
Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro65 70 75 80Glu Asp Ile Gly
Thr Tyr Phe Cys Gln Gln Tyr Gly Asp Leu Ser Pro 85 90 95Leu Thr Phe
Gly Gly Gly Thr Lys Val Asp Ile Arg Arg Thr Val 100 105
110114110PRTArtificial SequenceDescription of Artificial Sequence
Synthetic polypeptide 114Asp Ile Gln Met Thr Gln Ser Pro Ser Ser
Leu Ser Ala Ser Val Gly1 5 10 15Asp Arg Val Thr Ile Thr Cys Arg Ala
Ser Gln Ser Ile Ser Asn Tyr 20 25 30Leu Asn Trp Tyr Gln Gln Lys Pro
Gly Lys Ala Pro Lys Leu Leu Ile 35 40 45Tyr Ala Ala Ser Ser Leu Gln
Ser Gly Val Pro Ser Arg Phe Ser Gly 50 55 60Ser Gly Ser Gly Thr Asp
Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro65 70 75 80Glu Asp Ser Ala
Thr Tyr Tyr Cys Gln Gln Ser His Ser Thr Pro Arg 85 90 95Thr Phe Gly
Gln Gly Thr Lys Leu Glu Ile Lys Arg Thr Val 100 105
110115111PRTArtificial SequenceDescription of Artificial Sequence
Synthetic polypeptide 115Gln Ser Val Leu Thr Gln Pro Pro Ser Val
Ser Gly Ala Pro Gly Gln1 5 10 15Arg Val Thr Ile Ser Cys Thr Gly Thr
Ser Ser Asn Ile Gly Ala Gly 20 25 30Phe Asp Val His Trp Tyr Gln Gln
Phe Pro Gly Thr Ala Pro Lys Leu 35 40 45Leu Ile Tyr Asp Asn Val Lys
Arg Pro Ser Gly Val Pro Asp Arg Phe 50 55 60Ser Gly Ser Lys Ser Gly
Thr Ser Ala Ser Leu Ala Ile Thr Gly Leu65 70 75 80Arg Ala Glu Asp
Glu Ala Asp Tyr Tyr Cys Gln Ser Tyr Asp Thr Ser 85 90 95Leu Ser Arg
Tyr Val Phe Gly Thr Gly Thr Lys Val Thr Val Leu 100 105
110116111PRTArtificial SequenceDescription of Artificial Sequence
Synthetic polypeptide 116Glu Thr Thr Leu Thr Gln Ser Pro Gly Thr
Leu Ser Leu Ser Pro Gly1 5 10 15Glu Arg Ala Thr Leu Ser Cys Arg Ala
Ser Gln Ser Val Ser Ser Ile 20 25 30Tyr Leu Ala Trp Tyr Gln Gln Lys
Pro Gly Gln Ala Pro Arg Leu Val 35 40 45Ile His Gly Ala Ser Ser Arg
Ala Thr Gly Ile Pro Asp Arg Phe Ser 50 55 60Gly Ser Gly Ser Gly Thr
Asp Phe Thr Leu Thr Ile Ser Arg Leu Glu65 70 75 80Pro Glu Asp Phe
Ala Val Tyr Tyr Cys Gln Gln Tyr Gly Ser Ser Pro 85 90 95Leu Thr Phe
Gly Gly Gly Thr Lys Val Glu Ile Lys Arg Thr Val 100 105
110117111PRTArtificial SequenceDescription of Artificial Sequence
Synthetic polypeptide 117Glu Thr Thr Leu Thr Gln Ser Pro Gly Ile
Leu Ser Leu Ser Pro Gly1 5 10 15Glu Ser Ala Thr Leu Ser Cys Gly Ala
Ser Gln Thr Ile Ser Ser Arg 20 25 30Tyr Leu Ala Trp Tyr Gln Gln Arg
Pro Gly Gln Ala Pro Arg Leu Leu 35 40 45Ile Phe Asp Ala Ser Arg Arg
Ala Thr Gly Val Pro Asp Arg Phe Ser 50 55 60Gly Gly Gly Ser Gly Thr
Asp Phe Thr Leu Thr Ile Ser Arg Leu Glu65 70 75 80Pro Glu Asp Phe
Gly Val Phe Tyr Cys Gln Gln Tyr Gly Ile Ser Pro 85 90 95Tyr Thr Phe
Gly Gln Gly Thr Lys Leu Glu Ile Lys Arg Thr Val 100 105
110118111PRTArtificial SequenceDescription of Artificial Sequence
Synthetic polypeptide 118Glu Ile Val Leu Thr Gln Ser Pro Gly Thr
Leu Ser Leu Ser Pro Gly1 5 10 15Glu Arg Ala Thr Leu Ser Cys Arg Ala
Ser Gln Ser Val Ser Gly Asn 20 25 30Asn Leu Ala Trp Tyr Gln Gln Lys
Phe Gly Gln Ala Pro Arg Leu Leu 35 40 45Ile Tyr Ala
Ala Ser Ser Arg Ala Thr Asp Ile Pro Ala Arg Phe Ser 50 55 60Gly Ser
Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Arg Leu Glu65 70 75
80Pro Glu Asp Phe Ala Val Tyr Tyr Cys Gln Gln Tyr Val Asp Ser Pro
85 90 95Arg Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys Arg Thr Val
100 105 110119111PRTArtificial SequenceDescription of Artificial
Sequence Synthetic polypeptide 119Glu Ile Val Leu Thr Gln Ser Pro
Gly Thr Leu Ser Leu Ser Pro Gly1 5 10 15Glu Arg Ala Thr Leu Ser Cys
Arg Ala Ser Gln Ser Val Ser Ser Ser 20 25 30Tyr Leu Ala Trp Tyr Gln
Gln Lys Pro Gly Gln Ala Pro Arg Leu Leu 35 40 45Ile Tyr Gly Ala Ser
Ser Arg Ala Thr Gly Ile Pro Asp Arg Phe Ser 50 55 60Gly Ser Gly Ser
Gly Thr Asp Phe Thr Leu Thr Ile Ser Arg Leu Glu65 70 75 80Pro Glu
Asp Phe Ala Val Tyr Tyr Cys Gln Gln Tyr Gly Ser Ser Pro 85 90 95Leu
Thr Phe Gly Gly Gly Thr Lys Val Glu Ile Lys Arg Thr Val 100 105
110120111PRTArtificial SequenceDescription of Artificial Sequence
Synthetic polypeptide 120Glu Ile Val Leu Thr Gln Ser Pro Gly Thr
Leu Ser Leu Ser Pro Gly1 5 10 15Glu Arg Ala Thr Leu Ser Cys Arg Ala
Ser Gln Ser Val Ser Ser Ser 20 25 30Tyr Leu Ala Trp Tyr Gln Gln Lys
Pro Gly Gln Ala Pro Arg Leu Leu 35 40 45Ile Tyr Gly Ala Ser Ser Arg
Ala Ala Gly Ile Pro Asp Arg Phe Ser 50 55 60Gly Ser Gly Ser Gly Thr
Asp Phe Thr Leu Thr Ile Ser Arg Leu Glu65 70 75 80Pro Glu Asp Phe
Ala Val Tyr Tyr Cys Gln Gln Tyr Gly Ser Ser Pro 85 90 95Lys Thr Phe
Gly Gln Gly Thr Lys Val Glu Ile Lys Arg Thr Val 100 105
110121111PRTArtificial SequenceDescription of Artificial Sequence
Synthetic polypeptide 121Gln Ser Val Leu Thr Gln Pro Pro Ser Val
Ser Gly Ala Pro Gly Gln1 5 10 15Gly Val Thr Ile Ser Cys Ser Gly Ser
Ser Ser Asn Ile Gly Ala Asn 20 25 30Tyr Val Val His Trp Tyr Arg Gln
Leu Pro Gly Ala Ala Pro Lys Leu 35 40 45Leu Ile Tyr Asp Asp Ile His
Arg Pro Ser Gly Val Pro Asp Arg Phe 50 55 60Ser Gly Ser Arg Ser Gly
Thr Ser Ala Ser Leu Ala Ile Thr Gly Leu65 70 75 80Gln Pro Glu Asp
Glu Ala Asp Tyr Tyr Cys Gln Thr Tyr Asp Thr Ser 85 90 95Leu Arg Gly
Ser Val Phe Gly Gly Gly Thr Lys Leu Thr Val Leu 100 105
110122110PRTArtificial SequenceDescription of Artificial Sequence
Synthetic polypeptide 122Gln Ser Val Leu Thr Gln Pro Pro Ser Ala
Ser Gly Thr Pro Gly Gln1 5 10 15Arg Val Thr Ile Ser Cys Ser Gly Ser
Ser Ser Asn Ile Gly Ser Asn 20 25 30Tyr Val Tyr Trp Tyr Gln Gln Leu
Pro Gly Thr Ala Pro Lys Leu Leu 35 40 45Ile Tyr Arg Asn Asn Gln Arg
Pro Ser Gly Val Pro Asp Arg Phe Ser 50 55 60Gly Ser Lys Ser Gly Thr
Ser Ala Ser Leu Ala Ile Ser Gly Leu Arg65 70 75 80Ser Glu Asp Glu
Ala Asp Tyr Tyr Cys Ala Ala Trp Asp Asp Ser Leu 85 90 95Ser Gly Trp
Val Phe Gly Gly Gly Thr Lys Val Thr Val Leu 100 105
110123108PRTArtificial SequenceDescription of Artificial Sequence
Synthetic polypeptide 123Gln Ala Val Leu Thr Gln Pro Pro Ser Val
Ser Val Ala Pro Gly Lys1 5 10 15Thr Ala Thr Ile Thr Cys Gly Val Asn
Asn Leu Gly Arg Lys Ser Val 20 25 30His Trp Tyr Gln Gln Lys Pro Gly
Gln Ala Pro Val Leu Val Val Tyr 35 40 45Asp Ser Asn Asp Arg Pro Ser
Gly Ile Pro Glu Arg Phe Ser Gly Ser 50 55 60Asn Ser Gly Asn Thr Ala
Thr Leu Ile Ile Ser Arg Val Glu Ala Gly65 70 75 80Asp Glu Ala Asp
Tyr Ser Cys Gln Val Trp Asp Asn Asn Val Asp His 85 90 95Pro Val Phe
Gly Gly Gly Thr Lys Leu Thr Val Leu 100 105124108PRTArtificial
SequenceDescription of Artificial Sequence Synthetic polypeptide
124Ser Ser Glu Leu Thr Gln Asp Pro Ala Val Ser Val Ala Leu Gly Gln1
5 10 15Thr Val Arg Ile Thr Cys Gln Gly Asp Ser Leu Arg Ser Tyr Tyr
Ala 20 25 30Ser Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Val Leu Val
Ile Tyr 35 40 45Gly Lys Asn Asn Arg Pro Ser Gly Ile Pro Asp Arg Phe
Ser Gly Ser 50 55 60Ser Ser Gly Asn Thr Ala Ser Leu Thr Ile Thr Gly
Ala Gln Ala Glu65 70 75 80Asp Glu Ala Asp Tyr Tyr Cys Asn Ser Arg
Asp Ser Ser Gly Asn His 85 90 95Trp Val Phe Gly Gly Gly Thr Lys Val
Thr Val Leu 100 105125108PRTArtificial SequenceDescription of
Artificial Sequence Synthetic polypeptide 125Glu Ile Val Met Thr
Gln Ser Pro Gly Thr Leu Ser Leu Ser Pro Gly1 5 10 15Glu Arg Ala Thr
Leu Ser Cys Arg Ala Ser Gln Ser Val Ser Ser Ser 20 25 30Leu Ala Trp
Tyr Gln Gln Lys Pro Gly Gln Ala Pro Arg Leu Leu Ile 35 40 45Tyr Gly
Ala Ser Asn Arg Ala Thr Gly Ile Pro Asp Arg Phe Ser Gly 50 55 60Ser
Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Arg Leu Glu Pro65 70 75
80Glu Asp Phe Ala Val Tyr Tyr Cys Gln Gln Tyr Gly Gly Ser Pro Arg
85 90 95Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys Arg 100
105126108PRTArtificial SequenceDescription of Artificial Sequence
Synthetic polypeptide 126Glu Ile Val Leu Thr Gln Ser Pro Gly Thr
Leu Ser Leu Ser Pro Gly1 5 10 15Glu Arg Ala Thr Leu Ser Cys Arg Ala
Ser Gln Ser Val Ser Ser Ser 20 25 30Tyr Leu Ala Trp Tyr Gln Gln Lys
Pro Gly Gln Ala Pro Arg Leu Leu 35 40 45Ile Tyr Gly Ala Ser Ser Arg
Ala Thr Gly Ile Pro Asp Arg Phe Ser 50 55 60Gly Ser Gly Ser Gly Thr
Asp Phe Thr Leu Thr Ile Ser Arg Leu Glu65 70 75 80Pro Glu Asp Phe
Ala Val Tyr Tyr Cys Gln Gln Tyr Gly Ser Ser Pro 85 90 95Asn Phe Gly
Gly Gly Thr Lys Val Asp Ile Lys Arg 100 105127110PRTArtificial
SequenceDescription of Artificial Sequence Synthetic polypeptide
127Gln Ala Val Leu Thr Gln Pro Pro Ser Ala Ser Gly Thr Pro Gly Gln1
5 10 15Arg Val Thr Ile Ser Cys Ser Gly Ser Ser Ser Asn Ile Gly Ser
Asn 20 25 30Ser Val Tyr Trp Tyr Gln Gln Leu Pro Gly Thr Ala Pro Lys
Leu Leu 35 40 45Ile Tyr Arg Asn Asn Gln Arg Pro Ser Gly Val Pro Asp
Arg Phe Ser 50 55 60Gly Ser Lys Ser Gly Thr Ser Ala Ser Leu Ala Ile
Ser Gly Leu Gln65 70 75 80Ser Glu Asp Glu Ala Asp Tyr Tyr Cys Ala
Ala Trp Asp Asp Ser Leu 85 90 95Asn Gly Val Val Phe Gly Gly Gly Thr
Lys Leu Thr Val Leu 100 105 110128111PRTArtificial
SequenceDescription of Artificial Sequence Synthetic polypeptide
128Gln Ser Val Val Thr Gln Pro Pro Ser Val Ser Gly Ala Pro Gly Gln1
5 10 15Arg Val Thr Ile Ser Cys Thr Gly Ser Ser Ser Asn Ile Gly Ala
Gly 20 25 30Tyr Asp Val His Trp Tyr Gln Gln Leu Pro Gly Thr Ala Pro
Lys Leu 35 40 45Leu Ile Tyr Gly Asp Thr Asn Arg Pro Ser Gly Val Pro
Asp Arg Phe 50 55 60Ser Gly Ser Lys Ser Gly Thr Ser Ala Ser Leu Ala
Ile Thr Gly Leu65 70 75 80Gln Ala Glu Asp Glu Ala Asp Tyr Tyr Cys
Gln Ser Tyr Asp Gly Ser 85 90 95Arg Ser Gly Leu Val Phe Gly Gly Gly
Thr Lys Leu Thr Val Leu 100 105 110129111PRTArtificial
SequenceDescription of Artificial Sequence Synthetic polypeptide
129Glu Ile Val Leu Thr Gln Ser Pro Gly Thr Leu Ser Leu Ser Pro Gly1
5 10 15Glu Arg Ala Thr Leu Ser Cys Arg Ala Ser Gln Ser Leu Ser Asn
Ala 20 25 30Tyr Leu Ala Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Arg
Leu Leu 35 40 45Leu Tyr Gly Gly Ser Thr Arg Ala Thr Gly Ile Pro Asp
Arg Phe Ser 50 55 60Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile
Ser Ser Leu Glu65 70 75 80Ala Glu Asp Phe Ala Val Tyr Tyr Cys Gln
Gln Tyr Gly Ser Ser Pro 85 90 95Arg Thr Phe Gly Gln Gly Thr Lys Val
Glu Ile Lys Arg Thr Val 100 105 110130111PRTArtificial
SequenceDescription of Artificial Sequence Synthetic polypeptide
130Glu Ile Val Leu Thr Gln Ser Pro Gly Thr Leu Ser Leu Ser Pro Gly1
5 10 15Glu Arg Ala Thr Leu Ser Cys Arg Ala Ser Gln Ser Val Ser Ser
Ser 20 25 30Tyr Leu Ala Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Arg
Leu Leu 35 40 45Ile Tyr Gly Thr Ser Ser Arg Ala Thr Asp Ile Pro Asp
Arg Phe Ser 50 55 60Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile
Ser Arg Leu Glu65 70 75 80Pro Glu Asp Phe Ala Val Tyr Tyr Cys Gln
Gln Tyr Gly Arg Ser Pro 85 90 95Phe Thr Phe Gly Gly Gly Thr Lys Val
Glu Ile Lys Arg Thr Val 100 105 110131110PRTArtificial
SequenceDescription of Artificial Sequence Synthetic polypeptide
131Asp Ile Gln Leu Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly1
5 10 15Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Gly Ile Ser Asn
Tyr 20 25 30Leu Ala Trp Tyr Gln Gln Lys Pro Gly Lys Val Pro Lys Leu
Leu Ile 35 40 45Tyr Ala Ala Ser Thr Leu Gln Ser Gly Val Pro Ser Arg
Phe Ser Gly 50 55 60Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser
Ser Leu Gln Pro65 70 75 80Glu Asp Val Ala Thr Tyr Tyr Cys Gln Lys
Tyr Asn Ser Ala Pro Arg 85 90 95Thr Phe Gly Gln Gly Thr Lys Val Glu
Ile Lys Arg Thr Val 100 105 110132110PRTArtificial
SequenceDescription of Artificial Sequence Synthetic polypeptide
132Asp Ile Gln Leu Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly1
5 10 15Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Gly Ile Ser Asn
Tyr 20 25 30Leu Ala Trp Tyr Gln Gln Lys Pro Gly Lys Val Pro Lys Leu
Leu Ile 35 40 45Tyr Ala Ala Ser Thr Leu Gln Ser Gly Val Pro Ser Arg
Phe Ser Gly 50 55 60Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser
Ser Leu Gln Pro65 70 75 80Glu Asp Val Ala Thr Tyr Tyr Cys Gln Lys
Tyr Asn Ser Ala Pro Leu 85 90 95Thr Phe Gly Gly Gly Thr Lys Val Glu
Ile Lys Arg Thr Val 100 105 110133108PRTArtificial
SequenceDescription of Artificial Sequence Synthetic polypeptide
133Glu Ile Val Met Thr Gln Ser Pro Gly Thr Leu Ser Leu Ser Pro Gly1
5 10 15Glu Arg Ala Thr Leu Ser Cys Arg Ala Ser Gln Ser Val Ser Ser
Ser 20 25 30Tyr Leu Ala Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Arg
Leu Leu 35 40 45Ile Tyr Gly Ala Ser Ser Arg Ala Thr Gly Ile Pro Asp
Arg Phe Ser 50 55 60Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile
Ser Arg Leu Glu65 70 75 80Pro Glu Asp Phe Ala Val Tyr Tyr Cys Gln
Gln Tyr Gly Ser Ser Pro 85 90 95Tyr Thr Phe Gly Gln Gly Thr Lys Val
Glu Ile Lys 100 105134110PRTArtificial SequenceDescription of
Artificial Sequence Synthetic polypeptide 134Gln Ser Val Leu Thr
Gln Pro Pro Ser Ala Ser Gly Thr Pro Gly Gln1 5 10 15Arg Val Thr Ile
Ser Cys Ser Gly Ser Ser Ser Asn Ile Gly Ser Asn 20 25 30Phe Val Tyr
Trp Tyr Gln Gln Leu Pro Gly Thr Ala Pro Lys Leu Leu 35 40 45Ile Tyr
Lys Ser Asn Gln Arg Pro Ser Gly Val Pro Asp Arg Phe Ser 50 55 60Gly
Ser Lys Ser Gly Thr Ser Ala Ser Leu Ala Ile Ser Gly Leu Arg65 70 75
80Ser Glu Asp Glu Ala Asp Tyr Tyr Cys Ala Ala Trp Asp Asp Ser Leu
85 90 95Ser Gly Tyr Val Phe Gly Thr Gly Thr Gln Leu Thr Val Leu 100
105 110135112PRTArtificial SequenceDescription of Artificial
Sequence Synthetic polypeptide 135Glu Thr Thr Leu Thr Gln Ser Pro
Asp Thr Leu Ser Leu Ser Pro Gly1 5 10 15Glu Arg Ala Thr Leu Ser Cys
Arg Ala Ser Gln Ser Val Ser Ser Gly 20 25 30Ser Leu Ala Trp Tyr Gln
Gln Lys Pro Gly Gln Ala Pro Arg Leu Leu 35 40 45Ile Tyr Ala Ala Ser
Ser Arg Ala Ala Gly Ile Pro Asp Arg Phe Ser 50 55 60Gly Ser Gly Ser
Gly Thr Asp Phe Thr Leu Thr Ile Ser Arg Leu Glu65 70 75 80Pro Glu
Asp Phe Ala Val Tyr Tyr Cys Gln Gln Tyr Gly Ser Ser Pro 85 90 95Gly
Leu Thr Phe Gly Gly Gly Thr Gln Val Glu Ile Lys Arg Thr Val 100 105
110136111PRTArtificial SequenceDescription of Artificial Sequence
Synthetic polypeptide 136Glu Thr Thr Leu Thr Gln Ser Pro Gly Thr
Leu Ser Leu Ser Pro Gly1 5 10 15Glu Arg Ala Thr Leu Ser Cys Arg Ala
Ser Gln Ser Val Ser Ser Ser 20 25 30Tyr Leu Ala Trp Tyr Gln Gln Lys
Pro Gly Gln Ala Pro Arg Leu Leu 35 40 45Ile Tyr Gly Ala Ser Ser Arg
Ala Thr Gly Ile Pro Asp Arg Phe Ser 50 55 60Gly Ser Gly Ser Gly Thr
Asp Phe Thr Leu Thr Ile Ser Arg Leu Glu65 70 75 80Pro Glu Asp Phe
Ala Val Tyr Tyr Cys Gln Gln Tyr Gly Ser Ser Pro 85 90 95Tyr Thr Phe
Gly Gln Gly Thr Lys Leu Glu Ile Lys Arg Thr Val 100 105
110137110PRTArtificial SequenceDescription of Artificial Sequence
Synthetic polypeptide 137Glu Ile Val Leu Thr Gln Ser Pro Gly Thr
Leu Ser Leu Ser Pro Gly1 5 10 15Glu Arg Ala Thr Leu Ser Cys Arg Ala
Ser Gln Asn Ile Gly Trp Tyr 20 25 30Leu Ala Trp Tyr Gln His Lys Pro
Gly Gln Ala Pro Arg Leu Ile Met 35 40 45Tyr Asp Ala Ser Thr Arg Ala
Thr Gly Ile Pro Asp Arg Phe Ser Gly 50 55 60Ser Gly Ser Gly Thr Asp
Phe Thr Leu Thr Ile Ser Arg Leu Glu Pro65 70 75 80Glu Asp Phe Ala
Val Tyr Tyr Cys Gln Gln Tyr Asp Ser Val Pro Ser 85 90 95Thr Phe Gly
Gln Gly Thr Asn Leu Glu Ile Lys Arg Thr Val 100 105
110138111PRTArtificial SequenceDescription of Artificial Sequence
Synthetic polypeptide 138Gln Ser Val Leu Thr Gln Pro Pro Ser Val
Ser Gly Ala Pro Gly Gln1 5 10 15Arg Val Thr Ile Ser Cys Thr Gly Ser
Ser Ser Asn Ile Gly Ala Gly 20 25 30Tyr Asp Val His Trp Tyr Gln Gln
Leu Pro Gly Thr Thr Pro Lys Leu 35 40 45Leu Ile Tyr Asp Asn Thr Asn
Arg Pro Ser Gly Val Pro Asp Arg Phe 50
55 60Ser Ala Ser Lys Ser Gly Ala Ser Ala Ser Leu Ala Ile Thr Gly
Leu65 70 75 80Arg Asp Glu Asp Glu Ala Asp Tyr Tyr Cys Gln Ser Tyr
Asp Ser Ser 85 90 95Leu Ser Ala Ser Val Phe Gly Gly Gly Thr Lys Leu
Thr Val Leu 100 105 110139110PRTArtificial SequenceDescription of
Artificial Sequence Synthetic polypeptide 139Gln Ser Ala Leu Thr
Gln Pro Arg Ser Val Ser Gly Ser Pro Gly Gln1 5 10 15Ser Val Thr Ile
Ser Cys Thr Gly Thr Thr Ser Asp Val Gly Gly Tyr 20 25 30Asn Tyr Val
Ser Trp Tyr Gln Gln His Pro Gly Glu Ala Pro Lys Leu 35 40 45Ile Ile
Tyr Asp Val Ser Asn Arg Pro Ser Gly Val Ser Asn Arg Phe 50 55 60Ser
Gly Ser Lys Ser Gly Asn Thr Ala Ser Leu Thr Val Ser Gly Leu65 70 75
80Gln Ala Glu Asp Glu Ala Asp Tyr Tyr Cys Ser Ser Phe Ala Gly Ser
85 90 95Ser Asn Leu Ile Phe Gly Gly Gly Thr Lys Leu Thr Val Leu 100
105 110140110PRTArtificial SequenceDescription of Artificial
Sequence Synthetic polypeptide 140Glu Thr Thr Leu Thr Gln Ser Pro
Ala Thr Leu Ser Val Ser Pro Gly1 5 10 15Glu Arg Ala Thr Leu Ser Cys
Arg Ala Ser Gln Ser Val Ser Arg Asn 20 25 30Leu Ala Trp Tyr Gln Gln
Lys Pro Gly Gln Ala Pro Arg Leu Leu Ile 35 40 45Tyr Gly Ala Ser Ser
Arg Ala Thr Gly Ile Pro Asp Arg Phe Ser Gly 50 55 60Ser Gly Ser Gly
Thr Asp Phe Thr Leu Thr Ile Ser Arg Leu Glu Pro65 70 75 80Glu Asp
Phe Ala Val Tyr Tyr Cys Gln Gln Tyr Gly Ser Ser Ser Ile 85 90 95Thr
Phe Gly Gln Gly Thr Arg Leu Glu Ile Lys Arg Thr Val 100 105
110141110PRTArtificial SequenceDescription of Artificial Sequence
Synthetic polypeptide 141Ser Tyr Glu Leu Thr Gln Pro Pro Ser Ala
Ser Gly Thr Pro Gly Gln1 5 10 15Arg Val Thr Ile Ser Cys Ser Gly Ser
Ser Ser Asn Ile Gly Ser Asn 20 25 30Thr Val Asn Trp Tyr Gln Gln Leu
Pro Gly Thr Ala Pro Lys Leu Leu 35 40 45Ile Tyr Ser Asn Asn His Arg
Pro Ser Gly Val Pro Asp Arg Phe Ser 50 55 60Gly Ser Lys Ser Gly Thr
Ser Ala Ser Leu Ala Ile Ser Gly Leu Gln65 70 75 80Ser Glu Asp Glu
Ala Asp Tyr Tyr Cys Ala Thr Trp Asp Asp Ser Leu 85 90 95Asn Gly Trp
Val Phe Gly Gly Gly Thr Lys Val Thr Val Leu 100 105
110142110PRTArtificial SequenceDescription of Artificial Sequence
Synthetic polypeptide 142Gln Ser Val Leu Thr Gln Pro Pro Ser Ala
Ser Gly Thr Pro Gly Gln1 5 10 15Arg Val Thr Ile Ser Cys Ser Gly Ser
Ser Ser Asn Ile Gly Gly Asn 20 25 30Thr Val Asn Trp Tyr Gln Gln Val
Pro Gly Thr Ala Pro Lys Leu Leu 35 40 45Ile His Ser Asn Asn Gln Arg
Pro Ser Gly Val Pro Asp Arg Phe Ser 50 55 60Gly Ser Lys Ser Gly Thr
Ser Ala Ser Leu Ala Ile Ser Gly Leu Leu65 70 75 80Ser Glu Asp Glu
Ala Asp Tyr Tyr Cys Glu Val Trp Asp Asp Ser Leu 85 90 95Asn Gly Arg
Val Phe Gly Gly Gly Thr Lys Leu Thr Val Leu 100 105
110143108PRTArtificial SequenceDescription of Artificial Sequence
Synthetic polypeptide 143Asp Ile Gln Leu Thr Gln Ser Pro Ser Ser
Leu Ser Ala Ser Val Gly1 5 10 15Asp Arg Val Thr Ile Thr Cys Arg Ala
Ser Gln Gly Ile Ser Asn Tyr 20 25 30Leu Ala Trp Tyr Gln Gln Lys Pro
Gly Lys Val Pro Lys Leu Leu Ile 35 40 45Tyr Ala Ala Ser Thr Leu Gln
Ser Gly Val Pro Ser Arg Phe Ser Gly 50 55 60Ser Gly Ser Gly Thr Asp
Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro65 70 75 80Glu Asp Phe Ala
Thr Tyr Tyr Cys Gln Gln Ser Tyr Ser Thr Pro Arg 85 90 95Thr Phe Gly
Gln Gly Thr Lys Leu Glu Ile Lys Arg 100 105144110PRTArtificial
SequenceDescription of Artificial Sequence Synthetic polypeptide
144His Pro Glu Leu Thr Gln Pro Pro Ser Ala Ser Gly Thr Pro Gly Gln1
5 10 15Arg Val Thr Ile Ser Cys Ser Gly Ser Ser Ser Asn Ile Gly Ser
Asn 20 25 30Thr Val Asn Trp Tyr Gln Gln Leu Pro Gly Thr Gly Pro Lys
Leu Leu 35 40 45Ile Tyr Ser Asn Asn Gln Arg Pro Ser Gly Val Pro Asp
Arg Phe Ser 50 55 60Gly Ser Lys Ser Gly Thr Ser Ala Ser Leu Ala Ile
Ser Gly Leu Gln65 70 75 80Ser Glu Asp Glu Ala Asp Tyr Tyr Cys Ala
Ala Trp Asp Asp Ser Leu 85 90 95Asn Gly Trp Val Phe Gly Gly Gly Thr
Lys Val Thr Val Leu 100 105 110145109PRTArtificial
SequenceDescription of Artificial Sequence Synthetic polypeptide
145Glu Ile Val Leu Thr Gln Ser Pro Gly Thr Leu Ser Leu Ser Pro Gly1
5 10 15Glu Arg Ala Thr Leu Ser Cys Arg Ala Ser Gln Ser Val Ser Ser
Ser 20 25 30Tyr Leu Ala Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Arg
Leu Leu 35 40 45Ile Tyr Gly Ala Ser Ser Arg Ala Thr Gly Ile Pro Asp
Arg Phe Ser 50 55 60Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile
Ser Arg Leu Glu65 70 75 80Pro Glu Asp Phe Ala Val Tyr Tyr Cys Gln
Gln Phe Gly Ser Ser Gln 85 90 95Val Thr Phe Gly Gly Gly Thr Lys Val
Glu Ile Lys Arg 100 105146107PRTArtificial SequenceDescription of
Artificial Sequence Synthetic polypeptide 146His Val Ile Leu Thr
Gln Pro Pro Ser Val Ser Val Ala Pro Gly Met1 5 10 15Thr Ala Arg Met
Thr Cys Gly Gly Asp Asn Val Gly Arg Arg Asn His 20 25 30Trp Tyr Gln
Gln Lys Pro Gly Gln Ala Pro Val Leu Val Val Tyr Asp 35 40 45Asp Gly
Gly Arg Pro Ser Ala Ile Pro Ala Arg Phe Ser Gly Ser Lys 50 55 60Ser
Gly Asn Thr Ala Thr Leu Ile Ile Ser Arg Val Glu Ala Gly Asp65 70 75
80Glu Ala Asp Tyr Tyr Cys Gln Met Trp His Ser Ser Gly Asp Gln Trp
85 90 95Val Phe Gly Gly Gly Thr Lys Leu Thr Val Leu 100
105147110PRTArtificial SequenceDescription of Artificial Sequence
Synthetic polypeptide 147Gln Ala Val Leu Thr Gln Pro Pro Ser Ala
Ser Gly Thr Pro Gly Gln1 5 10 15Arg Val Thr Ile Ser Cys Ser Gly Ser
Ser Ser Asn Ile Gly Ser Asn 20 25 30Tyr Val Tyr Trp Tyr Gln His Leu
Pro Gly Thr Ala Pro Lys Leu Leu 35 40 45Ile Asp Arg Asn Asp Gln Arg
Pro Ser Gly Val Pro Asp Arg Phe Ser 50 55 60Gly Ser Lys Ser Gly Thr
Ser Ala Ser Leu Ala Ile Ser Gly Leu Arg65 70 75 80Ser Glu Asp Glu
Ala Asp Tyr Tyr Cys Ala Ala Trp Asp Asp Asn Leu 85 90 95Ser Gly Val
Val Phe Gly Gly Gly Thr Lys Val Thr Val Leu 100 105
110148109PRTArtificial SequenceDescription of Artificial Sequence
Synthetic polypeptide 148Gln Ser Val Val Thr Gln Pro Pro Ser Val
Ser Gly Ala Pro Gly Gln1 5 10 15Arg Val Thr Ile Ser Cys Thr Gly Ser
Ser Ile Gly Ala Gly Tyr Asp 20 25 30Val His Trp Tyr Gln Gln Leu Pro
Arg Thr Ala Pro Lys Leu Leu Ile 35 40 45Phe Gly Asn Thr Asn Arg Pro
Ser Gly Val Pro Asp Arg Phe Ser Gly 50 55 60Ser Lys Ser Gly Thr Ser
Ala Ser Leu Thr Ile Thr Gly Leu Gln Ala65 70 75 80Glu Asp Glu Ala
Asn Tyr Tyr Cys Gln Thr Tyr Asp Ser Ser Leu Ser 85 90 95Gly Trp Val
Phe Gly Gly Gly Thr Lys Leu Thr Val Leu 100 105149110PRTArtificial
SequenceDescription of Artificial Sequence Synthetic polypeptide
149Pro Glu Leu Thr Gln Pro Pro Ser Ala Ser Gly Thr Pro Gly Gln Arg1
5 10 15Val Thr Ile Ser Cys Ser Gly Ser Ser Ser Asn Ile Gly Ser Asn
His 20 25 30Val Tyr Trp Tyr Gln Gln Leu Pro Gly Thr Ala Pro Lys Leu
Leu Ile 35 40 45Tyr Arg Asn Asn Gln Arg Pro Ser Gly Val Pro Asp Arg
Phe Ser Gly 50 55 60Ser Lys Ser Gly Thr Ser Ala Ser Leu Ala Ile Ser
Gly Leu Arg Ser65 70 75 80Glu Asp Glu Ala Asp Tyr Tyr Cys Ala Thr
Trp Asp Asp Asn Leu Ser 85 90 95Gly Arg Leu Val Phe Gly Gly Gly Thr
Lys Leu Thr Val Leu 100 105 110150111PRTArtificial
SequenceDescription of Artificial Sequence Synthetic polypeptide
150Gln Ser Val Leu Thr Gln Pro Pro Ser Val Ser Gly Ala Pro Gly Gln1
5 10 15Arg Val Thr Ile Ser Cys Ile Gly Ser Asn Ser Asn Ile Gly Ala
Asn 20 25 30Phe Ala Val His Trp Tyr Gln Gln Leu Pro Gly Ala Ala Pro
Lys Leu 35 40 45Leu Ile Tyr Asp Asn Thr Asn Arg Pro Ser Gly Val Pro
Asp Arg Phe 50 55 60Ser Gly Ser Lys Ser Gly Thr Ser Ala Ser Leu Asp
Ile Thr Gly Leu65 70 75 80Gln Ala Asp Asp Glu Ala Asp Tyr Tyr Cys
Gln Ser Tyr Asp Ala Arg 85 90 95Leu Asn Gly Trp Val Phe Gly Gly Gly
Thr Lys Leu Thr Val Leu 100 105 110151110PRTArtificial
SequenceDescription of Artificial Sequence Synthetic polypeptide
151Gln Ser Ala Leu Thr Gln Pro Pro Ser Ala Ser Gly Ser Pro Gly Gln1
5 10 15Ser Val Thr Ile Ser Cys Ala Gly Ala Ser Ser Asp Ile Gly Thr
Tyr 20 25 30Asn Ser Val Ser Trp Tyr Gln Gln His Pro Gly Lys Ala Pro
Lys Leu 35 40 45Ile Ile Tyr Glu Val Thr Lys Arg Pro Ser Gly Val Pro
Asp Arg Phe 50 55 60Ser Gly Ser Lys Ser Gly Asn Thr Ala Ser Leu Thr
Val Ser Gly Leu65 70 75 80Gln Ala Glu Asp Glu Ala Asp Tyr Tyr Cys
Asn Ser Tyr Ala Gly Thr 85 90 95Lys Gly Tyr Val Phe Gly Ser Gly Thr
Lys Val Thr Val Leu 100 105 110152107PRTArtificial
SequenceDescription of Artificial Sequence Synthetic polypeptide
152Ser Ser Glu Leu Thr Gln Asp Pro Ala Val Ser Val Ala Leu Gly Gln1
5 10 15Thr Val Arg Ile Thr Cys Gln Gly Asp Ser Leu Arg Asn Glu Tyr
Ala 20 25 30Ser Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Val Leu Val
Met Lys 35 40 45Gly Asn Asn Asn Arg Pro Ser Val Ile Pro Asp Arg Phe
Ser Gly Ser 50 55 60Arg Ser Gly Asn Thr Ala Ser Leu Thr Ile Thr Gly
Ala Gln Ala Glu65 70 75 80Asp Glu Ala Asp Tyr Tyr Cys Ser Ser Arg
Asp Ser Ser Gly Asn Arg 85 90 95Phe Phe Gly Ser Gly Thr Lys Val Thr
Val Leu 100 105153107PRTArtificial SequenceDescription of
Artificial Sequence Synthetic polypeptide 153His Pro Glu Leu Thr
Gln Pro Pro Ser Leu Ser Val Ser Pro Gly Gln1 5 10 15Thr Ala Thr Ile
Ser Cys Ser Gly Glu Arg Leu Thr Asn Lys Tyr Thr 20 25 30Ser Trp Tyr
Gln Gln Arg Pro Gly Gln Ser Pro Ala Leu Val Ile Tyr 35 40 45Gln Asp
Asp Lys Arg Pro Ser Gly Ile Pro Glu Arg Phe Ser Gly Ser 50 55 60Asn
Ser Gly Asn Thr Ala Thr Leu Thr Ile Ser Gly Thr Gln Pro Met65 70 75
80Asp Glu Ala Val Tyr Tyr Cys Gln Ala Trp Asp Thr Asn Thr Gln Met
85 90 95Thr Phe Gly Gly Gly Thr Lys Leu Thr Val Leu 100
105154110PRTArtificial SequenceDescription of Artificial Sequence
Synthetic polypeptide 154Gln Ala Val Leu Thr Gln Pro Pro Ser Ala
Ser Gly Thr Pro Gly Gln1 5 10 15Arg Val Thr Ile Ser Cys Ser Gly Ser
Ser Ser Asn Ile Gly Thr Asn 20 25 30Thr Val Asn Trp Tyr Gln Gln Leu
Pro Gly Thr Ala Pro Lys Leu Leu 35 40 45Ile Tyr Arg Asn Ile Gln Arg
Pro Ser Gly Val Pro Asp Arg Phe Ser 50 55 60Gly Ser Lys Ser Gly Thr
Ser Ala Ser Leu Ala Ile Ser Gly Leu Gln65 70 75 80Ser Glu Asp Glu
Ala Asp Tyr Tyr Cys Ala Ala Trp Asp Asp Ser Leu 85 90 95Asn Gly Tyr
Val Phe Gly Thr Gly Thr Lys Leu Thr Val Leu 100 105
110155111PRTArtificial SequenceDescription of Artificial Sequence
Synthetic polypeptide 155Glu Thr Thr Leu Thr Gln Ser Pro Gly Thr
Val Ser Leu Ser Pro Gly1 5 10 15Glu Arg Ala Thr Leu Ser Cys Arg Ala
Ser Gln Ser Val Gly Gly Ser 20 25 30Asn Leu Ala Trp Tyr Gln Gln Lys
Pro Gly Gln Ala Pro Arg Leu Val 35 40 45Ile Tyr Ala Thr Ser Arg Lys
Ala Asn Gly Ile Pro Asp Arg Phe Ser 50 55 60Gly Ser Gly Ser Gly Thr
Asp Phe Thr Leu Thr Ile Ser Arg Leu Glu65 70 75 80Pro Glu Asp Phe
Ala Val Tyr Phe Cys Gln Gln Tyr Gly Thr Ser Pro 85 90 95Pro Ser Val
Thr Phe Gly Gly Gly Thr Lys Val Glu Ile Arg Arg 100 105
110156110PRTArtificial SequenceDescription of Artificial Sequence
Synthetic polypeptide 156Gln Ala Val Leu Thr Gln Pro Pro Ser Ala
Ser Gly Thr Pro Gly Gln1 5 10 15Arg Val Thr Ile Ser Cys Ser Gly Ser
Ser Ser Asn Ile Gly Ser Asn 20 25 30Pro Val Asn Trp Tyr Gln Gln Leu
Pro Gly Thr Ala Pro Lys Leu Leu 35 40 45Ile Tyr Ser Asn Asn Gln Arg
Pro Ser Gly Val Pro Asp Arg Phe Ser 50 55 60Gly Ser Lys Ser Gly Thr
Ser Ala Ser Leu Ala Ile Ser Gly Leu Gln65 70 75 80Ser Glu Asp Glu
Ala Asp Tyr Tyr Cys Ala Ala Trp Asp Asp Ser Leu 85 90 95Thr Gly Tyr
Val Phe Gly Thr Gly Thr Gln Leu Thr Val Leu 100 105
110157110PRTArtificial SequenceDescription of Artificial Sequence
Synthetic polypeptide 157Ser Tyr Val Leu Thr Gln Pro Pro Ser Ala
Ser Gly Thr Pro Asp Gln1 5 10 15Arg Val Thr Ile Ser Cys Ser Gly Ser
Ser Ser Asn Ile Gly Ser Asn 20 25 30Tyr Val Tyr Trp Tyr Gln Gln Phe
Pro Gly Ala Ala Pro Lys Leu Leu 35 40 45Met Ser Arg Asn Asn Gln Arg
Pro Ser Gly Val Pro Asp Arg Phe Ser 50 55 60Gly Ser Lys Ser Gly Thr
Ser Ala Ser Leu Ala Ile Ser Gly Leu Arg65 70 75 80Ser Glu Asp Glu
Ala Tyr Tyr Ser Cys Ala Ala Trp Asp Asp Ser Leu 85 90 95Asn Gly Leu
Val Phe Gly Gly Gly Thr Lys Val Thr Val Leu 100 105
110158109PRTArtificial SequenceDescription of Artificial Sequence
Synthetic polypeptide 158Asn Phe Met Leu Thr Gln Pro His Ser Val
Ser Glu Ser Pro Gly Lys1 5 10 15Thr Val Thr Ile Ser Cys Thr Gly Ser
Gly Gly Asn Ile Ala Arg Asn 20 25 30Tyr Val Gln Trp Tyr Gln Gln Arg
Pro Gly Ser Ala Pro Val Thr Val 35 40 45Ile Leu Glu Asp Asp Lys Arg
Pro Ser Gly Ile Pro Asp Arg Phe Ser 50 55 60Gly Ser Ile Asp Arg Ser
Ser Asn Ser Ala Ser Leu
Thr Ile Ser Gly65 70 75 80Leu Arg Thr Glu Asp Glu Ala Leu Tyr Tyr
Cys Gln Ser Tyr Asp Asp 85 90 95Ser Asp Leu Val Val Phe Gly Gly Gly
Thr Lys Leu Thr 100 105159111PRTArtificial SequenceDescription of
Artificial Sequence Synthetic polypeptide 159Asn Phe Met Leu Thr
Gln Pro His Ser Val Ser Glu Ser Pro Gly Lys1 5 10 15Thr Val Thr Ile
Ser Cys Thr Gly Ser Ser Gly Ser Ile Ala Ser Asn 20 25 30Tyr Val Gln
Trp Tyr Gln Gln Arg Pro Gly Ser Ala Pro Thr Thr Val 35 40 45Ile Tyr
Glu Asp Tyr Gln Arg Pro Ser Gly Val Pro Asp Arg Phe Ser 50 55 60Gly
Ser Ile Asp Ser Ser Ser Asn Ser Ala Ser Leu Thr Ile Ser Gly65 70 75
80Leu Lys Thr Glu Asp Glu Ala Asp Tyr Tyr Cys Gln Ser Tyr Asp Asp
85 90 95Ser Asp His Leu Ile Phe Gly Gly Gly Thr Lys Leu Thr Val Leu
100 105 110160110PRTArtificial SequenceDescription of Artificial
Sequence Synthetic polypeptide 160Gln Ser Val Leu Thr Gln Pro Pro
Ser Ala Ser Gly Thr Pro Gly Gln1 5 10 15Arg Val Thr Ile Ser Cys Ser
Gly Ser Ser Ser Asn Ile Gly Ser Asn 20 25 30Thr Val Asn Trp Tyr Lys
Gln Leu Pro Gly Thr Ala Pro Arg Leu Leu 35 40 45Ile Tyr Ser Asn Asp
Gln Arg Pro Ser Gly Val Pro Asp Arg Phe Ser 50 55 60Gly Ser Lys Ser
Gly Thr Ser Ala Ser Leu Ala Ile Ser Gly Leu Gln65 70 75 80Ser Glu
Asp Glu Ala Asn Tyr Tyr Cys Ala Ala Trp Asp Asp Ser Leu 85 90 95Ser
Gly Trp Val Phe Gly Gly Gly Thr Lys Leu Thr Val Leu 100 105
110161117PRTArtificial SequenceDescription of Artificial Sequence
Synthetic polypeptide 161Xaa Xaa Gln Leu Val Gln Ser Gly Xaa Glu
Val Xaa Lys Pro Gly Xaa1 5 10 15Ser Val Xaa Xaa Ser Cys Lys Xaa Ser
Gly Gly Xaa Phe Ser Ser Tyr 20 25 30Ala Xaa Xaa Trp Val Arg Gln Ala
Pro Gly Gln Gly Leu Glu Trp Met 35 40 45Gly Xaa Gly Ile Ile Xaa Xaa
Phe Gly Thr Thr Xaa Asn Tyr Ala Gln 50 55 60Lys Phe Gln Gly Arg Xaa
Thr Xaa Thr Ala Asp Xaa Xaa Thr Ser Thr65 70 75 80Ala Tyr Met Glu
Leu Ser Ser Leu Arg Ser Xaa Asp Thr Ala Val Tyr 85 90 95Tyr Cys Ala
Arg Gly Ser Tyr Tyr Tyr Glu Xaa Xaa Leu Asp Tyr Trp 100 105 110Gly
Xaa Gly Thr Xaa 115162109PRTArtificial SequenceDescription of
Artificial Sequence Synthetic polypeptide 162Pro Glu Leu Thr Gln
Pro Pro Ser Ala Ser Gly Thr Pro Gly Gln Arg1 5 10 15Val Thr Ile Ser
Cys Ser Gly Ser Ser Ser Asn Ile Gly Ser Asn Thr 20 25 30Val Asn Trp
Tyr Gln Gln Leu Pro Gly Thr Ala Pro Lys Leu Leu Ile 35 40 45Tyr Ser
Asn Asn Gln Arg Pro Ser Gly Val Pro Asp Arg Phe Ser Gly 50 55 60Ser
Lys Ser Gly Thr Ser Ala Ser Leu Ala Ile Ser Gly Leu Gln Ser65 70 75
80Glu Asp Glu Ala Asp Tyr Tyr Cys Ala Ala Trp Asp Asp Ser Leu Asn
85 90 95Gly Trp Val Phe Gly Gly Gly Thr Lys Leu Thr Val Leu 100
105163108PRTArtificial SequenceDescription of Artificial Sequence
Synthetic polypeptide 163Asp Ile Gln Met Thr Gln Ser Pro Ser Ser
Leu Ser Ala Phe Val Gly1 5 10 15Asp Arg Val Thr Ile Thr Cys Gln Ala
Ser Gln Asp Ile Ser Asn Tyr 20 25 30Leu Asn Trp Tyr Gln Gln Lys Pro
Gly Lys Ala Pro Lys Leu Leu Ile 35 40 45Tyr Asp Ala Thr Asn Leu Glu
Thr Gly Val Pro Ser Arg Phe Ser Gly 50 55 60Ser Gly Ser Gly Thr Asp
Phe Thr Phe Thr Ile Ser Ser Leu Gln Pro65 70 75 80Glu Asp Ile Ala
Thr Tyr Tyr Cys Gln Gln Tyr Asp Asn Leu Pro Leu 85 90 95Thr Phe Gly
Gly Gly Thr Lys Val Asp Ile Lys Arg 100 105164110PRTArtificial
SequenceDescription of Artificial Sequence Synthetic polypeptide
164Gln Ser Val Leu Thr Gln Pro Pro Ser Ala Ser Gly Thr Pro Gly Gln1
5 10 15Arg Val Thr Leu Ser Cys Ser Gly Ser Ser Ser Asn Ile Gly Gly
Asn 20 25 30Ser Val Asn Trp Tyr Gln His Val Pro Gly Thr Ala Pro Lys
Leu Leu 35 40 45Met His Ser Asp Asp Gln Arg Pro Ser Gly Val Pro Asp
Arg Phe Ser 50 55 60Gly Ser Lys Ser Gly Thr Ser Ala Ser Leu Ala Ile
Ser Gly Leu Gln65 70 75 80Ser Glu Asp Glu Ala Asp Tyr Tyr Cys Ala
Ala Trp Asp Asp Ser Leu 85 90 95Asn Ala Trp Val Phe Gly Gly Gly Thr
Lys Val Thr Val Leu 100 105 110165109PRTArtificial
SequenceDescription of Artificial Sequence Synthetic polypeptide
165Glu Thr Thr Leu Thr Gln Ser Pro Gly Thr Leu Ser Leu Ser Pro Gly1
5 10 15Glu Arg Ala Thr Leu Ser Cys Arg Ala Ser Gln Ser Val Ser Ser
Arg 20 25 30Tyr Leu Ala Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Arg
Leu Leu 35 40 45Ile Tyr Gly Ala Ser Asn Arg Ala Thr Gly Val Pro Asp
Arg Phe Ser 50 55 60Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile
Asn Arg Leu Glu65 70 75 80Pro Glu Asp Phe Ala Val Tyr Tyr Cys Gln
His Tyr Ser Arg Ser Leu 85 90 95Thr Phe Gly Gly Gly Thr Lys Val Glu
Ile Lys Arg Thr 100 105166110PRTArtificial SequenceDescription of
Artificial Sequence Synthetic polypeptide 166Gln Ser Val Leu Thr
Gln Pro Pro Ser Val Ser Ala Ala Pro Gly Gln1 5 10 15Met Val Thr Ile
Ser Cys Ser Gly Ser Asn Ser Asn Ile Gly Asn Asn 20 25 30Tyr Ile Ser
Trp Tyr Gln Gln Leu Pro Gly Ser Ala Pro Arg Leu Leu 35 40 45Ile Tyr
Asn Asn Tyr Lys Arg Pro Ser Gly Ile Pro Asp Arg Phe Ser 50 55 60Ala
Ser Lys Ser Gly Thr Ser Ala Thr Leu Gly Ile Thr Gly Leu Gln65 70 75
80Thr Gly Asp Glu Ala Asp Tyr Tyr Cys Gly Thr Trp Asp Ser Ser Leu
85 90 95Ser Ser Val Val Phe Gly Gly Gly Thr Lys Val Thr Val Leu 100
105 110167111PRTArtificial SequenceDescription of Artificial
Sequence Synthetic polypeptide 167Gln Ser Val Leu Thr Gln Pro Pro
Ser Val Ser Gly Ala Pro Gly Gln1 5 10 15Arg Val Thr Ile Ser Cys Thr
Gly Ser Ser Ser Asn Thr Gly Ala Gly 20 25 30Asn His Val His Trp Tyr
Gln Gln Val Ala Gly Ala Ala Pro Lys Leu 35 40 45Leu Ile Ser Asn Asn
Asn Asn Arg Pro Ser Gly Val Pro Asp Arg Phe 50 55 60Ser Ala Ser Lys
Ser Gly Thr Ser Ala Ser Leu Asp Ile Thr Gly Leu65 70 75 80Gln Ala
Glu Asp Glu Ala Asp Tyr Tyr Cys Gln Ser Tyr Asp Asn Ser 85 90 95Leu
Asn Asp Trp Val Phe Gly Gly Gly Thr Gln Leu Thr Val Leu 100 105
110168110PRTArtificial SequenceDescription of Artificial Sequence
Synthetic polypeptide 168Gln Ser Val Val Thr Gln Pro Pro Ser Glu
Ser Ala Ala Pro Gly Gln1 5 10 15Lys Val Thr Ile Ser Cys Ser Gly Ser
Ser Ser Asn Ile Gly Asn Asn 20 25 30Tyr Val Ser Trp Tyr Gln Gln Phe
Pro Gly Ala Ala Pro Lys Leu Leu 35 40 45Ile Phe Glu Asn Asn Lys Arg
His Ser Gly Ile Pro Asp Arg Phe Ser 50 55 60Gly Ser Lys Ser Gly Thr
Ser Ala Thr Leu Gly Ile Ala Glu Leu Gln65 70 75 80Thr Gly Asp Glu
Ala Asp Tyr Tyr Cys Gly Val Trp Asp Ser Ser Leu 85 90 95Ser Ala Trp
Val Phe Gly Gly Gly Thr Gln Leu Thr Val Leu 100 105
110169112PRTArtificial SequenceDescription of Artificial Sequence
Synthetic polypeptide 169Asn Phe Met Leu Thr Gln Pro His Ser Val
Ser Glu Ser Pro Gly Lys1 5 10 15Thr Val Thr Phe Ser Cys Thr Arg Ser
Ser Gly Ser Ile Ala Ser Lys 20 25 30Tyr Val Gln Trp Tyr Gln Gln Arg
Pro Gly Ser Ala Pro Thr Ile Val 35 40 45Ile Phe Glu Asn Thr Lys Arg
Pro Tyr Gly Val Pro Asp Arg Phe Ser 50 55 60Gly Ser Ile Asp Ser Ser
Ser Asn Ser Ala Ser Leu Thr Ile Ser Gly65 70 75 80Leu Lys Thr Glu
Asp Glu Ala Asp Tyr Tyr Cys Gln Ser Tyr Asp Ser 85 90 95Ser Asn His
Trp Val Phe Gly Gly Gly Thr Gln Leu Thr Val Leu Ser 100 105
1101705PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 170Val Thr Val Ser Ser1 51715PRTArtificial
SequenceDescription of Artificial Sequence Synthetic peptide 171Val
Arg Val Ser Ser1 517218DNAArtificial SequenceDescription of
Artificial Sequence Synthetic primer 172tttttttttt tttttttt
181736PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 173His His His His His His1 517410PRTArtificial
SequenceDescription of Artificial Sequence Synthetic peptide 174His
His His His His His His His His His1 5 1017513DNAArtificial
SequenceDescription of Artificial Sequence Synthetic
oligonucleotide 175ggccnnnnng gcc 13176109PRTArtificial
SequenceDescription of Artificial Sequence Synthetic polypeptide
176Pro Glu Leu Thr Gln Pro His Ser Val Ser Glu Ser Pro Gly Lys Thr1
5 10 15Val Thr Ile Ser Cys Thr Gly Ser Gly Gly Arg Ile Ala Thr Asn
His 20 25 30Val Gln Trp Tyr Gln Gln Arg Pro Gly Ser Ala Pro Thr Ile
Val Ile 35 40 45Tyr Glu Asn Asn Gln Arg Pro Ser Gly Val Pro Asn Arg
Phe Ser Gly 50 55 60Ser Ile Asp Asp Ser Ser Asn Ser Ala Ser Leu Thr
Ile Ser Ala Leu65 70 75 80Arg Thr Glu Asp Glu Ala Asp Tyr Tyr Cys
Gln Ser Ala Asp Ala Thr 85 90 95Asn Val Phe Phe Gly Gly Gly Thr Lys
Val Thr Val Leu 100 105177120PRTArtificial SequenceDescription of
Artificial Sequence Synthetic polypeptide 177Gln Val Gln Leu Val
Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ser1 5 10 15Ser Val Lys Val
Ser Cys Lys Ala Ser Gly Gly Thr Phe Ser Ser Tyr 20 25 30Ala Ile Ser
Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met 35 40 45Gly Gly
Ile Ile Pro Ile Phe Gly Thr Ala Asn Tyr Ala Gln Lys Phe 50 55 60Gln
Gly Arg Val Thr Ile Thr Ala Asp Lys Ser Thr Ser Thr Ala Tyr65 70 75
80Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys
85 90 95Ala Arg Gly Ser Tyr Tyr Tyr Glu Ser Ser Leu Asp Tyr Trp Gly
Gln 100 105 110Gly Thr Leu Val Thr Val Ser Ser 115
120178120PRTArtificial SequenceDescription of Artificial Sequence
Synthetic polypeptide 178Gln Val Gln Leu Val Gln Ser Gly Ala Glu
Val Lys Lys Pro Gly Ser1 5 10 15Ser Val Lys Val Ser Cys Lys Ala Ser
Gly Gly Thr Phe Ser Ser Tyr 20 25 30Ala Ile Ser Trp Val Arg Gln Ala
Pro Gly Gln Gly Leu Glu Trp Met 35 40 45Gly Gly Ile Ile Gly Met Phe
Gly Thr Thr Asn Tyr Ala Gln Lys Phe 50 55 60Gln Gly Arg Val Thr Ile
Thr Ala Asp Glu Leu Thr Ser Thr Ala Tyr65 70 75 80Met Glu Leu Ser
Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95Ala Arg Gly
Ser Tyr Tyr Tyr Glu Ser Ser Leu Asp Tyr Trp Gly Lys 100 105 110Gly
Thr Thr Val Thr Val Ser Ser 115 120179120PRTArtificial
SequenceDescription of Artificial Sequence Synthetic polypeptide
179Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ser1
5 10 15Ser Val Lys Val Ser Cys Lys Ala Ser Gly Gly Thr Phe Ser Ser
Tyr 20 25 30Ala Ile Ser Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu
Trp Met 35 40 45Gly Ala Ile Ile Gly Met Phe Gly Thr Thr Asn Tyr Ala
Gln Lys Phe 50 55 60Gln Gly Arg Val Thr Leu Thr Ala Asp Glu Leu Thr
Ser Thr Ala Tyr65 70 75 80Met Glu Leu Ser Ser Leu Arg Ser Glu Asp
Thr Ala Val Tyr Tyr Cys 85 90 95Ala Arg Gly Ser Tyr Tyr Tyr Glu Ser
Ser Leu Asp Tyr Trp Gly Lys 100 105 110Gly Thr Met Val Thr Val Ser
Ser 115 120180120PRTArtificial SequenceDescription of Artificial
Sequence Synthetic polypeptide 180Gln Val Gln Leu Val Gln Ser Gly
Ala Glu Val Lys Lys Pro Gly Ser1 5 10 15Ser Val Lys Val Ser Cys Lys
Ala Ser Gly Gly Thr Phe Ser Ser Tyr 20 25 30Ala Ile Ser Trp Val Arg
Gln Ala Pro Gly Gln Gly Leu Glu Trp Met 35 40 45Gly Ala Ile Ile Gly
Met Phe Gly Thr Thr Asn Tyr Ala Gln Lys Phe 50 55 60Gln Gly Arg Val
Thr Ile Thr Ala Asp Glu Leu Thr Ser Thr Ala Tyr65 70 75 80Met Glu
Leu Ser Ser Leu Arg Ser Asp Asp Thr Ala Val Tyr Tyr Cys 85 90 95Ala
Arg Gly Ser Tyr Tyr Tyr Glu Ser Ser Leu Asp Tyr Trp Gly Lys 100 105
110Gly Thr Met Val Thr Val Ser Ser 115 120181120PRTArtificial
SequenceDescription of Artificial Sequence Synthetic polypeptide
181Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ser1
5 10 15Ser Val Lys Val Ser Cys Lys Ala Ser Gly Gly Thr Phe Ser Ser
Tyr 20 25 30Ala Val Thr Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu
Trp Met 35 40 45Gly Ala Ile Ile Gly Met Phe Gly Thr Thr Asn Tyr Ala
Gln Lys Phe 50 55 60Gln Gly Arg Val Thr Leu Thr Ala Asp Glu Leu Thr
Ser Thr Ala Tyr65 70 75 80Met Glu Leu Ser Ser Leu Arg Ser Glu Asp
Thr Ala Val Tyr Tyr Cys 85 90 95Ala Arg Gly Ser Tyr Tyr Tyr Glu Ser
Ser Leu Asp Tyr Trp Gly Gln 100 105 110Gly Thr Leu Val Thr Val Ser
Ser 115 120182120PRTArtificial SequenceDescription of Artificial
Sequence Synthetic polypeptide 182Gln Val Gln Leu Val Gln Ser Gly
Ala Glu Val Lys Lys Pro Gly Ser1 5 10 15Ser Val Lys Val Ser Cys Lys
Ala Ser Gly Gly Thr Phe Ser Ser Tyr 20 25 30Ala Val Thr Trp Val Arg
Gln Ala Pro Gly Gln Gly Leu Glu Trp Met 35 40 45Gly Ala Ile Ile Gly
Met Phe Gly Thr Thr Asn Tyr Ala Gln Lys Phe 50 55 60Gln Gly Arg Val
Thr Ile Thr Ala Asp Glu Leu Thr Ser Thr Ala Tyr65 70 75 80Met Glu
Leu Ser Ser Leu Arg Ser Asp Asp Thr Ala Val Tyr Tyr Cys 85 90 95Ala
Arg Gly Ser Tyr Tyr Tyr Glu Ser Ser Leu Asp Tyr Trp Gly Gln 100 105
110Gly Thr Leu Val Thr Val Ser Ser 115 120183120PRTArtificial
SequenceDescription of Artificial Sequence Synthetic polypeptide
183Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ser1
5 10
15Ser Val Lys Val Ser Cys Lys Ala Ser Gly Gly Thr Phe Ser Ser Tyr
20 25 30Ala Val Thr Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp
Met 35 40 45Gly Ala Ile Ile Gly Met Phe Gly Thr Thr Asn Tyr Ala Gln
Lys Phe 50 55 60Gln Gly Arg Val Thr Leu Thr Ala Asp Glu Leu Thr Ser
Thr Ala Tyr65 70 75 80Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr
Ala Val Tyr Tyr Cys 85 90 95Ala Arg Gly Ser Tyr Tyr Tyr Glu Ser Ser
Leu Asp Tyr Trp Gly Lys 100 105 110Gly Thr Met Val Thr Val Ser Ser
115 120184120PRTArtificial SequenceDescription of Artificial
Sequence Synthetic polypeptide 184Gln Val Gln Leu Val Gln Ser Gly
Ala Glu Val Lys Lys Pro Gly Ser1 5 10 15Ser Val Lys Val Ser Cys Lys
Thr Ser Gly Gly Thr Phe Ser Ser Tyr 20 25 30Ala Val Thr Trp Val Arg
Gln Ala Pro Gly Gln Gly Leu Glu Trp Met 35 40 45Gly Gly Ile Ile Gly
Met Phe Gly Thr Thr Asn Tyr Ala Gln Lys Phe 50 55 60Gln Gly Arg Val
Thr Ile Thr Ala Asp Glu Leu Arg Ser Thr Ala Tyr65 70 75 80Met Glu
Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95Ala
Arg Gly Ser Tyr Tyr Tyr Glu Ser Ser Leu Asp Tyr Trp Gly Gln 100 105
110Gly Thr Leu Val Thr Val Ser Ser 115 120185120PRTArtificial
SequenceDescription of Artificial Sequence Synthetic polypeptide
185Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ser1
5 10 15Ser Val Lys Val Ser Cys Lys Thr Ser Gly Gly Thr Phe Ser Ser
Tyr 20 25 30Ala Val Thr Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu
Trp Met 35 40 45Gly Gly Ile Ile Gly Met Phe Gly Thr Thr Asn Tyr Ala
Gln Lys Phe 50 55 60Gln Gly Arg Val Thr Ile Thr Ala Asp Glu Leu Thr
Ser Thr Ala Tyr65 70 75 80Met Glu Leu Ser Ser Leu Arg Ser Glu Asp
Thr Ala Val Tyr Tyr Cys 85 90 95Ala Arg Gly Ser Tyr Tyr Tyr Glu Ser
Ser Leu Asp Tyr Trp Gly Arg 100 105 110Gly Thr Leu Val Thr Val Ser
Ser 115 120186120PRTArtificial SequenceDescription of Artificial
Sequence Synthetic polypeptide 186Gln Val Gln Leu Val Gln Ser Gly
Ala Glu Val Lys Lys Pro Gly Ser1 5 10 15Ser Val Lys Val Ser Cys Lys
Thr Ser Gly Gly Thr Phe Ser Ser Tyr 20 25 30Ala Val Thr Trp Val Arg
Gln Ala Pro Gly Gln Gly Leu Glu Trp Met 35 40 45Gly Ala Ile Ile Gly
Met Phe Gly Thr Thr Asn Tyr Ala Gln Lys Phe 50 55 60Gln Gly Arg Val
Thr Ile Thr Ala Asp Glu Leu Thr Ser Thr Ala Tyr65 70 75 80Met Glu
Leu Ser Ser Leu Arg Ser Asp Asp Thr Ala Val Tyr Tyr Cys 85 90 95Ala
Arg Gly Ser Tyr Tyr Tyr Glu Ser Ser Leu Asp Tyr Trp Gly Gln 100 105
110Gly Thr Leu Val Thr Val Ser Ser 115 120187120PRTArtificial
SequenceDescription of Artificial Sequence Synthetic polypeptide
187Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ser1
5 10 15Ser Val Lys Val Ser Cys Lys Thr Ser Gly Gly Thr Phe Ser Ser
Tyr 20 25 30Ala Val Thr Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu
Trp Met 35 40 45Gly Ala Ile Ile Gly Met Phe Gly Thr Thr Asn Tyr Ala
Gln Lys Phe 50 55 60Gln Gly Arg Val Thr Ile Thr Ala Asp Glu Leu Thr
Ser Thr Ala Tyr65 70 75 80Met Glu Leu Ser Ser Leu Arg Ser Asp Asp
Thr Ala Val Tyr Tyr Cys 85 90 95Ala Arg Gly Ser Tyr Tyr Tyr Glu Ser
Ser Leu Asp Tyr Trp Gly Arg 100 105 110Gly Thr Met Val Thr Val Ser
Ser 115 120188120PRTArtificial SequenceDescription of Artificial
Sequence Synthetic polypeptide 188Gln Val Gln Leu Val Gln Ser Gly
Ala Glu Val Lys Lys Pro Gly Ser1 5 10 15Ser Val Lys Val Ser Cys Lys
Thr Ser Gly Gly Thr Phe Ser Ser Tyr 20 25 30Ala Val Thr Trp Val Arg
Gln Ala Pro Gly Gln Gly Leu Glu Trp Met 35 40 45Gly Ala Ile Ile Gly
Met Phe Gly Thr Thr Asn Tyr Ala Gln Lys Phe 50 55 60Gln Gly Arg Val
Thr Ile Thr Ala Asp Glu Leu Thr Ser Thr Ala Tyr65 70 75 80Met Glu
Leu Ser Ser Leu Arg Ser Asp Asp Thr Ala Val Tyr Tyr Cys 85 90 95Ala
Arg Gly Ser Tyr Tyr Tyr Glu Ser Ser Leu Asp Tyr Trp Gly Gln 100 105
110Gly Thr Met Val Thr Val Ser Ser 115 120189120PRTArtificial
SequenceDescription of Artificial Sequence Synthetic polypeptide
189Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ser1
5 10 15Ser Val Lys Val Ser Cys Lys Thr Ser Gly Gly Thr Phe Ser Ser
Tyr 20 25 30Ala Val Thr Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu
Trp Met 35 40 45Gly Ala Ile Ile Gly Met Phe Gly Thr Thr Asn Tyr Ala
Gln Lys Phe 50 55 60Gln Gly Arg Val Thr Leu Thr Ala Asp Glu Leu Thr
Ser Thr Ala Tyr65 70 75 80Met Glu Leu Ser Ser Leu Arg Ser Glu Asp
Thr Ala Val Tyr Tyr Cys 85 90 95Ala Arg Gly Ser Tyr Tyr Tyr Glu Ser
Ser Leu Asp Tyr Trp Gly Gln 100 105 110Gly Thr Leu Val Thr Val Ser
Ser 115 120190120PRTArtificial SequenceDescription of Artificial
Sequence Synthetic polypeptide 190Gln Val Gln Leu Val Gln Ser Gly
Ala Glu Val Lys Lys Pro Gly Ser1 5 10 15Ser Val Lys Val Ser Cys Lys
Thr Ser Gly Gly Thr Phe Ser Ser Tyr 20 25 30Ala Val Thr Trp Val Arg
Gln Ala Pro Gly Gln Gly Leu Glu Trp Met 35 40 45Gly Ala Ile Ile Gly
Met Phe Gly Thr Thr Asn Tyr Ala Gln Lys Phe 50 55 60Gln Gly Arg Val
Thr Leu Thr Ala Asp Glu Leu Thr Ser Thr Ala Tyr65 70 75 80Met Glu
Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95Ala
Arg Gly Ser Tyr Tyr Tyr Glu Ser Ser Leu Asp Tyr Trp Gly Gln 100 105
110Gly Thr Met Val Thr Val Ser Ser 115 120191120PRTArtificial
SequenceDescription of Artificial Sequence Synthetic polypeptide
191Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ser1
5 10 15Ser Val Lys Val Ser Cys Lys Thr Ser Gly Gly Thr Phe Ser Ser
Tyr 20 25 30Ala Val Thr Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu
Trp Met 35 40 45Gly Ala Ile Ile Gly Met Phe Gly Thr Thr Asn Tyr Ala
Gln Lys Phe 50 55 60Gln Gly Arg Val Thr Leu Thr Ala Asp Glu Leu Thr
Ser Thr Ala Tyr65 70 75 80Met Glu Leu Ser Ser Leu Arg Ser Glu Asp
Thr Ala Val Tyr Tyr Cys 85 90 95Ala Arg Gly Ser Tyr Tyr Tyr Glu Ser
Ser Leu Asp Tyr Trp Gly Lys 100 105 110Gly Thr Met Val Thr Val Ser
Ser 115 120192120PRTArtificial SequenceDescription of Artificial
Sequence Synthetic polypeptide 192Gln Val Gln Leu Val Gln Ser Gly
Ala Glu Val Lys Lys Pro Gly Ser1 5 10 15Ser Val Lys Val Ser Cys Lys
Thr Ser Gly Gly Thr Phe Ser Ser Tyr 20 25 30Ala Val Thr Trp Val Arg
Gln Ala Pro Gly Gln Gly Leu Glu Trp Met 35 40 45Gly Ala Ile Ile Gly
Met Phe Gly Thr Thr Asn Tyr Ala Gln Lys Phe 50 55 60Gln Gly Arg Val
Thr Leu Thr Ala Asp Glu Leu Thr Ser Thr Ala Tyr65 70 75 80Met Glu
Leu Ser Ser Leu Arg Ser Xaa Asp Thr Ala Val Tyr Tyr Cys 85 90 95Ala
Arg Gly Ser Tyr Tyr Tyr Glu Ser Ser Leu Asp Tyr Trp Gly Gln 100 105
110Gly Thr Leu Val Thr Val Ser Ser 115 120193120PRTArtificial
SequenceDescription of Artificial Sequence Synthetic polypeptide
193Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ser1
5 10 15Ser Val Lys Val Ser Cys Lys Thr Ser Gly Gly Thr Phe Ser Ser
Tyr 20 25 30Ala Val Thr Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu
Trp Met 35 40 45Gly Ala Ile Ile Gly Met Phe Gly Thr Thr Asn Tyr Ala
Gln Lys Phe 50 55 60Gln Gly Arg Val Thr Leu Thr Ala Asp Glu Leu Thr
Ser Thr Ala Tyr65 70 75 80Met Glu Leu Ser Ser Leu Arg Ser Glu Asp
Thr Ala Val Tyr Tyr Cys 85 90 95Ala Arg Gly Ser Tyr Tyr Tyr Glu Ser
Ser Leu Asp Tyr Trp Gly Gln 100 105 110Gly Thr Leu Val Thr Val Ser
Ser 115 120194120PRTArtificial SequenceDescription of Artificial
Sequence Synthetic polypeptide 194Gln Val Gln Leu Val Gln Ser Gly
Ala Glu Val Lys Lys Pro Gly Ser1 5 10 15Ser Val Arg Val Ser Cys Lys
Thr Ser Gly Gly Thr Phe Ser Ser Tyr 20 25 30Ala Val Thr Trp Val Arg
Gln Ala Pro Gly Gln Gly Leu Glu Trp Met 35 40 45Gly Gly Ile Ile Gly
Met Phe Gly Thr Thr Asn Tyr Ala Gln Lys Phe 50 55 60Gln Gly Arg Leu
Thr Ile Thr Ala Asp Glu Met Thr Ser Thr Ala Tyr65 70 75 80Met Glu
Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95Ala
Arg Gly Ser Tyr Tyr Tyr Glu Thr Thr Leu Asp Tyr Trp Gly Arg 100 105
110Gly Thr Leu Val Thr Val Ser Ser 115 120195120PRTArtificial
SequenceDescription of Artificial Sequence Synthetic polypeptide
195Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ser1
5 10 15Ser Val Lys Val Ser Cys Lys Thr Ser Gly Gly Ser Phe Ser Ser
Tyr 20 25 30Ala Val Thr Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu
Trp Met 35 40 45Gly Gly Ile Ile Gly Met Phe Gly Thr Thr Asn Tyr Ala
Gln Lys Phe 50 55 60Gln Gly Arg Val Thr Ile Thr Ala Asp Glu Leu Thr
Ser Thr Ala Tyr65 70 75 80Met Glu Leu Ser Ser Leu Arg Ser Glu Asp
Thr Ala Val Tyr Tyr Cys 85 90 95Ala Arg Gly Ser Tyr Tyr Tyr Glu Ser
Ser Leu Asp Tyr Trp Gly Gln 100 105 110Gly Thr Met Val Thr Val Ser
Ser 115 120196120PRTArtificial SequenceDescription of Artificial
Sequence Synthetic polypeptide 196Gln Val Gln Leu Val Gln Ser Gly
Ala Glu Val Lys Lys Pro Gly Ser1 5 10 15Ser Val Lys Val Ser Cys Lys
Thr Thr Gly Gly Thr Phe Ser Ser Tyr 20 25 30Ala Val Thr Trp Val Arg
Gln Ala Pro Gly Gln Gly Leu Glu Trp Met 35 40 45Gly Gly Ile Ile Gly
Met Phe Gly Thr Thr Asn Tyr Ala Gln Lys Phe 50 55 60Gln Gly Arg Val
Thr Ile Thr Ala Asp Glu Met Thr Ser Thr Ala Tyr65 70 75 80Met Glu
Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95Ala
Arg Gly Ser Tyr Tyr Tyr Glu Ser Ser Leu Asp Tyr Trp Gly Lys 100 105
110Gly Thr Met Val Thr Val Ser Ser 115 120197120PRTArtificial
SequenceDescription of Artificial Sequence Synthetic polypeptide
197Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ser1
5 10 15Ser Val Lys Val Ser Cys Lys Thr Thr Gly Gly Thr Phe Ser Ser
Tyr 20 25 30Ala Val Thr Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu
Trp Met 35 40 45Gly Gly Ile Ile Gly Met Phe Gly Thr Thr Asn Tyr Ala
Gln Lys Phe 50 55 60Gln Gly Arg Val Thr Ile Thr Ala Asp Glu Met Thr
Ser Thr Ala Tyr65 70 75 80Met Glu Leu Ser Ser Leu Arg Ser Glu Asp
Thr Ala Val Tyr Tyr Cys 85 90 95Ala Arg Gly Ser Tyr Tyr Tyr Glu Ser
Ser Leu Asp Tyr Trp Gly Gln 100 105 110Gly Thr Leu Val Thr Val Ser
Ser 115 120198120PRTArtificial SequenceDescription of Artificial
Sequence Synthetic polypeptide 198Gln Val Gln Leu Val Gln Ser Gly
Ala Glu Val Lys Lys Pro Gly Ser1 5 10 15Ser Val Lys Val Ser Cys Lys
Thr Thr Gly Gly Thr Phe Ser Ser Tyr 20 25 30Ala Val Thr Trp Val Arg
Gln Ala Pro Gly Gln Gly Leu Glu Trp Met 35 40 45Gly Gly Ile Ile Gly
Met Phe Gly Thr Thr Asn Tyr Ala Gln Lys Phe 50 55 60Gln Gly Arg Val
Thr Ile Thr Ala Asp Glu Met Thr Ser Thr Ala Tyr65 70 75 80Met Glu
Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95Ala
Arg Gly Ser Tyr Tyr Tyr Glu Ser Ser Leu Asp Tyr Trp Gly Lys 100 105
110Gly Thr Leu Val Thr Val Ser Ser 115 120199120PRTArtificial
SequenceDescription of Artificial Sequence Synthetic polypeptide
199Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ser1
5 10 15Ser Val Lys Val Ser Cys Lys Thr Thr Gly Gly Thr Phe Ser Ser
Tyr 20 25 30Ala Val Thr Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu
Trp Met 35 40 45Gly Gly Ile Ile Gly Met Phe Gly Thr Thr Asn Tyr Ala
Gln Lys Phe 50 55 60Gln Gly Arg Val Thr Ile Thr Ala Asp Glu Met Thr
Ser Thr Ala Tyr65 70 75 80Met Glu Leu Ser Ser Leu Arg Ser Glu Asp
Thr Ala Val Tyr Tyr Cys 85 90 95Ala Arg Gly Ser Tyr Tyr Tyr Glu Ser
Ser Leu Asp Tyr Trp Gly Gln 100 105 110Gly Thr Met Val Thr Val Ser
Ser 115 120200120PRTArtificial SequenceDescription of Artificial
Sequence Synthetic polypeptide 200Gln Val Gln Leu Val Gln Ser Gly
Ala Glu Val Lys Lys Pro Gly Ser1 5 10 15Ser Val Lys Val Ser Cys Lys
Xaa Ser Gly Gly Thr Phe Ser Ser Tyr 20 25 30Ala Val Thr Trp Val Arg
Gln Ala Pro Gly Gln Gly Leu Glu Trp Met 35 40 45Gly Ala Ile Ile Gly
Met Phe Gly Thr Thr Asn Tyr Ala Gln Lys Phe 50 55 60Gln Gly Arg Val
Thr Ile Thr Ala Asp Glu Leu Thr Ser Thr Ala Tyr65 70 75 80Met Glu
Leu Ser Ser Leu Arg Ser Asp Asp Thr Ala Val Tyr Tyr Cys 85 90 95Ala
Arg Gly Ser Tyr Tyr Tyr Glu Ser Ser Leu Asp Tyr Trp Gly Gln 100 105
110Gly Thr Leu Val Thr Val Ser Ser 115 120201120PRTArtificial
SequenceDescription of Artificial Sequence Synthetic polypeptide
201Glu Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ser1
5 10 15Ser Val Lys Val Ser Cys Lys Ala Ser Gly Gly Thr Phe Ser Ser
Tyr 20 25 30Ala Ile Ser Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu
Trp Met 35 40 45Gly Ala Ile Ile Gly Met Phe Gly Thr Thr Asn Tyr Ala
Gln Lys Phe 50 55 60Gln Gly Arg Val Thr Ile Thr Ala Asp Glu Leu Thr
Ser Thr Ala Tyr65 70 75 80Met Glu Leu Ser Ser Leu Arg Ser Asp Asp
Thr Ala Val Tyr Tyr Cys 85 90
95Ala Arg Gly Ser Tyr Tyr Tyr Glu Ser Ser Leu Asp Tyr Trp Gly Gln
100 105 110Gly Thr Leu Val Thr Val Ser Ser 115
120202120PRTArtificial SequenceDescription of Artificial Sequence
Synthetic polypeptide 202Glu Val Gln Leu Val Gln Ser Gly Thr Glu
Val Lys Lys Pro Gly Ser1 5 10 15Ser Val Lys Leu Ser Cys Lys Ala Ser
Gly Gly Thr Phe Ser Ser Tyr 20 25 30Ala Val Thr Trp Val Arg Gln Ala
Pro Gly Gln Gly Leu Glu Trp Met 35 40 45Gly Gly Ile Ile Gly Met Phe
Gly Thr Thr Asn Tyr Ala Gln Lys Phe 50 55 60Gln Gly Arg Leu Thr Ile
Thr Ala Asp Glu Met Thr Ser Thr Ala Tyr65 70 75 80Met Glu Leu Ser
Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95Ala Arg Gly
Ser Tyr Tyr Tyr Glu Thr Thr Leu Asp Tyr Trp Gly Arg 100 105 110Gly
Thr Met Val Thr Val Ser Ser 115 120203120PRTArtificial
SequenceDescription of Artificial Sequence Synthetic polypeptide
203Glu Val Gln Leu Val Gln Ser Gly Thr Glu Val Lys Lys Pro Gly Ser1
5 10 15Ser Val Lys Val Ser Cys Lys Val Ser Gly Gly Thr Phe Ser Ser
Tyr 20 25 30Ala Val Thr Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu
Trp Met 35 40 45Gly Ala Ile Ile Gly Met Phe Gly Thr Thr Asn Tyr Ala
Gln Lys Phe 50 55 60Gln Gly Arg Val Thr Ile Thr Ala Asp Glu Leu Thr
Ser Thr Ala Tyr65 70 75 80Met Glu Leu Ser Ser Leu Arg Ser Asp Asp
Thr Ala Val Tyr Tyr Cys 85 90 95Ala Arg Gly Ser Tyr Tyr Tyr Glu Ser
Ser Leu Asp Tyr Trp Gly Gly 100 105 110Gly Thr Thr Val Thr Val Ser
Ser 115 120204120PRTArtificial SequenceDescription of Artificial
Sequence Synthetic polypeptide 204Glu Val Gln Leu Val Gln Ser Gly
Ala Glu Val Gln Lys Pro Gly Ser1 5 10 15Ser Val Lys Val Ser Cys Lys
Thr Ser Gly Gly Thr Phe Ser Ser Tyr 20 25 30Ala Val Thr Trp Val Arg
Gln Ala Pro Gly Gln Gly Leu Glu Trp Met 35 40 45Gly Ala Ile Ile Gly
Met Phe Gly Thr Thr Asn Tyr Ala Gln Lys Phe 50 55 60Gln Gly Arg Val
Thr Leu Thr Ala Asp Glu Leu Thr Ser Thr Ala Tyr65 70 75 80Met Glu
Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95Ala
Arg Gly Ser Tyr Tyr Tyr Glu Ser Ser Leu Asp Tyr Trp Gly Gln 100 105
110Gly Thr Leu Val Thr Val Ser Ser 115 120205120PRTArtificial
SequenceDescription of Artificial Sequence Synthetic polypeptide
205Glu Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala1
5 10 15Ser Val Lys Val Ser Cys Lys Ala Ser Gly Gly Ala Phe Ser Ser
Tyr 20 25 30Ala Val Thr Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu
Trp Met 35 40 45Gly Gly Ile Ile Gly Met Phe Gly Thr Thr Asn Tyr Ala
Gln Lys Phe 50 55 60Gln Gly Arg Val Thr Ile Thr Ala Asp Glu Leu Thr
Ser Thr Ala Tyr65 70 75 80Met Glu Leu Ser Ser Leu Arg Ser Glu Asp
Thr Ala Val Tyr Tyr Cys 85 90 95Ala Arg Gly Ser Tyr Tyr Tyr Glu Ser
Ser Leu Asp Tyr Trp Gly Gln 100 105 110Gly Thr Met Val Thr Val Ser
Ser 115 120206120PRTArtificial SequenceDescription of Artificial
Sequence Synthetic polypeptide 206Glu Val Gln Leu Val Gln Ser Gly
Ala Glu Val Lys Lys Pro Gly Ser1 5 10 15Ser Val Arg Val Ser Cys Lys
Thr Ser Gly Gly Thr Phe Ser Ser Tyr 20 25 30Ala Val Thr Trp Val Arg
Gln Ala Pro Gly Gln Gly Leu Glu Trp Met 35 40 45Gly Gly Ile Ile Gly
Met Phe Gly Thr Thr Asn Tyr Ala Gln Lys Phe 50 55 60Gln Gly Arg Leu
Thr Ile Thr Ala Asp Glu Met Thr Ser Thr Ala Tyr65 70 75 80Met Glu
Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95Ala
Arg Gly Ser Tyr Tyr Tyr Glu Thr Thr Leu Asp Tyr Trp Gly Gln 100 105
110Gly Thr Leu Val Thr Val Ser Ser 115 120207120PRTArtificial
SequenceDescription of Artificial Sequence Synthetic polypeptide
207Glu Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ser1
5 10 15Ser Val Arg Val Ser Cys Lys Thr Ser Gly Gly Thr Phe Ser Ser
Tyr 20 25 30Ala Val Thr Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu
Trp Met 35 40 45Gly Gly Ile Ile Gly Met Phe Gly Thr Thr Asn Tyr Ala
Gln Lys Phe 50 55 60Gln Gly Arg Leu Thr Ile Thr Ala Asp Glu Met Thr
Ser Thr Ala Tyr65 70 75 80Met Glu Leu Ser Ser Leu Arg Ser Glu Asp
Thr Ala Val Tyr Tyr Cys 85 90 95Ala Arg Gly Ser Tyr Tyr Tyr Glu Thr
Thr Leu Asp Tyr Trp Gly Lys 100 105 110Gly Thr Leu Val Thr Val Ser
Ser 115 120208120PRTArtificial SequenceDescription of Artificial
Sequence Synthetic polypeptide 208Glu Val Gln Leu Val Gln Ser Gly
Ala Glu Val Lys Lys Pro Gly Ser1 5 10 15Ser Val Arg Val Ser Cys Lys
Thr Ser Gly Gly Thr Phe Ser Ser Tyr 20 25 30Ala Val Thr Trp Val Arg
Gln Ala Pro Gly Gln Gly Leu Glu Trp Met 35 40 45Gly Gly Ile Ile Gly
Met Phe Gly Thr Thr Asn Tyr Ala Gln Lys Phe 50 55 60Gln Gly Arg Leu
Thr Ile Thr Ala Asp Glu Met Thr Ser Thr Ala Tyr65 70 75 80Met Glu
Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95Ala
Arg Gly Ser Tyr Tyr Tyr Glu Thr Thr Leu Asp Tyr Trp Gly Arg 100 105
110Gly Thr Met Val Thr Val Ser Ser 115 120209120PRTArtificial
SequenceDescription of Artificial Sequence Synthetic polypeptide
209Glu Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ser1
5 10 15Ser Val Lys Val Ser Cys Lys Thr Ser Gly Gly Thr Phe Ser Ser
Tyr 20 25 30Ala Val Thr Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu
Trp Met 35 40 45Gly Ala Ile Ile Gly Met Phe Gly Thr Thr Asn Tyr Ala
Gln Lys Phe 50 55 60Gln Gly Arg Val Thr Leu Thr Ala Asp Glu Leu Thr
Ser Thr Ala Tyr65 70 75 80Met Glu Leu Ser Ser Leu Arg Ser Glu Asp
Thr Ala Val Tyr Tyr Cys 85 90 95Ala Arg Gly Ser Tyr Tyr Tyr Glu Ser
Ser Leu Asp Tyr Trp Gly Arg 100 105 110Gly Thr Leu Val Thr Val Ser
Ser 115 120210120PRTArtificial SequenceDescription of Artificial
Sequence Synthetic polypeptide 210Glu Val Gln Leu Val Gln Ser Gly
Ala Glu Val Lys Lys Pro Gly Ser1 5 10 15Ser Val Lys Val Ser Cys Lys
Thr Ser Gly Gly Thr Phe Ser Ser Tyr 20 25 30Ala Val Thr Trp Val Arg
Gln Ala Pro Gly Gln Gly Leu Glu Trp Met 35 40 45Gly Ala Ile Ile Gly
Met Phe Gly Thr Thr Asn Tyr Ala Gln Lys Phe 50 55 60Gln Gly Arg Val
Thr Ile Thr Ala Asp Glu Leu Thr Ser Thr Ala Tyr65 70 75 80Met Glu
Leu Ser Ser Leu Arg Ser Asp Asp Thr Ala Val Tyr Tyr Cys 85 90 95Ala
Arg Gly Ser Tyr Tyr Tyr Glu Ser Ser Leu Asp Tyr Trp Gly Lys 100 105
110Gly Thr Leu Val Thr Val Ser Ser 115 120211120PRTArtificial
SequenceDescription of Artificial Sequence Synthetic polypeptide
211Glu Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ser1
5 10 15Ser Val Lys Val Ser Cys Lys Thr Ser Gly Gly Thr Phe Ser Ser
Tyr 20 25 30Ala Val Thr Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu
Trp Met 35 40 45Gly Ala Ile Ile Gly Met Phe Gly Thr Thr Asn Tyr Ala
Gln Lys Phe 50 55 60Gln Gly Arg Val Thr Ile Thr Ala Asp Glu Leu Thr
Ser Thr Ala Tyr65 70 75 80Met Glu Leu Ser Ser Leu Arg Ser Asp Asp
Thr Ala Val Tyr Tyr Cys 85 90 95Ala Arg Gly Ser Tyr Tyr Tyr Glu Ser
Ser Leu Asp Tyr Trp Gly Gln 100 105 110Gly Thr Leu Val Thr Val Ser
Ser 115 120212120PRTArtificial SequenceDescription of Artificial
Sequence Synthetic polypeptide 212Glu Val Gln Leu Val Gln Ser Gly
Ala Glu Val Lys Lys Pro Gly Ser1 5 10 15Ser Val Lys Val Ser Cys Lys
Thr Ser Gly Gly Thr Phe Ser Ser Tyr 20 25 30Ala Val Thr Trp Val Arg
Gln Ala Pro Gly Gln Gly Leu Glu Trp Met 35 40 45Gly Ala Ile Ile Gly
Met Phe Gly Thr Thr Asn Tyr Ala Gln Lys Phe 50 55 60Gln Gly Arg Val
Thr Ile Thr Ala Asp Glu Leu Thr Ser Thr Ala Tyr65 70 75 80Met Glu
Leu Ser Ser Leu Arg Ser Asp Asp Thr Ala Val Tyr Tyr Cys 85 90 95Ala
Arg Gly Ser Tyr Tyr Tyr Glu Ser Ser Leu Asp Tyr Trp Gly Gln 100 105
110Gly Thr Met Val Thr Val Ser Ser 115 120213120PRTArtificial
SequenceDescription of Artificial Sequence Synthetic polypeptide
213Glu Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ser1
5 10 15Ser Val Lys Val Ser Cys Lys Thr Ser Gly Gly Thr Phe Ser Ser
Tyr 20 25 30Ala Val Thr Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu
Trp Met 35 40 45Gly Ala Ile Ile Gly Met Phe Gly Thr Thr Asn Tyr Ala
Gln Lys Phe 50 55 60Gln Gly Arg Val Thr Ile Thr Ala Asp Glu Leu Thr
Ser Thr Ala Tyr65 70 75 80Met Glu Leu Ser Ser Leu Arg Ser Asp Asp
Thr Ala Val Tyr Tyr Cys 85 90 95Ala Arg Gly Ser Tyr Tyr Tyr Glu Ser
Ser Leu Asp Tyr Trp Gly Lys 100 105 110Gly Thr Leu Val Thr Val Ser
Ser 115 120214120PRTArtificial SequenceDescription of Artificial
Sequence Synthetic polypeptide 214Glu Val Gln Leu Val Gln Ser Gly
Ala Glu Val Lys Lys Pro Gly Ser1 5 10 15Ser Val Lys Val Ser Cys Lys
Thr Ser Gly Gly Thr Phe Ser Ser Tyr 20 25 30Ala Val Thr Trp Val Arg
Gln Ala Pro Gly Gln Gly Leu Glu Trp Met 35 40 45Gly Ala Ile Ile Gly
Met Phe Gly Thr Thr Asn Tyr Ala Gln Lys Phe 50 55 60Gln Gly Arg Val
Thr Ile Thr Ala Asp Glu Leu Thr Ser Thr Ala Tyr65 70 75 80Met Glu
Leu Ser Ser Leu Arg Ser Asp Asp Thr Ala Val Tyr Tyr Cys 85 90 95Ala
Arg Gly Ser Tyr Tyr Tyr Glu Ser Ser Leu Asp Tyr Trp Gly Lys 100 105
110Gly Thr Thr Val Thr Val Ser Ser 115 120215120PRTArtificial
SequenceDescription of Artificial Sequence Synthetic polypeptide
215Glu Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ser1
5 10 15Ser Val Lys Val Ser Cys Lys Thr Ser Gly Gly Thr Phe Ser Ser
Tyr 20 25 30Ala Val Thr Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu
Trp Met 35 40 45Gly Ala Ile Ile Gly Met Phe Gly Thr Thr Asn Tyr Ala
Gln Lys Phe 50 55 60Gln Gly Arg Val Thr Ile Thr Ala Asp Glu Leu Thr
Ser Thr Ala Tyr65 70 75 80Met Glu Leu Ser Ser Leu Arg Ser Asp Asp
Thr Ala Val Tyr Tyr Cys 85 90 95Ala Arg Gly Ser Tyr Tyr Tyr Glu Ser
Ser Leu Asp Tyr Trp Gly Lys 100 105 110Gly Thr Met Val Thr Val Ser
Ser 115 120216120PRTArtificial SequenceDescription of Artificial
Sequence Synthetic polypeptide 216Glu Val Gln Leu Val Gln Ser Gly
Ala Glu Val Lys Lys Pro Gly Ser1 5 10 15Ser Val Lys Val Ser Cys Lys
Thr Ser Gly Gly Thr Phe Ser Ser Tyr 20 25 30Ala Val Thr Trp Val Arg
Gln Ala Pro Gly Gln Gly Leu Glu Trp Met 35 40 45Gly Ala Ile Ile Gly
Met Phe Gly Thr Thr Asn Tyr Ala Gln Lys Phe 50 55 60Gln Gly Arg Val
Thr Leu Thr Ala Asp Glu Leu Thr Ser Thr Ala Tyr65 70 75 80Met Glu
Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95Ala
Arg Gly Ser Tyr Tyr Tyr Glu Ser Ser Leu Asp Tyr Trp Gly Gln 100 105
110Gly Thr Leu Val Thr Val Ser Ser 115 120217120PRTArtificial
SequenceDescription of Artificial Sequence Synthetic polypeptide
217Glu Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ser1
5 10 15Ser Val Lys Val Ser Cys Lys Thr Ser Gly Gly Thr Phe Ser Ser
Tyr 20 25 30Ala Val Thr Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu
Trp Met 35 40 45Gly Ala Ile Ile Gly Met Phe Gly Thr Thr Asn Tyr Ala
Gln Lys Phe 50 55 60Gln Gly Arg Val Thr Leu Thr Ala Asp Glu Leu Thr
Ser Thr Ala Tyr65 70 75 80Met Glu Leu Ser Ser Leu Arg Ser Glu Asp
Thr Ala Val Tyr Tyr Cys 85 90 95Ala Arg Gly Ser Tyr Tyr Tyr Glu Ser
Ser Leu Asp Tyr Trp Gly Lys 100 105 110Gly Thr Leu Val Thr Val Ser
Ser 115 120218120PRTArtificial SequenceDescription of Artificial
Sequence Synthetic polypeptide 218Glu Val Gln Leu Val Gln Ser Gly
Ala Glu Val Lys Lys Pro Gly Ser1 5 10 15Ser Val Lys Val Ser Cys Lys
Thr Ser Gly Gly Thr Phe Ser Ser Tyr 20 25 30Ala Val Thr Trp Val Arg
Gln Ala Pro Gly Gln Gly Leu Glu Trp Met 35 40 45Gly Ala Ile Ile Gly
Met Phe Gly Thr Thr Asn Tyr Ala Gln Lys Phe 50 55 60Gln Gly Arg Val
Thr Leu Thr Ala Asp Glu Leu Thr Ser Thr Ala Tyr65 70 75 80Met Glu
Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95Ala
Arg Gly Ser Tyr Tyr Tyr Glu Ser Ser Leu Asp Tyr Trp Gly Lys 100 105
110Gly Thr Met Val Thr Val Ser Ser 115 120219120PRTArtificial
SequenceDescription of Artificial Sequence Synthetic polypeptide
219Glu Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ser1
5 10 15Ser Val Lys Val Ser Cys Lys Thr Ser Gly Gly Thr Phe Ser Ser
Tyr 20 25 30Ala Val Thr Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu
Trp Met 35 40 45Gly Ala Ile Ile Gly Met Phe Gly Thr Thr Asn Tyr Ala
Gln Lys Phe 50 55 60Gln Gly Arg Val Thr Leu Thr Ala Asp Glu Leu Thr
Ser Thr Ala Tyr65 70 75 80Met Glu Leu Ser Ser Leu Arg Ser Glu Asp
Thr Ala Val Tyr Tyr Cys 85 90 95Ala Arg Gly Ser Tyr Tyr Tyr Glu Ser
Ser Leu Asp Tyr Trp Gly Arg 100 105 110Gly Thr Leu Val Thr Val Ser
Ser 115 120220120PRTArtificial SequenceDescription of Artificial
Sequence Synthetic polypeptide 220Glu Val Gln Leu Val Gln Ser Gly
Ala Glu Val Lys Lys Pro Gly Ser1 5 10 15Ser Val Lys Val Ser Cys Lys
Thr Ser Gly Gly Thr Phe Ser Ser Tyr 20 25 30Ala Val Thr Trp Val Arg
Gln Ala Pro Gly Gln Gly Leu
Glu Trp Met 35 40 45Gly Ala Ile Ile Gly Met Phe Gly Thr Thr Asn Tyr
Ala Gln Lys Phe 50 55 60Gln Gly Arg Val Thr Leu Thr Ala Asp Xaa Leu
Thr Ser Thr Ala Tyr65 70 75 80Met Glu Leu Ser Ser Leu Arg Ser Glu
Asp Thr Ala Val Tyr Tyr Cys 85 90 95Ala Arg Gly Ser Tyr Tyr Tyr Glu
Ser Ser Leu Asp Tyr Trp Gly Arg 100 105 110Gly Thr Leu Val Thr Val
Ser Ser 115 120221120PRTArtificial SequenceDescription of
Artificial Sequence Synthetic polypeptide 221Glu Val Gln Leu Val
Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ser1 5 10 15Ser Val Lys Val
Ser Cys Lys Thr Ser Gly Gly Thr Phe Ser Ser Tyr 20 25 30Ala Val Thr
Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met 35 40 45Gly Ala
Ile Ile Gly Met Phe Gly Thr Thr Asn Tyr Ala Gln Lys Phe 50 55 60Gln
Gly Arg Val Thr Leu Thr Ala Asp Glu Leu Thr Ser Thr Ala Tyr65 70 75
80Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys
85 90 95Ala Arg Gly Ser Tyr Tyr Tyr Glu Ser Ser Leu Asp Tyr Trp Gly
Arg 100 105 110Gly Thr Met Val Thr Val Ser Ser 115
120222120PRTArtificial SequenceDescription of Artificial Sequence
Synthetic polypeptide 222Glu Val Gln Leu Val Gln Ser Gly Ala Glu
Val Lys Lys Pro Gly Ser1 5 10 15Ser Val Lys Val Ser Cys Lys Thr Ser
Gly Gly Thr Phe Ser Ser Tyr 20 25 30Ala Val Thr Trp Val Arg Gln Ala
Pro Gly Gln Gly Leu Glu Trp Met 35 40 45Gly Gly Ile Ile Gly Met Phe
Gly Thr Thr Asn Tyr Ala Gln Lys Phe 50 55 60Gln Gly Arg Val Thr Met
Thr Ala Asp Glu Met Thr Ser Thr Ala Tyr65 70 75 80Met Glu Leu Ser
Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95Ala Arg Gly
Ser Tyr Tyr Tyr Glu Ser Ser Leu Asp Tyr Trp Gly Lys 100 105 110Gly
Thr Leu Val Thr Val Ser Ser 115 120223120PRTArtificial
SequenceDescription of Artificial Sequence Synthetic polypeptide
223Glu Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ser1
5 10 15Ser Val Lys Val Ser Cys Lys Thr Ser Gly Gly Thr Phe Ser Ser
Tyr 20 25 30Ala Val Thr Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu
Trp Met 35 40 45Gly Gly Ile Ile Gly Met Phe Gly Thr Thr Asn Tyr Ala
Gln Lys Phe 50 55 60Gln Gly Arg Val Thr Ile Thr Ala Asp Glu Leu Thr
Ser Thr Ala Tyr65 70 75 80Met Glu Leu Ser Ser Leu Arg Ser Glu Asp
Thr Ala Val Tyr Tyr Cys 85 90 95Ala Arg Gly Ser Tyr Tyr Tyr Glu Ser
Ser Leu Asp Tyr Trp Gly Gln 100 105 110Gly Thr Leu Val Thr Val Ser
Ser 115 120224120PRTArtificial SequenceDescription of Artificial
Sequence Synthetic polypeptide 224Glu Val Gln Leu Val Gln Ser Gly
Ala Glu Val Lys Lys Pro Gly Ser1 5 10 15Ser Val Lys Val Ser Cys Lys
Thr Ser Gly Gly Thr Phe Ser Ser Tyr 20 25 30Ala Val Thr Trp Val Arg
Gln Ala Pro Gly Gln Gly Leu Glu Trp Met 35 40 45Gly Gly Ile Ile Gly
Met Phe Gly Thr Thr Asn Tyr Ala Gln Lys Phe 50 55 60Gln Gly Arg Val
Thr Ile Thr Ala Asp Glu Leu Thr Ser Thr Ala Tyr65 70 75 80Met Glu
Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95Ala
Arg Gly Ser Tyr Tyr Tyr Glu Ser Ser Leu Asp Tyr Trp Gly Lys 100 105
110Gly Thr Leu Val Thr Val Ser Ser 115 120225120PRTArtificial
SequenceDescription of Artificial Sequence Synthetic polypeptide
225Glu Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ser1
5 10 15Ser Val Lys Val Ser Cys Lys Thr Ser Gly Gly Thr Phe Ser Ser
Tyr 20 25 30Ala Val Thr Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu
Trp Met 35 40 45Gly Gly Ile Ile Gly Met Phe Gly Thr Thr Asn Tyr Ala
Gln Lys Phe 50 55 60Gln Gly Arg Val Thr Ile Thr Ala Asp Glu Met Thr
Ser Thr Ala Tyr65 70 75 80Met Glu Leu Ser Ser Leu Arg Ser Glu Asp
Thr Ala Val Tyr Tyr Cys 85 90 95Ala Arg Gly Ser Tyr Tyr Tyr Glu Ser
Ser Leu Asp Tyr Trp Gly Arg 100 105 110Gly Thr Leu Val Thr Val Ser
Ser 115 120226120PRTArtificial SequenceDescription of Artificial
Sequence Synthetic polypeptide 226Glu Val Gln Leu Val Gln Ser Gly
Ala Glu Val Lys Lys Pro Gly Ser1 5 10 15Ser Val Lys Val Ser Cys Lys
Thr Ser Gly Gly Thr Phe Ser Ser Tyr 20 25 30Ala Val Thr Trp Val Arg
Gln Ala Pro Gly Gln Gly Leu Glu Trp Met 35 40 45Gly Gly Ile Ile Gly
Met Phe Gly Thr Thr Asn Tyr Ala Gln Lys Phe 50 55 60Gln Gly Arg Val
Thr Ile Thr Ala Asp Glu Met Thr Ser Thr Ala Tyr65 70 75 80Met Glu
Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95Ala
Arg Gly Ser Tyr Tyr Tyr Glu Ser Ser Leu Asp Tyr Trp Gly Arg 100 105
110Gly Thr Met Val Thr Val Ser Ser 115 120227120PRTArtificial
SequenceDescription of Artificial Sequence Synthetic polypeptide
227Glu Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ser1
5 10 15Ser Val Lys Val Ser Cys Lys Thr Thr Gly Gly Thr Phe Ser Ser
Tyr 20 25 30Ala Val Thr Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu
Trp Met 35 40 45Gly Gly Ile Ile Gly Met Phe Gly Thr Thr Asn Tyr Ala
Gln Lys Phe 50 55 60Gln Gly Arg Val Thr Ile Thr Ala Asp Glu Met Thr
Ser Thr Ala Tyr65 70 75 80Met Glu Leu Ser Ser Leu Arg Ser Glu Asp
Thr Ala Val Tyr Tyr Cys 85 90 95Ala Arg Gly Ser Tyr Tyr Tyr Glu Ser
Ser Leu Asp Tyr Trp Gly Gln 100 105 110Gly Thr Leu Val Thr Val Ser
Ser 115 120228120PRTArtificial SequenceDescription of Artificial
Sequence Synthetic polypeptide 228Glu Val Gln Leu Val Gln Ser Gly
Ala Glu Val Lys Lys Pro Gly Ser1 5 10 15Ser Val Lys Val Ser Cys Lys
Thr Thr Gly Gly Thr Phe Ser Ser Tyr 20 25 30Ala Val Thr Trp Val Arg
Gln Ala Pro Gly Gln Gly Leu Glu Trp Met 35 40 45Gly Gly Ile Ile Gly
Met Phe Gly Thr Thr Asn Tyr Ala Gln Lys Phe 50 55 60Gln Gly Arg Val
Thr Ile Thr Ala Asp Glu Met Thr Ser Thr Ala Tyr65 70 75 80Met Glu
Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95Ala
Arg Gly Ser Tyr Tyr Tyr Glu Ser Ser Leu Asp Tyr Trp Gly Gln 100 105
110Gly Thr Met Val Thr Val Ser Ser 115 120229120PRTArtificial
SequenceDescription of Artificial Sequence Synthetic polypeptide
229Gly Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ser1
5 10 15Ser Val Lys Val Ser Cys Lys Thr Ser Gly Gly Thr Phe Ser Ser
Tyr 20 25 30Ala Val Thr Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu
Trp Met 35 40 45Gly Ala Ile Ile Gly Met Phe Gly Thr Thr Asn Tyr Ala
Gln Lys Phe 50 55 60Gln Gly Arg Val Thr Leu Thr Ala Asp Glu Leu Thr
Ser Thr Ala Tyr65 70 75 80Met Glu Leu Ser Ser Leu Arg Ser Glu Asp
Thr Ala Val Tyr Tyr Cys 85 90 95Ala Arg Gly Ser Tyr Tyr Tyr Glu Ser
Ser Leu Asp Tyr Trp Gly Lys 100 105 110Gly Thr Met Val Thr Val Ser
Ser 115 120230120PRTArtificial SequenceDescription of Artificial
Sequence Synthetic polypeptide 230Gly Val Gln Leu Val Gln Ser Gly
Ala Glu Val Lys Lys Pro Gly Ser1 5 10 15Ser Val Lys Val Ser Cys Lys
Thr Ser Gly Gly Thr Phe Ser Ser Tyr 20 25 30Ala Val Thr Trp Val Arg
Gln Ala Pro Gly Gln Gly Leu Glu Trp Met 35 40 45Gly Ala Ile Ile Gly
Met Phe Gly Thr Thr Asn Tyr Ala Gln Lys Phe 50 55 60Gln Gly Arg Val
Thr Ile Thr Ala Asp Glu Leu Thr Ser Thr Ala Tyr65 70 75 80Met Glu
Leu Ser Ser Leu Arg Ser Asp Asp Thr Ala Val Tyr Tyr Cys 85 90 95Ala
Arg Gly Ser Tyr Tyr Tyr Glu Ser Ser Leu Asp Tyr Trp Gly Arg 100 105
110Gly Thr Leu Val Thr Val Ser Ser 115 120231120PRTArtificial
SequenceDescription of Artificial Sequence Synthetic polypeptide
231Gln Met Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ser1
5 10 15Ser Val Lys Val Ser Cys Lys Thr Ser Gly Gly Thr Phe Ser Ser
Tyr 20 25 30Ala Val Thr Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu
Trp Met 35 40 45Gly Ala Ile Ile Gly Met Phe Gly Thr Thr Asn Tyr Ala
Gln Lys Phe 50 55 60Gln Gly Arg Val Thr Ile Thr Ala Asp Glu Leu Thr
Ser Thr Ala Tyr65 70 75 80Met Glu Leu Ser Ser Leu Arg Ser Asp Asp
Thr Ala Val Tyr Tyr Cys 85 90 95Ala Arg Gly Ser Tyr Tyr Tyr Glu Ser
Ser Leu Asp Tyr Trp Gly Lys 100 105 110Gly Thr Met Val Thr Val Ser
Ser 115 120232120PRTArtificial SequenceDescription of Artificial
Sequence Synthetic polypeptide 232Gln Met Gln Leu Val Gln Ser Gly
Ala Glu Val Lys Lys Pro Gly Ser1 5 10 15Ser Val Lys Val Ser Cys Lys
Thr Ser Gly Gly Thr Phe Ser Ser Tyr 20 25 30Ala Val Thr Trp Val Arg
Gln Ala Pro Gly Gln Gly Leu Glu Trp Met 35 40 45Gly Ala Ile Ile Gly
Met Phe Gly Thr Thr Asn Tyr Ala Gln Lys Phe 50 55 60Gln Gly Arg Val
Thr Leu Thr Ala Asp Glu Leu Thr Ser Thr Ala Tyr65 70 75 80Met Glu
Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95Ala
Arg Gly Ser Tyr Tyr Tyr Glu Ser Ser Leu Asp Tyr Trp Gly Gln 100 105
110Gly Thr Leu Val Thr Val Ser Ser 115 120233120PRTArtificial
SequenceDescription of Artificial Sequence Synthetic polypeptide
233Gln Val Gln Leu Leu Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ser1
5 10 15Ser Val Lys Val Ser Cys Lys Thr Thr Gly Gly Thr Phe Ser Ser
Tyr 20 25 30Ala Val Thr Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu
Trp Met 35 40 45Gly Gly Ile Ile Gly Met Phe Gly Thr Thr Asn Tyr Ala
Gln Lys Phe 50 55 60Gln Gly Arg Val Thr Ile Thr Ala Asp Glu Met Thr
Ser Thr Ala Tyr65 70 75 80Met Glu Leu Ser Ser Leu Arg Ser Glu Asp
Thr Ala Val Tyr Tyr Cys 85 90 95Ala Arg Gly Ser Tyr Tyr Tyr Glu Ser
Ser Leu Asp Tyr Trp Gly Lys 100 105 110Gly Thr Leu Val Thr Val Ser
Ser 115 120234120PRTArtificial SequenceDescription of Artificial
Sequence Synthetic polypeptide 234Gln Val Gln Leu Gln Gln Ser Gly
Ala Glu Val Lys Lys Pro Gly Ser1 5 10 15Ser Val Lys Val Ser Cys Lys
Thr Ser Gly Gly Thr Phe Ser Ser Tyr 20 25 30Ala Val Thr Trp Val Arg
Gln Ala Pro Gly Gln Gly Leu Glu Trp Met 35 40 45Gly Ala Ile Ile Gly
Met Phe Gly Thr Thr Asn Tyr Ala Gln Lys Phe 50 55 60Gln Gly Arg Val
Thr Leu Thr Ala Asp Glu Leu Thr Ser Thr Ala Tyr65 70 75 80Met Glu
Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95Ala
Arg Gly Ser Tyr Tyr Tyr Glu Ser Ser Leu Asp Tyr Trp Gly Gln 100 105
110Gly Thr Leu Val Thr Val Ser Ser 115 120235120PRTArtificial
SequenceDescription of Artificial Sequence Synthetic polypeptide
235Gln Val Gln Leu Gln Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ser1
5 10 15Ser Val Lys Val Ser Cys Lys Thr Ser Gly Gly Thr Phe Ser Ser
Tyr 20 25 30Ala Val Thr Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu
Trp Met 35 40 45Gly Ala Ile Ile Gly Met Phe Gly Thr Thr Asn Tyr Ala
Gln Lys Phe 50 55 60Gln Gly Arg Val Thr Ile Thr Ala Asp Glu Leu Thr
Ser Thr Ala Tyr65 70 75 80Met Glu Leu Ser Ser Leu Arg Ser Asp Asp
Thr Ala Val Tyr Tyr Cys 85 90 95Ala Arg Gly Ser Tyr Tyr Tyr Glu Ser
Ser Leu Asp Tyr Trp Gly Lys 100 105 110Gly Thr Met Val Thr Val Ser
Ser 115 120236120PRTArtificial SequenceDescription of Artificial
Sequence Synthetic polypeptide 236Gln Val Gln Leu Gln Gln Ser Gly
Ala Glu Val Lys Lys Pro Gly Ser1 5 10 15Ser Val Lys Val Ser Cys Lys
Thr Ser Gly Gly Thr Phe Ser Ser Tyr 20 25 30Ala Val Thr Trp Val Arg
Gln Ala Pro Gly Gln Gly Leu Glu Trp Met 35 40 45Gly Gly Ile Ile Gly
Met Phe Gly Thr Thr Asn Tyr Ala Gln Lys Phe 50 55 60Gln Gly Arg Val
Thr Ile Thr Ala Asp Glu Leu Thr Ser Thr Ala Tyr65 70 75 80Met Glu
Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95Ala
Arg Gly Ser Tyr Tyr Tyr Glu Asn Ser Leu Asp Tyr Trp Gly Lys 100 105
110Gly Thr Leu Val Thr Val Ser Ser 115 120237120PRTArtificial
SequenceDescription of Artificial Sequence Synthetic polypeptide
237Gln Val Gln Leu Gln Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ser1
5 10 15Ser Val Arg Val Ser Cys Lys Thr Ser Gly Gly Thr Phe Ser Ser
Tyr 20 25 30Ala Val Thr Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu
Trp Met 35 40 45Gly Gly Ile Ile Gly Met Phe Gly Thr Thr Asn Tyr Ala
Gln Lys Phe 50 55 60Gln Gly Arg Leu Thr Ile Thr Ala Asp Glu Met Thr
Ser Thr Ala Tyr65 70 75 80Met Glu Leu Ser Ser Leu Arg Ser Glu Asp
Thr Ala Val Tyr Tyr Cys 85 90 95Ala Arg Gly Ser Tyr Tyr Tyr Glu Thr
Thr Leu Asp Tyr Trp Gly Gln 100 105 110Gly Thr Met Val Thr Val Ser
Ser 115 120238120PRTArtificial SequenceDescription of Artificial
Sequence Synthetic polypeptide 238Gln Val Gln Leu Gln Gln Ser Gly
Ala Glu Val Lys Lys Pro Gly Ser1 5 10 15Ser Val Arg Val Ser Cys Lys
Thr Ser Gly Gly Thr Phe Ser Ser Tyr 20 25 30Ala Val Thr Trp Val Arg
Gln Ala Pro Gly Gln Gly Leu Glu Trp Met 35 40 45Gly Gly Ile Ile Gly
Met Phe Gly Thr Thr Asn Tyr Ala Gln Lys Phe 50 55 60Gln Gly Arg Leu
Thr Ile Thr Ala Asp Glu Met Thr Ser Thr Ala Tyr65 70 75 80Met Glu
Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95Ala
Arg Gly Ser Tyr Tyr Tyr Glu Thr Thr Leu Asp Tyr Trp Gly Lys 100 105
110Gly Thr Leu Val Thr Val Ser Ser 115
120239120PRTArtificial SequenceDescription of Artificial Sequence
Synthetic polypeptide 239Gln Val Gln Leu Val Gln Ser Gly Ala Glu
Val Lys Lys Pro Gly Ser1 5 10 15Ser Val Lys Val Ser Cys Lys Thr Ser
Gly Gly Thr Phe Ser Ser Tyr 20 25 30Ala Val Thr Trp Val Arg Gln Ala
Pro Gly Gln Gly Leu Glu Trp Met 35 40 45Gly Gly Ile Ile Gly Met Phe
Gly Thr Thr Asn Tyr Ala Gln Lys Phe 50 55 60Gln Gly Arg Val Thr Met
Thr Ala Asp Glu Met Thr Ser Thr Ala Tyr65 70 75 80Met Glu Leu Ser
Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95Ala Arg Gly
Ser Tyr Tyr Tyr Glu Ser Ser Leu Asp Tyr Trp Gly Gln 100 105 110Gly
Thr Leu Val Thr Val Ser Ser 115 120240120PRTArtificial
SequenceDescription of Artificial Sequence Synthetic polypeptide
240Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ser1
5 10 15Ser Val Lys Val Ser Cys Lys Thr Ser Gly Gly Thr Phe Ser Ser
Tyr 20 25 30Ala Val Thr Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu
Trp Met 35 40 45Gly Gly Ile Ile Gly Met Phe Gly Thr Thr Asn Tyr Ala
Gln Lys Phe 50 55 60Gln Gly Arg Val Thr Met Thr Ala Asp Glu Met Thr
Ser Thr Ala Tyr65 70 75 80Met Glu Leu Ser Ser Leu Arg Ser Glu Asp
Thr Ala Val Tyr Tyr Cys 85 90 95Ala Arg Gly Ser Tyr Tyr Tyr Glu Ser
Ser Leu Asp Tyr Trp Gly Gln 100 105 110Gly Thr Met Val Thr Val Ser
Ser 115 120241120PRTArtificial SequenceDescription of Artificial
Sequence Synthetic polypeptide 241Gln Val Gln Leu Val Gln Ser Gly
Ala Glu Val Lys Lys Pro Gly Ser1 5 10 15Ser Val Lys Val Ser Cys Lys
Thr Ser Gly Gly Thr Phe Ser Ser Tyr 20 25 30Ala Val Thr Trp Val Arg
Gln Ala Pro Gly Gln Gly Leu Glu Trp Met 35 40 45Gly Gly Ile Ile Gly
Met Phe Gly Thr Thr Asn Tyr Ala Gln Lys Phe 50 55 60Gln Gly Arg Val
Thr Ile Thr Ala Asp Glu Met Thr Ser Thr Ala Tyr65 70 75 80Met Glu
Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95Ala
Arg Gly Ser Tyr Tyr Tyr Glu Ser Ser Leu Asp Tyr Trp Gly Gln 100 105
110Gly Thr Leu Val Thr Val Ser Ser 115 120242120PRTArtificial
SequenceDescription of Artificial Sequence Synthetic polypeptide
242Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ser1
5 10 15Ser Val Lys Val Ser Cys Lys Thr Ser Gly Gly Thr Phe Ser Ser
Tyr 20 25 30Ala Val Thr Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu
Trp Met 35 40 45Gly Ala Ile Ile Gly Met Phe Gly Thr Thr Asn Tyr Ala
Gln Lys Phe 50 55 60Gln Gly Arg Val Thr Ile Thr Ala Asp Glu Leu Thr
Ser Thr Ala Tyr65 70 75 80Met Glu Leu Ser Ser Leu Arg Ser Asp Asp
Thr Ala Val Tyr Tyr Cys 85 90 95Ala Arg Gly Ser Tyr Tyr Tyr Glu Ser
Ser Leu Asp Tyr Trp Gly Lys 100 105 110Gly Thr Leu Val Thr Val Ser
Ser 115 120243120PRTArtificial SequenceDescription of Artificial
Sequence Synthetic polypeptide 243Gln Val Gln Leu Val Gln Ser Gly
Ala Glu Val Lys Lys Pro Gly Ser1 5 10 15Ser Val Lys Val Ser Cys Lys
Thr Ser Gly Gly Thr Phe Ser Ser Tyr 20 25 30Ala Val Thr Trp Val Arg
Gln Ala Pro Gly Gln Gly Leu Glu Trp Met 35 40 45Gly Ala Ile Ile Gly
Met Phe Gly Thr Thr Asn Tyr Ala Gln Lys Phe 50 55 60Gln Gly Arg Val
Thr Leu Thr Ala Asp Glu Leu Thr Ser Thr Ala Tyr65 70 75 80Met Glu
Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95Ala
Arg Gly Ser Tyr Tyr Tyr Glu Ser Ser Leu Asp Tyr Trp Gly Gln 100 105
110Gly Thr Pro Val Thr Val Ser Ser 115 120244120PRTArtificial
SequenceDescription of Artificial Sequence Synthetic polypeptide
244Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ser1
5 10 15Ser Val Lys Val Ser Cys Lys Thr Ser Gly Gly Thr Phe Ser Ser
Tyr 20 25 30Ala Val Thr Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu
Trp Met 35 40 45Gly Ala Ile Ile Gly Met Phe Gly Thr Thr Asn Tyr Ala
Gln Lys Phe 50 55 60Gln Gly Arg Val Thr Leu Thr Ala Asp Glu Leu Thr
Ser Thr Ala Tyr65 70 75 80Met Glu Leu Ser Ser Leu Arg Ser Glu Asp
Thr Ala Val Tyr Tyr Cys 85 90 95Ala Arg Gly Ser Tyr Tyr Tyr Glu Ser
Ser Leu Asp Tyr Trp Gly Arg 100 105 110Gly Thr Leu Val Thr Val Ser
Ser 115 120245120PRTArtificial SequenceDescription of Artificial
Sequence Synthetic polypeptide 245Gln Val Gln Leu Val Gln Ser Gly
Ala Glu Val Lys Lys Pro Gly Ser1 5 10 15Ser Val Lys Val Ser Cys Lys
Thr Ser Gly Gly Thr Phe Ser Ser Tyr 20 25 30Ala Val Thr Trp Val Arg
Gln Ala Pro Gly Gln Gly Leu Glu Trp Met 35 40 45Gly Ala Ile Ile Gly
Met Phe Gly Thr Thr Asn Tyr Ala Gln Lys Phe 50 55 60Gln Gly Arg Val
Thr Leu Thr Ala Asp Glu Leu Thr Ser Thr Ala Tyr65 70 75 80Met Glu
Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95Ala
Arg Gly Ser Tyr Tyr Tyr Glu Ser Ser Leu Asp Tyr Trp Gly Arg 100 105
110Gly Thr Met Val Thr Val Ser Ser 115 120246120PRTArtificial
SequenceDescription of Artificial Sequence Synthetic polypeptide
246Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ser1
5 10 15Ser Val Lys Val Ser Cys Lys Thr Ser Gly Gly Thr Phe Ser Ser
Tyr 20 25 30Ala Val Thr Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu
Trp Met 35 40 45Gly Ala Ile Ile Gly Met Phe Gly Thr Thr Asn Tyr Ala
Gln Lys Phe 50 55 60Gln Gly Arg Val Thr Leu Thr Ala Asp Glu Leu Thr
Ser Thr Ala Tyr65 70 75 80Met Glu Leu Ser Ser Leu Arg Ser Glu Asp
Thr Ala Val Tyr Tyr Cys 85 90 95Ala Arg Gly Ser Tyr Tyr Tyr Glu Ser
Ser Leu Asp Tyr Trp Gly Lys 100 105 110Gly Thr Leu Val Thr Val Ser
Ser 115 120247120PRTArtificial SequenceDescription of Artificial
Sequence Synthetic polypeptide 247Gln Val Gln Leu Val Gln Ser Gly
Ala Glu Val Lys Lys Pro Gly Ser1 5 10 15Ser Val Lys Val Ser Cys Lys
Thr Ser Gly Gly Thr Phe Ser Ser Tyr 20 25 30Ala Val Thr Trp Val Arg
Gln Ala Pro Gly Gln Gly Leu Glu Trp Met 35 40 45Gly Gly Ile Ile Gly
Met Phe Gly Thr Thr Asn Tyr Ala Gln Lys Phe 50 55 60Gln Gly Arg Val
Thr Ile Thr Ala Asp Glu Ser Thr Ser Thr Ala Tyr65 70 75 80Met Glu
Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95Ala
Arg Gly Ser Tyr Tyr Tyr Glu Thr Ser Leu Asp Tyr Trp Gly Lys 100 105
110Gly Thr Leu Val Thr Val Ser Ser 115 120248120PRTArtificial
SequenceDescription of Artificial Sequence Synthetic polypeptide
248Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ser1
5 10 15Ser Val Lys Val Ser Cys Lys Thr Thr Gly Gly Thr Phe Ser Ser
Tyr 20 25 30Ala Val Thr Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu
Trp Met 35 40 45Gly Gly Ile Ile Gly Met Phe Gly Thr Thr Asn Tyr Ala
Gln Lys Phe 50 55 60Gln Gly Arg Val Thr Ile Thr Ala Asp Glu Met Thr
Ser Thr Ala Tyr65 70 75 80Met Glu Leu Ser Ser Leu Arg Ser Glu Asp
Thr Ala Val Tyr Tyr Cys 85 90 95Ala Arg Gly Ser Tyr Tyr Tyr Glu Ser
Ser Leu Asp Tyr Trp Gly Arg 100 105 110Gly Thr Met Val Thr Val Ser
Ser 115 120249120PRTArtificial SequenceDescription of Artificial
Sequence Synthetic polypeptide 249Glu Val Gln Leu Val Gln Ser Gly
Ala Glu Val Lys Lys Pro Gly Ala1 5 10 15Ser Val Lys Val Ser Cys Lys
Thr Ser Gly Gly Thr Phe Ser Ser Tyr 20 25 30Ala Val Thr Trp Val Arg
Gln Ala Pro Gly Gln Gly Leu Glu Trp Met 35 40 45Gly Gly Ile Ile Gly
Met Phe Gly Thr Thr Asn Tyr Ala Gln Lys Phe 50 55 60Gln Gly Arg Val
Thr Ile Thr Ala Asp Glu Met Thr Ser Thr Ala Tyr65 70 75 80Met Glu
Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95Ala
Arg Gly Ser Tyr Tyr Tyr Glu Ser Ser Leu Asp Tyr Trp Gly Gln 100 105
110Gly Thr Leu Val Thr Val Ser Ser 115 120250120PRTArtificial
SequenceDescription of Artificial Sequence Synthetic polypeptide
250Glu Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ser1
5 10 15Ser Val Lys Val Ser Cys Lys Thr Ser Gly Gly Thr Phe Ser Ser
Tyr 20 25 30Ala Val Thr Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu
Trp Met 35 40 45Gly Ala Ile Ile Gly Met Phe Gly Thr Thr Asn Tyr Ala
Gln Lys Phe 50 55 60Gln Gly Arg Val Thr Leu Thr Ala Asp Glu Leu Thr
Ser Thr Ala Tyr65 70 75 80Met Glu Leu Ser Ser Leu Arg Ser Glu Asp
Thr Ala Val Tyr Tyr Cys 85 90 95Ala Arg Gly Ser Tyr Tyr Tyr Glu Ser
Ser Leu Asp Tyr Trp Gly Lys 100 105 110Gly Thr Thr Val Thr Val Ser
Ser 115 120251120PRTArtificial SequenceDescription of Artificial
Sequence Synthetic polypeptide 251Glu Val Gln Leu Val Gln Ser Gly
Ala Glu Val Lys Lys Pro Gly Ser1 5 10 15Ser Val Lys Val Ser Cys Lys
Thr Ser Gly Gly Thr Phe Ser Ser Tyr 20 25 30Ala Val Thr Trp Val Arg
Gln Ala Pro Gly Gln Gly Leu Glu Trp Met 35 40 45Gly Gly Ile Ile Gly
Met Phe Gly Thr Thr Asn Tyr Ala Gln Lys Phe 50 55 60Gln Gly Arg Val
Thr Ile Thr Ala Asp Glu Leu Thr Ser Thr Ala Tyr65 70 75 80Met Glu
Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95Ala
Arg Gly Ser Tyr Tyr Tyr Glu Asn Ser Leu Asp Tyr Trp Gly Arg 100 105
110Gly Thr Leu Val Thr Val Ser Ser 115 120252120PRTArtificial
SequenceDescription of Artificial Sequence Synthetic polypeptide
252Glu Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ser1
5 10 15Ser Val Lys Val Ser Cys Lys Thr Thr Gly Gly Thr Phe Ser Ser
Tyr 20 25 30Ala Val Thr Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu
Trp Met 35 40 45Gly Gly Ile Ile Gly Met Phe Gly Thr Thr Asn Tyr Ala
Gln Lys Phe 50 55 60Gln Gly Arg Val Thr Ile Thr Ala Asp Glu Met Thr
Ser Thr Ala Tyr65 70 75 80Met Glu Leu Ser Ser Leu Arg Ser Glu Asp
Thr Ala Val Tyr Tyr Cys 85 90 95Ala Arg Gly Ser Tyr Tyr Tyr Glu Ser
Ser Leu Asp Tyr Trp Gly Arg 100 105 110Gly Thr Met Val Thr Val Ser
Ser 115 120253120PRTArtificial SequenceDescription of Artificial
Sequence Synthetic polypeptide 253Glu Val Gln Leu Val Gln Ser Gly
Ala Glu Val Lys Lys Pro Gly Ser1 5 10 15Ser Val Lys Val Ser Cys Lys
Thr Thr Gly Gly Thr Phe Ser Ser Tyr 20 25 30Ala Val Thr Trp Val Arg
Gln Ala Pro Gly Gln Gly Leu Glu Trp Met 35 40 45Gly Gly Ile Ile Gly
Met Phe Gly Thr Thr Asn Tyr Ala Gln Lys Phe 50 55 60Gln Gly Arg Val
Thr Ile Thr Ala Asp Glu Met Thr Ser Thr Ala Tyr65 70 75 80Met Glu
Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95Ala
Arg Gly Ser Tyr Tyr Tyr Glu Ser Ser Leu Asp Tyr Trp Gly Lys 100 105
110Gly Thr Met Val Thr Val Ser Ser 115 120254120PRTArtificial
SequenceDescription of Artificial Sequence Synthetic polypeptide
254Gln Val Gln Leu Glu Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ser1
5 10 15Ser Val Lys Val Ser Cys Lys Thr Ser Gly Gly Thr Phe Ser Ser
Tyr 20 25 30Ala Val Thr Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu
Trp Met 35 40 45Gly Ala Ile Ile Gly Met Phe Gly Thr Thr Asn Tyr Ala
Gln Lys Phe 50 55 60Gln Gly Arg Val Thr Leu Thr Ala Asp Glu Leu Thr
Ser Thr Ala Tyr65 70 75 80Met Glu Leu Ser Ser Leu Arg Ser Glu Asp
Thr Ala Val Tyr Tyr Cys 85 90 95Ala Arg Gly Ser Tyr Tyr Tyr Glu Ser
Ser Leu Asp Tyr Trp Gly Glu 100 105 110Gly Thr Thr Val Thr Val Ser
Ser 115 120255123PRTArtificial SequenceDescription of Artificial
Sequence Synthetic polypeptide 255Gln Val Gln Leu Gln Glu Ser Gly
Pro Gly Leu Val Lys Pro Ser Glu1 5 10 15Thr Leu Ser Leu Thr Cys Thr
Val Ser Gly Tyr Ser Phe Asp Ser Gly 20 25 30Tyr Tyr Trp Gly Trp Leu
Arg Gln Pro Pro Gly Lys Gly Leu Glu Trp 35 40 45Ile Gly Ser Ile Tyr
His Ser Arg Asn Thr Tyr Tyr Asn Pro Ser Leu 50 55 60Lys Ser Arg Val
Thr Ile Ser Val Asp Thr Ser Lys Asn Gln Phe Ser65 70 75 80Leu Gln
Leu Ser Ser Val Thr Ala Ala Asp Thr Ala Val Tyr Tyr Cys 85 90 95Ala
Arg Gly Thr Trp Tyr Ser Ser Asn Leu Arg Tyr Trp Phe Asp Pro 100 105
110Trp Gly Lys Gly Thr Leu Val Arg Val Ser Ser 115
120256108PRTArtificial SequenceDescription of Artificial Sequence
Synthetic polypeptide 256Phe Met Leu Thr Gln Pro His Ser Val Ser
Glu Ser Pro Gly Lys Thr1 5 10 15Val Thr Ile Ser Cys Thr Gly Ser Gly
Gly Asn Ile Ala Arg Asn Tyr 20 25 30Val Gln Trp Tyr Gln Gln Arg Pro
Gly Ser Ala Pro Val Thr Val Ile 35 40 45Leu Glu Asp Asp Lys Arg Pro
Ser Gly Ile Pro Asp Arg Phe Ser Gly 50 55 60Ser Ile Asp Arg Ser Ser
Asn Ser Ala Ser Leu Thr Ile Ser Gly Leu65 70 75 80Arg Thr Glu Asp
Glu Ala Leu Tyr Tyr Cys Gln Ser Tyr Asp Asp Ser 85 90 95Asp Leu Val
Val Phe Gly Gly Gly Thr Lys Leu Thr 100 105257110PRTArtificial
SequenceDescription of Artificial Sequence Synthetic polypeptide
257Phe Met Leu Thr Gln Pro His Ser Val Ser Glu Ser Pro Gly Lys Thr1
5 10 15Val Thr Ile Ser Cys Thr Gly Ser Ser Gly Ser Ile Ala Ser Asn
Tyr 20 25 30Val Gln Trp Tyr Gln Gln Arg Pro Gly Ser Ala Pro Thr Thr
Val Ile 35 40 45Tyr Glu Asp Tyr Gln Arg Pro Ser Gly Val Pro Asp Arg
Phe Ser Gly 50 55 60Ser Ile Asp Ser Ser Ser Asn Ser Ala Ser Leu Thr
Ile Ser Gly Leu65 70
75 80Lys Thr Glu Asp Glu Ala Asp Tyr Tyr Cys Gln Ser Tyr Asp Asp
Ser 85 90 95Asp His Leu Ile Phe Gly Gly Gly Thr Lys Leu Thr Val Leu
100 105 110258109PRTArtificial SequenceDescription of Artificial
Sequence Synthetic polypeptide 258Ser Val Leu Thr Gln Pro Pro Ser
Ala Ser Gly Thr Pro Gly Gln Arg1 5 10 15Val Thr Ile Ser Cys Ser Gly
Ser Ser Ser Asn Ile Gly Ser Asn Thr 20 25 30Val Asn Trp Tyr Lys Gln
Leu Pro Gly Thr Ala Pro Arg Leu Leu Ile 35 40 45Tyr Ser Asn Asp Gln
Arg Pro Ser Gly Val Pro Asp Arg Phe Ser Gly 50 55 60Ser Lys Ser Gly
Thr Ser Ala Ser Leu Ala Ile Ser Gly Leu Gln Ser65 70 75 80Glu Asp
Glu Ala Asn Tyr Tyr Cys Ala Ala Trp Asp Asp Ser Leu Ser 85 90 95Gly
Trp Val Phe Gly Gly Gly Thr Lys Leu Thr Val Leu 100
105259109PRTArtificial SequenceDescription of Artificial Sequence
Synthetic polypeptide 259Ser Val Leu Thr Gln Pro Pro Ser Ala Ser
Gly Thr Pro Gly Gln Arg1 5 10 15Val Thr Leu Ser Cys Ser Gly Ser Ser
Ser Asn Ile Gly Gly Asn Ser 20 25 30Val Asn Trp Tyr Gln His Val Pro
Gly Thr Ala Pro Lys Leu Leu Met 35 40 45His Ser Asp Asp Gln Arg Pro
Ser Gly Val Pro Asp Arg Phe Ser Gly 50 55 60Ser Lys Ser Gly Thr Ser
Xaa Ser Leu Ala Ile Ser Gly Leu Gln Ser65 70 75 80Glu Asp Glu Ala
Asp Tyr Tyr Cys Ala Xaa Trp Asp Asp Ser Leu Asn 85 90 95Ala Trp Val
Phe Gly Gly Xaa Thr Lys Val Thr Val Leu 100 105260109PRTArtificial
SequenceDescription of Artificial Sequence Synthetic polypeptide
260Pro Glu Leu Thr Gln Pro His Ser Val Ser Glu Ser Pro Gly Lys Thr1
5 10 15Val Thr Ile Ser Cys Thr Gly Ser Gly Gly Arg Ile Ala Thr Asn
His 20 25 30Val Gln Trp Tyr Gln Gln Arg Pro Gly Ser Ala Pro Thr Ile
Val Ile 35 40 45Tyr Glu Asn Asn Gln Arg Pro Ser Gly Val Pro Asn Arg
Phe Ser Gly 50 55 60Ser Ile Asp Asp Ser Ser Asn Ser Ala Ser Leu Thr
Ile Ser Ala Leu65 70 75 80Arg Thr Glu Asp Glu Ala Asp Tyr Tyr Cys
Gln Ser Ala Asp Ala Thr 85 90 95Asn Val Phe Phe Gly Gly Gly Thr Lys
Val Thr Val Leu 100 105261109PRTArtificial SequenceDescription of
Artificial Sequence Synthetic polypeptide 261Pro Glu Leu Thr Gln
Pro Pro Ser Ala Ser Gly Thr Pro Gly Gln Arg1 5 10 15Val Thr Ile Ser
Cys Ser Gly Ser Ser Ser Asn Ile Gly Ser Asn Thr 20 25 30Val Asn Trp
Tyr Gln Gln Leu Pro Gly Thr Ala Pro Lys Leu Leu Ile 35 40 45Tyr Ser
Asn Asn Gln Arg Pro Ser Gly Val Pro Asp Arg Phe Ser Gly 50 55 60Ser
Lys Ser Gly Thr Ser Ala Ser Leu Ala Ile Ser Gly Leu Gln Ser65 70 75
80Glu Asp Glu Ala Asp Tyr Tyr Cys Ala Ala Trp Asp Asp Ser Leu Asn
85 90 95Gly Trp Val Phe Gly Gly Xaa Thr Lys Leu Thr Val Leu 100
105262108PRTArtificial SequenceDescription of Artificial Sequence
Synthetic polypeptide 262Asp Ile Gln Met Thr Gln Ser Pro Ser Ser
Leu Ser Ala Phe Val Gly1 5 10 15Asp Arg Val Thr Ile Thr Cys Gln Ala
Ser Gln Asp Ile Ser Asn Tyr 20 25 30Leu Asn Trp Tyr Gln Gln Lys Pro
Gly Lys Ala Pro Lys Leu Leu Ile 35 40 45Tyr Asp Ala Thr Asn Leu Glu
Thr Gly Val Pro Ser Arg Phe Ser Gly 50 55 60Ser Gly Ser Gly Thr Asp
Phe Thr Phe Thr Ile Ser Ser Leu Gln Pro65 70 75 80Glu Asp Ile Ala
Thr Tyr Tyr Cys Gln Gln Tyr Asp Asn Leu Pro Leu 85 90 95Thr Phe Gly
Gly Gly Thr Lys Val Asp Ile Lys Arg 100 10526345DNAArtificial
SequenceDescription of Artificial Sequence Synthetic
oligonucleotide 263tgg atg gga agg atc atc cct atc ctt ggt ata gca
aac tac gca 45Trp Met Gly Arg Ile Ile Pro Ile Leu Gly Ile Ala Asn
Tyr Ala1 5 10 1526415PRTArtificial SequenceDescription of
Artificial Sequence Synthetic peptide 264Trp Met Gly Arg Ile Ile
Pro Ile Leu Gly Ile Ala Asn Tyr Ala1 5 10 1526545DNAArtificial
SequenceDescription of Artificial Sequence Synthetic
oligonucleotide 265tgg atg ggc gcg atc atc ggt atg ttt ggt aca aca
aac tac gca 45Trp Met Gly Ala Ile Ile Gly Met Phe Gly Thr Thr Asn
Tyr Ala1 5 10 1526615PRTArtificial SequenceDescription of
Artificial Sequence Synthetic peptide 266Trp Met Gly Ala Ile Ile
Gly Met Phe Gly Thr Thr Asn Tyr Ala1 5 10 1526745DNAArtificial
SequenceDescription of Artificial Sequence Synthetic
oligonucleotide 267tgg atg gga ggg atc atc ggt atg ttt gga aca acc
aac tat gca 45Trp Met Gly Gly Ile Ile Gly Met Phe Gly Thr Thr Asn
Tyr Ala1 5 10 1526815PRTArtificial SequenceDescription of
Artificial Sequence Synthetic peptide 268Trp Met Gly Gly Ile Ile
Gly Met Phe Gly Thr Thr Asn Tyr Ala1 5 10 1526945DNAArtificial
SequenceDescription of Artificial Sequence Synthetic
oligonucleotide 269tgg atg gga gcg atc atc ggt atg ttt ggt aca aca
aac tac gca 45Trp Met Gly Ala Ile Ile Gly Met Phe Gly Thr Thr Asn
Tyr Ala1 5 10 1527015PRTArtificial SequenceDescription of
Artificial Sequence Synthetic peptide 270Trp Met Gly Ala Ile Ile
Gly Met Phe Gly Thr Thr Asn Tyr Ala1 5 10 1527145DNAArtificial
SequenceDescription of Artificial Sequence Synthetic
oligonucleotide 271tgg atg ggc gcg atc atc ggt atg ttt ggt aca aca
aac tac gca 45Trp Met Gly Ala Ile Ile Gly Met Phe Gly Thr Thr Asn
Tyr Ala1 5 10 1527215PRTArtificial SequenceDescription of
Artificial Sequence Synthetic peptide 272Trp Met Gly Ala Ile Ile
Gly Met Phe Gly Thr Thr Asn Tyr Ala1 5 10 1527345DNAArtificial
SequenceDescription of Artificial Sequence Synthetic
oligonucleotide 273tgg atg gga ggg atc atc ggt atg ttt ggt aca acg
aac tat gca 45Trp Met Gly Gly Ile Ile Gly Met Phe Gly Thr Thr Asn
Tyr Ala1 5 10 1527415PRTArtificial SequenceDescription of
Artificial Sequence Synthetic peptide 274Trp Met Gly Gly Ile Ile
Gly Met Phe Gly Thr Thr Asn Tyr Ala1 5 10 1527545DNAArtificial
SequenceDescription of Artificial Sequence Synthetic
oligonucleotide 275tgg atg gga ggg atc atc ggt atg ttt ggt aca aca
aac tac gca 45Trp Met Gly Gly Ile Ile Gly Met Phe Gly Thr Thr Asn
Tyr Ala1 5 10 1527615PRTArtificial SequenceDescription of
Artificial Sequence Synthetic peptide 276Trp Met Gly Gly Ile Ile
Gly Met Phe Gly Thr Thr Asn Tyr Ala1 5 10 1527736DNAArtificial
SequenceDescription of Artificial Sequence Synthetic
oligonucleotide 277gtc acg att acc gcg gac aaa tcc acg agc aca gcc
36Val Thr Ile Thr Ala Asp Lys Ser Thr Ser Thr Ala1 5
1027812PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 278Val Thr Ile Thr Ala Asp Lys Ser Thr Ser Thr
Ala1 5 1027936DNAArtificial SequenceDescription of Artificial
Sequence Synthetic oligonucleotide 279gtc acg ctt acc gcg gac gaa
tta acg tcc aca gcc 36Val Thr Leu Thr Ala Asp Glu Leu Thr Ser Thr
Ala1 5 1028012PRTArtificial SequenceDescription of Artificial
Sequence Synthetic peptide 280Val Thr Leu Thr Ala Asp Glu Leu Thr
Ser Thr Ala1 5 1028136DNAArtificial SequenceDescription of
Artificial Sequence Synthetic oligonucleotide 281ctc aca atc acc
gcg gac gag atg acg tcc aca gcc 36Leu Thr Ile Thr Ala Asp Glu Met
Thr Ser Thr Ala1 5 1028212PRTArtificial SequenceDescription of
Artificial Sequence Synthetic peptide 282Leu Thr Ile Thr Ala Asp
Glu Met Thr Ser Thr Ala1 5 1028336DNAArtificial SequenceDescription
of Artificial Sequence Synthetic oligonucleotide 283gtc aca atc acc
gcg gac gaa tta acg tcc aca gcc 36Val Thr Ile Thr Ala Asp Glu Leu
Thr Ser Thr Ala1 5 1028412PRTArtificial SequenceDescription of
Artificial Sequence Synthetic peptide 284Val Thr Ile Thr Ala Asp
Glu Leu Thr Ser Thr Ala1 5 1028536DNAArtificial SequenceDescription
of Artificial Sequence Synthetic oligonucleotide 285gtc acg ctt acc
gcg gac gaa tta acg tcc aca gcc 36Val Thr Leu Thr Ala Asp Glu Leu
Thr Ser Thr Ala1 5 1028612PRTArtificial SequenceDescription of
Artificial Sequence Synthetic peptide 286Val Thr Leu Thr Ala Asp
Glu Leu Thr Ser Thr Ala1 5 1028736DNAArtificial SequenceDescription
of Artificial Sequence Synthetic oligonucleotide 287gtc acg att acc
gcg gac gag atg acg tcc aca gcc 36Val Thr Ile Thr Ala Asp Glu Met
Thr Ser Thr Ala1 5 1028812PRTArtificial SequenceDescription of
Artificial Sequence Synthetic peptide 288Val Thr Ile Thr Ala Asp
Glu Met Thr Ser Thr Ala1 5 1028936DNAArtificial SequenceDescription
of Artificial Sequence Synthetic oligonucleotide 289gtc acg att acc
gcg gac gaa tta acg tcc aca gcc 36Val Thr Ile Thr Ala Asp Glu Leu
Thr Ser Thr Ala1 5 1029012PRTArtificial SequenceDescription of
Artificial Sequence Synthetic peptide 290Val Thr Ile Thr Ala Asp
Glu Leu Thr Ser Thr Ala1 5 1029139DNAArtificial SequenceDescription
of Artificial Sequence Synthetic oligonucleotide 291acn aar gcn tcn
tay ctn agy acn agy agy agy ctn gay 39Thr Lys Ala Ser Tyr Leu Ser
Thr Ser Ser Ser Leu Asp1 5 1029213PRTArtificial SequenceDescription
of Artificial Sequence Synthetic peptide 292Thr Lys Ala Ser Tyr Leu
Ser Thr Ser Ser Ser Leu Asp1 5 1029339DNAArtificial
SequenceDescription of Artificial Sequence Synthetic
oligonucleotide 293gcn cgn ggn ath tay tty tay ggn acn acn tay tty
gay 39Ala Arg Gly Ile Tyr Phe Tyr Gly Thr Thr Tyr Phe Asp1 5
1029413PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 294Ala Arg Gly Ile Tyr Phe Tyr Gly Thr Thr Tyr
Phe Asp1 5 1029539DNAArtificial SequenceDescription of Artificial
Sequence Synthetic oligonucleotide 295rcn mrn gsn wyn tay ytn wry
rsn asn asn wry ytn gay 39Xaa Xaa Xaa Xaa Tyr Xaa Xaa Xaa Xaa Xaa
Xaa Xaa Asp1 5 1029613PRTArtificial SequenceDescription of
Artificial Sequence Synthetic peptide 296Xaa Xaa Xaa Xaa Tyr Xaa
Xaa Xaa Xaa Xaa Xaa Xaa Asp1 5 10
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References