U.S. patent application number 17/507482 was filed with the patent office on 2022-02-03 for neutralizing anti-influenza a antibodies and uses thereof.
The applicant listed for this patent is HUMABS BIOMED SA, MEDIMMUNE, LLC. Invention is credited to Ebony Benjamin, Davide Corti, Anna DeMarco, Barbara Guarino, Nicole Kallewaard-LeLay, Antonio Lanzavecchia, Josephine Mary McAuliffe, Frances Palmer-Hill, Leslie Wachter, Andy Yuan, Qing Zhu.
Application Number | 20220033480 17/507482 |
Document ID | / |
Family ID | |
Filed Date | 2022-02-03 |
United States Patent
Application |
20220033480 |
Kind Code |
A1 |
Benjamin; Ebony ; et
al. |
February 3, 2022 |
NEUTRALIZING ANTI-INFLUENZA A ANTIBODIES AND USES THEREOF
Abstract
The invention relates to antibodies and binding fragments
thereof that are capable of binding to influenza A virus
hemagglutinin and neutralizing at least one group 1 subtype and at
least 1 group 2 subtype of influenza A virus.
Inventors: |
Benjamin; Ebony;
(Gaithersburg, MD) ; Kallewaard-LeLay; Nicole;
(Gaithersburg, MD) ; McAuliffe; Josephine Mary;
(Gaithersburg, MD) ; Palmer-Hill; Frances;
(Gaithersburg, MD) ; Wachter; Leslie;
(Gaithersburg, MD) ; Yuan; Andy; (Gaithersburg,
MD) ; Zhu; Qing; (Gaithersburg, MD) ; Corti;
Davide; (Bellinzona, CH) ; Lanzavecchia; Antonio;
(Bellinzona, CH) ; Guarino; Barbara; (Bellinzona,
CH) ; DeMarco; Anna; (Bellinzona, CH) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
MEDIMMUNE, LLC
HUMABS BIOMED SA |
Gaithersburg
Bellinzona |
MD |
US
CH |
|
|
Appl. No.: |
17/507482 |
Filed: |
October 21, 2021 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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16596039 |
Oct 8, 2019 |
11186627 |
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17507482 |
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15026276 |
Mar 31, 2016 |
10494419 |
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PCT/US14/58652 |
Oct 1, 2014 |
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16596039 |
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61885808 |
Oct 2, 2013 |
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62002414 |
May 23, 2014 |
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International
Class: |
C07K 16/10 20060101
C07K016/10; A61K 31/13 20060101 A61K031/13; A61K 39/395 20060101
A61K039/395; A61K 31/7012 20060101 A61K031/7012; A61K 31/215
20060101 A61K031/215; A61K 39/42 20060101 A61K039/42; A61K 45/06
20060101 A61K045/06; C12P 21/00 20060101 C12P021/00; G01N 33/569
20060101 G01N033/569; G01N 33/577 20060101 G01N033/577 |
Claims
1. An isolated antibody or a binding fragment thereof that is
capable of binding to influenza A virus hemagglutinin and
neutralizing at least one group 1 subtype and at least 1 group 2
subtype of influenza A virus.
2. An antibody or binding fragment according to claim 1, wherein
the antibody or binding fragment is capable of neutralizing one or
more influenza A virus group 1 subtype selected from: H1, H2, H5,
H6, H8, H9, H11, H12, H13, H16 and variants thereof; and one or
more influenza A virus group 2 subtypes selected from: H3, H4, H7,
H10, H14 and H15 and variants thereof.
3. An antibody or binding fragment thereof according to any one of
the preceding claims, wherein the antibody or binding fragment is
capable of neutralizing group 1 subtypes: H1, H2, H5, H6 and H9 and
group 2 subtypes H3 and H7; or wherein the antibody or binding
fragment is capable of neutralizing group 1 subtypes: H1, H2, H5
and H6 and group 2 subtypes H3 and H7.
4. An antibody or binding fragment thereof according to any one of
the preceding claims, wherein the antibody or binding fragment has
high neutralizing potency expressed as 50% inhibitory concentration
(IC.sub.50 ug/ml) in the range of from about 0.01 ug/ml to about 50
ug/ml of antibody for neutralization of influenza A virus in a
microneutralization assay.
5. An antibody or binding fragment thereof according to according
to any one of the preceding claims, wherein the antibody or
fragment thereof includes a set of six CDRs: HCDR1, HCDR2, HCDR3,
LCDR1, LCDR2, LCDR3 in which the set of six CDRs is selected from
the group consisting of: (a) HCDR1 of SEQ ID NO.: 3, HCDR2 of SEQ
ID NO.: 4, HCDR3 of SEQ ID NO.: 5, LCDR1 of SEQ ID NO.: 8, LCDR2 of
SEQ ID NO.: 9 and LCDR3 of SEQ ID NO.: 10; (b) HCDR1 of SEQ ID NO.:
13, HCDR2 of SEQ ID NO.: 14, HCDR3 of SEQ ID NO.: 15, LCDR1 of SEQ
ID NO.: 18, LCDR2 of SEQ ID NO.: 19, LCDR3 of SEQ ID NO.: 20; (c)
HCDR1 of SEQ ID NO.: 23, HCDR2 of SEQ ID NO.: 24, HCDR3 of SEQ ID
NO.: 25, LCDR1 of SEQ ID NO.: 28, LCDR2 of SEQ ID NO.: 29 and LCDR3
of SEQ ID NO.: 30; (d) HCDR1 of SEQ ID NO.: 33, HCDR2 of SEQ ID
NO.: 34, HCDR3 of SEQ ID NO.: 35, LCDR1 of SEQ ID NO.: 38, LCDR2 of
SEQ ID NO.: 39 and LCDR3 of SEQ ID NO.: 40; (e) HCDR1 of SEQ ID
NO.: 43, HCDR2 of SEQ ID NO.: 44, HCDR3 of SEQ ID NO.: 45, LCDR1 of
SEQ ID NO.: 48, LCDR2 of SEQ ID NO.: 49 and LCDR3 of SEQ ID NO.:
50; (f) HCDR1 of SEQ ID NO.: 53, HCDR2 of SEQ ID NO.: 54, HCDR3 of
SEQ ID NO.: 55, LCDR1 of SEQ ID NO.: 58, LCDR2 of SEQ ID NO.: 59
and LCDR3 of SEQ ID NO.: 60; (g) HCDR1 of SEQ ID NO.: 63, HCDR2 of
SEQ ID NO.: 64, HCDR3 of SEQ ID NO.: 65, LCDR1 of SEQ ID NO.: 68,
LCDR2 of SEQ ID NO.: 69 and LCDR3 of SEQ ID NO.: 70; (h) HCDR1 of
SEQ ID NO.: 73, HCDR2 of SEQ ID NO.: 74, HCDR3 of SEQ ID NO.: 75,
LCDR1 of SEQ ID NO.: 78, LCDR2 of SEQ ID NO.: 79 and LCDR3 of SEQ
ID NO.: 80; (i) HCDR1 of SEQ ID NO.: 83, HCDR2 of SEQ ID NO.: 84,
HCDR3 of SEQ ID NO.: 85, LCDR1 of SEQ ID NO.: 88, LCDR2 of SEQ ID
NO.: 89, LCDR3 of SEQ ID NO.: 90; (j) HCDR1 of SEQ ID NO.: 93,
HCDR2 of SEQ ID NO.: 94, HCDR3 of SEQ ID NO.: 95, LCDR1 of SEQ ID
NO.: 98, LCDR2 of SEQ ID NO.: 99 and LCDR3 of SEQ ID NO.: 100; (k)
HCDR1 of SEQ ID NO.: 103, HCDR2 of SEQ ID NO.: 104, HCDR3 of SEQ ID
NO.: 105, LCDR1 of SEQ ID NO.: 108, LCDR2 of SEQ ID NO.: 109 and
LCDR3 of SEQ ID NO.: 110; (l) HCDR1 of SEQ ID NO.: 113, HCDR2 of
SEQ ID NO.: 114, HCDR3 of SEQ ID NO.: 115, LCDR1 of SEQ ID NO.:
118, LCDR2 of SEQ ID NO.: 119 and LCDR3 of SEQ ID NO.: 110; (m)
HCDR1 of SEQ ID NO.: 123, HCDR2 of SEQ ID NO.: 124, HCDR3 of SEQ ID
NO.: 125, LCDR1 of SEQ ID NO.: 128, LCDR2 of SEQ ID NO.: 129 and
LCDR3 of SEQ ID NO.: 130; (n) HCDR1 of SEQ ID NO.: 133, HCDR2 of
SEQ ID NO.: 134, HCDR3 of SEQ ID NO.: 135, LCDR1 of SEQ ID NO.:
138, LCDR2 of SEQ ID NO.: 139 and LCDR3 of SEQ ID NO.: 140; and (o)
HCDR1 of SEQ ID NO.: 143, HCDR2 of SEQ ID NO.: 144, HCDR3 of SEQ ID
NO.: 145, LCDR1 of SEQ ID NO.: 148, LCDR2 of SEQ ID NO.: 149 and
LCDR3 of SEQ ID NO.: 150 (p) a set of six CDRS according to any one
of (a) to (o) comprising one or more amino acid substitutions,
deletions or insertions; (q) a set of six CDRS according to any one
of (a) to (p) comprising 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13,
14, 15, 16, 17, 18, 19, 20, 21, 22, 23, or 24 or 25 amino acid
substitutions; (r) a set of six CDRs HCDR1, HCDR2, HCDR3, LCDR1,
LCDR2, LCDR3 according to any one of (a) to (q) comprising: (i) a
HCDR1 having an amino acid sequence identical to or comprising 3 or
fewer amino acid residue substitutions relative to SEQ ID NO: 3;
(ii) a HCDR2 having an amino acid sequence identical to or
comprising 5 or fewer amino acid residue substitutions relative to
SEQ ID NO:4; (iii) a HCDR3 having an amino acid sequence identical
to or comprising 6 or fewer amino acid residue substitutions
relative to SEQ ID NO:5; (iv) a LCDR1 having an amino acid sequence
identical to or comprising 5 or fewer amino acid residue
substitutions and/or one deletion relative to SEQ ID NO:6; (v) a
LCDR2 having an amino acid sequence identical to or comprising 5 or
fewer amino acid residue substitutions relative to SEQ ID NO:7; and
(vi) a LCDR3 having an amino acid sequence identical to or
comprising 1 or fewer amino acid residue substitutions relative to
SEQ ID NO:8; (s) a set of six CDRs HCDR1, HCDR2, HCDR3, LCDR1,
LCDR2, LCDR3 according to any one of (a) to (r) comprising: (i) a
HCDR1 in which: Kabat residue 31 is S, Kabat residue 32 is N or Y,
Kabat residue 33 is N, S, or R, Kabat residue 34 is A, Kabat
residue 35 is V or T, Kabat residue 35A is W Kabat residue 35B is
N; (ii) a HCDR2 in which: Kabat residue 50 is R, Kabat residue 51
is T, Kabat residue 52 is Y, Kabat residue 52A is Y, Kabat residue
53 is R, Kabat residue 54 is S, Kabat residue 55 is K or G, Kabat
residue 56 is W, Kabat residue 57 is Y, Kabat residue 58 is N or Y,
Kabat residue 59 is D, Kabat residue 60 is Y, Kabat residue 61 is
A, Kabat residue 62 is E, V or d, Kabat residue 63 is S or F, Kabat
residue 64 is V or L, Kabat residue 65 is K; (iii) a HCDR3 in
which: Kabat residue 95 is S or G, Kabat residue 96 is G, Kabat
residue 97 is H, Kabat residue 98 is I, Kabat residue 99 is T,
Kabat residue 100 is V or E, Kabat residue 100A is F, Kabat residue
1006 is G, Kabat residue 100C is V or L, Kabat residue 100D is N,
Kabat residue 100E is V or I, Kabat residue 100F is D, Kabat
residue 1000 is A, Kabat residue 100F is F or Y, Kabat residue 101
is D, Kabat residue 102 is M, I or V; (iv) a LCDR1 in which: Kabat
residue 24 is R, Kabat residue 25 is T, A or absent, Kabat residue
26 is S or A, Kabat residue 27 is Q, Kabat residue 28 is S or R,
Kabat residue 29 is L, Kabat residue 30 is S, N or R Kabat residue
31 is S, Kabat residue 32 is Y, Kabat residue 33 is L, T or D,
Kabat residue 34 is H; (v) a LCDR2 in which: Kabat residue 50 is A,
Kabat residue 51 is A, T or S, Kabat residue 52 is S or T, Kabat
residue 53 is S or T, Kabat residue 54 is L or R, Kabat residue 55
is Q, L or G, Kabat residue 56 is S, and, (vi) a LCDR3 in which:
Kabat residue 89 is Q, Kabat residue 90 is Q or L, Kabat residue 91
is S, Kabat residue 92 is R, and Kabat residue 93 is T.
6. An antibody or binding fragment thereof according to any one of
the preceding claims comprising a VH having at least 75% identity
and/or a VL having at least 75% identity to a VH and/or VL selected
from the group consisting of: (a) VH of SEQ ID NO.: 2 and VL of SEQ
ID NO.: 7, (b) VH of SEQ ID NO.: 12 and VL of SEQ ID NO.: 17, (c)
VH of SEQ ID NO.: 22 and VL of SEQ ID NO.: 27, (d) VH of SEQ ID
NO.: 32 and VL of SEQ ID NO.: 37, (e) VH of SEQ ID NO.: 42 and VL
of SEQ ID NO.: 47, (f) VH of SEQ ID NO.: 52 and VL of SEQ ID NO.:
57, (g) VH of SEQ ID NO.: 62 and VL of SEQ ID NO.: 67, (h) VH of
SEQ ID NO.: 72 and VL of SEQ ID NO.: 77, (i) VH of SEQ ID NO.: 82
and VL of SEQ ID NO.: 87, (j) VH of SEQ ID NO.: 92 and VL of SEQ ID
NO.: 97, (k) VH of SEQ ID NO.: 102 and VL of SEQ ID NO.: 107, (l)
VH of SEQ ID NO.: 112 and VL of SEQ ID NO.: 117, (m) VH of SEQ ID
NO.: 122 and VL of SEQ ID NO.: 127, (n) VH of SEQ ID NO.: 132 and
VL of SEQ ID NO.: 137, (o) VH of SEQ ID NO.: 144 and VL of SEQ ID
NO.: 147 and (p) VH of SEQ ID NO: 152 and VL of SEQ ID NO: 157.
7. An antibody or binding fragment thereof according to any one of
the preceding claims comprising a VH and a VL selected from the
group consisting of: (a) VH of SEQ ID NO.: 2 and VL of SEQ ID NO.:
7, (b) VH of SEQ ID NO.: 12 and VL of SEQ ID NO.: 17, (c) VH of SEQ
ID NO.: 22 and VL of SEQ ID NO.: 27, (d) VH of SEQ ID NO.: 32 and
VL of SEQ ID NO.: 37, (e) VH of SEQ ID NO.: 42 and VL of SEQ ID
NO.: 47, (f) VH of SEQ ID NO.: 52 and VL of SEQ ID NO.: 57, (g) VH
of SEQ ID NO.: 62 and VL of SEQ ID NO.: 67, (h) VH of SEQ ID NO.:
72 and VL of SEQ ID NO.: 77, (i) VH of SEQ ID NO.: 82 and VL of SEQ
ID NO.: 87, (j) VH of SEQ ID NO.: 92 and VL of SEQ ID NO.: 97, (k)
VH of SEQ ID NO.: 102 and VL of SEQ ID NO.: 107, (l) VH of SEQ ID
NO.: 112 and VL of SEQ ID NO.: 117, (m) VH of SEQ ID NO.: 122 and
VL of SEQ ID NO.: 127, (n) VH of SEQ ID NO.: 132 and VL of SEQ ID
NO.: 137, (o) VH of SEQ ID NO.: 144 and VL of SEQ ID NO.: 147 and
(p) VH of SEQ ID NO: 152 and VL of SEQ ID NO: 157.
8. An antibody or binding fragment thereof according to any one of
the preceding claims, wherein the antibody or binding fragment is
selected from the group consisting of: an immunoglobulin molecule,
a monoclonal antibody, a chimeric antibody, a CDR-grafted antibody,
a humanized antibody, a Fab, a Fab', a F(ab')2, a Fv, a disulfide
linked Fv, a scFv, a single domain antibody, a diabody, a
multispecific antibody, a dual-specific antibody, and a bispecific
antibody.
9. An antibody or binding fragment thereof according to any one of
the preceding claims, wherein the VH comprises human germline
framework VH6-1, the VL comprises human germline framework VK1-39,
and combination thereof.
10. An antibody or binding fragment thereof according to any one of
the preceding claims, comprising an Fc region.
11. An antibody or binding fragment thereof according to claim any
one of the preceding claims, wherein the antibody is an IgG1, IgG2
or IgG4 or fragment thereof.
12. An antibody to influenza A virus or a binding fragment thereof
that is capable of binding to influenza A virus hemagglutinin and
neutralizing at least one group 1 subtype and at least one group 2
subtype of influenza A virus, wherein the antibody or binding
fragment thereof binds an epitope that is conserved among one or
more influenza A virus group 1 subtypes selected from H1, H2, H5,
H6, H8, H9, H11, H12, H13 and H16 and one or more group 2 subtypes
selected from H3, H4, H7, H10, H14 and H15.
13. An antibody to influenza A virus or a binding fragment thereof
that is capable of binding to influenza A virus hemaglutinin and
neutralizing at least one group 1 subtype and at least one group 2
subtype of influenza A virus, wherein the antibody or binding
fragment thereof binds an epitope that is located in a conserved
stalk region of HA2.
14. The antibody or binding fragment thereof according to claim 12,
wherein the epitope includes one or more amino acids selected from:
positions 18, 19, 42 and 45 of HA2 according to a H3 numbering
system
15. An antibody to influenza A virus or a binding fragment thereof
that is capable of binding to influenza A virus hemagglutinin and
neutralizing at least one group 1 subtype and at least 1 group 2
subtype of influenza A virus that binds to the same epitope as or
competes for binding to influenza A virus hemagglutinin with an
antibody according to any one of the preceding claims.
16. The antibody or binding fragment thereof according to claim 14,
wherein the antibody or binding fragment binds to the same epitope
or competes for binding to influenza A virus hemagglutinin with an
antibody having an amino acid sequence shown in SEQ ID NO: 112.
17. An isolated nucleic acid encoding an antibody or binding
fragment thereof according to any one of claims 1 to 16.
18. A vector comprising an isolated nucleic acid according to claim
17.
19. A host cell comprising a nucleic acid according to claim 17 or
a vector according to claim 18.
20. A method for manufacturing an antibody or binding fragment
thereof according to any one of claims 1 to 16 comprising culturing
a host cell according to claim 19 under conditions suitable for
expression of the antibody or fragment thereof.
21. A method according to claim 20, further comprising isolating
the antibody or binding fragment thereof from the host cell
culture.
22. A composition comprising an antibody or binding fragment
thereof according to any one of claims 1 to 16 and a
pharmaceutically acceptable carrier.
23. A composition comprising an antibody or binding fragment
thereof according to any one of claims 1 to 16, 25 mM His and 0.15M
NaCl at pH 6.0
24. An antibody or binding fragment thereof according to any one of
claims 1 to 16 for use in the prophylaxis or treatment of Influenza
A infection in a subject.
25. The use of an antibody or binding fragment thereof according to
any one of claims 1 to 16 in the manufacture of a medicament for
the prophylaxis or treatment of Influenza A infection in a
subject.
26. A method for prophylaxis or treatment of Influenza A infection
in a subject comprising administering an effective amount of an
antibody or binding fragment thereof according to any one of claims
1 to 16 to the subject.
27. A method for prophylaxis or treatment of Influenza A infection
in a subject comprising administering an effective amount of an
antibody or binding fragment thereof according to any one of claims
1 to 16 in combination with a small molecule antiviral
composition.
28. The method according to claim 27, wherein the small molecule
antiviral composition is a neuramidase inhibitor or an
adamantane.
29. The method according to claim 27, wherein the small molecule
antiviral composition is selected from oseltamivir, zanamivir,
amantadine, rimantadine, and combinations thereof.
30. The use of an antibody or fragment thereof according to any one
of claims 1 to 16 for in vitro diagnosis of Influenza A infection
in a subject.
Description
CROSS-REFERENCE TO RELATED APPLICATIONS
[0001] This application is a Divisional application of U.S.
application Ser. No. 16/596,039, filed Oct. 8, 2019, which is a
Divisional application of U.S. application Ser. No. 15/026,276,
filed Mar. 31, 2016 and issued as U.S. Pat. No. 10,494,419, which
is a U.S. National Stage application of International Application
No. PCT/US2014/058652 filed on Oct. 1, 2014, said International
Application No. PCT/US2014/058652 claims benefit under 35 U.S.C.
.sctn. 119(e) of the U.S. Provisional Application Nos. 61/885,808,
filed Oct. 2, 2013 and 62/002,414, filed May 23, 2014. Each of the
above listed applications is incorporated by reference herein in
its entirety for all purposes.
REFERENCE TO THE SEQUENCE LISTING
[0002] This application incorporates by reference a Sequence
Listing submitted with this application as text file entitled
0098_0030 US3_SL, created on Oct. 20, 2021, and having a size of
92.0 kilobytes.
FIELD OF THE INVENTION
[0003] The invention relates to antibodies that have broad
neutralizing activity against influenza A virus and to uses of such
antibodies.
BACKGROUND TO THE INVENTION
[0004] Influenza viruses cause annual influenza epidemics and
occasional pandemics, which pose a significant threat to public
health worldwide. Seasonal influenza infection is associated with
200,000-500,000 deaths each year, particularly in young children,
immunocompromised patients and the elderly. Mortality rates
typically increase further during seasons with pandemic influenza
outbreaks. There remains a significant unmet medical need to
develop potent anti-viral therapeutics for preventing and treating
influenza infections, particularly in under-served populations.
[0005] There are three types of influenza viruses, types A, B and
C. Influenza A viruses can infect a wide variety of birds and
mammals, including humans, pigs, chickens and ferrets. Influenza A
viruses can be classified into subtypes based on allelic variations
in antigenic regions of two genes that encode surface glycoproteins
hemagglutinin (HA) and neuraminidase (NA). HA is the
receptor-binding and membrane fusion glycoprotein, which mediates
viral attachment and entry into target cells; HA is the primary
target of protective humoral immune responses. The HA protein is
trimeric in structure and is comprised of three identical copies of
a single polypeptide precursor, HA0, which upon proteolytic
maturation, is cleaved into a pH-dependent, metastable intermediate
containing the globular head (HA1) and the stalk region (HA2). The
membrane distal "globular head" constitutes the majority of the HA1
structure and contains the sialic acid binding pocket for viral
entry and major antigenic domains. The membrane proximal "stalk"
structure, assembled from HA2 and some HA1 residues, contains the
fusion machinery, which undergoes a conformational change in the
low pH environment of late endosomes to trigger membrane fusion and
penetration into cells. The degree of sequence homology between
influenza A subtypes is smaller in the HA1 (34%-59% homology
between subtypes) than in the HA2 region (51%-80% homology).
Neutralizing antibodies elicited by influenza virus infection are
normally targeted to the variable HA1 globular head to prevent
viral receptor binding and are usually strain-specific. Rarely,
broad cross-reactive monoclonal antibodies have been identified
that target the globular head of HA (Krause J. C. et al. 2011 J.
Virol. 85; Whittle J. et al., 2011 PNAS 108; Ekiert D C et al.,
2012 Nature 489; Lee P S et al., 2012 PNAS 109). In contrast, the
structure of the stalk region is relatively conserved and a handful
of broadly neutralizing antibodies have recently been identified
that bind to HA stalk to prevent the pH-triggered fusion step for
viral entry (Ekiert D. C. et al., 2009 Science 324; Sui J. et al.,
Nat Struct Mol Biol 16; Wrammert J et al., 2011 J Exp Med 208;
Ekiert D. C et al., 2011 Science 333; Corti D et al., 2010 J Clin
Invest 120; Throsby M., 2008 PLoS One 3). The majority of these
stalk reactive neutralizing antibodies are either specific to
influenza A group 1 viruses or specific to group 2 viruses. Very
recently, stalk binding antibodies were isolated that were
cross-reactive to both groups 1 and 2 viruses (Corti D. et al.,
2011 Science 333; Li G M et al., 2012 PNAS 109 and Cyrille D et
al., 2012 Science 337; Nakamura G et al., 2013, Cell Host &
Microbe 14).
[0006] To date, there are no marketed antibodies that broadly
neutralize or inhibit all influenza A virus infection or attenuate
disease caused by influenza A virus. Therefore, there remains a
need for new antibodies that protect against multiple group 1 and
group 2 subtypes of influenza A virus.
DESCRIPTION OF THE INVENTION
[0007] The invention provides an antibody to influenza A virus or a
binding fragment thereof that is capable of binding to influenza A
virus hemagglutinin and neutralizing at least one group 1 subtype
and at least 1 group 2 subtype of influenza A virus.
[0008] Preferably antibody or binding fragments of the invention
are capable of binding to influenza A virus hemagglutinin and
neutralizing at least 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 influenza A
virus group 1 subtype and at least 1, 2, 3, 4, 5, or 6 influenza A
virus group 2 subtypes. Further preferably, antibody or binding
fragments of the invention are capable of binding to influenza A
virus hemagglutinin and neutralizing at least 5 influenza A virus
group 1 subtypes and at least 1 or 2 influenza A virus group 2
subtypes.
[0009] The hemagglutinin subtypes of influenza A viruses fall into
two major phylogenetic groupings, identified as group 1, which
includes subtypes H1, H2, H5, H6, H8, H9, H11, H12, H13, H16 and
H17 and group 2, which includes subtypes H3, H4, H7, H10, H14, and
H15. In one embodiment, an antibody or binding fragment according
to the invention is capable of binding to and/or neutralizing one
or more influenza A virus group 1 subtypes selected from H1, H2,
H5, H6, H8, H9, H11, H12, H13, H16 and H17 and variants thereof and
one or more influenza A virus group 2 subtype selected from H3, H4,
H7, H10, H14 and H15 and variants thereof. In another embodiment,
an antibody or binding fragment according to the invention is
capable of binding to and/or neutralizing influenza A virus group 1
subtypes H1, H2, H5, H6, H8, H9, H11, H12, H13, H16 and H17 and
influenza A virus group 2 subtypes H3, H4, H7, H10, H14 and H15. In
another embodiment, the antibody or binding fragment is capable of
binding to and/or neutralizing group 1 subtypes H1, H2, H5, H6, and
H9 and group 2 subtypes H3 and H7. In a further embodiment, the
antibody or binding fragment is capable of binding to and/or
neutralizing group 1 subtypes H1, H2, H5 and H6 and group 2
subtypes H3 and H7.
[0010] The invention is based on isolation of a naturally-occurring
human monoclonal antibody (mAb) from IgG memory B cells that were
collected from individual donors as starting materials.
Optimization was used to generate antibody variants with improved
characteristics, as described herein. The optimized antibody
variants are not naturally occurring; they are generated using
recombinant techniques. Antibody or fragments thereof of the
invention bind to the stalk region of HA and neutralize infection
of more than one subtype of influenza A virus, selected from group
1 and group 2 subtypes, respectively. Antibodies of the invention,
which are anti-Influenza A HA stalk-binding antibodies,
demonstrated a broader breath of coverage or better neutralizing
activity against influenza A viruses compared to an antibody from
the published literature (Antibody Fl6v4, described in
WO2013/011347A1) and shown in Table 6 of Example 5. Additionally,
antibodies of the invention may be more effective than other mAb(s)
in blocking HA maturation as shown in FIGS. 1B, 1C and 1D of
Example 6.
[0011] In some embodiments, the antibody or binding fragment
thereof includes a set of six CDRs in which the set of six CDRs is
selected from the group consisting of:
(a) HCDR1 of SEQ ID NO.: 3, HCDR2 of SEQ ID NO.: 4, HCDR3 of SEQ ID
NO.: 5, LCDR1 of SEQ ID NO.: 8, LCDR2 of SEQ ID NO.: 9 and LCDR3 of
SEQ ID NO.: 10;
(b) HCDR1 of SEQ ID NO.: 13, HCDR2 of SEQ ID NO.: 14, HCDR3 of SEQ
ID NO.: 15, LCDR1 of SEQ ID NO.: 18, LCDR2 of SEQ ID NO.: 19, LCDR3
of SEQ ID NO.: 20;
(c) HCDR1 of SEQ ID NO.: 23, HCDR2 of SEQ ID NO.: 24, HCDR3 of SEQ
ID NO.: 25, LCDR1 of SEQ ID NO.: 28, LCDR2 of SEQ ID NO.: 29 and
LCDR3 of SEQ ID NO.: 30;
(d) HCDR1 of SEQ ID NO.: 33, HCDR2 of SEQ ID NO.: 34, HCDR3 of SEQ
ID NO.: 35, LCDR1 of SEQ ID NO.: 38, LCDR2 of SEQ ID NO.: 39 and
LCDR3 of SEQ ID NO.: 40;
(e) HCDR1 of SEQ ID NO.: 43, HCDR2 of SEQ ID NO.: 44, HCDR3 of SEQ
ID NO.: 45, LCDR1 of SEQ ID NO.: 48, LCDR2 of SEQ ID NO.: 49 and
LCDR3 of SEQ ID NO.: 50;
(f) HCDR1 of SEQ ID NO.: 53, HCDR2 of SEQ ID NO.: 54, HCDR3 of SEQ
ID NO.: 55, LCDR1 of SEQ ID NO.: 58, LCDR2 of SEQ ID NO.: 59 and
LCDR3 of SEQ ID NO.: 60;
(g) HCDR1 of SEQ ID NO.: 63, HCDR2 of SEQ ID NO.: 64, HCDR3 of SEQ
ID NO.: 65, LCDR1 of SEQ ID NO.: 68, LCDR2 of SEQ ID NO.: 69 and
LCDR3 of SEQ ID NO.: 70;
(h) HCDR1 of SEQ ID NO.: 73, HCDR2 of SEQ ID NO.: 74, HCDR3 of SEQ
ID NO.: 75, LCDR1 of SEQ ID NO.: 78, LCDR2 of SEQ ID NO.: 79 and
LCDR3 of SEQ ID NO.: 80;
(i) HCDR1 of SEQ ID NO.: 83, HCDR2 of SEQ ID NO.: 84, HCDR3 of SEQ
ID NO.: 85, LCDR1 of SEQ ID NO.: 88, LCDR2 of SEQ ID NO.: 89, LCDR3
of SEQ ID NO.: 90;
(j) HCDR1 of SEQ ID NO.: 93, HCDR2 of SEQ ID NO.: 94, HCDR3 of SEQ
ID NO.: 95, LCDR1 of SEQ ID NO.: 98, LCDR2 of SEQ ID NO.: 99 and
LCDR3 of SEQ ID NO.: 100;
(k) HCDR1 of SEQ ID NO.: 103, HCDR2 of SEQ ID NO.: 104, HCDR3 of
SEQ ID NO.: 105, LCDR1 of SEQ ID NO.: 108, LCDR2 of SEQ ID NO.: 109
and LCDR3 of SEQ ID NO.: 110;
(l) HCDR1 of SEQ ID NO.: 113, HCDR2 of SEQ ID NO.: 114, HCDR3 of
SEQ ID NO.: 115, LCDR1 of SEQ ID NO.: 118, LCDR2 of SEQ ID NO.: 119
and LCDR3 of SEQ ID NO.: 110;
(m) HCDR1 of SEQ ID NO.: 123, HCDR2 of SEQ ID NO.: 124, HCDR3 of
SEQ ID NO.: 125, LCDR1 of SEQ ID NO.: 128, LCDR2 of SEQ ID NO.: 129
and LCDR3 of SEQ ID NO.: 130;
(n) HCDR1 of SEQ ID NO.: 133, HCDR2 of SEQ ID NO.: 134, HCDR3 of
SEQ ID NO.: 135, LCDR1 of SEQ ID NO.: 138, LCDR2 of SEQ ID NO.: 139
and LCDR3 of SEQ ID NO.: 140; and
(o) HCDR1 of SEQ ID NO.: 143, HCDR2 of SEQ ID NO.: 144, HCDR3 of
SEQ ID NO.: 145, LCDR1 of SEQ ID NO.: 148, LCDR2 of SEQ ID NO.: 149
and LCDR3 of SEQ ID NO.: 150;
[0012] (p) a set of six CDRS according to any one of (a) to (o)
comprising one or more amino acid substitutions, deletions or
insertions; (q) a set of six CDRS according to any one of (a) to
(p) comprising 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15,
16, 17, 18, 19, 20, 21, 22, 23, or 24 or 25 amino acid
substitutions; (r) a set of six CDRs HCDR1, HCDR2, HCDR3, LCDR1,
LCDR2, LCDR3 according to any one of (a) to (q) comprising: [0013]
(i) a HCDR1 having an amino acid sequence identical to or
comprising 3 or fewer amino acid residue substitutions relative to
SEQ ID NO: 3; [0014] (ii) a HCDR2 having an amino acid sequence
identical to or comprising 5 or fewer amino acid residue
substitutions relative to SEQ ID NO:4; [0015] (iii) a HCDR3 having
an amino acid sequence identical to or comprising 6 or fewer amino
acid residue substitutions relative to SEQ ID NO:5; [0016] (iv) a
LCDR1 having an amino acid sequence identical to or comprising 5 or
fewer amino acid residue substitutions and/or one deletion relative
to SEQ ID NO:6; [0017] (v) a LCDR2 having an amino acid sequence
identical to or comprising 5 or fewer amino acid residue
substitutions relative to SEQ ID NO:7; and [0018] (vi) a LCDR3
having an amino acid sequence identical to or comprising 1 or fewer
amino acid residue substitutions relative to SEQ ID NO:8; (s) a set
of six CDRs HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, LCDR3 according to
any one of (a) to (r) comprising: [0019] (i) a HCDR1 in which:
[0020] Kabat residue 31 is S, [0021] Kabat residue 32 is N or Y,
[0022] Kabat residue 33 is N, S, or R, [0023] Kabat residue 34 is
A, [0024] Kabat residue 35 is V or T, [0025] Kabat residue 35A is W
[0026] Kabat residue 35B is N; [0027] (ii) a HCDR2 in which: [0028]
Kabat residue 50 is R, [0029] Kabat residue 51 is T, [0030] Kabat
residue 52 is Y, [0031] Kabat residue 52A is Y, [0032] Kabat
residue 53 is R, [0033] Kabat residue 54 is S, [0034] Kabat residue
55 is K or G, [0035] Kabat residue 56 is W, [0036] Kabat residue 57
is Y, [0037] Kabat residue 58 is N or Y, [0038] Kabat residue 59 is
D, [0039] Kabat residue 60 is Y, [0040] Kabat residue 61 is A,
[0041] Kabat residue 62 is E, V or d, [0042] Kabat residue 63 is S
or F, [0043] Kabat residue 64 is V or L, [0044] Kabat residue 65 is
K; [0045] (iii) a HCDR3 in which: [0046] Kabat residue 95 is S or
G, [0047] Kabat residue 96 is G, [0048] Kabat residue 97 is H,
[0049] Kabat residue 98 is I, [0050] Kabat residue 99 is T, [0051]
Kabat residue 100 is V or E, [0052] Kabat residue 100A is F, [0053]
Kabat residue 100B is G, [0054] Kabat residue 100C is V or L,
[0055] Kabat residue 100D is N, [0056] Kabat residue 100E is V or
I, [0057] Kabat residue 100F is D, [0058] Kabat residue 100G is A,
[0059] Kabat residue 100F is F or Y, [0060] Kabat residue 101 is D,
[0061] Kabat residue 102 is M, I or V; [0062] (iv) a LCDR1 in
which: [0063] Kabat residue 24 is R, [0064] Kabat residue 25 is T,
A or absent, [0065] Kabat residue 26 is S or A, [0066] Kabat
residue 27 is Q, [0067] Kabat residue 28 is S or R, [0068] Kabat
residue 29 is L, [0069] Kabat residue 30 is S, N or R [0070] Kabat
residue 31 is S, [0071] Kabat residue 32 is Y, [0072] Kabat residue
33 is L, T or D, [0073] Kabat residue 34 is H; [0074] (v) a LCDR2
in which: [0075] Kabat residue 50 is A, [0076] Kabat residue 51 is
A, T or S, [0077] Kabat residue 52 is S or T, [0078] Kabat residue
53 is S or T, [0079] Kabat residue 54 is L or R, [0080] Kabat
residue 55 is Q, L or G, [0081] Kabat residue 56 is S, and, [0082]
(vi) a LCDR3 in which: [0083] Kabat residue 89 is Q, [0084] Kabat
residue 90 is Q or L, [0085] Kabat residue 91 is S, [0086] Kabat
residue 92 is R, and [0087] Kabat residue 93 is T.
[0088] The invention provides antibodies and binding fragments
thereof comprising a set of six CDRs: HCDR1, HCDR2, HCDR3, LCDR1,
LCDR2, LCDR3, wherein the set of six CDRs is shown in Tables 11 and
13.
[0089] Variant antibody sequences of the invention may share 75% or
more (e.g., 80%, 85%, 90%, 95%, 97%, 98%, 99% or more) amino acid
sequence identity with the sequences recited in the application. In
some embodiments the sequence identity is calculated with regard to
the full length of the reference sequence (i.e. the sequence
recited in the application). In some further embodiments,
percentage identity, as referred to herein, is as determined using
BLAST version 2.1.3 using the default parameters specified by the
NCBI (the National Center for Biotechnology Information;
http://www.ncbi.nlm.nih.gov/) [Blosum 62 matrix; gap open penalty=l
1 and gap extension penalty=l].
[0090] Variant antibodies are also included within the scope of the
invention. Thus, variants of the sequences recited in the
application are also included within the scope of the invention.
Variants of the antibody sequences having improved affinity and/or
potency may be obtained using methods known in the art and are
included within the scope of the invention. For example, amino acid
substitutions may be used to obtain antibodies with further
improved affinity. Alternatively, codon optimization of the
nucleotide sequence may be used to improve the efficiency of
translation in expression systems for the production of the
antibody. Further, polynucleotides comprising a sequence optimized
for antibody specificity or neutralizing activity by the
application of a directed evolution method to any of the nucleic
acid sequences of the invention are also within the scope of the
invention.
[0091] The invention provides an antibody or binding fragment
thereof according to the invention comprising a VH having at least
75% identity and/or a VL having at least 75% identity to a VH
and/or VL selected from the group consisting of:
(a) VH of SEQ ID NO.: 2 and VL of SEQ ID NO.: 7,
(b) VH of SEQ ID NO.: 12 and VL of SEQ ID NO.: 17,
(c) VH of SEQ ID NO.: 22 and VL of SEQ ID NO.: 27,
(d) VH of SEQ ID NO.: 32 and VL of SEQ ID NO.: 37,
(e) VH of SEQ ID NO.: 42 and VL of SEQ ID NO.: 47,
(f) VH of SEQ ID NO.: 52 and VL of SEQ ID NO.: 57,
(g) VH of SEQ ID NO.: 62 and VL of SEQ ID NO.: 67,
(h) VH of SEQ ID NO.: 72 and VL of SEQ ID NO.: 77,
(i) VH of SEQ ID NO.: 82 and VL of SEQ ID NO.: 87,
(j) VH of SEQ ID NO.: 92 and VL of SEQ ID NO.: 97,
(k) VH of SEQ ID NO.: 102 and VL of SEQ ID NO.: 107,
(l) VH of SEQ ID NO.: 112 and VL of SEQ ID NO.: 117,
(m) VH of SEQ ID NO.: 122 and VL of SEQ ID NO.: 127,
(n) VH of SEQ ID NO.: 132 and VL of SEQ ID NO.: 137,
(o) VH of SEQ ID NO.: 144 and VL of SEQ ID NO.: 147 and
(p) VH of SEQ ID NO: 152 and VL of SEQ ID NO: 157.
[0092] An antibody or binding fragment thereof according to the
invention may comprise a VH and a VL selected from the group
consisting of:
(a) VH of SEQ ID NO.: 2 and VL of SEQ ID NO.: 7,
(b) VH of SEQ ID NO.: 12 and VL of SEQ ID NO.: 17,
(c) VH of SEQ ID NO.: 22 and VL of SEQ ID NO.: 27,
(d) VH of SEQ ID NO.: 32 and VL of SEQ ID NO.: 37,
(e) VH of SEQ ID NO.: 42 and VL of SEQ ID NO.: 47,
(f) VH of SEQ ID NO.: 52 and VL of SEQ ID NO.: 57,
(g) VH of SEQ ID NO.: 62 and VL of SEQ ID NO.: 67,
(h) VH of SEQ ID NO.: 72 and VL of SEQ ID NO.: 77,
(i) VH of SEQ ID NO.: 82 and VL of SEQ ID NO.: 87,
(j) VH of SEQ ID NO.: 92 and VL of SEQ ID NO.: 97,
(k) VH of SEQ ID NO.: 102 and VL of SEQ ID NO.: 107,
(l) VH of SEQ ID NO.: 112 and VL of SEQ ID NO.: 117,
(m) VH of SEQ ID NO.: 122 and VL of SEQ ID NO.: 127,
(n) VH of SEQ ID NO.: 132 and VL of SEQ ID NO.: 137,
(o) VH of SEQ ID NO.: 144 and VL of SEQ ID NO.: 147 and
(p) VH of SEQ ID NO: 152 and VL of SEQ ID NO: 157.
[0093] An antibody or binding fragment thereof according to the
invention may be selected from the group consisting of: an
immunoglobulin molecule, a monoclonal antibody, a chimeric
antibody, a CDR-grafted antibody, a humanized antibody, a Fab, a
Fab', a F(ab')2, a Fv, a disulfide linked Fv, a scFv, a single
domain antibody, a diabody, a multispecific antibody, a
dual-specific antibody, and a bispecific antibody.
[0094] An antibody or binding fragment thereof according to the
invention may comprise a VH comprising a human germline framework,
preferably VH6-1 and/or a VL comprising a human germline framework,
preferably VK1-39. Preferably an antibody or binding fragment
thereof according to the invention comprises a VH comprising human
germline framework VH6-1 and a VL comprising a human germline
framework VK1-39. The VH6 framework is rarely used in
antibodies.
[0095] An antibody or binding fragment thereof according to the
invention may comprise an Fc region, preferably the antibody is an
IgG1, IgG2 or IgG4 or a binding fragment thereof.
[0096] In one embodiment, an antibody of the invention comprises a
human IgG constant domain having one or more amino acid
substitutions relative to a wild-type human IgG constant domain. An
antibody of the invention may comprise a human IgG constant domain
having the M252Y, S254T, and T256E ("YTE") amino acid
substitutions, wherein amino acid residues are numbered according
to the EU index as in Kabat.
[0097] The invention also provides an antibody to influenza A virus
or a binding fragment thereof that is capable of binding to
influenza A virus hemagglutinin and neutralizing at least one group
1 subtype and at least one group 2 subtype of influenza A virus
characterized in that the antibody or binding fragment thereof
competes for binding to influenza A virus hemagglutinin with an
antibody of the invention, described above. Accordingly, the
invention comprises an antibody, or fragment thereof, that binds to
the same epitope as an antibody of the invention, or an antibody
that competes for binding with an antibody of the invention.
[0098] The invention further provides an isolated nucleic acid
encoding an antibody or fragment thereof according to the
invention. Preferably, the nucleic acid is a cDNA. The invention
also includes nucleic acid sequences encoding part or all of the
light and heavy chains and CDRs of the antibodies of the present
invention. Thus, provided herein are nucleic acid sequences
encoding part or all of the light and heavy chains and CDRs of
exemplary antibodies of the invention. The SEQ ID numbers for the
nucleic acid sequences encoding the CDRs, heavy chain and light
chain variable regions of the exemplary antibodies of the invention
are provided. Due to the redundancy of the genetic code, variants
of these sequences will exist that encode the same amino acid
sequences.
[0099] The invention yet further provides a vector comprising an
isolated nucleic acid according to the invention; preferably the
vector is an expression vector.
[0100] Additionally, the invention provides a host cell comprising
an isolated nucleic acid or a vector according to the invention.
Suitable host cells include mammalian cell lines, such as those
derived from HEK or CHO cells.
[0101] Further, the invention provides a method for manufacturing
an antibody or fragment of the invention comprising culturing a
host cell of the invention under conditions suitable for expression
of the antibody or fragment thereof.
[0102] Such methods may further comprise isolating the antibody or
fragment thereof from the host cell culture and optionally
formulating the isolated antibody or fragment into a
composition.
[0103] The invention yet further provides a composition comprising
an antibody or fragment thereof according to the invention and a
pharmaceutically acceptable carrier.
[0104] Also provided by the invention is a composition comprising
an antibody or fragment thereof according to the invention,
histidine and NaCl at a pH in the range of from about 5.5 to about
6.5, preferably at about pH 6.0; yet more preferably comprising an
antibody or fragment thereof according to the invention, about 20
to about 30 mM histidine and about 0.1 to about 0.2 M NaCl, at a pH
in the range of from about 5.5 to about 6.5, preferably at about pH
6.0; most preferably comprising 25 mM His and 0.15M NaCl at a pH in
the range of from about 5.5 to about 6.5, for example, at about pH
6.0
[0105] Additionally, the invention provides: [0106] an antibody or
fragment thereof according to the invention for use in the
prophylaxis or treatment of influenza A infection in a subject;
[0107] the use of an antibody or fragment thereof according to the
invention in the manufacture of a medicament for the prophylaxis or
treatment of Influenza A infection in a subject; [0108] a method
for prophylaxis or treatment of Influenza A infection in a subject
comprising administration of an antibody or fragment thereof
according to the invention; [0109] the use of an antibody or
fragment thereof according to the invention to prevent the
pH-triggered fusion step for Influenza A viral entry into cells; or
[0110] the use of an antibody or fragment thereof according to the
invention to inhibit Influenza A virus HA maturation.
[0111] Exemplary antibodies of the invention include, but are not
limited to: Antibody 3, Antibody 5, Antibody 6, Antibody 8,
Antibody 10, Antibody 11, Antibody 12, Antibody 13, Antibody 14,
and Antibody 15.
[0112] The invention also provides the use of an antibody or
binding fragment thereof according to the invention in in vitro
diagnosis of influenza A infection in a subject.
DETAILED DESCRIPTION
Introduction
[0113] The present invention provides antibodies, including human
forms, as well as fragments, derivatives/conjugates and
compositions thereof that bind to Influenza A virus hemagglutinin
(HA) stalk and neutralize influenza A virus infection group 1 and
group 2 subtypes as described herein; such anti-influenza A virus
HA stalk antibodies are referred to herein as antibodies of the
invention.
[0114] As used herein, the term "neutralize" refers to the ability
of an antibody, or binding fragment thereof, to bind to an
infectious agent, such as influenza A virus, and reduce the
biological activity, for example, virulence, of the infectious
agent. The minimal requirement for neutralization is the ability
for the antibody, or binding fragment thereof, to bind to the
infectious agent. In one embodiment, the antibody or binding
fragment thereof of the invention immunospecifically binds at least
one specified epitope or antigenic determinant of the Influenza A
virus. In a more particular embodiment, the antibody or binding
fragment thereof of the invention immunospecifically binds at least
one specified epitope or antigenic determinant of the Influenza A
virus HA stalk protein.
[0115] An antibody can neutralize the activity of an infectious
agent, such as Influenza A virus at various points during the
lifecycle of the virus. For example, an antibody may interfere with
viral attachment to a target cell by interfering with the
interaction of the virus and one or more cell surface receptors.
Alternately, an antibody may interfere with one or more
post-attachment interactions of the virus with its receptors, for
example, by interfering with viral internalization by
receptor-mediated endocytosis.
[0116] In one embodiment, the antibody or binding fragment thereof
neutralizes the activity of Influenza A by interfering with the
fusion process, for example, by interfering with fusion of the
viral and endosomal membranes. In another embodiment, the antibody
or binding fragment thereof interferes with protease mediated
cleavage of HA0, thus interfering with viral maturation and the
formation of the HA2 viral fusion peptide. For example, in one
embodiment, the antibody or binding fragment thereof interferes
with protease mediated HA0 cleavage, necessary for activation of
the Influenza A virus.
[0117] As used herein, the terms "antibody" and "antibodies", also
known as immunoglobulins, encompass monoclonal antibodies
(including full-length monoclonal antibodies), human antibodies,
humanized antibodies, camelid antibodies, chimeric antibodies,
single-chain Fvs (scFv), single-chain antibodies, single domain
antibodies, domain antibodies, Fab fragments, F(ab')2 fragments,
antibody fragments that exhibit the desired biological activity
(e.g. the antigen binding portion), disulfide-linked Fvs (dsFv),
and anti-idiotypic (anti-Id) antibodies (including, e.g., anti-Id
antibodies to antibodies of the invention), intrabodies, and
epitope-binding fragments of any of the above. In particular,
antibodies include immunoglobulin molecules and immunologically
active fragments of immunoglobulin molecules, i.e., molecules that
contain at least one antigen-binding site. Immunoglobulin molecules
can be of any isotype (e.g., IgG, IgE, IgM, IgD, IgA and IgY),
subisotype (e.g., IgG1, IgG2, IgG3, IgG4, IgA1 and IgA2) or
allotype (e.g., Gm, e.g., G1m(f, z, a or x), G2m(n), G3m(g, b, or
c), Am, Em, and Km(1, 2 or 3)).
[0118] Human 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 one covalent disulfide bond, 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 (VH) followed by a number of
constant domains (CH). Each light chain has a variable domain at
one end (VL) and a constant domain (CL) 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.
Light chains are classified as either lambda chains or kappa chains
based on the amino acid sequence of the light chain constant
region. The variable domain of a kappa light chain may also be
denoted herein as VK.
[0119] The antibodies of the invention include full length or
intact antibody, antibody fragments, including antigen binding
fragments, native sequence antibody or amino acid variants, human,
humanized, post-translationally modified, chimeric or fusion
antibodies, immunoconjugates, and functional fragments thereof. The
antibodies can be modified in the Fc region to provide desired
effector functions or serum half-life. As discussed in more detail
in the sections below, with the appropriate Fc regions, the naked
antibody bound on the cell surface can induce cytotoxicity, e.g.,
via antibody-dependent cellular cytotoxicity (ADCC) or by
recruiting complement in complement dependent cytotoxicity (CDC),
or by recruiting nonspecific cytotoxic cells that express one or
more effector ligands that recognize bound antibody on the
Influenza A virus HA stalk and subsequently cause phagocytosis of
the cell in antibody dependent cell-mediated phagocytosis (ADCP),
or some other mechanism. Alternatively, where it is desirable to
eliminate or reduce effector function, so as to minimize side
effects or therapeutic complications, certain other Fc regions may
be used. The Fc region of the antibodies of the invention can be
modified to increase the binding affinity for FcRn and thus
increase serum half-life. Alternatively, the Fc region can be
conjugated to PEG or albumin to increase the serum half-life, or
some other conjugation that results in the desired effect.
[0120] The present anti-Influenza A virus HA stalk antibodies are
useful for diagnosing, preventing, treating and/or alleviating one
or more symptoms of the Influenza A virus infection in a
mammal.
[0121] The invention provides a composition comprising an
anti-Influenza A virus HA stalk antibody of the invention and a
carrier. For the purposes of preventing or treating Influenza A
virus infection, compositions can be administered to the patient in
need of such treatment. The invention also provides formulations
comprising an anti-Influenza A virus HA stalk antibody of the
invention and a carrier. In one embodiment, the formulation is a
therapeutic formulation comprising a pharmaceutically acceptable
carrier.
[0122] In certain embodiments the invention provides methods useful
for preventing or treating Influenza A infection in a mammal,
comprising administering a therapeutically effective amount of the
antibody to the mammal. The antibody therapeutic compositions can
be administered short term (acutely), chronically, or
intermittently as directed by physician.
[0123] In certain embodiments the invention also provides articles
of manufacture comprising at least an anti-Influenza A virus HA
stalk antibody, such as sterile dosage forms and kits. Kits can be
provided which contain the antibodies for detection and
quantitation of Influenza A virus in vitro, e.g. in an ELISA or a
Western blot. Such antibody useful for detection may be provided
with a label such as a fluorescent or radiolabel.
Terminology
[0124] Before describing the present invention in detail, it is to
be understood that this invention is not limited to specific
compositions or process steps, as such may vary. It must be noted
that, as used in this specification and the appended claims, the
singular form "a", "an" and "the" include plural referents unless
the context clearly dictates otherwise.
[0125] Unless defined otherwise, all technical and scientific terms
used herein have the same meaning as commonly understood by one of
ordinary skill in the art to which this invention is related. For
example, the Concise Dictionary of Biomedicine and Molecular
Biology, Juo, Pei-Show, 2nd ed., 2002, CRC Press; The Dictionary of
Cell and Molecular Biology, 3rd ed., 1999, Academic Press; and the
Oxford Dictionary Of Biochemistry And Molecular Biology, Revised,
2000, Oxford University Press, provide one of skill with a general
dictionary of many of the terms used in this invention.
[0126] Amino acids may be referred to herein by either their
commonly known three letter symbols or by the one-letter symbols
recommended by the IUPAC-IUB Biochemical Nomenclature Commission.
Nucleotides, likewise, may be referred to by their commonly
accepted single-letter codes.
[0127] The numbering of amino acids in the variable domain,
complementarity determining region (CDRs) and framework regions
(FR), of an antibody follow, unless otherwise indicated, the Kabat
definition as set forth in Kabat et al. Sequences of Proteins of
Immunological Interest, 5th Ed. Public Health Service, National
Institutes of Health, Bethesda, Md. (1991). Using this numbering
system, the actual linear amino acid sequence may contain fewer or
additional amino acids corresponding to a shortening of, or
insertion into, a FR or CDR of the variable domain. For example, a
heavy chain variable domain may include a single amino acid
insertion (residue 52a according to Kabat) after residue 52 of H2
and inserted residues (e.g. residues 82a, 82b, and 82c, etc.,
according to Kabat) after heavy chain FR residue 82. The Kabat
numbering of residues may be determined for a given antibody by
alignment at regions of homology of the sequence of the antibody
with a "standard" Kabat numbered sequence. Maximal alignment of
framework residues frequently requires the insertion of "spacer"
residues in the numbering system, to be used for the Fv region. In
addition, the identity of certain individual residues at any given
Kabat site number may vary from antibody chain to antibody chain
due to interspecies or allelic divergence.
Anti-Influenza a Virus HA Stalk Antibodies
[0128] In certain embodiments, the antibodies are isolated and/or
purified and/or pyrogen free antibodies. The term "purified" as
used herein, refers to other molecules, e.g., polypeptide, nucleic
acid molecule that have been identified and separated and/or
recovered from a component of its natural environment. Thus, in one
embodiment the antibodies of the invention are purified antibodies
wherein they have been separated from one or more components of
their natural environment. The term "isolated antibody" as used
herein refers to an antibody which is substantially free of other
antibody molecules having different antigenic specificities (e.g.,
an isolated antibody that specifically binds to Influenza A virus
HA stalk is substantially free of antibodies that specifically bind
antigens other than those of Influenza A virus HA stalk). Thus, in
one embodiment the antibodies of the invention are isolated
antibodies wherein they have been separated from antibodies with a
different specificity. Typically an isolated antibody is a
monoclonal antibody. Moreover, an isolated antibody of the
invention may be substantially free of one or more other cellular
materials and/or chemicals and is herein referred to an isolated
and purified antibody. In one embodiment of the invention, a
combination of "isolated" monoclonal antibodies relates to
antibodies having different specificities and being combined in a
well-defined composition. Methods of production and
purification/isolation of antibodies are described below in more
detail.
[0129] The isolated antibodies of the present invention comprise
antibody amino acid sequences disclosed herein encoded by any
suitable polynucleotide, or any isolated or formulated
antibody.
[0130] The antibodies of the invention immunospecifically bind at
least one specified epitope specific to the Influenza A virus HA
stalk protein. The term "epitope" as used herein refers to a
protein determinant capable of binding to an antibody. Epitopes
usually include chemically active surface groupings of molecules
such as amino acids or sugar side chains and usually have specific
three dimensional structural characteristics, as well as specific
charge characteristics. Conformational and non-conformational
epitopes are distinguished in that the binding to the former but
not the latter is lost in the presence of denaturing solvents.
[0131] In one embodiment, the antibody or binding fragment thereof
binds to an epitope that is conserved among at least H1, H2, H3,
H4, H5, H6, H7, H8, H9, H10, H11, H12, H13, H14, H15, H16 or H17 or
all influenza A HA subtypes. In another embodiment, the antibody or
binding fragment thereof binds to an epitope that is conserved
among one or more, or at least 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10
influenza A virus group 1 subtypes selected from H1, H2, H5, H6,
H8, H9, H11, H12, H13 and H16 and one or more, or at least 1, 2, 3,
4, 5, or 6 group 2 subtypes selected from H3, H4, H7, H10, H14 and
H15.
[0132] In one embodiment, the antibody or binding fragment thereof
binds at least 17H1, H2, H3, H4, H5, H6, H7, H8, H9, H10, H11, H12,
H13, H14, H15, H16 or H17 or all influenza A subtypes with an ECK
of between about 0.01 ug/ml and about 5 ug/ml, or between about
0.01 ug/ml and about 0.5 ug/ml, or between about 0.01 ug/ml and
about 0.1 ug/ml, or less than about 5 ug/ml, 1 ug/ml, 0.5 ug/ml,
0.1 ug/ml, or 0.05 ug/ml. In another embodiment, the antibody or
binding fragment thereof binds one or more, or at least 1, 2, 3, 4,
5, 6, 7, 8, 9, or 10 influenza A virus group 1 subtypes selected
from H1, H2, H5, H6, H8, H9, H11, H12, H13 and H16 and one or more,
or at least 1, 2, 3, 4, 5, or 6 group 2 subtypes selected from H3,
H4, H7, H10, H14 and H15 with an ECK of between about 0.01 ug/ml
and about 5 ug/ml, or between about 0.01 ug/ml and about 0.5 ug/ml,
or between about 0.01 ug/ml and about 0.1 ug/ml, or less than about
5 ug/ml, 1 ug/ml, 0.5 ug/ml, 0.1 ug/ml, or 0.05 ug/ml.
[0133] In one embodiment, the antibody or binding fragment thereof
recognizes an epitope that is either a linear epitope, or
continuous epitope. In another embodiment, the antibody or binding
fragment thereof recognizes a non-linear or conformational epitope.
In one embodiment, the epitope is located in the highly conserved
stalk region of HA2. In a more particular embodiment, the antibody
or binding fragment binds to a conformational epitope in the highly
conserved stalk region of HA2. In one embodiment, the epitope
includes one or more amino acids selected from: positions 18, 19,
42, 45 in the stalk region of HA2 (positions are numbered according
to H3 numbering system as described in Weiss et al., J. Mol. Biol.
(1990) 212, 737-761 (1990)) as contact residues. In a more
particular embodiment, the epitope includes one or more amino acids
selected from 18, 19, 42 and 45 in the stalk region of HA2 as
contact residues. In a further embodiment, the epitope includes
amino acids 18, 19, 42 and 45 in the stalk region of HA2 as contact
residues. In yet a further embodiment, the epitope includes amino
acids 18, 19, and 42 in the stalk region of HA2 as contact
residues.
[0134] The epitope or epitopes recognized by the antibody or
binding fragment thereof of the invention may have a number of
uses. For example, the epitope in purified or synthetic form can be
used to raise immune responses (i.e., as a vaccine, or for the
production of antibodies for other uses) or for screening sera for
antibodies that immunoreact with the epitope. In one embodiment, an
epitope recognized by the antibody or binding fragment thereof of
the invention, or an antigen having such an epitope may be used as
a vaccine for raising an immune response. In another embodiment,
the antibodies and binding fragments of the invention can be used
to monitor the quality of vaccines, for example, by determining
whether the antigen in a vaccine contains the correct immunogenic
epitope in the correct conformation.
Variable Regions
[0135] As used herein, the term "parent antibody" refers to an
antibody which is encoded by an amino acid sequence used for the
preparation of the variant or derivative, defined herein. The
parent polypeptide may comprise a native antibody sequence (i.e., a
naturally occurring, including a naturally occurring allelic
variant) or an antibody sequence with pre-existing amino acid
sequence modifications (such as other insertions, deletions and/or
substitutions) of a naturally occurring sequence. The parent
antibody may be a humanized antibody or a human antibody. In
specific embodiments, antibodies of the invention are variants of
the parent antibody. As used herein, the term "variant" refers to
an antibody, which differs in amino acid sequence from a "parent"
antibody amino acid sequence by virtue of addition, deletion and/or
substitution of one or more amino acid residue(s) in the parent
antibody sequence.
[0136] The antigen-binding portion of an antibody comprises one or
more fragments of an antibody that retain the ability to
specifically bind to an antigen. It has been shown that the
antigen-binding function of an antibody can be performed by
fragments of a full-length antibody. Examples of binding fragments
encompassed within the term "antigen-binding portion" of an
antibody include (i) a Fab fragment, a monovalent fragment
consisting of the VL, VH, CL and CH1 domains; (ii) a F(ab')2
fragment, a bivalent fragment comprising two Fab fragments linked
by a disulfide bridge at the hinge region; (iii) a Fd fragment
consisting of the VH and CH1 domains; (iv) a Fv fragment consisting
of the VL and VH domains of a single arm of an antibody, (v) a dAb
fragment (Ward et al., (1989) Nature 341:544-546), which consists
of a VH domain; and (vi) an isolated complementarity determining
region (CDR). Furthermore, although the two domains of the Fv
fragment, VL and VH, are coded for by separate genes, they can be
joined, using recombinant methods, by a synthetic linker that
enables them to be made as a single protein chain in which the VL
and VH regions pair to form monovalent molecules (known as single
chain Fv (scFv); see e.g., Bird et al. (1988) Science 242:423-426;
and Huston et al. (1988) Proc. Natl. Acad. Sci. USA 85:5879-5883).
Such single chain antibodies are also intended to be encompassed
within the term "antigen-binding portion" of an antibody. These
antibody fragments are obtained using conventional techniques known
to those with skill in the art, and the fragments are screened for
utility in the same manner as are intact antibodies.
Antigen-binding portions can be produced by recombinant DNA
techniques, or by enzymatic or chemical cleavage of intact
immunoglobulins.
[0137] Antibodies of the invention comprise at least one antigen
binding domain, comprising a VH and a VL domain described
herein.
[0138] In certain embodiments, the purified antibodies comprise a
VH and/or VL that has a given percent identify to at least one of
the VH and/or VL sequences disclosed in Table 1 As used herein, the
term "percent (%) sequence identity", also including "homology" is
defined as the percentage of amino acid residues or nucleotides in
a candidate sequence that are identical with the amino acid
residues or nucleotides in the reference sequences, such as parent
antibody sequence, after aligning the sequences and introducing
gaps, if necessary, to achieve the maximum percent sequence
identity, and not considering any conservative substitutions as
part of the sequence identity. Optimal alignment of the sequences
for comparison may be produced, besides manually, by means of the
local homology algorithm of Smith and Waterman, 1981, Ads App.
Math. 2, 482, by means of the local homology algorithm of Neddleman
and Wunsch, 1970, J. Mol. Biol. 48, 443, by means of the similarity
search method of Pearson and Lipman, 1988, Proc. Natl Acad. Sci.
USA 85, 2444, or by means of computer programs which use these
algorithms (GAP, BESTFIT, FASTA, BLAST P, BLAST N and TFASTA in
Wisconsin Genetics Software Package, Genetics Computer Group, 575
Science Drive, Madison, Wis.).
[0139] Antibodies of the invention may comprise a VH amino acid
sequence having at least 65%, 70%, 75%, 80%, 85%, 90%, 95% or
having 100% identity to the VH amino acid sequences described
herein. The antibodies may have a VH amino acid sequence having at
least, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or having
100% identity to the amino acid sequence of the VH amino acid
sequences described herein.
[0140] Antibodies of the invention may comprise a VL amino acid
sequence having at least 65%, 70%, 75%, 80%, 85%, 90%, 95% or
having 100% identity to the VL amino acid sequences described
herein. The antibodies may have a VL amino acid sequence having at
least, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or having
100% identity to the VL amino acid sequences described herein.
[0141] Antibodies within the scope of the of the invention are
capable of neutralizing one or more group 1 subtype and one or more
group 2 subtype of Influenza A virus, as described herein.
Complementarity Determining Regions (CDRs)
[0142] While the variable domain (VH and VL) comprises the
antigen-binding region; the variability is not evenly distributed
through the variable domains of antibodies. It is concentrated in
segments called Complementarity Determining Regions (CDRs), both in
the light chain (VL or VK) and the heavy chain (VH) variable
domains. The more highly conserved portions of the variable domains
are called the framework regions (FR). The variable domains of
native heavy and light chains each comprise four FR, largely
adopting a .beta.-sheet configuration, connected by three CDRs,
which form loops connecting, and in some cases forming part of, the
.beta.-sheet structure. The CDRs in each chain are held together in
close proximity by the FR and, with the CDRs from the other chain,
contribute to the formation of the antigen-binding site of
antibodies (see, Kabat et al., Supra). The three CDRs of the heavy
chain are designated CDR-H1, CDR-H2, and CDR-H3, and the three CDRs
of the light chain are designated CDR-L1, CDR-L2, and CDR-L3. The
Kabat numbering system is used herein. As such, CDR-H1 begins at
approximately amino acid 31 (i.e., approximately 9 residues after
the first cysteine residue), includes approximately 5-7 amino
acids, and ends at the next tyrosine residue. CDR-H2 begins at the
fifteenth residue after the end of CDR-H1, includes approximately
16-19 amino acids, and ends at the next arginine or lysine residue.
CDR-H3 begins at approximately the thirty third amino acid residue
after the end of CDR-H2; includes 3-25 amino acids; and ends at the
sequence W-G-X-G, where X is any amino acid. CDR-L1 begins at
approximately residue 24 (i.e., following a cysteine residue);
includes approximately 10-17 residues; and ends at the next
tyrosine residue. CDR-L2 begins at approximately the sixteenth
residue after the end of CDR-L1 and includes approximately 7
residues. CDR-L3 begins at approximately the thirty third residue
after the end of CDR-L2; includes approximately 7-11 residues and
ends at the sequence F-G-X-G, where X is any amino acid. Note that
CDRs vary considerably from antibody to antibody (and by definition
will not exhibit homology with the Kabat consensus sequences).
[0143] The present invention encompasses neutralizing
anti-Influenza A HA stalk antibodies comprising amino acids in a
sequence that is substantially the same as an amino acid sequence
described herein. Amino acid sequences that are substantially the
same as the sequences described herein include sequences comprising
conservative amino acid substitutions, as well as amino acid
deletions and/or insertions in an amino acid sequence of for
example, Antibody 11, Antibody 12, Antibody 13, Antibody 14 or
Antibody 15, or in an amino acid sequence shown in SEQ ID NOs: 102,
112, 122, 132, or 142. A conservative amino acid substitution
refers to the replacement of a first amino acid by a second amino
acid that has chemical and/or physical properties (e.g, charge,
structure, polarity, hydrophobicity/hydrophilicity) that are
similar to those of the first amino acid. Conservative
substitutions include replacement of one amino acid by another
within the following groups: lysine (K), arginine (R) and histidine
(H); aspartate (D) and glutamate (E); asparagine (N), glutamine
(Q), serine (S), threonine (T), tyrosine (Y), K, R, H, D and E;
alanine (A), valine (V), leucine (L), isoleucine (I), proline (P),
phenylalanine (F), tryptophan (W), methionine (M), cysteine (C) and
glycine (G); F, W and Y; C, S and T.
Framework Regions
[0144] The variable domains of the heavy and light chains each
comprise four framework regions (FR1, FR2, FR3, FR4), which are the
more highly conserved portions of the variable domains. The four
FRs of the heavy chain are designated FR-H1, FR-H2, FR-H3 and
FR-H4, and the four FRs of the light chain are designated FR-L1,
FR-L2, FR-L3 and FR-L4. The Kabat numbering system is used herein,
See Table 1, Kabat et al, Supra. As such, FR-H1 begins at position
1 and ends at approximately amino acid 30, FR-H2 is approximately
from amino acid 36 to 49, FR-H3 is approximately from amino acid 66
to 94 and FR-H4 is approximately amino acid 103 to 113. FR-L1
begins at amino acid 1 and ends at approximately amino acid 23,
FR-L2 is approximately from amino acid 35 to 49, FR-L3 is
approximately from amino acid 57 to 88 and FR-L4 is approximately
from amino acid 98 to 107. In certain embodiments the framework
regions may contain substitutions according to the Kabat numbering
system, e.g., insertion at 106A in FR-L1. In addition to naturally
occurring substitutions, one or more alterations (e.g.,
substitutions) of FR residues may also be introduced in an antibody
of the invention, provided it retains neutralizing ability. In
certain embodiments, these result in an improvement or optimization
in the binding affinity of the antibody for Influenza A virus HA
stalk. Examples of framework region residues to modify include
those which non-covalently bind antigen directly (Amit et al.,
Science, 233:747-753 (1986)); interact with/effect the conformation
of a CDR (Chothia et al., J. Mol. Biol., 196:901-917 (1987));
and/or participate in the VL-VH interface (U.S. Pat. No.
5,225,539).
[0145] In another embodiment the FR may comprise one or more amino
acid changes for the purposes of "germlining". For example, the
amino acid sequences of selected antibody heavy and light chains
are compared to germline heavy and light chain amino acid sequences
and where certain framework residues of the selected VL and/or VH
chains differ from the germline configuration (e.g., as a result of
somatic mutation of the immunoglobulin genes used to prepare the
phage library), it may be desirable to "back-mutate" the altered
framework residues of the selected antibodies to the germline
configuration (i.e., change the framework amino acid sequences of
the selected antibodies so that they are the same as the germline
framework amino acid sequences). Such "back-mutation" (or
"germlining") of framework residues can be accomplished by standard
molecular biology methods for introducing specific mutations (e.g.,
site-directed mutagenesis; PCR-mediated mutagenesis, and the
like).
Nucleotide Sequences Encoding Antibodies of the Invention
[0146] In addition to the amino acid sequences described above, the
invention further provides nucleotide sequences corresponding to
the amino acid sequences and encoding for the human antibodies of
the invention. In one embodiment, the invention provides
polynucleotides comprising a nucleotide sequence encoding an
antibody described herein or fragments thereof. These include, but
are not limited to, nucleotide sequences that code for the above
referenced amino acid sequences. Thus, the present invention also
provides polynucleotide sequences encoding VH and VL framework
regions including CDRs and FRs of antibodies described herein as
well as expression vectors for their efficient expression in cells
(e.g. mammalian cells). Methods of making the antibodies using
polynucleotides are described below in more detail.
[0147] The invention also encompasses polynucleotides that
hybridize under stringent or lower stringency hybridization
conditions, e.g., as defined herein, to polynucleotides that encode
an antibody of the invention described herein. The term
"stringency" as used herein refers to experimental conditions (e.g.
temperature and salt concentration) of a hybridization experiment
to denote the degree of homology between the probe and the filter
bound nucleic acid; the higher the stringency, the higher percent
homology between the probe and filter bound nucleic acid.
[0148] Stringent hybridization conditions include, but are not
limited to, hybridization to filter-bound DNA in 6.times. sodium
chloride/sodium citrate (SSC) at about 45.degree. C. followed by
one or more washes in 0.2.times.SSC/0.1% SDS at about 50-65.degree.
C., highly stringent conditions such as hybridization to
filter-bound DNA in 6.times.SSC at about 45.degree. C. followed by
one or more washes in 0.1.times.SSC/0.2% SDS at about 65.degree.
C., or any other stringent hybridization conditions known to those
skilled in the art (see, for example, Ausubel, F. M. et al., eds.
1989 Current Protocols in Molecular Biology, vol. 1, Green
Publishing Associates, Inc. and John Wiley and Sons, Inc., NY at
pages 6.3.1 to 6.3.6 and 2.10.3).
[0149] Substantially identical sequences may be polymorphic
sequences, i.e., alternative sequences or alleles in a population.
An allelic difference may be as small as one base pair.
Substantially identical sequences may also comprise mutagenized
sequences, including sequences comprising silent mutations. A
mutation may comprise one or more residue changes, a deletion of
one or more residues, or an insertion of one or more additional
residues.
[0150] The polynucleotides may be obtained, and the nucleotide
sequence of the polynucleotides determined, by any method known in
the art. For example, if the nucleotide sequence of the antibody is
known, a polynucleotide encoding the antibody may be assembled from
chemically synthesized oligonucleotides (e.g., as described in
Kutmeier et al., BioTechniques 17:242 (1994)), which, briefly,
involves the synthesis of overlapping oligonucleotides containing
portions of the sequence encoding the antibody, annealing and
ligating of those oligonucleotides, and then amplification of the
ligated oligonucleotides by PCR.
[0151] A polynucleotide encoding an antibody may also be generated
from nucleic acid from a suitable source. If a clone containing a
nucleic acid encoding a particular antibody is not available, but
the sequence of the antibody molecule is known, a nucleic acid
encoding the immunoglobulin may be chemically synthesized or
obtained from a suitable source (e.g., an antibody cDNA library, or
a cDNA library generated from, or nucleic acid, preferably
polyA+RNA, isolated from, any tissue or cells expressing the
antibody, such as hybridoma cells selected to express an antibody)
by PCR amplification using synthetic primers hybridizable to the 3'
and 5' ends of the sequence or by cloning using an oligonucleotide
probe specific for the particular gene sequence to identify, e.g.,
a cDNA clone from a cDNA library that encodes the antibody.
Amplified nucleic acids generated by PCR may then be cloned into
replicable cloning vectors using any method well known in the
art.
[0152] Once the nucleotide sequence and corresponding amino acid
sequence of the antibody is determined, the nucleotide sequence of
the antibody may be manipulated using methods well known in the art
for the manipulation of nucleotide sequences, e.g., recombinant DNA
techniques, site directed mutagenesis, PCR, etc. (see, for example,
the techniques described in Sambrook et al., 1990, Molecular
Cloning, A Laboratory Manual, 2d Ed., Cold Spring Harbor
Laboratory, Cold Spring Harbor, N.Y. and Ausubel et al., eds.,
1998, Current Protocols in Molecular Biology, John Wiley &
Sons, NY), to generate antibodies having a different amino acid
sequence, for example to create amino acid substitutions,
deletions, and/or insertions.
Binding Characteristics
[0153] As described above, the anti-Influenza A virus HA stalk
antibodies of the invention immunospecifically bind at least one
specified epitope or antigenic determinants of the Influenza A
virus HA stalk protein, peptide, subunit, fragment, portion or any
combination thereof either exclusively or preferentially with
respect to other polypeptides. The term "epitope" or "antigenic
determinant" as used herein refers to a protein determinant capable
of binding to an antibody, wherein the term "binding" herein
preferably relates to a specific binding. These protein
determinants or epitopes usually consist of chemically active
surface groupings of molecules such as amino acids or sugar side
chains and usually have a specific three dimensional structural
characteristics, as well as specific charge characteristics.
Conformational and non-conformational epitopes are distinguished in
that the binding to the former but not the latter is lost in the
presence of denaturing solvents. The term "discontinuous epitope"
as used herein, refers to a conformational epitope on a protein
antigen which is formed from at least two separate regions in the
primary sequence of the protein.
[0154] The interactions between antigens and antibodies are the
same as for other non-covalent protein-protein interactions. In
general, four types of binding interactions exist between antigens
and antibodies: (i) hydrogen bonds, (ii) dispersion forces, (iii)
electrostatic forces between Lewis acids and Lewis bases, and (iv)
hydrophobic interactions. Hydrophobic interactions are a major
driving force for the antibody-antigen interaction, and are based
on repulsion of water by non-polar groups rather than attraction of
molecules (Tanford, 1978). However, certain physical forces also
contribute to antigen-antibody binding, for example, the fit or
complimentary of epitope shapes with different antibody binding
sites. Moreover, other materials and antigens may cross-react with
an antibody, thereby competing for available free antibody.
[0155] Measurement of the affinity constant and specificity of
binding between antigen and antibody is a pivotal element in
determining the efficacy of prophylactic, therapeutic, diagnostic
and research methods using the antibodies of the invention.
"Binding affinity" generally refers to the strength of the sum
total of the noncovalent interactions between a single binding site
of a molecule (e.g., an antibody) and its binding partner (e.g., an
antigen). Unless indicated otherwise, as used herein, "binding
affinity" refers to intrinsic binding affinity which reflects a 1:1
interaction between members of a binding pair (e.g., antibody and
antigen). The affinity of a molecule X for its partner Y can
generally be represented by the equilibrium dissociation constant
(Kd), which is calculated as the ratio k.sub.off/k.sub.on. See,
e.g., Chen, Y., et al., (1999) J. Mol Biol 293:865-881. Affinity
can be measured by common methods known in the art, including those
described and exemplified herein. An example of a commercially
available system for kinetic characterization includes the
OCTET.RTM. family of instruments. Low-affinity antibodies generally
bind antigen slowly and tend to dissociate readily, whereas
high-affinity antibodies generally bind antigen faster and tend to
remain bound longer. A variety of methods of measuring binding
affinity are known in the art, any of which can be used for
purposes of the present invention.
[0156] Determination of binding affinity can be measured using the
specific techniques described further in the Example section, and
methods well known in the art. One such method includes measuring
the disassociation constant "Kd" by a radiolabeled antigen binding
assay (RIA) performed with the Fab version of an antibody of
interest and its antigen as described by the following assay that
measures solution binding affinity of Fabs for antigen by
equilibrating Fab with a minimal concentration of
(.sup.125I)-labeled antigen in the presence of a titration series
of unlabeled antigen, then capturing bound antigen with an anti-Fab
antibody-coated plate (Chen, et al., (1999) J. Mol Biol
293:865-881). To establish conditions for the assay, microtiter
plates (Dynex) are coated overnight with 5 .mu.g/ml of a capturing
anti-Fab antibody (Cappel Labs) in 50 mM sodium carbonate (H 9.6),
and subsequently blocked with 2% (w/v) bovine serum albumin in PBS
for two to five hours at room temperature (approximately 23.degree.
C.). In a non-adsorbant plate (Nunc #269620), 100 pM or 26 pM
[.sup.125I]-antigen are mixed with serial dilutions of a Fab of
interest (e.g., consistent with assessment of an anti-VEGF
antibody, Fab-12, in Presta et al., (1997) Cancer Res.
57:4593-4599). The Fab of interest is then incubated overnight;
however, the incubation may continue for a longer period (e.g., 65
hours) to insure that equilibrium is reached. Thereafter, the
mixtures are transferred to the capture plate for incubation at
room temperature (e.g., for one hour). The solution is then removed
and the plate washed eight times with 0.1% Tween-20 in PBS. When
the plates have dried, 150 .mu.l/well of scintillant
(MicroScint-20; Packard) is added, and the plates are counted on a
Topcount gamma counter (Packard) for ten minutes. Concentrations of
each Fab that give less than or equal to 20% of maximal binding are
chosen for use in competitive binding assays.
[0157] In another instance the Kd value may be measured by using
surface plasmon resonance assays using a BIAcore.TM.-2000 or a
BIAcore.TM.-3000 (BIAcore, Inc., Piscataway, N.J.) at 25.degree. C.
with immobilized antigen CM5 chips at .sup..about.10 response units
(RU). Briefly, carboxymethylated dextran biosensor chips (CM5,
BIAcore Inc.) are activated with
N-ethyl-N'-(3-dimethylaminopropyl)-carbodiimide hydrochloride (EDC)
and N-hydroxysuccinimide (NHS) according to the supplier's
instructions. Antigen is diluted with 110 mM sodium acetate, pH
4.8, into 5 ug/ml (.sup..about.0.2 uM) before injection at a flow
rate of 5 ul/minute to achieve approximately 10 response units (RU)
of coupled protein. Following the injection of antigen, IM
ethanolamine is injected to block unreacted groups. For kinetics
measurements, two-fold serial dilutions of Fab (0.78 nM to 500 nM)
are injected in PBS with 0.05% Tween 20 (PBST) at 25.degree. C. at
a flow rate of approximately 25 ul/min. Association rates
(k.sub.on) and dissociation rates (k.sub.off) are calculated using
a simple one-to-one Langmuir binding model (BIAcore Evaluation
Software version 3.2) by simultaneously fitting the association and
dissociation sensorgram.
[0158] If the on-rate exceeds 10.sup.6 M.sup.-1 S.sup.-1 by the
surface plasmon resonance assay above, then the on-rate can be
determined by using a fluorescent quenching technique that measures
the increase or decrease in fluorescence emission intensity
(excitation=295 nm; emission=340 nm, 16 nm band-pass) at 25.degree.
C. of a 20 nM anti-antigen antibody (Fab form) in PBS, pH 7.2, in
the presence of increasing concentrations of antigen as measured in
a spectrometer, such as a stop-flow equipped spectrophometer (Aviv
Instruments) or a 8000-series SLM-Aminco spectrophotometer
(ThermoSpectronic) with a stir red cuvette. An "on-rate" or "rate
of association" or "association rate" or "k.sub.on" according to
this invention can also be determined with the same surface plasmon
resonance technique described above using a BIAcore.TM.-2000 or a
BIAcore.TM.-3000 (BIAcore, Inc., Piscataway, N.J.) as described
above.
[0159] Methods and reagents suitable for determination of binding
characteristics of an antibody of the present invention, or an
altered/mutant derivative thereof (discussed below), are known in
the art and/or are commercially available (U.S. Pat. Nos.
6,849,425; 6,632,926; 6,294,391; 6,143,574). Moreover, equipment
and software designed for such kinetic analyses are commercially
available (e.g. Biacore.RTM. A100, and Biacore.RTM. 2000
instruments; Biacore International AB, Uppsala, Sweden).
[0160] In one embodiment, antibodies of the present invention,
including binding fragments or variants thereof, may also be
described or specified in terms of their binding affinity for
Influenza A virus polypeptides. Typically, antibodies with high
affinity have Kd of less than 10.sup.-7 M. In one embodiment,
antibodies or binding fragments thereof bind Influenza A
polypeptides, or fragments or variants thereof, with a dissociation
constant or Kd of less than or equal to 5.times.10.sup.-7 M,
10.sup.-7 M, 5.times.10.sup.-8 M, 10.sup.-8 M, 5.times.10.sup.-9 M,
10.sup.-9 M, 5.times.10.sup.-1.degree. M, 10.sup.-10 M,
5.times.10.sup.-11 M, 10.sup.-11 M, 5.times.10.sup.-12 M,
10.sup.-12 M, 5.times.10.sup.-13 M, 10.sup.-13 M,
5.times.10.sup.-14 M, 10.sup.-14 M, 5.times.10.sup.-15 M or
10.sup.-15 M. Influenza A polypeptides can include HA polypeptides.
In a more particular embodiment, antibodies or binding fragments
thereof bind Influenza A polypeptides, or fragments or variants
thereof, with a dissociation constant or Kd of less than or equal
to 5.times.10.sup.-10 M, 10.sup.-10 M, 5.times.10.sup.-11 M,
10.sup.-11 M, 5.times.10.sup.-12 M or 10.sup.-12 M. The invention
encompasses antibodies that bind Influenza A polypeptides with a
dissociation constant or Kd that is within a range between any of
the individual recited values.
[0161] In another embodiment, antibodies or binding fragments
thereof of the invention bind Influenza A polypeptides or fragments
or variants thereof with an off rate (k.sub.off) of less than or
equal to 5.times.10.sup.-2 sec.sup.-1, 10.sup.-2 sec.sup.-1,
5.times.10.sup.-3 sec.sup.-1 or 10.sup.-3 sec.sup.-1,
5.times.10.sup.-4 sec.sup.-1, 10.sup.-4 sec.sup.-1,
5.times.10.sup.-5 sec.sup.-1, or 10.sup.-5 sec.sup.-1,
5.times.10.sup.-6 sec.sup.-1, 10.sup.-6 sec.sup.-1,
5.times.10.sup.-7 sec.sup.-1 or 10.sup.-7 sec.sup.-1. In a more
particular embodiment, antibodies or binding fragments thereof of
the invention bind Influenza A polypeptides or fragments or
variants thereof with an off rate (k.sub.off) less than or equal to
5.times.10.sup.-4 sec.sup.-1, 10.sup.-4 sec.sup.-1,
5.times.10.sup.-5 sec.sup.-1, or 10.sup.-5 sec.sup.-1,
5.times.10.sup.-6 sec.sup.-1, 10.sup.-6 sec.sup.-1,
5.times.10.sup.-7 sec.sup.-1 or 10.sup.-7 sec.sup.-1. The invention
also encompasses antibodies that bind Influenza A polypeptides with
an off rate (k.sub.off) that is within a range between any of the
individual recited values.
[0162] In another embodiment, antibodies or binding fragments
thereof of the invention bind Influenza A polypeptides or fragments
or variants thereof with an on rate (k.sub.on) of greater than or
equal to 10.sup.3 M.sup.-1 sec.sup.-1, 5.times.10.sup.3 M.sup.-1
sec.sup.-1, 10.sup.4 M.sup.-1 sec.sup.-1, 5.times.10.sup.4 M.sup.-1
sec.sup.-1, 10.sup.5 M.sup.-1 sec.sup.-1, 5.times.10.sup.5 M.sup.-1
sec.sup.-1, 10.sup.6 M.sup.-1 sec-1, 5.times.10.sup.6 M.sup.-1
sec.sup.-1, 10.sup.7 M.sup.-1 sec-1, or 5.times.10.sup.7 M.sup.-1
sec.sup.-1. In a more particular embodiment, antibodies or binding
fragments thereof of the invention bind Influenza A polypeptides or
fragments or variants thereof with an on rate (k.sub.on) greater
than or equal to 10.sup.5 M.sup.-1 sec.sup.-1, 5.times.10.sup.5
M.sup.-1 sec.sup.-1, 10.sup.6 M.sup.-1 sec-1, 5.times.10.sup.6
M.sup.-1 sec.sup.-1, 10.sup.7 M.sup.-1 sec.sup.-1 or
5.times.10.sup.7 M.sup.-1 sec.sup.-1. The invention encompasses
antibodies that bind Influenza A polypeptides with on rate
(k.sub.on) that is within a range between any of the individual
recited values.
[0163] In one embodiment, a binding assay may be performed either
as direct binding assays or as competition-binding assays. Binding
can be detected using standard ELISA or standard Flow Cytometry
assays. In a direct binding assay, a candidate antibody is tested
for binding to its cognate antigen. Competition-binding assay, on
the other hand, assess the ability of a candidate antibody to
compete with a known antibody or other compound that binds to the
Influenza A virus HA stalk. In general any method that permits the
binding of an antibody with the Influenza A virus HA stalk that can
be detected is encompassed with the scope of the present invention
for detecting and measuring the binding characteristics of the
antibodies. One of skill in the art will recognize these well-known
methods and for this reason are not provided in detail here. These
methods are also utilized to screen a panel of antibodies for those
providing the desired characteristics.
[0164] An antibody of the invention immunospecifically binds to
Influenza A virus HA stalk and is capable of neutralizing Influenza
A virus infection. Neutralization assays can be performed as
described herein in the Examples section or using other methods
known in the art. The term "inhibitory concentration 50%"
(abbreviated as "IC.sub.50") represents the concentration of an
inhibitor (e.g., an antibody of the invention) that is required for
50% neutralization of Influenza A virus. It will be understood by
one of ordinary skill in the art that a lower IC.sub.50 value
corresponds to a more potent inhibitor.
[0165] In one embodiment, an antibody or binding fragment thereof
according to the invention has a neutralizing potency expressed as
50% inhibitory concentration (IC.sub.50 ug/ml) in the range of from
about 0.01 ug/ml to about 50 ug/ml, or in the range of from about
0.01 ug/ml to about 5 ug/ml of antibody, or in the range of from
about 0.01 ug/ml to about 0.1 ug/ml of antibody for neutralization
of influenza A virus in a microneutralization assay. The highest
concentration of antibody used in microneutralization assay
described herein was 50 ug/ml. The high potency of antibodies of
the invention means that lower concentrations of antibody can be
used to attain 50% neutralization of influenza A virus.
[0166] In certain embodiments, the antibodies of the invention may
induce cell death. An antibody which "induces cell death" is one
which causes a viable cell to become nonviable. Cell death in vitro
may be determined in the absence of complement and immune effector
cells to distinguish cell death induced by antibody-dependent
cell-mediated cytotoxicity (ADCC) or complement dependent
cytotoxicity (CDC). Thus, the assay for cell death may be performed
using heat inactivated serum (i.e., in the absence of complement)
and in the absence of immune effector cells. To determine whether
the antibody is able to induce cell death, loss of membrane
integrity as evaluated by uptake of propidium iodide (PI), trypan
blue (see Moore et al. Cytotechnology 17:1-11 (1995)), 7AAD or
other methods well known in the art can be assessed relative to
untreated cells.
[0167] In a specific embodiment, the antibodies of the invention
may induce cell death via apoptosis. An antibody which "induces
apoptosis" is one which induces programmed cell death as determined
by binding of annexin V, fragmentation of DNA, cell shrinkage,
dilation of endoplasmic reticulum, cell fragmentation, and/or
formation of membrane vesicles (called apoptotic bodies). Various
methods are available for evaluating the cellular events associated
with apoptosis. For example, phosphatidyl serine (PS) translocation
can be measured by annexin binding; DNA fragmentation can be
evaluated through DNA laddering; and nuclear/chromatin condensation
along with DNA fragmentation can be evaluated by any increase in
hypodiploid cells. Preferably, the antibody which induces apoptosis
is one which results in about 2 to 50 fold, preferably about 5 to
50 fold, and most preferably about 10 to 50 fold, induction of
annexin binding relative to untreated cell in an annexin binding
assay.
[0168] In another specific embodiment, the antibodies of the
invention may induce cell death via antibody-dependent cellular
cytotoxicity (ADCC) and/or complement-dependent cell-mediated
cytotoxicity (CDC) and/or antibody dependent cell-mediated
phagocytosis (ADCP). Expression of ADCC activity and CDC activity
of the human IgG1 subclass antibodies generally involves binding of
the Fc region of the antibody to a receptor for an antibody
(hereinafter referred to as "Fc.gamma.R") existing on the surface
of effector cells such as killer cells, natural killer cells or
activated macrophages. Various complement components can be bound.
Regarding the binding, it has been suggested that several amino
acid residues in the hinge region and the second domain of C region
(hereinafter referred to as "Cy2 domain") of the antibody are
important (Eur. J. Immunol., 23, 1098 (1993), Immunology, 86, 319
(1995), Chemical Immunology, 65, 88 (1997)) and that a sugar chain
in the C.gamma.2 domain (Chemical Immunology, 65, 88 (1997)) is
also important.
[0169] To assess ADCC activity of an antibody of interest, an in
vitro ADCC assay can be used, such as that described in U.S. Pat.
No. 5,500,362. The assay may also be performed using a commercially
available kit, e.g. CytoTox 96.RTM. (Promega). Useful effector
cells for such assays include, but are not limited to peripheral
blood mononuclear cells (PBMC), Natural Killer (NK) cells, and NK
cell lines. NK cell lines expressing a transgenic Fc receptor (e.g.
CD16) and associated signaling polypeptide (e.g.
FC.sub..epsilon.RI-.gamma.) may also serve as effector cells (WO
2006/023148). For example, the ability of any particular antibody
to mediate lysis by complement activation and/or ADCC can be
assayed. The cells of interest are grown and labeled in vitro; the
antibody is added to the cell culture in combination with immune
cells which may be activated by the antigen antibody complexes;
i.e., effector cells involved in the ADCC response. The antibody
can also be tested for complement activation. In either case,
cytolysis is detected by the release of label from the lysed cells.
The extent of cell lysis may also be determined by detecting the
release of cytoplasmic proteins (e.g. LDH) into the supernatant. In
fact, antibodies can be screened using the patient's own serum as a
source of complement and/or immune cells. Antibodies that are
capable of mediating human ADCC in the in vitro test can then be
used therapeutically in that particular patient. ADCC activity of
the molecule of interest may also be assessed in vivo, e.g., in an
animal model such as that disclosed in Clynes et al., Proc. Natl.
Acad. Sci. (USA) 95:652-656 (1998). Moreover, techniques for
modulating (i.e., increasing or decreasing) the level of ADCC, and
optionally CDC activity, of an antibody are well-known in the art
(e.g., U.S. Pat. Nos. 5,624,821; 6,194,551; 7,317,091). Antibodies
of the present invention may be capable or may have been modified
to have the ability of inducing ADCC and/or CDC. Assays to
determine ADCC function can be practiced using human effector cells
to assess human ADCC function. Such assays may also include those
intended to screen for antibodies that induce, mediate, enhance,
block cell death by necrotic and/or apoptotic mechanisms. Such
methods including assays utilizing viable dyes, methods of
detecting and analyzing caspases, and assays measuring DNA breaks
can be used to assess the apoptotic activity of cells cultured in
vitro with an antibody of interest.
Production of Antibodies
[0170] The following describes exemplary techniques for the
production of the antibodies useful in the present invention.
Monoclonal Antibodies
[0171] Monoclonal antibodies can be prepared using a wide variety
of techniques known in the art including the use of hybridoma
(Kohler et al., Nature, 256:495 (1975); Harlow et al., Antibodies:
A Laboratory Manual, (Cold Spring Harbor Laboratory Press, 2nd ed.
1988); Hammerling et al., in: Monoclonal Antibodies and T-Cell
Hybridomas 563-681 (Elsevier, N.Y., 1981), recombinant, and phage
display technologies, or a combination thereof. The term
"monoclonal antibody" as used herein refers to an antibody obtained
from a population of substantially homogeneous or isolated
antibodies, e.g., the individual antibodies comprising the
population are identical except for possible naturally occurring
mutations that may be present in minor amounts. Monoclonal
antibodies are highly specific, being directed against a single
antigenic site. Furthermore, in contrast to polyclonal antibody
preparations which include different antibodies directed against
different determinants (epitopes), each monoclonal antibody is
directed against the same determinant on the antigen. In addition
to their specificity, monoclonal antibodies are advantageous in
that they may be synthesized uncontaminated by other antibodies.
The modifier "monoclonal" is not to be construed as requiring
production of the antibody by any particular method. Following is a
description of representative methods for producing monoclonal
antibodies which is not intended to be limiting and may be used to
produce, for example, monoclonal mammalian, chimeric, humanized,
human, domain, diabodies, vaccibodies, linear and multispecific
antibodies.
Hybridoma Techniques
[0172] Methods for producing and screening for specific antibodies
using hybridoma technology are routine and well known in the art.
In the hybridoma method, mice or other appropriate host animals,
such as hamster, are immunized as described above to elicit
lymphocytes that produce or are capable of producing antibodies
that will specifically bind to the antigen used for immunization.
Alternatively, lymphocytes may be immunized in vitro. After
immunization, lymphocytes are isolated and then fused with a
myeloma cell line using a suitable fusing agent or fusion partner,
such as polyethylene glycol, to form a hybridoma cell (Goding,
Monoclonal Antibodies: Principles and Practice, pp. 59-103
(Academic Press, 1986)). In certain embodiments, the selected
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 selective medium
that selects against the unfused parental cells. In one aspect, the
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
and derivatives e.g., 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)).
[0173] Once hybridoma cells that produce antibodies of the desired
specificity, affinity, and/or activity are identified, the clones
may be subcloned by limiting dilution procedures and grown by
standard methods (Goding, Supra). Suitable culture media for this
purpose include, for example, D-MEM or RPMI-1640 medium. In
addition, the hybridoma cells may be grown in vivo as ascites
tumors in an animal e.g., by i.p. injection of the cells into
mice.
[0174] The monoclonal antibodies secreted by the sub-clones are
suitably separated from the culture medium, ascites fluid, or serum
by conventional antibody purification procedures such as, for
example, affinity chromatography (e.g., using protein A or protein
G-Sepharose) or ion-exchange chromatography, affinity tags,
hydroxylapatite chromatography, gel electrophoresis, dialysis, etc.
Exemplary purification methods are described in more detail
below.
Recombinant DNA Techniques
[0175] Methods for producing and screening for specific antibodies
using recombinant DNA technology are routine and well known in the
art (e.g. U.S. Pat. No. 4,816,567). DNA encoding the monoclonal
antibodies may be readily isolated and/or sequenced using
conventional procedures (e.g., by using oligonucleotide probes that
are capable of binding specifically to genes encoding the heavy and
light chains of murine antibodies). Once isolated, the DNA may be
placed into expression vectors, which are then transfected into
host cells such as E. coli cells, simian COS cells, Chinese Hamster
Ovary (CHO) cells, or myeloma cells that do not otherwise produce
antibody protein, to obtain the synthesis of monoclonal antibodies
in the recombinant host cells. Review articles on recombinant
expression in bacteria of DNA encoding the antibody include Skerra
et al., Curr. Opinion in Immunol., 5:256-262 (1993) and Pluckthun,
Immunol. Revs., 130:151-188 (1992). As described below for
antibodies generated by phage display and humanization of
antibodies, DNA or genetic material for recombinant antibodies can
be obtained from source(s) other than hybridomas to generate
antibodies of the invention.
[0176] Recombinant expression of an antibody or variant thereof
generally requires construction of an expression vector containing
a polynucleotide that encodes the antibody. The invention, thus,
provides replicable vectors comprising a nucleotide sequence
encoding an antibody molecule, a heavy or light chain of an
antibody, a heavy or light chain variable domain of an antibody or
a portion thereof, or a heavy or light chain CDR, operably linked
to a promoter. Such vectors may include the nucleotide sequence
encoding the constant region of the antibody molecule (see, e.g.,
U.S. Pat. Nos. 5,981,216; 5,591,639; 5,658,759 and 5,122,464) and
the variable domain of the antibody may be cloned into such a
vector for expression of the entire heavy, the entire light chain,
or both the entire heavy and light chains.
[0177] Once the expression vector is transferred to a host cell by
conventional techniques, the transfected cells are then cultured by
conventional techniques to produce an antibody. Thus, the invention
includes host cells containing a polynucleotide encoding an
antibody of the invention or fragments thereof, or a heavy or light
chain thereof, or portion thereof, or a single-chain antibody of
the invention, operably linked to a heterologous promoter. In
certain embodiments for the expression of double-chained
antibodies, vectors encoding both the heavy and light chains may be
co-expressed in the host cell for expression of the entire
immunoglobulin molecule, as detailed below.
[0178] Mammalian cell lines available as hosts for expression of
recombinant antibodies are well known in the art and include many
immortalized cell lines available from the American Type Culture
Collection (ATCC), including but not limited to Chinese hamster
ovary (CHO) cells, HeLa cells, baby hamster kidney (BHK) cells,
monkey kidney cells (COS), human hepatocellular carcinoma cells
(e.g., Hep G2), human epithelial kidney 293 cells, and a number of
other cell lines. Different host cells have characteristic and
specific mechanisms for the post-translational processing and
modification of proteins and gene products. Appropriate cell lines
or host systems can be chosen to ensure the correct modification
and processing of the antibody or portion thereof expressed. To
this end, eukaryotic host cells which possess the cellular
machinery for proper processing of the primary transcript,
glycosylation, and phosphorylation of the gene product may be used.
Such mammalian host cells include but are not limited to CHO, VERY,
BHK, Hela, COS, MDCK, 293, 3T3, W138, BT483, Hs578T, HTB2, BT2O and
T47D, NS0 (a murine myeloma cell line that does not endogenously
produce any functional immunoglobulin chains), SP20, CRL7O3O and
HsS78Bst cells. Human cell lines developed by immortalizing human
lymphocytes can be used to recombinantly produce monoclonal
antibodies. The human cell line PER.C6. (Crucell, Netherlands) can
be used to recombinantly produce monoclonal antibodies.
[0179] Additional cell lines which may be used as hosts for
expression of recombinant antibodies include, but are not limited
to, insect cells (e.g. Sf21/Sf9, Trichoplusia ni Bti-Tn5b1-4) or
yeast cells (e.g. S. cerevisiae, Pichia, U.S. Pat. No. 7,326,681;
etc), plants cells (US20080066200); and chicken cells
(WO2008142124).
[0180] In certain embodiments, antibodies of the invention are
expressed in a cell line with stable expression of the antibody.
Stable expression can be used for long-term, high-yield production
of recombinant proteins. For example, cell lines which stably
express the antibody molecule may be generated. Host cells can be
transformed with an appropriately engineered vector comprising
expression control elements (e.g., promoter, enhancer,
transcription terminators, polyadenylation sites, etc.), and a
selectable marker gene. Following the introduction of the foreign
DNA, cells may be allowed to grow for 1-2 days in an enriched
media, and then are switched to a selective media. The selectable
marker in the recombinant plasmid confers resistance to the
selection and allows cells that stably integrated the plasmid into
their chromosomes to grow and form foci which in turn can be cloned
and expanded into cell lines. Methods for producing stable cell
lines with a high yield are well known in the art and reagents are
generally available commercially.
[0181] In certain embodiments, antibodies of the invention are
expressed in a cell line with transient expression of the antibody.
Transient transfection is a process in which the nucleic acid
introduced into a cell does not integrate into the genome or
chromosomal DNA of that cell. It is in fact maintained as an
extra-chromosomal element, e.g. as an episome, in the cell.
Transcription processes of the nucleic acid of the episome are not
affected and a protein encoded by the nucleic acid of the episome
is produced.
[0182] The cell line, either stable or transiently transfected, is
maintained in cell culture medium and conditions well known in the
art resulting in the expression and production of monoclonal
antibodies. In certain embodiments, the mammalian cell culture
media is based on commercially available media formulations,
including, for example, DMEM or Ham's F12. In other embodiments,
the cell culture media is modified to support increases in both
cell growth and biologic protein expression. As used herein, the
terms "cell culture medium," "culture medium," and "medium
formulation" refer to a nutritive solution for the maintenance,
growth, propagation, or expansion of cells in an artificial in
vitro environment outside of a multicellular organism or tissue.
Cell culture medium may be optimized for a specific cell culture
use, including, for example, cell culture growth medium which is
formulated to promote cellular growth, or cell culture production
medium which is formulated to promote recombinant protein
production. The terms nutrient, ingredient, and component are used
interchangeably herein to refer to the constituents that make up a
cell culture medium.
[0183] In one embodiment, the cell lines are maintained using a fed
batch method. As used herein, "fed batch method," refers to a
method by which a fed batch cell culture is supplied with
additional nutrients after first being incubated with a basal
medium. For example, a fed batch method may comprise adding
supplemental media according to a determined feeding schedule
within a given time period. Thus, a "fed batch cell culture" refers
to a cell culture wherein the cells, typically mammalian, and
culture medium are supplied to the culturing vessel initially and
additional culture nutrients are fed, continuously or in discrete
increments, to the culture during culturing, with or without
periodic cell and/or product harvest before termination of
culture.
[0184] The cell culture medium used and the nutrients contained
therein are known to one of skill in the art. In one embodiment,
the cell culture medium comprises a basal medium and at least one
hydrolysate, e.g., soy-based hydrolysate, a yeast-based
hydrolysate, or a combination of the two types of hydrolysates
resulting in a modified basal medium. In another embodiment, the
additional nutrients may include only a basal medium, such as a
concentrated basal medium, or may include only hydrolysates, or
concentrated hydrolysates. Suitable basal media include, but are
not limited to Dulbecco's Modified Eagle's Medium (DMEM), DME/F12,
Minimal Essential Medium (MEM), Basal Medium Eagle (BME), RPMI
1640, F-10, F-12, .alpha.-Minimal Essential Medium (.alpha.-MEM),
Glasgow's Minimal Essential Medium (G-MEM), PF CHO (see, e.g., CHO
protein free medium (Sigma) or EX-CELL.TM. 325 PF CHO Serum-Free
Medium for CHO Cells Protein-Free (SAFC Bioscience), and Iscove's
Modified Dulbecco's Medium. Other examples of basal media which may
be used in the invention include BME Basal Medium
(Gibco-Invitrogen, see also Eagle, H (1965) Proc. Soc. Exp. Biol.
Med. 89, 36); Dulbecco's Modified Eagle Medium (DMEM, powder)
(Gibco-Invitrogen (#31600); see also Dulbecco and Freeman (1959)
Virology 8, 396; Smith et al. (1960) Virology 12, 185. Tissue
Culture Standards Committee, In Vitro 6:2, 93); CMRL 1066 Medium
(Gibco-Invitrogen (#11530), see also Parker R. C. et al (1957)
Special Publications, N.Y. Academy of Sciences, 5, 303).
[0185] The basal medium may be serum-free, meaning that the medium
contains no serum (e.g., fetal bovine serum (FBS), horse serum,
goat serum, or any other animal-derived serum known to one skilled
in the art) or animal protein free media or chemically defined
media.
[0186] The basal medium may be modified in order to remove certain
non-nutritional components found in standard basal medium, such as
various inorganic and organic buffers, surfactant(s), and sodium
chloride. Removing such components from basal cell medium allows an
increased concentration of the remaining nutritional components,
and may improve overall cell growth and protein expression. In
addition, omitted components may be added back into the cell
culture medium containing the modified basal cell medium according
to the requirements of the cell culture conditions. In certain
embodiments, the cell culture medium contains a modified basal cell
medium, and at least one of the following nutrients, an iron
source, a recombinant growth factor; a buffer; a surfactant; an
osmolarity regulator; an energy source; and non-animal
hydrolysates. In addition, the modified basal cell medium may
optionally contain amino acids, vitamins, or a combination of both
amino acids and vitamins. In another embodiment, the modified basal
medium further contains glutamine, e.g, L-glutamine, and/or
methotrexate.
[0187] Antibody production can be conducted in large quantity by a
bioreactor process using fed-batch, batch, perfusion or continuous
feed bioreactor methods known in the art. Large-scale bioreactors
have at least 1000 liters of capacity, preferably about 1,000 to
100,000 liters of capacity. These bioreactors may use agitator
impellers to distribute oxygen and nutrients. Small scale
bioreactors refers generally to cell culturing in no more than
approximately 100 liters in volumetric capacity, and can range from
about 1 liter to about 100 liters. Alternatively, single-use
bioreactors (SUB) may be used for either large-scale or small-scale
culturing.
[0188] Temperature, pH, agitation, aeration and inoculum density
will vary depending upon the host cells used and the recombinant
protein to be expressed. For example, a recombinant protein cell
culture may be maintained at a temperature between 30 and
45.degree. C. The pH of the culture medium may be monitored during
the culture process such that the pH stays at an optimum level,
which may be for certain host cells, within a pH range of 6.0 to
8.0. An impellor driven mixing may be used for such culture methods
for agitation. The rotational speed of the impellor may be
approximately 50 to 200 cm/sec tip speed, but other airlift or
other mixing/aeration systems known in the art may be used,
depending on the type of host cell being cultured. Sufficient
aeration is provided to maintain a dissolved oxygen concentration
of approximately 20% to 80% air saturation in the culture, again,
depending upon the selected host cell being cultured.
Alternatively, a bioreactor may sparge air or oxygen directly into
the culture medium. Other methods of oxygen supply exist, including
bubble-free aeration systems employing hollow fiber membrane
aerators.
Phage Display Techniques
[0189] Monoclonal antibodies or antibody fragments can be isolated
from antibody phage libraries generated using the techniques
described in McCafferty et al., Nature, 348:552-554 (1990).
Clackson et al., Nature, 352:624-628 (1991) and Marks et al., J.
Mol. Biol., 222:581-597 (1991). In such methods antibodies can be
isolated by screening of a recombinant combinatorial antibody
library, preferably a scFv phage display library, prepared using
human VL and VH cDNAs prepared from mRNA derived from human
lymphocytes. Methodologies for preparing and screening such
libraries are known in the art. In addition to commercially
available kits for generating phage display libraries (e.g., the
Pharmacia Recombinant Phage Antibody System, catalog no.
27-9400-01; and the Stratagene SurfZAP.TM. phage display kit,
catalog no. 240612), examples of methods and reagents particularly
amenable for use in generating and screening antibody display
libraries can be found in, for example, U.S. Pat. Nos. 6,248,516;
6,545,142; 6,291,158; 6,291,159; 6,291,160; 6,291,161; 6,680,192;
5,969,108; 6,172,197; 6,806,079; 5,885,793; 6,521,404; 6,544,731;
6,555,313; 6,593,081; 6,582,915; 7,195,866. Thus, these techniques
are viable alternatives to traditional monoclonal antibody
hybridoma techniques for generation and isolation of monoclonal
antibodies.
[0190] In phage display methods, functional antibody domains are
displayed on the surface of phage particles which carry the
polynucleotide sequences encoding them. In a particular embodiment,
such phage can be utilized to display antigen-binding domains
expressed from a repertoire or combinatorial antibody library
(e.g., human or murine). Phage expressing an antigen binding domain
that binds the antigen of interest can be selected or identified
with antigen, e.g., using labeled antigen or antigen bound or
captured to a solid surface or bead. Phage used in these methods
are typically filamentous phage including fd and M13 binding
domains expressed from phage with Fab, Fv or disulfide stabilized
Fv antibody domains recombinantly fused to either the phage gene
Ill or gene VIII protein.
[0191] As described in the above references, after phage selection,
the antibody coding regions from the phage can be isolated and used
to generate whole antibodies, including human antibodies, humanized
antibodies, or any other desired antigen binding fragment, and
expressed in any desired host, including mammalian cells, insect
cells, plant cells, yeast, and bacteria, e.g., as described in
detail below. For example, techniques to recombinantly produce Fab,
Fab' and F(ab')2 fragments can also be employed using methods known
in the art such as those disclosed in PCT publication WO 92/22324;
Mullinax et al., BioTechniques 12(6):864-869 (1992); and Better et
al., Science 240:1041-1043 (1988).
[0192] Examples of techniques which can be used to produce
single-chain Fvs and antibodies include those described in U.S.
Pat. Nos. 4,946,778 and 5,258,498. Thus, techniques described above
and those well known in the art can be used to generate recombinant
antibodies wherein the binding domain, e.g. ScFv, was isolated from
a phage display library.
Antibody Purification and Isolation
[0193] Once an antibody molecule has been produced by recombinant
or hybridoma expression, it may be purified by any method known in
the art for purification of an immunoglobulin molecule, for
example, by chromatography (e.g., ion exchange, affinity,
particularly by affinity for the specific antigens Protein A or
Protein G, and sizing column chromatography), centrifugation,
differential solubility, or by any other standard technique for the
purification of proteins. Further, the antibodies of the present
invention or fragments thereof may be fused to heterologous
polypeptide sequences (referred to herein as "tags") to facilitate
purification.
[0194] When using recombinant techniques, the antibody can be
produced intracellularly, in the periplasmic space, or directly
secreted into the medium. If the antibody is produced
intracellularly, as a first step, the particulate debris, either
host cells or lysed fragments, is removed, for example, by
centrifugation or ultrafiltration. Carter et al., Bio/Technology,
10:163-167 (1992) describe a procedure for isolating antibodies
which are secreted into the periplasmic space of E. coli. Where the
antibody is secreted into the medium, supernatants from such
expression systems are generally first concentrated using a
commercially available protein concentration filter, for example,
an Amicon or Millipore Pellicon ultrafiltration unit. A protease
inhibitor such as PMSF may be included in any of the foregoing
steps to inhibit proteolysis and antibiotics may be included to
prevent the growth of adventitious contaminants.
[0195] The antibody composition prepared from the cells can be
purified using, for example, hydroxylapatite chromatography,
hydrophobic interaction chromatography, ion exchange
chromatography, gel electrophoresis, dialysis, and/or affinity
chromatography either alone or in combination with other
purification steps. The suitability of protein A as an affinity
ligand depends on the species and isotype of any immunoglobulin Fc
domain that is present in the antibody and will be understood by
one of skill in the art. The matrix to which the affinity ligand is
attached is most often agarose, but other matrices are available.
Mechanically stable matrices such as controlled pore glass or
poly(styrenedivinyl)benzene allow for faster flow rates and shorter
processing times than can be achieved with agarose. Where the
antibody comprises a CH.sub.3 domain, the Bakerbond ABX resin (J.
T. Baker, Phillipsburg, N.J.) is useful for purification. Other
techniques for protein purification such as fractionation on an
ion-exchange column, ethanol precipitation, Reverse Phase HPLC,
chromatography on silica, chromatography on heparin, SEPHAROSE
chromatography on an anion or cation exchange resin (such as a
polyaspartic acid column), chromatofocusing, SDS-PAGE, and ammonium
sulfate precipitation are also available depending on the antibody
to be recovered.
[0196] Following any preliminary purification step(s), the mixture
comprising the antibody of interest and contaminants may be
subjected to low pH hydrophobic interaction chromatography using an
elution buffer at a pH between about 2.5-4.5, and performed at low
salt concentrations (e.g., from about 0-0.25 M salt).
[0197] Thus, in certain embodiments is provided antibodies of the
invention that are substantially purified/isolated. In one
embodiment, these isolated/purified recombinantly expressed
antibodies may be administered to a patient to mediate a
prophylactic or therapeutic effect. A prophylactic is a medication
or a treatment designed and used to prevent a disease, disorder or
infection from occurring. A therapeutic is concerned specifically
with the treatment of a particular disease, disorder or infection.
A therapeutic dose is the amount needed to treat a particular
disease, disorder or infection. In another embodiment these
isolated/purified antibodies may be used to diagnose Influenza A
virus infection.
Human Antibodies
[0198] Human antibodies can be generated using methods well known
in the art. Human antibodies avoid some of the problems associated
with antibodies that possess murine or rat variable and/or constant
regions. The presence of such murine or rat derived proteins can
lead to the rapid clearance of the antibodies or can lead to the
generation of an immune response against the antibody by a
patient.
[0199] Human antibodies can be derived by in vitro methods.
Suitable examples include but are not limited to phage display
(MedImmune (formerly CAT), Morphosys, Dyax, Biosite/Medarex, Xoma,
Symphogen, Alexion (formerly Proliferon), Affimed) ribosome display
(MedImmune (formerly CAT)), yeast display, and the like. The phage
display technology (See e.g., U.S. Pat. No. 5,969,108) can be used
to produce human antibodies and antibody fragments in vitro, from
immunoglobulin variable (V) domain gene repertoires from
unimmunized donors. According to this technique, antibody V domain
genes are cloned in-frame into either a major or minor coat protein
gene of a filamentous bacteriophage, such as M13 or fd, and
displayed as functional antibody fragments on the surface of the
phage particle. Because the filamentous particle contains a
single-stranded DNA copy of the phage genome, selections based on
the functional properties of the antibody also result in selection
of the gene encoding the antibody exhibiting those properties.
Thus, the phage mimics some of the properties of the B-cell. Phage
display can be performed in a variety of formats, reviewed in,
e.g., Johnson, Kevin S. and Chiswell, David J., Current Opinion in
Structural Biology 3:564-571 (1993). Several sources of V-gene
segments can be used for phage display. Clackson et al., Nature,
352:624-628 (1991) isolated a diverse array of anti-oxazolone
antibodies from a small random combinatorial library of V genes
derived from the spleens of immunized mice. A repertoire of V genes
from unimmunized human donors can be constructed and antibodies to
a diverse array of antigens (including self-antigens) can be
isolated essentially following the techniques described by Marks et
al., J. Mol. Biol. 222:581-597 (1991), or Griffith et al., EMBO J.
12:725-734 (1993). See, also, U.S. Pat. Nos. 5,565,332 and
5,573,905.
[0200] As discussed above, human antibodies may also be generated
by in vitro activated B cells (see U.S. Pat. Nos. 5,567,610 and
5,229,275).
[0201] Immunoglobulin genes undergo various modifications during
maturation of the immune response, including recombination between
V, D and J gene segments, isotype switching, and hypermutation in
the variable regions. Recombination and somatic hypermutation are
the foundation for generation of antibody diversity and affinity
maturation, but they can also generate sequence liabilities that
may make commercial production of such immunoglobulins as
therapeutic agents difficult or increase the immunogenicity risk of
the antibody. In general, mutations in CDR regions are likely to
contribute to improved affinity and function, while mutations in
framework regions may increase the risk of immunogenicity. This
risk can be reduced by reverting framework mutations to germline
while ensuring that activity of the antibody is not adversely
impacted. The diversification processes may also generate some
structural liabilities or these structural liabilities may exist
within germline sequences contributing to the heavy and light chain
variable domains. Regardless of the source, it may be desirable to
remove potential structural liabilities that may result in
instability, aggregation, heterogeneity of product, or increased
immunogenicity. Examples of undesirable liabilities include
unpaired cysteines (which may lead to disulfide bond scrambling, or
variable sulfhydryl adduct formation), N-linked glycosylation sites
(resulting in heterogeneity of structure and activity), as well as
deamidation (e.g. NG, NS), isomerization (DG), oxidation (exposed
methionine), and hydrolysis (DP) sites.
[0202] Accordingly, in order to reduce the risk of immunogenicity
and improve pharmaceutical properties, it may be desirable to
revert a framework sequence to germline, revert a CDR to germline,
and/or remove a structural liability.
[0203] Thus, in one embodiment, where a particular antibody differs
from its respective germline sequence at the amino acid level, the
antibody sequence can be mutated back to the germline sequence.
Such corrective mutations can occur at one, two, three or more
positions, or a combination of any of the mutated positions, using
standard molecular biological techniques.
Antibody Fragments
[0204] In certain embodiments, the present antibodies are antibody
fragments or antibodies comprising these fragments. The antibody
fragment comprises a portion of the full length antibody, which
generally is the antigen binding or variable region thereof.
Examples of antibody fragments include Fab, Fab', F(ab').sub.2, Fd
and Fv fragments. Diabodies; linear antibodies (U.S. Pat. No.
5,641,870) and single-chain antibody molecules.
[0205] Traditionally, these fragments were derived via proteolytic
digestion of intact antibodies using techniques well known in the
art. However, these fragments can now be produced directly by
recombinant host cells. Fab, Fv and scFv antibody fragments can all
be expressed in and secreted from E. coli, thus allowing the facile
production of large amounts of these fragments. In one embodiment,
the antibody fragments can be isolated from the antibody phage
libraries discussed above. Alternatively, Fab'-SH fragments can
also be directly recovered from E. coli and chemically coupled to
form F(ab').sub.2 fragments (Carter et al., Bio/Technology,
10:163-167 (1992)). According to another approach, F(ab').sub.2
fragments can be isolated directly from recombinant host cell
culture. Other techniques for the production of antibody fragments
will be apparent to the skilled practitioner. In other embodiments,
the antibody of choice is a single-chain Fv fragment (scFv). In
certain embodiments, the antibody is not a Fab fragment. Fv and
scFv are the only species with intact combining sites that are
devoid of constant regions; thus, they are suitable for reduced
nonspecific binding during in vivo use. scFv fusion proteins may be
constructed to yield fusion of an effector protein at either the
amino or the carboxy terminus of an scFv.
[0206] In certain embodiments, the present antibodies are domain
antibodies, e.g., antibodies containing the small functional
binding units of antibodies, corresponding to the variable regions
of the heavy (VH) or light (VL) chains of human antibodies.
Examples of domain antibodies include, but are not limited to,
those of Domantis (see, for example, WO04/058821; WO04/081026;
WO04/003019; WO03/002609; U.S. Pat. Nos. 6,291,158; 6,582,915;
6,696,245; and 6,593,081).
[0207] In certain embodiments of the invention, the present
antibodies are linear antibodies. Linear antibodies comprise a pair
of tandem Fd segments (VH-CH1-VH-CH1) which form a pair of
antigen-binding regions. See, Zapata et al., Protein Eng.,
8(10):1057-1062 (1995).
Other Amino Acid Sequence Modifications
[0208] In addition to the above described human, humanized and/or
chimeric antibodies, the present invention also encompasses further
modifications and, their variants and fragments thereof, of the
antibodies of the invention comprising one or more amino acid
residues and/or polypeptide substitutions, additions and/or
deletions in the variable light (VL) domain and/or variable heavy
(VH) domain and/or Fc region and post translational modifications.
Included in these modifications are antibody conjugates wherein an
antibody has been covalently attached to a moiety. Moieties
suitable for attachment to the antibodies include but are not
limited to, proteins, peptides, drugs, labels, and cytotoxins.
These changes to the antibodies may be made to alter or fine tune
the characteristics (biochemical, binding and/or functional) of the
antibodies as is appropriate for treatment and/or diagnosis of
Influenza A infection. Methods for forming conjugates, making amino
acid and/or polypeptide changes and post-translational
modifications are well known in the art, some of which are detailed
below.
[0209] Amino acid changes to the antibodies necessarily results in
sequences that are less than 100% identical to the above identified
antibody sequences or parent antibody sequence. In certain
embodiments, in this context, the antibodies many have about 25% to
about 95% sequence identity to the amino acid sequence of either
the heavy or light chain variable domain of an antibody as
described herein. Thus, in one embodiment a modified antibody may
have an amino acid sequence having at least 25%, 35%, 45%, 55%,
65%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% amino acid
sequence identity or similarity with the amino acid sequence of
either the heavy or light chain variable domain of an antibody as
described herein. In another embodiment, an altered antibody may
have an amino acid sequence having at least 25%, 35%, 45%, 55%,
65%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% amino acid
sequence identity or similarity with the amino acid sequence of the
heavy or light chain CDR1, CDR2, or CDR3 of an antibody as
described herein. In another embodiment, an altered antibody may
have an amino acid sequence having at least 25%, 35%, 45%, 55%,
65%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% amino acid
sequence identity or similarity with the amino acid sequence of the
heavy or light chain FR1, FR2, FR3 or FR4 of an antibody as
described herein.
[0210] In certain embodiments, altered antibodies are generated by
one or more amino acid alterations (e.g., substitutions, deletion
and/or additions) introduced in one or more of the variable regions
of the antibody. In another embodiment, the amino acid alterations
are introduced in the framework regions. One or more alterations of
framework region residues may result in an improvement in the
binding affinity of the antibody for the antigen. This may be
especially true when these changes are made to humanized antibodies
wherein the framework region may be from a different species than
the CDR regions. Examples of framework region residues to modify
include those which non-covalently bind antigen directly (Amit et
al., Science, 233:747-753 (1986)); interact with/effect the
conformation of a CDR (Chothia et al., J. Mol. Biol., 196:901-917
(1987)); and/or participate in the VL-VH interface (U.S. Pat. Nos.
5,225,539 and 6,548,640). In one embodiment, from about one to
about five framework residues may be altered. Sometimes, this may
be sufficient to yield an antibody mutant suitable for use in
preclinical trials, even where none of the hypervariable region
residues have been altered. Normally, however, an altered antibody
will comprise additional hypervariable region alteration(s).
[0211] One useful procedure for generating altered antibodies is
called "alanine scanning mutagenesis" (Cunningham and Wells,
Science, 244:1081-1085 (1989)). In this method, one or more of the
hypervariable region residue(s) are replaced by alanine or
polyalanine residue(s) to alter the interaction of the amino acids
with the target antigen. Those hypervariable region residue(s)
demonstrating functional sensitivity to the substitutions then are
refined by introducing additional or other mutations at or for the
sites of substitution. Thus, while the site for introducing an
amino acid sequence variation is predetermined, the nature of the
mutation per se need not be predetermined. The Ala-mutants produced
this way are screened for their biological activity as described
herein.
[0212] In certain embodiments the substitutional variant involves
substituting one or more hypervariable region residues of a parent
antibody (e.g. a humanized or human antibody). Generally, the
resulting variant(s) selected for further development will have
improved biological properties relative to the parent antibody from
which they are generated. A convenient way for generating such
substitutional variants involves affinity maturation using phage
display (Hawkins et al., J. Mol. Biol., 254:889-896 (1992) and
Lowman et al., Biochemistry, 30(45):10832-10837 (1991)). Briefly,
several hypervariable region sites (e.g., 6-7 sites) are mutated to
generate all possible amino acid substitutions at each site. The
antibody mutants thus generated are displayed in a monovalent
fashion from filamentous phage particles as fusions to the gene III
product of M13 packaged within each particle. The phage-displayed
mutants are then screened for their biological activity (e.g.,
binding affinity) as herein disclosed.
[0213] Mutations in antibody sequences may include substitutions,
deletions, including internal deletions, additions, including
additions yielding fusion proteins, or conservative substitutions
of amino acid residues within and/or adjacent to the amino acid
sequence, but that result in a "silent" change, in that the change
produces a functionally-equivalent antibody. Conservative amino
acid substitutions may be made on the basis of similarity in
polarity, charge, solubility, hydrophobicity, hydrophilicity,
and/or the amphipathic nature of the residues involved. For
example, non-polar (hydrophobic) amino acids include alanine,
leucine, isoleucine, valine, proline, phenylalanine, tryptophan,
and methionine; polar neutral amino acids include glycine, serine,
threonine, cysteine, tyrosine, asparagine, and glutamine;
positively charged (basic) amino acids include arginine, lysine,
and histidine; and negatively charged (acidic) amino acids include
aspartic acid and glutamic acid. In addition, glycine and proline
are residues that can influence chain orientation. Non-conservative
substitutions will entail exchanging a member of one of these
classes for a member of another class. Furthermore, if desired,
non-classical amino acids or chemical amino acid analogs can be
introduced as a substitution or addition into the antibody
sequence. Non-classical amino acids include, but are not limited
to, the D-isomers of the common amino acids, .alpha.-amino
isobutyric acid, 4-aminobutyric acid, Abu, 2-amino butyric acid,
.gamma.-Abu, .epsilon.-Ahx, 6-amino hexanoic acid, Aib, 2-amino
isobutyric acid, 3-amino propionic acid, ornithine, norleucine,
norvaline, hydroxyproline, sarcosine, citrulline, cysteic acid,
t-butylglycine, t-butylalanine, phenylglycine, cyclohexylalanine,
.beta.-alanine, fluoro-amino acids, designer amino acids such as
.beta.-methyl amino acids, C.alpha.-methyl amino acids,
N.alpha.-methyl amino acids, and amino acid analogs in general.
[0214] In another embodiment, any cysteine residue not involved in
maintaining the proper conformation of the antibody also may be
substituted, generally with serine, to improve the oxidative
stability of the molecule and prevent aberrant crosslinking.
Conversely, cysteine bond(s) may be added to the antibody to
improve its stability (particularly where the antibody is an
antibody fragment such as an Fv fragment).
Variant Fc Regions
[0215] It is known that variants of the Fc region (e.g., amino acid
substitutions and/or additions and/or deletions) enhance or
diminish effector function of the antibody (See e.g., U.S. Pat.
Nos. 5,624,821; 5,885,573; 6,538,124; 7,317,091; 5,648,260;
6,538,124; WO 03/074679; WO 04/029207; WO 04/099249; WO 99/58572;
US Publication No. 2006/0134105; 2004/0132101; 2006/0008883) and
may alter the pharmacokinetic properties (e.g. half-life) of the
antibody (see, U.S. Pat. Nos. 6,277,375 and 7,083,784). Thus, in
certain embodiments, the antibodies of the invention comprise an
altered Fc region (also referred to herein as "variant Fc region")
in which one or more alterations have been made in the Fc region in
order to change functional and/or pharmacokinetic properties of the
antibodies. Such alterations may result in a decrease or increase
of Clq binding and complement dependent cytotoxicity (CDC) or of
Fc.gamma.R binding, for IgG, and antibody-dependent cellular
cytotoxicity (ADCC), or antibody dependent cell-mediated
phagocytosis (ADCP). The present invention encompasses the
antibodies described herein with variant Fc regions wherein changes
have been made to fine tune the effector function, enhancing or
diminishing, providing a desired effector function. Accordingly,
the antibodies of the invention comprise a variant Fc region (i.e.,
Fc regions that have been altered as discussed below). Antibodies
of the invention comprising a variant Fc region are also referred
to here as "Fc variant antibodies." As used herein native refers to
the unmodified parental sequence and the antibody comprising a
native Fc region is herein referred to as a "native Fc antibody".
Fc variant antibodies can be generated by numerous methods well
known to one skilled in the art. Non-limiting examples include,
isolating antibody coding regions (e.g., from hybridoma) and making
one or more desired substitutions in the Fc region of the isolated
antibody coding region. Alternatively, the antigen-binding portion
(e.g., variable regions) of an antibody may be sub-cloned into a
vector encoding a variant Fc region. In one embodiment, the variant
Fc region exhibits a similar level of inducing effector function as
compared to the native Fc region. In another embodiment, the
variant Fc region exhibits a higher induction of effector function
as compared to the native Fc. Some specific embodiments of variant
Fc regions are detailed infra. Methods for measuring effector
function are well known in the art.
[0216] The effector function of an antibody is modified through
changes in the Fc region, including but not limited to, amino acid
substitutions, amino acid additions, amino acid deletions and
changes in post-translational modifications to Fc amino acids (e.g.
glycosylation). The methods described below may be used to fine
tune the effector function of a present antibody, a ratio of the
binding properties of the Fc region for the FcR (e.g., affinity and
specificity), resulting in a therapeutic antibody with the desired
properties.
[0217] It is understood that the Fc region as used herein includes
the polypeptides comprising the constant region of an antibody
excluding the first constant region immunoglobulin domain. Thus Fc
refers to the last two constant region immunoglobulin domains of
IgA, IgD, and IgG, and the last three constant region
immunoglobulin domains of IgE and IgM, and the flexible hinge
N-terminal to these domains. For IgA and IgM Fc may include the J
chain. For IgG, Fc comprises immunoglobulin domains Cgamma2 and
Cgamma3 (C.gamma.2 and C.gamma.3) and the hinge between Cgamma1
(C.gamma.1) and Cgamma2 (C.gamma.2). Although the boundaries of the
Fc region may vary, the human IgG heavy chain Fc region is usually
defined to comprise residues C226 or P230 to its carboxyl-terminus,
wherein the numbering is according to the EU index as set forth in
Kabat. Fc may refer to this region in isolation, or this region in
the context of an antibody, antibody fragment, or Fc fusion
protein. Polymorphisms have been observed at a number of different
Fc positions, including but not limited to positions 270, 272, 312,
315, 356, and 358 as numbered by the EU index, and thus slight
differences between the presented sequence and sequences in the
prior art may exist.
[0218] In one embodiment, Fc variant antibodies exhibit altered
binding affinity for one or more Fc receptors including, but not
limited to FcRn, Fc.gamma.RI (CD64) including isoforms
Fc.gamma.RIA, Fc.gamma.RIB, and Fc.gamma.RIC, Fc.gamma.RII (CD32
including isoforms Fc.gamma.RIIA, Fc.gamma.RIIB, and
Fc.gamma.RIIC), and Fc.gamma.RIII (CD16, including isoforms
Fc.gamma.RIIIA and Fc.gamma.RIIIB) as compared to an native Fc
antibody.
[0219] In one embodiment, an Fc variant antibody has enhanced
binding to one or more Fc ligand relative to a native Fc antibody.
In another embodiment, the Fc variant antibody exhibits increased
or decreased affinity for an Fc ligand that is at least 2 fold, or
at least 3 fold, or at least 5 fold, or at least 7 fold, or a least
10 fold, or at least 20 fold, or at least 30 fold, or at least 40
fold, or at least 50 fold, or at least 60 fold, or at least 70
fold, or at least 80 fold, or at least 90 fold, or at least 100
fold, or at least 200 fold, or is between 2 fold and 10 fold, or
between 5 fold and 50 fold, or between 25 fold and 100 fold, or
between 75 fold and 200 fold, or between 100 and 200 fold, more or
less than a native Fc antibody. In another embodiment, Fc variant
antibodies exhibit affinities for an Fc ligand that are at least
90%, at least 80%, at least 70%, at least 60%, at least 50%, at
least 40%, at least 30%, at least 20%, at least 10%, or at least 5%
more or less than an native Fc antibody. In certain embodiments, an
Fc variant antibody has increased affinity for an Fc ligand. In
other embodiments, an Fc variant antibody has decreased affinity
for an Fc ligand.
[0220] In a specific embodiment, an Fc variant antibody has
enhanced binding to the Fc receptor Fc.gamma.RIIIA. In another
specific embodiment, an Fc variant antibody has enhanced binding to
the Fc receptor Fc.gamma.RIIB. In a further specific embodiment, an
Fc variant antibody has enhanced binding to both the Fc receptors
Fc.gamma.RIIIA and Fc.gamma.RIIB. In certain embodiments, Fc
variant antibodies that have enhanced binding to Fc.gamma.RIIIA do
not have a concomitant increase in binding the Fc.gamma.RIIB
receptor as compared to a native Fc antibody. In a specific
embodiment, an Fc variant antibody has reduced binding to the Fc
receptor Fc.gamma.RIIIA. In a further specific embodiment, an Fc
variant antibody has reduced binding to the Fc receptor
Fc.gamma.RIIB. In still another specific embodiment, an Fc variant
antibody exhibiting altered affinity for Fc.gamma.RIIIA and/or
Fc.gamma.RIIB has enhanced binding to the Fc receptor FcRn. In yet
another specific embodiment, an Fc variant antibody exhibiting
altered affinity for Fc.gamma.RIIIA and/or Fc.gamma.RIIB has
altered binding to C1q relative to a native Fc antibody.
[0221] In one embodiment, Fc variant antibodies exhibit affinities
for Fc.gamma.RIIIA receptor that are at least 2 fold, or at least 3
fold, or at least 5 fold, or at least 7 fold, or a least 10 fold,
or at least 20 fold, or at least 30 fold, or at least 40 fold, or
at least 50 fold, or at least 60 fold, or at least 70 fold, or at
least 80 fold, or at least 90 fold, or at least 100 fold, or at
least 200 fold, or are between 2 fold and 10 fold, or between 5
fold and 50 fold, or between 25 fold and 100 fold, or between 75
fold and 200 fold, or between 100 and 200 fold, more or less than
an native Fc antibody. In another embodiment, Fc variant antibodies
exhibit affinities for Fc.gamma.RIIIA that are at least 90%, at
least 80%, at least 70%, at least 60%, at least 50%, at least 40%,
at least 30%, at least 20%, at least 10%, or at least 5% more or
less than an native Fc antibody.
[0222] In one embodiment, Fc variant antibodies exhibit affinities
for Fc.gamma.RIIB receptor that are at least 2 fold, or at least 3
fold, or at least 5 fold, or at least 7 fold, or a least 10 fold,
or at least 20 fold, or at least 30 fold, or at least 40 fold, or
at least 50 fold, or at least 60 fold, or at least 70 fold, or at
least 80 fold, or at least 90 fold, or at least 100 fold, or at
least 200 fold, or are between 2 fold and 10 fold, or between 5
fold and 50 fold, or between 25 fold and 100 fold, or between 75
fold and 200 fold, or between 100 and 200 fold, more or less than
an native Fc antibody. In another embodiment, Fc variant antibodies
exhibit affinities for Fc.gamma.RIIB that are at least 90%, at
least 80%, at least 70%, at least 60%, at least 50%, at least 40%,
at least 30%, at least 20%, at least 10%, or at least 5% more or
less than an native Fc antibody.
[0223] In one embodiment, Fc variant antibodies exhibit increased
or decreased affinities to C1 q relative to a native Fc antibody.
In another embodiment, Fc variant antibodies exhibit affinities for
C1 q receptor that are at least 2 fold, or at least 3 fold, or at
least 5 fold, or at least 7 fold, or a least 10 fold, or at least
20 fold, or at least 30 fold, or at least 40 fold, or at least 50
fold, or at least 60 fold, or at least 70 fold, or at least 80
fold, or at least 90 fold, or at least 100 fold, or at least 200
fold, or are between 2 fold and 10 fold, or between 5 fold and 50
fold, or between 25 fold and 100 fold, or between 75 fold and 200
fold, or between 100 and 200 fold, more or less than an native Fc
antibody. In another embodiment, Fc variant antibodies exhibit
affinities for Clq that are at least 90%, at least 80%, at least
70%, at least 60%, at least 50%, at least 40%, at least 30%, at
least 20%, at least 10%, or at least 5% more or less than an native
Fc antibody. In still another specific embodiment, an Fc variant
antibody exhibiting altered affinity for Ciq has enhanced binding
to the Fc receptor FcRn. In yet another specific embodiment, an Fc
variant antibody exhibiting altered affinity for C1q has altered
binding to Fc.gamma.RIIIA and/or Fc.gamma.RIIB relative to a native
Fc antibody.
[0224] It is well known in the art that antibodies are capable of
directing the attack and destruction through multiple processes
collectively known in the art as antibody effector functions. One
of these processes, known as "antibody-dependent cell-mediated
cytotoxicity" or "ADCC" refers to a form of cytotoxicity in which
secreted Ig bound onto Fc receptors (FcRs) present on certain
cytotoxic cells (e.g., Natural Killer (NK) cells, neutrophils, and
macrophages) enables these cytotoxic effector cells to bind
specifically to an antigen-bearing cells and subsequently kill the
cells with cytotoxins. Specific high-affinity IgG antibodies
directed to the surface of cells "arm" the cytotoxic cells and are
required for such killing. Lysis of the cell is extracellular,
requires direct cell-to-cell contact, and does not involve
complement.
[0225] Another process encompassed by the term effector function is
complement dependent cytotoxicity (hereinafter referred to as
"CDC") which refers to a biochemical event of cell destruction by
the complement system. The complement system is a complex system of
proteins found in normal blood plasma that combines with antibodies
to destroy pathogenic bacteria and other foreign cells.
[0226] Still another process encompassed by the term effector
function is antibody dependent cell-mediated phagocytosis (ADCP)
which refers to a cell-mediated reaction wherein nonspecific
cytotoxic cells that express one or more effector ligands recognize
bound antibody on a cell and subsequently cause phagocytosis of the
cell.
[0227] It is contemplated that Fc variant antibodies are
characterized by in vitro functional assays for determining one or
more Fc.gamma.R mediated effector cell functions. In certain
embodiments, Fc variant antibodies have similar binding properties
and effector cell functions in in vivo models (such as those
described and disclosed herein) as those in in vitro based assays.
However, the present invention does not exclude Fc variant
antibodies that do not exhibit the desired phenotype in in vitro
based assays but do exhibit the desired phenotype in vivo.
[0228] In certain embodiments, an antibody comprising an Fc variant
has enhanced cytotoxicity or phagocytosis activity (e.g., ADCC, CDC
and ADCP) relative to an antibody comprising a native Fc region. In
a specific embodiment, an Fc variant antibody has cytotoxicity or
phagocytosis activity that is at least 2 fold, or at least 3 fold,
or at least 5 fold or at least 10 fold or at least 50 fold or at
least 100 fold, or at least 200 fold, or is between 2 fold and 10
fold, or between 5 fold and 50 fold, or between 25 fold and 100
fold, or between 75 fold and 200 fold, or between 100 and 200 fold,
greater than that of a native Fc antibody. Alternatively, an Fc
variant antibody has reduced cytotoxicity or phagocytosis activity
relative to a native Fc antibody. In a specific embodiment, an Fc
variant antibody has cytotoxicity or phagocytosis activity that is
at least 2 fold, or at least 3 fold, or at least 5 fold or at least
10 fold or at least 50 fold or at least 100 fold, or at least 200
fold, or is between 2 fold and 10 fold, or between 5 fold and 50
fold, or between 25 fold and 100 fold, or between 75 fold and 200
fold, or between 100 and 200 fold, lower than that of a native Fc
antibody.
[0229] In certain embodiments, Fc variant antibodies exhibit
decreased ADCC activities as compared to a native Fc antibody. In
another embodiment, Fc variant antibodies exhibit ADCC activities
that are at least 2 fold, or at least 3 fold, or at least 5 fold or
at least 10 fold or at least 50 fold or at least 100 fold, or at
least 200 fold, or is between 2 fold and 10 fold, or between 5 fold
and 50 fold, or between 25 fold and 100 fold, or between 75 fold
and 200 fold, or between 100 and 200 fold, less than that of a
native Fc antibody. In still another embodiment, Fc variant
antibodies exhibit ADCC activities that are reduced by at least
10%, or at least 20%, or by at least 30%, or by at least 40%, or by
at least 50%, or by at least 60%, or by at least 70%, or by at
least 80%, or by at least 90%, or by at least 100%, or by at least
200%, or by at least 300%, or by at least 400%, or by at least
500%, relative to a native Fc antibody. In certain embodiments, Fc
variant antibodies have no detectable ADCC activity. In specific
embodiments, the reduction and/or ablatement of ADCC activity may
be attributed to the reduced affinity Fc variant antibodies exhibit
for Fc ligands and/or receptors.
[0230] In an alternative embodiment, Fc variant antibodies exhibit
increased ADCC activities as compared to a native Fc antibody. In
another embodiment, Fc variant antibodies exhibit ADCC activities
that are at least 2 fold, or at least 3 fold, or at least 5 fold or
at least 10 fold or at least 50 fold or at least 100 fold greater
than that of a native Fc antibody. In still another embodiment, Fc
variant antibodies exhibit ADCC activities that are increased by at
least 10%, or at least 20%, or by at least 30%, or by at least 40%,
or by at least 50%, or by at least 60%, or by at least 70%, or by
at least 80%, or by at least 90%, or by at least 100%, or by at
least 200%, or by at least 300%, or by at least 400%, or by at
least 500% relative to a native Fc antibody. In specific
embodiments, the increased ADCC activity may be attributed to the
increased affinity Fc variant antibodies exhibit for Fc ligands
and/or receptors.
[0231] In a specific embodiment, an Fc variant antibody has
enhanced binding to the Fc receptor Fc.gamma.RIIIA and has enhanced
ADCC activity relative to a native Fc antibody. In other
embodiments, the Fc variant antibody has both enhanced ADCC
activity and enhanced serum half-life relative to a native Fc
antibody.
[0232] In another specific embodiment, an Fc variant antibody has
reduced binding to the Fc receptor Fc.gamma.RIIIA and has reduced
ADCC activity relative to a native Fc antibody. In other
embodiments, the Fc variant antibody has both reduced ADCC activity
and enhanced serum half-life relative to a native Fc antibody.
[0233] In certain embodiments, the cytotoxicity is mediated by CDC
wherein the Fc variant antibody has either enhanced or decreased
CDC activity relative to a native Fc antibody. The complement
activation pathway is initiated by the binding of the first
component of the complement system (C1q) to a molecule, an antibody
for example, complexed with a cognate antigen. To assess complement
activation, a CDC assay, e.g. as described in Gazzano-Santoro et
al., 1996, J. Immunol. Methods, 202:163, may be performed.
[0234] In one embodiment, antibodies of the invention exhibit
increased CDC activity as compared to a native Fc antibody. In
another embodiment, Fc variant antibodies exhibit CDC activity that
is at least 2 fold, or at least 3 fold, or at least 5 fold or at
least 10 fold or at least 50 fold or at least 100 fold, or at least
200 fold, or is between 2 fold and 10 fold, or between 5 fold and
50 fold, or between 25 fold and 100 fold, or between 75 fold and
200 fold, or between 100 and 200 fold more than that of an native
Fc antibody. In still another embodiment, Fc variant antibodies
exhibit CDC activity that is increased by at least 10%, or at least
20%, or by at least 30%, or by at least 40%, or by at least 50%, or
by at least 60%, or by at least 70%, or by at least 80%, or by at
least 90%, or by at least 100%, or by at least 200%, or by at least
300%, or by at least 400%, or by at least 500% relative to a native
Fc antibody. In specific embodiments, the increase of CDC activity
may be attributed to the increased affinity Fc variant antibodies
exhibit for Cl q.
[0235] Antibodies of the invention may exhibit increased CDC
activity as compared to a native Fc antibody by virtue of
COMPLEGENT.RTM. Technology (Kyowa Hakko Kirin Co., Ltd.), which
enhances one of the major mechanisms of action of an antibody, CDC.
With an approach called isotype chimerism, in which portions of
IgG3, an antibody's isotype, are introduced into corresponding
regions of IgG1, the standard isotype for therapeutic antibodies,
COMPLEGENT.RTM. Technology significantly enhances CDC activity
beyond that of either IgG1 or IgG3, while retaining the desirable
features of IgG1, such as ADCC, PK profile and Protein A binding.
In addition, it can be used together with POTELLIGENT.RTM.
Technology, creating an even superior therapeutic Mab
(ACCRETAMAB.RTM.) with enhanced ADCC and CDC activities
[0236] Fc variant antibody of the invention may have enhanced ADCC
activity and enhanced serum half-life relative to a native Fc
antibody.
[0237] Fc variant antibody of the invention may CDC activity and
enhanced serum half life relative to a native Fc antibody.
[0238] Fc variant antibody of the invention may have enhanced ADCC
activity, enhanced CDC activity and enhanced serum half-life
relative to a native Fc antibody.
[0239] The serum half-life of proteins comprising Fc regions may be
increased by increasing the binding affinity of the Fc region for
FcRn. The term "antibody half-life" as used herein means a
pharmacokinetic property of an antibody that is a measure of the
mean survival time of antibody molecules following their
administration. Antibody half-life can be expressed as the time
required to eliminate 50 percent of a known quantity of
immunoglobulin from the patient's body (or other mammal) or a
specific compartment thereof, for example, as measured in serum,
i.e., circulating half-life, or in other tissues. Half-life may
vary from one immunoglobulin or class of immunoglobulin to another.
In general, an increase in antibody half-life results in an
increase in mean residence time (MRT) in circulation for the
antibody administered.
[0240] The increase in half-life allows for the reduction in amount
of drug given to a patient as well as reducing the frequency of
administration. To increase the serum half-life of the antibody,
one may incorporate a salvage receptor binding epitope into the
antibody (especially an antibody fragment) as described in U.S.
Pat. No. 5,739,277, for example. As used herein, the term "salvage
receptor binding epitope" refers to an epitope of the Fc region of
an IgG molecule (e.g., IgG1, IgG2, IgG3, or IgG4) that is
responsible for increasing the in vivo serum half-life of the IgG
molecule.
[0241] Alternatively, antibodies of the invention with increased
half-lives may be generated by modifying amino acid residues
identified as involved in the interaction between the Fc and the
FcRn receptor (see, for examples, U.S. Pat. Nos. 6,821,505 and
7,083,784; and WO 09/058492). In addition, the half-life of
antibodies of the invention may be increased by conjugation to PEG
or Albumin by techniques widely utilized in the art. In some
embodiments antibodies comprising Fc variant regions of the
invention have an increased half-life of about 5%, about 10%, about
15%, about 20%, about 25%, about 30%, about 35%, about 40%, about
45%, about 50%, about 60%, about 65%, about 70%, about 80%, about
85%, about 90%, about 95%, about 100%, about 125%, about 150% or
more as compared to an antibody comprising a native Fc region. In
some embodiments antibodies comprising Fc variant regions have an
increased half-life of about 2 fold, about 3 fold, about 4 fold,
about 5 fold, about 10 fold, about 20 fold, about 50 fold or more,
or is between 2 fold and 10 fold, or between 5 fold and 25 fold, or
between 15 fold and 50 fold, as compared to an antibody comprising
a native Fc region.
[0242] In one embodiment, the present invention provides Fc
variants, wherein the Fc region comprises a modification (e.g.,
amino acid substitutions, amino acid insertions, amino acid
deletions) at one or more positions selected from the group
consisting of 221, 225, 228, 234, 235, 236, 237, 238, 239, 240,
241, 243, 244, 245, 247, 250, 251, 252, 254, 255, 256, 257, 262,
263, 264, 265, 266, 267, 268, 269, 279, 280, 284, 292, 296, 297,
298, 299, 305, 308, 313, 316, 318, 320, 322, 325, 326, 327, 328,
329, 330, 331, 332, 333, 334, 339, 341, 343, 370, 373, 378, 392,
416, 419, 421, 428, 433, 434, 435, 436, 440, and 443 as numbered by
the EU index as set forth in Kabat. Optionally, the Fc region may
comprise a modification at additional and/or alternative positions
known to one skilled in the art (see, e.g., U.S. Pat. Nos.
5,624,821; 6,277,375; 6,737,056; 7,083,784; 7,317,091; 7,217,797;
7,276,585; 7,355,008; 2002/0147311; 2004/0002587; 2005/0215768;
2007/0135620; 2007/0224188; 2008/0089892; WO 94/29351; and WO
99/58572). Additional, useful amino acid positions and specific
substitutions are exemplified in Tables 2, and 6-10 of U.S. Pat.
No. 6,737,056; the tables presented in FIG. 41 of US 2006/024298;
the tables presented in FIGS. 5, 12, and 15 of US 2006/235208; the
tables presented in FIGS. 8, 9 and 10 of US 2006/0173170 and the
tables presented in FIGS. 8-10, 13 and 14 of WO 09/058492.
[0243] In a specific embodiment, the present invention provides an
Fc variant, wherein the Fc region comprises at least one
substitution selected from the group consisting of 221K, 221Y,
225E, 225K, 225W, 228P, 234D, 234E, 234N, 234Q, 234T, 234H, 234Y,
234I, 234V, 234F, 235A, 235D, 235R, 235W, 235P, 235S, 235N, 235Q,
235T, 235H, 235Y, 235I, 235V, 235E, 235F, 236E, 237L, 237M, 237P,
239D, 239E, 239N, 239Q, 239F, 239T, 239H, 239Y, 240I, 240A, 240T,
240M, 241W, 241L, 241Y, 241E, 241R. 243W, 243L 243Y, 243R, 243Q,
244H, 245A, 247L, 247V, 247G, 250E, 250Q, 251F, 252L, 252Y, 254S,
254T, 255L, 256E, 256F, 256M, 257C, 257M, 257N, 262I, 262A, 262T,
262E, 263I, 263A, 263T, 263M, 264L, 264I, 264W, 264T, 264R, 264F,
264M, 264Y, 264E, 265A, 265G, 265N, 265Q, 265Y, 265F, 265V, 265I,
265L, 265H, 265T, 266I, 266A, 266T, 266M, 267Q, 267L, 268E, 269H,
269Y, 269F, 269R, 270E, 280A, 284M, 292P, 292L, 296E, 296Q, 296D,
296N, 296S, 296T, 296L, 296I, 296H, 296G, 297S, 297D, 297E, 298A,
298H, 298I, 298T, 298F, 299I, 299L, 299A, 299S, 299V, 299H, 299F,
299E, 305I, 308F, 313F, 316D, 318A, 318S, 320A, 320S, 322A, 322S,
325Q, 325L, 325I, 325D, 325E, 325A, 325T, 325V, 325H, 326A, 326D,
326E, 326G, 326M, 326V, 327G, 327W, 327N, 327L, 328S, 328M, 328D,
328E, 328N, 328Q, 328F, 328I, 328V, 328T, 328H, 328A, 329F, 329H,
329Q, 330K, 330G, 330T, 330C, 330L, 330Y, 330V, 330I, 330F, 330R,
330H, 331G, 331A, 331L, 331M, 331F, 331W, 331K, 331Q, 331E, 331S,
331V, 331I, 331C, 331Y, 331H, 331R, 331N, 331D, 331T, 332D, 332S,
332W, 332F, 332E, 332N, 332Q, 332T, 332H, 332Y, 332A, 333A, 333D,
333G, 333Q, 333S, 333V, 334A, 334E, 334H, 334L, 334M, 334Q, 334V,
334Y, 339T, 370E, 370N, 378D, 392T, 396L, 416G, 419H, 421K, 428L,
428F, 433K, 433L, 434A, 424F, 434W, 434Y, 436H, 440Y and 443W as
numbered by the EU index as set forth in Kabat. Optionally, the Fc
region may comprise additional and/or alternative amino acid
substitutions known to one skilled in the art including but not
limited to those exemplified in Tables 2, and 6-10 of U.S. Pat. No.
6,737,056; the tables presented in FIG. 41 of US 2006/024298; the
tables presented in FIGS. 5, 12, and 15 of US 2006/235208; the
tables presented in FIGS. 8, 9 and 10 of US 2006/0173170 and the
tables presented in FIGS. 8, 9 and 10 of WO 09/058492.
[0244] In a specific embodiment, the present invention provides an
Fc variant antibody, wherein the Fc region comprises at least one
modification (e.g., amino acid substitutions, amino acid
insertions, amino acid deletions) at one or more positions selected
from the group consisting of 228, 234, 235 and 331 as numbered by
the EU index as set forth in Kabat. In one embodiment, the
modification is at least one substitution selected from the group
consisting of 228P, 234F, 235E, 235F, 235Y, and 331S as numbered by
the EU index as set forth in Kabat.
[0245] In another specific embodiment, the present invention
provides an Fc variant antibody, wherein the Fc region is an IgG4
Fc region and comprises at least one modification at one or more
positions selected from the group consisting of 228 and 235 as
numbered by the EU index as set forth in Kabat. In still another
specific embodiment, the Fc region is an IgG4 Fc region and the
non-naturally occurring amino acids are selected from the group
consisting of 228P, 235E and 235Y as numbered by the EU index as
set forth in Kabat.
[0246] In another specific embodiment, the present invention
provides an Fc variant, wherein the Fc region comprises at least
one non-naturally occurring amino acid at one or more positions
selected from the group consisting of 239, 330 and 332 as numbered
by the EU index as set forth in Kabat. In one embodiment, the
modification is at least one substitution selected from the group
consisting of 239D, 330L, 330Y, and 332E as numbered by the EU
index as set forth in Kabat.
[0247] In a specific embodiment, the present invention provides an
Fc variant antibody, wherein the Fc region comprises at least one
non-naturally occurring amino acid at one or more positions
selected from the group consisting of 252, 254, and 256 as numbered
by the EU index as set forth in Kabat. In one embodiment, the
modification is at least one substitution selected from the group
consisting of 252Y, 254T and 256E as numbered by the EU index as
set forth in Kabat. In particularly preferred antibodies of the
invention, the modification is three substitutions 252Y, 254T and
256E as numbered by the EU index as set forth in Kabat (known as
"YTE"), see U.S. Pat. No. 7,083,784.
[0248] In certain embodiments the effector functions elicited by
IgG antibodies strongly depend on the carbohydrate moiety linked to
the Fc region of the protein (Claudia Ferrara et al., 2006,
Biotechnology and Bioengineering 93:851-861). Thus, glycosylation
of the Fc region can be modified to increase or decrease effector
function (see for examples, Umana et al., 1999, Nat. Biotechnol
17:176-180; Davies et al., 2001, Biotechnol Bioeng 74:288-294;
Shields et al., 2002, J Biol Chem 277:26733-26740; Shinkawa et al.,
2003, J Biol Chem 278:3466-3473; U.S. Pat. Nos. 6,602,684;
6,946,292; 7,064,191; 7,214,775; 7,393,683; 7,425,446; 7,504,256;
U.S. Publication. Nos. 2003/0157108; 2003/0003097; 2009/0010921;
Potillegent.TM. technology (Biowa, Inc. Princeton, N.J.);
GlycoMAb.TM. glycosylation engineering technology (GLYCART
biotechnology AG, Zurich, Switzerland)). Accordingly, in one
embodiment the Fc regions of antibodies of the invention comprise
altered glycosylation of amino acid residues. In another
embodiment, the altered glycosylation of the amino acid residues
results in lowered effector function. In another embodiment, the
altered glycosylation of the amino acid residues results in
increased effector function. In a specific embodiment, the Fc
region has reduced fucosylation. In another embodiment, the Fc
region is afucosylated (see for examples, U.S. Patent Application
Publication No. 2005/0226867). In one aspect, these antibodies with
increased effector function, specifically ADCC, as generated in
host cells (e.g., CHO cells, Lemna minor) engineered to produce
highly defucosylated antibody with over 100-fold higher ADCC
compared to antibody produced by the parental cells (Mori et al.,
2004, Biotechnol Bioeng 88:901-908; Cox et al., 2006, Nat
Biotechnol., 24:1591-7).
[0249] Addition of sialic acid to the oligosaccharides on IgG
molecules can enhance their anti-inflammatory activity and alters
their cytotoxicity (Keneko et al., Science, 2006, 313:670-673;
Scallon et al., Mol. Immuno. 2007 March; 44(7):1524-34). The
studies referenced above demonstrate that IgG molecules with
increased sialylation have anti-inflammatory properties whereas IgG
molecules with reduced sialylation have increased immunostimulatory
properties (e.g., increase ADCC activity). Therefore, an antibody
can be modified with an appropriate sialylation profile for a
particular therapeutic application (US Publication No. 2009/0004179
and International Publication No. WO 2007/005786).
[0250] In one embodiment, the Fc regions of antibodies of the
invention comprise an altered sialylation profile compared to the
native Fc region. In one embodiment, the Fc regions of antibodies
of the invention comprise an increased sialylation profile compared
to the native Fc region. In another embodiment, the Fc regions of
antibodies of the invention comprise a decreased sialylation
profile compared to the native Fc region.
[0251] In one embodiment, the Fc variants of the present invention
may be combined with other known Fc variants such as those
disclosed in Ghetie et al., 1997, Nat Biotech. 15:637-40; Duncan et
al., 1988, Nature 332:563-564; Lund et al., 1991, J. Immunol
147:2657-2662; Lund et al., 1992, Mol Immunol 29:53-59; Alegre et
al, 1994, Transplantation 57:1537-1543; Hutchins et al., 1995, Proc
Natl. Acad Sci USA 92:11980-11984; Jefferis et al., 1995, Immunol
Lett. 44:111-117; Lund et al., 1995, Faseb J 9:115-119; Jefferis et
al., 1996, Immunol Lett 54:101-104; Lund et al., 1996, J Immunol
157:4963-4969; Armour et al., 1999, Eur J Immunol 29:2613-2624;
Idusogie et al., 2000, J Immunol 164:4178-4184; Reddy et al., 2000,
J Immunol 164:1925-1933; Xu et al., 2000, Cell Immunol 200:16-26;
Idusogie et al., 2001, J Immunol 166:2571-2575; Shields et al.,
2001, J Biol Chem 276:6591-6604; Jefferis et al, 2002, Immunol Lett
82:57-65; Presta et al., 2002, Biochem Soc Trans 30:487-490); U.S.
Pat. Nos. 5,624,821; 5,885,573; 5,677,425; 6,165,745; 6,277,375;
5,869,046; 6,121,022; 5,624,821; 5,648,260; 6,528,624; 6,194,551;
6,737,056; 7,122,637; 7,183,387; 7,332,581; 7,335,742; 7,371,826;
6,821,505; 6,180,377; 7,317,091; 7,355,008; 2004/0002587; and WO
99/58572. Other modifications and/or substitutions and/or additions
and/or deletions of the Fc domain will be readily apparent to one
skilled in the art.
Glycosylation
[0252] In addition to the ability of glycosylation to alter the
effector function of antibodies, modified glycosylation in the
variable region can alter the affinity of the antibody for antigen.
In one embodiment, the glycosylation pattern in the variable region
of the present antibodies is modified. For example, an aglycoslated
antibody can be made (i.e., the antibody lacks glycosylation).
Glycosylation can be altered to, for example, increase the affinity
of the antibody for antigen. Such carbohydrate modifications can be
accomplished by, for example, altering one or more sites of
glycosylation within the antibody sequence. For example, one or
more amino acid substitutions can be made that result in
elimination of one or more variable region framework glycosylation
sites to thereby eliminate glycosylation at that site. Such
aglycosylation may increase the affinity of the antibody for
antigen. Such an approach is described in further detail in U.S.
Pat. Nos. 5,714,350 and 6,350,861. One or more amino acid
substitutions can also be made that result in elimination of a
glycosylation site present in the Fc region (e.g., Asparagine 297
of IgG). Furthermore, aglycosylated antibodies may be produced in
bacterial cells which lack the necessary glycosylation
machinery.
Antibody Conjugates
[0253] In certain embodiments, the antibodies of the invention are
conjugated or covalently attached to a substance using methods well
known in the art. In one embodiment, the attached substance is a
therapeutic agent, a detectable label (also referred to herein as a
reporter molecule) or a solid support. Suitable substances for
attachment to antibodies include, but are not limited to, an amino
acid, a peptide, a protein, a polysaccharide, a nucleoside, a
nucleotide, an oligonucleotide, a nucleic acid, a hapten, a drug, a
hormone, a lipid, a lipid assembly, a synthetic polymer, a
polymeric microparticle, a biological cell, a virus, a fluorophore,
a chromophore, a dye, a toxin, a hapten, an enzyme, an antibody, an
antibody fragment, a radioisotope, solid matrixes, semi-solid
matrixes and combinations thereof. Methods for conjugation or
covalently attaching another substance to an antibody are well
known in the art.
[0254] In certain embodiments, the antibodies of the invention are
conjugated to a solid support. Antibodies may be conjugated to a
solid support as part of the screening and/or purification and/or
manufacturing process. Alternatively antibodies of the invention
may be conjugated to a solid support as part of a diagnostic method
or composition. A solid support suitable for use in the present
invention is typically substantially insoluble in liquid phases. A
large number of supports are available and are known to one of
ordinary skill in the art. Thus, solid supports include solid and
semi-solid matrixes, such as aerogels and hydrogels, resins, beads,
biochips (including thin film coated biochips), microfluidic chip,
a silicon chip, multi-well plates (also referred to as microtitre
plates or microplates), membranes, conducting and non-conducting
metals, glass (including microscope slides) and magnetic supports.
More specific examples of solid supports include silica gels,
polymeric membranes, particles, derivatized plastic films, glass
beads, cotton, plastic beads, alumina gels, polysaccharides such as
Sepharose, poly(acrylate), polystyrene, poly(acrylamide), polyol,
agarose, agar, cellulose, dextran, starch, FICOLL, heparin,
glycogen, amylopectin, mannan, inulin, nitrocellulose,
diazocellulose, polyvinylchloride, polypropylene, polyethylene
(including poly(ethylene glycol)), nylon, latex bead, magnetic
bead, paramagnetic bead, superparamagnetic bead, starch and the
like.
[0255] In some embodiments, the solid support may include a
reactive functional group, including, but not limited to, hydroxyl,
carboxyl, amino, thiol, aldehyde, halogen, nitro, cyano, amido,
urea, carbonate, carbamate, isocyanate, sulfone, sulfonate,
sulfonamide, sulfoxide, etc., for attaching the antibodies of the
invention.
[0256] A suitable solid phase support can be selected on the basis
of desired end use and suitability for various synthetic protocols.
For example, where amide bond formation is desirable to attach the
antibodies of the invention to the solid support, resins generally
useful in peptide synthesis may be employed, such as polystyrene
(e.g., PAM-resin obtained from Bachem Inc., Peninsula Laboratories,
etc.), POLYHIPE.TM. resin (obtained from Aminotech, Canada),
polyamide resin (obtained from Peninsula Laboratories), polystyrene
resin grafted with polyethylene glycol (TentaGel.TM., Rapp
Polymere, Tubingen, Germany), polydimethyl-acrylamide resin
(available from Milligen/Biosearch, California), or PEGA beads
(obtained from Polymer Laboratories).
[0257] In certain embodiments, the antibodies of the invention are
conjugated to labels for purposes of diagnostics and other assays
wherein the antibody and/or its associated ligand may be detected.
A label conjugated to an antibody and used in the present methods
and compositions described herein, is any chemical moiety, organic
or inorganic, that exhibits an absorption maximum at wavelengths
greater than 280 nm, and retains its spectral properties when
covalently attached to an antibody. Labels include, without
limitation, a chromophore, a fluorophore, a fluorescent protein, a
phosphorescent dye, a tandem dye, a particle, a hapten, an enzyme
and a radioisotope.
[0258] In certain embodiments, the antibodies are conjugated to a
fluorophore. As such, fluorophores used to label antibodies of the
invention include, without limitation; a pyrene (including any of
the corresponding derivative compounds disclosed in U.S. Pat. No.
5,132,432), an anthracene, a naphthalene, an acridine, a stilbene,
an indole or benzindole, an oxazole or benzoxazole, a thiazole or
benzothiazole, a 4-amino-7-nitrobenz-2-oxa-1, 3-diazole (NBD), a
cyanine (including any corresponding compounds in U.S. Pat. Nos.
6,977,305 and 6,974,873), a carbocyanine (including any
corresponding compounds in U.S. Ser. No. 09/557,275; U.S. Pat. Nos.
4,981,977; 5,268,486; 5,569,587; 5,569,766; 5,486,616; 5,627,027;
5,808,044; 5,877,310; 6,002,003; 6,004,536; 6,008,373; 6,043,025;
6,127,134; 6,130,094; 6,133,445; and publications WO 02/26891, WO
97/40104, WO 99/51702, WO 01/21624; EP 1 065 250 A1), a
carbostyryl, a porphyrin, a salicylate, an anthranilate, an
azulene, a perylene, a pyridine, a quinoline, a borapolyazaindacene
(including any corresponding compounds disclosed in U.S. Pat. Nos.
4,774,339; 5,187,288; 5,248,782; 5,274,113; and 5,433,896), a
xanthene (including any corresponding compounds disclosed in U.S.
Pat. Nos. 6,162,931; 6,130,101; 6,229,055; 6,339,392; 5,451,343;
5,227,487; 5,442,045; 5,798,276; 5,846,737; 4,945,171; U.S. Ser.
Nos. 09/129,015 and 09/922,333), an oxazine (including any
corresponding compounds disclosed in U.S. Pat. No. 4,714,763) or a
benzoxazine, a carbazine (including any corresponding compounds
disclosed in U.S. Pat. No. 4,810,636), a phenalenone, a coumarin
(including an corresponding compounds disclosed in U.S. Pat. Nos.
5,696,157; 5,459,276; 5,501,980 and 5,830,912), a benzofuran
(including an corresponding compounds disclosed in U.S. Pat. Nos.
4,603,209 and 4,849,362) and benzphenalenone (including any
corresponding compounds disclosed in U.S. Pat. No. 4,812,409) and
derivatives thereof. As used herein, oxazines include resorufins
(including any corresponding compounds disclosed in 5,242,805),
aminooxazinones, diaminooxazines, and their benzo-substituted
analogs.
[0259] In a specific embodiment, the fluorophores conjugated to the
antibodies described herein include xanthene (rhodol, rhodamine,
fluorescein and derivatives thereof) coumarin, cyanine, pyrene,
oxazine and borapolyazaindacene. In other embodiments, such
fluorophores are sulfonated xanthenes, fluorinated xanthenes,
sulfonated coumarins, fluorinated coumarins and sulfonated
cyanines. Also included are dyes sold under the tradenames, and
generally known as, ALEXA FLUOR.RTM., DyLight, CY.RTM. Dyes,
BODIPY.RTM., OREGON GREEN.RTM., PACIFIC BLUE.TM., IRDYE.RTM., FAM,
FITC, and ROX.TM..
[0260] The choice of the fluorophore attached to the antibody will
determine the absorption and fluorescence emission properties of
the conjugated antibody. Physical properties of a fluorophore label
that can be used for antibody and antibody bound ligands include,
but are not limited to, spectral characteristics (absorption,
emission and stokes shift), fluorescence intensity, lifetime,
polarization and photo-bleaching rate, or combination thereof. All
of these physical properties can be used to distinguish one
fluorophore from another, and thereby allow for multiplexed
analysis. In certain embodiments, the fluorophore has an absorption
maximum at wavelengths greater than 480 nm. In other embodiments,
the fluorophore absorbs at or near 488 nm to 514 nm (particularly
suitable for excitation by the output of the argon-ion laser
excitation source) or near 546 nm (particularly suitable for
excitation by a mercury arc lamp). In other embodiment a
fluorophore can emit in the NIR (near infra red region) for tissue
or whole organism applications. Other desirable properties of the
fluorescent label may include cell permeability and low toxicity,
for example if labeling of the antibody is to be performed in a
cell or an organism (e.g., a living animal).
[0261] In certain embodiments, an enzyme is a label and is
conjugated to an antibody described herein. Enzymes are desirable
labels because amplification of the detectable signal can be
obtained resulting in increased assay sensitivity. The enzyme
itself does not produce a detectable response but functions to
break down a substrate when it is contacted by an appropriate
substrate such that the converted substrate produces a fluorescent,
colorimetric or luminescent signal. Enzymes amplify the detectable
signal because one enzyme on a labeling reagent can result in
multiple substrates being converted to a detectable signal. The
enzyme substrate is selected to yield the preferred measurable
product, e.g. colorimetric, fluorescent or chemiluminescence. Such
substrates are extensively used in the art and are well known by
one skilled in the art.
[0262] In one embodiment, colorimetric or fluorogenic substrate and
enzyme combination uses oxidoreductases such as horseradish
peroxidase and a substrate such as 3,3'-diaminobenzidine (DAB) and
3-amino-9-ethylcarbazole (AEC), which yield a distinguishing color
(brown and red, respectively). Other colorimetric oxidoreductase
substrates that yield detectable products include, but are not
limited to: 2,2-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid)
(ABTS), o-phenylenediamine (OPD), 3,3',5,5'-tetramethylbenzidine
(TMB), o-dianisidine, 5-aminosalicylic acid, 4-chloro-1-naphthol.
Fluorogenic substrates include, but are not limited to,
homovanillic acid or 4-hydroxy-3-methoxyphenylacetic acid, reduced
phenoxazines and reduced benzothiazines, including Amplex.RTM. Red
reagent and its variants (U.S. Pat. No. 4,384,042) and reduced
dihydroxanthenes, including dihydrofluoresceins (U.S. Pat. No.
6,162,931) and dihydrorhodamines including dihydrorhodamine 123.
Peroxidase substrates that are tyramides (U.S. Pat. Nos. 5,196,306;
5,583,001 and 5,731,158) represent a unique class of peroxidase
substrates in that they can be intrinsically detectable before
action of the enzyme but are "fixed in place" by the action of a
peroxidase in the process described as tyramide signal
amplification (TSA). These substrates are extensively utilized to
label antigen in samples that are cells, tissues or arrays for
their subsequent detection by microscopy, flow cytometry, optical
scanning and fluorometry.
[0263] In another embodiment, a colorimetric (and in some cases
fluorogenic) substrate and enzyme combination uses a phosphatase
enzyme such as an acid phosphatase, an alkaline phosphatase or a
recombinant version of such a phosphatase in combination with a
colorimetric substrate such as 5-bromo-6-chloro-3-indolyl phosphate
(BCIP), 6-chloro-3-indolyl phosphate, 5-bromo-6-chloro-3-indolyl
phosphate, p-nitrophenyl phosphate, or o-nitrophenyl phosphate or
with a fluorogenic substrate such as 4-methylumbelliferyl
phosphate, 6,8-difluoro-7-hydroxy-4-methylcoumarinyl phosphate
(DiFMUP, U.S. Pat. No. 5,830,912) fluorescein diphosphate,
3-O-methylfluorescein phosphate, resorufin phosphate,
9H-(1,3-dichloro-9,9-dimethylacridin-2-one-7-yl) phosphate (DDAO
phosphate), or ELF 97, ELF 39 or related phosphates (U.S. Pat. Nos.
5,316,906 and 5,443,986).
[0264] Glycosidases, in particular beta-galactosidase,
beta-glucuronidase and beta-glucosidase, are additional suitable
enzymes. Appropriate colorimetric substrates include, but are not
limited to, 5-bromo-4-chloro-3-indolyl beta-D-galactopyranoside
(X-gal) and similar indolyl galactosides, glucosides, and
glucuronides, o-nitrophenyl beta-D-galactopyranoside (ONPG) and
p-nitrophenyl beta-D-galactopyranoside. In one embodiment,
fluorogenic substrates include resorufin beta-D-galactopyranoside,
fluorescein digalactoside (FDG), fluorescein diglucuronide and
their structural variants (U.S. Pat. Nos. 5,208,148; 5,242,805;
5,362,628; 5,576,424 and 5,773,236), 4-methylumbelliferyl
beta-D-galactopyranoside, carboxyumbelliferyl
beta-D-galactopyranoside and fluorinated coumarin
beta-D-galactopyranosides (U.S. Pat. No. 5,830,912).
[0265] Additional enzymes include, but are not limited to,
hydrolases such as cholinesterases and peptidases, oxidases such as
glucose oxidase and cytochrome oxidases, and reductases for which
suitable substrates are known.
[0266] Enzymes and their appropriate substrates that produce
chemiluminescence are preferred for some assays. These include, but
are not limited to, natural and recombinant forms of luciferases
and aequorins. Chemiluminescence-producing substrates for
phosphatases, glycosidases and oxidases such as those containing
stable dioxetanes, luminol, isoluminol and acridinium esters are
additionally useful.
[0267] In another embodiment, haptens such as biotin, are also
utilized as labels. Biotin is useful because it can function in an
enzyme system to further amplify the detectable signal, and it can
function as a tag to be used in affinity chromatography for
isolation purposes. For detection purposes, an enzyme conjugate
that has affinity for biotin is used, such as avidin-HRP.
Subsequently a peroxidase substrate is added to produce a
detectable signal.
[0268] Haptens also include hormones, naturally occurring and
synthetic drugs, pollutants, allergens, affector molecules, growth
factors, chemokines, cytokines, lymphokines, amino acids, peptides,
chemical intermediates, nucleotides and the like.
[0269] In certain embodiments, fluorescent proteins may be
conjugated to the antibodies as a label. Examples of fluorescent
proteins include green fluorescent protein (GFP) and the
phycobiliproteins and the derivatives thereof. The fluorescent
proteins, especially phycobiliprotein, are particularly useful for
creating tandem dye labeled labeling reagents. These tandem dyes
comprise a fluorescent protein and a fluorophore for the purposes
of obtaining a larger stokes shift wherein the emission spectra is
farther shifted from the wavelength of the fluorescent protein's
absorption spectra. This is particularly advantageous for detecting
a low quantity of antigen in a sample wherein the emitted
fluorescent light is maximally optimized, in other words little to
none of the emitted light is reabsorbed by the fluorescent protein.
For this to work, the fluorescent protein and fluorophore function
as an energy transfer pair wherein the fluorescent protein emits at
the wavelength that the fluorophore absorbs at and the fluorphore
then emits at a wavelength farther from the fluorescent proteins
than could have been obtained with only the fluorescent protein. A
particularly useful combination is the phycobiliproteins disclosed
in U.S. Pat. Nos. 4,520,110; 4,859,582; 5,055,556 and the
sulforhodamine fluorophores disclosed in U.S. Pat. No. 5,798,276,
or the sulfonated cyanine fluorophores disclosed in U.S. Pat. Nos.
6,977,305 and 6,974,873; or the sulfonated xanthene derivatives
disclosed in U.S. Pat. No. 6,130,101 and those combinations
disclosed in U.S. Pat. No. 4,542,104. Alternatively, the
fluorophore functions as the energy donor and the fluorescent
protein is the energy acceptor.
[0270] In certain embodiments, the label is a radioactive isotope.
Examples of suitable radioactive materials include, but are not
limited to, iodine (.sup.121I, .sup.123I, .sup.125I, .sup.131I),
carbon (.sup.14C), sulfur (.sup.35S), tritium (.sup.3H), indium
(.sup.111In, .sup.112In, .sup.113mIn, .sup.115mIn), technetium
(.sup.99Tc, .sup.99mTc), thallium (.sup.201Ti), gallium (.sup.68Ga,
.sup.67Ga), palladium (.sup.163Pd), molybdenum (.sup.99Mo), xenon
(.sup.135Xe), fluorine (.sup.18F), .sup.153Sm, .sup.177Lu,
.sup.159Gd, .sup.149Pm, .sup.140La, .sup.175Yb, .sup.166Ho,
.sup.90Y, .sup.47Sc, .sup.186Re, .sup.188Re, .sup.142Pr,
.sup.105Rh, and .sup.97Ru.
Medical Treatments and Uses
[0271] The antibodies and binding fragments thereof of the
invention and variants thereof may be used for the treatment of
influenza A virus infection, for the prevention of influenza A
virus infection and/or for the detection, diagnosis and/or
prognosis of influenza A virus infection.
[0272] Methods of diagnosis may include contacting an antibody or
an antibody fragment with a sample. Such samples may be tissue
samples taken from, for example, nasal passages, sinus cavities,
salivary glands, lung, liver, pancreas, kidney, ear, eye, placenta,
alimentary tract, heart, ovaries, pituitary, adrenals, thyroid,
brain or skin. The methods of detection, diagnosis, and/or
prognosis may also include the detection of an antigen/antibody
complex.
[0273] In one embodiment, the invention provides a method of
treating a subject by administering to the subject an effective
amount of an antibody or an binding fragment thereof, according to
the invention, or a pharmaceutical composition that includes the
antibody or binding fragment thereof. In one embodiment, the
antibody or binding fragment thereof is substantially purified
(i.e., substantially free from substances that limit its effect or
produce undesired side-effects). In one embodiment, the antibody or
binding fragment thereof of the invention is administered
post-exposure, or after the subject has been exposed to influenza A
virus or is infected with influenza A virus. In another embodiment,
the antibody or binding fragment thereof of the invention is
administered pre-exposure, or to a subject that has not yet been
exposed to influenza A virus or is not yet infected with influenza
A virus. In one embodiment, the antibody or binding fragment
thereof of the invention is administered to a subject that is
sero-negative for one or more influenza A subtypes. In another
embodiment, the antibody or binding fragment thereof of the
invention is administered to a subject that is sero-positive for
one or more influenza A subtypes. In one embodiment, the antibody
or binding fragment thereof of the invention is administered to a
subject within 1, 2, 3, 4, 5 days of infection or symptom onset. In
another embodiment, the antibody or binding fragment thereof of the
invention can be administered to a subject after 1, 2, 3, 4, 5, 6,
or 7 days, and within 2, 3, 4, 5, 6, 7, 8, 9 or 10 days after
infection or symptom onset.
[0274] In one embodiment, the method reduces influenza A virus
infection in the subject. In another embodiment, the method
prevents, reduces the risk or delays influenza A virus infection in
the subject. In one embodiment, the subject is a mammal. In a more
particular embodiment, the subject is human. In one embodiment, the
subject includes, but is not limited to, one who is particularly at
risk of or susceptible to influenza A virus infection, including,
for example, an immunocompromised subject.
[0275] Treatment can be a single dose schedule or a multiple dose
schedule and the antibody or binding fragment thereof of the
invention can be used in passive immunization.
[0276] In one embodiment, the antibody or binding fragment thereof
of the invention is administered to a subject in combination with
one or more antiviral medications. In one embodiment, the antibody
or binding fragment thereof of the invention is administered to a
subject in combination with one or more small molecule antiviral
medications. Small molecule antiviral medications include
neuraminidase inhibitors such as oseltamivir (TAMIFLU.RTM.),
zanamivir (RELENZA.RTM.) and adamantanes such as Amantadine and
rimantadine.
[0277] In another embodiment, the invention provides a composition
for use as a medicament for the prevention or treatment of an
influenza A virus infection. In another embodiment, the invention
provides the use of an antibody or binding fragment thereof of the
invention and/or a protein comprising an epitope to which an
antibody or binding fragment thereof of the invention binds in the
manufacture of a medicament for treatment of a subject and/or
diagnosis in a subject.
[0278] Antibodies and fragments thereof as described in the present
invention may also be used in a kit for the diagnosis of influenza
A virus infection. Further, epitopes capable of binding an antibody
of the invention may be used in a kit for monitoring the efficacy
of vaccination procedures by detecting the presence of protective
anti-influenza A virus antibodies. Antibodies, antibody fragment,
or variants and derivatives thereof, as described in the present
invention may also be used in a kit for monitoring vaccine
manufacture with the desired immunogenicity.
[0279] The invention also provides a method of preparing a
pharmaceutical composition, which includes the step of admixing a
monoclonal antibody with one or more pharmaceutically-acceptable
carriers, wherein the antibody is a monoclonal antibody according
to the invention described herein.
[0280] Various delivery systems are known and can be used to
administer the antibody or binding fragment thereof of the
invention, including, but not limited to, encapsulation in
liposomes, microparticles, microcapsules, recombinant cells capable
of expressing the antibody or antibody fragment, receptor-mediated
endocytosis, construction of a nucleic acid as part of a retroviral
or other vector, delivery of naked nucleotide acids by
electroporation delivery technology (as described in Muthumani et
al., PLoS One. 2013 Dec. 31; 8(12):e84234. doi:
10.1371/journal.pone.0084234. eCollection 2013) etc. Methods of
introduction include, but are not limited to, intradermal,
intramuscular, intraperitoneal, intravenous, subcutaneous,
intranasal, epidural, and oral routes. The compositions may be
administered by any convenient route, for example by infusion or
bolus injection, by absorption through epithelial or mucutaneous
linings (e.g., oral mucosa, rectal and intestinal mucosa, etc.) and
may be administered together with other biologically active agents,
including, but not limited to small molecule antiviral
compositions. Administration can be systemic or local. Pulmonary
administration can also be employed, e.g., by use of an inhaler or
nebulizer, and formulation with an aerosolizing agent. In yet
another embodiment, the composition can be delivered in a
controlled release system.
[0281] The present invention also provides pharmaceutical
compositions. Such compositions include a therapeutically effective
amount of an antibody or binding fragment thereof of the invention,
and a pharmaceutically acceptable carrier. The term
"pharmaceutically acceptable" as used herein, means approved by a
regulatory agency of the Federal or a state government or listed in
the U.S. Pharmacopeia or other generally recognized pharmacopeia
for use in animals, and more particularly in humans. The term
"carrier" refers to a diluent, adjuvant, excipient, or vehicle with
which the therapeutic is administered. Such pharmaceutical carriers
can be sterile liquids, such as water and oils, including those of
petroleum, animal, vegetable or synthetic origin, such as peanut
oil, soybean oil, mineral oil, sesame oil and the like. Water is a
preferred carrier when the pharmaceutical composition is
administered intravenously. Saline solutions and aqueous dextrose
and glycerol solutions can also be employed as liquid carriers,
particularly for injectable solutions. Suitable pharmaceutical
excipients include starch, glucose, lactose, sucrose, gelatin,
malt, rice, flour, chalk, silica gel, sodium stearate, glycerol
monostearate, talc, sodium chloride, dried skim milk, glycerol,
propylene, glycol, water, ethanol and the like. The composition, if
desired, can also contain minor amounts of wetting or emulsifying
agents, or pH buffering agents. These compositions can take the
form of solutions, suspensions, emulsion, tablets, pills, capsules,
powders, sustained-release formulations and the like. The
composition can be formulated as a suppository, with traditional
binders and carriers such as triglycerides. Oral formulation can
include standard carriers such as pharmaceutical grades of
mannitol, lactose, starch, magnesium stearate, sodium saccharine,
cellulose, magnesium carbonate, etc. In one embodiment, the
pharmaceutical composition contains a therapeutically effective
amount of the antibody or binding fragment thereof, preferably in
purified form, together with a suitable amount of carrier so as to
provide the form for proper administration to the patient. The
formulation should suit the mode of administration.
[0282] Typically, for antibody therapeutics, the dosage
administered to a patient is between about 0.1 mg/kg to 100 mg/kg
of the patient's body weight.
LIST OF FIGURES
[0283] FIG. 1A shows the binding of Antibody 3 and Antibody 12 to
surface expressed HA protein of subtypes H11, H12, H13, H16, H17,
H4, H10, H14, and H15. Histograms depict the number of cells vs the
florescence intensity of antibody binding to HA transfected cells
in white or mock transfected cells in grey.
[0284] FIG. 1B shows the percent inhibition of low pH induced
fusion of chicken red blood cells and A/Puerto Rico/8/34 in the
presence of Antibody 3, Antibody 12, or MPE8v3 (non-relevant viral
fusion protein antibody) (Corti D et al., 2013, Nature 501).
[0285] FIG. 10 shows immunoblots of uncleaved (HA0), recombinant H1
HA after digestion with trypsin for 5, 10 or 20 minutes. Digestion
reactions contained either HA alone (input), or HA pre-treated with
Fl6v4 (disclosed in WO2013/011347A1), Antibody 3, FE17.23 (globular
head specific mAb) (Corti D et al., 2010, J Clin Invest 120) or
non-relevant control antibody (Ctrl. IgG).
[0286] FIG. 1D shows immunoblots of uncleaved (HA0), recombinant H1
HA after digestion with trypsin of 5, 10 or 20 minutes. Digestion
reactions contained either HA alone (input), or HA pre-treated with
Antibody 3, Antibody 12, Antibody 14, or non-relevant control
antibody (Ctrl. IgG).
[0287] FIG. 2 shows the percentage of NK cell mediated killing of
A/HK/8/68 infected MDCK cells in the presence of increasing amount
of Antibody 3, Antibody 11, Antibody 12, and Antibody 14.
[0288] FIG. 3 shows the percentage of macrophages that phagocytosed
A/HK/8/68 HA expressing MDCK target cells in the presence of
increasing amount of Antibody 3, Antibody 11, Antibody 12, and
Antibody 14, or non-relevant isotype control (Ctrl. IgG).
[0289] FIG. 4 shows the percentage of complement dependent killing
of A/PR/8/34 infected MDCK cells in the presence of increasing
amount of Antibody 3, Antibody 11, Antibody 12, and Antibody
14.
[0290] FIG. 5 shows the percentage of surviving animals in each
group of a study when different concentrations of Antibody 3 or a
non-relevant control antibody (Ctrl. IgG) were administered to mice
4 hours before infection with a lethal dose of H1N1 influenza
virus.
[0291] FIG. 6 shows the percentage of surviving animals in each
group of a study in which different concentrations of Antibody 3 or
a non-relevant control antibody (Ctrl. IgG) were administered to
mice 4 hours before infection with a lethal dose of H3 influenza
virus.
[0292] FIG. 7 shows the percentage of surviving animals in each
group when mice were infected with a lethal dose of H1N1 influenza
virus and treated at different time points (1 and 2 days
post-infection) with 30 mg/kg of Antibody 3 or a non-relevant
control antibody (Ctrl. IgG).
[0293] FIG. 8 shows the percentage of surviving animals in each
group of a study in which mice were infected with a lethal dose of
H3 influenza virus and treated at different time points (3, 4 and 5
days post-infection) with 30 mg/kg of Antibody 3 or a non-relevant
control antibody (Ctrl. IgG).
[0294] FIG. 9 shows the percentage of surviving animals in each
group of a study in which mice were infected with a lethal dose of
H1N1 influenza virus and treated at 1 day post infection with 2
mg/kg of Antibody 3, Antibody 11, Antibody 12, Antibody 14 or a
non-relevant control antibody (Ctrl. IgG).
[0295] FIG. 10 shows the percentage of surviving animals in each
group of a study in which mice were infected with a lethal dose of
H3 influenza virus and treated at 2 day post infection with 3 mg/kg
of Antibody 3, Antibody 11, Antibody 12, Antibody 14 or a
non-relevant control antibody (Ctrl. IgG).
[0296] FIG. 11 shows the percentage of surviving animals in each
group of a study in which mice were infected with a lethal dose of
H1N1 influenza virus and treatment of 25 mg/kg BID oseltamivir for
5 days, 10 mg/kg of Antibody 12, or 10 mg/kg of non-relevant
control antibody (Ctrl. IgG) was initiated at different time points
(4 hr before, 1 day post, or 2 days post infection).
[0297] FIG. 12 shows the percentage of surviving animals in each
group of a study in which mice were infected with a lethal dose of
H3 influenza virus and treatment of 25 mg/kg oraloseltamivir twice
daily (BID) for 5 days, or single dose 10 mg/kg of Antibody 12, or
10 mg/kg of a non-relevant control antibody (Ctrl. IgG) that was
initiated at various time points (1, 2, 3 or 4 days post
infection).
[0298] FIG. 13 shows the percentage of surviving animals in each
group in a study that mice were infected with a lethal dose of H3
influenza virus and treated with Antibody 12 at 2.5 mg/kg or 0.3
mg/kg single dose, oseltamivir at 25 mg/kg BID for 5 days, or a
combination of Antibody 12 at 2.5 mg/kg or 0.3 mg/kg and
oseltamivir at 25 mg/kg BID for 5 days at 2 days post
infection.
[0299] FIG. 14 shows the percentage of surviving ferrets in each
group of a study after infection with a lethal dose of H5N1
influenza virus and treatment with 25 mg/kg single dose Antibody
12, 25 mg/kg BID oseltamivir for 5 days, or a non-relevant control
antibody (Ctrl. IgG) at 1, 2, or 3 days post infection.
[0300] FIG. 15 shows an alignment of HA2 Protein of Influenza A
Strains Used in MARM selection.
[0301] FIG. 16 shows the VH percent identity of anti-HA Antibodies
1-15 and Antibody 3-GL.
[0302] FIG. 17 shows the VH alignment of anti-HA Antibodies 1-15
and Antibody 3-GL.
[0303] FIG. 18 shows the VL percent identity of anti-HA Antibodies
1-15 and Antibody 3-GL.
[0304] FIG. 19 shows the VL alignment of anti-HA Antibodies 1-15
and Antibody 3-GL.
EXAMPLES
Example 1. Construction and Optimization of Human Monoclonal
Antibodies Isolated from Memory B Cells
[0305] The CD22+ IgG+ B cells were sorted from cryopreserved
peripheral blood mononuclear cells (PBMCs) of a donor selected for
high titers of heterosubtypic antibodies and immortalized at 3
cells/well using Epstein Barr Virus (EBV) and CpG
oligodeoxynucleotide 2006 and feeder cells. Culture supernatants
containing antibodies were harvested after 14 days and screened by
ELISA binding assay to determine the binding activity against H5
(A/Vietnam/1203/04) and H7 (A/NLD/03) hemagglutinin (HA),
respectively. Four B cell clones (Antibody 1, Antibody 4, Antibody
7, and Antibody 9) were found to bind specifically to both HAs and
were therefore collected. The VH and VL genes of these clones were
sequenced and found to be clonally related according to the
homology analysis performed on VH and VL V, D and J fragments using
the Kabat database. Of note, the VH of Antibody 4 was found to have
a degenerate nucleotide site in the HCDR3 encoding for either
valine (encoded in Antibody 5) or glutamate (encoded in Antibody
6). The VH and VL genes of the four antibodies were cloned into
IgG1 expression vectors (minor sequence modifications to facilitate
cloning and or codon optimization resulted in the five antibodies;
Antibody 3, Antibody 5, Antibody 6, Antibody 8 and Antibody 10;
used in the following Examples) and recombinant antibodies were
produced by transient transfection of mammalian cell lines derived
from HEK or CHO cells. Supernatants from transfected cells were
collected after 7-10 days of culture, and IgGs were affinity
purified by Protein A chromatography, and dialyzed into PBS.
Antibody 3 was further optimized to create variants in which
non-germline encoded somatic mutations located in the framework
regions were changed to the germline encoded amino acid, and the
CDR regions were subjected to parsimonious mutagenesis. Full IgG
constructs containing different mutations were expressed as
described above and the crude supernatants were screened by ELISA
to select clones that had increased binding activity to H3 and H1
HA proteins. ELISA was performed using a coating concentration of
0.15 pg/ml of rabbit anti-human IgG in order to capture and
normalize IgG from the supernatants, and then 0.5 pg/ml of
biotinylated HA subtype H1 (A/California/7/04 (H1N1)) or subtype H3
(A/Perth/16/09 (H3N2)) was added and incubated for one hour.
Binding was detected by the addition of streptavidin-HRP (1:5000),
and development absorbance was read at 450 nm. The beneficial
single mutations conferring better binding were combined and cloned
into a combinatorial library, which were expressed and screened by
ELISA as described above. This library approach resulted in the
creation of 5 additional Antibody 3 variants that were further
characterized (Antibodies 11-15).
Example 2. Anti-HA Neutralizing Antibody (nAb) Binds to HA of
Different Subtypes
[0306] To test if the epitope of the anti-HA antibodies is
conserved among HAs of different subtypes, a HA cross-reactivity
ELISA binding assay was performed. A 384-well Maxisorb ELISA plate
(Nunc) was coated overnight at 4.degree. C. with 0.5 ug/ml
recombinant HA (rHA), subtype H1 (A/California/7/09 (H1N1)),
subtype H2 (A/Swine/MO/06 (H2N3)), subtype H3 (A/Perth/16/09
(H3N2)), subtype H5 (A/Vietnam/1203/04(H5N1)), subtype H6
(A/teal/HK/W312/97(H6N1)), subtype H7 (A/Netherlands/219/03 (H7N7))
and subtype H9 (A/chicken/HK/G9/97 (H9N2)) in PBS. The plate was
washed with PBS containing 0.1% v/v Tween-20 to remove uncoated
protein and subsequently blocking solution containing 1% (w/v)
casein (Thermo Scientific) was added and incubated for 1 hr at room
temperature. The blocking solution was discarded and 3-fold
serially diluted anti-HA antibodies in blocking solution
(Casein-PBS (Thermo Scientific) were added and incubated for 1 hr
at room temperature. The plate was washed three times and bound
antibodies were detected using a peroxidase-conjugated mouse
anti-human IgG antibody (Jackson). The binding activity of antibody
was calculated by either measuring the chemiluminescent signal
after addition of Supersignal Pico substrate (Thermo Scientific) or
by measuring the color change at 450 nm after incubation with
Tetramethylbenzidine (TMB) one component substrate (KPL) followed
by the addition of 2N sulfuric acid to stop the reaction.
TABLE-US-00001 TABLE 1 Binding to rHA by ELISA (EC.sub.50, ug/ml)
H1 H2 H5 H6 H9 H3 H7 A/CA/7/09 A/swine/MO/06 A/VN/1203/04
A/HK/W312/97 A/HK/G9/97 A/Perth/16/09 A/NLD/219/03 Antibody 3 0.026
0.028 0.022 0.043 0.012 0.019 0.020 Antibody 5 0.045 0.048 0.041
0.047 >6 0.030 0.024 Antibody 6 0.311 0.213 0.256 0.214 >6
0.064 0.116 Antibody 8 0.069 0.058 0.044 0.091 >6 0.067 0.015
Antibody 10 0.073 0.075 0.058 0.097 2.699 0.049 0.034
[0307] Table 1 shows that all anti-HA IgGs tested bound to
recombinant HA of subtypes H1, H2, H3, H5, H6, H9 and H7.
Recombinant HA of subtype H9 was recognized by Antibody 3 and
Antibody 10, but not by Antibody 5, Antibody 6 and Antibody 8 at
the highest concentration of antibody tested (6 ug/ml). This
indicates that the epitopes of the majority of these anti-HA IgGs
are conserved among HA molecules of different subtypes.
TABLE-US-00002 TABLE 2 Binding to rHA by ELISA (EC.sub.50, ug/ml)
H1 H2 H5 H6 H9 H3 H7 A/CA/7/09 A/swine/MO/06 A/VN/1203/04
A/HK/W312/97 A/HK/G9/97 A/Perth/16/09 A/NLD/219/03 Antibody 3 0.045
0.095 0.099 0.072 0.171 0.129 0.258 Antibody 11 0.085 0.126 0.168
0.129 0.164 0.176 0.553 Antibody 12 0.059 0.088 0.084 0.083 0.098
0.028 0.061 Antibody 13 0.050 0.062 0.080 0.097 0.161 0.023 0.049
Antibody 14 0.048 0.079 0.061 0.073 0.095 0.030 0.064 Antibody 15
0.028 0.042 0.035 0.043 0.065 0.032 0.035
[0308] Table 2 shows that all anti-HA IgGs variants tested bound to
recombinant HA of group 1 subtypes H1, H2, H5, H6 and H9 with
similar ECK values. All the variants bound to group 2 HA proteins
(H3 and H7), however, Antibody 11 and Antibody 3 showed decreased
activity with increased ECK values compared to the Antibodies
12-15.
[0309] To extend these binding results to include more diverse HA
subtypes, we performed additional binding studies using a flow
cytometry based binding to HA transfected cells. In this assay, HEK
cells were transiently transfected with full-length wild type HA
expressing plasmids of subtype H4 A/duck/Czechoslovakia/56 (H4N6)),
subtype H10 (A/chicken/Germany/N49 (H10N7)), subtype H11
(A/duck/Memphis/546/74 (H11N9)), subtype H12 (A/duck/Alberta/60/76
(H12N5)), subtype H13 (A/gull/Maryland/704/77 (H13N6)), subtype H14
(A/mallard/Astrakhan/263/82 (H14N5)), subtype H15
(A/shearwater/West Australia/2576/79 (H15N9)), subtype H16
(A/black-headed gull/Sweden/2/99 (H16N3)), and subtype H17
(A/little yellow-shouldered bat/Guatemala/164/2009 (H17N10)).
Forty-eight hours after transfection, cells were detached with
trypsin, and incubated with 5 ug/ml of Antibody 3 or Antibody 12 on
ice for 1 hour. After the hour incubation, the antibody bound to
cell-surface expressed HA protein was then stained with a goat
anti-human IgG Daylight 649 (Jackson ImmunoResearch), and which was
detected by flow cytometry. FIG. 1A shows the shift in fluorescence
intensity when antibody is bound to HA expressing cells (white) vs
mock-transfected cells (grey) from each of the subtypes. Antibody 3
bound to all HAs tested with the exception of H12, while Antibody
12 bound to all HAs tested from both groups (group 1 H11, H12, H13,
H16, and H17 and group 2 H4, H10, H14, and H15)
Example 3 Kinetic Characterization of the HA Binding to Antibody 3
and Antibody 5 IgG1 by Using Octet
[0310] Affinity measurements were performed using a ForteBio Octet
QK 384 Kinetic Analyzer (Menlo Park, Calif.) in 384 slanted-well
plates. All reagents were diluted in Octet Kinetics Buffer
(ForteBio). His-tagged HA of different subtypes: subtype H1
(A/California/7/04 (H1N1)) and subtype H3 (A/Perth/16/09 (H3N2))
were immobilized onto anti-His sensors at 10 .mu.g/mL. Anti-HA mAb
association/dissociation were then monitored in 2-fold dilutions
from 100 nM, plus a zero mAb control.
[0311] Association and dissociation raw data were corrected for any
drift in the zero mAb controls, and then exported to GraphPad Prism
(San Diego, Calif.) for affinity curve fitting. Data were fitted
using global association/dissociation fitting with an imposed limit
of >5.times.10.sup.-6 sec.sup.-1. As shown in Table 3, both
antibodies have a very high affinity binding to H1 at pM level with
a slow dissociation rate under limit of detection. Similar
K.sub.on, K.sub.off and Kd of both antibodies were observed with H3
trimer at sub-nM level.
TABLE-US-00003 TABLE 3 Kinetic Binding Analysis of Pan A mAbs on
rHA by Octet H1 A/CA/7/09 H3 A/Perth/16/09 K.sub.on K.sub.off Kd
K.sub.on K.sub.off Kd (e5 M.sup.-1s.sup.-1) (e.sup.-6 s.sup.-1)
(pM) (e5 M.sup.-1s.sup.-1) (e.sup.-6 s.sup.-1) (pM) Antibody 5.3
<5 11 3.3 62 188 3 Antibody 10 <5 5 2.6 88 338 5
Example 4 In Vitro Cross-Reactive Neutralizing Activity of Anti-HA
IgG1s Against Virus of Different Subtypes
[0312] The microneutralization assay (MNA) was modified from a
previously described accelerated viral inhibition assay using
neuraminidase activity (NA) as a read-out (Hassantoufighi, A. et
al. 2010, Vaccine 28:790). Briefly, MNA were performed on MDCK
cells that were cultured in MEM medium (Invitrogen) supplemented
with antibiotics, glutamine (complete MEM medium) and 10% (v/v)
fetal bovine serum. 60 TCID.sub.50 (50% tissue culture infectious
doses) of virus was added to three-fold dilutions of antibody in a
384-well plate in complete MEM medium containing 0.75 ug/ml Trypsin
(Worthington) in duplicate wells, after 30 minutes incubation at
room temperature, 2.times.10.sup.4 cells/well were added to the
plate. After incubation at 33.degree. C. 5% CO.sub.2 incubator for
approximately 40 hr, the NA activity was measured by adding a
fluorescently-labeled substrate, methylumbelliferyl-N-acetyl
neuraminic acid (MU-NANA) (Sigma) to each well and incubated at
37.degree. C. for 1 hr. Virus replication represented by NA
activity was quantified by reading fluorescence in Fluorometer
Envison (PerkinElmer) using the following settings: excitation 355
nm, emission 460 nm; 10 flashes per well. The neutralization titer
(50% inhibitory concentration [IC.sub.50]) is expressed as the
final antibody concentration that reduced the fluorescence signal
by 50% compared to cell control wells. Table 4 and 5 showed anti-HA
antibodies neutralized influenza A viruses of different subtypes
tested below: H1-PR34 (A/Puerto Rico/8/34 (H1N1)); H1-PR34-OR
(A/Puerto Rico/8/34 containing the NA 274Y (N2 numbering) mutation
confering oseltamivir resistance (H1N1)); H1-FM47 (A/Fort
Monmouth/1/47 (H1N1)); H1-NJ76 (A/New Jersey/8/76 (H1N1)); H1-Kaw86
(A/Kawasaki/9/86 (H1N1)); H1-TX91 (caA/Texas/36/91 (H1N1)): H1-BJ95
(ca A/Beijing/262/95 (H1N1)); H1-Ncal99 (ca A/New Caledonia/20/99
(H1N1)); H1-SD07 (ca A/South Dakota/6/07 (H1N1)); H1-CA09 (ca
A/California/7/09 (H1N1)); H1-CA09-OR (ca A/California/7/09
containing the NA 274Y (N2 numbering) mutation confering
oseltamivir resistance (H1N1)); H5-VN04 (ca A/Vietnam/1203/04
(H5N1)); H5-HK03 (ca A/Hong Kong/213/03 (H5N1)); H9-HK97 (ca
A/chicken/Hong Kong/G9/97 (H9N2); H2-JP57 (ca A/Japan/57 (H2N2));
H2-M006 (ca A/swine/Missouri/06 (H2N3)); H6-HK97 (ca A/teal/Hong
Kong/W312/97 (H6N1)); H6-AB85 (ca A/mallard/Alberta/89/85 (H6N2));
H3-HK68 (A/Hong Kong/8/68 (H3N2)); H3-Vic75 (A/Victoria/3/75
(H3N2)); H3-LA87 (A/Los Angeles/7/09 (H3N2)); H3-SD93 (A/Shan
dong/9/93 (H3N2)); H3-WH95 (ca A/Wuhan/359/95 (H3N2)); H3-Syd97 (ca
A/Sydney/5/97 (H3N2)); H3-WH95-OR (ca A/Wuhan/359/95 containing the
NA 274Y (N2 numbering) mutation confering oseltamivir resistance
(H3N2)); H3-Pa99 (ca A/Panama/2007/99 (H3N2)); H3-Wy03
(A/Wyoming/03/03 (H3N2)); H3-WI05 (A/Wisconsin/67/05 (H3N2));
H3-Perth09 (ca A/Perth/16/09 (H3N2)), H3-VC11 (A/Victoria/361/11
(H3N2)); H7-NLD03 (ca A/Netherlands/219/03 (H7N7)); H7-BC04 (ca
A/Brit. Columbia/CN-6/04 (H7N3-LP); H7-ANU13 (ca A/Anhui/1/13
(H7N9).
TABLE-US-00004 TABLE 4 Neutralization of infectious viruses
(IC.sub.50 ug/ml) Anti- Anti- Anti- Anti- Anti- Virus body 3 body 5
body 6 body 8 body 10 Group 1 H1-PR34 1.07 1.13 4.37 3.02 2.15
H1-FM47 0.92 0.86 3.04 1.37 1.11 H1-NJ76 1.41 1.64 2.60 2.26 0.15
H1-Kaw86 0.58 1.01 3.51 2.11 1.62 H1-TX91 0.60 0.76 2.20 0.70 0.48
H1-BJ95 3.41 5.06 20.86 10.60 4.46 H1-Ncal99 0.79 0.85 3.00 2.06
1.26 H1-SD07 0.97 1.61 6.27 2.62 1.37 H1-CA09 2.19 2.52 5.56 4.50
1.62 H2-MO06 2.27 2.38 2.90 2.62 1.04 H5-VM04 2.11 2.60 8.87 3.90
2.21 H5-HK03 4.64 1.18 10.45 1.82 1.60 H6-HK97 1.77 2.27 3.23 2.97
1.05 H9-HK97 1.79 2.43 16.47 26.39 1.76 Group 2 H3-HK68 0.68 0.39
2.04 2.82 0.85 H3-Vic75 0.75 0.57 1.09 3.83 0.91 H3-LA87 4.19 3.54
12.60 >50 4.59 H3-SD93 9.39 6.92 19.50 >50 11.65 H3-WH95 3.96
3.72 10.54 >50 8.70 H3-Syd97 3.75 3.03 6.54 >50 9.29 H3-Pa99
17.74 16.74 25.82 >50 18.71 H3-Wy03 0.63 0.77 4.70 >50 1.52
H3-WI05 2.44 2.83 6.76 >50 4.46 H3-Perth09 1.49 2.22 5.03 >50
2.56 H7-NLD03 4.78 4.14 >50 12.75 3.80 H7-BC04 4.72 5.35 >50
14.69 3.59
[0313] Table 4 shows that anti-HA antibodies neutralize all group 1
influenza A viruses tested. All anti-HA antibodies except Antibody
8 demonstrated neutralizing activity against all H3 influenza A
viruses tested and all anti-HA antibodies except Antibody 6
exhibited neutralizing activity against H7-NLD03 (ca
A/Netherlands/219/03 (H7N7)); H7-BC04 (ca A/Brit. Columbia/CN-6/04
(H7N3-LP).
TABLE-US-00005 TABLE 5 Neutralization of infectious viruses
(IC.sub.50 ug/ml) Antibody Antibody Antibody Antibody Antibody
Antibody Virus 3 11 12 13 14 15 Group 1 H1-PR34 2.17 0.88 1.07 1.30
1.25 1.47 H1-PR34-OR 1.39 0.73 0.69 0.88 0.83 0.90 H1-FM47 1.04
0.43 0.28 0.50 0.44 0.35 H1-NJ76 0.57 0.13 0.12 0.12 0.11 0.25
H1-Kaw86 1.01 0.53 0.28 0.41 0.35 0.48 H1-TX91 0.92 0.11 0.12 0.09
0.09 0.13 H1-BJ95 2.98 1.01 1.31 1.86 2.09 1.81 H1-Ncal99 1.16 0.66
0.61 0.77 0.67 0.79 H1-SD07 2.04 0.98 0.78 1.35 1.05 0.81 H1-CA09
2.07 0.90 0.98 1.23 1.07 1.17 H1-CA09-OR 2.10 0.87 0.84 1.05 1.23
1.35 H1-BS10 2.16 1.15 1.25 1.23 1.93 1.89 H2-JP57 0.46 0.31 0.35
0.47 0.67 0.33 H2-MO06 1.09 0.60 0.53 0.57 0.65 0.83 H5-VM04 1.19
0.57 0.31 0.56 0.33 0.28 H5-HK03 0.71 0.21 0.17 0.17 0.21 0.05
H6-AB85 0.69 0.24 0.32 0.29 0.26 0.19 H6-HK97 0.63 0.40 0.45 0.55
0.26 0.33 H9-HK97 1.18 0.36 0.31 0.29 0.44 0.35 Group 2 H3-HK68
1.37 0.46 0.42 0.44 0.65 0.50 H3-Vic75 1.12 0.46 0.32 0.43 0.44
0.35 H3-LA87 2.04 0.80 0.82 1.00 0.83 0.83 H3-SD93 3.57 1.11 1.32
1.56 1.57 1.43 H3-WH95 5.63 2.45 2.09 2.77 2.77 3.32 H3-WH95-OR
7.70 2.26 2.34 3.01 3.09 3.48 H3-Syd97 6.50 1.53 1.56 2.18 1.82
1.79 H3-Pa99 9.00 2.18 2.04 2.62 4.36 3.39 H3-WI05 2.62 1.07 1.09
1.19 1.19 1.30 H3-Perth09 1.30 0.17 0.25 0.28 0.47 0.50 H3-VC11
3.40 0.85 0.83 1.03 1.15 1.29 H7-NLD03 4.74 0.94 0.83 2.45 1.16
1.30 H7-BC04 2.95 0.71 0.78 0.96 0.86 1.25 H7-ANU13 4.26 nd 2.56 nd
2.12 nd
[0314] Table 5 shows that the Antibody variants (Antibodies 11-15)
are more effective than parental Antibody 3 in neutralizing all
group 1 and group 2 influenza A viruses tested with decreased
IC.sub.50 values. In addition, antibodies also neutralized 3
viruses which have a mutation engineered into the NA protein
conferring oseltamivir resistance (OR).
Example 5. Neutralizing Activity of Anti-HA IgGs Against Swine
Origin H3N2 Viruses
[0315] The neutralizing activity of anti-HA Antibody 3 and variants
(Antibodies 11-15) against newly emerged swine-origin H3N2 viruses
(A/Minnesota/11/2010 and A/Indiana/10/2011) was measured in a
microneutralization assay as described in Example 4. Antibody Fl6v4
(described in WO2013/011347A1) was used as a control antibody. As
shown in Table 6 from two independent experiments, Antibody 3 and
the antibody variants (Antibodies 11-15) were more effective than
Fl6v4 in neutralizing swine-origin A/Indiana/10/2011 H3N2 virus.
Antibody 3 and the antibody variants potently neutralized
swine-origin A/Minnesota/11/2010 H3N2 virus whereas Fl6v4 failed to
neutralize at the highest concentration (50 ug/ml) of antibody
tested.
TABLE-US-00006 TABLE 6 Neutralizing activity (IC.sub.50 ug/ml) FI6
Antibody Antibody Antibody Antibody Antibody Antibody H3N2 virus v4
3 11 12 13 14 15 swine-origin >50 2.2 1.6 1.1 1.6 1.4 0.9
A/Minnesota/ >50 4.2 1.5 1.2 1.4 2.3 2.7 11/2010 swine-origin
13.7 3.1 2.8 2.5 2.2 3.3 5.5 A/Indiana/ 29.3 3.7 2.1 1.8 3.9 2.9
3.9 10/2011
Example 6. Anti-HA Neutralizing Antibody Inhibits Influenza Fusion
and Protease-Mediated HA0 Cleavage
[0316] To test for the antibody mediated fusion inhibition, a low
pH induced red blood cell fusion assay was performed through a
modified protocol described previously (Wang T. T. et al., 2010
PLoS Pathog. 6). In brief, A/Puerto Rico/8/34 virus
(10.times.10.sup.6 TCID50) was incubated with human red blood cells
(2% final red cell concentration) on ice for 10 minutes. Dilutions
of Antibody 3, Antibody 12, and a non-relevant antibody MPE8v3 were
incubated with virus for 30 minutes at RT. The red blood cells were
then added to the virus-antibody mixture for 30 minutes at
37.degree. C. and finally sodium acetate buffer (0.5 M pH 5.0) was
added for additional 45 minutes at 37.degree. C. Samples were
centrifuged for 6 minutes at 400.times.g and incubated for
additional 45 minutes at RT and then centrifuged again for 6
minutes at 400.times.g to pellet red blood cells. Supernatants were
then transferred to an ELISA plate to determine the amount of
released NADPH by measuring absorbance at 540 nm (FIG. 1B). The
result showed that Antibody 3 and Antibody 12 potently inhibited
viral fusion whereas the MPE8v3, a human monoclonal antibody
against the fusion protein of a paramyxovirus (Corti et al., 2013
Nature 501), was not able to inhibit the low pH induced fusion.
[0317] To test for antibody mediated blockade of the HA maturation,
recombinant HA of A/New Caledonia/20/99 (H1N1) was incubated for 40
minutes with Antibody 3, Fl6v4, FE17.23 or an isotope control
antibody at molar ratio of 15:1 (mAb:HA). The antibody-HA mixture
was then exposed to 2.5 ug/ml of TPCK-treated trypsin and incubated
for 5, 10 and 20 minutes at 37.degree. C. The samples were
separated on a polyacrylamide gel and then transferred to
nitrocellulose membrane for Western blot analysis using a
biotinylated human mAb (F032) (Humabs) that recognizes HA2 and HA0
of influenza A strains (FIG. 1C). The result showed that Antibody 3
was more potent than Fl6v4 in blocking the protease-mediated HA0
cleavage. In contrast, FE17.23, a human monoclonal antibody that
recognizes the HA globular head and control antibody were not able
to inhibit protease-mediated HA0 cleavage. In a separate experiment
we compared the protease cleavage inhibition of Antibody 12 and
Antibody 14 in comparison to Antibody 3 using the same conditions
described above (FIG. 1D). The results showed that Antibody 12,
Antibody 13, had a similar ability to block the protease cleavage
as Antibody 3.
Example 7. Anti-HA Antibodies Exhibit Fc-Effector Function
[0318] Antibodies have the potential to clear virus infected cells
through Fc-effector function such as antibody dependent cellular
cytotoxicity (ADCC), antibody dependent cellular phagocytosis
(ADCP), and complement dependent killing (CDC). To confirm the
anti-HA antibodies exhibited ADCC activity; we tested their ability
to kill virus infected cells in the presence of human natural
killer (NK) cells. The ADCC assay was performed on MDCK cells
infected with A/Hong Kong/8/68 at an MOI of 20. Infected cells were
incubated with a dilution series of antibody, and then incubated
with purified NK cells that were negatively selected from human
PBMC (Miltenyi), at an effector to target ratio of 6:1. The
infected cells, antibody, and NK cells mixtures were incubated for
4 hours, and cell killing was measured by LDH release (Roche). FIG.
2 shows that all four anti-HA stalk antibodies exhibited dose
dependent killing of infected MDCK cells.
[0319] To measure the ability of the anti-HA antibodies to mediate
phagocytosis, we used MDCK cells stably transfected with the HA
derived from A/Hong Kong/8/68 as target cells. Human monocytes were
isolated from PBMCs, and cultured for 7 days in the presence of
M-CSF to differentiate into macrophages. The human macrophages and
HA-expressing target cells were fluorescently labelled violet and
green, respectively (CellTrace Violet or CSFE, Invitrogen).
Labelled effector and target cells were incubated at a 6:1 ratio in
the presence of a dilution series of antibody for 2 hours, and then
analyzed by flow cytometry. The percent phagocytosis was measured
as the percent of violet stained macrophages that also were
positive for the green target cells (double positive). FIG. 3 shows
that all the anti-HA antibodies showed similar levels of ADCP, as
expected the nonspecific control antibody showed no
phagocytosis.
[0320] To measure the ability of the anti-HA antibodies to work
with complement to mediate the killing of infected cells, we
performed CDC assay. In this this assay, MDCK cells were infected
with A/Puerto Rico/8/34 at an MOI of 2, incubated with a dilution
series of antibody, and complement derived from a rabbit
(Cedarlane) at an effector to target ratio of 1:18. Cell killing
was measured by LDH release (Roche). FIG. 4 shows that all the
anti-HA antibodies showed the ability to mediate cell killing in
the presence of complement.
Example 8. Prophylactic and Therapeutic Effect of Anti-HA
Antibodies
[0321] The protective efficacy of human neutralizing antibody
(nAbs) against influenza virus infection was evaluated in
six-to-eight weeks' old BALB/c (Harlan Laboratories) mouse model.
Mice were treated with different doses of nAb either before or
after lethal viral challenge.
[0322] Prophylactic activity (FIGS. 5 & 6) Mice in groups of 8
were administered with Antibody 3 as a single intraperitoneal
injection (IP) at doses of 0.1, 0.3, 1, 3 and 10 mg/kg, or with a
human isotype non-relevant control IgG at 10 mg/kg in 100 .mu.l
volumes. Four hours after dosing, mice were inoculated intranasally
with 7 times the fifty percent mouse lethal dose (7 MLD.sub.50) of
A/California/7/09 (H1N1) (H1-CA09) or 7:1 A/PR/8:A/HK/8/68 HA
(H3N1) (H3-HK68) reassortant in a 50 .mu.l volume. Mice were
weighed on the day or one day before virus challenge and monitored
daily for 14 days for weight loss and survival (mice with body
weight loss 25% were euthanized). Antibody 3 conferred protection
in a dose-dependent manner. IP injection of 1 mg/kg or greater of
Antibody 3 provided complete protection in animals challenged with
H1-CA09 (FIG. 5) and H3-HK68 (FIG. 6). A lower antibody dose (0.3
mg/kg) was also highly protective with 90% protection. As expected,
none of the mice that received the isotype control mAb at 10 mg/kg
survived lethal challenge of infection.
[0323] Therapeutic activity (FIGS. 7 & 8) Mice were inoculated
with 3 MLD.sub.50 of H1-CA09 and injected with Antibody 3 at 24 and
48 hours post infection (h.p.i.) (FIG. 7) or with 5 MLD.sub.50 of
H3-HK68 at 72, 96 and 120 h.p.i. (FIG. 8). IP treatment with 30
mg/kg of Antibody 3 at 24 and 48 h.p.i protected 75-100% of mice
challenged with H1-CA09, and at 72 and 96h.p.i protected 87.5-100%
of mice challenged with H3-HK68. Treatment with same dose of
non-relevant isotype control antibody at 0 or 24 h.p.i in H1 and H3
models failed to protect mice from lethal challenge with a survival
rate of 0 or 12.5%, respectively.
[0324] Therapeutic activity of Antibody 3 variants (FIGS. 9 &
10) Mice were inoculated with 3 MLD.sub.50 of H1-CA09 and injected
with antibodies 24 h.p.i. (FIG. 9) or inoculated with 7 MLD.sub.50
H3-HK68 and injected with antibodies 48 h.p.i. (FIG. 10). IP
treatment with 2 mg/kg of Antibody 3 and variant mAbs (Antibody 11,
Antibody 12, and Antibody 14) protected 87.5-100% of mice
challenged with H1-CA09, and 3 mg/kg dose of the different nAbs
protected 50-87.5% of mice challenged with H3-HK68 As expected,
treatment with same dose of non-relevant isotype control antibody
at 24 or 48 h.p.i in H1 and H3 models failed to protect mice with a
survival rate of 0 or 12.5%, respectively.
Example 9. Therapeutic Effect of Anti-HA Antibodies and Small
Molecule Inhibitor Oseltamivir
[0325] To directly compare the protective efficacy of anti-HA nAbs
to small molecule neuraminidase (NA) inhibitor, oseltamivir, and
the effect of combination therapy, we used the influenza murine
model of infection described in Example 8.
[0326] Therapeutic comparison of anti-HA nAbs and oseltamivir
(FIGS. 11 & 12) Mice were inoculated with 3 MLD.sub.50 of
H1-CA09 and treated with 10 mg/kg of Antibody 12 or 25 mg/kg BID
for 5 days of oseltamivir initiated either at 4 hrs prior, 1 day,
or 2 days post infection (FIG. 11). Treatment with Antibody 12
prior to and 1 day post infection protected 100% of mice challenged
with H1-CA09, whereas all animals treated with oseltamivir
succumbed to the infection. All animals treated with the same dose
of non-relevant isotype control 4 hours prior to infection died
with a survival rate of 0%. Additionally, mice were inoculated with
7 MLD.sub.50 of H3-HK68 then treated with 10 mg/kg of Antibody 12
or 25 mg/kg BID for 5 days of oseltamivir initiated either at 1, 2,
3, or 4 days post infection (FIG. 12). Animals treated with
Antibody 12 at 1, 2, or 3 days post infection showed a survival
rate of 100%, whereas treatment with oseltamivir at these same time
points showed only a 60%-20% survival rate. As expected, mice
treated with same dose of non-relevant isotype control antibody 1
day post infection succumbed to the infection with a survival rate
of 10%.
[0327] Therapeutic combination of anti-HA nAbs and oseltamivir
(FIG. 13) To assess the additive effect of the combination of
anti-HA mAb with oseltamivir, mice were inoculated with 7
MLD.sub.50 of H3-HK68 and treated with a suboptimal concentration
of Antibody 12 (2.5 or 0.3 mg/kg), oseltamivir at 25 mg/kg BID for
5 days, or a combination of Antibody 12 (2.5 or 0.3 mg/kg) and
oseltamivir at 25 mg/kg BID for 5 days, at day 3 post infection
(FIG. 13). Treatment with either Antibody 12 or oseltamivir alone
protected only 10-20% of the animals whereas treatment with the 2.5
mg/kg of Antibody 12 in combination with oseltamivir protected 80%,
and 0.3 mg/kg of Antibody 12 in combination with oseltamivir
protected 50% of the animals.
Example 10. Therapeutic Effect of Anti-HA Antibodies and Small
Molecule Inhibitor Against H5N1 Influenza Infection in the
Ferret
[0328] The protective efficacy of anti-HA nAbs and oseltamivir
against a highly pathogenic influenza virus infection was evaluated
in five-to-six months' old influenza sero-negative ferrets (Triple
F Farms). All ferrets were challenged intranasally with 1 LD.sub.90
of ANN/1203/04 (H5N1) highly pathogenic avian influenza virus in
1.0 mL (approximately 0.5 mL/nare), and then treated with either a
single dose of Antibody 12 at 25 mg/kg or oseltamivir at 25 mg/kg
BID for 5 days initiated at 1, 2, or 3 days post infection. Percent
survival was calculated for each group (n=7) (FIG. 14). Ferrets
treated with Antibody 12 initiated at 1, 2, and 3 days post
infection, as well as those treated with oseltamivir 1 day post
infection were protected, having a 100% survival rate. However,
when oseltamivir treatment was initiated at 2 and 3 days post
infection, ferrets only had 71% survival (mean day of death of 12)
and 29% survival (mean day of death 9), respectively. As expected
animals treated with 25 mg/kg of a non-relevant isotype control
antibody at 1 day post infection failed to live with a 0% survival
rate.
Example 11. Epitope Identification by Selection of Monoclonal
Antibody Resistant Mutants (MARMs)
[0329] Antibody resistant mutants were isolated using two different
methods from three H3N2 viruses. A/Aichi/2/68 (Aichi/68) H3N2 was
incubated with high concentrations of Antibody 12
(125.times.IC.sub.50) for 1 hour before the mixture of virus and
antibody was adsorbed to MDCK cells at 30,000 TCID50 per well in
10.times.96-well plates and cultured in the presence of Antibody 12
(10.times.IC.sub.50). 3 putative Antibody 12 HK2/68 MARMs
exhibiting the cytopathic effect (CPE) on the infected cells up to
3 days after infection were isolated. The HA gene were amplified by
RT-PCR and subsequently sequenced. Sequence analysis revealed 2
nonsynonymous substitutions compared with the parental sequence
(Table 7). These two nucleotide changes respectively code for
single amino acid substitutions from isoleucine (I) to arginine
(R); and from aspartic acid (D) to tyrosine (Y) at amino acid
position 18 and 19 in the highly conserved stalk region of HA2.
Alternatively, serial passage of influenza H3N2 viruses,
A/Wisconsin/67/2005 (W105), and ca A/Panama/2007/1999 (Pa99) were
propagated in the presence of increasing concentrations of Antibody
12 from 2-5.times.IC.sub.50 up to 100.times.IC.sub.50. Potential
escape mutants were subcloned by limited dilution and their cognate
HA genes were subjected to sequence analysis. The single amino acid
changes from D to Y at position 19 and from Glutamine(Q) to R at
position 42 in HA2 was identified. In addition, double mutations
were observed with amino acid substitution from Histine (H) to Q at
position 156 in HA1 in combination with D19Y, or from D to
asparagine (N) at position 19 in combination with amino acid change
from 1 to N at residue 45 in HA2; or from alanine (A) to threonine
(T) at position 196 in HA1 in combination with Q42R (Table 7).
Similarly, when Pa99 was serially passaged in the presence of
Antibody 12 concentrations up to 100.times.IC.sub.50, single amino
acid substitution was selected at HA2 residue 42 (Q42R) and 45
(145T) (Table 7). The representative MARM variants shown in Table 7
were used in a microneutralization assay to further evaluate the
phenotypic susceptibility of these MARMs to neutralization by
Antibody 12. The results showed that the in vitro-selected W105
MARMs containing mutations D19Y, H156Q/D19Y, D19N/145N, Q42R or
A196T/Q42R; Pa99 MARMs containing Q42R or 145T, and Aichi/68 MARMs
harboring mutations D19Y or I18R were less susceptible to antibody
neutralization, with increases in calculated IC.sub.50 values
ranging from >8-fold for Pa99 resistant clones to >180-fold
for W105 resistant variants when compared with their parental wild
type strains, respectively (Table 8). To assess the effect of these
amino acid substitutions on the susceptibility to neutralization by
Antibody 12, recombinant A/Hong Kong/1-5/68 (rHK68) H3 variants
encoding individual mutations were generated and evaluated using a
microneutralization assay. As shown in Table 9, the H3 rHK68_I18R
and rHK68_D19Y variants exhibited resistance to Antibody 12 at the
highest concentration tested (.about.200 .mu.g/mL) and conferred
>130-fold reduction in susceptibility to Antibody 12
neutralization compared with wild type rHK68 virus. The single
amino acid changes Q42R in rHK68 resulted in modest about 8-fold
reductions in susceptibility to neutralization by Antibody 12.
However, amino acid substitutions (K156Q, A196T, I45N or I45T)
identified in the HA proteins of selected MARMs did not alter the
susceptibility of recombinant HK68 viruses encoding such
substitutions to Antibody 12 in microneutralization assay. These
results suggest that Antibody 12 recognizes conformational epitopes
in a highly conserved stalk region of HA2 and amino acids at
positions 18, 19 42 or 45 are key contact residues.
TABLE-US-00007 TABLE 7 Amino acid substitutions identified in the
H3 HA of Antibody 12 resistant mutants Nucleotide Amino acid
Location in H3N2 Virus change change in HA HA subunits
A/Wisconsin/67/2005 G1090T D19Y HA2 C156A, G1090T H156Q, D19Y HA1,
HA2 A1160G Q42R HA2 G634A, A1160G A196T, Q42R HA1 ,HA2 G1090A,
T1169A D19N, I45N HA2, HA2 ca A/Panama/2007/99 A1160G Q42R HA2
T1169C I45T HA2 A/Aichi/2/68 G1090T D19Y HA2 T1088G I18R HA2
TABLE-US-00008 TABLE 8 Susceptibility of H3 resistant variants to
Antibody 12 neutralization (Neut) Amino acid Fold changes changes
in HA of Avg. Neut. relative to wild Parental H3N2 virus MARMs
tested (.mu.g/ml) type virus A/Wisconsin/67/2005 wild type 1.09
D19Y >200 >180 Q42R >200 >180 H156Q/D19Y >200
>180 D19N/I45N >200 >180 A196T/Q42R >200 >180 ca
A/Panama/2007/99 wild type 6.68 Q42R >600 >90 I45T 54.51 8.16
A/Aichi/2/68 wild type 3.98 D19Y >50 >12 I18R >50
>12
TABLE-US-00009 TABLE 9 Susceptibility of rHK68 H3 variants to
Antibody 12 Neutralization (Neut) Fold changes reassortant Avg.
Neut. relative to virus_mutation (.mu.g/ml) wild type virus rHK68
wild type 1.42 1 rHK68_H8R >200 >130 rHK68_D19N 3.04 2.01
rHK68_D19Y >200 >130 rHK68_Q42R 11.13 7.82 rHK68_I45N 1.94
1.28 rHK68_I45T 3.38 2.23 rHK68_K156Q 3.33 2.34 rHK68_A196T 4.06
2.85
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TABLE-US-00010 [0344] Sequence Listing Information Antibody 1
(original cDNA) SEQ ID NO: 1
cagatacagctgcaggagtcgggtccaggactggtgaagccctcgcagaccctctcactcacctgtgccat
ctccggggacagtgtctctagcaacaatgctgtttggaactggatcaggcagtccccatcgagaggccttga
gtggctgggaaggacatactacaggtccaagtggtataatgattatgcagaatctgtgaaaagtcgaataa
ccgtcaatccagacacatccaagaaccagttctccctgcacctgaagtctgtgactcccgaggacacggct
gtgttttactgtgtacgatctggccacattacggtttttggagtgaatgttgacgcttttgatatgtggggcca-
agg gacaatggtcaccgtctcttcag SEQ ID NO: 2
QIQLQESGPGLVKPSQTLSLTCAISGDSVSSNNAVWNWIRQSPSRGLEWLG
RTYYRSKWYNDYAESVKSRITVNPDTSKNQFSLHLKSVTPEDTAVFYCVRS
GHITVFGVNVDAFDMWGQGTMVTVSS SEQ ID NO: 3 HCDR1 SNNAVWN SEQ ID NO: 4
HCDR2 RTYYRSKWYNDYAESVKS SEQ ID NO: 5 HCDR3 SGHITVFGVNVDAFDM SEQ ID
NO: 6
gacatccagatcacccagtcgccatcctccctgtctgcatctgtaggagacagagtaaccatcacttgccgg
acaagtcagagccttagtagctatttacattggtatcagcagaaaccagggaaagcccctaagctcctgatc
tatgctgcatccagtttgcaaagtggggtcccatcaaggttcagtggcagtggatctgggacagatttcactct
caccatcagtagtctgcaacctgaagattttgcaacttactactgtcaacagagtcggacgttcggccaagg
gaccaaggtggaaatcaaa SEQ ID NO: 7
DIQITQSPSSLSASVGDRVTITCRTSQSLSSYLHWYQQKPGKAPKLLIYAASS
LQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQSRTFGQGTKVEIK SEQ ID NO: 8
LCDR1 RTSQSLSSYLH SEQ ID NO: 9 LCDR2 AASSLQS SEQ ID NO: 10 LCDR3
QQSRT Antibody 2 (expressed form of Antibody 1) SEQ ID NO: 11
caggtacagctgcaggagtcgggtccaggactggtgaagccctcgcagaccctctcactcacctgtgccat
ctccggggacagtgtctctagcaacaatgctgtttggaactggatcaggcagtccccatcgagaggccttga
gtggctgggaaggacatactacaggtccaagtggtataatgattatgcagaatctgtgaaaagtcgaataa
ccgtcaatccagacacatccaagaaccagttctccctgcacctgaagtctgtgactcccgaggacacggct
gtgttttactgtgtacgatctggccacattacggtttttggagtgaatgttgacgcttttgatatgtggggcca-
agg gacaatggtcaccgtctcttcag SEQ ID NO: 12
QVQLQESGPGLVKPSQTLSLTCAISGDSVSSNNAVWNWIRQSPSRGLEWL
GRTYYRSKWYNDYAESVKSRITVNPDTSKNQFSLHLKSVTPEDTAVFYCVR
SGHITVFGVNVDAFDMWGQGTMVTVSS SEQ ID NO: 13 HCDR1 SNNAVWN SEQ ID NO:
14 HCDR2 RTYYRSKWYNDYAESVKS SEQ ID NO: 15 HCDR3 SGHITVFGVNVDAFDM
SEQ ID NO: 16
gacatccagatgacccagtcgccatcctccctgtctgcatctgtaggagacagagtaaccatcacttgccgg
acaagtcagagccttagtagctatttacattggtatcagcagaaaccagggaaagcccctaagctcctgatc
tatgctgcatccagtttgcaaagtggggtcccatcaaggttcagtggcagtggatctgggacagatttcactct
caccatcagtagtctgcaacctgaagattttgcaacttactactgtcaacagagtcggacgttcggccaagg
gaccaaggtggaaatcaaa SEQ ID NO: 17
DIQMTQSPSSLSASVGDRVTITCRTSQSLSSYLHWYQQKPGKAPKLLIYAAS
SLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQSRTFGQGTKVEIK SEQ ID NO: 18
LCDR1 RTSQSLSSYLH SEQ ID NO: 19 LCDR2 AASSLQS SEQ ID NO: 20 LCDR3
QQSRT Antibody 3 (codon optimized Antibody 2) SEQ ID NO: 21
caggtccagctgcaggagagcggccccggactggtcaagccttcacagacactgagcctgacatgcgcc
attagcggagatagcgtgagctccaacaatgccgtgtggaactggatcaggcagtctccaagtcgaggac
tggagtggctgggacgaacatactatagatccaagtggtacaatgactatgctgaatcagtgaaaagccg
aattactgtcaaccccgatacctccaagaatcagttctctctgcacctgaaaagtgtgacccctgaggacac
agccgtgttctactgcgtcagaagcggccatatcaccgtctttggcgtcaatgtggatgctttcgatatgtggg
ggcaggggactatggtcaccgtgtcaagc SEQ ID NO: 22
QVQLQESGPGLVKPSQTLSLTCAISGDSVSSNNAVWNWIRQSPSRGLEWL
GRTYYRSKWYNDYAESVKSRITVNPDTSKNQFSLHLKSVTPEDTAVFYCVR
SGHITVFGVNVDAFDMWGQGTMVTVSS SEQ ID NO: 23 HCDR1 SNNAVWN SEQ ID NO:
24 HCDR2 RTYYRSKWYNDYAESVKS SEQ ID NO: 25 HCDR3 SGHITVFGVNVDAFDM
SEQ ID NO: 26
gatattcagatgacccagagcccttccagcctgtccgcttcagtgggggatcgagtgaccattacctgccga
accagccagagcctgagctcctacctgcactggtatcagcagaagcccggcaaagcccctaagctgctg
atctacgccgcttctagtctgcagtccggagtgccaagccggttctccggatctgggagtggaaccgacttta
ccctgacaatttcaagcctgcagcccgaggatttcgctacatactactgtcagcagagcagaactttcgggc
agggcactaaggtggagatcaaa SEQ ID NO: 27
DIQMTQSPSSLSASVGDRVTITCRTSQSLSSYLHWYQQKPGKAPKLLIYAAS
SLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQSRTFGQGTKVEIK SEQ ID NO: 28
LCDR1 RTSQSLSSYLH SEQ ID NO: 29 LCDR2 AASSLQS SEQ ID NO: 30 LCDR3
QQSRT Antibody 4 (original cDNA) degenerate nucleotide in HCDR3, t
or a SEQ ID NO: 31
caggtccagctgcagcagtcaggtccaggactggtgaagccctcgcagaccctctcactcacctgtgccat
ctccggggacagagtctctagcaacagtgctgtttggaactggatcaggcagtccccatcgagaggcctcg
agtggctgggaaggacatattacaggtccaaatggtattatgattatgcagaatctgtgaaaagtcgaatagt
tatcgacccagacacatccaagaaccaggtctccctgcagttgaattctgtgactcccgaggactcggctat
atattactgtgcaagaggtggccacattacggtgtttgggctgaatattgacgcttatgatatttggggccaag
gggcaaaggtcaccgtgtcttcag SEQ ID NO: 32
QVQLQQSGPGLVKPSQTLSLTCAISGDRVSSNSAVWNWIRQSPSRGLEWL
GRTYYRSKWYYDYAESVKSRIVIDPDTSKNQVSLQLNSVTPEDSAIYYCARG
GHITVFGLNIDAYDIWGQGAKVTVSS SEQ ID NO: 33 HCDR1 SNSAVWN SEQ ID NO:
34 HCDR2 RTYYRSKWYYDYAESVKS SEQ ID NO: 35 HCDR3 GGHITVFGLNIDAYDI
SEQ ID NO: 36
gacatccaggtgacccagtctccgtcctccctgtctgcatctgtaggagacagagtcaccatctcttgccggg
cacagagccttagcagctacttacattggtatcagcagaaaccagggcaaccccctaaactcctgatctat
gctgcaaccactttgcaaagtggggtcccatcacggttcagtggtagtggatctgggacagatttcactctca
ccatcagtactttccaagctgaagatgttgccacttactattgtcaacagagtcggacgttcggccaagggac
caaggttgaaatcaaac SEQ ID NO: 37
DIQVTQSPSSLSASVGDRVTISCRAQSLSSYLHWYQQKPGQPPKLLIYAATT
LQSGVPSRFSGSGSGTDFTLTISTFQAEDVATYYCQQSRTFGQGTKVEIK SEQ ID NO: 38
LCDR1 RAQSLSSYLH SEQ ID NO: 39 LCDR2 AATTLQS SEQ ID NO: 40 LCDR3
QQSRT Antibody 5 (expressed form of Antibody 4 HCDR3 V) SEQ ID NO:
41
caggtacagctgcagcagtcaggtccaggactggtgaagccctcgcagaccctctcactcacctgtgccat
ctccggggacagagtctctagcaacagtgctgtttggaactggatcaggcagtccccatcgagaggcctcg
agtggctgggaaggacatattacaggtccaaatggtattatgattatgcagaatctgtgaaaagtcgaatagt
tatcgacccagacacatccaagaaccaggtctccctgcagttgaattctgtgactcccgaggactcggctat
atattactgtgcaagaggtggccacattacggtgtttgggctgaatattgacgcttatgatatttggggccaag
gggcaatggtcaccgtctcttcag SEQ ID NO: 42
QVQLQQSGPGLVKPSQTLSLTCAISGDRVSSNSAVWNWIRQSPSRGLEWL
GRTYYRSKWYYDYAESVKSRIVIDPDTSKNQVSLQLNSVTPEDSAIYYCARG
GHITVFGLNIDAYDIWGQGAMVTVSS SEQ ID NO: 43 HCDR1 SNSAVWN SEQ ID NO:
44 HCDR2 RTYYRSKWYYDYAESVKS SEQ ID NO: 45 HCDR3 GGHITVFGLNIDAYDI
SEQ ID NO: 46
gacatccagatgacccagtctccgtcctccctgtctgcatctgtaggagacagagtcaccatctcttgccggg
cacagagccttagcagctacttacattggtatcagcagaaaccagggcaaccccctaaactcctgatctat
gctgcaaccactttgcaaagtggggtcccatcacggttcagtggtagtggatctgggacagatttcactctca
ccatcagtactttccaagctgaagatgttgccacttactattgtcaacagagtcggacgttcggccaagggac
caaggtggagatcaaac SEQ ID NO: 47
DIQMTQSPSSLSASVGDRVTISCRAQSLSSYLHWYQQKPGQPPKLLIYAATT
LQSGVPSRFSGSGSGTDFTLTISTFQAEDVATYYCQQSRTFGQGTKVEIK SEQ ID NO: 48
LCDR1 RAQSLSSYLH SEQ ID NO: 49 LCDR2 AATTLQS SEQ ID NO: 50 LCDR3
QQSRT Antibody 6 (expressed form of Antibody 4 HCDR3 E) SEQ ID NO:
51
caggtacagctgcagcagtcaggtccaggactggtgaagccctcgcagaccctctcactcacctgtgccat
ctccggggacagagtctctagcaacagtgctgtttggaactggatcaggcagtccccatcgagaggcctcg
agtggctgggaaggacatattacaggtccaaatggtattatgattatgcagaatctgtgaaaagtcgaatagt
tatcgacccagacacatccaagaaccaggtctccctgcagttgaattctgtgactcccgaggactcggctat
atattactgtgcaagaggtggccacattacggagtttgggctgaatattgacgcttatgatatttggggccaag
gggcaatggtcaccgtctcttcag SEQ ID NO: 52
QVQLQQSGPGLVKPSQTLSLTCAISGDRVSSNSAVWNWIRQSPSRGLEWL
GRTYYRSKWYYDYAESVKSRIVIDPDTSKNQVSLQLNSVTPEDSAIYYCARG
GHITEFGLNIDAYDIWGQGAMVTVSS SEQ ID NO: 53 HCDR1 SNSAVWN SEQ ID NO:
54 HCDR2 RTYYRSKWYYDYAESVKS SEQ ID NO: 55 HCDR3 GGHITEFGLNIDAYDI
SEQ ID NO: 56
gacatccagatgacccagtctccgtcctccctgtctgcatctgtaggagacagagtcaccatctcttgccggg
cacagagccttagcagctacttacattggtatcagcagaaaccagggcaaccccctaaactcctgatctat
gctgcaaccactttgcaaagtggggtcccatcacggttcagtggtagtggatctgggacagatttcactctca
ccatcagtactttccaagctgaagatgttgccacttactattgtcaacagagtcggacgttcggccaagggac
caaggtggagatcaaac SEQ ID NO: 57
DIQMTQSPSSLSASVGDRVTISCRAQSLSSYLHWYQQKPGQPPKLLIYAATT
LQSGVPSRFSGSGSGTDFTLTISTFQAEDVATYYCQQSRTFGQGTKVEIK SEQ ID NO: 58
LCDR1 RAQSLSSYLH SEQ ID NO: 59 LCDR2 AATTLQS SEQ ID NO: 60 LCDR3
QQSRT Antibody 7 (original cDNA) SEQ ID NO: 61
caggtacagctgcagcagtcaggtccaggactggtgaagccctcgcagaccctctccctcacctgtgtcat
ctccggagacactgtctctagcaacagagctacttggaattggatgaggcagtccccattgagaggccttga
gtggctgggaaggacatactacaggtccaagtggtataatgattacgcagtttctgtgaaaagtcgagtagt
catcaacccagacacatccaagaaccaagtctccctgcagttgaacactgtgactcccgatgactcgggtg
tatacttttgtgcaagaggtggccacatcacggtctttggagtgaatattgacgcttttgacatctggggcctc-
g ggacaaaggtcaccgtctcttcag SEQ ID NO: 62
QVQLQQSGPGLVKPSQTLSLTCVISGDTVSSNRATWNWMRQSPLRGLEWL
GRTYYRSKWYNDYAVSVKSRVVINPDTSKNQVSLQLNTVTPDDSGVYFCAR
GGHITVFGVNIDAFDIWGLGTKVTVSS SEQ ID NO: 63 HCDR1 SNRATWN
SEQ ID NO: 64 HCDR2 RTYYRSKWYNDYAVSVKS SEQ ID NO: 65 HCDR3
GGHITVFGVNIDAFDI SEQ ID NO: 66
gacatccaggtgacccagtctccatcctccctgtctgcatctgtaggagacagagttaccatctcttgccggg
caagtcagagacttaatagttatctacattggtatcagcagacaccagggcaagccccgaagctgctgatct
atgcaacgtccactttgcaaagtggggtctcaccaagattcagtggcagtggatctgggacagatttcactct
caccatcagcagtctccaacctgaagatgttgcaacttactactgtcaattgagtcggacgttcggccacgg
gaccaaggttgaaatcaaac SEQ ID NO: 67
DIQVTQSPSSLSASVGDRVTISCRASQRLNSYLHWYQQTPGQAPKLLIYATS
TLQSGVSPRFSGSGSGTDFTLTISSLQPEDVATYYCQLSRTFGHGTKVEIK SEQ ID NO: 68
LCDR1 RASQRLNSYLH SEQ ID NO: 69 LCDR2 ATSTLQS SEQ ID NO: 70 LCDR3
QLSRT Antibody 8 (expressed form of Antibody 7) SEQ ID NO: 71
caggtacagctgcagcagtcaggtccaggactggtgaagccctcgcagaccctctccctcacctgtgtcat
ctccggagacactgtctctagcaacagagctacttggaattggatgaggcagtccccattgagaggccttga
gtggctgggaaggacatactacaggtccaagtggtataatgattacgcagtttctgtgaaaagtcgagtagt
catcaacccagacacatccaagaaccaagtctccctgcagttgaacactgtgactcccgatgactcgggtg
tatacttttgtgcaagaggtggccacatcacggtctttggagtgaatattgacgcttttgacatctggggcctc-
g ggacaaaggtcaccgtctcttcag SEQ ID NO: 72
QVQLQQSGPGLVKPSQTLSLTCVISGDTVSSNRATWNWMRQSPLRGLEWL
GRTYYRSKWYNDYAVSVKSRVVINPDTSKNQVSLQLNTVTPDDSGVYFCAR
GGHITVFGVNIDAFDIWGLGTKVTVSS SEQ ID NO: 73 HCDR1 SNRATWN SEQ ID NO:
74 HCDR2 RTYYRSKWYNDYAVSVKS SEQ ID NO: 75 HCDR3 GGHITVFGVNIDAFDI
SEQ ID NO: 76
gacatccagatgacccagtctccatcctccctgtctgcatctgtaggagacagagttaccatctcttgccggg
caagtcagagacttaatagttatctacattggtatcagcagacaccagggcaagccccgaagctgctgatct
atgcaacgtccactttgcaaagtggggtctcaccaagattcagtggcagtggatctgggacagatttcactct
caccatcagcagtctccaacctgaagatgttgcaacttactactgtcaattgagtcggacgttcggccacgg
gaccaaggtggaaatcaaac SEQ ID NO: 77
DIQMTQSPSSLSASVGDRVTISCRASQRLNSYLHWYQQTPGQAPKLLIYATS
TLQSGVSPRFSGSGSGTDFTLTISSLQPEDVATYYCQLSRTFGHGTKVEIK SEQ ID NO: 78
LCDR1 RASQRLNSYLH SEQ ID NO: 79 LCDR2 ATSTLQS SEQ ID NO: 80 LCDR3
QLSRT Antibody 9 (original cDNA) SEQ ID NO: 81
caagtagagctgcagcagtcaggtccaggactggtgaagccctcgcagaccctctcactcacctgtgccat
ctccggggacagtgtctctagcaacagtgctacttggaactggatcaggcagtccccatcgagaggccttg
agtggctgggaaggacatactacaggtccaagtggtataatgattatgcagattttctgaaaaggcgaataa
ccatcaatccagacacatccaacaacgaggtctccctgcggctgacctctgtgactcccgacgacacggct
ttgtattactgtgcaagaggtggccacattacggtgtttggagtgaatattgacgcctttgacgtctggggcca-
a gggacaatggccaccgtctcttcag SEQ ID NO: 82
QVELQQSGPGLVKPSQTLSLTCAISGDSVSSNSATWNWIRQSPSRGLEWL
GRTYYRSKWYNDYADFLKRRITINPDTSNNEVSLRLTSVTPDDTALYYCARG
GHITVFGVNIDAFDVWGQGTMATVSS SEQ ID NO: 83 HCDR1 SNSATWN SEQ ID NO:
84 HCDR2 RTYYRSKWYNDYADFLKR SEQ ID NO: 85 HCDR3 GGHITVFGVNIDAFDV
SEQ ID NO: 86
gacatccaggtgacccagtctccatcctccctgtctgcatctgtaggagacagaatcaccatctcttgccgga
caagtcagagccttaggagctatttacattggtatcagcaaaaaccagggaaagcccctaagctcctgatct
atgcttcatccactttacaaagtggggtcccatcaaggttcagtggcagtggatctgggacagatttcactctc
accatcagcaatctccaacctgaagattttgcaacttactactgtcaactgagtcggacgttcggccaaggg
accaaggttgaaatcaaac SEQ ID NO: 87
DIQVTQSPSSLSASVGDRITISCRTSQSLRSYLHWYQQKPGKAPKLLIYASST
LQSGVPSRFSGSGSGTDFTLTISNLQPEDFATYYCQLSRTFGQGTKVEIK SEQ ID NO: 88
LCDR1 RTSQSLRSYLH SEQ ID NO: 89 LCDR2 ASSTLQS SEQ ID NO: 90 LCDR3
QLSRT Antibody 10 (expressed form of Antibody 9) SEQ ID NO: 91
caggtacagctgcagcagtcaggtccaggactggtgaagccctcgcagaccctctcactcacctgtgccat
ctccggggacagtgtctctagcaacagtgctacttggaactggatcaggcagtccccatcgagaggccttg
agtggctgggaaggacatactacaggtccaagtggtataatgattatgcagattttctgaaaaggcgaataa
ccatcaatccagacacatccaacaacgaggtctccctgcggctgacctctgtgactcccgacgacacggct
ttgtattactgtgcaagaggtggccacattacggtgtttggagtgaatattgacgcctttgacgtctggggcca-
a gggacaatggtcaccgtctcttcag SEQ ID NO: 92
QVQLQQSGPGLVKPSQTLSLTCAISGDSVSSNSATWNWIRQSPSRGLEWL
GRTYYRSKWYNDYADFLKRRITINPDTSNNEVSLRLTSVTPDDTALYYCARG
GHITVFGVNIDAFDVWGQGTMVTVSS SEQ ID NO: 93 HCDR1 SNSATWN SEQ ID NO:
94 HCDR2 RTYYRSKWYNDYADFLKR SEQ ID NO: 95 HCDR3 GGHITVFGVNIDAFDV
SEQ ID NO: 96
gacatccagatgacccagtctccatcctccctgtctgcatctgtaggagacagaatcaccatctcttgccgga
caagtcagagccttaggagctatttacattggtatcagcaaaaaccagggaaagcccctaagctcctgatct
atgcttcatccactttacaaagtggggtcccatcaaggttcagtggcagtggatctgggacagatttcactctc
accatcagcaatctccaacctgaagattttgcaacttactactgtcaactgagtcggacgttcggccaaggg
accaaggtggagatcaaac SEQ ID NO: 97
DIQMTQSPSSLSASVGDRITISCRTSQSLRSYLHWYQQKPGKAPKLLIYASS
TLQSGVPSRFSGSGSGTDFTLTISNLQPEDFATYYCQLSRTFGQGTKVEIK SEQ ID NO: 98
LCDR1 RTSQSLRSYLH SEQ ID NO: 99 LCDR2 ASSTLQS SEQ ID NO: 100 LCDR3
QLSRT Antibody 11 SEQ ID NO: 101
caggtccagctgcagcagagcggccccggactggtcaagccttcacagacactgagcctgacatgcgcc
attagcggagatagcgtgagctcctacaatgccgtgtggaactggatcaggcagtctccaagtcgaggact
ggagtggctgggacgaacatactatagatccgggtggtacaatgactatgctgaatcagtgaaaagccga
attactatcaaccccgatacctccaagaatcagttctctctgcagctgaacagtgtgacccctgaggacaca
gccgtgtactactgcgccagaagcggccatatcaccgtctttggcgtcaatgtggatgctttcgatatgtgggg
gcaggggactatggtcaccgtgtcaagc SEQ ID NO: 102
QVQLQQSGPGLVKPSQTLSLTCAISGDSVSSYNAVWNWIRQSPSRGLEWL
GRTYYRSGWYNDYAESVKSRITINPDTSKNQFSLQLNSVTPEDTAVYYCAR
SGHITVFGVNVDAFDMWGQGTMVTVSS SEQ ID NO: 103 HCDR1 SYNAVWN SEQ ID NO:
104 HCDR2 RTYYRSGWYNDYAESVKS SEQ ID NO: 105 HCDR3 SGHITVFGVNVDAFDM
SEQ ID NO: 106
gatattcagatgacccagagcccttccagcctgtccgcttcagtgggggatcgagtgaccattacctgccga
accagccagagcctgagctcctacacgcactggtatcagcagaagcccggcaaagcccctaagctgctg
atctacgccgcttctagtcggctgtccggagtgccaagccggttctccggatctgggagtggaaccgacttta
ccctgacaatttcaagcctgcagcccgaggatttcgctacatactactgtcagcagagcagaactttcgggc
agggcactaaggtggagatcaaa SEQ ID NO: 107
DIQMTQSPSSLSASVGDRVTITCRTSQSLSSYTHWYQQKPGKAPKLLIYAAS
SRLSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQSRTFGQGTKVE1K SEQ ID NO: 108
LCDR1 RTSQSLSSYTH SEQ ID NO: 109 LCDR2 AASSRLS SEQ ID NO: 110 LCDR3
QQSRT Antibody 12 SEQ ID NO: 111
caggtccagctgcagcagagcggccccggactggtcaagccttcacagacactgagcctgacatgcgcc
attagcggagatagcgtgagctcctacaatgccgtgtggaactggatcaggcagtctccaagtcgaggact
ggagtggctgggacgaacatactatagatccgggtggtacaatgactatgctgaatcagtgaaaagccga
attactatcaaccccgatacctccaagaatcagttctctctgcagctgaacagtgtgacccctgaggacaca
gccgtgtactactgcgccagaagcggccatatcaccgtctttggcgtcaatgtggatgctttcgatatgtgggg
gcaggggactatggtcaccgtgtcaagc SEQ ID NO: 112
QVQLQQSGPGLVKPSQTLSLTCAISGDSVSSYNAVWNWIRQSPSRGLEWL
GRTYYRSGWYNDYAESVKSRITINPDTSKNQFSLQLNSVTPEDTAVYYCAR
SGHITVFGVNVDAFDMWGQGTMVTVSS SEQ ID NO: 113 HCDR1 SYNAVWN SEQ ID NO:
114 HCDR2 RTYYRSGWYNDYAESVKS SEQ ID NO: 115 HCDR3 SGHITVFGVNVDAFDM
SEQ ID NO: 116
gatattcagatgacccagagcccttccagcctgtccgcttcagtgggggatcgagtgaccattacctgccga
accagccagagcctgagctcctacacgcactggtatcagcagaagcccggcaaagcccctaagctgctg
atctacgccgcttctagtcgggggtccggagtgccaagccggttctccggatctgggagtggaaccgacttt
accctgacaatttcaagcctgcagcccgaggatttcgctacatactactgtcagcagagcagaactttcggg
cagggcactaaggtggagatcaaa SEQ ID NO: 117
DIQMTQSPSSLSASVGDRVTITCRTSQSLSSYTHWYQQKPGKAPKLLIYAAS
SRGSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQSRTFGQGTKVEIK SEQ ID NO: 118
LCDR1 RTSQSLSSYTH SEQ ID NO: 119 LCDR2 AASSRGS SEQ ID NO: 120 LCDR3
QQSRT Antibody 13 SEQ ID NO: 121
caggtccagctgcagcagagcggccccggactggtcaagccttcacagacactgagcctgacatgcgcc
attagcggagatagcgtgagctcctacaatgccgtgtggaactggatcaggcagtctccaagtcgaggact
ggagtggctgggacgaacatactatagatccgggtggtacaatgactatgctgaatcagtgaaaagccga
attactatcaaccccgatacctccaagaatcagttctctctgcagctgaacagtgtgacccctgaggacaca
gccgtgtactactgcgccagaagcggccatatcaccgtctttggcgtcaatgtggatgctttcgatatgtgggg
gcaggggactatggtcaccgtgtcaagc SEQ ID NO: 122
QVQLQQSGPGLVKPSQTLSLTCAISGDSVSSYNAVWNWIRQSPSRGLEWL
GRTYYRSGWYNDYAESVKSRITINPDTSKNQFSLQLNSVTPEDTAVYYCAR
SGHITVFGVNVDAFDMWGQGTMVTVSS SEQ ID NO: 123 HCDR1 SYNAVWN SEQ ID NO:
124 HCDR2 RTYYRSGWYNDYAESVKS SEQ ID NO: 125 HCDR3 SGHITVFGVNVDAFDM
SEQ ID NO: 126
gatattcagatgacccagagcccttccagcctgtccgcttcagtgggggatcgagtgaccattacctgccga
accagccagagcctgagctcctacgaccactggtatcagcagaagcccggcaaagcccctaagctgctg
atctacgccgcttctagtcggctgtccggagtgccaagccggttctccggatctgggagtggaaccgacttta
ccctgacaatttcaagcctgcagcccgaggatttcgctacatactactgtcagcagagcagaactttcgggc
agggcactaaggtggagatcaaa SEQ ID NO: 127
DIQMTQSPSSLSASVGDRVTITCRTSQSLSSYDHWYQQKPGKAPKLLIYAAS
SRLSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQSRTFGQGTKVE1K SEQ ID NO: 128
LCDR1 RTSQSLSSYDH SEQ ID NO: 129 LCDR2 AASSRLS SEQ ID NO: 130 LCDR3
QQSRT
Antibody 14 SEQ ID NO: 131
caggtccagctgcagcagagcggccccggactggtcaagccttcacagacactgagcctgacatgcgcc
attagcggagatagcgtgagctccaacaatgccgtgtggaactggatcaggcagtctccaagtcgaggac
tggagtggctgggacgaacatactatagatccaagtggtacaatgactatgctgaatcagtgaaaagccg
aattactatcaaccccgatacctccaagaatcagttctctctgcagctgaacagtgtgacccctgaggacac
agccgtgtactactgcgccagaagcggccatatcaccgtctttggcgtcaatgtggatgctttcgatatgtggg
ggcaggggaccacagtcaccgtctcctca SEQ ID NO: 132
QVQLQQSGPGLVKPSQTLSLTCAISGDSVSSNNAVWNWIRQSPSRGLEWL
GRTYYRSKWYNDYAESVKSRITINPDTSKNQFSLQLNSVTPEDTAVYYCAR
SGHITVFGVNVDAFDMWGQGTTVTVSS SEQ ID NO: 133 HCDR1 SNNAVWN SEQ ID NO:
134 HCDR2 RTYYRSKWYNDYAESVKS SEQ ID NO: 135 HCDR3 SGHITVFGVNVDAFDM
SEQ ID NO: 136
gatattcagatgacccagagcccttccagcctgtccgcttcagtgggggatcgagtgaccattacctgccga
accagccagagcctgagctcctacacgcactggtatcagcagaagcccggcaaagcccctaagctgctg
atctacgccgcttctagtcggctgtccggagtgccaagccggttctccggatctgggagtggaaccgacttta
ccctgacaatttcaagcctgcagcccgaggatttcgctacatactactgtcagcagagcagaactttcgggc
agggcactaaggtggagatcaaa SEQ ID NO: 137
DIQMTQSPSSLSASVGDRVTITCRTSQSLSSYTHWYQQKPGKAPKLLIYAAS
SRLSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQSRTFGQGTKVE1K SEQ ID NO: 138
LCDR1 RTSQSLSSYTH SEQ ID NO: 139 LCDR2 AASSRLS SEQ ID NO: 140 LCDR3
QQSRT Antibody 15 SEQ ID NO: 141
caggtccagctgcagcagagcggccccggactggtcaagccttcacagacactgagcctgacatgcgcc
attagcggagatagcgtgagctccaacaatgccgtgtggaactggatcaggcagtctccaagtcgaggac
tggagtggctgggacgaacatactatagatccaagtggtacaatgactatgctgaatcagtgaaaagccg
aattactatcaaccccgatacctccaagaatcagttctctctgcagctgaacagtgtgacccctgaggacac
agccgtgtactactgcgccagaagcggccatatcaccgtctttggcgtcaatgtggatgctttcgatatgtggg
ggcaggggaccacagtcaccgtctcctca SEQ ID NO: 142
QVQLQQSGPGLVKPSQTLSLTCAISGDSVSSNNAVWNWIRQSPSRGLEWL
GRTYYRSKWYNDYAESVKSRITINPDTSKNQFSLQLNSVTPEDTAVYYCAR
SGHITVFGVNVDAFDMWGQGTTVTVSS SEQ ID NO: 143 HCDR1 SNNAVWN SEQ ID NO:
144 HCDR2 RTYYRSKWYNDYAESVKS SEQ ID NO: 145 HCDR3 SGHITVFGVNVDAFDM
SEQ ID NO: 146
gatattcagatgacccagagcccttccagcctgtccgcttcagtgggggatcgagtgaccattacctgccga
accagccagagcctgagytcctacacgcactggtatcagcagaagcccggcaaagcccctaagctgctg
atctacgccgcttctagtcgggggtccggagtgccaagccggttctccggatctgggagtggaaccgacttt
accctgacaatttcaagcctgcagcccgaggatttcgctacatactactgtcagcagagcagaactttcggg
cagggcactaaggtggagatcaaa SEQ ID NO: 147
DIQMTQSPSSLSASVGDRVTITCRTSQSLSSYTHWYQQKPGKAPKLLIYAAS
SRGSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQSRTFGQGTKVEIK SEQ ID NO: 148
LCDR1 RTSQSLSSYTH SEQ ID NO: 149 LCDR2 AASSRGS SEQ ID NO: 150 LCDR3
QQSRT Antibody 3-GL SEQ ID NO: 151
caggtccagctgcagcagagcggccccggactggtcaagccttcacagacactgagcctgacatgcgcc
attagcggagatagcgtgagctccaacaatgccgtgtggaactggatcaggcagtctccaagtcgaggac
tggagtggctgggacgaacatactatagatccaagtggtacaatgactatgctgaatcagtgaaaagccg
aattactatcaaccccgatacctccaagaatcagttctctctgcagctgaacagtgtgacccctgaggacac
agccgtgtactactgcgccagaagcggccatatcaccgtctttggcgtcaatgtggatgctttcgatatgtggg
ggcaggggaccacagtcaccgtctcctca SEQ ID NO: 152
QVQLQQSGPGLVKPSQTLSLTCAISGDSVSSNNAVWNWIRQSPSRGLEWL
GRTYYRSKWYNDYAESVKSRITINPDTSKNQFSLQLNSVTPEDTAVYYCAR
SGHITVFGVNVDAFDMWGQGTTVTVSS SEQ ID NO: 153 HCDR1 SNNAVWN SEQ ID NO:
154 HCDR2 RTYYRSKWYNDYAESVKS SEQ ID NO: 155 HCDR3 SGHITVFGVNVDAFDM
SEQ ID NO: 156
gatattcagatgacccagagcccttccagcctgtccgcttcagtgggggatcgagtgaccattacctgccga
accagccagagcctgagctcctacctgcactggtatcagcagaagcccggcaaagcccctaagctgctg
atctacgccgcttctagtctgcagtccggagtgccaagccggttctccggatctgggagtggaaccgacttta
ccctgacaatttcaagcctgcagcccgaggatttcgctacatactactgtcagcagagcagaactttcgggc
agggcactaaggtggagatcaaa SEQ ID NO: 157
DIQMTQSPSSLSASVGDRVTITCRTSQSLSSYLHWYQQKPGKAPKLLIYAAS
SLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQSRTFGQGTKVEIK SEQ ID NO: 158
LCDR1 RTSQSLSSYLH SEQ ID NO: 159 LCDR2 AASSLQS SEQ ID NO: 160 LCDR3
QQSRT
Sequence CWU 1
1
1721385DNAArtificial SequenceDescription of Artificial Sequence
Synthetic polynucleotide 1cagatacagc tgcaggagtc gggtccagga
ctggtgaagc cctcgcagac cctctcactc 60acctgtgcca tctccgggga cagtgtctct
agcaacaatg ctgtttggaa ctggatcagg 120cagtccccat cgagaggcct
tgagtggctg ggaaggacat actacaggtc caagtggtat 180aatgattatg
cagaatctgt gaaaagtcga ataaccgtca atccagacac atccaagaac
240cagttctccc tgcacctgaa gtctgtgact cccgaggaca cggctgtgtt
ttactgtgta 300cgatctggcc acattacggt ttttggagtg aatgttgacg
cttttgatat gtggggccaa 360gggacaatgg tcaccgtctc ttcag
3852128PRTArtificial SequenceDescription of Artificial Sequence
Synthetic polypeptide 2Gln Ile Gln Leu Gln Glu Ser Gly Pro Gly Leu
Val Lys Pro Ser Gln1 5 10 15Thr Leu Ser Leu Thr Cys Ala Ile Ser Gly
Asp Ser Val Ser Ser Asn 20 25 30Asn Ala Val Trp Asn Trp Ile Arg Gln
Ser Pro Ser Arg Gly Leu Glu 35 40 45Trp Leu Gly Arg Thr Tyr Tyr Arg
Ser Lys Trp Tyr Asn Asp Tyr Ala 50 55 60Glu Ser Val Lys Ser Arg Ile
Thr Val Asn Pro Asp Thr Ser Lys Asn65 70 75 80Gln Phe Ser Leu His
Leu Lys Ser Val Thr Pro Glu Asp Thr Ala Val 85 90 95Phe Tyr Cys Val
Arg Ser Gly His Ile Thr Val Phe Gly Val Asn Val 100 105 110Asp Ala
Phe Asp Met Trp Gly Gln Gly Thr Met Val Thr Val Ser Ser 115 120
12537PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 3Ser Asn Asn Ala Val Trp Asn1 5418PRTArtificial
SequenceDescription of Artificial Sequence Synthetic peptide 4Arg
Thr Tyr Tyr Arg Ser Lys Trp Tyr Asn Asp Tyr Ala Glu Ser Val1 5 10
15Lys Ser516PRTArtificial SequenceDescription of Artificial
Sequence Synthetic peptide 5Ser Gly His Ile Thr Val Phe Gly Val Asn
Val Asp Ala Phe Asp Met1 5 10 156309DNAArtificial
SequenceDescription of Artificial Sequence Synthetic polynucleotide
6gacatccaga tcacccagtc gccatcctcc ctgtctgcat ctgtaggaga cagagtaacc
60atcacttgcc ggacaagtca gagccttagt agctatttac attggtatca gcagaaacca
120gggaaagccc ctaagctcct gatctatgct gcatccagtt tgcaaagtgg
ggtcccatca 180aggttcagtg gcagtggatc tgggacagat ttcactctca
ccatcagtag tctgcaacct 240gaagattttg caacttacta ctgtcaacag
agtcggacgt tcggccaagg gaccaaggtg 300gaaatcaaa 3097103PRTArtificial
SequenceDescription of Artificial Sequence Synthetic polypeptide
7Asp Ile Gln Ile Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly1 5
10 15Asp Arg Val Thr Ile Thr Cys Arg Thr Ser Gln Ser Leu Ser Ser
Tyr 20 25 30Leu His 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 Phe Ala Thr Tyr Tyr Cys Gln Gln
Ser Arg Thr Phe Gly Gln 85 90 95Gly Thr Lys Val Glu Ile Lys
100811PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 8Arg Thr Ser Gln Ser Leu Ser Ser Tyr Leu His1 5
1097PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 9Ala Ala Ser Ser Leu Gln Ser1 5105PRTArtificial
SequenceDescription of Artificial Sequence Synthetic peptide 10Gln
Gln Ser Arg Thr1 511385DNAArtificial SequenceDescription of
Artificial Sequence Synthetic polynucleotide 11caggtacagc
tgcaggagtc gggtccagga ctggtgaagc cctcgcagac cctctcactc 60acctgtgcca
tctccgggga cagtgtctct agcaacaatg ctgtttggaa ctggatcagg
120cagtccccat cgagaggcct tgagtggctg ggaaggacat actacaggtc
caagtggtat 180aatgattatg cagaatctgt gaaaagtcga ataaccgtca
atccagacac atccaagaac 240cagttctccc tgcacctgaa gtctgtgact
cccgaggaca cggctgtgtt ttactgtgta 300cgatctggcc acattacggt
ttttggagtg aatgttgacg cttttgatat gtggggccaa 360gggacaatgg
tcaccgtctc ttcag 38512128PRTArtificial SequenceDescription of
Artificial Sequence Synthetic polypeptide 12Gln Val Gln Leu Gln Glu
Ser Gly Pro Gly Leu Val Lys Pro Ser Gln1 5 10 15Thr Leu Ser Leu Thr
Cys Ala Ile Ser Gly Asp Ser Val Ser Ser Asn 20 25 30Asn Ala Val Trp
Asn Trp Ile Arg Gln Ser Pro Ser Arg Gly Leu Glu 35 40 45Trp Leu Gly
Arg Thr Tyr Tyr Arg Ser Lys Trp Tyr Asn Asp Tyr Ala 50 55 60Glu Ser
Val Lys Ser Arg Ile Thr Val Asn Pro Asp Thr Ser Lys Asn65 70 75
80Gln Phe Ser Leu His Leu Lys Ser Val Thr Pro Glu Asp Thr Ala Val
85 90 95Phe Tyr Cys Val Arg Ser Gly His Ile Thr Val Phe Gly Val Asn
Val 100 105 110Asp Ala Phe Asp Met Trp Gly Gln Gly Thr Met Val Thr
Val Ser Ser 115 120 125137PRTArtificial SequenceDescription of
Artificial Sequence Synthetic peptide 13Ser Asn Asn Ala Val Trp
Asn1 51418PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 14Arg Thr Tyr Tyr Arg Ser Lys Trp Tyr Asn Asp Tyr
Ala Glu Ser Val1 5 10 15Lys Ser1516PRTArtificial
SequenceDescription of Artificial Sequence Synthetic peptide 15Ser
Gly His Ile Thr Val Phe Gly Val Asn Val Asp Ala Phe Asp Met1 5 10
1516309DNAArtificial SequenceDescription of Artificial Sequence
Synthetic polynucleotide 16gacatccaga tgacccagtc gccatcctcc
ctgtctgcat ctgtaggaga cagagtaacc 60atcacttgcc ggacaagtca gagccttagt
agctatttac attggtatca gcagaaacca 120gggaaagccc ctaagctcct
gatctatgct gcatccagtt tgcaaagtgg ggtcccatca 180aggttcagtg
gcagtggatc tgggacagat ttcactctca ccatcagtag tctgcaacct
240gaagattttg caacttacta ctgtcaacag agtcggacgt tcggccaagg
gaccaaggtg 300gaaatcaaa 30917103PRTArtificial SequenceDescription
of Artificial Sequence Synthetic polypeptide 17Asp 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 Thr Ser Gln Ser Leu Ser Ser Tyr 20 25 30Leu His 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 Phe Ala Thr Tyr Tyr Cys Gln Gln Ser Arg Thr Phe Gly Gln
85 90 95Gly Thr Lys Val Glu Ile Lys 1001811PRTArtificial
SequenceDescription of Artificial Sequence Synthetic peptide 18Arg
Thr Ser Gln Ser Leu Ser Ser Tyr Leu His1 5 10197PRTArtificial
SequenceDescription of Artificial Sequence Synthetic peptide 19Ala
Ala Ser Ser Leu Gln Ser1 5205PRTArtificial SequenceDescription of
Artificial Sequence Synthetic peptide 20Gln Gln Ser Arg Thr1
521384DNAArtificial SequenceDescription of Artificial Sequence
Synthetic polynucleotide 21caggtccagc tgcaggagag cggccccgga
ctggtcaagc cttcacagac actgagcctg 60acatgcgcca ttagcggaga tagcgtgagc
tccaacaatg ccgtgtggaa ctggatcagg 120cagtctccaa gtcgaggact
ggagtggctg ggacgaacat actatagatc caagtggtac 180aatgactatg
ctgaatcagt gaaaagccga attactgtca accccgatac ctccaagaat
240cagttctctc tgcacctgaa aagtgtgacc cctgaggaca cagccgtgtt
ctactgcgtc 300agaagcggcc atatcaccgt ctttggcgtc aatgtggatg
ctttcgatat gtgggggcag 360gggactatgg tcaccgtgtc aagc
38422128PRTArtificial SequenceDescription of Artificial Sequence
Synthetic polypeptide 22Gln Val Gln Leu Gln Glu Ser Gly Pro Gly Leu
Val Lys Pro Ser Gln1 5 10 15Thr Leu Ser Leu Thr Cys Ala Ile Ser Gly
Asp Ser Val Ser Ser Asn 20 25 30Asn Ala Val Trp Asn Trp Ile Arg Gln
Ser Pro Ser Arg Gly Leu Glu 35 40 45Trp Leu Gly Arg Thr Tyr Tyr Arg
Ser Lys Trp Tyr Asn Asp Tyr Ala 50 55 60Glu Ser Val Lys Ser Arg Ile
Thr Val Asn Pro Asp Thr Ser Lys Asn65 70 75 80Gln Phe Ser Leu His
Leu Lys Ser Val Thr Pro Glu Asp Thr Ala Val 85 90 95Phe Tyr Cys Val
Arg Ser Gly His Ile Thr Val Phe Gly Val Asn Val 100 105 110Asp Ala
Phe Asp Met Trp Gly Gln Gly Thr Met Val Thr Val Ser Ser 115 120
125237PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 23Ser Asn Asn Ala Val Trp Asn1 52418PRTArtificial
SequenceDescription of Artificial Sequence Synthetic peptide 24Arg
Thr Tyr Tyr Arg Ser Lys Trp Tyr Asn Asp Tyr Ala Glu Ser Val1 5 10
15Lys Ser2516PRTArtificial SequenceDescription of Artificial
Sequence Synthetic peptide 25Ser Gly His Ile Thr Val Phe Gly Val
Asn Val Asp Ala Phe Asp Met1 5 10 1526309DNAArtificial
SequenceDescription of Artificial Sequence Synthetic polynucleotide
26gatattcaga tgacccagag cccttccagc ctgtccgctt cagtggggga tcgagtgacc
60attacctgcc gaaccagcca gagcctgagc tcctacctgc actggtatca gcagaagccc
120ggcaaagccc ctaagctgct gatctacgcc gcttctagtc tgcagtccgg
agtgccaagc 180cggttctccg gatctgggag tggaaccgac tttaccctga
caatttcaag cctgcagccc 240gaggatttcg ctacatacta ctgtcagcag
agcagaactt tcgggcaggg cactaaggtg 300gagatcaaa 30927103PRTArtificial
SequenceDescription of Artificial Sequence Synthetic polypeptide
27Asp 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 Thr Ser Gln Ser Leu Ser Ser
Tyr 20 25 30Leu His 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 Phe Ala Thr Tyr Tyr Cys Gln Gln
Ser Arg Thr Phe Gly Gln 85 90 95Gly Thr Lys Val Glu Ile Lys
1002811PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 28Arg Thr Ser Gln Ser Leu Ser Ser Tyr Leu His1 5
10297PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 29Ala Ala Ser Ser Leu Gln Ser1 5305PRTArtificial
SequenceDescription of Artificial Sequence Synthetic peptide 30Gln
Gln Ser Arg Thr1 531385DNAArtificial SequenceDescription of
Artificial Sequence Synthetic polynucleotide 31caggtccagc
tgcagcagtc aggtccagga ctggtgaagc cctcgcagac cctctcactc 60acctgtgcca
tctccgggga cagagtctct agcaacagtg ctgtttggaa ctggatcagg
120cagtccccat cgagaggcct cgagtggctg ggaaggacat attacaggtc
caaatggtat 180tatgattatg cagaatctgt gaaaagtcga atagttatcg
acccagacac atccaagaac 240caggtctccc tgcagttgaa ttctgtgact
cccgaggact cggctatata ttactgtgca 300agaggtggcc acattacggt
gtttgggctg aatattgacg cttatgatat ttggggccaa 360ggggcaaagg
tcaccgtgtc ttcag 38532128PRTArtificial SequenceDescription of
Artificial Sequence Synthetic polypeptide 32Gln Val Gln Leu Gln Gln
Ser Gly Pro Gly Leu Val Lys Pro Ser Gln1 5 10 15Thr Leu Ser Leu Thr
Cys Ala Ile Ser Gly Asp Arg Val Ser Ser Asn 20 25 30Ser Ala Val Trp
Asn Trp Ile Arg Gln Ser Pro Ser Arg Gly Leu Glu 35 40 45Trp Leu Gly
Arg Thr Tyr Tyr Arg Ser Lys Trp Tyr Tyr Asp Tyr Ala 50 55 60Glu Ser
Val Lys Ser Arg Ile Val Ile Asp Pro Asp Thr Ser Lys Asn65 70 75
80Gln Val Ser Leu Gln Leu Asn Ser Val Thr Pro Glu Asp Ser Ala Ile
85 90 95Tyr Tyr Cys Ala Arg Gly Gly His Ile Thr Val Phe Gly Leu Asn
Ile 100 105 110Asp Ala Tyr Asp Ile Trp Gly Gln Gly Ala Lys Val Thr
Val Ser Ser 115 120 125337PRTArtificial SequenceDescription of
Artificial Sequence Synthetic peptide 33Ser Asn Ser Ala Val Trp
Asn1 53418PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 34Arg Thr Tyr Tyr Arg Ser Lys Trp Tyr Tyr Asp Tyr
Ala Glu Ser Val1 5 10 15Lys Ser3516PRTArtificial
SequenceDescription of Artificial Sequence Synthetic peptide 35Gly
Gly His Ile Thr Val Phe Gly Leu Asn Ile Asp Ala Tyr Asp Ile1 5 10
1536307DNAArtificial SequenceDescription of Artificial Sequence
Synthetic polynucleotide 36gacatccagg tgacccagtc tccgtcctcc
ctgtctgcat ctgtaggaga cagagtcacc 60atctcttgcc gggcacagag ccttagcagc
tacttacatt ggtatcagca gaaaccaggg 120caacccccta aactcctgat
ctatgctgca accactttgc aaagtggggt cccatcacgg 180ttcagtggta
gtggatctgg gacagatttc actctcacca tcagtacttt ccaagctgaa
240gatgttgcca cttactattg tcaacagagt cggacgttcg gccaagggac
caaggttgaa 300atcaaac 30737102PRTArtificial SequenceDescription of
Artificial Sequence Synthetic polypeptide 37Asp Ile Gln Val Thr Gln
Ser Pro Ser Ser Leu Ser Ala Ser Val Gly1 5 10 15Asp Arg Val Thr Ile
Ser Cys Arg Ala Gln Ser Leu Ser Ser Tyr Leu 20 25 30His Trp Tyr Gln
Gln Lys Pro Gly Gln Pro Pro Lys Leu Leu Ile Tyr 35 40 45Ala Ala Thr
Thr Leu Gln Ser Gly Val Pro Ser Arg Phe Ser Gly Ser 50 55 60Gly Ser
Gly Thr Asp Phe Thr Leu Thr Ile Ser Thr Phe Gln Ala Glu65 70 75
80Asp Val Ala Thr Tyr Tyr Cys Gln Gln Ser Arg Thr Phe Gly Gln Gly
85 90 95Thr Lys Val Glu Ile Lys 1003810PRTArtificial
SequenceDescription of Artificial Sequence Synthetic peptide 38Arg
Ala Gln Ser Leu Ser Ser Tyr Leu His1 5 10397PRTArtificial
SequenceDescription of Artificial Sequence Synthetic peptide 39Ala
Ala Thr Thr Leu Gln Ser1 5405PRTArtificial SequenceDescription of
Artificial Sequence Synthetic peptide 40Gln Gln Ser Arg Thr1
541385DNAArtificial SequenceDescription of Artificial Sequence
Synthetic polynucleotide 41caggtacagc tgcagcagtc aggtccagga
ctggtgaagc cctcgcagac cctctcactc 60acctgtgcca tctccgggga cagagtctct
agcaacagtg ctgtttggaa ctggatcagg 120cagtccccat cgagaggcct
cgagtggctg ggaaggacat attacaggtc caaatggtat 180tatgattatg
cagaatctgt gaaaagtcga atagttatcg acccagacac atccaagaac
240caggtctccc tgcagttgaa ttctgtgact cccgaggact cggctatata
ttactgtgca 300agaggtggcc acattacggt gtttgggctg aatattgacg
cttatgatat ttggggccaa 360ggggcaatgg tcaccgtctc ttcag
38542128PRTArtificial SequenceDescription of Artificial Sequence
Synthetic polypeptide 42Gln Val Gln Leu Gln Gln Ser Gly Pro Gly Leu
Val Lys Pro Ser Gln1 5 10 15Thr Leu Ser Leu Thr Cys Ala Ile Ser Gly
Asp Arg Val Ser Ser Asn 20 25 30Ser Ala Val Trp Asn Trp Ile Arg Gln
Ser Pro Ser Arg Gly Leu Glu 35 40 45Trp Leu Gly Arg Thr Tyr Tyr Arg
Ser Lys Trp Tyr Tyr Asp Tyr Ala 50 55 60Glu Ser Val Lys Ser Arg Ile
Val Ile Asp Pro Asp Thr Ser Lys Asn65 70 75 80Gln Val Ser Leu Gln
Leu Asn Ser Val Thr Pro Glu Asp Ser Ala Ile 85 90 95Tyr Tyr Cys Ala
Arg Gly Gly His Ile Thr Val Phe Gly Leu Asn Ile 100 105 110Asp Ala
Tyr Asp Ile Trp Gly Gln Gly Ala Met Val Thr Val Ser Ser 115 120
125437PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 43Ser Asn Ser Ala Val Trp Asn1 54418PRTArtificial
SequenceDescription of Artificial Sequence Synthetic peptide 44Arg
Thr Tyr Tyr Arg Ser Lys Trp Tyr Tyr Asp Tyr Ala Glu Ser Val1 5 10
15Lys Ser4516PRTArtificial SequenceDescription of Artificial
Sequence Synthetic peptide 45Gly Gly His Ile Thr Val Phe Gly Leu
Asn Ile Asp Ala Tyr Asp Ile1 5 10
1546307DNAArtificial SequenceDescription of Artificial Sequence
Synthetic polynucleotide 46gacatccaga tgacccagtc tccgtcctcc
ctgtctgcat ctgtaggaga cagagtcacc 60atctcttgcc gggcacagag ccttagcagc
tacttacatt ggtatcagca gaaaccaggg 120caacccccta aactcctgat
ctatgctgca accactttgc aaagtggggt cccatcacgg 180ttcagtggta
gtggatctgg gacagatttc actctcacca tcagtacttt ccaagctgaa
240gatgttgcca cttactattg tcaacagagt cggacgttcg gccaagggac
caaggtggag 300atcaaac 30747102PRTArtificial SequenceDescription of
Artificial Sequence Synthetic polypeptide 47Asp Ile Gln Met Thr Gln
Ser Pro Ser Ser Leu Ser Ala Ser Val Gly1 5 10 15Asp Arg Val Thr Ile
Ser Cys Arg Ala Gln Ser Leu Ser Ser Tyr Leu 20 25 30His Trp Tyr Gln
Gln Lys Pro Gly Gln Pro Pro Lys Leu Leu Ile Tyr 35 40 45Ala Ala Thr
Thr Leu Gln Ser Gly Val Pro Ser Arg Phe Ser Gly Ser 50 55 60Gly Ser
Gly Thr Asp Phe Thr Leu Thr Ile Ser Thr Phe Gln Ala Glu65 70 75
80Asp Val Ala Thr Tyr Tyr Cys Gln Gln Ser Arg Thr Phe Gly Gln Gly
85 90 95Thr Lys Val Glu Ile Lys 1004810PRTArtificial
SequenceDescription of Artificial Sequence Synthetic peptide 48Arg
Ala Gln Ser Leu Ser Ser Tyr Leu His1 5 10497PRTArtificial
SequenceDescription of Artificial Sequence Synthetic peptide 49Ala
Ala Thr Thr Leu Gln Ser1 5505PRTArtificial SequenceDescription of
Artificial Sequence Synthetic peptide 50Gln Gln Ser Arg Thr1
551385DNAArtificial SequenceDescription of Artificial Sequence
Synthetic polynucleotide 51caggtacagc tgcagcagtc aggtccagga
ctggtgaagc cctcgcagac cctctcactc 60acctgtgcca tctccgggga cagagtctct
agcaacagtg ctgtttggaa ctggatcagg 120cagtccccat cgagaggcct
cgagtggctg ggaaggacat attacaggtc caaatggtat 180tatgattatg
cagaatctgt gaaaagtcga atagttatcg acccagacac atccaagaac
240caggtctccc tgcagttgaa ttctgtgact cccgaggact cggctatata
ttactgtgca 300agaggtggcc acattacgga gtttgggctg aatattgacg
cttatgatat ttggggccaa 360ggggcaatgg tcaccgtctc ttcag
38552128PRTArtificial SequenceDescription of Artificial Sequence
Synthetic polypeptide 52Gln Val Gln Leu Gln Gln Ser Gly Pro Gly Leu
Val Lys Pro Ser Gln1 5 10 15Thr Leu Ser Leu Thr Cys Ala Ile Ser Gly
Asp Arg Val Ser Ser Asn 20 25 30Ser Ala Val Trp Asn Trp Ile Arg Gln
Ser Pro Ser Arg Gly Leu Glu 35 40 45Trp Leu Gly Arg Thr Tyr Tyr Arg
Ser Lys Trp Tyr Tyr Asp Tyr Ala 50 55 60Glu Ser Val Lys Ser Arg Ile
Val Ile Asp Pro Asp Thr Ser Lys Asn65 70 75 80Gln Val Ser Leu Gln
Leu Asn Ser Val Thr Pro Glu Asp Ser Ala Ile 85 90 95Tyr Tyr Cys Ala
Arg Gly Gly His Ile Thr Glu Phe Gly Leu Asn Ile 100 105 110Asp Ala
Tyr Asp Ile Trp Gly Gln Gly Ala Met Val Thr Val Ser Ser 115 120
125537PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 53Ser Asn Ser Ala Val Trp Asn1 55418PRTArtificial
SequenceDescription of Artificial Sequence Synthetic peptide 54Arg
Thr Tyr Tyr Arg Ser Lys Trp Tyr Tyr Asp Tyr Ala Glu Ser Val1 5 10
15Lys Ser5516PRTArtificial SequenceDescription of Artificial
Sequence Synthetic peptide 55Gly Gly His Ile Thr Glu Phe Gly Leu
Asn Ile Asp Ala Tyr Asp Ile1 5 10 1556307DNAArtificial
SequenceDescription of Artificial Sequence Synthetic polynucleotide
56gacatccaga tgacccagtc tccgtcctcc ctgtctgcat ctgtaggaga cagagtcacc
60atctcttgcc gggcacagag ccttagcagc tacttacatt ggtatcagca gaaaccaggg
120caacccccta aactcctgat ctatgctgca accactttgc aaagtggggt
cccatcacgg 180ttcagtggta gtggatctgg gacagatttc actctcacca
tcagtacttt ccaagctgaa 240gatgttgcca cttactattg tcaacagagt
cggacgttcg gccaagggac caaggtggag 300atcaaac 30757102PRTArtificial
SequenceDescription of Artificial Sequence Synthetic polypeptide
57Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly1
5 10 15Asp Arg Val Thr Ile Ser Cys Arg Ala Gln Ser Leu Ser Ser Tyr
Leu 20 25 30His Trp Tyr Gln Gln Lys Pro Gly Gln Pro Pro Lys Leu Leu
Ile Tyr 35 40 45Ala Ala Thr Thr Leu Gln Ser Gly Val Pro Ser Arg Phe
Ser Gly Ser 50 55 60Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Thr
Phe Gln Ala Glu65 70 75 80Asp Val Ala Thr Tyr Tyr Cys Gln Gln Ser
Arg Thr Phe Gly Gln Gly 85 90 95Thr Lys Val Glu Ile Lys
1005810PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 58Arg Ala Gln Ser Leu Ser Ser Tyr Leu His1 5
10597PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 59Ala Ala Thr Thr Leu Gln Ser1 5605PRTArtificial
SequenceDescription of Artificial Sequence Synthetic peptide 60Gln
Gln Ser Arg Thr1 561385DNAArtificial SequenceDescription of
Artificial Sequence Synthetic polynucleotide 61caggtacagc
tgcagcagtc aggtccagga ctggtgaagc cctcgcagac cctctccctc 60acctgtgtca
tctccggaga cactgtctct agcaacagag ctacttggaa ttggatgagg
120cagtccccat tgagaggcct tgagtggctg ggaaggacat actacaggtc
caagtggtat 180aatgattacg cagtttctgt gaaaagtcga gtagtcatca
acccagacac atccaagaac 240caagtctccc tgcagttgaa cactgtgact
cccgatgact cgggtgtata cttttgtgca 300agaggtggcc acatcacggt
ctttggagtg aatattgacg cttttgacat ctggggcctc 360gggacaaagg
tcaccgtctc ttcag 38562128PRTArtificial SequenceDescription of
Artificial Sequence Synthetic polypeptide 62Gln Val Gln Leu Gln Gln
Ser Gly Pro Gly Leu Val Lys Pro Ser Gln1 5 10 15Thr Leu Ser Leu Thr
Cys Val Ile Ser Gly Asp Thr Val Ser Ser Asn 20 25 30Arg Ala Thr Trp
Asn Trp Met Arg Gln Ser Pro Leu Arg Gly Leu Glu 35 40 45Trp Leu Gly
Arg Thr Tyr Tyr Arg Ser Lys Trp Tyr Asn Asp Tyr Ala 50 55 60Val Ser
Val Lys Ser Arg Val Val Ile Asn Pro Asp Thr Ser Lys Asn65 70 75
80Gln Val Ser Leu Gln Leu Asn Thr Val Thr Pro Asp Asp Ser Gly Val
85 90 95Tyr Phe Cys Ala Arg Gly Gly His Ile Thr Val Phe Gly Val Asn
Ile 100 105 110Asp Ala Phe Asp Ile Trp Gly Leu Gly Thr Lys Val Thr
Val Ser Ser 115 120 125637PRTArtificial SequenceDescription of
Artificial Sequence Synthetic peptide 63Ser Asn Arg Ala Thr Trp
Asn1 56418PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 64Arg Thr Tyr Tyr Arg Ser Lys Trp Tyr Asn Asp Tyr
Ala Val Ser Val1 5 10 15Lys Ser6516PRTArtificial
SequenceDescription of Artificial Sequence Synthetic peptide 65Gly
Gly His Ile Thr Val Phe Gly Val Asn Ile Asp Ala Phe Asp Ile1 5 10
1566310DNAArtificial SequenceDescription of Artificial Sequence
Synthetic polynucleotide 66gacatccagg tgacccagtc tccatcctcc
ctgtctgcat ctgtaggaga cagagttacc 60atctcttgcc gggcaagtca gagacttaat
agttatctac attggtatca gcagacacca 120gggcaagccc cgaagctgct
gatctatgca acgtccactt tgcaaagtgg ggtctcacca 180agattcagtg
gcagtggatc tgggacagat ttcactctca ccatcagcag tctccaacct
240gaagatgttg caacttacta ctgtcaattg agtcggacgt tcggccacgg
gaccaaggtt 300gaaatcaaac 31067103PRTArtificial SequenceDescription
of Artificial Sequence Synthetic polypeptide 67Asp Ile Gln Val Thr
Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly1 5 10 15Asp Arg Val Thr
Ile Ser Cys Arg Ala Ser Gln Arg Leu Asn Ser Tyr 20 25 30Leu His Trp
Tyr Gln Gln Thr Pro Gly Gln Ala Pro Lys Leu Leu Ile 35 40 45Tyr Ala
Thr Ser Thr Leu Gln Ser Gly Val Ser Pro 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 Leu Ser Arg Thr Phe Gly His
85 90 95Gly Thr Lys Val Glu Ile Lys 1006811PRTArtificial
SequenceDescription of Artificial Sequence Synthetic peptide 68Arg
Ala Ser Gln Arg Leu Asn Ser Tyr Leu His1 5 10697PRTArtificial
SequenceDescription of Artificial Sequence Synthetic peptide 69Ala
Thr Ser Thr Leu Gln Ser1 5705PRTArtificial SequenceDescription of
Artificial Sequence Synthetic peptide 70Gln Leu Ser Arg Thr1
571385DNAArtificial SequenceDescription of Artificial Sequence
Synthetic polynucleotide 71caggtacagc tgcagcagtc aggtccagga
ctggtgaagc cctcgcagac cctctccctc 60acctgtgtca tctccggaga cactgtctct
agcaacagag ctacttggaa ttggatgagg 120cagtccccat tgagaggcct
tgagtggctg ggaaggacat actacaggtc caagtggtat 180aatgattacg
cagtttctgt gaaaagtcga gtagtcatca acccagacac atccaagaac
240caagtctccc tgcagttgaa cactgtgact cccgatgact cgggtgtata
cttttgtgca 300agaggtggcc acatcacggt ctttggagtg aatattgacg
cttttgacat ctggggcctc 360gggacaaagg tcaccgtctc ttcag
38572128PRTArtificial SequenceDescription of Artificial Sequence
Synthetic polypeptide 72Gln Val Gln Leu Gln Gln Ser Gly Pro Gly Leu
Val Lys Pro Ser Gln1 5 10 15Thr Leu Ser Leu Thr Cys Val Ile Ser Gly
Asp Thr Val Ser Ser Asn 20 25 30Arg Ala Thr Trp Asn Trp Met Arg Gln
Ser Pro Leu Arg Gly Leu Glu 35 40 45Trp Leu Gly Arg Thr Tyr Tyr Arg
Ser Lys Trp Tyr Asn Asp Tyr Ala 50 55 60Val Ser Val Lys Ser Arg Val
Val Ile Asn Pro Asp Thr Ser Lys Asn65 70 75 80Gln Val Ser Leu Gln
Leu Asn Thr Val Thr Pro Asp Asp Ser Gly Val 85 90 95Tyr Phe Cys Ala
Arg Gly Gly His Ile Thr Val Phe Gly Val Asn Ile 100 105 110Asp Ala
Phe Asp Ile Trp Gly Leu Gly Thr Lys Val Thr Val Ser Ser 115 120
125737PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 73Ser Asn Arg Ala Thr Trp Asn1 57418PRTArtificial
SequenceDescription of Artificial Sequence Synthetic peptide 74Arg
Thr Tyr Tyr Arg Ser Lys Trp Tyr Asn Asp Tyr Ala Val Ser Val1 5 10
15Lys Ser7516PRTArtificial SequenceDescription of Artificial
Sequence Synthetic peptide 75Gly Gly His Ile Thr Val Phe Gly Val
Asn Ile Asp Ala Phe Asp Ile1 5 10 1576310DNAArtificial
SequenceDescription of Artificial Sequence Synthetic polynucleotide
76gacatccaga tgacccagtc tccatcctcc ctgtctgcat ctgtaggaga cagagttacc
60atctcttgcc gggcaagtca gagacttaat agttatctac attggtatca gcagacacca
120gggcaagccc cgaagctgct gatctatgca acgtccactt tgcaaagtgg
ggtctcacca 180agattcagtg gcagtggatc tgggacagat ttcactctca
ccatcagcag tctccaacct 240gaagatgttg caacttacta ctgtcaattg
agtcggacgt tcggccacgg gaccaaggtg 300gaaatcaaac
31077103PRTArtificial SequenceDescription of Artificial Sequence
Synthetic polypeptide 77Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu
Ser Ala Ser Val Gly1 5 10 15Asp Arg Val Thr Ile Ser Cys Arg Ala Ser
Gln Arg Leu Asn Ser Tyr 20 25 30Leu His Trp Tyr Gln Gln Thr Pro Gly
Gln Ala Pro Lys Leu Leu Ile 35 40 45Tyr Ala Thr Ser Thr Leu Gln Ser
Gly Val Ser Pro 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 Leu Ser Arg Thr Phe Gly His 85 90 95Gly Thr Lys Val
Glu Ile Lys 1007811PRTArtificial SequenceDescription of Artificial
Sequence Synthetic peptide 78Arg Ala Ser Gln Arg Leu Asn Ser Tyr
Leu His1 5 10797PRTArtificial SequenceDescription of Artificial
Sequence Synthetic peptide 79Ala Thr Ser Thr Leu Gln Ser1
5805PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 80Gln Leu Ser Arg Thr1 581385DNAArtificial
SequenceDescription of Artificial Sequence Synthetic polynucleotide
81caagtagagc tgcagcagtc aggtccagga ctggtgaagc cctcgcagac cctctcactc
60acctgtgcca tctccgggga cagtgtctct agcaacagtg ctacttggaa ctggatcagg
120cagtccccat cgagaggcct tgagtggctg ggaaggacat actacaggtc
caagtggtat 180aatgattatg cagattttct gaaaaggcga ataaccatca
atccagacac atccaacaac 240gaggtctccc tgcggctgac ctctgtgact
cccgacgaca cggctttgta ttactgtgca 300agaggtggcc acattacggt
gtttggagtg aatattgacg cctttgacgt ctggggccaa 360gggacaatgg
ccaccgtctc ttcag 38582128PRTArtificial SequenceDescription of
Artificial Sequence Synthetic polypeptide 82Gln Val Glu Leu Gln Gln
Ser Gly Pro Gly Leu Val Lys Pro Ser Gln1 5 10 15Thr Leu Ser Leu Thr
Cys Ala Ile Ser Gly Asp Ser Val Ser Ser Asn 20 25 30Ser Ala Thr Trp
Asn Trp Ile Arg Gln Ser Pro Ser Arg Gly Leu Glu 35 40 45Trp Leu Gly
Arg Thr Tyr Tyr Arg Ser Lys Trp Tyr Asn Asp Tyr Ala 50 55 60Asp Phe
Leu Lys Arg Arg Ile Thr Ile Asn Pro Asp Thr Ser Asn Asn65 70 75
80Glu Val Ser Leu Arg Leu Thr Ser Val Thr Pro Asp Asp Thr Ala Leu
85 90 95Tyr Tyr Cys Ala Arg Gly Gly His Ile Thr Val Phe Gly Val Asn
Ile 100 105 110Asp Ala Phe Asp Val Trp Gly Gln Gly Thr Met Ala Thr
Val Ser Ser 115 120 125837PRTArtificial SequenceDescription of
Artificial Sequence Synthetic peptide 83Ser Asn Ser Ala Thr Trp
Asn1 58418PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 84Arg Thr Tyr Tyr Arg Ser Lys Trp Tyr Asn Asp Tyr
Ala Asp Phe Leu1 5 10 15Lys Arg8516PRTArtificial
SequenceDescription of Artificial Sequence Synthetic peptide 85Gly
Gly His Ile Thr Val Phe Gly Val Asn Ile Asp Ala Phe Asp Val1 5 10
1586310DNAArtificial SequenceDescription of Artificial Sequence
Synthetic polynucleotide 86gacatccagg tgacccagtc tccatcctcc
ctgtctgcat ctgtaggaga cagaatcacc 60atctcttgcc ggacaagtca gagccttagg
agctatttac attggtatca gcaaaaacca 120gggaaagccc ctaagctcct
gatctatgct tcatccactt tacaaagtgg ggtcccatca 180aggttcagtg
gcagtggatc tgggacagat ttcactctca ccatcagcaa tctccaacct
240gaagattttg caacttacta ctgtcaactg agtcggacgt tcggccaagg
gaccaaggtt 300gaaatcaaac 31087103PRTArtificial SequenceDescription
of Artificial Sequence Synthetic polypeptide 87Asp Ile Gln Val Thr
Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly1 5 10 15Asp Arg Ile Thr
Ile Ser Cys Arg Thr Ser Gln Ser Leu Arg Ser Tyr 20 25 30Leu His Trp
Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile 35 40 45Tyr Ala
Ser 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 Asn Leu Gln Pro65 70 75
80Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Leu Ser Arg Thr Phe Gly Gln
85 90 95Gly Thr Lys Val Glu Ile Lys 1008811PRTArtificial
SequenceDescription of Artificial Sequence Synthetic peptide 88Arg
Thr Ser Gln Ser Leu Arg Ser Tyr Leu His1 5 10897PRTArtificial
SequenceDescription of Artificial Sequence Synthetic peptide 89Ala
Ser Ser Thr Leu Gln Ser1 5905PRTArtificial SequenceDescription of
Artificial Sequence Synthetic peptide 90Gln Leu Ser Arg Thr1
591385DNAArtificial SequenceDescription of Artificial Sequence
Synthetic polynucleotide 91caggtacagc tgcagcagtc aggtccagga
ctggtgaagc cctcgcagac cctctcactc 60acctgtgcca tctccgggga cagtgtctct
agcaacagtg ctacttggaa ctggatcagg 120cagtccccat cgagaggcct
tgagtggctg ggaaggacat actacaggtc caagtggtat 180aatgattatg
cagattttct gaaaaggcga ataaccatca atccagacac atccaacaac
240gaggtctccc tgcggctgac ctctgtgact cccgacgaca cggctttgta
ttactgtgca
300agaggtggcc acattacggt gtttggagtg aatattgacg cctttgacgt
ctggggccaa 360gggacaatgg tcaccgtctc ttcag 38592128PRTArtificial
SequenceDescription of Artificial Sequence Synthetic polypeptide
92Gln Val Gln Leu Gln Gln Ser Gly Pro Gly Leu Val Lys Pro Ser Gln1
5 10 15Thr Leu Ser Leu Thr Cys Ala Ile Ser Gly Asp Ser Val Ser Ser
Asn 20 25 30Ser Ala Thr Trp Asn Trp Ile Arg Gln Ser Pro Ser Arg Gly
Leu Glu 35 40 45Trp Leu Gly Arg Thr Tyr Tyr Arg Ser Lys Trp Tyr Asn
Asp Tyr Ala 50 55 60Asp Phe Leu Lys Arg Arg Ile Thr Ile Asn Pro Asp
Thr Ser Asn Asn65 70 75 80Glu Val Ser Leu Arg Leu Thr Ser Val Thr
Pro Asp Asp Thr Ala Leu 85 90 95Tyr Tyr Cys Ala Arg Gly Gly His Ile
Thr Val Phe Gly Val Asn Ile 100 105 110Asp Ala Phe Asp Val Trp Gly
Gln Gly Thr Met Val Thr Val Ser Ser 115 120 125937PRTArtificial
SequenceDescription of Artificial Sequence Synthetic peptide 93Ser
Asn Ser Ala Thr Trp Asn1 59418PRTArtificial SequenceDescription of
Artificial Sequence Synthetic peptide 94Arg Thr Tyr Tyr Arg Ser Lys
Trp Tyr Asn Asp Tyr Ala Asp Phe Leu1 5 10 15Lys
Arg9516PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 95Gly Gly His Ile Thr Val Phe Gly Val Asn Ile Asp
Ala Phe Asp Val1 5 10 1596310DNAArtificial SequenceDescription of
Artificial Sequence Synthetic polynucleotide 96gacatccaga
tgacccagtc tccatcctcc ctgtctgcat ctgtaggaga cagaatcacc 60atctcttgcc
ggacaagtca gagccttagg agctatttac attggtatca gcaaaaacca
120gggaaagccc ctaagctcct gatctatgct tcatccactt tacaaagtgg
ggtcccatca 180aggttcagtg gcagtggatc tgggacagat ttcactctca
ccatcagcaa tctccaacct 240gaagattttg caacttacta ctgtcaactg
agtcggacgt tcggccaagg gaccaaggtg 300gagatcaaac
31097103PRTArtificial SequenceDescription of Artificial Sequence
Synthetic polypeptide 97Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu
Ser Ala Ser Val Gly1 5 10 15Asp Arg Ile Thr Ile Ser Cys Arg Thr Ser
Gln Ser Leu Arg Ser Tyr 20 25 30Leu His Trp Tyr Gln Gln Lys Pro Gly
Lys Ala Pro Lys Leu Leu Ile 35 40 45Tyr Ala Ser 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 Asn Leu Gln Pro65 70 75 80Glu Asp Phe Ala Thr
Tyr Tyr Cys Gln Leu Ser Arg Thr Phe Gly Gln 85 90 95Gly Thr Lys Val
Glu Ile Lys 1009811PRTArtificial SequenceDescription of Artificial
Sequence Synthetic peptide 98Arg Thr Ser Gln Ser Leu Arg Ser Tyr
Leu His1 5 10997PRTArtificial SequenceDescription of Artificial
Sequence Synthetic peptide 99Ala Ser Ser Thr Leu Gln Ser1
51005PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 100Gln Leu Ser Arg Thr1 5101384DNAArtificial
SequenceDescription of Artificial Sequence Synthetic polynucleotide
101caggtccagc tgcagcagag cggccccgga ctggtcaagc cttcacagac
actgagcctg 60acatgcgcca ttagcggaga tagcgtgagc tcctacaatg ccgtgtggaa
ctggatcagg 120cagtctccaa gtcgaggact ggagtggctg ggacgaacat
actatagatc cgggtggtac 180aatgactatg ctgaatcagt gaaaagccga
attactatca accccgatac ctccaagaat 240cagttctctc tgcagctgaa
cagtgtgacc cctgaggaca cagccgtgta ctactgcgcc 300agaagcggcc
atatcaccgt ctttggcgtc aatgtggatg ctttcgatat gtgggggcag
360gggactatgg tcaccgtgtc aagc 384102128PRTArtificial
SequenceDescription of Artificial Sequence Synthetic polypeptide
102Gln Val Gln Leu Gln Gln Ser Gly Pro Gly Leu Val Lys Pro Ser Gln1
5 10 15Thr Leu Ser Leu Thr Cys Ala Ile Ser Gly Asp Ser Val Ser Ser
Tyr 20 25 30Asn Ala Val Trp Asn Trp Ile Arg Gln Ser Pro Ser Arg Gly
Leu Glu 35 40 45Trp Leu Gly Arg Thr Tyr Tyr Arg Ser Gly Trp Tyr Asn
Asp Tyr Ala 50 55 60Glu Ser Val Lys Ser Arg Ile Thr Ile Asn Pro Asp
Thr Ser Lys Asn65 70 75 80Gln Phe Ser Leu Gln Leu Asn Ser Val Thr
Pro Glu Asp Thr Ala Val 85 90 95Tyr Tyr Cys Ala Arg Ser Gly His Ile
Thr Val Phe Gly Val Asn Val 100 105 110Asp Ala Phe Asp Met Trp Gly
Gln Gly Thr Met Val Thr Val Ser Ser 115 120 1251037PRTArtificial
SequenceDescription of Artificial Sequence Synthetic peptide 103Ser
Tyr Asn Ala Val Trp Asn1 510418PRTArtificial SequenceDescription of
Artificial Sequence Synthetic peptide 104Arg Thr Tyr Tyr Arg Ser
Gly Trp Tyr Asn Asp Tyr Ala Glu Ser Val1 5 10 15Lys
Ser10516PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 105Ser Gly His Ile Thr Val Phe Gly Val Asn Val
Asp Ala Phe Asp Met1 5 10 15106309DNAArtificial SequenceDescription
of Artificial Sequence Synthetic polynucleotide 106gatattcaga
tgacccagag cccttccagc ctgtccgctt cagtggggga tcgagtgacc 60attacctgcc
gaaccagcca gagcctgagc tcctacacgc actggtatca gcagaagccc
120ggcaaagccc ctaagctgct gatctacgcc gcttctagtc ggctgtccgg
agtgccaagc 180cggttctccg gatctgggag tggaaccgac tttaccctga
caatttcaag cctgcagccc 240gaggatttcg ctacatacta ctgtcagcag
agcagaactt tcgggcaggg cactaaggtg 300gagatcaaa
309107103PRTArtificial SequenceDescription of Artificial Sequence
Synthetic polypeptide 107Asp 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 Thr
Ser Gln Ser Leu Ser Ser Tyr 20 25 30Thr His Trp Tyr Gln Gln Lys Pro
Gly Lys Ala Pro Lys Leu Leu Ile 35 40 45Tyr Ala Ala Ser Ser Arg Leu
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 Arg Thr Phe Gly Gln 85 90 95Gly Thr Lys
Val Glu Ile Lys 10010811PRTArtificial SequenceDescription of
Artificial Sequence Synthetic peptide 108Arg Thr Ser Gln Ser Leu
Ser Ser Tyr Thr His1 5 101097PRTArtificial SequenceDescription of
Artificial Sequence Synthetic peptide 109Ala Ala Ser Ser Arg Leu
Ser1 51105PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 110Gln Gln Ser Arg Thr1 5111384DNAArtificial
SequenceDescription of Artificial Sequence Synthetic polynucleotide
111caggtccagc tgcagcagag cggccccgga ctggtcaagc cttcacagac
actgagcctg 60acatgcgcca ttagcggaga tagcgtgagc tcctacaatg ccgtgtggaa
ctggatcagg 120cagtctccaa gtcgaggact ggagtggctg ggacgaacat
actatagatc cgggtggtac 180aatgactatg ctgaatcagt gaaaagccga
attactatca accccgatac ctccaagaat 240cagttctctc tgcagctgaa
cagtgtgacc cctgaggaca cagccgtgta ctactgcgcc 300agaagcggcc
atatcaccgt ctttggcgtc aatgtggatg ctttcgatat gtgggggcag
360gggactatgg tcaccgtgtc aagc 384112128PRTArtificial
SequenceDescription of Artificial Sequence Synthetic polypeptide
112Gln Val Gln Leu Gln Gln Ser Gly Pro Gly Leu Val Lys Pro Ser Gln1
5 10 15Thr Leu Ser Leu Thr Cys Ala Ile Ser Gly Asp Ser Val Ser Ser
Tyr 20 25 30Asn Ala Val Trp Asn Trp Ile Arg Gln Ser Pro Ser Arg Gly
Leu Glu 35 40 45Trp Leu Gly Arg Thr Tyr Tyr Arg Ser Gly Trp Tyr Asn
Asp Tyr Ala 50 55 60Glu Ser Val Lys Ser Arg Ile Thr Ile Asn Pro Asp
Thr Ser Lys Asn65 70 75 80Gln Phe Ser Leu Gln Leu Asn Ser Val Thr
Pro Glu Asp Thr Ala Val 85 90 95Tyr Tyr Cys Ala Arg Ser Gly His Ile
Thr Val Phe Gly Val Asn Val 100 105 110Asp Ala Phe Asp Met Trp Gly
Gln Gly Thr Met Val Thr Val Ser Ser 115 120 1251137PRTArtificial
SequenceDescription of Artificial Sequence Synthetic peptide 113Ser
Tyr Asn Ala Val Trp Asn1 511418PRTArtificial SequenceDescription of
Artificial Sequence Synthetic peptide 114Arg Thr Tyr Tyr Arg Ser
Gly Trp Tyr Asn Asp Tyr Ala Glu Ser Val1 5 10 15Lys
Ser11516PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 115Ser Gly His Ile Thr Val Phe Gly Val Asn Val
Asp Ala Phe Asp Met1 5 10 15116309DNAArtificial SequenceDescription
of Artificial Sequence Synthetic polynucleotide 116gatattcaga
tgacccagag cccttccagc ctgtccgctt cagtggggga tcgagtgacc 60attacctgcc
gaaccagcca gagcctgagc tcctacacgc actggtatca gcagaagccc
120ggcaaagccc ctaagctgct gatctacgcc gcttctagtc gggggtccgg
agtgccaagc 180cggttctccg gatctgggag tggaaccgac tttaccctga
caatttcaag cctgcagccc 240gaggatttcg ctacatacta ctgtcagcag
agcagaactt tcgggcaggg cactaaggtg 300gagatcaaa
309117103PRTArtificial SequenceDescription of Artificial Sequence
Synthetic polypeptide 117Asp 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 Thr
Ser Gln Ser Leu Ser Ser Tyr 20 25 30Thr His Trp Tyr Gln Gln Lys Pro
Gly Lys Ala Pro Lys Leu Leu Ile 35 40 45Tyr Ala Ala Ser Ser Arg Gly
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 Arg Thr Phe Gly Gln 85 90 95Gly Thr Lys
Val Glu Ile Lys 10011811PRTArtificial SequenceDescription of
Artificial Sequence Synthetic peptide 118Arg Thr Ser Gln Ser Leu
Ser Ser Tyr Thr His1 5 101197PRTArtificial SequenceDescription of
Artificial Sequence Synthetic peptide 119Ala Ala Ser Ser Arg Gly
Ser1 51205PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 120Gln Gln Ser Arg Thr1 5121384DNAArtificial
SequenceDescription of Artificial Sequence Synthetic polynucleotide
121caggtccagc tgcagcagag cggccccgga ctggtcaagc cttcacagac
actgagcctg 60acatgcgcca ttagcggaga tagcgtgagc tcctacaatg ccgtgtggaa
ctggatcagg 120cagtctccaa gtcgaggact ggagtggctg ggacgaacat
actatagatc cgggtggtac 180aatgactatg ctgaatcagt gaaaagccga
attactatca accccgatac ctccaagaat 240cagttctctc tgcagctgaa
cagtgtgacc cctgaggaca cagccgtgta ctactgcgcc 300agaagcggcc
atatcaccgt ctttggcgtc aatgtggatg ctttcgatat gtgggggcag
360gggactatgg tcaccgtgtc aagc 384122128PRTArtificial
SequenceDescription of Artificial Sequence Synthetic polypeptide
122Gln Val Gln Leu Gln Gln Ser Gly Pro Gly Leu Val Lys Pro Ser Gln1
5 10 15Thr Leu Ser Leu Thr Cys Ala Ile Ser Gly Asp Ser Val Ser Ser
Tyr 20 25 30Asn Ala Val Trp Asn Trp Ile Arg Gln Ser Pro Ser Arg Gly
Leu Glu 35 40 45Trp Leu Gly Arg Thr Tyr Tyr Arg Ser Gly Trp Tyr Asn
Asp Tyr Ala 50 55 60Glu Ser Val Lys Ser Arg Ile Thr Ile Asn Pro Asp
Thr Ser Lys Asn65 70 75 80Gln Phe Ser Leu Gln Leu Asn Ser Val Thr
Pro Glu Asp Thr Ala Val 85 90 95Tyr Tyr Cys Ala Arg Ser Gly His Ile
Thr Val Phe Gly Val Asn Val 100 105 110Asp Ala Phe Asp Met Trp Gly
Gln Gly Thr Met Val Thr Val Ser Ser 115 120 1251237PRTArtificial
SequenceDescription of Artificial Sequence Synthetic peptide 123Ser
Tyr Asn Ala Val Trp Asn1 512418PRTArtificial SequenceDescription of
Artificial Sequence Synthetic peptide 124Arg Thr Tyr Tyr Arg Ser
Gly Trp Tyr Asn Asp Tyr Ala Glu Ser Val1 5 10 15Lys
Ser12516PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 125Ser Gly His Ile Thr Val Phe Gly Val Asn Val
Asp Ala Phe Asp Met1 5 10 15126309DNAArtificial SequenceDescription
of Artificial Sequence Synthetic polynucleotide 126gatattcaga
tgacccagag cccttccagc ctgtccgctt cagtggggga tcgagtgacc 60attacctgcc
gaaccagcca gagcctgagc tcctacgacc actggtatca gcagaagccc
120ggcaaagccc ctaagctgct gatctacgcc gcttctagtc ggctgtccgg
agtgccaagc 180cggttctccg gatctgggag tggaaccgac tttaccctga
caatttcaag cctgcagccc 240gaggatttcg ctacatacta ctgtcagcag
agcagaactt tcgggcaggg cactaaggtg 300gagatcaaa
309127103PRTArtificial SequenceDescription of Artificial Sequence
Synthetic polypeptide 127Asp 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 Thr
Ser Gln Ser Leu Ser Ser Tyr 20 25 30Asp His Trp Tyr Gln Gln Lys Pro
Gly Lys Ala Pro Lys Leu Leu Ile 35 40 45Tyr Ala Ala Ser Ser Arg Leu
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 Arg Thr Phe Gly Gln 85 90 95Gly Thr Lys
Val Glu Ile Lys 10012811PRTArtificial SequenceDescription of
Artificial Sequence Synthetic peptide 128Arg Thr Ser Gln Ser Leu
Ser Ser Tyr Asp His1 5 101297PRTArtificial SequenceDescription of
Artificial Sequence Synthetic peptide 129Ala Ala Ser Ser Arg Leu
Ser1 51305PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 130Gln Gln Ser Arg Thr1 5131384DNAArtificial
SequenceDescription of Artificial Sequence Synthetic polynucleotide
131caggtccagc tgcagcagag cggccccgga ctggtcaagc cttcacagac
actgagcctg 60acatgcgcca ttagcggaga tagcgtgagc tccaacaatg ccgtgtggaa
ctggatcagg 120cagtctccaa gtcgaggact ggagtggctg ggacgaacat
actatagatc caagtggtac 180aatgactatg ctgaatcagt gaaaagccga
attactatca accccgatac ctccaagaat 240cagttctctc tgcagctgaa
cagtgtgacc cctgaggaca cagccgtgta ctactgcgcc 300agaagcggcc
atatcaccgt ctttggcgtc aatgtggatg ctttcgatat gtgggggcag
360gggaccacag tcaccgtctc ctca 384132128PRTArtificial
SequenceDescription of Artificial Sequence Synthetic polypeptide
132Gln Val Gln Leu Gln Gln Ser Gly Pro Gly Leu Val Lys Pro Ser Gln1
5 10 15Thr Leu Ser Leu Thr Cys Ala Ile Ser Gly Asp Ser Val Ser Ser
Asn 20 25 30Asn Ala Val Trp Asn Trp Ile Arg Gln Ser Pro Ser Arg Gly
Leu Glu 35 40 45Trp Leu Gly Arg Thr Tyr Tyr Arg Ser Lys Trp Tyr Asn
Asp Tyr Ala 50 55 60Glu Ser Val Lys Ser Arg Ile Thr Ile Asn Pro Asp
Thr Ser Lys Asn65 70 75 80Gln Phe Ser Leu Gln Leu Asn Ser Val Thr
Pro Glu Asp Thr Ala Val 85 90 95Tyr Tyr Cys Ala Arg Ser Gly His Ile
Thr Val Phe Gly Val Asn Val 100 105 110Asp Ala Phe Asp Met Trp Gly
Gln Gly Thr Thr Val Thr Val Ser Ser 115 120 1251337PRTArtificial
SequenceDescription of Artificial Sequence Synthetic peptide 133Ser
Asn Asn Ala Val Trp Asn1 513418PRTArtificial SequenceDescription of
Artificial Sequence Synthetic peptide 134Arg Thr Tyr Tyr Arg Ser
Lys Trp Tyr Asn Asp Tyr Ala Glu Ser Val1 5 10 15Lys
Ser13516PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 135Ser Gly His Ile Thr Val Phe Gly Val Asn Val
Asp Ala Phe Asp Met1 5 10 15136309DNAArtificial SequenceDescription
of Artificial Sequence Synthetic polynucleotide 136gatattcaga
tgacccagag cccttccagc ctgtccgctt cagtggggga tcgagtgacc 60attacctgcc
gaaccagcca gagcctgagc tcctacacgc actggtatca gcagaagccc
120ggcaaagccc ctaagctgct gatctacgcc
gcttctagtc ggctgtccgg agtgccaagc 180cggttctccg gatctgggag
tggaaccgac tttaccctga caatttcaag cctgcagccc 240gaggatttcg
ctacatacta ctgtcagcag agcagaactt tcgggcaggg cactaaggtg 300gagatcaaa
309137103PRTArtificial SequenceDescription of Artificial Sequence
Synthetic polypeptide 137Asp 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 Thr
Ser Gln Ser Leu Ser Ser Tyr 20 25 30Thr His Trp Tyr Gln Gln Lys Pro
Gly Lys Ala Pro Lys Leu Leu Ile 35 40 45Tyr Ala Ala Ser Ser Arg Leu
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 Arg Thr Phe Gly Gln 85 90 95Gly Thr Lys
Val Glu Ile Lys 10013811PRTArtificial SequenceDescription of
Artificial Sequence Synthetic peptide 138Arg Thr Ser Gln Ser Leu
Ser Ser Tyr Thr His1 5 101397PRTArtificial SequenceDescription of
Artificial Sequence Synthetic peptide 139Ala Ala Ser Ser Arg Leu
Ser1 51405PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 140Gln Gln Ser Arg Thr1 5141384DNAArtificial
SequenceDescription of Artificial Sequence Synthetic polynucleotide
141caggtccagc tgcagcagag cggccccgga ctggtcaagc cttcacagac
actgagcctg 60acatgcgcca ttagcggaga tagcgtgagc tccaacaatg ccgtgtggaa
ctggatcagg 120cagtctccaa gtcgaggact ggagtggctg ggacgaacat
actatagatc caagtggtac 180aatgactatg ctgaatcagt gaaaagccga
attactatca accccgatac ctccaagaat 240cagttctctc tgcagctgaa
cagtgtgacc cctgaggaca cagccgtgta ctactgcgcc 300agaagcggcc
atatcaccgt ctttggcgtc aatgtggatg ctttcgatat gtgggggcag
360gggaccacag tcaccgtctc ctca 384142128PRTArtificial
SequenceDescription of Artificial Sequence Synthetic polypeptide
142Gln Val Gln Leu Gln Gln Ser Gly Pro Gly Leu Val Lys Pro Ser Gln1
5 10 15Thr Leu Ser Leu Thr Cys Ala Ile Ser Gly Asp Ser Val Ser Ser
Asn 20 25 30Asn Ala Val Trp Asn Trp Ile Arg Gln Ser Pro Ser Arg Gly
Leu Glu 35 40 45Trp Leu Gly Arg Thr Tyr Tyr Arg Ser Lys Trp Tyr Asn
Asp Tyr Ala 50 55 60Glu Ser Val Lys Ser Arg Ile Thr Ile Asn Pro Asp
Thr Ser Lys Asn65 70 75 80Gln Phe Ser Leu Gln Leu Asn Ser Val Thr
Pro Glu Asp Thr Ala Val 85 90 95Tyr Tyr Cys Ala Arg Ser Gly His Ile
Thr Val Phe Gly Val Asn Val 100 105 110Asp Ala Phe Asp Met Trp Gly
Gln Gly Thr Thr Val Thr Val Ser Ser 115 120 1251437PRTArtificial
SequenceDescription of Artificial Sequence Synthetic peptide 143Ser
Asn Asn Ala Val Trp Asn1 514418PRTArtificial SequenceDescription of
Artificial Sequence Synthetic peptide 144Arg Thr Tyr Tyr Arg Ser
Lys Trp Tyr Asn Asp Tyr Ala Glu Ser Val1 5 10 15Lys
Ser14516PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 145Ser Gly His Ile Thr Val Phe Gly Val Asn Val
Asp Ala Phe Asp Met1 5 10 15146309DNAArtificial SequenceDescription
of Artificial Sequence Synthetic polynucleotide 146gatattcaga
tgacccagag cccttccagc ctgtccgctt cagtggggga tcgagtgacc 60attacctgcc
gaaccagcca gagcctgagy tcctacacgc actggtatca gcagaagccc
120ggcaaagccc ctaagctgct gatctacgcc gcttctagtc gggggtccgg
agtgccaagc 180cggttctccg gatctgggag tggaaccgac tttaccctga
caatttcaag cctgcagccc 240gaggatttcg ctacatacta ctgtcagcag
agcagaactt tcgggcaggg cactaaggtg 300gagatcaaa
309147103PRTArtificial SequenceDescription of Artificial Sequence
Synthetic polypeptide 147Asp 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 Thr
Ser Gln Ser Leu Ser Ser Tyr 20 25 30Thr His Trp Tyr Gln Gln Lys Pro
Gly Lys Ala Pro Lys Leu Leu Ile 35 40 45Tyr Ala Ala Ser Ser Arg Gly
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 Arg Thr Phe Gly Gln 85 90 95Gly Thr Lys
Val Glu Ile Lys 10014811PRTArtificial SequenceDescription of
Artificial Sequence Synthetic peptide 148Arg Thr Ser Gln Ser Leu
Ser Ser Tyr Thr His1 5 101497PRTArtificial SequenceDescription of
Artificial Sequence Synthetic peptide 149Ala Ala Ser Ser Arg Gly
Ser1 51505PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 150Gln Gln Ser Arg Thr1 5151384DNAArtificial
SequenceDescription of Artificial Sequence Synthetic polynucleotide
151caggtccagc tgcagcagag cggccccgga ctggtcaagc cttcacagac
actgagcctg 60acatgcgcca ttagcggaga tagcgtgagc tccaacaatg ccgtgtggaa
ctggatcagg 120cagtctccaa gtcgaggact ggagtggctg ggacgaacat
actatagatc caagtggtac 180aatgactatg ctgaatcagt gaaaagccga
attactatca accccgatac ctccaagaat 240cagttctctc tgcagctgaa
cagtgtgacc cctgaggaca cagccgtgta ctactgcgcc 300agaagcggcc
atatcaccgt ctttggcgtc aatgtggatg ctttcgatat gtgggggcag
360gggaccacag tcaccgtctc ctca 384152128PRTArtificial
SequenceDescription of Artificial Sequence Synthetic polypeptide
152Gln Val Gln Leu Gln Gln Ser Gly Pro Gly Leu Val Lys Pro Ser Gln1
5 10 15Thr Leu Ser Leu Thr Cys Ala Ile Ser Gly Asp Ser Val Ser Ser
Asn 20 25 30Asn Ala Val Trp Asn Trp Ile Arg Gln Ser Pro Ser Arg Gly
Leu Glu 35 40 45Trp Leu Gly Arg Thr Tyr Tyr Arg Ser Lys Trp Tyr Asn
Asp Tyr Ala 50 55 60Glu Ser Val Lys Ser Arg Ile Thr Ile Asn Pro Asp
Thr Ser Lys Asn65 70 75 80Gln Phe Ser Leu Gln Leu Asn Ser Val Thr
Pro Glu Asp Thr Ala Val 85 90 95Tyr Tyr Cys Ala Arg Ser Gly His Ile
Thr Val Phe Gly Val Asn Val 100 105 110Asp Ala Phe Asp Met Trp Gly
Gln Gly Thr Thr Val Thr Val Ser Ser 115 120 1251537PRTArtificial
SequenceDescription of Artificial Sequence Synthetic peptide 153Ser
Asn Asn Ala Val Trp Asn1 515418PRTArtificial SequenceDescription of
Artificial Sequence Synthetic peptide 154Arg Thr Tyr Tyr Arg Ser
Lys Trp Tyr Asn Asp Tyr Ala Glu Ser Val1 5 10 15Lys
Ser15516PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 155Ser Gly His Ile Thr Val Phe Gly Val Asn Val
Asp Ala Phe Asp Met1 5 10 15156309DNAArtificial SequenceDescription
of Artificial Sequence Synthetic polynucleotide 156gatattcaga
tgacccagag cccttccagc ctgtccgctt cagtggggga tcgagtgacc 60attacctgcc
gaaccagcca gagcctgagc tcctacctgc actggtatca gcagaagccc
120ggcaaagccc ctaagctgct gatctacgcc gcttctagtc tgcagtccgg
agtgccaagc 180cggttctccg gatctgggag tggaaccgac tttaccctga
caatttcaag cctgcagccc 240gaggatttcg ctacatacta ctgtcagcag
agcagaactt tcgggcaggg cactaaggtg 300gagatcaaa
309157103PRTArtificial SequenceDescription of Artificial Sequence
Synthetic polypeptide 157Asp 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 Thr
Ser Gln Ser Leu Ser Ser Tyr 20 25 30Leu His 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 Phe Ala
Thr Tyr Tyr Cys Gln Gln Ser Arg Thr Phe Gly Gln 85 90 95Gly Thr Lys
Val Glu Ile Lys 10015811PRTArtificial SequenceDescription of
Artificial Sequence Synthetic peptide 158Arg Thr Ser Gln Ser Leu
Ser Ser Tyr Leu His1 5 101597PRTArtificial SequenceDescription of
Artificial Sequence Synthetic peptide 159Ala Ala Ser Ser Leu Gln
Ser1 51605PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 160Gln Gln Ser Arg Thr1 51617PRTArtificial
SequenceDescription of Artificial Sequence Synthetic
peptideMOD_RES(2)..(2)Asn or TyrMOD_RES(3)..(3)Asn, Ser or
ArgMOD_RES(5)..(5)Val or Thr 161Ser Xaa Xaa Ala Xaa Trp Asn1
516217PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptideMOD_RES(7)..(7)Lys or GlyMOD_RES(10)..(10)Asn or
TyrMOD_RES(14)..(14)Glu, Val or AspMOD_RES(15)..(15)Ser or
PheMOD_RES(16)..(16)Val or Leu 162Arg Thr Tyr Tyr Arg Ser Xaa Trp
Tyr Xaa Asp Tyr Ala Xaa Xaa Xaa1 5 10 15Lys16316PRTArtificial
SequenceDescription of Artificial Sequence Synthetic
peptideMOD_RES(1)..(1)Ser or GlyMOD_RES(6)..(6)Val or
GluMOD_RES(9)..(9)Val or LeuMOD_RES(11)..(11)Val or
IleMOD_RES(14)..(14)Phe or TyrMOD_RES(16)..(16)Met, Ile or Val
163Xaa Gly His Ile Thr Xaa Phe Gly Xaa Asn Xaa Asp Ala Xaa Asp Xaa1
5 10 1516411PRTArtificial SequenceDescription of Artificial
Sequence Synthetic peptideMOD_RES(2)..(2)Thr, Ala or
absentMOD_RES(3)..(3)Ser or AlaMOD_RES(5)..(5)Ser or
ArgMOD_RES(7)..(7)Ser, Asn or ArgMOD_RES(10)..(10)Leu, Thr or Asp
164Arg Xaa Xaa Gln Xaa Leu Xaa Ser Tyr Xaa His1 5
101657PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptideMOD_RES(2)..(2)Ala, Thr or SerMOD_RES(3)..(4)Ser
or ThrMOD_RES(5)..(5)Leu or ArgMOD_RES(6)..(6)Gln, Leu or Gly
165Ala Xaa Xaa Xaa Xaa Xaa Ser1 51665PRTArtificial
SequenceDescription of Artificial Sequence Synthetic
peptideMOD_RES(2)..(2)Gln or Leu 166Gln Xaa Ser Arg Thr1
5167128PRTArtificial SequenceDescription of Artificial Sequence
Synthetic polypeptide 167Gln Val Gln Leu Gln Gln Ser Gly Pro Gly
Leu Val Lys Pro Ser Gln1 5 10 15Thr Leu Ser Leu Thr Cys Ala Ile Ser
Gly Asp Ser Val Ser Ser Asn 20 25 30Asn Ala Val Trp Asn Trp Ile Arg
Gln Ser Pro Ser Arg Gly Leu Glu 35 40 45Trp Leu Gly Arg Thr Tyr Tyr
Arg Ser Lys Trp Tyr Asn Asp Tyr Ala 50 55 60Glu Ser Val Lys Ser Arg
Ile Thr Ile Asn Pro Asp Thr Ser Lys Asn65 70 75 80Gln Phe Ser Leu
Gln Leu Asn Ser Val Thr Pro Glu Asp Thr Ala Val 85 90 95Tyr Tyr Cys
Ala Arg Ser Gly His Ile Thr Val Phe Gly Val Asn Val 100 105 110Asp
Ala Phe Asp Met Trp Gly Gln Gly Thr Met Val Thr Val Ser Ser 115 120
125168103PRTArtificial SequenceDescription of Artificial Sequence
Synthetic polypeptide 168Asp 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 Thr
Ser Gln Ser Leu Ser Ser Tyr 20 25 30Leu His 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 Phe Ala
Thr Tyr Tyr Cys Gln Gln Ser Arg Thr Phe Gly Gln 85 90 95Gly Thr Lys
Val Glu Ile Lys 100169221PRTInfluenza A virus 169Gly Ile Phe Gly
Ala Ile Ala Gly Phe Ile Glu Asn Gly Trp Glu Gly1 5 10 15Met Val Asp
Gly Trp Tyr Gly Phe Arg His Gln Asn Ser Glu Gly Ile 20 25 30Gly Gln
Ala Ala Asp Leu Lys Ser Thr Gln Ala Ala Ile Asn Gln Ile 35 40 45Asn
Gly Lys Leu Asn Arg Leu Ile Gly Lys Thr Asn Glu Lys Phe His 50 55
60Gln Ile Glu Lys Glu Phe Ser Glu Val Glu Gly Arg Ile Gln Asp Leu65
70 75 80Glu Lys Tyr Val Glu Asp Thr Lys Ile Asp Leu Trp Ser Tyr Asn
Ala 85 90 95Glu Leu Leu Val Ala Leu Glu Asn Gln His Thr Ile Asp Leu
Thr Asp 100 105 110Ser Glu Met Asn Lys Leu Phe Glu Arg Thr Lys Lys
Gln Leu Arg Glu 115 120 125Asn Ala Glu Asp Met Gly Asn Gly Cys Phe
Lys Ile Tyr His Lys Cys 130 135 140Asp Asn Ala Cys Ile Gly Ser Ile
Arg Asn Gly Thr Tyr Asp His Asp145 150 155 160Val Tyr Arg Asp Glu
Ala Leu Asn Asn Arg Phe Gln Ile Lys Gly Val 165 170 175Glu Leu Lys
Ser Gly Tyr Lys Asp Trp Ile Leu Trp Ile Ser Phe Ala 180 185 190Ile
Ser Cys Phe Leu Leu Cys Val Ala Leu Leu Gly Phe Ile Met Trp 195 200
205Ala Cys Gln Lys Gly Asn Ile Arg Cys Asn Ile Cys Ile 210 215
220170221PRTInfluenza A virus 170Gly Ile Phe Gly Ala Ile Ala Gly
Phe Ile Glu Asn Gly Trp Glu Gly1 5 10 15Met Val Asp Gly Trp Tyr Gly
Phe Arg His Gln Asn Ser Glu Gly Thr 20 25 30Gly Gln Ala Ala Asp Leu
Lys Ser Thr Gln Ala Ala Ile Asn Gln Ile 35 40 45Asn Gly Lys Leu Asn
Arg Leu Ile Glu Lys Thr Asn Glu Lys Phe His 50 55 60Gln Ile Glu Lys
Glu Phe Ser Glu Val Glu Gly Arg Ile Gln Asp Leu65 70 75 80Glu Lys
Tyr Val Glu Asp Thr Lys Ile Asp Leu Trp Ser Tyr Asn Ala 85 90 95Glu
Leu Leu Val Ala Leu Glu Asn Gln His Thr Ile Asp Leu Thr Asp 100 105
110Ser Glu Met Asn Lys Leu Phe Glu Arg Thr Lys Lys Gln Leu Arg Glu
115 120 125Asn Ala Glu Asp Met Gly Asn Gly Cys Phe Lys Ile Tyr His
Lys Cys 130 135 140Asp Asn Ala Cys Ile Gly Ser Ile Arg Asn Gly Thr
Tyr Asp His Asp145 150 155 160Val Tyr Arg Asp Glu Ala Leu Asn Asn
Arg Phe Gln Ile Lys Gly Val 165 170 175Glu Leu Lys Ser Gly Tyr Lys
Asp Trp Ile Leu Trp Ile Ser Phe Ala 180 185 190Ile Ser Cys Phe Leu
Leu Cys Val Val Leu Leu Gly Phe Ile Met Trp 195 200 205Ala Cys Gln
Lys Gly Asn Ile Arg Cys Asn Ile Cys Ile 210 215
220171221PRTInfluenza A virus 171Gly Leu Phe Gly Ala Ile Ala Gly
Phe Ile Glu Asn Gly Trp Glu Gly1 5 10 15Met Ile Asp Gly Trp Tyr Gly
Phe Arg His Gln Asn Ser Glu Gly Thr 20 25 30Gly Gln Ala Ala Asp Leu
Lys Ser Thr Gln Ala Ala Ile Asp Gln Ile 35 40 45Asn Gly Lys Leu Asn
Arg Val Ile Glu Lys Thr Asn Glu Lys Phe His 50 55 60Gln Ile Glu Lys
Glu Phe Ser Glu Val Glu Gly Arg Ile Gln Asp Leu65 70 75 80Glu Lys
Tyr Val Glu Asp Thr Lys Ile Asp Leu Trp Ser Tyr Asn Ala 85 90 95Glu
Leu Leu Val Ala Leu Glu Asn Gln His Thr Ile Asp Leu Thr Asp 100 105
110Ser Glu Met Asn Lys Leu Phe Glu Lys Thr Arg Arg Gln Leu Arg Glu
115 120 125Asn Ala Glu Glu Met Gly Asn Gly Cys Phe Lys Ile Tyr His
Lys Cys 130 135 140Asp Asn Ala Cys Ile Glu Ser Ile Arg Asn Gly Thr
Tyr Asp His Asp145 150 155 160Val Tyr Arg Asp Glu Ala Leu Asn Asn
Arg Phe Gln Ile Lys Gly Val 165 170 175Glu Leu Lys Ser Gly Tyr Lys
Asp Trp Ile Leu Trp Ile Ser Phe Ala 180 185 190Ile Ser Cys Phe Leu
Leu Cys Val Val Leu Leu Gly Phe Ile Met Trp 195
200 205Ala Cys Gln Arg Gly Asn Ile Arg Cys Asn Ile Cys Ile 210 215
220172221PRTInfluenza A virus 172Gly Leu Phe Gly Ala Ile Ala Gly
Phe Ile Glu Asn Gly Trp Glu Gly1 5 10 15Met Ile Asp Gly Trp Tyr Gly
Phe Arg His Gln Asn Ser Glu Gly Thr 20 25 30Gly Gln Ala Ala Asp Leu
Lys Ser Thr Gln Ala Ala Ile Asp Gln Ile 35 40 45Asn Gly Lys Leu Asn
Arg Val Ile Glu Lys Thr Asn Glu Lys Phe His 50 55 60Gln Ile Glu Lys
Glu Phe Ser Glu Val Glu Gly Arg Ile Gln Asp Leu65 70 75 80Glu Lys
Tyr Val Glu Asp Thr Lys Ile Asp Leu Trp Ser Tyr Asn Ala 85 90 95Glu
Leu Leu Val Ala Leu Glu Asn Gln His Thr Ile Asp Leu Thr Asp 100 105
110Ser Glu Met Asn Lys Leu Phe Glu Lys Thr Arg Arg Gln Leu Arg Glu
115 120 125Asn Ala Glu Asp Met Gly Asn Gly Cys Phe Lys Ile Tyr His
Lys Cys 130 135 140Asp Asn Ala Cys Ile Glu Ser Ile Arg Asn Gly Thr
Tyr Asp His Asp145 150 155 160Val Tyr Arg Asp Glu Ala Leu Asn Asn
Arg Phe Gln Ile Lys Gly Val 165 170 175Glu Leu Lys Ser Gly Tyr Lys
Asp Trp Ile Leu Trp Ile Ser Phe Ala 180 185 190Ile Ser Cys Phe Leu
Leu Cys Val Val Leu Leu Gly Phe Ile Met Trp 195 200 205Ala Cys Gln
Arg Gly Asn Ile Arg Cys Asn Ile Cys Ile 210 215 220
* * * * *
References