U.S. patent application number 13/061026 was filed with the patent office on 2012-05-24 for conserved hemagglutinin epitope, antibodies to the epitope and methods of use.
This patent application is currently assigned to Burnham Institute for Medical Research. Invention is credited to Robert C. Liddington, Wayne Marasco, Jianhua Sui.
Application Number | 20120128684 13/061026 |
Document ID | / |
Family ID | 41647157 |
Filed Date | 2012-05-24 |
United States Patent
Application |
20120128684 |
Kind Code |
A1 |
Marasco; Wayne ; et
al. |
May 24, 2012 |
Conserved Hemagglutinin Epitope, Antibodies to the Epitope and
Methods of Use
Abstract
Disclosed are antibodies that bind to the stem region of
influenza hemagglutinin in the neutral pH conformation,
hemagglutinin epitopes in the stem region, and methods of making
and using both.
Inventors: |
Marasco; Wayne; (Wellesley,
MA) ; Sui; Jianhua; (Boston, MA) ; Liddington;
Robert C.; (La Jolla, CA) |
Assignee: |
Burnham Institute for Medical
Research
La Jolla
CA
Dana-Farber Cancer Institute, Inc.
Boston
MA
|
Family ID: |
41647157 |
Appl. No.: |
13/061026 |
Filed: |
August 25, 2009 |
PCT Filed: |
August 25, 2009 |
PCT NO: |
PCT/US09/54950 |
371 Date: |
September 12, 2011 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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61091599 |
Aug 25, 2008 |
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61150231 |
Feb 5, 2009 |
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61154400 |
Feb 22, 2009 |
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Current U.S.
Class: |
424/147.1 ;
424/159.1; 424/192.1; 424/210.1; 435/5; 530/300; 530/350;
530/389.4; 530/402; 536/23.4; 536/23.72 |
Current CPC
Class: |
C07K 14/005 20130101;
A61K 2039/505 20130101; A61K 39/00 20130101; A61P 37/04 20180101;
A61P 31/16 20180101; C07K 16/1018 20130101; C07K 2317/92 20130101;
C12N 2760/16034 20130101; C07K 2317/76 20130101; C07K 2299/00
20130101; C07K 2319/40 20130101; C12N 2760/16022 20130101 |
Class at
Publication: |
424/147.1 ;
530/300; 536/23.72; 424/210.1; 424/159.1; 530/350; 536/23.4;
424/192.1; 530/402; 435/5; 530/389.4 |
International
Class: |
A61K 39/395 20060101
A61K039/395; C12N 15/44 20060101 C12N015/44; A61K 39/145 20060101
A61K039/145; C07K 19/00 20060101 C07K019/00; A61P 31/16 20060101
A61P031/16; C12Q 1/70 20060101 C12Q001/70; C07K 1/00 20060101
C07K001/00; C07K 16/10 20060101 C07K016/10; A61P 37/04 20060101
A61P037/04; C07K 2/00 20060101 C07K002/00; C12N 15/62 20060101
C12N015/62 |
Goverment Interests
STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH
[0002] This invention was made with government support under NIH
grants P41 RR-01081, U01-AI074518-01, and P01-AI055789. The
government has certain rights in the invention.
Claims
1. An immunogen comprising an epitope or epitope unit recognized by
a protective monoclonal antibody having the specificity for the
stem region of hemagglutinn protein of an influenza virus.
2. The immunogen of claim 1, wherein said antibody binds both the
HA1 and HA2 peptide.
3. The immunogen of claim 1, wherein said epitope is the F10
epitope.
4. The immunogen of claim 1, wherein the antibody is monoclonal
antibody D7, D8, F10, G17, H40, A66, D80, E88, E90, or H98 or a
monoclonal antibody that competes with the binding of monoclonal
antibody D7, D8, F10, G17, H40, A66, D80, E88, E90, or H98 to the
HA protein.
5. The immunogen of claim 1, wherein said hemagglutin protein is in
the neutral pH conformation.
6. The immunogen of claim 1, wherein said immunogen is a peptide or
a synthetic peptide.
7. The immunogen of claim 1, wherein the conformation of said
epitope is defined by amino acid residues 18, 38, 39, 40 and 291 of
HA1 and 18, 19, 20, 21, 38, 41, 42, 45, 49, 52, 53, and 56 of HA2
when said hemagglutinin in the neutral pH conformation.
8. The immunogen of claim 1, wherein said immunogen comprises a
peptide comprising one or more of the following amino acid
sequences TABLE-US-00037 (SEQ ID NO: 125) a)
[Xaa.sub.0].sub.m-Xaa.sub.1-Xaa.sub.2- [Xaa.sub.0].sub.p
wherein, m, and p are independently 0 or 1-10, Xaa.sub.0, is
independently any amino acid, Xaa.sub.1 is S, T, F H or Y, and
Xaa.sub.2 is H, Y, M, L or Q; TABLE-US-00038 (SEQ ID NO: 126) b)
[Xaa.sub.0].sub.m-Xaa.sub.1-Xaa.sub.2- [Xaa.sub.0].sub.p
wherein, m, and p are independently 0 or 1-10, Xaa.sub.0, is
independently any amino acid, and Xaa.sub.1 is H, Q, Y, S, D, N or
T, Xaa.sub.2 is Q, E, K, I, V, M, E, R or T; TABLE-US-00039 (SEQ ID
NO: 127) c)
[Xaa.sub.0].sub.m-Xaa.sub.1-Xaa.sub.2-Xaa.sub.3-Xaa.sub.4-
[Xaa.sub.0].sub.p
wherein, m, and p are independently 0 or 1-10, Xaa.sub.0, is
independently any amino acid, and Xaa.sub.1 is I, V, M, or L;
Xaa.sub.2 is D, N, H, Y, D, A, S or E, Xaa.sub.3 is G or A, and
Xaa.sub.4 is W, R, or G; or TABLE-US-00040 d) (SEQ ID NO: 128)
[Xaa.sub.0].sub.m-Xaa.sub.1-[Xaa.sub.0].sub.q
Xaa.sub.2-Xaa.sub.3-[Xaa.sub.0].sub.q Xaa.sub.4-[Xaa.sub.0].sub.r
Xaa.sub.5-[Xaa.sub.0].sub.q-Xaa.sub.6 Xaa.sub.7 - [Xaa.sub.0].sub.q
-Xaa.sub.8 -[Xaa.sub.0].sub.p
wherein, m, and p are independently 0 or 1-10, q is 2, and r is 3
Xaa.sub.0, is independently any amino acid, and Xaa.sub.1 is K, Q,
R, N, L, G, F, H or Y, Xaa.sub.2 is S or T, Xaa.sub.3 is Q or P,
Xaa.sub.4 is F, V, I, M, L, or T, Xaa.sub.5 is I, T, S, N, Q, D, or
A, Xaa.sub.6 is I, V, M, or L, Xaa.sub.7 is N, S, T, or D Xaa.sub.8
is I, F, V, A, or T; TABLE-US-00041 e) (SEQ ID NO: 129)
[Xaa.sub.0].sub.m-Xaa.sub.1-[Xaa.sub.0].sub.q
Xaa.sub.2-Xaa.sub.3-[Xaa.sub.0].sub.q Xaa.sub.4-[Xaa.sub.0].sub.r
Xaa.sub.5-[Xaa.sub.0].sub.q-Xaa.sub.6 Xaa.sub.7 - [Xaa.sub.0].sub.s
- [Xaa.sub.8].sub.t -[Xaa.sub.0].sub.p
wherein, m, and p are independently 0 or 1-10, q is 2, r is 3, s is
0 or 2, and t is 0 or 1, Xaa.sub.0, is independently any amino
acid, and Xaa.sub.1 is K, Q, R, N, L, G, F, H or Y, Xaa.sub.2 is S
or T, Xaa.sub.3 is Q or P, Xaa.sub.4 is F, V, I, M, L, or T,
Xaa.sub.5 is I, T, S, N, Q, D, or A, Xaa.sub.6 is I, V, M, or L,
Xaa.sub.7 is N, S, T, or D, Xaa.sub.8 I, F, V, A, or T;
9. A nucleic acid encoding the immunogen of claim 8.
10. The immunogen of claim 1, further comprising an adjuvant.
11. The immunogen of claim 1, wherein said immunogen is conjugated
to a carrier.
12. A composition comprising the immunogen of claim 1 together with
one or more pharmaceutically acceptable excipients, diluents,
and/or adjuvants.
13. The composition of claim 12, wherein said composition further
comprises a anti-influenza antibody of antigen binding fragment
thereof.
14. The composition of claim 13, wherein said antibody is
monoclonal antibody D7, D8, F10, G17, H40, A66, D80, E88, E90, or
H98 or a monoclonal antibody that competes with the binding of
monoclonal antibody D7, D8, F10, G17, H40, A66, D80, E88, E90, or
H98 to the HA protein.
15. A conjugate comprising one or more peptides or peptide
fragments optionally linked to a backbone wherein the peptides or
peptide fragments are spatially positioned relative to each other
so that they together form a non-linear sequence which mimics the
tertiary structure of an F10 epitope, wherein said conjugate
competes with the binding of monoclonal antibody F10 to the HA
protein.
16. The conjugate of claim 15, wherein said peptide or peptide
fragment comprises one or more of the following amino acids
sequences: TABLE-US-00042 (SEQ ID NO: 125) a)
[Xaa.sub.0].sub.m-Xaa.sub.1-Xaa.sub.2- [Xaa.sub.0].sub.p
wherein, m, and p are independently 0 or 1-10, Xaa.sub.0, is
independently any amino acid, Xaa.sub.1 is S, T, F H or Y, and
Xaa.sub.2 is H, Y, M, L or Q; TABLE-US-00043 (SEQ ID NO: 126) b)
[Xaa.sub.0].sub.m-Xaa.sub.1-Xaa.sub.2- [Xaa.sub.0].sub.p
wherein, m, and p are independently 0 or 1-10, Xaa.sub.0, is
independently any amino acid, and Xaa.sub.1 is H, Q, Y, S, D, N or
T, Xaa.sub.2 is Q, E, K, I, V, M, E, R or T; TABLE-US-00044 (SEQ ID
NO: 127) c)
[Xaa.sub.0].sub.m-Xaa.sub.1-Xaa.sub.2-Xaa.sub.3-Xaa.sub.4-
[Xaa.sub.0].sub.p
wherein, m, and p are independently 0 or 1-10, Xaa.sub.0, is
independently any amino acid, and Xaa.sub.1 is I, V, M, or L;
Xaa.sub.2 is D, N, H, Y, D, A, S or E, Xaa.sub.3 is G or A, and
Xaa.sub.4 is W, R, or G; or TABLE-US-00045 d) (SEQ ID NO: 128)
[Xaa.sub.0].sub.m-Xaa.sub.1-[Xaa.sub.0].sub.q
Xaa.sub.2-Xaa.sub.3-[Xaa.sub.0].sub.q Xaa.sub.4-[Xaa.sub.0].sub.r
Xaa.sub.5-[Xaa.sub.0].sub.q-Xaa.sub.6 Xaa.sub.7 - [Xaa.sub.0].sub.q
-Xaa.sub.8 -[Xaa.sub.0].sub.p
wherein, m, and p are independently 0 or 1-10, q is 2, and r is 3
Xaa.sub.0, is independently any amino acid, and Xaa.sub.1 is K, Q,
R, N, L, G, F, H or Y, Xaa.sub.2 is S or T, Xaa.sub.3 is Q or P,
Xaa.sub.4 is F, V, I, M, L, or T, Xaa.sub.5 is I, T, S, N, Q, D, or
A, Xaa.sub.6 is I, V, M, or L, Xaa.sub.7 is N, S, T, or D Xaa.sub.8
is I, F, V, A, or T; TABLE-US-00046 e) (SEQ ID NO: 129)
[Xaa.sub.0].sub.m-Xaa.sub.1-[Xaa.sub.0].sub.q
Xaa.sub.2-Xaa.sub.3-[Xaa.sub.0].sub.q Xaa.sub.4-[Xaa.sub.0].sub.r
Xaa.sub.5-[Xaa.sub.0].sub.q-Xaa.sub.6 Xaa.sub.7 - [Xaa.sub.0].sub.s
- [Xaa.sub.8].sub.t -[Xaa.sub.0].sub.p
wherein, m, and p are independently 0 or 1-10, q is 2, r is 3, s is
0 or 2, and t is 0 or 1, Xaa.sub.0, is independently any amino
acid, and Xaa.sub.1 is K, Q, R, N, L, G, F, H or Y, Xaa.sub.2 is S
or T, Xaa.sub.3 is Q or P, Xaa.sub.4 is F, V, I, M, L, or T,
Xaa.sub.5 is I, T, S, N, Q, D, or A, Xaa.sub.6 is I, V, M, or L,
Xaa.sub.7 is N, S, T, or D, Xaa.sub.8 I, F, V, A, or T;
17. A nucleic acid encoding the conjugate of claim 16.
18. The conjugate of claim 15, further comprising an adjuvant.
19. The conjugate of claim 15, wherein said conjugate is linked to
a carrier.
20. A composition comprising the conjugate of claim 15 together
with one or more pharmaceutically acceptable excipients, diluents,
and/or adjuvants.
21. The composition of claim 20, wherein said composition further
comprises an anti-influenza antibody of antigen binding fragment
thereof.
22. The composition of claim 21, wherein said antibody is
monoclonal antibody D7, D8, F10, G17, H40, A66, D80, E88, E90, or
H98 or a monoclonal antibody that competes with the binding of
monoclonal antibody D7, D8, F10, G17, H40, A66, D80, E88, E90, or
H98 to the HA protein.
24. A method preventing a disease or disorder caused by an
influenza virus, comprising administering to person at risk of
suffering from said disease or disorder the composition of claim
12.
25. The method of claim 24, wherein the method further comprises
administering an anti-viral drug, a viral entry inhibitor or a
viral attachment inhibitor.
26. The method of claim 25, wherein said anti-viral drug is a
neuraminidase inhibitor, a HA inhibitor, a sialic acid inhibitor or
an M2 ion channel.
27. The method of claim 26, wherein said M2 ion channel inhibitor
is amantadine or, rimantadine.
28. The method of claim 26, wherein said neuraminidase inhibitor
zanamivir, or oseltamivir phosphate.
29. The method of claim 24, wherein the method comprises further
administering one or more antibodies specific to a Group I
influenza virus and or an Group II influenza virus
30. The method of claim 24, wherein said antibody is administered
prior to or after exposure to influenza virus.
31. The method of claim 29, wherein said antibody is administered
at a dose sufficient to neutralize said influenza virus.
32. A method of treating a subject, the method comprising
administering to the subject the stem region of influenza
hemagglutinin in the neutral pH conformation in isolation from
other components of influenza virus, wherein the subject produces
an immune response to the stem region.
33. A method of treating a subject, the method comprising
administering to the subject the stem region of influenza
hemagglutinin in the neutral pH conformation in isolation from the
head region of hemagglutinin, wherein the subject produces an
immune response to the stem region.
34. A method of treating a subject, the method comprising
administering to the subject influenza hemagglutinin in the neutral
pH conformation in isolation from other components of influenza
virus, wherein the head region of the hemagglutinin is modified to
reduce the antigenicity of the head region, wherein the subject
produces an immune response to the stem region.
35. The method of claim 34, wherein the head region of the
hemagglutinin is modified by removing or replacing glycosylation
sites.
36. The method of claim 34, wherein the head region of the
hemagglutinin is modified by adding glycosylation sites.
37. The method of claim 34, wherein the head region of the
hemagglutinin is modified by removing all or a portion of the head
region.
38. The method of claim 34, wherein the subject produces an immune
response that prevents or reduces the severity of an influenza
infection.
39. The method claim 34, wherein the immune response is reactive to
influenza viruses within a subtype.
41. The method claim 34, wherein the immune response is reactive to
influenza viruses in each subtype within a cluster.
42. The method claim 34, wherein the immune response is reactive to
influenza viruses in each cluster within a group.
43. The method claim 34, wherein the immune response is reactive to
all influenza viruses in each subtype within a group.
44. The method of claim 43, wherein the immune response is reactive
to influenza viruses within group 1.
45. A method, the method comprising screening antibodies reactive
to hemagglutinin for binding to hemagglutinin immobilized on a
surface, thereby identifying antibodies of interest.
46. A method, the method comprising screening antibodies reactive
to hemagglutinin for binding to the stem region of influenza
hemagglutinin in the neutral pH conformation in isolation from the
head region of hemagglutinin, thereby identifying antibodies of
interest.
47. A method, the method comprising screening antibodies reactive
to hemagglutinin for binding to influenza hemagglutinin in the
neutral pH conformation in isolation from other components of
influenza virus, wherein the head region of the hemagglutinin is
modified to reduce the antigenicity of the head region, thereby
identifying antibodies of interest.
48. The method of claim 47, wherein the head region of the
hemagglutinin is modified by removing or replacing glycosylation
sites.
49. The method of claim 47, wherein the head region of the
hemagglutinin is modified by adding glycosylation sites.
50. The method of claim 47, wherein the head region of the
hemagglutinin is modified by removing all or a portion of the head
region.
51. The method of claim 47, further comprising screening the
antibodies of interest for competing with antibody F10 for binding
to hemagglutinin, thereby identifying F10-competing antibodies.
52. The method of claim 47, wherein the hemagglutinin is
hemagglutinin from a group 2 influenza virus.
53. The method of claim 47, wherein the hemagglutinin is
hemagglutinin from a group 1 influenza virus.
54. The method of claim 47, further comprising producing the
identified antibodies.
55. An antibody produced by the method of claim 54.
56. A method of screening a compound for binding to an F10 antibody
comprising contacting said F10 antibody with a compound of interest
and detecting a compound-antibody complex.
Description
RELATED APPLICATIONS
[0001] This application claims the benefit of provisional
applications U.S. Ser. No. 61/091,599, filed Aug. 25, 2008, U.S.
Ser. No. 61/150,231, filed Feb. 5, 2009, and U.S. Ser. No.
61/154,400, filed Feb. 22, 2009 the contents which are each herein
incorporated by reference in their entirety.
FIELD OF THE INVENTION
[0003] The disclosed invention is generally in the field of
influenza and specifically in the area of antibodies and immunogens
for the treatments for influenza.
BACKGROUND OF THE INVENTION
[0004] Seasonal influenza A is a scourge of the young and old,
killing more than 250,000 worldwide each year, while creating an
economic burden for millions (W.H.O. web site
who.int/mediacentre/factsheets/2003/fs211/en/. World Health
Organization factsheet 211: influenza (2003)). Pandemic influenza,
which occurs when a new virus emerges and infects people globally
that have little or no immunity, represents a grave threat to human
health: for example, the 1918 "Spanish Flu" pandemic caused an
estimated 50 million deaths (Webster, 1918 Spanish influenza: the
secrets remain elusive. Proc Natl Acad Sci USA 96, 1164-6 (1999);
de Wit & Fouchier, Emerging influenza. J Clin Virol 41, 1-6
(2008)). Vaccines have historically been the mainstay of infection
control. However, due to rapid antigenic drift, the vaccine antigen
needs to be updated annually based on global influenza surveillance
(W.H.O. web site
who.int/csr/disease/influenza/influenzanetwork/en/index.html.
(2008); Carrat & Flahault, Influenza vaccine: the challenge of
antigenic drift. Vaccine 25, 6852-62 (2007)), and it is not always
fully successful. In addition, some recent H5N1 vaccines have shown
promising results (Cinatl et al., The threat of avian influenza A
(H5N1). Part IV: Development of vaccines. Med Microbiol Immunol
196, 213-25 (2007); Subbarao & Luke H5N1 viruses and vaccines.
PLoS Pathog 3, e40 (2007); Leroux-Roels et al., Broad Clade 2
Cross-Reactive Immunity Induced by an Adjuvanted Clade 1 rH5N1
Pandemic Influenza Vaccine. PLoS ONE 3, e1665 (2008); Baras et al.,
Cross-Protection against Lethal H5N1 Challenge in Ferrets with an
Adjuvanted Pandemic Influenza Vaccine. PLoS ONE 3, e1401 (2008)),
but none has been reported to elicit a broad neutralizing response
in humans. Neuraminidase inhibitors, especially oseltamavir
(Tamiflu), remain the primary antiviral treatment, but they have
limited efficacy if administered late in infection, and widespread
use is likely to result in the emergence of resistant viral strains
(de Jong et al., Oseltamivir resistance during treatment of
influenza A (H5N1) infection. N Engl J Med 353, 2667-72 (2005);
W.H.O. Clinical management of human infection with avian influenza
A (H5N1) virus. web site
who.int/csr/disease/avian_influenza/guidelines/ClinicalManagement07.pdf).
[0005] Influenza A is sub-classified by its two major surface
proteins: hemagglutinin (HA or H), which mediates cell entry, first
by recognizing host proteins bearing sialic acid on their surface,
and second by triggering the fusion of viral and host membranes
following endocytosis, allowing viral RNA to enter the cytoplasm;
and neuraminidase (NA or N), which cleaves sialic acid from host
and viral proteins, facilitating cell exit (Wright et al.,
Orthomyxoviruses. in Fields Virology Vol. 2 (eds. Knipe, D.,
Howley, P., Griffin, D., Lamb, R. & Martin, M.) 1692-1740
(Lippincott Williams & Wilkins 2006)). There are 16 HA subtypes
and 9 NA subtypes which make up all known strains of influenza A
viruses by various combinations of HA and NA (Wright et al. (2006))
(See FIG. 8).
[0006] The recent spread of highly pathogenic avian influenza
(HPAI), H5N1, across Asia, Europe and Africa raises the specter of
a new pandemic, should the virus mutate to become readily
transmissible from person-to-person. The evolution of H5N1 into a
pandemic threat could occur through a single reassortment of its
segmented genome or through the slower process of genetic drift
(Wright et al. (2006); Fauci, Pandemic influenza threat and
preparedness. Emerg Infect Dis 12, 73-7 (2006)). Nearly 400 human
H5N1 infections have been reported since 1997 from 14 countries,
with a case mortality rate in the immunocompetent population above
60% (W.H.O. web site
who.int/csr/disease/influenza/influenzanetwork/en/index.html.
(2008)).
[0007] New therapeutic strategies that provide potent and broadly
cross-protective host immunity are therefore a global public health
priority. Human monoclonal antibody (mAb)-based "passive"
immunotherapy is now being used to treat a number of human
diseases, including Respiratory Syncytial Virus infection, and it
has been proposed how immunotherapy could be used strategically in
a viral outbreak setting (Marasco & Sui, The growth and
potential of human antiviral monoclonal antibody therapeutics. Nat
Biotechnol 25, 1421-34 (2007)).
BRIEF SUMMARY OF THE INVENTION
[0008] The present invention relates to an immunogen capable of
inducing antibodies against a target peptide of the stem region of
hemagglutinn protein of an influenza virus. The immunogen is a
peptide or a synthetic peptide. In particular, the immunogen of
this invention comprises one or more epitopes or epitope units.
Optionally, the immunogen further comprises a general immune
stimulator. These immunogens of the present invention are capable
of inducing antibodies against influenza A virus to prevent
infection by the virus.
[0009] In one aspect the invention provides an immunogen having an
epitope or epitope unit recognized by a protective monoclonal
antibody having the specificity for the stem region of hemagglutinn
protein of an influenza virus.
[0010] The antibody binds both the HA1 and HA2 peptide. In some
embodiments the epitope is recognized by monoclonal antibody D7,
D8, F10, G17, H40, A66, D80, E88, E90, or H98 or a monoclonal
antibody that competes with the binding of monoclonal antibody D7,
D8, F10, G17, H40, A66, D80, E88, E90, or H98 to the HA protein.
Preferably, the epitope is the F10 epitope.
[0011] In some embodiments the hemagglutinin protein is in the
neutral pH conformation.
[0012] The immunogen is a peptide or a synthetic peptide.
[0013] In some aspects the immunogen is a conjugate having one or
more peptides or peptide fragments that are spatially positioned
relative to each other so that they together form a non-linear
sequence which mimics the tertiary structure of an F10 epitope.
Optionally, the one or more peptides or peptide fragments are
linked to a backbone. The conjugate competes with the binding of
monoclonal antibody F10 to the HA protein.
[0014] The e conformation of the epitope is defined by amino acid
residues 18, 38, 39, 40 and 291 of HA1 and 18, 19, 20, 21, 38, 41,
42, 45, 49, 52, 53, and 56 of HA2 when the hemagglutinin in the
neutral pH conformation.
[0015] In some embodiments the immunogen is a peptide having one or
more of the following amino acid sequences:
TABLE-US-00001 (SEQ ID NO: 125)
[Xaa.sub.0].sub.m-Xaa.sub.1-Xaa.sub.2- [Xaa.sub.0].sub.p (SEQ ID
NO: 126) [Xaa.sub.0].sub.m-Xaa.sub.1-Xaa.sub.2- [Xaa.sub.0].sub.p
(SEQ ID NO: 127)
[Xaa.sub.0].sub.m-Xaa.sub.1-Xaa.sub.2-Xaa.sub.3-Xaa.sub.4-
[Xaa.sub.0].sub.p (SEQ ID NO: 128)
[Xaa.sub.0].sub.m-Xaa.sub.1-[Xaa.sub.0].sub.q
Xaa.sub.2-Xaa.sub.3-[Xaa.sub.0].sub.q Xaa.sub.4-[Xaa.sub.0].sub.r
Xaa.sub.5-[Xaa.sub.0].sub.q-Xaa.sub.6 Xaa.sub.7 - [Xaa.sub.0].sub.q
-Xaa.sub.8 -[Xaa.sub.0].sub.p (SEQ ID NO: 129)
[Xaa.sub.0].sub.m-Xaa.sub.1-[Xaa.sub.0].sub.q
Xaa.sub.2-Xaa.sub.3-[Xaa.sub.0].sub.q Xaa.sub.4-[Xaa.sub.0].sub.r
Xaa.sub.5-[Xaa.sub.0].sub.q-Xaa.sub.6 Xaa.sub.7 - [Xaa.sub.0].sub.s
- [Xaa.sub.8].sub.t -[Xaa.sub.0].sub.p
[0016] Wherein, m, and p are independently 0 or 1-100, preferably
about 1-90, 1-80, 1-70, 1-60, 1-50, 1-40, 1-30, 1-20 or 1-10; q is
2, r is 3, s is 0 or 2, and t is 0 or 1, and Xaa.sub.0, is
independently any amino acid. Preferably s is 2 and t is 1.
[0017] Preferably Xaa.sub.1 of SEQ ID NO: 125 is S, T, F H or Y and
Xaa.sub.2 of SEQ ID NO: 125 is H, Y, M, L or Q. Most preferably,
Xaa.sub.1 of SEQ ID NO: 125 is Y. Most preferably, Xaa.sub.2 of SEQ
ID NO: 125 is H.
[0018] Preferably, Xaa.sub.1 of SEQ ID NO: 126 is H, Q, Y, S, D, N
or T and Xaa.sub.2 of is SEQ ID NO: 126 is Q, E, K, I, V, M, E, R
or T. Most preferably, Xaa.sub.1 of SEQ ID NO: 126 is H. Most
preferably, Xaa.sub.2 of SEQ ID NO: 126 is Q.
[0019] Preferably, in SEQ ID NO; 127 Xaa.sub.1 is I, V, M, or L;
Xaa.sub.2 is D, N, H, Y, D, A, S or E, Xaa.sub.3 is G or A, and
Xaa.sub.4 is W, R, or G. Most preferably, in SEQ ID NO; 127
Xaa.sub.1 is V; Xaa.sub.2 is D, Xaa.sub.3 is G, and Xaa.sub.4 is
W.
[0020] Preferably in SEQ ID NO:128 or SEQ ID NO:129, Xaa.sub.1 is
K, Q, R, N, L, G, F, H or Y; Xaa.sub.2 is S or T, Xaa.sub.3 is Q or
P; Xaa.sub.4 is F, V, I, M, L, or T; Xaa.sub.5 is I, T, S, N, Q, D,
or A; Xaa.sub.6 is I, V, M, or L; Xaa.sub.7 is N, S, T, or D and
Xaa.sub.8 is I, F, V, A, or T. Most preferably, SEQ ID NO:128 or
SEQ ID NO:129, Xaa.sub.1 is K,; Xaa.sub.2 is T, Xaa.sub.3 is Q;
Xaa.sub.4 is I; Xaa.sub.5 is T; Xaa.sub.6 is V; Xaa.sub.7 is N, and
Xaa.sub.8 is I.
[0021] In some aspects of the inventions, one or more amino acids
are D-amino acids.
[0022] Optionally, the immunogen further comprises an adjuvant or
is conjugated to a carrier.
[0023] In various aspects the invention includes a composition
containing the immunogen together with one or more pharmaceutically
acceptable excipients, diluents, and/or adjuvants. In some
embodiments the composition further comprises an anti-influenza
antibody of antigen binding fragment thereof. Preferably, the
antibody is monoclonal antibody D7, D8, F10, G17, H40, A66, D80,
E88, E90, or H98 or a monoclonal antibody that competes with the
binding of monoclonal antibody D7, D8, F10, G17, H40, A66, D80,
E88, E90, or H98 to the HA protein.
[0024] Also provided by the invention is nucleic acids encoding the
immunogens of the invention and composition comprising the nucleic
acids.
[0025] The invention further comprises a method preventing a
disease or disorder caused by an influenza virus by administering
to person at risk of suffering from said disease or disorder an
immunogen composition described herein. Optionally, the method
includes further administering an anti-viral drug, a viral entry
inhibitor or a viral attachment inhibitor. The anti-viral drug is a
neuraminidase inhibitor, a HA inhibitor, a sialic acid inhibitor or
an M2 ion channel. The M2 ion channel inhibitor is amantadine or,
rimantadine. The neuraminidase inhibitor zanamivir, or oseltamivir
phosphate.
[0026] In another aspect the method includes further administering
one or more antibodies specific to a Group I influenza virus and or
a Group II influenza virus. The antibody is administered at a dose
sufficient to neutralize the influenza virus.
[0027] Administration is prior to or after exposure to influenza
virus.
[0028] Also disclosed are methods of treating subjects and methods
of screening and producing antibodies. For example, disclosed is a
method of treating a subject suffering or at risk of influenza
infection, the method comprising administering to the subject one
or more of the disclosed antibodies, such as the disclosed HA stem
antibodies. For example, disclosed is a method of treating a
subject, the method comprising administering to the subject the
stem region of influenza hemagglutinin in the neutral pH
conformation in isolation from other components of influenza virus,
wherein the subject produces an immune response to the stem region.
For example, disclosed is a method of treating a subject, the
method comprising administering to the subject the stem region of
influenza hemagglutinin in the neutral pH conformation in isolation
from the head region of hemagglutinin, wherein the subject produces
an immune response to the stem region. For example, disclosed is a
method of treating a subject, the method comprising administering
to the subject influenza hemagglutinin in the neutral pH
conformation in isolation from other components of influenza virus,
wherein the head region of the hemagglutinin is modified to reduce
the antigenicity of the head region, wherein the subject produces
an immune response to the stem region. For example, disclosed is a
method, the method comprising screening antibodies reactive to
hemagglutinin for binding to hemagglutinin immobilized on a
surface, thereby identifying antibodies of interest. For example,
disclosed is a method comprising screening antibodies reactive to
hemagglutinin for binding to the stem region of influenza
hemagglutinin in the neutral pH conformation in isolation from the
head region of hemagglutinin, thereby identifying antibodies of
interest. For example, disclosed is a method comprising screening
antibodies reactive to hemagglutinin for binding to influenza
hemagglutinin in the neutral pH conformation in isolation from
other components of influenza virus, wherein the head region of the
hemagglutinin is modified to reduce the antigenicity of the head
region, thereby identifying antibodies of interest.
[0029] In some forms, the head region of the hemagglutinin can be
modified by removing or replacing glycosylation sites. In some
forms, the head region of the hemagglutinin can be modified by
adding glycosylation sites. In some forms, the head region of the
hemagglutinin can be modified by removing all or a portion of the
head region.
[0030] In some forms, the disclosed antibodies, disclosed
hemagglutinins, and disclosed methods can produce an immune
reaction in a subject. For example, in some forms, the subject can
produce an immune response that prevents or reduces the severity of
an influenza infection. In some forms, the immune response can be
reactive to influenza viruses within a subtype. In some forms, the
immune response can be reactive to influenza viruses in each
subtype within a cluster. In some forms, the immune response can be
reactive to influenza viruses in each cluster within a group. In
some forms, the immune response can be reactive to all influenza
viruses in each subtype within a group. In some forms, the immune
response can be reactive to influenza viruses within group 1.
[0031] In some forms, the disclosed methods can further comprise
screening the antibodies of interest for competing with antibody
F10 for binding to hemagglutinin, thereby identifying F10-competing
antibodies. In some forms, the hemagglutinin can be hemagglutinin
from a group 2 influenza virus. In some forms, the hemagglutinin
can be hemagglutinin from a group 1 influenza virus. In some forms,
the disclosed methods can further comprising producing the
identified antibodies. Also disclosed are antibodies produced by
the disclosed methods. Also disclosed are antibodies identified by
the disclosed methods.
[0032] The disclosed compositions and methods are based upon the
discovery of monoclonal antibodies which neutralize the influenza
virus, e.g. influenza A virus. The influenza A virus is a Group I
influenza A virus such as a H1 cluster influenza virus. The H1
cluster influenza virus is an H1a cluster or an H1b cluster. The
monoclonal antibody is fully human. In some forms, the monoclonal
antibody can be a bivalent antibody, a monovalent antibody, a
single chain antibody or fragment thereof. Specifically, such
monoclonal can bind to an epitope on the stem region of the
hemagglutinin protein (HA), such as HA1 or HA2 polypeptide. The
epitope can be non-linear.
[0033] The epitope can comprise both the HA1 and HA2. The epitope
can be non-linear. In some forms the epitope can comprise the amino
acid position 18, 38, 40, 291 of the HA1 polypeptide and the amino
acid at position 18, 19, 20, 21, 38, 41, 42, 45, 49, 52, 53 and 56
of the HA2 polypeptide.
[0034] The disclosed compositions and methods are further based
upon the discovery of a protocol for generating broadly
neutralizing human antibodies that target a highly conserved
epitope in the stem region of HA. Using the trimeric H5 ectodomain
expressed in baculovirus which produces shorter N-glycans and
uncharged mannoses absorbed on a plastic surface, allowed for the
dominant presentation of the stem epitope while masking the
normally immunodominat globular head. Accordingly, also disclosed
is a method of producing an isolated antibody that specifically
binds a pathogenic enveloped virus by exposing a single chain or
Fab expression library to a membrane fusion protein of the virus,
identifying an antibody in the library that specifically binds said
protein; and isolating the antibody from the library. The fusion
protein can be immobilized on a solid surface, e.g. plastic. In
some forms the fusion protein can have modified glycosylations
compared to a wild type fusion protein. For example, the fusion can
be produced in a non-mammalian cell, such as an insect cell. The
fusion protein can be, for example, a trimeric hemagglutinin (HA)
protein.
[0035] Also disclosed is a method of vaccinating a subject against
pathogenic enveloped virus such as an influenza virus by
administering to the subject, for example, a membrane fusion
protein (e.g., a trimeric hemagglutinin (HA) protein coated) or
embedded in a biologically compatible matrix. In some forms the
fusion protein can have modified glycosylations compared to a wild
type fusion protein.
[0036] Also disclosed is a composition comprising a monoclonal
antibody as described herein and kits containing the composition in
one or more containers and instructions for use.
[0037] The invention further provides a method of screening a
compound for binding to an F10 antibody by contacting said F10
antibody with a compound of interest and detecting a
compound-antibody complex. Also included in the invention are the
compound identified by the method and their use as immunogens.
[0038] Unless otherwise defined, all technical and scientific terms
used herein have the same meaning as commonly understood by one of
ordinary skill in the art to which this disclosed subject matter
belongs. Although methods and materials similar or equivalent to
those described herein can be used in the practice or testing of
the disclosed subject matter, suitable methods and materials are
described below. All publications, patent applications, patents,
and other references mentioned herein are incorporated by reference
in their entirety. In the case of conflict, the present
specification, including definitions, will control. In addition,
the materials, methods, and examples are illustrative only and are
not intended to be limiting.
[0039] Additional advantages of the disclosed method and
compositions will be set forth in part in the description which
follows, and in part will be understood from the description, or
may be learned by practice of the disclosed method and
compositions. The advantages of the disclosed method and
compositions will be realized and attained by means of the elements
and combinations particularly pointed out in the appended claims.
It is to be understood that both the foregoing general description
and the following detailed description are exemplary and
explanatory only and are not restrictive of the invention as
claimed.
BRIEF DESCRIPTION OF THE DRAWINGS
[0040] The accompanying drawings, which are incorporated in and
constitute a part of this specification, illustrate several
embodiments of the disclosed method and compositions and together
with the description, serve to explain the principles of the
disclosed method and compositions.
[0041] FIGS. 1A to 1D show in vitro binding and neutralization of
anti-H5 antibodies. (A)
[0042] The 10 Abs were converted to soluble scFv-Fcs (scFv linked
to Hinge, CH2 and CH3 domains of human IgG1) and evaluated for
binding to trimeric H5-TH04 or monomeric HA1 of H5-TH04 coated on
an ELISA plate. The H5 scFv-Fcs recognizes trimeric H5 but not HA1.
An antibody raised against HA1 ("2A") recognized both. (B)
Neutralization of H5-TH04-pseudotyped viruses (virus-like particles
with HIV-1 only cores that display H5 on their surface). %
neutralization at 2 concentrations is shown with standard deviation
(s.d.) bars. The mAb 80R (Sui et al. (2004)) was used as a negative
control ("Ctrl."). (C-D) Neutralization of wild type H5-VN04 and
H5-IN05 by the 10 scFv-Fcs at three concentrations using a plaque
reduction assay. Results are consistent with those obtained from a
microneutralization assay.
[0043] FIGS. 2A to 2F show prophylactic and therapeutic efficacy of
anti-H5 neutralizing mAbs ("nAbs") in mice. (A and B) Prophylactic
efficacy. % survival of mice treated with anti-H5 nAbs or control
mAb 1 h before lethal challenge by i.n. inoculation with (A)
H5-VN04 or (B) H5-HK97 viruses. (C-F). Therapeutic efficacy. Mice
were inoculated with H5-VN04 and injected with nAbs at 24, 48, 72 h
post-inoculation (hpi) (C, E and F) or with H5-HK97 at 24 hpi
(D).
[0044] FIGS. 3A and 3B show neutralization mechanism. (A) nAbs do
not inhibit cell-binding of full-length HA from H5-TH04-pseudotyped
HIV-1 viruses. None of the 3 nAb-treated viruses inhibited cell
binding; mouse anti-H5 mAb, 17A2.1.2, and ferret anti-H5N1 serum,
which inhibit haemagglutination, were used as positive controls;
anti-SARS spike protein ("80R") and anti-HA1 ("2A") were used as
negative controls. Vertical bars represent s.d. (B) All 3 nAbs
inhibit cell fusion. HeLa cells were transfected with
H5-TH04-expressing plasmid and expose a pH 5.0 buffer for 4 mins in
the presence or absence of nAbs. Syncytia formation induced by
brief exposure to pH 5.0 was completely inhibited by D8, F10 and
A66, at 20 .mu.g ml.sup.-1 (.about.0.13 .mu.M), whereas controls
("80R" and anti-HA1 mAb ("2A")) at the same concentration had no
effect.
[0045] FIGS. 4A to 4D show structure of the H5-F10 complex. (A)
Structure of the H5 trimer bound to F10 (scFv). H5 is very similar
to the uncomplexed structure (Yamada et al. (2006)) (pairwise RMSD
(C.alpha.)=1.0 and 0.63 .ANG. for 2 independent trimers). HA1 is
depicted by the light colored ribbon diagram at the top of the
figure, HA2 is depicted by the 5.alpha. helices and 4.beta. strands
at the bottom of the figure closest to the membrane as well as the
3 long .alpha. helices in the middle of the figure approximately
5.5 cm high, the .alpha.A-helix of HA2 is represented by 3 vertical
.alpha. helices approximately 2 cm high (one is hidden toward the
back of the complex), the "fusion peptide" (FP) is a thin tubular
structure that wraps horizontally around the bottom of the 3 long
.alpha. helices of HA2 (identified by an arrow labeled Fusion
Protein), and F10 (VH and VL) represented by the ribbon diagrams on
either side of HA2 plus a third F10 molecule hidden behind the
stem. (B) Close-up of the epitope showing H5 as a molecular
surface, with selected epitope residues labeled. The fusion peptide
is represented by an approximately 1 cm in diameter light-shaded
area beginning underneath the Trp21 label and continuing down to
the end of the figure (seen underneath the Gly20 and Asp19 labels).
The light-shaded area seen underneath the Met54 and Thr41 labels
and everything to the right of those residues are not a part of the
fusion peptide. The tip of F10 (the ribbon structure) and selected
CDR side-chains are shown. Of 1500 .ANG..sup.2 buried surface at
the interface, 43% involves hydrophobic interactions. (C) Surface
of the central stem region, showing two H5 monomers. One monomer
has HA1 depicted by the light-shaded region on the far left of the
figure and HA2 depicted by the dark colored region without any
residues labeled; the path of FP through the epitope is outlined,
while mutations not affecting binding are the lightly shaded region
labeled with 50.sub.2, 54.sub.2, 57.sub.2, and 61.sub.2 (see FIG.
4D). The fusion peptides (FP and FP') are labeled in both monomers.
HA2 epitope residues are labeled 18.sub.2 19.sub.2 20.sub.2,
21.sub.2, 38.sub.2, 41.sub.2, 42.sub.2, 45.sub.2, 49.sub.2,
52.sub.2, 53.sub.2, and 56.sub.2 and HA1 epitope residues are
labeled 18.sub.1, 38.sub.1, 39.sub.1, 40.sub.1, 292.sub.1 and
291.sub.1. The position of buried residue H111.sub.2 is shown as a
black ball labeled "H". (D) Binding of the 3 nAbs to H5 mutants in
the .alpha.A helix. Note the very similar response to all mutants
tested. Mutations were made either to alanine or to the
corresponding H7 residue. 293T cells were transiently transfected
with mutants; 24 hours after transfection, nAbs or ferret anti-H5N1
serum were used to stain the transfected cells. Mean Fluorescent
Intensity (MFI) was normalized against ferret anti-serum (100%) to
account for different expression levels.
[0046] FIG. 5 shows sequence conservation in HA Groups, Clusters
and Subtypes at the F10 epitope. Circles below residue numbers
indicate estimated contribution to the binding energy at each
position. Positions 19, 21 and 45 of HA2 show a strong
contribution. Position 18 of HA1 and positions 18, 20, 41, 49, 52,
53 and 56 of HA2 show intermediate contribution. Positions 38 and
291 of HA1 and positions 38 and 42 of HA2 show neutral
contribution. Residues without a circle are not directly involved
in the epitope but are discussed in the text. Many residues are
conserved among groups, clusters and/or subtypes. Residues 18, 20,
21, 41, 42, 44-46, 48, 49, 51-53, 55 and 56 are all highly
conversed for all clusters/groups except H12 at residues 48 and 49
and H14 and H3 at residue 49. Group 1 has cluster/subtype specific
residues at 17 of HA1 and 38 and 111 of HA2. Subtype H3 and cluster
H7 are identical at HA1 residue 38 and cluster H1b and subtypes H8
and H12 and cluster H3 are similar at HA1 residue 292. Group 2 is
identical at residue 17 of HA1 and Group 1 cluster H9 is identical
at residue 18 of HA1. Group 1 cluster H1a and Group 2 are similar
at residue 18 of HA1 as well as clusters H1a and H9 at residue 38
of HA1. Subtypes H1 and H6 and cluster H1b are identical at residue
291 of HA1. Cluster H1a, subtype H8 and cluster H7 are identical at
residue 19 of HA2. Cluster H3 is identical at residue 38 of HA2 and
while residue 111 of HA2 is also identical within the cluster.
Cluster H1b and cluster H7 are identical at residues 18 and 292 of
HA1, respectively. Cluster H1b at residue 38 of HA1 and subtypes H2
and H5 and cluster H9 at residue 291 of HA1 are similar. Cluster H7
is identical at residues 38 of HA2 and 111 of HA2, respectively.
The network of inter-helical contacts that stabilize the fusogenic
structure (Bullough et al., Structure of influenza haemagglutinin
at the pH of membrane fusion. Nature 371, 37-43 (1994)) are
indicated below the HA2 sequences. Subtypes that can be
recognized/neutralized by F10 are indicated with "+" on the far
right. "(+) or (-)" indicates a predicted positive/negative
binding. Amino acid at positions 18-21 of HA2 are VDGW (SEQ ID
NO:20), IDGW (SEQ ID NO:21), INGW (SEQ ID NO:22), and VAGW (SEQ ID
NO:23).
[0047] FIGS. 6A to 6D show cross subtype neutralization by nAbs.
(A) nAbs D8, F10 and A66 all neutralized H5-TH04, H1-SC1918,
H1-PR34, H1-WSN33, H2-JP57, H6-NY98 and H11-MP74 (strains described
below) pseudotyped viruses. (B) Microneutralization assay.
Neutralization titers (0.1 mg ml.sup.-1 Ab stock solution) of nAb
F10 against two wild-type H5N1, three H1N1, one H2N2, one H6N1, one
H6N2, one H8N4, two H9N2 and one H3N2 virus. 80R is the negative
control. Vertical bars and whiskers represent the lowest and
highest neutralization titer (2.sup..chi., values of .chi. are
shown on the y-axis) of 2-3 independent experiments. (C-D)
Prophylactic efficacy against two H1N1 strains in mice. % survival
of mice treated with anti-H5 nAbs or control mAb are shown before
lethal challenge by i.n. inoculation with (C) H1-WSN33 or (D)
H1-PR34 viruses. Complete viral strain designations are: H1-OH83
(A/Ohio/83 (H1N1)), H1-PR34 (A/Puerto Rico/8/34 (H1N1)), H1-SC1918
((A/South Carolina/1/1918 (H1N1)), H1-WSN33 (A/WSN/1933 (H1N1)),
H2-AA60 (A/Ann Arbor/6/60 (H2N2)), H2-JP57 (A/Japan/305/57(H2N2)),
H3-SY97 (A/Sydney/5/97(H3N2)), H6-HK99 (A/quail/Hong
Kong/1721-30/99(H6N1)), H6-NY98 (A/chicken/New York/14677-13/1998
(H6N2)), H7-FP34 (A/FPV/Rostock/34 (H7N1)), H8-ON68
(A/turkey/Ontario/6118/68), H9-HK(G9)97 (A/chicken/HongKong/G9/97
(H9N2)), H9-HK99 (A/HongKong/1073/99 (H9N2)), H11-MP74
(A/duck/memphis/546/74 (H11N9)).
[0048] FIG. 7 shows 3-dimensional comparison of the F10 epitope in
Group 1 and Group 2 HAs. Stereo overlay of crystal structures of
the 5 known HA subtypes in the region of the F10 epitope, showing
conservation and differences between the 2 phylogenetic groups.
Shown are H1, H5 and H9 (Group 1) (PDB codes 1RU7, 21BX and 1JSD);
and H3 and H7 (Group 2) (PDB codes 1MQL and 1TI8). For 17.sub.1, Y
is in Group 1 and H is in Group 2. For 18.sub.1, the H/H/Q in the
lower left are in Group 1 and the two H in the upper right are in
Group 2. For 21.sub.2, the two vertical W are in Group 2 while the
three tilted W are in Group 1. For 38.sub.1, the N are in Group 2
and the H are in Group 1. For 111.sub.2, the H are in Group 1 and
the T are in Group 2. RMS differences for pair-wise overlays are
0.56.+-.0.11 .ANG. (observed range, Group 1); 0.75 .ANG.(Group 2);
and 1.21.+-.0.12 .ANG. between groups. Consistent differences
between phylogenetic groups include the orientation of W21.sub.2
and alternative side-chain directions at 18.sub.1 and 38.sub.1,
which are linked to the packing of buried His111.sub.2 (the
putative pH trigger in Group 1; absent in Group 2); and the burial
of the larger tyrosine (Group 1) versus histidine (the putative pH
trigger in Group 2) at 17.sub.1. Of particular note, N38.sub.1 is
glycosylated in 4 members of the Group 2 clusters. Other epitope
residues are indicated by numbered circles.
[0049] FIG. 8 shows phylogenetic relationships and sequence
comparison among HA subtypes. Phylogenetic tree of the 16 HA
subtypes of influenza A viruses based on amino-acid sequences. Four
clusters of HA subtypes are (1) Cluster H1: H2, H5, H1, H6, H13,
H16, and H11; (2) Cluster H9: H8, H12, and H9; (3) Cluster H3: H4,
H14, and H3; and (4) Cluster H7: H15, H7, and H10. Within Cluster
H1 are two subclusters: subcluster H1a: H2, H5, H1, and H6; and
subcluster H1b: H13, H16, and H11. The sequences used for analysis
were: H1 (A/South Carolina/1/1918), H2 (A/Japan/305/1957), H3
(A/Aichi/2/1968), H4 (A/duck/Czechoslovakia/56), H5
(A/VietNam1203/2004), H6 (A/chicken/California/431/00), H7
(A/Netherland/219/03), H8 (A/turkey/Ontario/6118/68), H9
(A/swine/HK/9/98), H10 (A/chicken/Germany/N49), H11
(A/duck/England/56), H12 (A/duck/Alberta/60/76), H13
(A/gull/Maryland/704/77), H14 (A/mallard/Astrakhan/263/1982), H15
(A/shearwater/West Australia/2576/79) and H16 (A/black-headed
gull/Sweden/2/99).
[0050] FIGS. 9A and 9B show SDS-PAGE and gel filtration analysis of
HA proteins. (A) Antibody 2A was obtained from a separate
HA1-targeted selection against the HA1 (residues 11-325) fragment
of H5-TH04 (left panel). H5 HA (H5-VN04 strain) used for library
selection is shown in the right panel, (B) H5-VN04 (H5) and scFv
F10 complex. HA0 was fully cleaved into HA1 and HA2 by
co-expression with furin (left panel). Complexes were formed by
first mixing H5 and F10 at a molar ratio of 1:10, and then purified
by gel filtration.
[0051] FIG. 10 shows binding of anti-H5 scFv-Fcs to H5 or HA1 by
competition ELISA. 10.sup.12 pfu of anti-H5 phage-scFvs were mixed
with 5 .mu.g/mL of anti-H5 scFv-Fcs and added to H5
(H5-VN04)-coated plates, washed, and followed by HRP-anti-M13 to
detect phage-scFvs bound to H5. mAb 2A-Fc did not compete for the
epitope recognized by the 10 H5-selected Abs. All H5-selected
scFv-Fcs cross-competed. Of these, Ab F10 (phage-scFv) binding to
the H5 trimer was the least inhibited by the other scFv-Fcs
suggesting that it has the highest affinity among all Abs
tested.
[0052] FIG. 11 shows kinetic and thermodynamic characterization of
the binding of H5 to nAbs D8, F10 and A66-IgG1s. nAbs were captured
on a CM4 chip via anti-human IgG1; trimeric H5 (H5-VN04) at various
concentrations (20, 10, 5, 2.5, 2.5, 1.25, 0.625 nM) was injected
over the chip surface. Binding kinetics were evaluated using a 1:1
Langmuir binding model. The recorded binding curves (with blank
reference subtracted) and the calculated curves are closely
superimposable. Each ka, kd and K.sub.D value represents the mean
and standard error of three experiments. Note: the middle graph
labeled "D10-IgG" is actually a graph of F10-IgG.
[0053] FIG. 12 shows viral titers in lung, spleen and brain of mice
treated with anti-H5 nAbs after H5-VN04 challenge. BALB/c mice
(n=5) were treated by i.p. injection of 15 mg/kg of mAb at 24, 48
or 72 hrs after i.n. infection with 10 MLD50 of H5-VN04. Viral
titers were determined in lung, brain, and spleen collected at 96
hpi. Data are displayed in box-and-whiskers form in which the box
extends from the 25th to the 75th percentile, with a horizontal
line at the median. Whiskers above and below the box indicate the
extreme values. Results of Student T-test statistic analysis are
noted with a single star (*) for p<0.05, and double stars (**)
for p<0.01. The arrows crossing the Y axis indicate the
detection limit of the titration.
[0054] FIG. 13A is an illustration of the structure of the
A/Vietnam 1203/04 trimer. The receptor binding site and antigenic
variation sites are highlighted on the monomer.
[0055] FIG. 13B is an illustration showing the location of amino
acid residues in the HA of H5N1 influenza viruses that are under
positive selection.
[0056] FIG. 14 is a schematic illustration of convergent
combination Immunotherapy for H5N1.
[0057] FIG. 15 shows in vitro binding and neutralization of anti-H5
antibodies. (a) The 10 Abs were converted to soluble scFv-Fcs (scFv
linked to Hinge, CH2 and CH3 domains of human IgG1) and evaluated
for binding to trimeric H5-TH04 or monomeric HA1 of H5-TH04 coated
on an ELISA plate. The H5 scFv-Fcs recognize trimeric H5 but not
HA1. An antibody raised against HA1 ("2A") recognized both. (b)
Neutralization of H5-TH04-pseudotyped viruses (virus-like particles
with HIV-1 only cores that display H5 on their surface). %
neutralization at 2 concentrations is shown with standard deviation
(s.d.) bars. The mAb 80R.sup.18 was used as a negative control
("Ctrl."). (c-d) Neutralization of wild type H5-VN04 and H5-1N05 by
the 10 scFv-Fcs at three concentrations using a plaque reduction
assay. Results are consistent with those obtained from a
microneutralization assay (data not shown).
[0058] FIG. 16 shows the prophylactic and therapeutic efficacy of
anti-H5 nAbs in mice. Mice were treated with different doses of nAb
either before or after lethal viral challenge. Prophylactic
efficiacy (a, b, g and h). Mice were treated with anti-H5 nAbs or
control mAb 24 hour before lethal challenge by intranasally (i.n.)
with 10 median lethal doses (MLD50) of the H5N1 or H1N1s. (a)
Intra-peritoneal (i.p.) injection of 10 mg/kg of any of the three
nAbs provided complete protection of mice challenged with H5-VN04
(A/Vietnam/1203/04 (H5N1), Clade 1). A lower antibody dose (2.5
mg/kg) was also highly protective. (b) Prophylactic protection
against H5-HK97 (A/HongKong/483/97 (H5N1), Clade 0) virus was
observed in 80-100% of the mice treated with 10 mg/kg of any of the
three nAbs. (g) Any of the three nAbs (at 10 mg/kg of single
injection) provided complete protection of mice challenged with
H1-WSN33 (A/WSN/1933(H1N1)) viruses. (h) D8 and F10 completely
protected mice challenged with H1-PR34 (A/Puerto Rico/8/34 (H1N1))
when given at 10 mg/kg of single injection. A66 provided complete
protection of mice when 25 mg/kg of antibody was given as a single
injection. Therapeutic efficacy (c-f). Mice were inoculated with
H5-VN04 and injected with nAbs at 24, 48, 72 hpi (c, e and f) or
with H5-HK97 at 24 hpi (d). I.p. treatment with 15 mg/kg (a
therapeutically achievable dose in humans) of any of the 3 nAbs at
24 h post-inoculation (hpi) protected 80-100% of mice challenged
with 10-times the MLD50 of either H5-VN04 or H5-HK97 virus.
[0059] FIG. 17 shows FACS analysis of anti-H5 nAbs binding to H1,
H2, H5, H6 (cluster H1a); H11, H13 and H16 (Cluster H1b); and H8
and H9 (Cluster H9). 293T cells were transiently transfected with
different HA-expressing plasmids, and mAb binding to the cells was
analyzed by FACS. H5-specific antibody 2A and 80R are negative
control. Lack of binding to Group 2 HAs, H4, H7, and H14, are also
shown. Complete viral strain designations are:
H1-PR34 (A/Puerto Rico/8/34 (H1N1));
H1-SC1918 ((A/South Carolina/1/1918 (H1N1));
H1-WSN33 (A/WSN/1933 (H1N1));
H5-TH04 (A/Thailand/2-SP-33/2004 (H5N1));
H2-JP57 (A/Japan/305/57(H2N2));
H4-CA07 (A/bufflehead/California/HKWF205/2007 (H4N8))
H6-NY98 (A/Chicken/New York/14677-13/1998 (H6N2));
H7-FP34 (A/FPV/Rostock/34 (H7N1));
H8-0N68 (A/turkey/Ontario/6118/68);
H9-HK(G9)97 (A/chicken/HongKong/G9/97 (H9N2));
H9-HK99 (A/HongKong/1073/99 (H9N2));
H11-MP74 (A/duck/memphis/546/74 (H11N9));
H13-MD77 (A/Gull/MD/704/77 (H13N6));
H14-CU82 (A/mallard/Gurjev/263/82(H14N5));
H16-DE06 (A/Shorebird/DE/172/06 (H16N3)).
[0060] FIG. 18 shows cross subtype neutralization by nAbs. (a) nAbs
D8, F10 and A66 all neutralized H5-TH04, H1-SC1918, H1-PR34,
H1-WSN33, H2-JP57, H6-NY98 and H11-MP74 pseudotyped viruses. (b)
Microneutralization assay. Neutralization titers (0.1 mg ml.sup.-1
Ab stock solution) of nAb F10 against two wild-type H5N1, three
H1N1, one H2N2, one H6N1, one H6N2, one H8N4, two H9N2 and one H3N2
virus. 80R is the negative control. Vertical bars and whiskers
represent the lowest and highest neutralization titer.
[0061] FIG. 19 shows binding to HA of Swine flu H1N1 viruses and
neutralization of Swine flu H1N1 viruses by nAbs. (a) FACS analysis
of nAbs D8, F10, and A66 binding to H1 proteins of three swine flu
H1N1 2009 pandemic strains. 293T cells were transiently transfected
with different HA-expressing plasmids, and mAb binding to the cells
was analyzed by FACS. Anti-SARS antibody 80R was used as negative
control. (b) Microneutralization assay. Neutralization titers (0.1
mg ml.sup.-1 Ab stock solution) of nAb F10 against H1N1-PR34, two
swine flu 2009 H1N1 strains, and a control H3N2 virus. 80R is the
negative control. Vertical bars and whiskers represent the lowest
and highest neutralization titer (2 values of are shown on the
y-axis) of 2-3 independent experiments.
DETAILED DESCRIPTION OF THE INVENTION
[0062] The present invention relates to an immunogenic composition
comprising peptide immunogens (natural or synthetic) capable of
inducing antibodies against the hemagglutinin (HA) protein of
influenza A virus. The present invention provides peptides that
bind human monoclonal antibodies specific against the hemagglutinin
(HA) protein of influenza A virus as well as antibodies specific
against the hemagglutinin (HA) protein of influenza A virus.
[0063] Influenza virus remains a constant public health threat,
owing to its ability to evade immune surveillance through rapid
genetic drift and reassortment.
[0064] Influenza A is a negative-sense, single-stranded RNA virus,
with an eight-segment genome encoding 10 proteins. It belongs to
the family Orthomyxoviridae which includes the genera of influenza
virus A, B and C as defined by the antigenicity of the nucleocapsid
and matrix proteins. Generally, influenza A virus is associated
with more severe disease in humans. Influenza A virus is further
subtyped by two surface proteins, hemagglutinin (HA) which attaches
the virion to the host cell for cell entry, and neuraminidase (NA)
which facilitates the spread of the progeny virus by cleaving the
host sialic acid attached to the progeny virus or cell surface.
[0065] There are 16 HA subtypes and 9 NA subtypes which make up all
subtypes of influenza A viruses by various combinations of HA and
NA. All combinations of the 16 HA and 9 NA virus subtypes are found
in water fowl. Of the hundreds of strains of avian influenza A
viruses, only four are known to have caused human infections: H5N1,
H7N3, H7N7 and H9N2. In general, human infection with these viruses
has resulted in mild symptoms and very little severe illness: there
has been only one fatal case of pneumonia caused by H7N7. However,
the exception is the highly pathogenic H5N1 virus, for which there
is no natural immunity in humans. The infidelity of the RNA
polymerase and the selective pressure of host immunity can lead to
the accumulation of mutations and change in surface antigenicity of
these proteins. This antigenic change is called antigenic drift. In
addition, as a result of its segmented genome, shuffling of gene
segments can occur if two different subtypes of influenza A virus
infect the same cell. For example, if a human H3N2 virus and an
avian H5N1 virus co-infect a human or other member of a mammalian
species, such an event can produce a novel H5N2. This novel virus
can then be efficiently transmitted from human to human because all
of most of the gene segments come from the human virus. Such
genetic reassortment would lead to a major antigen change, a
so-called antigenic shift, which would mean that most of the global
population would not have any neutralizing antibodies against the
reassortment virus. Such a situation, coupled with the high
mortality of influenza H5N1 pneumonia, is one of the most feared
scenarios in the field of public health.
[0066] Influenza virus hemagglutinin (HA) is the most variable
antigen of influenza virus, and is responsible for virus entry into
cells. It is synthesized as a trimeric precursor polypeptide HA0
which is post-translationally cleaved to two polypeptides HA1 and
HA2 linked by a single disulphide bond. The HA1 chain of HA is
responsible for the attachment of virus to the cell surface. HA2
mediates the fusion of viral and cell membranes in endosomes,
allowing the release of the ribonucleoprotein complex into the
cytoplasm. In contrast to HAL the HA2 molecule represents a
relatively conserved part of HA.
[0067] Both the HA1 and HA2 chains of HA are immunogenic and
antibodies reactive with both chains have been demonstrated after
natural infection in humans. While antibodies specific to HA1 are
mostly neutralizing, different mechanism of virus neutralization by
HA1 specific Mabs in vitro have been described including blocking
the receptor site on HA1, intracellular inhibition of virus-cell
fusion, or simultaneous attachment inhibition and virus-cell fusion
inhibition, depending on antibody concentration. Although less well
studied, inhibition of cell fusion by anti-HA2 antibodies has been
reported.
[0068] More than two decades ago, the HA molecule of the H3 subtype
was characterized by sequencing the HA of antigenic drift variants
and escape mutants, and the antigenic epitopes were mapped on the
molecule's three-dimensional structure. Since then, the antigenic
sites on H1, H2 and H5 of an avian pathogenic virus were mapped on
the three-dimensional structures of H3. After the outbreak of H5N1
infection in humans in Hong Kong in 1997 and the isolation of H9N2
virus from human cases in 1999, the X-ray structures of both
proteins were solved. However, antigenic drift of the 1997 swine
isolate (A/Duck/Singapore/3/97) that was used to solve the
structure, and more recently isolated highly pathogenic strains, is
significant. Indeed, there are 28 minor changes and two potentially
major changes between the swine isolate (A/Duck/Singapore/3/97) and
the HPAI H5N1 strain (A/Vietnam1203/04).
[0069] Phylogenetic analyses of the H5 HA genes from the 2004-2005
outbreak have shown two different lineages of HA genes, termed
clades 1 and 2. HPAI H5N1 strain (A/Vietnam1203/04) is a member of
clade 1. Viruses in each of these clades are distributed in
non-overlapping geographic regions of Asia. The H5N1 viruses from
Indochina are tightly clustered within clade 1, whereas H5N1
isolated from several surrounding countries are distinct from clade
1 isolates, and belong in a more divergent clade 2. Clade 1 viruses
were isolated from humans and birds in Vietnam, Thailand and
Cambodia but only from birds in Laos and Malaysia. The clade 2
viruses were found in viruses isolated exclusively from birds in
China, Indonesia, Japan, and South Korea. The most recent
epidemiologic studies analyzed 82 H5N1 viruses isolated from
poultry throughout Indonesia and Vietnam, as well as 11 human
isolates from southern Vietnam together with sequence data
available in public databases, to address questions relevant to
virus introduction, endemicity and evolution (Stevens, J. et al.
Structure of the uncleaved human H1 hemagglutinin from the extinct
1918 influenza virus. Science 303, 1866-70 (2004)). Phylogenetic
analysis showed that all viruses from Indonesia form a distinct
sublineage of H5N1 genotype Z viruses, suggesting that this
outbreak likely originated from a single introduction via spread
throughout the country during the past two years. Continued virus
activities in Indonesia were attributed to transmission via poultry
movement within the country, rather than through repeated
introductions by bird migration. Within Indonesia and Vietnam, H5N1
viruses have evolved over time into geographically distinct groups
within each country.
[0070] Recently, the structure of HA from A/Vietnam1203/4 was
solved. Comparison of its amino acid sequences with the HA genes
from HPAI 2004 and 2005 isolates from clade 1 and 2 viruses
identified 13 positions of antigenic variation that are mainly
clustered around the receptor binding domain, while the rest are
within the vestigual esterase domain. Regions of antigenic
variation have been identified in H1 and H3 serotypes (FIG. 13A).
For H1, these sites are designated Sa, Sb, Ca and Cb while for H3,
sites are designated A, B, C and D. Escape mutants of H5 HAs can be
clustered into three epitopes; site 1: an exposed loop (HA1
140-145) that overlaps antigenic sites A of H3 and Ca2 of H.sup.2;
site 2: HA1 residues 156 and 157 that corresponds to antigenic site
B in H3 serotypes; and 3) HA1 129-133, which is restricted to the
Sa site in H1 HAs and H9 serotypes. In the recent studies by Smith,
detection of positive selection at the amino acid level indicated
that eight residues in the HA proteins were under positive
selection (FIG. 13B). These residues include five in antigenic
sites A and E (positions 83, 86, 138, 140 and 141); two involved in
receptor binding (positions 129 and 175); and positions 156 is a
site for potential N-linked glycosylation that is near the
receptor-binding site. The results further revealed that three
residues in HA (Val 86, Ser 129 and Thr 156) were more frequently
observed in human isolates than in chicken or duck isolates and
likely represented early adaptation of H5N1 genotype Z to
humans.
[0071] Another important finding from these studies is that the
phylogenetic differences between the Indonesian and Vietnamese
sub-lineages was also reflected in significant differences in
antigenic cross-reactivity between these two group of viruses.
Specifically, viruses from Indonesia did not react to ferret
antisera against A/Vietnam1203/04, and representative viruses from
Vietnam did not react with ferret antisera against Indonesian
viruses IDN/5/06 and Dk/IDN/MS/04. These findings are in agreement
with earlier studies with immune human serum and human 1997 and
2003 H5N1 viruses that these strains were not only phylogenetically
but also antigenically distinct. Thus, natural variation as well as
escape mutants suggests that continued evolution of the virus
should impact the decision on which strain(s) should be used for
passive and active immunization.
[0072] High affinity, cross-subtype, broadly-neutralizing human
anti-HA mAbs have been identified. Specifically, a human Ab phage
display library and H5 hemagglutinin (HA) ectodomain was used to
select ten neutralizing mAbs (nAbs) with a remarkably broad range
among Group 1 influenza viruses, including the H5N1 "bird flu" and
the H1N1 "Spanish flu" and "Swine flu" strains. These nAbs inhibit
the post-attachment fusion process by recognizing a novel and
highly conserved neutralizing epitope within the stem region at a
point where key elements of the conformational change--the fusion
peptide and the exposed surface of helix .alpha.A--are brought into
close apposition. The crystal structure of one mAb (mAbF10) bound
to H5N1 HA reveals that only the heavy chain inserts into a highly
conserved pocket in the HA stem region, inhibiting the
conformational changes required for membrane fusion. It has been
discovered that nAbs targeting this pocket can provide broad
protection against both seasonal and pandemic influenza A
infections. The crystal structure further revealed that the epitope
to which the F10 mAb is defined by amino acid residues 18, 38, 39,
40 and 291 of HA1 and 18, 19, 20, 21, 38, 41, 42, 45, 49, 52, 53,
and 56 of HA2. This epitope is referred to herein as the F10
epitope. Structural and sequence analysis of all 16 HA subtypes
points to the existence of only two variants of this epitope,
corresponding to the two phylogenetic groupings of HA (Groups 1 and
2). This discovery indicates that a small cocktail of nAbs derived
from a subset of each group can provide broad protection against
both seasonal and pandemic influenza.
[0073] Remarkably, we repeatedly isolated nAbs that utilizes the
same VH germline gene, IGHV1-69*01, and encodes a CDR3 loop
containing a tyrosine at an equivalent position to Y102, from a
non-immune library. This indicates that broad anti-HA
cross-immunity pre-exists in the H5-naive population, possibly due
to previous exposure to H1, and, for library donors born before
1968, H2 subtypes. The recurrent use of this germline VH segment,
the commonality of the CDR3 tyrosine introduced through N insertion
and/or germline D gene assembly, and the promiscuous use of VL
genes by the discovered nAbs discovered indicate that the precursor
frequency of rearranged VH segments that could recognize this
epitope is significant. This indicates that with suitable exposure
to the F10 epitope identified here, these broad-spectrum nAbs can
be readily induced in vivo. These discoveries led to the disclosed
simple solution to provide universal protection against virus
subtypes in both groups.
[0074] Three unique anti-HA-1 scFvs were identified by sequencing
analysis of the 58 HA-1 positive clones. These scFvs were
designated as 38B and 1C. The VH and VL amino acid sequence of 2A
is shown in Table 2.
[0075] Ten unique anti-HA0 scFvs were identified by sequencing
analysis of the 97 HA0 positive clones. These scFvs were designated
as 7, 8, 10, 17, 40, 66, 80, 88, 90, and 98. Six different VH and
10 different VL genes were revealed. Some scFvs shared the same VH
gene. Five out of the six different VH genes belonged to the
IGHV1-69 gene family. Three out of ten VL genes were kappa
chain.
[0076] 2A scFv is a moderate neutralizing antibody, 38B and 1C are
non-neutralizing antibodies. Ten scFvs, 7, 8, 10, 17, 40, 66, 80,
88, 90, and 98 are potent neutralizing antibodies (Table 2). The
nucleic acid and amino acid sequence of the neutralizing influenza
antibodies are provided below:
TABLE-US-00002 TABLE 5A Antibody 2A Variable Region nucleic acid
sequences V.sub.H chain of 2A (SEQ ID NO: 41)
CAGGTGCAGCTGGTGCAGTCTGGGGCTGAGGTGAAGAAGCCTGGGTCCTCGGTGAAGGTCTCCTGCAAGGCTTC-
TGGAGG
CACCTTCAGTGACAATGCTATCAGCTGGGTGCGACAGGCCCCAGGACAAGGGCTTGAGTGGATGGGGGGCATCA-
TTCCTA
TCTTTGGAAAACCAAACTACGCACAGAAGTTCCAGGGCAGAGTCACGATTACTGCGGACGAATCCACGAGCACA-
GCCTAC
ATGGACCTGAGGAGCCTGAGATCTGAGGACACGGCCGTTTATTACTGTGCGAGAGATTCAGACGCGTATTACTA-
TGGTTC GGGGGGTATGGACGTCTGGGGCCAAGGCACCCTGGTCACCGTCTCCTCA V.sub.L
chain of 2A (SEQ ID NO: 44)
CTGCCTGTGCTGACTCAATCATCCTCTGCCTCTGCTTCCCTGGGATCCTCGGTCAAGCTCACCTGCACTCTGAG-
CAGTGG
GCATAGTAACTACATCATCGCATGGCATCAACAGCAGCCAGGGAAGGCCCCTCGGTACTTGATGAAGGTTAATA-
GTGATG
GCAGCCACACCAAGGGGGACGGGATCCCTGATCGCTTCTCAGGCTCCAGCTCTGGGGCTGACCGCTACCTCACC-
ATCTCC
AACCTCCAGTCTGAGGATGAGGCTAGTTATTTCTGTGAGACCTGGGACACTAAGATTCATGTCTTCGGAACTGG-
GACCAA GGTCTCCGTCCTCAG
TABLE-US-00003 TABLE 5B Antibody 2A Variable Region amino acid
sequences V.sub.H chain of 2A (SEQ ID NO: 43)
QVQLVQSGAEVKKPGSSVKVSCKASGGTFSDNAISWVRQAPGQGLEWMGGIIPIFGKPNYAQKFQGRVTITADE-
STSTAY MDLRSLRSEDTAVYYCARDSDAYYYGSGGMDVWGQGTLVTVSS V.sub.L chain of
2A (SEQ ID NO: 45)
LPVLTQSSSASASLGSSVKLTCTLSSGHSNYIIAWHQQQPGKAPRYLMKVNSDGSHTKGDGIPDRFSGSSSGAD-
RYLTIS NLQSEDEASYFCETWDTKIHVFGTGTKVSVL
TABLE-US-00004 TABLE 5C Antibody D7 Variable Region nucleic acid
sequences V.sub.H chain of D7 (SEQ ID NO: 46)
CAGGTGCAGCTGGTGCAGTCTGGGGCTGAGGTGAAGAAGCCTGGGTCCTCGGTGAAGGTCTCCTGCAAGGCTCC-
TGGAGG
TATCTTCAACACCAATGCTTTCAGCTGGGTCCGACAGGCCCCTGGACAAGGTCTTGAGTGGGTGGGAGGGGTCA-
TCCCTT
TGTTTCGAACAGCAAGCTACGCACAGAACGTCCAGGGCAGAGTCACCATTACCGCGGACGAATCCACGAACACA-
GCCTAC
ATGGAGCTTACCAGCCTGAGATCTGCGGACACGGCCGTGTATTACTGTGCGAGAAGTAGTGGTTACCATTTTAG-
GAGTCA CTTTGACTCCTGGGGCCTGGGAACCCTGGTCACCGTCTCCTCA V.sub.L chain of
D7 (SEQ ID NO: 50)
AATTTTATGCTGACTCAGCCCCACTCTGTGTCGGCGTCTCCGGGGAAGACGGTGACCATCTCCTGCACCGGCAG-
CAGTGG
CAACATTGCCGCCAACTATGTGCAGTGGTACCAACAACGTCCGGGCAGTGCCCCCACTACTGTGATCTATGAGG-
ATGACC
GAAGACCCTCTGGGGTCCCTGATCGGTTCTCTGGCTCCATCGACAGGTCCTCCAACTCTGCCTCCCTCACCATC-
TCAGGA
CTGAAGACTGAGGACGAGGCTGACTACTACTGTCAGACTTATGATACCAACAATCATGCTGTGTTCGGAGGAGG-
CACCCA CCTGACCGTCCTC
TABLE-US-00005 TABLE 5D Antibody H98 Variable Region nucleic acid
sequences V.sub.H chain of H98 (SEQ ID NO: 48)
CAGGTGCAGCTGGTGCAATCTGGGGCTGAGGTGAAGAAGCCTGGGTCCTCGGTGAAGGTCTCCTGCAAGGCTCC-
TGGAGG
TATCTTCAACACCAATGCTTTCAGCTGGGTCCGACAGGCCCCTGGACAAGGTCTTGAGTGGGTGGGAGGGGTCA-
TCCCTT
TGTTTCGAACAGCAAGCTACGCACAGAACGTCCAGGGCAGAGTCACCATTACCGCGGACGAATCCACGAACACA-
GCCTAC
ATGGAGCTTACCAGCCTGAGATCTGCGGACACGGCCGTGTATTACTGTGCGAGAAGTAGTGGTTACCATTTTAG-
GAGTCA CTTTGACTCCTGGGGCCTGGGAACCCTGGTCACCGTCTCCTCA V.sub.L chain of
H98 (SEQ ID NO: 52)
TCCTATGAGCTGACTCAGCCACCCTCAGCGTCTGGGAAACACGGGCAGAGGGTCACCATCTCTTGTTCTGGAGG-
CACCTC
CAACATCGGACGTAATCATGTTAACTGGTACCAGCAACTCCCAGGAACGGCCCCCAAACTCCTCATCTATAGTA-
ATGAAC
AGCGGCCCTCAGGGGTCCCTGACCGATTCTCTGGCTCCAAATCTGGCACCTCCGCCTCCCTGGCCGTGAGTGGG-
CTCCAG
TCTGAGGATGAGGCTGATTATTACTGTGCATCATGGGATGACAACTTGAGTGGTTGGGTGTTCGGCGGAGGGAC-
CAAGCT GACCGTCCTA
TABLE-US-00006 TABLE 5E Antibody D7 and H98 Variable Region chain
amino acid sequences V.sub.H chain of D7 and H98 (SEQ ID NO: 47)
QVQLVQSGAEVKKPGSSVKVSCKAPGGIFNTNAFSWVRQAPGQGLEWVGGVIPLFRTASYAQNVQGRVTITADE-
STNTAY MELTSLRSADTAVYYCARSSGYHFRSHFDSWGLGTLVTVSS V.sub.L chain of
D7 (SEQ ID NO: 49)
NFMLTQPHSVSASPGKTVTISCTGSSGNIAANYVQWYQQRPGSAPTTVIYEDDRRPSGVPDRFSGSIDRSSNSA-
SLTISG LKTEDEADYYCQTYDTNNHAVFGGGTHLTVL V.sub.L chain of H98 (SEQ ID
NO: 51)
SYELTQPPSASGKHGQRVTISCSGGTSNIGRNHVNWYQQLPGTAPKLLIYSNEQRPSGVPDRFSGSKSGTSASL-
AVSGLQ SEDEADYYCASWDDNLSGWVFGGGTKLTVL
TABLE-US-00007 TABLE 5F Antibody D8 Variable Region nucleic acid
sequences V.sub.H chain of D8 (SEQ ID NO: 54)
CAGGTGCAGCTGGTGCAGTCTGGGGCTGAGGTGAAGAAGCCTGGGTCCTCGGTGAAGGTCTCCTGCAAGGCTTC-
TGGAGG
CACCTTCAGCGCTTATGCTTTCACCTGGGTGCGGCAGGCCCCTGGACAAGGGCTTGAGTGGATGGGAGGCATCA-
CCGGAA
TGTTTGGCACAGCAAACTACGCACAGAAGTTCCAGGGCAGAGTCACGATTACCGCGGACGAACTCACGAGCACA-
GCCTAC
ATGGAGTTGAGCTCCCTGACATCTGAAGACACGGCCCTTTATTATTGTGCGAGAGGATTGTATTACTATGAGAG-
TAGTCT TGACTATTGGGGCCAGGGAACCCTGGTCACCGTCTCCTCAG V.sub.L chain of
D8 (SEQ ID NO: 58)
CAGTCTGTGCTGACTCAGCCACCCTCCGCGTCCGGGTCTCCTGGACAGTCAGTCACCATCTCCTGCACTGGAAC-
CAGCAG
TGACGTTGGTGGTTATAACTCTGTCTCCTGGTACCAACAGCACCCAGGCAAAGCCCCCAAACTCATGATTTATG-
AGGTCA
CTAAGCGGCCCTCAGGGGTCCCTGATCGCTTCTCTGCCTCCAAGTCTGGCAACACGGCCTCCCTGACCGTCTCT-
GGGCTC
CAGGCTGAGGATGAGGCTGATTATTTCTGCTGCTCATATGCAGGCCACAGTGCTTATGTCTTCGGAACTGGGAC-
CAAGGT CACCGTCCTG
TABLE-US-00008 TABLE 1G Antibody D80 Variable Region nucleic acid
sequences V.sub.H chain of D80 (SEQ ID NO: 56)
CAGGTGCAGCTGGTGCAGTCTGGGGCTGAGGTGAAGAAGCCTGGGTCCTCGGTGAAGGTCTCCTGCAGGGCTTC-
TGGAGG
CACCTTCAGCGCTTATGCTTTCACCTGGGTGCGGCAGGCCCCTGGACAAGGGCTTGAGTGGATGGGAGGCATCA-
CCGGAA
TGTTTGGCACAGCAAACTACGCACAGAAGTTCCAGGGCAGAGTCACGATTACCGCGGACGAACTCACGAGCACA-
GCCTAC
ATGGAGTTGAGCTCCCTGACATCTGAAGACACGGCCCTTTATTATTGTGCGAGAGGATTGTATTACTATGAGAG-
TAGTCT TGACTATTGGGGCCAGGGAACCCTGGTCACCGTCTCCTCAG V.sub.K chain of
D80 (SEQ ID NO: 60)
GAAATTGTGCTGACTCAGTCTCCAGGCACCCTGTCTTTGTCTCCAGGGGAAAGAGCCACCCTCTCCTGCAGGGC-
CAGTCA
GAGTCTTAGCAGCAAGTACTTAGCCTGGTATCAGCAGAAACCTGGCCAGGCTCCCAGGCTCCTCATCTATGGTG-
CATCCA
GCAGGGCCACTGGCATCCCAGACAGGTTCAGTGGCAGTGGGTCTGGGACAGACTTCACCCTCACCATCAGTAGA-
CTGGAG
CCTGAAGATTTTGCAGTGTATTCCTGTCAGCAGTATGATGGCGTACCTCGGACGTTCGGCCAAGGGACCACGGT-
GGAAAT CAAA
TABLE-US-00009 TABLE 5H Antibody D8 and D80 Variable Region chain
amino acid sequences V.sub.H chain of D8 and D80 (SEQ ID NO: 53)
QVQLVQSGAEVKKPGSSVKVSCKASGGTFSAYAFTWVRQAPGQGLEWMGGITGMFGTANYAQKFQGRVTITADE-
LTSTAY MELSSLTSEDTALYYCARGLYYYESSLDYWGQGTLVTVSS V.sub.L chain of D8
(SEQ ID NO: 55)
QSVLTQPPSASGSPGQSVTISCTGTSSDVGGYNSVSWYQQHPGKAPKLMIYEVTKRPSGVPDRFSASKSGNTAS-
LTVSGL QAEDEADYFCCSYAGHSAYVFGTGTKVTVL V.sub.K chain of D80 (SEQ ID
NO: 57)
EIVLTQSPGTLSLSPGERATLSCRASQSLSSKYLAWYQQKPGQAPRLLIYGASSRATGIPDRFSGSGSGTDFTL-
TISRLE PEDFAVYSCQQYDGVPRTFGQGTTVEIK
TABLE-US-00010 TABLE 1I Antibody F10 Variable Region nucleic acid
sequences V.sub.H chain of F10 (SEQ ID NO: 62)
CAGGTGCAGCTGGTGCAGTCAGGGGCTGAGGTGAAGAAGCCTGGGTCCTCGGTGAAGGTCTCCTGCACGTCCTC-
TGAAGT
CACCTTCAGTAGTTTTGCTATCAGCTGGGTGCGACAGGCCCCTGGACAAGGGCTTGAGTGGCTGGGAGGGATCA-
GCCCTA
TGTTTGGAACACCTAATTACGCGCAGAAGTTCCAAGGCAGAGTCACCATTACCGCGGACCAGTCCACGAGGACA-
GCCTAC
ATGGACCTGAGGAGCCTGAGATCTGAGGACACGGCCGTGTATTATTGTGCGAGATCTCCTTCTTACATTTGTTC-
TGGTGG AACCTGCGTCTTTGACCATTGGGGCCAGGGAACCCTGGTCACCGTCTCCTCA V.sub.L
chain of F10 (SEQ ID NO: 25)
CAGCCTGGGCTGACTCAGCCACCCTCGGTGTCCAAGGGCTTGAGACAGACCGCCACACTCACCTGCACTGGGAA-
CAGCAA
CAATGTTGGCAACCAAGGAGCAGCTTGGCTGCAGCAGCACCAGGGCCACCCTCCCAAACTCCTATCCTACAGGA-
ATAATG
ACCGGCCCTCAGGGATCTCAGAGAGATTCTCTGCATCCAGGTCAGGAAACACAGCCTCCCTGACCATTACTGGA-
CTCCAG
CCTGAGGACGAGGCTGACTATTACTGCTCAACATGGGACAGCAGCCTCAGTGCTGTGGTATTCGGCGGAGGGAC-
CAAGCT GACCGTCCTA
TABLE-US-00011 TABLE 5J Antibody E90 Variable Region nucleic acid
sequences V.sub.H chain of E90 (SEQ ID NO: 64)
CAGGTACAGCTGCAGCAGTCAGGGGCTGAGGTGAAGAAGCCTGGGTCCTCGGTGAAGGTCTCCTGCACGTCCTC-
TGAAGT
CACCTTCAGTAGTTTTGCTATCAGCTGGGTGCGACAGGCCCCTGGACAAGGGCTTGAGTGGCTGGGAGGGATCA-
GCCCTA
TGTTTGGAACACCTAATTACGCGCAGAAGTTCCAAGGCAGAGTCACCATTACCGCGGACCAGTCCACGAGGACA-
GCCTAC
ATGGACCTGAGGAGCCTGAGATCTGAGGACACGGCCGTGTATTATTGTGCGAGATCTCCTTCTTACATTTGTTC-
TGGTGG AACCTGCGTCTTTGACCATTGGGGCCAGGGAACCCTGGTCACCGTCTCCTCA V.sub.L
chain of E90 (SEQ ID NO: 27)
GACATCCAGATGACCCAGTCTCCATCCTCCCTGTCTGCATCTGTAGGAGACAGAGTCACCATCACTTGCCGGGC-
AAGTCA
GAGCATTAGCAGCTATTTAAATTGGTATCAGCAGAAACCAGGGAAAGCCCCTAAGCTCCTGATCTATGCTGCAT-
CCAGTT
TGCAAAGAGGGGTCCCATCAAGGTTCAGTGGCAGTGGATCTGGGACAGACTTCACTCTCACCATTAGCAGCCTG-
CAGCCT
GAAGATTTTGCAGTGTATTACTGTCAGCAGTATGATAGTTCACCGTACACTTTTGGCCAGGGGACCAAGGTAGA-
GATCAA A
TABLE-US-00012 TABLE 1K Antibody F10 and E90 Variable Region amino
acid sequences V.sub.H chain of F10 and E90 (SEQ ID NO: 59)
QVQLVQSGAEVKKPGSSVKVSCTSSEVTFSSFAISWVRQAPGQGLEWLGGISPMFGTPNYAQKFQGRVTITADQ-
STRTAY MDLRSLRSEDTAVYYCARSPSYICSGGTCVFDHWGQGTLVTVSS V.sub.L chain
of F10 (SEQ ID NO: 61)
QPGLTQPPSVSKGLRQTATLTCTGNSNNVGNQGAAWLQQHQGHPPKLLSYRNNDRPSGISERFSASRSGNTASL-
TITGLQ PEDEADYYCSTWDSSLSAVVFGGGTKLTVL V.sub.L chain of E90 (SEQ ID
NO: 63)
DIQMTQSPSSLSASVGDRVTITCRASQSISSYLNWYQQKPGKAPKLLIYAASSLQRGVPSRFSGSGSGTDFTLT-
ISSLQP EDFAVYYCQQYDSSPYTFGQGTKVEIK
TABLE-US-00013 TABLE 5L Antibody G17 Variable Region nucleic acid
sequences V.sub.H chain of G17 (SEQ ID NO: 29)
CAGGTGCAGCTGGTGCAATCTGGGGCTGAAGTGAAGAAGCCTGGGGCCTCAGTGAAGGTCTCCTGCAAGACTTC-
TGGAGT
CACCTTCAGCAGCTATGCTATCAGTTGGGTGCGACAGGCCCCTGGACAAGGGCTTGAGTGGATGGGAGGGATCA-
TCGGTG
TCTTTGGTGTACCAAAGTACGCGCAGAACTTCCAGGGCAGAGTCACAATTACCGCGGACAAACCGACGAGTACA-
GTCTAC
ATGGAGCTGAACAGCCTGAGAGCTGAGGACACGGCCGTGTATTACTGTGCGAGAGAGCCCGGGTACTACGTAGG-
AAAGAA TGGTTTTGATGTCTGGGGCCAAGGGACAATGGTCACCGTCTCTTCA V.sub.L chain
of G17 (SEQ ID NO: 31)
TCCTATGAGCTGACTCAGCCACCCTCGGTGTCCAAGGGCTTGAGACAGACCGCCATACTCACCTGCACTGGAGA-
CAGCAA
CAATGTTGGCCACCAAGGTACAGCTTGGCTGCAACAACACCAGGGCCACCCTCCCAAACTCCTATCCTACAGGA-
ATGGCA
ACCGGCCCTCAGGGATCTCAGAGAGATTCTCTGCATCCAGGTCAGGAAATACAGCCTCCCTGACCATTATTGGA-
CTCCAG
CCTGAGGACGAGGCTGACTACTACTGCTCAGTATGGGACAGCAGCCTCAGTGCCTGGGTGTTCGGCGGAGGGAC-
CAAGCT GACCGTCCTA
TABLE-US-00014 TABLE 5M Antibody G17 Variable Region amino acid
sequences V.sub.H chain of G17 (SEQ ID NO: 24)
QVQLVQSGAEVKKPGASVKVSCKTSGVTFSSYAISWVRQAPGQGLEWMGGIIGVFGVPKYAQNFQGRVTITADK-
PTSTVY MELNSLRAEDTAVYYCAREPGYYVGKNGFDVWGQGTMVTVSS V.sub.L chain of
G17 (SEQ ID NO: 26)
SYELTQPPSVSKGLRQTAILTCTGDSNNVGHQGTAWLQQHQGHPPKLLSYRNGNRPSGISERFSASRSGNTASL-
TIIGLQ PEDEADYYCSVWDSSLSAWVFGGGTKLTVL
TABLE-US-00015 TABLE 5N Antibody H40 Variable Region nucleic acid
sequences V.sub.H chain of H40 (SEQ ID NO: 33)
CAGGTGCAGCTGGTGCAGTCTGGGGCTGAGGTGAGGAAGCCTGGGGCCTCAGTGAAGGTCTCATGTAAGGCTTC-
TGGATA
CACCTTCACCGGTTATTATATTCACTGGGTGCGACAGGCCCCTGGACAAGGACTTGAGTGGATGGGTTGGATCA-
ACCCTA
TGACTGGTGGCACAAACTATGCACAGAAGTTTCAGGTCTGGGTCACCATGACCCGGGACACGTCCATCAACACA-
GCCTAC
ATGGAGGTGAGCAGGCTGACATCTGACGACACGGCCGTGTATTACTGTGCGAGGGGGGCTTCCGTATTACGATA-
TTTTGA CTGGCAGCCCGAGGCTCTTGATATCTGGGGCCTCGGGACCACGGTCACCGTCTCCTCA
V.sub.L chain of H40 (SEQ ID NO: 35)
CAGCCTGTGCTGACTCAGCCACCCTCGGTGTCAGTGGCCCCAGGACAGACGGCCAGCATTCCCTGTGGGGGGAA-
CAACAT
TGGAGGCTACAGTGTACACTGGTACCAACAAAAGCCGGGCCAGGCCCCCCTCTTGGTCATTTATGACGATAAAG-
ACCGGC
CCTCAGGGATCCCTGAGCGATTCTCTGGCGCCAACTCTGGGAGCACGGCCACCCTGACAATCAGCAGGGTCGAA-
GCCGGG
GATGAGGGCGACTACTACTGTCAGGTGTGGGATAGTGGTAATGATCGTCCGCTGTTCGGCGGAGGGACCAAGCT-
GACCGT CCTA
TABLE-US-00016 TABLE 5O Antibody H40 Variable Region amino acid
sequences V.sub.H chain of H40 (SEQ ID NO: 28)
QVQLVQSGAEVRKPGASVKVSCKASGYTFTGYYIHWVRQAPGQGLEWMGWINPMTGGTNYAQKFQVWVTMTRDT-
SINTAY MEVSRLTSDDTAVYYCARGASVLRYFDWQPEALDIWGLGTTVTVSS V.sub.L chain
of H40 (SEQ ID NO: 30)
QPVLTQPPSVSVAPGQTASIPCGGNNIGGYSVHWYQQKPGQAPLLVIYDDKDRPSGIPERFSGANSGSTATLTI-
SRVEAG DEGDYYCQVWDSGNDRPLFGGGTKLTVL
TABLE-US-00017 TABLE 5P Antibody A66 Variable Region nucleic acid
sequences V.sub.H chain of A66 (SEQ ID NO: 37)
CAGGTGCAGCTGGTGCAGTCTGGGGCTGAAGTGAAGAAGCCTGGCTCCTCGGTGAAGGTTTCCTGCAAGGCTTC-
TGGAGG
CCCCTTCAGCATGACTGCTTTCACCTGGCTGCGACAGGCCCCTGGACAAGGGCTTGAGTGGATGGGTGGGATCA-
GCCCTA
TCTTTCGTACACCGAAGTACGCACAGAAGTTCCAGGGCAGAGTCACGATTACCGCGGACGAATCCACGAACACA-
GCCAAC
ATGGAGCTGACCAGCCTGAAATCTGAGGACACGGCCGTGTATTACTGTGCGAGAACCCTTTCCTCCTACCAACC-
GAATAA TGATGCTTTTGCTATCTGGGGCCAAGGGACAATGGTCACCGTCTCTTCA V.sub.K
chain of A66 (SEQ ID NO: 39)
GAAATTGTGTTGACGCAGTCTCCAGCCACCCTGTCTTTGTCTCCAGGGGAAAGAGCCACCCTCTCCTGCAGGGC-
CAGTCA
GAGTGTTAGCAGCTACTTAGCCTGGTACCAACAGAAACCTGGCCAGGCTCCCAGGCTCCTCATCTATGATGCAT-
CCAACA
GGGCCACTGGCATCCCAGCCAGGTTCAGTGGCAGTGGGTCTGGGACAGACTTCACTCTCACCATCAGCAGACTG-
GAGCCT
GAAGATTTTGCAGTCTATTTCTGTCAGCAGTATGGTAGCTCACCTCAATTCGGCCAAGGGACACGACTGGAGAT-
TAAA
TABLE-US-00018 TABLE 5Q Antibody A66 Variable Region amino acid
sequences V.sub.H chain of A66 (SEQ ID NO: 32)
QVQLVQSGAEVKKPGSSVKVSCKASGGPFSMTAFTWLRQAPGQGLEWMGGISPIFRTPKYAQKFQGRVTITADE-
STNTAN MELTSLKSEDTAVYYCARTLSSYQPNNDAFAIWGQGTMVTVSS V.sub.K chain of
A66 (SEQ ID NO: 34)
EIVLTQSPATLSLSPGERATLSCRASQSVSSYLAWYQQKPGQAPRLLIYDASNRATGIPARFSGSGSGTDFTLT-
ISRLEP EDFAVYFCQQYGSSPQFGQGTRLEIK
TABLE-US-00019 TABLE 5R Antibody E88 Variable Region nucleic acid
sequences V.sub.H chain of E88 (SEQ ID NO: 40)
CAGGTGCAGCTGGTGCAGTCTGGGGCTGAAGTGAAGAAGCCTGGCTCCTCGGTGAAGGTTTCCTGCAAGGCTTC-
TGGAGG
CCCCTTCAGCATGACTGCTTTCACCTGGCTGCGACAGGCCCCTGGACAAGGGCTTGAGTGGATGGGTGGGATCA-
GCCCTA
TCTTTCGTACACCGAAGTACGCACAGAAGTTCCAGGGCAGAGTCACGATTACCGCGGACGAATCCACGAACACA-
GCCAAC
ATGGAGCTGACCAGCCTGAAATCTGAGGACACGGCCGTGTATTACTGTGCGAGAACCCTTTCCTCCTACCAACC-
GAATAA TGATGCTTTTGCTATCTGGGGCCAAGGGACAATGGTCACCGTCTCTTCA V.sub.L
chain of E88 (SEQ ID NO: 42)
CTGCCTGTGCTGACTCAGCCACCCTCAGCGTCTGGGACCCCCGGGCAGAGGGTCACCATCTCTTGTTCTGGAAG-
CAGCTC
CAACATCGGAAGTAATACTGTAAACTGGTACCAGCAGCTCCCAGGAACGGCCCCCAAACTCCTCATCTATAGTA-
ATAATC
AGCGGCCCTCAGGGGTCCCTGACCGATTCTCTGGCTCCAGGTCAGGCACCTCAGCCTCCCTGGCCATCATTGGA-
CTCCGG
CCTGAGGATGAAGCTGATTATTACTGTCAGTCGTATGACAGCAGGCTCAGTGCTTCTCTCTTCGGAACTGGGAC-
CACGGT CACCGTCCTC
TABLE-US-00020 TABLE 5S Antibody E88 Variable Region amino acid
sequences V.sub.H chain of E88 (SEQ ID NO: 36)
QVQLVQSGAEVKKPGSSVKVSCKASGGPFSMTAFTWLRQAPGQGLEWMGGISPIFRTPKYAQKFQGRVTITADE-
STNTAN MELTSLKSEDTAVYYCARTLSSYQPNNDAFAIWGQGTMVTVSS V.sub.L chain of
E88 (SEQ ID NO: 38)
LPVLTQPPSASGTPGQRVTISCSGSSSNIGSNTVNWYQQLPGTAPKLLIYSNNQRPSGVPDRFSGSRSGTSASL-
AIIGLR PEDEADYYCQSYDSRLSASLFGTGTTVTVL
[0077] The amino acid sequences of the heavy and light chain
complementary determining regions of the neutralizing influenza
antibodies are shown in Tables 2 and 6. Sequences in Table 6 are,
from left to right then top to bottom, SEQ ID NO:65-124.
TABLE-US-00021 TABLE 6 Antibody CDR1 CDR2 CDR3 CONSENSUS SYAFS
GIIPMFGTPNYAQKFQG SSGYYYG GGFDV D7/H98VH TNAFS GVIPLFRTASYAQNVQG
SSGYHFGRSHFDS D8/D80VH AYAFT GIIGMFGTANYAQKFQG GLYYYESSLDY F10/90VH
SFAIS GISPMFGTPNYAQKFQG SPSYICSGGTCVFDH G17VH SYAIS
GIIGVFGVPKYAQKFQG EPGYYVGKNGFDV H40VH GYYIH WINPMTGGTNYAQKFQV
GASVLRYFDWQPEALDI A66VH MTAFT GISPIFRTPKYAQKFQG TLSSYQPNNDAFAI 2AVH
DNAIS GIIPIFGKPNYAQKFQG DSDAYYYGSGGMDV CONSENSUS TGSSSNIGNYVA
SNSDRPS QSYDSLSAYV D7VL TGSSSNIAANYVQ EDDRRPS QSYDTNNHAV DD8VL
TGTSSDVGGYNSVS EVTKRPS CSYAGHSAYV F10VL TGNSNNVGNQGAA RNNDRPS
STWDSSLSAVV G17VH TGDSNNVGHQGTA RNGNRPS SVWDSSLSAWV H40VH
GGNNIGGYSVH DDKDRPS QVWDSGNDRPL A66VH RASQSVSSYLA DASNRAT QQYGSSPQV
D80VL RASQSLSSKYLA GASSRAT QQYDGVPRT E88VL TGSSSNIGNYVA SNNQRPS
QSYDSRLSASL E90VK SGSSSNIGSNTVN AASSLQR QQYDSSPYT H98VL RASQSISSYLN
SNEQRPS ASWDDNLSGWV 2AVL TLSSGHSNYIIA VNSDGSHTKGD ETWDTKIHV
[0078] Those skilled in the art will recognize that additional
scFvs and monoclonal antibodies having different binding affinities
can also be therapeutically effective. For example, antibodies and
scFvs having binding affinities ranging from about 1 pM to about
200 mM can also be therapeutically effective.
[0079] Disclosed are materials, compositions, and components that
can be used for, can be used in conjunction with, can be used in
preparation for, or are products of the disclosed method and
compositions. These and other materials are disclosed herein, and
it is understood that when combinations, subsets, interactions,
groups, etc. of these materials are disclosed that while specific
reference of each various individual and collective combinations
and permutation of these compounds may not be explicitly disclosed,
each is specifically contemplated and described herein. For
example, if an antibody is disclosed and discussed and a number of
modifications that can be made to a number of molecules including
the antibody are discussed, each and every combination and
permutation of antibody and the modifications that are possible are
specifically contemplated unless specifically indicated to the
contrary. Thus, if a class of molecules A, B, and C are disclosed
as well as a class of molecules D, E, and F and an example of a
combination molecule, A-D is disclosed, then even if each is not
individually recited, each is individually and collectively
contemplated. Thus, is this example, each of the combinations A-E,
A-F, B-D, B-E, B-F, C-D, C-E, and C-F are specifically contemplated
and should be considered disclosed from disclosure of A, B, and C;
D, E, and F; and the example combination A-D. Likewise, any subset
or combination of these is also specifically contemplated and
disclosed. Thus, for example, the sub-group of A-E, B-F, and C-E
are specifically contemplated and should be considered disclosed
from disclosure of A, B, and C; D, E, and F; and the example
combination A-D. This concept applies to all aspects of this
application including, but not limited to, steps in methods of
making and using the disclosed compositions. Thus, if there are a
variety of additional steps that can be performed it is understood
that each of these additional steps can be performed with any
specific embodiment or combination of embodiments of the disclosed
methods, and that each such combination is specifically
contemplated and should be considered disclosed.
[0080] Disclosed are antibodies that bind to the stem region of
influenza hemagglutinin in the neutral pH conformation. Such
antibodies can be referred to herein as HA stem antibodies. For
example, disclosed are antibodies that bind the epitope of
influenza hemagglutinin bound by antibody F10. For example,
disclosed are antibodies that bind the epitope of influenza
hemagglutinin in the neutral pH conformation defined by amino acid
residues 18, 38, 39, 40 and 291 of HA1 and 18, 19, 20, 21, 38, 41,
42, 45, 49, 52, 53, and 56 of HA2. For example, disclosed are
antibodies that bind the epitope of influenza hemagglutinin in the
neutral pH conformation defined by amino acid residues 17, 18, 38,
39, 40 and 291 of HA1 and 18, 19, 20, 21, 38, 41, 42, 45, 49, 52,
53, 56, and 111 of HA2. For example, disclosed are antibodies that
bind to every subtype within an influenza virus group.
[0081] In some forms, the antibody can compete with antibody F10
for binding to hemagglutinin. In some forms, the antibody can have
a VH CDR2 sequence that is the same as the VH CDR2 sequence of
antibody D7, D8, F10, G17, H40 or A66 or of the consensus VH
sequence SEQ ID NO:1. In some forms, the antibody can have a VH
CDR3 sequence that is the same as the VH CDR3 sequence of antibody
D7, D8, F10, G17, H40 or A66 or of the consensus VH sequence SEQ ID
NO:1. In some forms, the antibody can have a VH CDR1 sequence that
is the same as the VH CDR1 sequence of antibody D7, D8, F10, G17,
H40 or A66 or of the consensus VH sequence SEQ ID NO:1. In some
forms, the antibody can have a VH sequence that is the same as the
VH sequence of antibody D7, D8, F10, G17, H40 or A66 or of the
consensus VH sequence SEQ ID NO:1. In some forms, the antibody can
have a VL sequence that is the same as the VL sequence of antibody
D7, D8, F10, G17, H40, A66, D80, E88, E90, or H98 or of the
consensus VL sequence SEQ ID NO:2. In some forms, the antibody can
have any combination of the VH FR1, VH CDR1, VH FR2, VH CDR2, VH
FR3, VH CDR3, and VH FR4 sequences of antibodies D7, D8, F10, G17,
H40 and A66 and the consensus VH sequence SEQ ID NO:1, and any
combination of the VL FR1, VL CDR1, VL FR2, VL CDR2, VL FR3, VL
CDR3, and VL FR4 sequences of antibodies D7, D8, F10, G17, H40,
A66, D80, E88, E90, and H98 and the consensus VL sequence SEQ ID
NO:2. In some forms, the antibody can prevent or inhibit virus-host
membrane fusion. In some forms, the antibody can prevent or inhibit
cell fusion mediated by cell surface-expressed influenza
hemagglutinin.
[0082] Also disclosed are antibodies identified or produced in
disclosed methods. For example, disclosed is a method, the method
comprising screening antibodies reactive to hemagglutinin for
binding to hemagglutinin immobilized on a surface, thereby
identifying antibodies of interest. For example, disclosed is a
method comprising screening antibodies reactive to hemagglutinin
for binding to the stem region of influenza hemagglutinin in the
neutral pH conformation in isolation from the head region of
hemagglutinin, thereby identifying antibodies of interest. For
example, disclosed is a method comprising screening antibodies
reactive to hemagglutinin for binding to influenza hemagglutinin in
the neutral pH conformation in isolation from other components of
influenza virus, wherein the head region of the hemagglutinin is
modified to reduce the antigenicity of the head region, thereby
identifying antibodies of interest.
[0083] Also disclosed are different forms of hemagglutinins. These
forms of hemagglutinins are useful in the preparation of vaccines
for influenza. For example, disclosed is the stem region of
influenza hemagglutinin in the neutral pH conformation in isolation
from other components of influenza virus. For example, disclosed is
the stem region of influenza hemagglutinin in the neutral pH
conformation in isolation from the head region of hemagglutinin.
For example, disclosed is influenza hemagglutinin in the neutral pH
conformation in isolation from other components of influenza virus,
wherein the head region of the hemagglutinin is modified to reduce
the antigenicity of the head region. In some forms, the head region
of the hemagglutinin can be modified by removing or replacing
glycosylation sites. In some forms, the head region of the
hemagglutinin can be modified by adding glycosylation sites. In
some forms, the head region of the hemagglutinin can be modified by
removing all or a portion of the head region. Also disclosed is
influenza hemagglutinin bound to s surface, solid support or
substrate.
[0084] In some forms, the disclosed antibodies and disclosed
hemagglutinins can produce an immune reaction in a subject, i.e.
immunogenic. The disclosed antibodies and disclosed hemagglutinins
are formulated in compositions suitable for the use as a vaccine.
For example, the disclosed hemagglutinins are used for active
immunization of a subject to induce an immune reaction in a
subject. Alternatively, the disclosed antibodies are used for the
passive immunization of a subject to provide immediate protection
from an influenza infection.
[0085] For example, in some forms, the subject can produce an
immune response that prevents or reduces the severity of an
influenza infection. In some forms, the immune response can be
reactive to influenza viruses within a subtype. In some forms, the
immune response can be reactive to influenza viruses in each
subtype within a cluster. In some forms, the immune response can be
reactive to influenza viruses in each cluster within a group. In
some forms, the immune response can be reactive to all influenza
viruses in each subtype within a group. In some forms, the immune
response can be reactive to influenza viruses within group 1.
[0086] In some forms, the antibodies can be identified as competing
with antibody F10 for binding to hemagglutinin, thereby identifying
F10-competing antibodies. In some forms, the hemagglutinin can be
hemagglutinin from a group 2 influenza virus. In some forms, the
hemagglutinin can be hemagglutinin from a group 1 influenza virus.
Also disclosed are antibodies produced by the disclosed methods.
Also disclosed are antibodies identified by the disclosed
methods.
A. Antibodies
[0087] Disclosed are antibodies that bind to the stem region of
influenza hemagglutinin in the neutral pH conformation. Such
antibodies can be referred to herein as HA stem antibodies. For
example, disclosed are antibodies that bind the epitope of
influenza hemagglutinin bound by antibody F10. For example,
disclosed are antibodies that bind the epitope of influenza
hemagglutinin in the neutral pH conformation defined by amino acid
residues 18, 38, 39, 40 and 291 of HA1 and 18, 19, 20, 21, 38, 41,
42, 45, 49, 52, 53, and 56 of HA2. For example, disclosed are
antibodies that bind the epitope of influenza hemagglutinin in the
neutral pH conformation defined by amino acid residues 17, 18, 38,
39, 40 and 291 of HA1 and 18, 19, 20, 21, 38, 41, 42, 45, 49, 52,
53, 56, and 111 of HA2. For example, disclosed are antibodies that
bind to every subtype within an influenza virus group. Examples of
HA stem antibodies include antibodies D7, D8, F10, G17, H40, A66,
H98, D80, E90, and E8.
[0088] The terms "antibody" and "antibodies" are used herein in a
broad sense and includes both polyclonal and monoclonal antibodies.
In addition to intact immunoglobulin molecules, also included in
the term "antibodies" are fragments or polymers of those
immunoglobulin molecules, and human or humanized versions of
immunoglobulin molecules or fragments thereof, and can be chosen
for their ability to interact with hemagglutinin. Thus, antibody
refers to immunoglobulin molecules and immunologically active
portions of immunoglobulin (Ig) molecules, i.e., molecules that
contain an antigen binding site that specifically binds
(immunoreacts with) an antigen. Antibodies include, but are not
limited to, polyclonal, monoclonal, chimeric, dAb (domain
antibody), single chain, Fab, Fab' and F(ab')2 fragments, scFvs,
and Fab expression libraries.
[0089] The antibodies can be tested for their desired activity
using the in vitro assays described herein, or by analogous
methods, after which their in vivo therapeutic and/or prophylactic
activities are tested according to known clinical testing
methods.
[0090] The disclosed antibodies or antigen-binding fragments,
variants, or derivatives thereof include, but are not limited to,
polyclonal, monoclonal, multispecific, human, humanized,
primatized, or chimeric antibodies, single chain antibodies,
epitope-binding fragments, e.g., Fab, Fab' and F(ab').sub.2, Fd,
Fvs, single-chain Fvs (scFv), single-chain antibodies,
disulfide-linked Fvs (sdFv), fragments comprising either a V.sub.L
or V.sub.H domain, fragments produced by a Fab expression library,
and anti-idiotypic (anti-Id) antibodies (including, e.g., anti-Id
antibodies to HA stem antibodies disclosed herein). ScFv molecules
are known in the art and are described, e.g., in U.S. Pat. No.
5,892,019. Unless the context clearly indicates otherwise, the
terms "antibody" and "antibodies" include intact immunoglobulin
molecules and any epitope- or antigen-binding fragments, variants,
or derivatives thereof described herein or known in the art. The
disclosed immunoglobulin or antibody molecules can be of any type
(e.g., IgG, IgE, IgM, IgD, IgA, and IgY), class (e.g., IgG1, IgG2,
IgG3, IgG4, IgA1 and IgA2) or subclass of immunoglobulin.
[0091] A single chain Fv ("scFv") polypeptide molecule is a
covalently linked VH::VL heterodimer, which can be expressed from a
gene fusion including VH- and VL-encoding genes linked by a
peptide-encoding linker (See Huston et al. (1988) Proc Nat Acad Sci
USA 85(16):5879-5883). A number of methods have been described to
discern chemical structures for converting the naturally
aggregated, but chemically separated, light and heavy polypeptide
chains from an antibody V region into an scFv molecule, which will
fold into a three dimensional structure substantially similar to
the structure of an antigen binding site. See, e.g., U.S. Pat. Nos.
5,091,513; 5,132,405; and 4,946,778.
[0092] Very large naive human scFv libraries have been and can be
created to offer a large source of rearranged antibody genes
against a plethora of target molecules. Smaller libraries can be
constructed from individuals with infectious diseases in order to
isolate disease-specific antibodies. (See Barbas et al., Proc.
Natl. Acad. Sci. USA 89:9339-43 (1992); Zebedee et al., Proc. Natl.
Acad. Sci. USA 89:3175-79 (1992)).
[0093] The term "antigen binding site," or "binding portion" refers
to the part of the immunoglobulin molecule that participates in
antigen binding. The antigen binding site is formed by amino acid
residues of the N-terminal variable ("V") regions of the heavy
("H") and light ("L") chains. Three highly divergent stretches
within the V regions of the heavy and light chains, referred to as
"hypervariable regions," are interposed between more conserved
flanking stretches known as "framework regions," or "FRs". Thus,
the term "FR" refers to amino acid sequences which are naturally
found between, and adjacent to, hypervariable regions in
immunoglobulins. In an antibody molecule, the three hypervariable
regions of a light chain and the three hypervariable regions of a
heavy chain are disposed relative to each other in three
dimensional space to form an antigen-binding surface. The
antigen-binding surface is complementary to the three-dimensional
surface of a bound antigen, and the three hypervariable regions of
each of the heavy and light chains are referred to as
"complementarity-determining regions," or "CDRs."
[0094] Various procedures known within the art can be used for the
production of polyclonal or monoclonal antibodies directed against
a protein (such as the disclosed hemagglutinin compositions), or
against derivatives, fragments, analogs homologs or orthologs
thereof (See, for example, Antibodies: A Laboratory Manual, Harlow
E, and Lane D, 1988, Cold Spring Harbor Laboratory Press, Cold
Spring Harbor, N.Y., incorporated herein by reference).
[0095] Antibodies can be purified by well-known techniques, such as
affinity chromatography using protein A or protein G, which provide
primarily the IgG fraction of immune serum. Subsequently, or
alternatively, the specific antigen which is the target of the
immunoglobulin sought, or an epitope thereof, can be immobilized on
a column to purify the immune specific antibody by immunoaffinity
chromatography. Purification of immunoglobulins is discussed, for
example, by D. Wilkinson (The Scientist, published by The
Scientist, Inc., Philadelphia Pa., Vol. 14, No. 8 (Apr. 17, 2000),
pp. 25-28).
[0096] The terms "monoclonal antibody," "mAb" and "monoclonal
antibody composition" as used herein refers to an antibody obtained
from a substantially homogeneous population of antibodies, i.e.,
the individual antibodies within the population are identical
except for possible naturally occurring mutations that may be
present in a small subset of the antibody molecules. Thus, for
example, "monoclonal antibody" can refer to a population of
antibody molecules that contain only one molecular species of
antibody molecule consisting of a unique light chain gene product
and a unique heavy chain gene product. In particular, the
complementarity determining regions (CDRs) of the monoclonal
antibody can be identical in all the molecules of the population.
mAbs contain an antigen binding site capable of immunoreacting with
a particular epitope of the antigen characterized by a unique
binding affinity for it.
[0097] The monoclonal antibodies herein specifically include
"chimeric" antibodies in which a portion of the heavy and/or light
chain is identical with or homologous to corresponding sequences in
antibodies derived from a particular species or belonging to a
particular antibody class or subclass, while the remainder of the
chain(s) is identical with or homologous to corresponding sequences
in antibodies derived from another species or belonging to another
antibody class or subclass, as well as fragments of such
antibodies, as long as they exhibit the desired activity (See, U.S.
Pat. No. 4,816,567 and Morrison et al., Proc. Natl. Acad. Sci. USA,
81:6851-6855 (1984)).
[0098] The disclosed monoclonal antibodies can be made using any
procedure which produces monoclonal antibodies. For example,
disclosed monoclonal antibodies can be prepared using hybridoma
methods, such as those described by Kohler and Milstein, Nature,
256:495 (1975). In a hybridoma method, a mouse or other appropriate
host animal is typically immunized with an immunizing agent to
elicit lymphocytes that produce or are capable of producing
antibodies that will specifically bind to the immunizing agent.
Alternatively, the lymphocytes can be immunized in vitro. For
example, monoclonal antibodies can be prepared using hybridoma
methods, such as those described by Kohler and Milstein, Nature,
256:495 (1975). In a hybridoma method, a mouse, hamster, or other
appropriate host animal, is typically immunized with an immunizing
agent to elicit lymphocytes that produce or are capable of
producing antibodies that will specifically bind to the immunizing
agent.
[0099] Cells expressing cell surface localized versions of these
proteins can be used to immunize mice, rats or other species.
Traditionally, the generation of monoclonal antibodies has depended
on the availability of purified protein or peptides for use as the
immunogen. More recently DNA based immunizations have been used to
elicit strong immune responses and generate monoclonal antibodies.
In this approach, DNA-based immunization can be used, wherein DNA
encoding the epitope or antigen of interest (hemagglutinin, for
example) expressed either alone or as a fusion protein with human
IgG1 or an epitope tag is injected into the host animal according
to methods known in the art (e.g., Kilpatrick K E, et al.
Hybridoma. 1998 December; 17(6):569-76; Kilpatrick K E et al.
Hybridoma. 2000 August; 19(4):297-302, which are incorporated
herein by referenced in full for the methods of antibody
production) and as described in the examples. In particular, a
hemagglutinin composition comprising the stem region in properly
folded form can be used.
[0100] An alternate approach to immunizations with either purified
protein or DNA is to use antigen expressed in baculovirus. The
advantages to this system include ease of generation, high levels
of expression, and post-translational modifications that are highly
similar to those seen in mammalian systems. Use of this system
involves expressing the epitope or antigen of interest
(hemagglutinin, for example) as fusion proteins with a signal
sequence fragment. The antigen can be produced by inserting a gene
fragment in-frame between the signal sequence and the mature
protein domain of hemagglutinin (or an engineered portion of
hemagglutinin) nucleotide sequence. This results in the display of
the foreign proteins on the surface of the virion. This method
allows immunization with whole virus, eliminating the need for
purification of target antigens.
[0101] Generally, either peripheral blood lymphocytes ("PBLs") are
used in methods of producing monoclonal antibodies if cells of
human origin are desired, or spleen cells or lymph node cells are
used if non-human mammalian sources are desired. The lymphocytes
can then be fused with an immortalized cell line using a suitable
fusing agent, such as polyethylene glycol, to form a hybridoma cell
(Goding, "Monoclonal Antibodies: Principles and Practice" Academic
Press, (1986) pp. 59-103). Immortalized cell lines are usually
transformed mammalian cells, including myeloma cells of rodent,
bovine, equine, and human origin. Usually, rat or mouse myeloma
cell lines are employed. The hybridoma cells can be cultured in a
suitable culture medium that can, for example, contain one or more
substances that inhibit the growth or survival of the unfused,
immortalized cells. For example, if the parental cells lack the
enzyme hypoxanthine guanine phosphoribosyl transferase (HGPRT or
HPRT), the culture medium for the hybridomas typically will include
hypoxanthine, aminopterin, and thymidine ("HAT medium"), which
substances prevent the growth of HGPRT-deficient cells. Useful
immortalized cell lines are those that fuse efficiently, support
stable high level expression of antibody by the selected
antibody-producing cells, and are sensitive to a medium such as HAT
medium. An example of useful immortalized cell lines are murine
myeloma lines, which can be obtained, for instance, from the Salk
Institute Cell Distribution Center, San Diego, Calif. and the
American Type Culture Collection, Rockville, Md. 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); Brodeur et al., "Monoclonal Antibody Production
Techniques and Applications" Marcel Dekker, Inc., New York, (1987)
pp. 51-63). The culture medium in which the hybridoma cells are
cultured can then be assayed for the presence of monoclonal
antibodies directed against hemagglutinin stem region. In some
forms, the binding specificity of monoclonal antibodies produced by
the hybridoma cells can be determined by immunoprecipitation or by
an in vitro binding assay, such as radioimmunoassay (RIA) or
enzyme-linked immunoabsorbent assay (ELISA). Such techniques and
assays are known in the art, and are described further in the
Examples below or in Harlow and Lane "Antibodies, A Laboratory
Manual" Cold Spring Harbor Publications, New York, (1988). Useful
techniques for identifying and characterizing the binding of HA
stem antibodies are described in the examples. For example,
antibodies can be screened for binding to hemagglutinin bound to or
immobilized on a surface.
[0102] After the desired hybridoma cells are identified, the clones
can be subcloned by limiting dilution or FACS sorting procedures
and grown by standard methods. Suitable culture media for this
purpose include, for example, Dulbecco's Modified Eagle's Medium
and RPMI-1640 medium. Alternatively, the hybridoma cells can be
grown in vivo as ascites in a mammal.
[0103] The monoclonal antibodies secreted by the subclones can be
isolated or purified from the culture medium or ascites fluid by
conventional immunoglobulin purification procedures such as, for
example, protein A-Sepharose, protein G, hydroxylapatite
chromatography, gel electrophoresis, dialysis, or affinity
chromatography.
[0104] The monoclonal antibodies can also be made by recombinant
DNA methods, such as those described in U.S. Pat. No. 4,816,567
(Cabilly et al.). DNA encoding the disclosed monoclonal antibodies
can be readily isolated and 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). Libraries of antibodies or active antibody fragments
can also be generated and screened using phage display techniques,
e.g., as described in U.S. Pat. No. 5,804,440 to Burton et al. and
U.S. Pat. No. 6,096,441 to Barbas et al. The antibodies produced
can be screened for antibodies having the desired binding ability
in the ways described for identifying monoclonal antibodies
discussed above, described elsewhere herein, and in ways known to
those of skill in the art. The examples describe a useful way of
producing and identifying HA stem antibodies.
[0105] In vitro methods are also suitable for preparing monovalent
antibodies. Digestion of antibodies to produce fragments thereof,
particularly, Fab fragments, can be accomplished using routine
techniques known in the art. For instance, digestion can be
performed using papain. Examples of papain digestion are described
in WO 94/29348 published Dec. 22, 1994 and U.S. Pat. No. 4,342,566.
Papain digestion of antibodies typically produces two identical
antigen binding fragments, called Fab fragments, each with a single
antigen binding site, and a residual Fc fragment. Pepsin treatment
yields a fragment that has two antigen combining sites and is still
capable of cross-linking antigen.
[0106] The fragments, whether attached to other sequences or not,
can also include insertions, deletions, substitutions, or other
selected modifications of particular regions or specific amino
acids residues, provided the activity of the antibody or antibody
fragment is not significantly altered or impaired compared to the
non-modified antibody or antibody fragment. These modifications can
provide for some additional property, such as to remove/add amino
acids capable of disulfide bonding, to increase its bio-longevity,
to alter its secretory characteristics, etc. In any case, the
antibody or antibody fragment should possess a bioactive property,
such as specific binding to its cognate antigen. Functional or
active regions of the antibody or antibody fragment can be
identified by mutagenesis of a specific region of the protein,
followed by expression and testing of the expressed polypeptide.
Such methods are readily apparent to a skilled practitioner in the
art and can include site-specific mutagenesis of the nucleic acid
encoding the antibody or antibody fragment. (Zoller, M. J. Curr.
Opin. Biotechnol. 3:348-354, 1992).
[0107] The immunizing agent can include the protein antigen, a
fragment thereof or a fusion protein thereof. Generally, either
peripheral blood lymphocytes can be used if cells of human origin
are desired, or spleen cells or lymph node cells can be used if
non-human mammalian sources are desired. The lymphocytes can then
be fused with an immortalized cell line using a suitable fusing
agent, such as polyethylene glycol, to form a hybridoma cell
(Goding, Monoclonal Antibodies: Principles and Practice, Academic
Press, (1986) pp. 59-103). Immortalized cell lines are usually
transformed mammalian cells, particularly myeloma cells of rodent,
bovine and human origin. Usually, rat or mouse myeloma cell lines
are employed. The hybridoma cells can be cultured in a suitable
culture medium that can contain one or more substances that inhibit
the growth or survival of the unfused, immortalized cells. For
example, if the parental cells lack the enzyme hypoxanthine guanine
phosphoribosyl transferase (HGPRT or HPRT), the culture medium for
the hybridomas can include hypoxanthine, aminopterin, and thymidine
("HAT medium"), which substances prevent the growth of
HGPRT-deficient cells.
[0108] Useful immortalized cell lines include those that fuse
efficiently, support stable high level expression of antibody by
the selected antibody-producing cells, and are sensitive to a
medium such as HAT medium. Useful immortalized cell lines include
murine myeloma lines, which can be obtained, for instance, from the
Salk Institute Cell Distribution Center, San Diego, Calif. and the
American Type Culture Collection, Manassas, Va. Human myeloma and
mouse-human heteromyeloma cell lines also have been described for
the production of human monoclonal antibodies (See Kozbor, J.
Immunol., 133:3001 (1984); Brodeur et al., Monoclonal Antibody
Production Techniques and Applications, Marcel Dekker, Inc., New
York, (1987) pp. 51-63)).
[0109] The culture medium in which the hybridoma cells are cultured
can then be assayed for the presence of monoclonal antibodies
directed against the antigen. The binding specificity of monoclonal
antibodies produced by the hybridoma cells can be determined by
immunoprecipitation or by an in vitro binding assay, such as
radioimmunoassay (RIA) or enzyme-linked immunoabsorbent assay
(ELISA). Such techniques and assays are known in the art. The
binding affinity of the monoclonal antibody can, for example, be
determined by, for example, the Scatchard analysis of Munson and
Pollard, Anal. Biochem., 107:220 (1980). Moreover, in therapeutic
applications of the disclosed antibodies, it is useful to identify
antibodies having a high degree of specificity and a high binding
affinity for the target antigen.
[0110] After the desired hybridoma cells are identified, the clones
can be subcloned by limiting dilution procedures and grown by
standard methods. (See Goding, Monoclonal Antibodies: Principles
and Practice, Academic Press, (1986) pp. 59-103). Suitable culture
media for this purpose include, for example, Dulbecco's Modified
Eagle's Medium and RPMI-1640 medium. Alternatively, the hybridoma
cells can be grown in vivo as ascites in a mammal.
[0111] The monoclonal antibodies secreted by the subclones can be
isolated or purified from the culture medium or ascites fluid by
conventional immunoglobulin purification procedures such as, for
example, protein A-Sepharose, hydroxylapatite chromatography, gel
electrophoresis, dialysis, or affinity chromatography.
[0112] Monoclonal antibodies can also be made by recombinant DNA
methods, such as those described in U.S. Pat. No. 4,816,567. DNA
encoding the disclosed monoclonal antibodies can be readily
isolated and 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). The disclosed hybridoma cells can serve as a source of
such DNA. Once isolated, the DNA can be placed into expression
vectors, which are then transfected into host cells such as simian
COS cells, Chinese hamster ovary (CHO) cells, or myeloma cells that
do not otherwise produce immunoglobulin protein, to obtain the
synthesis of monoclonal antibodies in the recombinant host cells.
The DNA also can be modified, for example, by substituting the
coding sequence for human heavy and light chain constant domains in
place of the homologous murine sequences (see U.S. Pat. No.
4,816,567; Morrison, Nature 368, 812-13 (1994)) or by covalently
joining to the immunoglobulin coding sequence all or part of the
coding sequence for a non-immunoglobulin polypeptide. Such a
non-immunoglobulin polypeptide can be substituted for the constant
domains of an antibody, or can be substituted for the variable
domains of one antigen-combining site of an antibody to create a
chimeric bivalent antibody.
[0113] Fully human antibodies are antibody molecules in which the
entire sequence of both the light chain and the heavy chain,
including the CDRs, arise from human genes. Such antibodies are
termed "human antibodies" or "fully human antibodies" herein. Human
monoclonal antibodies can be prepared by, for example, using trioma
technique; the human B-cell hybridoma technique (see Kozbor, et
al., 1983 Immunol Today 4: 72); and the EBV hybridoma technique to
produce human monoclonal antibodies (see Cole, et al., 1985 In:
MONOCLONAL ANTIBODIES AND CANCER THERAPY, Alan R. Liss, Inc., pp.
77-96). Human monoclonal antibodies can be utilized and can be
produced by using human hybridomas (see Cote, et al., 1983. Proc
Natl Acad Sci USA 80: 2026-2030) or by transforming human B-cells
with Epstein Barr Virus in vitro (see Cole, et al., 1985 In:
MONOCLONAL ANTIBODIES AND CANCER THERAPY, Alan R. Liss, Inc., pp.
77-96).
[0114] In addition, human antibodies can also be produced using
additional techniques, including phage display libraries (See
Hoogenboom and Winter, J. Mol. Biol., 227:381 (1991); Marks et al.,
J. Mol. Biol., 222:581 (1991)). Similarly, human antibodies can be
made by introducing human immunoglobulin loci into transgenic
animals, e.g., mice in which the endogenous immunoglobulin genes
have been partially or completely inactivated. Upon challenge,
human antibody production is observed, which closely resembles that
seen in humans in all respects, including gene rearrangement,
assembly, and antibody repertoire. This approach is described, for
example, in U.S. Pat. Nos. 5,545,807; 5,545,806; 5,569,825;
5,625,126; 5,633,425; 5,661,016, and in Marks et al.,
Bio/Technology 10, 779-783 (1992); Lonberg et al., Nature 368
856-859 (1994); Morrison, Nature 368, 812-13 (1994); Fishwild et
al, Nature Biotechnology 14, 845-51 (1996); Neuberger, Nature
Biotechnology 14, 826 (1996); and Lonberg and Huszar, Intern. Rev.
Immunol. 13 65-93 (1995).
[0115] Human antibodies can additionally be produced using
transgenic nonhuman animals which are modified so as to produce
fully human antibodies rather than the animal's endogenous
antibodies in response to challenge by an antigen. (See PCT
publication WO94/02602). The endogenous genes encoding the heavy
and light immunoglobulin chains in the nonhuman host have been
incapacitated, and active loci encoding human heavy and light chain
immunoglobulins are inserted into the host's genome. The human
genes can be incorporated, for example, using yeast artificial
chromosomes containing the requisite human DNA segments. An animal
which provides all the desired modifications can then be obtained
as progeny by crossbreeding intermediate transgenic animals
containing fewer than the full complement of the modifications. A
useful form of such a nonhuman animal include, for example, a
mouse, and is termed the Xenomouse.TM. as disclosed in PCT
publications WO 96/33735 and WO 96/34096. This animal produces B
cells which secrete fully human immunoglobulins. The antibodies can
be obtained directly from the animal after immunization with an
immunogen of interest, as, for example, a preparation of a
polyclonal antibody, or alternatively from immortalized B cells
derived from the animal, such as hybridomas producing monoclonal
antibodies. Additionally, the genes encoding the immunoglobulins
with human variable regions can be recovered and expressed to
obtain the antibodies directly, or can be further modified to
obtain analogs of antibodies such as, for example, single chain Fv
(scFv) molecules.
[0116] An example of a method of producing a nonhuman host,
exemplified as a mouse, lacking expression of an endogenous
immunoglobulin heavy chain is disclosed in U.S. Pat. No. 5,939,598.
It can be obtained by a method, which includes deleting the J
segment genes from at least one endogenous heavy chain locus in an
embryonic stem cell to prevent rearrangement of the locus and to
prevent formation of a transcript of a rearranged immunoglobulin
heavy chain locus, the deletion being effected by a targeting
vector containing a gene encoding a selectable marker; and
producing from the embryonic stem cell a transgenic mouse whose
somatic and germ cells contain the gene encoding the selectable
marker.
[0117] One method for producing an antibody of interest, such as a
human antibody, is disclosed in U.S. Pat. No. 5,916,771. This
method includes introducing an expression vector that contains a
nucleotide sequence encoding a heavy chain into one mammalian host
cell in culture, introducing an expression vector containing a
nucleotide sequence encoding a light chain into another mammalian
host cell, and fusing the two cells to form a hybrid cell. The
hybrid cell expresses an antibody containing the heavy chain and
the light chain.
[0118] As another example, a method for identifying a clinically
relevant epitope on an immunogen, and a correlative method for
selecting an antibody that binds immunospecifically to the relevant
epitope with high affinity, are disclosed in PCT publication WO
99/53049.
[0119] The antibody can be expressed by a vector containing a DNA
segment encoding the single chain antibody described above. These
can include vectors, liposomes, naked DNA, adjuvant-assisted DNA,
gene gun, catheters, etc. Vectors can include chemical conjugates
such as described in WO 93/64701, which has targeting moiety (e.g.
a ligand to a cellular surface receptor), and a nucleic acid
binding moiety (e.g. polylysine), viral vector (e.g. a DNA or RNA
viral vector), fusion proteins such as described in PCT/US 95/02140
(WO 95/22618) which is a fusion protein containing a target moiety
(e.g. an antibody specific for a target cell) and a nucleic acid
binding moiety (e.g. a protamine), plasmids, phage, etc. The
vectors can be chromosomal, non-chromosomal or synthetic.
[0120] Useful vectors include viral vectors, fusion proteins and
chemical conjugates. Retroviral vectors include moloney murine
leukemia viruses. DNA viral vectors are an example. These vectors
include pox vectors such as orthopox or avipox vectors, herpesvirus
vectors such as a herpes simplex I virus (HSV) vector (see Geller,
A. I. et al., J. Neurochem, 64:487 (1995); Lim, F., et al., in DNA
Cloning: Mammalian Systems, D. Glover, Ed. (Oxford Univ. Press,
Oxford England) (1995); Geller, A. I. et al., Proc Natl. Acad.
Sci.: U.S.A. 90:7603 (1993); Geller, A. I., et al., Proc Natl.
Acad. Sci. USA 87:1149 (1990), Adenovirus Vectors (see LeGal
LaSalle et al., Science, 259:988 (1993); Davidson, et al., Nat.
Genet 3:219 (1993); Yang, et al., J. Virol. 69:2004 (1995) and
Adeno-associated Virus Vectors (see Kaplitt, M. G. et al., Nat.
Genet. 8:148 (1994).
[0121] Pox viral vectors introduce the gene into the cells
cytoplasm. Avipox virus vectors result in only a short term
expression of the nucleic acid. Adenovirus vectors,
adeno-associated virus vectors and herpes simplex virus (HSV)
vectors can be used for introducing the nucleic acid into neural
cells. The adenovirus vector results in a shorter term expression
(about 2 months) than adeno-associated virus (about 4 months),
which in turn is shorter than HSV vectors. The particular vector
chosen can depend upon the target cell and the condition being
treated. The introduction can be by standard techniques, e.g.
infection, transfection, transduction or transformation. Examples
of modes of gene transfer include e.g., naked DNA, CaPO.sub.4
precipitation, DEAE dextran, electroporation, protoplast fusion,
lipofection, cell microinjection, and viral vectors.
[0122] The vector can be employed to target essentially any desired
target cell. For example, stereotaxic injection can be used to
direct the vectors (e.g. adenovirus, HSV) to a desired location.
Additionally, the particles can be delivered by
intracerebroventricular (icy) infusion using a minipump infusion
system, such as a SynchroMed Infusion System. A method based on
bulk flow, termed convection, has also proven effective at
delivering large molecules to extended areas of the brain and can
be useful in delivering the vector to the target cell. (See Bobo et
al., Proc. Natl. Acad. Sci. USA 91:2076-2080 (1994); Morrison et
al., Am. J. Physiol. 266:292-305 (1994)). Other methods that can be
used include catheters, intravenous, parenteral, intraperitoneal
and subcutaneous injection, and oral or other known routes of
administration.
[0123] These vectors can be used to express large quantities of
antibodies that can be used in a variety of ways. For example, to
detect the presence of an influenza virus in a sample. The antibody
can also be used to try to bind to and disrupt influenza virus cell
membrane fusion.
[0124] Techniques can be adapted for the production of single-chain
antibodies specific to the disclosed antigenic protein (see e.g.,
U.S. Pat. No. 4,946,778). In addition, methods can be adapted for
the construction of F.sub.ab expression libraries (see e.g., Huse,
et al., 1989 Science 246: 1275-1281) to allow rapid and effective
identification of monoclonal F.sub.ab fragments with the desired
specificity for a protein or derivatives, fragments, analogs or
homologs thereof. Antibody fragments that contain the idiotypes to
a protein antigen can be produced by techniques known in the art
including, but not limited to: (i) an F.sub.(ab')2 fragment
produced by pepsin digestion of an antibody molecule; (ii) an
F.sub.ab fragment generated by reducing the disulfide bridges of an
F.sub.(ab')2 fragment; (iii) an F.sub.ab fragment generated by the
treatment of the antibody molecule with papain and a reducing agent
and (iv) F.sub.v fragments.
[0125] Also disclosed are heteroconjugate antibodies.
Heteroconjugate antibodies are composed of two covalently joined
antibodies. Such antibodies have, for example, been proposed to
target immune system cells to unwanted cells (see U.S. Pat. No.
4,676,980), and for treatment of HIV infection (see WO 91/00360; WO
92/200373; EP 03089). It is contemplated that the antibodies can be
prepared in vitro using known methods in synthetic protein
chemistry, including those involving crosslinking agents. For
example, immunotoxins can be constructed using a disulfide exchange
reaction or by forming a thioether bond. Examples of suitable
reagents for this purpose include iminothiolate and
methyl-4-mercaptobutyrimidate and those disclosed, for example, in
U.S. Pat. No. 4,676,980.
[0126] It can be desirable to modify the disclosed antibody with
respect to effector function, so as to enhance, e.g., the
effectiveness of the antibody in treating influenza. For example,
cysteine residue(s) can be introduced into the Fc region, thereby
allowing interchain disulfide bond formation in this region. The
homodimeric antibody thus generated can have improved
internalization capability and/or increased complement-mediated
cell killing and antibody-dependent cellular cytotoxicity (ADCC)
(See Caron et al., J. Exp Med., 176: 1191-1195 (1992) and Shopes,
J. Immunol., 148: 2918-2922 (1992)). Alternatively, an antibody can
be engineered that has dual Fc regions and can thereby have
enhanced complement lysis and ADCC capabilities (See Stevenson et
al., Anti-Cancer Drug Design, 3: 219-230 (1989)).
[0127] Also disclosed are immunoconjugates comprising an antibody
conjugated to a cytotoxic agent such as a toxin (e.g., an
enzymatically active toxin of bacterial, fungal, plant, or animal
origin, or fragments thereof), or a radioactive isotope (i.e., a
radioconjugate).
[0128] Enzymatically active toxins and fragments thereof that can
be used include diphtheria A chain, nonbinding active fragments of
diphtheria toxin, exotoxin A chain (from Pseudomonas aeruginosa),
ricin A chain, abrin A chain, modeccin A chain, alpha-sarcin,
Aleurites fordii proteins, dianthin proteins, Phytolaca americana
proteins (PAPI, PAPII, and PAP-S), momordica charantia inhibitor,
curcin, crotin, sapaonaria officinalis inhibitor, gelonin,
mitogellin, restrictocin, phenomycin, enomycin, and the
tricothecenes. A variety of radionuclides are available for the
production of radioconjugated antibodies. Examples include
.sup.212Bi, .sup.131I, .sup.131In, .sup.90Y, and .sup.186Re.
[0129] Conjugates of the antibody and cytotoxic agent can be made
using a variety of bifunctional protein-coupling agents such as
N-succinimidyl-3-(2-pyridyldithiol) propionate (SPDP),
iminothiolane (IT), bifunctional derivatives of imidoesters (such
as dimethyl adipimidate HCL), active esters (such as disuccinimidyl
suberate), aldehydes (such as glutareldehyde), bis-azido compounds
(such as bis (p-azidobenzoyl) hexanediamine), bis-diazonium
derivatives (such as bis-(p-diazoniumbenzoyl)-ethylenediamine),
diisocyanates (such as tolyene 2,6-diisocyanate), and bis-active
fluorine compounds (such as 1,5-difluoro-2,4-dinitrobenzene). For
example, a ricin immunotoxin can be prepared as described in
Vitetta et al., Science 238: 1098 (1987). Carbon-14-labeled
1-isothiocyanatobenzyl-3-methyldiethylene triaminepentaacetic acid
(MX-DTPA) is an exemplary chelating agent for conjugation of
radionucleotide to the antibody (See WO94/11026).
[0130] Those of ordinary skill in the art will recognize that a
large variety of possible moieties can be coupled to the disclosed
antibodies or to other molecules (See, for example, "Conjugate
Vaccines", Contributions to Microbiology and Immunology, J. M.
Cruse and R. E. Lewis, Jr (eds), Carger Press, New York, (1989),
the entire contents of which are incorporated herein by
reference).
[0131] Coupling can be accomplished by any chemical reaction that
will bind the two molecules so long as the antibody and the other
moiety retain their respective activities. This linkage can include
many chemical mechanisms, for instance covalent binding, affinity
binding, intercalation, coordinate binding and complexation.
Covalent binding is also useful. Covalent binding can be achieved
either by direct condensation of existing side chains or by the
incorporation of external bridging molecules. Many bivalent or
polyvalent linking agents are useful in coupling protein molecules,
such as the disclosed antibodies, to other molecules. For example,
representative coupling agents can include organic compounds such
as thioesters, carbodiimides, succinimide esters, diisocyanates,
glutaraldehyde, diazobenzenes and hexamethylene diamines. This
listing is not intended to be exhaustive of the various classes of
coupling agents known in the art but, rather, is exemplary of the
more common coupling agents (See Killen and Lindstrom, Jour. Immun.
133:1335-2549 (1984); Jansen et al., Immunological Reviews
62:185-216 (1982); and Vitetta et al., Science 238:1098 (1987)).
Examples of useful linkers are described in the literature (See,
for example, Ramakrishnan, S. et al., Cancer Res. 44:201-208 (1984)
describing use of MBS (M-maleimidobenzoyl-N-hydroxysuccinimide
ester). See also U.S. Pat. No. 5,030,719, describing use of
halogenated acetyl hydrazide derivative coupled to an antibody by
way of an oligopeptide linker. Useful linkers include: (i) EDC
(1-ethyl-3-(3-dimethylamino-propyl)carbodiimide hydrochloride; (ii)
SMPT
(4-succinimidyloxycarbonyl-alpha-methyl-alpha-(2-pridyl-dithio)-toluene
(Pierce Chem. Co., Cat. (21558G); (iii) SPDP (succinimidyl-6
[3-(2-pyridyldithio)propionamido]hexanoate (Pierce Chem. Co., Cat
#21651G); (iv) Sulfo-LC-SPDP (sulfosuccinimidyl 6
[3-(2-pyridyldithio)-propianamide]hexanoate (Pierce Chem. Co. Cat.
#2165-G); and (v) sulfo-NHS(N-hydroxysulfo-succinimide: Pierce
Chem. Co., Cat. #24510) conjugated to EDC.
[0132] The linkers described above can contain components that have
different attributes, thus leading to conjugates with differing
physio-chemical properties. For example, sulfo-NHS esters of alkyl
carboxylates are more stable than sulfo-NHS esters of aromatic
carboxylates. NHS-ester containing linkers are less soluble than
sulfo-NHS esters. Further, the linker SMPT contains a sterically
hindered disulfide bond, and can form conjugates with increased
stability. Disulfide linkages, are in general, less stable than
other linkages because the disulfide linkage is cleaved in vitro,
resulting in less conjugate available. Sulfo-NHS, in particular,
can enhance the stability of carbodimide couplings. Carbodimide
couplings (such as EDC) when used in conjunction with sulfo-NHS,
forms esters that are more resistant to hydrolysis than the
carbodimide coupling reaction alone.
[0133] The antibodies disclosed herein can also be formulated as
immunoliposomes. Liposomes containing the antibody can be prepared
by methods known in the art, such as described in Epstein et al.,
Proc. Natl. Acad. Sci. USA, 82: 3688 (1985); Hwang et al., Proc.
Natl. Acad. Sci. USA, 77: 4030 (1980); and U.S. Pat. Nos. 4,485,045
and 4,544,545. Liposomes with enhanced circulation time are
disclosed in U.S. Pat. No. 5,013,556.
[0134] Particularly useful liposomes can be generated by the
reverse-phase evaporation method with a lipid composition
comprising phosphatidylcholine, cholesterol, and PEG-derivatized
phosphatidylethanolamine (PEG-PE). Liposomes can be extruded
through filters of defined pore size to yield liposomes with the
desired diameter. Fab' fragments of the disclosed antibodies can be
conjugated to the liposomes as described in Martin et al., J. Biol.
Chem., 257: 286-288 (1982) via a disulfide-interchange
reaction.
[0135] As used herein, the term "antibody" or "antibodies" can also
refer to a human antibody and/or a humanized antibody. Many
non-human antibodies (e.g., those derived from mice, rats, or
rabbits) are naturally antigenic in humans, and thus can give rise
to undesirable immune responses when administered to humans.
Therefore, the use of human or humanized antibodies in the methods
serves to lessen the chance that an antibody administered to a
human will evoke an undesirable immune response.
[0136] In general, antibody molecules obtained from humans can
relate to any of the classes IgG, IgM, IgA, IgE and IgD, which
differ from one another by the nature of the heavy chain present in
the molecule. Certain classes have subclasses as well, such as
IgG.sub.1, IgG.sub.2, and others. Furthermore, in humans, the light
chain can be a kappa chain or a lambda chain.
[0137] As used herein, the term "antibody" encompasses, but is not
limited to, whole immunoglobulin (i.e., an intact antibody) of any
class. Native antibodies are usually heterotetrameric
glycoproteins, composed of two identical light (L) chains and two
identical heavy (H) chains. Typically, 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 (V(H)) followed by a number of
constant domains. Each light chain has a variable domain at one end
(V(L)) and a constant domain at its other end; the constant domain
of the light chain is aligned with the first constant domain of the
heavy chain, and the light chain variable domain is aligned with
the variable domain of the heavy chain. Particular amino acid
residues are believed to form an interface between the light and
heavy chain variable domains. The light chains of antibodies from
any vertebrate species can be assigned to one of two clearly
distinct types, called kappa (.kappa.) and lambda (.lamda.), based
on the amino acid sequences of their constant domains. Depending on
the amino acid sequence of the constant domain of their heavy
chains, immunoglobulins can be assigned to different classes. There
are five major classes of human immunoglobulins: IgA, IgD, IgE, IgG
and IgM, and several of these can be further divided into
subclasses (isotypes), e.g., IgG-1, IgG-2, IgG-3, and IgG-4; IgA-1
and IgA-2. One skilled in the art would recognize the comparable
classes for mouse. The heavy chain constant domains that correspond
to the different classes of immunoglobulins are called alpha,
delta, epsilon, gamma, and mu, respectively.
[0138] In the context of antibodies, the term "variable" is used
herein to describe certain portions of the variable domains that
differ in sequence among antibodies and are used in the binding and
specificity of each particular antibody for its particular antigen.
However, the variability is not usually evenly distributed through
the variable domains of antibodies. It is typically concentrated in
three segments called complementarity determining regions (CDRs) or
hypervariable regions both in the light chain and the heavy chain
variable domains. The more highly conserved portions of the
variable domains are called the framework (FR). The variable
domains of native heavy and light chains each comprise four FR
regions, 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 regions and, with
the CDRs from the other chain, contribute to the formation of the
antigen binding site of antibodies (see Kabat E. A. et al.,
"Sequences of Proteins of Immunological Interest," National
Institutes of Health, Bethesda, Md. (1987)). The constant domains
are not involved directly in binding an antibody to an antigen, but
exhibit various effector functions, such as participation of the
antibody in antibody-dependent cellular toxicity.
[0139] The term "antibody" as used herein is also meant to include
intact molecules as well as fragments thereof, such as, for
example, Fab and F(ab').sub.2, which are capable of binding the
epitopic determinant.
[0140] As used herein, the term "antibody" encompasses chimeric
antibodies and hybrid antibodies, with dual or multiple antigen or
epitope specificities, and fragments, such as F(ab').sub.2, Fab',
Fab and the like, including hybrid fragments. Thus, fragments of
the antibodies that retain the ability to bind their specific
antigens are provided. For example, fragments of antibodies which
maintain hemagglutinin binding activity, such as to the
hemagglutinin stem region, are included within the meaning of the
term "antibody." Such antibodies and fragments can be made by
techniques known in the art and can be screened for specificity and
activity according to the methods set forth in the Examples and in
general methods for producing antibodies and screening antibodies
for specificity and activity (See Harlow and Lane. Antibodies, A
Laboratory Manual. Cold Spring Harbor Publications, New York,
(1988)).
[0141] Also included within the meaning of "antibody" are
conjugates of antibody fragments and antigen binding proteins
(single chain antibodies) as described, for example, in U.S. Pat.
No. 4,704,692, the contents of which are hereby incorporated by
reference.
[0142] Antibody fragments and segments can be chemically linked
where the bond formed between the fragments and segments as a
result of the chemical ligation is an unnatural (non-peptide) bond
(Schnolzer, M et al. Science, 256:221 (1992)). This technique has
been used to synthesize analogs of protein domains as well as large
amounts of relatively pure proteins with full biological activity
(deLisle Milton R C et al., Techniques in Protein Chemistry IV.
Academic Press, New York, pp. 257-267 (1992)).
[0143] Also disclosed are fragments of antibodies which have
bioactivity. The fragments can be recombinant proteins obtained by
cloning nucleic acids encoding the fragments in an expression
system capable of producing the fragments, such as an adenovirus or
baculovirus expression system. For example, one can determine the
active domain of an antibody from a specific hybridoma that can
cause a biological effect associated with the interaction of the
antibody with, for example, hemagglutinin. For example, amino acids
found to not contribute to either the activity or the binding
specificity or affinity of the antibody can be deleted without a
loss in the respective activity. For example, amino or
carboxy-terminal amino acids can be sequentially removed from
either the native or the modified non-immunoglobulin molecule or
the immunoglobulin molecule and the respective activity assayed in
one of many available assays. In another example, a fragment of an
antibody can comprise a modified antibody wherein at least one
amino acid has been substituted for the naturally occurring amino
acid at a specific position, and a portion of either amino terminal
or carboxy terminal amino acids, or even an internal region of the
antibody, has been replaced with a polypeptide fragment or other
moiety, such as biotin, which can facilitate in the purification of
the modified antibody. For example, a modified antibody can be
fused to a maltose binding protein, through either peptide
chemistry or cloning the respective nucleic acids encoding the two
polypeptide fragments into an expression vector such that the
expression of the coding region results in a hybrid polypeptide.
The hybrid polypeptide can be affinity purified by passing it over
an amylose affinity column, and the modified antibody receptor can
then be separated from the maltose binding region by cleaving the
hybrid polypeptide with the specific protease factor Xa (see, for
example, New England Biolabs Product Catalog, 1996, pg. 164.).
Similar purification procedures are available for isolating hybrid
proteins from eukaryotic cells as well.
[0144] The fragments, whether attached to other sequences or not,
include insertions, deletions, substitutions, or other selected
modifications of particular regions or specific amino acids
residues, provided the activity of the fragment is not
significantly altered or impaired compared to the nonmodified
antibody or antibody fragment. These modifications can provide for
some additional property, such as to remove or add amino acids
capable of disulfide bonding, to increase its bio-longevity, to
alter its secretory characteristics, etc. In any case, the fragment
must possess a bioactive property, such as binding activity,
regulation of binding at the binding domain, etc. Functional or
active regions of the antibody can be identified by mutagenesis of
a specific region of the protein, followed by expression and
testing of the expressed polypeptide. Such methods are readily
apparent to a skilled practitioner in the art and can include
site-specific mutagenesis of the nucleic acid encoding the antigen.
(Zoller M J et al. Nucl. Acids Res. 10:6487-500 (1982).
[0145] Techniques can also be adapted for the production of
single-chain antibodies specific to an epitope or antigen of
interest (hemagglutinin, for example) (see e.g., U.S. Pat. No.
4,946,778). In addition, methods can be adapted for the
construction of F(ab) expression libraries (see e.g., Huse, et al.,
1989 Science 246: 1275-1281) to allow rapid and effective
identification of monoclonal F(ab) fragments with the desired
specificity for a protein or derivatives, fragments, analogs or
homologs thereof. Antibody fragments that contain the idiotypes to
a protein antigen can be produced by techniques known in the art
including, but not limited to: (i) an F(ab').sub.2 fragment
produced by pepsin digestion of an antibody molecule; (ii) an Fab
fragment generated by reducing the disulfide bridges of an
F(ab').sub.2 fragment; (iii) an Fab fragment generated by the
treatment of the antibody molecule with papain and a reducing agent
and (iv) Fv fragments.
[0146] Methods for the production of single-chain antibodies are
well known to those of skill in the art. The skilled artisan is
referred to U.S. Pat. No. 5,359,046, (incorporated herein by
reference) for such methods. A single chain antibody can be created
by fusing together the variable domains of the heavy and light
chains using a short peptide linker, thereby reconstituting an
antigen binding site on a single molecule. Single-chain antibody
variable fragments (scFvs) in which the C-terminus of one variable
domain is tethered to the N-terminus of the other variable domain
via a 15 to 25 amino acid peptide or linker have been developed
without significantly disrupting antigen binding or specificity of
the binding (Bedzyk et al., 1990; Chaudhary et al., 1990). The
linker can be chosen to permit the heavy chain and light chain to
bind together in their proper conformational orientation. See, for
example, Huston, J. S., et al., Methods in Enzym. 203:46-121
(1991), which is incorporated herein by reference. These Fvs lack
the constant regions (Fc) present in the heavy and light chains of
the native antibody.
[0147] In vitro methods are also suitable for preparing monovalent
antibodies. Digestion of antibodies to produce fragments thereof,
particularly, Fab fragments, can be accomplished using routine
techniques known in the art. For instance, digestion can be
performed using papain. Examples of papain digestion are described
in WO 94/29348 published Dec. 22, 1994, U.S. Pat. No. 4,342,566,
and Harlow and Lane, Antibodies, A Laboratory Manual, Cold Spring
Harbor Publications, New York, (1988). Papain digestion of
antibodies typically produces two identical antigen binding
fragments, called Fab fragments, each with a single antigen binding
site, and a residual Fc fragment. Pepsin treatment yields a
fragment, called the F(ab').sub.2 fragment, that has two antigen
combining sites and is still capable of cross-linking antigen.
[0148] Fab fragments produced in the antibody digestion also
contain the constant domains of the light chain and the first
constant domain of the heavy chain. Fab' fragments differ from Fab
fragments by the addition of a few residues at the carboxy terminus
of the heavy chain domain including one or more cysteines from the
antibody hinge region. A F(ab').sub.2 fragment is a bivalent
fragment comprising two Fab' fragments linked by a disulfide bridge
at the hinge region. Fab'-SH is the designation herein for Fab' in
which the cysteine residue(s) of the constant domains bear a free
thiol group. Antibody fragments originally were produced as pairs
of Fab' fragments which have hinge cysteines between them. Other
chemical couplings of antibody fragments are also known.
[0149] In hybrid antibodies, one heavy and light chain pair is
homologous to that found in an antibody raised against one antigen
recognition feature, e.g., epitope, while the other heavy and light
chain pair is homologous to a pair found in an antibody raised
against another epitope. This results in the property of
multi-functional valency, i.e., ability to bind at least two
different epitopes simultaneously. As used herein, the term "hybrid
antibody" refers to an antibody wherein each chain is separately
homologous with reference to a mammalian antibody chain, but the
combination represents a novel assembly so that two different
antigens are recognized by the antibody. Such hybrids can be
formed, for example, by fusion of hybridomas producing the
respective component antibodies, or by recombinant techniques. Such
hybrids can, of course, also be formed using chimeric chains.
[0150] The antibodies can be anti-idiotypic antibodies (antibodies
that bind other antibodies) as described, for example, in U.S. Pat.
No. 4,699,880. Such anti-idiotypic antibodies could bind endogenous
or foreign antibodies in a treated individual, thereby to
ameliorate or prevent pathological conditions associated with an
immune response, e.g., in the context of an autoimmune disease.
[0151] The targeting function of the antibody can be further used
therapeutically by coupling the antibody or a fragment thereof with
a therapeutic agent. Such coupling of the antibody or fragment
(e.g., at least a portion of an immunoglobulin constant region
(Fc)) with the therapeutic agent can be achieved by making an
immunoconjugate or by making a fusion protein, comprising the
antibody or antibody fragment and the therapeutic agent.
[0152] Also included within the meaning of "antibody" are
conjugates of antibody fragments and antigen binding proteins
(single chain antibodies) as described, for example, in U.S. Pat.
No. 4,704,692, the contents of which are hereby incorporated by
reference.
[0153] One method of producing proteins comprising the antibodies
is to link two or more peptides or polypeptides together by protein
chemistry techniques. For example, peptides or polypeptides can be
chemically synthesized using currently available laboratory
equipment using either Fmoc (9-fluorenylmethyloxycarbonyl) or Boc
(tert-butyloxycarbonoyl) chemistry. (Applied Biosystems, Inc.,
Foster City, Calif.). One skilled in the art can readily appreciate
that a peptide or polypeptide corresponding to the antibody, for
example, can be synthesized by standard chemical reactions. For
example, a peptide or polypeptide can be synthesized and not
cleaved from its synthesis resin whereas the other fragment of an
antibody can be synthesized and subsequently cleaved from the
resin, thereby exposing a terminal group which is functionally
blocked on the other fragment. By peptide condensation reactions,
these two fragments can be covalently joined via a peptide bond at
their carboxyl and amino termini, respectively, to form an
antibody. (Grant GA (1992) Synthetic Peptides: A User Guide. W.H.
Freeman and Co., N.Y. (1992); Bodansky M and Trost B., Ed. (1993)
Principles of Peptide Synthesis. Springer-Verlag Inc., NY.
Alternatively, the peptide or polypeptide can be independently
synthesized in vivo as described above. Once isolated, these
independent peptides or polypeptides can be linked to form an
antibody via similar peptide condensation reactions.
[0154] For example, enzymatic ligation of cloned or synthetic
peptide segments allow relatively short peptide fragments to be
joined to produce larger peptide fragments, polypeptides or whole
protein domains (Abrahmsen L et al., Biochemistry, 30:4151 (1991)).
Alternatively, native chemical ligation of synthetic peptides can
be utilized to synthetically construct large peptides or
polypeptides from shorter peptide fragments. This method consists
of a two step chemical reaction (Dawson et al. Synthesis of
Proteins by Native Chemical Ligation. Science, 266:776-779 (1994)).
The first step is the chemoselective reaction of an unprotected
synthetic peptide-alpha-thioester with another unprotected peptide
segment containing an amino-terminal Cys residue to give a
thioester-linked intermediate as the initial covalent product.
Without a change in the reaction conditions, this intermediate
undergoes spontaneous, rapid intramolecular reaction to form a
native peptide bond at the ligation site. Application of this
native chemical ligation method to the total synthesis of a protein
molecule is illustrated by the preparation of human interleukin 8
(IL-8) (Baggiolini M et al. (1992) FEBS Lett. 307:97-101;
Clark-Lewis I et al., J. Biol. Chem., 269:16075 (1994); Clark-Lewis
I et al., Biochemistry, 30:3128 (1991); Rajarathnam K et al.,
Biochemistry 33:6623-30 (1994)).
[0155] The antibody can be bound to a substrate or labeled with a
detectable moiety or both bound and labeled. The detectable
moieties contemplated with the present compositions include
fluorescent, enzymatic and radioactive markers.
[0156] In the case where there are two or more definitions of a
term which is used and/or accepted within the art, the definition
of the term as used herein is intended to include all such meanings
unless explicitly stated to the contrary. A specific example is the
use of the term "complementarity determining region" ("CDR") to
describe the non-contiguous antigen combining sites found within
the variable region of both heavy and light chain polypeptides.
This particular region has been described by Kabat et al., U.S.
Dept. of Health and Human Services, "Sequences of Proteins of
Immunological Interest" (1983) and by Chothia et al., J. Mol. Biol.
196:901-917 (1987), which are incorporated herein by reference,
where the definitions include overlapping or subsets of amino acid
residues when compared against each other. Nevertheless,
application of either definition to refer to a CDR of an antibody
or variants thereof is intended to be within the scope of the term
as defined and used herein. The appropriate amino acid residues
which encompass the CDRs as defined by each of the above cited
references are set forth below in Table 4 as a comparison. The
exact residue numbers which encompass a particular CDR will vary
depending on the sequence and size of the CDR. Those skilled in the
art can routinely determine which residues comprise a particular
CDR given the variable region amino acid sequence of the
antibody.
TABLE-US-00022 TABLE 4 CDR Definitions.sup.1 Kabat Chothia V.sub.H
CDR1 31-35 26-32 V.sub.H CDR2 50-65 52-58 V.sub.H CDR3 95-102
95-102 V.sub.L CDR1 24-34 26-32 V.sub.L CDR2 50-56 50-52 V.sub.L
CDR3 89-97 91-96 .sup.1Numbering of all CDR definitions in Table 1
is according to the numbering conventions set forth by Kabat et al.
(see below). Kabat et al. also defined a numbering system for
variable domain sequences that is applicable to any antibody. One
of ordinary skill in the art can unambigously assign this system of
"Kabat numbering" to any variable domain sequence, without reliance
on any experimental data beyond the sequence itself. As used
herein, "Kabat numbering" refers to the numbering system set forth
by Kabat et al., U.S. Dept. of Health and Human Services, "Sequence
of Proteins of Immunological Interest" (1983). Unless otherwise
specified, references to the numbering of specific amino acid
residue positions in an HA stem antibody or antigen-binding
fragment, variant, or derivative thereof of the disclosed
antibodies are according to the Kabat numbering system.
[0157] In camelid species, the heavy chain variable region,
referred to as V.sub.HH, forms the entire antigen-binding domain.
The main differences between camelid V.sub.HH variable regions and
those derived from conventional antibodies (V.sub.H) include (a)
more hydrophobic amino acids in the light chain contact surface of
V.sub.H as compared to the corresponding region in V.sub.HH, (b) a
longer CDR3 in V.sub.HH, and (c) the frequent occurrence of a
disulfide bond between CDR1 and CDR3 in V.sub.HH. immunoglobulin
molecule.
[0158] Antibody fragments, including single-chain antibodies, can
comprise the variable region(s) alone or in combination with the
entirety or a portion of the following: hinge region, C.sub.H1,
C.sub.H2, and C.sub.H3 domains. Also disclosed are antigen-binding
fragments also comprising any combination of variable region(s)
with a hinge region, C.sub.H1, C.sub.H2, and C.sub.H3 domains. For
example, antibody fragments comprising all or a portion of the
heavy chain of a HA stem antibody can be used. Such antibody
fragments can be effective because the heavy chain is inserted into
stem region pocket and is dominant in specifying binding to the
stem region (see Examples). Antibodies or immunospecific fragments
thereof for use in the disclosed methods can be from any animal
origin including birds and mammals. In some forms, the antibodies
can be human, murine, donkey, rabbit, goat, guinea pig, camel,
llama, horse, or chicken antibodies. In other forms, the variable
region can be condricthoid in origin (e.g., from sharks). As used
herein, "human" antibodies include antibodies having the amino acid
sequence of a human immunoglobulin and include antibodies isolated
from human immunoglobulin libraries or from animals transgenic for
one or more human immunoglobulins and that do not express
endogenous immunoglobulins, as described elsewhere herein and, for
example in, U.S. Pat. No. 5,939,598 by Kucherlapati et al.
[0159] As used herein, the term "heavy chain portion" includes
amino acid sequences derived from an immunoglobulin heavy chain. A
polypeptide comprising a heavy chain portion can comprise at least
one of: a C.sub.H1 domain, a hinge (e.g., upper, middle, and/or
lower hinge region) domain, a C.sub.H2 domain, a C.sub.H3 domain,
or a variant or fragment thereof. For example, an antibody for use
in the disclosed methods can comprise a polypeptide chain
comprising a C.sub.H1 domain; a polypeptide chain comprising a
C.sub.H1 domain, at least a portion of a hinge domain, and a
C.sub.H2 domain; a polypeptide chain comprising a C.sub.H1 domain
and a C.sub.H3 domain; a polypeptide chain comprising a C.sub.H1
domain, at least a portion of a hinge domain, and a C.sub.H3
domain, or a polypeptide chain comprising a C.sub.H1 domain, at
least a portion of a hinge domain, a C.sub.H2 domain, and a
C.sub.H3 domain. In some forms, the disclosed antibody can comprise
a polypeptide chain comprising a C.sub.H3 domain. Further, an for
use in the disclosed methods can lack at least a portion of a
C.sub.H2 domain (e.g., all or part of a C.sub.H2 domain). As
discussed elsewhere herein, it will be understood by one of
ordinary skill in the art that these domains (e.g., the heavy chain
portions) can be modified such that they vary in amino acid
sequence from the naturally occurring immunoglobulin molecule.
[0160] In some forms, the disclosed antibodies (such as, for
example, HA stem antibodies, and epitope- or antigen-binding
fragments, variants, or derivatives thereof), the heavy chain
portions of one polypeptide chain of a multimer can be identical to
those on a second polypeptide chain of the multimer. Alternatively,
heavy chain portion-containing monomers need not be identical. For
example, each monomer can comprise a different target binding site,
forming, for example, a bispecific antibody.
[0161] The heavy chain portions of an antibody can be derived from
different immunoglobulin molecules. For example, a heavy chain
portion of an antibody can comprise a C.sub.H1 domain derived from
an IgG1 molecule and a hinge region derived from an IgG3 molecule.
In another example, a heavy chain portion can comprise a hinge
region derived, in part, from an IgG1 molecule and, in part, from
an IgG3 molecule. In another example, a heavy chain portion can
comprise a chimeric hinge derived, in part, from an IgG1 molecule
and, in part, from an IgG4 molecule.
[0162] As used herein, the term "light chain portion" includes
amino acid sequences derived from an immunoglobulin light chain. In
some forms, the light chain portion can comprise at least one of a
V.sub.L or C.sub.L domain.
[0163] The disclosed antibodies (such as, for example, HA stem
antibodies, and epitope- or antigen-binding fragments, variants, or
derivatives thereof) can be described or specified in terms of the
epitope(s) or portion(s) of an antigen, for example, a target
protein or polypeptide (a hemagglutinin composition, hemagglutinin
fragment, or hemagglutinin stem portion, for example) that they
recognize or specifically bind. The portion of a target polypeptide
which specifically interacts with the antigen binding domain of an
antibody is an "epitope," or an "antigenic determinant." As used
herein, the term "epitope" includes any protein determinant capable
of specific binding to an immunoglobulin, an scFv, or a T-cell
receptor. Epitopic determinants usually consist of 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. For
example, antibodies can be raised against N-terminal or C-terminal
peptides of a polypeptide.
[0164] Particularly useful epitopes are, for example, the stem
region of hemagglutinin and the epitope for antibody F10. A target
polypeptide can comprise a single epitope, but typically comprises
at least two epitopes, and can include any number of epitopes,
depending on the size, conformation, and type of antigen.
Furthermore, it should be noted that an "epitope" on a target
polypeptide can be or include non-polypeptide elements, e.g., an
"epitope" can include a carbohydrate side chain. Although
antibodies, their generation and their binding properties are
described herein in alternatively and individually in terms of
epitope(s), antigen(s), peptide(s), polypeptide(s), protein(s),
etc., it is understood that use or mention of one or a subset of
these and related terms (which are intended to refer to epitopes
and various molecules that comprise such epitopes) should be
considered a reference to all such terms individually and in any
combination. Thus, for example, reference to binding of an antibody
to a protein should also be considered a reference to binding of
the antibody to the relevant antigen, epitope, etc. that the
protein comprises.
[0165] The minimum size of a peptide or polypeptide epitope for an
antibody is thought to be about four to five amino acids. Peptide
or polypeptide epitopes can contain, for example, at least seven,
at least nine, or between at least about 15 to about 30 amino
acids. Since a CDR can recognize an antigenic peptide or
polypeptide in its tertiary form, the amino acids comprising an
epitope need not be contiguous, and in some cases, may not even be
on the same peptide chain. The HA stem region epitope described
herein fall into this latter category. An epitope recognized by
antibodies can contain a sequence of at least 4, at least 5, at
least 6, at least 7, at least 8, at least 9, at least 10, at least
15, at least 20, at least 25, or between about 15 to about 30
contiguous or non-contiguous amino acids of the target protein or
polypeptide.
[0166] As used herein, the terms "immunological binding," and
"immunological binding properties" refer to the non-covalent
interactions of the type which occur between an immunoglobulin
molecule and an antigen for which the immunoglobulin is specific.
The strength, or affinity of immunological binding interactions can
be expressed in terms of the dissociation constant (K.sub.d) of the
interaction, wherein a smaller K.sub.d represents a greater
affinity. Immunological binding properties of selected polypeptides
can be quantified using methods well known in the art. One such
method entails measuring the rates of antigen-binding site/antigen
complex formation and dissociation, wherein those rates depend on
the concentrations of the complex partners, the affinity of the
interaction, and geometric parameters that equally influence the
rate in both directions. Thus, both the "on rate constant"
(K.sub.on) and the "off rate constant" (K.sub.off) can be
determined by calculation of the concentrations and the actual
rates of association and dissociation (See Nature 361:186-87
(1993)). The ratio of K.sub.off/K.sub.on enables the cancellation
of all parameters not related to affinity, and is equal to the
dissociation constant K.sub.d (See, generally, Davies et al. (1990)
Annual Rev Biochem 59:439-473).
[0167] By "specifically binds" or "immunoreacts with" is meant that
the antibody reacts with one or more antigenic determinants of the
desired antigen and does not react with other polypeptides. Thus,
"specifically binds" generally means that an antibody binds to an
epitope via its antigen binding domain, and that the binding
entails some complementarity between the antigen binding domain and
the epitope. According to this definition, an antibody is said to
"specifically bind" to an epitope when it binds to that epitope,
via its antigen binding domain more readily than it would bind to a
random, unrelated epitope. The term "specificity" is used herein to
qualify the relative affinity by which a certain antibody binds to
a certain epitope. For example, antibody "A" can be deemed to have
a higher specificity for a given epitope than antibody "B," or
antibody "A" can be said to bind to epitope "C" with a higher
specificity than it has for related epitope "D."
[0168] By "preferentially binds," it is meant that the antibody
specifically binds to an epitope more readily than it would bind to
a related, similar, homologous, or analogous epitope. Thus, an
antibody which "preferentially binds" to a given epitope would more
likely bind to that epitope than to a related epitope, even though
such an antibody may cross-react with the related epitope.
[0169] By way of non-limiting example, an antibody can be
considered to bind a first epitope preferentially if it binds said
first epitope with a dissociation constant (K.sub.D) that is less
than the antibody's K.sub.D for the second epitope. In another
non-limiting example, an antibody can be considered to bind a first
antigen preferentially if it binds the first epitope with an
affinity that is at least one order of magnitude less than the
antibody's K.sub.D for the second epitope. In another non-limiting
example, an antibody can be considered to bind a first epitope
preferentially if it binds the first epitope with an affinity that
is at least two orders of magnitude less than the antibody's
K.sub.D for the second epitope.
[0170] In another non-limiting example, an antibody can be
considered to bind a first epitope preferentially if it binds the
first epitope with an off rate (k(off)) that is less than the
antibody's k(off) for the second epitope. In another non-limiting
example, an antibody can be considered to bind a first epitope
preferentially if it binds the first epitope with an affinity that
is at least one order of magnitude less than the antibody's k(off)
for the second epitope. In another non-limiting example, an
antibody can be considered to bind a first epitope preferentially
if it binds the first epitope with an affinity that is at least two
orders of magnitude less than the antibody's k(off) for the second
epitope.
[0171] An antibody can be said to bind a target polypeptide with an
off rate (k(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. An antibody can be said to bind a target
polypeptide with an off rate (k(off)), for example, 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.
[0172] An antibody or antigen-binding fragment, variant, or
derivative disclosed herein can be said to bind a target
polypeptide with an on rate (k(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 or 5.times.10.sup.4 M.sup.-1
sec.sup.-1. An antibody can be said to bind a target polypeptide
with an on rate (k(on)) greater than or equal to 10.sup.5 M.sup.-1
sec.sup.-1, 5.times.10.sup.5M.sup.-1 sec.sup.-1 10.sup.6 M.sup.-1
sec.sup.-1, or 5.times.10.sup.6 M.sup.-1 sec.sup.-1 or 10.sup.7
M.sup.-1 sec.sup.-1.
[0173] An antibody can be said to specifically bind to a influenza
epitope when, for example, the equilibrium binding constant
(K.sub.d) can be, for example, .ltoreq.1 .mu.M, .ltoreq.100 nM,
.ltoreq.10 nM, and .ltoreq.100 pM to about 1 pM, as measured by
assays such as radioligand binding assays or similar assays known
to those skilled in the art.
[0174] An antibody is said to competitively inhibit binding of a
reference antibody to a given epitope if it preferentially binds to
that epitope to the extent that it blocks, to some degree, binding
of the reference antibody to the epitope. Competitive inhibition
can be determined by any method known in the art, for example,
competition ELISA assays. An antibody can be said to competitively
inhibit binding of the reference antibody to a given epitope by at
least 90%, at least 80%, at least 70%, at least 60%, or at least
50%.
[0175] As used herein, the term "affinity" refers to a measure of
the strength of the binding of an individual epitope with the CDR
of an immunoglobulin molecule. See, e.g., Harlow et al.,
Antibodies: A Laboratory Manual, (Cold Spring Harbor Laboratory
Press, 2nd ed. 1988) at pages 27-28. As used herein, the term
"avidity" refers to the overall stability of the complex between a
population of immunoglobulins and an antigen, that is, the
functional combining strength of an immunoglobulin mixture with the
antigen. See, e.g., Harlow at pages 29-34. Avidity is related to
both the affinity of individual immunoglobulin molecules in the
population with specific epitopes, and also the valencies of the
immunoglobulins and the antigen. For example, the interaction
between a bivalent monoclonal antibody and an antigen with a highly
repeating epitope structure, such as a polymer, would be one of
high avidity.
[0176] Antibodies can also be described or specified in terms of
their cross-reactivity. As used herein, the term "cross-reactivity"
refers to the ability of an antibody, specific for one antigen, to
react with a second antigen; a measure of relatedness between two
different antigenic substances. Thus, an antibody is cross reactive
if it binds to an epitope other than the one that induced its
formation. The cross reactive epitope generally contains many of
the same complementary structural features as the inducing epitope,
and in some cases, may actually fit better than the original.
[0177] For example, certain antibodies have some degree of
cross-reactivity, in that they bind related, but non-identical
epitopes, e.g., epitopes with at least 95%, at least 90%, at least
85%, at least 80%, at least 75%, at least 70%, at least 65%, at
least 60%, at least 55%, and at least 50% identity (as calculated
using methods known in the art and described herein) to a reference
epitope. An antibody can be said to have little or no
cross-reactivity if it does not bind epitopes with less than 95%,
less than 90%, less than 85%, less than 80%, less than 75%, less
than 70%, less than 65%, less than 60%, less than 55%, and less
than 50% identity (as calculated using methods known in the art and
described herein) to a reference epitope. An antibody can be deemed
"highly specific" for a certain epitope, if it does not bind any
other analog, ortholog, or homolog of that epitope.
[0178] Antibodies can also be described or specified in terms of
their binding affinity to a protein, polypeptide, epitope, antigen,
etc. Examples of binding affinities include those with a
dissociation constant or Kd less than 5.times.10.sup.-2 M,
10.sup.-2 M, 5.times.10.sup.-3 M, 10.sup.-3M, 5.times.10.sup.-4 M,
10.sup.-4 M, 5.times.10.sup.-5M, 10.sup.-5 M, 5.times.10.sup.-6 M,
10.sup.-6 M, 5.times.10.sup.-7M, 10.sup.-7 M, 5.times.10.sup.-8M,
10.sup.-8M, 5.times.10.sup.-9 M, 10.sup.-9M, 5.times.10.sup.-10 M,
10.sup.-10 M, 5.times.10.sup.-11M, 10.sup.-11M,
5.times.10.sup.-12M, 10.sup.-12M, 5.times.10.sup.-13 M, 10.sup.-13
M, 5.times.10.sup.-14 M, 10.sup.-14M, 5.times.10.sup.-15M, or
10.sup.-15M.
[0179] Antibodies can be "multispecific," e.g., bispecific,
trispecific or of greater multispecificity, meaning that it
recognizes and binds to two or more different epitopes present on
one or more different antigens (e.g., proteins) at the same time.
Thus, whether an antibody is "monospecfic" or "multispecific,"
e.g., "bispecific," refers to the number of different epitopes with
which an antibody reacts. Multispecific antibodies can be specific
for different epitopes of a target polypeptide or can be specific
for a target polypeptide as well as for a heterologous epitope,
such as a heterologous polypeptide or solid support material.
[0180] As used herein the term "valency" refers to the number of
potential binding domains, e.g., antigen binding domains, present
in an antibody. Each binding domain specifically binds one epitope.
When an antibody comprises more than one binding domain, each
binding domain can specifically bind the same epitope, for an
antibody with two binding domains, termed "bivalent monospecific,"
or to different epitopes, for an antibody with two binding domains,
termed "bivalent bispecific." An antibody can also be bispecific
and bivalent for each specificity (termed "bispecific tetravalent
antibodies"). In some forms, tetravalent minibodies or domain
deleted antibodies can be made.
[0181] Bispecific bivalent antibodies, and methods of making them,
are described, for instance in U.S. Pat. Nos. 5,731,168; 5,807,706;
5,821,333; and U.S. Appl. Publ. Nos. 2003/020734 and 2002/0155537,
the disclosures of all of which are incorporated by reference
herein. Bispecific tetravalent antibodies, and methods of making
them are described, for instance, in WO 02/096948 and WO 00/44788,
the disclosures of both of which are incorporated by reference
herein. See generally, PCT publications WO 93/17715; WO 92/08802;
WO 91/00360; WO 92/05793; Tutt et al., J. Immunol. 147:60-69
(1991); U.S. Pat. Nos. 4,474,893; 4,714,681; 4,925,648; 5,573,920;
5,601,819; Kostelny et al., J. Immunol. 148:1547-1553 (1992).
[0182] Those skilled in the art will recognize that it is possible
to determine, without undue experimentation, if an antibody (such
as a human antibody or human monoclonal antibody) has the same
specificity as a disclosed antibody (such as a human antibody or
human monoclonal antibody) by ascertaining whether the former
prevents the latter from binding to the HA protein of the influenza
virus. If the antibody being tested competes with the disclosed
antibody, as shown by a decrease in binding by the disclosed
antibody, then it is likely that the two antibodies bind to the
same, or to a closely related, epitope.
[0183] Another way to determine whether an antibody (such as a
human antibody or human monoclonal antibody) has the specificity of
a disclosed antibody (such as a human antibody or human monoclonal
antibody) is to pre-incubate the disclosed antibody with the
influenza HA protein, with which it is normally reactive, and then
add the antibody being tested to determine if the antibody being
tested is inhibited in its ability to bind the HA protein. If the
antibody being tested is inhibited then, in all likelihood, it has
the same, or functionally equivalent, epitopic specificity as the
disclosed antibody. Screening of disclosed antibodies can be also
carried out by utilizing the influenza virus and determining
whether the test antibody is able to neutralize the influenza
virus.
[0184] As previously indicated, the subunit structures and three
dimensional configuration of the constant regions of the various
immunoglobulin classes are well known. As used herein, the term
"V.sub.H domain" includes the amino terminal variable domain of an
immunoglobulin heavy chain and the term "C.sub.H domain" includes
the first (most amino terminal) constant region domain of an
immunoglobulin heavy chain. The C.sub.H1 domain is adjacent to the
V.sub.H domain and is amino terminal to the hinge region of an
immunoglobulin heavy chain molecule.
[0185] As used herein the term "C.sub.H2 domain" includes the
portion of a heavy chain molecule that extends, for example, from
about residue 244 to residue 360 of an antibody using conventional
numbering schemes (residues 244 to 360, Kabat numbering system; and
residues 231-340, EU numbering system; see Kabat E A et al. op.
cit. The C.sub.H2 domain is unique in that it is not closely paired
with another domain. Rather, two N-linked branched carbohydrate
chains are interposed between the two C.sub.H2 domains of an intact
native IgG molecule. It is also well documented that the C.sub.H3
domain extends from the C.sub.H2 domain to the C-terminal of the
IgG molecule and comprises approximately 108 residues.
[0186] As used herein, the term "hinge region" includes the portion
of a heavy chain molecule that joins the C.sub.H1 domain to the
C.sub.H2 domain. This hinge region comprises approximately 25
residues and is flexible, thus allowing the two N-terminal antigen
binding regions to move independently. Hinge regions can be
subdivided into three distinct domains: upper, middle, and lower
hinge domains (Roux et al., J. Immunol. 161:4083 (1998)).
[0187] As used herein the term "disulfide bond" includes the
covalent bond formed between two sulfur atoms. The amino acid
cysteine comprises a thiol group that can form a disulfide bond or
bridge with a second thiol group. In most naturally occurring IgG
molecules, the C.sub.H1 and C.sub.L regions are linked by a
disulfide bond and the two heavy chains are linked by two disulfide
bonds at positions corresponding to 239 and 242 using the Kabat
numbering system (position 226 or 229, EU numbering system).
[0188] As used herein, the term "chimeric antibody" means any
antibody where some regions, sites or sequences come form two or
more different species. For example, the immunoreactive region or
site can obtained or derived from a first species and the constant
region (which may be intact, partial or modified as described
herein) can be obtained from a second species. In some forms, the
target binding region or site can be from a non-human source (e.g.
mouse or primate) and the constant region can be human.
[0189] As used herein, the term "engineered antibody" refers to an
antibody in which the variable domain in either the heavy and light
chain or both is altered by at least partial replacement of one or
more CDRs from an antibody of known specificity and, optionally, by
partial framework region replacement and sequence changing.
Although the CDRs can be derived from an antibody of the same class
or even subclass as the antibody from which the framework regions
are derived, the CDRs can also be derived from an antibody of
different class and/or from an antibody from a different species.
An engineered antibody in which one or more "donor" CDRs from a
non-human antibody of known specificity is grafted into a human
heavy or light chain framework region is referred to herein as a
"humanized antibody." It may not be necessary to replace all of the
CDRs with the complete CDRs from the donor variable region to
transfer the antigen binding capacity of one variable domain to
another. Rather, it may only be necessary to transfer those
residues that are necessary to maintain the activity of the target
binding site. Given the explanations set forth in, e.g., U.S. Pat.
Nos. 5,585,089, 5,693,761, 5,693,762, and 6,180,370, it will be
well within the competence of those skilled in the art, either by
carrying out routine experimentation or by trial and error testing
to obtain a functional engineered or humanized antibody.
[0190] As used herein the term "properly folded" protein or
polypeptide includes proteins and polypeptides (e.g., HA and HA
stem region) in which all of the functional domains comprising the
protein or polypeptide are distinctly active. As used herein, the
term "improperly folded" protein or polypeptide includes proteins
and polypeptides in which at least one of the functional domains of
the protein or polypeptide is not active. In some forms, a properly
folded polypeptide comprises polypeptide chains linked by at least
one disulfide bond and, conversely, an improperly folded
polypeptide comprises polypeptide chains not linked by at least one
disulfide bond. In the context of hemagglutinin, properly folded
hemagglutinin or properly folder hemagglutinin stem region refers
hemagglutinin (or hemagglutinin region) that has a native
conformation. In the context of the disclosed HA stem antibodies
and methods, a properly folded hemagglutinin or properly folded
hemagglutinin stem region is, for example, one to which antibody
F10 can bind, specifically bind, preferentially bind, etc.
[0191] As used herein the term "engineered" includes manipulation
of nucleic acid or polypeptide molecules by synthetic means (e.g.
by recombinant techniques, in vitro peptide synthesis, by enzymatic
or chemical coupling of peptides or some combination of these
techniques).
[0192] As used herein, the terms "linked," "fused" or "fusion" are
used interchangeably. These terms refer to the joining together of
two more elements or components, by whatever means including
chemical conjugation or recombinant means. An "in-frame fusion"
refers to the joining of two or more polynucleotide open reading
frames (ORFs) to form a continuous longer ORF, in a manner that
maintains the correct translational reading frame of the original
ORFs. Thus, a recombinant fusion protein is a single protein
containing two ore more segments that correspond to polypeptides
encoded by the original ORFs (which segments are not normally so
joined in nature.) Although the reading frame is thus made
continuous throughout the fused segments, the segments can be
physically or spatially separated by, for example, in-frame linker
sequence. For example, polynucleotides encoding the CDRs of an
immunoglobulin variable region can be fused, in-frame, but be
separated by a polynucleotide encoding at least one immunoglobulin
framework region or additional CDR regions, as long as the "fused"
CDRs are co-translated as part of a continuous polypeptide.
[0193] In the context of polypeptides, a "linear sequence" or a
"sequence" is an order of amino acids in a polypeptide in an amino
to carboxyl terminal direction in which residues that neighbor each
other in the sequence are contiguous in the primary structure of
the polypeptide.
[0194] The term "expression" as used herein refers to a process by
which a gene produces a biochemical, for example, an RNA or
polypeptide. The process includes any manifestation of the
functional presence of the gene within the cell including, without
limitation, gene knockdown as well as both transient expression and
stable expression. It includes without limitation transcription of
the gene into messenger RNA (mRNA), transfer RNA (tRNA), small
hairpin RNA (shRNA), small interfering RNA (siRNA) or any other RNA
product, and the translation of such mRNA into polypeptide(s). If
the final desired product is a biochemical, expression includes the
creation of that biochemical and any precursors. Expression of a
gene produces a "gene product." As used herein, a gene product can
be either a nucleic acid, e.g., a messenger RNA produced by
transcription of a gene, or a polypeptide which is translated from
a transcript. Gene products described herein further include
nucleic acids with post transcriptional modifications, e.g.,
polyadenylation, or polypeptides with post translational
modifications, e.g., methylation, glycosylation, the addition of
lipids, association with other protein subunits, proteolytic
cleavage, and the like.
[0195] Any of the disclosed antibodies or other described
antibodies can be included or excluded from any group or genus of
antibodies and from use in any method, including the disclosed
methods. Thus, for example, antibodies D7, D8, F10, G17, H40, A66,
H98, D80, E90, and E8 described herein; C179 (described by Okuno et
al., A common neutralizing epitope conserved between the
hemagglutinins of influenza A virus H1 and H2 strains. J Virol 67,
2552-8 (1993)), and the antibodies described by Kashyap et al.,
Combinatorial antibody libraries from survivors of the Turkish H5N1
avian influenza outbreak reveal virus neutralization strategies.
Proc Natl Acad Sci USA 105, 5986-91 (2008), can be, individually of
in any combination, included or excluded.
B. Hemagglutinin Compositions
[0196] Disclosed are hemagglutinin compositions. The disclosed
hemagglutinin compositions are useful as immunogens, e.g. vaccines,
to stimulate a subjects immune response to influenza virus. For
example, disclosed are hemagglutinin compositions that comprise,
for example, the hemagglutinin stem region, either as part of a
full hemagglutinin complex, as part of a trimeric ectodomain of
hemagglutinin, as part of the extracellular portion of
hemagglutinin, or in isolation from other parts of hemagglutinin.
The hemagglutinin composition can be immobilized on a surface. For
example, the hemagglutinin composition can be the stem region of
hemagglutinin in the neutral pH conformation in isolation from the
head region of hemagglutinin, thereby identifying antibodies of
interest. For example, the hemagglutinin composition can be
hemagglutinin in the neutral pH conformation in isolation from
other components of influenza virus, where the head region of the
hemagglutinin is modified to reduce the antigenicity of the head
region. Because the stem region is formed in trimeric
hemagglutinin, hemagglutinin compositions that include the stem
region of trimeric hemagglutinin are particularly useful. For
example, the hemagglutinin composition includes an isolated peptide
(natural or synthetic) that includes the F10 epitope, an F10
epitope unit and any mimotope thereof. An F10 epitope or an F10
epitope unit for the purposes of the present invention is a portion
of an antigen molecule, e.g. hemagglutinin, which is delineated by
the area of interration with the F10 antibody. The meaning of
mimotope is defined as an entity which is sufficiently similar to
the native F10 epitope so as to be capable of being recognized by
antibodies which recognize the native F10 epitope; (Gheysen, H. M.,
et al., 1986, Synthetic peptides as antigens. Wiley, Chichester,
Ciba foundation symposium 119, p 130-149; Gheysen, H. M., 1986,
Molecular Immunology, 23, 7, 709-715); or are capable of raising
antibodies, when coupled to a suitable carrier, which antibodies
cross-react with the native F10 epitope.
[0197] In some forms the hemagglutinin compositions are immunogen
compositions. The immunogen compositions according to the invention
contain an epitope or epitope unit recognized by a protective
monoclonal antibody having the specificity for the stem region of
hemagglutinin protein of an influenza virus. A protective
monoclonal antibody having the specificity for the stem region of
hemagglutinin protein of an influenza virus includes monoclonal
antibody D7, D8, F10, G17, H40, A66, D80, E88, E90, or H98
disclosed herein or a monoclonal antibody that competes with the
binding of monoclonal antibody D7, D8, F10, G17, H40, A66, D80,
E88, E90, or H98 to the HA protein. The antibody binds both the HA1
and HA2 peptide.
[0198] An epitope or epitope unit is for example the F10 epitope.
By F10 epitope it is meant the epitope recognized by the F10
antibody disclosed herein. The epitope is a confirmation epitope
defined by amino acids of the HA1 and HA2 peptide of hemagglutinin
protein of the influenza virus. The hemagglutinin protein is in the
neutral pH conformation. Specifically, the F10 epitope is defined
by amino acid residues 18, 38, 39, 40 and 291 of HA1 and 18, 19,
20, 21, 38, 41, 42, 45, 49, 52, 53, and 56 of HA2 when the
hemagglutinin in the neutral pH conformation.
[0199] In some forms the hemagglutinin composition is conjugated
(e.g. a chimeric peptide) having one or more peptides or peptide
fragments linked to a backbone where peptides or peptide fragments
are spatially positioned relative to each other so that they
together form a non-linear sequence which mimics the tertiary
structure of an F10 epitope. wherein said conjugate competes with
the binding of monoclonal antibody F10 to the HA protein. The
backbone is a peptide backbone wherein peptides corresponding to
segments of native hemagglutinin are coupled to form an epitope
mimicking an the F10 epitope. Alternatively the peptide backbone
mimics the structure of the native hemagglutin protein such that
peptides are coupled to for form an epitope mimicking an the F10
epitope. Optionally, the backbone is a non-peptide backbone having
two or more attachement points onto which peptides are coupled.
See, WO9738011, the contents of which are incorporated by
reference.
[0200] The peptides or peptide fragments that make up the F10
epitope include one or more of the following peptides
TABLE-US-00023 (SEQ ID NO: 125) a)
[Xaa.sub.0].sub.m-Xaa.sub.1-Xaa.sub.2- [Xaa.sub.0].sub.p
wherein, m, and p are independently 0 or 1-10, Xaa.sub.0, is
independently any amino acid,
[0201] Xaa.sub.1 is S, T, F H or Y, and
[0202] Xaa.sub.2 is H, Y, M, L or Q;
TABLE-US-00024 (SEQ ID NO: 126) b)
[Xaa.sub.0].sub.m-Xaa.sub.1-Xaa.sub.2- [Xaa.sub.0].sub.p
wherein, m, and p are independently 0 or 1-10, Xaa.sub.0, is
independently any amino acid, and
[0203] Xaa.sub.1 is H, Q, Y, S, D, N or T,
[0204] Xaa.sub.2 is Q, E, K, I, V, M, E, R or T;
TABLE-US-00025 (SEQ ID NO: 127) c)
[Xaa.sub.0].sub.m-Xaa.sub.1-Xaa.sub.2-Xaa.sub.3-Xaa.sub.4-
[Xaa.sub.0].sub.p
wherein, m, and p are independently 0 or 1-10, Xaa.sub.0, is
independently any amino acid, and
[0205] Xaa.sub.1 is I, V, M, or L;
[0206] Xaa.sub.2 is D, N, H, Y, D, A, S or E,
[0207] Xaa.sub.3 is G or A, and
[0208] Xaa.sub.4 is W, R, or G; or
TABLE-US-00026 (SEQ ID NO: 128) d)
[Xaa.sub.0].sub.m-Xaa.sub.1-[Xaa.sub.0].sub.q
Xaa.sub.2-Xaa.sub.3-[Xaa.sub.0].sub.q Xaa.sub.4-[Xaa.sub.0].sub.r
Xaa.sub.5-[Xaa.sub.0].sub.q-Xaa.sub.6 Xaa.sub.7 -[Xaa.sub.0].sub.q
- Xaa.sub.8 -[Xaa.sub.0].sub.p
wherein, m, and p are independently 0 or 1-10, q is 2, and r is 3
Xaa.sub.0, is independently any amino acid, and
[0209] Xaa.sub.1 is K, Q, R, N, L, G, F, H or Y,
[0210] Xaa.sub.2 is S or T,
[0211] Xaa.sub.3 is Q or P,
[0212] Xaa.sub.0 is F, V, I, M, L, or T,
[0213] Xaa.sub.5 is I, T, S, N, Q, D, or A,
[0214] Xaa.sub.6 is I, V, M, or L,
[0215] Xaa.sub.7 is N, S, T, or D
[0216] Xaa.sub.8 is I, F, V, A, or T;
TABLE-US-00027 (SEQ ID NO: 129) e)
[Xaa.sub.0].sub.m-Xaa.sub.1-[Xaa.sub.0].sub.q
Xaa.sub.2-Xaa.sub.3-[Xaa.sub.0].sub.q Xaa.sub.4-[Xaa.sub.0].sub.r
Xaa.sub.5-[Xaa.sub.0].sub.q-Xaa.sub.6 Xaa.sub.7 -[Xaa.sub.0].sub.s
- [Xaa.sub.8].sub.t -[Xaa.sub.0].sub.p
wherein, m, and p are independently 0 or 1-10, q is 2, r is 3, s is
0 or 2, and t is 0 or 1, Xaa.sub.0, is independently any amino
acid, and
[0217] Xaa.sub.1 is K, Q, R, N, L, G, F, H or Y,
[0218] Xaa.sub.2 is S or T,
[0219] Xaa.sub.3 is Q or P,
[0220] Xaa.sub.4 is F, V, I, M, L, or T,
[0221] Xaa.sub.5 is I, T, S, N, Q, D, or A,
[0222] Xaa.sub.6 is I, V, M, or L,
[0223] Xaa.sub.7 is N, S, T, or D,
[0224] Xaa.sub.8 I, F, V, A, or T.
[0225] The peptide can be a linear peptide of a cyclic peptide.
Linear peptide can be prepared synthetically and then screened for
a particular characteristic in various biological assays. E.g.,
Scott, J. K. and G. P. Smith, Science 249:386, 1990; Devlin, J. J.,
et al., Science 24:404, 1990; Furka, A. et al., Int. J. Pept.
Protein Res. 37:487, 1991; Lam, K. S., et al., Nature 354:82,
1991.
[0226] Cyclized peptides are often found to possess superior
immunogenic activity compared to linear peptide immunogens. Linear
peptide immunogens comprising three or more core sequences are
found to bind with the terminal sequences only, while cyclization
allows binding by all core sequences present in the peptide
immunogens. Various methods for producing cyclic peptides have been
described. One involves solution or liquid phase peptide synthesis,
where amino acid residues in solution are linked by peptide bonds,
with reactive groups not involved in the peptide bond formation,
such as the amino group of the N-terminal residue, the carboxy
group of the C-terminal residue, sulfhydryl groups on cysteine
residues and similar or other reactive groups in the amino acid
side chains, protected by suitable protecting groups.
[0227] In one embodiment cyclic peptide immunogens are formed using
terminal cysteine residues by reduction of thiol groups to form
disulfide bridges.
[0228] Another approach involves solid phase peptide synthesis, in
which synthesis is carried out on an insoluble solid matrix.
Protecting groups are employed for reactive side chains. The
general methodology of solid phase synthesis is well known in the
art. Merrifield, R. B., Solid phase synthesis (Nobel lecture).
Angew Chem 24:799-810 (1985) and Barany et al., The Peptides,
Analysis, Synthesis and Biology, Vol. 2, Gross, E. and Meienhofer,
J., Eds. Academic Press 1-284 (1980). For example, chemical
reaction protocols, such as those described in U.S. Pat. Nos.
4,033,940 and 4,102,877, have been devised to produce circularized
peptides. In other techniques, biological and chemical methods are
combined to produce cyclic peptides. These latter methods involve
first expressing linear precursors of cyclic peptides in cells
(e.g., bacteria) to produce linear precursors of cyclic peptides
and then adding of an exogenous agent such as a protease or a
nucleophilic reagent to chemically convert these linear precursors
into cyclic peptides. See, e.g., Camerero, J. A., and Muir, T. W.,
J. Am. Chem. Society. 121:5597 (1999); Wu, H. et al, Proc. Natl.
Acad. Sci. USA, 95:9226 (1998).
[0229] Head-to-tail (backbone) peptide cyclization has been used to
rigidify structure and improve in vivo stability of small bioactive
peptides (see Camarero and Muir, J. Am. Chem. Soc., 121:5597-5598
(1999)). An important consequence of peptide cyclization is
retention of biological activity and/or the identification of new
classes of pharmacological agents. A chemical cross-linking
approach was used to prepare a backbone cyclized version of bovine
pancreatic trypsin inhibitor (Goldenburg and Creighton, J. Mol.
Biol., 165:407-413 (1983)). Other approaches include chemical
(Camarero et al., Angew. Chem. Int. Ed., 37:347-349 (1998); Tam and
Lu, Prot. Sci., 7:1583-1592 (1998); Camarero and Muir, Chem.
Commun., 1997:1369-1370 (1997); and Zhang and Tam, J. Am. Chem.
Soc. 119:2363-2370 (1997)) and enzymatic (Jackson et al., J. Am.
Chem. Soc., 117:819-820 (1995)) intramolecular ligation methods
which allow linear synthetic peptides to be efficiently cyclized
under aqueous conditions.
[0230] A native chemical ligation approach utilizes inteins
(internal proteins) to catalyze head-to-tail peptide and protein
ligation in vivo (see, for example, Evans et al., J. Biol. Chem.
274:18359-18363 (1999); Iwai and Pluckthun, FEBS Lett. 459.166-172
(1999); Wood et al., Nature Biotechnology 17:889-892 (1999);
Camarero and Muir, J. Am. Chem. Soc. 121:5597-5598 (1999); and
Scott et al., Proc. Natl. Acad. Sci. USA 96:13638-13643
(1999)).
[0231] Preparation of vaccines, which contain hemagglutinin
compositions as active ingredients, is generally well understood in
the art as exemplified by U.S. Pat. Nos. 4,608,251; 4,601,903;
4,599,231; 4,599,230, 4,596,792, and 4,578,770, all incorporated
herein by reference.
[0232] The hemagglutinin composition used in the vaccinal strategy
according to the present invention can also be obtained using
genetic engineering methods. The one skilled in the art can refer
to the known sequence of the phage insert that expresses a specific
epitope unit of an immunogenic polypeptide mimic of the invention
and also to the general literature to determine the appropriate
codons that can be used to synthesize the desired peptide. There is
no need to say that the expression of the polynucleotide that
encodes the immunogenic polypeptide mimic of interest may be
optimized, according to the organism in which the sequence has to
be expressed and the specific codon usage of this organism (mammal,
plant, bacteria, etc.). For bacteria and plant, respectively, the
general codon usages can be found in European patent application
No. EP 0 359 472 (Mycogen).
[0233] As an alternative embodiment, the epitope unit according to
the present invention is recombinantly expressed as a part of
longer polypeptide that serves as a carrier molecule. Specifically,
the polynucleotide coding for the immunogenic polypeptide of the
invention, for example a polypeptide having an amino acid length
between 10 and 200 amino acid residues, is inserted at least one
permissive site of the polynucleotide coding for the Bordetella
cyaA adenylate cyclase, for example, at a nucleotide position
located between amino acids 235 and 236 of the Bordetella adenylate
cyclase. Such a technique is fully described in the U.S. Pat. No.
5,503,829 granted on Apr. 2, 1996 (Leclerc et al.).
[0234] In another embodiment of the hemagglutinin composition
according to the present invention, the nucleotide sequence coding
for the desired immunogenic polypeptide carrying one or more
epitope units is inserted in the nucleotide sequence coding for
surface protein of Haemophilus influenza, such as described in PCT
Application No. PCT/US96/17698 (The Research Foundation of State
University of New York), which is incorporated by reference
herein.
[0235] In another embodiment of the hemagglutinin composition
according to the invention, the composition comprises a
polynucleotide coding for the immunogenic polypeptide or oligomeric
peptide of pharmaceutical interest.
[0236] For the purpose of the present invention, a specific
embodiment of comprises the in vivo production of a hemagglutinin
composition for example in an oligomeric form by the introduction
of the genetic information in the mammal organism, specifically in
the patient organism. This genetic information can be introduced in
vitro in a cell that has been previously extracted from the
organism, the modified cell being subsequently reintroduced in the
said organism directly in vivo into the appropriate tissue. The
method for delivering the corresponding protein or peptide to the
interior of a cell of a vertebrate in vivo comprises the step of
introducing a preparation comprising a pharmaceutically acceptable
injectable carrier and a polynucleotide operatively coding for the
polypeptide into the interstitial space of a tissue comprising the
cell, whereby the polynucleotide is taken up into the interior of
the cell and has a pharmaceutical effect.
[0237] In a specific embodiment, the invention provides a
hemagglutinin composition comprising a polynucleotide operatively
coding for the ypeptide of interest or one of its above-described
peptides in solution in a physiologically acceptable injectable
carrier and suitable for introduction interstitially into a tissue
to cause cells of the tissue to express the said protein or
polypeptide.
[0238] The polynucleotide operatively coding for the hemagglutinin
composition, mimic or oligomeric peptide can be a vector comprising
the genomic DNA or the complementary DNA (cDNA) coding for the
corresponding protein or its protein derivative and a promoter
sequence allowing the expression of the genomic DNA or the
complementary DNA in the desired eukaryotic cells, such as
vertebrate cells, specifically mammalian cells. The vector
component of a therapeutic composition according to the present
invention is advantageously a plasmid, a part of which is of viral
or bacterial origin, which carries a viral or a bacterial origin of
replication and a gene allowing its selection, such as an
antibiotic resistance gene. By "vector" according to this specific
embodiment of the invention is intended a circular or linear DNA
molecule. This vector can also contain an origin of replication
that allows it to replicate in the eukaryotic host cell, such as an
origin of replication from a bovine papillomavirus.
[0239] Therapeutic compositions comprising a polynucleotide are
described in PCT application No. WO 90/11092 (Vical Inc.), and also
in PCT application No. WO 95/11307 (Institut Pasteur, INSERM,
Universite d'Ottawa), as well as in the articles of Tacson et al.
(1996, Nature Medicine, 2(8):888-892) and of Huygen et al. (1996,
Nature Medicine, 2(8):893-898).
[0240] In another embodiment, the DNA to be introduced is complexed
with DEAE-dextran (Pagano et al., 1967, J. Virol., 1:891) or with
nuclear proteins (Kaneda et al., 1989, Science, 243:375), with
lipids (Felgner et al., 1987, Proc. Natl. Acad. Sci., 84:7413), or
encapsulated within liposomes (Fraley et al., 1980, J. Biol. Chem.,
255:10431).
[0241] In another embodiment, the therapeutic polynucleotide can be
included in a transfection system comprising polypeptides that
promote its penetration within the host cells as described in PCT
application No. WO 95/10534 (Seikagaku Corporation).
[0242] The therapeutic polynucleotide and vector according to the
present invention can advantageously be administered in the form of
a gel that facilitates transfection into the cells. Such a gel
composition can be a complex of poly-L-lysine and lactose as
described by Midoux (1993, Nucleic Acids Research, 21:871-878) or
also poloxamer 407 as described by Pastore (1994, Circulation,
90:1-517). The therapeutic polynucleotide and vector according to
the invention can also be suspended in a buffer solution or be
associated with liposomes.
[0243] Thus, the polynucleotide and vector according to the
invention are used to make pharmaceutical compositions for
delivering the DNA (genomic DNA or cDNA) coding for the immunogenic
polypeptide mimic of the invention at the site of the injection.
The amount of the vector to be injected varies according to the
site of injection. As an indicative dose, the vector can be
injected in an amount of about 0.1 and about 100 .mu.g of the
vector in a patient.
[0244] In another embodiment of the therapeutic polynucleotide
according to the invention, the polynucleotide can be introduced in
vitro into a host cell, preferably in a host cell previously
harvested from the patient to be treated, and more preferably a
somatic cell such as a muscle cell. In a subsequent step, the cell
that has been transformed with the vaccinal nucleotide coding for
the immunogenic polypeptide of the invention is implanted back into
the patient in order to deliver the recombinant protein within the
body either locally or systemically.
[0245] Consequently, the present invention also concerns an
immunogenic composition comprising a polynucleotide or an
expression vector as described hereinabove in combination with a
pharmaceutically acceptable vehicle allowing its administration to
the human or other animal. A further embodiment of the invention
comprises a vaccine composition comprising a polynucleotide or a
vector as described above in combination with a pharmaceutically
acceptable vehicle allowing its administration to the human or the
animal.
[0246] The approach of increasing immunogenicity of small
immunogenic molecules by conjugating these molecules to large
"carrier" molecules has been utilized successfully for decades
(see, e.g., Goebel et al. (1939) J. Exp. Med. 69: 53). For example,
many immunogenic compositions have been described in which purified
capsular polymers have been conjugated to carrier proteins to
create more effective immunogenic compositions by exploiting this
"carrier effect." Schneerson et al. (1984) Infect. Immun. 45:
582-591).
[0247] In one aspect of the invention, method for conjugating a
hemagglutinin composition, e.g. immunogen according to the
invention via a reactive group of an amino acid residue of the
peptide immunogen to a protein/polypeptide carrier having one or
more functional groups is provided. The protein/polypeptide carrier
may be human serum albumin, keyhole limpet hemocyanin (KLH),
immunoglobulin molecules, thyroglobulin, ovalbumin, influenza
hemagglutinin, PAN-DR binding peptide (PADRE polypeptide), malaria
circumsporozite (CS) protein, hepatitis B surface antigen
(HBSAg.sub.19-28, Heat Shock Protein (HSP) 65, Bacillus
Calmette-Guerin (BCG), cholera toxin, cholera toxin mutants with
reduced toxicity, diphtheria toxin, CRM.sub.197 protein that is
cross-reactive with diphtheria toxin, recombinant Streptococcal C5a
peptidase, Streptococcus pyogenes ORF1224, Streptococcus pyogenes
ORF1664, Streptococcus pyogenes ORF 2452, Chlamydia pneumoniae ORF
T367, Chlamydia pneumoniae ORF T858, Tetanus toxoid, HIV gp120 T1,
microbial surface components recognizing adhesive matrix molecules
(MSCRAMMS), growth factor/hormone, cytokines or chemokines
[0248] The hemagglutinin composition, e.g. immunogen can be used as
vaccines to generate an anti-influenza HA-mediated immune response
in order to prevent influenza infections. Synthetic peptides
require both stabilization and adjuvantation for the induction of
an effective immune response in vivo. Various methods have been
employed to protect synthetic peptide immunogens against
degradation in vitro and in vivo, mediated by various processes
including chemical and physical pathways. (Manning M C, et al.
Pharmaceutical Research, 1989, 6:903-918).
[0249] Numerous adjuvants and/or depot-based parenteral, mucosal or
transdermal delivery systems destined for use with human or
veterinary vaccines have been developed to enhance the immune
response. These include the use of mineral salts, water-in-oil
(w/o)-emulsions, liposomes, polymeric microparticles, nanoparticles
and gels/hydrogels. (Cox J C, et al. Vaccine, 1997, 15:248-256).
Freund's complete adjuvant (FCA), a suspension of heat-killed M.
tuberculosis mycobacteria in mineral oil containing a surfactant,
has been recognized as one of the most powerful adjuvants.
Adjuvants are well known in the art (Vaccine Design--The Subunit
and Adjuvant Approach, 1995, Pharmaceutical Biotechnology, Volume
6, Eds. Powell, M. F., and Newman, M. J., Plenum Press, New York
and London, ISBN 0-306-44867-X). Preferred adjuvants for use with
immunogens of the present invention include aluminium or calcium
salts (hydroxide or phosphate). Adjuvants may be selected from
GM-CSF, 529 SE, IL-12, aluminum phosphate, aluminum hydroxide,
Mycobacterium tuberculosis, Bordetella pertussis, bacterial
lipopolysaccharides, aminoalkyl glucosane phosphate compounds,
MPL.TM. (3-O-deacylated monophosphoryl lipid A), a polypeptide,
Quil A, STIMULON.TM. QS-21, a pertussis toxin (PT), an E. coli
heat-labile toxin (LT), IL-1alpha, IL-1.beta., IL-2, IL-4, IL-5,
IL-6, IL-7, IL-8, IL-10, IL-13, IL-14, IL-15, IL-16, IL-17, IL-18,
interferon-alpha, interferon-B, interferon-gamma, G-CSF, TNF-alpha
and TNF-B.
[0250] Still other adjuvants include mineral oil and water
emulsions, calcium salts such as calcium phosphate, aluminum salts
(alum), such as aluminum hydroxide, aluminum phosphate, etc.,
Amphigen, Avridine, L121/squalene, D-lactide-polylactide/glycoside,
pluronic acids, polyols, muramyl dipeptide, killed Bordetella,
saponins, such as Stimulon.TM. QS-21 (Antigenics, Framingham,
Mass.), described in U.S. Pat. No. 5,057,540, which is hereby
incorporated by reference3, and particles generated therefrom such
as ISCOMS (immunostimulating complexes), Mycobacterium
tuberculosis, bacterial lipopolysaccharides, synthetic
polynucleotides such as oligonucleotides containing a CpG motif
(U.S. Pat. No. 6,207,646, which is hereby incorporated by
reference), a pertussis toxin (PT), or an E. coli heat-labile toxin
(LT), particularly LT-K63, LT-R72, PT-K9/G129; see, e.g.,
International Patent Publication Nos. WO 93/13302 and WO 92/19265,
which are/incorporated herein by reference for all purposes.
[0251] Also useful as adjuvants are cholera toxins and mutants
thereof, including those described in published International
Patent Application No. WO 00/18434 (wherein the glutamic acid at
amino acid position 29 is replaced by another amino acid (other
than aspartic acid, preferably a histidine). Similar CT toxins or
mutants are described in published International Patent Application
number WO 02/098368 (wherein the isoleucine at amino acid position
16 is replaced by another amino acid, either alone or in
combination with the replacement of the serine at amino acid
position 68 by another amino acid; and/or wherein the valine at
amino acid position 72 is replaced by another amino acid). Other CT
toxins are described in published International Patent Application
number WO 02/098369 (wherein the arginine at amino acid position 25
is replaced by another amino acid; and/or an amino acid is inserted
at amino acid position 49; and/or two amino acids are inserted at
amino acid position 35 and 36).
[0252] Various methods may be employed to adjuvant synthetic
peptide-based immunogens, but normally a carrier or depot system is
required for effective long-term immunogenic responses. Notable
examples include adsorbing the immunogen onto a mineral salt or
gel. For example, encapsulating a peptide immunogen within a
polymeric matrix (monolithic matrix) or gel, or layering a
polymeric material around a peptide immunogen (core-shell) may be
an effective strategy. Or, an immunogen may be incorporated in a
liposome or vesicular type of formulation, with the immunogen
either embedded in the lipid matrix or physically entrapped in the
internal aqueous phase. Another strategy may employ a
mineral-based, vegetable-based or animal-based oil, with an aqueous
solution of the immunogen in various proportions, to prepare a
water-in-oil (w/o)-emulsion or a water-in-oil-in-water
(w/o/w)-double emulsion. Powell M F, et al., Pharmaceutical
Biotechnology, Vol. 6, Plenum Press, New York, 1995
[0253] In some forms, the head region of the hemagglutinin can be
modified by removing or replacing glycosylation sites. In some
forms, the head region of the hemagglutinin can be modified by
adding glycosylation sites. In some forms, the head region of the
hemagglutinin can be modified by removing all or a portion of the
head region.
[0254] In some forms, the hemagglutinin composition can be or be
derived from hemagglutinin from a group 2 influenza virus. In some
forms, the hemagglutinin composition can be or be derived form
hemagglutinin from a group 1 influenza virus. In some forms, the
hemagglutinin composition can be or be derived from hemagglutinin
from a particular cluster, subcluster or subtype of influenza
virus. In some forms, the hemagglutinin composition can be or be
derived from a combination of hemagglutinins from a combination of
particular clusters, subclusters and/or subtypes of influenza
virus.
[0255] In some forms, the disclosed hemagglutinin compositions can
produce an immune reaction in a subject. For example, in some
forms, the subject can produce an immune response that prevents or
reduces the severity of an influenza infection. In some forms, the
immune response can be reactive to influenza viruses within a
subtype. In some forms, the immune response can be reactive to
influenza viruses in each subtype within a cluster. In some forms,
the immune response can be reactive to influenza viruses in each
cluster within a group. In some forms, the immune response can be
reactive to all influenza viruses in each subtype within a group.
In some forms, the immune response can be reactive to influenza
viruses within group 1.
[0256] Disclosed are hemagglutinin compositions comprising, for
example, hemagglutinin, a trimeric ectodomain of hemagglutinin, the
extracellular portion of hemagglutinin, a trimeric stem region of
hemagglutinin lacking all or a portion or portions of the head
structure, or the hemagglutinin stem region in isolation from other
parts of hemagglutinin. Disclosed are hemagglutinin compositions
comprising, for example, hemagglutinin, a trimeric ectodomain of
hemagglutinin, the extracellular portion of hemagglutinin, or a
trimeric stem region of hemagglutinin lacking a portion or portions
of the head structure where the head region of the hemagglutinin is
modified by removing and/or replacing one or more glycosylation
sites. Disclosed are hemagglutinin compositions comprising, for
example, hemagglutinin, a trimeric ectodomain of hemagglutinin, the
extracellular portion of hemagglutinin, or a trimeric stem region
of hemagglutinin lacking a portion or portions of the head
structure where the head region of the hemagglutinin is modified by
adding one or more glycosylation sites. Disclosed are hemagglutinin
compositions comprising, for example, hemagglutinin, a trimeric
ectodomain of hemagglutinin, the extracellular portion of
hemagglutinin, or a trimeric stem region of hemagglutinin lacking a
portion or portions of the head structure where the head region of
the hemagglutinin is modified by removing and/or replacing one or
more glycosylation sites and by adding one or more glycosylation
sites. In some forms, the head region of the hemagglutinin can be
modified by removing all or a portion of the head region. For
example, some or all of the amino acids of the head regions in a
trimeric ectodomain of hemagglutinin can be removed to leave
portions of the head region of loops between the stem region
portions of the trimer.
[0257] It has been discovered that hemagglutinin bound or
immobilized on a substrate or surface exposes or presents the stem
region epitope(s) of hemagglutinin for effective binding by
antibodies. This allowed the identification of a number of
broad-spectrum neutralizing antibodies against hemagglutinin and
against influenza virus. Accordingly, disclosed are compositions
comprising hemagglutinin composition bound or immobilized on a
substrate or surface. Binding, attachment, and/or immobilization of
proteins to substrates, solid supports an surfaces is a well
established art and those of skill in the art can use any known
techniques, chemistries and materials to bind hemagglutinin and
hemagglutinin compositions to any suitable material.
[0258] Hemagglutinin and hemagglutinin compositions immobilized on
a solid support can also be used, for example, to generate an
immune response in a subject. For example, beads, microparticles or
nanoparticles with hemagglutinin composition bound to the surface
can be administered to subjects. Because of the discovery that
immobilized hemagglutinin can be bound by antibodies specific for
the hemagglutinin stem region, such compositions can be used to
generate an immune response in a subject. Such an immune response
can produce antibodies and immune system components that can
inhibit influenza virus.
[0259] A influenza protein (e.g., HA or neuramindase), or a
derivative, fragment, analog, homolog or ortholog thereof, can be
utilized as an immunogen in the generation of antibodies that
immunospecifically bind these protein components. The disclosed
hemagglutinin compositions are an example.
[0260] Solid supports or surface are solid-state substrates,
compositions, surfaces and/or supports with which molecules, such
as hemagglutinin, hemagglutinin compositions and/or antibodies, can
be associated. Molecules can be associated with solid supports
directly or indirectly. For example, molecules can be bound to the
surface of a solid support or associated with capture agents (e.g.,
compounds or molecules that bind an analyte) immobilized on solid
supports. An array is a solid support to which multiple molecules
have been associated in an array, grid, or other organized
pattern.
[0261] Solid-state substrates for use in solid supports can include
any solid material with which components can be associated,
directly or indirectly. This includes materials such as acrylamide,
agarose, cellulose, nitrocellulose, glass, gold, polystyrene,
polyethylene vinyl acetate, polypropylene, polymethacrylate,
polyethylene, polyethylene oxide, polysilicates, polycarbonates,
teflon, fluorocarbons, nylon, silicon rubber, polyanhydrides,
polyglycolic acid, polylactic acid, polyorthoesters, functionalized
silane, polypropylfumerate, collagen, glycosaminoglycans, and
polyamino acids. Solid-state substrates can have any useful form
including thin film, membrane, bottles, dishes, fibers, woven
fibers, shaped polymers, particles, beads, microparticles, or a
combination. Solid-state substrates and solid supports can be
porous or non-porous. A chip is a polygonal (e.g., rectangular,
square, triangular, circular) small piece of material. Useful forms
for solid-state substrates are plates, dishes, thin films, beads,
or chips. A useful form for a solid-state substrate is a microtiter
dish. In some embodiments, a multiwell glass slide can be
employed.
[0262] An array can include a plurality of molecules immobilized at
identified or predefined locations on the solid support. Each
predefined location on the solid support generally has one type of
component (that is, all the components at that location are the
same). Alternatively, multiple types of components can be
immobilized in the same predefined location on a solid support.
Each location will have multiple copies of the given components.
The spatial separation of different components on the solid support
allows separate detection and identification.
[0263] Although useful, it is not required that the solid support
be a single unit or structure. A set of molecules can be
distributed over any number of solid supports. For example, at one
extreme, each component can be immobilized in a separate reaction
tube or container, or on separate beads or microparticles.
[0264] Each of the components immobilized on the solid support can
be located in a different predefined region of the solid support.
The different locations can be different reaction chambers. Each of
the different predefined regions can be physically separated from
each other of the different regions. The distance between the
different predefined regions of the solid support can be either
fixed or variable. For example, in an array, each of the components
can be arranged at fixed distances from each other, while
components associated with beads will not be in a fixed spatial
relationship. In particular, the use of multiple solid support
units (for example, multiple beads) will result in variable
distances.
[0265] Components can be associated or immobilized on a solid
support at any density. Components can be immobilized to the solid
support at a density exceeding 400 different components per cubic
centimeter. Arrays of components can have any number of components.
For example, an array can have at least 1,000 different components
immobilized on the solid support, at least 10,000 different
components immobilized on the solid support, at least 100,000
different components immobilized on the solid support, or at least
1,000,000 different components immobilized on the solid
support.
C. Kits
[0266] The materials described above as well as other materials can
be packaged together in any suitable combination as a kit useful
for performing, or aiding in the performance of, the disclosed
method. It is useful if the kit components in a given kit are
designed and adapted for use together in the disclosed method. For
example disclosed are kits for identifying HA stem antibodies, the
kit comprising one or more of the antibodies D7, D8, F10, G17, H40,
A66, H98, D80, E90, and E8.
D. Mixtures
[0267] Disclosed are mixtures formed by performing or preparing to
perform the disclosed method. For example, disclosed are mixtures
comprising a cell and one or more of the antibodies D7, D8, F10,
G17, H40, A66, H98, D80, E90, and E8.
[0268] Whenever the method involves mixing or bringing into contact
compositions or components or reagents, performing the method
creates a number of different mixtures. For example, if the method
includes 3 mixing steps, after each one of these steps a unique
mixture is formed if the steps are performed separately. In
addition, a mixture is formed at the completion of all of the steps
regardless of how the steps were performed. The present disclosure
contemplates these mixtures, obtained by the performance of the
disclosed methods as well as mixtures containing any disclosed
reagent, composition, or component, for example, disclosed
herein.
E. Systems
[0269] Disclosed are systems useful for performing, or aiding in
the performance of, the disclosed method. Systems generally
comprise combinations of articles of manufacture such as
structures, machines, devices, and the like, and compositions,
compounds, materials, and the like. Such combinations that are
disclosed or that are apparent from the disclosure are
contemplated. For example, disclosed and contemplated are systems
comprising one or more of the antibodies D7, D8, F10, G17, H40,
A66, H98, D80, E90, and E8 and a machine to detect antibody
binding.
F. Data Structures and Computer Control
[0270] Disclosed are data structures used in, generated by, or
generated from, the disclosed method. Data structures generally are
any form of data, information, and/or objects collected, organized,
stored, and/or embodied in a composition or medium. An antibody
titre stored in electronic form, such as in RAM or on a storage
disk, is a type of data structure.
[0271] The disclosed method, or any part thereof or preparation
therefor, can be controlled, managed, or otherwise assisted by
computer control. Such computer control can be accomplished by a
computer controlled process or method, can use and/or generate data
structures, and can use a computer program. Such computer control,
computer controlled processes, data structures, and computer
programs are contemplated and should be understood to be disclosed
herein.
Uses
[0272] The disclosed methods and compositions are applicable to
numerous areas including, but not limited to, identifying and
confirming the potency of HA stem antibodies, passive immunization
with the HA stem antibodies and active immunization using the
disclosed hemagglutinin compositions. Other uses include as a
control for detecting HA stem antibodies in a body fluid. Other
uses are disclosed, apparent from the disclosure, and/or will be
understood by those in the art.
Method
[0273] Disclosed are methods of treating subjects, methods of
screening and producing antibodies and methods of screening for F10
mimitopes useful as immunogens. For example, disclosed is a method
of treating a subject suffering or at risk of influenza infection,
the method comprising administering to the subject one or more of
the disclosed hemagglutinin compositions and/or antibodies, such as
the disclosed HA stem antibodies. For example, disclosed is a
method of treating a subject, the method comprising administering
to the subject the stem region of influenza hemagglutinin in the
neutral pH conformation in isolation from other components of
influenza virus, wherein the subject produces an immune response to
the stem region. For example, disclosed is a method of treating a
subject, the method comprising administering to the subject the
stem region of influenza hemagglutinin in the neutral pH
conformation in isolation from the head region of hemagglutinin,
wherein the subject produces an immune response to the stem region.
For example, disclosed is a method of treating a subject, the
method comprising administering to the subject influenza
hemagglutinin in the neutral pH conformation in isolation from
other components of influenza virus, wherein the head region of the
hemagglutinin is modified to reduce the antigenicity of the head
region, wherein the subject produces an immune response to the stem
region. For example, disclosed is a method for treating a subject
by administering a hemagglutinin composition. The hemagglutinin
composition according to the invention contain an epitope or
epitope unit recognized by a protective monoclonal antibody having
the specificity for the stem region of hemagglutinin protein of an
influenza virus. A protective monoclonal antibody having the
specificity for the stem region of hemagglutinin protein of an
influenza virus includes monoclonal antibody D7, D8, F10, G17, H40,
A66, D80, E88, E90, or H98 disclosed herein or a monoclonal
antibody that competes with the binding of monoclonal antibody D7,
D8, F10, G17, H40, A66, D80, E88, E90, or H98 to the HA protein.
The antibody binds both the HA1 and HA2 peptide.
[0274] An epitope or epitope unit is for example the F10 epitope.
By F10 epitope it is meant the epitope recognized by the F10
antibody disclosed herein. The epitope is a confirmation epitope
defined by amino acids of the HA1 and HA2 peptide of hemagglutinin
protein of the influenza virus. The hemagglutinin protein is in the
neutral pH conformation. Specifically, the F10 epitope is defined
by amino acid residues 18, 38, 39, 40 and 291 of HA1 and 18, 19,
20, 21, 38, 41, 42, 45, 49, 52, 53, and 56 of HA2 when the
hemagglutinin in the neutral pH conformation.
[0275] In some forms the hemagglutinin composition is conjugated
(e.g. a chimeric peptide) having one or more peptides or peptide
fragments linked to a backbone where peptides or peptide fragments
are spatially positioned relative to each other so that they
together form a non-linear sequence which mimics the tertiary
structure of an F10 epitope. wherein said conjugate competes with
the binding of monoclonal antibody F10 to the HA protein. The
backbone is a peptide backbone wherein peptides corresponding to
segments of native hemagglutinin are coupled to form an epitope
mimicking an the F10 epitope. Alternatively the peptide backbone
mimics the structure of the native hemagglutin protein such that
peptides are coupled to for form an epitope mimicking an the F10
epitope. Optionally, the backbone is a non-peptide backbone having
two or more attachement points onto which peptides are coupled.
See, WO9738011, the contents of which are incorporated by
reference.
[0276] The peptides or peptide fragments that make up the F10
epitope include one or more of the following peptides
TABLE-US-00028 (SEQ ID NO: 125) a)
[Xaa.sub.0].sub.m-Xaa.sub.1-Xaa.sub.2- [Xaa.sub.0].sub.p
wherein, m, and p are independently 0 or 1-10, Xaa.sub.0, is
independently any amino acid,
[0277] Xaa.sub.1 is S, T, F H or Y, and
[0278] Xaa.sub.2 is H, Y, M, L or Q;
TABLE-US-00029 (SEQ ID NO: 126) b)
[Xaa.sub.0].sub.m-Xaa.sub.1-Xaa.sub.2- [Xaa.sub.0].sub.p
wherein, m, and p are independently 0 or 1-10, Xaa.sub.0, is
independently any amino acid, and
[0279] Xaa.sub.1 is H, Q, Y, S, D, N or T,
[0280] Xaa.sub.2 is Q, E, K, I, V, M, E, R or T;
TABLE-US-00030 (SEQ ID NO: 127) c)
[Xaa.sub.0].sub.m-Xaa.sub.1-Xaa.sub.2-Xaa.sub.3-Xaa.sub.4-
[Xaa.sub.0].sub.p
wherein, m, and p are independently 0 or 1-10, Xaa.sub.0, is
independently any amino acid, and
[0281] Xaa.sub.1 is I, V, M, or L;
[0282] Xaa.sub.2 is D, N, H, Y, D, A, S or E,
[0283] Xaa.sub.3 is G or A, and
[0284] Xaa.sub.4 is W, R, or G; or
TABLE-US-00031 d) (SEQ ID NO: 128)
[Xaa.sub.0].sub.m-Xaa.sub.1-[Xaa.sub.0].sub.q
Xaa.sub.2-Xaa.sub.3-[Xaa.sub.0].sub.q Xaa.sub.4-[Xaa.sub.0].sub.r
Xaa.sub.5-[Xaa.sub.0].sub.q-Xaa.sub.6 Xaa.sub.7 -[Xaa.sub.0].sub.q
- Xaa.sub.8 -[Xaa.sub.0].sub.p
wherein, m, and p are independently 0 or 1-10, q is 2, and r is 3
Xaa.sub.0, is independently any amino acid, and
[0285] Xaa.sub.1 is K, Q, R, N, L, G, F, H or Y,
[0286] Xaa.sub.2 is S or T,
[0287] Xaa.sub.3 is Q or P,
[0288] Xaa.sub.4 is F, V, I, M, L, or T,
[0289] Xaa.sub.5 is I, T, S, N, Q, D, or A,
[0290] Xaa.sub.6 is I, V, M, or L,
[0291] Xaa.sub.7 is N, S, T, or D
[0292] Xaa.sub.8 is I, F, V, A, or T;
TABLE-US-00032 e) (SEQ ID NO: 129)
[Xaa.sub.0].sub.m-Xaa.sub.1-[Xaa.sub.0].sub.q
Xaa.sub.2-Xaa.sub.3-[Xaa.sub.0].sub.q Xaa.sub.4-[Xaa.sub.0].sub.r
Xaa.sub.5-[Xaa.sub.0].sub.q-Xaa.sub.6 Xaa.sub.7 -[Xaa.sub.0].sub.s
- [Xaa.sub.8].sub.t -[Xaa.sub.0].sub.p
wherein, m, and p are independently 0 or 1-10, q is 2, r is 3, s is
0 or 2, and t is 0 or 1, Xaa.sub.0, is independently any amino
acid, and
[0293] Xaa.sub.1 is K, Q, R, N, L, G, F, H or Y,
[0294] Xaa.sub.2 is S or T,
[0295] Xaa.sub.3 is Q or P,
[0296] Xaa.sub.4 is F, V, I, M, L, or T,
[0297] Xaa.sub.5 is I, T, S, N, Q, D, or A,
[0298] Xaa.sub.6 is I, V, M, or L,
[0299] Xaa.sub.7 is N, S, T, or D,
[0300] Xaa.sub.8 I, F, V, A, or T.
[0301] Disclosed is a method, the method comprising screening
antibodies reactive to hemagglutinin for binding to hemagglutinin
immobilized on a surface, thereby identifying antibodies of
interest. For example, disclosed is a method comprising screening
antibodies reactive to hemagglutinin for binding to the stem region
of influenza hemagglutinin in the neutral pH conformation in
isolation from the head region of hemagglutinin, thereby
identifying antibodies of interest. For example, disclosed is a
method comprising screening antibodies reactive to hemagglutinin
for binding to influenza hemagglutinin in the neutral pH
conformation in isolation from other components of influenza virus,
wherein the head region of the hemagglutinin is modified to reduce
the antigenicity of the head region, thereby identifying antibodies
of interest.
[0302] In some forms, the head region of the hemagglutinin can be
modified by removing or replacing glycosylation sites. In some
forms, the head region of the hemagglutinin can be modified by
adding glycosylation sites. In some forms, the head region of the
hemagglutinin can be modified by removing all or a portion of the
head region.
[0303] Disclosed are antibodies that can be used, identified or
produced in the disclosed methods. For example, disclosed are
antibodies that bind to the stem region of influenza hemagglutinin
in the neutral pH conformation ("HA stem antibodies"). For example,
disclosed are antibodies that bind the epitope of influenza
hemagglutinin bound by antibody F10. For example, disclosed are
antibodies that bind the epitope of influenza hemagglutinin in the
neutral pH conformation defined by amino acid residues 18, 38, 39,
40 and 291 of HA1 and 18, 19, 20, 21, 38, 41, 42, 45, 49, 52, 53,
and 56 of HA2. For example, disclosed are antibodies that bind the
epitope of influenza hemagglutinin in the neutral pH conformation
defined by amino acid residues 17, 18, 38, 39, 40 and 291 of HA1
and 18, 19, 20, 21, 38, 41, 42, 45, 49, 52, 53, 56, and 111 of HA2.
For example, disclosed are antibodies that bind to every subtype
within an influenza virus group.
[0304] In some forms, the antibody can compete with antibody F10
for binding to hemagglutinin. In some forms, the antibody can have
a VH CDR2 sequence that is the same as the VH CDR2 sequence of
antibody D7, D8, F10, G17, H40 or A66 or of the consensus VH
sequence SEQ ID NO:1. In some forms, the antibody can have a VH
CDR3 sequence that is the same as the VH CDR3 sequence of antibody
D7, D8, F10, G17, H40 or A66 or of the consensus VH sequence SEQ ID
NO:1. In some forms, the antibody can have a VH CDR1 sequence that
is the same as the VH CDR1 sequence of antibody D7, D8, F10, G17,
H40 or A66 or of the consensus VH sequence SEQ ID NO:1. In some
forms, the antibody can have a VH sequence that is the same as the
VH sequence of antibody D7, D8, F10, G17, H40 or A66 or of the
consensus VH sequence SEQ ID NO:1. In some forms, the antibody can
have a VL sequence that is the same as the VL sequence of antibody
D7, D8, F10, G17, H40, A66, D80, E88, E90, or H98 or of the
consensus VL sequence SEQ ID NO:2. In some forms, the antibody can
have any combination of the VH FR1, VH CDR1, VH FR2, VH CDR2, VH
FR3, VH CDR3, and VH FR4 sequences of antibodies D7, D8, F10, G17,
H40 and A66 and the consensus VH sequence SEQ ID NO:1, and any
combination of the VL FR1, VL CDR1, VL FR2, VL CDR2, VL FR3, VL
CDR3, and VL FR4 sequences of antibodies D7, D8, F10, G17, H40,
A66, D80, E88, E90, and H98 and the consensus VL sequence SEQ ID
NO:2. In some forms, the antibody can prevent or inhibit virus-host
membrane fusion. In some forms, the antibody can prevent or inhibit
cell fusion mediated by cell surface-expressed influenza
hemagglutinin.
[0305] In some forms, the disclosed antibodies, the disclosed
hemagglutinins, and the disclosed methods can produce an immune
reaction in a subject. For example, in some forms, the subject can
produce an immune response that prevents or reduces the severity of
an influenza infection. In some forms, the immune response can be
reactive to influenza viruses within a subtype. In some forms, the
immune response can be reactive to influenza viruses in each
subtype within a cluster. In some forms, the immune response can be
reactive to influenza viruses in each cluster within a group. In
some forms, the immune response can be reactive to all influenza
viruses in each subtype within a group. In some forms, the immune
response can be reactive to influenza viruses within group 1.
[0306] In some forms, the disclosed methods can further comprise
screening the antibodies of interest for competing with antibody
F10 for binding to hemagglutinin, thereby identifying F10-competing
antibodies. In some forms, the hemagglutinin can be hemagglutinin
from a group 2 influenza virus. In some forms, the hemagglutinin
can be hemagglutinin from a group 1 influenza virus. In some forms,
the disclosed methods can further comprising producing the
identified antibodies. Also disclosed are antibodies produced by
the disclosed methods. Also disclosed are antibodies identified by
the disclosed methods.
[0307] In some forms, the disclosed methods can further comprise
screening a compound of interest, e.g., an F10 mimitope composition
for competing with antibody F10 for binding to hemagglutinin,
thereby identifying F10 mimitopes. The F10 mimitopes are useful as
immunogens. In some forms, the hemagglutinin can be hemagglutinin
from a group 2 influenza virus. In some forms, the hemagglutinin
can be hemagglutinin from a group 1 influenza virus. Also included
in the invention are F10 mimitopes identified by the disclosed
methods.
[0308] The disclosed antibodies and/or hemagglutinin compositions
can be administered to a subject alone or in combination prior to
and/or following exposure or possible exposure of the subject to
influenza virus. For example, the disclosed antibodies and/or
hemagglutinin compositions can be administered to a subject prior
to entering an area of infection or suspected infection, prior to
coming into the presence of infected subjects or subjects suspected
of infection, after entering an area of infection or suspected
infection, after coming into the presence of infected subjects or
subjects suspected of infection, prior to and after entering an
area of infection or suspected infection, or prior to and after
coming into the presence of infected subjects or subjects suspected
of infection.
[0309] As used herein the phrase "immune response" or its
equivalent "immunological response" refers to the development of a
humoral (antibody mediated) and/or a cellular (mediated by
antigen-specific T cells or their secretion products) response
directed against a target protein or polypeptide in a subject. Such
a response can be an active response induced by administration of
immunogen (such as the peptide immunogens described herein) or a
passive response induced by administration of antibody or primed
T-cells. A cellular immune response is elicited by the presentation
of polypeptide epitopes in association with Class I or Class II MHC
molecules, to activate antigen-specific CD4.sup.+ T helper cells
and/or CD8.sup.+ cytotoxic T cells. The response may also involve
activation of monocytes, macrophages, NK, cells, basophils,
dendritic cells, astrocytes, microglia cells, eosinophils or other
components of innate immunity.
[0310] As used herein "active immunity" refers to any immunity
conferred upon a subject by administration of an antigen, e.g. the
disclosed hemagglutinin compositions
[0311] As used herein "passive immunity" refers to any immunity
conferred upon a subject without administration of an antigen.
"Passive immunity" therefore includes, but is not limited to,
administration of a an antibody, such as the disclosed antibodies
and, for example, a HA stem antibody, or, for example, a
replicating display vehicle which includes an immunological portion
of an antibody presented on its surface to a subject. Although
replication of such a vehicle is active, the immune response is
passive from the standpoint of the subject.
[0312] Passive immunization has proven to be an effective and safe
strategy for the prevention and treatment of viral diseases (See
Keller et al., Clin. Microbiol. Rev. 13:602-14 (2000); Casadevall,
Nat. Biotechnol. 20:114 (2002); Shibata et al., Nat. Med. 5:204-10
(1999); and Igarashi et al., Nat. Med. 5:211-16 (1999), each of
which are incorporated herein by reference)). Passive immunization
using neutralizing human monoclonal antibodies can provide an
immediate treatment strategy for emergency prophylaxis and
treatment of influenza such as bird flu as an alternative or in
combination with the disclosed vaccines and drugs.
[0313] Subunit vaccines offer significant advantages over
conventional immunogens. They avoid the safety hazards inherent in
production, distribution, and delivery of conventional killed or
attenuated whole-pathogen vaccines. Furthermore, they can be
rationally designed to include only confirmed protective epitopes,
thereby avoiding suppressive T epitopes (see Steward et al., J.
Virol. 69:7668 (1995)) or immunodominant B epitopes that subvert
the immune system by inducing futile, non-protective responses
(e.g. "decoy" epitopes). (See Garrity et al., J. Immunol. 159:279
(1997)). For example, the subunit vaccine comprises the F10
epitope
[0314] Moreover, those skilled in the art will recognize that good
correlation exists between the antibody neutralizing activity in
vitro and the protection in vivo for many different viruses,
challenge routes, and animal models (See Burton, Natl. Rev.
Immunol. 2:706-13 (2002); Parren et al., Adv. Immunol. 77:195-262
(2001)). The data presented herein demonstrate that the D7, D8,
F10, G17, H40, A66, D80, E88, E90, and H98 human monoclonal
antibodies can be analyzed in in vivo animal studies to confirm its
clinical utility as a potent viral entry inhibitor for emergency
prophylaxis and treatment of influenza.
[0315] Methods for the screening of antibodies that possess the
desired specificity include, but are not limited to, enzyme linked
immunosorbent assay (ELISA) and other immunologically mediated
techniques known within the art.
[0316] Antibodies directed against a influenza virus protein such
as HA (or a fragment thereof) can be used in methods known within
the art relating to the localization and/or quantitation of a
influenza virus protein (e.g., for use in measuring levels of the
influenza virus protein within appropriate physiological samples,
for use in diagnostic methods, for use in imaging the protein, and
the like). In some forms, antibodies specific to an influenza virus
protein, or derivative, fragment, analog or homolog thereof, that
contain the antibody derived antigen binding domain, can be
utilized as pharmacologically active compounds (referred to herein
as "therapeutics").
[0317] An antibody specific for an influenza virus protein can be
used to isolate an influenza virus polypeptide by standard
techniques, such as immunoaffinity, chromatography or
immunoprecipitation. Antibodies directed against an influenza virus
protein (or a fragment thereof) can be used diagnostically to
monitor protein levels in tissue as part of a clinical testing
procedure, e.g., to, for example, determine the efficacy of a given
treatment regimen. Detection can be facilitated by coupling (i.e.,
physically linking) the antibody to a detectable substance.
Examples of detectable substances include various enzymes,
prosthetic groups, fluorescent materials, luminescent materials,
bioluminescent materials, and radioactive materials. Examples of
suitable enzymes include horseradish peroxidase, alkaline
phosphatase, .beta.-galactosidase, or acetylcholinesterase;
examples of suitable prosthetic group complexes include
streptavidin/biotin and avidin/biotin; examples of suitable
fluorescent materials include umbelliferone, fluorescein,
fluorescein isothiocyanate, rhodamine, dichlorotriazinylamine
fluorescein, dansyl chloride or phycoerythrin; an example of a
luminescent material includes luminol; examples of bioluminescent
materials include luciferase, luciferin, and aequorin, and examples
of suitable radioactive material include .sup.125I, .sup.131I,
.sup.35S or .sup.3H.
[0318] The disclosed antibodies, including polyclonal, monoclonal,
humanized and fully human antibodies, can be used as therapeutic
agents. Such agents can generally be employed to treat or prevent
an influenza virus-related disease or pathology (e.g., bird flu) in
a subject. An antibody preparation, such as, for example, one
having high specificity and high affinity for its target antigen,
can be administered to the subject and will generally have an
effect due to its binding with the target. Administration of the
antibody can abrogate or inhibit or interfere with the
internalization of the virus into a cell. In this case, the
antibody binds to the target and masks a binding site of the
naturally occurring ligand, thereby blocking fusion the virus to
the cell membrane inhibiting internalization of the virus.
[0319] A therapeutically effective amount of an antibody or
hemagglutinin composition relates generally to the amount needed to
achieve a therapeutic objective. As noted elsewhere herein, this
can be a binding interaction between the antibody and its target
antigen that, in certain cases, interferes with the functioning of
the target. The amount required to be administered will furthermore
depend on the binding affinity of the antibody for its specific
antigen, and will also depend on the rate at which an administered
antibody is depleted from the free volume other subject to which it
is administered. Common ranges for therapeutically effective dosing
of an antibody or antibody fragment can be, by way of nonlimiting
example, from about 0.1 mg/kg body weight to about 50 mg/kg body
weight. Common dosing frequencies can range, for example, from
twice daily to once a week.
[0320] Antibodies specifically binding an influenza virus protein
or a fragment thereof, as well as other molecules identified by the
screening assays disclosed herein, can be administered for the
treatment of an influenza virus-related disorders in the form of
pharmaceutical compositions. Principles and considerations involved
in preparing such compositions, as well as guidance in the choice
of components are provided, for example, in Remington: The Science
And Practice Of Pharmacy 19th ed. (Alfonso R. Gennaro, et al.,
editors) Mack Pub. Co., Easton, Pa., 1995; Drug Absorption
Enhancement: Concepts, Possibilities, Limitations, And Trends,
Harwood Academic Publishers, Langhorne, Pa., 1994; and Peptide And
Protein Drug Delivery (Advances In Parenteral Sciences, Vol. 4),
1991, M. Dekker, New York.
[0321] Where antibody fragments are used, it can be useful to use
the smallest inhibitory fragment that specifically binds to the
binding domain of the target protein. For example, based upon the
variable-region sequences of an antibody, peptide molecules can be
designed that retain the ability to bind the target protein
sequence. Such peptides can be synthesized chemically and/or
produced by recombinant DNA technology (See, e.g., Marasco et al.,
Proc. Natl. Acad. Sci. USA, 90: 7889-7893 (1993)).
[0322] The formulation can also contain more than one active
compound as necessary for the particular indication being treated,
such as, for example, those with complementary activities that do
not adversely affect each other. Alternatively, or in addition, the
composition can comprise an agent that enhances its function, such
as, for example, a cytotoxic agent, cytokine, chemotherapeutic
agent, or growth-inhibitory agent. Such molecules are suitably
present in combination in amounts that are effective for the
purpose intended.
[0323] The active ingredients can also be entrapped in
microcapsules prepared, for example, by coacervation techniques or
by interfacial polymerization, for example, hydroxymethylcellulose
or gelatin-microcapsules and poly-(methylmethacrylate)
microcapsules, respectively, in colloidal drug delivery systems
(for example, liposomes, albumin microspheres, microemulsions,
nano-particles, and nanocapsules) or in macro emulsions.
[0324] The formulations to be used for in vivo administration must
be sterile. This can readily be accomplished, for example, by
filtration through sterile filtration membranes.
[0325] Sustained-release preparations can be prepared. Suitable
examples of sustained-release preparations include semipermeable
matrices of solid hydrophobic polymers containing the antibody,
which matrices are in the form of shaped articles, e.g., films, or
microcapsules. Examples of sustained-release matrices include
polyesters, hydrogels (for example,
poly(2-hydroxyethyl-methacrylate), or poly(vinylalcohol)),
polylactides (U.S. Pat. No. 3,773,919), copolymers of L-glutamic
acid and .gamma. ethyl-L-glutamate, non-degradable ethylene-vinyl
acetate, degradable lactic acid-glycolic acid copolymers such as
the LUPRON DEPOT.TM. (injectable microspheres composed of lactic
acid-glycolic acid copolymer and leuprolide acetate), and
poly-D-(-)-3-hydroxybutyric acid. While polymers such as
ethylene-vinyl acetate and lactic acid-glycolic acid enable release
of molecules for over 100 days, certain hydrogels release proteins
for shorter time periods.
[0326] The disclosed antibodies can be used as agents for detecting
the presence of an influenza virus (or a protein or a protein
fragment thereof) in a sample. The antibody can contain a
detectable label. Antibodies can be, for example, polyclonal or
monoclonal. An intact antibody, or a fragment thereof (e.g.,
F.sub.ab, scFv, or F.sub.(ab)2) can be used. The term "labeled",
with regard to the probe or antibody, is intended to encompass
direct labeling of the probe or antibody by coupling (i.e.,
physically linking) a detectable substance to the probe or
antibody, as well as indirect labeling of the probe or antibody by
reactivity with another reagent that is directly labeled. Examples
of indirect labeling include detection of a primary antibody using
a fluorescently-labeled secondary antibody and end-labeling of a
DNA probe with biotin such that it can be detected with
fluorescently-labeled streptavidin. The term "biological sample" is
intended to include tissues, cells and biological fluids isolated
from a subject, as well as tissues, cells and fluids present within
a subject. Included within the usage of the term "biological
sample", therefore, is blood and a fraction or component of blood
including blood serum, blood plasma, or lymph. That is, the
disclosed detection method can be used to detect an analyte mRNA,
protein, or genomic DNA in a biological sample in vitro as well as
in vivo. For example, in vitro techniques for detection of an
analyte mRNA include Northern hybridizations and in situ
hybridizations. In vitro techniques for detection of an analyte
protein include enzyme linked immunosorbent assays (ELISAs),
Western blots, immunoprecipitations, and immunofluorescence. In
vitro techniques for detection of an analyte genomic DNA include
Southern hybridizations. Procedures for conducting immunoassays are
described, for example in "ELISA: Theory and Practice: Methods in
Molecular Biology", Vol. 42, J. R. Crowther (Ed.) Human Press,
Totowa, N.J., 1995; "Immunoassay", E. Diamandis and T.
Christopoulus, Academic Press, Inc., San Diego, Calif., 1996; and
"Practice and Theory of Enzyme Immunoassays", P. Tijssen, Elsevier
Science Publishers, Amsterdam, 1985. Furthermore, in vivo
techniques for detection of an analyte protein include introducing
into a subject a labeled anti-analyte protein antibody. For
example, the antibody can be labeled with a radioactive marker
whose presence and location in a subject can be detected by
standard imaging techniques.
[0327] As used herein the terms "immunogenic agent" or "immunogen"
or "antigen" are used interchangeably to describe a molecule
capable of inducing an immunological response against itself on
administration to a recipient, either alone, in conjunction with an
adjuvant, or presented on a solid support or display vehicle. For
example, beads, microparticles or nanoparticles with hemagglutinin
composition bound to the surface can be administered to subjects.
Because of the discovery that immobilized hemagglutinin can be
bound by antibodies specific for the hemagglutinin stem region,
such compositions can be used to generate an immune response in a
subject. Such an immune response can produce antibodies and immune
system components that can inhibit influenza virus.
[0328] As used herein the term "adjuvant" refers to a compound
that, when administered in conjunction with an antigen, augments
the immune response to the antigen, but when administered alone
does not generate an immune response to the antigen. Adjuvants can
augment an immune response by several mechanisms including
lymphocyte recruitment, stimulation of B and/or T cells, and
stimulation of macrophages.
[0329] A pharmaceutical preparation can include, as an active
ingredient, a composition comprising at least one epitope of a
target protein or polypeptide, the at least one epitope being
capable of eliciting antibodies capable of binding to the stem
region of hemagglutinin. Preferably, the at least one epitope is
the F10 epitope or F10 epitope unit disclosed herein,
Alternatively, a pharmaceutical composition can include, as an
active ingredient, a composition comprising at least an
immunological portion of an antibody being for binding at least one
epitope of the stem region of hemagglutinin.
[0330] The preparation can be administered to a subject or organism
per se, or in a pharmaceutical composition where it is mixed with
suitable carriers or excipients.
[0331] As used herein a "pharmaceutical composition" refers to a
preparation of one or more of the active ingredients described
herein with other chemical components such as physiologically
suitable carriers and excipients. The purpose of a pharmaceutical
composition is to facilitate administration of a compound to a
subject or organism.
[0332] Herein the term "active ingredient" refers to the
preparation accountable for the biological effect.
[0333] As used herein, the phrases "physiologically acceptable
carrier" and "pharmaceutically acceptable carrier" which can be
interchangeably used refer to a carrier or a diluent that does not
cause significant irritation to a subject or organism and does not
abrogate the biological activity and properties of the administered
compound. An adjuvant is included under these phrases.
[0334] Herein the term "excipient" refers to an inert substance
added to a pharmaceutical composition to further facilitate
administration of an active ingredient. Examples, without
limitation, of excipients include calcium carbonate, calcium
phosphate, various sugars and types of starch, cellulose
derivatives, gelatin, vegetable oils and polyethylene glycols.
[0335] Techniques for formulation and administration of drugs may
be found in Remington's Pharmaceutical Sciences, Mack Publishing
Co., Easton, Pa., latest edition, which is incorporated herein by
reference.
[0336] Suitable routes of administration can, for example, include
oral, rectal, transmucosal, especially transnasal, intestinal or
parenteral delivery, including intramuscular, subcutaneous and
intramedullary injections as well as intrathecal, direct
intraventricular, intravenous, intraperitoneal, intranasal, or
intraocular injections. Alternately, one can administer a
preparation in a local rather than systemic manner.
[0337] Pharmaceutical compositions can be manufactured by processes
well known in the art, e.g., by means of conventional mixing,
dissolving, granulating, dragee-making, levigating, emulsifying,
encapsulating, entrapping or lyophilizing processes.
[0338] Pharmaceutical compositions for use in the disclosed methods
thus can be formulated in conventional manner using one or more
physiologically acceptable carriers comprising excipients and
auxiliaries, which facilitate processing of the active ingredients
into preparations which, can be used pharmaceutically. Proper
formulation is dependent upon the route of administration
chosen.
[0339] For injection, the active ingredients can be formulated in
aqueous solutions, preferably in physiologically compatible buffers
such as Hank's solution, Ringer's solution, or physiological salt
buffer. For transmucosal administration, penetrants appropriate to
the barrier to be permeated are used in the formulation. Such
penetrants are generally known in the art.
[0340] For oral administration, the compounds can be formulated
readily by combining the active compounds with pharmaceutically
acceptable carriers well known in the art. Such carriers enable the
compounds to be formulated as tablets, pills, dragees, capsules,
liquids, gels, syrups, slurries, suspensions, and the like, for
oral ingestion by a patient. Pharmacological preparations for oral
use can be made using a solid excipient, optionally grinding the
resulting mixture, and processing the mixture of granules, after
adding suitable auxiliaries if desired, to obtain tablets or dragee
cores. Suitable excipients are, in particular, fillers such as
sugars, including lactose, sucrose, mannitol, or sorbitol;
cellulose preparations such as, for example, maize starch, wheat
starch, rice starch, potato starch, gelatin, gum tragacanth, methyl
cellulose, hydroxypropylmethyl-cellulose, sodium
carbomethylcellulose; and/or physiologically acceptable polymers
such as polyvinylpyrrolidone (PVP). If desired, disintegrating
agents can be added, such as cross-linked polyvinyl pyrrolidone,
agar, or alginic acid or a salt thereof such as sodium
alginate.
[0341] Dragee cores are provided with suitable coatings. For this
purpose, concentrated sugar solutions can be used which may
optionally contain gum arabic, talc, polyvinyl pyrrolidone,
carbopol gel, polyethylene glycol, titanium dioxide, lacquer
solutions and suitable organic solvents or solvent mixtures.
Dyestuffs or pigments can be added to the tablets or dragee
coatings for identification or to characterize different
combinations of active compound doses.
[0342] Pharmaceutical compositions, which can be used orally,
include push-fit capsules made of gelatin as well as soft, sealed
capsules made of gelatin and a plasticizer, such as glycerol or
sorbitol. The push-fit capsules can contain the active ingredients
in admixture with filler such as lactose, binders such as starches,
lubricants such as talc or magnesium stearate and, optionally,
stabilizers. In soft capsules, the active ingredients can be
dissolved or suspended in suitable liquids, such as fatty oils,
liquid paraffin, or liquid polyethylene glycols. In addition,
stabilizers can be added. All formulations for oral administration
should be in dosages suitable for the chosen route of
administration.
[0343] For buccal administration, the compositions can take the
form of tablets or lozenges formulated in conventional manner.
[0344] For administration by nasal inhalation, the active
ingredients for use in the disclosed methods can be conveniently
delivered in the form of an aerosol spray presentation from a
pressurized pack or a nebulizer with the use of a suitable
propellant, e.g., dichlorodifluoromethane, trichlorofluoromethane,
dichloro-tetrafluoroethane or carbon dioxide. In the case of a
pressurized aerosol, the dosage unit can be determined by providing
a valve to deliver a metered amount. Capsules and cartridges of,
e.g., gelatin for use in a dispenser can be formulated containing a
powder mix of the compound and a suitable powder base such as
lactose or starch.
[0345] The preparations described herein can be formulated for
parenteral administration, e.g., by bolus injection or continuous
infusion. Formulations for injection can be presented in unit
dosage form, e.g., in ampoules or in multidose containers with
optionally, an added preservative. The compositions can be
suspensions, solutions or emulsions in oily or aqueous vehicles,
and can contain formulatory agents such as suspending, stabilizing
and/or dispersing agents.
[0346] Pharmaceutical compositions for parenteral administration
include aqueous solutions of the active preparation in
water-soluble form. Additionally, suspensions of the active
ingredients can be prepared as appropriate oily or water based
injection suspensions. Suitable lipophilic solvents or vehicles
include fatty oils such as sesame oil, or synthetic fatty acids
esters such as ethyl oleate, triglycerides or liposomes. Aqueous
injection suspensions can contain substances, which increase the
viscosity of the suspension, such as sodium carboxymethyl
cellulose, sorbitol or dextran. Optionally, the suspension can also
contain suitable stabilizers or agents which increase the
solubility of the active ingredients to allow for the preparation
of highly concentrated solutions.
[0347] Alternatively, the active ingredient can be in powder form
for constitution with a suitable vehicle, e.g., sterile,
pyrogen-free water based solution, before use.
[0348] The preparations can also be formulated in rectal
compositions such as suppositories or retention enemas, using,
e.g., conventional suppository bases such as cocoa butter or other
glycerides.
[0349] Pharmaceutical compositions for use in the disclosed methods
include compositions wherein the active ingredients are contained
in an amount effective to achieve the intended purpose. More
specifically, a therapeutically effective amount means an amount of
active ingredients effective to prevent, alleviate or ameliorate
symptoms of disease or prolong the survival of the subject being
treated.
[0350] Determination of a therapeutically effective amount is well
within the capability of those skilled in the art, especially in
light of the detailed disclosure provided herein.
[0351] For any preparation used in the disclosed methods, the
therapeutically effective amount or dose can be estimated initially
from in vitro and cell culture assays. For example, a dose can be
formulated in animal models to achieve a desired circulating
antibody concentration or titer. Such information can be used to
more accurately determine useful doses in humans.
[0352] Toxicity and therapeutic efficacy of the active ingredients
described herein can be determined by standard pharmaceutical
procedures in vitro, in cell cultures or experimental animals. The
data obtained from these in vitro and cell culture assays and
animal studies can be used in formulating a range of dosage for use
in human. The dosage may vary depending upon the dosage form
employed and the route of administration utilized. The exact
formulation, route of administration and dosage can be chosen by
the individual physician in view of the patient's condition. (See
e.g., Fingl et al in The Pharmacological Basis of Therapeutics, Ch.
1 p. 1. (1975)).
[0353] Dosage amount and interval can be adjusted individually to
provide plasma of antibodies which are sufficient to prevent or
reduce viral entry (minimal effective concentration, MEC). The MEC
will vary for each preparation, but can be estimated from in vitro
data. Dosages necessary to achieve the MEC will depend on
individual characteristics and route of administration. Binding
assays can be used to determine plasma concentrations.
[0354] Dosage intervals can also be determined using the MEC value.
Preparations should be administered using a regimen, which
maintains plasma levels above the MEC for example, 10-90% of the
time, preferable between 30-90% of the time and most preferably
50-90%.
[0355] Depending on the severity and responsiveness of the
condition to be treated, dosing can be of a single or a plurality
of administrations, with course of treatment lasting from several
days to several weeks or until cure is effected or diminution of
the disease state is achieved.
[0356] The amount of a composition to be administered will, of
course, be dependent on the subject being treated, the severity of
the affliction, the manner of administration, the judgment of the
prescribing physician, etc.
[0357] The disclosed antibodies, hemagglutinin compositions or
agents (also referred to herein as "active compounds"), and
derivatives, fragments, analogs and homologs thereof, can be
incorporated into pharmaceutical compositions suitable for
administration. Such compositions typically comprise the antibody
or agent and a pharmaceutically acceptable carrier. As used herein,
the term "pharmaceutically acceptable carrier" is intended to
include any and all solvents, dispersion media, coatings,
antibacterial and antifungal agents, isotonic and absorption
delaying agents, and the like, compatible with pharmaceutical
administration. Suitable carriers are described in the most recent
edition of Remington's Pharmaceutical Sciences, a standard
reference text in the field, which is incorporated herein by
reference. Examples of such carriers or diluents include, but are
not limited to, water, saline, ringer's solutions, dextrose
solution, and 5% human serum albumin. Liposomes and non-aqueous
vehicles such as fixed oils can also be used. The use of such media
and agents for pharmaceutically active substances is well known in
the art. Except insofar as any conventional media or agent is
incompatible with the active compound, use thereof in the
compositions is contemplated. Supplementary active compounds can
also be incorporated into the compositions.
[0358] A pharmaceutical composition can be formulated to be
compatible with its intended route of administration. Examples of
routes of administration include parenteral, e.g., intravenous,
intradermal, subcutaneous, oral (e.g., inhalation), transdermal
(i.e., topical), transmucosal, and rectal administration. Solutions
or suspensions used for parenteral, intradermal, or subcutaneous
application can include the following components: a sterile diluent
such as water for injection, saline solution, fixed oils,
polyethylene glycols, glycerine, propylene glycol or other
synthetic solvents; antibacterial agents such as benzyl alcohol or
methyl parabens; antioxidants such as ascorbic acid or sodium
bisulfite; chelating agents such as ethylenediaminetetraacetic acid
(EDTA); buffers such as acetates, citrates or phosphates, and
agents for the adjustment of tonicity such as sodium chloride or
dextrose. The pH can be adjusted with acids or bases, such as
hydrochloric acid or sodium hydroxide. The parenteral preparation
can be enclosed in ampoules, disposable syringes or multiple dose
vials made of glass or plastic.
[0359] Pharmaceutical compositions suitable for injectable use
include sterile aqueous solutions (where water soluble) or
dispersions and sterile powders for the extemporaneous preparation
of sterile injectable solutions or dispersion. For intravenous
administration, suitable carriers include physiological saline,
bacteriostatic water, Cremophor ELO (BASF, Parsippany, N.J.) or
phosphate buffered saline (PBS). In all cases, the composition must
be sterile and should be fluid to the extent that easy
syringeability exists. It must be stable under the conditions of
manufacture and storage and must be preserved against the
contaminating action of microorganisms such as bacteria and fungi.
The carrier can be a solvent or dispersion medium containing, for
example, water, ethanol, polyol (for example, glycerol, propylene
glycol, and liquid polyethylene glycol, and the like), and suitable
mixtures thereof. The proper fluidity can be maintained, for
example, by the use of a coating such as lecithin, by the
maintenance of the required particle size in the case of dispersion
and by the use of surfactants. Prevention of the action of
microorganisms can be achieved by various antibacterial and
antifungal agents, for example, parabens, chlorobutanol, phenol,
ascorbic acid, thimerosal, and the like. In many cases, it can be
useful to include isotonic agents, for example, sugars,
polyalcohols such as manitol, sorbitol, sodium chloride in the
composition. Prolonged absorption of the injectable compositions
can be brought about by including in the composition an agent which
delays absorption, for example, aluminum monostearate and
gelatin.
[0360] Sterile injectable solutions can be prepared by
incorporating the active compound in the required amount in an
appropriate solvent with one or a combination of ingredients
enumerated above, as required, followed by filtered sterilization.
Generally, dispersions are prepared by incorporating the active
compound into a sterile vehicle that contains a basic dispersion
medium and the required other ingredients from those enumerated
above. In the case of sterile powders for the preparation of
sterile injectable solutions, methods of preparation are vacuum
drying and freeze drying that yields a powder of the active
ingredient plus any additional desired ingredient from a previously
sterile filtered solution thereof.
[0361] Oral compositions generally include an inert diluent or an
edible carrier. They can be enclosed in gelatin capsules or
compressed into tablets. For the purpose of oral therapeutic
administration, the active compound can be incorporated with
excipients and used in the form of tablets, troches, or capsules.
Oral compositions can also be prepared using a fluid carrier for
use as a mouthwash, wherein the compound in the fluid carrier is
applied orally and swished and expectorated or swallowed.
Pharmaceutically compatible binding agents, and/or adjuvant
materials can be included as part of the composition. The tablets,
pills, capsules, troches and the like can contain any of the
following ingredients, or compounds of a similar nature: a binder
such as microcrystalline cellulose, gum tragacanth or gelatin; an
excipient such as starch or lactose, a disintegrating agent such as
alginic acid, Primogel, or corn starch; a lubricant such as
magnesium stearate or Sterotes; a glidant such as colloidal silicon
dioxide; a sweetening agent such as sucrose or saccharin; or a
flavoring agent such as peppermint, methyl salicylate, or orange
flavoring.
[0362] For administration by inhalation, the compounds are
delivered in the form of an aerosol spray from pressured container
or dispenser which contains a suitable propellant, e.g., a gas such
as carbon dioxide, or a nebulizer.
[0363] Systemic administration can also be by transmucosal or
transdermal means. For transmucosal or transdermal administration,
penetrants appropriate to the barrier to be permeated are used in
the formulation. Such penetrants are generally known in the art,
and include, for example, for transmucosal administration,
detergents, bile salts, and fusidic acid derivatives. Transmucosal
administration can be accomplished through the use of nasal sprays
or suppositories. For transdermal administration, the active
compounds can be formulated into ointments, salves, gels, or creams
as generally known in the art. The compounds can also be prepared
in the form of suppositories (e.g., with conventional suppository
bases such as cocoa butter and other glycerides) or retention
enemas for rectal delivery.
[0364] In some forms, the active compounds can be prepared with
carriers that will protect the compound against rapid elimination
from the body, such as a controlled release formulation, including
implants and microencapsulated delivery systems. Biodegradable,
biocompatible polymers can be used, such as ethylene vinyl acetate,
polyanhydrides, polyglycolic acid, collagen, polyorthoesters, and
polylactic acid. Methods for preparation of such formulations will
be apparent to those skilled in the art. The materials can also be
obtained commercially from Alza Corporation and Nova
Pharmaceuticals, Inc. Liposomal suspensions (including liposomes
targeted to infected cells with monoclonal antibodies to viral
antigens) can also be used as pharmaceutically acceptable carriers.
These can be prepared according to methods known to those skilled
in the art, for example, as described in U.S. Pat. No.
4,522,811.
[0365] It is especially advantageous to formulate oral or
parenteral compositions in dosage unit form for ease of
administration and uniformity of dosage. Dosage unit form as used
herein refers to physically discrete units suited as unitary
dosages for the subject to be treated; each unit containing a
predetermined quantity of active compound calculated to produce the
desired therapeutic effect in association with the required
pharmaceutical carrier. The specification for the dosage unit forms
can be dictated by and directly dependent on the unique
characteristics of the active compound and the particular
therapeutic effect to be achieved, and the limitations inherent in
the art of compounding such an active compound for the treatment of
individuals.
[0366] The pharmaceutical compositions can be included in a
container, pack, or dispenser together with instructions for
administration.
[0367] The disclosed compositions can, if desired, be presented in
a pack or dispenser device, such as an FDA approved kit, which may
contain one or more unit dosage forms containing the active
ingredient. The pack can, for example, comprise metal or plastic
foil, such as a blister pack. The pack or dispenser device can be
accompanied by instructions for administration. The pack or
dispenser can also be accommodated by a notice associated with the
container in a form prescribed by a governmental agency regulating
the manufacture, use or sale of pharmaceuticals, which notice is
reflective of approval by the agency of the form of the
compositions or human or veterinary administration. Such notice,
for example, may be of labeling approved by the U.S. Food and Drug
Administration for prescription drugs or of an approved product
insert. Compositions comprising a preparation formulated in a
compatible pharmaceutical carrier can also be prepared, placed in
an appropriate container, and labeled for treatment of an indicated
condition, as if further detailed above.
[0368] The term "activity" as used herein refers to a measurable
result of the interaction of molecules. Some exemplary methods of
measuring these activities are provided herein. The term "modulate"
as used herein refers to the ability of a compound to change an
activity in some measurable way as compared to an appropriate
control. As a result of the presence of compounds in the assays,
activities can increase or decrease as compared to controls in the
absence of these compounds. Preferably, an increase in activity is
at least 25%, more preferably at least 50%, most preferably at
least 100% compared to the level of activity in the absence of the
compound. Similarly, a decrease in activity is preferably at least
25%, more preferably at least 50%, most preferably at least 100%
compared to the level of activity in the absence of the compound. A
compound that increases a known activity is an "agonist". One that
decreases, or prevents, a known activity is an "antagonist".
[0369] The term "inhibit" means to reduce or decrease in activity
or expression. This can be a complete inhibition or activity or
expression, or a partial inhibition. Inhibition can be compared to
a control or to a standard level. Inhibition can be 1, 2, 3, 4, 5,
6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23,
24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40,
41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57,
58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74,
75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91,
92, 93, 94, 95, 96, 97, 98, 99, or 100%.
[0370] The term "monitoring" as used herein refers to any method in
the art by which an activity can be measured.
[0371] The term "providing" as used herein refers to any means of
adding a compound or molecule to something known in the art.
Examples of providing can include the use of pipettes, pipettemen,
syringes, needles, tubing, guns, etc. This can be manual or
automated. It can include transfection by any mean or any other
means of providing nucleic acids to dishes, cells, tissue,
cell-free systems and can be in vitro or in vivo.
[0372] The term "preventing" as used herein refers to administering
a compound prior to the onset of clinical symptoms of a disease or
conditions so as to prevent a physical manifestation of aberrations
associated with the disease or condition.
[0373] By "treatment" is meant the medical management of a patient
with the intent to cure, ameliorate, stabilize, or prevent a
disease, pathological condition, or disorder. This term includes
active treatment, that is, treatment directed specifically toward
the improvement of a disease, pathological condition, or disorder,
and also includes causal treatment, that is, treatment directed
toward removal of the cause of the associated disease, pathological
condition, or disorder. In addition, this term includes palliative
treatment, that is, treatment designed for the relief of symptoms
rather than the curing of the disease, pathological condition, or
disorder; preventative treatment, that is, treatment directed to
minimizing or partially or completely inhibiting the development of
the associated disease, pathological condition, or disorder; and
supportive treatment, that is, treatment employed to supplement
another specific therapy directed toward the improvement of the
associated disease, pathological condition, or disorder.
[0374] The term "in need of treatment" as used herein refers to a
judgment made by a caregiver (e.g. physician, nurse, nurse
practitioner, or individual in the case of humans; veterinarian in
the case of animals, including non-human mammals) that a subject
requires or will benefit from treatment. This judgment is made
based on a variety of factors that are in the realm of a care
giver's expertise, but that include the knowledge that the subject
is ill, or will be ill, as the result of a condition that is
treatable by the compounds of the invention.
[0375] As used herein, "subject" includes, but is not limited to,
animals, plants, bacteria, viruses, parasites and any other
organism or entity that has nucleic acid. The subject may be a
vertebrate, more specifically a mammal (e.g., a human, horse, pig,
rabbit, dog, sheep, goat, non-human primate, cow, cat, guinea pig
or rodent), a fish, a bird or a reptile or an amphibian. The
subject may to an invertebrate, more specifically an arthropod
(e.g., insects and crustaceans). The term does not denote a
particular age or sex. Thus, adult and newborn subjects, as well as
fetuses, whether male or female, are intended to be covered. A
patient refers to a subject afflicted with a disease or disorder.
The term "patient" includes human and veterinary subjects. In the
context of endometriosis and endometriosis cells, it is understood
that a subject is a subject that has or can have endometriosis
and/or endometriosis cells.
[0376] The terms "higher," "increases," "elevates," or "elevation"
refer to increases above basal levels, e.g., as compared to a
control. The terms "low," "lower," "reduces," or "reduction" refer
to decreases below basal levels, e.g., as compared to a
control.
[0377] Disclosed are methods (also referred to herein as "screening
assays") for identifying modulators, i.e., candidate or test
compounds or agents (e.g., peptides, peptidomimetics, small
molecules or other drugs) that modulate or otherwise interfere with
the fusion of an influenza virus to the cell membrane. Also
disclosed are methods of identifying compounds useful to treat
influenza infection. Also disclosed are compounds identified using
the screening assays described herein.
[0378] For example, disclosed are assays for screening candidate or
test compounds which modulate the interaction between the influenza
virus and the cell membrane. Additionally, disclosed are assays for
screening candidate or test compounds that compete for binding of
hemaglutinin to the antibodies disclosed herein. Compound
identified in these methods are useful as immunogens for influenza
virus.
[0379] The test compounds can be obtained using any of the numerous
approaches in combinatorial library methods known in the art,
including: biological libraries; spatially addressable parallel
solid phase or solution phase libraries; synthetic library methods
requiring deconvolution; the "one-bead one-compound" library
method; and synthetic library methods using affinity chromatography
selection. The biological library approach is limited to peptide
libraries, while the other four approaches are applicable to
peptide, non-peptide oligomer or small molecule libraries of
compounds (See, e.g., Lam, 1997. Anticancer Drug Design 12:
145).
[0380] A "small molecule" as used herein, is meant to refer to a
composition that has a molecular weight of less than about 5 kD,
such as, for example, less than about 4 kD. Small molecules can be,
e.g., nucleic acids, peptides, polypeptides, peptidomimetics,
carbohydrates, lipids or other organic or inorganic molecules.
Libraries of chemical and/or biological mixtures, such as fungal,
bacterial, or algal extracts, are known in the art and can be
screened with any of the known or disclosed assays.
[0381] Examples of methods for the synthesis of molecular libraries
can be found in the art, for example in: DeWitt, et al., 1993.
Proc. Natl. Acad. Sci. U.S.A. 90: 6909; Erb, et al., 1994. Proc.
Natl. Acad. Sci. U.S.A. 91: 11422; Zuckermann, et al., 1994. J.
Med. Chem. 37: 2678; Cho, et al., 1993. Science 261: 1303; Carrell,
et al., 1994. Angew. Chem. Int. Ed. Engl. 33: 2059; Carell, et al.,
1994. Angew. Chem. Int. Ed. Engl. 33: 2061; and Gallop, et al.,
1994. J. Med. Chem. 37: 1233.
[0382] Libraries of compounds can be presented in solution (see
e.g., Houghten, 1992. Biotechniques 13: 412-421), or on beads (see
Lam, 1991. Nature 354: 82-84), on chips (see Fodor, 1993. Nature
364: 555-556), bacteria (see U.S. Pat. No. 5,223,409), spores (see
U.S. Pat. No. 5,233,409), plasmids (see Cull, et al., 1992. Proc.
Natl. Acad. Sci. USA 89: 1865-1869) or on phage (see Scott and
Smith, 1990. Science 249: 386-390; Devlin, 1990. Science 249:
404-406; Cwirla, et al., 1990. Proc. Natl. Acad. Sci. U.S.A. 87:
6378-6382; Felici, 1991. J. Mol. Biol. 222: 301-310; and U.S. Pat.
No. 5,233,409.).
[0383] In some forms, a candidate compound can be introduced to an
antibody-antigen complex and it can be determined whether the
candidate compound disrupts the antibody-antigen complex. In some
aspects a disruption of this complex indicates that the candidate
compound modulates the interaction between an influenza virus and
the cell membrane. For example, the antibody can be monoclonal
antibody D7, D8, F10, G17, H40, A66, D80, E88, E90, and H98 and the
antigen can be located on the HA protein of an influenza virus.
[0384] In some forms, at least one HA protein is provided, which is
exposed to at least one neutralizing monoclonal antibody. Formation
of an antibody-antigen complex is detected, and one or more
candidate compounds are introduced to the complex. If the
antibody-antigen complex is disrupted following introduction of the
one or more candidate compounds, the candidate compounds is useful
to treat a an influenza virus-related disease or disorder, e.g.
bird flu or swine flu. For example, the at least one influenza
virus protein can be provided as an influenza virus molecule.
[0385] Determining the ability of the test compound to interfere
with or disrupt the antibody-antigen complex can be accomplished,
for example, by coupling the test compound with a radioisotope or
enzymatic label such that binding of the test compound to the
antigen or biologically-active portion thereof can be determined by
detecting the labeled compound in a complex. For example, test
compounds can be labeled with .sup.125I, .sup.35S, .sup.14C, or
.sup.3H, either directly or indirectly, and the radioisotope
detected by direct counting of radioemission or by scintillation
counting. Alternatively, test compounds can be
enzymatically-labeled with, for example, horseradish peroxidase,
alkaline phosphatase, or luciferase, and the enzymatic label
detected by determination of conversion of an appropriate substrate
to product.
[0386] In some forms, the assay can comprise contacting an
antibody-antigen complex with a test compound, and determining the
ability of the test compound to interact with the antigen or
otherwise disrupt the existing antibody-antigen complex. In some
forms, determining the ability of the test compound to interact
with the antigen and/or disrupt the antibody-antigen complex can
comprise determining the ability of the test compound to
preferentially bind to the antigen or a biologically-active portion
thereof, as compared to the antibody.
[0387] In some forms, the assay can comprise contacting an
antibody-antigen complex with a test compound and determining the
ability of the test compound to modulate the antibody-antigen
complex. Determining the ability of the test compound to modulate
the antibody-antigen complex can be accomplished, for example, by
determining the ability of the antigen to bind to or interact with
the antibody, in the presence of the test compound.
[0388] Those skilled in the art will recognize that, in any of the
screening methods disclosed herein, the antibody can be a an
influenza virus neutralizing antibody, such as monoclonal antibody
D7, D8, F10, G17, H40, A66, D80, E88, E90, and H98. Additionally,
the antigen can be a HA protein, or a portion thereof. In any of
the assays described herein, the ability of a candidate compound to
interfere with the binding between the D7, D8, F10, G17, H40, A66,
D80, E88, E90, and H98 monoclonal antibody and the HA protein
indicates that the candidate compound will be able to interfere
with or modulate the fusion of the influenza virus and the cell
membrane Moreover, because the binding of the HA protein to cell is
responsible for influenza virus entry into cells such candidate
compounds will also be useful in the treatment of a influenza virus
related disease or disorder, e.g. bird flu.
[0389] The screening methods disclosed herein can be performed as a
cell-based assay or as a cell-free assay. The cell-free assays are
amenable to use of both the soluble form or the membrane-bound form
of the HA proteins and fragments thereof. In the case of cell-free
assays comprising the membrane-bound forms of the HA proteins, it
can be desirable to utilize a solubilizing agent such that the
membrane-bound form of the proteins are maintained in solution.
Examples of such solubilizing agents include non-ionic detergents
such as n-octylglucoside, n-dodecylglucoside, n-dodecylmaltoside,
octanoyl-N-methylglucamide, decanoyl-N-methylglucamide, Triton.RTM.
X-100, Triton.RTM. X-114, Thesit.RTM., Isotridecypoly(ethylene
glycol ether).sub.n, N-dodecyl-N,N-dimethyl-3-ammonio-1-propane
sulfonate, 3-(3-cholamidopropyl)dimethylamminiol-1-propane
sulfonate (CHAPS), or
3-(3-cholamidopropyl)dimethylamminiol-2-hydroxy-1-propane sulfonate
(CHAPSO).
[0390] In some forms, it can be desirable to immobilize either the
antibody or the antigen to facilitate separation of complexed from
uncomplexed forms of one or both following introduction of the
candidate compound, as well as to accommodate automation of the
assay. Observation of the antibody-antigen complex in the presence
and absence of a candidate compound, can be accomplished in any
vessel suitable for containing the reactants. Examples of such
vessels include microtiter plates, test tubes, and micro-centrifuge
tubes. In some forms, a fusion protein can be provided that adds a
domain that allows one or both of the proteins to be bound to a
matrix. For example, GST-antibody fusion proteins or GST-antigen
fusion proteins can be adsorbed onto glutathione sepharose beads
(Sigma Chemical, St. Louis, Mo.) or glutathione derivatized
microtiter plates, that are then combined with the test compound,
and the mixture is incubated under conditions conducive to complex
formation (e.g., at physiological conditions for salt and pH).
Following incubation, the beads or microtiter plate wells are
washed to remove any unbound components, the matrix immobilized in
the case of beads, complex determined either directly or
indirectly. Alternatively, the complexes can be dissociated from
the matrix, and the level of antibody-antigen complex formation can
be determined using standard techniques.
[0391] Other techniques for immobilizing proteins on matrices can
also be used in the screening assays. For example, either the
antibody or the antigen can be immobilized utilizing conjugation of
biotin and streptavidin. Biotinylated antibody or antigen molecules
can be prepared from biotin-NHS (N-hydroxy-succinimide) using
techniques well-known within the art (e.g., biotinylation kit,
Pierce Chemicals, Rockford, Ill.), and immobilized in the wells of
streptavidin-coated 96 well plates (Pierce Chemical).
Alternatively, other antibodies reactive with the antibody or
antigen of interest, but which do not interfere with the formation
of the antibody-antigen complex of interest, can be derivatized to
the wells of the plate, and unbound antibody or antigen trapped in
the wells by antibody conjugation. Methods for detecting such
complexes, in addition to those described above for the
GST-immobilized complexes, include immunodetection of complexes
using such other antibodies reactive with the antibody or
antigen.
[0392] Also disclosed are novel agents identified by any of the
disclosed screening assays and uses thereof for treatments as
described herein.
[0393] Antibodies can be detected by appropriate assays, e.g.,
conventional types of immunoassays. For example, an assay can be
performed in which a influenza protein (e.g., HAL HA 2 or
neurominidase) or fragment thereof is affixed to a solid phase.
Incubation can be maintained for a sufficient period of time to
allow the antibody in the sample to bind to the immobilized
polypeptide on the solid phase. After this first incubation, the
solid phase can be separated from the sample. The solid phase can
be washed to remove unbound materials and interfering substances
such as non-specific proteins which may also be present in the
sample. The solid phase containing the antibody of interest bound
to the immobilized polypeptide can subsequently be incubated with a
second, labeled antibody or antibody bound to a coupling agent such
as biotin or avidin. This second antibody can be another
anti-influenza antibody or another antibody. Labels for antibodies
are well-known in the art and include radionuclides, enzymes (e.g.
maleate dehydrogenase, horseradish peroxidase, glucose oxidase,
catalase), fluors (fluorescein isothiocyanate, rhodamine,
phycocyanin, fluorescarmine), biotin, and the like. The labeled
antibodies are incubated with the solid and the label bound to the
solid phase is measured. These and other immunoassays can be easily
performed by those of ordinary skill in the art.
[0394] An exemplary method for detecting the presence or absence of
a influenza virus (in a biological sample, for example) can involve
obtaining a biological sample from a test subject and contacting
the biological sample with a labeled monoclonal or scFv antibody
such that the presence of the influenza virus is detected in the
biological sample.
[0395] As used herein, the term "labeled", with regard to the probe
or antibody, is intended to encompass direct labeling of the probe
or antibody by coupling (i.e., physically linking) a detectable
substance to the probe or antibody, as well as indirect labeling of
the probe or antibody by reactivity with another reagent that is
directly labeled. Examples of indirect labeling include detection
of a primary antibody using a fluorescently-labeled secondary
antibody and end-labeling of a DNA probe with biotin such that it
can be detected with fluorescently-labeled streptavidin. The term
"biological sample" is intended to include tissues, cells and
biological fluids isolated from a subject, as well as tissues,
cells and fluids present within a subject. That is, the detection
method can be used to detect an influenza virus in a biological
sample in vitro as well as in vivo. For example, in vitro
techniques for detection of an influenza virus include enzyme
linked immunosorbent assays (ELISAs), Western blots,
immunoprecipitations, and immunofluorescence. Furthermore, in vivo
techniques for detection of an influenza virus include introducing
into a subject a labeled anti-influenza virus antibody. For
example, the antibody can be labeled with a radioactive marker
whose presence and location in a subject can be detected by
standard imaging techniques.
[0396] In some forms, the biological sample can contain protein
molecules from the test subject. The biological sample can be, for
example, a peripheral blood leukocyte sample isolated by
conventional means from a subject.
[0397] Also disclosed are kits for detecting the presence of an
influenza virus in a biological sample. For example, the kit can
comprise: a labeled compound or agent capable of detecting an
influenza virus (e.g., an anti-influenza scFv or monoclonal
antibody) in a biological sample; means, materials, and/or a system
for determining the amount of an influenza virus in the sample; and
means, materials, and/or a system for comparing the amount of an
influenza virus in the sample with a standard. The compound or
agent can be packaged in a suitable container. The kit can further
comprise instructions for using the kit to detect an influenza
virus in a sample.
[0398] It has been over a decade since the first antibodies were
used as scaffolds for the efficient presentation of antigenic
determinants to the immune systems. (See Zanetti, Nature 355:476-77
(1992); Zaghouani et al., Proc. Natl. Acad. Sci. USA 92:631-35
(1995)). When a peptide is included as an integral part of an IgG
molecule, the antigenicity and immunogenicity of the peptide
epitopes are greatly enhanced as compared to the free peptide. Such
enhancement can be due to the antigen-IgG chimeras longer
half-life, better presentation and constrained conformation, which
mimic their native structures.
[0399] Moreover, an added advantage of using an antigen-Ig chimera
is that either the variable or the Fc region of the antigen-Ig
chimera can be used for targeting professional antigen-presenting
cells (APCs). Recombinant Igs have been generated in which the
complementarity-determining regions (CDRs) of the heavy chain
variable gene (V.sub.H) are replaced with various antigenic
peptides recognized by B or T cells. Such antigen-Ig chimeras have
been used to induce both humoral and cellular immune responses (See
Bona et al., Immunol. Today 19:126-33 (1998)).
[0400] Chimeras with specific epitopes engrafted into the CDR3 loop
have been used to induce humoral responses to either HIV-1 gp120
V3-loop or the first extracellular domain (D1) of human CD4
receptor (See Lanza et al., Proc. Natl. Acad. Sci. USA 90:11683-87
(1993); Zaghouani et al., Proc. Natl. Acad. Sci. USA 92:631-35
(1995)). The immune sera were able to prevent infection of CD4
SupT1 cells by HIV-1MN (anti-gp120 V3C) or inhibit syncytia
formation (anti-CD4-D1). The CDR2 and CDR3 can be replaced with
peptide epitopes simultaneously, and the length of peptide inserted
can be up to 19 amino acids long. Alternatively, one group has
developed a "troybody" strategy in which peptide antigens are
presented in the loops of the Ig constant (C) region and the
variable region of the chimera can be used to target IgD on the
surface of B-cells or MHC class II molecules on professional APCs
including B-cells, dendritic cells (DC) and macrophages (See Lunde
et al., Biochem. Soc. Trans. 30:500-6 (2002)).
[0401] An antigen-Ig chimera can also be made by directly fusing
the antigen with the Fc portion of an IgG molecule. You et al.,
Cancer Res. 61:3704-11 (2001) were able to obtain all arms of
specific immune response, including very high levels of antibodies
to hepatitis B virus core antigen using this method.
[0402] DNA vaccines are stable, can provide the antigen an
opportunity to be naturally processed, and can induce a
longer-lasting response. Although a very attractive immunization
strategy, DNA vaccines often have very limited potency to induce
immune responses. Poor uptake of injected DNA by professional APCs,
such as dendritic cells (DCs), may be the main cause of such
limitation. Combined with the antigen-Ig chimera vaccines, a
promising new DNA vaccine strategy based on the enhancement of APC
antigen presentation has been reported (see Casares, et al., Viral
Immunol. 10:129-36 (1997); Gerloni et al., Nat. Biotech. 15:876-81
(1997); Gerloni et al., DNA Cell Biol. 16:611-25 (1997); You et
al., Cancer Res. 61:3704-11 (2001)), which takes advantage of the
presence of Fc receptors (Fc.gamma.Rs) on the surface of DCs.
[0403] It is possible to generate a DNA vaccine encoding an antigen
(Ag)-Ig chimera. Upon immunization, Ag-Ig fusion proteins can be
expressed and secreted by the cells taking up the DNA molecules.
The secreted Ag-Ig fusion proteins, while inducing B-cell
responses, can be captured and internalized by interaction of the
Fc fragment with Fc.gamma.Rs on DC surface, which will promote
efficient antigen presentation and greatly enhance antigen-specific
immune responses. Applying the same principle, DNA encoding
antigen-Ig chimeras carrying a functional anti-MHC II specific scFv
region gene can also target the immunogens to all three types of
APCs. The immune responses could be further boosted with use of the
same protein antigens generated in vitro (i.e., "prime and boost"),
if necessary. Using this strategy, specific cellular and humoral
immune responses against infection of influenza virus were
accomplished through intramuscular (i.m.) injection of a DNA
vaccine (See Casares et al., Viral. Immunol. 10:129-36 (1997)).
[0404] Therapeutic or prophylactic compositions are provided
herein, which can comprise, for example, mixtures of one or more
hemagglutinin compositions, monoclonal antibodies or ScFvs and
combinations thereof. The prophylactic vaccines can be used to
prevent an influenza virus infection and the therapeutic vaccines
can be used to treat individuals following an influenza virus
infection. Prophylactic uses include the provision of increased
antibody titer to an influenza virus in a vaccination subject and
or decrease influenza virus titer in a subject. In this manner,
subjects at high risk of contracting influenza can be provided with
passive immunity to an influenza virus.
[0405] These vaccine compositions can be administered in
conjunction with ancillary immunoregulatory agents. For example,
cytokines, lymphokines, and chemokines, including, but not limited
to, IL-2, modified IL-2 (Cys125.fwdarw.Ser125), GM-CSF, IL-12,
.gamma.-interferon, IP-10, MIP1.beta., and RANTES.
[0406] The disclosed vaccines have superior immunoprotective and
immunotherapeutic properties over other anti-viral vaccines. Also
disclosed is a method of immunization, e.g., inducing an immune
response, of a subject. A subject can be immunized by
administration to the subject a composition containing a membrane
fusion protein of a pathogenic enveloped virus. The fusion protein
can be coated or embedded in a biologically compatible matrix.
[0407] The fusion protein can be glycosylated, e.g. can contain a
carbohydrate moiety. The carbohydrate moiety can be in the form of
a monosaccharide, disaccharide(s). oligosaccharide(s),
polysaccharide(s), or their derivatives (e.g. sulfo- or
phospho-substituted). The carbohydrate can be linear or branched.
The carbohydrate moiety can be N-linked or O-linked to a
polypeptide. N-linked glycosylation is to the amide nitrogen of
asparagine side chains and O-linked glycosylation is to the hydroxy
oxygen of serine and threonine side chains.
[0408] The carbohydrate moiety can be endogenous to the subject
being vaccinated. Alternatively, the carbohydrate moiety can be
exogenous to the subject being vaccinated. The carbohydrate
moieties can be carbohydrate moieties that are not typically
expressed on polypeptides of the subject being vaccinated. For
example, the carbohydrate moieties can be plant-specific
carbohydrates. Plant specific carbohydrate moieties include for
example N-linked glycan having a core bound .alpha.1,3 fucose or a
core bound .beta.1,2 xylose. Alternatively, the carbohydrate
moieties can be carbohydrate moieties that are expressed on
polypeptides or lipids of the subject being vaccinate. For example
many host cells have been genetically engineered to produce human
proteins with human-like sugar attachments.
[0409] For example, the fusion protein can be a trimeric
hemagglutinin protein. Optionally, the hemagglutinin protein can be
produced in a non-mammalian cell such as a plant cell.
[0410] The subject can be at risk of developing or suffering from a
viral infection. Enveloped viruses include for example,
epstein-barr virus, herpes simplex virus, type 1 and 2, human
cytomegalovirus, human herpesvirus, type 8, varicella zoster virus,
hepatitis B virus, hepatitis C virus, human immunodeficiency virus,
influenza virus, measles virus, mumps virus, parainfluenza virus,
respiratory syncytial virus, rabies virus, and rubella virus.
[0411] The methods described herein lead to a reduction in the
severity or the alleviation of one or more symptoms of a viral
infection. Infections can be diagnosed and or monitored, typically
by a physician using standard methodologies A subject requiring
immunization can be identified by methods know in the art. For
example subjects can be immunized as outlined in the CDC's General
Recommendation on Immunization (51(RR02) pp 1-36) Cancer is
diagnosed for example by physical exam, biopsy, blood test, or
x-ray.
[0412] The subject can be e.g., any mammal, e.g., a human, a
primate, mouse, rat, dog, cat, cow, horse, pig, a fish or a bird.
The treatment ican be administered prior to diagnosis of the
infection. Alternatively, treatment can be administered after
diagnosis. Efficaciousness of treatment can be determined in
association with any known method for diagnosing or treating the
particular disorder or infection. Alleviation of one or more
symptoms of the disorder indicates that the compound confers a
clinical benefit.
[0413] A vaccine candidate targeting humoral immunity can fulfill
at least three criteria to be successful: provoke a strong antibody
response ("immunogenicity"); a significant fraction of the
antibodies it provokes must cross-react with the pathogen
("immunogenic fitness"); and the antibodies it provokes must be
protective. While immunogenicity can often be enhanced using
adjuvants or carriers, immunogenic fitness and the ability to
induce protection (as evidenced by neutralization) are intrinsic
properties of an antigen which will ultimately determine the
success of that antigen as a vaccine component.
[0414] "Immunogenic fitness" is defined as the fraction of
antibodies induced by an antigen that cross-react with the pathogen
(See Matthews et al., J. Immunol. 169:837 (2002)). It is distinct
from immunogenicity, which is gauged by the titer of all of the
antibodies induced by an antigen, including those antibodies that
do not cross-react with the pathogen. Inadequate immunogenic
fitness has probably contributed to the disappointing track record
of peptide vaccines to date. Peptides that bind with high affinity
to antibodies and provoke high antibody titers frequently lack
adequate immunogenic fitness, and, therefore, they fail as
potential vaccine components. Therefore, it can be useful to
include immunogenic fitness as one of the criteria for selecting
influenza vaccine candidates.
[0415] A common explanation for poor immunogenic fitness is the
conformational flexibility of most short peptides. Specifically, a
flexible peptide may bind well to antibodies from patients, and
elicit substantial antibody titers in naive subjects. However, if
the peptide has a large repertoire of conformations, a
preponderance of the antibodies it induces in naive subjects may
fail to cross-react with the corresponding native epitope on intact
pathogen.
[0416] Like short peptides, some APFs may be highly flexible and,
therefore may fail as vaccine components. The most immunogenically
fit APFs are likely to consist of self-folding protein subdomains
that are intrinsically constrained outside the context of the whole
protein.
[0417] Because immunogenic fitness is primarily a property of the
APF itself, and not of the responding immune system, immunogenic
fitness can be evaluated in an animal model (e.g. in mice) even
though ultimately the APF will have to perform in humans.
[0418] The immunogenic fitness achieved by APFs can be evaluated by
immunosorption of anti-APF sera with purified spike or membrane
protein, in a procedure analogous to that described in Matthews et
al., J. Immunol. 169:837 (2002). IgG is purified from sera
collected from mice that have been immunized. Purified,
biotinylated proteins (as appropriate, depending on the particular
APF with which the mice were immunized) can be mixed with the mouse
IgG and incubated. Streptavidin-coated sepharose beads can then be
added in sufficient quantity to capture all of the biotinylated
protein, along with any bound IgG. The streptavidin-coated beads
are removed by centrifugation at 13,000 rpm in a microcentrifuge,
leaving IgG that has been depleted of antibodies directed against
the protein, respectively. Mock immunoabsorptions can be performed
in parallel in the same way, except that biotinylated BSA will be
substituted for influenza protein as a mock absorbent.
[0419] To measure the immunogenic fitness of APFs, the absorbed
antibodies and the mock-absorbed antibodies can be titered
side-by-side in ELISA against the immunizing APF. For APFs affinity
selected from a phage display NPL, the antigen for these ELISAs can
be purified APF-GST fusion proteins. For the potentially
glycosylated APFs from the mammalian cell display NPL, the antigen
for these ELISAs can be APF-Fc fusion proteins secreted by
mammalian cells and purified with protein A. The percentage
decrease in the anti-APF titer of absorbed antibodies compared with
the mock-absorbed antibodies can provide a measure of the
immunogenic fitness of the APF.
[0420] Also disclosed are both prophylactic and therapeutic methods
of treating a subject at risk of (or susceptible to) an influenza
virus-related disease or disorder. Such diseases or disorders
include but are not limited to, e.g., bird flu.
[0421] Also disclosed are methods for preventing an influenza
virus-related disease or disorder in a subject by administering to
the subject a hemagglutinin composition, a monoclonal antibody or
scFv antibody of the invention or an agent identified according to
the methods of the invention. For example, hemagglutinin
compositions, scFv and/or monoclonal antibody D7, D8, F10, G17,
H40, A66, D80, E88, E90, and H98 can be administered in
therapeutically effective amounts. Optionally, two or more
anti-influenza antibodies are co-administered.
[0422] Subjects at risk for an influenza virus-related diseases or
disorders include patients who have come into contact with an
infected person or who have been exposed to the influenza virus in
some other way. Administration of a prophylactic agent can occur
prior to the manifestation of symptoms characteristic of the
influenza virus-related disease or disorder, such that a disease or
disorder is prevented or, alternatively, delayed in its
progression.
[0423] The appropriate agent can be determined based on screening
assays described herein. Alternatively, or in addition, the agent
to be administered can be a scFv or monoclonal antibody that
neutralizes an influenza virus that has been identified according
to the known or disclosed methods.
[0424] Also disclosed are methods of treating an influenza
virus-related disease or disorder in a patient. In some forms, the
method involves administering an agent (e.g., an agent identified
by a screening assay described herein and/or an scFv antibody or
monoclonal antibody identified according to known or disclosed
methods), or combination of agents that neutralize the influenza to
a patient suffering from the disease or disorder.
[0425] Also disclosed is a method of treating an influenza-related
disease or disorder, such as bird flu or swine flu in a patient by
administering two or more antibodies, such as D7, D8, F10, G17,
H40, A66, D80, E88, E90, and H98 that bind to the same epitope of
the HA protein.
[0426] The disclosed subject matter is further described in the
following examples, which do not limit the scope of the invention
as described in the claims.
EXAMPLES
A. Example 1
Isolation and Analysis of Neutralizing Antibodies to Hemagglutinin
Stem Region
[0427] In this example, a phage-display antibody library and
recombinant H5 trimeric ectodomain were used to isolate a group of
high-affinity neutralizing mAbs ("nAbs") that were potent
inhibitors of H5N1 viral infection in vitro and in vivo. Based on
crystallographic and functional studies, it was shown that the nAbs
bind to a common epitope--a highly conserved pocket in the stem
region of HA containing the "fusion peptide"--that rationalizes
their ability to block membrane fusion rather than cell attachment.
Sequence and structural analysis of all 16 HA subtypes points to
the existence of just two variants of this epitope, corresponding
to the two classic phylogenetic groupings of HA (Groups 1 and 2).
Eight further Group 1 HA subtypes were tested and demonstrated a
remarkable and unprecedented cross-subtype binding and/or
neutralization spectrum. Since a Group 1 subtype (H5) was used for
panning, the nAbs, as expected, failed to neutralize a Group 2
subtype, H7. These results nevertheless indicate that a cocktail
comprising a small subset of nAbs raised against representatives of
the two groups can provide broad protection against all seasonal
and pandemic influenza A viruses.
[0428] 1. Methods
[0429] i. Crystallization of the H5-F10 Complex
[0430] H5-F10 complexes were formed by incubating the two purified
components with an excess of F10, and isolated by Superdex 200 in
TBS buffer. Peak fractions were pooled and concentrated to
.about.11 mg ml.sup.-1. The integrity of the H5 trimer was examined
using Gel filtration and SDS-PAGE. Crystals grew at 22.degree. C.
by equilibrating equal volumes of protein and reservoir solution
(12.5% PEG 1K (w/v), 25% ethylene glycol (w/v), 100 mM Tris, pH
8.5) using the hanging drop vapor diffusion technique.
[0431] iI. Data Collection, Structure Determination, and
Refinement
[0432] Diffraction data were collected from crystals flash-frozen
at 100K in the reservoir buffer at the Stanford Synchrotron
Radiation Laboratory beam-line 9.2, set at a wavelength of 1.0
.ANG., and processed with XDS (Kabsch, Automatic processing of
rotation diffraction data from crystals of initially unknown
symmetry and cell constants. Journal of Applied Crystallography 26,
795-800 (1993)) and HKL2000 (Otwinowski & Minor, Processing of
X-ray diffraction data collected in oscillation mode. in Methods in
Enzymology, Volume 276: Macromolecular Crystallography, Part A
(eds. Carter Jr. & Sweet) 307-326 (Academic Press, New York,
1997)). The structure was solved at 3.2 .ANG. resolution by
molecular replacement with PHASER using the structures of H5
(A/Vietnam/1194/04; PDB code 21BX) and a homology model of F10
based on the structure of SARS nAb 80R (PDB code 2GHW) (Hwang et
al., Structural basis of neutralization by a human anti-severe
acute respiratory syndrome spike protein antibody, 80R. J Biol Chem
281, 34610-6 (2006); Rodriguez et al., Homology modeling, model and
software evaluation: three related resources. Bioinformatics 14,
523-8 (1998)) as starting models. The asymmetric unit contains two
H5 trimers and three F10 molecules per trimer, and was refined
using REFMAC5 (Murshudov et al., Refinement of macromolecular
structures by the maximum-likelihood method. Acta Crystallogr D
Biol Crystallogr 53, 240-55 (1997)) with simulated annealing in CNS
(Murshudov et al. (1997)) and manual rebuilding with Coot (Emsley
& Cowtan, Coot: model-building tools for molecular graphics.
Acta Crystallogr D Biol Crystallogr 60, 2126-32 (2004)) and
Xtalview (McRee, A visual protein crystallographic software system
for X11/Xview. Journal of Molecular Graphics 10, 44-46 (1992)). The
final maps are of high quality, and key features such as the F10
CDR loops and interfacial residues are unambiguous and consistent
in the 6 copies. The final model includes 503/503/503/497/497/497
residues for the 6 independent copies of H5,
235/235/236/233/234/234 residues for the 6 F10scFvs, 24
N-acetyl-D-glucosamine and 6 .beta.-D-mannose units, but no water
molecules. The R.sub.FREE is 0.29 with excellent geometry as
assessed with PROCHECK (Laskowski et al., PROCHECK: a program to
check the stereochemical quality of protein structures. Journal of
Applied Crystallography 26, 283-291 (1993)) and Rampage (Table 1):
percentage of residues in favored, allowed and outlier regions are
90.0%, 9.5%, and 0.5%, respectively.
TABLE-US-00033 TABLE 1 Data collection and refinement statistics
for H5-F10. Table 1A Native H5-F10 Data collection Space group C2
Cell dimensions a, b, c (.ANG.) 205., 119., 339. .alpha., .beta.,
.gamma. (.degree.) 90, 99.6, 90 Resolution (.ANG.) 3.2 (3.28-3.20)*
R.sub.merge 0.13 (0.81) I/.sigma.I 9.5 (2.0) Completeness (%) 85
(68) Redundancy 4.5 (4.5) Refinement Resolution (.ANG.) 50-3.2
(3.28-3.20) No. reflections 106885 R.sub.work/R.sub.free 0.23
(0.32)/0.29 (0.38) No. atoms Protein 34573 Carbohydrate 402 Water 0
B-factors Protein 83.5 Carbohydrate 123.7 Water N/A R.m.s.
deviations Bond lengths (.ANG.) 0.010 Bond angles (.degree.) 1.31
Table 1B H5-F10 Data Collection Cell parameters a = 205.3, b =
118.5, c = 338.9 .beta. = 99.6.degree. Space group C2 Resolution
(.ANG.)* 3.2 (3.28-3.20) Total reflections 509705 Unique
reflections 112570 Completeness (%)* 85.0 (68.4) Average
I/.sigma.(I)* 9.5 (2.0) R.sub.MERGE (%)* 12.8 (81.0) Redundancy*
4.5 (4.5) .sigma. cutoff -3 Refinement Resolution 50-3.2
(3.28-3.20) R.sub.WORK* 0.23 (0.32) R.sub.FREE (5% data)* 0.29
(0.38) RMSD bond distance (.ANG.) 0.01 RMSD bond angle (.degree.)
1.31 Average B value 75.7 Solvent atoms 0 .sigma. cutoff none
Ramachandran plot Residues in favored regions 90.0 (%) Residues in
allowed regions 9.5 (%) Residues in outlier regions 0.5 (%) A
single crystal was used for both structure determination at
3.2-.ANG. resolution and refinement. *Values in parentheses are for
highest-resolution shell.
[0433] iii. Phage Display Library Selection
[0434] Recombinant trimeric H5-VN04 ectodomain was produced for
crystallization studies (see below) except that furin co-infection
to ensure complete activation was not employed. Abs were identified
by two-rounds of selection of a 27 billion member human scFv phage
display library against recombinant trimeric H5 immobilized on
Immunotube (Nunc), followed by ELISA screening. Ten unique anti-H5
Abs were identified by sequence analysis of 97 H5-positive clones
out of 392 clones screened.
[0435] iv. Plaque Reduction Assay
[0436] H5-VN04, H5-IN05 or A/Netherland/219/03 (H7N7) (H7-NL03)
viruses (10,000 pfu) were incubated with anti-H5 scFv-Fcs at three
different concentrations (1, 10 or 100 .mu.g mL.sup.-1) at
37.degree. C. for 30 mins. The virus-Ab mixture was diluted
logarithmically and transferred onto MDCK cell monolayers in
12-well plates and incubated at 37.degree. C. for 1 h. Cells were
then washed and overlaid with agar. After 4 days of incubation, the
overlay was discarded, and plaques visualized by crystal violet
staining
[0437] v. Microneutralization Assay
[0438] The method was performed as described previously (Rowe et
al., Detection of antibody to avian influenza A (H5N1) virus in
human serum by using a combination of serologic assays. J Clin
Microbiol 37, 937-43 (1999)). Briefly, 100 TCID.sub.50 (median
tissue culture infectious doses) of virus were mixed in equal
volume with two-fold serial dilutions of Ab stock solution (0.1 mg
ml.sup.-1) in 96-well tissue culture plates, and incubated for 1 h
at 37.degree. C. Indicator MDCK cells (1.5.times.10.sup.4 cells per
well) were added to the plates, followed by incubation at
37.degree. C. for 20 h. To establish the endpoint, cell monolayers
were then washed with PBS, fixed in acetone, and viral antigen
detected by indirect ELISA with a mAb against influenza A NP (A-3,
Accurate).
[0439] vi. Viral Binding Inhibition Assay
[0440] 0.5.times.10.sup.6 293T cells were incubated with
H5-TH04-pseudotyped HIV viruses (.about.500 ng of p24) in the
presence of anti-H5 nAbs, control mAbs, or in the absence of
antibodies, in PBS buffer containing 0.5% (w/v) BSA and 0.02% (w/v)
NaN.sub.3 at 4.degree. C. After 1 h incubation, cells were spun
down. Supernatants were collected and tested for p24 levels using
an HIV-1 p24.sup.CA capture ELISA kit (NCI, Frederick, NIH) to
quantify unbound virus. Cells were then washed once or twice and
lysed to quantify the cell-bound virus using the same method.
[0441] vii. Cell Fusion Inhibition Assay
[0442] HeLa cells, .about.90% confluent in six-well plates, were
transfected with pcDNA3.1-H5-TH04 plasmid (3 .mu.g total DNA per
well) using lipofectamine 2000 (Invitrogen). After .about.30 hours
of transfection, the culture medium was supplemented with 1 ml of
anti-H5 or control mAbs for 1-2 hours, and cells were then washed
and incubated with low-pH fusion buffer (150 mM NaCl+10 mM HEPES,
adjusted to pH 5.0) for 4-5 mins. Cells were then returned to the
standard culture medium for 2-3 hours at 37.degree. C., and finally
fixed with 0.25% (v/v) glutaraldehyde and stained with 0.1% (w/v)
crystal violet. Photomicrographs were taken at 10.times.
magnification.
[0443] viii. Prophylactic and Therapeutic Efficacy Studies in
Mice
[0444] Female 8-10 weeks old BALB/c mice were used in all
experiments. Mice were weighed on the day of virus challenge and
then daily for 2 weeks. Body weight was used as the clinical
endpoint; mice with body weight loss .gtoreq.25% of pre-infection
values were euthanized. Animal studies were conducted per approved
Institutional Animal Care and Use Committee protocols.
[0445] a. Prophylactic Efficacy Study Three human nAbs (D8-IgG1,
F10-IgG1 and A66-IgG1) or control human mAb 80R-IgG1 (Sui et al.
(2004)) at 2.5 mg kg.sup.-1 or 10 mg kg.sup.-1 were administered
into 4 groups of 5 mice each by i.p. injection in 0.5 mL volume.
One hour after mAb administration, two groups of mice were
challenged with H5-VN04 and two groups with H5-HK97 by i.n.
inoculation with 10 MLD.sub.50 in 50 .mu.l volumes per mouse. Mice
were observed and weighed daily for two-weeks after infection.
Analogous studies were performed to evaluate the protective
efficacy of the nAbs against A/Puerto Rico/8/1934 (H1N1) or
A/WSN/1933 (H1N1) viruses.
[0446] b. Post-Exposure Therapy Efficacy Study
[0447] The experimental design recapitulates the prophylaxis study,
with the following exceptions. Twelve groups of 10 mice were first
inoculated i.n. with 10 MLD.sub.50 of VN04. At 24, 48 and 72 hours
after H5-VN04 infection, one group of mice received i.p injections
of 15 mg kg.sup.-1 body weight of one of the three nAbs or control
(80R).
[0448] ix. Expression and Preparation of Various HA Proteins for
Panning
[0449] HA1 is an N-terminal fragment of HA of H5N1
A/Thailand/2(SP-33)/2004 (H5-TH04), residues 11 to 325 (H3
numbering). The gene was codon-optimized and expressed as fusion
protein with a C-terminal 9 amino-acids tag (C9-tag:GTETSQVAPA).
The fusion protein HA1-C9 was expressed in 293T cells transiently
and the secreted proteins in supernatant were harvested 48 hours
after transfection and purified from the supernatant by affinity
chromatography using Protein A Sepharose that coupled covalently
with anti-C9 antibody 1D4 (National Cell Culture Center). The
method to produce HA0 protein of H5-VN04 is the same as described
below for crystallization of H5-F10 complex but without the
baculovirus coinfection for furin-cleavage.
[0450] x. Epitope Mapping of Anti-HAS Antibodies D8, F10 and
A66.
[0451] All the mutants of pcDNA3.1-H5-TH04 were constructed by the
QuikChange method (Stratagene, La Jolla, Calif.). Various
full-length wild type HA and HA mutants expressing plasmids of H1,
H5 or H7 were transfected transiently into 293T cells. 24 hours
after transfection, cells were harvested for immunostaining Anti-H5
or control mAb 80R (Sui et al., Potent neutralization of severe
acute respiratory syndrome (SARS) coronavirus by a human mAb to S1
protein that blocks receptor association. Proc Natl Acad Sci USA
101, 2536-41 (2004)) at 10 .mu.g/mL or ferret anti-H5N1 serum at
1:300 were incubated with transfected 293T cells at 4.degree. C.
for 1 hour. Cells were then washed three times with PBS containing
0.5% BSA and 0.1% NaN.sub.3. FITC-labeled goat anti-human IgG
(Pierce Biotech., Rockford, Ill.) or FITC-labeled goat anti-ferret
IgG (Bethyl, Montgomery, Tex.) were then added to cells and
incubated for 30 mins at 4.degree. C. Cells were washed as above,
and binding of antibodies to cells was analyzed using a Becton
Dickinson FACScalibur with CellQuest software.
[0452] xi. Expression and Preparation of Phage-scFvs, Soluble
scFv-Fcs and Full-Length Human IgG1
[0453] Phage-scFvs of individual clones were produced as described
previously (Sui et al., Potent neutralization of severe acute
respiratory syndrome (SARS) coronavirus by a human mAb to S1
protein that blocks receptor association. Proc Natl Acad Sci USA
101, 2536-41 (2004)). scFv-Fcs and whole human IgG1 were produced
as described previously (Sui et al., Potent neutralization of
severe acute respiratory syndrome (SARS) coronavirus by a human mAb
to S1 protein that blocks receptor association. Proc Natl Acad Sci
USA 101, 2536-41 (2004); Gould et al., Protective and therapeutic
capacity of human single-chain Fv-Fc fusion proteins against West
Nile virus. J Virol 79, 14606-13 (2005); Reff et al., Depletion of
B cells in vivo by a chimeric mouse human monoclonal antibody to
CD20. Blood 83, 435-445 (1994)). In brief, selected scFvs were
converted to scFv-Fcs by subcloning the scFv into an Fc expression
vector pcDNA 3.1-Hinge which contains the hinge, CH2, and CH3
domains of human IgG1 but lacks CH1. For whole human IgG1s, the VH
and VL gene fragments of scFv were separately subcloned into human
IgG1 kappa light chain or lambda light chain expression vector
TCAE5 or TCAE6 (Reff et al., Depletion of B cells in vivo by a
chimeric mouse human monoclonal antibody to CD20. Blood 83, 435-445
(1994)). scFv-Fcs or IgG1s were expressed in 293T or 293F cells
(Invitrogen) by transient transfection and purified by protein A
sepharose affinity chromatography.
[0454] xii. ELISA
[0455] 0.2 .mu.g of pure H5 HA proteins was coated onto 96-well
Maxisorb ELISA plate (Nunc, NY) at 2 .mu.g/mL in PBS at 4.degree.
C. overnight. The plate was washed with PBS for 3 times to remove
uncoated proteins. For regular ELISA, 1 .mu.g/mL of anti-H5
scFv-Fcs followed by HRP-anti-human IgG1 were used to detect the
binding of anti-H5 scFv-Fcs to H5 HA proteins. For competition
ELISA, 50 .mu.L (10.sup.12 pfu) of anti-H5 phage-scFvs were mixed
with 5 .mu.g/mL of anti-H5 scFv-Fcs and applied to H5-VN04 HA
coated ELISA plate. The competition of scFv-Fcs for the binding of
phage-scFvs to HA0 were determined by measuring the remaining
binding of phage-scFvs using HRP-anti-M13. The optical density at
450 nm was measured after incubation of peroxidase
tetramethylbenzidine (TMB) substrate system (KPL, Gaithersburg
Md.).
[0456] xiii. Surface Plasmon Resonance (SPR) Analysis
[0457] Kinetic analyses of H5 HA mAbs binding to recombinant
H5-VN04 HA0 trimer were performed on a Biacore T100 (Biacore,
Sweden) at 25.degree. C. Anti-human IgG Fc antibody (Biacore) was
covalently attached to individual flow cell surfaces of a CM4
sensor chip by amine-coupling using the amine coupling kit
(Biacore). HA mAbs were captured onto anti-human IgG Fc surfaces
(flow rate of 10 .mu.l/min in HBS buffer (Biacore)) to ensure that
mAb-H5 binding occurred as a homogenous 1:1 Langmuir interaction.
H5 was injected over each flow cell at a flow rate of 30 .mu.l/min
in HBS buffer, and at concentrations ranging from to 0.31 to 20 nM.
A buffer injection served as a negative control. All experiments
contained an additional anti-human IgG Fc antibody control surface
that served to account for changes in the buffer refractive index
and to test for potential nonspecific interactions between H5 and
anti-human IgG Fc. Upon completion of each association and
dissociation cycle, surfaces were regenerated with 3 M MgCl.sub.2
solution. The association rates (ka), dissociation rates (kd), and
affinity constants (K.sub.D) were calculated using Biacore T100
evaluation software. The quality of each fit was based on the
agreement between experimental data and the calculated fits, where
the Chi.sup.2 values were below 1.0. Surface densities of mAbs
against H5 were optimized to minimize mass transfer and avoid any
contribution of avidity effects. All ka, kd, K.sub.D values
reported here represent the mean and standard error of three
experiments.
[0458] xiv. Haemagglutination Inhibition (HI) Assay
[0459] The HI test was performed as previously described (Donald
& Isaacs, Counts of influenza virus particles. J Gen Microbiol
10, 457-64 (1954)). Briefly, H5N1/PR8 (Subbarao et al., Evaluation
of a genetically modified reassortant H5N1 influenza A virus
vaccine candidate generated by plasmid-based reverse genetics.
Virology 305, 192-200 (2003)), H5-VN04 and H1-PR34 viruses were
mixed with Log 2 antibody dilutions in PBS and incubated at
20-22.degree. C. for 30 minutes. A 0.5% suspension of turkey
erythrocytes was added to each well and the mixture incubated for
30 min at RT before visual scoring for haemagglutination
activity.
[0460] xv. Expression, Purification, and Crystallization of the
H5-F10 Complex
[0461] The gene encoding single chain (VH-linker-VL) F10 (scFv) was
cloned into pSynI vector containing an N-terminal periplasmic
secretion signal pelB, and a C-terminal 6.times.His tag. F10 scFv
was expressed in XL10 cells in 2YT media containing 0.1% glucose
(w/v) at 25.degree. C. for 15 hours with 0.5 mM IPTG. Protein was
purified first by Hisbind Ni-NTA (Novagen) according to the
manufacturer's instructions, and then by Superdex 200 (Amersham
Biosciences) in 50 mM Tris-HCl, 0.5 M NaCl, pH 8.
[0462] The ectodomain of H5-VN04 HA gene was expressed in insect
cells as a fusion protein by adapting the protocol described
previously (Stevens et al., Structure and receptor specificity of
the hemagglutinin from an H5N1 influenza virus. Science 312, 404-10
(2006)). This construct contains a C-terminal trimerizing `foldon`
sequence from the bacteriophage T4 fibritin to stabilize the
trimeric structure, followed by a thrombin site and a His.sub.6
tag. The cDNA of the fusion protein was cloned into the baculovirus
transfer vector, pAcGP67A (BD Biosciences, Bedford, Mass.), to
allow for efficient secretion of recombinant protein. To obtain
fully cleaved HA (as HA1-HA2 trimers), sf9 cells were co-infected
with baculovirus stocks of HA0 and furin at an empirically derived
ratio. The furin cDNA was a gift from Dr. Robert Fuller (University
of Michigan). Three days after infection, the cells were spun down
and the supernatant was incubated with Ni-NTA beads (Qiagen Inc.,
Valencia, Calif.). The beads were washed with TBS buffer (10 mM
Tris.HCl, 80 mM NaCl, pH8.0) with 10 mM imidazole, and eluted with
TBS with 250 mM Imidazole. The eluted H5 protein was dialyzed
against TBS buffer and further purified by ion-exchange using Mono
Q HR10/10 column
[0463] (GE Healthcare, Piscataway, N.J.). The purified H5 was
digested by thrombin overnight and further purified by Superdex 200
column in TBS buffer.
[0464] H5-F10 complexes were formed by mixing the two purified
components, and isolated by Superdex 200 in TBS buffer (FIG. 9B).
Peak fractions were pooled and concentrated to .about.11 mg/ml. The
integrity of the H5 trimer was examined using Gel filtration
(Superdex 200 column) and SDS-PAGE. Crystals grew by the hanging
drop vapor diffusion method at 22.degree. C. Two .mu.L of H5-F10
were mixed with an equal volume of 12.5% PEG 1K, 25% ethylene
glycol, 100 mM Tris, pH 8.5. Crystals were flash-frozen in liquid
nitrogen prior to data collection.
[0465] xvi. Data Collection, Structure Determination, and
Refinement
[0466] X-ray diffraction data were collected at the Stanford
Synchrotron Radiation Laboratory (SSRL) beam-lines 7.1 and 9.2.
Data were processed with XDS (Kabsch, Automatic processing of
rotation diffraction data from crystals of initially unknown
symmetry and cell constants. J. Appl Cryst 26, 795-800 (1993)) and
the HKL2000 package (Otwinowski & Minor, in Macromolecular
Crystallography, part A (ed. C. W. Carter, J. R. M. S.) 307-326.
(Academic Press (New York), 1997)).
[0467] The structure of the H5-F10 complex was determined by
molecular replacement with PHASER using the structure of H5
(A/Vietnam/1194/04; PDB code 21BX) and the scFv structure of SARS
nAb 80R (PDB code 2 GHW) as search models. The scFv structure
homology model was build with WHATIF (Rodriguez et al., Homology
modeling, model and software evaluation: three related resources.
CABIOS 14, 523-528 (1998)). The asymmetric unit contains two H5
trimers and six F10 molecules.
[0468] Solutions from molecular replacement were subjected to
several rounds of refinement with the program REFMAC5 (Murshudov et
al., Refinement of Macromolecular Structures by the
Maximum-Likelihood Method. Acta Cryst D53, 240-255 (1997)) with
simulated annealing in CNS (Murshudov et al. (1997)) and manual
model rebuilding with Coot (Emsley & Cowtan, Model-Building
Tools for Molecular Graphics. Acta Crystallographica Section
D--Biological Crystallography 60, 2126-2132 (2004)) and Xtalview
(McRee, A visual protein crystallographic software system for
X11/XView. J. Molecular Graphics 10, 44-46 (1992)). The final model
includes 506/503/503/496/495/496 residues for 6 independent copies
of HA, respectively, 233 residues for each nAb10, 24
N-acetyl-d-glucosamine and 6 .beta.-d-mannose, 0 water molecules.
Geometric parameters were assessed with PROCHECK (Morris et al., A
program to check the stereochemical quality of protein structures.
J. Appl Cryst 26, 283-291 (1993)) and Rampage.
[0469] Accession codes. Protein Data Bank: Coordinates and
structure factors for the H5-F10 complex have been deposited with
codes PDB ID 3FKU and RCSB ID RCSB050713.
[0470] 2. Results
[0471] i. Identification of nAbs Against H5N1
[0472] The current H5N1 epidemics involve viruses derived from a
single lineage of H5 HA. Within this lineage, four distinct clades
have been identified as major threats to public health (W.H.O. web
site who.int/csr/disease/avian
influenza/guidelines/summaryH520070403.pdf (2007); W.H.O. Evolution
of H5N1 avian influenza viruses in Asia. Emerg Infect Dis 11,
1515-21 (2005)). Recombinant trimeric ectodomain of H5 HA from one
of these viruses (strain A/Vietnam/1203/04 (H5N1), "H5-VN04", Clade
1) was expressed in insect cells (Stevens et al., Structure and
receptor specificity of the hemagglutinin from an H5N1 influenza
virus. Science 312, 404-10 (2006)) (FIG. 9), immobilized it on a
plastic surface, and selected Abs from a "non-immune" human Ab
phage display library (utilizing single-chain VH-VL fragments
("scFv")) (Sui et al., Potent neutralization of severe acute
respiratory syndrome (SARS) coronavirus by a human mAb to 51
protein that blocks receptor association. Proc Natl Acad Sci USA
101, 2536-41 (2004)). Two rounds of panning and the screening of
392 clones identified 10 unique Abs that were formed by six
distinct VH (variable region of heavy chain) fragments in
combination with seven different VL (variable region of light
chain) fragments (Table 2).
[0473] Table 2 shows framework regions 1-4 (FR1-4) and
complementarity-determining regions 1-3 (CDR1-3) for both the VH
and VL are shown. FR and CDR regions are defined using the Kabat
database. The VH and VL gene names are shown on the right (using
the IMGT database). Dots denote identity with the consensus
sequence, whereas hyphens denote gaps. Six different VH and 10
different VL genes were found. Some antibodies share the same VH
gene. Five out of the six different VH belong to one gene family,
IGHV1-69*01. The VL genes are more diverse than the VH genes, three
out of the 10 VL are .kappa. chain. Highlighted residues for F10
are critical for binding to H5, and their conservation is
indicated. VH sequences, from top to bottom: SEQ ID NO:1
(consensus) and SEQ ID NOs:3-9. VL sequences, from top to bottom:
SEQ ID NO:2 (consensus) and SEQ ID NOs:10-19.
TABLE-US-00034 TABLE 2 Amino acid sequences of variable regions of
anti-H5 mAbs VH FR1 CDR1 FR2 CDR2 Consensus IGHV1-69*01 D7/H98
D8/D80 F10/E90 G17 H40 A66/E8 QVQLVQSGAEVKKPGSSVKVSCKASGGTFS
.............................. ........................P..IFN
.............................. ......................TS.EV...
...............A.......T..V... ...........R...A..........Y..T
...........................P.. SYAIS ..... TN.F. AY.FT .F... .....
G.Y.H MT.FT WVRQAPGQGLEWMG .............. ............V.
.............. ............L. .............. ..............
.L............ ##STR00001## FR3 CDR3 FR4 IGHV gene Consensus
IGHV1-69*01 D7/H98 D8/D80 F10/E90 G17 H40 A66/E8
RVTITADESTSTAYMELSSLRSEDTAVYYCAR ................................
..........N......T....A......... ........L...........T.....L.....
.......Q..R....D.R.............. .......KP...V....N...A..........
W..M.R.T.IN.....VTR.T.D......... ..........N..N...T..K...........
##STR00002## WGQGTLVTVSS ----------- ..L........ ...........
........... .....M..... ..L..T..... .....M..... 1-69 1-69 1-69 1-69
1-2 1-69 VL FR1 CDR1 FR2 CDR2 Consensus QPVLTQPPSASGSPGQRVTISC
TGSSSNIG--NYVA WYQQKPGQAPKLLIY SNSD----RPS D7VL
NFM....H.V.A...KT..... .......A-A...Q ....R..S..TTV.. EDDR----...
D8VL .S..............S..... ..T..DV.GY.S.S ....H..K....M..
EVTK----... F10VL ..G......V.KGLR.TA.LT. ..N.N.V.-NQGA.
.L..HQ.HP....S. R.N.----... G17VL SYE......V.KGLR.TAILT.
..D.N.V.-HQGT. .L..HQ.HP....S. R.GN----... H40VL
.........V.VA...TAS.P. G.--NN..-GYS.H ..........L.... DDK.----...
A66VK EI....S.ATLL...E.A.L.. RA.Q.VSS---.L ..........R....
DA.N----.AT D80VK EI....S.GTLL...E.A.L.. RA.Q.LSSK--.L.
..........R.... GA.S----.AT E88VL L...........T...R.TI..
S.......-S.T.N ....L..T....... S.NQ----... E90VK
DIQM..S..SLSASVGD....T. RA.Q.ISS---.LN .......K....... AA.S----LQR
H98VL SYE...P.....KH..R..... S.GT....-R.H.N ....L..T.......
..EQ----... FR3 CDR3 FR4 IGLV gene Consensus
GIPDRFSGS--RSGTTASLTISGLQPEDEADYYC QSYDS-LSAYV FGGGTKLTVL D7VL
.V.......ID..SNS.........T........ ....T-NNHA. .....H.... LV6-57
D8VL .V.....A.--K..N.....V....A......F. C..AG-H.... ..T...V...
LV2-11 F10VL .ISE...A.--...N......T............ STW..S...V.
.......... LV10-54 G17VL .ISE...A.--...N......I............
SVW..S...W. .......... LV10-54 H40VL
.I.E.....--N..S..T....RVEAG..G.... .VW..GNFRPL .......... LV3-21
A66VK .I.A.....--G...DFT....R.E...F.V.F. .Q.G.--.PQ- ..Q..R.EIK
KV3-20 D80VK .I.D.....--G...DFT....R.E...F.V.S. .Q..G--VPRT
..Q..TVEIK KV3-20 E88VL .V.D.....--....S...A.I..R.........
.....R...SL ..T..TV... LV1-44 E90VL
.V.S.....--G...DFT....S.....F.V... QQ...--.PYT ..Q...VEIK KV1-39
H98VL .V.D.....--K...S...AV....S........ A.W.DN..GW. ..........
LV1-44
[0474] All 10 nAbs were found to bind trimeric H5-VN04 with similar
avidity, but did not bind monomeric HA1 (FIG. 1A). Presented as
scFv-Fc constructs, they potently neutralized the Clade 1 H5
pseudo-virus, A/Thailand/2-SP-33/2004 (H5N1) ("H5-TH04") (FIG. 1B);
and, in a stringent plaque-reduction assay, they all exhibited high
levels of neutralization against H5-VN04, as well as the more
divergent (Clade 2.1) A/Indonesia/5/2005 ("H5-IN05") (FIGS. 1C and
1D). It was also found that the nAbs cross-competed with each other
in a competition ELISA (FIG. 10), indicating that they share a
common epitope. Based on this finding, as well as VH sequence
diversity and neutralization potency, three of the nAbs (D8, F10
and A66) were converted into full-length human IgG1s for further
studies; all three IgG1s bound to recombinant H5-VN04 with high
affinity (Kd.about.100-200 pM) and very slow dissociation rates
(kd.about.10.sup.-4s.sup.-1) (FIG. 11).
[0475] ii. Prophylactic and Therapeutic Efficacy in Mice
[0476] The protective efficacy of the three IgG1s against H5N1
virus infection was evaluated in a BALB/c mouse model (FIG. 2).
Mice were treated with IgG1s before (prophylactically) or after
(therapeutically) lethal viral challenge. Prophylaxis using 10 mg
kg.sup.-1 of IgG1s effectively protected (80-100%) mice when
challenged with a high lethal dose of H5-VN04 (Clade 1) or
A/HongKong/483/97 (H5-HK97) (Clade 0) (FIGS. 2A and 2B).
Therapeutic treatment with 15 mg kg.sup.-1 (an achievable dose in
humans) of IgG1 at 24 h post-inoculation also protected 80-100% of
the mice challenged with either H5-VN04 or H5-HK97 virus (FIGS. 2C
and 2D). Mice treated at later times (48 or 72 h post-inoculation)
with H5-VN04 showed similar or higher levels of protection (FIGS.
2E and 2F). Furthermore, surviving mice remained healthy and showed
minimal body weight loss over the 2-week observation period.
[0477] While human influenza viruses are typically restricted to
the upper respiratory tract, systemic spread is a typical outcome
of H5N1 infection in mice, and has been reported in some humans. It
was found that the three IgG1s caused potent suppression of viral
replication in the lungs (measured 4 days post-challenge) of mice
treated within 48 hours of viral challenge; and that two IgG1s, F10
and A66, were effective when given at 72 hpi. The impact of
antibody therapy on systemic infection was dramatically
demonstrated by .gtoreq.1000-fold suppression of virus spread to
the spleen, even when given 72 hpi (FIG. 12). Suppression was also
seen in the brain, but in this case systemic spread was too low in
control animals for accurate quantitation.
[0478] iii. nAbs Inhibit Cell Fusion Rather than Receptor
Binding
[0479] Two ways in which anti-HA Abs can neutralize infection is by
blocking the initial binding of HA to its cellular receptor (sialic
acid) or by interfering with the subsequent step of HA-mediated
virus-host membrane fusion, which occurs in acidic endosomes
(Skehel & Wiley, Receptor binding and membrane fusion in virus
entry: the influenza hemagglutinin. Annu Rev Biochem 69, 531-69
(2000); Kida et al., Interference with a conformational change in
the haemagglutinin molecule of influenza virus by antibodies as a
possible neutralization mechanism. Vaccine 3, 219-22 (1985)). It
was found that none of the nAbs inhibited virus binding to cells
(FIG. 3A) or hemagglutination of red blood cells. However, it was
shown, using a model system of cell fusion, that the nAbs potently
inhibited membrane fusion (FIG. 3B).
[0480] iv. Structural Characterization of the nAb Epitope
[0481] In order to provide a structural basis for neutralization
and to establish that even broader-spectrum therapeutics can be
based on this discovery, the crystal structure of F10 (as the scFv
fragment) in complex with the H5 (H5-VN04) ectodomain (FIG. 4) was
determined. H5 activated by cleavage of the single chain precursor,
HA0, into two polypeptides, HA1 and HA2 was used. Cleavage leads to
the partial burial of the "fusion peptide" (the first .about.21
residues of each HA2) into the stem (Skehel & Wiley (2000); Ha
et al., H5 avian and H9 swine influenza virus haemagglutinin
structures: possible origin of influenza subtypes. Embo J 21,
865-75 (2002)), which also contributes to the formation of each of
three hydrophobic "pockets" located below the large trimeric
receptor-binding head. In the complex, one F10 nAb binds into each
pocket, burying .about.1500 .ANG..sup.2 of protein surface. Only
the heavy chain (VH) participates directly in binding, utilizing
all three of its complementarity-determining regions (CDRs). The
light chain (VL) points out into solution, and makes only
non-specific contacts with the distal end of the oligosaccharide of
glycosylated residue Asn33.sub.1 from a neighboring monomer. The
epitope on H5 encompasses the entire pocket, which is formed by the
HA2 fusion peptide, flanked by elements of HA1 on one side and
helix .alpha.A of HA2 on the other.
[0482] The key interactions are as follows (FIG. 4B): (i) CDR-H2
adopts the "type 2" conformation (Chothia et al., Structural
repertoire of the human VH segments. J Mol Biol 227, 799-817
(1992)), which is relatively rare in human Abs. Two hydrophobic
residues, Met54 and Phe55, from the tip of H2, insert into the
pocket. Phe55 lies across a flat hydrophobic surface formed by the
main-chain of the fusion peptide, residues 18.sub.2-21.sub.2; it
also makes favorable orthogonal aromatic interactions (Samanta et
al., Packing of aromatic rings against tryptophan residues in
proteins. Acta Crystallogr D Biol Crystallogr 55, 1421-7 (1999))
with the side-chains of Trp21.sub.2 at the back of the pocket, and
His18.sub.1 at the front (subscripts 1 or 2 refer to HA1 or HA2,
and the numbering scheme follows the structure of H3 (pdb:2hmg)
(Stevens et al. (2006); Weis et al., Refinement of the influenza
virus hemagglutinin by simulated annealing. J Mol Biol 212, 737-61
(1990))). The Met54 sulfur makes .pi.-aromatic interactions (Pal
& Chakrabarti, Non-hydrogen bond interactions involving the
methionine sulfur atom. J Biomol Struct Dyn 19, 115-28 (2001)) with
the Trp21.sub.2 ring, hydrophobic interactions with Ile45.sub.2
from helix .alpha.A, an a H-bond between Met54 C.dbd.O and the
His38.sub.1 side-chain; s. (ii) Tyr102 from CDR-H3 extends from the
apex of the H3 loop, to a location only .about.3 .ANG. from Phe55,
and complements CDR-H2 by cementing together the fusion peptide
(via a main-chain H-bond to Asp19.sub.2) and the .alpha.A helix of
HA2 (by intercalating between Thr41.sub.2 and Ile45.sub.2). A large
hydrophobic residue at the neighboring position 103 supports the
side-chain conformation of Tyr102; and (iii) the CDR-H1 loop is
characterized by small hydrophobic/polar side-chains (notably
Val27, Thr 28 and Ser31) such that CDR-H1 fits snugly beneath the
HA head while packing against helix .alpha.A. A somatic mutation of
conserved Gly26=>Glu generates a non-canonical conformation for
H1, with Thr27 pointing outward and making contact with H5.
[0483] An N-terminal hairpin (residues 129.sub.2 and M30.sub.2)
from HA2 of the counterclockwise neighbor packs against the other
side of helix .alpha.A at this point, wrapping around its fusion
peptide and further locking it into place (FIGS. 4A and 4C). Thus,
F10 stabilizes the fusion peptide of more than one subunit. One
framework (FR3) residue, Gln74, appears to be especially important
in stabilizing the CDR-H1 and CDR-H2 loop conformations, by forming
H-bonds to the main chain C.dbd.O groups of Pro53 and Met54, as
well as the side-chain of Ser30. The FR3 residue at position 72 is
the major determinant of the choice between two distinct
conformations of the H2 loop (Chothia et al. (1992)).
[0484] Consistent with the structural data, mutations in three H5
residues on HA2 .alpha.A that make important interactions with
F10--Val52.sub.2, Asn53.sub.2 and Ile56.sub.2--greatly reduce or
ablate nAb binding, while the conservative mutation, Val52Leu, has
no effect (FIGS. 4C and 4D). Mutations to other surfaces of the
.alpha.A helix either have no effect (typically exposed residues)
or lead to increased nAb binding, perhaps by subtly increasing the
flexibility of the epitope (FIG. 4D). Significantly, the nine other
nAbs show very similar mutant binding profiles. Together with the
cross-competition noted above, this indicates that the epitopes for
all 10 nAbs overlap very closely, and that the nAbs bind in a
similar location and orientation.
[0485] v. Structural Basis of H5 Neutralization by the nAb
Panel
[0486] The broad neutralizing behavior against H5 can be attributed
in part to the exclusive role of VH in antigen binding and the use
of a common germline gene, VH1-69, in five out of the six
VHs--although their CDR3 loops are variable in sequence and length
(13-17 residues) (Table 2). In addition, free energy calculations
(Champ & Camacho, FastContact: a free energy scoring tool for
protein-protein complex structures. Nucleic Acids Res 35, W556-60
(2007)) point to dominant binding contributions (.about.70% of the
total favorable free energy) of the three conserved residues in the
VH segment (highlighted residues in Table 2). In CDR-H2 derived
from germline V1-69, position 55 is always Phe, and position 54 is
always hydrophobic (M/I/L/V). In the nAbs used in this example,
CDR-H3 always has a Tyr predicted to lie at the tip of the CDR3
loop (conserved at the 6th position). The conformation and sequence
of the CDR1 loop does not seem to be critical, since the other Abs
that were isolated do not contain the somatic mutation
(Gly26=>Glu) found in F10, and are predicted to have canonical
structures. The sixth VH gene that was isolated is derived from the
germline gene, VH1-2; its H2 loop has the same length as VH1-69,
but by virtue of a change from Ala to Arg at position 72 (Chothia
et al. (1992)) it is predicted to adopt a distinct conformation
("type 3"), which presents loop residues 3 and 4 to the antigen
(rather than residues 4 and 5 in type 2 loops). The specific
somatic mutation at position 4, from Asn to Met, presumably
promotes H5 binding. It is not possible to predict the structure of
the larger H3 loop, but a tyrosine is located at the center of the
loop that may play an analogous role to that in VH1-69.
[0487] Thus, the F10-H5 crystal structure indicates a common
mechanism of H5 virus neutralization for the discovered nAbs. They
make no contact with the receptor-binding sites in the head and so
do not inhibit cell attachment. Rather, they lock the fusion
peptide and helix .alpha.A in place, thereby preventing the large
structural reorganizations that are required for membrane fusion
(Stevens et al., Structure and receptor specificity of the
hemagglutinin from an H5N1 influenza virus. Science 312, 404-10
(2006); Skehel & Wiley (2000); Stevens et al., Structure of the
uncleaved human H1 hemagglutinin from the extinct 1918 influenza
virus. Science 303, 1866-70 (2004); Daniels et al., Fusion mutants
of the influenza virus hemagglutinin glycoprotein. Cell 40, 431-9
(1985); Thoennes et al., Analysis of residues near the fusion
peptide in the influenza hemagglutinin structure for roles in
triggering membrane fusion. Virology 370, 403-14 (2008); Earp et
al., The many mechanisms of viral membrane fusion proteins. Curr
Top Microbiol Immunol 285, 25-66 (2005)). The data point to this
event occurring at an early step in the infectious process,
although it cannot be ruled out that the nAbs act at a later stage,
given the close packing of molecules on the surface of the mature
virion which might restrict early access to the epitope. The only
previously published crystal structure of an HA-nAb complex that
inhibits membrane fusion utilizes a different mechanism: it
prevents conformational changes by cross-linking the upper surfaces
of adjacent subunits in the head (Barbey-Martin et al., An antibody
that prevents the hemagglutinin low pH fusogenic transition.
Virology 294, 70-4 (2002)).
[0488] vi. Anti-H5 nAbs Bind and Neutralize a Broad Range of Group
1 Viruses In Vitro and In Vivo
[0489] Next, all of the available HA sequences (total 6360) in the
public influenza sequence database (Table 3) were examined. Of
note, the sequences of the F10 epitope are nearly always conserved
within the H5 subtype. Indeed, many epitope residues, especially in
HA2, are highly conserved across all 16 HA subtypes (FIG. 5). This
high sequence conservation provides a rationale for the
cross-neutralization of the H5N1 virus clades described above. This
was then confirmed by test the antibodies against a broader range
of HA subtypes.
TABLE-US-00035 TABLE 3 ##STR00003## ##STR00004## Highlights: top `(
)/`, (amino acid variant(s)) / amino acid consensus at the
position; bottom `( )/`, (number of amino acid variants)/ number of
consensus amino acids. Non-highlighted amino acids are 100%
conserved or variants are observed .ltoreq.5 times at those
positions for subtypes H4, H6, H9, H10, H11. Histidines H17 (HA1)
and H111 (HA2) that can play a role in pH-trigger are in bold
underline.
[0490] Group 1 viruses, which contain 10 of the 16 subtypes, are
further classified into 3 "clusters", H1a, H1b, and H9 (Russell et
al., H1 and H7 influenza haemagglutinin structures extend a
structural classification of haemagglutinin subtypes. Virology 325,
287-96 (2004); Fouchier et al., Characterization of a novel
influenza A virus hemagglutinin subtype (H16) obtained from
black-headed gulls. J Virol 79, 2814-22 (2005)) (FIG. 5). nAb
binding to eight members of Clusters H1a, H1b and H9, which include
avian H5 as well as the most common human influenza subtypes (the
major exception is the Group 2 subtype, H3), were tested. In
addition to H5, it was found that all three IgG1s bound to cells
expressing full-length H1 from two different strains of H1N1,
including the 1918 "Spanish flu"; H2 from H2N2; and H6 from H6N2;
the Cluster 1b subtypes: H11 from H11N9; H13 from H13N6; and H16
from H16N3; as well as Cluster H9 subtypes from two H9N2 strains.
However, none of them bound to a Group 2 subtype, H7 from H7N1
(FIG. 1D).
[0491] The IgG1s also neutralized H5-, H1-, H2-, H6- and
H11-pseudotyped virus infections (FIG. 6A). In a
micro-neutralization assay, F10-IgG1 also neutralized H5N1, H1N1,
H2N2, H6N1, H6N2, H8N4, and H9N2 influenza viruses (FIG. 6B).
However, none of the nAbs neutralized Group 2 viruses, e.g. H3N2
(FIG. 6B). Thus, these nAbs recognize an epitope on HA that is
conserved among H5 clades as well as in all members of Group 1
viruses. Finally, the in vivo protective efficacy of two of the
IgG1s was demonstrated against two lethal H1N1 viral strains in a
BALB/c mouse model, using the same protocol as for the H5N1 studies
(FIGS. 6C and 6D).
[0492] vii. Structural Basis of the Group-Specific Broad-Spectrum
Virus Neutralization
[0493] The ability of the nAbs to recognize all Group 1 (cluster
H1a/b and H9) viruses (H12 was not tested) can be attributed to the
key conserved features of the nAbs described above in combination
with the highly conserved pocket on HA (FIGS. 4 and 5). The epitope
can be divided into 3 elements: (i) at its center, the sequence of
the N-terminal segment of HA2-fusion peptide residues
18.sub.2-21.sub.2--is conserved across all HA subtypes (note that
the side-chain at position 19.sub.2 does not participate in
binding); (ii) a downstream segment of HA2 adopts part of the
.alpha.A helix (residues 39.sub.2-56.sub.2), which is nearly
invariant; the only significant difference is a Thr to Gln change
at position 49.sub.2 in the untested H9 cluster subtype, H12.
Thr49.sub.2 lies at the periphery of the epitope and makes one long
H-bond (3.5 .ANG.) to Ser31. Simple modeling suggests there is
plenty of space to accommodate the larger Gln side-chain and that
it can make comparable H-bonds; and (iii) smaller contributions
from segments of the HA1 chain (residues 18.sub.1 and 38.sub.1) and
a loop at the base of the head (residues 291.sub.1 and
292.sub.1).
[0494] 3-dimensional comparisons of the epitope in the 5 known
crystal structure subtypes (three Group 1 (H1, H5 and H9) and two
Group 2 (H3 and H7) (Russell et al., H1 and H7 influenza
haemagglutinin structures extend a structural classification of
haemagglutinin subtypes. Virology 325, 287-96 (2004); Ha et al., H5
avian and H9 swine influenza virus haemagglutinin structures:
possible origin of influenza subtypes. Embo J 21, 865-75 (2002);
Gamblin et al., The structure and receptor binding properties of
the 1918 influenza hemagglutinin. Science 303, 1838-42 (2004);
Yamada et al., Haemagglutinin mutations responsible for the binding
of H5N1 influenza A viruses to human-type receptors. Nature 444,
378-82 (2006); Ha et al., X-ray structure of the hemagglutinin of a
potential H3 avian progenitor of the 1968 Hong Kong pandemic
influenza virus. Virology 309, 209-18 (2003)) show that they adopt
two distinct structural classes consistent with the phylogenetic
groupings (Russell et al. (2004); Fouchier et al. (2005)) (FIG. 7).
These differences arise from group-specific differences in the
location of buried residues, notably histidines (H111.sub.2 is
unique to Group 1; H17.sub.1 is unique to Group 2) that have been
proposed to be the "triggers" for pH-induced conformational changes
(Thoennes et al. (2008)). The differences cause the side-chain of
Trp21.sub.2 to turn through 90.degree. in Group 2 subtypes,
eliminating favorable binding to Phe55 from the tested nAbs. In
addition, four out of six Group 2 subtypes are glycosylated at
position 38.sub.1, at the periphery of the F10 epitope; modeling
studies predict steric clashes with the CDR-H1 loop. These
structural differences rationalize the observed lack of
binding/neutralization of Group 2 HA subtypes and viruses.
[0495] viii. Prospects for Immune Escape
[0496] The remarkable transformation to the fusogenic state
includes repacking of the central helices of three HA2 protomers to
form a new triple-helical bundle, in which residues 34-37 form an
N-terminal cap, as well as the creation of C-terminal arms that
extend to the N-terminus of the new bundle (Chen et al., N- and
C-terminal residues combine in the fusion-pH influenza
hemagglutinin HA(2) subunit to form an N cap that terminates the
triple-stranded coiled coil. Proc Natl Acad Sci USA 96, 8967-72
(1999)). It is straightforward to model the locations of the F10
epitope residues in this model of the fusogenic state. All 8
epitope residues, which were fully exposed in the neutral pH
structure, become either part of the new hydrophobic bundle core
(Thr41.sub.2, Ile45.sub.2, Val52.sub.2 and Ile56.sub.2), or they
make networks of H-bonds with the C-terminal arms and other
elements that stabilize the new bundle (Lys 38.sub.2, Gln42.sub.2,
Thr49.sub.2, Asn53.sub.2). The requirement for adopting two
entirely different conformations, each with a distinct hydrophobic
core and H-bonding network may place powerful evolutionary
constraints on the sequence of the helix, as evidenced by the
almost complete lack of genetic drift within helix .alpha.A among
the 16 HA subtypes.
[0497] To test this hypothesis, an attempt was made to select
neutralization escape mutants. VN/04 (H5N1) virus was propagated in
MDCK cells for 72 h in the presence of 40 .mu.g ml.sup.-1 of each
of the 3 nAbs as well as a murine Ab, 22F, that targets the
receptor-binding head. Following three in vitro passages, a mutant
VN04 virus (K193E) that was resistant to 22F was isolated. In
contrast, no viruses resistant to any of the 3 IgG1s (D8, F10, or
A66) were identified. While these experiments cannot prove that
escape mutants with unimpaired viral fitness will never arise, they
clearly support the notion that the pocket is more refractory than
epitopes in the head. Notwithstanding, if such mutants should
arise, new reactive nAbs can be identified using the disclosed
methods, or other of the disclosed HA stem antibodies that are
engineered to have even broader spectrum reactivity (Sui et al.,
Broadening of neutralization activity to directly block a dominant
antibody-driven SARS-coronavirus evolution pathway. PLoS Pathog 4,
e1000197 (2008)) can be used.
[0498] 3. Discussion
[0499] Prior to the present study, the vast majority of nAbs
isolated against influenza A virus have targeted the
receptor-binding head and lacked broad cross-neutralizing activity.
However, a murine nAb, termed C179 (Okuno et al., A common
neutralizing epitope conserved between the hemagglutinins of
influenza A virus H1 and H2 strains. J Virol 67, 2552-8 (1993)),
was positively selected on the basis of its cross-neutralization
properties (of H1 and H2 subtypes), and subsequently shown to
neutralize H5, but not Group 2 subtypes (Okuno et al., A common
neutralizing epitope conserved between the hemagglutinins of
influenza A virus H1 and H2 strains. J Virol 67, 2552-8 (1993);
Smirnov et al., An epitope shared by the hemagglutinins of H1, H2,
H5, and H6 subtypes of influenza A virus. Acta Virol 43, 237-44
(1999)). Moreover, C179 was shown to block membrane fusion rather
than cell attachment and to protect mice against viral challenge
(Smirnov et al., Prevention and treatment of bronchopneumonia in
mice caused by mouse-adapted variant of avian H5N2 influenza A
virus using monoclonal antibody against conserved epitope in the HA
stem region. Arch Virol 145, 1733-41 (2000)), although a detailed
mechanism was not reported. The activities of C179 and F10 were
compared and found that both showed similar binding towards H5. F10
was found to efficiently compete with C179 for binding to H5, but
not vice versa. Furthermore, the point mutant V52.sub.2E abrogated
binding to both Abs, while T318.sub.1K only affected C179 binding.
These results suggest that F10 and C179 have partially overlapping
epitopes and that their modes of action are similar.
[0500] The manner in which HA was presented to the antibody phage
display library in this study seems to have been helpful in
presenting the stem portion, since similar attempts to isolate
broadly nAbs using cell-surface expressed HA showed only partial
success against H5, and most Abs recognized linear epitopes (Lim et
al., Neutralizing human monoclonal antibody against H5N1 influenza
HA selected from a Fab-phage display library. Virol J 5, 130
(2008)). As noted above, nAbs that utilize the same VH germline
gene (IGHV1-69 or "VH1-69") were repeatedly isolated. Huang et al.
(Structural basis of tyrosine sulfation and VH-gene usage in
antibodies that recognize the HIV type 1 coreceptor-binding site on
gp120. Proc Natl Acad Sci USA 101, 2706-11 (2004)) have pointed out
that this is the only VH gene that consistently encodes 2
hydrophobic residues at the tip of its CDR-H2 loop; indeed, it is
the only germline gene to encode a Phe at this position, which
makes several critical interactions with H5. Moreover, the "type 2"
H2 loop, which is long and compact, is only predicted to occur in 4
out of the .about.50 human germline genes. These factors can
explain at least in part the remarkable ability of nAbs derived
from this germline gene to cross-react with viral epitopes: their
unusual ability to bind to conserved hydrophobic pockets. Such
pockets are likely to have an important function and for this
reason they are often cryptic in the unactivated state of the
antigen. For example, VH1-69 is the predominant gene utilized by a
group of CD4-induced ("CD41") nAbs raised against the HIV-1 surface
glycoprotein, gp120, where the "pocket" is part of a conserved
co-receptor binding site that is only exposed transiently upon
binding to its primary receptor, CD4 (Huang et al. (2004)).
Similarly, an antibody raised against the HIV gp41 trimeric
"inner-core" fusion protein intermediate utilizes the hydrophobic
tip of its VH1-69 CDR-H2 loop to insert into a conserved
hydrophobic pocket that blocks further assembly to the
fusion-competent 6-helix structure (Luftig et al., Structural basis
for HIV-1 neutralization by a gp41 fusion intermediate-directed
antibody. Nat Struct Mol Biol 13, 740-7 (2006)). In vivo, B cells
carrying the VH1-69 gene are the primary mediators of innate
defense against HCV infection, generating antibodies against its
membrane fusion glycoprotein, E2 (Chan et al., V(H)1-69 gene is
preferentially used by hepatitis C virus-associated B cell
lymphomas and by normal B cells responding to the E2 viral antigen.
Blood 97, 1023-6 (2001)), although the epitope and mode of action
have not been determined. Notably, as disclosed herein, VH1-69 is
not the only germline that is suitable for achieving neutralization
in a similar manner. Another recent example is a nAb against Ebola
virus surface glycoprotein, KZ52, which uses the VH3-21 germline
(Lee et al., Structure of the Ebola virus glycoprotein bound to an
antibody from a human survivor. Nature 454, 177-82 (2008)).
However, the realization and discovery here of their common ability
to lock viral envelope proteins into a non-fusogenic conformation
represents the discovery of a general strategy for broad-spectrum
and/or potent viral neutralization.
[0501] Recent work using immune-based phage-display libraries
generated from B cell populations of patients who survived H5N1
infection resulted in the isolation of three human nAbs that
neutralized both H1 and H5 viral strains. The authors postulated
that the reason for survival was an effective humoral immune
response mediated by such nAb-generating B cells in vivo (Kashyap
et al., Combinatorial antibody libraries from survivors of the
Turkish H5N1 avian influenza outbreak reveal virus neutralization
strategies. Proc Natl Acad Sci USA 105, 5986-91 (2008)), although
no control populations were studied. Analysis of their data
indicates that the antibodies are also derived from the VH1-69
germline gene, and share other key characteristics, including the
Met-Phe pair in CDR-H2 and a tyrosine at the tip of CDR-H3.
[0502] As discussed earlier, the broad-spectrum nAbs described
herein are not generated/expanded during successive rounds of
influenza infection and repeated vaccination. Only the disclosed
methods have been effective to produce such antibodies consistently
and repeatedly. It is unlikely that the F10 epitope provokes
self-tolerance mechanism(s) via auto-antigen mimicry (Scherer et
al., Difficulties in eliciting broadly neutralizing anti-HIV
antibodies are not explained by cardiolipin autoreactivity. Aids
21, 2131-9 (2007)). Rather, an immunodominant Ab response to the
highly-exposed globular head can overwhelm the Ab response to the
F10-epitope. Thus, eliminating or reducing the antigenicity of the
head region of HA provides a composition that can be used to
generate an immune to the HA stem region in vivo. It is not
surprising that many viruses are highly adept at keeping their most
critical (and conserved) determinants of pathogenesis cryptic, in
which case subunit-based vaccines as described herein, such as
those utilizing properly presented fragments of F10 or F10-like
epitopes, for example, can provide distinct advantages over
whole-virus-based approaches for the induction of broad spectrum
nAbs in vivo (Selvarajah et al., Focused dampening of antibody
response to the immunodominant variable loops by engineered soluble
gp140. AIDS Res Hum Retroviruses 24, 301-14 (2008); Scheerlinck et
al., Redistribution of a murine humoral immune response following
removal of an immunodominant B cell epitope from a recombinant
fusion protein. Mol Immunol 30, 733-9 (1993)).
[0503] In summary, in vitro methodologies were used to isolate a
family of high affinity broad-spectrum human nAbs against HA that
show potent in vitro and in vivo efficacy against both highly
pathogenic H5N1s and H1N1s. The nAbs inhibit the post-attachment
fusion process by recognizing a highly conserved epitope within the
stem region of HA at a point where key elements of the
conformational change are brought into close apposition. This
region was shown to be recalcitrant to the generation of escape
mutants. Thus, the disclosed antibodies can be used for passive
immunotherapy, either alone or in combination with small molecule
inhibitors. Finally, structural work pinpoints the reasons why
Group 2 HAs do not bind the nAbs described here: despite surface
sequence similarities, they form a structurally distinct group, but
one that is also highly conserved and therefore can be targeted for
production of Group 2-reactive antibodies.
TABLE-US-00036 TABLE 7 Contact residues at the H5-F10 interface
##STR00005##
Contact residues defined by having a closest interatomic distance
of <4.5 .ANG.. The color scheme indicates contributions of
individual residues to the binding free energy: very favorable
(outline (V27, T28, I45, D19, W21)), favorable (italic (S25, S291,
M292, I56, V52, N53, T49, H18, V18, G20, T57, T41)), negligible
(highlighted (S30, S31, Q40, H38), unfavorable (bold (Q42, K38)),
very unfavorable (bold underline (M54, F55, Y102, S105)).
[0504] Based on energy calculations using the server at the web
site structure.pitt.edu/servers/fastcontact (W.H.O. at the web site
who.int/mediacentre/factsheets/2003/fs211/en/. World Health
Organization factsheet 211: influenza (2003)).
[0505] It is understood that the disclosed method and compositions
are not limited to the particular methodology, protocols, and
reagents described as these may vary. It is also to be understood
that the terminology used herein is for the purpose of describing
particular embodiments only, and is not intended to limit the scope
of the present invention which will be limited only by the appended
claims.
[0506] It must be noted that as used herein and in the appended
claims, the singular forms "a", "an", and "the" include plural
reference unless the context clearly dictates otherwise. Thus, for
example, reference to "an antibody" includes a plurality of such
antibodies, reference to "the antibody" is a reference to one or
more antibodies and equivalents thereof known to those skilled in
the art, and so forth.
[0507] "Optional" or "optionally" means that the subsequently
described event, circumstance, or material may or may not occur or
be present, and that the description includes instances where the
event, circumstance, or material occurs or is present and instances
where it does not occur or is not present.
[0508] Ranges may be expressed herein as from "about" one
particular value, and/or to "about" another particular value. When
such a range is expressed, also specifically contemplated and
considered disclosed is the range from the one particular value
and/or to the other particular value unless the context
specifically indicates otherwise. Similarly, when values are
expressed as approximations, by use of the antecedent "about," it
will be understood that the particular value forms another,
specifically contemplated embodiment that should be considered
disclosed unless the context specifically indicates otherwise. It
will be further understood that the endpoints of each of the ranges
are significant both in relation to the other endpoint, and
independently of the other endpoint unless the context specifically
indicates otherwise. Finally, it should be understood that all of
the individual values and sub-ranges of values contained within an
explicitly disclosed range are also specifically contemplated and
should be considered disclosed unless the context specifically
indicates otherwise. The foregoing applies regardless of whether in
particular cases some or all of these embodiments are explicitly
disclosed.
[0509] Unless defined otherwise, all technical and scientific terms
used herein have the same meanings as commonly understood by one of
skill in the art to which the disclosed method and compositions
belong. Although any methods and materials similar or equivalent to
those described herein can be used in the practice or testing of
the present method and compositions, the particularly useful
methods, devices, and materials are as described. Publications
cited herein and the material for which they are cited are hereby
specifically incorporated by reference. Nothing herein is to be
construed as an admission that the present invention is not
entitled to antedate such disclosure by virtue of prior invention.
No admission is made that any reference constitutes prior art. The
discussion of references states what their authors assert, and
applicants reserve the right to challenge the accuracy and
pertinency of the cited documents. It will be clearly understood
that, although a number of publications are referred to herein,
such reference does not constitute an admission that any of these
documents forms part of the common general knowledge in the
art.
[0510] Throughout the description and claims of this specification,
the word "comprise" and variations of the word, such as
"comprising" and "comprises," means "including but not limited to,"
and is not intended to exclude, for example, other additives,
components, integers or steps.
[0511] Those skilled in the art will recognize, or be able to
ascertain using no more than routine experimentation, many
equivalents to the specific embodiments of the method and
compositions described herein. Such equivalents are intended to be
encompassed by the following claims.
[0512] Although particular embodiments have been disclosed herein
in detail, this has been done by way of example for purposes of
illustration only, and is not intended to be limiting with respect
to the scope of the appended claims, which follow. In particular,
it is contemplated by the inventors that various substitutions,
alterations, and modifications may be made to the disclosed subject
matter without departing from the spirit and scope of the disclosed
subject matter as defined by the claims. Other aspects, advantages,
and modifications considered to be within the scope of the
following claims. The claims presented are representative of the
subject matter disclosed herein. Other, unclaimed subject matter is
also contemplated. Applicants reserve the right to pursue such
subject matter in later claims.
Sequence CWU 1
1
1291121PRTArtificial SequenceConsensus Sequence 1Gln Val Gln Leu
Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ser1 5 10 15Ser Val Lys
Val Ser Cys Lys Ala Ser Gly Gly Thr Phe Ser Ser Tyr 20 25 30Ala Ile
Ser Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met 35 40 45Gly
Gly Ile Ile Pro Met Phe Gly Thr Pro Asn Tyr Ala Gln Lys Phe 50 55
60Gln Gly Arg Val Thr Ile Thr Ala Asp Glu Ser Thr Ser Thr Ala Tyr65
70 75 80Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr
Cys 85 90 95Ala Arg Ser Ser Gly Tyr Tyr Tyr Gly Gly Gly Phe Asp Val
Trp Gly 100 105 110Gln Gly Thr Leu Val Thr Val Ser Ser 115
1202108PRTArtificial SequenceConsensus Sequence 2Gln Pro Val Leu
Thr Gln Pro Pro Ser Ala Ser Gly Ser Pro Gly Gln1 5 10 15Arg Val Thr
Ile Ser Cys Thr Gly Ser Ser Ser Asn Ile Gly Asn Tyr 20 25 30Val Ala
Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Lys Leu Leu Ile 35 40 45Tyr
Ser Asn Ser Asp Arg Pro Ser Gly Ile Pro Asp Arg Phe Ser Gly 50 55
60Ser Arg Ser Gly Thr Thr Ala Ser Leu Thr Ile Ser Gly Leu Gln Pro65
70 75 80Glu Asp Glu Ala Asp Tyr Tyr Cys Gln Ser Tyr Asp Ser Leu Ser
Ala 85 90 95Tyr Val Phe Gly Gly Gly Thr Lys Leu Thr Val Leu 100
105398PRTHomo sapiens 3Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val
Lys Lys Pro Gly Ser1 5 10 15Ser Val Lys Val Ser Cys Lys Ala Ser Gly
Gly Thr Phe Ser Ser Tyr 20 25 30Ala Ile Ser Trp Val Arg Gln Ala Pro
Gly Gln Gly Leu Glu Trp Met 35 40 45Gly Gly Ile Ile Pro Ile Phe Gly
Thr Pro Asn Tyr Ala Gln Lys Phe 50 55 60Gln Gly Arg Val Thr Ile Thr
Ala Asp Glu Ser Thr Ser Thr Ala Tyr65 70 75 80Met Glu Leu Ser Ser
Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95Ala
Arg4122PRTHomo sapiens 4Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val
Lys Lys Pro Gly Ser1 5 10 15Ser Val Lys Val Ser Cys Lys Ala Pro Gly
Gly Ile Phe Asn Thr Asn 20 25 30Ala Phe Ser Trp Val Arg Gln Ala Pro
Gly Gln Gly Leu Glu Trp Val 35 40 45Gly Gly Val Ile Pro Leu Phe Arg
Thr Ala Ser Tyr Ala Gln Asn Val 50 55 60Gln Gly Arg Val Thr Ile Thr
Ala Asp Glu Ser Thr Asn Thr Ala Tyr65 70 75 80Met Glu Leu Thr Ser
Leu Arg Ser Ala Asp Thr Ala Val Tyr Tyr Cys 85 90 95Ala Arg Ser Ser
Gly Tyr His Phe Gly Arg Ser His Phe Asp Ser Trp 100 105 110Gly Leu
Gly Thr Leu Val Thr Val Ser Ser 115 1205121PRTHomo sapiens 5Gln Val
Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ser1 5 10 15Ser
Val Lys Val Ser Cys Lys Ala Ser Gly Gly Thr Phe Ser Ala Tyr 20 25
30Ala Phe Thr Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met
35 40 45Gly Gly Ile Ile Gly Met Phe Gly Thr Ala Asn Tyr Ala Gln Lys
Phe 50 55 60Gln Gly Arg Val Thr Ile Thr Ala Asp Glu Leu Thr Ser Thr
Ala Tyr65 70 75 80Met Glu Leu Ser Ser Leu Thr Ser Glu Asp Thr Ala
Leu Tyr Tyr Cys 85 90 95Ala Arg Ser Gly Leu Tyr Tyr Tyr Glu Ser Ser
Leu Asp Tyr Trp Gly 100 105 110Gln Gly Thr Leu Val Thr Val Ser Ser
115 1206124PRTHomo sapiens 6Gln Val Gln Leu Val Gln Ser Gly Ala Glu
Val Lys Lys Pro Gly Ser1 5 10 15Ser Val Lys Val Ser Cys Thr Ser Ser
Glu Val Thr Phe Ser Ser Phe 20 25 30Ala Ile Ser Trp Val Arg Gln Ala
Pro Gly Gln Gly Leu Glu Trp Leu 35 40 45Gly Gly Ile Ser Pro Met Phe
Gly Thr Pro Asn Tyr Ala Gln Lys Phe 50 55 60Gln Gly Arg Val Thr Ile
Thr Ala Asp Gln Ser Thr Arg Thr Ala Tyr65 70 75 80Met Asp Leu Arg
Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95Ala Arg Ser
Pro Ser Tyr Ile Cys Ser Gly Gly Thr Cys Val Phe Asp 100 105 110His
Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser 115 1207122PRTHomo
sapiens 7Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro
Gly Ala1 5 10 15Ser Val Lys Val Ser Cys Lys Thr Ser Gly Val Thr Phe
Ser Ser Tyr 20 25 30Ala Ile Ser Trp Val Arg Gln Ala Pro Gly Gln Gly
Leu Glu Trp Met 35 40 45Gly Gly Ile Ile Gly Val Phe Gly Val Pro Lys
Tyr Ala Gln Lys Phe 50 55 60Gln Gly Arg Val Thr Ile Thr Ala Asp Lys
Pro Thr Ser Thr Val Tyr65 70 75 80Met Glu Leu Asn Ser Leu Arg Ala
Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95Ala Arg Glu Pro Gly Tyr Tyr
Val Gly Lys Asn Gly Phe Asp Val Trp 100 105 110Gly Gln Gly Thr Met
Val Thr Val Ser Ser 115 1208126PRTHomo sapiens 8Gln Val Gln Leu Val
Gln Ser Gly Ala Glu Val Arg Lys Pro Gly Ala1 5 10 15Ser Val Lys Val
Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Gly Tyr 20 25 30Tyr Ile His
Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met 35 40 45Gly Trp
Ile Asn Pro Met Thr Gly Gly Thr Asn Tyr Ala Gln Lys Phe 50 55 60Gln
Val Trp Val Thr Met Thr Arg Asp Thr Ser Ile Asn Thr Ala Tyr65 70 75
80Met Glu Val Thr Arg Leu Thr Ser Asp Asp Thr Ala Val Tyr Tyr Cys
85 90 95Ala Arg Gly Ala Ser Val Leu Arg Tyr Phe Asp Trp Gln Pro Glu
Ala 100 105 110Leu Asp Ile Trp Gly Leu Gly Thr Thr Val Thr Val Ser
Ser 115 120 1259124PRTHomo sapiens 9Gln Val Gln Leu Val Gln Ser Gly
Ala Glu Val Lys Lys Pro Gly Ser1 5 10 15Ser Val Lys Val Ser Cys Lys
Ala Ser Gly Gly Pro Phe Ser Met Thr 20 25 30Ala Phe Thr Trp Leu Arg
Gln Ala Pro Gly Gln Gly Leu Glu Trp Met 35 40 45Gly Gly Ile Ser Pro
Ile Phe Arg Thr Pro Lys Asn Tyr Ala Gln Lys 50 55 60Phe Gln Gly Arg
Val Thr Ile Thr Ala Asp Glu Ser Thr Asn Thr Ala65 70 75 80Asn Met
Glu Leu Thr Ser Leu Lys Ser Glu Asp Thr Ala Val Tyr Tyr 85 90 95Cys
Ala Arg Thr Leu Ser Ser Tyr Gln Pro Asn Asn Asp Ala Phe Ala 100 105
110Ile Trp Gly Gln Gly Thr Met Val Thr Val Ser Ser 115
12010111PRTHomo sapiens 10Asn Phe Met Leu Thr Gln Pro His Ser Val
Ser Ala Ser Pro Gly Lys1 5 10 15Thr Val Thr Ile Ser Cys Thr Gly Ser
Ser Ser Asn Ile Ala Ala Asn 20 25 30Tyr Val Gln Trp Tyr Gln Gln Arg
Pro Gly Ser Ala Pro Thr Thr Val 35 40 45Ile Tyr Glu Asp Asp Arg Arg
Pro Ser Gly Val Pro Asp Arg Phe Ser 50 55 60Gly Ser Ile Asp Arg Ser
Ser Asn Ser Ala Ser Leu Thr Ile Ser Gly65 70 75 80Leu Gln Thr Glu
Asp Glu Ala Asp Tyr Tyr Cys Gln Ser Tyr Asp Thr 85 90 95Asn Asn His
Ala Val Phe Gly Gly Gly Thr His Leu Thr Val Leu 100 105
11011110PRTHomo sapiens 11Gln Ser Val Leu Thr Gln Pro Pro Ser Ala
Ser Gly Ser Pro Gly Gln1 5 10 15Ser Val Thr Ile Ser Cys Thr Gly Thr
Ser Ser Asp Val Gly Gly Tyr 20 25 30Asn Ser Val Ser Trp Tyr Gln Gln
His Pro Gly Lys Ala Pro Lys Leu 35 40 45Met Ile Tyr Glu Val Thr Lys
Arg Pro Ser Gly Val Pro Asp Arg Phe 50 55 60Ser Ala Ser Lys Ser Gly
Asn Thr Ala Ser Leu Thr Val Ser Gly Leu65 70 75 80Gln Ala Glu Asp
Glu Ala Asp Tyr Phe Cys Cys Ser Tyr Ala Gly His 85 90 95Ser Ala Tyr
Val Phe Gly Thr Gly Thr Lys Val Thr Val Leu 100 105 11012110PRTHomo
sapiens 12Gln Pro Gly Leu Thr Gln Pro Pro Ser Val Ser Lys Gly Leu
Arg Gln1 5 10 15Thr Ala Thr Leu Thr Cys Thr Gly Asn Ser Asn Asn Val
Gly Asn Gln 20 25 30Gly Ala Ala Trp Leu Gln Gln Gly Gln Gly His Pro
Pro Lys Leu Leu 35 40 45Ile Tyr Arg Asn Asn Asp Arg Pro Ser Gly Ile
Ser Glu Arg Phe Ser 50 55 60Ala Ser Arg Ser Gly Asn Thr Ala Ser Leu
Thr Ile Thr Gly Leu Gln65 70 75 80Pro Glu Asp Glu Ala Asp Tyr Tyr
Cys Ser Thr Trp Asp Ser Ser Leu 85 90 95Ser Ala Val Val Phe Gly Gly
Gly Thr Lys Leu Thr Val Leu 100 105 11013110PRTHomo sapiens 13Ser
Tyr Glu Leu Thr Gln Pro Pro Ser Ala Ser Lys Gly Leu Arg Gln1 5 10
15Thr Ala Ile Leu Thr Cys Thr Gly Asp Ser Asn Asn Val Gly His Gln
20 25 30Gly Thr Ala Trp Leu Gln Gln His Gln Gly His Pro Pro Lys Leu
Leu 35 40 45Ser Tyr Arg Asn Gly Asn Arg Pro Ser Gly Ile Ser Glu Arg
Phe Ser 50 55 60Ala Ser Arg Ser Gly Asn Thr Ala Ser Leu Thr Ile Ile
Gly Leu Gln65 70 75 80Pro Glu Asp Glu Ala Asp Tyr Tyr Cys Ser Val
Trp Asp Ser Ser Leu 85 90 95Ser Ala Trp Val Phe Gly Gly Gly Thr Lys
Leu Thr Val Leu 100 105 11014110PRTHomo sapiens 14Gln Pro Val Leu
Thr Gln Pro Pro Ser Val Ser Val Ala Pro Gly Gln1 5 10 15Thr Ala Ser
Ile Pro Cys Gly Gly Ser Ser Asn Asn Ile Gly Gly Tyr 20 25 30Ser Val
His Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Leu Leu Leu 35 40 45Ile
Tyr Asp Asp Lys Asp Arg Pro Ser Gly Ile Pro Glu Arg Phe Ser 50 55
60Gly Ser Asn Ser Gly Ser Thr Ala Thr Leu Thr Ile Ser Arg Val Glu65
70 75 80Ala Gly Asp Glu Gly Asp Tyr Tyr Cys Gln Val Trp Asp Ser Gly
Asn 85 90 95Asp Arg Pro Leu Phe Gly Gly Gly Thr Lys Leu Thr Val Leu
100 105 11015107PRTHomo sapiens 15Glu Ile Val Leu Thr Gln Ser Pro
Ala Thr Leu Leu Ser Pro Gly Glu1 5 10 15Arg Ala Thr Leu Ser Cys Arg
Ala Ser Gln Ser Val Ser Ser Tyr Phe 20 25 30Ala Trp Tyr Gln Gln Lys
Pro Gly Gln Ala Pro Arg Leu Leu Ile Tyr 35 40 45Asp Ala Ser Asn Arg
Ala Thr Gly Ile Pro Ala Arg Phe Ser Gly Ser 50 55 60Gly Ser Gly Thr
Asp Phe Thr Leu Thr Ile Ser Arg Leu Glu Pro Glu65 70 75 80Asp Phe
Ala Val Tyr Phe Cys Gln Gln Tyr Gly Ser Leu Ser Pro Gln 85 90 95Val
Phe Gly Gln Gly Thr Arg Leu Glu Ile Lys 100 10516108PRTHomo sapiens
16Glu Ile Val Leu Thr Gln Ser Pro Gly Thr Leu Leu Ser Pro Gly Glu1
5 10 15Arg Ala Thr Leu Ser Cys Arg Ala Ser Gln Ser Leu Ser Ser Lys
Tyr 20 25 30Leu Ala Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Arg Leu
Leu Ile 35 40 45Tyr Gly Ala Ser Ser Arg Ala Thr Gly Ile Pro Asp Arg
Phe Ser Gly 50 55 60Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser
Arg Leu Glu Pro65 70 75 80Glu Asp Phe Ala Val Tyr Ser Cys Gln Gln
Tyr Asp Gly Leu Val Pro 85 90 95Arg Thr Phe Gly Gln Gly Thr Thr Val
Glu Ile Lys 100 10517110PRTHomo sapiens 17Leu Pro Val Leu Thr Gln
Pro Pro Ser Ala Ser Gly Thr Pro Gly Gln1 5 10 15Arg Val Thr Ile Ser
Cys Ser Gly Ser Ser Ser Asn Ile Gly Ser Asn 20 25 30Thr Val Asn Trp
Tyr Gln Gln Leu Pro Gly Thr Ala Pro Lys Leu Leu 35 40 45Ile Tyr Ser
Asn Asn Gln Arg Pro Ser Gly Val Pro Asp Arg Phe Ser 50 55 60Gly Ser
Arg Ser Gly Thr Ser Ala Ser Leu Ala Ile Ile Gly Leu Arg65 70 75
80Pro Glu Asp Glu Ala Asp Tyr Tyr Cys Gln Ser Tyr Asp Ser Arg Leu
85 90 95Ser Ala Ser Leu Phe Gly Thr Gly Thr Thr Val Thr Val Leu 100
105 11018107PRTHomo sapiens 18Asp Ile Gln Met Thr Gln Ser Pro Ser
Ser Leu Ser Ala Ser Val Gly1 5 10 15Asp Val Thr Ile Ser Thr Arg Ala
Ser Gln Ser Ile Ser Ser Tyr Leu 20 25 30Asn Trp Tyr Gln Gln Lys Pro
Gly Lys Ala Pro Lys Leu Leu Ile Tyr 35 40 45Ala Ala Ser Ser Leu Arg
Gln Gly Val Pro Ser Arg Phe Ser Gly Ser 50 55 60Gly Ser Gly Thr Asp
Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro Glu65 70 75 80Asp Phe Ala
Val Tyr Tyr Cys Gln Gln Tyr Asp Ser Leu Ser Pro Tyr 85 90 95Thr Phe
Gly Gln Gly Thr Lys Val Glu Ile Lys 100 10519110PRTHomo sapiens
19Ser Tyr Glu Leu Thr Gln Pro Pro Ser Ala Ser Gly Lys His Gly Gln1
5 10 15Arg Val Thr Ile Ser Cys Ser Gly Gly Thr Ser Asn Ile Gly Arg
Asn 20 25 30His Val Asn Trp Tyr Gln Gln Leu Pro Gly Thr Ala Pro Lys
Leu Leu 35 40 45Ile Tyr Ser Asn Glu Gln Arg Pro Ser Gly Val Pro Asp
Arg Phe Ser 50 55 60Gly Ser Lys Ser Gly Thr Ser Ala Ser Leu Ala Val
Ser Gly Leu Gln65 70 75 80Ser Glu Asp Glu Ala Asp Tyr Tyr Cys Ala
Ser Trp Asp Asp Asn Leu 85 90 95Ser Gly Trp Val Phe Gly Gly Gly Thr
Lys Leu Thr Val Leu 100 105 110204PRTHomo sapiens 20Val Asp Gly
Trp1214PRTHomo sapiens 21Ile Asp Gly Trp1224PRTHomo sapiens 22Ile
Asn Gly Trp1234PRTHomo sapiens 23Val Ala Gly Trp124122PRTHomo
sapiens 24Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro
Gly Ala1 5 10 15Ser Val Lys Val Ser Cys Lys Thr Ser Gly Val Thr Phe
Ser Ser Tyr 20 25 30Ala Ile Ser Trp Val Arg Gln Ala Pro Gly Gln Gly
Leu Glu Trp Met 35 40 45Gly Gly Ile Ile Gly Val Phe Gly Val Pro Lys
Tyr Ala Gln Asn Phe 50 55 60Gln Gly Arg Val Thr Ile Thr Ala Asp Lys
Pro Thr Ser Thr Val Tyr65 70 75 80Met Glu Leu Asn Ser Leu Arg Ala
Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95Ala Arg Glu Pro Gly Tyr Tyr
Val Gly Lys Asn Gly Phe Asp Val Trp 100 105 110Gly Gln Gly Thr Met
Val Thr Val Ser Ser 115 12025330DNAHomo sapiens 25cagcctgggc
tgactcagcc accctcggtg tccaagggct tgagacagac cgccacactc 60acctgcactg
ggaacagcaa caatgttggc aaccaaggag cagcttggct gcagcagcac
120cagggccacc ctcccaaact cctatcctac aggaataatg accggccctc
agggatctca 180gagagattct ctgcatccag gtcaggaaac acagcctccc
tgaccattac tggactccag 240cctgaggacg aggctgacta ttactgctca
acatgggaca gcagcctcag tgctgtggta 300ttcggcggag ggaccaagct
gaccgtccta 33026110PRTHomo sapiens 26Ser Tyr Glu Leu Thr Gln Pro
Pro Ser Val Ser Lys Gly Leu Arg Gln1 5 10 15Thr Ala Ile Leu Thr Cys
Thr Gly Asp Ser Asn Asn Val Gly His Gln 20 25 30Gly Thr Ala Trp Leu
Gln Gln His Gln Gly His Pro Pro Lys Leu Leu 35 40 45Ser Tyr Arg
Asn Gly Asn Arg Pro Ser Gly Ile Ser Glu Arg Phe Ser 50 55 60Ala Ser
Arg Ser Gly Asn Thr Ala Ser Leu Thr Ile Ile Gly Leu Gln65 70 75
80Pro Glu Asp Glu Ala Asp Tyr Tyr Cys Ser Val Trp Asp Ser Ser Leu
85 90 95Ser Ala Trp Val Phe Gly Gly Gly Thr Lys Leu Thr Val Leu 100
105 11027321DNAHomo sapiens 27gacatccaga tgacccagtc tccatcctcc
ctgtctgcat ctgtaggaga cagagtcacc 60atcacttgcc gggcaagtca gagcattagc
agctatttaa attggtatca gcagaaacca 120gggaaagccc ctaagctcct
gatctatgct gcatccagtt tgcaaagagg ggtcccatca 180aggttcagtg
gcagtggatc tgggacagac ttcactctca ccattagcag cctgcagcct
240gaagattttg cagtgtatta ctgtcagcag tatgatagtt caccgtacac
ttttggccag 300gggaccaagg tagagatcaa a 32128126PRTHomo sapiens 28Gln
Val Gln Leu Val Gln Ser Gly Ala Glu Val Arg Lys Pro Gly Ala1 5 10
15Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Gly Tyr
20 25 30Tyr Ile His Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp
Met 35 40 45Gly Trp Ile Asn Pro Met Thr Gly Gly Thr Asn Tyr Ala Gln
Lys Phe 50 55 60Gln Val Trp Val Thr Met Thr Arg Asp Thr Ser Ile Asn
Thr Ala Tyr65 70 75 80Met Glu Val Ser Arg Leu Thr Ser Asp Asp Thr
Ala Val Tyr Tyr Cys 85 90 95Ala Arg Gly Ala Ser Val Leu Arg Tyr Phe
Asp Trp Gln Pro Glu Ala 100 105 110Leu Asp Ile Trp Gly Leu Gly Thr
Thr Val Thr Val Ser Ser 115 120 12529366DNAHomo sapiens
29caggtgcagc tggtgcaatc tggggctgaa gtgaagaagc ctggggcctc agtgaaggtc
60tcctgcaaga cttctggagt caccttcagc agctatgcta tcagttgggt gcgacaggcc
120cctggacaag ggcttgagtg gatgggaggg atcatcggtg tctttggtgt
accaaagtac 180gcgcagaact tccagggcag agtcacaatt accgcggaca
aaccgacgag tacagtctac 240atggagctga acagcctgag agctgaggac
acggccgtgt attactgtgc gagagagccc 300gggtactacg taggaaagaa
tggttttgat gtctggggcc aagggacaat ggtcaccgtc 360tcttca
36630108PRTHomo sapiens 30Gln Pro Val Leu Thr Gln Pro Pro Ser Val
Ser Val Ala Pro Gly Gln1 5 10 15Thr Ala Ser Ile Pro Cys Gly Gly Asn
Asn Ile Gly Gly Tyr Ser Val 20 25 30His Trp Tyr Gln Gln Lys Pro Gly
Gln Ala Pro Leu Leu Val Ile Tyr 35 40 45Asp Asp Lys Asp Arg Pro Ser
Gly Ile Pro Glu Arg Phe Ser Gly Ala 50 55 60Asn Ser Gly Ser Thr Ala
Thr Leu Thr Ile Ser Arg Val Glu Ala Gly65 70 75 80Asp Glu Gly Asp
Tyr Tyr Cys Gln Val Trp Asp Ser Gly Asn Asp Arg 85 90 95Pro Leu Phe
Gly Gly Gly Thr Lys Leu Thr Val Leu 100 10531330DNAHomo sapiens
31tcctatgagc tgactcagcc accctcggtg tccaagggct tgagacagac cgccatactc
60acctgcactg gagacagcaa caatgttggc caccaaggta cagcttggct gcaacaacac
120cagggccacc ctcccaaact cctatcctac aggaatggca accggccctc
agggatctca 180gagagattct ctgcatccag gtcaggaaat acagcctccc
tgaccattat tggactccag 240cctgaggacg aggctgacta ctactgctca
gtatgggaca gcagcctcag tgcctgggtg 300ttcggcggag ggaccaagct
gaccgtccta 33032123PRTHomo sapiens 32Gln Val Gln Leu Val Gln Ser
Gly Ala Glu Val Lys Lys Pro Gly Ser1 5 10 15Ser Val Lys Val Ser Cys
Lys Ala Ser Gly Gly Pro Phe Ser Met Thr 20 25 30Ala Phe Thr Trp Leu
Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met 35 40 45Gly Gly Ile Ser
Pro Ile Phe Arg Thr Pro Lys Tyr Ala Gln Lys Phe 50 55 60Gln Gly Arg
Val Thr Ile Thr Ala Asp Glu Ser Thr Asn Thr Ala Asn65 70 75 80Met
Glu Leu Thr Ser Leu Lys Ser Glu Asp Thr Ala Val Tyr Tyr Cys 85 90
95Ala Arg Thr Leu Ser Ser Tyr Gln Pro Asn Asn Asp Ala Phe Ala Ile
100 105 110Trp Gly Gln Gly Thr Met Val Thr Val Ser Ser 115
12033378DNAHomo sapiens 33caggtgcagc tggtgcagtc tggggctgag
gtgaggaagc ctggggcctc agtgaaggtc 60tcatgtaagg cttctggata caccttcacc
ggttattata ttcactgggt gcgacaggcc 120cctggacaag gacttgagtg
gatgggttgg atcaacccta tgactggtgg cacaaactat 180gcacagaagt
ttcaggtctg ggtcaccatg acccgggaca cgtccatcaa cacagcctac
240atggaggtga gcaggctgac atctgacgac acggccgtgt attactgtgc
gaggggggct 300tccgtattac gatattttga ctggcagccc gaggctcttg
atatctgggg cctcgggacc 360acggtcaccg tctcctca 37834106PRTHomo
sapiens 34Glu Ile Val Leu Thr Gln Ser Pro Ala Thr Leu Ser Leu Ser
Pro Gly1 5 10 15Glu Arg Ala Thr Leu Ser Cys Arg Ala Ser Gln Ser Val
Ser Ser Tyr 20 25 30Leu Ala Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro
Arg Leu Leu Ile 35 40 45Tyr Asp Ala Ser Asn Arg Ala Thr Gly Ile Pro
Ala Arg Phe Ser Gly 50 55 60Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr
Ile Ser Arg Leu Glu Pro65 70 75 80Glu Asp Phe Ala Val Tyr Phe Cys
Gln Gln Tyr Gly Ser Ser Pro Gln 85 90 95Phe Gly Gln Gly Thr Arg Leu
Glu Ile Lys 100 10535324DNAHomo sapiens 35cagcctgtgc tgactcagcc
accctcggtg tcagtggccc caggacagac ggccagcatt 60ccctgtgggg ggaacaacat
tggaggctac agtgtacact ggtaccaaca aaagccgggc 120caggcccccc
tcttggtcat ttatgacgat aaagaccggc cctcagggat ccctgagcga
180ttctctggcg ccaactctgg gagcacggcc accctgacaa tcagcagggt
cgaagccggg 240gatgagggcg actactactg tcaggtgtgg gatagtggta
atgatcgtcc gctgttcggc 300ggagggacca agctgaccgt ccta 32436123PRTHomo
sapiens 36Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro
Gly Ser1 5 10 15Ser Val Lys Val Ser Cys Lys Ala Ser Gly Gly Pro Phe
Ser Met Thr 20 25 30Ala Phe Thr Trp Leu Arg Gln Ala Pro Gly Gln Gly
Leu Glu Trp Met 35 40 45Gly Gly Ile Ser Pro Ile Phe Arg Thr Pro Lys
Tyr Ala Gln Lys Phe 50 55 60Gln Gly Arg Val Thr Ile Thr Ala Asp Glu
Ser Thr Asn Thr Ala Asn65 70 75 80Met Glu Leu Thr Ser Leu Lys Ser
Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95Ala Arg Thr Leu Ser Ser Tyr
Gln Pro Asn Asn Asp Ala Phe Ala Ile 100 105 110Trp Gly Gln Gly Thr
Met Val Thr Val Ser Ser 115 12037369DNAHomo sapiens 37caggtgcagc
tggtgcagtc tggggctgaa gtgaagaagc ctggctcctc ggtgaaggtt 60tcctgcaagg
cttctggagg ccccttcagc atgactgctt tcacctggct gcgacaggcc
120cctggacaag ggcttgagtg gatgggtggg atcagcccta tctttcgtac
accgaagtac 180gcacagaagt tccagggcag agtcacgatt accgcggacg
aatccacgaa cacagccaac 240atggagctga ccagcctgaa atctgaggac
acggccgtgt attactgtgc gagaaccctt 300tcctcctacc aaccgaataa
tgatgctttt gctatctggg gccaagggac aatggtcacc 360gtctcttca
36938110PRTHomo sapiens 38Leu Pro Val Leu Thr Gln Pro Pro Ser Ala
Ser Gly Thr Pro Gly Gln1 5 10 15Arg Val Thr Ile Ser Cys Ser Gly Ser
Ser Ser Asn Ile Gly Ser Asn 20 25 30Thr Val Asn Trp Tyr Gln Gln Leu
Pro Gly Thr Ala Pro Lys Leu Leu 35 40 45Ile Tyr Ser Asn Asn Gln Arg
Pro Ser Gly Val Pro Asp Arg Phe Ser 50 55 60Gly Ser Arg Ser Gly Thr
Ser Ala Ser Leu Ala Ile Ile Gly Leu Arg65 70 75 80Pro Glu Asp Glu
Ala Asp Tyr Tyr Cys Gln Ser Tyr Asp Ser Arg Leu 85 90 95Ser Ala Ser
Leu Phe Gly Thr Gly Thr Thr Val Thr Val Leu 100 105 11039318DNAHomo
sapiens 39gaaattgtgt tgacgcagtc tccagccacc ctgtctttgt ctccagggga
aagagccacc 60ctctcctgca gggccagtca gagtgttagc agctacttag cctggtacca
acagaaacct 120ggccaggctc ccaggctcct catctatgat gcatccaaca
gggccactgg catcccagcc 180aggttcagtg gcagtgggtc tgggacagac
ttcactctca ccatcagcag actggagcct 240gaagattttg cagtctattt
ctgtcagcag tatggtagct cacctcaatt cggccaaggg 300acacgactgg agattaaa
31840369DNAHomo sapiens 40caggtgcagc tggtgcagtc tggggctgaa
gtgaagaagc ctggctcctc ggtgaaggtt 60tcctgcaagg cttctggagg ccccttcagc
atgactgctt tcacctggct gcgacaggcc 120cctggacaag ggcttgagtg
gatgggtggg atcagcccta tctttcgtac accgaagtac 180gcacagaagt
tccagggcag agtcacgatt accgcggacg aatccacgaa cacagccaac
240atggagctga ccagcctgaa atctgaggac acggccgtgt attactgtgc
gagaaccctt 300tcctcctacc aaccgaataa tgatgctttt gctatctggg
gccaagggac aatggtcacc 360gtctcttca 36941369DNAHomo sapiens
41caggtgcagc tggtgcagtc tggggctgag gtgaagaagc ctgggtcctc ggtgaaggtc
60tcctgcaagg cttctggagg caccttcagt gacaatgcta tcagctgggt gcgacaggcc
120ccaggacaag ggcttgagtg gatggggggc atcattccta tctttggaaa
accaaactac 180gcacagaagt tccagggcag agtcacgatt actgcggacg
aatccacgag cacagcctac 240atggacctga ggagcctgag atctgaggac
acggccgttt attactgtgc gagagattca 300gacgcgtatt actatggttc
ggggggtatg gacgtctggg gccaaggcac cctggtcacc 360gtctcctca
36942330DNAHomo sapiens 42ctgcctgtgc tgactcagcc accctcagcg
tctgggaccc ccgggcagag ggtcaccatc 60tcttgttctg gaagcagctc caacatcgga
agtaatactg taaactggta ccagcagctc 120ccaggaacgg cccccaaact
cctcatctat agtaataatc agcggccctc aggggtccct 180gaccgattct
ctggctccag gtcaggcacc tcagcctccc tggccatcat tggactccgg
240cctgaggatg aagctgatta ttactgtcag tcgtatgaca gcaggctcag
tgcttctctc 300ttcggaactg ggaccacggt caccgtcctc 33043123PRTHomo
sapiens 43Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro
Gly Ser1 5 10 15Ser Val Lys Val Ser Cys Lys Ala Ser Gly Gly Thr Phe
Ser Asp Asn 20 25 30Ala Ile Ser Trp Val Arg Gln Ala Pro Gly Gln Gly
Leu Glu Trp Met 35 40 45Gly Gly Ile Ile Pro Ile Phe Gly Lys Pro Asn
Tyr Ala Gln Lys Phe 50 55 60Gln Gly Arg Val Thr Ile Thr Ala Asp Glu
Ser Thr Ser Thr Ala Tyr65 70 75 80Met Asp Leu Arg Ser Leu Arg Ser
Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95Ala Arg Asp Ser Asp Ala Tyr
Tyr Tyr Gly Ser Gly Gly Met Asp Val 100 105 110Trp Gly Gln Gly Thr
Leu Val Thr Val Ser Ser 115 12044335DNAHomo sapiens 44ctgcctgtgc
tgactcaatc atcctctgcc tctgcttccc tgggatcctc ggtcaagctc 60acctgcactc
tgagcagtgg gcatagtaac tacatcatcg catggcatca acagcagcca
120gggaaggccc ctcggtactt gatgaaggtt aatagtgatg gcagccacac
caagggggac 180gggatccctg atcgcttctc aggctccagc tctggggctg
accgctacct caccatctcc 240aacctccagt ctgaggatga ggctagttat
ttctgtgaga cctgggacac taagattcat 300gtcttcggaa ctgggaccaa
ggtctccgtc ctcag 33545111PRTHomo sapiens 45Leu Pro Val Leu Thr Gln
Ser Ser Ser Ala Ser Ala Ser Leu Gly Ser1 5 10 15Ser Val Lys Leu Thr
Cys Thr Leu Ser Ser Gly His Ser Asn Tyr Ile 20 25 30Ile Ala Trp His
Gln Gln Gln Pro Gly Lys Ala Pro Arg Tyr Leu Met 35 40 45Lys Val Asn
Ser Asp Gly Ser His Thr Lys Gly Asp Gly Ile Pro Asp 50 55 60Arg Phe
Ser Gly Ser Ser Ser Gly Ala Asp Arg Tyr Leu Thr Ile Ser65 70 75
80Asn Leu Gln Ser Glu Asp Glu Ala Ser Tyr Phe Cys Glu Thr Trp Asp
85 90 95Thr Lys Ile His Val Phe Gly Thr Gly Thr Lys Val Ser Val Leu
100 105 11046363DNAHomo sapiens 46caggtgcagc tggtgcagtc tggggctgag
gtgaagaagc ctgggtcctc ggtgaaggtc 60tcctgcaagg ctcctggagg tatcttcaac
accaatgctt tcagctgggt ccgacaggcc 120cctggacaag gtcttgagtg
ggtgggaggg gtcatccctt tgtttcgaac agcaagctac 180gcacagaacg
tccagggcag agtcaccatt accgcggacg aatccacgaa cacagcctac
240atggagctta ccagcctgag atctgcggac acggccgtgt attactgtgc
gagaagtagt 300ggttaccatt ttaggagtca ctttgactcc tggggcctgg
gaaccctggt caccgtctcc 360tca 36347121PRTHomo sapiens 47Gln Val Gln
Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ser1 5 10 15Ser Val
Lys Val Ser Cys Lys Ala Pro Gly Gly Ile Phe Asn Thr Asn 20 25 30Ala
Phe Ser Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Val 35 40
45Gly Gly Val Ile Pro Leu Phe Arg Thr Ala Ser Tyr Ala Gln Asn Val
50 55 60Gln Gly Arg Val Thr Ile Thr Ala Asp Glu Ser Thr Asn Thr Ala
Tyr65 70 75 80Met Glu Leu Thr Ser Leu Arg Ser Ala Asp Thr Ala Val
Tyr Tyr Cys 85 90 95Ala Arg Ser Ser Gly Tyr His Phe Arg Ser His Phe
Asp Ser Trp Gly 100 105 110Leu Gly Thr Leu Val Thr Val Ser Ser 115
12048363DNAHomo sapiens 48caggtgcagc tggtgcaatc tggggctgag
gtgaagaagc ctgggtcctc ggtgaaggtc 60tcctgcaagg ctcctggagg tatcttcaac
accaatgctt tcagctgggt ccgacaggcc 120cctggacaag gtcttgagtg
ggtgggaggg gtcatccctt tgtttcgaac agcaagctac 180gcacagaacg
tccagggcag agtcaccatt accgcggacg aatccacgaa cacagcctac
240atggagctta ccagcctgag atctgcggac acggccgtgt attactgtgc
gagaagtagt 300ggttaccatt ttaggagtca ctttgactcc tggggcctgg
gaaccctggt caccgtctcc 360tca 36349111PRTHomo sapiens 49Asn Phe Met
Leu Thr Gln Pro His Ser Val Ser Ala Ser Pro Gly Lys1 5 10 15Thr Val
Thr Ile Ser Cys Thr Gly Ser Ser Gly Asn Ile Ala Ala Asn 20 25 30Tyr
Val Gln Trp Tyr Gln Gln Arg Pro Gly Ser Ala Pro Thr Thr Val 35 40
45Ile Tyr Glu Asp Asp Arg Arg Pro Ser Gly Val Pro Asp Arg Phe Ser
50 55 60Gly Ser Ile Asp Arg Ser Ser Asn Ser Ala Ser Leu Thr Ile Ser
Gly65 70 75 80Leu Lys Thr Glu Asp Glu Ala Asp Tyr Tyr Cys Gln Thr
Tyr Asp Thr 85 90 95Asn Asn His Ala Val Phe Gly Gly Gly Thr His Leu
Thr Val Leu 100 105 11050333DNAHomo sapiens 50aattttatgc tgactcagcc
ccactctgtg tcggcgtctc cggggaagac ggtgaccatc 60tcctgcaccg gcagcagtgg
caacattgcc gccaactatg tgcagtggta ccaacaacgt 120ccgggcagtg
cccccactac tgtgatctat gaggatgacc gaagaccctc tggggtccct
180gatcggttct ctggctccat cgacaggtcc tccaactctg cctccctcac
catctcagga 240ctgaagactg aggacgaggc tgactactac tgtcagactt
atgataccaa caatcatgct 300gtgttcggag gaggcaccca cctgaccgtc ctc
33351110PRTHomo sapiens 51Ser Tyr Glu Leu Thr Gln Pro Pro Ser Ala
Ser Gly Lys His Gly Gln1 5 10 15Arg Val Thr Ile Ser Cys Ser Gly Gly
Thr Ser Asn Ile Gly Arg Asn 20 25 30His Val Asn Trp Tyr Gln Gln Leu
Pro Gly Thr Ala Pro Lys Leu Leu 35 40 45Ile Tyr Ser Asn Glu Gln Arg
Pro Ser Gly Val Pro Asp Arg Phe Ser 50 55 60Gly Ser Lys Ser Gly Thr
Ser Ala Ser Leu Ala Val Ser Gly Leu Gln65 70 75 80Ser Glu Asp Glu
Ala Asp Tyr Tyr Cys Ala Ser Trp Asp Asp Asn Leu 85 90 95Ser Gly Trp
Val Phe Gly Gly Gly Thr Lys Leu Thr Val Leu 100 105 11052330DNAHomo
sapiens 52tcctatgagc tgactcagcc accctcagcg tctgggaaac acgggcagag
ggtcaccatc 60tcttgttctg gaggcacctc caacatcgga cgtaatcatg ttaactggta
ccagcaactc 120ccaggaacgg cccccaaact cctcatctat agtaatgaac
agcggccctc aggggtccct 180gaccgattct ctggctccaa atctggcacc
tccgcctccc tggccgtgag tgggctccag 240tctgaggatg aggctgatta
ttactgtgca tcatgggatg acaacttgag tggttgggtg 300ttcggcggag
ggaccaagct gaccgtccta 33053120PRTHomo sapiens 53Gln Val Gln Leu Val
Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ser1 5 10 15Ser Val Lys Val
Ser Cys Lys Ala Ser Gly Gly Thr Phe Ser Ala Tyr 20 25 30Ala Phe Thr
Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met 35 40 45Gly Gly
Ile Thr Gly Met Phe Gly Thr Ala Asn Tyr Ala Gln Lys Phe 50 55 60Gln
Gly Arg Val Thr Ile Thr Ala Asp Glu Leu Thr Ser Thr Ala Tyr65 70 75
80Met Glu Leu Ser Ser Leu Thr Ser Glu Asp Thr Ala Leu Tyr Tyr Cys
85 90 95Ala Arg Gly Leu Tyr Tyr Tyr Glu Ser Ser Leu
Asp Tyr Trp Gly Gln 100 105 110Gly Thr Leu Val Thr Val Ser Ser 115
12054361DNAHomo sapiens 54caggtgcagc tggtgcagtc tggggctgag
gtgaagaagc ctgggtcctc ggtgaaggtc 60tcctgcaagg cttctggagg caccttcagc
gcttatgctt tcacctgggt gcggcaggcc 120cctggacaag ggcttgagtg
gatgggaggc atcaccggaa tgtttggcac agcaaactac 180gcacagaagt
tccagggcag agtcacgatt accgcggacg aactcacgag cacagcctac
240atggagttga gctccctgac atctgaagac acggcccttt attattgtgc
gagaggattg 300tattactatg agagtagtct tgactattgg ggccagggaa
ccctggtcac cgtctcctca 360g 36155110PRTHomo sapiens 55Gln Ser Val
Leu Thr Gln Pro Pro Ser Ala Ser Gly Ser Pro Gly Gln1 5 10 15Ser Val
Thr Ile Ser Cys Thr Gly Thr Ser Ser Asp Val Gly Gly Tyr 20 25 30Asn
Ser Val Ser Trp Tyr Gln Gln His Pro Gly Lys Ala Pro Lys Leu 35 40
45Met Ile Tyr Glu Val Thr Lys Arg Pro Ser Gly Val Pro Asp Arg Phe
50 55 60Ser Ala Ser Lys Ser Gly Asn Thr Ala Ser Leu Thr Val Ser Gly
Leu65 70 75 80Gln Ala Glu Asp Glu Ala Asp Tyr Phe Cys Cys Ser Tyr
Ala Gly His 85 90 95Ser Ala Tyr Val Phe Gly Thr Gly Thr Lys Val Thr
Val Leu 100 105 11056361DNAHomo sapiens 56caggtgcagc tggtgcagtc
tggggctgag gtgaagaagc ctgggtcctc ggtgaaggtc 60tcctgcaggg cttctggagg
caccttcagc gcttatgctt tcacctgggt gcggcaggcc 120cctggacaag
ggcttgagtg gatgggaggc atcaccggaa tgtttggcac agcaaactac
180gcacagaagt tccagggcag agtcacgatt accgcggacg aactcacgag
cacagcctac 240atggagttga gctccctgac atctgaagac acggcccttt
attattgtgc gagaggattg 300tattactatg agagtagtct tgactattgg
ggccagggaa ccctggtcac cgtctcctca 360g 36157108PRTHomo sapiens 57Glu
Ile Val Leu Thr Gln Ser Pro Gly Thr Leu Ser Leu Ser Pro Gly1 5 10
15Glu Arg Ala Thr Leu Ser Cys Arg Ala Ser Gln Ser Leu Ser Ser Lys
20 25 30Tyr Leu Ala Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Arg Leu
Leu 35 40 45Ile Tyr Gly Ala Ser Ser Arg Ala Thr Gly Ile Pro Asp Arg
Phe Ser 50 55 60Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser
Arg Leu Glu65 70 75 80Pro Glu Asp Phe Ala Val Tyr Ser Cys Gln Gln
Tyr Asp Gly Val Pro 85 90 95Arg Thr Phe Gly Gln Gly Thr Thr Val Glu
Ile Lys 100 10558330DNAHomo sapiens 58cagtctgtgc tgactcagcc
accctccgcg tccgggtctc ctggacagtc agtcaccatc 60tcctgcactg gaaccagcag
tgacgttggt ggttataact ctgtctcctg gtaccaacag 120cacccaggca
aagcccccaa actcatgatt tatgaggtca ctaagcggcc ctcaggggtc
180cctgatcgct tctctgcctc caagtctggc aacacggcct ccctgaccgt
ctctgggctc 240caggctgagg atgaggctga ttatttctgc tgctcatatg
caggccacag tgcttatgtc 300ttcggaactg ggaccaaggt caccgtcctg
33059124PRTHomo sapiens 59Gln Val Gln Leu Val Gln Ser Gly Ala Glu
Val Lys Lys Pro Gly Ser1 5 10 15Ser Val Lys Val Ser Cys Thr Ser Ser
Glu Val Thr Phe Ser Ser Phe 20 25 30Ala Ile Ser Trp Val Arg Gln Ala
Pro Gly Gln Gly Leu Glu Trp Leu 35 40 45Gly Gly Ile Ser Pro Met Phe
Gly Thr Pro Asn Tyr Ala Gln Lys Phe 50 55 60Gln Gly Arg Val Thr Ile
Thr Ala Asp Gln Ser Thr Arg Thr Ala Tyr65 70 75 80Met Asp Leu Arg
Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95Ala Arg Ser
Pro Ser Tyr Ile Cys Ser Gly Gly Thr Cys Val Phe Asp 100 105 110His
Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser 115 12060324DNAHomo
sapiens 60gaaattgtgc tgactcagtc tccaggcacc ctgtctttgt ctccagggga
aagagccacc 60ctctcctgca gggccagtca gagtcttagc agcaagtact tagcctggta
tcagcagaaa 120cctggccagg ctcccaggct cctcatctat ggtgcatcca
gcagggccac tggcatccca 180gacaggttca gtggcagtgg gtctgggaca
gacttcaccc tcaccatcag tagactggag 240cctgaagatt ttgcagtgta
ttcctgtcag cagtatgatg gcgtacctcg gacgttcggc 300caagggacca
cggtggaaat caaa 32461110PRTHomo sapiens 61Gln Pro Gly Leu Thr Gln
Pro Pro Ser Val Ser Lys Gly Leu Arg Gln1 5 10 15Thr Ala Thr Leu Thr
Cys Thr Gly Asn Ser Asn Asn Val Gly Asn Gln 20 25 30Gly Ala Ala Trp
Leu Gln Gln His Gln Gly His Pro Pro Lys Leu Leu 35 40 45Ser Tyr Arg
Asn Asn Asp Arg Pro Ser Gly Ile Ser Glu Arg Phe Ser 50 55 60Ala Ser
Arg Ser Gly Asn Thr Ala Ser Leu Thr Ile Thr Gly Leu Gln65 70 75
80Pro Glu Asp Glu Ala Asp Tyr Tyr Cys Ser Thr Trp Asp Ser Ser Leu
85 90 95Ser Ala Val Val Phe Gly Gly Gly Thr Lys Leu Thr Val Leu 100
105 11062372DNAHomo sapiens 62caggtgcagc tggtgcagtc aggggctgag
gtgaagaagc ctgggtcctc ggtgaaggtc 60tcctgcacgt cctctgaagt caccttcagt
agttttgcta tcagctgggt gcgacaggcc 120cctggacaag ggcttgagtg
gctgggaggg atcagcccta tgtttggaac acctaattac 180gcgcagaagt
tccaaggcag agtcaccatt accgcggacc agtccacgag gacagcctac
240atggacctga ggagcctgag atctgaggac acggccgtgt attattgtgc
gagatctcct 300tcttacattt gttctggtgg aacctgcgtc tttgaccatt
ggggccaggg aaccctggtc 360accgtctcct ca 37263107PRTHomo sapiens
63Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly1
5 10 15Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Ser Ile Ser Ser
Tyr 20 25 30Leu Asn Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu
Leu Ile 35 40 45Tyr Ala Ala Ser Ser Leu Gln Arg 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 Val Tyr Tyr Cys Gln Gln
Tyr Asp Ser Ser Pro Tyr 85 90 95Thr Phe Gly Gln Gly Thr Lys Val Glu
Ile Lys 100 10564372DNAHomo sapiens 64caggtacagc tgcagcagtc
aggggctgag gtgaagaagc ctgggtcctc ggtgaaggtc 60tcctgcacgt cctctgaagt
caccttcagt agttttgcta tcagctgggt gcgacaggcc 120cctggacaag
ggcttgagtg gctgggaggg atcagcccta tgtttggaac acctaattac
180gcgcagaagt tccaaggcag agtcaccatt accgcggacc agtccacgag
gacagcctac 240atggacctga ggagcctgag atctgaggac acggccgtgt
attattgtgc gagatctcct 300tcttacattt gttctggtgg aacctgcgtc
tttgaccatt ggggccaggg aaccctggtc 360accgtctcct ca
372655PRTArtificial SequenceConsensus Sequence 65Ser Tyr Ala Phe
Ser1 56617PRTArtificial SequenceConsensus Sequence 66Gly Ile Ile
Pro Met Phe Gly Thr Pro Asn Tyr Ala Gln Lys Phe Gln1 5 10
15Gly6712PRTArtificial SequenceConsensus Sequence 67Ser Ser Gly Tyr
Tyr Tyr Gly Gly Gly Phe Asp Val1 5 10685PRTHomo sapiens 68Thr Asn
Ala Phe Ser1 56917PRTHomo sapiens 69Gly Val Ile Pro Leu Phe Arg Thr
Ala Ser Tyr Ala Gln Asn Val Gln1 5 10 15Gly7013PRTHomo sapiens
70Ser Ser Gly Tyr His Phe Gly Arg Ser His Phe Asp Ser1 5
10715PRTHomo sapiens 71Ala Tyr Ala Phe Thr1 57217PRTHomo sapiens
72Gly Ile Ile Gly Met Phe Gly Thr Ala Asn Tyr Ala Gln Lys Phe Gln1
5 10 15Gly7311PRTHomo sapiens 73Gly Leu Tyr Tyr Tyr Glu Ser Ser Leu
Asp Tyr1 5 10745PRTHomo sapiens 74Ser Phe Ala Ile Ser1 57517PRTHomo
sapiens 75Gly Ile Ser Pro Met Phe Gly Thr Pro Asn Tyr Ala Gln Lys
Phe Gln1 5 10 15Gly7615PRTHomo sapiens 76Ser Pro Ser Tyr Ile Cys
Ser Gly Gly Thr Cys Val Phe Asp His1 5 10 15775PRTHomo sapiens
77Ser Tyr Ala Ile Ser1 57817PRTHomo sapiens 78Gly Ile Ile Gly Val
Phe Gly Val Pro Lys Tyr Ala Gln Lys Phe Gln1 5 10 15Gly7913PRTHomo
sapiens 79Glu Pro Gly Tyr Tyr Val Gly Lys Asn Gly Phe Asp Val1 5
10805PRTHomo sapiens 80Gly Tyr Tyr Ile His1 58117PRTHomo sapiens
81Trp Ile Asn Pro Met Thr Gly Gly Thr Asn Tyr Ala Gln Lys Phe Gln1
5 10 15Val8217PRTHomo sapiens 82Gly Ala Ser Val Leu Arg Tyr Phe Asp
Trp Gln Pro Glu Ala Leu Asp1 5 10 15Ile835PRTHomo sapiens 83Met Thr
Ala Phe Thr1 58417PRTHomo sapiens 84Gly Ile Ser Pro Ile Phe Arg Thr
Pro Lys Tyr Ala Gln Lys Phe Gln1 5 10 15Gly8514PRTHomo sapiens
85Thr Leu Ser Ser Tyr Gln Pro Asn Asn Asp Ala Phe Ala Ile1 5
10865PRTHomo sapiens 86Asp Asn Ala Ile Ser1 58717PRTHomo sapiens
87Gly Ile Ile Pro Ile Phe Gly Lys Pro Asn Tyr Ala Gln Lys Phe Gln1
5 10 15Gly8814PRTHomo sapiens 88Asp Ser Asp Ala Tyr Tyr Tyr Gly Ser
Gly Gly Met Asp Val1 5 108912PRTArtificial SequenceConsensus
Sequence 89Thr Gly Ser Ser Ser Asn Ile Gly Asn Tyr Val Ala1 5
10907PRTArtificial SequenceConsensus Sequence 90Ser Asn Ser Asp Arg
Pro Ser1 59110PRTArtificial SequenceConsensus Sequence 91Gln Ser
Tyr Asp Ser Leu Ser Ala Tyr Val1 5 109213PRTHomo sapiens 92Thr Gly
Ser Ser Ser Asn Ile Ala Ala Asn Tyr Val Gln1 5 10937PRTHomo sapiens
93Glu Asp Asp Arg Arg Pro Ser1 59410PRTHomo sapiens 94Gln Ser Tyr
Asp Thr Asn Asn His Ala Val1 5 109514PRTHomo sapiens 95Thr Gly Thr
Ser Ser Asp Val Gly Gly Tyr Asn Ser Val Ser1 5 10967PRTHomo sapiens
96Glu Val Thr Lys Arg Pro Ser1 59710PRTHomo sapiens 97Cys Ser Tyr
Ala Gly His Ser Ala Tyr Val1 5 109813PRTHomo sapiens 98Thr Gly Asn
Ser Asn Asn Val Gly Asn Gln Gly Ala Ala1 5 10997PRTHomo sapiens
99Arg Asn Asn Asp Arg Pro Ser1 510011PRTHomo sapiens 100Ser Thr Trp
Asp Ser Ser Leu Ser Ala Val Val1 5 1010113PRTHomo sapiens 101Thr
Gly Asp Ser Asn Asn Val Gly His Gln Gly Thr Ala1 5 101027PRTHomo
sapiens 102Arg Asn Gly Asn Arg Pro Ser1 510311PRTHomo sapiens
103Ser Val Trp Asp Ser Ser Leu Ser Ala Trp Val1 5 1010411PRTHomo
sapiens 104Gly Gly Asn Asn Ile Gly Gly Tyr Ser Val His1 5
101057PRTHomo sapiens 105Asp Asp Lys Asp Arg Pro Ser1 510611PRTHomo
sapiens 106Gln Val Trp Asp Ser Gly Asn Asp Arg Pro Leu1 5
1010711PRTHomo sapiens 107Arg Ala Ser Gln Ser Val Ser Ser Tyr Leu
Ala1 5 101087PRTHomo sapiens 108Asp Ala Ser Asn Arg Ala Thr1
51099PRTHomo sapiens 109Gln Gln Tyr Gly Ser Ser Pro Gln Val1
511012PRTHomo sapiens 110Arg Ala Ser Gln Ser Leu Ser Ser Lys Tyr
Leu Ala1 5 101117PRTHomo sapiens 111Gly Ala Ser Ser Arg Ala Thr1
51129PRTHomo sapiens 112Gln Gln Tyr Asp Gly Val Pro Arg Thr1
511312PRTHomo sapiens 113Thr Gly Ser Ser Ser Asn Ile Gly Asn Tyr
Val Ala1 5 101147PRTHomo sapiens 114Ser Asn Asn Gln Arg Pro Ser1
511511PRTHomo sapiens 115Gln Ser Tyr Asp Ser Arg Leu Ser Ala Ser
Leu1 5 1011613PRTHomo sapiens 116Ser Gly Ser Ser Ser Asn Ile Gly
Ser Asn Thr Val Asn1 5 101177PRTHomo sapiens 117Ala Ala Ser Ser Leu
Gln Arg1 51189PRTHomo sapiens 118Gln Gln Tyr Asp Ser Ser Pro Tyr
Thr1 511911PRTHomo sapiens 119Arg Ala Ser Gln Ser Ile Ser Ser Tyr
Leu Asn1 5 101207PRTHomo sapiens 120Ser Asn Glu Gln Arg Pro Ser1
512111PRTHomo sapiens 121Ala Ser Trp Asp Asp Asn Leu Ser Gly Trp
Val1 5 1012212PRTHomo sapiens 122Thr Leu Ser Ser Gly His Ser Asn
Tyr Ile Ile Ala1 5 1012311PRTHomo sapiens 123Val Asn Ser Asp Gly
Ser His Thr Lys Gly Asp1 5 101249PRTArtificial SequenceChemically
Synthesized 124Glu Thr Trp Asp Thr Lys Ile His Val1
51254PRTArtificial SequenceChemically Synthesized 125Xaa Tyr His
Xaa11264PRTArtificial SequenceChemically Synthesized 126Xaa His Gln
Xaa11276PRTArtificial SequenceChemically Synthesized 127Xaa Val Asp
Gly Trp Xaa1 512815PRTArtificial SequenceChemically Synthesized
128Xaa Lys Xaa Thr Gln Xaa Ile Xaa Thr Xaa Val Asn Xaa Ile Xaa1 5
10 1512915PRTArtificial SequenceChemically Synthesized 129Xaa Lys
Xaa Thr Gln Xaa Ile Xaa Thr Xaa Val Asn Xaa Ile Xaa1 5 10 15
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