U.S. patent application number 15/628544 was filed with the patent office on 2018-08-16 for broadly neutralizing human antibody that recognizes the receptor-binding pocket of influenza hemagglutinin.
The applicant listed for this patent is Children's Hospital Boston, Duke University. Invention is credited to Stephen C. Harrison, Barton F. Haynes, Thomas B. Kepler, Hua-Xin Liao, Michael Anthony Moody, Aaron G. Schmidt, James Whittle.
Application Number | 20180230201 15/628544 |
Document ID | / |
Family ID | 47629934 |
Filed Date | 2018-08-16 |
United States Patent
Application |
20180230201 |
Kind Code |
A1 |
Whittle; James ; et
al. |
August 16, 2018 |
BROADLY NEUTRALIZING HUMAN ANTIBODY THAT RECOGNIZES THE
RECEPTOR-BINDING POCKET OF INFLUENZA HEMAGGLUTININ
Abstract
The invention features a novel influenza antibody that
specifically binds to influenza hemagglutinin and reduces or
inhibits hemagglutinin binding to sialic acid. The invention also
provides methods, compositions, and kits featuring the novel
antibody and its use in preventing or treating influenza
infection.
Inventors: |
Whittle; James; (Chevy
Chase, MD) ; Harrison; Stephen C.; (Brighton, MA)
; Haynes; Barton F.; (Durham, NC) ; Liao;
Hua-Xin; (Durham, NC) ; Moody; Michael Anthony;
(Durham, NC) ; Kepler; Thomas B.; (Boston, MA)
; Schmidt; Aaron G.; (Boston, MA) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
Children's Hospital Boston
Duke University |
Boston
Durham |
MA
NC |
US
US |
|
|
Family ID: |
47629934 |
Appl. No.: |
15/628544 |
Filed: |
June 20, 2017 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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14171322 |
Feb 3, 2014 |
9718873 |
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15628544 |
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PCT/US2012/049573 |
Aug 3, 2012 |
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14171322 |
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61514662 |
Aug 3, 2011 |
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Current U.S.
Class: |
1/1 |
Current CPC
Class: |
C07K 2317/33 20130101;
A61P 43/00 20180101; C07K 16/1018 20130101; A61P 31/00 20180101;
A61K 51/1006 20130101; A61K 31/713 20130101; A61P 31/16 20180101;
C07K 2299/00 20130101; C07K 2317/55 20130101; C07K 2317/565
20130101; A61K 38/02 20130101; A61K 47/6841 20170801; C07K 2317/76
20130101 |
International
Class: |
C07K 16/10 20060101
C07K016/10; A61K 31/713 20060101 A61K031/713; A61K 47/68 20060101
A61K047/68; A61K 51/10 20060101 A61K051/10; A61K 38/02 20060101
A61K038/02 |
Goverment Interests
STATEMENT OF RIGHTS TO INVENTIONS MADE UNDER FEDERALLY SPONSORED
RESEARCH
[0002] This work was supported by grant nos. U19 AI067854 and
P01GM62580 from the National Institutes of Health. The Government
has certain rights in this invention.
Claims
1.-33. (canceled)
34. A method of treating or preventing an influenza virus infection
in a subject in need thereof, wherein the method comprises
administering to the subject an effective amount of i) an
anti-influenza antibody or antibody fragment that specifically
binds influenza hemagglutinin (HA) at residues 136, 137 and 226 of
the HA polypeptide sequence and thereby reduces or inhibits
influenza hemagglutinin binding to sialic acid, wherein the
anti-influenza antibody or antibody fragment comprises at least one
sequence selected from the group consisting of: a variable heavy
(V.sub.H) chain sequence SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO:
11, SEQ ID NO: 12, or one or more heavy chain CDR regions present
in a variable heavy (V.sub.H) chain amino acid sequence of SEQ ID
NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, or SEQ ID NO: 12; and a
variable light (V.sub.L) chain sequence SEQ ID NO: 13, SEQ ID NO:
14, SEQ ID NO: 15, SEQ ID NO: 16, or one or more light chain CDR
regions present in a variable light (V.sub.L) chain amino acid
sequence of SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, or SEQ ID
NO: 16 or ii) a pharmaceutical composition comprising said
anti-influenza antibody or antibody fragment, thereby treating or
preventing influenza virus infection in the subject.
35.-38. (canceled)
39. The method of claim 34, wherein the influenza is H1N1, H2N2,
H3N2, or a human adapted H5 strain.
40. The method of claim 34, wherein the subject has or is at risk
of developing an influenza infection.
41. The method of claim 40, wherein the subject is a mammal.
42. The method of claim 41, wherein the subject is human.
43. The method of claim 40, wherein the subject is susceptible to
viral infection.
44. The method of claim 43, wherein the subject is a pregnant
female.
45. The method of claim 43, wherein the subject is a young subject
or an infant subject.
46. The method of claim 43, wherein the subject is an elderly
subject.
47. The method of claim 34, wherein i) the anti-influenza antibody
or antibody fragment, or ii) the pharmaceutical composition is
administered by intramuscular injection, intravenous injection,
subcutaneous injection, or inhalation.
48.-58. (canceled)
59. An anti-influenza Fab that specifically binds influenza
hemagglutinin (HA) at residues 136, 137 and 226 of the HA
polypeptide sequence and thereby reduces or inhibits influenza
hemagglutinin binding to sialic acid.
60. The anti-influenza Fab of claim 59, wherein the HA polypeptide
sequence is selected from the group consisting of SEQ ID NOS:
17-44.
61. The anti-influenza Fab of claim 59, wherein the HA is selected
from the group consisting of H1 HA, H2 HA, H3 HA, or H5 HA.
62. The anti-influenza Fab of claim 61, wherein the Fab further
binds HA at one or more HA residues selected from the group
consisting of: HA residues 158-160; HA residues 190-195; HA
residues 222, 225, and 227; and A/Solomon Islands/3/2006 HA
residues 187 and 189.
63. The anti-influenza Fab of claim 62, wherein the Fab further
contacts an HA residue selected from the group consisting of HA
residues 190-195.
64. The anti-influenza Fab of claim 61, wherein the antibody
complementary determining region (CDR) H1 region contacts HA
residue 158; the CDR H2 region contacts HA residues 158-160; the
CDR H3 region contacts HA residues 135-136, 190-194 and 226; the
CDR L1 region contacts HA residues 222, 225 and 227; or the CDR L3
region contacts HA residues 187 and 198.
Description
CROSS-REFERENCE TO RELATED APPLICATIONS
[0001] This application is a Continuation of U.S. application Ser.
No. 14/171,322, filed Feb. 3, 2014, which is a Continuation-in-Part
filing of PCT/US2012/49573, filed on Aug. 3, 2012, which claims the
benefit of U.S. Provisonal Patent Application No. 61/514,662, filed
Aug. 3, 2011, the contents of all of which are incorporated herein
by reference in their entirety.
BACKGROUND OF THE INVENTION
[0003] The well-known seasonal drift of influenza virus
antigenicity accounts for the absence of long-term immune
protection in previously infected individuals. The hemagglutinin
(HA), a trimeric surface glycoprotein that binds the viral receptor
and promotes fusion and penetration from low-pH endosomes, is the
principal surface antigen on influenza virions. HA presents
conserved as well as variable epitopes, but neutralizing antibodies
against the latter dominate the response to immunization and
infection.
[0004] Accordingly, there is a need for developing broadly
neutralizing therapeutics that can effectively treat or prevent
drifted strains of influenza.
SEQUENCE SUBMISSION
[0005] The present application is being filed along with a Sequence
Listing in electronic format. The Sequence Listing is entitled
"048218-519NO1US_Seq_Listing_03FEB2014", was created on Feb. 3,
2014, with a file size of 85 KB. The information in the electronic
format of the Sequence Listing is part of the present application
and is incorporated herein by reference in its entirety.
SUMMARY OF THE INVENTION
[0006] As described below, the present invention is based upon the
discovery of novel antibodies that broadly neutralize influenza
antigenic variants. The invention features compositions and kits
containing the novel antibodies, as well as methods for using these
novel therapeutic molecules to treat or prevent influenza viral
infection.
[0007] In aspects, the invention provides isolated anti-influenza
antibody or antibody fragment that specifically binds to an epitope
of an influenza hemagglutinin (HA). Binding of the antibody or
antibody fragment to the influenza HA epitope reduces or inhibits
influenza HA binding to sialic acid.
[0008] The present invention also provides in other aspects an
isolated anti-influenza antibody or antibody fragment that
specifically binds to a sialic acid binding domain of a surface
antigen of influenza virus. In one embodiment, the surface antigen
of influenza virus is HA.
[0009] In embodiments, the epitope of influenza HA comprises a
sialic-acid binding domain.
[0010] In embodiments, the HA is H1 HA, H2 HA, H3 HA, or H5 HA (an
HA from a human adapted H5 strain).
[0011] In related embodiments, the antibody contacts one or more
residues in influenza HA epitope comprising residue 158; residues
158-160; 135-136, 190-195, and 226; 222, 225, and 227; and residues
187 and 189 where the numbering refers to the one used in
structures such as that for A/Solomon Islands/3/2006 (Protein Data
Bank accession number 3SM5).
[0012] In related embodiments, the influenza HA epitope comprises
the amino acids set forth in any one of SEQ ID NOs:17-44.
[0013] In related embodiments, the influenza HA epitope comprises
the CH65-CH67 binding residues in any one of SEQ ID NOs:17-44
(e.g., the CH65-CH67 binding residues identified in FIG. 15).
[0014] In other embodiments, the anti-influenza antibody or
antibody fragment contacts one or more residues in the sialic acid
binding pocket of the HA epitope selected from the group consisting
of residues: 134-136, 190-195 and 226. In another embodiment, the
antibody or antibody fragment further contacts one or more residues
in the HA epitope selected from the group consisting of residues:
158, 158-160, 135-136, 190-195 and 226, and 187 and 189, where the
numbering refers to the one used in structures such as that for
A/Solomon Islands/3/2006 (Protein Data Bank accession number
3SM5).
[0015] In another embodiment, the antibody CDR H1 region contacts
residue 158 of the HA epitope; the CDR H2 region contacts residues
158-160 of the HA epitope; the CDR H3 region contacts residues
135-136, 190-194 and 226 of the HA epitope; the CDR L1 region
contacts residues 222, 225 and 227 of the HA epitope; or the CDR L3
region contacts residues 187 and 198 of the HA epitope.
[0016] In embodiments, the anti-influenza antibody or antibody
fragment comprises a variable heavy (V.sub.H) chain, and wherein
the V.sub.H chain comprises an amino acid sequence set forth in SEQ
ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, or SEQ ID NO: 12.
[0017] In embodiments, the anti-influenza antibody or antibody
fragment comprises one or more heavy chain CDR regions present in a
variable heavy (V.sub.H) chain amino acid sequence of SEQ ID NO: 9,
SEQ ID NO: 10, SEQ ID NO: 11, or SEQ ID NO: 12. In related
embodiments, the one or more heavy chain CDR regions comprises a
CDR3 region present in the variable heavy (V.sub.H) chain amino
acid sequence of SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, or SEQ
ID NO: 12.
[0018] In embodiments, the anti-influenza antibody or antibody
fragment comprises a variable light (V.sub.L) chain, and wherein
the V.sub.L chain comprises an amino acid sequence set forth in SEQ
ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, or SEQ ID NO: 16.
[0019] In embodiments, the anti-influenza antibody or antibody
fragment comprises one or more light chain CDR regions present in a
variable light (V.sub.L) chain amino acid sequence of SEQ ID NO:
13, SEQ ID NO: 14, SEQ ID NO: 15, or SEQ ID NO: 16. In related
embodiments, the one or more light chain CDR regions comprises a
CDR3 region present in the variable heavy (V.sub.L) chain amino
acid sequence of SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, or
SEQ ID NO: 16.
[0020] In embodiments, the anti-influenza antibody or antibody
fragment comprises i) a variable heavy (V.sub.H) chain amino
comprising an amino acid sequence set forth in SEQ ID NO: 10, and
ii) a variable light (V.sub.L) chain comprising an amino acid
sequence set forth in SEQ ID NO: 14.
[0021] In embodiments, the anti-influenza antibody or antibody
fragment comprises a variable heavy (V.sub.H) chain, wherein the
CDR3 region of the V.sub.H chain comprises Arg104, Ser105, Val106,
Asp107, Tyr109, Tyr110, Tyr112, or a combination thereof.
[0022] In embodiments, the anti-influenza antibody is a monoclonal
antibody or antibody fragment thereof.
[0023] In embodiments, the anti-influenza antibody is a humanized
antibody.
[0024] In embodiments, the antibody fragment is an Fab fragment, an
Fab' fragment, an Fd fragment, a Fd' fragment, an Fv fragment, a
dAb fragment, an F(ab')2 fragment, a single chain fragment, a
diabody, or a linear antibody.
[0025] In embodiments, the anti-influenza antibody or antibody
fragment further comprises an agent conjugated to the
anti-influenza antibody or antibody fragment thereof. In related
embodiments, the agent conjugated to the antibody or antibody
fragment thereof is a therapeutic agent or detectable label.
[0026] The therapeutic agent can be any therapeutic agent suitable
for use with the novel antibodies. Such agents are well known in
the art and include small molecules, nanoparticles, polypeptides,
radioisotopes, inhibitory nucleic acids, and the like. In
embodiments, the therapeutic agent is an antiviral agent or a
toxin. In embodiments, the therapeutic agent is an siRNA, shRNA, or
antisense nucleic acid molecule that reduces influenza virus
production.
[0027] The detectable label can be any detectable label suitable
for use with the novel antibodies. Such labels are well known in
the art and include labels that are detected by spectroscopic,
photochemical, biochemical, immunochemical, physical, or chemical
means. In embodiments, the detectable label is an enzyme, a
fluorescent molecule, a particle label, an electron-dense reagent,
a radiolabel, a microbubble, biotin, digoxigenin, or a hapten or a
protein that has been made detectable.
[0028] In aspects, the invention provides pharmaceutical
compositions containing at least one of the anti-influenza antibody
or antibody fragments described herein. In embodiments, the
pharmaceutical compositions contain a pharmaceutically acceptable
carrier, diluent, or excipient.
[0029] In aspects, the invention provides isolated polynucleotides
encoding an anti-influenza antibody or antibody fragments described
herein. In related aspects, the invention provides expression
vectors comprising such an isolated polynucleotide. In further
related aspects, the invention provides host cells comprising such
an expression vector.
[0030] In aspects, the invention provides methods for treating or
preventing an influenza virus infection in a subject in need
thereof. The methods involve administering to the subject an
effective amount of an anti-influenza antibody or antibody fragment
described herein, or a pharmaceutical composition containing the
antibody or antibody fragment. The methods treat or prevent
influenza virus infection in the subject, including reducing or
alleviating symptoms associated with infection.
[0031] In aspects, the invention provides methods for neutralizing
an influenza virus in a subject in need thereof. The methods
involve administering to the subject an effective amount of an
anti-influenza antibody or antibody fragment described herein, or a
pharmaceutical composition containing the antibody or antibody
fragment. The methods neutralize the influenza virus in the
subject, thereby treating or prevent influenza virus infection in
the subject, including reducing or alleviating symptoms associated
with infection.
[0032] In aspects, the invention provides methods for establishment
of influenza virus infection in a subject in need thereof. The
methods involve administering to the subject an effective amount of
an anti-influenza antibody or antibody fragment described herein,
or a pharmaceutical composition containing the antibody or antibody
fragment. The methods inhibit establishment of influenza virus
infection in the subject, thereby preventing symptoms associated
with infection.
[0033] In aspects, the invention provides methods for inhibiting
dissemination of influenza virus infection in a subject in need
thereof. The methods involve administering to the subject an
effective amount of an anti-influenza antibody or antibody fragment
described herein, or a pharmaceutical composition containing the
antibody or antibody fragment. The methods inhibit dissemination of
influenza virus infection in the subject, thereby reducing or
alleviating symptoms associated with infection.
[0034] In aspects, the invention provides methods for inhibiting
influenza virus entry into a cell in a subject. The methods involve
administering to the subject an effective amount of an
anti-influenza antibody or antibody fragment described herein, or a
pharmaceutical composition containing the antibody or antibody
fragment. The methods inhibit influenza virus entry into a cell in
the subject, thereby preventing symptoms associated with infection
or reducing or alleviating symptoms associated with infection.
[0035] In aspects, the invention provides methods for inhibiting
influenza virus entry into a cell. The methods involve contacting a
cell having or at risk of developing influenza virus infection with
an anti-influenza antibody or antibody fragment described herein,
or a pharmaceutical composition containing the antibody or antibody
fragment. The methods inhibit influenza virus entry into the
cell.
[0036] In any of the above aspects, the subject has or is at risk
of developing an influenza infection. In related embodiments, the
subject is a mammal (e.g., human). In related embodiments, the
subject is susceptible to viral infection (e.g., a pregnant female,
a young subject or an infant subject, an elderly subject).
[0037] In any of the above aspects and embodiments, the
anti-influenza antibody or antibody fragment, or the pharmaceutical
composition is administered by intramuscular injection, intravenous
injection, subcutaneous injection, or inhalation.
[0038] In aspects, the invention provides kits for treating or
preventing influenza virus infection; kits for neutralizing
influenza virus; kits for inhibiting establishment of influenza
virus infection; kits for inhibiting dissemination of influenza
virus infection; and kits for inhibiting influenza virus entry into
a cell.
[0039] In embodiments, the kits contain an anti-influenza antibody
or antibody fragment described herein.
[0040] In embodiments, the kits also contain a therapeutic agent.
In related embodiments, the therapeutic agent inhibits influenza
infection.
[0041] In embodiments, the kits also contain directions for using
the kits in any of the methods described herein.
[0042] In any of the above embodiments, the influenza can be H1N1,
H2N2, H3N2, or a human adapted H5 influenza strain (i.e., an H5
influenza that has acquired human-receptor specificity; see FIG.
14D for exemplary strains).
[0043] Additional objects and advantages of the invention will be
set forth in part in the description which follows, and in part
will be obvious from the description, or may be learned by practice
of the invention. The objects and advantages of the invention will
be realized and attained by means of the elements and combinations
disclosed herein, including those 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. The accompanying drawings, which are incorporated herein
and constitute a part of this specification, illustrate several
embodiments of the invention and, together with the description,
serve to explain the principles of the invention.
Definitions
[0044] To facilitate an understanding of the present invention, a
number of terms and phrases are defined below.
[0045] As used herein, the singular forms "a", "an", and "the"
include plural forms unless the context clearly dictates otherwise.
Thus, for example, reference to "an influenza antibody" includes
reference to more than one influenza antibody.
[0046] Unless specifically stated or obvious from context, as used
herein, the term "or" is understood to be inclusive.
[0047] As used herein, the terms "comprises," "comprising,"
"containing," "having" and the like can have the meaning ascribed
to them in U.S. Patent law and can mean "includes," "including,"
and the like; "consisting essentially of" or "consists essentially"
likewise has the meaning ascribed in U.S. Patent law and the term
is open-ended, allowing for the presence of more than that which is
recited so long as basic or novel characteristics of that which is
recited is not changed by the presence of more than that which is
recited, but excludes prior art embodiments.
[0048] The term "antibody" means an immunoglobulin molecule that
recognizes and specifically binds to a target, such as a protein,
polypeptide, peptide, carbohydrate, polynucleotide, lipid, or
combinations of the foregoing through at least one antigen
recognition site within the variable region of the immunoglobulin
molecule. As used herein, the term "antibody" encompasses intact
polyclonal antibodies, intact monoclonal antibodies, antibody
fragments (such as Fab, Fab', F(ab')2, and Fv fragments), single
chain Fv (scFv) mutants, multispecific antibodies such as
bispecific antibodies generated from at least two intact
antibodies, chimeric antibodies, humanized antibodies, human
antibodies, fusion proteins comprising an antigen determination
portion of an antibody, and any other modified immunoglobulin
molecule comprising an antigen recognition site so long as the
antibodies exhibit the desired biological activity. An antibody can
be of any the five major classes of immunoglobulins: IgA, IgD, IgE,
IgG, and IgM, or subclasses (isotypes) thereof (e.g. IgG1, IgG2,
IgG3, IgG4, IgA1 and IgA2), based on the identity of their
heavy-chain constant domains referred to as alpha, delta, epsilon,
gamma, and mu, respectively. The different classes of
immunoglobulins have different and well known subunit structures
and three-dimensional configurations. Antibodies can be naked or
conjugated to other molecules such as toxins, radioisotopes, and
the like.
[0049] The basic four-chain antibody unit is a heterotetrameric
glycoprotein composed of two identical light (L) chains and two
identical heavy (H) chains. An IgM antibody consists of 5 of the
basic heterotetramer unit along with an additional polypeptide
called J chain, and therefore contains 10 antigen binding sites,
while secreted IgA antibodies can polymerize to form polyvalent
assemblages comprising 2-5 of the basic 4-chain units along with J
chain. In the case of IgGs, the 4-chain unit is generally about
150,000 daltons. Each L chain is linked to an H chain by one
covalent disulfide bond, while the two H chains are linked to each
other by one or more disulfide bonds depending on the H chain
isotype. Each H and L chain also has regularly spaced intrachain
disulfide bridges. Each H chain has at the N-terminus, a variable
domain (V.sub.H) followed by three constant domains (C.sub.H) for
each of the .alpha. and .gamma. chains and four C.sub.H domains for
.mu. and .epsilon. isotypes. Each L chain has at the N-terminus, a
variable domain (V.sub.L) followed by a constant domain (C.sub.L)
at its other end. The V.sub.L is aligned with the V.sub.H and the
C.sub.L is aligned with the first constant domain of the heavy
chain (CH1). Particular amino acid residues are believed to form an
interface between the light chain and heavy chain variable domains.
The pairing of a V.sub.H and V.sub.L together forms a single
antigen-binding site. For the structure and properties of the
different classes of antibodies, see, e.g., Basic and Clinical
Immunology, 8th edition, Daniel P. Stites, Abba I. Terr and
Tristram G. Parslow (eds.), Appleton & Lange, Norwalk, Conn.,
1994, page 71, and Chapter 6.
[0050] An "isolated antibody" is one that has been separated and/or
recovered from a component of its natural environment. Contaminant
components of its natural environment are materials that would
interfere with diagnostic or therapeutic uses for the antibody, and
may include enzymes, hormones, and other proteinaceous or
nonproteinaceous solutes. In embodiments, the antibody is purified:
(1) to 80%, 85%, 90%, 95%, 99% or more by weight of antibody as
determined by the Lowry method; (2) to a degree sufficient to
obtain at least 15 residues of N-terminal or internal amino acid
sequence by use of a spinning cup sequenator; or (3) to homogeneity
by SDS-PAGE under reducing or non-reducing conditions using
Coomassie blue, silver stain, and the like. Isolated antibody
includes the antibody in situ within recombinant cells since at
least one component of the antibody's natural environment will not
be present. In embodiments, an isolated antibody will be prepared
by at least one purification step.
[0051] The term "antibody fragment" refers to a portion of an
intact antibody and refers to the antigenic determining variable
regions of an intact antibody. Examples of antibody fragments
include, but are not limited to Fab, Fab', F(ab')2, and Fv
fragments, linear antibodies, single chain antibodies, and
multispecific antibodies formed from antibody fragments.
[0052] Papain digestion of antibodies produces two identical
antigen-binding fragments, called "Fab" fragments, and a residual
"Fc" fragment, a designation reflecting the ability to crystallize
readily. The Fab fragment consists of an entire L chain along with
the variable region domain of the H chain (V.sub.H), and the first
constant domain of one heavy chain (CH1). Each Fab fragment is
monovalent with respect to antigen binding, i.e., it has a single
antigen-binding site. Pepsin treatment of an antibody yields a
single large F(ab')2 fragment that roughly corresponds to two
disulfide linked Fab fragments having divalent antigen-binding
activity and is still capable of cross-linking antigen. Fab'
fragments differ from Fab fragments by having additional few
residues at the carboxy terminus of the CH1 domain including one or
more cysteines from the antibody 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. F(ab')2 antibody
fragments originally were produced as pairs of Fab' fragments that
have hinge cysteines between them. Other chemical couplings of
antibody fragments are also known.
[0053] The "Fc" fragment comprises the carboxy-terminal portions of
both H chains held together by disulfides. The effector functions
of antibodies are determined by sequences in the Fc region, which
region is also the part recognized by Fc receptors (FcR) found on
certain types of cells.
[0054] An "Fv antibody" refers to the minimal antibody fragment
that contains a complete antigen-recognition and -binding site
either as two-chains, in which one heavy and one light chain
variable domain form a non-covalent dimer, or as a single-chain
(scFv or sFv), in which one heavy and one light chain variable
domain are covalently linked by a flexible peptide linker so that
the two chains associate in a similar dimeric structure. In this
configuration the complementary determining regions (CDRs) of each
variable domain interact to define the antigen-binding specificity
of the Fv dimer. Alternatively a single variable domain (or half of
an Fv) can be used to recognize and bind antigen, although
generally with lower affinity.
[0055] The term "diabodies" refers to small antibody fragments
prepared by constructing sFv fragments (see preceding paragraph)
with short linkers (about 5-10 residues) between the V.sub.H and
V.sub.L domains such that inter-chain but not intra-chain pairing
of the V domains is achieved, resulting in a bivalent fragment,
i.e., fragment having two antigen-binding sites. Bispecific
diabodies are heterodimers of two "crossover" sFv fragments in
which the V.sub.H and V.sub.L domains of the two antibodies are
present on different polypeptide chains. Diabodies are described
more fully in, for example, EP 404,097; WO 93/11161; and Hollinger
et al., Proc. Natl. Acad. Sci. USA, 90:6444-6448 (1993).
[0056] A "monoclonal antibody" refers to homogenous antibody
population involved in the highly specific recognition and binding
of a single antigenic determinant, or epitope. This is in contrast
to polyclonal antibodies that typically include different
antibodies directed against different antigenic determinants. The
term "monoclonal antibody" encompasses both intact and full-length
monoclonal antibodies as well as antibody fragments (such as Fab,
Fab', F(ab')2, Fv), single chain (scFv) mutants, fusion proteins
comprising an antibody portion, and any other modified
immunoglobulin molecule comprising an antigen recognition site.
Furthermore, "monoclonal antibody" refers to such antibodies made
in any number of manners including but not limited to by hybridoma,
phage selection, recombinant expression, and transgenic
animals.
[0057] The term "humanized antibody" refers to forms of non-human
(e.g., murine) antibodies that are specific immunoglobulin chains,
chimeric immunoglobulins, or fragments thereof that contain minimal
non-human (e.g., murine) sequences. Typically, humanized antibodies
are human immunoglobulins in which residues from the complementary
determining region (CDR) are replaced by residues from the CDR of a
non-human species (e.g. mouse, rat, rabbit, hamster, and the like)
that have the desired specificity, affinity, and capability (Jones
et al., 1986, Nature, 321:522-525; Riechmann et al., 1988, Nature,
332:323-327; Verhoeyen et al., 1988, Science, 239:1534-1536). In
some instances, the Fv framework region (FR) residues of a human
immunoglobulin are replaced with the corresponding residues in an
antibody from a non-human species that has the desired specificity,
affinity, and capability. The humanized antibody can be further
modified by the substitution of additional residue either in the Fv
framework region and/or within the replaced non-human residues to
refine and optimize antibody specificity, affinity, and/or
capability. In general, the humanized antibody will comprise
substantially all of at least one, and typically two or three,
variable domains containing all or substantially all of the CDR
regions that correspond to the non-human immunoglobulin whereas all
or substantially all of the FR regions are those of a human
immunoglobulin consensus sequence. The humanized antibody can also
comprise at least a portion of an immunoglobulin constant region or
domain (Fc), typically that of a human immunoglobulin. Examples of
methods used to generate humanized antibodies are described in U.S.
Pat. No. 5,225,539.
[0058] The term "human antibody" means an antibody produced by a
human or an antibody having an amino acid sequence corresponding to
an antibody produced by a human made using any technique known in
the art. This definition of a human antibody includes intact or
full-length antibodies, fragments thereof, and/or antibodies
comprising at least one human heavy and/or light chain polypeptide
such as, for example, an antibody comprising murine light chain and
human heavy chain polypeptides.
[0059] "Hybrid antibodies" are immunoglobulin molecules in which
pairs of heavy and light chains from antibodies with different
antigenic determinant regions are assembled together so that two
different epitopes or two different antigens can be recognized and
bound by the resulting tetramer.
[0060] The term "chimeric antibodies" refers to antibodies wherein
the amino acid sequence of the immunoglobulin molecule is derived
from two or more species. Typically, the variable region of both
light and heavy chains corresponds to the variable region of
antibodies derived from one species of mammals (e.g., mouse, rat,
rabbit, etc) with the desired specificity, affinity, and capability
while the constant regions are homologous to the sequences in
antibodies derived from another (usually human) to avoid eliciting
an immune response in that species.
[0061] A "variable region" of an antibody refers to the variable
region of the antibody light chain or the variable region of the
antibody heavy chain, either alone or in combination. The variable
regions of the heavy and light chain each consist of four framework
regions (FR) connected by three complementarity determining regions
(CDRs) also known as hypervariable regions. The CDRs in each chain
are held together in close proximity by the FRs and, with the CDRs
from the other chain, contribute to the formation of the
antigen-binding site of antibodies. There are at least two
techniques for determining CDRs: (1) an approach based on
cross-species sequence variability (see Kabat et al., Sequences of
Proteins of Immunological Interest (5th ed., 1991, National
Institutes of Health, Bethesda Md.)); and (2) an approach based on
crystallographic studies of antigen-antibody complexes (see
Al-lazikani et al. J. Molec. Biol. 273:927-948 (1997)). In
addition, combinations of these two approaches are sometimes used
in the art to determine CDRs.
[0062] "Administering" is defined herein as a means of providing an
agent or a composition containing the agent to a subject in a
manner that results in the agent being inside the subject's body.
Such an administration can be by any route including, without
limitation, oral, transdermal (e.g., vagina, rectum, oral mucosa),
by injection (e.g., subcutaneous, intravenous, parenterally,
intraperitoneally, intrathecal), or by inhalation (e.g., oral or
nasal). Pharmaceutical preparations are, of course, given by forms
suitable for each administration route.
[0063] By "agent" is meant any small molecule chemical compound,
antibody, nucleic acid molecule, or polypeptide, or fragments
thereof.
[0064] By "analog" is meant a molecule that is not identical, but
has analogous functional or structural features. For example, an
amide, ester, carbamate, carbonate, ureide, or phosphate analog of
an influenza antibody is a molecule that either: 1) does not
destroy the biological activity of the influenza antibody and
confers upon that influenza antibody advantageous properties in
vivo, such as uptake, duration of action, or onset of action; or 2)
is itself biologically inactive but is converted in vivo to a
biologically active compound. Analogs include prodrug forms of an
influenza antibody. Such a prodrug is any compound that when
administered to a biological system generates the influenza
antibody as a result of spontaneous chemical reaction(s), enzyme
catalyzed chemical reaction(s), and/or metabolic chemical
reaction(s).
[0065] By "control" is meant a standard or reference condition.
[0066] The term "derivative" means a pharmaceutically active
compound with equivalent or near equivalent physiological
functionality to a given agent (e.g., an influenza antibody). As
used herein, the term "derivative" includes any pharmaceutically
acceptable salt, ether, ester, prodrug, solvate, stereoisomer
including enantiomer, diastereomer or stereoisomerically enriched
or racemic mixture, and any other compound which upon
administration to the recipient, is capable of providing (directly
or indirectly) such a compound or an antivirally active metabolite
or residue thereof.
[0067] By "disease" is meant any condition or disorder that damages
or interferes with the normal function of a cell, tissue, or
organ.
[0068] The term "epitope" or "antigenic determinant" are used
interchangeably herein and refer to that portion of an antigen
capable of being recognized and specifically bound by a particular
antibody. When the antigen is a polypeptide, epitopes can be formed
both from contiguous amino acids and noncontiguous amino acids
juxtaposed by tertiary folding of a protein. Epitopes formed from
contiguous amino acids are typically retained upon protein
denaturing, whereas epitopes formed by tertiary folding are
typically lost upon protein denaturing. An epitope typically
includes at least 3, and more usually, at least 5 or 8-10 amino
acids in a unique spatial conformation.
[0069] Competition between antibodies is determined by an assay in
which the immunoglobulin under test inhibits specific binding of a
reference antibody to a common antigen. Numerous types of
competitive binding assays are known, for example: solid phase
direct or indirect radioimmunoassay (RIA), solid phase direct or
indirect enzyme immunoassay (EIA), sandwich competition assay (see
Stahli et al., Methods in Enzymology 9:242-253 (1983)); solid phase
direct biotin-avidin EIA (see Kirkland et al., J. Immunol.
137:3614-3619 (1986)); solid phase direct labeled assay, solid
phase direct labeled sandwich assay (see Harlow and Lane,
"Antibodies, A Laboratory Manual," Cold Spring Harbor Press
(1988)); solid phase direct label RIA using I-125 label (see Morel
et al., Molec. Immunol. 25(1):7-15 (1988)); solid phase direct
biotin-avidin EIA (Cheung et al., Virology 176:546-552 (1990)); and
direct labeled RIA (Moldenhauer et al., Scand. J. Immunol. 32:77-82
(1990)). Typically, such an assay involves the use of purified
antigen bound to a solid surface or cells bearing either of these,
an unlabeled test immunoglobulin and a labeled reference
immunoglobulin. Competitive inhibition is measured by determining
the amount of label bound to the solid surface or cells in the
presence of the test immunoglobulin. Usually the test
immunoglobulin is present in excess. Antibodies identified by
competition assay (competing antibodies) include antibodies binding
to the same epitope as the reference antibody and antibodies
binding to an adjacent epitope sufficiently proximal to the epitope
bound by the reference antibody for steric hindrance to occur. When
a competing antibody is present in excess, it will inhibit specific
binding of a reference antibody to a common antigen by at least
25%, 50, 75%, or more.
[0070] By "enhances" or "increases" is meant a positive alteration
of at least about 10%, 25%, 50%, 75%, or 100% relative to a
reference.
[0071] By "fragment" is meant a portion of a polypeptide or nucleic
acid molecule. This portion contains at least 10%, 20%, 30%, 40%,
50%, 60%, 70%, 80%, or 90% of the entire length of the reference
nucleic acid molecule or polypeptide. A fragment may contain 10,
20, 30, 40, 50, 60, 70, 80, 90, or 100, 200, 300, 400, 500, 600,
700, 800, 900, or 1000 nucleotides or amino acids.
[0072] "Hybridization" means hydrogen bonding, which may be
Watson-Crick, Hoogsteen, or reversed Hoogsteen hydrogen bonding
between complementary nucleobases. For example, adenine and thymine
are complementary nucleobases that pair through the formation of
hydrogen bonds.
[0073] By "isolated polynucleotide" is meant a nucleic acid (e.g.,
a DNA) that is free of the genes which, in the naturally-occurring
genome of the organism from which the nucleic acid molecule of the
invention is derived, flank the gene. The term therefore includes,
for example, a recombinant DNA that is incorporated into a vector;
into an autonomously replicating plasmid or virus; or into the
genomic DNA of a prokaryote or eukaryote; or that exists as a
separate molecule (for example, a cDNA or a genomic or cDNA
fragment produced by PCR or restriction endonuclease digestion)
independent of other sequences. In addition, the term includes an
RNA molecule that is transcribed from a DNA molecule, as well as a
recombinant DNA that is part of a hybrid gene encoding additional
polypeptide sequence.
[0074] By an "isolated polypeptide" is meant a polypeptide of the
invention that has been separated from components that naturally
accompany it. Typically, the polypeptide is isolated when it is at
least 60%, by weight, free from the proteins and
naturally-occurring organic molecules with which it is naturally
associated. In embodiments, the preparation is at least 75%, at
least 90%, or at least 99%, by weight, a polypeptide of the
invention. An isolated polypeptide of the invention may be
obtained, for example, by extraction from a natural source, by
expression of a recombinant nucleic acid encoding such a
polypeptide; or by chemically synthesizing the protein. Purity can
be measured by any appropriate method, for example, column
chromatography, polyacrylamide gel electrophoresis, HPLC analysis,
and the like.
[0075] The terms "identical" or "percent identity" in the context
of two or more nucleic acids or polypeptides, refer to two or more
sequences or subsequences that are the same or have a specified
percentage of nucleotides or amino acid residues that are the same,
when compared and aligned (introducing gaps, if necessary) for
maximum correspondence, not considering any conservative amino acid
substitutions as part of the sequence identity. The percent
identity may be measured using sequence comparison software or
algorithms or by visual inspection. Various algorithms and software
are known in the art that may be used to obtain alignments of amino
acid or nucleotide sequences. One such non-limiting example of a
sequence alignment algorithm is the algorithm described in Karlin
et al., Proc. Natl. Acad. Sci., 87:2264-2268 (1990), as modified in
Karlin et al., Proc. Natl. Acad. Sci., 90:5873-5877 (1993), and
incorporated into the NBLAST and XBLAST programs (Altschul et al.,
Nucleic Acids Res., 25:3389-3402 (1991)). In certain embodiments,
Gapped BLAST may be used as described in Altschul et al., Nucleic
Acids Res. 25:3389-3402 (1997). BLAST-2, WU-BLAST-2 (Altschul et
al., Methods in Enzymology, 266:460-480 (1996)), ALIGN, ALIGN-2
(Genentech, South San Francisco, Calif.) or Megalign (DNASTAR) are
additional publicly available software programs that can be used to
align sequences. In certain embodiments, the percent identity
between two nucleotide sequences is determined using the GAP
program in GCG software (e.g., using a NWSgapdna.CMP matrix and a
gap weight of 40, 50, 60, 70, or 90 and a length weight of 1, 2, 3,
4, 5, or 6). In certain embodiments, the GAP program in the GCG
software package, which incorporates the algorithm of Needleman and
Wunsch (J. Mol. Biol. (48):444-453 (1970)) may be used to determine
the percent identity between two amino acid sequences (e.g., using
either a Blossum 62 matrix or a PAM250 matrix, and a gap weight of
16, 14, 12, 10, 8, 6, or 4 and a length weight of 1, 2, 3, 4, 5).
In certain embodiments, the percent identity between nucleotide or
amino acid sequences is determined using the algorithm of Myers and
Miller (CABIOS, 4:11-17 (1989)). For example, the percent identity
may be determined using the ALIGN program (version 2.0) and using a
PAM120 with residue table, a gap length penalty of 12 and a gap
penalty of 4. Appropriate parameters for maximal alignment by
particular alignment software can be determined by one skilled in
the art. In certain embodiments, the default parameters of the
alignment software are used. In certain embodiments, the percentage
identity "X" of a first amino acid sequence to a second sequence
amino acid is calculated as 100.times.(Y/Z), where Y is the number
of amino acid residues scored as identical matches in the alignment
of the first and second sequences (as aligned by visual inspection
or a particular sequence alignment program) and Z is the total
number of residues in the second sequence. If the length of a first
sequence is longer than the second sequence, the percent identity
of the first sequence to the second sequence will be longer than
the percent identity of the second sequence to the first
sequence.
[0076] As a non-limiting example, whether any particular
polynucleotide has a certain percentage sequence identity (e.g., is
at least 80% identical, at least 85% identical, at least 90%
identical, and in some embodiments, at least 95%, 96%, 97%, 98%, or
99% identical) to a reference sequence can, in certain embodiments,
be determined using the Bestfit program (Wisconsin Sequence
Analysis Package, Version 8 for Unix, Genetics Computer Group,
University Research Park, 575 Science Drive, Madison, Wis. 53711).
Bestfit uses the local homology algorithm of Smith and Waterman,
Advances in Applied Mathematics 2: 482 489 (1981), to find the best
segment of homology between two sequences. When using Bestfit or
any other sequence alignment program to determine whether a
particular sequence is, for instance, 95% identical to a reference
sequence according to the present invention, the parameters are set
such that the percentage of identity is calculated over the full
length of the reference nucleotide sequence and that gaps in
homology of up to 5% of the total number of nucleotides in the
reference sequence are allowed.
[0077] In some embodiments, two nucleic acids or polypeptides of
the invention are substantially identical, meaning they have at
least 70%, at least 75%, at least 80%, at least 85%, at least 90%,
and in some embodiments at least 95%, 96%, 97%, 98%, 99% nucleotide
or amino acid residue identity, when compared and aligned for
maximum correspondence, as measured using a sequence comparison
algorithm or by visual inspection. Identity can exist over a region
of the sequences that is at least about 5, at least about 10, about
20, about 40-60 residues in length or any integral value
therebetween, or over a longer region than 60-80 residues, at least
about 90-100 residues, or the sequences are substantially identical
over the full length of the sequences being compared.
[0078] A "conservative amino acid substitution" is one in which one
amino acid residue is replaced with another amino acid residue
having a similar side chain. Families of amino acid residues having
similar side chains have been defined in the art, including basic
side chains (e.g., lysine, arginine, histidine), acidic side chains
(e.g., aspartic acid, glutamic acid), uncharged polar side chains
(e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine,
cysteine), nonpolar side chains (e.g., alanine, valine, leucine,
isoleucine, proline, phenylalanine, methionine, tryptophan),
beta-branched side chains (e.g., threonine, valine, isoleucine) and
aromatic side chains (e.g., tyrosine, phenylalanine, tryptophan,
histidine). For example, substitution of a phenylalanine for a
tyrosine is a conservative substitution. Preferably, conservative
substitutions in the sequences of the polypeptides and antibodies
of the invention do not abrogate the binding of the polypeptide or
antibody containing the amino acid sequence, to the antigen(s).
Methods of identifying nucleotide and amino acid conservative
substitutions which do not eliminate antigen binding are well-known
in the art (see, e.g., Brummell et al., Biochem. 32: 1180-1 187
(1993); Kobayashi et al. Protein Eng. 12(10):879-884 (1999); and
Burks et al. Proc. Natl. Acad. Sci. USA 94:412-417 (1997)).
[0079] "Pharmaceutically acceptable" refers to approved or
approvable by a regulatory agency of the Federal or a state
government or listed in the U.S. Pharmacopeia or other generally
recognized pharmacopeia for use in animals, including humans.
[0080] "Pharmaceutically acceptable excipient, carrier or diluent"
refers to an excipient, carrier or diluent that can be administered
to a subject, together with an agent, and which does not destroy
the pharmacological activity thereof and is nontoxic when
administered in doses sufficient to deliver a therapeutic amount of
the agent.
[0081] A "pharmaceutically acceptable salt" of an influenza
antibody recited herein is an acid or base salt that is generally
considered in the art to be suitable for use in contact with the
tissues of human beings or animals without excessive toxicity,
irritation, allergic response, or other problem or complication.
Such salts include mineral and organic acid salts of basic residues
such as amines, as well as alkali or organic salts of acidic
residues such as carboxylic acids. Specific pharmaceutical salts
include, but are not limited to, salts of acids such as
hydrochloric, phosphoric, hydrobromic, malic, glycolic, fumaric,
sulfuric, sulfamic, sulfanilic, formic, toluenesulfonic,
methanesulfonic, benzene sulfonic, ethane disulfonic,
2-hydroxyethylsulfonic, nitric, benzoic, 2-acetoxybenzoic, citric,
tartaric, lactic, stearic, salicylic, glutamic, ascorbic, pamoic,
succinic, fumaric, maleic, propionic, hydroxymaleic, hydroiodic,
phenylacetic, alkanoic such as acetic, HOOC--(CH2).sub.n--COOH
where n is 0-4, and the like. Similarly, pharmaceutically
acceptable cations include, but are not limited to sodium,
potassium, calcium, aluminum, lithium and ammonium. Those of
ordinary skill in the art will recognize further pharmaceutically
acceptable salts for the antibodies provided herein, including
those listed by Remington's Pharmaceutical Sciences, 17th ed., Mack
Publishing Company, Easton, Pa., p. 1418 (1985). In general, a
pharmaceutically acceptable acid or base salt can be synthesized
from a parent compound that contains a basic or acidic moiety by
any conventional chemical method. Briefly, such salts can be
prepared by reacting the free acid or base forms of these compounds
with a stoichiometric amount of the appropriate base or acid in an
appropriate solvent.
[0082] The term "patient" or "subject" refers to an animal which is
the object of treatment, observation, or experiment. By way of
example only, a subject includes, but is not limited to, a mammal,
including, but not limited to, a human or a non-human mammal, such
as a non-human primate, bovine, equine, canine, ovine, or
feline.
[0083] As used herein, the terms "prevent," "preventing,"
"prevention," "prophylactic treatment," and the like, refer to
reducing the probability of developing a disease or condition in a
subject, who does not have, but is at risk of or susceptible to
developing a disease or condition.
[0084] By "reduces" is meant a negative alteration of at least
about 10%, 25%, 50%, 75%, or 100% relative to a reference.
[0085] By "reference" is meant a standard or control condition.
[0086] By "specifically binds" is meant a compound or antibody that
recognizes and binds a polypeptide of the invention, but which does
not substantially recognize and bind other molecules in a sample,
for example, a biological sample, which naturally includes a
polypeptide of the invention.
[0087] That an antibody "specifically binds" to an epitope or
protein means that the antibody reacts or associates more
frequently, more rapidly, with greater duration, with greater
affinity, or with some combination of the above to an epitope or
protein than with alternative substances, including unrelated
proteins. In certain embodiments, "specifically binds" means, for
instance, that an antibody binds to a protein with a K.sub.D of
about 0.1 mM or less, but more usually less than about 1 .mu.M. In
certain embodiments, "specifically binds" means that an antibody
binds to a protein at times with a K.sub.D of at least about 0.1
.mu.M or less, and at other times at least about 0.01 .mu.M or
less. Because of the sequence identity between homologous proteins
in different species, specific binding can include an antibody that
recognizes a particular protein in more than one species. It is
understood that an antibody or binding moiety that specifically
binds to a first target may or may not specifically bind to a
second target. As such, "specific binding" does not necessarily
require (although it can include) exclusive binding, i.e. binding
to a single target. Generally, but not necessarily, reference to
binding means specific binding.
[0088] As used herein, "substantially pure" refers to material
which is at least 50% pure (i.e., free from contaminants), more
preferably at least 90% pure, more preferably at least 95% pure,
more preferably at least 98% pure, more preferably at least 99%
pure.
[0089] As used herein, the terms "treat," treating," "treatment,"
and the like refer to reducing or ameliorating a disorder and/or
symptoms associated therewith. By "ameliorate" is meant decrease,
suppress, attenuate, diminish, arrest, or stabilize the development
or progression of a disease. It will be appreciated that, although
not precluded, treating a disorder or condition does not require
that the disorder, condition or symptoms associated therewith be
completely eliminated.
[0090] The term "therapeutic effect" refers to some extent of
relief of one or more of the symptoms of a disorder or its
associated pathology. The term refers to both therapeutic treatment
and prophylactic or preventative measures, wherein the object is to
prevent or slow down (lessen) the targeted pathologic condition or
disorder. Those in need of treatment include those already with the
disorder as well as those prone to have the disorder or those in
whom the disorder is to be prevented. A subject or mammal is
successfully "treated" for an infection if, after receiving a
therapeutic amount of an antibody according to the methods of the
present invention, the patient shows observable and/or measurable
reduction in or absence of one or more of the following: reduction
in the number of infected cells or absence of the infected cells;
reduction in the percent of total cells that are infected; relief
to some extent of one or more of the symptoms associated with the
specific infection (e.g., symptoms associated with influenza
infection); reduced morbidity and mortality, and improvement in
quality of life issues. The above parameters for assessing
successful treatment and improvement in the disease are readily
measurable by routine procedures familiar to a physician.
[0091] "Therapeutically effective amount" is intended to qualify
the amount required to achieve a therapeutic effect. A physician or
veterinarian having ordinary skill in the art can readily determine
and prescribe the "therapeutically effective amount" (e.g.,
ED.sub.50) of the pharmaceutical composition required. For example,
the physician or veterinarian could start doses of the compounds of
the invention employed in a pharmaceutical composition at levels
lower than that required in order to achieve the desired
therapeutic effect and gradually increase the dosage until the
desired effect is achieved.
[0092] The phrase "combination therapy" embraces the administration
of an influenza antibody and a second therapeutic agent as part of
a specific treatment regimen intended to provide a beneficial
effect from the co-action of these therapeutic agents. The
beneficial effect of the combination includes, but is not limited
to, pharmacokinetic or pharmacodynamic co-action resulting from the
combination of therapeutic agents. Administration of these
therapeutic agents in combination typically is carried out over a
defined time period (usually minutes, hours, days, or weeks
depending upon the combination selected). "Combination therapy"
generally is not intended to encompass the administration of two or
more of these therapeutic agents as part of separate monotherapy
regimens that incidentally and arbitrarily result in the
combinations of the present invention. "Combination therapy" is
intended to embrace administration of these therapeutic agents in a
sequential manner, that is, wherein each therapeutic agent is
administered at a different time, as well as administration of
these therapeutic agents, or at least two of the therapeutic
agents, in a substantially simultaneous manner. Substantially
simultaneous administration can be accomplished, for example, by
administering to the subject a single capsule having a fixed ratio
of each therapeutic agent or in multiple, single capsules for each
of the therapeutic agents. For example, one combination of the
present invention comprises an influenza antibody and at least one
additional therapeutic agent (e.g., antiviral agent, including
anti-influenza agents) at the same or different times or they can
be formulated as a single, co-formulated pharmaceutical composition
comprising the two compounds. As another example, a combination of
the present invention (e.g., an influenza antibody and at least one
additional therapeutic agent, such as an antiviral agent) is
formulated as separate pharmaceutical compositions that can be
administered at the same or different time. Sequential or
substantially simultaneous administration of each therapeutic agent
can be effected by any appropriate route including, but not limited
to, oral routes, intravenous routes, intramuscular routes, and
direct absorption through mucous membrane tissues (e.g., nasal,
mouth, vaginal, and rectal). The therapeutic agents can be
administered by the same route or by different routes. For example,
one component of a particular combination may be administered by
intravenous injection while the other component(s) of the
combination may be administered orally. The components may be
administered in any therapeutically effective sequence.
[0093] The phrase "combination" embraces groups of compounds or
non-drug therapies useful as part of a combination therapy.
[0094] The term "vector" means a construct that is capable of
delivering and expressing, one or more gene(s) or sequence(s) of
interest in a host cell. Examples of vectors include, but are not
limited to, viral vectors, naked DNA or RNA expression vectors,
plasmid, cosmid or phage vectors, DNA or RNA expression vectors
associated with cationic condensing agents, DNA or RNA expression
vectors encapsulated in liposomes, and certain eukaryotic cells,
such as producer cells.
[0095] Unless specifically stated or obvious from context, as used
herein, the term "about" is understood as within a range of normal
tolerance in the art, for example within 2 standard deviations of
the mean. About can be understood as within 10%, 9%, 8%, 7%, 6%,
5%, 4%, 3%, 2%, 1%, 0.5%, 0.1%, 0.05%, or 0.01% of the stated
value. Unless otherwise clear from context, all numerical values
provided herein are modified by the term about.
[0096] Ranges provided herein are understood to be shorthand for
all of the values within the range. For example, a range of 1 to 50
is understood to include any number, combination of numbers, or
sub-range from the group consisting 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, or 50.
[0097] The recitation of a listing of chemical groups in any
definition of a variable herein includes definitions of that
variable as any single group or combination of listed groups. The
recitation of an embodiment for a variable or aspect herein
includes that embodiment as any single embodiment or in combination
with any other embodiments or portions thereof.
[0098] Any compositions or methods provided herein can be combined
with one or more of any of the other compositions and methods
provided herein.
DESCRIPTION OF THE DRAWINGS
[0099] The patent or application file contains at least one drawing
executed in color. Copies of this patent or patent application
publication with color drawings will be provided by the Office upon
request and payment of the necessary fee.
[0100] FIG. 1A: Inferred lineage of clone 860. Left: the unmutated
common ancestor (UCA) of the three antibodies (shown by their
numbers, right) isolated from the donor. (FIG. 1B) Alignment of
heavy-chain (top) (SEQ ID NOS 51 and 10-12, respectively, in order
of appearance) and light-chain (bottom) (SEQ ID NOS 13-16,
respectively, in order of appearance) sequences in the lineage.
(FIG. 1C) Contact of the Fab from CH65 with HA1. Heavy chain in
dark blue; light chain in light blue; CDRs in colors as labeled in
(FIG. 1B); HA in red, with the atomic surface shown as a partly
transparent overlay. Residues that have mutated from the UCA are in
green stick representation.
[0101] FIG. 2A: HA trimer with bound CH65 Fab. One HA chain is in
red (HA1) and green (HA2); the other two chains are in gray;
glycans are in yellow. The Fab bound to the colored HA chain is in
dark blue (heavy chain) and light blue (light chain), with the
contacting CDRs in colors as labeled in FIG. 1B. (FIG. 2B) Blow up
of the Fv region and its contact with HA1. Colors as in FIGS.
1A-1C. Note that the heavy-chain CDR3 (magenta) projects into the
receptor-binding pocket on HA1, while the remaining CDRs have more
limited surface contacts. (FIG. 2C) and (FIG. 2D) Surface
representation of the contact between Fab CH65 and HA1, opened up
as shown by the arrows. The sialic-acid pocket on one HA subunit is
in dark red; the rest of the subunit, in dull red; the remaining
two subunits, in gray; glycans, in yellow.
[0102] FIGS. 3A and 3B: Comparison of interactions from CH65 (FIG.
3A) and .alpha.-2,6-sialyl lactose (FIG. 3B).
[0103] FIGS. 4A-4F: Enzyme-linked immunoabsorbent assay (ELISA) of
reactivity of CH65-CH67 lineage members to H1 and H3 influenza
strains. 293 T cells were transfected with full-length HA from
strain X31 (H3) (top panel, FIGS. 4A-4C) or with cell-surface
expressed globular head from A/Solomon Islands/3/2006 [H1] (bottom
panel, FIGS. 4D-4F). Cells were fixed with formaldehyde and probed
with CH65 Fab (FIGS. 4B and 4E) or CH66 full-length antibody (FIGS.
4C and 4F), followed by a FITC-conjugated secondary antibody
specific for the human Fab. Cells were imaged by FITC emission (532
nm). As a control, transfected cells were probed with secondary
antibody only (FIGS. 4A and 4D).
[0104] FIG. 5: Sequences (SEQ ID NOS 52-56, respectively, in order
of appearance) at the VDJ recombination site of CH65. The key
indicates the origin of the heavy-chain coding segments (V, D, J,
and n-nucleotide).
[0105] FIG. 6: Heavy chain DNA sequences of CH65-CH67 HA
antibodies.
[0106] FIG. 7: Light chain DNA sequences of CH65-CH67 HA
antibodies.
[0107] FIG. 8: Heavy chain amino acid sequences of CH65-CH67 HA
antibodies.
[0108] FIG. 9: Light chain amino acid sequences of CH65-CH67 HA
antibodies.
[0109] FIG. 10: Alignment of VH DNA sequences of CL860UCA (SEQ ID
NO: 1), CH65 (SEQ ID NO: 2), CH66 (SEQ ID NO: 3) and CH67 (SEQ ID
NO: 4).
[0110] FIG. 11: Alignment of VL DNA sequences of CL860UCA (SEQ ID
NO: 5), CH65 (SEQ ID NO: 6), CH66 (SEQ ID NO: 7) and CH67 (SEQ ID
NO: 8).
[0111] FIG. 12: Alignment of VH amino acid sequences of CL860UCA
(SEQ ID NO: 9), CH65 (SEQ ID NO: 10), CH66 (SEQ ID NO: 11) and CH67
(SEQ ID NO: 12).
[0112] FIG. 13: Alignment of VL amino acid sequences of CL860UCA
(SEQ ID NO: 13), CH65 (SEQ ID NO: 14), CH66 (SEQ ID NO: 15) and
CH67 (SEQ ID NO: 16).
[0113] FIGS. 14A-14D: Representative receptor binding domains from
H1 (FIG. 14A; SEQ ID NOS 17-34, respectively, in order of
appearance), H2 (FIG. 14B; SEQ ID NOS 35-38, respectively, in order
of appearance), H3 (FIG. 14C; SEQ ID NOS 39-43, respectively, in
order of appearance), and H5 (FIG. 14D; (SEQ ID NOS 44-45,
respectively, in order of appearance) hemagglutinin. The CH65-CH67
antibody binding epitopes are underlined.
[0114] FIG. 15: Sequence alignment of representative receptor
binding domains from H1, H2, H3, and H5 hemagglutinin (SEQ ID NOS
57-61, respectively, in order of appearance). The amino acid
residues that interact with the CH65-CH67 antibodies are
underlined.
DETAILED DESCRIPTION OF THE INVENTION
[0115] The invention features novel antibodies that broadly
neutralize influenza antigenic variants. The invention also
provides compositions and kits containing the novel antibodies, as
well as methods for using these novel therapeutic molecules to
treat or prevent (e.g., vaccinate) influenza infection.
[0116] The receptor for influenza virus is sialic acid, attached by
terminal .alpha.-2,3 or .alpha.-2,6 linkage to glycans on
glycoproteins or glycolipids (reviewed in Wiley, D. C. and Skehel,
J. J. Annu. Rev. Biochem. 56:365-394 (1987)). Most neutralizing
antibodies block cell attachment, either because their footprint
overlaps the receptor-binding site or because they exert steric
interference when bound elsewhere on the HA surface (Knossow, M.
and Skehel, J. J. Immunology 119:1-7 (2006)). Two mouse monoclonal
neutralizing antibodies, for which structures of Fab:HA complexes
have been determined, have loops that project into the sialic-acid
binding pocket on HA and present an aspartic-acid side chain
roughly where the sialic-acid carboxylate would be (Fleury, D. et
al., Nat. Struct. Biol. 5:119-123 (1998); and Barbey-Martin, C. et
al., Virology 294:70-74 (2002)). But both of these antibodies also
have extensive contacts with other surface regions, in which escape
mutations could occur more readily than in the receptor site.
[0117] The invention is based, at least in part, on the discovery
of novel antibodies having principal contacts in the receptor
pocket. One such antibody, designated CH65, was found by isolating
rearranged heavy- and light-chain genes from sorted single plasma
cells, obtained from a subject who had received the 2007 trivalent
vaccine. CH65 neutralizes a remarkably broad range of H1 seasonal
isolates spanning more than three decades. Its 19-residue
heavy-chain complementarity-determining region 3 (CDR-H3) inserts
into the receptor pocket, mimicking many of the interactions made
by sialic acid.
[0118] Both heavy- and light-chain CDRs participate in more
restricted, additional contacts with the outward-facing surface of
HA1. The inferred, unmutated ancestor of CH65 differs from the
affinity matured antibody at 12 positions in the heavy-chain
variable domain, and at 6 in the light-chain variable domain. The
human B-cell repertoire thus includes the potential to generate
antibodies directed primarily at the receptor binding site. The
large number of seasonal H1 viruses neutralized by antibody CH65
suggests that such responses are ordinarily too rare to select for
resistance, or that resistance comes at too great a fitness
cost--as would be the case if potential escape mutations were to
compromise receptor binding. Thus, it is surprising that the
inventors have discovered that broad neutralization of influenza
virus can be achieved by antibodies with contacts that mimic those
of the receptor. Accordingly, the invention provides novel
antibodies that mimic the contact between influenza HA and the
sialic acid receptor. These novel antibodies can effectively treat
and/or prevent infection by drifted strains of influenza. As such,
the invention features compositions and kits containing the novel
antibodies, as well as methods for using these therapeutic
molecules to treat and/or prevent influenza infection. The
invention also relates to combination therapies including the novel
antibodies.
CH65-CH67 Hemagglutinin Antibodies
[0119] The present invention provides novel anti-influenza
antibodies that specifically bind to an epitope of an influenza
hemagglutinin (HA). Binding of the antibodies to the HA reduces or
inhibits influenza hemagglutinin binding to sialic acid.
[0120] As stated above, the invention features in certain
embodiments, an anti-influenza antibody or antibody fragment that
specifically binds to a sialic acid binding domain of a surface
antigen of influenza virus. Preferably, the surface antigen of
influenza virus is HA.
[0121] In embodiments, the epitope of influenza HA comprises a
sialic-acid binding domain.
[0122] In embodiments, the HA is H1 HA, H2 HA, H3 HA, or H5 HA (an
HA from a human adapted H5 strain).
[0123] In related embodiments, the antibody or antibody fragmenr
contacts one or more residues in the the influenza HA epitope
comprising residue:158; 158-160; 135-136, 190-195, and 226; 222,
225, and 227; and 187 and 189 where the numbering refers to the one
used in structures such as that for A/Solomon Islands/3/2006
(Protein Data Bank accession number 3SM5).
[0124] In one embodiment, and as stated above, the antibody
contacts one or more residues in the sialic acid binding pocket of
the HA epitope selected from the group consisting of residue:
134-136, 190-195, and 226.
[0125] In another embodiment, and as stated above, the antibody
further contacts one or more residues in the HA epitope selected
from the group consisting of residue: 158, 158-160, 135-136,
190-195 and 226, and 187 and 189.
[0126] As described herein, in certain embodiments, the antibody
CDR H1 region contacts residue 158 of the HA epitope; the CDR H2
region contacts residues 158-160 of the HA epitope; the CDR H3
region contacts residues 135-136, 190-194 and 226 of the HA
epitope; the CDR L1 region contacts residues 222, 225 and 227 of
the HA epitope; or the CDR L3 region contacts residues 187 and 198
of the HA epitope.
[0127] In related embodiments, the influenza HA epitope comprises
the amino acids set forth in any one of SEQ ID NOs:17-44.
[0128] In related embodiments, the influenza HA epitope comprises
the CH65-CH67 binding residues in any one of SEQ ID NOs:17-44
(e.g., the CH65-CH67 binding residues identified in FIG. 15).
[0129] In embodiments, the anti-influenza antibody or antibody
fragment comprises a variable heavy (V.sub.H) chain, and wherein
the V.sub.H chain comprises an amino acid sequence set forth in SEQ
ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, or SEQ ID NO: 12.
[0130] In embodiments, the anti-influenza antibody or antibody
fragment comprises one or more heavy chain CDR regions present in a
variable heavy (V.sub.H) chain amino acid sequence of SEQ ID NO: 9,
SEQ ID NO: 10, SEQ ID NO: 11, or SEQ ID NO: 12. In related
embodiments, the one or more heavy chain CDR regions comprises a
CDR3 region present in the variable heavy (V.sub.H) chain amino
acid sequence of SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, or SEQ
ID NO: 12.
[0131] In embodiments, the anti-influenza antibody or antibody
fragment comprises a variable light (V.sub.L) chain, and wherein
the V.sub.L chain comprises an amino acid sequence set forth in SEQ
ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, or SEQ ID NO: 16.
[0132] In embodiments, the anti-influenza antibody or antibody
fragment comprises one or more light chain CDR regions present in a
variable light (V.sub.L) chain amino acid sequence of SEQ ID NO:
13, SEQ ID NO: 14, SEQ ID NO: 15, or SEQ ID NO: 16. In related
embodiments, the one or more light chain CDR regions comprises a
CDR3 region present in the variable heavy (V.sub.L) chain amino
acid sequence of SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, or
SEQ ID NO: 16.
[0133] In embodiments, the anti-influenza antibody or antibody
fragment comprises i) a variable heavy (V.sub.H) chain amino
comprising an amino acid sequence set forth in SEQ ID NO: 10, and
ii) a variable light (V.sub.L) chain comprising an amino acid
sequence set forth in SEQ ID NO: 14.
[0134] In embodiments, the anti-influenza antibody or antibody
fragment comprises a variable heavy (V.sub.H) chain, wherein the
CDR3 region of the V.sub.H chain comprises Arg104, Ser105, Val106,
Asp107, Tyr109, Tyr110, Tyr112, or a combination thereof.
[0135] In embodiments, the anti-influenza antibody is a monoclonal
antibody or antibody fragment thereof.
[0136] In embodiments, the anti-influenza antibody is a humanized
antibody.
[0137] In embodiments, the antibody fragment is an Fab fragment, an
Fab' fragment, an Fd fragment, a Fd' fragment, an Fv fragment, a
dAb fragment, an F(ab')2 fragment, a single chain fragment, a
diabody, or a linear antibody.
[0138] In embodiments, the anti-influenza antibody or antibody
fragment further comprises an agent conjugated to the
anti-influenza antibody or antibody fragment thereof. In related
embodiments, the agent conjugated to the antibody or antibody
fragment thereof is a therapeutic agent or detectable label.
[0139] The therapeutic agent can be any therapeutic agent suitable
for use with the novel antibodies. Such agents are well known in
the art and include small molecules, nanoparticles, polypeptides,
radioisotopes, inhibitory nucleic acids, and the like. In
embodiments, the therapeutic agent is an antiviral agent or a
toxin. In embodiments, the therapeutic agent is an siRNA, shRNA, or
antisense nucleic acid molecule that reduces influenza virus
production.
[0140] The detectable label can be any detectable label suitable
for use with the novel antibodies. Such labels are well known in
the art and include labels that are detected by spectroscopic,
photochemical, biochemical, immunochemical, physical, or chemical
means. In embodiments, the detectable label is an enzyme, a
fluorescent molecule, a particle label, an electron-dense reagent,
a radiolabel, a microbubble, biotin, digoxigenin, or a hapten or a
protein that has been made detectable.
[0141] In any of the above embodiments, the influenza can be H1N1,
H2N2, H3N2, or a human adapted H5 strain.
[0142] The antibodies of the invention can be prepared by any
conventional means known in the art. For example, polyclonal
antibodies can be prepared by immunizing an animal (e.g., a rabbit,
rat, mouse, donkey, goat, hamster, guinea pig, sheep, ungulate,
cow, camel, fowl, chicken, and the like) by multiple subcutaneous
or intraperitoneal injections of the relevant antigen (e.g., a
purified peptide fragment, full-length recombinant protein, fusion
protein, and the like) optionally conjugated to suitable hapten
(e.g., keyhole limpet hemocyanin (KLH), serum albumin, and the
like). The antigens can be diluted in any suitable vehicle (e.g.,
sterile saline) and combined with an adjuvant (e.g., Complete or
Incomplete Freund's Adjuvant) to form a stable emulsion. The
polyclonal antibody is then recovered from blood, ascites and the
like, of an animal so immunized. Collected blood is clotted, and
the serum decanted, clarified by centrifugation, and assayed for
antibody titer. The polyclonal antibodies can be purified from
serum or ascites according to standard methods in the art including
affinity chromatography, ion-exchange chromatography, gel
electrophoresis, dialysis, and the like.
[0143] Monoclonal antibodies can be prepared using hybridoma
methods, such as those described by Kohler and Milstein, Nature
256:495 (1975). Using the hybridoma method, an appropriate host
animal is immunized as described above to elicit the production by
lymphocytes of antibodies that will specifically bind to an
immunizing antigen. Lymphocytes can also be immunized in vitro.
Following immunization, the lymphocytes are isolated and fused with
a suitable myeloma cell line using, for example, polyethylene
glycol, to form hybridoma cells that can then be selected away from
unfused lymphocytes and myeloma cells. Hybridomas that produce
monoclonal antibodies directed specifically against a chosen
antigen as determined by immunoprecipitation, immunoblotting, or by
an in vitro binding assay (e.g., radioimmunoassay (RIA),
enzyme-linked immunosorbent assay (ELISA), and the like) can then
be propagated either in vitro culture using standard methods (see
Goding, Monoclonal Antibodies: Principles and Practice, Academic
Press, 1986) or in vivo as ascites tumors in an animal. The
monoclonal antibodies can then be purified from the culture medium
or ascites fluid as described for polyclonal antibodies above.
[0144] Alternatively monoclonal antibodies can also be made using
recombinant DNA methods as described in U.S. Pat. No. 4,816,567.
The polynucleotides encoding a monoclonal antibody are isolated
from mature B-cells or hybridoma cell, such as by RT-PCR using
oligonucleotide primers that specifically amplify the genes
encoding the heavy and light chains of the antibody, and their
sequence is determined using conventional procedures. The isolated
polynucleotides (including the isolated polynucleotides described
herein) encoding the heavy and light chains are then cloned into
suitable expression vectors, which when transfected into host cells
such as E. coli cells, simian COS cells, Chinese hamster ovary
(CHO) cells, or myeloma cells that do not otherwise produce
immunoglobulin protein, monoclonal antibodies are generated by the
host cells. Also, recombinant monoclonal antibodies or fragments
thereof of the desired species can be isolated from phage display
libraries expressing CDRs of the desired species as described (see
McCafferty et al., 1990, Nature, 348:552-554; Clackson et al.,
1991, Nature, 352:624-628; and Marks et al., 1991, J. Mol. Biol.,
222:581-597).
[0145] The polynucleotide(s) encoding a monoclonal antibody can
further be modified in a number of different manners using
recombinant DNA technology to generate alternative antibodies. In
some embodiments, the constant domains of the light and heavy
chains of, for example, a mouse monoclonal antibody can be
substituted 1) for those regions of, for example, a human antibody
to generate a chimeric antibody or 2) for a non-immunoglobulin
polypeptide to generate a fusion antibody. In some embodiments, the
constant regions are truncated or removed to generate the desired
antibody fragment of a monoclonal antibody. Site-directed or
high-density mutagenesis of the variable region can be used to
optimize specificity, affinity, etc. of a monoclonal antibody.
[0146] In some embodiments of the present invention, the monoclonal
antibody is a humanized antibody. Humanized antibodies are
antibodies that contain minimal sequences from non-human (e.g.,
rodent) antibodies within the antigen determination or
hypervariable region that comprise the three complementary
determination regions (CDRs) within each antibody chain. Such
antibodies are used therapeutically to reduce antigenicity and HAMA
(human anti-mouse antibody) responses when administered to a human
subject. In practice, humanized antibodies are typically human
antibodies with minimum to virtually no non-human sequences. A
human antibody is an antibody produced by a human or an antibody
having an amino acid sequence corresponding to an antibody produced
by a human.
[0147] Humanized antibodies can be produced using various
techniques known in the art. An antibody can be humanized by
substituting the CDRs of a human antibody with those of a non-human
antibody (e.g., mouse, rat, rabbit, hamster, and the like) having
the desired specificity, affinity, and capability (see, e.g., the
methods of Jones et al., Nature 321:522-525 (1986); Riechmann et
al., Nature 332:323-327 (1988); and Verhoeyen et al., Science
239:1534-1536 (1988). The humanized antibody can be further
modified by the substitution of additional residue either in the
variable human framework region and/or within the replaced
non-human residues to refine and optimize antibody specificity,
affinity, and/or capability.
[0148] The choice of human heavy and/or light chain variable
domains to be used in making humanized antibodies can be important
for reducing antigenicity. According to the "best-fit" method, the
sequence of the variable domain of a non-human antibody is screened
against the entire library of known human variable-domain amino
acid sequences. Thus in certain embodiments, the human amino acid
sequence which is most homologous to that of the non-human antibody
from which the CDRs are taken is used as the human framework region
(FR) for the humanized antibody (see Sims et al., J. Immunol. 151:
2296 (1993); Chothia et al., J. Mol. Biol. 196:901 (1987)). Another
method uses a particular FR derived from the consensus sequence of
all human antibodies of a particular subgroup of light or heavy
chains and can be used for several difference humanized antibodies
(see Carter et al., PNAS 89; 4285 (1992); Presta et al., J.
Immunol. 151: 2623 (1993)). In embodiments, a combination of
methods is used to pick the human variable FR to use in generation
of humanized antibodies.
[0149] It is further understood that antibodies to be humanized
must retain high affinity for the antigen as well as other
favorable biological properties. To achieve this goal, humanized
antibodies can be prepared by a process of analysis of the parental
sequence from the non-human antibody to be humanized and the
various candidate humanizing sequences. Three-dimensional
immunoglobulin models are available and familiar to those skilled
in the art. Computer programs can be used to illustrate and display
probable three-dimensional conformational structures of selected
candidate antibody sequences. Use of such models permits analysis
of the likely role of the residues in the function of the antibody
to be humanized, i.e., the analysis of residues that influence the
ability of the candidate antibody to bind its antigen. In this way,
FR residues can be selected and combined from the parental antibody
to the recipient humanized antibody so that the desired antibody
characteristics are achieved. In general, the residues in the CDRs
of the antigen determination region (or hypervariable region) are
retained from the parental antibody (e.g. the non-human antibody
with the desired antigen binding properties) in the humanized
antibody for antigen binding. In certain embodiments, at least one
additional residue within the variable FR is retained from the
parental antibody in the humanized antibody. In certain
embodiments, up to six additional residues within the variable FR
are retained from the parental antibody in the humanized
antibody.
[0150] Amino acids from the variable regions of the mature heavy
and light chains of immunoglobulins are designated Hx and Lx
respectively, where x is a number designating the position of an
amino acid according to the scheme of Kabat, Sequences of Proteins
of Immunological Interest, U.S. Department of Health and Human
Services, 1987, 1991. Kabat lists many amino acid sequences for
antibodies for each subgroup, and lists the most commonly occurring
amino acid for each residue position in that subgroup to generate a
consensus sequence. Kabat uses a method for assigning a residue
number to each amino acid in a listed sequence, and this method for
assigning residue numbers has become standard in the field. Kabat's
scheme is extendible to other antibodies not included in his
compendium by aligning the antibody in question with one of the
consensus sequences in Kabat by reference to conserved amino acids.
The use of the Kabat numbering system readily identifies amino
acids at equivalent positions in different antibodies. For example,
an amino acid at the L50 position of a human antibody occupies the
equivalent position to an amino acid position L50 of a mouse
antibody. Moreover, any two antibody sequences can be uniquely
aligned, for example to determine percent identity, by using the
Kabat numbering system so that each amino acid in one antibody
sequence is aligned with the amino acid in the other sequence that
has the same Kabat number. After alignment, if a subject antibody
region (e.g., the entire mature variable region of a heavy or light
chain) is being compared with the same region of a reference
antibody, the percentage sequence identity between the subject and
reference antibody regions is the number of positions occupied by
the same amino acid in both the subject and reference antibody
region divided by the total number of aligned positions of the two
regions, with gaps not counted, multiplied by 100 to convert to
percentage.
[0151] In addition to humanized antibodies, fully human antibodies
can be directly prepared using various techniques known in the art.
Immortalized human B lymphocytes immunized in vitro or isolated
from an immunized individual that produce an antibody directed
against a target antigen can be generated (See, e.g., Cole et al.,
Monoclonal Antibodies and Cancer Therapy, Alan R. Liss, p. 77
(1985); Boemer et al., J. Immunol. 147:86-95 (1991); and U.S. Pat.
No. 5,750,373). Also, the human antibody can be selected from a
phage library, where that phage library expresses human antibodies
(see Vaughan et al., Nat. Biotech. 14:309-314 (1996); Sheets et
al., Proc. Nat'l. Acad. Sci. 95:6157-6162 (1998); Hoogenboom and
Winter, J. Mol. Biol. 227:381 (1991); and Marks et al., J. Mol.
Biol. 222:581 (1991)). Human antibodies can also be made in
transgenic mice containing human immunoglobulin loci that are
capable upon immunization of producing the full repertoire of human
antibodies in the absence of endogenous immunoglobulin production.
This approach is described in U.S. Pat. Nos. 5,545,807; 5,545,806;
5,569,825; 5,625,126; 5,633,425; and 5,661,016.
[0152] This invention also encompasses bispecific antibodies.
Bispecific antibodies are antibodies that are capable of
specifically recognizing and binding at least two different
epitopes. The different epitopes can either be within the same
molecule (e.g., influenza HA) or on different molecules such that
the bispecific antibody can specifically recognize and bind an
epitope in an antigen of interest (e.g., influenza HA) as well as,
for example, another viral protein (e.g., neurominidase, M2, and
the like). Bispecific antibodies can be intact antibodies or
antibody fragments. Techniques for making bispecific antibodies are
common in the art (see Millstein et al., Nature 305:537-539 (1983);
Brennan et al., Science 229:81 (1985); Suresh et al, Methods in
Enzymol. 121:120 (1986); Traunecker et al., EMBO J. 10:3655-3659
(1991); Shalaby et al., J. Exp. Med. 175:217-225 (1992); Kostelny
et al., J. Immunol. 148:1547-1553 (1992); Gruber et al., J.
Immunol. 152:5368 (1994); and U.S. Pat. No. 5,731,168). Antibodies
with more than two valencies are also contemplated. For example,
trispecific antibodies can be prepared (see Tutt et al., J.
Immunol. 147:60 (1991)).
[0153] In embodiments, the antibodies of the invention are antibody
fragments. Various techniques are known for the production of
antibody fragments: Traditionally, these fragments are derived via
proteolytic digestion of intact antibodies (see, e.g., Morimoto et
al., Journal of Biochemical and Biophysical Methods 24:107-117
(1993); and Brennan et al., Science 229:81 (1985)). Antibody
fragments can also be produced recombinantly. Fab, Fv, and scFv
antibody fragments can all be expressed in and secreted from E.
coli or other host cells, thus allowing the production of large
amounts of these fragments. Such antibody fragments can also be
isolated from antibody phage libraries as discussed above. The
antibody fragment can also be linear antibodies as described in
U.S. Pat. No. 5,641,870, for example, and can be monospecific or
bispecific. Other techniques for the production of antibody
fragments will be readily apparent to the skilled practitioner.
[0154] According to the present invention, techniques can be
adapted for the production of single-chain antibodies specific to a
polypeptide of the invention (see U.S. Pat. No. 4,946,778). In
addition, methods can be adapted for the construction of Fab
expression libraries (see Huse et al., Science 246:1275-1281
(1989)) to allow rapid and effective identification of monoclonal
Fab fragments with the desired specificity for influenza HA.
[0155] Antibody fragments that contain the idiotypes to a
polypeptide of the invention may be produced by techniques in the
art including, but not limited to: (a) an F(ab')2 fragment produced
by pepsin digestion of an antibody molecule; (b) an Fab fragment
generated by reducing the disulfide bridges of an F(ab')2 fragment,
(c) an Fab fragment generated by the treatment of the antibody
molecule with papain and a reducing agent, and (d) Fv
fragments.
[0156] It can further be desirable, especially in the case of
antibody fragments, to modify an antibody in order to increase its
serum half-life. This can be achieved, for example, by
incorporation of a salvage receptor binding epitope into the
antibody fragment by mutation of the appropriate region in the
antibody fragment or by incorporating the epitope into a peptide
tag that is then fused to the antibody fragment at either end or in
the middle (e.g., by DNA or peptide synthesis).
[0157] Heteroconjugate antibodies are also within the scope of the
present invention. Heteroconjugate antibodies are composed of two
covalently joined antibodies. Such antibodies have, for example,
been proposed to target immune cells to unwanted cells (U.S. Pat.
No. 4,676,980). 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.
[0158] Another alteration contemplated by the present invention is
that the variable domains in both the heavy and light chains are
altered by at least partial replacement of one or more CDRs and, if
necessary, by partial framework region replacement and sequence
changing. Although the CDRs may be derived from an antibody of the
same class or even subclass as the antibody from which the
framework regions are derived, it is envisaged that the CDRs will
be derived from an antibody of different class and preferably from
an antibody from a different species. 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 antigen binding site. Given the explanations set forth in
U.S. Pat. Nos. 5,585,089, 5,693,761 and 5,693,762, 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 funcpQE-9tional antibody with reduced
immunogenicity.
[0159] Alterations to the variable region notwithstanding, those
skilled in the art will appreciate that the modified antibodies of
this invention can comprise antibodies, or immunoreactive fragments
thereof, in which at least a fraction of one or more of the
constant region domains has been deleted or otherwise altered so as
to provide desired biochemical characteristics such as increased
tumor localization or reduced serum half-life when compared with an
antibody of approximately the same immunogenicity comprising a
native or unaltered constant region. In some embodiments, the
constant region of the modified antibodies will comprise a human
constant region. Modifications to the constant region compatible
with this invention comprise additions, deletions or substitutions
of one or more amino acids in one or more domains. That is, the
modified antibodies disclosed herein may comprise alterations or
modifications to one or more of the three heavy chain constant
domains (CH1, CH2 or CH3) and/or to the light chain constant domain
(CL). In some embodiments of the invention modified constant
regions wherein one or more domains are partially or entirely
deleted are contemplated. In some embodiments the modified
antibodies will comprise domain deleted constructs or variants
wherein the entire CH2 domain has been removed (.DELTA.CH2
constructs). In some embodiments the omitted constant region domain
will be replaced by a short amino acid spacer (e.g., 10 residues)
that provides some of the molecular flexibility typically imparted
by the absent constant region.
[0160] The present invention further embraces variants and
equivalents which are substantially homologous to the chimeric,
humanized and human antibodies, or antibody fragments thereof, set
forth herein. These can contain, for example, conservative
substitution mutations, i.e., the substitution of one or more amino
acids by similar amino acids. For example, conservative
substitution refers to the substitution of an amino acid with
another within the same general class such as, for example, one
acidic amino acid with another acidic amino acid, one basic amino
acid with another basic amino acid or one neutral amino acid by
another neutral amino acid. What is intended by a conservative
amino acid substitution is well known in the art.
[0161] The antibodies of the present invention can be assayed for
immunospecific binding by any method known in the art. The
immunoassays which can be used include, but are not limited to,
competitive and non-competitive assay systems using techniques such
as BIAcore analysis, FACS analysis, immunofluorescence,
immunocytochemistry, Western blots, radioimmunoassays, ELISA,
"sandwich" immunoassays, immunoprecipitation assays, precipitin
reactions, gel diffusion precipitin reactions, immunodiffusion
assays, agglutination assays, complement-fixation assays,
immunoradiometric assays, fluorescent immunoassays, protein A
immunoassays, and the like. Such assays are routine and well known
in the art (see, e.g., Ausubel et al, eds, 1994, Current Protocols
in Molecular Biology, Vol. 1, John Wiley & Sons, Inc., New
York, which is incorporated by reference herein in its
entirety).
[0162] In embodiments, the immunospecificity of an antibody against
a influenza HA is determined using ELISA. An ELISA assay comprises
preparing antigen, coating wells of a microtiter plate with
antigen, adding the antibody conjugated to a detectable compound
such as an enzymatic substrate (e.g., horseradish peroxidase or
alkaline phosphatase) to the well, incubating for a period of time
and detecting the presence of the antigen. In some embodiments, the
antibody is not conjugated to a detectable compound, but instead a
second conjugated antibody that recognizes the antibody against the
influenza HA antigen is added to the well. In some embodiments,
instead of coating the well with the antigen, the antibody can be
coated to the well and a second antibody conjugated to a detectable
compound can be added following the addition of the antigen to the
coated well. One of skill in the art would be knowledgeable as to
the parameters that can be modified to increase the signal detected
as well as other variations of ELISAs known in the art (see e.g.
Ausubel et al, eds, 1994, Current Protocols in Molecular Biology,
Vol. 1, John Wiley & Sons, Inc., New York at 11.2.1).
[0163] The binding affinity of an antibody to influenza HA and the
off-rate of an antibody-antigen interaction can be determined by
competitive binding assays. One example of a competitive binding
assay is a radioimmunoassay comprising the incubation of labeled
antigen (e.g. .sup.3H or .sup.125I), or fragment or variant
thereof, with the antibody of interest in the presence of
increasing amounts of unlabeled antigen followed by the detection
of the antibody bound to the labeled antigen. The affinity of the
antibody against an antigen and the binding off-rates can be
determined from the data by scatchard plot analysis. In some
embodiments, BIAcore kinetic analysis is used to determine the
binding on and off rates of antibodies against a cancer stem cell
marker. BIAcore kinetic analysis comprises analyzing the binding
and dissociation of antibodies from chips with immobilized cancer
stem cell marker antigens on their surface.
Influenza Hemagglutinin Antibody Polypeptides and
Polynucleotides
[0164] The present invention also encompasses isolated
polynucleotides that encode a polypeptide comprising an influenza
hemagglutinin (HA) antibody or fragment thereof.
[0165] The term "polynucleotide encoding a polypeptide" encompasses
a polynucleotide which includes only coding sequences for the
polypeptide as well as a polynucleotide which includes additional
coding and/or non-coding sequences. The polynucleotides of the
invention can be in the form of RNA or in the form of DNA. DNA
includes cDNA, genomic DNA, and synthetic DNA; and can be
double-stranded or single-stranded, and if single stranded can be
the coding strand or non-coding (anti-sense) strand.
[0166] The present invention further relates to variants of the
polynucleotides, for example, fragments, analogs, and derivatives.
The variant of the polynucleotide can be a naturally occurring
allelic variant of the polynucleotide or a non-naturally occurring
variant of the polynucleotide. In certain embodiments, the
polynucleotide can have a coding sequence which is a naturally
occurring allelic variant of the coding sequence of the disclosed
polypeptides. As known in the art, an allelic variant is an
alternate form of a polynucleotide sequence that have, for example,
a substitution, deletion, or addition of one or more nucleotides,
which does not substantially alter the function of the encoded
polypeptide.
[0167] In embodiments, the polynucleotides can comprise the coding
sequence for the mature polypeptide fused in the same reading frame
to a polynucleotide which aids, for example, in expression and
secretion of a polypeptide from a host cell (e.g., a leader
sequence which functions as a secretory sequence for controlling
transport of a polypeptide from the cell). The polypeptide having a
leader sequence is a preprotein and can have the leader sequence
cleaved by the host cell to form the mature form of the
polypeptide. The polynucleotides can also encode for a proprotein
which is the mature protein plus additional 5' amino acid residues.
A mature protein having a prosequence is a proprotein and is an
inactive form of the protein. Once the prosequence is cleaved an
active mature protein remains.
[0168] In embodiments, the polynucleotides can comprise the coding
sequence for the mature polypeptide fused in the same reading frame
to a marker sequence that allows, for example, for purification of
the encoded polypeptide. For example, the marker sequence can be a
hexa-histidine tag (SEQ ID NO: 46) supplied by a pQE-9 vector to
provide for purification of the mature polypeptide fused to the
marker in the case of a bacterial host, or the marker sequence can
be a hemagglutinin (HA) tag derived from the influenza
hemagglutinin protein when a mammalian host (e.g., COS-7 cells) is
used. Additional tags include, but are not limited to, Calmodulin
tags, FLAG tags, Myc tags, S tags, SBP tags, Softag 1, Softag 3, V5
tag, Xpress tag, Isopeptag, SpyTag, Biotin Carboxyl Carrier Protein
(BCCP) tags, GST tags, fluorescent protein tags (e.g., green
fluorescent protein tags), maltose binding protein tags, Nus tags,
Strep-tag, thioredoxin tag, TC tag, Ty tag, and the like.
[0169] In embodiments, the present invention provides isolated
nucleic acid molecules having a nucleotide sequence at least 80%
identical, at least 85% identical, at least 90% identical, at least
95% identical, or at least 96%, 97%, 98% or 99% identical to a
polynucleotide encoding a polypeptide comprising an influenza HA
antibody or antibody fragment of the present invention.
[0170] By a polynucleotide having a nucleotide sequence at least,
for example, 95% "identical" to a reference nucleotide sequence is
intended that the nucleotide sequence of the polynucleotide is
identical to the reference sequence except that the polynucleotide
sequence can include up to five point mutations per each 100
nucleotides of the reference nucleotide sequence. In other words,
to obtain a polynucleotide having a nucleotide sequence at least
95% identical to a reference nucleotide sequence, up to 5% of the
nucleotides in the reference sequence can be deleted or substituted
with another nucleotide, or a number of nucleotides up to 5% of the
total nucleotides in the reference sequence can be inserted into
the reference sequence. These mutations of the reference sequence
can occur at the amino- or carboxy-terminal positions of the
reference nucleotide sequence or anywhere between those terminal
positions, interspersed either individually among nucleotides in
the reference sequence or in one or more contiguous groups within
the reference sequence.
[0171] As a practical matter, whether any particular nucleic acid
molecule is at least 80% identical, at least 85% identical, at
least 90% identical, and in some embodiments, at least 95%, 96%,
97%, 98%, or 99% identical to a reference sequence can be
determined conventionally using known computer programs such as the
Bestfit program (Wisconsin Sequence Analysis Package, Version 8 for
Unix, Genetics Computer Group, University Research Park, 575
Science Drive, Madison, Wis. 53711). Bestfit uses the local
homology algorithm of Smith and Waterman, Advances in Applied
Mathematics 2: 482 489 (1981), to find the best segment of homology
between two sequences. When using Bestfit or any other sequence
alignment program to determine whether a particular sequence is,
for instance, 95% identical to a reference sequence according to
the present invention, the parameters are set such that the
percentage of identity is calculated over the full length of the
reference nucleotide sequence and that gaps in homology of up to 5%
of the total number of nucleotides in the reference sequence are
allowed.
[0172] The polynucleotide variants can contain alterations in the
coding regions, non-coding regions, or both. In some embodiments,
the polynucleotide variants contain alterations which produce
silent substitutions, additions, or deletions, but do not alter the
properties or activities of the encoded polypeptide. In some
embodiments, nucleotide variants are produced by silent
substitutions due to the degeneracy of the genetic code.
Polynucleotide variants can be produced for a variety of reasons,
e.g., to optimize codon expression for a particular host (change
codons in the human mRNA to those preferred by a bacterial host
such as E. coli).
[0173] The polypeptides of the present invention can be recombinant
polypeptides, natural polypeptides, or synthetic polypeptides
comprising an antibody, or fragment thereof, against influenza HA.
It will be recognized in the art that some amino acid sequences of
the invention can be varied without significant effect of the
structure or function of the protein. Thus, the invention further
includes variations of the polypeptides which show substantial
activity or which include regions of a humanized antibody, or
fragment thereof, against influenza HA. Such mutants include
deletions, insertions, inversions, repeats, and type
substitutions.
[0174] The polypeptides and analogs can be further modified to
contain additional chemical moieties not normally part of the
protein. Those derivatized moieties can improve the solubility, the
biological half life or absorption of the protein. The moieties can
also reduce or eliminate any desirable side effects of the proteins
and the like. An overview for those moieties can be found in
Remington's Pharmaceutical Sciences, 20th ed., Mack Publishing Co.,
Easton, Pa. (2000).
[0175] The isolated polypeptides described herein can be produced
by any suitable method known in the art. Such methods range from
direct protein synthetic methods to constructing a DNA sequence
encoding isolated polypeptide sequences and expressing those
sequences in a suitable transformed host. In some embodiments, a
DNA sequence is constructed using recombinant technology by
isolating or synthesizing a DNA sequence encoding a wild-type
protein of interest. Optionally, the sequence can be mutagenized by
site-specific mutagenesis to provide functional analogs thereof.
See, e.g. Zoeller et al., Proc. Nat'l. Acad. Sci. USA 81:5662-5066
(1984) and U.S. Pat. No. 4,588,585.
[0176] In embodiments, a DNA sequence encoding a polypeptide of
interest would be constructed by chemical synthesis using an
oligonucleotide synthesizer. Such oligonucleotides can be designed
based on the amino acid sequence of the desired polypeptide and
selecting those codons that are favored in the host cell in which
the recombinant polypeptide of interest will be produced. Standard
methods can be applied to synthesize an isolated polynucleotide
sequence encoding an isolated polypeptide of interest. For example,
a complete amino acid sequence can be used to construct a
back-translated gene. Further, a DNA oligomer containing a
nucleotide sequence coding for the particular isolated polypeptide
can be synthesized. For example, several small oligonucleotides
coding for portions of the desired polypeptide can be synthesized
and then ligated. The individual oligonucleotides typically contain
5' or 3' overhangs for complementary assembly.
[0177] Once assembled (e.g., by synthesis, site-directed
mutagenesis, or another method), the polynucleotide sequences
encoding a particular isolated polypeptide of interest will be
inserted into an expression vector and optionally operatively
linked to an expression control sequence appropriate for expression
of the protein in a desired host. Proper assembly can be confirmed
by nucleotide sequencing, restriction mapping, and expression of a
biologically active polypeptide in a suitable host. As well known
in the art, in order to obtain high expression levels of a
transfected gene in a host, the gene can be operatively linked to
transcriptional and translational expression control sequences that
are functional in the chosen expression host.
[0178] Recombinant expression vectors are used to amplify and
express DNA encoding the influenza HA antibodies. Recombinant
expression vectors are replicable DNA constructs which have
synthetic or cDNA-derived DNA fragments encoding an influenza HA
antibody or a bioequivalent analog operatively linked to suitable
transcriptional or translational regulatory elements derived from
mammalian, microbial, viral or insect genes. A transcriptional unit
generally comprises an assembly of (1) a genetic element or
elements having a regulatory role in gene expression, for example,
transcriptional promoters or enhancers, (2) a structural or coding
sequence which is transcribed into mRNA and translated into
protein, and (3) appropriate transcription and translation
initiation and termination sequences, as described in detail below.
Such regulatory elements can include an operator sequence to
control transcription. The ability to replicate in a host, usually
conferred by an origin of replication, and a selection gene to
facilitate recognition of transformants can additionally be
incorporated. DNA regions are operatively linked when they are
functionally related to each other. For example, DNA for a signal
peptide (secretory leader) is operatively linked to DNA for a
polypeptide if it is expressed as a precursor which participates in
the secretion of the polypeptide; a promoter is operatively linked
to a coding sequence if it controls the transcription of the
sequence; or a ribosome binding site is operatively linked to a
coding sequence if it is positioned so as to permit translation.
Generally, operatively linked means contiguous, and in the case of
secretory leaders, means contiguous and in reading frame.
Structural elements intended for use in yeast expression systems
include a leader sequence enabling extracellular secretion of
translated protein by a host cell. Alternatively, where recombinant
protein is expressed without a leader or transport sequence, it can
include an N-terminal methionine residue. This residue can
optionally be subsequently cleaved from the expressed recombinant
protein to provide a final product.
[0179] The choice of expression control sequence and expression
vector will depend upon the choice of host. A wide variety of
expression host/vector combinations can be employed. Useful
expression vectors for eukaryotic hosts, include, for example,
vectors comprising expression control sequences from SV40, bovine
papilloma virus, adenovirus and cytomegalovirus. Useful expression
vectors for bacterial hosts include known bacterial plasmids, such
as plasmids from Escherichia coli, including pCR 1, pBR322, pMB9
and their derivatives, wider host range plasmids, such as M13 and
filamentous single-stranded DNA phages.
[0180] Suitable host cells for expression of a polypeptide include
prokaryotes, yeast, insect or higher eukaryotic cells under the
control of appropriate promoters. Prokaryotes include gram negative
or gram positive organisms, for example E. coli or bacilli. Higher
eukaryotic cells include established cell lines of mammalian
origin. Cell-free translation systems could also be employed.
Appropriate cloning and expression vectors for use with bacterial,
fungal, yeast, and mammalian cellular hosts are well known in the
art (see Pouwels et al., Cloning Vectors: A Laboratory Manual,
Elsevier, N.Y., 1985).
[0181] Various mammalian or insect cell culture systems are also
advantageously employed to express recombinant protein. Expression
of recombinant proteins in mammalian cells can be performed because
such proteins are generally correctly folded, appropriately
modified and completely functional. Examples of suitable mammalian
host cell lines include the COS-7 lines of monkey kidney cells,
described by Gluzman (Cell 23:175, 1981), and other cell lines
capable of expressing an appropriate vector including, for example,
L cells, C127, 3T3, Chinese hamster ovary (CHO), HeLa and BHK cell
lines. Mammalian expression vectors can comprise nontranscribed
elements such as an origin of replication, a suitable promoter and
enhancer linked to the gene to be expressed, and other 5' or 3'
flanking nontranscribed sequences, and 5' or 3' nontranslated
sequences, such as necessary ribosome binding sites, a
polyadenylation site, splice donor and acceptor sites, and
transcriptional termination sequences. Baculovirus systems for
production of heterologous proteins in insect cells are reviewed by
Luckow and Summers, Bio/Technology 6:47 (1988).
[0182] The proteins produced by a transformed host can be purified
according to any suitable method. Such standard methods include
chromatography (e.g., ion exchange, affinity and sizing column
chromatography, and the like), centrifugation, differential
solubility, or by any other standard technique for protein
purification. Affinity tags such as hexahistidine, maltose binding
domain, influenza coat sequence, glutathione-S-transferase, and the
like can be attached to the protein to allow easy purification by
passage over an appropriate affinity column. Isolated proteins can
also be physically characterized using such techniques as
proteolysis, nuclear magnetic resonance and x-ray
crystallography.
[0183] For example, supernatants from systems which secrete
recombinant protein into culture media can be first concentrated
using a commercially available protein concentration filter, for
example, an Amicon or Millipore Pellicon ultrafiltration unit.
Following the concentration step, the concentrate can be applied to
a suitable purification matrix. Alternatively, an anion exchange
resin can be employed, for example, a matrix or substrate having
pendant diethylaminoethyl (DEAE) groups. The matrices can be
acrylamide, agarose, dextran, cellulose or other types commonly
employed in protein purification. Alternatively, a cation exchange
step can be employed. Suitable cation exchangers include various
insoluble matrices comprising sulfopropyl or carboxymethyl groups.
Finally, one or more reversed-phase high performance liquid
chromatography (RP-HPLC) steps employing hydrophobic RP-HPLC media,
e.g., silica gel having pendant methyl or other aliphatic groups,
can be employed to further purify a cancer stem cell protein-Fc
composition. Some or all of the foregoing purification steps, in
various combinations, can also be employed to provide a homogeneous
recombinant protein.
[0184] Recombinant protein produced in bacterial culture can be
isolated, for example, by initial extraction from cell pellets,
followed by one or more concentration, salting-out, aqueous ion
exchange or size exclusion chromatography steps. High performance
liquid chromatography (HPLC) can be employed for final purification
steps. Microbial cells employed in expression of a recombinant
protein can be disrupted by any convenient method, including
freeze-thaw cycling, sonication, mechanical disruption, or use of
cell lysing agents.
Methods of Treatment
[0185] The present invention provides methods for treating or
preventing influenza infection.
[0186] In aspects, the invention provides methods for treating or
preventing an influenza virus infection in a subject in need
thereof. The methods involve administering to the subject an
effective amount of an anti-influenza antibody or antibody fragment
described herein, or a pharmaceutical composition containing the
antibody or antibody fragment. The methods treat or prevent
influenza virus infection in the subject, including reducing or
alleviating symptoms associated with infection.
[0187] In aspects, the invention provides methods for neutralizing
an influenza virus in a subject in need thereof. The methods
involve administering to the subject an effective amount of an
anti-influenza antibody or antibody fragment described herein, or a
pharmaceutical composition containing the antibody or antibody
fragment. The methods neutralize the influenza virus in the
subject, thereby treating or prevent influenza virus infection in
the subject, including reducing or alleviating symptoms associated
with infection.
[0188] In aspects, the invention provides methods for establishment
of influenza virus infection in a subject in need thereof. The
methods involve administering to the subject an effective amount of
an anti-influenza antibody or antibody fragment described herein,
or a pharmaceutical composition containing the antibody or antibody
fragment. The methods inhibit establishment of influenza virus
infection in the subject, thereby preventing symptoms associated
with infection.
[0189] In aspects, the invention provides methods for inhibiting
dissemination of influenza virus infection in a subject in need
thereof. The methods involve administering to the subject an
effective amount of an anti-influenza antibody or antibody fragment
described herein, or a pharmaceutical composition containing the
antibody or antibody fragment. The methods inhibit dissemination of
influenza virus infection in the subject, thereby reducing or
alleviating symptoms associated with infection.
[0190] In aspects, the invention provides methods for inhibiting
influenza virus entry into a cell in a subject. The methods involve
administering to the subject an effective amount of an
anti-influenza antibody or antibody fragment described herein, or a
pharmaceutical composition containing the antibody or antibody
fragment. The methods inhibit influenza virus entry into a cell in
the subject, thereby preventing symptoms associated with infection
or reducing or alleviating symptoms associated with infection.
[0191] In aspects, the invention provides methods for inhibiting
influenza virus entry into a cell. The methods involve contacting a
cell having or at risk of developing influenza virus infection with
an anti-influenza antibody or antibody fragment described herein,
or a pharmaceutical composition containing the antibody or antibody
fragment. The methods inhibit influenza virus entry into the
cell.
[0192] In any of the above aspects, the influenza can be H1N1,
H2N2, H3N2, or a human adapted H5 strain.
[0193] In any of the above aspects, the subject has or is at risk
of developing an influenza infection. In related embodiments, the
subject is a mammal (e.g., human). In related embodiments, the
subject is susceptible to viral infection (e.g., a pregnant female,
a young subject or an infant subject, an elderly subject).
[0194] In any of the above aspects and embodiments, the
anti-influenza antibody or antibody fragment, or the pharmaceutical
composition is administered by intramuscular injection, intravenous
injection, subcutaneous injection, or inhalation.
Pharmaceutical Compositions
[0195] The invention provides for pharmaceutical compositions
containing the novel influenza HA antibodies described herein. In
embodiments, the pharmaceutical compositions contain a
pharmaceutically acceptable carrier, excipient, or diluent, which
includes any pharmaceutical agent that does not itself induce the
production of an immune response harmful to a subject receiving the
composition, and which may be administered without undue toxicity.
As used herein, the term "pharmaceutically acceptable" means being
approved by a regulatory agency of the Federal or a state
government or listed in the U.S. Pharmacopia, European Pharmacopia
or other generally recognized pharmacopia for use in mammals, and
more particularly in humans. These compositions can be useful for
treating and/or preventing influenza infection.
[0196] A thorough discussion of pharmaceutically acceptable
carriers, diluents, and other excipients is presented in
Remington's Pharmaceutical Sciences (17th ed., Mack Publishing
Company) and Remington: The Science and Practice of Pharmacy (21st
ed., Lippincott Williams & Wilkins), which are hereby
incorporated by reference. The formulation of the pharmaceutical
composition should suit the mode of administration. In embodiments,
the pharmaceutical composition is suitable for administration to
humans, and can be sterile, non-particulate and/or
non-pyrogenic.
[0197] Pharmaceutically acceptable carriers, excipients, or
diluents include, but are not limited, to saline, buffered saline,
dextrose, water, glycerol, ethanol, sterile isotonic aqueous
buffer, and combinations thereof.
[0198] Wetting agents, emulsifiers and lubricants, such as sodium
lauryl sulfate and magnesium stearate, as well as coloring agents,
release agents, coating agents, sweetening, flavoring and perfuming
agents, preservatives, and antioxidants can also be present in the
compositions.
[0199] Examples of pharmaceutically-acceptable antioxidants
include, but are not limited to: (1) water soluble antioxidants,
such as ascorbic acid, cysteine hydrochloride, sodium bisulfate,
sodium metabisulfite, sodium sulfite and the like; (2) oil-soluble
antioxidants, such as ascorbyl palmitate, butylated hydroxyanisole
(BHA), butylated hydroxytoluene (BHT), lecithin, propyl gallate,
alpha-tocopherol, and the like; and (3) metal chelating agents,
such as citric acid, ethylenediamine tetraacetic acid (EDTA),
sorbitol, tartaric acid, phosphoric acid, and the like.
[0200] In embodiments, the pharmaceutical composition is provided
in a solid form, such as a lyophilized powder suitable for
reconstitution, a liquid solution, suspension, emulsion, tablet,
pill, capsule, sustained release formulation, or powder.
[0201] In embodiments, the pharmaceutical composition is supplied
in liquid form, for example, in a sealed container indicating the
quantity and concentration of the active ingredient in the
pharmaceutical composition. In related embodiments, the liquid form
of the pharmaceutical composition is supplied in a hermetically
sealed container.
[0202] Methods for formulating the pharmaceutical compositions of
the present invention are conventional and well-known in the art
(see Remington and Remington's). One of skill in the art can
readily formulate a pharmaceutical composition having the desired
characteristics (e.g., route of administration, biosafety, and
release profile).
[0203] Methods for preparing the pharmaceutical compositions
include the step of bringing into association the active ingredient
with a pharmaceutically acceptable carrier and, optionally, one or
more accessory ingredients. The pharmaceutical compositions can be
prepared by uniformly and intimately bringing into association the
active ingredient with liquid carriers, or finely divided solid
carriers, or both, and then, if necessary, shaping the product.
Additional methodology for preparing the pharmaceutical
compositions, including the preparation of multilayer dosage forms,
are described in Ansel's Pharmaceutical Dosage Forms and Drug
Delivery Systems (9th ed., Lippincott Williams & Wilkins),
which is hereby incorporated by reference.
Methods of Delivery
[0204] The pharmaceutical compositions of the invention can be
administered to a subject by oral and non-oral means (e.g.,
topically, transdermally, or by injection). Such modes of
administration and the methods for preparing an appropriate
pharmaceutical composition for use therein are described in
Gibaldi's Drug Delivery Systems in Pharmaceutical Care (1st ed.,
American Society of Health-System Pharmacists), which is hereby
incorporated by reference.
[0205] In embodiments, the pharmaceutical compositions are
administered orally in a solid form.
[0206] Pharmaceutical compositions suitable for oral administration
can be in the form of capsules, cachets, pills, tablets, lozenges
(using a flavored basis, usually sucrose and acacia or tragacanth),
powders, granules, or as a solution or a suspension in an aqueous
or non-aqueous liquid, or as an oil-in-water or water-in-oil liquid
emulsion, or as an elixir or syrup, or as pastilles (using an inert
base, such as gelatin and glycerin, or sucrose and acacia) and/or
as mouth washes and the like, each containing a predetermined
amount of a compound(s) described herein, a derivative thereof, or
a pharmaceutically acceptable salt or prodrug thereof as the active
ingredient(s). The active ingredient can also be administered as a
bolus, electuary, or paste.
[0207] In solid dosage forms for oral administration (e.g.,
capsules, tablets, pills, dragees, powders, granules and the like),
the active ingredient is mixed with one or more pharmaceutically
acceptable carriers, excipients, or diluents, such as sodium
citrate or dicalcium phosphate, and/or any of the following: (1)
fillers or extenders, such as starches, lactose, sucrose, glucose,
mannitol, and/or silicic acid; (2) binders, such as, for example,
carboxymethylcellulose, alginates, gelatin, polyvinyl pyrrolidone,
sucrose and/or acacia; (3) humectants, such as glycerol; (4)
disintegrating agents, such as agar-agar, calcium carbonate, potato
or tapioca starch, alginic acid, certain silicates, and sodium
carbonate; (5) solution retarding agents, such as paraffin; (6)
absorption accelerators, such as quaternary ammonium compounds; (7)
wetting agents, such as, for example, acetyl alcohol and glycerol
monostearate; (8) absorbents, such as kaolin and bentonite clay;
(9) lubricants, such a talc, calcium stearate, magnesium stearate,
solid polyethylene glycols, sodium lauryl sulfate, and mixtures
thereof; and (10) coloring agents. In the case of capsules,
tablets, and pills, the pharmaceutical compositions can also
comprise buffering agents. Solid compositions of a similar type can
also be prepared using fillers in soft and hard-filled gelatin
capsules, and excipients such as lactose or milk sugars, as well as
high molecular weight polyethylene glycols and the like.
[0208] A tablet can be made by compression or molding, optionally
with one or more accessory ingredients. Compressed tablets can be
prepared using binders (for example, gelatin or hydroxypropylmethyl
cellulose), lubricants, inert diluents, preservatives,
disintegrants (for example, sodium starch glycolate or cross-linked
sodium carboxymethyl cellulose), surface-actives, and/or dispersing
agents. Molded tablets can be made by molding in a suitable machine
a mixture of the powdered active ingredient moistened with an inert
liquid diluent.
[0209] The tablets and other solid dosage forms, such as dragees,
capsules, pills, and granules, can optionally be scored or prepared
with coatings and shells, such as enteric coatings and other
coatings well-known in the art.
[0210] The pharmaceutical compositions can also be formulated so as
to provide slow, extended, or controlled release of the active
ingredient therein using, for example, hydroxypropylmethyl
cellulose in varying proportions to provide the desired release
profile, other polymer matrices, liposomes and/or microspheres. The
pharmaceutical compositions can also optionally contain opacifying
agents and may be of a composition that releases the active
ingredient(s) only, or preferentially, in a certain portion of the
gastrointestinal tract, optionally, in a delayed manner. Examples
of embedding compositions include polymeric substances and waxes.
The active ingredient can also be in micro-encapsulated form, if
appropriate, with one or more pharmaceutically acceptable carriers,
excipients, or diluents well-known in the art (see, e.g., Remington
and Remington's).
[0211] The pharmaceutical compositions can be sterilized by, for
example, filtration through a bacteria-retaining filter, or by
incorporating sterilizing agents in the form of sterile solid
compositions which can be dissolved in sterile water, or some other
sterile injectable medium immediately before use.
[0212] In embodiments, the pharmaceutical compositions are
administered orally in a liquid form.
[0213] Liquid dosage forms for oral administration of an active
ingredient include pharmaceutically acceptable emulsions,
microemulsions, solutions, suspensions, syrups and elixirs. In
addition to the active ingredient, the liquid dosage forms can
contain inert diluents commonly used in the art, such as, for
example, water or other solvents, solubilizing agents and
emulsifiers, such as ethyl alcohol, isopropyl alcohol, ethyl
carbonate, ethyl acetate, benzyl alcohol, benzyl benzoate,
propylene glycol, 1,3-butylene glycol, oils (in particular,
cottonseed, groundnut, corn, germ, olive, castor and sesame oils),
glycerol, tetrahydrofuryl alcohol, polyethylene glycols and fatty
acid esters of sorbitan, and mixtures thereof. In addition to inert
diluents, the liquid pharmaceutical compositions can include
adjuvants such as wetting agents, emulsifying and suspending
agents, sweetening, flavoring, coloring, perfuming and preservative
agents, and the like.
[0214] Suspensions, in addition to the active ingredient(s) can
contain suspending agents such as, but not limited to, ethoxylated
isostearyl alcohols, polyoxyethylene sorbitol and sorbitan esters,
microcrystalline cellulose, aluminum metahydroxide, bentonite,
agar-agar and tragacanth, and mixtures thereof.
[0215] In embodiments, the pharmaceutical compositions are
administered by non-oral means such as by topical application,
transdermal application, injection, and the like. In related
embodiments, the pharmaceutical compositions are administered
parenterally by injection, infusion, or implantation (e.g.,
intravenous, intramuscular, intraarticular, subcutaneous, and the
like).
[0216] Compositions for parenteral use can be presented in unit
dosage forms, e.g. in ampoules or in vials containing several
doses, and in which a suitable preservative can be added. Such
compositions can be in form of a solution, a suspension, an
emulsion, an infusion device, a delivery device for implantation,
or it can be presented as a dry powder to be reconstituted with
water or another suitable vehicle before use. One or more
co-vehicles, such as ethanol, can also be employed. Apart from the
active ingredient(s), the compositions can contain suitable
parenterally acceptable carriers and/or excipients or the active
ingredient(s) can be incorporated into microspheres, microcapsules,
nanoparticles, liposomes, or the like for controlled release.
Furthermore, the compositions can also contain suspending,
solubilising, stabilising, pH-adjusting agents, and/or dispersing
agents.
[0217] The pharmaceutical compositions can be in the form of
sterile injections. To prepare such a composition, the active
ingredient is dissolved or suspended in a parenterally acceptable
liquid vehicle. Exemplary vehicles and solvents include, but are
not limited to, water, water adjusted to a suitable pH by addition
of an appropriate amount of hydrochloric acid, sodium hydroxide or
a suitable buffer, 1,3-butanediol, Ringer's solution and isotonic
sodium chloride solution. The pharmaceutical composition can also
contain one or more preservatives, for example, methyl, ethyl or
n-propyl p-hydroxybenzoate. To improve solubility, a dissolution
enhancing or solubilising agent can be added or the solvent can
contain 10-60% w/w of propylene glycol or the like.
[0218] The pharmaceutical compositions can contain one or more
pharmaceutically acceptable sterile isotonic aqueous or nonaqueous
solutions, dispersions, suspensions or emulsions, or sterile
powders, which can be reconstituted into sterile injectable
solutions or dispersions just prior to use. Such pharmaceutical
compositions can contain antioxidants; buffers; bacteriostats;
solutes, which render the formulation isotonic with the blood of
the intended recipient; suspending agents; thickening agents;
preservatives; and the like.
[0219] Examples of suitable aqueous and nonaqueous carriers, which
can be employed in the pharmaceutical compositions of the invention
include water, ethanol, polyols (such as glycerol, propylene
glycol, polyethylene glycol, and the like), and suitable mixtures
thereof, vegetable oils, such as olive oil, and injectable organic
esters, such as ethyl oleate. Proper fluidity can be maintained,
for example, by the use of coating materials, such as lecithin, by
the maintenance of the required particle size in the case of
dispersions, and by the use of surfactants.
[0220] In some embodiments, in order to prolong the effect of an
active ingredient, it is desirable to slow the absorption of the
compound from subcutaneous or intramuscular injection. This can be
accomplished by the use of a liquid suspension of crystalline or
amorphous material having poor water solubility. The rate of
absorption of the active ingredient then depends upon its rate of
dissolution which, in turn, can depend upon crystal size and
crystalline form. Alternatively, delayed absorption of a
parenterally-administered active ingredient is accomplished by
dissolving or suspending the compound in an oil vehicle. In
addition, prolonged absorption of the injectable pharmaceutical
form can be brought about by the inclusion of agents that delay
absorption such as aluminum monostearate and gelatin.
[0221] Controlled release parenteral compositions can be in form of
aqueous suspensions, microspheres, microcapsules, magnetic
microspheres, oil solutions, oil suspensions, emulsions, or the
active ingredient can be incorporated in biocompatible carrier(s),
liposomes, nanoparticles, implants or infusion devices.
[0222] Materials for use in the preparation of microspheres and/or
microcapsules include biodegradable/bioerodible polymers such as
polyglactin, poly-(isobutyl cyanoacrylate),
poly(2-hydroxyethyl-L-glutamine) and poly(lactic acid).
[0223] Biocompatible carriers which can be used when formulating a
controlled release parenteral formulation include carbohydrates
such as dextrans, proteins such as albumin, lipoproteins or
antibodies.
[0224] Materials for use in implants can be non-biodegradable,
e.g., polydimethylsiloxane, or biodegradable such as, e.g.,
poly(caprolactone), poly(lactic acid), poly(glycolic acid) or
poly(ortho esters).
[0225] In embodiments, the active ingredient(s) are administered by
aerosol. This is accomplished by preparing an aqueous aerosol,
liposomal preparation, or solid particles containing the compound.
A nonaqueous (e.g., fluorocarbon propellant) suspension can be
used. The pharmaceutical composition can also be administered using
a sonic nebulizer, which would minimize exposing the agent to
shear, which can result in degradation of the compound.
[0226] Ordinarily, an aqueous aerosol is made by formulating an
aqueous solution or suspension of the active ingredient(s) together
with conventional pharmaceutically-acceptable carriers and
stabilizers. The carriers and stabilizers vary with the
requirements of the particular compound, but typically include
nonionic surfactants (Tweens, Pluronics, or polyethylene glycol),
innocuous proteins like serum albumin, sorbitan esters, oleic acid,
lecithin, amino acids such as glycine, buffers, salts, sugars or
sugar alcohols. Aerosols generally are prepared from isotonic
solutions.
[0227] Dosage forms for topical or transdermal administration of an
active ingredient(s) includes powders, sprays, ointments, pastes,
creams, lotions, gels, solutions, patches and inhalants. The active
ingredient(s) can be mixed under sterile conditions with a
pharmaceutically acceptable carrier, and with any preservatives,
buffers, or propellants as appropriate.
[0228] Transdermal patches suitable for use in the present
invention are disclosed in Transdermal Drug Delivery: Developmental
Issues and Research Initiatives (Marcel Dekker Inc., 1989) and U.S.
Pat. Nos. 4,743,249, 4,906,169, 5,198,223, 4,816,540, 5,422,119,
5,023,084, which are hereby incorporated by reference. The
transdermal patch can also be any transdermal patch well-known in
the art, including transscrotal patches. Pharmaceutical
compositions in such transdermal patches can contain one or more
absorption enhancers or skin permeation enhancers well-known in the
art (see, e.g., U.S. Pat. Nos. 4,379,454 and 4,973,468, which are
hereby incorporated by reference). Transdermal therapeutic systems
for use in the present invention can be based on iontophoresis,
diffusion, or a combination of these two effects.
[0229] Transdermal patches have the added advantage of providing
controlled delivery of active ingredient(s) to the body. Such
dosage forms can be made by dissolving or dispersing the active
ingredient(s) in a proper medium. Absorption enhancers can also be
used to increase the flux of the active ingredient across the skin.
The rate of such flux can be controlled by either providing a rate
controlling membrane or dispersing the active ingredient(s) in a
polymer matrix or gel.
[0230] Such pharmaceutical compositions can be in the form of
creams, ointments, lotions, liniments, gels, hydrogels, solutions,
suspensions, sticks, sprays, pastes, plasters and other kinds of
transdermal drug delivery systems. The compositions can also
include pharmaceutically acceptable carriers or excipients such as
emulsifying agents, antioxidants, buffering agents, preservatives,
humectants, penetration enhancers, chelating agents, gel-forming
agents, ointment bases, perfumes, and skin protective agents.
[0231] Examples of emulsifying agents include, but are not limited
to, naturally occurring gums, e.g. gum acacia or gum tragacanth,
naturally occurring phosphatides, e.g. soybean lecithin and
sorbitan monooleate derivatives.
[0232] Examples of antioxidants include, but are not limited to,
butylated hydroxy anisole (BHA), ascorbic acid and derivatives
thereof, tocopherol and derivatives thereof, and cysteine.
[0233] Examples of preservatives include, but are not limited to,
parabens, such as methyl or propyl p-hydroxybenzoate and
benzalkonium chloride.
[0234] Examples of humectants include, but are not limited to,
glycerin, propylene glycol, sorbitol and urea.
[0235] Examples of penetration enhancers include, but are not
limited to, propylene glycol, DMSO, triethanolamine,
N,N-dimethylacetamide, N,N-dimethylformamide, 2-pyrrolidone and
derivatives thereof, tetrahydrofurfuryl alcohol, propylene glycol,
diethylene glycol monoethyl or monomethyl ether with propylene
glycol monolaurate or methyl laurate, eucalyptol, lecithin,
Transcutol, and Azone.RTM..
[0236] Examples of chelating agents include, but are not limited
to, sodium EDTA, citric acid and phosphoric acid.
[0237] Examples of gel forming agents include, but are not limited
to, Carbopol, cellulose derivatives, bentonite, alginates, gelatin
and polyvinylpyrrolidone.
[0238] In addition to the active ingredient(s), the ointments,
pastes, creams, and gels of the present invention can contain
excipients, such as animal and vegetable fats, oils, waxes,
paraffins, starch, tragacanth, cellulose derivatives, polyethylene
glycols, silicones, bentonites, silicic acid, talc and zinc oxide,
or mixtures thereof.
[0239] Powders and sprays can contain excipients such as lactose,
talc, silicic acid, aluminum hydroxide, calcium silicates and
polyamide powder, or mixtures of these substances. Sprays can
additionally contain customary propellants, such as
chlorofluorohydrocarbons, and volatile unsubstituted hydrocarbons,
such as butane and propane.
[0240] Injectable depot forms are made by forming microencapsule
matrices of compound(s) of the invention in biodegradable polymers
such as polylactide-polyglycolide. Depending on the ratio of
compound to polymer, and the nature of the particular polymer
employed, the rate of compound release can be controlled. Examples
of other biodegradable polymers include poly(orthoesters) and
poly(anhydrides). Depot injectable formulations are also prepared
by entrapping the drug in liposomes or microemulsions which are
compatible with body tissue.
[0241] Subcutaneous implants are well-known in the art and are
suitable for use in the present invention. Subcutaneous
implantation methods are preferably non-irritating and mechanically
resilient. The implants can be of matrix type, of reservoir type,
or hybrids thereof. In matrix type devices, the carrier material
can be porous or non-porous, solid or semi-solid, and permeable or
impermeable to the active compound or compounds. The carrier
material can be biodegradable or may slowly erode after
administration. In some instances, the matrix is non-degradable but
instead relies on the diffusion of the active compound through the
matrix for the carrier material to degrade. Alternative
subcutaneous implant methods utilize reservoir devices where the
active compound or compounds are surrounded by a rate controlling
membrane, e.g., a membrane independent of component concentration
(possessing zero-order kinetics). Devices consisting of a matrix
surrounded by a rate controlling membrane also suitable for
use.
[0242] Both reservoir and matrix type devices can contain materials
such as polydimethylsiloxane, such as Silastic.TM., or other
silicone rubbers. Matrix materials can be insoluble polypropylene,
polyethylene, polyvinyl chloride, ethylvinyl acetate, polystyrene
and polymethacrylate, as well as glycerol esters of the glycerol
palmitostearate, glycerol stearate, and glycerol behenate type.
Materials can be hydrophobic or hydrophilic polymers and optionally
contain solubilising agents.
[0243] Subcutaneous implant devices can be slow-release capsules
made with any suitable polymer, e.g., as described in U.S. Pat.
Nos. 5,035,891 and 4,210,644, which are hereby incorporated by
reference.
[0244] In general, at least four different approaches are
applicable in order to provide rate control over the release and
transdermal permeation of a drug compound. These approaches are:
membrane-moderated systems, adhesive diffusion-controlled systems,
matrix dispersion-type systems and microreservoir systems. It is
appreciated that a controlled release percutaneous and/or topical
composition can be obtained by using a suitable mixture of these
approaches.
[0245] In a membrane-moderated system, the active ingredient is
present in a reservoir which is totally encapsulated in a shallow
compartment molded from a drug-impermeable laminate, such as a
metallic plastic laminate, and a rate-controlling polymeric
membrane such as a microporous or a non-porous polymeric membrane,
e.g., ethylene-vinyl acetate copolymer. The active ingredient is
released through the rate controlling polymeric membrane. In the
drug reservoir, the active ingredient can either be dispersed in a
solid polymer matrix or suspended in an unleachable, viscous liquid
medium such as silicone fluid. On the external surface of the
polymeric membrane, a thin layer of an adhesive polymer is applied
to achieve an intimate contact of the transdermal system with the
skin surface. The adhesive polymer is preferably a polymer which is
hypoallergenic and compatible with the active drug substance.
[0246] In an adhesive diffusion-controlled system, a reservoir of
the active ingredient is formed by directly dispersing the active
ingredient in an adhesive polymer and then by, e.g., solvent
casting, spreading the adhesive containing the active ingredient
onto a flat sheet of substantially drug-impermeable metallic
plastic backing to form a thin drug reservoir layer.
[0247] A matrix dispersion-type system is characterized in that a
reservoir of the active ingredient is formed by substantially
homogeneously dispersing the active ingredient in a hydrophilic or
lipophilic polymer matrix. The drug-containing polymer is then
molded into disc with a substantially well-defined surface area and
controlled thickness. The adhesive polymer is spread along the
circumference to form a strip of adhesive around the disc.
[0248] A microreservoir system can be considered as a combination
of the reservoir and matrix dispersion type systems. In this case,
the reservoir of the active substance is formed by first suspending
the drug solids in an aqueous solution of water-soluble polymer and
then dispersing the drug suspension in a lipophilic polymer to form
a multiplicity of unleachable, microscopic spheres of drug
reservoirs.
[0249] Any of the above-described controlled release, extended
release, and sustained release compositions can be formulated to
release the active ingredient in about 30 minutes to about 1 week,
in about 30 minutes to about 72 hours, in about 30 minutes to 24
hours, in about 30 minutes to 12 hours, in about 30 minutes to 6
hours, in about 30 minutes to 4 hours, and in about 3 hours to 10
hours. In embodiments, an effective concentration of the active
ingredient(s) is sustained in a subject for 4 hours, 6 hours, 8
hours, 10 hours, 12 hours, 16 hours, 24 hours, 48 hours, 72 hours,
or more after administration of the pharmaceutical compositions to
the subject.
Dosages
[0250] When the agents described herein are administered as
pharmaceuticals to humans and animals, they can be given per se or
as a pharmaceutical composition containing active ingredient in
combination with a pharmaceutically acceptable carrier, excipient,
or diluent.
[0251] Actual dosage levels and time course of administration of
the active ingredients in the pharmaceutical compositions of the
invention can be varied so as to obtain an amount of the active
ingredient which is effective to achieve the desired therapeutic
response for a particular patient, composition, and mode of
administration, without being toxic to the patient. Generally,
agents or pharmaceutical compositions of the invention are
administered in an amount sufficient to reduce or eliminate
symptoms associated with influenza infection.
[0252] Exemplary dose ranges include 0.01 mg to 250 mg per day,
0.01 mg to 100 mg per day, 1 mg to 100 mg per day, 10 mg to 100 mg
per day, 1 mg to 10 mg per day, and 0.01 mg to 10 mg per day. A
preferred dose of an agent is the maximum that a patient can
tolerate and not develop serious or unacceptable side effects. In
embodiments, the agent is administered at a concentration of about
10 micrograms to about 100 mg per kilogram of body weight per day,
about 0.1 to about 10 mg/kg per day, or about 1.0 mg to about 10
mg/kg of body weight per day.
[0253] In embodiments, the pharmaceutical composition comprises an
agent in an amount ranging between 1 and 10 mg, such as 1, 2, 3, 4,
5, 6, 7, 8, 9, or 10 mg.
[0254] In embodiments, the therapeutically effective dosage
produces a serum concentration of an agent of from about 0.1 ng/ml
to about 50-100 .mu.g/ml. The pharmaceutical compositions typically
should provide a dosage of from about 0.001 mg to about 2000 mg of
compound per kilogram of body weight per day. For example, dosages
for systemic administration to a human patient can range from 1-10
.mu.g/kg, 20-80 .mu.g/kg, 5-50 .mu.g/kg, 75-150 .mu.g/kg, 100-500
.mu.g/kg, 250-750 .mu.g/kg, 500-1000 .mu.g/kg, 1-10 mg/kg, 5-50
mg/kg, 25-75 mg/kg, 50-100 mg/kg, 100-250 mg/kg, 50-100 mg/kg,
250-500 mg/kg, 500-750 mg/kg, 750-1000 mg/kg, 1000-1500 mg/kg,
1500-2000 mg/kg, 5 mg/kg, 20 mg/kg, 50 mg/kg, 100 mg/kg, 500 mg/kg,
1000 mg/kg, 1500 mg/kg, or 2000 mg/kg. Pharmaceutical dosage unit
forms are prepared to provide from about 1 mg to about 5000 mg, for
example from about 100 to about 2500 mg of the compound or a
combination of essential ingredients per dosage unit form.
[0255] In embodiments, about 50 nM to about 1 .mu.M of an agent is
administered to a subject. In related embodiments, about 50-100 nM,
50-250 nM, 100-500 nM, 250-500 nM, 250-750 nM, 500-750 nM, 500 nM
to 1 .mu.M, or 750 nM to 1 .mu.M of an agent is administered to a
subject.
[0256] Determination of an effective amount is well within the
capability of those skilled in the art, especially in light of the
detailed disclosure provided herein. Generally, an efficacious or
effective amount of an agent is determined by first administering a
low dose of the agent(s) and then incrementally increasing the
administered dose or dosages until a desired effect (e.g., reduced
symptoms associated with influenza infection) is observed in the
treated subject, with minimal or acceptable toxic side effects.
Applicable methods for determining an appropriate dose and dosing
schedule for administration of a pharmaceutical composition of the
present invention are described, for example, in Goodman and
Gilman's The Pharmacological Basis of Therapeutics, Goodman et al.,
eds., 11th Edition, McGraw-Hill 2005, and Remington: The Science
and Practice of Pharmacy, 20th and 21st Editions, Gennaro and
University of the Sciences in Philadelphia, Eds., Lippencott
Williams & Wilkins (2003 and 2005), each of which is hereby
incorporated by reference.
Combination Therapies
[0257] The agents and pharmaceutical compositions described herein
can also be administered in combination with another therapeutic
molecule. The therapeutic molecule can be any compound used to
treat influenza infection. Examples of such compounds include, but
are not limited to, inhibitory nucleic acids that reduce influenza
virus production, antiviral agents (e.g., amantadine, rimantadine,
zanamivir, oseltamivir, and the like), toxins, and agents that
reduce the symptoms associated with influenza infection (e.g.,
anti-inflammatories).
[0258] The influenza HA antibody can be administered before,
during, or after administration of the additional therapeutic
agent. In embodiments, the antibody is administered before the
first administration of the additional therapeutic agent. In
embodiments, the antibody is administered after the first
administration of the additional therapeutic agent (e.g., 1, 2, 3,
4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14 days or more). In embodiments,
the antibody is administered simultaneously with the first
administration of the additional therapeutic agent.
[0259] The amount of therapeutic agent administered to a subject
can readily be determined by the attending physician or
veterinarian. Generally, an efficacious or effective amount of an
antibody and an additional therapeutic is determined by first
administering a low dose of one or both active agents and then
incrementally increasing the administered dose or dosages until a
desired effect is observed (e.g., reduced influenza infection
symptoms), with minimal or no toxic side effects. Applicable
methods for determining an appropriate dose and dosing schedule for
administration of a combination of the present invention are
described, for example, in Goodman and Gilman's The Pharmacological
Basis of Therapeutics, 11th Edition, supra, and in Remington: The
Science and Practice of Pharmacy, 20th and 21st Editions,
supra.
Kits
[0260] The invention provides for kits for preventing or treating
influenza infection; neutralizing an influenza virus; inhibiting
establishment of influenza virus infection; inhibiting
dissemination of influenza virus infection; as well as inhibiting
influenza virus entry into a cell. In embodiments, the kit contains
one or more agents or pharmaceutical compositions described herein.
In embodiments, the kit provides instructions for use. The
instructions for use can pertain to any of the methods described
herein. In related embodiments, the instructions pertain to using
the agent(s) or pharmaceutical composition(s) for treating or
preventing influenza infection. Kits according to this aspect of
the invention may comprise a carrier means, such as a box, carton,
tube or the like, having in close confinement therein one or more
container means, such as vials, tubes, ampules, bottles and the
like. In embodiments, the kit provides a notice in the form
prescribed by a governmental agency regulating the manufacture,
use, or sale of pharmaceuticals or biological products, which
notice reflects approval by the agency of manufacture, use or sale
of the kit and the components therein for human administration.
Examples
[0261] It should be appreciated that the invention should not be
construed to be limited to the examples that are now described;
rather, the invention should be construed to include any and all
applications provided herein and all equivalent variations within
the skill of the ordinary artisan.
Example 1: The Clonal Lineage of a Broadly Neutralizing
Antibody
[0262] Rearranged Ig V.sub.H and V.sub.L genes were isolated by
RT/PCR from peripheral blood mononuclear cells, collected from a
subject one week after vaccination with the 2007 trivalent
inactivated vaccine (TIV) (Liao, H. X. et al. J. Virol. Methods
158:171-179 (2009)). Among the clonal lineages detected by
sequencing the rearranged genes was the three-member clone (mAbs
CH65, CH66 and CH67) shown in FIG. 1A. The inferred sequence of the
unmutated common ancestor (UCA) of the clonal lineage of antibodies
CH65, CH66 and CH67 is unambiguous, except at position 99 of the
heavy chain, which might be either glycine or alanine. FIG. 1B
shows an alignment of the amino acid sequences of each antibody to
the UCA. All three mature antibodies bind the H1 hemagglutinin (HA)
present in the vaccine (A/Solomon Islands/3/2006) with about equal
affinity; the UCA binds much more weakly.
Example 2: Breadth of Neutralizing Activity
[0263] The heavy chain of CH65 differs from the UCA at 12 positions
in the variable domain; and at 6 positions its light chain. CH65
IgG1 and its Fab were expressed in 293T cells by transient
transfection and purified as described below. Neutralization was
tested against a large panel of H1 isolates from the past 30 years,
including vaccine strains from 1977, 1991 and 1995, and observed
strikingly broad potency (Table 1). CH67 was also tested against a
subset of this panel (Table 1).
TABLE-US-00001 TABLE 1 Broad neutralization of seasonal influenza
strains A/H1N1 by human MAb CH65 and CH67 mAb minimum effective
concentration (ug/ml) H1N1 virus strain CH65 CH67 A/USSR/90/1977*
100 25 A/Kawasaki/6/1986 0.098 0.39 A/Texas/36/1991 Neg Neg
A/Wellington/47/1992 Neg Neg A/Florida/2/1993 0.012 0.012
A/Beijing/262/1995* 0.098 0.098 A/Shengzhen/227/1995 0.012 0.024
A/Shanghai/8/1996 Neg Neg A/Johannesburg/159/1997 0.098 0.39
A/Shanghai/2/1997 0.195 0.195 A/Moscow/13/1998 0.012 0.012
A/Ostrava/801/1998 12.5 Neg A/New Caledonia/22/1999 0.391 0.195
A/Bangkok/163/2000 0.195 0.098 A/Fujian/156/2000 0.488 0.049
A/Chile/8885/2001 0.195 0.098 A/Auckland/65/2001 0.195 0.098
A/Neimenggu/52/2002.sup.# 0.098 0.098 A/Brazil/1403/2003 3.125
0.195 A/Canada/59/2004 0.098 0.098 A/Solomon
Islands/03/2006*.sup.,+ 0.024 0.098 A/Brisbane/59/2007 0.098 0.98
A/California/07/2009 (swine)* Neg 6.25 *Strains that were included
in seasonal vaccines. H1 component of the vaccine received by the
subject. .sup.#Originally reported as insensitive to mAb CH65.
indicates data missing or illegible when filed
[0264] The antibody neutralized H1N1 strains isolated as early as
1986, covering 21 years of antigenic drift. As expected, it
neutralized A/Solomon Islands/3/2006, the H1 component of the 2007
vaccine. Of the 36 strains tested, it failed to neutralize only
six, including the 2009 pandemic strain, A/Texas/36/1991 and
A/USSR/90/1977. The CH67 antibody has a similar breadth; it also
neutralizes (weakly) the 2009 pandemic strain. There is also
evidence that CH66 (and likely CH65) binds HA from an H3 virus
(X31, a lab strain derived from the 1968 pandemic. Too few
HA-directed human monoclonal antibodies have been characterized for
systematic comparison, but neutralization by serum samples does not
ordinarily exhibit this degree of breadth.
Example 3: Structure of CH65:HA
[0265] A complex of the mAb CH65 Fab with the HA ectodomain from
A/Solomon Islands/03/2006 (HA.sup.SI) was crystallized, recorded
diffraction to a minimum Bragg spacing of 3.2-.ANG. (Table 2), and
the structure was determined by molecular replacement as outlined
below.
TABLE-US-00002 TABLE 2 Crystallographic statistics Data collection
Resolution (last shell), .ANG. 30.0-3.20 (3.31-3.20) Wavelength,
.ANG. 0.980 Space group I212121 Unit cell dimensions (a, b, c),
.ANG. 155.0, 191.8, 332.1 Unit cell angles (.alpha., .beta.,
.gamma.), .degree. 90, 90, 90 I/.sigma. (last shell) 16.7 (2.1)
Rsym (last shell), % 9.8 (63.7) Completeness (last shell), % 98.9
(94.4) Number of refections 368947 unique 80377 Redundancy 4.6
Refinement Resolution, .ANG. 30.0-3.20 Number of refections 80336
working 78354 free 1982 Rwork, % 21.1 Rfree, % 24.8 Ramachandran
plot, % 88.5/9.3/2.2 (favored/additional/disallowed) Number of
atoms: protein 21794 other (sulfate ions) 214 rmsd bond lengths,
.ANG. 0.010 rmsd bond angles, .degree. 1.287
[0266] The asymmetric unit of the crystal contains a single copy of
the HA trimer, with three bound Fabs (FIGS. 2A-2D). The final model
includes all HA1 and HA2 residues in the expressed protein, except
four disordered residues at the C-terminus of HA1. The electron
density maps showed evidence for N-linked glycosylation at all
eight potential sites on each monomer, and one or more sugar
residues at five of these positions could be modeled. The Fab is
well-ordered, except residue 1 of the light chain and residues
141-147 of the heavy chain; these residues are all far from the
binding site.
[0267] A/Solomon Islands/03/2006 (this work) and A/Puerto
Rico/8/1934 (Gamblin, S. J. et al., Science 303:1838-1842 (2004))
are, to the inventors' knowledge, the only seasonal H1N1 strains
for which a structure of the HA has been determined; others are
either pandemic strains or animal influenza strains. Comparison,
using the program DALI, shows that HA.sup.SI is similar to other
H1N1 HAs, such as those of the pandemic isolates from 2009
(C.alpha. RMSD 0.9 .ANG. over 495 aligned residues, 79% sequence
identity; PDB IDs 3LZG (Xu, R. et al., Science 328:357-360 (2010))
and 3LYJ (Zhang, W. et al., Protein Cell 1:459-467 (2010))) and
1918 (C.alpha. RMSD 1.5 .ANG. over 495 aligned residues, 85%
sequence identity; PDB IDs 3LZF (Xu, R et al., Science 328:357-360
(2010)) and 1RUZ (Gamblin, S. J. et al., Science 303:1838-1842
(2004))) and the seasonal isolate from 1934 (C.alpha. RMSD 1.5
.ANG. over 482 aligned residues, 86% sequence identity; PDB ID 1RVZ
(Gamblin, S. J. et al., Science 303:1838-1842 (2004))). The
vestigial esterase domain of HA.sup.SI resembles that of the 2009
HA more closely than it does those from the 1918 and 1934 HAs.
[0268] MAb CH65 binds the globular head of the HA trimer (FIGS. 1C
and 2A). The epitope includes both the receptor site and the
antigenic site designated Sb in an early analysis of H1 sequences
(Caton, A. J. et al., Cell 31:417-427 (1982)). The contact buries
858 .ANG..sup.2 on the antibody and 748 .ANG..sup.2 on HA1. All
three CDRs of the heavy chain, as well as CDR-L1 and L3 of the
light chain, participate in the interface (FIGS. 1C and 2B-D).
CDR-H3 inserts into the receptor site. Seven of its nineteen
residues contribute 402 .ANG..sup.2 of buried surface area, or 47%
of the complete interface. The other CDRs form flanking
interactions. CDR-L3 contacts the N-terminal end of the short
.alpha.-helix, site Sb, at the edge of the receptor pocket, and
CDR-H1 and -H2 contact a loop that protrudes from HA1 adjacent to
the C-terminus of the short .alpha.-helix.
[0269] Tables 3 and 4 summarize several of the critical
interactions between the antibody and HA.
TABLE-US-00003 TABLE 3 Residues in CDR H3 of CH65 that contact HA
(Table discloses residues 104-107 of SEQ ID NO: 10) Arg104* Ser105
Val106 Asp107 Tyr109 Tyr 110 Tyr 112 *for some influenza strains
(not Solomon Islands), Arg104 might contact HA
TABLE-US-00004 TABLE 4 Residues in HA that contact CH65 CDR HA
residue contacted CDR H1 158 CDR H2 158-160 CDR H3 135-136;
190-195; 226 CDR L1 222; 225; 227 CDR L2 (none) CDR L3 187; 189
Example 4: CDR-H3 of mAb CH65 Compared to the Receptor
[0270] Because CDR-H3 inserts into the receptor site, this
structure was compared to that of the human receptor analog LSTc
(sialic-acid-.alpha.2,6-galactose-.beta.1,4-N-acteylglucosamine)
bound to 1934 HA (PDB ID 1RVZ: reference (Gamblin, S. J. et al.,
Science 303:1838-1842 (2004)))(FIGS. 3A and 3B). In CH65, Asp107 at
the tip of CDR-H3 accepts hydrogen bonds from the backbone amide of
HA1 Ala137, the side chain hydroxyl of Ser136, and the side chain
N.epsilon. of Arg226. (Arginine is found only rarely at position
226: glutamine is more common. Arg226 adopts a kinked conformation
in the crystal structure; a glutamine would fit readily, with its
N.epsilon. in the same position as the corresponding atom of the
arginine side chain.) The backbone amide of Val106 in the antibody
donates a hydrogen bond to the carboxyl oxygen of HA1 Val135 on
HA1, and the nonpolar side chain of Val106 is in van der Waals
contact with HA1 Trp153 and Leu194. In receptor analog LSTc, the
carboxylate group of sialic acid has the same contacts with HA1 as
does the (chemically analogous) side chain of Asp107, and the
N-acetyl group interacts with HA in the same way as just described
for the amide and side chain of Val106. In short, mAb CH65 mimics
most of the chemical groups on the human receptor that interact
with HA.
Example 5: Glycosylation
[0271] Glycosylation at antigenic sites is an important mechanism
of immune evasion by influenza virus (Knossow, M. and Skehel, J. J.
Immunology 119:1-7 (2006); Wiley, D. C. and Skehel, J. J. Annu.
Rev. Biochem. 56:365-394 (1987); and Wei, C. J. et al., Sci.
Transl. Med. 2:24ra21 (2010)). In HA.sup.SI, glycosylation leaves
sites Sb and Cb exposed, partially obscures site Ca, and entirely
masks antigenic site Sa. Site Sa is the epitope recognized by
antibody 2D1, the prototype for Ig-mediated immunity to 2009 H1N1
in survivors of the 1918 epidemic (Xu, R. et al., Science
328:357-360 (2010)). Of the side chains in contact with 2D1, 7/16
differ between HA.sup.SI and 1918 HA; in comparison, only 3/16
differ between 2009 pandemic HA and 1918 H A. Because the HA of
A/Solomon Islands/03/2006 is glycosylated at site Sa, neither
vaccination with TIV-2007, nor prior infection with an A/Solomon
Islands/03/2006-like strain could have elicited a 2D1-like immune
response.
Example 6: Affinity Maturation
[0272] The amino-acid sequence of CH65 is the result of affinity
maturation from its UCA. Analysis of the structure in light of its
clonal lineage (FIG. 1B) shows that the central interactions of the
antibodies with HA have remained unchanged by affinity maturation.
The CDR-H3 has not mutated, nor has the contact of the light-chain
CDR-L3 with the N-terminal end of the short .alpha.-helix, site Sb.
(Ser93 of CDR-L3 is Asp in lineage member CH67; substitution to Asp
may allow CH67 to accept a hydrogen-bond from HA Asn187.) Elsewhere
on the interaction surface of the antibody, changes to two residues
create additional hydrogen bonds between the antibody and
HA.sup.SI. Light-chain residues Asp26 and Arg29 in CDR-L1 have
mutated from their respective germline counterparts, Asn and Ser.
Asp26 accepts a hydrogen-bond from HA Lys222. Arg29 is positioned
to donate two hydrogen-bonds to HA Asp225. Other changes, including
those at position 31 (Gly to Asp) and positions 33-35 (Trp-Met-His
to His-Ile-Asn) may exert subtle effects on the conformation of
CDR-H3.
Example 7: CH65-CH67 Lineage Reactivity to Different Influenza
Strains
[0273] The ability of CH65-CH67 to react with other influenza
strains was assessed. 293 T cells were transfected with full-length
HA from strain X31 (H3 influenza strain) (FIGS. 4A-4C, top panel)
or with cell-surface expressed globular head from A/Solomon
Islands/3/2006 (H1 influenza strain) (FIGS. 4D-4F, bottom panel).
Cells were fixed with formaldehyde and probed with CH65 Fab (FIGS.
4B and 4E) or CH66 full-length antibody (FIGS. 4C and 4F), followed
by a FITC-conjugated secondary antibody specific for the human Fab.
Cells were imaged by FITC emission (532 nm). As a control,
transfected cells were probed with secondary antibody only (FIGS.
4A and 4D). These results indicate that the CH65-CH67 antibodies
(e.g., CH66) also bind other influenza serotypes (e.g., H3).
[0274] As discussed in detail in the above examples, CH65 comes
from an adult subject in the US, who received the 2007 TIV one week
before donating a plasma sample. It was assumed that the subject
had been exposed to H1N1 influenza strains in the past, so that the
antibodies obtained by screening with a panel of recombinant
hemagglutinins (rHAs) were from a secondary response. Indeed, the
number of mutations (overall frequency about 5%) is too great to
have occurred within just one week of a primary exposure. In the
setting of TIV, a large fraction of the circulating antibody
secreting plasma cells one week post vaccination are influenza
specific (Wrammert, J. et al., Nature 453:667-671 (2008)). Unlike
other methods (e.g., phage display) for high-throughput analysis of
human B-cell responses, the procedure described herein to isolate
CH65 detects paired rearranged V.sub.H and V.sub.L regions, and
hence reconstructs the complete antigen combining site of the
native antibody (Liao, H. X. et al. J. Virol. Methods 158:171-179
(2009)).
[0275] The CH65 antibody belongs to a relatively small clonal
lineage of detected sequences, but there were presumably other
members not represented among the expressed antibodies. Because the
plasma cell from which it came probably derived from a
vaccine-stimulated memory cell, most of the mutations that separate
it from the UA probably occurred during the earlier primary
response. The breadth of infectivity neutralization by CH65 implies
that it might have arisen during nearly any of the seasonal
outbreaks of the two decades preceding 2007, as only a small number
of mutations during the secondary response could have produced a
very tightly binding antibody from one of somewhat lower
affinity.
[0276] The antigen combining site of CH65 has no markedly atyptical
structural features. It has contributions from V.sub.H 1.about.2,
D.sub.H 1.about.1, and J.sub.H 6 and from V.kappa.3-.about.21 and
J.kappa.2 (Table 5).
TABLE-US-00005 TABLE 5 Gene usage in CH65 VH VL CDR3 CDR3 Mutation
length No. Mutation length No. ID V D J frequency of a.a Isotype ID
V J frequency of a.a H0082 1~2 1~1 6 5.0% 19 G1 L0024 3~21 2 4.2%
11 H1226 1~2 1~1 6 4.8% 19 G1 L0408 3~21 2 4.3% 11 H2250 1~2 1~1 6
4.7% 19 G1 L0797 3~21 2 3.3% 11
[0277] The 19-residue heavy-chain CDR3 is of roughly average length
(Volpe, J. M. and Kepler, T. B., Immunome Res. 4:3 (2008)). Its
sequence in the mature antibody is the same as in the UCA. The VDJ
recombination that gave rise to the coding sequence of the UCA
included 17 n-nucleotides (FIG. 5), so that six of the fourteen
CDR3 residues are encoded by the random insertions produced by
imprecise joining. These six residues include Val106 and Asp107,
which together make the most critical contacts within the
sialic-acid pocket. The predicted n-nucleotide additions are
somewhat fewer than average at the V-D junction and somewhat
greater than average at the D-J junction (Volpe, J. M. and Kepler,
T. B., Immunome Res. 4:3 (2008)).
[0278] The tip of the CH65 heavy-chain CDR3 is a strikingly
faithful mimic of the sialic-acid surface that contacts HA. Early
work on influenza virus antigenic variation led to discussion of an
apparent conflict between escape from neutralization and
conservation of an exposed receptor binding site. The HA structure
resolved the issue, by showing that the sialic-acid binding site is
smaller than the footprint of a typical antibody and hence that
mutations in the periphery of the receptor pocket can interfere
with neutralization without blocking receptor attachment (Wiley, D.
C. et al., Nature 289:373-378 (1981); and Wilson, I. A. et al.,
Nature 289:366-373 (1981)). Indeed, variations affecting the
susceptibility to neutralization by Ab CH65 map to sites that flank
the receptor pocket but avoid any direct receptor contacts.
[0279] Two published structures of murine mAbs bound with H3 HAs
show some degree of penetration into the receptor site--in both
cases, by the heavy-chain CDR3. Neither mAb mimics the sialic-acid
interaction as extensively as does CH65. In one (PDB ID 1KEN
(Barbey-Martin, C. et al., Virology 294:70-74 (2002))), an aspartic
acid side chain approaches the location of the sialic-acid
carboxylate, but in an orientation that can accept a hydrogen bond
only from the hydroxyl of Ser136 and not from the main-chain NH of
Asn 137. In the other (2VIR (Fleury, D. et al., Nat. Struct. Biol.
5:119-123 (1998))), a Tyr-Asp pair at the tip of the CDR3 has an
orientation related to that of the Val-Asp pair in our CH65:HA
complex, and the aspartic acid side chain has the same
hydrogen-bonding pattern, but the mimicry does not extend to any of
the interactions of the receptor N-acetyl group. H3 HAs have
leucine, rather than glutamine or arginine at position 226, so that
additional polar contact is not available.
[0280] Sites of mutations in naturally occurring, seasonal
antigenic variants of HA are largely on the outward facing surface
of HA1. Some relatively rare antibodies that bind a conserved site
along the "stem" of the HA have come from phage-displayed libraries
of unrelated, rearranged human V.sub.H genes (all from
V.sub.H1-69). The structure and characteristics of CH65 show that
it is also possible to elicit broadly neutralizing,
receptor-binding site antibodies. A parallel can be drawn with the
broadly neutralizing, receptor-site antibodies against HIV-1 (e.g.,
antibody VRC01), which are reasonably close mimics of the
functional receptor, CD4 (Zhou T. et al., Science 329:811-817
(2010)).
[0281] An immunogen with an enhanced probability of eliciting a
CH65-like response may protect against series of seasonal strains.
A strategy for designing such an immunogen, based on analysis of
both the structure and the lineage, could include (in addition to
the native HA) a component to induce a UCA-like primary response.
Inspection of the differences between CH65 and its UCA suggests
that the principal changes affecting affinity are in the
light-chain CDR1, where mutations at positions 26 and 29 have
introduced salt bridges with HA (Table 6).
TABLE-US-00006 TABLE 6 Potential influence of residues in CH65 that
have changed from UCA CH65 UCA potential effect Heavy chain E1 Q
distant from contact D31 G near a contact, but no salt bridge or
strong polar H-bond H33 Y no obvious likely perturbation I 34 M ''
N35 H '' H52 N might compensate for Y->H at 33 D57 G no obvious
likely perturbation A75 S distant V83 L '' N84 S '' G85 R '' K87 R
'' Light chain D26 N adds salt bridge R29 S adds salt bridge N35 Y
might affect hc:lc interface C48 Y changes at 48 and 49 would
compensate for ech other Y49 D '' I 98 V distant
[0282] A modified HA, in which the same contacts instead gain
stability from mutations in the antigen, might have the desired
properties.
[0283] The lack of common resistance mutations among the many
strains tested suggests that Ab CH65 will be a useful template for
a therapeutic antibody. Oseltamivir-resistant H1N1, which emerged
rapidly beginning in 2007-8, has become the predominant strain of
seasonal influenza, and management of severe infection could
benefit from a broadly reacting, immune-based therapeutic (Dharan,
N. J. et al. JAMA 301:1034-1041 (2009)). Previous studies with a
human mAb targeting the globular head of H5N1 indicate that the
effective neutralizing concentrations for CH65 will be protective
in vivo (Simmons, C. P. et al. PLoS Med. 4:e178 (2007)).
[0284] Accordingly, described herein are novel influenza HA
antibodies that will be extremely effective in treating and
preventing influenza infection. As seasonal antigenic drift of
circulating influenza virus leads to a requirement for frequent
changes in vaccine composition, because exposure or vaccination
elicits human antibodies with limited cross-neutralization of
drifted strains, there is a significant unmet need for an effective
therapy that can broadly neutralize influenza drifted strains. The
above results clearly demonstrate that use of the novel antibodies
provides a solution to this unmet need. Therapy with the novel
antibodies described herein is therefore a significant advance in
the treatment of patients suffering from influenza infection.
[0285] The results reported herein were obtained using the
following methods and materials.
Clinical Sample
[0286] MAbs CH65, CH66 and CH67 were obtained from a subject
vaccinated with the 2007 TIV under a Duke Institutional Board
approved human subjects protocol. The subject received the
2007-2008 Fluzone.degree. (Sanofi Pasteur, Swiftwater, Pa.), which
contained A/Solomon Islands/3/2006(H1N1),
A/Wisconsin/67/2005(H3N2), and B/Malaysia/2506/2004. Blood was
drawn on day 7 post-vaccination, and PBMC were isolated and
cryopreserved on the same day. Single plasmablasts were sorted into
96-well plates, using a panel of antibodies as described (Moody M
A, et al., PLoS One 6:e25797 (2011)). Single-cell RT/PCR was
carried out to obtain DNA for sequencing (Liao, H. X. et al. J.
Virol. Methods 158:171-179 (2009)), which was done in both forward
and reverse directions using a BigDye.RTM. sequencing kit on an ABI
3700 (Ewing, B. et al., Genome Res. 8:175-185 (1998)) and assembled
with a method based on quality scores at each position (Kepler, T.
B. et al. BMC Genomics 11:444 (2010)). Ig isotype was determined by
local alignment with known sequences; V, D, and J region genes,
CDR3 loop lengths, and mutation frequencies were determined by
comparison with the inferred unmutated ancestor.
Lineage Analysis
[0287] The UCA was inferred using a Bayesian method by first
determining the clonal tree by maximum likelihood using DNAML
(Felsenstein, J. J. Mol. Evol. 17:368-376 (1981)), and then
computing the posterior joint distribution on gene segments and
recombination sites, conditional on the inferred ML tree. The
posterior probability mass function on nucleotides at each position
was then obtained directly.
Expression and Purification of IgG and Fab
[0288] The variable regions of immunoglobulin heavy- and
light-chain genes were isolated by RT/PCR from single plasma cells
as described above (Liao, H. X. et al. J. Virol. Methods
158:171-179 (2009)). For production of purified full-length IgG
antibody, the V.sub.H and V.kappa. genes of CH65, CH66 and CH67
were cloned into a pcDNA 3.1 expression vector containing either
the human IgG1 constant region gene or the .kappa.-chain constant
region gene (Liao, H. X. et al. J. Virol. Methods 158:171-179
(2009)). To produce the CH65 Fab, a 5' primer, HV13274-F1
(5'-AAGCTTACCATGCCGATGGGCTCC-3' (SEQ ID NO: 47)), was designed to
contain a restriction site (Hind III) and sequences to anneal to
the 5' sequences of the Ig signal peptide, and a 3' primer,
HV13221H-R474 (5'-GAGCCCAAATCTTGTGACAAATGATCTAGA-3' (SEQ ID NO:
48)) was designed to contain a restriction site (XbaI) and to
introduce a stop codon after the sequence (5'TCTTGTGACAAA3' (SEQ ID
NO: 49)), encoding amino acid residues, SCDK (SEQ ID NO: 50), just
before the hinge of the human IgG1 constant region. PCR
amplification, using these primers and the full-length IgG1 heavy
chain gene as template, yielded the Fab gene, which was cloned into
pcDNA3.1/hygro (Nicely, N. I. et al., Nat. Struct. Mol. Biol.
17:1492-1494 (2010)). Recombinant, intact, CH65 IgG1 and its Fab
were produced in 293T cells by co-transfection with the genes
encoding heavy and light chains. The intact antibody was purified
using anti-human IgG beads (Sigma, St. Louis, Mo.); the Fab, using
anti-L chain beads (Sigma, St. Louis, Mo.) followed by FPLC gel
filtration (Liao, H. X. et al. J. Virol. Methods 158:171-179
(2009); and Nicely, N. I. et al., Nat. Struct. Mol. Biol.
17:1492-1494 (2010)).
Infectivity Neutralization
[0289] Infectivity neutralization was analyzed in a
microneutralization assay based on the methods of the influenza
reference laboratories at the Centers for Disease Control and
Prevention (CDC) (Hancock, K. et al., N. Engl. J. Med.
361:1945-1952 (2009)). H1N1 historical virus stocks were provided
by Vladimir Lugovtsev (Div. of Viral Products, CBER, FDA). All
viruses were titrated on MDCK cells and used at 100 TCID.sub.50 per
well (in triplicate). Two-fold serial dilutions of mAb CH65,
starting at 100 .mu.g/ml, were mixed with virus stocks before
addition to MDCK cell monolayers. The minimum concentrations that
completely inhibited virus replication (EC.sub.99) are reported in
Table 1.
HA Expression and Purification
[0290] Codon-optimized cDNA of the ectodomain of HA A/Solomon
Islands/03/2006 was synthesized by GeneArt and subcloned into a pET
vector modified for ligation-independent cloning (LIC). The
synthetic gene encoded a secretion signal at the N terminus, and,
in place of the transmembrane domain, a thrombin cleavage site, a
T4-fibritin "foldon" to promote proper trimerization, and a His6
tag (SEQ ID NO: 46) at the C terminus. Trichoplusia ni (Hi-5) cells
were infected with recombinant baculovirus. The supernatant was
harvested at 48 hours post-infection by centrifugation,
concentrated and diafiltered against phosphate-buffered saline with
40 mM imidazole, and loaded onto Ni-NTA resin. The protein was
eluted, dialyzed, and incubated overnight with TPCK-treated trypsin
at 1:500 mass ratio to remove the trimerization and His6 tags (SEQ
ID NO: 46) and to cleave the HA0 precursor peptide. The protein was
further purified by gel filtration chromatography on Superdex 200
(GE Healthcare).
Crystallization
[0291] The CH65 Fab and the A/Solomon Islands/03/2006 HA were
incubated in 4.5:1 molar ratio, and the resulting 3:1 complex was
separated from excess Fab by gel filtration chromatography on
Superdex 200 in 10 mM Hepes pH 7.5, 150 mM NaCl. The complex was
concentrated to an absorbance of 10 at 280 nm (approximately 6
mg/mL). Crystals were grown in hanging drops over a reservoir
containing 2.2 M ammonium sulfate, 100 mM Tris pH 7.5, and 5%
PEG-400 at 18 degrees C. Crystallization was improved by
microseeding. After 3-14 days, crystals were cryoprotected by
adding reservoir solution supplemented with 15% glycerol to the
drop, then harvested and flash cooled in liquid nitrogen.
Structure Determination and Refinement
[0292] Diffraction experiments were performed at beamline 24-ID-E
at the Advanced Photon Source. A dataset at 3.2-.ANG. resolution
was collected from a single .about.50.times.50.times.300 .mu.m rod
and processed using HKL2000 (Table 2). Molecular replacement (MR)
calculations were performed with PHASER (McCoy, A. J. et al., J.
Appl. Crystallogr. 40:658-674 (2007)), using 1934 H1 HA (PDB ID
1RVZ (Gamblin, S. J. et al., Science 303:1838-1842 (2004))) as the
starting model. Initial phases from MR enabled a search for Fab
molecules by phased molecular replacement in MOLREP, using a
library of Fab structures. The model was refined in CNS (Brunger,
A. T., Nat. Protoc. 2:2728-2733 (2007)) by simulated annealing
using deformable elastic network restraints and rebuilt in COOT
(Emsley, P. and Cowtan, K., Acta Crystallogr. D. Biol. Crystallogr.
60:2126-2132 (2004)). N-linked glycans from a high-resolution
structure were fitted into experimental electron density maps where
appropriate. Strong 3-fold non-crystallographic symmetry restraints
were applied to HA and to each domain of the Fab throughout
refinement, allowing variation in the angle between the conserved
domain and the variable domain of the Fab. Finally, 3 cycles of
individual positional and B-factor refinement in PHENIX (Adams, P.
D. et al., Acta Crystallogr. D. Biol. Crystallogr. 58:1948-1954
(2002)) resulted in a model in good agreement with observed
intensities (R/Rfree=21.1/24.8%) (Table 2). Coordinates and
diffraction data have been submitted to the PDB, accession number
3SM5.
Other Embodiments
[0293] From the foregoing description, it will be apparent that
variations and modifications may be made to the invention described
herein to adopt it to various usages and conditions. Such
embodiments are also within the scope of the following claims.
[0294] The recitation of a listing of elements in any definition of
a variable herein includes definitions of that variable as any
single element or combination (or subcombination) of listed
elements. The recitation of an embodiment herein includes that
embodiment as any single embodiment or in combination with any
other embodiments or portions thereof.
INCORPORATION BY REFERENCE
[0295] All patents and publications mentioned in this specification
are herein incorporated by reference to the same extent as if each
independent patent and publication was specifically and
individually indicated to be incorporated by reference.
Sequence CWU 1
1
611378DNAArtificial SequenceDescription of Artificial Sequence
Synthetic polynucleotide 1caggtgcagc tggtgcagtc tggggctgag
gtgaagaagc ctggggcctc agtgaaggtc 60tcctgcaagg cttctggata caccttcacc
ggctactata tgcactgggt gcgacaggcc 120cctggacaag ggcttgagtg
gatgggatgg atcaacccta acagtggtgg cacaaactat 180gcacagaagt
ttcagggctg ggtcaccatg accagggaca cgtccatcag cacagcctac
240atggagctga gcaggctgag atctgacgac acggccgtgt attactgtgc
gagaggggga 300ctggaacccc gatctgtaga ctactactac tacggtatgg
acgtctgggg ccaagggacc 360acggtcaccg tctcctca 3782378DNAArtificial
SequenceDescription of Artificial Sequence Synthetic polynucleotide
2gaagtgcagc tggtgcagtc tggagctgag gtgaagaagc ctggggcctc agtgaaagtc
60tcctgcaagg cttctggata caccttcacc gactatcata taaactgggt gcgacaggcc
120cctggacaag ggcttgagtg gatgggatgg atccacccta acagtggtga
cacaaactat 180gcacagaagt ttcagggctg ggtcaccatg accagggaca
cggccatcag cacagcctac 240atggaggtga atggcttgaa atctgacgac
acggccgtgt attattgtgc gagaggggga 300ctggaacccc gatctgtaga
ctactactat tatggtatgg acgtctgggg ccaagggacc 360acggtcaccg tctcctca
3783378DNAArtificial SequenceDescription of Artificial Sequence
Synthetic polynucleotide 3caggtgcagc tggtgcagtc tggggctgag
gtgaagaagc ctggggcctc agtgaaagtc 60tcctgcaagg cttctggata caccttcacc
gactatcata taaactgggt gcgacaggcc 120cctggacaag ggcttgagtg
gatgggatgg atccacccta acagtggtga cacaaactat 180gcacagaagt
ttcagggctg ggtcaccatg accagggaca cgtccatcag cacagcctac
240atggaggtga atggcttgaa atctgacgac acggccgtgt attattgtgc
gagaggggga 300ctggaacctc gatctgtaga ctactactat tatggtatgg
acgtctgggg ccaagggacc 360acggtcaccg tctcctca 3784378DNAArtificial
SequenceDescription of Artificial Sequence Synthetic polynucleotide
4caggtgcagc tggtgcagtc tggggctgag gtgaggaagc ctggggcctc agtgaaggtc
60tcctgcaagg cttctggata caccttcacc gacaactata tacactgggt gcgacaggcc
120cctggacaag ggcttgagtg gatgggatgg atccacccta acagtggtgc
cacaaagtat 180gcacagaagt ttgagggctg ggtcaccatg accagggaca
cgtcaatcag cacagtctac 240atggaactga gcagatcgag atctgacgac
acggccgtat attactgtgc gagagcggga 300ctggaaccac gatccgtaga
ctactacttc tacggtttgg acgtctgggg ccaagggacc 360gcggtcaccg tctcctca
3785324DNAArtificial SequenceDescription of Artificial Sequence
Synthetic polynucleotide 5cagtctgtgc tgactcagcc accctcggtg
tcagtggccc caggacagac ggccaggatt 60acctgtgggg gaaacaacat tggaagtaaa
agtgtgcact ggtaccagca gaagccaggc 120caggcccctg tgctggtcgt
ctatgatgat agcgaccggc cctcagggat ccctgagcga 180ttctctggct
ccaactctgg gaacacggcc accctgacca tcagcagggt cgaagccggg
240gatgaggccg actattactg tcaggtgtgg gatagtagta gtgatcatgt
ggtattcggc 300ggagggacca agctgaccgt ccta 3246324DNAArtificial
SequenceDescription of Artificial Sequence Synthetic polynucleotide
6cagtctgtgc tgactcagcc accctcggtg tcagtggccc cagggcagac ggccaggatt
60acctgtgggg gaaatgatat tggaaggaag agtgtgcact ggaaccagca gaagccaggc
120caggcccctg tgctggtcgt ctgttatgat agcgaccggc cctcagggat
ccctgagcga 180ttctctggct ccaattcagg gaacacggcc accctgacca
tcagtagggt cgaagccggg 240gatgaggccg actattattg tcaggtgtgg
gatagtagta gtgatcatgt gatattcggc 300ggagggacca agctgaccgt ccta
3247324DNAArtificial SequenceDescription of Artificial Sequence
Synthetic polynucleotide 7cagtctgccc tgactcagcc accctcggtg
tcagtggccc cagggcagac ggccaggatt 60acctgtgggg gaaatgatat tggaaggaag
agtgtgcact ggaaccagca gaagccaggc 120caggcccctg tgctggtcgt
ctgttatgat agtgaccggc cctcagggat ccctgagcga 180ttctctggct
ccaattcagg gaacacggcc accctgacca tcagcagggt cgaagccggg
240gatgaggccg actattattg tcaggtgtgg gatagtagta gtgatcatgt
ggtattcggc 300ggagggacca agctgaccgt ccta 3248324DNAArtificial
SequenceDescription of Artificial Sequence Synthetic polynucleotide
8cagtctgccc tgactcagcc accctcggtg tcagtggccc caggacagac ggccacgatt
60acctgtgggg gaaacaacat tggacgtaaa agagtggact ggttccagca gaagccaggc
120caggcccctg tgctggtcgt ctatgaggat agcgaccggc cctcagggat
ccctgagcga 180ttctctgact ccaactctgg gaccacggcc accctgacca
tcagcagggt cgaagccggg 240gatgaggccg actattactg tcaggtgtgg
gatagtgata gtgatcatgt ggtattcggc 300ggagggacca aactgaccgt ccta
3249126PRTArtificial SequenceDescription of Artificial Sequence
Synthetic polypeptide 9Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val
Lys Lys Pro Gly Ala 1 5 10 15 Ser Val Lys Val Ser Cys Lys Ala Ser
Gly Tyr Thr Phe Thr Gly Tyr 20 25 30 Tyr Met His Trp Val Arg Gln
Ala Pro Gly Gln Gly Leu Glu Trp Met 35 40 45 Gly Trp Ile Asn Pro
Asn Ser Gly Gly Thr Asn Tyr Ala Gln Lys Phe 50 55 60 Gln Gly Trp
Val Thr Met Thr Arg Asp Thr Ser Ile Ser Thr Ala Tyr 65 70 75 80 Met
Glu Leu Ser Arg Leu Arg Ser Asp Asp Thr Ala Val Tyr Tyr Cys 85 90
95 Ala Arg Gly Gly Leu Glu Pro Arg Ser Val Asp Tyr Tyr Tyr Tyr Gly
100 105 110 Met Asp Val Trp Gly Gln Gly Thr Thr Val Thr Val Ser Ser
115 120 125 10126PRTArtificial SequenceDescription of Artificial
Sequence Synthetic polypeptide 10Glu Val Gln Leu Val Gln Ser Gly
Ala Glu Val Lys Lys Pro Gly Ala 1 5 10 15 Ser Val Lys Val Ser Cys
Lys Ala Ser Gly Tyr Thr Phe Thr Asp Tyr 20 25 30 His Ile Asn Trp
Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met 35 40 45 Gly Trp
Ile His Pro Asn Ser Gly Asp Thr Asn Tyr Ala Gln Lys Phe 50 55 60
Gln Gly Trp Val Thr Met Thr Arg Asp Thr Ala Ile Ser Thr Ala Tyr 65
70 75 80 Met Glu Val Asn Gly Leu Lys Ser Asp Asp Thr Ala Val Tyr
Tyr Cys 85 90 95 Ala Arg Gly Gly Leu Glu Pro Arg Ser Val Asp Tyr
Tyr Tyr Tyr Gly 100 105 110 Met Asp Val Trp Gly Gln Gly Thr Thr Val
Thr Val Ser Ser 115 120 125 11126PRTArtificial SequenceDescription
of Artificial Sequence Synthetic polypeptide 11Gln Val Gln Leu Val
Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala 1 5 10 15 Ser Val Lys
Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Asp Tyr 20 25 30 His
Ile Asn Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met 35 40
45 Gly Trp Ile His Pro Asn Ser Gly Asp Thr Asn Tyr Ala Gln Lys Phe
50 55 60 Gln Gly Trp Val Thr Met Thr Arg Asp Thr Ser Ile Ser Thr
Ala Tyr 65 70 75 80 Met Glu Val Asn Gly Leu Lys Ser Asp Asp Thr Ala
Val Tyr Tyr Cys 85 90 95 Ala Arg Gly Gly Leu Glu Pro Arg Ser Val
Asp Tyr Tyr Tyr Tyr Gly 100 105 110 Met Asp Val Trp Gly Gln Gly Thr
Thr Val Thr Val Ser Ser 115 120 125 12126PRTArtificial
SequenceDescription of Artificial Sequence Synthetic polypeptide
12Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Arg Lys Pro Gly Ala 1
5 10 15 Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Asp
Asn 20 25 30 Tyr Ile His Trp Val Arg Gln Ala Pro Gly Gln Gly Leu
Glu Trp Met 35 40 45 Gly Trp Ile His Pro Asn Ser Gly Ala Thr Lys
Tyr Ala Gln Lys Phe 50 55 60 Glu Gly Trp Val Thr Met Thr Arg Asp
Thr Ser Ile Ser Thr Val Tyr 65 70 75 80 Met Glu Leu Ser Arg Ser Arg
Ser Asp Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Ala Gly Leu
Glu Pro Arg Ser Val Asp Tyr Tyr Phe Tyr Gly 100 105 110 Leu Asp Val
Trp Gly Gln Gly Thr Ala Val Thr Val Ser Ser 115 120 125
13108PRTArtificial SequenceDescription of Artificial Sequence
Synthetic polypeptide 13Gln Ser Val Leu Thr Gln Pro Pro Ser Val Ser
Val Ala Pro Gly Gln 1 5 10 15 Thr Ala Arg Ile Thr Cys Gly Gly Asn
Asn Ile Gly Ser Lys Ser Val 20 25 30 His Trp Tyr Gln Gln Lys Pro
Gly Gln Ala Pro Val Leu Val Val Tyr 35 40 45 Asp Asp Ser Asp Arg
Pro Ser Gly Ile Pro Glu Arg Phe Ser Gly Ser 50 55 60 Asn Ser Gly
Asn Thr Ala Thr Leu Thr Ile Ser Arg Val Glu Ala Gly 65 70 75 80 Asp
Glu Ala Asp Tyr Tyr Cys Gln Val Trp Asp Ser Ser Ser Asp His 85 90
95 Val Val Phe Gly Gly Gly Thr Lys Leu Thr Val Leu 100 105
14108PRTArtificial SequenceDescription of Artificial Sequence
Synthetic polypeptide 14Gln Ser Val Leu Thr Gln Pro Pro Ser Val Ser
Val Ala Pro Gly Gln 1 5 10 15 Thr Ala Arg Ile Thr Cys Gly Gly Asn
Asp Ile Gly Arg Lys Ser Val 20 25 30 His Trp Asn Gln Gln Lys Pro
Gly Gln Ala Pro Val Leu Val Val Cys 35 40 45 Tyr Asp Ser Asp Arg
Pro Ser Gly Ile Pro Glu Arg Phe Ser Gly Ser 50 55 60 Asn Ser Gly
Asn Thr Ala Thr Leu Thr Ile Ser Arg Val Glu Ala Gly 65 70 75 80 Asp
Glu Ala Asp Tyr Tyr Cys Gln Val Trp Asp Ser Ser Ser Asp His 85 90
95 Val Ile Phe Gly Gly Gly Thr Lys Leu Thr Val Leu 100 105
15108PRTArtificial SequenceDescription of Artificial Sequence
Synthetic polypeptide 15Gln Ser Ala Leu Thr Gln Pro Pro Ser Val Ser
Val Ala Pro Gly Gln 1 5 10 15 Thr Ala Arg Ile Thr Cys Gly Gly Asn
Asp Ile Gly Arg Lys Ser Val 20 25 30 His Trp Asn Gln Gln Lys Pro
Gly Gln Ala Pro Val Leu Val Val Cys 35 40 45 Tyr Asp Ser Asp Arg
Pro Ser Gly Ile Pro Glu Arg Phe Ser Gly Ser 50 55 60 Asn Ser Gly
Asn Thr Ala Thr Leu Thr Ile Ser Arg Val Glu Ala Gly 65 70 75 80 Asp
Glu Ala Asp Tyr Tyr Cys Gln Val Trp Asp Ser Ser Ser Asp His 85 90
95 Val Val Phe Gly Gly Gly Thr Lys Leu Thr Val Leu 100 105
16108PRTArtificial SequenceDescription of Artificial Sequence
Synthetic polypeptide 16Gln Ser Ala Leu Thr Gln Pro Pro Ser Val Ser
Val Ala Pro Gly Gln 1 5 10 15 Thr Ala Thr Ile Thr Cys Gly Gly Asn
Asn Ile Gly Arg Lys Arg Val 20 25 30 Asp Trp Phe Gln Gln Lys Pro
Gly Gln Ala Pro Val Leu Val Val Tyr 35 40 45 Glu Asp Ser Asp Arg
Pro Ser Gly Ile Pro Glu Arg Phe Ser Asp Ser 50 55 60 Asn Ser Gly
Thr Thr Ala Thr Leu Thr Ile Ser Arg Val Glu Ala Gly 65 70 75 80 Asp
Glu Ala Asp Tyr Tyr Cys Gln Val Trp Asp Ser Asp Ser Asp His 85 90
95 Val Val Phe Gly Gly Gly Thr Lys Leu Thr Val Leu 100 105
17216PRTInfluenza A virus 17Ala Pro Leu Gln Leu Gly Asn Cys Ser Val
Ala Gly Trp Ile Leu Gly 1 5 10 15 Asn Pro Glu Cys Glu Leu Leu Ile
Ser Arg Glu Ser Trp Ser Tyr Ile 20 25 30 Val Glu Lys Pro Asn Pro
Glu Asn Gly Thr Cys Tyr Pro Gly His Phe 35 40 45 Ala Asp Tyr Glu
Glu Leu Arg Glu Gln Leu Ser Ser Val Ser Ser Phe 50 55 60 Glu Arg
Phe Glu Ile Phe Pro Lys Glu Ser Ser Trp Pro Asn His Thr 65 70 75 80
Thr Thr Gly Val Ser Ala Ser Cys Ser His Asn Gly Glu Ser Ser Phe 85
90 95 Tyr Lys Asn Leu Leu Trp Leu Thr Gly Lys Asn Gly Leu Tyr Pro
Asn 100 105 110 Leu Ser Lys Ser Tyr Ala Asn Asn Lys Glu Lys Glu Val
Leu Val Leu 115 120 125 Trp Gly Val His His Pro Pro Asn Ile Gly Asp
Gln Arg Ala Leu Tyr 130 135 140 His Thr Glu Asn Ala Tyr Val Ser Val
Val Ser Ser His Tyr Ser Arg 145 150 155 160 Lys Phe Thr Pro Glu Ile
Ala Lys Arg Pro Lys Val Arg Asp Arg Glu 165 170 175 Gly Arg Ile Asn
Tyr Tyr Trp Thr Leu Leu Glu Pro Gly Asp Thr Ile 180 185 190 Ile Phe
Glu Ala Asn Gly Asn Leu Ile Ala Pro Arg Tyr Ala Phe Ala 195 200 205
Leu Ser Arg Gly Phe Gly Ser Gly 210 215 18217PRTInfluenza A virus
18Ala Pro Leu Gln Leu Gly Asn Cys Ser Val Ala Gly Trp Ile Leu Gly 1
5 10 15 Asn Pro Glu Cys Glu Ser Leu Phe Ser Lys Glu Ser Trp Ser Tyr
Ile 20 25 30 Ala Glu Thr Pro Asn Pro Glu Asn Gly Thr Cys Tyr Pro
Gly His Phe 35 40 45 Ala Asp Tyr Glu Glu Leu Arg Glu Gln Leu Ser
Ser Val Ser Ser Phe 50 55 60 Glu Arg Phe Glu Ile Phe Pro Lys Glu
Ser Ser Trp Pro Asn His Thr 65 70 75 80 Val Thr Lys Gly Val Thr Ala
Ser Cys Ser His Asn Gly Lys Ser Ser 85 90 95 Phe Tyr Lys Asn Leu
Leu Trp Leu Thr Glu Lys Asn Gly Leu Tyr Pro 100 105 110 Asn Leu Ser
Lys Ser Tyr Val Asn Asn Lys Glu Lys Glu Val Leu Val 115 120 125 Leu
Trp Gly Val His His Pro Ser Asn Ile Gly Asp Gln Arg Ala Ile 130 135
140 Tyr His Thr Glu Asn Ala Tyr Val Ser Val Val Ser Ser His Tyr Ser
145 150 155 160 Arg Arg Phe Thr Pro Glu Ile Ala Lys Arg Pro Lys Val
Arg Gly Gln 165 170 175 Glu Gly Arg Ile Asn Tyr Tyr Trp Thr Leu Leu
Glu Pro Gly Asp Thr 180 185 190 Ile Ile Phe Glu Ala Asn Gly Asn Leu
Ile Ala Pro Trp Tyr Ala Phe 195 200 205 Ala Leu Ser Arg Gly Phe Gly
Ser Gly 210 215 19217PRTInfluenza A virus 19Ala Pro Leu Gln Leu Gly
Asn Cys Ser Val Ala Gly Trp Ile Leu Gly 1 5 10 15 Asn Pro Glu Cys
Glu Ser Leu Phe Ser Lys Glu Ser Trp Ser Tyr Ile 20 25 30 Ala Glu
Thr Pro Asn Pro Glu Asn Gly Thr Cys Tyr Pro Gly Tyr Phe 35 40 45
Ala Asp Tyr Glu Glu Leu Arg Glu Gln Leu Ser Ser Val Ser Ser Phe 50
55 60 Glu Arg Phe Glu Ile Phe Pro Lys Glu Ser Ser Trp Pro Lys His
Thr 65 70 75 80 Val Thr Lys Gly Val Thr Ala Ser Cys Ser His Asn Gly
Lys Ser Ser 85 90 95 Phe Tyr Lys Asn Leu Leu Trp Leu Thr Glu Lys
Asn Gly Leu Tyr Pro 100 105 110 Asn Leu Ser Lys Ser Tyr Val Asn Asn
Lys Glu Lys Glu Val Leu Val 115 120 125 Leu Trp Gly Val His His Pro
Ser Asn Ile Gly Asp Gln Arg Ala Ile 130 135 140 Tyr His Thr Glu Asn
Ala Tyr Val Ser Val Val Ser Ser His Tyr Ser 145 150 155 160 Arg Arg
Phe Thr Pro Glu Ile Ala Lys Arg Pro Lys Val Arg Gly Gln 165 170 175
Glu Gly Arg Ile Asn Tyr Tyr Trp Thr Leu Leu Glu Pro Gly Asp Thr 180
185 190 Ile Ile Phe Glu Ala Asn Gly Asn Leu Ile Ala Pro Trp Tyr Ala
Phe 195 200 205 Ala Leu Ser Arg Gly Phe Gly Ser Gly 210 215
20217PRTInfluenza A virus 20Ala Pro Leu Gln Leu Gly Asn Cys Ser Val
Ala Gly Trp Ile Leu Gly 1 5 10 15 Asn Pro Glu Cys Glu Ser Leu Phe
Thr Lys Glu Ser Trp Ser Tyr Ile 20 25
30 Ala Glu Thr Pro Asn Pro Glu Asn Gly Thr Cys Tyr Pro Gly Tyr Phe
35 40 45 Ala Asp Tyr Glu Glu Leu Arg Glu Gln Leu Ser Ser Val Ser
Ser Phe 50 55 60 Glu Arg Phe Glu Ile Phe Pro Lys Glu Ser Ser Trp
Pro Asn His Thr 65 70 75 80 Val Thr Lys Gly Val Thr Ala Ser Cys Ser
His Asn Gly Lys Ser Ser 85 90 95 Phe Tyr Arg Asn Leu Leu Trp Leu
Thr Glu Lys Asn Gly Leu Tyr Pro 100 105 110 Asn Leu Ser Lys Ser Tyr
Val Asn Asn Lys Glu Lys Glu Val Leu Val 115 120 125 Leu Trp Gly Val
His His Pro Ser Asn Met Gly Asp Gln Arg Ala Ile 130 135 140 Tyr His
Thr Glu Asn Ala Tyr Val Ser Val Val Ser Ser His Tyr Ser 145 150 155
160 Arg Arg Phe Thr Pro Glu Ile Ala Lys Arg Pro Lys Val Arg Asp Gln
165 170 175 Glu Gly Arg Ile Asn Tyr Tyr Trp Thr Leu Leu Glu Pro Gly
Asp Thr 180 185 190 Ile Ile Phe Glu Ala Asn Gly Asn Leu Ile Ala Pro
Arg Tyr Ala Phe 195 200 205 Ala Leu Ser Arg Gly Phe Gly Ser Gly 210
215 21217PRTInfluenza A virus 21Ala Pro Leu Gln Leu Gly Asn Cys Ser
Ile Ala Gly Trp Ile Leu Gly 1 5 10 15 Asn Pro Glu Cys Glu Ser Leu
Phe Ser Lys Glu Ser Trp Ser Tyr Ile 20 25 30 Ala Glu Thr Pro Asn
Pro Glu Asn Gly Thr Cys Tyr Pro Gly Tyr Phe 35 40 45 Ala Asp Tyr
Glu Glu Leu Arg Glu Gln Leu Ser Ser Val Ser Ser Phe 50 55 60 Glu
Arg Phe Glu Ile Phe Pro Lys Glu Ser Ser Trp Pro Asn His Thr 65 70
75 80 Val Thr Lys Gly Val Thr Ala Ser Cys Ser His Asn Gly Lys Ser
Ser 85 90 95 Phe Tyr Lys Asn Leu Leu Trp Leu Thr Glu Lys Asn Gly
Leu Tyr Pro 100 105 110 Asn Leu Ser Lys Ser Tyr Val Asn Asn Lys Lys
Lys Glu Val Leu Val 115 120 125 Leu Trp Gly Val His His Pro Ser Asn
Ile Gly Asp Gln Arg Ala Ile 130 135 140 Tyr His Thr Glu Asn Ala Tyr
Val Ser Val Val Ser Ser His Tyr Ser 145 150 155 160 Arg Arg Phe Thr
Pro Glu Ile Ala Lys Arg Pro Lys Val Arg Asn Gln 165 170 175 Glu Gly
Arg Ile Asn Tyr Tyr Trp Thr Leu Leu Glu Pro Gly Asp Thr 180 185 190
Ile Ile Phe Glu Ala Asn Gly Asn Leu Ile Ala Pro Trp Tyr Ala Phe 195
200 205 Ala Leu Ser Arg Gly Phe Glu Ser Gly 210 215
22216PRTInfluenza A virus 22Ala Pro Leu Gln Leu Gly Asn Cys Ser Val
Ala Gly Trp Ile Leu Gly 1 5 10 15 Asn Pro Glu Cys Glu Ser Leu Ile
Phe Lys Glu Ser Trp Ser Tyr Ile 20 25 30 Val Glu Thr Pro Asn Pro
Glu Asn Gly Thr Cys Tyr Pro Gly Tyr Phe 35 40 45 Ala Asp Tyr Glu
Glu Leu Arg Glu Gln Leu Ser Ser Val Ser Ser Phe 50 55 60 Glu Arg
Phe Glu Ile Phe Pro Lys Glu Ser Ser Trp Pro Asn His Thr 65 70 75 80
Val Thr Gly Val Ser Ala Ser Cys Ser His Asn Gly Lys Ser Ser Phe 85
90 95 Tyr Arg Asn Leu Leu Trp Leu Thr Glu Lys Asn Gly Leu Tyr Pro
Asn 100 105 110 Leu Ser Lys Ser Tyr Val Asn Asn Lys Glu Lys Glu Val
Leu Val Leu 115 120 125 Trp Gly Val His His Pro Ser Asn Ile Arg Asp
Gln Arg Ala Ile Tyr 130 135 140 His Thr Glu Asn Ala Tyr Val Ser Val
Val Ser Ser His Tyr Ser Arg 145 150 155 160 Arg Phe Thr Pro Glu Ile
Ala Lys Arg Pro Lys Val Arg Gly Gln Glu 165 170 175 Gly Arg Ile Asn
Tyr Tyr Trp Thr Leu Leu Glu Pro Gly Asp Thr Ile 180 185 190 Ile Phe
Glu Ala Asn Gly Asn Leu Ile Ala Pro Trp Tyr Ala Phe Ala 195 200 205
Leu Ser Arg Gly Phe Gly Ser Gly 210 215 23216PRTInfluenza A virus
23Ala Pro Leu Gln Leu Gly Asn Cys Ser Val Ala Gly Trp Ile Leu Gly 1
5 10 15 Asn Pro Glu Cys Glu Leu Leu Ile Ser Lys Glu Ser Trp Ser Tyr
Ile 20 25 30 Val Glu Lys Pro Asn Pro Glu Asn Gly Thr Cys Tyr Pro
Gly His Phe 35 40 45 Ala Asp Tyr Glu Glu Leu Arg Glu Gln Leu Ser
Ser Val Ser Ser Phe 50 55 60 Glu Arg Phe Glu Ile Phe Pro Lys Glu
Ser Ser Trp Pro Asn His Thr 65 70 75 80 Val Thr Gly Val Ser Ala Ser
Cys Ser His Asn Gly Glu Ser Ser Phe 85 90 95 Tyr Arg Asn Leu Leu
Trp Leu Thr Gly Lys Asn Gly Leu Tyr Pro Asn 100 105 110 Leu Ser Lys
Ser Tyr Ala Asn Asn Lys Glu Lys Glu Val Leu Val Leu 115 120 125 Trp
Gly Val His His Pro Pro Asn Ile Gly Asp Gln Lys Ala Leu Tyr 130 135
140 His Thr Glu Asn Ala Tyr Val Ser Val Val Ser Ser His Tyr Ser Arg
145 150 155 160 Lys Phe Thr Pro Glu Ile Ala Lys Arg Pro Lys Val Arg
Asp Gln Glu 165 170 175 Gly Arg Ile Asn Tyr Tyr Trp Thr Leu Leu Glu
Pro Gly Asp Thr Ile 180 185 190 Ile Phe Glu Ala Asn Gly Asn Leu Ile
Ala Pro Arg Tyr Ala Phe Ala 195 200 205 Leu Ser Arg Gly Phe Gly Ser
Gly 210 215 24216PRTInfluenza A virus 24Ala Pro Leu Gln Leu Gly Asn
Cys Ser Val Ala Gly Trp Ile Leu Gly 1 5 10 15 Asn Pro Glu Cys Glu
Ser Leu Ile Ser Lys Glu Ser Trp Ser Tyr Ile 20 25 30 Val Glu Thr
Pro Asn Pro Glu Asn Gly Thr Cys Tyr Pro Gly Tyr Phe 35 40 45 Ala
Asp Tyr Glu Glu Leu Arg Glu Gln Leu Ser Ser Val Ser Ser Phe 50 55
60 Glu Arg Phe Glu Ile Phe Pro Lys Glu Ser Ser Trp Pro Asn His Thr
65 70 75 80 Val Thr Gly Val Thr Ala Ser Cys Ser His Asn Gly Lys Ser
Ser Phe 85 90 95 Tyr Arg Asn Leu Leu Trp Leu Thr Glu Lys Asn Gly
Leu Tyr Pro Asn 100 105 110 Leu Ser Asn Ser Tyr Val Asn Asn Lys Glu
Lys Glu Val Leu Val Leu 115 120 125 Trp Gly Val His His Pro Ser Asn
Ile Gly Val Gln Arg Ala Ile Tyr 130 135 140 His Thr Glu Asn Ala Tyr
Val Ser Val Val Ser Ser His Tyr Ser Arg 145 150 155 160 Arg Phe Thr
Pro Glu Ile Ala Lys Arg Pro Lys Val Arg Gly Gln Glu 165 170 175 Gly
Arg Ile Asn Tyr Tyr Trp Thr Leu Leu Glu Pro Gly Asp Thr Ile 180 185
190 Ile Phe Glu Ala Asn Gly Asn Leu Ile Ala Pro Trp Tyr Ala Phe Ala
195 200 205 Leu Ser Arg Gly Phe Gly Ser Gly 210 215
25216PRTInfluenza A virus 25Ala Pro Leu Gln Leu Gly Asn Cys Ser Val
Ala Gly Trp Ile Leu Gly 1 5 10 15 Asn Pro Glu Cys Glu Leu Leu Ile
Ser Lys Glu Ser Trp Ser Tyr Ile 20 25 30 Val Glu Thr Pro Asn Pro
Glu Asn Gly Thr Cys Tyr Pro Gly Tyr Phe 35 40 45 Ala Asp Tyr Glu
Glu Leu Arg Glu Gln Leu Ser Ser Val Ser Ser Phe 50 55 60 Glu Arg
Phe Glu Ile Phe Pro Lys Gly Ser Ser Trp Pro Asn His Thr 65 70 75 80
Val Thr Gly Val Ser Ala Ser Cys Ser His Asn Gly Lys Ser Ser Phe 85
90 95 Tyr Arg Asn Leu Leu Trp Leu Thr Gly Lys Asn Gly Leu Tyr Pro
Asn 100 105 110 Leu Ser Met Ser Tyr Val Asn Asn Lys Glu Lys Glu Val
Leu Val Leu 115 120 125 Trp Gly Val His His Pro Pro Asn Ile Gly Asp
Gln Arg Ala Leu Tyr 130 135 140 His Thr Glu Asn Ala Tyr Val Ser Val
Val Ser Ser His Tyr Ser Arg 145 150 155 160 Arg Phe Thr Pro Glu Ile
Ala Lys Arg Pro Lys Val Arg Asp Gln Glu 165 170 175 Gly Arg Ile Asn
Tyr Tyr Trp Thr Leu Leu Glu Pro Gly Asp Thr Ile 180 185 190 Ile Phe
Glu Ala Asn Gly Asn Leu Ile Ala Pro Trp Tyr Ala Phe Ala 195 200 205
Leu Ser Arg Gly Phe Gly Ser Gly 210 215 26217PRTInfluenza A virus
26Ala Pro Leu Gln Leu Gly Asn Cys Ser Ile Ala Gly Trp Ile Leu Gly 1
5 10 15 Asn Pro Glu Cys Glu Ser Leu Phe Ser Lys Lys Ser Trp Ser Tyr
Ile 20 25 30 Ala Glu Thr Pro Asn Ser Glu Asn Gly Thr Cys Tyr Pro
Gly Tyr Phe 35 40 45 Ala Asp Tyr Glu Glu Leu Arg Glu Gln Leu Ser
Ser Val Ser Ser Phe 50 55 60 Glu Arg Phe Glu Ile Phe Pro Lys Glu
Ser Ser Trp Pro Asn His Thr 65 70 75 80 Val Thr Lys Gly Val Thr Ala
Ser Cys Ser His Lys Gly Arg Ser Ser 85 90 95 Phe Tyr Arg Asn Leu
Leu Trp Leu Thr Glu Lys Asn Gly Leu Tyr Pro 100 105 110 Asn Leu Ser
Lys Ser Tyr Val Asn Asn Lys Glu Lys Glu Val Leu Val 115 120 125 Leu
Trp Gly Val His His Pro Ser Asn Ile Gly Asp Gln Arg Ala Ile 130 135
140 Tyr His Thr Glu Asn Ala Tyr Val Ser Val Val Ser Ser His Tyr Asn
145 150 155 160 Arg Arg Phe Thr Pro Glu Ile Ala Lys Arg Pro Lys Val
Arg Gly Gln 165 170 175 Glu Gly Arg Ile Asn Tyr Tyr Trp Thr Leu Leu
Glu Pro Gly Asp Thr 180 185 190 Ile Ile Phe Glu Ala Asn Gly Asn Leu
Ile Ala Pro Trp Tyr Ala Phe 195 200 205 Ala Leu Ser Arg Gly Phe Gly
Ser Gly 210 215 27216PRTInfluenza A virus 27Ala Pro Leu Gln Leu Gly
Asn Cys Ser Val Ala Gly Trp Ile Leu Gly 1 5 10 15 Asn Pro Glu Cys
Glu Leu Leu Ile Ser Lys Glu Ser Trp Ser Tyr Ile 20 25 30 Val Glu
Thr Pro Asn Pro Glu Asn Gly Thr Cys Tyr Pro Gly Tyr Phe 35 40 45
Ala Asp Tyr Glu Glu Leu Arg Glu Gln Leu Ser Ser Val Ser Ser Phe 50
55 60 Glu Arg Phe Glu Ile Phe Pro Lys Glu Ser Ser Trp Pro Asn His
Thr 65 70 75 80 Val Thr Gly Val Ser Ala Ser Cys Ser His Asn Gly Lys
Ser Ser Phe 85 90 95 Tyr Arg Asn Leu Leu Trp Leu Thr Gly Lys Asn
Gly Leu Tyr Pro Asn 100 105 110 Leu Ser Met Ser Tyr Val Asn Asn Lys
Glu Lys Glu Val Leu Val Leu 115 120 125 Trp Gly Val His His Pro Pro
Asn Ile Gly Asn Gln Arg Ala Leu Tyr 130 135 140 His Thr Glu Asn Ala
Tyr Val Ser Val Val Ser Ser His Tyr Ser Arg 145 150 155 160 Arg Phe
Thr Pro Glu Ile Ala Lys Arg Pro Lys Val Arg Asp Gln Glu 165 170 175
Gly Arg Ile Asn Tyr Tyr Trp Thr Leu Leu Glu Pro Gly Asp Thr Ile 180
185 190 Ile Phe Glu Ala Asn Gly Asn Leu Ile Ala Pro Trp Tyr Ala Phe
Ala 195 200 205 Leu Ser Arg Gly Phe Gly Ser Gly 210 215
28216PRTInfluenza A virus 28Ala Pro Leu Gln Leu Gly Asn Cys Ser Val
Ala Gly Trp Ile Leu Gly 1 5 10 15 Asn Pro Glu Cys Glu Leu Leu Ile
Ser Lys Gly Ser Trp Ser Tyr Ile 20 25 30 Val Glu Thr Pro Asn Pro
Glu Asn Gly Thr Cys Tyr Pro Gly Tyr Phe 35 40 45 Ala Asp Tyr Glu
Glu Leu Arg Glu Gln Leu Ser Ser Val Ser Ser Phe 50 55 60 Glu Arg
Phe Glu Ile Phe Pro Lys Gly Ser Ser Trp Pro Asn His Thr 65 70 75 80
Val Thr Gly Val Ser Ala Ser Cys Ser His Asn Gly Lys Ser Ser Phe 85
90 95 Tyr Arg Asn Leu Leu Trp Leu Thr Gly Lys Asn Gly Leu Tyr Pro
Asn 100 105 110 Leu Ser Met Ser Tyr Val Asn Asn Lys Glu Lys Glu Val
Leu Val Leu 115 120 125 Trp Gly Val His His Pro Pro Asn Ile Gly Asn
Gln Arg Ala Leu Tyr 130 135 140 His Thr Glu Asn Ala Tyr Val Ser Val
Val Ser Ser His Tyr Ser Arg 145 150 155 160 Arg Phe Thr Pro Glu Ile
Ala Lys Arg Pro Lys Val Arg Asp Gln Glu 165 170 175 Gly Arg Ile Asn
Tyr Tyr Trp Thr Leu Leu Glu Pro Gly Asp Thr Ile 180 185 190 Ile Phe
Glu Ala Asn Gly Asn Leu Ile Ala Pro Trp Tyr Ala Phe Ala 195 200 205
Leu Ser Arg Gly Phe Gly Ser Gly 210 215 29216PRTInfluenza A virus
29Ala Pro Leu Gln Leu Gly Asn Cys Ser Val Ala Gly Trp Ile Leu Gly 1
5 10 15 Asn Pro Glu Cys Glu Leu Leu Ile Ser Lys Glu Ser Trp Ser Tyr
Ile 20 25 30 Val Glu Thr Pro Asn Pro Glu Asn Gly Thr Cys Tyr Pro
Gly Tyr Phe 35 40 45 Ala Asp Tyr Glu Glu Leu Arg Glu Gln Leu Ser
Ser Val Ser Ser Phe 50 55 60 Glu Arg Phe Glu Ile Phe Pro Lys Glu
Ser Ser Trp Pro Asn His Thr 65 70 75 80 Val Thr Gly Val Ser Ala Ser
Cys Ser His Asn Gly Lys Ser Ser Phe 85 90 95 Tyr Arg Asn Leu Leu
Trp Leu Thr Gly Lys Asn Gly Leu Tyr Pro Asn 100 105 110 Leu Ser Lys
Ser Tyr Ala Asn Asn Lys Lys Lys Glu Val Leu Val Leu 115 120 125 Trp
Gly Val His His Pro Pro Asn Ile Gly Asn Gln Arg Ala Leu Tyr 130 135
140 His Thr Glu Asn Ala Tyr Val Ser Val Val Ser Ser His Tyr Ser Arg
145 150 155 160 Arg Phe Thr Pro Glu Ile Ala Lys Arg Pro Lys Val Arg
Asp Gln Glu 165 170 175 Gly Arg Ile Asn Tyr Tyr Trp Thr Leu Leu Glu
Pro Gly Asp Thr Ile 180 185 190 Ile Phe Glu Ala Asn Gly Asn Leu Ile
Ala Pro Arg Tyr Ala Phe Ala 195 200 205 Leu Ser Arg Gly Phe Gly Ser
Gly 210 215 30216PRTInfluenza A virus 30Ala Pro Leu Gln Leu Gly Asn
Cys Ser Val Ala Gly Trp Ile Leu Gly 1 5 10 15 Asn Pro Glu Cys Glu
Leu Leu Ile Ser Lys Glu Ser Trp Ser Tyr Ile 20 25 30 Val Glu Thr
Pro Asn Pro Glu Asn Gly Ala Cys Tyr Pro Gly Tyr Phe 35 40 45 Ala
Asp Tyr Glu Glu Leu Arg Glu Gln Leu Ser Ser Val Ser Ser Phe 50 55
60 Glu Arg Phe Glu Ile Phe Pro Lys Lys Ser Ser Trp Pro Asn His Thr
65 70 75 80 Val Thr Gly Val Ser Ala Ser Cys Ser His Asn Gly Lys Ser
Ser Phe 85 90 95 Tyr Arg Asn Leu Leu Trp Leu Thr Gly Lys Asn Gly
Leu Tyr Pro Asn 100 105 110 Leu Ser Lys Ser Tyr Ala Asn Asn Lys Lys
Lys Glu Val Leu Ile Leu 115 120 125 Trp Gly Val His His Pro Pro
Asn
Ile Gly Asp Gln Arg Thr Leu Tyr 130 135 140 His Thr Glu Asn Ala Tyr
Val Ser Val Val Ser Ser His Tyr Ser Arg 145 150 155 160 Arg Phe Thr
Pro Glu Ile Thr Lys Arg Pro Lys Val Arg Asp Gln Glu 165 170 175 Gly
Arg Ile Asn Tyr Tyr Trp Thr Leu Leu Glu Pro Gly Asp Thr Ile 180 185
190 Ile Phe Glu Ala Asn Gly Asn Leu Ile Ala Pro Trp Tyr Ala Phe Ala
195 200 205 Leu Ser Arg Gly Phe Gly Ser Gly 210 215
31216PRTInfluenza A virus 31Ala Pro Leu Gln Leu Gly Asn Cys Ser Val
Ala Gly Trp Ile Leu Gly 1 5 10 15 Asn Pro Glu Cys Glu Leu Leu Ile
Ser Lys Glu Ser Trp Ser Tyr Ile 20 25 30 Val Glu Thr Pro Asn Pro
Glu Asn Gly Ala Cys Tyr Pro Gly Tyr Phe 35 40 45 Ala Asp Tyr Glu
Glu Leu Arg Glu Gln Leu Ser Ser Val Ser Ser Phe 50 55 60 Glu Arg
Phe Glu Ile Phe Pro Lys Lys Ser Ser Trp Pro Asn His Thr 65 70 75 80
Val Thr Gly Val Ser Ala Ser Cys Ser His Asn Gly Lys Ser Ser Phe 85
90 95 Tyr Arg Asn Leu Leu Trp Leu Thr Gly Lys Asn Gly Leu Tyr Pro
Asn 100 105 110 Leu Ser Lys Ser Tyr Ala Asn Asn Lys Lys Lys Glu Val
Leu Ile Leu 115 120 125 Trp Gly Val His His Pro Pro Asn Ile Gly Asp
Gln Arg Thr Leu Tyr 130 135 140 His Thr Glu Asn Ala Tyr Val Ser Val
Val Ser Ser His Tyr Ser Arg 145 150 155 160 Arg Phe Thr Pro Glu Ile
Thr Lys Arg Pro Lys Val Arg Asp Gln Glu 165 170 175 Gly Arg Ile Asn
Tyr Tyr Trp Thr Leu Leu Glu Pro Gly Asp Thr Ile 180 185 190 Ile Phe
Glu Ala Asn Gly Asn Leu Ile Ala Pro Trp Tyr Ala Phe Ala 195 200 205
Leu Ser Arg Gly Phe Gly Ser Gly 210 215 32216PRTInfluenza A virus
32Ala Pro Leu Gln Leu Gly Asn Cys Ser Val Ala Gly Trp Ile Leu Gly 1
5 10 15 Asn Pro Glu Cys Glu Leu Leu Ile Ser Lys Glu Ser Trp Ser Tyr
Ile 20 25 30 Val Glu Thr Pro Asn Pro Glu Asn Gly Thr Cys Tyr Pro
Gly Tyr Phe 35 40 45 Ala Asp Tyr Glu Glu Leu Arg Glu Gln Leu Ser
Ser Val Ser Ser Phe 50 55 60 Glu Arg Phe Glu Ile Phe Pro Lys Glu
Ser Ser Trp Pro Asn His Thr 65 70 75 80 Val Thr Gly Val Ser Ala Ser
Cys Ser His Asn Gly Lys Ser Ser Phe 85 90 95 Tyr Arg Asn Leu Leu
Trp Leu Thr Gly Lys Asn Gly Leu Tyr Pro Asn 100 105 110 Leu Ser Lys
Ser Tyr Ala Asn Asn Lys Glu Lys Glu Val Leu Val Leu 115 120 125 Trp
Gly Val His His Pro Pro Asn Ile Gly Asn Gln Arg Ala Leu Tyr 130 135
140 His Thr Glu Asn Ala Tyr Val Ser Val Val Ser Ser His Tyr Ser Arg
145 150 155 160 Arg Phe Thr Pro Glu Ile Ala Lys Arg Pro Lys Val Arg
Asp Gln Glu 165 170 175 Gly Arg Ile Asn Tyr Tyr Trp Thr Leu Leu Glu
Pro Gly Asp Thr Ile 180 185 190 Ile Phe Glu Ala Asn Gly Asn Leu Ile
Ala Pro Arg Tyr Ala Phe Ala 195 200 205 Leu Ser Arg Gly Phe Gly Ser
Gly 210 215 33216PRTInfluenza A virus 33Ala Pro Leu Gln Leu Gly Asn
Cys Ser Val Ala Gly Trp Ile Leu Gly 1 5 10 15 Asn Pro Glu Cys Glu
Ser Leu Ile Ser Lys Glu Ser Trp Ser Tyr Ile 20 25 30 Val Glu Thr
Pro Asn Pro Glu Asn Gly Thr Cys Tyr Pro Gly Tyr Phe 35 40 45 Ala
Asp Tyr Glu Glu Leu Arg Glu Gln Leu Ser Ser Val Ser Ser Phe 50 55
60 Glu Arg Phe Glu Ile Phe Pro Lys Glu Ser Ser Trp Pro Asn His Thr
65 70 75 80 Val Thr Gly Val Ser Ala Ser Cys Ser His Asn Gly Lys Ser
Ser Phe 85 90 95 Tyr Arg Asn Leu Leu Trp Leu Thr Lys Lys Asn Gly
Leu Tyr Pro Asn 100 105 110 Leu Ser Lys Ser Tyr Val Asn Asn Lys Glu
Lys Glu Val Leu Val Leu 115 120 125 Trp Gly Val His His Pro Ser Asn
Ile Gly Asp Gln Arg Thr Ile Tyr 130 135 140 His Thr Glu Asn Ala Tyr
Val Ser Val Val Ser Ser His Tyr Ser Arg 145 150 155 160 Arg Phe Thr
Pro Glu Ile Ala Lys Arg Pro Lys Val Arg Asp Gln Glu 165 170 175 Gly
Arg Ile Asn Tyr Tyr Trp Thr Leu Leu Glu Pro Gly Asp Thr Ile 180 185
190 Ile Phe Glu Ala Asn Gly Asn Leu Ile Ala Pro Trp Tyr Ala Phe Ala
195 200 205 Leu Ser Arg Gly Phe Gly Ser Gly 210 215
34217PRTInfluenza A virus 34Ala Pro Leu His Leu Gly Lys Cys Asn Ile
Ala Gly Trp Ile Leu Gly 1 5 10 15 Asn Pro Glu Cys Glu Ser Leu Ser
Thr Ala Ser Ser Trp Ser Tyr Ile 20 25 30 Val Glu Thr Pro Ser Ser
Asp Asn Gly Thr Cys Tyr Pro Gly Asp Phe 35 40 45 Ile Asp Tyr Glu
Glu Leu Arg Glu Gln Leu Ser Ser Val Ser Ser Phe 50 55 60 Glu Arg
Phe Glu Ile Phe Pro Lys Thr Ser Ser Trp Pro Asn His Asp 65 70 75 80
Ser Asn Lys Gly Val Thr Ala Ala Cys Pro His Ala Gly Ala Lys Ser 85
90 95 Phe Tyr Lys Asn Leu Ile Trp Leu Val Lys Lys Gly Asn Ser Tyr
Pro 100 105 110 Lys Leu Ser Lys Ser Tyr Ile Asn Asp Lys Gly Lys Glu
Val Leu Val 115 120 125 Leu Trp Gly Ile His His Pro Ser Thr Ser Ala
Asp Gln Gln Ser Leu 130 135 140 Tyr Gln Asn Ala Asp Ala Tyr Val Phe
Val Gly Ser Ser Arg Tyr Ser 145 150 155 160 Lys Lys Phe Lys Pro Glu
Ile Ala Ile Arg Pro Lys Val Arg Asp Gln 165 170 175 Glu Gly Arg Met
Asn Tyr Tyr Trp Thr Leu Val Glu Pro Gly Asp Lys 180 185 190 Ile Thr
Phe Glu Ala Thr Gly Asn Leu Val Val Pro Arg Tyr Ala Phe 195 200 205
Ala Met Glu Arg Asn Ala Gly Ser Gly 210 215 35223PRTInfluenza A
virus 35Asn Gly Lys Leu Cys Lys Leu Asn Gly Ile Pro Pro Leu Glu Leu
Gly 1 5 10 15 Asp Cys Ser Ile Ala Gly Trp Leu Leu Gly Asn Pro Glu
Cys Asp Arg 20 25 30 Leu Leu Ser Val Pro Glu Trp Ser Tyr Ile Met
Glu Lys Glu Asn Pro 35 40 45 Arg Asn Gly Leu Cys Tyr Pro Gly Ser
Phe Asn Asp Tyr Glu Glu Leu 50 55 60 Lys His Leu Leu Ser Ser Val
Lys His Phe Glu Lys Val Lys Ile Leu 65 70 75 80 Pro Lys Asp Arg Trp
Thr Gln His Thr Thr Thr Gly Gly Ser Gln Ala 85 90 95 Cys Ala Val
Ser Gly Asn Pro Ser Phe Phe Arg Asn Met Val Trp Leu 100 105 110 Thr
Lys Lys Gly Ser Asp Tyr Pro Val Ala Lys Gly Ser Tyr Asn Asn 115 120
125 Thr Ser Gly Glu Gln Met Leu Ile Ile Trp Gly Val His His Pro Ile
130 135 140 Asp Glu Thr Glu Gln Arg Thr Leu Tyr Gln Asn Val Gly Thr
Tyr Val 145 150 155 160 Ser Val Gly Thr Ser Thr Leu Asn Lys Arg Ser
Thr Pro Glu Ile Ala 165 170 175 Thr Arg Pro Lys Val Asn Gly Leu Gly
Ser Arg Met Glu Phe Ser Trp 180 185 190 Thr Leu Leu Asp Met Trp Asp
Thr Ile Asn Phe Glu Ser Thr Gly Asn 195 200 205 Leu Ile Ala Pro Glu
Tyr Gly Phe Lys Ile Lys Arg Gly Ser Ser 210 215 220
36224PRTInfluenza A virus 36Asn Gly Lys Leu Cys Lys Leu Asn Gly Ile
Pro Pro Leu Glu Leu Gly 1 5 10 15 Asp Cys Ser Ile Ala Gly Trp Leu
Leu Gly Asn Pro Glu Cys Asp Arg 20 25 30 Leu Leu Arg Val Pro Glu
Trp Ser Tyr Ile Met Glu Lys Glu Asn Pro 35 40 45 Arg Asp Gly Leu
Cys Tyr Pro Gly Ser Phe Asn Asp Tyr Glu Glu Leu 50 55 60 Lys His
Leu Leu Ser Ser Val Lys His Phe Glu Lys Val Arg Ile Leu 65 70 75 80
Pro Lys Asp Arg Trp Thr Gln His Thr Thr Thr Gly Gly Ser Arg Ala 85
90 95 Cys Ala Val Ser Gly Asn Pro Ser Phe Phe Arg Asn Met Ile Trp
Leu 100 105 110 Thr Lys Lys Gly Ser Asn Tyr Pro Val Ala Lys Gly Ser
Tyr Asn Asn 115 120 125 Thr Ser Gly Glu Gln Met Leu Ile Ile Trp Gly
Val His His Pro Ile 130 135 140 Asp Glu Thr Glu Gln Arg Thr Leu Tyr
Gln Asn Val Glu Thr Tyr Val 145 150 155 160 Ser Val Val Thr Ser Thr
Leu Asn Lys Arg Ser Thr Pro Lys Ile Ala 165 170 175 Thr Arg Pro Lys
Val Asn Gly Leu Gly Gly Arg Met Glu Phe Ser Trp 180 185 190 Thr Leu
Leu Asp Met Trp Asp Thr Ile Asn Phe Glu Ser Thr Gly Asn 195 200 205
Leu Ile Ala Pro Glu Tyr Gly Phe Lys Ile Ser Lys Arg Gly Ser Ser 210
215 220 37224PRTInfluenza A virus 37Asn Gly Lys Leu Cys Lys Leu Asn
Gly Ile Pro Pro Leu Glu Leu Gly 1 5 10 15 Asp Cys Ser Ile Ala Gly
Trp Leu Leu Gly Asn Pro Glu Cys Asp Arg 20 25 30 Leu Leu Ser Val
Pro Glu Trp Ser Tyr Ile Met Glu Lys Glu Asn Pro 35 40 45 Arg Asp
Gly Leu Cys Tyr Pro Gly Ser Phe Asn Asp Tyr Glu Glu Leu 50 55 60
Lys His Leu Leu Ser Ser Val Lys His Phe Glu Lys Val Lys Ile Leu 65
70 75 80 Pro Lys Asp Arg Trp Thr Gln His Thr Thr Thr Gly Gly Ser
Arg Ala 85 90 95 Cys Ala Val Ser Gly Asn Pro Ser Phe Phe Arg Asn
Met Val Trp Leu 100 105 110 Thr Glu Lys Gly Ser Asn Tyr Pro Val Ala
Lys Gly Ser Tyr Asn Asn 115 120 125 Thr Ser Gly Glu Gln Met Leu Ile
Ile Trp Gly Val His His Pro Asn 130 135 140 Asp Glu Thr Glu Gln Arg
Thr Leu Tyr Gln Asn Val Gly Thr Tyr Val 145 150 155 160 Ser Val Gly
Thr Ser Thr Leu Asn Lys Arg Ser Thr Pro Glu Ile Ala 165 170 175 Thr
Arg Pro Lys Val Asn Gly Leu Gly Gly Arg Met Glu Phe Ser Trp 180 185
190 Thr Leu Leu Asp Met Trp Asp Thr Ile Asn Phe Glu Ser Thr Gly Asn
195 200 205 Leu Ile Ala Pro Glu Tyr Gly Phe Lys Ile Ser Lys Arg Gly
Ser Ser 210 215 220 38224PRTInfluenza A virus 38Asn Gly Lys Leu Cys
Lys Leu Asn Gly Ile Pro Pro Leu Glu Leu Gly 1 5 10 15 Asp Cys Ser
Ile Ala Gly Trp Leu Leu Gly Asn Pro Glu Cys Asp Arg 20 25 30 Leu
Leu Arg Val Pro Glu Trp Ser Tyr Ile Met Glu Lys Glu Asn Pro 35 40
45 Arg Tyr Ser Leu Cys Tyr Pro Gly Ser Phe Asn Asp Tyr Glu Glu Leu
50 55 60 Lys His Leu Leu Ser Ser Val Lys His Phe Glu Lys Val Arg
Ile Leu 65 70 75 80 Pro Lys Asp Arg Trp Thr Gln His Thr Thr Thr Gly
Asp Ser Lys Ala 85 90 95 Cys Ala Val Ser Gly Lys Pro Ser Phe Phe
Arg Asn Met Val Trp Leu 100 105 110 Thr Lys Lys Gly Pro Asn Tyr Pro
Val Ala Lys Gly Ser Tyr Asn Asn 115 120 125 Thr Ser Gly Glu Gln Met
Leu Ile Ile Trp Gly Val His His Pro Lys 130 135 140 Asp Glu Ala Glu
Gln Arg Ala Leu Tyr Gln Asn Val Gly Thr Tyr Val 145 150 155 160 Ser
Ala Ser Thr Ser Thr Leu Asn Lys Arg Ser Ile Pro Glu Ile Ala 165 170
175 Thr Arg Pro Glu Val Asn Gly Leu Gly Ser Arg Met Glu Phe Ser Trp
180 185 190 Thr Leu Leu Asp Ala Trp Asp Thr Ile Asn Phe Glu Ser Thr
Gly Asn 195 200 205 Leu Val Ala Pro Glu Tyr Gly Phe Lys Ile Ser Lys
Arg Gly Ser Ser 210 215 220 39211PRTInfluenza A virus 39His Gln Ile
Leu Asp Gly Glu Asn Cys Thr Leu Ile Asp Ala Leu Leu 1 5 10 15 Gly
Asp Pro Gln Cys Asp Gly Phe Gln Asn Lys Lys Trp Asp Leu Phe 20 25
30 Val Glu Arg Ser Lys Ala Tyr Ser Asn Cys Tyr Pro Tyr Asp Val Pro
35 40 45 Asp Tyr Ala Ser Leu Arg Ser Leu Val Ala Ser Ser Gly Thr
Leu Glu 50 55 60 Phe Asn Asp Glu Ser Phe Asn Trp Thr Gly Val Thr
Gln Asn Gly Thr 65 70 75 80 Ser Ser Ser Cys Lys Arg Arg Ser Asn Asn
Ser Phe Phe Ser Arg Leu 85 90 95 Asn Trp Leu Thr His Leu Lys Phe
Lys Tyr Pro Ala Leu Asn Val Thr 100 105 110 Met Pro Asn Asn Glu Lys
Phe Asp Lys Leu Tyr Ile Trp Gly Val His 115 120 125 His Pro Val Thr
Asp Asn Asp Gln Ile Phe Leu Tyr Ala Gln Ala Ser 130 135 140 Gly Arg
Ile Thr Val Ser Thr Lys Arg Ser Gln Gln Thr Val Ile Pro 145 150 155
160 Asn Ile Gly Ser Arg Pro Arg Ile Arg Asn Ile Pro Ser Arg Ile Ser
165 170 175 Ile Tyr Trp Thr Ile Val Lys Pro Gly Asp Ile Leu Leu Ile
Asn Ser 180 185 190 Thr Gly Asn Leu Ile Ala Pro Arg Gly Tyr Phe Lys
Ile Arg Ser Gly 195 200 205 Lys Ser Ser 210 40211PRTInfluenza A
virus 40His Gln Ile Leu Asp Gly Lys Asn Cys Thr Leu Ile Asp Ala Leu
Leu 1 5 10 15 Gly Asp Pro Gln Cys Asp Gly Phe Gln Asn Lys Lys Trp
Asp Leu Phe 20 25 30 Val Glu Arg Ser Lys Ala Tyr Ser Asn Cys Tyr
Pro Tyr Asp Val Pro 35 40 45 Asp Tyr Ala Ser Leu Arg Ser Leu Val
Ala Ser Ser Gly Thr Leu Glu 50 55 60 Phe Asn Asn Glu Ser Phe Asn
Trp Thr Gly Val Thr Gln Asn Gly Thr 65 70 75 80 Ser Ser Ala Cys Ile
Arg Arg Ser Lys Asn Ser Phe Phe Ser Arg Leu 85 90 95 Asn Trp Leu
Thr His Leu Asn Phe Lys Tyr Pro Ala Leu Asn Val Thr 100 105 110 Met
Pro Asn Asn Glu Gln Phe Asp Lys Leu Tyr Ile Trp Gly Val His 115 120
125 His Pro Gly Thr Asp Lys Asp Gln Ile Phe Pro Tyr Ala Gln Ala Ser
130 135 140 Gly Arg Ile Thr Val Ser Thr Lys Arg Ser Gln Gln Thr Ala
Ile Pro 145 150 155 160 Asn Ile Gly Ser Arg Pro Arg Val Arg Asn Ile
Pro Ser Arg Ile Ser 165 170 175 Ile Tyr Trp Thr Ile Val Lys Pro Gly
Asp Ile Leu Leu Ile Asn Ser 180 185 190 Thr Gly Asn Leu Ile Ala Pro
Arg Gly Tyr Phe Lys Ile Arg Ser Gly 195 200 205 Lys Ser Ser 210
41211PRTInfluenza A virus
41His Arg Ile Leu Asp Gly Ile Asp Cys Thr Leu Ile Asp Ala Leu Leu 1
5 10 15 Gly Asp Pro His Cys Asp Val Phe Gln Asn Glu Thr Trp Asp Leu
Phe 20 25 30 Val Glu Arg Ser Lys Ala Phe Ser Asn Cys Tyr Pro Tyr
Asp Val Pro 35 40 45 Asp Tyr Ala Ser Leu Arg Ser Leu Val Ala Ser
Ser Gly Thr Leu Glu 50 55 60 Phe Ile Thr Glu Gly Phe Thr Trp Thr
Gly Val Thr Gln Asn Gly Gly 65 70 75 80 Ser Asn Ala Cys Lys Arg Gly
Pro Gly Ser Gly Phe Phe Ser Arg Leu 85 90 95 Asn Trp Leu Thr Lys
Ser Gly Ser Thr Tyr Pro Val Leu Asn Val Thr 100 105 110 Met Pro Asn
Asn Asp Asn Phe Asp Lys Leu Tyr Ile Trp Gly Ile His 115 120 125 His
Pro Ser Thr Asp Gln Glu Gln Thr Ser Leu Tyr Val Gln Ala Ser 130 135
140 Gly Arg Val Thr Val Ser Thr Arg Arg Ser Gln Gln Thr Ile Ile Pro
145 150 155 160 Asn Ile Gly Ser Arg Pro Trp Val Arg Gly Leu Ser Ser
Arg Ile Ser 165 170 175 Ile Tyr Trp Thr Ile Val Lys Pro Gly Asp Val
Leu Val Ile Asn Ser 180 185 190 Asn Gly Asn Leu Ile Ala Pro Arg Gly
Tyr Phe Lys Met Arg Thr Gly 195 200 205 Lys Ser Ser 210
42211PRTInfluenza A virus 42His Gln Ile Leu Asp Gly Glu Asn Cys Thr
Leu Ile Asp Ala Leu Leu 1 5 10 15 Gly Asp Pro Gln Cys Asp Gly Phe
Gln Asn Lys Lys Trp Asp Leu Phe 20 25 30 Val Glu Arg Ser Lys Ala
Tyr Ser Asn Cys Tyr Pro Tyr Asp Val Pro 35 40 45 Asp Tyr Ala Ser
Leu Arg Ser Leu Val Ala Ser Ser Gly Thr Leu Glu 50 55 60 Phe Asn
Asn Glu Ser Phe Asn Trp Thr Gly Val Thr Gln Asn Gly Thr 65 70 75 80
Ser Ser Ala Cys Ile Arg Arg Ser Asn Asn Ser Phe Phe Ser Arg Leu 85
90 95 Asn Trp Leu Thr His Leu Lys Phe Lys Tyr Pro Ala Leu Asn Val
Thr 100 105 110 Met Pro Asn Asn Glu Lys Phe Asp Lys Leu Tyr Ile Trp
Gly Val His 115 120 125 His Pro Gly Thr Asp Asn Asp Gln Ile Phe Pro
Tyr Ala Gln Ala Ser 130 135 140 Gly Arg Ile Thr Val Ser Thr Lys Arg
Ser Gln Gln Thr Val Ile Pro 145 150 155 160 Asn Ile Gly Ser Arg Pro
Arg Val Arg Asn Ile Pro Ser Arg Ile Ser 165 170 175 Ile Tyr Trp Thr
Ile Val Lys Pro Gly Asp Ile Leu Leu Ile Asn Ser 180 185 190 Thr Gly
Asn Leu Ile Ala Pro Arg Gly Tyr Phe Lys Ile Arg Ser Gly 195 200 205
Lys Ser Ser 210 43211PRTInfluenza A virus 43His Gln Ile Leu Asp Gly
Glu Asn Cys Thr Leu Ile Asp Ala Leu Leu 1 5 10 15 Gly Asp Pro His
Cys Asp Gly Phe Gln Asn Lys Glu Trp Asp Leu Phe 20 25 30 Val Glu
Arg Ser Lys Ala Tyr Ser Asn Cys Tyr Pro Tyr Asp Val Pro 35 40 45
Asp Tyr Ala Ser Leu Arg Ser Leu Val Ala Ser Ser Gly Thr Leu Glu 50
55 60 Phe Asn Asn Glu Ser Phe Asn Trp Thr Gly Val Ala Gln Asn Gly
Thr 65 70 75 80 Ser Ser Ser Cys Lys Arg Arg Ser Ile Lys Ser Phe Phe
Ser Arg Leu 85 90 95 Asn Trp Leu His Gln Leu Lys Tyr Arg Tyr Pro
Ala Leu Asn Val Thr 100 105 110 Met Pro Asn Asn Asp Lys Phe Asp Lys
Leu Tyr Ile Trp Gly Val His 115 120 125 His Pro Ser Thr Asp Ser Asp
Gln Thr Ser Leu Tyr Thr Gln Ala Ser 130 135 140 Gly Arg Val Thr Val
Ser Thr Lys Arg Ser Gln Gln Thr Val Ile Pro 145 150 155 160 Asn Ile
Gly Ser Arg Pro Trp Val Arg Gly Ile Ser Ser Arg Ile Ser 165 170 175
Ile Tyr Trp Thr Ile Val Lys Pro Gly Asp Ile Leu Leu Ile Asn Ser 180
185 190 Thr Gly Asn Leu Ile Ala Pro Arg Gly Tyr Phe Lys Ile Arg Ser
Gly 195 200 205 Lys Ser Ser 210 44215PRTInfluenza A virus 44Val Lys
Pro Leu Ile Leu Arg Asp Cys Ser Val Ala Gly Trp Leu Leu 1 5 10 15
Gly Asn Pro Met Cys Asp Glu Phe Ile Asn Val Pro Glu Trp Ser Tyr 20
25 30 Ile Val Glu Lys Ala Asn Pro Thr Asn Asp Leu Cys Tyr Pro Gly
Ser 35 40 45 Phe Asn Asp Tyr Glu Glu Leu Lys His Leu Leu Ser Arg
Ile Asn His 50 55 60 Phe Glu Lys Ile Gln Ile Ile Pro Lys Ser Ser
Trp Ser Asp His Glu 65 70 75 80 Ala Ser Ser Gly Val Ser Ser Ala Cys
Pro Tyr Leu Gly Ser Pro Ser 85 90 95 Phe Phe Arg Asn Val Val Trp
Leu Ile Lys Lys Asn Ser Thr Tyr Pro 100 105 110 Thr Ile Lys Lys Ser
Tyr Asn Asn Thr Asn Gln Glu Asp Leu Leu Val 115 120 125 Leu Trp Gly
Ile His His Pro Lys Asp Ala Ala Glu Gln Thr Arg Leu 130 135 140 Tyr
Gln Asn Pro Thr Thr Tyr Ile Ser Ile Gly Thr Ser Thr Leu Asn 145 150
155 160 Gln Arg Leu Val Pro Lys Ile Ala Thr Arg Ser Lys Val Asn Gly
Leu 165 170 175 Ser Ser Arg Met Glu Phe Phe Trp Thr Ile Leu Lys Pro
Asn Asp Ala 180 185 190 Ile Asn Phe Glu Ser Asn Gly Asn Phe Ile Ala
Pro Glu Tyr Ala Tyr 195 200 205 Lys Ile Val Lys Lys Gly Asp 210 215
45216PRTInfluenza A virus 45Val Lys Pro Leu Ile Leu Arg Asp Cys Ser
Val Ala Gly Trp Leu Leu 1 5 10 15 Gly Asn Pro Met Cys Asp Glu Phe
Ile Asn Val Pro Glu Trp Ser Tyr 20 25 30 Ile Val Glu Lys Ala Asn
Pro Thr Asn Asp Leu Cys Tyr Pro Gly Ser 35 40 45 Phe Asn Asp Tyr
Glu Glu Leu Lys His Leu Leu Ser Arg Ile Asn His 50 55 60 Phe Glu
Lys Ile Gln Ile Ile Pro Lys Ser Ser Trp Ser Asp His Glu 65 70 75 80
Ala Ser Ser Gly Val Ser Ser Ala Cys Pro Tyr Leu Gly Ser Pro Ser 85
90 95 Phe Phe Arg Asn Val Val Trp Leu Ile Lys Lys Asn Ser Thr Tyr
Pro 100 105 110 Thr Ile Lys Lys Ser Tyr Asn Asn Thr Asn Gln Glu Asp
Leu Leu Val 115 120 125 Leu Trp Gly Ile His His Pro Asn Asp Ala Ala
Glu Gln Thr Arg Leu 130 135 140 Tyr Gln Asn Pro Thr Thr Tyr Ile Ser
Ile Gly Thr Ser Thr Leu Asn 145 150 155 160 Gln Arg Leu Val Pro Lys
Ile Ala Thr Arg Ser Lys Val Asn Gly Gln 165 170 175 Ser Gly Arg Met
Glu Phe Phe Trp Thr Ile Leu Lys Pro Asn Asp Ala 180 185 190 Ile Asn
Phe Glu Ser Asn Gly Asn Phe Ile Ala Pro Glu Tyr Ala Tyr 195 200 205
Lys Ile Val Lys Lys Gly Asp Ser 210 215 466PRTArtificial
SequenceDescription of Artificial Sequence Synthetic 6xHis tag
46His His His His His His 1 5 4724DNAArtificial SequenceDescription
of Artificial Sequence Synthetic primer 47aagcttacca tgccgatggg
ctcc 244830DNAArtificial SequenceDescription of Artificial Sequence
Synthetic primer 48gagcccaaat cttgtgacaa atgatctaga
304912DNAArtificial SequenceDescription of Artificial Sequence
Synthetic oligonucleotide 49tcttgtgaca aa 12504PRTArtificial
SequenceDescription of Artificial Sequence Synthetic peptide 50Ser
Cys Asp Lys 1 51126PRTHomo sapiensMOD_RES(99)..(99)Gly or Ala 51Gln
Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala 1 5 10
15 Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Gly Tyr
20 25 30 Tyr Met His Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu
Trp Met 35 40 45 Gly Trp Ile Asn Pro Asn Ser Gly Gly Thr Asn Tyr
Ala Gln Lys Phe 50 55 60 Gln Gly Trp Val Thr Met Thr Arg Asp Thr
Ser Ile Ser Thr Ala Tyr 65 70 75 80 Met Glu Leu Ser Arg Leu Arg Ser
Asp Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Xaa Gly Leu Glu
Pro Arg Ser Val Asp Tyr Tyr Tyr Tyr Gly 100 105 110 Met Asp Val Trp
Gly Gln Gly Thr Thr Val Thr Val Ser Ser 115 120 125
5268DNAArtificial SequenceDescription of Artificial Sequence
Synthetic oligonucleotide 52attattgtgc gagaggggga ctggaacccc
gatctgtaga ctactactat tatggtatgg 60acgtctgg 685368DNAArtificial
SequenceDescription of Artificial Sequence Synthetic
oligonucleotide 53attactgtgc gagaggggga ctggaacccc gatctgtaga
ctactactac tacggtatgg 60acgtctgg 685414DNAArtificial
SequenceDescription of Artificial Sequence Synthetic
oligonucleotide 54attactgtgc gaga 145517DNAArtificial
SequenceDescription of Artificial Sequence Synthetic
oligonucleotide 55ggtacaactg gaacgac 175632DNAArtificial
SequenceDescription of Artificial Sequence Synthetic
oligonucleotide 56attactacta ctactacggt atggacgtct gg
3257214PRTInfluenza A virus 57Lys Pro Leu Ile Leu Arg Asp Cys Ser
Val Ala Gly Trp Leu Leu Gly 1 5 10 15 Asn Pro Met Cys Asp Glu Phe
Ile Asn Val Pro Glu Trp Ser Tyr Ile 20 25 30 Val Glu Lys Ala Asn
Pro Thr Asn Asp Leu Cys Tyr Pro Gly Ser Phe 35 40 45 Asn Asp Tyr
Glu Glu Leu Lys His Leu Leu Ser Arg Ile Asn His Phe 50 55 60 Glu
Lys Ile Gln Ile Ile Pro Lys Ser Ser Trp Ser Asp His Glu Ala 65 70
75 80 Ser Ser Gly Val Ser Ser Ala Cys Pro Tyr Leu Gly Ser Pro Ser
Phe 85 90 95 Phe Arg Asn Val Val Trp Leu Ile Lys Lys Asn Ser Thr
Tyr Pro Thr 100 105 110 Ile Lys Lys Ser Tyr Asn Asn Thr Asn Gln Glu
Asp Leu Leu Val Leu 115 120 125 Trp Gly Ile His His Pro Lys Asp Ala
Ala Glu Gln Thr Arg Leu Tyr 130 135 140 Gln Asn Pro Thr Thr Tyr Ile
Ser Ile Gly Thr Ser Thr Leu Asn Gln 145 150 155 160 Arg Leu Val Pro
Lys Ile Ala Thr Arg Ser Lys Val Asn Gly Leu Ser 165 170 175 Ser Arg
Met Glu Phe Phe Trp Thr Ile Leu Lys Pro Asn Asp Ala Ile 180 185 190
Asn Phe Glu Ser Asn Gly Asn Phe Ile Ala Pro Glu Tyr Ala Tyr Lys 195
200 205 Ile Val Lys Lys Gly Asp 210 58213PRTInfluenza A virus 58Pro
Pro Leu Glu Leu Gly Asp Cys Ser Ile Ala Gly Trp Leu Leu Gly 1 5 10
15 Asn Pro Glu Cys Asp Arg Leu Leu Ser Val Pro Glu Trp Ser Tyr Ile
20 25 30 Met Glu Lys Glu Asn Pro Arg Asn Gly Leu Cys Tyr Pro Gly
Ser Phe 35 40 45 Asn Asp Tyr Glu Glu Leu Lys His Leu Leu Ser Ser
Val Lys His Phe 50 55 60 Glu Lys Val Lys Ile Leu Pro Lys Asp Arg
Trp Thr Gln His Thr Thr 65 70 75 80 Thr Gly Gly Ser Gln Ala Cys Ala
Val Ser Gly Asn Pro Ser Phe Phe 85 90 95 Arg Asn Met Val Trp Leu
Thr Lys Lys Gly Ser Asp Tyr Pro Val Ala 100 105 110 Lys Gly Ser Tyr
Asn Asn Thr Ser Gly Glu Gln Met Leu Ile Ile Trp 115 120 125 Gly Val
His His Pro Ile Asp Glu Thr Glu Gln Arg Thr Leu Tyr Gln 130 135 140
Asn Val Gly Thr Tyr Val Ser Val Gly Thr Ser Thr Leu Asn Lys Arg 145
150 155 160 Ser Thr Pro Glu Ile Ala Thr Arg Pro Lys Val Asn Gly Leu
Gly Ser 165 170 175 Arg Met Glu Phe Ser Trp Thr Leu Leu Asp Met Trp
Asp Thr Ile Asn 180 185 190 Phe Glu Ser Thr Gly Asn Leu Ile Ala Pro
Glu Tyr Gly Phe Lys Ile 195 200 205 Lys Arg Gly Ser Ser 210
59215PRTInfluenza A virus 59Ala Pro Leu His Leu Gly Lys Cys Asn Ile
Ala Gly Trp Ile Leu Gly 1 5 10 15 Asn Pro Glu Cys Glu Ser Leu Ser
Thr Ala Ser Ser Trp Ser Tyr Ile 20 25 30 Val Glu Thr Pro Ser Ser
Asp Asn Gly Thr Cys Tyr Pro Gly Asp Phe 35 40 45 Ile Asp Tyr Glu
Glu Leu Arg Glu Gln Leu Ser Ser Val Ser Ser Phe 50 55 60 Glu Arg
Phe Glu Ile Phe Pro Lys Thr Ser Ser Trp Pro Asn His Asp 65 70 75 80
Ser Asn Lys Gly Val Thr Ala Ala Cys Pro His Ala Gly Ala Lys Ser 85
90 95 Phe Tyr Lys Asn Leu Ile Trp Leu Val Lys Lys Gly Asn Ser Tyr
Pro 100 105 110 Lys Leu Ser Lys Ser Tyr Ile Asn Asp Lys Gly Lys Glu
Val Leu Val 115 120 125 Leu Trp Gly Ile His His Pro Ser Thr Ser Ala
Asp Gln Gln Ser Leu 130 135 140 Tyr Gln Asn Ala Asp Ala Tyr Val Phe
Val Gly Ser Ser Arg Tyr Ser 145 150 155 160 Lys Lys Phe Lys Pro Glu
Ile Ala Ile Arg Pro Lys Val Arg Asp Gln 165 170 175 Glu Gly Arg Met
Asn Tyr Tyr Trp Thr Leu Val Glu Pro Gly Asp Lys 180 185 190 Ile Thr
Phe Glu Ala Thr Gly Asn Leu Val Val Pro Arg Tyr Ala Phe 195 200 205
Ala Met Glu Arg Asn Ala Gly 210 215 60214PRTInfluenza A virus 60Ala
Pro Leu Gln Leu Gly Asn Cys Ser Val Ala Gly Trp Ile Leu Gly 1 5 10
15 Asn Pro Glu Cys Glu Leu Leu Ile Ser Arg Glu Ser Trp Ser Tyr Ile
20 25 30 Val Glu Lys Pro Asn Pro Glu Asn Gly Thr Cys Tyr Pro Gly
His Phe 35 40 45 Ala Asp Tyr Glu Glu Leu Arg Glu Gln Leu Ser Ser
Val Ser Ser Phe 50 55 60 Glu Arg Phe Glu Ile Phe Pro Lys Glu Ser
Ser Trp Pro Asn His Thr 65 70 75 80 Thr Thr Gly Val Ser Ala Ser Cys
Ser His Asn Gly Glu Ser Ser Phe 85 90 95 Tyr Lys Asn Leu Leu Trp
Leu Thr Gly Lys Asn Gly Leu Tyr Pro Asn 100 105 110 Leu Ser Lys Ser
Tyr Ala Asn Asn Lys Glu Lys Glu Val Leu Val Leu 115 120 125 Trp Gly
Val His His Pro Pro Asn Ile Gly Asp Gln Arg Ala Leu Tyr 130 135 140
His Thr Glu Asn Ala Tyr Val Ser Val Val Ser Ser His Tyr Ser Arg 145
150 155 160 Lys Phe Thr Pro Glu Ile Ala Lys Arg Pro Lys Val Arg Asp
Arg Glu 165 170 175 Gly Arg Ile Asn Tyr Tyr Trp Thr Leu Leu Glu Pro
Gly Asp Thr Ile 180 185 190 Ile Phe Glu Ala Asn Gly Asn Leu Ile Ala
Pro Arg Tyr Ala Phe Ala 195 200 205 Leu Ser Arg Gly Phe Gly 210
61209PRTInfluenza A virus 61Arg Ile Leu Asp Gly Ile Asp Cys Thr Leu
Ile
Asp Ala Leu Leu Gly 1 5 10 15 Asp Pro His Cys Asp Val Phe Gln Asn
Glu Thr Trp Asp Leu Phe Val 20 25 30 Glu Arg Ser Lys Ala Phe Ser
Asn Cys Tyr Pro Tyr Asp Val Pro Asp 35 40 45 Tyr Ala Ser Leu Arg
Ser Leu Val Ala Ser Ser Gly Thr Leu Glu Phe 50 55 60 Ile Thr Glu
Gly Phe Thr Trp Thr Gly Val Thr Gln Asn Gly Gly Ser 65 70 75 80 Asn
Ala Cys Lys Arg Gly Pro Gly Ser Gly Phe Phe Ser Arg Leu Asn 85 90
95 Trp Leu Thr Lys Ser Gly Ser Thr Tyr Pro Val Leu Asn Val Thr Met
100 105 110 Pro Asn Asn Asp Asn Phe Asp Lys Leu Tyr Ile Trp Gly Ile
His His 115 120 125 Pro Ser Thr Asp Gln Glu Gln Thr Ser Leu Tyr Val
Gln Ala Ser Gly 130 135 140 Arg Val Thr Val Ser Thr Arg Arg Ser Gln
Gln Thr Ile Ile Pro Asn 145 150 155 160 Ile Gly Ser Arg Pro Trp Val
Arg Gly Leu Ser Ser Arg Ile Ser Ile 165 170 175 Tyr Trp Thr Ile Val
Lys Pro Gly Asp Val Leu Val Ile Asn Ser Asn 180 185 190 Gly Asn Leu
Ile Ala Pro Arg Gly Tyr Phe Lys Met Arg Thr Gly Lys 195 200 205
Ser
* * * * *