U.S. patent application number 15/645399 was filed with the patent office on 2017-11-02 for influenza a virus specific antibodies.
The applicant listed for this patent is AIMM THERAPEUTICS B.V.. Invention is credited to Arjen Quirinus BAKKER, Tim BEAUMONT, Mark Jeroen KWAKKENBOS, Hergen SPITS.
Application Number | 20170313766 15/645399 |
Document ID | / |
Family ID | 47297373 |
Filed Date | 2017-11-02 |
United States Patent
Application |
20170313766 |
Kind Code |
A1 |
SPITS; Hergen ; et
al. |
November 2, 2017 |
INFLUENZA A VIRUS SPECIFIC ANTIBODIES
Abstract
The invention relates to isolated, synthetic or recombinant
antibodies and functional parts thereof specific for multiple
influenza A virus subtypes. The invention further relates to the
use of such antibodies for diagnosis of an influenza A virus
infection and as a medicament and/or prophylactic agent for at
least in part treating or alleviating symptoms of an influenza A
virus infection.
Inventors: |
SPITS; Hergen; (Amsterdam,
NL) ; BEAUMONT; Tim; (Amsterdam, NL) ;
KWAKKENBOS; Mark Jeroen; (Amsterdam, NL) ; BAKKER;
Arjen Quirinus; (Amsterdam, NL) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
AIMM THERAPEUTICS B.V. |
AMSTERDAM |
|
NL |
|
|
Family ID: |
47297373 |
Appl. No.: |
15/645399 |
Filed: |
July 10, 2017 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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14362210 |
Jun 2, 2014 |
9718874 |
|
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PCT/NL2012/050851 |
Dec 3, 2012 |
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15645399 |
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Current U.S.
Class: |
1/1 |
Current CPC
Class: |
A61P 31/16 20180101;
A61K 2039/505 20130101; C07K 2317/21 20130101; C07K 2317/565
20130101; G01N 33/56983 20130101; C07K 2317/31 20130101; C07K
2317/76 20130101; C07K 2317/92 20130101; C07K 2317/33 20130101;
C07K 16/1018 20130101 |
International
Class: |
C07K 16/10 20060101
C07K016/10; G01N 33/569 20060101 G01N033/569 |
Foreign Application Data
Date |
Code |
Application Number |
Dec 2, 2011 |
EP |
111991783.7 |
Claims
1. A synthetic or recombinant antibody or functional part thereof,
or immunoglobulin chain or functional equivalent thereof: said
synthetic or recombinant antibody or functional part thereof, or
immunoglobulin chain or functional equivalent thereof having an in
vitro H3N2 influenza A virus neutralizing activity with an IC50
value of less than 1 .mu.g/ml; and wherein said synthetic or
recombinant antibody or functional part thereof or immunoglobulin
chain or functional equivalent is capable of specifically binding
at least one other influenza A virus subtype.
2. The synthetic or recombinant antibody or functional part
thereof, or immunoglobulin chain or functional equivalent thereof
of claim 1, wherein said at least one other influenza A virus
subtype is a group 2 influenza A virus subtype.
3. The synthetic or recombinant antibody or functional part
thereof, or immunoglobulin chain or functional equivalent thereof
of claim 1, wherein said at least one other influenza A virus
subtype is a group 1 influenza A virus subtype.
4. The synthetic or recombinant antibody or functional part
thereof, or immunoglobulin chain or functional equivalent thereof
of claim 1, comprising: a heavy chain CDR1 sequence comprising a
sequence which is at least 85% identical to a sequence selected
from the group consisting of SEQ ID NO's:1-5, and/or a heavy chain
CDR2 sequence comprising a sequence which is at least 85% identical
to a sequence selected from the group consisting of SEQ ID
NO's:6-10, and/or a heavy chain CDR3 sequence comprising a sequence
which is at least 85% identical to a sequence selected from the
group consisting of SEQ ID NO's:11-15, and/or a light chain CDR1
sequence comprising a sequence which is at least 85% identical to a
sequence selected from the group consisting of SEQ ID NO's:16-20,
and/or a light chain CDR2 sequence comprising a sequence which is
at least 85% identical to a sequence selected from the group
consisting of SEQ ID NO's:21-25, and/or a light chain CDR3 sequence
comprising a sequence which is at least 85% identical to a sequence
selected from the group consisting of SEQ ID NO's:26-30.
5. The synthetic or recombinant antibody or functional part
thereof, or immunoglobulin chain or functional equivalent thereof
of claim 4, having a heavy chain sequence comprising a sequence
which is at least 85% identical to a sequence selected from the
group consisting of SEQ ID NO's:31-35 and/or having a light chain
sequence which is at least 85% identical to a sequence selected
from the group consisting of SEQ ID NO's:36-40.
6. The synthetic or recombinant antibody or functional part
thereof, or immunoglobulin chain or functional equivalent thereof
of claim 4, wherein said synthetic or recombinant antibody or
functional part thereof, or immunoglobulin chain or functional
equivalent thereof is a nucleic acid molecule or a functional
equivalent with a length of at least 15 nucleotides, or a
functional equivalent thereof, encoding at least one CDR sequence
of an antibody or functional part thereof or immunoglobulin chain
or functional equivalent.
7. The synthetic or recombinant antibody or functional part
thereof, or immunoglobulin chain or functional equivalent thereof
of claim 6, comprising a sequence which has at least 85% sequence
identity to a sequence selected from the group consisting of: SEQ
ID NO's:41-45, and SEQ ID NO's:46-50, and SEQ ID NO's:51-55, and
SEQ ID NO's:56-60, and SEQ ID NO's:61-65, and SEQ ID
NO's:66-70.
8. The synthetic or recombinant antibody or functional part
thereof, or immunoglobulin chain or functional equivalent thereof
of claim 6, comprising a sequence which has at least 85% sequence
identity to a sequence selected from the group consisting of SEQ ID
NO's:71-75 and/or comprising a sequence which has at least 85%
sequence identity to a sequence selected from the group consisting
of SEQ ID NO's:76-80.
9. A vector comprising the nucleic acid molecule or functional
equivalent of claim 6.
10. An isolated or recombinant cell comprising the nucleic acid
molecule or functional equivalent of claim 6, and/or a vector of
claim 9.
11. A pharmaceutical composition comprising: at least one of:
synthetic or recombinant antibody or functional part thereof, or
immunoglobulin chain or functional equivalent thereof of claim 4;
the nucleic acid molecule or functional equivalent of claim 6, or
the vector of claim 9; and a pharmaceutical acceptable carrier,
diluent and/or excipient.
12. The synthetic or recombinant antibody or functional part
thereof, or immunoglobulin chain or functional equivalent thereof
of claim 4 or a vector thereof, for use as a medicament and/or
prophylactic agent.
13. The synthetic or recombinant antibody or functional part
thereof, or immunoglobulin chain or functional equivalent thereof
of claim 12, wherein said use is treating and/or inhibiting an
influenza A virus infection.
14. A method for producing the synthetic or recombinant antibody or
functional part thereof, or immunoglobulin chain or functional
equivalent thereof of claim 1 comprising: providing a cell with the
nucleic acid molecule or functional equivalent of claim 6, or the
vector of claim 9, and allowing said cell to translate said nucleic
acid molecule or functional equivalent or vector, thereby producing
said antibody or functional part or immunoglobulin chain or
functional equivalent; and harvesting, purifying and/or isolating
said antibody or functional part or immunoglobulin chain or
functional equivalent.
15. The synthetic or recombinant antibody or functional part
thereof, or immunoglobulin chain or functional equivalent thereof
of claim 1 for use in diagnosis of an influenza A virus
infection.
16. A method for determining whether an influenza A virus is
present in a sample comprising: contacting said sample with the
synthetic or recombinant antibody or functional part thereof, or
immunoglobulin chain or functional equivalent thereof of claim 1,
allowing said synthetic or recombinant antibody or functional part
thereof, or immunoglobulin chain or functional equivalent thereof
to bind said influenza A virus, if present, and determining whether
influenza A virus is bound to said synthetic or recombinant
antibody or functional part thereof, or immunoglobulin chain or
functional equivalent thereof, thereby determining whether an
influenza A virus is present in said sample.
17. A synthetic or recombinant antibody or functional part thereof,
or immunoglobulin chain or functional equivalent thereof, having an
in vitro H7N1 influenza A virus neutralizing activity with an IC50
value of less than 5.0 .mu.g/ml.
18. A synthetic or recombinant antibody or functional part thereof,
or immunoglobulin chain or functional equivalent thereof, having an
in vitro H7N7 influenza A virus neutralizing activity with an IC50
value of less than 0.5 .mu.g/ml.
19. A synthetic or recombinant antibody or functional part thereof,
or immunoglobulin chain or functional equivalent thereof, having an
in vitro H1N1 influenza A virus neutralizing activity with an IC50
value of less than 5.0 .mu.g/ml.
20. A synthetic or recombinant antibody or functional part thereof,
or immunoglobulin chain or functional equivalent thereof, having an
in vitro H5N1 influenza A virus neutralizing activity with an IC50
value of less than 5.0 .mu.g/ml.
21. The synthetic or recombinant antibody or functional part
thereof or claim 17, which is capable of specifically binding at
least one other influenza A virus subtype.
22. A synthetic or recombinant multimeric antibody, multimeric
immunoglobulin or functional equivalent thereof, comprising: i) at
least two different heavy chain CDR sequences and at least two
different light chain CDR sequences of an antibody selected from
the group consisting of AT10_001 and AT10_002 and AT10_003 and
AT10_004 and AT10_005; and ii) at least two different heavy chain
CDR sequences and at least two different light chain CDR sequences
of an antibody selected from the group consisting of AT10_001 and
AT10_002 and AT10_003 and AT10_004 and AT10_005, wherein said
antibody selected in i) is different from said antibody selected in
ii).
23. The synthetic or recombinant multimeric antibody, multimeric
immunoglobulin or functional equivalent thereof of claim 22,
comprising: i) heavy chain CDR1, CDR2 and CDR3 and light chain
CDR1, CDR2 and CDR3 sequences of an antibody selected from the
group consisting of AT10_001 and AT10_002 and AT10_003 and AT10_004
and AT10_005; and ii) heavy chain CDR1, CDR2 and CDR3 and light
chain CDR1, CDR2 and CDR3 sequences of an antibody selected from
the group consisting of AT10_001 and AT10_002 and AT10_003 and
AT10_004 and AT10_005, wherein said antibody selected in i) is
different from said antibody selected in ii).
24. The synthetic or recombinant multimeric antibody, multimeric
immunoglobulin or functional equivalent thereof of claim 22,
comprising: i) the heavy chain sequence and the light chain
sequence of an antibody selected from the group consisting of
AT10_001 and AT10_002 and AT10_003 and AT10_004 and AT10_005, or a
sequences that is at least 85% identical thereto; and ii) the heavy
chain sequence and the light chain sequence of an antibody selected
from the group consisting of AT10_001 and AT10_002 and AT10_003 and
AT10_004 and AT10_005, or a sequence that is at least 85% identical
thereto, wherein said antibody selected in i) is different from
said antibody selected in ii).
25. The synthetic or recombinant multimeric antibody, multimeric
immunoglobulin or functional equivalent thereof of claim 22,
comprising: i) at least two different heavy chain CDR sequences and
at least two different light chain CDR sequences of antibody
AT10_002; and ii) at least two different heavy chain CDR sequences
and at least two different light chain CDR sequences of antibody
AT10_005.
26. The synthetic or recombinant multimeric antibody, multimeric
immunoglobulin or functional equivalent thereof of claim 22,
comprising: i) heavy chain CDR1, CDR2 and CDR3 and light chain
CDR1, CDR2 and CDR3 sequences of antibody AT10_002; and ii) heavy
chain CDR1, CDR2 and CDR3 and light chain CDR1, CDR2 and CDR3
sequences of antibody AT10_005.
27. The synthetic or recombinant multimeric antibody, multimeric
immunoglobulin or functional equivalent thereof of claim 22,
comprising: i) the heavy chain sequence and the light chain
sequence of antibody AT10_002, or sequences that are at least 85%
identical thereto; and ii) the heavy chain sequence and the light
chain sequence of antibody AT10_005, or sequences that are at least
85% identical thereto.
28. The synthetic or recombinant multimeric antibody, multimeric
immunoglobulin or functional equivalent thereof of claim 22, which
is a dimeric antibody.
29. An isolated or recombinant cell or a pharmaceutical composition
comprising the synthetic or recombinant multimeric antibody,
multimeric immunoglobulin or functional equivalent thereof of claim
22.
30. The synthetic or recombinant multimeric antibody, multimeric
immunoglobulin or functional equivalent thereof of claim 22 for use
as a medicament and/or prophylactic agent.
31. The synthetic or recombinant multimeric antibody, multimeric
immunoglobulin or functional equivalent thereof of claim 22 for use
as a medicament and/or prophylactic agent for at least in part
treating and/or preventing and/or alleviating the symptoms of an
influenza A infection.
32. The synthetic or recombinant multimeric antibody, multimeric
immunoglobulin or functional equivalent thereof of claim 22 for use
in diagnosis of an influenza A infection.
33. A method for at least in part treating and/or preventing an
influenza A virus infection, comprising administering to an
individual in need thereof a therapeutically effective amount of
the synthetic or recombinant multimeric antibody, multimeric
immunoglobulin or functional equivalent thereof of claim 22 and/or
the cell or pharmaceutical composition of claim 29.
34. A method for neutralizing a H1N1 influenza A virus and/or an
H3N2 influenza A virus, comprising contacting said H1N1 influenza A
virus and/or said H3N2 influenza A virus with The synthetic or
recombinant multimeric antibody, multimeric immunoglobulin or
functional equivalent thereof of claim 22, resulting in
neutralization of said virus.
35. A method for determining whether an influenza A virus is
present in a sample comprising: contacting said sample with The
synthetic or recombinant multimeric antibody, multimeric
immunoglobulin or functional equivalent thereof of claim 22,
allowing said multimeric antibody, multimeric immunoglobulin or
functional equivalent to bind said influenza A virus, if present,
and determining whether influenza A virus is bound to said
synthetic or recombinant multimeric antibody, multimeric
immunoglobulin or functional equivalent thereof of, thereby
determining whether an influenza A virus is present in said
sample.
36. A synthetic or recombinant antibody or functional part thereof,
or immunoglobulin chain or functional equivalent thereof, capable
of interacting with amino acids at positions A38, A40, A41, A42,
A291, A292, A293, A318, B18, B19, B20, B21, B38, B41, B42, B45,
B46, B48, B49, B52, B53, and B56 of influenza A virus group 1
hemagglutinin (H1/H5).
37. A synthetic or recombinant antibody or functional part thereof,
or immunoglobulin chain or functional equivalent thereof, capable
of interacting with amino acids at positions A21, A324, A325, A327,
B12, B14, B15, B16, B17, B18, B19, B25, B26, B30, B31, B32, B33,
B34, B35, B36, B38, B146, B150, B153, and B154 of influenza A virus
group 2 hemagglutinin (H3/H7).
38. A synthetic or recombinant antibody or functional part thereof,
or immunoglobulin chain or functional equivalent thereof, capable
of interacting with amino acids at positions A38, A48, A275, A276,
A277, A278, A289, A291, A318, B19, B20, B21, B36, B38, B39, B41,
B42, B45, B46, B48, B49, B50, B52, B53, B56, B57, B58, B150 of
influenza A virus group 2 hemagglutinin (H3/H7).
Description
CROSS REFERENCE TO RELATED APPLICATIONS
[0001] This application is a continuation of and claims priority to
Ser. No. 14/362,210 filed on Jun. 2, 2014 which is a national stage
entry of and claims the priority to PCT/NL2012/050851, filed on
Dec. 3, 2012, which claims priority to European Application No.
11191783.7, filed Dec. 29, 2011, the entire contents of each of
which are hereby incorporated in total by reference.
FIELD OF THE INVENTION
[0002] The invention relates to the fields of biology, immunology
and medicine. In particular, the invention relates to influenza A
virus specific antibodies.
SEQUENCE LISTING
[0003] This application incorporates by reference the Sequence
Listing contained in an ASCII text file named
"362346_00045_SeqList" submitted via EFS-Web. The text file was
created on 7/7/2017, and is 30 kb in size.
BACKGROUND
[0004] Influenza is an infectious disease of birds and mammals that
can be caused by three types of influenza viruses, types A, B and
C. Influenza viruses are RNA viruses belonging to the family of
Orthomyxoviridae. Influenza viruses are RNA viruses consisting of
seven negative single-stranded RNA-segments encoding nine proteins
(influenza C), or eight negative single-stranded RNA-segments
encoding eleven proteins (influenza A and B). Influenza viruses
infect millions of people every year. Symptoms of influenza include
symptoms comparable with the common cold, such as fever, headache,
chills, muscle pains and soar throat. However, influenza can also
lead to life-threatening complications, such as pneumonia, and
death, in high-risk groups such as young children, the elderly, and
immune compromised or chronically ill individuals.
[0005] The influenza A virus can be subdivided into different types
based on envelope protein expression. Currently 16 hemagglutinin
(HA) serotypes (H1-H16) and 9 neuraminidase (NA) serotypes (N1-N9)
have been identified, which are used to classify influenza viruses
(e.g. H1N1). HA consists of two subunits, HA1 and HA2, linked by
disulphide bonds. HA must be cleaved by host proteases to yield the
two polypeptides HA1 and HA2 in order to be infectious. The major
part of HA1 forms the globular head region of HA and HA2 mainly
forms the stem region of HA. The globular head region differs
considerable between different HA subtypes, whereas the stem region
is more conserved. HA is needed for host cell entry. Following
cleavage, the exposed N-terminus of the HA2 polypeptide acts to
mediate fusion of the viral membrane with the host cell membrane,
allowing the virus to infect the host cell. NA is needed for the
release of new virions. NA catalyses the hydrolysis of terminal
sialic acid residues of glycoproteins of the host cell, thereby
preventing binding of HA to these proteins. NA thus facilitates
release of the virus from a cell and consequently spreading of the
virus. In FIG. 1 a schematic representation of an influenza virus
is shown.
[0006] Influenza virus infections are most prevalent in winter. In
annual influenza epidemics 5-15% of the population are affected
with upper respiratory tract infections. Hospitalization and deaths
mainly occur in high-risk groups (very young children, elderly,
immuno compromised and chronically ill individuals). Annual
epidemics are thought to result in between three and five million
cases of severe illness and between 250 000 and 500 000 deaths
every year around the world. The estimated costs of influenza
epidemics to the US economy are 71-167 billion per year, resulting
from health care costs and lost productivity. Seasonal influenza
vaccines need to be developed each year as a result of antigenic
drift of influenza virus. Mutations in the influenza genome may
induce amino acid substitution(s) that cause antigenic changes in
the HA and NA protein, resulting in the escape of immunity of a
host. So, even though influenza strains may have high homology, a
specific vaccine may not protect against different strains from the
same influenza A subtype. In addition, because the newly developed
influenza vaccines are based on a prediction of the dominant
subtypes for the coming year, the vaccines not always protect
against the influenza subtype that actually arises.
[0007] Additionally, a process called antigenic shift results in
the formation of new virus subtypes through combination of HA and
NA from different influenza virus subtypes. Mutations and genetic
mixing of human and avian and/or swine influenza can lead to a
pandemic. According to the World Health Organization (WHO), a
pandemic can start when three conditions have been met, namely
emergence of a disease new to a population, agents that infect
humans, causing serious illness, and agents that spread easily and
sustainably among humans. In the past, several pandemic influenza
outbreaks have occurred, such as the 1889 Asiatic pandemic (H2N8),
the 1918 Spanish Flu pandemic (H1N1), the 1957 Asian Flue pandemic
(H2N2), the 1968 Hong Kong Flu (H3N2) and the 2009 pandemic (H1N1).
These pandemics were responsible for the death of millions of
people.
[0008] Antiviral drugs can be effective for the prevention and
treatment of influenza. Two classes of antiviral drugs are
available: M2 protein inhibitors and Neuramidase inhibitors.
However, the number of influenza strains that show resistance
against those inhibitors is increasing.
[0009] An alternative approach to prevent and treat influenza
infection is the administration of antibodies directed against the
influenza proteins. Broadly cross-neutralizing antibodies have been
described for influenza viruses belonging to phylogenetic group 1
(Throsby et al. PLoS ONE, 2008 & Sui et al. Nature structural
& molecular biology, 2009). These antibodies recognize a
conserved region in the stem of the HA protein and are capable of
treating influenza infection in mice. A mouse monoclonal antibody
(mAb) has been described that recognizes a conserved epitope in the
region containing the receptor binding domain of the HA1 subunit.
This antibody neutralizes H1N1, H2N2 and H3N2 influenza viruses
(Yoshida et al. PLoS Pathogen. 2009). However, escape mutants have
been reported to arise. This antibody is a mouse antibody which has
the disadvantage of possible side effects when used in humans.
[0010] WO 2009/115972 discloses a human monoclonal antibody having
neutralizing activity against H1N1 and H3N2. However, neutralizing
activity against both H1N1 and H3N2 is inefficient, with IC50
values of around 10 .mu.g/ml. In WO 2010/010466 a human antibody,
FI6, is described that neutralizes H5N1 (group 1) and H7N1 (group
2) pseudotyped influenza viruses and H1N1 and H3N2 infectious
viruses. Again, neutralizing activity against both infectious
viruses is inefficient, with IC50 values between 2 and 12.5
.mu.g/ml. Human antibodies disclosed in WO 2010/130636 have H3 and
H7 cross-binding activity. H3 and H7 are both group 2 influenza
viruses. Some of these antibodies are, in addition, capable of
binding H1 (group 1). However, none of these antibodies was capable
of neutralizing influenza viruses of both group 1 and group 2. As a
result, a cocktail of antibodies is necessary for the
neutralization of both group 1 and group 2 influenza subtypes.
Furthermore, the H3N2 neutralizing activity of antibodies capable
of neutralizing both H3 and H7 influenza virus subtypes is above 1
.mu.g/ml. It is for instance shown in the Examples and Table 7 that
antibody CR8020, described in WO 2010/130636, has an inefficient
neutralizing activity against H3N2 A/swine/Neth/St. Oedenrode/96,
with an IC50 value of more than 15 .mu.g/ml.
[0011] For these reasons, there is a need for additional influenza
A virus antibodies and therapies against influenza A virus
infection.
BRIEF DESCRIPTION OF THE DRAWINGS
[0012] FIG. 1. Schematic representation of an influenza virus
(Subbarao K. and Joseph T. Nature Reviews Immunology 2007: 7,
267-278).
[0013] FIG. 2. Cell sorting of H3 and H7 binding B cells following
incubation of Alexa Fluor 647 labeled Influenza H3
(A/Wyoming/03/2003) and H7 (A/Netherlands/219/2003) HA proteins
with Bcl6 and Bcl-xL transduced polyclonal cultured B cells.
[0014] FIG. 3. Antibody binding to HA transfected 293T cells. 293T
cells were transfected with DNA encoding for the HA of H1 (A/New
Caledonia/20/199), H3 (A/Wisconsin/67/2005), H5 (A/Thailand/Vietnam
Consensus/2004) and H7 (A/Netherlands/219/2003) and incubated with
mAb. Antibody binding was detected with anti-human IgG-PE.
[0015] FIG. 4. Antibody binding to virus infected cells. MDCK-SIAT
cells were infected with Influenza H1N1 (A/Hawaii/31/2007) and H3N2
(A/Netherlands/177/2008) and incubated with mAb. Antibody binding
was detected with anti-human IgG-PE.
[0016] FIG. 5. Antibody binding to virus infected cells. MDCK cells
were infected with Influenza H1N1 (A/Neth/602/2009), H3N2
(A/Swine/St. Oedenrode/1996), high pathogenic H5N1
(A/Turkey/Turkey/2004), high pathogenic H7N7 (A/Ch/Neth/621557/03)
and low pathogenic H7N1 (A/Ch/Italy/1067/1999) and incubated with
mAb. Antibody binding was detected with anti-human IgG-PE.
[0017] FIGS. 6A and 6B provide an antibody competition assay.
Labeled antibodies AT10_001, AT10_002 and AT10_004 were tested for
binding to H3N2 (A/Netherlands/177/2008) infected MDCK-SIAT cells
in the presence of non-labeled competitor antibody FIG. 6A shows
labeled AT10_004 was also tested for binding to H1N1
(A/Hawaii/31/2007) infected MDCK-SIAT cells in the presence of
non-labeled competitor antibody FIG. 6B shows rituximab (Ritux,
CD20 antibody) was used as a negative control.
[0018] FIG. 7. Binding of AT10 antibodies to different HA
conformations by in vitro pH-shift experiment. 293T cells were
transfected with DNA encoding for the HA of H3
(A/Wisconsin/67/2005), detached from the plastic with Trypsin-EDTA
and treated with either 500 mM Dithiothreitol (DTT), PBS pH5, or
left untreated. Cells were subsequently incubated with recombinant
AT10_001, AT10_002, AT10_003 or AT10_004. Antibody binding was
detected using anti-human-IgG-PE.
[0019] FIG. 8. Survival (A) and body weight change (B) of C57Bl/6J
mice challenged intranasally with increasing amounts of influenza
A/HKx-31 (H3N2).
[0020] FIG. 9. Survival (A) and body weight change (B,C,D) of mice
intravenously injected with 1 or 5 mg/kg antibody AT10_001,
AT10_002 or AT10_004 one day before intranasal challenge with 10
Lethal Dose 50 (20,000 TCID50) of influenza A/HKx-31 (H3N2).
[0021] FIG. 10. Survival (A) and body weight change (B) of mice
intravenously injected with 15 mg/kg antibody AT10_002 one day
before intranasal challenge or 2, 3 or 4 days post intranasal
challenge with 10 Lethal Dose 50 (20,000 TCID50) of influenza
A/HKx-31 (H3N2)
[0022] FIG. 11. SPR plot showing the dual specificity of BiFlu for
H3 and H1 and the presence of both kappa and lambda light chain on
BiFlu. Antibodies AT10_002 (lambda light chain), AT10_005 (kappa
light chain) and BiFlu (contains AT10_002 and AT10_005) were
captured on an anti-IgG (A) or anti-lambda (B) coated sensor chip.
In subsequent incubation cycles captured antibodies were tested for
their ability to bind hemagglutinin H3 and H1 and light chain
antibodies directed against kappa and/or lambda. An increase in the
SPR shift indicates proteins binding to the captured
antibodies.
[0023] FIG. 12. Antibody binding to virus infected cells. MDCK-SIAT
cells were infected with Influenza H1N1 (A/Hawaii/31/2007) and H3N2
(A/Netherlands/177/2008) and incubated with several concentrations
of AT10_002, AT10_005 or BiFlu mAb. Antibody binding was detected
with anti-human IgG-PE.
[0024] FIG. 13. Neutralization curves of antibodies AT10_002,
AT10_005 and BiFlu for Influenza H1N1 (A/Hawaii/31/2007) (A) and
H3N2 (A/Netherlands/177/2008) (B). Each virus was incubated with
different amounts of antibody and then added to a confluent
monolayer of MDCK-SIAT cells. Following an 24 hr incubation period
cells were washed, fixed and, stained for DAPI and Influenza
nuclear protein. The percentage of infected cells (relative to the
no antibody control) is shown for each concentration of the
antibody tested.
[0025] FIG. 14. Survival (A) and body weight change (B) of mice
intravenously injected with 1 mg/kg antibody AT10_002, AT10_005 or
Rituximab, 2 mg/kg AT10_002/AT10_005 mix (1 mg/kg for each
antibody) or 2 mg/kg BiFlu one day before intranasal challenge with
10 Lethal Dose 50 of H1N1 Influenza A/PR/8/34.
DESCRIPTION OF THE INVENTION
[0026] It is an object of the present invention to provide
additional antibodies specific for multiple influenza A virus
subtypes, or functional equivalents of such antibodies and
compositions comprising such antibodies. Preferably antibodies are
provided that have a high influenza virus neutralizing activity.
Furthermore, preferably antibodies are provided which are capable
of neutralizing at least two influenza virus subtypes.
[0027] The present invention provides such antibodies specific for
multiple influenza A virus subtypes. As demonstrated in the
Examples, antibodies are provided that are capable of binding at
least two influenza A virus subtypes, preferably both group 1 and
group 2 influenza A virus subtypes. Furthermore, antibodies are
provided that have a high influenza A virus neutralizing
capacity.
[0028] The invention provides in one embodiment an isolated,
synthetic or recombinant antibody or functional part thereof or
immunoglobulin chain or functional equivalent thereof, having an in
vitro H3N2 influenza A virus neutralizing activity with an IC50
value of less than 1 .mu.g/ml, preferably of less than 0.7
.mu.g/ml, more preferably of equal to or less than 0.3 .mu.g/ml,
more preferably of less than 0.2 .mu.g/ml, which antibody or
functional part or immunoglobulin chain or functional equivalent is
capable of specifically binding at least one other influenza A
virus subtype. Said H3N2 influenza A virus preferably comprises a
H3N2 A/Ned/177/2008, 113N2 HKX-31 or H3N2
A/swine/Neth/St.Oedenrode/96 strain, most preferably a H3N2
A/Ned/177/2008 strain.
[0029] H3N2 influenza virus is one of the influenza viruses capable
of infecting humans. H3N2 can be transferred from human to other
humans. Antibodies capable of neutralizing H3N2 influenza virus are
therefore particularly important for application in humans.
[0030] In another preferred embodiment the invention provides an
isolated, synthetic or recombinant antibody or functional part
thereof, or immunoglobulin chain or functional equivalent thereof,
having an in vitro H7N1 influenza A virus neutralizing activity
with an IC50 value of less than 5.0 .mu.g/ml, preferably of less
than 4.0 .mu.g/ml, more preferably of less than 1.0 .mu.g/ml, more
preferably equal to or less than about 0.6 .mu.g/ml. Said H7N1
influenza A virus preferably comprises a H7N1 A/ck/Italy/1067/99
strain. In a particularly preferred embodiment said antibody or
functional part or immunoglobulin chain or functional equivalent is
also capable of specifically binding at least one other influenza A
virus subtype, so that protection against multiple strains can be
obtained. Although to date, no cases have been reported of
transmission of H7N1 from birds to humans, mutations may occur
making this virus infectious for humans.
[0031] In another preferred embodiment the invention provides an
isolated, synthetic or recombinant antibody or functional part
thereof, or immunoglobulin chain or functional equivalent thereof,
having an in vitro H7N7 influenza A virus neutralizing activity
with an IC50 value of less than 0.5 .mu.g/ml, preferably equal to
or less than about 0.4 .mu.g/ml, more preferably equal to or less
than about 0.2 .mu.g/ml, most preferably equal to or less than
about 0.1 .mu.g/ml. Said H7N7 influenza A virus preferably
comprises a H7N7 A/ck/Neth/621557/03 strain. In a particularly
preferred embodiment said antibody or functional part or
immunoglobulin chain or functional equivalent is also capable of
specifically binding at least one other influenza A virus subtype,
so that protection against multiple strains can be obtained. H7N7
influenza virus is one of the influenza viruses capable of
infecting humans following bird to human transmission. Antibodies
capable of neutralizing H7N7 influenza virus are therefore
particularly important for application in humans.
[0032] In another preferred embodiment the invention provides an
isolated, synthetic or recombinant antibody or functional part
thereof, or immunoglobulin chain or functional equivalent thereof,
having an in vitro H1N1 influenza A virus neutralizing activity
with an IC50 value of less than 5.0 .mu.g/ml, preferably of less
than 4.0 .mu.g/ml, more preferably of less than 3.0 .mu.g/ml, more
preferably equal to or less than about 2.7 .mu.g/ml. Said H1N1
influenza A virus preferably comprises a H1N1 A/Neth/602/2009
strain or, most preferably, a H1N1 A/Hawaii/31/2007 strain. In a
particularly preferred embodiment said antibody or functional part
or immunoglobulin chain or functional equivalent is also capable of
specifically binding at least one other influenza A virus subtype,
so that protection against multiple strains can be obtained. H1N1
influenza virus is one of the influenza viruses capable of
infecting humans following human to human transmission. Antibodies
capable of neutralizing H1N1 influenza virus are therefore
particularly important for application in humans.
[0033] In another preferred embodiment the invention provides an
isolated, synthetic or recombinant antibody or functional part
thereof, or immunoglobulin chain or functional equivalent thereof,
having an in vitro H5N1 influenza A virus neutralizing activity
with an IC50 value of less than 5.0 .mu.g/ml, preferably of less
than 4.0 .mu.g/ml, more preferably of less than 3.0 .mu.g/ml, more
preferably of less than 2.0 .mu.g/ml, more preferably equal to or
less than about 1.3 .mu.g/ml. Said H5N1 influenza A virus
preferably comprises a H5N1 A/turkey/Turkey/05 strain. In a
particularly preferred embodiment said antibody or functional part
or immunoglobulin chain or functional equivalent is also capable of
specifically binding at least one other influenza A virus subtype,
so that protection against multiple strains can be obtained. H5N1
influenza virus is one of the influenza viruses capable of
infecting humans following human to human transmission. Antibodies
capable of neutralizing H5N1 influenza virus are therefore
particularly important for application in humans.
[0034] A "functional part of an antibody" is defined herein as a
part that has at least one shared property as said antibody in
kind, not necessarily in amount. Said functional part is capable of
binding the same antigen as said antibody, albeit not necessarily
to the same extent. A functional part of an antibody preferably
comprises a single domain antibody, a single chain antibody, a
nanobody, an unibody, a single chain variable fragment (scFv), a
Fab fragment or a F(ab').sub.2 fragment.
[0035] A functional part of an antibody is also produced by
altering an antibody such that at least one property--preferably an
antigen-binding property--of the resulting compound is essentially
the same in kind, not necessarily in amount. This is done in many
ways, for instance through conservative amino acid substitution,
whereby an amino acid residue is substituted by another residue
with generally similar properties (size, hydrophobicity, etc), such
that the overall functioning is not seriously affected.
[0036] A "functional equivalent of an immunoglobulin chain" is
defined herein as an artificial binding compound, comprising at
least one CDR sequence of an immunoglobulin chain.
[0037] "Neutralizing activity" as used herein is defined as the
inhibition or reduction of an influenza virus' capacity of
infecting a host cell. Neutralizing activity can be measured by any
method known in the art. One of such methods is detailed in the
Examples of this application and involves the prevention of
influenza infection of cultured cells by monoclonal antibodies. In
this method, influenza virus is mixed with an antibody and after 1
hour of incubation added to cells. After 24 hours influenza
infection of the cells can be measured by the detection of
expression of the nuclear protein of influenza in the target cells.
Potent antibodies will prevent or reduce influenza infection and
subsequent influenza nuclear protein expression in the target cell.
"IC50" is a term well known in the art and refers herein to the
concentration of influenza A neutralizing antibody necessary to
inhibit or reduce influenza A virus infectivity of host cells by
half.
[0038] A "group 2 subtype influenza A virus" is an influenza A
virus having a HA serotype of group 2 influenza A viruses.
Currently, viruses having a H3, H4, H7, H10, H14 and H15 serotype
are the group 2 influenza A viruses. A "group 1 subtype influenza A
virus" is an influenza A virus having a HA serotype of group 1
influenza A viruses. Currently, viruses having a H1, H2, H5, H6,
H8, H9, H11, H12, H13 and H16 serotype are the group 1 influenza A
viruses.
[0039] As used herein "specifically binding" refers to the
interaction between an antibody and its epitope, indicating that
said antibody preferentially binds to said epitope. Thus, although
the antibody may non-specifically bind to other antigens or amino
acid sequences, the binding affinity of said antibody for its
epitope is significantly higher than the non-specific binding
affinity of said antibody for any other antigen or amino acid
sequence.
[0040] An "influenza A virus subtype" as used herein refers to
different influenza A viruses, for example H1N1, H1N2, H1N7, H2N2,
H3N2, H3N8, H4N8, H5N1, H5N2, H5N9, H6N2, H6N5, H7N2, H7N3, H7N7,
H8N4, H9N2, H10N7, H11N6, H12N5 or H13N6.
[0041] An "influenza A virus strain" as used herein refers to
different influenza A viruses belonging to the same subtype, for
example H3N2 A/Ned/177/2008, H3N2 A/Wyoming/03/2003 and H3N2
A/Panama/2007/99.
[0042] Isolated, synthetic or recombinant antibodies or functional
parts thereof or immunoglobulin chains or functional equivalents
thereof according to the present invention are herein also referred
to as "antibody according to the invention".
[0043] Preferred influenza A neutralizing antibodies according to
the invention are AT10_004, AT10_002 and AT10_001, because these
antibodies have been demonstrated to have particularly desired
cross-binding and/or neutralizing characteristics. AT10_004,
AT10_002 and AT10_001 have heavy chain sequences of SEQ ID NO's:31,
33 and 34 as depicted in table 1, respectively, and light chain
sequences of SEQ ID NO's:36, 38 and 39 as depicted in table 1,
respectively. The heavy and light chain CDR sequences of these
preferred antibodies are also depicted in table 1. SEQ ID NO's:1, 3
and 4 are the heavy chain CDR1 sequences of antibodies AT10_004,
AT10_002 and AT10_001 respectively, SEQ ID NO's:6, 8 and 9 are the
heavy chain CDR2 sequences of these antibodies, and SEQ ID NO's:11,
13 and 14 are the heavy chain CDR3 sequences of these antibodies.
SEQ ID NO's:16, 18 and 19 are the light chain CDR1 sequences of
antibodies AT10_004, AT10_002 and AT10_001 respectively, SEQ ID
NO's:21, 23 and 24 are the light chain CDR2 sequences of these
antibodies, and SEQ ID NO's:26, 28 and 29 are the light chain CDR3
sequences of these antibodies.
[0044] Antibody AT10_004 is a preferred antibody because it is
capable of specifically binding both group 1 and group 2 influenza
A viruses. As shown in the Examples, antibody AT10_004 has
cross-binding activity to at least H1, 113 and H7 subtype influenza
A viruses. AT10_004 is capable of binding to a wide variety of
recombinant HA subtypes and influenza A viruses. It is capable of
binding at least human influenza H1N1 (A/Hawaii/31/2007) infected
cells and human influenza H3N2 (A/Netherlands/177/2008) infected
cells and HA of human influenza H1N1 (A/New Caledonia/20/1999),
H3N2 (A/Wyoming/03/2003, A/Aichi/2/1968 and A/Wisconsin/67/2005),
H7N7 (A/Netherlands/219/2003) and H9N2 (A/Hong Kong/1073/1999).
Antibody AT10_004 is furthermore preferred because, in addition to
recognizing HA of human influenza viruses and human influenza virus
infected cells, it is also capable of recognizing cells infected
with several influenza viruses infecting non-human animals, namely
cells infected with turkey H5N1 (A/Turkey/Turkey/2004), swine H3N2
(A/swine/St.oedenrode/1996), chicken H7N1 (A/Ch/Italy/1067/1999)
and chicken H7N7 (A/Ch/Neth/621557/2003) and binding to HA of swine
H4N6 (A/Swine/Ontario/01911-1/1999) and HA of duck H15N8
(A/duck/AUS/341-1983). Antibody AT10_004 is also preferred because
it has a high neutralizing activity for H3N2 viruses, having an in
vitro H3N2 A/Ned/177/2008 neutralizing activity with an IC50 value
of about 0.17 .mu.g/ml, and having an in vitro H3N2
A/swine/Neth/St. Oedenrode/96 neutralizing activity with an IC50
value of about 2.3 .mu.g/ml, and even having an in vitro H3N2
HKX-31 neutralizing activity with an IC50 value of about 0.017
.mu.g/ml. AT10_004 also has protective activity against H3N2 virus
(influenza A/HKx-31) in vivo. Antibody AT10_004 furthermore has a
particularly high neutralizing activity for H7N1 viruses, having an
in vitro H7N1 A/ck/Italy/1067/99 neutralizing activity with an IC50
value of about 0.6 .mu.g/ml. As shown in Table 7, the protective
effect of antibody AT10_004 against H7N1 A/ck/Italy/1067/99 is even
higher, meaning that a lower IC50 value is obtained, as compared to
the protective effect of antibody AT10_004 against H3N2
A/swine/Neth/St. Oedenrode/96. Antibody AT10_004 furthermore has a
particularly high neutralizing activity for H7N7 viruses, having an
in vitro H7N7 A/ck/Neth/621557/03 neutralizing activity with an
IC50 value of about 0.2 .mu.g/ml. Antibody AT10_004 is further
preferred because it binds to an epitope in the conserved stem
region of the HA protein. Because limited variation is present in
this region, an antibody of which the epitope is located in the
stem region is capable of binding to a broad range of influenza
viruses. One embodiment therefore provides an antibody or
functional part or immunoglobulin chain or functional equivalent
which has heavy chain CDR1, CDR2 and CDR3 sequences and light chain
CDR1, CDR2 and CDR3 sequences of antibody AT10_004, comprising the
sequence of SEQ ID NO:1, SEQ ID NO:6, SEQ ID NO:11, SEQ ID NO:16,
SEQ ID NO:21 and SEQ ID NO:26, or sequences that are at least 70%
identical thereto.
[0045] In another embodiment an antibody or functional part or
immunoglobulin chain or functional equivalent is provided that
comprises heavy chain CDR1, CDR2 and CDR3 sequences and light chain
CDR1, CDR2 and CDR3 sequences of antibody AT10_002, comprising the
sequence of SEQ ID NO:3, SEQ ID NO:8, SEQ ID NO:13, SEQ ID NO:18,
SEQ ID NO:23 and SEQ ID NO:28 or sequences that are at least 70%
identical thereto. Antibody AT10_002 is a preferred antibody
because it has cross-binding activity to at least H3 and H7 subtype
influenza A viruses. AT10_002 is capable of binding to a wide
variety of recombinant HA subtypes and influenza A viruses. It is
capable of binding at least human influenza H3N2
(A/Netherlands/177/2008) infected cells, and HA of human influenza
H3N2 (A/Wyoming/03/2003, A/Aichi/2/1968 and A/Wisconsin/67/2005)
and H7N7 (A/Netherlands/219/2003). Antibody AT10_002 is furthermore
preferred because, in addition to recognizing HA of human influenza
viruses and human influenza virus infected cells, it is also
capable of recognizing cells infected with several influenza
viruses infecting non-human animals, namely cells infected with
swine H3N2 (A/swine/St.oedenrode/1996), chicken H7N1
(A/Ch/Italy/1067/1999) and chicken H7N7 (A/Ch/Neth/621557/2003) and
binding to HA of duck H10N3 (A/duck/Hong Kong/786/1979) and HA of
duck H15N8 (A/duck/AUS/341-1983). Furthermore, antibody AT10_002
neutralizes at least one H3 subtype influenza A virus. Antibody
AT10_002 is further preferred because it has a high neutralizing
activity for H3N2 viruses, having an in vitro H3N2 A/Ned/177/2008
neutralizing activity with an IC50 value of about 0.18 .mu.g/ml,
and having an in vitro H3N2 A/swine/Neth/St. Oedenrode/96
neutralizing activity with an IC50 value of about 0.3 .mu.g/ml, and
having an in vitro H3N2 HKX-31 neutralizing activity with an IC50
value of about 0.25 .mu.g/ml. AT10_002 also has protective activity
against H3N2 virus (influenza A/HKx-31) in vivo. As demonstrated in
the Example antibody AT10_002 provides the best protective activity
of the antibodies tested and is therefore particularly preferred.
Antibody AT10_002 furthermore has a particularly high neutralizing
activity for H7N1 viruses, having an in vitro H7N1
A/ck/Italy/1067/99 neutralizing activity with an IC50 value of
about 3.6 .mu.g/ml. Antibody AT10_002 furthermore has a
particularly high neutralizing activity for H7N7 viruses, having an
in vitro H7N7 A/ck/Neth/621557/03 neutralizing activity with an
IC50 value of about 0.1 .mu.g/ml. Antibody AT10_002 is further
preferred because it binds to an epitope in the conserved stem
region of the HA protein. Because limited variation is present in
this region, an antibody of which the epitope is located in the
stem region is capable of binding to a broad range of influenza
viruses.
[0046] In another embodiment an antibody or functional part or
immunoglobulin chain or functional equivalent is provided that
comprises heavy chain CDR1, CDR2 and CDR3 sequences and light chain
CDR1, CDR2 and CDR3 sequences of antibody AT10_001, comprising the
sequence of SEQ ID NO:4, SEQ ID NO:9, SEQ ID NO: 14, SEQ ID NO:19,
SEQ ID NO:24 and SEQ ID NO:29 or sequences that are at least 70%
identical thereto. Antibody AT10_001 is a preferred antibody
because it has cross-binding activity to at least H3 and H7 subtype
influenza A viruses. AT10_001 is capable of binding to a wide
variety of recombinant HA subtypes and influenza A viruses. It is
capable of binding at least human influenza H1N1 (A/Neth/602/2009)
infected cells, human influenza H3N2 (A/Netherlands/177/2008)
infected cells and HA of human influenza H3N2 (A/Wyoming/03/2003,
A/Aichi/2/1968 and A/Wisconsin/67/2005) and H7N7
(A/Netherlands/219/2003). Antibody AT10_001 is furthermore
preferred because, in addition to recognizing HA of human influenza
viruses and human influenza virus infected cells, it is also
capable of recognizing cells infected with several influenza
viruses infecting non-human animals, such as cells infected with
chicken H7N1 (A/Ch/Italy/1067/1999) and chicken H7N7
(A/Ch/Neth/621557/2003) and binding to HA of swine H4N6
(A/Swine/Ontario/01911-1/1999). Furthermore, antibody AT10_001
neutralizes at least one H3 subtype influenza A virus. Antibody
AT10_001 is further preferred because it has a high neutralizing
activity for H3N2 viruses, having an in vitro H3N2 A/Ned/177/2008
neutralizing activity with an IC50 value of about 0.64 .mu.g/ml,
and having an in vitro H3N2 HKX-31 neutralizing activity with an
IC50 value of about 2.1 .mu.g/ml. AT10_001 also has protective
activity against H3N2 virus (influenza A/HKx-31) in vivo. Antibody
AT10_001 furthermore has a particularly high neutralizing activity
for H7N7 viruses, having an in vitro H7N7 A/ck/Neth/621557/03
neutralizing activity with an IC50 value of about 0.4 .mu.g/ml.
Antibody AT10_001 is further preferred because binds to an epitope
in the conserved stem region of the HA protein. Because limited
variation is present in this region, an antibody of which the
epitope is located in the stem region is capable of binding to a
broad range of influenza viruses.
[0047] Preferably, an influenza A neutralizing antibody according
to the invention comprises heavy chain CDR1, CDR2 and CDR3
sequences and light chain CDR1, CDR2 and CDR3 sequences that are at
least 75%, more preferably at least 80%, more preferably at least
85%, more preferably at least 86%, more preferably at least 87%,
more preferably at least 88%, more preferably at least 89%, more
preferably at least 90%, more preferably at least 91%, more
preferably at least 92%, more preferably at least 93%, more
preferably at least 94%, more preferably at least 95%, more
preferably at least 96%, more preferably at least 97%, more
preferably at least 98%, more preferably at least 99% identical to
the sequences depicted in table 1.
[0048] The terms "AT10_004", "AT10_002" and "AT10_001" as used
herein encompass all antibodies and functional equivalents with the
indicated heavy chain and light chain sequences, for instance
isolated and/or purified or recombinantly produced.
[0049] As is well known by the skilled person, a heavy chain of an
antibody is the larger of the two types of chains making up an
immunoglobulin molecule. A heavy chain comprises constant domains
and a variable domain, which variable domain is involved in antigen
binding. A light chain of an antibody is the smaller of the two
types of chains making up an immunoglobulin molecule. A light chain
comprises a constant domain and a variable domain. The variable
domain is often, together with the variable domain of the heavy
chain, involved in antigen binding.
[0050] Complementary-determining regions (CDRs) are the
hypervariable regions present in heavy chain variable domains and
light chain variable domains. In case of whole antibodies, the CDRs
of a heavy chain and the connected light chain of an antibody
together form the antigen-binding site.
[0051] Based on the antibodies depicted in table 1, it is possible
to produce an immunoglobulin chain or functional equivalent thereof
comprising at least one CDR sequence of an immunoglobulin variable
domain depicted in table 1 which is specific for and capable of
neutralizing influenza A virus. Further provided is thus an
isolated, recombinant or synthetic immunoglobulin chain or
functional equivalent thereof comprising at least one CDR sequence
of an immunoglobulin variable region depicted in table 1.
Preferably, antibodies are provided which comprises at least two
CDR's, more preferably at least three CDR's, of the same antibody
indicated in table 1. Hence, preferably at least two or three CDR's
of AT10_004, or AT10_003, or AT10_002 or AT10_001 or AT10_005, are
jointly present in one antibody or functional part according to the
invention. In a preferred embodiment, a human antibody is provided
because the use of a human antibody diminishes the chance of
side-effects due to an immunological reaction in a human
individual. Optionally, said at least one CDR sequence is
optimized, preferably in order to improve binding efficacy or
stability. This is for instance done by mutagenesis experiments
where after the stability and/or binding efficacy of the resulting
compounds are preferably tested and an improved influenza A
neutralizing antibody is selected.
[0052] A skilled person is well capable of generating variants
comprising at least one altered CDR sequence according to the
invention. For instance, conservative amino acid substitution is
applied. It is also possible to alter at least one CDR sequence
depicted in table 1 in order to generate a variant antibody, or a
functional part thereof, with at least one altered property as
compared to the original antibody. Preferably, an antibody or
functional part is provided comprising a CDR sequence which is at
least 70% identical to a CDR sequence as depicted in table 1, so
that the favourable binding and neutralizing characteristics of an
influenza A neutralizing antibody according to the invention are at
least in part maintained or even improved. A CDR sequence as
depicted in table 1 is preferably altered such that the resulting
antibody or functional part comprises at least one improved
property, such as for instance an improved binding affinity,
selectivity and/or stability, as compared to the original antibody.
Variant antibodies or functional parts thereof comprising an amino
acid sequence which is at least 70% identical to a CDR sequence as
depicted in table 1 are therefore also within the scope of the
present invention. Various methods are available in the art for
altering an amino acid sequence. For instance, a heavy chain or
light chain sequence with a desired CDR sequence is artificially
synthesized. Preferably, a nucleic acid molecule encoding a CDR
sequence according to the invention is mutated, for instance using
random--or site-directed--mutagenesis.
[0053] Besides optimizing CDR sequences in order to improve binding
efficacy or stability, it is often advantageous to optimize at
least one sequence in at least one of the framework regions. This
is preferably done in order to improve binding efficacy or
stability. Framework sequences are for instance optimized by
mutating a nucleic acid molecule encoding such framework sequence
where after the characteristics of the resulting antibody--or
functional part--are preferably tested. This way, it is possible to
obtain improved antibodies or functional parts. In a preferred
embodiment, human germline sequences are used for framework regions
in antibodies or functional parts thereof or immunoglobulin chains
or functional equivalents according to the invention. The use of
germline sequences preferably minimizes the risk of immunogenicity
of said antibodies or functional parts, immunoglobulin chains or
functional equivalents, because these sequences are less likely to
contain somatic alterations which are unique to individuals from
which the framework regions are derived, and may cause an
immunogenic response when applied to another human individual.
[0054] The invention thus provides an isolated, synthetic or
recombinant antibody or functional part thereof, or immunoglobulin
chain or functional equivalent thereof comprising: [0055] a heavy
chain CDR1 sequence comprising a sequence which is at least 70%
identical to a sequence selected from the group consisting of SEQ
ID NO's:1, 3 and 4, and/or [0056] a heavy chain CDR2 sequence
comprising a sequence which is at least 70% identical to a sequence
selected from the group consisting of SEQ ID NO's:6, 8 and 9,
and/or [0057] a heavy chain CDR3 sequence comprising a sequence
which is at least 70% identical to a sequence selected from the
group consisting of SEQ ID NO's:11, 13 and 14, and/or [0058] a
light chain CDR1 sequence comprising a sequence which is at least
70% identical to a sequence selected from the group consisting of
SEQ ID NO's:16, 18 and 19, and/or [0059] a light chain CDR2
sequence comprising a sequence which is at least 70% identical to a
sequence selected from the group consisting of SEQ ID NO's:21, 23
and 24, and/or [0060] a light chain CDR3 sequence comprising a
sequence which is at least 70% identical to a sequence selected
from the group consisting of SEQ ID NO's:26, 28 and 29. Preferably,
said antibody or functional part or immunoglobulin chain or
functional equivalent comprises heavy chain CDR1, CDR2 and/or CDR3
sequences and/or light chain CDR1, CDR2 and/or CDR3 sequences that
are at least 75%, more preferably at least 80%, more preferably at
least 85%, more preferably at least 86%, more preferably at least
87%, more preferably at least 88%, more preferably at least 89%,
more preferably at least 90%, more preferably at least 91%, more
preferably at least 92%, more preferably at least 93%, more
preferably at least 94%, more preferably at least 95%, more
preferably at least 96%, more preferably at least 97%, more
preferably at least 98%, more preferably at least 99%, most
preferably 100% identical to these sequences.
[0061] In another embodiment an antibody according to the invention
comprises heavy chain CDR1, CDR2 and CDR3 sequences and light chain
CDR1, CDR2 and CDR3 sequences of antibody AT10_003, which has a
heavy chain sequence of SEQ ID NO:32 as depicted in table 1, and a
light chain sequence of SEQ ID NO:37 as depicted in table 1. SEQ ID
NO:2 is the heavy chain CDR1 sequence, SEQ ID NO:7 is the heavy
chain CDR2 sequence, SEQ ID NO:12 is the heavy chain CDR3 sequence,
SEQ ID NO:17 is the light chain CDR1 sequence, SEQ ID NO:22 is the
light chain CDR2 sequence, and SEQ ID NO:27 is the light chain CDR3
sequence of antibody AT10_003. Antibody AT10_003 is a preferred
antibody because it is capable of specifically binding both group 1
and group 2 influenza A viruses. Antibody AT10_003 has
cross-binding activity to at least H3, H5 and H7 subtype influenza
A viruses. AT10_003 is capable of binding to a wide variety of
recombinant HA subtypes and influenza A viruses. It is capable of
binding at least human influenza H1N1 (A/Hawaii/31/2007) infected
cells and human influenza H3N2 (A/Netherlands/177/2008) infected
cells, and HA of human influenza H3N2 (A/Wyoming/03/2003,
A/Aichi/2/1968 and A/Wisconsin/67/2005), H5N1 (A/Vietnam/1203/2004
and A/Thailand/Vietnam Consensus/2004), H7N7
(A/Netherlands/219/2003) and H9N2 (A/Hong Kong/1073/1999). Antibody
AT10_003 is furthermore preferred because, in addition to
recognizing HA of human influenza viruses and human influenza virus
infected cells, it is also capable of recognizing cells infected
with several influenza viruses infecting non-human animals, such as
cells infected with chicken H7N7 (A/Ch/Neth/621557/2003) and swine
H3N2 (A/swine/St.oedenrode/1996), and it is capable of binding to
HA of swine H4N6 (A/Swine/Ontario/01911-1/1999), HA of duck H10N3
(A/duck/Hong Kong/786/1979) and HA of duck H15N8
(A/duck/AUS/341/1983). The term "AT10_003" as used herein encompass
all antibodies and functional equivalents with the AT10_003heavy
chain and light chain sequences depicted in table 1, for instance
isolated and/or purified or recombinantly produced.
[0062] As described above, a skilled person is well capable of
producing an immunoglobulin chain or functional equivalent thereof
comprising at least one CDR sequence of an immunoglobulin variable
domain depicted in table 1 which is specific for influenza A virus
and of generating variants comprising at least one altered CDR
sequence according to the invention.
[0063] The invention therefore provides an isolated, synthetic or
recombinant antibody or functional part thereof, or immunoglobulin
chain or functional equivalent thereof comprising: [0064] a heavy
chain CDR1 sequence comprising a sequence which is at least 70%
identical to SEQ ID NO:2, and/or [0065] a heavy chain CDR2 sequence
comprising a sequence which is at least 70% identical to SEQ ID
NO:7, and/or [0066] a heavy chain CDR3 sequence comprising a
sequence which is at least 70% identical to SEQ ID NO:12, and/or
[0067] a light chain CDR1 sequence comprising a sequence which is
at least 70% identical to SEQ ID NO:17, and/or [0068] a light chain
CDR2 sequence comprising a sequence which is at least 70% identical
to SEQ ID NO:22, and/or [0069] a light chain CDR3 sequence
comprising a sequence which is at least 70% identical to SEQ ID
NO:27. Preferably, said antibody or functional part or
immunoglobulin chain or functional equivalent comprises heavy chain
CDR1, CDR2 and/or CDR3 sequences and/or light chain CDR1, CDR2
and/or CDR3 sequences that are at least 75%, more preferably at
least 80%, more preferably at least 85%, more preferably at least
86%, more preferably at least 87%, more preferably at least 88%,
more preferably at least 89%, more preferably at least 90%, more
preferably at least 91%, more preferably at least 92%, more
preferably at least 93%, more preferably at least 94%, more
preferably at least 95%, more preferably at least 96%, more
preferably at least 97%, more preferably at least 98%, more
preferably at least 99%, most preferably 100% identical to these
sequences.
[0070] In another embodiment an antibody according to the invention
comprises at least one of heavy chain CDR1, CDR2 and CDR3 sequences
and light chain CDR1, CDR2 and CDR3 sequences of antibody AT10_005,
which has a heavy chain sequence of SEQ ID NO:35 as depicted in
table 1, and a light chain sequence of SEQ ID NO:40 as depicted in
table 1. SEQ ID NO:5 is the heavy chain CDR1 sequence, SEQ ID NO:10
is the heavy chain CDR2 sequence, SEQ ID NO:15 is the heavy chain
CDR3 sequence, SEQ ID NO:20 is the light chain CDR1 sequence, SEQ
ID NO:25 is the light chain CDR2 sequence, and SEQ ID NO:30 is the
light chain CDR3 sequence of antibody AT10_005. Antibody AT10_005
is a preferred antibody because it has cross-binding activity to at
least H1, H5 and H9 subtype influenza A viruses. AT10_005 is
capable of binding to a wide variety of recombinant HA subtypes and
influenza A viruses. It is capable of binding at least human
influenza H1N1 (A/Neth/602/2009) infected cells, and HA of human
influenza H1N1 (A/California/07/2009, and A/New Caledonia/20/1999),
H5N1 (A/Vietnam/1203/2004), and H9N2 (A/Hong Kong/1073/1999).
Antibody AT10_005 is furthermore preferred because, in addition to
recognizing HA of human influenza viruses and human influenza virus
infected cells, it is also capable of recognizing cells infected
with several influenza viruses infecting non-human animals, such as
cells infected with turkey H5N1 (A/Turkey/Turkey/2004). Antibody
AT10_005 is also preferred because it has a high neutralizing
activity for H1N1 viruses, having an in vitro H1N1 A/Hawaii/31/2007
neutralizing activity with an IC50 value of about 0.24 .mu.g/ml,
and having an in vitro H1N1 A/Neth/602/2009 (swine origin)
neutralizing activity with an IC50 value of about 2.7 .mu.g/ml.
AT10_005 also has protective activity against H1N1 in vivo.
Antibody AT10_005 furthermore has a particularly high neutralizing
activity for H5N1 viruses, having an in vitro H5N1
A/turkey/Turkey/05 neutralizing activity with an IC50 value of
about 1.3 .mu.g/ml. Antibody AT10_005 is further preferred because
it binds to an epitope in the conserved stem region of the HA
protein. Because limited variation is present in this region, an
antibody of which the epitope is located in the stem region is
capable of binding to a broad range of influenza viruses. The term
"AT10_005" as used herein encompass all antibodies and functional
equivalents with the indicated heavy chain and light chain
sequences, for instance isolated and/or purified or recombinantly
produced.
[0071] As described above, a skilled person is well capable of
producing an immunoglobulin chain or functional equivalent thereof
comprising at least one CDR sequence of an immunoglobulin variable
domain depicted in table 1 which is specific for influenza A virus
and of generating variants comprising at least one altered CDR
sequence according to the invention.
[0072] The invention therefore provides an isolated, synthetic or
recombinant antibody or functional part thereof, or immunoglobulin
chain or functional equivalent thereof comprising: [0073] a heavy
chain CDR1 sequence comprising a sequence which is at least 70%
identical to SEQ ID NO:5, and/or [0074] a heavy chain CDR2 sequence
comprising a sequence which is at least 70% identical to SEQ ID
NO:10, and/or [0075] a heavy chain CDR3 sequence comprising a
sequence which is at least 70% identical to SEQ ID NO:15, and/or
[0076] a light chain CDR1 sequence comprising a sequence which is
at least 70% identical to SEQ ID NO:20, and/or [0077] a light chain
CDR2 sequence comprising a sequence which is at least 70% identical
to SEQ ID NO:25, and/or [0078] a light chain CDR3 sequence
comprising a sequence which is at least 70% identical to SEQ ID
NO:30. Preferably, said antibody or functional part or
immunoglobulin chain or functional equivalent comprises heavy chain
CDR1, CDR2 and/or CDR3 sequences and/or light chain CDR1, CDR2
and/or CDR3 sequences that are at least 75%, more preferably at
least 80%, more preferably at least 85%, more preferably at least
86%, more preferably at least 87%, more preferably at least 88%,
more preferably at least 89%, more preferably at least 90%, more
preferably at least 91%, more preferably at least 92%, more
preferably at least 93%, more preferably at least 94%, more
preferably at least 95%, more preferably at least 96%, more
preferably at least 97%, more preferably at least 98%, more
preferably at least 99%, most preferably 100% identical to these
sequences.
[0079] In a preferred embodiment an antibody according to the
invention comprises both the heavy and light chain CDR sequences of
one of the above mentioned antibodies. Provided are thus antibodies
which have heavy chain CDR1, CDR2 and CDR3 sequences and light
chain CDR1, CDR2 and CDR3 sequences of antibody AT10_004,
comprising the sequence of SEQ ID NO:1, SEQ ID NO:6, SEQ ID NO:11,
SEQ ID NO:16, SEQ ID NO:21 and SEQ ID NO:26, or sequences that are
at least 70% identical thereto.
[0080] In another embodiment antibodies which have heavy chain
CDR1, CDR2 and CDR3 sequences and light chain CDR1, CDR2 and CDR3
sequences of antibody AT10_003 are provided, comprising the
sequence of SEQ ID NO:2, SEQ ID NO:7, SEQ ID NO:12, SEQ ID NO:17,
SEQ ID NO:22 and SEQ ID NO:27, or sequences that are at least 70%
identical thereto.
[0081] In another embodiment antibodies which have heavy chain
CDR1, CDR2 and CDR3 sequences and light chain CDR1, CDR2 and CDR3
sequences of antibody AT10_002 are provided, comprising the
sequence of SEQ ID NO:3, SEQ ID NO:8, SEQ ID NO:13, SEQ ID NO:18,
SEQ ID NO:23 and SEQ ID NO:28, or sequences that are at least 70%
identical thereto.
[0082] In another embodiment antibodies which have heavy chain
CDR1, CDR2 and CDR3 sequences and light chain CDR1, CDR2 and CDR3
sequences of antibody AT10_001 are provided, comprising the
sequence of SEQ ID NO:4, SEQ ID NO:9, SEQ ID NO:14, SEQ ID NO:19,
SEQ ID NO:24 and SEQ ID NO:29, or sequences that are at least 70%
identical thereto.
[0083] In another embodiment antibodies which have heavy chain
CDR1, CDR2 and CDR3 sequences and light chain CDR1, CDR2 and CDR3
sequences of antibody AT10_005 are provided, comprising the
sequence of SEQ ID NO:5, SEQ ID NO:10, SEQ ID NO:15, SEQ ID NO:20,
SEQ ID NO:25 and SEQ ID NO:30, or sequences that are at least 70%
identical thereto.
[0084] As described herein before, the term "antibodies" also
encompasses functional parts, immunoglobulin chains or functional
equivalents thereof.
[0085] Preferably, said antibody or functional part or
immunoglobulin chain or functional equivalent comprises heavy chain
CDR1, CDR2 and CDR3 sequences and light chain CDR1, CDR2 and CDR3
sequences that are at least 75%, more preferably at least 80%, more
preferably at least 85%, more preferably at least 86%, more
preferably at least 87%, more preferably at least 88%, more
preferably at least 89%, more preferably at least 90%, more
preferably at least 91%, more preferably at least 92%, more
preferably at least 93%, more preferably at least 94%, more
preferably at least 95%, more preferably at least 96%, more
preferably at least 97%, more preferably at least 98%, more
preferably at least 99%, most preferably 100% identical to the
above mentioned CDR sequences.
[0086] In a preferred embodiment, an antibody according to the
invention comprises a heavy chain sequence and/or light chain
sequence, or a sequence which has at least 70% sequence identity
thereto, as depicted in table 1. Also provided is therefore an
antibody or functional part or immunoglobulin chain or functional
equivalent, having a heavy chain sequence comprising a sequence
which is at least 70% identical to a sequence selected from the
group consisting of SEQ ID NO's:31-35 and/or having a light chain
sequence which is at least 70% identical to a sequence selected
from the group consisting of SEQ ID NO's:36-40, or sequences that
are at least at least 75%, more preferably at least 80%, more
preferably at least 85%, more preferably at least 86%, more
preferably at least 87%, more preferably at least 88%, more
preferably at least 89%, more preferably at least 90%, more
preferably at least 91%, more preferably at least 92%, more
preferably at least 93%, more preferably at least 94%, more
preferably at least 95%, more preferably at least 96%, more
preferably at least 97%, more preferably at least 98%, more
preferably at least 99%, most preferably 100% identical to any one
of these heavy chain or light chain sequences.
[0087] Preferably, an antibody according to the invention comprises
a heavy chain sequence which is at least 75%, more preferably at
least 80%, more preferably at least 85%, more preferably at least
86%, more preferably at least 87%, more preferably at least 88%,
more preferably at least 89%, more preferably at least 90%
identical to a sequence selected from the group consisting of SEQ
ID NO's:31-35 and/or a light chain sequence which is at least 75%,
more preferably at least 80%, more preferably at least 85%, more
preferably at least 86%, more preferably at least 87%, more
preferably at least 88%, more preferably at least 89%, more
preferably at least 90% identical to a sequence selected from the
group consisting of SEQ ID NO's:36-40. Most preferably, an antibody
according to the invention comprises a heavy chain sequence which
is at least 91%, more preferably at least 92%, more preferably at
least 93%, more preferably at least 94%, more preferably at least
95%, more preferably at least 96%, more preferably at least 97%,
more preferably at least 98%, more preferably at least 99%,
identical to a sequence selected from the group consisting of SEQ
ID NO's:31-35 and/or a light chain sequence which is at least 91%,
more preferably at least 92%, more preferably at least 93%, more
preferably at least 94%, more preferably at least 95%, more
preferably at least 96%, more preferably at least 97%, more
preferably at least 98%, more preferably at least 99%, most
preferably 100% identical to a sequence selected from the group
consisting of SEQ ID NO's:36-40. The higher the identity, the more
closely an antibody resembles an antibody depicted in table 1.
[0088] An antibody or functional part or immunoglobulin chain or
functional equivalent according to the invention preferably
comprises a heavy chain as well as a light chain which resemble the
heavy and the light chain of the same antibody depicted in table 1.
Thus, in a preferred embodiment an antibody according to the
invention comprises a heavy chain sequence of a given antibody,
preferably antibody AT10_004, comprising the sequence of SEQ ID
NO:31 and a light chain sequence of the same antibody, preferably
AT10_004, comprising the sequence of SEQ ID NO:36, or sequences
that are at least 70%, preferably at least 75%, more preferably at
least 80%, more preferably at least 85%, more preferably at least
86%, more preferably at least 87%, more preferably at least 88%,
more preferably at least 89%, more preferably at least 90%, more
preferably at least 91%, more preferably at least 92%, more
preferably at least 93%, more preferably at least 94%, more
preferably at least 95%, more preferably at least 96%, more
preferably at least 97%, more preferably at least 98%, more
preferably at least 99%, identical thereto.
[0089] In another embodiment an antibody according to the invention
or functional part thereof comprises a heavy chain sequence of
antibody AT10_003, comprising the sequence of SEQ ID NO:32 and a
light chain sequence of antibody AT10_003, comprising the sequence
of SEQ ID NO:37 or sequences that are at least 70%, preferably at
least 75%, more preferably at least 80%, more preferably at least
85%, more preferably at least 86%, more preferably at least 87%,
more preferably at least 88%, more preferably at least 89%, more
preferably at least 90%, more preferably at least 91%, more
preferably at least 92%, more preferably at least 93%, more
preferably at least 94%, more preferably at least 95%, more
preferably at least 96%, more preferably at least 97%, more
preferably at least 98%, more preferably at least 99%, identical
thereto.
[0090] In another embodiment an antibody according to the invention
or functional part thereof comprises a heavy chain sequence of
antibody AT10_002, comprising the sequence of SEQ ID NO:33 and the
light chain sequence of antibody AT10_002, comprising the sequence
of SEQ ID NO:38, or sequences that are at least 70%, preferably at
least 75%, more preferably at least 80%, more preferably at least
85%, more preferably at least 86%, more preferably at least 87%,
more preferably at least 88%, more preferably at least 89%, more
preferably at least 90%, more preferably at least 91%, more
preferably at least 92%, more preferably at least 93%, more
preferably at least 94%, more preferably at least 95%, more
preferably at least 96%, more preferably at least 97%, more
preferably at least 98%, more preferably at least 99%, identical
thereto.
[0091] In another embodiment an antibody according to the invention
or functional part thereof comprises a heavy chain sequence of
antibody AT10_001, comprising the sequence of SEQ ID NO:34 and the
light chain sequence of antibody AT10_001, comprising the sequence
of SEQ ID NO:39, or sequences that are at least 70%, preferably at
least 75%, more preferably at least 80%, more preferably at least
85%, more preferably at least 86%, more preferably at least 87%,
more preferably at least 88%, more preferably at least 89%, more
preferably at least 90%, more preferably at least 91%, more
preferably at least 92%, more preferably at least 93%, more
preferably at least 94%, more preferably at least 95%, more
preferably at least 96%, more preferably at least 97%, more
preferably at least 98%, more preferably at least 99%, identical
thereto.
[0092] In another embodiment an antibody according to the invention
or functional part thereof comprises a heavy chain sequence of
antibody AT10_005, comprising the sequence of SEQ ID NO:35, and the
light chain sequence of antibody AT10_005, comprising the sequence
of SEQ ID NO:40, or sequences that are at least 70%, preferably at
least 75%, more preferably at least 80%, more preferably at least
85%, more preferably at least 86%, more preferably at least 87%,
more preferably at least 88%, more preferably at least 89%, more
preferably at least 90%, more preferably at least 91%, more
preferably at least 92%, more preferably at least 93%, more
preferably at least 94%, more preferably at least 95%, more
preferably at least 96%, more preferably at least 97%, more
preferably at least 98%, more preferably at least 99%, identical
thereto.
[0093] The invention provides antibodies having an in vitro H3N2
influenza A virus neutralizing activity with an IC50 value of less
than 1 .mu.g/ml. An advantage of such antibodies is that a low
dosis of said antibody is needed in order to obtain neutralizing
capacity. Therefore, less of said influenza A neutralizing antibody
has to be administered to an individual for treatment and/or
prevention of an influenza A infection. It is favourable to use an
amount as low as possible to achieve a desired effect from both a
health care point of view and from an economical point of view. It
is preferred to administer to a subject as less as possible of a
therapeutic antibody, because this reduces the chance of undesired
effects, such as immunological reactions to the antibody.
Furthermore, if a lower amount of antibody is used, the cost of
treatment of an individual to prevent of counteract influenza
infection is reduced.
[0094] Generally, the higher the neutralizing activity of an
antibody, the lower the amount of antibody necessary for treatment
of an individual. As shown in the examples, antibody AT10_001 has
an in vitro H3N2 A/Ned/177/2008 virus neutralizing activity with an
IC50 value of about 0.64 .mu.g/ml, antibody AT10_004 has an in
vitro H3N2 A/Ned/177/2008 virus neutralizing activity with an IC50
value of about 0.17 .mu.g/ml, and antibody AT10_002 has an in vitro
H3N2 A/Ned/177/2008 virus neutralizing activity with an IC50 value
of about 0.18 .mu.g/ml. Therefore, preferably an antibody according
to the invention has an in vitro H3N2 influenza A virus
neutralizing activity with an IC50 value of less than 0.8 .mu.g/ml,
more preferably of less than 0.6 .mu.g/ml, more preferably of less
than 0.5 .mu.g/ml, more preferably of less than 0.4 .mu.g/ml, more
preferably of less than 0.3 .mu.g/ml, more preferably of less than
0.2 .mu.g/ml. The example further demonstrates that antibodies
AT10_001, AT10_002 and AT10_004 have in vivo H3N2 neutralizing
activity. These antibodies were shown to protect mice against
influenza A virus H3N2 HKx-31. All mice receiving antibody
AT10_001, AT10_002 or AT10_004 treatment survived a challenge with
H3N2 virus, whereas all control mice receiving treatment with a
control antibody lost more than 25% of their body weight and had to
be removed from the study. In a preferred embodiment, an antibody
according to the invention therefore has in vivo H3N2
neutralization activity, for instance as measured by protective
activity against influenza H3N2 infection in a mouse model as
described in the Example.
[0095] Preferably an influenza A neutralizing antibody according to
the invention has said in vitro neutralizing activity as determined
in a neutralization assay as described in the examples.
[0096] Several strains of influenza A virus of the same subtype
exist. Different strains of the same influenza A virus subtype may
have differences in host infectivity. Therefore, in a preferred
embodiment, an influenza A neutralizing antibody according to the
invention neutralizes at least one H3N2 influenza virus strain with
the indicated neutralizing activity, more preferably at least two,
more preferably at least three, more preferably at least four, more
preferably at least five different H3N2 influenza virus strains. In
a preferred embodiment, an influenza A neutralizing antibody
according to the invention neutralizes at least H3N2 A/Ned/177/2008
influenza virus strain, and/or H3N2 HKx-31, and/or H3N2
A/swine/Neth/St. Oedenrode/96.
[0097] Antibodies provided by the invention are capable of binding
at least two different influenza subtypes. In one embodiment, an
antibody is provided that is capable of binding H3N2 and at least
one other group 2 influenza A virus subtype. In another embodiment,
an antibody is provided that is capable of binding H1N1 and at
least one other group 1 influenza A virus subtype. An advantage of
such antibodies is that they thus have cross-binding activity, i.e.
are capable of binding at least two different influenza A virus
subtypes. In a preferred embodiment, an influenza A neutralizing
antibody is provided that is further capable of neutralizing said
at least one other group subtype influenza A virus. Such antibodies
have cross-neutralizing activity, i.e. have neutralizing activity
for at least two different influenza A virus subtypes. Such
antibodies have the advantage that the use of a single antibody
allows neutralization of multiple influenza subtypes. Such
antibodies thus have broad neutralizing activity.
[0098] In another preferred embodiment, an influenza A neutralizing
antibody is provided that is capable of binding at least one group
2 subtype influenza A virus and at least one group 1 subtype
influenza A virus. In a more preferred embodiment, an influenza A
neutralizing antibody is provided that is further capable of
neutralizing said at least one group 2 and/or said at least one
group 1 subtype influenza A virus.
[0099] An influenza A neutralizing antibody according to the
invention capable of specifically binding at least two group 2
influenza A virus subtypes, or at least one group 1 and one group 2
influenza A virus subtype, is preferably capable of binding to an
epitope within a hemagglutinin protein of an influenza A virus
protein that is shared between influenza subtypes.
[0100] Preferably, said epitope is located in a conserved region of
the hemagglutinin protein of influenza A virus. As described above,
H3, H4, H7, H10, H14 and H15 are currently known influenza viruses
from group 2. Said at least two group 2 subtype influenza A virus
subtypes are thus preferably selected from the group consisting of
H3, H4, H7, H10, H14 and H15 containing influenza A virus subtypes.
Provided is in one embodiment an antibody according to the
invention capable of binding and/or neutralizing a H3N2 influenza A
virus and capable of binding a H4, H7, H10, H14 or H15 containing
influenza A virus. Preferably, such antibody is capable of binding
a H7 containing influenza A virus subtype. H7 containing influenza
viruses frequently infect poultry. Because humans are in direct
contact with infected poultry, there is considerable risk of
infection of humans with H7 influenza viruses and mixing of avian
H7 and human influenza viruses. Infection of humans with H7
containing influenza virus resulting in death has been reported.
Therefore, in a preferred embodiment, the invention provides an
influenza A neutralizing antibody capable of binding a H3 and a H7
subtype influenza A virus. Preferably, said antibody is further
capable of neutralizing both H3 and H7 subtype influenza A
virus.
[0101] As described above, H1, H2, H5, H6, H8, H9, H11, H12, H13
and H16 are currently known influenza viruses from group 1. The
above mentioned at least one group 1 subtype influenza A virus is
therefore preferably selected from the group consisting of H1, H2,
H5, H6, H8, H9, H11, H12, H13 and H16 containing influenza A virus
subtypes. Provided is therefore in one embodiment an antibody
according to the invention capable of binding and/or neutralizing a
H3N2 influenza A virus and capable of binding a H1, H2, H5, H6, H8,
H9, H11, H12, H13 or H16 containing influenza A virus. Preferably
said at least one group 1 subtype influenza A virus is selected
from the group consisting of H1 and H5 containing influenza A virus
subtypes. H1N1 is one of the influenza A viruses capable of
infecting humans and generally the seasonal influenza epidemic
comprises at least one H1N1 influenza virus. H5 containing viruses,
such as H5N1, H5N3, H5N4 and H5N9, mainly infect birds. However,
some of H5 influenza subtypes can be transferred from birds to
human. Infection of humans with H5 influenza subtypes is
particularly dangerous because of a risk of life-threatening
complications, such as pneumonia, and of death.
[0102] In a particularly preferred embodiment, the invention
provides an influenza A neutralizing antibody capable of binding
and/or neutralizing a H3, a H7 and a H1 subtype influenza A
virus.
[0103] In one embodiment, antibodies according to the invention are
capable of binding at least one influenza virus which infects
non-human animals, including, but not limited to birds such as
chickens, ducks, geese, turkeys, and pheasants, and swine, ferrets,
rabbits, cats, dogs and horses. Such antibodies can be used to
counteract influenza virus infection in said non-human animals, for
instance, but not limited to, animals that are kept as livestock or
pet. Furthermore, because humans are in direct contact with such
animals, there is considerable risk of infection of humans with
influenza viruses that have infected said animals. Another risk is
the mixing of influenza viruses capable of infecting non-human
animals and influenza viruses capable of infecting humans resulting
in new, potentially highly pathogenic, influenza viruses.
Therefore, preferably an antibody according to the invention is
provided which is capable of binding an influenza A virus subtype
that infects non-human animals. In a preferred embodiment, said
antibody is capable of binding an avian and/or swine influenza A
virus subtype. Examples of such avian and/or swine influenza A
virus subtype include, but are not limited to, H4, H10, H15, H5 and
H7 containing influenza viruses such as H4N6, H10N3, H15N8, H7N1,
H7N7 and/or H5N1.
[0104] As indicated above, H7 containing influenza viruses
frequently infect poultry and there is a considerable risk of
infection of humans with H7 influenza viruses and mixing of avian
H7 and human influenza viruses. Therefore, in one embodiment an
antibody according to the invention is provided which is capable of
binding a H7 subtype influenza A virus. More preferably, an
antibody is provided which is capable of neutralizing a H7 subtype
influenza A virus. For example, such antibody has an in vitro H7N7
(such as A/Ch/Neth/621557/03) and/or H7N1 (such as
A/Ch/Italy/1067/99) influenza A virus neutralizing activity.
Preferably, said antibody has an in vitro H7N1 and/or H7N7
influenza A virus neutralizing activity with an IC50 value of less
than 10 .mu.g/ml, more preferably of less than 5 .mu.g/ml, more
preferably of less than 4 .mu.g/ml, more preferably of less than 3
.mu.g/ml, more preferably of less than 2 .mu.g/ml, more preferably
of less than 1 .mu.g/ml, more preferably of less than 0.8 .mu.g/ml,
more preferably of less than 0.6 .mu.g/ml, more preferably of less
than 0.5 .mu.g/ml, more preferably of less than 0.4 .mu.g/ml, more
preferably of less than 0.3 .mu.g/ml, more preferably of less than
0.2 .mu.g/ml. Preferably such influenza A neutralizing antibody
according to the invention has said in vitro neutralizing activity
as determined in a neutralization assay as described in the
examples. In a preferred embodiment, an influenza A neutralizing
antibody according to the invention neutralizes at least one H7N1
and/or H7N7 influenza virus strain with the indicated neutralizing
activity, more preferably at least two, more preferably at least
three, more preferably at least four, more preferably at least five
different H7N1 and/or H7N7 influenza virus strains. In a preferred
embodiment, an influenza A neutralizing antibody according to the
invention neutralizes at least H7N7 (A/Ch/Neth/621557/03) and/or
H7N1 (A/Ch/Italy/1067/99).
[0105] A particularly preferred antibody of the invention capable
of binding H7 subtype influenza A viruses is AT10_004, which has a
heavy chain sequence of SEQ ID NO:31 as depicted in table 1, and a
light chain sequence of SEQ ID NO:36 as depicted in table 1.
Another particularly preferred antibody of the invention capable of
binding H7 subtype influenza A viruses is AT10_002, which has a
heavy chain sequence of SEQ ID NO:33 as depicted in table 1, and a
light chain sequence of SEQ ID NO:38 as depicted in table 1.
Another particularly preferred antibody of the invention capable of
binding H7 subtype influenza A viruses is AT10_001, which has a
heavy chain sequence of SEQ ID NO:34 as depicted in table 1, and a
light chain sequence of SEQ ID NO:39 as depicted in table 1.
[0106] The heavy and light chain CDR sequences of these preferred
antibodies are also depicted in table 1. SEQ ID NO:1 is the heavy
chain CDR1 sequence, SEQ ID NO:6 is the heavy chain CDR2 sequence,
SEQ ID NO:11 is the heavy chain CDR3 sequence, SEQ ID NO:16 is the
light chain CDR1 sequence, SEQ ID NO:21 is the light chain CDR2
sequence, and SEQ ID NO:26 is the light chain CDR3 sequence of
antibody AT10_004. SEQ ID NO:3 is the heavy chain CDR1 sequence,
SEQ ID NO:8 is the heavy chain CDR2 sequence, SEQ ID NO:131 is the
heavy chain CDR3 sequence, SEQ ID NO:18 is the light chain CDR1
sequence, SEQ ID NO:23 is the light chain CDR2 sequence, and SEQ ID
NO:28 is the light chain CDR3 sequence of antibody AT10_002. SEQ ID
NO:4 is the heavy chain CDR1 sequence, SEQ ID NO:9 is the heavy
chain CDR2 sequence, SEQ ID NO:14 is the heavy chain CDR3 sequence,
SEQ ID NO:19 is the light chain CDR1 sequence, SEQ ID NO:24 is the
light chain CDR2 sequence, and SEQ ID NO:29 is the light chain CDR3
sequence of antibody AT10_001.
[0107] The invention thus provides an isolated, synthetic or
recombinant antibody or functional part thereof, or immunoglobulin
chain or functional equivalent thereof comprising: [0108] a heavy
chain CDR1 sequence comprising a sequence which is at least 70%
identical to SEQ ID NO:1, and [0109] a heavy chain CDR2 sequence
comprising a sequence which is at least 70% identical to SEQ ID
NO:6, and [0110] a heavy chain CDR3 sequence comprising a sequence
which is at least 70% identical to SEQ ID NO:11, and [0111] a light
chain CDR1 sequence comprising a sequence which is at least 70%
identical to SEQ ID NO:16, and [0112] a light chain CDR2 sequence
comprising a sequence which is at least 70% identical to SEQ ID
NO:21, and [0113] a light chain CDR3 sequence comprising a sequence
which is at least 70% identical to SEQ ID NO:26.
[0114] The invention further provides an isolated, synthetic or
recombinant antibody or functional part thereof, or immunoglobulin
chain or functional equivalent thereof comprising: [0115] a heavy
chain CDR1 sequence comprising a sequence which is at least 70%
identical to SEQ ID NO:3, and [0116] a heavy chain CDR2 sequence
comprising a sequence which is at least 70% identical to SEQ ID
NO:8, and [0117] a heavy chain CDR3 sequence comprising a sequence
which is at least 70% identical to SEQ ID NO:13, and [0118] a light
chain CDR1 sequence comprising a sequence which is at least 70%
identical to SEQ ID NO:18, and [0119] a light chain CDR2 sequence
comprising a sequence which is at least 70% identical to SEQ ID
NO:23, and [0120] a light chain CDR3 sequence comprising a sequence
which is at least 70% identical to SEQ ID NO:28.
[0121] The invention further provides an isolated, synthetic or
recombinant antibody or functional part thereof, or immunoglobulin
chain or functional equivalent thereof comprising: [0122] a heavy
chain CDR1 sequence comprising a sequence which is at least 70%
identical to SEQ ID NO:4, and [0123] a heavy chain CDR2 sequence
comprising a sequence which is at least 70% identical to SEQ ID
NO:9, and [0124] a heavy chain CDR3 sequence comprising a sequence
which is at least 70% identical to SEQ ID NO:14, and [0125] a light
chain CDR1 sequence comprising a sequence which is at least 70%
identical to SEQ ID NO:19, and [0126] a light chain CDR2 sequence
comprising a sequence which is at least 70% identical to SEQ ID
NO:24, and [0127] a light chain CDR3 sequence comprising a sequence
which is at least 70% identical to SEQ ID NO:29.
[0128] Preferably, said antibody or functional part or
immunoglobulin chain or functional equivalent comprises heavy chain
CDR1, CDR2 and/or CDR3 sequences and/or light chain CDR1, CDR2
and/or CDR3 sequences that are at least 75%, more preferably at
least 80%, more preferably at least 85%, more preferably at least
86%, more preferably at least 87%, more preferably at least 88%,
more preferably at least 89%, more preferably at least 90%, more
preferably at least 91%, more preferably at least 92%, more
preferably at least 93%, more preferably at least 94%, more
preferably at least 95%, more preferably at least 96%, more
preferably at least 97%, more preferably at least 98%, more
preferably at least 99%, most preferably 100% identical to these
sequences.
[0129] As described above, some H5 influenza subtypes can infect
humans. Infection of humans with H5 influenza subtypes is
particularly dangerous because of a risk of life-threatening
complications, such as pneumonia, and of death. Therefore, in one
embodiment an antibody according to the invention is provided which
is capable of binding a H5 subtype influenza A virus. More
preferably, an antibody is provided which is capable of
neutralizing a H5 subtype influenza A virus. For example such
antibody has an in vitro H5N1 (such as A/Turkey/Turkey/04)
influenza A virus neutralizing activity. Preferably, said antibody
has an in vitro H5N1 influenza A virus neutralizing activity with
an IC50 value of less than 10 .mu.g/ml, more preferably of less
than 5 .mu.g/ml, more preferably of less than 4 .mu.g/ml, more
preferably of less than 3 .mu.g/ml, more preferably of less than 2
.mu.g/ml, more preferably of less than 1 .mu.g/ml, more preferably
of less than 0.8 .mu.g/ml, more preferably of less than 0.6
.mu.g/ml, more preferably of less than 0.5 .mu.g/ml, more
preferably of less than 0.4 .mu.g/ml, more preferably of less than
0.3 .mu.g/ml, more preferably of less than 0.2 .mu.g/ml. Preferably
such influenza A neutralizing antibody according to the invention
has said in vitro neutralizing activity as determined in a
neutralization assay as described in the examples. In a preferred
embodiment, an influenza A neutralizing antibody according to the
invention is provided that neutralizes at least one H5N1 influenza
virus strain with the indicated neutralizing activity, more
preferably at least two, more preferably at least three, more
preferably at least four, more preferably at least five different
H5N1 influenza virus strains. In a preferred embodiment, an
influenza A neutralizing antibody according to the invention
neutralizes at least H5N1 (A/Turkey/Turkey/04)).
[0130] A particularly preferred antibody according to the invention
capable of binding a H5 subtype influenza A virus is AT10_003,
Another particularly preferred antibody according to the invention
capable of binding a H5 subtype influenza A virus is AT10_005.
Antibodies or functional parts having sequences that are at least
70% identical to the CDR sequences of AT10_003 or AT10_005 are
therefore preferred for counteracting a H5 subtype influenza A
virus.
[0131] An antibody according to the invention is preferably a human
antibody. The use of human antibodies for prophylaxis and therapy
in humans diminishes the chance of side-effects due to an
immunological reaction in a human individual against non-human
sequences. In another embodiment an antibody according to the
invention is a humanized antibody. Humanized antibodies are made by
incorporating non-human hypervariable domains into human antibodies
and therefore immunogenic properties are diminished as compared to
fully non-human antibodies. In another embodiment an antibody
according to the invention is a chimeric antibody. In a chimeric
antibody, sequences of interest, such as for instance a binding
site of interest, are included into an antibody according to the
invention.
[0132] Preferred antibodies according to the invention have a high
binding affinity for the hemagglutinin protein. Measurement of the
affinity constant and specificity of binding between antigen and
antibody is preferred in determining the efficacy of prophylactic,
therapeutic, diagnostic and research methods using anti-influenza A
antibodies of the invention. "Binding affinity" generally refers to
the strength of the total sum of the noncovalent interactions
between a single binding site of a molecule (e.g., an antibody) and
its binding partner (e.g., an antigen). Unless indicated otherwise,
as used herein, "binding affinity" refers to intrinsic binding
affinity which reflects a 1:1 interaction between members of a
binding pair (e.g., antibody and antigen). The affinity can
generally be represented by the equilibrium dissociation constant
(K.sub.D), which is calculated as the k.sub.a to k.sub.d ratio,
see, e.g., Chen, Y., et al., (1999) J. Mol Biol 293:865-881.
Affinity can be measured by common methods known in the art, such
as for instance a surface plasmon resonance (SPR) assay such as
BiaCore or IBIS-iSPR instrument at IBIS Technologies BV (Hengelo,
the Netherlands) or solution phase assays, such as Kinexa.
Preferably an antibody according to the invention has a binding
affinity for an epitope on the influenza HA protein characterized
by a dissociation constant (K.sub.D) of at most 100 nM, more
preferably at most 50 nM, more preferably at most 25 nM, more
preferably at most 10 nM, more preferably at most 5 nM, more
preferably at most 2 nM, more preferably at most 1 nM, more
preferably at most 0.5 nM, more preferably at most 0.3 nM, more
preferably at most 0.1 nM.
[0133] The invention further provides an isolated, synthetic or
recombinant nucleic acid molecule with a length of at least 15
nucleotides, or a functional equivalent thereof, encoding at least
one CDR sequence of an antibody or functional part or
immunoglobulin chain or functional equivalent according to the
invention. Preferably a nucleic acid according to the invention has
a length of at least 30 nucleotides, more preferably at least 50
nucleotides, more preferably at least 75 nucleotides. A nucleic
acid according to the invention is for instance isolated from a
B-cell which is capable of producing an antibody according to the
invention. In a preferred embodiment a nucleic acid encoding an
antibody according to the invention is provided.
[0134] As used herein "an isolated, synthetic or recombinant
nucleic acid molecule with a length of at least 15 nucleotides, or
a functional equivalent thereof, encoding at least one CDR sequence
of an antibody or functional part thereof or immunoglobulin chain
or functional equivalent thereof according to the invention" is
herein also referred to as "a nucleic acid molecule or functional
equivalent thereof according to the invention".
[0135] As used herein, a nucleic acid molecule or nucleic acid
sequence of the invention preferably comprises a chain of
nucleotides, more preferably DNA and/or RNA. In other embodiments a
nucleic acid molecule or nucleic acid sequence of the invention
comprises other kinds of nucleic acid structures such as for
instance a DNA/RNA helix, peptide nucleic acid (PNA), locked
nucleic acid (LNA) and/or a ribozyme. Such other nucleic acid
structures are referred to as functional equivalents of a nucleic
acid sequence. The term "functional equivalent of a nucleic acid
molecule" also encompasses a chain comprising non-natural
nucleotides, modified nucleotides and/or non-nucleotide building
blocks which exhibit the same function as natural nucleotides.
[0136] Nucleic acid sequences encoding preferred heavy chain and
light chain CDR's of antibodies AT10_004, AT10_003, AT10_002,
AT10_001 and AT10_005 are depicted in table 1. Nucleic acid
molecules encoding a heavy or light chain CDR of an antibody
according to the invention which differ from the CDR nucleic acid
sequences depicted in table 1 but have nucleic acid codons encoding
for the same amino acids of said heavy or light chain CDR are also
encompassed by the invention. Nucleic acid molecules encoding a
heavy or light chain CDR of an antibody depicted in table 1 which
has been altered, for instance through conservative amino acid
substitution, whereby an amino acid residue is substituted by
another residue with generally similar properties (size,
hydrophobicity, etc), are also encompassed by the invention, as
long as the resulting CDR has at least 70% sequence identity with a
CDR depicted in table 1.
[0137] A preferred nucleic acid molecule according to the invention
comprises: [0138] a heavy chain CDR1 encoding sequence which has at
least 70% sequence identity to a sequence which is selected from
the group consisting of SEQ ID NO's:41-45, and/or [0139] a heavy
chain CDR2 encoding sequence which has at least 70% sequence
identity to a sequence which is selected from the group consisting
of SEQ ID NO's:46-50, and/or [0140] a heavy chain CDR3 encoding
sequence which has at least 70% sequence identity to a sequence
which is selected from the group consisting of SEQ ID NO's:51-55,
and/or [0141] a light chain CDR1 encoding sequence which has at
least 70% sequence identity to a sequence which is selected from
the group consisting of SEQ ID NO's:56-60, and/or [0142] a light
chain CDR2 encoding sequence which has at least 70% sequence
identity to a sequence which is selected from the group consisting
of SEQ ID NO's:61-65, and/or [0143] a light chain CDR3 encoding
sequence which has at least 70% sequence identity to a sequence
which is selected from the group consisting of SEQ ID
NO's:66-70.
[0144] A nucleic acid molecule according to the invention
preferably comprises a sequence which has at least 75%, more
preferably at least 80%, more preferably at least 85%, more
preferably at least 90%, most preferably at least 95% sequence,
most preferably 100% identity to said sequences. Preferably, said
nucleic acid molecule comprises at least one CDR encoding sequence.
Further provided is a nucleic acid molecule or functional
equivalent thereof comprising a sequence which has at least 70%
sequence identity, preferably at least 75%, more preferably at
least 80%, more preferably at least 85%, more preferably at least
90%, most preferably at least 95%, most preferably 100% sequence
identity to a nucleic acid molecule selected from SEQ ID
NO's:41-70, said nucleic acid molecule or functional equivalent
having at least 15 nucleotides.
[0145] A nucleic acid molecule or functional equivalent thereof
according to the present invention preferably encodes a region
which has at least 70% sequence identity to a heavy chain and/or a
light chain as depicted in table 1. Thus, a preferred nucleic acid
molecule or a functional equivalent comprises a sequence which has
at least 70% sequence identity to a sequence selected from the
group consisting of SEQ ID NO's:71-75 and/or a sequence which has
at least 70% sequence identity to a sequence selected from the
group consisting of SEQ ID NO's:76-80. More preferably, a nucleic
acid molecule or a functional equivalent according to the invention
comprises a heavy chain encoding sequence as well as a light chain
encoding sequence which resemble the heavy and the light chain
encoding sequences of the same antibody depicted in table 1. Thus,
in a preferred embodiment a nucleic acid or functional equivalent
according to the invention comprises a heavy chain encoding
sequence of antibody AT10_004, comprising the sequence of SEQ ID
NO:71 and a light chain encoding sequence of antibody AT10_004,
comprising the sequence of SEQ ID NO:76 or sequences that are at
least 70%, preferably at least 75%, more preferably at least 80%,
more preferably at least 85%, more preferably at least 86%, more
preferably at least 87%, more preferably at least 88%, more
preferably at least 89%, more preferably at least 90%, more
preferably at least 91%, more preferably at least 92%, more
preferably at least 93%, more preferably at least 94%, more
preferably at least 95%, more preferably at least 96%, more
preferably at least 97%, more preferably at least 98%, more
preferably at least 99%, identical thereto.
[0146] In another embodiment a nucleic acid or functional
equivalent according to the invention comprises a heavy chain
encoding sequence of antibody AT10_003, comprising the sequence of
SEQ ID NO:72 and a light chain encoding sequence of antibody
AT10_003, comprising the sequence of SEQ ID NO:77, or sequences
that are at least 70%, preferably at least 75%, more preferably at
least 80%, more preferably at least 85%, more preferably at least
86%, more preferably at least 87%, more preferably at least 88%,
more preferably at least 89%, more preferably at least 90%, more
preferably at least 91%, more preferably at least 92%, more
preferably at least 93%, more preferably at least 94%, more
preferably at least 95%, more preferably at least 96%, more
preferably at least 97%, more preferably at least 98%, more
preferably at least 99%, identical thereto.
[0147] In another embodiment a nucleic acid or functional
equivalent according to the invention comprises a heavy chain
encoding sequence of antibody AT10_002, comprising the sequence of
SEQ ID NO:73 and a light chain encoding sequence of antibody
AT10_002, comprising the sequence of SEQ ID NO:78, or sequences
that are at least 70%, preferably at least 75%, more preferably at
least 80%, more preferably at least 85%, more preferably at least
86%, more preferably at least 87%, more preferably at least 88%,
more preferably at least 89%, more preferably at least 90%, more
preferably at least 91%, more preferably at least 92%, more
preferably at least 93%, more preferably at least 94%, more
preferably at least 95%, more preferably at least 96%, more
preferably at least 97%, more preferably at least 98%, more
preferably at least 99%, identical thereto.
[0148] In another embodiment a nucleic acid or functional
equivalent according to the invention comprises a heavy chain
encoding sequence of antibody AT10_001, comprising the sequence of
SEQ ID NO:74 and a light chain encoding sequence of antibody
AT10_001, comprising the sequence of SEQ ID NO:79, or sequences
that are at least 70%, preferably at least 75%, more preferably at
least 80%, more preferably at least 85%, more preferably at least
86%, more preferably at least 87%, more preferably at least 88%,
more preferably at least 89%, more preferably at least 90%, more
preferably at least 91%, more preferably at least 92%, more
preferably at least 93%, more preferably at least 94%, more
preferably at least 95%, more preferably at least 96%, more
preferably at least 97%, more preferably at least 98%, more
preferably at least 99%, identical thereto.
[0149] In another embodiment a nucleic acid or functional
equivalent according to the invention comprises a heavy chain
encoding sequence of antibody AT10_005, comprising the sequence of
SEQ ID NO:75 and a light chain encoding sequence of antibody
AT10_005, comprising the sequence of SEQ ID NO:80, or sequences
that are at least 70%, preferably at least 75%, more preferably at
least 80%, more preferably at least 85%, more preferably at least
86%, more preferably at least 87%, more preferably at least 88%,
more preferably at least 89%, more preferably at least 90%, more
preferably at least 91%, more preferably at least 92%, more
preferably at least 93%, more preferably at least 94%, more
preferably at least 95%, more preferably at least 96%, more
preferably at least 97%, more preferably at least 98%, more
preferably at least 99%, identical thereto.
[0150] The percentage of identity of an amino acid or nucleic acid
sequence, or the term "% sequence identity", is defined herein as
the percentage of residues in a candidate amino acid or nucleic
acid sequence that is identical with the residues in a reference
sequence after aligning the two sequences and introducing gaps, if
necessary, to achieve the maximum percent identity. Methods and
computer programs for the alignment are well known in the art.
[0151] Further provided is a vector comprising a nucleic acid
molecule or sequence or functional equivalent according to the
invention. As used herein "a vector comprising a nucleic acid
sequence or molecule or functional equivalent according to the
invention" is also referred to as "a vector according to the
invention". Methods for constructing a vector with a nucleic acid
or functional equivalent according to the invention are well known
in the art. Non-limiting examples of vectors suitable for
generating a vector of the invention are retroviral and lentiviral
vectors. Such vector is suitable for a variety of applications. For
instance, a vector of the invention comprising a therapeutically
beneficial nucleic acid sequence is suitable for prophylactic or
therapeutic applications against influenza. Administration of such
vector to an individual, preferably a human, in need thereof
results in expression of said prophylactic or therapeutic nucleic
acid sequence in vivo resulting in at least partial treatment or
prophylaxis against influenza. Said vector can also be used in
applications involving in vitro expression of a nucleic acid
molecule of interest, for instance for (commercial) production of
antibodies or functional equivalents according to the invention.
Also provided is therefore an isolated or recombinant cell
comprising a nucleic acid molecule or functional equivalent a
vector according to the invention.
[0152] A nucleic acid molecule or vector according to the invention
is particularly useful for generating antibodies or functional
parts, or immunoglobulin chains or functional equivalents, which
are specific for influenza A virus HA protein. This is for instance
done by introducing such nucleic acid molecule or vector into a
cell so that the cell's nucleic acid translation machinery will
produce the encoded antibodies or functional parts, immunoglobulin
chains or functional equivalents. In one embodiment, a nucleic acid
molecule or vector encoding a heavy and/or light chain according to
the invention is expressed in so called producer cells, such as for
instance cells of a Chinese hamster ovary (CHO), NSO (a mouse
myeloma) or 293(T) cell line, some of which are adapted to
commercial antibody production. Proliferation of said producer
cells results in a producer cell line capable of producing
antibodies according to the invention. Preferably, said producer
cell line is suitable for producing antibodies for use in humans.
Hence, said producer cell line is preferably free of pathogenic
agents such as pathogenic micro-organisms. Most preferably,
antibodies consisting of human sequences are generated using at
least one nucleic acid molecule or vector according to the
invention.
[0153] An isolated or recombinant antibody producing cell capable
of producing an antibody according to the invention is therefore
also provided. An antibody producing cell is defined herein as a
cell which is capable of producing and/or secreting antibodies or
functional equivalents thereof, and/or which is capable of
developing into a cell which is capable of producing and/or
secreting antibodies or functional equivalents thereof. An antibody
producing cell according to the invention is preferably a producer
cell which is adapted to commercial antibody production.
Preferably, said producer cell is suitable for producing antibodies
for use in humans. A method for producing an antibody according to
the invention is also provided, said method comprising providing a
cell, preferably an antibody producing cell, with a nucleic acid
molecule or functional equivalent or a vector according to the
invention, and allowing said cell to translate said nucleic acid
molecule or functional equivalent or vector, thereby producing
antibodies according to the invention. A method according to the
invention preferably further comprises a step of harvesting,
purifying and/or isolating antibodies according to the invention.
Obtained antibodies according to the invention are preferably used
in human therapy, optionally after additional purifying, isolation
or processing steps.
[0154] In one embodiment, an antibody according to the invention is
coupled to another moiety to form an antibody-drug conjugate. An
antibody according to the invention is for instance coupled to an
antiviral agent, such as acyclovir, penciclovar, lamivudine,
ribavirin, zanamivir, laninamivir, peramivir, idoxuridine,
oseltamivir, amantadine, remantidine, maxamine, peramivir, or
thymalfasin. The term "antiviral agent" as used herein refers to
any substance that reduces or blocks the function, or growth, of a
virus and/or causes destruction of a virus. In another embodiment,
a moiety that is coupled to an antibody according to the invention
is an antimicrobial peptide. The term "antimicrobial peptide" as
used herein refers to small amphipathic peptides of variable length
(typically 6 to 100 amino acids), sequence and structure with
activity against microorganisms such as for instance bacteria,
protozoa, yeast, fungi and/or viruses. Antimicrobial peptides
usually act through relatively non-specific mechanisms resulting in
membranolytic activity but several antimicrobial peptides can also
stimulate the innate immune response. In a preferred embodiment,
said antimicrobial peptide has anti-viral activity. Non-limiting
examples of suitable antimicrobial peptides are magainins, PGLa,
cathelicidins (such as LL-37 and cathelicidin-related antimicrobial
peptide (CRAMP)), alamethicin, mellitin and cecropin, hydramacin-1,
pexiganan, MSI-78, MSI-843, MSI-594, polyphemusin, human
antimicrobial peptide, defensins, protegrins and indolicidin. In
yet another embodiment, a moiety that is coupled to an antibody
according to the invention is an immunomodulatory molecule such as
an CD3 antibody. Such CD3 antibody is capable of binding T cells
and, if coupled to an antibody according to the invention,
targeting T cells to influenza A virus infected cells.
[0155] Said other moiety, for example a cytotoxic agent, is
preferably coupled to an antibody according to the invention via a
linker such as for instance an acid-labile hydrazone linker, or via
a peptide linker like citruline-valine, or through a thioether
linkage, or by sortase catalized transamidation, which is described
in detail in WO 2010/087994.
[0156] Sortase catalized transamidation involves engineering of a
sortase recognition site (LPETGG) on the heavy chain of an
antibody, preferably on the C-terminal part of the heavy chain, and
on the moiety to be coupled to said antibody. The antibody and the
moiety further typically contain a GGGGS sequence and a tag for
purification purposes, such as a HIS tag. Subsequently sortase
mediated transamidation is performed followed by click chemistry
linkage. In a sortase catalized transaminidation, "click chemistry
linkage" typically involves chemical coupling of, for instance, an
alkyne-containing reagent and, for instance, an azide-containing
reagent which are added by sortase through addition of glycines to
the sortase motif on the heavy chain of the antibody and to a
sortase motif on the moiety (such as a protein, peptide or
antibody) to be coupled to the antibody. In one embodiment, the
invention therefore provides an antibody according to the invention
wherein a sortase recognition site (LPETGG) is engineered on the
heavy chain of the antibody, preferably on the C-terminal part of
the heavy chain, the antibody preferably further containing a GGGGS
sequence and a purification tag, such as a HIS tag.
[0157] In another embodiment an antibody according to the invention
is coupled to another moiety via a thioether linkage. In such case,
one or more cysteines are preferably incorporated into an antibody
according to the invention. Cysteines contain a thiol group and,
therefore, incorporation of one or more cysteines into an antibody
according to the invention, or replacement of one or more amino
acids by one or more cysteines of an antibody according to the
invention, enable coupling of said antibody to another moiety. Said
one or more cysteines are preferably introduced into an antibody
according to the invention at a position where it does not
significantly influence folding of said antibody, and does not
significantly alter antigen binding or effector function. The
invention therefore also provides an antibody according to the
invention wherein at least one amino acid other than cysteine has
been replaced by a cysteine.
[0158] Influenza specific antibodies described herein have
different (cross-) binding and neutralizing capacities. An antibody
according to the invention, such as AT10_001, AT10_002, AT10_003,
AT10_004 or AT10_005 can be advantageously used in combination with
another antibody according to the invention. Such combination
provides an even stronger anti-influenza effect. In one embodiment
an antibody according to the invention is combined with another
antibody according to the invention that is capable of binding
and/or neutralizing at least one other influenza A subtype.
Combination of antibodies according to the invention which bind
and/or neutralize different influenza A virus subtypes enables
counteracting a wider range of influenza A subtypes in a single
treatment. Such combination is thus useful in counteracting a broad
range of influenza viruses. Furthermore, it is also advantageous to
combine an antibody according to the invention with a known
antibody capable of binding and/or neutralizing an influenza A
virus subtype. Such combination for instance provides a stronger
response against an influenza A virus and/or provides a response
against a wide range of influenza subtypes. Yet another example is
a combination of an antibody according to the invention and a known
antibody specific for influenza B. In another embodiment, the
invention provides an influenza A virus bispecific antibody with
specificity for at least two different influenza A virus subtypes,
preferably at least three influenza A virus subtypes, more
preferably at least four influenza A subtypes. An "influenza A
virus bispecific antibody" as used herein is defined as an antibody
capable of simultaneously binding at least two different influenza
A virus subtypes, such as two, three or four subtypes, and is also
referred to as an "influenza A virus bispecific antibody according
to the invention" or a "bispecific antibody according to the
invention". The term "influenza A virus bispecific antibody" also
encompasses functional parts of such influenza A virus bispecific
antibody which has retained its capability of binding at least two
different influenza A virus subtypes simultaneously, such as
bispecific single chain variable fragments (scFv), bispecific Fab
fragments and bispecific F(ab').sub.2 fragments. Also provided is a
pharmaceutical composition comprising an influenza A virus
bispecific antibody according to the invention.
[0159] In one embodiment, a bispecific antibody according to the
invention comprises two non-identical heavy chain-light chain
combinations, thus having two antigen-binding regions which
recognize two different influenza A virus subtypes, preferably two
different HA subtypes. For instance, in one embodiment, an
influenza A virus bispecific antibody comprises a heavy and light
chain of an antibody according to the invention as depicted in
table 1 and a heavy and light chain of another antibody according
to the invention as depicted in table 1. Bispecific single chain
variable fragments (scFv), bispecific Fab fragments and bispecific
F(ab').sub.2 fragments comprise for instance a scFv or Fab or
F(ab').sub.2 fragment of an antibody according to the invention and
a scFv or Fab or F(ab').sub.2 fragment of another antibody
according to the invention. In a preferred embodiment, an influenza
A virus bispecific antibody according to the invention comprises a
heavy and light chain of two antibodies selected from the group
consisting of AT10_001, AT10_002, AT10_003, AT10_004 and AT10_005
as depicted in table 1, or a scFv or Fab fragment thereof.
Preferably said bispecific antibody comprises a heavy and light
chain of antibody AT10_003 or AT10_005, preferably of antibody
AT10_005, and a heavy and light chain of an antibody selected from
the group consisting of AT10_001, AT10_002 and AT10_004.
[0160] In another embodiment, two antibodies according to the
invention are coupled to each other or an antibody according to the
invention is coupled to a known influenza specific antibody. This
is in a preferred embodiment done by sortase catalized
transamidation, which is described herein before and in detail in
WO 2010/087994. For this purpose, sortase catalized transamidation
involves engineering of a sortase recognition site (LPETGG) on the
heavy chains of both antibodies to be coupled, preferably on the
C-terminal part of the heavy chains. The antibodies further
typically contain a GGGGS sequence and a purification tag, such as
a HIS tag. Thus, if two antibodies according to the invention are
coupled, both said antibodies are preferably engineered as
described herein before and in detail in WO 2010/087994.
Subsequently sortase mediated transamidation is preferably
performed followed by click chemistry linkage to couple both
antibodies via their heavy chains. As herein explained before,
"click chemistry linkage" involves chemical coupling of, for
instance, an alkyne-containing reagent and, for instance, an
azide-containing reagent which are added by sortase through
addition of glycines to the sortase motif on the heavy chain of a
first antibody and to the heavy chain of a second antibody that is
to be coupled to the first antibody. One embodiment of the
invention therefore provides a synthetic or recombinant multimeric
antibody, multimeric immunoglobulin or functional equivalent
thereof, comprising:
i) at least two, preferably three, different heavy chain CDR
sequences and at least two, preferably three, different light chain
CDR sequences of an antibody selected from the group consisting of
AT10_001 and AT10_002 and AT10_003 and AT10_004 and AT10_005; and
ii) at least two, preferably three, different heavy chain CDR
sequences and at least two, preferably three, different light chain
CDR sequences of another antibody. Said other antibody is
preferably another influenza specific antibody, although this is
not necessary. In a particularly preferred embodiment, at least two
antibodies according to the invention are coupled to each other by
sortase catalized transamidation, whereby said at least two
antibodies are preferably selected from the group consisting of
AT10_001, AT10_002, AT10_003, AT10_004 and AT10_005 as depicted in
Table 1.
[0161] One preferred embodiment of the invention therefore provides
a synthetic or recombinant multimeric antibody, multimeric
immunoglobulin or functional equivalent thereof, comprising:
i) at least two different heavy chain CDR sequences and at least
two different light chain CDR sequences of an antibody selected
from the group consisting of AT10_001 and AT10_002 and AT10_003 and
AT10_004 and AT10_005; and ii) at least two different heavy chain
CDR sequences and at least two different light chain CDR sequences
of an antibody selected from the group consisting of AT10_001 and
AT10_002 and AT10_003 and AT10_004 and AT10_005, wherein said
antibody selected in i) is different from said antibody selected in
ii).
[0162] Preferably, a multimeric antibody, multimeric immunoglobulin
or functional equivalent according to the invention comprises the
heavy chain CDR1, CDR2 and CDR3 and light chain CDR1, CDR2 and CDR3
sequences of at least two antibodies according to the invention.
Further provided is therefore a synthetic or recombinant multimeric
antibody, multimeric immunoglobulin or functional equivalent
thereof, comprising:
i) heavy chain CDR1, CDR2 and CDR3 and light chain CDR1, CDR2 and
CDR3 sequences of an antibody selected from the group consisting of
AT10_001 and AT10_002 and AT10_003 and AT10_004 and AT10_005; and
ii) heavy chain CDR1, CDR2 and CDR3 and light chain CDR1, CDR2 and
CDR3 sequences of an antibody selected from the group consisting of
AT10_001 and AT10_002 and AT10_003 and AT10_004 and AT10_005,
wherein said antibody selected in i) is different from said
antibody selected in ii).
[0163] In one embodiment a multimeric antibody, multimeric
immunoglobulin or functional equivalent according to the invention
comprises the heavy chain sequence and the light chain sequence of
at least two antibodies according to the invention, or sequences
that are at least 70% identical thereto. The invention thus also
provides a synthetic or recombinant multimeric antibody, multimeric
immunoglobulin or functional equivalent thereof, comprising:
i) the heavy chain sequence and the light chain sequence of an
antibody selected from the group consisting of AT10_001 and
AT10_002 and AT10_003 and AT10_004 and AT10_005, or a sequence that
is at least 70%, more preferably at least 80%, more preferably at
least 85%, more preferably at least 86%, more preferably at least
87%, more preferably at least 88%, more preferably at least 89%,
more preferably at least 90%, more preferably at least 91%, more
preferably at least 92%, more preferably at least 93%, more
preferably at least 94%, more preferably at least 95%, more
preferably at least 96%, more preferably at least 97%, more
preferably at least 98%, more preferably at least 99%, identical
thereto; and ii) the heavy chain sequence and the light chain
sequence of an antibody selected from the group consisting of
AT10_001 and AT10_002 and AT10_003 and AT10_004 and AT10_005, or a
sequence that is at least 70%, more preferably at least 80%, more
preferably at least 85%, more preferably at least 86%, more
preferably at least 87%, more preferably at least 88%, more
preferably at least 89%, more preferably at least 90%, more
preferably at least 91%, more preferably at least 92%, more
preferably at least 93%, more preferably at least 94%, more
preferably at least 95%, more preferably at least 96%, more
preferably at least 97%, more preferably at least 98%, more
preferably at least 99%, identical thereto, wherein said antibody
selected in i) is different from said antibody selected in ii).
[0164] Such multimeric antibody, multimeric immunoglobulin or
functional equivalent is typically a hetero multimeric complex,
comprising at least one heavy chain of one antibody and at least
one heavy chain of another antibody. In one embodiment, the heavy
chain of one kind of antibody is paired with the heavy chain of
another kind of antibody. In a preferred embodiment, said hetero
multimeric complex comprises two paired heavy chains of one kind of
antibody, coupled to two paired heavy chains of another kind of
antibody. Preferably, the corresponding light chains of said
antibodies are also bound to said paired heavy chains, thus forming
two coupled antibodies. As used herein, the term "dimeric antibody"
refers to two antibodies that are coupled to each other (wherein
each antibody contains two heavy chains and two light chains). The
term "multimeric antibody" refers to at least two, such as for
instance two, three, four or five, antibodies that are coupled to
each other. The term "multimeric immunoglobulin" refers to at least
two immunoglobulin chains (such as for instance single domain
antibodies, single chain antibodies, nanobodies, unibodies or
single chain variable fragments (scFv)) that are coupled to each
other.
[0165] In one embodiment, antibody AT10_003 or AT10_005 is coupled
to an antibody selected from the group consisting of AT10_001,
AT10_002 and AT10_004 by sortase catalized transamidation. Such
combination of antibodies is preferred because antibodies AT10_003
and AT10_005 have specificity against at least influenza A virus
subtypes H1 and H5 and antibodies AT10_001, AT10_002 and AT10_004
have specificity against at least influenza A virus subtypes H3 and
H7 and are capable of neutralizing at least H3N2 with high
neutralizing capacity. Therefore, such combinations provide
activity against a broad range of influenza A virus subtypes.
Preferably, antibody AT10_005 is coupled to antibody AT10_001,
AT10_002 or AT10_004 by sortase catalized transamidation, because
antibody AT10_005 is capable of neutralizing at least H1N1
influenza A virus with high neutralizing capacity. The invention
therefore in one embodiment provides an influenza A virus
bispecific antibody according to the invention comprising at least
part of the sequence, preferably the heavy and/or light chain, of
antibody AT10_003 or antibody AT10_005 as depicted in table 1,
preferably of antibody AT10_005, and comprising at least part of
the sequence, preferably the heavy and/or light chain, of antibody
AT10_001, AT10_002 or AT10_004 as depicted in table 1, whereby said
part of the sequence preferably comprises at least 70% of the
sequence of said antibody, more preferably at least 75%, more
preferably at least 80%, more preferably at least 85%, more
preferably at least 90%, more preferably at least 95%, more
preferably at least 95% of the sequence of said antibody. In a
particularly preferred embodiment, an influenza A virus bispecific
antibody according to the invention comprising essentially the
whole sequence of antibody AT10_003 or antibody AT10_005 as
depicted in table 1, preferably antibody AT10_005, and comprising
essentially the whole sequence of antibody AT10_001, AT10_002 or
AT10_004 as depicted in table 1 is provided. For instance,
preferably an influenza A virus bispecific antibody according to
the invention comprises the heavy chain and the light chain of
antibody AT10_003 or of antibody AT10_005 and the heavy chain and
the light chain of antibody AT10_001, of antibody AT10_002 or of
antibody AT10_004. Preferably said antibodies are coupled by
sortase catalized transamidation as herein described.
[0166] In another embodiment, antibody AT10_003 is coupled to an
antibody selected from the group consisting of AT10_001, AT10_002,
AT10_004 and AT10_005 by sortase catalized transamidation. Such
combination of antibodies is preferred because the AT10_003 epitope
is located on the HA1 subunit of the HA protein, whereas the
binding epitope of antibodies AT10_001, AT10_002, AT10_004 and
AT10_005 is, at least partly, located on the HA2 subunit of the
protein. Therefore, such combinations target different epitopes
within the HA protein and therefore such combination provides a
strong response against influenza A virus. The invention therefore
in one embodiment provides an influenza A virus bispecific antibody
according to the invention comprising at least part of the
sequence, preferably the heavy and/or light chain, of antibody
AT10_003 as depicted in table 1 and comprising at least part of the
sequence, preferably the heavy and/or light chain, of antibody
AT10_001, AT10_002, AT10_004 or AT10_005 as depicted in table 1,
whereby said part of the sequence preferably comprises at least 70%
of the sequence of said antibody, more preferably at least 75%,
more preferably at least 80%, more preferably at least 85%, more
preferably at least 90%, more preferably at least 95%, more
preferably at least 95% of the sequence of said antibody. In a
particularly preferred embodiment, an influenza A virus bispecific
antibody according to the invention comprising essentially the
whole sequence of antibody AT10_003 as depicted in table 1, and
comprising essentially the whole sequence of antibody AT10_001,
AT10_002, AT10_004 or AT10_005 as depicted in table 1 is provided.
Preferably said antibodies are coupled by sortase catalized
transamidation as herein described.
[0167] Yet another embodiment of the invention provides a synthetic
or recombinant multimeric antibody, multimeric immunoglobulin or
functional equivalent thereof, comprising:
i) at least two different heavy chain CDR sequences and at least
two different light chain CDR sequences of antibody AT10_002; and
ii) at least two different heavy chain CDR sequences and at least
two different light chain CDR sequences of antibody AT10_005.
Preferably, said multimeric antibody, multimeric immunoglobulin or
functional equivalent according to the invention comprises: i)
heavy chain CDR1, CDR2 and CDR3 and light chain CDR1, CDR2 and CDR3
sequences of antibody AT10_002; and ii) heavy chain CDR1, CDR2 and
CDR3 and light chain CDR1, CDR2 and CDR3 sequences of antibody
AT10_005. In a particularly preferred embodiment, said multimeric
antibody, multimeric immunoglobulin or functional equivalent
comprises: i) the heavy chain sequence and the light chain sequence
of antibody AT10_002, or sequences that are at least 70%, more
preferably at least 80%, more preferably at least 85%, more
preferably at least 86%, more preferably at least 87%, more
preferably at least 88%, more preferably at least 89%, more
preferably at least 90%, more preferably at least 91%, more
preferably at least 92%, more preferably at least 93%, more
preferably at least 94%, more preferably at least 95%, more
preferably at least 96%, more preferably at least 97%, more
preferably at least 98%, more preferably at least 99%, identical
thereto; and ii) the heavy chain sequence and the light chain
sequence of antibody AT10_005, or sequences that are at least 70%,
more preferably at least 80%, more preferably at least 85%, more
preferably at least 86%, more preferably at least 87%, more
preferably at least 88%, more preferably at least 89%, more
preferably at least 90%, more preferably at least 91%, more
preferably at least 92%, more preferably at least 93%, more
preferably at least 94%, more preferably at least 95%, more
preferably at least 96%, more preferably at least 97%, more
preferably at least 98%, more preferably at least 99% identical
thereto.
[0168] As shown in the Examples, a multimeric antibody or
immunoglobulin based on antibodies AT10_002 and AT10_005 provide
excellent influenza neutralizing activity, both in vitro and in
vivo. Since AT10_002 is capable of neutralizing H3N2 and AT10_005
is capable of neutralizing H1N1, a multimeric antibody or
immunoglobulin based on antibodies AT10_002 and AT10_005 is
particularly suitable for neutralizing both H3N2 and H1N1. Further
provided is therefore a method for neutralizing a H1N1 influenza A
virus and/or an H3N2 influenza A virus, comprising contacting said
H1N1 influenza A virus and/or said H3N2 influenza A virus with a
multimeric antibody, multimeric immunoglobulin or functional
equivalent comprising at least two, preferably three, different
heavy chain CDR sequences and at least two, preferably three,
different light chain CDR sequences of antibodies AT10_002 and
AT10_005, resulting in neutralization of said virus.
[0169] In one embodiment, a multimeric antibody, multimeric
immunoglobulin or functional equivalent according to the invention
is a dimeric antibody or dimeric immunoglobulin. However, the
invention also encompasses other multimeric antibodies or
immunoglobulins, such as for instance trimeric, tetrameric or
pentameric antibodies or immunoglobulins.
[0170] Further provided is an isolated or recombinant cell or a
pharmaceutical composition comprising a multimeric antibody,
multimeric immunoglobulin or functional equivalent according to the
invention, as well as a synthetic or recombinant multimeric
antibody, multimeric immunoglobulin or functional equivalent
according to the invention for use as a medicament and/or
prophylactic agent. As shown in the Examples, such multimeric
antibodies or immunoglobulins are particularly suitable for
treating and/or preventing and/or alleviating the symptoms of an
influenza A infection. The invention therefore also provides a
synthetic or recombinant multimeric antibody, multimeric
immunoglobulin or functional equivalent according to the invention
for use as a medicament and/or prophylactic agent for at least in
part treating and/or preventing and/or alleviating the symptoms of
an influenza A infection, as well as a method for at least in part
treating and/or preventing an influenza A virus infection,
comprising administering to an individual in need thereof a
therapeutically effective amount of a multimeric antibody,
multimeric immunoglobulin or functional equivalent according to the
invention and/or a cell or pharmaceutical composition comprising a
multimeric antibody, multimeric immunoglobulin or functional
equivalent according to the invention.
[0171] A multimeric antibody, multimeric immunoglobulin or
functional equivalent according to the invention is also suitable
for use in diagnosis of an influenza A virus. This is for instance
done by contacting a sample with a multimeric antibody, multimeric
immunoglobulin or functional equivalent according to the invention
and subsequently determining whether influenza A virus is bound to
said multimeric antibody, multimeric immunoglobulin or functional
equivalent. The invention therefore also provides a method for
determining whether an influenza A virus is present in a sample
comprising: [0172] contacting said sample with a multimeric
antibody, multimeric immunoglobulin or functional equivalent
according to the invention, [0173] allowing said multimeric
antibody, multimeric immunoglobulin or functional equivalent to
bind said influenza A virus, if present, and [0174] determining
whether influenza A virus is bound to said multimeric antibody,
multimeric immunoglobulin or functional equivalent, thereby
determining whether an influenza A virus is present in said
sample.
[0175] A synthetic or recombinant multimeric antibody, multimeric
immunoglobulin or functional equivalent according to the invention
for use in diagnosis of an influenza A infection is also provided
herewith.
[0176] A major advantage of a multimeric antibody, multimeric
immunoglobulin or functional equivalent according to the invention
over a mixture of separately produced antibodies is the fact that
for pharmaceutical uses, only one registration procedure is
required for a multimeric antibody, multimeric immunoglobulin or
functional equivalent according to the invention, whereas a mixture
of antibodies requires multiple registration procedures, usually
one procedure for each individual antibody and one separate
procedure for such mixture as a whole. The use of a multimeric
antibody, multimeric immunoglobulin or functional equivalent
according to the invention is therefore more time and cost
effective.
[0177] Antibodies according to the invention are capable of
counteracting influenza A viruses. Antibodies according to the
invention are therefore particularly suitable for use as a medicine
or prophylactic agent. Preferably, antibodies according to the
invention are used which consist of human sequences, in order to
reduce the chance of adverse side effects when human individuals
are treated. Such human sequences can be isolated from a human or
synthetically or recombinantly produced based on the sequence of
human antibodies. Provided is therefore an antibody according to
the invention for use as a medicament and/or prophylactic agent.
Also provided is a nucleic acid molecule or functional equivalent
thereof according to the invention or a vector according to the
invention comprising such nucleic acid or functional equivalent for
use as a medicament and/or prophylactic agent. When a nucleic acid
or functional equivalent according to the invention is
administered, it will be translated in situ by the host's machinery
into an antibody according to the invention. Produced antibodies
according to the invention are capable of preventing and/or
counteracting an influenza A infection. Antibodies according to the
invention are particularly suitable for use as a medicament because
they are (heterosubtype) cross-binding antibodies, capable of
binding several influenza A virus subtypes. In a particularly
preferred embodiment said antibody comprises antibody AT10_004,
AT10_003, AT10_002, AT10_001, AT10_005 or a functional part
thereof. Provided is thus antibody AT10_004, comprising a heavy
chain sequence of SEQ ID NO:31 and a light chain sequence of SEQ ID
NO:36, for use as a medicament and/or prophylactic agent. Also
provided is antibody AT10_003, comprising a heavy chain sequence of
SEQ ID NO:32 and a light chain sequence of SEQ ID NO:37, for use as
a medicament and/or prophylactic agent. Also provided is antibody
AT10_002, comprising a heavy chain sequence of SEQ ID NO:33 and a
light chain sequence of SEQ ID NO:38, for use as a medicament
and/or prophylactic agent. Also provided is antibody AT10_001,
comprising a heavy chain sequence of SEQ ID NO:34 and a light chain
sequence of SEQ ID NO:39, for use as a medicament and/or
prophylactic agent. Also provided is antibody AT10_005, comprising
a heavy chain sequence of SEQ ID NO:35 and a light chain sequence
of SEQ ID NO:40, for use as a medicament and/or prophylactic
agent.
[0178] More preferably, said antibody according to the invention
for use as a medicament and/or prophylactic agent is selected from
the group consisting of AT10_002 and AT10_004 and AT10_001 and
AT10_005. As shown in the Examples, these antibodies are
particularly effective in counteracting influenza. Most preferably,
the invention provides AT10_002 for use as a medicament and/or
prophylactic agent, because this antibody is very effective in
counteracting influenza.
[0179] An antibody according to the invention, or a nucleic acid
molecule or functional equivalent thereof according to the
invention is preferably used for at least in part treating and/or
preventing an influenza A virus infection. As used herein "at least
in part treating an influenza A virus infection" includes
counteracting an influenza A virus infection, alleviating symptoms
resulting from an influenza A virus infection and/or counteracting
inflammation resulting from an influenza A virus infection.
Examples of symptoms resulting from an influenza A virus infection
include, but are not limited to, fever, respiratory symptoms such
as cough, sore throat, runny or stuffy nose, breathing problems and
pneumonia, muscle aches, headache, fatigue and conjunctivitis. Also
provided is therefore an antibody according to the invention, or a
nucleic acid molecule or functional equivalent thereof according to
the invention, or a vector according to the invention, for use in a
method of at least in part treating and/or preventing an influenza
A virus infection. Further provided is a use of an antibody or
functional part or immunoglobulin chain or functional equivalent or
a nucleic acid molecule or functional equivalent according to the
invention or a vector according to the invention for the
preparation of a medicament and/or prophylactic agent for at least
in part treating and/or preventing an influenza A virus infection.
Preferred antibodies are antibodies AT10_004, AT10_003, AT10_002,
AT10_001 and AT10_005, which have heavy chain and light chain
sequences as depicted in table 1.
[0180] The invention further provides a pharmaceutical composition
comprising an antibody according to the invention, and/or a
bispecific antibody according to the invention, and a
pharmaceutical acceptable carrier, diluent and/or excipient. Also
provided is a pharmaceutical composition comprising a nucleic acid
molecule or functional equivalent according to the invention, or a
vector according to the invention comprising such nucleic acid or
functional equivalent, and a pharmaceutical acceptable carrier,
diluent and/or excipient. Examples of suitable carriers for
instance comprise keyhole limpet haemocyanin (KLH), serum albumin
(e.g. BSA or RSA) and ovalbumin. In one preferred embodiment said
suitable carrier comprises a solution, like for example saline. A
pharmaceutical composition according to the invention is preferably
suitable for human use.
[0181] The invention further provides a method for at least in part
treating and/or preventing an influenza A virus infection,
comprising administering to an individual in need thereof a
therapeutically effective amount of an antibody according to the
invention, and/or a bispecific antibody according to the invention,
and/or a nucleic acid molecule or functional equivalent thereof
according to the invention, and/or a vector according to the
invention, and/or a pharmaceutical composition according to the
invention. As used herein, an "individual" is a human or an animal,
preferably an animal that can be infected by influenza virus, such
as birds and mammals. Individuals include, but are not limited to,
chickens, ducks, geese, turkeys, swans, emus, guinea fowls and
pheasants, humans, pigs, ferrets, seals, rabbits, cats, dogs and
horses. In a preferred embodiment of the invention an individual is
a human.
[0182] In order to at least in part treat or prevent a influenza A
virus infection, an antibody, a nucleic acid molecule or functional
equivalent thereof, a vector, and/or a pharmaceutical composition
according to the invention is preferably administered to an
individual before an influenza A virus infection has taken place.
Alternatively, an antibody, a nucleic acid molecule or functional
equivalent thereof, a vector, and/or a pharmaceutical composition
according to the invention is administered when an individual is
already infected. In that case, an influenza A virus infection is
counteracted, symptoms resulting from an influenza A virus
infection are alleviated and/or inflammation resulting from an
influenza A virus infection is counteracted. Said antibody or
functional equivalent is particularly suitable for administered to
individuals with an increased risk of complications, such as
hospitalized individuals, for instance infants, individuals with
compromised immunity and/or elderly people. An antibody, a nucleic
acid molecule or functional equivalent thereof, a vector, and/or a
pharmaceutical composition according to the invention is preferably
administered via one or more injections. Typical doses of
administration of an antibody according to the invention or
combinations of at least two thereof are between 0.1 and 10 mg per
kg body weight. For prophylactic or therapeutic application
antibodies according to the invention are preferably combined with
a pharmaceutically acceptable carrier, diluent and/or
excipient.
[0183] An antibody according to the invention is also particularly
suitable for diagnostic uses. For instance, if an individual,
preferably a human, is suspected of suffering from an influenza A
virus infection, a sample, such as a saliva, sputum, blood, or
tissue sample, can be obtained from said individual. Subsequently,
said sample can be tested for the presence of influenza A virus,
using an antibody according to the invention. Preferably, said
sample is mixed with an antibody according to the invention, which
will specifically bind to a HA protein of influenza A virus. The
presence of HA proteins of influenza A virus in a sample is
indicative for the presence of an influenza A virus infection. HA
proteins of influenza A virus and/or influenza A virus comprising a
HA protein bound to an antibody according to the invention can be
isolated from the sample and/or detected using any method known in
the art, for example, but not limited to, isolation using magnetic
beads, streptavidin-coated beads, or isolation through the use of
secondary antibodies immobilized on a column. Alternatively, or
additionally, an antibody according to the invention is labeled in
order to be able to detect said antibody, for instance, but not
limited to, fluorescently labeled, or radioactively labeled.
Alternatively, an antibody according to the invention is detected
using a labeled secondary antibody which is directed against said
antibody. If binding of said antibody is detected, HA protein of
influenza A virus is present, which is indicative for the presence
of an influenza A virus infection. The invention thus provides an
antibody according to the invention for use in diagnosis of an
influenza A virus infection.
[0184] The invention thus further provides a method for determining
whether an influenza A virus is present in a sample comprising:
[0185] contacting said sample with an antibody according to the
invention, [0186] allowing said antibody to bind said influenza A
virus, if present, and [0187] determining whether influenza A virus
is bound to said antibody thereby determining whether an influenza
A virus is present.
[0188] In a preferred embodiment it is determined whether an
individual is suffering from an influenza A virus infection.
Provided is therefore a method for determining whether an
individual is suffering from an influenza A virus infection
comprising: [0189] contacting a sample from said individual with an
antibody according to the invention, [0190] allowing said antibody
to bind said influenza A virus, if present, and [0191] determining
whether influenza A virus is bound to said antibody thereby
determining whether said individual is suffering from an influenza
A virus infection. Preferably said individual is a human.
[0192] In yet another embodiment, the invention provides an
isolated, synthetic or recombinant antibody or functional part
thereof, or immunoglobulin chain or functional equivalent thereof,
capable of interacting with amino acids at positions A38, A40, A41,
A42, A291, A292, A293, A318, B18, B19, B20, B21, B38, B41, B42,
B45, B46, B48, B49, B52, B53, and B56 of influenza A virus group 1
hemagglutinin (H1/H5). These are hemagglutinin amino acids that
interact with antibody AT10_005. Antibodies, immunoglobulins or
functional parts or functional equivalents thereof, capable of
specifically interacting with said hemagglutinin amino acids, are
therefore herewith provided.
[0193] Yet another embodiment provides an isolated, synthetic or
recombinant antibody or functional part thereof, or immunoglobulin
chain or functional equivalent thereof, capable of interacting with
amino acids at positions A21, A324, A325, A327, B12, B14, B15, B16,
B17, B18, B19, B25, B26, B30, B31, B32, B33, B34, B35, B36, B38,
B146, B150, B153, and B154 of influenza A virus group 2
hemagglutinin (H3/H7). These are hemagglutinin amino acids that
interact with antibody AT10_004. Antibodies, immunoglobulins or
functional parts or functional equivalents thereof, capable of
specifically interacting with said hemagglutinin amino acids, are
therefore herewith provided.
[0194] Yet another embodiment provides an isolated, synthetic or
recombinant antibody or functional part thereof, or immunoglobulin
chain or functional equivalent thereof, capable of interacting with
amino acids at positions A38, A48, A275, A276, A277, A278, A289,
A291, A318, B19, B20, B21, B36, B38, B39, B41, B42, B45, B46, B48,
B49, B50, B52, B53, B56, B57, B58, B150 of influenza A virus group
2 hemagglutinin (H3/H7). These are hemagglutinin amino acids that
interact with antibody AT10_002. Antibodies, immunoglobulins or
functional parts or functional equivalents thereof, capable of
specifically interacting with said hemagglutinin amino acids, are
therefore herewith provided.
[0195] The above mentioned amino acid numbering for hemagglutinin
is according to Wilson et al. 1981 Nature 289, 366-373 and Nobusawa
et al. 1991 Virology 182, 475-485.
[0196] Yet another embodiment provides an isolated, synthetic or
recombinant antibody or functional part thereof, or immunoglobulin
chain or functional equivalent thereof, capable of competing with
AT10_001 or AT10_002 or AT10_003 or AT10_004 or AT10_005 for at
least part of the same epitope on influenza A virus hemagglutinin,
said antibody, immunoglobulin, functional part or equivalent having
at least the same affinity for said influenza A virus hemagglutinin
(typically having the same or a lower Km value as compared to
AT10_001 or AT10_002 or AT10_003 or AT10_004 or AT10_005),
resulting in a decreased binding between said influenza A virus
hemagglutinin and AT10_001 or AT10_002 or AT10_003 or AT10_004 or
AT10_005. Said epitope preferably comprises the amino acids at
positions A38, A40, A41, A42, A291, A292, A293, A318, B18, B19,
B20, B21, B38, B41, B42, B45, B46, B48, B49, B52, B53 and B56 of
influenza A virus hemagglutinin. In another preferred embodiment
said epitope comprises the amino acids at positions A21, A324,
A325, A327, B12, B14, B15, B16, B17, B18, B19, B25, B26, B30, B31,
B32, B33, B34, B35, B36, B38, B146, B150, B153, B154 of influenza A
virus hemagglutinin. In yet another preferred embodiment said
epitope comprises A38, A48, A275, A276, A277, A278, A289, A291,
A318, B19, B20, B21, B36, B38, B39, B41, B42, B45, B46, B48, B49,
B50, B52, B53, B56, B57, B58, B150 of influenza A virus
hemagglutinin.
[0197] The invention is further explained in the following
examples. These examples do not limit the scope of the invention,
but merely serve to clarify the invention.
Examples
Generation of Immortalized B Cells
[0198] Human memory B cells were immortalized using the BCL6/Bcl-xL
technology described by Kwakkenbos et al. (Generation of stable
monoclonal antibody-producing B cell receptor--positive human
memory B cells by genetic programming. Nature Medicine (2010) vol.
16 (1) pp. 123-8 and patent application WO 2007/067046). In brief,
human memory B cells from Influenza vaccinated donors were
transduced with a retroviral vector containing BCL6 and Bcl-xL.
Transduced B cells can be maintained in culture with CD40Ligand
expressing L-cells and interleukin (IL)-21 (R&D systems).
Selection of Heterosubtypic Mabs
[0199] To identify B cells that secrete heterosubtypic
cross-binding mAbs two approaches were used. i) The Influenza H3
(A/Wyoming/03/2003) and H7 (A/Netherlands/219/2003) HA proteins
(Protein Sciences) were labeled with Alexa Fluor 647 Molecular
Probes) and incubated with Bcl6 and Bcl-xL transduced polyclonal
cultured B cells. HA binding B cells were sorted single cell per
well by FACSAria (FIG. 2) and maintained in culture for 2 to 3
weeks before the supernatant of the B cell clones were screened for
HA binding by ELISA or binding to H3N2 (A/Netherlands/177/2008)
infected cells and/or H7 (A/Netherlands/219/2003) transfected HEK
cells. Cells were seeded in small pools, e.g. 40 cells per well and
maintained in culture for 2-3 weeks. The supernatant of these pools
was screened for binding to H7 transfected HEK cells. The B cells
of the double positive tested wells were seeded 1 cell per well.
The culture supernatant of these monoclonal B cell lines was used
to screen for HA binding by ELISA.
[0200] B cells that showed reactivity to more than 1 HA type were
further cultured and characterized for HA recognition by ELISA
(Table 2) and binding to HA expressing HEK cells (FIG. 3).
HA ELISA
[0201] The B cell supernatant of cross-reactive B cell clones was
tested for binding to different HA antigens by ELISA. Recombinant
HA of H1 (A/New Caledonia/20/1999), H3 (A/Wyoming/03/2003), H5
(A/Vietnam/1203/2004) and H7 (A/Netherlands/219/2003) (Protein
Sciences) were coated to ELISA plates at 1 .mu.g/ml. After coating,
the plates were washed 1.times. with PBS and 350 .mu.l blocking
buffer, PBS/4% Protivar, was added and incubated 1 hr at RT. The
plates were then washed 3.times. with PBST (PBS/0.05% Tween20) and
the antibodies/culture supernatants were added to the wells.
Incubation was allowed to proceed for 1 hr at RT, then the plates
were washed 3.times. with PBST. Samples were then incubated with a
goat anti-human IgG-HRP antibody (Jackson) for 1 hr at RT. Bound
antibodies were detected using TMB (3,3', 5,5' tetramethyl
benzidine) substrate buffer, the reaction was stopped using H2504.
OD 450 nm was measured on an Envision (PerkinElmer). AT10_001 and
AT10_002 recognized both H3 and H7 proteins but not the HA proteins
of H1 and H5. AT10_003 recognized H3, H5 and H7 protein while
AT10_004 recognized H1, H3 and H7 proteins (Table 2).
Antibody Binding HA Transfected 293T Cells
[0202] To test heterosubtypic binding of the AT10 mAbs to cell
surface expressed HA, 293T cells were transfected with different
full length HA constructs. Using Fugene (Roche) 293T cells were
transfected with DNA encoding the HA of H1 (A/New
Caledonia/20/1999), H3 (A/Wisconsin/67/2005), H5
(A/Thailand/Vietnam Consensus/2004) and H7
(A/Netherlands/219/2003). The transfected cells were incubated with
B cell supernatant containing IgG antibodies for 30 minutes at
4.degree. C. and then washed 2.times. with 150 .mu.l PBS/2%
FCS.
[0203] Antibody binding was detected with anti-human IgG-PE
(Southern Biotech) and analyzed on a FACScanto (Becton, Dickinson
and Company) (FIG. 3). As a control untransfected 293T cells were
used. AT10_001 and AT10_002 recognized both H3 and H7 cell surface
expressed proteins but not the HA proteins of H1 and H5. AT10_003
recognized H3, H5 and H7 protein while AT10_004 recognized the H1,
H3 and H7 HA proteins.
Cloning of Selected Antibodies.
[0204] We isolated total RNA with the RNeasy.RTM. mini kit
(Qiagen), generated cDNA, performed PCR and cloned the heavy and
light chain variable regions into the pCR2.1 TA cloning vector
(Invitrogen). To rule out reverse transcriptase or DNA polymerase
induced mutations, we performed several independent cloning
experiments. To produce recombinant mAb we cloned the heavy and
light variable regions in frame with human IgG1 and Kappa constant
regions into a pcDNA3.1 (Invitrogen) based vector and transiently
transfected 293T cells. We purified recombinant mAb from the
culture supernatant with an AKTA (GE healthcare).
Cross Binding Specificity of AT10 Antibodies
[0205] Eleven different recombinant HA proteins (Sino Biological
Inc and Protein Sciences) were used to test the potential of the
antibodies to bind different HA subtypes. Reactivity to these HA
proteins (table 3) was tested in an ELISA, as described above. None
of the mAbs showed reactivity with Influenza B. AT10_001, AT10_002,
AT10_003 and AT10_004 showed binding to all human group 2 HA
proteins. AT10_001, AT10_003 and AT10_004 also showed reactivity to
Swine H4N6 (A/Swine/Ontario/01911-1/1999). AT10_002 and AT10_003
recognized Duck H10N3 (A/duck/Hong Kong/786/1979) and Duck H15N8
(A/duck/AUS/341/1983), AT10_004 also showed some activity to H15N8
(A/duck/AUS/341/1983). AT10_003 recognized the group 1 HA molecules
from H9N2 (A/Hong Kong/1073/1999) and H5N1 (A/Vietnam/1203/2004)
while AT10_004 also showed binding to the HAs of H1N1
(A/California/07/2009) and H9N2 (A/Hong Kong/1073/1999). AT10_005
bound exclusively to the group 1 HA proteins tested.
Antibody Binding to Virus Infected Cells
[0206] To test the binding capacity of the AT10 antibodies
AT10_001, AT10_002, AT10_003 and AT10_004 to virus infected cells
we performed FACS analysis on Influenza H1N1 (A/Hawaii/31/2007) and
H3N2 (A/Netherlands/177/2008) infected cells (both virus strains
were obtained from the Department of Medical Microbiology, AMC,
Amsterdam). MDCK-SIAT cells were grown in a T175 culture flask to
80-100% confluency in DMEM/FCS/PS/G418. The cell layer was washed
2.times. with 10 ml PBS after which 15 ml of
Optimem/PS/G418/Trypsin was added. Subsequently 0.5 ml of 100.000
TCID50 Influenza virus (H1N1 or H3N2) was added to the flask and
cells were cultured at 37.degree. C. After 24-48 hr the cells were
washed 2.times. with 10 ml PBS and detached from the plastic using
Trypsin-EDTA. Cells were counted and frozen at -150.degree. C.
until use. The infected cells were defrosted and incubated with IgG
antibodies/B cell supernatant for 30 minutes at 4.degree. C. and
then washed 2.times. with 150 .mu.l PBS/2% FCS. Antibody binding
was detected with anti-human IgG-PE and analyzed on a FACScanto
(Becton, Dickinson and Company). As a control non-infected cells
were used (FIG. 4). All mAbs showed binding to H3N2 infected cells
but not to non-infected cells. Antibodies AT10_004 and AT10_003
also showed some binding to H1N1 infected cells.
[0207] Similar experiments were performed for the AT10 antibodies
AT10_001, AT10_002, AT10_003, AT10_004 and AT10_005 with Influenza
H1N1 (A/Neth/602/2009), H3N2 (A/Swine/St. Oedenrode/1996), high
pathogenic H5N1 (A/Turkey/Turkey/2004), high pathogenic H7N7
(A/Ch/Neth/621557/03) and low pathogenic H7N1
(A/Ch/Italy/1067/1999) infected cells (Central Veterinary
Institute, Lelystad). MDCK cells were infected with virus as
described above, only the cells were fixated with 4%
paraformaldehyde for 20 minutes at 4.degree. C., washed 1.times.
with PBS and then frozen. As a control non-infected cells were
used. FACS staining and analysis was done a described above (FIG. 5
and Table 4). AT10_001 recognized both H7 viruses but failed to
recognize H3N2 (A/Swine/St.Oedenrode/1996) infected cells. AT10_001
showed some reactivity to H1N1 (A/Neth/602/2009). Antibodies
AT10_002 and AT10_004 recognized all three group 2 Influenza
infected cell batches, AT10_004 also showed some reactivity to H5N1
(A/Turkey/Turkey/2004) Influenza. AT10_003 only showed some low
binding to H3N2 (A/Swine/St.Oedenrode/1996) and H7N7
(A/Ch/Neth/621557/2003) infected cells. AT10_005 bound to group 1
Influenza infected cells and not to group 2 Influenza infected
cells.
Virus Neutralization
[0208] To determine whether the obtained antibodies were capable of
blocking Influenza A virus infection, an in vitro neutralization
assay was performed. The assay was performed on MDCK-SIAT cells
(Journal of Virology August 2003; pp. 8418-25). MDCK-SIAT cells
were grown in DMEM/8% FCS/PS/G418 in an 96 well plate (CellCarrier
Plate, PerkinElmer) to 80-100% confluency. Neutralization assays
are performed in Optimem/PS/G418/Trypsin medium without FCS or BSA.
Fifty .mu.l of recombinant mAb was mixed with 50 .mu.l of virus
suspension (100TCID50/50 .mu.l) of H3N2 (A/Ned/177/2008) or H1N1
(A/Hawaii/31/2007) Influenza and incubated for 1 hr at 37.degree.
C. The suspension was then transferred in multiply into 96-well
plates containing MDCK-SIAT cells in 100 .mu.l
Optimem/PS/G418/Trypsin. Prior to use the MDCK-SIAT cells were
washed twice with 150 .mu.l PBS. The plates were then centrifuged
for 15 minutes at RT at 2500 rpm and placed at 37.degree. C./5%
CO2. After 24 hr cells were washed twice with PBS, fixed with
Formalin (37% formaldehyde in water) for 10 minutes at RT, washed
twice with 150 .mu.l PBS and stained with DAPI and an antibody
against the nuclear protein of the Influenza virus (NP-FITC, Abcam)
at RT. After 30 minutes cells were washed twice with 150 .mu.l PBS
and 100 .mu.l of PBS/50% Glycerol was added to the wells. Viral
infection of the MDCK-SIAT cells was measured and analyzed on the
Operetta (PerkinElmer) using an 20.times. objective. To quantify
neutralizing capacity of the mAbs the number of infected cells was
counted (positive for DAPI and NP-FITC) (Table 5). IC50 values were
calculated in Prism, values are from 1 representative experiment,
assay points performed in quadruplicate. AT10_001, AT10_002 and
AT10_004 showed potent inhibition of H3N2 (A/Ned/177/2008) and H3N2
HKX-31 Influenza virus infection in vitro. Neutralization of H1N1
(A/Hawaii/31/2007) was not observed for AT10_001, AT10_002,
AT10_003 and AT10_004.
[0209] To determine whether the obtained AT10 antibodies were
capable of blocking multiple Influenza A virus strains, additional
in vitro neutralization assays were performed. Influenza viruses
A/swine/Neth/St. Oedenrode/96 (H3N2; de Jong et al. 1999),
A/ck/Neth/621557/03 (H7N7; van der Goot et al. 2005),
A/ck/Italy/1067/99 (H7N1), A/turkey/Turkey/05 (H5N1; Londt et al.
2008) and A/Neth/602/2009 (swine-origin H1N1; Munster et al. 2009)
were used in this assay. Madin-Darby canine kidney (MDCK) cells
were cultured in Optimem (Gibco BRL Life Technologies) containing
5% FBS (Integro) and 1% Pen Streptomycine (Gibco BRL Life
Technologies).
[0210] Cells were seeded at a density of 3.times.10.sup.4 cells per
well in 96-well plates and incubated O/N at 37.degree. C.
Three-fold serial dilutions of the mAbs were made in PBS starting
with a concentration of 15 .mu.g/ml. Rituximab mAb was taken along
as negative control. Virus dilutions were prepared in virus
infection medium consisting of Optimem supplemented with
antibiotics and, in case of LPAI viruses, 1 .mu.g/ml trypsin/TPCK
(Sigma). Each mAb dilution was mixed with an equal volume of virus
followed by 1 hour incubation at 37.degree. C. After washing of the
cells with PBS, the mAb/virus mixture (.about.100-1000 TCID.sub.50)
was inoculated onto the cell monolayers. Cells were incubated for
24-32 hours at 37.degree. C., after which they were washed twice
with PBS, fixed with 4% formalin for 20 min and then washed again
with PBS. Cells incubated with medium only were included as
negative control and cells incubated with virus only as positive
control. The assay was performed in quadruplicate. Cells were
stained with 1 hour with DAPI and an antibody against the nuclear
protein of the Influenza virus (NP-FITC, Abcam or HB65 followed by
Goat-anti-mouse IgG Alexa-647, Invitrogen) at RT. Cells were washed
twice with 150 .mu.l PBS after staining and 100 .mu.l of PBS/50%
Glycerol was added to the wells. Viral infection of the MDCK cells
was measured and analysed on the Operetta (PerkinElmer) using an
10.times. objective. To quantify neutralizing capacity of the mAbs
the number of infected cells was counted (positive for DAPI and
NP-FITC/HB65-Alexa-647). IC50 values were calculated in Prism,
values are from 1 representative experiment. The results are shown
in Table 7. AT10_002 and AT10_004 showed potent inhibition of the
group 2 influenza virus infection in vitro but did not prevent
infection with group 1 viruses. Antibody AT10_005 prevents
infection with group 1 Influenza A viruses but has no effect on
group 2 viruses. Antibody CR8020 (WO 2010 130636) does not show any
neutralizing capacity for H3N2 A/Swine/Neth/St. Oedenrode/96 and
H7N1 A/ck/Italy/1067/99 at 15 .mu.g/ml while AT10_002 and AT10_004
show IC50 values below 4 .mu.g/ml.
Antibody Competition
[0211] Antibody AT10_001 and AT10_003 were labeled with Alexa Fluor
555 (Molecular Probes) and antibody AT10_002 and AT10_004 were
labeled with Alexa Fluor 647 (Molecular Probes). Labeled antibodies
were tested for binding to H3N2 (A/Netherlands/177/2008) infected
MDCK-SIAT cells to determine if they maintained their binding
capacity. For competition experiments H3N2 (A/Netherlands/177/2008)
infected cells were incubated with increasing amounts of
non-labeled competitor antibody for 10 minutes at 4.degree. C.
before the addition of Alexa Fluor-labeled antibody. Cell-antibody
mix was incubated for another 15 minutes at 4.degree. C. and washed
2.times. with PBS/2% FCS before analysis on the Guava easyCyte 8
(Millipore). AT10_001, AT10_002 and AT10_004 all bind to a similar
region on the HA protein as they all block each other's binding
(FIG. 6A). Antibody competition was also performed on H1N1 infected
cells (A/Hawaii/31/2007). AT10_004-Alexa-647 antibody binding was
blocked by unlabeled AT10_004 and AT10_005 (FIG. 6B). The AT10_005
antibody recognize the stem region of the group 1 HA molecules. As
AT10_004 competes with AT10_005 for binding it is likely that
AT10_004 also recognizes the HA stem region. Because AT10_001,
AT10_002 and AT10_004 all bind to a similar region on the HA
protein (FIG. 6A) AT10_001 and AT10_002 therefore also have their
binding epitope on the stem region.
HA1 Subunit ELISA
[0212] To test whether the HA1 subunit is essential for the binding
of the antibodies to the HA protein an HA1 subunit specific ELISA
was done. Recombinant HA of full length H3 (A/Aichi/2/1968, full
length) and H3 HA1 subunit (A/Aichi/2/1968, HA1 subunit, Met-1-Arg
345) were coated to ELISA plates at 1 .mu.g/ml. After coating, the
plates were washed 1.times. with PBS and 300 .mu.l blocking buffer,
PBS/4% Protivar, was added and incubated 1 hr at RT. The plates
were then washed 3.times. with PBST (PBS/0.05% Tween20) and the
recombinant antibodies were added to the wells. Incubation was
allowed to proceed for 1 hr at RT, then the plates were washed
3.times. with PBST. Samples were then incubated with a goat
anti-human IgG-HRP antibody (Jackson) for 1 hr at RT. Bound
antibodies were detected using TMB substrate buffer, the reaction
was stopped using H2504. OD 450 nm was measured on an Envision
(PerkinElmer) (Table 6). AT10_001, AT10_002 and AT10_004 recognized
full-length H3 HA protein but not the HA1 subunit of this protein
indicating their binding epitope is, at least partly, located on
the HA2 subunit of the protein. AT10_003 recognized both the
full-length HA protein and the HA1 subunit indicating that the
AT10_003 epitope is located on the HA1 subunit of the HA
protein.
Binding of AT10 Antibodies to Different HA Conformations
[0213] Upon endocytic uptake of virions, the acidic environment of
the endosome triggers HA-driven fusion of the viral and the
endosomal membrane. This fusion is mediated by a conformational
change of the HA protein (triggered by the low pH) from a
pre-fusion state to a post-fusion state. We performed an in vitro
pH-shift experiment to test to which conformational configuration
of HA the antibodies can bind.
[0214] Using Fugene (Roche) 293T cells were transfected with DNA
encoding the HA of H3 (A/Wisconsin/67/2005). 48 hours post
transfection the cells were harvested using trypsin-EDTA and stored
at -150.degree. C. until further use. For the pH-shift experiment,
cells were washed 2.times. with PBS and then incubated for 30
minutes at room temperature with 10 .mu.g/ml trypsin-EDTA in PBS.
Cells were washed 2.times. with PBS and a fraction was set aside as
trypsin condition. Remaining cells were split to two tubes and
treated with either 500 mM Dithiothreitol (DTT) for 20 minutes at
room temperature or incubated for 5 minutes at 37.degree. C. with
PBS pH5. Cells were washed 2.times. with PBS and incubated with
recombinant AT10_001, AT10_002, AT10_003 or AT10_004. Antibody
binding was detected using anti-human-IgG-PE (southern Biotech)
antibody and analyzed on a Guava easyCyte 8 (Millipore) (FIG. 7).
AT10_001, AT10_002, AT10_003 and AT10_004 all bind the trypsin
treated cells. Binding of AT10_001, AT10_002 and AT10_004 is lost
upon treatment of the transfected cells with pH5 buffer, indicating
that these antibodies recognize the pre-fusion but not the
post-fusion conformation of the HA protein. Treatment of the cells
with DTT, which induces dissociation of the HA1 subunit from the
HA2 subunit, has no effect on binding of these antibodies
indicating that the binding epitope is located on the HA2 subunit.
AT10_003 recognizes both the pre-fusion and post-fusion
conformation but binding is lost upon DTT treatment, indicating
that the binding epitope is only available when is the HA1 subunit
is present.
Prophylactic and Therapeutic Efficacy of AT10 Antibodies In
Vivo
[0215] The AT10 antibodies were tested in a mouse influenza
challenge model to determine their efficacy. Male C57Bl/6J mice (4
per group) were intranasally challenged with increasing amounts of
influenza A/HKx-31 (H3N2) and body weight changes were monitored
twice a day for 14 days to determine the viral dose response.
Twenty-five percent bodyweight loss was used as humane endpoint;
mice loosing more than 25% of their body weight were removed from
the study. In the highest dose group (20000 TCID.sub.50) all
animals lost 25% of their bodyweight within 8 days while in the
2000 TCID50 group only 50% of the mice reached this bodyweight loss
(FIG. 8). Based on these results a viral dose of 10 LD50 (20,000
TCID50) was used in subsequent antibody experiments.
[0216] Antibodies AT10_001, AT10_002, AT10_004 and a negative
control antibody (Rituximab) were tested for prophylactic efficacy
in the influenza model. Mice were intravenously injected with 1 or
5 mg/kg antibody one day before challenge with 10 LD50 influenza
A/HK.times.31. Bodyweight was monitored for 10 days after which the
experiment was terminated. All control mice lost 25% bodyweight
within 8 days and were removed from the study, however none of the
mice that received prophylactic AT10 antibody had to be removed
from the study demonstrating a protective effect of the antibodies
(P=<0.000.1, Mantel-Cox, FIG. 9A). For all AT10 antibody groups
a dose dependent effect could be seen, e.g. mice that received a
dose of 1 mg/kg antibody lost more body weight than the mice that
received 5 mg/kg of the same antibody (FIGS. 9B,C,D). Treatment
with AT10_002 at 1 mg/kg is significantly more protective than
treatment with 1 mg/kg of AT10_001 from day 4 post infection to the
end of the experiment (P=<0.05, 2 way ANOVA). There is no
significant difference in weight loss between the groups of mice
that received AT10_002 at 1 mg/kg and mice that received AT10_004
at 1 mg/kg although a trend towards a better protective activity of
AT10_002 can clearly be seen. Based on the weight loss graphs the
antibodies can be ranked for activity as follows:
AT10_002>AT10_004>AT10_001.
[0217] The AT10 antibody that showed the best protective activity
in the prophylactic Influenza experiment, AT10_002, was tested for
therapeutic efficacy in the influenza model. Mice were
intravenously injected with 15 mg/kg antibody two, three, or four
days post challenge with 10 LD50 influenza A/HK.times.31. As
controls, mice were injected with 15 mg/kg AT10_002 or a negative
control antibody (Rituximab, 15 mg/kg) one day before 10 LD50
influenza A/HK.times.31 challenge. Bodyweight was monitored for 10
days after which the experiment was terminated. The results are
shown in FIG. 10. All control mice that received Rituximab lost 25%
bodyweight within 8.5 days and were removed from the study, none of
the mice that received prophylactic AT10_002 antibody (day-1)
showed loss of bodyweight and had to be removed from the study,
confirming the protective effect of the AT10_002 antibodies.
Intravenous administration of AT10_002 at days two or three post
Influenza challenge prevented lethal bodyweight loss in all the
mice, showing the therapeutic effect of the AT10_002 antibody.
Treatment of Influenza challenged mice with AT10_002 antibodies
four days post infection protected 40% of the mice against lethal
bodyweight loss. These findings show that AT10_002 antibodies can
be used to prevent lethality up to several days after an Influenza
infection.
Generation of Pan-Specific Anti-Influenza a IgG Multimeric
Antibody
[0218] To generate a multimeric antibody complex that recognises
most Influenza A viruses we coupled AT10_002 and AT10_005 together
(BiFlu) using the sortase technology, described in detail in WO
2010/087994. To be able to link AT10_002 and AT10_005 a tag (named
ST) containing a sortase recognition site plus a His6 tag, with
sequence GGGGSLPETGGGHHHHHH, is attached to the C-terminus of the
heavy chain of the antibodies via genetic fusion.
The sortase reaction was performed by mixing 10.0 mg AT10-002 ST
antibody in 2000 .mu.l reaction buffer (25 mM Tris, pH 7.5, 150 mM
NaCl, 10 mM CaCl.sub.2) containing 60 .mu.M sortase and 500 .mu.M
GGG-Dibenzo-azacyclo-octyn (DIBAC). Similarly, 10.0 mg AT10-005 ST
antibody was mixed with 2000 .mu.l sortase-buffer containing 90
.mu.M sortase and 1 mM GGG-azide. Both samples were incubated 16 h
at 37.degree. C. After incubation sortase was deactivated by the
addition of 50 mM EDTA. Before loading the sample on a gel
filtration column, the reaction mixture was centrifuged (3 min,
13.200 rpm) to pellet any aggregates. Gel filtration chromatography
of the two sortase-tagged antibodies was performed on a HiLoad
Superdex 200 16/60 column (GE Healthcare, Piscataway, N.J., United
States) in coupling buffer (25 mM Tris, pH 7.5+150 mM NaCl). Before
loading samples, the column was equilibrated with 1.0 CV (column
volumes) coupling buffer. After loading, the column was run with
1.5 CV equilibration buffer.
[0219] Next, the purified antibodies were subjected to
click-chemistry coupling. 3.0 mg AT10-002-DIBAC was mixed with 2.9
mg AT10-005-azide and incubated at 25.degree. C. in 3.0 ml coupling
buffer (25 mM Tris, pH 7.5, 150 mM NaCl). After 16 h the sample was
subjected to gel filtration (as above) in PBS (Fresenius Kabi, Bad
Homburg, Germany). Fractions containing the IgG dimers were
collected, pooled and concentrated with 50 kDa cut-off membranes
AMICON centrifugal filter devices (Millipore, Billerica, Mass.,
United States).
Qualitative SPR Analysis of the BiFlu Preparation
[0220] Surface plasma resonance (SPR) analysis was performed on the
BiFlu preparation to determine if dimeric BiFlu was formed (e.g.
dimers consisting of both AT10_002 (lambda light chain) and
AT10_005 (kappa light chain)) and if the preparation consists of
AT10_002 AT10_005 heterodimers SPR analysis was performed on an
IBIS MX96 SPR imaging system (IBIS Technologies BV., Enschede, The
Netherlands) as described (Lokate et al., 2007, J. Am. Chem. Soc.
129:14013-140318). In short, one SPR analysis cycle consists of one
or more incubation steps in which analytes are flushed over a
coated sensor. This is followed by a regeneration step in which any
bound analyte is removed from the sensor. Multiple cycles can be
performed in one experiment. In our SPR capture-binding assay the
antibodies of interest are first captured on an isotype-specific
antibody (i.e. anti-human IgG, anti-human kappa light chain or
anti-human lambda light chain), which is immobilized on the SPR
sensor and then incubated with analytes. Obtained data was analyzed
using Sprint software (version 1.6.8.0, IBIS Technologies BV,
Enschede, The Netherlands).
[0221] The SPR sensor was coated by immobilization of isotype
specific antibodies anti-human IgG (Jackson Immunoresearch, West
Grove, Pa., USA), anti-human kappa light chain (Dako, Glostrup,
Denmark) and anti-human lambda light chain (Dako, Glostrup,
Denmark) on an amine-specific EasySpot gold-film gel-type SPR-chip
(Ssens BV, Enschede, The Netherlands) by spotting them on the
sensor surface using a continuous flow microspotter device (Wasatch
Microfluidics, Salt Lake City, Utah, USA) in coupling buffer (10 mM
NaAc, pH 4.5, 0.03% Tween20).
[0222] After spotting for 45 minutes the sensor is deactivated with
0.1 M ethanolamine, pH 8.5 and washed three times with system
buffer (PBS+0.03% Tween20+0.05% NaN.sub.3). Before starting the
analysis, the coupled sensor was incubated for two minutes with
regeneration buffer (10 mM glycine-HCl, pH 2), followed by three
wash steps with system buffer.
[0223] Then the coated SPR chip is injected either with AT10_002,
AT10_005 (2 .mu.g/ml in system buffer) or BiFlu (4 .mu.g/ml in
system buffer) and incubated for 30 min. Subsequently, non-captured
IgG is removed by a 5 minute incubation with system buffer. Next,
the sensor is injected with influenza H3-hemagglutinin protein
(H3N2, Wyoming, 03/2003, Sino Biological inc., Beijing, P.R. China,
0.25 to 2.0 .mu.g/ml) in system buffer and incubated for 30 min to
measure association. To measure complex dissociation the sensor is
washed with system buffer and incubated for 40 min. The injection
of H3 is followed by injections with influenza H1-hemagglutinin
(H1N1, New Caledonia, 20/1999, Sin Biological inc., Beijing, P.R.
China, 1.0 ug/ml) and anti-human light chain antibody (anti-kappa
or anti-lambda) in a similar fashion as described above. When the
single antibodies and BiFlu are captured on anti-human IgG (FIG.
11A) and on anti-lambda light chain (FIG. 11B), BiFlu binds both H1
and H3 with the same affinity as the single antibodies.
Furthermore, the results demonstrate that BiFlu is heterodimer with
two different light chains. The two monomeric antibodies (AT10_002
and AT10_005) bind only one analyte and have one type of light
chain.
[0224] Altogether, the SPR analysis demonstrates that BiFlu is a
heterodimer of AT10_002 and AT10_005, which binds H3 and H1 with
equal affinity as the single antibodies.
Antibody Binding to Virus Infected Cells
[0225] To test if the binding capacity of the BiFlu antibodies is
maintained and if the BiFlu has the combined binding properties of
AT10_002 and AT10_005 we performed FACS analysis on Influenza H1N1
(A/Hawaii/31/2007) and H3N2 (A/Netherlands/177/2008) infected
cells. Influenza A infected MDCK-SIAT cells were generated as
described above. The infected cells were defrosted and incubated
with different concentrations of AT10_002, AT10_005 or BiFlu
antibodies for 30 minutes at 4.degree. C. and then washed 2.times.
with 150 .mu.l IMDM/2% FCS. Antibody binding was detected with
anti-human IgG-APC and analyzed on a Guava easyCyte 8HT
(Millepore). The results are shown in FIG. 12. BiFlu and AT10_005
showed concentration dependent binding to H1N1 infected cells while
AT10_002 did not bind to these cells. H3N2 infected cells were
bound by BiFlu and AT10_002 but not by AT10_005 antibodies. For
both virus subtypes the BiFlu antibodies show similar binding
affinity (as shown by MFI of the APC signal) as the relevant single
control antibody. Together these results show that BiFlu antibodies
have the combined binding properties of AT10_002 and AT10_005.
Virus Neutralization
[0226] To determine whether BiFlu is also capable of blocking
Influenza A virus infection, an in vitro neutralization assay was
performed. The assay was performed on MDCK-SIAT cells as described
above. To quantify neutralizing capacity of the mAbs the number of
infected cells was counted (positive for DAPI and NP-FITC). Shown
in FIG. 13 are the neutralization curves for H1N1
(A/Hawaii/31/2007) and H3N2 (A/Ned/177/2008) neutralization for
AT10_002, AT10_005 and BiFlu. The concentration depicted for BiFlu
has been adjusted to represent the same available binding
opportunities (e.g. concentration shown is half of the actual
concentration BiFlu as BiFlu has the double molecular weight
compared to the single antibodies). BiFlu neutralizes H1N1 and H3N2
as well as its relevant single components.
Prophylactic Efficacy of BiFlu Antibodies In Vivo (FIG. 14)
[0227] Antibodies AT10_002, AT10_005, BiFlu (AT10_002-AT10_005
dimer), AT10_002/AT10_005 mix and a negative control antibody
(Rituximab) were tested for prophylactic efficacy in the influenza
model. Male C57Bl/6J mice (6 per group) were intranasally
challenged with 10 LD50 influenza A/HK.times.31 or 10 LD50 H1N1
Influenza A/PR/8/34 and body weight changes were monitored for 10
days. Twenty-five percent bodyweight loss was used as humane
endpoint; mice loosing more than 25% of their body weight were
removed from the study.
[0228] Mice were intravenously injected with 1 mg/kg AT10_002, 1
mg/kg AT10_005, a mix of AT10_002 and AT10_005 1 mg/kg each, 2
mg/kg BiFlu or 1 mg/kg Rituximab antibody one day before viral
challenge. All control mice (Rituximab) lost 25% bodyweight within
8 days and were removed from the study. In the H1N1 challenge model
AT10_005 antibody showed a protective effect e.g. none of the mice
had to be removed from the study. In addition, the mice that
received the BiFlu preparation and the AT10_002/AT10_005 antibody
mix were also protected (FIG. 14). No statistical difference in
bodyweight loss is observed between the groups of mix that received
the AT10_002/AT10_005 antibody mix and the BiFlu group (P>0.05,
2 way ANOVA). Similar results were obtained in the H3N2 in vivo
model. AT10_002 antibody, the antibody mix (AT10_002/AT10_005) and
BiFlu showed protection in the H3N2 model. Together these data show
that the BiFlu antibody complex retains its functionality in vivo
and has similar protective activity as a mix of its single
components.
Protein Modelling to Determine the Amino Acids Involved in the
Antibody Hemagglutinin Interaction. (Table 8, 9 and 10)
[0229] The multiple sequence alignments were done by Clustal.OMEGA.
and further processed by showalign, part of EMBOSS. All the
structural work was done with Pymol. Minimisation was done using
the software NAMD with the force field CHARMM.
[0230] The first step to build a 3D model of the antibody is to
select the best 3D template. This is done by using a global
alignment (Needleman and Wunsch) of the query sequence against a
databank of all sequences of antibodies present in the protein
database (PDB). Then one structure is chosen amongst the structure
with the highest percentage of identity in the sequence. The next
step is to highlight the regions where substitutions occurred and
modify the sequence and the structure in such a way that the final
model resembles the antibody to analyse. Two techniques are
applied: 1) Substitution of amino acid, this method keeps the main
chain in place and only replaces the side chain. 2) Grafting of
loop, this method modifies the main chain and is necessary when
there are insertion or deletion in a loop, when the sequence is too
far or when substitutions may affect the main chain conformation,
e.g. substitution of Glycine or Proline.
[0231] To generate the complex antibody-haemagglutinin with the
antibodies AT10_005 and AT10_004 the structure of experimentally
determined complexes were used as template. The model of the
antibody is superimposed on the antibody of the crystal determined
structure, the haemagglutinin is kept intact. For AT10_002 the
docking procedure was to: (i) analyse the stem of haemagglutinin to
restrict the area where actual binding were tested, (ii) manual
positioning of the antibody in the remaining area of point (i),
(iii) evaluation of the quality of the complex by checking the
structure for short contacts, hydrogen bond capable groups missing
hydrogen bonds in the complex, size of the contact area.
AT10_005:
[0232] The amino acids of influenza A virus group 1 haemagglutinin
(H1/H5) in contact with AT10_005 are: A38, A40, A41, A42, A291,
A292, A293, A318, B18, B19, B20, B21, B38, B41, B42, B45, B46, B48,
B49, B52, B53, B56.
AT10_004:
[0233] The amino acids of influenza A virus group 2 haemagglutinin
(H3/H7) in contact with AT10_004 are: A21, A324, A325, A327, B12,
B14, B15, B16, B17, B18, B19, B25, B26, B30, B31, B32, B33, B34,
B35, B36, B38, B146, B150, B153, B154.
AT10_002:
[0234] The amino acids of influenza A virus group 2 haemagglutinin
(H3/H7) in contact with AT10_002 are: A38, A48, A275, A276, A277,
A278, A289, A291, A318, B19, B20, B21, B36, B38, B39, B41, B42,
B45, B46, B48, B49, B50, B52, B53, B56, B57, B58, B150.
[0235] Amino acid numbering for the HA molecule was done according
to: Wilson et al. 1981 Nature 289, 366-373 and Nobusawa et al. 1991
Virology 182, 475-485.
Interactions Antibody-Haemagglutinin
[0236] AT10_005 interacts with the conserved hydrophobic pocket
demonstrated by the crystal of the complex of CR6261 or F10
antibodies with haemagglutinin. The interaction is mainly
hydrophobic as for all antibodies binding this pocket.
[0237] AT10_004 interacts with the same beta strand as CR8020 in
its crystal complex with haemagglutinin but AT10_004 binds in a
stronger way by, among other interactions, continuing the beta
sheet of haemagglutinin. This interaction is mediated via the main
chain and thus it allows cross-reactivity between H1 and H3 even in
the absence of conservation (because the main chain is conserved
between amino acids).
[0238] AT10_002 interacts with the conserved hydrophobic patch in a
new way since except for the CDR3 of VH, all interactions come from
the VL domain.
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TABLE-US-00001 [0258] TABLE 1 Preferred influenza A virus specific
antibodies according to the invention SEQ ID NO Antibody Identity
Sequence 1 AT10- Heavy chain RHGIS 004 CDR1 2 AT10- Heavy chain
ELSIH 003 CDR1 3 AT10- Heavy chain SSNYY 002 CDR1 4 AT10- Heavy
chain TYAMS 001 CDR1 5 AT10- Heavy chain NYAIS 005 CDR1 6 AT10-
Heavy chain WISAYTGDTDYAQKFQG 004 CDR2 7 AT10- Heavy chain
SFDPEDGETIYAQKFQG 003 CDR2 8 AT10- Heavy chain TIYHSGSTYYNPSLKS 002
CDR2 9 AT10- Heavy chain GISGSGESTYYADSVKG 001 CDR2 10 AT10- Heavy
chain GIIPIFGTTNYAQKFQG 005 CDR2 11 AT10- Heavy chain
LRLQGEVVVPPSQSNWFDP 004 CDR3 12 AT10- Heavy chain GWGAVTSPFDF 003
CDR3 13 AT10- Heavy chain GGGFGWSQTYFGY 002 CDR3 14 AT10- Heavy
chain QGDHIAWLLRGINFDY 001 CDR3 15 AT10- Heavy chain
HGGVYYYGSASSGWFDP 005 CDR3 16 AT10- Light chain RASQSVSRYLA 004
CDR1 17 AT10- Light chain RSSQSLLHSNGHIYFD 003 CDR1 18 AT10- Light
chain TGTSSDVGAYNYVS 002 CDR1 19 AT10- Light chain RASQSVSSSYLA 001
CDR1 20 AT10- Light chain RASQSVSSSYLA 005 CDR1 21 AT10- Light
chain DASNRAT 004 CDR2 22 AT10- Light chain LVSKRAS 003 CDR2 23
AT10- Light chain DVTYRPS 002 CDR2 24 AT10- Light chain GASTRAT 001
CDR2 25 AT10- Light chain GASTRAT 005 CDR2 26 AT10- Light chain
QQRSNWLK 004 CDR3 27 AT10- Light chain MQALETP 003 CDR3 28 AT10-
Light chain SSQSRSST 002 CDR3 29 AT10- Light chain QNYGSPF 001 CDR3
30 AT10- Light chain QQYGSLP 005 CDR3 31 AT10- Heavy chain
QVQLVQSGAEVRKPGASVKVSCKASGYTFTRHGISWVRQAPGQGLEWMGWISA 004
YTGDTDYAQKFQGRVTMTTDTSTNTAYMELRSLRSDDAAVYYCARLRLQGEVVV
PPSQSNWFDPWGQGTLVTVSS 32 AT10- Heavy chain
QVHLVQSGAEVRKPGASVKVSCKVSGYTLNELSIHWLRQAPGRGLEWMGSFDP 003
EDGETIYAQKFQGRVTMTGDTSTDTAYLELTSLRSEDTALYYCARGWGAVTSPF
DFWGQGTLVTVSS 33 AT10- Heavy chain
QLQLQESGPRLVKPSETLSLTCSVSGVSISSSNYYVVGWIRQPPGKGLEWIGTIYHS 002
GSTYYNPSLKSRLIISVDTSKNQFYLQLTSLTAADSAVYYCATGGGFGWSQTYFG
YVVGQGTLVTASS 34 AT10- Heavy chain
EVQLLESGGGLVQPGGSLRLSCAASGFSFSTYAMSWVRQAPGKGLEWVSGISGS 001
GESTYYADSVKGRFTVSRDNSKNTLYLQMNSLRAEDTAVYYCAKQGDHIAWLLR
GINFDYVVGQGVLVTVSS 35 AT10- Heavy chain
QVQLVQSGAEVKKPGSSVKVSCKASGGAFSNYAISWVRQAPGQGLEWMGGIIPIF 005
GTTNYAQKFQGRVTITADKFTTIAYMELRSLRSEDTAVYYCARHGGVYYYGSASS
GWFDPWGQGTLVTVSS 36 AT10- Light chain
EIVLTQSPATLSLYPGERATLSCRASQSVSRYLAWYQQKPGQAPRLLIYDASNRAT 004
GIPARFSGSGSGTDFTLTISSLEPEDFAVYYCCDR3 QQRSNWLKITFGQGTRLEIKGTV 37
AT10- Light chain
DIVMTQSPLSLPVTPGEPASISCRSSQSLLHSNGHIYFDWYLQKPGQSPQLLIYLV 003
SKRASGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCMQALETPFTFGPGTKVHI KRTV 38
AT10- Light chain
QSALTQPASVSGSPGQSITISCTGTSSDVGAYNYVSWYQHHPGKAPKLMIYDVTY 002
RPSGVSTRFSGSKSGNTASLTISGLQAEDEADYYCSSQSRSSTLVIFGGGTKLTVL GQPK 39
AT10- Light chain
EIVLTQSPGTLSLSPGERATLSCRASQSVSSSYLAWYQQKPGQAPRLLIYGASTRA 001
TGIPDRFSGRGSGTDFTLTISSLEPEDFAVYYCQNYGSPFLFTFGPGTKVDIKRTV 40 AT10-
Light chain
EIVLTQSPGTLSLSPGERATLSCRASQSVSSSYLAWYQQKPGQAPRLLIFGASTRA 005
TGIPDRFSGSGSGTDFTLTISRLEPEDFAVFYCQQYGSLPLTFGGGTKVEIKGTV 41 AT10-
Heavy chain agg cat ggt atc agc 004 CDR1 42 AT10- Heavy chain gaa
tta tcc att cac 003 CDR1 43 AT10- Heavy chain agt agt aat tat tac
002 CDR1 44 AT10- Heavy chain acc tat gcc atg agc 001 CDR1 45 AT10-
Heavy chain aac tat gct atc agc 005 CDR1 46 AT10- Heavy chain tgg
atc agc gct tac act ggt gac aca gac tat gca cag aaa ttc cag ggg 004
CDR2 47 AT10- Heavy chain agt ttt gat cct gaa gat ggt gaa aca atc
tac gcg cag aag ttc cag ggc 003 CDR2 48 AT10- Heavy chain act atc
tat cac agt ggc agc acc tac tac aac ccg tcc ctc aag agt 002 CDR2 49
AT10- Heavy chain ggt att agt ggt agt ggt gag agc aca tac tac gca
gac tcc gtg aag ggc 001 CDR2 50 AT10- Heavy chain ggg atc atc cct
atc ttt gga aca aca aac tac gca cag aag ttc cag ggc 005 CDR2 51
AT10- Heavy chain ctt cgt ttg cag ggt gaa gtg gtg gtc cct cct agt
caa tcc aat tgg ttc gac ccc 004 CDR3 52 AT10- Heavy chain ggt tgg
ggg gcg gtg act tca ccc ttt gac ttc 003 CDR3 53 AT10- Heavy chain
ggg ggg ggg ttt ggc tgg tct caa acc tac ttt ggc tac 002 CDR3 54
AT10- Heavy chain caa ggg gat cat att gcc tgg tta tta agg ggg att
aac ttt gac tac 001 CDR3 55 AT10- Heavy chain cat ggg gga gtg tat
tat tat ggg tcg gcg agt tcg gga tgg ttc gac ccc 005 CDR3 56 AT10-
Light chain agg gcc agt cag agt gtt agc agg tac tta gcc 004 CDR1 57
AT10- Light chain agg tct agt cag agc ctc ctg cat agt aat ggg cac
atc tat ttc gat 003 CDR1 58 AT10- Light chain act gga acc agc agt
gac gtt ggt gct tat aac tat gtt tct 002 CDR1 59 AT10- Light chain
agg gcc agt cag agt gtt agc agc agt tac tta gcc 001 CDR1 60 AT10-
Light chain agg gcc agt cag agt gtt agt agc agc tac tta 005 CDR1 61
AT10- Light chain gat gca tcc aac agg gcc act 004 CDR2 62 AT10-
Light chain ttg gtt tct aag cgg gcc tcc 003 CDR2 63 AT10- Light
chain gat gtc act tat cgg ccc tca 002 CDR2 64 AT10- Light chain ggt
gca tcc acc agg gcc act 001 CDR2 65 AT10- Light chain ggt gca tcc
acc agg gcc act 005 CDR2 66 AT10- Light chain cag cag cgt agc aac
tgg ctt aag 004 CDR3 67 AT10- Light chain atg caa gct cta gaa act
cca 003 CDR3 68 AT10- Light chain agt tca cag tca cgc agc agc act
002 CDR3 69 AT10- Light chain cag aac tat ggt agt cca ttt 001 CDR3
70 AT10- Light chain cag cag tat ggt agc tta cct 005 CDR3 71 AT10-
Heavy chain cag gtt cag ctg gtg cag tct gga gct gag gtg agg aag cct
ggg gcc tca gtg aag gtc 004 tcc tgc aag gct tcc ggt tac acg ttt acc
agg cat ggt atc agc tgg gtg cga cag gcc
cct gga caa ggg ctt gag tgg atg gga tgg atc agc gct tac act ggt gac
aca gac tat gca cag aaa ttc cag ggg cga gtc acc atg acc aca gat aca
tcc acg aac aca gcc tac atg gaa ctg agg agc ctg aga tct gac gac gcg
gcc gta tat tac tgt gcg aga ctt cgt ttg cag ggt gaa gtg gtg gtc cct
cct agt caa tcc aat tgg ttc gac ccc tgg ggc cag gga acc ctg gtc acc
gtc tcc tca 72 AT10- Heavy chain cag gtc cac ctg gta cag tct ggg
gct gag gtg agg aag cct ggg gcc tca gtg aag gtc 003 tcc tgc aaa gtt
tcc gga tac aca ctc aat gaa tta tcc att cac tgg ctg cga cag gct cct
gga aga ggg ctt gag tgg atg gga agt ttt gat cct gaa gat ggt gaa aca
atc tac gcg cag aag ttc cag ggc aga gtc acc atg acc ggg gac aca tct
aca gac aca gcc tac ctg gag ctg acc agc ctg aga tct gag gac acg gcc
ctc tat tac tgt gca aga ggt tgg ggg gcg gtg act tca ccc ttt gac ttc
tgg ggc cag gga aca ctg gtc acc gtc tcc tca 73 AT10- Heavy chain
cag ctg cag ctg cag gag tcg ggc cca cga ctg gtg aag ccc tcg gag acc
ctg tcc ctc 002 acc tgc tct gtc tcc ggt gtc tcc atc agc agt agt aat
tat tac tgg ggc tgg atc cgc cag ccc cca ggg aag ggg ctg gag tgg att
ggg act atc tat cac agt ggc agc acc tac tac aac ccg tcc ctc aag agt
cga ctc atc atc tcc gtc gac acg tcc aag aat cag ttc tac ctg cag ttg
acc tct ctg acc gcc gca gac tcg gct gtc tat tac tgt gcg acc ggg ggg
ggg ttt ggc tgg tct caa acc tac ttt ggc tac tgg ggc cag gga acc ctg
gtc acc gcc tcc tca 74 AT10- Heavy chain gag gtg cag ctg ttg gag
tct ggg gga ggc ttg gta cag cct ggg ggg tcc ctg aga ctc 001 tcc tgt
gca gcc tcc gga ttc agc ttt agc acc tat gcc atg agc tgg gtc cgc cag
gct cca gga aag ggg ctg gag tgg gtc tca ggt att agt ggt agt ggt gag
agc aca tac tac gca gac tcc gtg aag ggc cgg ttc acc gtc tcc aga gac
aat tcc aag aac acc ctg tat ctg caa atg aac agc ctg aga gcc gag gac
acg gcc gtc tat tac tgt gcg aaa caa ggg gat cat att gcc tgg tta tta
agg ggg att aac ttt gac tac tgg ggc cag gga gtc ctt gtc acc gtc tcc
tca 75 AT10- Heavy chain cag gtg cag ctg gtg cag tct ggg gct gaa
gtg aag aag cct ggg tcc tcg gtg aag gtc 005 tcc tgc aag gct tct gga
ggc gcc ttc agc aac tat gct atc agc tgg gtg cga cag gcc cct gga caa
ggg ctt gag tgg atg gga ggg atc atc cct atc ttt gga aca aca aac tac
gca cag aag ttc cag ggc aga gtc acg att acc gcg gac aaa ttc acg acc
ata gcc tac atg gag ttg cgc agc ctg aga tct gag gac acg gcc gtt tat
tac tgt gcg agg cat ggg gga gtg tat tat tat ggg tcg gcg agt tcg gga
tgg ttc gac ccc tgg ggc cag gga acc ctg gtc acc gtc tcc tca 76
AT10- Light chain gaa att gtg ttg aca cag tct cca gcc acc ctg tct
ttg tat cca ggg gaa aga gcc acc 004 ctc tct tgc agg gcc agt cag agt
gtt agc agg tac tta gcc tgg tac caa cag aaa cct ggc cag gct ccc agg
ctc ctc atc tat gat gca tcc aac agg gcc act ggc atc cca gcc agg ttc
agt ggc agt ggg tct ggg aca gac ttc acc ctc acc atc agc agc cta gag
cct gaa gat ttt gca gtt tat tac tgt cag cag cgt agc aac tgg ctt aag
atc acc ttc ggc caa ggg aca cga ctg gaa att aaa gga act gtg 77
AT10- Light chain gat att gtg atg act cag tct cca ctc tcc ctg ccc
gtc acc cct gga gag ccg gcc tcc 003 atc tcc tgc agg tct agt cag agc
ctc ctg cat agt aat ggg cac atc tat ttc gat tgg tac ctg cag aag cca
ggg cag tct cca cag ctc ctg atc tat ttg gtt tct aag cgg gcc tcc ggg
gtc cct gac agg ttc agt ggc agt gga tca ggc aca gat ttt aca ctg aaa
atc agc aga gtg gag gct gag gat gtt ggg gtt tat tac tgc atg caa gct
cta gaa act cca ttc act ttc ggc cct ggg acc aaa gtg cat atc aaa cga
act gtg 78 AT10- Light chain cag tct gcc ctg act cag cct gcc tcc
gtg tct ggg tct cct ggc cag tcg atc acc atc 002 tcc tgc act gga acc
agc agt gac gtt ggt gct tat aac tat gtt tct tgg tac caa cac cac cca
ggc aaa gcc ccc aaa ctc atg att tat gat gtc act tat cgg ccc tca ggg
gtt tct act cgc ttc tct ggc tcc aag tct ggc aac acg gcc tcc ctg acc
atc tct ggg ctc cag gct gag gac gag gct gat tat tat tgc agt tca cag
tca cgc agc agc act ctc gtg att ttc ggc ggg ggg acc aag ttg acc gtc
cta ggt cag ccc aag 79 AT10- Light chain gaa att gtg ttg acg cag
tct cca ggc acc ctg tct ttg tct cca ggt gaa aga gcc acc 001 ctc tcc
tgc agg gcc agt cag agt gtt agc agc agt tac tta gcc tgg tac cag cag
aaa cct ggc cag gct ccc agg ctc ctc atc tat ggt gca tcc acc agg gcc
act ggc atc cca gac agg ttc agt ggc cgt ggg tct ggg aca gac ttc act
ctc acc atc agc agc ctg gag cct gaa gat ttt gca gtg tat tac tgt cag
aac tat ggt agt cca ttt tta ttc act ttc ggc cct ggg acc aaa gtg gat
atc aaa cga act gtg 80 AT10- Light chain gaa att gtg ttg acg cag
tct cca ggc acc ctg tct ttg tct cca ggg gaa aga gcc acc 005 ctc tcc
tgc agg gcc agt cag agt gtt agt agc agc tac tta gcc tgg tac cag cag
aaa cct ggc cag gct ccc agg ctc ctc atc ttt ggt gca tcc acc agg gcc
act ggc atc cca gac agg ttc agc ggc agt ggg tct ggg aca gac ttc act
ctc acc atc agc aga ctg gag cct gaa gat ttt gca gtg ttt tac tgt cag
cag tat ggt agc tta cct ctc act ttc ggc gga ggg acc aag gtg gag atc
aaa gga act gtg
TABLE-US-00002 TABLE 2 Recombinant human HA recognition by B cells
that secrete heterosubtypic cross-binding mAbs. Group Host Virus
Strain AT10_001 AT10_002 AT10_003 AT10_004 1 Human H1N1 A/New
Caledonia/20/1999 Negative Negative Negative Positive 2 Human H3N2
A/Wyoming/03/2003 Positive Positive Positive Positive 1 Human H5N1
A/Vietnam/1203/2004 Negative Negative Positive Negative 2 Human
H7N7 A/Netherland/219/2003 Positive Positive Positive Positive
TABLE-US-00003 TABLE 3 Recombinant human, swine and duck infecting
Influenza HA protein recognition by heterosubtypic cross-binding
mAbs. Neg ctrl Group Host Virus Strain AT10_001 AT10_002 AT10_003
AT10_004 AT10_005 mAb 1 Human H1N1 A/California/07/2009 Negative
Negative Negative Negative Positive Negative 1 Human H1N1 A/New
Negative Negative Negative Positive Positive Negative 1 Human H5N1
Caledonia/20/1999 Negative Negative Positive Negative Positive
Negative 1 Human H9N2 A/Vietnam/1203/2004 Negative Negative
Positive Positive Positive Negative A/Hong Kong/1073/1999 2 Human
H3N2 A/Aichi/2/1968 Positive Positive Positive Positive Negative
Negative 2 Human H3N2 A/Wyoming/03/2003 Positive Positive Positive
Positive Negative Negative 2 Swine H4N6 A/Swine/Ontario/01911- Low
Negative Positive Low Negative Negative 2 Human H7N7 1/1999
Positive Positive Positive Positive Negative Negative 2 Duck H10N3
A/Netherlands/219/2003 Positive Positive Positive Positive Negative
Negative 2 Duck H15N8 A/duck/Hong Negative Positive Positive
Negative Negative Negative Kong/786/1979 Negative Low
A/duck/AUS/341/1983 Positive Human Influenza B B/Florida/4/2006
Negative Negative Negative Negative Negative Negative
TABLE-US-00004 TABLE 4 Antibody binding to virus infected MDCK
cells. Neg ctrl Group Host Virus Strain AT10_001 AT10_002 AT10_003
AT10_004 AT10_005 mAb 1 Human H1N1 A/Neth/602/2009 Low Negative
Negative Negative Positive Negative 1 Turkey H5N1
A/Turkey/Turkey/2004 Positive Negative Negative Low Positive
Negative (HPAI) Negative Positive 2 Swine H3N2
A/swine/St.oedenrode/ Negative Positive Low Positive Negative
Negative 2 Chicken H7N1 1996 (LPAI) Positive Positive Positive
Positive Negative Negative 2 Chicken H7N7 A/Ch/Italy/1067/1999
Positive Positive Negative Positive Negative Negative (LPAI) Low
A/Ch/Neth/621557/2003 Positive (HPAI)
TABLE-US-00005 TABLE 5 In vitro influenza A virus neutralization of
virus infected MDCK-SIAT cells by recombinant antibodies. AT10_001
AT10_002 AT10_003 AT10_004 AT10_005 H3N2 A/Ned/177/2008 0.64 0.18
>50 0.17 ND H3N2 HKX-31 2.1 0.25 >15 0.017 ND H1N1
A/Hawaii/31/2007 >15 >15 >15 >50 0.24 ND = Not done
IC50 values displayed in .mu.g/ml
TABLE-US-00006 TABLE 6 Recombinant HA and HA1 subunit recognition
by recombinant antibodies. AT10_001 AT10_002 AT10_003 AT10_004
AT10_005 H3N2 A/Aichi/2/1968 Full length 0.953 0.920 1.319 0.491
-0.003 H3N2 A/Aichi/2/1968 HA1 subunit 0.010 -0.006 1.277 0.096
-0.007
Sequence CWU 1
1
8315PRTArtificialAT10-004 Heavy chain CDR1 1Arg His Gly Ile Ser 1 5
25PRTArtificialAT10-003 Heavy chain CDR1 2Glu Leu Ser Ile His 1 5
35PRTArtificialAT10-002 Heavy chain CDR1 3Ser Ser Asn Tyr Tyr 1 5
45PRTArtificialAT10-001 Heavy chain CDR1 4Thr Tyr Ala Met Ser 1 5
55PRTArtificialAT10-005 Heavy chain CDR1 5Asn Tyr Ala Ile Ser 1 5
617PRTArtificialAT10-004 Heavy chain CDR2 6Trp Ile Ser Ala Tyr Thr
Gly Asp Thr Asp Tyr Ala Gln Lys Phe Gln 1 5 10 15 Gly
717PRTArtificialAT10-003 Heavy chain CDR2 7Ser Phe Asp Pro Glu Asp
Gly Glu Thr Ile Tyr Ala Gln Lys Phe Gln 1 5 10 15 Gly
816PRTArtificialAT10-002 Heavy chain CDR2 8Thr Ile Tyr His Ser Gly
Ser Thr Tyr Tyr Asn Pro Ser Leu Lys Ser 1 5 10 15
917PRTArtificialAT10-001 Heavy chain CDR2 9Gly Ile Ser Gly Ser Gly
Glu Ser Thr Tyr Tyr Ala Asp Ser Val Lys 1 5 10 15 Gly
1017PRTArtificialAT10-005 Heavy chain CDR2 10Gly Ile Ile Pro Ile
Phe Gly Thr Thr Asn Tyr Ala Gln Lys Phe Gln 1 5 10 15 Gly
1119PRTArtificialAT10-004 Heavy chain CDR3 11Leu Arg Leu Gln Gly
Glu Val Val Val Pro Pro Ser Gln Ser Asn Trp 1 5 10 15 Phe Asp Pro
1211PRTArtificialAT10-003 Heavy chain CDR3 12Gly Trp Gly Ala Val
Thr Ser Pro Phe Asp Phe 1 5 10 1313PRTArtificialAT10-002 Heavy
chain CDR3 13Gly Gly Gly Phe Gly Trp Ser Gln Thr Tyr Phe Gly Tyr 1
5 10 1416PRTArtificialAT10-001 Heavy chain CDR3 14Gln Gly Asp His
Ile Ala Trp Leu Leu Arg Gly Ile Asn Phe Asp Tyr 1 5 10 15
1517PRTArtificialAT10-005 Heavy chain CDR3 15His Gly Gly Val Tyr
Tyr Tyr Gly Ser Ala Ser Ser Gly Trp Phe Asp 1 5 10 15 Pro
1611PRTArtificialAT10-004 Light chain CDR1 16Arg Ala Ser Gln Ser
Val Ser Arg Tyr Leu Ala 1 5 10 1716PRTArtificialAT10-003 Light
chain CDR1 17Arg Ser Ser Gln Ser Leu Leu His Ser Asn Gly His Ile
Tyr Phe Asp 1 5 10 15 1814PRTArtificialAT10-002 Light chain CDR1
18Thr Gly Thr Ser Ser Asp Val Gly Ala Tyr Asn Tyr Val Ser 1 5 10
1912PRTArtificialAT10-001 Light chain CDR1 19Arg Ala Ser Gln Ser
Val Ser Ser Ser Tyr Leu Ala 1 5 10 2012PRTArtificialAT10-005 Light
chain CDR1 20Arg Ala Ser Gln Ser Val Ser Ser Ser Tyr Leu Ala 1 5 10
217PRTArtificialAT10-004 Light chain CDR2 21Asp Ala Ser Asn Arg Ala
Thr 1 5 227PRTArtificialAT10-003 Light chain CDR2 22Leu Val Ser Lys
Arg Ala Ser 1 5 237PRTArtificialAT10-002 Light chain CDR2 23Asp Val
Thr Tyr Arg Pro Ser 1 5 247PRTArtificialAT10-001 Light chain CDR2
24Gly Ala Ser Thr Arg Ala Thr 1 5 257PRTArtificialAT10-005 Light
chain CDR2 25Gly Ala Ser Thr Arg Ala Thr 1 5
268PRTArtificialAT10-004 Light chain CDR3 26Gln Gln Arg Ser Asn Trp
Leu Lys 1 5 277PRTArtificialAT10-003 Light chain CDR3 27Met Gln Ala
Leu Glu Thr Pro 1 5 288PRTArtificialAT10-002 Light chain CDR3 28Ser
Ser Gln Ser Arg Ser Ser Thr 1 5 297PRTArtificialAT10-001 Light
chain CDR3 29Gln Asn Tyr Gly Ser Pro Phe 1 5
307PRTArtificialAT10-005 Light chain CDR3 30Gln Gln Tyr Gly Ser Leu
Pro 1 5 31128PRTArtificialAT10-004 Heavy chain 31Gln 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 Arg His 20 25 30
Gly Ile Ser Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met 35
40 45 Gly Trp Ile Ser Ala Tyr Thr Gly Asp Thr Asp Tyr Ala Gln Lys
Phe 50 55 60 Gln Gly Arg Val Thr Met Thr Thr Asp Thr Ser Thr Asn
Thr Ala Tyr 65 70 75 80 Met Glu Leu Arg Ser Leu Arg Ser Asp Asp Ala
Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Leu Arg Leu Gln Gly Glu Val
Val Val Pro Pro Ser Gln Ser 100 105 110 Asn Trp Phe Asp Pro Trp Gly
Gln Gly Thr Leu Val Thr Val Ser Ser 115 120 125
32120PRTArtificialAT10-003 Heavy chain 32Gln Val His Leu Val Gln
Ser Gly Ala Glu Val Arg Lys Pro Gly Ala 1 5 10 15 Ser Val Lys Val
Ser Cys Lys Val Ser Gly Tyr Thr Leu Asn Glu Leu 20 25 30 Ser Ile
His Trp Leu Arg Gln Ala Pro Gly Arg Gly Leu Glu Trp Met 35 40 45
Gly Ser Phe Asp Pro Glu Asp Gly Glu Thr Ile Tyr Ala Gln Lys Phe 50
55 60 Gln Gly Arg Val Thr Met Thr Gly Asp Thr Ser Thr Asp Thr Ala
Tyr 65 70 75 80 Leu Glu Leu Thr Ser Leu Arg Ser Glu Asp Thr Ala Leu
Tyr Tyr Cys 85 90 95 Ala Arg Gly Trp Gly Ala Val Thr Ser Pro Phe
Asp Phe Trp Gly Gln 100 105 110 Gly Thr Leu Val Thr Val Ser Ser 115
120 33123PRTArtificialAT10-002 Heavy chain 33Gln Leu Gln Leu Gln
Glu Ser Gly Pro Arg Leu Val Lys Pro Ser Glu 1 5 10 15 Thr Leu Ser
Leu Thr Cys Ser Val Ser Gly Val Ser Ile Ser Ser Ser 20 25 30 Asn
Tyr Tyr Trp Gly Trp Ile Arg Gln Pro Pro Gly Lys Gly Leu Glu 35 40
45 Trp Ile Gly Thr Ile Tyr His Ser Gly Ser Thr Tyr Tyr Asn Pro Ser
50 55 60 Leu Lys Ser Arg Leu Ile Ile Ser Val Asp Thr Ser Lys Asn
Gln Phe 65 70 75 80 Tyr Leu Gln Leu Thr Ser Leu Thr Ala Ala Asp Ser
Ala Val Tyr Tyr 85 90 95 Cys Ala Thr Gly Gly Gly Phe Gly Trp Ser
Gln Thr Tyr Phe Gly Tyr 100 105 110 Trp Gly Gln Gly Thr Leu Val Thr
Ala Ser Ser 115 120 34125PRTArtificialAT10-001 Heavy chain 34Glu
Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10
15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Ser Phe Ser Thr Tyr
20 25 30 Ala Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu
Trp Val 35 40 45 Ser Gly Ile Ser Gly Ser Gly Glu Ser Thr Tyr Tyr
Ala Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Val Ser Arg Asp Asn
Ser Lys Asn Thr Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala
Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Lys Gln Gly Asp His
Ile Ala Trp Leu Leu Arg Gly Ile Asn Phe 100 105 110 Asp Tyr Trp Gly
Gln Gly Val Leu Val Thr Val Ser Ser 115 120 125
35126PRTArtificialAT10-005 Heavy chain 35Gln Val Gln Leu Val Gln
Ser Gly Ala Glu Val Lys Lys Pro Gly Ser 1 5 10 15 Ser Val Lys Val
Ser Cys Lys Ala Ser Gly Gly Ala Phe Ser Asn Tyr 20 25 30 Ala Ile
Ser Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met 35 40 45
Gly Gly Ile Ile Pro Ile Phe Gly Thr Thr Asn Tyr Ala Gln Lys Phe 50
55 60 Gln Gly Arg Val Thr Ile Thr Ala Asp Lys Phe Thr Thr Ile Ala
Tyr 65 70 75 80 Met Glu Leu Arg Ser Leu Arg Ser Glu Asp Thr Ala Val
Tyr Tyr Cys 85 90 95 Ala Arg His Gly Gly Val Tyr Tyr Tyr Gly Ser
Ala Ser Ser Gly Trp 100 105 110 Phe Asp Pro Trp Gly Gln Gly Thr Leu
Val Thr Val Ser Ser 115 120 125 36114PRTArtificialAT10-004 Light
chain 36Glu Ile Val Leu Thr Gln Ser Pro Ala Thr Leu Ser Leu Tyr Pro
Gly 1 5 10 15 Glu Arg Ala Thr Leu Ser Cys Arg Ala Ser Gln Ser Val
Ser Arg Tyr 20 25 30 Leu Ala Trp Tyr Gln Gln Lys Pro Gly Gln Ala
Pro Arg Leu Leu Ile 35 40 45 Tyr Asp Ala Ser Asn Arg Ala Thr Gly
Ile Pro Ala Arg Phe Ser Gly 50 55 60 Ser Gly Ser Gly Thr Asp Phe
Thr Leu Thr Ile Ser Ser Leu Glu Pro 65 70 75 80 Glu Asp Phe Ala Val
Tyr Tyr Cys Cys Asp Arg Gln Gln Arg Ser Asn 85 90 95 Trp Leu Lys
Ile Thr Phe Gly Gln Gly Thr Arg Leu Glu Ile Lys Gly 100 105 110 Thr
Val 37115PRTArtificialAT10-003 Light chain 37Asp Ile Val Met Thr
Gln Ser Pro Leu Ser Leu Pro Val Thr Pro Gly 1 5 10 15 Glu Pro Ala
Ser Ile Ser Cys Arg Ser Ser Gln Ser Leu Leu His Ser 20 25 30 Asn
Gly His Ile Tyr Phe Asp Trp Tyr Leu Gln Lys Pro Gly Gln Ser 35 40
45 Pro Gln Leu Leu Ile Tyr Leu Val Ser Lys Arg Ala Ser Gly Val Pro
50 55 60 Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu
Lys Ile 65 70 75 80 Ser Arg Val Glu Ala Glu Asp Val Gly Val Tyr Tyr
Cys Met Gln Ala 85 90 95 Leu Glu Thr Pro Phe Thr Phe Gly Pro Gly
Thr Lys Val His Ile Lys 100 105 110 Arg Thr Val 115
38115PRTArtificialAT10-002 Light chain 38Gln Ser Ala Leu Thr Gln
Pro Ala Ser Val Ser Gly Ser Pro Gly Gln 1 5 10 15 Ser Ile Thr Ile
Ser Cys Thr Gly Thr Ser Ser Asp Val Gly Ala Tyr 20 25 30 Asn Tyr
Val Ser Trp Tyr Gln His His Pro Gly Lys Ala Pro Lys Leu 35 40 45
Met Ile Tyr Asp Val Thr Tyr Arg Pro Ser Gly Val Ser Thr Arg Phe 50
55 60 Ser Gly Ser Lys Ser Gly Asn Thr Ala Ser Leu Thr Ile Ser Gly
Leu 65 70 75 80 Gln Ala Glu Asp Glu Ala Asp Tyr Tyr Cys Ser Ser Gln
Ser Arg Ser 85 90 95 Ser Thr Leu Val Ile Phe Gly Gly Gly Thr Lys
Leu Thr Val Leu Gly 100 105 110 Gln Pro Lys 115
39112PRTArtificialAT10-001 Light chain 39Glu Ile Val Leu Thr Gln
Ser Pro Gly Thr Leu Ser Leu Ser Pro Gly 1 5 10 15 Glu Arg Ala Thr
Leu Ser Cys Arg Ala Ser Gln Ser Val Ser Ser Ser 20 25 30 Tyr Leu
Ala Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Arg Leu Leu 35 40 45
Ile Tyr Gly Ala Ser Thr Arg Ala Thr Gly Ile Pro Asp Arg Phe Ser 50
55 60 Gly Arg Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu
Glu 65 70 75 80 Pro Glu Asp Phe Ala Val Tyr Tyr Cys Gln Asn Tyr Gly
Ser Pro Phe 85 90 95 Leu Phe Thr Phe Gly Pro Gly Thr Lys Val Asp
Ile Lys Arg Thr Val 100 105 110 40111PRTArtificialAT10-005 Light
chain 40Glu Ile Val Leu Thr Gln Ser Pro Gly Thr Leu Ser Leu Ser Pro
Gly 1 5 10 15 Glu Arg Ala Thr Leu Ser Cys Arg Ala Ser Gln Ser Val
Ser Ser Ser 20 25 30 Tyr Leu Ala Trp Tyr Gln Gln Lys Pro Gly Gln
Ala Pro Arg Leu Leu 35 40 45 Ile Phe Gly Ala Ser Thr Arg Ala Thr
Gly Ile Pro Asp Arg Phe Ser 50 55 60 Gly Ser Gly Ser Gly Thr Asp
Phe Thr Leu Thr Ile Ser Arg Leu Glu 65 70 75 80 Pro Glu Asp Phe Ala
Val Phe Tyr Cys Gln Gln Tyr Gly Ser Leu Pro 85 90 95 Leu Thr Phe
Gly Gly Gly Thr Lys Val Glu Ile Lys Gly Thr Val 100 105 110
4115DNAArtificialAT10-004 Heavy chain CDR1 41aggcatggta tcagc
154215DNAArtificialAT10-003 Heavy chain CDR1 42gaattatcca ttcac
154315DNAArtificialAT10-002 Heavy chain CDR1 43agtagtaatt attac
154415DNAArtificialAT10-001 Heavy chain CDR1 44acctatgcca tgagc
154515DNAArtificialAT10-005 Heavy chain CDR1 45aactatgcta tcagc
154651DNAArtificialAT10-004 Heavy chain CDR2 46tggatcagcg
cttacactgg tgacacagac tatgcacaga aattccaggg g
514751DNAArtificialAT10-003 Heavy chain CDR2 47agttttgatc
ctgaagatgg tgaaacaatc tacgcgcaga agttccaggg c
514848DNAArtificialAT10-002 Heavy chain CDR2 48actatctatc
acagtggcag cacctactac aacccgtccc tcaagagt
484951DNAArtificialAT10-001 Heavy chain CDR2 49ggtattagtg
gtagtggtga gagcacatac tacgcagact ccgtgaaggg c
515051DNAArtificialAT10-005 Heavy chain CDR2 50gggatcatcc
ctatctttgg aacaacaaac tacgcacaga agttccaggg c
515157DNAArtificialAT10-004 Heavy chain CDR3 51cttcgtttgc
agggtgaagt ggtggtccct cctagtcaat ccaattggtt cgacccc
575233DNAArtificialAT10-003 Heavy chain CDR3 52ggttgggggg
cggtgacttc accctttgac ttc 335339DNAArtificialAT10-002 Heavy chain
CDR3 53gggggggggt ttggctggtc tcaaacctac tttggctac
395448DNAArtificialAT10-001 Heavy chain CDR3 54caaggggatc
atattgcctg gttattaagg gggattaact ttgactac
485551DNAArtificialAT10-005 Heavy chain CDR3 55catgggggag
tgtattatta tgggtcggcg agttcgggat ggttcgaccc c
515633DNAArtificialAT10-004 Light chain CDR1 56agggccagtc
agagtgttag caggtactta gcc 335748DNAArtificialAT10-003 Light chain
CDR1 57aggtctagtc agagcctcct gcatagtaat gggcacatct atttcgat
485842DNAArtificialAT10-002 Light chain CDR1 58actggaacca
gcagtgacgt tggtgcttat aactatgttt ct 425936DNAArtificialAT10-001
Light chain CDR1 59agggccagtc agagtgttag cagcagttac ttagcc
366033DNAArtificialAT10-005 Light chain CDR1 60agggccagtc
agagtgttag tagcagctac tta 336121DNAArtificialAT10-004 Light chain
CDR2 61gatgcatcca acagggccac t 216221DNAArtificialAT10-003 Light
chain CDR2 62ttggtttcta agcgggcctc c 216321DNAArtificialAT10-002
Light chain CDR2 63gatgtcactt atcggccctc a
216421DNAArtificialAT10-001 Light chain CDR2 64ggtgcatcca
ccagggccac t 216521DNAArtificialAT10-005 Light chain CDR2
65ggtgcatcca ccagggccac t 216624DNAArtificialAT10-004 Light chain
CDR3 66cagcagcgta gcaactggct taag 246721DNAArtificialAT10-003 Light
chain CDR3 67atgcaagctc tagaaactcc a 216824DNAArtificialAT10-002
Light chain CDR3 68agttcacagt cacgcagcag cact
246921DNAArtificialAT10-001 Light chain CDR3 69cagaactatg
gtagtccatt t 217021DNAArtificialAT10-005 Light chain CDR3
70cagcagtatg gtagcttacc t 2171384DNAArtificialAT10-004 Heavy chain
71caggttcagc tggtgcagtc tggagctgag gtgaggaagc ctggggcctc agtgaaggtc
60tcctgcaagg cttccggtta cacgtttacc aggcatggta tcagctgggt gcgacaggcc
120cctggacaag ggcttgagtg gatgggatgg atcagcgctt acactggtga
cacagactat 180gcacagaaat tccaggggcg agtcaccatg accacagata
catccacgaa cacagcctac 240atggaactga ggagcctgag atctgacgac
gcggccgtat attactgtgc
gagacttcgt 300ttgcagggtg aagtggtggt ccctcctagt caatccaatt
ggttcgaccc ctggggccag 360ggaaccctgg tcaccgtctc ctca
38472360DNAArtificialAT10-003 Heavy chain 72caggtccacc tggtacagtc
tggggctgag gtgaggaagc ctggggcctc agtgaaggtc 60tcctgcaaag tttccggata
cacactcaat gaattatcca ttcactggct gcgacaggct 120cctggaagag
ggcttgagtg gatgggaagt tttgatcctg aagatggtga aacaatctac
180gcgcagaagt tccagggcag agtcaccatg accggggaca catctacaga
cacagcctac 240ctggagctga ccagcctgag atctgaggac acggccctct
attactgtgc aagaggttgg 300ggggcggtga cttcaccctt tgacttctgg
ggccagggaa cactggtcac cgtctcctca 36073369DNAArtificialAT10-002
Heavy chain 73cagctgcagc tgcaggagtc gggcccacga ctggtgaagc
cctcggagac cctgtccctc 60acctgctctg tctccggtgt ctccatcagc agtagtaatt
attactgggg ctggatccgc 120cagcccccag ggaaggggct ggagtggatt
gggactatct atcacagtgg cagcacctac 180tacaacccgt ccctcaagag
tcgactcatc atctccgtcg acacgtccaa gaatcagttc 240tacctgcagt
tgacctctct gaccgccgca gactcggctg tctattactg tgcgaccggg
300ggggggtttg gctggtctca aacctacttt ggctactggg gccagggaac
cctggtcacc 360gcctcctca 36974375DNAArtificialAT10-001 Heavy chain
74gaggtgcagc tgttggagtc tgggggaggc ttggtacagc ctggggggtc cctgagactc
60tcctgtgcag cctccggatt cagctttagc acctatgcca tgagctgggt ccgccaggct
120ccaggaaagg ggctggagtg ggtctcaggt attagtggta gtggtgagag
cacatactac 180gcagactccg tgaagggccg gttcaccgtc tccagagaca
attccaagaa caccctgtat 240ctgcaaatga acagcctgag agccgaggac
acggccgtct attactgtgc gaaacaaggg 300gatcatattg cctggttatt
aagggggatt aactttgact actggggcca gggagtcctt 360gtcaccgtct cctca
37575378DNAArtificialHeavy chain 75caggtgcagc tggtgcagtc tggggctgaa
gtgaagaagc ctgggtcctc ggtgaaggtc 60tcctgcaagg cttctggagg cgccttcagc
aactatgcta tcagctgggt gcgacaggcc 120cctggacaag ggcttgagtg
gatgggaggg atcatcccta tctttggaac aacaaactac 180gcacagaagt
tccagggcag agtcacgatt accgcggaca aattcacgac catagcctac
240atggagttgc gcagcctgag atctgaggac acggccgttt attactgtgc
gaggcatggg 300ggagtgtatt attatgggtc ggcgagttcg ggatggttcg
acccctgggg ccagggaacc 360ctggtcaccg tctcctca
37876333DNAArtificialAT10-004 Light chain 76gaaattgtgt tgacacagtc
tccagccacc ctgtctttgt atccagggga aagagccacc 60ctctcttgca gggccagtca
gagtgttagc aggtacttag cctggtacca acagaaacct 120ggccaggctc
ccaggctcct catctatgat gcatccaaca gggccactgg catcccagcc
180aggttcagtg gcagtgggtc tgggacagac ttcaccctca ccatcagcag
cctagagcct 240gaagattttg cagtttatta ctgtcagcag cgtagcaact
ggcttaagat caccttcggc 300caagggacac gactggaaat taaaggaact gtg
33377345DNAArtificialAT10-003 Light chain 77gatattgtga tgactcagtc
tccactctcc ctgcccgtca cccctggaga gccggcctcc 60atctcctgca ggtctagtca
gagcctcctg catagtaatg ggcacatcta tttcgattgg 120tacctgcaga
agccagggca gtctccacag ctcctgatct atttggtttc taagcgggcc
180tccggggtcc ctgacaggtt cagtggcagt ggatcaggca cagattttac
actgaaaatc 240agcagagtgg aggctgagga tgttggggtt tattactgca
tgcaagctct agaaactcca 300ttcactttcg gccctgggac caaagtgcat
atcaaacgaa ctgtg 34578345DNAArtificialAT10-002 Light chain
78cagtctgccc tgactcagcc tgcctccgtg tctgggtctc ctggccagtc gatcaccatc
60tcctgcactg gaaccagcag tgacgttggt gcttataact atgtttcttg gtaccaacac
120cacccaggca aagcccccaa actcatgatt tatgatgtca cttatcggcc
ctcaggggtt 180tctactcgct tctctggctc caagtctggc aacacggcct
ccctgaccat ctctgggctc 240caggctgagg acgaggctga ttattattgc
agttcacagt cacgcagcag cactctcgtg 300attttcggcg gggggaccaa
gttgaccgtc ctaggtcagc ccaag 34579336DNAArtificialAT10-001 Light
chain 79gaaattgtgt tgacgcagtc tccaggcacc ctgtctttgt ctccaggtga
aagagccacc 60ctctcctgca gggccagtca gagtgttagc agcagttact tagcctggta
ccagcagaaa 120cctggccagg ctcccaggct cctcatctat ggtgcatcca
ccagggccac tggcatccca 180gacaggttca gtggccgtgg gtctgggaca
gacttcactc tcaccatcag cagcctggag 240cctgaagatt ttgcagtgta
ttactgtcag aactatggta gtccattttt attcactttc 300ggccctggga
ccaaagtgga tatcaaacga actgtg 33680333DNAArtificialAT10-005 Light
chain 80gaaattgtgt tgacgcagtc tccaggcacc ctgtctttgt ctccagggga
aagagccacc 60ctctcctgca gggccagtca gagtgttagt agcagctact tagcctggta
ccagcagaaa 120cctggccagg ctcccaggct cctcatcttt ggtgcatcca
ccagggccac tggcatccca 180gacaggttca gcggcagtgg gtctgggaca
gacttcactc tcaccatcag cagactggag 240cctgaagatt ttgcagtgtt
ttactgtcag cagtatggta gcttacctct cactttcggc 300ggagggacca
aggtggagat caaaggaact gtg 333816PRTArtificialsortase recognition
site 81Leu Pro Glu Thr Gly Gly 1 5 825PRTArtificialantibody tag
sequence 82Gly Gly Gly Gly Ser 1 5 8318PRTArtificial SequenceST tag
83Gly Gly Gly Gly Ser Leu Pro Glu Thr Gly Gly Gly His His His His 1
5 10 15 His His
* * * * *