U.S. patent application number 13/897843 was filed with the patent office on 2015-06-25 for human binding molecules capable of neutralizing influenza virus h3n2 and uses thereof.
The applicant listed for this patent is Crucell Holland B.V.. Invention is credited to Robert H.E. Friesen, Mandy A. C. Jongeneelen, Theodorus H.J. Kwaks, Mark Throsby.
Application Number | 20150175677 13/897843 |
Document ID | / |
Family ID | 42271865 |
Filed Date | 2015-06-25 |
United States Patent
Application |
20150175677 |
Kind Code |
A9 |
Throsby; Mark ; et
al. |
June 25, 2015 |
HUMAN BINDING MOLECULES CAPABLE OF NEUTRALIZING INFLUENZA VIRUS
H3N2 AND USES THEREOF
Abstract
Described are binding molecules, e.g., human monoclonal
antibodies, that bind to influenza virus comprising HA of the H3
subtype, e.g., H3N2, and have a broad neutralizing activity against
such influenza virus. Described are polynucleotides encoding the
binding molecules, their sequences and compositions comprising the
binding molecules and methods of identifying or producing the
binding molecules. The binding molecules can be used in the
diagnosis, prophylaxis, and/or treatment of influenza virus H3N2
infection. The binding molecules may provide cross-subtype
protection, such that infections with H3, H7, and/or H10-based
influenza subtypes can be prevented and/or treated.
Inventors: |
Throsby; Mark; (Utrecht,
NL) ; Friesen; Robert H.E.; (Leiden, NL) ;
Kwaks; Theodorus H.J.; (Voorschoten, NL) ;
Jongeneelen; Mandy A. C.; (Leiden, NL) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
Crucell Holland B.V. |
Leiden |
|
NL |
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|
Prior
Publication: |
|
Document Identifier |
Publication Date |
|
US 20130309248 A1 |
November 21, 2013 |
|
|
Family ID: |
42271865 |
Appl. No.: |
13/897843 |
Filed: |
May 20, 2013 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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13138941 |
Oct 27, 2011 |
8470327 |
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PCT/EP2010/056217 |
May 6, 2010 |
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13897843 |
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61215890 |
May 11, 2009 |
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Current U.S.
Class: |
424/159.1 ;
435/320.1; 435/339; 435/5; 435/69.6; 530/388.3; 530/389.4;
530/391.3; 536/23.53 |
Current CPC
Class: |
A61K 2039/505 20130101;
C12N 2760/16111 20130101; A61K 39/12 20130101; A61K 2039/545
20130101; A61P 31/16 20180101; C07K 2317/21 20130101; C07K 2317/622
20130101; C07K 2317/33 20130101; C07K 2317/76 20130101; A61K
2039/507 20130101; C07K 16/1018 20130101; C07K 2317/92 20130101;
C07K 2317/34 20130101; C07K 2317/55 20130101 |
International
Class: |
C07K 16/10 20060101
C07K016/10 |
Foreign Application Data
Date |
Code |
Application Number |
May 11, 2009 |
EP |
09159947.2 |
Jan 20, 2010 |
EP |
10151155.8 |
Claims
1. An isolated human binding molecule able to specifically
recognize and bind to an epitope in influenza hemagglutinin protein
(HA), having neutralizing activity against influenza viruses
comprising HA of the H3 subtype and having cross-neutralizing
activity against at least an influenza virus comprising HA of the
H7 subtype, and/or an influenza virus comprising HA of the H10
subtype.
2. The binding molecule of claim 1 having neutralizing activity
against one or more of the H3N2 strains selected from the group
consisting of A/Wisconsin/67/2005, A/Hiroshima/52/2005,
A/Panama/2007/99, and A/Johannesburg/33/94.
3. The binding molecule of claim 2, wherein the binding molecule
further has neutralizing activity against the H3N2 strain A/Hong
Kong/1/68.
4. The binding molecule of claim 2, wherein the binding molecule
has neutralizing activity against all naturally occurring isolates
of influenza virus H3N2 known before Jan. 20, 2010.
5. The binding molecule of claim 1, wherein the binding molecule
binds to an epitope comprising the amino acid at position 19, 25,
27, 33 and/or 34 of the HA2 polypeptide of the H3 HA protein.
6. The binding molecule of claim 5, wherein the binding molecule
binds to the epitope on HA2, when the amino acid on position 19 is
aspartic acid (D), the amino acid on position 25 is glutamine (Q),
the amino acid on position 27 is glycine (G), the amino acid at
position 33 is glycine (G) and/or the amino acid at position 34 is
glutamine.
7. The binding molecule of claim 6, wherein the binding molecule
does not bind to the epitope on HA2 when one or more of the amino
acids have changed.
8. The binding molecule of claim 1, wherein the binding molecule is
able to prevent in vitro the trypsin cleavage of the H3 HA
precursor molecule HA0 in HA 1 and HA2.
9. The binding molecule of claim 1, wherein the binding molecule is
able to prevent the conformational change of the H3 HA protein
required for fusion of the viral membrane with the endosomal
membrane of an infected cell.
10. The binding molecule of claim 1, wherein the binding molecule
is not capable of binding to and neutralizing influenza virus A
comprising HA of the H1 subtype.
11. The binding molecule of claim 1, wherein the binding molecule
has cross-neutralizing activity against at least an influenza virus
comprising HA of the H7 subtype, and/or an influenza virus
comprising HA of the H10 subtype.
12. The binding molecule of claim 1, wherein the binding molecule
has cross-neutralizing activity against all influenza virus
subtypes of phylogenetic group 2.
13. The binding molecule of claim 1, wherein the binding molecule
is selected from the group consisting of: a) a binding molecule
comprising a heavy chain CDR1 region of SEQ ID NO:81, a heavy chain
CDR2 region of SEQ ID NO:82, and a heavy chain CDR3 region of SEQ
ID NO:83, b) a binding molecule comprising a heavy chain CDR1
region of SEQ ID NO:87, a heavy chain CDR2 region of SEQ ID NO:88,
and a heavy chain CDR3 region of SEQ ID NO:89, c) a binding
molecule comprising a heavy chain CDR1 region of SEQ ID NO:103, a
heavy chain CDR2 region of SEQ ID NO:104, and a heavy chain CDR3
region of SEQ ID NO:105, d) a binding molecule comprising a heavy
chain CDR1 region of SEQ ID NO:109, a heavy chain CDR2 region of
SEQ ID NO:110, and a heavy chain CDR3 region of SEQ ID NO:111, e) a
binding molecule comprising a heavy chain CDR1 region of SEQ ID
NO:115, a heavy chain CDR2 region of SEQ ID NO:116, and a heavy
chain CDR3 region of SEQ ID NO:117, f) a binding molecule
comprising a heavy chain CDR1 region of SEQ ID NO:121, a heavy
chain CDR2 region of SEQ ID NO:122, and a heavy chain CDR3 region
of SEQ ID NO:123, g) a binding molecule comprising a heavy chain
CDR1 region of SEQ ID NO:126, a heavy chain CDR2 region of SEQ ID
NO:127, and a heavy chain CDR3 region of SEQ ID NO:128, h) a
binding molecule comprising a heavy chain CDR1 region of SEQ ID
NO:132, a heavy chain CDR2 region of SEQ ID NO:133, and a heavy
chain CDR3 region of SEQ ID NO:134, i) a binding molecule
comprising a heavy chain CDR1 region of SEQ ID NO:138, a heavy
chain CDR2 region of SEQ ID NO:139, and a heavy chain CDR3 region
of SEQ ID NO:140, j) a binding molecule comprising a heavy chain
CDR1 region of SEQ ID NO:144, a heavy chain CDR2 region of SEQ ID
NO:145, and a heavy chain CDR3 region of SEQ ID NO:146, k) a
binding molecule comprising a heavy chain CDR1 region of SEQ ID
NO:150, a heavy chain CDR2 region of SEQ ID NO:151, and a heavy
chain CDR3 region of SEQ ID NO:152, l) a binding molecule
comprising a heavy chain CDR1 region of SEQ ID NO:156, a heavy
chain CDR2 region of SEQ ID NO:157, and a heavy chain CDR3 region
of SEQ ID NO:158, m) a binding molecule comprising a heavy chain
CDR1 region of SEQ ID NO:162, a heavy chain CDR2 region of SEQ ID
NO:163, and a heavy chain CDR3 region of SEQ ID NO:164, n) a
binding molecule comprising a heavy chain CDR1 region of SEQ ID
NO:168, a heavy chain CDR2 region of SEQ ID NO:169, and a heavy
chain CDR3 region of SEQ ID NO:170, o) a binding molecule
comprising a heavy chain CDR1 region of SEQ ID NO:173, a heavy
chain CDR2 region of SEQ ID NO:174, and a heavy chain CDR3 region
of SEQ ID NO:175, and p) a binding molecule comprising a heavy
chain CDR1 region of SEQ ID NO:179, a heavy chain CDR2 region of
SEQ ID NO:180, and a heavy chain CDR3 region of SEQ ID NO:181.
14. The binding molecule of claim 1, wherein the binding molecule
is selected from the group consisting of: a) a binding molecule
comprising a heavy chain CDR1 region of SEQ ID NO:81, a heavy chain
CDR2 region of SEQ ID NO:82, and a heavy chain CDR3 region of SEQ
ID NO:83, b) a binding molecule comprising a heavy chain CDR1
region of SEQ ID NO:109, a heavy chain CDR2 region of SEQ ID
NO:110, and a heavy chain CDR3 region of SEQ ID NO:111, c) a
binding molecule comprising a heavy chain CDR1 region of SEQ ID
NO:138, a heavy chain CDR2 region of SEQ ID NO:139, and a heavy
chain CDR3 region of SEQ ID NO:140, d) a binding molecule
comprising a heavy chain CDR1 region of SEQ ID NO:144, a heavy
chain CDR2 region of SEQ ID NO:145, and a heavy chain CDR3 region
of SEQ ID NO:146, and e) a binding molecule comprising a heavy
chain CDR1 region of SEQ ID NO:173, a heavy chain CDR2 region of
SEQ ID NO:174, and a heavy chain CDR3 region of SEQ ID NO:175.
15. The binding molecule of claim 1, wherein the binding molecule
is a human monoclonal antibody.
16. A method of diagnosing influenza infection in a subject, the
method comprising: utilizing the binding molecule of claim 1 to
diagnose the influenza infection in the subject.
17. A method of treating or prophylaxing against influenza
infection in a subject, the method comprising: utilizing the
binding molecule of claim 1 to treat or prophylax the subject
against an influenza infection caused by influenza virus comprising
HA of the H3 subtype.
18. A non-naturally occurring peptide of the type comprising a
heavy chain CDR1 region, a heavy chain CDR2 region, and a heavy
chain CDR3 region, said peptide being able to specifically bind to
an epitope in influenza hemagglutinin protein (HA), wherein said
peptide has neutralizing activity against influenza viruses
comprising HA of the H3 subtype and having cross-neutralizing
activity against at least an influenza virus comprising HA of the
H7 subtype, and/or an influenza virus comprising HA of the H10
subtype.
19. An immunoconjugate comprising: the binding molecule of claim 1,
and at least one tag associated therewith.
20. A composition comprising: the binding molecule of claim 1, and
a pharmaceutically acceptable excipient.
21. The composition of claim 20, further comprising at least one
additional binding molecule.
22. The composition of claim 21, wherein the additional binding
molecule is able to neutralize influenza virus comprising HA of the
H1 and H5 subtype.
23. A composition comprising: at least two influenza virus
neutralizing binding molecules, wherein at least one binding
molecule is able to neutralize one or more influenza virus subtypes
of phylogenetic group 1 and wherein at least one binding molecule
is able to neutralize one or more influenza virus subtypes of
phylogenetic group 2.
24. A pharmaceutical composition comprising at least two influenza
virus neutralizing binding molecules, wherein at least one binding
molecules is able to neutralize influenza viruses comprising HA of
the H1 and/or H5 subtype, and wherein at least one binding molecule
is able to neutralize influenza viruses comprising HA of the H3, H7
and/or H10 subtype.
25. A polynucleotide encoding the binding molecule of claim 1.
26. A vector comprising at least one polynucleotide of claim
25.
27. A host comprising at least one vector of claim 28.
28. The host of claim 27, wherein the host is a human cell.
29. A process for producing a molecule having neutralizing activity
against influenza viruses comprising hemagglutinin protein (HA) of
the H3 subtype and having cross-neutralizing activity against at
least an influenza virus comprising HA of the H7 subtype, and/or an
influenza virus comprising HA of the H10 subtype, wherein the
process comprises: culturing the host of claim 27 under conditions
conducive to the expression of the molecule, and, optionally,
recovering the expressed molecule.
Description
CROSS-REFERENCE TO RELATED APPLICATIONS
[0001] This application is a continuation of co-pending U.S. patent
application Ser. No. 13/138,941, filed Oct. 27, 2011, which
application is a national phase entry under 35 U.S.C. .sctn.371 of
International Patent Application PCT/EP2010/056217, filed May 6,
2010, published in English as International Patent Publication WO
2010/130636 Al on Nov. 18, 2010, which claims the benefit under
Article 8 of the Patent Cooperation Treaty to EP Application Serial
No. 10151155.8, filed Jan. 20, 2010, which itself claims priority
under Article 8 of the PCT to European Patent Application Serial
No. 09159947.2 filed May 11, 2009, and also under 35 U.S.C.
.sctn.119(e) to U.S. Provisional Patent Application 61/215,890,
filed May 11, 2009, each of which is incorporated herein by this
reference.
STATEMENT ACCORDING TO 37 C.F.R. .sctn.1.821(c) or (e)--SEQUENCE
LISTING SUBMITTED AS PDF FILE WITH A REQUEST TO TRANSFER CRF FROM
PARENT APPLICATION
[0002] Pursuant to 37 C.F.R. .sctn.1.821(c) or (e), a file
containing a PDF version of the Sequence Listing has been submitted
concomitant with this application, the contents of which are hereby
incorporated by reference. The transmittal documents of this
application include a Request to Transfer CRF from the prior
application.
TECHNICAL FIELD
[0003] The disclosure relates to biotechnology and medicine,
particularly, to human binding molecules able to neutralize various
influenza A subtypes, including neutralizing binding molecules
against influenza viruses comprising HA of the H3 subtype, such as
influenza virus H3N2. In particular, it relates to the diagnosis,
prophylaxis and/or treatment of an infection by an influenza virus
comprising HA of the H3 subtype, in particular influenza virus
H3N2.
BACKGROUND
[0004] Influenza viruses are RNA orthomyxoviruses and consist of
three types, A, B and C. Whereas influenza viruses of types B and C
are predominantly human pathogens, influenza A viruses infect a
wide variety of birds and mammals, including humans, horses, marine
mammals, pigs, ferrets, and chickens. In animals, most influenza A
viruses cause mild localized infections of the respiratory and
intestinal tract. However, highly pathogenic influenza A subtypes,
such as H5N1, also exist that cause systemic infections in poultry
in which mortality may reach 100%. Several subtypes of influenza A
viruses also may cause severe illness in man.
[0005] Influenza A viruses can be classified into subtypes based on
allelic variations in antigenic regions of two genes that encode
surface glycoproteins, namely, hemagglutinin (HA) and neuraminidase
(NA), which are required for viral attachment and cellular release.
Other major viral proteins include the nucleoprotein, the
nucleocapsid structural protein, membrane proteins (M1 and M2),
polymerases (PA, PB and PB2) and non-structural proteins (NS1 and
NS2). Currently, sixteen subtypes of HA (H1-H16) and nine NA
(N1-N9) antigenic variants are known in influenza A virus.
Influenza virus subtypes can further be classified by reference to
their phylogenetic group. Phylogenetic analysis (Fouchier et al.,
2005) has demonstrated a subdivision of HAs that falls into two
main groups (Air, 1981): inter alia the H1, H2, H5 and H9 subtypes
in phylogenetic group 1 and inter alia the H3, H4 and H7 subtypes
in phylogenetic group 2 (FIG. 1).
[0006] Only some of the influenza A subtypes (i.e., H1N1, H1N2 and
H3N2) circulate among people, but all combinations of the 16 HA and
9 NA subtypes have been identified in avian species Animals
infected with influenza A often act as a reservoir for the
influenza viruses and certain subtypes have been shown to cross the
species barrier to humans, such as the highly pathogenic influenza
A strain H5N1.
[0007] Influenza infection is one of the most common diseases known
to man, causing between three and five million cases of severe
illness and between 250,000 and 500,000 deaths every year around
the world. Influenza rapidly spreads in seasonal epidemics
affecting 5-15% of the population and the burden on health care
costs and lost productivity are extensive (World Healthcare
Organization (WHO)). Hospitalization and deaths mainly occur in
high-risk groups (elderly, chronically ill).
[0008] Annual epidemics of influenza occur when the antigenic
properties of the viral surface protein HA and NA are altered. The
mechanism of altered antigenicity is twofold: antigenic shift,
caused by genetic rearrangement between human and animal viruses
after double infection of host cells, which can cause a pandemic;
and antigenic drift, caused by small changes in the HA and NA
proteins on the virus surface, which can cause influenza epidemics.
The emergence of variant virus strains by these two mechanisms is
the cause of influenza epidemics. Three times in the last century,
the influenza A viruses have undergone major genetic changes,
mainly in their HA-component, resulting in global pandemics and
large tolls in terms of both disease and deaths. The most infamous
pandemic was "Spanish Flu," caused by influenza virus H1N1, which
affected large parts of the world population and is thought to have
killed at least 40 million people in 1918-1919. More recently, two
other influenza A pandemics occurred, in 1957 ("Asian influenza,"
caused by influenza virus H2N2) and 1968 ("Hong Kong influenza,"
caused by influenza virus H3N2), and caused significant morbidity
and mortality globally. In contrast to current seasonal influenza
epidemics, these pandemics were associated with severe outcomes
also among healthy younger persons.
[0009] Current approaches to dealing with annual influenza
epidemics include annual vaccination, preferably generating
heterotypic cross-protection. However, as indicated above,
circulating influenza viruses in humans are subject to permanent
antigenic changes, which require annual adaptation of the influenza
vaccine formulation to ensure the closest possible match between
the influenza vaccine strains and the circulating influenza
strains.
[0010] Although yearly vaccination with the flu vaccine is the best
way to prevent the flu, antiviral drugs, such as oseltamivir
(TAMIFLU.RTM.), can be effective for prevention and treatment of
the flu. However, the number of influenza virus strains showing
resistance against such oseltamivir is increasing.
[0011] An alternative approach is the development of antibody-based
prophylactic or therapeutic means to neutralize various seasonal
influenza viruses. The primary target of neutralizing antibodies
that protect against influenza virus infection is the globular head
(HA1 part) of the viral HA protein, which contains the receptor
binding site, but is subject to continuing genetic evolution with
amino acid substitutions in antibody-binding sites (antigenic
drift). Cross-neutralizing antibodies recognizing the more
conserved stem-region of HA of influenza A viruses of phylogenetic
group 1 (e.g., H1 and H5) have recently been identified (e.g.,
WO2008/028946). There has, however, been limited success in
identifying antibodies that neutralize one or more influenza A
virus subtypes of phylogenetic group 2, such as H3 viruses, and
their breadth of neutralization is narrow and their potency
low.
[0012] Antibodies specifically recognizing H3N2 influenza virus
strains have been described. Thus, a human monoclonal antibody,
C28, capable of binding to and neutralizing several H3N2 influenza
virus strains from the years between 1968 and 1980 has been
described by Ostberg and Pursch (1983). Wang et al. (2010) have
described an anti-HA2 murine antibody neutralizing H3 viruses
spanning several decades, but which was shown not to neutralize any
non-H3 subtype viruses.
[0013] Cross-reactive anti-HA2 murine antibodies recognizing HA of
the H3 subtype, as well as of the H4 and H7 subtype, and capable of
in vitro reducing influenza virus replication of H3 and H4
influenza viruses have been described by Stropkovska et al. (2009).
It was demonstrated that the accessibility of the HA2 epitopes to
these antibodies in the native virus was low, and that the
antibodies have a higher reactivity with HA after its trypsin
cleavage and pH 5 treatment (Vare{hacek over (c)}kova et al.,
2003a), which may explain the observation that the in vitro
inhibition of virus replication (Vare{hacek over (c)}kova et al.,
2003b), as well as in vivo potency of these antibodies was
relatively low (Gocnik et al., 2007).
[0014] In WO2009/115972, a human monoclonal antibody, Fab28, is
disclosed that recognizes an epitope on the stem region of HA and
displays a neutralizing activity against H1N1, but less
neutralizing activity against H3N2.
SUMMARY
[0015] In view of the severity of the respiratory illness caused by
certain influenza A viruses, and the always existing threat of a
potential pandemic, as well has the high economic impact of the
seasonal epidemics, an ongoing need exists for effective means for
preventing and treating the various influenza A subtypes. Thus, a
need exist for binding molecules, such as broadly neutralizing
human binding molecules, able to neutralize seasonal influenza
virus subtypes, including influenza viruses comprising HA of the H3
subtype, e.g., H3N2, and that have little or none of the drawbacks
of the antibodies known in the prior art.
[0016] Provided are binding molecules that can be used in medicine,
in particular, for diagnosis, prevention, and/or treatment of
infection with influenza virus comprising HA of the H3 subtype,
such as H3N2 infections. Some of the binding molecules described
herein are unique in their breadth of neutralizing activity within
the H3 subtype. Thus, some of the binding molecules identified
herein are able to neutralize several, including at least one or
more recent, strains within the H3N2 subtype and may be used as a
universal prophylactic and/or treatment agent for seasonal
influenza, irrespective of the causative influenza H3N2 strain. At
least some of the binding molecules are able to prevent in vitro
the cleavage of the HA precursor molecule HA0 by trypsin.
Furthermore, at least some of the binding molecules hereof are able
to prevent the conformational change of the HA protein, thought to
be involved in membrane fusion of the influenza viral membrane and
the endosomal membrane of an infected cell. Furthermore, at least
some binding molecules hereof are unique in that they are also able
to cross-neutralizing influenza viruses of at least one other
subtype, including influenza viruses comprising HA of the H7 and/or
H10 subtypes, and thus can be used as a universal prophylactic,
diagnostic and/or treatment agent for influenza viruses, even
irrespective of the causative influenza subtype.
BRIEF DESCRIPTION OF THE DRAWINGS
[0017] FIG. 1 shows a phylogenetic tree of amino acid sequences at
the subtype level. Division of subtypes by group is indicated. The
H1 clade, comprising inter alia the H1 subtypes, and the H9 clade,
comprising the H9 subtypes, form phylogenetic group 1, and the H7,
comprising inter alia the H7 subtypes, and the H3 clade, comprising
inter alia the H3 subtypes, form phylogenetic group 2.
[0018] FIG. 2 is a bar diagram showing binding of IgG1 to
surface-expressed H3 rHA, measured by FACS analysis, after
sequential treatment with trypsin (striped bars), pH 4.9 buffered
medium (solid white bars) and DTT (crossed bars) and expressed as
percentage binding to untreated rHA (solid black bars).
[0019] FIG. 3 shows the results of an in vitro protease
susceptibility assay. Samples were run on a 4-12% BisTris gel in
1.times. MOPS buffer. Protein bands were visualized by colloidal
blue staining
[0020] FIG. 4 is a schematic representation of the different
conformations of the HA protein during the infection process.
[0021] FIG. 5 is a bar diagram showing binding of the H3 mAbs to
HA-expressing cells after different treatments measured by FACS
analysis, after sequential treatment with trypsin (striped bars),
pH 4.9 buffered medium (solid white bars) and DTT (crossed bars)
and expressed as percentage binding to untreated rHA (solid black
bars).
[0022] FIG. 6 shows the result of the time course experiment
described in Example 11 to determine the incubation time of HA with
trypsin to achieve cleavage of H3 HA.
[0023] FIG. 7 shows the results of trypsin digestion of H3 HA
samples pre-incubated with mAbs, as described in Example 11.
[0024] FIG. 8 is a bar diagram demonstrating that CR8043 inhibits
pH-induced conformational change in H3 HA.
[0025] FIG. 9 shows that CR8020 and CR8041 are also capable of
blocking the pH-induced conformational change of HA: Panel A. mAbs
added before Trypsin cleavage; Panel B. mAbs added after trypsin
cleavage; Panel C. mAbs added after all treatments.
[0026] FIG. 10 shows the Kaplan-Meier survival probability curves.
Antibody was administered intravenously at day -1 before challenge
using a dose range from 30 down to 1 mg/kg. Control Ab was
administered at 30 mg/kg (grey), followed by a lethal challenge at
day 0 with 25 LD50 A/HK/1/68-MA20 (H3N2). CR8020 (A) was tested in
a separate study from CR8041 (B) and CR8043 (C), which were
evaluated in one experiment. Therefore, the same control antibody
group is used for B and C.
[0027] FIG. 11 shows the mean body weight change (%) relative to
day 0. Antibody was administered intravenously at day -1 before
challenge using a dose range from 30 down to 1 mg/kg. Control Ab
was administered at 30 mg/kg (grey), followed by a lethal challenge
at day 0 with 25 LD50 A/HK/1/68-MA20 (H3N2). Bars represent the 95%
CI of the mean. If a mouse died or was euthanized during follow-up
of the study, the last observed body weight was carried forward.
CR8020 (A) was tested in a separate study from CR8041 (B) and
CR8043 (C), which were evaluated in one experiment. Therefore, the
same control antibody group is used for B and C.
[0028] FIG. 12 shows the median clinical score. Antibody was
administered intravenously at day -1 before challenge using a dose
range from 30 down to 1 mg/kg. Control Ab was administered at 30
mg/kg (grey), followed by a lethal challenge at day 0 with 25 LD50
A/HK1/68-MA20 (H3N2). Bars represent interquartile ranges. CR8020
(A) was tested in a separate study from CR8041 (B) and CR8043 (C),
which were evaluated in one experiment. Therefore, the same control
antibody group is used for B and C. Clinical score explanation:
0=no clinical signs; 1=rough coat; 2=rough coat, less reactive,
passive during handling; 3=rough coat, rolled up, labored
breathing, passive during handling; 4=rough coat, rolled up,
labored breathing, does not roll back on stomach when laid down on
its back. Mice observed with clinical score 4 were euthanized on
the same day.
[0029] FIG. 13 demonstrates the therapeutic efficacy of mAb CR8020
in the mouse lethal challenge model with influenza A/HK/1/68-MA20
(H3N2). A single dose of mAb CR8020 (15 mg/kg) was administered
intravenously either at day -1 pre-challenge or at day 1, 2, 3, 4,
5, or 6 post-challenge in 129X1/SvJ mice (n=10/group). Control mAb
(15 mg/kg) was administered at day 1 post-challenge. Mice were
challenged at day 0 with 25 LD50 A/HK/1/68-MA20 (H3N2) and
monitored for 21 days. Panel A: Kaplan-Meier survival probability
curves. Panel B: Mean body weight change (%) relative to day 0.
Bars represent the 95% CI of the mean. If a mouse died or was
euthanized during follow-up of the study, the last observed body
weight was carried forward. Panel C: Median clinical score. Bars
represent interquartile ranges. 0=no clinical signs; 1=rough coat;
2=rough coat, less reactive, passive during handling; 3=rough coat,
rolled up, labored breathing, passive during handling; 4=rough
coat, rolled up, labored breathing, does not roll back on stomach
when laid down on its back. Mice observed with clinical score 4
were euthanized on the same day.
[0030] FIG. 14 shows the prophylactic efficacy of mAb CR8020 in the
mouse lethal challenge model with mouse-adapted influenza
A/CH/NL/621557/03 (H7N7). Panel A: Kaplan-Meier survival
probability curves. Panel B: Mean body weight change (%) relative
to day 0. Bars represent the 95% CI of the mean. If a mouse died or
was euthanized during follow-up of the study, the last observed
body weight was carried forward. Panel C: Median clinical score.
Bars represent interquartile ranges. 0=no clinical signs; 1=rough
coat; 2=rough coat, less reactive, passive during handling; 3=rough
coat, rolled up, labored breathing, passive during handling;
4=rough coat, rolled up, labored breathing, does not roll back on
stomach when laid down on its back. Mice observed with clinical
score 4 were euthanized on the same day.
[0031] FIG. 15 shows the prophylactic efficacy of mAbs CR8020,
CR8041 and CR8043 in the mouse lethal challenge model with
mouse-adapted influenza A/CH/NL/621557/03 (H7N7). MAbs were
administered intravenously at day -1 before challenge in female
Balb/c mice (n=8/group) using a dose range from 10 down to 1 mg/kg
(CR8020) or 30 down to 1 mg/kg (CR8041 and CR8043). Control mAb was
administered at day -1 at 30 mg/kg (grey). At day 0, a lethal
challenge was given by intranasal inoculation with 25 LD.sub.50
mouse-adapted A/CH/NL/621557/03 (H7N7) and the mice were
subsequently monitored for 21 days. Panel A: Kaplan-Meier survival
probability curves. Panel B: Mean body weight change (%) relative
to day 0. Bars represent the 95% CI of the mean. If a mouse died or
was euthanized during follow-up of the study, the last observed
body weight was carried forward. Panel C: Median Clinical score.
Bars represent interquartile ranges. 0=no clinical signs; 1=rough
coat; 2=rough coat, less reactive, passive during handling; 3=rough
coat, rolled up, labored breathing, passive during handling;
4=rough coat, rolled up, labored breathing, does not roll back on
stomach when laid down on its back. Mice observed with clinical
score 4 were euthanized on the same day.
[0032] FIG. 16 shows the therapeutic efficacy of mAb CR8020 in the
mouse lethal challenge model with mouse-adapted influenza
A/CH/NL/621557/03 (H7N7). A single dose of mAb CR8020 (15 mg/kg)
was administered intravenously either at day -1 pre-challenge or at
day 1, 2, 3, 4, 5, or 6 post-challenge in female Balb/c mice
(n=8/group). Control mAb (15 mg/kg) was administered at day 1
post-challenge. Mice were challenged at day 0 with 25 LD.sub.50
mouse-adapted A/CH/NL/621557/03 (H7N7) and monitored for 21 days.
Panel A: Kaplan-Meier survival probability curves. Panel B: Mean
body weight change (%) relative to day 0. Bars represent the 95% CI
of the mean. If a mouse died or was euthanized during follow-up of
the study, the last observed body weight was carried forward. Panel
C: Median clinical score. Bars represent interquartile ranges. 0=no
clinical signs; 1=rough coat; 2=rough coat, less reactive, passive
during handling; 3=rough coat, rolled up, labored breathing,
passive during handling; 4=rough coat, rolled up, labored
breathing, does not roll back on stomach when laid down on its
back. Mice observed with clinical score 4 were euthanized on the
same day.
DETAILED DESCRIPTION
[0033] Unless another usage is indicated, the term "included" or
"including" as used herein is deemed to be followed by the words
"without limitation."
[0034] As used herein, the term "binding molecule" refers to an
intact immunoglobulin including monoclonal antibodies, such as
chimeric, humanized or human monoclonal antibodies, or to an
antigen-binding and/or variable domain-comprising fragment of an
immunoglobulin that competes with the intact immunoglobulin for
specific binding to the binding partner of the immunoglobulin,
e.g., H3. Regardless of structure, the antigen-binding fragment
binds with the same antigen that is recognized by the intact
immunoglobulin. An antigen-binding fragment can comprise a peptide
or polypeptide comprising an amino acid sequence of at least 2, 5,
10, 15, 20, 25, 30, 35, 40, 50, 60, 70, 80, 90, 100, 125, 150, 175,
200, or 250 contiguous amino acid residues of the amino acid
sequence of the binding molecule.
[0035] The term "binding molecule," as used herein, includes all
immunoglobulin classes and subclasses known in the art. Depending
on the amino acid sequence of the constant domain of their heavy
chains, binding molecules can be divided into the five major
classes of intact antibodies: IgA, IgD, IgE, IgG, and IgM, and
several of these may be further divided into subclasses (isotypes),
e.g., IgA1, IgA2, IgG1, IgG2, IgG3 and IgG4.
[0036] Antigen-binding fragments include, inter alia, Fab, F(ab'),
F(ab')2, Fv, dAb, Fd, complementarity-determining region (CDR)
fragments, single-chain antibodies (scFv), bivalent single-chain
antibodies, single-chain phage antibodies, diabodies, triabodies,
tetrabodies, (poly)peptides that contain at least a fragment of an
immunoglobulin that is sufficient to confer specific antigen
binding to the (poly)peptide, etc. The above fragments may be
produced synthetically or by enzymatic or chemical cleavage of
intact immunoglobulins or they may be genetically engineered by
recombinant DNA techniques. The methods of production are well
known in the art and are described, for example, in Antibodies: A
Laboratory Manual, edited by E. Harlow and D. Lane (1988), Cold
Spring Harbor Laboratory, Cold Spring Harbor, N.Y., which is
incorporated herein by reference. A binding molecule or
antigen-binding fragment thereof may have one or more binding
sites. If there is more than one binding site, the binding sites
may be identical to one another or they may be different.
[0037] The binding molecule can be a naked or unconjugated binding
molecule, but can also be part of an immunoconjugate. A naked or
unconjugated binding molecule is intended to refer to a binding
molecule that is not conjugated, operatively linked or otherwise
physically or functionally associated with an effector moiety or
tag, such as inter alia a toxic substance, a radioactive substance,
a liposome, or an enzyme. It will be understood that naked or
unconjugated binding molecules do not exclude binding molecules
that have been stabilized, multimerized, humanized or in any other
way manipulated, other than by the attachment of an effector moiety
or tag. Accordingly, all post-translationally modified naked and
unconjugated binding molecules are included herewith, including
where the modifications are made in the natural binding
molecule-producing cell environment, by a recombinant binding
molecule-producing cell, and are introduced by the hand of man
after initial binding molecule preparation. Of course, the term
"naked or unconjugated binding molecule" does not exclude the
ability of the binding molecule to form functional associations
with effector cells and/or molecules after administration to the
body, as some of such interactions are necessary in order to exert
a biological effect. The lack of associated effector group or tag
is, therefore, applied in definition to the naked or unconjugated
binding molecule in vitro, not in vivo.
[0038] As used herein, the term "biological sample" encompasses a
variety of sample types, including blood and other liquid samples
of biological origin, solid tissue samples such as a biopsy
specimen or tissue cultures, or cells derived therefrom and the
progeny thereof. The term also includes samples that have been
manipulated in any way after their procurement, such as by
treatment with reagents, solubilization, or enrichment for certain
components, such as proteins or polynucleotides. The term
encompasses various kinds of clinical samples obtained from any
species, and also includes cells in culture, cell supernatants and
cell lysates.
[0039] The term "complementarity-determining regions" (CDR) as used
herein means sequences within the variable regions of binding
molecules, such as immunoglobulins, that usually contribute to a
large extent to the antigen binding site, which is complementary in
shape and charge distribution to the epitope recognized on the
antigen. The CDR regions can be specific for linear epitopes,
discontinuous epitopes, or conformational epitopes of proteins or
protein fragments, either as present on the protein in its native
conformation or, in some cases, as present on the proteins as
denatured, e.g., by solubilization in SDS. Epitopes may also
consist of post-translational modifications of proteins.
[0040] The term "deletion," as used herein, denotes a change in
either amino acid or nucleotide sequence in which one or more amino
acid or nucleotide residues, respectively, are absent as compared
to the reference, often the naturally occurring, molecule.
[0041] The term "expression-regulating nucleic acid sequence (or
polynucleotide)" as used herein refers to polynucleotides necessary
for and/or affecting the expression of an operably linked coding
sequence in a particular host organism. The expression-regulating
polynucletides, such as inter alia appropriate transcription
initiation, termination, promoter, enhancer sequences; repressor or
activator sequences; efficient RNA processing signals such as
splicing and polyadenylation signals; sequences that stabilize
cytoplasmic mRNA; sequences that enhance translation efficiency
(e.g., ribosome binding sites); sequences that enhance protein
stability; and, when desired, sequences that enhance protein
secretion, can be any nucleic acid sequence showing activity in the
host organism of choice and can be derived from genes encoding
proteins, which are either homologous or heterologous to the host
organism. The identification and employment of
expression-regulating sequences is routine to the person skilled in
the art.
[0042] The term "functional variant," as used herein, refers to a
binding molecule that comprises a nucleotide and/or amino acid
sequence that is altered by one or more nucleotides and/or amino
acids compared to the nucleotide and/or amino acid sequences of the
reference binding molecule and that is still capable of competing
for binding to the binding partner, e.g., H3N2, with the reference
binding molecule. In other words, the modifications in the amino
acid and/or nucleotide sequence of the reference binding molecule
do not significantly affect or alter the binding characteristics of
the binding molecule encoded by the nucleotide sequence or
containing the amino acid sequence, i.e., the binding molecule is
still able to recognize and bind its target. The functional variant
may have conservative sequence modifications including nucleotide
and amino acid substitutions, additions and deletions. These
modifications can be introduced by standard techniques known in the
art, such as site-directed mutagenesis and random PCR-mediated
mutagenesis, and may comprise natural as well as non-natural
nucleotides and amino acids.
[0043] Conservative amino acid substitutions include the ones in
which the amino acid residue is replaced with an amino acid residue
having similar structural or chemical properties. Families of amino
acid residues having similar side chains are known in the art.
These families include amino acids with basic side chains (e.g.,
lysine, arginine, histidine), acidic side chains (e.g., aspartic
acid, glutamic acid), uncharged polar side chains (e.g.,
asparagine, glutamine, serine, threonine, tyrosine, cysteine,
tryptophan), non-polar side chains (e.g., glycine, alanine, valine,
leucine, isoleucine, proline, phenylalanine, methionine),
beta-branched side chains (e.g., threonine, valine, isoleucine) and
aromatic side chains (e.g., tyrosine, phenylalanine, tryptophan).
It will be clear to the skilled artisan that other classifications
of amino acid residue families than the one used above can also be
employed. Furthermore, a variant may have non-conservative amino
acid substitutions, e.g., replacement of an amino acid with an
amino acid residue having different structural or chemical
properties. Similar minor variations may also include amino acid
deletions or insertions, or both. Guidance in determining which
amino acid residues may be substituted, inserted, or deleted
without abolishing immunological activity may be found using
computer programs well known in the art.
[0044] A mutation in a polynucleotide can be a single alteration
made at a locus (a point mutation), such as transition or
transversion mutations, or alternatively, multiple nucleotides may
be inserted, deleted or changed at a single locus. In addition, one
or more alterations may be made at any number of loci within a
nucleotide sequence. The mutations may be performed by any suitable
method known in the art.
[0045] The term "influenza virus subtype" as used herein refers to
influenza A virus variants that are characterized by various
combinations of the hemagglutinin (H) and neuramidase (N) viral
surface proteins. Hereof, influenza virus subtypes may be referred
to by their H number, such as, for example, "influenza virus
comprising HA of the H3 subtype," or "H3 influenza," or by a
combination of an H number and an N number, such as, for example,
"influenza virus subtype H3N2" or "H3N2." The term "subtype"
specifically includes all individual "strains" within each subtype,
which usually result from mutations and show different pathogenic
profiles. Such strains may also be referred to as various
"isolates" of a viral subtype. Accordingly, as used herein, the
terms "strains" and "isolates" may be used interchangeably. The
current nomenclature for human influenza virus strains or isolates
includes the geographical location of the first isolation, strain
number and year of isolation, usually with the antigenic
description of HA and NA given in brackets, e.g., A/Moscow/10/00
(H3N2). Non-human strains also include the host of origin in the
nomenclature.
[0046] The influenza virus subtypes can further be classified by
reference to their phylogenetic group. Phylogenetic analysis
(Fouchier et al., 2005) has demonstrated a subdivision of HAs that
falls into two main groups (Air, 1981): inter alia the H1, H2, H5
and H9 subtypes in phylogenetic group 1 and inter alia the H3, H4
and H7 subtypes in phylogenetic group 2 (FIG. 1).
[0047] The term "neutralizing" as used herein in relation to the
binding molecules hereof refers to binding molecules that inhibit
an influenza virus from replicatively infecting a target cell,
regardless of the mechanism by which neutralization is achieved.
Thus, neutralization can, e.g., be achieved by inhibiting the
attachment or adhesion of the virus to the cell surface, or by
inhibition of the fusion of viral and cellular membranes following
attachment of the virus to the target cell, and the like.
[0048] The term "cross-neutralizing" or "cross-neutralization" as
used herein in relation to binding molecules refers to the ability
of the binding molecules hereof to neutralize influenza A viruses
of different subtypes, such as, for example, influenza viruses
comprising HA of the H3, H7 and/or H10 subtype.
[0049] The term "host," is intended to refer to an organism or a
cell into which a vector such as a cloning vector or an expression
vector has been introduced. The organism or cell can be prokaryotic
or eukaryotic. The hosts may be isolated host cells, e.g., host
cells in culture. The term "host cells" merely signifies that the
cells are modified for the (over)-expression of the binding
molecules hereof and include B-cells that originally express these
binding molecules and which cells have been modified to
over-express the binding molecule by immortalization,
amplification, enhancement of expression, etc. It should be
understood that the term "host" is intended to refer not only to
the particular subject organism or cell, but to the progeny of such
an organism or cell as well. Because certain modifications may
occur in succeeding generations due to either mutation or
environmental influences, such progeny may not, in fact, be
identical to the parent organism or cell, but are still included
within the scope of the term "host" as used herein.
[0050] The term "human," when applied to binding molecules as
defined herein, refers to molecules that are either directly
derived from a human or based upon a human sequence. When a binding
molecule is derived from or based on a human sequence and
subsequently modified, it is still to be considered human as used
throughout the specification. In other words, the term "human,"
when applied to binding molecules, is intended to include binding
molecules having variable and constant regions derived from human
germline immunoglobulin sequences or based on variable or constant
regions occurring in a human or human lymphocyte and modified in
some form. Thus, the human binding molecules may include amino acid
residues not encoded by human germline immunoglobulin sequences,
comprise substitutions and/or deletions (e.g., mutations introduced
by, for instance, random or site-specific mutagenesis in vitro or
by somatic mutation in vivo). "Based on" as used herein refers to
the situation that a nucleic acid sequence may be exactly copied
from a template, or with minor mutations, such as by error-prone
PCR methods, or synthetically made matching the template exactly or
with minor modifications. Semi-synthetic molecules based on human
sequences are also considered to be human as used herein.
[0051] The term "insertion," also known as the term "addition,"
denotes a change in an amino acid or nucleotide sequence resulting
in the addition of one or more amino acid or nucleotide residues,
respectively, as compared to the parent sequence.
[0052] The term "isolated," when applied to binding molecules as
defined herein, refers to binding molecules that are substantially
free of other proteins or polypeptides, particularly free of other
binding molecules having different antigenic specificities, and are
also substantially free of other cellular material and/or
chemicals. For example, when the binding molecules are
recombinantly produced, they may be substantially free of culture
medium components and, when the binding molecules are produced by
chemical synthesis, they may be substantially free of chemical
precursors or other chemicals, i.e., they are separated from
chemical precursors or other chemicals that are involved in the
synthesis of the protein. The term "isolated" when applied to
nucleic acid molecules encoding binding molecules as defined
herein, is intended to refer to nucleic acid molecules in which the
nucleotide sequences encoding the binding molecules are free of
other nucleotide sequences, particularly nucleotide sequences
encoding binding molecules that bind binding partners other than
H5N1. Furthermore, the term "isolated" refers to nucleic acid
molecules that are substantially separated from other cellular
components that naturally accompany the native nucleic acid
molecule in its natural host, e.g., ribosomes, polymerases, or
genomic sequences with which it is naturally associated. Moreover,
"isolated" nucleic acid molecules, such as cDNA molecules, can be
substantially free of other cellular material or culture medium
when produced by recombinant techniques, or substantially free of
chemical precursors or other chemicals when chemically
synthesized.
[0053] The term "monoclonal antibody" as used herein refers to a
preparation of antibody molecules of single specificity. A
monoclonal antibody displays a single binding specificity and
affinity for a particular epitope. Accordingly, the term "human
monoclonal antibody" refers to an antibody displaying a single
binding specificity that has variable and constant regions derived
from or based on human germline immunoglobulin sequences or derived
from completely synthetic sequences. The method of preparing the
monoclonal antibody is not relevant for the binding
specificity.
[0054] The term "naturally occurring" as used herein as applied to
an object refers to the fact that an object can be found in nature.
For example, a polypeptide or polynucleotide sequence that is
present in an organism that can be isolated from a source in nature
and that has not been intentionally modified by man in the
laboratory is naturally occurring.
[0055] The term "nucleic acid molecule" as used herein refers to a
polymeric form of nucleotides and includes both sense and
anti-sense strands of RNA, cDNA, genomic DNA, and synthetic forms
and mixed polymers of the above. A nucleotide refers to a
ribonucleotide, deoxynucleotide or a modified form of either type
of nucleotide. The term also includes single- and double-stranded
forms of DNA. In addition, a polynucleotide may include either or
both naturally occurring and modified nucleotides linked together
by naturally occurring and/or non-naturally occurring nucleotide
linkages. The nucleic acid molecules may be modified chemically or
biochemically or may contain non-natural or derivatized nucleotide
bases, as will be readily appreciated by those of skill in the art.
Such modifications include, for example, labels, methylation,
substitution of one or more of the naturally occurring nucleotides
with an analog, internucleotide modifications such as uncharged
linkages (e.g., methyl phosphonates, phosphotriesters,
phosphoramidates, carbamates, etc.), charged linkages (e.g.,
phosphorothioates, phosphorodithioates, etc.), pendent moieties
(e.g., polypeptides), intercalators (e.g., acridine, psoralen,
etc.), chelators, alkylators, and modified linkages (e.g., alpha
anomeric nucleic acids, etc.). The above term is also intended to
include any topological conformation, including single-stranded,
double-stranded, partially duplexed, triplex, hairpinned, circular
and padlocked conformations. Also included are synthetic molecules
that mimic polynucleotides in their ability to bind to a designated
sequence via hydrogen bonding and other chemical interactions. Such
molecules are known in the art and include, for example, those in
which peptide linkages substitute for phosphate linkages in the
backbone of the molecule. A reference to a nucleic acid sequence
encompasses its complement unless otherwise specified. Thus, a
reference to a nucleic acid molecule having a particular sequence
should be understood to encompass its complementary strand, with
its complementary sequence. The complementary strand is also
useful, e.g., for anti-sense therapy, hybridization probes and PCR
primers.
[0056] The term "operably linked" refers to two or more nucleic
acid sequence elements that are usually physically linked and are
in a functional relationship with each other. For instance, a
promoter is operably linked to a coding sequence if the promoter is
able to initiate or regulate the transcription or expression of a
coding sequence, in which case, the coding sequence should be
understood as being "under the control of the promoter.
[0057] By "pharmaceutically acceptable excipient" is meant any
inert substance that is combined with an active molecule such as a
drug, agent, or binding molecule for preparing an agreeable or
convenient dosage form. The "pharmaceutically acceptable excipient"
is an excipient that is non-toxic to recipients at the dosages and
concentrations employed, and is compatible with other ingredients
of the formulation comprising the drug, agent or binding molecule.
Pharmaceutically acceptable excipients are widely applied in the
art.
[0058] The term "specifically binding," as used herein, in
reference to the interaction of a binding molecule, e.g., an
antibody, and its binding partner, e.g., an antigen, means that the
interaction is dependent upon the presence of a particular
structure, e.g., an antigenic determinant or epitope, on the
binding partner. In other words, the antibody preferentially binds
or recognizes the binding partner even when the binding partner is
present in a mixture of other molecules or organisms. The binding
may be mediated by covalent or non-covalent interactions or a
combination of both. In yet other words, the term "specifically
binding" means immunospecifically binding to an antigenic
determinant or epitope and not immunospecifically binding to other
antigenic determinants or epitopes. A binding molecule that
immunospecifically binds to an antigen may bind to other peptides
or polypeptides with lower affinity as determined by, e.g.,
radioimmunoassays (RIA), enzyme-linked immunosorbent assays
(ELISA), BIACORE, or other assays known in the art. Binding
molecules or fragments thereof that immunospecifically bind to an
antigen may be cross-reactive with related antigens, carrying the
same epitope. In certain embodiments, binding molecules or
fragments thereof that immunospecifically bind to an antigen do not
cross-react with other antigens.
[0059] A "substitution," as used herein, denotes the replacement of
one or more amino acids or nucleotides by different amino acids or
nucleotides, respectively.
[0060] The term "therapeutically effective amount" refers to an
amount of the binding molecule as defined herein that is effective
for preventing, ameliorating and/or treating a condition resulting
from infection with influenza of the H3 subtype. "Amelioration" as
used herein may refer to the reduction of visible or perceptible
disease symptoms, viremia, or any other measurable manifestation of
influenza infection.
[0061] The term "treatment" or "treating" refers to therapeutic
treatment as well as prophylactic or preventative measures to cure
or halt or at least retard disease progress. Those in need of
treatment include those already inflicted with a condition
resulting from infection with influenza virus comprising HA of the
H3 subtype as well as those in which infection with influenza virus
comprising HA of the H3 subtype is to be prevented. Subjects
partially or totally recovered from infection with H3 influenza
might also be in need of treatment. Prevention encompasses
inhibiting or reducing the spread of influenza virus comprising HA
of the H3 subtype or inhibiting or reducing the onset, development
or progression of one or more of the symptoms associated with
infection with H3 influenza.
[0062] The term "vector" denotes a nucleic acid molecule into which
a second nucleic acid molecule can be inserted for introduction
into a host where it will be replicated, and in some cases,
expressed. In other words, a vector is capable of transporting a
nucleic acid molecule to which it has been linked. Cloning as well
as expression vectors are contemplated by the term "vector," as
used herein. Vectors include, but are not limited to, plasmids,
cosmids, bacterial artificial chromosomes (BAC) and yeast
artificial chromosomes (YAC) and vectors derived from
bacteriophages or plant or animal (including human) viruses.
Vectors comprise an origin of replication recognized by the
proposed host and in case of expression vectors, promoter and other
regulatory regions recognized by the host. A vector containing a
second nucleic acid molecule is introduced into a cell by
transformation, transfection, or by making use of viral entry
mechanisms. Certain vectors are capable of autonomous replication
in a host into which they are introduced (e.g., vectors having a
bacterial origin of replication can replicate in bacteria). Other
vectors can be integrated into the genome of a host upon
introduction into the host, and thereby are replicated along with
the host genome.
[0063] Provided are human binding molecules able to specifically
bind to influenza virus strains comprising HA of the H3 subtype,
including H3N2, and exhibiting neutralizing activity against such
influenza virus. In certain embodiments, the binding molecules
hereof are unique in that they are able to neutralize several,
including at least one or more recent, strains, such as all known
strains, of influenza virus subtype H3, the most common epidemic
subtype in humans, with high potency. In certain embodiments, the
binding molecules bind to a conserved epitope in the stem region of
the H3 HA protein. In certain embodiments, the binding molecules
have hemagglutination-inhibiting activity. In certain embodiments,
the binding molecules are able to prevent in vitro cleavage of the
HA precursor molecule HA0. In certain embodiments, the binding
molecules hereof are able to prevent the conformational change of
the HA protein required for fusion of the influenza viral membrane
with the endosomal membrane of an infected cell.
[0064] Also provided are binding molecules that bind to an epitope
in the hemagglutinin protein that is shared between influenza
subtypes within the phylogenetic group 2 to which H3 subtypes
belong and, therefore, relates to binding molecules that
cross-react between H3-, H7-, and/or H10 influenza-based subtypes,
and other influenza subtypes that contain the HA protein with these
particular epitopes, such as all subtypes of phylogenetic group 2.
Several binding molecules hereof are thus unique in that they
possess cross-neutralizing activity against one or more other
influenza virus A subtypes, such as influenza viruses comprising HA
of the H7 and/or the H10 subtype. The binding molecules hereof may
be able to cross-neutralize all influenza virus subtypes of
phylogenetic group 2, encompassing the H3, H7 and H10 subtypes, and
thus can be used as a universal prophylactic, diagnostic and/or
treatment agent for influenza viruses belonging to phylogenetic
group 2, irrespective of the causative influenza subtype within
that phylogenetic group.
[0065] It is surmised that these binding molecules bind to hitherto
unknown conserved epitopes that are not or much less prone to
antigenic drift or shift. Hence, it is also encompassed to use the
binding molecules hereof to identify and/or characterize these
epitopes. Also described are nucleic acid molecules encoding at
least the binding region of the human binding molecules. Further
described is the use of the human binding molecules hereof in the
prophylaxis and/or treatment of a subject having, or at risk of
developing, an H3 influenza infection, such as a H3N2 influenza
infection. Furthermore, disclosed is the use of the human binding
molecules hereof in the diagnosis/detection of such influenza
infection.
[0066] Provided are binding molecules that specifically bind to and
have neutralizing activity against influenza virus A, particularly
influenza virus A comprising HA of the H3 subtype, in particular,
H3N2. The binding molecules may be human binding molecules. In
certain embodiments, the binding molecules hereof are able to
specifically bind to and/or have neutralizing activity against
several influenza virus H3N2 strains, preferably two or more
different H3N2 strains, more preferably three or more, more
preferably four or more, more preferably five or more, different
H3N2 strains. The strains may be obtained from both humans or from
non-human animals, e.g., birds. In certain embodiments, the binding
molecules bind to and neutralize at least one or more of the recent
H3N2 strains selected from the group consisting of
A/Wisconsin/67/2005, A/Hiroshima/52/2005, A/Panama/2007/99, and
A/Johannesburg/33/94. In another embodiment, the binding molecules
also bind to and neutralize the H3N2 strain A/Hong Kong/1/68. Most
preferably, the binding molecules bind to and have neutralizing
activity against all influenza H3N2 strains from the years between
1968 and 2005. The binding molecules may have neutralizing activity
against at least all naturally occurring isolates of influenza
virus H3N2 known before Jan. 20, 2010.
[0067] The binding molecules hereof may be able to specifically
bind to the HA0, HA1 and/or HA2 subunit of the HA protein. They may
be able to specifically bind to linear or structural and/or
conformational epitopes on the HA0, HA1 and/or HA2 subunit of the
HA protein. The HA molecule may be purified from viruses or
recombinantly produced and optionally isolated before use.
Alternatively, HA may be expressed on the surface of cells. In
certain embodiments, the binding molecules hereof bind to an
epitope comprising one or more of the amino acids at positions 19,
25, 27, 33 and 34 of the HA2 polypeptide of the H3 HA protein. In
certain embodiments, the binding molecules bind to the epitope on
HA2, when the amino acid on position 19 is aspartic acid (D), the
amino acid on position 25 is glutamine (Q), the amino acid on
position 27 is glycine (G), the amino acid at position 33 is
glycine (G) and/or the amino acid at position 34 is glutamine
(numbering of HA2 starting at position 1 just following the
Arginine residue that constitutes the cleavage site between HA1 and
HA2). In certain embodiments, the binding molecules do not bind to
the epitope on HA2 when one or more of the amino acids have
changed.
[0068] In another aspect, also described are binding molecules that
are capable of, at least in vitro, preventing the trypsin cleavage
of the H3 HA precursor molecule HA0 in HA1 and HA2.
[0069] In another aspect, described are binding molecules that are
able to prevent the conformational change of the H3 HA protein,
required for membrane fusion of the influenza viral membrane and
the endosomal membrane of an infected cell, at least in vitro.
[0070] In another aspect, the binding molecules have some or all of
the properties listed above, i.e., cross-neutralizing activity,
binding to a conserved epitope on the stem region of HA2,
inhibiting in vitro trypsin cleavage of HA0, and/or inhibiting
conformational change.
[0071] In certain embodiments, the binding molecules hereof have
all or some of the properties above and, in addition, are not
capable of binding to and/or neutralizing influenza virus A
comprising HA of the H1 subtype, such as H1N1.
[0072] The binding molecules hereof may be able to specifically
bind to, e.g., influenza virus H3N2 that are viable, living and/or
infective or that are in inactivated/attenuated form. Methods for
inactivating/attenuating virus, e.g., influenza virus H3N2, are
well known in the art and include, but are not limited to,
treatment with formalin, .beta.-propiolactone (BPL), merthiolate,
and/or ultraviolet light.
[0073] The binding molecules hereof may also be able to
specifically bind to one or more fragments of the influenza
viruses, such as inter alia a preparation of one or more proteins
and/or (poly)peptides derived from subtype H3N2 or one or more
recombinantly produced proteins and/or polypeptides of H3N2. For
methods of treatment and/or prevention of H3N2 infections, the
binding molecules are preferably able to specifically bind to
surface accessible proteins of H3N2 such as the surface
glycoproteins, hemagglutinin (HA), which is required for viral
attachment and cellular release.
[0074] The nucleotide and/or amino acid sequence of proteins of
various H3N2 strains can be found in the GenBank-database, NCBI
Influenza Virus Sequence Database, Influenza Sequence Database
(ISD), EMBL-database and/or other databases. It is well within the
reach of the skilled person to find such sequences in the
respective databases.
[0075] In another embodiment, the binding molecules hereof are able
to specifically bind to a fragment of the above-mentioned proteins
and/or polypeptides, wherein the fragment at least comprises an
epitope recognized by the binding molecules hereof. An "epitope" as
used herein is a moiety that is capable of binding to a binding
molecule hereof with sufficiently high affinity to form a
detectable antigen-binding molecule complex.
[0076] The binding molecules hereof may or may not be able to
specifically bind to the extracellular part of HA (also called
herein soluble HA (sHA)).
[0077] The binding molecules hereof can be intact immunoglobulin
molecules, such as polyclonal or monoclonal antibodies, or the
binding molecules can be antigen-binding fragments including, but
not limited to, Fab, F(ab'), F(ab').sub.2, Fv, dAb, Fd,
complementarity-determining region (CDR) fragments, single-chain
antibodies (scFv), bivalent single-chain antibodies, single-chain
phage antibodies, diabodies, triabodies, tetrabodies, and
(poly)peptides that contain at least a fragment of an
immunoglobulin that is sufficient to confer specific antigen
binding to influenza virus H3N2 strains or a fragment thereof. In a
preferred embodiment, the binding molecules hereof are human
monoclonal antibodies.
[0078] The binding molecules hereof can be used in non-isolated or
isolated form. Furthermore, the binding molecules hereof can be
used alone or in a mixture comprising at least one binding molecule
(or variant or fragment thereof) hereof. In other words, the
binding molecules can be used in combination, e.g., as a
pharmaceutical composition comprising two or more binding molecules
hereof, variants or fragments thereof. For example, binding
molecules having different, but complementary activities can be
combined in a single therapy to achieve a desired prophylactic,
therapeutic or diagnostic effect, but alternatively, binding
molecules having identical activities can also be combined in a
single therapy to achieve a desired prophylactic, therapeutic or
diagnostic effect. Optionally, the mixture further comprises at
least one other therapeutic agent. In certain embodiments, the
therapeutic agent such as, e.g., M2 inhibitors (e.g., amantidine,
rimantadine) and/or neuraminidase inhibitors (e.g., zanamivir,
oseltamivir) is useful in the prophylaxis and/or treatment of an
influenza virus H3N2 infection
[0079] Typically, binding molecules described herein can bind to
their binding partners, i.e., influenza virus H3N2 or fragments
thereof, with an affinity constant (K.sub.d-value) that is lower
than 0.2.times.10.sup.-4 M, 1.0.times.10.sup.-5 M,
1.0.times.10.sup.-6 M, 1.0.times.10.sup.-7 M, preferably lower than
1.0.times.10.sup.-8 M, more preferably lower than
1.0.times.10.sup.-9 M, more preferably lower than
1.0.times.10.sup.-1.degree. M, even more preferably lower than
1.0.times.10.sup.-11 M, and, in particular, lower than
1.0.times.10.sup.-12 M. The affinity constants can vary for
antibody isotypes. For example, affinity binding for an IgM isotype
refers to a binding affinity of at least about 1.0.times.10.sup.-7
M. Affinity constants can, for instance, be measured using surface
plasmon resonance, for example, using the BIACORE system (Pharmacia
Biosensor AB, Uppsala, Sweden).
[0080] Typically, the binding molecules hereof have a neutralizing
activity of 10 .mu.g/ml or less, preferably 5 .mu.g/ml or less,
more preferably 2 .mu.g/ml or less, even more preferably 1 .mu.g/ml
or less, as determined in an in vitro virus neutralization assay
(VNA) as described in Example 6.
[0081] The binding molecules hereof may bind to influenza virus
H3N2 or a fragment thereof in soluble form, such as, for instance,
in a sample or in suspension or may bind to influenza virus H3N2 or
a fragment thereof bound or attached to a carrier or substrate,
e.g., microtiter plates, membranes and beads, etc. Carriers or
substrates may be made of glass, plastic (e.g., polystyrene),
polysaccharides, nylon, nitrocellulose, or Teflon, etc. The surface
of such supports may be solid or porous and of any convenient
shape. Furthermore, the binding molecules may bind to influenza
virus H3N2 in purified/isolated or non-purified/non-isolated
form.
[0082] The binding molecules hereof exhibit neutralizing activity.
Neutralizing activity can, for instance, be measured as described
herein. Alternative assays measuring neutralizing activity are
described in, for instance, WHO Manual on Animal Influenza
Diagnosis and Surveillance, Geneva: World Health Organisation,
2005, version 2002.5.
[0083] Described is an isolated human binding molecule that is able
to recognize and bind to an epitope in the influenza hemagglutinin
protein (HA), characterized in that the binding molecule has
neutralizing activity against an influenza virus A, comprising HA
of the H3 subtype. An example of an influenza subtype that contains
HA of the H3 subtype is H3N2. Particularly preferred are binding
molecules that neutralize the H3N2 influenza subtype. In certain
embodiments, the binding molecules neutralize at least one or more
of the recent H3N2 strains. In certain embodiments, the binding
molecules thus at least bind to and neutralize one or more H3N2
strains selected from the group consisting of A/Wisconsin/67/2005,
A/Hiroshima/52/2005, A/Panama/2007/99, and A/Johannesburg/33/94. In
another embodiment, the binding molecules also bind to and
neutralize the H3N2 strain A/Hong Kong/1/68. Most preferably, the
binding molecules bind to and have neutralizing activity against
all influenza H3N2 strains from the years between 1968 and 2005,
preferably all known strains of the influenza virus subtype.
[0084] In another embodiment, the binding molecules hereof also
have neutralizing activity against influenza viruses of other
influenza virus A subtypes, preferably at least influenza viruses
comprising HA of the H7 subtype, such as the strain
A/Mallard/Netherlands/12/2000, and/or H10 subtype, such as the
strain A/chick/Germany/N/49. It thus has been shown that some of
the binding molecules described herein cross-neutralize these
influenza subtypes. Also provided are binding molecules that bind
to an epitope in the hemagglutinin protein that is shared and
conserved between influenza subtypes and, therefore, relates to
binding molecules that cross-react between H3-, H7-, and/or H10
influenza-based subtypes, and other influenza subtypes that contain
the HA protein with these particular epitopes, preferably all
influenza viruses of phylogenetic group 2. The cross-neutralizing
binding molecules preferably bind to and neutralize several strains
of the H3-, H7, and/or H10-subtypes. In certain embodiments, these
cross-neutralizing binding molecules bind to and neutralize at
least one or more of the recent H3N2 strains selected from the
group consisting of A/Wisconsin/67/2005, A/Hiroshima/52/2005,
A/Johannesburg/33/94, and A/Panama/2007/99. In another embodiment,
the binding molecules also bind to and neutralize the H3N2 strain
A/Hong Kong/1/68. Most preferably, the binding molecules bind to
and have neutralizing activity against all influenza H3N2 strains
from the years between 1968 and 2005, preferably all known and, In
certain embodiments, also future H3N2 strains. In a further
embodiment, the binding molecules neutralize substantially all
isolates of the other influenza virus subtypes.
[0085] In certain embodiments, the binding molecules bind to and
neutralize all influenza virus subtypes of phylogenetic group
2.
[0086] The skilled person, based on what has been disclosed herein,
can determine whether an antibody indeed cross-reacts with HA
proteins from different subtypes and also determine whether they
are able to neutralize influenza viruses of different subtypes in
vivo.
[0087] Influenza viruses infect cells by binding to sialic acid
residues on the cell surface of target cells, and following
transfer into endosomes, by fusing their membranes with the
endosomal membranes and releasing the genome-transcriptase complex
into the cell. Both receptor binding and membrane fusion processes
are mediated by the HA glycoprotein. The HA of influenza virus A
comprises two structurally distinct regions, i.e., a globular head
region, which contains a receptor binding site that is responsible
for virus attachment to the target cell, and is involved in the
hemagglutination activity of HA, and a stem region, containing a
fusion peptide, which is necessary for membrane fusion between the
viral envelope and the endosomal membrane of the cell. The HA
protein is a trimer in which each monomer consists of two
disulphide-linked glycopolypeptides, HA1 and HA2, that are produced
during infection by proteolytic cleavage of a precursor (HA0).
Cleavage is necessary for virus infectivity since it is required to
prime the HA for membrane fusion to allow conformational change.
Activation of the primed molecule occurs at low pH in endosomes,
between pH5 and pH6, and requires extensive changes in HA
structure. The three-dimensional structure of the pre-fusion
uncleaved (I), pre-fusion cleaved (II) and post-fusion HA (III)
conformations are schematically shown in FIG. 4. Each of the stages
in the priming and activation of HA for its participation in the
membrane fusion process presents a different target for inhibition,
e.g., by monoclonal antibodies.
[0088] In certain embodiments, the binding molecules are at least
able to prevent the cleavage of the HA precursor molecule HA0 in an
in vitro assay, e.g., an assay as described below in the Examples.
As explained above, cleavage of the HA precursor molecule HA0 into
HA1 and HA2 by host proteases is required to activate virus
infectivity. The prevention of cleavage of the HA precursor
molecule HA0 by the binding molecules hereof thus may prevent
infection by the influenza virus.
[0089] In certain embodiments, the binding molecules bind to an
epitope comprising the amino acid at position 19, 25, 27, 33 and/or
34 of the HA2 polypeptide of the H3 HA protein. In certain
embodiments, the binding molecules bind to the epitope on HA2, when
the amino acid on position 19 is aspartic acid (D), the amino acid
on position 25 is glutamine (Q), the amino acid on position 27 is
glycine (G), the amino acid at position 33 is glycine (G) and/or
the amino acid at position 34 is glutamine. In certain embodiments,
the binding molecules do not bind to the epitope on HA2 when one or
more of the amino acids have changed. The numbering of the amino
acids is based on the hemagglutinin sequence from Uniprot database
number Q91MA7 (SEQ ID NO:193 of the incorporated herein SEQUENCE
LISTING). Q91MA7 gives the full-length sequence of immature HA from
A/Hong Kong/1/1968. The HA2 sequence starts at G346 of the
uncleaved HA immature protein. In the numbering above, the G346 is
G1 in HA2 sequence.
[0090] Preferred is a binding molecule that is selected from the
group consisting of: [0091] a) a binding molecule comprising a
heavy chain CDR1 region of SEQ ID NO:81, a heavy chain CDR2 region
of SEQ ID NO:82, and a heavy chain CDR3 region of SEQ ID NO:83,
[0092] b) a binding molecule comprising a heavy chain CDR1 region
of SEQ ID NO:87, a heavy chain CDR2 region of SEQ ID NO:88, and a
heavy chain CDR3 region of SEQ ID NO:89, [0093] c) a binding
molecule comprising a heavy chain CDR1 region of SEQ ID NO:103, a
heavy chain CDR2 region of SEQ ID NO:104, and a heavy chain CDR3
region of SEQ ID NO:105, [0094] d) a binding molecule comprising a
heavy chain CDR1 region of SEQ ID NO:109, a heavy chain CDR2 region
of SEQ ID NO:110, and a heavy chain CDR3 region of SEQ ID NO:111,
[0095] e) a binding molecule comprising a heavy chain CDR1 region
of SEQ ID NO:115, a heavy chain CDR2 region of SEQ ID NO:116, and a
heavy chain CDR3 region of SEQ ID NO:117, [0096] f) a binding
molecule comprising a heavy chain CDR1 region of SEQ ID NO:121, a
heavy chain CDR2 region of SEQ ID NO:122, and a heavy chain CDR3
region of SEQ ID NO:123, [0097] g) a binding molecule comprising a
heavy chain CDR1 region of SEQ ID NO:126, a heavy chain CDR2 region
of SEQ ID NO:127, and a heavy chain CDR3 region of SEQ ID NO:128,
[0098] h) a binding molecule comprising a heavy chain CDR1 region
of SEQ ID NO:132, a heavy chain CDR2 region of SEQ ID NO:133, and a
heavy chain CDR3 region of SEQ ID NO:134, [0099] i) a binding
molecule comprising a heavy chain CDR1 region of SEQ ID NO:138, a
heavy chain CDR2 region of SEQ ID NO:139, and a heavy chain CDR3
region of SEQ ID NO:140, [0100] j) a binding molecule comprising a
heavy chain CDR1 region of SEQ ID NO:144, a heavy chain CDR2 region
of SEQ ID NO:145, and a heavy chain CDR3 region of SEQ ID NO:146,
[0101] k) a binding molecule comprising a heavy chain CDR1 region
of SEQ ID NO:150, a heavy chain CDR2 region of SEQ ID NO:151, and a
heavy chain CDR3 region of SEQ ID NO:152, [0102] l) a binding
molecule comprising a heavy chain CDR1 region of SEQ ID NO:156, a
heavy chain CDR2 region of SEQ ID NO:157, and a heavy chain CDR3
region of SEQ ID NO:158, [0103] m) a binding molecule comprising a
heavy chain CDR1 region of SEQ ID NO:162, a heavy chain CDR2 region
of SEQ ID NO:163, and a heavy chain CDR3 region of SEQ ID NO:164,
[0104] n) a binding molecule comprising a heavy chain CDR1 region
of SEQ ID NO:168, a heavy chain CDR2 region of SEQ ID NO:169, and a
heavy chain CDR3 region of SEQ ID NO:170, [0105] o) a binding
molecule comprising a heavy chain CDR1 region of SEQ ID NO:173, a
heavy chain CDR2 region of SEQ ID NO:174, and a heavy chain CDR3
region of SEQ ID NO:175, and [0106] p) a binding molecule
comprising a heavy chain CDR1 region of SEQ ID NO:179, a heavy
chain CDR2 region of SEQ ID NO:180, and a heavy chain CDR3 region
of SEQ ID NO:181.
[0107] In a preferred embodiment, the binding molecule is for a use
as a medicament and preferably for the diagnostic, therapeutic
and/or prophylactic treatment of influenza infection. In certain
embodiments, the influenza virus that causes the influenza
infection and that can be treated by the binding molecules hereof,
is influenza virus subtype H3N2. Also described is a pharmaceutical
composition comprising a binding molecule hereof, and a
pharmaceutically acceptable excipient.
[0108] In yet another embodiment, also described is the use of a
binding molecule hereof in the preparation of a medicament for the
diagnosis, prophylaxis, and/or treatment of an influenza virus
infection. Such infections can occur in small populations, but can
also spread around the world in seasonal epidemics or, worse, in
global pandemics where millions of individuals are at risk.
Provided are binding molecules that can neutralize the infection of
influenza strains that cause such seasonal epidemics, as well as
potential pandemics. Importantly, protection and treatment can be
envisioned now with the binding molecules hereof in relation to
various influenza subtypes as it has been disclosed that the
binding molecules hereof are able to cross-neutralizing various
influenza subtypes of phylogenetic group 2, encompassing subtypes
H3, H7 and H10.
[0109] In a preferred embodiment, the human binding molecules
hereof are characterized in that the human binding molecules are
selected from the group consisting of: [0110] a) a binding molecule
comprising a heavy chain CDR1 region of SEQ ID NO:81, a heavy chain
CDR2 region of SEQ ID NO:82, and a heavy chain CDR3 region of SEQ
ID NO:83, a light chain CDR1 region comprising the peptide of SEQ
ID NO:84, a light chain CDR2 region comprising the peptide of SEQ
ID NO:85, and a light chain CDR3 region comprising the peptide of
SEQ ID NO:86, [0111] b) a binding molecule comprising a heavy chain
CDR1 region of SEQ ID NO:87, a heavy chain CDR2 region of SEQ ID
NO:88, and a heavy chain CDR3 region of SEQ ID NO:89, a light chain
CDR1 region comprising the peptide of SEQ ID NO:90, a light chain
CDR2 region comprising the peptide of SEQ ID NO:91, and a light
chain CDR3 region comprising the peptide of SEQ ID NO:92, [0112] c)
a binding molecule comprising a heavy chain CDR1 region of SEQ ID
NO:87, a heavy chain CDR2 region of SEQ ID NO:88, and a heavy chain
CDR3 region of SEQ ID NO:89, a light chain CDR1 region comprising
the peptide of SEQ ID NO:93, a light chain CDR2 region comprising
the peptide of SEQ ID NO:94, and a light chain CDR3 region
comprising the peptide of SEQ ID NO:95, [0113] d) a binding
molecule comprising a heavy chain CDR1 region of SEQ ID NO:87, a
heavy chain CDR2 region of SEQ ID NO:88, and a heavy chain CDR3
region of SEQ ID NO:89, a light chain CDR1 region comprising the
peptide of SEQ ID NO:96, a light chain CDR2 region comprising the
peptide of SEQ ID NO:97, and a light chain CDR3 region comprising
the peptide of SEQ ID NO:98, [0114] e) a binding molecule
comprising a heavy chain CDR1 region of SEQ ID NO:87, a heavy chain
CDR2 region of SEQ ID NO:88, and a heavy chain CDR3 region of SEQ
ID NO:89, a light chain CDR1 region comprising the peptide of SEQ
ID NO:99, a light chain CDR2 region comprising the peptide of SEQ
ID NO:100, and a light chain CDR3 region comprising the peptide of
SEQ ID NO:101, [0115] f) a binding molecule comprising a heavy
chain CDR1 region of SEQ ID NO:87, a heavy chain CDR2 region of SEQ
ID NO:88, and a heavy chain CDR3 region of SEQ ID NO:89, a light
chain CDR1 region comprising the peptide of SEQ ID NO:102, a light
chain CDR2 region comprising the peptide of SEQ ID NO:85, and a
light chain CDR3 region comprising the peptide of SEQ ID NO:86,
[0116] g) a binding molecule comprising a heavy chain CDR1 region
of SEQ ID NO:103, a heavy chain CDR2 region of SEQ ID NO:104, and a
heavy chain CDR3 region of SEQ ID NO:105, a light chain CDR1 region
comprising the peptide of SEQ ID NO:106, a light chain CDR2 region
comprising the peptide of SEQ ID NO:107, and a light chain CDR3
region comprising the peptide of SEQ ID NO:108, [0117] h) a binding
molecule comprising a heavy chain CDR1 region of SEQ ID NO:109, a
heavy chain CDR2 region of SEQ ID NO:110, and a heavy chain CDR3
region of SEQ ID NO:111, a light chain CDR1 region comprising the
peptide of SEQ ID NO:112, a light chain CDR2 region comprising the
peptide of SEQ ID NO:113, and a light chain CDR3 region comprising
the peptide of SEQ ID NO:114, [0118] i) a binding molecule
comprising a heavy chain CDR1 region of SEQ ID NO:115, a heavy
chain CDR2 region of SEQ ID NO:116, and a heavy chain CDR3 region
of SEQ ID NO:117, a light chain CDR1 region comprising the peptide
of SEQ ID NO:118, a light chain CDR2 region comprising the peptide
of SEQ ID NO:119, and a light chain CDR3 region comprising the
peptide of SEQ ID NO:120, [0119] j) a binding molecule comprising a
heavy chain CDR1 region of SEQ ID NO:121, a heavy chain CDR2 region
of SEQ ID NO:122, and a heavy chain CDR3 region of SEQ ID NO:123, a
light chain CDR1 region comprising the peptide of SEQ ID NO:124, a
light chain CDR2 region comprising the peptide of SEQ ID NO:119,
and a light chain CDR3 region comprising the peptide of SEQ ID
NO:125, [0120] k) a binding molecule comprising a heavy chain CDR1
region of SEQ ID NO:126, a heavy chain CDR2 region of SEQ ID
NO:127, and a heavy chain CDR3 region of SEQ ID NO:128, a light
chain CDR1 region comprising the peptide of SEQ ID NO:129, a light
chain CDR2 region comprising the peptide of SEQ ID NO:130, and a
light chain CDR3 region comprising the peptide of SEQ ID NO:131,
[0121] l) a binding molecule comprising a heavy chain CDR1 region
of SEQ ID NO:132, a heavy chain CDR2 region of SEQ ID NO:133, and a
heavy chain CDR3 region of SEQ ID NO:134, a light chain CDR1 region
comprising the peptide of SEQ ID NO:135, a light chain CDR2 region
comprising the peptide of SEQ ID NO:136, and a light chain CDR3
region comprising the peptide of SEQ ID NO:137, [0122] m) a binding
molecule comprising a heavy chain CDR1 region of SEQ ID NO:138, a
heavy chain CDR2 region of SEQ ID NO:139, and a heavy chain CDR3
region of SEQ ID NO:140, a light chain CDR1 region comprising the
peptide of SEQ ID NO:141, a light chain CDR2 region comprising the
peptide of SEQ ID NO:142, and a light chain CDR3 region comprising
the peptide of SEQ ID NO:143, [0123] n) a binding molecule
comprising a heavy chain CDR1 region of SEQ ID NO:144, a heavy
chain CDR2 region of SEQ ID NO:145, and a heavy chain CDR3 region
of SEQ ID NO:146, a light chain CDR1 region comprising the peptide
of SEQ ID NO:147, a light chain CDR2 region comprising the peptide
of SEQ ID NO:148, and a light chain CDR3 region comprising the
peptide of SEQ ID NO:149, [0124] o) a binding molecule comprising a
heavy chain CDR1 region of SEQ ID NO:150, a heavy chain CDR2 region
of SEQ ID NO:151, and a heavy chain CDR3 region of SEQ ID NO:152, a
light chain CDR1 region comprising the peptide of SEQ ID NO:153, a
light chain CDR2 region comprising the peptide of SEQ ID NO:154,
and a light chain CDR3 region comprising the peptide of SEQ ID
NO:155, [0125] p) a binding molecule comprising a heavy chain CDR1
region of SEQ ID NO:156, a heavy chain CDR2 region of SEQ ID
NO:157, and a heavy chain CDR3 region of SEQ ID NO:158, a light
chain CDR1 region comprising the peptide of SEQ ID NO:159, a light
chain CDR2 region comprising the peptide of SEQ ID NO:160, and a
light chain CDR3 region comprising the peptide of SEQ ID NO:161,
[0126] q) a binding molecule comprising a heavy chain CDR1 region
of SEQ ID NO:162, a heavy chain CDR2 region of SEQ ID NO:163, and a
heavy chain CDR3 region of SEQ ID NO:164, a light chain CDR1 region
comprising the peptide of SEQ ID NO:165, a light chain CDR2 region
comprising the peptide of SEQ ID NO:166, and a light chain CDR3
region comprising the peptide of SEQ ID NO:167, [0127] r) a binding
molecule comprising a heavy chain CDR1 region of SEQ ID NO:168, a
heavy chain CDR2 region of SEQ ID NO:169, and a heavy chain CDR3
region of SEQ ID NO:170, a light chain CDR1 region comprising the
peptide of SEQ ID NO:171, a light chain CDR2 region comprising the
peptide of SEQ ID NO:172, and a light chain CDR3 region comprising
the peptide of SEQ ID NO:137, [0128] s) a binding molecule
comprising a heavy chain CDR1 region of SEQ ID NO:173, a heavy
chain CDR2 region of SEQ ID NO:174, and a heavy chain CDR3 region
of SEQ ID NO:175, a light chain CDR1 region comprising the peptide
of SEQ ID NO:176, a light chain CDR2 region comprising the peptide
of SEQ ID NO:177, and a light chain CDR3 region comprising the
peptide of SEQ ID NO:178, and [0129] t) a binding molecule
comprising a heavy chain CDR1 region of SEQ ID NO:179, a heavy
chain CDR2 region of SEQ ID NO:180, and a heavy chain CDR3 region
of SEQ ID NO:181, a light chain CDR1 region comprising the peptide
of SEQ ID NO:182, a light chain CDR2 region comprising the peptide
of SEQ ID NO:183, and a light chain CDR3 region comprising the
peptide of SEQ ID NO:184.
[0130] In a specific embodiment, the binding molecules are selected
from the group consisting of a binding molecule comprising a heavy
chain CDR1 region comprising the peptide of SEQ ID NO:81, a heavy
chain CDR2 region comprising the peptide of SEQ ID NO:82 and a
heavy chain CDR3 region comprising the peptide of SEQ ID NO:83; a
binding molecule comprising a heavy chain CDR1 region comprising
the peptide of SEQ ID NO:109, a heavy chain CDR2 region comprising
the peptide of SEQ ID NO:110 and a heavy chain CDR3 region
comprising the peptide of SEQ ID NO:111; a binding molecule
comprising a heavy chain CDR1 region comprising the peptide of SEQ
ID NO:138, a heavy chain CDR2 region comprising the peptide of SEQ
ID NO:139 and a heavy chain CDR3 region comprising the peptide of
SEQ ID NO:140; a binding molecule comprising a heavy chain CDR1
region comprising the peptide of SEQ ID NO:144, a heavy chain CDR2
region comprising the peptide of SEQ ID NO:145 and a heavy chain
CDR3 region comprising the peptide of SEQ ID NO:146; and a binding
molecule comprising a heavy chain CDR1 region comprising the
peptide of SEQ ID NO:173, a heavy chain CDR2 region comprising the
peptide of SEQ ID NO:174 and a heavy chain CDR3 region comprising
the peptide of SEQ ID NO:175.
[0131] The CDR regions of the binding molecules are shown in Table
1. CDR regions are according to Kabat et al. (1991) as described in
Sequences of Proteins of Immunological Interest. The binding
molecules may comprise one, two, three, four, five or all six CDR
regions as disclosed herein. In certain embodiments, a binding
molecule comprises at least two of the CDRs disclosed herein.
[0132] In yet another embodiment, the binding molecules comprise a
heavy chain variable region comprising an amino acid sequence
selected from the group consisting of SEQ ID NO:2, SEQ ID NO:6, SEQ
ID NO:10, SEQ ID NO:14, SEQ ID NO:18, SEQ ID NO:22, SEQ ID NO:26,
SEQ ID NO:30, SEQ ID NO:34, SEQ ID NO:38, SEQ ID NO:42, SEQ ID
NO:46, SEQ ID NO:50, SEQ ID NO:54, SEQ ID NO:58, SEQ ID NO:62, SEQ
ID NO:66, SEQ ID NO:70, SEQ ID NO:74, and SEQ ID NO:78. In a
further embodiment, the binding molecules comprise a light chain
variable region comprising an amino acid sequence selected from the
group consisting of SEQ ID NO:4, SEQ ID NO:8, SEQ ID NO:12, SEQ ID
NO:16, SEQ ID NO:20, SEQ ID NO:24, SEQ ID NO:28, SEQ ID NO:32, SEQ
ID NO:36, SEQ ID NO:40, SEQ ID NO:44, SEQ ID NO:48, SEQ ID NO:52,
SEQ ID NO:56, SEQ ID NO:60, SEQ ID NO:64, SEQ ID NO:68, SEQ ID
NO:72, SEQ ID NO:76, and SEQ ID NO:80.
[0133] Another aspect includes functional variants of the binding
molecules as defined herein. Molecules are considered to be
functional variants of a binding molecule hereof, if the variants
are capable of competing for specifically binding to influenza
virus H3N2 or a fragment thereof with the "parental" or "reference"
binding molecules; in other words, when the functional variants are
still capable of binding to the same or overlapping epitope of the
influenza virus H3N2 or a fragment thereof. For the sake of this
application, "parental" and "reference" will be used as synonyms
meaning that the information of the reference or parental molecule,
or the physical molecule itself may form the basis for the
variation. In certain embodiments, the functional variants are
capable of competing for specifically binding to at least two (or
more) different influenza virus H3N2 strains or fragments thereof
that are specifically bound by the reference binding molecules.
[0134] Furthermore, molecules are considered to be functional
variants of a binding molecule hereof, if they have neutralizing
activity against influenza virus H3N2, preferably against the at
least two (or more) influenza virus H3N2 strains against which the
parental binding molecule exhibits neutralizing activity.
Functional variants include, but are not limited to, derivatives
that are substantially similar in primary structural sequence,
including those that have modifications in the Fc receptor or other
regions involved with effector functions, and/or which contain,
e.g., in vitro or in vivo modifications, chemical and/or
biochemical, that are not found in the parental binding molecule.
Such modifications include inter alia acetylation, acylation,
covalent attachment of a nucleotide or nucleotide derivative,
covalent attachment of a lipid or lipid derivative, cross-linking,
disulfide bond formation, glycosylation, hydroxylation,
methylation, oxidation, pegylation, proteolytic processing,
phosphorylation, and the like.
[0135] Alternatively, functional variants can be binding molecules
as defined herein comprising an amino acid sequence containing
substitutions, insertions, deletions or combinations thereof of one
or more amino acids compared to the amino acid sequences of the
parental binding molecules. Furthermore, functional variants can
comprise truncations of the amino acid sequence at either or both
the amino or carboxyl termini. Functional variants may have the
same or different, either higher or lower, binding affinities
compared to the parental binding molecule, but are still capable of
binding to influenza virus H3N2 or a fragment thereof. For
instance, functional variants hereof may have increased or
decreased binding affinities for influenza virus H3N2 or a fragment
thereof compared to the parental binding molecules. In certain
embodiments, the amino acid sequences of the variable regions,
including, but not limited to, framework regions, hypervariable
regions, in particular, the CDR3 regions, are modified. Generally,
the light chain and the heavy chain variable regions comprise three
hypervariable regions, comprising three CDRs, and more conserved
regions, the so-called framework regions (FRs). The hypervariable
regions comprise amino acid residues from CDRs and amino acid
residues from hypervariable loops. Functional variants intended to
fall within the scope of the disclosure have at least about 50% to
about 99%, preferably at least about 60% to about 99%, more
preferably at least about 70% to about 99%, even more preferably at
least about 80% to about 99%, most preferably at least about 90% to
about 99%, in particular, at least about 95% to about 99%, and, in
particular, at least about 97% to about 99% amino acid sequence
homology with the parental binding molecules as defined herein.
Computer algorithms such as inter alia Gap or Bestfit known to a
person skilled in the art can be used to optimally align amino acid
sequences to be compared and to define similar or identical amino
acid residues. Functional variants can be obtained by altering the
parental binding molecules or parts thereof by general molecular
biology methods known in the art including, but not limited to,
error-prone PCR, oligonucleotide-directed mutagenesis,
site-directed mutagenesis and heavy and/or light chain shuffling.
In certain embodiments, the functional variants hereof have
neutralizing activity against influenza virus H3N2. The
neutralizing activity may either be identical, or be higher or
lower compared to the parental binding molecules. Henceforth, when
the term (human) binding molecule is used, this also encompasses
functional variants of the (human) binding molecule.
[0136] In yet a further aspect, described are immunoconjugates,
i.e., molecules comprising at least one binding molecule as defined
herein and further comprising at least one tag, such as inter alia
a detectable moiety/agent. Also contemplated are mixtures of
immunoconjugates hereof or mixtures of at least one immunoconjugate
hereof and another molecule, such as a therapeutic agent or another
binding molecule or immunoconjugate. In a further embodiment, the
immunoconjugates may comprise more than one tag. These tags can be
the same or distinct from each other and can be joined/conjugated
non-covalently to the binding molecules. The tag(s) can also be
joined/conjugated directly to the human binding molecules through
covalent bonding. Alternatively, the tag(s) can be
joined/conjugated to the binding molecules by means of one or more
linking compounds. Techniques for conjugating tags to binding
molecules are well known to the skilled artisan.
[0137] The tags of the immunoconjugates hereof may be therapeutic
agents, but they can also be detectable moieties/agents. Tags
suitable in therapy and/or prevention may be toxins or functional
parts thereof, antibiotics, enzymes, or other binding molecules
that enhance phagocytosis or immune stimulation. Immunoconjugates
comprising a detectable agent can be used diagnostically to, for
example, assess if a subject has been infected with an influenza
virus H3N2 strain or monitor the development or progression of an
influenza virus H3N2 infection as part of a clinical testing
procedure to, e.g., determine the efficacy of a given treatment
regimen. However, they may also be used for other detection and/or
analytical and/or diagnostic purposes. Detectable moieties/agents
include, but are not limited to, enzymes, prosthetic groups,
fluorescent materials, luminescent materials, bioluminescent
materials, radioactive materials, positron-emitting metals, and
non-radioactive paramagnetic metal ions. The tags used to label the
binding molecules for detection and/or analytical and/or diagnostic
purposes depend on the specific detection/analysis/diagnosis
techniques and/or methods used such as inter alia
immunohistochemical staining of (tissue) samples, flow cytometric
detection, scanning laser cytometric detection, fluorescent
immunoassays, enzyme-linked immunosorbent assays (ELISAs),
radioimmunoassays (RIAs), bioassays (e.g., phagocytosis assays),
Western blotting applications, etc. Suitable labels for the
detection/analysis/diagnosis techniques and/or methods known in the
art are well within the reach of the skilled artisan.
[0138] The human binding molecules or immunoconjugates hereof can
also be attached to solid supports, which are particularly useful
for in vitro immunoassays or purification of influenza virus H3N2
or a fragment thereof. Such solid supports might be porous or
nonporous, planar or non-planar. The binding molecules hereof can
be fused to marker sequences, such as a peptide to facilitate
purification. Examples include, but are not limited to, the
hexa-histidine tag, the hemagglutinin (HA) tag, the myc tag or the
flag tag. Alternatively, an antibody can be conjugated to a second
antibody to form an antibody heteroconjugate. In another aspect,
the binding molecules hereof may be conjugated/attached to one or
more antigens. In certain embodiments, these antigens are antigens
that are recognized by the immune system of a subject to which the
binding molecule-antigen conjugate is administered. The antigens
may be identical, but may also differ from each other. Conjugation
methods for attaching the antigens and binding molecules are well
known in the art and include, but are not limited to, the use of
cross-linking agents. The binding molecules hereof will bind to
influenza virus H3N2 and the antigens attached to the binding
molecules will initiate a powerful T-cell attack on the conjugate,
which will eventually lead to the destruction of the influenza
virus H3N2.
[0139] Next to producing immunoconjugates chemically by
conjugating, directly or indirectly, via, for instance, a linker,
the immunoconjugates can be produced as fusion proteins comprising
the binding molecules hereof and a suitable tag. Fusion proteins
can be produced by methods known in the art such as, e.g.,
recombinantly by constructing nucleic acid molecules comprising
nucleotide sequences encoding the binding molecules in frame with
nucleotide sequences encoding the suitable tag(s) and then
expressing the nucleic acid molecules.
[0140] Also provided is a polynucleotide encoding at least a
binding molecule, functional variant or immunoconjugate hereof.
Such nucleic acid molecules can be used as intermediates for
cloning purposes, e.g., in the process of affinity maturation as
described above. In a preferred embodiment, the nucleic acid
molecules are isolated or purified.
[0141] The skilled person will appreciate that functional variants
of these nucleic acid molecules are also intended to be a part of
the disclosure. Functional variants are polynucletides that can be
directly translated, using the standard genetic code, to provide an
amino acid sequence identical to that translated from the parental
nucleic acid molecules.
[0142] In certain embodiments, the polynucleotides encode binding
molecules comprising the CDR regions as described above. In a
further embodiment, polynucleotides encode binding molecules
comprising two, three, four, five or even all six CDR regions of
the binding molecules hereof.
[0143] In another embodiment, the polynucleotides encode binding
molecules comprising a heavy chain comprising the variable heavy
chain sequences as described above. In another embodiment, the
polynucleotides encode binding molecules comprising a light chain
comprising the variable light chain sequences as described above.
The nucleotide sequences and the amino acid sequences of the heavy
and light chain variable regions of the binding molecules hereof
are given below.
[0144] Also provided are vectors, i.e., nucleic acid constructs,
comprising one or more nucleic acid molecules hereof. Vectors can
be derived from plasmids such as inter alia F, R1, RP1, Col,
pBR322, TOL, Ti, etc.; cosmids; phages such as lambda, lambdoid,
M13, Mu, P1, P22, Q13, T-even, T-odd, T2, T4, T7, etc.; and plant
viruses. Vectors can be used for cloning and/or for expression of
the binding molecules hereof and might even be used for gene
therapy purposes. Vectors comprising one or more nucleic acid
molecules hereof operably linked to one or more
expression-regulating nucleic acid molecules are also covered by
the disclosure. The choice of the vector is dependent on the
recombinant procedures followed and the host used. Introduction of
vectors in host cells can be effected by inter alia calcium
phosphate transfection, virus infection, DEAE-dextran-mediated
transfection, lipofectamin transfection or electroporation. Vectors
may be autonomously replicating or may replicate together with the
chromosome into which they have been integrated. In certain
embodiments, the vectors contain one or more selection markers. The
choice of the markers may depend on the host cells of choice,
although this is not critical to the disclosure as is well known to
persons skilled in the art. They include, but are not limited to,
kanamycin, neomycin, puromycin, hygromycin, zeocin, thymidine
kinase gene from Herpes simplex virus (HSV-TK), dihydrofolate
reductase gene from mouse (dhfr) Vectors comprising one or more
nucleic acid molecules encoding the human binding molecules as
described above operably linked to one or more nucleic acid
molecules encoding proteins or peptides that can be used to isolate
the human binding molecules are also covered. These proteins or
peptides include, but are not limited to,
glutathione-S-transferase, maltose binding protein, metal-binding
polyhistidine, green fluorescent protein, luciferase and
beta-galactosidase.
[0145] Hosts containing one or more copies of the vectors mentioned
above are an additional subject hereof. The hosts may be host
cells. Host cells include, but are not limited to, cells of
mammalian, plant, insect, fungal or bacterial origin. Bacterial
cells include, but are not limited to, cells from Gram-positive
bacteria or Gram-negative bacteria such as several species of the
genera Escherichia, such as E. coli, and Pseudomonas. In the group
of fungal cells, preferably, yeast cells are used. Expression in
yeast can be achieved by using yeast strains such as inter alia
Pichia pastoris, Saccharomyces cerevisiae and Hansenula polymorpha.
Furthermore, insect cells such as cells from Drosophila and Sf9 can
be used as host cells. Besides that, the host cells can be plant
cells such as inter alia cells from crop plants such as forestry
plants, or cells from plants providing food and raw materials such
as cereal plants, or medicinal plants, or cells from ornamentals,
or cells from flower bulb crops. Transformed (transgenic) plants or
plant cells are produced by known methods, for example,
Agrobacterium-mediated gene transfer, transformation of leaf discs,
protoplast transformation by polyethylene glycol-induced DNA
transfer, electroporation, sonication, microinjection or bolistic
gene transfer. Additionally, a suitable expression system can be a
baculovirus system. Expression systems using mammalian cells such
as Chinese Hamster Ovary (CHO) cells, COS cells, BHK cells, NSO
cells or Bowes melanoma cells are preferred herein. Mammalian cells
provide expressed proteins with post-translational modifications
that are most similar to natural molecules of mammalian origin.
Since the disclosure deals with molecules that may have to be
administered to humans, a completely human expression system would
be particularly preferred. Therefore, even more preferably, the
host cells are human cells. Examples of human cells are inter alia
HeLa, 911, AT1080, A549, 293 and HEK293T cells. In preferred
embodiments, the human producer cells comprise at least a
functional part of a nucleic acid sequence encoding an adenovirus
E1 region in expressible format. In even more preferred
embodiments, the host cells are derived from a human retina and
immortalized with nucleic acids comprising adenoviral E1 sequences,
such as 911 cells or the cell line deposited at the European
Collection of Cell Cultures (ECACC), CAMR, Salisbury, Wiltshire SP4
OJG, Great Britain on 29 February 1996 under number 96022940 and
marketed under the trademark PER.C6.RTM. (PER.C6.RTM. is a
registered trademark of Crucell Holland B.V.) For the purposes of
this application "PER.C6.RTM. cells" refers to cells deposited
under number 96022940 or ancestors, passages upstream or
downstream, as well as descendants from ancestors of deposited
cells, as well as derivatives of any of the foregoing. Production
of recombinant proteins in host cells can be performed according to
methods well known in the art. The use of the cells marketed under
the trademark PER.C6.RTM. as a production platform for proteins of
interest has been described in WO 00/63403, the disclosure of which
is incorporated herein by reference in its entirety.
[0146] Binding molecules can be prepared by various means. A method
of producing a binding molecule hereof is an additional part of the
disclosure. The method comprises the steps of a) culturing a host
hereof under conditions conducive to the expression of the binding
molecule, and b) optionally, recovering the expressed binding
molecule. The expressed binding molecules can be recovered from the
cell-free extract, but preferably they are recovered from the
culture medium. The above method of producing can also be used to
make functional variants of the binding molecules and/or
immunoconjugates hereof. Methods to recover proteins, such as
binding molecules, from cell-free extracts or culture medium are
well known to the man skilled in the art. Binding molecules,
functional variants and/or immunoconjugates as obtainable by the
above-described method are also a part hereof.
[0147] Alternatively, next to the expression in hosts, such as host
cells, the binding molecules and immunoconjugates hereof can be
produced synthetically by conventional peptide synthesizers or in
cell-free translation systems using RNA nucleic acid derived from
DNA molecules hereof. Binding molecules and immunoconjugates as
obtainable by the above-described synthetic production methods or
cell-free translation systems are also a part hereof.
[0148] In certain embodiments, binding molecules can also be
produced in transgenic, non-human, mammals such as inter alia
rabbits, goats or cows, and secreted into, for instance, the milk
thereof.
[0149] In yet another alternative embodiment, binding molecules
hereof, preferably human binding molecules specifically binding to
influenza virus H3N2 or a fragment thereof, may be generated by
transgenic non-human mammals, such as, for instance, transgenic
mice or rabbits, that express human immunoglobulin genes. In
certain embodiments, the transgenic non-human mammals have a genome
comprising a human heavy chain transgene and a human light chain
transgene encoding all or a portion of the human binding molecules
as described above. The transgenic non-human mammals can be
immunized with a purified or enriched preparation of influenza
virus H3N2 or a fragment thereof. Protocols for immunizing
non-human mammals are well established in the art. See Using
Antibodies: A Laboratory Manual, edited by E. Harlow, D. Lane
(1998), Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y.,
and Current Protocols in Immunology, edited by J. E. Coligan, A. M.
Kruisbeek, D. H. Margulies, E. M. Shevach, W. Strober (2001), John
Wiley & Sons Inc., New York, the disclosures of which are
incorporated herein by reference. Immunization protocols often
include multiple immunizations, either with or without adjuvants
such as Freund's complete adjuvant and Freund's incomplete
adjuvant, but may also include naked DNA immunizations. In another
embodiment, the human binding molecules are produced by B-cells,
plasma and/or memory cells derived from the transgenic animals. In
yet another embodiment, the human binding molecules are produced by
hybridomas, which are prepared by fusion of B-cells obtained from
the above-described transgenic non-human mammals to immortalized
cells. B-cells, plasma cells and hybridomas as obtainable from the
above-described transgenic non-human mammals and human binding
molecules as obtainable from the above-described transgenic
non-human mammals, B-cells, plasma and/or memory cells and
hybridomas are also a part hereof.
[0150] In a further aspect, provided is a method of identifying a
binding molecule, such as a human binding molecule, e.g., a human
monoclonal antibody or fragment thereof, specifically binding to
influenza virus H3N2 or nucleic acid molecules encoding such
binding molecules and comprises the steps of: (a) contacting a
collection of binding molecules on the surface of replicable
genetic packages with influenza virus H3N2 or a fragment thereof
under conditions conducive to binding, (b) selecting at least once
for a replicable genetic package binding to influenza virus H3N2 or
a fragment thereof, (c) separating and recovering the replicable
genetic package binding to influenza virus H3N2 or a fragment
thereof from replicable genetic packages that do not bind to
influenza virus H3N2 or a fragment thereof. A replicable genetic
package as used herein can be prokaryotic or eukaryotic and
includes cells, spores, yeasts, bacteria, viruses, (bacterio)phage,
ribosomes and polysomes. A preferred replicable genetic package is
a phage. The binding molecules, such as, for instance, single chain
Fvs, are displayed on the replicable genetic package, i.e., they
are attached to a group or molecule located at an exterior surface
of the replicable genetic package. The replicable genetic package
is a screenable unit comprising a binding molecule to be screened
linked to a nucleic acid molecule encoding the binding molecule.
The nucleic acid molecule should be replicable either in vivo
(e.g., as a vector) or in vitro (e.g., by PCR, transcription and
translation). In vivo replication can be autonomous (as for a
cell), with the assistance of host factors (as for a virus) or with
the assistance of both host and helper virus (as for a phagemid).
Replicable genetic packages displaying a collection of binding
molecules is formed by introducing nucleic acid molecules encoding
exogenous binding molecules to be displayed into the genomes of the
replicable genetic packages to form fusion proteins with endogenous
proteins that are normally expressed from the outer surface of the
replicable genetic packages. Expression of the fusion proteins,
transport to the outer surface and assembly results in display of
exogenous binding molecules from the outer surface of the
replicable genetic packages.
[0151] The selection step(s) in the methods hereof can be performed
with influenza H3N2 viruses that are live and still infective or
inactivated. Inactivation of influenza virus H3N2 may be performed
by viral inactivation methods well known to the skilled artisan
such as inter alia treatment with formalin, .beta.-propiolactone
(BPL), merthiolate, and/or ultraviolet light. Methods to test, if
influenza virus H3N2 is still alive, infective and/or viable or
partly or completely inactivated, are well known to the person
skilled in the art. The influenza virus H3N2 used in the above
method does not need to be in purified form and, e.g., may be
present in serum and/or blood of an infected individual. The
influenza virus H3N2 used may also be isolated from cell culture in
a suitable medium.
[0152] In certain embodiments, the influenza virus H3N2 is in
suspension when contacted with the replicable genetic packages.
Alternatively, they may also be coupled to a carrier when contact
takes place. In certain embodiments, a first and further selection
may take place against one influenza virus H3N2 strain.
Alternatively, first and further selection rounds may be performed
against different influenza virus H3N2 strains. Alternatively, the
selection step(s) may be performed in the presence of a fragment of
influenza virus H3N2 such as, e.g., cell membrane preparations,
recombinant H3N2 proteins or polypeptides, fusion proteins
comprising H3N2 proteins or polypeptides, cells expressing
recombinant H3N2 proteins or polypeptides, and the like.
Extracellularly exposed parts of these proteins or polypeptides can
also be used as selection material. The fragments of influenza
virus H3N2 may be immobilized to a suitable material before use or
may be used in suspension. In certain embodiments, the selection
can be performed on different fragments of influenza virus H3N2 or
fragments of different influenza virus H3N2 strains. Finding
suitable selection combinations are well within the reach of the
skilled artisan. Selections may be performed by ELISA or FACS.
[0153] In yet a further aspect, provided is a method of obtaining a
binding molecule specifically binding to an influenza virus H3N2
strain or fragment thereof or a nucleic acid molecule encoding such
a binding molecule, wherein the method comprises the steps of a)
performing the above-described method of identifying binding
molecules, and b) isolating from the recovered replicable genetic
package the binding molecule and/or the nucleic acid molecule
encoding the binding molecule. The collection of binding molecules
on the surface of replicable genetic packages can be a collection
of scFvs or Fabs. Once a new scFv or Fab has been established or
identified with the above-mentioned method of identifying binding
molecules or nucleic acid molecules encoding the binding molecules,
the DNA encoding the scFv or Fab can be isolated from the bacteria
or phages and combined with standard molecular biological
techniques to make constructs encoding scFvs, bivalent scFvs, Fabs
or complete human immunoglobulins of a desired specificity (e.g.,
IgG, IgA or IgM). These constructs can be transfected into suitable
cell lines and complete human monoclonal antibodies can eventually
be produced (see Huls et al., 1999; Boel et al., 2000).
[0154] As mentioned before, the preferred replicable genetic
package is a phage. Phage display methods for identifying and
obtaining (human) binding molecules, e.g., (human) monoclonal
antibodies, are by now well-established methods known by the person
skilled in the art. They are, e.g., described in U.S. Pat. No.
5,696,108; Burton and Barbas, 1994; de Kruif et al., 1995b; and
Phage Display: A Laboratory Manual, edited by C. F. Barbas, D. R.
Burton, J. K. Scott and G. J. Silverman (2001), Cold Spring Harbor
Laboratory Press, Cold Spring Harbor, N.Y. All these references are
herewith incorporated herein in their entirety. For the
construction of phage display libraries, collections of human
monoclonal antibody heavy and light chain variable region genes are
expressed on the surface of bacteriophage, preferably filamentous
bacteriophage, particles, in, for example, single-chain Fv (scFv)
or in Fab format (see de Kruif et al., 1995b). Large libraries of
antibody fragment-expressing phages typically contain more than
1.0.times.10.sup.9 antibody specificities and may be assembled from
the immunoglobulin V-regions expressed in the B-lymphocytes of
immunized or non-immunized individuals. In a specific embodiment
hereof, the phage library of binding molecules, preferably scFv
phage library, is prepared from RNA isolated from cells obtained
from a subject that has been vaccinated against influenza virus,
recently vaccinated against an unrelated pathogen, recently
suffered from an influenza virus H3N2 infection or from a healthy
individual. RNA can be isolated from inter alia bone marrow or
peripheral blood, preferably peripheral blood lymphocytes or
isolated B-cells or even subpopulations of B-cells such as memory
B-cells, identified as CD24+/CD27+ B-cells. The subject can be an
animal, preferably a human. In a preferred embodiment, the
libraries may be assembled from the immunoglobulin V-regions
expressed by IgM memory B-cells, identified as IgM+/CD24+/CD27+
cells.
[0155] Alternatively, phage display libraries may be constructed
from immunoglobulin variable regions that have been partially
assembled in vitro to introduce additional antibody diversity in
the library (semi-synthetic libraries). For example, in
vitro-assembled variable regions contain stretches of synthetically
produced, randomized or partially randomized DNA in those regions
of the molecules that are important for antibody specificity, e.g.,
CDR regions. Phage antibodies specific for influenza virus H3N2 can
be selected from the library by exposing the virus or fragment
thereof to a phage library to allow binding of phages expressing
antibody fragments specific for the virus or fragment thereof.
Non-bound phages are removed by washing and bound phages eluted for
infection of E. coli bacteria and subsequent propagation. Multiple
rounds of selection and propagation are usually required to
sufficiently enrich for phages binding specifically to the virus or
fragment thereof. If desired, before exposing the phage library to
the virus or fragment thereof, the phage library can first be
subtracted by exposing the phage library to non-target material
such as viruses or fragments thereof of a different strain, i.e.,
non-H3N2 influenza viruses. These subtractor viruses or fragments
thereof can be bound to a solid phase or can be in suspension.
Phages may also be selected for binding to complex antigens such as
complex mixtures of H3N2 proteins or (poly)peptides optionally
supplemented with other material. Host cells expressing one or more
proteins or (poly)peptides of influenza virus H3N2 may also be used
for selection purposes. A phage display method using these host
cells can be extended and improved by subtracting non-relevant
binders during screening by addition of an excess of host cells
comprising no target molecules or non-target molecules that are
similar, but not identical, to the target, and thereby strongly
enhance the chance of finding relevant binding molecules. Of
course, the subtraction may be performed before, during or after
the screening with virus or fragments thereof. The process is
referred to as the MABSTRACT.RTM. process (MABSTRACT.RTM. is a
registered trademark of Crucell Holland B.V., see also, U.S. Pat.
No. 6,265,150, which is incorporated herein by reference).
[0156] In yet another aspect, provided is a method of obtaining a
binding molecule potentially having neutralizing activity against
influenza virus H3N2, wherein the method comprises the steps of (a)
performing the method of obtaining a binding molecule specifically
binding to influenza virus H3N2 or a fragment thereof or a nucleic
acid molecule encoding such a binding molecule as described above,
and (b) verifying if the binding molecule isolated has neutralizing
activity against the virus, preferably against at least one or more
influenza virus H3N2 strains selected from the group consisting of
A/Hong Kong/1/68, A/Johannesburg/33/94, A/Panama/2007/99,
A/Wisconsin/67/2005 and A/Hiroshima/52/2005, preferably all strains
of H3N2, in particular, all known and future H3N2 strains. Assays
for verifying if a binding molecule has neutralizing activity are
well known in the art (see WHO Manual on Animal Influenza Diagnosis
and Surveillance, Geneva: World Health Organisation, 2005 version
2002.5).
[0157] In a further aspect, provided is a human binding molecule
having neutralizing activity against at least influenza virus A
comprising HA of the H3 subtype, obtainable by one of the methods
as described above.
[0158] In yet a further aspect, provided are compositions
comprising at least a binding molecule, such as a human monoclonal
antibody, at least a functional variant thereof, at least an
immunoconjugate hereof and/or a combination thereof. In addition to
that, the compositions may comprise inter alia stabilizing
molecules, such as albumin or polyethylene glycol, or salts. In
certain embodiments, the salts used are salts that retain the
desired biological activity of the binding molecules and do not
impart any undesired toxicological effects. If necessary, the human
binding molecules may be coated in or on a material to protect them
from the action of acids or other natural or non-natural conditions
that may inactivate the binding molecules.
[0159] In yet a further aspect, provided are compositions
comprising at least a polynucleotide as defined herein. The
compositions may comprise aqueous solutions such as aqueous
solutions containing salts (e.g., NaCl or salts as described
above), detergents (e.g., SDS) and/or other suitable
components.
[0160] Furthermore, also described are pharmaceutical compositions
comprising at least a binding molecule such as a human monoclonal
antibody hereof (or functional fragment or variant thereof), at
least an immunoconjugate hereof, at least a composition hereof, or
combinations thereof. The pharmaceutical composition further
comprises at least one pharmaceutically acceptable excipient.
Pharmaceutically acceptable excipients are well known to the
skilled person. The pharmaceutical composition may further comprise
at least one other therapeutic agent. Suitable agents are also well
known to the skilled artisan.
[0161] In a preferred embodiment, such pharmaceutical composition
comprises at least one additional binding molecule, i.e., the
pharmaceutical composition can be a cocktail or mixture of binding
molecules. The pharmaceutical composition may comprise at least two
binding molecules hereof, or at least one binding molecule hereof
and at least one further influenza virus binding and/or
neutralizing molecule. In another embodiment, the additional
binding molecule may be formulated for simultaneous separate or
sequential administration.
[0162] In certain embodiments, the pharmaceutical compositions may
comprise two or more binding molecules that have neutralizing
activity against influenza virus A comprising HA of the H3 subtype,
such as H3N2. In certain embodiments, the binding molecules exhibit
synergistic neutralizing activity when used in combination. In
other words, the compositions may comprise at least two binding
molecules having neutralizing activity, characterized in that the
binding molecules act synergistically in neutralizing influenza
virus H3N2. As used herein, the term "synergistic" means that the
combined effect of the binding molecules when used in combination
is greater than their additive effects when used individually. The
synergistically acting binding molecules may bind to different
structures on the same or distinct fragments of influenza virus
H3N2. A way of calculating synergy is by means of the combination
index. The concept of the combination index (CI) has been described
by Chou and Talalay (1984). The compositions may, e.g., comprise
one binding molecule having neutralizing activity and one
non-neutralizing H3N2-specific binding molecule. The
non-neutralizing and neutralizing H3N2-specific binding molecules
may also act synergistically in neutralizing influenza virus
H3N2.
[0163] In certain embodiments, the pharmaceutical composition may
comprise at least two influenza virus neutralizing binding
molecules, wherein at least one binding molecule is able to
neutralize one or more influenza virus subtypes of phylogenetic
group 1 and wherein at least one binding molecule is able to
neutralize one or more influenza virus subtypes of phylogenetic
group 2.
[0164] In certain embodiments, the pharmaceutical composition may
comprise at least one binding molecule hereof and at least one
further influenza virus neutralizing binding molecule.
[0165] In another embodiment, the further influenza virus
neutralizing binding molecule preferably is capable of binding to
and neutralizing an influenza virus of a different subtype,
preferably an influenza virus comprising HA of the H1, such as
H1N1, and/or HA of the H5 subtype, such as H5N1, such as the
binding molecules as disclosed in WO 2008/028946. In certain
embodiments, the further binding molecule is a cross-neutralizing
binding molecule against (all) influenza virus subtypes of
phylogenetic group 1, including H1, H2, H5, H9. In a preferred
embodiment, the further binding molecule is the binding molecule
identified as CR6261 in WO 2008/028946, comprising a heavy chain
variable region comprising amino acids 1-121 of amino acid sequence
of SEQ ID NO:186, or a functional variant thereof, and/or a light
chain variable region comprising amino acids 1-112 of SEQ ID
NO:188. In yet another embodiment, the binding molecule comprises a
heavy and light chain comprising the amino acid sequences of SEQ ID
NO:186 and SEQ ID NO:188, respectively. The binding molecules in
the pharmaceutical composition thus preferably are capable of
reacting with influenza viruses of different subtypes. The binding
molecules should be of high affinity and should have a broad
specificity. In certain embodiments, both binding molecules are
cross-neutralizing molecules in that they each neutralize influenza
viruses of different subtypes. In addition, they preferably
neutralize as many strains of each of the different influenza virus
subtypes as possible.
[0166] A pharmaceutical composition hereof can further comprise at
least one other therapeutic, prophylactic and/or diagnostic agent.
The pharmaceutical composition may comprise at least one other
prophylactic and/or therapeutic agent. The further therapeutic
and/or prophylactic agents may be agents able to prevent and/or
treat an influenza virus H3N2 infection and/or a condition
resulting from such an infection. Therapeutic and/or prophylactic
agents include, but are not limited to, anti-viral agents. Such
agents can be binding molecules, small molecules, organic or
inorganic compounds, enzymes, polynucleotide sequences, anti-viral
peptides, etc. Other agents that are currently used to treat
patients infected with influenza virus H3N2 are M2 inhibitors
(e.g., amantidine, rimantadine) and/or neuraminidase inhibitors
(e.g., zanamivir, oseltamivir). These can be used in combination
with the binding molecules hereof. "In combination" herein means
simultaneously, as separate formulations, or as one single combined
formulation, or according to a sequential administration regimen as
separate formulations, in any order. Agents able to prevent and/or
treating an infection with influenza virus H3N2 and/or a condition
resulting from such an infection that are in the experimental phase
might also be used as other therapeutic and/or prophylactic agents
useful herein.
[0167] The binding molecules or pharmaceutical compositions can be
tested in suitable animal model systems prior to use in humans.
Such animal model systems include, but are not limited to, mouse,
ferret, and monkey.
[0168] Typically, pharmaceutical compositions must be sterile and
stable under the conditions of manufacture and storage. The binding
molecules, immunoconjugates, nucleic acid molecules or compositions
can be in powder form for reconstitution in the appropriate
pharmaceutically acceptable excipient before or at the time of
delivery. In the case of sterile powders for the preparation of
sterile injectable solutions, the preferred methods of preparation
are vacuum drying and freeze-drying (lyophilization) that yield a
powder of the active ingredient plus any additional desired
ingredient from a previously sterile-filtered solution thereof.
[0169] Alternatively, the binding molecules, immunoconjugates,
nucleic acid molecules or compositions can be in solution and the
appropriate pharmaceutically acceptable excipient can be added
and/or mixed before or at the time of delivery to provide a unit
dosage injectable form. In certain embodiments, the
pharmaceutically acceptable excipient used herein is suitable to
high drug concentration, can maintain proper fluidity and, if
necessary, can delay absorption.
[0170] The choice of the optimal route of administration of the
pharmaceutical compositions will be influenced by several factors
including the physico-chemical properties of the active molecules
within the compositions, the urgency of the clinical situation and
the relationship of the plasma concentrations of the active
molecules to the desired therapeutic effect. For instance, if
necessary, the binding molecules can be prepared with carriers that
will protect them against rapid release, such as a controlled
release formulation, including implants, transdermal patches, and
microencapsulated delivery systems. Biodegradable, biocompatible
polymers can inter alia be used, such as ethylene vinyl acetate,
polyanhydrides, polyglycolic acid, collagen, polyorthoesters, and
polylactic acid. Furthermore, it may be necessary to coat the
binding molecules with, or co-administer the binding molecules
with, a material or compound that prevents the inactivation of the
human binding molecules. For example, the binding molecules may be
administered to a subject in an appropriate carrier, for example,
liposomes or a diluent.
[0171] The routes of administration can be divided into two main
categories, oral and parenteral administration, such as intravenous
or by inhalation.
[0172] Oral dosage forms can be formulated inter alia as tablets,
troches, lozenges, aqueous or oily suspensions, dispersible powders
or granules, emulsions, hard capsules, soft gelatin capsules,
syrups or elixirs, pills, dragees, liquids, gels, or slurries.
These formulations can contain pharmaceutical excipients including,
but not limited to, inert diluents, granulating and disintegrating
agents, binding agents, lubricating agents, preservatives,
coloring, flavoring or sweetening agents, vegetable or mineral
oils, wetting agents, and thickening agents.
[0173] The pharmaceutical compositions can also be formulated for
parenteral administration. Formulations for parenteral
administration can be inter alia in the form of aqueous or
non-aqueous isotonic sterile non-toxic injection or infusion
solutions or suspensions. The solutions or suspensions may comprise
agents that are non-toxic to recipients at the dosages and
concentrations employed such as 1,3-butanediol, Ringer's solution,
Hank's solution, isotonic sodium chloride solution, oils, fatty
acids, local anesthetic agents, preservatives, buffers, viscosity-
or solubility-increasing agents, water-soluble antioxidants,
oil-soluble antioxidants and metal chelating agents.
[0174] In a further aspect, the binding molecules, such as human
monoclonal antibodies, (functional fragments and variants thereof),
immunoconjugates, compositions, or pharmaceutical compositions
hereof can be used as a medicament. So, a method of diagnosis,
treatment and/or prevention of an influenza virus H3N2 infection
using the binding molecules, immunoconjugates, compositions, or
pharmaceutical compositions hereof is another part hereof. The
above-mentioned molecules can inter alia be used in the diagnosis,
prophylaxis, treatment, or combination thereof, of an influenza
virus H3N2 infection. They are suitable for treatment of yet
untreated patients suffering from an influenza virus H3N2 infection
and patients who have been or are treated for an influenza virus
H3N2 infection.
[0175] The above-mentioned molecules or compositions may be
employed in conjunction with other molecules useful in diagnosis,
prophylaxis and/or treatment. They can be used in vitro, ex vivo or
in vivo. For instance, the binding molecules such as human
monoclonal antibodies (or functional variants thereof),
immunoconjugates, compositions or pharmaceutical compositions
hereof can be co-administered with a vaccine against influenza
virus H3N2 (if available). Alternatively, the vaccine may also be
administered before or after administration of the molecules
hereof. Instead of a vaccine, anti-viral agents can also be
employed in conjunction with the binding molecules hereof. Suitable
anti-viral agents are mentioned above.
[0176] The molecules are typically formulated in the compositions
and pharmaceutical compositions hereof in a therapeutically or
diagnostically effective amount. Alternatively, they may be
formulated and administered separately. For instance, the other
molecules, such as the anti-viral agents, may be applied
systemically, while the binding molecules hereof may be applied
intravenously.
[0177] Treatment may be targeted at patient groups that are
susceptible to H3N2 infection. Such patient groups include, but are
not limited to, e.g., the elderly (e.g., .gtoreq.50 years old,
.gtoreq.60 years old, and preferably .gtoreq.65 years old), the
young (e.g., .ltoreq.5 years old, .ltoreq.1 year old), hospitalized
patients and patients who have been treated with an antiviral
compound, but have shown an inadequate antiviral response.
[0178] Dosage regimens can be adjusted to provide the optimum
desired response (e.g., a therapeutic response). A suitable dosage
range may, for instance, be 0.1-100 mg/kg body weight, preferably
1-50 mg/kg body weight, preferably 0.5-15 mg/kg body weight. For
example, a single bolus may be administered, several divided doses
may be administered over time or the dose may be proportionally
reduced or increased as indicated by the exigencies of the
therapeutic situation. The molecules and compositions hereof are
preferably sterile. Methods to render these molecules and
compositions sterile are well known in the art. The other molecules
useful in diagnosis, prophylaxis and/or treatment can be
administered in a similar dosage regimen as proposed for the
binding molecules hereof. If the other molecules are administered
separately, they may be administered to a patient prior to (e.g., 2
minutes, 5 minutes, 10 minutes, 15 minutes, 30 minutes, 45 minutes,
60 minutes, 2 hours, 4 hours, 6 hours, 8 hours, 10 hours, 12 hours,
14 hours, 16 hours, 18 hours, 20 hours, 22 hours, 24 hours, 2 days,
3 days, 4 days, 5 days, 7 days, 2 weeks, 4 weeks or 6 weeks
before), concomitantly with, or subsequent to (e.g., 2 minutes, 5
minutes, 10 minutes, 15 minutes, 30 minutes, 45 minutes, 60
minutes, 2 hours, 4 hours, 6 hours, 8 hours, 10 hours, 12 hours, 14
hours, 16 hours, 18 hours, 20 hours, 22 hours, 24 hours, 2 days, 3
days, 4 days, 5 days, 7 days, 2 weeks, 4 weeks or 6 weeks after)
the administration of one or more of the human binding molecules or
pharmaceutical compositions hereof. The exact dosing regimen is
usually sorted out during clinical trials in human patients.
[0179] Human binding molecules and pharmaceutical compositions
comprising the human binding molecules are particularly useful, and
often preferred, when they are to be administered to human beings
as in vivo therapeutic agents, since recipient immune response to
the administered antibody will often be substantially less than
that occasioned by administration of a monoclonal murine, chimeric
or humanized binding molecule.
[0180] In another aspect, described is the use of the binding
molecules such as neutralizing human monoclonal antibodies
(functional fragments and variants thereof), immunoconjugates,
nucleic acid molecules, compositions or pharmaceutical compositions
hereof in the preparation of a medicament for the diagnosis,
prophylaxis, treatment, or combination thereof, of an influenza
virus H3N2 infection.
[0181] Next to that, kits comprising at least a binding molecule
such as a neutralizing human monoclonal antibody (functional
fragments and variants thereof), at least an immunoconjugate, at
least a nucleic acid molecule, at least a composition, at least a
pharmaceutical composition, at least a vector, at least a host
hereof or a combination thereof are also a part hereof. Optionally,
the above-described components of the kits hereof are packed in
suitable containers and labeled for diagnosis, prophylaxis and/or
treatment of the indicated conditions. The above-mentioned
components may be stored in unit or multi-dose containers as an
aqueous, preferably sterile, solution or as a lyophilized,
preferably sterile, formulation for reconstitution. The containers
may be formed from a variety of materials such as glass or plastic
and may have a sterile access port (for example, the container may
be an intravenous solution bag or a vial having a stopper to be
pierced by a hypodermic injection needle). The kit may further
comprise more containers comprising a pharmaceutically acceptable
buffer. It may further include other materials desirable from a
commercial and user standpoint, including other buffers, diluents,
filters, needles, syringes, culture medium for one or more of the
suitable hosts and, possibly, even at least one other therapeutic,
prophylactic or diagnostic agent. Associated with the kits can be
instructions customarily included in commercial packages of
therapeutic, prophylactic or diagnostic products that contain
information about, for example, the indications, usage, dosage,
manufacture, administration, contra-indications and/or warnings
concerning the use of such therapeutic, prophylactic or diagnostic
products.
[0182] The binding molecules can also be advantageously used as a
diagnostic agent in an in vitro method for the detection of
phylogenetic group 2 subtype influenza virus. Thus also disclosed
is a method of detecting influenza virus phylogenetic group 2
subtype influenza virus in a sample, wherein the method comprises
the steps of (a) contacting a sample with a diagnostically
effective amount of a binding molecule (functional fragments and
variants thereof) or an immunoconjugate hereof, and (b) determining
whether the binding molecule or immunoconjugate specifically binds
to a molecule of the sample. The sample may be a biological sample
including, but not limited to blood, serum, stool, sputum,
nasopharyngeal aspirates, bronchial lavages, urine, tissue or other
biological material from (potentially) infected subjects, or a
non-biological sample such as water, drink, etc. The (potentially)
infected subjects may be human subjects, but also animals that are
suspected as carriers of influenza virus phylogenetic group 2
subtype influenza virus might be tested for the presence of the
virus using the human binding molecules or immunoconjugates hereof.
The sample may first be manipulated to make it more suitable for
the method of detection. Manipulation means inter alia treating the
sample suspected to contain and/or containing the virus in such a
way that the virus will disintegrate into antigenic components such
as proteins, (poly)peptides or other antigenic fragments. In
certain embodiments, the human binding molecules or
immunoconjugates hereof are contacted with the sample under
conditions that allow the formation of an immunological complex
between the human binding molecules and the virus or antigenic
components thereof that may be present in the sample. The formation
of an immunological complex, if any, indicating the presence of the
virus in the sample, is then detected and measured by suitable
means. Such methods include, inter alia, homogeneous and
heterogeneous binding immunoassays, such as radio-immunoassays
(RIA), ELISA, immunofluorescence, immunohistochemistry, FACS,
BIACORE and Western blot analyses.
[0183] Preferred assay techniques, especially for large-scale
clinical screening of patient sera and blood and blood-derived
products are ELISA and Western blot techniques. ELISA tests are
particularly preferred. For use as reagents in these assays, the
binding molecules or immunoconjugates hereof are conveniently
bonded to the inside surface of microtiter wells. The binding
molecules or immunoconjugates hereof may be directly bonded to the
microtiter well. However, maximum binding of the binding molecules
or immunoconjugates hereof to the wells might be accomplished by
pre-treating the wells with polylysine prior to the addition of the
binding molecules or immunoconjugates hereof. Furthermore, the
binding molecules or immunoconjugates hereof may be covalently
attached by known means to the wells. Generally, the binding
molecules or immunoconjugates are used between 0.01 to 100 .mu.g/ml
for coating, although higher as well as lower amounts may also be
used. Samples are then added to the wells coated with the binding
molecules or immunoconjugates hereof.
[0184] Furthermore, binding molecules hereof can be used to
identify specific binding structures of influenza virus H3N2. The
binding structures can be epitopes on proteins and/or polypeptides.
They can be linear, but also structural and/or conformational. In
one embodiment, the binding structures can be analyzed by means of
PEPSCAN analysis (see inter alia WO 84/03564, WO 93/09872,
Slootstra et al., 1996). Alternatively, a random peptide library
comprising peptides from a protein of influenza virus H3N2 can be
screened for peptides capable of binding to the binding molecules
hereof. The binding structures/peptides/epitopes found can be used
as vaccines and for the diagnosis of influenza virus H3N2
infections. In case fragments other than proteins and/or
polypeptides are bound by the binding molecules, binding structures
can be identified by mass spectrometry, high performance liquid
chromatography and nuclear magnetic resonance.
[0185] In a further aspect, provided is a method of screening a
binding molecule (or a functional fragment or variant thereof) for
specific binding to the same epitope of influenza virus H3N2, as
the epitope bound by a human binding molecule hereof, wherein the
method comprises the steps of (a) contacting a binding molecule to
be screened, a binding molecule hereof and influenza virus H3N2 or
a fragment thereof, (b) measure if the binding molecule to be
screened is capable of competing for specifically binding to
influenza virus H3N2 or a fragment thereof with the binding
molecule hereof. In a further step, it may be determined if the
screened binding molecules that are capable of competing for
specifically binding to influenza virus H3N2 or a fragment thereof
have neutralizing activity. A binding molecule that is capable of
competing for specifically binding to influenza virus H3N2 or a
fragment thereof with the binding molecule hereof is another part
hereof. In the above-described screening method, "specifically
binding to the same epitope" also contemplates specific binding to
substantially or essentially the same epitope as the epitope bound
by the binding molecule hereof. The capacity to block, or compete
with, the binding of the binding molecules hereof to influenza
virus H3N2 typically indicates that a binding molecule to be
screened binds to an epitope or binding site on influenza virus
H3N2 that structurally overlaps with the binding site on influenza
virus H3N2 that is immunospecifically recognized by the binding
molecules hereof. Alternatively, this can indicate that a binding
molecule to be screened binds to an epitope or binding site that is
sufficiently proximal to the binding site immunospecifically
recognized by the binding molecules hereof to sterically or
otherwise inhibit binding of the binding molecules hereof to
influenza virus H3N2.
[0186] In general, competitive inhibition is measured by means of
an assay, wherein an antigen composition, i.e., a composition
comprising influenza virus H3N2 or fragments thereof, is admixed
with reference binding molecules, i.e., the binding molecules
hereof, and binding molecules to be screened. Usually, the binding
molecules to be screened are present in excess. Protocols based
upon ELISAs and Western blotting are suitable for use in such
simple competition studies. By using species or isotype secondary
antibodies, one will be able to detect only the bound reference
binding molecules, the binding of which will be reduced by the
presence of a binding molecule to be screened that recognizes
substantially the same epitope. In conducting a binding molecule
competition study between a reference binding molecule and any
binding molecule to be screened (irrespective of species or
isotype), one may first label the reference binding molecule with a
detectable label, such as, e.g., biotin, an enzymatic, a
radioactive or other label to enable subsequent identification.
Binding molecules identified by these competition assays
("competitive binding molecules" or "cross-reactive binding
molecules") include, but are not limited to, antibodies, antibody
fragments and other binding agents that bind to an epitope or
binding site bound by the reference binding molecule, i.e., a
binding molecule hereof, as well as antibodies, antibody fragments
and other binding agents that bind to an epitope or binding site
sufficiently proximal to an epitope bound by the reference binding
molecule for competitive binding between the binding molecules to
be screened and the reference binding molecule to occur. In certain
embodiments, competitive binding molecules hereof will, when
present in excess, inhibit specific binding of a reference binding
molecule to a selected target species by at least 10%, preferably
by at least 25%, more preferably by at least 50%, and most
preferably by at least 75%-90% or even greater. The identification
of one or more competitive binding molecules that bind to about,
substantially, essentially or at the same epitope as the binding
molecules hereof is a straightforward technical matter. As the
identification of competitive binding molecules is determined in
comparison to a reference binding molecule, i.e., a binding
molecule hereof, it will be understood that actually determining
the epitope to which the reference binding molecule and the
competitive binding molecule bind is not in any way required in
order to identify a competitive binding molecule that binds to the
same or substantially the same epitope as the reference binding
molecule. The disclosure is further illustrated in the following
Examples and figures. The Examples are not intended to limit the
scope hereof in any way.
EXAMPLES
Example 1
Construction of scFv Phage Display Libraries Using RNA Extracted
from Memory B Cells
[0187] Peripheral blood was collected from normal healthy donors by
venapuncture in EDTA anti-coagulation sample tubes. scFv phage
display libraries were obtained as described in WO 2008/028946,
which is incorporated by reference herein. Memory B cells
(CD24+/CD27+) were separated from naive B cells (CD24+/CD27-) and
memory T cells (CD24-/CD27+) and in a next step, IgM memory B cells
(IgM+) were separated from switch memory B cells (IgM-) using IgM
expression. RNA was isolated from the IgM memory B cells and cDNA
prepared.
[0188] A two-round PCR amplification approach was applied using the
primer sets shown in Tables 1 and 2 to isolate the immunoglobulin
VH and VL regions from the respective donor repertoire.
[0189] First-round amplification on the respective cDNA using the
primer sets mentioned in Table 1 yielded seven, six and nine
products of about 650 base pairs for, respectively, VH, Vkappa and
Vlambda regions. For IgM memory B cell VH region amplification, the
OCM constant primer was used in combination with OH1 to OH7. The
thermal cycling program for first-round amplifications was: 2
minutes 96.degree. C. (denaturation step), 30 cycles of 30 seconds
96.degree. C./30 seconds 55.degree. C./60 seconds 72.degree. C., 10
minutes 72.degree. C. final elongation and 4.degree. C.
refrigeration. The products were loaded on and isolated from a 1%
agarose gel using gel-extraction columns (Qiagen) and eluted in 50
.mu.l 1 mM Tris-HCl pH 8.0. Ten percent of first-round products (5
.mu.l) was subjected to second-round amplification using the
primers mentioned in Table 2. These primers were extended with
restriction sites enabling the directional cloning of the
respective VL and VH regions into phage display vector PDV-006. The
PCR program for second-round amplifications was as follows: 2
minutes 96.degree. C. (denaturation step), 30 cycles of 30 seconds
96.degree. C./30 seconds 60.degree. C./60 seconds 72.degree. C., 10
minutes 72.degree. C. final elongation and 4.degree. C.
refrigeration. The second-round products (.about.350 base pairs)
were first pooled according to natural occurrence of J segments
found in immunoglobulin gene products, resulting in seven, six and
nine pools for, respectively, the VH, Vkappa and Vlambda variable
regions (see Tables 3 and 4).
[0190] To obtain a normalized distribution of immunoglobulin
sequences in the immune library, the six Vkappa and nine Vlambda
light chain pools were mixed according to the percentages mentioned
in Table 3. This single final VL pool (3 .mu.g) was digested
overnight with SalI and NotI restriction enzymes, loaded on and
isolated from a 1.5% agarose gel (.about.350 base pairs) using
Qiagen gel-extraction columns and ligated in SalI-NotI cut PDV-006
vector (.about.5000 base pairs) as follows: 10 .mu.l PDV-006 vector
(50 ng/.mu.l), 7 .mu.l VL insert (10 ng/.mu.l), 5 .mu.l 10.times.
ligation buffer (NEB), 2.5 T4 DNA Ligase (400 U/.mu.l) (NEB), 25.5
.mu.l ultrapure water (vector to insert ratio was 1:2). Ligation
was performed overnight in a water bath of 16.degree. C. Next, the
volume was doubled with water, extracted with an equal volume of
phenol-chloroform-isoamylalcohol (75:24:1) (Invitrogen) followed by
chloroform (Merck) extraction and precipitated with 1 .mu.l Pellet
Paint (Novogen), 10 .mu.l sodium acetate (3 M pH 5.0) and 100 .mu.l
isopropanol for two hours at -20.degree. C.
[0191] The obtained sample was subsequently centrifuged at
20,000.times.g for 30 minutes at 4.degree. C. The obtained
precipitate was washed with 70% ethanol and centrifuged for 10
minutes at 20,000.times.g at room temperature. Ethanol was removed
by vacuum aspiration and the pellet was air dried for several
minutes and then dissolved in 50 .mu.l buffer containing 10 mM
Tris-HCl, pH 8.0. One .mu.l ligation mixture was used for the
transformation of 40 .mu.l TG-1 electro-competent cells
(Stratagene) in a chilled 0.1 cm electroporation cuvette (Biorad)
using a Genepulser II apparatus (Biorad) set at 1.7 kV, 200 Ohm, 25
.mu.F (time constant .about.4.5 msec). Directly after pulse, the
bacteria were flushed from the cuvette with 1000 .mu.l SOC medium
(Invitrogen) containing 5% (w/v) glucose (Sigma) at 37.degree. C.
and transferred to a 15 ml round bottom culture tube. Another 500
.mu.l SOC/glucose was used to flush residual bacteria from the
cuvette and was added to the culture tube. Bacteria were recovered
by culturing for exactly one hour at 37.degree. C. in a shaker
incubator at 220 rpm. The transformed bacteria were plated over
large 240 mm square petri dishes (NUNC) containing 200 ml 2TY agar
(16 g/l bacto-tryptone, 10 g/l bacto-yeast extract, 5 g/l NaCl, 15
g/l agar, pH 7.0) supplemented with 50 .mu.g/ml ampicillin and 5%
(w/v) glucose (Sigma). A 1 to 1000 dilution was plated for counting
purposes on 15 cm petri dishes containing the same medium.
[0192] This transformation procedure was repeated sequentially
twenty times and the complete library was plated over a total of
thirty large square petri dishes and grown overnight in a
37.degree. C. culture stove. Typically, around 1.times.10.sup.7 cfu
were obtained using the above protocol. The intermediate VL light
chain library was harvested from the plates by mildly scraping the
bacteria into 10 ml 2TY medium per plate. The cell mass was
determined by OD600 measurement and two times 500 OD of bacteria
was used for maxi plasmid DNA preparation using two P500 maxiprep
columns (Qiagen) according to manufacturer's instructions.
[0193] Analogous to the VL variable regions, the second round VH-JH
products were first mixed together to obtain the normal J segment
usage distribution (see Table 4), resulting in seven VH subpools
called PH1 to PH7. The pools were mixed to acquire a normalized
sequence distribution using the percentages depicted in Table 4,
obtaining one VH fraction that was digested with SfiI and XhoI
restriction enzymes and ligated in SfiI-XhoI cut PDV-VL
intermediate library obtained as described above. The ligation
set-up, purification method, subsequent transformation of TG1 and
harvest of bacteria was exactly as described for the VL
intermediate library (see above). The final library (approximately
5.times.10.sup.6 cfu) was checked for insert frequency with a
colony PCR using a primer set flanking the inserted VH-VL regions.
More than 95% of the colonies showed a correct length insert (see
Table 5). The colony PCR products were used for subsequent DNA
sequence analysis to check sequence variation and to assess the
percentage of colonies showing a complete ORF. This was typically
above 70% (see Table 5). The frequency of mutations in the V genes
was also analyzed. Out of 50 sequences, 47 (94%) were not in
germline configuration indicative of a maturation process and
consistent with the memory phenotype of the B cells used as an RNA
source for the library. Finally, the library was rescued and
amplified by using CT helper phages (see WO 02/103012) and was used
for phage antibody selection by panning methods as described
below.
Example 2
Selection of Phages Carrying Single Chain Fv Fragments Against
Influenza A Subtypes H3 and H7 and Influenza B
[0194] Antibody fragments were selected using antibody phage
display libraries constructed essentially as described above and
general phage display technology and MABSTRACT.RTM. technology
essentially as described in U.S. Pat. No. 6,265,150 and in WO
98/15833 (both of which are incorporated by reference herein).
Furthermore, the methods and helper phages as described in WO
02/103012 (which is incorporated by reference herein) were used
herein.
[0195] Selection was performed against recombinant hemagglutinin
(HA) of influenza A subtype H3 (A/Wisconsin/67/2005) and H7
(A/Netherlands/219/2003) or influenza B (B/Ohio/01/2005). HA
antigens were diluted in PBS (5.0 .mu.g/ml), added to MaxiSorp.TM.
Nunc-Immuno Tubes (Nunc) and incubated overnight at 4.degree. C. on
a rotating wheel. The immunotubes were emptied and washed three
times in block buffer (2% non-fat dry milk (ELK) in PBS).
Subsequently, the immunotubes were filled completely with block
buffer and incubated for one to two hours at room temperature.
Aliquots of phage display library (500-1000 .mu.l,
0.5.times.10.sup.13-1.times.10.sup.13 cfu, amplified using CT
helper phage (see WO 02/103012)) were blocked in blocking buffer
supplemented with 10% non-heat inactivated fetal bovine serum and
2% mouse serum for one to two hours at room temperature. The
blocked phage library was added to the immunotubes, incubated for
two hours at room temperature, and washed with wash buffer (0.05%
(v/v) TWEEN.RTM.-20 in PBS) to remove unbound phages. Bound phages
were eluted from the respective antigen by incubation with 1 ml of
100 mM triethylamine (TEA) for 10 minutes at room temperature.
Subsequently, the eluted phages were mixed with 0.5 ml of 1 M
Tris-HCl pH 7.5 to neutralize the pH. This mixture was used to
infect 5 ml of an XL1-Blue E. coli culture that had been grown at
37.degree. C. to an OD 600 nm of approximately 0.3. The phages were
allowed to infect the XL1-Blue bacteria for 30 minutes at
37.degree. C. Then, the mixture was centrifuged for 10 minutes at
3000.times.g at room temperature and the bacterial pellet was
resuspended in 0.5 ml 2-trypton yeast extract (2TY) medium. The
obtained bacterial suspension was divided over two 2TY agar plates
supplemented with tetracycline, ampicillin and glucose.
[0196] After incubation overnight of the plates at 37.degree. C.,
the colonies were scraped from the plates and used to prepare an
enriched phage library, essentially as described by De Kruif et al.
(1995a) and WO 02/103012. Briefly, scraped bacteria were used to
inoculate 2TY medium containing ampicillin, tetracycline and
glucose and grown at a temperature of 37.degree. C. to an OD 600 nm
of .about.0.3. CT helper phages were added and allowed to infect
the bacteria after which the medium was changed to 2TY containing
ampicillin, tetracycline and kanamycin. Incubation was continued
overnight at 30.degree. C. The next day, the bacteria were removed
from the 2TY medium by centrifugation after which the phages in the
medium were precipitated using polyethylene glycol (PEG) 6000/NaCl.
Finally, the phages were dissolved in 2 ml of PBS with 1% bovine
serum albumin (BSA), filter-sterilized and used for the next round
of selection. The second round of selection is performed either on
the same HA subtype or on HA of a different subtype.
[0197] Two consecutive rounds of selections were performed before
isolation of individual single-chain phage antibodies. After the
second round of selection, individual E. coli colonies were used to
prepare monoclonal phage antibodies. Essentially, individual
colonies were grown to log-phase in 96-well plate format and
infected with VCS-M13 helper phages, after which phage antibody
production was allowed to proceed overnight. The supernatants
containing phage antibodies were used directly in ELISA for binding
to HA antigens. Alternatively, phage antibodies were
PEG/NaCl-precipitated and filter-sterilized for both elisa and flow
cytometry analysis.
Example 3
Validation of the HA Specific Single-Chain Phage Antibodies
[0198] Selected supernatants containing single-chain phage
antibodies that were obtained in the screenings described above
were validated in ELISA for specificity, i.e., binding to different
HA antigens. For this purpose, baculovirus-expressed recombinant H3
(A/Wisconsin/67/2005), H7 (A/Netherlands/219/2003) and B
(B/Ohio/01/2005) HAs (Protein Sciences, CT, USA) were coated to
Maxisorp.TM. ELISA plates. After coating, the plates were washed
three times with PBS containing 0.1% v/v TWEEN.RTM.-20 and blocked
in PBS containing 3% BSA or 2% ELK for one hour at room
temperature. The selected single-chain phage antibodies were
incubated for one hour in an equal volume of PBS containing 4% ELK
to obtain blocked phage antibodies. The plates were emptied, washed
three times with PBS/0.1% TWEEN.RTM.-20 and the blocked
single-chain phage antibodies were added to the wells. Incubation
was allowed to proceed for one hour, the plates were washed with
PBS/0.1% TWEEN.RTM.-20 and bound phage antibodies were detected
(using OD 492 nm measurement) using an anti-M13 antibody conjugated
to peroxidase. As a control, the procedure was performed
simultaneously without single-chain phage antibody and with an
unrelated negative control single-chain phage antibody. From the
selections on the different HA antigens with the IgM memory B cell
libraries, six unique single-chain phage antibodies specific for
both recombinant H3 HA and H7 HA were obtained (SC08-001, SC08-003,
SC08-006, SC08-014, SC08-017 and SC08-018). In addition, two unique
single-chain phage antibodies specific for recombinant H3 HA
(SC08-015 and SC08-016) and five for recombinant H7 HA (SC08-007,
SC08-009, SC08-010, SC08-011 and SC08-013) were isolated. See,
Table 6.
[0199] Alternatively, PEG/NaCl-precipitated and filter-sterilized
phage antibodies were used to validate elisa binding and
specificity. For this purpose, baculovirus-expressed recombinant
influenza A H1 (A/New Caledonia/20/1999), H3 (A/Wisconsin/67/2005),
H5 (A/Vietnam/1203/2004), H7 (A/Netherlands/219/2003) and influenza
B (B/Ohio/01/2005, B/Malaysia/2506/2004, B/Jilin/219/2003) HAs
(Protein Sciences, CT, USA) were coated to Maxisorp.TM. ELISA
plates. After coating, the plates were washed three times with PBS
containing 0.1% v/v TWEEN.RTM.-20 and blocked in PBS containing 3%
BSA or 2% ELK for one hour at room temperature. The selected
single-chain phage antibodies were incubated for one hour in an
equal volume of PBS containing 4% ELK to obtain blocked phage
antibodies. The plates were emptied, washed three times with
PBS/0.1% TWEEN.RTM.-20 and the blocked single-chain phage
antibodies were added to the wells. Incubation was allowed to
proceed for one hour, the plates were washed with PBS/0.1%
TWEEN.RTM.-20 and bound phage antibodies were detected (using OD
492 nm measurement) using an anti-M13 antibody conjugated to
peroxidase. As a control, the procedure was performed
simultaneously without single-chain phage antibody and with a
negative control single-chain phage antibody. From the selections
on the different HA antigens with the IgM memory B cell libraries,
two unique single-chain phage antibodies specific for recombinant
H1, H3 and H7 HA were obtained (SC08-001 and SC08-014). In
addition, six unique single-chain phage antibodies specific for
recombinant H3 HA (SC08-003, SC08-006, SC08-015, SC08-016, SC08-017
and SC08-018), and five for recombinant H7 HA (SC08-007, SC08-009,
SC08-010, SC08-011 and SC08-013) were isolated. See, Table 7.
[0200] Alternatively, PEG/NaCl-precipitated and filter-sterilized
phage antibodies were used to validate binding and specificity by
FACS analysis. For this purpose, full-length recombinant influenza
A subtypes H1 (A/New Caledonia/20/1999), H3 (A/Wisconsin/67/2005),
H5(TV), H7 (A/Netherlands/219/2003) and influenza B
(B/Ohio/o1/2005) HAs were expressed on the surface of PER.C6.RTM.
cells. The cells were incubated with single-chain phage antibodies
for one hour followed by three wash steps with PBS+0.1% BSA. Bound
phages were detected using FITC-conjugated M13-antibody. From the
selections on the different HA antigens with the IgM memory B cell
libraries, one single-chain phage antibody specific for influenza A
subtypes H1, H3 and H7 HA was isolated (SC08-001). In addition, six
unique single-chain phage antibodies specific for H3 HA (SC08-003,
SC08-006, SC08-015, SC08-016, SC08-017 and SC08-018), four unique
single-chain phage antibodies specific for H7 HA (SC08-007,
SC08-010, SC08-011 and SC08-013) were isolated. See Table 8. Of
these, six phage antibodies (SC08-001, SC08-003, SC08-015,
SC08-016, SC08-017, SC08-018) were used for construction of fully
human immunoglobulins for further characterization (see Example
5).
Example 4
Selection and Validation of Influenza A (H3N2) HA Specific
Immortalized B-Cell Clones
[0201] In addition to phage display, the binding molecules hereof
can also be isolated by other methods, for example, using
immortalized B cells, as described in, e.g., WO 2007067046.
Immortalized IgM memory cells (CD19+/CD27+, IgD+), derived from
vaccinated donors, were stained with APC-labeled H3 HA and single
cells sorted into limiting dilution culture. After recovery and
cell expansion, the supernatants of the H3 HA sorted cells were
measured by solid phase ELISA for H1, H3 and H7
immunoreactivity.
[0202] Subsequently, the target-specific B cells were characterized
for binding activity and neutralization. The B cells were cloned by
limiting dilution to yield single clones. The clones were seeded
into culture plates and the cells cultured for 14 days.
Supernatants of the clones were screened for production of anti-HA
monoclonal antibodies that bind to HA-transfected 293 cells
expressing H1, H3, H5 and H7 derived HA. As a control for aspecific
or background staining, untransfected 293 cells were used.
[0203] In order to determine whether the selected B-cell clone
supernatants containing either IgM or IgG antibodies that were
obtained in the screenings described above were capable of blocking
influenza A (H3N2) infection, an in vitro virus neutralization
assay (VNA) was performed. The VNA was performed on MDCK cells
(ATCC CCL-34). MDCK cells were cultured in MDCK cell culture medium
(MEM medium supplemented with antibiotics, 20 mM Hepes and 0.15%
(w/v) sodium bicarbonate (complete MEM medium), supplemented with
10% (v/v) fetal bovine serum). The H3N2 (A/Wisconsin/67/2005)
strain that was used in the assay was diluted to a titer of
5.7.times.10.sup.3 TCID50/ml (50% tissue culture infective dose per
ml), with the titer calculated according to the method of Spearman
and Karber. The IgG or IgM preparations were serially two-fold
diluted (1:2-1:64) in complete MEM medium in quadruplicate wells.
25 .mu.l of the respective IgG dilution was mixed with 25 .mu.l of
virus suspension (100 TCID50/25 .mu.l) and incubated for one hour
at 37.degree. C. The suspension was then transferred in
quadruplicate onto 96-well plates containing confluent MDCK
cultures in 50 .mu.l complete MEM medium. Prior to use, MDCK cells
were seeded at 3.times.10.sup.4 cells per well in MDCK cell culture
medium, grown until cells had reached confluence, washed with
300-350 .mu.l PBS, pH 7.4 and finally 50 .mu.l complete MEM medium
was added to each well. The inoculated cells were cultured for
three to four days at 37.degree. C. and observed daily for the
development of cytopathogenic effect (CPE). CPE was compared to the
positive control.
[0204] Of the 187 IgG supernatants tested, 43 were found to
neutralize the H3N2 (A/Wisconsin/67/2005) strain used in this
assay. Of these, 14 were used for construction of human IgG
immunoglobulins as described in Example 5.
Example 5
Construction of Fully Human Immunoglobulin Molecules (Human
Monoclonal Antibodies) from the Selected Single Chain Fvs and
B-Cell Clones
[0205] From the selected specific single-chain phage antibody
(scFv) clones, plasmid DNA was obtained and nucleotide and amino
acid sequences were determined according to standard techniques.
Heavy and light chain variable regions of the scFvs were cloned
directly by restriction digest for expression in the IgG expression
vectors pIg-C911-HCgamma1 (see SEQ ID NO:189), pIG-C909-Ckappa (see
SEQ ID NO:190), or pIg-C910-Clambda (see SEQ ID NO:191). Heavy and
light chain variable regions of the B-cell clones were
PCR-amplified and cloned directly by restriction digest for
expression in the IgG expression vectors pIg-C911-HCgamma1 (see SEQ
ID NO:190), pIG-C909-Ckappa (see SEQ ID NO:191), or
pIg-C910-Clambda (see SEQ ID NO:192). The VH and VL gene identity
(see I. M. Tomlinson et al., V-BASE Sequence Directory, Cambridge
United Kingdom: MRC Centre for Protein Engineering (1997)) of the
scFvs were determined (see Table 9).
[0206] Nucleotide sequences for all constructs were verified
according to standard techniques known to the skilled artisan. The
resulting expression constructs encoding the human IgG1 heavy and
light chains were transiently expressed in combination in 293T
cells and supernatants containing human IgG1 antibodies were
obtained and produced using standard purification procedures. The
human IgG1 antibodies were titrated in a concentration range of
between 10 and 0.003 .mu.g/ml against H3, H7 or B antigen (data not
shown). An unrelated antibody was included as a control
antibody.
[0207] The amino acid sequence of the CDRs of the heavy and light
chains of the selected immunoglobulin molecules is given in Table
9. The nucleotide sequence and amino acid sequence of the heavy and
light chain variable regions are given below. The immunoglobulins
comprise the heavy and light chain constant region of CR6261, as
given below.
Example 6
In Vitro Neutralization of Influenza Virus by H3N2 Binding IgGs
(Virus Neutralization Assay)
[0208] In order to determine whether the selected IgGs were capable
of blocking influenza A (H3N2) infection, an in vitro virus
neutralization assay (VNA) was performed. The VNA was performed on
MDCK cells (ATCC CCL-34). MDCK cells were cultured in MDCK cell
culture medium (MEM medium supplemented with antibiotics, 20 mM
Hepes and 0.15% (w/v) sodium bicarbonate (complete MEM medium),
supplemented with 10% (v/v) fetal bovine serum). The H3N2
(A/Wisconsin/67/2005) strain that was used in the assay was diluted
to a titer of 5.7.times.10.sup.3 TCID50/ml (50% tissue culture
infective dose per ml), with the titer calculated according to the
method of Spearman and Karber. The IgG preparations (200 .mu.g/ml)
were serially two-fold diluted (1:2-1:512) in complete MEM medium
in quadruplicate wells. 25 .mu.l of the respective IgG dilution was
mixed with 25 .mu.l of virus suspension (100 TCID50/25 .mu.l) and
incubated for one hour at 37.degree. C. The suspension was then
transferred in quadruplicate onto 96-well plates containing
confluent MDCK cultures in 50 .mu.l complete MEM medium. Prior to
use, MDCK cells were seeded at 3.times.10.sup.4 cells per well in
MDCK cell culture medium, grown until cells had reached confluence,
washed with 300-350 .mu.l PBS, pH 7.4 and finally 50 .mu.l complete
MEM medium was added to each well. The inoculated cells were
cultured for three to four days at 37.degree. C. and observed daily
for the development of cytopathogenic effect (CPE). CPE was
compared to the positive control.
[0209] The human anti-H3 HA and/or anti-H7 HA antibodies of Example
5 were subjected to the above-described VNA. Of these antibodies,
all antibodies, except CR8040, CR8052 and CR8069, neutralized the
A/Wisconsin/67/2005 H3N2 strain. The concentrations (in .mu.g/ml)
at which these antibodies protect MDCK cultures against CPE are
given in Table 11.
Example 7
Cross-Binding Reactivity of Anti-H3N2 IgGs
[0210] The H3N2 neutralizing IgG antibodies described above were
validated in ELISA for binding specificity, i.e., binding to
different HA antigens. For this purpose, baculovirus-expressed
recombinant H1 (A/New Caledonia/20/1999), H3 (A/Wisconsin/67/2005,
A/New York/55/2004, A/Wyoming/3/2003) and H7
(A/Netherlands/219/2003) HAs (Protein Sciences, CT, USA) were
coated to Maxisorp.TM. ELISA plates. After coating, the plates were
washed three times with PBS containing 0.1% v/v TWEEN.RTM.-20 and
blocked in PBS containing 3% BSA or 2% ELK for one hour at room
temperature. The plates were emptied, washed three times with
PBS/0.1% TWEEN.RTM.-20 and the IgG antibodies were added to the
wells. Incubation was allowed to proceed for one hour, the plates
were washed with PBS/0.1% TWEEN.RTM.-20 and bound antibodies were
detected (using OD 492 nm measurement) using an anti-human IgG
antibody conjugated to peroxidase. As a control, an unrelated IgG
CR4098 was used.
[0211] From the selected H3N2 neutralizing antibodies, CR8001 shows
heterosubtypic cross-binding to all the recombinant HAs tested,
CR8020, CR8021, CR8041, CR8043 and CR8057 show heterosubtypic
cross-binding to all three tested H3 HAs, as well as the H7 HA.
CR8003, CR8015, CR8016, CR8017, CR8018, CR8038, CR8039, CR8040,
CR8049, CR8050, CR8052 and CR8069 show cross-binding to all three
tested H3 HAs. One antibody, CR8019, shows binding to only two of
the H3 HAs. See Table 12.
[0212] Additionally, the selected H3N2-neutralizing antibodies were
used to test heterosubtypic binding by FACS analysis. For this
purpose, full-length recombinant influenza A subtypes H1 (A/New
Caledonia/20/1999), H3 (A/Wisconsin/67/2005) and H7
(A/Netherlands/219/2003) HAs were expressed on the surface of
PER.C6.RTM. cells. The cells were incubated with IgG antibodies for
one hour followed by three wash steps with PBS+0.1% BSA. Bound
antibodies were detected using PE-conjugated anti-human antibody.
As a control, untransfected PER.C6.RTM. cells were used.
[0213] From the H3N2-neutralizing antibodies, CR8001 shows
cross-binding activity to influenza A subtypes H1, H3 and H7 HA,
but not wild-type PER.C6.RTM. cells. In addition, CR8020 and CR8041
show strong binding to both H3 and H7 HA. CR8043 and CR8057 show
strong binding to H3 HA and weak binding to H7 HA. CR8055 showed
low levels of background staining on PER.C6.RTM. cells. The
remaining 13 antibodies show binding to H3 transfected cells only.
See Table 12.
Example 8
Cross-Neutralizing Activity of Anti-H3N2 IgGs
[0214] In order to determine whether the selected IgGs were capable
of blocking multiple influenza A strains, additional in vitro virus
neutralization assays (VNA) were performed. The VNA were performed
on MDCK cells (ATCC CCL-34). MDCK cells were cultured in MDCK cell
culture medium (MEM medium supplemented with antibiotics, 20 mM
Hepes and 0.15% (w/v) sodium bicarbonate (complete MEM medium),
supplemented with 10% (v/v) fetal bovine serum). The H1N1 (A/New
Caledonia/20/1999 A/Brisbane/59/2007 and A/Solomon
Islands/IVR-145), H3N2 (A/Hong Kong/1/68, A/Johannesburg/33/94,
A/Panama/2000/1999, A/Hiroshima/52/2005 and A/Wisconsin/67/2005),
H7N3 (A/Mallard/Netherlands/12/2000) and H10 (A/Chick/Germany/N/49)
strains that were used in the assay were all diluted to a titer of
5.7.times.10.sup.3 TCID50/ml (50% tissue culture infective dose per
ml), with the titer calculated according to the method of Spearman
and Karber. The IgG preparations (80 .mu.g/ml) were serially
two-fold diluted (1:2-1:512) in complete MEM medium in
quadruplicate wells. 25 .mu.l of the respective IgG dilution was
mixed with 25 .mu.l of virus suspension (100 TCID50/25 .mu.l) and
incubated for one hour at 37.degree. C. The suspension was then
transferred in quadruplicate onto 96-well plates containing
confluent MDCK cultures in 50 .mu.l complete MEM medium. Prior to
use, MDCK cells were seeded at 3.times.10.sup.4 cells per well in
MDCK cell culture medium, grown until cells had reached confluence,
washed with 300-350 .mu.l PBS, pH 7.4 and finally 50 .mu.l complete
MEM medium was added to each well. The inoculated cells were
cultured for three to four days at 37.degree. C. and observed daily
for the development of cytopathogenic effect (CPE). CPE was
compared to the positive control.
[0215] From the panel of H3N2-neutralizing antibodies, CR8020 and
CR8041 show heterosubtypic cross-neutralizing activity to all
tested influenza A subtypes H3, H7 and H10 viruses, but not H1
viruses. In addition, CR8043 shows cross-neutralization to all
tested H3 and H10 virus strains. CR8039, CR8041, CR8043 and CR8057
show cross-neutralization of all tested H3 virus strains. An
additional 13 antibodies show cross-neutralization to more than one
of the tested H3 virus strains. See Table 13.
Example 9
Anti-H3N2 Antibodies Bind to the Pre-Fusion Conformation of HA
[0216] In order to determine whether the selected IgGs were capable
of binding the pre- or post-fusion conformation of the HA molecule,
an in vitro pH-shift experiment was performed.
[0217] For this purpose, full-length recombinant influenza A
subtype H3 (A/Wisconsin/67/2005) HA was expressed on the surface of
PER.C6.RTM. cells. To assay for specific reactivity at different
structural HA conformations, 3.times.10.sup.5 cells were treated
with 10 .mu.g/ml trypsin-EDTA in DMEM for 30 minutes at RT, washed
and incubated for 5 minutes in acidified PBS (pH 4.9), washed and
then incubated for 20 minutes in the presence of 20 mM DTT at RT.
Cells were split at each step and untreated adherent cells were
resuspended in 0.05% EDTA. Cell fractions of each treatment were
incubated with anti-H3N2 IgGs CR8001, CR8020, CR8041, CR8043 and
CR8057 for 30 minutes. Cells were then incubated for 30 minutes
with phycoerythrin-conjugated anti-IgG (Southern Biotech). Stained
cells were analyzed using a FACS Calibur with CELLQuest Pro
software (Becton Dickinson). FACS binding of IgG1 to
surface-expressed H3 rHA was measured after sequential treatment
with trypsin (striped bars), pH 4.9 buffered medium (solid white
bars) and DTT (crossed bars) and expressed as percentage binding to
untreated rHA (solid black bars). See FIG. 2.
[0218] Antibodies CR8001, CR8020, CR8041 and CR8043 all show a
marked decrease in binding after pH-shift indicating specificity
for an epitope present only before the low PH-induced
conformational change of the HA molecule. Antibody CR8057 showed a
decrease in binding only after DTT treatment indicating specificity
for a conformation-independent epitope available only when HA1 is
present.
Example 10
Anti-H3N2 Antibody CR8041 Prevents Cleavage of HA0
[0219] In order to determine whether the selected IgGs were capable
of protecting the HA molecule from protease cleavage, an in vitro
protease susceptibility assay was performed.
[0220] For this purpose, 7.5 .mu.g recombinant soluble influenza A
subtype H3 (A/Wisconsin/67/2005) HA (Protein Sciences, CT, USA) was
subjected to different pH (4.9, 5.3 and 8.0) treatments for one
hour at 37.degree. C. After incubation, reactions were neutralized.
The samples were digested overnight with 0.5 .mu.g trypsin in the
presence and absence of 7.5 .mu.g CR8041 or CR8057 Fab fragments.
Reactions were quenched by addition of SDS loading buffer. Three
.mu.l Nupage reducing agent (Invitrogen) was added to each sample.
Samples were run on a 4-12% BisTris gel in 1.times. MOPS buffer.
Protein bands were visualized by colloidal blue staining (see FIG.
3). In the absence of Fab fragments, the H3 HA molecule is readily
converted to its protease-susceptible post-fusion form at pH 4.9 or
5.3, but not at pH 8.0. In the presence of Fab fragment CR8057, the
degradation of H3 HA and thus the conformational change at pH 4.9
is not inhibited. In contrast, the presence of Fab CR8041 not only
prevents H3 HA conformational change and degradation at low pH, but
also the pH-independent cleavage of HA0 into HA1 and HA2. These
results point towards an epitope for CR8041 on, or close to, the
cleavage site. Competition experiments (results not shown) with the
anti-H3N2 antibody panel indicate an overlapping epitope and a
similar working mechanism for the CR8001, CR8020 and CR8043
antibodies.
Example 11
Mechanism of Action of the Binding Molecules
[0221] The HA glycoprotein is a trimer in which each monomer
consists of two disulphide-linked glycopolypeptides (named HA1 and
HA2) that are produced during infection by proteolytic cleavage of
a precursor (HA0). Cleavage is necessary for virus infectivity
since it is required to prime the HA for membrane fusion, to allow
conformational change.
[0222] Activation of the primed molecule occurs at low pH in
endosomes, between pH5 and pH6, and requires extensive changes in
HA structure. The three-dimensional structure of the pre-fusion
uncleaved (I), pre-fusion cleaved (II) and post-fusion HA (III)
conformations are schematically shown in FIG. 4.
[0223] In vitro, the conformational changes of the HA molecule can
be mimicked using HA surface-expressed mammalian cells. First, the
proteolytic cleavage can be triggered by adding trypsin to the
cells. Second, the pre- to post-fusion conformational change can be
achieved by lowering the pH. Additionally, the HA1 part of the
molecule can be removed by adding a reducing agent like DTT. In
this way and by addition of the antibodies at specific stages, it
is possible to investigate at what stage the antibody interferes
with the infection process. Hereto, PER.C6.RTM. cells were
transfected with an H3 HA expression construct harboring HA from
A/Wisconsin/67/2005 and subjected to different treatments as
described in Example 10.
[0224] For this experiment, cells were first incubated with anti-H3
mAbs before trypsin cleavage and subsequently treated as described
above (see FIG. 5).
[0225] Binding of anti-H3 mAbs was detected with PE-conjugated
anti-human antibody according to standard protocols. Fluorescence
signals were measured by FACS analysis. "Cells only" means the
signal obtained after mAb binding to untreated cells and was set at
100%. As can be seen in FIG. 5, the mAbs are still bound to HA
following the different treatments. Since it was shown in Example
10 above that the H3 mAbs CR8020, CR8041 and CR8043 only bind to
the pre-fusion state (i.e., before the conformational shift due to
lower pH), it was concluded that binding of the antibody in fact
inhibits the trypsin cleavage (see also Example 10), at least in
vitro, and thus also the subsequent steps leading to the
conformational change and fusion. Antibody CR8057, which binds the
HA1 part of the HA molecule near the receptor attachment site is
capable of binding to HA after conformational shift and, as
expected, is lost when the HA1 part is removed following disruption
of the disulphide bonds between HA1 and HA2 domains by DTT
treatment.
[0226] The inhibition of trypsin cleavage was subsequently
confirmed in a different in vitro experiment. First, a time course
experiment was done to determine how long H3 HA should be incubated
with trypsin to achieve proper cleavage of HA0 in HA1 and HA2.
Hereto, recombinant soluble H3 HA (A/Wisconsin/67/2005; Protein
Sciences, CT, USA) was incubated in 4 mM Tris.HCl buffer at pH 8.0
containing 6.7 .mu.g/ml Trypsin and 1% N-dodecyl-.beta.-demaltosid.
Trypsin digestion was stopped at several time points by addition of
1% BSA. Samples were run on SDS-page gel (reduced) and blotted
according to standard methods. HA0, HA1 and HA2 bands were detected
using a rabbit anti-H3HA polyclonal antibody (Protein Sciences, CT,
USA). FIG. 6 shows that two hours' incubation is enough for near
complete cleavage evidenced by appearance of the HA1 and HA2 bands
on the reducing gel. Next, recombinant soluble H3 HA was incubated
with either CR8020, CR8041, CR8043 or CR8057 and subsequently
subjected to trypsin cleavage at pH 8.0. Trypsin digestion was
again stopped at several time points by adding 1% BSA. Samples were
run on SDS-page (reduced) and blotted. HA0, HA1 and HA2 bands were
detected using an anti-H3 polyclonal antibody. The results show
that all three mAbs CR8020, CR8041 and CR8043 prevent trypsin
cleavage in vitro since incubation of the H3 HA bound to the
antibody with trypsin results in protection of the HA0 form of HA
on the gel (FIG. 7). In contrast, incubation of H3 HA with a
control mAb (CR8057) at the same conditions results in
disappearance of the HA0 band. This experiment confirms the data
discussed in Example 10 for CR8041 and extends this observation to
CR8020 and CR8043 antibodies. The binding molecules hereof thus
prevent at least trypsin cleavage of the HA0 molecule, at least in
vitro. It is, however, noted that this does not exclude that
additional inhibitory effects are also mediated by the CR8020,
CR8041 and CR8043 mAbs that are more downstream in the process of
infection and result in interference with the pH-induced
conformational shift and/or fusion process.
[0227] To investigate whether this could be the case, the
experiment discussed above was repeated, but now the antibody
CR8043, or the antibody CR8057 as a control, was added to the cells
expressing H3 HA only after trypsin cleavage. Following incubation,
the cells were subsequently incubated in low pH buffer as described
in Example 10 and treated with DTT as described. If the mechanism
of action would be restricted to inhibition of trypsin cleavage, it
is expected that the mAb CR8043 loses binding after pH treatment
since we have established in Example 10 that the antibodies do not
bind to the post-fusion conformation of HA. In contrast, as can be
seen from FIG. 8, mAb CR8043 binding is still detected after
exposure to low pH and subsequent DTT treatment indicating that the
pH-induced conformational shift is also inhibited by CR8043, at
least in vitro. CR8057, which has been shown to bind to the HA1
region of HA, behaves as expected and is no longer detectable when
the HA1 part is lost following DTT treatment.
[0228] To investigate whether antibodies CR8020 and CR8041 are also
capable of blocking the pH-induced conformational change of HA, the
experiments discussed above were repeated. Now the antibodies
CR8020, CR8041 and CR8043, or the antibody CR8057 as a control,
were added to cells expressing either A/Hong Kong/1/1968, A/Hong
Kong/24/1985 or A/Wisconsin/67/2005 subtype H3 HA, either after all
treatments described in above, before low pH incubation or before
trypsin cleavage.
[0229] As shown earlier for A/Wisconsin/67/2005 H3 HA, the CR8020,
CR8041 and CR8043 antibodies recognize an epitope present only
before low pH treatment. This epitope is conserved in the three HAs
used in this experiment as can be seen in FIG. 9, Panel C.
[0230] If the mechanism of action would be restricted to inhibition
of trypsin cleavage, it is expected that the mAbs CR8020, CR8041
and CR8043 lose binding of already cleaved HA after pH treatment
since we have established in Example 10 that the antibodies do not
bind to the post-fusion conformation of HA. In contrast, as can be
seen from FIG. 9, Panel B, mAb binding is still detected after
exposure to low pH and subsequent DTT treatment on all three
different H3 HAs indicating that the pH-induced conformational
shift is also inhibited by CR8020, CR8041 and CR8043, at least in
vitro. CR8057, which has been shown to bind to the highly variable
HA1 region of HA, shows no binding to A/Hong Kong/1/1968 and A/Hong
Kong/24/1985 HAs.
Example 12
In Vitro-Generated Escape Mutants Indicate that the Position of the
Epitope Coincides with a Conserved Sequence in H3 HA
[0231] To investigate to which region in HA CR8020, CR8041 and
CR8043 binds, it was attempted to generate escape mutants in in
vitro cultures. A/Hong Kong/1/1968 viruses were passaged in MDCK
cell cultures in the presence of limiting amounts of monoclonal
antibodies. First, it was determined what concentration of antibody
resulted in a 3 log reduction of virus infection following
inoculation of MDCK cells with 100 TCID50 units mixed with
different amounts of monoclonal antibody and incubation for three
days. This concentration of antibody was added to the inoculum in
serial passages and after each passage, the virus was plaque
titrated in the absence and presence of different amounts of
antibody to determine whether the viruses are still sensitive to
antibody-mediated neutralization. This procedure was followed for
each of the mAbs CR8020, CR8041 and CR8043. From each culture,
escape viruses could be isolated by plaque assay and, of two
isolates of each, viral RNA was extracted and used to determine the
HA sequence. The observed mutated amino acids were as follows:
[0232] CR8020: D19N and Q27L in both analyzed plaques;
[0233] CR8041: G33E in two plaques;
[0234] CR8043: R25M in one and Q34R in the other plaque.
[0235] All three monoclonal antibodies show escape mutations in a
similar domain in the HA2 part of the HA stem region adjacent to
the fusion peptide. Comparison of amino acid sequences of H3N2
viruses present in the NCBI influenza database (on the World Wide
Web at ncbi.nlm.nih.gov/genomes/FLU/Database/select.cgi) in this
region reveals a striking conservation of the sequence. Table 14
depicts the sequence variation in the HA2 region between amino
acids W14 and K39 with the observed escape mutations highlighted.
N=number of strains having a specific sequence. In addition, the
year of isolation (years) is indicated as well as the strains
tested positive in neutralization experiments with the H3
antibodies (Pa=A/Panama/2000/1999; Wis=A/Wisconsin/67/2005;
Hs=A/Hiroshima/52/2005; HK=A/HongKong/1/1968). Of the 1363 H3
viruses present in the database that contained the mentioned HA2
sequence, the majority (81%) had sequences that are present in
virus strains that were shown to be neutralized. Of the remaining
sequences, most have amino acids that can be considered conserved
changes. For the other mutations, a functional neutralization test
will be needed to establish whether the change affects the
functionality of the antibody. Importantly, three amino acid
changes that came up in the escape virus experiment (R25, G33 and
Q34) do not occur in natural influenza sequences and the other two
mutations appeared only in combination (D19 and Q27), a combination
that is also not present in the natural sequences. This could mean
that the mutations have a negative effect on the virus fitness.
Altogether, it is concluded that the antibodies interact with an
epitope on HA2 that is highly conserved between H3 subtype viruses
confirming the broad neutralization capability of the monoclonal
antibodies.
Example 13
Preparation of Monoclonal Antibodies for In Vivo Experiments
[0236] To enable characterization and the subsequent validation of
the IgGs as potential therapeutic antibodies in vivo, they need to
be manufactured and purified in sufficient quantities. The IgGs
were produced in PER.C6.RTM. cells in a 25 L Wave-bag and the
culture was harvested. From the clarified harvest, IgG was purified
using Protein A affinity chromatography and a buffer exchange step.
The monomer content of the purified buffer exchanged IgG is
.about.99% both before and after 0.2 .mu.m sterile filtration.
Additional in vitro virus neutralization assays (VNA) were
performed with the different antibody preparations obtained, as
described above. The results are shown in Table 15.
Example 14
Prophylactic Activity of Human IgG Monoclonal Antibodies Against
Lethal H3N2 Challenge In Vivo
[0237] MAbs CR8020, CR8041 and CR8043 were tested for prophylactic
efficacy in a mouse lethal challenge model with influenza
A/HK/1/68-MA20 (H3N2) virus in female 129X1/SvJ mice (Jackson Labs)
(MA=mouse adapted). A/HK/1/68-MA20 virus was obtained from Prof. E.
G. Brown, University of Ottawa, Ottawa, Ontario, Canada; E. G.
Brown et al. (2001). The virus was passaged once in embryonated
chicken eggs before use in the mice experiments. All mice were
acclimatized and maintained for a period of at least four days
prior to the start of the experiment.
[0238] MAbs were intravenously dosed at 30, 10, 3 and 1 mg/kg in
the tail vein (vena coccygeus) at day -1 before challenge, assuming
an average weight of 18 g per mouse and a fixed dose volume of 0.2
mL. The mice (n=8 per group) were then challenged at day 0 with 25
LD50 A/HK/1/68-MA20 (H3N2) virus by intranasal inoculation. The
actual dose of the virus administered was estimated by titrating a
few replicate samples from the inoculum remaining after inoculation
of the animals was completed. Virus titers (TCID50/mL) of the
inoculum were determined on MDCK cells. The results showed that no
inactivation of virus had unintentionally occurred during
preparation or administration of the inoculum. Clinical signs and
body weights were determined daily from day -1 before challenge
until the end of the study at day 21. Clinical signs were scored
with a scoring system (0=no clinical signs; 1=rough coat; 2=rough
coat, less reactive, passive during handling; 3=rough coat, rolled
up, labored breathing, passive during handling; 4=rough coat,
rolled up, labored breathing, does not roll back on stomach when
laid down on its back). At a score of 4, the animal was euthanized.
To analyze mAb plasma levels at day 0 and determine the presence of
hemagglutination-inhibiting (HI) antibodies at day 21, blood
samples were drawn from all mice on D0, just before challenge, and
on D21 post-infection.
[0239] The mAbs were tested in two separate experiments. In each
experiment, a negative control antibody (CR3014) group was taken
along, dosed at 30 mg/kg. MAb CR8020 was tested in the first
experiment; mAbs CR8041 and CR8043 in the second.
[0240] All mice were active and appeared healthy without showing
signs of disease during the acclimatization period. FIG. 10 shows
the survival rates of the mice following mAb administration. A
clear dose-response relationship was observed, with all groups
dosed with CR8020, CR8041 or CR8043 at 30, 10 or 3 mg/kg showing
100% survival, whereas at 1 mg/kg CR8020, 25% of the mice survived
and none of the mice survived in the 1 mg/kg CR8041 and CR8043
groups. The two control mAb groups showed 0% survival. In the first
experiment, administration of mAb CR8020 resulted in a
statistically significant difference in survival time at all four
concentrations tested, compared to the control group (p<0.005;
Log Rank Test). In the second experiment, administration of mAbs
CR8041 and CR8043 also resulted in a statistically significant
difference in survival time at all four concentrations tested,
compared to the control group (p<0.001 for both mAbs; Log Rank
Test).
[0241] In FIG. 11, the mean body weight change of the mice during
the 21-day study period following mAb administration is shown. Like
with the survival rates, there is a clear inverse relationship
between the weight loss and dose of antibody used. When the
concentration of antibody was increased, the weight loss decreased:
mice in groups dosed with CR8020, CR8041 or CR8043 at 30, 10 or 3
mg/kg showed an increase in mean body weight of approximately
10-15% from day 0 to day 21, consistent with age-related weight
gain, whereas in the 1-mg/kg groups and in the control mAb groups,
the mean body weight of the mice declined in the study period.
[0242] Body weight changes were analyzed in more detail with Area
under the Curve (AUC) analysis. For the purpose of this analysis,
the last observed body weight was carried forward to day 21 if a
mouse died or was euthanized during follow-up of the study.
Briefly, the weight per mouse at day 0 was used as baseline value
and weight change from day 0 to day 21 was determined relative to
baseline. The AUC was defined as the summation of the area above
and the area below the baseline. Mean AUC values of the mAb dose
groups were compared with the respective control groups using
analysis of variance with Dunnet's adjustment for multiple
comparisons (Table 16).
[0243] The analysis showed that the mean AUC of the 3-, 10- and
30-mg/kg groups from CR8020, CR8041 and CR8043 differed
statistically significantly (P<0.001) from that of the
corresponding control groups (Table 16). Both for the CR8041 as
well as for the CR8043 1-mg/kg dose groups, a statistically
significant difference was found when compared to the control group
(p=0.004 and p<0.001 respectively). However, due to the two
surviving mice in the CR8020 1-mg/kg dose groups, an increase in
variation of body weight was observed and, therefore, no
statistical significant difference could be demonstrated when
compared to the control group.
[0244] Additional analysis was performed to investigate a dose
response in the reduction of weight loss by comparing mean AUC
values per antibody concentration for each antibody using analysis
of variance with Tukey's adjustment for multiple comparisons (Table
16). Both for mAbs CR8020 and CR8041, the body weight loss in the
1-mg/kg groups is statistically significantly higher (p<0.001)
than in the respective 3-mg/kg groups, whereas there is no
statistically significant difference between the 3-, 10- and
30-mg/kg groups (p>0.05). For mAb CR8043, both the weight loss
in the 1-mg/kg group was statistically significantly higher than in
the 3-mg/kg group (p<0.001) and that of the 3-mg/kg group was
significantly higher than that of the 10-mg/kg group (p<0.001).
The mean AUC of the 10- and 30-mg/kg groups of CR8043 did not
significantly differ (p=0.997).
[0245] Median clinical scores of the mice are depicted in FIG. 12.
The mice dosed with CR8020, CR8041 or CR8043 at 30 and 10 mg/kg did
not show any clinical signs, as indicated by a median clinical
score of 0 throughout the 21-day study period of the two studies.
MAb 8020 also showed no clinical score in the 3-mg/kg dose group,
whereas in the 3-mg/kg dose groups of mAb 8041 and 8043, increases
in clinical score were observed to a median score of 1 and 3,
respectively. In the 1-mg/kg dose groups of all three mAbs,
clinical scores were increased reaching a median score of 4 in all
groups. Mice observed with clinical score 4 were euthanized on the
same day. The two surviving mice in the CR8020 1-mg/kg dose group
became ill at day 7 of the study and showed a maximum clinical
score of 1 and 3, respectively. Both mice recovered completely. Of
the CR8041 and CR8043 3-mg/kg dose groups, the body weight loss
profile shows a similar pattern as the clinical score profile.
[0246] These results show that at least three human anti-H3N2
antibodies, identified and developed as disclosed herein (CR8020,
CR8041 and CR8043), are each separately able to provide protection
against a lethal dose of influenza H3N2 in vivo. A clear
dose-response relationship between the amount of each antibody
administered and survival rate was observed. The results show that
anti-H3N2 IgG antibody CR8041 and 8043 were able to prevent
clinical manifestation of H3N2 infection in mice when administered
one day prior to infection at a dose of 10 mg/kg or higher. MAb
CR8020 was able to prevent clinical manifestation of H3N2 infection
in mice when administered one day prior to infection at a dose of 3
mg/kg or higher.
Example 15
Protective and Therapeutic Activity of Human IgG Monoclonal
Antibodies Against Lethal H3N2 Challenge In Vivo
[0247] A study was performed to test the therapeutic effect of the
monoclonal antibodies as disclosed herein, exemplified by CR8020,
in a post-infection model, against a lethal H3N2 A/HK/1/68-MA20
influenza virus challenge in vivo.
[0248] Mice (n=10 per group) were intravenously dosed with mAb
CR8020 at 15 mg/kg in the tail vein (vena coccygeus) at day -1
before challenge (group 1; prophylaxis positive control) or at day
1, 2, 3, 4, 5 or 6 after challenge (groups 2-7), assuming an
average weight of 18 g per mouse and a fixed dose volume of 0.2 mL.
Group 8 received negative control mAb CR3014 (15 mg/kg) at day 1
after challenge. The mice were challenged at day 0 with 25 LD50
(2.8 log TCID50) A/HK/1/68-MA20 (H3N2) virus by intranasal
inoculation. The virus batch, type, and age of mice were the same
as used in Example 14. Clinical signs and body weights were
determined daily from day -1 before challenge until the end of the
study at day 21.
[0249] FIG. 13, Panel A, shows the survival rates of the mice
following intravenous administration of mAb CR8020 (15 mg/kg in all
groups) or control mAb (15 mg/kg). When 15 mg/kg mAb CR8020 was
administered at day -1 pre-challenge or day 1 or 2 post-challenge,
all animals survived the viral challenge, whereas the survival rate
in the control mAb group was 0%. When 15 mg/kg mAb CR8020 was
administered at day 3 or 4 after challenge, 50% and 10% survival
was observed, respectively. The survival time of each of these
groups was statistically significantly different compared to the
control group (day 3 group, p<0.001, and day 4 group, p=0.002;
Log Rank Test). Groups treated with 15 mg/kg CR8020 at day 5 or 6
showed a survival rate of 0%. There was no statistically
significant difference in survival time of the day 5 or 6 treated
groups compared to the control group (p=0.648 and p=0.342,
respectively; Log Rank Test).
[0250] In FIG. 13, Panel B, the mean body weight change relative to
day 0 of the mice during the 21-day study period is shown. Like
with the survival rates, there is a clear relationship between
weight loss and time of 15 mg/kg mAb CR8020 administration; when
treatment with 15 mg/kg mAb CR8020 is administered at later time
points, the weight loss increased.
[0251] Body weight changes were statistically analyzed in more
detail using Area under the Curve (AUC) analysis (Table 17). For
area under the curve analysis, the last observed body weight was
carried forward to day 21 if a mouse died or was euthanized during
follow-up of the study. Briefly, the weight per mouse at day 0 was
used as baseline value and weight change from day 0 to day 21 was
determined relative to baseline. The AUC was defined as the
summation of the area above and the area below the baseline.
[0252] Median clinical scores of the mice are depicted in FIG. 13,
Panel C. Of the mice treated with 15 mg/kg CR8020 at day -1
pre-challenge, all survived and none showed any clinical signs
during the observation period. Mice treated with 15 mg/kg CR8020 at
day 1 post-challenge showed a 100% survival, however, four out of
ten animals showed clinical signs, reaching a maximum clinical
score between 1 and 3. Of the animals treated with 15 mg/kg CR8020
at day 2 post-challenge, all survived. However, nine out of ten
animals showed clinical signs reaching a maximum clinical score of
2 or 3. Animals treated with 15 mg/kg CR8020 at day 3
post-challenge showed a 50% survival. Of the survivors (n=5), all
animals showed clinical signs with a maximum clinical score of 3.
Of the animals treated with 15 mg/kg CR8020 at day 4
post-challenge, all, but one mouse died. The surviving mouse showed
clinical signs reaching a maximum clinical score of 2. All mice
that survived across the treatment arms were free from symptoms at
day 21.
[0253] Clinical scores were analyzed using the GENMOD procedure
(SAS) to fit a model for repeated measures with mice as subjects
and data measured on an ordinal scale (Table 18). Since the curves
do have different patterns, the variable "day" was entered as a
class variable in this model. From the groups treated with 15 mg/kg
mAb CR8020 at day -1 before challenge and days 1 and 2
post-challenge in which 100% of the mice survived, the median
clinical score was significantly different from the control mAb
group during most of the study period of 21 days (p.ltoreq.0.001
for all three groups). From the groups treated with 15 mg/kg mAb
CR8020 at day 3 or day 4 post-challenge in which, respectively, 50%
and 10% of the mice survived, the median clinical score was also
significantly different from the control mAb group during most of
the study period of 21 days (p<0.05 for both groups). From the
groups treated with 15 mg/kg mAb CR8020 at day 5 or day 6
post-challenge, the median clinical score was significantly
different from the control mAb group at day 3 only
(p.ltoreq.0.001). This difference, although statistically
significant, is not considered relevant.
[0254] In conclusion, therapy with 15 mg/kg of mAb CR8020 provides
100% protection up to day 2 after challenge in a lethal H3N2 mouse
model. When administered at day 3 or day 4 after challenge,
treatment with 15 mg/kg mAb CR8020 provides partial protection.
When administered at day 5 or day 6 after challenge, no protective
effect of 15 mg/kg mAb CR8020 was observed in the lethal H3N2 mouse
model.
[0255] These results show that a post-infection treatment with a
monoclonal antibody directed against H3N2 influenza virus, as
disclosed herein and exemplified by antibody CR8020, can rescue
mammalian subjects, as showed herein in mice, after challenge with
a lethal dose of H3N2 influenza virus. Even at a late stage, i.e.,
four days post-infection, the antibody is able to partially protect
mice from lethal infection with influenza H3N2 virus. Strikingly,
at day 21 post-infection, all surviving antibody-treated animals
reached normal body weight levels and did not show any remaining
clinical signs.
Example 16
Prophylactic Activity of Human IgG Monoclonal Antibodies Against
Lethal H7N7 Challenge In Vivo
[0256] A study was performed to test the prophylactic effect of the
monoclonal antibodies as disclosed herein, exemplified by CR8020,
against a lethal challenge with H7N7 influenza virus in vivo. MAb
CR8020 was tested for prophylactic efficacy in a mouse lethal
challenge model with mouse-adapted influenza
A/Chicken/Netherlands/621557/2003 (H7N7) virus (Central Veterinary
Institute (CVI), Lelystad, The Netherlands). The A/CH/NL/621557/03
(H7N7) virus was adapted to mice after three lung-to-lung passages.
The mouse-adapted H7N7 Passage 3 virus was propagated in
embryonated chicken eggs in CVI's laboratory. All mice (Balb/c,
female, age six to eight weeks, n=8 per group) were acclimatized
and maintained for a period of at least four days prior to the
start of the experiment. MAb CR8020 was intravenously dosed at 30,
10, 3 or 1 mg/kg in the tail vein (vena coccygeus) at day -1 before
challenge, assuming an average weight of 18 g per mouse and a fixed
dose volume of 0.2 mL. A control group was taken along and dosed
with 30 mg/kg negative control mAb CR3014. The mice were then
challenged at day 0 with 25 LD.sub.50 A/CH/NL/621557/03 (H7N7)
virus by intranasal inoculation. The actual dose of the virus
administered was estimated by titrating a few replicate samples
from the inoculum remaining after inoculation of the animals was
completed. Virus titers (TCID.sub.50/mL) of the inoculum were
determined on MDCK cells. Clinical signs and body weights were
determined daily from day -1 before challenge until the end of the
study at day 21 in the same manner as described in Example 14. To
analyze mAb plasma levels at day 0 and determine the presence of
hemagglutination-inhibiting (HI) antibodies at day 21, blood
samples were drawn from all mice on D0, just before challenge, and
on D21 post-infection.
[0257] All mice were active and appeared healthy without showing
signs of disease during the acclimatization period. FIG. 14, Panel
A, shows the survival rates of the mice, following mAb
administration. Mice dosed with 1 mg/kg mAb CR8020 or more showed a
survival rate of 100%, whereas in the control mAb group, 0%
survived.
[0258] In FIG. 14, Panel B, the mean body weight change of the mice
during the 21-day study period following mAb administration is
shown. In the mAb CR8020 3-, 10- and 30-mg/kg groups, the mice did
not lose weight over the 21-day study period, whereas in the mAb
CR8020 1-mg/kg and control mAb groups, weight loss was observed,
with the mean body weight of the mice in the mAb CR8020 1-mg/kg
group recovering to baseline level at day 21. Body weight changes
were analyzed in more detail with Area under the Curve (AUC)
analysis (Table 19). For area under the curve analysis, the last
observed body weight was carried forward to day 21 if a mouse died
or was euthanized during follow-up of the study. Briefly, the
weight per mouse at day 0 was used as baseline value and weight
change from day 0 to day 21 was determined relative to baseline.
The AUC was defined as the summation of the area above and the area
below the baseline.
[0259] There is a clear inverse relationship between the weight
loss and dose of antibody used. When the concentration of antibody
was increased, the weight loss decreased. The mean difference in
weight loss, as compared to the control mAb, was 47.44, 79.75,
86.71 and 80.48 g*day in the mAb CR8020 1-, 3-, 10- and 30-mg/kg
groups, respectively. All differences were statistically
significant (p<0.001).
[0260] Median clinical scores of the mice are depicted in FIG. 14,
Panel C. All, except one or two, animals within each group showed
clinical signs (score=1) at day 1 post-challenge. This is probably
not related to the viral challenge, since a non-challenged group
taken along in the study showed a similar effect at day 1 (data not
shown).
[0261] Of the mice treated with 3, 10 or 30 mg/kg mAb CR8020 at day
-1 pre-challenge, all survived and none of the animals showed any
clinical signs during the observation period (from day 2 to day 21
post-infection). Mice treated with 1 mg/kg mAb CR8020 at day -1
pre-challenge, showed a 100% survival rate, but all eight mice
showed clinical signs reaching a maximum clinical score of 3.
[0262] These results show that human anti-H3N2 antibody CR8020,
identified and developed as disclosed herein (CR8020), is able to
provide heterosubtypic protection against a lethal dose of
influenza H7N7 in vivo. When administered one day prior to
infection at a dose of 3 mg/kg or higher, mAb CR8020 was able to
completely prevent clinical manifestation of H7N7 infection in
mice. At a dose of 1 mg/kg CR8020 administered one day prior to
infection, all mice survived the lethal challenge, and the body
weight loss and clinical signs observed fully resolved at the end
of the 21-day study period.
[0263] A second study was performed to assess and compare the
prophylactic efficacy of mAb CR8020, CR8041 and CR8043 in the H7N7
mouse model. MAb CR8020, CR8041 and CR8043 (produced in PER.C6.RTM.
cells) were tested for prophylactic efficacy in the mouse lethal
challenge model with mouse-adapted influenza
A/Chicken/Netherlands/621557/2003 (H7N7) virus (Central Veterinary
Institute (CVI), Lelystad, The Netherlands). Briefly, all mice
(Balb/c, female, age six to eight weeks, n=8 per group) were
acclimatized and maintained for a period of at least four days
prior to the start of the experiment. MAb CR8020 was intravenously
dosed at 10, 3 or 1 mg/kg in the tail vein (vena coccygeus) at day
-1 before challenge, assuming an average weight of 18 g per mouse
and a fixed dose volume of 0.2 mL. Mabs CR8041 and CR8043 were
dosed in the same manner at 30, 10, 3 or 1 mg/kg. A control group
was taken along and dosed with 30 mg/kg negative control mAb
CR3014. After mAb administration, the mice were challenged at day 0
with 25 LD.sub.50 mouse-adapted A/CH/NL/621557/03 (H7N7) virus by
intranasal inoculation. Clinical signs and body weights were
determined daily from day -1 before challenge until the end of the
study at day 21.
[0264] In FIG. 15, the survival rates, the percentage of body
weight change and the clinical scores of the mice are depicted,
following prophylactic administration of the mAbs. As shown in FIG.
15, Panel A, 100% survival was observed in the groups receiving 3
or 10 mg/kg CR8020, 10 or 30 mg/kg CR8041 and in the group
receiving 30 mg/kg CR8043. In the control mAb group, the survival
rate was 0%. Prophylactic administration of CR8020 at all three
dose levels and CR8041 at all four dose levels provided a
statistically significant improvement of survival time, compared to
the control mAb group (log-rank, p<0.002). Prophylactic
administration of 1 mg/kg of CR8043 did not result in a
statistically significant improvement of survival time, compared to
the control mAb group (log-rank, p=0.692). Increasing the CR8043
dose to 3 mg/kg or more, resulted in a statistically significant
improvement of survival time, compared to the control mAb group
(log-rank, p.ltoreq.0.034).
[0265] In a post-hoc analysis, the survival times were compared of
the lowest dose groups of mAbs CR8020, CR8041 and CR8043.
Prophylactic administration of 1 mg/kg CR8020 resulted in a
statistically significant improvement of survival time, compared to
1 mg/kg of CR8041 and 1 mg/kg of CR8043 (log-rank, respectively
p=0.029 and p<0.001). In addition, prophylactic administration
of 1 mg/kg CR8041 resulted in a statistically significant
improvement of survival time when compared to 1 mg/kg CR8043
(log-rank, p=0.004).
[0266] In FIG. 15, Panel B, the mean body weight change of the mice
during the 21-day study period following prophylactic
administration of the mAbs is shown. In the mAb CR8020 and mAb
CR8041 1-mg/kg groups, severe weight loss was observed comparable
to that of the control mAb group. In the higher dose groups of mAb
CR8020 and CR8041, weight loss during the 21-day study was limited
or absent. In the groups dosed with mAb CR8043, severe weight loss
was observed in all groups, with the mean body weight of the group
dosed at 30 mg/kg recovering almost to the baseline level at day
21. Body weight changes were analyzed in more detail with Area
under the Curve (AUC) analysis (Table 21). There is a clear inverse
relationship between the weight loss and dose of antibody used.
When the concentration of antibody was increased, the weight loss
decreased. With 1 mg/kg of CR8020, there was no statistically
significant reduction in weight loss compared to the control group
(p=0.356). Increasing the dosing to 3 or 10 mg/kg resulted in a
statistically significant reduction in weight loss, compared to the
control group (p<0.001 in both cases). With 1 mg/kg of CR8041,
there was no statistically significant reduction in weight loss
compared to the control group (p=1).
[0267] Increasing the dosing to 3, 10 or 30 mg/kg CR8041 resulted
in a statistically significant reduction in weight loss, compared
to the control group (p<0.001 in all three cases). With 1, 3 or
10 mg/kg of CR8043, there was no statistically significant
reduction in weight loss compared to the control group (p=0.997,
0.510 and 0.992, respectively). Increasing the dosing to 30 mg/kg
resulted in a statistically significant reduction in weight loss,
compared to the control group (p<0.001). In an additional
analysis of the mean AUC of body weight change data, mAbs CR8020,
CR8041 and CR8043 were compared using a univariate analysis of
variance with antibody and doses included in the model as fixed
factors. Since a dose of 30 mg/kg of CR8020 was not included in the
study, the comparison was limited to antibody doses of 1, 3 and 10
mg/kg. Differences between antibodies were estimated by using
marginal means with Sidak adjustment for multiple comparisons. Over
the three doses of antibody considered, treatment with CR8020
resulted in a statistically significant improved reduction of
weight loss compared to CR8041 and CR8043 (mean difference in
marginal means of 23.73 and 68.29 g*day, respectively, p=0.013 and
p<0.001). In addition, treatment with CR8041 resulted in a
statistically significant improved reduction of weight loss when
compared to CR8043 (difference in marginal means of 44.56 g*day,
p<0.001).
[0268] Median clinical scores of the mice are depicted in FIG. 15,
Panel C. All mice, except one at day 0 (3-mg/kg CR8020 group),
showed clinical signs (score=1, rough coat)) from day 0-day 3. This
increase was not observed in the acclimatization period and at day
-1. The cause of this increased clinical score is not precisely
clear. Of the groups treated with 3 or 10 mg/kg mAb CR8020 at day
-1 pre-challenge, the median clinical score returned to 0 at day 9
post-challenge, whereas in the control group, the median clinical
score reached 4 at day 8, with all mice dead or euthanized at day
9. The CR8020 1-mg/kg group showed a median clinical score of 3
from days 4-13, returning to score 0 at day 15. Of the groups
treated with 3, 10 or 30 mg/kg mAb CR8041 at day -1 pre-challenge,
the median clinical score returned to 0 at day 9, 10 or 12
post-challenge, respectively. The CR8041 1-mg/kg group reached a
median clinical score of 4 at day 10 after challenge. Of the groups
treated with 1, 3 or 10 mg/kg CR8043, the median clinical score
reached 4 at day 9, 9 or 12, respectively, whereas the median
clinical score of the 30-mg/kg CR8043 group reached 3 from days 6
to 13 and returned to 0 at day 14.
[0269] The above results clearly show that human anti-H3N2
antibodies CR8020, CR8041 and CR8043 are able to provide
heterosubtypic protection against a lethal dose of influenza H7N7
in vivo. Mab CR8020 was found to be the most potent of the three
mAbs against the mouse-adapted influenza A/CH/NL/621557/03 (H7N7)
virus, based on the outcome of the post-hoc analyses of survival
times and body weight change. At a dose of 3 or 10 mg/kg mAb CR8020
administered one day prior to infection, 100% of the mice survived
the lethal challenge and clinical manifestation of the H7N7
infection was strongly reduced. At a dose of 1 mg/kg CR8020
administered one day prior to infection, 75% of the mice survived
the lethal challenge in this experiment, and the clinical signs of
the surviving mice resolved completely at day 15 of the 21-day
study period.
Example 17
Therapeutic Activity of Human IgG Monoclonal Antibodies Against
Lethal H7N7 Challenge In Vivo
[0270] This study was performed to assess the therapeutic efficacy
and window of mAb CR8020 in the H7N7 model. MAb CR8020 (produced in
PER.C6.RTM. cells) was tested for therapeutic efficacy in the mouse
lethal challenge model with mouse-adapted influenza
A/Chicken/Netherlands/621557/2003 (H7N7) virus (Central Veterinary
Institute (CVI), Lelystad, The Netherlands). Briefly, all mice
(Balb/c, female, age six to eight weeks, n=8 per group) were
acclimatized and maintained for a period of at least four days
prior to the start of the experiment. MAb CR8020 was intravenously
dosed at 15 mg/kg in the tail vein (vena coccygeus) at day -1
before challenge, (group 1; prophylaxis positive control) or at day
1, 2, 3, 4, 5 or 6 after challenge (groups 2-7), assuming an
average weight of 18 g per mouse and a fixed dose volume of 0.2 mL.
Group 8 received negative control mAb CR3014 (15 mg/kg) at day 1
after challenge. The mice were challenged at day 0 with 25
LD.sub.50 mouse-adapted A/CH/NL/621557/03 (H7N7) virus by
intranasal inoculation. Clinical signs and body weights were
determined daily from day -1 before challenge until the end of the
study at day 21.
[0271] FIG. 16, Panel A, shows the survival rates of the mice,
following intravenous administration of mAb CR8020 (15 mg/kg in all
groups) or control mAb (15 mg/kg). When 15 mg/kg mAb CR8020 was
administered at day 1 pre-challenge or day 1 or 3 post-challenge,
all animals survived the viral challenge, whereas in the control
mAb group, the survival rate was 0%. When 15 mg/kg mAb CR8020 was
administered at days 2 and 4, respectively, 87.5% and 50% survival
was observed. The survival time of these groups was statistically
significantly different from that of the control mAb group (p=0.002
and p=0.014, respectively). Groups treated with 15 mg/kg CR8020 at
days 5 and 6 experienced a survival rate of 0% and there was no
statistically significant difference in survival time of these
groups compared to the control mAb group (p=0.837 and p=0.876,
respectively).
[0272] In FIG. 16, Panel B, the mean body weight change relative to
day 0 of the mice during the 21-day study period is shown. In
general, mean body weight loss increases when mAb CR8020 is
administered at later time points following challenge. However, the
mean body weight curves of the mAb CR8020 day-2 and -3 treatment
groups cross at day 10, due to the single non-surviving mouse in
the day-2 treatment group. Area under the curve analysis of the
body weight changes shows a sharp transition in the mean weight
loss between the treatments at day -1 to day 3 compared to
treatment at days 4 to 6 (Table 22). Treatment with 15 mg/kg of
CR8020 at day -1 pre-challenge or day 1, 2 or 3 post-challenge
resulted in a statistically significant reduction in weight loss
compared to the control group (p<0.001 for all four groups).
Treatment with 15 mg/kg of CR8020 at days 4, 5 or day 6 did not
result in a statistically significant reduction in weight loss
compared to the control group (p=0.566, p=0.979 and p=0.858,
respectively).
[0273] Median clinical scores of the mice are depicted in FIG. 16,
Panel C. Of the animals treated with 15 mg/kg CR8020 at day -1
pre-challenge, all survived and none of the animals showed any
clinical signs during the observation period. Animals treated at
day 1 post-challenge showed a 100% survival, however, seven out of
eight animals showed clinical signs reaching a maximum clinical
score of 1. The eighth animal reached a maximum clinical score of
3. Of the animals treated at day 2 post-challenge, all, but one
animal survived. The surviving animals (seven out of eight) showed
clinical signs reaching a maximum clinical score of 1 (n=4) or 3
(n=3) Animals treated at day 3 post-challenge showed a 100%
survival and all animals showed clinical signs with a maximum
clinical score of 3. Of the animals treated at day 4
post-challenge, 50% survived the lethal challenge. The surviving
animals showed clinical signs reaching a maximum clinical score of
3. Animals treated at day 5 or 6 post-challenge did not survive.
Clinical scores were analyzed using the GENMOD procedure (SAS) to
fit a model to repeated measures with mice as subjects and data
measured on an ordinal scale (Table 23). From the groups treated
with 15 mg/kg mAb CR8020 at day -1 before challenge and day 1, 2, 3
or 4 post-challenge, the median clinical score was statistically
significantly different from the control mAb group during most of
the study period of 21 days (days 8-21; p.ltoreq.0.038 for all four
groups). From the group treated with 15 mg/kg mAb CR8020 at day 5
post-challenge, the median clinical score was significantly
different from the control mAb group at day 8 only
(p.ltoreq.0.001). This difference, although statistically
significant, is not considered relevant. The median clinical score
of the 15-mg/kg mAb CR8020 day 6 treatment group was not
statistically different from the control group.
[0274] This study clearly shows that therapy with 15 mg/kg of mAb
CR8020 provides 87.5%-100% protection when administered up to day 3
after challenge in a lethal H7N7 mouse model. When administered at
day 4 after challenge, treatment with 15 mg/kg mAb CR8020 provides
partial protection. When administered at day 5 or day 6 after
challenge, no protective effect of 15 mg/kg mAb CR8020 was observed
in the lethal H7N7 mouse model. In other words, when administered
four days or more before death, CR8020 provided protection in this
lethal mouse model.
Example 18
Cocktail of Monoclonal Antibodies that Efficiently Neutralizes
Multiple Influenza Subtypes from Phylogenetic Groups 1 and 2
[0275] The seasonal influenza vaccine each year consists of two
different preparations inducing immunity to influenza A strains, a
representative for the circulating H1 subtype and a representative
for the circulating H3 strain. The underlying reason for this is
that influenza strains from the H1 and H3 subtypes are so much
different that the vaccines prepared from either type does not
induce protection against the other subtype. Ideally, a broadly
protective monoclonal antibody preparation to treat influenza would
be effective against influenza strains from both the phylogenetic
group 1 (H1) and group 2 (H3). However, again due to the sequence
differences between the HA molecules, such a single antibody is
hard to find. For example, the Fab28 antibody described in WO
2009/115972 binds and neutralizes H1 subtypes much better than H3
subtype viruses, probably due to the lesser conservation of the
epitope between the group 1 and group 2 viruses compared to viruses
within a phylogenetic group. To reach the goal of a single product
effective against multiple influenza subtypes from both
phylogenetic groups, one may thus have to combine two or more
different antibodies in a cocktail. In order to be successful, such
preparation should consist of antibodies that do not interfere with
each other.
[0276] Antibodies that efficiently neutralize viruses from H1, H5
and H9 subtypes have been described in WO2008/028946, with
antibodies CR6261 and CR6323 as typical examples. The binding
region (epitope) of CR6261 has been elucidated in detail using
co-crystallization of H1 or H5 HA molecules and CR6261 (see also
PDB database entries 3GBM and 3GBM at http://www.pdb.org and Ekiert
et al., 2009). To investigate whether the monoclonal antibodies
hereof can be used in combination with the previously described
CR6261 antibodies, it was tested whether the antibodies were able
to bind to subtypes from both phylogenetic groups 1 and 2. Hereto,
ELISA and FACS binding experiments were done as described in
Example 7 using HA molecules of H1 and H5 subtypes, as well as H3
and H7 subtypes with CR6261, CR6323, CR8001, CR8020, CR8041 and
CR8043. The results are summarized in Table 20 and show that the
antibodies that broadly neutralize viruses of group 1 do not bind
to viruses of group 2 and vice versa. Since the antibodies do not
interfere with each other, it can be expected that the neutralizing
potency of the antibodies against the respective subtypes will be
maintained, resulting in efficient neutralization of both group 1
and 2 subtypes.
[0277] Therefore, a cocktail comprising CR6261 and/or CR6323 on the
one hand and CR8020, CR8041, and/or CR8043 on the other hand will
be active against viruses of at least both subtypes H1 and H3.
Thus, efficient protection is possible to influenza subtypes from
phylogenetic groups 1 and 2 using one preparation.
Example 19
Binding Kinetics of the Binding Molecules
[0278] The affinities of papain-cleaved Fab fragments of CR8020 and
CR8043 were measured using the Octet RED system and streptavidin
biosensors from ForteBio. Influenza hemagglutinin antigens of the
H3 subtypes A/Wisconsin/67/2005 (Protein Science) and
A/Brisbane/10/2007 (Protein Science) were biotinylated for
immobilization to streptavidin biosensors (ForteBio). Fab binding
experiments were repeated five times using a concentration range
between 2.3-150 nM and 0.16-30 nM for CR8020 and CR8043,
respectively, in kinetic buffer (ForteBio, 18.5032). The
experimental set-up for affinity measurements on the Octet was as
follows: Immobilization of the biotinylated hemagglutinin to
streptavidin biosensors for 1800 seconds, followed by association
of the serial diluted Fabs CR8020 and CR8043 for 1200 seconds, and
subsequent dissociation in kinetic buffer for 1800 seconds. Binding
data were analyzed with Octet Analysis software using the 1:1
model.
[0279] The affinity constant (K.sub.d-value) of the binding
molecules for HA of the H3 subtype are shown in Table 24.
TABLE-US-00001 TABLE 1 First round Vkappa, Vlambda and VH
amplifications Primer name Primer nucleotide sequence SEQ ID NO:
OK1 (HuVK1B) GAC ATC CAG WTG ACC CAG TCT CC 192 OK2 (HuVK2) GAT GTT
GTG ATG ACT CAG TCT CC 193 OK3 (HuVK2B2) GAT ATT GTG ATG ACC CAG
ACT CC 194 OK4 (HuVK3B) GAA ATT GTG WTG ACR CAG TCT CC 195 OK5
(HuVK5) GAA ACG ACA CTC ACG CAG TCT CC 196 OK6 (HuVK6) GAA ATT GTG
CTG ACT CAG TCT CC 197 OCK (HuCK) ACA CTC TCC CCT GTT GAA GCT CTT
198 OL1 (HuVL1A) * CAG TCT GTG CTG ACT CAG CCA CC 199 OL1 (HuVL1B)
* CAG TCT GTG YTG ACG CAG CCG CC 200 OL1 (HuVL1C) * CAG TCT GTC GTG
ACG CAG CCG CC 201 OL2 (HuVL2B) CAG TCT GCC CTG ACT CAG CC 202 OL3
(HuVL3A) TCC TAT GWG CTG ACT CAG CCA CC 203 OL4 (HuVL3B) TCT TCT
GAG CTG ACT CAG GAC CC 204 OL5 (HuVL4B) CAG CYT GTG CTG ACT CAA TC
205 OL6 (HuVL5) CAG GCT GTG CTG ACT CAG CCG TC 206 OL7 (HuVL6) AAT
TTT ATG CTG ACT CAG CCC CA 207 OL8 (HuVL7/8) CAG RCT GTG GTG ACY
CAG GAG CC 208 OL9 (HuVL9) # CWG CCT GTG CTG ACT CAG CCM CC 209 OL9
(HuVL10) # CAG GCA GGG CTG ACT CAG 210 OCL (HuCL2) X TGA ACA TTC
TGT AGG GGC CAC TG 211 OCL (HuCL7) X AGA GCA TTC TGC AGG GGC CAC TG
212 OH1 (HuVH1B7A) + CAG RTG CAG CTG GTG CAR TCT GG 213 OH1
(HuVH1C) + SAG GTC CAG CTG GTR CAG TCT GG 214 OH2 (HuVH2B) CAG RTC
ACC TTG AAG GAG TCT GG 215 OH3 (HuVH3A) GAG GTG CAG CTG GTG GAG 216
OH4 (HuVH3C) GAG GTG CAG CTG GTG GAG WCY GG 217 OH5 (HuVH4B) CAG
GTG CAG CTA CAG CAG TGG GG 218 OH6 (HuVH4C) CAG STG CAG CTG CAG GAG
TCS GG 219 OH7 (HuVH6A) CAG GTA CAG CTG CAG CAG TCA GG 220 OCM
(HuCIgM) TGG AAG AGG CAC GTT CTT TTC TTT 221 * Mix in 1:1:1 ratio #
Mix in 1:1 ratio X Mix in 1:1 ratio + Mix in 1:1 ratio
TABLE-US-00002 TABLE 2 Second round Vkappa, Vlambda and VH
amplifications Primer name Primer nucleotide sequence SEQ ID NO
OK1S (HuVK1B-SAL) TGA GCA CAC AGG TCG ACG GAC ATC CAG WTG ACC 222
CAG TCT CC OK2S (HuVK2-SAL) TGA GCA CAC AGG TCG ACG GAT GTT GTG ATG
ACT 223 CAG TCT CC OK3S (HuVK2B2-SAL) TGA GCA CAC AGG TCG ACG GAT
ATT GTG ATG ACC 224 CAG ACT CC OK4S (HuVK3B-SAL) TGA GCA CAC AGG
TCG ACG GAA ATT GTG WTG ACR 225 CAG TCT CC OK5S (HuVK5-SAL) TGA GCA
CAC AGG TCG ACG GAA ACG ACA CTC ACG 226 CAG TCT CC OK6S (HuVK6-SAL)
TGA GCA CAC AGG TCG ACG GAA ATT GTG CTG ACT 227 CAG TCT CC OJK1
(HuJK1-NOT) GAG TCA TTC TCG ACT TGC GGC CGC ACG TTT GAT 228 TTC CAC
CTT GGT CCC OJK2 (HuJK2-NOT) GAG TCA TTC TCG ACT TGC GGC CGC ACG
TTT GAT 229 CTC CAG CTT GGT CCC OJK3 (HuJK3-NOT) GAG TCA TTC TCG
ACT TGC GGC CGC ACG TTT GAT 230 ATC CAC TTT GGT CCC OJK4
(HuJK4-NOT) GAG TCA TTC TCG ACT TGC GGC CGC ACG TTT GAT 231 CTC CAC
CTT GGT CCC OJK5 (HuJK5-NOT) GAG TCA TTC TCG ACT TGC GGC CGC ACG
TTT AAT 232 CTC CAG TCG TGT CCC OL1S (HuVL1A-SAL) * TGA GCA CAC AGG
TCG ACG CAG TCT GTG CTG ACT 233 CAG CCA CC OL1S (HuVL1B-SAL) * TGA
GCA CAC AGG TCG ACG CAG TCT GTG YTG ACG 234 CAG CCG CC OL1S
(HuVL1C-SAL) * TGA GCA CAC AGG TCG ACG CAG TCT GTC GTG ACG 235 CAG
CCG CC OL2S (HuVL2B-SAL) TGA GCA CAC AGG TCG ACG CAG TCT GCC CTG
ACT 236 CAG CC OL3S (HuVL3A-SAL) TGA GCA CAC AGG TCG ACG TCC TAT
GWG CTG ACT 237 CAG CCA CC OL4S (HuVL3B-SAL) TGA GCA CAC AGG TCG
ACG TCT TCT GAG CTG ACT 238 CAG GAC CC OL5S (HuVL4B-SAL) TGA GCA
CAC AGG TCG ACG CAG CYT GTG CTG ACT 239 CAA TC OL6S (HuVL5-SAL) TGA
GCA CAC AGG TCG ACG CAG GCT GTG CTG ACT 240 CAG CCG TC OL7S
(HuVL6-SAL) TGA GCA CAC AGG TCG ACG AAT TTT ATG CTG ACT 241 CAG CCC
CA OL8S (HuVL7/8-SAL) TGA GCA CAC AGG TCG ACG CAG RCT GTG GTG ACY
242 CAG GAG CC OL9S (HuVL9-SAL) # TGA GCA CAC AGG TCG ACG CWG CCT
GTG CTG ACT 243 CAG CCM CC OL9S (HuVL10-SAL) # TGA GCA CAC AGG TCG
ACG CAG GCA GGG CTG ACT 244 CAG OJL1 (HuJL1-NOT) GAG TCA TTC TCG
ACT TGC GGC CGC ACC TAG GAC 245 GGT GAC CTT GGT CCC OJL2
(HuJL2/3-NOT) GAG TCA TTC TCG ACT TGC GGC CGC ACC TAG GAC 246 GGT
CAG CTT GGT CCC OJL3 (HuJL7-NOT) GAG TCA TTC TCG ACT TGC GGC CGC
ACC GAG GAC 247 GGT CAG CTG GGT GCC OH1S (HuVH1B-SFI) + GTC CTC GCA
ACT GCG GCC CAG CCG GCC ATG GCC 248 CAG RTG CAG CTG GTG CAR TCT GG
OH1S (HuVH1C-SFI) + GTC CTC GCA ACT GCG GCC CAG CCG GCC ATG GCC 249
SAG GTC CAG CTG GTR CAG TCT GG OH2S (HuVH2B-SFI) GTC CTC GCA ACT
GCG GCC CAG CCG GCC ATG GCC 250 CAG RTC ACC TTG AAG GAG TCT GG OH3S
(HuVH3A-SFI) GTC CTC GCA ACT GCG GCC CAG CCG GCC ATG GCC 251 GAG
GTG CAG CTG GTG GAG OH4S (HuVH3C-SFI) GTC CTC GCA ACT GCG GCC CAG
CCG GCC ATG GCC 252 GAG GTG CAG CTG GTG GAG WCY GG OH5S
(HuVH4B-SFI) GTC CTC GCA ACT GCG GCC CAG CCG GCC ATG GCC 253 CAG
GTG CAG CTA CAG CAG TGG GG OH6S (HuVH4C-SFI) GTC CTC GCA ACT GCG
GCC CAG CCG GCC ATG GCC 254 CAG STG CAG CTG CAG GAG TCS GG OH7S
(HuVH6A-SFI) GTC CTC GCA ACT GCG GCC CAG CCG GCC ATG GCC 255 CAG
GTA CAG CTG CAG CAG TCA GG OJH1 (HuJH1/2-XHO) GAG TCA TTC TCG ACT
CGA GAC RGT GAC CAG GGT 256 GCC OJH2 (HuJH3-XHO) GAG TCA TTC TCG
ACT CGA GAC GGT GAC CAT TGT 257 CCC OJH3 (HuJH4/5-XHO) GAG TCA TTC
TCG ACT CGA GAC GGT GAC CAG GGT 258 TCC OJH4 (HuJH6-XHO) GAG TCA
TTC TCG ACT CGA GAC GGT GAC CGT GGT 259 CCC * Mix in 1:1:1 ratio #
Mix in 1:1 ratio + Mix in 1:1 ratio
TABLE-US-00003 TABLE 3 Second round VL regions amplification
overview Share in Share in Template 5' primer 3' primer Product
PK/PL (%) Pool VL (%) K1 OK1S OJK1 K1J1 25 PK1 30 OK1S OJK2 K1J2 25
OK1S OJK3 K1J3 10 OK1S OJK4 K1J4 25 OK1S OJK5 K1J5 15 K2 OK2S OJK1
K2J1 25 PK2 4 OK2S OJK2 K2J2 25 OK2S OJK3 K2J3 10 OK2S OJK4 K2J4 25
OK2S OJK5 K2J5 15 K3 OK3S OJK1 K3J1 25 PK3 1 OK3S OJK2 K3J2 25 OK3S
OJK3 K3J3 10 OK3S OJK4 K3J4 25 OK3S OJK5 K3J5 15 K4 OK4S OJK1 K4J1
25 PK4 19 OK4S OJK2 K4J2 25 OK4S OJK3 K4J3 10 OK4S OJK4 K4J4 25
OK4S OJK5 K4J5 15 K5 OK5S OJK1 K5J1 25 PK5 1 OK5S OJK2 K5J2 25 OK5S
OJK3 K5J3 10 OK5S OJK4 K5J4 25 OK5S OJK5 K5J5 15 K6 OK6S OJK1 K6J1
25 PK6 5 OK6S OJK2 K6J2 25 OK6S OJK3 K6J3 10 OK6S OJK4 K6J4 25 OK6S
OJK5 K6J5 15 L1 OL1S OJL1 L1J1 30 PL1 14 OL1S OJL2 L1J2 60 OL1S
OJL3 L1J3 10 L2 OL2S OJL1 L2J1 30 PL2 10 OL2S OJL2 L2J2 60 OL2S
OJL3 L2J3 10 L3 OL3S OJL1 L3J1 30 PL3 10 OL3S OJL2 L3J2 60 OL3S
OJL3 L3J3 10 L4 OL4S OJL1 L4J1 30 PL4 1 OL4S OJL2 L4J2 60 OL4S OJL3
L4J3 10 L5 OL5S OJL1 L5J1 30 PL5 1 OL5S OJL2 L5J2 60 OL5S OJL3 L5J3
10 L6 OL6S OJL1 L6J1 30 PL6 1 OL6S OJL2 L6J2 60 OL6S OJL3 L6J3 10
L7 OL7S OJL1 L7J1 30 PL7 1 OL7S OJL2 L7J2 60 OL7S OJL3 L7J3 10 L8
OL8S OJL1 L8J1 30 PL8 1 OL8S OJL2 L8J2 60 OL8S OJL3 L8J3 10 L9 OL9S
OJL1 L9J1 30 PL9 1 OL9S OJL2 L9J2 60 OL9S OJL3 L9J3 10 VL 100%
TABLE-US-00004 TABLE 4 Second round VH regions amplification
overview Share in Share in Template 5' primer 3' primer Product
PK/PL (%) Pool VH (%) H1 OH1S OJH1 H1J1 10 PH1 25 OH1S OJH2 H1J2 10
OH1S OJH3 H1J3 60 OH1S OJH4 H1J4 20 H2 OH2S OJH1 H2J1 10 PH2 2 OH2S
OJH2 H2J2 10 OH2S OJH3 H2J3 60 OH2S OJH4 H2J4 20 H3 OH3S OJH1 H3J1
10 PH3 25 OH3S OJH2 H3J2 10 OH3S OJH3 H3J3 60 OH3S OJH4 H3J4 20 H4
OH4S OJH1 H4J1 10 PH4 25 OH4S OJH2 H4J2 10 OH4S OJH3 H4J3 60 OH4S
OJH4 H4J4 20 H5 OH5S OJH1 H5J1 10 PH5 2 OH5S OJH2 H5J2 10 OH5S OJH3
H5J3 60 OH5S OJH4 H5J4 20 H6 OH6S OJH1 H6J1 10 PH6 20 OH6S OJH2
H6J2 10 OH6S OJH3 H6J3 60 OH6S OJH4 H6J4 20 H7 OH7S OJH1 H7J1 10
PH7 1 OH7S OJH2 H7J2 10 OH7S OJH3 H7J3 60 OH7S OJH4 H7J4 20 VH
100%
TABLE-US-00005 TABLE 5 Characteristics of the individual IgM memory
B cell libraries. IgM memory libraries Cells Libraries Total PBL %
memory Size % Insert % % Donor (.times.10.sup.6) B cells
(.times.10.sup.6) frequency ORF Unique Individual 1 3 96 74 98
Individual 2 72.5 1.7 5 98 79 98 Individual 3 67.5 1.4 3 96 79 98
Individual 4 132.5 2.3 6 98 69 99
TABLE-US-00006 TABLE 6 Cross-binding activity of single-chain phage
antibodies to HA molecules of different HA subtypes as measured by
ELISA (ELISA titer; OD 492 nm). SC # H3 H7 HB sc08-001 0.885 2.451
x sc08-003 1.320 0.222 x sc08-006 0.511 0.227 x sc08-007 0.074
2.365 x sc08-009 0.095 1.130 x sc08-010 0.165 1.242 x sc08-011
0.090 1.802 x sc08-013 0.078 1.400 x sc08-014 0.239 0.834 x
sc08-015 0.727 0.165 x sc08-016 1.112 0.164 x sc08-017 1.158 0.285
x sc08-018 0.711 0.221 x x = not determined; H3 = HA of H3 subtype;
H7 = HA of H7 subtype; HB = HA of influenza virus B.
TABLE-US-00007 TABLE 7 Cross-binding activity of
PEG/NACl-precipitated and filter- sterilized phage antibodies to HA
molecules of different HA subtypes as measured by ELISA (OD 492
nm). SC # H1 H3 H5 H7 B(o) sc08-001 + + - + - sc08-003 - + - - -
sc08-006 - + - - - sc08-007 - - - + - sc08-009 - - - + - sc08-010 -
- - + - sc08-011 - - - + - sc08-013 - - - + - sc08-014 + + - + -
sc08-015 - + - - - sc08-016 - + - - - sc08-017 - + - - - sc08-018 -
+ - - - H1 = HA of H1 subtype, H3 = HA of H3 subtype; H5 = HA of H5
subtype; H7 = HA of H7 subtype; B(o) = HA of influenza virus
B/Ohio/01/2005.
TABLE-US-00008 TABLE 8 FACS analysis of PEG/NACl-precipitated and
filter-sterilized phage antibodies (expressed as MFI = mean
fluorescence intensity). PER.C6 = untransfected PER.C6 .RTM. cells
(control); mH1, mH3, mH5, mH7, mHB = membrane bound HA of the
subtypes H1, H3, H5, H7 and influenza B subtypes respectively. SC #
PER.C6 .RTM. mH1 mH3 mH5 mH7 mHB sc08-001 2 27 68 5 62 x sc08-003 5
9 77 7 7 x sc08-006 2 6 69 5 6 x sc08-007 1 5 4 4 73 x sc08-009 11
12 11 10 15 x sc08-010 2 4 3 4 60 x sc08-011 1 3 4 4 73 x sc08-013
2 5 3 7 61 x sc08-014 10 26 82 17 32 x sc08-015 3 7 79 7 6 x
sc08-016 1 7 82 5 5 x sc08-017 1 6 81 5 5 x sc08-018 2 6 74 6 7
x
TABLE-US-00009 TABLE 9 Data of the CDR regions of the HA specific
immunoglobulins (SEQ ID NO:). Vh VI IgG# locus HCDR1 HCDR2 HCDR3
locus LCDR1 LCDR2 LCDR3 CR8001 3-53 SNYVS (81) LIYTGGTTYYADSVK
VSALRFLQWPNYAM 1-4 SGTRSDVGGHNY EVSHRPS (85) SSYTGEGPLGV G (82) DV
(83) VS (84) (86) CR8003 3-7 SYWMS (87) NMKQDGSEKYYVDS
GSCDDSWTGCHDA 2-14 GGNNIGSKSVH DSARPS (91) QVWESGSDLR VKG (88) FDI
(89) (90) LL (92) CR8015 3-7 SYWMS (87) NMKQDGSEKYYVDS
GSCDDSWTGCHDA 2-14 GGDNIGRKSVH DNSDRPS (94) HVWGSSRDHY VKG (88) FDI
(89) (93) V (95) CR8016 3-7 SYWMS (87) NMKQDGSEKYYVDS GSCDDSWTGCHDA
1-13 TGSSSNIGAGYD GNN (97)RPS QSYDSSLSVYV VKG (88) FDI (89) VH (96)
(98) CR8017 3-7 SYWMS (87) NMKQDGSEKYYVDS GSCDDSWTGCHDA 2-13
QGDSLRSYYAS AKTNRPS (100) NSRDSSGNHV VKG (88) FDI (89) (99) V (101)
CR8018 3-7 SYWMS (87) NMKQDGSEKYYVDS GSCDDSWTGCHDA 1-4 TGTSSDVGGYNY
EVSHRPS (85) SSYTGEGPLGV VKG (88) FDI (89) VS (102) (86) CR8019
3-23 TSAMS (103) GISGSGATTYYAGSV DTSLFEYDTSGFTAP O12 RASQSISGYLN
GASTLQS (107) QQTYTSPPYA KG (104) GNAFDI (105) (106) (108) CR8020
1-18 RFGVS (109) WISAYNGDTYYAQK EPPLFYSSWSLDN A27 ARASQSVSMNYL
GASRRAT (113) QQYGTSPRT FQA (110) (111) A (112) (114) CR8021 3-23
AYAMN (115) AIGGSGGSTYYADS GRDWTGGYFFDS B3 KSSQSIFYSSNNK WASTRES
(119) QQYYSIPYT VKG (116) (117) NYLT (118) (120) CR8038 3-23 GYAMS
(121) DIGGSGGGTYYADS SSSWDRAYFFDS B3 KSSQSVLYSSIHK WASTRES (119)
QQYYRSPPT VKG (122) (123) NYLA (124) (125) CR8039 4-59 SYYWS (126)
YIYYRGGTSYNPSLK KDWGSAAGSVWYF 1-2 TGTSSDVGGYNY EVSKRPS (130)
SSYAGSNNLI S (127) DL (128) VS (129) (131) CR8040 3-33 SYGMH (132)
FIWYDGSNKHYADS DGGYSTWEWYFDL A26 RASQGIGSNLH YASQSIT (136)
HQSSSLPLT MKG (133) (134) (135) (137) CR8041 1-18 SFGLS (138)
WISAYNGEIKYAQKF EPPLYFSSWSLDF A27 ARASQSVSSNYL GASRRAT (142)
QQYDSSPRT QG (139) (140) A (141) (143) CR8043 1-03 AYSMH (144)
WINTAIGNTQYSQK GASWDARGWSGY B3 KSSQSVFSSSTN WSSTRES (148) HQYYTAPWT
FQD (145) (146) KNYLA (147) (149) CR8049 2-26 NTRMGVS
HIFSNDETSYRTSLK IGSGYESSAYSTWL 2-14 EGDTIGSKSVH NDRDRPS (154)
QVWESGGDQT (150) R (151) DP (152) (153) V (155) CR8050 4-34 DHYWS
EVVHSGDTNYTPSL GRNVAVVGAIQRHY A27 RASQSVSRNYLA GASSRAT (160)
QHYGSVLVA (156) RN (157) DY (158) (159) (161) CR8052 4-61 SGTYYWS
DISYSGSTNYNPSLK AMAAYNYDRGGYN O12 RASQGINTYLN AASTLQS (166)
QQSYSTAIT (162) S (163) DYYYMDV (164) (165) (167) CR8055 3-33 TYGMH
(168) FIWYDGSNKHYQDS DGGYSTWEWYFDL A26 RASRSIGSDLH FASQSMS (172)
HQSSSLPLT VKG (169) (170) (171) (137) CR8057 3-53 VIFMS (173)
IIYIDDSTYYADSVK ESGDFGDQTGPYHY 2-14 TGSSGDIGGYNA EVTSRPS (177)
CSFADSNILI G (174) YAMDV (175) VS (176) (178) CR8069 3-43 DYTMH
(179) LISWDGGMSNYADS DIRPRMPARHFMDV L2 RASQNVNYNLA VASTRAT (183)
QQYNNWPPA VKG (180) (181) (182) IT (184)
TABLE-US-00010 TABLE 10 Data of the HA-specific IgGs. SEQ ID NOs of
the nucleotide and amino acid sequences of the variable regions of
the heavy and light chains SEQ ID SEQ ID SEQ ID SEQ ID NO of NO of
NO of NO of nucleotide amino acid nucleotide amino acid sequence
sequence sequence sequence heavy chain heavy chain light chain
light chain variable variable variable variable Name IgG region
region region region CR8001 1 2 3 4 CR8003 5 6 7 8 CR8015 9 10 11
12 CR8016 13 14 15 16 CR8017 17 18 19 20 CR8018 21 22 23 24 CR8019
25 26 27 28 CR8020 29 30 31 32 CR8021 33 34 35 36 CR8038 37 38 39
40 CR8039 41 42 43 44 CR8040 45 46 47 48 CR8041 49 50 51 52 CR8043
53 54 55 56 CR8049 57 58 59 60 CR8050 61 62 63 64 CR8052 65 66 67
68 CR8055 69 70 71 72 CR8057 73 74 75 76 CR8069 77 78 79 80
TABLE-US-00011 TABLE 11 In vitro neutralization of influenza virus
H3N2 by selected IgGs Neutralization titer SK50 (.mu.g/ml) IgG #
A/Wisconsin/67/2005 CR8001 11.95 CR8003 5.31 CR8015 23.78 CR8016
1.77 CR8017 2.82 CR8018 6.03 CR8019 1.98 CR8020 8.45 CR8021 1.77
CR8038 3.54 CR8039 1.8 CR8040 >40 CR8041 3.99 CR8043 1.49 CR8049
3.26 CR8050 1.77 CR8052 >40 CR8055 1.07 CR8057 0.011 CR8069
ND
TABLE-US-00012 TABLE 12 Cross-binding reactivity of anti-H3N2 IgGs.
NCal. = A/New Caledonia/20/1999 (H1N1); Wisc. = A/Wisconsin/67/2005
(H3N2); NY. = A/New York/55/2004 (H3N2), Wyo. = A/Wyoming/3/2003
(H3N2); Neth. = A/Netherlands/219/2003 (H7N7); ND = not done. IgG
Elisa binding (titration) Facs binding, [IgG] = 5 .mu.g/ml, H1 H3
H3 H3 H7 MFI IgG # NCal. Wisc NY Wyo Neth PerC6 H1 H3 H7 CR8001 + +
+ + + 4 100 763 106 CR8003 - + + + - 3 3 657 5 CR8015 - + + + - 3 4
600 4 CR8016 - + + + - 3 3 840 5 CR8017 - + + + - 3 3 558 4 CR8018
- + + + - 3 3 348 4 CR8019 - + - + - 3 4 685 6 CR8020 - + + + + 4 3
657 140 CR8021 - + + + + 4 4 678 4 CR8038 - + + + - ND ND ND ND
CR8039 - + + + - 4 4 503 4 CR8040 - + + + - 4 5 446 4 CR8041 - + +
+ + 4 4 364 120 CR8043 - + + + + 4 4 646 11 CR8049 - + + + - 3 3
542 4 CR8050 - + + + - 6 8 282 6 CR8052 - + + + - 4 4 364 5 CR8055
- - - - - 21 31 433 26 CR8057 - + + + low 7 8 943 15 CR8069 - + + +
- 4 6 447 5
TABLE-US-00013 TABLE 13 Cross-neutralizing activity of anti-H3N2
IgGs; ND = not done Neutralization titer SK50 ( .mu.g/ml) H1 H2 A/
A/ H7 H10 A/New A/ Solomon A/ A/ A/ A/ Hong A/Mallard/ A/Chick/
Caledonia/ Brisbane/ islands/ Wisconsin/ Hiroshima/ Panama/
Johannesburg/ Kong/ Netherlands/ Germany/ IgG # 20/1999 59/1007
IVR-145 67/2005 52/2005 2000/1999 33/1994 1/1968 12/2000 N/49
CR8001 >40 >40 >40 11.95 13.02 >40 6.51 7.07 >40
>40 CR8003 >40 >40 >40 5.31 4.27 >40 >40 ND
>40 >40 CR8015 >40 >40 >40 23.78 28.28 >40 >40
ND >40 >40 CR8016 >40 >40 >40 1.77 8.84 28.28 >40
ND >40 >40 CR8017 >40 >40 >40 2.82 13.55 >40
>40 ND >40 >40 CR8018 >40 >40 >40 6.03 8.45
>40 >40 ND >40 >40 CR8019 >40 >40 >40 1.98
0.88 >40 0.88 ND >40 >40 CR8020 >40 >40 >40 8.45
11.95 7.74 7.07 1.77 0.028 17.68 CR8021 ND >40 >40 1.77 2.5
>40 3.54 14.14 >40 >40 CR8038 ND ND ND 3.54 7.07 >40
5.95 ND >40 >40 CR8039 >40 >40 >40 1.8 3.26 4.6 1.33
2.97 >40 >40 CR8040 >40 >40 >40 >40 >40 >40
6.77 ND >40 >40 CR8041 >40 >40 >40 3.99 4.75 2.99
1.69 1.05 1.105 25 CR8043 >40 >40 >40 1.49 3.54 10.15 2.66
4.2 >40 14.87 CR8049 >40 >40 >40 3.26 3.54 >40
>40 ND >40 >40 CR8050 ND ND ND 1.77 ND 6.5 1.49 ND >40
>40 CR8052 >40 >40 >40 >40 >40 21.89 >40 ND
>40 >40 CR8055 >40 >40 >40 1.07 1.15 >40 3.38 ND
>40 >40 CR8057 >40 >40 >40 0.011 0.0068 0.022 2.17
2.17 >40 >40 CR8069 >40 >40 >40 ND 3.54 11.89 3.54
11.89 >40 >40
TABLE-US-00014 TABLE 14 Sequence conservation around the binding
region of the H3 mAbs CR8020, CR8041 and CR8043 HA2 position 14 15
16 17 18 19 20 21 22 23 24 25 26 27 28 29 Consensus W E G M V D G W
Y G F R H Q N S Group_l -- -- -- -- -- -- -- -- -- -- -- -- -- --
-- -- Group_2 -- -- -- -- -- -- -- -- -- -- -- -- -- -- -- --
Group_3 -- -- -- -- M -- -- -- -- -- -- -- -- -- -- -- Group_4 --
-- -- -- -- -- -- -- -- -- -- -- -- -- -- -- Group_5 -- -- -- -- I
-- -- -- -- -- -- -- -- -- -- -- Group_6 -- -- -- -- -- -- -- -- --
-- -- -- -- -- -- -- Group_7 -- -- -- -- -- -- -- -- -- -- -- -- --
L -- -- Group_8 -- -- -- -- M -- -- -- -- -- -- -- -- -- -- --
Group_9 -- -- -- -- -- -- -- -- -- -- -- -- -- -- -- A Group_10 --
-- -- -- K -- -- -- -- -- -- -- -- -- -- -- Group_11 -- -- -- -- --
-- -- -- -- -- -- -- -- -- -- -- Group_12 -- -- -- -- -- -- -- --
-- -- -- -- -- -- -- Y Group_13 -- -- -- -- -- -- -- -- -- -- -- --
-- -- -- -- Group_14 -- -- -- -- -- -- -- -- -- -- -- -- -- -- --
-- Group_15 -- -- -- -- -- -- -- -- -- -- -- -- -- -- -- X Group_16
-- -- -- -- -- -- -- -- -- -- -- -- -- -- -- Y Group_17 -- -- -- --
-- -- -- -- N -- -- -- -- -- -- -- Group_18 -- -- -- -- -- -- -- C
-- -- -- -- -- -- -- -- Group_19 -- -- -- -- -- N -- -- -- -- -- --
-- -- -- -- Group_20 -- -- -- -- I -- -- -- -- -- -- -- -- R -- --
Group_21 -- -- -- -- M -- -- -- -- -- -- -- -- -- -- -- Group_22 --
-- -- -- M -- -- -- -- -- -- -- -- H -- -- Group_23 -- K -- -- --
-- -- R -- -- -- -- -- -- -- -- HA2 position 30 31 32 33 34 35 36
37 38 39 Tested Consensus E G T G Q A A D L K N Years strains
Group_l -- -- -- -- -- -- -- -- -- -- 655 1972-2008 Pa Group_2 --
-- I -- -- -- -- -- -- -- 380 2004-2008 Hs-Wi Group_3 -- -- -- --
-- -- -- -- -- -- 127 1999-2004 Group_4 -- -- R -- -- -- -- -- --
-- 91 2007-2009 Group_5 -- -- -- -- -- -- -- -- -- -- 69 1968-1997
HK Group_6 -- -- M -- -- -- -- -- -- -- 10 2007 Group_7 -- -- -- --
-- -- -- -- -- -- 6 1999-2004 Group_8 -- -- I -- -- -- -- -- -- --
4 2002-2007 Group_9 -- -- -- -- -- -- -- -- -- -- 3 2004 Group_10
-- -- -- -- -- -- -- -- -- -- 3 1999 Group_11 -- -- R -- -- -- --
-- F -- 2 2009 Group_12 -- -- -- -- -- -- -- -- -- -- 2 2003-2004
Group_13 -- -- I -- -- -- -- -- F -- 1 2006 Group_14 -- -- V -- --
-- -- -- -- -- 1 2007 Group_15 -- -- I -- -- -- -- -- -- -- 1 2007
Group_16 -- -- R -- -- -- -- -- -- -- 1 2008 Group_17 -- -- -- --
-- -- -- -- -- -- 1 2003 Group_18 -- -- -- -- -- -- -- -- -- -- 1
2001 Group_19 -- -- -- -- -- -- -- -- -- -- 1 1999 Group_20 -- --
-- -- -- -- -- -- -- -- 1 1975 Group_21 -- -- R -- -- -- -- -- --
-- 1 2008 Group_22 -- -- -- -- -- -- -- -- -- -- 1 2002 Group_23 --
-- -- -- -- -- -- -- -- -- 1 2002
TABLE-US-00015 TABLE 15 Neutralization titers on various Influenza
A strains H1 H3 H7 H10 3 A/ A/ A/ A/ A/ A/Chick/ A/ H1N1 Wisconsin/
Hiroshima/ Panama/ A/ Hong Mallard/ NL/ Chick/ strains 67/ 52/
2000/ Johannesburg/ Kong/ A/HK/1/ A/HK/1/ Netherlands/ 621557/
Germany/ IgG # (1999-2007) 2005 2005 1999 33/1994 1/1968 68-M20
68-M2c 12/2000 03-ma N/49 CR8001 >40 11.9 13.0 >40 6.5 7.1
5.26 tbd >40 39.8 >40 CR8020 >40 3.5 3.5 5.0 2.0 1.8 1.8
1.8 2.5 27.6 6.6 CR8041 >40 3.3 3.5 5.0 1.6 1.7 1.8 1.7 38.0
258.0 37.2 CR8043 >40 1.6 1.8 4.2 1.2 0.8 1.8 1.2 >40 >40
6.6 CR8057 >40 0.005 0.003 0.01 >40 >40 >40 >40
>40 -- >250 All SK50 titers in ug/ml; Mouse-adapted strains;
Ma "pandemic" H7 strain
TABLE-US-00016 TABLE 16 Mean area under the curve of body weight
change from baseline at day 0. Mean p-value.sup.a AUC SD (mAb vs
Study Group (g * day) (g * day) control) p-value.sup.b 1 30 mg/kg
control -82.86 14.15 1 mg/kg CR8020 -63.21 30.91 0.09 3 mg/kg
CR8020 16.95 8.20 <0.001 <0.001 (1 vs 3 mg/kg) 10 mg/kg
CR8020 31.44 9.09 <0.001 0.454 (3 vs 10 mg/kg) 30 mg/kg CR8020
25.62 12.94 <0.001 ns 2 30 mg/kg control -86.51 8.83 1 mg/kg
CR8041 -68.26 11.41 0.004 3 mg/kg CR8041 19.51 13.82 <0.001
<0.001 (1 vs 3 mg/kg) 10 mg/kg CR8041 35.23 11.06 <0.001
0.061 (3 vs 10 mg/kg) 30 mg/kg CR8041 28.21 7.89 <0.001 ns 1
mg/kg CR8043 -66.19 8.74 <0.001 3 mg/kg CR8043 8.48 11.81
<0.001 <0.001 (1 vs 3 mg/kg) 10 mg/kg CR8043 31.57 7.90
<0.001 <0.001 (3 vs 10 mg/kg) 30 mg/kg CR8043 27.72 6.61
<0.001 0.997 (10 vs 30 mg/kg) .sup.aMean AUC values of the mAb
dose groups were compared to the control Ab groups using analysis
of variance with Dunnet's adjustment for multiple comparisons.
.sup.bMean AUC values per antibody concentration were compared for
each antibody using analysis of variance with Tukey's adjustment
for multiple comparisons. ns = not statistically significant
TABLE-US-00017 TABLE 17 Mean area under the curve of body weight
change from baseline at day 0. Mean AUC SD Group (g * day) (g *
day) p-value.sup.a 15 mg/kg CR8020 at day -1 33.44 10.06 <0.001
15 mg/kg CR8020 at day 1 10.70 16.23 <0.001 15 mg/kg CR8020 at
day 2 -15.23 11.60 <0.001 15 mg/kg CR8020 at day 3 -65.45 35.90
0.003 15 mg/kg CR8020 at day 4 -85.95 23.14 0.742 15 mg/kg CR8020
at day 5 -100.88 12.78 0.986 15 mg/kg CR8020 at day 6 -84.91 12.28
0.653 Control mAb at day 1 -95.76 11.55 .sup.aMean AUC values of
the 15 mg/kg mAb CR8020 dosed groups were compared to the control
mAb group using analysis of variance with Dunnet's adjustment for
multiple comparisons in the post-hoc analysis. Prophylactic
treatment with 15 mg/kg mAb CR8020 resulted in a statistically
significant reduction in weight loss compared to the control group
(p < 0.001). Therapeutic treatment at day 1, day 2 or day 3 with
15 mg/kg mAb CR8020 also resulted in a statistically significant
reduction in weight loss compared to the control group (p <
0.001, p < 0.001 and p = 0.003, respectively). Treatment at days
4, day 5 or day 6 with 15 mg/kg mAb CR8020 did not result in a
statistically significant reduction in weight loss compared to the
control group (p > 0.05 for all three groups).
TABLE-US-00018 TABLE 18 Median clinical scores. The interval with
significant difference between clinical scores compared to the
control mAb group are listed (e.g., between 15 mg/kg at day -1 and
the control group the difference in clinical scores is significant
from day 4 onwards). Relative to control Interval Group (day(s) p
15 mg/kg CR8020 at day -1 4-21 .ltoreq.0.001 15 mg/kg CR8020 at day
1 2-21 .ltoreq.0.001 15 mg/kg CR8020 at day 2 3, 5-21 .ltoreq.0.001
15 mg/kg CR8020 at day 3 3, 5-21 .ltoreq.0.012 15 mg/kg CR8020 at
day 4 3, 5-21 .ltoreq.0.034 15 mg/kg CR8020 at day 5 3
.ltoreq.0.001 15 mg/kg CR8020 at day 6 3 .ltoreq.0.001
TABLE-US-00019 TABLE 19 Mean area under the curve of body weight
change from baseline at day 0. p-value.sup.a Mean AUC (mAb CR8020
vs Group (g * day) SD (g * day) control) 30 mg/kg control -93.06
10.88 1 mg/kg CR8020 -45.61 15.05 <0.001 3 mg/kg CR8020 -13.31
9.51 <0.001 10 mg/kg CR8020 -6.35 12.40 <0.001 30 mg/kg
CR8020 -12.59 7.35 <0.001 .sup.aMean AUC values of the mAb
CR8020 dosed groups were compared to the control mAb group using
analysis of variance with Dunnet's adjustment for multiple
comparisons in the post-hoc analysis.
TABLE-US-00020 TABLE 20 Summary of binding and neutralization
properties of monoclonal antibodies specific for influenza virus
HA. H1 H3 Binding VNA Binding VNA CR6261 + + - - CR6323 + + - -
CR8001 + - + + CR8020 - - + + CR8041 - - + + CR8043 - - + +
TABLE-US-00021 TABLE 21 Mean area under the curve of body weight
change from baseline at day 0. Mean AUC SD p-value (mAb Group (g *
day) (g * day) vs control).sup.a 30 mg/kg control -101.38 11.67 1
mg/kg CR8020 -82.58 34.71 0.356 3 mg/kg CR8020 -5.70 23.97
<0.001 10 mg/kg CR8020 2.13 13.13 <0.001 1 mg/kg CR8041
-105.05 17.04 1 3 mg/kg CR8041 -32.22 30.87 <0.001 10 mg/kg
CR8041 -20.06 17.92 <0.001 30 mg/kg CR8041 -10.01 10.11
<0.001 1 mg/kg CR8043 -107.75 11.04 0.997 3 mg/kg CR8043 -117.88
5.91 0.510 10 mg/kg CR8043 -94.00 23.23 0.992 30 mg/kg CR8043
-56.82 17.55 <0.001 .sup.aMean AUC values of the mAb CR8020
dosed groups were compared to the control mAb group using analysis
of variance with Dunnet's adjustment for multiple comparisons in
the post-hoc analysis.
TABLE-US-00022 TABLE 22 Mean area under the curve of body weight
change from baseline at day 0. Mean AUC SD p-value (mAb Group (g *
day) (g * day) vs control).sup.a 15 mg/kg CR8020 at day -1 -7.68
8.17 <0.001 15 mg/kg CR8020 at day 1 -20.43 8.41 <0.001 15
mg/kg CR8020 at day 2 -38.18 37.35 <0.001 15 mg/kg CR8020 at day
3 -28.27 9.63 <0.001 15 mg/kg CR8020 at day 4 -99.11 37.90 0.566
15 mg/kg CR8020 at day 5 -93.62 10.29 0.979 15 mg/kg CR8020 at day
6 -94.06 7.65 0.858 Control antibody at day 1 -93.33 10.58
.sup.aMean AUC values were compared using the RobustReg procedure
(SAS) which allocates less weight to outliers.
TABLE-US-00023 TABLE 23 Median clinical scores. Relative to control
Interval Group (day(s) p 15 mg/kg CR8020 at day -1 4-21
.ltoreq.0.001 15 mg/kg CR8020 at day 1 2, 5-21 .ltoreq.0.012 15
mg/kg CR8020 at day 2 6-21 .ltoreq.0.038 15 mg/kg CR8020 at day 3
7-21 .ltoreq.0.035 15 mg/kg CR8020 at day 4 5, 6, 8-21
.ltoreq.0.016 15 mg/kg CR8020 at day 5 8 <0.001 15 mg/kg CR8020
at day 6 -- .gtoreq.0.449
TABLE-US-00024 TABLE 24 Binding kinetics Fab kon (1/Ms) Kdis (1/s)
KD (nM) A/Wisconsin CRF8020 2.03E+05 2.08E-03 11.2 CRF8043 4.08E+05
9.86E-05 0.3 A/Brisbane CRF8020 1.81E+05 1.43E-03 8.9 CRF8043
3.12E+05 8.69E-05 0.3
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Sequence CWU 1
1
2581339DNAArtificialSC08-001 VH DNA 1gaggtgcagc tggtggagtc
tggaggaggc ctgatccagc cgggggggtc cctgagactc 60tcctgtgcag cctctggatt
caccgtcagt agcaactacg tgagctgggt ccgccaggcc 120ccagggaagg
ggctggagtg gctctcactt atttacacgg gtggtaccac atactacgca
180gactccgtga agggccgatt caccatctcc agagacaact ccaagaatac
ggtgtttctt 240caaatgaaca gcctgagagc cgaggacgcg gccatgtatt
actgtgcgag agtgtcagca 300ttacggtttt tgcagtggcc aaactacgcg atggacgtc
3392113PRTArtificialSC08-001 VH PROTEIN 2Glu Val Gln Leu Val Glu
Ser Gly Gly Gly Leu Ile Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu
Ser Cys Ala Ala Ser Gly Phe Thr Val Ser Ser Asn 20 25 30 Tyr Val
Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Leu 35 40 45
Ser Leu Ile Tyr Thr Gly Gly Thr Thr Tyr Tyr Ala Asp Ser Val Lys 50
55 60 Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Val Phe
Leu 65 70 75 80 Gln Met Asn Ser Leu Arg Ala Glu Asp Ala Ala Met Tyr
Tyr Cys Ala 85 90 95 Arg Val Ser Ala Leu Arg Phe Leu Gln Trp Pro
Asn Tyr Ala Met Asp 100 105 110 Val 3303DNAArtificialSC08-001 VL
DNA 3cagtctgccc tgactcagcc tgcctccgtg tctgggtctc ctggacggtc
gatcaccatc 60tcctgctctg gaacccgcag tgacgttggt ggtcataatt atgtctcctg
gtaccaacaa 120cacccaggca aagcccccaa actcatgatt tatgaggtca
gtcatcggcc ctcaggggtt 180tctaatcgct tctctggctc caagtctggc
agcacggcct ccctgaccat ctctggcctc 240cagtctgagg acgaggctga
ttattactgc agctcttata caggtgaagg ccccctagga 300gtg
3034101PRTArtificialSC08-001 VL PROTEIN 4Gln Ser Ala Leu Thr Gln
Pro Ala Ser Val Ser Gly Ser Pro Gly Arg 1 5 10 15 Ser Ile Thr Ile
Ser Cys Ser Gly Thr Arg Ser Asp Val Gly Gly His 20 25 30 Asn Tyr
Val Ser Trp Tyr Gln Gln His Pro Gly Lys Ala Pro Lys Leu 35 40 45
Met Ile Tyr Glu Val Ser His Arg Pro Ser Gly Val Ser Asn Arg Phe 50
55 60 Ser Gly Ser Lys Ser Gly Ser Thr Ala Ser Leu Thr Ile Ser Gly
Leu 65 70 75 80 Gln Ser Glu Asp Glu Ala Asp Tyr Tyr Cys Ser Ser Tyr
Thr Gly Glu 85 90 95 Gly Pro Leu Gly Val 100
5342DNAArtificialSC08-003 VH DNA 5gaggtgcagc tggtggagac cgggggagac
ttggtccagc ctggggggtc cctgagactc 60tcctgttcag cctctgaatt cagcttcagt
agttattgga tgagctgggt ccgccaggct 120ccagggaaag ggctggagtg
ggtggccaac atgaagcaag atggaagtga gaagtactat 180gtggactctg
tgaagggccg gttcaccatc tccagagaca acgccaagaa ctcattatat
240ctgcaaatga acagcctgag aggcgaggac acggctgtgt attactgtgc
gaggggttcc 300tgtgacgatt cttggactgg ttgtcatgat gcttttgaca tc
3426114PRTArtificialSC08-003 VH PROTEIN 6Glu Val Gln Leu Val Glu
Thr Gly Gly Asp Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu
Ser Cys Ser Ala Ser Glu Phe Ser Phe Ser Ser Tyr 20 25 30 Trp Met
Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45
Ala Asn Met Lys Gln Asp Gly Ser Glu Lys Tyr Tyr Val Asp Ser Val 50
55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Ser Leu
Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Gly Glu Asp Thr Ala Val
Tyr Tyr Cys 85 90 95 Ala Arg Gly Ser Cys Asp Asp Ser Trp Thr Gly
Cys His Asp Ala Phe 100 105 110 Asp Ile 7291DNAArtificialSC08-003
VL DNA 7gtgttgacgc agccgccctc ggtgtcagtg gccccaggac agacggccag
gattgcctgt 60gggggaaaca acattgggag taaaagtgtg cactggtacc agcagaagcc
aggccaggcc 120cctgtgctgg tcgtctatga tgatagcgcc cggccctcag
ggatccctga gcgattctct 180ggctccaatt ctgggaacac ggccaccctg
accatcagca gggtcgaggc cggggatgaa 240gccgactatt actgtcaggt
gtgggagagt ggtagtgatc tacgactgct t 291897PRTArtificialSC08-003 VL
PROTEIN 8Val Leu Thr Gln Pro Pro Ser Val Ser Val Ala Pro Gly Gln
Thr Ala 1 5 10 15 Arg Ile Ala Cys Gly Gly Asn Asn Ile Gly Ser Lys
Ser Val His Trp 20 25 30 Tyr Gln Gln Lys Pro Gly Gln Ala Pro Val
Leu Val Val Tyr Asp Asp 35 40 45 Ser Ala Arg Pro Ser Gly Ile Pro
Glu Arg Phe Ser Gly Ser Asn Ser 50 55 60 Gly Asn Thr Ala Thr Leu
Thr Ile Ser Arg Val Glu Ala Gly Asp Glu 65 70 75 80 Ala Asp Tyr Tyr
Cys Gln Val Trp Glu Ser Gly Ser Asp Leu Arg Leu 85 90 95 Leu
9342DNAArtificialSC08-015 VH DNA 9caggtgcagc tgcaggagtc ggggggagac
ttggtccagc ctggggggtc cctgagactc 60tcctgttcag cctctgaatt cagcttcagt
agttattgga tgagctgggt ccgccaggct 120ccagggaaag ggctggagtg
ggtggccaac atgaagcaag atggaagtga gaagtactat 180gtggactctg
tgaagggccg gttcaccatc tccagagaca acgccaagaa ctcattatat
240ctgcaaatga acagcctgag aggcgaggac acggctgtgt attactgtgc
gaggggttcc 300tgtgacgatt cttggactgg ttgtcatgat gcttttgaca tc
34210114PRTArtificialSC08-015 VH PROTEIN 10Gln Val Gln Leu Gln Glu
Ser Gly Gly Asp Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu
Ser Cys Ser Ala Ser Glu Phe Ser Phe Ser Ser Tyr 20 25 30 Trp Met
Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45
Ala Asn Met Lys Gln Asp Gly Ser Glu Lys Tyr Tyr Val Asp Ser Val 50
55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Ser Leu
Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Gly Glu Asp Thr Ala Val
Tyr Tyr Cys 85 90 95 Ala Arg Gly Ser Cys Asp Asp Ser Trp Thr Gly
Cys His Asp Ala Phe 100 105 110 Asp Ile 11288DNAArtificialSC08-015
VL DNA 11gtgttgacgc agccgccctc ggtgtcagtg gccccaggac agacggccaa
gattacctgt 60gggggagaca acattggaag aaaaagtgtg cactggtacc agcagaagcc
aggcctggcc 120cctgtgctgg tcgtcaatga taatagcgac cggccctcag
ggatccctgc gcgattctct 180ggctccaact ctgggaacac ggccaccctg
accatcagca gggtcgaagc cggggatgag 240gccgactatt actgtcacgt
gtggggtagt agtcgtgacc attatgtc 2881296PRTArtificialSC08-015 VL
PROTEIN 12Val Leu Thr Gln Pro Pro Ser Val Ser Val Ala Pro Gly Gln
Thr Ala 1 5 10 15 Lys Ile Thr Cys Gly Gly Asp Asn Ile Gly Arg Lys
Ser Val His Trp 20 25 30 Tyr Gln Gln Lys Pro Gly Leu Ala Pro Val
Leu Val Val Asn Asp Asn 35 40 45 Ser Asp Arg Pro Ser Gly Ile Pro
Ala Arg Phe Ser Gly Ser Asn Ser 50 55 60 Gly Asn Thr Ala Thr Leu
Thr Ile Ser Arg Val Glu Ala Gly Asp Glu 65 70 75 80 Ala Asp Tyr Tyr
Cys His Val Trp Gly Ser Ser Arg Asp His Tyr Val 85 90 95
13342DNAArtificialSC08-016 VH DNA 13gaggtgcagc tggtggagtc
tgggggagac ttggtccagc ctggggggtc cctgagactc 60tcctgttcag cctctgaatt
cagcttcagt agttattgga tgagctgggt ccgccaggct 120ccagggaaag
ggctggagtg ggtggccaac atgaagcaag atggaagtga gaagtactat
180gtggactctg tgaagggccg gttcaccatc tccagagaca acgccaagaa
ctcattatat 240ctgcaaatga acagcctgag aggcgaggac acggctgtgt
attactgtgc gaggggttcc 300tgtgacgatt cttggactgg ttgtcatgat
gcttttgaca tc 34214114PRTArtificialSC08-016 VH PROTEIN 14Glu Val
Gln Leu Val Glu Ser Gly Gly Asp Leu Val Gln Pro Gly Gly 1 5 10 15
Ser Leu Arg Leu Ser Cys Ser Ala Ser Glu Phe Ser Phe Ser Ser Tyr 20
25 30 Trp Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp
Val 35 40 45 Ala Asn Met Lys Gln Asp Gly Ser Glu Lys Tyr Tyr Val
Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala
Lys Asn Ser Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Gly Glu
Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Gly Ser Cys Asp Asp
Ser Trp Thr Gly Cys His Asp Ala Phe 100 105 110 Asp Ile
15303DNAArtificialSC08-016 VL DNA 15cagtctgtcg tgacgcagcc
gccctcagtg tctggggccc cagggcagag ggtcaccatc 60tcctgcactg ggagcagctc
caacatcggg gcaggttatg atgtacactg gtaccagcag 120cttccaggaa
cagcccccaa actcctcatc tatggtaaca acaatcggcc ctcaggggtc
180cctgaccgat tctctggatc caggtctggc cctttagccc tcctggccat
cactgggctc 240caggctgagg atgaggctga ttattactgc cagtcctatg
acagcagcct gagtgtttat 300gtc 30316101PRTArtificialSC08-016 VL
PROTEIN 16Gln Ser Val Val Thr Gln Pro Pro Ser Val Ser Gly Ala Pro
Gly Gln 1 5 10 15 Arg Val Thr Ile Ser Cys Thr Gly Ser Ser Ser Asn
Ile Gly Ala Gly 20 25 30 Tyr Asp Val His Trp Tyr Gln Gln Leu Pro
Gly Thr Ala Pro Lys Leu 35 40 45 Leu Ile Tyr Gly Asn Asn Asn Arg
Pro Ser Gly Val Pro Asp Arg Phe 50 55 60 Ser Gly Ser Arg Ser Gly
Pro Leu Ala Leu Leu Ala Ile Thr Gly Leu 65 70 75 80 Gln Ala Glu Asp
Glu Ala Asp Tyr Tyr Cys Gln Ser Tyr Asp Ser Ser 85 90 95 Leu Ser
Val Tyr Val 100 17342DNAArtificialSC08-017 VH DNA 17gaggtgcagc
tggtggagac tgggggagac ttggtccagc ctggggggtc cctgagactc 60tcctgttcag
cctctgaatt cagcttcagt agttattgga tgagctgggt ccgccaggct
120ccagggaaag ggctggagtg ggtggccaac atgaagcaag atggaagtga
gaagtactat 180gtggactctg tgaagggccg gttcaccatc tccagagaca
acgccaagaa ctcattatat 240ctgcaaatga acagcctgag aggcgaggac
acggctgtgt attactgtgc gaggggttcc 300tgtgacgatt cttggactgg
ttgtcatgat gcttttgaca tc 34218114PRTArtificialSC08-017 VH PROTEIN
18Glu Val Gln Leu Val Glu Thr Gly Gly Asp Leu Val Gln Pro Gly Gly 1
5 10 15 Ser Leu Arg Leu Ser Cys Ser Ala Ser Glu Phe Ser Phe Ser Ser
Tyr 20 25 30 Trp Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu
Glu Trp Val 35 40 45 Ala Asn Met Lys Gln Asp Gly Ser Glu Lys Tyr
Tyr Val Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp
Asn Ala Lys Asn Ser Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg
Gly Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Gly Ser Cys
Asp Asp Ser Trp Thr Gly Cys His Asp Ala Phe 100 105 110 Asp Ile
19294DNAArtificialSC08-017 VL DNA 19tcttctgagc tgactcagga
ccctgctgtg tctgtggcct tgggacagac agtcaggatc 60acatgccaag gagacagcct
cagaagctat tatgcaagct ggtaccagca gaagccagga 120caggcccctg
tacttgtcat ctatgctaaa accaaccggc cctcagggat cccagaccga
180ttctctggct ccacctcagg aaacactgct tccttgacca tcactggggc
tcaggcggag 240gatgaggctg actattactg taactcccgg gacagcagtg
gtaaccatgt ggta 2942098PRTArtificialSC08-017 VL PROTEIN 20Ser Ser
Glu Leu Thr Gln Asp Pro Ala Val Ser Val Ala Leu Gly Gln 1 5 10 15
Thr Val Arg Ile Thr Cys Gln Gly Asp Ser Leu Arg Ser Tyr Tyr Ala 20
25 30 Ser Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Val Leu Val Ile
Tyr 35 40 45 Ala Lys Thr Asn Arg Pro Ser Gly Ile Pro Asp Arg Phe
Ser Gly Ser 50 55 60 Thr Ser Gly Asn Thr Ala Ser Leu Thr Ile Thr
Gly Ala Gln Ala Glu 65 70 75 80 Asp Glu Ala Asp Tyr Tyr Cys Asn Ser
Arg Asp Ser Ser Gly Asn His 85 90 95 Val Val
21342DNAArtificialSC08-018 VH DNA 21gaggtgcagc tggtggagac
tgggggagac ttggtccagc ctggggggtc cctgagactc 60tcctgttcag cctctgaatt
cagcttcagt agttattgga tgagctgggt ccgccaggct 120ccagggaaag
ggctggagtg ggtggccaac atgaagcaag atggaagtga gaagtactat
180gtggactctg tgaagggccg gttcaccatc tccagagaca acgccaagaa
ctcattatat 240ctgcaaatga acagcctgag aggcgaggac acggctgtgt
attactgtgc gaggggttcc 300tgtgacgatt cttggactgg ttgtcatgat
gcttttgata tc 34222114PRTArtificialSC08-018 VH PROTEIN 22Glu Val
Gln Leu Val Glu Thr Gly Gly Asp Leu Val Gln Pro Gly Gly 1 5 10 15
Ser Leu Arg Leu Ser Cys Ser Ala Ser Glu Phe Ser Phe Ser Ser Tyr 20
25 30 Trp Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp
Val 35 40 45 Ala Asn Met Lys Gln Asp Gly Ser Glu Lys Tyr Tyr Val
Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala
Lys Asn Ser Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Gly Glu
Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Gly Ser Cys Asp Asp
Ser Trp Thr Gly Cys His Asp Ala Phe 100 105 110 Asp Ile
23303DNAArtificialSC08-018 VL DNA 23cagtctgccc tgactcagcc
tgcctccgtg tctgggtctc ctggacagtc gatcaccatc 60tcctgcactg gaaccagcag
tgacgttggt ggttataact atgtctcctg gtaccaacaa 120cacccaggca
aagcccccaa actcatgatt tatgaggtca gtcatcggcc ctcaggggtt
180tctaatcgct tctctggctc caagtctggc agcacggcct ccctgaccat
ctctggcctc 240cagtctgagg acgaggctga ttattactgc agctcttata
caggtgaagg ccccctagga 300gtg 30324101PRTArtificialSC08-018 VL
PROTEIN 24Gln 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 Gly Tyr 20 25 30 Asn Tyr Val Ser Trp Tyr Gln Gln His Pro
Gly Lys Ala Pro Lys Leu 35 40 45 Met Ile Tyr Glu Val Ser His Arg
Pro Ser Gly Val Ser Asn Arg Phe 50 55 60 Ser Gly Ser Lys Ser Gly
Ser Thr Ala Ser Leu Thr Ile Ser Gly Leu 65 70 75 80 Gln Ser Glu Asp
Glu Ala Asp Tyr Tyr Cys Ser Ser Tyr Thr Gly Glu 85 90 95 Gly Pro
Leu Gly Val 100 25357DNAArtificialSC08-019 VH DNA 25gaggtgcagc
tggtggagtc tgggggaggc ttggtacagc ctggggggtc cctgagactc 60tcctgtggag
cctctggaat cagcgttagc acttctgcca tgagctgggt ccgccaggtt
120ccagggaagg ggctggagtg ggtctcaggt attagtggta gtggtgctac
cacatactac 180gcaggctccg tgaagggtcg attcaccatc tccagagaca
aatccaagaa cacactgcat 240ctgcaaatga gcagactgag agccgaggac
acggccattt actactgtgc gaaagatacc 300tccttgtttg agtatgatac
aagtggtttt acggctcccg gcaatgcttt tgatatc
35726119PRTArtificialSC08-019 VH PROTEIN 26Glu Val Gln Leu Val Glu
Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu
Ser Cys Gly Ala Ser Gly Ile Ser Val Ser Thr Ser 20 25 30 Ala Met
Ser Trp Val Arg Gln Val Pro Gly Lys Gly Leu Glu Trp Val 35 40 45
Ser Gly Ile Ser Gly Ser Gly Ala Thr Thr Tyr Tyr Ala Gly Ser Val 50
55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Lys Ser Lys Asn Thr Leu
His 65 70 75 80 Leu Gln Met Ser Arg Leu Arg Ala Glu Asp Thr Ala Ile
Tyr Tyr Cys 85 90 95 Ala Lys Asp Thr Ser Leu Phe Glu Tyr Asp Thr
Ser Gly Phe Thr Ala 100 105 110 Pro Gly Asn Ala Phe Asp Ile 115
27294DNAArtificialSC08-019 VL DNA 27gacatccagw tgacccagtc
tccatcctcc ctgtctgcat ctgtagatga cagagtcacc 60atcacttgcc gggcaagtca
gagcattagc ggctatttaa attggtatca acagaaacca 120gggaaagccc
ctaacctcct gatctatggt gcatccactt tgcagagtgg ggtcccatca
180aggttcagtg gcagtggatc tgggacagat ttcactctca ccatcaccag
tctgcaacct 240gaagactatg caacttacta ctgtcaacag acttacacct
cccctccgta cgct 2942898PRTArtificialSC08-019 VL PROTEIN 28Asp Ile
Gln Xaa Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Asp 1 5 10 15
Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Ser Ile Ser Gly Tyr
20
25 30 Leu Asn Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Asn Leu Leu
Ile 35 40 45 Tyr Gly Ala Ser Thr Leu Gln Ser Gly Val Pro Ser Arg
Phe Ser Gly 50 55 60 Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile
Thr Ser Leu Gln Pro 65 70 75 80 Glu Asp Tyr Ala Thr Tyr Tyr Cys Gln
Gln Thr Tyr Thr Ser Pro Pro 85 90 95 Tyr Ala
29333DNAArtificialSC08-020 VH DNA 29caggtacagc tgcagcagtc
aggagctgag gtgaagaccc ctggggcctc agtgaaggtc 60tcctgcaagg cctctggata
cacctttacc aggtttggtg tcagctggat acgacaggcc 120cctggacaag
ggcttgagtg gattggatgg atcagcgctt acaatggtga cacatactat
180gcacagaagt tccaggccag agtcaccatg accacagaca catccacgac
cacagcctac 240atggagatga ggagcctgag atctgacgac acggccgtgt
attactgtgc gagagaaccc 300cccctttttt acagcagctg gtctcttgac aac
33330111PRTArtificialSC08-020 VH PROTEIN 30Gln Val Gln Leu Gln Gln
Ser Gly Ala Glu Val Lys Thr Pro Gly Ala 1 5 10 15 Ser Val Lys Val
Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Arg Phe 20 25 30 Gly Val
Ser Trp Ile Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Ile 35 40 45
Gly Trp Ile Ser Ala Tyr Asn Gly Asp Thr Tyr Tyr Ala Gln Lys Phe 50
55 60 Gln Ala Arg Val Thr Met Thr Thr Asp Thr Ser Thr Thr Thr Ala
Tyr 65 70 75 80 Met Glu Met Arg Ser Leu Arg Ser Asp Asp Thr Ala Val
Tyr Tyr Cys 85 90 95 Ala Arg Glu Pro Pro Leu Phe Tyr Ser Ser Trp
Ser Leu Asp Asn 100 105 110 31294DNAArtificialSC08-020 VL DNA
31gaaattgtgw tgacrcagtc tccaggcacc ctgtctttgt ctccagggga aagagccacc
60ctctcctgca gggccagtca gagtgttagc atgaactact tagcctggtt ccagcagaaa
120cctggccagg ctcccaggct cctcatctat ggtgcgtccc gcagggccac
tggcatcccc 180gacaggatca gtggcagtgg gtctgggaca gacttcactc
tcaccatcag cagactggag 240cctgcagatt ttgcagtgta ttactgtcag
cagtatggta cctcacctcg gacg 2943298PRTArtificialSC08-020 VL PROTEIN
32Glu Ile Val Xaa 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 Met
Asn 20 25 30 Tyr Leu Ala Trp Phe Gln Gln Lys Pro Gly Gln Ala Pro
Arg Leu Leu 35 40 45 Ile Tyr Gly Ala Ser Arg Arg Ala Thr Gly Ile
Pro Asp Arg Ile 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 Ala Asp Phe Ala Val Tyr
Tyr Cys Gln Gln Tyr Gly Thr Ser Pro 85 90 95 Arg Thr
33330DNAArtificialSC08-021 VH DNA 33gaggtgcagc tggtggagtc
tgggggaggc ttgatacagc ctggggggtc cctgagactc 60tcctgtgcag cctctggatt
cacctttagc gcctatgcca tgaactgggt ccgccaggct 120ccagggaagg
ggctggagtg ggtctcagct attggtggta gtggcggtag cacatactac
180gcagactccg tgaagggccg gttcaccatc tccagagaca actccaagaa
gatcctgtat 240ctgcaaatga acggcctgag agccgaggac acggccatat
attactgtgc gaaaggccgg 300gattggactg ggggttactt ctttgactcc
33034110PRTArtificialSC08-021 VH PROTEIN 34Glu Val Gln Leu Val Glu
Ser Gly Gly Gly Leu Ile Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu
Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ala Tyr 20 25 30 Ala Met
Asn Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45
Ser Ala Ile Gly Gly Ser Gly Gly Ser Thr Tyr Tyr Ala Asp Ser Val 50
55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Lys Ile Leu
Tyr 65 70 75 80 Leu Gln Met Asn Gly Leu Arg Ala Glu Asp Thr Ala Ile
Tyr Tyr Cys 85 90 95 Ala Lys Gly Arg Asp Trp Thr Gly Gly Tyr Phe
Phe Asp Ser 100 105 110 35309DNAArtificialSC08-021 VL DNA
35gacatccagw tgacccagtc tccagactcc ctggctgtgt ctctgggcga gagggccacc
60atcaactgca agtccagcca gagtattttc tacagctcca acaataagaa ctacttaact
120tggtaccagc agaaaccagg acagcctcct aagctgctca tttactgggc
atctacccgg 180gaatccggag tccctgaccg attcagtggc agcgggtctg
ggacagattt cactctcacc 240atcagcagcc tgcaggctga agatgtggca
gtttattact gtcagcaata ctatagtatt 300ccctacact
30936103PRTArtificialSC08-021 VL PROTEIN 36Asp Ile Gln Xaa Thr Gln
Ser Pro Asp Ser Leu Ala Val Ser Leu Gly 1 5 10 15 Glu Arg Ala Thr
Ile Asn Cys Lys Ser Ser Gln Ser Ile Phe Tyr Ser 20 25 30 Ser Asn
Asn Lys Asn Tyr Leu Thr Trp Tyr Gln Gln Lys Pro Gly Gln 35 40 45
Pro Pro Lys Leu Leu Ile Tyr Trp Ala Ser Thr Arg Glu Ser Gly Val 50
55 60 Pro Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu
Thr 65 70 75 80 Ile Ser Ser Leu Gln Ala Glu Asp Val Ala Val Tyr Tyr
Cys Gln Gln 85 90 95 Tyr Tyr Ser Ile Pro Tyr Thr 100
37330DNAArtificialSC08-038 VH DNA 37gaggtgcagc tggtggactc
tgggggaggc ttggtacagc cgggggggtc cctgagactc 60tcctgtgcag cctctggatt
cgcctttagc ggctatgcca tgagctgggt ccgccaggct 120ccagggaagg
ggctggagtg ggtctcagat attggtggta gtggtggtgg cacatactac
180gcagactccg tgaagggccg gttcaccatc tccagagaca atgccaagaa
cacgctgtat 240ctgcaaatga atagcctgag agccgaggac acggccgtat
attactgtgc gaaaagcagt 300agctgggacc gggcctactt ctttgactcc
33038109PRTArtificialSC08-038 VH PROTEIN 38Val Gln Leu Val Asp Ser
Gly Gly Gly Leu Val Gln Pro Gly Gly Ser 1 5 10 15 Leu Arg Leu Ser
Cys Ala Ala Ser Gly Phe Ala Phe Ser Gly Tyr Ala 20 25 30 Met Ser
Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val Ser 35 40 45
Asp Ile Gly Gly Ser Gly Gly Gly Thr Tyr Tyr Ala Asp Ser Val Lys 50
55 60 Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Thr Leu Tyr
Leu 65 70 75 80 Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr
Tyr Cys Ala 85 90 95 Lys Ser Ser Ser Trp Asp Arg Ala Tyr Phe Phe
Asp Ser 100 105 39309DNAArtificialSC08-038 VL DNA 39gatattgtga
tgacccagac tccagactcc ctggctgtgt ctctgggcga gagggccacc 60atcaactgca
agtccagcca gagtgtttta tacagctcca tccataagaa ctacttagcc
120tggtaccagc aaaaaccagg acagcctcct aagctgctca tttactgggc
atctacccgg 180gaatccgggg tccctgaccg attcagtggc agcgggtctg
ggacagattt cactctcacc 240atcagcagcc tgcaggctga agatgtggca
gtttattact gtcagcaata ttatagatct 300cctccaact
30940103PRTArtificialSC08-038 VL PROTEIN 40Asp Ile Val Met Thr Gln
Thr Pro Asp Ser Leu Ala Val Ser Leu Gly 1 5 10 15 Glu Arg Ala Thr
Ile Asn Cys Lys Ser Ser Gln Ser Val Leu Tyr Ser 20 25 30 Ser Ile
His Lys Asn Tyr Leu Ala Trp Tyr Gln Gln Lys Pro Gly Gln 35 40 45
Pro Pro Lys Leu Leu Ile Tyr Trp Ala Ser Thr Arg Glu Ser Gly Val 50
55 60 Pro Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu
Thr 65 70 75 80 Ile Ser Ser Leu Gln Ala Glu Asp Val Ala Val Tyr Tyr
Cys Gln Gln 85 90 95 Tyr Tyr Arg Ser Pro Pro Thr 100
41336DNAArtificialSC08-039 VH DNA 41cagctgcagc tgcaggagtc
gggcccagga ctggtgaagc cttcggagac cctgtccctc 60acgtgcactg tctctggcgg
ctccatcggt agttactact ggagctggat acggcagccc 120ccagggaagg
gactggagtg gattggatat atctattacc gtgggggtac cagttacaac
180ccctccctca agagtcgagt caccatatca gtcgacacgt ccaagagcca
gttcaccttg 240aagctgaact ctgtgaccgc tgcggacacg gccgtgtatt
actgtgcgag aaaggactgg 300ggatcagcgg ccggaagtgt ctggtacttc gatctc
33642112PRTArtificialSC08-039 VH PROTEIN 42Gln Leu Gln Leu Gln Glu
Ser Gly Pro Gly Leu Val Lys Pro Ser Glu 1 5 10 15 Thr Leu Ser Leu
Thr Cys Thr Val Ser Gly Gly Ser Ile Gly Ser Tyr 20 25 30 Tyr Trp
Ser Trp Ile Arg Gln Pro Pro Gly Lys Gly Leu Glu Trp Ile 35 40 45
Gly Tyr Ile Tyr Tyr Arg Gly Gly Thr Ser Tyr Asn Pro Ser Leu Lys 50
55 60 Ser Arg Val Thr Ile Ser Val Asp Thr Ser Lys Ser Gln Phe Thr
Leu 65 70 75 80 Lys Leu Asn Ser Val Thr Ala Ala Asp Thr Ala Val Tyr
Tyr Cys Ala 85 90 95 Arg Lys Asp Trp Gly Ser Ala Ala Gly Ser Val
Trp Tyr Phe Asp Leu 100 105 110 43300DNAArtificialSC08-039 VL DNA
43cagtctgccc tgactcagcc tccctccgcg tccgggtctc ctggacagtc agtcaccatc
60tcctgcactg gaaccagcag tgacgttggt ggttataatt atgtctcctg gtaccaacaa
120cacccaggca aagcccccaa actcatgatt cgtgaggtca gtaagcggcc
ctcaggggtc 180cctgatcgct tctctggttc caagtctggc aacacggcct
ccctgaccgt ctctgggctc 240caggctgagg atgaggctga atactactgc
agctcgtatg caggcagcaa caatctgata 30044100PRTArtificialSC08-039 VL
PROTEIN 44Gln Ser Ala Leu Thr Gln Pro Pro Ser Ala Ser Gly Ser Pro
Gly Gln 1 5 10 15 Ser Val Thr Ile Ser Cys Thr Gly Thr Ser Ser Asp
Val Gly Gly Tyr 20 25 30 Asn Tyr Val Ser Trp Tyr Gln Gln His Pro
Gly Lys Ala Pro Lys Leu 35 40 45 Met Ile Arg Glu Val Ser Lys Arg
Pro Ser Gly Val Pro Asp Arg Phe 50 55 60 Ser Gly Ser Lys Ser Gly
Asn Thr Ala Ser Leu Thr Val Ser Gly Leu 65 70 75 80 Gln Ala Glu Asp
Glu Ala Glu Tyr Tyr Cys Ser Ser Tyr Ala Gly Ser 85 90 95 Asn Asn
Leu Ile 100 45333DNAArtificialSC08-040 VH DNA 45gaggtgcagc
tggtggagtc agggggaggc gtggtccagc ctgggaggtc cctgagactc 60tcctgtgcag
cgtctggatt cgctttcagt agctatggca tgcactgggt ccgccaggct
120ccaggcaagg gactggagtg ggtgaccttt atatggtatg atggaagtaa
taaacactat 180gcagactcca tgaagggccg attcaccatc tccagagaca
attccaagaa cacgctgtat 240ctgcaaatga gcagcctgag agccgaggac
acggctgttt attactgtgc gagagatggg 300ggatatagca cctgggaatg
gtacttcgat ctc 33346111PRTArtificialSC08-040 VH PROTEIN 46Glu Val
Gln Leu Val Glu Ser Gly Gly Gly Val Val Gln Pro Gly Arg 1 5 10 15
Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Ala Phe Ser Ser Tyr 20
25 30 Gly Met His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp
Val 35 40 45 Thr Phe Ile Trp Tyr Asp Gly Ser Asn Lys His Tyr Ala
Asp Ser Met 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser
Lys Asn Thr Leu Tyr 65 70 75 80 Leu Gln Met Ser Ser Leu Arg Ala Glu
Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Asp Gly Gly Tyr Ser
Thr Trp Glu Trp Tyr Phe Asp Leu 100 105 110
47291DNAArtificialSC08-040 VL DNA 47gaaattgtgc tgactcagtc
tccggacttt cagtctgtga ctccaaagga gagagtcacc 60atcacctgcc gggccagtca
gggcattggc agtaacttac actggtacca gcagaaacca 120gatcagtctc
caaagctcct catcaagtat gcttcccagt ccatcacagg ggtcccctcg
180aggttcagtg gcaggggatc tgggacagat ttcaccctca ccatcaatag
cctggaagtt 240gaagatgctg cagtgtatta ctgtcatcag agtagtagtt
taccgctcac t 2914897PRTArtificialSC08-040 VL PROTEIN 48Glu Ile Val
Leu Thr Gln Ser Pro Asp Phe Gln Ser Val Thr Pro Lys 1 5 10 15 Glu
Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Gly Ile Gly Ser Asn 20 25
30 Leu His Trp Tyr Gln Gln Lys Pro Asp Gln Ser Pro Lys Leu Leu Ile
35 40 45 Lys Tyr Ala Ser Gln Ser Ile Thr Gly Val Pro Ser Arg Phe
Ser Gly 50 55 60 Arg Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Asn
Ser Leu Glu Val 65 70 75 80 Glu Asp Ala Ala Val Tyr Tyr Cys His Gln
Ser Ser Ser Leu Pro Leu 85 90 95 Thr 49333DNAArtificialSC08-041 VH
DNA 49caggtgcagc tggtgcagtc tggcgctgag gtgaagaagc ctggggcctc
agtgaaggtc 60tcctgccagg cttcgggtta cacctttacc tcctttggtc tcagctgggt
gcgacaggcc 120cctggacaag ggcctgagtg gatgggatgg atcagcgctt
acaatggtga aataaagtat 180gcacagaagt tccagggcag agtctccatg
accacagaca catcaacgag gacagcctac 240atggaggtgc ggagcctcag
acctgacgac acggccgtat actactgtgc gagagagccc 300cccctgtatt
tcagtagctg gtctctcgac ttc 33350111PRTArtificialSC08-041 VH PROTEIN
50Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala 1
5 10 15 Ser Val Lys Val Ser Cys Gln Ala Ser Gly Tyr Thr Phe Thr Ser
Phe 20 25 30 Gly Leu Ser Trp Val Arg Gln Ala Pro Gly Gln Gly Pro
Glu Trp Met 35 40 45 Gly Trp Ile Ser Ala Tyr Asn Gly Glu Ile Lys
Tyr Ala Gln Lys Phe 50 55 60 Gln Gly Arg Val Ser Met Thr Thr Asp
Thr Ser Thr Arg Thr Ala Tyr 65 70 75 80 Met Glu Val Arg Ser Leu Arg
Pro Asp Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Glu Pro Pro
Leu Tyr Phe Ser Ser Trp Ser Leu Asp Phe 100 105 110
51294DNAArtificialSC08-041 VL DNA 51gaaattgtgt tgacgcagtc
tccaggcacc ctgtctttgt ctccagggga aagagccacc 60ctctcctgca gggccagtca
gagtgttagc agcaactact tagcctggtt ccagcagaaa 120cctggccagg
ctcccaggct cctcatctat ggtgcatcaa ggagggccac tggcatccca
180gacaggttca gtggcagtgg gtctgggaca gacttcactc tcaccatcag
cagactggag 240cctgaagatt ttgcagtgta ttactgtcag cagtatgata
gctcacctcg gacg 2945298PRTArtificialSC08-041 VL PROTEIN 52Glu 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 Asn 20
25 30 Tyr Leu Ala Trp Phe Gln Gln Lys Pro Gly Gln Ala Pro Arg Leu
Leu 35 40 45 Ile Tyr Gly Ala Ser Arg 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 Tyr Tyr Cys
Gln Gln Tyr Asp Ser Ser Pro 85 90 95 Arg Thr
53330DNAArtificialSC08-043 VH DNA 53caggtgcagc tggtgcagtc
tggggctgag gtgaagaagc ctggggcctc agtgaagctt 60tcctgcaagg cttctggata
caccttcact gcctattcta tgcattgggt gcgccaggcc 120cccggacaaa
gccttgagtg gttgggatgg atcaacactg ccatcggtaa cacacaatat
180tcacagaagt tccaggacag agtcaccatt accagggaca catctgcgcg
cacatcgtac 240atggaactga gcagcctgag atctggagac acggctgtct
atttctgtgc gagaggggcc 300tcttgggacg cccgtgggtg gtctggctac
33054110PRTArtificialSC08-043 VH PROTEIN 54Gln Val Gln Leu Val Gln
Ser Gly Ala Glu Val Lys Lys Pro Gly Ala 1 5 10 15 Ser Val Lys Leu
Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Ala Tyr 20 25 30 Ser Met
His Trp Val Arg Gln Ala Pro Gly Gln Ser Leu Glu Trp Leu 35 40 45
Gly Trp Ile Asn Thr Ala Ile Gly Asn Thr Gln Tyr Ser Gln Lys Phe 50
55 60 Gln Asp Arg Val Thr Ile Thr Arg Asp Thr Ser Ala Arg Thr Ser
Tyr 65 70 75 80 Met Glu Leu Ser Ser Leu Arg Ser Gly Asp Thr Ala Val
Tyr Phe Cys 85 90 95 Ala Arg Gly Ala Ser Trp Asp Ala Arg Gly Trp
Ser Gly Tyr 100 105 110 55309DNAArtificialSC08-043 VL DNA
55gacatccagw tgacccagtc tccagactcc ctggctgtgt ctctgggcga gagggccacc
60atcaactgca agtccagcca gagtgttttt tccagctcca ccaataagaa ctacttagct
120tggtaccagc agaaaccagg
acagcctcct aaggtgctaa tttactggtc atctacccgg 180gaatccgggg
tccctgaccg attcagtgcc agcgggtctg ggacagattt cactctcacc
240atcagcagcc tgcaggctgc agatgtggca gtttattact gtcaccaata
ttatactgct 300ccgtggacg 30956103PRTArtificialSC08-043 VL PROTEIN
56Asp Ile Gln Xaa Thr Gln Ser Pro Asp Ser Leu Ala Val Ser Leu Gly 1
5 10 15 Glu Arg Ala Thr Ile Asn Cys Lys Ser Ser Gln Ser Val Phe Ser
Ser 20 25 30 Ser Thr Asn Lys Asn Tyr Leu Ala Trp Tyr Gln Gln Lys
Pro Gly Gln 35 40 45 Pro Pro Lys Val Leu Ile Tyr Trp Ser Ser Thr
Arg Glu Ser Gly Val 50 55 60 Pro Asp Arg Phe Ser Ala Ser Gly Ser
Gly Thr Asp Phe Thr Leu Thr 65 70 75 80 Ile Ser Ser Leu Gln Ala Ala
Asp Val Ala Val Tyr Tyr Cys His Gln 85 90 95 Tyr Tyr Thr Ala Pro
Trp Thr 100 57345DNAArtificialSC08-049 VH DNA 57caggtcacct
tgaaggagtc tggtcctgta ctggtgaagc ccaaagagac cctcacgctg 60acctgcaccg
tctctgggtt ctcactcagc aacactagaa tgggtgtgag ttggatccgt
120cagcccccag ggaaggccct ggagtggctt gcgcacatct tttcgaacga
cgaaacatcc 180tacaggacat ctctgaagag gaggctcacc atctcccagg
acatctccaa aagtcaggtg 240gtcctttcta tgaccaacgt ggaccctgca
gacacagcca catatttttg tgcacggatc 300gggtctggct atgagagtag
tgcttactcc acctggctcg acccc 34558115PRTArtificialSC08-049 VH
PROTEIN 58Gln Val Thr Leu Lys Glu Ser Gly Pro Val Leu Val Lys Pro
Lys Glu 1 5 10 15 Thr Leu Thr Leu Thr Cys Thr Val Ser Gly Phe Ser
Leu Ser Asn Thr 20 25 30 Arg Met Gly Val Ser Trp Ile Arg Gln Pro
Pro Gly Lys Ala Leu Glu 35 40 45 Trp Leu Ala His Ile Phe Ser Asn
Asp Glu Thr Ser Tyr Arg Thr Ser 50 55 60 Leu Lys Arg Arg Leu Thr
Ile Ser Gln Asp Ile Ser Lys Ser Gln Val 65 70 75 80 Val Leu Ser Met
Thr Asn Val Asp Pro Ala Asp Thr Ala Thr Tyr Phe 85 90 95 Cys Ala
Arg Ile Gly Ser Gly Tyr Glu Ser Ser Ala Tyr Ser Thr Trp 100 105 110
Leu Asp Pro 115 5998PRTArtificialSC08-049 VL PROTEIN 59Gln Ser Val
Leu Thr Gln Pro Pro Ser Val Ser Val Ala Pro Gly Gln 1 5 10 15 Thr
Ala Arg Leu Thr Cys Glu Gly Asp Thr Ile Gly Ser Lys Ser Val 20 25
30 His Trp Tyr Gln Gln Arg Pro Gly Gln Ala Pro Val Leu Val Val Tyr
35 40 45 Asn Asp Arg Asp Arg Pro Ser Gly Ile Pro Glu Arg Phe Ser
Gly Ser 50 55 60 Asn Ser Gly Arg Thr Ala Thr Leu Thr Ile Ser Arg
Val Glu Ala Gly 65 70 75 80 Asp Glu Ala Asp Tyr Phe Cys Gln Val Trp
Glu Ser Gly Gly Asp Gln 85 90 95 Thr Val 60339DNAArtificialSC08-050
VH DNA 60caggtgcagc tacagcagtg gggcgcagga ctattgaagc cttcggagac
cctgtccctc 60acctgcgctg tgtatggtgg gtcgttcact gatcactact ggagctggat
ccgccagtcc 120ccagggaagg ggctggagtg gattggtgaa gtcgttcata
gtggagacac caactacacc 180ccgtccctca gaaatcgagt ttccatatcg
gtcgactcgt ccaagaatca gttctccctg 240aggctggggt ctgtgaccgc
cgcggacacg gctgtctatt actgtgcgag aggcaggaat 300gttgcggtag
ttggtgctat tcagaggcac tatgactac 33961113PRTArtificialSC08-050 VH
PROTEIN 61Gln Val Gln Leu Gln Gln Trp Gly Ala Gly Leu Leu Lys Pro
Ser Glu 1 5 10 15 Thr Leu Ser Leu Thr Cys Ala Val Tyr Gly Gly Ser
Phe Thr Asp His 20 25 30 Tyr Trp Ser Trp Ile Arg Gln Ser Pro Gly
Lys Gly Leu Glu Trp Ile 35 40 45 Gly Glu Val Val His Ser Gly Asp
Thr Asn Tyr Thr Pro Ser Leu Arg 50 55 60 Asn Arg Val Ser Ile Ser
Val Asp Ser Ser Lys Asn Gln Phe Ser Leu 65 70 75 80 Arg Leu Gly Ser
Val Thr Ala Ala Asp Thr Ala Val Tyr Tyr Cys Ala 85 90 95 Arg Gly
Arg Asn Val Ala Val Val Gly Ala Ile Gln Arg His Tyr Asp 100 105 110
Tyr 62294DNAArtificialSC08-050 VL DNA 62gaaattgtga tgacgcagtc
tccaggcacc ctgtctttgt ctccagggga aagagccacc 60ctctcctgca gggccagtca
gagtgttagc agaaactact tagcctggta ccagcagaag 120cctggcctgg
ctcccaggct cctcatctct ggtgcatcga gcagggccac tggcgtccca
180gacaggttca gtggcagggg gtctgacaca gacttcactc tcaccatcag
cagactggag 240cctgaagatt ttgccgtgta ttactgtcag cactatggtt
cggtccttgt agct 2946398PRTArtificialSC08-050 VL PROTEIN 63Glu Ile
Val Met 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 Arg Asn 20
25 30 Tyr Leu Ala Trp Tyr Gln Gln Lys Pro Gly Leu Ala Pro Arg Leu
Leu 35 40 45 Ile Ser Gly Ala Ser Ser Arg Ala Thr Gly Val Pro Asp
Arg Phe Ser 50 55 60 Gly Arg Gly Ser Asp Thr Asp Phe Thr Leu Thr
Ile Ser Arg Leu Glu 65 70 75 80 Pro Glu Asp Phe Ala Val Tyr Tyr Cys
Gln His Tyr Gly Ser Val Leu 85 90 95 Val Ala
64357DNAArtificialSC08-052 VH DNA 64caggtgcagc tgcaggagtc
gggcccagga ctggtgaagc cttcggagac cctgtccctc 60acctgcactg tctctggtgg
ctccgtcagc agtggtactt actactggag ctggatccgg 120cagcccccag
ggaagggact ggagtggatt ggggatatct cttacagtgg gagcaccaac
180tacaacccct ccctcaagag tcgagtcacc atttctagag acacgtccaa
gaacctggtc 240tccctgaagc tgacctctgt gaccgctgcg gacacggccg
tgcattactg tgcgagagcg 300atggcggctt ataattatga caggggtggt
tataacgact actactacat ggacgtc 35765119PRTArtificialSC08-052 VH
PROTEIN 65Gln Val Gln Leu Gln Glu Ser Gly Pro Gly Leu Val Lys Pro
Ser Glu 1 5 10 15 Thr Leu Ser Leu Thr Cys Thr Val Ser Gly Gly Ser
Val Ser Ser Gly 20 25 30 Thr Tyr Tyr Trp Ser Trp Ile Arg Gln Pro
Pro Gly Lys Gly Leu Glu 35 40 45 Trp Ile Gly Asp Ile Ser Tyr Ser
Gly Ser Thr Asn Tyr Asn Pro Ser 50 55 60 Leu Lys Ser Arg Val Thr
Ile Ser Arg Asp Thr Ser Lys Asn Leu Val 65 70 75 80 Ser Leu Lys Leu
Thr Ser Val Thr Ala Ala Asp Thr Ala Val His Tyr 85 90 95 Cys Ala
Arg Ala Met Ala Ala Tyr Asn Tyr Asp Arg Gly Gly Tyr Asn 100 105 110
Asp Tyr Tyr Tyr Met Asp Val 115 66291DNAArtificialSC08-052 VL DNA
66gacatccagt tgacccagtc tccatcctcc ctgtctgcat ctgtcggaga cagagtcacc
60atcacttgcc gggcaagtca gggcattaac acctatttaa attggtatca gcaaaaacca
120gggaaggccc ctaaggtcct gatctttgct gcatccactt tgcaaagtgg
agtcccatca 180aggttcagtg gcagtggttc tgggacagaa ttcactctca
acatcaacaa tctgcaacct 240gaagattttg caacttacta ctgtcaacag
agttacagta ctgcgatcac t 2916797PRTArtificialSC08-052 VL PROTEIN
67Asp Ile Gln Leu Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly 1
5 10 15 Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Gly Ile Asn Thr
Tyr 20 25 30 Leu Asn Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys
Val Leu Ile 35 40 45 Phe Ala Ala Ser Thr Leu Gln Ser Gly Val Pro
Ser Arg Phe Ser Gly 50 55 60 Ser Gly Ser Gly Thr Glu Phe Thr Leu
Asn Ile Asn Asn Leu Gln Pro 65 70 75 80 Glu Asp Phe Ala Thr Tyr Tyr
Cys Gln Gln Ser Tyr Ser Thr Ala Ile 85 90 95 Thr
68333DNAArtificialSC08-055 VH DNA 68gaggtgcagc tggtggagtc
tgggggaggc gtggtccagc ctgggaggtc cctgagactc 60tcctgtgcgg cgtctggatt
cagcttcacc acctatggca tgcactgggt ccgccaggct 120ccaggcaagg
ggctggagtg ggtggccttt atttggtatg atggaagtaa caaacactat
180caagactccg tgaagggccg attcaccatc tccaaggaca attccaacaa
catgttgtat 240ctgcaaatgg acagcctgag agtcgccgac acggctgttt
attactgtgt gagagatggg 300ggatatagca cctgggaatg gtacttcgat ctc
33369111PRTArtificialSC08-055 VH PROTEIN 69Glu Val Gln Leu Val Glu
Ser Gly Gly Gly Val Val Gln Pro Gly Arg 1 5 10 15 Ser Leu Arg Leu
Ser Cys Ala Ala Ser Gly Phe Ser Phe Thr Thr Tyr 20 25 30 Gly Met
His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45
Ala Phe Ile Trp Tyr Asp Gly Ser Asn Lys His Tyr Gln Asp Ser Val 50
55 60 Lys Gly Arg Phe Thr Ile Ser Lys Asp Asn Ser Asn Asn Met Leu
Tyr 65 70 75 80 Leu Gln Met Asp Ser Leu Arg Val Ala Asp Thr Ala Val
Tyr Tyr Cys 85 90 95 Val Arg Asp Gly Gly Tyr Ser Thr Trp Glu Trp
Tyr Phe Asp Leu 100 105 110 70291DNAArtificialSC08-055 VL DNA
70gaaattgtgc tgactcagtc tccagacttt cagtctgtgg ctccaaagga gaaagtcacc
60atcacctgcc gggccagtcg gagcattggt agtgacttgc actggtttca gcagaggcca
120gatcagtctc caaagctcct catcaagttt gcttcccagt ccatgtcagg
ggtcccctcg 180aggttcagtg gcagtgggtc tgggagagat ttcaccctca
ccatcagtag cctggaggct 240gaagatgctg ctacgtatta ctgtcatcag
agtagtagtt taccgctcac t 2917197PRTArtificialSC08-055 VL PROTEIN
71Glu Ile Val Leu Thr Gln Ser Pro Asp Phe Gln Ser Val Ala Pro Lys 1
5 10 15 Glu Lys Val Thr Ile Thr Cys Arg Ala Ser Arg Ser Ile Gly Ser
Asp 20 25 30 Leu His Trp Phe Gln Gln Arg Pro Asp Gln Ser Pro Lys
Leu Leu Ile 35 40 45 Lys Phe Ala Ser Gln Ser Met Ser Gly Val Pro
Ser Arg Phe Ser Gly 50 55 60 Ser Gly Ser Gly Arg Asp Phe Thr Leu
Thr Ile Ser Ser Leu Glu Ala 65 70 75 80 Glu Asp Ala Ala Thr Tyr Tyr
Cys His Gln Ser Ser Ser Leu Pro Leu 85 90 95 Thr
72348DNAArtificialSC08-057 VH DNA 72gaggtgcagc tggtggagtc
tggaggaggc ttggtccaac ctggggggtc cctgagactc 60tcctgtgcag cctctgggtt
caccgacagt gtcatcttca tgagttgggt ccgccaggct 120ccagggaagg
ggctggagtg tgtctcaatt atttatatcg atgattccac atactacgca
180gactccgtga agggccgatt caccatctcc agacacaatt ccatgggcac
agtgtttctt 240gaaatgaaca gcctgagacc tgacgacacg gccgtctatt
actgtgcgac agagagcgga 300gactttggtg accaaacggg tccctatcat
tactacgcta tggacgtc 34873116PRTArtificialSC08-057 VH PROTEIN (
73Glu Val Gln Leu Val 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 Thr Asp Ser Val
Ile 20 25 30 Phe Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu
Glu Cys Val 35 40 45 Ser Ile Ile Tyr Ile Asp Asp Ser Thr Tyr Tyr
Ala Asp Ser Val Lys 50 55 60 Gly Arg Phe Thr Ile Ser Arg His Asn
Ser Met Gly Thr Val Phe Leu 65 70 75 80 Glu Met Asn Ser Leu Arg Pro
Asp Asp Thr Ala Val Tyr Tyr Cys Ala 85 90 95 Thr Glu Ser Gly Asp
Phe Gly Asp Gln Thr Gly Pro Tyr His Tyr Tyr 100 105 110 Ala Met Asp
Val 115 74300DNAArtificialSC08-057 VL DNA 74cagtctgccc tgactcagcc
tgcctccgtg tctgggtctc ctggacagtc gatcaccatc 60tcctgcactg gaagcagcgg
tgacattggt ggttataacg ctgtctcctg gtaccaacac 120cacccaggca
aagcccccaa actgatgatt tatgaggtca ctagtcggcc ctcaggggtt
180tccgatcgct tctctgcgtc caggtctggc gacacggcct ccctgactgt
ctctggtctc 240caggctgagg acgaggctca ctattactgc tgctcatttg
cagacagcaa cattttgatt 30075100PRTArtificialSC08-057 VL PROTEIN
75Gln 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 Ser Ser Gly Asp Ile Gly Gly
Tyr 20 25 30 Asn Ala Val Ser Trp Tyr Gln His His Pro Gly Lys Ala
Pro Lys Leu 35 40 45 Met Ile Tyr Glu Val Thr Ser Arg Pro Ser Gly
Val Ser Asp Arg Phe 50 55 60 Ser Ala Ser Arg Ser Gly Asp Thr Ala
Ser Leu Thr Val Ser Gly Leu 65 70 75 80 Gln Ala Glu Asp Glu Ala His
Tyr Tyr Cys Cys Ser Phe Ala Asp Ser 85 90 95 Asn Ile Leu Ile 100
76336DNAArtificialSC08-069 VH DNA 76gaggtgcagc tggtggagac
tgggggagtc gtggtacagc ctggggggtc cctgagactc 60tcctgtgcag cctctggctt
cacgtttgag gattatacca tgcactgggt ccgtcaagtt 120ccggggaagg
gtctggagtg ggtcgcgctc attagttggg atggcggtat gtcaaactat
180gcagactctg tgaagggccg attcaccatc tccagagaca acagcaaaaa
ctctctgtat 240ctgcaagtga gcagtctgag aagtgaagac accgccctgt
attactgtgc aaaagatata 300cgaccccgta tgccagctcg tcactttatg gacgtc
33677112PRTArtificialSC08-069 VH PROTEIN 77Glu Val Gln Leu Val Glu
Thr Gly Gly Val Val Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu
Ser Cys Ala Ala Ser Gly Phe Thr Phe Glu Asp Tyr 20 25 30 Thr Met
His Trp Val Arg Gln Val Pro Gly Lys Gly Leu Glu Trp Val 35 40 45
Ala Leu Ile Ser Trp Asp Gly Gly Met Ser Asn Tyr Ala Asp Ser Val 50
55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Ser Leu
Tyr 65 70 75 80 Leu Gln Val Ser Ser Leu Arg Ser Glu Asp Thr Ala Leu
Tyr Tyr Cys 85 90 95 Ala Lys Asp Ile Arg Pro Arg Met Pro Ala Arg
His Phe Met Asp Val 100 105 110 78297DNAArtificialSC08-069 VL DNA
78gaaattgtgt tgacgcagtc tccagccacc ctgtctgtgt ctccggggga aagagccacc
60ctctcctgca gggccagtca gaatgtcaac tacaacttag cctggtacca gcagaaacct
120ggccaggctc ccaggctcct catctatgtt gcatccacca gggccactgg
tatcccagac 180aggttcagtg gcagtgggtc tgggacagag ttcactctca
ccatcagcag tctgcagtct 240gaagattttg cagtttatta ctgtcagcag
tataataact ggcctccggc gatcact 2977999PRTArtificialSC08-069 VL
PROTEIN 79Glu Ile Val Leu Thr Gln Ser Pro Ala Thr Leu Ser Val Ser
Pro Gly 1 5 10 15 Glu Arg Ala Thr Leu Ser Cys Arg Ala Ser Gln Asn
Val Asn Tyr Asn 20 25 30 Leu Ala Trp Tyr Gln Gln Lys Pro Gly Gln
Ala Pro Arg Leu Leu Ile 35 40 45 Tyr Val Ala Ser Thr Arg Ala Thr
Gly Ile Pro Asp Arg Phe Ser Gly 50 55 60 Ser Gly Ser Gly Thr Glu
Phe Thr Leu Thr Ile Ser Ser Leu Gln Ser 65 70 75 80 Glu Asp Phe Ala
Val Tyr Tyr Cys Gln Gln Tyr Asn Asn Trp Pro Pro 85 90 95 Ala Ile
Thr 805PRTArtificialHCDR1 80Ser Asn Tyr Val Ser 1 5
8116PRTArtificialHCDR2 81Leu Ile Tyr Thr Gly Gly Thr Thr Tyr Tyr
Ala Asp Ser Val Lys Gly 1 5 10 15 8216PRTArtificialHCDR3 82Val Ser
Ala Leu Arg Phe Leu Gln Trp Pro Asn Tyr Ala Met Asp Val 1 5 10 15
8314PRTArtificialLCDR1 83Ser Gly Thr Arg Ser Asp Val Gly Gly His
Asn Tyr Val Ser 1 5 10 847PRTArtificialLCDR2 84Glu Val Ser His Arg
Pro Ser 1 5 8511PRTArtificialLCDR3 85Ser Ser Tyr Thr Gly Glu Gly
Pro Leu Gly Val 1 5 10 865PRTArtificialHCDR1 86Ser Tyr Trp Met Ser
1 5 8717PRTArtificialHCDR2 87Asn Met Lys Gln Asp Gly Ser Glu Lys
Tyr Tyr Val Asp Ser Val Lys 1 5 10 15 Gly 8816PRTArtificialHCDR3
88Gly Ser Cys Asp Asp Ser Trp Thr Gly Cys His Asp Ala Phe Asp Ile 1
5 10 15 8911PRTArtificialLCDR1 89Gly Gly Asn Asn Ile Gly Ser Lys
Ser Val His 1 5 10 906PRTArtificialLCDR2 90Asp Ser Ala Arg Pro Ser
1 5 9112PRTArtificialLCDR3 91Gln Val Trp Glu Ser Gly Ser Asp Leu
Arg Leu Leu 1 5 10 9211PRTArtificialLCDR1 92Gly Gly Asp Asn Ile Gly
Arg Lys Ser Val His 1 5 10
937PRTArtificialLCDR2 93Asp Asn Ser Asp Arg Pro Ser 1 5
9411PRTArtificialLCDR3 94His Val Trp Gly Ser Ser Arg Asp His Tyr
Val 1 5 10 9514PRTArtificialLCDR1 95Thr Gly Ser Ser Ser Asn Ile Gly
Ala Gly Tyr Asp Val His 1 5 10 967PRTArtificialLCDR2 96Gly Asn Asn
Asn Arg Pro Ser 1 5 9711PRTArtificialLCDR3 97Gln Ser Tyr Asp Ser
Ser Leu Ser Val Tyr Val 1 5 10 9811PRTArtificialLCDR1 98Gln Gly Asp
Ser Leu Arg Ser Tyr Tyr Ala Ser 1 5 10 997PRTArtificialLCDR2 99Ala
Lys Thr Asn Arg Pro Ser 1 5 10011PRTArtificialLCDR3 100Asn Ser Arg
Asp Ser Ser Gly Asn His Val Val 1 5 10 10114PRTArtificialLCDR1
101Thr Gly Thr Ser Ser Asp Val Gly Gly Tyr Asn Tyr Val Ser 1 5 10
1025PRTArtificialHCDR1 102Thr Ser Ala Met Ser 1 5
10317PRTArtificialHCDR2 103Gly Ile Ser Gly Ser Gly Ala Thr Thr Tyr
Tyr Ala Gly Ser Val Lys 1 5 10 15 Gly 10421PRTArtificialHCDR3
104Asp Thr Ser Leu Phe Glu Tyr Asp Thr Ser Gly Phe Thr Ala Pro Gly
1 5 10 15 Asn Ala Phe Asp Ile 20 10511PRTArtificialLCDR1 105Arg Ala
Ser Gln Ser Ile Ser Gly Tyr Leu Asn 1 5 10 1067PRTArtificialLCDR2
106Gly Ala Ser Thr Leu Gln Ser 1 5 10710PRTArtificialLCDR3 107Gln
Gln Thr Tyr Thr Ser Pro Pro Tyr Ala 1 5 10 1085PRTArtificialHCDR1
108Arg Phe Gly Val Ser 1 5 10917PRTArtificialHCDR2 109Trp Ile Ser
Ala Tyr Asn Gly Asp Thr Tyr Tyr Ala Gln Lys Phe Gln 1 5 10 15 Ala
11013PRTArtificialHCDR3 110Glu Pro Pro Leu Phe Tyr Ser Ser Trp Ser
Leu Asp Asn 1 5 10 11113PRTArtificialLCDR1 111Ala Arg Ala Ser Gln
Ser Val Ser Met Asn Tyr Leu Ala 1 5 10 1127PRTArtificialLCDR2
112Gly Ala Ser Arg Arg Ala Thr 1 5 1139PRTArtificialLCDR3 113Gln
Gln Tyr Gly Thr Ser Pro Arg Thr 1 5 1145PRTArtificialHCDR1 114Ala
Tyr Ala Met Asn 1 5 11517PRTArtificialHCDR2 115Ala Ile Gly Gly Ser
Gly Gly Ser Thr Tyr Tyr Ala Asp Ser Val Lys 1 5 10 15 Gly
11612PRTArtificialHCDR3 116Gly Arg Asp Trp Thr Gly Gly Tyr Phe Phe
Asp Ser 1 5 10 11717PRTArtificialLCDR1 117Lys Ser Ser Gln Ser Ile
Phe Tyr Ser Ser Asn Asn Lys Asn Tyr Leu 1 5 10 15 Thr
1187PRTArtificialLCDR2 118Trp Ala Ser Thr Arg Glu Ser 1 5
1199PRTArtificialLCDR3 119Gln Gln Tyr Tyr Ser Ile Pro Tyr Thr 1 5
1205PRTArtificialHCDR1 120Gly Tyr Ala Met Ser 1 5
12117PRTArtificialHCDR2 121Asp Ile Gly Gly Ser Gly Gly Gly Thr Tyr
Tyr Ala Asp Ser Val Lys 1 5 10 15 Gly 12212PRTArtificialHCDR3
122Ser Ser Ser Trp Asp Arg Ala Tyr Phe Phe Asp Ser 1 5 10
12317PRTArtificialLCDR1 123Lys Ser Ser Gln Ser Val Leu Tyr Ser Ser
Ile His Lys Asn Tyr Leu 1 5 10 15 Ala 1249PRTArtificialLCDR3 124Gln
Gln Tyr Tyr Arg Ser Pro Pro Thr 1 5 1255PRTArtificialHCDR1 125Ser
Tyr Tyr Trp Ser 1 5 12616PRTArtificialHCDR2 126Tyr Ile Tyr Tyr Arg
Gly Gly Thr Ser Tyr Asn Pro Ser Leu Lys Ser 1 5 10 15
12715PRTArtificialHCDR3 127Lys Asp Trp Gly Ser Ala Ala Gly Ser Val
Trp Tyr Phe Asp Leu 1 5 10 15 12814PRTArtificialLCDR1 128Thr Gly
Thr Ser Ser Asp Val Gly Gly Tyr Asn Tyr Val Ser 1 5 10
1297PRTArtificialLCDR2 129Glu Val Ser Lys Arg Pro Ser 1 5
13010PRTArtificialLCDR3 130Ser Ser Tyr Ala Gly Ser Asn Asn Leu Ile
1 5 10 1315PRTArtificialHCDR1 131Ser Tyr Gly Met His 1 5
13217PRTArtificialHCDR2 132Phe Ile Trp Tyr Asp Gly Ser Asn Lys His
Tyr Ala Asp Ser Met Lys 1 5 10 15 Gly 13313PRTArtificialHCDR3
133Asp Gly Gly Tyr Ser Thr Trp Glu Trp Tyr Phe Asp Leu 1 5 10
13411PRTArtificialLCDR1 134Arg Ala Ser Gln Gly Ile Gly Ser Asn Leu
His 1 5 10 1357PRTArtificialLCDR2 135Tyr Ala Ser Gln Ser Ile Thr 1
5 1369PRTArtificialLCDR3 136His Gln Ser Ser Ser Leu Pro Leu Thr 1 5
1375PRTArtificialHCDR1 137Ser Phe Gly Leu Ser 1 5
13817PRTArtificialHCDR2 138Trp Ile Ser Ala Tyr Asn Gly Glu Ile Lys
Tyr Ala Gln Lys Phe Gln 1 5 10 15 Gly 13913PRTArtificialHCDR3
139Glu Pro Pro Leu Tyr Phe Ser Ser Trp Ser Leu Asp Phe 1 5 10
14013PRTArtificialLCDR1 140Ala Arg Ala Ser Gln Ser Val Ser Ser Asn
Tyr Leu Ala 1 5 10 1417PRTArtificialLCDR2 141Gly Ala Ser Arg Arg
Ala Thr 1 5 1429PRTArtificialLCDR3 142Gln Gln Tyr Asp Ser Ser Pro
Arg Thr 1 5 1435PRTArtificialHCDR1 143Ala Tyr Ser Met His 1 5
14417PRTArtificialHCDR2 144Trp Ile Asn Thr Ala Ile Gly Asn Thr Gln
Tyr Ser Gln Lys Phe Gln 1 5 10 15 Asp 14512PRTArtificialHCDR3
145Gly Ala Ser Trp Asp Ala Arg Gly Trp Ser Gly Tyr 1 5 10
14617PRTArtificialLCDR1 146Lys Ser Ser Gln Ser Val Phe Ser Ser Ser
Thr Asn Lys Asn Tyr Leu 1 5 10 15 Ala 1477PRTArtificialLCDR2 147Trp
Ser Ser Thr Arg Glu Ser 1 5 1489PRTArtificialLCDR3 148His Gln Tyr
Tyr Thr Ala Pro Trp Thr 1 5 1497PRTArtificialHCDR1 149Asn Thr Arg
Met Gly Val Ser 1 5 15016PRTArtificialHCDR2 150His Ile Phe Ser Asn
Asp Glu Thr Ser Tyr Arg Thr Ser Leu Lys Arg 1 5 10 15
15116PRTArtificialHCDR3 151Ile Gly Ser Gly Tyr Glu Ser Ser Ala Tyr
Ser Thr Trp Leu Asp Pro 1 5 10 15 15211PRTArtificialLCDR1 152Glu
Gly Asp Thr Ile Gly Ser Lys Ser Val His 1 5 10
1537PRTArtificialLCDR2 153Asn Asp Arg Asp Arg Pro Ser 1 5
15411PRTArtificialLCDR3 154Gln Val Trp Glu Ser Gly Gly Asp Gln Thr
Val 1 5 10 1555PRTArtificialHCDR1 155Asp His Tyr Trp Ser 1 5
15616PRTArtificialHCDR2 156Glu Val Val His Ser Gly Asp Thr Asn Tyr
Thr Pro Ser Leu Arg Asn 1 5 10 15 15716PRTArtificialHCDR3 157Gly
Arg Asn Val Ala Val Val Gly Ala Ile Gln Arg His Tyr Asp Tyr 1 5 10
15 15812PRTArtificialLCDR1 158Arg Ala Ser Gln Ser Val Ser Arg Asn
Tyr Leu Ala 1 5 10 1597PRTArtificialLCDR2 159Gly Ala Ser Ser Arg
Ala Thr 1 5 1609PRTArtificialLCDR3 160Gln His Tyr Gly Ser Val Leu
Val Ala 1 5 1617PRTArtificialHCDR1 161Ser Gly Thr Tyr Tyr Trp Ser 1
5 16216PRTArtificialHCDR2 162Asp Ile Ser Tyr Ser Gly Ser Thr Asn
Tyr Asn Pro Ser Leu Lys Ser 1 5 10 15 16320PRTArtificialHCDR3
163Ala Met Ala Ala Tyr Asn Tyr Asp Arg Gly Gly Tyr Asn Asp Tyr Tyr
1 5 10 15 Tyr Met Asp Val 20 16411PRTArtificialLCDR1 164Arg Ala Ser
Gln Gly Ile Asn Thr Tyr Leu Asn 1 5 10 1657PRTArtificialLCDR2
165Ala Ala Ser Thr Leu Gln Ser 1 5 1669PRTArtificialLCDR3 166Gln
Gln Ser Tyr Ser Thr Ala Ile Thr 1 5 1675PRTArtificialHCDR1 167Thr
Tyr Gly Met His 1 5 16817PRTArtificialHCDR2 168Phe Ile Trp Tyr Asp
Gly Ser Asn Lys His Tyr Gln Asp Ser Val Lys 1 5 10 15 Gly
16913PRTArtificialHCDR3 169Asp Gly Gly Tyr Ser Thr Trp Glu Trp Tyr
Phe Asp Leu 1 5 10 17011PRTArtificialLCDR1 170Arg Ala Ser Arg Ser
Ile Gly Ser Asp Leu His 1 5 10 1717PRTArtificialLCDR2 171Phe Ala
Ser Gln Ser Met Ser 1 5 1725PRTArtificialHCDR1 172Val Ile Phe Met
Ser 1 5 17316PRTArtificialHCDR2 173Ile Ile Tyr Ile Asp Asp Ser Thr
Tyr Tyr Ala Asp Ser Val Lys Gly 1 5 10 15 17419PRTArtificialHCDR3
174Glu Ser Gly Asp Phe Gly Asp Gln Thr Gly Pro Tyr His Tyr Tyr Ala
1 5 10 15 Met Asp Val 17514PRTArtificialLCDR1 175Thr Gly Ser Ser
Gly Asp Ile Gly Gly Tyr Asn Ala Val Ser 1 5 10
1767PRTArtificialLCDR2 176Glu Val Thr Ser Arg Pro Ser 1 5
17710PRTArtificialLCDR3 177Cys Ser Phe Ala Asp Ser Asn Ile Leu Ile
1 5 10 1785PRTArtificialHCDR1 178Asp Tyr Thr Met His 1 5
17917PRTArtificialHCDR2 179Leu Ile Ser Trp Asp Gly Gly Met Ser Asn
Tyr Ala Asp Ser Val Lys 1 5 10 15 Gly 18014PRTArtificialHCDR3
180Asp Ile Arg Pro Arg Met Pro Ala Arg His Phe Met Asp Val 1 5 10
18111PRTArtificialLCDR1 181Arg Ala Ser Gln Asn Val Asn Tyr Asn Leu
Ala 1 5 10 1827PRTArtificialLCDR2 182Val Ala Ser Thr Arg Ala Thr 1
5 18311PRTArtificialLCDR3 183Gln Gln Tyr Asn Asn Trp Pro Pro Ala
Ile Thr 1 5 10 1841353DNAArtificialCR6261 HC DNA 184gaggtgcagc
tggtggagtc tggggctgag gtgaagaagc ctgggtcctc ggtgaaagtc 60tcttgcaagg
cttctggagg ccccttccgc agctatgcta tcagctgggt gcgacaggcc
120cctggacaag ggcctgagtg gatgggaggg atcatcccta tttttggtac
aacaaaatac 180gcaccgaagt tccagggcag agtcacgatt accgcggacg
atttcgcggg cacagtttac 240atggagctga gcagcctgcg atctgaggac
acggccatgt actactgtgc gaaacatatg 300gggtaccagg tgcgcgaaac
tatggacgtc tggggcaaag ggaccacggt caccgtctcg 360agtgctagca
ccaagggccc cagcgtgttc cccctggccc ccagcagcaa gagcaccagc
420ggcggcacag ccgccctggg ctgcctggtg aaggactact tccccgagcc
cgtgaccgtg 480agctggaaca gcggcgcctt gaccagcggc gtgcacacct
tccccgccgt gctgcagagc 540agcggcctgt acagcctgag cagcgtggtg
accgtgccca gcagcagcct gggcacccag 600acctacatct gcaacgtgaa
ccacaagccc agcaacacca aggtggacaa acgcgtggag 660cccaagagct
gcgacaagac ccacacctgc cccccctgcc ctgcccccga gctgctgggc
720ggaccctccg tgttcctgtt cccccccaag cccaaggaca ccctcatgat
cagccggacc 780cccgaggtga cctgcgtggt ggtggacgtg agccacgagg
accccgaggt gaagttcaac 840tggtacgtgg acggcgtgga ggtgcacaac
gccaagacca agccccggga ggagcagtac 900aacagcacct accgggtggt
gagcgtgctc accgtgctgc accaggactg gctgaacggc 960aaggagtaca
agtgcaaggt gagcaacaag gccctgcctg cccccatcga gaagaccatc
1020agcaaggcca agggccagcc ccgggagccc caggtgtaca ccctgccccc
cagccgggag 1080gagatgacca agaaccaggt gtccctcacc tgtctggtga
agggcttcta ccccagcgac 1140atcgccgtgg agtgggagag caacggccag
cccgagaaca actacaagac caccccccct 1200gtgctggaca gcgacggcag
cttcttcctg tacagcaagc tcaccgtgga caagagccgg 1260tggcagcagg
gcaacgtgtt cagctgcagc gtgatgcacg aggccctgca caaccactac
1320acccagaaga gcctgagcct gagccccggc aag
1353185451PRTArtificialCR6261 HC PROTEIN 185Glu Val Gln Leu Val Glu
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 Pro Phe Arg Ser Tyr 20 25 30 Ala Ile
Ser Trp Val Arg Gln Ala Pro Gly Gln Gly Pro Glu Trp Met 35 40 45
Gly Gly Ile Ile Pro Ile Phe Gly Thr Thr Lys Tyr Ala Pro Lys Phe 50
55 60 Gln Gly Arg Val Thr Ile Thr Ala Asp Asp Phe Ala Gly Thr Val
Tyr 65 70 75 80 Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Met
Tyr Tyr Cys 85 90 95 Ala Lys His Met Gly Tyr Gln Val Arg Glu Thr
Met Asp Val Trp Gly 100 105 110 Lys Gly Thr Thr Val Thr Val Ser Ser
Ala Ser Thr Lys Gly Pro Ser 115 120 125 Val Phe Pro Leu Ala Pro Ser
Ser Lys Ser Thr Ser Gly Gly Thr Ala 130 135 140 Ala Leu Gly Cys Leu
Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val 145 150 155 160 Ser Trp
Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala 165 170 175
Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val 180
185 190 Pro Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn
His 195 200 205 Lys Pro Ser Asn Thr Lys Val Asp Lys Arg Val Glu Pro
Lys Ser Cys 210 215 220 Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala
Pro Glu Leu Leu Gly 225 230 235 240 Gly Pro Ser Val Phe Leu Phe Pro
Pro Lys Pro Lys Asp Thr Leu Met 245 250 255 Ile Ser Arg Thr Pro Glu
Val Thr Cys Val Val Val Asp Val Ser His 260 265 270 Glu Asp Pro Glu
Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val 275 280 285 His Asn
Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr 290 295 300
Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly 305
310 315 320 Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala
Pro Ile 325 330 335 Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg
Glu Pro Gln Val 340 345 350 Tyr Thr Leu Pro Pro Ser Arg Glu Glu Met
Thr Lys Asn Gln Val Ser 355 360 365 Leu Thr Cys Leu Val Lys Gly Phe
Tyr Pro Ser Asp Ile Ala Val Glu 370 375 380 Trp Glu Ser Asn Gly Gln
Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro 385 390 395 400 Val Leu Asp
Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val 405 410 415 Asp
Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met 420 425
430 His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser
435 440 445 Pro Gly Lys 450 186663DNAArtificialCR6261 LC DNA
186cagtctgtgt tgacgcagcc gccctcagtg tctgcggccc caggacagaa
ggtcaccatc 60tcctgctctg gaagcagctc caacattggg aatgattatg tatcctggta
ccagcagctc 120ccaggaacag cccccaaact cctcatttat gacaataata
agcgaccctc agggattcct 180gaccgattct ctggctccaa gtctggcacg
tcagccaccc tgggcatcac cggactccag 240actggggacg aggccaacta
ttactgcgca acatgggatc gccgcccgac tgcttatgtt 300gtcttcggcg
gagggaccaa gctgaccgtc ctaggtgcgg ccgcaggcca gcccaaggcc
360gctcccagcg tgaccctgtt ccccccctcc tccgaggagc tgcaggccaa
caaggccacc 420ctggtgtgcc tcatcagcga cttctaccct ggcgccgtga
ccgtggcctg gaaggccgac 480agcagccccg tgaaggccgg cgtggagacc
accaccccca gcaagcagag caacaacaag 540tacgccgcca gcagctacct
gagcctcacc cccgagcagt ggaagagcca ccggagctac 600agctgccagg
tgacccacga gggcagcacc gtggagaaga ccgtggcccc caccgagtgc 660agc
663187221PRTArtificialCR6261 LC PROTEIN 187Gln Ser Val Leu Thr Gln
Pro Pro Ser Val Ser Ala Ala Pro Gly Gln 1 5 10 15 Lys Val Thr Ile
Ser Cys Ser Gly Ser Ser Ser Asn Ile Gly Asn Asp 20 25 30 Tyr Val
Ser Trp Tyr Gln Gln Leu Pro Gly Thr Ala Pro Lys Leu Leu 35 40 45
Ile Tyr Asp Asn Asn Lys Arg Pro Ser Gly Ile Pro Asp Arg Phe Ser 50
55 60 Gly Ser Lys Ser Gly Thr Ser Ala Thr Leu Gly Ile Thr Gly Leu
Gln 65 70 75 80 Thr Gly Asp Glu Ala Asn Tyr Tyr Cys Ala Thr Trp Asp
Arg Arg Pro 85 90 95 Thr Ala Tyr Val Val Phe Gly Gly Gly Thr Lys
Leu Thr Val Leu Gly 100 105 110 Ala Ala Ala Gly Gln Pro Lys Ala Ala
Pro Ser Val Thr Leu Phe Pro 115 120 125 Pro Ser Ser Glu Glu Leu Gln
Ala Asn Lys Ala Thr Leu Val Cys Leu 130 135 140 Ile Ser Asp Phe Tyr
Pro Gly Ala Val Thr Val Ala Trp Lys Ala Asp 145 150 155 160 Ser Ser
Pro Val Lys Ala Gly Val Glu Thr Thr Thr Pro Ser Lys Gln 165 170
175 Ser Asn Asn Lys Tyr Ala Ala Ser Ser Tyr Leu Ser Leu Thr Pro Glu
180 185 190 Gln Trp Lys Ser His Arg Ser Tyr Ser Cys Gln Val Thr His
Glu Gly 195 200 205 Ser Thr Val Glu Lys Thr Val Ala Pro Thr Glu Cys
Ser 210 215 220 18810515DNAArtificialVector pIg-C911-HCgamma1
188tcgacggatc gggagatctc ccgatcccct atggtgcact ctcagtacaa
tctgctctga 60tgccgcatag ttaagccagt atctgctccc tgcttgtgtg ttggaggtcg
ctgagtagtg 120cgcgagcaaa atttaagcta caacaaggca aggcttgacc
gacaattgca tgaagaatct 180gcttagggtt aggcgttttg cgctgcttcg
ctaggtggtc aatattggcc attagccata 240ttattcattg gttatatagc
ataaatcaat attggctatt ggccattgca tacgttgtat 300ccatatcata
atatgtacat ttatattggc tcatgtccaa cattaccgcc atgttgacat
360tgattattga ctagttatta atagtaatca attacggggt cattagttca
tagcccatat 420atggagttcc gcgttacata acttacggta aatggcccgc
ctggctgacc gcccaacgac 480ccccgcccat tgacgtcaat aatgacgtat
gttcccatag taacgccaat agggactttc 540cattgacgtc aatgggtgga
gtatttacgg taaactgccc acttggcagt acatcaagtg 600tatcatatgc
caagtacgcc ccctattgac gtcaatgacg gtaaatggcc cgcctggcat
660tatgcccagt acatgacctt atgggacttt cctacttggc agtacatcta
cgtattagtc 720atcgctatta ccatggtgat gcggttttgg cagtacatca
atgggcgtgg atagcggttt 780gactcacggg gatttccaag tctccacccc
attgacgtca atgggagttt gttttggcac 840caaaatcaac gggactttcc
aaaatgtcgt aacaactccg ccccattgac gcaaatgggc 900ggtaggcgtg
tacggtggga ggtctatata agcagagctc gtttagtgaa ccgtcagatc
960gcctggagac gccatccacg ctgttttgac ctccatagaa gacaccggga
ccgatccagc 1020ctccgcggcc gggaacggtg cattggaagc tggcctggat
atcctgactc tcttaggtag 1080ccttgcagaa gttggtcgtg aggcactggg
caggtaagta tcaaggttac aagacaggtt 1140taaggagatc aatagaaact
gggcttgtcg agacagagaa gactcttgcg tttctgatag 1200gcacctattg
gtcttactga catccacttt gcctttctct ccacaggtgt ccactcccag
1260ttcaattaca gctcgccacc atgggatgga gctgtatcat cctcttcttg
gtactgctgc 1320tggcccagcc ggccagtgac cttgaccggt gcaccacttt
tgatgatgtt caagctccta 1380attacactca acatacttca tctatgaggg
gggtttacta tcctgatgaa atttttagat 1440cggacactct ttatttaact
caggatttat ttcttccatt ttattctaat gttacagggt 1500ttcatactat
taatcatacg tttggcaacc ctgtcatacc ttttaaggat ggtatttatt
1560ttgctgccac agagaaatca aatgttgtcc gtggttgggt ttttggttct
accatgaaca 1620acaagtcaca gtcggtgatt attattaaca attctactaa
tgttgttata cgagcatgta 1680actttgaatt gtgtgacaac cctttctttg
ctgtttctaa acccatgggt acacagacac 1740atactatgat attcgataat
gcatttaatt gcactttcga gtacatatct gatgcctttt 1800cgcttgatgt
ttcagaaaag tcaggtaatt ttaaacactt acgagagttt gtgtttaaaa
1860ataaagatgg gtttctctat gtttataagg gctatcaacc tatagatgta
gttcgtgatc 1920taccttctgg ttttaacact ttgaaaccta tttttaagtt
gcctcttggt attaacatta 1980caaattttag agccattctt acagcctttt
cacctgctca agacatttgg ggcacgtcag 2040ctgcagccta ttttgttggc
tatttaaagc caactacatt tatgctcaag tatgatgaaa 2100atggtacaat
cacagatgct gttgattgtt ctcaaaatcc acttgctgaa ctcaaatgct
2160ctgttaagag ctttgagatt gacaaaggaa tttaccagac ctctaatttc
agggttgttc 2220cctcaggaga tgttgtgaga ttccctaata ttacaaactt
gtgtcctttt ggagaggttt 2280ttaatgctac taaattccct tctgtctatg
catgggagag aaaaaaaatt tctaattgtg 2340ttgctgatta ctctgtgctc
tacaactcaa catttttttc aacctttaag tgctatggcg 2400tttctgccac
taagttgaat gatctttgct tctccaatgt ctatgcagat tcttttgtag
2460tcaagggaga tgatgtaaga caaatagcgc caggacaaac tggtgttatt
gctgattata 2520attataaatt gccagatgat ttcatgggtt gtgtccttgc
ttggaatact aggaacattg 2580atgctacttc aactggtaat tataattata
aatataggta tcttagacat ggcaagctta 2640ggccctttga gagagacata
tctaatgtgc ctttctcccc tgatggcaaa ccttgcaccc 2700cacctgctct
taattgttat tggccattaa atgattatgg tttttacacc actactggca
2760ttggctacca accttacaga gttgtagtac tttcttttga acttttaaat
gcaccggcca 2820cggtttgtgg accaaaatta tccactgacc ttattaagaa
ccagtgtgtc aattttaatt 2880ttaatggact cactggtact ggtgtgttaa
ctccttcttc aaagagattt caaccatttc 2940aacaatttgg ccgtgatgtt
tctgatttca ctgattccgt tcgagatcct aaaacatctg 3000aaatattaga
catttcacct tgctcttttg ggggtgtaag tgtaattaca cctggaacaa
3060atgcttcatc tgaagttgct gttctatatc aagatgttaa ctgcactgat
gtttctacag 3120caattcatgc agatcaactc acaccagctt ggcgcatata
ttctactgga aacaatgtat 3180tccagactca ggcaggctgt cttataggag
ctgagcatgt cgacacttct tatgagtgcg 3240acattcctat tggagctggc
atttgtgcta gttaccatac agtttcttta ttacgtagta 3300ctagccaaaa
atctattgtg gcttatacta tgtctttagg tgctgatagt tcaattgctt
3360actctaataa caccattgct atacctacta acttttcaat tagcattact
acagaagtaa 3420tgcctgtttc tatggctaaa acctccgtag attgtaatat
gtacatctgc ggagattcta 3480ctgaatgtgc taatttgctt ctccaatatg
gtagcttttg cacacaacta aatcgtgcac 3540tctcaggtat tgctgctgaa
caggatcgca acacacgtga agtgttcgct caagtcaaac 3600aaatgtacaa
aaccccaact ttgaaatatt ttggtggttt taatttttca caaatattac
3660ctgaccctct aaagccaact aagaggtctt ttattgagga cttgctcttt
aataaggtga 3720cactcgctga tgctggcttc atgaagcaat atggcgaatg
cctaggtgat attaatgcta 3780gagatctcat ttgtgcgcag aagttcaatg
gacttacagt gttgccacct ctgctcactg 3840atgatatgat tgctgcctac
actgctgctc tagttagtgg tactgccact gctggatgga 3900catttggtgc
tggcgctgct cttcaaatac cttttgctat gcaaatggca tataggttca
3960atggcattgg agttacccaa aatgttctct atgagaacca aaaacaaatc
gccaaccaat 4020ttaacaaggc gattagtcaa attcaagaat cacttacaac
aacatcaact gcattgggca 4080agctgcaaga cgttgttaac cagaatgctc
aagcattaaa cacacttgtt aaacaactta 4140gctctaattt tggtgcaatt
tcaagtgtgc taaatgatat cctttcgcga cttgataaag 4200tcgaggcgga
ggtacaaatt gacaggttaa ttacaggcag acttcaaagc cttcaaacct
4260atgtaacaca acaactaatc agggctgctg aaatcagggc ttctgctaat
cttgctgcta 4320ctaaaatgtc tgagtgtgtt cttggacaat caaaaagagt
tgacttttgt ggaaagggct 4380accaccttat gtccttccca caagcagccc
cgcatggtgt tgtcttccta catgtcacgt 4440atgtgccatc ccaggagagg
aacttcacca cagcgccagc aatttgtcat gaaggcaaag 4500catacttccc
tcgtgaaggt gtttttgtgt ttaatggcac ttcttggttt attacacaga
4560ggaacttctt ttctccacaa ataattacta cagacaatac atttgtctca
ggaaattgtg 4620atgtcgttat tggcatcatt aacaacacag tttatgatcc
tctgcaacct gagcttgact 4680cattcaaaga agagctggac aagtacttca
aaaatcatac atcaccagat gttgattttg 4740gcgacatttc aggcattaac
gcttctgtcg tcaacattca aaaagaaatt gaccgcctca 4800atgaggtcgc
taaaaattta aatgaatcac tcattgacct tcaagaactg ggaaaatatg
4860agcaatatat taaatggcct ctcgacgaac aaaaactcat ctcagaagag
gatctgaatg 4920ctgtgggcca ggacacgcag gaggtcatcg tggtgccaca
ctccttgccc tttaaggtgg 4980tggtgatctc agccatcctg gccctggtgg
tgctcaccat catctccctt atcatcctca 5040tcatgctttg gcagaagaag
ccacgttagg cggccgctcg agtgctagca ccaagggccc 5100cagcgtgttc
cccctggccc ccagcagcaa gagcaccagc ggcggcacag ccgccctggg
5160ctgcctggtg aaggactact tccccgagcc cgtgaccgtg agctggaaca
gcggcgcctt 5220gaccagcggc gtgcacacct tccccgccgt gctgcagagc
agcggcctgt acagcctgag 5280cagcgtggtg accgtgccca gcagcagcct
gggcacccag acctacatct gcaacgtgaa 5340ccacaagccc agcaacacca
aggtggacaa acgcgtggag cccaagagct gcgacaagac 5400ccacacctgc
cccccctgcc ctgcccccga gctgctgggc ggaccctccg tgttcctgtt
5460cccccccaag cccaaggaca ccctcatgat cagccggacc cccgaggtga
cctgcgtggt 5520ggtggacgtg agccacgagg accccgaggt gaagttcaac
tggtacgtgg acggcgtgga 5580ggtgcacaac gccaagacca agccccggga
ggagcagtac aacagcacct accgggtggt 5640gagcgtgctc accgtgctgc
accaggactg gctgaacggc aaggagtaca agtgcaaggt 5700gagcaacaag
gccctgcctg cccccatcga gaagaccatc agcaaggcca agggccagcc
5760ccgggagccc caggtgtaca ccctgccccc cagccgggag gagatgacca
agaaccaggt 5820gtccctcacc tgtctggtga agggcttcta ccccagcgac
atcgccgtgg agtgggagag 5880caacggccag cccgagaaca actacaagac
caccccccct gtgctggaca gcgacggcag 5940cttcttcctg tacagcaagc
tcaccgtgga caagagccgg tggcagcagg gcaacgtgtt 6000cagctgcagc
gtgatgcacg aggccctgca caaccactac acccagaaga gcctgagcct
6060gagccccggc aagtgataat ctagagggcc cgtttaaacc cgctgatcag
cctcgactgt 6120gccttctagt tgccagccat ctgttgtttg cccctccccc
gtgccttcct tgaccctgga 6180aggtgccact cccactgtcc tttcctaata
aaatgaggaa attgcatcgc attgtctgag 6240taggtgtcat tctattctgg
ggggtggggt ggggcaggac agcaaggggg aggattggga 6300agacaatagc
aggcatgctg gggatgcggt gggctctatg gcttctgagg cggaaagaac
6360cagctggggc tctagggggt atccccacgc gccctgtagc ggcgcattaa
gcgcggcggg 6420tgtggtggtt acgcgcagcg tgaccgctac acttgccagc
gccctagcgc ccgctccttt 6480cgctttcttc ccttcctttc tcgccacgtt
cgccggcttt ccccgtcaag ctctaaatcg 6540ggggctccct ttagggttcc
gatttagtgc tttacggcac ctcgacccca aaaaacttga 6600ttagggtgat
ggttcacgta gtgggccatc gccctgatag acggtttttc gccctttgac
6660gttggagtcc acgttcttta atagtggact cttgttccaa actggaacaa
cactcaaccc 6720tatctcggtc tattcttttg atttataagg gattttgccg
atttcggcct attggttaaa 6780aaatgagctg atttaacaaa aatttaacgc
gaattaattc tgtggaatgt gtgtcagtta 6840gggtgtggaa agtccccagg
ctccccagca ggcagaagta tgcaaagcat gcatctcaat 6900tagtcagcaa
ccaggtgtgg aaagtcccca ggctccccag caggcagaag tatgcaaagc
6960atgcatctca attagtcagc aaccatagtc ccgcccctaa ctccgcccat
cccgccccta 7020actccgccca gttccgccca ttctccgccc catggctgac
taattttttt tatttatgca 7080gaggccgagg ccgcctctgc ctctgagcta
ttccagaagt agtgaggagg cttttttgga 7140ggcctaggct tttgcaaaaa
gctcccggga gcttgtatat ccattttcgg atctgatcaa 7200gagacaggat
gaggatcgtt tcgcatgatt gaacaagatg gattgcacgc aggttctccg
7260gccgcttggg tggagaggct attcggctat gactgggcac aacagacaat
cggctgctct 7320gatgccgccg tgttccggct gtcagcgcag gggcgcccgg
ttctttttgt caagaccgac 7380ctgtccggtg ccctgaatga actgcaggac
gaggcagcgc ggctatcgtg gctggccacg 7440acgggcgttc cttgcgcagc
tgtgctcgac gttgtcactg aagcgggaag ggactggctg 7500ctattgggcg
aagtgccggg gcaggatctc ctgtcatctc accttgctcc tgccgagaaa
7560gtatccatca tggctgatgc aatgcggcgg ctgcatacgc ttgatccggc
tacctgccca 7620ttcgaccacc aagcgaaaca tcgcatcgag cgagcacgta
ctcggatgga agccggtctt 7680gtcgatcagg atgatctgga cgaagagcat
caggggctcg cgccagccga actgttcgcc 7740aggctcaagg cgcgcatgcc
cgacggcgag gatctcgtcg tgacccatgg cgatgcctgc 7800ttgccgaata
tcatggtgga aaatggccgc ttttctggat tcatcgactg tggccggctg
7860ggtgtggcgg accgctatca ggacatagcg ttggctaccc gtgatattgc
tgaagagctt 7920ggcggcgaat gggctgaccg cttcctcgtg ctttacggta
tcgccgctcc cgattcgcag 7980cgcatcgcct tctatcgcct tcttgacgag
ttcttctgag cgggactctg gggttcgaaa 8040tgaccgacca agcgacgccc
aacctgccat cacgagattt cgattccacc gccgccttct 8100atgaaaggtt
gggcttcgga atcgttttcc gggacgccgg ctggatgatc ctccagcgcg
8160gggatctcat gctggagttc ttcgcccacc ccaacttgtt tattgcagct
tataatggtt 8220acaaataaag caatagcatc acaaatttca caaataaagc
atttttttca ctgcattcta 8280gttgtggttt gtccaaactc atcaatgtat
cttatcatgt ctgtataccg tcgacctcta 8340gctagagctt ggcgtaatca
tggtcatagc tgtttcctgt gtgaaattgt tatccgctca 8400caattccaca
caacatacga gccggaagca taaagtgtaa agcctggggt gcctaatgag
8460tgagctaact cacattaatt gcgttgcgct cactgcccgc tttccagtcg
ggaaacctgt 8520cgtgccagct gcattaatga atcggccaac gcgcggggag
aggcggtttg cgtattgggc 8580gctcttccgc ttcctcgctc actgactcgc
tgcgctcggt cgttcggctg cggcgagcgg 8640tatcagctca ctcaaaggcg
gtaatacggt tatccacaga atcaggggat aacgcaggaa 8700agaacatgtg
agcaaaaggc cagcaaaagg ccaggaaccg taaaaaggcc gcgttgctgg
8760cgtttttcca taggctccgc ccccctgacg agcatcacaa aaatcgacgc
tcaagtcaga 8820ggtggcgaaa cccgacagga ctataaagat accaggcgtt
tccccctgga agctccctcg 8880tgcgctctcc tgttccgacc ctgccgctta
ccggatacct gtccgccttt ctcccttcgg 8940gaagcgtggc gctttctcat
agctcacgct gtaggtatct cagttcggtg taggtcgttc 9000gctccaagct
gggctgtgtg cacgaacccc ccgttcagcc cgaccgctgc gccttatccg
9060gtaactatcg tcttgagtcc aacccggtaa gacacgactt atcgccactg
gcagcagcca 9120ctggtaacag gattagcaga gcgaggtatg taggcggtgc
tacagagttc ttgaagtggt 9180ggcctaacta cggctacact agaagaacag
tatttggtat ctgcgctctg ctgaagccag 9240ttaccttcgg aaaaagagtt
ggtagctctt gatccggcaa acaaaccacc gctggtagcg 9300gtttttttgt
ttgcaagcag cagattacgc gcagaaaaaa aggatctcaa gaagatcctt
9360tgatcttttc tacggggtct gacgctcagt ggaacgaaaa ctcacgttaa
gggattttgg 9420tcatgagatt atcaaaaagg atcttcacct agatcctttt
aaattaaaaa tgaagtttta 9480aatcaatcta aagtatatat gagtaaactt
ggtctgacag ttaccaatgc ttaatcagtg 9540aggcacctat ctcagcgatc
tgtctatttc gttcatccat agttgcctga ctccccgtcg 9600tgtagataac
tacgatacgg gagggcttac catctggccc cagtgctgca atgataccgc
9660gagacccacg ctcaccggct ccagatttat cagcaataaa ccagccagcc
ggaagggccg 9720agcgcagaag tggtcctgca actttatccg cctccatcca
gtctattaat tgttgccggg 9780aagctagagt aagtagttcg ccagttaata
gtttgcgcaa cgttgttgcc attgctacag 9840gcatcgtggt gtcacgctcg
tcgtttggta tggcttcatt cagctccggt tcccaacgat 9900caaggcgagt
tacatgatcc cccatgttgt gcaaaaaagc ggttagctcc ttcggtcctc
9960cgatcgttgt cagaagtaag ttggccgcag tgttatcact catggttatg
gcagcactgc 10020ataattctct tactgtcatg ccatccgtaa gatgcttttc
tgtgactggt gagtactcaa 10080ccaagtcatt ctgagaatag tgtatgcggc
gaccgagttg ctcttgcccg gcgtcaatac 10140gggataatac cgcgccacat
agcagaactt taaaagtgct catcattgga aaacgttctt 10200cggggcgaaa
actctcaagg atcttaccgc tgttgagatc cagttcgatg taacccactc
10260gtgcacccaa ctgatcttca gcatctttta ctttcaccag cgtttctggg
tgagcaaaaa 10320caggaaggca aaatgccgca aaaaagggaa taagggcgac
acggaaatgt tgaatactca 10380tactcttcct ttttcaatat tattgaagca
tttatcaggg ttattgtctc atgagcggat 10440acatatttga atgtatttag
aaaaataaac aaataggggt tccgcgcaca tttccccgaa 10500aagtgccacc tgacg
105151898777DNAArtificialVector pIg-C909-Ckappa 189tcgacggatc
gggagatctc ccgatcccct atggtgcact ctcagtacaa tctgctctga 60tgccgcatag
ttaagccagt atctgctccc tgcttgtgtg ttggaggtcg ctgagtagtg
120cgcgagcaaa atttaagcta caacaaggca aggcttgacc gacaattgtt
aattaacatg 180aagaatctgc ttagggttag gcgttttgcg ctgcttcgct
aggtggtcaa tattggccat 240tagccatatt attcattggt tatatagcat
aaatcaatat tggctattgg ccattgcata 300cgttgtatcc atatcataat
atgtacattt atattggctc atgtccaaca ttaccgccat 360gttgacattg
attattgact agttattaat agtaatcaat tacggggtca ttagttcata
420gcccatatat ggagttccgc gttacataac ttacggtaaa tggcccgcct
ggctgaccgc 480ccaacgaccc ccgcccattg acgtcaataa tgacgtatgt
tcccatagta acgccaatag 540ggactttcca ttgacgtcaa tgggtggagt
atttacggta aactgcccac ttggcagtac 600atcaagtgta tcatatgcca
agtacgcccc ctattgacgt caatgacggt aaatggcccg 660cctggcatta
tgcccagtac atgaccttat gggactttcc tacttggcag tacatctacg
720tattagtcat cgctattacc atggtgatgc ggttttggca gtacatcaat
gggcgtggat 780agcggtttga ctcacgggga tttccaagtc tccaccccat
tgacgtcaat gggagtttgt 840tttggcacca aaatcaacgg gactttccaa
aatgtcgtaa caactccgcc ccattgacgc 900aaatgggcgg taggcgtgta
cggtgggagg tctatataag cagagctcgt ttagtgaacc 960gtcagatcgc
ctggagacgc catccacgct gttttgacct ccatagaaga caccgggacc
1020gatccagcct ccgcggccgg gaacggtgca ttggaatcga tgactctctt
aggtagcctt 1080gcagaagttg gtcgtgaggc actgggcagg taagtatcaa
ggttacaaga caggtttaag 1140gagatcaata gaaactgggc ttgtcgagac
agagaagact cttgcgtttc tgataggcac 1200ctattggtct tactgacatc
cactttgcct ttctctccac aggtgtccac tcccagttca 1260attacagctc
gccaccatgc ggctgcccgc ccagctgctg ggccttctca tgctgtgggt
1320gcccgcctcg agatctatcg atgcatgcca tggtaccaag cttgccacca
tgagcagcag 1380ctcttggctg ctgctgagcc tggtggccgt gacagccgcc
cagagcacca tcgaggagca 1440ggccaagacc ttcctggaca agttcaacca
cgaggccgag gacctgttct accagagcag 1500cctggccagc tggaactaca
acaccaacat caccgaggag aacgtgcaga acatgaacaa 1560cgccggcgac
aagtggagcg ccttcctgaa ggagcagagc acactggccc agatgtaccc
1620cctgcaggag atccagaacc tgaccgtgaa gctgcagctg caggccctgc
agcagaacgg 1680cagcagcgtg ctgagcgagg acaagagcaa gcggctgaac
accatcctga acaccatgtc 1740caccatctac agcaccggca aagtgtgcaa
ccccgacaac ccccaggagt gcctgctgct 1800ggagcccggc ctgaacgaga
tcatggccaa cagcctggac tacaacgagc ggctgtgggc 1860ctgggagagc
tggcggagcg aagtgggcaa gcagctgcgg cccctgtacg aggagtacgt
1920ggtgctgaag aacgagatgg ccagggccaa ccactacgag gactacggcg
actactggag 1980aggcgactac gaagtgaacg gcgtggacgg ctacgactac
agcagaggcc agctgatcga 2040ggacgtggag cacaccttcg aggagatcaa
gcctctgtac gagcacctgc acgcctacgt 2100gcgggccaag ctgatgaacg
cctaccccag ctacatcagc cccatcggct gcctgcccgc 2160ccacctgctg
ggcgacatgt ggggccggtt ctggaccaac ctgtacagcc tgaccgtgcc
2220cttcggccag aagcccaaca tcgacgtgac cgacgccatg gtggaccagg
cctgggacgc 2280ccagcggatc ttcaaggagg ccgagaagtt cttcgtgagc
gtgggcctgc ccaacatgac 2340ccagggcttt tgggagaaca gcatgctgac
cgaccccggc aatgtgcaga aggccgtgtg 2400ccaccccacc gcctgggacc
tgggcaaggg cgacttccgg atcctgatgt gcaccaaagt 2460gaccatggac
gacttcctga ccgcccacca cgagatgggc cacatccagt acgacatggc
2520ctacgccgcc cagcccttcc tgctgcggaa cggcgccaac gagggctttc
acgaggccgt 2580gggcgagatc atgagcctga gcgccgccac ccccaagcac
ctgaagagca tcggcctgct 2640gagccccgac ttccaggagg acaacgagac
cgagatcaac ttcctgctga agcaggccct 2700gaccatcgtg ggcaccctgc
ccttcaccta catgctggag aagtggcggt ggatggtgtt 2760taagggcgag
atccccaagg accagtggat gaagaagtgg tgggagatga agcgggagat
2820cgtgggcgtg gtggagcccg tgccccacga cgagacctac tgcgaccccg
ccagcctgtt 2880ccacgtgagc aacgactact ccttcatccg gtactacacc
cggaccctgt accagttcca 2940gttccaggag gccctgtgcc aggccgccaa
gcacgagggc cccctgcaca agtgcgacat 3000cagcaacagc accgaggccg
gacagaaact gttcaacatg ctgcggctgg gcaagagcga 3060gccctggacc
ctggccctgg agaatgtggt gggcgccaag aacatgaatg tgcgccccct
3120gctgaactac ttcgagcccc tgttcacctg gctgaaggac cagaacaaga
acagcttcgt 3180gggctggagc accgactgga gcccctacgc cgaccagagc
atcaaagtgc ggatcagcct 3240gaagagcgcc ctgggcgaca aggcctacga
gtggaacgac aacgagatgt acctgttccg 3300gagcagcgtg gcctatgcca
tgcggcagta cttcctgaaa gtgaagaacc agatgatcct 3360gttcggcgag
gaggacgtga gagtggccaa cctgaagccc cggatcagct tcaacttctt
3420cgtgaccgcc cccaagaacg tgagcgacat catcccccgg accgaagtgg
agaaggccat 3480ccggatgagc cggagccgga tcaacgacgc cttccggctg
aacgacaact ccctggagtt 3540cctgggcatc cagcccaccc tgggccctcc
caaccagccc cccgtgagca tctggctgat 3600cgtgtttggc gtggtgatgg
gcgtgatcgt ggtgggaatc gtgatcctga tcttcaccgg 3660catccgggac
cggaagaaga agaacaaggc ccggagcggc gagaacccct acgccagcat
3720cgatatcagc aagggcgaga acaaccccgg cttccagaac accgacgacg
tgcagaccag 3780cttctgataa tctagaacga gctcgaattc gaagcttctg
cagacgcgtc gacgtcatat 3840ggatccgata tcgccgtggc ggccgcaccc
agcgtgttca tcttcccccc ctccgacgag 3900cagctgaaga gcggcaccgc
cagcgtggtg tgcctgctga acaacttcta cccccgggag 3960gccaaggtgc
agtggaaggt ggacaacgcc ctgcagagcg gcaacagcca ggagagcgtg
4020accgagcagg acagcaagga ctccacctac agcctgagca gcaccctcac
cctgagcaag 4080gccgactacg agaagcacaa
ggtgtacgcc tgcgaggtga cccaccaggg cctgagcagc 4140cccgtgacca
agagcttcaa ccggggcgag tgttaataga cttaagttta aaccgctgat
4200cagcctcgac tgtgccttct agttgccagc catctgttgt ttgcccctcc
cccgtgcctt 4260ccttgaccct ggaaggtgcc actcccactg tcctttccta
ataaaatgag gaaattgcat 4320cgcattgtct gagtaggtgt cattctattc
tggggggtgg ggtggggcag gacagcaagg 4380gggaggattg ggaagacaat
agcaggcatg ctggggatgc ggtgggctct atggcttctg 4440aggcggaaag
aaccagctgg ggctctaggg ggtatcccca cgcgccctgt agcggcgcat
4500taagcgcggc gggtgtggtg gttacgcgca gcgtgaccgc tacacttgcc
agcgccctag 4560cgcccgctcc tttcgctttc ttcccttcct ttctcgccac
gttcgccggc tttccccgtc 4620aagctctaaa tcgggggctc cctttagggt
tccgatttag tgctttacgg cacctcgacc 4680ccaaaaaact tgattagggt
gatggttcac gtagtgggcc atcgccctga tagacggttt 4740ttcgcccttt
gacgttggag tccacgttct ttaatagtgg actcttgttc caaactggaa
4800caacactcaa ccctatctcg gtctattctt ttgatttata agggattttg
gccatttcgg 4860cctattggtt aaaaaatgag ctgatttaac aaaaatttaa
cgcgaattaa ttctgtggaa 4920tgtgtgtcag ttagggtgtg gaaagtcccc
aggctcccca gcaggcagaa gtatgcaaag 4980catgcatctc aattagtcag
caaccaggtg tggaaagtcc ccaggctccc cagcaggcag 5040aagtatgcaa
agcatgcatc tcaattagtc agcaaccata gtcccgcccc taactccgcc
5100catcccgccc ctaactccgc ccagttccgc ccattctccg ccccatggct
gactaatttt 5160ttttatttat gcagaggccg aggccgcctc tgcctctgag
ctattccaga agtagtgagg 5220aggctttttt ggaggcctag gcttttgcaa
aaagctcccg ggagcttgta tatccatttt 5280cggatctgat cagcacgtga
tgaaaaagcc tgaactcacc gcgacgtctg tcgagaagtt 5340tctgatcgaa
aagttcgaca gcgtctccga cctgatgcag ctctcggagg gcgaagaatc
5400tcgtgctttc agcttcgatg taggagggcg tggatatgtc ctgcgggtaa
atagctgcgc 5460cgatggtttc tacaaagatc gttatgttta tcggcacttt
gcatcggccg cgctcccgat 5520tccggaagtg cttgacattg gggaattcag
cgagagcctg acctattgca tctcccgccg 5580tgcacagggt gtcacgttgc
aagacctgcc tgaaaccgaa ctgcccgctg ttctgcagcc 5640ggtcgcggag
gccatggatg cgatcgctgc ggccgatctt agccagacga gcgggttcgg
5700cccattcgga ccacaaggaa tcggtcaata cactacatgg cgtgatttca
tatgcgcgat 5760tgctgatccc catgtgtatc actggcaaac tgtgatggac
gacaccgtca gtgcgtccgt 5820cgcgcaggct ctcgatgagc tgatgctttg
ggccgaggac tgccccgaag tccggcacct 5880cgtgcacgcg gatttcggct
ccaacaatgt cctgacggac aatggccgca taacagcggt 5940cattgactgg
agcgaggcga tgttcgggga ttcccaatac gaggtcgcca acatcttctt
6000ctggaggccg tggttggctt gtatggagca gcagacgcgc tacttcgagc
ggaggcatcc 6060ggagcttgca ggatcgccgc ggctccgggc gtatatgctc
cgcattggtc ttgaccaact 6120ctatcagagc ttggttgacg gcaatttcga
tgatgcagct tgggcgcagg gtcgatgcga 6180cgcaatcgtc cgatccggag
ccgggactgt cgggcgtaca caaatcgccc gcagaagcgc 6240ggccgtctgg
accgatggct gtgtagaagt actcgccgat agtggaaacc gacgccccag
6300cactcgtccg agggcaaagg aatagcacgt gctacgagat ttcgattcca
ccgccgcctt 6360ctatgaaagg ttgggcttcg gaatcgtttt ccgggacgcc
ggctggatga tcctccagcg 6420cggggatctc atgctggagt tcttcgccca
ccccaacttg tttattgcag cttataatgg 6480ttacaaataa agcaatagca
tcacaaattt cacaaataaa gcattttttt cactgcattc 6540tagttgtggt
ttgtccaaac tcatcaatgt atcttatcat gtctgtatac cgtcgacctc
6600tagctagagc ttggcgtaat catggtcata gctgtttcct gtgtgaaatt
gttatccgct 6660cacaattcca cacaacatac gagccggaag cataaagtgt
aaagcctggg gtgcctaatg 6720agtgagctaa ctcacattaa ttgcgttgcg
ctcactgccc gctttccagt cgggaaacct 6780gtcgtgccag ctgcattaat
gaatcggcca acgcgcgggg agaggcggtt tgcgtattgg 6840gcgctcttcc
gcttcctcgc tcactgactc gctgcgctcg gtcgttcggc tgcggcgagc
6900ggtatcagct cactcaaagg cggtaatacg gttatccaca gaatcagggg
ataacgcagg 6960aaagaacatg tgagcaaaag gccagcaaaa ggccaggaac
cgtaaaaagg ccgcgttgct 7020ggcgtttttc cataggctcc gcccccctga
cgagcatcac aaaaatcgac gctcaagtca 7080gaggtggcga aacccgacag
gactataaag ataccaggcg tttccccctg gaagctccct 7140cgtgcgctct
cctgttccga ccctgccgct taccggatac ctgtccgcct ttctcccttc
7200gggaagcgtg gcgctttctc atagctcacg ctgtaggtat ctcagttcgg
tgtaggtcgt 7260tcgctccaag ctgggctgtg tgcacgaacc ccccgttcag
cccgaccgct gcgccttatc 7320cggtaactat cgtcttgagt ccaacccggt
aagacacgac ttatcgccac tggcagcagc 7380cactggtaac aggattagca
gagcgaggta tgtaggcggt gctacagagt tcttgaagtg 7440gtggcctaac
tacggctaca ctagaagaac agtatttggt atctgcgctc tgctgaagcc
7500agttaccttc ggaaaaagag ttggtagctc ttgatccggc aaacaaacca
ccgctggtag 7560cggttttttt gtttgcaagc agcagattac gcgcagaaaa
aaaggatctc aagaagatcc 7620tttgatcttt tctacggggt ctgacgctca
gtggaacgaa aactcacgtt aagggatttt 7680ggtcatgaga ttatcaaaaa
ggatcttcac ctagatcctt ttaaattaaa aatgaagttt 7740taaatcaatc
taaagtatat atgagtaaac ttggtctgac agttaccaat gcttaatcag
7800tgaggcacct atctcagcga tctgtctatt tcgttcatcc atagttgcct
gactccccgt 7860cgtgtagata actacgatac gggagggctt accatctggc
cccagtgctg caatgatacc 7920gcgagaccca cgctcaccgg ctccagattt
atcagcaata aaccagccag ccggaagggc 7980cgagcgcaga agtggtcctg
caactttatc cgcctccatc cagtctatta attgttgccg 8040ggaagctaga
gtaagtagtt cgccagttaa tagtttgcgc aacgttgttg ccattgctac
8100aggcatcgtg gtgtcacgct cgtcgtttgg tatggcttca ttcagctccg
gttcccaacg 8160atcaaggcga gttacatgat cccccatgtt gtgcaaaaaa
gcggttagct ccttcggtcc 8220tccgatcgtt gtcagaagta agttggccgc
agtgttatca ctcatggtta tggcagcact 8280gcataattct cttactgtca
tgccatccgt aagatgcttt tctgtgactg gtgagtactc 8340aaccaagtca
ttctgagaat agtgtatgcg gcgaccgagt tgctcttgcc cggcgtcaat
8400acgggataat accgcgccac atagcagaac tttaaaagtg ctcatcattg
gaaaacgttc 8460ttcggggcga aaactctcaa ggatcttacc gctgttgaga
tccagttcga tgtaacccac 8520tcgtgcaccc aactgatctt cagcatcttt
tactttcacc agcgtttctg ggtgagcaaa 8580aacaggaagg caaaatgccg
caaaaaaggg aataagggcg acacggaaat gttgaatact 8640catactcttc
ctttttcaat attattgaag catttatcag ggttattgtc tcatgagcgg
8700atacatattt gaatgtattt agaaaaataa acaaataggg gttccgcgca
catttccccg 8760aaaagtgcca cctgacg 87771908792DNAArtificialVector
pIg-C910-Clambda 190tcgacggatc gggagatctc ccgatcccct atggtgcact
ctcagtacaa tctgctctga 60tgccgcatag ttaagccagt atctgctccc tgcttgtgtg
ttggaggtcg ctgagtagtg 120cgcgagcaaa atttaagcta caacaaggca
aggcttgacc gacaattgtt aattaacatg 180aagaatctgc ttagggttag
gcgttttgcg ctgcttcgct aggtggtcaa tattggccat 240tagccatatt
attcattggt tatatagcat aaatcaatat tggctattgg ccattgcata
300cgttgtatcc atatcataat atgtacattt atattggctc atgtccaaca
ttaccgccat 360gttgacattg attattgact agttattaat agtaatcaat
tacggggtca ttagttcata 420gcccatatat ggagttccgc gttacataac
ttacggtaaa tggcccgcct ggctgaccgc 480ccaacgaccc ccgcccattg
acgtcaataa tgacgtatgt tcccatagta acgccaatag 540ggactttcca
ttgacgtcaa tgggtggagt atttacggta aactgcccac ttggcagtac
600atcaagtgta tcatatgcca agtacgcccc ctattgacgt caatgacggt
aaatggcccg 660cctggcatta tgcccagtac atgaccttat gggactttcc
tacttggcag tacatctacg 720tattagtcat cgctattacc atggtgatgc
ggttttggca gtacatcaat gggcgtggat 780agcggtttga ctcacgggga
tttccaagtc tccaccccat tgacgtcaat gggagtttgt 840tttggcacca
aaatcaacgg gactttccaa aatgtcgtaa caactccgcc ccattgacgc
900aaatgggcgg taggcgtgta cggtgggagg tctatataag cagagctcgt
ttagtgaacc 960gtcagatcgc ctggagacgc catccacgct gttttgacct
ccatagaaga caccgggacc 1020gatccagcct ccgcggccgg gaacggtgca
ttggaatcga tgactctctt aggtagcctt 1080gcagaagttg gtcgtgaggc
actgggcagg taagtatcaa ggttacaaga caggtttaag 1140gagatcaata
gaaactgggc ttgtcgagac agagaagact cttgcgtttc tgataggcac
1200ctattggtct tactgacatc cactttgcct ttctctccac aggtgtccac
tcccagttca 1260attacagctc gccaccatgc ggttctccgc tcagctgctg
ggccttctgg tgctgtggat 1320tcccggcgtc tcgagatcta tcgatgcatg
ccatggtacc aagcttgcca ccatgagcag 1380cagctcttgg ctgctgctga
gcctggtggc cgtgacagcc gcccagagca ccatcgagga 1440gcaggccaag
accttcctgg acaagttcaa ccacgaggcc gaggacctgt tctaccagag
1500cagcctggcc agctggaact acaacaccaa catcaccgag gagaacgtgc
agaacatgaa 1560caacgccggc gacaagtgga gcgccttcct gaaggagcag
agcacactgg cccagatgta 1620ccccctgcag gagatccaga acctgaccgt
gaagctgcag ctgcaggccc tgcagcagaa 1680cggcagcagc gtgctgagcg
aggacaagag caagcggctg aacaccatcc tgaacaccat 1740gtccaccatc
tacagcaccg gcaaagtgtg caaccccgac aacccccagg agtgcctgct
1800gctggagccc ggcctgaacg agatcatggc caacagcctg gactacaacg
agcggctgtg 1860ggcctgggag agctggcgga gcgaagtggg caagcagctg
cggcccctgt acgaggagta 1920cgtggtgctg aagaacgaga tggccagggc
caaccactac gaggactacg gcgactactg 1980gagaggcgac tacgaagtga
acggcgtgga cggctacgac tacagcagag gccagctgat 2040cgaggacgtg
gagcacacct tcgaggagat caagcctctg tacgagcacc tgcacgccta
2100cgtgcgggcc aagctgatga acgcctaccc cagctacatc agccccatcg
gctgcctgcc 2160cgcccacctg ctgggcgaca tgtggggccg gttctggacc
aacctgtaca gcctgaccgt 2220gcccttcggc cagaagccca acatcgacgt
gaccgacgcc atggtggacc aggcctggga 2280cgcccagcgg atcttcaagg
aggccgagaa gttcttcgtg agcgtgggcc tgcccaacat 2340gacccagggc
ttttgggaga acagcatgct gaccgacccc ggcaatgtgc agaaggccgt
2400gtgccacccc accgcctggg acctgggcaa gggcgacttc cggatcctga
tgtgcaccaa 2460agtgaccatg gacgacttcc tgaccgccca ccacgagatg
ggccacatcc agtacgacat 2520ggcctacgcc gcccagccct tcctgctgcg
gaacggcgcc aacgagggct ttcacgaggc 2580cgtgggcgag atcatgagcc
tgagcgccgc cacccccaag cacctgaaga gcatcggcct 2640gctgagcccc
gacttccagg aggacaacga gaccgagatc aacttcctgc tgaagcaggc
2700cctgaccatc gtgggcaccc tgcccttcac ctacatgctg gagaagtggc
ggtggatggt 2760gtttaagggc gagatcccca aggaccagtg gatgaagaag
tggtgggaga tgaagcggga 2820gatcgtgggc gtggtggagc ccgtgcccca
cgacgagacc tactgcgacc ccgccagcct 2880gttccacgtg agcaacgact
actccttcat ccggtactac acccggaccc tgtaccagtt 2940ccagttccag
gaggccctgt gccaggccgc caagcacgag ggccccctgc acaagtgcga
3000catcagcaac agcaccgagg ccggacagaa actgttcaac atgctgcggc
tgggcaagag 3060cgagccctgg accctggccc tggagaatgt ggtgggcgcc
aagaacatga atgtgcgccc 3120cctgctgaac tacttcgagc ccctgttcac
ctggctgaag gaccagaaca agaacagctt 3180cgtgggctgg agcaccgact
ggagccccta cgccgaccag agcatcaaag tgcggatcag 3240cctgaagagc
gccctgggcg acaaggccta cgagtggaac gacaacgaga tgtacctgtt
3300ccggagcagc gtggcctatg ccatgcggca gtacttcctg aaagtgaaga
accagatgat 3360cctgttcggc gaggaggacg tgagagtggc caacctgaag
ccccggatca gcttcaactt 3420cttcgtgacc gcccccaaga acgtgagcga
catcatcccc cggaccgaag tggagaaggc 3480catccggatg agccggagcc
ggatcaacga cgccttccgg ctgaacgaca actccctgga 3540gttcctgggc
atccagccca ccctgggccc tcccaaccag ccccccgtga gcatctggct
3600gatcgtgttt ggcgtggtga tgggcgtgat cgtggtggga atcgtgatcc
tgatcttcac 3660cggcatccgg gaccggaaga agaagaacaa ggcccggagc
ggcgagaacc cctacgccag 3720catcgatatc agcaagggcg agaacaaccc
cggcttccag aacaccgacg acgtgcagac 3780cagcttctga taatctagaa
cgagctcgaa ttcgaagctt ctgcagacgc gtcgacgtca 3840tatggatccg
atatcgccgt ggcggccgca ggccagccca aggccgctcc cagcgtgacc
3900ctgttccccc cctcctccga ggagctgcag gccaacaagg ccaccctggt
gtgcctcatc 3960agcgacttct accctggcgc cgtgaccgtg gcctggaagg
ccgacagcag ccccgtgaag 4020gccggcgtgg agaccaccac ccccagcaag
cagagcaaca acaagtacgc cgccagcagc 4080tacctgagcc tcacccccga
gcagtggaag agccaccgga gctacagctg ccaggtgacc 4140cacgagggca
gcaccgtgga gaagaccgtg gcccccaccg agtgcagcta atagacttaa
4200gtttaaaccg ctgatcagcc tcgactgtgc cttctagttg ccagccatct
gttgtttgcc 4260cctcccccgt gccttccttg accctggaag gtgccactcc
cactgtcctt tcctaataaa 4320atgaggaaat tgcatcgcat tgtctgagta
ggtgtcattc tattctgggg ggtggggtgg 4380ggcaggacag caagggggag
gattgggaag acaatagcag gcatgctggg gatgcggtgg 4440gctctatggc
ttctgaggcg gaaagaacca gctggggctc tagggggtat ccccacgcgc
4500cctgtagcgg cgcattaagc gcggcgggtg tggtggttac gcgcagcgtg
accgctacac 4560ttgccagcgc cctagcgccc gctcctttcg ctttcttccc
ttcctttctc gccacgttcg 4620ccggctttcc ccgtcaagct ctaaatcggg
ggctcccttt agggttccga tttagtgctt 4680tacggcacct cgaccccaaa
aaacttgatt agggtgatgg ttcacgtagt gggccatcgc 4740cctgatagac
ggtttttcgc cctttgacgt tggagtccac gttctttaat agtggactct
4800tgttccaaac tggaacaaca ctcaacccta tctcggtcta ttcttttgat
ttataaggga 4860ttttggccat ttcggcctat tggttaaaaa atgagctgat
ttaacaaaaa tttaacgcga 4920attaattctg tggaatgtgt gtcagttagg
gtgtggaaag tccccaggct ccccagcagg 4980cagaagtatg caaagcatgc
atctcaatta gtcagcaacc aggtgtggaa agtccccagg 5040ctccccagca
ggcagaagta tgcaaagcat gcatctcaat tagtcagcaa ccatagtccc
5100gcccctaact ccgcccatcc cgcccctaac tccgcccagt tccgcccatt
ctccgcccca 5160tggctgacta atttttttta tttatgcaga ggccgaggcc
gcctctgcct ctgagctatt 5220ccagaagtag tgaggaggct tttttggagg
cctaggcttt tgcaaaaagc tcccgggagc 5280ttgtatatcc attttcggat
ctgatcagca cgtgatgaaa aagcctgaac tcaccgcgac 5340gtctgtcgag
aagtttctga tcgaaaagtt cgacagcgtc tccgacctga tgcagctctc
5400ggagggcgaa gaatctcgtg ctttcagctt cgatgtagga gggcgtggat
atgtcctgcg 5460ggtaaatagc tgcgccgatg gtttctacaa agatcgttat
gtttatcggc actttgcatc 5520ggccgcgctc ccgattccgg aagtgcttga
cattggggaa ttcagcgaga gcctgaccta 5580ttgcatctcc cgccgtgcac
agggtgtcac gttgcaagac ctgcctgaaa ccgaactgcc 5640cgctgttctg
cagccggtcg cggaggccat ggatgcgatc gctgcggccg atcttagcca
5700gacgagcggg ttcggcccat tcggaccgca aggaatcggt caatacacta
catggcgtga 5760tttcatatgc gcgattgctg atccccatgt gtatcactgg
caaactgtga tggacgacac 5820cgtcagtgcg tccgtcgcgc aggctctcga
tgagctgatg ctttgggccg aggactgccc 5880cgaagtccgg cacctcgtgc
acgcggattt cggctccaac aatgtcctga cggacaatgg 5940ccgcataaca
gcggtcattg actggagcga ggcgatgttc ggggattccc aatacgaggt
6000cgccaacatc ttcttctgga ggccgtggtt ggcttgtatg gagcagcaga
cgcgctactt 6060cgagcggagg catccggagc ttgcaggatc gccgcggctc
cgggcgtata tgctccgcat 6120tggtcttgac caactctatc agagcttggt
tgacggcaat ttcgatgatg cagcttgggc 6180gcagggtcga tgcgacgcaa
tcgtccgatc cggagccggg actgtcgggc gtacacaaat 6240cgcccgcaga
agcgcggccg tctggaccga tggctgtgta gaagtactcg ccgatagtgg
6300aaaccgacgc cccagcactc gtccgagggc aaaggaatag cacgtgctac
gagatttcga 6360ttccaccgcc gccttctatg aaaggttggg cttcggaatc
gttttccggg acgccggctg 6420gatgatcctc cagcgcgggg atctcatgct
ggagttcttc gcccacccca acttgtttat 6480tgcagcttat aatggttaca
aataaagcaa tagcatcaca aatttcacaa ataaagcatt 6540tttttcactg
cattctagtt gtggtttgtc caaactcatc aatgtatctt atcatgtctg
6600tataccgtcg acctctagct agagcttggc gtaatcatgg tcatagctgt
ttcctgtgtg 6660aaattgttat ccgctcacaa ttccacacaa catacgagcc
ggaagcataa agtgtaaagc 6720ctggggtgcc taatgagtga gctaactcac
attaattgcg ttgcgctcac tgcccgcttt 6780ccagtcggga aacctgtcgt
gccagctgca ttaatgaatc ggccaacgcg cggggagagg 6840cggtttgcgt
attgggcgct cttccgcttc ctcgctcact gactcgctgc gctcggtcgt
6900tcggctgcgg cgagcggtat cagctcactc aaaggcggta atacggttat
ccacagaatc 6960aggggataac gcaggaaaga acatgtgagc aaaaggccag
caaaaggcca ggaaccgtaa 7020aaaggccgcg ttgctggcgt ttttccatag
gctccgcccc cctgacgagc atcacaaaaa 7080tcgacgctca agtcagaggt
ggcgaaaccc gacaggacta taaagatacc aggcgtttcc 7140ccctggaagc
tccctcgtgc gctctcctgt tccgaccctg ccgcttaccg gatacctgtc
7200cgcctttctc ccttcgggaa gcgtggcgct ttctcatagc tcacgctgta
ggtatctcag 7260ttcggtgtag gtcgttcgct ccaagctggg ctgtgtgcac
gaaccccccg ttcagcccga 7320ccgctgcgcc ttatccggta actatcgtct
tgagtccaac ccggtaagac acgacttatc 7380gccactggca gcagccactg
gtaacaggat tagcagagcg aggtatgtag gcggtgctac 7440agagttcttg
aagtggtggc ctaactacgg ctacactaga agaacagtat ttggtatctg
7500cgctctgctg aagccagtta ccttcggaaa aagagttggt agctcttgat
ccggcaaaca 7560aaccaccgct ggtagcggtt tttttgtttg caagcagcag
attacgcgca gaaaaaaagg 7620atctcaagaa gatcctttga tcttttctac
ggggtctgac gctcagtgga acgaaaactc 7680acgttaaggg attttggtca
tgagattatc aaaaaggatc ttcacctaga tccttttaaa 7740ttaaaaatga
agttttaaat caatctaaag tatatatgag taaacttggt ctgacagtta
7800ccaatgctta atcagtgagg cacctatctc agcgatctgt ctatttcgtt
catccatagt 7860tgcctgactc cccgtcgtgt agataactac gatacgggag
ggcttaccat ctggccccag 7920tgctgcaatg ataccgcgag acccacgctc
accggctcca gatttatcag caataaacca 7980gccagccgga agggccgagc
gcagaagtgg tcctgcaact ttatccgcct ccatccagtc 8040tattaattgt
tgccgggaag ctagagtaag tagttcgcca gttaatagtt tgcgcaacgt
8100tgttgccatt gctacaggca tcgtggtgtc acgctcgtcg tttggtatgg
cttcattcag 8160ctccggttcc caacgatcaa ggcgagttac atgatccccc
atgttgtgca aaaaagcggt 8220tagctccttc ggtcctccga tcgttgtcag
aagtaagttg gccgcagtgt tatcactcat 8280ggttatggca gcactgcata
attctcttac tgtcatgcca tccgtaagat gcttttctgt 8340gactggtgag
tactcaacca agtcattctg agaatagtgt atgcggcgac cgagttgctc
8400ttgcccggcg tcaatacggg ataataccgc gccacatagc agaactttaa
aagtgctcat 8460cattggaaaa cgttcttcgg ggcgaaaact ctcaaggatc
ttaccgctgt tgagatccag 8520ttcgatgtaa cccactcgtg cacccaactg
atcttcagca tcttttactt tcaccagcgt 8580ttctgggtga gcaaaaacag
gaaggcaaaa tgccgcaaaa aagggaataa gggcgacacg 8640gaaatgttga
atactcatac tcttcctttt tcaatattat tgaagcattt atcagggtta
8700ttgtctcatg agcggataca tatttgaatg tatttagaaa aataaacaaa
taggggttcc 8760gcgcacattt ccccgaaaag tgccacctga cg
879219123DNAArtificialOK1 (HuVK1B) 191gacatccagw tgacccagtc tcc
2319223DNAArtificialOK2 (HuVK2) 192gatgttgtga tgactcagtc tcc
2319323DNAArtificialOK3 (HuVK2B2) 193gatattgtga tgacccagac tcc
2319423DNAArtificialOK4 (HuVK3B) 194gaaattgtgw tgacrcagtc tcc
2319523DNAArtificialOK5 (HuVK5) 195gaaacgacac tcacgcagtc tcc
2319623DNAArtificialOK6 (HuVK6) 196gaaattgtgc tgactcagtc tcc
2319724DNAArtificialOCK (HuCK) 197acactctccc ctgttgaagc tctt
2419823DNAArtificialOL1 (HuVL1A)* 198cagtctgtgc tgactcagcc acc
2319923DNAArtificialOL1 (HuVL1B)* 199cagtctgtgy tgacgcagcc gcc
2320023DNAArtificialOL1 (HuVL1C)* 200cagtctgtcg tgacgcagcc gcc
2320120DNAArtificialOL2 (HuVL2B) 201cagtctgccc tgactcagcc
2020223DNAArtificialOL3 (HuVL3A) 202tcctatgwgc tgactcagcc acc
2320323DNAArtificialOL4 (HuVL3B) 203tcttctgagc tgactcagga ccc
2320420DNAArtificialOL5 (HuVL4B) 204cagcytgtgc tgactcaatc
2020523DNAArtificialOL6 (HuVL5) 205caggctgtgc tgactcagcc gtc
2320623DNAArtificialOL7 (HuVL6) 206aattttatgc tgactcagcc cca
2320723DNAArtificialOL8 (HuVL7/8)
207cwgcctgtgc tgactcagcc mcc 2320823DNAArtificialOL9 (HuVL9)#
208cwgcctgtgc tgactcagcc mcc 2320918DNAArtificialOL9 (HuVL10)#
209caggcagggc tgactcag 1821023DNAArtificialOCL (HuCL2)X
210tgaacattct gtaggggcca ctg 2321123DNAArtificialOCL (HuCL7)X
211agagcattct gcaggggcca ctg 2321223DNAArtificialOH1(HuVH1B7A)+
212cagrtgcagc tggtgcartc tgg 2321323DNAArtificialOH1 (HuVH1C)+
213saggtccagc tggtrcagtc tgg 2321423DNAArtificialOH2 (HuVH2B)
214cagrtcacct tgaaggagtc tgg 2321518DNAArtificialOH3 (HuVH3A)
215gaggtgcagc tggtggag 1821623DNAArtificialOH4 (HuVH3C)
216gaggtgcagc tggtggagwc ygg 2321723DNAArtificialOH5 (HuVH4B)
217caggtgcagc tacagcagtg ggg 2321823DNAArtificialOH6 (HuVH4C)
218cagstgcagc tgcaggagtc sgg 2321923DNAArtificialOH7 (HuVH6A)
219caggtacagc tgcagcagtc agg 2322024DNAArtificialOCM (HuCIgM)
220tggaagaggc acgttctttt cttt 2422141DNAArtificialOK1S (HuVK1B-SAL)
221tgagcacaca ggtcgacgga catccagwtg acccagtctc c
4122241DNAArtificialOK2S (HuVK2-SAL) 222tgagcacaca ggtcgacgga
tgttgtgatg actcagtctc c 4122341DNAArtificialOK3S (HuVK2B2-SAL)
223tgagcacaca ggtcgacgga tattgtgatg acccagactc c
4122441DNAArtificialOK4S (HuVK3B-SAL) 224tgagcacaca ggtcgacgga
aattgtgwtg acrcagtctc c 4122541DNAArtificialOK5S (HuVK5-SAL)
225tgagcacaca ggtcgacgga aacgacactc acgcagtctc c
4122641DNAArtificialOK6S (HuVK6-SAL) 226tgagcacaca ggtcgacgga
aattgtgctg actcagtctc c 4122748DNAArtificialOJK1 (HuJK1-NOT)
227gagtcattct cgacttgcgg ccgcacgttt gatttccacc ttggtccc
4822848DNAArtificialOJK2 (HuJK2-NOT) 228gagtcattct cgacttgcgg
ccgcacgttt gatctccagc ttggtccc 4822948DNAArtificialOJK3 (HuJK3-NOT)
229gagtcattct cgacttgcgg ccgcacgttt gatatccact ttggtccc
4823048DNAArtificialOJK4 (HuJK4-NOT) 230gagtcattct cgacttgcgg
ccgcacgttt gatctccacc ttggtccc 4823148DNAArtificialOJK5 (HuJK5-NOT)
231gagtcattct cgacttgcgg ccgcacgttt aatctccagt cgtgtccc
4823241DNAArtificialOL1S (HuVL1A-SAL)* 232tgagcacaca ggtcgacgca
gtctgtgctg actcagccac c 4123341DNAArtificialOL1S (HuVL1B-SAL)*
233tgagcacaca ggtcgacgca gtctgtgytg acgcagccgc c
4123441DNAArtificialOL1S (HuVL1C-SAL)* 234tgagcacaca ggtcgacgca
gtctgtcgtg acgcagccgc c 4123538DNAArtificialOL2S (HuVL2B-SAL)
235tgagcacaca ggtcgacgca gtctgccctg actcagcc
3823641DNAArtificialOL3S (HuVL3A-SAL) 236tgagcacaca ggtcgacgtc
ctatgwgctg actcagccac c 4123741DNAArtificialOL4S (HuVL3B-SAL)
237tgagcacaca ggtcgacgtc ttctgagctg actcaggacc c
4123838DNAArtificialOL5S (HuVL4B-SAL) 238tgagcacaca ggtcgacgca
gcytgtgctg actcaatc 3823941DNAArtificialOL6S (HuVL5-SAL)
239tgagcacaca ggtcgacgca ggctgtgctg actcagccgt c
4124041DNAArtificialOL7S (HuVL6-SAL) 240tgagcacaca ggtcgacgaa
ttttatgctg actcagcccc a 4124141DNAArtificialOL8S (HuVL7/8-SAL)
241tgagcacaca ggtcgacgca grctgtggtg acycaggagc c
4124241DNAArtificialOL9S (HuVL9-SAL)# 242tgagcacaca ggtcgacgcw
gcctgtgctg actcagccmc c 4124336DNAArtificialOL9S (HuVL10-SAL)#
243tgagcacaca ggtcgacgca ggcagggctg actcag 3624448DNAArtificialOJL1
(HuJL1-NOT) 244gagtcattct cgacttgcgg ccgcacctag gacggtgacc ttggtccc
4824548DNAArtificialOJL2 (HuJL2/3-NOT) 245gagtcattct cgacttgcgg
ccgcacctag gacggtcagc ttggtccc 4824648DNAArtificialOJL3 (HuJL7-NOT)
246gagtcattct cgacttgcgg ccgcaccgag gacggtcagc tgggtgcc
4824756DNAArtificialOH1S (HuVH1B-SFI)+ 247gtcctcgcaa ctgcggccca
gccggccatg gcccagrtgc agctggtgca rtctgg 5624856DNAArtificialOH1S
(HuVH1C-SFI)+ 248gtcctcgcaa ctgcggccca gccggccatg gccsaggtcc
agctggtrca gtctgg 5624956DNAArtificialOH2S (HuVH2B-SFI)
249gtcctcgcaa ctgcggccca gccggccatg gcccagrtca ccttgaagga gtctgg
5625051DNAArtificialOH3S (HuVH3A-SFI) 250gtcctcgcaa ctgcggccca
gccggccatg gccgaggtgc agctggtgga g 5125156DNAArtificialOH4S
(HuVH3C-SFI) 251gtcctcgcaa ctgcggccca gccggccatg gccgaggtgc
agctggtgga gwcygg 5625256DNAArtificialOH5S (HuVH4B-SFI)
252gtcctcgcaa ctgcggccca gccggccatg gcccaggtgc agctacagca gtgggg
5625356DNAArtificialOH6S (HuVH4C-SFI) 253gtcctcgcaa ctgcggccca
gccggccatg gcccagstgc agctgcagga gtcsgg 5625456DNAArtificialOH7S
(HuVH6A-SFI) 254gtcctcgcaa ctgcggccca gccggccatg gcccaggtac
agctgcagca gtcagg 5625536DNAArtificialOJH1 (HuJH1/2-XHO)
255gagtcattct cgactcgaga crgtgaccag ggtgcc 3625636DNAArtificialOJH2
(HuJH3-XHO) 256gagtcattct cgactcgaga cggtgaccat tgtccc
3625736DNAArtificialOJH3 (HuJH4/5-XHO) 257gagtcattct cgactcgaga
cggtgaccag ggttcc 3625836DNAArtificialOJH4 (HuJH6-XHO)
258gagtcattct cgactcgaga cggtgaccgt ggtccc 36
* * * * *
References