U.S. patent application number 14/375675 was filed with the patent office on 2014-12-25 for human monoclonal antibodies broadly protective against influenza b virus and methods of using the same.
This patent application is currently assigned to OSAKA UNIVERSITY. The applicant listed for this patent is DEPARTMENT OF MEDICAL SCIENCES, MEDICAL AND BIOLOGICAL LABORATORIES CO., LTD., OSAKA UNIVERSITY, THE RESEARCH FOUNDATION FOR MICROBIAL DISEASES OF OSAKA UNIVERSITY. Invention is credited to Jotika Boon-Long, Kazuhito Fujiyama, Kazuyoshi Ikuta, Ritsuko Koketsu, Motoki Kuhara, Mayo Yasugi.
Application Number | 20140377262 14/375675 |
Document ID | / |
Family ID | 48904929 |
Filed Date | 2014-12-25 |
United States Patent
Application |
20140377262 |
Kind Code |
A1 |
Yasugi; Mayo ; et
al. |
December 25, 2014 |
HUMAN MONOCLONAL ANTIBODIES BROADLY PROTECTIVE AGAINST INFLUENZA B
VIRUS AND METHODS OF USING THE SAME
Abstract
Materials and methods are provided for treating influenza B
infections in humans. Anti-human influenza virus monoclonal
antibodies and antigen-binding fragments thereof having a
neutralization activity against a human influenza B virus are
provided. Methods for producing anti-human influenza B virus
monoclonal antibodies are also provided. The antibodies and
antigen-binding fragments thereof can be effective against a wide
range of influenza B viral strains. Methods of inhibiting or
treating a human influenza B infection are provided. The
anti-influenza B therapeutics can also be used to manufacture
medicaments effective against influenza B infections, to detect
human influenza B in a human subject, for use in pharmaceutical
compositions, and for use in kits for at least one of the
prevention, the treatment, and the detection of human influenza B
in a human subject.
Inventors: |
Yasugi; Mayo; (Suita-shi,
JP) ; Kuhara; Motoki; (Ina-shi, JP) ;
Boon-Long; Jotika; (Nonthaburi, TH) ; Fujiyama;
Kazuhito; (Suita-shi, JP) ; Koketsu; Ritsuko;
(Suita-shi, JP) ; Ikuta; Kazuyoshi; (Suita-shi,
JP) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
OSAKA UNIVERSITY
THE RESEARCH FOUNDATION FOR MICROBIAL DISEASES OF OSAKA
UNIVERSITY
MEDICAL AND BIOLOGICAL LABORATORIES CO., LTD.
DEPARTMENT OF MEDICAL SCIENCES |
Suita-shi, Osaka
Suita-shi, Osaka
Nagoya-shi, Aichi
Nonthaburi |
|
JP
JP
JP
TH |
|
|
Assignee: |
OSAKA UNIVERSITY
Suita-shi, Osaka
JP
THE RESEARCH FOUNDATION FOR MICROBIAL DISEASES OF OSAKA
UNIVERSITY
Suita-shi, Osaka
JP
MEDICAL AND BIOLOGICAL LABORATORIES CO., LTD.
Nagoya-shi, Aichi
JP
DEPARTMENT OF MEDICAL SCIENCES
Nonthaburi
TH
|
Family ID: |
48904929 |
Appl. No.: |
14/375675 |
Filed: |
January 31, 2013 |
PCT Filed: |
January 31, 2013 |
PCT NO: |
PCT/JP2013/000538 |
371 Date: |
July 30, 2014 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
|
61592657 |
Jan 31, 2012 |
|
|
|
Current U.S.
Class: |
424/133.1 ;
424/142.1; 435/5; 435/70.21; 530/387.3; 530/388.15 |
Current CPC
Class: |
A61P 31/16 20180101;
G01N 33/56983 20130101; C07K 2317/24 20130101; C07K 2317/54
20130101; C07K 2317/76 20130101; A61K 2039/505 20130101; C07K
2317/92 20130101; C07K 2317/34 20130101; C07K 16/1018 20130101;
C07K 2317/565 20130101; C07K 2317/21 20130101; G01N 2333/11
20130101 |
Class at
Publication: |
424/133.1 ;
530/388.15; 530/387.3; 424/142.1; 435/5; 435/70.21 |
International
Class: |
C07K 16/10 20060101
C07K016/10; G01N 33/569 20060101 G01N033/569 |
Goverment Interests
GOVERNMENT FUNDING
[0002] The subject matter described herein was supported, at least
in part, by the Japan Science and Technology Agency/Japan
International Cooperation Agency, Science and Technology Research
Partnership for Sustainable Development (JST/JICA, SATREPS); and a
Grant-in-Aid for Young Scientists (B) from the Japan Society for
the Promotion of Science to Mayo Yasugi.
Claims
1. An anti-human influenza virus monoclonal antibody or an
antigen-binding fragment thereof comprising a neutralization
activity against a human influenza B virus, wherein the monoclonal
antibody comprises a human monoclonal antibody or a humanized
monoclonal antibody.
2. The anti-human influenza virus monoclonal antibody or
antigen-binding fragment thereof of claim 1, wherein the monoclonal
antibody or antigen-binding fragment thereof has a neutralization
activity against at least a B/Florida/4/2006 strain, a
B/Shanghai/361/2002 strain, a B/Johannesburg/5/1999 strain, a
B/Yamanashi/166/1998 strain, and a B/Mie/1/1993 strain.
3. The anti-human influenza virus monoclonal antibody or
antigen-binding fragment thereof of claim 1, wherein the monoclonal
antibody or antigen-binding fragment thereof has a neutralization
activity against at least a B/Florida/4/2006 strain, a
B/Shanghai/361/2002 strain, a B/Johannesburg/5/1999 strain, a
B/Yamanashi/166/1998 strain, a B/Mie/1/1993 strain, a
B/Malaysia/2506/04 strain, a B/Shandong/7/1997 strain, and a
B/Victoria/2/1987 strain.
4. The anti-human influenza virus human monoclonal antibody of
claim 1, wherein the human monoclonal antibody is produced by a
hybridoma made by fusing a peripheral blood mononuclear cell (PBMC)
from a human being having an influenza B virus infection with a
fusion partner cell capable of efficient cell fusion.
5. The anti-human influenza virus human monoclonal antibody of
claim 4, wherein the influenza B virus comprises at least one of a
B/Florida/4/2006 strain, a B/Shanghai/361/2002 strain, a
B/Johannesburg/5/1999 strain, a B/Yamanashi/166/1998 strain, a
B/Mie/1/1993 strain, a B/Malaysia/2506/04 strain, a
B/Shandong/7/1997 strain, and a B/Victoria/2/1987 strain.
6. The anti-human influenza B virus human monoclonal antibody of
claim 4, wherein the fusion partner cell is a SPYMEG cell.
7. The anti-human influenza B virus monoclonal antibody or
antigen-binding fragment thereof of claim 1 comprising an IgG, a
Fab, a Fab', a F(ab')2, a scFv, or a dsFv.
8. The anti-human influenza virus monoclonal antibody or
antigen-binding fragment thereof of claim 1, comprising: a heavy
chain variable region comprising a first complementarity
determining region (CDR1) having a first amino acid sequence
comprising SEQ ID NO: 1, 7, or 13, a second complementarity
determining region (CDR2) having a second amino acid sequence
comprising SEQ ID NO: 2, 8, or 14, and a third complementarity
determining region (CDR3) having a third amino acid sequence
comprising SEQ ID NO: 3, 9, or 15, and a light chain variable
region comprising a first complementarity determining region (CDR1)
having a fourth amino acid sequence comprising SEQ ID NO: 4, 10, or
16; a second complementarity determining region (CDR2) having a
fifth amino acid sequence comprising SEQ ID NO: 5, 11, or 17, and a
third complementarity determining region (CDR3) having a sixth
amino acid sequence comprising SEQ ID NO: 6, 12, or 18.
9. The anti-human influenza virus monoclonal antibody or
antigen-binding fragment thereof of claim 8, wherein the first
amino acid sequence comprises SEQ ID NO: 1, the second amino acid
sequence comprises SEQ ID NO: 2, the third amino acid sequence
comprises SEQ ID NO: 3, the fourth amino acid sequence comprises
SEQ ID NO: 4, the fifth amino acid sequence comprises SEQ ID NO: 5,
and the sixth amino acid sequence comprises SEQ ID NO: 6.
10. The anti-human influenza virus monoclonal antibody or
antigen-binding fragment thereof of claim 8, wherein the first
amino acid sequence comprises SEQ ID NO: 7, the second amino acid
sequence comprises SEQ ID NO: 8, the third amino acid sequence
comprises SEQ ID NO: 9, the fourth amino acid sequence comprises
SEQ ID NO: 10, the fifth amino acid sequence comprises SEQ ID NO:
11, and the sixth amino acid sequence comprises SEQ ID NO: 12.
11. The anti-human influenza virus monoclonal antibody or
antigen-binding fragment thereof of claim 8, wherein the first
amino acid sequence comprises SEQ ID NO: 13, the second amino acid
sequence comprises SEQ ID NO: 14, the third amino acid sequence
comprises SEQ ID NO: 15, the fourth amino acid sequence comprises
SEQ ID NO: 16, the fifth amino acid sequence comprises SEQ ID NO:
17, and the sixth amino acid sequence comprises SEQ ID NO: 18.
12. A pharmaceutical composition comprising the anti-human
influenza virus human monoclonal antibody or antigen-binding
fragment thereof of claim 1 and a pharmacologically acceptable
carrier.
13. A kit for at least one of the prevention, the treatment, and
the detection of human influenza B in a human subject comprising
the anti-human influenza virus human monoclonal antibody or
antigen-binding fragment thereof of claim 1.
14. Use of the anti-human influenza B virus human monoclonal
antibody or antigen-binding fragment thereof of claim 1 to
manufacture a medicament for inhibiting or treating a human
influenza B infection in a human subject.
15. A method of detecting human influenza B in a human subject
comprising; contacting a sample from the human subject with the
anti-human influenza B virus antibody or antigen-binding fragment
thereof of claim 1; and detecting the presence or absence of a
human influenza B virus in the human subject based on whether the
antibody binds a hemagglutinin (HA) protein of the human influenza
B virus.
16. A method for producing an anti-human influenza B virus human
monoclonal antibody comprising: producing a hybridoma by fusing a
peripheral blood mononuclear cell (PBMC) from a human being in an
influenza B virus infection with a fusion partner cell capable of
efficient cell fusion; and obtaining an anti-human influenza virus
monoclonal antibody from the hybridoma.
17. The method for producing an anti-human influenza B virus human
monoclonal antibody of claim 16, wherein the influenza B virus
comprises at least one of a B/Florida/4/2006 strain, a
B/Shanghai/361/2002 strain, a B/Johannesburg/5/1999 strain, a
B/Yamanashi/166/1998 strain, a B/Mie/1/1993 strain, a
B/Malaysia/2506/04 strain, a B/Shandong/7/1997 strain, and
B/Victoria/2/1987 strain.
18. The method for producing an anti-human influenza virus human
monoclonal antibody of claim 16, wherein the fusion partner cell is
a SPYMEG cell.
19. An anti-human influenza virus human monoclonal antibody
produced by the method of claim 16.
Description
RELATED APPLICATIONS
[0001] The present application claims the benefit of U.S.
Provisional Patent Application No. 61/592,657, filed on Jan. 31,
2012, which is incorporated herein by reference in its
entirety.
TECHNICAL FIELD
[0003] The present invention relates to materials and methods for
the treatment of influenza B viral infections in humans.
BACKGROUND ART
[0004] The ability of influenza virus to evade immune surveillance
through rapid genetic drift and reassortment means that it remains
a continuous public health threat. During annual epidemics five to
fifteen percent of the worldwide population are typically infected,
resulting in three million to five million cases of severe illness
and between 250,000 to 500,000 deaths per year (Lambert et al., The
New England Journal of Medicine 363:2036-2044; WHO, Influenza
(Seasonal) Fact Sheet No. 211). Influenza B virus, like H1 and H3
subtypes of influenza A virus, has caused epidemics in humans (WHO,
Influence (Seasonal) Fact Sheet No. 211). In contrast to influenza
A, influenza B virus is found almost exclusively in humans and has
a much slower mutational rate than that observed for influenza A
virus (Carrat et al., Vaccine 25:6852-6862; Nobusawa et al.,
Journal of Virology 80:3675-3678; Webster et al, The Journal of
General Virology 54:243-251). However, co-circulation of two
phylogenetically and antigenically distinct lineages, represented
by the B/Yamagata/16/88 and B/Victoria/2/87, has caused antigenic
variation through genetic reassortment and antigenic drift from
cumulative mutations, leading to annual endemics (Hay et al.,
Philosophical Transactions of the Royal Society of London
356:1861-1870; Lin et al., Virus Research 103:47-52).
[0005] The development of vaccines producing broadly reactive
antibodies and therapeutic strategies using human monoclonal
antibodies (HuMAbs) with global reactivity has recently been
gathering great interest. Neuraminidase inhibitors oseltamivir
(Tamiflu) and zanamivir (Relenza) have been widely used for the
treatment of influenza viral infection. However, they have limited
efficacy when administered more than 48 hours after the onset of
illness (Aoki et al., The Journal of Antimicrobial Chemotherapy 51:
123-129), and widespread use has resulted in the emergence of
resistant viral strains (Kiso et al, Lancet 364:759-765; Lowen et
al., Infectious Disorders Drug Targets 7:318-328; Reece, Journal of
Medical Virology 79:1577-1586). Therefore, new therapeutic
strategies that provide potent and broadly cross-protective host
immunity are a global public health priority. Thus, the development
of novel antibody-based therapies is of great interest (Marasco et
al., Nature Biotechnology 25:1421-1434).
[0006] Several human monoclonal antibodies (HuMAbs) with broad
neutralizing activities were identified against the hemagglutinin
(HA) protein in influenza A viruses, including C6261 and F10, which
react with group 1 viruses (Ekiert et al., Science 324:246-251; Sui
et al., Nature Structural & Molecular Biology 16:265-273), and
CR8020, which neutralizes group 2 viruses (Ekiert et al., Science
333:843-850). Another HuMAb, FI6v3, which neutralized both group 1
and group 2 influenza A viruses, was isolated in 2011 (Corti et
al., Science 333: 850-856). Although influenza B virus has a much
slower mutational rate than that observed for influenza A virus
like H1 and H3 subtypes, it has annually caused epidemics in humans
(NPL1: Hay et al., Philosophical Transactions of the Royal Society
of London 256:1861-1870; NPL2: Lin et al., Virus Research
103:47-52). For influenza B virus, however, broadly neutralizing
HuMAbs, CR8033, CR8071 and CR9114, have firstly reported on
September 2012 (NPL3: Dreyfus et al., Science 337: 1343-1348).
Accordingly, a need exists for broadly neutralizing HuMAb against
the influenza B virus.
CITATION LIST
Non Patent Literature
[0007] [NPL 1] Hay et al., Philos Trans R Soc Lond B Biol Sci. 2001
Dec. 29; 356(1416): 1861-1870. [0008] [NPL 2] Lin et al., Virus
Res. 2004 July; 103(1-2):47-52. [0009] [NPL 3] Dreyfus et al.,
Science. 2012 Sep. 14; 337(6100):1343-8. doi:
10.1126/science.1222908. Epub 2012 Aug. 9.
SUMMARY OF INVENTION
Technical Problem
[0010] In the current study, three HuMAbs were prepared that
broadly reacted to the HA protein in influenza B virus. Three
HuMAbs designated 5A7, 3A2, and 10C4 against influenza B virus were
prepared using peripheral lymphocytes from vaccinated volunteers.
In vitro, HuMAb 5A7 broadly neutralized influenza B strains
isolated from 1985 to 2006, whereas 3A2 and 10C4 reacted to the
Yamagata lineage only. Epitope mapping revealed that 3A2 and 10C4
recognized the 190-helix region near the receptor binding site in
the hemagglutinin (HA) protein. Amino acid residues of the
190-helix readily mutate. 5A7 recognized amino acid positions 315
to 324 near the C terminal of HA 1, a highly conserved region in
influenza B viruses. Moreover, 5A7 showed therapeutic efficacy in
mice even if HuMAb was injected 72 hours post-infection. HuMAb 5A7
synthesized from full-length variable gene-transfected CHO-K1 cells
also showed neutralizing activity against influenza B viruses.
These results indicate that the antibodies of the invention,
including 5A7, can be used as therapeutics against influenza B
virus.
[0011] It is therefore a feature of the present invention to
provide therapeutics including antibodies and antigen-binding
fragments thereof capable of preventing, inhibiting, and treating
an influenza B infection.
[0012] Another feature of the present invention is to provide
methods for producing such therapeutics.
[0013] A further feature of the present invention is to provide
therapeutics effective against a wide range of influenza B viral
strains.
[0014] Additional features and advantages of the present invention
will be set forth in part in the description that follows, and in
part will be apparent from the description, or may be learned by
practice of the present invention. The objectives and other
advantages of the present invention will be realized and attained
by means of the elements and combinations particularly pointed out
in the description and appended claims.
Solution to Problem
[0015] To achieve these and other advantages, and in accordance
with the purposes of the present invention, as embodied and broadly
described herein, the present invention relates to an anti-human
influenza virus monoclonal antibody or an antigen-binding fragment
thereof having a neutralization activity against a human influenza
B virus, wherein the monoclonal antibody can include a human
monoclonal antibody and/or a humanized monoclonal antibody.
[0016] The present invention provides a method for producing an
anti-human influenza B virus monoclonal antibody. The method can
include producing a hybridoma by fusing a peripheral blood
mononuclear cell (PBMC) from a human being, for example a patient
and/or a vaccine in an influenza B virus infection with a fusion
partner cell capable of efficient cell fusion. The method can also
include obtaining an anti-human influenza virus monoclonal antibody
from the hybridoma. The influenza B virus in the method can include
at least one of a B/Florida/4/2006 strain, a
B/Shanghai/361/2002/strain, a B/Johannesburg/5/1999 strain, a
B/Yamanashi/166/1998 strain, a B/Mie/1/1993 strain, a
B/Malaysia/2506/04 strain, a B/Shandong/7/1997 strain, and
B/Victoria/2/1987 strain. The fusion partner cell is a SPYMEG cell.
Thus, an anti-human influenza virus monoclonal antibody produced by
the method is further provided.
[0017] The present invention provides a method of inhibiting or
treating a human influenza B infection in a human subject including
administering a therapeutically effective amount of the anti-human
influenza B virus human antibody or antigen-binding fragment
thereof of the invention to the human subject. The method can
further include diagnosing the patient with an influenza B
infection. The method can further include monitoring for a decrease
in at least one symptom of an influenza B infection.
[0018] The present invention provides use of an anti-human
influenza B virus monoclonal antibody or antigen-binding fragment
thereof of the present invention to manufacture a medicament for
inhibiting or treating a human influenza B infection in a human
subject. The present invention also provides a method of detecting
human influenza B in a human subject including contacting a sample
from the human subject with an anti-human influenza B virus
monoclonal antibody or antigen-binding fragment thereof of the
invention. The present invention further provides a pharmaceutical
composition containing an anti-human influenza virus monoclonal
antibody or antigen-binding fragment thereof of the present
invention and a pharmacologically acceptable carrier. The present
invention still further provides a kit for at least one of the
prevention, the treatment, and the detection of human influenza B
in a human subject containing an anti-human influenza virus
monoclonal antibody or antigen-binding fragment thereof of the
present invention. The kit can include the pharmaceutical
composition and/or one or more additional influenza B or other
antagonists.
[0019] The accompanying drawings, which are incorporated in and
constitute a part of this application, illustrate some of the
embodiments of the present invention and together with the
description, serve to explain the principles of the present
invention.
BRIEF DESCRIPTION OF DRAWINGS
[0020] FIG. 1 shows the sequences of escape mutants selected by the
incubation of B/Florida/4/2006 with HuMAbs. The amino acid
sequences of the HA protein in the escape mutants are compared with
the original B/Florida/4/2006. Asterisks indicate the amino acid
residues different between the original virus and the escape
mutants.
[0021] FIG. 2 shows a series of truncated forms of HA (a to f) in
an expression plasmid, which were prepared as depicted in the
diagram. Transfected 293T cells were subjected to Western blotting
with 5A7. The serum from a mouse infected with B/Ibaraki/2/1985 was
used as a control (Ms serum).
[0022] FIG. 3 shows expression plasmids bearing chimeric HA protein
prepared with B/Shanghai/361/2002 (Sh/02) and B/Florida/4/2006
(Flo/06). 293T cells expressing the chimeric protein were subjected
to an immunofluorescence assay (IFA) with 3A2 (left panels). White
bars represent the amino acid sequence in Sh/02, and black bars
represent the amino acid sequence in Flo/06. The different amino
acid residues in the HA protein from each of the two viral strains
are shown in the top and bottom bars.
[0023] FIG. 4 shows an epitope map of the HuMAbs in
three-dimensional structure of the HA protein.
[0024] FIG. 5 shows the therapeutic efficacy of 5A7 in mice. Mice
were treated intraperitoneally with HuMAb at 5, 10, or 15 mg/kg or
with PBS at 4 hours post-challenge with a lethal dose
(2.5.times.10.sup.4 FFU/mouse) of mouse-adapted B/Ibaraki/2/1985.
Survival and body weight were checked daily. Each group consists of
five mice. Body weight is shown as the mean+ or -SEM of five
mice.
[0025] FIG. 6 shows the therapeutic efficacy of 5A7 in mice. Mice
were treated intraperitoneally with HuMAb at 5, 10, or 15 mg/kg or
with PBS at 4 hours post-challenge with a lethal dose
(2.5.times.10.sup.4 FFU/mouse) of mouse-adapted B/Ibaraki/2/1985.
Survival and body weight were checked daily. Each group consists of
five mice. Body weight is shown as the mean+ or -SEM of five
mice.
[0026] FIG. 7 shows mice treated with 5A7 at 10 mg/kg or with PBS
at 4 hours post-challenge with 2.5.times.10.sup.4 FFU/mouse
mouse-adapted B/Ibaraki/2/1985 (left panel) and
5.0.times.10.sup.3FFU/mouse B/Florida/4/2006 (right panel). The
titers in lungs were calculated at 3 and 6 days post-infection.
Each group consists of three mice. Black bars are mean values.
Asterisks denote P<0.05 compared to the PBS-treated group.
[0027] FIG. 8 shows that mice were given 10 mg/kg HuMAb or PBS at
4, 24, 48, or 72 hours post-infection (hpi) with mouse-adapted
B/Ibaraki/2/1985 (2.5.times.10.sup.4 FFU/mouse). Survival and body
weight were checked daily. Each group consists of ten mice. Body
weight is shown as the mean+ or -SEM of ten mice.
[0028] FIG. 9 shows that mice were given 10 mg/kg HuMAb or PBS at
4, 24, 48, or 72 hours post-infection (hpi) with mouse-adapted
B/Ibaraki/2/1985 (2.5.times.10.sup.4 FFU/mouse). Survival and body
weight were checked daily. Each group consists of ten mice. Body
weight is shown as the mean+ or -SEM of ten mice.
[0029] FIG. 10 shows an in vitro neutralization assay using
synthesized 5A7 from CHO-K1 cells. The infectivity of
B/Florida/4/2006 and B/Malaysia/2506/2004 were measured in the
presence of synthesized 5A7 from CHO-K1 (5A7/CHO; solid lines) and
compared with infectivity in the presence of 5A7 from hybridoma
supernatant (5A7/hybridoma; dashed lines). HuMAbs (100 mcg/ml
(microgram/ml)) were serially diluted four-fold.
[0030] FIG. 11 shows sequences of the V.sub.H and V.sub.L region of
the three HuMAbs. These three HuMAbs were derived from different
germ lines except D region V.sub.H of 3A2 and 10C4.
[0031] FIG. 12 shows sequences of the V.sub.H and V.sub.L region of
the three HuMAbs. These three HuMAbs were derived from different
germ lines except D region V.sub.H of 3A2 and 10C4.
[0032] FIG. 13 shows sequences of the V.sub.H and V.sub.L region of
the three HuMAbs. These three HuMAbs were derived from different
germ lines except D region V.sub.H of 3A2 and 10C4. The
accompanying drawings, which are incorporated into and constitute a
part of the specification, illustrate specific features/embodiments
of the present invention, and taken in conjunction with the
detailed description, serve to explain the principles of the
present invention. It is to be understood that both the foregoing
general description and the following detailed description are
exemplary and explanatory only and are intended to provide a
further explanation of the present invention, as claimed.
DESCRIPTION OF EMBODIMENTS
Detailed Description of the Present Invention
[0033] The present invention provides an anti-human influenza virus
monoclonal antibody or an antigen-binding fragment thereof having a
neutralization activity against a human influenza B virus, wherein
the monoclonal antibody includes a human monoclonal antibody and/or
a humanized monoclonal antibody. The monoclonal antibody or
antigen-binding fragment thereof can have a neutralization activity
against at least a B/Florida/4/2006 strain, a B/Shanghai/361/2002
strain, a B/Johannesburg/5/1999 strain, a B/Yamanashi/166/1998
strain, and a B/Mie/1/1993 strain. The monoclonal antibody or
antigen-binding fragment thereof can have a neutralization activity
against at least a B/Florida/4/2006 strain, a B/Shanghai/361/2002
strain, a B/Johannesburg/5/1999 strain, a B/Yamanashi/166/1998
strain, a B/Mie/1/1993 strain, a B/Malaysia/2506/04 strain, a
B/Shandong/7/1997 strain, and a B/Victoria/2/1987 strain. The
anti-human influenza B virus monoclonal antibody or antigen-binding
fragment thereof can include an IgG, a Fab, a Fab', a F(ab')2, a
scFv, a dsFv, or any combination thereof.
[0034] The anti-human influenza virus monoclonal antibody or
antigen-binding fragment thereof can include at least one heavy
chain variable region and/or at least one light chain variable
region. The heavy chain variable region can include at least one of
a first complementarity determining region (CDR1), a second
complementarity determining region (CDR2), and a third
complementarity determining region (CDR3). The CDR1 of the heavy
chain variable region can have a first amino acid sequence
including SEQ ID NO: 1, 7, or 13. The CDR2 of the heavy chain
variable region can have a second amino acid sequence including SEQ
ID NO: 2, 8, or 14. The CDR3 of the heavy chain variable region can
have a third amino acid sequence including SEQ ID NO: 3, 9, or 15.
The light chain variable region can also include at least one of a
first complementarity determining region (CDR1), a second
complementarity determining region (CDR2), and a third
complementarity determining region (CDR3). The CDR1 of the light
chain variable region can have a fourth amino acid sequence
including SEQ ID NO: 4, 10, or 16. The CDR2 of the light chain
variable region can have a fifth amino acid sequence including SEQ
ID NO: 5, 11, or 17. The CDR3 of the light chain variable region
can have a sixth amino acid sequence including SEQ ID NO: 6, 12, or
18.
[0035] The anti-human influenza virus monoclonal antibody or
antigen-binding fragment can have the first amino acid sequence
include SEQ ID NO: 1, the second amino acid sequence include SEQ ID
NO: 2, the third amino acid sequence include SEQ ID NO: 3, the
fourth amino acid sequence include SEQ ID NO: 4, the fifth amino
acid sequence include SEQ ID NO: 5, and the sixth amino acid
sequence include SEQ ID NO: 6. For example, the anti-human
influenza virus monoclonal antibody can include antibody 5A7. The
anti-human influenza virus monoclonal antibody or antigen-binding
fragment thereof can have the first amino acid sequence include SEQ
ID NO: 7, the second amino acid sequence include SEQ ID NO: 8, the
third amino acid sequence include SEQ ID NO: 9, the fourth amino
acid sequence include SEQ ID NO: 10, the fifth amino acid sequence
include SEQ ID NO: 11 and the sixth amino acid sequence include SEQ
ID NO: 12. For example, the anti-human influenza virus monoclonal
antibody can include antibody 3A2. The anti-human influenza virus
monoclonal antibody or antigen-binding fragment thereof can have
the first amino acid sequence include SEQ ID NO: 13, the second
amino acid sequence include SEQ ID NO: 14, the third amino acid
sequence include SEQ ID NO: 15, the fourth amino acid sequence
include SEQ ID NO: 16, the fifth amino acid sequence include SEQ ID
NO: 17, and the sixth amino acid sequence include SEQ ID NO: 18.
For example, the anti-human influenza virus monoclonal antibody can
include antibody 10C4.
[0036] The monoclonal antibody can be produced by a hybridoma made
by fusing a peripheral blood mononuclear cell (PBMC) from a human
being, for example a patient and/or a vaccine having an influenza B
virus infection with a fusion partner cell capable of efficient
cell fusion. The influenza B virus of the infection can include at
least one of a B/Florida/4/2006 strain, a B/Shanghai/361/2002
strain, a B/Johannesburg/5/1999 strain, a B/Yamanashi/166/1998
strain, a B/Mie/1/1993 strain, a B/Malaysia/2506/04 strain, a
B/Shandong/7/1997 strain, and a B/Victoria/2/1987 strain. The
fusion partner cell can be a SPYMEG cell.
[0037] Accordingly, the present invention provides a method for
producing an anti-human influenza B virus monoclonal antibody. The
method can include producing a hybridoma by fusing a peripheral
blood mononuclear cell (PBMC) from a human being, for example a
patient and/or a vaccine in an influenza B virus infection with a
fusion partner cell capable of efficient cell fusion. The method
can also include obtaining an anti-human influenza virus monoclonal
antibody from the hybridoma. The influenza B virus in the method
can include at least one of a B/Florida/4/2006 strain, a
B/Shanghai/361/2002 strain, a B/Johannesburg/5/1999 strain, a
B/Yamanashi/166/1998 strain, a B/Mie/1/1993 strain, a
B/Malaysia/2506/04 strain, a B/Shandong/7/1997 strain, and
B/Victoria/2/1987 strain. The fusion partner cell can be a SPYMEG
cell. Thus, an anti-human influenza virus monoclonal antibody
produced by the method is further provided. The monoclonal antibody
can include, for example, a human monoclonal antibody and/or a
humanized monoclonal antibody.
[0038] Anti-influenza antibodies and polypeptides containing
antigen binding fragments thereof are provided as well as methods,
uses, compositions, and kits employing the same. A method of
forming an antibody specific to an influenza or a polypeptide or a
fragment thereof is provided. Such a method can contain providing a
nucleic acid encoding a influenza antigen polypeptide or a
polypeptide containing an immunologically specific epitope thereof;
expressing the polypeptide containing the antigen amino acid
sequence or a polypeptide containing an immunologically specific
epitope thereof from the isolated nucleic acid; and generating an
antibody specific to the polypeptide obtained or a polypeptide
containing an antigen binding fragment thereof. An antibody or
polypeptide containing an antigen binding fragment thereof produced
by the aforementioned method is provided. An isolated antibody or
isolated polypeptide containing an antigen binding fragment thereof
that specifically binds an influenza antigen is provided. Such an
antibody can be generated using any acceptable method(s) known in
the art. The antibodies as well as kits, methods, and/or other
aspects of the present invention employing antibodies can include
one or more of the following: a polyclonal antibody, a monoclonal
antibody, a chimeric antibody, a single-chain antibody, a
monovalent antibody, a diabody, and/or a humanized antibody.
[0039] Naturally occurring antibody structural units typically
contain a tetramer. Each such tetramer can be composed of two
identical pairs of polypeptide chains, each pair having one
full-length light" (for example, about 25 kDa) and one full-length
"heavy" chain (for example, about 50-70 kDa). The amino-terminal
portion of each chain typically includes a variable region of about
100 to 110 or more amino acids that typically is responsible for
antigen recognition. The carboxy-terminal portion of each chain
typically defines a constant region that may be responsible for
effector function. Human light chains are typically classified as
kappa and lambda light chains. Heavy chains are typically
classified as mu, delta, gamma, alpha, or epsilon, and define the
antibody's isotype as IgM, IgD, IgG, IgA, and IgE, respectively.
IgG has several subclasses, including, but not limited to, IgG1,
IgG2, IgG3, and IgG4. IgM has subclasses including, but not limited
to, IgM1 and IgM2. IgA is similarly subdivided into subclasses
including, but not limited to, IgA1 and IgA2. In light and heavy
chains, the variable and constant regions can be joined by a "J"
region of about 12 or more amino acids, with the heavy chain also
including a "D" region of about 10 or more amino acids. See, e.g.,
Fundamental Immunology Ch. 7 (Paul, W., ed., 2nd ed. Raven Press,
N. Y. (1989)) (incorporated by reference in its entirety for all
purposes). The variable regions of each light/heavy chain pair
typically form the antigen binding site.
[0040] The variable regions typically exhibit the same general
structure of relatively conserved framework regions (FR) joined by
three hyper variable regions, also called complementarity
determining regions or CDRs. The CDRs from the two chains of each
pair typically are aligned by the framework regions, which can
enable binding to a specific epitope. From N-terminal to
C-terminal, both light and heavy chain variable regions typically
contain the domains FR1, CDR1, FR2, CDR2, FR3, CDR3 and FR4. The
assignment of amino acids to each domain is typically in accordance
with the definitions of Kabat Sequences of Proteins of
Immunological Interest (National Institutes of Health, Bethesda,
Md. (1987 and 1991)), or Chothia & Lesk J. MoI. Biol.
196:901-917 (1987); Chothia et al., Nature 342:878-883 (1989).
[0041] "Antibody fragments" include a portion of an intact
antibody, such as the antigen binding or variable region of the
intact antibody. Examples of antibody fragments include Fab, Fab 1,
F(ab')2, and Fv fragments; diabodies; linear antibodies (Zapata et
al., Protein Eng. 8(10): 1057-1062 [1995]); single-chain antibody
molecules; and multispecific antibodies formed from antibody
fragments. Papain digestion of antibodies produces two identical
antigen-binding fragments, called "Fab" fragments, each with a
single antigen-binding site, and a residual "Fc" fragment, a
designation reflecting the ability to crystallize readily. Pepsin
treatment yields an F(ab')2 fragment that has two antigen-combining
sites and is still capable of cross-linking antigen. "Fv" is an
antibody fragment which contains a complete antigen-recognition and
-binding site. This region includes a dimer of one heavy- and one
light-chain variable domain in tight, non-covalent association. A
single variable domain (or half of an Fv containing only three CDRs
specific for an antigen) can recognize and bind an antigen.
"Single-chain Fv" or "sFv" antibody fragments include the V.sub.H
and V.sub.L domains of the antibody, wherein these domains are
present in a single polypeptide chain. The Fv polypeptide can
further contain a polypeptide linker between the V.sub.H and
V.sub.L domains which enables the sFv to form the desired structure
for antigen binding. For a review of sFv, see Pluckthun in The
Pharmacology of Monoclonal Antibodies, vol. 113, Rosenburg and
Moore eds., Springer-Verlag, New York, pp. 269-315 (1994).
[0042] Antibodies can be used as probes, therapeutic treatments and
other uses. Antibodies can be made by injecting mice, rabbits,
goats, or other animals with the translated product or synthetic
peptide fragments thereof. These antibodies are useful in
diagnostic assays or as an active ingredient in a pharmaceutical
composition.
[0043] The antibody or polypeptide administered can be conjugated
to a functional agent to form an immunoconguate. The functional
agent can be a cytotoxic agent such as a chemotherapeutic agent, a
toxin (e.g., an enzymatically active toxin of bacterial, fungal,
plant, or animal origin, or fragments thereof), or a radioactive
isotope (i.e., a radioconjugate), an antibiotic, a nucleolytic
enzyme, or any combination thereof. Chemotherapeutic agents can be
used in the generation of immunoconjugates, e.g., methotrexate,
adriamicin, vinca alkaloids (vincristine, vinblastine, etoposide),
doxorubicin, melphalan, mitomycin C, chlorambucil, daunorubicin or
other intercalating agents, enzymes, and/or fragments thereof, such
as nucleolytic enzymes, antibiotics, and toxins such as small
molecule toxins or enzymatically active toxins of bacterial,
fungal, plant or animal origin, including fragments and/or variants
thereof, and the various antitumor or anticancer agents disclosed
below. Enzymatically active toxins and fragments thereof that can
be used include, for example, diphtheria A chain, nonbinding active
fragments of diphtheria toxin, exotoxin A chain (from Pseudomonas
aeruginosa), ricin A chain, abrin A chain, modeccin A chain,
alpha-sarcin, Aleurites fordii proteins, dianthin proteins,
Phytolaca americana proteins (PAPI, PAPII, and PAP-S), momordica
charantia inhibitor, curcin, crotin, sapaonaria officinalis
inhibitor, gelonin, mitogellin, restrictocin, phenomycin, enomycin,
and the tricotheeenes. Any appropriate radionucleotide or
radioactive agent known in the art or are otherwise available can
be used to produce radioconjugated antibodies.
[0044] Conjugates of the antibody and cytotoxic agent can be made
using a variety of bifunctional protein-coupling agents such as
N-succinimidyl-3-(2-pyridyldithiol)propionate (SPDP); iminothiolane
(IT); bifunctional derivatives of imidoesters (such as dimethyl
adipimidate HCL); active esters (such as disuccinimidyl suberate);
aldehydes (such as glutareldehyde); bis-azido compounds (such as
bis(p-azidobenzoyl) hexanediamine); bis-diazonium derivatives (such
as bis-(p-diazo-niumbenzoyl)-ethylenediamine); diisocyanates (such
as tolyene 2,6-diisocyanate); bis-active fluorine compounds (such
as 1,5-difluoro-2,4-dinitrobenzene); maleimidocaproyl (MC);
valine-citrulline, dipeptide site in protease cleavable linker
(VC); 2-amino-5-ureido pentanoic acid PAB=p-aminobenzylcarbamoyl
("self immolative" portion of linker) (Citrulene); N-methyl-valine
citrulline where the linker peptide bond has been modified to
prevent its cleavage by cathepsin B (Me);
maleimidocaproyl-polyethylene glycol, attached to antibody
cysteines; N-Succinimidyl 4-(2-pyridylthio)pentanoate (SPP); and
N-succinimidyl 4-(N-maleimidomethyl)cyclohexane-1 carboxylate
(SMCC). For example, a ricin immunotoxin can be prepared as
described in Vitetta et al., Science, 238: 1098 (1987).
Carbon-14-labeled 1-isothiocyanatobenzyl-3-methyldiethylene
triaminepentaacctic acid (MX-DTPA) is an exemplary chelating agent
for conjugation of radionucleotide to the antibody, see WO
94/11026. The antibody can be conjugated to a "receptor" (such as
streptavidin) for utilization in tumor pre-targeting wherein the
antibody-receptor conjugate is administered to the subject,
followed by removal of unbound conjugate from the circulation using
a clearing agent and then administration of a "ligand" (e.g.,
avidin) that is conjugated to a cytotoxic agent (e.g., a
radionucleotide).
[0045] The antibodies of the present invention can be coupled
directly or indirectly to a detectable marker by techniques well
known in the art. A detectable marker is an agent detectable, for
example, by spectroscopic, photochemical, biochemical,
immunochemical, or chemical means. Useful detectable markers
include, but are not limited to, fluorescent dyes, chemiluminescent
compounds, radioisotopes, electron-dense reagents, enzymes, colored
particles, biotin, or dioxigenin. A detectable marker often
generates a measurable signal, such as radioactivity, fluorescent
light, color, or enzyme activity. Antibodies conjugated to
detectable agents may be used for diagnostic or therapeutic
purposes. Examples of detectable agents include various enzymes,
prosthetic groups, fluorescent materials, luminescent materials,
bioluminescent materials, radioactive materials, positron emitting
metals using various positron emission tomographies, and
nonradioactive paramagnetic metal ions. The detectable substance
can be coupled or conjugated either directly to the antibody or
indirectly, through an intermediate such as, for example, a linker
known in the art, using techniques known in the art. See, e.g.,
U.S. Pat. No. 4,741,900, describing the conjugation of metal ions
to antibodies for diagnostic use. Examples of suitable enzymes
include horseradish peroxidase, alkaline phosphatase,
beta-galactosidase, and acetyl-cholinesterase; examples of suitable
prosthetic group complexes include streptavidin/biotin and
avidin/biotin; examples of suitable fluorescent materials include
umbelliferone, fluorescein, fluorescein isothiocyanate, rhodamine,
dichlorotriazinylamine fluorescein, dansyl chloride, and
phycoerythrin; an example of a luminescent material includes
luminol; examples of bioluminescent materials include luciferin,
and aequorin.
[0046] Antibodies useful in practicing the present invention can be
prepared in laboratory animals or by recombinant DNA techniques
using the following methods. Polyclonal antibodies can be raised in
animals by multiple subcutaneous (sc) or intraperitoneal (ip)
injections of the gene product molecule or fragment thereof in
combination with an adjuvant such as Freund's adjuvant (complete or
incomplete). To enhance immuno-genicity, it can be useful to first
conjugate the gene product molecule or a fragment containing the
target amino acid sequence to a protein that is immunogenic in the
species to be immunized, e.g., keyhole limpet hemocyanin, serum
albumin, bovine thyroglobulin, or soybean trypsin inhibitor using a
bifunctional or derivatizing agent, for example, maleimidobenzoyl
sulfosuccinimide ester (conjugation through cysteine residues),
N-hydroxysuccinimide (through lysine residues), glutaraldehyde,
succinic anhydride, SOCl, etc. Alternatively, immunogenic
conjugates can be produced recombinantly as fusion proteins.
[0047] Animals can be immunized against the immunogenic conjugates
or derivatives (such as a fragment containing the target amino acid
sequence) by combining about 1 mg or about 1 microgram of conjugate
(for rabbits or mice, respectively) with about 3 volumes of
Freund's complete adjuvant and injecting the solution intradermally
at multiple sites. Approximately 7 to 14 days later, animals are
bled and the serum is assayed for antibody titer. Animals are
boosted with antigen repeatedly until the titer plateaus. The
animal can be boosted with the same molecule or fragment thereof as
was used for the initial immunization, but conjugated to a
different protein and/or through a different cross-linking agent.
In addition, aggregating agents such as alum can be used in the
injections to enhance the immune response.
[0048] The antibody administered can include a chimeric antibody.
The antibody administered can include a humanized antibody. The
antibody administered can include a completely humanized antibody.
The antibodies can be humanized or partially humanized. Non-human
antibodies can be humanized using any applicable method known in
the art. A humanized antibody can be produced using a transgenic
animal whose immune system has been partly or fully humanized. Any
antibody or fragment thereof of the present invention can be
partially or fully humanized. Chimeric antibodies can be produced
using any known technique in the art. See, e.g., U.S. Pat. Nos.
5,169,939; 5,750,078; 6,020,153; 6,420,113; 6,423,511; 6,632,927;
and 6,800,738.
[0049] The antibody administered can include a monoclonal antibody,
that is, the anti-influenza antibodies of the present invention
that can be monoclonal antibodies. Monoclonal antibodies can be
prepared using hybridoma methods, such as those described by Kohler
and Milstein, Nature, 256:495 (1975). In a hybridoma method, a
mouse, hamster, or other appropriate host animal, is typically
immunized with an immunizing agent to elicit lymphocytes that
produce or are capable of producing antibodies that will
specifically bind to the immunizing agent. Alternatively, the
lymphocytes can be immunized in vitro. Monoclonal antibodies can be
screened as are described, for example, in Harlow & Lane,
Antibodies, A Laboratory Manual, Cold Spring Harbor Press, New York
(1988); Goding, Monoclonal Antibodies, Principles and Practice (2d
ed.) Academic Press, New York (1986). Monoclonal antibodies can be
tested for specific immunoreactivity with a translated product and
lack of immunoreactivity to the corresponding prototypical gene
product.
[0050] Monoclonal antibodies can be prepared by recovering spleen
cells from immunized animals and immortalizing the cells in
conventional fashion, e.g., by fusion with myeloma cells. The
clones are then screened for those expressing the desired antibody.
The monoclonal antibody preferably does not cross-react with other
gene products. After the desired hybridoma cells are identified,
the clones can be subcloned by limiting dilution procedures and
grown by standard methods. Suitable culture media for this purpose
include, for example, Dulbecco's Modified Eagle's Medium and
RPMI-1640 medium. Alternatively, the hybridoma cells can be grown
in vivo as ascites in a mammal. The monoclonal antibodies secreted
by the subclones can be isolated or purified from the culture
medium or ascites fluid by conventional immunoglobulin purification
procedures such as, for example, protein A-Sepharose,
hydroxylapatite chromatography, gel electrophoresis, dialysis, or
affinity chromatography.
[0051] The monoclonal antibodies can also be made by recombinant
DNA methods, such as those described in U.S. Pat. No. 4,816,567.
DNA encoding the monoclonal antibodies of the present invention can
be readily isolated and sequenced using conventional procedures
(e.g., by using oligonucleotide probes that are capable of binding
specifically to genes encoding the heavy and light chains of murine
antibodies). The hybridoma cells of the present invention can serve
as a preferred source of such DNA. Once isolated, the DNA can be
placed into expression vectors, which are then transfected into
host cells such as simian COS cells, Chinese hamster ovary (CHO)
cells, or myeloma cells that do not otherwise produce
immunoglobulin protein, to obtain the synthesis of monoclonal
antibodies in the recombinant host cells. The DNA also can be
modified, for example, by substituting the coding sequence for
human heavy and light chain constant domains in place of the
homologous murine sequences or by covalently joining to the
immunoglobulin coding sequence all or part of the coding sequence
for a non-immunoglobulin polypeptide. Such a non-immunoglobulin
polypeptide can be substituted for the constant domains of an
antibody of the present invention, or can be substituted for the
variable domains of one antigen-combining site of an antibody of
the invention to create a chimeric bivalent antibody. Preparation
of antibodies using recombinant DNA methods such as the phagemid
display method, can be accomplished using commercially available
kits, as for example, the Recombinant Phagemid Antibody System
available from Pharmacia (Uppsala, Sweden), or the SurfZAP.TM.
phage display system (Stratagene Inc., La Jolla, Calif.).
[0052] Also included in the present invention are hybridoma cell
lines, transformed B cell lines, and host cells that produce the
monoclonal antibodies of the present invention; the progeny or
derivatives of these hybridomas, transformed B cell lines, and host
cells; and equivalent or similar hybridomas, transformed B cell
lines, and host cells.
[0053] The antibodies can be diabodies. The term "diabodies` refers
to small antibody fragments with two antigen-binding sites, which
fragments include a heavy-chain variable domain (V.sub.H) connected
to a light-chain variable domain (V.sub.L) in the same polypeptide
chain (Vn-V.sub.L). By using a linker that is too short to allow
pairing between the two domains on the same chain, the domains can
be forced to pair with the complementary domains of another chain
and create two antigen-binding sites. Diabodies are described more
fully in, for example, EP 404,097; WO 93/11161; and Hollinger et
al., Proc. Natl. Acad. Sci. USA, 90:6444-6448 (1993).
[0054] The antibody administered can include a single-chain
antibody. The antibodies can be monovalent antibodies. Methods for
preparing monovalent antibodies are well known in the art. For
example, one method involves recombinant expression of
immunoglobulin light chain and modified heavy chain. The heavy
chain can be truncated generally at any point in the Fc region so
as to prevent heavy chain crosslinking. Alternatively, the relevant
cysteine residues are substituted with another amino acid residue
or are deleted so as to prevent crosslinking. In vitro methods are
also suitable for preparing monovalent antibodies. Digestion of
antibodies to produce fragments thereof, particularly, Fab
fragments, can be accomplished using routine techniques known in
the art.
[0055] The antibodies can be bispecific. Bispecific antibodies that
specifically bind to one protein and that specifically bind to
other antigens relevant to pathology and/or treatment are produced,
isolated, and tested using standard procedures that have been
described in the literature. [See, e.g., Pluckthun & Pack,
Immunotechnology, 3:83-105 (1997); Carter, et al., J.
Hematotherapy, 4:463-470 (1995); Renner & Pfreundschuh,
Immunological Reviews, 1995, No. 145, pp. 179-209; Pfreundschuh
U.S. Pat. No. 5,643,759; Segal, et al., J. Hematotherapy, 4:377-382
(1995); Segal, et al., Immunobiology, 185:390-402 (1992); and
Bolhuis, et al., Cancer Immunol. Immunother., 34:1-8 (1991)].
[0056] The antibodies disclosed herein can be formulated as
immunoliposomes. Liposomes containing the antibody are prepared by
methods known in the art. such as described in Epstein et al.,
Proc. Natl. Acad. Sci. USA, 82: 3688 (1985); Hwang et al., Proc.
Natl. Acad. Sci. USA 77: 4030 (1980); and U.S. Pat. Nos. 4,485,045
and 4,544,545. Liposomes with enhanced circulation time are
disclosed in U.S. Pat. No. 5,013,556. Particularly useful liposomes
can be generated by the reverse-phase evaporation method with a
lipid composition containing phosphatidylcholine, cholesterol, and
PEG-derivatized phosphatidylethanolamine (PEG-PE). Liposomes can be
extruded through filters of defined pore size to yield liposomes
with the desired diameter. Fab' fragments of the antibody of the
present invention can be conjugated to the liposomes as described
in Martin et al., J. Biol. Chem., 257:286-288 (1982) via a
disulfide-interchange reaction. A chemotherapeutic agent (such as
Doxorubicin) is optionally contained within the liposome. See
Gabizon et al, J. National Cancer Inst., 81(19): 1484 (1989).
[0057] The influenza antagonist can include an aptamer that binds
the hemagglutinin (HA) protein of an influenza B virus. The aptamer
can bind HA in the same location/epitope as the antibodies
described herein and/or to other locations/epitopes. The aptamer
can contain one or more of a nucleic acid, a RNA, a DNA, and an
amino acid. Aptamers can be selected and produced using any
suitable technique or protocol. For example, oligonucleotide
libraries with variable regions ranging from 18 to 50 nucleotides
in length can be used as templates for run-off transcription
reactions to generate random pools of RNA aptamers. This aptamer
pool can then be exposed to unconjugated matrix to remove
non-specific interacting species. The remaining pool is then
incubated with an immobilized target. The majority of aptamer
species in this pool can have low affinity, for the target can be
washed away leaving a smaller, more specific pool bound to the
matrix. This pool can then be eluted, precipitated, reverse
transcribed, and used as a template for run-off transcription.
After five rounds of selection, aliquots can be removed that are
cloned and sequenced. Selection can be continued until similar
sequences are reproducibly recovered.
[0058] Aptamer production can be performed using a bead-based
selection system. In this process, a library of beads is generated
in which each bead is coated with a population of aptamers with
identical sequences composed of natural and modified nucleotides.
This bead library, which can contain greater than 100,000,000
unique sequences, can be incubated with a peptide that corresponds
to hemagglutinin (HA) protein, or a portion thereof, e.g., an
extracellular domain, that is conjugated with a tag such as a
fluorescent dye. After washing, beads that demonstrate the highest
binding affinity can be isolated and aptamer sequences can be
determined for subsequent synthesis.
[0059] The present invention provides a method of inhibiting or
treating a human influenza B infection in a human subject including
administering a therapeutically effective amount of the anti-human
influenza B virus monoclonal antibody or antigen-binding fragment
thereof of the invention to the human subject. The method can
further include diagnosing the patient with an influenza B
infection. Anti-influenza antibodies or antigen-binding fragment
thereof of the present invention can be administered to a subject
before, during, and/or after diagnosing the patient as having an
influenza infection.
[0060] The method can further include monitoring for a decrease in
at least one symptom of an influenza B infection. For example, the
at least one symptom can include fever, headache, fatigue, chills,
malaise, myalgia, arthralgia, nasal congestion, sore throat, cough,
respiratory distress, stomach pain, or any combination thereof. The
anti-human influenza virus monoclonal antibody or antigen-binding
administered in combination with one or more additional therapies
directed to influenza B and/or other influenzas such as influenza A
and/or influenza C. The combination can act synergistically to
inhibit or treat the influenza B infection. The one or more
additional therapies can include, for example, a neuraminidase
inhibitor, a hemagglutinin inhibitor, an anti-inflammatory agent,
or any combination thereof. The neuraminidase inhibitor can
include, for example, zanamivir, oseltamivir, peramivir,
laninamivir, any pharmaceutically acceptable salt thereof, or any
combination thereof.
[0061] In accordance with the present invention, two or more
influenza antagonists can be administered. At least one of the
influenza antagonists can include an influenza B antagonist. The at
least one influenza B antagonist can be combined with one or more
influenza A antagonists and/or one or more influenza C antagonists.
At least one influenza antagonist can be administered in
combination with one or more additional therapies directed against
an influenza viral infection. The administration of two or more
therapies, including one or more influenza antagonists, can be
simultaneous, sequential, or in combination. Accordingly, when two
or more therapies are administered, they need not be administered
simultaneously or in the same way or in the same dose. When
administered simultaneously, the two or more therapies can be
administered in the same composition or in different compositions.
The two or more therapies can be administered using the same route
of administration or different routes of administration. When
administered at different times, the therapies can be administered
before or after each other. Administration order of the two or more
therapies can be alternated. The respective doses of the one or
more therapies can be varied over time. The type of one or more
therapy can be varied over time. When administered at separate
times, the separation of the two or more administrations can be any
time period. If administered multiple times, the length of the time
period can vary. The separation between administration of the two
or more two or more therapies can be 0 seconds, 1 second, 5
seconds, 10 seconds, 30 seconds, 1 minute, 5 minutes, 10 minutes,
15 minutes, 20 minutes, 30, minutes, 45 minutes, 1 hour, 1.5 hours,
2 hours, 2.5 hours, 3 hours, 4 hours, 5 hours, 7.5 hours, 10 hours,
12 hours, 15 hours, 18 hours, 21 hours, 24 hours, 1.5 days, 2 days,
3 days, 4 days, 5 days, 6 days, 7 days, 10 days, 2 weeks, 3 weeks,
4 weeks, one month, 6 weeks, 8 weeks, three months, six months, 1
year or longer.
[0062] Two or more influenza antagonists can act synergistically to
treat or reduce an influenza infection or a symptom of the same,
for example, fever. An influenza antagonist can be one or more
anti-influenza antibody alone or in combination with one or more
other influenza antagonist, for example, a small drug
pharmaceutical, or other anti-influenza therapy. Two or more
anti-influenza antibodies, or at least one anti-influenza antibody
and one or more additional therapies can act synergistically to
treat or reduce an influenza B viral infection. Two or more
therapies, including one or more anti-influenza antibody, can be
administered in synergistic amounts. Accordingly, the
administration of two or more therapies can have a synergistic
effect on the decrease in one or more symptoms of an influenza
infection, whether administered simultaneously, sequentially, or in
any combination. A first therapy can increase the efficacy of a
second therapy greater than if second therapy was employed alone,
or a second therapy increases the efficacy of a first therapy, or
both. The effect of administering two or more therapies can be such
that the effect on decreasing one or more symptoms of an influenza
infection is greater than the additive effect of each being
administered alone. When given in synergistic amounts, one therapy
can enhance the efficacy of one or more other therapy on the
decrease in one or more symptoms of an influenza infection, even if
the amount of one or more therapy alone would have no substantial
effect on one or more symptom of an influenza infection.
Measurements and calculations of synergism can be performed as
described in Teicher, "Assays for In Vitro and In Vivo Synergy," in
Methods in Molecular Medicine, vol. 85: Novel Anticancer Drug
Protocols, pp. 297-321 (2003) and/or by calculating the combination
index (CI) using CalcuSyn software.
[0063] The present invention provides use of an anti-human
influenza B virus monoclonal antibody or antigen-binding fragment
thereof of the present invention to manufacture a medicament for
inhibiting or treating a human influenza B infection in a human
subject. The present invention also provides a method of detecting
human influenza B in a human subject. The method can include
contacting a sample from the human subject with an anti-human
influenza B virus human antibody or antigen-binding fragment
thereof of the invention. The method can further include detecting
the presence or absence of a human influenza B virus in the human
subject based on whether the antibody binds a hemagglutinin (HA)
protein of the human influenza B virus. The present invention
further provides a pharmaceutical composition containing an
anti-human influenza virus human antibody or antigen-binding
fragment thereof of the present invention and a pharmacologically
acceptable carrier. The present invention still further provides a
kit for at least one of the prevention, the treatment, and the
detection of human influenza B in a human subject containing an
anti-human influenza virus monoclonal antibody or antigen-binding
fragment thereof of the present invention. The kit can include the
pharmaceutical composition and/or one or more additional
anti-influenza B or other antagonists.
[0064] Exact formulation, route of administration and dosage can be
chosen by the individual physician in view of the patient's
condition. [See, e.g., Fingl et. al., in The Pharmacological Basis
of Therapeutics, 1975, Ch. 1 p. I.] The attending physician can
determine when to terminate, interrupt, or adjust administration
due to toxicity, or to organ dysfunctions. Conversely, the
attending physician can also adjust treatment to higher levels if
the clinical response were not adequate, precluding toxicity. The
magnitude of an administrated dose in the management of disorder of
interest will vary with the severity of the disorder to be treated
and the route of administration. The severity of the disorder can,
for example, be evaluated, in part, by standard prognostic
evaluation methods. The dose and dose frequency, can vary according
to the age, body weight, and response of the individual patient. A
program comparable to that discussed above can be used in
veterinary medicine.
[0065] Use of pharmaceutically acceptable carriers to formulate the
compounds herein disclosed for the practice of the invention into
dosages suitable for systemic administration is within the scope of
the present invention. With proper choice of carrier and suitable
manufacturing practice, the compositions relevant to the present
invention, in particular, those formulated as solutions, can be
administered parenterally, such as by intravenous injection. The
compounds can be formulated readily using pharmaceutically
acceptable carriers well known in the art into dosages suitable for
oral administration. Such carriers enable the compounds relevant to
the present invention to be formulated as tablets, pills, capsules,
liquids, gels, syrups, slurries, tablets, dragees, solutions,
suspensions and the like, for oral ingestion by a patient to be
treated.
[0066] The therapeutic agent can be prepared in a depot form to
allow for release into the body to which it is administered is
controlled with respect to time and location within the body (see,
for example, U.S. Pat. No. 4,450,150). Depot forms of therapeutic
agents can be, for example, an implantable composition containing
the therapeutic agent and a porous or non-porous material, such as
a polymer, wherein the therapeutic agent is encapsulated by or
diffused throughout the material and/or degradation of the
non-porous material. The depot is then implanted into the desired
location within the body and the therapeutic agent is released from
the implant at a predetermined rate.
[0067] The therapeutic agent that is used in the present invention
can be formed as a composition, such as a pharmaceutical
composition containing a carrier and a therapeutic compound.
Pharmaceutical compositions containing the therapeutic agent can
include more than one therapeutic agent. The pharmaceutical
composition can alternatively contain a therapeutic agent in
combination with other pharmaceutically active agents or drugs.
[0068] The carrier can be any suitable carrier. For example, the
carrier can be a pharmaceutically acceptable carrier. With respect
to pharmaceutical compositions, the carrier can be any of those
conventionally used with consideration of chemico-physical
considerations, such as solubility and lack of reactivity with the
active compound(s), and by the route of administration. In addition
to, or in the alternative to, the following described
pharmaceutical compositions, the therapeutic compounds of the
present inventive methods can be formulated as inclusion complexes,
such as cyclodextrin inclusion complexes, or liposomes.
[0069] The pharmaceutically acceptable carriers described herein,
for example, vehicles, adjuvants, excipients, and diluents; are
well-known to those skilled in the art and are readily available to
the public. The pharmaceutically acceptable carrier can be
chemically inert to the active agent(s) and one which has no
detrimental side effects or toxicity under the conditions of use.
The choice of carrier can be determined in part by the particular
therapeutic agent, as well as by the particular method used to
administer the therapeutic compound. There are a variety of
suitable formulations of the pharmaceutical composition of the
present invention. The following formulations for oral, aerosol,
parenteral, subcutaneous, transdermal, transmucosal, intestinal,
intramedullary injections, direct intraventricular, intravenous,
intranasal, intraocular, intramuscular, intraarterial, intrathecal,
intraperitoneal, rectal, and vaginal administration are exemplary
and are in no way limiting. More than one route can be used to
administer the therapeutic agent, and in some instances, a
particular route can provide a more immediate and more effective
response than another route. Depending on the specific disorder
being treated, such agents can be formulated and administered
systemically or locally. Techniques for formulation and
administration may be found in Remington's Pharmaceutical Sciences,
18th ed., Mack Publishing Co., Easton, Pa. (1990).
[0070] Formulations suitable for oral administration can include
(a) liquid solutions, such as an effective amount of the inhibitor
dissolved in diluents, such as water, saline, or orange juice; (b)
capsules, sachets, tablets, lozenges, and troches, each containing
a predetermined amount of the active ingredient, as solids or
granules; (c) powders; (d) suspensions in an appropriate liquid;
and (e) suitable emulsions. Liquid formulations can include
diluents, such as water and alcohols, for example, ethanol, benzyl
alcohol, and the polyethylene alcohols, either with or without the
addition of a pharmaceutically acceptable surfactant. Capsule forms
can be of the ordinary hard or soft shelled gelatin type
containing, for example, surfactants, lubricants, and inert
fillers, such as lactose, sucrose, calcium phosphate, and corn
starch. Tablet forms can include one or more of lactose, sucrose,
mannitol, corn starch, potato starch, alginic acid,
microcrystalline cellulose, acacia, gelatin, guar gum, colloidal
silicon dioxide, croscarmellose sodium, talc, magnesium stearate,
calcium stearate, zinc stearate, stearic acid, and other
excipients, colorants, diluents, buffering agents, disintegrating
agents, moistening agents, preservatives, flavoring agents, and
other pharmacologically compatible excipients. Lozenge forms can
contain the inhibitor in a flavor, usually sucrose and acacia or
tragacanth, as well as pastilles containing the inhibitor in an
inert base, such as gelatin and glycerin, or sucrose and acacia,
emulsions, gels, and the like containing, in addition to, such
excipients as are known in the art.
[0071] Pharmaceutical preparations that can be used orally include
push-fit capsules made of gelatin, as well as soft, sealed capsules
made of gelatin and a plasticizer, such as glycerol or sorbitol.
The push-fit capsules can contain the active ingredients in
admixture with filler such as lactose, binders such as starches,
and/or lubricants such as talc or magnesium stearate and,
optionally, stabilizers. In soft capsules, the active compounds may
be dissolved or suspended in suitable liquids, such as fatty oils,
liquid paraffin, or liquid polyethylene glycols. In addition,
stabilizers can be added.
[0072] The therapeutic agent, alone or in combination with other
suitable components, can be made into aerosol formulations to be
administered via inhalation. These aerosol formulations can be
placed into pressurized acceptable propellants, such as
dichlorodifluoromethane, propane, nitrogen, and the like. They also
can be formulated as pharmaceuticals for non-pressurized
preparations, such as in a nebulizer or an atomizer. Such spray
formulations also may be used to spray mucosa. Topical formulations
are well known to those of skill in the art. Such formulations are
particularly suitable in the context of the invention for
application to the skin.
[0073] Injectable formulations are in accordance with the present
invention. The parameters for effective pharmaceutical carriers for
injectable compositions are well-known to those of ordinary skill
in the art [see, e.g., Pharmaceutics and Pharmacy Practice, J. B.
Lippincott Company, Philadelphia, Pa., Banker and Chalmers, eds.,
pages 238250 (1982), and ASHP Handbook on Injectable Drugs,
Toissel, 4th ed., pages 622 630 (1986)]. For injection, the agents
of the present invention can be formulated in aqueous solutions,
preferably in physiologically compatible buffers such as Hanks's
solution, Ringer's solution, or physiological saline buffer. For
such transmucosal administration, penetrants appropriate to the
barrier to be permeated are used in the formulation. Such
penetrants are generally known in the art.
[0074] Formulations suitable for parenteral administration can
include aqueous and non-aqueous, isotonic sterile injection
solutions, which can contain anti-oxidants, buffers, bacteriostats,
and solutes that render the formulation isotonic with the blood of
the intended recipient, and aqueous and non-aqueous sterile
suspensions that can include suspending agents, solubilizers,
thickening agents, stabilizers, and preservatives. The therapeutic
agent can be administered in a physiologically acceptable diluent
in a pharmaceutical carrier, such as a sterile liquid or mixture of
liquids, including water, saline, aqueous dextrose and related
sugar solutions, an alcohol, such as ethanol or hexadecyl alcohol,
a glycol, such as propylene glycol or polyethylene glycol,
poly(ethyleneglycol) 400, glycerol, dimethylsulfoxide, ketals such
as 2,2-dimethyl-1,3-dioxolane-4-methanol, ethers, oils, fatty
acids, fatty acid esters or glycerides, or acetylated fatty acid
glycerides with or without the addition of a pharmaceutically
acceptable surfactant, such as a soap or a detergent, suspending
agent, such as pectin, carbomers, methylcellulose,
hydroxypropylmethylcellulose, or carboxymethyl-cellulose, or
emulsifying agents and other pharmaceutical adjuvants.
[0075] Oils, which can be used in parenteral formulations, include
petroleum, animal, vegetable, or synthetic oils. Specific examples
of oils include peanut, soybean, sesame, cottonseed, corn, olive,
petrolatum, and mineral. Suitable fatty acids for use in parenteral
formulations include oleic acid, stearic acid, and isostearic acid.
Ethyl oleate and isopropyl myristate are examples of suitable fatty
acid esters.
[0076] Suitable soaps for use in parenteral formulations include
fatty alkali metal, ammonium, and triethanolamine salts, and
suitable detergents include (a) cationic detergents such as, for
example, dimethyl dialkyl ammonium halides, and alkyl pyridinium
halides, (b) anionic detergents such as, for example, alkyl, aryl,
and olefin sulfonates, alkyl, olefin, ether, and monoglyceride
sulfates, and sulfosuccinates, (c) nonionic detergents such as, for
example, fatty amine oxides, fatty acid alkanolamides, and
polyoxyethylenepolypropylene copolymers, (d) amphoteric detergents
such as, for example, alkyl-beta-aminopropionates, and
2-alkyl-imidazoline quaternary ammonium salts, and (e) mixtures
thereof.
[0077] The parenteral formulations can contain from about 0.5% to
about 25% by weight of the drug in solution. Preservatives and
buffers can be used. In order to minimize or eliminate irritation
at the site of injection, such compositions may contain one or more
nonionic surfactants having a hydrophilic-lipophilic balance (HLB)
of from about 12 to about 17. The quantity of surfactant in such
formulations will typically range from about 5% to about 15% by
weight. Suitable surfactants include polyethylene glycol sorbitan
fatty acid esters, such as sorbitan monooleate and the high
molecular weight adducts of ethylene oxide with a hydrophobic base,
formed by the condensation of propylene oxide with propylene
glycol. The parenteral formulations can be presented in unit-dose
or multi-dose sealed containers, such as ampoules and vials, and
can be stored in a freeze-dried (lyophilized) condition requiring
only the addition of the sterile liquid excipient, for example,
water, for injections, immediately prior to use. Extemporaneous
injection solutions and suspensions can be prepared from sterile
powders, granules, and tablets of the kind previously
described.
[0078] The therapeutic agent can be made into suppositories by
mixing with a variety of bases, such as emulsifying bases or
water-soluble bases. Formulations suitable for vaginal
administration can be presented as pessaries, tampons, creams,
gels, pastes, foams, or spray formulas containing, in addition to
the active ingredient, such carriers as are known in the art to be
appropriate.
[0079] Agents intended to be administered intracellularly may be
administered using techniques well known to those of ordinary skill
in the art. For example, such agents can be encapsulated into
liposomes. Liposomes are spherical lipid bilayers with aqueous
interiors. Molecules present in an aqueous solution at the time of
liposome formation are incorporated into the aqueous interior. The
liposomal contents are both protected from the external
microenvironment and, because liposomes fuse with cell membranes,
are efficiently delivered into the cell cytoplasm. Additionally,
due to their hydrophobicity, small organic molecules may be
directly administered intracellularly. Materials and methods
described for one aspect of the present invention can also be
employed in other aspects of the present invention. For example, a
material such a nucleic acid or antibody described for use in
screening assays can also be employed as therapeutic agents and
vice versa.
[0080] The present invention includes the following
aspects/embodiments/features in any order and/or in any
combination:
[0081] 1. The present invention relates to an anti-human influenza
virus monoclonal antibody or an antigen-binding fragment thereof
comprising a neutralization activity against a human influenza B
virus, wherein the monoclonal antibody comprises a human monoclonal
antibody or a humanized monoclonal antibody.
[0082] 2. The anti-human influenza virus monoclonal antibody or
antigen-binding fragment thereof of any preceding or following
embodiment/feature/aspect, wherein the monoclonal antibody or
antigen-binding fragment thereof has a neutralization activity
against at least a B/Florida/4/2006 strain, a B/Shanghai/361/2002
strain, a B/Johannesburg/5/1999 strain, a B/Yamanashi/166/1998
strain, and a B/Mie/1/1993 strain.
[0083] 3. The anti-human influenza virus monoclonal antibody or
antigen-binding fragment thereof of any preceding or following
embodiment/feature/aspect, wherein the monoclonal antibody or
antigen-binding fragment thereof has a neutralization activity
against at least a B/Florida/4/2006 strain, a B/Shanghai/361/2002
strain, a B/Johannesburg/5/1999 strain, a B/Yamanashi/166/1998
strain, a B/Mie/1/1993 strain, a B/Malaysia/2506/04 strain, a
B/Shandong/7/1997 strain, and a B/Victoria/2/1987 strain.
[0084] 4. The anti-human influenza virus human monoclonal antibody
of any preceding or following embodiment/feature/aspect, wherein
the human monoclonal antibody is produced by a hybridoma made by
fusing a peripheral blood mononuclear cell (PBMC) from a human
being having an influenza B virus infection with a fusion partner
cell capable of efficient cell fusion.
[0085] 5. The anti-human influenza virus human monoclonal antibody
of any preceding or following embodiment/feature/aspect, wherein
the influenza B virus comprises at least one of a B/Florida/4/2006
strain, a B/Shanghai/361/2002 strain, a B/Johannesburg/5/1999
strain, a B/Yamanashi/166/1998 strain, a B/Mie/1/1993 strain, a
B/Malaysia/2506/04 strain, a B/Shandong/7/1997 strain, and a
B/Victoria/2/1987 strain.
[0086] 6. The anti-human influenza B virus human monoclonal
antibody of any preceding or following embodiment/feature/aspect,
wherein the fusion partner cell is a SPYMEG cell.
[0087] 7. The anti-human influenza B virus monoclonal antibody or
antigen-binding fragment thereof of any preceding or following
embodiment/feature/aspect comprising an IgG, a Fab, a Fab', a
F(ab')2, a scFv, or a dsFv.
[0088] 8. The anti-human influenza virus monoclonal antibody or
antigen-binding fragment thereof of any preceding or following
embodiment/feature/aspect, comprising:
a heavy chain variable region comprising a first complementarity
determining region (CDR1) having a first amino acid sequence
comprising SEQ ID NO: 1, 7, or 13, a second complementarity
determining region (CDR2) having a second amino acid sequence
comprising SEQ ID NO: 2, 8, or 14, and a third complementarity
determining region (CDR3) having a third amino acid sequence
comprising SEQ ID NO: 3, 9, or 15, and a light chain variable
region comprising a first complementarity determining region (CDR1)
having a fourth amino acid sequence comprising SEQ ID NO: 4, 10, or
16; a second complementarity determining region (CDR2) having a
fifth amino acid sequence comprising SEQ ID NO: 5, 11, or 17, and a
third complementarity determining region (CDR3) having a sixth
amino acid sequence comprising SEQ ID NO: 6, 12, or 18.
[0089] 9. The anti-human influenza virus monoclonal antibody or
antigen-binding fragment thereof of any preceding or following
embodiment/feature/aspect, wherein the first amino acid sequence
comprises SEQ ID NO: 1, the second amino acid sequence comprises
SEQ ID NO: 2, the third amino acid sequence comprises SEQ ID NO: 3,
the fourth amino acid sequence comprises SEQ ID NO: 4, the fifth
amino acid sequence comprises SEQ ID NO: 5, and the sixth amino
acid sequence comprises SEQ ID NO: 6.
[0090] 10. The anti-human influenza virus monoclonal antibody or
antigen-binding fragment thereof of any preceding or following
embodiment/feature/aspect, wherein the anti-human influenza virus
monoclonal antibody comprises antibody 5A7.
[0091] 11. The anti-human influenza virus monoclonal antibody or
antigen-binding fragment thereof of any preceding or following
embodiment/feature/aspect, wherein the first amino acid sequence
comprises SEQ ID NO: 7, the second amino acid sequence comprises
SEQ ID NO: 8, the third amino acid sequence comprises SEQ ID NO: 9,
the fourth amino acid sequence comprises SEQ ID NO: 10, the fifth
amino acid sequence comprises SEQ ID NO: 11, and the sixth amino
acid sequence comprises SEQ ID NO: 12.
[0092] 12. The anti-human influenza virus monoclonal antibody or
antigen-binding fragment thereof of any preceding or following
embodiment/feature/aspect, wherein the anti-human influenza virus
monoclonal antibody comprises antibody 3A2.
[0093] 13. The anti-human influenza virus monoclonal antibody or
antigen-binding fragment thereof of any preceding or following
embodiment/feature/aspect, wherein the first amino acid sequence
comprises SEQ ID NO: 13, the second amino acid sequence comprises
SEQ ID NO: 14, the third amino acid sequence comprises SEQ ID NO:
15, the fourth amino acid sequence comprises SEQ ID NO: 16, the
fifth amino acid sequence comprises SEQ ID NO: 17, and the sixth
amino acid sequence comprises SEQ ID NO: 18.
[0094] 14. The anti-human influenza virus monoclonal antibody or
antigen-binding fragment thereof of any preceding or following
embodiment/feature/aspect, wherein the anti-human influenza virus
monoclonal antibody comprises antibody 10C4.
[0095] 15. A pharmaceutical composition comprising the anti-human
influenza virus human monoclonal antibody or antigen-binding
fragment thereof of any preceding or following
embodiment/feature/aspect and a pharmacologically acceptable
carrier.
[0096] 16. A kit for at least one of the prevention, the treatment,
and the detection of human influenza B in a human subject
comprising the anti-human influenza virus human monoclonal antibody
or antigen-binding fragment thereof of any preceding or following
embodiment/feature/aspect.
[0097] 17. A method of inhibiting or treating a human influenza B
infection in a human subject comprising administering a
therapeutically effective amount of the anti-human influenza B
virus monoclonal antibody or antigen-binding fragment thereof of
any preceding or following embodiment/feature/aspect to the human
subject.
[0098] 18. The method of inhibiting or treating a human influenza B
infection in a human subject of any preceding or following
embodiment/feature/aspect, further comprising diagnosing the
patient with an influenza B infection.
[0099] 19. The method of inhibiting or treating a human influenza B
infection in a human subject of any preceding or following
embodiment/feature/aspect, further comprising monitoring for a
decrease in at least one symptom of an influenza B infection.
[0100] 20. The method of inhibiting or treating a human influenza B
infection in a human subject of any preceding or following
embodiment/feature/aspect, wherein the at least one symptom
comprises fever, headache, fatigue, chills, malaise, myalgia,
arthralgia, nasal congestion, sore throat, cough, respiratory
distress, or stomach pain, or any combination thereof.
[0101] 21. The method of inhibiting or treating a human influenza B
infection in a human subject of any preceding or following
embodiment/feature/aspect, wherein the anti-human influenza virus
human monoclonal antibody or antigen-binding fragment thereof of
claim 1 is administered in combination with one or more additional
therapies directed to influenza B.
[0102] 22. The method of inhibiting or treating a human influenza B
infection in a human subject of any preceding or following
embodiment/feature/aspect, wherein the combination acts
synergistically to inhibit or treat the influenza B infection.
[0103] 23. The method of inhibiting or treating a human influenza B
infection in a human subject of any preceding or following
embodiment/feature/aspect, wherein the one or more additional
therapies comprise a neuraminidase inhibitor, a hemagglutinin
inhibitor, an anti-inflammatory agent, or any combination
thereof.
[0104] 24. The method of inhibiting or treating a human influenza B
infection in a human subject of any preceding or following
embodiment/feature/aspect, wherein the neuraminidase inhibitor
comprises zanamivir, oseltamivir, peramivir, laninamivir, any
pharmaceutically acceptable salt thereof, or any combination
thereof.
[0105] 25. Use of the anti-human influenza B virus human monoclonal
antibody or antigen-binding fragment thereof of any preceding or
following embodiment/feature/aspect to manufacture a medicament for
inhibiting or treating a human influenza B infection in a human
subject.
[0106] 26. A method of detecting human influenza B in a human
subject comprising; contacting a sample from the human subject with
the anti-human influenza B virus antibody or antigen-binding
fragment thereof of any preceding or following
embodiment/feature/aspect; and
detecting the presence or absence of a human influenza B virus in
the human subject based on whether the antibody binds a
hemagglutinin (HA) protein of the human influenza B virus.
[0107] 27. A method for producing an anti-human influenza B virus
human monoclonal antibody comprising:
producing a hybridoma by fusing a peripheral blood mononuclear cell
(PBMC) from a human being having an influenza B virus infection
with a fusion partner cell capable of efficient cell fusion; and
obtaining an anti-human influenza virus monoclonal antibody from
the hybridoma.
[0108] 28. The method for producing an anti-human influenza B virus
human monoclonal antibody of any preceding or following
embodiment/feature/aspect, wherein the influenza B virus comprises
at least one of a B/Florida/4/2006 strain, a B/Shanghai/361/2002
strain, a B/Johannesburg/5/1999 strain, a B/Yamanashi/166/1998
strain, a B/Mie/1/1993 strain, a B/Malaysia/2506/04 strain, a
B/Shandong/7/1997 strain, and B/Victoria/2/1987 strain.
[0109] 29. The method for producing an anti-human influenza virus
human monoclonal antibody of any preceding or following
embodiment/feature/aspect, wherein the fusion partner cell is a
SPYMEG cell.
[0110] 30. An anti-human influenza virus human monoclonal antibody
produced by the method of any preceding or following
embodiment/feature/aspect.
[0111] The present invention can include any combination of these
various features or embodiments above and/or below as set forth in
sentences and/or paragraphs. Any combination of disclosed features
herein is considered part of the present invention and no
limitation is intended with respect to combinable features.
[0112] The present invention will be further clarified by the
following examples, which are intended to be exemplary of, but not
limiting, the present invention. Human materials were collected
using protocols approved by the Institutional Review Boards of the
Research Institute for Microbial Diseases, Osaka University
(#19-8-6). Animal studies were conducted under the applicable laws
and guidelines for the care and use of laboratory animals in the
Research Institute for Microbial Diseases, Osaka University. They
were approved by the Animal Experiment Committee of the Research
Institute for Microbial Diseases, Osaka University (#H21-24-0), as
specified in the Fundamental Guidelines for the Proper Conduct of
Animal Experiment and Related Activities in Academic Research
Institutions under the jurisdiction of the Ministry of Education,
Culture, Sports, Science and Technology, Japan, 2006. The National
Institute of Infectious Diseases and Dr. Shin-ichi Tamura (the
National Institute of Infectious Diseases) provided viral strains.
Natsuko Fukura, Azusa Asai, Tadahiro Sasaki, and Yohei Watanabe
provided helpful advice and technical assistance. Data are
expressed as the means+ or -standard errors of the means (SEM).
Statistical analysis was performed by Student's t test. A P value
of <0.05 was considered significant.
[0113] Eight influenza B vaccine strains (B/Victoria/2/1987,
B/Mie/1/1993, B/Shandong/7/1997, B/Yamanashi/166/1998,
B/Johannesburg/5/1999, B/Shanghai/361/2002, B/Malaysia/2506/2004,
and B/Florida/4/2006) and the mouse-adapted strain B/Ibaraki/2/1985
were used. The B/Malaysia/2506/2004 and B/Florida/4/2006 strains
were kindly provided by the National Institute of Infectious
Diseases, Tokyo, Japan. Mouse-adapted B/Ibaraki/2/1985 strain was
provided by Dr. S. Tamura, National Institute of Infectious
Diseases (Chen et al., Vaccine 19:1446-1455). Viruses were
propagated either in Madin-Darby canine kidney (MDCK) cells or in
9-day-old embryonated chicken eggs. Infectivity was titrated by
focus-forming assay.
EXAMPLES
[0114] The present invention will be further clarified by the
following examples, which are intended to be exemplary of, but not
limiting, the present invention.
Example 1
[0115] Human monoclonal antibodies (HuMAbs) were prepared in
accordance with the procedure described in Kubota-Koketsu et al.,
Biochemical and Biophysical Research Communications 387:180-185
(2009). Healthy volunteers were vaccinated with the HA split
vaccine including A/Brisbane/59/2007 (H1N1), A/Uruguay/716/2007
(H3N2), and B/Florida/4/2006 strains. One to two weeks later, the
vaccine-derived PBMCs were fused with SPYMEG cells and after
screening and cloning, three hybridoma clones producing HuMAbs,
designated 5A7, 3A2, and 10C4, were established. The reactivity of
the HuMAbs was tested by IFA and Western blotting.
[0116] Briefly, 10 ml blood was drawn from a healthy volunteer
vaccinated in the 2008/2009 winter season with trivalent HA split
vaccine, which included A/Brisbane/59/2007, A/Uruguay/716/2007, and
B/Florida/4/2006 (The Research Foundation for Microbial Diseases of
Osaka University, Kagawa, Japan), and then the PBMCs were collected
by density gradient centrifugation through Ficoll-Paque Plus (GE
Healthcare, Uppsala, Sweden). SPYMEG cells, established from mouse
myeloma cell line SP2/0-Ag14 and human megakaryoblastic cell line
MEG-01, were used as fusion partner cells. SPYMEG cells are
non-secretors of human and murine immunoglobulin. The PBMCs were
fused with SPYMEG cells using polyethylene glycol #1500 (Roche
Diagnostics, Mannheim, Germany). The fused cells were cultured in
Dulbecco's modified Eagle medium (DMEM; Invitrogen, Carlsbad,
Calif.) supplemented with 15% fetal bovine serum and
hypoxanthine-aminopterin-thymidine. The first screening for MAb
specific for influenza viruses was performed by immunofluorescence
assay (IFA). For the IFA, the infected cells were fixed with
absolute ethanol and then reacted with hybridoma supernatant for 30
min at 37 deg C., followed by incubation with FITC-conjugated
anti-human IgG for 45 min at 37 deg C. The cells in the specific
MAb-positive wells were cloned by limiting dilution, then followed
by a second screening by IFA. Hybridoma cells taken from
IFA-positive wells that had a single colony per well were cultured
and expanded in Hybridoma-SFM (Invitrogen). MAb was purified from
100 ml hybridoma culture supernatant by affinity chromatography
using HiTrap Protein G HP Columns (GE Healthcare) and then dialyzed
into phosphate buffered saline (PBS) using Slide-A-Lyzer.RTM.
Dialysis Cassettes (Thermo Scientific, Waltham, Mass.).
[0117] For IgG isotyping, ELISA microplates (Maxsorp; Nunc,
Copenhagen, Denmark) were coated overnight at 4 deg C. with goat
anti-human IgG (Jackson ImmunoResearch Laboratories, Inc, West
Grove, Pa.) in 0.05 M sodium bicarbonate buffer (pH 8.6). After
washing with PBS including 0.1% Tween-20, the wells were blocked
with 0.5% BSA in PBS for 1 hour at 37 deg C. After washing again,
the wells were incubated with hybridoma supernatants or control
serum for 2 hours at 37 deg C. Following further washing, the wells
were incubated with HRP-conjugated anti-human IgG1, IgG2, IgG3, or
IgG4 (SouthernBiotech, Birmingham, Ala.) for 1 hour at 37 deg C.
The wells were washed five times followed by incubation with TMB
peroxidase substrate (KPL, Gaithersburg, Md.) at room temperature
in the dark. After 20 minutes, the reaction was stopped with 2N
H2SO4 solution. The color development was read at 450 nm in an
ELISA Photometer (Biotek ELISA Reader; Biotek, Winooski, Vt.). All
samples were run in triplicate.
[0118] For the sequencing of HuMAb variable regions, total RNA
extracted from the hybridoma using an RNeasy Mini Kit (Qiagen) was
subjected to RT-PCR using a PrimeScript RT reagent Kit (Takara,
Shiga, Japan) with an oligo (dT) primer. The coding region of the
H- and L-chains of HuMAb was amplified by PCR with the following
primers: 5'-ATGGAGTTTGGGCTGAGCTGGGTT-3' (H-chain-forward) (SEQ ID
NO. 19) and 5'-CTCCCGCGGCTTTGTCTTGGCATTA-3' (H-chain-reverse) (SEQ
ID NO. 20); and 5'-ATGGCCTGGRYCYCMYTCYWCCTM-3' (L-chain-forward)
(SEQ ID NO. 21) and 5'-TGGCAGCTGTAGCTTCTGTGGGACT-3'
(L-chain-reverse) (SEQ ID NO. 22). PCR products were ligated into
pGEM-T Easy Vector (Promega) and their sequences were analyzed
using a BigDye Terminator v3.1 Cycle Sequencing Kit and an ABI
Prism 3100 Genetic Analyzer (Applied Biosystems, Foster City,
Calif.).
[0119] IgG plasmids were constructed using T Easy Vectors with the
variable region gene of H- and L-chains were subjected to PCR to
add restriction enzyme sites and a Kozak sequence with the
following primer sets (restriction enzyme sites are underlined):
5'-ATTTGCGGCCGCCATGGAGTTTGGGCTGAG-3' (HC.sub.--5Fw_NotI;
H-chain-forward) (SEQ ID NO. 23) and
5'-ATACTCGAGGGTGCCAGGGGGAAGACCGATG-3' (HC_Reverse_XhoI;
H-chain-reverse) (SEQ ID NO. 24); and
5'-ATTTGCGGCCGCCATGGCCTGGGCTCTGCT-3' (5A7Lambda.sub.--18 Fw_NotI;
L-chain-forward) (SEQ ID NO. 25) and
5'-ATACTCGAGGGCGGGAACAGAGTGACCGTGG-3' (Lambda_Reverse_XhoI;
L-chain-reverse) (SEQ ID NO. 26). PCR products of the coding region
of H- and L-chains were digested by restriction enzymes, Not I and
Xho I, and then ligated to expression vectors, pQCXIP-hCH and
pQCXIH-hC lambda, which have a human immunoglobulin-constant region
of gamma and lambda chains (MBL), respectively.
[0120] All of three HuMAbs reacted with the HA protein in influenza
B virus (Table 1). HuMAbs, 5A7 and 10C4 were IgG1 isotype, and 3A2
was IgG3 (Table 1). Sequencing analysis of the V.sub.H and V.sub.L
region of the three HuMAbs revealed that each had different amino
acid residues in antigenic regions including the
complementarity-determining regions (CDRs) (Tables 2 and 3).
TABLE-US-00001 TABLE 1 Table 1: Pattern of Reactivity of HuMAbs.
Target Isotype IFA Reducing WB 5A7 HA of influenza B IgG1 +.sup.1 +
3A2 HA of influenza B IgG3 + - .sup.2 10C4 HA of influenza B IgG1 +
- .sup.1Positive result .sup.2 Negative result
TABLE-US-00002 TABLE2 Table 2: Deduced Amino Acid Sequences of CDRs
in the V.sub.H of three HuMAbs. SEQ. SEQ. SEQ. ID ID ID V.sub.H
CDR1 No. CDR2 No. CDR3 No. 5A7 NYGMH 1 VVWYDGLIKY 2 DLQPPHSPYGM 3
YADSVKG DV 3A2 SYYWS 7 YVYNSGSTR 8 APDDYYDSVGYY 9 YNPSLKS YGCPYFDS
10C4 NYAMS 13 AISGGGDWT 14 DVTYLYDSSGYY 15 YYADSVKG
YGGADRDYYFDY
TABLE-US-00003 TABLE3 Table 3: Deduced Amino Acid Sequences of CDRs
in the Y.sub.L of three HuMAbs. SEQ. SEQ. SEQ. ID ID ID V.sub.L
CDR1 No. CDR2 No. CDR3 No. 5A7 SGSSSNI 4 NNNQRPS 5 AAWDDSLTVS 6
GSNDVY 3A2 RASPSIA 10 GASTRAT 11 QQYSNWPRT 12 DNLA 10C4 SGGSSNI 16
SNNQRPL 17 QQWDDSLNGWV 18 GSNYVN
[0121] The neutralizing activities of HuMAbs were determined as
follows. The virus neutralization (VN) assay was carried out in
accordance with Okuno et al. 28:1308-1313 (1990), with minor
modification. MAb at a concentration of 100 mcg/ml was serially
diluted four-fold with Minimum Essential Medium (MEM; Invitrogen)
and incubated with 200 focus-forming units (FFU) of viruses at 37
C. for 1 hour. Then, MDCK cells were adsorbed with the mixtures at
37 C. for 1 hour. After incubation for 12 hours, the cells were
fixed and subjected to IFA. The lowest concentration of MAb that
inhibited 50% of viral growth was designated the VN.sub.50 titer.
In the focus formation assay, MDCK cells in a 96-well plate were
adsorbed with viruses diluted serially 10-fold at 37 deg C. for 1
hour. The cells were then washed with PBS and incubated at 37 deg
C. for 12 hours. The cells were fixed and subjected to IFA.
[0122] VN assays were performed with the three HuMAbs. HuMAb 5A7
had a lower VN.sub.50 (6.25 to 25 mcg/ml) compared with 3A2 and
10C4; however, 5A7 neutralized the Yamagata and Victoria lineages
isolated during 1985 to 2006. HuMAbs 3A2 and 10C4 had a VN.sub.50
of 0.02 to 6.25 mcg/ml for Yamagata lineage, whereas they hardly
neutralized any Victoria lineage except mouse-adapted
B/Ibaraki/2/1985, which was neutralized slightly by 3A2 (Table 4).
To clarify the mechanism of neutralization by the three HuMAbs, HI
and fusion inhibition assays were also performed. In the
hemagglutinin inhibition (HI) assay, viral titers were determined
with a hemagglutination assay. Briefly, the viruses were serially
diluted two-fold with PBS and mixed with 0.7% (v/v) human O-type
red blood cells. After incubation at room temperature for 1 hour,
hemagglutination units (HAUs) were estimated. Next, HI titration
was performed as follows. MAb at a concentration of 100 mcg/ml was
serially diluted two-fold and mixed with 8 HAU per 50 l of viral
sample. After incubation at 37 C. for 1 hour, the mixtures were
further incubated with 0.7% (v/v) human red blood cells for 1 hour
at room temperature. The lowest concentration of MAb that
completely inhibited hemagglutination was designated the HI
titer.
[0123] For the fusion inhibition assay, cell-cell fusion was
accomplished as described previously (Okuno et al., Journal of
Virology 67:2552-2558). Briefly, monkey kidney cell line CV-1 cells
were infected with B/Florida/4/2006 at an MOI of 0.3. After
incubation for 24 hours, the cells were washed with MEM and then
incubated for 15 min at 37 deg C. in MEM supplemented with 2.5
mcg/ml of acetylated trypsin (Sigma, St. Louis, Mo.). After
washing, the cells were incubated for 30 min with diluted HuMAbs.
Thereafter, the cells were treated for 2 min at 37 deg C. with MEM
supplemented with 10 mM MES and 10 mM HEPES (pH 5.5). After the
medium was completely removed by washing, the cells were incubated
for 3 hours. Then they were fixed with absolute methanol and
stained with Giemsa (Wako, Osaka, Japan).
TABLE-US-00004 TABLE 4 Table 4. Characterization of HuMAbs HuMAb
5A7 3A2 10C4 VN.sub.50 (.mu.g/ml).sup.1 Yamagata lineage
B/Florida/4/2006 6.25 0.10 0.39 B/Shanghai/361/2002 6.25 6.25 0.39
R/Johannesburg/5/1999 6.25 0.10 0.39 B/Yamanashi/166/1998 6.25 0.10
0.39 B/Mie/1/1993 6.25 0.10 0.39 Victoria lineage
B/Malaysia/2506/2004 6.25 >100 >100 B/Shandong/7/1997 25
>100 >100 B/Victria/2/1987 25 >100 >100 Mouse-adapted
B/Ibaraki/2/1985 25 100 >100 HI (.mu.g/ml).sup.2
B/Florida/4/2006 25 0.39 0.39 Fusion inhibition (.mu.g/ml).sup.3
B/Florida/4/2006 100 25 25 .sup.1The results are shown as the
lowest concentrations of purified HuMAbs that inhibited 50% of
viral growth in vitro. .sup.2The results are shown as the lowest
concentrations of purified HuMAbs that completely inhibited
hemagglulination. .sup.3The results are shown as the lowest
concentrations of purified HuMAbs that showed cell fusion
inhibition.
[0124] Accordingly, all three HuMAbs had HI activity and also
inhibited cell-cell fusion (Table 4). However, 3A2 and 10C4 showed
markedly higher HI titers (0.39 mcg/ml) than 5A7 (25 mcg/ml). These
results indicate that all three HuMAbs should inhibit viral binding
to the cell membrane.
Example 2
[0125] To determine the epitope regions of B/Florida/4/2006
recognized by the three HuMAbs, escape mutants were selected. The
escape mutants were selected by the incubation of B/Florida/4/2006
with HuMAbs. Escape mutants were selected by culturing
B/Florida/4/2006 in the presence of MAb as described previously
(Gulati et al., Journal of Virology 76:12274-12280), with minor
modification. Viruses were incubated with MAb serially diluted
10-fold (to give final concentrations of 0.0025 to 2.5 mcg/ml) at
37 C. for 1 hour. Then MDCK cells were inoculated with the mixtures
and cultured in DMEM/F-12+GlutaMAX.TM.-I supplemented with 0.4%
BSA, antibiotics, and 2 mcg/ml acetylated trypsin. After culturing
for 72 hours, the supernatants were collected and subjected to VN
and HI assays. Those viral samples that showed a reduced VN.sub.50
and HI titer were subjected to direct sequencing analysis of the
entire HA gene.
[0126] Direct sequencing analysis was performed as follows. Viral
RNA extracted with QIAamp Viral RNA Mini Kit (Qiagen, Hilden,
Germany) was subjected to one step RT-PCR (Superscript.TM. III
One-Step RT-PCR System with Platinum.RTM. Taq High Fidelity;
Invitrogen) with the following HA primer set:
5'-CAGAATTCATGAAGGCAATAATTGTACTAC-3' forward (SEQ ID NO. 27) and
5'-CTCCGCGGCCGCTTATAGACAGATGGAGCATGAAACG-3' reverse (SEQ ID NO:
28). The PCR products were purified with Qiaquick PCR Purification
Kit (Qiagen). After electrophoresis, the discrete band was
extracted using the Qiaquick Gel Extraction Kit (Qiagen) and
sequenced.
[0127] HA plasmids were constructed as follows. The HA gene of
B/Florida/4/2006 was amplified by one step RT-PCR and inserted into
the pGEM-T Easy Vector (Promega, Madison, Wis.). Mutant and
truncated HA genes were generated by site-directed mutagenic PCR
(GeneTailor.TM. Site-Directed Mutagenesis System; Invitrogen) and
conventional PCR (Expand High Fidelity.sup.PLUS PCR System; Roche),
respectively, using the HA plasmid of B/Florida/4/2006 inserted
into pGEM-T easy vector. Each of the plasmids was subcloned into
the expression vector pCAGGS/MCSII (Ueda et al., Journal of
Virology 84: 3068-3078). The expression plasmids were transfected
into human embryonic kidney 293T cells with lipofectamine2000
(Invitrogen) according to the manufacturer's instructions.
[0128] The amino acid sequences of the HA protein in the escape
mutants were compared with the original B/Florida/4/2006. Asterisks
in FIG. 1 indicate the amino acid residues different between the
original virus and the escape mutants. Interestingly, each escape
mutant of 3A2 and 10C4 had amino acid substitutions at identical
positions 194D and 196T; amino acid numbering was started after the
signal peptide (Wang et al., Journal of Virology 82:3011-3020).
These positions are located at the 190-helix antigenic site near
the receptor-binding site (Wang et al., Journal of Virology
82:3011-3020). Remarkably, in the presence of serially diluted 5A7,
escape mutants were not established even after the virus was
passaged ten times, implying that the amino acid sequence
recognized by 5A7 was essential for viral survival. HuMAb 5A7
reacted to the HA0 protein by Western blotting under reducing
conditions, suggesting that 5A7 had a sequential epitope. Thus, the
epitope region of 5A7 was further investigated using HA truncation
vectors containing HA segments of varying length. Western blotting
with 5A7 was carried out on 293T cells transfected with the
truncated HA expression vectors. HuMAb 5A7 reacted with truncated
HA segments that included amino acid residues 1 to 324 but not to
those with residues 1 to 314 (FIG. 2). These results indicate that
5A7 recognizes amino acid residues between 315 to 324, IGNCPIWVKT
(SEQ. ID NO. 44) in the HA protein, which locates near the C
terminal of the HA 1 protein. Notably, this region is a highly
conserved domain in influenza B viruses.
Example 3
[0129] HuMAb 3A2 showed low reactivity against B/Shanghai/361/2002
and was therefore examined for an additional distinct epitope
region. To do this, various chimeric sequences of HA were
constructed from B/Florida/4/2006 and B/Shanghai/361/2002, which
differ at seven residues (positions 37, 40, 88, 131, 227, 249,
456), expressed in plasmids, and transfected into 293T cells. IFA
of the chimeric HA proteins expressed in 293T cells showed that
131P and 227S were essential for the reaction with 3A2 (FIG. 3).
These results indicate that the epitope of 3A2 is dependent on
residues at positions 131, 194, 196, and 227, and the epitope of
10C4 is dependent on residues at positions 194 and 196. The epitope
regions to which the three HuMAbs map are shown in a HA trimer
three-dimensional model in FIG. 4. HuMAbs 3A2 and 10C4 recognized
the top of the globular head including the 190-helix antigenic
site, whereas 5A7 reacted with the stalk region distant from the
viral membrane.
[0130] HuMAb, 5A7 recognizes the stalk region of the HA protein, as
do almost all broadly neutralizing MAbs for influenza A viruses.
The amino acid sequence in the stalk region is highly conserved,
implying that the amino acid residues would not easily mutate.
Indeed, the amino acid residues in the epitope region did not
mutate even when the virus was passaged ten times under
5A7-treatment conditions, whereas mutants developed quickly in the
presence of 3A2 or 10C4. Failure to establish escape mutants in the
presence of 5A7 is an advantage for this HuMAb as a therapeutic
candidate. Although 5A7 reacted with both of Yamagata and Victoria
lineages, the concentration required for VN.sub.50 was higher than
those of 3A2 and 10C4. Such results can be explained by either a
difference in binding affinity or in physical accessibility of
HuMAbs to the epitope region. Although binding affinity was not
examined in this study, accessibility can be estimated using
epitope mapping. HuMAbs 3A2 and 10C4 recognized the 190-helix site
distant from the viral membrane, whereas the epitope region of 5A7
localized to the stalk region, indicating that 5A7 would have more
difficulty physically accessing the HA protein. Modification of the
HuMAb structure and improvement of binding affinity should lead to
the development of better therapeutic antibodies.
[0131] MAbs recognizing the globular head show strong HI activity,
whereas those against the stalk region usually do not show any.
Thus, it is considered that MAbs against the globular head inhibit
the receptor binding step and that MAbs against the stalk region
inhibit the fusion step in viral replication. The HuMAbs that
recognize the 190-helix in the globular head near the receptor
binding site, 3A2 and 10C4, did in fact have strong HI activities,
suggesting that these HuMAbs inhibited binding to the receptor.
Surprisingly, 5A7 also showed weak HI activity, implying that 5A7
would also inhibit the binding step. It would be attributed that
5A7 recognizes the stalk region distal from the viral membrane,
whereas MAbs that do not show HI activity recognize a more proximal
position of the stalk. All three HuMAbs also showed fusion
inhibition activity. HuMAbs bound to the HA protein could
secondarily disturb the structural change of HA depending on low
pH.
Example 4
[0132] The activity of 5A7 as a passive transfer therapy in
influenza B viral infection was examined in mice. In the passive
transfer experiments, before infection, mice were anesthetized by
intraperitoneal administration of pentobarbital sodium
(Somnopentyl; Kyoritsu Seiyaku Corporation, Tokyo, Japan). Six-week
old female BALB/c mice from Japan SLC Inc. were used. Mice were
given 5, 10, or 15 mg/kg HuMAb intraperitoneally at 4, 24, 48, or
72 hours after challenge with 25 mcl (microlitre) mouse-adapted
B/Ibaraki/2/1985 virus at a lethal dose (2.5.times.10.sup.4
FFU/mouse). Mice were weighed daily and sacrificed if they fell to
60% of starting weight. To titrate the viruses in the infected
lungs, B/Ibaraki/2/1985 or B/Florida/4/2006 were infected at
2.5.times.10.sup.4 or 5.0.times.10.sup.3 FFU/mouse, respectively.
The lungs were harvested 3 and 6 days post-infection and virus
titers in lung homogenates were determined by focus-forming
assay.
[0133] Mice were treated intraperitoneally with 5, 10, or 15 mg/kg
5A7 at 4 hours post-challenge with a lethal dose of mouse-adapted
B/Ibaraki/2/1985. Survival rate and weight change were checked
daily and when the body weight had dropped to less than 60% of
starting weight, mice were sacrificed. Complete therapeutic
efficacy against the virus was seen with each dose of 5A7 tested
(FIG. 5). The weight change was particularly mild in the groups
treated with 5A7 at 10 or 15 mg/kg (FIG. 6). In mice treated with
10 mg/kg 5A7, the viral load in the lungs was titrated three and
six days post-infection with mouse-adapted B/Ibaraki/2/1985 or
B/Florida/4/2006. The viral titers were significantly lower in
5A7-treated mice compared to untreated controls for both viral
infections (FIG. 7). Finally, mice were given 5A7 at 10 mg/kg
intraperitoneally at 4, 24, 48, or 72 hours post-challenge with a
lethal dose of mouse-adapted B/Ibaraki/2/1985, and the survival
rate and weight change were monitored. When injected four hours
post-infection, 5A7 treatment showed complete therapeutic efficacy.
Notably, most of the mice were alive in the 5A7-treated group at 24
hours post-infection. Moreover, several mice survived even after
treatment with 5A7 more than 48 hours post-infection (FIGS. 8 and
9). These results indicate that 5A7 has the potential for
development as a therapy against influenza B viral infection.
[0134] Mouse-adapted B/Ibaraki/2/1985 was used to examine the
kinetics of survival rate and weight change in the passive transfer
experiments as other viral strains are not lethal to mice even if
passaged several times in vivo. HuMAb 5A7 protected mice against
mouse-adapted B/Ibaraki/2/1985 challenge even when administered 72
hours post-infection, although 5A7 had shown the lowest sensitivity
to mouse-adapted B/Ibaraki/2/1985 in vitro. These results suggested
that 5A7 would have therapeutic efficacy against a wide spectrum of
influenza B viruses, and in fact the lung viral titers of both
mouse-adapted B/Ibaraki/2/1985 and B/Florida/4/2006 were reduced
significantly under 5A7-treatment conditions.
Example 5
[0135] The neutralizing efficacy of CHO-K1-derived 5A7 was examined
in vitro. 5A7 was synthesized in CHO-K1 cells and examined for
neutralizing efficacy against influenza B viruses. CHO-K1 cells
were transfected with full-length variable region genes of 5A7 in
the pQC vector to establish a stable cell line secreting 5A7
(5A7/CHO-K1). For stable expression using mammalian cells, CHO-K1
cells were cultured in 5% CO.sub.2 at 37 deg C. in DMEM containing
10% fetal bovine serum. Cells grown on 6-well plates (Corning,
Corning, N.Y.) were used for transfection with pQCXIP-hCH and
pQCXIH-hC lambda expression vectors using Lipofectamine2000
transfection reagent (Invitrogen). Transfected cells were incubated
in 5% CO.sub.2 at 37 deg C. in DMEM with 1 mcg/ml of puromycin and
100 mcg/ml of hygromycin for 3 weeks. Cells were then replated to
15-cm dishes (Corning) and incubated, and colonies were picked and
cultured as putative cell lines stably expressing IgG. The
transformants at 90% confluence on 15-cm dishes had the medium
changed to serum-free Nutrient Mixture F-12 Ham's (Sigma), cells
were then cultured in 5% CO.sub.2 at 37 deg C. for 1 week.
Recombinant IgGs were purified from culture medium using HiTrap
Protein G HP Columns. The purified IgGs were dialyzed against PBS
and then concentrated using an Amicon Ultra Centrifugal Filter
(Millipore, Billerica, Mass.). In vitro viral neutralization tests
were performed using purified 5A7/CHO-K1, and neutralizing activity
was found against both the viral strains examined (B/Florida/4/2006
and B/Malaysia/2506/2004). Notably, the neutralizing activity of
5A7/CHO-K1 was similar to that of 5A7 produced by the hybridoma
(FIG. 10).
Example 6
[0136] HuMabs 5A7, 3A2 and 10C4 were subjected to surface plasmon
resonance analysis to examine their binding affinities. Each HuMab
was immobilized on the surface of the sensor chip. The vaccine
antigen, Ha protein of B/Florida/4/2006, at concentrations 12.5,
25, 50, 100 and 200 nM was consecutively injected on the chip
surface and the association and dissociation phases were monitored.
KD value could not be calculated for 5A7 precisely as it was
difficult to dissociate from HA (Table 5).
TABLE-US-00005 TABLE 5 Kinetic constants of HuMAbs binding to
influenza B virus-derived HA. kon.sup.1 (S.sup.-1) koff.sup.2
(M.sup.-1S.sup.-1) K.sub.D (M) 5A7 1.8 .times. 10.sup.3 <1.0
.times. 10.sup.-5 <5.6 .times. 10.sup.-9 3A2 5.3 .times.
10.sup.4 2.1 .times. 10.sup.-5 .sup. 4.0 .times. 10.sup.-10 10C4
1.6 .times. 10.sup.4 2.8 .times. 10.sup.-5 1.8 .times. 10.sup.-9
.sup.1Association rate constant. .sup.2Dissociation rate
constant.
[0137] Sequences of the V.sub.H and V.sub.L region of the three
HuMAbs were compared and analyzed to the closest germline sequences
using IgBlast software in NCBI database. These three HuMAbs were
derived from different germ lines except D region V.sub.H of 3A2
and 10C4 (FIG. 11-13).
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[0163] Applicants specifically incorporate the entire contents of
all cited references in this disclosure. Further, when an amount,
concentration, or other value or parameter is given as either a
range, preferred range, or a list of upper preferable values and
lower preferable values, this is to be understood as specifically
disclosing all ranges formed from any pair of any upper range limit
or preferred value and any lower range limit or preferred value,
regardless of whether ranges are separately disclosed. Where a
range of numerical values is recited herein, unless otherwise
stated, the range is intended to include the endpoints thereof, and
all integers and fractions within the range. It is not intended
that the scope of the invention be limited to the specific values
recited when defining a range.
[0164] Other embodiments of the present invention will be apparent
to those skilled in the art from consideration of the present
specification and practice of the present invention disclosed
herein. It is intended that the present specification and examples
be considered as exemplary only with a true scope and spirit of the
invention being indicated by the following claims and equivalents
thereof.
Sequence CWU 1
1
4415PRTArtificial SequenceAmino acid sequence of IgG (5A7 VH CDR1)
1Asn Tyr Gly Met His 1 5 217PRTArtificial SequenceAmino acid
sequence of IgG (5A7 VH CDR2) 2Val Val Trp Tyr Asp Gly Leu Ile Lys
Tyr Tyr Ala Asp Ser Val Lys 1 5 10 15 Gly 313PRTArtificial
SequenceAmino acid sequence of IgG (5A7 VH CDR3) 3Asp Leu Gln Pro
Pro His Ser Pro Tyr Gly Met Asp Val 1 5 10 413PRTArtificial
SequenceAmino acid sequence of IgG (5A7 VL CDR1) 4Ser Gly Ser Ser
Ser Asn Ile Gly Ser Asn Asp Val Tyr 1 5 10 57PRTArtificial
SequenceAmino acid sequence of IgG (5A7 VL CDR2) 5Asn Asn Asn Gln
Arg Pro Ser 1 5 610PRTArtificial SequenceAmino acid sequence of IgG
(5A7 VL CDR3) 6Ala Ala Trp Asp Asp Ser Leu Thr Val Ser 1 5 10
75PRTArtificial SequenceAmino acid sequence of IgG (3A2 VH CDR1)
7Ser Tyr Tyr Trp Ser 1 5 816PRTArtificial SequenceAmino acid
sequence of IgG (3A2 VH CDR2) 8Tyr Val Tyr Asn Ser Gly Ser Thr Arg
Tyr Asn Pro Ser Leu Lys Ser 1 5 10 15 920PRTArtificial
SequenceAmino acid sequence of IgG (3A2 VH CDR3) 9Ala Pro Asp Asp
Tyr Tyr Asp Ser Val Gly Tyr Tyr Tyr Gly Cys Pro 1 5 10 15 Tyr Phe
Asp Ser 20 1011PRTArtificial SequenceAmino acid sequence of IgG
(3A2 VL CDR1) 10Arg Ala Ser Pro Ser Ile Ala Asp Asn Leu Ala 1 5 10
117PRTArtificial SequenceAmino acid sequence of IgG (3A2 VL CDR2)
11Gly Ala Ser Thr Arg Ala Thr 1 5 129PRTArtificial SequenceAmino
acid sequence of IgG (3A2 VL CDR3) 12Gln Gln Tyr Ser Asn Trp Pro
Arg Thr 1 5 135PRTArtificial SequenceAmino acid sequence of IgG
(10C4 VH CDR1) 13Asn Tyr Ala Met Ser 1 5 1417PRTArtificial
SequenceAmino acid sequence of IgG (10C4 VH CDR2) 14Ala Ile Ser Gly
Gly Gly Asp Trp Thr Tyr Tyr Ala Asp Ser Val Lys 1 5 10 15 Gly
1524PRTArtificial SequenceAmino acid sequence of IgG (10C4 VH CDR3)
15Asp Val Thr Tyr Leu Tyr Asp Ser Ser Gly Tyr Tyr Tyr Gly Gly Ala 1
5 10 15 Asp Arg Asp Tyr Tyr Phe Asp Tyr 20 1613PRTArtificial
SequenceAmino acid sequence of IgG (10C4 VL CDR1) 16Ser Gly Gly Ser
Ser Asn Ile Gly Ser Asn Tyr Val Asn 1 5 10 177PRTArtificial
SequenceAmino acid sequence of IgG (10C4 VL CDR2) 17Ser Asn Asn Gln
Arg Pro Leu 1 5 1811PRTArtificial SequenceAmino acid sequence of
IgG (10C4 VL CDR3) 18Gln Gln Trp Asp Asp Ser Leu Asn Gly Trp Val 1
5 10 1924DNAArtificial SequencePrimer sequence of H-chain-forward
19atggagtttg ggctgagctg ggtt 242025DNAArtificial SequencePrimer
sequence of H-chain-reverse 20ctcccgcggc tttgtcttgg catta
252124DNAArtificial SequencePrimer sequence of L-chain-forward
21atggcctggr ycycmytcyw cctm 242225DNAArtificial SequencePrimer
sequence of L-chain-reverse 22tggcagctgt agcttctgtg ggact
252330DNAArtificial SequencePrimer sequence of HC_5Fw_NotI;
H-chain-forward 23atttgcggcc gccatggagt ttgggctgag
302431DNAArtificial SequencePrimer sequence of HC_Reverse_XhoI;
H-chain- reverse 24atactcgagg gtgccagggg gaagaccgat g
312530DNAArtificial SequencePrimer sequence of 5A7Lambda_18Fw_NotI;
L-chain-forward 25atttgcggcc gccatggcct gggctctgct
302631DNAArtificial SequencePrimer sequence of Lambda_Reverse_XhoI;
L-chain-reverse 26atactcgagg gcgggaacag agtgaccgtg g
312730DNAArtificial SequencePrimer sequence of HA primer forward
27cagaattcat gaaggcaata attgtactac 302837DNAArtificial
SequencePrimer sequence of HA primer reverse 28ctccgcggcc
gcttatagac agatggagca tgaaacg 3729569PRTInfluenza B virus 29Asp Arg
Ile Cys Thr Gly Ile Thr Ser Ser Asn Ser Pro His Val Val 1 5 10 15
Lys Thr Ala Thr Gln Gly Glu Val Asn Val Thr Gly Val Ile Pro Leu 20
25 30 Thr Thr Thr Pro Thr Lys Ser Tyr Phe Ala Asn Leu Lys Gly Thr
Arg 35 40 45 Thr Arg Gly Lys Leu Cys Pro Asp Cys Leu Asn Cys Thr
Asp Leu Asp 50 55 60 Val Ala Leu Gly Arg Pro Met Cys Val Gly Thr
Thr Pro Ser Ala Lys 65 70 75 80 Ala Ser Ile Leu His Glu Val Lys Pro
Val Thr Ser Gly Cys Phe Pro 85 90 95 Ile Met His Asp Arg Thr Lys
Ile Arg Gln Leu Pro Asn Leu Leu Arg 100 105 110 Gly Tyr Glu Asn Ile
Arg Leu Ser Thr Gln Asn Val Ile Asp Ala Glu 115 120 125 Lys Ala Pro
Gly Gly Pro Tyr Arg Leu Gly Thr Ser Gly Ser Cys Pro 130 135 140 Asn
Ala Thr Ser Lys Ser Gly Phe Phe Ala Thr Met Ala Trp Ala Val 145 150
155 160 Pro Lys Asp Asn Asn Lys Asn Ala Thr Asn Pro Leu Thr Val Glu
Val 165 170 175 Pro Tyr Ile Cys Thr Glu Gly Glu Asp Gln Ile Thr Val
Trp Gly Phe 180 185 190 His Ser Asp Asp Lys Thr Gln Met Lys Asn Leu
Tyr Gly Asp Ser Asn 195 200 205 Pro Gln Lys Phe Thr Ser Ser Ala Asn
Gly Val Thr Thr His Tyr Val 210 215 220 Ser Gln Ile Gly Ser Phe Pro
Asp Gln Thr Glu Asp Gly Gly Leu Pro 225 230 235 240 Gln Ser Gly Arg
Ile Val Val Asp Tyr Met Met Gln Lys Pro Gly Lys 245 250 255 Thr Gly
Thr Ile Val Tyr Gln Arg Gly Val Leu Leu Pro Gln Lys Val 260 265 270
Trp Cys Ala Ser Gly Arg Ser Lys Val Ile Lys Gly Ser Leu Pro Leu 275
280 285 Ile Gly Glu Ala Asp Cys Leu His Glu Lys Tyr Gly Gly Leu Asn
Lys 290 295 300 Ser Lys Pro Tyr Tyr Thr Gly Glu His Ala Lys Ala Ile
Gly Asn Cys 305 310 315 320 Pro Ile Trp Val Lys Thr Pro Leu Lys Leu
Ala Asn Gly Thr Lys Tyr 325 330 335 Arg Pro Pro Ala Lys Leu Leu Lys
Glu Arg Gly Phe Phe Gly Ala Ile 340 345 350 Ala Gly Phe Leu Glu Gly
Gly Trp Glu Gly Met Ile Ala Gly Trp His 355 360 365 Gly Tyr Thr Ser
His Gly Ala His Gly Val Ala Val Ala Ala Asp Leu 370 375 380 Lys Ser
Thr Gln Glu Ala Ile Asn Lys Ile Thr Lys Asn Leu Asn Ser 385 390 395
400 Leu Ser Glu Leu Glu Val Lys Asn Leu Gln Arg Leu Ser Gly Ala Met
405 410 415 Asp Glu Leu His Asn Glu Ile Leu Glu Leu Asp Glu Lys Val
Asp Asp 420 425 430 Leu Arg Ala Asp Thr Ile Ser Ser Gln Ile Glu Leu
Ala Val Leu Leu 435 440 445 Ser Asn Glu Gly Ile Ile Asn Ser Glu Asp
Glu His Leu Leu Ala Leu 450 455 460 Glu Arg Lys Leu Lys Lys Met Leu
Gly Pro Ser Ala Val Glu Ile Gly 465 470 475 480 Asn Gly Cys Phe Glu
Thr Lys His Lys Cys Asn Gln Thr Cys Leu Asp 485 490 495 Arg Ile Ala
Ala Gly Thr Phe Asn Ala Gly Glu Phe Ser Leu Pro Thr 500 505 510 Phe
Asp Ser Leu Asn Ile Thr Ala Ala Ser Leu Asn Asp Asp Gly Leu 515 520
525 Asp Asn His Thr Ile Leu Leu Tyr Tyr Ser Thr Ala Ala Ser Ser Leu
530 535 540 Ala Val Thr Leu Met Leu Ala Ile Phe Ile Val Tyr Met Val
Ser Arg 545 550 555 560 Asp Asn Val Ser Cys Ser Ile Cys Leu 565
30552PRTInfluenza B virus 30Arg Ile Cys Thr Gly Ile Thr Ser Ser Asn
Ser Pro His Val Val Lys 1 5 10 15 Thr Ala Thr Gln Gly Glu Val Asn
Val Thr Gly Val Ile Pro Leu Thr 20 25 30 Thr Thr Pro Thr Lys Ser
Tyr Phe Ala Asn Leu Lys Gly Thr Arg Thr 35 40 45 Arg Gly Lys Leu
Cys Pro Asp Cys Leu Asn Cys Thr Asp Leu Asp Val 50 55 60 Ala Leu
Gly Arg Pro Met Cys Val Gly Thr Thr Pro Ser Ala Lys Ala 65 70 75 80
Ser Ile Leu His Glu Val Lys Pro Val Thr Ser Gly Cys Phe Pro Ile 85
90 95 Met His Asp Arg Thr Lys Ile Arg Gln Leu Pro Asn Leu Leu Arg
Gly 100 105 110 Tyr Glu Asn Ile Arg Leu Ser Thr Gln Asn Val Ile Asp
Ala Glu Lys 115 120 125 Ala Pro Gly Gly Pro Tyr Arg Leu Gly Thr Ser
Gly Ser Cys Pro Asn 130 135 140 Ala Thr Ser Lys Ser Gly Phe Phe Ala
Thr Met Ala Trp Ala Val Pro 145 150 155 160 Lys Asp Asn Asn Lys Asn
Ala Thr Asn Pro Leu Thr Val Glu Val Pro 165 170 175 Tyr Ile Cys Thr
Glu Gly Glu Asp Gln Ile Thr Val Trp Gly Phe His 180 185 190 Ser Asp
Asn Lys Ile Gln Met Lys Asn Leu Tyr Gly Asp Ser Asn Pro 195 200 205
Gln Lys Phe Thr Ser Ser Ala Asn Gly Val Thr Thr His Tyr Val Ser 210
215 220 Gln Ile Gly Ser Phe Pro Asp Gln Thr Glu Asp Gly Gly Leu Pro
Gln 225 230 235 240 Ser Gly Arg Ile Val Val Asp Tyr Met Met Gln Lys
Pro Gly Lys Thr 245 250 255 Gly Thr Ile Val Tyr Gln Arg Gly Val Leu
Leu Pro Gln Lys Val Trp 260 265 270 Cys Ala Ser Gly Arg Ser Lys Val
Ile Lys Gly Ser Leu Pro Leu Ile 275 280 285 Gly Glu Ala Asp Cys Leu
His Glu Lys Tyr Gly Gly Leu Asn Lys Ser 290 295 300 Lys Pro Tyr Tyr
Thr Gly Glu His Ala Lys Ala Ile Gly Asn Cys Pro 305 310 315 320 Ile
Trp Val Lys Thr Pro Leu Lys Leu Ala Asn Gly Thr Lys Tyr Arg 325 330
335 Pro Pro Ala Lys Leu Leu Lys Glu Arg Gly Phe Phe Gly Ala Ile Ala
340 345 350 Gly Phe Leu Glu Gly Gly Trp Glu Gly Met Ile Ala Gly Trp
His Gly 355 360 365 Tyr Thr Ser His Gly Ala His Gly Val Ala Val Ala
Ala Asp Leu Lys 370 375 380 Ser Thr Gln Glu Ala Ile Asn Lys Ile Thr
Lys Asn Leu Asn Ser Leu 385 390 395 400 Ser Glu Leu Glu Val Lys Asn
Leu Gln Arg Leu Ser Gly Ala Met Asp 405 410 415 Glu Leu His Asn Glu
Ile Leu Glu Leu Asp Glu Lys Val Asp Asp Leu 420 425 430 Arg Ala Asp
Thr Ile Ser Ser Gln Ile Glu Leu Ala Val Leu Leu Ser 435 440 445 Asn
Glu Gly Ile Ile Asn Ser Glu Asp Glu His Leu Leu Ala Leu Glu 450 455
460 Arg Lys Leu Lys Lys Met Leu Gly Pro Ser Ala Val Glu Ile Gly Asn
465 470 475 480 Gly Cys Phe Glu Thr Lys His Lys Cys Asn Gln Thr Cys
Leu Asp Arg 485 490 495 Ile Ala Ala Gly Thr Phe Asn Ala Gly Glu Phe
Ser Leu Pro Thr Phe 500 505 510 Asp Ser Leu Asn Ile Thr Ala Ala Ser
Leu Asn Asp Asp Gly Leu Asp 515 520 525 Asn His Thr Ile Leu Leu Tyr
Tyr Ser Thr Ala Ala Ser Ser Leu Ala 530 535 540 Val Thr Leu Met Leu
Ala Ile Phe 545 550 31552PRTInfluenza B virus 31Arg Ile Cys Thr Gly
Ile Thr Ser Ser Asn Ser Pro His Val Val Lys 1 5 10 15 Thr Ala Thr
Gln Gly Glu Val Asn Val Thr Gly Val Ile Pro Leu Thr 20 25 30 Thr
Thr Pro Thr Lys Ser Tyr Phe Ala Asn Leu Lys Gly Thr Arg Thr 35 40
45 Arg Gly Lys Leu Cys Pro Asp Cys Leu Asn Cys Thr Asp Leu Asp Val
50 55 60 Ala Leu Gly Arg Pro Met Cys Val Gly Thr Thr Pro Ser Ala
Lys Ala 65 70 75 80 Ser Ile Leu His Glu Val Lys Pro Val Thr Ser Gly
Cys Phe Pro Ile 85 90 95 Met His Asp Arg Thr Lys Ile Arg Gln Leu
Pro Asn Leu Leu Arg Gly 100 105 110 Tyr Glu Asn Ile Arg Leu Ser Thr
Gln Asn Val Ile Asp Ala Glu Lys 115 120 125 Ala Pro Gly Gly Pro Tyr
Arg Leu Gly Thr Ser Gly Ser Cys Pro Asn 130 135 140 Ala Thr Ser Lys
Ser Gly Phe Phe Ala Thr Met Ala Trp Ala Val Pro 145 150 155 160 Lys
Asp Asn Asn Lys Asn Ala Thr Asn Pro Leu Thr Val Glu Val Pro 165 170
175 Tyr Ile Cys Thr Glu Gly Glu Asp Gln Ile Thr Val Trp Gly Phe His
180 185 190 Ser Asp Asn Lys Asn Gln Met Lys Asn Leu Tyr Gly Asp Ser
Asn Pro 195 200 205 Gln Lys Phe Thr Ser Ser Ala Asn Gly Val Thr Thr
His Tyr Val Ser 210 215 220 Gln Ile Gly Ser Phe Pro Asp Gln Thr Glu
Asp Gly Gly Leu Pro Gln 225 230 235 240 Ser Gly Arg Ile Val Val Asp
Tyr Met Met Gln Lys Pro Gly Lys Thr 245 250 255 Gly Thr Ile Val Tyr
Gln Arg Gly Val Leu Leu Pro Gln Lys Val Trp 260 265 270 Cys Ala Ser
Gly Arg Ser Lys Val Ile Lys Gly Ser Leu Pro Leu Ile 275 280 285 Gly
Glu Ala Asp Cys Leu His Glu Lys Tyr Gly Gly Leu Asn Lys Ser 290 295
300 Lys Pro Tyr Tyr Thr Gly Glu His Ala Lys Ala Ile Gly Asn Cys Pro
305 310 315 320 Ile Trp Val Lys Thr Pro Leu Lys Leu Ala Asn Gly Thr
Lys Tyr Arg 325 330 335 Pro Pro Ala Lys Leu Leu Lys Glu Arg Gly Phe
Phe Gly Ala Ile Ala 340 345 350 Gly Phe Leu Glu Gly Gly Trp Glu Gly
Met Ile Ala Gly Trp His Gly 355 360 365 Tyr Thr Ser His Gly Ala His
Gly Val Ala Val Ala Ala Asp Leu Lys 370 375 380 Ser Thr Gln Glu Ala
Ile Asn Lys Ile Thr Lys Asn Leu Asn Ser Leu 385 390 395 400 Ser Glu
Leu Glu Val Lys Asn Leu Gln Arg Leu Ser Gly Ala Met Asp 405 410 415
Glu Leu His Asn Glu Ile Leu Glu Leu Asp Glu Lys Val Asp Asp Leu 420
425 430 Arg Ala Asp Thr Ile Ser Ser Gln Ile Glu Leu Ala Val Leu Leu
Ser 435 440 445 Asn Glu Gly Ile Ile Asn Ser Glu Asp Glu His Leu Leu
Ala Leu Glu 450 455 460 Arg Lys Leu Lys Lys Met Leu Gly Pro Ser Ala
Val Glu Ile Gly Asn 465 470 475 480 Gly Cys Phe Glu Thr Lys His Lys
Cys Asn Gln Thr Cys Leu Asp Arg 485 490 495 Ile Ala Ala Gly Thr Phe
Asn Ala Gly Glu Phe Ser Leu Pro Thr Phe 500 505 510 Asp Ser Leu Asn
Ile Thr Ala Ala Ser Leu Asn Asp Asp Gly Leu Asp 515 520 525 Asn His
Thr Ile Leu Leu Tyr Tyr Ser Thr Ala Ala Ser Ser Leu Ala 530 535 540
Val Thr Leu Met Leu Ala Ile Phe 545 550
32441DNAArtificialCDS(1)..(441)The variable region of the heavy
chain (5A7) 32atg aaa cac ctg tgg ttc ttc ctc ctc ctg gtg gca gct
ccc aga tgg 48Met Lys His Leu Trp Phe Phe Leu Leu Leu Val Ala Ala
Pro Arg Trp 1 5 10 15 gtc ctg tcc cag gtg cag ctg gtg gag tcg ggc
cca gga ctg gtg aag 96Val Leu Ser Gln Val Gln Leu Val Glu Ser Gly
Pro Gly Leu Val Lys 20 25 30
cct tct gag acc ctg tcc ctc acc tgc act gtc tct agt ggc tcc atc
144Pro Ser Glu Thr Leu Ser Leu Thr Cys Thr Val Ser Ser Gly Ser Ile
35 40 45 agt agt tac tac tgg agc tgg atc cgg cag ccc ccc ggg aag
gga ctg 192Ser Ser Tyr Tyr Trp Ser Trp Ile Arg Gln Pro Pro Gly Lys
Gly Leu 50 55 60 gag tgg att ggg tat gtc tat aac agt ggg agt acc
agg tac aac ccc 240Glu Trp Ile Gly Tyr Val Tyr Asn Ser Gly Ser Thr
Arg Tyr Asn Pro 65 70 75 80 tcc ctc aag agt cgc ctc acc atg tca gtg
gac gcg tcc agg aag cag 288Ser Leu Lys Ser Arg Leu Thr Met Ser Val
Asp Ala Ser Arg Lys Gln 85 90 95 gtc tcc ctg aag ttg agc tct gtg
agt gct gcg gac acg gcc gtg tat 336Val Ser Leu Lys Leu Ser Ser Val
Ser Ala Ala Asp Thr Ala Val Tyr 100 105 110 tac tgt gcg aga gcc ccg
gac gat tac tat gat agt gtt ggt tat tac 384Tyr Cys Ala Arg Ala Pro
Asp Asp Tyr Tyr Asp Ser Val Gly Tyr Tyr 115 120 125 tac gga tgt ccg
tac ttc gac tcc tgg ggc cag gga acc ctg gtc acc 432Tyr Gly Cys Pro
Tyr Phe Asp Ser Trp Gly Gln Gly Thr Leu Val Thr 130 135 140 gtc tcc
tca 441Val Ser Ser 145 33147PRTArtificialThe variable region of the
heavy chain (5A7) 33Met Lys His Leu Trp Phe Phe Leu Leu Leu Val Ala
Ala Pro Arg Trp 1 5 10 15 Val Leu Ser Gln Val Gln Leu Val Glu Ser
Gly Pro Gly Leu Val Lys 20 25 30 Pro Ser Glu Thr Leu Ser Leu Thr
Cys Thr Val Ser Ser Gly Ser Ile 35 40 45 Ser Ser Tyr Tyr Trp Ser
Trp Ile Arg Gln Pro Pro Gly Lys Gly Leu 50 55 60 Glu Trp Ile Gly
Tyr Val Tyr Asn Ser Gly Ser Thr Arg Tyr Asn Pro 65 70 75 80 Ser Leu
Lys Ser Arg Leu Thr Met Ser Val Asp Ala Ser Arg Lys Gln 85 90 95
Val Ser Leu Lys Leu Ser Ser Val Ser Ala Ala Asp Thr Ala Val Tyr 100
105 110 Tyr Cys Ala Arg Ala Pro Asp Asp Tyr Tyr Asp Ser Val Gly Tyr
Tyr 115 120 125 Tyr Gly Cys Pro Tyr Phe Asp Ser Trp Gly Gln Gly Thr
Leu Val Thr 130 135 140 Val Ser Ser 145
34381DNAArtificialCDS(1)..(381)The variable region of the lamda
chain (5A7) 34atg gaa gcc cca gcg cag ctt ctc ttc ctc ctg cta ctc
tgg ctc cca 48Met Glu Ala Pro Ala Gln Leu Leu Phe Leu Leu Leu Leu
Trp Leu Pro 1 5 10 15 gat acc agt gga gaa ata ggg atg acg cag tct
cca gcc acc ctg tct 96Asp Thr Ser Gly Glu Ile Gly Met Thr Gln Ser
Pro Ala Thr Leu Ser 20 25 30 gtg tct cca ggg gaa aga gcc acc ctc
ttt tgc agg gcc agt ccg agt 144Val Ser Pro Gly Glu Arg Ala Thr Leu
Phe Cys Arg Ala Ser Pro Ser 35 40 45 att agc gac aac tta gcc tgg
tac cag cag aaa cct ggc cag gct ccc 192Ile Ser Asp Asn Leu Ala Trp
Tyr Gln Gln Lys Pro Gly Gln Ala Pro 50 55 60 agg ctc ctc ttc tat
ggt gca tcc acc agg gcc act ggt atc cca gcc 240Arg Leu Leu Phe Tyr
Gly Ala Ser Thr Arg Ala Thr Gly Ile Pro Ala 65 70 75 80 agg ttc agc
ggc agt ggg tct ggg aca gag ttc act ctc acc atc agc 288Arg Phe Ser
Gly Ser Gly Ser Gly Thr Glu Phe Thr Leu Thr Ile Ser 85 90 95 agt
gtg cag tct gaa gat att gga gtt tat tat tgt cag cag tat agt 336Ser
Val Gln Ser Glu Asp Ile Gly Val Tyr Tyr Cys Gln Gln Tyr Ser 100 105
110 aac tgg cct cgt act ttt ggc cag ggg acc aag ctg cag atc aaa
381Asn Trp Pro Arg Thr Phe Gly Gln Gly Thr Lys Leu Gln Ile Lys 115
120 125 35127PRTArtificialThe variable region of the lamda chain
(5A7) 35Met Glu Ala Pro Ala Gln Leu Leu Phe Leu Leu Leu Leu Trp Leu
Pro 1 5 10 15 Asp Thr Ser Gly Glu Ile Gly Met Thr Gln Ser Pro Ala
Thr Leu Ser 20 25 30 Val Ser Pro Gly Glu Arg Ala Thr Leu Phe Cys
Arg Ala Ser Pro Ser 35 40 45 Ile Ser Asp Asn Leu Ala Trp Tyr Gln
Gln Lys Pro Gly Gln Ala Pro 50 55 60 Arg Leu Leu Phe Tyr Gly Ala
Ser Thr Arg Ala Thr Gly Ile Pro Ala 65 70 75 80 Arg Phe Ser Gly Ser
Gly Ser Gly Thr Glu Phe Thr Leu Thr Ile Ser 85 90 95 Ser Val Gln
Ser Glu Asp Ile Gly Val Tyr Tyr Cys Gln Gln Tyr Ser 100 105 110 Asn
Trp Pro Arg Thr Phe Gly Gln Gly Thr Lys Leu Gln Ile Lys 115 120 125
36423DNAArtificialCDS(1)..(423)The variable region of the heavy
chain (3A2) 36atg gag ttt ggg ctg agc tgg gtt ctc ctc gtt gct ctt
tta aga ggt 48Met Glu Phe Gly Leu Ser Trp Val Leu Leu Val Ala Leu
Leu Arg Gly 1 5 10 15 gtc cag tgt cag gtg cag ctg gtg gag tct ggg
gga gac gtg gtc caa 96Val Gln Cys Gln Val Gln Leu Val Glu Ser Gly
Gly Asp Val Val Gln 20 25 30 cct ggg agg tcc ctg aga ctc tcc tgc
gca gcg tct gga ttc acc ttc 144Pro Gly Arg Ser Leu Arg Leu Ser Cys
Ala Ala Ser Gly Phe Thr Phe 35 40 45 aat aac tat ggc atg cac tgg
gtc cgc cag gct cca ggc aag ggg ctg 192Asn Asn Tyr Gly Met His Trp
Val Arg Gln Ala Pro Gly Lys Gly Leu 50 55 60 gag tgg gtg gca gtt
gtc tgg tat gat gga ctt att aaa tac tat gcg 240Glu Trp Val Ala Val
Val Trp Tyr Asp Gly Leu Ile Lys Tyr Tyr Ala 65 70 75 80 gac tcc gtg
aag ggc cga ttc acc atc tcc aga gac aat tcg aaa aac 288Asp Ser Val
Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn 85 90 95 acc
ctg tat ctg caa atg aac acc ctg aga gcc gag gac atg ggt gtc 336Thr
Leu Tyr Leu Gln Met Asn Thr Leu Arg Ala Glu Asp Met Gly Val 100 105
110 tat tac tgt gcg aga gat cta cag cct ccc cat tca ccc tac ggt atg
384Tyr Tyr Cys Ala Arg Asp Leu Gln Pro Pro His Ser Pro Tyr Gly Met
115 120 125 gac gtc tgg ggc caa ggg acc acg gtc acc gtc tcc tca
423Asp Val Trp Gly Gln Gly Thr Thr Val Thr Val Ser Ser 130 135 140
37141PRTArtificialThe variable region of the heavy chain (3A2)
37Met Glu Phe Gly Leu Ser Trp Val Leu Leu Val Ala Leu Leu Arg Gly 1
5 10 15 Val Gln Cys Gln Val Gln Leu Val Glu Ser Gly Gly Asp Val Val
Gln 20 25 30 Pro Gly Arg Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly
Phe Thr Phe 35 40 45 Asn Asn Tyr Gly Met His Trp Val Arg Gln Ala
Pro Gly Lys Gly Leu 50 55 60 Glu Trp Val Ala Val Val Trp Tyr Asp
Gly Leu Ile Lys Tyr Tyr Ala 65 70 75 80 Asp Ser Val Lys Gly Arg Phe
Thr Ile Ser Arg Asp Asn Ser Lys Asn 85 90 95 Thr Leu Tyr Leu Gln
Met Asn Thr Leu Arg Ala Glu Asp Met Gly Val 100 105 110 Tyr Tyr Cys
Ala Arg Asp Leu Gln Pro Pro His Ser Pro Tyr Gly Met 115 120 125 Asp
Val Trp Gly Gln Gly Thr Thr Val Thr Val Ser Ser 130 135 140
38387DNAArtificialCDS(1)..(387)The variable region of the kappa
chain (3A2) 38atg gcc tgg gtc tca ttc tac ctc acc ctc ctc act cac
tgt gca ggg 48Met Ala Trp Val Ser Phe Tyr Leu Thr Leu Leu Thr His
Cys Ala Gly 1 5 10 15 tcc tgg gcc cag tct gtg ctg act cag cca ccc
tca gcg tct ggg acc 96Ser Trp Ala Gln Ser Val Leu Thr Gln Pro Pro
Ser Ala Ser Gly Thr 20 25 30 ccc ggg cag agg gtc acc atc tct tgt
tct gga agc agc tcc aac atc 144Pro Gly Gln Arg Val Thr Ile Ser Cys
Ser Gly Ser Ser Ser Asn Ile 35 40 45 gga agt aat gat gtc tat tgg
tac cag aac ctc cca gga acg gcc ccc 192Gly Ser Asn Asp Val Tyr Trp
Tyr Gln Asn Leu Pro Gly Thr Ala Pro 50 55 60 aaa ctc ctc atc tat
aat aat aat caa cgg ccc tca ggg gtc cct gac 240Lys Leu Leu Ile Tyr
Asn Asn Asn Gln Arg Pro Ser Gly Val Pro Asp 65 70 75 80 cga ttc tct
ggc tcc aag tct ggc acc tca gcc tcc ctg gcc atc agt 288Arg Phe Ser
Gly Ser Lys Ser Gly Thr Ser Ala Ser Leu Ala Ile Ser 85 90 95 ggg
ctc cgg tcc gag gat gag gct gat tat tat tgt gca gca tgg gat 336Gly
Leu Arg Ser Glu Asp Glu Ala Asp Tyr Tyr Cys Ala Ala Trp Asp 100 105
110 gac agc ctg act gtc tcc ttc gga act ggg acc aag gtc acc gtc cta
384Asp Ser Leu Thr Val Ser Phe Gly Thr Gly Thr Lys Val Thr Val Leu
115 120 125 ggt 387Gly 39129PRTArtificialThe variable region of the
kappa chain (3A2) 39Met Ala Trp Val Ser Phe Tyr Leu Thr Leu Leu Thr
His Cys Ala Gly 1 5 10 15 Ser Trp Ala Gln Ser Val Leu Thr Gln Pro
Pro Ser Ala Ser Gly Thr 20 25 30 Pro Gly Gln Arg Val Thr Ile Ser
Cys Ser Gly Ser Ser Ser Asn Ile 35 40 45 Gly Ser Asn Asp Val Tyr
Trp Tyr Gln Asn Leu Pro Gly Thr Ala Pro 50 55 60 Lys Leu Leu Ile
Tyr Asn Asn Asn Gln Arg Pro Ser Gly Val Pro Asp 65 70 75 80 Arg Phe
Ser Gly Ser Lys Ser Gly Thr Ser Ala Ser Leu Ala Ile Ser 85 90 95
Gly Leu Arg Ser Glu Asp Glu Ala Asp Tyr Tyr Cys Ala Ala Trp Asp 100
105 110 Asp Ser Leu Thr Val Ser Phe Gly Thr Gly Thr Lys Val Thr Val
Leu 115 120 125 Gly 40456DNAArtificialCDS(1)..(456)The variable
region of the heavy chain (10C4) 40atg gag ttt ggg ctg agc tgg ctt
ttt ctt gtg gct att tta aaa ggt 48Met Glu Phe Gly Leu Ser Trp Leu
Phe Leu Val Ala Ile Leu Lys Gly 1 5 10 15 gtc cag tgt gag gtg cag
ctg ttg gag tct ggg gga ggc ttg gtc cag 96Val Gln Cys Glu Val Gln
Leu Leu Glu Ser Gly Gly Gly Leu Val Gln 20 25 30 ccg ggg ggg tcc
ctg aga ctc tcc tgt gca gcc tct gga ttc acc ttt 144Pro Gly Gly Ser
Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe 35 40 45 agc aac
tat gcc atg agc tgg gtc cgc cag gct cca ggg aag ggg ctg 192Ser Asn
Tyr Ala Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu 50 55 60
gag tgg gtc tca gct att agt ggt ggt ggt gat tgg aca tac tac gca
240Glu Trp Val Ser Ala Ile Ser Gly Gly Gly Asp Trp Thr Tyr Tyr Ala
65 70 75 80 gac tcc gtg aag ggc cga ttc tcc atc tcc agc gac aat tcc
aag aac 288Asp Ser Val Lys Gly Arg Phe Ser Ile Ser Ser Asp Asn Ser
Lys Asn 85 90 95 acg ctg tat ctg caa atg aac agc ctg aga gcc gag
gac acg gcc gta 336Thr Leu Tyr Leu Gln Met Asn Ser Leu Arg Ala Glu
Asp Thr Ala Val 100 105 110 tat tac tgt gcg aaa gat gtc acg tat ttg
tat gac agt agt ggt tat 384Tyr Tyr Cys Ala Lys Asp Val Thr Tyr Leu
Tyr Asp Ser Ser Gly Tyr 115 120 125 tac tac ggg gga gcc gac cgc gat
tat tac ttt gac tac tgg ggc cag 432Tyr Tyr Gly Gly Ala Asp Arg Asp
Tyr Tyr Phe Asp Tyr Trp Gly Gln 130 135 140 gga acc ctg gtc acc gtc
tcc tca 456Gly Thr Leu Val Thr Val Ser Ser 145 150
41152PRTArtificialThe variable region of the heavy chain (10C4)
41Met Glu Phe Gly Leu Ser Trp Leu Phe Leu Val Ala Ile Leu Lys Gly 1
5 10 15 Val Gln Cys Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val
Gln 20 25 30 Pro Gly Gly Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly
Phe Thr Phe 35 40 45 Ser Asn Tyr Ala Met Ser Trp Val Arg Gln Ala
Pro Gly Lys Gly Leu 50 55 60 Glu Trp Val Ser Ala Ile Ser Gly Gly
Gly Asp Trp Thr Tyr Tyr Ala 65 70 75 80 Asp Ser Val Lys Gly Arg Phe
Ser Ile Ser Ser Asp Asn Ser Lys Asn 85 90 95 Thr Leu Tyr Leu Gln
Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val 100 105 110 Tyr Tyr Cys
Ala Lys Asp Val Thr Tyr Leu Tyr Asp Ser Ser Gly Tyr 115 120 125 Tyr
Tyr Gly Gly Ala Asp Arg Asp Tyr Tyr Phe Asp Tyr Trp Gly Gln 130 135
140 Gly Thr Leu Val Thr Val Ser Ser 145 150
42390DNAArtificialCDS(1)..(390)The variable region of the lamda
chain (10C4) 42atg ccc tgg gct ctt ctc ctc ctc acc ctc ctc act cac
tgt gca ggg 48Met Pro Trp Ala Leu Leu Leu Leu Thr Leu Leu Thr His
Cys Ala Gly 1 5 10 15 tcc tgg gcc cag tct gtg ctg act cag cca ccc
tca gcg tct ggg acc 96Ser Trp Ala Gln Ser Val Leu Thr Gln Pro Pro
Ser Ala Ser Gly Thr 20 25 30 ccc ggg cag agg gtc tcc atc tct tgt
tct gga ggc agc tcc aac atc 144Pro Gly Gln Arg Val Ser Ile Ser Cys
Ser Gly Gly Ser Ser Asn Ile 35 40 45 gga agt aat act gta aac tgg
tac cag cag ctc cca gga acg gcc ccc 192Gly Ser Asn Thr Val Asn Trp
Tyr Gln Gln Leu Pro Gly Thr Ala Pro 50 55 60 aga ctc ctc atc tat
agc aat aat cag cgg ccc tta ggg gtc cct gac 240Arg Leu Leu Ile Tyr
Ser Asn Asn Gln Arg Pro Leu Gly Val Pro Asp 65 70 75 80 cga ttc tct
gag tcc aag tct ggc acc tca gcc tcc ctg gcc atc agt 288Arg Phe Ser
Glu Ser Lys Ser Gly Thr Ser Ala Ser Leu Ala Ile Ser 85 90 95 ggg
ctc cgg tct gag gat gag gct gat tat tac tgt gct gca tgg gat 336Gly
Leu Arg Ser Glu Asp Glu Ala Asp Tyr Tyr Cys Ala Ala Trp Asp 100 105
110 gac agc ctg aat ggt tgg gtg ttc ggc gga ggg acc agg ctg acc gtc
384Asp Ser Leu Asn Gly Trp Val Phe Gly Gly Gly Thr Arg Leu Thr Val
115 120 125 cta ggt 390Leu Gly 130 43130PRTArtificialThe variable
region of the lamda chain (10C4) 43Met Pro Trp Ala Leu Leu Leu Leu
Thr Leu Leu Thr His Cys Ala Gly 1 5 10 15 Ser Trp Ala Gln Ser Val
Leu Thr Gln Pro Pro Ser Ala Ser Gly Thr 20 25 30 Pro Gly Gln Arg
Val Ser Ile Ser Cys Ser Gly Gly Ser Ser Asn Ile 35 40 45 Gly Ser
Asn Thr Val Asn Trp Tyr Gln Gln Leu Pro Gly Thr Ala Pro
50 55 60 Arg Leu Leu Ile Tyr Ser Asn Asn Gln Arg Pro Leu Gly Val
Pro Asp 65 70 75 80 Arg Phe Ser Glu Ser Lys Ser Gly Thr Ser Ala Ser
Leu Ala Ile Ser 85 90 95 Gly Leu Arg Ser Glu Asp Glu Ala Asp Tyr
Tyr Cys Ala Ala Trp Asp 100 105 110 Asp Ser Leu Asn Gly Trp Val Phe
Gly Gly Gly Thr Arg Leu Thr Val 115 120 125 Leu Gly 130
4410PRTArtificialepitope region of 5A7 44Ile Gly Asn Cys Pro Ile
Trp Val Lys Thr 1 5 10
* * * * *