U.S. patent application number 13/728056 was filed with the patent office on 2013-05-02 for fully human influenza m2 specific antibodies.
This patent application is currently assigned to Intercell AG. The applicant listed for this patent is Intercell AG. Invention is credited to Martin F. Bachmann, Monika Bauer, Roger Beerli, Nicole Schmitz.
Application Number | 20130109845 13/728056 |
Document ID | / |
Family ID | 42232909 |
Filed Date | 2013-05-02 |
United States Patent
Application |
20130109845 |
Kind Code |
A1 |
Bachmann; Martin F. ; et
al. |
May 2, 2013 |
FULLY HUMAN INFLUENZA M2 SPECIFIC ANTIBODIES
Abstract
The present invention relates to human antibodies, preferably to
fully human antibodies, which are specifically binding to influenza
M2e antigen. The invention further relates to individual light-
and/or heavy chains of such antibodies, to nucleic acids encoding
said antibodies or their light- and/or heavy chain, and to
expression vectors for the expression of said antibodies.
Furthermore, the invention relates to the use of said antibodies in
the treatment and/or prevention of influenza A virus infection,
preferably in humans.
Inventors: |
Bachmann; Martin F.;
(Ramismuhle, CH) ; Bauer; Monika; (Zurich, CH)
; Beerli; Roger; (Adlikon b. Regensdorf, CH) ;
Schmitz; Nicole; (Urdorf, CH) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
Intercell AG; |
Vienna |
|
AT |
|
|
Assignee: |
Intercell AG
Vienna
AT
|
Family ID: |
42232909 |
Appl. No.: |
13/728056 |
Filed: |
December 27, 2012 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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13132658 |
Jun 3, 2011 |
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PCT/EP09/66052 |
Nov 30, 2009 |
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13728056 |
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Current U.S.
Class: |
530/388.15 ;
435/320.1; 435/455; 536/23.53 |
Current CPC
Class: |
C07K 2317/92 20130101;
C07K 2317/56 20130101; A61K 2039/505 20130101; C07K 2317/565
20130101; A61P 31/16 20180101; C07K 16/1018 20130101; C07K 2317/21
20130101; C07K 2317/732 20130101; C07K 2317/34 20130101; C07K
2317/76 20130101; C07K 2317/622 20130101 |
Class at
Publication: |
530/388.15 ;
536/23.53; 435/320.1; 435/455 |
International
Class: |
C07K 16/10 20060101
C07K016/10 |
Foreign Application Data
Date |
Code |
Application Number |
Dec 4, 2008 |
EP |
08170749.9 |
Oct 20, 2009 |
EP |
09173548.0 |
Claims
1. A method of producing an isolated monoclonal antibody, wherein
said monoclonal antibody is specifically binding influenza M2e
antigen, and wherein said monoclonal antibody is a human monoclonal
antibody, and wherein said antibody comprises at least one antigen
binding site, wherein said antigen binding site comprises: (a) one
light chain variable region (LCVR), wherein said LCVR comprises:
(i) one light chain complementarity determining region 1 (LC CDR1),
wherein said LC CDR1 consists of the peptide of any one of SEQ ID
NOs 1, 2, 3, 4, 5, and 6; (ii) one LC CDR2, wherein said LC CDR2
consists of the peptide of SEQ ID NO: 7; and (iii) one LC CDR3,
wherein said LC CDR3 consists of the peptide of any one of SEQ ID
NOs 8, 9, 10, and 11; and (b) one heavy chain variable region
(HCVR), wherein said HCVR comprises: (i) one heavy chain
complementarity determining region 1 (HC CDR1), wherein said HC
CDR1 consists of the peptide of SEQ ID NO: 12; (ii) one HC CDR2,
wherein said HC CDR2 consists of the peptide of any one of SEQ ID
NOs 13 and 14; and (iii) one HC CDR3, wherein said HC CDR3 consists
of the peptide of any one of SEQ ID NOs 15, 16, 17, 18, and 19.
2. The method of claim 1, wherein said at least one antigen binding
site recognizes an epitope comprised by the amino acid sequence
LLTEVETP (SEQ ID NO: 93).
3. The method of claim 1, wherein said LC CDR1 consists of the
peptide of any one of SEQ ID NOs 1, 4 and 6, said LC CDR2 consists
of the peptide of SEQ ID NO: 7, said LC CDR3 consists of the
peptide of SEQ ID NO: 8, said HC CDR1 consists of the peptide of
SEQ ID NO: 12, said HC CDR2 consists of the peptide of SEQ ID NO:
13, and said HC CDR3 consists of the peptide of SEQ ID NO: 15.
4. The method of claim 3, wherein said LC CDR1 consists of the
peptide of SEQ ID NO: 1, said LC CDR2 consists of the peptide of
SEQ ID NO: 7, said LC CDR3 consists of the peptide of SEQ ID NO: 8,
said HC CDR1 consists of the peptide of SEQ ID NO: 12, said HC CDR2
consists of the peptide of SEQ ID NO: 13, and said HC CDR3 consists
of the peptide of SEQ ID NO: 15.
5. The method of claim 1, wherein position 5 to 113 of said LCVR
consists of the peptide of any one of SEQ ID NOs 20, 21 and 22.
6. The method of claim 1, wherein position 5 to 113 of said LCVR
consists of the peptide of any one of SEQ ID NOs 20, 21 and 22, and
wherein position 1 to 4 of said LCVR consists of the peptide of SEQ
ID NO: 24.
7. The method of claim 1, wherein position 7 to 121 of said HCVR
consists of the peptide of SEQ ID NO: 23.
8. The method of claim 1, wherein position 7 to 121 of said HCVR
consists of the peptide of SEQ ID NO: 23, and wherein position 1 to
6 of said HCVR consists of the peptide of SEQ ID NO: 25.
9. The method of claim 1, wherein position 5 to 113 of said LCVR
consists of the peptide of any one of SEQ ID NOs 20, 21 and 22, and
wherein position 7 to 121 of said HCVR consists of the peptide of
SEQ ID NO: 23.
10. The method of claim 1, wherein position 5 to 113 of said LCVR
consists of the peptide of SEQ ID NO: 20, and wherein position 7 to
121 of said HCVR consists of the peptide of SEQ ID NO: 23, and
wherein position 1 to 4 of said LCVR consists of the peptide of SEQ
ID NO: 24, and wherein position 1 to 6 of said HCVR consists of the
peptide of SEQ ID NO: 25.
11. The method of claim 1, wherein said monoclonal antibody
comprises at least one light chain, and wherein said monoclonal
antibody further comprises at least one heavy chain, wherein said
light chain comprises the amino acid sequence of any one of SEQ ID
NOs 26, 27, and 28, and wherein said heavy chain comprises of the
amino acid of SEQ ID NO: 29.
12. The method of claim 1, wherein said monoclonal antibody
comprises at least one light chain, and wherein said monoclonal
antibody further comprises at least one heavy chain, wherein said
light chain consists of the amino acid sequence of any one of SEQ
ID NOs 26, 27, and 28, and wherein said heavy chain consists of the
amino acid of SEQ ID NO: 29.
13. The method of claim 1, wherein said influenza M2e antigen is
the extracellular domain of the influenza A M2 protein, and wherein
said influenza M2e antigen is the peptide of any one of SEQ ID NOs
48 to 83 and 90 to 92.
14. The method of claim 1, wherein said influenza M2e antigen is
the extracellular domain of the influenza A M2 protein, and wherein
said influenza M2e antigen is the peptide of any one of SEQ ID NO:
48.
15. The method of claim 11, wherein the dissociation constant (Kd)
of said monoclonal antibody and said influenza M2e antigen is at
most 100 nM or less.
16. The method of claim 11, wherein the dissociation constant (Kd)
of said monoclonal antibody and said influenza M2e antigen is at
most 10 nM or less.
17. A method of producing an isolated polynucleotide comprising a
nucleic acid encoding the monoclonal antibody of the method of
claim 1.
18. A method of producing an expression vector comprising a nucleic
acid encoding the monoclonal antibody of the method of claim 1.
19. A method of producing an isolated host cell comprising at least
one expression vector comprising a nucleic acid encoding the
monoclonal antibody of the method of claim 1.
Description
RELATED APPLICATIONS
[0001] This application is a continuation of U.S. application Ser.
No. 13/132,658, filed Jun. 3, 2011, which is a national stage
filing under 35 U.S.C. .sctn.371 of international application
PCT/EP2009/066052, filed Nov. 30, 2009, which was published under
PCT Article 21(2) in English, and claims priority to EP 09173548,
filed Oct. 20, 2009 and EP 08170749, filed Dec. 4, 2008.
FIELD OF THE INVENTION
[0002] The present invention relates to human antibodies,
preferably to fully human antibodies, which are specifically
binding to influenza M2e antigen. The invention further relates to
individual light- and/or heavy chains of such antibodies, to
nucleic acids encoding said antibodies or their light- and/or heavy
chain, and to expression vectors for the expression of said
antibodies. Furthermore, the invention relates to the use of said
antibodies in the treatment and/or prevention of influenza A virus
infection, preferably in humans.
RELATED ART
[0003] Influenza A virus still is a major cause of disease in
humans, accounting for three to five million cases of severe
illness and 250,000-500,000 deaths each year. Efficient influenza A
vaccines are available, which act by inducing neutralizing
antibodies against hemagglutinin (HA). Since HA undergoes
continuous change due to mutations (antigenic drift), new antigenic
variants of influenza A arise every year requiring constant update
of the vaccines. Effective vaccination is further complicated by
the occasional re-assortment of the segmented viral genome leading
to the replacement of HA or neuraminidase (NA) from one subtype by
another subtype, a process called antigenic shift. Passive
immunization with monoclonal antibodies (mAbs) targeting HA is very
efficient, however, suffers the same disadvantages as the current
vaccines due to antigenic shift and drift.
[0004] An ideal target for active and passive immunization
strategies would therefore be a conserved viral protein. The matrix
protein 2 (M2) fits the bill and has received considerable
attention as a potential target against influenza infection over
the past decades (Zebedee S L, Lamb R A: Influenza A virus M2
protein: monoclonal antibody restriction of virus growth and
detection of M2 in virions. J Virol 1988, 62:2762-2772). M2 is a
tetrameric ion channel which is involved in virus uncoating in the
endosome and in virus maturation in the trans-Golgi network. Its 23
amino acid extracellular domain has remained remarkably conserved
in human influenza A virus isolates over the last hundred years, at
least in part due to the fact that the M2 protein is co-transcribed
with the matrix protein 1 (M1). Whereas M2 is abundantly expressed
on infected cells, only very few M2 molecules are present in
Influenza A virus membranes. In accordance with these observations
M2 specific antibodies were shown to protect by the elimination of
infected cells by ADCC rather than by preventing infection through
neutralization of the virus (Jegerlehner A, Schmitz N, Storni T,
Bachmann M F: Influenza A vaccine based on the extracellular domain
of M2: weak protection mediated via antibody-dependent NK cell
activity. J. Immunol. 2004, 172:5598-5605).
[0005] Passive immunization with monoclonal antibodies has several
advantages over vaccination. In particular, it allows treating
people which poorly respond to vaccines, such as the elderly, young
children or immune compromised individuals. In addition, passive
immunization is the treatment option of choice in situations where
rapid protection is crucial, such as for post-exposure treatment or
prophylaxis for the acutely exposed. A number of M2 ectodomain
(M2e)-specific mAbs have been reported to protect mice from a
lethal challenge in a prophylactic setting. While these mAbs
include fully human antibodies derived from transchromosomic mice
(Wang R et al., Antiviral Res. 2008, 80:168-177; WO2006/061723A2;
and WO03/078600A2), no natural human M2e-specific antibodies have
been reported to date. However, for application in human subjects,
natural human antibodies are the preferred choice. In contrast to
humanized and fully human antibodies derived from phage display or
transchromosomic mice, natural human antibodies combine the
advantage of minimal immunogenicity with the smallest possible
off-target reactivity and toxicity. Furthermore, human derived
antibodies have the advantage of having gone through the affinity
maturation process, resulting in high affinity antibodies.
SUMMARY OF THE INVENTION
[0006] A library-based screening led to the identification,
isolation and cloning of 53 human scFv which showed high affinity
to the extracellular domain of the influenza A M2 protein. Fully
human monoclonal IgG1 antibodies have been generated from
representative scFv clones. Human, and in particular fully human
antibodies, are advantageous because they show less severe side
effects when administered to a human subject. Without being bound
to any theory this is because human, and in particular fully human,
antibodies are typically and preferably not recognized by the human
immune system. It has surprisingly been found that the selected
antibody clones show different combinations of identical and/or
highly similar CDR sequences in their light chain variable regions
(LCVR) and in their heavy chain variable regions (HCVR). Based on
the particular combination of CDRs, different types of LCVRs and
HCVRs can be distinguished (cf. Tables 1 and 2). However, the LCVRs
and HCVRs of all clones are highly similar as can be deduced from
the sequence information which is provided in Tables 1 and 2. It
was therefore concluded that the antibodies of the invention are
clonally related. Table 3 provides an overview of the different
combinations of LCVR and HCVR types as defined by their CDRs which
were found in the selected clones. The table also indicates the
abundance of each of these combinations among the 53 clones.
Further surprisingly it has been found that the antibodies of the
invention show a very high affinity towards influenza M2e antigen,
and in particular towards the extracellular domain of the influenza
A M2 protein. The dissociation constants (Kd) between the
antibodies and RNAse--influenza A M2e antigen conjugate was found
to be in the low picomolar range. An epitope mapping revealed that
the minimal epitope which is recognized by antibody clone D005 is
comprised in the amino acid sequence LLTEVETP (SEQ ID NO:93). This
epitope is comprised by the M2 protein of most known influenza A
strains. Consequently, it has been found, that the antibodies of
the invention show a similarly high affinity to different variants
of the extracellular domain of influenza A M2 protein which are
derived from different strains of influenza A virus. It has also
been demonstrated that the antibodies of the invention are
specifically binding to cells which are expressing recombinant
influenza A M2 protein on their surface. Moreover, antibodies of
the invention were found to exhibit a preferential binding to
cell-associated influenza A M2 protein. Furthermore it was found
that antibodies of the invention are capable of specifically
binding influenza A M2 protein in the context of influenza A virus
particles. Most importantly, it has been demonstrated in a mouse
model for influenza A virus infection that the antibodies of the
invention are highly effective in the treatment and/or prevention
of influenza A virus infection. Contrary to other influenza A M2
specific human antibodies which are known in the art, the
antibodies of the invention have therapeutic activity when
administered as a single dose on day one or day two after
infection. The antibodies disclosed herein are therefore useful as
a medicament against influenza A virus infection in a therapeutic
as well as in a prophylactic setting.
TABLE-US-00001 TABLE 1 CDRs of the LCVRs of fully human M2 specific
monoclonal antibodies. Nine types of LCVRs can be distinguished
based on the combination of their CDR sequences (1A to 3B). Based
on sequence similarities in the LC CDRs 3 groups of LCVRs can be
distinguished: 1A to 1E, 2A to 2B, and 3A to 3B). Type Clones LC
CDR1 LC CDR2 LC CDR 3 1A D005 and qsvlytsnnkny was qqyfmtpit 31
others (SEQ ID NO: 1) (SEQ ID NO: 7) (SEQ ID NO: 8) 1B F048, F084
qsvlntsnnkny was qqyfmtpit (SEQ ID NO: 2) (SEQ ID NO: 7) (SEQ ID
NO: 8) 1C E011 qsvlhtsnnkny was qqyfmtpit (SEQ ID NO: 3) (SEQ ID
NO: 7) (SEQ ID NO: 8) 1D E036 qsvlytsnnkny was qqyfmapit (SEQ ID
NO: 1) (SEQ ID NO: 7) (SEQ ID NO: 9) 1E F076 qsvlytsnnkny was
qqyfvtpit (SEQ ID NO: 1) (SEQ ID NO: 7) (SEQ ID NO: 10) 2A E040 and
qsvlyssnneny was qqyfmtpit 10 others (SEQ ID NO: 4) (SEQ ID NO: 7)
(SEQ ID NO: 8) 2B E043 qsvlyssnnedy was qqyfmtpit (SEQ ID NO: 5)
(SEQ ID NO: 7) (SEQ ID NO: 8) 3A F052, F015, qsllyssnnkny was
qqyfmtpit F077 (SEQ ID NO: 6) (SEQ ID NO: 7) (SEQ ID NO: 8) 3B F027
qsllyssnnkny was qqyfmtpia (SEQ ID NO: 1) (SEQ ID NO: 7) (SEQ ID
NO: 11)
TABLE-US-00002 TABLE 2 CDRs of the HCVRs of fully human M2 specific
monoclonal antibodies. Six types of HCVRs can be distinguished
based on the combination of their CDR sequences. Type Clones HC
CDR1 HC CDR2 HC CDR 3 1A D005 and 45 glnfgdyp iksksygvtt
tsssgflyyfdy others (SEQ ID NO: 12) (SEQ ID NO: 13) (SEQ ID NO: 15)
1B E031, F023, glnfgdyp iksksygvtt tsssgflyyfdh F057 (SEQ ID NO:
12) (SEQ ID NO: 13) (SEQ ID NO: 16) 1C E005 glnfgdyp iksksygvtt
tssssflyyfdy (SEQ ID NO: 12) (SEQ ID NO: 13) (SEQ ID NO: 17) 1D
E034 glnfgdyp iksksygvtt tsnsgflyyfdy (SEQ ID NO: 12) (SEQ ID NO:
13) (SEQ ID NO: 18) 1E E044 glnfgdyp ikskpygvtt tsssgflyyfdy (SEQ
ID NO: 12) (SEQ ID NO: 14) (SEQ ID NO: 15) 1F F071 glnfgdyp
iksksygvtt tsssgfsyyfdy (SEQ ID NO: 12) (SEQ ID NO: 13) (SEQ ID NO:
19)
TABLE-US-00003 TABLE 3 Combination of LCVRs and HCVRs as occurring
in 53 independent clones of fully human M2 specific monoclonal
antibodies. Clones D005, E040 and F052 (highlighted in bold)
represent three of most abundant combinations of LCVR and HCVR and
were thus chosen as representative clones for further analysis.
Combinations of CDR types (LC Number of VR - HC VR) Clones Clones
1A-1A D005, D013, D019, D033, D037, 26 E007, E012, E021, E028,
E029, E033, E035, E049, E051, F004, F005, F025, F037, F039, F040,
F045, F046, F054, F065, F075, F087 1A-1B E031, F023, F057 3 1A-1D
E034 1 1A-1E E044 1 1A-1F F071 1 1B-1A F048, F084 2 1C-1A E011 1
1D-1A E036 1 1E-1A F076 1 2A-1A E006, E023, E030, E040, E048, 10
F003, F020, F044, F062, F066 2A-1C E005 1 2B-1A E043 1 3A-1A F015,
F052, F077 3 3B-1A F027 1
[0007] In one aspect, the invention relates to a monoclonal
antibody, preferably to an isolated monoclonal antibody, wherein
said monoclonal antibody is specifically binding influenza M2e
antigen, and wherein said monoclonal antibody is a human monoclonal
antibody, preferably a fully human monoclonal antibody, wherein
preferably the EC50 value and/or the dissociation constant (Kd) of
said monoclonal antibody and said influenza M2e antigen is at most
1000 nM, preferably at most 100 nM, more preferably at most 10 nM,
still more preferably at most 1 nM, still more preferably at most
100 pM, still more preferably at most 10 pM, and most preferably at
most 1 pM.
[0008] In a preferred embodiment, (i) said monoclonal antibody
comprises at least one LCVR, wherein said LCVR comprises: (a) one
LC CDR1, wherein said LC CDR1 consists of the peptide of any one of
SEQ ID NOs 1, 2, 3, 4, 5, and 6; (b) one LC CDR2, wherein said LC
CDR2 consists of the peptide of SEQ ID NO:7; (c) one LC CDR3,
wherein said LC CDR3 consists of the peptide of any one of SEQ ID
NOs 8, 9, 10, and 11; and/or (ii) said monoclonal antibody
comprises at least one HCVR, wherein said HCVR comprises: (a) one
HC CDR1, wherein said HC CDR1 consists of the peptide of SEQ ID
NO:12; (b) one HC CDR2, wherein said HC CDR2 consists of the
peptide of any one of SEQ ID NOs 13 and 14; and (c) one HC CDR3,
wherein said HC CDR3 consists of the peptide of any one of SEQ ID
NOs 15, 16, 17, 18, and 19.
[0009] In a further aspect the invention relates to an isolated
monoclonal antibody, wherein said monoclonal antibody is
specifically binding influenza M2e antigen, and wherein preferably
said monoclonal antibody is a human monoclonal antibody, most
preferably a fully human monoclonal antibody, and wherein said
antibody comprises at least one antigen binding site, wherein said
antigen binding site comprises: (a) one LCVR, wherein said LCVR
comprises: (i) one LC CDR1, wherein said LC CDR1 consists of the
peptide of any one of SEQ ID NOs 1, 2, 3, 4, 5, and 6; (ii) one LC
CDR2, wherein said LC CDR2 consists of the peptide of SEQ ID NO:7;
and (iii) one LC CDR3, wherein said LC CDR3 consists of the peptide
of any one of SEQ ID NOs 8, 9, 10, and 11; and (b) one HCVR,
wherein said HCVR comprises: (i) one HC CDR1, wherein said HC CDR1
consists of the peptide of SEQ ID NO:12; (ii) one HC CDR2, wherein
said HC CDR2 consists of the peptide of any one of SEQ ID NOs 13
and 14; and (iii) one HC CDR3, wherein said HC CDR3 consists of the
peptide of any one of SEQ ID NOs 15, 16, 17, 18, and 19.
[0010] A further aspect of the invention is a LCVR of a monoclonal
antibody, wherein said monoclonal antibody is a human monoclonal
antibody, most preferably a fully human monoclonal antibody, and
wherein said monoclonal antibody is specifically binding influenza
M2e antigen, and wherein said LCVR comprises: (a) one LC CDR1,
wherein said LC CDR1 consists of the peptide of any one of SEQ ID
NOs 1, 2, 3, 4, 5, and 6; (b) one LC CDR2, wherein said LC CDR2
consists of the peptide of SEQ ID NO:7; (c) one LC CDR3, wherein
said LC CDR3 consists of the peptide of any one of SEQ ID NOs 8, 9,
10, and 11.
[0011] A further aspect of the invention is a HCVR of a monoclonal
antibody, wherein said monoclonal antibody is a human monoclonal
antibody, preferably a fully human monoclonal antibody, and wherein
said monoclonal antibody is specifically binding influenza M2e
antigen, and wherein said HCVR comprises: one HC CDR1, wherein said
HC CDR1 consists of the peptide of SEQ ID NO:12; (b) one HC CDR2,
wherein said HC CDR2 consists of the peptide of any one of SEQ ID
NOs 13 and 14; and (c) one HC CDR3, wherein said HC CDR3 consists
of the peptide of any one of SEQ ID NOs 15, 16, 17, 18, and 19.
[0012] In a further aspect, the invention relates a nucleic acid
molecule encoding a HCVR or a LCVR of the invention, a monoclonal
antibody of the invention or an individual chain thereof.
[0013] In a further aspect, the invention relates to an expression
vector for the recombinant expression of an antibody of the
invention.
[0014] In a further aspect, the invention relates to a host cell
comprising at least one nucleic acid molecule or at least one
expression vector of the invention.
[0015] In a further aspect, the invention relates to a
pharmaceutical composition comprising at least one monoclonal
antibody of the invention.
[0016] In a further aspect, the invention relates to a method of
passive immunization, preferably against influenza A virus, said
method comprising administering to a subject an effective amount of
the monoclonal antibody of the invention or an effective amount of
the pharmaceutical composition of the invention, wherein preferably
said monoclonal antibody is an IgG1.
[0017] In a further aspect, the invention relates to a method of
treating and/or preventing influenza A virus infection, said method
comprising administering to a subject an effective amount of the
monoclonal antibody of the invention or an effective amount of the
pharmaceutical composition of the invention, wherein preferably
said subject is a human, and wherein further preferably said
monoclonal antibody is an IgG1.
[0018] In a further aspect, the invention relates to a monoclonal
antibody of the invention or to the pharmaceutical composition of
the invention, for use in passive immunization, preferably against
influenza A virus, preferably in a human, and wherein further
preferably said monoclonal antibody is an IgG1.
[0019] In a further aspect, the invention relates to the use of the
monoclonal antibody of the invention in the manufacture of a
medicament for the treatment and/or prophylaxis of influenza A
virus infection, preferably in a human, and wherein further
preferably said monoclonal antibody is an IgG1.
DESCRIPTION OF THE FIGURES
[0020] FIG. 1. Alignment of LCVR sequences (amino acids 5 to 113)
of M2-specific human antibodies. Identical amino acids are shown as
dots. LC CDRs 1-3 are boxed.
[0021] FIG. 2. Alignment of HCVR sequences (amino acids 7 to 121)
of M2-specific human antibodies (the same antibodies as in FIG. 1).
Identical amino acids are shown as dots. HC CDRs 1-3 are boxed.
[0022] FIG. 3. Effect of M2-specific antibodies on
Influenza-induced morbidity and mortality. Mice were treated with
500 .mu.g of the indicated scFv-msFc.gamma.2c antibody on day -2,
infected with Influenza A virus PR8 on day 0, and weight (A), body
temperature (B) and survival (C) were monitored on the indicated
days.
[0023] FIG. 4. Effect of scFv-D005-msFc.gamma.2c on survival of
Influenza-infected mice. (A) Dose titration. Mice were treated with
the indicated amounts of antibody on day -2, infected with
Influenza A virus PR8 on day 0, and survival was monitored for 21
days. (B) Therapeutic application of antibody. Mice were infected
with Influenza A virus PR8 on day 0, treated with 200 .mu.g of the
antibody on the indicated days, and survival was monitored for 21
days. Control, mouse IgG on day -2.
[0024] FIG. 5. Effect of M2-specific antibodies on
Influenza-induced morbidity and mortality. Mice were treated with
the antibody D005 in the indicated format on day -2, infected with
Influenza A virus PR8 on day 0, and weight (A), body temperature
(B) and survival (C) were monitored on the indicated days.
[0025] FIG. 6. Binding to cell surface M2 in the presence of
soluble M2e peptide. L929/M2#E9 cells were stained with antibody
14C2 (A) or D005 (B) in the presence (solid line) or absence (heavy
line) of soluble M2e peptide and analyzed by FACS. Dotted line,
staining with fluorescently labelled secondary antibody alone.
[0026] FIG. 7. Direct binding of antibody D005 to Influenza A PR8
virus particles. The indicated amount of virus was captured on
wells of an ELISA plate previously coated with anti-HA antibody.
Virus was detected with hIgG1k-D005 or an irrelevant isotype
control.
[0027] FIG. 8. Characterization of binding specificity of antibody
D005. (A) Reactivity of IgG1k-D005 with M2e-derived peptides from
different strains of Influenza A. (B) Epitope mapping.
DETAILED DESCRIPTION OF THE INVENTION
[0028] "Antibody": As used herein, the term "antibody" refers to a
molecule, preferably a protein, which is capable of specifically
binding an antigen, typically and preferably by binding an epitope
or antigenic determinant of said antigen, or a hapten. Preferably,
the term antibody refers to an antigen or hapten binding molecule
comprising at least one variable region, wherein preferably said
molecule comprises least one HCVR and/or at least one LCVR. Further
preferably, the term antibody refers to an antigen or hapten
binding molecule comprising at least one, preferably exactly two
antigen binding sites, wherein each of said antigen binding site(s)
is formed by one HCVR and one LCVR. Furthermore, the term antibody
refers to whole antibodies, preferably of the IgG, IgA, IgE, IgM,
or IgD class, more preferably of the IgG class, most preferably
IgG1, IgG2, IgG3, and IgG4, and to antigen binding fragments
thereof. In a preferred embodiment said whole antibodies comprise
either a kappa or a lambda light chain. The term "antibody" also
refers to antigen or hapten binding antibody fragments, preferably
to proteolytic fragments and their recombinant analogues. most
preferably to Fab, Fab' and F(ab')2, and Fv. The term antibody
further encompasses a protein comprising at least one, preferably
two variable regions, wherein further preferably said protein
comprises exactly one HCVR and exactly one LCVR. In a preferred
embodiment the term antibody refers to a single chain antibody,
preferably to scFv. Thus, preferred antibodies are single chain
antibodies, preferably scFvs, disulfide-linked Fvs (sdFv) and
fragments comprising either a light chain variable region (LCVR) or
a heavy chain variable region (HCVR). In the context of the
invention the term "antibody" preferably refers to recombinant
antibodies, including recombinant proteins consisting of a single
polypeptide, wherein said polypeptide comprises at least one,
preferably exactly one, variable region. In the context of the
invention recombinant antibodies may further comprise functional
elements, such as, for example, a linker region, a signal peptide
or hydrophobic leader sequence, a detection tag and/or a
purification tag (e.g. Fc).
[0029] "recognizing": An antibody is said to be "recognizing" an
epitope when said antibody is specifically binding an antigen
comprising said epitope in a position which is available for
interaction with said antibody, and when said antibody does not
specifically bind an otherwise identical antigen which does not
comprise said epitope, or wherein said epitope is located in a
position which is not available for interaction with said antibody.
Similarly, an antigen binding site is said to be recognizing an
epitope, when an antibody comprising said antigen binding site is
recognizing said epitope, wherein typically and preferably said
antibody does not comprise a second antigen binding site having a
different structure.
[0030] "Fv": The term Fv refers to the smallest proteolytic
fragment of an antibody capable of binding an antigen or hapten and
to recombinant analogues of said fragment.
[0031] "single chain antibody": A single chain antibody is an
antibody consisting of a single polypeptide. Preferred single chain
antibodies consist of a polypeptide comprising at least one,
preferably exactly one VR, wherein preferably said VR is a HCVR.
More preferred single chain antibodies consist of a polypeptide
comprising a at least one, preferably exactly one, HCVR and at
least one, preferably exactly one, LCVR. Still more preferred
single chain antibodies comprise exactly one HCVR and exactly one
LCVR. Typically and preferably said HCVR and said LCVR are forming
an antigen binding site. Most preferred single chain antibodies are
scFv, wherein said scFv consist of a single polypeptide comprising
exactly one HCVR and exactly one LCVR, wherein said HCVR and said
LCVR are linked to each other by a linker region, wherein
preferably said linker region consists of at least 15, preferably
of 15 to 20 amino acids (Bird et al. (1988) Science,
242(4877):423-426). Further preferred single chain antibodies are
scFv, wherein said scFv are encoded by a coding region, wherein
said coding region, in 5' to 3' direction, comprises in the
following order: (1) a light chain variable region (LCVR)
consisting of light chain framework (LC FR) 1, complementary
determining region (LC CDR) 1, LC FR2, LC CDR 2, LC FR3, LC CDR3
and LC FR4 from a .kappa. or .lamda. light chain; (2) a flexible
linker (L), and (3) a heavy chain variable region (HCVR) consisting
of framework (HC FR) 1, complementary determining region (HC CDR)
1, HC FR2, HC CDR2, HC FR3, HC CDR3 and HC FR4. Alternatively,
single chain antibodies are scFv, wherein said scFv are encoded by
a coding region, wherein said coding region, in 5' to 3' direction,
comprises in the following order: (1) a heavy chain variable region
(HCVR) consisting of framework (HC FR) 1, complementary determining
region (HC CDR) 1, HC FR2, HC CDR2, HC FR3, HC CDR3 and HC FR4; (2)
a flexible linker (L), and (3) a light chain variable region (LCVR)
consisting of light chain framework (LC FR) 1, complementary
determining region (LC CDR) 1, LC FR2, LC CDR2, LC FR3, LC DR3 and
LC FR4 from a .kappa. or .lamda. light chain.
[0032] "diabody": The term "diabody" refers to an antibody
comprising two polypeptide chains, preferably two identical
polypeptide chains, wherein each polypeptide chain comprises a HCVR
and a LCVR, wherein said HCVR and said LCVR are linked to each
other by a linker region, wherein preferably said linker region
comprises at most 10 amino acids (Huston et al. (1988), PNAS
85(16):587958-83; Holliger et al. (1993), PNAS 90(14):6444-6448,
Hollinger & Hudson, 2005, Nature Biotechnology 23(9):1126-1136;
Arndt et al. (2004) FEBS Letters 578(3):257-261). Preferred linker
regions of diabodies comprise 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10
amino acids.
[0033] "human antibody": As used herein, the term "human antibody"
refers to an antibody, preferably a recombinant antibody,
essentially having the amino acid sequence of a human
immunoglobulin, or a fragment thereof, and includes antibodies
isolated from human immunoglobulin libraries. In the context of the
invention "human antibodies" may comprise a limited number of amino
acid exchanges as compared to the sequence of a native human
antibody. Such amino acid exchanges can, for example, be caused by
cloning procedures. However, the number of such amino acid
exchanges in human antibodies of the invention is preferably
minimized Preferably, the amino acid sequence of human antibodies
is at least 85%, preferably 90%, more preferably 95%, even more
preferably at least 96%, still more preferably 97%, again still
more preferably 98%, again still more preferably 99% and most
preferably 100% identical to that of native human antibodies. More
preferably, the amino acid sequence of human antibodies is at least
85%, preferably 90%, more preferably 95%, even more preferably at
least 96%, still more preferably 97%, again still more preferably
98%, again still more preferably 99% and most preferably 100%
identical to that of native human antibodies which are specifically
binding to the antigen or hapten of interest. Most preferably, the
amino acid sequence of human antibodies is at least 85%, preferably
90%, more preferably 95%, even more preferably at least 96%, still
more preferably 97%, again still more preferably 98%, again still
more preferably 99% and most preferably 100% identical to that of
native human antibodies which are specifically binding influenza
M2e antigen, wherein preferably said influenza M2e antigen is
selected from any one of SEQ ID NOs 48 to 52, and wherein most
preferably said influenza M2e antigen is SEQ ID NO:48.
[0034] Preferred recombinant human antibodies differ from native
human antibodies in at most 20, 15, 10, 9, 8, 7, 6, 5, 4, 3, 2, or
1 amino acid. Very preferably, differences in the amino acid
sequence of recombinant human antibodies and native human
antibodies are eliminated my means of molecular cloning, and thus,
most preferably, the amino acid sequence of a recombinant human
antibodies and native human antibodies are identical. Such
antibodies are also referred to as "fully human antibodies". An
illustrative example how a fully human antibody may be obtained
from a human antibody selected from a human antibody library is
provided in Example 8. Typically and preferably, fully human
antibodies are not immunogenic in humans.
[0035] Preferred human antibodies comprise (a) least one,
preferably one, HCVR, (b) at least one, preferably one, HCCR, (c)
at least one, preferably one, LCVR, and (d) at least one,
preferably one, LCCR, wherein said at least one HCVR, and/or said
at least one HCCR, and/or said at least one LCVR, and/or said at
least one LCCR are at least 85%, preferably 90%, more preferably
95%, still more preferably at least 96%, again still more
preferably 97%, again still more preferably 98%, again still more
preferably 99%, and most preferably 100% identical to the
respective native human regions.
[0036] It is well established that the constant regions of
immunoglobulins, including human immunoglobulins, exist in various
allotypes, i.e. that the amino acid sequence of said constant
regions may differ to a certain extend between individuals of a
population. Allotypes of the constant regions of human
immunoglobulins are very well studied and the sequence information
is readily available to the artisan from various sources, including
the Immuno Genetics Information System. It is to be understood that
different allotypes of the constant regions of one immunoglobulin
are interchangeable for the purpose of the invention. For example,
the human gamma 1 heavy chain of a monoclonal antibody of the
invention may comprise any existing allotype of a human gamma 1
HCCR.
[0037] "monoclonal antibody": As used herein, the term "monoclonal
antibody" refers to an antibody population comprising only one
single antibody species, i.e. antibodies having an identical amino
acid sequence.
[0038] "constant region (CR)": The term "constant region" refers to
a light chain constant region (LCCR) or a heavy chain constant
region (HCCR) of an antibody. Typically and preferably, said CR
comprises one to four immunoglobulin domains characterized by
disulfide stabilized loop structures. Preferred CRs are CRs of
immunoglobulins, preferably of human immunoglobulins, wherein
further preferably said immunoglobulins, preferably said human
immunoglobulins are selected from the group consisting of IgG1,
IgG2, IgG3, IgG4, IgA, IgE, IgM, and IgD. Very preferred CRs are
human CRs comprising or consisting of an amino acid sequence
available from public databases, including, for example the
Immunogenetic Information System.
[0039] light chain constant region (LCCR): The LCCR, more
specifically the kappa LCCR or the lambda LCCR, typically
represents the C-terminal half of a native kappa or lambda light
chain of an native antibody. A LCCR typically comprises about 110
amino acids representing one immunoglobulin domain.
[0040] heavy chain constant region (HCCR): The constant region of a
heavy chain comprises about three quarters or more of the heavy
chain of an antibody and is situated at its C-terminus Typically,
the HCCR comprises either three or four immunoglobulin domains.
Preferred HCCRs are selected from gamma HCCR, alpha HCCR, epsilon
HCCR, my HCCR, and delta HCCR. Very preferred are gamma HCCR,
wherein preferably said gamma HCCR is selected from gamma 1 HCCR,
gamma 2 HCCR, gamma 3 HCCR, and gamma 4 HCCR, wherein most
preferably said gamma HCCR is a gamma 1 HCCR.
[0041] "variable region (VR)": Refers to the variable region or
variable domain of an antibody, more specifically to the heavy
chain variable region (HCVR) or to the light chain variable region
(LCVR). Typically and preferably, a VR comprises a single
immunoglobulin domain. Preferred VRs are VRs of immunoglobulins,
preferably of human immunoglobulins, wherein further preferably
said immunoglobulins, preferably said human immunoglobulins, are
selected from the group consisting of IgG1, IgG2, IgG3, IgG4, IgA,
IgE, IgM, and IgD. VRs of various species are known in the art.
Preferred VRs are human VRs, wherein the framework of said human
VRs exhibit at least 80%, preferably at least 85%, more preferably
90%, again more preferably at least 95%, most preferably at least
99% sequence identity with the framework of any known human VR
sequence. Preferred VRs are human VRs, wherein the framework of
said human VRs exhibit at least 80%, preferably at least 85%, more
preferably 90%, again more preferably at least 95%, most preferably
at least 99% sequence identity with the framework of any human VR
sequence available from public databases, most preferably with any
human VR sequence available from the Immunogenetics Information
System.
[0042] Each VR comprises so called complementarity determining
regions (CDRs) which are determining the binding characteristics of
the antibody and which are embedded in the so called framework.
Typically and preferably, VRs comprise three CDRs, preferably CDR1,
CDR2, and CDR3, which are embedded into the framework (FR 1-4).
Thus, in a preferred embodiment, a VR comprises the following
elements in the following order from the N-- to the C-terminus:
FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4.
[0043] Generally VRs comprise or preferably consist of a
polypeptide, wherein said polypeptide is a product of a member of a
family of V-gene segments in combination with further gene segments
as, for example, D and J gene segments (HCVR) or J gene segments
(LCDR).
[0044] "light chain variable region (LCVR)": Light chain variable
regions are encoded by rearranged nucleic acid molecules and are
either a kappa LCVR or a lambda LCVR. Human kappa LCVRs comprise a
polypeptide, wherein said polypeptide is a product of a member of
family 1 to 7 of human kappa V-gene segments. In the context of the
invention preferred kappa LCVRs are human kappa LCVRs, preferably
human kappa LCVRs which are encoded by a DNA which can be amplified
from human B cells using a primer combination of any one of the
oligonucleotides disclosed as SEQ ID NO:49 to 52 of WO2008/055795A1
with any one oligonucleotide disclosed as SEQ ID NO:53 to 56 of
WO2008/055795A1, and further preferably, PCR conditions described
in Example 3 or of WO2008/055795A1.
[0045] Human lambda LCVRs comprise a polypeptide, wherein said
polypeptide is a product of a member of family 1 to 11 of human
lambda V-gene segments. In the context of the invention preferred
lambda LCVRs are human lambda LCVRs, preferably human lambda LCVRs
which are encoded by a DNA which can be amplified from human B
cells using a primer combination of any one of SEQ ID NO:57 to 65
of WO2008/055795A1 with any one of SEQ ID NO:66 to 68 of
WO2008/055795A1, and further preferably, PCR conditions described
in Example 3 of WO2008/055795A1.
[0046] Typically and preferably, LCVRs comprise three LC CDRs,
preferably LC CDR1, LC CDR2, and LC CDR3, which are embedded into
the light chain framework (LC FR 1-4). Thus, in a preferred
embodiment, a LCVR comprises the following elements in the
following order from the N- to the C-terminus: LC FR1-LC CDR1-LC
FR2-LC CDR2-LC FR3-LC CDR3-LC FR4.
[0047] "heavy chain variable region (HCVR)": Heavy chain variable
regions are encoded by rearranged nucleic acid molecules. Human
HCVRs comprise a polypeptide, wherein said polypeptide is a product
of a member of family 1 to 7 of human lambda V-gene segments. In
the context of the invention preferred HCVRs are human HCVRs,
preferably human HCVRs which are encoded by a DNA which can be
amplified from human B cells using a primer combination of any one
of SEQ ID NO:42 to 47 of WO2008/055795A1 with SEQ ID NO:48 of
WO2008/055795A1 and, further preferably, PCR conditions described
in Example 3 of WO2008/055795A1.
[0048] Typically and preferably, HCVRs comprise three HC CDRs,
preferably HC CDR1, HC CDR2, and HC CDR3, which are embedded into
the heavy chain framework (HC FR 1-4). Thus, in a preferred
embodiment, a HCVR comprises the following elements in the
following order from the N- to the C-terminus: HC FR1-HC CDR1-HC
FR2-HC CDR2-HC FR3-HC CDR3-HC FR4.
[0049] "CDR": The complementarity determining region (CDR) 1, 2 and
3 of the HCVR and of the LCVR, respectively, of an antibody can be
identified by methods generally known in the art. For the purpose
of this application, CDR and FR boundaries are defined as set forth
by Scavinger et al. 1999 (Exp Clin Immunogenet., Vol. 16 pp.
234-240), or by Lefranc et al. 2003 (Developmental and Comparative
Immunology Vol. 27 pp. 55-77).
[0050] "antigen": As used herein, the term "antigen" refers to a
molecule which is bound by an antibody. An antigen is recognized by
the immune system and/or by a humoral immune response and can have
one or more epitopes, preferably B-cell epitopes, or antigenic
determinants.
[0051] "extracellular domain of the influenza A virus M2 protein":
As used herein, the term "extracellular domain of the influenza A
virus M2 protein" refers to the N-terminal extracellular domain of
the M2 protein of influenza A virus, or to any consecutive stretch
of at least 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17,
18, 19, 20, 21, 22, or 23, amino acids thereof. Preferably,
extracellular domain of the influenza A virus M2 protein refers to
amino acid residues 2 to 24 of the influenza A virus M2 protein, or
to any consecutive stretch of at least of at least 3, 4, 5, 6, 7,
8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, or 22 amino
acids thereof. In a more preferred embodiment the extracellular
domain of the influenza A virus M2 protein comprises or consists of
a peptide selected from any one of SEQ ID NOs 48 to 83 and 90 to
92, or to any consecutive stretch of at least of at least 3, 4, 5,
6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, or 22
amino acids thereof. In a very preferred embodiment the
extracellular domain of the influenza A virus M2 protein comprises
or consists of a peptide selected from any one of SEQ ID NOs 48 to
50. Most preferably, the extracellular domain of the influenza A
virus M2 protein is the M2e consensus sequence (SEQ ID NO:48).
TABLE-US-00004 TABLE 4 Variants of the extracellular domain of
influenza A virus M2 protein. Sequences are shown without the
N-terminal Methionine. Influenza A M2e variant aa Sequence SEQ ID
NO aa 2-24 of SEQ ID NO: 84 (PR8) SLLTEVETPIRNEWGCRCNGSSD 51 M2-T:
SLLTEVETPTRNEWGCRCNDSSD 52 M2e consensus SLLTEVETPIRNEWGCRCNDSSD 48
M2e-short SLLTEVETPIRNEWGC 49 M2-KE: SLLTEVETPTKNEWECRCNDSSD 53
M2-K: A/Wisconsin/3523/88 (H1N1) SLLTEVETPIRNEWGCKCNDSSD 54 M2-E:
SLLTEVETPIRNEWECRCNDSSD 55 M2-GE: SLLTEVETPIRNGWECRCNDSSD 56 M2-S:
SLLTEVETPIRSEWGCRCNDSSD 57 M2-FP: SFLPEVETPIRNEWGCRCNDSSD 58
M2-DSSN: SLLTEVETPIRNEWGCRCNDSSN 59 M2-K: SLLTEVETPIRKEWGCRCNDSSD
61 M2-F: FLLTEVETPIRNEWGCRCNDSSD 62 M2-EG:
A/NewYork/687/1995/(H3N2) SLLTEVETPIRNEWECRCNGSSD 63 M2-PS:
SLPTEVETPIRSEWGCRCNDSSD 64 M2-P: SLLPEVETPIRNEWGCRCNDSSD 65 M2-PG:
SLLPEVETPIRNGWGCRCNDSSD 66 M2-TGE: A/DK/ST/5048/2001 (H3N8)
SLLTEVETPTRNGWECRCNDSSD 67 M2-FG: A/X-31 (H3N2)
SFLTEVETPIRNEWGCRCNGSSD 68 M2-EYS: SLLTEVETPIRNEWEYRCSDSSD 69
M2-LTGS: A/HK/156/97 (H9N2) SLLTEVETLTRNGWGCRCSDSSD 70 M2-LTKGS:
A/HK/542/97 (H5N1) SLLTEVETLTKNGWGCRCSDSSD 71 M2-HTES:
SLLTEVETHTRNEWECRCNDSSD 72 M2-TES: A/VN/1203/2004 (H5N1) M2e-VN
SLLTEVETPTRNEWECRCSDSSD 50 M2-TGEK: A/Neth/33/03 (H7N1)
SLLTEVETPTRNGWECKCNDSSD 73 M2-FLTGEKS: SFLTEVETLTRNGWECRCSDSSD 74
M2-LTGEKS: A/HK/1074/99 (H9N2) SLLTEVETLTRNGWECKCSDSSD 75 M2-DLTGS:
A/HK/485/97/(H5N1) SLLTEVDTLTRNGWGCRCSDSSD 76 M2-TGS:
A/chicken/SH/F/98/(H9N2) SLLTEVETPTRNGWGCRCSDSSD 77 M2-KTGEKS:
A/Quail/AR/16309-7/94 SLLTEVKTPTRNGWECKCSDSSD 78 (H7N3NSA)
M2-TDGEKS: A/Chick/Pen/13552-1/98 SLLTEVETPTRDGWECKCSDSSD 79
(H7N2NSB) M2-HTGEKS: A/Chick/CA/1002a/00 SLLTEVETHTRNGWECKCSDSSD 80
(H6N2) M2-P: A/swine/Quebec/192/81/(H1N1) SLPTEVETPIRNEWGCRCNDSSD
81 M2-SG: A/swine/Tenn/25/77/(H1N1) SLLTEVETPIRSEWGCRCNDSGD 82
M2-KGENS: A/Turkey/VA/158512/02 SLLTEVETPIRKGWECNCSDSSD 83 (H7N2)
M2TGEKS: A/Canada/rv504/2004 (H7N3) SLLTEVETPTRNGWECKCSDSSD 90
M2GHTGKS: A/chicken/HongKong/SF1/03 SLLTGVETHTRNGWGCKCSDSSD 91
(H9N2) M2PHTGS: A/chicken/HongKong/YU427/03 SLLPEVETHTRNGWGCRCSDSSD
92 (H9N2)
[0052] "influenza M2e antigen": As used herein the expression
"influenza M2e antigen" refers to an antigen comprising at least
one epitope of the extracellular domain of the influenza A virus M2
protein. In a preferred embodiment, the term influenza M2e antigen
refers to an antigen comprising at least one epitope of any one of
the peptides of SEQ ID NOs 48 to 83 and 90 to 92. More preferably,
the term influenza M2e antigen refers to an antigen comprising or
consisting of the extracellular domain of the influenza A M2
protein. Still more preferably, the term influenza M2e antigen
refers to an antigen comprising or consisting of the peptide of any
one of SEQ ID NOs 48 to 83 and 90 to 92, or to any consecutive
stretch of at least of at least 3, 4, 5, 6, 7, 8, 9, 10, 11, 12,
13, 14, 15, 16, 17, 18, 19, 20, 21, or 22 amino acids thereof. In a
very preferred embodiment, the influenza M2e antigen comprises or
preferably consists of the peptide of any one of SEQ ID NOs 48 to
50. Most preferably, said influenza M2e antigen comprises or
preferably consists of the M2e consensus sequence (SEQ ID
NO:48).
[0053] The term "influenza M2e antigen" also includes conjugates,
fusion or coupling products comprising at least one epitope of the
extracellular domain of the influenza A virus M2 protein. This
includes conjugates of the extracellular domain of the influenza A
virus M2 protein with a carrier, wherein preferably said carrier is
RNAse A. In particular, the term "influenza M2e antigen" also
includes virus-like particles, preferably virus like particles of
RNA bacteriophages, wherein an extracellular domain of the
influenza A virus M2 protein is coupled to said virus like
particle, preferably to said virus-like particle of a RNA
bacteriophage. Preferred virus-like particles are virus-like
particles of RNA bacteriophages Q.beta. or AP205, most preferably
of RNA bacteriophage Q.beta.. A very preferred influenza M2e
antigen is a virus-like particle of RNA bacteriophage Q.beta.,
wherein the peptide of SEQ ID NO:48 is coupled to said virus-like
particle, wherein preferably said peptide is coupled to said
virus-like particle by means of a covalent non-peptide bond. The
term influenza M2e antigen also includes fusion proteins comprising
the extracellular domain of the influenza A virus M2 protein or at
least one epitope thereof. In particular, the term influenza M2e
antigen includes fusion proteins comprising a peptide of any one of
SEQ ID NOs 48 to 83 and 90 to 92. Preferred fusion proteins are
chimeric proteins, wherein said chimeric proteins comprise an
extracellular domain of a first influenza A virus M2 protein and
the transmembrane- and intercellular domain of a second influenza A
virus M2 protein. A very preferred chimeric protein is the protein
of SEQ ID NO:85.
[0054] Furthermore, the term influenza M2e antigen includes virus
particles or virus-like particles comprising the extracellular
domain of the influenza A virus M2 protein or at least one epitope
thereof. Thus, influenza M2e antigen also refers to influenza virus
particles or influenza virus-like particles, preferably to
influenza A virus particles or influenza A virus-like
particles.
[0055] In a preferred embodiment, the term influenza M2e antigen
refers to cells, preferably to eukaryotic cells, comprising the
extracellular domain of the influenza A virus M2 protein on their
cell surface. This includes cells which are infected by influenza
virus, preferably by influenza A virus. This also includes stably
transformed or transfected cells expressing a recombinant protein,
wherein said protein comprises the extracellular domain of the
influenza A virus M2 protein or at least one epitope thereof, and
wherein preferably said recombinant protein comprises a domain
which allows the integration of said recombinant protein into the
cell membrane. Preferred recombinant proteins in this context are
influenza A M2 proteins or chimeric proteins, wherein said chimeric
proteins comprise an extracellular domain of a first influenza A
virus M2 protein and the transmembrane- and intercellular domain of
a second influenza A virus M2 protein. In a preferred embodiment
said recombinant protein is the protein of any one of SEQ ID NOs 84
or 85, preferably SEQ ID NO:84, wherein further preferably the
N-terminal methionine residue of said recombinant proteins is
cleaved off.
[0056] In a further preferred embodiment influenza M2e antigen
refers to a eukaryotic cell, preferably to a L929 cell or to a 293T
cell, wherein said cell is expressing a recombinant protein,
wherein said protein comprises the extracellular domain of the
influenza A virus M2 protein or at least one epitope thereof, and
wherein preferably said recombinant protein comprises a domain
which allows the integration of said recombinant protein into the
cell membrane. Preferred recombinant proteins in this context are
influenza A M2 proteins or chimeric proteins, wherein said chimeric
proteins comprise an extracellular domain of a first influenza A
virus M2 protein and the transmembrane- and intercellular domain of
a second influenza A virus M2 protein. In a preferred embodiment
said recombinant protein is the protein of any one of SEQ ID NOs 84
or 85, wherein further preferably the N-terminal methionine residue
of said recombinant proteins is cleaved off.
[0057] "specifically binding": The specificity of an antibody
relates to the antibody's capability of specifically binding an
antigen. The specificity of this interaction between the antibody
and the antigen (affinity) is characterized by a binding constant
or, inversely, by a dissociation constant (Kd). It is to be
understood that the apparent affinity of an antibody to an antigen
depends on the structure of the antibody and of the antigen, and on
the actual assay conditions. The apparent affinity of an antibody
to an antigen in a multivalent interaction may be significantly
higher than in a monovalent interaction due to avidity. Thus,
affinity is preferably determined under conditions favoring
monovalent interactions. Kd can be determined by methods known in
the art. Preferably, Kd of a given combination of antibody and
antigen is determined by Friguet ELISA essentially as described
(Friguet B. et al., 1985, J. Immunol. Meth. 77, 305-319), wherein a
constant amount of purified antibody, for example scFv or Fab
fragment, is contacted with a serial dilution of a known
concentration of antigen.
[0058] Very preferably, Kd of an antibody and an antigen in
solution is determined by Friguet ELISA, wherein preferably said
antibody is an scFv antibody, most preferably a scFv-msFc.gamma.2c
fusion, and wherein further preferably said antigen is an influenza
M2e antigen, and wherein still more preferably said influenza M2e
antigen comprises or consists of the peptide of any one of SEQ ID
NOs 48 to 83 and 90 to 92, most preferably of the peptide of SEQ ID
NO:48. In one embodiment, said influenza M2e antigen is a conjugate
of RNAse-A and of the peptide of SEQ ID NO:48. In a preferred
embodiment said Friguet-ELISA is performed under conditions
essentially as described in Example 11 herein. In a very preferred
embodiment said Friguet-ELISA is performed under conditions
essentially as described in Example 11, wherein said influenza M2e
antigen is in solution, and wherein said influenza M2e antigen is
SEQ ID NO:48. The affinity of a given combination of antibody and
antigen may also be determined by ELISA, wherein a constant amount
of immobilized antigen is contacted with a serial dilution of a
known concentration of a purified antibody, preferably a scFv or
Fab fragment. The affinity is then determined as the concentration
of the antibody where half-maximal binding is observed (EC50). Very
preferably, EC50 of an antibody and an immobilized antigen is
determined by ELISA, wherein preferably said antibody is an scFv
antibody, most preferably a scFv-msFc.gamma.2c fusion, and wherein
further preferably said antigen is an influenza M2e antigen,
wherein preferably said influenza M2e antigen comprises or
preferably consists of the peptide of any one of SEQ ID NOs 48 to
83, most preferably of the peptide of SEQ ID NO:48. Most
preferably, said influenza M2e antigen is a conjugate of RNAse-A
and of the peptide of SEQ ID NO:48. In a very preferred embodiment
said ELISA is performed as described in the first paragraph of
Example 4 herein. Alternatively, Kd of an interaction of an
antibody and an antigen is determined by Biacore analysis as the
ratio of on rate (k.sub.on) and off rate (k.sub.off). Kd may also
be determined by equilibrium dialysis.
[0059] Lower values of Kd indicate a more specific binding of the
antibody to the antigen than higher values. In the context of the
application, an antibody is considered to be "specifically binding
an antigen", when the dissociation constant (Kd) as determined by
Friguet ELISA as described above is at most 10 nM (<=10.sup.-8
M), preferably at most 1 nM (<=10.sup.-9 M), more preferably at
most 100 pM (<=10.sup.-10 M), still more preferably at most 10
pM (<=10.sup.11 M), most preferably at most 1 pM
(<=10.sup.-12 M). Very preferred are antibodies capable of
binding an antigen with a Kd of less than 20 pM, wherein further
preferably said Kd is determined in solution. In the context of the
application, an antibody is further considered to be "specifically
binding an antigen", when the EC50, preferably determined as
described above is at most 1000 nM (<=10.sup.-6 M), preferably
at most 100 nM (<=10.sup.-7 M), more preferably at most 10 nM
(<=10.sup.-8 M), still more preferably at most 1 nM
(<=10.sup.-9 M), still more preferably at most 100 pM
(<=10.sup.-10 M), still more preferably at most 10 pM
(<=10.sup.11 M), and most preferably at most 1 pM
(<=10.sup.-12 M). Very preferred are antibodies capable of
binding an antigen with a EC50 of less than 100 pM, wherein further
preferably said EC50 is determined with immobilized antigen. In
this context, Kd and/or EC50 values are referred to as being in the
"low picomolar range" when these values are below 100 pM.
[0060] The affinity of an antibody to influenza M2e antigen may
also be determined in an experimental set-up, wherein said
influenza M2e antigen is a cell, typically and preferably a living
cell, and wherein said cell comprises the extracellular domain of
the influenza A virus M2 protein or an epitope thereof on the cell
surface. The affinity of an antibody to a cell is preferably
determined by FACS technology, preferably in an experimental set-up
essentially as disclosed in Examples 5 and 9 herein. It is to be
understood that the EC50 values for a specific combination of
antibody and cells are comparable only within the same experimental
set-up relative to a control antibody. An antibody is regarded as
specifically binding an influenza M2e antigen, wherein said
influenza M2e antigen is a cell, when the EC50 value for the
interaction between a control antibody and said influenza M2e
antigen is at least 10.sup.3-fold, preferably at least
10.sup.4-fold, more preferably at least 10.sup.5-fold, and most
preferably at least 10.sup.6-fold higher than the EC50 value for
the interaction between said antibody and said influenza M2e
antigen.
[0061] "effective amount": A therapeutically effective amount of a
monoclonal antibody of the invention or of a pharmaceutical
composition of the invention generally refers to an amount
necessary to achieve, at dosages and periods of time necessary, the
desired therapeutic result, wherein preferably said result is
preventing, reducing or ameliorating infection with influenza
virus, preferably with influenza A virus. With respect to a
therapeutic treatment of a human, an "effective amount" typically
refers to an amount of 1 mg to 1000 mg, preferably 10 mg to 500 mg,
more preferably 10 mg to 300 mg, still more preferably 50 mg to 200
mg, and most preferably about 100 mg of said monoclonal
antibody.
[0062] "Tag": The term tag, preferably a purification or detection
tag, refers to a polypeptide segment that can be attached to a
second polypeptide to provide for purification or detection of the
second polypeptide or provides sites for attachment of the second
polypeptide to a substrate. In principle, any peptide or protein
for which an antibody or other specific binding agent is available
can be used as an affinity tag. Tags include haemagglutinin tag,
myc tag, poly-histidine tag, protein A, glutathione S transferase,
Glu-Glu affinity tag, substance P, FLAG peptide, streptavidin
binding peptide, or other antigenic epitope or binding domain
(mostly taken from U.S. Pat. No. 6,686,168).
[0063] A library-based screening method for the identification,
isolation and cloning of scFv specifically binding an antigen of
interest is disclosed in WO2008/055795A1. In particular, said
method allows for the identification, isolation and cloning of
human scFv and for the subsequent generation of fully human
antibodies, including Fab fragments and whole IgG. Applying said
technology, human monoclonal antibodies specifically binding
influenza M2e antigen have been identified and cloned.
[0064] In one aspect, the invention provides a monoclonal antibody,
preferably an isolated monoclonal antibody, specifically binding
influenza M2e antigen, wherein said monoclonal antibody is a human
monoclonal antibody, preferably a fully human monoclonal antibody.
In a preferred embodiment said monoclonal antibody is a recombinant
monoclonal antibody.
[0065] In a preferred embodiment said human monoclonal antibody,
preferably said fully human monoclonal antibody, is not recognized
by the human immune system.
[0066] The specificity of an antibody is mainly determined by the
amino acid sequence of the complementarity determining regions
(CDRs) in the heavy chain variable regions (HCVR) of said antibody
and/or by the CDRs in the light chain variable regions said
antibody (LCVR). The invention discloses CDRs of HCVRs (HC CDRs)
and CDRs of the LCVRs (LC CDRs) of monoclonal antibodies, wherein
said monoclonal antibodies are capable of specifically binding
influenza A M2, and wherein typically and preferably said HCVRs and
said LCVRs are forming an antigen binding site.
[0067] It has surprisingly been found that the monoclonal
antibodies of the invention which are capable of specifically
binding influenza M2e antigen share identical or closely related HC
CDRs and/or LC CDRs in different combinations, wherein the closely
related HC CDRs and/or LC CDRs differ in at most a few amino acid
residues (cf. Tables 1-3). It is therefore concluded that all
antibodies of Tables 1-3 are clonally related.
[0068] Thus, in one embodiment said monoclonal antibody comprises
(i) at least one, preferably exactly one, LCVR, wherein said LCVR
comprises: (a) one LC CDR1, wherein said LC CDR1 consists of the
peptide of any one of SEQ ID NOs 1, 2, 3, 4, 5, and 6, and wherein
preferably said LC CDR1 is located at CDR1 position within the
framework of said LCVR; (b) one LC CDR2, wherein said LC CDR2
consists of the peptide of SEQ ID NO:7, and wherein preferably said
LC CDR2 is located at CDR2 position within the framework of said
LCVR; (c) one LC CDR3, wherein said LC CDR3 consists of the peptide
of any one of SEQ ID NOs 8, 9, 10, and 11, and wherein preferably
said LC CDR3 is located at CDR3 position within the framework of
said LCVR; and/or said monoclonal comprises (ii) at least one,
preferably exactly one HCVR, wherein said HCVR comprises: (a) one
HC CDR1, wherein said HC CDR1 consists of the peptide of SEQ ID
NO:12, and wherein preferably said HC CDR1 is located at CDR1
position within the framework of said HCVR; (b) one HC CDR2,
wherein said HC CDR2 consists of the peptide of any one of SEQ ID
NOs 13 and 14, and wherein preferably said HC CDR2 is located at
CDR2 position within the framework of said HCVR; and (c) one HC
CDR3, wherein said HC CDR3 consists of the peptide of any one of
SEQ ID NOs 15, 16, 17, 18, and 19, and wherein preferably said HC
CDR3 is located at CDR3 position within the framework of said
HCVR.
[0069] In a further embodiment said monoclonal antibody comprises
(i) at least one, preferably exactly one, LCVR, wherein said LCVR
comprises: (a) one LC CDR1, wherein said LC CDR1 consists of the
peptide of any one of SEQ ID NOs 1, 2, 3, 4, 5, and 6; (b) one LC
CDR2, wherein said LC CDR2 consists of the peptide of SEQ ID NO:7;
(c) one LC CDR3, wherein said LC CDR3 consists of the peptide of
any one of SEQ ID NOs 8, 9, 10, and 11; and wherein said monoclonal
comprises (ii) at least one, preferably exactly one HCVR, wherein
said HCVR comprises: (a) one HC CDR1, wherein said HC CDR1 consists
of the peptide of SEQ ID NO:12; (b) one HC CDR2, wherein said HC
CDR2 consists of the peptide of any one of SEQ ID NOs 13 and 14;
and (c) one HC CDR3, wherein said HC CDR3 consists of the peptide
of any one of SEQ ID NOs 15, 16, 17, 18, and 19.
[0070] Typically and preferably, said monoclonal antibody comprises
exactly one LCVR and exactly one HCVR, which are preferably forming
an antigen binding site. In case of an antibody in the IgG format,
said monoclonal antibody comprises exactly two LCVR and exactly two
HCVR. However, the antibody of the invention may also comprise or
alternatively consist of only one LCVR or of only one HCVR. Thus,
in a further preferred embodiment said monoclonal antibody
comprises or alternatively consists of at least one, preferably
exactly one, LCVR, wherein said LCVR comprises: (a) one LC CDR1,
wherein said LC CDR1 consists of the peptide of any one of SEQ ID
NOs 1, 2, 3, 4, 5, and 6; (b) one LC CDR2, wherein said LC CDR2
consists of the peptide of SEQ ID NO:7; (c) one LC CDR3, wherein
said LC CDR3 consists of the peptide of any one of SEQ ID NOs 8, 9,
10, and 11.
[0071] In a further preferred embodiment said monoclonal antibody
comprises or alternatively consists of at least one, preferably
exactly one HCVR, wherein said HCVR comprises: (a) one HC CDR1,
wherein said HC CDR1 consists of the peptide of SEQ ID NO:12; (b)
one HC CDR2, wherein said HC CDR2 consists of the peptide of any
one of SEQ ID NOs 13 and 14; and (c) one HC CDR3, wherein said HC
CDR3 consists of the peptide of any one of SEQ ID NOs 15, 16, 17,
18, and 19.
[0072] Typically and preferably the antibodies of the invention
comprise at least on antigen binding site comprising one LCVR and
one HCVR, wherein said antigen binding site recognizes an epitope
of influenza A M2e antigen. One aspect of the invention therefore
is an isolated monoclonal antibody, wherein said monoclonal
antibody is specifically binding influenza M2e antigen, and wherein
preferably said monoclonal antibody is a human monoclonal antibody,
more preferably a fully human monoclonal antibody, and wherein said
antibody comprises at least one antigen binding site, wherein said
antigen binding site comprises: (a) one LCVR, wherein said LCVR
comprises: (i) one LC CDR1, wherein said LC CDR1 consists of the
peptide of any one of SEQ ID NOs 1, 2, 3, 4, 5, and 6; (ii) one LC
CDR2, wherein said LC CDR2 consists of the peptide of SEQ ID NO:7;
and (iii) one LC CDR3, wherein said LC CDR3 consists of the peptide
of any one of SEQ ID NOs 8, 9, 10, and 11; and (b) one HCVR,
wherein said HCVR comprises: (i) one HC CDR1, wherein said HC CDR1
consists of the peptide of SEQ ID NO:12; (ii) one HC CDR2, wherein
said HC CDR2 consists of the peptide of any one of SEQ ID NOs 13
and 14; and (iii) one HC CDR3, wherein said HC CDR3 consists of the
peptide of any one of SEQ ID NOs 15, 16, 17, 18, and 19.
[0073] Based on sequence similarities between the amino acid
sequences of LC CDR1 and LC CDR3 three closely relates groups of
antibodies can be distinguished (cf. Table 1). Thus, in one
embodiment said LCVR comprises: (a) one LC CDR1, wherein said LC
CDR1 consists of the peptide of any one of SEQ ID NOs 1, 2, and 3;
(b) one LC CDR2, wherein said LC CDR2 consists of the peptide of
SEQ ID NO:7; (c) one LC CDR3, wherein said LC CDR3 consists of the
peptide of any one of SEQ ID NOs 8, 9 and 10; and wherein
preferably said HCVR comprises: (a) one HC CDR1, wherein said HC
CDR1 consists of the peptide of SEQ ID NO:12; (b) one HC CDR2,
wherein said HC CDR2 consists of the peptide of any one of SEQ ID
NOs 13 and 14; and (c) one HC CDR3, wherein said HC CDR3 consists
of the peptide of any one of SEQ ID NOs 15, 16, 17, 18, and 19.
[0074] In a further embodiment LCVR comprises: (a) one LC CDR1,
wherein said LC CDR1 consists of the peptide of any one of SEQ ID
NOs 4 and 5; (b) one LC CDR2, wherein said LC CDR2 consists of the
peptide of SEQ ID NO:7; (c) one LC CDR3, wherein said LC CDR3
consists of the peptide of SEQ ID NO:8; and wherein preferably said
HCVR comprises: (a) one HC CDR1, wherein said HC CDR1 consists of
the peptide of SEQ ID NO:12; (b) one HC CDR2, wherein said HC CDR2
consists of the peptide of any one of SEQ ID NOs 13 and 14; and (c)
one HC CDR3, wherein said HC CDR3 consists of the peptide of any
one of SEQ ID NOs 15, 16, 17, 18, and 19.
[0075] In a further embodiment, said LCVR comprises: (a) one LC
CDR1, wherein said LC CDR1 consists of the peptide of any one of
SEQ ID NOs 1 and 6; (b) one LC CDR2, wherein said LC CDR2 consists
of the peptide of SEQ ID NO:7; (c) one LC CDR3, wherein said LC
CDR3 consists of the peptide of SEQ ID NO:8 and 11; and wherein
preferably said HCVR comprises: (a) one HC CDR1, wherein said HC
CDR1 consists of the peptide of SEQ ID NO:12; (b) one HC CDR2,
wherein said HC CDR2 consists of the peptide of any one of SEQ ID
NOs 13 and 14; and (c) one HC CDR3, wherein said HC CDR3 consists
of the peptide of any one of SEQ ID NOs 15, 16, 17, 18, and 19.
[0076] Among the antibodies of the invention nine different types
of LCVRs were identified comprising different combinations of LC
CDR sequences (cf. Table 1). Thus, in a preferred embodiment said
LCVR is selected from: (a) a LCVR, wherein (i) said LC CDR1
consists of the peptide of SEQ ID NO:1, (ii) said LC CDR2 consists
of the peptide of SEQ ID NO:7, and (iii) said LC CDR3 consists of
the peptide of SEQ ID NO:8; (b) a LCVR, wherein (i) said LC CDR1
consists of the peptide of SEQ ID NO:2, (ii) said LC CDR2 consists
of the peptide of SEQ ID NO:7, and (iii) said LC CDR3 consists of
the peptide of SEQ ID NO:8; (c) a LCVR, wherein (i) said LC CDR1
consists of the peptide of SEQ ID NO:3, (ii) said LC CDR2 consists
of the peptide of SEQ ID NO:7, and (iii) said LC CDR3 consists of
the peptide of SEQ ID NO:8; (d) a LCVR, wherein (i) said LC CDR1
consists of the peptide of SEQ ID NO:1, (ii) said LC CDR2 consists
of the peptide of SEQ ID NO:7, and (iii) said LC CDR3 consists of
the peptide of SEQ ID NO:9; (e) a LCVR, wherein (i) said LC CDR1
consists of the peptide of SEQ ID NO:1, (ii) said LC CDR2 consists
of the peptide of SEQ ID NO:7, and (iii) said LC CDR3 consists of
the peptide of SEQ ID NO:10; (f) a LCVR, wherein (i) said LC CDR1
consists of the peptide of SEQ ID NO:4, (ii) said LC CDR2 consists
of the peptide of SEQ ID NO:7, and (iii) said LC CDR3 consists of
the peptide of SEQ ID NO:8; (g) a LCVR, wherein (i) said LC CDR1
consists of the peptide of SEQ ID NO:5, (ii) said LC CDR2 consists
of the peptide of SEQ ID NO:7, and (iii) said LC CDR3 consists of
the peptide of SEQ ID NO:8; (h) a LCVR, wherein (i) said LC CDR1
consists of the peptide of SEQ ID NO:6, (ii) said LC CDR2 consists
of the peptide of SEQ ID NO:7, and (iii) said LC CDR3 consists of
the peptide of SEQ ID NO:8; and (i) a LCVR, wherein (i) said LC
CDR1 consists of the peptide of SEQ ID NO:1, (ii) said LC CDR2
consists of the peptide of SEQ ID NO:7, and (iii) said LC CDR3
consists of the peptide of SEQ ID NO:11.
[0077] Among the antibodies of the invention the most abundant
combinations of LC CDRs are types 1A, 1A and 3A (cf. Table 1).
Thus, in a preferred embodiment said LCVR is selected from: (a) a
LCVR, wherein (i) said LC CDR1 consists of the peptide of SEQ ID
NO:1, (ii) said LC CDR2 consists of the peptide of SEQ ID NO:7, and
(iii) said LC CDR3 consists of the peptide of SEQ ID NO:8; (b) a
LCVR, wherein (i) said LC CDR1 consists of the peptide of SEQ ID
NO:4, (ii) said LC CDR2 consists of the peptide of SEQ ID NO:7, and
(iii) said LC CDR3 consists of the peptide of SEQ ID NO:8; and (c)
a LCVR, wherein (i) said LC CDR1 consists of the peptide of SEQ ID
NO:6, (ii) said LC CDR2 consists of the peptide of SEQ ID NO:7, and
(iii) said LC CDR3 consists of the peptide of SEQ ID NO:8. In a
very preferred embodiment, said LCVR is selected from: (a) a LCVR,
wherein (i) said LC CDR1 consists of the peptide of SEQ ID NO:1,
(ii) said LC CDR2 consists of the peptide of SEQ ID NO:7, and (iii)
said LC CDR3 consists of the peptide of SEQ ID NO:8.
[0078] Antibodies obtained from library screening may differ from
fully human antibodies in certain amino acid positions of the VRs
due to cloning artifacts, wherein typically these positions are
located near the N- and/or C-terminus of the variable region.
Preferably, these artifacts are removed from the antibody in order
to obtain fully human antibodies. For example, in order to obtain
fully human LCVRs, position 1 to 4 of the LCVRs is replaced by SEQ
ID NO:24. Thus, in a preferred embodiment position 1 to 4 of said
LCVR consists of SEQ ID NO:24.
[0079] In a preferred embodiment said LCVR comprises or preferably
consists of any one of the amino acid sequences depicted in FIG. 1.
In a further preferred embodiment position 5 to 113 of said LCVR
consists of the peptide of any one of SEQ ID NOs 20, 21 and 22,
wherein preferably position 1 to 4 of said LCVR consists of the
peptide of SEQ ID NO:24.
[0080] In a further preferred embodiment position 5 to 113 of said
LCVR consists of a peptide, wherein said peptide is encoded by the
nucleic acid sequence of any one of SEQ ID NOs 86, 87, and 88,
wherein preferably position 1 to 4 of said LCVR consists of the
peptide of SEQ ID NO:24.
[0081] Furthermore, among the antibodies of the invention six
different types of HCVRs were identified comprising different
combinations of HC CDR sequences (cf. Table 2). Thus, in a
preferred embodiment said HCVR is selected from: (a) a HCVR,
wherein (i) said HC CDR1 consists of the peptide of SEQ ID NO:12,
(ii) said HC CDR2 consists of the peptide of SEQ ID NO:13, and
(iii) said HC CDR3 consists of the peptide of SEQ ID NO:15; (b) a
HCVR, wherein (i) said HC CDR1 consists of the peptide of SEQ ID
NO:12, (ii) said HC CDR2 consists of the peptide of SEQ ID NO:13,
and (iii) said HC CDR3 consists of the peptide of SEQ ID NO:16; (c)
a HCVR, wherein (i) said HC CDR1 consists of the peptide of SEQ ID
NO:12, (ii) said HC CDR2 consists of the peptide of SEQ ID NO:13,
and (iii) said HC CDR3 consists of the peptide of SEQ ID NO:17; (d)
a HCVR, wherein (i) said HC CDR1 consists of the peptide of SEQ ID
NO:12, (ii) said HC CDR2 consists of the peptide of SEQ ID NO:13,
and (iii) said HC CDR3 consists of the peptide of SEQ ID NO:18; (e)
a HCVR, wherein (i) said HC CDR1 consists of the peptide of SEQ ID
NO:12, (ii) said HC CDR2 consists of the peptide of SEQ ID NO:14,
and (iii) said HC CDR3 consists of the peptide of SEQ ID NO:15; and
(f) a HCVR, wherein (i) said HC CDR1 consists of the peptide of SEQ
ID NO:12, (ii) said HC CDR2 consists of the peptide of SEQ ID
NO:13, and (iii) said HC CDR3 consists of the peptide of SEQ ID
NO:19.
[0082] Among the antibodies of the invention the most abundant
combination of HC CDRs is type 1A (cf. Table 2). Thus, in a
preferred embodiment said HCVR is selected from: (a) a HCVR,
wherein (i) said HC CDR1 consists of the peptide of SEQ ID NO:12,
(ii) said HC CDR2 consists of the peptide of SEQ ID NO:13, and
(iii) said HC CDR3 consists of the peptide of SEQ ID NO:15;
[0083] In order to remove cloning artifacts and to obtain fully
human HCVRs, typically and preferably position 1 to 6 of the HCVRs
is replaced by SEQ ID NO:25. Thus, in a preferred embodiment
position 1 to 6 of said HCVR consists of SEQ ID NO:25.
[0084] In a preferred embodiment said HCVR comprises or preferably
consists of any one of the amino acid sequences depicted in FIG. 2.
In a further preferred embodiment position 7 to 121 of said HCVR
consists of the peptide of SEQ ID NO:23, wherein preferably
position 1 to 6 of said HCVR consists of the peptide of SEQ ID
NO:25. In a further preferred embodiment position 7 to 121 of said
HCVR consists of a peptide, wherein said peptide is encoded by the
nucleic acid sequence of SEQ ID NO:89, wherein preferably position
1 to 6 of said HCVR consists of the peptide of SEQ ID NO:25.
[0085] Among the antibodies of the invention fourteen different
combinations of LC CDRs and HC CDRs were identified (cf. Table 2).
Thus in one embodiment said monoclonal antibody comprises at least
one, preferably exactly one antigen binding site, wherein the
combination of LC CDRs and HC CDRs is selected from any one of the
combinations of Table 3. Preferably said combination is chosen from
one of the most abundant combinations 1A-1A, 1A-1B, 2A-1A, and
3A-1A.
[0086] In a very preferred embodiment said LC CDR1 consists of the
peptide of any one of SEQ ID NOs 1, 4 and 6, said LC CDR2 consists
of the peptide of SEQ ID NO:7, said LC CDR3 consists of the peptide
of SEQ ID NO:8, said HC CDR1 consists of the peptide of SEQ ID
NO:12, said HC CDR2 consists of the peptide of SEQ ID NO:13, and
said HC CDR3 consists of the peptide of any one of SEQ ID NOs 15
and 16.
[0087] In a very preferred embodiment said LC CDR1 consists of the
peptide of SEQ ID NO:1, said LC CDR2 consists of the peptide of SEQ
ID NO:7, said LC CDR3 consists of the peptide of SEQ ID NO:8, said
HC CDR1 consists of the peptide of SEQ ID NO:12, said HC CDR2
consists of the peptide of SEQ ID NO:13, and said HC CDR3 consists
of the peptide of SEQ ID NO:15.
[0088] In a very preferred embodiment said LC CDR1 consists of the
peptide of SEQ ID NO:1, said LC CDR2 consists of the peptide of SEQ
ID NO:7, said LC CDR3 consists of the peptide of SEQ ID NO:8, said
HC CDR1 consists of the peptide of SEQ ID NO:12, said HC CDR2
consists of the peptide of SEQ ID NO:13, and said HC CDR3 consists
of the peptide of SEQ ID NO:16.
[0089] In a very preferred embodiment said LC CDR1 consists of the
peptide of SEQ ID NO:4, said LC CDR2 consists of the peptide of SEQ
ID NO:7, said LC CDR3 consists of the peptide of SEQ ID NO:8, said
HC CDR1 consists of the peptide of SEQ ID NO:12, said HC CDR2
consists of the peptide of SEQ ID NO:13, and said HC CDR3 consists
of the peptide of SEQ ID NO:15.
[0090] In a very preferred embodiment said LC CDR1 consists of the
peptide of SEQ ID NO:6, said LC CDR2 consists of the peptide of SEQ
ID NO:7, said LC CDR3 consists of the peptide of SEQ ID NO:8, said
HC CDR1 consists of the peptide of SEQ ID NO:12, said HC CDR2
consists of the peptide of SEQ ID NO:13, and said HC CDR3 consists
of the peptide of SEQ ID NO:15.
[0091] In a preferred embodiment said LCVR comprises a peptide,
wherein said peptide consists of any one of the amino acid
sequences depicted in FIG. 1; and said HCVR comprises a peptide,
wherein said peptide consists of any one of the amino acid
sequences depicted in FIG. 2.
[0092] In a very preferred embodiment LCVR comprises a peptide,
wherein said peptide consists of any one of the amino acid
sequences depicted in FIG. 1; and said HCVR comprises a peptide,
wherein said peptide consists the amino acid sequence depicted in
FIG. 2, wherein said amino acid sequence depicted in FIG. 2 has the
same designator as the amino acid sequence depicted in FIG. 1. The
designator is a character followed by three digits (e.g. D005).
[0093] In a preferred embodiment said antigen binding site
comprises one LCVR and one HCVR, (a) wherein position 5 to 113 of
said LCVR consists of the peptide of any one of SEQ ID NOs 20, 21
and 22, and (b) wherein position 7 to 121 of said HCVR consists of
the peptide of SEQ ID NO:23, and wherein preferably position 1 to 4
of said LCVR consists of the peptide of SEQ ID NO:24, and/or
wherein further preferably position 1 to 6 of said HCVR consists of
the peptide of SEQ ID NO:25.
[0094] In a preferred embodiment said antigen binding site
comprises one LCVR and one HCVR, (a) wherein position 5 to 113 of
said LCVR consists of the peptide of SEQ ID NO:20, and (b) wherein
position 7 to 121 of said HCVR consists of the peptide of SEQ ID
NO:23, and wherein preferably position 1 to 4 of said LCVR consists
of the peptide of SEQ ID NO:24, and/or wherein further preferably
position 1 to 6 of said HCVR consists of the peptide of SEQ ID
NO:25.
[0095] In a preferred embodiment said antigen binding site
comprises one LCVR and one HCVR, (a) wherein position 5 to 113 of
said LCVR consists of the peptide of SEQ ID NO:21, and (b) wherein
position 7 to 121 of said HCVR consists of the peptide of SEQ ID
NO:23, and wherein preferably position 1 to 4 of said LCVR consists
of the peptide of SEQ ID NO:24, and/or wherein further preferably
position 1 to 6 of said HCVR consists of the peptide of SEQ ID
NO:25.
[0096] In a preferred embodiment said antigen binding site
comprises one LCVR and one HCVR, (a) wherein position 5 to 113 of
said LCVR consists of the peptide of SEQ ID NO:22, and (b) wherein
position 7 to 121 of said HCVR consists of the peptide of SEQ ID
NO:23, and wherein preferably position 1 to 4 of said LCVR consists
of the peptide of SEQ ID NO:24, and/or wherein further preferably
position 1 to 6 of said HCVR consists of the peptide of SEQ ID
NO:25.
[0097] In a further preferred embodiment said antigen binding site
comprises one LCVR and one HCVR, (a) wherein position 5 to 113 of
said LCVR consists of a peptide, wherein said peptide is encoded by
the nucleic acid sequence of any one of SEQ ID NOs 86, 87 and 88,
and (b) wherein position 7 to 121 of said HCVR consists of a
peptide, wherein said peptide is encoded by the nucleic acid
sequence of SEQ ID NO:89, and wherein preferably position 1 to 4 of
said LCVR consists of the peptide of SEQ ID NO:24, and/or wherein
further preferably position 1 to 6 of said HCVR consists of the
peptide of SEQ ID NO:25.
[0098] In a further preferred embodiment position 5 to 113 of said
LCVR is at least 80%, preferably 85%, more preferably 90%, again
more preferably 95%, even more preferably at least 96%, still more
preferably 97%, again still more preferably 98%, again still more
preferably 99% and most preferably 100% identical to the peptide of
any one of SEQ ID NOs 20, 21, and 22, preferably to the peptide of
SEQ ID NO:24; and position 7 to 121 of said HCVR is at least 80%,
preferably 85%, more preferably 90%, again more preferably 95%,
even more preferably at least 96%, still more preferably 97%, again
still more preferably 98%, again still more preferably 99% and most
preferably 100% identical to the peptide of SEQ ID NO:23.
[0099] In a further preferred embodiment said LCVR is at least 80%,
preferably 85%, more preferably 90%, again more preferably 95%,
even more preferably at least 96%, still more preferably 97%, again
still more preferably 98%, again still more preferably 99% and most
preferably 100% identical to any one of the amino acid sequences
depicted in FIG. 1; and said HCVR is at least 80%, preferably 85%,
more preferably 90%, again more preferably 95%, even more
preferably at least 96%, still more preferably 97%, again still
more preferably 98%, again still more preferably 99% and most
preferably 100% identical to any one of the amino acid sequences
depicted in FIG. 2.
[0100] In a further preferred embodiment said LCVR is at least 95%,
preferably at least 96%, more preferably 97%, still more preferably
98%, still more preferably 99%, and most preferably 100% identical
to any one of the amino acid sequences depicted in FIG. 1; and
wherein said HCVR is at least 95%, preferably at least 96%, more
preferably 97%, still more preferably 98%, still more preferably
99%, and most preferably 100% identical to any one of the amino
acid sequences depicted in FIG. 2.
[0101] In a further preferred embodiment the differences in the
amino acid sequences referred to above are located outside of the
CDR positions of said LCVR and/or of said HCVR. Whether the amino
acid sequence of a peptide or polypeptide has an amino acid
sequence that is at least 80%, 85%, 90%, 95%, 97% or 99% identical
to another, can be determined conventionally using known computer
programs such the Bestfit program.
[0102] A monoclonal antibody of the invention can be recombinantly
produced in any naturally occurring or synthetic format. The
following embodiments thus explicitly refer to all aspects and
embodiments of the invention. In one embodiment said monoclonal
antibody is a recombinant antibody. In a preferred embodiment the
monoclonal antibody of the invention is an antibody selected from
the group consisting of: (a) single chain antibody, preferably
scFv; (b) Fab fragment; (c) F(ab')2 fragment; (d) scFv-Fc fusion;
(e) IgG1; (f) IgG2; (g) IgG3; (h) IgG4; (i) IgA; (j) IgE; (k) IgM;
(l) IgD; and (m) diabody. In a preferred embodiment said monoclonal
antibody comprises or preferably consists of exactly one LCVR
and/or of exactly one HCVR.
[0103] In a further preferred embodiment said monoclonal antibody
is a single chain antibody, wherein preferably said single chain
antibody is an scFv. In a further preferred embodiment said single
chain antibody comprises or preferably consists of a peptide,
wherein said peptide is encoded by the nucleic acid sequence of any
one of SEQ ID NOs 38 to 40. In a further preferred embodiment said
single chain antibody is an Fc-fusion, preferably a Fc.gamma.2c
fusion, wherein further preferably said Fc.gamma.2c fusion
comprises or preferably consists of the a peptide, wherein said
peptide is encoded by any one of SEQ ID NOs 42, 44, and 46. In a
further preferred embodiment said single chain antibody comprises
the peptide of any one of SEQ ID NOs 43, 45, and 47.
[0104] In a further preferred embodiment said monoclonal antibody
is an IgG, preferably a human IgG. In a further preferred
embodiment said monoclonal antibody is a IgG1, preferably a human
IgG1. In a further preferred embodiment said monoclonal antibody
comprises at least one, preferably exactly two, kappa LC(s),
wherein preferably said kappa LC(s) comprise(s) or more preferably
consist(s) of a peptide, wherein said peptide is at least 80%,
preferably 85%, more preferably 90%, again more preferably 95%,
even more preferably at least 96%, still more preferably 97%, again
still more preferably 98%, again still more preferably 99% and most
preferably 100% identical to any one of SEQ ID NOs 26 to 28. In a
further preferred embodiment said monoclonal antibody is a human
IgG1, wherein said human IgG1 comprises at least one, preferably
exactly two, kappa LC(s), wherein said kappa LC(s) comprise(s) or
preferably consist(s) of the peptide of any one of SEQ ID NOs 26 to
28.
[0105] In a further preferred embodiment said monoclonal antibody
comprises at least one, preferably exactly two, gamma 1 HC(s),
wherein preferably said gamma 1 HC(s) comprise(s) or more
preferably consist(s) of a peptide, wherein said peptide is at
least 80%, preferably 85%, more preferably 90%, again more
preferably 95%, even more preferably at least 96%, still more
preferably 97%, again still more preferably 98%, again still more
preferably 99% and most preferably 100% identical to SEQ ID NO:29.
In a further preferred embodiment said gamma 1 HC(s) comprise(s) or
preferably consist(s) of the peptide of SEQ ID NO:29.
[0106] In a very preferred embodiment said monoclonal antibody is a
human IgG1, wherein said human IgG1 comprises at least one,
preferably exactly two, kappa LC(s), wherein said kappa LC(s)
comprise(s) or more preferably consist(s) of a peptide of any one
of SEQ ID NOs 26 to 28, and wherein said monoclonal antibody
comprises at least one, preferably exactly two, gamma 1 HC(s),
wherein said gamma 1 HC(s) comprise(s) or more preferably
consist(s) of the of SEQ ID NO:29.
[0107] In a very preferred embodiment said monoclonal antibody is a
human IgG1, wherein said human IgG1 comprises at least one,
preferably exactly two, kappa LC(s), wherein said kappa LC(s)
comprise(s) or more preferably consist(s) of the peptide of SEQ ID
NO:26, and wherein said monoclonal antibody comprises at least one,
preferably exactly two, gamma 1 HC(s), wherein said gamma 1 HC(s)
comprise(s) or more preferably consist(s) of the of SEQ ID
NO:29.
[0108] In a very preferred embodiment said monoclonal antibody is a
human IgG1, wherein said human IgG1 comprises at least one,
preferably exactly two, kappa LC(s), wherein said kappa LC(s)
comprise(s) or more preferably consist(s) of the peptide of SEQ ID
NO:27, and wherein said monoclonal antibody comprises at least one,
preferably exactly two, gamma 1 HC(s), wherein said gamma 1 HC(s)
comprise(s) or more preferably consist(s) of the of SEQ ID
NO:29.
[0109] In a very preferred embodiment said monoclonal antibody is a
human IgG1, wherein said human IgG1 comprises at least one,
preferably exactly two, kappa LC(s), wherein said kappa LC(s)
comprise(s) or more preferably consist(s) of the peptide of SEQ ID
NO:28, and wherein said monoclonal antibody comprises at least one,
preferably exactly two, gamma 1 HC(s), wherein said gamma 1 HC(s)
comprise(s) or more preferably consist(s) of the of SEQ ID
NO:29.
[0110] Preferably, the monoclonal antibody of the invention is an
IgG1, preferably a human IgG1, most preferably a fully human IgG1.
Thus, in a preferred embodiment said monoclonal antibody comprises
two, preferably exactly two, of said gamma 1 HCs, wherein further
preferably said two, preferably said exactly two of said gamma 1
HCs are identical.
[0111] In a further preferred embodiment said monoclonal antibody
comprises two, preferably exactly two LCs, wherein preferably said
LCs are selected from (a) lambda LC; and (b) kappa LC, most
preferably kappa LC; wherein still further preferably said two,
preferably said exactly two of said LCs are identical.
[0112] In a preferred embodiment said monoclonal antibody,
preferably said isolated monoclonal antibody, is specifically
binding influenza M2e antigen, wherein said influenza M2e antigen
comprises or consists of at least one epitope of the extracellular
domain of the influenza A M2 protein. In a further preferred
embodiment said influenza M2e antigen comprises or consists of the
extracellular domain of the influenza A M2 protein, preferably of
amino acids 2 to 24 of the influenza A M2 protein. In a further
preferred embodiment, said influenza M2e antigen comprises or
preferably consists of the peptide of any one of SEQ ID NOs 48 to
83 and 90 to 92, wherein preferably said influenza M2e antigen
comprises or preferably consists of the peptide of any one of SEQ
ID NOs 48 to 50, and wherein most preferably said influenza M2e
antigen comprises or preferably consists of the peptide of SEQ ID
NO:48.
[0113] In a further preferred embodiment, said influenza M2e
antigen comprises or consists of at least one epitope of SEQ ID
NO:48, preferably said influenza M2e antigen comprises or consists
of an epitope comprised by the amino acid sequence of SEQ ID
NO:93.
[0114] In a further preferred embodiment said monoclonal antibody
is specifically binding influenza M2e antigen, wherein the EC50
value and/or the dissociation constant (Kd) of said monoclonal
antibody and said influenza M2e antigen is at most 1000 nM
(<=10.sup.-6 M), preferably at most 100 nM (<=10.sup.-7 M),
more preferably at most 10 nM (<=10.sup.-8 M), still more
preferably at most 1 nM (<=10.sup.-9 M), still more preferably
at most 100 pM (<=10.sup.-10 M), still more preferably at most
10 pM (<=10.sup.-11 M), and most preferably at most 1 pM
(<=10.sup.-12 M), wherein preferably said influenza M2e antigen
is the extracellular domain of influenza A M2 protein, wherein
preferably said influenza M2e antigen is the peptide of any one of
SEQ ID NOs 48 to 83, more preferably of any one of SEQ ID NOs 48 to
50, and most preferably of SEQ ID NO:48, and wherein still further
preferably said Kd is determined by Friguet-ELISA, most preferably
under conditions essentially as described in Example 11, and/or
wherein said EC50 value is determined by ELISA, preferably under
conditions essentially as described in the first paragraph of
Example 4 herein.
[0115] In a further preferred embodiment said monoclonal antibody
is specifically binding influenza M2e antigen, wherein the
dissociation constant (Kd) of said monoclonal antibody and said
influenza M2e antigen is at most 100 nM, preferably at most 10 nM,
more preferably at most 6 nM and most preferably at most 5 nM,
wherein preferably said influenza M2e antigen is SEQ ID NO:48, most
preferably in solution, and wherein still further preferably said
Kd is determined by Friguet-ELISA, most preferably under conditions
essentially as described in Example 11.
[0116] In a further preferred embodiment said monoclonal antibody
is specifically binding influenza M2e antigen, the dissociation
constant (Kd) of said monoclonal antibody and said influenza M2e
antigen is 1 to 100 nM, preferably 1 to 10 nM, more preferably 1 to
6 nM, still more preferably 3 to 6 nM, and most preferably 4 to 5
nM, wherein further preferably said influenza M2e antigen is SEQ ID
NO:48, most preferably in solution, and wherein still further
preferably said Kd is determined by Friguet-ELISA, most preferably
under conditions essentially as described in Example 11.
[0117] In a further preferred embodiment said monoclonal antibody
is specifically binding influenza M2e antigen, wherein the
dissociation constant (Kd) of said monoclonal antibody and said
influenza M2e antigen is 0.01 pM to 1000 nM, preferably 0.1 pM to
100 nM, more preferably 0.1 pM to 10 nM, still more preferably 0.1
pM to 1 nM, still more preferably 0.1 pM to 100 pM, still more
preferably 0.1 pM to 50 pM, still more preferably 0.1 pM to 20 pM,
still more preferably 0.1 pM to 15 pM, still more preferably 1 pM
to 15 pM, and most preferably 1 pM to 10 pM, wherein preferably
said influenza M2e antigen is an RNAse conjugate of the
extracellular domain of influenza A M2 protein, most preferably in
solution, wherein further preferably said influenza M2e antigen
comprises the peptide of any one of SEQ ID NOs 48 to 83 and 90 to
92, more preferably of any one of SEQ ID NOs 48 to 50, and most
preferably of SEQ ID NO:48, and wherein still further preferably
said Kd is determined by Friguet-ELISA, most preferably under
conditions essentially as described in Example 11.
[0118] In a further preferred embodiment the said monoclonal
antibody is specifically binding influenza M2e antigen, wherein
said influenza M2e antigen is a cell comprising at least one
epitope of the extracellular domain of influenza A M2 protein on
its cell surface.
[0119] In a further preferred embodiment the said monoclonal
antibody is specifically binding influenza M2e antigen, wherein
said influenza M2e antigen is a cell comprising the extracellular
domain of influenza A M2 protein on its cell surface.
[0120] In a further preferred embodiment said monoclonal antibody
is specifically binding influenza M2e antigen, wherein the
dissociation constant (Kd) of said monoclonal antibody and said
influenza M2e antigen is 1 to 100 nM, preferably 1 to 10 nM, more
preferably 1 to 6 nM, still more preferably 3 to 6 nM, and most
preferably 4 to 5 nM, and wherein preferably said influenza M2e
antigen is SEQ ID NO:48, most preferably in solution, and wherein
still further preferably said Kd is determined by Friguet-ELISA,
most preferably under conditions essentially as described in
Example 11, and wherein further preferably said LC CDR1 consists of
the peptide of any one of SEQ ID NOs 1, 4 and 6, said LC CDR2
consists of the peptide of SEQ ID NO:7, said LC CDR3 consists of
the peptide of SEQ ID NO:8, said HC CDR1 consists of the peptide of
SEQ ID NO:12, said HC CDR2 consists of the peptide of SEQ ID NO:13,
and said HC CDR3 consists of the peptide of SEQ ID NO:15.
[0121] In a further preferred embodiment said monoclonal antibody
is specifically binding influenza M2e antigen, wherein the
dissociation constant (Kd) of said monoclonal antibody and said
influenza M2e antigen is 1 to 100 nM, preferably 1 to 10 nM, more
preferably 1 to 6 nM, still more preferably 3 to 6 nM, and most
preferably 4 to 5 nM, and wherein preferably said influenza M2e
antigen is SEQ ID NO:48, most preferably in solution, and wherein
still further preferably said Kd is determined by Friguet-ELISA,
most preferably under conditions essentially as described in
Example 11, and wherein further preferably said LC CDR1 consists of
the peptide of SEQ ID NO:1, said LC CDR2 consists of the peptide of
SEQ ID NO:7, said LC CDR3 consists of the peptide of SEQ ID NO:8,
said HC CDR1 consists of the peptide of SEQ ID NO:12, said HC CDR2
consists of the peptide of SEQ ID NO:13, and said HC CDR3 consists
of the peptide of SEQ ID NO:15.
[0122] In a further preferred embodiment said monoclonal antibody
is specifically binding influenza M2e antigen, wherein the
dissociation constant (Kd) of said monoclonal antibody and said
influenza M2e antigen is 1 to 100 nM, preferably 1 to 10 nM, more
preferably 1 to 6 nM, still more preferably 3 to 6 nM, and most
preferably 4 to 5 nM, and wherein preferably said influenza M2e
antigen is SEQ ID NO:48, most preferably in solution, and wherein
still further preferably said Kd is determined by Friguet-ELISA,
most preferably under conditions essentially as described in
Example 11, and wherein position 5 to 113 of said LCVR consists of
the peptide of any one of SEQ ID NOs 20, 21 and 22.
[0123] In a further preferred embodiment said monoclonal antibody
is specifically binding influenza M2e antigen, wherein the
dissociation constant (Kd) of said monoclonal antibody and said
influenza M2e antigen is 1 to 100 nM, preferably 1 to 10 nM, more
preferably 1 to 6 nM, still more preferably 3 to 6 nM, and most
preferably 4 to 5 nM and wherein preferably said influenza M2e
antigen is SEQ ID NO:48, most preferably in solution, and wherein
still further preferably said Kd is determined by Friguet-ELISA,
most preferably under conditions essentially as described in
Example 11, and wherein position 7 to 121 of said HCVR consists of
the peptide of SEQ ID NO:23.
[0124] In a further preferred embodiment said monoclonal antibody
is specifically binding influenza M2e antigen, wherein the
dissociation constant (Kd) of said monoclonal antibody and said
influenza M2e antigen is 1 to 100 nM, preferably 1 to 10 nM, more
preferably 1 to 6 nM, still more preferably 3 to 6 nM, and most
preferably 4 to 5 nM, and wherein preferably said influenza M2e
antigen is SEQ ID NO:48, most preferably in solution, and wherein
still further preferably said Kd is determined by Friguet-ELISA,
most preferably under conditions essentially as described in
Example 11, and wherein position 5 to 113 of said LCVR consists of
the peptide of any one of SEQ ID NOs 20, 21 and 22, and wherein
position 7 to 121 of said HCVR consists of the peptide of SEQ ID
NO:23.
[0125] In a further preferred embodiment said monoclonal antibody
is specifically binding influenza M2e antigen, wherein the
dissociation constant (Kd) of said monoclonal antibody and said
influenza M2e antigen is 1 to 100 nM, preferably 1 to 10 nM, more
preferably 1 to 6 nM, still more preferably 3 to 6 nM, and most
preferably 4 to 5, and wherein preferably said influenza M2e
antigen is SEQ ID NO:48, most preferably in solution, and wherein
still further preferably said Kd is determined by Friguet-ELISA,
most preferably under conditions essentially as described in
Example 11, and wherein position 5 to 113 of said LCVR consists of
the peptide of SEQ ID NO:20, and wherein position 7 to 121 of said
HCVR consists of the peptide of SEQ ID NO:23.
[0126] In a further preferred embodiment said monoclonal antibody
is specifically binding influenza M2e antigen, wherein the
dissociation constant (Kd) of said monoclonal antibody and said
influenza M2e antigen is 1 to 100 nM, preferably 1 to 10 nM, more
preferably 1 to 6 nM, still more preferably 3 to 6 nM, and most
preferably 4 to 5, and wherein preferably said influenza M2e
antigen is SEQ ID NO:48, most preferably in solution, and wherein
still further preferably said Kd is determined by Friguet-ELISA,
most preferably under conditions essentially as described in
Example 11, and wherein said monoclonal antibody is a human IgG,
preferably a human IgG1, wherein preferably said human IgG1
comprises at least one, preferably exactly two, kappa LC(s),
wherein said kappa LC(s) comprise(s) or more preferably consist(s)
of a peptide of any one of SEQ ID NOs 26 to 28, preferably SEQ ID
NO:20, and wherein said monoclonal antibody comprises at least one,
preferably exactly two, gamma 1 HC(s), wherein said gamma 1 HC(s)
comprise(s) or more preferably consist(s) of the of SEQ ID
NO:29.
[0127] An epitope mapping revealed that the minimal epitope which
is recognized by an antibody of the invention is comprised in the
amino acid sequence LLTEVETP (SEQ ID NO:93) of the M2e consensus
sequence (SEQ ID NO:48). It has been shown that an antibody of the
invention is capable of recognizing variants of this epitope
occurring in M2e of other influenza A genotypes (see FIG. 8A).
[0128] In a further preferred embodiment said at least one antigen
binding site recognizes an epitope comprised by the amino acid
sequence LLTEVETP (SEQ ID NO:93), and wherein further preferably
said LC CDR1 consists of the peptide of SEQ ID NO:1, said LC CDR2
consists of the peptide of SEQ ID NO:7, said LC CDR3 consists of
the peptide of SEQ ID NO:8, said HC CDR1 consists of the peptide of
SEQ ID NO:12, said HC CDR2 consists of the peptide of SEQ ID NO:13,
and said HC CDR3 consists of the peptide of SEQ ID NO:15.
[0129] In a further preferred embodiment said at least one antigen
binding site recognizes an epitope comprised by the amino acid
sequence LLTEVETP (SEQ ID NO:93), and wherein position 5 to 113 of
said LCVR consists of the peptide of SEQ ID NO:20, and wherein
position 7 to 121 of said HCVR consists of the peptide of SEQ ID
NO:23.
[0130] In a further preferred embodiment said at least one antigen
binding site recognizes an epitope comprised by the amino acid
sequence LLTEVETP (SEQ ID NO:93), and wherein said monoclonal
antibody is a human IgG, preferably a human IgG1, wherein
preferably said human IgG1 comprises at least one, preferably
exactly two, kappa LC(s), wherein said kappa LC(s) comprise(s) or
more preferably consist(s) of the peptide of SEQ ID NO:26, and
wherein said monoclonal antibody comprises at least one, preferably
exactly two, gamma 1 HC(s), wherein said gamma 1 HC(s) comprise(s)
or more preferably consist(s) of the of SEQ ID NO:29.
[0131] In a further preferred embodiment said at least one antigen
binding site recognizes an epitope comprised by the amino acid
sequence LLTEVETP (SEQ ID NO:93), and wherein further preferably
said LC CDR1 consists of the peptide of SEQ ID NO:4, said LC CDR2
consists of the peptide of SEQ ID NO:7, said LC CDR3 consists of
the peptide of SEQ ID NO:8, said HC CDR1 consists of the peptide of
SEQ ID NO:12, said HC CDR2 consists of the peptide of SEQ ID NO:13,
and said HC CDR3 consists of the peptide of SEQ ID NO:15.
[0132] In a further preferred embodiment said at least one antigen
binding site recognizes an epitope comprised by the amino acid
sequence LLTEVETP (SEQ ID NO:93), and wherein position 5 to 113 of
said LCVR consists of the peptide of SEQ ID NO:21, and wherein
position 7 to 121 of said HCVR consists of the peptide of SEQ ID
NO:23.
[0133] In a further preferred embodiment said at least one antigen
binding site recognizes an epitope comprised by the amino acid
sequence LLTEVETP (SEQ ID NO:93), and wherein said monoclonal
antibody is a human IgG, preferably a human IgG1, wherein
preferably said human IgG1 comprises at least one, preferably
exactly two, kappa LC(s), wherein said kappa LC(s) comprise(s) or
more preferably consist(s) of the peptide of SEQ ID NO:27, and
wherein said monoclonal antibody comprises at least one, preferably
exactly two, gamma 1 HC(s), wherein said gamma 1 HC(s) comprise(s)
or more preferably consist(s) of the of SEQ ID NO:29.
[0134] In a further preferred embodiment said at least one antigen
binding site recognizes an epitope comprised by the amino acid
sequence LLTEVETP (SEQ ID NO:93), and wherein further preferably
said LC CDR1 consists of the peptide of SEQ ID NO:6, said LC CDR2
consists of the peptide of SEQ ID NO:7, said LC CDR3 consists of
the peptide of SEQ ID NO:8, said HC CDR1 consists of the peptide of
SEQ ID NO:12, said HC CDR2 consists of the peptide of SEQ ID NO:13,
and said HC CDR3 consists of the peptide of SEQ ID NO:15.
[0135] In a further preferred embodiment said at least one antigen
binding site recognizes an epitope comprised by the amino acid
sequence LLTEVETP (SEQ ID NO:93), and wherein position 5 to 113 of
said LCVR consists of the peptide of SEQ ID NO:22, and wherein
position 7 to 121 of said HCVR consists of the peptide of SEQ ID
NO:23.
[0136] In a further preferred embodiment said at least one antigen
binding site recognizes an epitope comprised by the amino acid
sequence LLTEVETP (SEQ ID NO:93), and wherein said monoclonal
antibody is a human IgG, preferably a human IgG1, wherein
preferably said human IgG1 comprises at least one, preferably
exactly two, kappa LC(s), wherein said kappa LC(s) comprise(s) or
more preferably consist(s) of the peptide of SEQ ID NO:28, and
wherein said monoclonal antibody comprises at least one, preferably
exactly two, gamma 1 HC(s), wherein said gamma 1 HC(s) comprise(s)
or more preferably consist(s) of the of SEQ ID NO:29.
[0137] It is to be understood that it is well within the skill of
the artisan to use a HCVR of a first antibody specifically binding
influenza M2e antigen, to select a corresponding LCVR from a
suitable source, and to create a second antibody, wherein said
second antibody comprises said HCVR of said first antibody and the
selected LCVR, and wherein said second antibody is capable of
binding influenza A Me2 antigen with about the same specificity as
said first antibody ("chain shuffling"). It is furthermore apparent
for the artisan that in an analogous manner the LCVR of a first
antibody can be used to select a corresponding HCVR from a suitable
source. Suitable sources for the amplification of LCVRs and/or
HCVRs are, for example, cDNA from naive human B cells, cDNA from B
cells of a human subject immunized with an influenza M2e antigen,
and fully synthetic libraries, such as Morphosys' HuCAL library.
These methods are described in detail in Kang A S et al. (Proc Natl
Acad Sci USA 88, 11120-11123, 1991), Marks J D et al.
(Biotechnology (N Y) 10, 779-783, 1992), and Jespers et al.
(Biotechnology (NY) 12, 899-903, 1994).
[0138] Thus, a further aspect of the invention is a LCVR of a
monoclonal antibody, wherein said monoclonal antibody is a human
monoclonal antibody, most preferably a fully human monoclonal
antibody, and wherein said monoclonal antibody is specifically
binding influenza M2e antigen, and wherein said LCVR comprises: (a)
one LC CDR1, wherein said LC CDR1 consists of the peptide of any
one of SEQ ID NOs 1, 2, 3, 4, 5, and 6; (b) one LC CDR2, wherein
said LC CDR2 consists of the peptide of SEQ ID NO:7; (c) one LC
CDR3, wherein said LC CDR3 consists of the peptide of any one of
SEQ ID NOs 8, 9, 10, and 11.
[0139] In a preferred embodiment said LCVR is selected from: (a) a
LCVR, wherein (i) said LC CDR1 consists of the peptide of SEQ ID
NO:1, (ii) said LC CDR2 consists of the peptide of SEQ ID NO:7, and
(iii) said LC CDR3 consists of the peptide of SEQ ID NO:8; (b) a
LCVR, wherein (i) said LC CDR1 consists of the peptide of SEQ ID
NO:2, (ii) said LC CDR2 consists of the peptide of SEQ ID NO:7, and
(iii) said LC CDR3 consists of the peptide of SEQ ID NO:8; (c) a
LCVR, wherein (i) said LC CDR1 consists of the peptide of SEQ ID
NO:3, (ii) said LC CDR2 consists of the peptide of SEQ ID NO:7, and
(iii) said LC CDR3 consists of the peptide of SEQ ID NO:8; (d) a
LCVR, wherein (i) said LC CDR1 consists of the peptide of SEQ ID
NO:1, (ii) said LC CDR2 consists of the peptide of SEQ ID NO:7, and
(iii) said LC CDR3 consists of the peptide of SEQ ID NO:9; (e) a
LCVR, wherein (i) said LC CDR1 consists of the peptide of SEQ ID
NO:1, (ii) said LC CDR2 consists of the peptide of SEQ ID NO:7, and
(iii) said LC CDR3 consists of the peptide of SEQ ID NO:10; (f) a
LCVR, wherein (i) said LC CDR1 consists of the peptide of SEQ ID
NO:4, (ii) said LC CDR2 consists of the peptide of SEQ ID NO:7, and
(iii) said LC CDR3 consists of the peptide of SEQ ID NO:8; (g) a
LCVR, wherein (i) said LC CDR1 consists of the peptide of SEQ ID
NO:5, (ii) said LC CDR2 consists of the peptide of SEQ ID NO:7, and
(iii) said LC CDR3 consists of the peptide of SEQ ID NO:8; (h) a
LCVR, wherein (i) said LC CDR1 consists of the peptide of SEQ ID
NO:6, (ii) said LC CDR2 consists of the peptide of SEQ ID NO:7, and
(iii) said LC CDR3 consists of the peptide of SEQ ID NO:8; and (i)
a LCVR, wherein (i) said LC CDR1 consists of the peptide of SEQ ID
NO:1, (ii) said LC CDR2 consists of the peptide of SEQ ID NO:7, and
(iii) said LC CDR3 consists of the peptide of SEQ ID NO:11.
[0140] In a very preferred embodiment position 5 to 113 of said
LCVR consists of the peptide of any one of SEQ ID NOs 20, 21 and
22, most preferably SEQ ID NO:20, wherein preferably position 1 to
4 of said LCVR consists of the peptide of SEQ ID NO:24.
[0141] A further aspect of the invention is a HCVR of a monoclonal
antibody, wherein said monoclonal antibody is a human monoclonal
antibody, preferably a fully human monoclonal antibody, and wherein
said monoclonal antibody is specifically binding influenza M2e
antigen, and wherein said HCVR comprises: one HC CDR1, wherein said
HC CDR1 consists of the peptide of SEQ ID NO:12; (b) one HC CDR2,
wherein said HC CDR2 consists of the peptide of any one of SEQ ID
NOs 13 and 14; and (c) one HC CDR3, wherein said HC CDR3 consists
of the peptide of any one of SEQ ID NOs 15, 16, 17, 18, and 19.
[0142] In a further preferred embodiment, said HCVR comprises: one
HC CDR1, wherein said HC CDR1 consists of the peptide of SEQ ID
NO:12; (b) one HC CDR2, wherein said HC CDR2 consists of the
peptide of any one of SEQ ID NO:13; and (c) one HC CDR3, wherein
said HC CDR3 consists of the peptide of SEQ ID NO:15. In a
preferred embodiment position 7 to 121 of said HCVR consists of the
peptide of SEQ ID NO:23, wherein preferably position 1 to 6 of said
HCVR consists of the peptide of SEQ ID NO:25.
[0143] All aspects of the invention, and hereby in particular the
pharmaceutical compositions, methods and uses, which are disclosed
in the following, relate to any one of the monoclonal antibodies
disclosed herein. However, embodiments which relate to the antibody
clones D005, E040 and F052 are preferred, and hereby in particular
clone D005. Especially preferred are therefore embodiments, wherein
said monoclonal antibody comprises at least one antigen binding
site, wherein said antigen binding site comprises: (a) one LCVR,
wherein said LCVR comprises: (i) one LC CDR1, wherein said LC CDR1
consists of the peptide of any one of SEQ ID NOs 1, 4, and 6,
preferably SEQ ID NO:1; (ii) one LC CDR2, wherein said LC CDR2
consists of the peptide of SEQ ID NO:7; and (iii) one LC CDR3,
wherein said LC CDR3 consists of the peptide of SEQ ID NO:8; and
(b) one HCVR, wherein said HCVR comprises: (i) one HC CDR1, wherein
said HC CDR1 consists of the peptide of SEQ ID NO:12; (ii) one HC
CDR2, wherein said HC CDR2 consists of the peptide of SEQ ID NO:13;
and (iii) one HC CDR3, wherein said HC CDR3 consists of the peptide
of SEQ ID NO:15. Still more preferred are embodiments, wherein
position 5 to 113 of said LCVR consists of the peptide of any one
of SEQ ID NOs 20, 21 and 22, preferably of SEQ ID NO:20, and
wherein position 7 to 121 of said HCVR consists of the peptide of
SEQ ID NO:23. Still more preferred are embodiments, wherein said
monoclonal antibody is an IgG1, and wherein said monoclonal
antibody comprises at least one light chain, and wherein said
monoclonal antibody further comprises at least one heavy chain,
wherein said light chain comprises or preferably consists of the
amino acid sequence of any one of SEQ ID NOs 26, 27, and 28,
preferably of SEQ ID NO:26, and wherein said heavy chain comprises
or preferably consists of the amino acid of SEQ ID NO:29.
[0144] In a further aspect, the invention relates a nucleic acid
molecule encoding a HCVR or a LCVR of the invention, a monoclonal
antibody of the invention or an individual chain thereof. In a
preferred embodiment said nucleic acid molecule is encoding a
peptide selected from (a) a LCVR of the invention, wherein
preferably said LCVR comprises or preferably consists of any one of
the peptides depicted in FIG. 1; (b) a LCVR, wherein said LCVR
comprises or preferably consists of the peptide of any one of SEQ
ID NOs 20, 21, and 22; (c) a HCVR of the invention, wherein
preferably said HCVR comprises or preferably consists of any one of
the peptides depicted in FIG. 2; (d) a HCVR, wherein said HCVR
comprises or preferably consists of the peptide of SEQ ID NO:23;
(e) a single chain antibody of the invention, wherein preferably
said single chain antibody comprises or preferably consists of the
peptide of any one of SEQ ID NOs 43, 45 and 47; (f) a kappa LC of
the invention, wherein preferably said kappa LC comprises or
preferably consists of the peptide of any one of SEQ ID NOs 26, 27,
and 28; (g) a gamma 1 HC of the invention, wherein preferably said
gamma 1 HC comprises or preferably consists of the peptide of SEQ
ID NO:29; and (h) a monoclonal antibody of the invention.
[0145] In a further preferred embodiment said nucleic acid molecule
comprises or preferably consists of the nucleotide sequence of any
one of SEQ ID NOs 86 to 89. In a further preferred embodiment said
nucleic acid molecule comprises the nucleotide sequence of any one
of SEQ ID NOs 86 to 88, and wherein said nucleic acid molecule
further comprises the nucleotide sequence of SEQ ID NO:89.
[0146] In a further preferred embodiment said nucleic acid molecule
comprises or preferably consists of the nucleotide sequence of any
one of SEQ ID NOs 30, 32, 34, and 36. In a further preferred
embodiment said nucleic acid molecule comprises the nucleotide
sequence of any one of SEQ ID NOs 32, 34, and 36, and wherein said
nucleic acid molecule further comprises the nucleotide sequence of
SEQ ID NO:30.
[0147] In a further preferred embodiment said nucleic acid molecule
comprises or preferably consists of the nucleotide sequence of any
one of SEQ ID NOs 38, 39, and 40.
[0148] In a further preferred embodiment said nucleic acid molecule
comprises or preferably consists of the nucleotide sequence of SEQ
ID NO:41.
[0149] In a further preferred embodiment said nucleic acid molecule
comprises or preferably consists of the nucleotide sequence of any
one of SEQ ID NOs 42, 44, and 46. In a further preferred embodiment
said nucleic acid molecule comprises or preferably consists of the
nucleotide sequence of any one of SEQ ID NOs 42, 44, and 46.
[0150] In a further aspect, the invention relates to an expression
vector for the recombinant expression of an antibody of the
invention. In a preferred embodiment, said expression vector
comprises at least one nucleic acid molecule of the invention.
Expression vectors suitable for the expression of the monoclonal
antibodies of the invention are disclosed, for example, in
WO2008/055795A1. In a preferred embodiment said expression vector
comprises the nucleotide sequence of any one of SEQ ID NOs 86 to
89. In a further preferred embodiment said expression vector
comprises the nucleotide sequence of any one of SEQ ID NOs 86 to
88, wherein preferably said expression vector further comprises the
nucleotide sequence of SEQ ID NO:89. In a further preferred
embodiment said expression vector comprises the nucleotide sequence
of SEQ ID NO:41.
[0151] In a further aspect, the invention relates to a host cell
comprising at least one nucleic acid molecule or at least one
expression vector of the invention, wherein preferably said host
cell is a bacteria cell or an eukaryotic cell. In a preferred
embodiment said host cell is a eukaryotic cell selected from (a)
yeast cell, (b) insect cell; and (c) mammalian cell, wherein
preferably said mammalian cell is selected from HEK-293T cell, CHO
cell, and COS cell. Very preferably, said mammalian cells is a
HEK-293T cell.
[0152] In a further aspect, the invention relates to the monoclonal
antibody of the invention for use as a pharmaceutical.
[0153] The monoclonal antibody of the invention can be incorporated
into compositions suitable for administration to a subject. Thus,
in a further aspect, the invention relates to a pharmaceutical
composition comprising at least one monoclonal antibody of the
invention, wherein preferably said pharmaceutical composition
further comprises a pharmaceutically acceptable carrier, diluent or
excipient. Pharmaceutically acceptable carriers, diluents and
excipients are disclosed, for example, in Remington, The Science
and Practice of Pharmacy, 19.sup.th edition, Gennaro (ed.), Mack
publishing Co., Easton, Pa., 1995. Pharmaceutical compositions of
the invention are administered in a single dose or in multiple
doses.
[0154] In a preferred embodiment said pharmaceutical composition
further comprises at least one further antibody, wherein preferably
said at least one further antibody is specifically binding an
influenza antigen, preferably an influenza M2e antigen.
[0155] In a further preferred embodiment said pharmaceutical
composition further comprises at least one further antibody,
wherein preferably said at least one further antibody is
specifically binding an influenza antigen, wherein said influenza
antigen is an antigen of influenza A virus HA protein or an antigen
of influenza A virus NA protein.
[0156] The monoclonal antibodies of the invention may be used in
passive immunization, preferably of humans, and further preferably
against influenza A virus. The monoclonal antibodies of the
invention are therefore useful in the treatment and/or prophylaxis
of influenza A infection. In a further aspect, the invention
relates to a method of passive immunization, preferably against
influenza A virus, said method comprising administering to a
subject an effective amount of the monoclonal antibody of the
invention or an effective amount of the pharmaceutical composition
of the invention.
[0157] The monoclonal antibody and/or the pharmaceutical
composition of the invention are preferably administered to a
subject, preferably to a human, using standard administration
techniques, preferably selected from oral administration,
intravenous administration, intraperitoneal administration,
subcutaneous administration, pulmonary administration, transdermal
administration, intramuscular administration, intranasal
administration, buccal administration, sublingual administration,
and suppository administration.
[0158] In a further aspect, the invention relates to a method of
treating influenza A virus infection, said method comprising
administering to a subject an effective amount of the monoclonal
antibody of the invention or an effective amount of the
pharmaceutical composition of the invention, wherein preferably
said subject is a human, and wherein further preferably said
subject suffers from influenza A virus infection.
[0159] In a further aspect, the invention relates to a method of
preventing influenza A virus infection, said method comprising
administering to a subject an effective amount of the monoclonal
antibody of the invention or an effective amount of the
pharmaceutical composition of the invention, wherein preferably
said subject is a human, and wherein further preferably said
subject is not infected with influenza A virus.
[0160] In a further aspect, the invention relates to the monoclonal
antibody of the invention or to the pharmaceutical composition of
the invention, for use in passive immunization, preferably against
influenza A virus, preferably in a human, wherein further
preferably said monoclonal antibody is to be administered to said
human.
[0161] In a further aspect, the invention relates to the monoclonal
antibody of the invention or to the pharmaceutical composition of
the invention, for use in a method of treatment and/or prophylaxis
of influenza A virus infection, preferably in a human.
[0162] In a further aspect, the invention relates to the use of the
monoclonal antibody of the invention in the manufacture of a
medicament for passive immunization, preferably against influenza A
virus.
[0163] In a further aspect, the invention relates to a monoclonal
antibody of the invention for use in passive immunization,
preferably against influenza A virus.
[0164] In a further aspect, the invention relates to the use of the
monoclonal antibody of the invention in the manufacture of a
medicament for the treatment and/or prophylaxis of influenza A
virus infection, preferably in a human.
[0165] In a further aspect, the invention relates to a monoclonal
antibody of the invention for use in a method of treating or
preventing influenza A virus infection, preferably in a human.
[0166] A further aspect of the invention is the use of an antibody
of the invention in a method of quantitative and/or qualitative
detection of influenza A virus M2 protein, preferably in a blood
sample and most preferably by ELISA.
[0167] It is to be understood that the all aspects of the invention
relate to any monoclonal antibody which is disclosed herein.
Example 1
Identification of M2-Specific Single-Chain Antibodies by Mammalian
Cell Display
[0168] Peripheral blood mononuclear cells (PBMC) were isolated from
10 ml of heparinized blood of an individual with high M2-titers
using the BD Vacutainer.TM. CPT.TM. Tube method (BD Biosciences,
Franklin Lakes, N.J.). PBMC were pre-incubated with Alexa 647
nm-labeled Q.beta. (5.5 .mu.g/ml) and human gamma globulin (11
.mu.g/ml; Jackson ImmunoResearch) and then stained with: (1) an M2
extracellular domain consensus peptide (M2e; SEQ ID NO:48, cf.
Table 7) coupled to Q.beta. (2.4 .mu.g/ml) in combination with a
Alexa 488 nm-labeled Q.beta.-specific mouse mAb (2 .mu.g/ml), as
well as a M2-specific mouse mAb (0.5 .mu.g/ml) in combination with
FITC-labeled donkey anti-mouse IgG antibody (1 .mu.g/ml; Jackson
ImmunoResearch); (2) PE-labeled mouse anti-human IgM (diluted 1:50;
BD Biosciences/Pharmingen), mouse anti-human IgD (diluted 1:100; BD
Biosciences/Pharmingen), mouse anti-human CD14 (diluted 1:50; BD
Biosciences/Pharmingen), and mouse anti-human CD3 (diluted 1:50; BD
Biosciences/Pharmingen) antibodies; and (3) PE-TexasRed-labeled
mouse anti-human CD19 antibody (diluted 1:50; Caltag Laboratories).
After staining, cells were washed and filtered, and 334
M2e-specific B cells (FL1-positive, FL2-negative, FL3-positive,
FL4-negative) were sorted on a FACSVantage.RTM. SE flow cytometer
(Becton Dickinson).
[0169] Antigen-specific B cells were used for the construction of a
Sindbis-based scFv cell surface display library essentially as
described (see WO 1999/025876 A1 for Sindbis-based screening in
general and WO 2008/055795 A1 for its application in antibody
screening, the entirety of which is incorporated herein by
reference). Cells displaying M2e-specific scFv antibodies were
isolated using M2e coupled to RNase A (5 .mu.g/ml) in combination
with an RNase-specific rabbit polyclonal antibody (2.5 .mu.g/ml;
Abcam) and a FITC-labeled donkey anti-rabbit IgG antibody (1.5
.mu.g/ml; Jackson ImmunoResearch) or using (Q.beta.-M2e (1
.mu.g/ml) in combination with a M2-specific mouse mAb (0.5
.mu.g/ml) and FITC-labeled donkey anti-mouse IgG antibody (1
.mu.g/ml; Jackson ImmunoResearch). Each cell was sorted into a well
of a 24-well plate containing 50% confluent BHK feeder cells. Upon
virus spread (2 days post sorting), the infected cells were tested
by FACS analysis for M2e-binding to identify virus clones encoding
M2e-specific scFv antibodies.
Example 2
Gene Rescue, ELISA Screening and Sequencing of M2-Specific
Antibodies
[0170] The supernatants of BHK cells encoding putative M2e-specific
antibodies, each containing a monoclonal recombinant Sindbis virus,
were subjected to RT-PCR as described (see WO 2008/055795 A1). The
resulting PCR products, each comprising a scFv coding region, were
digested with the restriction endonuclease Sfi1 and cloned into the
expression vector pCEP-SP-Sfi-Fc (disclosed as SEQ ID NO:37 in WO
2008/055795 A1), allowing for expression of scFv proteins fused to
a C-terminal human Fc-.gamma.1 domain under the control of a CMV
promoter.
[0171] For ELISA analysis, each of the clones was transfected into
HEK-293T cells in a 24-well plate format, using Lipofectamine 2000
(Invitrogen) according to the manufacturer's recommendations. 2-3
days post transfection, supernatants containing transiently
expressed scFv-Fc fusion proteins were harvested. To check for
M2e-specific binding, ELISA plates were coated with M2e-conjugated
RNAse A at a concentration of 4 .mu.g/ml in phosphate-buffered
saline (PBS) over night at 4.degree. C. In parallel, scFv-Fc
expression levels were monitored in by sandwich ELISA. For this, an
identical set of plates was coated with Fc.gamma.-specific, goat
anti-human F(ab')2 antibody (Jackson ImmunoResearch Laboratories
109-006-098) at a concentration of 2.5 .mu.g/ml. The plates were
then washed with wash buffer (PBS/0.05 Tween) and blocked for 2 h
at room temperature with 3% BSA in wash buffer. The plates were
then washed again and incubated with 3-fold serial dilutions of the
cell culture supernatants, starting at a dilution of 1/10. All
dilutions were done in wash buffer. Plates were incubated at room
temperature for 2 h and then extensively washed with wash buffer.
Bound scFv-Fc fusion proteins were then detected by a 1 h
incubation with a HRPO-labeled, Fc.gamma.-specific, goat anti-human
IgG antibody (Jackson ImmunoResearch Laboratories 109-035-098).
After extensive washing with wash buffer, plates were developed
with OPD solution (1 OPD tablet, 25 ml OPD buffer and 8 .mu.l 30%
H.sub.2O.sub.2) for 5 to 10 min and the reaction was stopped with
5% H.sub.2SO.sub.4 solution. Plates were then read at OD 450 nm on
an ELISA reader (Biorad Benchmark).
[0172] In total, 53 ELISA-positive clones encoding M2e-specific
scFv antibodies, each binding with an EC50 in the range of 2 to 10
ng/ml (approximately 18 to 90 pM), were sequenced as described (see
WO 2008/055795 A1). All antibody sequences were very similar and
obviously clonally related, with heavy chain variable regions
comprising VH3 family sequences and light chain variable regions
comprising VK4 family sequences. The amino acid sequences of the
light chains of all 53 clones are depicted in FIG. 1. The amino
acid sequences of the heavy chains of the same clones are depicted
in FIG. 2. An overview about the CDR sequences, including
references to the SEQ ID NOs, of the light and heavy chains of
these antibodies is provided in Tables 1 and 2. The combinations of
LCVRs and HCVRs observed in the clones, and the frequency of each
of these combinations, are disclosed in Table 3.
[0173] The clones D005, E040 and F052 represent three of most
abundant combinations of LCVR and HCVR (1A-1A, 2A-1A, and 3A-1A,
cf. Table 3) and were thus chosen as representative clones for
further analysis.
Example 3
Expression and Purification of M2-Specific scFv-msFc.gamma.2c
Fusion Proteins
[0174] To investigate the effect of M2e-specific human antibodies
on Influenza A infection in a mouse model, clones D005, E040 and
F052 were expressed and purified as scFv-mouse Fc-.gamma.2c
(msFc.gamma.2c) fusion proteins. The nucleotide sequences encoding
the D005, E040 and F052 scFv-mouse Fc-.gamma.2c (msFc.gamma.2c)
fusion proteins correspond to SEQ ID NOs:42, 44 and 46,
respectively. The amino acid sequences of the D005, E040 and F052
scFv-mouse Fc-.gamma.2c (msFc.gamma.2c) fusion proteins correspond
to SEQ ID NOs: 43, 45 and 47, respectively. The corresponding scFv
coding regions were excised from the pCEP-SP-Sfi-Fc expression
vectors with the restriction endonuclease Sfi1 and the resulting
fragments were cloned into the expression vector
pCEP-SP-Sfi-msFc.gamma.2c (SEQ ID NO:41). The Sfi-digested
fragments of monoclonal antibodies D005, E042, and F052 correspond
to SEQ ID NOs 38, 39 and 40, respectively. These Sfi-fragments
encode the entire scFv fragment of the corresponding antibody,
including the linker sequence, but do not include the Fc
domain.
[0175] Large-scale expression of scFv-msFc.gamma.2c fusion proteins
was done in HEK-293T cells. One day before transfection, 10.sup.7
293T cells were plated onto a 14 cm tissue culture plate for each
protein to be expressed. Cells were then transfected with the
respective scFv-msFc.gamma.2c fusion construct using Lipofectamine
Plus (Invitrogen) according to the manufacturer's recommendations,
incubated one day, and replated on three 14 cm dishes in the
presence of 1 .mu.g/ml puromycin. After three days of selection,
puromycin-resistant cells were transferred to six 14 cm plates and
grown to confluency. Finally, cells were transferred to a
poly-L-lysine coated roller bottle. After 1-2 days medium was
replaced by serum-free medium and supernatants containing the
respective scFv-msFc.gamma.2c fusion protein was collected every 3
days and filtered through a 0.22 .mu.m Millex GV sterile filter
(Millipore).
[0176] For each of the scFv-msFc.gamma.2c fusion proteins, the
consecutive harvests were pooled and applied to a protein
A-sepharose column. The column was washed with 10 column volumes of
phosphate-buffered saline (PBS), and bound protein eluted with 0.1
M Glycine pH 3.6. 1 ml fractions were collected in tubes containing
0.1 ml of 1 M Tris pH 7.5 for neutralization. Protein-containing
fractions were analyzed by SDS-PAGE and pooled. The buffer was
exchanged with PBS by dialysis using 10,000 MWCO Slide-A-Lyzer
dialysis cassettes (Pierce). The purified proteins in PBS were then
filtered through 0.22 .mu.m Millex GV sterile filters (Millipore)
and aliquotted. Working stocks were kept at 4.degree. C., whereas
aliquots for long-term storage were flash-frozen in liquid nitrogen
and kept -80.degree. C.
Example 4
ELISA Analysis of scFv-msFc.gamma.2c Fusion Protein Binding to
M2-Derived Peptides
[0177] To confirm binding of scFv-msFc.gamma.2c fusion proteins to
M2e, an ELISA analysis was carried out with purified
scFv-D005-msFc.gamma.2c, scFv-E040-msFc.gamma.2c and
scFv-F052-msFc.gamma.2c. Thus, ELISA plates were coated with M2e
(SEQ ID NO:48) conjugated to RNAse A at a concentration of 4
.mu.g/ml in phosphate-buffered saline (PBS), one hour at 37.degree.
C. The plates were then washed with wash buffer (PBS/0.05% Tween)
and blocked for 1 h at 37.degree. C. with 3% BSA in wash buffer.
The plates were then washed again and incubated with serial
dilutions of purified scFv-D005-msFc.gamma.2c,
scFv-E040-msFc.gamma.2c and scFv-F052-msFc.gamma.2c. Plates were
incubated at room temperature for 1.5 h at 37.degree. C. and then
extensively washed with wash buffer. Bound scFv-Fc fusion proteins
were then detected by a 1 h incubation at room temperature with a
HRPO-labeled, Fc.gamma.-specific, goat anti-mouse IgG antibody
(Jackson ImmunoResearch Laboratories 115-035-071). After extensive
washing with wash buffer, plates were developed with OPD solution
(1 OPD tablet, 25 ml OPD buffer and 8 .mu.l 30% H.sub.2O.sub.2) for
10 min and the reaction was stopped with 5% H.sub.2SO.sub.4
solution. Plates were then read at OD 450 nm on an ELISA reader
(Biorad Benchmark). The antibodies scFv-D005-msFc.gamma.2c,
scFv-E040-msFc.gamma.2c and scFv-F052-msFc.gamma.2c were found to
bind immobilized M2e with a high apparent affinity. The apparent
affinity of the same antibodies towards immobilized M2e-short (SEQ
ID NO:49) and M2e-VN (SEQ ID NO:50) was determined using the same
experimental set-up. The apparent affinities for each combination
of antibody and antigen are provided in Table 5. These values
correspond well with the ones obtained with unpurified culture
supernatants (described in Example 2).
TABLE-US-00005 TABLE 5 Apparent affinities of
scFv-D005-msFc.gamma.2c, scFv- E040-msFc.gamma.2c and
scFv-F052-msFc.gamma.2c towards different versions of influenza M2
extracellular domain. Antibody M2e M2e-short M2e-VN D005 81.5 pM
80.9 pM 51.5 pM E040 88.3 pM 90.2 pM 46.4 pM F052 50.6 pM 62.8 pM
45.9 pM
[0178] To further investigate binding of the scFv-msFc.gamma.2c
fusion proteins to different M2-derived peptides, also a
competition ELISA was carried out. Thus, ELISA plates were coated
with M2e conjugated to RNAse A at a concentration of 4 .mu.g/ml in
phosphate-buffered saline (PBS), over night at 4.degree. C. The
plates were then washed with wash buffer (PBS/0.05% Tween) and
blocked for 2 h at 37.degree. C. with 3% BSA in wash buffer. The
plates were then washed again and incubated with purified
scFv-D005-msFc.gamma.2c, scFv-E040-msFc.gamma.2c and
scFv-F052-msFc.gamma.2c at a concentration of 100 ng/ml in the
absence or presence of increasing concentrations of M2e, an M2
extracellular domain peptide derived from H5N1 Influenza A VN1203
(M2e-VN; SEQ ID NO:50), or a shortened M2e peptide (M2e-short; SEQ
ID NO:49) (Table 7). Plates were incubated at room temperature for
2 h and then extensively washed with wash buffer. Bound scFv-Fc
fusion proteins were then detected by a 1 h incubation with a
HRPO-labeled, Fc.gamma.-specific, goat anti-mouse IgG antibody
(Jackson ImmunoResearch Laboratories 115-035-071). After extensive
washing with wash buffer, plates were developed with OPD solution
and read on an ELISA reader as described above. Binding of each of
the antibodies to immobilized M2e was inhibited by all three
peptides to a similar extent, indicating that all three peptides
were recognized equally well (Table 6).
TABLE-US-00006 TABLE 6 Inhibition of scFv-msFc.gamma.2c binding to
immobilized M2e by soluble M2 peptides. EC50 values (.mu.M) of
inhibition of binding are shown. scFv-msFc.gamma.2c M2e M2e-VN
M2e-short D005 18.8 32.5 27.5 E040 21.3 43.2 35.5 F052 22.0 50.8
46.8
TABLE-US-00007 TABLE 7 M2 variants used in this study. Sub- SEQ M2e
variant Abbreviation type ID NO M2 Sequence.sup.(2)
Consensus.sup.(1) M2e n/a 48 SLLTEVETPIRNEWGCRCNDSSD Short
M2e-short n/a 49 SLLTEVETPIRNEWGC A/VN/1203/04 M2e-VN H5N1 50
SLLTEVETPTRNEWECRCSDSSD A/PR/8/34 M2-PR H1N1 84
SLLTEVETPIRNEWGCRCNGSSDPLTIAANIIGI LHLTLWILDRLFFKCIYRRFKYGLKGGPSTE
GVPKSMREEYRKEQQSAVDADDGHFVSIELE A/VN/1203/04 M2- H5N1 85
SLLTEVETPTRNEWECRCSDSSDPLTIAANIIGI VN/PR
LHLTLWILDRLFFKCIYRRFKYGLKGGPSTE GVPKSMREEYRKEQQSAVDADDGHFVSIELE
.sup.(1)M2e consensus sequence derived from H1, H2, and H3 subtypes
of human Influenza A viruses. .sup.(2)Variations from M2 consensus
sequence are shown in bold (Tompkins et al. 2007, Emerging
Infectious Diseases Vol. 13, No. 3, pp. 426-435, cf. Table
therein). All sequences are shown without the N-terminal
Methionine, which is removed upon expression in vivo.
Example 5
Binding of M2-Specific scFv-msFc.gamma.2c Fusion Proteins to
M2-Expressing L929 Cells
[0179] The ability of the recombinant antibodies to recognize
native M2 was assessed by analyzing their reactivity with
L929-M2#E9 cells, a clone of L929 cells expressing full-length M2
derived from mouse-adapted H.sub.1N.sub.1 Influenza A PR8 (M2-PR;
SEQ ID NO:84) (Table 7). Thus, L929-M2#E9 cells were brought to a
single-cell suspension and incubated with 2-fold serial dilutions
of, respectively, scFv-D005-msFc.gamma.2c, scFv-E040-msFc.gamma.2c
or scFv-F052-msFc.gamma.2c in FACS.RTM. buffer (phosphate-buffered
saline containing 1% FCS). After a 1 h incubation on ice, cells
were washed in FACS.RTM. buffer and bound antibodies detected by a
half hour incubation on ice with Cy5-labeled goat anti-mouse
antibody (Jackson Immuno, Cat No 115-176-071) in FACS buffer. After
a final wash, the fluorescence intensity of the stained cells was
analyzed by flow cytometry using a FACScalibur.RTM. (Becton
Dickinson). The antibodies scFv-D005-msFc.gamma.2c,
scFv-E040-msFc.gamma.2c and scFv-F052-msFc.gamma.2c were found to
bind native, cell surface-expressed M2-PR with a high affinity,
with an EC50 of 0.68, 0.73 and 0.46 nM, respectively.
Example 6
Protective Effect of M2-Specific scFv-msFc.gamma.2c Fusion Proteins
in a Mouse Model of Influenza A Infection
[0180] The efficacy anti-M2 scFv-msFc.gamma.2c antibodies in a
prophylactic setting was tested in a mouse model of Influenza A
infection. This model reflects most of the immunological and
histological aspects of Influenza infection in humans and is
therefore routinely used to assess the efficacy of anti-viral
agents. Thus, six week old female C57BL/6 mice were injected
intraperitoneally with 500 .mu.g of scFv-D005-msFc.gamma.2c,
scFv-E040-msFc.gamma.2c, scFv-F052-msFc.gamma.2c or mouse IgG in
PBS (6 mice per group). One day later, mice were bled in order to
verify the presence of the antibodies in the blood by ELISA.
Antibodies were readily detectable in the sera of all mice, except
for one mouse receiving clone D005, which was subsequently removed
from the analysis (not shown). Another day later (day 0), mice were
infected intranasally with a lethal dose of mouse-adapted Influenza
A virus PR8 (4.times.LD50) followed by monitoring of weight-loss
and fever (temperature drop) for 12 days. Control mice treated with
mouse IgG developed severe signs of morbidity, characterized by
dramatic temperature drop and weight loss, within 5 to 6 days and
invariably died on days 7 or 8 (FIG. 3C). In contrast, animals
treated with M2-specific antibodies were almost completely
protected from any signs of morbidity, developed hardly any signs
of fever and lost weight only temporarily (FIGS. 3A and B).
[0181] To determine the minimal amount of antibody required to
achieve full protection of mice in a prophylactic setting,
scFv-msFc-.gamma.2c D005 was titrated. To this end, six week old
female C57BL/6 mice were injected intraperitoneally with decreasing
amounts of the antibody (200, 60, 20, or 6 .mu.g per mouse) or, as
a control, with 200 .mu.g of mouse IgG (6 mice per group). One day
later, mice were bled in order to verify the presence of the
antibodies in the blood by ELISA. Antibodies were readily
detectable in the sera of all mice, except for one mouse receiving
the 60 .mu.g dose, which was subsequently removed from the analysis
(not shown). Another day later (day 0), animals were infected
intranasally with a lethal dose of influenza A virus strain PR/8/34
(4.times.LD50) and monitored for 21 days (FIG. 4A). As expected,
control mice quickly succumbed to the disease and were dead in less
than two weeks. In contrast, antibody D005 showed protective
activity at all doses tested. In groups of mice receiving 200, 60
or 20 .mu.g antibody, all animals survived the lethal challenge.
Even at the lowest dose of 6 .mu.g D005, half of the mice recovered
and survived infection, indicating that the M2-specific antibody is
a potent prophylactic agent.
Example 7
Therapeutic Activity of a M2-Specific scFv-msFc.gamma.2c Fusion
Protein in a Mouse Model of Influenza A Infection
[0182] In view of the similarities of clones D005, E040 and F052 in
sequence, affinity and prophylactic activity, only
scFv-D005-msFc.gamma.2c was tested in a therapeutic setting. Thus,
six weeks old female C57BL/6 mice were infected intranasally with a
lethal dose of mouse-adapted Influenza A virus PR8
(4.times.LD.sub.50). 1, 2, or 3 days later, groups of mice were
injected intraperitoneally with 200 .mu.g of
scFv-D005-msFc.gamma.2c in PBS (6 mice per group). As controls, one
group of mice each was injected with scFv-D005-msFc.gamma.2c or
mouse IgG two days prior to infection. Each mouse was bled one day
after receiving antibody in order to verify the presence of the
antibodies in the blood (not shown). The mice were observed closely
to monitor signs of morbidity as well as mortality for a total of
21 days (FIG. 4B and not shown). All mice receiving the control
antibody eventually died, whereas all mice receiving a prophylactic
injection of scFv-D005-msFc.gamma.2c two days prior to infection
survived for at least 3 weeks. Significantly, therapeutic efficacy
of the antibody could be shown in groups of mice treated as much as
3 days after infection. Survival rates of mice treated 1, 2 and 3
days after infection were 100, 83 and 17%, respectively. The
measurement of survival rates generally corresponds to mortality
(the proportion of deaths to a specified population), whereas
morbidity general corresponds to the incidence of disease or the
rate of sickness (in a specified population).
Example 8
Construction, Expression, and Purification of Fully Human
M2-Specific IgG1
[0183] Expression vectors allowing for expression of the clones
D005, E040 and F052 as fully human IgG1.kappa. were generated.
Thus, DNA sequences encoding a human .gamma.1 heavy chain shared by
clones D005, E040 and F052, as well as each of the unique .kappa.
light chains were produced by total gene synthesis (SEQ ID NOs 30,
32, 34, and 36, by GeneArt AG, Germany). The heavy chain coding
sequence was flanked by a Asc1 recognition site upstream and a Pac1
recognition site downstream of the open reading frame (cf. SEQ ID
NO:30). The light chain coding sequences were flanked by Nhe1
recognition sites upstream and Pme1 recognition sites downstream of
the respective open reading frame (cf. SEQ ID NOs 32, 34, and 36).
The amino acid sequence of the entire human .gamma.1 heavy chain,
including the signal peptide, is depicted in SEQ ID NO:31. The
amino acid sequences of the .kappa. light chains of D005, E040 and
F052 are depicted in SEQ ID NOs 33, 35 and 37, respectively.
[0184] Heavy and light chain coding regions were then combined into
the EBNA-based expression vector pCB15 (disclosed as SEQ ID NO:104
of WO 2008/055795 A1). Thus, the heavy chain coding region was
digested with the restriction enzymes Asc1 and Pac1, and ligated
into Asc1-Pac1 digested pCB15, generating the plasmid
pCB15-fh-HC-.gamma.1-D005. This plasmid was then digested with the
restriction enzymes Nhe1 and Pme1 and ligated to each of the
Nhe1-Pme1 digested light chain coding regions, generating the
plasmids pCB15-fh-IgG1.kappa.-D005, pCB15-fh-IgG1.kappa.-E040 and
pCB15-fh-IgG1.kappa.-F052. Expression of IgG1.kappa.-D005,
IgG1.kappa.-E040 and IgG1.kappa.-F052 in HEK-293T cells, as well as
purification by protein A-sepharose chromatography, was done as
described for the scFv-Fc fusion proteins (Example 2).
Example 9
Binding of a M2-Specific Fully Human IgG1 to M2-Expressing 293T
Cells
[0185] In view of the similarities of clones D005, E040 and F052 in
sequence, affinity and prophylactic activity, only IgG1-D005 was
analyzed in more detail. The ability of the fully human mAb
IgG1-D005 to recognize native, cell surface-expressed M2 was
assessed by analyzing its reactivity with 293T cells expressing
full-length M2 variants. Thus, 293T cells were transfected with a
recombinant expression vector encoding the M2 protein of A/PR/8/34
(M2-PR, SEQ ID NO:84) or the fusion protein M2-VN/PR (SEQ ID
NO:85), respectively. M2-VN/PR comprises an extracellular domain
derived from H5N1 Influenza A VN1203 (M2e-VN, SEQ ID NO:50) which
is fused to the N-terminus of the transmembrane and intercellular
regions of M2-PR (cf. Table 7, the part of SEQ ID NO:84 which is
shown in italics).
[0186] Transfected 293T cells were then brought to a single-cell
suspension and incubated with 2-fold serial dilutions of IgG1-D005
in FACS buffer (phosphate-buffered saline containing 1% FCS). After
a 1 h incubation on ice, cells were washed in FACS buffer and bound
antibodies detected by a half hour incubation on ice with
Cy5-labeled goat anti-human antibody in FACS buffer. After a final
wash, the fluorescence-intensity of the stained cells was analyzed
by flow cytometry using a FACScalibur (Becton Dickinson). The mAb
IgG1-D005 bound native, cell surface-expressed M2-PR and M2-VN/PR
with identical affinities, with an EC50 of 3.9 nM.
Example 10
M2-Specific Fully Human IgG1 is Protective in a Mouse Model of
Influenza a Infection and Requires Interaction with Fc Receptor
[0187] In view of the similarities of clones D005, E040 and F052 in
sequence, affinity and prophylactic activity, only antibody D005
was analyzed in more detail in a mouse model of Influenza A
infection. On one hand, the prophylactic activity of fully human
IgG1k-D005 was tested and compared to the scFv-D005-msFc.gamma.2c
fusion protein. On the other hand, the involvement of
antibody-dependent cellular cytotoxicity (ADCC) was investigated,
by using a scFv-D005 antibody fused to a mutated human Fc.gamma.1
incapable of binding to Fc receptors (scFv-D005-hFcm). Thus, six
week old female C57BL/6 mice were injected intraperitoneally with
equimolar amounts of IgG1-D005 (200 .mu.g), scFv-D005-msFc.gamma.2c
(144 .mu.g), scFv-D005-hFcm (144 .mu.g), or human IgG (200 .mu.g)
in PBS (6 mice per group). One day later, mice were bled in order
to verify the presence of the antibodies in the blood by ELISA.
Antibodies were readily detectable in the sera of all mice (not
shown). Another day later (day 0), mice were infected intranasally
with a lethal dose of mouse-adapted Influenza A virus PR8
(4.times.LD.sub.50) followed by monitoring of weight-loss and fever
(temperature drop) for 16 days. Control mice treated with mouse IgG
developed severe signs of morbidity, characterized by dramatic
temperature drop and weight loss, within 6 to 7 days and died
between days 8 and 11 (FIG. 5). In contrast, similar to animals
treated with scFv-D005-msFc.gamma.2c, those treated with
hIgG1k-D005 were almost completely protected from any signs of
morbidity, developed hardly any signs of fever and lost weight only
temporarily (FIGS. 5A and B). Thus, fully human IgG1k-D005 has a
strong prophylactic activity in mice and is equipotent to
scFv-D005-msFc.gamma.2c. Significantly, mice treated with
scFv-D005-hFcm were not protected and developed severe signs of
morbidity similar to mice treated with the control IgG (FIGS. 5A
and B). Consequently, all animals treated with scFv-D005-hFcm
succumbed to disease and were dead by day 9 (FIG. 5C). Thus, Fc
receptor interaction is required for protection, suggesting that
ADCC is a major component of the prophylactic activity.
Example 11
Determination of Affinities by Friguet-ELISA
[0188] The dissociation constants (Kd) of antibody binding to M2e
in solution were determined using an ELISA-based method essentially
as described (Friguet B. et al., 1985, J. Immunol. Meth. 77,
305-319). Briefly, a 10 ng/ml solution of, respectively,
scFv-D005-msFc.gamma.2c, scFv-E040-msFc.gamma.2c or
scFv-F052-msFc.gamma.2c, was incubated in the presence of different
concentrations of RNAse conjugated to influenza A M2e (SEQ ID
NO:48) (3-fold serial dilutions ranging from 10 nM to 0.17 pM with
respect to the content of influenza A M2e) in PBS/1% BSA. After 2 h
at room temperature, free antibody was detected by a classical
ELISA similar to the one described in Example 4. For this, ELISA
plates that had been coated with RNAse-M2e conjugate at a
concentration of 20 ng/ml at 4.degree. C. overnight were washed
with wash buffer (PBS/0.05% Tween) and blocked for 1 h at
37.degree. C. with 3% BSA in wash buffer. The plates were then
washed again and incubated with the solution binding reactions for
30 min at room temperature. After extensive washing with wash
buffer, bound scFv-Fc.gamma.2c fusion proteins were detected by a 1
h incubation at room temperature with a HRPO-labeled,
Fc.gamma.-specific, goat anti-mouse IgG antibody (Jackson
ImmunoResearch Laboratories 115-035-071). After extensive washing
with wash buffer, plates were developed with OPD solution (1 OPD
tablet, 25 ml OPD buffer and 8 .mu.l 30% H.sub.2O.sub.2) for 15 min
and the reaction was stopped with 5% H.sub.2SO.sub.4 solution.
Plates were then read at OD 450 nm on an ELISA reader (Biorad
Benchmark). The Kd values were determined as the EC50 of the ELISA
signal as a function of the RNAse-M2e concentration present in the
solution binding reaction. The antibodies scFv-D005-msFc.gamma.2c,
scFv-E040-msFc.gamma.2c and scFv-F052-msFc.gamma.2c were found to
bind soluble RNase-M2e with a high affinity. Kd values were found
to be 4 pM (D005), 13 pM (E040) and 6 pM (F052).
[0189] The same assay was repeated under otherwise identical
conditions using free M2e peptide (SEQ ID NO:48) instead of the
RNAse-M2e conjugate. The antibodies scFv-D005-msFc.gamma.2c,
scFv-E040-msFc.gamma.2c and scFv-F052-msFc.gamma.2c were found to
bind soluble M2e peptide with a high affinity. Kd values were found
to be 4 nM (D005, E040) and 5 nM (F052).
Example 12
Preferential Binding of Antibody D005 to Cell-Associated M2
[0190] The ability of antibody D005 to distinguish between native,
cell-associated M2 and an unstructured, soluble M2e peptide was
assessed by flow cytometry. For comparison, mouse monoclonal
antibody 14C2 was also analyzed (Zebedee et al., 1988, J. Virol.
62(8): 2762-2772). Thus, scFv-D005-msFc.gamma.2c and mAb 14C2 were
incubated in FACS buffer at a concentration of 0.5 .mu.g/ml in the
presence or absence of 12 nM soluble M2e peptide for 1 hour on ice.
The prebound antibodies were then used for staining of L929-M2#E9
cells. Thus, L929-M2#E9 cells were brought to a single-cell
suspension and incubated with the prebound antibodies or, as a
control, with FACS buffer. After a 50 min incubation on ice, cells
were washed in FACS buffer and bound antibodies detected by a 20
min incubation on ice with Cy5-labeled goat anti-mouse antibody
(Jackson Immuno, Cat No 115-176-071) in FACS buffer. After a final
wash, the fluorescence intensity of the stained cells was analyzed
by flow cytometry using a FACScalibur.RTM. (Becton Dickinson) (FIG.
6). Both antibodies were found to bind native, cell
surface-expressed M2 with similar efficiency in the absence of
peptide. However, in the presence of peptide, mouse mAb 14C2 did
not efficiently recognize L292/M2#E9 cells. In contrast, antibody
scFv-D005-msFc.gamma.2c efficiently stained the cells despite the
presence of an excess of competing peptide. This indicates that
antibody D005, but not mAb 14C2, may bind a conformational epitope
only present in native, cell-surface M2.
Example 13
Direct Recognition of Virus Particles by Antibody D005
[0191] To investigate the ability of antibody D005 to directly bind
to Influenza A virus particles, a capture ELISA was carried out.
Thus, wells of an ELISA plate were coated overnight at 4.degree. C.
with HA-specific mouse mAb H37-80 at a concentration of 10 .mu.g/ml
in coating buffer. The plate was then washed with wash buffer
(PBS/0.05% Tween) and blocked for 3 h at 37.degree. C. with 5% BSA
in wash buffer. The plate was then washed again and incubated with
10.sup.8, 5.times.10.sup.7, 2.5.times.10.sup.7, or
1.25.times.10.sup.7 pfu Influenza A PR8 per well in wash buffer/1%
BSA. After 1 hour incubation at room temperature, the plate was
washed again and incubated with 1 .mu.g/ml fully-human IgG1k-D005
or an isotype-matched control antibody (human IgG1k, Sigma, Cat.
No. 15154) in wash buffer/1% BSA. After 1 hour incubation at room
temperature, the plate was washed again and incubated with a 1:1000
dilution of HRPO-conjugated goat anti-human IgG (Jackson Immuno,
Cat No 109-035-098) in wash buffer/1% BSA. After 1 hour incubation
at room temperature, the plate was washed extensively with wash
buffer, developed with OPD solution and read on an ELISA reader as
described above. Whereas no detectable ELISA signal was obtained
with the control antibody, hIgG1k-D005 readily detected captured
Influenza A particles in a dose-dependent manner (FIG. 7).
Example 14
Analysis of Crossreactivity and Fine Mapping of Epitope Recognized
by Antibody D005
[0192] Crossreactivity of antibody D005 with M2 sequences derived
from different Influenza A strains was analyzed by testing the
binding of IgG1k-D005 to solid phase bound peptide variants
(analysis performed by Pepscan Presto BV, Lelystad, the
Netherlands). Since it was previously found that the epitope
recognized by D005 is comprised within the 16 amino acid peptide
M2e-short (SEQ ID NO:49) (Example 4), only peptides spanning this
region were synthesized. In doing so, 35 of the 23-mer peptides
listed in Table 4 (SEQ IDs NO:48, 50-59, 61-83, and 90-92) were
covered by 28 different 16-mer peptides. Binding was assayed at two
antibody concentrations, 0.5 .mu.g/ml and 0.05 .mu.g/ml, revealing
that antibody D005 is broadly cross-reactive. Significantly, all
peptides tested were recognized, and 19 of the peptides were
recognized as well as the one corresponding to the M2e consensus
sequence (FIG. 8A).
[0193] The minimal epitope was determined in a similar manner, by
testing the binding of IgG1k-D005 to solid phase bound variants of
a peptide corresponding to peptide M2e-short (SEQ ID NO:49)
(analysis performed by Pepscan Presto BV, Lelystad, the
Netherlands). Three types of analyses were done: first, N- and
C-terminal deletions; second, epitope scanning by synthesizing all
105 different 3 to 16 mer variants; and third, point mutations by
synthesiszing all 304 possible single positional variants. The 3
analyses yielded comparable results (FIG. 8B). N-/C-terminal
deletion and epitope scanning analyses narrowed down the region
recognized by D005 to the 8-mer peptide LLTEVETP (SEQ ID NO:93).
Sequence CWU 1
1
93112PRTHomo sapiens 1Gln Ser Val Leu Tyr Thr Ser Asn Asn Lys Asn
Tyr 1 5 10 212PRTHomo sapiens 2Gln Ser Val Leu Asn Thr Ser Asn Asn
Lys Asn Tyr 1 5 10 312PRTHomo sapiens 3Gln Ser Val Leu His Thr Ser
Asn Asn Lys Asn Tyr 1 5 10 412PRTHomo sapiens 4Gln Ser Val Leu Tyr
Ser Ser Asn Asn Glu Asn Tyr 1 5 10 512PRTHomo sapiens 5Gln Ser Val
Leu Tyr Ser Ser Asn Asn Glu Asp Tyr 1 5 10 612PRTHomo sapiens 6Gln
Ser Leu Leu Tyr Ser Ser Asn Asn Lys Asn Tyr 1 5 10 73PRTHomo
sapiens 7Trp Ala Ser 1 89PRTHomo sapiens 8Gln Gln Tyr Phe Met Thr
Pro Ile Thr 1 5 99PRTHomo sapiens 9Gln Gln Tyr Phe Met Ala Pro Ile
Thr 1 5 109PRTHomo sapiens 10Gln Gln Tyr Phe Val Thr Pro Ile Thr 1
5 119PRTHomo sapiens 11Gln Gln Tyr Phe Met Thr Pro Ile Ala 1 5
128PRTHomo sapiens 12Gly Leu Asn Phe Gly Asp Tyr Pro 1 5
1310PRTHomo sapiens 13Ile Lys Ser Lys Ser Tyr Gly Val Thr Thr 1 5
10 1410PRTHomo sapiens 14Ile Lys Ser Lys Pro Tyr Gly Val Thr Thr 1
5 10 1512PRTHomo sapiens 15Thr Ser Ser Ser Gly Phe Leu Tyr Tyr Phe
Asp Tyr 1 5 10 1612PRTHomo sapiens 16Thr Ser Ser Ser Gly Phe Leu
Tyr Tyr Phe Asp His 1 5 10 1712PRTHomo sapiens 17Thr Ser Ser Ser
Ser Phe Leu Tyr Tyr Phe Asp Tyr 1 5 10 1812PRTHomo sapiens 18Thr
Ser Asn Ser Gly Phe Leu Tyr Tyr Phe Asp Tyr 1 5 10 1912PRTHomo
sapiens 19Thr Ser Ser Ser Gly Phe Ser Tyr Tyr Phe Asp Tyr 1 5 10
20109PRTHomo sapiens 20Thr Gln Ser Pro Asp Ser Leu Ala Val Ser Leu
Gly Glu Arg Ala Thr 1 5 10 15 Ile Asn Cys Lys Ser Ser Gln Ser Val
Leu Tyr Thr Ser Asn Asn Lys 20 25 30 Asn Tyr Leu Gly Trp Tyr Gln
Gln Lys Pro Gly Gln Pro Pro Asn Leu 35 40 45 Leu Ile Tyr Trp Ala
Ser Thr Arg Glu Ser Gly Val Pro Asp Arg Phe 50 55 60 Ser Gly Ser
Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Asn Ser Val 65 70 75 80 Gln
Ala Glu Asp Val Ala Val Tyr Tyr Cys Gln Gln Tyr Phe Met Thr 85 90
95 Pro Ile Thr Phe Gly Gln Gly Thr Arg Leu Glu Ile Lys 100 105
21109PRTHomo sapiens 21Thr Gln Ser Pro Asp Ser Leu Ala Val Ser Leu
Gly Glu Arg Ala Thr 1 5 10 15 Ile Asn Cys Lys Ser Ser Gln Ser Val
Leu Tyr Ser Ser Asn Asn Glu 20 25 30 Asn Tyr Leu Ala Trp Tyr Gln
Gln Lys Pro Gly Gln Pro Pro Lys Leu 35 40 45 Leu Ile Tyr Trp Ala
Ser Thr Arg Glu Ser Gly Val Pro Asp Arg Phe 50 55 60 Ser Gly Ser
Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Asn Ser Val 65 70 75 80 Gln
Ala Glu Asp Val Ala Val Tyr Tyr Cys Gln Gln Tyr Phe Met Thr 85 90
95 Pro Ile Thr Phe Gly Gln Gly Thr Arg Leu Glu Ile Lys 100 105
22109PRTHomo sapiens 22Thr Gln Ser Pro Asp Ala Leu Ala Val Ser Leu
Gly Glu Arg Ala Thr 1 5 10 15 Ile Asn Cys Lys Ser Ser Gln Ser Leu
Leu Tyr Ser Ser Asn Asn Lys 20 25 30 Asn Tyr Leu Ala Trp Tyr Gln
Lys Lys Pro Gly Gln Pro Pro Lys Leu 35 40 45 Leu Ile Tyr Trp Ala
Ser Thr Arg Glu Ser Gly Val Pro Asp Arg Phe 50 55 60 Ser Gly Ser
Gly Ser Gly Thr Glu Phe Thr Leu Thr Ile Asn Ser Val 65 70 75 80 Gln
Ala Glu Asp Val Ala Val Tyr Tyr Cys Gln Gln Tyr Phe Met Thr 85 90
95 Pro Ile Thr Phe Gly Gln Gly Thr Arg Leu Glu Ile Lys 100 105
23115PRTHomo sapiens 23Ser Gly Gly Ala Leu Val Gln Pro Gly Arg Ser
Leu Arg Leu Ser Cys 1 5 10 15 Arg Thr Ser Gly Leu Asn Phe Gly Asp
Tyr Pro Ile Asn Trp Val Arg 20 25 30 Gln Ala Pro Gly Lys Gly Leu
Glu Trp Val Gly Phe Ile Lys Ser Lys 35 40 45 Ser Tyr Gly Val Thr
Thr Glu Phe Ala Ala Ser Val Glu Gly Arg Phe 50 55 60 Thr Ile Ser
Arg Asp Asp Ser Arg Gly Ile Ala Tyr Leu Gln Met Asn 65 70 75 80 Ser
Leu Lys Thr Glu Asp Thr Ala Val Tyr Tyr Cys Thr Ser Ser Ser 85 90
95 Gly Phe Leu Tyr Tyr Phe Asp Tyr Trp Gly Gln Gly Thr Leu Val Thr
100 105 110 Val Ser Ser 115 244PRTHomo sapiens 24Asp Ile Val Met 1
256PRTHomo sapiens 25Glu Val Gln Leu Val Glu 1 5 26220PRTHomo
sapiens 26Asp Ile Val Met Thr Gln Ser Pro Asp Ser Leu Ala Val Ser
Leu Gly 1 5 10 15 Glu Arg Ala Thr Ile Asn Cys Lys Ser Ser Gln Ser
Val Leu Tyr Thr 20 25 30 Ser Asn Asn Lys Asn Tyr Leu Gly Trp Tyr
Gln Gln Lys Pro Gly Gln 35 40 45 Pro Pro Asn Leu Leu Ile Tyr Trp
Ala Ser Thr Arg Glu Ser Gly Val 50 55 60 Pro Asp Arg Phe Ser Gly
Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr 65 70 75 80 Ile Asn Ser Val
Gln Ala Glu Asp Val Ala Val Tyr Tyr Cys Gln Gln 85 90 95 Tyr Phe
Met Thr Pro Ile Thr Phe Gly Gln Gly Thr Arg Leu Glu Ile 100 105 110
Lys Arg Thr Val Ala Ala Pro Ser Val Phe Ile Phe Pro Pro Ser Asp 115
120 125 Glu Gln Leu Lys Ser Gly Thr Ala Ser Val Val Cys Leu Leu Asn
Asn 130 135 140 Phe Tyr Pro Arg Glu Ala Lys Val Gln Trp Lys Val Asp
Asn Ala Leu 145 150 155 160 Gln Ser Gly Asn Ser Gln Glu Ser Val Thr
Glu Gln Asp Ser Lys Asp 165 170 175 Ser Thr Tyr Ser Leu Ser Ser Thr
Leu Thr Leu Ser Lys Ala Asp Tyr 180 185 190 Glu Lys His Lys Val Tyr
Ala Cys Glu Val Thr His Gln Gly Leu Ser 195 200 205 Ser Pro Val Thr
Lys Ser Phe Asn Arg Gly Glu Cys 210 215 220 27220PRTHomo sapiens
27Asp Ile Val Met Thr Gln Ser Pro Asp Ser Leu Ala Val Ser Leu Gly 1
5 10 15 Glu Arg Ala Thr Ile Asn Cys Lys Ser Ser Gln Ser Val Leu Tyr
Ser 20 25 30 Ser Asn Asn Glu Asn Tyr Leu Ala Trp Tyr Gln Gln Lys
Pro Gly Gln 35 40 45 Pro Pro Lys Leu Leu Ile Tyr Trp Ala Ser Thr
Arg Glu Ser Gly Val 50 55 60 Pro Asp Arg Phe Ser Gly Ser Gly Ser
Gly Thr Asp Phe Thr Leu Thr 65 70 75 80 Ile Asn Ser Val Gln Ala Glu
Asp Val Ala Val Tyr Tyr Cys Gln Gln 85 90 95 Tyr Phe Met Thr Pro
Ile Thr Phe Gly Gln Gly Thr Arg Leu Glu Ile 100 105 110 Lys Arg Thr
Val Ala Ala Pro Ser Val Phe Ile Phe Pro Pro Ser Asp 115 120 125 Glu
Gln Leu Lys Ser Gly Thr Ala Ser Val Val Cys Leu Leu Asn Asn 130 135
140 Phe Tyr Pro Arg Glu Ala Lys Val Gln Trp Lys Val Asp Asn Ala Leu
145 150 155 160 Gln Ser Gly Asn Ser Gln Glu Ser Val Thr Glu Gln Asp
Ser Lys Asp 165 170 175 Ser Thr Tyr Ser Leu Ser Ser Thr Leu Thr Leu
Ser Lys Ala Asp Tyr 180 185 190 Glu Lys His Lys Val Tyr Ala Cys Glu
Val Thr His Gln Gly Leu Ser 195 200 205 Ser Pro Val Thr Lys Ser Phe
Asn Arg Gly Glu Cys 210 215 220 28220PRTHomo sapiens 28Asp Ile Val
Met Thr Gln Ser Pro Asp Ala Leu Ala Val Ser Leu Gly 1 5 10 15 Glu
Arg Ala Thr Ile Asn Cys Lys Ser Ser Gln Ser Leu Leu Tyr Ser 20 25
30 Ser Asn Asn Lys Asn Tyr Leu Ala Trp Tyr Gln Lys Lys Pro Gly Gln
35 40 45 Pro Pro Lys Leu Leu Ile Tyr Trp Ala Ser Thr Arg Glu Ser
Gly Val 50 55 60 Pro Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Glu
Phe Thr Leu Thr 65 70 75 80 Ile Asn Ser Val Gln Ala Glu Asp Val Ala
Val Tyr Tyr Cys Gln Gln 85 90 95 Tyr Phe Met Thr Pro Ile Thr Phe
Gly Gln Gly Thr Arg Leu Glu Ile 100 105 110 Lys Arg Thr Val Ala Ala
Pro Ser Val Phe Ile Phe Pro Pro Ser Asp 115 120 125 Glu Gln Leu Lys
Ser Gly Thr Ala Ser Val Val Cys Leu Leu Asn Asn 130 135 140 Phe Tyr
Pro Arg Glu Ala Lys Val Gln Trp Lys Val Asp Asn Ala Leu 145 150 155
160 Gln Ser Gly Asn Ser Gln Glu Ser Val Thr Glu Gln Asp Ser Lys Asp
165 170 175 Ser Thr Tyr Ser Leu Ser Ser Thr Leu Thr Leu Ser Lys Ala
Asp Tyr 180 185 190 Glu Lys His Lys Val Tyr Ala Cys Glu Val Thr His
Gln Gly Leu Ser 195 200 205 Ser Pro Val Thr Lys Ser Phe Asn Arg Gly
Glu Cys 210 215 220 29451PRTHomo sapiens 29Glu Val Gln Leu Val Glu
Ser Gly Gly Ala Leu Val Gln Pro Gly Arg 1 5 10 15 Ser Leu Arg Leu
Ser Cys Arg Thr Ser Gly Leu Asn Phe Gly Asp Tyr 20 25 30 Pro Ile
Asn Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45
Gly Phe Ile Lys Ser Lys Ser Tyr Gly Val Thr Thr Glu Phe Ala Ala 50
55 60 Ser Val Glu Gly Arg Phe Thr Ile Ser Arg Asp Asp Ser Arg Gly
Ile 65 70 75 80 Ala Tyr Leu Gln Met Asn Ser Leu Lys Thr Glu Asp Thr
Ala Val Tyr 85 90 95 Tyr Cys Thr Ser Ser Ser Gly Phe Leu Tyr Tyr
Phe Asp Tyr Trp Gly 100 105 110 Gln Gly Thr Leu Val Thr Val Ser Ser
Ala Ser Thr Lys Gly Pro Ser 115 120 125 Val Phe Pro Leu Ala Pro Ser
Ser Lys Ser Thr Ser Gly Gly Thr Ala 130 135 140 Ala Leu Gly Cys Leu
Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val 145 150 155 160 Ser Trp
Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala 165 170 175
Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val 180
185 190 Pro Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn
His 195 200 205 Lys Pro Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro
Lys Ser Cys 210 215 220 Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala
Pro Glu Leu Leu Gly 225 230 235 240 Gly Pro Ser Val Phe Leu Phe Pro
Pro Lys Pro Lys Asp Thr Leu Met 245 250 255 Ile Ser Arg Thr Pro Glu
Val Thr Cys Val Val Val Asp Val Ser His 260 265 270 Glu Asp Pro Glu
Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val 275 280 285 His Asn
Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr 290 295 300
Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly 305
310 315 320 Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala
Pro Ile 325 330 335 Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg
Glu Pro Gln Val 340 345 350 Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu
Thr Lys Asn Gln Val Ser 355 360 365 Leu Thr Cys Leu Val Lys Gly Phe
Tyr Pro Ser Asp Ile Ala Val Glu 370 375 380 Trp Glu Ser Asn Gly Gln
Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro 385 390 395 400 Val Leu Asp
Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val 405 410 415 Asp
Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met 420 425
430 His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser
435 440 445 Pro Gly Lys 450 301413DNAHomo
sapienssig_peptide(1)..(57) 30atggaattcg gcctgagctg ggtgttcctg
gtggccatcc tgaagggcgt gcagtgcgag 60gtgcagctgg tcgagagcgg cggagccctg
gtgcagcccg gcagaagcct gagactgagc 120tgccggacca gcggcctgaa
cttcggcgac taccccatca actgggtgcg gcaggctcca 180gggaaaggac
tcgaatgggt gggcttcatc aagagcaaga gctacggcgt gaccaccgag
240ttcgccgcca gcgtggaggg ccggttcacc atcagccggg acgacagccg
gggcattgcc 300tacctgcaga tgaacagcct gaaaaccgag gacaccgccg
tgtactactg caccagcagc 360agcggctttc tgtactactt cgactactgg
ggacagggca ccctggtgac cgtgagcagc 420gccagcacca agggccccag
cgtgttcccc ctggccccca gcagcaagag taccagcgga 480ggcactgctg
ccctcggatg cctggtgaag gactacttcc ccgagcccgt gaccgtgtcc
540tggaactctg gcgccctgac ctccggcgtg cacaccttcc ccgccgtgct
ccagtctagt 600ggcctgtata gcctgagcag cgtggtgaca gtccctagca
gttccctggg aacccagacc 660tacatctgca acgtgaacca caagcccagc
aacaccaagg tggacaagaa ggtggagccc 720aagagctgcg acaagaccca
cacctgcccc ccctgccctg cccctgagct gctgggcgga 780ccctccgtgt
tcctgttccc ccccaagccc aaggacaccc tgatgatcag ccggaccccc
840gaggtgacct gcgtggtggt ggatgtgagt catgaggatc ctgaggtgaa
gttcaattgg 900tacgtggacg gcgtggaggt gcacaacgcc aagaccaagc
cccgggagga acagtacaac 960agcacctacc gggtggtgtc cgtgctgacc
gtgctgcacc aggactggct gaacggcaaa 1020gaatacaagt gcaaggtgtc
caacaaggcc ctgcctgccc ccatcgagaa aaccatcagc 1080aaggccaagg
gccagcctag agaaccccag gtgtacacac tgcctccatc ccgggacgag
1140ctgaccaaga accaggtgtc cctgacctgt ctggtgaagg gcttctaccc
cagcgatatc 1200gccgtggagt gggagagcaa cggccagccc gagaacaact
acaagaccac cccccctgtg 1260ctggacagcg acggcagctt cttcctgtac
agcaagctga ccgtggacaa gagccggtgg 1320cagcagggca acgtgttcag
ctgcagcgtg atgcacgagg ccctgcacaa ccactacacc 1380cagaagagcc
tgagcctgtc ccccggcaag tga 141331470PRTHomo sapiensSIGNAL(1)..(19)
31Met Glu Phe Gly Leu Ser Trp Val Phe Leu Val Ala Ile Leu Lys Gly 1
5 10 15 Val Gln Cys Glu Val Gln Leu Val Glu Ser Gly Gly Ala Leu Val
Gln 20 25 30 Pro Gly Arg Ser Leu Arg Leu Ser Cys Arg Thr Ser Gly
Leu Asn Phe 35 40 45 Gly Asp Tyr Pro Ile Asn Trp Val Arg Gln Ala
Pro Gly Lys Gly Leu 50 55 60 Glu Trp Val Gly Phe Ile Lys Ser Lys
Ser Tyr Gly Val Thr Thr Glu 65 70 75 80 Phe Ala Ala Ser Val Glu Gly
Arg Phe Thr Ile Ser Arg Asp Asp Ser 85 90 95 Arg Gly Ile Ala Tyr
Leu Gln Met Asn Ser Leu Lys Thr Glu Asp Thr 100 105 110 Ala Val Tyr
Tyr Cys Thr Ser Ser Ser Gly Phe Leu Tyr Tyr Phe Asp 115 120 125 Tyr
Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser Ala Ser Thr Lys 130 135
140 Gly Pro Ser Val Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly
145 150 155 160 Gly Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe
Pro Glu Pro 165 170 175 Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr
Ser Gly Val His Thr 180 185 190 Phe Pro Ala Val Leu Gln Ser Ser Gly
Leu Tyr Ser Leu Ser Ser Val 195 200 205 Val Thr Val Pro Ser Ser Ser
Leu Gly Thr Gln Thr Tyr Ile Cys Asn 210 215 220 Val Asn His Lys Pro
Ser
Asn Thr Lys Val Asp Lys Lys Val Glu Pro 225 230 235 240 Lys Ser Cys
Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu 245 250 255 Leu
Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp 260 265
270 Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp
275 280 285 Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val
Asp Gly 290 295 300 Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu
Glu Gln Tyr Asn 305 310 315 320 Ser Thr Tyr Arg Val Val Ser Val Leu
Thr Val Leu His Gln Asp Trp 325 330 335 Leu Asn Gly Lys Glu Tyr Lys
Cys Lys Val Ser Asn Lys Ala Leu Pro 340 345 350 Ala Pro Ile Glu Lys
Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu 355 360 365 Pro Gln Val
Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn 370 375 380 Gln
Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile 385 390
395 400 Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys
Thr 405 410 415 Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu
Tyr Ser Lys 420 425 430 Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly
Asn Val Phe Ser Cys 435 440 445 Ser Val Met His Glu Ala Leu His Asn
His Tyr Thr Gln Lys Ser Leu 450 455 460 Ser Leu Ser Pro Gly Lys 465
470 32723DNAHomo sapienssig_peptide(1)..(60) 32atggtgctgc
agacccaggt gttcatcagc ctgctgctgt ggatcagcgg cgcctacggc 60gacatcgtga
tgacccagag ccccgacagc ctggccgtga gcctgggcga gcgggccacc
120atcaactgca agagcagcca gagcgtgctg tacaccagca acaacaagaa
ctacctgggc 180tggtatcagc agaagcccgg ccagcccccc aacctgctga
tctactgggc cagcacccgg 240gagagcggcg tgcccgaccg gtttagcggc
agcggctccg gcaccgactt caccctgacc 300atcaacagcg tgcaggccga
ggacgtggcc gtgtactact gccagcagta cttcatgacc 360cccatcacct
tcggccaggg cacccggctg gaaatcaagc gtacggtggc cgccccctcc
420gtgttcatct tcccccccag cgacgagcag ctgaagagcg gcaccgccag
cgtggtgtgc 480ctgctgaaca acttctaccc ccgggaggcc aaggtgcagt
ggaaggtgga caacgccctg 540cagagcggca acagccagga aagcgtcacc
gagcaggaca gcaaggactc cacctacagc 600ctgagcagca ccctgaccct
gagcaaggcc gactacgaga agcacaaggt gtacgcctgc 660gaggtgaccc
accagggcct gtccagcccc gtgaccaaga gcttcaaccg gggcgagtgc 720tga
72333240PRTHomo sapiensSIGNAL(1)..(20) 33Met Val Leu Gln Thr Gln
Val Phe Ile Ser Leu Leu Leu Trp Ile Ser 1 5 10 15 Gly Ala Tyr Gly
Asp Ile Val Met Thr Gln Ser Pro Asp Ser Leu Ala 20 25 30 Val Ser
Leu Gly Glu Arg Ala Thr Ile Asn Cys Lys Ser Ser Gln Ser 35 40 45
Val Leu Tyr Thr Ser Asn Asn Lys Asn Tyr Leu Gly Trp Tyr Gln Gln 50
55 60 Lys Pro Gly Gln Pro Pro Asn Leu Leu Ile Tyr Trp Ala Ser Thr
Arg 65 70 75 80 Glu Ser Gly Val Pro Asp Arg Phe Ser Gly Ser Gly Ser
Gly Thr Asp 85 90 95 Phe Thr Leu Thr Ile Asn Ser Val Gln Ala Glu
Asp Val Ala Val Tyr 100 105 110 Tyr Cys Gln Gln Tyr Phe Met Thr Pro
Ile Thr Phe Gly Gln Gly Thr 115 120 125 Arg Leu Glu Ile Lys Arg Thr
Val Ala Ala Pro Ser Val Phe Ile Phe 130 135 140 Pro Pro Ser Asp Glu
Gln Leu Lys Ser Gly Thr Ala Ser Val Val Cys 145 150 155 160 Leu Leu
Asn Asn Phe Tyr Pro Arg Glu Ala Lys Val Gln Trp Lys Val 165 170 175
Asp Asn Ala Leu Gln Ser Gly Asn Ser Gln Glu Ser Val Thr Glu Gln 180
185 190 Asp Ser Lys Asp Ser Thr Tyr Ser Leu Ser Ser Thr Leu Thr Leu
Ser 195 200 205 Lys Ala Asp Tyr Glu Lys His Lys Val Tyr Ala Cys Glu
Val Thr His 210 215 220 Gln Gly Leu Ser Ser Pro Val Thr Lys Ser Phe
Asn Arg Gly Glu Cys 225 230 235 240 34723DNAHomo
sapienssig_peptide(1)..(60) 34atggtgctgc agacccaggt gttcatcagc
ctgctgctgt ggatcagcgg cgcctacggc 60gacatcgtga tgacccagag ccccgacagc
ctggccgtga gcctgggcga gcgggccacc 120atcaactgca agagcagcca
gagcgtgctg tacagcagca acaacgagaa ctacctggcc 180tggtatcagc
agaagcccgg ccagcccccc aagctgctga tctactgggc cagcacccgg
240gagagcggcg tgcccgaccg gtttagcggc agcggctccg gcaccgactt
caccctgacc 300atcaacagcg tgcaggccga ggacgtggcc gtgtactact
gccagcagta cttcatgacc 360cccatcacct tcggccaggg cacccggctg
gaaatcaagc gtacggtggc cgccccctcc 420gtgttcatct tcccccccag
cgacgagcag ctgaagagcg gcaccgccag cgtggtgtgc 480ctgctgaaca
acttctaccc ccgggaggcc aaggtgcagt ggaaggtgga caacgccctg
540cagagcggca acagccagga aagcgtcacc gagcaggaca gcaaggactc
cacctacagc 600ctgagcagca ccctgaccct gagcaaggcc gactacgaga
agcacaaggt gtacgcctgc 660gaggtgaccc accagggcct gtccagcccc
gtgaccaaga gcttcaaccg gggcgagtgc 720tga 72335240PRTHomo
sapiensSIGNAL(1)..(20) 35Met Val Leu Gln Thr Gln Val Phe Ile Ser
Leu Leu Leu Trp Ile Ser 1 5 10 15 Gly Ala Tyr Gly Asp Ile Val Met
Thr Gln Ser Pro Asp Ser Leu Ala 20 25 30 Val Ser Leu Gly Glu Arg
Ala Thr Ile Asn Cys Lys Ser Ser Gln Ser 35 40 45 Val Leu Tyr Ser
Ser Asn Asn Glu Asn Tyr Leu Ala Trp Tyr Gln Gln 50 55 60 Lys Pro
Gly Gln Pro Pro Lys Leu Leu Ile Tyr Trp Ala Ser Thr Arg 65 70 75 80
Glu Ser Gly Val Pro Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp 85
90 95 Phe Thr Leu Thr Ile Asn Ser Val Gln Ala Glu Asp Val Ala Val
Tyr 100 105 110 Tyr Cys Gln Gln Tyr Phe Met Thr Pro Ile Thr Phe Gly
Gln Gly Thr 115 120 125 Arg Leu Glu Ile Lys Arg Thr Val Ala Ala Pro
Ser Val Phe Ile Phe 130 135 140 Pro Pro Ser Asp Glu Gln Leu Lys Ser
Gly Thr Ala Ser Val Val Cys 145 150 155 160 Leu Leu Asn Asn Phe Tyr
Pro Arg Glu Ala Lys Val Gln Trp Lys Val 165 170 175 Asp Asn Ala Leu
Gln Ser Gly Asn Ser Gln Glu Ser Val Thr Glu Gln 180 185 190 Asp Ser
Lys Asp Ser Thr Tyr Ser Leu Ser Ser Thr Leu Thr Leu Ser 195 200 205
Lys Ala Asp Tyr Glu Lys His Lys Val Tyr Ala Cys Glu Val Thr His 210
215 220 Gln Gly Leu Ser Ser Pro Val Thr Lys Ser Phe Asn Arg Gly Glu
Cys 225 230 235 240 36723DNAHomo sapienssig_peptide(1)..(60)
36atggtgctgc agacccaggt gttcatcagc ctgctgctgt ggatcagcgg cgcctacggc
60gacatcgtga tgacccagag ccccgacgcc ctggccgtga gcctgggcga gcgggccacc
120atcaactgca agagcagcca gagcctgctg tacagcagca acaacaagaa
ctacctggcc 180tggtatcaga aaaagcccgg ccagcccccc aagctgctga
tctactgggc cagcacccgg 240gagagcggcg tgcccgaccg gtttagcggc
agcggctccg gcaccgagtt caccctgacc 300atcaacagcg tgcaggccga
ggacgtggcc gtgtactact gccagcagta cttcatgacc 360cccatcacct
tcggccaggg cacccggctg gaaatcaagc gtacggtggc cgccccctcc
420gtgttcatct tcccccccag cgacgagcag ctgaagagcg gcaccgccag
cgtggtgtgc 480ctgctgaaca acttctaccc ccgggaggcc aaggtgcagt
ggaaggtgga caacgccctg 540cagagcggca acagccagga aagcgtcacc
gagcaggaca gcaaggactc cacctacagc 600ctgagcagca ccctgaccct
gagcaaggcc gactacgaga agcacaaggt gtacgcctgc 660gaggtgaccc
accagggcct gtccagcccc gtgaccaaga gcttcaaccg gggcgagtgc 720tga
72337240PRTHomo sapiensSIGNAL(1)..(20) 37Met Val Leu Gln Thr Gln
Val Phe Ile Ser Leu Leu Leu Trp Ile Ser 1 5 10 15 Gly Ala Tyr Gly
Asp Ile Val Met Thr Gln Ser Pro Asp Ala Leu Ala 20 25 30 Val Ser
Leu Gly Glu Arg Ala Thr Ile Asn Cys Lys Ser Ser Gln Ser 35 40 45
Leu Leu Tyr Ser Ser Asn Asn Lys Asn Tyr Leu Ala Trp Tyr Gln Lys 50
55 60 Lys Pro Gly Gln Pro Pro Lys Leu Leu Ile Tyr Trp Ala Ser Thr
Arg 65 70 75 80 Glu Ser Gly Val Pro Asp Arg Phe Ser Gly Ser Gly Ser
Gly Thr Glu 85 90 95 Phe Thr Leu Thr Ile Asn Ser Val Gln Ala Glu
Asp Val Ala Val Tyr 100 105 110 Tyr Cys Gln Gln Tyr Phe Met Thr Pro
Ile Thr Phe Gly Gln Gly Thr 115 120 125 Arg Leu Glu Ile Lys Arg Thr
Val Ala Ala Pro Ser Val Phe Ile Phe 130 135 140 Pro Pro Ser Asp Glu
Gln Leu Lys Ser Gly Thr Ala Ser Val Val Cys 145 150 155 160 Leu Leu
Asn Asn Phe Tyr Pro Arg Glu Ala Lys Val Gln Trp Lys Val 165 170 175
Asp Asn Ala Leu Gln Ser Gly Asn Ser Gln Glu Ser Val Thr Glu Gln 180
185 190 Asp Ser Lys Asp Ser Thr Tyr Ser Leu Ser Ser Thr Leu Thr Leu
Ser 195 200 205 Lys Ala Asp Tyr Glu Lys His Lys Val Tyr Ala Cys Glu
Val Thr His 210 215 220 Gln Gly Leu Ser Ser Pro Val Thr Lys Ser Phe
Asn Arg Gly Glu Cys 225 230 235 240 38812DNAartificial
sequencesynthetic 38ggcccaggcg gccgagctcg tgatgaccca gtctcctgac
tccctggctg tgtccctggg 60cgagagggcc accatcaact gcaagtccag ccagagtgtt
ctatacacct ccaacaataa 120gaactactta ggttggtacc agcagaaacc
agggcagccg cctaatttac tcatttattg 180ggcatctacc cgggaatccg
gggtccctga ccgattcagt ggcagcgggt ctgggacaga 240tttcactctc
accatcaaca gcgtgcaggc tgaggatgtg gcagtttatt actgccagca
300gtattttatg actcccatca ccttcggcca agggacacga ctggagatta
aaggtggttc 360ctctagatct tcctcctctg gtggcggtgg ctcgggcggt
ggtggggagg tgcagctggt 420ggagtctggg ggggccttgg tacagccagg
gcggtccctg agactctcct gtagaacctc 480tggactcaat tttggagatt
atcctataaa ctgggtccgc caggctccag ggaaggggct 540ggagtgggta
gggttcatca aaagcaagtc ttatggtgtg acaacagaat tcgccgcgtc
600tgtggagggc agattcacca tctcgaggga tgattccaga ggcatcgcct
atctgcagat 660gaacagcctg aaaaccgagg acacagccgt ctattactgt
acgtccagta gtggtttttt 720gtactacttt gactactggg gccagggaac
cctggtcacc gtctcctcag cctccaccaa 780gggcccatcg gtcactagtg
gccaggccgg cc 81239812DNAartificial sequencesynthetic 39ggcccaggcg
gccgagctcg tgatgactca gtctccagac tccctggctg tgtctctggg 60cgagagggcc
accatcaact gcaagtccag ccagagtgtt ttatacagct ccaacaatga
120gaactactta gcttggtacc agcagaaacc aggacagcct cctaaactgc
tcatttactg 180ggcatctacc cgggaatccg gggtccctga ccgattcagt
ggcagcgggt ctgggacaga 240tttcactctc accatcaaca gcgtgcaggc
tgaggatgtg gcagtttatt actgccagca 300gtattttatg actcccatca
ccttcggcca agggacacga ctggagatta aaggtggttc 360ctctagatct
tcctcctctg gtggcggtgg ctcgggcggt ggtggggagg tgcagctgtt
420ggagtctggg ggggccttgg tacagccagg gcggtccctg agactctcct
gtagaacctc 480tggactcaat tttggagatt atcctataaa ctgggtccgc
caggctccag ggaaggggct 540ggagtgggta gggttcatca aaagcaagtc
ttatggtgtg acaacagaat tcgccgcgtc 600tgtggagggc agattcacca
tctcaaggga tgattccaga ggcatcgcct atctgcagat 660gaacagcctg
aaaaccgagg acacagccgt ctattactgt acgtccagta gtggtttttt
720gtactacttt gactactggg gccagggaac cctggtcacc gtctcctcag
cttccaccaa 780gggcccatca gtcactagtg gccaggccgg cc
81240812DNAartificial sequencesynthetic 40ggcccaggcg gccgagcgcg
tgatgacaca gtctccagac gccctggctg tgtctctggg 60cgagagggcc accatcaact
gcaagtccag ccagagtctt ttatacagct ccaataataa 120gaactactta
gcttggtatc agaagaaacc aggacagcct cctaagctgc tcatttactg
180ggcatctacc cgggaatccg gggtccctga ccgattcagt ggcagcgggt
ctgggacaga 240gttcactctc accatcaaca gcgtgcaggc tgaggatgtg
gcagtttatt actgccagca 300gtattttatg actcccatca ccttcggcca
agggaccaag ctggagatca aaggtggttc 360ctctagatct tcctcctctg
gtggcggtgg ctcgggcggt ggtgggcagg tgcagctgca 420ggagtcgggg
ggggccttgg tacagccagg gcggtccctg agactctcct gtagaacctc
480tggactcaat tttggagatt atcctataaa ctgggtccgc caggctccag
ggaaggggct 540ggagtgggta gggttcatca aaagcaagtc ttatggtgtg
acaacagaat tcgccgcgtc 600tgtggagggc agattcacca tctcaaggga
tgattccaga ggcatcgcct atctgcagat 660gaacagcctg aaaaccgagg
acacagccgt ctattactgt acgtccagta gtggtttttt 720gtactacttt
gactactggg gccagggaac cctggtcacc gtctcctcag cttccaccaa
780gggcccatcg gtcactagtg gccaggccgg cc 8124110348DNAartificial
sequencecloning vector 41ggtaccatgg agacagacac actcctgcta
tgggtactgc tgctctgggt tccaggttcc 60actggtgacg cggatccggc ccaggcggcc
ttaattaaag gtttaaacgg ccaggccggc 120cgcaagcttg agcccagagt
gcccataaca cagaacccct gtcctccact caaagagtgt 180cccccatgcg
cagctccaga cctcttgggt ggaccatccg tcttcatctt ccctccaaag
240atcaaggatg tactcatgat ctccctgagc cccatggtca catgtgtggt
ggtggatgtg 300agcgaggatg acccagacgt ccagatcagc tggtttgtga
acaacgtgga agtacacaca 360gctcagacac aaacccatag agaggattac
aacagtactc tccgggtggt cagtgccctc 420cccatccagc accaggactg
gatgagtggc aaggagttca aatgcaaggt caacaacaga 480gccctcccat
cccccatcga gaaaaccatc tcaaaaccca gagggccagt aagagctcca
540caggtatatg tcttgcctcc accagcagaa gagatgacta agaaagagtt
cagtctgacc 600tgcatgatca caggcttctt acctgccgaa attgctgtgg
actggaccag caatgggcgt 660acagagcaaa actacaagaa caccgcaaca
gtcctggact ctgatggttc ttacttcatg 720tacagcaagc tcagagtaca
aaagagcact tgggaaagag gaagtctttt cgcctgctca 780gtggtccacg
agggtctgca caatcacctt acgactaaga ccatctcccg gtctctgggt
840aaatgactcg aggcccgaac aaaaactcat ctcagaagag gatctgaata
gcgccgtcga 900ccatcatcat catcatcatt gagtttaacg atccagacat
gataagatac attgatgagt 960ttggacaaac cacaactaga atgcagtgaa
aaaaatgctt tatttgtgaa atttgtgatg 1020ctattgcttt atttgtaacc
attataagct gcaataaaca agttaacaac aacaattgca 1080ttcattttat
gtttcaggtt cagggggagg tggggaggtt ttttaaagca agtaaaacct
1140ctacaaatgt ggtatggctg attatgatcc ggctgcctcg cgcgtttcgg
tgatgacggt 1200gaaaacctct gacacatgca gctcccggag acggtcacag
cttgtctgta agcggatgcc 1260gggagcagac aagcccgtca gggcgcgtca
gcgggtgttg gcgggtgtcg gggcgcagcc 1320atgaggtcga ctctagagga
tcgatccccg ccgccggacg aactaaacct gactacggca 1380tctctgcccc
ttcttcgcgg ggcagtgcat gtaatccctt cagttggttg gtacaacttg
1440ccaactgggc cctgttccac atgtgacacg gggggggacc aaacacaaag
gggttctctg 1500actgtagttg acatccttat aaatggatgt gcacatttgc
caacactgag tggctttcat 1560cctggagcag actttgcagt ctgtggactg
caacacaaca ttgcctttat gtgtaactct 1620tggctgaagc tcttacacca
atgctggggg acatgtacct cccaggggcc caggaagact 1680acgggaggct
acaccaacgt caatcagagg ggcctgtgta gctaccgata agcggaccct
1740caagagggca ttagcaatag tgtttataag gcccccttgt taaccctaaa
cgggtagcat 1800atgcttcccg ggtagtagta tatactatcc agactaaccc
taattcaata gcatatgtta 1860cccaacggga agcatatgct atcgaattag
ggttagtaaa agggtcctaa ggaacagcga 1920tatctcccac cccatgagct
gtcacggttt tatttacatg gggtcaggat tccacgaggg 1980tagtgaacca
ttttagtcac aagggcagtg gctgaagatc aaggagcggg cagtgaactc
2040tcctgaatct tcgcctgctt cttcattctc cttcgtttag ctaatagaat
aactgctgag 2100ttgtgaacag taaggtgtat gtgaggtgct cgaaaacaag
gtttcaggtg acgcccccag 2160aataaaattt ggacgggggg ttcagtggtg
gcattgtgct atgacaccaa tataaccctc 2220acaaacccct tgggcaataa
atactagtgt aggaatgaaa cattctgaat atctttaaca 2280atagaaatcc
atggggtggg gacaagccgt aaagactgga tgtccatctc acacgaattt
2340atggctatgg gcaacacata atcctagtgc aatatgatac tggggttatt
aagatgtgtc 2400ccaggcaggg accaagacag gtgaaccatg ttgttacact
ctatttgtaa caaggggaaa 2460gagagtggac gccgacagca gcggactcca
ctggttgtct ctaacacccc cgaaaattaa 2520acggggctcc acgccaatgg
ggcccataaa caaagacaag tggccactct tttttttgaa 2580attgtggagt
gggggcacgc gtcagccccc acacgccgcc ctgcggtttt ggactgtaaa
2640ataagggtgt aataacttgg ctgattgtaa ccccgctaac cactgcggtc
aaaccacttg 2700cccacaaaac cactaatggc accccgggga atacctgcat
aagtaggtgg gcgggccaag 2760ataggggcgc gattgctgcg atctggagga
caaattacac acacttgcgc ctgagcgcca 2820agcacagggt tgttggtcct
catattcacg aggtcgctga gagcacggtg ggctaatgtt 2880gccatgggta
gcatatacta cccaaatatc tggatagcat atgctatcct aatctatatc
2940tgggtagcat aggctatcct aatctatatc tgggtagcat atgctatcct
aatctatatc 3000tgggtagtat atgctatcct aatttatatc tgggtagcat
aggctatcct aatctatatc 3060tgggtagcat atgctatcct aatctatatc
tgggtagtat atgctatcct aatctgtatc 3120cgggtagcat atgctatcct
aatagagatt agggtagtat atgctatcct aatttatatc 3180tgggtagcat
atactaccca aatatctgga tagcatatgc tatcctaatc tatatctggg
3240tagcatatgc tatcctaatc tatatctggg tagcataggc tatcctaatc
tatatctggg 3300tagcatatgc tatcctaatc tatatctggg tagtatatgc
tatcctaatt tatatctggg 3360tagcataggc tatcctaatc tatatctggg
tagcatatgc tatcctaatc tatatctggg 3420tagtatatgc tatcctaatc
tgtatccggg tagcatatgc tatcctcatg catatacagt 3480cagcatatga
tacccagtag tagagtggga gtgctatcct ttgcatatgc cgccacctcc
3540caagggggcg tgaattttcg ctgcttgtcc ttttcctgca tgctggttgc
tcccattctt 3600aggtgaattt aaggaggcca ggctaaagcc gtcgcatgtc
tgattgctca ccaggtaaat 3660gtcgctaatg ttttccaacg cgagaaggtg
ttgagcgcgg agctgagtga cgtgacaaca 3720tgggtatgcc caattgcccc
atgttgggag gacgaaaatg gtgacaagac agatggccag 3780aaatacacca
acagcacgca tgatgtctac tggggattta ttctttagtg cgggggaata
3840cacggctttt aatacgattg agggcgtctc ctaacaagtt acatcactcc
tgcccttcct 3900caccctcatc tccatcacct ccttcatctc cgtcatctcc
gtcatcaccc tccgcggcag 3960ccccttccac cataggtgga aaccagggag
gcaaatctac tccatcgtca aagctgcaca 4020cagtcaccct gatattgcag
gtaggagcgg gctttgtcat aacaaggtcc ttaatcgcat 4080ccttcaaaac
ctcagcaaat atatgagttt gtaaaaagac catgaaataa cagacaatgg
4140actcccttag cgggccaggt tgtgggccgg gtccaggggc cattccaaag
gggagacgac 4200tcaatggtgt aagacgacat tgtggaatag caagggcagt
tcctcgcctt aggttgtaaa 4260gggaggtctt actacctcca tatacgaaca
caccggcgac ccaagttcct tcgtcggtag 4320tcctttctac gtgactccta
gccaggagag ctcttaaacc ttctgcaatg ttctcaaatt 4380tcgggttgga
acctccttga ccacgatgct ttccaaacca ccctcctttt ttgcgcctgc
4440ctccatcacc ctgaccccgg ggtccagtgc ttgggccttc tcctgggtca
tctgcggggc 4500cctgctctat cgctcccggg ggcacgtcag gctcaccatc
tgggccacct tcttggtggt 4560attcaaaata atcggcttcc cctacagggt
ggaaaaatgg ccttctacct ggagggggcc 4620tgcgcggtgg agacccggat
gatgatgact gactactggg actcctgggc ctcttttctc 4680cacgtccacg
acctctcccc ctggctcttt cacgacttcc ccccctggct ctttcacgtc
4740ctctaccccg gcggcctcca ctacctcctc gaccccggcc tccactacct
cctcgacccc 4800ggcctccact gcctcctcga ccccggcctc cacctcctgc
tcctgcccct cctgctcctg 4860cccctcctcc tgctcctgcc cctcctgccc
ctcctgctcc tgcccctcct gcccctcctg 4920ctcctgcccc tcctgcccct
cctgctcctg cccctcctgc ccctcctcct gctcctgccc 4980ctcctgcccc
tcctcctgct cctgcccctc ctgcccctcc tgctcctgcc cctcctgccc
5040ctcctgctcc tgcccctcct gcccctcctg ctcctgcccc tcctgctcct
gcccctcctg 5100ctcctgcccc tcctgctcct gcccctcctg cccctcctgc
ccctcctcct gctcctgccc 5160ctcctgctcc tgcccctcct gcccctcctg
cccctcctgc tcctgcccct cctcctgctc 5220ctgcccctcc tgcccctcct
gcccctcctc ctgctcctgc ccctcctgcc cctcctcctg 5280ctcctgcccc
tcctcctgct cctgcccctc ctgcccctcc tgcccctcct cctgctcctg
5340cccctcctgc ccctcctcct gctcctgccc ctcctcctgc tcctgcccct
cctgcccctc 5400ctgcccctcc tcctgctcct gcccctcctc ctgctcctgc
ccctcctgcc cctcctgccc 5460ctcctgcccc tcctcctgct cctgcccctc
ctcctgctcc tgcccctcct gctcctgccc 5520ctcccgctcc tgctcctgct
cctgttccac cgtgggtccc tttgcagcca atgcaacttg 5580gacgtttttg
gggtctccgg acaccatctc tatgtcttgg ccctgatcct gagccgcccg
5640gggctcctgg tcttccgcct cctcgtcctc gtcctcttcc ccgtcctcgt
ccatggttat 5700caccccctct tctttgaggt ccactgccgc cggagccttc
tggtccagat gtgtctccct 5760tctctcctag gccatttcca ggtcctgtac
ctggcccctc gtcagacatg attcacacta 5820aaagagatca atagacatct
ttattagacg acgctcagtg aatacaggga gtgcagactc 5880ctgccccctc
caacagcccc cccaccctca tccccttcat ggtcgctgtc agacagatcc
5940aggtctgaaa attccccatc ctccgaacca tcctcgtcct catcaccaat
tactcgcagc 6000ccggaaaact cccgctgaac atcctcaaga tttgcgtcct
gagcctcaag ccaggcctca 6060aattcctcgt cccccttttt gctggacggt
agggatgggg attctcggga cccctcctct 6120tcctcttcaa ggtcaccaga
cagagatgct actggggcaa cggaagaaaa gctgggtgcg 6180gcctgtgagg
atcagcttat cgatgataag ctgtcaaaca tgagaattct tgaagacgaa
6240agggcctcgt gatacgccta tttttatagg ttaatgtcat gataataatg
gtttcttaga 6300cgtcaggtgg cacttttcgg ggaaatgtgc gcggaacccc
tatttgttta tttttctaaa 6360tacattcaaa tatgtatccg ctcatgagac
aataaccctg ataaatgctt caataatatt 6420gaaaaaggaa gagtatgagt
attcaacatt tccgtgtcgc ccttattccc ttttttgcgg 6480cattttgcct
tcctgttttt gctcacccag aaacgctggt gaaagtaaaa gatgctgaag
6540atcagttggg tgcacgagtg ggttacatcg aactggatct caacagcggt
aagatccttg 6600agagttttcg ccccgaagaa cgttttccaa tgatgagcac
ttttaaagtt ctgctatgtg 6660gcgcggtatt atcccgtgtt gacgccgggc
aagagcaact cggtcgccgc atacactatt 6720ctcagaatga cttggttgag
tactcaccag tcacagaaaa gcatcttacg gatggcatga 6780cagtaagaga
attatgcagt gctgccataa ccatgagtga taacactgcg gccaacttac
6840ttctgacaac gatcggagga ccgaaggagc taaccgcttt tttgcacaac
atgggggatc 6900atgtaactcg ccttgatcgt tgggaaccgg agctgaatga
agccatacca aacgacgagc 6960gtgacaccac gatgcctgca gcaatggcaa
caacgttgcg caaactatta actggcgaac 7020tacttactct agcttcccgg
caacaattaa tagactggat ggaggcggat aaagttgcag 7080gaccacttct
gcgctcggcc cttccggctg gctggtttat tgctgataaa tctggagccg
7140gtgagcgtgg gtctcgcggt atcattgcag cactggggcc agatggtaag
ccctcccgta 7200tcgtagttat ctacacgacg gggagtcagg caactatgga
tgaacgaaat agacagatcg 7260ctgagatagg tgcctcactg attaagcatt
ggtaactgtc agaccaagtt tactcatata 7320tactttagat tgatttaaaa
cttcattttt aatttaaaag gatctaggtg aagatccttt 7380ttgataatct
catgaccaaa atcccttaac gtgagttttc gttccactga gcgtcagacc
7440ccgtagaaaa gatcaaagga tcttcttgag atcctttttt tctgcgcgta
atctgctgct 7500tgcaaacaaa aaaaccaccg ctaccagcgg tggtttgttt
gccggatcaa gagctaccaa 7560ctctttttcc gaaggtaact ggcttcagca
gagcgcagat accaaatact gtccttctag 7620tgtagccgta gttaggccac
cacttcaaga actctgtagc accgcctaca tacctcgctc 7680tgctaatcct
gttaccagtg gctgctgcca gtggcgataa gtcgtgtctt accgggttgg
7740actcaagacg atagttaccg gataaggcgc agcggtcggg ctgaacgggg
ggttcgtgca 7800cacagcccag cttggagcga acgacctaca ccgaactgag
atacctacag cgtgagctat 7860gagaaagcgc cacgcttccc gaagggagaa
aggcggacag gtatccggta agcggcaggg 7920tcggaacagg agagcgcacg
agggagcttc cagggggaaa cgcctggtat ctttatagtc 7980ctgtcgggtt
tcgccacctc tgacttgagc gtcgattttt gtgatgctcg tcaggggggc
8040ggagcctatg gaaaaacgcc agcaacgcgg cctttttacg gttcctggcc
ttttgctgcg 8100ccgcgtgcgg ctgctggaga tggcggacgc gatggatatg
ttctgccaag ggttggtttg 8160cgcattcaca gttctccgca agaattgatt
ggctccaatt cttggagtgg tgaatccgtt 8220agcgaggcca tccagcctcg
cgtcgaacta gatgatccgc tgtggaatgt gtgtcagtta 8280gggtgtggaa
agtccccagg ctccccagca ggcagaagta tgcaaagcat gcatctcaat
8340tagtcagcaa ccaggtgtgg aaagtcccca ggctccccag caggcagaag
tatgcaaagc 8400atgcatctca attagtcagc aaccatagtc ccgcccctaa
ctccgcccat cccgccccta 8460actccgccca gttccgccca ttctccgccc
catggctgac taattttttt tatttatgca 8520gaggccgagg ccgcggcctc
tgagctattc cagaagtagt gaggaggctt ttttggaggg 8580tgaccgccac
gaggtgccgc caccatcccc tgacccacgc ccctgacccc tcacaaggag
8640acgaccttcc atgaccgagt acaagcccac ggtgcgcctc gccacccgcg
acgacgtccc 8700ccgggccgta cgcaccctcg ccgccgcgtt cgccgactac
cccgccacgc gccacaccgt 8760cgaccccgac cgccacatcg aacgcgtcac
cgagctgcaa gaactcttcc tcacgcgcgt 8820cgggctcgac atcggcaagg
tgtgggtcgc ggacgacggc gccgcggtgg cggtctggac 8880cacgccggag
agcgtcgaag cgggggcggt gttcgccgag atcggcccgc gcatggccga
8940gttgagcggt tcccggctgg ccgcgcagca acagatggaa ggcctcctgg
cgccgcaccg 9000gcccaaggag cccgcgtggt tcctggccac cgtcggcgtc
tcgcccgacc accagggcaa 9060gggtctgggc agcgccgtcg tgctccccgg
agtggaggcg gccgagcgcg ccggggtgcc 9120cgccttcctg gagacctccg
cgccccgcaa cctccccttc tacgagcggc tcggcttcac 9180cgtcaccgcc
gacgtcgagt gcccgaagga ccgcgcgacc tggtgcatga cccgcaagcc
9240cggtgcctga cgcccgcccc acgacccgca gcgcccgacc gaaaggagcg
cacgacccgg 9300tccgacggcg gcccacgggt cccagggggg tcgacctcga
aacttgttta ttgcagctta 9360taatggttac aaataaagca atagcatcac
aaatttcaca aataaagcat ttttttcact 9420gcattctagt tgtggtttgt
ccaaactcat caatgtatct tatcatgtct ggatcgatcc 9480gaaccccttc
ctcgaccaat tctcatgttt gacagcttat catcgcagat ccgggcaacg
9540ttgttgcatt gctgcaggcg cagaactggt aggtatggaa gatctataca
ttgaatcaat 9600attggcaatt agccatatta gtcattggtt atatagcata
aatcaatatt ggctattggc 9660cattgcatac gttgtatcta tatcataata
tgtacattta tattggctca tgtccaatat 9720gaccgccatg ttgacattga
ttattgacta gttattaata gtaatcaatt acggggtcat 9780tagttcatag
cccatatatg gagttccgcg ttacataact tacggtaaat ggcccgcctg
9840gctgaccgcc caacgacccc cgcccattga cgtcaataat gacgtatgtt
cccatagtaa 9900cgccaatagg gactttccat tgacgtcaat gggtggagta
tttacggtaa actgcccact 9960tggcagtaca tcaagtgtat catatgccaa
gtccgccccc tattgacgtc aatgacggta 10020aatggcccgc ctggcattat
gcccagtaca tgaccttacg ggactttcct acttggcagt 10080acatctacgt
attagtcatc gctattacca tggtgatgcg gttttggcag tacaccaatg
10140ggcgtggata gcggtttgac tcacggggat ttccaagtct ccaccccatt
gacgtcaatg 10200ggagtttgtt ttggcaccaa aatcaacggg actttccaaa
atgtcgtaat aaccccgccc 10260cgttgacgca aatgggcggt aggcgtgtac
ggtgggaggt ctatataagc agagctcgtt 10320tagtgaaccg tcagatctct
agaagctg 10348421608DNAartificial sequencesynthetic 42atggagacag
acacactcct gctatgggta ctgctgctct gggttccagg ttccactggt 60gacgcggatc
cggcccaggc ggccgagctc gtgatgaccc agtctcctga ctccctggct
120gtgtccctgg gcgagagggc caccatcaac tgcaagtcca gccagagtgt
tctatacacc 180tccaacaata agaactactt aggttggtac cagcagaaac
cagggcagcc gcctaattta 240ctcatttatt gggcatctac ccgggaatcc
ggggtccctg accgattcag tggcagcggg 300tctgggacag atttcactct
caccatcaac agcgtgcagg ctgaggatgt ggcagtttat 360tactgccagc
agtattttat gactcccatc accttcggcc aagggacacg actggagatt
420aaaggtggtt cctctagatc ttcctcctct ggtggcggtg gctcgggcgg
tggtggggag 480gtgcagctgg tggagtctgg gggggccttg gtacagccag
ggcggtccct gagactctcc 540tgtagaacct ctggactcaa ttttggagat
tatcctataa actgggtccg ccaggctcca 600gggaaggggc tggagtgggt
agggttcatc aaaagcaagt cttatggtgt gacaacagaa 660ttcgccgcgt
ctgtggaggg cagattcacc atctcgaggg atgattccag aggcatcgcc
720tatctgcaga tgaacagcct gaaaaccgag gacacagccg tctattactg
tacgtccagt 780agtggttttt tgtactactt tgactactgg ggccagggaa
ccctggtcac cgtctcctca 840gcctccacca agggcccatc ggtcactagt
ggccaggccg gccgcaagct tgagcccaga 900gtgcccataa cacagaaccc
ctgtcctcca ctcaaagagt gtcccccatg cgcagctcca 960gacctcttgg
gtggaccatc cgtcttcatc ttccctccaa agatcaagga tgtactcatg
1020atctccctga gccccatggt cacatgtgtg gtggtggatg tgagcgagga
tgacccagac 1080gtccagatca gctggtttgt gaacaacgtg gaagtacaca
cagctcagac acaaacccat 1140agagaggatt acaacagtac tctccgggtg
gtcagtgccc tccccatcca gcaccaggac 1200tggatgagtg gcaaggagtt
caaatgcaag gtcaacaaca gagccctccc atcccccatc 1260gagaaaacca
tctcaaaacc cagagggcca gtaagagctc cacaggtata tgtcttgcct
1320ccaccagcag aagagatgac taagaaagag ttcagtctga cctgcatgat
cacaggcttc 1380ttacctgccg aaattgctgt ggactggacc agcaatgggc
gtacagagca aaactacaag 1440aacaccgcaa cagtcctgga ctctgatggt
tcttacttca tgtacagcaa gctcagagta 1500caaaagagca cttgggaaag
aggaagtctt ttcgcctgct cagtggtcca cgagggtctg 1560cacaatcacc
ttacgactaa gaccatctcc cggtctctgg gtaaatga 160843535PRTartificial
sequencesynthetic 43Met Glu Thr Asp Thr Leu Leu Leu Trp Val Leu Leu
Leu Trp Val Pro 1 5 10 15 Gly Ser Thr Gly Asp Ala Asp Pro Ala Gln
Ala Ala Glu Leu Val Met 20 25 30 Thr Gln Ser Pro Asp Ser Leu Ala
Val Ser Leu Gly Glu Arg Ala Thr 35 40 45 Ile Asn Cys Lys Ser Ser
Gln Ser Val Leu Tyr Thr Ser Asn Asn Lys 50 55 60 Asn Tyr Leu Gly
Trp Tyr Gln Gln Lys Pro Gly Gln Pro Pro Asn Leu 65 70 75 80 Leu Ile
Tyr Trp Ala Ser Thr Arg Glu Ser Gly Val Pro Asp Arg Phe 85 90 95
Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Asn Ser Val 100
105 110 Gln Ala Glu Asp Val Ala Val Tyr Tyr Cys Gln Gln Tyr Phe Met
Thr 115 120 125 Pro Ile Thr Phe Gly Gln Gly Thr Arg Leu Glu Ile Lys
Gly Gly Ser 130 135 140 Ser Arg Ser Ser Ser Ser Gly Gly Gly Gly Ser
Gly Gly Gly Gly Glu 145 150 155 160 Val Gln Leu Val Glu Ser Gly Gly
Ala Leu Val Gln Pro Gly Arg Ser 165 170 175 Leu Arg Leu Ser Cys Arg
Thr Ser Gly Leu Asn Phe Gly Asp Tyr Pro 180 185 190 Ile Asn Trp Val
Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val Gly 195 200 205 Phe Ile
Lys Ser Lys Ser Tyr Gly Val Thr Thr Glu Phe Ala Ala Ser 210 215 220
Val Glu Gly Arg Phe Thr Ile Ser Arg Asp Asp Ser Arg Gly Ile Ala 225
230 235 240 Tyr Leu Gln Met Asn Ser Leu Lys Thr Glu Asp Thr Ala Val
Tyr Tyr 245 250 255 Cys Thr Ser Ser Ser Gly Phe Leu Tyr Tyr Phe Asp
Tyr Trp Gly Gln 260 265 270 Gly Thr Leu Val Thr Val Ser Ser Ala Ser
Thr Lys Gly Pro Ser Val 275 280 285 Thr Ser Gly Gln Ala Gly Arg Lys
Leu Glu Pro Arg Val Pro Ile Thr 290 295 300 Gln Asn Pro Cys Pro Pro
Leu Lys Glu Cys Pro Pro Cys Ala Ala Pro 305 310 315 320 Asp Leu Leu
Gly Gly Pro Ser Val Phe Ile Phe Pro Pro Lys Ile Lys 325 330 335 Asp
Val Leu Met Ile Ser Leu Ser Pro Met Val Thr Cys Val Val Val 340 345
350 Asp Val Ser Glu Asp Asp Pro Asp Val Gln Ile Ser Trp Phe Val Asn
355 360 365 Asn Val Glu Val His Thr Ala Gln Thr Gln Thr His Arg Glu
Asp Tyr 370 375 380 Asn Ser Thr Leu Arg Val Val Ser Ala Leu Pro Ile
Gln His Gln Asp 385 390 395 400 Trp Met Ser Gly Lys Glu Phe Lys Cys
Lys Val Asn Asn Arg Ala Leu 405 410 415 Pro Ser Pro Ile Glu Lys Thr
Ile Ser Lys Pro Arg Gly Pro Val Arg 420 425 430 Ala Pro Gln Val Tyr
Val Leu Pro Pro Pro Ala Glu Glu Met Thr Lys 435 440 445 Lys Glu Phe
Ser Leu Thr Cys Met Ile Thr Gly Phe Leu Pro Ala Glu 450 455 460 Ile
Ala Val Asp Trp Thr Ser Asn Gly Arg Thr Glu Gln Asn Tyr Lys 465 470
475 480 Asn Thr Ala Thr Val Leu Asp Ser Asp Gly Ser Tyr Phe Met Tyr
Ser 485 490 495 Lys Leu Arg Val Gln Lys Ser Thr Trp Glu Arg Gly Ser
Leu Phe Ala 500 505 510 Cys Ser Val Val His Glu Gly Leu His Asn His
Leu Thr Thr Lys Thr 515 520 525 Ile Ser Arg Ser Leu Gly Lys 530 535
441608DNAartificial sequencesynthetic 44atggagacag acacactcct
gctatgggta ctgctgctct gggttccagg ttccactggt 60gacgcggatc cggcccaggc
ggccgagctc gtgatgactc agtctccaga ctccctggct 120gtgtctctgg
gcgagagggc caccatcaac tgcaagtcca gccagagtgt tttatacagc
180tccaacaatg agaactactt agcttggtac cagcagaaac caggacagcc
tcctaaactg 240ctcatttact gggcatctac ccgggaatcc ggggtccctg
accgattcag tggcagcggg 300tctgggacag atttcactct caccatcaac
agcgtgcagg ctgaggatgt ggcagtttat 360tactgccagc agtattttat
gactcccatc accttcggcc aagggacacg actggagatt 420aaaggtggtt
cctctagatc ttcctcctct ggtggcggtg gctcgggcgg tggtggggag
480gtgcagctgt tggagtctgg gggggccttg gtacagccag ggcggtccct
gagactctcc 540tgtagaacct ctggactcaa ttttggagat tatcctataa
actgggtccg ccaggctcca 600gggaaggggc tggagtgggt agggttcatc
aaaagcaagt cttatggtgt gacaacagaa 660ttcgccgcgt ctgtggaggg
cagattcacc atctcaaggg atgattccag aggcatcgcc 720tatctgcaga
tgaacagcct gaaaaccgag gacacagccg tctattactg tacgtccagt
780agtggttttt tgtactactt tgactactgg ggccagggaa ccctggtcac
cgtctcctca 840gcttccacca agggcccatc agtcactagt ggccaggccg
gccgcaagct tgagcccaga 900gtgcccataa cacagaaccc ctgtcctcca
ctcaaagagt gtcccccatg cgcagctcca 960gacctcttgg gtggaccatc
cgtcttcatc ttccctccaa agatcaagga tgtactcatg 1020atctccctga
gccccatggt cacatgtgtg gtggtggatg tgagcgagga tgacccagac
1080gtccagatca gctggtttgt gaacaacgtg gaagtacaca cagctcagac
acaaacccat 1140agagaggatt acaacagtac tctccgggtg gtcagtgccc
tccccatcca gcaccaggac 1200tggatgagtg gcaaggagtt caaatgcaag
gtcaacaaca gagccctccc atcccccatc 1260gagaaaacca tctcaaaacc
cagagggcca gtaagagctc cacaggtata tgtcttgcct 1320ccaccagcag
aagagatgac taagaaagag ttcagtctga cctgcatgat cacaggcttc
1380ttacctgccg aaattgctgt ggactggacc agcaatgggc gtacagagca
aaactacaag 1440aacaccgcaa cagtcctgga ctctgatggt tcttacttca
tgtacagcaa gctcagagta 1500caaaagagca cttgggaaag aggaagtctt
ttcgcctgct cagtggtcca cgagggtctg 1560cacaatcacc ttacgactaa
gaccatctcc cggtctctgg gtaaatga 160845535PRTartificial
sequencesynthetic 45Met Glu Thr Asp Thr Leu Leu Leu Trp Val Leu Leu
Leu Trp Val Pro 1 5 10 15 Gly Ser Thr Gly Asp Ala Asp Pro Ala Gln
Ala Ala Glu Leu Val Met 20 25 30 Thr Gln Ser Pro Asp Ser Leu Ala
Val Ser Leu Gly Glu Arg Ala Thr 35 40 45 Ile Asn Cys Lys Ser Ser
Gln Ser Val Leu Tyr Ser Ser Asn Asn Glu 50 55 60 Asn Tyr Leu Ala
Trp Tyr Gln Gln Lys Pro Gly Gln Pro Pro Lys Leu 65 70 75 80 Leu Ile
Tyr Trp Ala Ser Thr Arg Glu Ser Gly Val Pro Asp Arg Phe 85 90 95
Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Asn Ser Val 100
105 110 Gln Ala Glu Asp Val Ala Val Tyr Tyr Cys Gln Gln Tyr Phe Met
Thr 115 120 125 Pro Ile Thr Phe Gly Gln Gly Thr Arg Leu Glu Ile Lys
Gly Gly Ser 130 135 140 Ser Arg Ser Ser Ser Ser Gly Gly Gly Gly Ser
Gly Gly Gly Gly Glu 145 150 155 160 Val Gln Leu Leu Glu Ser Gly Gly
Ala Leu Val Gln Pro Gly Arg Ser 165 170 175 Leu Arg Leu Ser Cys Arg
Thr Ser Gly Leu Asn Phe Gly Asp Tyr Pro 180 185 190 Ile Asn Trp Val
Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val Gly 195 200
205 Phe Ile Lys Ser Lys Ser Tyr Gly Val Thr Thr Glu Phe Ala Ala Ser
210 215 220 Val Glu Gly Arg Phe Thr Ile Ser Arg Asp Asp Ser Arg Gly
Ile Ala 225 230 235 240 Tyr Leu Gln Met Asn Ser Leu Lys Thr Glu Asp
Thr Ala Val Tyr Tyr 245 250 255 Cys Thr Ser Ser Ser Gly Phe Leu Tyr
Tyr Phe Asp Tyr Trp Gly Gln 260 265 270 Gly Thr Leu Val Thr Val Ser
Ser Ala Ser Thr Lys Gly Pro Ser Val 275 280 285 Thr Ser Gly Gln Ala
Gly Arg Lys Leu Glu Pro Arg Val Pro Ile Thr 290 295 300 Gln Asn Pro
Cys Pro Pro Leu Lys Glu Cys Pro Pro Cys Ala Ala Pro 305 310 315 320
Asp Leu Leu Gly Gly Pro Ser Val Phe Ile Phe Pro Pro Lys Ile Lys 325
330 335 Asp Val Leu Met Ile Ser Leu Ser Pro Met Val Thr Cys Val Val
Val 340 345 350 Asp Val Ser Glu Asp Asp Pro Asp Val Gln Ile Ser Trp
Phe Val Asn 355 360 365 Asn Val Glu Val His Thr Ala Gln Thr Gln Thr
His Arg Glu Asp Tyr 370 375 380 Asn Ser Thr Leu Arg Val Val Ser Ala
Leu Pro Ile Gln His Gln Asp 385 390 395 400 Trp Met Ser Gly Lys Glu
Phe Lys Cys Lys Val Asn Asn Arg Ala Leu 405 410 415 Pro Ser Pro Ile
Glu Lys Thr Ile Ser Lys Pro Arg Gly Pro Val Arg 420 425 430 Ala Pro
Gln Val Tyr Val Leu Pro Pro Pro Ala Glu Glu Met Thr Lys 435 440 445
Lys Glu Phe Ser Leu Thr Cys Met Ile Thr Gly Phe Leu Pro Ala Glu 450
455 460 Ile Ala Val Asp Trp Thr Ser Asn Gly Arg Thr Glu Gln Asn Tyr
Lys 465 470 475 480 Asn Thr Ala Thr Val Leu Asp Ser Asp Gly Ser Tyr
Phe Met Tyr Ser 485 490 495 Lys Leu Arg Val Gln Lys Ser Thr Trp Glu
Arg Gly Ser Leu Phe Ala 500 505 510 Cys Ser Val Val His Glu Gly Leu
His Asn His Leu Thr Thr Lys Thr 515 520 525 Ile Ser Arg Ser Leu Gly
Lys 530 535 461608DNAartificial sequencesynthetic 46atggagacag
acacactcct gctatgggta ctgctgctct gggttccagg ttccactggt 60gacgcggatc
cggcccaggc ggccgagcgc gtgatgacac agtctccaga cgccctggct
120gtgtctctgg gcgagagggc caccatcaac tgcaagtcca gccagagtct
tttatacagc 180tccaataata agaactactt agcttggtat cagaagaaac
caggacagcc tcctaagctg 240ctcatttact gggcatctac ccgggaatcc
ggggtccctg accgattcag tggcagcggg 300tctgggacag agttcactct
caccatcaac agcgtgcagg ctgaggatgt ggcagtttat 360tactgccagc
agtattttat gactcccatc accttcggcc aagggaccaa gctggagatc
420aaaggtggtt cctctagatc ttcctcctct ggtggcggtg gctcgggcgg
tggtgggcag 480gtgcagctgc aggagtcggg gggggccttg gtacagccag
ggcggtccct gagactctcc 540tgtagaacct ctggactcaa ttttggagat
tatcctataa actgggtccg ccaggctcca 600gggaaggggc tggagtgggt
agggttcatc aaaagcaagt cttatggtgt gacaacagaa 660ttcgccgcgt
ctgtggaggg cagattcacc atctcaaggg atgattccag aggcatcgcc
720tatctgcaga tgaacagcct gaaaaccgag gacacagccg tctattactg
tacgtccagt 780agtggttttt tgtactactt tgactactgg ggccagggaa
ccctggtcac cgtctcctca 840gcttccacca agggcccatc ggtcactagt
ggccaggccg gccgcaagct tgagcccaga 900gtgcccataa cacagaaccc
ctgtcctcca ctcaaagagt gtcccccatg cgcagctcca 960gacctcttgg
gtggaccatc cgtcttcatc ttccctccaa agatcaagga tgtactcatg
1020atctccctga gccccatggt cacatgtgtg gtggtggatg tgagcgagga
tgacccagac 1080gtccagatca gctggtttgt gaacaacgtg gaagtacaca
cagctcagac acaaacccat 1140agagaggatt acaacagtac tctccgggtg
gtcagtgccc tccccatcca gcaccaggac 1200tggatgagtg gcaaggagtt
caaatgcaag gtcaacaaca gagccctccc atcccccatc 1260gagaaaacca
tctcaaaacc cagagggcca gtaagagctc cacaggtata tgtcttgcct
1320ccaccagcag aagagatgac taagaaagag ttcagtctga cctgcatgat
cacaggcttc 1380ttacctgccg aaattgctgt ggactggacc agcaatgggc
gtacagagca aaactacaag 1440aacaccgcaa cagtcctgga ctctgatggt
tcttacttca tgtacagcaa gctcagagta 1500caaaagagca cttgggaaag
aggaagtctt ttcgcctgct cagtggtcca cgagggtctg 1560cacaatcacc
ttacgactaa gaccatctcc cggtctctgg gtaaatga 160847535PRTartificial
sequencesynthetic 47Met Glu Thr Asp Thr Leu Leu Leu Trp Val Leu Leu
Leu Trp Val Pro 1 5 10 15 Gly Ser Thr Gly Asp Ala Asp Pro Ala Gln
Ala Ala Glu Arg Val Met 20 25 30 Thr Gln Ser Pro Asp Ala Leu Ala
Val Ser Leu Gly Glu Arg Ala Thr 35 40 45 Ile Asn Cys Lys Ser Ser
Gln Ser Leu Leu Tyr Ser Ser Asn Asn Lys 50 55 60 Asn Tyr Leu Ala
Trp Tyr Gln Lys Lys Pro Gly Gln Pro Pro Lys Leu 65 70 75 80 Leu Ile
Tyr Trp Ala Ser Thr Arg Glu Ser Gly Val Pro Asp Arg Phe 85 90 95
Ser Gly Ser Gly Ser Gly Thr Glu Phe Thr Leu Thr Ile Asn Ser Val 100
105 110 Gln Ala Glu Asp Val Ala Val Tyr Tyr Cys Gln Gln Tyr Phe Met
Thr 115 120 125 Pro Ile Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys
Gly Gly Ser 130 135 140 Ser Arg Ser Ser Ser Ser Gly Gly Gly Gly Ser
Gly Gly Gly Gly Gln 145 150 155 160 Val Gln Leu Gln Glu Ser Gly Gly
Ala Leu Val Gln Pro Gly Arg Ser 165 170 175 Leu Arg Leu Ser Cys Arg
Thr Ser Gly Leu Asn Phe Gly Asp Tyr Pro 180 185 190 Ile Asn Trp Val
Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val Gly 195 200 205 Phe Ile
Lys Ser Lys Ser Tyr Gly Val Thr Thr Glu Phe Ala Ala Ser 210 215 220
Val Glu Gly Arg Phe Thr Ile Ser Arg Asp Asp Ser Arg Gly Ile Ala 225
230 235 240 Tyr Leu Gln Met Asn Ser Leu Lys Thr Glu Asp Thr Ala Val
Tyr Tyr 245 250 255 Cys Thr Ser Ser Ser Gly Phe Leu Tyr Tyr Phe Asp
Tyr Trp Gly Gln 260 265 270 Gly Thr Leu Val Thr Val Ser Ser Ala Ser
Thr Lys Gly Pro Ser Val 275 280 285 Thr Ser Gly Gln Ala Gly Arg Lys
Leu Glu Pro Arg Val Pro Ile Thr 290 295 300 Gln Asn Pro Cys Pro Pro
Leu Lys Glu Cys Pro Pro Cys Ala Ala Pro 305 310 315 320 Asp Leu Leu
Gly Gly Pro Ser Val Phe Ile Phe Pro Pro Lys Ile Lys 325 330 335 Asp
Val Leu Met Ile Ser Leu Ser Pro Met Val Thr Cys Val Val Val 340 345
350 Asp Val Ser Glu Asp Asp Pro Asp Val Gln Ile Ser Trp Phe Val Asn
355 360 365 Asn Val Glu Val His Thr Ala Gln Thr Gln Thr His Arg Glu
Asp Tyr 370 375 380 Asn Ser Thr Leu Arg Val Val Ser Ala Leu Pro Ile
Gln His Gln Asp 385 390 395 400 Trp Met Ser Gly Lys Glu Phe Lys Cys
Lys Val Asn Asn Arg Ala Leu 405 410 415 Pro Ser Pro Ile Glu Lys Thr
Ile Ser Lys Pro Arg Gly Pro Val Arg 420 425 430 Ala Pro Gln Val Tyr
Val Leu Pro Pro Pro Ala Glu Glu Met Thr Lys 435 440 445 Lys Glu Phe
Ser Leu Thr Cys Met Ile Thr Gly Phe Leu Pro Ala Glu 450 455 460 Ile
Ala Val Asp Trp Thr Ser Asn Gly Arg Thr Glu Gln Asn Tyr Lys 465 470
475 480 Asn Thr Ala Thr Val Leu Asp Ser Asp Gly Ser Tyr Phe Met Tyr
Ser 485 490 495 Lys Leu Arg Val Gln Lys Ser Thr Trp Glu Arg Gly Ser
Leu Phe Ala 500 505 510 Cys Ser Val Val His Glu Gly Leu His Asn His
Leu Thr Thr Lys Thr 515 520 525 Ile Ser Arg Ser Leu Gly Lys 530 535
4823PRTInfluenza A virus 48Ser Leu Leu Thr Glu Val Glu Thr Pro Ile
Arg Asn Glu Trp Gly Cys 1 5 10 15 Arg Cys Asn Asp Ser Ser Asp 20
4916PRTInfluenza A virus 49Ser Leu Leu Thr Glu Val Glu Thr Pro Ile
Arg Asn Glu Trp Gly Cys 1 5 10 15 5023PRTInfluenza A virus 50Ser
Leu Leu Thr Glu Val Glu Thr Pro Thr Arg Asn Glu Trp Glu Cys 1 5 10
15 Arg Cys Ser Asp Ser Ser Asp 20 5123PRTInfluenza A virus 51Ser
Leu Leu Thr Glu Val Glu Thr Pro Ile Arg Asn Glu Trp Gly Cys 1 5 10
15 Arg Cys Asn Gly Ser Ser Asp 20 5223PRTInfluenza A virus 52Ser
Leu Leu Thr Glu Val Glu Thr Pro Thr Arg Asn Glu Trp Gly Cys 1 5 10
15 Arg Cys Asn Asp Ser Ser Asp 20 5323PRTInfluenza A virus 53Ser
Leu Leu Thr Glu Val Glu Thr Pro Thr Lys Asn Glu Trp Glu Cys 1 5 10
15 Arg Cys Asn Asp Ser Ser Asp 20 5423PRTInfluenza A virus 54Ser
Leu Leu Thr Glu Val Glu Thr Pro Ile Arg Asn Glu Trp Gly Cys 1 5 10
15 Lys Cys Asn Asp Ser Ser Asp 20 5523PRTInfluenza A virus 55Ser
Leu Leu Thr Glu Val Glu Thr Pro Ile Arg Asn Glu Trp Glu Cys 1 5 10
15 Arg Cys Asn Asp Ser Ser Asp 20 5623PRTInfluenza A virus 56Ser
Leu Leu Thr Glu Val Glu Thr Pro Ile Arg Asn Gly Trp Glu Cys 1 5 10
15 Arg Cys Asn Asp Ser Ser Asp 20 5723PRTInfluenza A virus 57Ser
Leu Leu Thr Glu Val Glu Thr Pro Ile Arg Ser Glu Trp Gly Cys 1 5 10
15 Arg Cys Asn Asp Ser Ser Asp 20 5823PRTInfluenza A virus 58Ser
Phe Leu Pro Glu Val Glu Thr Pro Ile Arg Asn Glu Trp Gly Cys 1 5 10
15 Arg Cys Asn Asp Ser Ser Asp 20 5923PRTInfluenza A virus 59Ser
Leu Leu Thr Glu Val Glu Thr Pro Ile Arg Asn Glu Trp Gly Cys 1 5 10
15 Arg Cys Asn Asp Ser Ser Asn 20 6023PRTInfluenza A virus 60Ser
Leu Leu Thr Glu Val Glu Thr Pro Ile Arg Asn Glu Trp Gly Cys 1 5 10
15 Arg Cys Asn Asp Ser Ser Asp 20 6123PRTInfluenza A virus 61Ser
Leu Leu Thr Glu Val Glu Thr Pro Ile Arg Lys Glu Trp Gly Cys 1 5 10
15 Arg Cys Asn Asp Ser Ser Asp 20 6224PRTInfluenza A virus 62Met
Phe Leu Leu Thr Glu Val Glu Thr Pro Ile Arg Asn Glu Trp Gly 1 5 10
15 Cys Arg Cys Asn Asp Ser Ser Asp 20 6323PRTInfluenza A virus
63Ser Leu Leu Thr Glu Val Glu Thr Pro Ile Arg Asn Glu Trp Glu Cys 1
5 10 15 Arg Cys Asn Gly Ser Ser Asp 20 6423PRTInfluenza A virus
64Ser Leu Pro Thr Glu Val Glu Thr Pro Ile Arg Ser Glu Trp Gly Cys 1
5 10 15 Arg Cys Asn Asp Ser Ser Asp 20 6523PRTInfluenza A virus
65Ser Leu Leu Pro Glu Val Glu Thr Pro Ile Arg Asn Glu Trp Gly Cys 1
5 10 15 Arg Cys Asn Asp Ser Ser Asp 20 6623PRTInfluenza A virus
66Ser Leu Leu Pro Glu Val Glu Thr Pro Ile Arg Asn Gly Trp Gly Cys 1
5 10 15 Arg Cys Asn Asp Ser Ser Asp 20 6723PRTInfluenza A virus
67Ser Leu Leu Thr Glu Val Glu Thr Pro Thr Arg Asn Gly Trp Glu Cys 1
5 10 15 Arg Cys Asn Asp Ser Ser Asp 20 6823PRTInfluenza A virus
68Ser Phe Leu Thr Glu Val Glu Thr Pro Ile Arg Asn Glu Trp Gly Cys 1
5 10 15 Arg Cys Asn Gly Ser Ser Asp 20 6923PRTInfluenza A virus
69Ser Leu Leu Thr Glu Val Glu Thr Pro Ile Arg Asn Glu Trp Glu Tyr 1
5 10 15 Arg Cys Ser Asp Ser Ser Asp 20 7023PRTInfluenza A virus
70Ser Leu Leu Thr Glu Val Glu Thr Leu Thr Arg Asn Gly Trp Gly Cys 1
5 10 15 Arg Cys Ser Asp Ser Ser Asp 20 7123PRTInfluenza A virus
71Ser Leu Leu Thr Glu Val Glu Thr Leu Thr Lys Asn Gly Trp Gly Cys 1
5 10 15 Arg Cys Ser Asp Ser Ser Asp 20 7223PRTInfluenza A virus
72Ser Leu Leu Thr Glu Val Glu Thr His Thr Arg Asn Glu Trp Glu Cys 1
5 10 15 Arg Cys Asn Asp Ser Ser Asp 20 7323PRTInfluenza A virus
73Ser Leu Leu Thr Glu Val Glu Thr Pro Thr Arg Asn Gly Trp Glu Cys 1
5 10 15 Lys Cys Asn Asp Ser Ser Asp 20 7423PRTInfluenza A virus
74Ser Phe Leu Thr Glu Val Glu Thr Leu Thr Arg Asn Gly Trp Glu Cys 1
5 10 15 Arg Cys Ser Asp Ser Ser Asp 20 7523PRTInfluenza A virus
75Ser Leu Leu Thr Glu Val Glu Thr Leu Thr Arg Asn Gly Trp Glu Cys 1
5 10 15 Lys Cys Ser Asp Ser Ser Asp 20 7623PRTInfluenza A virus
76Ser Leu Leu Thr Glu Val Asp Thr Leu Thr Arg Asn Gly Trp Gly Cys 1
5 10 15 Arg Cys Ser Asp Ser Ser Asp 20 7723PRTInfluenza A virus
77Ser Leu Leu Thr Glu Val Glu Thr Pro Thr Arg Asn Gly Trp Gly Cys 1
5 10 15 Arg Cys Ser Asp Ser Ser Asp 20 7824PRTInfluenza A virus
78Met Ser Leu Leu Thr Glu Val Lys Thr Pro Thr Arg Asn Gly Trp Glu 1
5 10 15 Cys Lys Cys Ser Asp Ser Ser Asp 20 7923PRTInfluenza A virus
79Ser Leu Leu Thr Glu Val Glu Thr Pro Thr Arg Asp Gly Trp Glu Cys 1
5 10 15 Lys Cys Ser Asp Ser Ser Asp 20 8024PRTInfluenza A virus
80Met Ser Leu Leu Thr Glu Val Glu Thr His Thr Arg Asn Gly Trp Glu 1
5 10 15 Cys Lys Cys Ser Asp Ser Ser Asp 20 8123PRTInfluenza A virus
81Ser Leu Pro Thr Glu Val Glu Thr Pro Ile Arg Asn Glu Trp Gly Cys 1
5 10 15 Arg Cys Asn Asp Ser Ser Asp 20 8223PRTInfluenza A virus
82Ser Leu Leu Thr Glu Val Glu Thr Pro Ile Arg Ser Glu Trp Gly Cys 1
5 10 15 Arg Cys Asn Asp Ser Gly Asp 20 8323PRTInfluenza A virus
83Ser Leu Leu Thr Glu Val Glu Thr Pro Ile Arg Lys Gly Trp Glu Cys 1
5 10 15 Asn Cys Ser Asp Ser Ser Asp 20 8497PRTInfluenza A virus
84Met Ser Leu Leu Thr Glu Val Glu Thr Pro Ile Arg Asn Glu Trp Gly 1
5 10 15 Cys Arg Cys Asn Gly Ser Ser Asp Pro Leu Thr Ile Ala Ala Asn
Ile 20 25 30 Ile Gly Ile Leu His Leu Thr Leu Trp Ile Leu Asp Arg
Leu Phe Phe 35 40 45 Lys Cys Ile Tyr Arg Arg Phe Lys Tyr Gly Leu
Lys Gly Gly Pro Ser 50 55 60 Thr Glu Gly Val Pro Lys Ser Met Arg
Glu Glu Tyr Arg Lys Glu Gln 65 70 75 80 Gln Ser Ala Val Asp Ala Asp
Asp Gly His Phe Val Ser Ile Glu Leu 85 90 95 Glu 8597PRTInfluenza A
virus 85Met Ser Leu Leu Thr Glu Val Glu Thr Pro Thr Arg Asn Glu Trp
Glu 1 5 10 15 Cys Arg Cys Ser Asp Ser Ser Asp Pro Leu Thr Ile Ala
Ala Asn Ile 20 25 30 Ile Gly Ile Leu His Leu Thr Leu Trp Ile Leu
Asp Arg Leu Phe Phe 35 40 45 Lys Cys Ile Tyr Arg Arg Phe Lys Tyr
Gly Leu Lys Gly Gly Pro Ser 50 55 60 Thr Glu Gly Val Pro Lys Ser
Met Arg Glu Glu Tyr Arg Lys Glu Gln 65 70 75 80 Gln Ser Ala Val Asp
Ala Asp Asp Gly His Phe Val Ser Ile Glu Leu 85 90 95 Glu
86327DNAHomo sapiens 86acccagagcc ccgacagcct ggccgtgagc ctgggcgagc
gggccaccat caactgcaag 60agcagccaga gcgtgctgta caccagcaac aacaagaact
acctgggctg gtatcagcag 120aagcccggcc agccccccaa cctgctgatc
tactgggcca gcacccggga gagcggcgtg 180cccgaccggt ttagcggcag
cggctccggc accgacttca ccctgaccat caacagcgtg 240caggccgagg
acgtggccgt gtactactgc cagcagtact tcatgacccc catcaccttc
300ggccagggca cccggctgga aatcaag 32787327DNAHomo sapiens
87acccagagcc ccgacagcct ggccgtgagc ctgggcgagc gggccaccat caactgcaag
60agcagccaga gcgtgctgta cagcagcaac aacgagaact acctggcctg gtatcagcag
120aagcccggcc agccccccaa gctgctgatc tactgggcca gcacccggga
gagcggcgtg 180cccgaccggt ttagcggcag cggctccggc accgacttca
ccctgaccat caacagcgtg 240caggccgagg acgtggccgt gtactactgc
cagcagtact tcatgacccc catcaccttc 300ggccagggca cccggctgga aatcaag
32788327DNAHomo sapiens 88acccagagcc ccgacgccct ggccgtgagc
ctgggcgagc gggccaccat caactgcaag 60agcagccaga gcctgctgta cagcagcaac
aacaagaact acctggcctg gtatcagaaa 120aagcccggcc agccccccaa
gctgctgatc tactgggcca gcacccggga gagcggcgtg 180cccgaccggt
ttagcggcag cggctccggc accgagttca ccctgaccat caacagcgtg
240caggccgagg acgtggccgt gtactactgc cagcagtact tcatgacccc
catcaccttc 300ggccagggca cccggctgga aatcaag 32789345DNAHomo sapiens
89agcggcggag ccctggtgca gcccggcaga agcctgagac tgagctgccg gaccagcggc
60ctgaacttcg gcgactaccc catcaactgg gtgcggcagg ctccagggaa aggactcgaa
120tgggtgggct tcatcaagag caagagctac ggcgtgacca ccgagttcgc
cgccagcgtg 180gagggccggt tcaccatcag ccgggacgac agccggggca
ttgcctacct gcagatgaac 240agcctgaaaa ccgaggacac cgccgtgtac
tactgcacca gcagcagcgg ctttctgtac 300tacttcgact actggggaca
gggcaccctg gtgaccgtga gcagc 3459023PRTInfluenza A virus 90Ser Leu
Leu Thr Glu Val Glu Thr Pro Thr Arg Asn Gly Trp Glu Cys 1 5 10 15
Lys Cys Ser Asp Ser Ser Asp 20 9123PRTInfluenza A virus 91Ser Leu
Leu Thr Gly Val Glu Thr His Thr Arg Asn Gly Trp Gly Cys 1 5 10 15
Lys Cys Ser Asp Ser Ser Asp 20 9223PRTInfluenza A virus 92Ser Leu
Leu Pro Glu Val Glu Thr His Thr Arg Asn Gly Trp Gly Cys 1 5 10 15
Arg Cys Ser Asp Ser Ser Asp 20 938PRTInfluenza A virus 93Leu Leu
Thr Glu Val Glu Thr Pro 1 5
* * * * *