Patent application title: FULLY HUMAN INFLUENZA M2 SPECIFIC ANTIBODIES
Inventors:
Martin F. Bachmann (Ramismuhle, CH)
Monika Bauer (Zurich, CH)
Roger Beerli (Adlikon B. Regensdorf, CH)
Nicole Schmitz (Urdorf, CH)
Assignees:
Intercell AG
IPC8 Class: AC07K1610FI
USPC Class:
53038815
Class name: Immunoglobulin, antibody, or fragment thereof, other than immunoglobulin antibody, or fragment thereof that is conjugated or adsorbed monoclonal human
Publication date: 2013-05-02
Patent application number: 20130109845
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.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. §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 μg of the indicated scFv-msFcγ2c 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-msFcγ2c 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 μg 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 κ or λ 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 κ or λ 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 Qβ or AP205, most preferably of RNA bacteriophage Qβ. A very preferred influenza M2e antigen is a virus-like particle of RNA bacteriophage Qβ, 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-msFcγ2c 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-msFcγ2c 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 (kon) and off rate (koff). 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 (<=10-8 M), preferably at most 1 nM (<=10-9 M), more preferably at most 100 pM (<=10-10 M), still more preferably at most 10 pM (<=1011 M), most preferably at most 1 pM (<=10-12 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 (<=10-6 M), preferably at most 100 nM (<=10-7 M), more preferably at most 10 nM (<=10-8 M), still more preferably at most 1 nM (<=10-9 M), still more preferably at most 100 pM (<=10-10 M), still more preferably at most 10 pM (<=1011 M), and most preferably at most 1 pM (<=10-12 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 103-fold, preferably at least 104-fold, more preferably at least 105-fold, and most preferably at least 106-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 Fcγ2c fusion, wherein further preferably said Fcγ2c 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 (<=10-6 M), preferably at most 100 nM (<=10-7 M), more preferably at most 10 nM (<=10-8 M), still more preferably at most 1 nM (<=10-9 M), still more preferably at most 100 pM (<=10-10 M), still more preferably at most 10 pM (<=10-11 M), and most preferably at most 1 pM (<=10-12 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, 19th 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® CPT® Tube method (BD Biosciences, Franklin Lakes, N.J.). PBMC were pre-incubated with Alexa 647 nm-labeled Qβ (5.5 μg/ml) and human gamma globulin (11 μg/ml; Jackson ImmunoResearch) and then stained with: (1) an M2 extracellular domain consensus peptide (M2e; SEQ ID NO:48, cf. Table 7) coupled to Qβ (2.4 μg/ml) in combination with a Alexa 488 nm-labeled Qβ-specific mouse mAb (2 μg/ml), as well as a M2-specific mouse mAb (0.5 μg/ml) in combination with FITC-labeled donkey anti-mouse IgG antibody (1 μg/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® 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 μg/ml) in combination with an RNase-specific rabbit polyclonal antibody (2.5 μg/ml; Abcam) and a FITC-labeled donkey anti-rabbit IgG antibody (1.5 μg/ml; Jackson ImmunoResearch) or using (Qβ-M2e (1 μg/ml) in combination with a M2-specific mouse mAb (0.5 μg/ml) and FITC-labeled donkey anti-mouse IgG antibody (1 μg/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-γ1 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 μg/ml in phosphate-buffered saline (PBS) over night at 4° C. In parallel, scFv-Fc expression levels were monitored in by sandwich ELISA. For this, an identical set of plates was coated with Fcγ-specific, goat anti-human F(ab')2 antibody (Jackson ImmunoResearch Laboratories 109-006-098) at a concentration of 2.5 μg/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, Fcγ-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 μl 30% H2O2) for 5 to 10 min and the reaction was stopped with 5% H2SO4 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-msFcγ2c 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-γ2c (msFcγ2c) fusion proteins. The nucleotide sequences encoding the D005, E040 and F052 scFv-mouse Fc-γ2c (msFcγ2c) 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-γ2c (msFcγ2c) 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-msFcγ2c (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-msFcγ2c fusion proteins was done in HEK-293T cells. One day before transfection, 107 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-msFcγ2c 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 μg/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-msFcγ2c fusion protein was collected every 3 days and filtered through a 0.22 μm Millex GV sterile filter (Millipore).
[0176] For each of the scFv-msFcγ2c 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 μm Millex GV sterile filters (Millipore) and aliquotted. Working stocks were kept at 4° C., whereas aliquots for long-term storage were flash-frozen in liquid nitrogen and kept -80° C.
Example 4
ELISA Analysis of scFv-msFcγ2c Fusion Protein Binding to M2-Derived Peptides
[0177] To confirm binding of scFv-msFcγ2c fusion proteins to M2e, an ELISA analysis was carried out with purified scFv-D005-msFcγ2c, scFv-E040-msFcγ2c and scFv-F052-msFcγ2c. Thus, ELISA plates were coated with M2e (SEQ ID NO:48) conjugated to RNAse A at a concentration of 4 μg/ml in phosphate-buffered saline (PBS), one hour at 37° C. The plates were then washed with wash buffer (PBS/0.05% Tween) and blocked for 1 h at 37° C. with 3% BSA in wash buffer. The plates were then washed again and incubated with serial dilutions of purified scFv-D005-msFcγ2c, scFv-E040-msFcγ2c and scFv-F052-msFcγ2c. Plates were incubated at room temperature for 1.5 h at 37° 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, Fcγ-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 μl 30% H2O2) for 10 min and the reaction was stopped with 5% H2SO4 solution. Plates were then read at OD 450 nm on an ELISA reader (Biorad Benchmark). The antibodies scFv-D005-msFcγ2c, scFv-E040-msFcγ2c and scFv-F052-msFcγ2c 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-msFcγ2c, scFv- E040-msFcγ2c and scFv-F052-msFcγ2c 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-msFcγ2c 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 μg/ml in phosphate-buffered saline (PBS), over night at 4° C. The plates were then washed with wash buffer (PBS/0.05% Tween) and blocked for 2 h at 37° C. with 3% BSA in wash buffer. The plates were then washed again and incubated with purified scFv-D005-msFcγ2c, scFv-E040-msFcγ2c and scFv-F052-msFcγ2c 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, Fcγ-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-msFcγ2c binding to immobilized M2e by soluble M2 peptides. EC50 values (μM) of inhibition of binding are shown. scFv-msFcγ2c 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-msFcγ2c 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 H1N1 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-msFcγ2c, scFv-E040-msFcγ2c or scFv-F052-msFcγ2c 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-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® (Becton Dickinson). The antibodies scFv-D005-msFcγ2c, scFv-E040-msFcγ2c and scFv-F052-msFcγ2c 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-msFcγ2c Fusion Proteins in a Mouse Model of Influenza A Infection
[0180] The efficacy anti-M2 scFv-msFcγ2c 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 μg of scFv-D005-msFcγ2c, scFv-E040-msFcγ2c, scFv-F052-msFcγ2c 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×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-γ2c 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 μg per mouse) or, as a control, with 200 μg 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 μg 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×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 μg antibody, all animals survived the lethal challenge. Even at the lowest dose of 6 μg 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-msFcγ2c 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-msFcγ2c 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×LD50). 1, 2, or 3 days later, groups of mice were injected intraperitoneally with 200 μg of scFv-D005-msFcγ2c in PBS (6 mice per group). As controls, one group of mice each was injected with scFv-D005-msFcγ2c 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-msFcγ2c 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 IgG1κ were generated. Thus, DNA sequences encoding a human γ1 heavy chain shared by clones D005, E040 and F052, as well as each of the unique κ 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 γ1 heavy chain, including the signal peptide, is depicted in SEQ ID NO:31. The amino acid sequences of the κ 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-γ1-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-IgG1κ-D005, pCB15-fh-IgG1κ-E040 and pCB15-fh-IgG1κ-F052. Expression of IgG1κ-D005, IgG1κ-E040 and IgG1κ-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-msFcγ2c 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 Fcγ1 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 μg), scFv-D005-msFcγ2c (144 μg), scFv-D005-hFcm (144 μg), or human IgG (200 μg) 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×LD50) 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-msFcγ2c, 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-msFcγ2c. 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-msFcγ2c, scFv-E040-msFcγ2c or scFv-F052-msFcγ2c, 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° C. overnight were washed with wash buffer (PBS/0.05% Tween) and blocked for 1 h at 37° 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-Fcγ2c fusion proteins were detected by a 1 h incubation at room temperature with a HRPO-labeled, Fcγ-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 μl 30% H2O2) for 15 min and the reaction was stopped with 5% H2SO4 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-msFcγ2c, scFv-E040-msFcγ2c and scFv-F052-msFcγ2c 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-msFcγ2c, scFv-E040-msFcγ2c and scFv-F052-msFcγ2c 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-msFcγ2c and mAb 14C2 were incubated in FACS buffer at a concentration of 0.5 μg/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® (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-msFcγ2c 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° C. with HA-specific mouse mAb H37-80 at a concentration of 10 μg/ml in coating buffer. The plate was then washed with wash buffer (PBS/0.05% Tween) and blocked for 3 h at 37° C. with 5% BSA in wash buffer. The plate was then washed again and incubated with 108, 5×107, 2.5×107, or 1.25×107 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 μg/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 μg/ml and 0.05 μg/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
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