# Patent

## Title
Human M2e peptide immunogens

## Abstract

The present invention provides novel peptide immunogens comprising influenza virus matrix 2 protein epitopes and related compositions and methods. The present invention relates to a composition comprising a peptide immunogen useful for the prevention and treatment of an influenza virus-mediated disease. The invention also relates to vaccines, immunogenic products and immunogenic compositions containing the peptide immunogens.

## Background
RELATED APPLICATIONS
This application is related to provisional application U.S. Ser. No. 61/113,880, filed Nov. 12, 2008, the contents of which are herein incorporated by reference in their entirety.


FIELD OF THE INVENTION
The present invention relates generally to vaccines and therapeutics for influenza virus infection. The invention specifically relates to peptide immunogens suitable for generating influenza matrix 2 protein-specific antibodies and their manufacture and use.
BACKGROUND OF THE INVENTION
Influenza virus infects 5-20% of the population and results in 30,000-50,000 deaths each year in the U.S. Although the influenza vaccine is the primary method of infection prevention, four antiviral drugs are also available in the U.S.: amantadine, rimantadine, oseltamivir and zanamivir. As of December 2005, only oseltamivir (TAMIFLUTM) is recommended for treatment of influenza A due to the increasing resistance of the virus to amantadine and rimantidine resulting from an amino acid substitution in the M2 protein of the virus. Recently, a drug resistant avian virus was found in a 14-year-girl in Viet Nam. Resistance to Tamiflu has also been found in human influenza as well (Mai Le et al., Nature 437:1108 (2005)).
Influenza vaccines have been demonstrated to have a protective effect against influenza infection. However, yearly emerging antigenic variants of influenza viruses necessitate surveillance to contemporary of circulating virus strains. In some cases, difficulty in the prediction of new variant strains has prevented the timely production of the vaccine (Frace et al., Vaccine 17:2237 (1999)). Recently, pandemic avian influenza has become a serious threat due to the emergence of avian influenza viruses such as H5N1 in southern Asia. The currently available vaccines would be ineffective against avian viruses (Lipatov et al., J. Virology 78:8951 (2004); Osterholm et al., N Engl. Med. 352:1839 (2005)). A third problem with the current vaccine is the ineffectiveness in certain populations with compromised immune systems, for instance premature infants, the elderly, AIDS and transplant patients.
Disease caused by influenza A viral infections is typified by its cyclical nature. Antigenic drift and shift allow for different A strains to emerge every year. Added to that, the threat of highly pathogenic strains entering into the general population has stressed the need for novel therapies for flu infections.
SUMMARY OF THE INVENTION
The present invention relates to a synthetic peptide immunogen capable of inducing antibodies against a M2e target peptide of influenza A virus. In particular, the peptide immunogen of this invention comprises one or more epitopes. Optionally, the peptide immunogen further comprises a general immune stimulator. These peptide immunogens of the present invention are effective, capable of inducing antibodies against influenza A virus to prevent infection by the virus.
The peptide immunogen of this invention is represented by the following formula: [Xaa0]m-Xaa1-Xaa2-Xaa3-Xaa4-Xaa5-[Xaa6]p-[Xaa7]q-[Xaa8-[Xaa0]m-Xaa1-Xaa2-Xaa3-Xaa4-Xaa5-[Xaa6]p-[Xaa7]q]n; wherein, m, p and q are independently 0 or 1, n is any number between 0 and 4, Xaa0 is any amino acid, preferably C; Xaa6 is any amino acid, preferably V or C; Xaa7 is any amino acid, preferably E; Xaa8 is any amino acid not including proline, preferably G or A; Xaa1-Xaa2-Xaa3-Xaa4-Xaa5- is S-L-L-T-E or, a peptide having a single substitution to the sequence S-L-L-T-E (SEQ ID NO: 47), the substitution selected from the group consisting of: Xaa1 is C or T; Xaa2 is A, C, F or K, Xaa3 is A, C, E, F, I, K, M, Q, S, T or V, and Xaa5 is D or C.
In another aspect the peptide immunogen of this invention is represented by the following formula: [Xaa0]m-Xaa1-Xaa2-Xaa3-Xaa4-Xaa5-[Xaa6]p-[Xaa7]q-[Xaa8-[Xaa0]m-Xaa1-Xaa2-Xaa3-Xaa4-Xaa5-[Xaa6]p-[Xaa7]q]n; wherein, m, p and q are independently 0 or 1, n is any number between 0 and 4, Xaa0 is any amino acid, preferably C; Xaa6 is any amino acid, preferably V or C; Xaa2 is any amino acid, preferably E; Xaa8 is any amino acid not including proline, preferably G or A; Xaa1-Xaa2-Xaa3-Xaa4-Xaa5- is S-L-L-T-E or, a peptide having a single substitution to the sequence S-L-L-T-E (SEQ ID NO: 47), the substitution selected from the group consisting of: Xaa1 is A, C, D, L, T or V, Xaa2 is A, C, F, H, I, K, M, N, Q, R, T, W, or Y, Xaa3 is any amino acid, Xaa4 is M, N, Q, S, or W, and Xaa5 is A, D, F, H, I, K, M, N, Q, S, W, Y, or C.
In some aspects of the inventions, one or more amino acids are D-amino acids.
In some aspects of the inventions, the peptide immunogens are cyclic. Cyclization of peptide immunogens can be performed by cross-linking cysteine residues present in the peptide or by chemical means.
In some aspects of the inventions, the peptide immunogens are conjugated to carrier proteins such as KLH through intermolecular crosslinking.
The invention relates to compositions comprising the peptide immunogen and a pharmaceutically acceptable adjuvant and/or carrier selected from the group consisting of alum, liposyn, saponin, squalene, L121, emulsigen monophosphyryl lipid A (MPL), polysorbate 80, QS21, Montanide ISA51, ISA35, ISA206 and ISA 720.
The invention relates to preventing or treating a disease associated with influenza virus infection by administering compositions comprising the peptide immunogens of the invention.
The invention relates to methods for generating antibodies reactive to influenza matrix 2 (M2) protein by administering compositions comprising the peptide immunogens of the invention. It is an object of the invention to develop an immunogen that will enable the generation of high levels of high affinity antibodies against M2 protein.
One aspect of this invention provides a vaccine comprising an immunologically effective amount of a peptide immunogen composition in accordance with this invention and one or more pharmaceutically acceptable carriers. The vaccine when administered at an appropriate dosage will generate immunotherapeutic antibodies directed against influenza A virus.
The present invention provides a vaccine delivery vehicle that is suitable for human or veterinary use for the prophylaxis and treatment of influenza.
The present invention and other objects, features, and advantages of the present invention will become further apparent in the following Detailed Description of the invention and the accompanying figures and embodiments.



BRIEF DESCRIPTION OF THE DRAWINGS
FIG. 1 shows the binding of three anti-M2 antibodies and control hu14C2 antibody to 293-HEK cells transfected with an M2 expression construct or control vector, in the presence or absence of free M2 peptide.
FIGS. 2A and 2B are graphs showing human monoclonal antibody binding to influenza A/PR/32.
FIG. 3A is a chart showing amino acid sequences of extracellular domains of M2 variants.
FIGS. 3B and 3C are bar charts showing binding of human monoclonal anti-influenza antibody binding to M2 variants shown in FIG. 3A.
FIG. 4 is a series of bar charts showing binding of MAbs 8I10 and 23K12 to M2 protein representing influenza strain A/HK/483/1997 sequence that was stably expressed in the CHO cell line DG44.
FIG. 5 is an illustration showing the anti-M2 antibodies bind a highly conserved region in the N-Terminus of M2e.
FIGS. 6A and 6B are schematic diagrams that depict a core peptide immunogen and amino acid variants that are effective in binding anti-M2e huMAbs 8I10 and 23K12 under high (6A) and low (6B) stringency conditions.
FIG. 6C is a schematic diagram that depicts variants of the core sequence.
FIGS. 6D, 6E and 6F are schematic diagrams that depict specific linear and cyclized peptide immunogens containing the core sequences of the invention.



DETAILED DESCRIPTION
The present invention relates to an immunogenic composition comprising synthetic peptide immunogens capable of inducing antibodies against the extracellular domain of the matrix 2 (M2) polypeptide of influenza A virus. The present invention provides peptides that bind human monoclonal antibodies specific against the extracellular domain of the matrix 2 (M2) polypeptide of influenza A virus.
The predominant fraction of neutralizing antibodies is directed to the polymorphic regions of the hemagglutinin and neuraminidase proteins. A third transmembrane protein of type A influenza virus, matrix protein 2 (M2), is abundantly expressed by virus-infected cells, where it is believed to provide an obligatory transmembrane proton flux for viral replication (Ciampor et al., Virus Research 22:247 (1992); Grambas and Hay, Virology 190:11 (1992); Sugrue et al., EMBO J. 9:3469 (1990)). Unlike HA and NA, M2 is conserved and may represent a target for the development of antibody-based passive immunotherapies for influenza patients (Ito et al., J. Virology 65:5491 (1991); Slepushkin et al., Vaccine 13:1399 (1995); Neirynck et al., Nature Med. 5:1157 (1999)). Thus, such a neutralizing MAb would presumably target only one or a few strains. A recent focus has been on the relatively invariant matrix 2 (M2) protein. Potentially, a neutralizing MAb to M2 would be an adequate therapy for all influenza A strains.
The M2 protein is found in a homotetramer that forms an ion channel and is thought to aid in the uncoating of the virus upon entering the cell. After infection, M2 can be found in abundance at the cell surface. It is subsequently incorporated into the virion coat, where it only comprises about 2% of total coat protein. The M2 extracellular domain (M2e) is short, with the amino terminal 2-24 amino acids displayed outside of the cell.
Anti-M2 monoclonal antibodies to date have been directed towards this linear sequence. Thus, they may not exhibit desired binding properties to cellularly expressed M2, including conformational determinants on native M2.
Recent vaccine development has used immunogenic peptides conjugated to carrier proteins. However, carrier proteins are too complex for use in driving antibody responses to site-specific targets. The mass of the carrier molecule is much greater than that of the functionally important target peptide site. Consequently, the major immune response is directed to the carrier protein rather than to the target site of the peptide immunogen. Moreover, immunization with hapten-carrier conjugates frequently leads to carrier-induced immune suppression (Schutze et al., J Immunol, 1985, 135:2319). A disadvantage with the peptide-carrier protein conjugates is that these molecules are highly complex and are difficult to characterize and it is difficult to develop effective quality control procedures for the manufacturing process.
To be effective, a peptide immunogen must do more than merely evoke an anti-peptide response. An effective peptide immunogen must also evoke a functional immune response, i.e., the antibody produced must have immunological cross-reactivity to the authentic target. It is known that peptide immunogens generally do not retain a preferred structure. Therefore, it is important in designing a peptide target site to introduce structural constraints. However, the imposed structural constraint must be able to mimic the conformation of the targeted epitope so that antibodies evoked will be cross-reactivities to that site on the authentic molecule (Moore, Chapter 2 in Synthetic Peptides A User's guide, ed Grant, WH Freeman and Company: New York, 1992, pp 63-67). Peptide immunogens have been designed employing promiscuous Th epitopes, the invasin domain, and with imposed structural constraint for a peptide-based vaccine for HIV (U.S. Pat. No. 6,090,388).
A long-felt need exists in the art for new antibodies that bind to the cell-expressed M2 and conformational determinants on the native M2. Accordingly, a suitable peptide-based immunogen that mimic M2 is needed for generating vaccines and therapeutics against influenza virus. It would be desirable to provide a synthetic peptide immunogen that generates a site-specific immune response without epitopic suppression by undesirable T cell responses. The peptide-based anti-M2e immunogen should provoke an early and strong immune response in humans for protective immunity without the adverse carrier-induced immune suppression. The peptide immunogen should also be stable and well defined chemically with no need of elaborate downstream processing for ease of manufacture and quality control to avoid the need of an elaborate production plant.
M2 is a 96 amino acid transmembrane protein present as a homotetramer on the surface of influenza virus and virally infected cells. M2 contains a 23 amino acid ectodomain (M2e) that is highly conserved across influenza A strains. Few amino acid changes have occurred since the 1918 pandemic strain thus M2e is an attractive target for influenza therapies. Peptides that incorporate immunogenic epitopes of M2e form a preferred aspect of the present invention.
Mimotopes which have the same characteristics as these epitopes, and immunogens comprising such mimotopes which generate an immune response which cross-react with the IgE epitope in the context of the IgE molecule, also form part of the present invention.
The present invention, therefore, includes isolated peptides encompassing these IgE epitopes themselves, and any mimotope thereof. The meaning of mimotope is defined as an entity which is sufficiently similar to the native M2e epitope so as to be capable of being recognized by antibodies which recognize the native M2e epitope; (Gheysen, H. M., et al., 1986, Synthetic peptides as antigens. Wiley, Chichester, Ciba foundation symposium 119, p 130-149; Gheysen, H. M., 1986, Molecular Immunology, 23, 7, 709-715); or are capable of raising antibodies, when coupled to a suitable carrier, which antibodies cross-react with the native M2e epitope.
Monoclonal Antibodies for Screening M2e Peptide Immunogens
The antibodies used for screening the peptide immunogens are referred to herein as huM2e antibodies. Monoclonal antibodies used are specific to the M2 ectodomain (M2e) and derived from full-length M2 is expressed in cell lines. The huM2e antibodies bind conformational determinants on the M2-transfected cells, as well as native M2, either on influenza infected cells, or on the virus itself. The huM2e antibodies do not bind the linear M2e peptide, but they do bind several natural M2 variants expressed upon cDNA transfection into cell lines. The human monoclonal antibodies exhibit specificity for a very broad range of influenza A virus strains.
The huM2e antibodies have one or more of the following characteristics: the huM2e antibody binds a) to an epitope in the extracellular domain of the matrix 2 (M2) polypeptide of an influenza virus; b) binds to influenza A infected cells; and/or c) binds to influenza A virus (i.e., virons). The huM2e antibodies of the invention eliminate influenza infected cells through immune effector mechanisms such as ADCC and promotes direct viral clearance by binding to influenza virons. The huM2e antibodies of the invention bind to the amino-terminal region of the M2e polypeptide. Preferably, the huM2e antibodies of the invention bind to the amino-terminal region of the M2e polypeptide wherein the N-terminal methionine residue is absent. Exemplary M2e sequences include those sequences listed on Table 1 below.







TABLE 1







Exemplary M2e sequences















SEQ ID


Type
Name
Subtype
M2E Sequence
NO:





A
BREVIG MISSION.1.1918
H1N1
MSLLTEVETPTRNEWGCRCNDSSD
48


 


A
FORT MONMOUTH.1.1947
H1N1
MSLLTEVETPTKNEWECRCNDSSD
49


 


A
.SINGAPORE.02.2005
H3N2
MSLLTEVETPIRNEWECRCNDSSD
50


 


A
WISCONSIN.10.98
H1N1
MSLLTEVETPIRNGWECKCNDSSD
51


 


A
WISCONSIN.301.1976
H1N1
MSLLTEVETPIRSEWGCRCNDSSD
52


 


A
PANAMA.1.66
H2N2
MSFLPEVETPIRNEWGCRCNDSSD
53


 


A
NEW YORK.321.1999
H3N2
MSLLTEVETPIRNEWGCRCNDSSN
54


 


A
CARACAS.1.71
H3N2
MSLLTEVETPIRKEWGCRCNDSSD
55


 


A
TAIWAN.3.71
H3N2
MSFLTEVETPIRNEWGCRCNDSSD
56


 


A
WUHAN.359.95
H3N2
MSLPTEVETPIRSEWGCRCNDSSD
57


 


A
HONG KONG.1144.99
H3N2
MSLLPEVETPIRNEWGCRCNDSSD
58


 


A
HONG KONG.1180.99
H3N2
MSLLPEVETPIRNGWGCRCNDSSD
59


 


A
HONG KONG.1774.99
H3N2
MSLLTEVETPTRNGWECRCSGSSD
60


 


A
NEW YORK.217.02
H1N2
MSLLTEVETPIRNEWEYRCNDSSD
61


 


A
NEW YORK.300.2003
H1N2
MSLLTEVETPIRNEWEYRCSDSSD
62


 


A
SWINE.SPAIN.54008.2004
H3N2
MSLLTEVETPTRNGWECRYSDSSD
63


 


A
GUANGZHOU.333.99
H9N2
MSFLTEVETLTRNGWECRCSDSSD
64


 


A
HONG KONG.1073.99
H9N2
MSLLTEVETLTRNGWECKCRDSSD
65


 


A
HONG KONG.1.68
H3N2
MSLLTEVETPIRNEWGCRCNDSSD
66


 


A
SWINE.HONG KONG.126.1982
H3N2
MSLLTEVETPIRSEWGCRCNDSGD
67


 


A
NEW YORK.703.1995
H3N2
MSLLTEVETPIRNEWECRCNGSSD
68


 


A
SWINE.QUEBEC.192.81
H1N1
MSLPTEVETPIRNEWGCRCNDSSD
69


 


A
PUERTO RICO.8.34
H1N1
MSLLTEVETPIRNEWGCRCNGSSD
70


 


A
HONG KONG.485.97
H5N1
MSLLTEVDTLTRNGWGCRCSDSSD
71


 


A
HONG KONG.542.97
H5N1
MSLLTEVETLTKNGWGCRCSDSSD
72


 


A
SILKY CHICKEN.SHANTOU.1826.2004
H9N2
MSLLTEVETPTRNGWECKCSDSSD
73


 


A
CHICKEN.TAIWAN.0305.04
H6N1
MSLLTEVETHTRNGWECKCSDSSD
74


 


A
QUAIL.ARKANSAS.16309-7.94
H7N3NSA
MSLLTEVKTPTRNGWECKCSDSSD
75


 


A
HONG KONG.486.97
H5N1
MSLLTEVETLTRNGWGCRCSDSSD
76


 


A
CHICKEN.PENNSYLVANIA.13552-1.98
H7N2NSB
MSLLTEVETPTRDGWECKCSDSSD
77


 


A
CHICKEN.HEILONGJIANG.48.01
H9N2
MSLLTEVETPTRNGWGCRCSDSSD
78


 


A
SWINE.KOREA.S5.2005
H1N2
MSLLTEVETPTRNGWECKCNDSSD
79


 


A
HONG KONG.1073.99
H9N2
MSLLTEVETLTRNGWECKCSDSSD
80


 


A
WISCONSIN.3523.88
H1N1
MSLLTEVETPIRNEWGCKCNDSSD
81


 


A
X-31 VACCINE STRAIN
H3N2
MSFLTEVETPIRNEWGCRCNGSSD
82


 


A
CHICKEN.ROSTOCK.8.1934
H7N1
MSLLTEVETPTRNGWECRCNDSSD
83


 


A
ENVIRONMENT.NEW YORK.16326-
H7N2
MSLLTEVETPIRKGWECNCSDSSD
84



1.2005





 


A
INDONESIA.560H.2006
H5N1
MSLLTEVETPTRNEWECRCSDSSD
85


 


A
CHICKEN.HONG KONG.SF1.03
H9N2
MSLLTGVETHTRNGWGCKCSDSSD
86


 


A
CHICKEN.HONGKONG.YU427.03
H9N2
MSLLPEVETHTRNGWGCRCSDSSD
87









In one embodiment, the peptide immunogens of the invention comprise a M2e peptide that wholly or partially includes the amino acid residues from position 2 to position 7 of M2e (SLLTEV). The huM2e antibodies bind wholly or partially to the amino acid sequence SLLTE (SEQ ID NO: 47) comprising the peptide immunogens of the invention.
Exemplary huM2e monoclonal antibodies that bind to the peptide immunogens are the 8I10, 21B15 and 23K12 antibodies described herein.
The 8I10 antibody includes a heavy chain variable region (SEQ ID NO: 88) encoded by the nucleic acid sequence shown below in SEQ ID NO: 89, and a light chain variable region (SEQ ID NO: 90) encoded by the nucleic acid sequence shown in SEQ ID NO: 91.
The amino acids encompassing the CDRs as defined by Chothia, C. et al. (1989, Nature, 342: 877-883) are underlined and those defined by Kabat E. A. et al. (1991, Sequences of Proteins of Immunological Interest, 5th edit., NIH Publication no. 91-3242 U.S. Department of Heath and Human Services.) are highlighted in bold in the sequences below.
The heavy chain CDRs of the 8I10 antibody have the following sequences per Kabat definition: NYYWS (SEQ ID NO: 92), FIYYGGNTKYNPSLKS (SEQ ID NO: 93) and ASCSGGYCILD (SEQ ID NO: 94). The light chain CDRs of the 8I10 antibody have the following sequences per Kabat definition: RASQNIYKYLN (SEQ ID NO: 95), AA SGLQS (SEQ ID NO: 96) and QQSYSPPLT (SEQ ID NO: 97).
The heavy chain CDRs of the 8I10 antibody have the following sequences per Chothia definition: GSSISN (SEQ ID NO: 98), FIYYGGNTK (SEQ ID NO: 99) and ASCSGGYCILD (SEQ ID NO: 94). The light chain CDRs of the 8I10 antibody have the following sequences per Chothia definition: RASQNIYKYLN (SEQ ID NO: 95), AASGLQS (SEQ ID NO: 96) and QQSYSPPLT (SEQ ID NO: 97).








>8I10 VH nucleotide sequence:



(SEQ ID NO: 89)



CAGGTGCAATTGCAGGAGTCGGGCCCAGGACTGGTGAAGCCTTCGGAGACCCTGTCCCTCACCTGCACTGTCT



 


CTGGTTCGTCCATCAGTAATTACTACTGGAGCTGGATCCGGCAGTCCCCAGGGAAGGGACTGGAGTGGATTGG


 


GTTTATCTATTACGGTGGAAACACCAAGTACAATCCCTCCCTCAAGAGCCGCGTCACCATATCACAAGACACT


 


TCCAAGAGTCAGGTCTCCCTGACGATGAGCTCTGTGACCGCTGCGGAATCGGCCGTCTATTTCTGTGCGAGAG


 


CGTCTTGTAGTGGTGGTTACTGTATCCTTGACTACTGGGGCCAGGGAACCCTGGTCACCGTCTCG


 


>8I10 VH amino acid sequence:


Kabat Bold, Chothia underlined


(SEQ ID NO: 88)



Q V Q L Q E S G P G L V K P S E T L S L T C T V S



 


G S S I S N Y Y W S W I R Q S P G K G L E W I G F


 


I Y Y G G N T K Y N P S L K S R V T I S Q D T S K


 


S Q V S L T M S S V T A A E S A V Y F C A R A S C


 


S G G Y C I L D Y W G Q G T L V T V S


 


>8I10 VL nucleotide sequence:


(SEQ ID NO: 91)



GACATCCAGATGACCCAGTCTCCATCCTCCCTGTCTGCATCTGTAGGAGACAGAGTCACCATCACTTGCCGGG



 


CGAGTCAGAACATTTACAAGTATTTAAATTGGTATCAGCAGAGACCAGGGAAAGCCCCTAAGGGCCTGATCTC


 


TGCTGCATCCGGGTTGCAAAGTGGGGTCCCATCAAGGTTCAGTGGCAGTGGATCTGGGACAGATTTCACTCTC


 


ACCATCACCAGTCTGCAACCTGAAGATTTTGCAACTTACTACTGTCAACAGAGTTACAGTCCCCCTCTCACTT


 


TCGGCGGAGGGACCAGGGTGGAGATCAAAC


 


>8I10 VL amino acid sequence:


Kabat Bold, Chothia underlined


(SEQ ID NO: 90)



D I Q M T Q S P S S L S A S V G D R V T I T C R A



 


S Q N I Y K Y L N W Y Q Q R P G K A P K G L I S A


 


A S G L Q S G V P S R F S G S G S G T D F T L T I


 


T S L Q P E D F A T Y Y C Q Q S Y S P P L T F G G


 


G T R V E I K






The 21B15 antibody includes antibody includes a heavy chain variable region (SEQ ID NO: 100) encoded by the nucleic acid sequence shown below in SEQ ID NO: 101, and a light chain variable region (SEQ ID NO: 102) encoded by the nucleic acid sequence shown in SEQ ID NO: 103.
The amino acids encompassing the CDRs as defined by Chothia et al. 1989, are underlined and those defined by Kabat et al., 1991 are highlighted in bold in the sequences below.
The heavy chain CDRs of the 21B15 antibody have the following sequences per Kabat definition: NYYWS (SEQ ID NO: 92), FIYYGGNTKYNPSLKS (SEQ ID NO: 93) and ASCSGGYCILD (SEQ ID NO: 94). The light chain CDRs of the 21B15 antibody have the following sequences per Kabat definition: RASQNIYKYLN (SEQ ID NO: 95), AASGLQS (SEQ ID NO: 96) and QQSYSPPLT (SEQ ID NO: 97).
The heavy chain CDRs of the 21B15 antibody have the following sequences per Chothia definition: GSSISN (SEQ ID NO: 98), FIYYGGNTK (SEQ ID NO: 93) and ASCSGGYCILD (SEQ ID NO: 94). The light chain CDRs of the 21B15 antibody have the following sequences per Chothia definition: RASQNIYKYLN (SEQ ID NO: 95), AASGLQS (SEQ ID NO: 96) and QQSYSPPLT (SEQ ID NO: 97).








>21B15 VH nucleotide sequence:



(SEQ ID NO: 101)



CAGGTGCAATTGCAGGAGTCGGGCCCAGGACTGGTGAAGCCTTCGGAGACCCTGTCCCTCACCTGCACTGTCT



 


CTGGTTCGTCCATCAGTAATTACTACTGGAGCTGGATCCGGCAGTCCCCAGGGAAGGGACTGGAGTGGATTGG


 


GTTTATCTATTACGGTGGAAACACCAAGTACAATCCCTCCCTCAAGAGCCGCGTCACCATATCACAAGACACT


 


TCCAAGAGTCAGGTCTCCCTGACGATGAGCTCTGTGACCGCTGCGGAATCGGCCGTCTATTTCTGTGCGAGAG


 


CGTCTTGTAGTGGTGGTTACTGTATCCTTGACTACTGGGGCCAGGGAACCCTGGTCACCGTCTCG


 


>21B15 VH amino acid sequence:


Kabat Bold, Chothia underlined


(SEQ ID NO: 100)



Q V Q L Q E S G P G L V K P S E T L S L T C T V S



 


G S S I S N Y Y W S W I R Q S P G K G L E W I G F


 


I Y Y G G N T K Y N P S L K S R V T I S Q D T S K


 


S Q V S L T M S S V T A A E S A V Y F C A R A S C


 


S G G Y C I L D Y W G Q G T L V T V S


 


>21B15 VL nucleotide sequence:


(SEQ ID NO: 103)



GACATCCAGGTGACCCAGTCTCCATCCTCCCTGTCTGCATCTGTAGGAGACAGAGTCACCATCACTTGCCGCG



 


CGAGTCAGAACATTTACAAGTATTTAAATTGGTATCAGCAGAGACCAGGGAAAGCCCCTAAGGGCCTGATCTC


 


TGCTGCATCCGGGTTGCAAAGTGGGGTCCCATCAAGGTTCAGTGGCAGTGGATCTGGGACAGATTTCACTCTC


 


ACCATCACCAGTCTGCAACCTGAAGATTTTGCAACTTACTACTGTCAACAGAGTTACAGTCCCCCTCTCACTT


 


TCGGCGGAGGGACCAGGGTGGATATCAAAC


 


>21B15 VL amino acid sequence:


Kabat Bold, Chothia underlined


(SEQ ID NO: 102)



D I Q V T Q S P S S L S A S V G D R V T I T C R A



 


S Q N I Y K Y L N W Y Q Q R P G K A P K G L I S A


 


A S G L Q S G V P S R F S G S G S G T D F T L T I


 


T S L Q P E D F A T Y Y C Q Q S Y S P P L T F G G


 


G T R V D I K






The 23K12 antibody includes antibody includes a heavy chain variable region (SEQ ID NO: 104) encoded by the nucleic acid sequence shown below in SEQ ID NO: 105, and a light chain variable region (SEQ ID NO: 106) encoded by the nucleic acid sequence shown in SEQ ID NO: 107.
The amino acids encompassing the CDRs as defined by Chothia et al., 1989 are underlined and those defined by Kabat et al., 1991 are highlighted in bold in the sequences below.
The heavy chain CDRs of the 23K12 antibody have the following sequences per Kabat definition: SNYMS (SEQ ID NO: 108), VIYSGGSTYYADSVK (SEQ ID NO: 109) and CLSRMRGYGLDV (SEQ ID NO: 110). The light chain CDRs of the 23K12 antibody have the following sequences per Kabat definition: RTSQSISSYLN (SEQ ID NO: 111), AASSLQSGVPSRF (SEQ ID NO: 112) and QQSYSMPA (SEQ ID NO: 113).
The heavy chain CDRs of the 23K12 antibody have the following sequences per Chothia definition: GFTVSSN (SEQ ID NO: 114), VIYSGGSTY (SEQ ID NO: 115) and CLSRMRGYGLDV (SEQ ID NO: 110). The light chain CDRs of the 23K12 antibody have the following sequences per Chothia definition: RTSQSISSYLN (SEQ ID NO: 111), AASSLQSGVPSRF (SEQ ID NO: 112) and QQSYSMPA (SEQ ID NO: 113).








>23K12 VH nucleotide sequence:



(SEQ ID NO: 105)



GAGGTGCAGCTGGTGGAGTCTGGGGGAGGCTTGGTCCAGCCTGGGGGGTCCCTGAGAATCTCCTGTGCAGCCT



 


CTGGATTCACCGTCAGTAGCAACTACATGAGTTGGGTCCGCCAGGCTCCAGGGAAGGGGCTGGAGTGGGTCTC


 


AGTTATTTATAGTGGTGGTAGCACATACTACGCAGACTCCGTGAAGGGCAGATTCTCCTTCTCCAGAGACAAC


 


TCCAAGAACACAGTGTTTCTTCAAATGAACAGCCTGAGAGCCGAGGACACGGCTGTGTATTACTGTGCGAGAT


 


GTCTGAGCAGGATGCGGGGTTACGGTTTAGACGTCTGGGGCCAAGGGACCACGGTCACCGTCTCG


 


>23K12 VH amino acid sequence:


Kabat Bold, Chothia underlined


(SEQ ID NO: 104)



E V Q L V E S G G G L V Q P G G S L R I S C A A S



 


G F T V S S N Y M S W V R Q A P G K G L E W V S V


 


I Y S G G S T Y Y A D S V K G R F S F S R D N S K


 


N T V F L Q M N S L R A E D T A V Y Y C A R C L S


 


R M R G Y G L D V W G Q G T T V T V S


 


>23K12 VL nucleotide sequence:


(SEQ ID NO: 107)



GACATCCAGATGACCCAGTCTCCATCCTCCCTGTCTGCATCTGTAGGAGACAGAGTCACCATCACTTGCCGGA



 


CAAGTCAGAGCATTAGCAGCTATTTAAATTGGTATCAGCAGAAACCAGGGAAAGCCCCTAAACTCCTGATCTA


 


TGCTGCATCCAGTTTGCAAAGTGGGGTCCCATCAAGGTTCAGTGGCAGTGGATCTGGGACAGATTTCACTCTC


 


ACCATCAGCGGTCTGCAACCTGAAGATTTTGCAACCTACTACTGTCAACAGAGTTACAGTATGCCTGCCTTTG


 


GCCAGGGGACCAAGCTGGAGATCAAA


 


>23K12 VL amino acid sequence:


Kabat Bold, Chothia underlined


(SEQ ID NO: 106)



D I Q M T Q S P S S L S A S V G D R V T I T C R T



 


S Q S I S S Y L N W Y Q Q K P G K A P K L L I Y A


 


A S S L Q S G V P S R F S G S G S G T D F T L T I


 


S G L Q P E D F A T Y Y C Q Q S Y S M P A F G Q G


 


T K L E I K






Unless otherwise defined, scientific and technical terms used in connection with the present invention shall have the meanings that are commonly understood by those of ordinary skill in the art. Further, unless otherwise required by context, singular terms shall include pluralities and plural terms shall include the singular. Generally, nomenclatures utilized in connection with, and techniques of, cell and tissue culture, molecular biology, and protein and oligo- or polynucleotide chemistry and hybridization described herein are those well known and commonly used in the art. Standard techniques are used for recombinant DNA, oligonucleotide synthesis, and tissue culture and transformation (e.g., electroporation, lipofection). Enzymatic reactions and purification techniques are performed according to manufacturer's specifications or as commonly accomplished in the art or as described herein. The practice of the present invention will employ, unless indicated specifically to the contrary, conventional methods of virology, immunology, microbiology, molecular biology and recombinant DNA techniques within the skill of the art, many of which are described below for the purpose of illustration. Such techniques are explained fully in the literature. See, e.g., Sambrook, et al. Molecular Cloning: A Laboratory Manual (2nd Edition, 1989); Maniatis et al. Molecular Cloning: A Laboratory Manual (1982); DNA Cloning: A Practical Approach, vol. I & II (D. Glover, ed.); Oligonucleotide Synthesis (N. Gait, ed., 1984); Nucleic Acid Hybridization (B. Hames & S. Higgins, eds., 1985); Transcription and Translation (B. Hames & S. Higgins, eds., 1984); Animal Cell Culture (R. Freshney, ed., 1986); Perbal, A Practical Guide to Molecular Cloning (1984).
The nomenclatures utilized in connection with, and the laboratory procedures and techniques of, analytical chemistry, synthetic organic chemistry, and medicinal and pharmaceutical chemistry described herein are those well known and commonly used in the art. Standard techniques are used for chemical syntheses, chemical analyses, pharmaceutical preparation, formulation, and delivery, and treatment of patients.
Definitions
The following definitions are useful in understanding the present invention:
The term “antibody” (Ab) as used herein includes monoclonal antibodies, polyclonal antibodies, multispecific antibodies (e.g., bispecific antibodies), and antibody fragments, so long as they exhibit the desired biological activity. The term “immunoglobulin” (Ig) is used interchangeably with “antibody” herein.
An “isolated antibody” is one that has been separated and/or recovered from a component of its natural environment. Contaminant components of its natural environment are materials that would interfere with diagnostic or therapeutic uses for the antibody, and may include enzymes, hormones, and other proteinaceous or nonproteinaceous solutes. In preferred embodiments, the antibody is purified: (1) to greater than 95% by weight of antibody as determined by the Lowry method, and most preferably more than 99% by weight; (2) to a degree sufficient to obtain at least 15 residues of N-terminal or internal amino acid sequence by use of a spinning cup sequenator; or (3) to homogeneity by SDS-PAGE under reducing or non-reducing conditions using Coomassie blue or, preferably, silver stain. Isolated antibody includes the antibody in situ within recombinant cells since at least one component of the antibody's natural environment will not be present. Ordinarily, however, isolated antibody will be prepared by at least one purification step.
The basic four-chain antibody unit is a heterotetrameric glycoprotein composed of two identical light (L) chains and two identical heavy (H) chains. An IgM antibody consists of 5 of the basic heterotetramer unit along with an additional polypeptide called J chain, and therefore contain 10 antigen binding sites, while secreted IgA antibodies can polymerize to form polyvalent assemblages comprising 2-5 of the basic 4-chain units along with J chain. In the case of IgGs, the 4-chain unit is generally about 150,000 daltons. Each L chain is linked to an H chain by one covalent disulfide bond, while the two H chains are linked to each other by one or more disulfide bonds depending on the H chain isotype. Each H and L chain also has regularly spaced intrachain disulfide bridges. Each H chain has at the N-terminus, a variable domain (VH) followed by three constant domains (CH) for each of the α and γ chains and four CH domains for μ and c isotypes. Each L chain has at the N-terminus, a variable domain (VL) followed by a constant domain (CL) at its other end. The VL is aligned with the VH and the CL is aligned with the first constant domain of the heavy chain (CH1). Particular amino acid residues are believed to form an interface between the light chain and heavy chain variable domains. The pairing of a VH and VL together forms a single antigen-binding site. For the structure and properties of the different classes of antibodies, see, e.g., Basic and Clinical Immunology, 8th edition, Daniel P. Stites, Abba I. Terr and Tristram G. Parslow (eds.), Appleton & Lange, Norwalk, Conn., 1994, page 71, and Chapter 6.
The L chain from any vertebrate species can be assigned to one of two clearly distinct types, called kappa (κ) and lambda (λ), based on the amino acid sequences of their constant domains (CL). Depending on the amino acid sequence of the constant domain of their heavy chains (CH), immunoglobulins can be assigned to different classes or isotypes. There are five classes of immunoglobulins: IgA, IgD, IgE, IgG, and IgM, having heavy chains designated alpha (), delta (δ), epsilon (), gamma (γ) and mu (μ), respectively. The γ and  classes are further divided into subclasses on the basis of relatively minor differences in CH sequence and function, e.g., humans express the following subclasses: IgG1, IgG2, IgG3, IgG4, IgA 1, and IgA2.
The term “variable” refers to the fact that certain segments of the V domains differ extensively in sequence among antibodies. The V domain mediates antigen binding and defines specificity of a particular antibody for its particular antigen. However, the variability is not evenly distributed across the 110-amino acid span of the variable domains. Instead, the V regions consist of relatively invariant stretches called framework regions (FRs) of 15-30 amino acids separated by shorter regions of extreme variability called “hypervariable regions” that are each 9-12 amino acids long. The variable domains of native heavy and light chains each comprise four FRs, largely adopting a β-sheet configuration, connected by three hypervariable regions, which form loops connecting, and in some cases forming part of, the β-sheet structure. The hypervariable regions in each chain are held together in close proximity by the FRs and, with the hypervariable regions from the other chain, contribute to the formation of the antigen-binding site of antibodies (see Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, Md. (1991)). The constant domains are not involved directly in binding an antibody to an antigen, but exhibit various effector functions, such as participation of the antibody in antibody dependent cellular cytotoxicity (ADCC).
The term “hypervariable region” when used herein refers to the amino acid residues of an antibody that are responsible for antigen binding. The hypervariable region generally comprises amino acid residues from a “complementarity determining region” or “CDR” (e.g., around about residues 24-34 (L1), 50-56 (L2) and 89-97 (L3) in the VL, and around about 1-35 (H1), 50-65 (H2) and 95-102 (H3) in the VH; Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, Md. (1991)) and/or those residues from a “hypervariable loop” (e.g., residues 26-32 (L1), 50-52 (L2) and 91-96 (L3) in the VL, and 26-32 (H1), 53-55 (H2) and 96-101 (H3) in the VH; Chothia and Lesk, J. Mol. Biol. 196:901-917 (1987)).
The term “monoclonal antibody” as used herein refers to an antibody obtained from a population of substantially homogeneous antibodies, i.e., the individual antibodies comprising the population are identical except for possible naturally occurring mutations that may be present in minor amounts. Monoclonal antibodies are highly specific, being directed against a single antigenic site. Furthermore, in contrast to polyclonal antibody preparations that include different antibodies directed against different determinants (epitopes), each monoclonal antibody is directed against a single determinant on the antigen. In addition to their specificity, the monoclonal antibodies are advantageous in that they may be synthesized uncontaminated by other antibodies. The modifier “monoclonal” is not to be construed as requiring production of the antibody by any particular method. For example, the monoclonal antibodies useful in the present invention may be prepared by the hybridoma methodology first described by Kohler et al., Nature, 256:495 (1975), or may be made using recombinant DNA methods in bacterial, eukaryotic animal or plant cells (see, e.g., U.S. Pat. No. 4,816,567). The “monoclonal antibodies” may also be isolated from phage antibody libraries using the techniques described in Clackson et al., Nature, 352:624-628 (1991) and Marks et al., J. Mol. Biol., 222:581-597 (1991), for example.
The monoclonal antibodies herein include “chimeric” antibodies in which a portion of the heavy and/or light chain is identical with or homologous to corresponding sequences in antibodies derived from a particular species or belonging to a particular antibody class or subclass, while the remainder of the chain(s) is identical with or homologous to corresponding sequences in antibodies derived from another species or belonging to another antibody class or subclass, as well as fragments of such antibodies, so long as they exhibit the desired biological activity (see U.S. Pat. No. 4,816,567; and Morrison et al., Proc. Natl. Acad. Sci. USA, 81:6851-6855 (1984)). The present invention provides variable domainantigen-binding sequences derived from human antibodies. Accordingly, chimeric antibodies of primary interest herein include antibodies having one or more human antigen binding sequences (e.g., CDRs) and containing one or more sequences derived from a non-human antibody, e.g., an FR or C region sequence. In addition, chimeric antibodies of primary interest herein include those comprising a human variable domain antigen binding sequence of one antibody class or subclass and another sequence, e.g., FR or C region sequence, derived from another antibody class or subclass. Chimeric antibodies of interest herein also include those containing variable domain antigen-binding sequences related to those described herein or derived from a different species, such as a non-human primate (e.g., Old World Monkey, Ape, etc). Chimeric antibodies also include primatized and humanized antibodies.
Furthermore, chimeric antibodies may comprise residues that are not found in the recipient antibody or in the donor antibody. These modifications are made to further refine antibody performance. For further details, see Jones et al., Nature 321:522-525 (1986); Riechmann et al., Nature 332:323-329 (1988); and Presta, Curr. Op. Struct. Biol. 2:593-596 (1992).
A “humanized antibody” is generally considered to be a human antibody that has one or more amino acid residues introduced into it from a source that is non-human. These non-human amino acid residues are often referred to as “import” residues, which are typically taken from an “import” variable domain. Humanization is traditionally performed following the method of Winter and co-workers (Jones et al., Nature, 321:522-525 (1986); Reichmann et al., Nature, 332:323-327 (1988); Verhoeyen et al., Science, 239:1534-1536 (1988)), by substituting import hypervariable region sequences for the corresponding sequences of a human antibody. Accordingly, such “humanized” antibodies are chimeric antibodies (U.S. Pat. No. 4,816,567) wherein substantially less than an intact human variable domain has been substituted by the corresponding sequence from a non-human species.
A “human antibody” is an antibody containing only sequences present in an antibody naturally produced by a human. However, as used herein, human antibodies may comprise residues or modifications not found in a naturally occurring human antibody, including those modifications and variant sequences described herein. These are typically made to further refine or enhance antibody performance.
An “intact” antibody is one that comprises an antigen-binding site as well as a CL and at least heavy chain constant domains, CH1, CH2 and CH3. The constant domains may be native sequence constant domains (e.g., human native sequence constant domains) or amino acid sequence variant thereof. Preferably, the intact antibody has one or more effector functions.
An “antibody fragment” comprises a portion of an intact antibody, preferably the antigen binding or variable region of the intact antibody. Examples of antibody fragments include Fab, Fab′, F(ab′)2, and Fv fragments; diabodies; linear antibodies (see U.S. Pat. No. 5,641,870; Zapata et al., Protein Eng. 8(10): 1057-1062 [1995]); single-chain antibody molecules; and multispecific antibodies formed from antibody fragments.
The phrase “functional fragment or analog” of an antibody is a compound having qualitative biological activity in common with a full-length antibody. For example, a functional fragment or analog of an anti-IgE antibody is one that can bind to an IgE immunoglobulin in such a manner so as to prevent or substantially reduce the ability of such molecule from having the ability to bind to the high affinity receptor, FcεRI.
Papain digestion of antibodies produces two identical antigen-binding fragments, called “Fab” fragments, and a residual “Fc” fragment, a designation reflecting the ability to crystallize readily. The Fab fragment consists of an entire L chain along with the variable region domain of the H chain (VH), and the first constant domain of one heavy chain (CH1). Each Fab fragment is monovalent with respect to antigen binding, i.e., it has a single antigen-binding site. Pepsin treatment of an antibody yields a single large F(ab′)2 fragment that roughly corresponds to two disulfide linked Fab fragments having divalent antigen-binding activity and is still capable of cross-linking antigen. Fab′ fragments differ from Fab fragments by having additional few residues at the carboxy terminus of the CH1 domain including one or more cysteines from the antibody hinge region. Fab′-SH is the designation herein for Fab′ in which the cysteine residue(s) of the constant domains bear a free thiol group. F(ab′)2 antibody fragments originally were produced as pairs of Fab′ fragments that have hinge cysteines between them. Other chemical couplings of antibody fragments are also known.
The “Fc” fragment comprises the carboxy-terminal portions of both H chains held together by disulfides. The effector functions of antibodies are determined by sequences in the Fc region, which region is also the part recognized by Fc receptors (FcR) found on certain types of cells.
“Fv” is the minimum antibody fragment that contains a complete antigen-recognition and -binding site. This fragment consists of a dimer of one heavy- and one light-chain variable region domain in tight, non-covalent association. From the folding of these two domains emanate six hypervariable loops (three loops each from the H and L chain) that contribute the amino acid residues for antigen binding and confer antigen binding specificity to the antibody. However, even a single variable domain (or half of an Fv comprising only three CDRs specific for an antigen) has the ability to recognize and bind antigen, although at a lower affinity than the entire binding site.
“Single-chain Fv” also abbreviated as “sFv” or “scFv” are antibody fragments that comprise the VH and VL antibody domains connected into a single polypeptide chain. Preferably, the sFv polypeptide further comprises a polypeptide linker between the VH and VL, domains that enables the sFv to form the desired structure for antigen binding. For a review of sFv, see Pluckthun in The Pharmacology of Monoclonal Antibodies, vol. 113, Rosenburg and Moore eds., Springer-Verlag, New York, pp. 269-315 (1994); Borrebaeck 1995, infra.
The term “diabodies” refers to small antibody fragments prepared by constructing sFv fragments (see preceding paragraph) with short linkers (about 5-10 residues) between the VH and VL domains such that inter-chain but not intra-chain pairing of the V domains is achieved, resulting in a bivalent fragment, i.e., fragment having two antigen-binding sites. Bispecific diabodies are heterodimers of two “crossover” sFv fragments in which the VH and VL domains of the two antibodies are present on different polypeptide chains. Diabodies are described more fully in, for example, EP 404,097; WO 93/11161; and Hollinger et al., Proc. Natl. Acad. Sci. USA, 90:6444-6448 (1993).
As used herein, an antibody that “internalizes” is one that is taken up by (i.e., enters) the cell upon binding to an antigen on a mammalian cell (e.g., a cell surface polypeptide or receptor). The internalizing antibody will of course include antibody fragments, human or chimeric antibody, and antibody conjugates. For certain therapeutic applications, internalization in vivo is contemplated. The number of antibody molecules internalized will be sufficient or adequate to kill a cell or inhibit its growth, especially an infected cell. Depending on the potency of the antibody or antibody conjugate, in some instances, the uptake of a single antibody molecule into the cell is sufficient to kill the target cell to which the antibody binds. For example, certain toxins are highly potent in killing such that internalization of one molecule of the toxin conjugated to the antibody is sufficient to kill the infected cell.
As used herein, an antibody is said to be “immunospecific,” “specific for” or to “specifically bind” an antigen if it reacts at a detectable level with the antigen, preferably with an affinity constant, Ka, of greater than or equal to about 104 M1, or greater than or equal to about 105 M−1, greater than or equal to about 106 M−1, greater than or equal to about 107 M−1, or greater than or equal to 108 Affinity of an antibody for its cognate antigen is also commonly expressed as a dissociation constant KD, and in certain embodiments, HuM2e antibody specifically binds to M2e if it binds with a KD of less than or equal to 10−4 M, less than or equal to about 10−5 M, less than or equal to about 10−6 M, less than or equal to 10−7 M, or less than or equal to 10−8 M. Affinities of antibodies can be readily determined using conventional techniques, for example, those described by Scatchard et al. (Ann. N.Y. Acad. Sci. USA 51:660 (1949)).
Binding properties of an antibody to antigens, cells or tissues thereof may generally be determined and assessed using immunodetection methods including, for example, immunofluorescence-based assays, such as immuno-histochemistry (IHC) and/or fluorescence-activated cell sorting (FACS).
An antibody having a “biological characteristic” of a designated antibody is one that possesses one or more of the biological characteristics of that antibody which distinguish it from other antibodies. For example, in certain embodiments, an antibody with a biological characteristic of a designated antibody will bind the same epitope as that bound by the designated antibody and/or have a common effector function as the designated antibody.
The term “antagonist” antibody is used in the broadest sense, and includes an antibody that partially or fully blocks, inhibits, or neutralizes a biological activity of an epitope, polypeptide, or cell that it specifically binds. Methods for identifying antagonist antibodies may comprise contacting a polypeptide or cell specifically bound by a candidate antagonist antibody with the candidate antagonist antibody and measuring a detectable change in one or more biological activities normally associated with the polypeptide or cell.
An “antibody that inhibits the growth of infected cells” or a “growth inhibitory” antibody is one that binds to and results in measurable growth inhibition of infected cells expressing or capable of expressing an M2e epitope bound by an antibody. Preferred growth inhibitory antibodies inhibit growth of infected cells by greater than 20%, preferably from about 20% to about 50%, and even more preferably, by greater than 50% (e.g., from about 50% to about 100%) as compared to the appropriate control, the control typically being infected cells not treated with the antibody being tested. Growth inhibition can be measured at an antibody concentration of about 0.1 to 30 μg/ml or about 0.5 nM to 200 nM in cell culture, where the growth inhibition is determined 1-10 days after exposure of the infected cells to the antibody. Growth inhibition of infected cells in vivo can be determined in various ways known in the art. The antibody is growth inhibitory in vivo if administration of the antibody at about 1 μg/kg to about 100 mg/kg body weight results in reduction the percent of infected cells or total number of infected cells within about 5 days to 3 months from the first administration of the antibody, preferably within about 5 to 30 days.
An antibody that “induces apoptosis” is one which induces programmed cell death as determined by binding of annexin V, fragmentation of DNA, cell shrinkage, dilation of endoplasmic reticulum, cell fragmentation, and/or formation of membrane vesicles (called apoptotic bodies). Preferably the cell is an infected cell. Various methods are available for evaluating the cellular events associated with apoptosis. For example, phosphatidyl serine (PS) translocation can be measured by annexin binding; DNA fragmentation can be evaluated through DNA laddering; and nuclear/chromatin condensation along with DNA fragmentation can be evaluated by any increase in hypodiploid cells. Preferably, the antibody that induces apoptosis is one that results in about 2 to 50 fold, preferably about 5 to 50 fold, and most preferably about 10 to 50 fold, induction of annexin binding relative to untreated cell in an annexin binding assay.
Antibody “effector functions” refer to those biological activities attributable to the Fc region (a native sequence Fc region or amino acid sequence variant Fc region) of an antibody, and vary with the antibody isotype. Examples of antibody effector functions include: C1q binding and complement dependent cytotoxicity; Fc receptor binding; antibody-dependent cell-mediated cytotoxicity (ADCC); phagocytosis; down regulation of cell surface receptors (e.g., B cell receptor); and B cell activation.
“Antibody-dependent cell-mediated cytotoxicity” or “ADCC” refers to a form of cytotoxicity in which secreted Ig bound to Fc receptors (FcRs) present on certain cytotoxic cells (e.g., Natural Killer (NK) cells, neutrophils, and macrophages) enable these cytotoxic effector cells to bind specifically to an antigen-bearing target cell and subsequently kill the target cell with cytotoxins. The antibodies “arm” the cytotoxic cells and are required for such killing. The primary cells for mediating ADCC, NK cells, express FcγRIII only, whereas monocytes express FcγRI, FcγRII and FcγRIII. FcR expression on hematopoietic cells is summarized in Table 3 on page 464 of Ravetch and Kinet, Annu. Rev. Immunol 9:457-92 (1991). To assess ADCC activity of a molecule of interest, an in vitro ADCC assay, such as that described in U.S. Pat. Nos. 5,500,362 or 5,821,337 may be performed. Useful effector cells for such assays include peripheral blood mononuclear cells (PBMC) and Natural Killer (NK) cells. Alternatively, or additionally, ADCC activity of the molecule of interest may be assessed in vivo, e.g., in a animal model such as that disclosed in Clynes et al., PNAS (USA) 95:652-656 (1998).
“Fc receptor” or “FcR” describes a receptor that binds to the Fc region of an antibody. In certain embodiments, the FcR is a native sequence human FcR. Moreover, a preferred FcR is one that binds an IgG antibody (a gamma receptor) and includes receptors of the FcγRI, FcγRII, and FcγRIII subclasses, including allelic variants and alternatively spliced forms of these receptors. FCγRII receptors include FcγRIIA (an “activating receptor”) and FcγRIIB (an “inhibiting receptor”), which have similar amino acid sequences that differ primarily in the cytoplasmic domains thereof. Activating receptor FcγRIIA contains an immunoreceptor tyrosine-based activation motif (ITAM) in its cytoplasmic domain. Inhibiting receptor FcγRIIB contains an immunoreceptor tyrosine-based inhibition motif (ITIM) in its cytoplasmic domain. (see review M. in Daeron, Annu. Rev. Immunol. 15:203-234 (1997)). FcRs are reviewed in Ravetch and Kinet, Annu. Rev. Immunol 9:457-92 (1991); Capel et al., Immunomethods 4:25-34 (1994); and de Haas et al., J. Lab. Clin. Med. 126:330-41 (1995). Other FcRs, including those to be identified in the future, are encompassed by the term “FcR” herein. The term also includes the neonatal receptor, FcRn, which is responsible for the transfer of maternal IgGs to the fetus (Guyer et al., J. Immunol. 117:587 (1976) and Kim et al., J. Immunol. 24:249 (1994)).
“Human effector cells” are leukocytes that express one or more FcRs and perform effector functions. Preferably, the cells express at least FcγRIII and perform ADCC effector function. Examples of human leukocytes that mediate ADCC include PBMC, NK cells, monocytes, cytotoxic T cells and neutrophils; with PBMCs and NK cells being preferred. The effector cells may be isolated from a native source, e.g., from blood.
“Complement dependent cytotoxicity” or “CDC” refers to the lysis of a target cell in the presence of complement. Activation of the classical complement pathway is initiated by the binding of the first component of the complement system (C1q) to antibodies (of the appropriate subclass) that are bound to their cognate antigen. To assess complement activation, a CDC assay, e.g., as described in Gazzano-Santoro et al., J. Immunol. Methods 202:163 (1996), may be performed.
The terms “influenza A” and “Influenzavirus A” refer to a genus of the Orthomyxoviridae family of viruses. Influenzavirus A includes only one species: influenza A virus which cause influenza in birds, humans, pigs, and horses. Strains of all subtypes of influenza A virus have been isolated from wild birds, although disease is uncommon. Some isolates of influenza A virus cause severe disease both in domestic poultry and, rarely, in humans.
A “mammal” for purposes of treating n infection, refers to any mammal, including humans, domestic and farm animals, and zoo, sports, or pet animals, such as dogs, cats, cattle, horses, sheep, pigs, goats, rabbits, etc. Preferably, the mammal is human.
“Treating” or “treatment” or “alleviation” refers to both therapeutic treatment and prophylactic or preventative measures; wherein the object is to prevent or slow down (lessen) the targeted pathologic condition or disorder. Those in need of treatment include those already with the disorder as well as those prone to have the disorder or those in whom the disorder is to be prevented. A subject or mammal is successfully “treated” for an infection if, after receiving a therapeutic amount of an antibody according to the methods of the present invention, the patient shows observable and/or measurable reduction in or absence of one or more of the following: reduction in the number of infected cells or absence of the infected cells; reduction in the percent of total cells that are infected; and/or relief to some extent, one or more of the symptoms associated with the specific infection; reduced morbidity and mortality, and improvement in quality of life issues. The above parameters for assessing successful treatment and improvement in the disease are readily measurable by routine procedures familiar to a physician.
The term “therapeutically effective amount” refers to an amount of an antibody or a drug effective to “treat” a disease or disorder in a subject or mammal. See preceding definition of “treating.”
“Chronic” administration refers to administration of the agent(s) in a continuous mode as opposed to an acute mode, so as to maintain the initial therapeutic effect (activity) for an extended period of time. “Intermittent” administration is treatment that is not consecutively done without interruption, but rather is cyclic in nature.
Administration “in combination with” one or more further therapeutic agents includes simultaneous (concurrent) and consecutive administration in any order.
“Carriers” as used herein include pharmaceutically acceptable carriers, excipients, or stabilizers that are nontoxic to the cell or mammal being exposed thereto at the dosages and concentrations employed. Often the physiologically acceptable carrier is an aqueous pH buffered solution. Examples of physiologically acceptable carriers include buffers such as phosphate, citrate, and other organic acids; antioxidants including ascorbic acid; low molecular weight (less than about 10 residues) polypeptide; proteins, such as serum albumin, gelatin, or immunoglobulins; hydrophilic polymers such as polyvinylpyrrolidone; amino acids such as glycine, glutamine, asparagine, arginine or lysine; monosaccharides, disaccharides, and other carbohydrates including glucose, mannose, or dextrins; chelating agents such as EDTA; sugar alcohols such as mannitol or sorbitol; salt-forming counterions such as sodium; and/or nonionic surfactants such as TWEENTM polyethylene glycol (PEG), and PLURONICSTM.
“Label” as used herein refers to a detectable compound or composition that is conjugated directly or indirectly to the antibody so as to generate a “labeled” antibody. The label may be detectable by itself (e.g., radioisotope labels or fluorescent labels) or, in the case of an enzymatic label, may catalyze chemical alteration of a substrate compound or composition that is detectable.
The term “epitope tagged” as used herein refers to a chimeric polypeptide comprising a polypeptide fused to a “tag polypeptide.” The tag polypeptide has enough residues to provide an epitope against which an antibody can be made, yet is short enough such that it does not interfere with activity of the polypeptide to which it is fused. The tag polypeptide is also preferably fairly unique so that the antibody does not substantially cross-react with other epitopes. Suitable tag polypeptides generally have at least six amino acid residues and usually between about 8 and 50 amino acid residues (preferably, between about 10 and 20 amino acid residues).
A “small molecule” is defined herein to have a molecular weight below about 500 Daltons.
The terms “nucleic acid” and “polynucleotide” are used interchangeably herein to refer to single- or double-stranded RNA, DNA, or mixed polymers. Polynucleotides may include genomic sequences, extra-genomic and plasmid sequences, and smaller engineered gene segments that express, or may be adapted to express polypeptides.
An “isolated nucleic acid” is a nucleic acid that is substantially separated from other genome DNA sequences as well as proteins or complexes such as ribosomes and polymerases, which naturally accompany a native sequence. The term embraces a nucleic acid sequence that has been removed from its naturally occurring environment, and includes recombinant or cloned DNA isolates and chemically synthesized analogues or analogues biologically synthesized by heterologous systems. A substantially pure nucleic acid includes isolated forms of the nucleic acid. Of course, this refers to the nucleic acid as originally isolated and does not exclude genes or sequences later added to the isolated nucleic acid by the hand of man.
The term “polypeptide” is used in its conventional meaning, i.e., as a sequence of amino acids. The polypeptides are not limited to a specific length of the product. Peptides, oligopeptides, and proteins are included within the definition of polypeptide, and such terms may be used interchangeably herein unless specifically indicated otherwise. This term also does not refer to or exclude post-expression modifications of the polypeptide, for example, glycosylations, acetylations, phosphorylations and the like, as well as other modifications known in the art, both naturally occurring and non-naturally occurring. A polypeptide may be an entire protein, or a subsequence thereof. Particular polypeptides of interest in the context of this invention are amino acid subsequences comprising CDRs and being capable of binding an antigen or Influenza A-infected cell.
An “isolated polypeptide” is one that has been identified and separated and/or recovered from a component of its natural environment. In preferred embodiments, the isolated polypeptide will be purified (1) to greater than 95% by weight of polypeptide as determined by the Lowry method, and most preferably more than 99% by weight, (2) to a degree sufficient to obtain at least 15 residues of N-terminal or internal amino acid sequence by use of a spinning cup sequenator, or (3) to homogeneity by SDS-PAGE under reducing or non-reducing conditions using Coomassie blue or, preferably, silver stain. Isolated polypeptide includes the polypeptide in situ within recombinant cells since at least one component of the polypeptide's natural environment will not be present. Ordinarily, however, isolated polypeptide will be prepared by at least one purification step.
A “native sequence” polynucleotide is one that has the same nucleotide sequence as a polynucleotide derived from nature. A “native sequence” polypeptide is one that has the same amino acid sequence as a polypeptide (e.g., antibody) derived from nature (e.g., from any species). Such native sequence polynucleotides and polypeptides can be isolated from nature or can be produced by recombinant or synthetic means.
A polynucleotide “variant,” as the term is used herein, is a polynucleotide that typically differs from a polynucleotide specifically disclosed herein in one or more substitutions, deletions, additions and/or insertions. Such variants may be naturally occurring or may be synthetically generated, for example, by modifying one or more of the polynucleotide sequences of the invention and evaluating one or more biological activities of the encoded polypeptide as described herein and/or using any of a number of techniques well known in the art.
A polypeptide “variant,” as the term is used herein, is a polypeptide that typically differs from a polypeptide specifically disclosed herein in one or more substitutions, deletions, additions and/or insertions. Such variants may be naturally occurring or may be synthetically generated, for example, by modifying one or more of the above polypeptide sequences of the invention and evaluating one or more biological activities of the polypeptide as described herein and/or using any of a number of techniques well known in the art.
Modifications may be made in the structure of the polynucleotides and polypeptides of the present invention and still obtain a functional molecule that encodes a variant or derivative polypeptide with desirable characteristics. When it is desired to alter the amino acid sequence of a polypeptide to create an equivalent, or even an improved, variant or portion of a polypeptide of the invention, one skilled in the art will typically change one or more of the codons of the encoding DNA sequence.
For example, certain amino acids may be substituted for other amino acids in a protein structure without appreciable loss of its ability to bind other polypeptides (e.g., antigens) or cells. Since it is the binding capacity and nature of a protein that defines that protein's biological functional activity, certain amino acid sequence substitutions can be made in a protein sequence, and, of course, its underlying DNA coding sequence, and nevertheless obtain a protein with like properties. It is thus contemplated that various changes may be made in the peptide sequences of the disclosed compositions, or corresponding DNA sequences that encode said peptides without appreciable loss of their biological utility or activity.
In many instances, a polypeptide variant will contain one or more conservative substitutions. A “conservative substitution” is one in which an amino acid is substituted for another amino acid that has similar properties, such that one skilled in the art of peptide chemistry would expect the secondary structure and hydropathic nature of the polypeptide to be substantially unchanged.
In making such changes, the hydropathic index of amino acids may be considered. The importance of the hydropathic amino acid index in conferring interactive biologic function on a protein is generally understood in the art (Kyte and Doolittle, 1982). It is accepted that the relative hydropathic character of the amino acid contributes to the secondary structure of the resultant protein, which in turn defines the interaction of the protein with other molecules, for example, enzymes, substrates, receptors, DNA, antibodies, antigens, and the like. Each amino acid has been assigned a hydropathic index on the basis of its hydrophobicity and charge characteristics (Kyte and Doolittle, 1982). These values are: isoleucine (+4.5); valine (+4.2); leucine (+3.8); phenylalanine (+2.8); cysteine/cystine (+2.5); methionine (+1.9); alanine (+1.8); glycine (−0.4); threonine (−0.7); serine (−0.8); tryptophan (−0.9); tyrosine (−1.3); proline (−1.6); histidine (−3.2); glutamate (−3.5); glutamine (−3.5); aspartate (−3.5); asparagine (−3.5); lysine (−3.9); and arginine (−4.5).
It is known in the art that certain amino acids may be substituted by other amino acids having a similar hydropathic index or score and still result in a protein with similar biological activity, i.e. still obtain a biological functionally equivalent protein. In making such changes, the substitution of amino acids whose hydropathic indices are within ±2 is preferred, those within ±1 are particularly preferred, and those within ±0.5 are even more particularly preferred. It is also understood in the art that the substitution of like amino acids can be made effectively on the basis of hydrophilicity. U.S. Pat. No. 4,554,101 states that the greatest local average hydrophilicity of a protein, as governed by the hydrophilicity of its adjacent amino acids, correlates with a biological property of the protein.
As detailed in U.S. Pat. No. 4,554,101, the following hydrophilicity values have been assigned to amino acid residues: arginine (+3.0); lysine (+3.0); aspartate (+3.0±1); glutamate (+3.0±1); serine (+0.3); asparagine (+0.2); glutamine (+0.2); glycine (0); threonine (−0.4); proline (−0.5±1); alanine (−0.5); histidine (−0.5); cysteine (−1.0); methionine (−1.3); valine (−1.5); leucine (−1.8); isoleucine (−1.8); tyrosine (−2.3); phenylalanine (−2.5); tryptophan (−3.4). It is understood that an amino acid can be substituted for another having a similar hydrophilicity value and still obtain a biologically equivalent, and in particular, an immunologically equivalent protein. In such changes, the substitution of amino acids whose hydrophilicity values are within ±2 is preferred, those within ±1 are particularly preferred, and those within ±0.5 are even more particularly preferred.
As outlined above, amino acid substitutions are generally therefore based on the relative similarity of the amino acid side-chain substituents, for example, their hydrophobicity, hydrophilicity, charge, size, and the like. Exemplary substitutions that take various of the foregoing characteristics into consideration are well known to those of skill in the art and include: arginine and lysine; glutamate and aspartate; serine and threonine; glutamine and asparagine; and valine, leucine and isoleucine.
Amino acid substitutions may further be made on the basis of similarity in polarity, charge, solubility, hydrophobicity, hydrophilicity and/or the amphipathic nature of the residues. For example, negatively charged amino acids include aspartic acid and glutamic acid; positively charged amino acids include lysine and arginine; and amino acids with uncharged polar head groups having similar hydrophilicity values include leucine, isoleucine and valine; glycine and alanine; asparagine and glutamine; and serine, threonine, phenylalanine and tyrosine. Other groups of amino acids that may represent conservative changes include: (1) ala, pro, gly, glu, asp, gln, asn, ser, thr; (2) cys, ser, tyr, thr; (3) val, ile, leu, met, ala, phe; (4) lys, arg, his; and (5) phe, tyr, trp, his. A variant may also, or alternatively, contain nonconservative changes. In a preferred embodiment, variant polypeptides differ from a native sequence by substitution, deletion or addition of five amino acids or fewer. Variants may also (or alternatively) be modified by, for example, the deletion or addition of amino acids that have minimal influence on the immunogenicity, secondary structure and hydropathic nature of the polypeptide.
Polypeptides may comprise a signal (or leader) sequence at the N-terminal end of the protein, which co-translationally or post-translationally directs transfer of the protein. The polypeptide may also be conjugated to a linker or other sequence for ease of synthesis, purification or identification of the polypeptide (e.g., poly-His), or to enhance binding of the polypeptide to a solid support. For example, a polypeptide may be conjugated to an immunoglobulin Fc region.
When comparing polynucleotide and polypeptide sequences, two sequences are said to be “identical” if the sequence of nucleotides or amino acids in the two sequences is the same when aligned for maximum correspondence, as described below. Comparisons between two sequences are typically performed by comparing the sequences over a comparison window to identify and compare local regions of sequence similarity. A “comparison window” as used herein, refers to a segment of at least about 20 contiguous positions, usually 30 to about 75, 40 to about 50, in which a sequence may be compared to a reference sequence of the same number of contiguous positions after the two sequences are optimally aligned.
Optimal alignment of sequences for comparison may be conducted using the Megalign program in the Lasergene suite of bioinformatics software (DNASTAR, Inc., Madison, Wis.), using default parameters. This program embodies several alignment schemes described in the following references: Dayhoff, M. O. (1978) A model of evolutionary change in proteins—Matrices for detecting distant relationships. In Dayhoff, M. O. (ed.) Atlas of Protein Sequence and Structure, National Biomedical Research Foundation, Washington D.C. Vol. 5, Suppl. 3, pp. 345-358; Hein J. (1990) Unified Approach to Alignment and Phylogenes pp. 626-645 Methods in Enzymology vol. 183, Academic Press, Inc., San Diego, Calif.; Higgins, D. G. and Sharp, P. M. (1989) CABIOS 5:151-153; Myers, E. W. and Muller W. (1988) CABIOS 4:11-17; Robinson, E. D. (1971) Comb. Theor 11:105; Santou, N. Nes, M. (1987) Mol. Biol. Evol. 4:406-425; Sneath, P. H. A. and Sokal, R. R. (1973) Numerical Taxonomy—the Principles and Practice of Numerical Taxonomy, Freeman Press, San Francisco, Calif.; Wilbur, W. J. and Lipman, D. J. (1983) Proc. Natl. Acad., Sci. USA 80:726-730.
Alternatively, optimal alignment of sequences for comparison may be conducted by the local identity algorithm of Smith and Waterman (1981) Add. APL. Math 2:482, by the identity alignment algorithm of Needleman and Wunsch (1970) J. Mol. Biol. 48:443, by the search for similarity methods of Pearson and Lipman (1988) Proc. Natl. Acad. Sci. USA 85: 2444, by computerized implementations of these algorithms (GAP, BESTFIT, BLAST, FASTA, and TFASTA in the Wisconsin Genetics Software Package, Genetics Computer Group (GCG), 575 Science Dr., Madison, Wis.), or by inspection.
One preferred example of algorithms that are suitable for determining percent sequence identity and sequence similarity are the BLAST and BLAST 2.0 algorithms, which are described in Altschul et al. (1977) Nucl. Acids Res. 25:3389-3402 and Altschul et al. (1990) J. Mol. Biol. 215:403-410, respectively. BLAST and BLAST 2.0 can be used, for example with the parameters described herein, to determine percent sequence identity for the polynucleotides and polypeptides of the invention. Software for performing BLAST analyses is publicly available through the National Center for Biotechnology Information.
In one illustrative example, cumulative scores can be calculated using, for nucleotide sequences, the parameters M (reward score for a pair of matching residues; always >0) and N (penalty score for mismatching residues; always <0). Extension of the word hits in each direction are halted when: the cumulative alignment score falls off by the quantity X from its maximum achieved value; the cumulative score goes to zero or below, due to the accumulation of one or more negative-scoring residue alignments; or the end of either sequence is reached. The BLAST algorithm parameters W, T and X determine the sensitivity and speed of the alignment. The BLASTN program (for nucleotide sequences) uses as defaults a wordlength (W) of 11, and expectation (E) of 10, and the BLOSUM62 scoring matrix (see Henikoff and Henikoff (1989) Proc. Natl. Acad. Sci. USA 89:10915) alignments, (B) of 50, expectation (E) of 10, M=5, N=−4 and a comparison of both strands.
For amino acid sequences, a scoring matrix can be used to calculate the cumulative score. Extension of the word hits in each direction are halted when: the cumulative alignment score falls off by the quantity X from its maximum achieved value; the cumulative score goes to zero or below, due to the accumulation of one or more negative-scoring residue alignments; or the end of either sequence is reached. The BLAST algorithm parameters W, T and X determine the sensitivity and speed of the alignment.
In one approach, the “percentage of sequence identity” is determined by comparing two optimally aligned sequences over a window of comparison of at least 20 positions, wherein the portion of the polynucleotide or polypeptide sequence in the comparison window may comprise additions or deletions (i.e., gaps) of 20 percent or less, usually 5 to 15 percent, or 10 to 12 percent, as compared to the reference sequences (which does not comprise additions or deletions) for optimal alignment of the two sequences. The percentage is calculated by determining the number of positions at which the identical nucleic acid bases or amino acid residues occur in both sequences to yield the number of matched positions, dividing the number of matched positions by the total number of positions in the reference sequence (i.e., the window size) and multiplying the results by 100 to yield the percentage of sequence identity.
“Homology” refers to the percentage of residues in the polynucleotide or polypeptide sequence variant that are identical to the non-variant sequence after aligning the sequences and introducing gaps, if necessary, to achieve the maximum percent homology. In particular embodiments, polynucleotide and polypeptide variants have at least 70%, at least 75%, at least 80%, at least 90%, at least 95%, at least 98%, or at least 99% polynucleotide or polypeptide homology with a polynucleotide or polypeptide described herein.
“Vector” includes shuttle and expression vectors. Typically, the plasmid construct will also include an origin of replication (e.g., the ColE1 origin of replication) and a selectable marker (e.g., ampicillin or tetracycline resistance), for replication and selection, respectively, of the plasmids in bacteria. An “expression vector” refers to a vector that contains the necessary control sequences or regulatory elements for expression of the antibodies including antibody fragment of the invention, in bacterial or eukaryotic cells. Suitable vectors are disclosed below.
As used in this specification and the appended claims, the singular forms “a,” “an” and “the” include plural references unless the content clearly dictates otherwise.
Preferred methods for determining mAb specificity and affinity by competitive inhibition can be found in Harlow, et al., Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., 1988), Colligan et al., eds., Current Protocols in Immunology, Greene Publishing Assoc. and Wiley Interscience, N.Y., (1992, 1993), and Muller, Meth. Enzymol. 92:589-601 (1983), which references are entirely incorporated herein by reference.
The techniques to raise antibodies of the present invention to small peptide sequences that recognize and bind to those sequences in the free or conjugated form or when presented as a native sequence in the context of a large protein are well known in the art. Such antibodies include murine, murine-human and human-human antibodies produced by hybridoma or recombinant techniques known in the art.
Screening Peptide Immunogens for Specific Binding to 8i10, or 23K12.
The present invention relates to peptide immunogens that bind HuM2e antibodies. In one embodiment, the antibody is an antibody designated herein as 8i10, 21B15, or 23K12. These antibodies are known to display preferential or specific binding to influenza A infected cells as compared to uninfected control cells of the same cell type.
In particular embodiments, the HuM2e antibodies bind to epitopes within M2e that are only present in the native conformation, i.e., as expressed in cells. In particular embodiments, these antibodies fail to specifically bind to an isolated M2e polypeptide, e.g., the 23 amino acid residue M2e fragment. It is understood that these antibodies recognize non-linear (i.e. conformational) epitope(s) of the M2 peptide. M2 ectodomain (M2e) includes or consists of the amino acid sequence SLLTEVETPIRNEWGCRCNDSSD (SEQ ID NO: 326) and variants thereof (The Center for Disease Control (CDC) influenza A database at www.flu.lanl.gov/).
The specific conformational epitopes within the M2 protein, and particularly within M2e, were identified as peptide immunogens which can be used as vaccines to prevent the development of influenza infection within a subject.
The peptide immunogen sequences were identified by discontinuous epitope mapping using CLIPSTM (Chemically Linked Immunogenic Peptides on Scaffolds; PepScan) technology, which has been developed to improve the biological function of synthetic peptides. CLIPS uses small, chemical “scaffolds” onto which one or more peptides can be attached. In vaccine development, these are ideal for mapping conformational (or ‘discontinuous’) epitopes because they closely resemble the native structure of proteins.
Binding activity of anti-M2 antibodies to mutant M2 peptides was analyzed with an ELISA assay using different M2 peptides. Human anti-M2 antibody nos. Z3G1, 8I10 and 23K12 were used in the study. Peptides comprising M2e sequences were screened for the ability to specifically bind HuM2e antibodies 8I10 and 23K12 and distinguished from the ability to bind the anti-M2 human monoclonal antibody Z3G1 (ATCC Deposit No. PTA-5967) which has a broad M2 binding spectrum. 8I10 and 23K12 have been characterized for the ability to bind M2 and M2e under conditions resembling native conformation. The assays were performed at both 0.01 μg/mL and 0.001 μg/mL concentrations of peptides. At 0.01 μg/mL, signal levels of 1000 or greater were selected and at 0.001 μg/mL, signal levels of 300 or greater were selected as significant. Most peptides that bind strongly to 8I10 and 23K12 also bind Z3G1.







TABLE 2







Peptides that specifically bind for 23K21 and


8I10 (A numeral “1” in the peptide sequence


indicates a differentially protected cystein


allowing for selective CLIP attachment at


certain residues)









SEQ ID




NO:
Peptides that bind to 23K21 and 8I10
Signal












116
CLTEVETPIRNEWGSRCSLLTEVETPIRNEWGC
1922


 


117
CLLTEVETPIRNEWGSCSLLTEVETPIRC
1865


 


118
CSLLTEVETPIRNECSLLTEVETC
1829


 


119
CLTEVETPIRNEWGSRCSLLTEVETPIRNC
1825


 


120
CSLLTEVETPIRNCSLLTEVETPIRC
1781


 


121
CSLLTEVETPIRCSLLTEVETPIRC
1767


 


122
CSLLTEVETPIRNEWCSLLTEVETC
1752


 


123
CSLLTEVETPIRNECLTEVETPIRNEWGSRC
1702


 


124
CSLLTEVETPCSLLTEVETPIRC
1693


 


125
CTEVETPIRNEWGSRSCSLLTEVETPIRNEWGC
1659


 


126
CETPIRNEWGSRSNDSCSLLTEVETPIRNC
1650


 


127
CSLLTEVETCSLLTEVETC
1647


 


128
CSLLTEVETCSLLTEVETPIC
1638


 


129
CSLLTEVETPIRNCSLLTEVETPIRNC
1602


 


130
CSLLTEVETPIRNECSLLTEVETPIRC
1588


 


131
CSLLTEVETPIRCSLLTEVETPIRNC
1549


 


132
CVETPIRNEWGSRSNDCSLLTEVETPIC
1548


 


133
CSLLTEVETPIRNECSLLTEVETPIRNC
1534


 


134
CSLLTEVETCSLLTEVETPIRNC
1480


 


135
CSLLTEVETPIRNCSLLTEVETPIRNEWC
1476


 


136
CLTEVETPIRNEWGSRCSLLTEVETC
1470


 


137
CSLLTEVETPCSLLTEVETPIC
1458


 


138
CSLLTEVETPIRNEWGCLLTEVETPIRNEWGSC
1456


 


139
CSLLTEVETPIRNCSLLTEVETPIC
1449


 


140
CSLLTEVETPIRNEWGCSLLTEVETPIRNEWGC
1434


 


141
CSLLTEVETPIRNEWCSLLTEVETPIRNEWGC
1427


 


142
CSLLTEVETCSLLTEVETPC
1424


 


143
CSLLTEVETPIRNEWGCSLLTEVETPIRNC
1418


 


144
CSLLTEVETPIRNCSLLTEVETPC
1405


 


145
CSLLTEVETPIRNECSLLTEVETPIC
1395


 


146
CSLLTEVETPIRNECSLLTEVETPIRNEWC
1394


 


147
CSLLTEVETPIRCSLLTEVETC
1388


 


148
CSLLTEVETPIRNCSLLTEVETPIRNEWGC
1356


 


149
CTEVETPIRNEWGSRSCLTEVETPIRNEWGSRC
1348


 


150
CSLLTEVETCSLLTEVETPIRNEWC
1345


 


151
CSLLTEVETPIRCSLLTEVETPIC
1340


 


152
CSLLTEVETPIRNECSLLTEVETPIRNEWGC
1331


 


153
CSLLTEVETPCSLLTEVETPIRNC
1320


 


154
CETPIRNEWGSRSNDSCSLLTEVETPIRNEC
1313


 


155
CSLLTEVETPIRNEWCSLLTEVETPIRC
1293


 


156
CSLLTEVETPCSLLTEVETPC
1286


 


157
CSLLTEVETPIRNEWGCSLLTEVETPIRC
1278


 


158
CSLLTEVETPCSLLTEVETPIRNEWC
1269


 


159
CSLLTEVETPIRNEWGCVETPIRNEWGSRSNDC
1241


 


160
CSLLTEVETPIRCLLTEVETPIRNEWGSC
1211


 


161
CPIRNEWGSRSNDSSDCSLLTEVETC
1206


 


162
CLTEVETPIRNEWGSRCSLLTEVETPIRC
1203


 


163
CSLLTEVETCSLLTEVETPIRC
1200


 


164
CSLLTEVETPIRNECSLLTEVETPC
1194


 


165
CSLLTEVETPIRCSLLTEVETPIRNEC
1194


 


166
CSLLTEVETPIRNCSLLTEVETC
1192


 


167
CSLLTEVETPIRCSLLTEVETPIRNEWGC
1188


 


168
CVETPIRNEWGSRSNDCSLLTEVETPIRC
1186


 


169
CTPIRNEWGSRSNDSSCSLLTEVETPIRC
1180


 


170
CSLLTEVETPIRCLTEVETPIRNEWGSRC
1175


 


171
CSLLTEVETCSLLTEVETPIRNEC
1172


 


172
CEVETPIRNEWGSRSNCSLLTEVETPIRC
1168


 


173
CSLLTEVETPIRCSLLTEVETPIRNEWC
1167


 


174
CSLLTEVETPIRNEWCSLLTEVETPIRNEWC
1165


 


175
CSLLTEVETPIRNEWGCLTEVETPIRNEWGSRC
1148


 


176
CPIRNEWGSRSNDSSDCSLLTEVETPIRC
1146


 


177
CSLLTEVETPIRNEWCSLLTEVETPIC
1144


 


178
CLLTEVETPIRNEWGSCSLLTEVETPIRNC
1141


 


179
CSLLTEVETCLLTEVETPIRNEWGSC
1141


 


180
CSLLTEVETPIRNEWCSLLTEVETPIRNC
1138


 


181
CSLLTEVETPIRCSLLTEVETPC
1115


 


182
CSLLTEVETPCLLTEVETPIRNEWGSC
1111


 


183
CSLLTEVETPIRNCSLLTEVETPIRNEC
1110


 


184
CTEVETPIRNEWGSRSCSLLTEVETPIRC
1104


 


185
CTPIRNEWGSRSNDSSCSLLTEVETPIRNC
1103


 


186
CSLLTEVETPCSLLTEVETPIRNEWGC
1103


 


187
CSLLTEVETPICSLLTEVETC
1089


 


188
CLTEVETPIRNEWGSRCSLLTEVETPIRNEWC
1079


 


189
CSLLTEVETPIRNEWCSLLTEVETPC
1074


 


190
CETPIRNEWGSRSNDSCSLLTEVETPIRC
1069


 


191
CLTEVETPIRNEWGSRCSLLTEVETPIC
1040


 


192
CSLLTEVETPIRNCLLTEVETPIRNEWGSC
1036


 


193
CSLLTEVETPCSLLTEVETPIRNEC
1032


 


194
CVETPIRNEWGSRSNDCSLLTEVETPIRNC
1028


 


195
CSLLTEVETPIRNCLTEVETPIRNEWGSRC
1027


 


196
CEVETPIRNEWGSRSNCSLLTEVETPIRNC
1026


 


197
CSLLTEVETPIRNEWGCSLLTEVETPIC
1023


 


198
CSLLTEVETPCSLLTEVETC
1018


 


199
CSLLTEVETPIRCTEVETPIRNEWGSRSC
1017


 


200
CSLLTEVETPIRNEWGCSLLTEVETC
1014


 


201
CEVETPIRNEWGSRSNCSLLTEVETC
1007


 


202
CPIRNEWGSRSNDSSDCSLLTEVETPIRNC
1004


 


203
CSLLTEVETPICSLLTEVETPIRC
1001


 


204
CTEVETPIRNEWGSRSCSLLTEVETPIRNC
1000


 


205
CLLTEVETPIRNEWGSCSLLTEVETPIRNEWGC
1000


 


206
CSLLTEVETPIRNEWGCSLLTEVETPIRNEC
994


 


207
CSLLTEVETPICSLLTEVETPC
986


 


208
CSLLTEVETPICSLLTEVETPIRNC
981


 


209
CSLLTEVETPIRCEVETPIRNEWGSRSNC
980


 


210
CSLLTEVETPIRNCTEVETPIRNEWGSRSC
977


 


211
CSLLTEVETPICSLLTEVETPIRNEWC
975


 


212
CSLLTEVETPIRNEWGCSLLTEVETPIRNEWC
972


 


213
CETPIRNEWGSRSNDSCSLLTEVETC
971


 


214
CSLLTEVETPIRNEWCLLTEVETPIRNEWGSC
961


 


215
CSLLTEVETCLTEVETPIRNEWGSRC
958


 


216
CTEVETPIRNEWGSRSCSLLTEVETPIC
957


 


217
CSLLTEVETPIRNEWGCSLLTEVETPC
955


 


218
CLLTEVETPIRNEWGSCSLLTEVETPIRNEWC
922


 


219
CEVETPIRNEWGSRSNCSLLTEVETPIC
912


 


220
CSLLTEVETPIRNECSLLTEVETPIRNEC
910


 


221
CSLLTEVETPCLTEVETPIRNEWGSRC
907


 


222
CSLLTEVETPCTEVETPIRNEWGSRSC
907


 


223
CVETPIRNEWGSRSNDCSLLTEVETC
905


 


224
CSLLTEVETPICLLTEVETPIRNEWGSC
901


 


225
CTEVETPIRNEWGSRSCSLLTEVETPIRNEWC
900


 


226
CTEVETPIRNEWGSRSCSLLTEVETC
891


 


227
CSLLTEVETPIRNECLLTEVETPIRNEWGSC
870


 


228
CLTEVETPIRNEWGSRCSLLTEVETPC
867


 


229
CEVETPIRNEWGSRSNCSLLTEVETPC
862


 


230
CTPIRNEWGSRSNDSSCSLLTEVETC
861


 


231
CTPIRNEWGSRSNDSSCSLLTEVETPIC
854


 


232
CETPIRNEWGSRSNDSCSLLTEVETPC
842


 


233
CEVETPIRNEWGSRSNCSLLTEVETPIRNEWGC
838


 


234
CVETPIRNEWGSRSNDCSLLTEVETPIRNEWC
837


 


235
CSLLTEVETPICLTEVETPIRNEWGSRC
835


 


236
CSLLTEVETPICSLLTEVETPIRNEWGC
826


 


237
CETPIRNEWGSRSNDSCSLLTEVETPIC
825


 


238
CEVETPIRNEWGSRSNCSLLTEVETPIRNEWC
825


 


239
CSLLTEVETCEVETPIRNEWGSRSNC
821


 


240
CLTEVETPIRNEWGSRCSLLTEVETPIRNEC
817


 


241
CSLLTEVETPIRNEWCSLLTEVETPIRNEC
815


 


242
CVETPIRNEWGSRSNDCSLLTEVETPC
814


 


243
CTPIRNEWGSRSNDSSCSLLTEVETPIRNEWGC
812


 


244
CLLTEVETPIRNEWGSCSLLTEVETPIRNEC
808


 


245
CSLLTEVETPICSLLTEVETPIC
797


 


246
CTEVETPIRNEWGSRSCSLLTEVETPC
796


 


247
CTPIRNEWGSRSNDSSCSLLTEVETPC
794


 


248
CPIRNEWGSRSNDSSDCSLLTEVETPIRNEC
787


 


249
CPIRNEWGSRSNDSSDCSLLTEVETPIRNEWC
786


 


250
CSLLTEVETCTEVETPIRNEWGSRSC
786


 


251
CETPIRNEWGSRSNDSCSLLTEVETPIRNEWC
784


 


252
CLLTEVETPIRNEWGSCSLLTEVETPIC
762


 


253
CSLLTEVETPIRNCEVETPIRNEWGSRSNC
761


 


254
CLLTEVETPIRNEWGSCSLLTEVETC
752


 


255
CETPIRNEWGSRSNDSCSLLTEVETPIRNEWGC
749


 


256
CPIRNEWGSRSNDSSDCSLLTEVETPIRNEWGC
742


 


257
CVETPIRNEWGSRSNDCSLLTEVETPIRNEWGC
737


 


258
CSLLTEVETPIRNECTEVETPIRNEWGSRSC
736


 


259
CPIRNEWGSRSNDSSDCSLLTEVETPC
733


 


260
CSLLTEVETPICEVETPIRNEWGSRSNC
731


 


261
CTEVETPIRNEWGSRSCSLLTEVETPIRNEC
721


 


262
CSLLTEVETPIRNCVETPIRNEWGSRSNDC
721


 


263
CSLLTEVETPIRNEWGCTEVETPIRNEWGSRSC
715


 


264
CSLLTEVETCVETPIRNEWGSRSNDC
707


 


265
CSLLTEVETPCVETPIRNEWGSRSNDC
704


 


266
CTPIRNEWGSRSNDSSCSLLTEVETPIRNEC
699


 


267
CPIRNEWGSRSNDSSDCSLLTEVETPIC
691


 


268
SLLTEVETPIRNECGCRCNDSSD
682


 


269
CVETPIRNEWGSRSNDCSLLTEVETPIRNEC
679


 


270
CLLTEVETPIRNEWGSCSLLTEVETPC
678


 


271
CSLLTEVETPIRCVETPIRNEWGSRSNDC
678


 


272
CTPIRNEWGSRSNDSSCSLLTEVETPIRNEWC
677


 


273
CEVETPIRNEWGSRSNCSLLTEVETPIRNEC
669


 


274
CSLLTEVETPCEVETPIRNEWGSRSNC
650


 


275
CSLLTEVETPIRNEWGCEVETPIRNEWGSRSNC
607


 


276
CSLLTEVETPIRNECEVETPIRNEWGSRSNC
591


 


277
CSLLTEVETPIRNEWCTEVETPIRNEWGSRSC
575


 


278
CSLLTEVETPIRNEWCLTEVETPIRNEWGSRC
561


 


279
CSLLTEVETPIRNECVETPIRNEWGSRSNDC
538


 


280
CSLLTEVETPICTEVETPIRNEWGSRSC
526


 


281
CSLLTEVETCSLLTEVETPIRNEWGC
328


 


282
CSCLCEVGMSCLCEC
2705


 


283
CSLLTEVGSLLTEV
2494


 


284
ASLLTEVGSLLTCV
2443


 


285
MSLLTEVGMSLLTCV
2389


 


286
CSLLTEVGMSLLTCV
2311


 


287
MSLLTEVGMSLLTEV
2267


 


288
CSCLCEVGMSLLTEV
2154


 


289
MSLCTEVGMSLCTEV
2153


 


290
CSLLTEVGSLLTCV
2142


 


291
MSLLTEVGMCLCTCV
2110


 


292
CSLLTEVGSLLTEC
2094


 


293
CSLLTEVGMSLLTEV
2039


 


294
MSCLCECGMSLLTEV
2039


 


295
MSLCTEVGMSCLTEV
2026


 


296
MSLLTEVGMCLLTEV
2023


 


297
CSLLTEVGMCLCTCV
1972


 


298
SLCTEVGSCLCEC
1967


 


299
SLLTEVGCLCTCV
1963


 


300
SLLTEVETKIRNEWGCRCNDSSD
1960


 


301
SLLCEVGCSLLTEC
1959


 


302
ASLLTEVGSCLTEV
1944


 


303
CSLLTEVGMCLLTEV
1941


 


304
MSLCTEVGMSLLCEV
1935


 


305
CSCLCEVGMSLLTEC
1925


 


306
CSLLTECGMSLLTCV
1918


 


307
SLCTEVGCLCTCV
1907


 


308
MSLCTEVGMCLCTCV
1902


 


309
CSLLTEVGMSLLCEV
1891


 


310
MSCLCECGMSLLTEC
1887


 


311
SLLTEVGCLLTEV
1859


 


312
TLLTEVETPIRNEWGCRCNDSSD
1855


 


313
SLLTEVETPIRNEWGCRCNDSGD
1852


 


314
SLLTEVGSLLCEV
1834


 


315
MCLCTCVGCSLLTEC
1823


 


316
MSLLTEVGCSLLTEV
1809


 


317
MSLLTEVGMSLCTEV
1804


 


318
SLLTEVETPIRNEWGCRCKDSSD
1802


 


319
SLLTECGSLLTCV
1800


 


320
SLLTEVGSLLTEV
1794


 


321
ALLTEVETPIRNEWGCRCNDSSD
1794


 


322
SKLTEVETPIRNEWGCRCNDSSD
1784


 


323
SLCTEVGSLLTCV
1781


 


324
SLCTEVGSLLTEV
1772


 


325
SLLTECGSLLTEV
1770


 


326
SLLTEVETPIRNEWGCRCNDSSD
1749


 


327
SLMTEVETPIRNEWGCRCNDSSD
1729


 


328
SLETEVETPIRNEWGCRCNDSSD
1720


 


329
SLLTEVGSCLCEC
1708


 


330
SLLTEVETPIRNEWGCRCNDYSD
1694


 


331
MSLLTECGMSLLTCV
1690


 


332
SALTEVETPIRNEWGCRCNDSSD
1689


 


333
SLLTCVGSLLTEC
1683


 


334
CSLLTEVGMSLCTEV
1682


 


335
CSCLCEVGCSLLTEC
1677


 


336
SLLCEVGSLLTEV
1674


 


337
SLLTEVETPIRNEWGCRCNYSSD
1666


 


338
SLLTEVETPIRNEWGCRCNDGSD
1666


 


339
MSLLTECGMSLLTEV
1658


 


340
MSCLCECGCSLLTEC
1652


 


341
CSLLTEVCSLLTEC
1644


 


342
SLLTEVETPIRNEWGCRCNSSSD
1637


 


343
MSLLTEVGMSLLTEC
1630


 


344
SMLTEVETPIRNEWGCRCNDSSD
1622


 


345
CSLLTEVGSCLTEV
1616


 


346
SLLMEVETPIRNEWGCRCNDSSD
1616


 


347
SLLTEVETPIRNEWICRCNDSSD
1599


 


348
SLLTECGCSLLTEV
1597


 


349
SLATEVETPIRNEWGCRCNDSSD
1583


 


350
SLLTEVETPIRNEWGCRCNDSND
1577


 


351
SLLCEVGSLLTEC
1564


 


352
SLQTEVETPIRNEWGCRCNDSSD
1561


 


353
SLLTEVGCSLLTEC
1556


 


354
SLLTEVGCSLLTEV
1555


 


355
SCLCECGCSLLTEV
1555


 


356
SLLTEVETPIRNEWGCRCNDSDD
1553


 


357
SLLTEVETPIPNEWGCRCNDSSD
1548


 


358
SFLTEVETPIRNEWGCRCNDSSD
1546


 


359
MSLLTEVGMSCLTEV
1535


 


360
CSLLTECGCSLLTEV
1534


 


361
SCLCECGCSLLTEC
1520


 


362
CSLLTECGMCLLTEV
1517


 


363
MSLLTECGCSLLTEV
1500


 


364
CSLLTEVGCSLLTEV
1499


 


365
CSLLTEVCMSLLTEC
1498


 


366
SLLTEVETPIRNEWWCRCNDSSD
1498


 


367
SLCTEVGSLLTEC
1495


 


368
SLLTEVETPIRNEWG
1486


 


369
SLLTEAETPIRNEWGCRCNDSSD
1481


 


370
SLLTEVETPIRNEWGERCNDSSD
1474


 


371
MSLCTEVGMSLLTEV
1468


 


372
CSLLTEVGMSCLTEV
1466


 


373
CSLLTECGMSLLTEV
1458


 


374
SLLTECGCSLLTEC
1451


 


375
SLLTEVETPIRNEWGCRVNDSSD
1446


 


376
CSLLTECGMSLLTEC
1445


 


377
SLKTEVETPIRNEWGCRCNDSSD
1434


 


378
SLLTECGSLLTEC
1426


 


379
SLCTEVGCSCLCEV
1421


 


380
SLLTEVETPIRNTWGCRCNDSSD
1409


 


381
SLLQEVETPIRNEWGCRCNDSSD
1399


 


382
CSLLTECGSLLTEV
1393


 


383
CSLLTEVGCSLLTEC
1380


 


384
SLLTEVETPIRNEYGCRCNDSSD
1377


 


385
CSLLTEVGCLCTCV
1375


 


386
SLLTEVETPIRNEVGCRCNDSSD
1371


 


387
SLLTEVETPIRTEWGCRCNDSSD
1365


 


388
CSLLTEVGCLLTEV
1351


 


389
SLLTEVETPYRNEWGCRCNDSSD
1350


 


390
CLLTEVGSLLTEV
1349


 


391
SNLTEVETPIRNEWGCRCNDSSD
1345


 


392
SLLTEVETPKRNEWGCRCNDSSD
1342


 


393
SLLTEVETPIRNEWGCRCNLSSD
1334


 


449
SLNTEVETPIRNEWGCRCNDSSD
1333


 


450
SLLTEVEHPIRNEWGCRCNDSSD
1331


 


451
CSLLTEVGMSLLTEC
1326


 


452
SLLTEVGSLLTEC
1326


 


453
SLLTEVETPIRNEAGCRCNDSSD
1311


 


454
SLLTEVETPIMNEWGCRCNDSSD
1308


 


455
CLCTCVGSLLTEC
1303


 


456
CSLLTECGMSLLCEV
1294


 


457
SLLTEVETPMRNEWGCRCNDSSD
1281


 


458
CSLLTECGSCLTEV
1278


 


459
SLITEVETPIRNEWGCRCNDSSD
1277


 


460
MSLLTECGMSLLCEV
1268


 


461
SLLTEVETPLRNEWGCRCNDSSD
1268


 


462
SLLTECGCLLTEV
1259


 


463
CSLLTECGSLLTCV
1258


 


464
SLLTEVGSLCTEV
1256


 


465
SLLTEVETPIRNEWGCRCNKSSD
1254


 


466
SCLTEVGCSLLTEV
1250


 


467
SLLTEVENPIRNEWGCRCNDSSD
1245


 


468
SCLCECGSLLTEC
1244


 


469
MSLLTECGMSLLTEC
1236


 


470
MSLLTEVGCSLLTEC
1227


 


471
SLLTEEETPIRNEWGCRCNDSSD
1220


 


472
SLLTECGSLCTEV
1218


 


473
SLSTEVETPIRNEWGCRCNDSSD
1217


 


474
SLLTECETPIRNEWGCRCNDSSD
1214


 


475
MSLLTECGMSLCTEV
1212


 


476
CSLLTEVGCSCLCEV
1206


 


477
SLLTECGSLLCEV
1206


 


478
SLTTEVETPIRNEWGCRCNDSSD
1205


 


407
SLLTEVETPIRNEWGCRCNDHSD
1200


 


479
SLLTEDETPIRNEWGCRCNDSSD
1196


 


480
SLLTECGCLCTCV
1193


 


481
CSLLTECGMSLCTEV
1187


 


482
CLCTCVGSLLTEV
1178


 


483
CSLLTECGCSLLTEC
1177


 


484
SLVTEVETPIRNEWGCRCNDSSD
1175


 


485
CSLLTECGMCLCTCV
1168


 


486
SLFTEVETPIRNEWGCRCNDSSD
1156


 


487
SLLTNVETPIRNEWGCRCNDSSD
1142


 


394
SLLTEVETPIRNEWGCRCNMSSD
1133


 


488
SLLTEVETPIHNEWGCRCNDSSD
1112


 


489
MSLLTECGMCLLTEV
1111


 


490
SVLTEVETPIRNEWGCRCNDSSD
1109


 


491
SLLTEVETLIRNEWGCRCNDSSD
1109


 


492
MSLLTECGMCLCTCV
1107


 


493
SLLTEVETMIRNEWGCRCNDSSD
1104


 


494
SLLTEQETPIRNEWGCRCNDSSD
1102


 


495
SLLTEVETPGRNEWGCRCNDSSD
1097


 


496
SCLTEVGSLLCEV
1095


 


436
SLLTEVETPI
1084


 


497
SLLTECGSCLCEC
1077


 


498
SLLTEMETPIRNEWGCRCNDSSD
1076


 


499
CSLLTEVGMSCLCEC
1066


 


500
SCLTEVGSLLTEV
1066


 


501
SLLTEVETPIRNEWGCCCNDSSD
1065


 


502
CSCLCEVGSLLTEC
1064


 


503
SLLTEVETPIRNEWGCWCNDSSD
1061


 


504
SLLTEVETPIRNEWGFRCNDSSD
1057


 


505
SLLTEKETPIRNEWGCRCNDSSD
1055


 


506
SLCTEVGSLLCEV
1050


 


507
SLCTEVETPIRNEWGCRCNDSSD
1050


 


508
MSLCTEVGCSLLTEV
1049


 


509
SLLTEVETPIYNEWGCRCNDSSD
1049


 


510
SLRTEVETPIRNEWGCRCNDSSD
1047


 


511
SLLCEVGCSLLTEV
1044


 


512
SLLTEVETPIRNQWGCRCNDSSD
1039


 


513
MCLLTEVGMSLLTEV
1032


 


514
SLLTEGETPIRNEWGCRCNDSSD
1029


 


515
SLLTEWETPIRNEWGCRCNDSSD
1025


 


516
SLLTEVETPIRNEWGCRCPDSSD
1024


 


517
SLLTEVETPIRNEMGCRCNDSSD
1020


 


518
SYLTEVETPIRNEWGCRCNDSSD
1019


 


519
SLLTEVETPIRNEWGCRSNDSSD
1016


 


520
VLLTEVETPIRNEWGCRCNDSSD
1014


 


437
SLLTEVETPIR
1012


 


521
SLLTAVETPIRNEWGCRCNDSSD
1012


 


522
SLLTMVETPIRNEWGCRCNDSSD
1011


 


523
SLLTEFETPIRNEWGCRCNDSSD
1009


 


524
SLLTEVETPIDNEWGCRCNDSSD
1001


 


401
SLLTEVETPIRNEWGCRCNWSSD
999


 


525
SLLTEVGCSCLCEV
996


 


526
MSLLTECGMSCLCEC
992


 


527
SLLTEVEQPIRNEWGCRCNDSSD
986


 


528
MSLLTCVGCSLLTEV
978


 


529
SLLTEVETNIRNEWGCRCNDSSD
978


 


445
SLLTEVETPIRNEWGCRCN
977


 


530
SLLTESETPIRNEWGCRCNDSSD
974


 


433
SLLTEVETPIRNEWGCRCNDSWD
971


 


531
MSLCTEVGMSLLTEC
970


 


532
MSCLTEVGMSLLTEV
968


 


533
CSLLTEVGSCLCEC
963


 


534
SLLTEVEKPIRNEWGCRCNDSSD
963


 


535
SLLTEVETPIRNEWGCRCNFSSD
963


 


408
SLLTEVETPIRNEWGCRCNDISD
962


 


536
MCLCTCVGCSLLTEV
954


 


537
SLLTEVEPPIRNEWGCRCNDSSD
953


 


538
LLLTEVETPIRNEWGCRCNDSSD
950


 


539
SLLTEYETPIRNEWGCRCNDSSD
950


 


422
SLLTEVETPIRNEWGCRCNDSFD
947


 


421
SLLTEVETPIRNEWGCRCNDSED
944


 


540
SLLTEHETPIRNEWGCRCNDSSD
942


 


541
SLLTEVETPIRNEWKCRCNDSSD
932


 


418
SLLTEVETPIRNEWGCRCNDWSD
929


 


542
MSLCTEVGMSLLTCV
927


 


395
SLLTEVETPIRNEWGCRCNNSSD
926


 


543
MSLLTCVGMSLLTEV
925


 


544
SLLTEVETPIRNEWGCRCRDSSD
924


 


545
SLLTEVSTPIRNEWGCRCNDSSD
923


 


546
SLLTEVETPIRNRWGCRCNDSSD
921


 


547
SLLTETETPIRNEWGCRCNDSSD
919


 


548
CSLLTECGSLCTEV
917


 


549
SLLTEVETFIRNEWGCRCNDSSD
916


 


550
SLLTENETPIRNEWGCRCNDSSD
914


 


551
SLLTEVATPIRNEWGCRCNDSSD
914


 


552
SLLTEVETPIRNEWGCYCNDSSD
913


 


553
SLLTEVTTPIRNEWGCRCNDSSD
909


 


554
CLLTEVGSLLTCV
902


 


555
SLLTEVETPIRNEWGCRCWDSSD
901


 


556
SLLTELETPIRNEWGCRCNDSSD
900


 


557
MSCLTEVGCSLLTEV
899


 


435
SLLTEVETP
899


 


409
SLLTEVETPIRNEWGCRCNDKSD
898


 


444
SLLTEVETPIRNEWGCRC
898


 


558
CSLLTECGMSCLCEC
897


 


559
SLLTEVETAIRNEWGCRCNDSSD
895


 


560
SLLTEVETPIRNEWGCRCFDSSD
894


 


561
SCLTEVETPIRNEWGCRCNDSSD
892


 


562
SLLTEVETPIRWEWGCRCNDSSD
890


 


398
SLLTEVETPIRNEWGCRCNRSSD
890


 


563
SLLTEVETPIRNEWGGRCNDSSD
887


 


564
SLLTEVELPIRNEWGCRCNDSSD
886


 


565
SLLTEVETPIRVEWGCRCNDSSD
884


 


566
SLLTEVCTPIRNEWGCRCNDSSD
881


 


567
SLLTECGCSCLCEV
879


 


568
SLLTEVETPIRNEWGCRCQDSSD
877


 


569
SLLTEVETPIRNEWLCRCNDSSD
876


 


570
MCLLTEVGCSLLTEV
875


 


571
SLLTEVETPIRNLWGCRCNDSSD
870


 


572
SLLTEVETPIRNEKGCRCNDSSD
868


 


573
SLLTEVETPIRNEWGCRTNDSSD
867


 


574
CLLTEVGSLLTEC
866


 


575
SLLTEVRTPIRNEWGCRCNDSSD
866


 


400
SLLTEVETPIRNEWGCRCNVSSD
865


 


443
SLLTEVETPIRNEWGCR
864


 


576
SLLTEVETPIFNEWGCRCNDSSD
862


 


577
SLLTEVETPIRNIWGCRCNDSSD
862


 


434
SLLTEVETPIRNEWGCRCNDSYD
862


 


415
SLLTEVETPIRNEWGCRCNDRSD
860


 


578
SLLTERETPIRNEWGCRCNDSSD
859


 


579
SLLTEVETPIRNEWGCRWNDSSD
859


 


580
SLCTEVGCSLLTEV
858


 


581
SLLTEVETYIRNEWGCRCNDSSD
857


 


582
SLLTEVETPIRAEWGCRCNDSSD
856


 


583
SLLTEVWTPIRNEWGCRCNDSSD
855


 


584
SLLTEVETPIRNEWGCRRNDSSD
855


 


585
SLLTEVETPIRLEWGCRCNDSSD
853


 


586
SLLTEVETPIRNEWGCRCNISSD
851


 


587
SLLTEVETPIRCEWGCRCNDSSD
850


 


396
SLLTEVETPIRNEWGCRCNPSSD
849


 


588
SLLTEVETPIRNEWGCRCNCSSD
848


 


589
CSLLTECGMSCLTEV
846


 


590
SLLTEVEMPIRNEWGCRCNDSSD
845


 


591
SLLTEVETPHRNEWGCRCNDSSD
844


 


397
SLLTEVETPIRNEWGCRCNQSSD
842


 


592
MSLCTEVGCSLLTEC
840


 


593
SLLTEVEIPIRNEWGCRCNDSSD
839


 


594
SLLTEVQTPIRNEWGCRCNDSSD
838


 


595
SLLTEVETPIRNEWGWRCNDSSD
838


 


596
SLLTEVETPIRNEWFCRCNDSSD
837


 


597
SLLTEVETGIRNEWGCRCNDSSD
835


 


598
SLLTEVETPIRNEWGCRKNDSSD
835


 


599
SLLTEVERPIRNEWGCRCNDSSD
834


 


430
SLLTEVETPIRNEWGCRCNDSRD
833


 


448
SLLTEVETPIRNEWGCRCNDSS
833


 


600
SLLTEVETPIRNEWGLRCNDSSD
831


 


601
SLLTECGSCLTEV
830


 


602
SLLTEVETEIRNEWGCRCNDSSD
828


 


603
SLLTEVETPIRNEWGCRCNGSSD
828


 


604
SLCTEVGSCLTEV
824


 


605
SLLTEVETPIRNMWGCRCNDSSD
820


 


606
SLLTEVETQIRNEWGCRCNDSSD
819


 


607
SLLTEVESPIRNEWGCRCNDSSD
817


 


447
SLLTEVETPIRNEWGCRCNDS
817


 


608
SLLTEIETPIRNEWGCRCNDSSD
816


 


609
SLLTEVETHIRNEWGCRCNDSSD
816


 


610
SLLTEVETDIRNEWGCRCNDSSD
813


 


611
SLLTEVETRIRNEWGCRCNDSSD
813


 


612
SLLTEVETPIRNEWGCRCIDSSD
812


 


613
SLLTEVETPIRNEWGCRCNHSSD
812


 


614
SLLTEVETVIRNEWGCRCNDSSD
810


 


615
SLLTEVETPIRNEWGCDCNDSSD
810


 


616
SLLTEVETPIRNPWGCRCNDSSD
808


 


617
SLLTEVETPIRNSWGCRCNDSSD
807


 


618
SLLTEVETPIRNEWGCRCLDSSD
807


 


619
SLLTEVETPIRNEWGCRCNASSD
807


 


620
SLLTEVETPIRFEWGCRCNDSSD
806


 


621
SLLTEVEFPIRNEWGCRCNDSSD
805


 


622
SLLTEVETPIRMEWGCRCNDSSD
805


 


623
SLLTEVETCIRNEWGCRCNDSSD
800


 


624
SLLTEVETPIRNEWVCRCNDSSD
799


 


413
SLLTEVETPIRNEWGCRCNDPSD
797


 


399
SLLTEVETPIRNEWGCRCNTSSD
795


 


625
SLLTEVETPIRNEWYCRCNDSSD
794


 


416
SLLTEVETPIRNEWGCRCNDTSD
794


 


626
SLLTEVETPIRNEWGCRCYDSSD
791


 


627
SLLTEVETPIRIEWGCRCNDSSD
789


 


406
SLLTEVETPIRNEWGCRCNDFSD
788


 


628
SLLTEVETPIRNEWGCRCMDSSD
786


 


629
SLLTEVMTPIRNEWGCRCNDSSD
783


 


630
SLLTEVETPERNEWGCRCNDSSD
783


 


631
SLLTEVETPIRKEWGCRCNDSSD
782


 


423
SLLTEVETPIRNEWGCRCNDSHD
782


 


426
SLLTEVETPIRNEWGCRCNDSLD
779


 


632
SLLTEVETPIRNEWGCRCVDSSD
775


 


633
SLLTEVETPIRNEWHCRCNDSSD
772


 


403
SLLTEVETPIRNEWGCRCNDCSD
772


 


417
SLLTEVETPIRNEWGCRCNDVSD
772


 


428
SLLTEVETPIRNEWGCRCNDSPD
772


 


634
SLLTEVETPIRNEWGCRANDSSD
770


 


425
SLLTEVETPIRNEWGCRCNDSKD
770


 


635
SLLTEVEAPIRNEWGCRCNDSSD
769


 


410
SLLTEVETPIRNEWGCRCNDLSD
769


 


636
SLLTEVETPIRNEIGCRCNDSSD
768


 


637
SLLTEVETPIRNENGCRCNDSSD
768


 


638
SLLTEVETPIRNEWGTRCNDSSD
767


 


639
SLLTEVETPIRNEWGCFCNDSSD
767


 


640
SLLTEVETPIRNKWGCRCNDSSD
765


 


641
SLLTEVETPIRNELGCRCNDSSD
765


 


642
SLLTEVETPIRNEWMCRCNDSSD
765


 


643
SLLTEVETPIRNGWGCRCNDSSD
764


 


644
SLLTEVETPIRNEWGVRCNDSSD
764


 


645
SLLTEVVTPIRNEWGCRCNDSSD
763


 


646
SLLTEVEGPIRNEWGCRCNDSSD
763


 


647
SLLTEVETPIRNEWGHRCNDSSD
763


 


648
SLLTEVETPIRNEWGKRCNDSSD
762


 


649
SLLTEVETTIRNEWGCRCNDSSD
760


 


650
SLLTEVETPVRNEWGCRCNDSSD
758


 


651
SLLTEVETPIRNEWGCRCHDSSD
758


 


652
SLLTEVEEPIRNEWGCRCNDSSD
757


 


653
SLLTEVETIIRNEWGCRCNDSSD
756


 


654
SLLTEVETPIRYEWGCRCNDSSD
755


 


655
SLYTEVETPIRNEWGCRCNDSSD
753


 


656
SLLTEVETPIWNEWGCRCNDSSD
753


 


657
SLLTEVDTPIRNEWGCRCNDSSD
751


 


658
SLLTEVYTPIRNEWGCRCNDSSD
751


 


438
SLLTEVETPIRN
751


 


659
SLLTEVETPIRNEWGARCNDSSD
750


 


440
SLLTEVETPIRNEW
750


 


660
SLLTEVETPDRNEWGCRCNDSSD
749


 


661
SLLTEVETPIRQEWGCRCNDSSD
748


 


662
SLLTEVETPIRNFWGCRCNDSSD
747


 


663
MCLCTCVGMSLLTEV
746


 


664
SLLTEVETPIRNEWGNRCNDSSD
746


 


665
SLLTEVETPFRNEWGCRCNDSSD
745


 


411
SLLTEVETPIRNEWGCRCNDMSD
744


 


666
SLLTEVETPIRNAWGCRCNDSSD
743


 


667
SLLTEVETPIRNEWGIRCNDSSD
739


 


668
SLLTEVETPIRNEWGYRCNDSSD
739


 


414
SLLTEVETPIRNEWGCRCNDQSD
739


 


669
SLLTEVETPIRNEWTCRCNDSSD
738


 


446
SLLTEVETPIRNEWGCRCND
738


 


670
SLLTEVETWIRNEWGCRCNDSSD
736


 


671
SLLTEVETPIRNEPGCRCNDSSD
735


 


672
SLLTEVETPIRNEWGRRCNDSSD
735


 


673
SLLTEVETPSRNEWGCRCNDSSD
733


 


674
SLLTEVETPIRNEWGCRCTDSSD
733


 


675
SLLTEVNTPIRNEWGCRCNDSSD
732


 


676
SLLTEVETPIENEWGCRCNDSSD
731


 


677
SLLTEVETPIQNEWGCRCNDSSD
731


 


678
SLLTEVETPIRSEWGCRCNDSSD
730


 


679
SLLTEVETPIRNEWGCECNDSSD
730


 


680
SLLTEVETPIVNEWGCRCNDSSD
729


 


681
SLLTEVETPIRNEWACRCNDSSD
729


 


682
SLLTEVETPIRNEWGSRCNDSSD
729


 


683
SLLTEVETPIRNEWGCACNDSSD
729


 


684
SLLTEVETPIRNHWGCRCNDSSD
728


 


685
SLLTEVETPIRNVWGCRCNDSSD
728


 


686
SLLTEVETPIRNEWGCRLNDSSD
728


 


432
SLLTEVETPIRNEWGCRCNDSVD
728


 


420
SLLTEVETPIRNEWGCRCNDSCD
725


 


687
SLLTEVETPCRNEWGCRCNDSSD
724


 


688
SLLTEVETPIRNETGCRCNDSSD
724


 


689
SLLTEVETPIRNEWGCRCGDSSD
724


 


690
SLLTEVHTPIRNEWGCRCNDSSD
722


 


691
SLLTEVETPIRNEWGCRMNDSSD
722


 


692
SLLTEVETPILNEWGCRCNDSSD
721


 


693
SLLTEVETPWRNEWGCRCNDSSD
720


 


694
SLLTEVETPIRNEWGCRCNESSD
720


 


695
SLLTEVEVPIRNEWGCRCNDSSD
719


 


696
SLLTEVETPIRNEHGCRCNDSSD
719


 


697
SLLTEVETPIRNEWGCRINDSSD
718


 


698
SLLTEVETPIRDEWGCRCNDSSD
716


 


699
SLLTEVETPIRNEWGCRYNDSSD
716


 


700
SLLTEVETSIRNEWGCRCNDSSD
715


 


701
SLLTEVETPIRNEWGCKCNDSSD
715


 


702
SLLTEVETPIRNYWGCRCNDSSD
714


 


703
SLLTEVETPIRNWWGCRCNDSSD
713


 


704
SLLTEVETPIRNEWRCRCNDSSD
713


 


705
SLLTEVKTPIRNEWGCRCNDSSD
712


 


706
SLLTEVETPIRNEWGCRCADSSD
712


 


707
SLLTEVETPIANEWGCRCNDSSD
708


 


708
SLLTEVETPIRNEGGCRCNDSSD
708


 


412
SLLTEVETPIRNEWGCRCNDNSD
708


 


709
SLLTEVLTPIRNEWGCRCNDSSD
707


 


710
SLLTEVETPIRREWGCRCNDSSD
707


 


711
CSLLTEVGSLCTEV
707


 


712
SLLTEVETPIRNDWGCRCNDSSD
706


 


713
SLLTEVETPIRNEWGQRCNDSSD
705


 


714
SLLTEVETPIRNEWCCRCNDSSD
704


 


715
SLLTEVETPARNEWGCRCNDSSD
702


 


716
SLLTEVETPIRNEFGCRCNDSSD
702


 


717
SLLTEVETPIRNEWDCRCNDSSD
701


 


431
SLLTEVETPIRNEWGCRCNDSTD
701


 


718
SLLTEVECPIRNEWGCRCNDSSD
700


 


719
SLLTEVETPIRNEWGCTCNDSSD
700


 


720
SLLTEVETPIINEWGCRCNDSSD
699


 


721
SLLTEVETPIRHEWGCRCNDSSD
699


 


722
SLLTEVETPIRNEWGCICNDSSD
699


 


723
SLLTEVETPIRNEWGCRFNDSSD
698


 


724
MCLLTEVGMSLLTEC
697


 


725
SLLTEVETPICNEWGCRCNDSSD
697


 


726
SLLTEVETPIRNEWECRCNDSSD
697


 


727
SLLTEVETPIRNEWGPRCNDSSD
696


 


728
SLLTEVETPIRNEWGCRCCDSSD
696


 


729
SLLTEVETPIRNEWGCRCSDSSD
696


 


730
SLLTEVETPIRNEWGCMCNDSSD
695


 


429
SLLTEVETPIRNEWGCRCNDSQD
695


 


731
SLLTEVFTPIRNEWGCRCNDSSD
694


 


732
SLLTEVETPIRNEWGMRCNDSSD
693


 


733
SLLTEVETPIRNCWGCRCNDSSD
692


 


734
SLLTEVETPIRNEWGCRHNDSSD
692


 


424
SLLTEVETPIRNEWGCRCNDSID
692


 


735
SLLTEVETPIRNEWSCRCNDSSD
691


 


736
SLLTEVETPIRNEWGCSCNDSSD
691


 


737
SLLTEVETPIRNERGCRCNDSSD
690


 


738
SLLTEVETPPRNEWGCRCNDSSD
689


 


739
SLLTEVETPIRNEWGCRCEDSSD
687


 


740
SHLTEVETPIRNEWGCRCNDSSD
686


 


741
SLLTEVETPIRNNWGCRCNDSSD
686


 


742
SLLTEVETPIRNEWGCVCNDSSD
684


 


404
SLLTEVETPIRNEWGCRCNDDSD
684


 


743
SLLTEVETPIRNEWGCNCNDSSD
683


 


402
SLLTEVETPIRNEWGCRCNDASD
683


 


744
SLLTEVETPIGNEWGCRCNDSSD
681


 


745
SLLTEVETPIRNESGCRCNDSSD
681


 


746
SLLTEVETPIRNEWGCRNNDSSD
681


 


747
SLCTEVGCSLLTEC
680


 


748
SLLTEVETPIRNEWGCLCNDSSD
680


 


442
SLLTEVETPIRNEWGC
680


 


749
MSLLCEVGMSLLTEV
677


 


750
SLLTEVETPRRNEWGCRCNDSSD
677


 


405
SLLTEVETPIRNEWGCRCNDESD
676


 


751
SLLTEVETPIRNEDGCRCNDSSD
675


 


752
SLLTEVETPIRNEWGCRGNDSSD
674


 


753
SLLTEVETPIRPEWGCRCNDSSD
673


 


754
SLLTEVETPISNEWGCRCNDSSD
672


 


755
SLLTQVETPIRNEWGCRCNDSSD
671


 


756
SLLTEVETPIRNEWGCQCNDSSD
671


 


757
SLLTEVETPIRNEWGCRPNDSSD
669


 


758
SLLTEVETPTRNEWGCRCNDSSD
668


 


427
SLLTEVETPIRNEWGCRCNDSMD
668


 


759
SLHTEVETPIRNEWGCRCNDSSD
667


 


760
SLLTEVETPIRNEWQCRCNDSSD
667


 


761
MSLLTEVGCSCLCEV
663


 


762
SLLTEVETPIRNEWGCRCDDSSD
662


 


763
SLLTEVEWPIRNEWGCRCNDSSD
660


 


764
SLLTEPETPIRNEWGCRCNDSSD
659


 


765
SLLTEVETPIRNEQGCRCNDSSD
657


 


766
SLLTEVETPITNEWGCRCNDSSD
653


 


767
SLLTEVETPIRNEWGCPCNDSSD
653


 


768
MSCLTEVGMSLLTEC
650


 


769
MCLLTEVGCSLLTEC
650


 


770
SLLTEVETPNRNEWGCRCNDSSD
649


 


771
SLLTEVETPIREEWGCRCNDSSD
648


 


772
SLLTEVETPIRNEWGCRQNDSSD
646


 


773
SLLTEVETPIRGEWGCRCNDSSD
644


 


774
SLLTEVETPIRNEWPCRCNDSSD
644


 


775
SLLTEVETPIRNEWGCGCNDSSD
644


 


776
SLLTEVETPIRNEWGCHCNDSSD
640


 


777
SLLTEVPTPIRNEWGCRCNDSSD
639


 


419
SLLTEVETPIRNEWGCRCNDSAD
639


 


778
SLLTEVETPIKNEWGCRCNDSSD
638


 


779
SLLTEVEDPIRNEWGCRCNDSSD
635


 


780
SLLTEVETPINNEWGCRCNDSSD
632


 


781
MSLLCEVGCSLLTEV
631


 


782
SLLTEVETPQRNEWGCRCNDSSD
630


 


783
SLLTEVEYPIRNEWGCRCNDSSD
628


 


784
SLLTEVGTPIRNEWGCRCNDSSD
627


 


785
SLLTEVETPIRNEWNCRCNDSSD
618


 


786
SWLTEVETPIRNEWGCRCNDSSD
615


 


787
SLLTEVETPIRNEEGCRCNDSSD
613


 


788
SQLTEVETPIRNEWGCRCNDSSD
609


 


789
SLCTEVGSLCTEV
607


 


790
SLLTEVETPIRNEWGDRCNDSSD
603


 


791
MSLCTEVGMCLLTEV
597


 


792
CSLLTEVGSLLCEV
593


 


793
MSLLTEVGMSLLCEV
591


 


794
SLLTEVETPIRNEWGCRDNDSSD
589


 


795
SLLTEVITPIRNEWGCRCNDSSD
587


 


796
SLLTEVETPIRNEWGCRENDSSD
579


 


797
SCLTEVGSLLTCV
573


 


798
CLCTCVGCSLLTEV
567


 


799
MSCLTEVGCSLLTEC
557


 


800
MSLLTECGCSCLCEV
556


 


801
MSLLTECGMSCLTEV
554


 


802
SLGTEVETPIRNEWGCRCNDSSD
542


 


803
SLLSEVETPIRNEWGCRCNDSSD
532


 


804
SLWTEVETPIRNEWGCRCNDSSD
525


 


805
MSLLTCVGMSLLTEC
524


 


806
SLCTEVGCLLTEV
522


 


807
CLLTEVGCSLLTEV
520


 


808
SCLTEVGCSLLTEC
514


 


809
CSLLTECGCLLTEV
507


 


810
SLLTCVGCSLLTEV
504


 


811
CSLLTECGCLCTCV
458


 


812
SLLTCVGCSLLTEC
447


 


813
CSLLTECGSLLTEC
441


 


814
CSLLTECGSLLCEV
430


 


815
CSLLTECGSCLCEC
419


 


816
CLLTEVGCSLLTEC
409


 


817
CSLLTECGCSCLCEV
404


 


818
MSLLTECGCSLLTEC
386


 


819
CLCTCVGCSLLTEC
340


 


820
MSLLTCVGCSLLTEC
330


 


821
CSCLCEVGCSLLTEV
325


 


822
SLLTDVETPIRNEWGCRCNDSSD
324


 


823
SCLTEVGSLLTEC
321


 


824
SLLTCVGSLLTEV
320


 


825
MSLLCEVGMSLLTEC
314


 


826
MSLLCEVGCSLLTEC
312


 


827
MSCLCECGCSLLTEV
306


 


828
MCLCTCVGMSLLTEC
306


 


829
MSLLTEVGMSCLCEC
304


 


830
1CSLLTEVETP1SLLTEVETPCLLTEVETPI1
2303


 


831
1CSLLTEVETP1SLLTEVETPCSLLTEVETP1
2147


 


832
1CLTEVETPIR1SLLTEVETPCLLTEVETPI1
2145


 


833
SLLTEVETCSLLTEVETATPIRNEWGCRC
2133


 


834
SLLTEVET1CSLLTEVET1SLLTEVETCEWG1R
2107


 


835
SLLTEVET1CSLLTEVET1SLLTEVETAWGCR1
2103


 


836
1CLTEVETPIR1SLLTEVETPCSLLTEVETP1
1936


 


837
1CETPIRNEWG1SLLTEVETPCLLTEVETPI1
1912


 


838
1CTEVETPIRN1SLLTEVETPCLLTEVETPI1
1887


 


839
1CTEVETPIRN1SLLTEVETPCSLLTEVETP1
1796


 


840
SLLTEVET1CSLLTEVET1SLLTEVETCEWGSR1
1765


 


841
1CSLLTEVETP1LTEVETPIRCSLLTEVETP1
1732


 


842
1CSLLTEVETP1TEVETPIRNCSLLTEVETP1
1717


 


843
1CVETPIRNEW1SLLTEVETPCLTEVETPIR1
1715


 


844
SLLTEVET1CSLLTEVET1SLLTEVETAGCR1N
1712


 


845
1CSLLTEVETP1SLLTEVETPCETPIRNEWG1
1704


 


846
SLLTEVETCSLLTEVETCNEWGSRSNDSSC
1697


 


847
1CLLTEVETPI1SLLTEVETPCLLTEVETPI1
1665


 


848
1CEVETPIRNE1SLLTEVETPCSLLTEVETP1
1655


 


849
1CLLTEVETPI1LTEVETPIRCSLLTEVETP1
1651


 


850
1CSLLTEVETP1SLLTEVETPCVETPIRNEW1
1649


 


851
1CSLLTEVETP1VETPIRNEWCSLLTEVETP1
1634


 


852
SLLTEVET1CSLLTEVET1SLLTEVETCIRNEW1
1598


 


853
SLLTEVET1CSLLTEVET1SLLTEVETCLLTEV1
1592


 


854
1CTEVETPIRN1LTEVETPIRCSLLTEVETP1
1590


 


855
1CEVETPIRNE1SLLTEVETPCLLTEVETPI1
1584


 


856
SLLTEVETCSLLTEVETCVETPIRNEWGC
1584


 


857
1CLTEVETPIR1SLLTEVETPCLTEVETPIR1
1576


 


858
1CETPIRNEWG1SLLTEVETPCSLLTEVETP1
1559


 


859
1CSLLTEVETP1SLLTEVETPCLTEVETPIR1
1552


 


860
1CVETPIRNEW1SLLTEVETPCSLLTEVETP1
1531


 


861
SLLTEVET1CSLLTEVET1SLLTEVETCSLLTE1
1490


 


862
SLLTEVET1CSLLTEVET1SLLTEVETCTPIRN1
1481


 


863
1CLTEVETPIR1LTEVETPIRCSLLTEVETP1
1458


 


864
1CTEVETPIRN1SLLTEVETPCLTEVETPIR1
1452


 


865
SLLTEVETCSLLTEVETAEWGCRCNDSSD
1451


 


866
1CVETPIRNEW1SLLTEVETPCETPIRNEWG1
1446


 


867
1CLLTEVETPI1SLLTEVETPCSLLTEVETP1
1438


 


868
1CEVETPIRNE1SLLTEVETPCETPIRNEWG1
1425


 


869
1CSLLTEVETP1EVETPIRNECSLLTEVETP1
1405


 


870
1CEVETPIRNE1EVETPIRNECSLLTEVETP1
1400


 


871
1CVETPIRNEW1SLLTEVETPCLLTEVETPI1
1396


 


872
1CEVETPIRNE1LTEVETPIRCSLLTEVETP1
1382


 


873
1CSLLTEVETP1VETPIRNEWCLLTEVETPI1
1382


 


874
SLLTEVETCSLLTEVETCEWGSRSNDSSDC
1375


 


875
SLLTEVETCSLLTEVETCSLLTEVETPIRC
1369


 


876
1CVETPIRNEW1LTEVETPIRCSLLTEVETP1
1327


 


877
1CVETPIRNEW1ETPIRNEWGCSLLTEVETP1
1317


 


878
1CSLLTEVETP1TEVETPIRNCLLTEVETPI1
1313


 


879
SLLTEVETCSLLTEVETCETPIRNEWGCR
1306


 


880
1CLTEVETPIR1TEVETPIRNCSLLTEVETP1
1290


 


881
CSLLTEVETPIRNEWGCETPIRNEWGSRSNDSC
1289


 


882
1CETPIRNEWG1LTEVETPIRCSLLTEVETP1
1286


 


883
1CLLTEVETPI1TEVETPIRNCSLLTEVETP1
1270


 


884
SLLTEVETCSLLTEVETCRNEWGSRSNDSC
1255


 


885
1CSLLTEVETP1ETPIRNEWGCSLLTEVETP1
1250


 


886
1CSLLTEVETP1LTEVETPIRCLTEVETPIR1
1241


 


887
1CTEVETPIRN1TEVETPIRNCSLLTEVETP1
1229


 


888
CSLLTEVETCPIRNEWGSRSNDSSDC
1224


 


889
1CSLLTEVETP1EVETPIRNECLLTEVETPI1
1221


 


890
SLLTEVET1CSLLTEVET1SLLTEVETCETPIR1
1208


 


891
CSLLTEVETPIRNCETPIRNEWGSRSNDSC
1204


 


892
1CSLLTEVETP1LLTEVETPICLTEVETPIR1
1203


 


893
SLLTEVET1CSLLTEVET1SLLTEVETCVETPI1
1200


 


894
CSLLTEVETCTPIRNEWGSRSNDSSC
1196


 


895
SLLTEVET1CSLLTEVET1SLLTEVETCRNEWG1
1195


 


896
1CETPIRNEWG1ETPIRNEWGCSLLTEVETP1
1180


 


897
1CEVETPIRNE1SLLTEVETPCLTEVETPIR1
1169


 


898
SLLTEVET1CSLLTEVET1SLLTEVETACR1ND
1169


 


899
SLLTEVETCSLLTEVETAPIRNEWGCRCN
1165


 


900
1CSLLTEVETP1LLTEVETPICSLLTEVETP1
1157


 


901
SLLTEVET1CSLLTEVET1SLLTEVETCLTEVE1
1144


 


902
1CLTEVETPIR1ETPIRNEWGCSLLTEVETP1
1125


 


903
1CEVETPIRNE1ETPIRNEWGCSLLTEVETP1
1124


 


904
SLLTEVETCSLLTEVETAIRNEWGCRCND
1120


 


905
CSLLTEVETPICETPIRNEWGSRSNDSC
1120


 


906
CSLLTEVETPIRNEWGCPIRNEWGSRSNDSSDC
1118


 


907
SLLTEVET1CSLLTEVET1SLLTEVETCPIRNE1
1104


 


908
SLLTEVET1CSLLTEVET1SLLTEVETCNEWG1
1103


 


909
SLLTEVETCSLLTEVETCLLTEVETPIRNC
1101


 


910
1CETPIRNEWG1SLLTEVETPCLTEVETPIR1
1099


 


911
CSLLTEVETPIRCETPIRNEWGSRSNDSC
1087


 


912
SLLTEVETCSLLTEVETCTPIRNEWGSRSC
1081


 


913
1CTEVETPIRN1SLLTEVETPCEVETPIRNE1
1079


 


914
SLLTEVET1CSLLTEVET1SLLTEVETCEVETP1
1062


 


915
SLLTEVET1CSLLTEVET1SLLTEVETCTEVET1
1053


 


916
1CSLLTEVETP1LTEVETPIRCLLTEVETPI1
1048


 


917
CSLLTEVETPIRNECETPIRNEWGSRSNDSC
1037


 


918
1CSLLTEVETP1SLLTEVETPCTEVETPIRN1
1022


 


919
1CETPIRNEWG1TEVETPIRNCSLLTEVETP1
1021


 


920
CSLLTEVETPIRNECTPIRNEWGSRSNDSSC
1019


 


921
1CEVETPIRNE1TEVETPIRNCSLLTEVETP1
1014


 


922
CSLLTEVETPCETPIRNEWGSRSNDSC
1010


 


923
1CSLLTEVETP1LLTEVETPICLLTEVETPI1
1009


 


924
CSLLTEVETPIRCTPIRNEWGSRSNDSSC
1000


 


925
SLLTEVETCSLLTEVETCVETPIRNEWGSC
979


 


926
SLLTEVETCSLLTEVETARNEWGCRCNDS
963


 


927
SLLTEVETCSLLTEVETCETPIRNEWGSRC
935


 


928
SLLTEVETCSLLTEVETANEWGCRCNDSS
897


 


929
1CETPIRNEWG1VETPIRNEWCSLLTEVETP1
890


 


930
SLLTEVETCSLLTEVETCEVETPIRNEWGC
867


 


931
CSLLTEVETPCPIRNEWGSRSNDSSDC
853


 


932
1CLTEVETPIR1VETPIRNEWCSLLTEVETP1
852


 


933
SLLTEVETCSLLTEVETCPIRNEWGSRSNC
840


 


934
1CEVETPIRNE1VETPIRNEWCSLLTEVETP1
834


 


935
1CVETPIRNEW1SLLTEVETPCVETPIRNEW1
815


 


936
1CETPIRNEWG1EVETPIRNECSLLTEVETP1
809


 


937
1CSLLTEVETP1ETPIRNEWGCLLTEVETPI1
805


 


938
CSLLTEVETCETPIRNEWGSRSNDSC
796


 


939
1CSLLTEVETP1LLTEVETPICETPIRNEWG1
777


 


940
SLLTEVETCSLLTEVETCTEVETPIRNEWC
775


 


941
1CEVETPIRNE1SLLTEVETPCTEVETPIRN1
772


 


942
1CETPIRNEWG1SLLTEVETPCTEVETPIRN1
749


 


943
1CLLTEVETPI1VETPIRNEWCSLLTEVETP1
743


 


944
1CSLLTEVETP1TEVETPIRNCLTEVETPIR1
740


 


945
1CLTEVETPIR1SLLTEVETPCVETPIRNEW1
733


 


946
1CLTEVETPIR1SLLTEVETPCTEVETPIRN1
729


 


947
1CVETPIRNEW1SLLTEVETPCTEVETPIRN1
724


 


948
SLLTEVETCSLLTEVETCIRNEWGSRSNDC
721


 


949
1CLLTEVETPI1SLLTEVETPCLTEVETPIR1
717


 


950
1CETPIRNEWG1SLLTEVETPCVETPIRNEW1
715


 


951
1CTEVETPIRN1SLLTEVETPCVETPIRNEW1
711


 


952
1CTEVETPIRN1VETPIRNEWCSLLTEVETP1
710


 


953
1CVETPIRNEW1TEVETPIRNCSLLTEVETP1
708


 


954
CSLLTEVETPICPIRNEWGSRSNDSSDC
700


 


955
1CVETPIRNEW1VETPIRNEWCSLLTEVETP1
699


 


956
1CTEVETPIRN1SLLTEVETPCETPIRNEWG1
691


 


957
1CTEVETPIRN1SLLTEVETPCTEVETPIRN1
684


 


958
1CSLLTEVETP1SLLTEVETPCEVETPIRNE1
684


 


959
1CEVETPIRNE1SLLTEVETPCVETPIRNEW1
674


 


960
SLLTEVETCSLLTEVETCLTEVETPIRNEC
666


 


961
1CLTEVETPIR1EVETPIRNECSLLTEVETP1
665


 


962
1CLTEVETPIR1SLLTEVETPCETPIRNEWG1
657


 


963
1CVETPIRNEW1EVETPIRNECSLLTEVETP1
652


 


964
1CETPIRNEWG1SLLTEVETPCETPIRNEWG1
645


 


965
CSLLTEVETPCTPIRNEWGSRSNDSSC
645


 


966
CSLLTEVETPICTPIRNEWGSRSNDSSC
622


 


967
1CLLTEVETPI1EVETPIRNECSLLTEVETP1
617


 


968
1CLLTEVETPI1ETPIRNEWGCSLLTEVETP1
608


 


969
CSLLTEVETPIRCPIRNEWGSRSNDSSDC
606


 


970
CSLLTEVETPIRNCPIRNEWGSRSNDSSDC
589


 


971
1CSLLTEVETP1LTEVETPIRCTEVETPIRN1
571


 


972
1CSLLTEVETP1LTEVETPIRCETPIRNEWG1
564


 


973
CSLLTEVETPIRNECPIRNEWGSRSNDSSDC
553


 


974
1CSLLTEVETP1LLTEVETPICVETPIRNEW1
551


 


975
1CLLTEVETPI1SLLTEVETPCVETPIRNEW1
539


 


976
1CLTEVETPIR1SLLTEVETPCEVETPIRNE1
538


 


977
1CVETPIRNEW1LLTEVETPICSLLTEVETP1
537


 


978
1CTEVETPIRN1EVETPIRNECSLLTEVETP1
535


 


979
CSLLTEVETPIRNCTPIRNEWGSRSNDSSC
532


 


980
CSLLTEVETPIRNEWGCTPIRNEWGSRSNDSSC
508


 


981
1CSLLTEVETP1TEVETPIRNCTEVETPIRN1
505


 


982
1CTEVETPIRN1ETPIRNEWGCSLLTEVETP1
504


 


983
SLLTEVET1CSLLTEVET1SLLTEVETCNEWGS1
346


 


984
CSLLTEVETPIRNC
2154


 


985
SLLTEVCTCIRNEWG
2047


 


986
SLLTEVETCSLLTEVETPIRNEWGSRC
1883


 


987
SLLTEVCTPICNEWG
1857


 


988
SLLTEVCTPIRCEWG
1853


 


989
SLLTEVECPIRCEWG
1849


 


990
CSLLTEVCTPIRNE
1721


 


991
SLLTEVETCIRNEWGC
1707


 


992
SLLTEVETCIRNCWG
1691


 


993
SLLTEVETCCRNEWG
1675


 


994
SLLTEVETPIRCCWG
1674


 


995
SLLTEVETSLLTEVETPIRNEWGCRCND
1663


 


996
SLLTEVCTPCRNEWG
1551


 


997
CSLLTEVETPIRCE
1512


 


998
SLLTEVETCIRCEWG
1495


 


999
SLLTEVETCSLLTEVETPIRNEWGSRSC
1488


 


1000
SLLTEVCTPIRNCWG
1440


 


1001
SLLTEVECPIRNCWG
1438


 


1002
SLCTEVETCIRNEWG
1385


 


1003
SLLTECETCIRNEWG
1384


 


1004
SLLTEVECCIRNEWG
1377


 


1005
SLLTEVETPCRNCWG
1377


 


1006
SLLTEVETCICNEWG
1360


 


1007
SLLTEVECPIRNC
1341


 


1008
SLLTECETPIRNCWG
1298


 


1009
SLLTEVETCIRNEWG
1286


 


1010
SLLTEVETPICNCWG
1255


 


1011
SLLTEVETCSLLTEVETPIRNEWGSRSNC
1239


 


1012
CSLLTEVETPICNE
1232


 


1013
CSLLTEVETCIRNE
1220


 


1014
SLLTEVCCPIRNEWG
1195


 


1015
SLLTEVETPIRNCWGC
1177


 


1016
SLLTEVCTPIRNC
1163


 


1017
SLLTEVECPIRNEWC
1161


 


1018
CSLLTEVETPIRNE
1159


 


1019
SLLTEVCTPIRNEWG
1156


 


1020
CSLLTEVETPCRNE
1148


 


1021
SLLTEVETPIRCEWGC
1135


 


1022
SLLTEVETCIRNC
1134


 


1023
SLLTEVETPIRCECG
1121


 


1024
CSLLTEVECPIRNE
1116


 


1025
SLLTEVECPCRNEWG
1110


 


1026
SCLTEVETPCRNEWG
1083


 


1027
CSLLTECETPIRNE
1082


 


1028
SLLTEVECPICNEWG
1079


 


1029
SLLTECETPIRCEWG
1052


 


1030
SLLTEVETCIRNEWC
1049


 


1031
SLCTEVETPIRNC
1035


 


1032
SLLTEVETCSLLTEVETPIRNEWGCR
1032


 


1033
SLLTECECPIRNEWG
1031


 


1034
SLLTEVETPICCEWG
1029


 


1035
SLLTEVECPIRCE
1027


 


1036
SLLTEVETPIRNCWG
1021


 


1037
SCLTEVCTPIRNE
1019


 


1038
SCLTEVETCIRNEWG
1015


 


1039
SLLTEVETPIRNCWC
1002


 


1040
SLLTEVETCSLLTEVETPIRC
995


 


1041
SLLTEVECPIRNEWG
980


 


1042
SLLTEVETSLLTEVETPIRNEWGCRC
979


 


1043
SLLTEVETSLLTEVETPIRNEWGCRCN
970


 


1044
SLLTEVCTCIRNE
949


 


1045
SLCTEVETPIRCE
945


 


1046
SLLTEVETCIRNECG
941


 


1047
SCLTEVETPIRNC
940


 


1048
SLLTEVETCSLLTEVETPIRNEWGC
938


 


1049
SLLTEVCTPIRNEWC
937


 


1050
SLLTEVECPIRNE
927


 


1051
SLLTEVETPICNEWGC
923


 


1052
SLLTEVETCSLLTEVETPIRNEWGSRSNDSSC
916


 


1053
SLLTEVETPIRCEWG
902


 


1054
SLCTEVETPIRNCWG
899


 


1055
SLLTEVETCIRCE
898


 


1056
SLLTECETPCRNEWG
897


 


1057
SLLTEVETPICNEWG
890


 


1058
SLLTEVETCSLLTEVETPIRNC
890


 


1059
SLLTECETPICNEWG
885


 


1060
SLLTECETPIRNEWG
880


 


1061
SLLTEVETPCRCEWG
875


 


1062
SLLTEVETCSLLTEVETPIRNEWGSRSNDC
874


 


1063
SLLTECCTPIRNEWG
866


 


1064
SLLTEVETCSLLTEVETPIRNEWGSRSNDSC
861


 


1065
SLLTEVCTPCRNE
853


 


1066
SCLTECETPIRNEWG
847


 


1067
SLLTEVETCSLLTEVETPC
844


 


995
SLLTEVETSLLTEVETPIRNEWGCRCNDS
836


 


1068
SLLTEVETCSLLTEVETPIRNEWGSRSNDSSDC
831


 


1069
SLLTEVETCSLLTEVETPIC
830


 


1070
SLLTEVETCSLLTEVETC
820


 


1071
SLLTEVETPCRNEWG
813


 


1072
CSLLTEVETPIRNEWGC
798


 


1073
SLLTEVCTPIRCE
789


 


1074
SLLTECETCIRNE
785


 


1075
SCLTECETPIRNE
780


 


1076
SLLTEVETCSLLTEVETPIRNEWC
768


 


1077
SLLTEVETPCCNEWG
766


 


1078
SLCTEVECPIRNEWG
750


 


1079
SLCTEVETPIRCEWG
750


 


1080
SLLTECETPIRNEWC
741


 


1081
SLLTEVCTPIRNEWGC
739


 


1082
SLLTEVETPIRCEWC
732


 


1083
SCLTEVETCIRNE
727


 


1084
SLLTEVETCICNE
727


 


1085
SLLTEVETPICNEWC
724


 


1086
SLLTEVECPIRNEWGC
715


 


1087
SLCTEVETCIRNE
712


 


1088
SLLTEVETCSLLTEVETPIRNEWGSC
712


 


1089
SLLTEVETSLLTEVETPIRNEWGCRCNDSS
711


 


1090
SCLTEVECPIRNE
705


 


1091
SLLTEVCTPICNE
699


 


1092
SLLTEVETPCRNEWGC
696


 


1093
SLLTEVETCIRNE
675


 


1094
SLLTEVCTPIRNECG
674


 


1095
SLLTEVETSLLTEVETPIRNEWGCRCNDSSD
669


 


1096
SLLTEVETPICNC
665


 


1097
SCLTEVETPIRNCWG
662


 


1098
SLLTEVETPICNECG
660


 


1099
SLLTEVECPIRNECG
656


 


1100
SLCTEVCTPIRNE
653


 


1101
SLLTECETPIRCE
648


 


1102
SLCTECETPIRNEWG
648


 


1103
SLCTEVECPIRNE
645


 


1104
SLLTECETPIRNC
643


 


1105
SLCTEVCTPIRNEWG
641


 


1106
SLLTEVETPIRNCCG
638


 


1107
SLLTEVETCSLLTEVETPIRNEC
629


 


1108
SLLTECETPIRNEWGC
607


 


1109
SLCTEVETPCRNEWG
607


 


1110
SLLTEVETPCRNEWC
589


 


1111
SLLTEVETPCRNECG
587


 


1112
SLLTEVECPICNE
582


 


1113
SLLTECETPIRNE
578


 


1114
SLCTEVETPICNEWG
578


 


1115
SLLTEVETPCRNC
571


 


1116
SLLTEVCTPIRNE
568


 


1117
SLLTEVETPIRNEWC
558


 


1118
SCLTEVECPIRNEWG
542


 


1119
SLLTEVECPCRNE
538


 


1120
SLCTEVETPCRNE
535


 


1121
SLCTEVETPIRNEWC
535


 


1122
SLLTECETPICNE
531


 


1123
SLLTECETPIRNECG
528


 


1124
SLCTEVETPIRNEWG
510


 


1125
SLLTECECPIRNE
507


 


1126
CMSLLTEVETPIRNC
1864


 


1127
MSLLTEVEKPIRNEWGCRCN
1854


 


1128
MSLLTEVETPCRNC
1834


 


1129
MSLLTEVECPIRNC
1752


 


1130
MSLLTEVCTPIRNC
1738


 


1131
DSLLTEVETPIRNEWGCRCN
1713


 


1132
MSLLTEVETPIRNEAGCRCN
1700


 


1133
MSLLTEVETPCRCE
1651


 


1134
MSLLTEVETPCRNE
1638


 


1135
MSLLTEVEMPIRNEWGCRCN
1580


 


1136
MSLLTEVEAPIRNEWGCRCN
1544


 


1137
MSLLTEVELPIRNEWGCRCN
1521


 


1138
MSLLTELETPIRNEWGCRCN
1497


 


1139
MSLLTEVETPIRNAWGCRCN
1480


 


1140
MSLTTEVETPIRNEWGCRCN
1474


 


1141
MTLLTEVETPIRNEWGCRCN
1470


 


1142
MSLLTEVETPICNC
1451


 


1143
MSLLTEVCTPIRCE
1428


 


1144
MSLLTEVETAIRNEWGCRCN
1398


 


1145
MSLCTEVETPIRNC
1395


 


1146
MSLLTEVETCIRNE
1387


 


1147
MSLLTEVETCICNE
1378


 


1148
MSLLTEVEEPIRNEWGCRCN
1361


 


1149
MSLLTEVETPIRNEWGCRCN
1360


 


1150
MSLLTEVEQPIRNEWGCRCN
1345


 


1151
MSLLTEVECPIRCE
1336


 


1152
MSLLTEKETPIRNEWGCRCN
1324


 


1153
ESLLTEVETPIRNEWGCRCN
1310


 


1154
MSLCTEVETCIRNE
1305


 


1155
MSLLTETETPIRNEWGCRCN
1279


 


1156
MSLLTEIETPIRNEWGCRCN
1255


 


1157
MSLLTEVETPIRNEWGCRAN
1252


 


1158
MSLLTEVCTPCRNE
1244


 


1159
TSLLTEVETPIRNEWGCRCN
1243


 


1160
MSLCTEVCTPIRNE
1243


 


1161
MSLLTEVETPIRNEWGCRCA
1233


 


1162
MSLVTEVETPIRNEWGCRCN
1202


 


1163
MSCLTEVETPIRCE
1192


 


1164
MSLATEVETPIRNEWGCRCN
1188


 


1165
MSLLTEVVTPIRNEWGCRCN
1183


 


1166
MSLLTEVQTPIRNEWGCRCN
1172


 


1167
MSLITEVETPIRNEWGCRCN
1167


 


1168
MSLLTEVECPIRNE
1158


 


1169
MSLLTEVCTPICNE
1157


 


1170
MSLLTEPETPIRNEWGCRCN
1153


 


1171
MSLLTEVEFPIRNEWGCRCN
1149


 


1172
MSLLTEVETPARNEWGCRCN
1149


 


1173
MSLLTEVETPIRNEWGARCN
1149


 


1174
MSLLTEVMTPIRNEWGCRCN
1133


 


1175
MSLLTECETPIRCE
1131


 


1176
CMSLLTEVECPIRNE
1122


 


1177
ASLLTEVETPIRNEWGCRCN
1111


 


1178
MSLLTEVETCIRCE
1105


 


1179
MSCLTEVETPIRNC
1102


 


1180
MSLLTEVEGPIRNEWGCRCN
1099


 


1181
MSLLTEVETPIRNEWACRCN
1087


 


1182
MSLLTEMETPIRNEWGCRCN
1075


 


1183
KSLLTEVETPIRNEWGCRCN
1068


 


1184
MSLLTEVEVPIRNEWGCRCN
1064


 


1185
MSLCTEVETPCRNE
1064


 


1186
MSCLTEVCTPIRNE
1055


 


1187
CMSLLTEVCTPIRNE
1054


 


1188
VSLLTEVETPIRNEWGCRCN
1054


 


1189
MSLLTEVESPIRNEWGCRCN
1051


 


1190
MSLLTEVSTPIRNEWGCRCN
1045


 


1191
MSLLTEFETPIRNEWGCRCN
1044


 


1192
CMSLLTEVETPIRCE
1034


 


1193
MSLCTEVECPIRNE
1023


 


1194
MSLLTECETPICNE
1016


 


1195
MSLLTEVTTPIRNEWGCRCN
1013


 


1196
RSLLTEVETPIRNEWGCRCN
1012


 


1197
MSLLTEVEDPIRNEWGCRCN
1004


 


1198
MSLLTEAETPIRNEWGCRCN
1003


 


1199
CMSLLTEVETPCRNE
992


 


1200
MSLLTEVETPIRAEWGCRCN
978


 


1201
MSLLTEVCTCIRNE
959


 


1202
MSLLTECECPIRNE
958


 


1203
HSLLTEVETPIRNEWGCRCN
956


 


1204
CMSLLTECETPIRNE
937


 


1205
MSLLTEVENPIRNEWGCRCN
936


 


1206
MSLLTECETPCRNE
932


 


1207
MSLLTEVERPIRNEWGCRCN
926


 


1208
MSLLTEQETPIRNEWGCRCN
913


 


1209
MSLLTEVETCIRNC
908


 


1210
MSFLTEVETPIRNEWGCRCN
907


 


1211
CMSLLTEVETCIRNE
896


 


1212
PSLLTEVETPIRNEWGCRCN
841


 


1213
MSLETEVETPIRNEWGCRCN
838


 


1214
MSLLTEVEHPIRNEWGCRCN
838


 


1215
YSLLTEVETPIRNEWGCRCN
835


 


1216
MSLLTEVCTPIRNE
824


 


1217
MSLLTEVECPICNE
821


 


1218
MSLLTEVETPIANEWGCRCN
814


 


1219
LSLLTEVETPIRNEWGCRCN
801


 


1220
WSLLTEVETPIRNEWGCRCN
742


 


1221
MSLLTEVETPIRNEWGCACN
739


 


1222
MSLLTEVECPCRNE
736


 


1223
MSCLTEVECPIRNE
730


 


1224
FSLLTEVETPIRNEWGCRCN
707


 


1225
NSLLTEVETPIRNEWGCRCN
706


 


1226
MSLLTEVEYPIRNEWGCRCN
685


 


1227
MSLLTDVETPIRNEWGCRCN
679


 


1228
MSKLTEVETPIRNEWGCRCN
672


 


1229
MSCLTEVETCIRNE
649


 


1230
MSLSTEVETPIRNEWGCRCN
648


 


1231
MSLLTECETPIRNE
630


 


1232
ISLLTEVETPIRNEWGCRCN
629


 


1233
MSCLTEVETPCRNE
625


 


1234
MSLCTEVETPIRNE
618


 


1235
CMSLLTEVETPICNE
615


 


1236
MSLLTEVEPPIRNEWGCRCN
610


 


1237
MSCLTEVETPIRNE
602


 


1238
GSLLTEVETPIRNEWGCRCN
584


 


1239
MSLCTEVETPIRCE
568


 


1240
MSLKTEVETPIRNEWGCRCN
567


 


1241
MSLLTEEETPIRNEWGCRCN
545


 


1242
CSLCTEVETPIRNE
518


 


1243
QSLLTEVETPIRNEWGCRCN
440


 


1244
MSLLTEVEIPIRNEWGCRCN
409


 


1245
MSLLTESETPIRNEWGCRCN
369


 


1246
MSLLTEGETPIRNEWGCRCN
357


 


1247
MSLLTECETPIRNC
344


 


1248
MSLLTECETCIRNE
312


 


1249
SSLLTEVETPIRNEWGCRCN
303
















TABLE 3







Peptides that show specific binding to Z3G1 (A


numeral “1” in the peptide sequence


indicates a differentially protected cystein


allowing for selective CLIP attachment at


certain residues)









SEQ ID




NO:
Peptides that bind to Z3G1
Signal












1177
ASLLTEVETPIRNEWGCRCN
2816


 


1131
DSLLTEVETPIRNEWGCRCN
2814


 


1153
ESLLTEVETPIRNEWGCRCN
2816


 


1224
FSLLTEVETPIRNEWGCRCN
2665


 


1238
GSLLTEVETPIRNEWGCRCN
2635


 


1203
HSLLTEVETPIRNEWGCRCN
2490


 


1232
ISLLTEVETPIRNEWGCRCN
2438


 


1183
KSLLTEVETPIRNEWGCRCN
2618


 


1219
LSLLTEVETPIRNEWGCRCN
2506


 


1149
MSLLTEVETPIRNEWGCRCN
2711


 


1225
NSLLTEVETPIRNEWGCRCN
2485


 


1212
PSLLTEVETPIRNEWGCRCN
2694


 


1243
QSLLTEVETPIRNEWGCRCN
2619


 


1196
RSLLTEVETPIRNEWGCRCN
2550


 


1249
SSLLTEVETPIRNEWGCRCN
2404


 


1159
TSLLTEVETPIRNEWGCRCN
2834


 


1188
VSLLTEVETPIRNEWGCRCN
2816


 


1220
WSLLTEVETPIRNEWGCRCN
2693


 


1215
YSLLTEVETPIRNEWGCRCN
2650


 


1250
MALLTEVETPIRNEWGCRCN
2604


 


1251
MDLLTEVETPIRNEWGCRCN
2726


 


1252
MELLTEVETPIRNEWGCRCN
2679


 


1253
MFLLTEVETPIRNEWGCRCN
1707


 


1254
MGLLTEVETPIRNEWGCRCN
2635


 


1255
MHLLTEVETPIRNEWGCRCN
2449


 


1256
MILLTEVETPIRNEWGCRCN
1634


 


1257
MKLLTEVETPIRNEWGCRCN
2629


 


1258
MLLLTEVETPIRNEWGCRCN
2091


 


1259
MMLLTEVETPIRNEWGCRCN
2666


 


1260
MNLLTEVETPIRNEWGCRCN
2733


 


1261
MPLLTEVETPIRNEWGCRCN
2521


 


1262
MQLLTEVETPIRNEWGCRCN
2526


 


1263
MRLLTEVETPIRNEWGCRCN
2519


 


1141
MTLLTEVETPIRNEWGCRCN
2809


 


1264
MVLLTEVETPIRNEWGCRCN
2465


 


1265
MWLLTEVETPIRNEWGCRCN
2615


 


1266
MYLLTEVETPIRNEWGCRCN
1960


 


1210
MSFLTEVETPIRNEWGCRCN
1087


 


1267
MSILTEVETPIRNEWGCRCN
2582


 


1268
MSMLTEVETPIRNEWGCRCN
1329


 


1269
MSVLTEVETPIRNEWGCRCN
2111


 


1164
MSLATEVETPIRNEWGCRCN
2747


 


1270
MSLDTEVETPIRNEWGCRCN
2594


 


1213
MSLETEVETPIRNEWGCRCN
2768


 


1271
MSLFTEVETPIRNEWGCRCN
2115


 


1272
MSLHTEVETPIRNEWGCRCN
2058


 


1167
MSLITEVETPIRNEWGCRCN
2684


 


1240
MSLKTEVETPIRNEWGCRCN
2691


 


1273
MSLMTEVETPIRNEWGCRCN
2804


 


1274
MSLNTEVETPIRNEWGCRCN
2787


 


1275
MSLPTEVETPIRNEWGCRCN
2007


 


1276
MSLQTEVETPIRNEWGCRCN
2792


 


1277
MSLRTEVETPIRNEWGCRCN
2554


 


1230
MSLSTEVETPIRNEWGCRCN
2768


 


1140
MSLTTEVETPIRNEWGCRCN
2742


 


1162
MSLVTEVETPIRNEWGCRCN
2776


 


1278
MSLYTEVETPIRNEWGCRCN
1313


 


1279
MSLLAEVETPIRNEWGCRCN
2807


 


1280
MSLLDEVETPIRNEWGCRCN
2763


 


1281
MSLLEEVETPIRNEWGCRCN
2805


 


1282
MSLLFEVETPIRNEWGCRCN
2581


 


1283
MSLLGEVETPIRNEWGCRCN
2717


 


1284
MSLLHEVETPIRNEWGCRCN
2691


 


1285
MSLLIEVETPIRNEWGCRCN
2371


 


1286
MSLLKEVETPIRNEWGCRCN
2825


 


1287
MSLLLEVETPIRNEWGCRCN
2804


 


1288
MSLLMEVETPIRNEWGCRCN
2847


 


1289
MSLLNEVETPIRNEWGCRCN
2824


 


1290
MSLLPEVETPIRNEWGCRCN
2728


 


1291
MSLLQEVETPIRNEWGCRCN
2792


 


1292
MSLLREVETPIRNEWGCRCN
2699


 


1293
MSLLSEVETPIRNEWGCRCN
2790


 


1294
MSLLVEVETPIRNEWGCRCN
2732


 


1295
MSLLWEVETPIRNEWGCRCN
2531


 


1296
MSLLYEVETPIRNEWGCRCN
2464


 


1227
MSLLTDVETPIRNEWGCRCN
2476


 


1297
MSLLTQVETPIRNEWGCRCN
1697


 


1298
MSLLTTVETPIRNEWGCRCN
2699


 


1156
MSLLTEIETPIRNEWGCRCN
2799


 


1138
MSLLTELETPIRNEWGCRCN
2589


 


1155
MSLLTETETPIRNEWGCRCN
2641


 


1299
MSLLTEVATPIRNEWGCRCN
2633


 


1300
MSLLTEVDTPIRNEWGCRCN
2856


 


1301
MSLLTEVFTPIRNEWGCRCN
1858


 


1302
MSLLTEVGTPIRNEWGCRCN
2789


 


1303
MSLLTEVHTPIRNEWGCRCN
2751


 


1304
MSLLTEVITPIRNEWGCRCN
2668


 


1305
MSLLTEVKTPIRNEWGCRCN
2708


 


1306
MSLLTEVLTPIRNEWGCRCN
2714


 


1174
MSLLTEVMTPIRNEWGCRCN
2813


 


1307
MSLLTEVNTPIRNEWGCRCN
2807


 


1308
MSLLTEVPTPIRNEWGCRCN
2380


 


1166
MSLLTEVQTPIRNEWGCRCN
2813


 


1309
MSLLTEVRTPIRNEWGCRCN
2668


 


1190
MSLLTEVSTPIRNEWGCRCN
2783


 


1195
MSLLTEVTTPIRNEWGCRCN
2791


 


1165
MSLLTEVVTPIRNEWGCRCN
2732


 


1310
MSLLTEVWTPIRNEWGCRCN
2546


 


1311
MSLLTEVYTPIRNEWGCRCN
2181


 


1136
MSLLTEVEAPIRNEWGCRCN
2833


 


1197
MSLLTEVEDPIRNEWGCRCN
2826


 


1148
MSLLTEVEEPIRNEWGCRCN
2835


 


1171
MSLLTEVEFPIRNEWGCRCN
2728


 


1180
MSLLTEVEGPIRNEWGCRCN
2802


 


1214
MSLLTEVEHPIRNEWGCRCN
2753


 


1244
MSLLTEVEIPIRNEWGCRCN
2742


 


1127
MSLLTEVEKPIRNEWGCRCN
2837


 


1137
MSLLTEVELPIRNEWGCRCN
2797


 


1135
MSLLTEVEMPIRNEWGCRCN
2839


 


1205
MSLLTEVENPIRNEWGCRCN
2805


 


1236
MSLLTEVEPPIRNEWGCRCN
2797


 


1150
MSLLTEVEQPIRNEWGCRCN
2826


 


1207
MSLLTEVERPIRNEWGCRCN
2753


 


1189
MSLLTEVESPIRNEWGCRCN
2712


 


1184
MSLLTEVEVPIRNEWGCRCN
2768


 


1312
MSLLTEVEWPIRNEWGCRCN
2541


 


1226
MSLLTEVEYPIRNEWGCRCN
2571


 


1144
MSLLTEVETAIRNEWGCRCN
2722


 


1172
MSLLTEVETPARNEWGCRCN
2774


 


1218
MSLLTEVETPIANEWGCRCN
2683


 


1200
MSLLTEVETPIRAEWGCRCN
2742


 


1139
MSLLTEVETPIRNAWGCRCN
2803


 


1132
MSLLTEVETPIRNEAGCRCN
2831


 


1181
MSLLTEVETPIRNEWACRCN
2732


 


1173
MSLLTEVETPIRNEWGARCN
2760


 


1221
MSLLTEVETPIRNEWGCACN
2662


 


1157
MSLLTEVETPIRNEWGCRAN
2769


 


1161
MSLLTEVETPIRNEWGCRCA
2770


 


1126
CMSLLTEVETPIRNC
2767


 


1192
CMSLLTEVETPIRCE
2453


 


1235
CMSLLTEVETPICNE
2331


 


1199
CMSLLTEVETPCRNE
2789


 


1211
CMSLLTEVETCIRNE
2713


 


1176
CMSLLTEVECPIRNE
2730


 


1187
CMSLLTEVCTPIRNE
2633


 


1313
CMSLLCEVETPIRNE
2582


 


1314
CMSLCTEVETPIRNE
1705


 


1315
CMCLLTEVETPIRNE
2347


 


984
CSLLTEVETPIRNC
2782


 


997
CSLLTEVETPIRCE
2516


 


1012
CSLLTEVETPICNE
2444


 


1020
CSLLTEVETPCRNE
2470


 


1013
CSLLTEVETCIRNE
2301


 


1024
CSLLTEVECPIRNE
2226


 


990
CSLLTEVCTPIRNE
1942


 


1316
CSLLCEVETPIRNE
2527


 


1242
CSLCTEVETPIRNE
1832


 


1317
MCLLTEVETPIRNC
2743


 


1318
MCLLTEVETPIRCE
2307


 


1319
MCLLTEVETPICNE
2379


 


1320
MCLLTEVETPCRNE
2404


 


1321
MCLLTEVETCIRNE
1948


 


1322
MCLLTEVECPIRNE
2421


 


1323
MCLLTEVCTPIRNE
1407


 


1324
MCLLCEVETPIRNE
2038


 


1145
MSLCTEVETPIRNC
2601


 


1239
MSLCTEVETPIRCE
1555


 


1185
MSLCTEVETPCRNE
1673


 


1193
MSLCTEVECPIRNE
1538


 


1160
MSLCTEVCTPIRNE
1906


 


1325
MSLLCEVETPIRNC
2823


 


1326
MSLLCEVETPIRCE
2772


 


1327
MSLLCEVETPICNE
2704


 


1328
MSLLCEVETPCRNE
2652


 


1329
MSLLCEVETCIRNE
2589


 


1330
MSLLCEVECPIRNE
2606


 


1331
MSLLCEVCTPIRNE
2601


 


1130
MSLLTEVCTPIRNC
2787


 


1143
MSLLTEVCTPIRCE
2667


 


1169
MSLLTEVCTPICNE
2526


 


1158
MSLLTEVCTPCRNE
2572


 


1201
MSLLTEVCTCIRNE
2143


 


1168
MSLLTEVECPIRNE
2613


 


1129
MSLLTEVECPIRNC
2755


 


1151
MSLLTEVECPIRCE
2675


 


1217
MSLLTEVECPICNE
2440


 


1222
MSLLTEVECPCRNE
2361


 


1146
MSLLTEVETCIRNE
2129


 


1209
MSLLTEVETCIRNC
2309


 


1178
MSLLTEVETCIRCE
2739


 


1147
MSLLTEVETCICNE
2721


 


1134
MSLLTEVETPCRNE
2695


 


1128
MSLLTEVETPCRNC
2810


 


1133
MSLLTEVETPCRCE
2597


 


1142
MSLLTEVETPICNC
2758


 


1018
CSLLTEVETPIRNE
2211


 


1332
MCLLTEVETPIRNE
2364


 


1234
MSLCTEVETPIRNE
2054


 


1333
MSLLCEVETPIRNE
2454


 


1216
MSLLTEVCTPIRNE
2529


 


287
MSLLTEVGMSLLTEV
2878


 


293
CSLLTEVGMSLLTEV
2827


 


513
MCLLTEVGMSLLTEV
2697


 


532
MSCLTEVGMSLLTEV
2336


 


371
MSLCTEVGMSLLTEV
2691


 


749
MSLLCEVGMSLLTEV
2786


 


543
MSLLTCVGMSLLTEV
2627


 


339
MSLLTECGMSLLTEV
2170


 


288
CSCLCEVGMSLLTEV
1812


 


663
MCLCTCVGMSLLTEV
2509


 


294
MSCLCECGMSLLTEV
1006


 


373
CSLLTECGMSLLTEV
1235


 


316
MSLLTEVGCSLLTEV
2650


 


364
CSLLTEVGCSLLTEV
2503


 


570
MCLLTEVGCSLLTEV
2717


 


557
MSCLTEVGCSLLTEV
2506


 


508
MSLCTEVGCSLLTEV
2832


 


781
MSLLCEVGCSLLTEV
2761


 


528
MSLLTCVGCSLLTEV
2467


 


363
MSLLTECGCSLLTEV
1235


 


536
MCLCTCVGCSLLTEV
2241


 


360
CSLLTECGCSLLTEV
1427


 


296
MSLLTEVGMCLLTEV
2809


 


303
CSLLTEVGMCLLTEV
2618


 


1334
MSLLCEVGMCLLTEV
2676


 


359
MSLLTEVGMSCLTEV
2793


 


372
CSLLTEVGMSCLTEV
2554


 


1335
MCLLTEVGMSCLTEV
1869


 


1336
MSLLCEVGMSCLTEV
2846


 


317
MSLLTEVGMSLCTEV
2895


 


334
CSLLTEVGMSLCTEV
2857


 


1337
MCLLTEVGMSLCTEV
2513


 


289
MSLCTEVGMSLCTEV
1093


 


1338
MSLLCEVGMSLCTEV
2595


 


793
MSLLTEVGMSLLCEV
2819


 


309
CSLLTEVGMSLLCEV
2734


 


1339
MCLLTEVGMSLLCEV
2234


 


1340
MSCLTEVGMSLLCEV
2341


 


304
MSLCTEVGMSLLCEV
2616


 


1341
MSLLCEVGMSLLCEV
2851


 


1342
MSLLTCVGMSLLCEV
2712


 


460
MSLLTECGMSLLCEV
2373


 


1343
CSCLCEVGMSLLCEV
1945


 


1344
MCLCTCVGMSLLCEV
2431


 


1345
MSCLCECGMSLLCEV
1307


 


456
CSLLTECGMSLLCEV
1640


 


285
MSLLTEVGMSLLTCV
2872


 


286
CSLLTEVGMSLLTCV
2768


 


1346
MCLLTEVGMSLLTCV
1098


 


542
MSLCTEVGMSLLTCV
2722


 


1347
MSLLCEVGMSLLTCV
2784


 


343
MSLLTEVGMSLLTEC
2831


 


451
CSLLTEVGMSLLTEC
2613


 


825
MSLLCEVGMSLLTEC
2617


 


761
MSLLTEVGCSCLCEV
2053


 


476
CSLLTEVGCSCLCEV
1680


 


1348
MSLCTEVGCSCLCEV
2001


 


1349
MSLLCEVGCSCLCEV
2586


 


291
MSLLTEVGMCLCTCV
2842


 


297
CSLLTEVGMCLCTCV
2649


 


1350
MSLLCEVGMCLCTCV
2664


 


829
MSLLTEVGMSCLCEC
2444


 


499
CSLLTEVGMSCLCEC
2101


 


1351
MSLLCEVGMSCLCEC
2492


 


470
MSLLTEVGCSLLTEC
2349


 


383
CSLLTEVGCSLLTEC
2486


 


592
MSLCTEVGCSLLTEC
1971


 


826
MSLLCEVGCSLLTEC
2573


 


365
CSLLTEVCMSLLTEC
1891


 


1352
CCLLTEVETPIRNE
2155


 


1353
CLLTEVETPIRNC
1996


 


1354
CLLTEVETPIRCE
1813


 


1355
CLLTEVETPICNE
1647


 


1356
CLLTEVETPCRNE
1991


 


1357
CLLTEVETCIRNE
1661


 


1358
CLLTEVECPIRNE
2326


 


1359
CLLTEVCTPIRNE
1585


 


1360
CLLCEVETPIRNE
2314


 


1100
SLCTEVCTPIRNE
1674


 


1361
SLLCEVETPIRNC
2460


 


1362
SLLCEVETPIRCE
2307


 


1363
SLLCEVETPICNE
1921


 


1364
SLLCEVETPCRNE
2035


 


1365
SLLCEVETCIRNE
2305


 


1366
SLLCEVECPIRNE
2377


 


1367
SLLCEVCTPIRNE
2283


 


1016
SLLTEVCTPIRNC
2308


 


1073
SLLTEVCTPIRCE
1873


 


1091
SLLTEVCTPICNE
1659


 


1065
SLLTEVCTPCRNE
2016


 


1044
SLLTEVCTCIRNE
1695


 


1050
SLLTEVECPIRNE
2469


 


1007
SLLTEVECPIRNC
2635


 


1035
SLLTEVECPIRCE
2426


 


1112
SLLTEVECPICNE
2022


 


1119
SLLTEVECPCRNE
1840


 


1093
SLLTEVETCIRNE
1792


 


1022
SLLTEVETCIRNC
2289


 


1055
SLLTEVETCIRCE
2161


 


1084
SLLTEVETCICNE
1953


 


1368
SLLTEVETPCRNE
1665


 


1115
SLLTEVETPCRNC
1816


 


1369
SLLTEVETPCRCE
1546


 


1096
SLLTEVETPICNC
2075


 


1370
CLLTEVETPIRNE
1524


 


1371
SLLCEVETPIRNE
2511


 


1116
SLLTEVCTPIRNE
1854


 


1072
CSLLTEVETPIRNEWGC
2232


 


1372
CLLTEVETPIRNEWGC
1866


 


1373
SLLCEVETPIRNEWGC
2281


 


1081
SLLTEVCTPIRNEWGC
1866


 


1086
SLLTEVECPIRNEWGC
2142


 


991
SLLTEVETCIRNEWGC
2284


 


1092
SLLTEVETPCRNEWGC
1873


 


1051
SLLTEVETPICNEWGC
2101


 


1021
SLLTEVETPIRCEWGC
2435


 


1015
SLLTEVETPIRNCWGC
2513


 


1374
SLLTEVETPIRNECGC
1660


 


1375
SLLTEVETPIRNEWCC
1813


 


1376
CLLTEVETPIRNEWC
1743


 


1377
CLLTEVETPIRNECG
1501


 


1378
CLLTEVETPIRNCWG
2388


 


1379
CLLTEVETPIRCEWG
2169


 


1380
CLLTEVETPICNEWG
2120


 


1381
CLLTEVETPCRNEWG
2074


 


1382
CLLTEVETCIRNEWG
2296


 


1383
CLLTEVECPIRNEWG
2157


 


1384
CLLCEVETPIRNEWG
2388


 


1385
CLLTEVETPIRNEWG
2142


 


1105
SLCTEVCTPIRNEWG
1477


 


1386
SLCCEVETPIRNEWG
1603


 


1387
SLLCEVETPIRNEWC
2406


 


1388
SLLCEVETPIRNECG
2107


 


1389
SLLCEVETPIRNCWG
2789


 


1390
SLLCEVETPIRCEWG
2601


 


1391
SLLCEVETPICNEWG
2536


 


1392
SLLCEVETPCRNEWG
2465


 


1393
SLLCEVETCIRNEWG
2531


 


1394
SLLCEVECPIRNEWG
2529


 


1395
SLLCEVCTPIRNEWG
2384


 


1396
SLLCEVETPIRNEWG
2384


 


1049
SLLTEVCTPIRNEWC
1883


 


1094
SLLTEVCTPIRNECG
1782


 


1000
SLLTEVCTPIRNCWG
2420


 


988
SLLTEVCTPIRCEWG
2090


 


987
SLLTEVCTPICNEWG
1883


 


996
SLLTEVCTPCRNEWG
1841


 


985
SLLTEVCTCIRNEWG
1546


 


1014
SLLTEVCCPIRNEWG
2253


 


1019
SLLTEVCTPIRNEWG
2692


 


1397
CLLTEVCTPIRNEWG
1634


 


1017
SLLTEVECPIRNEWC
2141


 


1099
SLLTEVECPIRNECG
2001


 


1001
SLLTEVECPIRNCWG
2589


 


989
SLLTEVECPIRCEWG
2509


 


1028
SLLTEVECPICNEWG
2449


 


1025
SLLTEVECPCRNEWG
2303


 


1004
SLLTEVECCIRNEWG
2507


 


1041
SLLTEVECPIRNEWG
2267


 


1030
SLLTEVETCIRNEWC
2337


 


1046
SLLTEVETCIRNECG
2018


 


992
SLLTEVETCIRNCWG
2616


 


998
SLLTEVETCIRCEWG
2566


 


1006
SLLTEVETCICNEWG
2498


 


993
SLLTEVETCCRNEWG
2389


 


1009
SLLTEVETCIRNEWG
2462


 


1110
SLLTEVETPCRNEWC
1867


 


1111
SLLTEVETPCRNECG
1470


 


1005
SLLTEVETPCRNCWG
2602


 


1061
SLLTEVETPCRCEWG
2155


 


1077
SLLTEVETPCCNEWG
2208


 


1071
SLLTEVETPCRNEWG
2167


 


1085
SLLTEVETPICNEWC
2488


 


1098
SLLTEVETPICNECG
2041


 


1010
SLLTEVETPICNCWG
2624


 


1034
SLLTEVETPICCEWG
2525


 


1057
SLLTEVETPICNEWG
2395


 


1082
SLLTEVETPIRCEWC
2255


 


1023
SLLTEVETPIRCECG
2464


 


994
SLLTEVETPIRCCWG
2755


 


1053
SLLTEVETPIRCEWG
2365


 


1039
SLLTEVETPIRNCWC
2321


 


1106
SLLTEVETPIRNCCG
1923


 


1036
SLLTEVETPIRNCWG
2350


 


1398
SLLTEVETPIRNECC
1493


 


1399
SLLTEVETPIRNECG
1545


 


1117
SLLTEVETPIRNEWC
1856


 


1070
SLLTEVETCSLLTEVETC
2104


 


1067
SLLTEVETCSLLTEVETPC
2082


 


1069
SLLTEVETCSLLTEVETPIC
2169


 


1040
SLLTEVETCSLLTEVETPIRC
2402


 


1058
SLLTEVETCSLLTEVETPIRNC
2295


 


1107
SLLTEVETCSLLTEVETPIRNEC
2025


 


1076
SLLTEVETCSLLTEVETPIRNEWC
2169


 


1048
SLLTEVETCSLLTEVETPIRNEWGC
2010


 


1088
SLLTEVETCSLLTEVETPIRNEWGSC
2180


 


986
SLLTEVETCSLLTEVETPIRNEWGSRC
2757


 


999
SLLTEVETCSLLTEVETPIRNEWGSRSC
2792


 


1011
SLLTEVETCSLLTEVETPIRNEWGSRSNC
2673


 


1062
SLLTEVETCSLLTEVETPIRNEWGSRSNDC
2483


 


1064
SLLTEVETCSLLTEVETPIRNEWGSRSNDSC
2335


 


1052
SLLTEVETCSLLTEVETPIRNEWGSRSNDSSC
2404


 


1068
SLLTEVETCSLLTEVETPIRNEWGSRSNDSSDC
2294


 


1032
SLLTEVETCSLLTEVETPIRNEWGCR
2395


 


1042
SLLTEVETSLLTEVETPIRNEWGCRC
2324


 


1043
SLLTEVETSLLTEVETPIRNEWGCRCN
2390


 


995
SLLTEVETSLLTEVETPIRNEWGCRCND
2393


 


995
SLLTEVETSLLTEVETPIRNEWGCRCNDS
2260


 


1089
SLLTEVETSLLTEVETPIRNEWGCRCNDSS
2212


 


1095
SLLTEVETSLLTEVETPIRNEWGCRCNDSSD
1980


 


831
1CSLLTEVETP1SLLTEVETPCSLLTEVETP1
2714


 


867
1CLLTEVETPI1SLLTEVETPCSLLTEVETP1
2593


 


836
1CLTEVETPIR1SLLTEVETPCSLLTEVETP1
2645


 


839
1CTEVETPIRN1SLLTEVETPCSLLTEVETP1
2420


 


848
1CEVETPIRNE1SLLTEVETPCSLLTEVETP1
2460


 


860
1CVETPIRNEW1SLLTEVETPCSLLTEVETP1
2474


 


858
1CETPIRNEWG1SLLTEVETPCSLLTEVETP1
2566


 


900
1CSLLTEVETP1LLTEVETPICSLLTEVETP1
2437


 


1400
1CLLTEVETPI1LLTEVETPICSLLTEVETP1
2220


 


1401
1CLTEVETPIR1LLTEVETPICSLLTEVETP1
2316


 


1402
1CTEVETPIRN1LLTEVETPICSLLTEVETP1
2258


 


1403
1CEVETPIRNE1LLTEVETPICSLLTEVETP1
2133


 


977
1CVETPIRNEW1LLTEVETPICSLLTEVETP1
1946


 


1404
1CETPIRNEWG1LLTEVETPICSLLTEVETP1
2288


 


841
1CSLLTEVETP1LTEVETPIRCSLLTEVETP1
2521


 


849
1CLLTEVETPI1LTEVETPIRCSLLTEVETP1
2601


 


863
1CLTEVETPIR1LTEVETPIRCSLLTEVETP1
2476


 


854
1CTEVETPIRN1LTEVETPIRCSLLTEVETP1
2469


 


872
1CEVETPIRNE1LTEVETPIRCSLLTEVETP1
2304


 


876
1CVETPIRNEW1LTEVETPIRCSLLTEVETP1
2350


 


882
1CETPIRNEWG1LTEVETPIRCSLLTEVETP1
2282


 


842
1CSLLTEVETP1TEVETPIRNCSLLTEVETP1
2591


 


883
1CLLTEVETPI1TEVETPIRNCSLLTEVETP1
2492


 


880
1CLTEVETPIR1TEVETPIRNCSLLTEVETP1
2396


 


887
1CTEVETPIRN1TEVETPIRNCSLLTEVETP1
2405


 


921
1CEVETPIRNE1TEVETPIRNCSLLTEVETP1
2272


 


953
1CVETPIRNEW1TEVETPIRNCSLLTEVETP1
2118


 


919
1CETPIRNEWG1TEVETPIRNCSLLTEVETP1
2223


 


869
1CSLLTEVETP1EVETPIRNECSLLTEVETP1
2582


 


967
1CLLTEVETPI1EVETPIRNECSLLTEVETP1
2220


 


961
1CLTEVETPIR1EVETPIRNECSLLTEVETP1
2331


 


978
1CTEVETPIRN1EVETPIRNECSLLTEVETP1
2295


 


870
1CEVETPIRNE1EVETPIRNECSLLTEVETP1
2618


 


963
1CVETPIRNEW1EVETPIRNECSLLTEVETP1
2365


 


936
1CETPIRNEWG1EVETPIRNECSLLTEVETP1
2368


 


851
1CSLLTEVETP1VETPIRNEWCSLLTEVETP1
2708


 


943
1CLLTEVETPI1VETPIRNEWCSLLTEVETP1
2422


 


932
1CLTEVETPIR1VETPIRNEWCSLLTEVETP1
2344


 


952
1CTEVETPIRN1VETPIRNEWCSLLTEVETP1
2380


 


934
1CEVETPIRNE1VETPIRNEWCSLLTEVETP1
2448


 


955
1CVETPIRNEW1VETPIRNEWCSLLTEVETP1
2307


 


929
1CETPIRNEWG1VETPIRNEWCSLLTEVETP1
2419


 


885
1CSLLTEVETP1ETPIRNEWGCSLLTEVETP1
2591


 


968
1CLLTEVETPI1ETPIRNEWGCSLLTEVETP1
2377


 


902
1CLTEVETPIR1ETPIRNEWGCSLLTEVETP1
2296


 


1405
1CTEVETPIRN1ETPIRNEWGCSLLTEVETP1
2268


 


903
1CEVETPIRNE1ETPIRNEWGCSLLTEVETP1
2140


 


877
1CVETPIRNEW1ETPIRNEWGCSLLTEVETP1
2349


 


896
1CETPIRNEWG1ETPIRNEWGCSLLTEVETP1
2425


 


830
1CSLLTEVETP1SLLTEVETPCLLTEVETPI1
2786


 


847
1CLLTEVETPI1SLLTEVETPCLLTEVETPI1
2722


 


832
1CLTEVETPIR1SLLTEVETPCLLTEVETPI1
2711


 


838
1CTEVETPIRN1SLLTEVETPCLLTEVETPI1
2616


 


855
1CEVETPIRNE1SLLTEVETPCLLTEVETPI1
2576


 


871
1CVETPIRNEW1SLLTEVETPCLLTEVETPI1
2544


 


837
1CETPIRNEWG1SLLTEVETPCLLTEVETPI1
2653


 


923
1CSLLTEVETP1LLTEVETPICLLTEVETPI1
2534


 


1406
1CLLTEVETPI1LLTEVETPICLLTEVETPI1
2350


 


1407
1CLTEVETPIR1LLTEVETPICLLTEVETPI1
2316


 


1408
1CTEVETPIRN1LLTEVETPICLLTEVETPI1
2446


 


1409
1CEVETPIRNE1LLTEVETPICLLTEVETPI1
2194


 


1410
1CVETPIRNEW1LLTEVETPICLLTEVETPI1
2331


 


1411
1CETPIRNEWG1LLTEVETPICLLTEVETPI1
2405


 


916
1CSLLTEVETP1LTEVETPIRCLLTEVETPI1
2722


 


1412
1CLLTEVETPI1LTEVETPIRCLLTEVETPI1
2436


 


1413
1CLTEVETPIR1LTEVETPIRCLLTEVETPI1
1445


 


1414
1CTEVETPIRN1LTEVETPIRCLLTEVETPI1
1793


 


1415
1CEVETPIRNE1LTEVETPIRCLLTEVETPI1
1407


 


1416
1CVETPIRNEW1LTEVETPIRCLLTEVETPI1
1369


 


1417
1CETPIRNEWG1LTEVETPIRCLLTEVETPI1
1474


 


878
1CSLLTEVETP1TEVETPIRNCLLTEVETPI1
2664


 


1418
1CLLTEVETPI1TEVETPIRNCLLTEVETPI1
2371


 


1419
1CLTEVETPIR1TEVETPIRNCLLTEVETPI1
1907


 


1420
1CTEVETPIRN1TEVETPIRNCLLTEVETPI1
1905


 


1421
1CEVETPIRNE1TEVETPIRNCLLTEVETPI1
1469


 


1422
1CVETPIRNEW1TEVETPIRNCLLTEVETPI1
1452


 


1423
1CETPIRNEWG1TEVETPIRNCLLTEVETPI1
1735


 


889
1CSLLTEVETP1EVETPIRNECLLTEVETPI1
2595


 


1424
1CLLTEVETPI1EVETPIRNECLLTEVETPI1
2331


 


1425
1CLTEVETPIR1EVETPIRNECLLTEVETPI1
1049


 


1426
1CTEVETPIRN1EVETPIRNECLLTEVETPI1
1107


 


873
1CSLLTEVETP1VETPIRNEWCLLTEVETPI1
2767


 


1427
1CLLTEVETPI1VETPIRNEWCLLTEVETPI1
2410


 


1428
1CLTEVETPIR1VETPIRNEWCLLTEVETPI1
1465


 


1429
1CTEVETPIRN1VETPIRNEWCLLTEVETPI1
1559


 


1430
1CEVETPIRNE1VETPIRNEWCLLTEVETPI1
1477


 


1431
1CVETPIRNEW1VETPIRNEWCLLTEVETPI1
1588


 


1432
1CETPIRNEWG1VETPIRNEWCLLTEVETPI1
1453


 


937
1CSLLTEVETP1ETPIRNEWGCLLTEVETPI1
2646


 


1433
1CLLTEVETPI1ETPIRNEWGCLLTEVETPI1
2361


 


1434
1CLTEVETPIR1ETPIRNEWGCLLTEVETPI1
1699


 


1435
1CTEVETPIRN1ETPIRNEWGCLLTEVETPI1
1573


 


1436
1CEVETPIRNE1ETPIRNEWGCLLTEVETPI1
1434


 


1437
1CVETPIRNEW1ETPIRNEWGCLLTEVETPI1
1582


 


1438
1CETPIRNEWG1ETPIRNEWGCLLTEVETPI1
1968


 


1439
1CSLLTEVETP1SLLTEVETPCLTEVETPIR1
2738


 


949
1CLLTEVETPI1SLLTEVETPCLTEVETPIR1
2403


 


857
1CLTEVETPIR1SLLTEVETPCLTEVETPIR1
2755


 


1440
1CTEVETPIRN1SLLTEVETPCLTEVETPIR1
2705


 


897
1CEVETPIRNE1SLLTEVETPCLTEVETPIR1
2565


 


843
1CVETPIRNEW1SLLTEVETPCLTEVETPIR1
2498


 


910
1CETPIRNEWG1SLLTEVETPCLTEVETPIR1
2473


 


892
1CSLLTEVETP1LLTEVETPICLTEVETPIR1
2546


 


1441
1CLLTEVETPI1LLTEVETPICLTEVETPIR1
2288


 


1442
1CLTEVETPIR1LLTEVETPICLTEVETPIR1
2057


 


1443
1CTEVETPIRN1LLTEVETPICLTEVETPIR1
2213


 


1444
1CEVETPIRNE1LLTEVETPICLTEVETPIR1
2000


 


1445
1CVETPIRNEW1LLTEVETPICLTEVETPIR1
2280


 


1446
1CETPIRNEWG1LLTEVETPICLTEVETPIR1
2367


 


886
1CSLLTEVETP1LTEVETPIRCLTEVETPIR1
2623


 


1447
1CLLTEVETPI1LTEVETPIRCLTEVETPIR1
2283


 


944
1CSLLTEVETP1TEVETPIRNCLTEVETPIR1
2646


 


1448
1CLLTEVETPI1TEVETPIRNCLTEVETPIR1
2440


 


1449
1CSLLTEVETP1EVETPIRNECLTEVETPIR1
2404


 


1450
1CLLTEVETPI1EVETPIRNECLTEVETPIR1
2188


 


1451
1CSLLTEVETP1VETPIRNEWCLTEVETPIR1
2549


 


1452
1CLLTEVETPI1VETPIRNEWCLTEVETPIR1
2607


 


1453
1CSLLTEVETP1ETPIRNEWGCLTEVETPIR1
2465


 


1454
1CLLTEVETPI1ETPIRNEWGCLTEVETPIR1
2218


 


918
1CSLLTEVETP1SLLTEVETPCTEVETPIRN1
2618


 


1455
1CLLTEVETPI1SLLTEVETPCTEVETPIRN1
2283


 


946
1CLTEVETPIR1SLLTEVETPCTEVETPIRN1
2434


 


957
1CTEVETPIRN1SLLTEVETPCTEVETPIRN1
2402


 


941
1CEVETPIRNE1SLLTEVETPCTEVETPIRN1
2692


 


947
1CVETPIRNEW1SLLTEVETPCTEVETPIRN1
2733


 


942
1CETPIRNEWG1SLLTEVETPCTEVETPIRN1
2601


 


1456
1CSLLTEVETP1LLTEVETPICTEVETPIRN1
2103


 


1457
1CLLTEVETPI1LLTEVETPICTEVETPIRN1
1884


 


1458
1CLTEVETPIR1LLTEVETPICTEVETPIRN1
1756


 


1459
1CTEVETPIRN1LLTEVETPICTEVETPIRN1
1749


 


1460
1CEVETPIRNE1LLTEVETPICTEVETPIRN1
1620


 


1461
1CVETPIRNEW1LLTEVETPICTEVETPIRN1
1835


 


1462
1CETPIRNEWG1LLTEVETPICTEVETPIRN1
1710


 


971
1CSLLTEVETP1LTEVETPIRCTEVETPIRN1
2360


 


1463
1CLLTEVETPI1LTEVETPIRCTEVETPIRN1
2188


 


1464
1CSLLTEVETP1TEVETPIRNCTEVETPIRN1
2755


 


1465
1CLLTEVETPI1TEVETPIRNCTEVETPIRN1
2488


 


1466
1CSLLTEVETP1EVETPIRNECTEVETPIRN1
2134


 


1467
1CLLTEVETPI1EVETPIRNECTEVETPIRN1
1851


 


1468
1CSLLTEVETP1VETPIRNEWCTEVETPIRN1
2301


 


1469
1CLLTEVETPI1VETPIRNEWCTEVETPIRN1
1713


 


1470
1CSLLTEVETP1ETPIRNEWGCTEVETPIRN1
2331


 


1471
1CLLTEVETPI1ETPIRNEWGCTEVETPIRN1
1983


 


958
1CSLLTEVETP1SLLTEVETPCEVETPIRNE1
2203


 


1472
1CLLTEVETPI1SLLTEVETPCEVETPIRNE1
2057


 


976
1CLTEVETPIR1SLLTEVETPCEVETPIRNE1
2216


 


913
1CTEVETPIRN1SLLTEVETPCEVETPIRNE1
2139


 


1473
1CEVETPIRNE1SLLTEVETPCEVETPIRNE1
2006


 


1474
1CVETPIRNEW1SLLTEVETPCEVETPIRNE1
1986


 


1475
1CETPIRNEWG1SLLTEVETPCEVETPIRNE1
2642


 


1476
1CSLLTEVETP1LLTEVETPICEVETPIRNE1
2472


 


1477
1CLLTEVETPI1LLTEVETPICEVETPIRNE1
2022


 


1478
1CLTEVETPIR1LLTEVETPICEVETPIRNE1
1681


 


1479
1CTEVETPIRN1LLTEVETPICEVETPIRNE1
1627


 


1480
1CEVETPIRNE1LLTEVETPICEVETPIRNE1
1394


 


1481
1CVETPIRNEW1LLTEVETPICEVETPIRNE1
1592


 


1482
1CETPIRNEWG1LLTEVETPICEVETPIRNE1
1648


 


1483
1CSLLTEVETP1LTEVETPIRCEVETPIRNE1
2040


 


1484
1CLLTEVETPI1LTEVETPIRCEVETPIRNE1
1777


 


1485
1CSLLTEVETP1TEVETPIRNCEVETPIRNE1
2036


 


1486
1CLLTEVETPI1TEVETPIRNCEVETPIRNE1
1798


 


1487
1CSLLTEVETP1EVETPIRNECEVETPIRNE1
1917


 


1488
1CLLTEVETPI1EVETPIRNECEVETPIRNE1
1631


 


1489
1CSLLTEVETP1VETPIRNEWCEVETPIRNE1
2359


 


1490
1CLLTEVETPI1VETPIRNEWCEVETPIRNE1
1730


 


1491
1CSLLTEVETP1ETPIRNEWGCEVETPIRNE1
2338


 


1492
1CLLTEVETPI1ETPIRNEWGCEVETPIRNE1
1781


 


850
1CSLLTEVETP1SLLTEVETPCVETPIRNEW1
2491


 


975
1CLLTEVETPI1SLLTEVETPCVETPIRNEW1
2316


 


945
1CLTEVETPIR1SLLTEVETPCVETPIRNEW1
2366


 


951
1CTEVETPIRN1SLLTEVETPCVETPIRNEW1
2460


 


959
1CEVETPIRNE1SLLTEVETPCVETPIRNEW1
2441


 


935
1CVETPIRNEW1SLLTEVETPCVETPIRNEW1
2470


 


950
1CETPIRNEWG1SLLTEVETPCVETPIRNEW1
2465


 


974
1CSLLTEVETP1LLTEVETPICVETPIRNEW1
2285


 


1493
1CLLTEVETPI1LLTEVETPICVETPIRNEW1
2661


 


1494
1CLTEVETPIR1LLTEVETPICVETPIRNEW1
2443


 


1495
1CTEVETPIRN1LLTEVETPICVETPIRNEW1
2088


 


1496
1CEVETPIRNE1LLTEVETPICVETPIRNEW1
1625


 


1497
1CVETPIRNEW1LLTEVETPICVETPIRNEW1
1817


 


1498
1CETPIRNEWG1LLTEVETPICVETPIRNEW1
1817


 


1499
1CSLLTEVETP1LTEVETPIRCVETPIRNEW1
2319


 


1500
1CLLTEVETPI1LTEVETPIRCVETPIRNEW1
2171


 


1501
1CSLLTEVETP1TEVETPIRNCVETPIRNEW1
2497


 


1502
1CLLTEVETPI1TEVETPIRNCVETPIRNEW1
2229


 


1503
1CSLLTEVETP1EVETPIRNECVETPIRNEW1
2469


 


1504
1CLLTEVETPI1EVETPIRNECVETPIRNEW1
2081


 


1505
1CSLLTEVETP1VETPIRNEWCVETPIRNEW1
2398


 


1506
1CLLTEVETPI1VETPIRNEWCVETPIRNEW1
2074


 


1507
1CSLLTEVETP1ETPIRNEWGCVETPIRNEW1
2778


 


1508
1CLLTEVETPI1ETPIRNEWGCVETPIRNEW1
2495


 


845
1CSLLTEVETP1SLLTEVETPCETPIRNEWG1
2417


 


1509
1CLLTEVETPI1SLLTEVETPCETPIRNEWG1
2057


 


962
1CLTEVETPIR1SLLTEVETPCETPIRNEWG1
2230


 


956
1CTEVETPIRN1SLLTEVETPCETPIRNEWG1
2352


 


868
1CEVETPIRNE1SLLTEVETPCETPIRNEWG1
2326


 


866
1CVETPIRNEW1SLLTEVETPCETPIRNEWG1
2328


 


964
1CETPIRNEWG1SLLTEVETPCETPIRNEWG1
2357


 


939
1CSLLTEVETP1LLTEVETPICETPIRNEWG1
2446


 


1510
1CLLTEVETPI1LLTEVETPICETPIRNEWG1
1999


 


1511
1CLTEVETPIR1LLTEVETPICETPIRNEWG1
2175


 


1512
1CTEVETPIRN1LLTEVETPICETPIRNEWG1
2645


 


1513
1CEVETPIRNE1LLTEVETPICETPIRNEWG1
2466


 


1514
1CVETPIRNEW1LLTEVETPICETPIRNEWG1
2575


 


1515
1CETPIRNEWG1LLTEVETPICETPIRNEWG1
2427


 


972
1CSLLTEVETP1LTEVETPIRCETPIRNEWG1
2583


 


1516
1CLLTEVETPI1LTEVETPIRCETPIRNEWG1
2320


 


1517
1CSLLTEVETP1TEVETPIRNCETPIRNEWG1
2299


 


1518
1CLLTEVETPI1TEVETPIRNCETPIRNEWG1
1972


 


1519
1CSLLTEVETP1EVETPIRNECETPIRNEWG1
2692


 


1520
1CLLTEVETPI1EVETPIRNECETPIRNEWG1
2433


 


1521
1CSLLTEVETP1VETPIRNEWCETPIRNEWG1
2569


 


1522
1CLLTEVETPI1VETPIRNEWCETPIRNEWG1
1931


 


1523
1CSLLTEVETP1ETPIRNEWGCETPIRNEWG1
2340


 


1524
1CLLTEVETPI1ETPIRNEWGCETPIRNEWG1
2007


 


861
SLLTEVET1CSLLTEVET1SLLTEVETCSLLTE1
2691


 


853
SLLTEVET1CSLLTEVET1SLLTEVETCLLTEV1
2690


 


901
SLLTEVET1CSLLTEVET1SLLTEVETCLTEVE1
2396


 


915
SLLTEVET1CSLLTEVET1SLLTEVETCTEVET1
2218


 


914
SLLTEVET1CSLLTEVET1SLLTEVETCEVETP1
2268


 


893
SLLTEVET1CSLLTEVET1SLLTEVETCVETPI1
2422


 


890
SLLTEVET1CSLLTEVET1SLLTEVETCETPIR1
2507


 


862
SLLTEVET1CSLLTEVET1SLLTEVETCTPIRN1
2690


 


907
SLLTEVET1CSLLTEVET1SLLTEVETCPIRNE1
2435


 


852
SLLTEVET1CSLLTEVET1SLLTEVETCIRNEW1
2663


 


895
SLLTEVET1CSLLTEVET1SLLTEVETCRNEWG1
2476


 


908
SLLTEVET1CSLLTEVET1SLLTEVETCNEWG1
2461


 


834
SLLTEVET1CSLLTEVET1SLLTEVETCEWG1R
2855


 


835
SLLTEVET1CSLLTEVET1SLLTEVETAWGCR1
2870


 


844
SLLTEVET1CSLLTEVET1SLLTEVETAGCR1N
2814


 


898
SLLTEVET1CSLLTEVET1SLLTEVETACR1ND
2686


 


908
SLLTEVET1CSLLTEVET1SLLTEVETCNEWGS1
2726


 


840
SLLTEVET1CSLLTEVET1SLLTEVETCEWGSR1
2730


 


1525
CLLTEVETPIRNEWGSCVETPIRNEWGSRSNDC
2278


 


938
CSLLTEVETCETPIRNEWGSRSNDSC
1916


 


922
CSLLTEVETPCETPIRNEWGSRSNDSC
2170


 


905
CSLLTEVETPICETPIRNEWGSRSNDSC
2041


 


911
CSLLTEVETPIRCETPIRNEWGSRSNDSC
2024


 


891
CSLLTEVETPIRNCETPIRNEWGSRSNDSC
1931


 


917
CSLLTEVETPIRNECETPIRNEWGSRSNDSC
1808


 


1526
CSLLTEVETPIRNEWCETPIRNEWGSRSNDSC
2012


 


881
CSLLTEVETPIRNEWGCETPIRNEWGSRSNDSC
2501


 


1527
CLLTEVETPIRNEWGSCETPIRNEWGSRSNDSC
2414


 


894
CSLLTEVETCTPIRNEWGSRSNDSSC
2068


 


965
CSLLTEVETPCTPIRNEWGSRSNDSSC
2351


 


966
CSLLTEVETPICTPIRNEWGSRSNDSSC
2125


 


924
CSLLTEVETPIRCTPIRNEWGSRSNDSSC
2162


 


979
CSLLTEVETPIRNCTPIRNEWGSRSNDSSC
1927


 


920
CSLLTEVETPIRNECTPIRNEWGSRSNDSSC
1988


 


1528
CSLLTEVETPIRNEWCTPIRNEWGSRSNDSSC
1959


 


980
CSLLTEVETPIRNEWGCTPIRNEWGSRSNDSSC
2116


 


1529
CLLTEVETPIRNEWGSCTPIRNEWGSRSNDSSC
2268


 


888
CSLLTEVETCPIRNEWGSRSNDSSDC
2246


 


931
CSLLTEVETPCPIRNEWGSRSNDSSDC
2377


 


954
CSLLTEVETPICPIRNEWGSRSNDSSDC
2267


 


969
CSLLTEVETPIRCPIRNEWGSRSNDSSDC
2228


 


970
CSLLTEVETPIRNCPIRNEWGSRSNDSSDC
2089


 


973
CSLLTEVETPIRNECPIRNEWGSRSNDSSDC
1955


 


1530
CSLLTEVETPIRNEWCPIRNEWGSRSNDSSDC
1983


 


906
CSLLTEVETPIRNEWGCPIRNEWGSRSNDSSDC
1954


 


1531
CLLTEVETPIRNEWGSCPIRNEWGSRSNDSSDC
1420


 


875
SLLTEVETCSLLTEVETCSLLTEVETPIRC
2643


 


909
SLLTEVETCSLLTEVETCLLTEVETPIRNC
2618


 


960
SLLTEVETCSLLTEVETCLTEVETPIRNEC
2157


 


940
SLLTEVETCSLLTEVETCTEVETPIRNEWC
2302


 


930
SLLTEVETCSLLTEVETCEVETPIRNEWGC
2300


 


925
SLLTEVETCSLLTEVETCVETPIRNEWGSC
2347


 


927
SLLTEVETCSLLTEVETCETPIRNEWGSRC
2311


 


912
SLLTEVETCSLLTEVETCTPIRNEWGSRSC
2434


 


933
SLLTEVETCSLLTEVETCPIRNEWGSRSNC
2192


 


948
SLLTEVETCSLLTEVETCIRNEWGSRSNDC
1961


 


884
SLLTEVETCSLLTEVETCRNEWGSRSNDSC
2620


 


846
SLLTEVETCSLLTEVETCNEWGSRSNDSSC
2640


 


874
SLLTEVETCSLLTEVETCEWGSRSNDSSDC
2509


 


856
SLLTEVETCSLLTEVETCVETPIRNEWGC
2671


 


879
SLLTEVETCSLLTEVETCETPIRNEWGCR
2631


 


833
SLLTEVETCSLLTEVETATPIRNEWGCRC
2640


 


899
SLLTEVETCSLLTEVETAPIRNEWGCRCN
2576


 


904
SLLTEVETCSLLTEVETAIRNEWGCRCND
2538


 


926
SLLTEVETCSLLTEVETARNEWGCRCNDS
2463


 


928
SLLTEVETCSLLTEVETANEWGCRCNDSS
2358


 


865
SLLTEVETCSLLTEVETAEWGCRCNDSSD
2254


 


1532
CSLLTEVETPIRNEWGCRCNDSSD
2284


 


1533
CLLTEVETPIRNEWGCRCNDSSD
1979


 


1534
SLLCEVETPIRNEWGCRCNDSSD
2161


 


566
SLLTEVCTPIRNEWGCRCNDSSD
1210


 


718
SLLTEVECPIRNEWGCRCNDSSD
1974


 


623
SLLTEVETCIRNEWGCRCNDSSD
1992


 


687
SLLTEVETPCRNEWGCRCNDSSD
2050


 


725
SLLTEVETPICNEWGCRCNDSSD
2005


 


587
SLLTEVETPIRCEWGCRCNDSSD
1956


 


733
SLLTEVETPIRNCWGCRCNDSSD
2006


 


268
SLLTEVETPIRNECGCRCNDSSD
2554


 


714
SLLTEVETPIRNEWCCRCNDSSD
2388


 


127
CSLLTEVETCSLLTEVETC
2555


 


198
CSLLTEVETPCSLLTEVETC
2341


 


187
CSLLTEVETPICSLLTEVETC
2554


 


147
CSLLTEVETPIRCSLLTEVETC
2662


 


166
CSLLTEVETPIRNCSLLTEVETC
2538


 


118
CSLLTEVETPIRNECSLLTEVETC
2489


 


122
CSLLTEVETPIRNEWCSLLTEVETC
2454


 


200
CSLLTEVETPIRNEWGCSLLTEVETC
2371


 


254
CLLTEVETPIRNEWGSCSLLTEVETC
2177


 


136
CLTEVETPIRNEWGSRCSLLTEVETC
2026


 


226
CTEVETPIRNEWGSRSCSLLTEVETC
2140


 


201
CEVETPIRNEWGSRSNCSLLTEVETC
2288


 


223
CVETPIRNEWGSRSNDCSLLTEVETC
2233


 


213
CETPIRNEWGSRSNDSCSLLTEVETC
1997


 


230
CTPIRNEWGSRSNDSSCSLLTEVETC
2168


 


161
CPIRNEWGSRSNDSSDCSLLTEVETC
2759


 


142
CSLLTEVETCSLLTEVETPC
2716


 


156
CSLLTEVETPCSLLTEVETPC
2673


 


207
CSLLTEVETPICSLLTEVETPC
2440


 


181
CSLLTEVETPIRCSLLTEVETPC
2448


 


144
CSLLTEVETPIRNCSLLTEVETPC
2673


 


164
CSLLTEVETPIRNECSLLTEVETPC
2582


 


189
CSLLTEVETPIRNEWCSLLTEVETPC
2505


 


217
CSLLTEVETPIRNEWGCSLLTEVETPC
2445


 


270
CLLTEVETPIRNEWGSCSLLTEVETPC
2307


 


228
CLTEVETPIRNEWGSRCSLLTEVETPC
2237


 


246
CTEVETPIRNEWGSRSCSLLTEVETPC
2182


 


229
CEVETPIRNEWGSRSNCSLLTEVETPC
2290


 


242
CVETPIRNEWGSRSNDCSLLTEVETPC
2379


 


232
CETPIRNEWGSRSNDSCSLLTEVETPC
2425


 


247
CTPIRNEWGSRSNDSSCSLLTEVETPC
2249


 


259
CPIRNEWGSRSNDSSDCSLLTEVETPC
2441


 


128
CSLLTEVETCSLLTEVETPIC
2876


 


137
CSLLTEVETPCSLLTEVETPIC
2815


 


245
CSLLTEVETPICSLLTEVETPIC
2492


 


151
CSLLTEVETPIRCSLLTEVETPIC
2561


 


139
CSLLTEVETPIRNCSLLTEVETPIC
2666


 


145
CSLLTEVETPIRNECSLLTEVETPIC
2684


 


177
CSLLTEVETPIRNEWCSLLTEVETPIC
2548


 


197
CSLLTEVETPIRNEWGCSLLTEVETPIC
2481


 


252
CLLTEVETPIRNEWGSCSLLTEVETPIC
2333


 


191
CLTEVETPIRNEWGSRCSLLTEVETPIC
2405


 


216
CTEVETPIRNEWGSRSCSLLTEVETPIC
2308


 


219
CEVETPIRNEWGSRSNCSLLTEVETPIC
2363


 


132
CVETPIRNEWGSRSNDCSLLTEVETPIC
2372


 


237
CETPIRNEWGSRSNDSCSLLTEVETPIC
2436


 


231
CTPIRNEWGSRSNDSSCSLLTEVETPIC
2460


 


267
CPIRNEWGSRSNDSSDCSLLTEVETPIC
2227


 


163
CSLLTEVETCSLLTEVETPIRC
2531


 


124
CSLLTEVETPCSLLTEVETPIRC
2839


 


203
CSLLTEVETPICSLLTEVETPIRC
2628


 


121
CSLLTEVETPIRCSLLTEVETPIRC
2767


 


120
CSLLTEVETPIRNCSLLTEVETPIRC
2751


 


130
CSLLTEVETPIRNECSLLTEVETPIRC
2751


 


155
CSLLTEVETPIRNEWCSLLTEVETPIRC
2646


 


157
CSLLTEVETPIRNEWGCSLLTEVETPIRC
2619


 


117
CLLTEVETPIRNEWGSCSLLTEVETPIRC
2622


 


162
CLTEVETPIRNEWGSRCSLLTEVETPIRC
2476


 


184
CTEVETPIRNEWGSRSCSLLTEVETPIRC
2419


 


172
CEVETPIRNEWGSRSNCSLLTEVETPIRC
2467


 


168
CVETPIRNEWGSRSNDCSLLTEVETPIRC
2484


 


190
CETPIRNEWGSRSNDSCSLLTEVETPIRC
2514


 


169
CTPIRNEWGSRSNDSSCSLLTEVETPIRC
2518


 


176
CPIRNEWGSRSNDSSDCSLLTEVETPIRC
2586


 


134
CSLLTEVETCSLLTEVETPIRNC
2633


 


153
CSLLTEVETPCSLLTEVETPIRNC
2691


 


208
CSLLTEVETPICSLLTEVETPIRNC
2548


 


131
CSLLTEVETPIRCSLLTEVETPIRNC
2753


 


129
CSLLTEVETPIRNCSLLTEVETPIRNC
2748


 


133
CSLLTEVETPIRNECSLLTEVETPIRNC
2724


 


180
CSLLTEVETPIRNEWCSLLTEVETPIRNC
2538


 


143
CSLLTEVETPIRNEWGCSLLTEVETPIRNC
2657


 


178
CLLTEVETPIRNEWGSCSLLTEVETPIRNC
2574


 


119
CLTEVETPIRNEWGSRCSLLTEVETPIRNC
2459


 


204
CTEVETPIRNEWGSRSCSLLTEVETPIRNC
2358


 


196
CEVETPIRNEWGSRSNCSLLTEVETPIRNC
2429


 


194
CVETPIRNEWGSRSNDCSLLTEVETPIRNC
2440


 


126
CETPIRNEWGSRSNDSCSLLTEVETPIRNC
2440


 


185
CTPIRNEWGSRSNDSSCSLLTEVETPIRNC
2492


 


202
CPIRNEWGSRSNDSSDCSLLTEVETPIRNC
2500


 


171
CSLLTEVETCSLLTEVETPIRNEC
2424


 


193
CSLLTEVETPCSLLTEVETPIRNEC
2387


 


1535
CSLLTEVETPICSLLTEVETPIRNEC
1851


 


165
CSLLTEVETPIRCSLLTEVETPIRNEC
2648


 


183
CSLLTEVETPIRNCSLLTEVETPIRNEC
2580


 


220
CSLLTEVETPIRNECSLLTEVETPIRNEC
2522


 


241
CSLLTEVETPIRNEWCSLLTEVETPIRNEC
2349


 


206
CSLLTEVETPIRNEWGCSLLTEVETPIRNEC
2419


 


244
CLLTEVETPIRNEWGSCSLLTEVETPIRNEC
2327


 


240
CLTEVETPIRNEWGSRCSLLTEVETPIRNEC
2221


 


261
CTEVETPIRNEWGSRSCSLLTEVETPIRNEC
2074


 


273
CEVETPIRNEWGSRSNCSLLTEVETPIRNEC
2008


 


269
CVETPIRNEWGSRSNDCSLLTEVETPIRNEC
2140


 


154
CETPIRNEWGSRSNDSCSLLTEVETPIRNEC
2122


 


266
CTPIRNEWGSRSNDSSCSLLTEVETPIRNEC
2080


 


248
CPIRNEWGSRSNDSSDCSLLTEVETPIRNEC
2249


 


150
CSLLTEVETCSLLTEVETPIRNEWC
2577


 


158
CSLLTEVETPCSLLTEVETPIRNEWC
2567


 


211
CSLLTEVETPICSLLTEVETPIRNEWC
2423


 


173
CSLLTEVETPIRCSLLTEVETPIRNEWC
2566


 


135
CSLLTEVETPIRNCSLLTEVETPIRNEWC
2820


 


146
CSLLTEVETPIRNECSLLTEVETPIRNEWC
2720


 


174
CSLLTEVETPIRNEWCSLLTEVETPIRNEWC
2647


 


212
CSLLTEVETPIRNEWGCSLLTEVETPIRNEWC
2545


 


218
CLLTEVETPIRNEWGSCSLLTEVETPIRNEWC
2541


 


188
CLTEVETPIRNEWGSRCSLLTEVETPIRNEWC
2522


 


225
CTEVETPIRNEWGSRSCSLLTEVETPIRNEWC
2482


 


238
CEVETPIRNEWGSRSNCSLLTEVETPIRNEWC
2391


 


234
CVETPIRNEWGSRSNDCSLLTEVETPIRNEWC
2412


 


251
CETPIRNEWGSRSNDSCSLLTEVETPIRNEWC
2374


 


272
CTPIRNEWGSRSNDSSCSLLTEVETPIRNEWC
2313


 


249
CPIRNEWGSRSNDSSDCSLLTEVETPIRNEWC
2397


 


281
CSLLTEVETCSLLTEVETPIRNEWGC
2479


 


186
CSLLTEVETPCSLLTEVETPIRNEWGC
2523


 


236
CSLLTEVETPICSLLTEVETPIRNEWGC
2345


 


167
CSLLTEVETPIRCSLLTEVETPIRNEWGC
2600


 


148
CSLLTEVETPIRNCSLLTEVETPIRNEWGC
2553


 


152
CSLLTEVETPIRNECSLLTEVETPIRNEWGC
2732


 


141
CSLLTEVETPIRNEWCSLLTEVETPIRNEWGC
2669


 


140
CSLLTEVETPIRNEWGCSLLTEVETPIRNEWGC
2678


 


205
CLLTEVETPIRNEWGSCSLLTEVETPIRNEWGC
2593


 


116
CLTEVETPIRNEWGSRCSLLTEVETPIRNEWGC
2492


 


125
CTEVETPIRNEWGSRSCSLLTEVETPIRNEWGC
2449


 


233
CEVETPIRNEWGSRSNCSLLTEVETPIRNEWGC
2442


 


257
CVETPIRNEWGSRSNDCSLLTEVETPIRNEWGC
2400


 


255
CETPIRNEWGSRSNDSCSLLTEVETPIRNEWGC
2414


 


243
CTPIRNEWGSRSNDSSCSLLTEVETPIRNEWGC
2466


 


256
CPIRNEWGSRSNDSSDCSLLTEVETPIRNEWGC
2460


 


179
CSLLTEVETCLLTEVETPIRNEWGSC
2416


 


182
CSLLTEVETPCLLTEVETPIRNEWGSC
2540


 


224
CSLLTEVETPICLLTEVETPIRNEWGSC
2388


 


160
CSLLTEVETPIRCLLTEVETPIRNEWGSC
2520


 


192
CSLLTEVETPIRNCLLTEVETPIRNEWGSC
2519


 


227
CSLLTEVETPIRNECLLTEVETPIRNEWGSC
2484


 


214
CSLLTEVETPIRNEWCLLTEVETPIRNEWGSC
2661


 


138
CSLLTEVETPIRNEWGCLLTEVETPIRNEWGSC
2624


 


1536
CLLTEVETPIRNEWGSCLLTEVETPIRNEWGSC
2616


 


1537
CLTEVETPIRNEWGSRCLLTEVETPIRNEWGSC
2667


 


1538
CTEVETPIRNEWGSRSCLLTEVETPIRNEWGSC
2425


 


1539
CEVETPIRNEWGSRSNCLLTEVETPIRNEWGSC
2461


 


1540
CVETPIRNEWGSRSNDCLLTEVETPIRNEWGSC
2218


 


1541
CETPIRNEWGSRSNDSCLLTEVETPIRNEWGSC
2255


 


1542
CTPIRNEWGSRSNDSSCLLTEVETPIRNEWGSC
2363


 


1543
CPIRNEWGSRSNDSSDCLLTEVETPIRNEWGSC
2260


 


215
CSLLTEVETCLTEVETPIRNEWGSRC
2328


 


221
CSLLTEVETPCLTEVETPIRNEWGSRC
2513


 


235
CSLLTEVETPICLTEVETPIRNEWGSRC
2351


 


170
CSLLTEVETPIRCLTEVETPIRNEWGSRC
2587


 


195
CSLLTEVETPIRNCLTEVETPIRNEWGSRC
2585


 


123
CSLLTEVETPIRNECLTEVETPIRNEWGSRC
2496


 


278
CSLLTEVETPIRNEWCLTEVETPIRNEWGSRC
2429


 


175
CSLLTEVETPIRNEWGCLTEVETPIRNEWGSRC
2641


 


1544
CLLTEVETPIRNEWGSCLTEVETPIRNEWGSRC
2500


 


250
CSLLTEVETCTEVETPIRNEWGSRSC
2474


 


222
CSLLTEVETPCTEVETPIRNEWGSRSC
2703


 


280
CSLLTEVETPICTEVETPIRNEWGSRSC
2241


 


199
CSLLTEVETPIRCTEVETPIRNEWGSRSC
2623


 


210
CSLLTEVETPIRNCTEVETPIRNEWGSRSC
2643


 


258
CSLLTEVETPIRNECTEVETPIRNEWGSRSC
2553


 


277
CSLLTEVETPIRNEWCTEVETPIRNEWGSRSC
2388


 


263
CSLLTEVETPIRNEWGCTEVETPIRNEWGSRSC
2496


 


1545
CLLTEVETPIRNEWGSCTEVETPIRNEWGSRSC
2592


 


239
CSLLTEVETCEVETPIRNEWGSRSNC
2549


 


274
CSLLTEVETPCEVETPIRNEWGSRSNC
2701


 


260
CSLLTEVETPICEVETPIRNEWGSRSNC
2680


 


209
CSLLTEVETPIRCEVETPIRNEWGSRSNC
2699


 


253
CSLLTEVETPIRNCEVETPIRNEWGSRSNC
2699


 


276
CSLLTEVETPIRNECEVETPIRNEWGSRSNC
2620


 


1546
CSLLTEVETPIRNEWCEVETPIRNEWGSRSNC
2519


 


275
CSLLTEVETPIRNEWGCEVETPIRNEWGSRSNC
2397


 


1547
CLLTEVETPIRNEWGSCEVETPIRNEWGSRSNC
2074


 


264
CSLLTEVETCVETPIRNEWGSRSNDC
2374


 


265
CSLLTEVETPCVETPIRNEWGSRSNDC
2484


 


1548
CSLLTEVETPICVETPIRNEWGSRSNDC
2174


 


271
CSLLTEVETPIRCVETPIRNEWGSRSNDC
2480


 


262
CSLLTEVETPIRNCVETPIRNEWGSRSNDC
2566


 


279
CSLLTEVETPIRNECVETPIRNEWGSRSNDC
2465


 


1549
CSLLTEVETPIRNEWCVETPIRNEWGSRSNDC
2433


 


159
CSLLTEVETPIRNEWGCVETPIRNEWGSRSNDC
2415


 


320
SLLTEVGSLLTEV
2530


 


283
CSLLTEVGSLLTEV
2903


 


390
CLLTEVGSLLTEV
2494


 


500
SCLTEVGSLLTEV
1610


 


324
SLCTEVGSLLTEV
1780


 


336
SLLCEVGSLLTEV
2475


 


824
SLLTCVGSLLTEV
1772


 


482
CLCTCVGSLLTEV
1953


 


354
SLLTEVGCSLLTEV
2511


 


807
CLLTEVGCSLLTEV
1558


 


580
SLCTEVGCSLLTEV
2382


 


511
SLLCEVGCSLLTEV
2562


 


810
SLLTCVGCSLLTEV
1992


 


798
CLCTCVGCSLLTEV
1489


 


311
SLLTEVGCLLTEV
1924


 


388
CSLLTEVGCLLTEV
2427


 


1550
SLLCEVGCLLTEV
1997


 


302
ASLLTEVGSCLTEV
2770


 


345
CSLLTEVGSCLTEV
2703


 


1551
SLLCEVGSCLTEV
2499


 


464
SLLTEVGSLCTEV
2219


 


711
CSLLTEVGSLCTEV
2409


 


1552
SLLCEVGSLCTEV
2722


 


314
SLLTEVGSLLCEV
2392


 


792
CSLLTEVGSLLCEV
2594


 


1553
CLLTEVGSLLCEV
2168


 


496
SCLTEVGSLLCEV
1896


 


506
SLCTEVGSLLCEV
1863


 


1554
SLLCEVGSLLCEV
2399


 


1555
SLLTCVGSLLCEV
2264


 


477
SLLTECGSLLCEV
2403


 


1556
CSCLCEVGSLLCEV
1583


 


1557
CLCTCVGSLLCEV
2246


 


1558
SCLCECGSLLCEV
1747


 


814
CSLLTECGSLLCEV
2256


 


284
ASLLTEVGSLLTCV
2358


 


290
CSLLTEVGSLLTCV
2503


 


1559
SLLCEVGSLLTCV
1972


 


452
SLLTEVGSLLTEC
1862


 


292
CSLLTEVGSLLTEC
2213


 


351
SLLCEVGSLLTEC
2438


 


525
SLLTEVGCSCLCEV
1829


 


379
SLCTEVGCSCLCEV
1500


 


1560
SLLCEVGCSCLCEV
2112


 


299
SLLTEVGCLCTCV
2346


 


385
CSLLTEVGCLCTCV
2474


 


1561
SLLCEVGCLCTCV
2331


 


329
SLLTEVGSCLCEC
2558


 


533
CSLLTEVGSCLCEC
2625


 


1562
SLLCEVGSCLCEC
2614


 


353
SLLTEVGCSLLTEC
2380


 


747
SLCTEVGCSLLTEC
2050


 


301
SLLCEVGCSLLTEC
2637


 


341
CSLLTEVCSLLTEC
1122


 


321
ALLTEVETPIRNEWGCRCNDSSD
2225


 


1563
DLLTEVETPIRNEWGCRCNDSSD
2320


 


1564
ELLTEVETPIRNEWGCRCNDSSD
2201


 


1565
FLLTEVETPIRNEWGCRCNDSSD
1018


 


1566
GLLTEVETPIRNEWGCRCNDSSD
2321


 


1567
HLLTEVETPIRNEWGCRCNDSSD
1946


 


1568
ILLTEVETPIRNEWGCRCNDSSD
1044


 


1569
KLLTEVETPIRNEWGCRCNDSSD
1826


 


538
LLLTEVETPIRNEWGCRCNDSSD
1399


 


1570
MLLTEVETPIRNEWGCRCNDSSD
2126


 


1571
NLLTEVETPIRNEWGCRCNDSSD
2135


 


1572
PLLTEVETPIRNEWGCRCNDSSD
1641


 


1573
QLLTEVETPIRNEWGCRCNDSSD
1627


 


1574
RLLTEVETPIRNEWGCRCNDSSD
1338


 


326
SLLTEVETPIRNEWGCRCNDSSD
2476


 


312
TLLTEVETPIRNEWGCRCNDSSD
2510


 


520
VLLTEVETPIRNEWGCRCNDSSD
1758


 


1575
WLLTEVETPIRNEWGCRCNDSSD
2180


 


1576
YLLTEVETPIRNEWGCRCNDSSD
1882


 


349
SLATEVETPIRNEWGCRCNDSSD
2145


 


1577
SLDTEVETPIRNEWGCRCNDSSD
1505


 


328
SLETEVETPIRNEWGCRCNDSSD
1963


 


459
SLITEVETPIRNEWGCRCNDSSD
1996


 


377
SLKTEVETPIRNEWGCRCNDSSD
1453


 


327
SLMTEVETPIRNEWGCRCNDSSD
2149


 


449
SLNTEVETPIRNEWGCRCNDSSD
1908


 


352
SLQTEVETPIRNEWGCRCNDSSD
2025


 


510
SLRTEVETPIRNEWGCRCNDSSD
1034


 


473
SLSTEVETPIRNEWGCRCNDSSD
2052


 


478
SLTTEVETPIRNEWGCRCNDSSD
1804


 


484
SLVTEVETPIRNEWGCRCNDSSD
2029


 


1578
SLLAEVETPIRNEWGCRCNDSSD
2356


 


1579
SLLDEVETPIRNEWGCRCNDSSD
2276


 


1580
SLLEEVETPIRNEWGCRCNDSSD
2420


 


1581
SLLFEVETPIRNEWGCRCNDSSD
2137


 


1582
SLLGEVETPIRNEWGCRCNDSSD
2301


 


1583
SLLHEVETPIRNEWGCRCNDSSD
2462


 


1584
SLLIEVETPIRNEWGCRCNDSSD
1899


 


1585
SLLKEVETPIRNEWGCRCNDSSD
2611


 


1586
SLLLEVETPIRNEWGCRCNDSSD
2344


 


346
SLLMEVETPIRNEWGCRCNDSSD
2477


 


1587
SLLNEVETPIRNEWGCRCNDSSD
2425


 


381
SLLQEVETPIRNEWGCRCNDSSD
2432


 


1588
SLLREVETPIRNEWGCRCNDSSD
2286


 


803
SLLSEVETPIRNEWGCRCNDSSD
2305


 


1589
SLLVEVETPIRNEWGCRCNDSSD
1932


 


1590
SLLWEVETPIRNEWGCRCNDSSD
2221


 


1591
SLLYEVETPIRNEWGCRCNDSSD
2181


 


822
SLLTDVETPIRNEWGCRCNDSSD
1113


 


522
SLLTMVETPIRNEWGCRCNDSSD
1244


 


608
SLLTEIETPIRNEWGCRCNDSSD
2016


 


556
SLLTELETPIRNEWGCRCNDSSD
1380


 


547
SLLTETETPIRNEWGCRCNDSSD
1594


 


551
SLLTEVATPIRNEWGCRCNDSSD
2093


 


657
SLLTEVDTPIRNEWGCRCNDSSD
2095


 


731
SLLTEVFTPIRNEWGCRCNDSSD
1708


 


784
SLLTEVGTPIRNEWGCRCNDSSD
2005


 


690
SLLTEVHTPIRNEWGCRCNDSSD
2057


 


795
SLLTEVITPIRNEWGCRCNDSSD
1438


 


705
SLLTEVKTPIRNEWGCRCNDSSD
1905


 


709
SLLTEVLTPIRNEWGCRCNDSSD
1743


 


629
SLLTEVMTPIRNEWGCRCNDSSD
2331


 


675
SLLTEVNTPIRNEWGCRCNDSSD
2150


 


594
SLLTEVQTPIRNEWGCRCNDSSD
2289


 


575
SLLTEVRTPIRNEWGCRCNDSSD
1024


 


545
SLLTEVSTPIRNEWGCRCNDSSD
2288


 


553
SLLTEVTTPIRNEWGCRCNDSSD
2102


 


645
SLLTEVVTPIRNEWGCRCNDSSD
1492


 


583
SLLTEVWTPIRNEWGCRCNDSSD
1889


 


658
SLLTEVYTPIRNEWGCRCNDSSD
1092


 


635
SLLTEVEAPIRNEWGCRCNDSSD
2325


 


779
SLLTEVEDPIRNEWGCRCNDSSD
2216


 


652
SLLTEVEEPIRNEWGCRCNDSSD
2362


 


621
SLLTEVEFPIRNEWGCRCNDSSD
2297


 


646
SLLTEVEGPIRNEWGCRCNDSSD
2341


 


450
SLLTEVEHPIRNEWGCRCNDSSD
2241


 


593
SLLTEVEIPIRNEWGCRCNDSSD
2365


 


534
SLLTEVEKPIRNEWGCRCNDSSD
2505


 


564
SLLTEVELPIRNEWGCRCNDSSD
2407


 


590
SLLTEVEMPIRNEWGCRCNDSSD
2516


 


467
SLLTEVENPIRNEWGCRCNDSSD
2549


 


537
SLLTEVEPPIRNEWGCRCNDSSD
2522


 


527
SLLTEVEQPIRNEWGCRCNDSSD
2344


 


599
SLLTEVERPIRNEWGCRCNDSSD
2131


 


607
SLLTEVESPIRNEWGCRCNDSSD
2156


 


695
SLLTEVEVPIRNEWGCRCNDSSD
2286


 


763
SLLTEVEWPIRNEWGCRCNDSSD
2178


 


783
SLLTEVEYPIRNEWGCRCNDSSD
2113


 


559
SLLTEVETAIRNEWGCRCNDSSD
2415


 


610
SLLTEVETDIRNEWGCRCNDSSD
2392


 


602
SLLTEVETEIRNEWGCRCNDSSD
2368


 


549
SLLTEVETFIRNEWGCRCNDSSD
2301


 


597
SLLTEVETGIRNEWGCRCNDSSD
2366


 


609
SLLTEVETHIRNEWGCRCNDSSD
2288


 


653
SLLTEVETIIRNEWGCRCNDSSD
2096


 


300
SLLTEVETKIRNEWGCRCNDSSD
2485


 


491
SLLTEVETLIRNEWGCRCNDSSD
2225


 


493
SLLTEVETMIRNEWGCRCNDSSD
2320


 


529
SLLTEVETNIRNEWGCRCNDSSD
2253


 


606
SLLTEVETQIRNEWGCRCNDSSD
2189


 


611
SLLTEVETRIRNEWGCRCNDSSD
2159


 


700
SLLTEVETSIRNEWGCRCNDSSD
2151


 


649
SLLTEVETTIRNEWGCRCNDSSD
2295


 


614
SLLTEVETVIRNEWGCRCNDSSD
2264


 


670
SLLTEVETWIRNEWGCRCNDSSD
2146


 


581
SLLTEVETYIRNEWGCRCNDSSD
2290


 


715
SLLTEVETPARNEWGCRCNDSSD
2244


 


660
SLLTEVETPDRNEWGCRCNDSSD
2390


 


630
SLLTEVETPERNEWGCRCNDSSD
2480


 


665
SLLTEVETPFRNEWGCRCNDSSD
2229


 


495
SLLTEVETPGRNEWGCRCNDSSD
2676


 


591
SLLTEVETPHRNEWGCRCNDSSD
2425


 


392
SLLTEVETPKRNEWGCRCNDSSD
2300


 


461
SLLTEVETPLRNEWGCRCNDSSD
2239


 


457
SLLTEVETPMRNEWGCRCNDSSD
2297


 


770
SLLTEVETPNRNEWGCRCNDSSD
2299


 


738
SLLTEVETPPRNEWGCRCNDSSD
2302


 


782
SLLTEVETPQRNEWGCRCNDSSD
2341


 


750
SLLTEVETPRRNEWGCRCNDSSD
2376


 


673
SLLTEVETPSRNEWGCRCNDSSD
2396


 


758
SLLTEVETPTRNEWGCRCNDSSD
2417


 


650
SLLTEVETPVRNEWGCRCNDSSD
2331


 


693
SLLTEVETPWRNEWGCRCNDSSD
2190


 


389
SLLTEVETPYRNEWGCRCNDSSD
2303


 


707
SLLTEVETPIANEWGCRCNDSSD
2302


 


524
SLLTEVETPIDNEWGCRCNDSSD
2337


 


676
SLLTEVETPIENEWGCRCNDSSD
2252


 


576
SLLTEVETPIFNEWGCRCNDSSD
2176


 


744
SLLTEVETPIGNEWGCRCNDSSD
2073


 


488
SLLTEVETPIHNEWGCRCNDSSD
2121


 


720
SLLTEVETPIINEWGCRCNDSSD
2262


 


778
SLLTEVETPIKNEWGCRCNDSSD
2322


 


692
SLLTEVETPILNEWGCRCNDSSD
2363


 


454
SLLTEVETPIMNEWGCRCNDSSD
2369


 


780
SLLTEVETPINNEWGCRCNDSSD
2277


 


357
SLLTEVETPIPNEWGCRCNDSSD
2515


 


677
SLLTEVETPIQNEWGCRCNDSSD
2372


 


754
SLLTEVETPISNEWGCRCNDSSD
2292


 


766
SLLTEVETPITNEWGCRCNDSSD
2358


 


680
SLLTEVETPIVNEWGCRCNDSSD
2366


 


656
SLLTEVETPIWNEWGCRCNDSSD
2250


 


509
SLLTEVETPIYNEWGCRCNDSSD
2296


 


582
SLLTEVETPIRAEWGCRCNDSSD
2306


 


698
SLLTEVETPIRDEWGCRCNDSSD
2187


 


771
SLLTEVETPIREEWGCRCNDSSD
2156


 


620
SLLTEVETPIRFEWGCRCNDSSD
2364


 


773
SLLTEVETPIRGEWGCRCNDSSD
2228


 


721
SLLTEVETPIRHEWGCRCNDSSD
2296


 


627
SLLTEVETPIRIEWGCRCNDSSD
2444


 


631
SLLTEVETPIRKEWGCRCNDSSD
2437


 


585
SLLTEVETPIRLEWGCRCNDSSD
2468


 


622
SLLTEVETPIRMEWGCRCNDSSD
2385


 


753
SLLTEVETPIRPEWGCRCNDSSD
2301


 


661
SLLTEVETPIRQEWGCRCNDSSD
2367


 


710
SLLTEVETPIRREWGCRCNDSSD
2278


 


678
SLLTEVETPIRSEWGCRCNDSSD
2322


 


387
SLLTEVETPIRTEWGCRCNDSSD
2360


 


565
SLLTEVETPIRVEWGCRCNDSSD
2300


 


562
SLLTEVETPIRWEWGCRCNDSSD
2302


 


654
SLLTEVETPIRYEWGCRCNDSSD
2202


 


666
SLLTEVETPIRNAWGCRCNDSSD
2334


 


712
SLLTEVETPIRNDWGCRCNDSSD
2324


 


662
SLLTEVETPIRNFWGCRCNDSSD
2417


 


643
SLLTEVETPIRNGWGCRCNDSSD
2408


 


684
SLLTEVETPIRNHWGCRCNDSSD
2377


 


577
SLLTEVETPIRNIWGCRCNDSSD
2490


 


640
SLLTEVETPIRNKWGCRCNDSSD
2402


 


571
SLLTEVETPIRNLWGCRCNDSSD
2415


 


605
SLLTEVETPIRNMWGCRCNDSSD
2455


 


741
SLLTEVETPIRNNWGCRCNDSSD
2319


 


616
SLLTEVETPIRNPWGCRCNDSSD
2288


 


512
SLLTEVETPIRNQWGCRCNDSSD
2402


 


546
SLLTEVETPIRNRWGCRCNDSSD
2257


 


617
SLLTEVETPIRNSWGCRCNDSSD
2300


 


380
SLLTEVETPIRNTWGCRCNDSSD
2314


 


685
SLLTEVETPIRNVWGCRCNDSSD
2281


 


703
SLLTEVETPIRNWWGCRCNDSSD
2293


 


702
SLLTEVETPIRNYWGCRCNDSSD
2311


 


453
SLLTEVETPIRNEAGCRCNDSSD
2383


 


751
SLLTEVETPIRNEDGCRCNDSSD
2359


 


787
SLLTEVETPIRNEEGCRCNDSSD
2330


 


716
SLLTEVETPIRNEFGCRCNDSSD
2444


 


708
SLLTEVETPIRNEGGCRCNDSSD
2486


 


696
SLLTEVETPIRNEHGCRCNDSSD
2398


 


636
SLLTEVETPIRNEIGCRCNDSSD
2402


 


572
SLLTEVETPIRNEKGCRCNDSSD
2580


 


641
SLLTEVETPIRNELGCRCNDSSD
2436


 


517
SLLTEVETPIRNEMGCRCNDSSD
2398


 


637
SLLTEVETPIRNENGCRCNDSSD
2265


 


671
SLLTEVETPIRNEPGCRCNDSSD
2279


 


765
SLLTEVETPIRNEQGCRCNDSSD
2264


 


737
SLLTEVETPIRNERGCRCNDSSD
2328


 


745
SLLTEVETPIRNESGCRCNDSSD
2346


 


688
SLLTEVETPIRNETGCRCNDSSD
2401


 


386
SLLTEVETPIRNEVGCRCNDSSD
2411


 


384
SLLTEVETPIRNEYGCRCNDSSD
2426


 


681
SLLTEVETPIRNEWACRCNDSSD
2431


 


717
SLLTEVETPIRNEWDCRCNDSSD
2322


 


726
SLLTEVETPIRNEWECRCNDSSD
2364


 


596
SLLTEVETPIRNEWFCRCNDSSD
2493


 


633
SLLTEVETPIRNEWHCRCNDSSD
2327


 


347
SLLTEVETPIRNEWICRCNDSSD
2359


 


541
SLLTEVETPIRNEWKCRCNDSSD
2328


 


569
SLLTEVETPIRNEWLCRCNDSSD
2379


 


642
SLLTEVETPIRNEWMCRCNDSSD
2378


 


785
SLLTEVETPIRNEWNCRCNDSSD
2236


 


774
SLLTEVETPIRNEWPCRCNDSSD
2328


 


760
SLLTEVETPIRNEWQCRCNDSSD
2338


 


704
SLLTEVETPIRNEWRCRCNDSSD
2371


 


735
SLLTEVETPIRNEWSCRCNDSSD
2374


 


669
SLLTEVETPIRNEWTCRCNDSSD
2401


 


624
SLLTEVETPIRNEWVCRCNDSSD
2418


 


366
SLLTEVETPIRNEWWCRCNDSSD
2406


 


625
SLLTEVETPIRNEWYCRCNDSSD
2362


 


659
SLLTEVETPIRNEWGARCNDSSD
2396


 


790
SLLTEVETPIRNEWGDRCNDSSD
2267


 


370
SLLTEVETPIRNEWGERCNDSSD
2286


 


504
SLLTEVETPIRNEWGFRCNDSSD
2349


 


563
SLLTEVETPIRNEWGGRCNDSSD
2270


 


647
SLLTEVETPIRNEWGHRCNDSSD
2113


 


667
SLLTEVETPIRNEWGIRCNDSSD
2211


 


648
SLLTEVETPIRNEWGKRCNDSSD
2295


 


600
SLLTEVETPIRNEWGLRCNDSSD
2389


 


732
SLLTEVETPIRNEWGMRCNDSSD
2269


 


664
SLLTEVETPIRNEWGNRCNDSSD
2328


 


727
SLLTEVETPIRNEWGPRCNDSSD
2334


 


713
SLLTEVETPIRNEWGQRCNDSSD
2369


 


672
SLLTEVETPIRNEWGRRCNDSSD
2372


 


682
SLLTEVETPIRNEWGSRCNDSSD
2346


 


638
SLLTEVETPIRNEWGTRCNDSSD
2404


 


644
SLLTEVETPIRNEWGVRCNDSSD
2444


 


595
SLLTEVETPIRNEWGWRCNDSSD
2439


 


668
SLLTEVETPIRNEWGYRCNDSSD
2410


 


683
SLLTEVETPIRNEWGCACNDSSD
2301


 


501
SLLTEVETPIRNEWGCCCNDSSD
2515


 


615
SLLTEVETPIRNEWGCDCNDSSD
2207


 


679
SLLTEVETPIRNEWGCECNDSSD
2148


 


639
SLLTEVETPIRNEWGCFCNDSSD
2312


 


775
SLLTEVETPIRNEWGCGCNDSSD
2186


 


776
SLLTEVETPIRNEWGCHCNDSSD
2134


 


722
SLLTEVETPIRNEWGCICNDSSD
2269


 


701
SLLTEVETPIRNEWGCKCNDSSD
2290


 


748
SLLTEVETPIRNEWGCLCNDSSD
2292


 


730
SLLTEVETPIRNEWGCMCNDSSD
2413


 


743
SLLTEVETPIRNEWGCNCNDSSD
2340


 


767
SLLTEVETPIRNEWGCPCNDSSD
2295


 


756
SLLTEVETPIRNEWGCQCNDSSD
2294


 


736
SLLTEVETPIRNEWGCSCNDSSD
2348


 


719
SLLTEVETPIRNEWGCTCNDSSD
2347


 


742
SLLTEVETPIRNEWGCVCNDSSD
2334


 


503
SLLTEVETPIRNEWGCWCNDSSD
2437


 


552
SLLTEVETPIRNEWGCYCNDSSD
2397


 


634
SLLTEVETPIRNEWGCRANDSSD
2265


 


794
SLLTEVETPIRNEWGCRDNDSSD
2033


 


796
SLLTEVETPIRNEWGCRENDSSD
2097


 


723
SLLTEVETPIRNEWGCRFNDSSD
2303


 


752
SLLTEVETPIRNEWGCRGNDSSD
2345


 


734
SLLTEVETPIRNEWGCRHNDSSD
2353


 


697
SLLTEVETPIRNEWGCRINDSSD
2409


 


598
SLLTEVETPIRNEWGCRKNDSSD
2435


 


686
SLLTEVETPIRNEWGCRLNDSSD
2363


 


691
SLLTEVETPIRNEWGCRMNDSSD
2356


 


746
SLLTEVETPIRNEWGCRNNDSSD
2384


 


757
SLLTEVETPIRNEWGCRPNDSSD
2326


 


772
SLLTEVETPIRNEWGCRQNDSSD
2327


 


584
SLLTEVETPIRNEWGCRRNDSSD
2383


 


519
SLLTEVETPIRNEWGCRSNDSSD
2344


 


573
SLLTEVETPIRNEWGCRTNDSSD
2374


 


375
SLLTEVETPIRNEWGCRVNDSSD
2316


 


579
SLLTEVETPIRNEWGCRWNDSSD
2364


 


699
SLLTEVETPIRNEWGCRYNDSSD
2169


 


706
SLLTEVETPIRNEWGCRCADSSD
2329


 


728
SLLTEVETPIRNEWGCRCCDSSD
2377


 


762
SLLTEVETPIRNEWGCRCDDSSD
2289


 


739
SLLTEVETPIRNEWGCRCEDSSD
2273


 


560
SLLTEVETPIRNEWGCRCFDSSD
2538


 


689
SLLTEVETPIRNEWGCRCGDSSD
2343


 


651
SLLTEVETPIRNEWGCRCHDSSD
2350


 


612
SLLTEVETPIRNEWGCRCIDSSD
2450


 


318
SLLTEVETPIRNEWGCRCKDSSD
2549


 


618
SLLTEVETPIRNEWGCRCLDSSD
2480


 


628
SLLTEVETPIRNEWGCRCMDSSD
2470


 


516
SLLTEVETPIRNEWGCRCPDSSD
2449


 


568
SLLTEVETPIRNEWGCRCQDSSD
2438


 


544
SLLTEVETPIRNEWGCRCRDSSD
2447


 


729
SLLTEVETPIRNEWGCRCSDSSD
2141


 


674
SLLTEVETPIRNEWGCRCTDSSD
2253


 


632
SLLTEVETPIRNEWGCRCVDSSD
2366


 


555
SLLTEVETPIRNEWGCRCWDSSD
2462


 


626
SLLTEVETPIRNEWGCRCYDSSD
2385


 


619
SLLTEVETPIRNEWGCRCNASSD
2454


 


588
SLLTEVETPIRNEWGCRCNCSSD
2505


 


694
SLLTEVETPIRNEWGCRCNESSD
2282


 


535
SLLTEVETPIRNEWGCRCNFSSD
2509


 


603
SLLTEVETPIRNEWGCRCNGSSD
2418


 


613
SLLTEVETPIRNEWGCRCNHSSD
2439


 


586
SLLTEVETPIRNEWGCRCNISSD
2517


 


465
SLLTEVETPIRNEWGCRCNKSSD
2597


 


393
SLLTEVETPIRNEWGCRCNLSSD
2634


 


394
SLLTEVETPIRNEWGCRCNMSSD
2574


 


395
SLLTEVETPIRNEWGCRCNNSSD
2502


 


396
SLLTEVETPIRNEWGCRCNPSSD
2445


 


397
SLLTEVETPIRNEWGCRCNQSSD
2376


 


398
SLLTEVETPIRNEWGCRCNRSSD
2514


 


342
SLLTEVETPIRNEWGCRCNSSSD
2440


 


399
SLLTEVETPIRNEWGCRCNTSSD
2483


 


400
SLLTEVETPIRNEWGCRCNVSSD
2476


 


401
SLLTEVETPIRNEWGCRCNWSSD
2556


 


337
SLLTEVETPIRNEWGCRCNYSSD
2475


 


402
SLLTEVETPIRNEWGCRCNDASD
2267


 


403
SLLTEVETPIRNEWGCRCNDCSD
2318


 


404
SLLTEVETPIRNEWGCRCNDDSD
2237


 


405
SLLTEVETPIRNEWGCRCNDESD
2203


 


406
SLLTEVETPIRNEWGCRCNDFSD
2395


 


338
SLLTEVETPIRNEWGCRCNDGSD
2226


 


407
SLLTEVETPIRNEWGCRCNDHSD
2537


 


408
SLLTEVETPIRNEWGCRCNDISD
2527


 


409
SLLTEVETPIRNEWGCRCNDKSD
2484


 


410
SLLTEVETPIRNEWGCRCNDLSD
2494


 


411
SLLTEVETPIRNEWGCRCNDMSD
2405


 


412
SLLTEVETPIRNEWGCRCNDNSD
2389


 


413
SLLTEVETPIRNEWGCRCNDPSD
2450


 


414
SLLTEVETPIRNEWGCRCNDQSD
2349


 


415
SLLTEVETPIRNEWGCRCNDRSD
2502


 


416
SLLTEVETPIRNEWGCRCNDTSD
2394


 


417
SLLTEVETPIRNEWGCRCNDVSD
2389


 


418
SLLTEVETPIRNEWGCRCNDWSD
2491


 


330
SLLTEVETPIRNEWGCRCNDYSD
2445


 


419
SLLTEVETPIRNEWGCRCNDSAD
2219


 


420
SLLTEVETPIRNEWGCRCNDSCD
2269


 


356
SLLTEVETPIRNEWGCRCNDSDD
2181


 


421
SLLTEVETPIRNEWGCRCNDSED
2527


 


422
SLLTEVETPIRNEWGCRCNDSFD
2492


 


313
SLLTEVETPIRNEWGCRCNDSGD
2485


 


423
SLLTEVETPIRNEWGCRCNDSHD
2353


 


424
SLLTEVETPIRNEWGCRCNDSID
2320


 


425
SLLTEVETPIRNEWGCRCNDSKD
2361


 


426
SLLTEVETPIRNEWGCRCNDSLD
2400


 


427
SLLTEVETPIRNEWGCRCNDSMD
2304


 


350
SLLTEVETPIRNEWGCRCNDSND
2299


 


428
SLLTEVETPIRNEWGCRCNDSPD
2344


 


429
SLLTEVETPIRNEWGCRCNDSQD
2175


 


430
SLLTEVETPIRNEWGCRCNDSRD
2373


 


431
SLLTEVETPIRNEWGCRCNDSTD
2205


 


432
SLLTEVETPIRNEWGCRCNDSVD
2232


 


433
SLLTEVETPIRNEWGCRCNDSWD
2477


 


434
SLLTEVETPIRNEWGCRCNDSYD
2479


 


435
SLLTEVETP
2380


 


436
SLLTEVETPI
2465


 


437
SLLTEVETPIR
2297


 


438
SLLTEVETPIRN
2130


 


439
SLLTEVETPIRNE
1819


 


440
SLLTEVETPIRNEW
2334


 


368
SLLTEVETPIRNEWG
2429


 


442
SLLTEVETPIRNEWGC
2369


 


443
SLLTEVETPIRNEWGCR
2444


 


444
SLLTEVETPIRNEWGCRC
2478


 


445
SLLTEVETPIRNEWGCRCN
2506


 


446
SLLTEVETPIRNEWGCRCND
2276


 


447
SLLTEVETPIRNEWGCRCNDS
2348


 


448
SLLTEVETPIRNEWGCRCNDSS
2325









Further, the peptides were screened for the ability to specifically bind 23K21 and 8I10 and NOT Z3G. In one set, peptides with high binding values for 23K21 and 8I10 that are NOT recognized by Z3G1 were identified. In a second set, peptides with high binding values for Z3G1 that are NOT recognized by 23K21 and 8I10 were identified.
Peptide sequences with high binding values for 23K12 and BI10 with no binding to Z3G1 were identified for low stringency (0.01 μg/mL) conditions (Table 4A) and high stringency (0.001 μg/mL) conditions (Table 4B).







TABLE 4A



























TABLE 4B




















Properties of the peptides that bind specifically to HuM2e antibodies 23K12/BI10 at different stringency conditions were analyzed. Antibodies 8i10 and 23k12 bind a conformational epitope with SLLTE as its core sequence. The best 23k12 binder is SLLTEVGSLLTEV (SEQ ID NO: 320), which is also recognized by Z3G1. The best 8i10 binder is CSLLTEVGSLLTEV (SEQ ID NO: 283), which is also recognized by Z3G1. The very best specific binder is CSLLTECGSLLTCV (SEQ ID NO: 463). The binding sequences contain a remarkably high number of cysteines.
Peptide sequences with high binding values for Z3G1 with no binding to 23K12 and BI10 were identified for low stringency (0.01 μg/mL) conditions (Table 5A) and high stringency (0.001 μg/mL) conditions (Table 5B).







TABLE 5A



























TABLE 5B




















There is a clear difference between binding specificity between antibodies 8i10/23k12 versus Z3G1. The very best specific binder is a longer sequence than with 23k12/8i10, has no dimer topology and also no CLIPS. Among the very best specific binder is mostly the whole native sequence, MSLLTEVETPIRNEWGCRCN (SEQ ID NO: 1149). The result with 0.01 μg/ml shows that LLXEVEXPIRN (SEQ ID NO: 1594) is the core of the Z3G1 binding epitope.
Specific binding motifs can derived from 0.001 μg/ml screening. Thus, mAbs 23k12/8i10 recognize M2e peptides with SLLTE as the core of the epitope. The T residue in S-L-L-T-E differentiates binding between 23k12/8i10 and Z3G1. In contrast, mAb Z3G1 recognize M2e with LLXEVEXPIRN (SEQ ID NO: 1594) as the core of the epitope. (Tables 6A and 6B).







TABLE 6A



























TABLE 6B




















Peptide Immunogens

In particular, a peptide immunogen of this invention that binds the HuM2e 23k12/8i10 comprises a core sequence of S-L-L-T-E as well as variants, modifications and multimers thereof. Core sequences for low stringency (0.01 μg/mL) conditions (FIG. 6A) and high stringency (0.001 μg/mL) conditions (FIG. 6B) and variants thereof derived from the binding data are shown in FIGS. 6A and 6B.
In some embodiments, the core sequence comprises a first additional amino acid at the C terminal end of S-L-L-T-E-Xaa6, wherein Xaa6 is any amino acid, preferably V or C. (FIG. 6C)
Further, in some embodiments, the core sequence comprises a first additional amino acid at the C terminal end of S-L-L-T-E-Xaa6-Xaa7, wherein Xaa7 is any amino acid, but preferably E. (FIG. 6C)
In some embodiments, the peptide immunogens comprise a plurality of core sequences linked by an amino acid, preferably a small amino acid such as G or A. However, amino acids such as proline, which can act as a structural disrupter in the middle of regular secondary structure elements such as alpha helices and beta sheets, are less desirable. The number of core sequences in peptide immunogens of the invention can be 1, 2, 3, 4, 5 or more. (FIG. 6D).
In some embodiments, an N-terminal amino acid Xaa0 is present in the peptide immunogens. Xaa0 can be any amino acid, but preferably is a cysteine. In some embodiments a N-terminal cysteine is used to cyclize the peptide immunogen. (FIG. 6E)
Peptide immunogens (linear or cyclized) with particularly strong affinity for 23K12/8I10 binding are shown in FIG. 6F.
The peptide immunogens of this invention that bind the huM2e monoclonal antibodies under high stringency conditions are represented by the following formula:







[Xaa0]m-Xaa1-Xaa2-Xaa3-Xaa4-Xaa5-[Xaa6]p-[Xaa7]q-


 


[Xaa8-[Xaa0]m-Xaa1-Xaa2-Xaa3-Xaa4-Xaa5-[Xaa6]p-


 


[Xaa7]q]n






    wherein, m, p and q are independently 0 or 1,
    n is any number between 0 and 4,
    Xaa0 is any amino acid, preferably C;
    Xaa6 is any amino acid, preferably V or C;
    Xaa7 is any amino acid, preferably E;
    Xaa8 is any amino acid not including proline, preferably G or A;
    Xaa1-Xaa2-Xaa3-Xaa4-Xaa5 is S-L-L-T-E,

or a peptide having a single substitution to the sequence S-L-L-T-E, the substitution selected from the group consisting of:

    Xaa1 is C or T;
    Xaa2 is A, C, F or K,
    Xaa3 is A, C, E, F, I, K, M, Q, S, T or V, and
    Xaa5 is D or C.


The peptide immunogens of this invention that bind the huM2e monoclonal antibodies under low stringency conditions are represented by the following formula:







[Xaa0]m-Xaa1-Xaa2-Xaa3-Xaa4-Xaa5-[Xaa6]p-[Xaa7]q-


 


[Xaa8-[Xaa0]m-Xaa1-Xaa2-Xaa3-Xaa4-Xaa5-[Xaa6]p-


 


[Xaa7]q]n






    wherein, m, p and q are independently 0 or 1,
    n is any number between 0 and 4,
    Xaa0 is any amino acid, preferably C;
    Xaa6 is any amino acid, preferably V or C;
    Xaa7 is any amino acid, preferably E;
    Xaa8 is any amino acid not including proline, preferably G or A;
    Xaa1-Xaa2-Xaa3-Xaa4-Xaa5 is S-L-L-T-E or,

a peptide having a single substitution to the sequence S-L-L-T-E, the substitution selected from the group consisting of:

    Xaa1 is A, C, D, L, T or V,
    Xaa2 is A, C, F, H, I, K, M, N, Q, R, T, W, or Y,
    Xaa3 is any amino acid,
    Xaa4 is M, N, Q, S, or W, and
    Xaa5 is A, D, F, H, I, K, M, N, Q, S, W, Y, or C.

Preparation of Linear and Cyclic Peptide Immunogens



Linear peptide immunogens can be prepared synthetically and then screened for a particular characteristic in various biological assays. E.g., Scott, J. K. and G. P. Smith, Science 249:386, 1990; Devlin, J. J., et al., Science 24:404, 1990; Furka, A. et al., Int. J. Pept. Protein Res. 37:487, 1991; Lam, K. S., et al., Nature 354:82, 1991.
Cyclized peptides are often found to possess superior immunogenic activity compared to linear peptide immunogens. Linear peptide immunogens comprising three or more core sequences are found to bind with the terminal sequences only, while cyclization allows binding by all core sequences present in the peptide immunogens. Various methods for producing cyclic peptides have been described. One involves solution or liquid phase peptide synthesis, where amino acid residues in solution are linked by peptide bonds, with reactive groups not involved in the peptide bond formation, such as the amino group of the N-terminal residue, the carboxy group of the C-terminal residue, sulfhydryl groups on cysteine residues and similar or other reactive groups in the amino acid side chains, protected by suitable protecting groups.
In one embodiment cyclic peptide immunogens are formed using terminal cysteine residues by reduction of thiol groups to form disulfide bridges.
Another approach involves solid phase peptide synthesis, in which synthesis is carried out on an insoluble solid matrix. Protecting groups are employed for reactive side chains. The general methodology of solid phase synthesis is well known in the art. Merrifield, R. B., Solid phase synthesis (Nobel lecture). Angew Chem 24:799-810 (1985) and Barany et al., The Peptides, Analysis, Synthesis and Biology, Vol. 2, Gross, E. and Meienhofer, J., Eds. Academic Press 1-284 (1980). For example, chemical reaction protocols, such as those described in U.S. Pat. Nos. 4,033,940 and 4,102,877, have been devised to produce circularized peptides. In other techniques, biological and chemical methods are combined to produce cyclic peptides. These latter methods involve first expressing linear precursors of cyclic peptides in cells (e.g., bacteria) to produce linear precursors of cyclic peptides and then adding of an exogenous agent such as a protease or a nucleophilic reagent to chemically convert these linear precursors into cyclic peptides. See, e.g., Camerero, J. A., and Muir, T. W., J. Am. Chem. Society. 121:5597 (1999); Wu, H. et al, Proc. Natl. Acad. Sci. USA, 95:9226 (1998).
Head-to-tail (backbone) peptide cyclization has been used to rigidify structure and improve in vivo stability of small bioactive peptides (see Camarero and Muir, J. Am. Chem. Soc., 121:5597-5598 (1999)). An important consequence of peptide cyclization is retention of biological activity and/or the identification of new classes of pharmacological agents. A chemical cross-linking approach was used to prepare a backbone cyclized version of bovine pancreatic trypsin inhibitor (Goldenburg and Creighton, J. Mol. Biol., 165:407-413 (1983)). Other approaches include chemical (Camarero et al., Angew. Chem. Int. Ed., 37:347-349 (1998); Tam and Lu, Prot. Sci., 7:1583-1592 (1998); Camarero and Muir, Chem. Commun., 1997:1369-1370 (1997); and Zhang and Tam, J. Am. Chem. Soc. 119:2363-2370 (1997)) and enzymatic (Jackson et al., J. Am. Chem. Soc., 117:819-820 (1995)) intramolecular ligation methods which allow linear synthetic peptides to be efficiently cyclized under aqueous conditions.
A native chemical ligation approach utilizes inteins (internal proteins) to catalyze head-to-tail peptide and protein ligation in vivo (see, for example, Evans et al., J. Biol. Chem. 274:18359-18363 (1999); Iwai and Pluckthun, FEBS Lett. 459.166-172 (1999); Wood et al., Nature Biotechnology 17:889-892 (1999); Camarero and Muir, J. Am. Chem. Soc. 121:5597-5598 (1999); and Scott et al., Proc. Natl. Acad. Sci. USA 96:13638-13643 (1999)).
The invention also encompasses isolated nucleic acid molecules comprising a sequences that encode M2e peptide immunogens of the invention. Also provided by the present invention are nucleic acid expression constructs, and host cells containing such nucleic acids, which encode M2e peptides, and variants thereof, which have at least one epitope characteristic of M2e peptide immunogens. This aspect of the invention pertains to isolated nucleic sequences encoding an M2e sequence or M2e peptide immunogen sequence as described herein, as well as those sequences readily derived from isolated nucleic molecules such as, for example, complementary sequences, reverse sequences and complements of reverse of sequences.
A related embodiment includes a nucleic acid expression construct comprising a promoter operably linked to the isolated nucleic acid molecule such that a M2e peptide immunogen or fusion protein comprising a M2e peptide immunogen as described herein is expressed in a host cell. In another embodiment, the invention provides a host cell containing such a nucleic acid expression construct. In a related embodiment, the invention provides a method for producing a peptide immunogen, comprising growing the described host cells for a time sufficient to express the peptide immunogen encoded by the nucleic acid expression construct.
Conjugated Peptide Immunogens
The approach of increasing immunogenicity of small immunogenic molecules by conjugating these molecules to large “carrier” molecules has been utilized successfully for decades (see, e.g., Goebel et al. (1939) J. Exp. Med. 69: 53). For example, many immunogenic compositions have been described in which purified capsular polymers have been conjugated to carrier proteins to create more effective immunogenic compositions by exploiting this “carrier effect.” Schneerson et al. (1984) Infect. Immun. 45: 582-591).
In one aspect of the invention, method for conjugating a M2e peptide immunogen via a reactive group of an amino acid residue of the peptide immunogen to a protein/polypeptide carrier having one or more functional groups is provided. The protein/polypeptide carrier may be human serum albumin, keyhole limpet hemocyanin (KLH), immunoglobulin molecules, thyroglobulin, ovalbumin, influenza hemagglutinin, PAN-DR binding peptide (PADRE polypeptide), malaria circumsporozite (CS) protein, hepatitis B surface antigen (HBSAg19-28, Heat Shock Protein (HSP) 65, Bacillus Calmette-Guerin (BCG), cholera toxin, cholera toxin mutants with reduced toxicity, diphtheria toxin, CRM197 protein that is cross-reactive with diphtheria toxin, recombinant Streptococcal C5a peptidase, Streptococcus pyogenes ORF1224, Streptococcus pyogenes ORF1664, Streptococcus pyogenes ORF 2452, Chlamydia pneumoniae ORF T367, Chlamydia pneumoniae ORF T858, Tetanus toxoid, HIV gp120 T1, microbial surface components recognizing adhesive matrix molecules (MSCRAMMS), growth factor/hormone, cytokines or chemokines.
Methods for protecting a subject from infection or decreasing susceptibility of a subject to infection by one or more influenza strains/isolates or subtypes, i.e., prophylactic methods, are additionally provided. In one embodiment, a method includes administering to the subject an amount of M2e peptide immunogens that specifically bind influenza M2 effective to protect the subject from infection, or effective to decrease susceptibility of the subject to infection, by one or more influenza strains/isolates or subtypes.
Symptoms or complications of influenza infection that can be reduced or decreased include, for example, chills, fever, cough, sore throat, nasal congestion, sinus congestion, nasal infection, sinus infection, body ache, head ache, fatigue, pneumonia, bronchitis, ear infection, ear ache or death.
Peptide Immunogens as Vaccines
The peptide immunogens can be used as vaccines to generate an anti-influenza M2-mediated immune response in order to prevent influenza infections. Synthetic peptides require both stabilization and adjuvantation for the induction of an effective immune response in vivo. Various methods have been employed to protect synthetic peptide immunogens against degradation in vitro and in vivo, mediated by various processes including chemical and physical pathways. (Manning M C, et al. Pharmaceutical Research, 1989, 6:903-918).
Numerous adjuvants and/or depot-based parenteral, mucosal or transdermal delivery systems destined for use with human or veterinary vaccines have been developed to enhance the immune response. These include the use of mineral salts, water-in-oil (w/o)-emulsions, liposomes, polymeric microparticles, nanoparticles and gels/hydrogels. (Cox J C, et al. Vaccine, 1997, 15:248-256). Freund's complete adjuvant (FCA), a suspension of heat-killed M. tuberculosis mycobacteria in mineral oil containing a surfactant, has been recognized as one of the most powerful adjuvants. Adjuvants are well known in the art (Vaccine Design—The Subunit and Adjuvant Approach, 1995, Pharmaceutical Biotechnology, Volume 6, Eds. Powell, M. F., and Newman, M. J., Plenum Press, New York and London, ISBN 0-306-44867-X). Preferred adjuvants for use with immunogens of the present invention include aluminium or calcium salts (hydroxide or phosphate). Adjuvants may be selected from GM-CSF, 529 SE, IL-12, aluminum phosphate, aluminum hydroxide, Mycobacterium tuberculosis, Bordetella pertussis, bacterial lipopolysaccharides, aminoalkyl glucosane phosphate compounds, MPLTM (3-O-deacylated monophosphoryl lipid A), a polypeptide, Quil A, STIMULONTM QS-21, a pertussis toxin (PT), an E. coli heat-labile toxin (LT), IL-1alpha, IL-1β, IL-2, IL-4, IL-5, IL-6, IL-7, IL-8, IL-10, IL-13, IL-14, IL-15, IL-16, IL-17, IL-18, interferon-alpha, interferon-β, interferon-gamma, G-CSF, TNF-alpha and TNF-β.
Still other adjuvants include mineral oil and water emulsions, calcium salts such as calcium phosphate, aluminum salts (alum), such as aluminum hydroxide, aluminum phosphate, etc., Amphigen, Avridine, L121/squalene, D-lactide-polylactide/glycoside, pluronic acids, polyols, muramyl dipeptide, killed Bordetella, saponins, such as StimulonTM QS-21 (Antigenics, Framingham, Mass.), described in U.S. Pat. No. 5,057,540, which is hereby incorporated by reference 3, and particles generated therefrom such as ISCOMS (immunostimulating complexes), Mycobacterium tuberculosis, bacterial lipopolysaccharides, synthetic polynucleotides such as oligonucleotides containing a CpG motif (U.S. Pat. No. 6,207,646, which is hereby incorporated by reference), a pertussis toxin (PT), or an E. coli heat-labile toxin (LT), particularly LT-K63, LT-R72, PT-K9/G129; see, e.g., International Patent Publication Nos. WO 93/13302 and WO 92/19265, which are/incorporated herein by reference for all purposes.
Also useful as adjuvants are cholera toxins and mutants thereof, including those described in published International Patent Application No. WO 00/18434 (wherein the glutamic acid at amino acid position 29 is replaced by another amino acid (other than aspartic acid, preferably a histidine). Similar CT toxins or mutants are described in published International Patent Application number WO 02/098368 (wherein the isoleucine at amino acid position 16 is replaced by another amino acid, either alone or in combination with the replacement of the serine at amino acid position 68 by another amino acid; and/or wherein the valine at amino acid position 72 is replaced by another amino acid). Other CT toxins are described in published International Patent Application number WO 02/098369 (wherein the arginine at amino acid position 25 is replaced by another amino acid; and/or an amino acid is inserted at amino acid position 49; and/or two amino acids are inserted at amino acid position 35 and 36).
Various methods may be employed to adjuvant synthetic peptide-based immunogens, but normally a carrier or depot system is required for effective long-term immunogenic responses. Notable examples include adsorbing the immunogen onto a mineral salt or gel. For example, encapsulating a peptide immunogen within a polymeric matrix (monolithic matrix) or gel, or layering a polymeric material around a peptide immunogen (core-shell) may be an effective strategy. Or, an immunogen may be incorporated in a liposome or vesicular type of formulation, with the immunogen either embedded in the lipid matrix or physically entrapped in the internal aqueous phase. Another strategy may employ a mineral-based, vegetable-based or animal-based oil, with an aqueous solution of the immunogen in various proportions, to prepare a water-in-oil (w/o)-emulsion or a water-in-oil-in-water (w/o/w)-double emulsion. Powell M F, et al., Pharmaceutical Biotechnology, Vol. 6, Plenum Press, New York, 1995.
Kits
It is particularly useful to use antibody binding sequences in the kit, which correspond to defined epitope sequences known to be specific for the immunogen under investigation. This kit will lead to a more specific answer than those kits used today, and hence to a better selection of immunogen vaccine therapy for the individual patient.
In an extension of this approach, one could also characterize the patient's serum by identifying the corresponding antibody binding peptides among a random display library using the aforementioned methods. This again may lead to optimisation of the epitope information, and thus to a better diagnosis.
Further, one could use the individual antibody binding sequences as (immunogen) vaccines leading to more specific (immunogen) vaccines. These antibody binding sequences could be administered in an isolated form or fused to a membrane protein of the phage display system, or to another carrier protein, which may have beneficial effect for the immunoprotective effect of the antibody binding peptide (Dalum et al., Nature Biotechnology, Vol. 17, pp. 666-669 (1999)).
The present invention relates to a kit for predicting binding of a specific antibody to at least one potential immunogen. The kit of the invention would also be useful for other screening purposes where it is desirable to test for antibody binding to peptide sequences, such as epitope variant.
In one embodiment the peptide immunogen may be immobilized on a solid support. Suitable solid support could be any chemical support, including micro titer plates, beads, capillary tubing or membranes. Each of these supports could be activated, supporting covalent, ionic or hydrophobic binding, chelation or affinity binding, or inactivated, promoting ionic or hydrophobic binding. Immobilisation could take place by attachment through covalent binding, ionic or hydrophobic binding, chelation, affinity binding, or through van der Waal bonds. A solid support could also be biological in nature, such as phages, bacteria, red blood cells or any related system allowing display of heterologous proteins or peptides.
The kit also can be used for screening different antigenic peptide sequences corresponding to structural epitopes at the same time. The kit above also can be used in a high throughput screening method for screening many samples, obtained e.g. from humans or animals, at the same time and thereby predicting which humans or animals will display an immunogenic response towards particular immunogens. Any practical combination of the number of antigenic peptide sequences and the number of humans or animals would be possible.
The following examples illustrate embodiments of the invention. It will be appreciated by one of skill in the art that the techniques disclosed in the examples which follow represent techniques discovered by the inventors to function well in the practice of the invention. However, those of skill in the art should, in light of the present disclosure, appreciate that many changes can be made in the specific embodiments which are disclosed and still obtain a like or similar result without departing from the spirit and scope of the invention.
EXAMPLES
Example 1
Identification of M2-Specific Antibodies
Mononuclear or B cells expressing three of the MAbs identified in human serum were diluted into clonal populations and induced to produce antibodies. Antibody containing supernatants were screened for binding to 293 FT cells stably transfected with the full length M2E protein from influenza strain Influenza subtype H3N2. Supernatants which showed positive staining/binding were re-screened again on 293 FT cells stably transfected with the full length M2E protein from influenza strain Influenza subtype H3N2 and on vector alone transfected cells as a control.
The variable regions of the antibodies were then rescue cloned from the B cell wells whose supernatants showed positive binding. Transient transfections were performed in 293 FT cells to reconstitute and produce these antibodies. Reconstituted antibody supernatants were screened for binding to 293 FT cells stably transfected with the full length M2E protein as detailed above to identify the rescued anti-M2E antibodies. Three different antibodies were identified: 8i10, 21B15 and 23K12.
Antibodies 21B15, 23K12, and 8I10 bound to the surface of 293-HEK cells stably expressing the M2 protein, but not to vector transfected cells (see FIG. 1). In addition, binding of these antibodies was not competed by the presence of 5 mg/ml 24-mer M2 peptide, whereas the binding of the control chimeric mouse V-region/human IgG1 kappa 14C2 antibody (hu14C2) generated against the linear M2 peptide was completely inhibited by the M2 peptide (see FIG. 1). These data confirm that these antibodies bind to conformational epitopes present in M2e expressed on the cell or virus surface, as opposed to the linear M2e peptide.
Example 2
Viral Binding of Human Anti-influenza Monoclonal Antibodies
UV-inactivated influenza A virus (A/PR/8/34) (Applied Biotechnologies) was plated in 384-well MaxiSorp plates (Nunc) at 1.2 μg/ml in PBS, with 25 μl/well, and was incubated at 4° C. overnight. The plates were then washed three times with PBS, and blocked with 1% Nonfat dry milk in PBS, 50 μl/well, and then were incubated at room temp for 1 hr. After a second wash with PBS, MAbs were added at the indicated concentrations in triplicate, and the plates were incubated at room temp for 1 hour. After another wash with PBS, to each well was added 25 μl of a 1/5000 dilution of horseradish peroxidase (HRP) conjugated goat anti-human IgG Fc (Pierce) in PBS/1% Milk, and the plates were left at room temp for 1 hr. After the final PBS wash, the HRP substrate 1-StepTM Ultra-TMB-ELISA (Pierce) was added at 25 μl/well, and the reaction proceeded in the dark at room temp. The assay was stopped with 25 μl/well 1N H2SO4, and light absorbance at 450 nm (A450) was read on a SpectroMax Plus plate reader. Data are normalized to the absorbance of MAb 8I10 binding at 10 μ/ml. Results are shown in FIGS. 2A and 2B.
Example 3
Binding of Human Anti-Influenza Monoclonal Antibodies to Full-Length M2 Variants
M2 variants (including those with a high pathology phenotype in vivo) were selected for analysis. See FIG. 3A for sequences.
M2 cDNA constructs were transiently transfected in HEK293 cells and analyzed as follows: To analyze the transient transfectants by FACS, cells on 10 cm tissue culture plates were treated with 0.5 ml Cell Dissociation Buffer (Invitrogen), and harvested. Cells were washed in PBS containing 1% FBS, 0.2% NaN3 (FACS buffer), and resuspended in 0.6 ml FACS buffer supplemented with 100 μg/ml rabbit IgG. Each transfectant was mixed with the indicated MAbs at 1 μg/ml in 0.2 ml FACS buffer, with 5×105 to 106 cells per sample. Cells were washed three times with FACS buffer, and each sample was resuspended in 0.1 ml containing 1 μg/ml alexafluor (AF) 647-anti human IgG H&L (Invitrogen). Cells were again washed and flow cytometry was performed on a FACSCanto device (Becton-Dickenson). The data is expressed as a percentage of the mean fluorescence of the M2-D20 transient transfectant. Data for variant binding are representative of 2 experiments. Data for alanine mutants are average readouts from 3 separate experiments with standard error. Results are shown in FIGS. 3B and 3C.
Example 4
Epitope Blocking
To determine whether the MAbs 8I10 and 23K12 bind to the same site, M2 protein representing influenza strain A/HK/483/1997 sequence was stably expressed in the CHO (Chinese Hamster Ovary) cell line DG44. Cells were treated with Cell Dissociation Buffer (Invitrogen), and harvested. Cells were washed in PBS containing 1% FBS, 0.2% NaN3 (FACS buffer), and resuspended at 107 cells/ml in FACS buffer supplemented with 100 μg/ml rabbit IgG. The cells were pre-bound by either MAb (or the 2N9 control) at 10 μg/ml for 1 hr at 4° C., and were then washed with FACS buffer. Directly conjugated AF647-8I10 or −23K12 (labeled with the AlexaFluor® 647 Protein Labeling kit (Invitrogen) was then used to stain the three pre-blocked cell samples at 1 μg/ml for 106 cells per sample. Flow cytometric analyses proceeded as before with the FACSCanto. Data are average readouts from 3 separate experiments with standard error. Results are shown in FIG. 4.
Example 5
Binding of Human Anti-influenza Monoclonal Antibodies to M2 Variants and Truncated M2 Peptides
The cross reactivity of mAbs 8i10 and 23K12 to other M2 peptide variants was assessed by ELISA. Peptide sequences are shown in Tables 7A and 7B. Additionally, a similar ELISA assay was used to determine binding activity to M2 truncated peptides.
In brief, each peptide was coated at 2 μg/mL to a flat bottom 384 well plate (Nunc) in 25 μL/well of PBS buffer overnight at 4° C. Plates were washed three times and blocked with 1% Milk/PBS for one hour at room temperature. After washing three times, MAb titers were added and incubated for one hour at room temperature. Diluted HRP conjugated goat anti-human immunoglobulin FC specific (Pierce) was added to each well after washing three times. Plates were incubated for one hour at room temperature and washed three times. 1-StepTM Ultra-TMB-ELISA (Pierce) was added at 25 μl/well, and the reaction proceeded in the dark at room temp. The assay was stopped with 25 μl/well 1N H2SO4, and light absorbance at 450 nm (A450) was read on a SpectroMax Plus plate reader. Results are shown in Tables 7A and 7B.








TABLE 7A







Cross reactivity binding of anti-M2 antibodies to



variant M2 peptides.









ELISA (OD 450)














seqNo
Name
Size
Description
14C2
8i10
23K12
2N9





 1
M2
23 aa
SLLTEVETPIRNEWGCRCNDSSD
+
−
−
−


 


 2
M2SG
23 aa
SLLTEVETPIRSEWGCRCNDSGD
+
−
−
−


 


 3
M2EG
23 aa
SLLTEVETPIRNEWECRCNGSSD
+
−
−
−


 


 4
M2P
23 aa
SLPTEVETPIRNEWGCRCNDSSD
+
−
−
−


 


 5
M2G
23 aa
SLLTEVETPIRNEWGCRCNGSSD
+
−
−
−


 


 6
M2DLTGS
23 aa
SLLTEVDTLTRNGWGCRCSDSSD
−
−
+
−


 


 7
M2KNS
23 aa
SLLTEVETPIRKEWGCNCSDSSD
+
−
−
−


 


 8
M2LGS
23 aa
SLLTEVETLIRNGWGCRCSDSSD
−
−
−
−


 


 9
M2LTKGS
23 aa
SLLTEVETLTKNGWGCRCSDSSD
−
−
−
−


 


10
M2SY
23 aa
SLLTEVETPIRSEWGCRYNDSSD
+
−
−
−


 


11
M2TGEKS
23 aa
SLLTEVETPTRNGWECKCSDSSD
+
−
−
−


 


12
M2HTGEKS
23 aa
SLLTEVETHTRNGWECKCSDSSD
−
−
−
−


 


13
M2KTGEKS
23 aa
SLLTEVKTPTRNGWECKCSDSSD
−
−
−
−


 


14
M2LTGS
23 aa
SLLTEVETLTRNGWGCRCSDSSD
−
−
+
−


 


15
M2TDGEKS
23 aa
SLLTEVETPTRDGWECKCSDSSD
+
−
−
−


 


16
M2TGS
23 aa
SLLTEVETPTRNGWGCRCSDSSD
+
−
W
−


 


17
M2TGEK
23 aa
SLLTEVETPTRNGWECKCNDSSD
+
−
−
−


 


18
M2LTGEKS
23 aa
SLLTEVETLTRNGWECKCSDSSD
−
−
W
−


 


19
M2K
23 aa
SLLTEVETPIRNEWGCKCNDSSD
+
W
+
−


 


20
M2FG
23 aa
SFLTEVETPIRNEWGCRCNGSSD
+
W
−
−


 


21
M2TGE
23 aa
SLLTEVETPTRNGWECRCNDSSD
+
−
−
−


 


22
M2KGENS
23 aa
SLLTEVETPIRKGWECNCSDSSD
+
−
−
−


 


23
M2TES
23 aa
SLLTEVETPTRNEWECRCSDSSD
+
−
−
−


 


24
M2GHTGKS
23 aa
SLLTGVETHTRNGWGCKCSDSSD
−
−
−
−


 


25
M2PHTGS
23 aa
SLLPEVETHTRNGWGCRCSDSSD
−
−
−
−











Percentage compared relative to binding to wild-type peptide (Seq 1)



Note:


mAbs were tested at 5 μg/mL










>25%
−
no binding



25-40%
W
weak binding


>40%
+
positive binding













TABLE 7B







Binding activity of M2 antibodies to truncated M2 peptides.














seqNo
Name
Size
Description
14C2
8i10
23K12
2N9


















 1
M2
23 aa
SLLTEVETPIRNEWGCRCNDSSD
3.85
0.11
0.22
0.06



 


26
M16
16 aa
LLTEVETPIRNEWGCR
3.94
0.09
0.21
0.09


 


27
M15
15 aa
LTEVETPIRNEWGCR
3.95
0.09
0.21
0.09


 


28
M12
12 aa
VETPIRNEWGCR
0.15
0.09
0.20
0.09


 


29
CM17
17 aa
ETPIRNEWGCRCNDSSD
0.19
0.11
0.34
0.11


 


30
CM16
16 aa
TPIRNEWGCRCNDSSD
0.23
0.13
0.35
0.12


 


31
CM15
15 aa
PIRNEWGCRCNDSSD
0.19
0.12
0.34
0.11


 


32
CM14
14 aa
IRNEWGCRCNDSSD
0.23
0.14
0.36
0.13


 


33
CM13
13 aa
RNEWGCRCNDSSD
0.22
0.14
0.34
0.13


 


34
CM12
12 aa
NEWGCRCNDSSD
0.27
0.14
0.39
0.14


 


35
NM17
17 aa
SLLTEVETPIRNEWGCR
3.99
0.26
0.58
0.10


 


36
NM16
16 aa
SLLTEVETPIRNEWGC
3.90
0.29
0.62
0.09


 


37
NM15
15 aa
SLLTEVETPIRNEWG
3.97
0.12
0.30
0.11


 


38
NM14
14 aa
SLLTEVETPIRNEW
3.97
0.11
0.24
0.09


 


39
NM13
13 aa
SLLTEVETPIRNE
0.18
0.11
0.25
0.10


 


40
NM12
12 aa
SLLTEVETPIRN
0.20
0.10
0.24
0.09


 


41
NM11
11 aa
SLLTEVETPIR
0.21
0.13
0.30
0.12


 


42
NM10
10 aa
SLLTEVETPI
0.17
0.10
0.24
0.10


 


43
NM8
 8 aa
SLLTEVET
0.15
0.10
0.20
0.09


 


44
NM7
 7 aa
SLLTEVE
0.14
0.10
0.20
0.08


 


45
NM9
 9 aa
SLLTEVETP
0.21
0.12
0.30
0.19


 


46
M2e
24 aa
MSLLTEVETPIRNEWGCRCNDSSD
3.98
0.13
0.43
0.10


 


CMV
HVIR1


0.16
0.11
0.21
3.99





Note:


mAbs were tested at 5 μg/mL






OTHER EMBODIMENTS
All publications and patent applications cited in this specification are herein incorporated by reference as if each individual publication or patent application is specifically and individually indicated to be incorporated by reference.
Although the foregoing invention has been described in some detail by way of illustration and example for purposes of clarity of understanding, it will be readily apparent to those of ordinary skill in the art in light of the teachings of this invention that certain changes and modifications may be made thereto without departing from the spirit or scope of the appended claims.

## Claims

1. A peptide immunogen consisting of the sequence of:









[Xaa0]m-Xaa1-Xaa2-Xaa3-Xaa4-Xaa5-[Xaa6]p-


 



[Xaa7]q-[Xaa8-[Xaa0]m-Xaa1-Xaa2-Xaa3-Xaa4-Xaa5-


 



[Xaa6]p-[Xaa7]q]n





wherein, m, p and q are independently 0 or 1,
n is any number between 1 and 4,
Xaa0, Xaa6, Xaa7 and Xaa8 is independently any amino acid,
Xaa1-Xaa2-Xaa3-Xaa4-Xaa5 is S-L-L-T-E.

2. The peptide immunogen of claim 1, wherein Xaa0 is C.

3. The peptide immunogen of claim 1, wherein Xaa6 is V or C.

4. The peptide immunogen of claim 1, wherein Xaa7 is E.

5. The peptide immunogen of claim 1, wherein Xaa8 is G or A.

6. The peptide immunogen of claim 1, wherein said peptide is non-linear, and wherein said peptide is cyclic, and n is any number between 1 and 4.

7. The peptide immunogen of claim 1, wherein said peptide is a Linear or cyclized peptide having the sequence SLLTEVGSLLTEV (SEQ ID NO: 320).