DELIVERY OF SIALIDASE TO CANCER CELLS, IMMUNE CELLS AND THE TUMOR MICROENVIRONMENT

Abstract

Recombinant oncolytic viruses for expression of sialidase and their use in the treatment of cancer, particularly solid tumors, are described.

Claims

1. A recombinant oncolytic virus comprising a nucleotide sequence encoding a polypeptide comprising a sialidase domain, wherein the nucleotide sequence encoding the sialidase is operably linked to a promoter.

2. The oncolytic virus of claim 1, wherein said oncolytic virus is selected from the group consisting of: vaccinia virus, reovirus, Seneca Valley virus (SVV), vesicular stomatitis virus (VSV), Newcastle disease virus (NDV), herpes simplex virus (HSV), morbillivirus virus, retrovirus, influenza virus, Sinbis virus, poxvirus, measles virus, cytomegalovirus (CMV), lentivirus, and adenovirus.

3. The oncolytic virus of claim 2, wherein said oncolytic virus is a poxvirus, and said poxvirus is a vaccinia virus.

4. The oncolytic virus of claim 3, wherein the vaccinia virus is selected from among Dryvax; Lister; M63; LIVP; Tian Tan; Modified Vaccinia Ankara; New York City Board of Health, Dairen, Ikeda, LC16M8, Tashkent, IHD-J, Brighton, Dairen I, Connaught, Elstree, Wyeth, Copenhagen, and Western Reserve strains; vaccinia virus strain Elstree, vaccinia virus strain CL, vaccinia virus strain Lederle-Chorioallantoic, vaccinia virus strain AS.

5. The oncolytic virus of claim 1, wherein the sialidase is a human sialidase or a bacterial sialidase.

6. The oncolytic virus of claim 1, wherein the sialidase is a Neu5Ac alpha(2,6)-Gal sialidase or a Neu5Ac alpha(2,3)-Gal sialidase.

7. The oncolytic virus of claim 1, wherein the human or bacterial sialidase is selected from the group consisting of: Clostridium perfringens sialidase, Actinomyces viscosus sialidase, Arthrobacter ureafaciens sialidase, NEU2, and NEU4.

8. The recombinant oncolytic virus of claim 1, wherein the promotor is a viral early promoter.

9. The recombinant oncolytic of claim 2, wherein the oncolytic virus is a poxvirus and the promoter is a poxvirus early promoter.

10. The recombinant oncolytic virus of claim 1, wherein the oncolytic virus is Talimogene Laherparepvec.

11. The recombinant oncolytic virus of claim 1 wherein the virus is a reovirus.

12. The recombinant oncolytic virus of claim 1, wherein the virus is an adenovirus having an E1ACR2 deletion.

13. The recombinant oncolytic virus of claim 1, wherein the sialidase is DAS181.

14. The recombinant oncolytic virus of claim 1, wherein the sialidase comprises the amino acid sequence of SEQ ID NO:1 or SEQ ID NO:2.

15. The recombinant oncolytic virus of claim 1 wherein the virus is vaccinia virus Western Reserve.

16. A method of treating solid tumor comprising administering to a patient in need thereof the recombinant virus of claim 1.

Description

DESCRIPTION OF THE DRAWINGS

[0013] FIG. 1: Detection of 2,6 sialic acid (by FITC-SNA) on A549 and MCF cells by fluorescence microscopy. A549 and MCF cells were fixed and incubated with FITC-SNA for one hour at 37° C. before imaged under fluorescence microscope to show the FITC-SNA labeled cells (left) and overlay with brightfield cells (right)

[0014] FIG. 2: Effective removal of 2,6 sialic acid, 2,3 sialic acid, and exposure of galactose on A549 cells by DAS181 treatment. A549 were treated with DAS181 for two hours at 37° C. and incubated with staining reagents one hour before imaged under fluorescence microscope to show effective removal of sialic acids on tumor cells.

[0015] FIG. 3: Effective removal of 2,6 sialic acid on A549 cells by DAS181 but not DAS185 treatment. A549 were treated with DAS181 for 30 minutes or two hours at 37° C. and incubated with FITC-SNA for one hour before examined using flow cytometry to show effective removal of 2,6 sialic acids on tumor cells.

[0016] FIG. 4: Effective removal of 2,3 sialic acid on A549 cells by DAS181 but not DAS185 treatment. A549 were treated with DAS181 for 30 minutes or two hours at 37° C. and incubated with FITC-MALII for one hour before examined using flow cytometry to show effective removal of 2,3 sialic acids on tumor cells

[0017] FIG. 5: Effective exposure of galactose on A549 cells by DAS181 but not DAS185 treatment. A549 were treated with DAS181 for 30 minutes or two hours at 37° C. and incubated with FITC-PNA for one hour before examined using flow cytometry to show effective exposure of galactose on tumor cells

[0018] FIG. 6: DAS181 treatment and PBMC stimulation regimen do not affect A549-red cell proliferation. A549-Red cells were seeded at 2 k/well overnight, followed by replacement of medium containing reagents listed on the left. Scan by IncuCyte was initiated immediately after the reagents were added (0 hr) and scheduled for every 3 hr. A549-red cell proliferation is monitored by analyzing the nuclear (red) counts. Kinetic readouts reveal no effect on A549 cell proliferation by vehicle, DAS181, or various stimulation reagents, without the presence of PBMCs.

[0019] FIG. 7: Detection of cytotoxicity in A549-red cells following co-culturing with PBMCs from Donor 1 with or without DAS181 treatment. These results showed that DAS181 treatment significantly boost anti-tumor cytotoxicity by PBMCs from Donor 1. A549-Red cells were seeded at 2 k/well overnight, followed by co-culturing with 100K/well Donor-1 PBMCs (E:T=50:1) in the presence of medium (no activation), CD3+CD28+IL-2 (T cell activation), or CD3+CD29+IL-2+IL-15+IL-21 (T and NK cell activation). Representative images were taken by IncuCyte at 0 hr and 72 hrs post adding PBMCs.

[0020] FIG. 8: Detection of cytotoxicity in A549-red cells following co-culturing with PBMCs from Donor 2 with or without DAS181 treatment. These results showed that DAS181 treatment significantly boost anti-tumor cytotoxicity by PBMCs from Donor 2. A549-Red cells were seeded at 2 k/well overnight, followed by co-culturing with 100 k/well Donor-1 PBMCs (E:T=50:1) in the presence of medium (no activation), CD3+CD28+IL-2 (T cell activation), or CD3+CD29+IL-2+IL-15+IL-21 (T and NK cell activation). Representative images were taken by IncuCyte at 0 hr and 72 hrs post adding PBMCs.

[0021] FIGS. 9A-9C: Detection of cytotoxicity in A549-red cells following co-culturing with PBMCs from Donor 1 with or without DAS181 treatment. These results showed that DAS181 treatment significantly boost anti-tumor cytotoxicity by PBMCs from Donor 1. The green lines indicate conditions without DAS181, and the blue lines indicate conditions with the DAS-181 treatment. A549-red tumor cells were seeded at 2 k cells/well in 96-well plate. After overnight incubation, PBMCs from Donor 1 mixed with (A) medium (B) CD3/CD28/IL-2, or (C) CD3/CD28/IL-2/IL-15/IL-21 were added into each well as indicated E:T ratio. At mean time, DAS181 (100 nM) was added. Plates were scanned by IncuCyte every 3 hr for total 72 hrs. Proliferation is monitored by analyzing RFP cell counts.

[0022] FIGS. 10A-10C: Detection of cytotoxicity in A549-red cells following co-culturing with PBMCs from Donor 2 with or without DAS181 treatment. These results showed that DAS181 treatment significantly boost anti-tumor cytotoxicity by PBMCs from Donor 2. The green lines indicate conditions without DAS181, and the blue lines indicate conditions with the DAS-181 treatment. A549-red tumor cells were seeded at 2 k cells/well in 96-well plate. After overnight incubation, PBMCs from Donor 2 mixed with (A) medium, (B) CD3/CD28/IL-2, or (C) CD3/CD28/IL-2/IL-15/IL-21 were added into each well as indicated E:T ratio. At mean time, DAS 181 (100 nM) was added. Plates were scanned by IncuCyte every 3 hr for total 72 hrs. Proliferation is monitored by analyzing RFP cell counts.

[0023] FIG. 11: DAS181 enhances NK-mediated tumor lysis by vaccinia virus, measured by MTS assay. =T-test P value<0.05, suggesting that DAS181 alone boosts NK cell-mediated U87 tumor killing in vitro, compared to enzyme-dead DAS185. *=T-Test P value<0.05.

[0024] FIG. 12: DAS181 increases NK-mediated tumor killing by vaccinia virus as measured by MTS assay. *=T-test P value<0.05, suggesting that DAS181 increases NK cell-mediated killin of U87 cells by VV in vitro.

[0025] FIG. 13: DAS181 significantly enhanced expression of maturation markers (CD80, CD86, HLA) in human DC cells that were cultured alone or exposed to VV-infected tumor cells. *=T-test P value<0.05.

[0026] FIG. 14: DAS181 significantly enhanced TNF-alpha production by THP-1 derived macrophages. *=T-test P value<0.05

[0027] FIG. 15: DAS181 treatment promotes oncolytic adenovirus-mediated tumor cell killing and growth prohibition. A549-red tumor cells were seeded at 2K cells/well in 96-well plates. After overnight incubation, DAS181 vehicle, oncolytic adenovirus, and DAS181 were added as indicated. CD3/CD28/IL-2 were also added into each well with the amount described previously. Graph showed that DAS181 plus oncolytic adenovirus effectively reduced tumor cell proliferation.

[0028] FIGS. 16A-16B: DAS181 treatment enhances PBMC-mediated tumor cell killing by vaccinia virus. A549-red tumor cells were seeded at 2K cells/well in 96-well plate. After overnight incubation, fresh PBMCs were added at densities of 10K/well (A) or 40K/well (B). CD3, CD28, IL-2, DAS181, and oncolytic adenovirus were added as indicated in the graph following with the timed scans by IncuCyte. Graph showed that DAS181 plus oncolytic adenovirus dramatically enhanced human PBMC-mediated tumor cell eradication.

[0029] FIG. 17: Schematic of a portion of a vaccinia virus construct for expressing a sialidase.

[0030] FIG. 18: Sequence of certain elements in a vaccinia virus construct for expressing a sialidase (DAS181).

[0031] FIG. 19: Sequence of a portion of a vaccinia virus construct for expressing a sialidase (DAS181).

[0032] FIGS. 20A-20B: DAS181 expressed by Sialidase-VV has in vitro activity towards sialic acid-containing substrates. (A) Standard curve of DAS181 activity at 0.5 nM, 1 nM, and 2 nM. (B) 1×10.sup.6 cells infected with Sialidase-VV express DAS181 equivalent to 0.78 nM-1.21 nM DAS181 in 1 ml medium in vitro.

[0033] FIG. 21: Sialidase-VV enhances Dendritic cell maturation. GM-CSF/IL4 derived human DC were cultured with Sial-VV or VV infected U87 tumor cell lysate for 24 hours. DAS181 of LPS was used as control DC were collected and stained with antibodies against CD80, CD86, HLA-DR, and HLA-ABC. The expression of DC maturation markers was determined by flow analysis. The results suggested that Sial-VV enhanced DC maturation. *=T-test P value<0.05

[0034] FIG. 22: Sialidase-VV induced IFN-gamma and IL2 expression by T cells. CD3 antibody-activated human T cells were co-cultured with A594 tumor cells in the presence of Sial-VV or VV-infected tumor cells lysate for 24 hours, and cytokine IFNr or IL-2 expression was measured by ELISA. The results suggested that Sial-VV cell lysate induced IFNr and IL2 expression by human T cells. *=T-test P value<0.05

[0035] FIG. 23: Sialidase-VV enhances T cell-mediated tumor cell lytic activity. CD3 Ab activated human T cells were co-cultured with Sial-VV or VV-infected A594 tumor cells for 24 hours, and tumor cell viability was determined by MTS assay. The results suggested that Sial-VV infection of tumor cells resulted in enhanced tumor killing. *=T-test P value<0.05

DETAILED DESCRIPTION

Oncolytic Viruses

[0036] Numerous oncolytic viruses, including Vaccina virus, Coxsackie virus, Adenovirus, Measles, Newcastle disease virus, Seneca Valley virus, Coxsackie A21, Vesicular stomatitis virus, Parvovirus H1, Reovirus, Herpes virus, Lentivirus, and Poliovirus, and Parvovirus. Vaccinia Virus Western Reserve, GLV-1h68, ACAM2000, and OncoVEX GFP, are available. The genomes of these oncolytic virus can be genetically modified to insert a nucleotide sequence encoding a protein that includes all or a catalytic portion of a sialidase. The nucleotide sequence encoding a protein that includes all or a catalytically active portion of a sialidase is placed under the control of a viral expression cassette so that the sialidase is expressed by infected cells.

Vesicular Stomatitis Virus (VSV)

[0037] VSV has been used in multiple oncolytic virus applications. In addition, VSV has been engineered to express an antigenic protein of human papilloma virus (HPV) as a method to treat HPV positive cervical cancers via vaccination (REF 18337377, 29998190) and to express pro-inflammatory factors to increase the immune reaction to tumors (REF 12885903). Various methods for engineering VSV to encode an additional gene have been described (REF 7753828). Briefly, the VSV RNA genome is reverse transcribed to a complementary, doubled stranded-DNA with an upstream T7 RNA polymerase promoter and an appropriate location within the VSV genome for gene insertion is identified (e.g., within the noncoding 5′ or 3′ regions flanking VSV glycoprotein (G) (REF 12885903). Restriction enzyme digestion can be accomplished, e.g., with Mlu I and Nhe I, yielding a linearized DNA molecule. An insert consisting of a DNA molecule encoding the gene of interest flanked by appropriate restriction sites can be ligated into the linearized VSV genomic DNA. The resulting DNA can be transcribed with T7 polymerase, yielding a complete VSV genomic RNA containing the inserted gene of interest. Introduction of this RNA molecule to a mammalian cell, e.g., via transfection and incubation results in viral progeny expressing the protein encoded by the gene of interest.

Adenovirus Serotype 5 (Ad5)

[0038] Ad5 contains a human E2F-1 promoter, which is a retinoblastoma (Rb) pathway—defective tumor specific transcription regulatory element that drives expression of the essential Ela viral genes, restricting viral replication and cytotoxicity to Rb pathway-defective tumor cells (REF 16397056). A hallmark of tumor cells is Rb pathway defects. Engineering a gene of interest into Ad5 is accomplished through ligation into Ad5 genome. A plasmid containing the gene of interest is generated via and digested, e.g., with AsiSI and PacI. An Ad5 DNA plasmid, e.g., PSF-AD5 (REF Sigma OGS268) is digested with AsiSI and PacI and ligated with recombinant bacterial ligase or co-transformed with RE digested gene of interest into permissive E. coli as has been reported for the generation of human granulocyte macrophage colony stimulating factor (GM-CSF) expressing Ad5 (REF 16397056). Recovery of the DNA and transfection into a permissive host, e.g., human embryonic kidney cells (HEK293) or HeLa yields virus expressing the gene of interest.

Vaccinia Virus (VV)

[0039] Various strains of VV have been used as templates for OV therapeutics; the unifying feature is deletion of the viral thymidine kinase (TK) gene, rendering a virus dependent upon actively replicating cells, i.e. neoplastic cells, for productive replication and thus these VVs have preferential infectivity of cancer cells exemplified by the Western Reserve (WR) strain of VV (REF 25876464). Production of VV's with a gene of interest inserted in the genome is accomplished with homologous recombination utilizing lox sites, as described in greater detail below.

Polypeptides with Sialidase Activity for Expression by an Oncolytic Virus

[0040] The recombinant oncolytic virus expresses a polypeptide that includes all or a catalytic portion of a sialidase that is capable of removing sialic acid (N-acetylneuraminic acid (Neu5Ac)) from a glycan on a human cell. In general, Neu5Ac is linked via an alpha 2,3, an alpha 2,6 or alpha 2,8 linkage to the penultimate sugar in glycan on a protein by any of a variety of sialyl transferases. The common human sialyltransferases are summarized in Table 1.

TABLE-US-00001 TABLE I Nomenclature of Neu5Ac sialyltransferases EC Abbreviation Resulting Group Substrate Number HGNC ST3Gal I Neu5Ac-α-(2,3) Gal Gal-β-1,3-GalNAc 2.4.99.4 10862 ST3Gal II Neu5Ac-α-(2,3) Gal Gal-β-1,3-GalNAc 2.4.99.4 10863 ST3Gal III Neu5Ac-α-(2,3) Gal Gal-β-1,3 2.4.99.6 10866 (4)-GlcNAc ST3Gal IV Neu5Ac-α-(2,3) Gal Gal-β-1,4-GlcNAc 2.4.99.9 10864 ST3Gal V Neu5Ac-α-(2,3) Gal Gal-β-1,4-Glc 2.4.99.9 10872 ST3Gal VI Neu5Ac-α-(2,3) Gal Gal-β-1,4-GlcNAc 2.4.99.9 18080 ST6Gal I Neu5Ac-α-(2,6) Gal Gal-β-1,4-GlcNAc 2.4.99.1 10860 ST6Gal II Neu5Ac-α-(2,6) Gal Gal-β-1,4-GlcNAc 2.4.99.2 10861 ST6GalNAc Neu5Ac-α-(2,6) GalNAc-α-1, 2.4.99.7 23614 I GalNAc O-Ser/Thr ST6GalNAc Neu5Ac-α-(2,6) c Gal-β-1,3-GalNAc- 2.4.99.7 10867 II GalNA α-1,O-Ser/Tbr ST6GalNAc Neu5Ac-α-(2,6) Neu5Ac-α-2,3-Gal- 2.4.99.7 19343 III GalNAc β-1,3-GalNAc ST6GalNAc Neu5Ac-α-(2,6) Neu5Ac-α-2,3Gal- 2.4.99.7 17846 IV GalNAc β-1,3-GalNAc ST6GalNAc Neu5Ac-α-(2,6) Neu5Ac-α-2, 2.4.99.7 19342 V GalNAc 6-GalNAc- β-1,3-GalNAc ST6GalNAc Neu5Ac-α-(2,6) All α-series 2.4.99.7 23364 VI GalNAc gangliosides ST8Sia I Neu5Ac-α-(2,8)- Neu5Ac-α-2, 2.4.99.8 10869 Neu5Ac 3-Gal-β-1, 4-Glc-β-1, 1Cer (GM3) ST8Sia II Neu5Ac-α-(2,8)- Neu5Ac-α- 2.4.99.8 10870 Neu5Ac 2,3-Gal-β-1, 4-GlcNAc ST8Sia III Neu5Ac-α-(2,8)- Neu5Ac-α-2, 2.4.99.8 14269 Neu5Ac 3-Gal-β-1, 4-GlcNAc ST8Sia IV Neu5Ac-α-(2,8)- (Neu5Ac-α-2,8) 2.4.99.8 10871 Neu5Ac nNeu5Ac- α-2,3-Gal-β-1-R ST8Sia V Neu5Ac-α-(2,8)- GM1b, GT1b, 2.4.99.8 17827 Neu5Ac GD1a, GD3 ST8Sia VI Neu5Ac-α-(2,8)- Neu5Ac-α- 2.4.99.8 23317 Neu5Ac 2,3(6)-Gal HGNC: Hugo Gene Community Nomenclature (www.genenames.org)
Domains within Polypeptides Having Sialidase Activity

[0041] The expressed polypeptide, in addition to the sialidase or catalytic portion thereof can, optionally, include peptide or protein sequences that contribute to the therapeutic activity of the protein. For example, the protein can include an anchoring domain that promotes interaction between the protein and a cell surface. The anchoring domain and sialidase domain can be arranged in any appropriate way that allows the protein to bind at or near a target cell membrane such that the therapeutic sialidase can exhibit an extracellular activity that removes sialic acid residues. The protein can have more than one anchoring domains. In cases in which the polypeptide has more than one anchoring domain, the anchoring domains can be the same or different. The protein can have more than one sialidase domain. In cases in which a compound has more than one sialidase domain, the sialidase domains can be the same or different. Where the protein comprises multiple anchoring domains, the anchoring domains can be arranged in tandem (with or without linkers) or on alternate sides of other domains, such as sialidase domains. Where a compound comprises multiple sialidase domains, the sialidase domains can be arranged in tandem (with or without linkers) or on alternate sides of other domains.

Sialidase Domain

[0042] The sialidase domain expressed by the oncolytic virus can be specific for Neu5Ac linked via alpha 2,3 linkage, specific for Neu5Ac linked via an alpha 2,6 or can cleave Neu5Ac linked via an alpha 2,3 linkage or an alpha 2,6 linkage. A variety of sialidases are described in Tables 2-5.

[0043] A sialidase that can cleave more than one type of linkage between a sialic acid residue and the remainder of a substrate molecule, in particular, a sialidase that can cleave both alpha(2, 6)-Gal and alpha(2, 3)-Gal linkages can be used in the compounds of the disclosure. Sialidases included are the large bacterial sialidases that can degrade the receptor sialic acids Neu5Ac alpha(2,6)-Gal and Neu5Ac alpha(2,3)-Gal. For example, the bacterial sialidase enzymes from Clostridium perfringens (Genbank Accession Number X87369), Actinomyces viscosus (GenBankX62276), Arthrobacter ureafaciens GenBank (AY934539), or Micromonospora viridifaciens (Genbank Accession Number D01045) can be used. Sialidase domains of compounds of the present disclosure can comprise all or a portion of the amino acid sequence of a large bacterial sialidase or can comprise amino acid sequences that are substantially homologous to all or a portion of the amino acid sequence of a large bacterial sialidase. In one preferred embodiment, a sialidase domain comprises a sialidase encoded by Actinomyces viscosus, such as that of SEQ ID NO: 1 or 2, or such as sialidase sequence substantially homologous to SEQ ID NO: 12. In yet another preferred embodiment, a sialidase domain comprises the catalytic domain of the Actinomyces viscosus sialidase extending from amino acids 274-666 of SEQ ID NO: or a substantially homologous sequence.

[0044] Additional sialidases include the human sialidases such as those encoded by the genes NEU2 (SEQ ID NO:8; Genbank Accession Number Y16535; Monti, E, Preti, Rossi, E., Ballabio, A and Borsani G. (1999) Genomics 57:137-143) and NEU4 (SEQ ID NO:9; Genbank Accession Number NM080741; Monti et al. (2002) Neurochem Res 27:646-663). Sialidase domains of compounds of the present disclosure can comprise all or a portion of the amino acid sequences of a sialidase or can comprise amino acid sequences that are substantially homologous to all or a portion of the amino acid sequences of a sialidase. Preferably, where a sialidase domain comprises a portion of the amino acid sequences of a naturally occurring sialidase, or sequences substantially homologous to a portion of the amino acid sequences of a naturally occurring sialidase, the portion comprises essentially the same activity as the intact sialidase. The present disclosure also includes sialidase catalytic domain proteins. As used herein a “sialidase catalytic domain protein” comprises a catalytic domain of a sialidase but does not comprise the entire amino acid sequence of the sialidase from which the catalytic domain is derived. A sialidase catalytic domain protein has sialidase activity. Preferably, a sialidase catalytic domain protein comprises at least 10%, at least 20%, at least 50%, at least 70% of the activity of the sialidase from which the catalytic domain sequence is derived. More preferably, a sialidase catalytic domain protein comprises at least 90% of the activity of the sialidase from which the catalytic domain sequence is derived.

[0045] A sialidase catalytic domain protein can include other amino acid sequences, such as but not limited to additional sialidase sequences, sequences derived from other proteins, or sequences that are not derived from sequences of naturally occurring proteins. Additional amino acid sequences can perform any of a number of functions, including contributing other activities to the catalytic domain protein, enhancing the expression, processing, folding, or stability of the sialidase catalytic domain protein, or even providing a desirable size or spacing of the protein.

[0046] A preferred sialidase catalytic domain protein is a protein that comprises the catalytic domain of the A. viscosus sialidase. Preferably, an A. viscosus sialidase catalytic domain protein comprises amino acids 270-666 of the A. viscosus sialidase sequence (SEQ ID NO:12). Preferably, an A. Viscosus sialidase catalytic domain protein comprises an amino acid sequence that begins at any of the amino acids from amino acid 270 to amino acid 290 of the A. viscosus sialidase sequence (SEQ ID NO: 12) and ends at any of the amino acids from amino acid 665 to amino acid 901 of said A. viscosus sialidase sequence (SEQ ID NO: 12), and lacks any A. viscosus sialidase protein sequence extending from amino acid 1 to amino acid 269.

[0047] In some preferred embodiments, an A. viscosus sialidase catalytic domain protein comprises amino acids 274-681 of the A. viscosus sialidase sequence (SEQ ID NO: 12) and lacks other A. viscosus sialidase sequence. In some preferred embodiments, an A. viscosus sialidase catalytic domain protein comprises amino acids 274-666 of the A. viscosus sialidase sequence (SEQ ID NO: 12) and lacks any other A. viscosus sialidase sequence. In some preferred embodiments, an A. viscosus sialidase catalytic domain protein comprises amino acids 290-666 of the A. viscosus sialidase sequence (SEQ ID NO: 12) and lacks any other A. viscosus sialidase sequence. In yet other preferred embodiments, an A. viscosus sialidase catalytic domain protein comprises amino acids 290-681 of the A. viscosus sialidase sequence (SEQ ID NO: 12) and lacks any other A. viscosus sialidase sequence.

TABLE-US-00002 TABLE 2 Engineered Sialidases Name Sequence AvCD MGDHPQATPAPAPDASTELPASMSQAQHLAANTATDNYRIPAI TTAPNGDLLISYDERPKDNGNGGSDAPNPNHIVQRRSTDGGKT WSAPTYIHQGTETGKKVGYSDPSYVVDHQTGTIFNFHVKSYDQ GWGGSRGGTDPENRGIIQAEVSTSTDNGWTWTHRTITADITKD KPWTARFAASGQGIQIQHGPHAGRLVQQYTIRTAGGAVQAVSV YSDDHGKTWQAGTPIGTGMDENKVVELSDGSLMLNSRASDGSG FRKVAHSTDGGQTWSEPVSDKNLPDSVDNAQIIRAFPNAAPDD PRAKVLLLSHSPNPRPWSRDRGTISMSCDDGASWTTSKVFHEP FVGYTTIAVQSDGSIGLLSEDAHNGADYGGIWYRNFTMNWLGE QCGQKPAE (SEQ ID NO: 1) DAS181 MGDHPQATPAPAPDASTELPASMSQAQHLAANTATDNYRIPAI TTAPNGDLLISYDERPKDNGNGGSDAPNPNHIVQRRSTDGGKT WSAPTYIHQGTETGKKVGYSDPSYVVDHQTGTIFNFHVKSYDQ GWGGSRGGTDPENRGIIQAEVSTSTDNGWTWTHRTITADITKD KPWTARFAASGQGIQIQHGPHAGRLVQQYTIRTAGGAVQAVSV YSDDHGKTWQAGTPIGTGMDENKVVELSDGSLMLNSRASDGSG FRKVAHSTDGGQTWSEPVSDKNLPDSVDNAQIIRAFPNAAPDD PRAKVLLLSHSPNPRPWSRDRGTISMSCDDGASWTTSKVFHEP FVGYTTIAVQSDGSIGLLSEDAHNGADYGGIWYRNFTMNWLGE QCGQKPAKRKKKGGKNGKNRRNRKKKNP (SEQ ID NO: 2)

TABLE-US-00003 TABLE 3 Human Sialidases Uniprot Name Identifier SEQ ID NO Human Neu 1 Q99519 3 Human Neu 2 Q9Y3R4 4 Human Neu 3 Q9UQ49 5 Human Neu 4 Q8WWR8 6 Human Neu 4 Q8WWR8 7 Isoform 2 Human Neu 4 Q8WWR8 8 Isoform 3

TABLE-US-00004 TABLE 4 Sialidases in organisms that are largely commensal with humans Uniprot/ Genbank Gene SEQ Organism ID name ID NO Actinomyces viscosus Q59164 nanH 9 Actinomyces viscosus A0A448PLN7 nanA 10 Streptococcus oralis A0A081R4G6 nanA 11 Streptococcus oralis D4FUA3 nanH 12 Streptococcus mitis A0A081Q0I6 nanA 13 Streptococcus mitis A0A3R9LET9 nanA_1 14 Streptococcus mitis A0A3R9J1C3 nanA_2 15 Streptococcus mitis A0A3R9IIK2 nanA_3 16 Streptococcus mitis A0A3R9IXG7 nanA_4 17 Streptococcus mitis A0A3R9K5C5 nanA_5 18 Streptococcus mitis J1H2U0 nanH 19 Porphyromonas gingivalis B2RL82 20 Tannerella forsythia Q84BM9 siaHI 21 Tannerella forsythia A0A1D3USB1 nanH 22 Akkermansia Muciniphila B2UPI5 23 Akkermansia Muciniphila B2UN42 24 Bacteroides thetaiotaomicron Q8AAK9 25

TABLE-US-00005 TABLE 5 Additional sialidases Uniprot/Genbank Organism ID Actinotignum schaalii S2VK03 Anaerotruncus colihominis B0PE27 Ruminococcus gnavus A0A2N5NZH2 Clostridium difficile Q185B3 Clostridium septicum P29767 Clostridium perfringens P10481 Clostridium perfringens Q8XMY5 Clostridium perfringens A0A2Z3TZA2 Vibrio cholerae P0C6E9 Salmonella typhimurium P29768 Paeniclostridium sordellii A0A446I8A2 Streptococcus pneumoniae (NanA) P62576 Streptococcus pneumoniae (NanB) Q54727 Pseudomonas aeruginosa A0A2X4HZU8 Aspergillus fumigatus Q4WQS0 Arthrobacter ureafaciens Q5W7Q2 Micromonospora viridifaciens Q02834

Anchoring Domain

[0048] As used herein, an “extracellular anchoring domain” or “anchoring domain” is any moiety that interacts with an entity that is at or on the exterior surface of a target cell or is in close proximity to the exterior surface of a target cell. An anchoring domain serves to retain a compound of the present disclosure at or near the external surface of a target cell. An extracellular anchoring domain preferably binds 1) a molecule expressed on the surface of a cancer cell, or a moiety, domain, or epitope of a molecule expressed on the surface of a cancer cell, 2) a chemical entity attached to a molecule expressed on the surface of a cancer cell, or 3) a molecule of the extracellular matrix surrounding a cancer cell.

[0049] Useful anchoring domains bind to heparin/sulfate, a type of GAG that is ubiquitously present on cell membranes. Many proteins specifically bind to heparin/heparan sulfate, and the GAG-binding sequences in these proteins have been identified (Meyer, F A, King, M and Gelman, R A. (1975) Biochimica et BiophysicaActa 392: 223-232; Schauer, S. ed., pp 233. Sialic Acids Chemistry, Metabolism and Function. Springer-Verlag, 1982). For example, the GAG-binding sequences of human platelet factor 4 (PF4) (SEQ ID NO:2), human interleukin 8 (IL8) (SEQ ID NO:3), humanantithrombin III (AT III) (SEQ ID NO:4), human apoprotein E (ApoE) (SEQ ID NO:5), human angio-associated migratory cell protein (AAMP) (SEQ ID NO:6), or human amphiregulin (SEQ ID NO:7) have been shown to have very high affinity to heparin.

Linkers

[0050] A protein that includes a sialidase or a catalytic domain thereof can optionally include one or more polypeptide linkers that can join domains of the compound. Linkers can be used to provide optimal spacing or folding of the domains of a protein. The domains of a protein joined by linkers can be sialidase domains, anchoring domains, or any other domains or moieties of the compound that provide additional functions such as enhancing protein stability, facilitating purification, etc. Some preferred linkers include the amino acid glycine. For example, linkers having the sequence: (GGGGS (SEQ ID NO:10))n, where n is 1-20.

EXAMPLES

Example 1: DAS181 Treatment Reduces Surface Sialic Acid on Tumor Cells

[0051] In this study the impact of DAS181 on the sialic acid burden of certain tumor cells was examined. Briefly, FACs and image-based quantitation of α-2,3 and α-2,6 sialic acid modifications on A549 (human alveolar basal epithelial adenocarcinoma) and MCF (human mamillary epithelial adenocarcinoma) tumor cells were conducted. Galatose exposure after sialic acid removal in A549 and MCF7 cells was detected by PNA-FITC using flow cytometry analysis and imaging approaches. As discussed above, there are two sialic acid is most often attached to the penultimate sugar by an α-2,3 linkage or an α-2,6 linkage, which can that can be detected by Maackia Amurensis Lectin II (MAL II) and Sambucus Nigra Lectin (SNA), respectively. In addition, surface galactose (e.g., galactose exposed after sialic acid removal) can be detected using Peanut Agglutinin (PNA).

[0052] FIG. 1 depicts the detection of 2,6 sialic acid by FITC-SNA on A549 and MCF cells by fluorescence imaging.

[0053] A549 cells were treated with various concentrations of DAS181 and them stained to image 2,6 linked sialic acid (FITC-SNA), 2,3 linked sialic acid (FITC-MALII) or galactose (FITC-PNA). As can be seen in FIG. 2, DAS181 effectively removed both 2,3 and 2,6 linked sialic acid and exposed galactose.

[0054] In contrast, DAS185, a variant of DAS181 lacking sialidase activity due to Y348F mutation, was not able to remove 2,6 linked sialic acid or 2,3 linked sialic acid. As shown in FIG. 3, incubation of A549 cells with DAS185 had essentially no impact on surface 2,3 linked sialic acid, while DAS181 reduced surface 2,3 linked sialic acid in a concentration dependent manner. Similarly, incubation of A549 cells with DAS185 had essentially no impact on surface 2,6 linked sialic acid, while DAS181 reduced surface 2,6 linked sialic acid in a concentration dependent manner (FIG. 4). Consistent with these results, incubation of A549 cells with DAS185 had essentially no impact on surface galactose, while DAS181 increased surface galactose in a concentration dependent manner.

Example 2: DAS181 Treatment Increases PDMC-Mediated Tumor Cell Killing

[0055] A549 cells were genetically labelled with a red fluorescent protein (A549-red). Fresh human PMBCs were harvested and stimulated with various cytokine and antibody combinations to activate effector T cells (CD3, CD38 and IL-2) or, in some cases, T cells and NK cells (CD3, CD28, IL-15 and IL-21). Activated PBMCs were then co-cultured with A549-red cells that had been exposed to DAS181 (100 nM). Tumor cell killing by PBMCs was monitored by live cell imaging and quantification with IncuCyte. The cell culture medium was collected and analyzed by ELISA to assess cytokine production by PBMCs.

[0056] FIG. 6 shows that neither the treatments used to stimulate PBMC nor DAS181 in combination with treatment used to stimulate PBMC impact A549-red cell proliferation.

[0057] FIG. 7 shows that DAS181 significantly increases tumor cell toxicity mediated by PBMC (Donor 1), both T cell mediated and NK cell mediated, compared to a vehicle only control. Similar results were observed using PBMC from a different donor (Donor 2; FIG. 8). FIG. 9 and FIG. 10 present a quantification of the data presented in FIG. 7 and FIG. 8, respectively.

Example 3: NK Cell Mediated Killing of Tumor Cells by Oncolytic Vaccinia Virus and DAS181

[0058] In this study the impact of an oncolytic vaccina virus (Western Reserve) and DAS181 on NK cell-mediated killing was examined. DAS185, a variant protein lacking sialidase activity was used as a control.

[0059] Briefly, tumor cells (U87-GFP) were plated in a 96-well tissue culture plate at 5×10.sup.4 cells per well (100 ul) in DMEM and incubated overnight at 37° C. On Day 2 the cells were infected with VV at MOI 0.5, 1, or 2 in fetal bovine serum-free medium for 2 hours and then exposed to 1 nM DAS181 or 1 mM DAS185. Tumor cells were then mixed with purified NK cells at Effector:Tumor (E:T)=1:1, 5:1, 10:1. The cells were cultured in medium supplemented with 2% FBS in order to decrease neuraminidase/sialidase background. After 24 hrs, tumor killing were measured by MTS assay (96 well plate), and cell culture medium was collected. Expression of IFN gamma were measured by ELISA. The results of this study are shown in FIG. 11 and FIG. 12 where it can be seen the DAS181, but not inactive DAS185, increased tumor cell killing by oncolytic vaccinia virus.

Example 4: Impact of DAS181 on DC Maturation and Antigen Presenting Activity in the Presence of Tumor Cells

[0060] In this study, the impact of DAS181 on monocyte-derived dendritic cell was examined DAS185, a variant protein lacking sialidase activity was used as a control.

[0061] Briefly, monocyte-derived dendritic cells (DC) were prepared by resuspending 5×10.sup.6 adherent PBMC in 3 ml of medium supplemented with 100 ng/ml of GM-CSF and 50 ng/ml of IL-4. After 48 hrs, 2 ml of fresh medium supplemented with 100 ng/ml of GM-CSF and 50 ng/ml of IL-4 was added to each well. After another 72 hrs, tumor cell (U87-GFP) were plated in 24-well plates in DMEM. The tumor cells were infected with VV at various MOI in FBS free medium for 2 hours. DC cultured in the presence of 1 nM DAS181 or DAS185 were mixed with tumor cells at 1:1 tumor cell:DC ratio. Dendritic cell maturation (expression of CD86, CD80, MHC-I) and production of pro-inflammatory cytokines (TNF-alpha) was then measured and quantified by flow cytometry and ELISA, respectively.

[0062] As can be seen in FIG. 13, DAS181 significant enhanced expression of dendritic cell maturation markers whether the cells were cultured alone or with vaccinia virus infected tumor cells.

[0063] The results of this study demonstrate that exposure to DAS181 increased and increased TNF-alpha secretion by dendritic cells (FIG. 14).

Example 5: DAS181 Increases Oncolytic Adenovirus Tumor Cell Killing in the Absence of Immune Cells

[0064] A549 cells were genetically labelled with red fluorescent protein (A549-red). Tumor cell proliferation and killing by oncolytic adenovirus (Ad5) in the presence or absence of DAS181 was monitored by live cell imaging and quantification with IncuCyte. The cell culture medium was collected for ELISA measurement of cytokine production by PBMCs. As shown in FIG. 15, DAS181 increased oncolytic adenovirus-mediated tumor cell killing and growth inhibition.

Example 6: DAS181 Increases Oncolytic Adenovirus Tumor Cell Killing in the Presence of PBMC

[0065] A549 cells were genetically labelled by a red fluorescent protein (A549-red). Fresh human PMBCs were harvested and stimulated with proper cytokine and antibody combinations to activate effector T cells. Activated PBMCs were then co-cultured with A549-red cells that have been treated with DAS181 with or without the oncolytic adenovirus (Ad5). Tumor cell killing by PBMCs was monitored by live cell imaging and quantification with IncuCyte. The cell culture medium was collected for ELISA measurement of cytokine production by PBMCs. As shown in FIG. 16, DAS181 significantly increased tumor cell killing when present together with oncolytic adenovirus in the presence of PBMC.

Example 7: Construction and Characterization of an Oncolytic Virus Expressing DAS181

[0066] A construct designed for expression of DAS181 is depicted schematically in FIG. 17.

[0067] To generate a recombinant VV expressing DAS181, a pSEM-1 vector was modified to include a sequence encoding DAS181 as well as two loxP sites with the same orientation flanking the sequence encoding the GFP protein (pSEM-1-TK-DAS181-GFP). DAS181 expression is under the transcriptional control of the F17R late promoter in order to limit the expression within tumor tissue. The sequences certain of the components and a portion of the construct and are shown in FIG. 18 and FIG. 19.

[0068] Western Reserve VV was used as the parental virus. VV expressing DAS181 was generated by recombination with pSEM-1-TK-DAS181-GFP into the TK gene of Western Reserve VV to generated VV-DAS181.

[0069] Recombinant Virus can be Generated as Follows.

Transfection:

[0070] Seed CV-1 cells in 6-well plate at 5×10.sup.5 cells/2 ml DMEM-10% FBS/well and grow overnight. Prepare parent VV virus (1 ml/well) by diluting a virus stock in DMEM/2% FBS at MOI 0.05. Remove medium from CV-1 wells and immediately add VV, and culture for 1-2 hours. CV-1 cells should be 60-80% confluent at this point. Transfection mix in 1.5 ml tubes. For each Transfection, dilute 9 ul Genejuice in 91 ul serum-free DMEM and incubate at room temperature for 5 min. Add 3 ug pSEM-1-TK-DAS181-GFP DNA gently by pipetting up and down two or three times. Leave at room temperature for 15 min. Aspirate VV virus from the CV-1 well and wash the cells once with 2 ml serum-free DMEM. Add 2 ml DMEM-2% FBS and add the DNA-genejuice solution drop-by-drop. Incubate at 37° C. for 48-72 hr or until all the cells round up. Harvest the cells by pipetting repeatedly. Release the virus from cells by repeated freeze-thawing of the harvested cells by first placing them in dry-ice/ethanol bath and then thawing them in a 37° C. water bath and vortexing. Repeat the freeze-thaw cycling three times. The cell lysate can be stored at −80° C.

Plaque Isolation:

[0071] Seed CV-1 cells in 6-well plates at 5×10.sup.5 cells/2 ml DMEM-10% FBS/well and grow overnight. CV-1 cells should be 60-80% confluent when receiving cell lysate. Sonicate the cell lysate on ice using sonic dismembrator with an ultrasonic convertor probe for 4 cycles of 30 s until the material in the suspension is dispersed. Make 10-fold serial dilutions of the cell lysate in DMEM-2% FBS. Add 1 ml of the cell lysate-medium per well at dilutions 10.sup.−2, 10.sup.−3, 10.sup.−4, incubate at 37° C. Pick well-separated GFP+ plaques using pipet tip. Rock the pipet tip slightly to scrape and detach cells in the plaque. Gently transfer to a microcentrifuge tube containing 0.5 ml DMEM medium. Freeze-thaw three times and sonicate. Repeat the same process of plaque isolation 3-5 times.

Virus Amplification:

[0072] Seed CV-1 cells 5×10.sup.5 cells/2 ml DMEM-10% FBS/well and grow overnight in 6-well plate. CV-1 should be confluent when starting the experiment. Infect 1 well with 250 ul of plaque lysate/1 ml DMEM-2% FBS, and incubate at 37° C. for 2 h. Remove the plaque lysate and add 2 ml fresh DMEM-2% FBS, and incubate for 48-72 hr until cells round up. Collect the cells by repeatedly pipetting, freeze-thaw 3 times and sonicate. Add half of the cell lysate in 4 ml DMEM-2% FBS and infect CV-1 cells in 75-CM2 flask, after 2 h, remove virus and add 12 ml DMEM-2% FBS and culture 48-72 h (until cell round up). Harvest the cells, spin down 5 min at 1800 G, and discard supernatant and resuspend in 1 ml DMEM-2.5% FBS.

Virus Titration:

[0073] Seed CV-1 cells 5×10.sup.5 cells/2 ml DMEM-10% FBS/well and grow overnight in 6-well plate. Dilute virus in DMEM-2% FBS, 50 ul virus/4950 ul DMEM-2% FBS (A, 10.sup.−2), 500 ul A/4500 ul medium (B, 10.sup.−3), and 500 ul B/4500 ul medium (C, 10.sup.−4), 10.sup.−7 to 10.sup.−10 for virus stock. Remove medium and wash 1× with PBS, and cells were infected with 1 ml virus dilution in duplicate. Incubate the cells for 1 h, rock the plate every 10 min. 1 h later, remove the virus and add 2 ml DMEM-10% FBS and incubate 48 h. Remove the medium, add 1 ml of 0.1% crystal violet in 20% ethanol for 15 min at room temperature. Remove the medium and allow to dry at room temperature for 24 hr. Count the plaque and express as plaque forming units (pfu) per ml.

Detection of DAS181 Expression by VV-RAS181:

[0074] CV-1 cells were infected with VV-DAS181 at MOI 0.2. 48 hours later, CV-1 cells were collected. DNA was extracted using Wizard SV Genomic DAN Purification System and used as template for DAS181 PCR amplification. PCR was conducted using standard PCR protocol and primer sequences (SialF: GGCGACCACCCACAGGCAACACCAGCACCTGCCCCA and SialR: CCGGTTGCGCCTATTCTTGCCGTTCTTGCCGCC). The expected PCR product (1251 bp) was found.

Example 8: DAS181 Expressed by Vaccinia Virus is Active In Vitro

[0075] CV-1 cells were plated in six well plate. The cells were transduced with Sialidase-VV or control VV at MOI 0.1 or MOI 1. After 24 hrs, transfected cells were collected, and single cell suspension were made in PBS at 3×10.sup.6/500 ul. Cell lysate was prepared using Sigma's Mammalian cell lysis kit for protein extraction (Sigma, MCL1-1KT), and supernatant was collected. The sialidase (DAS181) activity was measured using Neuraminidase Assay Kit (Abcam, ab138888) according to manufacturer's instruction. 1 nM, 2 nM, and 10 nM DAS181 was added to the VV-cell lysate as control and generated the standard curve. 1×10{circumflex over ( )}6 cells infected with Sialidase-VV express DAS181 equivalent to 0.78 nM-1.21 nM of DAS181 in 1 ml medium. As shown in FIG. 20, the DAS181 has sialidase activity in vitro.

Example 9: Vaccinia Virus-Sialidase Promotes Dendritic Cell Maturation

[0076] To determine if Sialidase-VV can promote DC activation and maturation, adherent human PBMC were re-suspend at 5×10.sup.6 cells in 3 ml medium supplemented with 100 ng/ml of GM-CSF and 50 ng/ml of IL-4 then cultured in 6-well plates with 2 ml per well of fresh medium supplemented with same concentrations of GM-CSF and IL-4. After 48 hrs, the cells were cultured in the presence of Sialidase-VV infected tumor cell lysate, VV-infected tumor cell lysate, VV-infected tumor cell lysate plus synthetic DAS181 protein, or LPS (positive control). After another 24 hrs, expression of CD86, CD80, MHC-II, MHC-I were determined by flow cytometry. As shown in FIG. 21, Sialidase-VV promotes the expression of markers indicative of dendritic cell activation and maturation.

Example 10: Sialidase-VV Enhances T Lymphocyte-Mediated Cytokine Production and Oncolytic Activity

[0077] To assess whether DAS181 can activate human T cells by inducing IFN-gamma (IFNr) and IL-2 expressing, human PBMCs were activated by adding CD3 antibody at 10 ug/ml, proliferation was further stimulated by adding IL-2 by every 48 hrs. On day 15, tumor cells (A549) were infected with VVs at MOI 0.5, 1, or 2 in 2.5% FBS medium for 2 hours. Activated T cells were added to the culture at effector:target ratio of 5:1 or 10:1 in the presence of CD3 antibody at 1 ug/ml. After another 24 hrs, tumor cytotoxicity was measured and cell culture medium was collected for cytokine array. As can be seen in FIG. 22, Sialidase-VV induces a significantly greater IL2 and IFN-gamma expression by CD3 activated T cells than does VV. In addition, as can be seen in FIG. 23, Sialidase-VV elicits stronger anti-tumor response than VV at and E;T of 5:1.

TABLE-US-00006 Sequences of certain Sialidases SEQ ID NO: 3         10         20         30         40         50         60         70         80 MTGERPSTAL PDRRWGPRIL GFWGGCRVWV FAAIFLLLSL AASWSKAEND FGLVQPLVTM EQLLWVSGRQ IGSVDTFRIP         90        100        110        120        130        140        150        160 LITATPRGTL LAFAEARKMS SSDEGAKFIA LRRSMDQGST WSPTAFIVND GDVPDGLNLG AVVSDVETGV VFLVYSLCAH        170        180        190        200        210        220        230        240 KAGCQVASTM LWVSKKDGVS WSTPRNLSLD IGTEVFAPGP GSGIQKQREP RKGRLIVCGH GTLERDGVFC LLSDDHGASW        250        260        270        280        290        300        310        320 RYGSGVSGIP YGQPKQENDF NPDECQPYEL PDGSVVINAR NQNNYHCHCR IVLRSYDACD TLRPRDVTFD PELVDPVVAA        330        340        350        360        370        380        390        400 GAVVTSSGIV FFSNPAHPEF RVNLTLRWSF SNGTSWRKET VQLWPGPSGY SSLATLEGSM DGEEQAPQLY VLYEKGRNHY        410 TESISVAKIS VYGTL SEQ ID NO: 4         10         20         30         40         50         60         70         80 MASLPVLQKE SVFQSGAHAY RIPALLYLPG QQSLLAFAEQ RASKKDEHAE LIVLRRGDYD APTHQVQWQA QEVVAQRALD         90        100        110        120        130        140        150        160 GHRSMNPCPL YDAQTGTLFL FFIAIPGQVT EQQQLQTRAN VTRLCQVTST DHGRTWSSPR DLTDAAIGPA YREWSTFAVG        170        180        190        200        210        220        230        240 PGHCLQLHDR ARSLVVPAYA YRKLHPIQRP IPSAFCFLSH DHGRTWARGH FVAQDTLECQ VAEVETGEQR VVTLNARSHL        250        260        270        280        290        300        310        320 RARVQAQSTN DGLDFQESQL VKKLVEPPPQ GCQGSVISFP SPRSGPGSPA QWLLYTHPTH SWQRADLGAY LNPRPPAPEA        330        340        350        360        370        380 WSEPVLLAKG SCAYSDLQSM GTGPDGSPLF GCLYEANDYE EVIFLMFTLK QAFPAEYLPQ SEQ ID NO: 5         10         20         30         40         50         60         70         80 MEEVTTCSFN SPLFRQEDDR GITYRIPALL YIPPTHTFLA FAEKRSTRRD EDALHLVLRR GLRIGQLVQW GPLKPLMEAT         90        100        110        120        130        140        150        160 LPGHRTMNPC PVWEQKSGCV FLFFICVRGH VTERQQIVSG RNAARLCFIY SQDAGCSWSE VRDLTEEVIG SELKHWATFA        170        180        190        200        210        220        230        240 VGPGHGIQLQ SGRLVIPAYT YYIPSWFFCF QLPCKTRPHS LMIYSDDLGV TWHHGRLIRP MVTVECEVAE VTGRAGHPVL        250        260        270        280        290        300        310        320 YCSARTPNRC RAEALSTDHG EGFQRLALSR QLCEPPHGCQ GSVVSFRPLE IPHRCQDSSS KDAPTIQQSS PGSSLRLEEE        330        340        350        360        370        380        390        400 AGTPSESWLL YSHPTSRKQR VDLGIYLNQT PLEAACWSRP WILHCGPCGY SDLAALEEEG LFGCLFECGT KQECEQIAFR        410        420 LFTHREILSH LQGDCTSPGR NPSQFKSN SEQ ID NO: 6         10         20         30         40         50         60         70         80 MGVPRTPSRT VLFERERTGL TYRVPSLLPV PPGPTLLAFV EQRLSPDDSH AHRLVLRRGT LAGGSVRWGA LHVLGTAALA         90        100        110        120        130        140        150        160 EHRSMNPCPV HDAGTGTVFL FFIAVLGHTP EAVQIATGRN AARLCCVASR DAGLSWGSAR DLTEEAIGGA VQDWATFAVG        170        180        190        200        210        220        230        240 PGHGVQLPSG RLLVPAYTYR VDRRECFGKI CRTSPHSFAF YSDDHGRTWR CGGLVPNLRS GECQLAAVDG GQAGSFLYCN        250        260        270        280        290        300        310        320 ARSPLGSRVQ ALSTDEGTSF LPAERVASLP ETAWGCQGSI VGFPAPAPNR PRDDSWSVGP GSPLQPPLLG PGVHEPPEEA        330        340        350        360        370        380        390        400 AVDPRGGQVP GGPFSRLQPR GDGPRQPGPR PGVSGDVGSW TLALPMPFAA PPQSPTWLLY SHPVGRRARL HMGIRLSQSP        410        420        430        440        450        460        470        480 LDPRSWTEPW VIYEGPSGYS DLASIGPAPE GGLVFACLYE SGARTSYDEI SFCTFSLREV LENVPASPKP PNLGDKPRGC CPWS SEQ ID NO: 7         10         20         30         40         50         60         70         80 MMSSAAFPRW LSMGVPRTPS RTVLFERERT GLTYRVPSLL PVPPGPTLLA FVEQRLSPDD SHAHRLVLRR GTLAGGSVRW         90        100        110        120        130        140        150        160 GALHVLGTAA LAEHRSMNPC PVHDAGTGTV FLFFIAVLGH TPEAVQIATG RNAARLCCVA SRDAGLSWGS ARDLTEEAIG        170        180        190        200        210        220        230        240 GAVQDWATFA VGPGHGVQLP SGRLLVPAYT YRVDRRECFG KICRTSPHSF AFYSDDHGRT WRCGGLVPNL RSGECQLAAV        250        260        270        280        290        300        310        320 DGGQAGSFLY CNARSPLGSR VQALSTDEGT SFLPAERVAS LPETAWGCQG SIVGFPAPAP NRPRDDSWSV GPGSPLQPPL        330        340        350        360        370        380        390        400 LGPGVHEPPE EAAVDPRGGQ VPGGPFSRLQ PRGDGPRQPG PRPGVSGDVG SWTLALPMPF AAPPQSPTWL LYSHPVGRRA        410        420        430        440        450        460        470        480 RLHMGIRLSQ SPLDPRSWTE PWVIYEGPSG YSDLASIGPA PEGGLVFACL YESGARTSYD EISFCTFSLR EVLENVPASP        490 KPPNLGDKPR GCCWPS SEQ ID NO: 8         10         20         30         40         50         60         70         80 MMSSAAFPRW LQSMGVPRTP SRTVLFERER TGLTYRVPSL LPVPPGPTLL AFVEQRLSPD DSHAHRLVLR RGTLAGGSVR         90        100        110        120        130        140        150        160 WGALHVLGTA ALAEHRSMNP CPVHDAGTGT VFLFFIAVLG HTPEAVQIAT GRNAARLCCV ASRDAGLSWG SARDLTEEAI        170        180        190        200        210        220        230        240 GGAVQSWATF AVGPGHGVQL PSGRLLVPAY TYRVDRRECF GKICRTSPHS FAFYSDDHGR TWRCGGLVPN LRSGECQLAA        250        260        270        280        290        300        310        320 VDGGQAGSFL YCNARSPLGS RVQALSTDEG TSFLPAERVA SLPETAWGCQ GSIVGFPAPA PNRPRDDSWS VGPGSPLQPP        330        340        350        360        370        380        390        400 LLGPGVHEPP EEAAVDPRGG QVPGGPFSRL QPRGDGPRQP GPRPGVSGDV GSWTLALPMP FAAPPQSPTW LLYSHPVGRR        410        420        430        440        450        460        470        480 ARLHMGIRLS QSPLDPRSWT EPWVIYEGPS GYSDLASIGP APEGGLVFAC LYESGARTSY DEISFCTFSL REVLENVPAS        490 PKPPNLGDKP RGCCWPS SEQ ID NO: 9         10         20         30         40         50         60         70         80 MTSHSPFSRR RLPALLGSLP LAATGLIAAA PPAHAVPTSD GLADVTITQV NAPADGLYSV GDVMTFNITL TNTSGEAHSY         90        100        110        120        130        140        150        160 APASTNLSGN VSKCRWRNVP AGTTKTDCTG LATHTVTAED LKAGGFTPQI AYEVKAVEYA GKALSTPETI KGATSPVKAN        170        180        190        200        210        220        230        240 SLRVESITPS SSQENYKLGD TVSYTVRVRS VSDKTINVAA TESSFDDLGR QCHWGGLKPG KGAVYNCKPL THTITQADVD        250        260        270        280        290        300        310        320 AGRWTPSITL TATGTDGATL QTLTATGNPI NVVGDHPQAT PAPAPDASTE LPASMSQAQH LAANTATDNY RIPAIPPPPM        330        340        350        360        370        380        390        400 GTCSSPTTSA RRTTATAAAT TPNPNHIVQR RSTDGGKTWS APTYIHQGTE TGKKVYGSDP SYVVDHQTGT IFNFHVKSYD         410        420        430        440        450        460        470        480 QGWGGSRGGT DPENRGIIQA EVSTSTDNGW TWTHRTITAD ITKDKPWTAR FAASGQGIQI QHGPHAGRLV QQYTIRTAGG        490        500        510        520        530        540        550        560 PVQAVSVYSD DHGKTWQAGT PIGTGMDENK VVELSDGSLM LNSRASDGSG FRKVAHSTDG GQTWSEPVSD KNLPDSVDNA        570        580        590        600        610        620        630        640 QIIRAFPNAA PDDPRAKVLL LSHSPNPRPW CRDRGTISMS CDDGASWTTS KVFHEPFVGY TTIAVQSDGS IGLLSEDAHN        650        660        670        680        690        700        710        720 GADYGGIWYR NFTMNWLGEQ CGQKPAEPSP GRRRRRHPQR HRRRSRPRRP RRALSPRRHR HHPPRPSRAL RPSRAGPGAG        730        740        750        760        770        780        790        800 AHDRSEHGAH TGSCAQSAPE QTDGPTAAPA PETSSAPAAE PTQAPTVAPS VEPTQAPGAQ PSSAPKPGAT GRAPSVVNPK        810        820        830        840        850        860        870        880 ATGAATEPGT PSSSASPAPS RNAAPTPKPG MEPDEIDRPS DGTMAQPTGA PARRVPRRRR RRRPAAGCLA RDQRAADPGP        890        900        910 CGCRGCRRVP AAAGSPFEEL NTRRAGHPAL STD SEQ ID NO: 10         10         20         30         40         50         60         70         80 MTTTKSSALR RLSALAGSLA LAVTGIIAAA PPAHATPTSD GLADVTITQT HAPADGIYAV GDVMTFDITL TNTSGQARSF         90        100        110        120        130        140        150        160 APASTNLSGN VLKCRWSNVA AGATKTDCTG LATHTVTAED LKAGGFTPQI AYEVKAVGYK GEALNKPEPV TGPTSQIKPA        170        180        190        200        210        220        230        240 SLKVESFTLA SPKETYTVGD VVSYTVRIRS LSDQTINVAA TDSSFDDLAR QCHWGNLKPG QGAVYNCKPL THTITQADAD        250        260        270        280        290        300        310        320 HGTWTPSITL AATGTDGAAL QTLAATGEPL SVVVERPKAD PAPAPDASTE LPASMSDAQH LAENTATDNY RIPAITTAPN        330        340        350        360        370        380        390        400 GDLLVSYDER PRDNGNNGGD SPNPNHIVQR RSTDGGKTWS APSYIHQGVE TGRKVGYSDP SYVVDNQTGT IFNFHVKSFD        410        420        430        440        450        460        470        480 QGWGHSQAGT DPEDRSVIQA EVSTSTDNGW SWTHRTITAD ITRDNPWTAR FAASGQGIQI HQGPHAGRLV QQYTIRTADG        490        500        510        520        530        540        550        560 VVQAVSVYSD DHGQTWQAGT PTGTGMDENK VVELSDGSLM LNSRASDGTG FRKVATSTDG GQTWSEPVPD KNLPDSVDNA        570        580        590        600        610        620        630        640 QIIRPFPNAA PSDPRAKVLL LSHSPNPRPW SRDRGTISMS CDNGASWVTG RVFNEKFVGY TTIAVQSDGS IGLLSEDGNY        650        660        670        680        690        700        710        720 GGIWYRNFTM GWVGDQCSQP RPEPSPSPTP SAAPSAEPTS EPTTAPAPEP TTAPSSEPSV SPEPSSSAIP APSQSSSATS        730        740        750        760        770        780        790 GPSTEPDEID RPSDGAMAQP TGGAGRPSTS VTGATSRNGL SRTGTNALLV LGVAAAAAAG GYLVLRIRRA RTE SEQ ID NO: 11         10         20         30         40         50         60         70         80 MNYKSLDRKQ RYGIRKFAVG AASVVIGTVV FGANPVLAQE QANAAGNATE TVEPGQGLSE LPKEASSGDL AHLDKDLAGK         90        100        110        120        130        140        150        160 LAAAQDNGVE VDQDHLKKNE SAESETPSST ETPAEEANKE EESEDQGAIP RDYYSRDLKN ANPVLEKEDV ETNAANGQRV        170        180        190        200        210        220        230        240 DLSNELDKLK QLKNATVHME FKPDASAPRF YNLFSVSSDT KENEYFTMSV LDNTALIEGR GANGEQFYDK YTDAPLKVRP        250        260        270        280        290        300        310        320 GQWNSVTFTV EQPTTELPHG RVRLYVNGVL SRTSLKSGNF IKDMPDVNQA QLGATKRGNK TVWASNLQVR NLTVYDRALS        330        340        350        360        370        380        390        400 PDEVQTRSQL FERGELEQKL PEGAKVTEKE DVFEGGRNNQ PNKDGIDSYR IPALLKTDKG TLIAGTDERR LHHSDWGDIG        410        420        430        440        450        460        470        480 MVVRRSSDNG KTWGDRIVIS NPRDNEHAKH ADWPSPVNID MALVQDPETK RIFAIYDMFL ESKAVFSLPG QAPKAYEQVG        490        500        510        520        530        540        550        560 DKVYQVLYKQ GESGRYTIRE NGEVFDPQNR KTDYRVVVDP KKPAYSDKGD LYKGNELIGN IYFEYSEKNI FRVSNTNYLW        570        580        590        600        610        620        630        640 MSYSDDDGKT WSAPKDITHG IRKDWMHFLG TGPGTGIALR TGPHKGRLVI PVYTTNNVSY LSGSQSSRVI YSDDHGETWQ        650        660        670        680        690        700        710        720 AGEAVNDNRP VGNQTIHSST MNNPGAQNTE STVVQLNNGD LKLFMRGLTG DLQVATSHDG GATWDKEIKR YPQVKDVYVQ        730        740        750        760        770        780        790        800 MSAIHTMHEG KEYILLSNAG GPGRNNGLVH LARVEENGEL GKFAYNSLQE LGNGEYGLLY EHADGNQNDY EHADGNQNDY        810        820        830        840        850        860        870        880 TLSYKKFNWD FLSRDRISPK EAKVKYAIQK WPGIIAMEFD SEVLVNKAPT LQLANGKTAT FMTQYDTKTL LFTIDPEDMG        890        900        910        920        930        940        950        960 QRITGLAEGA IESMHNLPVS LAGSKLSDGI NGSEAAIHEV PEFTGGVNAE EAAVAEIPEY TGPLATVGEE VAPTVEKPEF        970        980        990       1000       1010       1020       1030       1040 TGGVNAEEAP VAEMPEYTGP LSTVGEEVAP TVEKPEFTGG VNAVEAAVHE LPEFKGGVNA VLAASNELPE YRGGANFVLA       1050       1060       1070       1080       1090       1100       1110       1120 ASNDLPEYIG GVNGAEAAVH ELPEYKGDTN LVLAAADNKL SLGQDVTYQA PAAKQAGLPN TGSKETHSLI SLGLAGVLLS       1130 LFAFGKKRKE SEQ ID NO: 12         10         20         30         40         50         60         70         80 MSDLKKYEGV IPAFYACYDD QGEVSPERTR ALVQYFIDKG VQGLYVNGSS GECIYQSVED RKLILEEVMA VAKGKLTIIA         90        100        110        120        130        140        150        160 HVACNNTKDS MELARHAESL GVDAIATIPP IYFRLPEYSV AKYWNDISAA APNTDYVIYN IPQLAGVALT PSLYTEMLKN        170        180        190        200        210        220        230        240 PRVIGVKNSS MPVQDIQTFV SLGGEDHIVF NGPDEQFLGG RLMGAKAGIG GTYGAMPELF LKLNQLIAEK DLETARELQY        250        260        270        280        290        300 AINAIIGKLT SAHGNMYGVI KEVLKINEGL NIGSVRSPLT PVTEEDRPVV EAAAQLIRET KERFL SEQ ID NO: 13         10         20         30         40         50         60         70         80 MNQRHFDRKQ RYGIRKFTVG AASVVIGAVV FGVAPALAQE APSTNGETAG QSLPELPKEV ETGNLTNLDK ELADKLSTAT         90        100        110        120        130        140        150        160 DKGTEVNREE LQANPGSEKA AETEASNETP ATESEDEKED GNIPRDFYAR ELENVNTVVE KEDVETNPSN GQRVDMKEEL        170        180        190        200        210        220        230        240 DKLKKLQNAT IHMEFKPDAS APRFYNLFSV SSDTKVNEYF TMAILDNTAI VEGRDANGNQ FYGDYKTAPL KIKPGEWNSV        250        260        270        280        290        300        310        320 TFTVERPNAD QPKGQVRVYV NGVLSRTSPQ SGRFIKDMPD VNQVQIGTTK RTGKNFWGSN LKVRNLTVYD RALSPEEVKK        330        340        350        360        370        380        390        400 RSQLFERGEL EKKLPEGAKV TDKLDVFQGG ENRKPNKDGI ASYRIPALLK TDKGTLIAGA DERRLHHSDW GDIGMVVRRS        410        420        430        440        450        460        470        480 DDKGKTWGDR IVISNPRDNE NARRAHAGSP VNIDMALVQD PKTKRIFSIF DMFVEGEAVR DLPGAKPQAY EQIGNKVYQV        490        500        510        520        530        540        550        560 KYKKGEAGHY TIRENGEVFD PENRKTEYRV VVDPKKPAYS DKGDLYKGEE LIGNVYFDYS DKNIFRVSNT NYLWMSYSDD        570        580        590        600        610        620        630        640 DGKTWSAPKD ITYGIRKDWM HFLGTGPGTG IALHSGPHKG RLVIPAYTTN NVSYLGGSQS SRVIYSDDHG ETWHAGEAVN        650        660        670        680        690        700        710        720 DNRPIGNQTI HSSTMNNPGA QNTESTVVQL NNGDLKLFMR GLTGDLQVAT SKDGGATWEK DVKRYADVKD VYVQMSAIHT        730        740        750        760        770        780        790        800 VQEGKEYIIL SNAGGPGRYN GLVHVARVEA NGDLTWIKHN PIQSGKFAYN SLQDLGNGEF GLLYEHATAT QNEYTLSYKK        810        820        830        840        850        860        870        880 FNWDFLSKDG VAPTKATVKN AVEMSKNVIA LEFDSEVLVN QPPVLKLANG NFATFLTQYD SKTLLFAASK EDIGQEITEI        890        900        910        920        930        940        950        960 IDGAIESMHN LPVSLEGAGV PGGKNGAKAA IHEVPEFTGA VNGEGTVHED PAFEGGINGE EAAVHDVPDF SGGVNGEVAA        970        980        990       1000       1010       1020       1030       1040 IHEVPEFTGG INGEEAAKLE LPSYEGGANA VEAAKSELPS YEGGANAVEA AKLELPSYES GAHEVQPASS LNPTLADSVN       1050       1060       1070       1080       1090       1100       1110 SEQ ID NO: 14         10         20         30         40         50         60         70         80 MNQSSLNRKN RYGIRKFTIG VASVAIGSVL FGITPALAQE TTTNIDVSKV ETSLESGAPV SEPVTEVVSG DLNHLDKDLA         90        100        110        120        130        140        150        160 DKLALATNQG VDVNKHNLKE ETSKPEGNSE HLPVESNTGS EESIEHHPAK IEGADDAVVP PRDFFARELT NVKTVFERED        170        180        190        200        210        220        230        240 LATNTGNGQR VDLAEELDQL KQLQNATIHN EFKPDANAPQ FYNLFSVSSD KKKDEYFSMS VNKGTAMVEA RGADGSHFYG        250        260        270        280        290        300        310        320 SYSDAPLKIK PGQWNSVTFT VERPKADQPN GQVRLYVNGV LSRTNTKSGR FIKDMPDVNK VQIGATRRAN QTMWGSNLQI        330        340        350        360        370        380        390        400 RNLTVYNRAL TIEEVKKRSH LFERNDLEKK LPEGAEVTEK KDIFESGRNN QPNGEGINSY RIPALLKTDK GTLIAGGDER        410        420        430        440        450        460        470        480 RLHHFDYGDI GMVIRRSQDN GKTWGDKLTI SNLRDNPEAT DKTATSPLNI DMVLVQDPTT KRIFSIYDMF PETRAVFGMP        490        500        510        520        530        540        550        560 NQPEKAYEEI GDKTYQVLYK QGETERYTLRDNGEIFNSQN KKTEYRVVVN PTEAGFRDKG DLYKNQEILIG NIYFKQSDKN        570        580        590        600        610        620        630        640 PFRVANTSYL WMSYSDDDGK TWSAPKDITP GIRQDWMKFL GTGPGTGIVL RTGAHKGRIL VPAYTTNNIS HLGGSQSSRL        650        660        670        680        690        700        710        720 IYSDDHGQTW HAGESPNDNR PVGNSVIHSSNMNKSSAQNT IESTVLQLNNG DVKLFMRGLT GDLQVATSKD GGVTWEKTIK        730        740        750        760        770        780        790        800 RYPEVKDAYV QMSAIHTMHD GKEYILLSNA AGPGRERKNG LVHLARVEEN GELTWLHNNP IQNGEFAYNS LQELGGGEYG        810        820        830        840        850        860        870        880 LLYEHRENGQ NYYTLSYKKF NWDFVSDKLI SPTEAKVSQA YEMGKGVFGL EFDSEVLVNR APILRLANGR TAVFMTQYDS        890        900        910        920        930        940        950        960 KTLLFAVDKK DIGQEITGIV DGSIESMHNL TVNLAGAGIP GGMNAAESVE HYTEEYTGVL GTSGVEGVPT ISVPEYEGGV        970        980        990       1000       1010       1020       1030       1040 NSELALVSEK EDYRGGVNSA SSVVTEVLEY TGPLSTVGSE DAPTVSVLEY EGGVNIDSPE VTEAPEYKEP IGTSGYELAP       1050       1060       1070       1080       1090       1100       1110       1120 TVDKPAYTGT IEPLEKEENS GAIIEEGNVS YITENNNKPL ENNNVTTSSI ISESSKLKHT LKNATGSVQI HASEEVLKNV       1130       1140       1150       1160       1170       1180       1190       1200 KDVKIQEVKV SSLSSLNYKA YDIQLNDASG KAVQPKGTVI VTFAAEQSVE NVYYVDSKGN LHTLEFLQKD GEVTFETNHF       1210       1220       1230       1240       1250       1260       1270       1280 SIYAMTFQLS LDNVVLDNHR EDKNGEVNSA SPKLLSINGH SQSSQLENKV SNNEQSKLPN TGEDKSISTV LLGFVGVILG       1290 AMIFYRRKDS EG SEQ ID NO: 15         10         20         30         40         50         60         70         80 MDKKKIILTS LASVAVLGAA LAASQPSLVK AEEQPTASQP AGETGTKSEV TSPEIKQAEA DAKAAEAKVT EAQAKVDTTT         90        100        110        120        130        140        150        160 PVADEAAKKL ETEKKEADEA DAAKTKAEEA KKTADDELAA AKEKAAEADA KAKEEAKKEE DAKKEEADSK EALTEALKQL        170        180        190        200        210        220        230        240 PDNELLDKKA KEDLLKAVEA GDLKASDILA ELADDDKKAE ANKETEKKLR NKDQANEANV ATTPAEEAKS KDQLPADIKA        250        260        270        280        290        300        310        320 GIDKAEKADA ARPASEKLQD KADDLGENVD ELKKEADALK AEEDKKAETL KKQEDTLXEA KEALKSAKDN GFGEDITAPL        330        340        350        360        370        380        390        400 EKAVTAIEKE RDAAQNAFDQ AASDTKAVAD ELNKLTDEYN KTLEEVKAAK EKEANEPAKP VEEEPAKPAE KTEAEKAAEA        410        420        430        440        450        460        470        480 KTEADAKVAE LQKKADEAKT KADEATAKAT KEAEDVKAAE KAKEEADKAK TDAEAELAKA KEEAEKAKAK VEELKKEEKD        490        500        510        520        530        540        550        560 NLEALKAALD QLEKDIDADA TITNKEEAKK ALGKEDILAA VEKGDLTAGD VLKELENQNA TAEATKDQDP QADEIGATKQ        570        580        590        600        610        620        630        640 EGKPLSELPA ADKEKLDAAY NKEASKPIVK KLQDIADDLV EKIEKLTKVA DKDKADATEK AKAVEEKNAA LKKQKETLDK        650        660        670        680        690        700        710        720 AKAALETAKK NQADQAIDQG LQDAVTKLEA SFASAKTAAD EAQAKFDEVN EVVKAYKAAI DELTDDYNAT LGHIENLKEV        730        740        750        760        770        780        790        800 PKGEEPKDFS GGVNDDEAPS STPNTNEFTG GANADAPATA PNANEFAGGV NDEEAPTTEN KEPFNGGVND EEAPTVPNKP        810        820        830        840        850        860        870        880 EGEAPKPTGE NAKDAPVVKL PEFGANNPEI KKILDEIAKV KEQIKDGEEN GSEDYYVEGL KERLADLEEA FDTLSKNLPA        890        900        910        920        930        940        950        960 VNKVPEYTGP VTPENGQTQP AVNTPGGQQG GSSQQTPAVQ QGGSGQQAPA VQQGGSNQQV PAVQQTNTPA VAGTSQDNTY        970        980        990       1000 QAPAAKEEDK KELPNTGGQE SAALASVGFL GLLLGALPFV SEQ ID NO: 16         10         20         30         40         50         60         70         80 MKYRDFDRKR RYGIRKFAVG AASVVIGTVV FGANPVLAQE QANAAGANTE TVEPGQGSLE LPKEASSGDL AHLDKDLAGK         90        100        110        120        130        140        150        160 LAAAQDNGVE VDQDHLKKNE SAESETPSST ETPAEGTNKE EESEDQGAIP RDYYSRDLKN ANPVLEKEDV ETNAANGQRV        170        180        190        200        210        220        230        240 DLSNELDKLK QLKNATVHME FKPDASAPRF YNLFSVSSDT KENEYFTISV LDNTALIEGR GANGEQFYDK YTDAPLKVRP        250        260        270        280        290        300        310        320 GQWNSVFTFV EQPTTELPHG RVRLYVNGVL SRTSLKSGNF IKDMPDVNQA QLGATKRGNK TVWASNLQVR NLTVYDRALS        330        340        350        360        370        380        390        400 PDEVQTRSQL FERGELEQKL PEGAKVTEKE DVFEGGRNNQ PNKDGIKSRY IPALLKTDKG TLIAGTDERR LHHSDWGDID        410        420        430        440        450        460        470        480 MVVRRSSDNG KTWGDRIVIS NPRDNEHAKH ADWPSPVNID MALVQDPETK RIFAIYDMFL ESKAVFSLPG QAPKAYEQVG        490        500        510        520        530        540        550        560 DKVYQVLYKQ GESGRYTIRE NGEVFDPQNR KTDYRVVVDP KKPAYDDKGD LYKGNELIGN IYFEYSEKNI FRVSNTNYLW        570        580        590        600        610        620        630        640 MSYSDDDGKT WSAPKDITHG IRKDWMHFLG TGPGTGIALR TGPHKGRLVI PVYTTNNVSY LSGSQSSRVI YSDDHGETWQ        650        660        670        680        690        700        710        720 AGEAVNDNRP VGNQTIHSST NMMPGAQNTE STVVQLNNGD LKLFMRGLTG DLQVATSHDG GATWDKEIKR YPQVKDVYVQ        730        740        750        760        770        780        790        800 MSAIHTMHEG KEYILLSNAG GPGRNNGLVH LARVEENGEL TWLKHNPIQS GKFAYNSLQD LGNGEYGLLY EHADGNQNDY        810        820        830        840        850        860        870        880 TLSYKKFNWD FLTKDWISPK EAKVKYAIEK WPGILAMEFD SEVLVNKAPT LQLANGKTAR FMTQYDTKTL LFTVDSEDMG        890        900        910        920        930        940        950        960 QKVTGLAEGA IESMHNLPVS VAGTKLSNGM NGSEAAVHEV PEYTGPLGTA GEEPAPTVEK PEFTGGVNGE EAAVHEVPEY        970        980        990       1000       1010       1020       1030       1040 TGPLGTSGEE PAPTVEKPEF TGGVNAVEAA AHEVPEYTGP LGTSGKEPAP TVEKPEYTGG VNAVEAAVHE VEPYTGPLAT       1050       1060       1070       1080       1090       1100       1110       1120 VGEEAAPKVD KPEFTGGVNA VEAAVHELPE YTGGVNAADA AVHEIAEYKG ADSLVTLAAE DYTYKAPLAQ QTLPDTGNKE       1130       1140 SSLLASLGLT AFFLGLFAMG KKREK SEQ ID NO: 17         10         20         30         40         50         60         70         80 MEKIWREKSC RYSIRKLTVG TASVLLGAVF LASHTVSADT IKVKQNESTL EKTTAKTDTV TKTTESTEHT QPSEAIDHSK         90        100        110        120        130        140        150        160 QVLANNSSSE SKPTEAKVAS ATTNQASTEA IVKPNENKET EKQELPVTEQ SNYQLNYDRP TAPSYDGWEK QALPVGNGEM        170        180        190        200        210        220        230        240 GAKVFGLIGE ERIQYNEKTL WSGGPRPDST DYNGGNYRER YKILAEIRKA LEDGDRQKAK RLAEQNLVGP NNAQYGRYLA        250        260        270        280        290        300        310        320 FGDIFMVFNN QKKGLDTVTD YHRGLDITEA TTTTSYTQDG TTFKRETFSS YPDDVTVTHL TQKGDKKLDF TVWNSLTEDL        330        340        350        360        370        380        390        400 LANGDYSAEY SNYKSGHVTT DPNGILLKGT VKDNGLQFAS YLGIKTDGKV TVHEDSLTIT GASYATLLLS AKTNFAQNPK        410        420        430        440        450        460        470        480 TNYRKDIDLE KTVKGIVEAA QGKYYETLKR NHIKDYQSLF NRVKLNLGGS NIAQTTKEAL QTYNPTKGQK LEELFFQYGR        490        500        510        520        530        540        550        560 YLLISSSRSR TDALPANLQG VWNAVDNPPW NADYHLNVNL QMNYWPAYMS NLAETAKPMI NYIDDMRYYG RIAAKEYAGI        570        580        590        600        610        620        630        640 ESKDGQENGW LVHTQATPFG WTTPGWNYYW GWSPAANAWM MQNVYDYYKF TKDETYLKEK IYPMLKETAK FWNSFLHYDQ        650        660        670        680        690        700        710        720 ASDRWVSSPS YSPEHGTITI GNTFDQSLVW QLFHDYMEVA NHLNVDKDLV TEVKAKFDKL KPLHINKEGT IKEWYEEDSP        730        740        750        760        770        780        790        800 QFTNEGIENN HRHVSHLVGL FPGTLFSKDQ AEYLEAARAT LNHRGDGGTG WSKANKINLW ARLLDGNRAH RLLAEQLKYS        810        820        830        840        850        860        870        880 TLENLWDTHA PFQIDGNFGA TSGIAEMLLQ SHTGYIAPLP ALPDAWKDGQ VSGLVARGNF EVSMQWKDKN LQSLSFLSNV        890        900        910        920        930        940        950        960 GGDLVVDYPN IEASQVKVNG KPVKATVLKD GRIQLATQKG DVITFEHFSG RVTSLTAVRQ NGVTAELTFN QVEGATHYVI        970        980        990       1000       1010       1020       1030       1040 QRQVKDESGQ TSATREFVTN QTHFIDRSLD PQLAYTYTVK AMLGNVSTQV SEKANVETYN QLMDDRDSRI QYGSAFGNWA       1050       1060       1070       1080       1090       1100       1110       1120 DSELFGGTEK FADLSLGNYT DKDATATIPF NGVGIEYIGL KSSQLGIAEV KIDGKSVGEL DFYTAGATEK GSLIGRFTGL       1130       1140       1150       1160       1170       1180       1190       1200      SDGAHVMTIT VKQEHKHRGS ERSKISLDYF KVLPGQGTTI EKMDDRDSRI QYGSQFKDWS DTELYKSTEK YADINNSDPS       1210       1220       1230       1240       1250       1260       1270       1280 TASEAQATIP FTGTGIRIYG LKTSALGKAL VTLDGKEMPS LDFYTAGATQ KATLIGEFTN LTDGNHILTL KVDPNSPAGR       1290       1300       1310       1320       1330       1340       1350       1360 KKISLDSFDV IKSPAVSLDS PSIAPLKKGD KNISLTLPAG DWEAIAVTFP GIKDPLVLRR IDDNHLVTTG DQTVLSIQDN       1370       1380       1390       1400       1410       1420       1430       1440 QVQIPIPDET NRKIGNAIEA YSIQGNTTSS PVVAVFTKKD EKKVENQQPT TSKGDDPAPI VEIPEYTKPI GTAGLEQPPT       1450       1460       1470       1480       1490       1500       1510       1520 VSIPEYTQPI GTAGLEQAPT VSIPEYTKPV GTAGIEQAPT VSIPEYTKPI GTAGLEQAPT VSIPEYTQPI GTAGLEQPPT       1530       1540       1550       1560       1570       1580       1590       1600 VSIPEYTKSI GTAGLEQPPV VNVPEYTQPI GTAGIEQPPT VSIPEYTKPI GTAGQEQALT VSIPEYTKPI GTAGQEQAPT       1610       1620       1630       1640       1650       1660       1670       1680 VSVPEYKLRV LKDERTGVEI IGGATDLEGI SHISSRRVLA QELFGKTYDA YDLHLKNSTD QSLQPKGSVL VRLPISSAVE       1690       1700       1710       1720       1730       1740       1750       1760 NVYYLTPSKE LQALDFTIRE GMAEFTTSHF STYAVVYQAN GASTTAEQKP SETDIKPLAN SSEQVSSSPD LVQSTNDSPK       1770       1780       1790 EQLPATGETS NPLLFLSGLS LVLTATFLLK SKKDESN SEQ ID NO: 18         10         20         30         40         50         60         70         80 MKQYFLEKGR IFSIRKLTVG VASVAVGLTF FASGNVAASE LVTEPKLEVD GQSKEVADVK HEKEEAVKEE AVKEEVTEKT         90        100        110        120        130        140        150        160 ELTAEKATEE AKTAEVAGDV LPEEIPDRAY PDTPVKKVDT AAIVSEQESP QVETKSILKP TAVAPTEGEK ENRAVINGGQ        170        180        190        200        210        220        230        240 DLKRINYEGQ PATSAAMVYT IFSSPLAGGG SRRYLNSGSG IFVAPNIMLT VAHNFLVKDA DTNAGSIRGG DTTKFYYNVG        250        260        270        280        290        300        310        320 SNTAKNNSLP TSGNTVLFKE KDIHFWNKEK FGEGIKNDLA LVVAPVPLSI ASPNKAATFT PLAEHREYKA GEPVSTIGYP        330        340        350        360        370        380        390        400 TDSTSPELKE PIVPGQLYKA DGVVKGTEKL DDKGAVGITY RLTSVSGLSG GGIINGDGKV IGIHQHGTVD NMNIAEKDRF        410        420        430        440        450        460        470        480 GGGLVLSPEQ LAWVKEIIDK YGVKGWYQGD NGNRYYFTPE GEMIRNKTAV IGKNKYSFDQ NGIATLLEGV DYGRVVVEHL        490        500        510        520        530        540        550        560 DQKDNPVKEN DTFVEKTEVG TQFDYNYKTE IEKTDFYKKN KEKYEIVSID GKAVNKQLKD TWGEDYSVVS KAPAGTRVIK        570        580        590        600        610        620        630        640 VVYKVNKGSF DLRYRLKGTD QELAPATVDN NDGKEYEVSF VHRFQAKEIT GYRAVNASQE ATIQHKGVNQ VIFEYEKIED        650        660        670        680        690        700        710        720 PKPATPATPV VDPKDEETEI GNYGPLPSKA QLDYHKEELA AFIHYGMNTY TNSEWGNGRE NPQNFNPTNL DTDQWIKTLK        730        740        750        760        770        780        790        800 DAGFKRTIMV VKHHDGFVIY PSQYTKHTVA ASPWKDGKGD LLEEISKSAT KYDMNMGVYL SPWDANNPKY HVSTEKEYNE        810        820        830        840        850        860        870        880 YYLNQLEKIL GNPKYGNKGK FIEVWMDGAR GSGAQKVTYT FDEWFKYIKK AEGDIAIFSA QPTSVRWIGN ERGIAGDPVW        890        900        910        920        930        940        950        960 HKVKKAKITD DVKNEYLNHG DPEGDMYSVG EADVSIRSGW FYHDNQQPKS IKDLMDIYFK SVGRGTPLLL NIPPNKEGKF        970        980        990       1000       1010       1020       1030       1040 ADADVARLKE FRATLDQMYA TDFAKGATVT ASSTRKNHLY QASNLTDGKD DTSWALSNDA KTGEFTVDLG QKRRFDVVEL       1050       1060       1070       1080       1090       1100       1110       1120 KEDIAKGQRI SGFKVEVELN GRWVPYGEGS TVGYRRLVQG QPVEAQKIRV TITNSQATPI LTNFSVYKTP SSIEKTDGYP       1130       1140       1150       1160       1170       1180       1190       1200      LGLDYHSNTT ADKANTTWYD ESEGIRGTSM WTNKKDASVT YRFNGTKAYV VSTVDPNHGE MSVYVDGQKV ADVQTNNAAR       1210       1220       1230       1240       1250       1260       1270       1280 KRSQMVYETD DLAPGEHTIK LVNKTGKAIA TEGIYTLNNA GKGMFELKET TYEVQKGQPV TVTIKRVGGS KGAATVHVVT       1290       1300       1310       1320       1330       1340       1350       1360 EPGTGVHGKV YKDTTADLTF QDGETEKTLT IPTIDFTEQA DSIFDFKVKM TSASDNALLG FASEATVRVM KADLLQKDQV       1370       1380       1390       1400       1410       1420       1430       1440 SHDDQASQLD YSPGWHHETN SAGKYQNTES WASFGRLNEE QKKNASVTAY FYGTGLEIKG FVDPGHGIYK VTLDGKELEY       1450       1460       1470       1480       1490       1500       1510       1520 QDGQGNATDV NGKKYFSGTA TTRQGDQTLV RLTGLEEGWH AVTLQLDPRK NDTSRNIGIQ VDKFITRGED SALYTKEELV       1530       1540       1550       1560       1570       1580       1590       1600 QAMKNWKDEL AKFDQTSLKN TEPARQAFKS NLDKLSEQLS ASPANAQEIL KIATALQAIL DKEENYGKED TPTSEQPEEP       1610       1620       1630       1640       1650       1660       1670       1680 NYDKAMASLS EAIQNKSKEL SSDKEAKKKL VELSEQALTA IQEAKTQDAV DKALQAALTS INQLQATPKE EVKPSQPEEP       1690       1700       1710       1720       1730       1740       1750       1760 NYDKAMASLA EAIQNKSKEL GSDKESKKKL VELSEQALTA IQEAKTQDAV DKALQAALTS INQLQATPKE EAKPSQPEEP       1770       1780       1790       1800       1810       1820       1830       1840 NYDKAMASLA EAIQNKSKEL GSDKEAKKKL VELSEQALTA IQEAKTQDAV DKALQAALTS INQLQATPKE EVKHSIVPTD       1850       1860       1870       1880       1890       1900       1910       1920 GDKELVQPQP SLEVVEKVIN FKKVKQEDSS LPKGETRVTQ VGRAGKERIL TEVAPDGSRT IKLREVVEVA QDEIVLVGTK       1930       1940       1950       1960       1970       1980       1990       2000   KEESGKIASS VHEVPEFTGG VIDSEATIHN LPEFTGGVTD SEAAIHNLPE FTGGVTDSEA AIHNLPEFTG GMTDSEAAIH       2010       2020       2030       2040       2050       2060       2070       2080 NLPEFTGGMT DSEGVAHGVS NVEEGVPSGE ATSHQESGFT SDVTDSETTM NEIVYKNDEK SYVVPPMLED KTYQAPANRQ       2090       2100       2110 EVLPKTGSED GSAFASVGII GMFLGMIGIV KRKKD SEQ ID NO: 19         10         20         30         40         50         60         70         80 MSGLKKYEGV IPAFYACYDD AGEVSPERTR ALVQYFIDKG VQGLYVNGSS GECIYQSVED RKLILEEVMA VAKGKLTIIA         90        100        110        120        130        140        150        160 HVACNNTKDS IELARHAESL GVDAIATIPP IYFRLPEYSV AKYWNDISAA APNTDYVIYN IPQLAGVALT PSLYTEMLKN        170        180        190        200        210        220        230        240 PRVIGVKNSS MPVQDIQTFV SLGGDDHIVF NGPDEQFLGG RLMGAKAGIG GTYGAMPELF LKLNQLIADK DLETARELQY        250        260        270        280        290        300 AINAIIGKLT AAHGNMYCVI KEVLKINEGL NIGSVRSPLT PVTEEDRPVV EAAAQILIRES KERFL SEQ ID NO: 20         10         20         30         40         50         60         70         80 MANNTLLAKT RRYVCLVVFC CLMAMMHLSG QEVTMWGDSH GVAPNQVRRT LVKVALSESL PPGAKQIRIG FSLPKETEEK         90        100        110        120        130        140        150        160 VTALYLLVSD SLAVRDLPDY KGRVSYDSFP ISKEDRTTAL SADSVAGRCF FYLAADIGPV ASFSRSDTLT ARVEELAVDG        170        180        190        200        210        220        230        240 RPLPLKELSP ASRRLYREYE ALFVPGDGGS RNYRIPSILK TANGTLIAMA DRRKYNQTDL PEDIDIVMRR STDGGKSWSD        250        260        270        280        290        300        310        320 PRIIVQGEGR NHGFGDVALV QTQAGKLLMI FVGGVGLWQS TPDRPQRTYI SESRDEGLTW SPPRDITHIF FGKDCADPGR        330        340        350        360        370        380        390        400 SRWLASFCAS GQGLVLPSGR VMFVAAIRES GQEYVLNNYV LYSDDEGGTW QLSDCAYHRG DEAKLSLMPD GRVLMSVRNQ        410        420        430        440        450        460        470        480 GRQESRQRFF ALSSDDGLTW ERAKQFEGIH DPGCNGAMLQ VKRNGRNQML HSLPLGPDGR RDGAVYLFDH VSGRWSAPVV        490        500        510        520 VNSGSSAYSD MTLLADGTIG YFVEEDDEIS LVFIRFVLDD LFDARQ SEQ ID NO: 21         10         20         30         40         50         60         70         80 MTKKSSISRR SFLKSTALAG AAGMVGTGGA ATLLTSCGGG ASSNENANAA NKPLKEPGTY YVPELPDMAA DGKELKAGII         90        100        110        120        130        140        150        160 GCGGRGSGAA MNFLAAANGV SIVALGDTFQ DRVDSLAQKL KDEKNIDIPA DKRFVGLDAY KQVIDSDVDV VIVATPPNFR        170        180        190        200        210        220        230        240 PIHFQYAVEK SKHCFLEKPI CVDAVGYRTI MATAKQAQAK NLCVITGTQR HHQRSYIASY QQIMNGIAGE ITGGTVYWNQ        250        260        270        280        290        300        310        320 SMLWYRERQA GWSDCEWMIR DWVNWKWLSG DHIVEQHVHN IDVFTWFSGL KPVKAVGFGS RQRRITGDQY DNFSIDFTME        330        340        350        360        370        380        390        400 NGIHLHSMCR QIDGCANNVS EFIQGTKGSW NSTDMGIKDL AGNVIWKYDV EAEKASFKQN DPYTLEHVNW INTIRAGKSI        410        420        430        440        450        460 DQASETAVSN MAAIMGRESA YTGEETTWEA MTAAALDYTP ADLNLGKMDM KPFVVPVPGK PLEKK SEQ ID NO: 22         10         20         30         40         50         60         70         80 MKKFFWIIGL FISMLTTRAA DSVYVQNPQI PILIDRTDNV LFRIRIPDAT KGDVLNRLTI RFGNEDKLSE VKAVRLFYAG         90        100        110        120        130        140        150        160 TEAGTKGRSR FAPVTYVSSH NIRNTRSANP SYSVRQDEVT TVANTLTLKT RQPMVKGINY FWVSVEMDRN TSLLSKLTPT        170        180        190        200        210        220        230        240 VTEAVINDKP AVIAGEQAAV RRMGIGVRHA GDDGSASFRI PGLVTTNEGT LLGVYDVRYN NSVDLQEHID VGLSRSTDKG        250        260        270        280        290        300        310        320 QTWEPMRIAM SFGETDGLPS GQNGVGDPSI LVDERTNTVW VVAAWTHGMG NARAWTNSMP GMTPDETAQL MMVKSTDDGR        330        340        350        360        370        380        390        400 TWSEPINITS QVKDPSWCFL LQGPGRGITM RDGTLVFPIQ FIDSLRVPHA GIMYSKDRGE TWHISQPART NTTEAQVAEV        410        420        430        440        450        460        470        480 EPGVLMLNMR DNRGGSRAVS ITRDLGKSWT EHSSNRSALP ESICMASLIS VKAKDNIIGK DLLFFSNPNT TEGRHHITIK        490        500        510        520        530 ASLDGGVTWL PAHQVLLDEE DGWGYSCLSM IDRETVGIFY ESSVAHMTFQ AVKIKDLIR SEQ ID NO: 23         10         20         30         40         50         60         70         80 MTWLLCGRGK WNKVKRMMNS VFKCLMSAVC AVALPAFGQE EKTGFPTDRA VTVFSAGEGN PYASIRIPAL LSIGKGQLLA         90        100        110        120        130        140        150        160 FAEGRYKNTD QGENDIIMSV SKNGGKTWSR PRAIAKAHGA TFNNPCPVYD AKTRTVTVVF QRYPAGVKER QPNIPDGWDD        170        180        190        200        210        220        230        240 EKCIRNFMIQ SRNGGSSWTK PQEITKTTKR PSGVDIMASG PNAGTQLKSG AHKGRLVIPM NEGPFGKWVI SCIYSDDGGK        250        260        270        280        290        300        310        320 SWKLGQPTAN MKGMVNETSI AETDNGGVVM VARHWGAGNC RRIAWSQDGG ETWGQVEDAP ELFCDSTQNS LMTYSLSDQP        330        340        350        360        370        380        390        400 AYGGKSRILF SGPSAGRRIK GQVAMSYDNG KTWPVKKLLG EGGFAYSSLA MVEPGIVGVL YEENQEHIKK LKFVPITMEW        410 LTDGEDTGLA PGKKAPVLK SEQ ID NO: 24         10         20         30         40         50         60         70         80 MGLGLLCALG LSIPSVLGKE SFEQARRGKF TTLSTKYGLM SCRNGVAEIG GGGKSGEASL RMFGGQDAEL KLDLKDTPSR         90        100        110        120        130        140        150        160 EVRLSAWAER WTGQAPFEFS IVAIGPNGEK KIYDGKDIRT GGFHTRIEAS VPAGTRSLVF RLTSPENKGM KLDDLFLVPC        170        180        190        200        210        220        230        240 IPMKVNPQVE MASSAYPVMV RIPCSPVLSL NVRTDGCLNP QFLTAVNLDF TGTTKLSDIE SVAVIRGEEA PIIHHGEEPF        250        260        270        280        290        300        310        320 PKDSSQVLGT VKLAGSARPQ ISVKGKMELE PGDNYLWACV TMKEGASLDG RVVVRPASVV AGNKPVRVAN AAPVAQRIGV        330        340        350        360        370        380        390        400 AVVRHGDFKS KFYRIPGLAR SRKGTLLAVY DIRYNHSGDL PANIDVGVSR STDGGRTWSD VKIAIDDSKI SPSLGATRGV        410        420        430        440        450        460        470        480 GDPAILVDEK TGRIWVAAIW SHRHSIWGSK SGDNSPEACG QLVLAYSDDD GLTWSSPINI TEQTKNKDWR ILFNGPGNGI        490        500        510        520        530        540        550        560 CMKDGTLVFA AQYWDGKGVP WSTIVYSKDR GKTWHCGTGV NQQTTEAQVI ELEDGSVMIN ARCNWGGSRI VGVTKDLGQT        570        580        590        600        610        620        630        640 WEKHPTNRTA QLKEPVCQGS LLAVDGVPGA GRVVLFSNPN TTSGRSHMTL KASTNDAGSW PEDKWLLYDA RKGWGYSCLA        650        660        670 PVDKNHVGVL YESQGALNFL KIPYKDVLNA KNAR SEQ ID NO: 25         10         20         30         40         50         60         70         80 MKRNHYLFTL ILLLGCSIFV KASDTVFVHQ TQIPILIERQ DNVLFYFRLD AKESRMMDEI VLDFGKSVNL SDVQAVKLYY         90        100        110        120        130        140        150        160 GGTEALQDKG KKRFAPVDYI SSHRPGNTLA AIPSYSIKCA EALQPSAKVV LKSHYKLFPG INFFWISLQM KPETSLFTKI        170        180        190        200        210        220        230        240 SSELQSVKID GKEAICEERS PKDIIHRMAV GVRHAGDDGS ASFRIPGLVT SNKGTLLGVY DVRYNSSVDL QEYVDVGLSR        250        260        270        280        290        300        310        320 STDGGKTWEK MRLPLSFGEY DGLPAAQNGV GDPSILVDTQ TNTIWVVAAW THGMGNQRAW WSSHPGMDLY QTAQLVMAKS        330        340        350        360        370        380        390        400 TDDGKTWSKP INITEQVKDP SWYFLLQGPG RGITMSDGTL VFPTQFIDST RVPNAGIMYS KDRGKTWKMH NMARTNTTEA        410        420        430        440        450        460        470        480 QVVETEPGVL MLNMRDNRGG SRAVIATKDL GKTWTEHPSS RKALQEPVCM ASLIHVEAED NVLDKDILLF SNPNTTRGRN        490        500        510        520        530        540 HITIKASLDD GLTWPLEHQL MLDEGEGWGY SCLTMIDRET IGILYESSAA HMTFQAVKLK DLIR

[0078] While certain embodiments have been shown and described herein, it will be obvious to those skilled in the art that such embodiments are provided by way of example only. Numerous variations, changes, and substitutions will now occur to those skilled in the art without departing from the disclosure. It should be understood that various alternatives to the embodiments of the disclosure described herein may be employed in practicing the disclosure. It is intended that the following claims define the scope of the disclosure and that methods and structures within the scope of these claims and their equivalents be covered thereby.