AU2007201172A1 - Integrin antagonists - Google Patents

Description

AUSTRALIA

Patents Act COMPLETE SPECIFICATION

(ORIGINAL)

Class Int. Class Application Number: Lodged: Complete Specification Lodged: Accepted: Published: Priority Related Art: Name of Applicant: Immunex Corporation Actual Inventor(s): Roy Alvin Black, William C Fanslow, Douglas Pat Cerretti, Kurt Matthew Poindexter Address for Service and Correspondence: PHILLIPS ORMONDE FITZPATRICK Patent and Trade Mark Attorneys 367 Collins Street Melbourne 3000 AUSTRALIA Invention Title: INTEGRIN ANTAGONISTS Our Ref: 796779 POF Code: 461115/44735 The following statement is a full description of this invention, including the best method of performing it known to applicant(s): INTEGRIN ANTAGONISTS The present application is a divisional application from Australian patent application number 2001247219, the entire disclosure of which is incorporated herein by reference.

FIELD OF THE INVENTION This invention relates to methods and compositions that are useful for antagonizing the interaction between integrins and their ligands. In particular, the invention relates to the use of ADAM disintegrin domains for antagonizing the interaction between integrins and their ligands.

BACKGROUND OF THE INVENTION A. Integrins and Disintegrins Integrins are a family of cell surface proteins that mediate adhesion between cells (cell-cell adhesion) and between cells and extracellular matrix proteins (cell-ECM adhesion). Integrins are heterodimeric structures composed of noncovalently bound a and B subunits. In humans, at least fifteen different a subunits and eight different B subunits combine to form integrins with diverse biological activities and ligand specificities. Integrins play important roles in biological processes including embryonic development, platelet aggregation, immune reactions, tissue repair and remodeling, bone resorption, and tumor invasion and metastasis. Integrins are, therefore, important targets for therapeutic intervention in human disease.

The disintegrins are a family of low molecular weight, soluble, cysteine-rich peptides which have been isolated from snake venom (reviewed in Niewiarowski et al., Seminars in Hematology 31 289, 1994). The snake venom disintegrins typically contain an RGD (Arg- Gly-Asp, SEQ ID NO 19) motif. The RGD motif is recognized by many integrins, and is present in several integrin ligands including fibronectin, vitronectin, and von Willebrand factor.

Disintegrins disrupt normal adhesion processes by inhibiting the binding of cell surface integrins to their ligands.

Disintegrin-like domains have been identified in cellular proteins from both invertebrates and vertebrates (see, e. Westcamp and Blobel, Proc. Natl. Acad. Sci. USA 91 2748, 1994; Wolfsberg et al., Dev. Biol. 169 378, 1995 Alfandari et al., Dev. Biol. 182 314, 1997), including the ADAM family of transmembrane proteins.

B. ADAMs The ADAMs, which have also been called MDCs, are a family of type I transmembrane cysteine-rich glycoproteins (Weskamp et al., Proc. Natl. Acad. Sci. USA, 91 2748, 1994 Wolfsberg et al., Dev. Biol. 169 378, 1995). The multidomain structure of the ADAMs typically includes an amino- terminal metalloprotease domain, a disintegrin domain, a cysteinerich region (the region between the 899gM74751%74751 Div page I WoC disintegrin domain and the transmembrane domain), a transmembrane region, and a cytoplasmic domain. At least 30 ADAM family members have been identified, in a variety of animal species. The structure of the ADAMs suggests that they may be involved in a variety of biological processes, including cell adhesion, cell fusion, signal transduction, and proteolysis. Members of the ADAM family have, in fact, been shown to play roles in sperm-egg binding and fusion, myotube formation, neurogenesis, and proteolysis.

also called MDC-15 or metargidin, is the only ADAM identified to date which contains an RGD motif within its disintegrin domain. Zhang et al. Biol. Chem. 273(13):7345, 1998) have reported that the isolated disintegrin domain of ADAM-15, expressed in E. coli as a glutathione S-transferase fusion protein, specifically interacts with avP 3 integrin and that the interaction is mediated by the RGD tripeptide sequence. The recombinant fusion protein did not interact with other integrins tested, including aln,13 and a 1 Nath et al. Cell Science 112:579, 1999) have reported that the entire ADAM-15 extracellular domain, expressed as an Fc fusion protein in COS cells, interacts with avp3 and as 5 P integrins on hematopoietic cells and that the interaction is mediated by the RGD tripeptide sequence. Zhang et al. and Nath et al. commented that the RGDdependent interaction between ADAM-15 and av i integrin suggests a role in processes such as malignancy and angiogenesis.

C. Angiogenesis Angiogenesis, the generation of new blood vessels, is a spatially and temporally regulated process in which endothelial and smooth muscle cells proliferate, migrate, and assemble into tubes, in response to endogenous positive and negative regulatory molecules. Angiogenesis plays important roles in both normal and pathological physiology.

Under normal physiological conditions, angiogenesis is involved in fetal and embryonic development, wound healing, organ regeneration, and female reproductive remodeling processes including formation of the endometrium, corpus luteum, and placenta. Angiogenesis is stringently regulated under normal conditions, especially in adult animals, and perturbation of the regulatory controls can lead to pathological angiogenesis.

Pathological angiogenesis has been implicated in the manifestation and/or progression of inflammatory diseases, certain eye disorders, and cancer. In particular, several lines of evidence support the concept that angiogenesis is essential for the growth and persistence of solid tumors and their metastases (see, Folkman, N. Engl. J. Med. 285:1182, 1971; Folkman et al., Nature 339:58, 1989; Kim et al., Nature 362:841, 1993; Hori et al., Cancer Res., 51:6180, 1991; Zetter, Annu. Rev.

Med. 49:407, 1998). The formation of new blood vessels provides a growing tumor with oxygen, nutrients, waste removal, and a conduit by which invasive cells can enter the circulatory system and establish distant metastases. Various classes of angiogenesis inhibitors are presently being developed and tested for the prevention treatment of premalignant conditions), intervention treatment of small tumors), and regression treatment of large tumors) of cancers (see, Bergers et al., O Science 284:808, 1999) and other forms of pathological angiogenesis. Because many steps in the C' angiogenic process, including endothelial cell migration, proliferation, and morphogenesis require vascular cell adhesion, certain integrin antagonists have been tested as anti-angiogenic agents.

Several integrins are expressed on the surface of cultured endothelial and smooth muscle cells, including av3 integrin. The ,y3 integrin is an endothelial cell receptor for von Willebrand factor, fibrin, fibrinogen, and fibronectin, and a marker of angiogenic vascular tissue. Brooks et al.

have reported that monoclonal antibodies to avp3 integrin, as well as cyclic peptide inhibitors, disrupt angiogenesis and that a03 3 antibodies promote tumor regression (Science 264:569, 1994; Cell 79:1157, 1994). These results suggest that av 3 integrin is a useful therapeutic target for diseases characterized S 10 by pathological angiogenesis.

There is great need for additional compositions and methods of antagonizing the interaction 0 between integrins and their ligands. In particular, there is great need for additional compositions and methods of inhibiting angiogenesis for the prevention, abrogation, and mitigation of disease processes that are dependent upon pathological angiogenesis.

SUMMARY OF THE INVENTION The present invention is based upon the discovery that ADAM disintegrin domains are useful for inhibiting the biological activity of integrins and for inhibiting endothelial cell migration and angiogenesis, including the unexpected discovery that these inhibitory activities reside in ADAM disintegrin domains that lack an RGD motif.

The invention is directed to methods of antagonizing the binding of an integrin to its ligands, and thereby inhibiting the biological activity of the integrin, comprising contacting the integrin with an effective amount of an ADAM disintegrin domain polypeptide. The invention is further directed to methods of inhibiting endothelial cell migration and methods of inhibiting angiogenesis comprising administering an effective amount of an ADAM disintegrin domain polypeptide. In some embodiments the ADAM disintegrin domain polypeptide is in the form of a multimer, preferably a leucine zipper multimer or Fc polypeptide. In some embodiments the ADAM disintegrin domain is from a human ADAM, and preferably from ADAM-8, ADAM-9, ADAM-10, ADAM-15, ADAM-17, ADAM-21, ADAM-22, ADAM-23, or ADAM-29. The ADAM disintegrin domain is preferably produced in a recombinant cell, and is preferably present in a composition comprising a pharmaceutically acceptable carrier.

In some preferred embodiments the ADAM disintegrin domain polypeptide comprises an amino acid sequence selected from the group consisting of: amino acids 23-264 of SEQ ID NO:2, amino acids 23-303 of SEQ ID NO:4, amino acids 23-235 of SEQ ID NO:6. amino acids 23-292 of SEQ ID NO:8, amino acids 23-216 of SEQ ID NO:10, amino acids 23-305 of SEQ ID NO:12, amino acids 23-293 of SEQ ID NO:14, amino acids 23-312 of SEQ ID NO: 16, amino acids 23-310 of SEQ ID NO: 18, and amino acids 23-298 of SEQ ID NO:22. In some more preferred embodiments the ADAM disintegrin domain polypeptide comprises an amino acid sequence selected from the group consisting of: amino acids 34-91 of SEQ ID NO:2, amino acids 34-92 of SEQ ID NO:4, amino acids 34-99 of SEQ ID NO:6, amino acids 34-92 of SEQ ID NO:8, amino acids 34-93 of SEQ ID NO: amino acids 34-91 of SEQ ID NO: 12, amino acids 34-91 of SEQ ID NO: 14, amino acids 34-92 of SEQ ID NO: 16, amino acids 34-91 of SEQ ID NO: 18, and amino acids 34-91 of SEQ ID NO:22. In some most preferred embodiments the ADAM disintegrin domain polypeptide comprises an amino acid sequence selected from the group consisting of: amino acids 78-91 of SEQ ID NO:2, amino acids 79-92 of SEQ ID NO:4, amino acids 87-99 of SEQ ID NO:6, amino acids 79-92 of SEQ ID NO:8, amino acids 79-93 of SEQ ID NO:10. amino acids 78-91 of SEQ ID NO: 12, amino acids 78-91 of SEQ ID NO:14, amino acids 79-92 of SEQ ID NO:16. amino acids 78-91 of SEQ ID NO:18, and amino acids 78-91 of SEQ ID NO:22.

In some embodiments a therapeutically effective amount of the ADAM disintegrin domain is administered to a mammal in need of such treatment. In preferred embodiments the mammal is afflicted with a condition mediated by angiogenesis, an ocular disorder, malignant or metastatic condition, inflammatory disease, osteoporosis and other conditions mediated by accelerated bone resorption, restenosis, inappropriate platelet activation, recruitment, or aggregation, thrombosis, or a condition requiring tissue repair or wound healing. The ADAM disintegrin domain is, in some embodiments, administered in combination with radiation therapy and/or in combination with one or more additional therapeutic agents.

The invention also encompasses methods for identifying compounds that modulate integrin biological activity, that modulate the interaction between an integrin and an ADAM disintegrin domain, that inhibit endothelial cell migration, or that inhibit angiogenesis, comprising combining a test compound with an integrin or with endothelial cells and with an ADAM disintegrin domain polypeptide that binds to the integrin or endothelial cells and determining whether the test compound alters the binding of the ADAM disintegrin domain polypeptide to the integrin or endothelial cells.

These and other aspects of the present invention will become evident upon reference to the following detailed description, examples, and claims.

DETAILED DESCRIPTION OF THE INVENTION A. Abbreviations and Terminology Used in the Specification "4-1BB" and "4-1BB ligand" (4-1BB-L) are polypeptides described, inter alia, in U.S. Patent No. 5,674,704, including soluble forms thereof.

"ADAMs" are a family of transmembrane glycoproteins having disintegrin and metalloproteinase domains, also called MDC, metalloprotease/disintegrin/cysteine-rich proteins.

"Dis" is a disintegrin domain; "ADAMdis" is an ADAM disintegrin domain.

"CD40 ligand" (CD40L) is a polypeptide described, inter alia, in U.S. Patent No. 5,716,805, including soluble forms thereof.

"CD148" is a protein tyrosine phosphatase, also called DEP-1, ECRTP, and PTPRJ. CDI48 binding proteins are described in Daniel et al., PCT Publication No. WO 00/15258, 23 March 2000.

"DMEM" is Dulbecco's Modified Eagle Medium.

"FACS" is fluorescence activated cell sorting.

"Flt3L" is Flt3 ligand, a polypeptide described, inter alia, in U.S. Patent No. 5,554,512, including soluble forms thereof.

"HRMEC" are human renal microvascular endothelial cells.

"HMVEC-d" are human dermal microvascular endothelial cells.

"mAb" is a monoclonal antibody.

"MDC" is a family of cysteine-rich proteins having metalloprotease and disintegrin domains, also called ADAM.

"Nectin-3" is a cell adhesion molecule in the nectin family (which is described, inter alia, in Satoh-Horikawa et al., J. Biol. Chem. 275(14):10291, 2000). The GenBank accession numbers of human nectin-3 nucleic acid and polypeptide sequences are AF282874 and AAF97597 respectively (Reymond et al., 2000).

"PMA" is phorbol- 2-myristate-13-acetate.

"Tek." which has also been called Tie2 and ork, is an receptor tyrosine kinase (RTK) that is predominantly expressed in vascular endothelium: The molecular cloning of human Tek (ork) has been described by Ziegler, U.S. Patent No. 5,447,860. 'Tek antagonists" are described, inter alia, in Cerretti et al., PCT Publication No. WO 00/75323, 14 December 2000.

"TNF" is tumor necrosis factor. "TNFR" is a tumor necrosis factor receptor, including soluble forms thereof. "TNFR/Fc" is a tumor necrosis factor receptor-Fc fusion polypeptide.

"TRAIL" is TNF-related apoptosis-inducing ligand, a type II transmembrane polypeptide in the TNF family described, inter alia, in U.S. Patent No. 5,763,223, including soluble forms thereof.

"TWEAK" is TNF-weak effector of apoptosis, a type II transmembrane polypeptide in the TNF family described, inter alia, in Chicheportiche et al., J. Biol. Chem., 272(51):32401, 1997, including soluble forms thereof. "TWEAK-R" is the "TWEAK receptor," which is described, inter alia, in U.S. Serial Numbers 60/172,878 and 60/203,347 and Feng et al., Am. J. Pathol. 156(4):1253.

2000, including soluble forms thereof. TWEAK-R/Fc is a TWEAK receptor-Fc fusion polypeptide.

"VEGF" is vascular endothelial growth factor, also known as VPF or vascular permeability factor.

B. ADAM Polvpeptides and ADAM Disintegrin Domain Polvpeptides At least thirty ADAMs have been described. Table 1 provides reference information for selected human ADAMs.

ADAM disintegrin domains show sequence homology to the snake venom disintegrins, and are characterized by a framework of cysteines. For example, a typical disintegrin sequence comprises a framework such as: CDCX3.CX36CC2-4X7C4.6cx24CXCXS7CX-SC(SEQ ID The sequences of several ADAM disintegrin domains are shown in Table 2 and in the Sequence Listing.

The present invention encompasses the use of various forms of ADAM disintegrin domains that retain at least one activity selected from the group consisting of integrin binding activity, inhibition of endothelial cell migration, and inhibition of angiogenesis. The term "ADAM disintegrin domain polypeptide" is intended to encompass polypeptides containing all or part of a native ADAM disintegrin domain, with or without other ADAM domains (such as the cysteine-rich region), as well as related forms including, but not limited to: fragments, variants, derivatives, fusion polypeptides, and multimeric forms (multimers). The ability of these related forms to inhibit integrin binding. endothelial cell migration, and/or inhibition of angiogenesis may be determined in vitro or in vivo by using methods such as those exemplified below or by using other assays known in the art.

Table 1 Selected Members of the ADAM Famijly ADAM Other Names GS akAceso Published Description Number_(Human) ADAM-8 MS2, CD 156 D26579 Genomics 41(l):56, 1997 ADAM-9 MDC9, meltrin gamma U41766 J. Cell. Biol.

132(4):717, 1996 kuzbanian, AF091 J. Biol. Chem.

ADAM- 10reprolysin AF06I272(39):24588, 1997 U460027](9):4593, 1996 ADAM-17 TAE~rii,MD 2875 W~O96/4162 SVPHI-26 AF029899 WO 99/23228 ADAM-21 SVPHI1-8 AF029900 I WO 99/36549 ADAM-22 SVPH3-13, MDC2 AB009671 I WO 99/41388 ADAM-23 SVPH3-17, MDC3 ABOO 9672 tWO 99/41388 Biochem. Biophys.

ADAM-29 SVPHI AFI 71929 Res. Cornmun.

263:810, 1999 0 The term "variant" includes polypeptides that are substantially homologous to native ADAM disintegrin domains, but which have an amino acid sequence different from that of a native ADAM disintegrin domain because of one or more deletions, insertions or substitutions. Particular Sembodiments include, but are not limited to, ADAM disintegrin domain polypeptides that comprise from one to ten deletions, insertions or substitutions of amino acid residues, when compared to a native ADAM disintegrin domain sequence. Included as variants of ADAM disintegrin domain polypeptides are those variants that are naturally occurring, such as allelic forms and alternatively 1. spliced forms, as well as variants that have been constructed by modifying the amino acid sequence of Sa ADAM disintegrin domain polypeptide or the nucleotide sequence of a nucleic acid encoding a 0 10 ADAM disintegrin domain polypeptide.

Generally, substitutions for one or more amino acids present in the native polypeptide should 0 be made conservatively. Examples of conservative substitutions include substitution of amino acids outside of the active domain(s), and substitution of amino acids that do not alter the secondary and/or tertiary structure of the ADAM disintegrin domain. Additional examples include substituting one aliphatic residue for another, such as Ile, Val, Leu, or Ala for one another, or substitutions of one polar residue for another, such as between Lys and Arg; Glu and Asp; or Gin and Asn, or substitutions of one aromatic residue for another, such as Phe, Trp, or Tyr for one another. Other such conservative substitutions, for example, substitutions of entire regions having similar hydrophobicity characteristics, are known in the art.

In some preferred embodiments the ADAM disintegrin domain variant is at least about identical in amino acid sequence to the amino acid sequence of a native ADAM disintegrin domain; in some preferred embodiments the ADAM disintegrin domain variant is at least about 80% identical in amino acid sequence to the amino acid sequence of a native ADAM disintegrin domain. In some more preferred embodiments the ADAM disintegrin domain variant is at least about 90% identical in amino acid sequence to the amino acid sequence of a native ADAM disintegrin domain; in some more preferred embodiments the ADAM disintegrin domain variant is at least about 95% identical in amino acid sequence to the amino acid sequence of a native ADAM disintegrin domain. In some most preferred embodiments the ADAM disintegrin domain variant is at least about 98% identical in amino acid sequence to the amino acid sequence of a native ADAM disintegrin domain; in some most preferred embodiments the ADAM disintegrin domain variant is at least about 99% identical in amino acid sequence to the amino acid sequence of a native ADAM disintegrin domain.

Percent identity, in the case of both polypeptides and nucleic acids, may be determined by visual inspection. Percent identity may be determined using the alignment method of Needleman and Wunsch Mol. Biol. 48:443, 1970) as revised by Smith and Waterman (Adv. Appl. Math 2:482, 1981. Preferably, percent identity is determined by using a computer program, for example, the GAP computer program version 10.x available from the Genetics Computer Group (GCG; Madison, WI, see also Devereux et al., Nucl. Acids Res. 12:387, 1984). The preferred default parameters for the GAP program include: a unary comparison matrix (containing a value of 1 for identities and 0 for non- 0 identities) for nucleotides, and the weighted comparison matrix of Gribskov and Burgess, NucL Acids Res. 14:6745, 1986, as described by Schwartz and Dayhoff, eds., Atlas of Protein Sequence and Structure, National Biomedical Research Foundation, pp. 353-358, 1979 for amino acids; a Spenalty of 30 (amino acids) or 50 (nucleotides) for each gap and an additional I (amino acids) or 3 (nucleotides) penalty for each symbol in each gap; no penalty for end gaps; and no maximum penalty for long gaps. Other programs used by one skilled in the art of sequence comparison may also be used. For fragments of ADAM disintegrin domains, the percent identity is calculated based on that portion of ADAM disintegrin domain that is present in the fragment.

When a deletion or insertion strategy is adopted, the potential effect of the deletion or insertion on biological activity (such as integrin binding activity, inhibition of endothelial cell migration, or inhibition of angiogenesis) must be considered. Subunits of the inventive polypeptides 0 may be constructed by deleting terminal or internal residues or sequences. Additional guidance as to the types of mutations that can be made is provided by a comparison of the sequence of ADAM disintegrin domain polypeptides to polypeptides that have similar structures, as well as by performing structural analysis of the inventive polypeptides.

The term "variant" also includes ADAM disintegrin domain polypeptides that are encoded by nucleic acids capable of hybridizing under moderately stringent conditions prewashing solution of 5 X SSC, 0.5% SDS, 1.0 mM EDTA (pH 8.0) and hybridization conditions of 50 0 C, 5 X SSC, overnight) or higher stringency conditions to DNA sequences encoding ADAM disintegrin domain polypeptides, and which encode polypeptides that retain at least one activity selected from the group consisting of integrin binding activity, inhibition of endothelial cell migration, and inhibition of angiogenesis. The skilled artisan can determine additional combinations of salt and temperature that constitute moderate hybridization stringency. Conditions of higher stringency include higher temperatures for hybridization and post-hybridization washes, and/or lower salt concentration.

Mutations can be introduced into nucleic acids by synthesizing oligonucleotides containing a mutant sequence, flanked by restriction sites enabling ligation to fragments of the native sequence.

Following ligation, the resulting reconstructed sequence encodes a variant having the desired amino acid insertion, substitution, or deletion. Alternatively, oligonucleotide-directed site-specific mutagenesis procedures can be employed to provide an altered gene having particular codons altered according to the substitution, deletion, or insertion required. The well known polymerase chain reaction (PCR) procedure also may be employed to generate and amplify a DNA sequence encoding a desired polypeptide or fragment thereof. Oligonucleotides that define the desired termini of the DNA fragment are employed as 5' and 3' primers. The oligonucleotides may additionally contain recognition sites for restriction endonucleases to facilitate insertion of the amplified DNA fragment into an expression vector.

The present invention further encompasses the use of ADAM disintegrin domain polypeptides with or without associated native-pattern glycosylation. ADAM disintegrin domain expressed in yeast or mammalian expression systems COS-I or COS-7 cells) may be similar to or significantly different from a native ADAM disintegrin domain polypeptide in molecular weight and glycosylation pattern, depending upon the choice of expression system. Expression of ADAM disintegrin domain polypeptides in bacterial expression systems, such as E. coli, provides non-glycosylated molecules.

Different host cells may also process polypeptides differentially, resulting in heterogeneous mixtures of polypeptides with variable N- or C-termini.

The primary amino acid structure of ADAM disintegrin domain polypeptides may be modified to create derivatives by forming covalent or aggregative conjugates with other chemical moieties, such as glycosyl groups, lipids, phosphate, acetyl groups and the like. Covalent derivatives of ADAM disintegrin domain polypeptides may be prepared by linking particular functional groups to ADAM disintegrin domain amino acid side chains or at the N-terminus or C-terminus of a ADAM disintegrin domain polypeptide.

Fusion polypeptides of ADAM disintegrin domains that are useful in practicing the invention include covalent or aggregative conjugates of ADAMdis or its fragments with other polypeptides, such as by synthesis in recombinant culture as N-terminal or C-terminal fusions. One class of fusion polypeptides are discussed below in connection with ADAM disintegrin oligomers. As another example, a fusion polypeptide may comprise a signal peptide (which is also variously referred to as a signal sequence, signal, leader peptide, leader sequence, or leader) at the N-terminal region or Cterminal region of an ADAM disintegrin domain polypeptide which co-translationally or posttranslationally directs transfer of the polypeptide from its site of synthesis to a site inside or outside of the cell membrane or cell wall. It is particularly advantageous to fuse a signal peptide that promotes extracellular secretion to the N-terminus of a soluble ADAMdis polypeptide. In this case, the signal peptide is typically cleaved upon secretion of the soluble polypeptide from the cell.

Secreted soluble polypeptides may be identified (and distinguished from its non-soluble membrane-bound counterparts) by separating intact cells which express the desired polypeptide from the culture medium, by centrifugation, and assaying the medium (supernatant) for the presence of the desired polypeptide. The presence of the desired polypeptide in the medium indicates that the polypeptide was secreted from the cells and thus is a soluble form of the polypeptide. Soluble polypeptides may be prepared by any of a number of conventional techniques. A DNA sequence encoding a desired soluble polypeptide may be subcloned into an expression vector for production of the polypeptide, or the desired encoding DNA fragment may be chemically synthesized.

Soluble ADAM disintegrin domain polypeptides comprise all or part of the ADAM disintegrin domain, with or without additional segments from the extracellular portion of the ADAM (such as the cysteine-rich region) but generally lack a transmembrane domain that would cause retention of the polypeptide at the cell surface. Soluble polypeptides may include part of the transmembrane domain or all or part of the cytoplasmic domain as long as the polypeptide is secreted from the cell in which it is produced. Examples of soluble ADAM disintegrin domain polypeptides are provided in the examples. In some preferred embodiments of the present invention, a multimeric form of a soluble ADAM disintegrin domain polypeptide is used to inhibit integrin binding to ligands and, hence, integrin biological activity. In some most preferred embodiments the soluble ADAM disintegrin domain polypeptide is used to inhibit endothelial cell migration and/or inhibit angiogenesis.

These inhibitory activities may include both integrin-mediated and integrin-independent mechanisms.

ADAM disintegrin domain multimers are covalently-linked or non-covalently-linked multimers, including dimers, trimers, and higher multimers. Oligomers may be linked by disulfide bonds formed between cysteine residues on different ADAM disintegrin domain polypeptides. One embodiment of the invention is directed to multimers comprising multiple ADAM disintegrin domain polypeptides joined via covalent or non-covalent interactions between peptide moieties fused to the ADAM disintegrin domain polypeptides. Such peptides may be peptide linkers (spacers), or peptides that have the property of promoting multimerization. Leucine zippers and certain polypeptides derived from antibodies are among the peptides that can promote multimerization of ADAM disintegrin domain polypeptides attached thereto, as described in more detail below. In particular embodiments, the multimers comprise from two to four ADAM disintegrin domain polypeptides.

In some embodiments, a ADAM disintegrin domain multimer is prepared using polypeptides derived from immunoglobulins. Preparation of fusion proteins comprising certain heterologous polypeptides fused to various portions of antibody-derived polypeptides (including the Fc domain) has been described, by Ashkenazi et al. (Proc. Natl. Acad. Sci. USA 88:10535, 1991); Bym et al.

(Nature 344:677, 1990); and Hollenbaugh and Aruffo ("Construction of Immunoglobulin Fusion Proteins", in Current Protocols in Immunology, Suppl. 4, pages 10.19.1-10.19.11, 1992).

A preferred embodiment of the present invention is directed to an ADAM disintegrin domain (ADAMdis) dimer comprising two fusion polypeptides created by fusing an ADAM disintegrin domain to an Fc polypeptide. A gene fusion encoding the ADAMdis-Fc fusion polypeptide is inserted into an appropriate expression vector. ADAMdis-Fc fusion polypeptides are expressed in host cells transformed with the recombinant expression vector, and allowed to assemble much like antibody molecules, whereupon interchain disulfide bonds form between the Fc moieties to yield divalent soluble ADAMdis polypeptides. The term "Fc polypeptide" as used herein includes native and mutein forms of polypeptides derived from the Fc region of an antibody. Truncated forms of such polypeptides containing the hinge region that promotes dimerization are also included.

One suitable Fc polypeptide, described in PCT application WO 93/10151, is a single chain polypeptide extending from the N-terminal hinge region to the native C-terminus of the Fc region of a human IgGI antibody. Another useful Fc polypeptide is the Fc mutein described in U.S. Patent 5,457,035 and by Baum et al., EMBO J. 13:3992, 1994. The amino acid sequence of this mutein is identical to that of the native Fc sequence presented in WO 93/10151, except that amino acid 19 has been changed from Leu to Ala, amino acid 20 has been changed from Leu to Glu, and amino acid 22 has been changed from Gly to Ala. The mutein exhibits reduced affinity for Fc receptors. Fusion polypeptides comprising Fc moieties, and multimers formed therefrom, offer an advantage of facile purification by affinity chromatography over Protein A or Protein G columns, and Fc fusion Spolypeptides may provide a longer in vivo half life, which is useful in therapeutic applications, than Ci unmodified polypeptides.

t In other embodiments, a soluble ADAM disintegrin domain polypeptide may be substituted Sfor the variable portion of an antibody heavy or light chain. If fusion proteins are made with both 0 5 heavy and light chains of an antibody, it is possible to form an ADAM disintegrin domain multimer with as many as four soluble ADAM disintegrin domain polypeptides.

Alternatively, the ADAM disintegrin domain multimer is a fusion polypeptide comprising multiple ADAM disintegrin domain polypeptides, with or without peptide linkers (spacers), or peptides that have the property of promoting multimerization.. Among the suitable peptide linkers are those described in U.S. Patents 4,751,180 and 4,935,233. A DNA sequence encoding a desired peptide linker may be inserted between, and in the same reading frame as, the DNA sequences Sencoding ADAMdis, using conventional techniques known in the art. For example, a chemically synthesized oligonucleotide encoding the linker may be ligated between sequences encoding ADAMdis. In particular embodiments, a fusion protein comprises from two to four ADAM disintegrin domain polypeptides, separated by peptide linkers.

Another method for preparing ADAM disintegrin domain multimers involves use of a leucine zipper domain. Leucine zipper domains are peptides that promote multimerization of the proteins in which they are found. Leucine zippers were originally identified in several DNA-binding proteins (Landschulz et al., Science 240:1759, 1988), and have since been found in a variety of different proteins. Among the known leucine zippers are naturally occurring peptides and derivatives thereof that dimerize or trimerize. Examples of leucine zipper domains suitable for producing soluble oligomeric proteins are described in PCT application WO 94/10308, and the leucine zipper derived from lung surfactant protein D (SPD) described in Hoppe et al. FEBS Lett. 344:191, 1994. The use of a modified leucine zipper that allows for stable trimerization of a heterologous protein fused thereto is described in Fanslow et al., Semin. Immunol. 6:267, 1994. Recombinant fusion polypeptides comprising an ADAM disintegrin domain polypeptide fused to a leucine zipper peptide are expressed in suitable host cells, and the ADAM disintegrin domain multimer that forms is recovered from the culture supernatant.

C. Recombinant Production of ADAM Disintegrin Domain Polvpeptides The ADAM disintegrin domain polypeptides used in the present invention may be prepared using a recombinant expression system. Host cells transformed with a recombinant expression vector encoding the ADAM disintegrin domain polypeptide are cultured under conditions that promote expression of ADAM disintegrin domain and the ADAM disintegrin domain is recovered. ADAM disintegrin domain polypeptides can also be produced in transgenic plants or animals.

Any suitable expression system may be employed. Recombinant expression vectors include DNA encoding an ADAM disintegrin domain polypeptide operably linked to suitable transcriptional and translational regulatory nucleotide sequences, such as those derived from a mammalian, microbial, viral, or insect gene. Nucleotide sequences are operably linked when the regulatory sequence functionally relates to the ADAM disintegrin domain DNA sequence. Thus, a promoter nucleotide sequence is operably linked to an ADAM disintegrin domain DNA sequence if the promoter nucleotide sequence controls the transcription of the ADAM disintegrin domain DNA sequence.

Examples of regulatory sequences include transcriptional promoters, operators, or enhancers, an mRNA ribosomal binding site, and appropriate sequences which control transcription and translation initiation and termination. A sequence encoding an appropriate signal peptide (native or heterologous) can be incorporated into expression vectors. A DNA sequence for a signal peptide (secretory leader) may be fused in frame to the ADAM disintegrin domain sequence so that the ADAM disintegrin domain polypeptide is initially translated as a fusion protein comprising the signal peptide. A signal peptide that is functional in the intended host cells promotes extracellular secretion of the ADAM disintegrin domain polypeptide. The signal peptide is cleaved from the ADAM disintegrin domain polypeptide upon secretion from the cell. Suitable host cells for expression of ADAM disintegrin domain polypeptides include prokaryotes, yeast and higher eukaryotic cells, including insect and mammalian cells. Appropriate cloning and expression vectors for use with bacterial, fungal, yeast, insect, and mammalian cellular hosts are known in the art.

Using the techniques of recombinant DNA including mutagenesis and the polymerase chain reaction (PCR), the skilled artisan can produce DNA sequences that encode ADAM disintegrin domain polypeptides comprising various additions or substitutions of amino acid residues or sequences, or deletions of terminal or internal residues or sequences, including ADAM disintegrin domain fragments, variants, derivatives, multimers, and fusion polypeptides.

The procedures for purifying expressed ADAM disintegrin domain polypeptides will vary according to the host system employed, and whether or not the recombinant polypeptide is secreted.

ADAM disintegrin domain polypeptides may be purified using methods known in the art, including one or more concentration, salting-out, ion exchange, hydrophobic interaction, affinity purification, HPLC, or size exclusion chromatography steps. Fusion polypeptides comprising Fc moieties (and multimers formed therefrom) offer the advantage of facile purification by affinity chromatography over Protein A or Protein G columns.

D. Therapeutic Methods The disclosed methods may be used to inhibit integrin binding and integrin biological activity, and to inhibit endothelial cell migration, and/or angiogenesis in a mammal in need of such treatment.

The treatment is advantageously administered in order to prevent the onset or the recurrence of a disease or condition mediated by an integrin, or to treat a mammal that has a disease or condition mediated by an integrin.

Examples of the therapeutic uses of ADAM disintegrin domain polypeptides and compositions thereof include the treatment of individuals afflicted with conditions mediated by angiogenesis such as ocular disorders, dermatological disorders, and malignant or metastatic conditions, inflammatory diseases, osteoporosis and other conditions mediated by accelerated bone resorption, restenosis, inappropriate platelet activation, recruitment, or aggregation, thrombosis, or a condition requiring tissue repair or wound healing.

Among the ocular disorders that can be treated according to the present invention are eye diseases characterized by ocular neovascularization including, but not limited to, diabetic retinopathy (a major complication of diabetes), retinopathy of prematurity (this devastating eye condition, that frequently leads to chronic vision problems and carries a high risk of blindness, is a severe complication during the care of premature infants), neovascular glaucoma, retinoblastoma, retrolental fibroplasia, rubeosis, uveitis, macular degeneration, and corneal graft neovascularization. Other eye inflammatory diseases, ocular tumors, and diseases associated with choroidal or iris neovascularization can also be treated according to the present invention.

The present invention can also be used to treat malignant and metastatic conditions such as solid tumors. Solid tumors include both primary and metastatic sarcomas and carcinomas.

The present invention can also be used to treat inflammatory diseases including, but not limited to, arthritis, rheumatism, inflammatory bowel disease, and psoriasis.

Among the conditions mediated by inappropriate platelet activation, recruitment, aggregation, or thrombosis that can be treated according to the present invention are coronary artery disease or injury, myocardial infarction or injury following myocardial infarction, stroke, unstable angina, atherosclerosis, arteriosclerosis, preeclampsia, embolism, platelet-associated ischemic disorders including lung ischemia, coronary ischemia, and cerebral ischemia, restenosis following percutaneous coronary intervention including angioplasty, atherectomy, stent placement, and bypass surgery, thrombotic disorders including coronary artery thrombosis, cerebral artery thrombosis, intracardiac thrombosis, peripheral artery thrombosis, venous thrombosis, thrombosis and coagulopathies associated with exposure to a foreign or injured tissue surface, and reocclusion following thrombosis, deep venous thrombosis (DVT), pulmonary embolism transient ischemic attacks (TIAs), and another conditions where vascular occlusion is a common underlying feature. In some embodiments the methods according to the invention are used in individuals at high risk for thrombus formation or reformation, advanced coronary artery disease, or for occlusion, reocclusion, stenosis and/or restenosis of blood vessels, or stroke. In some embodiments the methods according to the invention are used in combination with angioplasty procedures, such as balloon angioplasty, laser angioplasty, coronary atherectomy or similar techniques, carotid endarterectomy, anastomosis of vascular grafts, surgery having a high risk of thrombus formation coronary bypass surgery, insertion of a prosthetic valve or vessel and the like), atherectomy, stent placement, placement of a chronic cardiovascular device such as an in-dwelling catheter or prosthetic valve or vessel, organ transplantation, or bypass surgery.

Other diseases and conditions that can be treated according to the present invention include benign tumors and preneoplastic conditions, myocardial angiogenesis, hemophilic joints, scleroderma, vascular adhesions, asthma and allergy, eczema and dermatitis, graft versus host disease, sepsis, adult respirator distress syndrome, telangiectasia, and wound granulation.

The methods according to the present invention can be tested in in vivo animal models for the desired prophylactic or therapeutic activity, as well as to determine the optimal therapeutic dosage, prior to administration to humans.

The amount of a particular ADAM disintegrin domain polypeptide that will be effective in a particular method of treatment depends upon age, type and severity of the condition to be treated, body weight, desired duration of treatment, method of administration, and other parameters. Effective dosages are determined by a physician or other qualified medical professional. Typical effective dosages are about 0.01 mg/kg to about 100 mg/kg body weight. In some preferred embodiments the dosage is about 0.1-50 mg/kg; in some preferred embodiments the dosage is about 0.5-10 mg/kg. The dosage for local administration is typically lower than for systemic administration. In some embodiments a single administration is sufficient; in some embodiments the ADAM disintegrin domain is administered as multiple doses over one or more days.

The ADAM disintegrin domain polypeptides are typically administered in the form of a pharmaceutical composition comprising one or more pharmacologically acceptable carriers.

Pharmaceutically acceptable carriers include diluents, fillers, adjuvants, excipients, and vehicles which are pharmaceutically acceptable for the route of administration, and may be aqueous or oleaginous suspensions formulated using suitable dispersing, wetting, and suspending agents.

Pharmaceutically acceptable carriers are generally sterile and free of pyrogenic agents, and may include water, oils, solvents, salts, sugars and other carbohydrates, emulsifying agents, buffering agents, antimicrobial agents, and chelating agents. The particular pharmaceutically acceptable carrier and the ratio of active compound to carrier are determined by the solubility and chemical properties of the composition, the mode of administration, and standard pharmaceutical practice.

The ADAM disintegrin domain polypeptides are administered to the patient in a manner appropriate to the indication. Thus, for example, ADAM disintegrin domain polypeptides, or pharmaceutical compositions thereof, may be administered by intravenous, transdermal, intradermal, intraperitoneal, intramuscular, intranasal, epidural, oral, topical, subcutaneous, intracavity, sustained release from implants, peristaltic routes, or by any other suitable technique. Parenteral administration is preferred.

In certain embodiments of the claimed invention, the treatment further comprises treating the mammal with one or more additional therapeutic agents. The additional therapeutic agent(s) may be administered prior to, concurrently with, or following the administration of the ADAM disintegrin domain polypeptide. The use of more than one therapeutic agent is particularly advantageous when the mammal that is being treated has a solid tumor. In some embodiments of the claimed invention, the treatment further comprises treating the mammal with radiation. Radiation, including brachytherapy and teletherapy, may be administered prior to, concurrently with, or following the administration of the ADAM disintegrin domain polypeptide and/or additional therapeutic agent(s).

0 In some preferred embodiments the method includes the administration of, in addition to an C,1 ADAM disintegrin domain polypeptide, one or more therapeutics selected from the group consisting cof alkylating agents, antimetabolites, vinca alkaloids and other plant-derived chemotherapeutics, antitumor antibiotics, antitumor enzymes, topoisomerase inhibitors, platinum analogs, adrenocortical suppressants, hormones and antihormones, antibodies, immunotherapeutics, radiotherapeutics, and biological response modifiers.

In some preferred embodiments the method includes administration of, in addition to an ADAM disintegrin domain polypeptide, one or more therapeutics selected from the group consisting ofcisplatin, cyclophosphamide, mechloretamine, melphalan, bleomycin, carboplatin, fluorouracil, N 10 fluorodeoxyuridine, methotrexate, taxol, asparaginase, vincristine, and vinblastine, lymphokines and cytokines such as interleukins, interferons (alpha., beta. or delta.) and TNF, chlorambucil, busulfan, 0carmustine, lomustine, semustine, streptozocin, dacarbazine, cytarabine, mercaptopurine, thioguanine, vindesine, etoposide, teniposide, dactinomycin, daunorubicin, doxorubicin, bleomycin, plicamycin, mitomycin, L-asparaginase, hydroxyurea, methylhydrazine, mitotane, tamoxifen, fluoxymesterone, IL-8 inhibitors, angiostatin, endostatin, kringle 5, angiopoietin-2 or other antagonists of angiopoietin-1, antagonists of platelet-activating factor, antagonists of basic fibroblast growth factor.

and COX-2 inhibitors.

In some preferred embodiments the method includes administration of, in addition to an ADAM disintegrin domain polypeptide, one or more therapeutic polypeptides, including soluble forms thereof, selected from the group consisting of Flt3 ligand, CD40 ligand, interleukin-2, interleukin-12, 4-1BB ligand, anti-4- I BB antibodies, TRAIL, TNF antagonists and TNF receptor antagonists including TNFR/Fc, Tek antagonists, TWEAK antagonists and TWEAK-R antagonists including TWEAK-R/Fc, VEGF antagonists including anti-VEGF antibodies, VEGF receptor (including VEGF- R I and VEGF-R2. also known as Flt and Flkl or KDR) antagonists, CD148 (also referred to as DEP- I, ECRTP, and PTPRJ. see Takahashi et al., J. Am. Soc. Nephrol. 10:2135-45, 1999; and PCT Publication No. WO 00/15258, 23 March 2000) binding proteins, and nectin-3 antagonists.

In some preferred embodiments the ADAM disintegrin domain polypeptides of the invention are used as a component of, or in combination with. "metronomic therapy," such as that described by Browder et al. and Klement et al. (Cancer Research.60:1878, 2000; J. Clin. Invest. 105(8):R15, 2000; see also Barinaga, Science 288:245, 2000).

As used herein, the terms "therapy," "therapeutic," "treat," and "treatment" generally include prophylaxis, i.e. prevention, in addition to therapy or treatment for an extant disease or condition. The methods of the present invention may be used as a first line treatment, for the treatment of residual disease following primary therapy, or as an adjunct to other therapies. Methods of measuring biological effectiveness are known in the art and are illustrated in the Examples below.

EXAMPLES

The following examples are intended to illustrate particular embodiments and not to limit the scope of the invention.

EXAMPLE 1 ADAM Disintegrin Domain Polypeptides This example describes one method for the recombinant production of ADAM disintegrin domain polypeptides.

Expression cassettes encoding an IgKappa leader sequence, ADAM disintegrin domain, and C-terminal Fc region were constructed in bacterial plasmids then transferred into eukaryotic expression vectors (pDC409, EMBO J. 10:2821, 1991, or another mammalian expression vector). The coding regions of the various constructs are summarized in Table 2. In addition to the disintegrin domain, these constructs encode additional portions of the extracellular portion of the ADAM cysteine-rich region and EGF-like domain).

The expression vectors were transfected into COS-1, CV-I/EBNA, or 293/EBNA cells. Two days after transfection the cells were "S labeled for four hours. Superatants and total cell lysates were prepared and aliquots were immunoprecipitated using protein A-sepharose beads to capture the Fc tagged polypeptides. "S labeled ADAM disintegrin-Fc polypeptides were run on 8-16% reducing gels and detected via autoradiography.

The cell type that produced the most soluble protein in the supernatant was used in a large scale (T-175 format, 20 flasks) transient transfection, and approximately one liter of supernatant was harvested after one week. ADAM disintegrin-Fc polypeptides were purified from the supematants using affinity chromatography (protein A column). The polypeptides were characterized by determining the N-terminal amino acid sequence, amino acid composition, and protein integrity (SDS- PAGE under reducing and non-reducing conditions) before the polypeptides were used in FACS, immunoprecipitations, and biological assays such as those described below.

Table 2 ADAM Disinteitrin Domain Polypeptide Construcs SEQ 11) 1,2ADAM ,1 Construct S DN NOlptid IgK Leader" disintegrin 14 Fc Region' is Framework)'* ADAM-8dis-Fc 1/2 1-20 23264 267-494 (34-91) ADAM-9dis-Fc 3/4 1-20 23-303 306533 (34-92) ADAM-l0dis-Fc 5/6 1-20 2325238-465 ADAM- I Sdis-Fc 7/8 1-20 2329 295-522 ADAM-I 7dis-Fc 9/10 1-20 2321 219-446 11/12 1-20 2330 308-535 ADAM-2]dis-Fc 13/14 1-20 (49)296&523 4 3-1 ADAM-22dis-Fc 15/16 1-20 (34-92) 315-542 ADAM-23dis-Fc 17/18 I 1 20 234-91)0 313-540 ADAM-29dis-Fc 21/22 1-20 23-298) 301-528 2residues in the polypeptide sequence .1the predicted cleavage site is after residue 4segment of the construct that includes ADAMdis, but may also contain additional ADAM sequences 4 disintegrin framework, SEQ ID EXAMPLE 2 Binding of ADAM Disintegrin Domain Polypeptides to Cells A. Binding to Endothelial cells This example describes a flow cytometric integrin mAb based binding inhibition assay, which is used to show binding of ADAM disintegrin-Fc polypeptides to integrins expressed on the surface of endoffielial cells. Human endothelial cells express cc4 3 ct. f. f3 4 a 1 a 2 cE, a 4 a 5 and a, in Legnins.

Primary human dermal microvascular endothelial cells (HMVEC-d) were maintained in supplemented endothelial growth medium (Clonetics Corporation, Walkersville, MD). The ADAM disintegrin-Fc polypeptides produced in Example I were shown to bind specifically to H-MVEC-d.

Monoclonal antibodies specific for human integrins a3 (LM609, anti CD51/61, Chemicon, Temecula, CA Brooks et al., Science 264:569, 1994), a 2 p 1 (BHA2.1 anti CD49b, Chemicon, Wang et al., Mol. Biol. of the Cell 9:865, 1998), asI (SAM-I anti CD49e, Biodesign, A. te Velde et al., J.

Immunol. 140:1548, 1988), aC3S (ASC-6 anti-CD49c, Chemicon, Pattaramalai et al., Exp. Cell. Res.

222: 281, 1996), ia4p (HP2/1 anti CD49d, Immunotech, Marseilles, France. Workshop of the 4 h International Conference on Human Leukocyte Differentiation Antigens, Vienna Austria, 1989, workshop number p 0 9 1 as 6 p (GoH3 anti CD49f, Immunotech, Workshop 4 h International Conference on Human Leukocyte Differentiation Antigens, workshop number p055), asp4 (439-9B anti CD104, Pharmingen, San Diego, CA., Schlossman et al., 1995 Leukocyte Typing V: White Cell Differntiation Antigens. Oxford University Press, New York), and a,05 (MAB 1961, Chemicon International, monoclonal anti-human integrin avPs mAb, IgGI isotype, inhibits avP5 mediated binding/adhesion to vitronectin/fibronectin; Weinaker, et al., J. Biol. Chem. 269:6940, 1994) were also shown to bind specifically to HMVEC-d. Each of these antibodies is known to specifically block binding of the indicated integrin to its ligands fibronectin, vitronectin, fibrinogen). The ability of integrin mAbs to inhibit the binding of ADAM disintegrin-Fc polypeptides reveals which integrins the disintegrin domains bind and, indirectly, which integrin binding activities the disintegrin domains are able to antagonize. The ability of the antibodies to inhibit binding of the ADAM disintegrin-Fc polypeptides to endothelial cells was tested as described below.

Prior to performing binding studies, HMVEC-d were removed from culture vessels using trypsin-EDTA. The cells were washed in media containing serum and resuspended in binding medium which consisted of PBS containing 1 mM Ca2+, 1 mM Mg2+ and 0.5 mM Mn2+, 0.1% sodium azide, Normal goat serum, 2% rabbit serum and 2% fetal bovine serum. Under these binding conditions, ADAM-8, -10, -15, -17, -20, -21.-22, -23, and -29dis-Fc all bind to human endothelial cells.

One hundred microliters of cell suspension, containing 200,000 to 500,000 HMVEC-d, were added to 12x75mm plastic test tubes. Monoclonal antibodies specific for one of the integrins, or a control monoclonal antibody (CD29 or M15), were added to the cell suspensions at a concentration of 100 gg/ml (5-8 fold mass excess) 15 minutes prior to addition of disintegrin-Fc fusion proteins.

ADAM disintegrin-Fc polypeptides and control Fc fusion polypeptides (P7.5II.Fc) were added, at various concentrations from 12.5 to 20 pg/ml, to the cell suspensions and incubated for 1 hour at C. Unbound Fc polypeptides were washed away by centrifugation of cells in 2 mis of binding media.

The washed cell pellets were resuspended in binding medium and then incubated at 30' C for minutes with goat anti-human Fc-specific biotinylated antibody at a concentration of 2.5 pg/ml for minutes. After centrifugation and washing of the cell pellets, the cells were resuspended in binding medium and bound anti-human Fc-biotin was detected by adding streptavidin-phycoerythrin conjugate to the cell suspension at a 1:1000 dilution (1 pg/ml) and incubating at 30' C for 30 minutes. The unbound streptavidin-phycoerythrin was washed away and the cells were resuspended in binding 0 medium containing propidum iodide. The level of fluorescent binding (disintegrin-Fc binding) was C determined by flow cytometry.

The level of binding of each ADAM disintegrin-Fc polypeptide was determined in the Spresence of anti-integrin specific mAb and in the presence of control mAb. Both the intensity of binding (MFI) and the percentage of cells binding were determined. Percent inhibition was calculated using the formula [I (MFI control-MFI integrin mAb) MFI control. The results of these studies are summarized in Table 3.

-17, -20 and -22 disintegrin domain polypeptides bound to avN; ADAM 23 disintegrin domain polypeptide bound to a2z ADAM-15, -21, -22 and -23 disintegrin domain polypeptides bound to a 5 s 1 ADAM-10, -17, -22 and -23 disintegrin domain polypeptides bound to the a 6 integrins; ADAM-10 and -15 disintegrin domain polypeptides bound to avp.. An excess of a 0 non blocking av3 5 antibody did significantly affect the binding of ADAM-10, -22, and -23 disintegrin polypeptides to endothelial cells, suggesting that these ADAMdis polypeptides interact with integrin sites other than or in addition to the ligand fibronectin, vitronectin) binding site. Based upon results from a different type of assay, Cal et al. have reported that the ADAM-23 disintegrin domain interacts with the avp3 integrin through an RGD-independent mechanism (Molec. Biol. of the Cell S1:1457, 2000).

Binding experiments are repeated using other ADAM disintegrin domains and other monoclonal antibodies. ADAM disintegrin-Fc polypeptides that bind to selected integrins are further tested for the ability to disrupt integrin-ligand interactions and to modulate endothelial cell function, angiogenesis, and other biological activities in vitro and in vivo.

2007201172 19 Mar 2007 Table 3 Binding of ADAM Disintegrin-Fc PolYeotides to Integrins Expressed on Human Endothelial Cells Integrin Binding' or or ND. not done) and Percent Binding 2 ADAM I CL3j174PI uP3 a6P4 a34P ADAM-8 ND ND ND ND ADAM-9 ADAM-O (48) (60) ADAM-17- (30) (69) I.M2 (50) AA-1 10) ADAM-22 (42) (36) (32) ADAM-23 (42) (<10)9 (32) j I positive binding defined as >20% binding inhibition; normal background variation 5-10%, baseline positive approx. 2X over background 2prent inhibition of binding by ADAM-dis-Fc in the presence of 5-8 fold excess integrin mAb as compared to control mAb 0 B. Binding to Primary Human T-Cells Primary human T-cells were purified from whole blood. These cells were used in FACS experiments to assess cell surface binding of purified ADAMdis-Fc polypeptides. ADAMdis-Fc Sbinding was assessed with and without Con A (5 ugg/ml) or immobilized OTK3 antibody (1 mg/ml, S 5 immobilized for 1 hour, 37*C) stimulation. ADAMdis-Fc polypeptides (20 pg/ml) were bound at either 4* C or 30° C in the presence of cations 0.5 mM each). Cell surface C integrin expression was assessed using a panel of murine and rat anti-human integrin antibodies. apds.

a3, aC, as, P, and p7 integrins were detected on the surface of these cells. ADAMdis-Fc polypeptides did not bind to primary human T-cells at 4* C. ADAM-8-, ADAM-9-, 1 10 ADAM-20-. ADAM-21-, ADAM-22-, and ADAM-23-dis-Fc polypeptides did bind primary T-cells at 030* C with Con A stimulation. ADAMdis-Fc binding was not inhibited by a three-fold molar excess of antibodies to the integrins listed above.

C. Binding to Resting Platelets Binding of ADAMdis-Fc polypeptides to citrated washed resting platelets was performed at 4 0 C or 30 0 C. Binding was analyzed by flow cytometry using a biotinylated-anti-human Fc specific antibody and streptavidin-PE. Resting platelets express the integrins CD41/CD61 and CD49e.

ADAM-9dis-Fc and ADAM-8dis-Fc bound resting platelets at 30 0 C but not at 4°C. ADAM-9dis-Fc binding to resting platelets at 30 0 C was not inhibited by a ten-fold excess of CD41a mAb.

EXAMPLE 3 Activity of ADAM Disintegrin Domain Polypeptides In a Wound Closure Assay A planar endothelial cell migration (wound closure) assay was used to quantitate the inhibition of angiogenesis by ADAM disintegrin-Fc polypeptides in vitro. In this assay, endothelial cell migration is measured as the rate of closure of a circular wound in a cultured cell monolayer. The rate of wound closure is linear, and is dynamically regulated by agents that stimulate and inhibit angiogenesis in vivo.

Primary human renal microvascular endothelial cells, HRMEC, were isolated, cultured, and used at the third passage after thawing, as described in Martin et al., In Vitro Cell Dev Biol 33:261, 1997. Replicate circular lesions, "wounds," (600-800 micron diameter) were generated in confluent HRMEC monolayers using a silicon-tipped drill press. At the time of wounding the medium (DMEM 1% BSA) was supplemented with 20 ng/ml PMA (phorbol- 2-myristate-13-acetate), a range of concentrations of ADAM disintegrin-Fc polypeptide, or combinations of PMA and ADAM disintegrin-Fc polypeptide. The residual wound area was measured as a function of time (0-12 hours) using a microscope and image analysis software (Bioquant, Nashville, TN). The relative migration rate was calculated for each agent and combination of agents by linear regression of residual wound Ct area plotted over time. The inhibition of PMA-induced endothelial migration by ADAM disintegrin- Fc polypeptides is shown in Table 4.

The effect of ADAM-dis-Fc polypeptides on EGF-induced migration was also determined.

For these experiments EGF (epidermal growth factor, 40 nglml) was added to the medium, instead of PMA, at the time of wounding. The results are shown in Table Table 4 Effect of ADAM-IS5 -17, -20, and -23dis-Fc Polypeptides in PMA-Induced Endothelial Cell Wound Clo~ur~ Mi~rarinn A~q~av 110 -nohla -el Won Clour -irto As- No PMA PMA PMA PMA PMA PMA Expt. ED Addition 20 nglml IgG ADAM- ADAM- ADAM- ADAM- -I dis-Fc l7dis-Fc 20dis-Fc 23dis-Fc HL-H- 142 0.0499 jig/mI 0.0436' 0.0655 (0.0009) dis-Fc (0.0016)2 (0.0004) 72%" l-LL-H-l147 0.0449 0.0357 0.0225 jig/mid 0.0244 0.0424 (0.0012) (0.0007) (0.0022) dis-Fc (0.0023) (0.0002) 0% 37% 100% HL-H-l53 0.0491 0.0392 0.0388 0.0317 Vg/mi 0.0253 0.0460 (0.006) (0.0016) (0.005) (0.005) dis-Fc 0.00013 (0.0022) 0% 33% 36% HL--H-l 54 0.0283 0.0160 pgmJd 0.0119 0.0312 (0.0008) (0.0017) dis-Fc (0.0012) (0.0016) 1 15% 79% 'Slopes to average triplicate Y values and treat as a single data point in order to test whether the slopes are significantly different 2 3Data in parentheses is the standard error of slopes Percent inhibition compared to migration rate observed in the presence of PMA Table Effect of ADAM- 17. -20, and -23dis-Fc Polylpeptides in EGF-IndUced Endothelial Cell Wound Closure Migration Assay No EGF EGF EGF EGF EGF Expt. ED Addition 410 ng/ml lgG ADAM- ADAM- ADAMl7dis-Fc 20dis-Fc 23dis-Fc HL-H- 154 0.0242 0.0172 0.0310 pg/mI 0.0119 0.037 8 (0.0029) (0.0031) (0.0036) dis-Fc (0.0012) (0.0061) 53% 80% 26% HL-H-l 55 0.0454 0.0412 0.0227 0.0207 9 Ag/mi 0.0164 0.0468 (0.0052) (0.0107) (0.0035) (0.0016) dis-Ec (0-0010) (0.0059) 5% 18% 79% 86% Slopes to average triplicate Y values and treat as a single data point in order to test whether the.

slopes are significantly different 2 Data in parentheses is the standard error of slopes 3Percent inhibition compared to migration rate observed in the presence of FOF alone and -23dis-Fc polypeptides showed the greatest inhibition of both EGF- and PMA-induced endothelial migration at 15 pg/mI. ADAM-I15 and I7dis-Fc polypeptides were less effective at inhibiting endothelial cell migration at 15 gg/ml. Hu IgG did not inhibite EGF- or PMAinduced endothelial cell migration in any of the experiments performed where it was included as a control Fc protein.

EXAMPLE 4 Activity of ADAM Disintegrin Domain Polypeptides In a Corneal Pocket Assay A mouse comeal pocket assay is used to quantitate the inhibition of angiogenesis by ADAM disintegrin-Fc polypeptides in vivo. In this assay, agents to be tested for angiogenic or anti-angiogenic activity are immobilized in a slow release form in a hydron pellet, which is implanted into micropockets created in the corneal epithelium of anesthetized mice. Vascularization is measured as the appearance, density, and extent of vessel ingrowth from the vascularized corneal limbus into the normally avascular cornea.

Hydron pellets, as described in Kenyon et al., Invest Opthamol. Visual Science 37:1625, 1996, incorporate sucralfate with bFGF (90 ng/pellet), bFGF and IgG (11 pg/pellet, control), or bFGF and a range of concentrations of ADAM disintegrin-Fc polypeptide. The pellets are surgically implanted into corneal stromal micropockets created by micro-dissection 1 mm medial to the lateral comeal limbus of 6-8 week old male C57BL mice. After five days, at the peak of neovascular response to bFGF, the corneas are photographed, using a Zeiss slit lamp, at an incipient angle of from the polar axis in the meridian containing the pellet. Images are digitized and processed by subtractive color filters (Adobe Photoshop 4.0) to delineate established microvessels by hemoglobin content. Image analysis software (Bioquant, Nashville, TN) is used to calculate the fraction of the corneal image that is vascularized, the vessel density within the vascularized area, and the vessel density within the total cornea. The inhibition of bFGF-induced corneal angiogenesis, as a function of the dose of ADAM disintegrin-Fc polypeptide, is determined.

EXAMPLE Inhibition of Neovascularization by ADAM Disintegrin Domain Polypeptides in a Murine Transplant Model Survival of heterotopically transplanted cardiac tissue from one mouse donor to the ear skin of another genetically similar mouse requires adequate neovascularization by the transplanted heart and the surrounding tissue, to promote survival and energy for cardiac muscle function. Inadequate vasculature at the site of transplant causes excessive ischemia to the heart, tissue damage, and failure of the tissue to engraft. Agents that antagonize factors involved in endothelial cell migration and vessel formation can decrease angiogenesis at the site of transplant, thereby limiting graft tissue function and ultimately engraftment itself. A murine heterotopic cardiac isograft model is used to demonstrate the antagonistic effects of ADAM disintegrin-Fc polypeptides on neovascularization.

Female BALB/c (=12 weeks of age) recipients are given neonatal heart grafts from donor mice of the same strain. The donor heart tissue is grafted into the left ear pinnae of the recipient on day 0 and the mice are divided into two groups. The control group receives human IgG (Hu IgG) while the other 1 group receives ADAM disintegrin-Fc polypeptide, both intraperitoneally. The treatments are ccontinued for five consecutive days. The functionality of the grafts is determined by monitoring Svisible pulsatile activity on days 7 and 14 post-engraftment. The inhibition of functional engraftment, as a function of the dose of ADAM disintegrin-Fc polypeptide, is determined. The histology of the transplanted hearts is examined is order to visualize the effects of ADAM disintegrin-Fc polypeptides on edema at the site of transplant and host and donor tissue vasculature (using, Factor VIII staining).

EXAMPLE 6 Treatment of Tumors With ADAM Disintegrin Domain Polypeptides ADAM disintegrin-Fc polypeptides are tested in animal models of solid tumors. The effect of the ADAM disintegrin-Fc polypeptides is determined by measuring tumor frequency and tumor growth.

The biological activity of ADAM disintegrin-Fc polypeptides is also demonstrated in other in vitro, ex vivo, and in vivo assays known to the skilled artisan, such as calcium mobilization assays and assays to measure platelet activation, recruitment, or aggregation.

The relevant disclosures of publications cited herein are specifically incorporated by reference. The examples presented above are not intended to be exhaustive or to limit the scope of the invention. The skilled artisan will understand that variations and modifications and variations are possible in light of the above teachings, and such modifications and variations are intended to be within the scope of the invention.

Claims (12)

1. A method of antagonizing the binding of an integrin to its ligands comprising contacting a Scell that expresses the integrin with an effective amount of an ADAM disintegrin domain polypeptide.

2. A method of antagonizing the binding of an integrin to its ligands in a mammal in need of such treatment comprising administering an effective amount of an ADAM disintegrin domain polypeptide. S3. The method of claim 2 wherein the mammal is afflicted with a condition selected from the group consisting of ocular disorders, malignant and metastatic conditions, inflammatory diseases, Sosteoporosis and other conditions mediated by accelerated bone resorption, restenosis, inappropriate platelet activation, recruitment, or aggregation, thrombosis, or a condition requiring tissue repair or Swound healing.

4. A method of inhibiting angiogenesis in a mammal in need of such treatment, comprising administering to the mammal an inhibition-effective amount of an ADAM disintegrin domain polypeptide, wherein the disintegrin domain does not contain an RGD sequence. The method of one of claims 1-4 wherein the ADAM disintegrin domain is in the form of a multimer.

6. The method of claim 5 wherein the multimer is a dimer or trimer.

7. The method of claim 5 wherein the multimer comprises an Fc polypeptide or a leucine zipper.

8. The method of one of claims 1-7 wherein the ADAM disintegrin domain is from a human ADAM.

9. The method of claim 8 wherein the ADAM disintegrin domain is from an ADAM selected from the group consisting of ADAM-8, ADAM-9, ADAM-10, ADAM-15, ADAM- 17, ADAM-21, ADAM-22, ADAM-23, and ADAM-29. The method of claim 9 wherein the ADAM disintegrin domain is from ADAM- 17, or ADAM-23. I The method of one of claims 1-10 wherein the ADAM disintegrin domain polypeptide comprises an amino acid sequence selected from the group consisting of: amino acids 1-494 of SEQ ID NO:2, amino acids 23-264 of SEQ ID NO:2, amino acids 1- 533 of SEQ ID NO:4, amino acids 23-303 of SEQ ID NO:4, amino acids 1-465 of SEQ ID NO:6, amino acids 23-235 of SEQ ID NO:6, amino acids 1-522 of SEQ ID NO:8, amino acids 23-292 of SEQ ID NO:8, amino acids 1-446 of SEQ ID NO:10, amino acids 23-216 of SEQ ID NO:10, amino acids 1-535 of SEQ ID NO:12, amino acids 23-305 of SEQ ID NO:12, amino acids 1-523 of SEQ ID NO: 14, amino acids 23-293 of SEQ ID NO: 14, amino acids 1-542 of SEQ ID NO: 16, amino acids 23- 312 of SEQ ID NO:16, amino acids 1-540 of SEQ ID NO:18, amino acids 23-310 of SEQ ID NO:18. amino acids 1-528 of SEQ ID NO:22, amino acids 23-298 of SEQ ID NO:22; fragments of the polypeptides of wherein said fragments retain at least one ADAMdis activity; variants of the polypeptides of or wherein said variants retain at least one ADAMdis activity; and fusion polypeptides comprising the polypeptides of or wherein said fusion polypeptides retain at least one ADAMdis activity.

12. The method of claim 1 I wherein the ADAM disintegrin domain comprises an amino acid sequence selected from the group consisting of amino acids 34-91 of SEQ ID NO:2, 34-92 of SEQ ID NO:4, 34-99 of SEQ ID NO:6, 34-92 of SEQ ID NO:8, 34-93 of SEQ ID NO: 10, 34-91 of SEQ ID NO:12, 34-91 of SEQ ID NO:14, 34-92 of SEQ ID NO:16, 34-91 of SEQ ID NO:18, or 34-91 of SEQ ID NO:22.

13. The method of one of claims 1-12 wherein the ADAM disintegrin domain polypeptide is a variant that is at least 70%, 80%, 90%, 95%, 98%, or 99% identical in amino acid sequence to a polypeptide selected from the group consisting of: amino acids 1-494 of SEQ ID NO:2, amino acids 23-264 of SEQ ID NO:2, amino acids 1- 533 of SEQ ID NO:4, amino acids 23-303 of SEQ ID NO:4, amino acids 1-465 of SEQ ID NO:6, amino acids 23-235 of SEQ ID NO:6, amino acids 1-522 of SEQ ID NO:8, amino acids 23-292 of SEQ ID NO:8, amino acids 1-446 of SEQ ID NO:10, amino acids 23-216 of SEQ ID NO:10, amino acids 1-535 of SEQ ID NO:12, amino acids 23-305 of SEQ ID NO:12, amino acids 1-523 of SEQ ID NO: 14, amino acids 23-293 of SEQ ID NO: 14, amino acids 1-542 of SEQ ID NO: 16, amino acids 23- 312 of SEQ ID NO:16, amino acids 1-540 of SEQ ID NO: 18, amino acids 23-310 of SEQ ID NO: 18, amino acids 1-528 of SEQ ID NO:22, amino acids 23-298 of SEQ ID NO:22; and fragments of the polypeptides of wherein said variant polypeptide retains at least one ADAMdis activity.

14. The method of one of claims 1-10 wherein the ADAM disintegrin domain polypeptide is encoded by a nucleic acid comprising a sequence selected from the group consisting of: nucleotides 118-1599 of SEQ ID NO:1, nucleotides 184-909 of SEQ ID NO: 1, nucleotides

46-1644 of SEQ ID NO:3, nucleotides 112-954 of SEQ ID NO:3, nucleotides 25-1419 of SEQ ID nucleotides 91-729 of SEQ ID NO:5, nucleotides 41-1606 of SEQ ID NO:7, nucleotides 107- 916 of SEQ ID NO:7, nucleotides 25-1362 of SEQ ID NO:9, nucleotides 91-672 of SEQ ID NO:9, nucleotides 25-1629 of SEQ ID NO:1 1, nucleotides 91-939 of SEQ ID NO: 11, nucleotides 25-1593 of SEQ ID NO:13, nucleotides 91-903 of SEQ ID NO: 13, nucleotides 25-1650 of SEQ ID nucleotides 91-960 of SEQ ID NO: 15, nucleotides 25-1644 of SEQ ID NO: 17, nucleotides 91-954 of SEQ ID NO:17, nucleotides 118-1701 of SEQ ID NO:21, nucleotides 184-1011 of SEQ ID NO:21; sequences which, due to the degeneracy of the genetic code, encode a polypeptide encoded by a nucleic acid of and sequences that hybridize under conditions of moderate or high stringency to a sequence of or and that encode a polypeptide that retains at least one ADAMdis activity. The method of one of claim 11-14 wherein the ADAMdis activity is selected from the group consisting of integrin binding activity, inhibition of endothelial cell migration, and inhibition of angiogenesis. 16. The method of one of claims 1-15 wherein the ADAM disintegrin domain polypeptide has been produced by culturing a recombinant cell that encodes the ADAM disintegrin domain polypeptide under conditions permitting expression of the ADAM disintegrin domain polypeptide, and recovering the ADAM disintegrin domain polypeptide. 17. The method of one of claims 1-16 wherein the ADAM disintegrin domain polypeptide is present in a composition comprising a pharmaceutically acceptable carrier. 18. The method of claim 2 wherein the mammal has a disease or condition mediated by angiogenesis. 19. The method of claim 18 wherein the disease or condition is characterized by ocular neovascularization. The method of claim 18 wherein the disease or condition is a solid tumor. 21. The method of one of claims 1-20 wherein the method further comprises treating the mammal with radiation. 22. The method of one of claims 1-21 wherein the method further comprises treating the mammal with a second therapeutic agent. 23. The method of claim 22 wherein the second therapeutic agent is selected from the group consisting of alkylating agents, antimetabolites, vinca alkaloids and other plant-derived chemotherapeutics, antitumor antibiotics, antitumor enzymes, topoisomerase inhibitors, platinum analogs, adrenocortical suppressants, hormones and antihormones, antibodies, immunotherapeutics, radiotherapeutics, and biological response modifiers. 24. The method of claim 22 wherein the second therapeutic agent is selected from the group consisting of cisplatin, cyclophosphamide, bleomycin, carboplatin, fluorouracil, 5-fluorouracil, fluorodeoxyuridine, methotrexate, taxol, asparaginase, vincristine, vinblastine, mechloretamine, melphalan, 5-fluorodeoxyuridine, lymphokines and cytokines such as interleukins, interferons (alpha., beta. or delta.) and TNF, chlorambucil, busulfan, carmustine, lomustine, semustine, streptozocin, dacarbazine, cytarabine, mercaptopurine, thioguanine, vindesine, etoposide, teniposide, dactinomycin, daunorubicin, doxorubicin, bleomycin, plicamycin, mitomycin, L-asparaginase, hydroxyurea, methylhydrazine. mitotane, tamoxifen, fluoxymesterone, and COX-2 inhibitors. The method of claim 22 wherein the second therapeutic agent is a polypeptide, including soluble forms thereof, selected from the group consisting of Flt3 ligand, CD40 ligand, interleukin-2, interleukin-12, 4-1BB ligand, anti-4-1BB antibodies, TRAIL, TNF antagonists and TNF receptor antagonists including TNFR/Fc, Tek antagonists, TWEAK antagonists and TWEAK-R antagonists including TWEAK-R/Fc, VEGF antagonists including anti-VEGF antibodies, VEGF receptor antagonists, CD148 binding proteins, and nectin-3 antagonists. 26. The method of claim 2 wherein the ADAM disintegrin domain is administered parenterally. 27. A method for inhibiting the biological activity of an integrin selected from the group consisting of av3, a 5 ai, as4, and avs5 comprising contacting the integrin with an inhibition-effective amount of an ADAM disintegrin domain polypeptide. 28. The method of claim 27 wherein the integrin is a,3 3 and wherein the ADAM disintegrin domain does not contain an RGD sequence. 29. The method of claim 28 wherein the ADAM is ADAM- 17, ADAM-20, or ADAM-22. The method of claim 27 wherein the integrin is a 2 z 1 and the ADAM is ADAM-23. 31. The method of claim 27 wherein the integrin is a 5 cs and the ADAM is ADAM-21, ADAM-22, or ADAM-23. 32. The method of claim 27 wherein the integrin is a6s3 or a6p4 and the ADAM is ADAM-17, ADAM-22, or ADAM-23. 33. The method of claim 27 wherein the integrin is av3s and the ADAM is ADAM-lO, or ADAM-23. 34. A method for identifying a compound that modulates integrin biological activity comprising: combining a test compound with an integrin and an ADAM disintegrin domain polypeptide that binds to the integrin; and determining whether the test compound alters the binding of the ADAM disintegrin domain polypeptide to the integrin. A method for identifying a compound that modulates the interaction between an integrin and an ADAM disintegrin domain comprising: combining a test compound with the integrin and an ADAM disintegrin domain polypeptide that binds to the integrin; and determining whether the test compound alters the binding of the ADAM disintegrin domain polypeptide to the integrin. 36. The method of claim 34 or 35 wherein the integrin is present on a cell surface. 37. The method of claim 36 wherein the cell is an endothelial cell. 38. The method of one of claims 34-37 wherein the integrin is selected from the group consisting of av3, a a 2 1 3p, ctP, ca 6 P 4 and 39. The method of one of claims 34-38 wherein the integrin biological activity or integrin binding activity is at least partially inhibited. A method for identifying a compound that inhibits endothelial cell migration and/or angiogenesis comprising: combining a test compound with endothelial cells and with an ADAM disintegrin domain polypeptide that binds to endothelial cells; and 4' p determining whether the test compound alters the binding of the ADAM disintegrn domain polypeptide to the endothelial cells. 41. The method of one of claims 34-40 wherein the ADAM disintegrin domain polypeptide comprises an ADAM disintegrin domain from ADAM-8, ADAM-9, ADAM-JO0. ADAM- 15, ADAM- 17, ADA M-20, ADAM-2 1, ADAM-22, ADAM-23, or ADAM-29. 42. The method of claim 41 wherein the ADAM disintegrin domain polypeptide comprises an ADAM disintegrin domain from ADAM-17, ADAM-20, or ADAM-23. SEQUENCE LISTING <110> Irnmunex Corporation Fanslow, William C. Poindexter, Kurt Cerretti, Douglas P. Black, Roy A. <120> IN'rEGRIN ANTAGONISTS <130> 2958-WO <140> <141> <150> 60/184,865 <151> 2000-02-25 <160> 22 <170> Patentln Ver. 2.1 <210> <211> <212> <213> <220> <223> <220> <221> <222> 1 1700 DNA Artificial Sequence Description of Artificial Sequence: fusion polypeptide CDS (118) .(1602) <400> 1 gggttttccc agtcacgacg ttgtaaaacg acggccagtg aattgtaata cgactcacta tagggcgaat tgggtaccgg gccccccctc gaggtcgacc caagctggct agccacc atg gag aca gac Met Glu Thr Asp 1 ctc ctg cta tgg Leu Leu Leu Trp ctg ctg ctc tgg Leu Leu Leu Trp gtt cca Val Pro 165 ggt tcc act Gly Ser Thr cag tgc gac Gln Cys Asp gg t Gly act agt. tgt ggg Thr Ser Cys Gly aac Asn 25 ctg ttt gtg gag Leu Phe Val Glu cgt ggg gag Arg Gly Glu tgc tgc aac Cys Cys Asn tgc ggc ccc ccc Cys Gly Pro Pro gac tgc cgg aac Asp Cys Arg Asn tct. acc Ser Thr acc tgc cag ctg Thr Cys Gln Leu gc t Ala 55 gag ggg gcc cag Glu Gly Ala Gln tgt Cys gcg cac ggt acc Ala His Gly Thr tgc Cys tgc cag gag tgc Cys Gln Glu Cys aag Lys gtg aag ccg gct Val Lys Pro Ala gag ctg tgc cgt Glu Leu Cys Arg 309 357 405 aag aag gac atg Lys Lys Asp Met gac ctc gag gag Asp Leu Glu Glu tgt gac ggc cgg Cys Asp Gly Arg cac cct His Pro gag tgc ccg gaa gac gcc ttc cag gag aac ggc acg ccc tgc tcc ggg Glu Cys Pro Glu Asp Ala Phe Gin Giu Asn Gly Thr Pro Cys Ser Gly 100 gg G1: gcc Ala tat Tyz 145 tgt Cys ata Ile gcg Ala tgc Cys tgc Cys 225 tgc Cys act Thr tgc Cys ctc Leu gag Glu 305 aag Lys aag Lys tac r Tyr -ttc Phe 130 gac *Asp gc Gly tgt Cys tat Tyr tgg Trp 210 tct Ser cac His gag Glu1 cca Pro ttc Phe 290 gtc E Val IJ ttc E Phe I~ ccg c Pro A tg( Cy~ 11 tg5 Trj atc I le gtt Val att Ile gaa Giu 195 aaa Lys gcc Ala tgc Cys gtg Val :cg Pro 275 cc ?ro ~ca hr ~ac ~sn 9gg Lrg -tac STyr 99gg Gly cta Leu *ctg *Leu gtc Val 180 cca Pro gga Gly cag Gin cac His cac His 260 tgc Cys cca Pro tgC Cys1 tgg Trp gag GiuC 340 aac Asr cce PrC cca Pro c aa Gin 165 gac Asp g tg Val cgt Arg tgc Cys gcg Ala 245 gca Ala cca Pro aaa Lys gtg Val1 tac ['yr 325 lag lu 99gg 1Gly ggt Giy ggc Gly 150 *tgt Cys gtg Val ccc Pro tgc Cys cac His 230 ggC Gly gcg Ala gca Ala ccc Pro gtg Val 310 gtg Val cag t Gin gcc Ala ggg Giy 135 tgc Cys aaa Lys tgc Cys gag Giu cag Gin 215 aac Asn tgg Trp tcc Ser cct Pro aag Lys 295 Itg /al jac ~sp :ac z .'yr I tgt Cys 120 cag Gln aag Lys gg t G ly c ac His ggc Giy 200 gac Asp cat His gcC Ala ggg Giy gaa Glu 280 gac A.sp gac Asp ;gc iac ~sn 105 ccc Pro gct Ala gcc Ala gg t Gly gcg Ala 185 acc Thr t ta Leu ggg Gly ccg Pro aga Arg 265 gcc Ala acc Thr gtg Val gtg S Val C agc a Ser '1 345 110 aca ctg gcE Thr Leu Al, gcC Ala agc Sex caa Glr 170 c tc Leu cgg Arg cac His gtg Val ccc Pro 250 tct Ser gag G1u ctc xgc 3er jag ;lu ~30 icg hr gag Glu cgg Arg 155 caa Gin acc Thr tgt Cys gtt Val1 tgc Cys 235 cac His tgt Cys ggc Gly atg Met cac His 315 gtg Val tac c Tyr ga~ G1~ 14( tac cct Prc ace Thy. gga Gli tac 220 aac Asn tgc Cys gac Asp gcg Ala atc Ile 300 gaa .lu -a t iis ~gt ~rg ccag a Gin 125 j tcc .i Ser 01 agg Arg tta Leu gag Giu cca Pro 205 aga *Arg *cac His gcg Ala aaa Lys ccg Pro 285 tcc Ser gac Asp aat Asn I gtg E Val 3 ca G12 tg Cy gc Al ggt Glj ga t Asr 190 gag Giu tcC Ser aag Lys aag Lys act Thr 270 tca Ser C9g krg ct Pro ;cc itc ra1 gtgc n~ Cys *ttc Phe :gac iAsp aga rArg 175 ggc Gly aag Lys agc Ser cag Gin c tg Leu 255 cac His gtc Val acc Thr gag Giu aag Lys 335 agc Ser N cag Gin tcc Ser atg Met 160 gc t Ala act Thr gtt Val1 aac Asn gag Giu 240 c tg Leu aca Thr ttc Phe cct Pro g tc 61a1 320 5Lca P'hr t c tal 501 549 597 645 693 741 789 837 885 933 981 1029 1077 1125 1173 1221 ctc acc gtc ctg Leu Thr Val Leu 355 cac His cag gac tgg Gin Asp Trp 360 ctg aat ggc aag Leu Asn Gly Lys gag Giu 365 tac aag tgc Tyr Lys Cys aag gtc Lys Val 370 aaa gcc Lys Ala 385 tcc cgg Ser Arg tcc aac aaa Ser Asn Lys aaa. ggg cag Lys Gly Gin gcc ctc Ala ccc Pro 390 acc Thr Leu 375 cga gaa Arg Glu aag aac Lys Asn 380 cca cag gtg tac Pro Gin Val Tyr 395 cag gtc agc ctg Gin Val Ser Leu acc ctg ccc Thr Leu Pro cca Pro 400 cca gcc ccc atc gag aaa acc atc tcc Pro Ala Pro Ile Glu Lys Thr Ile Ser gag gag Glu Glu atg Met 405 ccc Pro acc tgc Thr Cys 410 gtg Val1 ctg gtc Leu Val 415 aaa ggc ttc Lys Giy Phe cag ccg gag Gin Pro Giu. 435 ggc tcc ttc Gly Ser Phe 450 cag cag ggg Gin Gin Gly 465 tat Tyr 420 aac Asn agc gac atc Ser Asp Ile gcc Ala 425 acg Thr gag tgg gag Giu Trp Glu aac tac aag Asn Tyr Lys acc Thr 440 cct ccc gtg Pro Pro Val agc aat ggg Ser Asn Gly 430 gac tcc gac Asp Ser Asp agc agg tgg Ser Arg Trp 1269 1317 1365 1413 1461 1509 1557 1602 1662 1700 ttc ctc tat agc Phe Leu Tyr Ser 455 aac gtc ttc tca Asn Val Phe Ser 470 aag ctc acc gtg gac Lys Leu Thr Val Asp 460 tgc tcc gtg atg cat Cys Ser Val Met His 475 gag gct ctg Glu Ala Leu cac His 480 aac cac tac acg cag aag agc ctc tcc ctg tct Asn His Tyr Thr Gin Lys Ser Leu Ser Leu Ser 485 490 actagagcgg ccgccaccgc ggtggagctc cagcttttgt ttcgagcttg gcgtaatcat ggtcatagct gtttcctg ccg ggt aaa tga Pro Giy Lys 495 tccctttagt gagggttaat <210> 2 <211> 494 <212> PRT <213> Artificial Sequence <223> Description of Artificial polypeptide Sequence: fusion <400> 2 Met Giu Thr 1 Gly Ser Thr Gin Cys Asp Ser Thr Thr Cys Cys Gin Lys Lys Asp Giu Cys Pro Gly Tyr Cys 115 Ala Phe Trp 130 Tyr Asp Ile 145 Asp Gly Cys Cys Glu Met Giu 100 Tyr Gly Leu Thr Thr Gly Gin Cys Cys Asp Asn Pro Pro Gin 165 Leu Ser Pro Leu Lys 70 Asp Ala Gly Gly Gly 150 Leu Cys Pro Ala 55 Val1 Leu Phe Ala Gly 135 Cys Leu Gly Giu 40 Giu Lys Giu Gin Cys 120 Gin Lys Trp Val Leu Leu Leu Trp Val Pro Asn 25 Asp Gly Pro Giu Glu 105 Pro Ala Ala Leu Cys Ala Ala Phe 90 Asn Thr Ala Ser Gin 170 Phe Arg Gin Gly 75 Cys Gly Leu Giu Arg 155 Val1 Asn Cys Glu Asp Thr Ala Giu 140 Tyr Glu Arg Ala Leu Gly Pro Gin 125 Ser Arg Arg Cys His Cys Arg Cys 110 Gin Cys Ala Gly Cys Gly Arg His Ser Cys Phe Asp Arg, 175 Glu Asn Thr Pro Pro Gly Gln Ser Met 160 Ala Cys Gly Val Leu Cys Lys Gly Gly Gin Pro Leu Gly Ile Ala Cys Cys 225 Cys Thr Cys Leu G lu. 305 Lys Lys Leu Lys Lys 385 Ser Lys Gin Gly Gin 465 Asn Cys Tyr Trp 210 Ser His G iu Pro Phe 290 Val Phe Pro Thr Val 370 Ala Arg G ly Pro Ser 450 Gin His Ile Giu. 195 Lys Ala Cys Val Pro 275 Pro Thr Asn Arg Val 355 Ser Lys Glu Phe Glu 435 Phe 3 ly T'yr Val 180 Pro Gly Gin His His 260 Cys Pro Cys Trp Glu 340 Leu. Asn Gly Giu Tyr 420 Asn Phe Asn Thr Asp Val Arg Cys Ala 245 Ala Pro Lys Val1 Tyr 325 Glu. His Lys Gin Met 405 Pro Asn Leu Val1 Gin 485 Val Pro Cys His 230 Gly Ala Ala Pro Val 310 Val Gin Gin Ala Pro 390 Thr Ser Tyr Tyr Phe 470 Lys Cys His Ala 185 Glu. Gly Thr 200 Gin Asp Leu. 215 Asn His Giy Trp Ala Pro Ser Gly Arg 265 Pro Glu Ala 280 Lys Asp Thr 295 Val Asp Val Asp Gly Val Tyr Asn Ser 345 Asp Trp Leu 360 Leu Pro Ala 375 Arg Giu Pro Lys Asn Gin Asp Ile Ala 425 Lys Thr Thr 440 Ser Lys Leu 455 Ser Cys Ser Ser Leu Ser Arg Cys His Val Val Cys 235 Pro His 250 Ser Cys Giu Gly Leu. Met Ser His 315 Glu Val 330 Thr Tyr Asn Gly Pro Ile Gin Val 395 Val Ser 410 Val Giu Pro Pro Thr Val Val Met 475 Gly Tyr 220 Asn Cys Asp Ala Ile 300 Giu His Arg Lys Giu. 380 Tyr Leu Trp Val1 Asp 460 His Pro 205 Arg His Ala Lys Pro 285 Ser Asp Asn Val Glu 365 Lys Thr Thr Giu Leu 445 Lys Glu Leu Thr Thr Glu Asp Gly Thr 190 Giu Lys Val Ser Ser Asn Lys Gin Giu 240 Lys Leu Leu 255 Thr His Thr 270 Ser Val Phe Arg Thr Pro Pro Glu Vai 320 Ala Lys Thr 335 Val Ser Val 350 Tyr Lys Cys Thr Ile Ser Leu Pro Pro 400 Cys Leu Val 415 Ser Asn Gly 430 Asp Ser Asp Ser Arg Trp Ala Leu His Ser Pro Gly Lys <210> 3 <211> 1668 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: fusion polypeptide <220> <221> CDS <222> (1647) <400> 3 ggtaccgggc cccccctcga ggtcgaccca agctggctag ccacc atg gag aca gac 57 Met Glu Thr Asp aca ctc ctg cta tgg gta ctg ctg ctc tgg gtt cca ggt tcc act ggt 105 Thr Leu Leu Leu Trp Val Lau Leu Leu Trp, Val Pro Gly Ser Thr Gly 10 15 act agt tgt ggt aat aag ttg gtg gac gct ggg gaa gag tgt gac tgt 153 Thr Ser Cys Gly Asn Lys Leu Val Asp Ala Gly Glu Giu Cys Asp Cys 30 ggt act cca Gly Thr Pro aag gaa Lys Glu tgt gaz Cys G11L ttt gct Phe Ala tgt aag ctt aaa tca I Cys gac Asp gag Giu cca Pro tat Tyr atc Ile gtg Vai gaa Giu 165 tgt Cys att Ile cta LeuC tgt Cys C gtt C Val L 245 gta t Val C cca a Pro A Lys tgt Cys tgt Cys ga t Asp tgc Cys ttt Phe aat Asn 150 tac Tyr gag Giu caa Leu cgg Arg ga t Asp gtt Val1 tac Tyr ggc Gly 135 tc t Ser aag Lys aat Asn1 acg Lys t tc Phe gtt Val ttt Phe aac Asn 120 tca Ser aaa Lys dag Lys gta Val cct Ser ctt Leu cca Pro att Ile 105 ggc Gly aaa Lys ggt Gly tgt Cys caa Gin 185* agt CCE Prc gag Giu 90 cag Gin atg Met gcc Ala gac Asp gcc Ala 170 gag Giu cga gga Giy 75 tac Tyr aat Asn tgc Cys aag Lys aga Arg 155 act Thr ata Ile ggc Gly gat Asp tgt Cys I t t Lei ga~ ggt G11 tgC Cys gga Gly cag Gin gc t Ala 140 ttt Phe ggg Gly cct Pro acc I gac cct i Asp Pro 45 1 tgt gca aCys, Ala act tta Thr Leu aat ggt Asn Gly tat cct Tyr Pro 110 tat tat Tyr Tyr 125 gcc ccc Ala Pro ggc aat Gly Asn aat gct Asn Ala gta ttt Val Phe 190 aaa tat tgC CyE tat Tyr tgc Cys tct Ser 95 tgc Cys gat Asp aaa Lys tgt Cys t tg Leu 17 5 iga 'rp rtt al1 'ag in ag ys 55 aa iu gt S er N tgc gaa gga Cys Glu Gly 50 ggt gac tgt Gly Asp Cys 65 cga gga aaa Arg Gly Lys 80 tct cag ttc Ser Gin Phe cag aat aac Gin Asn Asn gct caa tgt Ala Gin Cys 130 gat tgt ttc Asp Cys Phe 145 ggt ttc tct Gly Phe Ser 160 tgt gga aag Cys Gly LysI att gtg cct Ile Val Pro Pa agt acc Ser Thr tgt aaa Cys Lys acc agt Thr Ser tgt cag Cys Gin 100 aaa gcc Lys Ala 115 caa gtc Gln Val att gaa Ile Giu ggc aat 31y Asn -tt cag ',eu Gin 180 ~ct att lia Ile 201 249 297 345 393 441 489 537 585 633 681 729 777 825 873 921 31n Thr Pro Ser Arg ;ga rgt ;iy :tg ~eu gt 'ys at sn tca Ser 215 gc t Ala 200 ga t Asp gga Gly gtt Val1 aag Lys cca Pro atc Ile rhr Lys Cys 205 :ct ggg atg ro Gly Met ~20 ~ga aac ttc ~rg Asn Phe 235 'I C G a L 2 g G a! SI t C 2 t 1gt gtg 'iy Vai lac gaa ~sn Giu 225 gt gta :ys Vai ga t Asp 210 ggc Gly gat Asp Phe aca Thr gc t Ala Gin aaa Lys tc t Ser 195 ttc cag aat tat Asn Tyr aat agc Asn Ser tgt gag Cys Glu 280 gat Asp aat Asn gga Giy gt t Val1 tgt Cys tac Tyr cag Gin cac His gga Gly 285 aaa Lys tgt Cys 270 gga Gly gt ys at sn tg al cat His ggc Gly gac Asp gga Gly tgg Trp agt Ser 290 cat His gc t Ala 275 gga Gly ggg Gly 260 ccc Pro cc t Pro aca tac aat gaa atg aat act gca ttg agg gac gga tct tgt gac aaa Thr act Thr tca Ser 325 egg Arg cct Pro gce Ala gtc Val1 tac Tyr 405 acc Thr c tg Leu tgC Cys age Ser gac Asp 485 age Ser gc t Ala Tyr eac His 310 gte Val1 acc Thr gag Glu aag Lys agc Ser 390 aag Lys ate Ilie ccc Pro ctg Leu aa t Asn 470 tcc Ser agg Arg ctg Asn 295 aca Thr ttC Phe cct Pro gtc Val1 aca Thr 375 gte Val1 tgC Cys tCC Ser cca Pro gtc Val1 455 ggg Gly gac Asp tgg Trp cac Glu Met Asn Thr Ala Leu Arg Asp Gly Ser Cys Asp Lys tgC Cys etc Leu gag Glu aag Lys 360 aag Lys etc Leu aag Lys aaa Lys tcc Ser 440 aaa Lys cag Gin ggc Gly cag Gin aac cca ccg Pro Pro ttc ccc Phe Pro 330 gte aca Val Thr 345 ttc aac Phe Asn ccg cgg Pro Arg ace gtc Thr Val gtC tcc Val Ser 410 gcc aaa Ala Lys 425 cgg gat Arg Asp ggc ttc Gly Phe ccg gag Pro Glu tce ttc Ser Phe 490 cag ggg Gin Gly 505 cac tac tgC Cys 315 cca Pro tgc Cys tgg Trp gag Glu ctg Leu 395 aac Asn ggg Gly gag Glu tat Tyr a ac Asn 475 ttc Phe aac Asn acg 300 ca Pro aaa Lys gtg Val tac Tyr gag Glu 380 cac His aaa Lys eag Gin ctg Leu ccc Pro 460 aac Asn etc Leu gte Val cag gca Ala ccc Pro gtg Val gtg Val1 365 cag Gin cag Gin gce Ala ccc Pro acc Thr 445 agc Ser tac Tyr tac Tyr ttc Phe aag Lys 525 cc t Pro aag Lys gtg Val 350 gac Asp tac Tyr gac Asp etc Leu cga Arg 430 aag Lys gac Asp aag Lys age Ser tea Ser 510 age Ser gaa Glu gac Asp 335 gac Asp ggc Gly aac Asn tgg Trp cca Pro 415 gaa Glu aae Asn ate Ile ac Thr aag Lys 495 tge Cys etc Leu gee Ala 320 ac Thr gtg Val gtg Val1 age Ser e tg Leu 400 gee Ala cca Pro cag Gin gee Ala aeg Thr 480 etc Leu tee Ser tee Ser 305 gag Giu etc Leu agc Ser gag Giu aeg Thr 385 aat Asn ccc Pro cag Gin gte Val1 gtg Val 465 ce t Pro ace Thr gtg Val etg Leu ggc geg Gly Ala atg ate Met Ile cac gaa His Glu 355 gtg eat Val His 370 tac egg Tyr Arg ggc aag Gly Lys ate gag Ile Glu gtg tac Val Tyr 435 age etg Ser Leu 450 gag tgg Glu Trp Ccc gtg Pro Val gtg gac Val. Asp atg cat Met His 515 tet eeg Ser Pro eeg Pro tee Ser 340 gac Asp aat Asn gtg Val gag Giu aaa Lys 420 ace Thr ace Thr gag Giu Ctg Leu aag Lys 500 gag Giu ggt Gly 1017 1065 1113 1161 1209 1257 1305 1353 1401 1449 1497 1545 1593 1641 1668 Leu His Asn His Tyr Thr Gin Lys <210> 4 <211> 533 <212> PRT <213> Artificial Sequence <223> Description of Artificial polypeptide Sequence: fusion <400> 4 Met 1 Gly Glu Glu Asp Gly Gin Asn Gin Cys 145 Phe Gly Val Val Glu 225 Val J His C Gly Asp S 2 Ser C 305 Glu G Leu M Ser H Glu V 3 Thr 'r 385 Asn G Pro I: Gin V Gli Se CYr Gil Cy LyE PhE Asr Cys 130 Phe Ser Lys Pro ksp 210 ,ly ksp ;ly rp ;er .90 ,ys ly et is al 70 yr ly le al Thr r Thr Asp r Ser 3 Cys Thr Cys Lys 115 Gin Ile Gly Leu Ala 195 Phe I Thr Ala His Ala I 275 Gly I Asp I Ala P Ile S 3 Glu A 355 His A Arg V Lys G Glu L 4 Tyr T' 435 As Gi 2 Cy Th Ly Se Gli Ale Val Gl. Asr Gin 180 Ile Gln Lys Ser 31y 260 ?ro ?ro lys ~ro er 40 ~sp sn al lu ys 20 hr p Thr 5 y Thr 0 s Gly Cys s Asp Glu i Pro 0 i Tyr Ile Val Glu 165 Cys Ile Leu I Cys Val 245 Val Pro I Thr 'J Thr F 3 Ser 1v 325 Arg T Pro G Ala L Val S 3 Tyr L 405 Thr I Leu P Le Se Th Ly: Cy: 7' Cy As; Cys Phe Asr 15C Tyr Glu Gin Gly Gly 230 Leu -ys ksn 'yr lis 10 ra1 'hr lu ys er 90 ys le ro u Le r Cy r Pr s Le 5 s Ar s As Va 3Ty s Gil 13 i Se Lys Asr Thi Ser 215 Ala Asn Asn Cys Asn 295 Thr Phe Pro Va1 Thr 375 Va1 Cys Ser Pro *u Leu s Gly o Lys 40 u Lys 5 g Phe p Val 1 Phe r Asn 120 Ser r Lys 3 Lys 1 Val Pro 200 Asp Gly Tyr Ser Glu 280 Glu P Cys I Leu I Glu 'v 3 Lys P 360 L y s P Leu T Lys V Lys A 4 Ser A 440 Tr As 2 G1 Se Le' Pre 11( 10 Gi] Lys Gl) Cys Gin 185 Ser Val Lys Asp ksn 265 hr 4et ?ro ,he ral '45 'he 'ro 'hr al la 25 rg p Va 1 n Ly 5 u Cy r Ph Pr 3 Gb1 9' e Gl Me 3 Ai #As; SAle 17C Gli. Arc Prc Ile Cys 250 Lys Lys Asn Pro Pro 330 Thr Asn Arg Va1 Ser 410 Lys Asp 1 Leu 0 s Leu s Glu e Ala o Gly 75 u Tyr 0 n Asn t Cys i Lys Arg 155 Thr 1 Ile Gly Asp Cys 235 Asp Asn Gly Thr I Cys I 315 Pro I Cys IV Trp 'I Glu G 3 Leu H 395 Asn L Gly G Glu L Le Va Le G1 6 G1 Cy: Gl1 Glr Ale 14( Ph Gil Pro Thr Pro 220 Arg V1al :ys 'yr kla )00 'ro ys ral 'yr lu is ys In eu u Leu 1 Asp u Asp Cys 0 y Thr s Asn I Tyr 1 Tyr 125 i Ala Gly Asn Val Lys 205 Gly Asn Gin I His Gly C 285 Leu I Ala I Pro L Val V .3 Val A 365 Gin r Gin A Ala L Pro A 4 Thr L 445 Tr Al 3 Pr Ali Le Gi] Pr 11 Ty: Prc Asr Ala Phe 190 Cys Met Phe Lys ys ;ly Lrg 'ro ,ys ral sp yr sp eu rg ss p Va 1 a Gi 0 o Cy Ty Cy Se 9' Cyr As; Lyt 1 Cys Leu 175 i Gl Trp Val Gin Lys 255 Glu Ser Asp Glu Asp 335 Asp Gly Asn Trp Pro 415 Glu Asn 1 Pro S y Glu s Cys r Gly s Arg r Ser s Gin Ala Asp Gly 160 I Cys Ile Gly Asn Cys 240 Cys Asn Val Gly Ala 320 Thr Va1 Val Ser Leu 400 Ala Pro Gin Val Ser 450 Leu Thr Cys Leu Val Lys Gly Phe Tyr 455 Pro 460 Ser Asp Ile Ala Val Glu, Trp Glu 465 Pro Pro Val Leu Ser Asn Gly 470 Asp Ser Asp Gin Pro Glu Asn Asri 475 Phe Leu Tyr Lys Thr Thr 480 Gly Ser Tyr Ser 485 Ser Lys Leu 495 Thr Val Asp Val Met His Arg Trp Gin Gin 505 His Asn Val'Phe Ser Cys Ser 510 Ser Leu Ser Ala Leu His Asn 520 Tyr Thr Gin 515 Leu, Ser Pro Gly Lys 530 Lys 525 <210> <211> 1443 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: fusion polypeptide <220> <221> CDS <222> (25)..(1422) <400> gtcgacccaa gctggctagc cacc atg gag aca. gac aca ctc ctg cta tgg Met Glu Thr Asp Thr Leu Leu Leu Trp gta Val ctg ctg ctc tgg Leu Leu Leu Trp gtt Val1 cca. ggt tcc act Pro Gly Ser Thr act agt tgt gga Thr Ser Cys Gly gga, atg gta gaa Gly Met Val Glu ggt gaa. gaa.tgt Gly Glu Glu Cys ga t Asp tgt ggc tat agt Cys Gly Tyr Ser gac cag Asp Gin tgt aaa. gat Cys Lys Asp tgc aaa ctg Cys Lys Leu gaa Glu tgc tgc ttc gat Cys Cys Phe Asp aat caa cca gag Asn Gin Pro Glu gga aga. aaa Giy Arg Lys ggt cct tgt Gly Pro Cys aaa. ect ggg aaa Lys Pro Gly Lys cag Gin 65 tgc agt cca. agt Cys Ser Pro Ser caa Gin tgt aca Cys Thr gca cag tgt gca, Ala Gin Cys Ala t tc Phe aag tca aag tct Lys Ser Lys Ser aag tgt cgg gat Lys Cys Arg Asp 195 243 291 339 387 gat Asp tca gac tgt gca. Ser Asp Cys Ala agg Arg 95 gaa gga ata, tgt Glu Gly Ile Cys aa t Asn 100 ggc ttc aca. gct Gly Phe Thr Ala tgc cca gca tct Cys Pro Ala Ser gac Asp 110 cct aaa. cca aac Pro Lys Pro Asn aca gac tgt aat Thr Asp Cys Asn agg cat Arg His 120 aca caa gtg Thr Gin Val tgc Cys 125 att aat ggg caa, Ile Asn Gly Gin tgt Cys 130 gca ggt tct atc Ala Gly Ser Ile tgt gag aaa Cys Glu Lys 135 aaa. gat gat Lys Asp Asp tat ggc tta. gag gag tgt acg tgt Tyr Gly Leu Glu Glu Cys Thr Cys 140 145 gcc agt tct gat Ala Ser Ser Asp aaa gaa tta tgc cat gta. tgc tgt atg aag aaa atg gac cca tca act Lys Glu Leu Cys His Val Cys Cys Met Lys Lys Met Asp Pro Ser Thr 155 tgl Cy~ 17( acc Th2 tgt CyE gct Ala gct Ala cct Pro 250 aag Lys gtg Val gac Asp tac Tyr gac Asp 330 ctc Leu cga. Arg tgcc agt aca s Ala Ser Thr atc acc ctg le Thr Leu gat gtt ttc Asp Val Phe 205 agg ctt aaa Arg Leu Lys 220 *gaa aga tct *Giu Arg Ser 235 gaa gcc gag Glu Ala Giu gac acc ctc Asp Thr Leu gac gtg agc Asp Val Ser 285 ggc gtg gag Gly Val Giu 300 aac agc acg Asn Ser Thr 315 tgg ctg aat Trp Leu Asn cca gcc cccz Pro Ala Pro gaa cca cag q Glu Pro Gin Iv GI' ca GlI 19( atc Met aae LyE tgt Cys ggc Gly atg Met 270 cac His gtg V'al tac T'yr Jgc ltc le 150 ~tg al1 g tct y Ser 175 I cct 'I Pro 0 Arg gca Ala gac Asp gcg Ala 255 ate Ile gaa Glu cat His cgg Arg aag LysC 335 gag a Glu z tac a Tyr TI gt~ Va gga GI~ tgC Cys att I le aaa Lys 240 ccg Pro tcc Ser gac Asp aa t Asn gtg la 1 320 jag ;lu ~aa Ays Icc ~hr j cag IGin I tcC r' Ser aga Arg ttt Phe 225 act Thr tca Ser cgg Arg cct Pro gcc Ala 305 gtc i Val tac a Tyr L acc a Thr I ctg c Leu P tgg Trp cct Pro t ta Leu 210 agt Ser cac His gtc Vai ac c Thr gag Glu 290 Fag -,ys ~gc er ~agt JySC 1tc t le S 3 cc c ro P agi Se2 tg( Cysa 191 gte Val cca Pro aca Thr t tc Phe cct Pro 275 gtc Val aca rhr jtC Iai :gc .YS :cc er '55 ca ro tagg r Arg 180 aac Asn gat Asp gag Giu tgC Cys c to Leu 260 gag Glu aag1 Lys aag Lys ctc z Leu 'J aag g Lys 340 aaa g Lys A tcc c Ser A aaa g 165 cac His gat Asp gc t Ala ctc Leu cca Pro 245 t tc Phe gtc Val ttc Phe ::cg Pro ~cc rhr '1 125 rtc t ~al S t t( Phc ttt Phe gat Asp tat Tyr 230 cog Pro ccc Pro aca Thr aac Asn krg 310 ;tc Tal :cc er agi SSe, age ~Arc ggt Giy 215 ga Giu tgc Cys cca Pro tgc Cys tgg Trp 295 gag Glu Ctg Leu aac Asn Gil G1l ct Prc *aac *Asn Pro aaa Lys gtg Val 280 tac Tyr gag Glu cac His aaa Lys t. cga. SArg 185 tac r Tyr cta Leu att Ile gca 'Ala ccc Pro 265 gtg Val gtg Val cag Gin cag Gin gcc Ala 579 627 675 723 771 819 867 915 963 loll 1059 1107 1155 1203 1251 1299 rcc aa la Lys gg gat rg Asp gc ttc ggg cag ccc Gly gag Giu 375 tat Gin 360 ctg Leu ccc Pro acc Thr agc 365 gtc agc Val Ser aag aac cag Lys Asn Gin gac 380 ctg acc tgc ctg gtc Leu Thr Cys 385 Leu Val Lys Gly Phe Tyr Pro Ser 390 acto Asp aag atc Ile 395 acc gcc gtg gag tgg gag agc aat Ala acg Val1 cc t Glu ccc Trp gtg 400 otg Ser gac Asn tcc Gin 9gC Asn Tyr ctc tac Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr 410 415 420 425 agc aag ctc acc gtg gac aag agc Ser Lys Leu Thr Val Asp Lys Ser 430 tca tgc tcc gtg atg cat gag gct Ser Cys Ser Val Met His Glu Ala 445 agc ctc tcc ctg tct ccg ggt aaa Ser Leu Ser Leu Ser Pro Gly Lys 460 465 <210> 6 <211> 465 <212> PRT <213> Artificial Sequence <223> Description of Artificial polypeptide agg tgg cag cag ggg aac gtc ttc Arg Trp Gin Gin Gly Asn Val Phe 435 440 ctg cac aac cac tac acg cag aag Leu His Asn His Tyr Thr Gin Lys 450 455 tga actagagcgg ccgctacaga t Sequence: fusion 1347 1395 1443 <400> 6 Met Gly Glu Asp Gin Lys Gly Pro Gin Cys 145 Cys Gin Ser Arg Phe 225 Thr Ser Arg Pro Ala 305 Val Giu Ser Cys Ala Cys Ser Ile Asn Cys 130 Ala Met Trp, Pro Leu 210 Ser His Val Thr Glu 290 Lys Ser Thr Thr Asp Asn Ser Lys Cys Phe 115 Ala Ser Lys Ser Cys 195 Val1 Pro Thr Phe Pro 275 Val1 Thr VJal Asp Gly Cys Gin Pro Ser Asn 100 Thr Giy Ser Lys Arg 180 Asn Asp Glu Cys Leu 260 Glu Lys Lys Leu Thr Thr Sly Pro Ser Glu Sly Asp Ser Asp Met 165 His Asp Ala Leu Pro 245 Phe Val1 Phe Pro Thr 325 Leu Ser TPyr Glu Gin .70 Lys Phe Cys Ile Sly 150 Asp Phe Phe Asp Tyr 230 Pro Pro Thr Asn Arg 310 Val Leu Cys Ser Gly 55 Gly Cys Thr Asn Cys 135 Lys Pro Ser Arg Gly 215 Glu Cys Pro Cys Trp 295 Glu Leu. Leu Gly Asp 40 Arg Pro Arg Aia Arg 120 Glu Asp Ser Gly Sly 200 Pro Asn Pro Lys Val1 280 Tyr Glu. His Trp Asn 25 Gin Lys Cys Asp Leu 105 His Lys Asp Thr Arg 185 Tyr Leu Ile Aia Pro 265 Val Val1 Gin Gin Val 10 Sly Cys Cys Cys Asp 90 Cys Thr Tyr Lys Cys 170 Thr Cys Ala Ala Pro 250 Lys Val Asp Tyr Asp 330 Leu Met Lys Lys Thr 75 Ser Pro Gin Gly Glu 155 Ala Ile Asp Arg Slu 235 Glu Asp Asp Gly Asn 315 Trp, Leu Val1 Asp Leu Ala Asp Ala Val1 Leu 140 Leu Ser Thr Val Leu 220 Arg Ala Thr Val1 Val1 300 Ser Leu Leu Giu Glu Lys Gin Cys Ser Cys 125 Glu Cys Thr Leu Phe 205 Lys Ser Glu Leu Ser 285 Giu Thr Asn Trp Gin Cys Pro Cys Ala Asp 110 Ile Glu His Sly Gin 190 Met Lys Cys Gly Met 270 His Val1 Tyr Giy Val Sly Cys Gly Ala Arg Pro Asn Cys Val Ser 175 Pro Arg Ala Asp Ala 255 Ile Glu His Arg Lys 335 Pro Glu Phe Lys Phe Slu Lys Gly Thr Cys 160 Val Sly Cys Ile Lys 240 Pro Ser Asp Asn Val 320 Giu Tyr Lys Cys Lys 340 Thr Ile Ser Lys 355 Leu Pro Pro Ser Val Ser Asn Lys Ala Lys Gly Gin 360 Arg Asp Glu Leu 375 Gly Phe Tyr Pro Ala Leu 345 Pro Ala Pro Ile Giu Lys Pro Thr Ser Arg Giu Pro Gin 365 Lys Asn Gin Val 380 Asp Ile Ala Val Val Tyr Thr Ser Leu Thr 370 Cys Leu 385 Ser Asn Vai Lys Giu Trp 390 Gly Gin Pro Giu 395 Thr Giu 400 Leu Asn Asn Tyr Thr Pro Pro 405 Ser Vai 415 Asp,,$er Asp Ser Arg Trp 435 Ala Leu His 450 Lys 465 <210> 7 <211> 1638 <212> DNA Gly 420 Gin Phe Phe Leu Lys Leu Thr Asp Lys His Glu Gin Gly Asn Val 440 Gin Ser Cys Ser Vai 445 Leu Asn His Tyr Thr 455 Lys Ser Leu Ser Pro Gly <213> <220> <223> <220> <221> <222> Artificial Sequence Description of Artificial Sequence: fusion polypeptide CDS (41)..(1609) <400> 7 cgggcccccc ctcgaggtcg acccaagctg gctagccacc atg gag aca gac Met Giu Thr Asp 1 aca Thr ctc ctg cta tgg gta ctg Ctg ctc Leu Leu Leu Trp Vai Leu Leu Leu tgg gtt Trp Val 15 cca ggt tcc act Pro Gly Ser Thr ggt act Gly Thr agt tgc gga Ser Cys Gly ttc ctg gat Phe Leu Asp atg ttt gtg gag Met Phe Val Giu ggc gag cag tgt Gly Glu Gin Cys gac tgt ggc Asp Cys Gly acc tgc cag Thr Cys Gin gac tgc gtc gat Asp Cys Val Asp tgc tgt gat tct Cys Cys Asp Ser ctg agg Leu Arg cca ggt gca cag Pro Gly Ala Gin gca tct gac gga Ala Ser Asp Gly tgt tgt caa aat Cys Cys Gin Asn tgc Cys cag ctg cgc ccg Gin Leu Arg Pro te t Ser 75 ggc tgg cag tgt Gly Trp Gin Cys cgt Arg cct acc aga ggg Pro Thr Arg Gly 247 295 343 tgt gac ttg cct Cys Asp Leu Pro gaa Giu ttc tgc cca gga Phe Cys Pro Gly agc tcc cag tgt Ser Ser Gin Cys ccc cct Pro Pro 100 gat gtc agc Asp Vai Ser cta Leu 105 ggg gat ggc gag Gly Asp Gly Giu tgc gct ggc ggg Cys Ala Gly Gly caa gct gtg Gin Ala Val 115 tgc atg cac ggg cgt tgt gcc tgcatgcacgggcgttgtgcctcc tat gcc cag cag tgc cag tca ctt Cys Met His Gly Arg Cys Ala Ser Tyr Ala Gin Gin 120 125 Gin Ser Leu tgg Trp aa t Asn 150 agt Ser tgc Cys C tC Leu gtg Val1 cct Pro 230 cag Gin gga Gly tgg Trp tctt Ser gag g Giu G~ 310 ctc a Leu M~ agc c Ser H gag g Giu V acg t, gga Gly 135 act Thr tat Tyr cag Gin tgg Trp cac His 215 ggc Gly cgt Arg cat H1isC 1ca c kia 1 2 :gt g ~ys A rgc g ;iy A .tg a [et I ac g is G tg ci al H: 3' ac c( ccl Pr cg~ ArS gt Val aca Thr gag Glu 200 Ctg Leu ace rhr gtg la 1 ,iy :cc 'ro 80 rac .sp cg l1a tc aa lu at is 60 t gga )o Gly ;gga ;Gly tcc *Ser ggt *Gly 185 aca Thr gac Asp gcc Ala gat Asp gtc Val 265 cct Pro I aaa a Lys T1 Ccg t Pro S tcc c Ser A gC Al aa As) tg4 Cy 17( ag~ Ar~ a te I lf ctq Leu tgt Cys ctc Leu 250 :g t :ys jac ~sp Lct ~hr ca er gg C a t n c cag ccc Gin Pro 140 gc Al~ 15~ acc Thi tttt i Phe cct Pro jacc cac I Thr Gir Igat gtS Asp Val ggc agt Gly Sex 220 *ggc cct *Gly Pro 235 ctg ggg Leu Gly gac agc Asp Ser tgc aoc Cys Thr cac aca His Thr 300 gtc ttc Val Phe 315 acc cct 1 A.le ggS G1 aga Arg cc t Pro aa t Asn 205 ga t Asp ggc Gly gca Ala aac Asn act rhr 285 tgC Ala Pro Leu Cys Leu 145 agc tgt ggg cgc aac Ser Cys Gly Arg Asn 160 gat gcc att tgt ggg Asp Ala Ile Cys Giy 175 ctg ctg ggc tcc atc Leu Leu Giy cr Ile 190 ggg act gag ctg aac Gly Thr Glu Leu Asn 210 gtg gcc cag ccc ctc Vai Ala Gin Pro Leu 225 ctg gtg tgt ata gac Leu Val Cys Ile Asp 240 cag gaa tgt cga agc Gin Glu Cys Arg Ser 255 agg cac tgc tac tgt Arg His Cys Tyr Cys 270 cag ctc aaa gca acc Gin Leu Lys Ala Thr 290 cca ccg tgc cca gca c I gct gcg cca ctt tgc ctc cag aca Gin Thr ccc agt Pro Ser cag ctc Gin Leu 180 cgg gat Arg Asp 195 tgc agc Cys Ser ctg act Leu Thr cat cga His Arg aaa tgc Lys Cys 260 gag gag 31u Glu 275 Igc tc gc t Ala ggc Gly 165 cag Gin cta Leu tgg Trp c tg Leu tgc Cys 245 cat Hs iga 487 535 583 631 679 727 775 823 871 919 967 1015 1063 1111 1159 1207 3er Scr Arg ct Cys Pro ctc Leu gag Giu t tc Phe gtc Val Pro Cys Pro 305 ccc cca aaa Pro Pro Lys 320 aca tgc gtg Thr Cys Vai 335 aac tgg tac Ala ccc Pro gtg Val gtg rg Thr Pro Pro aag Lys gtg Val1 gac Asp 355 tac Tyr gac gaa gcc Glu Ala gac acc Asp Thr 325 gac gtg Asp Val 340 ggc gtg Gly Val aac agc Asn Ser tgg ctg gac cct gag gtc aag ttc Asp Pro Giu Val Lys Phe 345 350 aat gcc aag aca aag ccg Asn Ala Lys Thr Lys Pro 365 gtg gtc agc gtC ctc acc Asn Trp Tyr Val Arg gtc cag Gin 370 cag Thr Tyr Arg 375 Val Val Ser Val Leu. Thi- Val Leu 380 His Gin Asp Trp Leu 385 aaa gcc ctc cca gcc Lys Ala Leu Pro Ala aat Asn 390 ggc aag gag tac Gly Lys Giu Tyr tgc aag gtc tcc aac Cys Lys Val Ser Asn 400 ccc atc gag aaa Pro Ile Glu Lys acc Thr 410 atc tcc aaa gcc Ile Ser Lys Ala aaa Lys 415 ggg cag ccc cga Gly Gin Pro Arg gaa cca Glu Pro 420 cag gtg tac Gin Val Tyr gtc agc ctg Val Ser Leu. 440 acc Thr 425 ctg ccc cca tcc Leu. Pro Pro Ser gag gag atg acc Glu Glu. Met Thr aag aac cag Lys Asn Gin 435 gac atc gcc Asp Ile Ala acc tgc ctg gtc Thr Cys Leu Val aaa Lys 445 ggc ttc tat ccc Gly Phe Tyr Pro gtg gag Val Giu 455 tgg gag agc aat Trp Giu Ser Asn ggg Gly 460 cag ccg gag aac Gin Pro Giu. Asn aac Asn 465 tac aag acc acg Tyr Lys Thr Thr 1255 1303 1351 1399 1447 1495 1543 1591 1638 cct Pro 470 ccc gtg ctg gac Pro Val Leu Asp tcc Ser 475 gac ggc tcc ttc Asp Gly Ser Phe t tc Phe 480 ctc tat agc aag Leu Tyr Ser Lys ctc Leu. 485 acc gtg gac aag Thr Val Asp Lys agg tgg cag cag Arg Trp Gin Gin aac gtc ttc tca Asn Val Phe Ser tgc tcc Cys Ser 500 gtg atg cat Val Met His gct ctg cac aac Aia Leu His Asn c ac His 510 tac acg cag aag Tyr Thr Gin Lys agc ctc tcc Ser Leu Ser 515 ctg tct ccg ggt aaa Leu Ser Pro Giy Lys 520 tga actagagcgg ccgccaccgc ggtggagct <210> 8 <211> 522 <212> PRT <213> Artificial Sequence <223> Description of Artificial Sequence: fusion polypeptide <400> 8 Met Giu Thr Asp Thr Leu Leu Leu Trp Val Leu Leu Leu Trp Val Pro Gly Ser Thr Gly Thr Ser Cys Gly Asn Met Phe Val Giu Pro Gly Giu 25 Gin Cys Asp cys Gly Phe Leu Asp Asp Cys Val Asp Pro Cys Cys Asp 40 Ser Leu Thr Cys Gin Leu Arg Pro Gly Ala Gin Cys Aia Ser Asp Giy 55 Pro Cys Cys Gin Asn Cys Gin Leu Arg Pro Ser Gly Trp, Gin Cys Arg 70 75 Pro Thr Arg Gly Asp Cys Asp Leu Pro Giu Phe Cys Pro Giy Asp Ser 90 9 Ser Gin Cys Gly Gly Gin 115 Gln Cys Gin 130 Pro 100 Ala Pro Asp Val Ser Val Cys Met His 120 Pro Leu 105 Gly Gly Gly Asp Gly Arg Cys Ala Giu Pro Cys Ala 110 Ser Tyr Ala Gin 125 Ala Ala Pro Leu Ser Leu Trp Ala Gin Pro 140 Cys Leu Gin Thr Ala Asn 145 150 Thr Arg Giy Asn Ala Phe Gly Ser Cy: Arg Cys Ser Leu Pro 225 Ile Arg Tyr Ala Pro 305 Lys Val1 Tyr Glu His 385 Lys Gin Met Pro Asn 465 Leu Val E Asi GI~ Asr 210C LeAsp Ser Cys Thr 290 Ala Pro Val1 Val1 Gin 370 Gin Ala Pro rhr 3er 150 ['yr [yr ~he 1i Pri ~*Gli lArs 19! iCys Let His Lys Glu 275 Ser Pro Lys Vai Asp 355 Tyr Asp Leu Arg Lys 435 Asp Lys Ser Ser oSer .i Leu. 180 1 Asp Ser Thr Arg *Cys 260 Glu Ser *Glu Asp Asp 340 Gly Asn Trp Pro 1 Giu 1 420 Asn C Ile A~ Thr TI Lys L 4 Cys S 500 G13 161 Gir Leu Trp Leu Cys 245 His Gly Arg Ala Thr 325 V'al Val Ser eu 105 ~ro ;ln lia hr eu 85 er Ser 1Cys Leu Val Pro 230 Gin Gly Trp Ser Giu 310 Leu Ser Glu Thr Asn 390 Pro Gin Val Val C Pro F 470 Thr Val M' Gli Trj His 215 G11 Arg His Ala Cys 295 Gly Met His Val1 T'yr 375 Gly I le la 1 3er 1u L55 ~ro ral let rVa i Th~ Git 20( Lei Thi Val Gi Pro 280 Asp Ala Ile Glu His 360 Arg Lys Glu Tyr Leu 440 Trp Val Asp His 1 Sen r Gly 185 .1 Thr i Asp Ala *Asp Val 265 Pro Lys *Pro Ser Asp 345 Asn Val Glu Lys ThrI 425 ThrC Glu S Leu A Lys S 4 Giu A 505 Cy: 17( Ar IlE Let Cys Leu 250 Cys Asp Thr Ser Arg 330 Pro Ala Val1 1'yr ['hr 110 ,eu ~ys er er 90 lia 3 Thr I Thr Asp Gly Gly 235 Leu. Asp Cys His Val 315 Thr Glu Lys Ser Lys 395 Ile Pro Leu Asn Ser 475 Ang IJ Leu 1 Pr Gi, Va Se~ 22( Pr Gl) Sex Thr Thr 300 Phe Pro Val Thr Val1 380 Cys Ser Pro lai ,iy 160 k.sp rrp Fis oAr n1 Pr 1. Asr 201 r* Asj )Glj SAla -Asn *Thr 285 *Cys Leu Glu Lys Lys 365 Leu Lys Lys Ser Lys 445 Gin Gly Gin Asn Asp Leu 190 i Giy Val Leu Gin Arg 270 Gin Pro Phe Val Phe 350 Pro Thr Val AlaI Arg 430 GlyF Pro G Ser P Gin G 4 HisT 510 Al 17! Le~ Thi Ala Val Glu 255 His Leu Pro Pro Thr 335 PAsn Arg Val Ser .sys 115 ~he ;iu ~he l 1 y lyr sGly 160 i Gly -Glu Gin Cys 240 Cys Cys Lys Cys Pro 320 Cys Trp Glu Leu Asn 400 Gly Giu Tyr Asn Phe 480 Asn Thr Gin Lys Sen 515 Leu Ser Leu Ser Pro Gly Lys 520 <210> 9 <211> 1386 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: fusion polypeptide <220> <221> CDS <222> (25)..(1365) <400> 9 gtcgacccaa gctggctagc cacc atg gag aca gac aca ctc ctg cta tgg Met Glu Thr Asp Thr Leu Leu Leu Trp gta ctg otg ctc tgg gtt Val Leu Leu Leu Trp Val 15 1 cca ggt toi Pro Giy Se: tog Ser aac Asn cag Gin act Thr gtg Val gc t Ala aaa Lys tgt Cys 9gc Gly agg Arg 170 tgt CysC gac c Asp C tct t SerC gag g Glu G 2 ag Ar~ aa( Asr tgc Cys gcc Ala tc Ser gaa Giu tgc Cys aat PAsn cgo Arg 155 aaa ys lag liu :ag ;in gt .Ys 'go ny 35 gtg ;Val gac i Asp agt Ser cag Gin tac *Tyr *gat Asp atc Ile gaa Glu 140 tgt Cys gga Gly aaac Lys ;z ctg a Leu S 2 gac a Asp L 220 gog c Ala P gal Asi acc Thi gac Asp aag Lys tgc Cys gac Asp cc t Pro 125 act rhr gtg 1a 1 aag *,ys :ga ~rg Lgc er ~05 .aa ,ys cg ro gae tgC Cys agg Arg aag Lys ac a Thr act Thr 110 ttc Phe gac Asp ccc Pro ccc Pro gta Vai 190 ato Ile act Thr tca Ser igga gaa Gly Giu tgc aac Cys Asn aac agt Asn Ser tgc cag Cys Gin 80 ggt aat Gly Asn

95. gtt tgc Val Cys tgc gag Cys Giu aac too Asn Ser tat gtc Tyr Val 1160 tgt aca Cys Thr 175 cag gat Gin Asp aat act Asn Thr cac aca His ThrC gtc ttc c Val Phe L 240 gag Giu agc Ser cc t Pro 65 gag Giu agc Ser ttg Leu agg Arg tgc Cys 145 gat Asp gta Val gta z ValI tt t Phe C ~25 :to t ~eu P tgl Cy ga Asl tgC C yE gcc Ala agt Ser gat Asp gaa Giu 130 aag Lys gc t Ala jga fly t t 1 e ~ga ;iy ~10 c a 4 ro toC 'he *act Thr t gat s Asp tgc ,Cys tgt Cys att Ile gag Giu ott Leu 115 cag Gin gtg Vail gaa Giu ttt Phe gaac Giu 195 aagt Lys I cog t Pro C 000 0 Pro P G1~ ccl Pr acc Thi aae Lys aat Asn tgc Cys 100 ggc Gly cag Gin tgc caa 31n :gt 180 ~ga rg :tt ~he .gc ,ys ca t act agt tgt SThr Ser Cys 0 tggc ato atg )Gly Ile Met ;ttg aag gaa Leu Lys Glu aac tgt cag Asn Cys Gin got act tgo Ala Thr Cys cog cot oca Pro Pro Pro aag tgt aag Lys Cys Lys ctg gag too Leu Giu Ser 135 tgo agg gao Cys Arg Asp 150 aag aao tta Lys Asn Leu 165 gao atg aat Asp Met Asn ttt tgg gat Phe Trp Asp tta goa gao Leu Ala Asp 215 ooa gca cot S Pro Ala ProC 230 aaa 000 aag g ggg aac Giy Asn tat otg Tyr Leu ggt gto Gly Val ttt gag Phe Giu aaa ggo Lys Gly gga aat Gly Asn 105 gat ggg Asp Giy 120 tgt gca Cys Ala ott tcc Leu Ser ttt ttg Phe Leu ggc aaa Gly Lys 185 ttc att Phe Ile 200 xao aga k.sn Arg ~aa gc ;iu Ala 99 147 195 243 291 339 387 435 483 531 579 627 675 723 771 'ro Lys 245 Pro Lys Asp Thr oto atg ato too cgg soc cot gag gto aca Leu Met Ile Ser Arg Thr Pro Giu Vai Thr 250 255 tgc Cys 260 gtg gtg gtg gao Val Val Val Asp gtg Val 265 819 age cac gaa gac cct gag gtc aag ttc aac tgg tac Ser His Giu Asp Pro Glu Val Lys Phe Asn Trp Tyr 270 275 gtg Val gac ggc gtg Asp Gly Val 280 gag Glu acg Thr aat Asn ccc Pro 330 cag Gin gtc Val gtg Val cct Pro acc Thr 410 gtg Val gtg Val tac Tgr Gly 315 atc Ile gtg Val age Ser gag Glu. ccc Pro 395 gtg Val atg Met cat His cgg Arg 300 aag Lys gag Glu tac Tyr ctg Leu tgg Trp 380 gtg Val gac Asp cat Hius aat Asn 285 gtg Val gag Giu aaa Lys ac Thr ace Thr 365 gag Giu ctg Leu aag Lys gag Giu gee aag Ala Lys gte age Val Ser tac aag Tyr Lys ace ate Thr Ile 335 etg ccc Leu Pro 350 tge etg Cys Leu age aat Ser Asri gac tee Asp Ser age agg Ser Arg 415 get ctg Ala Leu 430 aca Thr gtC Val1 tge Cys 320 tee Ser eca Pro gte Val1 ggg Gly gac Asp 400 tgg Trp cac aag Lys etc Leu 305 aag Lys aaa Lys tee Ser aaa Lys eag Gin 385 gge Gly eag Gin aac ceg Pro 290 ace Thr gte Val gee Aia egg Arg ggc Gly 370 ceg Pro tcC Ser cag Gin c ac egg Arg gte Val tee Ser aaa Lys ga t Asp 355 tte Phe gag Giu. t te Phe ggg Giy tac Tyr 435 gag gag eag tac Glu Glu Gin Tyr 295 ctg cac cag gac Leu His Gin Asp 310 aac aaa gee etc Asn Lys Ala Leu 325 ggg eag cee ega Gly Gin Pro Arg 340 gag ctg ace aag Giu Leu Thr Lys tat ce age gac Tyr Pro Ser Asp 375 aac aae tae aag Asn Asn Tyr Lys 390 tte etc tac age Phe Leu Tyr Ser 405 aac gte tte tca Asn Val Phe Ser 420 aeg cag aag age Thr Gin Lys Ser aac age Asn Ser tgg ctg Trp Leu eca gee Pro Ala gaa eca Giu Pro 345 aae cag Asn Gin 360 ate gee Ile Ala ace aeg Thr Thr aag etc Lys Leu tgC tee Cys Ser 425 etc, tee Leu Ser 915 963 1011 1059 1107 1155 1203 1251 1299 1347 1386 His Asn His 440 etg tet ceg ggt aaa tga actagagcgg cegetacaga t Leu Ser Pro Gly Lys 445 <210> <211> 446 <212> PRT <213> Artificial Sequence <223> Description of Artificial Sequence: fusion polypept ide <400> Met Glu Thr Asp Thr Leu Leu Leu Trp Val 1 5 10 Gly Ser Thr Gly Thr Ser Cys Gly Asn Ser .25 Glu Cys Asp Pro Gly Ile Met Tyr Lau Asn 40 Ser Asp Cys Thr Leu Lys Giu Gly Val Gin 55 Leu Leu Leu Trp Val Pro Arg Val Asp Giu. Gly Giu Asn Asp Thr Cys Cys Asn Cys Ser Asp Arg Asn Ser Pro Giu Ser Leu Arg Cys 145 Asp Val Val Phe Cys 225 Leu Giu Lys Lys Leu 305 Lys Lys Sex Lys C 3 Gin P 385 Gly S Gin G Asn H Cys Cys Lys ASx I Al Se: Asj Git 13( Ly~ Aiz G ij IlE Gli 210 Prc Phe Vtal Phe P ro 290 P'hr Tai Lrg ny 70 'ro er in is Giu p Leu 115 .i Gin 3 Val rPhe Glu 195 Lys Pro Pro Thr Asn 275 Arg Val Sex Lys( AspC 355 Phe Glu P1 Phe P Gly A 4 Tyr T 435 Asi Cy~ GI3 Gir Cys Gir Cys 180 Arg Phe Cys Pro Cys 260 rrp Alu Leu ksn 340 flu .yr Lsf ~he ,sn 20 hr a Aia s Pro 0 r' Lys iLeu Cys Lys 165 Asp Phe Leu Pro Lys 245 Vai Tyr Glu His Lys 325 Gin Leu 9I Pro Asn 3 Leu.' 405 Val P Gin L CY~ 7' Th Prc Cy~ Arc Asr Met Trp Ala Ala 230 Pro Vai Val1 Aln 31n 310 ro 'hr er 'y r .90 yr 'he s Gin Phe Giu Thr Ala Gin Lys Lys Cys Gin Cys 3 Pro Lys 1Ser 135 Asp Leu Asn Asp Asp 215 Pro Lys Val Asp Tyr 295 Asp LeuI Arg Lys 1 Asp I 375 Lys I1 Sex L Sex Ly G13 Asi 12C CyEc Leu Phe Gly Phe 200 Asn Giu Asp Asp Gly 280 Asn rrp Pro 3iu tsn 360 le 'hr lys ,ys s Giy r Asn 105 Giy Ala ISer Leu Lys 185 Ile Arg Ala Thr Vai 265 Val1 Sex Leu Ala Pro 345 Gin Ala Thr I Leu '1 Ser T 425 90 Ala Lys Cys Giy Arg 170 Cys Asp Sex Glu Leu 250 Ser Glu Thr Asn Pro 330 31n la 1 fa 1 ~ro 'hr ~10 -Ser Giu Cys Asn Arg 155 Lys Giu Gin Cys Gly 235 Met His Val Tyr Gly 315 Ile Vai Sex Glu 9) Pro N 395 Val P, Met H~ Ty: As~ Ii( Gli 14( CyE Glj Lys Leu Asp 220 Ala Ile Giu His Arg 300 Lys Glu ['yr 'eu rp, 380 Tal ISP [is ~CY p As] IPr 12' ITh: Ly~c Ar Ser 205 Lys Pro Sex Asp Asn 285 Val1 Glu Lys Thr Thr 365 Giu Leu Lys Glu s Thr p Thr 110 o Phe r* Asp L Pro Pro Val 190 Ile Thr Sex *Arg *Pro 270 Al a Val1 Tyr Thr Leu 350 Cys Sex Asp Sex Ala L Gi1 9, Va. Cy~ Asi Cyl 175~ Gir Asr His Val1 Thr 255 Glu Lys Ser Lys Ile 335 Pro Zeu ksn ~er ~rg 115 ~eu y Asn 1 Cys s Glu i Sex Val 160 Thr Asp Thr Thx Phe 240 Pro Val Thr Val Cys 320 Sex Pro Val Gly Asp 400 Trp His ys Sex Leu 440 Ser Leu Sex Pro Gly Lys 445 <210> 11 <211> 1653 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: fusion polypeptide <220> <221> CDS <222> (25)..(1632) <400> 11 gtcgacccaa gctggctagc cacc atg gag aca gac aca ctc ctg cta tgg Met Giu Thr Asp Thr Leu Leu Leu Trp 1 cca ggt tcc act Pro Giy Ser Thr gta Val ctg ctg ctc tgg Leu Leu Leu Trp ggt act agt tgt Gly Thr Ser Cys 20 tgt gga acc ata Cys Gly Thr Ile ggg aat Giy Asn cgg cag Arg Gin cta gtg gtt gaa Leu Val Val Giu gaa Giu ggg gag gaa tgt Gly Giu Giu Cys gac Asp 35 tgt gca aaa Cys Ala Lys gct gct tgt Ala Aia Cys ccc tgt tgt ctg Pro Cys Cys Leu aac tgt act cta Asn Cys Thr Leu cat cct ggg His Pro Gly ttt ctg cca Phe Leu Pro gct ttt gga ata Ala Phe Gly Ile tgt Cys. 65 tgc aaa gac tgc Cys Lys Asp Cys aaa Lys tca gga. Ser Gly act tta tgt aga. Thr Leu Cys Arg caa gtt ggt gaa Gin Val Giy Giu gac ctt cca gag Asp Leu Pro Giu tgg Trp tgc aat ggg aca Cys Asn Gly Thr tcc Ser 95 cat caa tgc cca His Gin Cys Pro gat Asp 100 gat gtg tat gtg Asp Val Tyr Vai gac ggg atc tcc Asp Gly Ile Ser aat gtg aat gcc Asn Vai Asn Ala tgc tat gaa aag Cys Tyr Giu Lys acg tgt Thr Cys 120 aat sac cat Asn Asn His agt gcs tct Ser Ala Ser 140 ga t Asp 125 ats csa tgt ass Ile Gin Cys Lys gag Giu 130 att ttt ggc caa Ile Phe Gly Gin gat gca agg Asp Ala Arg 135 gga sac cgt Gly Asn Arg 435 483 cag agt tgc tac Gin Ser Cys Tyr c as Gin 145 gaa atc sac acc Giu Ile Asn Thr ttc ggt Phe Gly 155 cac tgt ggt att His Cys Giy Ile g ta Val 160 ggc acs aca tat Gly Thr Thr Tyr gta Vai 165 as tgt tgg acc Lys Cys Trp Thr cct Pro 170 gat stc atg tgt Asp Ile Met Cys agg gtt cag tgt Arg Val Gin Cys sat gtg gga gta Asn Val. Gly Val a tt Ile 185 ccc sat ctg ats Pro Asn Leu Ile gag Giu 190 cat tct acs gtg His Ser Thr Val cag Gin 195 cag ttt cac ctc Gin Phe His Leu sat gac Asn Asp 200 acc act tgc Thr Thr Cys att ggt gag Ile Gly Giu 220 ggc act gat tat Giy Thr Asp Tyr tta ggg atg gct Leu Giy Met Ala ata cct gat Ile Pro Asp 215 aag atc tgc Lys Ile Cys gtg ass gat ggc Vai Lys Asp Gly acs Thr 225 gta tgt ggt ccs Val. Cys Giy Pro gas Glu 230 atc cgt Ile Arg 235 sag aag tgt gcc Lys Lys Cys Ala agt Ser 240 atg gtt cat ctg Met Val His Leu caa gcc tgt cag Gin Ala Cys Gin cct Pro 250 sag acc tgc sac Lys Thr Cys Asn atg Met 255 sgg gga atc tgc Arg Gly Ile Cys aac Asn 260 sac aaa caa cac Asn Lys Gin His tgt Cys 265 cac tgc aac cat gaa tgg gca ccc cca His Cys Asn His Glu Trp, Ala Pro Pro 270 tac Tyr 275 tgc aag gac aaa Cys Lys Asp Lys ggc tat Gly Tyr 280 gg9 Gl~ ttz Let tgc Cys c tc Leu 330 gag Giu aag Lys aag Lys C tc Leu aag Lys 410 aaa Lys tcc Ser aaa Lys cag Gin ggc Gly 490 cag Gin aggt agt f' Gly Ser Saat gtg Asn Val 300 cca ccg Pro Pro 315 ttc ccc Phe Pro gtc aca Val Thr ttc aac Phe Asn ccg cgg Pro Arg 380 acc gtc Thr Val 395 gtc tcc Val Ser gcc aaa Ala Lys cgg gat Arg Asp ggc ttc Gly Phe 460 ccg gag a Pro Giu A 475 tcc ttc t Ser Phe P cag ggg a Gin Gly A gc Al 28! at Met tg( Cys cca Pro tgc Cys t gg Trp 365 gag Glu ctg Leu aac Asn 9gg 3iy lag 3lu 145 :at .yr Lac hsf .tc he ac t gat a Asp 5 1 gga tGly cca Pro aaa Lys geg Val 350 tac PTyr gag Glu cac His aaa Lys cag Gin 430 ctg Leu ccc a Pro aac 1 Asn ctc t Leu 4 gtc t 4 agt ggc Ser Gly aag ttg Lys Leu gca cct Ala Pro 320 ccc aag Pro Lys 335 gtg gtg V'al Val gtg gac ltal Asp ::ag tac aln Tyr ag gac 71n Asp 400 ~cc Ctc lia Leu 15 .cc cga ~ro Arg cc aag hr Lys gc gac E er AspI ac aag a yr Lys T1 480 ac agc a rr Ser L 95 tc tca t cc' Pr cgl Ar~ 3 0 gaE Git gac Asr gac Asp ggc Gly aac Asn 385 tgg Trp cca Pro gaa G1u iac ksn Itc [le L65 'cc 'hr ~ag lys *cct Pro 290 gga I Gly i gcc Ala acc Thr gtg Val gtg Val 370 agc Ser ctg Leu gcc Ala cca ProC cag S Gin N 450 gcc g Ala V acg c Thr P ctc a Leu T cc, Pri tcl Se~ ga~ Gli ctc Let agc Ser 355 gag Giu acg Thr aat pAsn -cc Pro :ag 1n 135 jtc Pal rtg al1 Ct ro cc hr taag aac o Lys Asn t tgt gac r Cys Asp I ggc gcg aGly Ala 325 atg atc Met Ile 340 cac gaa His Glu gtg cat Val His tac cgg Tyr Arg ggc aag Gly Lys 405 atc gag Ile Glu 420 gtg tac Val Tyr ago ctg Ser Leu gag tgg Giu Trp C ccc gtg c Pro Val 1 485 gtg gac a Val Asp L 500 atg cat g Met His G aac Asn aaa Lys 310 ccg Pro tcc Ser gao Asp aa t Asn gtg Val1 390 gag Glu aaa Lys acc rhr acc rhr jag flu 170 ~tg ~eu Lag a ,ys S *ag g lu A atg Met 295 act Thr tca. Ser cgg Arg cct Pro gcc Ala 375 gtc Val1 tac Tyr aco Thr Ctg Leu Lgc Cy 455 agc z Ser jac t ksp S ~gc a er A rct c la L ga G1 ca Hi gt Va. ac( Thi ga~ Gli 36( aaS Ly~c agc Ser aag Lys atc Ile :ccc Pro 440 :tg eu lat .sn :cc ;er Lgg .rg tg eu a gga u Gly c aca Thr ttc 1. Phe ct Pro 345 jgtc j Val ;aca Thr gtC Val tgc Cys tcc Ser 425 cca Pro gtc Val1 ggg Gly gac Asp tgg Trp 505 cac His 915 963 1011 1059 1107 1155 1203 1251 1299 1347 1395 1443 1491 1539 1587 1632 Sn Val Phe Ser C gc tcc gtg ys Ser Val 510 aac cac tac acg cag aag agc ctc tcc ctg tct ccg ggt aaa tga Ct Asn His Tyr Thr Gin Lys Ser Leu Ser Leu Ser Pro Gly Lys 525 530 535 actagagcgg ccgctacaga t 1653 <210> 12 <211> 535 <212> PRT <213> Artificial Sequence <223> Description of Artificial polypeptide Sequence: fusion <400> 12 Met Gly Glu Leu Cys Gin Gin Asn Lys Gin 145 Gly Val Thr Tyr Thr 225 Met Gly Pro Pro AMg 305 Giu Asp Asp Gly Asn 385 Trp, Glu Ser Cys Leu Cys Val1 Cys Ala Glu 130 Glu Thr Gin Val His 210 Val Val1 Ile Pro Pro 290 Gly Ala Thr Val Val 370 Ser Leu Thr Thr Asp Asn Lys Gly Pro Phe 115 Ile Ile Thr cys Gin 195 Leu Cys His Cys Tyr 275 Pro Ser Giu Leu Ser 355 Glu Thr Asn Asp Gly Cys Cys Asp Glu Asp 100 Cys Phe Asn Tyr Glu 180 Gin Giy Gly Leu Asn 260 Cys Lys Cys Gly Met 340 His Val1 Tyr Gly Thr Thr Gly Thr Cys Cys Asp Tyr Gly Thr Val1 165 Asn Phe Met Pro Ser 245 Asn Lys Asn Asp Ala 325 Ile Giu His Arg Lys 405 Leu Ser Thr Leu Lys 70 Asp Val Glu Gin Gin 150 Lys Val1 His Ala Giu 230 Gin Lys Asp Asn Lys 310 Pro Ser Asp Asn Val1 390 Glu Leu Cys Ile His 55 Phe Leu Tyr Lys Asp 135 Gly Cys Gly Leu Ile 215 Lys Ala Gin Lys Met 295 Thr Ser Arg Pro Ala 375 Val1 Tyr Leu Gly Arg 40 Pro Leu Pro Val Thr 120 Ala Asn Trp Val Asn 200 Pro Ile Cys His Gly 280 Glu His Val1 Thr Giu 360 Lys Ser Lys Trp Asn 25 Gin Gly Pro Giu Gin 105 Cys Arg Arg Thr Ile 185 Asp Asp Cys Gin Cys 265 Tyr Gly Thr Phe Pro 345 Val1 Thr Val1 Cys Val 10 Leu Cys Ala Ser Trp 90 Asp Asn Ser Phe Pro 170 Pro Thr Ile Ile Pro 250 His Gly Leu Cys Leu. 330 Giu Lys Lys Leu Lys 410 Leu Val1 Ala Ala Gly 75 Cys Gly Asn Ala Gly 155 Asp Asn Thr Gly Arg 235 Lys Cys Gly Asn Pro 315 Phe Val1 Phe Pro Thr 395 Val1 Leu Val1 Lys Cys Thr Asn Ile His Ser 140 His Ile Leu Cys Giu 220 Lys Thr Asn Ser Val 300 Pro Pro Thr Asn Arg 380 Val Ser Leu Giu Asp Ala Leu Gly Ser Asp 125 Gin Cys Met Ile Trp 205 Val Lys Cys His Ala 285 Met Cys Pro Cys Trp 365 Glu Leu Asn Trp Glu Pro Phe Cys Thr Cys 110 Ile Ser Gly CYS Glu 190 Gly Lys Cys Asn Giu 270 Asp G ly Pro Lys Val1 350 Tyr Glu His Lys Val Gly Cys Gly Arg Ser Asn Gin Cys Ile Gly 175 His Thr Asp Ala Met 255 Trp Ser Lys Ala Pro 335 Val Val Gin Gin Ala2 415 Pro Glu Cys Ile Gin s0 His Val Cys Tyr Val1 160 Arg Ser Asp Gly Ser 240 Arg Ala Gly Leu Pro 320 Lys Val Asp Tyr Asp 400 Leu Pro Ala Pro Glu Pro Gin 435 Ile Glu Lys Thr Ile Ser 420 425 Val Tyr Thr Leu Pro Pro Lys Ala Lys Gly Ser Arg Asp 440 Leu Giu 445 Tyr Gin Pro Arg 430 Leu Thr Lys Pro Ser Asp Asn Gin 450 Ile Ala 465 Thr Val Ser Leu Thr Val Glu Trp Giu 470 Pro Pro Val Leu 485 Thr Val Asp Lys Cys 455 Ser Asn Giy Gin Glu Asn Asn Tyr Lys 480 Val Lys Gly Thr Asp Ser Asp Gly 490 Gin Phe Phe Leu Tyr Ser 495 Lys Leu Ser Arg Trp 505 His Gin Giy Asn Val Phe Ser 510 Gin Lys Ser Cys Ser Val Met 515 Leu Ser Leu Ser 530 His Giu Ala Pro Gly Lys 535 Asn His Tyr <210> 13 <211> 1617 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: fusion polypeptide <220> <221> CDS <222> .(1596) <400> 13 gtcgacccaa gctggctagc cacc atg gag aca gac aca ctc ctg cta tgg Met Giu Thr Asp Thr Leu Leu Leu Trp gta Val ctg ctg ctc tgg Leu Leu Leu Trp gt t Val1 cca ggt tcc act Pro Gly Ser Thr act agt tgt ggg Thr Ser Cys Gly ggt gtg gtt gaa Gly Val Val Glu gaa gag cag tgt Giu Giu Gin Cys gac tgt gga tcc gta cag cag Asp Cys Gly Ser Val Gin Gin tgt gaa caa Cys Glu Gin gct gcc tgt Ala Ala Cys gac Asp gcc tgt tgt ctg Ala Cys Cys Leu aac tgc act cta Asn Cys Thr Leu agg cct ggg Arg Pro Gly ttc atg cca Phe Met Pro gct ttt ggg ctt Ala Phe Gly Leu tgc aaa gac tgc Cys Lys Asp Cys aag Lys tca ggg Ser Gly gaa ctc tgt aga Glu Leu Cys Arg gag gtc aat gaa Glu Val Asn Giu gac ctt cca gaa Asp Leu Pro Giu 195 243 291 339 387 tgg Trp tgc aat gga aca Cys Asn Gly Thr cat cag tgt cca His Gin Cys Pro gaa Glu 100 gat aga tat gtg Asp Arg Tyr Val gac ggg atc ccc Asp Gly Ile Pro tgt Cys 110 agt gac agt gcc Ser Asp Ser Ala tgc tat caa aag Cys Tyr Gln Lys agg tgt Arg Cys 120 aat aac cat gac cag cat tgc agg gag att ttt ggt aaa gat gca aaa Asi agt Se2 ttt PhE tct Ser 170 cot Pro gtc Val att Ile atc Ile cct Pro 250 cac His "i Asri gca r Ala ggt Gly 155 gat Asp *ctt Leu acc Thr ggt GJly cat His 235 gag Glu tgt Cys C His tc t Ser 140 cac His Asp 125 cag Gin tgt Cys Gin aa t Asn ggt Gly His tgC Cys ata Ile Cy. tat Tyi aat Asr 160 gtC Val ctc Leu tgc Cys gaa Giu 220 aag Lys Acc Phr ~gc ttt tgt Phe Cys caa gat Gin Asp 190 tgg ggt Trp Gly 205 gtg aaa Val Lys aag tgt Lys Cys tgc aat Cys Asn tat ggg Tyr Gly 270 ggg Gly 175 cat His att Ile gat Asp gtc Val1 atg Met 255 tgg Trp aga Arg ttt Phe gac Asp ggt Gly agt Ser 240 aag Lys tcc sArg taaa -Lys 145 ggc 1Gly gtt Val *act *Thr tat Tyr act Thr 225 c tg Leu ggg Gly cca Gl. 13( gaa Git aca Thr caa Gin ttg Leu cat His 210 gtg Vai tc t Ser atc Ile :cc Sle atc Ile aca Thr tgt Cys cag Gin 195 tta Leu tgt Cys gtc Vail tgc e Cys ;z tac t Ph aac Asr tac TPyr gag Giu 180 cac His agg Arg 9gC Gly ttg eu Iat .sn ~60 :gc GI~ tct 1Se3 cta *Let 165 aat *Asn act Thr atg Met c ca Pro tca Ser 245 aac Asn 'Lys cag Gin 150 aaa ILys gtg Val cat His aac Asn gga Gly 230 cat His aaac Lys F Asj 13! 994 Gl) tgt CyE aga Arg atc Ile ata Ile 215 aag Lys gtC lai at is iga ~rg Ala aac Asn cat His Lys cgt Arg atc Ile gac att Asp Ile 185 aat ggt Asn Gly 200 tct gac Ser Asp atc tgc Ile Cys tgc ctt Cys Leu cac tgc His Cys 265 ggc tat Gly Tyr 280 483 531 579 627 675 723 771 819 867 915 963 1011 1059 1107 1155 1203 Ser Pro Pro Tyr Cys Gin His A 275 ggg Gly aaa Lys ccg Pro tcc Ser 330 gac Asp aat Asn gtg Val ggC Gly act Thr tca Ser 315 cgg Arg cct Pro gcc Ala gtc Val1 agt Ser cac His 300 gtc Val1 acc Thr gag Giu aag Lys agc Ser 380 att Ile 285 aca Thr ttc Phe cc t Pro gtc Val aca Thr 365 gtc Val gac Asp tgc Cys ctc Leu gag Giu aag Lys 350 aag Lys ctc Leu agt Ser cca Pro ttc Phe gtc Val1 335 ttc Phe ccg Pro ggC Gly ccg Pro ccc Pro 320 aca Thr aac Asn Cgg cca Pro tgc Cys 305 c ca Pro tgc Cys tgg Trp gag Giu ctg Leu 385 gca Ala 290 cca Pro aaa Lys gtg Val1 tac Tyr gag Giu 370 cac H-i s tc t Ser gca Ala ccc Pro gtg Val1 gtg Val 355 cag Gin cag Gin gca aag Ala Lys cct gaa Pro Giu aag gac Lys Asp 325 gtg gac Val Asp 340 gac ggc Asp Gly tac aac Tyr Asn gac tgg Asp TrpI aga Arg gcc Ala 310 acc Thr gtg Val gtg Val agc Ser ctg Leu 390 tct tgt Ser Cys 295 gag ggc Giu Gly ctc atg Leu Met agc cac Ser His gag gtg Giu Val 360 acg tac Thr Tyr 375 aat ggc Asn Giy gac Asp gcg Ala atc Ile gaa Giu 345 cat His cgg Arg aag Lys Arar acc gtc Thr Val gag tac Giu Tyr 395 aag tgc aag Lys Cys Lys gtc tcc Val Ser 400 aac aaa gcc ctc Asn Lys Ala Leu cca Pro 405 gcc ccc atc gag Ala Pro Ile Glu aaa Lys 410 acc atc tcc aaa Thr Ile Ser Lys gcc Ala 415 aaa ggg cag ccc Lys Gly Gin Pro gaa cca cag gtg Giu Pro Gin Val tac Tyr 425 acc ctg ccc cca tcc cgg gat gag ctg Thr Leu Pro Pro Ser Arg Asp Giu Leu 430 acc Thr 435 aag aac cag gtc Lys Asn Gin Val agc ctg Ser Leu 440 acc tgc ctg Thr Cys Leu. gag agc aat Giu Ser Asn 460 gtc Val 445 aaa ggc ttc tat Lys Giy Phe Tyr ccc Pro 450 agc gac atc gcc Ser Asp Ile Ala gtg gag tgg Val Glu Trp 455 cct ccc gtg Pro Pro Val ggg cag ccg gag Gly Gin Pro Giu aac Asn 465 aac tac aag acc Asn Tyr Lys Thr acg Thr 470 1251 1299 1347 1395 1443 1491 1539 1587 1617 ctg gac Leu Asp 475 tcc gac ggc tcc Ser Asp Gly Ser ttc Phe 480 ttc ctc tac agc Phe Leu Tyr Ser ctc acc gtg gac Leu Thr Val Asp aag Lys 490 agc agg tgg cag Ser Arg Trp Gin cag Gin 495 ggg aac gtc ttc Gly Asn Val Phe tgc tcc gtg atg Cys Ser Val Met cat His 505 gag gct ctg Glu Ala Leu cac aac His Asn 510 cac tac acg cag His Tyr Thr Gin agc ctc tcc ctg Ser Leu Ser Leu tct ccg Ser Pro 520 ggt aaa Giy Lys tga actagagcgg ccgctacaga t <210> 14 <211> 523 <212> PRT <213> Artificial Sequence <223> Description of Artificial polypept ide Sequence: fusion <400> 14 Met Glu Thr Asp Thr Gly Ser Gin Cys Leu Leu Leu Ser Cys Gly Trp Val Leu 10 Asn Giy Val Leu Leu Trp Val Pro Thr Gly Thr Val Giu Arg Giu Glu Gin Asp Ala Cys Cys Asp Cys Asn Leu Leu Cys Cys Cys Gly Ser Val Gin Thr Leu Arg Pro Cys Lys Phe Met 70 Cys Giu Gly Ala Ala Phe Gly Leu Lys Asp Pro Ser Glu Gly Cys Leu Cys Arg Val Asn Giu Asp Leu Pro Glu Asn Giy Thr Ser His Gin Cys Pro Ser Ala Tyr 115 Arg Giu Ile Giu 100 Arg Tyr Val Gin 105 Cys Gly Ile Pro Cys Tyr Gin Lys Arg 120 Phe Gly Lys Asp Ala Asn Asn His Asp 125 Gin Cys Ser Asp Gin His Cys Asn Cys Tyr Lys Ser Ala 13 0 Lys Glu 145 Gly Thr Ile Asn Ser Thr Tyr Leu 165 135 Gin Giy 150 Lys Cys Ser 140 Asn Arg Phe His Ile Ser 170 Gly His 155 Asp Val Cys Gly Ile Phe Cys Gly 175 Asn 160 Arg Val Gin Cys Glu Asn Val Arg Asp Ile Pro Leu Leu Gir 180 Thr Tyr Thr 225 Leu Gly Pro Pro Cys 305 Pro Cys Trp, Glu Leu 385 Asn Gly Glu Tyr Asn 465 Phe I Asn Leu Hisa 210 Val Ser Ile Pro Ala 290 Pro Lys Val Tyr Giu 370 Hlis Lays 31n eu ro 150 ~sn ~eu al1 Gin 195 Leu Cys Val1 Cys Tyr 275 Ser Ala Pro Val' Val 355 Gin Gin Ala Pro2 Thr 435 Ser I Tyr I Tyr S Phe S His, Arc Gl Leu Asn 260 Cys Ala Pro Lys Val 340 A~sp T'yr Asp k.rg 420 .sys ~sp ~ys ~er ~er 3Th~ Met Pr< ISe2: 245 Asr Gln Lys Glu Asp 325 Asp Gly Asn Trp Pro 405 Glu Asn Ile Thr Lys 485 Cys rHis -Asn )Giy 230 His Lys His Arg *Ala 310 *Thr Val Val Ser Leu 390 Aia Pro Gin Ala ThrI 470 Leu Ser N 211 Lysc Val His Arg Ser 295 Glu Leu Ser Giu Thr 375 Asn Pro Gln Val1 la 1 455 Pro ['hr Tal SAsn 200 Ser Ile *Cys, IHis Gly 280 Cys *Gly Met His Val1 360 Tyr Gly Ile Val Ser 440 Glu Pro Val MetI Ser 520 185 Asp Cys Leu Cys 265 Tyr Asp Ala Ile Glu 345 His Arg Lys Giu Tyr 425 Leu Trp V/al Asp H'is 505 Pro Val Ile Ile Pro 250 His Gly Lys Pro Ser 330 Asp Asn Val Glu Lys 410 Thr Thr Giu Leu Lys 490 Giu Gly *Thr Gly His 235 Giu Cys Gly Thr Ser 315 Arg Pro Ala Val1 Tyr 395 Thr Leu Cys Ser Asp 475 Ser Ala I Lys Cys Glu 220 Lys Thr Gly Ser His 300 Vai Thr Glu Lys Ser 380 L*ys Ile Pro Leu ksn 460 3er 1 rg .eu Trj 201 Val Lys Cys Tlyr Ile 285 Thr Phe Pro Val Thr 365 Val1 Cys Ser Pro VIal 445 k~sp rrp -'is 1Asp His Phe 190 )Gly Ile Asp *Lys Asp Giy Cys Val Ser 240 Asn Met Lys 255 -Gly Trp Ser 270 *Asp Ser Gly Cys Pro Pro Leu Phe Pro 320 Giu Val Thr 335 Lys Phe Asn 350 Lys Pro Arg Leu Thr Val Lys Val Ser 400 Lys Ala Lys 415 Ser Arg Asp 430 Lys Gly Phe Gin Pro Glu Gly Ser Phe 480 Gin Gin Gly 495 Asn His Tyr 510 500 Thr Gin Lys Ser Leu Ser Leu 515 <210> <211> i674 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: fusion polypeptide <220> <221> CDS <222> (25)..(1653) <400> gtcgacccaa gctggctagc cacc atg gag aca gac aca ctc ctg cta tgg Met Giu Thr Asp Thr Leu Leu Leu Trp 1 gta ctg ctg ctc tgg gtt cca ggt tcc. act ggt act agt tgt ggc aat Val1 ggc Gly tgt Cys gao Asp cot Pro gaa Glu atg Met aga Arg gtg Val acg Thr aac Asn 170 aat Asn gtt Val Ott Leu ggt Gly ttc Phe 250 gga z Gly ;z Leu Leu Leu TrI t tc Phe gtc Val1 tct Ser atg Met acg Thr gat Asp tgc Cys aca Thr gag Giu 155 aaa Lys atc Ile gtg Val1 gaa 3lu :cc P ro iac ksn att Ilie ctt Leu caa Gin ggc Gly tgc Cys gga Gly aaa Lys gca Ala 140 aag Lys cgg Arg cca Pro cag Gin gaa Glu 220 caa Gin ttt i Phe gaa Glu gaa Glu tgc Cys act Thr tca Ser tat Tyr acc Thr 125 tca Ser gg t Gly ga t Asp agg Arg caa Gln 205 ga t ksp Itg 4e t g t er *act *Thr gga Giy agt Ser gtg Val1 gga Gly tca Ser 110 aga Arg gac Asp aac Asn gtg Val Ott Leu 190 gga Gly gta Val atg Met act Thr Val 15 gga *Gly gca Ala gao Asp *tgc Cys aa t Asn 95 tgt Cys ga t Asp aaa Lys tgt Cys ctt Leu 175 gga Gly aga Arg gat Asp Pro gag Glu gag Glu ggt Gly cga Arg 80 toa Ser ga t Asp aga Arg tat Tyr ggg Gly 160 tgt Cys gaa Gl1u aca rhr Ctt Lieu Gly gag Glu tgt Cys Ott Leu 65 gaa Giu ago Ser ggt Gly caa Gin tgc Cys 145 aaa Lys ggt Gly O to Leu t ta Leu ggc Gly 225 Ser tgt Cys tgt Cys 50 tgc Cys gca Ala cag Gin gtt Val tgc Cys 130 tat Tyr gao Asp tao Tyr gat Asp aac Asn 210 tat T'yr Thr gat Asp 35 *aag Lys tgt Cys *gta Val tgt Cys cag Gin 115 aaa Lys gag Giu aaa Lys ott Leu gg t Gly 195 tgc Cys gtg Val1 *Gl) 20 tgt Cys aaa Lys aaa Lys aa t Asn gc Ala 100 gga Gly tao Tyr aaa Lys gao Asp t tg Leu 180 gaa Glu agt Ser gaa Giu Thr gga Gly tgo Cys aag Lys gat Asp ct Pro att Ile att Ile ctg Leu ac a Thr 165 tgt Cys ato Ile ggt Gly gat Asp Ser aoo Thr aco Thr tgo Cys tgt Cys aat Asn tgc Cys tgg Trp aat Asn 150 tgg Trp acc Thr ao a Thr 9gg G 1 y G 1 y 230 Cys ccc Prc t tc Leu aag Lys ga t Asp att Ile ttt Phe ggg Gly 135 ati Ile ata Ile aat Asn tot Ser oat His 215 aca Thr Gly gc Ala act Thr ttt Phe att Ile cat His gga Gly 120 caa Gin gaa Giu cag Gin att Ile act Thr 200 gtt Val1 cot Pro got Ala tgo Cys Asri gaa Giu caa Gin cag Gin ogt Arg aaa Lys 105 gga Giy aag Lys ggg Gly tgo Cys ggc Gly 185 t ta Leu aag Lys tgt :Cys tot Ser tca 3er ?6 147 195 243 291 339 387 435 483 531 579 627 675 723 771 819 867 tgc tta gaa cac agg tgt Ott cot gtg Cys Leu Giu His Arg Cys Leu Pro Val 240 245 tgc ttg ago agt aaa gaa ggc act att Cys Leu Ser Ser Lys Giu Gly Thr Ile 255 260 agt aat gag ctg aag tgt gtg tgt aac Ser Asn Giu Leu Lys Cys Val Cys Asn 275 iat gga gtt tgo ~sn Gly Val Cys 270 aga cac Arg His 280 Ct tgg ata ggt Trp Ile Gly aag act ggt Lys Thr Gly 300 tct Ser 285 gat tgc aac act Asp Cys Asri Thr ttc cct cac aat Phe Pro His Asn gat gat gca Asp Asp Ala 295 acc aat gga Thy Asn Gly atc act ctg tct Ile Thr Leu Ser ggc Gly 305 aat ggt gtt gct Asn Gly Val Ala ggc Gly 310 tct tgt Ser Cys 315 gac aaa act cac Asp Lys Thr His aca Thr 320 tgc cca ccg tgc Cys Pro Pro Cys cca Pro 325 gca cct gaa gcc Ala Pro Glu Ala gag Glu 330 ggc gcg ccg tca Gly Ala Pro Ser gtc Val 335 ttc ctc ttc ccc Phe Leu Phe Pro cca Pro 340 aaa ccc aag gac Lys Pro Lys Asp acc Thr 345 ctc atg atc tcc Leu Met Ile Ser acc cct gag gtc Thr Pro Giu Val aca Thr 355 tgc gtg gtg gtg Cys Val Val Val gac gtg Asp Val 360 agc cac gaa Ser His Giu gag gtg cat Giu Val His 380 gac Asp 365 cct gag gtc aag Pro Giu Val Lys aac tgg tac gtg Asn Trp Tyr Val gac ggc gtg Asp Gly Val 375 tac aac agc Tyr Asn Ser aat gcc aag aca Asn Ala Lys Thr ccg cgg gag gag Pro Arg Glu Giu cag Gin 390 acg tac Thr Tyr 395 cgg gtg gtc agc Arg Val Val Ser gtc Val1 400 ctc acc gtc ctg Leu Thr Val Leu cac His 405 cag gac tgg ctg Gin Asp Trp Leu 915 963 loll 1059 1107 1155 1203 1251 1299 1347 1395 1443 1491 1539 1587 aat Asn 410 ggc aag gag tac Gly Lys Giu Tyr aag Lys 415 tgc aag gtc tcc Cys Lys Val Ser aac Asn 420 aaa gcc ctc cca Lys Ala Leu Pro gcc Ala 425 ccc atc gag aaa Pro Ile Giu Lys atc tcc aaa gcc Ile Ser Lys Ala ggg cag ccc cga Gly Gin Pro Arg gaa cca Glu Pro 440 cag gtg tac Gin Val Tyr gtc agc ctg Val Ser Leu 460 acc Thr 445 ctg ccc cca tcc Leu Pro Pro Ser cgg Arg 450 gat gag ctg acc Asp Giu Leu Thr aag aac cag Lys Asn Gin 455 gac atc gcc Asp Ile Ala acc tgc ctg gtc Thr Cys Leu Vai ggc ttc tat ccc Gly Phe Tyr Pro gtg gag Val Glu 475 tgg gag agc sat Trp Giu Ser Asn ggg Gly 480 cag ccg gag aac Gin Pro Giu Asn aac Asn 485 tac aag acc acg Tyr Lys Thr Thr cct Pro 490 ccc gtg ctg gac Pro Val Leu Asp tcc Ser 495 gac ggc tcc ttc Asp Gly Ser Phe t tc Phe 500 ctc tac agc sag Leu Tyr Ser Lys ctc Leu 505 acc gtg gac sag Thr Val Asp Lys agg tgg cag cag Arg Trp Gin Gin ggg Gly 515 aac gtc ttc tca Asn Val Phe Ser tgc tcc Cys Ser 520 gtg atg cat Val Met His gag Glu 525 gct ctg cac aac Ala Leu His Ann cac His 530 tac acg cag aag Tyr Thr Gin Lys agc ctc tcc Ser Leu Ser 535 1635 ctg tct ccg ggt aaa tga actsgagcgg ccgctscaga t 1674 Lau Ser Pro Gly Lys 540 Ct <210> 16 <211> 542 <212> PRT <213> Artificial Sequence <223> Description of Artificial Sequence: fusion polypeptide K1 <400> 16 Met Glu Thr Asp Thr Leu Leu Leu Trp Val Leu Leu Leu Trp Val Pro 1 5 10 Gly Ser Thr Gly Thr Ser Cys Gly Asn Gly Phe Ile Giu Thr Gly Glu 25 Cl Giu Cys Asp Cys Gly Thr Pro Ala Glu Cys Val Leu Glu Gly Ala Giu 40 Cys Cys Lys Lys Cys Thr Leu Thr Gin Asp Ser Gin Cys Ser Asp Gly 55 Leu Cys Cys Lys Lys Cys Lys Phe Gin Pro Met Gly Thr Val Cys Arg 70 75 Glu Ala Val Asn Asp Cys Asp Ile Arg Giu Thr Cys Ser Gly Asn Ser 90 Ser Gin Cys Ala Pro Asn Ile His Lys Met Asp Gly Tyr Ser Cys Asp 100 105 110 Gly Val Gin Gly Ile Cys Phe Gly Gly Arg Cys Lys Thr Arg Asp Arg 115 120 125 Gin Cys Lys Tyr Ile Trp Gly Gin Lys Val Thr Ala Ser Asp Lys Tyr 130 135 140 Cys Tyr Glu Lys Leu Asn Ile Glu Gly Thr Giu Lys Gly Asn Cys Giy 145 150 155 160 Lys Asp Lys Asp Thr Trp Ile Gin Cys Asn Lys Arg Asp Val Leu Cys 165 170 175 Gly Tyr Leu Leu Cys Thr Asn Ile Gly Aen Ile Pro Arg Leu Gly Glu 180 185 190 Leu Asp Gly Giu Ile Thr Ser Thr Leu Val Val Gln Gin Gly Arg Thr 195 200 205 Leu Asn Cys Ser Gly Gly His Val Lys Leu Glu Giu Asp Val Asp Leu 210 215 220 Gly Tyr Val Giu Asp Gly Thr Pro Cys Giy Pro Gin Met Met Cys Leu 225 230 235 240 Giu His Arg Cys Lau Pro Val Ala Ser Phe Asn Phe Ser Thr Cys Leu 245 250 255 Ser Ser Lys Giu Gly Thr Ile Cys Ser Gly Asn Gly Val Cys Ser Asn 260 265 270 Giu Leu Lys Cys Vai Cys Asn Arg His Trp Ile Gly Ser Asp Cys Asn 275 280 285 Thr Tyr Phe Pro His Asn Asp Asp Ala Lys Thr Gly Ile Thr Leu Ser 290 295 300 Gly Asn Giy Val Ala Gly Thr Asn Gly Ser Cys Asp Lys Thr His Thr 305 310 315 320 Cys Pro Pro Cys Pro Ala Pro Glu Ala Glu Gly Ala Pro Ser Val Phe 325 330 335 Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro 340 345 350 Giu Val Thr Cys Val Val Val Asp Val Ser His Giu Asp Pro Giu Val 355 360 365 Lys Phe Asn Trp Tyr Val Asp Gly Val Giu Val His Asn Ala Lys Thr 370 375 380 Lys Pro Arg Glu Glu Gin Tyr Asn Ser Thr Tyr Arg Val Val Ser Val 385 390 395 400 Leu Thr Val Leu His Gin Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys 405 410 415 Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser 420 425 430 r- Ct Lys Ala Lys 435 Ser Arg Asp Gly Gin Pro Arg Glu Leu Thr Lys 455 Tyr Pro Ser Asp Gin Val Ser Pro Gin Val Tyr Thr Leu Pro Pro 445 Leu. Thr 460 450 Lys Gly Phe Ile Ala Val 465 Gin Glu Trp Giu. 475* Pro Val Leu Cys Leu. Val Ser Asn Gly 480 Asp Ser Asp Pro Giu Asn Asn Lys Thr Thr 485 Leu Pro 490 Thr 495 Gly Ser Phe Phe 500 Gin Gin Gly Asn 515 Asn His Tyr Thr 530 Tyr Ser Lys Leu 505 Ser Val Asp Lys Glu Ser Arg Trp, 510 Ala Leu His Val Phe Ser Cys 520 Leu Val Met His Gin Lys Ser 535 525 Gly Lys Ser Leu Ser Pro 540 <210> 17 <211> 1668 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: fusion polypept ide <220> <221> CDS <222> (25)..(1647) <400> 17 gtcgacccaa gctggctagc cacc atg gag aca gac aca ctc Met Glu Thr Asp Thr Leu ctg cta tgg Leu. Leu Trp gta Val1 ctg ctg ctc tgg gtt cca ggt tcc act Leu. Leu Leu. Trp Val Pro Gly Ser Thr 15 ggt Gly act agt tgt gga Thr Ser Cys Gly gga tac gtc gaa Gly Tyr Val Glu gc t Ala ggg gag gag tgt Gly Glu. Giu, Cys tgt ggt ttt cat gtg gaa Cys Gly Phe His Val Giu 147 tgc tat gga Cys Tyr Gly tgc agc gac Cys Ser Asp tta Leu. tgc tgt aag aaa Cys Cys Lys Lys tgt Cys 50 tcc ctc tcc aac Ser Leu Ser Asn ggg gct cac Gly Ala His ttt cag cca Phe Gin Pro ggg ccc tgc tgt Gly Pro Cys Cys aac Asn 65 aat acc tca tgt Asn Thr Ser Cys ctt Leu. cga ggg Arg Gly tat gaa tgc cgg Tyr Giu Cys Arg gct gtg aac gag Ala Val Asn Glu gat att act gaa Asp Ile Thr Glu tat Tyr tgt act gga gac Cys Thr Gly Asp ggt cag tgc cca Gly Gin Cys Pro aat ctt cat aag Asn Leu His Lys caa Gin 105 291 339 387 gac gga tat gca Asp Gly Tyr Ala aat caa aat cag Asn Gin Asn Gin ggc Giy 115 cgc tgc tac aat Arg Cys Tyr Asn ggc gag Gly Glu 120 tgc aag gcc Cys Lys Ala gac aac cag tgt Asp Asn Gin Cys tac atc tgg gga Tyr Ile Trp Gly aca aag gct Thr Lys Ala 135 gca ggg tct gac aag ttc tgc tat gaa aag ctg aat aca gaa Ala Gly Ser Asp Lys Phe Cys Ty-r Glu Lys Leu Asn Thr Glu 140 145 150 ggc act Gly Thr gag aag gga Glu Lys Gly 155 aaa cat gat Lys His Asp 170 gct cca cgt Ala Pro Arg tac cat caa Tyr His Gin gat gat gat Asp Asp Asp 220 ccg tct atg Pro Ser Met 235 aat atg agc Asn Met Ser 250 cat ggg gtg His Gly Val gca ggg aca Ala Gly Thr 1 ccc aag gat g Pro Lys Asp G 300 gac aaa act c Asp Lys Thr H 315 gcg ccg tca g Ala Pro Ser V 330 atc tcc cgg a aac tgc ggg aag As gt4 Va ati I1( 99C G1j acC Thr atg Met agc Ser tgt -ys a t ~sp raa 1u ac is tc al cc n Cys Gly Lys 160 9 ttC tgt gga 1. Phe Cys Gly 175 tggt caa ctt 2 Gly Gin Leu 190 cgg gtg att SArg Val Ile gat gtg ggc Asp Val Gly tgt tta gat Cys Leu Asp 240 tgt cca ctc Cys Pro Leu 255 agt aat gaa Ser Asn Glu 270 tgc agt atc Cys Ser Ile gga ccc aag Gly Pro Lys aca tgc cca c Thr Cys Pro 1 320 ttc ctc ttc C Phe Leu Phe P gat gga gac cgg Asp Gly Asp Arg tgg Trp 165 att cag tgc agc Ile Gin Cys Ser ttc tta ctc tgt acc aat ctt act cga Phe Leu Leu cag ggt Gir gac Asj tat Tyr 225 Cgg Arg gat Asp gcc Ala A.rg ggt W05 ~Cg ~ro 'cc i Gly tgC Cys 210 gta Val aag Lys t cc Ser acc Thr ga t Asp 290 cct Pro tgc Cys cca gag Glu 195 agt Ser gaa Glu tgC Cys aag Lys tgc Cys 275 cca Pro agt Ser cca P~ro iaa Cys Thr 180 atc att Ile Ile ggt gcc Gly Ala gat gga Asp Gly cta caa Leu Gin 245 ggt aaa Gly Lys 260 att tgt Ile Cys Asn cca Pro cat His acg Thr 230 att Ile gtc Val ga t Asp aac ksn ia t ksn raa lu Leu Thr Arg 185 act tcc ttc Thr Ser Phe 200 gta gtt tta Val Val Leu 215 cca tgt ggc Pro Cys Gly caa gcc cta Gin Ala Leu tgt tcg ggc Cys Ser Gly 265 ttc acc tgg Phe Thr Trp gtt Val1 gcc Ala gca Ala agg Arg acc Thr cc t Pro 325 aag 3 ctt Leu 295 aga Arg gcc Ala 280 531 579 627 675 723 771 819 867 915 963 1011 1059 1107 1155 1203 1251 cac His tct Ser gag Glu ccc Pro tgt Cys ggc Gly ccc cct 335 grc9 cCt t ~ro Pro Lys Pro Lys Asp Thr Leu Met 340 345 Ile Ser Arg Thr Pro 350 gag gtc aca Glu Val Thr tgc gtg gtg gtg gac gtg agc cac Cys Val Val Val Asp Val Ser His 355 360 gaa Giu cat His Cgg cct Pro gcc Ala 380 gtC gtc Val1 aca Thr gtc ttc Phe ccg Pro acc aac Asn cgg Arg 385 gtC tgg Trp 370 gag Giu c tg tac Tyr gag Glu gac Asp tac Tyr ggc Gly aac Asn 390 tgg gag Glu acg Thr aat gtg Val tac Tyr 9gc cac cag gac Arg Val 395 Val Ser Val Leu. Thr Val Leu His Gin Asp Trp Leu Asn Giy gag tac aag tgc Giu Tyr Lys Cys 400 aag gtc tcc aac aaa gcc Lys Val Ser Asn Lys Ala 415 420 405 ctc cca gcc ccc Leu. Pro Ala Pro atc Ile gag aaa acc atc Giu Lys Thr Ile tcc Ser 430 aaa gcc aaa ggg Lys Ala Lys Gly cag Gin 435 ccc cga gaa cca Pro Arg Glu Pro 425 cag gtg Gin Val 440 tac acc ctg Tyr Thr Leu ctg acc tgc Leu Thr Cys 460 ccc Pro 445 cca tcc cgg gat Pro Ser Arg Asp gag Glu 450 ctg acc aag aac cag gtc agc Leu. Thr Lys Asn Gin Val Ser 455 ctg gtc aaa ggc Leu. Val Lys Giy tat ccc agc gac Tyr Pro Ser Asp gcc gtg gag Ala Val Glu tgg gag Trp Giu 475 agc aat ggg cag Ser Asn Giy Gin ccg Pro 480 gag aac aac tac Giu Asn Asn Tyr aag acc acg cct ccc Lys Thr Thr Pro Pro 485 agc aag ctc acc gtg Ser Lys Leu Thr Val 1299 1347 1395 1443 1491 1539 1587 1635 1668 gtg Vali 490 ctg gac tcc gac Leu Asp Ser Asp ggc Gly 495 tcc ttc ttc ctc Ser Phe Phe Leu gac aag agc agg Asp Lys Ser Arg tgg Trp 510 cag cag ggg aac Gin Gin Gly Asn gtc Val 515 ttc tca tgc tcc Phe Ser Cys Ser gtg atg Val Met 520 cat gag gct His Giu Ala cac aac cac tac His Asn His Tyr cag aag agc ctc Gin Lys Ser Leu tcc ctg tct Ser Leu Ser 535 ccg ggt aaa tga actagagcgg ccgc Pro Gly Lys 540 <210> 18 <211> 540 <212> PRT <213> Artificial Sequence <223> Description of Artificial polypept ide tacaga t Sequence: fusion <400> 18 Met Giu Thr 1 Giy Ser Thr Giu Cys Asp Lys Cys Ser Asp Gly Cys Leu Thr 5 Leu Leu Leu Trp Val Leu Leu Leu Trp Val Pro Thr Ser Cys Gly Gly Phe His Val Ser Asn Gly Ala 55 Cys Leu Phe Gin Asn 25 Glu Tyr Val Glu Cys Tyr Gly Ala Gly Giu Cys Cys Lys Pro Cys Cys Asn Asn Thr Ser His Cys Ser Asp Tyr Pro Am Ala Val Asn Giu Cys Gin Cys Pro Pro Asn 70 Asp Ile Thr Giu Tyr 90 Leu His Lys Gin Asp Gly Giu Cys Arg Cys Thr Gly Gly Tyr Ala Asp Ser Gly Cvs Asn Gin Asn Gin Gly 115 100 Arg Cys Tyr Asn Gly 120 Cys Lys Ala 110 Arg Asp Asn Gin 125 Cys Gin 130 Tyr Ile Trp Gly Thr Lys Ala Ala 135 Gly Ser Asp Lys Phe Cys 140 Glu Lys Leu Asn Thr Glu Gly Thr Git Asp Phe Gin Asp Tyr 225 Arg Asp Ala Arg Gly 305 Pro Pro Thr Asn Arg 385 Val Ser Lys Asp Phe 465 Glu I~ Phe I Gly P~ GI' Lei Gl~ CY~ 21( Val LyE Sez Thr Asp 290 Pro Cys Pro Cys Trp 370 Glu Leu Asn G ly 450 ['yr sn ~he ~sn y Asp u1 Leu ~Glu 195 5 Ser LGlu Cys Lys Cys 275 Pro Ser Pro Lys Val 355 Tyr Glu His Lys Gin 435 Leu Pro Asn Leu r Val P 515 Ar~ Cy~ 18( I l Gl) Asr Let Gl- 260 Ile Val1 Ala Ala Pro 340 Val1 Val1 Gln G1n kla 420 Pro ['hr ;er 'yr 'r 00O ~he ;Trj 161 s Thi SAla Gly Gin 245 Lys Cys Arg Thr Pro 325 Lys Val1 Asp Tyr Asp 405 Leu Arg Lys Asp Lys 485 Ser Ser )Iii Asi Prc Hi~ Thi 23( IlE Val Asr Asn Asn 310 Glu Asp Asp Gly Asn 390 Trp Pro Glu Asn Ile 470 Thr Lys Cys eGin -i Leu Thr 5 Val 215 Pro Gin *Cys Phe Leu 295 Arg *Ala Thr Val Val 375 Ser Leu Ala Pro Gin 455 Ala Thr Leu 'I Ser X 5 Cys Thr Ser 200 Val Cys Ala Ser Thr 280 His Ser Glu Leu Ser 360 Giu Thr Asn Pro Aln 440 lal fal ?ro 'hr al1 20 Se: AZ 18~ *PhE Let G13 Let Gl 265 Trp Pro Cys Gly Met 345 His Val Tyr Giy Ile 425 Val Ser Giu Pro Val 505 Met rLys 170 SAla 5 STyr zAsp rPro Asn 250 His Ala Pro Asp Ala 330 Ile Glu His Arg Lys 410 Giu Tyr Leu Trp Val 490 AspI His C Ly~ Hi~ Pri Hi~ Asi Se2 231 Met Glj Glj Lys Lys 315 Pro Ser Asp Asn Val1 395 Giu Lys rhr rhr 'lu 475 eu lu a Gly Asn Cys Giy Lys s Asp 0 Arg s Gin SAsp 220 Met Ser Val Thr Asp 300 Thr pSer Arg Pro Ala 380 Val Tyr Thr Leu Cys 460 Ser Asp I Ser Ala L Lys 540 Va I l Glj 2 01 Thl Met Ser Cys Asp 285 Giu His Vali Thr Glu 365 Lys5 Ser Lys Ile Pro 445 eu k.sn ~er ~rg 4 eu 2 1 Phe a Gly 190 tArg Asp Cys Cys Ser 270 ICys Gly Thr Phe Pro 350 Val Thr Val Cys Ser 430 Pro ValI Gly C Asp C Trp G 510 His A Cys 175 Gin Val Val Leu Pro 255 Asn Ser Pro Cys Leu 335 Glu Leu Lys 115 'er ,ys fln ;iy ;in sn Gly Leu Ile Gly Asp 240 Leu Glu Ile Lys Pro 320 Phe Vali Phe Pro Thr 400 Val1 Ala Arg Gly Pro 480 Ser Gln His Tyr Thr Gin Lys Ser Leu Ser Leu Ser Pro Gly 530 535 <210> 19 <211> 3 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Consensus binding motif <400> 19 Arg Gly Asp I <210> <211> 67 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: consensus disintegrin domain <220> <221> VARIANT <222> <223> 3-5 varying residues in a consensus sequence <220> <221> VARIANT <222> <223> 3-6 varying residues in a consensus sequence <220> <221> VARIANT <222> <223> 2-4 varying residues in a consensus sequence <220> <221> VARIANT <222> <223> 7 varying residues in a consensus sequence <220> <221> VARIANT <222> <223> 4-6 varying residues in a consensus sequence <220> <221> VARIANT <222> <223> 2-4 varying residues in a consensus sequence <220> <221> VARIANT <222> <223> 8 varying residues in a consensus sequence <220> <221> VARIANT <222> <223> 5-7 varying residues in a consensus sequence <220> <221> VARIANT <222> <223> 3-5 varying residues in a consensus sequence <400> Cys Asp Cys Gly Xaa Xaa Xaa Xaa Xaa Cys Xaa Xaa Xaa Xaa Xaa Xaa 1 5 10 Cys Cys Xaa Xaa Xaa Xaa Cys Xaa Xaa Xaa Xaa Xaa Xaa Xaa Cys Xaa 25 Xaa Xaa Xaa Xaa Xaa Cys Cys Xaa Xaa Xaa Xaa Cys Xaa Xaa Xaa Xaa 40 Xaa Xaa Xaa Xaa Cys Xaa Xaa Xaa Xaa Xaa Xaa Xaa Cys Xaa Xaa Xaa 55 Ct Xaa Xaa Cys <210> 21 <211> 1725 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: fusion polypeptide <220> <221> CDS <222> (118)..(1704) <400> 21 gggttttecc agteacgaeg ttgtaaaaeg acggceagtg aattgtaata egactcacta tagggegaat tgggtaccgg gcccccectc gaggtegaec caagctgget agecace 11' atg gag aca gac aca etc etg eta tgg gta ctg ctg etc tgg Met Giu Thr Asp Thr Leu Leu Leu Trp Val Leu Leu Leu Trp gtt eca Val Pro 1 ggt tee Gly Ser gag tgt Glu Cys etg tea Leu Ser tgt tge Cys Cys gag gte Glu Val aag tgc Lys Cys agg gge Arg Gly agg agg Arg Arg 130 aaa gaa Lys GluI 145 act Thr gac Asp 35 aa t Asn aaa Lys aa t Asn cca Pro tac T'yr 115 att Ile ttg *ggt *Gly 20 *tgt Cys tgc Cys gac Asp gaa Glu ga t Asp 100 tgc Cys ttt Phe aac 5 act Thr gga Gly act Thr tgc Cys tgt Cys gac Asp tat Tyr ggt Gly ace agt Ser ect Pro etg Leu aag Lys 70 ga t Asp ttt Phe gaa Glu tgt Cys tta Leu act Thr 55 tte Phe ett Leu tat Tyr aag Lys 9g9 Gly aag Lys 40 ga t Asp eta Leu eca Pro gtg Val1 age Ser 120 gea Ala gac Asp 10 aat ggt gtg Asn Gly Val 25 cat tgt gca His Cys Ala ggt tet act Gly Ser Thr eca tea ggg Pro Ser Gly 75 gag tgg tgc Glu Trp Cys 90 gaa gat gga Glu Asp Gly 105 tgt cat gac Cys His Asp aat act gca Asn Thr Ala cgt gtt ggt Arg Val Gly gtt gaa gaa Val Glu Glu 30 aaa gat ccc Lys Asp Pro 45 tgt get ttt Cys Ala Phe 60 aaa gtg tgt Lys Val Cys aat ggt act Asn Gly Thr att ccc tgt Ile Pro Cys 110 ege aat gaa Arg Asn Glu 125 agt gag act Ser Glu Thr 140 cae tgt ggt Hlis Cys Gly gga Gly tgC Cys ggg Gly aga Arg tee Ser aag Lys eag Gin tge Cys atc Ile gaa Glu tgt Cys ett Leu aag Lys eat His gag Glu tgt Cys tac T'yr ~aaa ys 165 213 261 309 357 405 453 501 549 597 gca ggc Ala Gly 135 :ta ggt Leu Gly ieu Asn ThrI aat get aca tat Asn Ala Thr Tyr ata Ile 165 aag tgt aat ate Lys Cys Asn Ile tea gat gte cag Ser 170 Asp Val Gin tgt gga aga Cys Gly Arg 175 att cag tgt Ile Gin Cys act gtg cat Thr Val His 195 gag Glu 180 aat gtg aca gaa Asn Val Thr Glu att Ile 185 ccc aat atg agt Pro Asn Met Ser gat cat act Asp His Thr 190 agt act gat Ser Thr Asp tgg gct cgc ttc Trp Ala Arg Phe aat Asn 200 gac ata atg tgc Asp Ile Met Cys 693 741 789 tac cat Tyr His 210 ttg ggg atg aag Leu Gly Met Lys cct gat att ggt Pro Asp Ile Gly gaa Glu 220 gtg aaa gat gga Val Lys Asp Gly aca Thr 225 gag tgt ggg ata Glu Cys Gly Ile gat Asp 230 cat ata tgc atc His Ile Cys Ile cac His 235 agg cac tgt gtc Arg His Cys Val ata acc atc ttg Ile Thr Ile Leu aat Asn 245 agt aat tgc tca Ser Asn Cys Ser cct Pro 250 gca ttt tgt aac Ala Phe Cys Asn aag agg Lys Arg 255 ggc atc tgc Gly Ile Cys cct ccc aac Pro Pro Asn 275 aac Asri 260 aat aaa cat cac Asn Lys His His cat tgc aat tat His Cys Asn Tyr ctg tgg gac Leu Trp Asp 270 gac agt ggc Asp Ser Gly tgc ctg ata aaa Cys Leu Ile Lys tat gga ggt agt Tyr Gly Gly Ser gtt Val 285 cca ccc Pro Pro 290 cct aag aga aag Pro Lys Arg Lys aag Lys 295 aaa aag aag aga Lys Lys Lys Arg tgt gac aaa act Cys Asp Lys Thr cac His 305 aca tgc cca ccg Thr Cys Pro Pro cca gca cct gaa Pro Ala Pro Giu gag ggc gcg ccg Giu Gly Ala Pro gtc ttc ctc ttc Val Phe Leu Phe ccc Pro 325 cca aaa ccc aag Pro Lys Pro Lys gac Asp 330 acc ctc atg atc Thr Leu Met Ile tcc cgg Ser Arg 335 acc cct gag Thr Pro Giu gag gtc aag Glu Val Lys 355 gtc Val1 340 aca tgc gtg gtg gtg gac gtg agc cac Thr Cys Val Val Val Asp Val Ser His 345 gaa gac cct Glu Asp Pro 350 cat aat gcc His Asn Ala ttc aac tgg tac Phe Asn Trp Tyr gtg Val1 360 gac ggc gtg gag Asp Gly Val Giu 1029 1077 1125 1173 1221 1269 1317 1365 1413 1461 aag aca Lys Thr 370 aag ccg cgg gag Lys Pro Arg Glu cag tac aac agc Gin Tyr Asn Ser acg Thr 380 tac cgg gtg gtc TPyr Arg Val Val agc Ser 385 gtc ctc acc gtc Val Leu Thr Val ctg Leu 390 cac cag gac tgg His Gin Asp Trp ctg Leu 395 aat ggc aag gag Asn Gly Lys Glu aag tgc aag gtc Lys Cys Lys Val tcc Ser 405 aac aaa gcc ctc cca gcc ccc atc gag Asn Lys Ala Leu Pro Ala Pro Ile Glu 410 aaa acc Lys Thr 415 atc tcc aaa Ile Ser Lys gcc Ala 420 aaa ggg cag ccc Lys Gly Gin Pro gaa cca cag gtg Giu Pro Gin Val tac acc ctg Tyr Thr Leu 430 ccc cca tcc cgg gat gag ctg acc aag aac cag gtc agc ctg acc tgc Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gin Val Ser Leu Thr Cys ctg gtc Leu Val 450 aat ggg Asn Gly 435 aaa ggc ttc tat Lys Gly Phe Tyr 440 agc gac atc gcc Ser Asp Ile Ala 445 gag tgg gag agc Giu Trp Glu. Ser ccc Pro 455 aac Asri gtg Val 460 cc t Pro cag ccg gag Gin Pro Glu 465 tcc Ser aac Asn 470 ttc Phe tac aag acc Tyr Lys Thr acg Thr 475 C tc Leu ccc gtg ctg Pro Val Leu gac Asp 480 gac ggc tcc Asp Gly Ser t tc Phe 485 ctc tac agc Leu Tyr Ser aag Lys 490 tgc Cys acc gtg gac Thr Val Asp agg tgg cag cag ggg aac gtc ttc Arg Trp Gin Gin Gly Asn Val Phe 500 ctg cac aac cac tac acg cag aag Leu His Asn 1-us Tyr Thr Gin Lys 515 520 tga actagagcgg ccgctacaga t <210> 22 <211> 528 <212> PRT <213> Artificial Sequence <223> Description of Artificial polypeptide tca Ser 505 tcc gtg atg Ser Val Met cat His 510 aag agc Lys Ser 495 gag gct Giu Ala ggt aaa Gly Lys 1509 1557 1605 1653 1701 1725 agc ctc tcc ctg tct Ser Leu Ser Leu Ser Pro Sequence: fusion Trp Val Leu Leu Leu. Trp Val Pro <400> 22 Met Gly Giu Leu Cys Glu Lys Arg Arg Lys 145 Asn Ile Thr Tyr Thr 225 lSer Cys Ser Cys Va 1 Cys Gly Arg 130 Giu Ala Gin Val1 His 210 Glu. Thr Thr Asp Asn Lys Asn Pro Tyr 115 Ile Leu Thr Cys His 195 Leu Cys Asp Thr Leu 5 Gly Thr Ser Cys Giy Pro Cys Thr Leu Asp Cys Lys 70 Glu Cys Asp Asp Asp Phe 100 Cys Tyr Giu Phe Gly Ala Asn Thr Leu 150 Tyr Ile Lys 165 Giu Asn Val 180 Trp Ala Arg Gly Met Lys Gly Ile Asp 230 Leu Cys Leu Thr 55 Phe Leu Tyr Lys Gly 135 Gly Cys Thr Phe 215 His Leu Gly Lys 40 Asp Leu Pro Val1 Ser 120 Ala Asp Asn Glu Asn, 200 Pro Ile Asn 25 His Gly Pro Giu Giu 105 Cys Asn Arg Ile Ile 185 Asp Asp :ys Gly Val Val Cys Ala Lys Ser Thr Cys Ser Gly Lys 75 Trp Cys Asn 90 Asp Gly Ile His Asp Arg Thr Ala Ser 140 Val Gly His 155 Ser Asp Val 170 Pro Asn Met Ile Met Cys Ile Gly Glu 220 Ile His Arg Giu Asp Ala Val1 Gly Pro Asn 125 Glu Cys Gin Ser Trp 205 Val1 Giu. Pro Phe Cys Thr Cys 110 Giu Thr Gly Cys Asp 190 Ser Lys Gly Cys Gly Arg Ser Lys Gin Cys Ile Gly 175 His rhr Asp Giu Cys Leu Lys His Glu Cys Tyr Lys 160 Arg Thr Asp Gly His Cys Val His Ile Thr Ile Leu Asn Ser Asn Cys Ser 245 235 240 Pro Ala Phe Cys Asn Lys Arg 250 255 ct Gly Ile Cys s 260 Asn Lys His His Cys His Cys Asn 265 Pro Pro Pro Pro 290 His Thr 305 Val Phe Thr Pro Giu Val Lays Thr 370 Ser Val 385 Lys Cys Ile Ser Pro Pro Leu Val 450 Asn Gly 465 Ser Asp Arg Trp Asn 275 Pro Cys Leu Giu Lys 355 Lys Leu Lys Lys Ser 435 Lys Gin Gly Gin Cys Lys Pro Phe Val1 340 Phe Pro Thr Val1 Ala 420 Arg Gly Pro Ser Gin 500 Tyr Leu Trp Asp 270 Leu Arg Pro Pro 325 Thr Asn Arg Val1 Ser 405 Lys Asp Phe Glu Phe 485 Gly Ile Lys Lys Lys 295 Cys Pro 310 Pro Lys Cys Val Trp Tyr Glu Giu 375 Leu His 390 Asn Lys Giy Gin Giu Leu Tyr Pro 455 Asn Asn 470 Phe Leu Asn Val Gly 280 Lys Ala Pro Val1 Val1 360 Gin Gin Ala Pro Thr 440 Ser Tyr Tyr Phe Tyr Lys Pro Lys Val 345 Asp Tyr Asp Leu Arg 425 Lys Asp Lys Ser Ser 505 Gly Lys Giu Asp 330 Asp Gly Asn Trp Pro 410 Giu Asn Ile Thr Lys 490 Cys Gly Arg Ala 315 Thr Val1 Val1 Ser Leu 395 Ala Pro Gin Ala Thr 475 Leu Ser Ser Ser 300 Giu Leu Ser Giu Thr 380 Asn Pro Gin Val Val1 460 Pro Thr Val1 Val1 285 Cys Gly Met His Val1 365 Tyr G ly Ile Val1 Ser 445 Giu Pro Val1 Met Ser 525 Asp Asp Al a Ile Giu 350 His Arg Lys Giu Tyr 430 Leu Trp Val1 Asp His 510 Pro Ser Lys Pro Ser 335 Asp Asn Val1 Giu Lys 415 Thr Thr Glu Leu Lys 495 Giu Gly Gly Thr Ser 320 Arg Pro Ala Val1 Tyr 400 Thr Leu Cys Ser Asp 480 Ser Ala Lys Leu His Asn His Tyr Thr Gin Lys Ser Leu Ser Leu 515 520