Purified soluble vascular endothelial cell growth factor (vegf) inhibitor encoding purified dna, a process for its preparation and pharmaceutical compositions comprising it

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Purified soluble vascular endothelial cell growth factor (VEGF) inhibitor encoding purified DNA, a process for its preparation and pharmaceutical compositions comprising thereof

Merck & Co. Inc. C.092553 - 1 - 109016/2

BACKGROUND OF THE DISCLOSURE

Recently a new class of cell-derived dimeric mitogens with selectivity for vascular endothelial cells has been identified and designated vascular 5 endothelial cell growth factor (VEGF). VEGF has been purified from conditioned growth media of rat glioma cells [Conn et al., (1990), Proc. Natl. Acad. Sci. U.S.A., 87, pp 2628-2632, Duan D.S. et al., J. Biol. Chem. (1991), 266, 413-418]; and conditioned growth 10 media or bovine pituitary folliculo stellate cells [Ferrara and Henzel, (1989), Biochem. Biophys. Res. Comm., 161, pp. 851-858; Gozpadorowicz et al., (1989),

Proc. Natl. Acad. Sci. U.S.A., 86, pp. 7311-7315] and conditioned growth medium from human U937 cells 15 [Connolly, D.T. et al. (1989), Science, 246, pp. 1309-1312]. VEGF is a dimmer with an apparent molecular mass of about 46 kDa with each subunit having an apparent molecular mass of about 23 kDa. VEGF has some structural similarities to 20 platelet derived growth factor (PDGF), which is a mitogen for connective tissue cells but not mitogenic for vascular endothelial cells from large vessels.

The membrane-bound tyrosine kinase receptor, known as FLT, was shown to be a VEGF receptor [DeVries, 25 C. etal., (1992), Science, 255, pp. 989-991, Shibuya,

M. et al., (1990, Oncogene, 5, 519-524]. The FLT receptor specifically binds VEGF which induces mitogenesis. Another form of the VEGF receptor, designated KDR, is also known to bind VEGF and induce 30 mitogenesis. The partial cDNA sequence and nearly full length protein sequence of KDR is known as well [Terman, B.I. et al., (1991) Oncogene 6, pp. 1677-1683;

Terman, B.I. et al., (1992) Biochem. Biophys. Res.

Comm. 187, pp. 1^79-1586, Terman, B.I., WO 92/14748. -2- 109016.3

Persistent angiogenesis may cause or exacerbate certain diseases such as psoriasis, rheumatoid arthritis, hemangiomas, angiofibromas, diabetic retinopathy and neovascular glaucoma. An 5 inhibitor of VEGF activity would be useful as a treatment for such diseases and other VEGF-induced pathological angiogenesis and vascular permeability conditions, such as tumor vascularization.

SUMMARY OF THE DISCLOSURE

10 The present invention provides a purified DNA molecule encoding a soluble VEGF inhibitor protein wherein said protein comprises the amino acid sequence set forth in SEQ ID NO: 6. In one embodiment, the purified DNA molecule comprises the nucleotide sequence 15 of SEQ ID NO:5. In another embodiment, the DNA molecule consists of SEQ ID NO:5.

Further, there is provided an expression vector for expressing a soluble VEGF inhibitor protein in a recombinant host cell wherein said expression 20 vector comprises the DNA molecule which purified DNA molecule which encodes said VEGF inhibitor protein.

In one embodiment the expression vector contains a DNA molecule which comprises the nucleotide sequence as set forth in SEQ ID NO:5. 25 In another embodiment the expression vector contains a DNA molecule which contains the nucleotide sequence as set forth in SEQ ID NO:5.

Further, there is provided a host cell which expresses a recombinant soluble VEGF inhibitor protein 30 wherein said host cell ' contains the expression vector for expressing said VEGF inhibitor protein.

There is also provided a process for the expression of a .soluble VEGF inhibitor protein which comprising: -3- 109016/3 (a) transfecting the said expression vector into a soluble host cell; and

(b) culturing the host cells under conditions suitable for expression of said soluble VEGF 5 inhibitor protein from said expression vector.

Also provided is a process for the expression of a soluble VEGF inhibitor-protein, comprising: (a) transfecting the said expression vector into 10 a soluble host cell; and (b) culturing the host cells under conditions suitable for expression of said soluble VEGF inhibitor protein from said expression vector.

15 Further provided is a purified soluble VEGF inhibitor protein which comprises the amino acid sequence as set forth in SEQ ID No. 6. The inhibitor is used in inhibiting the VEGF receptor function. In another embodiment, the inhibitor is used for 20 inhibiting angiogenesis.

There is also provided a pharmaceutical composition comprising a VEGF inhibitor which consists or comprises the amino acid sequence as set forth in SEQ ID NO:6.

25 BRIEF DESCRIPTION OF THE DRAWINGS

Figure 1 - A schematic diagram of full length VEGF receptors (FLT and KDR), the soluble VEGF receptors (sVEGF-RI and sVEGF-RII) and the soluble receptors 30 -3a- 109016/1 containing the C-terminal transmembrane region (sVEGF-RTMI and sVEGF-RTMII) are shown with the protein domains of each.

5 Figure 2 - The DNA sequence of the sVEGF-RI soluble VEGF receptor/VEGF inhibitor is shown.

Figure 3 - The amino acid sequence of the sVEGF-RI soluble VEGF receptor/VEGF inhibitor is shown.

Figure 4 - Demonstration that recombinant 15 host cells express sVEGF-RI is shown by

I 117/JWW45 18888 the formation of high molecular weight complexes of sVEGF-RI and [125IJVEGF and separated by size exclusion chromatography.

Figure 5 - A 12.5% polyacrylamide electrophoretic gel is shown which demonstrates the high degree of purity obtained for sVEGF-RI.

Figure 6 - Cross-linked products of sVEGF-RI and [^^^ijveGF are shown at about 145 kDa, and at about 245 kDa.

Figure 7A and 7B - Analysis of VEGF binding to sVEGF-RI (A) and corresponding Scatchard plot (B).

Figure 8 - Inhibition of [^2^I]VEGF binding to HUVECs by sVEGF-RI is demonstrated.

Figure 9 - Inhibition of VEGF-mediated mitogenesis on HUVECs is shown using sVEGF-RI.

Figure 10 - The nucleotide sequence encoding sVEGF-RII is shown.

Figure 11 - The amino acid sequence for sVEGF-RII is shown. 117/JWW45 5 18888 -, Figure 12 - The nucleotide sequence encoding sVEGF-RTMII is shown. 5 Figure 13 - The amino acid sequence for sVEGF-RTMII is shown.

Figure 14 - The nucleotide sequence encoding sVEGF-RTMI is shown. 10

Figure 15 - The amino acid sequence for sVEGF-RTMI is shown.

Figure 16 - A diagram of pmFLT is shown. 15

Figure 17 - A diagram of pKDRA is shown.

DETAILED DESCRIPTION OF THE DISCLOSURE

The present invention relates to cDNA 20 encoding a soluble VEGF receptor protein (sVEGF-R) which is isolated from VEGF receptor producing cells or is recombinantly engineered from VEGF receptor-encoding DNA. sVEGF-R, as used herein, refers to a protein which can specifically bind to a vascular endothelial 25 cell growth factor without stimulating mitogenesis of vascular endothelial cells.

The amino acid sequence of FLT is known, [Shibuya, M. et al.. (1990), Oncogene, £, pp.519-524] and corresponds to the full length cell-associated VEGF 30 tyrosine kinase receptor. Other VEGF receptors are known to exist. Other known VEGF receptors include, 117/JWW45 18888 but are, not limited to KDR [Terman (1991), supra., and Terman (1992), supra.]. Mammalian cells capable of producing FLT, KDR and other VEGF receptors include, but are not limited to, vascular endothelial cells. Mammalian cell lines which produce FLT or KDR and other VEGF receptors include, but are not limited to, human endothelial cells. The preferred cells for the present invention include human umbilical vein endothelial cells (HUVEC).

Other cells and cell lines may also be suitable for use to isolate sVEGF-R cDNA. Selection of suitable cells may be done by screening for sVEGF-R binding activity on cell surfaces, in cell extracts or conditioned medium or by screening for gene expression by PCR or hybridization. Methods for detecting soluble receptor activity are well known in the art [Duan, D-S. R. et al., (1991) J.Biol.Chem., 266. pp.413-418] and measure the binding of labelled VEGF. Cells which possess VEGF binding activity in this assay may be suitable for the isolation of sVEGF-R cDNA.

Full length FLT producing cells such as human HUVEC cells (American Type Culture Collection, ATCC CRL 1730) [Hoshi, H. and McKeehan, W.L., Proc. Natl. Acad. Sci. U.S.A., (1984) 81. pp. 6413-6417] are grown according to the recommended culture conditions of the ATCC. Full length FLT, and KDR VEGF receptors as well as extracellular region (sVEGF-RI and sVEGF-RII) and extracellular region plus transmembrane region forms (sVEGF-RTMI and sVEGF-RTMII) are shown in Figure 1.

The full length receptor has an extracellular ligand 117/JWW45 18888 bindings region composed of about seven immunoglobulin-like domains, a membrane spanning sequence (transmembrane domain) and intracellular 5 tyrosine kinase domains. The inhibitory forms of this receptor, which are the subject of the present invention, are also shown in Figure 1 and lack the intracellular kinase domains, and for some inhibitors, the transmembrane sequence and the C-terminal most 10 Ig-like extracellular domain.

Any of a variety of procedures may be used to molecularly clone sVEGF-R cDNA. These methods include, but are not limited to, direct functional expression of the sVEGF-R gene following the construction of an 15 sVEGF-R-containing cDNA library in an appropriate expression vector system.

Another method is to screen a sVEGF-R-containing cDNA library constructed in a bacteriophage or plasmid shuttle vector with a labelled 20 oligonucleotide probe designed from the predicted amino acid sequence of sVEGF-R. The preferred method consists of screening a sVEGF-R-containing cDNA library constructed in a bacteriophage or plasmid shuttle vector with a partial cDNA encoding at least part of 25 the full length FLT protein. This partial cDNA is obtained by the specific PCR amplification of sVEGF-R DNA fragments through the design of oligonucleotide primers from the known sequence of the full length FLT-encoding DNA. 30 it is readily apparent to those skilled in the art that other types of libraries, as well as 117/JWW45 18888 libraries constructed from other cells or cell types, may be useful for isolating sVEGF-R-encoding DNA.

Other types of libraries include, but are not limited to, cDNA libraries derived from other cells or cell lines other than HUVECs and genomic DNA libraries.

It is readily apparent to those skilled in the art that suitable cDNA libraries may be prepared from cells or cell lines which have sVEGF-R activity. The selection of cells or cell lines for use in preparing a cDNA library to isolate sVEGF-R cDNA may be done by first measuring secreted sVEGF-R activity using the VEGF binding assay described fully herein.

Preparation of cDNA libraries can be performed by standard techniques well known in the art. Well known cDNA library construction techniques can be found for example, in Maniatis, T., Fritsch, E.F., Sambrook, J., Molecular Cloning: A Laboratory Manual (Cold Spring Harbor Laboratory, Cold Spring Harbor, New York, 1982).

It is also readily apparent to those skilled in the art that DNA encoding sVEGF-R may also be isolated from a suitable genomic DNA library. Construction of genomic DNA libraries can be performed by standard techniques well known in the art. Well known genomic DNA library construction techiques can be found in Maniatis, T., Fritsch, E.F., Sambrook, J. in Molecular Cloning: A Laboratory Manuel (Cold Spring Harbor Laboratory, Cold Spring Harbor, New York, 1982).

Another means of obtaining sVEGF-R molecules is to recombinantly engineer them from DNA encoding the

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117/JWW45 18888

partial, or complete amino acid sequence of a VEGF receptor. Examples of other VEGF receptors include, but are not limited to, KDR. Using recombinant DNA 5 techniques, DNA molecules are constructed which encode at least a portion of the VEGF receptor capable of binding VEGF without stimulating mitogenesis. Standard recombinant DNA techniques are used such as those found in Maniatis, et al.. supra. 10 Using one of the preferred methods of the present invention, cDNA clones encoding sVEGF-R are isolated in a two-stage approach employing polymerase chain reaction (PCR) based technology and cDNA library screening. In the first stage, DNA oligonucleotides 15 derived from the extracellular domain sequence information from the known full length FLT, KDR or other VEGF receptor is used to design degenerate oligonucleotide primers for the amplification of sVEGF-R-specific DNA fragments. In the second stage, 20 these fragments are cloned to serve as probes for the isolation of complete sVEGF-R cDNA from a commercially available lambda gtlO cDNA library (Clontech) derived from HUVEC cells (ATCC CRL 1730).

These PCR derived products were used as 25 hybridization probes for screening a lambda gtlO cDNA library derived from HUVECs (Clontech). Plating and plaque lifts of the library were performed by standard methods (T. Maniatis, E.F. Fritsch, J. Sambrook, Molecular Cloning: A Laboratory Manual (Cold Spring 30 Harbor Laboratory, Cold Spring Harbor, New York, 1982). The probes were random-primed labelled with

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117/JWW45 10 18888 32P-dCTP to high specific activity and a separate screening of the library (1 x 10^ plaques per screen) was conducted with each probe. The probes were 5 added to hybridization buffer (50% formamide, 5X Denhardts, 6X SSC (IX SSC = 0.15 M NaCl, 0.015 M Na3citrate-2H20, pH 7.0), 0.1% SDS, 100 μg/ml salmon sperm DNA) at 1 x 10^ cpm/ml.

Four positively hybridizing phage were 10 detected using the fit-specific probe. These positively hybridizing phage were observed to be less than full length fit.

Two fit cDNA clones of about 2.0 kb and 2.7 kb in length were subcloned into pGEM vectors (Promega) 15 and bi-directionally sequenced in their entirety by the chain termination method (Sanger et al., (1977) P.N.A.S. USA, 24, pp. 5463-5467,) and shown to contain a single open reading frame of about 569 amino acids. Sequence analysis demonstrated that a portion of the 5’ 20 fit coding region was missing from these clones. The remainder of the 5’ end was cloned using PCR and combined with the DNA of the clones lacking the 5‘ end to yield a single open reading frame encoding about 687 amino acids. 25 The sequence for the cDNA encoding fit-derived sVEGF-RI is shown in Table 1, and was identified in clones 7 and 11. The deduced amino acid sequence of sVEGF-RI from the cloned cDNA is shown in Table 2. Inspection of the deduced amino acid sequence 30 reveals the presence of a single, large open reading frame of 687 amino acids. By comparison with amino

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117/JWW45 11 18888 acid sequence of the full length FLT VEGF receptor, 31 amino acids are encoded at the C-terminal end of the cDNA which are different from those of FLT. 5 Using another of the preferred methods of the present invention, DNA encoding sVEGF-R is constructed from a DNA sequence encoding a VEGF receptor. For purposes of illustration, DNA encoding the VEGF receptor known as KDR was utilized. Using the receptor 10 DNA sequence, a DNA molecule is constructed which encodes the extracellular domain of the receptor, or the VEGF binding domain only and is denoted sVEGF-RII. Restriction endonuclease cleavage sites are identified within the receptor DNA and can be utilized directly to 15 excise the extracellular-encoding portion. In addition, PCR techniques as described above may be utilized to produce the desired portion of DNA. It is readily apparent to those skilled in the art that other techniques, which are standard in the art, may be 20 utilized to produce sVEGF-R molecules in a manner analagous to those described above. Such techniques are found, for example, in Maniatis et al.. supra.

Additional truncated forms of the VEGF receptor are constructed which contain the 25 transmembrane region. Retention of the transmembrane may facilitate orientation of the inhibitor molecule at the target cell surface. Examples of transmembrane region containing inhibitor molecules include but are not limited to those shown in Figure 1. sVEGF-RTMI and 30 sVEGF-RTMII, as shown in Figure 1, are FLT-related and KDR-related, respectively, transmembrane region

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117/JWW45 12 18888 containing receptor inhibitors. Construction of transmembrane region containing molecules, such as sVEGF-RTMI and sVEGF-RTMII, is done by standard 5 techniques known in the art including but not limited to utilizing convenient restriction endonuclease cleavage sites or PCR techniques as described herein.

It is readily understood by those skilled in the art that various forms of the inhibitors of a VEGF 10 receptor, as disclosed herein, containing only the extracellular region or containing, in addition, the transmembrane region may be constructed which have substantially the same activity.

The cloned sVEGF-R cDNA obtained through the 15 methods described above may be recombinantly expressed by molecular cloning into an expression vector containing a suitable promoter and other appropriate transcription regulatory elements, and transferred into prokaryotic or eukaryotic host cells to produce 20 recombinant sVEGF-R. Techniques for such manipulations are fully described in Maniatis, T, et al.. supra, and are well known in the art.

Expression vectors are defined herein as DNA sequences that are required for the transcription of 25 cloned copies of genes and the translation of their mRNAs in an appropriate host. Such vectors can be used to express eukaryotic genes in a variety of hosts such as bacteria, bluegreen algae, fungal cells, yeast cells, plant cells, insect cells and animal cells.

Specifically designed vectors allow the shuttling of DNA between hosts such as bacteria-yeast 30 117/JWW45 13 18888 or bactfria-animal or bacteria-insect cells. An appropriately constructed expression vector should contain: an origin of replication for autonomous 5 replication in host cells, selectable markers, a limited number of useful restriction enzyme sites, a potential for high copy number, and active promoters.

A promoter is defined as a DNA sequence that directs RNA polymerase to bind to DNA and initiate RNA 10 synthesis. A strong promoter is one which causes mRNAs to be initiated at high frequency. Expression vectors may include, but are not limited to, cloning vectors, modified cloning vectors, specifically designed plasmids or viruses. 15 A variety of mammalian expression vectors may be used to express recombinant sVEGF-R in mammalian cells. Commercially available mammalian expression vectors which may be suitable for recombinant sVEGF-R expression, include but are not limited to, pMClneo 20 (Stratagene), pXTl (Stratagene), pSG5 (Stratagene), EB0-pSV2-neo (ATCC 37593) pBPV-l(8-2) (ATCC 37110), pdBPV-MMTneo(342-12) (ATCC 37224), pRSVgpt (ATCC 37199), pRSVneo (ATCC 37198), pSV2-dhfr (ATCC 37146), pUCTag (ATCC 37460), and gZD35 (ATCC 37565). 25 DNA encoding sVEGF-R may also be cloned into an expression vector for expression in a recombinant host cell. Recombinant host cells may be prokaryotic or eukaryotic, including but not limited to bacteria, yeast, mammalian cells including but not limited to 30 cell lines of human, bovine, porcine, monkey and rodent origin, and insect cells including but not limited to 117/JWW45 14 - 18888 drosophila, moth, mosquito and armyworm derived cell lines. Cell lines derived from mammalian species which may be suitable and which are commercially available, include but are not limited to, CV-1 (ATCC CCL 70), COS-1 (ATCC CRL 1650), COS-7 (ATCC CRL 1651), CHO-Kl (ATCC CCL 61), 3T3 (ATCC CCL 92), NIH/3T3 (ATCC CRL 1658), HeLa (ATCC CCL 2), C127I (ATCC CRL 1616), BS-C-1 (ATCC CCL 26) and MRC-5 (ATCC CCL 171). Insect cell lines which may be suitable and are commercially available include but are not limited to 3M-S (ATCC CRL 8851) moth (ATCC CCL 80) mosquito (ATCC CCL 194 and 195; ATCC CRL 1660 and 1591) and armyworm (Sf9, ATCC CRL 1711).

The expression vector may be introduced into host cells via any one of a number of techniques including but not limited to transformation, transfection, liposome or protoplast fusion, and electroporation. The expression vector-containing cells are clonally propagated and individually analyzed to determine whether they produce sVEGF-R protein. Identification of sVEGF-R expressing host cell clones may be done by several means, including but not limited to immunological reactivity with anti-sVEGF-R antibodies, binding to radiolabelled VEGF, and the presence of host cell-secreted sVEGF-R activity.

Expression of sVEGF-R DNA may also be performed using in vitro produced synthetic mRNA. Synthetic mRNA can be efficiently translated in various cell-free systems, including but not limited to wheat germ extracts and reticulocyte extracts, as well as 117/JWW45 - 15 - 18888 efficiently translated in cell based systems, including but not limited to microinjection into frog oocytes, with microinjection into frog oocytes being preferred. 5 Levels of sVEGF-R protein produced by host

cells may be quantitated by immunoaffinity and/or ligand affinity techniques. sVEGF-R-specific affinity beads or sVEGF-R-specific antibodies are used to isolate 35s_methionine labelled or unlabelled sVEGF-R 10 protein. Labelled sVEGF-R protein is analyzed by SDS-PAGE. Unlabelled sVEGF-R protein is detected by Western blotting, ELISA or RIA assays employing sVEGF-R specific antibodies, or by ligand blotting with labelled VEGF. 15 Following expression of sVEGF-R in a recombinant host cell, sVEGF-R protein may be recovered to provide sVEGF-R in active form, capable of binding VEGF without stimulating mitogenesis. Several sVEGF-R purification procedures are available and suitable for 20 use. sVEGF-R may be purified from cell lysates and extracts, or from conditioned culture medium, by various combinations of, or individual application of salt fractionation, ion exchange chromatography, size exclusion chromatography, hydroxylapatite adsorption 25 chromatography, reversed phase chromatography, heparin sepharose chromatography, VEGF ligand affinity chromatography, and hydrophobic interaction chromatography.

In addition, recombinant sVEGF-R can be 30 separated from other cellular proteins by use of an immuno-affinity column made with monoclonal or 117/JWW45 16 18888 polyclonal antibodies specific for full length sVEGF-R, or polypeptide fragments of sVEGF-R. 5 Identification of sVEGF-RI - In an attempt to clone the VEGF receptor cDNA (fit) a HUVEC XgtlO cDNA library was screened with a DNA probe derived from the extracellular domain of the membrane bound or full length form of this receptor as shown in Figure 1. 10 Four incomplete clones, all lacking various lengths of 5’ coding sequence, were isolated from screening a total of 1 x 10θ plaques. Two of these isolates represent partial clones that were identical to full length fit, one of which contained the complete 3’ 15 coding region of the form described by Shibuya et al., supra. The other two clones were identical to full length fit up to base pair number 2219 (Table 1 and Figure 2) where they then diverged from full length fit. These clones (clone 7 and 11) coded for an 20 additional unique 31 amino acids before the open reading frame is terminated by a TAA codon (Table 2 and Figure 3).

Clone 7 and 11 coded for a protein with a predicted molecular mass of about 75 kDa containing 12 25 putative N-linked glycosylation sites. This version of the receptor was missing the transmembrane and intracellular kinase domains and thus coded for a natural soluble form of the VEGF receptor (sVEGF-RI). Further, the protein molecule predicted by sVEGF-RI has 30 only the first six Ig-like domains, missing the one closest to the transmembrane sequence (Figure 1). The 117/JWW45 17 18888 31 amino acids at the C-terminal end of sVEGF-RI contain two cysteine residues, but does not resemble an Ig domain. 5

Expression of sVEGF-RI in Sf9 cells - To analyze the binding and biological properties of this form of the receptor, the protein was expressed using a baculovirus expression system. Clone 7 was missing about 350 base 10 pairs of coding sequence at the 5’ end. This region was cloned by PCR using the primers described above and in Example 1. A clone containing the complete coding region of sVEGF-RI was constructed by combining the 5’ PCR fragment with sVEGF-RI clone 7 which overlapped at 15 a Sacl site. The 5’ EcoRI site was then changed to a BamHI site and the full length sVEGF-RI was cloned into pBluebac III (Invitrogen) as a BamHI/BamHI fragment. A recombinant baculovirus P-3 stock containing the sVEGF-RI gene 3’ in relation to the polyhedrin promoter 20 was then prepared as described herein.

Culture media from small scale infections were tested for the ability to form high molecular weight complexes with The labeled ligand and culture media from the baculovirus infected cells 25 were combined and incubated. The reactions were then analyzed by size exclusion chromatography. When the wild-type infected culture medium was mixed with the radioactive ligand (Figure 4) a single radioactive peak was observed. However, when the sVEGF-RI infected 30 culture medium was used, a high molecular weight complex was formed, as evident by the appearance of a

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117/JWW45 18 18888 second peak in this reaction eluting near the void volume of the column. This experiment showed that the natural soluble form of the FLT VEGF receptor, 5 sVEGF-RI, forms a high molecular weight complex with VEGF.

The recombinantly produced sVEGF-R is purified from the recombinant host cell extracts or cell culture fluid using heparin-sepharose column

10 chromatography which specifically binds the sVEGF-R protein. The heparin-sepharose bound VEGF-R column is washed using a suitable buffer containing between 0.1M and 0.6M NaCl which removes contaminating proteins without significant loss of sVEGF-R. The sVEGF-R is 15 eluted from the heparin-sepharose column using a suitable buffer containing about 1M NaCl, yielding substantially purified sVEGF-R.

Binding of the sVEGF-RI to VEGF - The binding of 20 125I-labelled VEGF to sVEGF-RI was characterized by crosslinking, and by complex formation with sVEGF-RI absorbed to 96 well plates.

The crosslinked products are shown in Figure 6. The sVEGF-RI was cross-linked to [125I]VEGF (lane 25 1); in the presence of unlabelled VEGF (lane 2) and unlabelled bFGF (lane 3). Two high molecular weight bands (about 145 kDa and 245 kDa) were formed in the sVEGF-RI and [^25I]VEGF containing reaction, and in the sVEGF-RI and [^-2^I]VEGF plus an excess of unlabelled 30 bFGF reaction. The two high molecular weight bands were not present when sVEGF-RI was 117/JWW45 19 18888 incubated with [^25i]VEGF plus an excess of unlabelled VEGF, demonstrating the specificity of sVEGF-RI for VEGF, and the ability of sVEGF-RI to form a dimer. The 5 145 kDa band is presumably a crosslinked complex

containing one receptor molecule (about 100 kDa) and a VEGF dimer (about 46 kDa). As shown in Figure 6 complexes containing two receptor molecules (about 245 kDA) were also observed. This suggests that each VEGF 10 dimer can bind one or two receptor molecules and that the soluble form of the VEGF receptor may undergo ligand-induced dimerization.

The affinity of sVEGF-RI for VEGF was evaluated by absorbing sVEGF-RI to the surface of a 96 15 well plate, followed by blocking the nonspecific sites with 0.5% gelatin. Variable amounts of labeled ligand were added to each well. These results demonstrate that sVEGF-RI binds VEGF with high affinity with an apparent K&lt;j of about 20pM (Figure 7). Since the 20 soluble form of the receptor is missing the Ig domain closest to the transmembrane spanning region, this domain is not required for ligand binding.

The sVEGF-RI is shown to inhibit binding of VEGF to HUVECs by incubating cultured HUVECs with 25 [125i]VEGF and various amounts of sVEGF-RI. Following incubation, the cells are washed to remove unbound [12^i]VEGF. The cells are then solubilized and the amount of cell-associated ig determined by gamma

counter, which demonstrates the amount of [425l]VEGF 30 which was capable of binding to the cellular VEGF receptor in the presence of sVEGF-RI. Using this 117/JWW45 20 18888 method,, it is demonstrated that sVEGF-RI was capable of inhibiting [^23I]VEGF binding to HUVECs VEGF receptor (see Figure 8).

5 Since sVEGF-RI was able to inhibit VEGF binding to cell receptors, it was then determined that sVEGF-RI could inhibit VEGF induced mitogenesis. Cells are preincubated with sVEGF-RI and then incubated with VEGF in the presence of [3H]thymidine. Following 10 incubation, the amount of cellular DNA-incorporated [3H]thymidine is measured which indicates whether VEGF has induced mitogenesis and caused [3H]thymidine to be incorporated into cellular DNA. The presence of sVEGF-RI inhibits the ability of VEGF to stimulate 15 mitogenesis as shown in Figure 9.

The inhibitor of the present invention can be used for the inhibition of VEGF activity. The inhibitor can be used either topically or intravascularly. For topical applications the 20 formulation would be applied directly at a rate of about 10 ng to about 1 mg/cm2/day. For intravaneous applications, the inhibitor is used at a rate of about 1 μg to about 10 mg/kg/day of body weight. For internal use, the formulation may be released directly 25 into the region to be treated either from implanted slow release polymeric material or from slow release pumps or repeated injections. The release rate in either case is about 100 ng to about 100 μg/day/cm3.

For non-topical application the VEGF 30 inhibitor is administered in combination with pharmaceutically acceptable carriers or diluents such 117/JWW45 21 18888 as phosphate buffer, saline, phosphate buffered saline, Ringer’s solution, and the like, in a pharmaceutical composition, according to standard pharmaceutical practice. For topical application, various pharmaceutical formulations are useful for the administration of the active compound of this invention. Such formulations include, but are not limited to, the following: ointments such as hydrophilic petrolatum or polyethylene glycol ointment; pastes which may contain gums such as xanthan gum; solutions such as alcoholic or aqueous solutions; gels such as aluminum hydroxide or sodium alginate gels; albumins such as human or animal albumins; collagens such as human or animal collagens; celluloses such as alkyl celluloses, hydroxy alkyl celluloses and alkylhydroxyalkyl celluloses, for example methylcellulose, hydroxyethyl cellulose, carboxymethyl cellulose, hydroxypropyl methylcellulose, and hydroxypropyl cellulose; polyoxamers such as Pluronic® Polyols exemplified by Pluronic® F-127; tetronics such as tetronic 1508; and alginates such as sodium alginate.

The following examples are provided as illustrative of the present invention without, however, limiting the same thereto.

Cloning fit-related sVEGF-RI - A 580 base pair DNA probe for fit was obtained by PCR of the HUVEC phage library using the primers 5’ GCACCTTGGTTGTGGCTGAC 3’ 117/JWW45 22 - 18888 (SEQ. TD. No.: 1) and 5’ TGGAATTCGTGCTGCTTCCTGGTCC 3’(SEQ. ID. No.: 2). The resulting DNA fragment was cloned into pGEM3Z as a Xbal/EcoRI fragment. The 5 probe was prepared by the random priming method [Feinberg, A.P. and Vogelstein, B., (1983)

Anal.Biochem., 132. pp.6-13] using the megaprime kit (Amersham) at a specific activity of 1 X 10? cpm/ng. The HUVEC cDNA library was plated at a density of 5 X 10 10^ plaques/150 cm plate then about 1 X 10θ plaques were screened by hybridization as previously described [Maniatis, T. et al., supra]. Briefly, following prehybridization at 42°C for 2 hours in 50% formamide, 5X SSC, 5X Denhardt’s solution, 0.17» SDS, 100 μg/ml 15 salmon sperm DNA (hybridization buffer) the filters were hybridized with the probe for 16 hours at 42eC in hybridization buffer. The filters were washed one time for 15 min at room temperature in 2X SSC then three times at 55’C in 0.1 X SSC. Four positive 20 plaques were identified and rescreened two additional times to obtain homogeneous isolates. Inserts were cloned into pGEM3Z for DNA sequence analysis. Two of these clones were identified which contained less than the full length fit coding region. DNA sequence 25 analysis showed that these clones lacked the 5’ coding region of fit. The DNA sequence is shown in Table 1 and Figure 2, and the deduced amino acid sequence is shown in Table 2 and Figure 3. The 5’ end of fit was cloned by PCR using the primers 5’ 30 GGAATTCCGCGCTCACCATGGTCAGC 3’ (SEQ.ID.NO.:3) and 5’ TTTGAATTCACCCGGCAGGGAATGACG 3’ (SEQ.ID.NO.:4). The PCR fragment generated with this set of primers was cloned into fit clone 7 as an EcoRI/SacI fragment. 117/JWW45 23 18888 , TABLE 1

GCGGACACTCCTCTCGGCTCCTCCCCGGCAGCGGCGGCGGCTCGGAGCGGGCTCCGGGG 5

CTCGGGTGCAGCGGCCAGCGGGCCTGGCGGCGAGGATTACCCGGGGAAGTGGTTGTCTC

CTGGCTGGAGCCGCGAGACGGGCGCTCAGGGCGCGGGGCCGGCGGCGGCGAACGAGAGG

10 ACGGACTCTGGCGGCCGGGTCGTTGGCCGGGGGAGCGCGGGCACCGGGCGAGCAGGCCG

CGTCGCGCTCACC ATG GTC AGC TAC TGG GAC ACC GGG GTC CTG CTG

TGC GCG CTG CTC AGC TGT CTG CTT CTC ACA GGA TCT AGT TCA GGT TCA AAA TTA AAA GAT CCT GAA CTG AGT TTA AAA GGC ACC CAG CAC ATC ATG CAA GCA GGC CAG ACA CTG CAT CTC CAA TGC AGG GGG GAA GCA GCC CAT AAA TGG TCT TTG CCT GAA ATG GTG AGT AAG GAA AGC GAA AGG CTG AGC ATA ACT AAA TCT GCC TGT GGA AGA AAT GGC AAA 25 30 117/JWW45 24 18888

CAA TTC TGC AGT ACT

ACT GGC TTC TAC AGC

AAG AAG GAA ACA GAA

GGT AGA CCT TTC GTA

CAC ATG ACT GAA GGA

TCA CCT AAC ATC ACT

TTG ATC CCT GAT GGA

TTC ATC ATA TCA AAT

TGT GAA GCA ACA GTC

ACA CAT CGA CAA ACC

TTA ACC TTG AAC ACA

TGC AAA TAT CTA GCT

TCT GCA ATC TAT ATA

GAG ATG TAC AGT GAA

AGG GAG CTC GTC ATT

GTT ACT TTA AAA AAG

AAA CGC ATA ATC TGG

GCA ACG TAC AAA GAA

AAT GGG CAT TTG TAT

AAT ACA ATC ATA GAT

GCT CAA GCA AAC CAC

GTA CCT ACT TCA AAG

TTT ATT AGT GAT ACA

ATC CCC GAA ATT ATA

CCC TGC CGG GTT ACG

TTT CCA CTT GAC ACT

GAC AGT AGA AAG GGC

ATA GGG CTT CTG ACC

AAG ACA AAC TAT CTC

GTC CAA ATA AGC ACA 117/JWW45 25 18888

CCA CGC CCA GTC AAA TTA CTT AGA GGC CAT ACT CTT GTC CTC AAT

TGT ACT GCT ACC ACT CCC TTG AAC ACG AGA GTT CAA ATG ACC TGG

AGT TAC CCT GAT GAA AAA AAT AAG AGA GCT TCC GTA AGG CGA CGA

ATT GAC CAA AGC AAT TCC CAT GCC AAC ATA TTC TAC AGT GTT CTT

ACT ATT GAC AAA ATG CAG AAC AAA GAC AAA GGA CTT TAT ACT TGT

CGT GTA AGG AGT GGA CCA TCA TTC AAA TCT GTT AAC ACC TCA GTG

CAT ATA TAT GAT AAA GCA TTC ATC ACT GTG AAA CAT CGA AAA CAG

CAG GTG CTT GAA ACC GTA GCT GGC AAG CGG TCT TAC CGG CTC TCT

ATG AAA GTG AAG GCA TTT CCC TCG CCG GAA GTT GTA TGG TTA AAA

GAT GGG TTA CCT GCG ACT GAG AAA TCT GCT CGC TAT TTG ACT CGT 117/JWW45 26 18888

GGC TAC i;CG TTA ATT ATC AAG GAC GTA ACT GAA GAG GAT GCA GGG

' AAT TAT ACA ATC TTG CTG AGC ATA AAA CAG TCA AAT GTG TTT AAA 5

AAC CTC ACT GCC ACT CTA ATT GTC AAT GTG AAA CCC CAG ATT TAC

GAA AAG GCC GTG TCA TCG TTT CCA GAC CCG GCT CTC TAC CCA CTG

10 GGC AGC AGA CAA ATC CTG ACT TGT ACC GCA TAT GGT ATC CCT CAA

CCT ACA ATC AAG TGG TTC TGG CAC CCC TGT AAC CAT AAT CAT TCC

GAA GCA AGG TGT GAC TTT TGT TCC AAT AAT GAA GAG TCC TTT ATC 15

CTG GAT GCT GAC AGC AAC ATG GGA AAC AGA ATT GAG AGC ATC ACT

CAG CGC ATG GCA ATA ATA GAA GGA AAG AAT AAG ATG GCT AGC ACC

20 TTG GTT GTG GCT GAC TCT AGA ATT TCT GGA ATC TAC ATT TGC ATA 25 30 117/JWW45 27 18888

GCT TCC AAT AAA GTT GGG ACT

ATC ACA GAT GTG CCA AAT GGG

CCG ACG GAA GGA GAG GAC CTG

TTC TTA TAC AGA GAC GTT ACT

AAC AGA ACA ATG CAC TAC AGT

ACT AAG GAG CAC TCC ATC ACT

TCC CTG CAA GAT TCA GGC ACC

TAC ACA GGG GAA GAA ATC CTC

GGT GAG CAC TGC AAC AAA AAG

TTT AAA AGC ACA AGG AAT GAT

CAT TAA

GTG GGA AGA AAC ATA AGC TTT TAT

TTT CAT GTT AAC TTG GAA AAA ATG

AAA CTG TCT TGC ACA GTT AAC AAG

TGG ATT TTA CTG CGG ACA GTT AAT

ATT AGC AAG CAA AAA ATG GCC ATC

CTT AAT CTT ACC ATC ATG AAT GTT

TAT GCC TGC AGA GCC AGG AAT GTA

CAG AAG AAA GAA ATT ACA ATC AGA

GCT GTT TTC TCT CGG ATC TCC AAA

TGT ACC ACA CAA AGT AAT GTA AAA 117/JWW45 28 18888

AGGACTCATTAAAAAGTAACAGTTGTCTCATATCATCTTGATTTATTGTCACTGTTG

CTAACTTTCAGGCTCGGAGGAGATGCTCCTCCCAAAATGAGTTCGGAGATGATAGCA

GTAATAATGAGACCCCCGGGCTCCAGCTCTGGGCCCCCCATTCAGGCCGAGGGGGCT

GCTCCGGGGGGCCGACTTGGTGCACGTTTGGATTTGGAGGATCCCTGCACTGCCTTC

TCTGTGTTTGTTGCTCTTGCTGTTTTCTCCTGCCTGATAAACAACAACTTGGGATGA TCCTTTCCATTTTGATGCCAACCTCTTTTTATTTTTAAGCGGCGCCCTATAGT (SEQ. ID. NO.: 5) 117/JWW45 29 18888 TABLE 2

Met Val Ser Tyr Trp Asp Thr Gly Val Leu Leu Cys Ala Leu Leu Ser Cys Leu Leu Leu Thr Gly Ser Ser Ser Gly Ser Lys Leu Lys Asp Pro Glu Leu Ser Leu Lys Gly Thr Gin His He Met Gin Ala Gly Gin Thr Leu His Leu Gin Cys Arg Gly Glu Ala Ala His Lys Trp Ser Leu Pro Glu Met Val Ser Lys Glu Ser Glu Arg Leu Ser lie Thr Lys Ser Ala Cys Gly Arg Asn Gly Lys Gin Phe Cys Ser Thr Leu Thr Leu Asn Thr Ala Gin Ala Asn His Thr Gly Phe Tyr Ser Cys Lys Tyr Leu Ala Val Pro Thr Ser Lys Lys Lys Glu Thr Glu Ser Ala lie Tyr lie Phe lie Ser Asp Thr Gly Arg Pro Phe Val Glu Met Tyr Ser Glu lie Pro Glu He He 25 30 117/JWW45 30 18888

His Met Thr Glu Gly Arg Glu Leu Val He Pro Cys Arg Val Thr Ser Pro Asn He Thr Val Thr Leu Lys Lye Phe Pro Leu Asp Thr Leu lie Pro Asp Gly Lys Arg He lie Trp Asp Ser Arg Lys Gly Phe lie He Ser Asn Ala Thr Tyr Lys Glu He Gly Leu Leu Thr Cys Glu Ala Thr Val Asn Gly His Leu Tyr Lys Thr Asn Tyr Leu Thr His Arg Gin Thr Asn Thr He He Asp Val Gin He Ser Thr Pro Arg Pro Val Lys Leu Leu Arg Gly His Thr Leu Val Leu Asn Cys Thr Ala Thr Thr Pro Leu Asn Thr Arg Val Gin Met Thr Trp Ser Tyr Pro Asp Glu Lys Asn Lys Arg Ala Ser Val Arg Arg Arg He Asp Gin Ser Asn Ser His Ala Asn He Phe Tyr Ser Val Leu 117/JWW45 31 18888

Thr Tie Asp Lys Met Gin Asn Lys Asp Lys Gly Leu Tyr Thr Cys Arg Val Arg Ser Gly Pro Ser Phe Lys Ser Val Asn Thr Ser Val His Ile Tyr Asp Lys Ala Phe Ile Thr Val Lys His Arg Lys Gin Gin Val Leu Glu Thr Val Ala Gly Lys Arg Ser Tyr Arg Leu Ser Met Lys Val Lys Ala Phe Pro Ser Pro Glu Val Val Trp Leu Lys Asp Gly Leu Pro Ala Thr Glu Lys Ser Ala Arg Tyr Leu Thr Arg Gly Tyr Ser Leu Ile Ile Lys Asp Val Thr Glu Glu Asp Ala Gly Asn Tyr Thr Ile Leu Leu Ser Ile Lys Gin Ser Asn Val Phe Lys Asn Leu Thr Ala Thr Leu Ile Val Asn Val Lys Pro Gin Ile Tyr Glu Lys Ala Val Ser Ser Phe Pro Asp Pro Ala Leu Tyr Pro Leu 117/JWW45 32 18888

Gly Sfir Arg Gin lie Leu Thr Cys Thr Ala Tyr Gly lie Pro Gin Pro Thr lie Lys Trp Phe Trp His Pro Cys Asn His Asn His Ser Glu Ala Arg Cys Asp Phe Cys Ser Asn Asn Glu Glu Ser Phe lie Leu Asp Ala Asp Ser Asn Met Gly Asn Arg He Glu Ser lie Thr Gin Arg Met Ala lie lie Glu Gly Lys Asn Lys Met Ala Ser Thr Leu Val Val Ala Asp Ser Arg lie Ser Gly lie Tyr lie Cys lie Ala Ser Asn Lys Val Gly Thr Val Gly Arg Asn lie Ser Phe Tyr lie Thr Asp Val Pro Asn Gly Phe His Val Asn Leu Glu Lys Met Pro Thr Glu Gly Glu Asp Leu Lys Leu Ser Cys Thr Val Asn Lys Phe Leu Tyr Arg Asp Val Thr Trp lie Leu Leu Arg Thr Val Asn 117/JWW45 - 33 18888

Asn Arg Thr Met His Tyr Ser lie Ser Lys Gin Lys Met Ala lie Thr Lys Glu His Ser lie Thr Leu Asn Leu Thr lie Met Asn Val Ser Leu Gin Asp Ser Gly Thr Tyr Ala Cys Arg Ala Arg Asn Val Tyr Thr Gly Glu Glu He Leu Gin Lys Lys Glu lie Thr lie Arg Gly Glu His Cys Asn Lys Lys Ala Val Phe Ser Arg lie Ser Lys Phe Lys Ser Thr Arg Asn Asp Cys Thr Thr Gin Ser Asn Val Lys His ··· (SEQ. ID. NO .: 6) 15 EXAMPLE 2

Expression of sVEGF-RI in Sf9_insect cells - The full 20 length sequence encoding sVEGF-RI was cloned as an

EcoRI/BamHI fragment into pGEM3Z. The EcoRI site was then modified to a BamHI site and cloned into pBlueBac III 3’ of the polyhedrin promoter (psFLTblue). This plasmid was transfected into Sf9 armyworm cells using 25 liposomes. After 48 hours the medium from the transfected cells which contains recombinant polyhedrin virus particles, was harvested. Dilutions (103 - 104 fold) of the virus were prepared and plaque purified in soft agar containing 150 μg/ml 5-bromo-4-chloro-3- 30 117/JWW45 34 - 18888 indolyl-B-D-galactoside. Recombinant plaques were identified by blue color and used to infect Sf9 cells (5 X IO» cells/well) in 12 well plates. Medium &lt;100 5 μΐ) from polyhedrin minus infections was used to prepare P-2 viral stocks by infecting 2.5 X 10^ cells in a T-25 flask. Large scale high titer P-3 viral stocks were then prepared by infecting Sf9 cells (500 ml at 2 X 10θ cells/ml) with 5 ml of the P-2 stock then 10 incubating at 27°C for 5-6 days and the medium was harvested by centrifugation. Protein expression was accomplished by infecting cells at a density of 2- 2.5 X 10^ cells/ml with a multiplicity of infection of 5 - 10. Twenty four hours after infection the cells were 15 changed to a serum free medium (SP900II, Gibco BRL), incubated for an additional 48 hours and the medium was collected. This conditioned medium contains the recombinantly expressed sVEGF-RI protein. 20 EXAMPLE 3

Iodination of VEGF - 125I-labeled human recombinant VEGF was prepared by the chloramine T method (Hunter, W.M. and Greenwood, F.C., (1962) Nature (London), 194. 25 pp. 495-496). Briefly, 1 μg of VEGF in 30% acetonitrile/Ο.ΙΧ trifluroacetic acid was adjusted to pH 7.1 by the addition of 1/3 volume of 0.4 M sodium phosphate buffer, pH 7.1. Freshly dissolved chloramine T (4 μΐ of a 2 mg/ml stock in 0.1 M sodium phosphate 30 buffer, pH 7.1) was added to the VEGF solution and reacted for 45 seconds at room temperature (total 117/JWW45 - 35 18888 volume of 150 μϊ). The reaction was stopped by the addition of 50 μϊ of 10 mM KI and 50 μϊ of 2 mg/ml meta bisufite. The labeled ligand was separated from the 5 free by gel filtration on a 0.7 X 15 cm Sephadex G-25 column equilibrated in PBS with 1 mg/ml gelatin. Fractions were counted in a Packard γ counter, aliquoted and stored at -70eC. VEGF was labeled to a specific activity of 5 x 10^ to 1 x 10^ cpm/ng. 10

Gel Filtration Chromatography - Receptor-ligand complex was formed by incubating 10 μΐ of 125i_iabeie&lt;j VEGF (105 cpm) with 100 μΐ of either wild-type or baculovirus sVEGF-RI-containing, infected Sf9 cell 15 culture medium overnight at room temperature. The reaction products were separated on a Sephacryl S200 gel filtration column (0.7 X 25 cm) equilibrated in PBS, 1 mg/ml gelatin, at a flow rate of 15 ml/hr. Fractions (0.75 ml) were collected and analyzed in a γ 20 counter. Receptor-ligand complexes pass quickly through the column while the free labelled VEGF passes through more slowly. The results of this experiment shown in Figure 4 demonstrate the formation of a high molecular weight complex between labelled VEGF and 25 sVEGF-RI protein. This shows that sVEGF-RI binds VEGF.

Crosslinking - Purified sVEGF-RI (l-10ng) was added to 25 μΐ of binding buffer (Dulbecco's Modified Eagle’s medium (DME), 25 mM HEPES, pH 7.5, 0.3% gelatin), and 1

30 x 105 cpm of [425i]_vEGF wag added (Figure 6, lane 1) with either 200ng of unlabelled VEGF (lane 2) or bFGF 117/JWW45 36 18888 (lane 3), then incubated 2 to 16 hours at room temperature. Bis(sulfosuccinimidyl)suberate (Pierce) crosslinker was added to a final concentration of 1 5 mM. The reaction was stopped after 15 min by the addition of boiling SDS PAGE sample buffer. The crosslinked products were separated by SDS PAGE on a 7.5% acrylamide gel and analyzed either by autoradiography or a phosphoimager. The results are

10 shown in Figure 6 and demonstrate that sVEGF-RI binds labelled VEGF by the appearance of two bands of about 145 kDa and 245 kDa. The 145 kDa band consists of one sVEGF-RI molecule and one VEGF molecule (Monomer, M.). The 245 kDa band apparently consists of two sVEGF-RI 15 molecules and one VEGF dimer (D). Free VEGF ligand (L) dimers migrated at about 45 kDA.

Binding assay - The binding of sVEGF-RI to VEGF was analyzed using a 96 well plate assay as described by

20 Duan, D-S. R. et al.. supra. Briefly, sVEGF-RI, 50 to 200 μΐ partially purified by Mono Q chromatography (Pharmacia), was diluted to 10 ml.in 25 mM TRIS, pH 7.4, 100 mM NaCl, 20 mM NH4HCO3. Aliquots (100 μΐ) were absorbed to the surface of a 96 well plate for 18 25 hours at 4eC, the plates were then washed twice with blocking buffer (DME, 25 mM HEPES, pH 7.5, 0.5% gelatin) and the nonspecific sites were blocked in the same buffer for 6 hours at 4*C. The plate was then washed twice in binding buffer. Various amounts of 30 [123I]VEGF were added to the wells in a final volume of 100 μΐ/well and incubated for 2 hours at room 117/JWW45 37 18888 temperature. The wells were washed three times with 100 μϊ of binding buffer, the bound protein was solubilized with 100 μϊ of 1% SDS, 0.5% BSA and counted in a γ counter. The results, shown in Figure 7, were analyzed by the method of Scatchard [Scatchard, G., (1949) Ann. N.Y. Acad. Sci., Σ1, pp. 660-672]. The analysis demonstrates that sVEGF-RI retains high affinity binding for VEGF with a K&lt;j value of about 20 pM. This clearly demonstrates that sVEGF-RI, lacking the transmembrane region and adjacent Ig-like domain, binds VEGF with high affinity and that these regions are not required for VEGF binding. EXAMPLE 4

Inhibition of VEGF binding by sVEGF-RI - The ability of sVEGF-RI to inhibit VEGF binding to HUVECs was tested. HUVECs were plated at 50,000 cells/well in 24 well plates precoated with gelatin, and allowed to grow to confluence. A constant amount of [l25l]VEGF (100,000 cpm) was mixed with various amounts of partially purified sVEGF-RI in binding buffer, in a total volume of 200 μϊ and preincubated at room temperature for 1 hour. Samples were added to the cells and incubated for 4 hours at 4eC with shaking. The medium was then aspirated and the cells were washed three times with binding buffer. The bound radioactivity was solubilized with 50 mM TRIS-HC1, pH 8.0, 150 mM NaCl, 17ο NP40, 1% BSA and counted in a γ counter. 117/JWW45 38 18888

The results are shown in Figure 8. At the highest concentration of sVEGF-RI, VEGF binding to HUVECs was reduced by 70%. It may, however, be difficult to completely inhibit binding to the cellular membrane bound receptor since one molecule of sVEGF-R bound to a VEGF dimer may be able to bind to cell associated receptor to form an inactive (sVEGF-RI)-VEGF-(membrane spanning VEGF receptor) complex. EXAMPLE 5

Inhibition of VEGF mediated mitogenesis by sVEGF-RI

Mitogenic inhibition - Since sVEGF-RI was able to inhibit VEGF binding to endothelial cells, it was then determined that the soluble receptor could inhibit VEGF induced mitogenesis in HUVECs. HUVECs were plated in gelatin coated 96 well plates at a density of 4000 cells/well in 100 μΐ of DME supplemented with 10% heat inactivated fetal calf serum plus antibiotics (penicillin G, 100 units/ml; streptomycin sulfate, 100 pg/ml). After 16 hours the medium was changed and test samples were added, cells were preincubated with a variable amount of purified sVEGF-RI for 15 minutes at 37°C before growth factor (10 ng/ml) was added. The cells were incubated for 24 hours then [methyl-3H]thymidine (0.8 μϋί/νβΐΐ; 20 Ci/mmol: ICi = 37 GBq, final specific activity of 0.8 μθί/nmole) was added followed by incubated for an additional 72 hours at 37°C under 5% C02. The cells were then washed twice with Hank's balanced salt solution adjusted to pH 7.5 117/JWW45 39 18888 with 25, mM Hepes, 0.1% BSA. The cells were then lysed, the DNA was solubilized with 0.2 M Na2C03, 0.1 M NaOH, and [3H]thymidine incorporation was quantified by 5 scintillation counting. The results are shown in

Figure 9. sVEGF-RI was able to completely inhibit VEGF induced [3H]thymidine incorporation in HUVECs. EXAMPLE 6 10

Purification of baculovirus expressed aVEGF-RI from_Sf9 cells - Culture medium from Sf9 cells infected with a baculovirus construct designed to express sVEGF-RI (Example 2) was chromatographed through a heparin 15 Sepharose CL-6B (Pharmacia) column (0.7 X 4 cm). The column was washed with 5 volumes of 10 mM Na-phosphate buffer, pH 6.2, 0.1 M NaCl, followed by 6 ml of 10 mM Na-phosphate buffer, pH 6.2, 0.6 M NaCl. The sVEGF-RI was eluted with 10 mM Na-phosphate buffer, pH 6.2, 1.0 20 M NaCl. Polyacrylamide gel electrophoresis was performed which demonstrated greater than 90% purity (as judged by coomassie blue staining) of the recombinantly produced sVEGF-R (Figure 5). The identity of the protein was confirmed by N-terminal 25 protein sequence analysis. The actual N-terminus (Ser Lys Leu ...) of the recombinant protein differs by two amino acids from that predicted by Shibuya et al., supra. (Ser-Ser-Ser...). The peptidase cleavage site in sVEGF-RI produced in Sf9 cells was between residues 30 gly-26 and ser-27. 117/JWW45 - 40 - 18888 EXAMPLE 7

Construction of KDR-related sVEGF-R - Soluble forms of 5 KDR (a known VEGF receptor) [Terman, B.I. et al., (1991) Oncogene 6., pp. 1677-1683; Terman, B.I. et al., (1992) Biochem. Biophys. Res. Comm. 187. pp. 1579-1586] may exist naturally but have not yet been identified. A soluble form of KDR is recombinantly constructed by 10 modifying its coding sequence by PCR using the primers 1) 5’ TTTTGGATCCCTGCAGACAGATCTACGTTTGAGAACC 3’ (SEQ. ID. NO.: 7) and 2) 5’ TTTTGGATCCTTAACGCTCTAGGACTGTGAGC 3’ (SEQ. ID. NO.: 8), and pKDRA (the Xhol/EcoRI fragment coding for the extracellular and transmembrane

15 domain of KDR cloned into the EcoRI site of pGEM 7Z obtained from Promega) as a template (Figure 17). This generated a translation stop codon after amino acid residue number 663 of KDR which corresponds to the extracellular domain of full length KDR. This modified 20 fragment is then used to replace the Pstl/BamHl fragment of pKDRA generating a truncated form of the KDR gene (Figure 10) which codes for a soluble receptor denoted sVEGF-RII (Figure 11). The Xhol site at base pair number 257 is then changed to a BamHI site by 25 standard cloning techniques. Another truncated form of the KDR receptor is created with primer 1 shown above, and primer 3) 5’ TTTTGGATCCAACGGTCCCTAGGATGATGAC 3’ , (SEQ. ID. NO.: 9) (Figure 12). This form of KDR, denoted sVEGF-RTMII, is truncated at the C-terminal 30 side of the transmembrane domain and therefore retains the transmembrane region (Figure 13). A similar form of the FLT receptor is generated by PCR using the -41 - 109016/2 primers, 4) 5’ AGCACCTTGGTTGTGGCTGACTC 3’ (SEQ. ID. NO.: 10) and 5) 5’ TTTTGGATCCTTAGATAAGGAGGGTTAATAGG 3‘ (SEQ. ID. NO.: 11) and plasmid pmFLT (full length fit cloned into the EcoRI site of pGEM3Z obtained from Promega) as a template (Figure 16). The 780 base pair PCR fragment can then he cloned together‘with the EcoRl/Xbal fragment from pmFLT to produce an EcoRl/BAMHl fragment (Figure 14) encoding a truncated form of FLT (denoted sVEGF-RTMI) which retains the transmembrane domain but lacks the cytoplasmic domain (Figure 15). The EcoRI site at the 5’ end of the gene is then modified to a BamHI site. The resulting truncated forms of KDR and FLT are then cloned into pBluebaclll (Stratagene) for expression in Sf9 insect cells. Characterization of these constructed truncated forms of VEGF receptors is accomplished by the techniques used to characterize sVEGF-RI as in Examples 2, 3, 4, 5, and 6. ******

Passages of the description which are not within the scope of the claims do not constitute part of the invention.

<img img-format="tif" img-content="drawing" file="IL109016AD00026.tif" id="idf0006" />

42 109016/2

SEQUENCE. LISTING

(1) GENERAL INFORMATION (i) APPLICANT: MERCK &amp;amp; CO., INC.

(ii) TITLE OF THE INVENTION: INHIBITOR OF VASCULAR ENDOTHELIAL CELL GROWTH FACTOR (iii) NUMBER OF SEQUENCES: 18 (iv) CORRESPONDENCE ADDRESS: (A) ADDRESSEE: Merck &amp;amp; Co., Inc. (B) STREET: P.O. Box 2000, 126 E. Lincoln Ave. (C) CITY: Rahway

(D) STATE: NJ

(E) COUNTRY: USA (F) ZIP: 07065-0900 (v) COMPUTER READABLE FORM: (A) MEDIUM TYPE: Diskette (B) COMPUTER: IBM Compatible

(C) OPERATING SYSTEM: DOS (D) SOFTWARE: FastSEQ for Windows Version 2.0 (vi) CURRENT APPLICATION DATA: (A) APPLICATION NUMBER: (B) FILING DATE: (C) CLASSIFICATION: (vii) PRIOR APPLICATION DATA: (A) APPLICATION NUMBER: (B) FILING DATE: (viii) ATTORNEY/AGENT INFORMATION: (A) NAME: Hand, J. Mark (B) REGISTRATION NUMBER: 36,545 (C) REFERENCE/DOCKET NUMBER: 18888 (ix) TELECOMMUNICATION INFORMATION: (A) TELEPHONE: 732-594-3905 (B) TELEFAX:- 732-594-4720 (C) TELEX: (2) INFORMATION FOR SEQ ID NO:1: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 20 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear

(ii) MOLECULE TYPE: cDNA (Xi) SEQUENCE DESCRIPTION: SEQ ID NO:1: 18888 43 109016/2

GCGGACACTC CTCTCGGCTC CTCCCCGGCA GCGGCGGCGG CTCGGAGCGG GCTCCGGGGC TCGGGTGCAG CGGCCAGCGG GCCTGGCGGC GAGGATTACC CGGGGAAGTG GTTGTCTCCT GGCTGGAGCC GCGAGACGGG CGCTCAGGGC GCGGGGCCGG CGGCGGCGAA CGAGAGGACG GACTCTGGCG GCCGGGTCGT TGGCCGGGGG AGCGCGGGCA CCGGGCGAGC AGGCCGCGTC GCGCTCACCA TGGTCAGCTA CTGGGACACC GGGGTCCTGC TGTGCGCGCT GCTCAGCTGT CTGCTTCTCA CAGGATCTAG TTCAGGTTCA AAATTAAAAG ATCCTGAACT GAGTTTAAAA GGCACCCAGC ACATCATGCA AGCAGGCCAG ACACTGCATC TCCAATGCAG GGGGGAAGCA GCCCATAAAT GGTCTTTGCC TGAAATGGTG AGTAAGGAAA GCGAAAGGCT GAGCATAACT AAATCTGCCT GTGGAAGAAA TGGCAAACAA TTCTGCAGTA CTTTAACCTT GAACACAGCT CAAGCAAACC ACACTGGCTT CTACAGCTGC AAATATCTAG CTGTACCTAC TTCAAAGAAG AAGGAAACAG AATCTGCAAT CTATATATTT ATTAGTGATA CAGGTAGACC TTTCGTAGAG ATGTACAGTG AAATCCCCGA AATTATACAC ATGACTGAAG GAAGGGAGCT CGTCATTCCC . TGCCGGGTTA CGTCACCTAA CATCACTGTT ACTTTAAAAA AGTTTCCACT TGACACTTTG' ATCCCTGATG GAAAACGCAT AATCTGGGAC AGTAGAAAGG GCTTCATCAT ATCAAATGCA ACGTACAAAG AAATAGGGCT TCTGACCTGT GAAGCAACAG TCAATGGGCA TTTGTATAAG ACAAACTATC TCACACATCG ACAAACCAAT ACAATCATAG ATGTCCAAAT AAGCACACCA CGCCCAGTCA AATTACTTAG AGGCCATACT CTTGTCCTCA ATTGTACTGC TACCACTCCC TTGAACACGA GAGTTCAAAT GACCTGGAGT TACCCTGATG AAAAAAATAA GAGAGCTTCC GTAAGGCGAC GAATTGACCA AAGCAATTCC CATGCCAACA TATTCTACAG TGTTCTTACT ATTGACAAAA TGCAGAACAA AGACAAAGGA CTTTATACTT GTCGTGTAAG GAGTGGACCA TCATTCAAAT CTGTTAACAC CTCAGTGCAT ATATATGATA AAGCATTCAT CACTGTGAAA CATCGAAAAC AGCAGGTGCT TGAAACCGTA GCTGGCAAGC GGTCTTACCG GCTCTCTATG AAAGTGAAGG CATTTCCCTC GCCGGAAGTT GTATGGTTAA AAGATGGGTT ACCTGCGACT GAGAAATCTG CTCGCTATTT GACTCGTGGC TACTCGTTAA TTATCAAGGA CGTAACTGAA GAGGATGCAG GGAATTATAC AATCTTGCTG AGCATAAAAC AGTCAAATGT GTTTAAAAAC CTCACTGCCA CTCTAATTGT CAATGTGAAA CCCCAGATTT ACGAAAAGGC CGTGTCATCG TTTCCAGACC CGGCTCTCTA CCCACTGGGC AGCAGACAAA TCCTGACTTG TACCGCATAT GGTATCCCTC AACCTACAAT CAAGTGGTTC TGGCACCCCT GTAACCATAA TCATTCCGAA GCAAGGTGTG ACTTTTGTTC CAATAATGAA GAGTCCTTTA TCCTGGATGC TGACAGCAAC ATGGGAAACA GAATTGAGAG CATCACTCAG CGCATGGCAA TAATAGAAGG AAAGAATAAG ATGGCTAGCA CCTTGGTTGT GGCTGACTCT AGAATTTCTG GAATCTACAT TTGCATAGCT TCCAATAAAG TTGGGACTGT GGGAAGAAAC ATAAGCTTTT ATATCACAGA TGTGCCAAAT GGGTTTCATG TTAACTTGGA AAAAATGCCG ACGGAAGGAG AGGACCTGAA ACTGTCTTGC ACAGTTAACA AGTTCTTATA CAGAGACGTT ACTTGGATTT TACTGCGGAC AGTTAATAAC AGAACAATGC ACTACAGTAT TAGCAAGCAA AAAATGGCCA TCACTAAGGA GCACTCCATC ACTCTTAATC TTACCATCAT GAATGTTTCC CTGCAAGATT CAGGCACCTA TGCCTGCAGA GCCAGGAATG TATACACAGG GGAAGAAATC CTCCAGAAGA AAGAAATTAC AATCAGAGGT GAGCACTGCA ACAAAAAGGC TGTTTTCTCT CGGATCTCCA AATTTAAAAG CACAAGGAAT GATTGTACCA CACAAAGTAA TGTAAAACAT TAAAGGACTC ATTAAAAAGT AACAGTTGTC TCATATCATC TTGATTTATT GTCACTGTTG CTAACTTTCA GGCTCGGAGG AGATGCTCCT CCCAAAATGA GTTCGGAGAT GATAGCAGTA ATAATGAGAC CCCCGGGCTC CAGCTCTGGG CCCCCCATTC AGGCCGAGGG GGCTGCTCCG GGGGGCCGAC TTGGTGCACG TTTGGATTTG GAGGATCCCT GCACTGCCTT CTCTGTGTTT GTTGCTCTTG CTGTTTTCTC CTGCCTGATA AACAACAACT TGGGATGATC CTTTCCATTT TGATGCCAAC CTCTTTTTAT TTTTAAGCGG CGCCCTATAG T (2) INFORMATION FOR SEQ ID NO: 6: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 687 amino acids (B) TYPE: amino acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear 60 120 180 240 300 360 420 480 540 600 660 720 780 840 900 960 1020 1080 1140 1200 1260 1320 1380 1440 1500 1560 1620 1680 1740 1800 1860 1920 1980 2040 2100 2160 2220 2280 2340 2400 2460 2520 2580 2640 2651 (ii) MOLECULE TYPE: protein 18888 44 ' 109016/2 GCACCTTGGT TGTGGCTGAC 20 (2) INFORMATION FOR SEQ ID NO:2: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 25 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear

(ii) MOLECULE TYPE: cDNA (xi) SEQUENCE DESCRIPTION: SEQ ID N0:2: TGGAATTCGT GCTGCTTCCT GGTCC 25 (2) INFORMATION FOR SEQ ID NO:3: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 26 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear

(ii) MOLECULE TYPE: cDNA (xi) SEQUENCE DESCRIPTION: SEQ ID NO:3: GGAATTCCGC GCTCACCATG GTCAGC 26 (2) INFORMATION FOR SEQ ID NO:4: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 27 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear

(ii) MOLECULE TYPE: cDNA (xi) SEQUENCE DESCRIPTION: SEQ ID NO:4: TTTGAATTCA CCCGGCAGGG AATGACG 27 (2) INFORMATION FOR SEQ ID NO:5: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 2651 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single - (D) TOPOLOGY: linear

(ii) MOLECULE TYPE: cDNA (xi) SEQUENCE DESCRIPTION: SEQ ID NO:5: 18888 45 109016/2 (xi) SEQUENCE DESCRIPTION: SEQ ID NO :6: Met 1 Val Ser Tyr Trp 5 Asp Thr Gly Val Leu 10 Leu Cys Ala Leu Leu 15 Ser Cys Leu Leu Leu 20 Thr Gly Ser Ser Ser 25 Gly Ser Lys Leu Lys 30 Asp Pro Glu Leu Ser 35 Leu Lys Gly Thr Gin 40 His He Met Gin Ala 45 Gly Gin Thr Leu His 50 Leu Gin Cys Arg Gly 55 Glu Ala Ala His Lys 60 Trp Ser Leu Pro Glu 65 Met Val Ser Lys Glu 70 Ser Glu Arg Leu Ser 75 lie Thr Lys Ser Ala 80 Cys Gly Arg Asn Gly 85 Lys Gin Phe Cys Ser 90 Thr Leu Thr Leu Asn 95 Thr Ala Gin Ala Asn 100 His Thr Gly Phe Tyr 105 Ser Cys Lys Tyr Leu 110 Ala Val Pro Thr Ser 115 Lys Lys Lys Glu Thr 120 Glu Ser Ala lie Tyr 125 He Phe He Ser Asp 130 Thr Gly Arg Pro Phe 135 Val Glu Met Tyr Ser 140 Glu lie Pro Glu lie 145 lie His Met Thr Glu 150 Gly Arg Glu Leu Val 155 lie Pro Cys Arg Val 160 Thr Ser Pro Asn He 165 Thr Val Thr Leu Lys 170 Lys Phe Pro Leu Asp 175 Thr Leu He Pro Asp 180 Gly Lys Arg lie He 185 Trp Asp Ser Arg Lys 190 Gly Phe lie lie Ser 195 Asn Ala Thr Tyr Lys 200 Glu He Gly Leu Leu 205 Thr Cys Glu Ala Thr 210 Val Asn Gly His Leu 215 Tyr Lys Thr Asn Tyr 220 Leu Thr His Arg Gin 225 Thr Asn Thr lie lie 230 Asp Val Gin lie Ser 235 Thr. Pro Arg Pro Val 240 Lys Leu Leu Arg Gly 245 His Thr Leu Val Leu 250 Asn Cys Thr Ala Thr 255 Thr Pro Leu Asn Thr 260 Arg Val Gin Met Thr 265 Trp Ser Tyr Pro Asp 270 Glu Lys Asn Lys Arg 275 Ala Ser Val Arg Arg 280 Arg He Asp Gin Ser 285 Asn Ser His Ala Asn 290 lie Phe Tyr Ser -&lt; Val 295 Leu Thr lie Asp Lys 300 Met Gin Asn Lys Asp 305 Lys Gly Leu Tyr Thr 310 Cys Arg Val Arg Ser 315 Gly Pro Ser Phe Lys 320 Ser Val Asn Thr Ser 325 Val His He Tyr Asp 330 Lys Ala Phe lie Thr 335 Val Lys His Arg Lys 340 Gin Gin Val Leu Glu 345 Thr Val Ala Gly Lys 350 Arg Ser Tyr Arg Leu 355 Ser Met Lys Val Lys 360 Ala Phe Pro Ser Pro 365 Glu Val Val Trp Leu 370 Lys Asp Gly Leu Pro 375 Ala Thr Glu Lys Ser 380 Ala Arg Tyr Leu Thr 385 Arg Gly Tyr Ser Leu 390 lie He Lys Asp Val 395 Thr Glu Glu Asp Ala 400 18888

<img img-format="tif" img-content="drawing" file="IL109016AD00027.tif" id="idf0007" />

46 109016/2

Gly Asn Tyr Thr Ile 405 Leu Leu Ser Ile Lys 410 Gin Ser Asn Val Phe 415 Lys Asn Leu Thr Ala 420 Thr Leu Ile Val Asn 425 Val Lys Pro Gin Ile 430 Tyr Glu Lys Ala Val 435 Ser Ser Phe Pro Asp 440 Pro Ala Leu Tyr Pro 445 Leu Gly Ser Arg Gin 450 lie Leu Thr Cys Thr 455 Ala Tyr Gly Ile Pro 460 Gin Pro Thr lie Lys 465 Trp Phe Trp His Pro 470 Cys Asn His Asn His 475 Ser Glu Ala Arg Cys 480 Asp Phe Cys Ser Asn 485 Asn Glu Glu Ser Phe 490 Ile Leu Asp Ala Asp 495 Ser Asn Met Gly Asn 500 Arg Ile Glu Ser Ile 505 Thr Gin Arg Met Ala 510 Ile Ile Glu Gly Lys 515 Asn Lys Met Ala Ser 520 Thr Leu Val Val Ala 525 Asp Ser Arg Ile Ser 530 Gly lie Tyr Ile Cys 535 Ile Ala Ser Asn Lys 540 Val Gly Thr Val Gly 545 Arg Asn lie Ser Phe 550 Tyr Ile Thr Asp Val 555 Pro Asn Gly Phe His 560 Val Asn Leu Glu Lys 565 Met Pro Thr Glu Gly 570 Glu Asp Leu Lys Leu 575 Ser Cys Thr Val Asn 580 Lys Phe Leu Tyr Arg 585 Asp Val Thr Trp Ile 590 Leu Leu Arg Thr Val 595 Asn Asn Arg Thr Met 600 His Tyr Ser Ile Ser 605 Lys Gin Lys Met Ala 610 Ile Thr Lys Glu His 615 Ser Ile Thr Leu Asn 620 Leu Thr Ile Met Asn 625 Val Ser Leu Gin Asp 630 Ser Gly Thr Tyr Ala 635 Cys Arg Ala Arg Asn 640 Val Tyr Thr Gly Glu 645 Glu Ile Leu Gin Lys 650 Lys Glu Ile Thr Ile 655 Arg Gly Glu His Cys 660 Asn Lys Lys Ala Val 665 Phe Ser Arg Ile Ser 670 Lys Phe Lys Ser Thr 675 Arg Asn Asp Cys Thr 680 Thr Gin Ser Asn Val 685 Lys His (2) INFORMATION FOR SEQ ID NO:7: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 3‘7 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear

(ii) MOLECULE TYPE: Genomic DNA (xi) SEQUENCE DESCRIPTION: SEQ ID NO:7:

TTTTGGATCC CTGCAGACAG ATCTACGTTT GAGAACC (2) INFORMATION FOR SEQ ID NO:8: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 32 base pairs 37 18888, 47 109016/2 (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear

(ii) MOLECULE TYPE: Genomic DNA (xi) SEQUENCE DESCRIPTION: SEQ ID NO:8: TTTTGGATCC TTAACGCTCT AGGACTGTGA GC 32 (2) INFORMATION FOR SEQ ID NO:9: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 31 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear

(ii) MOLECULE TYPE: Genomic DNA (xi) SEQUENCE DESCRIPTION: SEQ ID NO:9: TTTTGGATCC AACGGTCCCT AGGATGATGA C 31 (2) INFORMATION FOR SEQ ID NO:10: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 23 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear

(ii) MOLECULE TYPE: Genomic DNA (xi) SEQUENCE DESCRIPTION: SEQ ID NO:10: AGCACCTTGG TTGTGGCTGA CTC 23 (2) INFORMATION FOR SEQ ID NO:11: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 3"2 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear

(ii) MOLECULE TYPE: Genomic DNA (xi) SEQUENCE DESCRIPTION: SEQ ID NO:11: TTTTGGATCC TTAGATAAGG AGGGTTAATA GG - 32 ' (2) INFORMATION FOR SEQ ID NO:12: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 661 amino acids 18888» 48 109016/2 (B) TYPE: amino acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: protein (xi) SEQUENCE DESCRIPTION: SEQ ID NO:12:

Ser 1 Lys Leu Lys Asp 5 Pro Glu Leu Ser Leu 10 Lys Gly Thr Gin His 15 lie Met Gin Ala Gly 20 Gin Thr Leu His Leu 25 Gin Cys Arg Gly Glu 30 Ala Ala His Lys Trp 35 Ser Leu Pro Glu Met 40 Val Ser Lys Glu Ser 45 Glu Arg Leu Ser lie 50 Thr Lys Ser Ala Cys 55 Gly Arg Asn Gly Lys 60 Gin Phe Cys Ser Thr 65 Leu Thr Leu Asn Thr 70 Ala Gin Ala Asn His 75 Thr Gly Phe Tyr Ser 80 Cys Lys Tyr Leu Ala 85 Val Pro Thr Ser Lys 90 Lys Lys Glu Thr Glu 95 Ser Ala lie Tyr He 100 Phe He Ser Asp Thr 105 Gly Arg Pro Phe Val 110 Glu Met Tyr Ser Glu 115 He Pro Glu He lie 120 His Met Thr Glu Gly 125 Arg Glu Leu Val lie 130 Pro Cys Arg Val Thr 135 Ser Pro Asn He Thr 140 Val Thr Leu Lys Lys 145 Phe Pro Leu Asp Thr 150 Leu lie Pro Asp Gly 155 Lys Arg lie He Trp 160 Asp Ser Arg Lys Gly 165 Phe lie lie Ser Asn 170 Ala Thr Tyr Lys Glu 175 lie Gly Leu Leu Thr 180 Cys Glu Ala Thr Val 185 Asn Gly His Leu Tyr 190 Lys Thr Asn Tyr Leu 195 Thr His Arg Gin Thr 200 Asn Thr He He Asp 205 Val Gin lie Ser Thr 210 Pro Arg Pro Val Lys 215 Leu Leu Arg Gly His 220 Thr Leu Val Leu Asn 225 cys Thr Ala Thr Thr 230 Pro Leu Asn Thr Arg 235 Val Gin Met Thr Trp 240 Ser Tyr Pro Asp Glu 245 Lys Asn Lys Arg Ala 250 Ser Val Arg Arg Arg 255 lie Asp Gin Ser Asn 260 Ser His Ala Asn lie 265 Phe Tyr Ser Val Leu 270 Thr lie Asp Lys Met 275 Gin Asn Lys Asp Lys 280 Gly Leu Tyr Thr Cys 285 Arg Val Arg Ser Gly 290 Pro Ser Phe Lys Ser 295 Val Asn Thr Ser Val 300 His He Tyr Asp Lys 305 Ala Phe He Thr Val 310 Lys His Arg Lys Gin 315 Gin Val Leu Glu Thr 320 Val Ala Gly Lys Arg 325 Ser Tyr Arg Leu Ser 330 Met Lys Val Lys Ala 335 Phe Pro Ser Pro Glu 340 Val Val Trp Leu Lys 345 Asp Gly Leu Pro Ala 350 Thr Glu Lys Ser Ala 355 Arg Tyr Leu Thr Arg 360 Gly Tyr Ser Leu lie 365 He Lys Asp 18888 49 109016/2

Val Thr 370 Glu Glu Asp Ala Gly 375 Asn Tyr Thr Ile Leu 380 Leu Ser Ile Lys Gin 385 Ser Asn Val Phe Lys 390 Asn Leu Thr Ala Thr 395 Leu Ile Val Asn Val 400 Lys Pro Gin Ile Tyr 405 Glu Lys Ala Val Ser 410 Ser Phe Pro Asp Pro 415 Ala Leu Tyr Pro Leu 420 Gly Ser Arg Gin Ile 425 Leu Thr Cys Thr Ala 430 Tyr Gly Ile Pro Gin 435 Pro Thr Ile Lys Trp 440 Phe Trp His Pro Cys 445 Asn His Asn His Ser 450 Glu Ala Arg Cys Asp 455 Phe Cys Ser Asn Asn 460 Glu Glu Ser Phe Ile 465 Leu Asp Ala Asp Ser 470 Asn Met Gly Asn Arg 475 Ile Glu Ser Ile Thr 480 Gin Arg Met Ala Ile 485 lie Glu Gly Lys Asn 490 Lys Met Ala Ser Thr 495 Leu Val Val Ala Asp 500 Ser Arg Ile Ser Gly 505 Ile Tyr Ile Cys Ile 510 Ala Ser Asn Lys Val 515 Gly Thr Val Gly Arg 520 Asn Ile Ser Phe Tyr 525 Ile Thr Asp Val Pro 530 Asn Gly Phe His Val 535 Asn Leu Glu Lys Met 540 Pro Thr Glu Gly Glu 545 Asp Leu Lys Leu Ser 550 Cys Thr Val Asn Lys 555 Phe Leu Tyr Arg Asp 560 Val Thr Trp Ile Leu 565 Leu Arg Thr Val Asn 570 Asn Arg Thr Met His 575 Tyr Ser Ile Ser Lys 580 Gin Lys Met Ala Ile 585 Thr Lys Glu His Ser 590 Ile Thr Leu Asn Leu 595 Thr Ile Met Asn Val 600 Ser Leu Gin Asp Ser 605 Gly Thr Tyr Ala Cys 610 Arg Ala Arg Asn Val 615 Tyr Thr Gly Glu Glu 620 Ile Leu Gin Lys Lys 625 Glu Ile Thr Ile Arg 630 Gly Glu His Cys Asn 635 Lys Lys Ala Val Phe 640 Ser Ser Arg Asn Ile Val Ser Lys Lys 645 His Phe Lys Ser Thr Arg 650 Asn Asp Cys Thr Thr 655 Gin 660 (2) INFORMATION FOR SEQ ID NO:13: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 668 amino acids (B) TYPE: amino acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: protein (xi) SEQUENCE DESCRIPTION: SEQ ID NO:13:

Ser Glu Gin Asn Met Gin Ser Lys Val Leu Leu Ala Val Ala Leu Trp 15 10 15 18888 50 1090162

Leu Cys Val Glu 20 Thr Arg Ala Ala Ser 25 Val Gly Leu Pro Ser 30 Val Ser Leu Asp Leu 35 Pro Arg Leu Ser He 40 Gin Lys Asp He Leu 45 Thr He Lys Ala Asn 50 Thr Thr Leu Gin He 55 Thr Cys Arg Gly Gin 60 Arg Asp Leu Asp Trp 65 Leu Trp Pro Asn Asn 70 Gin Ser Gly Ser Glu 75 Gin Arg Val Glu Val 80 Thr Glu Cys Ser Asp 85 Gly Leu Phe Cys Lys 90 Thr Leu Thr He Pro 95 Lys Val lie Gly Asn 100 Asp Thr Gly Ala Tyr 105 Lys Cys Phe Tyr Arg 110 Glu Thr Asp Leu Ala 115 Ser Val lie Tyr Val 120 Tyr Val Gin Asp Tyr 125 Arg Ser Pro Phe lie 13 0 Ala Ser Val Ser Asp 135 Gin His Gly Val Val 140 Tyr He Thr Glu Asn 145 Lys Asn Lys Thr Val 150 Val lie Pro Cys Leu 155 Gly Ser He Ser Asn 160 Leu Asn Val Ser Leu 165 Cys Ala Arg Tyr Pro 170 Glu Lys Arg Phe Val 175 Pro Asp Gly Asn Arg 180 He Ser Trp Asp Ser 185 Lys Lys Gly Phe Thr 190 He Pro Ser Tyr Met 195 He Ser Tyr Ala Gly 200 Met Val Phe Cys Glu 205 Ala Lys He Asn Asp 210 Glu Ser Tyr Gin Ser 215 lie Met Tyr lie Val 220 Val Val Val Gly Tyr 225 Arg He Tyr Asp Val 230 Val Leu Ser Pro Ser 235 His Gly lie Glu Leu 240 Ser Val Gly Glu Lys 245 Leu Val Leu Asn Cys 250 Thr Ala Arg Thr Glu 255 Leu Asn Val Gly He 260 Asp Phe Asn Trp Glu 265 Tyr Pro Ser Ser Lys 270 His Gin His Lys Lys 275 Leu Val Asn Arg Asp 280 Leu Lys Thr Gin Ser 285 Gly Ser Glu Met Lys 290 Lys Phe Leu Ser Thr 295 Leu Thr He Asp Gly 300 Val Thr Arg Ser Asp 305 Gin Gly Leu Tyr Thr 310 Cys Ala Ala Ser Ser 315 Gly Leu Met Thr Lys 320 Lys Asn Ser Thr Phe 325 Val Arg Val His Glu 330 Lys Pro Phe Val Ala 335 Phe Gly Ser Gly Met 340 Glu Ser Leu Val Glu 345 Ala Thr Val Gly Glu 350 Arg Val Arg He Pro 355 Ala Lys Tyr Leu Gly 360 Tyr Pro Pro Pro Glu 365 He Lys Trp Tyr Lys 370 Asn Gly He Pro Leu 375 Glu Ser Asn His Thr 380 He Lys Ala Gly His 385 Val Leu Thr lie Met 390 Glu Val Ser Glu Arg 395 Asp Thr Gly Asn Tyr 400 Thr Val He Leu Thr 405 Asri Pro He Ser Lys 410 Glu Lys Gin Ser His 415 Val 'Val Ser Leu Val 420 Val Tyr Val Pro Pro 425 Gin He Gly Glu Lys 430 Ser Leu lie Ser Pro 435 Val Asp Ser Tyr Gin 440 Tyr Gly Thr Thr Gin 445 Thr Leu Thr 18888; 51 109016/2

Cys Thr Val Tyr Ala Ile Pro Pro Pro His His Ile His Trp Tyr Trp 450 455 460 Gin Leu Glu Glu Glu Cys Ala Asn Glu Pro Ser Gin Ala Val Ser Val 465 470 475 480 Thr Asn Pro Tyr Pro Cys Glu Glu Trp Arg Ser Val Glu Asp Phe Gin 485 490 495 Gly Gly Asn Lys Ile Ala Val Asn Lys Asn Gin Phe Ala Leu Ile Glu 500 505 510 Gly Lys Asn Lys Thr Val Ser Thr Leu Val Ile Gin Ala Ala Asn Val 515 520 525 Ser Ala Leu Tyr Lys Cys Glu Ala Val Asn Lys Val Gly Arg Gly Glu 530 535 540 Arg Val Ile Ser Phe His Val Thr Arg Gly Pro Glu Ile Thr Leu Gin 545 550 555 560 Pro Asp Met Gin Pro Thr Glu Gin Glu Ser Val Ser Leu Trp Cys Thr 565 570 575 Ala Asp Arg Ser Thr Phe Glu Asn Leu Thr Trp Tyr Lys Leu Gly Pro 580 585 590 Gin Pro Leu Pro Ile His Val Gly Glu Leu Pro Thr Pro Val Cys Lys 595 600 605 Asn Leu Asp Thr Leu Trp Lys Leu Asn Ala Thr Met Phe Ser Asn Ser 610 615 620 Thr Asn Asp Ile Leu Ile Met Glu Leu Lys Asn Ala Ser Leu Gin Asp 625 630 635 640 Gin Gly Asp Tyr Val Cys Leu Ala Gin Asp Arg Lys Thr Lys Lys Arg 645 650 655 His Cys Val Val Arg Gin Leu Thr Val Leu Glu Arg 660 665 (2) INFORMATION FOR SEQ ID NO:14: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 780 amino acids (B) TYPE: amino acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: protein (xi) SEQUENCE DESCRIPTION: SEQ ID NO:14:

Met Val Ser Tyr Trp Asp Thr Gly Val Leu Leu Cys Ala Leu Leu Ser 1 5 10 15

Cys Leu Leu Leu Thr Gly Ser Ser Ser Gly Ser Lys Leu Lys Asp Pro 20 25 30

Glu Leu Ser Leu Lys Gly Thr Gin His Ile Met Gin Ala Gly Gin Thr 35 40 45

Leu His Leu Gin Cys Arg Gly Glu Ala Ala His Lys Trp Ser Leu Pro 50 55 60

Glu Met Val Ser Lys Glu Ser Glu Arg Leu Ser Ile Thr Lys Ser Ala 65 70' 75 - 80

Cys Gly Arg Asn Gly Lys Gin Phe Cys Ser Thr Leu Thr Leu Asn Thr 85 90 95

Ala Gin Ala Asn His Thr Gly Phe Tyr Ser Cys Lys Tyr Leu Ala Val 100 105 110 1888! 52 109016/2

Pro Thr Ser 115 Lys Lys Lys Glu Thr 120 Glu Ser Ala lie Tyr 125 He Phe He Ser Asp 130 Thr Gly Arg Pro Phe 135 Val Glu Met Tyr Ser 140 Glu He Pro Glu lie 145 He His Met Thr Glu 150 Gly Arg Glu Leu Val 155 lie Pro Cys Arg Val 160 Thr Ser Pro Asn lie 165 Thr Val Thr Leu Lys 170 Lys Phe Pro Leu Asp 175 Thr Leu He Pro Asp 180 Gly Lys Arg He He 185 Trp Asp Ser Arg Lys 190 Gly Phe lie He Ser 195 Asn Ala Thr Tyr Lys 200 Glu He Gly Leu Leu 205 Thr Cys Glu Ala Thr 210 Val Asn Gly His Leu 215 Tyr Lys Thr Asn Tyr 220 Leu Thr His Arg Gin 225 Thr Asn Thr He lie 230 Asp Val Gin He Ser 235 Thr Pro Arg Pro Val 240 Lys Leu Leu Arg Gly 245 His Thr Leu Val Leu 250 Asn Cys Thr Ala Thr 255 Thr Pro Leu Asn Thr 260 Arg Val Gin Met Thr 265 Trp Ser Tyr Pro Asp 270 Glu Lys Asn Lys Arg 275 Ala Ser Val Arg Arg 280 Arg He Asp Gin Ser 285 Asn Ser His Ala Asn 290 He Phe Tyr Ser Val 295 Leu Thr He Asp Lys 300 Met Gin Asn Lys Asp 305 Lys Gly Leu Tyr Thr 310 Cys Arg Val Arg Ser 315 Gly Pro Ser Phe Lys 320 Ser Val Asn Thr Ser 325 Val His He Tyr Asp 330 Lys Ala Phe He Thr 335 Val Lys His Arg Lys 340 Gin Gin Val Leu Glu 345 Thr Val Ala Gly Lys Arg 350 Ser Tyr Arg Leu 355 Ser Met Lys Val Lys 360 Ala Phe Pro Ser Pro 365 Glu Val Val Trp Leu 370 Lys Asp Gly Leu Pro 375 Ala Thr Glu Lys Ser 380 Ala Arg Tyr Leu Thr 385 Arg Gly Tyr Ser Leu 390 lie He Lys Asp Val 395 Thr Glu Glu Asp Ala 400 Gly Asn Tyr Thr He 405 Leu Leu Ser He Lys 410 Gin Ser Asn Val Phe 415 Lys Asn Leu Thr Ala 420 Thr Leu lie Val Asn 425 Val Lys Pro Gin He 430 Tyr Glu Lys Ala Val 435 Ser Ser Phe Pro Asp 440 Pro Ala Leu Tyr Pro 445 Leu Gly Ser Arg Gin 450 He Leu Thr Cys Thr 455 Ala Tyr Gly He Pro 460 Gin Pro Thr He Lys 465 Trp Phe Trp His Pro 470 Cys Asn His Asn His 475 Ser Glu Ala Arg Cys 480 Asp Phe Cys Ser Asn 485 Asn Glu Glu Ser Phe 490 He Leu Asp Ala Asp 495 Ser Asn Met Gly Asn 500 Arg He Glu Ser lie 505 Thr Gin Arg Met Ala 510 He lie 'Glu Gly Lys 515 Asn Lys Met Ala Ser 520 Thr Leu Val Val Ala 525 Asp Ser Arg lie Ser Gly He Tyr He Cys He Ala Ser Asn Lys Val Gly Thr Val 530 535 540 1888; 53 109016/2

Gly Arg Asn He Ser Phe Tyr He Thr Asp Val Pro Asn Gly Phe His 545 550 555 560 Val Asn Leu Glu Lys Met Pro Thr Glu Gly Glu Asp Leu Lys Leu Ser 565 570 575 Cys Thr Val Asn Lys Phe Leu Tyr Arg Asp Val Thr Trp He Leu Leu 580 585 590 Arg Thr Val Asn Asn Arg Thr Met His Tyr Ser He Ser Lys Gin Lys 595 600 605 Met Ala lie Thr Lys Glu His Ser He Thr Leu Asn Leu Thr lie Met 610 615 620 Asn Val Ser Leu Gin Asp Ser Gly Thr Tyr Ala Cys Arg Ala Arg Asn 625 630 635 640 Val Tyr Thr Gly Glu Glu He Leu Gin Lys Lys Glu lie Thr lie Arg 645 650 655 Asp Gin Glu Ala Pro Tyr Leu Leu Arg Asn Leu Ser Asp His Thr Val 660 665 670 Ala lie Ser Ser Ser Thr Thr Leu Asp Cys His Ala Asn Gly Val Pro 675 680 685 Glu Pro Gin He Thr Trp Phe Lys Asn Asn His Lys He Gin Gin Glu 690 695 700 Pro Gly lie He Leu Gly Pro Gly Ser Ser Thr Leu Phe lie Glu Arg 705 710 715 720 Val Thr Glu Glu Asp Glu Gly Val Tyr His Cys Lys Ala Thr Asn Gin 725 730 735 Lys Gly Ser Val Glu Ser Ser Ala Tyr Leu Thr Val Gin Gly Thr Ser 740 745 750 Asp Lys Ser Asn Leu Glu Leu He Thr Leu Thr Cys Thr Cys Val Ala 755 760 765 Ala Thr Leu Phe Trp Leu Leu Leu Thr Leu Leu lie 770 775 780 (2) INFORMATION FOR SEQ ID NO:: 15: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 788 amino acids (B) TYPE: amino acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: protein (Xi) SEQUENCE DESCRIPTION: SEQ ID NO:15: Met Gin Ser Lys Val Leu Leu Ala Val Ala Leu Trp Leu Cys Val Glu 1 5 10 15 Thr Arg Ala Ala Ser Val Gly Leu Pro Ser Val Ser Leu Asp Leu Pro 20 25 30 Arg Leu Ser He Gin Lys Asp He Leu Thr lie Lys Ala Asn Thr Thr 35 40 45 Leu Gin lie Thr Cys Arg Gly Gin Arg Asp Leu Asp Trp Leu Trp Pro 50 55 60 Asn Asn Gin Ser Gly Ser Glu Gin Arg Val Glu Val Thr Glu Cys Ser ?65 70 75 80 Asp Gly Leu Phe Cys Lys Thr Leu Thr He Pro Lys Val He Gly Asn 85 90 95 Asp Thr Gly Ala Tyr Lys Cys Phe Tyr Arg Glu Thr Asp Leu Ala Ser 100 105 110 18888 54 109016/2

Val Ile Tyr 115 Val Tyr Val Gin Asp 120 Tyr Arg Ser Pro Phe 125 Ile Ala Ser Val Ser 130 Asp Gin His Gly Val 135 Val Tyr lie Thr Glu 140 Asn Lys Asn Lys Thr 145 Val Val Ile Pro Cys 150 Leu Gly Ser lie Ser 155 Asn Leu Asn Val Ser 160 Leu Cys Ala Arg Tyr 165 Pro Glu Lys Arg Phe 170 Val Pro Asp Gly Asn 175 Arg Ile Ser Trp Asp 180 Ser Lys Lys Gly Phe 185 Thr He Pro Ser Tyr 190 Met lie Ser Tyr Ala 195 Gly Met Val Phe Cys 200 Glu Ala Lys Ile Asn 205 Asp Glu Ser Tyr Gin 210 Ser Ile Met Tyr Ile 215 Val Val Val Val Gly 220 Tyr Arg Ile Tyr Asp 225 Val Val Leu Ser Pro 230 Ser His Gly Ile Glu 235 Leu Ser Val Gly Glu 240 Lys Leu Val Leu Asn 245 Cys Thr Ala Arg Thr 250 Glu Leu Asn Val Gly 255 Ile Asp Phe Asn Trp 260 Glu Tyr Pro Ser Ser 265 Lys His Gin His Lys 270 Lys Leu Val Asn Arg 275 Asp Leu Lys Thr Gin 280 Ser Gly Ser Glu Met 285 Lys Lys Phe Leu Ser 290 Thr Leu Thr Ile Asp 295 Gly Val Thr Arg Ser 300 Asp Gin Gly Leu Tyr 305 Thr Cys Ala Ala Ser 310 Ser Gly Leu Met Thr 315 Lys Lys Asn Ser Thr 320 Phe Val Arg Val His 325 Glu Lys Pro Phe Val 330 Ala Phe Gly Ser Gly 335 Met Glu Ser Leu Val 340 Glu Ala Thr Val Gly 345 Glu Arg Val Arg Ile 350 Pro Ala Lys Tyr Leu 355 Gly Tyr Pro Pro Pro 360 Glu Ile Lys Trp Tyr 365 Lys Asn Gly Ile Pro 370 Leu Glu Ser Asn His 375 Thr Ile Lys Ala Gly 380 His Val Leu Thr Ile 385 Met Glu Val Ser Glu 390 Arg Asp Thr Gly Asn 395 Tyr Thr Val Ile Leu 400 Thr Asn Pro Ile Ser 405 Lys Glu Lys Gin Ser 410 His Val Val Ser Leu 415 Val Val Tyr Val Pro 420 Pro Gin Ile Gly Glu 425 Lys Ser Leu Ile Ser 43 0 Pro Val Asp Ser Tyr 435 Gin Tyr (Sly Thr Thr 440 Gin Thr Leu Thr Cys 445 Thr Val Tyr Ala Ile 450 Pro Pro Pro His His 455 Ile His Trp Tyr Trp 460 Gin Leu Glu Glu Glu 465 Cys Ala Asn Glu Pro 470 Ser Gin Ala Val Ser 475 Val Thr Asn Pro Tyr 480 Pro Cys Glu Glu Trp 485 Arg Ser Val Glu Asp 490 Phe Gin Gly Gly Asn 495 Lys Ile Ala Val Asn 500 Lys Asn Gin Phe Ala 505 Leu Ile Glu Gly Lys 510 Asn Lys Thr Val Ser 515 Thr Leu Val Ile Gin 520 Ala Ala Asn Val Ser 525 Ala Leu Tyr Lys Cys 530 Glu Ala Val Asn Lys 535 Val Gly Arg Gly Glu 540 Arg Val Ile Ser 18888 55 109016/2 K-'

Phe 545 His Val Thr Arg Gly 550 Pro Glu He Thr Leu 555 Gin Pro Asp Met Gin 560 Pro Thr Glu Gin Glu 565 Ser Val Ser Leu Trp 570 Cys Thr Ala Asp Arg 575 Ser Thr Phe Glu Asn 580 Leu Thr Trp Tyr Lys 585 Leu Gly Pro Gin Pro 590 Leu Pro lie His Val 595 Gly Glu Leu Pro Thr 600 Pro Val Cys Lys Asn 605 Leu Asp Thr Leu Trp 610 Lys Leu Asn Ala Thr 615 Met Phe Ser Asn Ser 620 Thr Asn Asp He Leu 625 He Met Glu Leu Lys 630 Asn Ala Ser Leu Gin 635 Asp Gin Gly Asp Tyr 640 Val Cys Leu Ala Gin 645 Asp Arg Lys Thr Lys 650 Lys Arg His Cys Val 655 Val Arg Gin Leu Thr 660 Val Leu Glu Arg Val 665 Ala Pro Thr He Thr 670 Gly Asn Leu Glu Asn 675 Gin Thr Thr Ser lie 680 Gly Glu Ser lie Glu 685 Val Ser Cys Thr Ala 690 Ser Gly Asn Pro Pro 695 Pro Gin He Met Trp 700 Phe Lys Asp Asn Glu 705 Thr Leu Val Glu Asp 710 Ser Gly He Val Leu 715 Lys Asp Gly Asn Arg 720 Asn Leu Thr He Arg 725 Arg Val Arg Lys Glu 730 Asp Glu Gly Leu Tyr 735 Cys Gin Ala Cys Ser 740 Val Leu Gly Cys Ala 745 Lys Val Glu Ala Phe 750 Phe He He Glu Gly 755 Ala Gin Glu Lys Thr 760 Asn Leu Glu He He 765 He Leu Val Gly Leu Thr 770 Gly Thr Thr Val Val He Ala Met 775 Phe Phe Trp Leu Leu 780 Leu Val He lie 785 (2) INFORMATION FOR SEQ ID NO:16: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 2264 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear

(ii) MOLECULE TYPE: Genomic DNA (xi) SEQUENCE DESCRIPTION: SEQ ID NO:16: GGTGTGGTCG CTGCGTTTCC TCTGCCTGCG CCGGGCATCA CTTGCGCGCC GCAGAAAGTC 60 CGTCTGGCAG CCTGGATATC CTCTCCTACC GGCACCCGCA GACGCCCCTG CAGCCGCGGT 120 CGGCGCCCGG GCTCCCTAGC CCTGTGCGCT CAACTGTCCT GCGCTGCGGG GTGCCGCGAG 180 TTCCACCTCC GCGCCTCCTT CTCTAGACAG GCGCTGGGAG AAAGAACCGG CTCCCGAGTT 240 CCGGCATTTC GCCCGGCTCG AGGTGCAGGA TGCAGAGCAA GGTGCTGCTG GCCGTCGCCC 300 TGTGGCTCTG CGTGGAGACC CGGGCCGCCT CTGTGGGTTT GCCTAGTGTT TCTCTTGATC 360 TGCCCAGGCT CAGCATACAA AAAGACATAC TTACAATTAA GGCTAATACA ACTCTTCAAA 420 TTACTTGCAG GGGACAGAGG GACTTGGACT GGCTTTGGCC CAATAATCAG AGTGGCAGTG 480 AGCAAAGGGT GGAGGTGACT GAGTGCAGCG ATGGCCTCTT CTGTAAGACA CTCACAATTC 540 CAAAAGTGAT CGGAAATGAC ACTGGAGCCT ACAAGTGCTT CTACCGGGAA ACTGACTTGG 600 CCTCGGTCAT TTATGTCTAT GTTCAAGATT ACAGATCTCC ATTTATTGCT TCTGTTAGTG 660 18888 56

ACCAACATGG AGTCGTGTAC ATTACTGAGA TCGGGTCCAT TTCAAATCTC AACGTGTCAC TTCCTGATGG TAACAGAATT TCCTGGGACA TGATCAGCTA TGCTGGCATG GTCTTCTGTG CTATTATGTA CATAGTTGTC GTTGTAGGGT CTCATGGAAT TGAACTATCT GTTGGAGAAA AACTAAATGT GGGGATTGAC TTCAACTGGG AACTTGTAAA CCGAGACCTA AAAACCCAGT CCTTAACTAT AGATGGTGTA ACCCGGAGTG GTGGGCTGAT GACCAAGAAG AACAGCACAT CTTTTGGAAG TGGCATGGAA TCTCTGGTGG CTGCGAAGTA CCTTGGTTAC CCACCCCCAG TTGAGTCCAA TCACACAATT AAAGCGGGGC GAGACACAGG AAATTACACT GTCATCCTTA ATGTGGTCTC TCTGGTTGTG TATGTCCCAC CTGTGGATTC CTACCAGTAC GGCACCACTC CTCCCCCGCA TCACATCCAC TGGTATTGGC GCCAAGCTGT CTCAGTGACA AACCCATACC TCCAGGGAGG AAATAAAATT GCCGTTAATA ACAAAACTGT AAGTACCCTT GTTATCCAAG AAGCGGTCAA CAAAGTCGGG AGAGGAGAGA CTGAAATTAC TTTGCAACCT GACATGCAGC GCACTGCAGA CAGATCTACG TTTGAGAACC TGCCAATCCA TGTGGGAGAG TTGCCCACAC AATTGAATGC CACCATGTTC TCTAATAGCA ATGCATCCTT GCAGGACCAA GGAGACTATG AAAGACATTG CGTGGTCAGG CAGCTCACAG

ACAAAAACAA

TTTGTGCAAG

GCAAGAAGGG

AAGCAAAAAT

ATAGGATTTA

AGCTTGTCTT

AATACCCTTC

CTGGGAGTGA

ACCAAGGATT

TTGTCAGGGT

AAGCCACGGT

AAATAAAATG

ATGTACTGAC

CCAATCCCAT

CCCAGATTGG

AAACGCTGAC

AGTTGGAGGA

CTTGTGAAGA

AAAATCAATT

CGGCAAATGT

GGGTGATCTC

CCACTGAGCA

TCACATGGTA

CTGTTTGCAA

CAAATGACAT

TCTGCCTTGC

TCCTAGAGCG 109016/2 AACTGTGGTG ATTCCATGTC 720 ATACCCAGAA AAGAGATTTG 780 CTTTACTATT CCCAGCTACA 840 TAATGATGAA AGTTACCAGT 900 TGATGTGGTT CTGAGTCCGT 960 AAATTGTACA GCAAGAACTG 1020 TTCGAAGCAT CAGCATAAGA 1080 GATGAAGAAA TTTTTGAGCA 1140 GTACACCTGT GCAGCATCCA 1200 CCATGAAAAA CCTTTTGTTG 1260 GGGGGAGCGT GTCAGAATCC 1320 GTATAAAAAT GGAATACCCC 1380 GATTATGGAA GTGAGTGAAA 1440 TTCAAAGGAG AAGCAGAGCC . 1500 TGAGAAATCT CTAATCTCTC 1560 ATGTACGGTC TATGCCATTC 1620 AGAGTGCGCC AACGAGCCCA 1680 ATGGAGAAGT GTGGAGGACT 1740 TGCTCTAATT GAAGGAAAAA 1800 GTCAGCTTTG TACAAATGTG 1860 CTTCCACGTG ACCAGGGGTC 1920 GGAGAGCGTG TCTTTGTGGT 1980 CAAGCTTGGC CCACAGCCTC 2040 GAACTTGGAT ACTCTTTGGA 2100 TTTGATCATG GAGCTTAAGA 2160 TCAAGACAGG AAGACCAAGA 2220 TTAA 2264 (2) INFORMATION FOR SEQ ID NO:17: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 2352 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear

(ii) MOLECULE TYPE: Genomic DNA (xi) SEQUENCE DESCRIPTION:

GCGCTCACCA TGGTCAGCTA CTGGGACACC CTGCTTCTCA CAGGATCTA&amp;amp; TTCAGGTTCA GGCACCCAGC ACATCATGCA AGCAGGCCAG GCCCATAAAT GGTCTTTGCC TGAAATGGTG AAATCTGCCT GTGGAAGAAA TGGCAAACAA CAAGCAAACC ACACTGGCTT CTACAGCTGC AAGGAAACAG AATCTGCAAT CTATATATTT ATGTACAGTG AAATCCCCGA AATTATACAC TGCCGGGTTA CGTCACCTAA CATCACTGTT ATCCCTGATG GAAAACGCAT AATCTGGGAC ACGTACAAAG AAATAGGGCT TCTGACCTGT ACAAACTATC TCACACATCG ACAAACCAAT CGCCCAGTCA AATTACTTAG AGGCCATACT TTGAACACGA GAGTTCAAAT GACCTGGAGT GTAAGGCGAC GAATTGACCA AAGCAATTCC ATTGACAAAA TGCAGAACAA AGACAAAGGA SEQ ID NO :17: GGGGTCCTGC TGTGCGCGCT GCTCAGCTGT 60 AAATTAAAAG ATCCTGAACT GAGTTTAAAA 120 ACACTGCATC TCCAATGCAG GGGGGAAGCA 180 AGTAAGGAAA GCGAAAGGCT GAGCATAACT 240 TTCTGCAGTA CTTTAACCTT GAACACAGCT 300 AAATATCTAG CTGTACCTAC TTCAAAGAAG 360 ATTAGTGATA CAGGTAGACC TTTCGTAGAG 420 ATGACTGAAG GAAGGGAGCT CGTCATTCCC 480 ACTTTAAAAA AGTTTCCACT TGACACTTTG 540 AGTAGAAAGG GCTTCATCAT ATCAAATGCA 600 GAAGCAACAG TCAATGGGCA TTTGTATAAG 660 ACAATCATAG ATGTCCAAAT AAGCACACCA 720 CTTGTCCTCA ATTGTACTGC TACCACTCCC 780 TACCCTGATG AAAAAAATAA GAGAGCTTCC 840 CATGCCAACA TATTCTACAG TGTTCTTACT 900 CTTTATACTT GTCGTGTAAG GAGTGGACCA 960 18888

<img img-format="tif" img-content="drawing" file="IL109016AD00028.tif" id="idf0008" />

109016/2 TCATTCAAAT CTGTTAACAC CTCAGTGCAT ATATATGATA AAGCATTCAT CACTGTGAAA 1020 CATCGAAAAC AGCAGGTGCT TGAAACCGTA GCTGGCAAGC GGTCTTACCG GCTCTCTATG 1080 AAAGTGAAGG CATTTCCCTC GCCGGAAGTT GTATGGTTAA AAGATGGGTT ACCTGCGACT 1140 GAGAAATCTG CTCGCTATTT GACTCGTGGC TACTCGTTAA TTATCAAGGA CGTAACTGAA 1200 GAGGATGCAG GGAATTATAC AATCTTGCTG AGCATAAAAC AGTCAAATGT GTTTAAAAAC 1260 CTCACTGCCA CTCTAATTGT CAATGTGAAA CCCCAGATTT ACGAAAAGGC CGTGTCATCG 1320 TTTCCAGACC CGGCTCTCTA CCCACTGGGC AGCAGACAAA TCCTGACTTG TACCGCATAT 1380 GGTATCCCTC AACCTACAAT CAAGTGGTTC TGGCACCCCT GTAACCATAA TCATTCCGAA 1440 GCAAGGTGTG ACTTTTGTTC CAATAATGAA GAGTCCTTTA TCCTGGATGC TGACAGCAAC 1500 ATGGGAAACA GAATTGAGAG CATCACTCAG CGCATGGCAA TAATAGAAGG AAAGAATAAG 1560 ATGGCTAGCA CCTTGGTTGT GGCTGACTCT AGAATTTCTG GAATCTACAT TTGCATAGCT 1620 TCCAATAAAG TTGGGACTGT GGGAAGAAAC ATAAGCTTTT ATATCACAGA TGTGCCAAAT 1680 GGGTTTCATG TTAACTTGGA AAAAATGCCG ACGGAAGGAG AGGACCTGAA ACTGTCTTGC , 1740 ACAGTTAACA AGTTCTTATA CAGAGACGTT ACTTGGATTT TACTGCGGAC AGTTAATAAC : 1800 AGAACAATGC ACTACAGTAT TAGCAAGCAA AAAATGGCCA TCACTAAGGA GCACTCCATC 1860 ACTCTTAATC TTACCATCAT GAATGTTTCC CTGCAAGATT CAGGCACCTA TGCCTGCAGA 1920 GCCAGGAATG TATACACAGG GGAAGAAATC CTCCAGAAGA AAGAAATTAC AATCAGAGAT 1980 CAGGAAGCAC CATACCTCCT GCGAAACCTC AGTGATCACA CAGTGGCCAT CAGCAGTTCC 2040 ACCACTTTAG ACTGTCATGC TAATGGTGTC CCCGAGCCTC AGATCACTTG GTTTAAAAAC 2100 AACCACAAAA TACAACAAGA GCCTGGAATT ATTTTAGGAC CAGGAAGCAG CACGCTGTTT 2160 ATTGAAAGAG TCACAGAAGA GGATGAAGGT GTCTATCACT GCAAAGCCAC CAACCAGAAG 2220 GGCTCTGTGG AAAGTTCAGC ATACCTCACT GTTCAAGGAA CCTCGGACAA GTCTAATCTG 2280 GAGCTGATCA CTCTAACATG CACCTGTGTG GCTGCGACTC TCTTCTGGCT CCTATTAACC 2340 CTCCTTATCT AA 2352 (2) INFORMATION FOR SEQ ID NO:18: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 2383 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear

(ii) MOLECULE TYPE: Genomic DNA (xi) SEQUENCE DESCRIPTION: SEQ ID NO:18: CTCGAGGTGC AGGATGCAGA GCAAGGTGCT GCTGGCCGTC GCCCTGTGGC TCTGCGTGGA 60 GACCCGGGCC GCCTCTGTGG GTTTGCCTAG TGTTTCTCTT GATCTGCCCA GGCTCAGCAT 120 ACAAAAAGAC ATACTTACAA TTAAGGCTAA TACAACTCTT CAAATTACTT GCAGGGGACA 180 GAGGGACTTG GACTGGCTTT GGCCCAATAA TCAGAGTGGC AGTGAGCAAA GGGTGGAGGT 240 GACTGAGTGC AGCGATGGCCf TCTTCTGTAA GACACTCACA ATTCCAAAAG TGATCGGAAA 300 TGACACTGGA GCCTACAAGT GCTTCTACCG GGAAACTGAC TTGGCCTCGG TCATTTATGT 360 CTATGTTCAA GATTACAGAT CTCCATTTAT TGCTTCTGTT AGTGACCAAC ATGGAGTCGT 420 GTACATTACT GAGAACAAAA ACAAAACTGT GGTGATTCCA TGTCTCGGGT CCATTTCAAA 480 TCTCAACGTG TCACTTTGTG CAAGATACCC AGAAAAGAGA TTTGTTCCTG ATGGTAACAG 540 AATTTCCTGG GACAGCAAGA AGGGCTTTAC TATTCCCAGC TACATGATCA GCTATGCTGG 600 CATGGTCTTC TGTGAAGCAA AAATTAATGA TGAAAGTTAC CAGTCTATTA TGTACATAGT 660 TGTCGTTGTA GGGTATAGGA TTTATGATGT GGTTCTGAGT CCGTCTCATG GAATTGAACT 720 ATCTGTTGGA GAAAAGCTTG TCTTAAATTG TACAGCAAGA ACTGAACTAA ATGTGGGGAT 780 TGACTTCAAC TGGGAATACC CTTCTTCGAA GCATCAGCAT AAGAAACTTG TAAACCGAGA 840 GCTAAAAACC CAGTCTGGGA GTGAGATGAA GAAATTTTTG AGCACCTTAA CTATAGATGG 900 TGTAACCCGG AGTGACCAAG GATTGTACAC CTGTGCAGCA TCCAGTGGGC TGATGACCAA 960 GAAGAACAGC ACATTTGTCA GGGTCCATGA AAAACCTTTT GTTGCTTTTG GAAGTGGCAT 1020 GGAATCTCTG GTGGAAGCCA CGGTGGGGGA GCGTGTCAGA ATCCCTGCGA AGTACCTTGG 1080 TTACCCACCC CCAGAAATAA AATGGTATAA AAATGGAATA CCCCTTGAGT CCAATCACAC 1140 18888

<img img-format="tif" img-content="drawing" file="IL109016AD00029.tif" id="idf0009" />

109016/2

AATTAAAGCG GGGCATGTAC TGACGATTAT CACTGTCATC CTTACCAATC CCATTTCAAA TGTGTATGTC CCACCCCAGA TTGGTGAGAA GTACGGCACC ACTCAAACGC TGACATGTAC CCACTGGTAT TGGCAGTTGG AGGAAGAGTG GACAAACCCA TACCCTTGTG AAGAATGGAG AATTGCCGTT AATAAAAATC AATTTGCTCT CCTTGTTATC CAAGCGGCAA ATGTGTCAGC CGGGAGAGGA GAGAGGGTGA TCTCCTTCCA ACCTGACATG CAGCCCACTG AGCAGGAGAG TACGTTTGAG AACCTCACAT GGTACAAGCT AGAGTTGCCC ACACCTGTTT GCAAGAACTT GTTCTCTAAT AGCACAAATG ACATTTTGAT CCAAGGAGAC TATGTCTGCC TTGCTCAAGA CAGGCAGCTC ACAGTCCTAG AGCGTGTGGC GACGACAAGT ATTGGGGAAA GCATCGAAGT ACAGATCATG TGGTTTAAAG ATAATGAGAC GGATGGGAAC CGGAACCTCA CTATCCGCAG CTGCCAGGCA TGCAGTGTTC TTGGCTGTGC TGCCCAGGAA AAGACGAACT TGGAAATCAT GTTCTTCTGG CTACTTCTTG TCATCATCCT GGAAGTGAGT GAAAGAGACA CAGGAAATTA 1200 GGAGAAGCAG AGCCATGTGG TCTCTCTGGT 1260 ATCTCTAATC TCTCCTGTGG ATTCCTACCA 1320 GGTCTATGCC ATTCCTCCCC CGCATCACAT 1380 CGCCAACGAG CCCAGCCAAG CTGTCTCAGT 1440 AAGTGTGGAG GACTTCCAGG GAGGAAATAA 1500 AATTGAAGGA AAAAACAAAA CTGTAAGTAC 1560 TTTGTACAAA TGTGAAGCGG TCAACAAAGT 1620 CGTGACCAGG GGTCCTGAAA TTACTTTGCA 1680 CGTGTCTTTG TGGTGCACTG CAGACAGATC 1740 TGGCCCACAG CCTCTGCCAA TCCATGTGGG 1800 GGATACTCTT TGGAAATTGA ATGCCACCAT 1860 CATGGAGCTT AAGAATGCAT CCTTGCAGGA : 1920 CAGGAAGACC AAGAAAAGAC ATTGCGTGGT 1980 ACCCACGATC ACAGGAAACC TGGAGAATCA 2040 CTCATGCACG GCATCTGGGA ATCCCCCTCC 2100 CCTTGTAGAA GACTCAGGCA TTGTATTGAA 2160 AGTGAGGAAG GAGGACGAAG GCCTCTACAC 2220 AAAAGTGGAG GCATTTTTCA TAATAGAAGG 2280 TATTCTAGTA GGCACGACGG TGATTGCCAT 2340 AGGGACCGTT TAA 2383