AU755708B2 - Receptor ligand VEGF-C - Google Patents

Description

Receptor Ligand VEGF-C Field of the Invention The present invention generally relates to the field of genetic engineering and more particularly to growth factors for endothelial cells and growth factor genes.

Background of the Invention Developmental growth, the remodelling and regeneration of adult tissues, as well as solid tumour growth, can only occur when accompanied by blood vessel formation.

Angioblasts and hematopoietic precursor cells differentiate from'the mesoderm and form the blood islands of the yolk sac and the primary vascular system of the embryo. The development of blood vessels from these early (in situ) differentiating endothelial cells is termed vasculogenesis. Major embryonic blood vessels are believed to arise vis vasculogenesis, whereas the formation of the rest of the vascular tree is thought to occur as a result of vascular sprouting from pre-existing vessels, a process called angiogenesis, Risau et at., Devel. Biol., 125:441-450 (1988).

15 Endothelial cells give rise to several types of functionally and morphologically distinct vessels. When o•* *oo• *000 [n:\libc]03237:MEF 2 organs differentiate and begin to perform their specific functions, the phenotypic heterogeneity of endothelial cells increases. Upon angiogenic stimulation, endothelial cells may re-enter the cell cycle, migrate, withdraw from the cell cycle and subsequently differentiate again to form new vessels that are functionally adapted to their tissue environment.

Endothelial cells undergoing angiogenesis degrade the underlying basement membrane and migrate, forming capillary sprouts that project into the perivascular stroma. Ausprunk et al., Microvasc. Rev., 14:51-65 (1977). Angiogenesis during tissue development and regeneration depends on the tightly controlled processes of endothelial cell proliferation, migration, differentiation, and survival. Dysfunction of the endothelial cell regulatory system is a key feature of many diseases. Most significantly, tumor growth and metastasis have been shown to be angiogenesis dependent.

Folkman et al., J. Biol. Chem., 267:10931-10934 (1992).

Key signals regulating cell growth and differentiation are mediated by polypeptide growth factors and their transmembrane receptors, many of which are tyrosine kinases. Autophosphorylated peptides within the tyrosine kinase insert and carboxyl-terminal S 25 sequences of activated receptors are commonly recognized by kinase substrates involved in signal transduction for the readjustment of gene expression in responding cells.

Several families of receptor tyrosine kinases have been characterized. Van der Geer et al., Ann. Rev. Cell Biol., 10:251-337 (1994). The major growth factors and receptors transducing angiogenic stimuli are schematically shown in Fig. i.

Fibroblast growth factors are also known to be involved in the regulation of angiogenesis. They have been shown to be mitogenic and chemotactic for cultured endothelial cells. Fibroblast growth factors also stimulate the production of proteases, such as

A

3 collagenases and plasminogen activators, and induce tube formation by endothelial cells. Saksela et al., Ann.

Rev. Cell Biol., 4:93-126 (1988). There are two general classes of fibroblast growth factors, FGF-1 and FGF-2, both of which lack conventional signal peptides. Both types have an affinity for heparin and FGF-2 is bound to heparin sulfate proteoglycans in the subendothelial extracellular matrix from which it may be released after injury. Heparin potentiates the stimulation of endothelial cell proliferation by angiogenic FGFs, both by protecting against denaturation and degradation and dimerizing the FGFs. Cultured endothelial cells express the FGF-1 receptor but no significant levels of other high-affinity fibroblast growth factor receptors.

15 Among other ligands for receptor tyrosine kinases, the platelet derived growth factor, PDGF-BB, has been shown to be weakly angiogenic in the chick chorioallantoic membrane. Risau et al., Growth Factors, 7:261-266 (1992). Transforming growth factor a (TGFa) is 20 an angiogenic factor secreted by several tumor cell types and by macrophages. Hepatocyte growth factor (HGF), the ligand of the c-met proto-oncogene-encoded receptor, also i. s strongly angiogenic.

Recent evidence shows that there are 25 endothelial cell specific growth factors and receptors that may be primarily responsible for the stimulation of endothelial cell growth, differentiation and certain differentiated functions. The best studied of these is vascular endothelial growth factor (VEGF), a member of the PDGF family. Vascular endothelial growth factor is a dimeric glycoprotein of disulfide-linked 23 kD subunits.

Other reported effects of VEGF include the mobilization of intracellular calcium, the induction of plasminogen activator and plasminogen activator inhibitor-l synthesis, stimulation of hexose transport in endothelial cells, and promotion of monocyte migration in vitro.

Four VEGF isoforms, encoded by distinct mRNA splice 4 variants, appear to be equally capable of stimulating mitogenesis in endothelial cells. However, each isoform has a different affinity for cell surface proteoglycans, which behave as low affinity receptors for VEGF. The 121 and 165 amino acid isoforms of VEGF (VEGF121 and VEGF165) are secreted in a soluble form, whereas the isoforms of 189 and 206 amino acid residues remain cell surfaceassociated and have a strong affinity for heparin.

VEGF

was originally purified from several sources on the basis of its mitogenic activity toward endothelial cells, and also by its ability to induce microvascular permeability, hence it is also called vascular permeability factor

(VPF).

The pattern of VEGF expression suggests its involvement in the development and maintenance of the normal vascular system and in tumor angiogenesis. During murine development, the entire 7.5 day post-coital endoderm expresses VEGF and the ventricular neuroectoderm produces VEGF at the capillary ingrowth stage. See Breier et al., Development, 114:521-523 (1992). On day two of quail development, the vascularized area of the yolk sac as well as the whole embryo show expression of VEGF. In addition, epithelial cells next to fenestrated endothelia in adult mice show persistent VEGF expression, 25 suggesting a role in the maintenance of this specific endothelial phenotype and function.

Two high affinity receptors for VEGF have been characterized. These are VEGFR-I/Flt-I (fms-like tyrosine kinase-l) and VEGFR-2/Kdr/Flk-1 (kinase insert domain containing receptor/fetal liver kinase-l). Those receptors are classified in the PDGF-receptor family, but they have seven rather than five immunoglobulin-like loops in their extracellular domain (see Fig. 1) and they possess a longer kinase insert than normally observed in this family. The expression of VEGF receptors occurs mainly in vascular endothelial cells, although some may be present on hematopoietic progenitor cells, monocytes, -h 5 and melanoma cells. Only endothelial cells have been reported to proliferate in response to VEGF, and endothelial cells from different sources show different responses. Thus, the signals mediated through VEGFR-1 and VEGFR-2 appear to be cell type specific. The VEGFrelated placenta growth factor (PlGF) was recently shown to bind to VEGFR-1 with high affinity. PlGF was able to enhance the growth factor activity of VEGF, but it did not stimulate endothelial cells on its own. Naturally occurring VEGF/P1GF heterodimers were nearly as potent mitogens as VEGF homodimers for endothelial cells. Cao et al., J. Biol. Chem., 271:3154-62 (1996).

The Flt4 receptor tyrosine kinase (VEGFR-3) is closely related in structure to the products of the VEGFR-1 and VEGFR-2 genes. Despite this similarity, the mature form of Flt4 differs from the VEGF receptors in that it is proteolytically cleaved in the extracellular domain into two disulfide-linked polypeptides. Pajusola et al., Cancer Res., 52:5738-5743 (1992). The 4.5 and 20 5.8 kb Flt4 mRNAs encode polypeptides which differ in their C-termini due to the use of alternative 3' exons.

Isoforms of VEGF or P1GF do not show specific binding to Flt4 or induce its autophosphorylation.

Expression of Flt4 appears to be more 25 restricted than the expression of VEGFR-1 or VEGFR-2.

The expression of Flt4 first becomes detectable by in situ hybridization in the angioblasts of head mesenchyme, the cardinal vein, and extraembryonically in the allantois of 8.5 day p.c. mouse embryos. In 12.5 day p.c. embryos, the Flt4 signal is observed in developing venous and presumptive lymphatic endothelia, but arterial endothelia appear negative. During later stages of development, Flt4 mRNA becomes restricted to developing lymphatic vessels. The lymphatic endothelia and some high endothelial venules express Flt4 mRNA in adult human tissues and increased expression occurs in lymphatic sinuses in metastatic lymph nodes and in lymphangioma.

a -6- These results support the theory of the venous origin of 0 lymphatic vessels.

Five endothelial cell specific receptor tyrosine kinases, Fit-i (VEGFR-1), KDR/Flk-1 (VEGFR-2) Flt4 (VEGFR-3), Tie, and Tek/Tie-2 have so far been described, which possess the intrinsic tyrosine kinase activity essential for signal transduction. Targeted mutations inactivating Flt-1, Flk-l, Tie, and Tek in mouse embryos have indicated their essential and specific roles in vasculogenesis and angiogenesis at the molecular level. VEGFR-1 and VEGFR-2 bind VEGF with high affinity (Kd 16 pM and 760 pM, respectively) and VEGFR-1 also binds the related placenta growth factor (PlGF; Kd about 200 pM). A ligand for Tek is reported in PCT patent 15 publication WO 96/11269.

SUMMARY OF THE INVENTION The present invention provides a ligand, designated VEGF-C, for the Flt4 receptor tyrosine kinase (VEGFR-3). Thus, the invention provides a purified and isolated polypeptide which is capable of binding to the Flt4 receptor tyrosine kinase. Preferably, an Flt4 ligand of the invention is capable of stimulating tyrosine phosphorylation of Flt4 receptor tyrosine kinase in a host cell expressing the Flt4 receptor tyrosine 25 kinase. Preferred ligands of the invention are mammalian polypeptides. Highly preferred ligands are human polypeptides. As explained in detail below, dimers and multimers comprising polypeptides of the invention linked to each other or to other polypeptides are specifically contemplated as ligands of the invention.

In one embodiment, an FLT4 ligand polypeptide has a molecular weight of approximately 23 kD as determined by SDS-PAGE under reducing conditions. For example, the invention includes a ligand composed of one or more polypeptides of approximately 23 kD which is purifyable from conditioned media from a PC-3 prostatic

-'P

7 adenocarcinoma cell line, the cell line having ATCC Acc.

No. CRL 1435. Amino acid sequencing of this PC-3 cell derived ligand polypeptide revealed that the ligand polypeptide comprises an amino terminal amino acid sequence set forth in SEQ ID NO: 13. A conditioned medium comprising an Flt4 ligand is itself an aspect of the invention. The present invention also provides a new use for the PC-3 prostatic adenocarcinoma cell line which produces an Flt4 ligand. In a preferred embodiment, the ligand may be purified and isolated directly from the PC- 3 cell culture medium.

In a highly preferred embodiment, the ligand polypeptide comprises a fragment of the amino acid sequence shown in SEQ ID NO: 33 which specifically binds- 15 to the human Flt4 receptor tyrosine kinase. Exemplary fragments include: a polypeptide comprising an amino acid sequence set forth in SEQ ID NO: 33 from about residue 112 to about residue 213; a polypeptide comprising an amino acid sequence from about residue 104 to about residue 227 of SEQ ID NO: 33; and a polypeptide comprising an amino acid sequence from about residue 112 to about residue 227 of SEQ ID NO: 33. Other exemplary fragments include polypeptides comprising amino acid sequences of SEQ ID NO: 33 that span, approximately, the 25 following residues: 31-213, 31-227, 32-227, 103-217, 103-225, 104-213, 113-213, 103-227, 113-227, 131-211, 161-211, 103-225, 227-419, 228-419, 31-419, and 1-419, as described in greater detail below.

The present invention also provides one or more polypeptide precursors of an Flt4 ligand, wherein one such precursor (designated "prepro-VEGF-C") comprises the complete amino acid sequence (amino acid residues 1 to 419) shown in SEQ ID NO: 33. Thus, the invention includes a purified and isolated polypeptide having the amino acid sequence of residues 1 to 419 shown in SEQ ID NO: 33. Ligand precursors according to the invention, when expressed in an appropriate host cell, produce, via b 8 cleavage, a polypeptide which binds specifically to the Flt4 receptor tyrosine kinase. A putative 102 amino acid leader (prepro) peptide has been identified in the amino acid sequence shown in SEQ ID NO: 33. Thus, in a related aspect, the invention includes a purified and isolated polypeptide having the amino acids sequence of residues 103-419 shown in SEQ ID NO: 33.

In one embodiment, an expressed Flt4 ligand polypeptide precursor is proteolytically cleaved upon expression to produce an approximately 23 kD Flt4 ligand polypeptide. Thus, an Flt4 ligand polypeptide is provided which is the cleavage product of the precursor polypeptide shown in SEQ ID NO: 33 and which has a e..

molecular weight of approximately 23 kD under reducing conditions.

A putative VEGF-C precursor or splice variant, consisting of polypeptides with molecular weights of o* °about 29 and 32 kD, also is considered an aspect of the invention.

20 In another embodiment, an expressed Flt4 ligand polypeptide precursor is proteolytically cleaved upon expression to produce an approximately 21 kD VEGF-C polypeptide. Sequence analysis has indicated that an observed 21 kD form has an amino terminus approximately 9 amino acids downstream from the amino terminus of the 23 kD form, suggesting that alternative cleavage sites exist.

From the foregoing, it will be apparent that an aspect of the invention includes a fragment of the purified and isolated polypeptide having the amino acid sequence of residues 1 to 419 shown in SEQ ID NO: 33, the fragment being capable of specifically binding to Flt4 receptor tyrosine kinase. Preferred embodiments include fragments having an apparent molecular weight of approximately 21/23 kD and 29/32 kD as assessed by SDS- PAGE under reducing conditions.

Evidence suggests that the amino acids 9 essential for retaining Flt4 ligand activity are contained within approximately amino acids 103/112- 226/227 of SEQ ID NO: 33, and that a carboxy-terminal proteolytic cleavage to produce a mature, naturallyoccurring Flt4 ligand occurs at the approximate position of amino acids 226-227 of SEQ ID NO: 33. Accordingly, a preferred Flt4 ligand comprises approximately amino acids 103-227 of SEQ ID NO: 33.

VEGF-C mutational analysis described herein indicates that a naturally occurring VEGF-C polypeptide spanning amino acids 103-227 of SEQ ID NO: 33, produced by a natural processing cleavage that defines the Cterminus, exists and is biologically active as an Flt4 ligand. A polypeptide fragment consisting of residues o 15 104-213 of SEQ ID NO: 33 has been shown to retain

VEGF-C

biological activity. Additional mutational analyses indicate that a polypeptide spanning only amino acids 113-213 of SEQ ID NO: 33 retains Flt4 ligand activity.

Accordingly, preferred polypeptides comprise sequences 20 spanning, approximately, amino acid residues 103-227, 104-213, or 113-213, of SEQ ID NO: 33.

Moreover, sequence comparisons of members of the VEGF family of polypeptides provide an indication that still smaller fragments will retain biological 25 activity, and such smaller fragments are intended as aspects of the invention. In particular, eight highly conserved cysteine residues of the VEGF family of polypeptides define a region from residue 131 to residue 211 of SEQ ID NO: 33 (see Figures 10 31); therefore, a polypeptide spanning from about residue 131 to about residue 211 is expected to retain VEGF-C biological activity. In fact, a polypeptide comprising approximately residues 161-211, which retains an evolutionarily-conserved RCXXCC motif, is postulated to retain VEGF-C activity, and therefore is intended as an aspect of the invention. Some of the conserved cysteine residues in VEGF-C participate in interchain disulfide 10 bonding to make homo- and heterodimers of the various naturally occurring VEGF-C polypeptides. Beyond the preceding considerations, evidence exists that

VEGF-C

polypeptides lacking interchain disulfide bonds retain VEGF-C biological activity. Consequently, the materials and methods of the invention include all VEGF-C fragments that retain at least one biological activity of VEGF-C, regardless of the presence or absence of interchain disulfide bonds. The invention also includes multimers (including dimers) comprising such fragments linked to each other or to other polypeptides. Fragment linkage may be by way of covalent bonding disulfide bonding) or non-covalent bonding of polypeptide chains hydrogen bonding, bonding due to stable or induced 15 dipole-dipole interactions, bonding due to hydrophobic or hydrophilic interactions, combinations of these bonding mechanisms, and the like).

In yet another related aspect, the invention includes variants and analogs of the aforementioned 20 polypeptides, including VEGF-C, precursors of VEGF-C, and fragments of VEGF-C. The variants contemplated by the invention include purified and isolated polypeptides having amino acid sequences that differ from the amino acid sequences of VEGF-C, VEGF-C precursors and VEGF-C S 25 fragments by conservative substitutions, as recognized by those of skill in the art, or by additions or deletions of amino acid residues that are compatible with the retention of at least one biological activity of VEGF-C.

Analogs contemplated by the invention include polypeptides having modifications to one or more amino acid residues that differ from the modifications found in VEGF-C, VEGF-C precursors, or VEGF-C fragments, but are compatible with the retention of at least one biological activity of VEGF-C, VEGF-C precursors, or VEGF-C fragments. For example, analogs within the scope of the invention include glycosylation variants and conjugants (attachment of the aforementioned polypeptides to 11 compounds such as labels, toxins, etc.) The present invention also provides purified and isolated polynucleotides nucleic acids) encoding polypeptides of the invention, for example a cDNA or corresponding genomic DNA encoding a VEGF-C precursor, VEGF-C, or biologically active fragments or variants thereof. A preferred nucleic acid of the invention comprises a DNA encoding amino acid residues 1 to 419 of SEQ ID NO: 33 or one of the aforementioned fragments thereof. Due to the degeneracy of the genetic code, numerous such coding sequences are possible, each having in common the coding of the amino acid sequence shown in SEQ ID NO: 33 or fragment thereof. The invention also comprehends analogs of these 15 polynucleotides, or derivatives of any one of these polynucleotides that encodes a polypeptide which retains at least one biological activity of VEGF-C. DNA polynucleotides according to the invention include genomic DNAs, cDNAs, and oligonucleotides comprising the 20 coding sequence for a fragment of VEGF-C, or an analog of a VEGF-C fragment that retains at least one of the biological activities of VEGF-C. Distinct polynucleotides encoding a polypeptide of the invention by virtue of the degeneracy of the genetic code are 25 within the scope of the invention. In one embodiment, the invention contemplates polynucleotides having sequences that differ from polynucleotides encoding a VEGF-C fragment in a manner that results in conservative amino acid differences between the encoded polypeptides, as understood by those of skill in the art.

A preferred polynucleotide according to the invention comprises the human VEGF-C cDNA sequence set forth in SEQ ID NO: 32 from nucleotide 352 to 1611.

Other polynucleotides according to the invention encode a VEGF-C polypeptide from, mammals other than humans, birds avian quails), and others. Still other polynucleotides of the invention comprise a coding u 12 sequence for a VEGF-C fragment, and allelic variants of those DNAs encoding part or all of VEGF-C.

The invention further comprises polynucleotides that hybridize to the aforementioned polynucleotides under standard stringent hybridization conditions.

Exemplary stringent hybridization conditions are as follows: hybridization at 42"C in 50% formamide, 5X SSC, mM Na*PO,, pH 6.8 and washing in 0.2X SSC at 55 C. It is understood by those of skill in the art that variation in these conditions occurs based on the length and GC nucleotide content of the sequences to be hybridized.

Formulas standard in the art are appropriate for determining appropriate hybridization conditions. See Sambrook et al., Molecular Cloning: A Laboratory Manual 15 (Second ed., Cold Spring Harbor Laboratory Press 1989)

SS

9.47-9.51. These polynucleotides, capable of hybridizing 4.C to polynucleotides encoding VEGF-C, VEGF-C fragments, or VEGF-C analogs, are useful as nucleic acid probes for identifying, purifying and isolating polynucleotides 20 encoding other (non-human) mammalian forms of VEGF-C.

Additionally, these polynucleotides are useful in screening methods of the invention, as described below.

.444* Preferred nucleic acid probes of the invention comprise nucleic acid sequences of at least about 16 25 continuous nucleotides of SEQ ID NO: 32. More preferably, these nucleic acid probes would have at least about 20 nucleotides found in a subsequence of SEQ ID NO: 32. In using these nucleic acids as probes, it is preferred that the nucleic acids specifically hybridize to a portion of the sequence set forth in SEQ ID NO: 32.

Specific hybridization is herein defined as hybridization under standard stringent hybridization conditions. To identify and isolate other mammalian VEGF-C genes specifically, nucleic acid probes preferably are selected such that they fail to hybridize to genes related to VEGF-C fail to hybridize to human VEGF or to human VEGF-B genes).

A

-4« 13 O Thus, the invention comprehends polynucleotides comprising at least about 16 nucleotides wherein the polynucleotides are capable of specifically hybridizing to a gene encoding VEGF-C, a human gene. The specificity of hybridization ensures that a polynucleotide of the invention is able to hybridize to a nucleic acid encoding a VEGF-C under hybridization conditions that do not support hybridization of the polynucleotide to nucleic acids encoding, VEGF or VEGF-B. In one embodiment, polynucleotides of at least about 16 nucleotides, and preferably at least about nucleotides, are selected as continuous nucleotide sequences found in SEQ ID NO: 32 or the complement of the nucleotide sequence set forth in SEQ ID NO: 32.

Additional aspects of the invention include vectors which comprise nucleic acids of the invention; f and host cells transformed or transfected with nucleic acids or vectors of the invention. Preferred vectors of the invention are expression vectors wherein nucleic 20 acids of the invention are operatively connected to appropriate promoters and other control sequences, such that appropriate host cells transformed or transfected with the vectors are capable of expressing the Flt4 soot ligand. A preferred vector of the invention is plasmid 25 pFLT4-L, having ATCC accession no. 97231. Such vectors and host cells are useful for recombinantly producing VEGF-C polypeptides.

The invention further includes a method of making polypeptides of the invention. In a preferred method, a nucleic acid or vector of the invention is expressed in a host cell, and a polypeptide of the invention is purified from the host cell or the host cell's growth medium.

In a related embodiment, the invention includes a method of making a polypeptide capable of specifically binding to Flt4 receptor tyrosine kinase, comprising the steps of: transforming or transfecting a host cell r 14 with a nucleic acid of the invention; cultivating the host cell to express the nucleic acid; and purifying a polypeptide capable of specifically binding to Flt4 receptor tyrosine kinase from the host cell or from the host cell!s growth media.

The invention also is intended to include purified and isolated polypeptide ligands of Flt4 produced by methods of the invention. In one preferred embodiment, the invention includes a human

VEGF-C

polypeptide or biologically active fragment thereof that is substantially free of other human polypeptides.

In another aspect, the invention includes an antibody which is specifically reactive with polypeptides of the invention, or with polypeptides multimers of the invention. Antibodies, both monoclonal and polyclonal, may be made against a polypeptide of the invention g.o. according to standard techniques in the art. Such S" antibodies may be used in diagnostic applications to monitor angiogenesis, vascularization, lymphatic vessels 20 and their disease states, wound healing, or certain tumor cells, hematopoietic, or leukemia cells. The antibodies also may be used to block the ligand from activating the 0. Flt4 receptor.

Ligands according to the invention may be o 25 labeled with a detectable label and used to identify their corresponding receptors in situ. Labeled Flt4 ligand and anti-Flt4 ligand antibodies may be used as imaging agents in the detection of lymphatic vessels, high endothelial venules and their disease states, and Flt4 receptors expressed in histochemical tissue sections. The ligand or antibody may be covalently or non-covalently coupled to a suitable supermagnetic, paramagnetic, electron dense, echogenic, or radioactive agent for imaging. Other, non-radioactive labels, such as biotin and avidin, may also be used.

A related aspect of the invention is a method for the detection of specific cells, endothelial b- 15 O cells. These cells may be found in vivo, or in ex vivo biological tissue samples. The method of detection comprises the steps of exposing a biological tissue comprising, endothelial cells, to a polypeptide according to the invention which is capable of binding to Flt4 receptor tyrosine kinase, under conditions wherein the polypeptide binds to the cells, optionally washing the biological tissue, and detecting the polypeptide bound to the cells in the biological tissue, thereby detecting the cells.

The present invention also provides diagnostic and clinical applications for claimed ligands. In a preferred embodiment, Flt4 ligands or precursors are used to accelerate angiogenesis, during wound healing, 15 or to promote the endothelial functions of lymphatic vessels. A utility for VEGF-C is suggested as an inducer .of angiogenesis also in tissue transplantation, in eye diseases, in the formation of collateral vessels around arterial stenoses and into injured tissues after 20 infarction. Polypeptides according to the invention may be administered in any suitable manner using an appropriate pharmaceutically-acceptable vehicle, a pharmaceutically-acceptable diluent, adjuvant, excipient or carrier. VEGF-C polypeptides also may be used to quantify future metastatic risk by assaying biopsy material for the presence of active receptors or ligands in a binding assay or kit using detectably-labeled

VEGF-

C. An Flt4 ligand according to the invention also may be used to promote re-growth or permeability of lymphatic vessels in, for example, organ transplant patients. In addition, an Flt4 ligand may be used to mitigate the loss of axillary lymphatic vessels following surgical interventions in the treatment of cancer breast cancer). Ligands according to the invention also may be used to treat or prevent inflammation, edema, aplasia of the lymphatic vessels, lymphatic obstruction, elephantiasis, and Milroy's disease. Finally, Flt4 16 O ligands may be used to stimulate lymphocyte production and maturation, and to promote or inhibit trafficking of leukocytes between tissues and lymphatic vessels or to affect migration in and out of the thymus.

The invention includes a method of screening for an endothelial cell disorder in a mammalian subject.

The method comprises providing a sample of endothelial cells from the subject, contacting the sample of endothelial cells with a polypeptide according to claim 4, determining the growth rate of the cells, and correlating the growth rate with a disorder. In a preferred embodiment, the endothelial cells are lymphatic endothelial cells. In another preferred embodiment, the mammalian subject is a human being and the endothelial 15 cells are human cells. In yet another preferred embodiment, the disorder is a vessel disorder, a lymphatic vessel disorder, such as the loss of lymphatic vessels through surgery or the reduction in function of existing lymphatic vessels due to blockages. In another embodiment, the endothelial cells are contacted with the polypeptide in vitro. The growth rate determined in the method is the rate of cell division per unit time, determined by any one of a number of techniques known in the art. The correlation of the growth rate with a disorder can involve a positive or negative correlation, whether the polypeptide has Flt4 ligand activity or is an inhibitor of such activity, as described below.

Inhibitors of the Flt4 ligand may be used to control endothelial cell proliferation, lymphangiomas, and metastatic cancer. For example, such inhibitors may be used to arrest metastatic growth or spread, or to control other aspects of endothelial cell expression and growth. Inhibitors include antibodies, antisense oligonucleotides, and polypeptides which block the Flt4 receptor, all of which are intended as aspects of the invention.

In another embodiment, the invention provides a 17 method for modulating the growth of endothelial cells in a mammalian subject comprising the steps of exposing mammalian endothelial cells to a polypeptide according to the invention in an amount effective to modulate the growth of the mammalian endothelial cells. In one embodiment, the modulation of growth is effected by using a polypeptide capable of stimulating tyrosine phosphorylation of Flt4 receptor tyrosine kinase in a host cell expressing the Flt4 receptor tyrosine kinase.

In modulating the growth of endothelial cells, the invention contemplates the modulation of endothelial cell-related disorders. Endothelial cell disorders contemplated by the invention include, but are not limited to, physical loss of lymphatic vessels surgical removal of axillary lymph tissue), lymphatic vessel occlusion elephantiasis), and lymphangiomas. In a preferred embodiment, the subject, and endothelial cells, are human. The endothelial cells may be provided in vitro or in vivo, and they may be contained in a tissue graft. An effective amount of a polypeptide is defined herein as that amount of polypeptide empirically determined to be necessary to achieve a reproducible change in cell growth rate (as determined by microscopic or macroscopic visualization and estimation of cell doubling time, or nucleic acid synthesis assays), as would be understood by one of ordinary skill in the art.

The present invention also provides methods for using the claimed nucleic acids polynucleotides) in screening for endothelial cell disorders. In a preferred embodiment, the invention provides a method for screening an endothelial cell disorder in a mammalian subject comprising the steps of providing a sample of endothelial cell nucleic acids from the subject, contacting the sample of endothelial cell nucleic acids with a polynucleotide of the invention which is capable of hybridizing to a gene encoding VEGF-C (and preferably b-- 18 capable of hybridizing to VEGF-C mRNA), determining the level of hybridization between the endothelial cell nucleic acids and the polynucleotide, and correlating the level of hybridization with a disorder. A preferred mammalian subject, and source of endothelial cell nucleic acids, is a human. The disorders contemplated by the method of screening with polynucleotides include, but are not limited to, vessel disorders such as the aforementioned lymphatic vessel disorders, and hypoxia.

Purified and isolated polynucleotides encoding other (non-human) VEGF-C forms also are aspects of the invention, as are the polypeptides encoded thereby, and antibodies that are specifically immunoreactive with the non-human VEGF-C variants. Preferred non-human forms of 15 VEGF-C are forms derived from other vertebrate species, including avian and mammalian species. Mammalian forms are highly preferred. Thus, the invention includes a ~purified and isolated mammalian VEGF-C polypeptide, and also a purified and isolated polynucleotide encoding such a polypeptide.

In one embodiment, the invention includes a purified and isolated polypeptide having the amino acid sequence of residues 1 to 415 of SEQ ID NO: 41, which sequence corresponds to a putative mouse VEGF-C precursor. The putative mouse VEGF-C precursor is believed to be processed into a mature mouse VEGF-C in a manner analogous to the processing of the human prepropolypeptide. Thus, in a related aspect, the invention includes a purified and isolated polypeptide capable of specifically binding to an Flt4 receptor tyrosine kinase a human or mouse Flt-4 receptor tyrosine kinase), the polypeptide comprising a fragment of the purified and isolated polypeptide having the amino acid sequence of residues 1 to 415 of SEQ ID NO: 41, the fragment being capable of specifically binding to the Flt4 receptor tyrosine kinase. The invention further includes multimers of the foregoing polypeptides and purified and 19 isolated nucleic acids encoding the foregoing polypeptides, such as a nucleic acid comprising all or a portion of the sequence shown in SEQ ID NO: In another embodiment, the invention includes a purified and isolated quail VEGF- C polypeptide, biologically active fragments and multimers thereof, and polynucleotides encoding the foregoing polypeptides.

In yet another embodiment, the invention includes a DNA comprising a VEGF-C promoter, that is capable of promoting expression of a VEGF-C gene or another operatively-linked, protein-encoding gene in native host cells, under conditions wherein VEGF-C is expressed in such cells.

Accordingly, in a first embodiment there is provided a purified and isolated polypeptide comprising an amino acid sequence encoded by plasmid pFLT4-L, deposited as ATCC Accession Number 97231, wherein the polypeptide binds to the extracellular domain of Flt4 receptor tyrosine kinase (Flt4).

According to a second embodiment there is provided a purified and isolated polypeptide comprising an amino acid sequence encoded by plasmid pFLT4-L, deposited i as ATCC Accession Number 97231, wherein said polypeptide lacks the carboxyl terminal portion of the amino acid sequence encoded by plasmid pFLT4-L that contains cysteine motifs of a Balbiani ring 3 protein (BR3P).

According to a third embodiment there is provided a purified and isolated polypeptide that binds to the extracellular domain of human Flt4 receptor tyrosine kinase, said polypeptide comprising an amino acid sequence that includes a contiguous portion of the amino acid sequence in SEQ ID NO:33 effective to permit said binding, S* wherein said polypeptide lacks any portion of SEQ ID NO:33 that has one or more cysteine motifs of a Balbiani ring 3 protein (BR3P).

According to a fourth embodiment there is provided a purified and isolated polypeptide that binds to the extracellular domain of human Flt4 receptor tyrosine kinase, said polypeptide comprising a portion of the amino acid sequerce in SEQ ID NO:33 effective to permit said binding, said polypeptide lacking at least carboxy-terminal residues of SEQ ID NO:33 beyond residue 227.

According to a fifth embodiment there is provided a purified polypeptide that binds to the extracellular domain of human Flt4 receptor tyrosine kinase, said polypeptide produced by a method comprising the steps of: expressing a nucleic acid in a host cell, wherein said nucleic acid comprises a nucleotide sequence encoding a portion of the amino acid sequence in SEQ D NO:33 effective to permit such binding; and '.doc:mrr 19a purifying a polypeptide that binds to the extracellular domain of human Flt4 from said host cell or from a growth medium of said host cell, wherein said polypeptide lacks at least the carboxy-terminal portion of SEQ ID NO:33 that is characterized by cysteine motifs of a Balbiani ring 3 protein.

According to a sixth embodiment there is provided a purified and isolated polypeptide that binds to the extracellular domain of Flt4 receptor tyrosine kinase (Flt4), wherein the polypeptide comprises a portion of SEQ ID NO:33 effective to permit such binding, and wherein the polypeptide has an apparent molecular weight of approximately 23 kD as assessed by SDS-PAGE under reducing conditions.

According to a seventh embodiment there is provided a purified and isolated polypeptide comprising a human polypeptide that binds to the extracellular domain of Flt4 receptor tyrosine kinase and has an apparent molecular weight of approximately 23 kD as assessed by SDS-PAGE under reducing conditions, wherein amino terminal amino acids 2 through 18 of said human polypeptide have an amino acid sequence i. 15 corresponding to amino acids 2 through 18 set forth in SEQ ID NO: 13.

According to an eighth embodiment there is provided a purified and isolated polypeptide that binds to the extracellular domain of Flt4 receptor tyrosine kinase, wherein said polypeptide has an apparent molecular weight of approximately 23 kD as assessed by SDS-PAGE under reducing conditions and is purifyable from conditioned media from a PC-3 prostatic adenocarcinoma cell line, said cell line having ATCC CRL 1435, using an affinity purification procedure wherein the afffinity purification matrix comprises a polypeptide comprising the extracellular domain of Flt4 receptor tyrosine kinase.

According to a ninth embodiment there is provided a purified and isolated polypeptide that binds to the extracellular domain of human Flt4 receptor tyrosine kinase (Flt4EC), wherein said polypeptide comprises an amino acid sequence encoded by a DNA which hybridizes to a non-coding strand complementary to nucleotides 352 to 1611 of SEQ ID NO:32 under the following hybridization conditions: hybridization at 42°C in a hybridization solution comprising 50% formamide, 5 X SSC, 20 mM NaoP0 4 pH 6.8; and washing in 0.2 X SSC at and wherein said polypeptide lacks any cysteine motifs of a Balbiani ring 3 p _rotein (BR3P).

[R:\LIBZZ]04999.doc:mr 19b According to a tenth embodiment there is provided a purified and isolated mammalian polypeptide that binds to the extracellular domain of human Flt4 receptor tyrosine kinase (Flt4EC), said polypeptide including a contiguous portion of a mammalian Flt4 ligand precursor polypeptide that is sufficient to bind human Flt4EC, wherein said mammalian Flt4 ligand precursor polypeptide comprises an amino acid sequence that is encoded by a DNA which hybridizes to a non-coding strand complementary to nucleotides 352 to 1611 of SEQ ID NO: 32 under the following hybridization conditions: hybridization at 42°C in a hybridization solution comprising o0 50% formamide, 5 X SSC, 20 mM Na*P0 4 pH 6.8; and washing in 0.2 X SSC at wherein said contiguous portion includes eight cysteine residues that are conserved in human vascular endothelial growth factor (VEGF), human platelet derived growth factors A and B (PDGF-A, PDGF-B), human placenta growth factor (PIGF-1), and human vascular endothelial growth factor B (VEGF-B), is and wherein said polypeptide lacks any portion of the mammalian Flt4 ligand i precursor polypeptide that has one or more cysteine motifs of a Balbiani ring 3 protein (BR3P).

According to an eleventh embodiment there is provided a non-human mammalian polypeptide according to the ninth or tenth embodiment.

According to a twelfth embodiment there is provided a human polypeptide according to the ninth or tenth embodiment.

According to a thirteenth embodiment there is provided a purified and isolated polypeptide which is capable of binding the extracellular domain of Kdr receptor tyrosine kinase (VEGFR-2), said polypeptide having an amino acid sequence comprising a portion of SEQ ID NO:33 effective to permit such binding.

According to a fourteenth embodiment there is a composition comprising a polypeptide according to any one of the first to thirteenth embodiments in a pharmaceutically acceptable diluent, adjuvant, excipient, or carrier.

According to a fifteen embodiment there is provided a purified protein comprising a first polypeptide linked to a second polypeptide, wherein at least one of said first polypeptide and said second polypeptide is a polypeptide according to any one of the first to thirteenth embodiments, and wherein said protein is capable of binding to Flt4 receptor tyrosine kinase.

[R:\LIBZZ]04999.doc:nr 19c According to a sixteenth embodiment there is provided a purified protein comprising a first polypeptide linked to a second polypeptide, wherein each of said first polypeptide and said second polypeptide is a polypeptide according to any one of the first to thirteenth embodiments.

According to a seventeenth embodiment there is provided a pharmaceutical composition comprising a protein according to the fifteenth or sixteenth embodiment in a pharmaceutically-acceptable diluent, adjuvant, excipient, or carrier.

According to an eighteenth embodiment there is provided a purified and isolated nucleic acid comprising a nucleotide sequence encoding a polypeptide according to any one of the first to thirteenth embodiments.

According to a nineteenth embodiment there is provided a purified and isolated nucleic acid comprising a nucleotide sequence that encodes a polypeptide that binds to human Flt4 receptor tyrosine kinase, said polypeptide having an amino acid sequence comprising a portion of the amino acid sequence shown in SEQ ID NO:33 effective to 15 permit such binding, said nucleic acid lacking a nucleotide sequence that encodes the portion of the amino acid sequence shown in SEQ ID NO:33 that has cysteine motifs of a Balbiani ring 3 protein.

According to a twentieth embodiment there is provided a purified and isolated nucleic acid encoding a polypeptide that binds the extracellular domain of human Flt4 receptor tyrosine kinase and stimulates tyrosine phosphorylation of Flt4 receptor tyrosine kinase, said polypeptide having an amino acid sequence consisting of a continuous portion of the sequence shown in SEQ ID NO:33, said continuous portion commencing at S° residue number 103 of SEQ ID NO:33 and lacking at least carboxy terminal residues of SEQ ID NO:33 beyond residue 227.

According to a twenty-first embodiment there is provided a purified and isolated nucleic acid comprising a nucleotide sequence that encodes a polypeptide that binds human Flt4 receptor tyrosine kinase, said polypeptide having an amino acid sequence comprising a continuous portion of the amino acid sequence shown in SEQ ID NO:33 effective to permit such binding, said nucleic acid lacking a nucleotide sequence that encodes the carboxy-terminal portion of the amino acid sequence shown in SEQ ID NO:33 beyond residue 227.

According to a twenty-second embodiment there is provided a nucleic acid according to any one of the eighteenth to twenty-first embodiments, wherein the nucleic RA L a cid further comprises an expression control sequence operatively linked to the encoding Ssequence.

[R:\LIBZZ)04999.doc:mrr 19d According to a twenty-third embodiment there is provided an expression construct comprising the nucleic acid according to any one of the eighteenth to twenty-first embodiments oepratively linked to an expression control sequence, said expression construct lacking a nucleotide sequence that encodes the carboxy-terminal portion of the amino acid sequence shown in SEQ ID NO:33 beyond residue 227.

According to a twenty-fourth embodiment there is provided a vector comprising a nucleic acid according to any one of the eighteenth to twenty-first embodiments, wherein said vector lacks a nucleotide sequence that encodes the portion of the amino acid sequence shown in SEQ ID NO:33 that has cysteine motifs of a Balbiani ring 3 protein.

According to a twenty-fifth embodiment there is provided a host cell transformed or transfected with a nucleic acid according to any of the eighteenth to twenty-first embodiments.

According to a twenty-sixth embodiment there is provided a host cell transformed or transfected with an expression construct or vector according to the twenty-third or 15 twenty-fourth embodiment.

According to a twenty-seventh embodiment there is provided a host cell transformed or transfected with a polynucleotide, wherein said polynucleotide includes a strand that hybridizes to a DNA comprising :i the non-coding strand complementary to SEQ ID NO:32, under the following hybridization conditions: hybridization at 42 0 C for 20 hours in a solution containing formamide, 5 x SSPE, 5 x Denhardt's solution, 0.1% SDS and 0.1 mg/ml denatured salmon sperm DNA; and washing the filter twice for thirty minutes at room temperature and twice for thirty minutes at 65 0 C with a wash solution containing 1 x SSC, and 0.1% SDS; and wherein said host cell expresses a polypeptide encoded by said polynucleotide, wherein said polypeptide includes a domain defined by eight cysteine residues that are conserved in human vascular endothelial growth factor (VEGF), human platelet derived growth factor A (PDGF-A), and human platelet derived growth factor B (PDGF-

B),

wherein said polypeptide lacks any domain that has one or more cysteine motifs of a Balbiani ring 3 protein (BR3P), and wherein said polypeptide binds to the extracellular domain of human Flt4 receptor 41 3 y-tvrosine kinase.

[R:\LIBZZ]04999.doc:mrr 19e According to a twenty-eighth embodiment there is provided a host cell transformed or transfected with a polynucleotide comprising a nucleotide sequence that encodes a polypeptide that is capable of binding to the extracellular domain of human Flt4 receptor tyrosine kinase, wherein said polynucleotide includes a strand that hybridizes to a DNA comprising the non-coding strand complementary to SEQ ID NO:32, under the following hybridization conditions: hybridization at 42 0 C for 20 hours in a solution containing formamide, 5 x SSPE, 5 x Denhardt's solution, 0.1% SDS and 0.1 mg/ml denatured salmon sperm DNA; and washing the filter twice for thirty minutes at room temperature and twice for thirty minutes at 65 0 C with a wash solution containing 1 x SSC, and 0.1% SDS; and wherein said host cell expresses and secretes a polypeptide encoded by said 15 polynucleotide, and wherein said polypeptide binds the extracellular domain of human Flt4 receptor tyrosine kinase and has a molecular weight of about 23 kD or about 32 kD as assessed by SDS-PAGE under reducing conditions.

According to a twenty-ninth embodiment there is provided a host cell transformed or transfected with a polynucleotide comprising a nucleotide sequence that encodes the amino acid sequence shown in SEQ ID NO:33, wherein said host cell expresses a polypeptide encoded by said polynucleotide, said polypeptide including a contiguous :::portion of SEQ ID NO:33 that is sufficient to bind to the extracellular domain of human Flt4 receptor tyrosine kinase (Flt4EC), wherein said contiguous portion includes eight cysteine residues that are conserved in human vascular endothelial growth factor (VEGF), human platelet derived growth factor A (PDGF-A), and human platelet derived growth factor B DGF-B), wherein said polypeptide lacks any portion of SEQ ID NO:33 that has one or more cysteine motifs of a Balbiani ring 3 portein (BR3P), and wherein said polypeptide binds to Flt4EC.

According to a thirtieth embodiment there is provided a host cell comprising a polynucleotide that includes the insert of plasmid pFLT4-L, deposited as ATCC accession No. 97231, wherein said host cell expresses and secretes a polypeptide encoded by said ST insert, 'LIBZZ 999.doc:rr [R:\LIBZZ]04999.doc:mrr 19f wherein said secreted polypeptide binds to human Flt4 receptor tyrosine kinase and includes a domain defined by eight cysteine residues that are conserved in human vascular endothelial growth factor (VEGF), human platelet derived growth factor A (PDGF-A), and human platelet derived growth factor B (PDGF-B), and s wherein said secreted polypeptide lacks any domain that has one or more cysteine motifs ofa Balbiani ring 3 protein (BR3P).

According to a thirty-first embodiment there is provided a host cell transformed or transfected with a polynucleotide comprising a nucleotide sequence that encodes the amino acid sequence shown in SEQ ID NO:33, wherein said host cell expresses a 0o polypeptide encoded by said polynucleotide, said polypeptide including a contiguous portion of SEQ ID NO:33 that is sufficient to bind to the extracellular domain of human Flt4 receptor tyrosine kinase (Flt4EC), wherein said contiguous portion includes eight cysteine residues that are conserved in human vascular endothelial growth factor (VEGF), human platelet derived growth S 15 factor A (PDGF-A), and human platelet derived growth factor B (PDGF-B), wherein said polypeptide lacks any portion of SEQ ID NO:33 that precedes position 103 and lacks any portion of SEQ ID NO:33 that has one or more cysteine motifs of a Balbiani ring 3 protein (BR3P), and wherein said polypeptide has a molecular weight of about 23 kD as assessed by SDS PAGE under reducing conditions and binds to Flt4EC.

According to a thirty-second embodiment there is provided a eukaryotic host cell according to any one of the twenty-seventh to thirty-first embodiments that secretes said polypeptide.

According to a thirty-third embodiment there is provided a method of making a polypeptide that binds to the extracellular domain of Flt4 receptor tyrosine kinase, said method comprising the steps of: growing a host cell transformed or transfected with a nucleic acid according to any one of the eighteenth to twenty-second embodiments under conditions in which the cell expresses a polypeptide encoded by the nucleic acid; and purifying the polypeptide from said host cell or from a growth medium of said host cell, wherein the polypeptide binds to the extracellular domain of Flt4 receptor tyrosine kinase.

According to a thirty-fourth embodiment there is provided a method for producing a i olypeptide that is capable of binding the extracellular domain of human Flt4 receptor [R:\LIBZZ]04999.doc:mnT 19g tyrosine kinase and stimulating tyrosine phosphorylation of Flt4 receptor tyrosine kinase, comprising the steps of: growing a host cell according to any one of the twenty-fifth to thirty-second embodiments under conditions which permit expression in said host cell of a polypeptide encoded by said polynucleotide; and isolating said polypeptide from the host cell or the growth medium of the host cell, wherein said polypeptide binds to the extracellular domain of human Flt4 receptor tyrosine kinase and stimulates phosphorylation of Flt4 receptor tyrosine kinase.

According to a thirty-fifth embodiment there is provided a method for producing a to polypeptide that binds to the extracellular domain (EC) of human Flt4 receptor tyrosine kinase (Flt4), comprising steps of: growing a host cell comprising a polynucleotide that comprises a nucleotide sequence that encodes the amino acid sequence set forth in SEQ ID NO:33, under conditions in which the host cell expresses and secretes a polypeptide encoded by the 15 polynucleotide; and isolating a polypeptide that binds Flt4 EC from the growth medium of the host cell, said polypeptide having a molecular weight of approximately 23 kD as assessed by SDS- PAGE under reducing conditions and having an amino acid sequence comprising a portion of SEQ ID NO:33 effective to bind Flt4 EC.

According to a thirty-sixth embodiment there is provided a purified polypeptide that binds to the extracellular domain of Flt4 receptor tyrosine kinase, said polypeptide produced by a method according to any one of the thirty-third to thirty-fifth embodiments.

*According to a thirty-seventh embodiment there is provided a purified polypeptide that binds Flt4 receptor tyrosine kinase, said polypeptide comprising the amino acid sequence set forth in SEQ ID NO:41 or a portion thereof effective to permit such binding.

According to a thirty-eighth embodiment there is provided a purified and isolated murine Flt4 ligand precursor polypeptide comprising the amino acd sequence set forth in SEQ ID NO:41.

According to a thirty-ninth embodiment there is provided a composition comprising a polypeptide according to any one of the thirty-sixth to thirty-eighth embodiments, and further comprising a pharmaceutically acceptable diluent, adjuvant, excipient, or carrier.

According to a fortieth embodiment there is provided a purified polypeptide capable of binding to the extracellular domain of human Flt4 receptor tyrosine kinase, said S\L I polypeptide produced by a method comprising the steps of: SR:\LIBZZ]04999.doc: n, 19h expressing a nucleic acid in a host cell, said nucleic acid comprising a nucleotide sequence encoding the amino acid sequence in SEQ ID NO:41 or a portion thereof effective to permit such.binding; and purifying a polypeptide that binds to the extracellular domain of human Flt4 receptor tyrosine kinase from said host cell or from a growth medium of said host cell, wherein said polypeptide lacks at least the carboxy-terminal portion of SEQ ID NO:41 that is characterized by cysteine motifs of a Balbiani ring 3 protein.

According to a forty-first embodiment there is provided a composition comprising polyclonal antibodies raised against a polypeptide according to any one of the first to thirteenth and thirty-sixth to thirty-eighth embodiments.

According to a forty-second embodiment there is provided a purified antibody that binds to a peptide consisting of the amino acid sequence set forth in SEQ ID NO:39.

According to a forty-third embodiment there is provided a polyclonal antiserum produced by a process comprising the steps of: immunizing a mammal with a composition comprising a polypeptide according to any one of the first to thirteenth and thirty-sixth to thirty-eighth embodiments; and obtaining antiserum from said mammal after said immunizing step, said antiserum containing antibodies that bind to said polypeptide.

According to a forty-fourth embodiment there is provided polyclonal antibodies purified from an antiserum according to the forty-third embodiment, said polyclonal antibodies including antibodies that bind to said polypeptide.

According to a forty-fifth embodiment there is provided a method for detecting endothelial cells in a biological tissue sample comprising the steps of: exposing a biological tissue sample comprising endothelial cells to a polypeptide according to any one of the first to thirteenth and thirty-sixth to thirty-eighth embodiments, under conditions wherein said polypeptide binds to endothelial cells; and detecting said polypeptide bound to endothelial cells in 'said biological tissue, thereby detecting said endothelial cells.

According to a forty-sixth embodiment there is provided a method of modulating the growth of mammalian endothelial cells, comprising the step of: contacting mammalian endothelial cells with a polypeptide according to any one of the first to thirteenth and thirty-sixth to thirty-eighth embodiments, in an amount effective to modulate the growth of the mammalian endothelial cells.

(R:\LIBZZ)04999.doc:mn- 19i According to a forty-seventh embodiment there is provided use of a polypeptide according to any one of the first to thirteenth and thirty-sixth to thirty-eighth embodiments in the preparation of a medicament for modulating the growth of mammalian endothelial cells.

According to a forty-eighth embodiment there is provided a method of modulating the activity of human Flt4 receptor tyrosine kinase comprising administering to a person in need of modulation of Flt4 receptor tyrosine kinase activity a composition according to the fourteenth or thirty-ninth embodiment.

According to a forty-ninth embodiment there is provided a method of modulating the activity of human Flt4 receptor tyrosine kinase comprising contacting cells that express human Flt4 receptor tyrosine kinase with a polypeptide according to any one of the first to thirteenth and thirty-sixth to thirty-eighth embodiments.

According to a fiftieth embodiment there is provided use of a polypeptide according to any one of the first to thirteenth and thirty-sixth to thirty-eighth embodiments in the 15 preparation of a medicament for modulating the activity of Flt4 receptor tyrosine kinase.

a 000According to a fifty-first embodiment there is provided a method of modulating the growth of lymphatic endothelia, comprising steps of: identifying a patient in need of modulation of the growth of lymphatic endothelia; and 9T9 20 administering to the patient a composition comprising a polypeptide in an amount effective to modulate the growth of lymphatic endothelial cells, wherein said 9 9polypeptide is a polypeptide according to any one of the first to thirteenth and thirty-sixth to thirty-eighth embodiments.

According to a fifty-second embodiment there is provided the use of a polypeptide according to any one of the first to thirteenth and thirty-sixth to thirty-eighth embodiments in the manufacture of a medicament for modulating the growth of lymphatic endothelia.

According to a fifty-third embodiment there is provided the use of a polypeptide according to any one of the first to thirteenth and thirty-sixth to thirty-eighth embodiments in the manufacture of a medicament for treatment of a disease or condition selected from the group consisting of lymphatic vessel loss or damage due to surgical intervention, edema, lymphatic vessel aplasia, lymphatic vessel occlusion, and elephantiasis.

[R:\LIBZ]044999.doc:r.

19j According to a fifty-fourth embodiment there is provided a method of modulating the activity of Flt4 receptor tyrosine kinase (Flt4) in Flt4-expressing cells, comprising steps of: providing a nucleic acid according to any one of the eighteenth to twentysecond embodiments; transforming or transfecting a cell with the nucleic acid such that the cell expresses and secretes a polypeptide encoded by said nucleic acid, wherein said secreted polypeptide binds the extracellular domain of human Flt4 and has a molecular weight of about 23 kD as assessed by SDS-PAGE under reducing conditions; and contacting Flt4-expressing cells with the secreted 23 kD polypeptide.

According to a fifty-fifth embodiment there is provided use of a nucleic acid according to any one of the eighteenth to twenty-second embodiments in the manufacture of a medicament for the modulation of the growth of lymphatic endothelia.

According to a fifty-sixth embodiment there is provided a purified nucleic acid 15 comprising a vertebrate VEGF-C promoter nucleotide sequence.

According to a fifty-seventh embodiment there is provided a purified and isolated nucleic acid comprising a portion of SEQ ID NO:54, wherein said portion is capable of promoting expression of a protein encoding gene operatively linked thereto.

According to a fifty-eighth embodiment there is provided a purified and isolated mammalian nucleic acid which hybridizes to a nucleic acid complementary to SEQ ID NO:54 under the following hybridization conditions: hybridization at 42 0 C in a 0 hybridization solution comprising 50% formamide, 5 X SSC, 20 mM NaoP0 4 pH 6.8; and washing in 0.2 x SSC at 00** wherein said nucleic acid is capable of promoting expression of a protein encoding gene operatively linked thereto.

According to a fifty-ninth embodiment there is provided a polypeptide according to any one of the first to thirteenth and thirty-sixth to thirty-eighth embodiments when used for detecting endothelial cells in a biological tissue sample.

According to a sixtieth embodiment there is provided a polypeptide according to any one of the first to thirteenth and thirty-sixth to thirty-eighth embodiments when used for modulating the growth of mammalian cells.

According to a sixtieth-first embodiment there is provided a polypeptide according to any one of the first to thirteenth and thirty-sixth to thirty-eighth embodiments when Sused for modulating the activity of human Flt4 receptor tyrosine kinase.

(R:\LIBZZ]04999.doc:m r 19k According to a sixty-second embodiment there is provided a polypeptide according to any one of the first to thirteenth and thirty-sixth to thirty-eighth embodiments when used for modulating the growth of lymphatic endothelia.

According to a sixty-third embodiment there is provided a polypeptide according to any one of the first to thirteenth and thirty-sixth to thirty-eighth embodiments when used for treatment of a disease or condition selected from the group consisting of lymphatic vessel loss or damage due to surgical intervention, edema, lymphatic vessel aplasia, lymphatic vessel occlusion, and elephantiasis.

According to a sixty-fourth embodiment there is provided a nucleic acid according to any one of the eighteenth to twenty-second embodiments, when used for modulating the activity of Flt4 receptor tyrosine kinase (Flt4) in Flt4-expressing cells.

Brief description of the Drawing Figure 1 schematically depicts major endothelial cell receptor tyrosine kinases and growth factors involved in vasculogenesis and angiogenesis. Major structural domains 15 are depicted, including immunoglobulin-like domains (IGH), epidermal growth factor homology domains (EGFH), fibronectin type III domains (FNIII), transmembrane (TM) and juxtamembrane (JM) domains, tyrosine kinase (TK2, TK2) domains, kinase insert domains and carboxy-terminal domains (CT).

Figure 2 schematically depicts the construction of the pLTRFlt41 expression vector.

Figure 3 schematicaly depicts the constructions of the baculovirus vector encoding a secreted soluble Flt4 extracellular domain (Flt4EC).

Figure 4 shows results of stimulation of Flt4 autophosphorylation by conditioned medium from PC-3 cell cultures.

Figures 5A, 5B, and 5C show that the major tyrosyl phosphorylated polypeptide of Flt4-transfected cells stimulated with PC-3 conditioned medium is the 125 kD Flt4 polypeptide (VEGFR-3), and also that the Flt4 stimulating activity is not adsorbed to heparin- [R:\LIBZZ]04999.doc:mrr 20 sepharose.

Figure 6 shows Western immunoblotting analysis of the Flt4 ligand activity isolated from PC-3 conditioned medium.

.Figure 7 shows results of gel electrophoresis of chromatographic fractions from the affinity purification of Flt4 ligand (VEGF-C) isolated from concentrated PC-3 conditioned medium.

Figure 8 depicts the amino acid sequences of human, murine, and quail VEGF-C polypeptides, aligned to show similarity. Residues conserved in all three species are depicted in bold.

Figure 9 schematically depicts the cloning and structure of the Flt4 ligand, VEGF-C. The VEGF homologous region (dark shaded box) and amino and carboxyl terminal propeptides (light shaded and unshaded boxes, respectively) as well as putative signal sequence (ss) are depicted between 5' and 3' untranslated (ut) nucleic acid regions. The cleavage sites for the signal sequence and the amino and carboxyl terminal propeptides are indicated with triangles.

Figure 10 shows a comparison of the deduced 'i amino acid sequences of PDGF-A, PlGF-1, VEGF-B,67 SVEGF165, and Flt4 ligand

(VEGF-C).

Figure 11 depicts the exon-intron organization of the human VEGF-C gene. Seven exons are depicted as open boxes, with exon size depicted in base pairs.

Introns are depicted as lines, with intron size (base pairs) depicted above the lines. 5' and 3' untranslated sequences of a putative 2.4 kb mature mRNA are depicted as shaded boxes. The location of genomic clones used to characterize the VEGF-C gene are depicted below the map of the gene.

Figure 12 shows Northern blotting analysis of the genes encoding VEGF, VEGF-B, AND VEGF-C (indicated by "FLT4-L") in two human tumor cell lines and in brain tissue.

21 w Figure 13A is an autoradiograph showing recombinant VEGF-C isolated following a pulse-chase experiment and electrophoresed via SDS-PAGE under reducing conditions.

.Figure 13B is a photograph of polyacrylamide gel showing that recombinant VEGF-C forms are disulfidelinked in nonreducing conditions.

Figure 14A and 14B depict Western blots showing that VEGF-C stimulates autophosphorylation of VEGFR-2 (KDR) but has no effect on PDGFR-# phosphorylation.

Figure 15A shows that VEGF-C stimulates endothelial cell migration in a three-dimensional collagen gel assay.

Figure 16A shows the expression of VEGF-C mRNA 15 in human adult tissues.

Figure 16B shows the expression of VEGF, VEGF- B, and VEGF-C in selected human fetal tissues.

Figure 17 depicts the genomic structure of the human and murine VEGF-C genes. Sequences of exon-intron 20 junctions are depicted together with exon and intron lengths. Intron sequences are depicted in lower case letters. Nucleotides of the open reading frame observed in VEGF-C cDNAs are indicated as upper case letters in triplets (corresponding to the codons encoded at the junctions).

Figure 18 presents a schematic illustration of VEGF-C processing, including the major forms of VEGF-C.

Figure 19 depicts autoradiograms from a pulsechase immunoprecipitation experiment wherein cells transfected with a VEGF-C expression vector (VEGF-C) and mock transfected cells were pulse-labeled with radioactive amino acids and chased for varying lengths of time.

Figure 20 is a schematic map of the K14-VEGF-C vector construct.

Figures 21A-C depict electrophoretic fractionations of the various forms of recombinant

VEGF-C

22 produced by transfected 293 EBNA cells. Figure 21B depicts the electrophoretic fractionation, under nonreducing conditions, of polypeptides produced from mock transfected cells, cells transfected with wild type (wt) VEGFTC cDNA, and cells transfected with a cDNA variant encoding VEGF-C-R102S. Each of the bands identified in Figure 21B was excised and electrophoretically fractionated in a separate lane under reducing conditions. Fractionation of bands corresponding to wt VEGF-C are depicted in Figure 21A; fractionation of bands corresponding to the R102S variant are depicted in Figure 21C.

Figures 22A-B depict the forms and sizes of wild type and mutant recombinant VEGF-Cs, as revealed by 15 non-reducing gel electrophoresis. Figure 22A shows the VEGF-C forms secreted into the media; Figure 22B shows Sthe VEGF-C forms retained by the cells. Mock (M) transfected cells served as a control.

Figures 23A-B present a comparison of the 20 pattern of immunoprecipitated, labelled VEGF-C forms using antisera 882 and antisera 905. Adjacent lanes contain immunoprecipitates that were (lanes marked or were not (lanes marked subjected to reduction and alkylation.

Figures 24A-B present Northern blots of total RNA isolated from cells grown in the presence or absence of interleukin-l (IL-I) and/or dexamethasone (DEX) for the indicated times. For Figure 24B, the Northern blot was probed with radiolabeled DNA from a VEGF 581 bp cDNA covering bps 57-638 (Genbank Acc. No. X15997), and a human VEGF-B67 cDNA fragment (nucleotides 1/382, Genbank Acc. No. U48800). For Figure 24A, the Northern blot was probed with radiolabeled DNA from a human full-length VEGF-C cDNA (Genbank Acc. No. X94216). 18S and 28S- rRNA markers.

Figure 25 shows VEGF-C expression in P.

pastoris cultures transfected with a VEGF-C cDNA, with 23 Svector alone, or mock- transfected, following induction with methanol for various periods of time as indicated. About 10 Al of medium was analyzed by gel electrophoresis followed by Western blotting and detection with anti-VEGF-C antiserum.

Figure 26 depicts the results of a Western blot wherein NIH 3T3 cells expressing VEGFR-3 (Flt4), and PAE cells expressing VEGFR-2 (KDR), were stimulated with concentrated medium from Pichia yeast transfected with a VEGF-C cDNA-containing vector with a vector lacking an insert or stimulated with the positive control vanadate. The stimulated cells were lysed and immunoprecipitated with VEGFR-specific antibodies, and the immunoprecipitates were blotted and probed with anti- 15 phosphotyrosine antibodies.

Figures 27A-B present gel electrophoretograms of human VEGF-C (wt) and VEGF-C variants secreted (Figure 27A) or retained (Figure 27B) by the host 293 EBNA cells.

Mock transfected cells served as a control.

Molecular weight markers are indicated on the left in kilodaltons (kD).

Figures 28A-B show Western blots of VEGFRs that were stimulated to autophosphorylate by wild type (wt) VEGF-C, as well as three VEGF-C polypeptide variants.

Cell lysates (NIH 3T3 for VEGFR-3 and PAE for VEGFR-2) were subjected to receptor-specific antisera and the receptors were immunoprecipitated. Immunoprecipitates were then gel-fractionated and blotted for Western analyses. Western blots were probed with antiphosphotyrosine antibodies.

Figures 29A-D are photomicrographs of hematoxylin-eosin stained sections of K14-VEGF-C transgenic and control mouse littermate tissues. Areas shown are from the dorsal skin and snout, as indicated.

The white arrows show the endothelium-lined margin of the lacunae devoid of red cells.

Figure 30 presents a Northern blot of 24 polyadenylated RNA from the indicated tissues, hybridized with a pool of VEGF,

VEGF-B

167 and VEGF-C probes.

Estimated transcript sizes are shown on the right in kilobases (kb).

Figure 31 presents a comparison of the human and mouse VEGF-C amino acid sequences. The amino acid sequence of mouse VEGF-C is presented on the top line and differences in the human sequence are marked below it.

The sequences have been labeled to depict the regions shown in Figure 9. The arrow indicates the putative cleavage site for the signal peptidase; BR3P motifs, as well as a CR/SC motif, are boxed; and conserved cysteine residues are marked in bold above the sequence. Arginine residue 158 is also marked in bold. The numbering refers 15 to mouse VEGF-C residues.

Figure 32 presents SDS-PAGE-fractionated samples immunoprecipitated or affinity-purified from various 35 S-labeled media. In the left panel, control medium from Bosc23 cells containing vector only, medium from cells expressing human VEGF-C, and medium from cells expressing mouse VEGF-C were independently precipitated with human VEGFR-3-Extracellular Domain coupled to sepharose. In the right panel, similar conditioned media were subjected to precipitation with anti-VEGF-C antibodies. mwm: molecular weight markers; m- mouse; hhuman; a- anti.

Figure 33 shows Western blots of gelfractionated immunoprecipitates from lysates made from NIH 3T3 cells expressing VEGFR-3 or VEGFR-2, as indicated, that had been stimulated by contact with VEGF- C-containing lysates (or a vector control), as a measure of VEGF-C-induced receptor autophosphorylation. Western blots were probed with anti-phosphotyrosine (a-PTyr) or anti-receptor antisera (anti-VEGFR-3 and anti-VEGFR-2), as indicated. As a control, receptor autophosphorylation was induced by pervanadate treatment (V04). The arrows and numbers refer to the apparent molecular weights of 25 the tyrosyl phosphorylated receptor polypeptide bands.

bVEGF: human baculoviral VEGF-C protein; C-FGEVm: lysate from cells harboring a mouse VEGF-C cDNA cloned into the vector in an antisense orientation.

.Figures 34A-D depict photomicrographs of in situ hybridizations revealing the expression of VEGF-C and VEGF-B mRNAs in a parasagittal section of a 12.5 day mouse embryo. Figure 34A: VEGF-C probe; j- jugular veins, mn- metanephros, m- mesenterium (arrowheads), vcintervertebral vessels, lu- lung (arrowheads). Figure 34B: VEGF-B probe; h- heart, nasopharyngeal area (arrowheads). Figure 34C: VEGF-C sense strand probe serving as a control. Figure 34D: bright-field photomicrograph of the same field shown in Figure 34C.

Figures 35A-H depict sections of mouse embryos providing comparisons of VEGF-C and VEGFR-3 expression in the jugular vessels and the mesenteric area. Figures and 35C show expression of VEGF-C transcripts in the mesenchyme around the large sac-like structures in the 20 jugular area (arrowheads). Figures 35B and 35D show expression of VEGFR-3 transcripts in the jugular venous sacs. Figures 35E and 35G show VEGF-C mRNA distribution in the mesenteric region of a 14.5 day p.c. embryo, as well as around the gut. Figures 35F and 35H show VEGFR-3 mRNA in the mesenteric region of a 14.5 day embryo, as well as the gut area, developing lymphatic vessels, and venules.

S

Figures 36A-D depict photomicrographs showing FLT4 and VEGF-C in situ hybridization of the cephalic region of a 16-day p.c. mouse embryo. A section of the cephalic region hybridized with the Flt4 probe (Figure 36A) shows the developing snout, nasal structures and eyes. A more caudally located section shows hybridization with the VEGF-C probe (Figure 36B). The round structures on both sides in the upper part represent the developing molars.

In the upper (dorsal) part on both sides of the midline, the caudal portion of the developing conchae are seen.

26 W These structures also are shown in higher magnification darkfield (Figure 36C) and lightfield (Figure 36D) microscopy.

DETAILED DESCRIPTION OF THE INVENTION Described herein is the isolation of a novel vascular endothelial growth factor and the cloning of a DNA encoding this novel growth factor from a cDNA library prepared from the human prostatic adenocarcinoma cell line PC-3. The isolated cDNA encodes a protein which is proteolytically processed and secreted to cell culture medium. The secreted protein, designated VEGF-C, binds to the extracellular domain of Flt4 and induces tyrosine autophosphorylation of Flt4 and VEGFR-2. VEGF-C also stimulates the migration of endothelial cells in collagen 15 gel.

00. Data reported herein indicates that VEGF-C is o.0 expressed as a larger precursor which is cleaved to produce the ligand. A coexpressed region in some cases results from alternative splicing of RNA of the ligand gene. Such a co-expressed region may be a function of the particular expression system used to obtain the ligand. The skilled artisan understands that in recombinant production of proteins, additional sequence may be expressed along with a functional polypeptide S. 25 depending upon the particular recombinant construct used to express the protein, and subsequently removed to obtain the desired ligand. In some cases the recombinant ligand can be made lacking certain residues of the endogenous/natural ligand. Moreover, it is well-known in that conservative replacements may be made in a protein which do not alter the function of the protein.

Accordingly, it is anticipated that such alterations are within the scope of the invention. Moreover, it is anticipated that one or more VEGF-C precursors (the largest putative native secreted VEGF-C precursor having the complete amino acid sequence from residue 32 to 27 residue 419 of SEQ ID NO: 33) is capable of stimulating the Flt4 ligand without any further processing, in a manner similar to that in which VEGF stimulates its receptor in its unprocessed form after the secretion and concomitant release of the signal sequence.

Results reported herein show that Flt4 (VEGFR- 3) transmits signals for VEGF-C. This conclusion is based on the specific binding of VEGF-C to recombinant Flt4EC (Flt4 extracellular domain) protein and the induction of VEGFR-3 autophosphorylation by medium from VEGF-C transfected cells. In contrast, neither VEGF nor P1GF showed specific binding to VEGFR-3 or induced its autophosphorylation.

As set forth in greater detail below, the 15 putative prepro-VEGF-C has a deduced molecular mass of 46,883; a putative prepro-VEGF-C processing intermediate has an observed molecular weight of about 32 kD; and mature VEGF-C isolated form conditioned media has a molecular weight of about 23 kD as assessed by SDS-PAGE 20 under reducing conditions. A major part of the S''difference in the observed molecular mass of the purified and recombinant VEGF-C and the deduced molecular mass of the prepro-VEGF-C encoded by the VEGF-C open reading frame (ORF) is attributable to proteolytic removal of sequences at the amino-terminal and carboxyl-terminal regions of the prepro-VEGF-C polypeptide. However, proteolytic cleavage of the putative 102 amino acid leader sequence is not believed to account for the entire difference between the deduced molecular mass of 46,883 and the observed mass of about 23 kD, because the deduced molecular weight of a polypeptide consisting of amino acids 103-419 of SEQ ID NO: 33 is 35,881 kD. Evidence indicates that a portion of the observed difference in molecular weights is attributable to proteolytic removal of amino acid residues in both the amino and carboxyl terminal regions of the VEGF-C precursor. Extrapolation from studies of the structure of PDGF (Heldin et al., 28 Growth Factors, 8:245-52 (1993)) suggests that the region critical for receptor binding and activation by VEGF-C is contained within amino acids residues 104-213, which are found in the secreted form of the VEGF-C protein the form lacking the putative prepro leader sequence and some carboxyterminal sequences). The 23 kD polypeptide binding VEGFR-3 is likely to represent the VEGFhomologous domain. After biosynthesis, the nascent

VEGF-

C polypeptide may be glycosylated at three putative

N-

linked glycosylation sites identified in the deduced VEGF-C amino acid sequence. Polypeptides containing modifications, such as N-linked glycosylations, are intended as aspects of the invention.

The carboxyl terminal amino acid sequences, 15 which increase the length of the VEGF-C polypeptide in comparison with other ligands of this family, show a pattern of spacing of cysteine residues reminiscent of the Balbiani ring 3 protein (BR3P) sequence (Dignam et al., Gene, 88:133-40 (1990); Paulsson et al., J. Mol.

20 Biol., 211:331-49 (1990)). This novel C-terminal silk protein-like structural motif of VEGF-C may fold into an independent domain, which, on the basis of the considerations above, is at least partially cleaved off after biosynthesis. Interestingly, at least one cysteine motif of the BR3P type is also found in the carboxyl terminus of VEGF. In our experiments both the putative precursor and cleaved ligand were detected in the cell culture media, suggesting cleavage by cellular proteases.

The determination of amino-terminal and carboxy-terminal sequences of VEGF-C isolates allows the identification of the proteolytic processing sites. The generation of antibodies against different parts of the pro-VEGF-C molecule allows the exact determination of the precursorproduct relationship and ratio, their cellular distribution, and the kinetics of processing and secretion.

VEGF-C has a conserved pattern of eight 29 cysteine residues, which may participate in the formation of intra- and interchain disulfide bonds, creating an antiparallel dimeric biologically active molecule, similar to PDGF. Mutational analysis of the cysteine residues involved in the interchain disulfide bridges has shown that, in contrast to PDGF, VEGF dimers need to be held together by these covalent interactions in order to maintain biological activity. Disulfide linking of the VEGF-C polypeptide chains was evident in the analysis of VEGF-C in nonreducing conditions, although recombinant protein also contained ligand-active VEGF-C forms which lacked disulfide bonds between the polypeptides.

VEGFR-3, which distinguishes between VEGF and VEGF-C, is closely related in structure to VEGFR-1 and S 15 VEGFR-2. Finnerty et al., Oncogene, 8:2293-98 (1993); Galland et al., Oncogene, 8:1233-40 (1993); Pajusola et Sal., Cancer Res., 52:5738-43 (1992). Besides VEGFR-3, VEGFR-2 tyrosine kinase also is activated in response to VEGF-C. VEGFR-2 mediated signals cause striking changes 20 in the morphology, actin reorganization and membrane ruffling of porcine aortic endothelial cells overexpressing this receptor. In these cells, VEGFR-2 .also mediated ligand-induced chemotaxis and mitogenicity.

Waltenberger et al., J. Biol. Chem., 269:26988-95 (1994).

Similarly, the receptor chimera CSF-1R/VEGFR-3 was mitogenic when ectopically expressed in NIH 3T3 fibroblastic cells, but not in porcine aortic endothelial cells (Pajusola et al., 1994). Consistent with such results, the bovine capillary endothelial (BCE) cells, which express VEGFR-2 mRNA but very little or no VEGFR-1 or VEGFR-3 mRNAs, showed enhanced migration when stimulated with VEGF-C. Light microscopy of the BCE cell cultures in collagen gel also suggested that VEGF-C stimulated the proliferation of these cells. The data thus indicate that the VEGF ligands and receptors show a great specificity in their signalling, which may be celltype-dependent.

30 O The expression pattern of the VEGFR-3 (Kaipainen et al., Proc. Natl. Acad. Sci. (USA), 92:3566- (1995)) suggests that VEGF-C may function in the formation of the venous and lymphatic vascular systems during embryogenesis. Constitutive expression of VEGF-C in adult tissues shown herein further suggests that this gene product also is involved in the maintenance of the differentiated functions of the lymphatic and certain venous endothelia where VEGFR-3 is expressed (Kaipainen et al., 1995). Lymphatic capillaries do not have wellformed basal laminae and an interesting possibility remains that the silk-like BR3P motif is involved in producing a supramolecular structure which could regulate the availability of VEGF-C in tissues. However, as shown 15 here, VEGF-C also activates VEGFR-2, which is abundant in Proliferating endothelial cells of vascular sprouts and branching vessels of embryonic tissues, but not so abundant in adult tissues. Millauer et al., Nature, 367:576-78 (1993). These data have suggested that VEGFR-2 is a major regulator of vasculogenesis and angiogenesis. VEGF-C may thus have a unique effect on lymphatic endothelium and a more redundant function, shared with VEGF, in angiogenesis and possibly in regulating the permeability of several types of endothelia. Because VEGF-C stimulates VEGFR-2 and promotes endothelial migration, VEGF-C may be useful as an inducer of angiogenesis of blood and lymphatic vessels in wound healing, in tissue transplantation, in eye diseases, and in the formation of collateral vessels around arterial stenoses and into injured tissues after infarction.

Taken together, these results show an increased complexity of signalling in the vascular endothelium.

They reinforce the concept that when organs differentiate and begin to perform their specific functions, the phenotypic heterogeneity of endothelial cells increases in several types of functionally and morphologically 31 distinct vessels. However, upon stimulation by suitable angiogenic stimuli, endothelial cells can re-enter the cell cycle, migrate, withdraw from the cell cycle and subsequently differentiate again to form new vessels that are functionally adapted to their tissue environment.

This process of angiogenesis, concurrent with tissue development and regeneration, depends on the tightly controlled balance between positive and negative signals for endothelial cell proliferation, migration, differentiation and survival.

Previously-identified growth factors promoting angiogenesis include the fibroblast growth factors, hepatocyte growth factor/scatter factor, PDGF and TGF-a.

(See Folkman, Nature Med., 1:27-31 (1995); Friesel 15 et al., FASEB 9:919-25 (1995); Mustonen et al., J.

Cell. Biol., 129:895-98 (1995). However, VEGF has been the only growth factor relatively specific for endothelial cells. The newly identified factors VEGF-B [Olofsson et al., Proc. Natl. Acad. Sci., 93:2578-81 (1996)] and VEGF-C thus increase our understanding of the complexity of the specific and redundant positive signals for endothelial cells involved in vasculogenesis, angiogenesis, permeability, and perhaps also other endothelial functions. Expression studies using Northern blotting show abundant VEGF-C expression in heart and skeletal muscle; other tissues, such as placenta, ovary, small intestine, thyroid gland, kidney, prostate, spleen, testis and large intestine also express this gene.

Whereas P1GF is predominantly expressed in the placenta, the expression patterns of VEGF, VEGF-B and VEGF-C overlap in many tissues, which suggests that members of the VEGF family may form heterodimers and interact to exert their physiological functions.

Targeted mutagenesis leading to inactivation of the VEGF receptor loci in the mouse genome has shown that VEGFR-1 is necessary for the proper organization of endothelial cells forming the vascular endothelium, while 32 VEGFR-2 is necessary for the generation of both endothelial and hematopoietic cells. This suggests that the four genes of the VEGF family can be targets for mutations leading to vascular malformations or cardiovascular diseases.

The following Examples illustrate preferred embodiments of the invention, wherein the isolation, characterization, and function of Flt4 ligands and ligand-encoding nucleic acids according to the invention are shown.

EXAMPLE 1 Production of pLTRFlt41 expression vector Construction of the LTR-Flt41 vector, encoding S. the long form of Flt4 receptor tyrosine kinase, is 15 schematically shown in Fig. 2. The full-length Flt4s (Flt4 short form) cDNA (Genbank Accession No. X68203,

SEQ

ID NO: 36) was assembled by first subcloning the fragment, reported in Pajusola et al., Cancer Res., 52:5738-5743 (1992), incorporated by reference herein, 20 containing base pairs 56-2534 of the Flt4s into the EcoRI site of the pSP73 vector (Promega, Madison,

WI).

Since cDNA libraries used for screening of Flt4 cDNAs did not contain the extreme 5' protein-coding sequences, inverse PCR was used for the amplification of the 5' end of Flt4 corresponding to the first 12 amino acid residues (MQRGAALCLRLW). Poly(A)* RNA was isolated from human HEL erythroleukemia cells and double-stranded cDNA, were synthesized using an Amersham cDNA Synthesis System Plus kit (Amersham Corp., Buckinghamshire,

U.K.)

and a gene-specific primer: 5'-TGTCCTCGCTGTCCTTGTCT-3' (SEQ ID NO: which was located 195 bp downstream of the 5' end of clone S2.5. Double-stranded cDNA was treated with T4 DNA polymerase to blunt the ends and cDNA was purified by filtration with Centricon 100 filters (Amicon Inc., Beverly, MA). Circularization of the blunt-ended cDNA was performed by ligation in a total 33 volume of 150 microliters. The reaction mixture contained a standard ligation buffer, 5% PEG-8000, 1 mM DTT and 8 U of T4 DNA ligase (New England Biolabs, Beverly, MA). Ligation was carried out at 16'C for 16 hours. Fifteen microliters of this reaction mix were used in a standard PCR reaction (100 tl total volume) containing 100 ng of Flt4-specific primers introducing SacI and PstI restriction sites, and 1 unit of Taq DNA polymerase (Perkin Elmer Cetus). Two rounds of PCR were performed using 33 cycles per round (denaturation at 95 C for 1 minute, annealing at 55C for 2 minutes, and elongation at 72"C for 4 minutes). The PCR mixture was treated sequentially with the SacI and PstI restriction enzymes, and after purification with MagicPCR Preps 15 (Promega), DNA fragments were subcloned into the pGEM3Zf(+) vector for sequencing (Promega). The sequence corresponded to the 5' end of the Flt4s cDNA clone deposited in the Genbank Database as Accession No.

X68203.

20 The sequence encoding the first 12 amino acid residues was added to the expression construct by ligating an SphI-digested PCR fragment amplified using reverse transcription-PCR of poly(A) RNA isolated from the HEL cells. The forward primer had the following sequence: 5'-ACATGCATGC CACCATGCAG CGGGGCGCCG

CGCTGTGCCT

GCGACTGTGG CTCTGCCTGG GACTCCTGGA-3' (SEQ ID NO: 2) (SphI site underlined, translational start codon marked in bold). The translation start codon is immediately downstream from an optimized Kozak consensus sequence.

(Kozak, Nucl. Acids Res., 15: 8125-8148 (1987).) The reverse primer, 5'-ACATGCATGC CCCGCCGGT CATCC-3' (SEQ ID NO: 3) (SphI site underlined), to the 5' end of the fragment, thus replacing the unique SphI fragment of the plasmid. The resulting vector was digested with EcoRI and ClaI and ligated to a 138 bp PCR fragment amplified from the 0.6 kb EcoRI fragment (base pairs 3789 to 4416 in the Genbank X68203 sequence) which encodes the 34 3' end of Flt4s shown in Figure 1 of Pajusola et al., Cancer Res., 52:5738-5743 (1992), using the oligonucleotides 5'-CGGAATTCCC CATGACCCCA AC-3' (SEQ ID NO: 4) (forward primer, EcoRI site underlined) and CCATCGATGG ATCCTACCTG AAGCCGCTTT CTT-3' (SEQ ID NO: (reverse primer, Clal site underlined). The coding domain was completed by ligation of the 1.2 kb EcoRI fragment (base pairs 2535-3789 of the sequence found in Gen Bank Acc. No. X68203) into the above construct. The complete cDNA was subcloned as a HindIII-ClaI(blunted) fragment (this Clal site was also included in the 3' primer used to construct the 3' end of the coding sequence) to the pLTRpoly expression vector reported in Mkela et al., Gene, 118: 293-294 (1992) (Genbank 15 accession number X60280, SEQ ID NO: 37), incorporated by reference herein, using its HindIII-AccI(blunted) restriction sites.

The long form of Flt4 (Flt41) was produced by replacing the 3'-end of the short form as follows: The 3' 20 region of the Flt41 cDNA was PCR-amplified using a genespecific oligonucleotide (SEQ ID NO: 7, see below) and a pGEM 3Z vector-specific (SP6 promoter) oligonucleotide 5'-ATTTAGGTGACACTATA-31 (SEQ ID NO: 6) as reverse and forward primers, respectively. The template for PCR was an Flt41 cDNA clone containing a 495 bp EcoRI fragment extending downstream of the EcoRI site at nucleotide 3789 of the Genbank X68203 sequence (the sequence downstream of this EcoRI site is deposited as the Flt4 long form 3' sequence having Genbank accession number S66407 (SEQ ID NO: The gene-specific oligonucleotide contains a BamHI restriction site located right after the end of the coding region and has the following sequence: CCATCGATGGATCCCGATGCTGCTTAGTAGCTGT-3, (SEQ ID NO: 7) (BamHI site is underlined). The PCR product was digested with EcoRI and BamHI and transferred in frame to the LTRFlt4s vector fragment from which the coding sequences downstream of the EcoRI site at base pair 2535 (see.

35 W sequence X68203) had been removed by EcoRI-BamHI digestion. The resulting clone was designated pLTRFlt41. Again, the coding domain was completed by ligation of the 1.2 kb EcoRI fragment (base pairs 2535- 3789 of sequence X68203) back into the resulting construct.

EXAMPLE 2 Production and analysis of Flt41 transfected cells NIH 3T3 cells (60 confluent) were cotransfected with 5 micrograms of the pLTRFlt41 construct and 0.25 micrograms of the pSV2neo vector containing the neomycin phosphotransferase gene (Southern et al., J.

Mol. Appl. Genet., 1:327 (1982)), using the DOTAP liposome-based transfection reagents (Boehringer- 15 Mannheim, Mannheim, Germany). One day after transfection, the cells were transferred into selection i media containing 0.5 mg/ml geneticin (GIBCO, Grand Island, Colonies of geneticin-resistant cells were isolated and analyzed for expression of the Flt4 20 proteins. Cells were lysed in boiling lysis buffer containing 3.3% SDS 125 mM Tris, pH 6.8. Protein concentrations of the samples were measured by the BCA method (Pierce, Rockford, IL). About 50 micrograms of protein from each lysate were analyzed for the presence 25 of Flt4 by 6% SDS-polyacrylamide gel electrophoresis (SDS-PAGE) and immunoblotting using antisera against the carboxyl terminus of Flt4. Signals on Western blots were revealed using the ECL method (Amersham).

For production of anti-Flt4 antiserum, the Flt4 cDNA fragment encoding the 40 carboxy-terminal amino acid residues of the short form: NH2-PMTPTTYKG SVDNQTDSGM VLASEEFEQI ESRHRQESGFR-COOH (SEQ ID NO: 8) was cloned as a 657 bp EcoRI-fragment into the pGEX-IXT bacterial expression vector (Pharmacia-LKB, Inc., Uppsala, Sweden) in frame with the glutathione-S-transferase coding region. The resultant GST-Flt4S fusion protein was 36 W produced in E. coli and purified by affinity chromatography using a glutathione-Sepharose 4B column.

The purified protein was lyophilized, dissolved in phosphate-buffered saline (PBS), mixed with Freund's adjuvant and used for immunization of rabbits at biweekly intervals using methods standard in the art (Harlow et al., Antibodies: A Laboratory Manual (Cold Spring Harbor Laboratory Press, 1988)). Antisera were used, after the fourth booster immunization, for immunoprecipitation of Flt4 from transfected cells. Cell clones expressing Flt4 were also used for ligand stimulation analysis.

EXAMPLE 3 Construction of a Flt4 EC baculovirus vector 15 and expression and purification of its product The construction of an Flt4 extracellular domain (EC) baculovirus vector is schematically depicted in Fig. 3. The Flt4-encoding cDNA was prepared in both a long form and a short form, each being incorporated in a o. 20 vector under control of the Moloney murine leukemia virus LTR promoter. The nucleotide sequence of the short form of the Flt4 receptor is available from the Genbank S. database as Accession No. X68203 and the specific 3' segment of the long form cDNA is available under GenBank 25 Accession No. S66407.

The ends of a cDNA segment encoding the Flt4 extracellular domain (EC) were modified as follows: The 3' end of the Flt4 cDNA (Genbank Accession Number X68203) extracellular domain sequence was amplified using primer 1116 (5'-CTGGAGTCGACTTGGCGGACT-3'; SEQ ID NO: 9, SalI site underlined) and primer 1315

CGCGGATCCCTAGTGATGGTGATGGTGATGTCTACCTTCGATCATGCTGCCCTTAT

CCTC-3'; (SEQ ID NO: 10, BamHI site underlined). The sequence at the 5' end of primer 1315 is not complementary to the Flt4 coding region. Inspection of the sequence that is complementary to this region of 7 37 W primer 1315 reveals in a 5' to 3' order, a stop codon, six contiguous histidine codons (for subsequent chromatographic purification of the encoded polypeptide using a Ni-NTA column; Qiagen, Hilden, Germany), and an added BamHI site. The amplified fragment was digested with SalI and BamHI and used to replace a unique SalI- BamHI fragment in the LTRFlt4 vector shown in Fig. 3.

The SalI-BamHI fragment that was replaced encodes the Flt4 transmembrane and cytoplasmic domains. The result was a modified LTRFlt4 vector.

The 5' end without the Flt4 signal sequence encoding region was amplified by PCR using the primer 1335 (5'-CCCAAGCTTGGATCCAAGTGGCTACTCCATGACC-3'; (SEQ ID 1 NO: 11) the primer contains added HindIII (AAGCTT) and 15 BamHI (GGATCC) restriction sites, which are underlined).

The second primer used to amplify the region encoding the Flt4 signal sequence was primer 1332 GTTGCCTGTGATGTGCACCA-3'; SEQ ID NO: 12). The amplified fragment was digested with HindIII and SphI (the HindIII e. 20 site (AAGCTT) is underlined in primer 1335 and the SphI site is within the amplified region of the Flt4 cDNA).

The resultant HindIII-SphI fragment was used to replace a HindIII-SphI fragment.in the modified LTRFlt4 vector •described immediately above (the HindIII site is in the go* 5' junction of the Flt4 insert with the pLTRpoly portion of the vector, the SphI site is in the Flt4 cDNA). The resultant Flt4EC insert was then ligated as a BamHI fragment into the BamHI site in the pVTBac plasmid described in Tessier et al., Gene 98:177-183 (1991), incorporated herein by reference. The relative orientation of the insert was confirmed by partial sequencing so that the open reading frame of the signal sequence-encoding portion of the vector was adjacent to, and in frame with, the Flt4 coding region sequence. The Flt4EC construct was transfected together with baculovirus genomic DNA into SF-9 cells by lipofection.

Recombinant virus was purified, amplified and used for 38 infection of High-Five cells (Invitrogen, San Diego,

CA)

using methods standard in the art. The Flt4 extracellular domain (Flt4EC) was purified from the culture medium of the infected High-Five cells using Ni- NTA affinity chromatography according to manufacturer's instructions (Qiagen) for binding and elution of the 6xHis tag encoded in the COOH-terminus of the recombinant Flt4 extracellular domain.

EXAMPLE 4 Isolation of an Flt4 Ligand from Conditioned Media A human Flt4 ligand according to the invention was isolated from media conditioned by a PC-3 prostatic adenocarcinoma cell line (ATCC CRL 1435) in Ham's F-12 Nutrient mixture (GIBCO) containing 7% fetal calf serum (FCS). The cells were grown according to the supplier's instructions. In order to prepare the conditioned media, confluent PC-3 cells were cultured for 7 days in Ham's

F-

12 Nutrient mixture (GIBCO) in the absence of fetal bovine serum (FBS). Medium was then cleared by centrifugation at 10,000 g for 20 minutes. The medium was then screened to determine its ability to induce tyrosine phosphorylation of Flt4 by exposure to NIH 3T3 cells which had been transfected with Flt4-encoding cDNA using the pLTRFlt41 vector. For receptor stimulation 25 experiments, subconfluent NIH 3T3 cells were starved overnight in serum-free DMEM medium (GIBCO) containing 0.2% bovine serum albumin (BSA). The cells were stimulated with the conditioned media for 5 minutes, washed twice with cold PBS containing 100 micromolar vanadate, and lysed in RIPA buffer (10 mM Tris pH 7.5, mM NaC1, 0.5% sodium deoxycholate, 0.5% Nonidet P40 (BDH, Poole, England), 0.1% SDS, 0.1 U/ml Aprotinin (Boehringer Mannheim), 1 mM vanadate) for receptor immunoprecipitation analysis. The lysates were centrifuged for 20 minutes at 15,000 x g. The supernatants were incubated for 2 hours on ice with 3

-V-

I 39 microliters of the antiserum against the Flt4 C-terminus Sdescribed in Example 2. See also Pajusola et al., Oncogene, 8:2931-2937 (1993), incorporated by reference herein.

After a two hour incubation in the presence of anti-Flt4 antiserum, protein A-Sepharose (Pharmacia) was added and incubation was continued for 45 minutes with rotation. The immunoprecipitates were washed three times with the immunoprecipitation buffer and twice with 10 mM Tris, pH 7.5, before analysis by SDS-PAGE. Polypeptides were transferred to nitrocellulose and analyzed by Western blotting using Flt4- or phosphotyrosine-specific antisera and the ECL method (Amersham Corp.). Antiphosphotyrosine monoclonal antibodies (anti-PTyr; 15 were purchased from Transduction Laboratories (Lexington, Kentucky). In some cases, the filters were restained with a second antibody after stripping. The stripping of the filters was done for 30 minutes at 50 C in 100 mM 2mercaptoethanol, 2% SDS, 62.5 mM Tris-HCl, pH 6.7, with occasional agitation.

As shown in Fig. 4, the PC-3 conditioned medium (PC-3CM), stimulated tyrosine phosphorylation of a 125 kD polypeptide when Flt4- expressing NIH 3T3 cells were treated with the indicated preparations of media, lysed, and the lysates were immunoprecipitated with anti-Flt4 antiserum followed by SDS-PAGE, Western blotting, and staining using anti-PTyr antibodies. The resulting band was weakly phosphorylated upon stimulation with unconcentrated PC-3 conditioned medium (lane The 125 kD band comigrated with the tyrosine phosphorylated, processed form of the mature Flt4 from pervanadatetreated cells (compare lanes 2 and 7 of Fig. 4, see also Fig. 5A). Comigration was confirmed upon restaining with anti-Flt4 antibodies as is also shown in Fig. 5A (panel on the right). In order to show that the 125 kD polypeptide is not a non-specific component of the conditioned medium reactive with anti-phosphotyrosine

L-

40 antibodies, 15 microliters of conditioned medium were separated by SDS-PAGE, blotted on nitrocellulose, and the blot was stained with anti-PTyr antibodies. No signal was obtained (Fig. 5B). Also, unconditioned medium failed to stimulate Flt4 phosphorylation, as shown in Fig. 4, lane 1.

Fig. 5C shows a comparison of the effects of PC-3 CM stimulation on untransfected (lanes 4 and FGFR-4-transfected (lanes 8 and 9) and Flt4-transfected NIH 3T3 cells (lanes 1-3, 6 and These results indicate that neither untransfected NIH 3T3 cells nor NIH 3T3 cells transfected with FGFR-4 showed tyrosine phosphorylation of a protein of about 125 kD upon stimulation with the conditioned medium from PC-3 cells.

15 Analysis of stimulation by PC-3 CM pretreated with Heparin-Sepharose CL-6B (Pharmacia) for 2 hours at room temperature (lane 3) showed that the Flt4 ligand does not bind to heparin.

As shown in Fig. 4, lane 3, stimulating 20 activity was considerably increased when the PC-3 conditioned medium was concentrated four-fold using a concentrator (Amicon). Fig. 4, lane 4, shows that pretreatment of the concentrated PC-3 conditioned medium with 50 microliters of the Flt4 extracellular domain coupled to CNBr-activated sepharose CL-4B (Pharmacia; about 1mg of Flt4EC domain/ml sepharose resin) completely abolished Flt4 tyrosine phosphorylation. Similar pretreatment of the conditioned medium with unsubstituted sepharose CL-4B did not affect stimulatory activity, as shown in Fig. 4, lane 5. Also, the flow through obtained after concentration, which contained proteins of less than 10,000 molecular weight, did not stimulate Flt4 phosphorylation, as shown in Fig.

4, lane 6.

In another experiment, a comparison of Flt4 autophosphorylation in transformed NIH 3T3 cells expressing LTRFlt41 was conducted, using unconditioned 41 medium, medium from PC-3 cells expressing the Flt4 ligand, or unconditioned medium containing either ng/ml of VEGF165 or 50 ng/ml of PlGF-1. The cells were lysed, immunoprecipitated using anti-Flt4 antiserum and analyzed by Western blotting using anti-phosphotyrosine antibodies. Only the PC-3 conditioned medium expressing the Flt4 ligand (lane Flt-4L) stimulated Flt4 autophosphorylation.

The foregoing data show that PC-3 cells produce a ligand which binds to the extracellular domain of Flt4 and activates this receptor.

EXAMPLE Purification of the Flt4 Ligand The ligand expressed by human PC-3 cells as 15 characterized in Example 4 was purified and isolated .using a recombinantly-produced Flt4 extracellular domain (Flt4EC) in affinity chromatography.

Two harvests of serum-free conditioned medium, comprising a total of 8 liters, were collected from 500 20 confluent 15 cm diameter culture dishes containing confluent layers of PC-3 cells. The conditioned medium was clarified by centrifugation at 10,000 x g and concentrated 80-fold using an Ultrasette Tangential Flow Device (Filtron, Northborough, MA) with a 10 kD cutoff S: 25 Omega Ultrafiltration membrane according to the manufacturer's instructions. Recombinant Flt4 extracellular domain was expressed in a recombinant baculovirus cell system and purified by affinity chromatography on Ni-agarose (Ni-NTA affinity column obtained from Qiagen). The purified extracellular domain was coupled to CNBr-activated Sepharose CL-4B at a concentration of 5 mg/ml and used as an affinity matrix for ligand affinity chromatography.

Concentrated conditioned medium was incubated with 2 ml of the recombinant Flt4 extracellular domain- Sepharose affinity matrix in a rolling tube at room 42 temperature for 3 hours. All subsequent purification steps were at +4 C. The affinity matrix was then transferred to a column with an inner diameter of 15 mm and washed successively with 100 ml of PBS and 50 ml of 10 mM Na-phosphate buffer (pH Bound material was eluted step-wise with 100 mM glycine-HCl, successive 6 ml elutions having pHs of 4.0, 2.4, and 1.9. Several 2 ml fractions of the eluate were collected in tubes containing 0.5 ml 1 M Na-phosphate (pH Fractions were mixed immediately and dialyzed in 1 mM Tris-HCl (pH Aliquots of 75 ml each were analyzed for their ability to stimulate tyrosine phosphorylation of Flt4.

The ultrafiltrate, 100 il aliquots of the concentrated conditioned medium before and after ligand affinity 15 chromatography, as well as 15-fold concentrated fractions *of material released from the Flt4 extracellular domain- Sepharose matrix during the washings were also analyzed for their ability to stimulate Flt4 tyrosine phosphorylation.

As shown in Fig. 6, lane 3, the concentrated conditioned medium induced prominent tyrosine phosphorylation of Flt4 in transfected NIH 3T3 cells overexpressing Flt4. This activity was not observed in conditioned medium taken after medium was exposed to the Flt4 Sepharose affinity matrix described above (Fig. 6, lane The specifically-bound Flt4-stimulating material was retained on the affinity matrix after washing in PBS, 10 mM Na-phosphate buffer (pH and at pH 4.0 (Fig. 6, lanes 5-7, respectively), and it was eluted in the first two 2 ml aliquots at pH 2.4 (lanes 8 and A further decrease of the pH of the elution buffer did not cause release of additional Flt4stimulating material (Fig. 6, lane 11). Fig. 6, lane 1 depicts a control wherein Flt4-expressing cells were treated with unconditioned medium; lane 2 depicts the results following treatment of Flt4-expressing cells with the ultrafiltrate fraction of conditioned medium 43 containing polypeptides of less than 10 kD molecular weight.

Small aliquots of the chromatographic fractions were concentrated in a SpeedVac concentrator (Savant, Farmingdale, and subjected to SDS-PAGE under reducing conditions with subsequent silver staining of the gel, a standard technique in the art. As shown in Fig. 7, the major polypeptide, having a molecular weight of approximately 23 kD (reducing conditions), was detected in the fractions containing Flt4 stimulating activity (corresponding to lanes 8 and 9 in Fig. 6).

That polypeptide was not found in the other chromatographic fractions. On the other hand, besides S. these bands and a very faint band having a 32 kD 15 mobility, all other components detected in the two active fractions were also distributed in the starting material and in small amounts in the other washing and eluting steps after their concentration. Similar results were obtained in three independent affinity purifications, 20 indicating that the 23 kD polypeptide specifically binds to Flt4 and induces tyrosine phosphorylation of Flt4.

Fractions containing the 23 kD polypeptide were combined, dried in a SpeedVac concentrator and subjected to SDS-PAGE in a 12.5% gel. The proteins from the gel were then electroblotted to Immobilon-P (PVDF) transfer membrane (Millipore, Marlborough, MA) and visualized by staining of the blot with Coomassie Blue R-250. The region containing only the stained 23 kD band was cut from the blot and subjected to N-terminal amino acid sequence analysis in a Prosite Protein Sequencing System (Applied Biosystems, Foster City, CA). The data were analyzed using a 610A Data Analysis System (Applied Biosystems). Analysis revealed a single N-terminal sequence of NH 2 -XEETIKFAAAHYNTEILK-COOH (SEQ ID NO: 13).

44 EXAMPLE 6 Construction of PC-3 cell cDNA library in a eukaryotic expression vector Human poly(A)* RNA was isolated from five 15 cm diameter dishes of confluent PC-3 cells by a single step method using oligo(dT) (Type III, Collaborative Biomedical Products, Becton-Dickinson Labware, Bedford, MA) cellulose affinity chromatography (Sambrook et al., 1989). The yield was 70 micrograms. Six micrograms of the Poly(A) RNA were used to prepare an oligo(dT)-primed cDNA library in the mammalian expression vector pcDNA I and the Librarian kit of Invitrogen according to the instructions included in the kit. The library was estimated to contain about 10 6 independent recombinants 15 with an average insert size of approximately 1.8 kb.

EXAMPLE 7 Amplification of a unique nucleotide sequence encoding the Flt4 ligand amino terminus Degenerate oligonucleotides were designed based on the N-terminal amino acid sequence of the isolated human Flt4 ligand and were used as primers in a olymerase chain reaction (PCR) to amplify cDNA encoding the Flt4 ligand from the PC-3 cDNA library. The overall strategy described in Examples 7 and 8 is schematically i 25 depicted in Fig. 9, where the different primers have been marked with arrows.

The PCR was carried out using 1 microgram of DNA from the amplified PC-3 cDNA library and a mixture of 48 sense-strand primers present in equal proportions, the primer sequences collectively comprising the sequence GCAGARGARACNATHAA-3' (SEQ ID NO: 14) (wherein R is A or G, N is A,G,C or T and H is A, C or encoding amino acid residues 2-6 (EETIK, SEQ ID NO: 15) and 384 antisense-strand primers present in equal proportions, the anti-sense strand primers collectively comprising the sequence 5'-GCAYTTNARDATYTCNGT-3' (SEQ ID NO: 16) .r 45 (wherein Y is C or T and D is A, G or corresponding to amino acid residues 14-18 (TEILK, SEQ ID NO: 17).

Three extra nucleotides (GCA) were added to the terminus of each primer to increase annealing stability.

Two successive PCR runs were carried out using 1 U per reaction of DynaZyme (F-500L, Finnzymes, Espoo, Finland), a thermostable DNA polymerase, in a buffer supplied by the manufacturer (10 mM Tris-HCl, pH 8.8 at 25 C, 1.5 mM MgCI2. 50 mM KC1, 0.1% Triton-X100), at an extension temperature of 72 C. The first PCR run was carried out for 43 cycles. The first three cycles were run at an annealing temperature of 33'C for 2 minutes, and the remaining cycles were run at 42'C for 1 minute.

The region of the gel containing a weak band of 15 the expected size (57 bp) was cut out from the gel and eluted. The eluted material was reamplified for cycles using the same primer pairs described above at 42 C for 1 minute. The amplified fragment was cloned into a pCR II vector (Invitrogen) using the TA cloning kit 20 (Invitrogen) and sequenced using the radioactive dideoxynucleotide sequencing method of Sanger. Six clones were analyzed and all six clones contained the sequence encoding the expected peptide (amino acid residues 104-120 of the Flt4 ligand precursor).

Nucleotide sequence spanning the region from the third nucleotide of codon 6 to the third nucleotide of codon 13 (the extension region) was identical in all six clones: 3 (SEQ ID NO: 18) and thus represented an amplified product from the unique sequence encoding part of the amino terminus of the Flt4 ligand.

EXAMPLE 8 Amplification of the 5'-end of the cDNA encoding the Flt4 ligand Based on the unique nucleotide sequence encoding the N-terminus of the isolated human Flt4 ligand, two pairs of nested primers were designed to 46 amplify, in two nested PCR reactions, the complete 5 '-end of the corresponding cDNAs from one microgram of DNA of the above-described PC-3 cDNA library. First, amplification was performed with an equal mixture of 4 primers collectively defining the sequence

TCNGTGTTGTAGTGTGCTG-

3 (SEQ ID NO: 19), which is the antisense-strand primer corresponding to amino acid residues 9-15 (AAHYNTE, SEQ ID NO: 20), and sense-strand primer 5'-TAATACGACTCACTATAGGG-3' (SEQ ID NO: 21), corresponding to the T7 RNA promoter of the pcDNAI vector used for construction of the library. "Touchdown"

PCR

was used as disclosed in Don et al., Nucl. Acids Res., 19:4008 (1991), incorporated by reference herein. The annealing temperature of the two first cycles was 62 C and 15 subsequently the annealing temperature was decreased in every other cycle by 1 C until a final temperature of 53 C was reached, at which temperature 16 additional cycles i were conducted. Annealing time was 1 minute and extension at each cycle was conducted at 72"C for 1 minute. Multiple amplified DNA fragments were obtained in the first reaction. The products of the first amplification (1 pl of a 1:100 dilution in water) were S. used in the second amplification reaction employing a pair of nested primers comprising an antisense-strand primer 5'-GTTGTAGTGTGCTGCAGCGAATTT-3'; SEQ ID NO: 22) encoding amino acid residues 6-13 (KFAAAHYN, SEQ ID NO: 23) of the Flt4 ligand, and a sense-strand primer TCACTATAGGGAGACCCAAGC-3,; SEQ ID NO: 24), corresponding to nucleotides 2179-2199 of the pcDNAI vector. The sequences of these sense and antisense primers overlapped with the 3' ends of the corresponding primers used in the first PCR. "Touchdown" PCR was carried out by decreasing the annealing temperature from 72 C to 66 C and continuing with 18 additional cycles at 66'C. The annealing time was 1 minute and extension at each cycle was carried out at 72 C for 2 minutes. One major product of about 220 bp and three minor products of about 270 bp, 150 bp, and 100 bp

-VI

47 were obtained.

The amplified fragment of approximately 220 bp was excised from an agarose gel, cloned into a pCRII vector using the TA cloning kit (Invitrogen), and sequenced. Three recombinant clones were analyzed and they contained the sequence

TCACTATAGGGAGACCCAAGCTTGGTACCGAGCTCGGATCCACTAGTAACGGCCGCC

AGTGTGGTGGAATTCGACGAACTCATGACTGTACTCTACCCAGAATATTGGAAAATG

TACGAAG

CAAAGGCATGGCAACATAACAGAGAACAGGCCAACCTC

AACTCAAGGACAGAAGAGACTATAAAATTCGCTGCAGCACACTACAAC- 3' (SEQ ID NO: 25). The beginning of the sequence represents the pcDNAI vector and the underlined sequence represents the amplified product of the 5'-end of the cDNA insert.

EXAMPLE 9 15 Amplification of the 3'-end of **CDNA encoding the Flt4 ligand Based upon the amplified 5'-sequence of the clones encoding the amino terminus of the 23 kD human Flt4 ligand, two pairs of non-overlapping nested primers 20 were designed to amplify the 3'-portion of the Flt-4ligand-encoding cDNA clones. The sense-strand primer ACAGAGAACAGGCCAACC-3 (SEQ ID NO: 26), corresponding to nucleotides 152-169 of the amplified 5'-sequences of the Flt4 ligand (SEQ ID NO: 25), and antisense-strand primer 25 5'-TCTAGCATTTAGGTGACAC-3' (SEQ ID NO: 27) corresponding to nucleotides 2311-2329 of the pcDNAI vector were used in a first "touchdown" PCR. The annealing temperature of the reaction was decreased 1'C every two cycles from 72"C to 52 C, at which temperature 15 additional cycles were carried out. The annealing time was 1 minute and extension at each cycle was carried out at 72C for 3 minutes. DNA fragments of several sizes were obtained in the first amplification. Those products were diluted 1:200 in water and reamplified in PCR using the second pair of primers: 5'-AAGAGACTATAAAATTCGCTGCAGC-3' (SEQ ID NO: 28) and 5'-CCCTCTAGATGCATGCTCGA-3' (SEQ ID NO: 29) (antisense-strand primer corresponding to nucleotides 48 2279-2298 of the pcDNAI vector). Two DNA fragments were obtained, having sizes of 1350 bp and 570 bp. Those fragments were cloned into a pCRII vector and the inserts of the clones were sequenced. Both of these fragments were found to contain sequences encoding an amino acid sequence homologous to the VEGF sequence.

EXAMPLE Screening the PC-3 cell CDNA library using the PCR fragment of Flt4 ligand cDNA A 219 bp 5'-terminal fragment of human Flt4 ligand cDNA was amplified by PCR using the 5' PCR fragment described above and primers GTTGTAGTGTGCTGCAGCGAATTT-3 (antisense-strand primer,

SEQ

ID NO: 30) and 5'-TCACTATAGGGAGACCCAAGC-3' (SEQ ID NO: 5* 15 31) (sense-primer corresponding to nucleotides 2179-2199 Soo* of the pcDNAI vector). The amplified product was subjected to digestion with EcoRI (Boehringer Mannheim) to remove the portion of the DNA sequence amplified from the pcDNAI vector and the resulting 153 bp fragment 20 encoding the 5' end of the Flt4 ligand was labeled with 32 P]-dCTP using the Klenow fragment of E. coli DNA S' polymerase I (Boehringer Mannheim). That fragment was used as a probe for hybridization screening of the amplified PC-3 cell cDNA library.

Filter replicas of the library were hybridized with the radioactively labeled probe at 42"C for 20 hours in a solution containing 50% formamide, 5x SSPE, Denhardt's solution, 0.1% SDS and 0.1 mg/ml denatured salmon sperm DNA. Filters were washed twice in ix SSC, 0.1% SDS for 30 minutes at room temperature, then twice for 30 minutes at 65*C and exposed overnight.

On the basis of autoradiography, 10 positive recombinant bacterial colonies hybridizing with the probe were chosen from the library. Plasmid DNA was purified from these colonies and analyzed by EcoRI and NotI digestion and agarose gel electrophoresis followed by 49 ethidium bromide staining. The ten plasmid clones were divided into three groups on the basis of the presence of insert sizes of approximately 1.7, 1.9 and 2.1 kb, respectively. Inserts of plasmids from each group were sequenced using the T7 oligonucleotide as a primer and walking primers for subsequent sequencing reactions.

Sequence analysis showed that all clones contain the open reading frame encoding the NH2-terminal sequence of the 23 kD human Flt4 ligand. Dideoxy sequencing was continued using walking primers in the downstream direction. A complete human cDNA sequence and deduced amino acid sequence from a 2 kb clone is set forth in SEQ ID NOs: 32 and 33, respectively. A putative cleavage site of a "prepro" leader sequence is located 15 between residues 102 and 103 of SEQ ID NO: 33. When compared with sequences in the GenBank Database, the predicted protein product of this reading frame was found to be homologous with the predicted amino acid sequences of the PDGF/VEGF family of growth factors, as shown in 20 Fig. Plasmid pFLT4-L, containing the 2.1 kb human cDNA clone in pcDNAI vector, has been deposited with the American Type Culture Collection, 12301 Parklawn Drive, Rockville, MD 20852 as accession number 97231.

25 EXAMPLE 11 .0 Stimulation of Flt4 autophosphorylation by the protein product of the Flt4 ligand vector The 2.1 kb human cDNA insert of plasmid pFlt4- L, which contains the open reading frame encoding the sequence shown in SEQ ID NOs: 32 and 33; human

VEGF-C,

see below), was cut out from the pcDNAI vector using HindIII and NotI restriction enzymes, isolated from a preparative agarose gel, and ligated to the corresponding sites in the pREP7 expression vector (Invitrogen). The pREP7 vector containing the pFlt4-L insert was transfected into 293-EBNA cells (Invitrogen) using.the &i 50 calcium phosphate transfection method (Sambrook et a., 1989). About 48 hours after transfection the medium of the transfected cells was changed to DMEM medium lacking fetal calf serum and incubated for 36 h. The conditioned medium was then collected, centrifuged at 5000 x g for minutes, the supernatant was concentrated 5-fold using Centriprep 10 (Amicon) and used to stimulate NIH 3T3 cells expressing LTRFlt41 (the Flt4 receptor), as in Example 4. The cells were lysed, immunoprecipitated using anti-Flt4 antiserum and analyzed by Western blotting using anti-phosphotyrosine antibodies.

The conditioned medium from two different dishes of the transfected cells stimulated Flt4 autophosphorylation in comparison with the medium from 15 mock-transfected cells, which gave only background levels of phosphorylation of the Flt4 receptor. When the concentrated conditioned medium was pre-absorbed with S..microliters of a slurry of Flt4EC domain coupled to Sepharose (see example no phosphorylation was 20 obtained, showing that the activity responsible for Flt4 autophosphorylation was indeed the Flt4 ligand. Thus these results demonstrate that an expression vector having an approximately 2.1 kb insert and containing an open reading frame as shown in SEQ ID NO: 32 is expressed as a biologically active Flt4 ligand (VEGF-C) in transfected cells. The sequence encoded by that open reading frame is shown in SEQ ID NO: 33.

The deduced molecular weight of a polypeptide consisting of the complete amino acid sequence in SEQ ID NO: 33 (residues 1 to 419) is 46,883. The deduced molecular weight of a polypeptide consisting of amino acid residues 103 to 419 of SEQ ID NO: 33 is 35,881. The Flt4 ligand purified from PC-3 cultures had an observed molecular weight of about 23 kD as assessed by SDS-PAGE under reducing conditions. Thus, it appears that the Flt4 ligand mRNA is translated into a precursor polypeptide, from which the mature ligand is derived by 51 proteolytic cleavage. Also, the Flt4 ligand may be glycosylated at three putative N-linked glycosylation sites conforming to the consensus which can be identified in the deduced Flt4 ligand amino acid sequence

(N-

residues underlined in Fig. The carboxyl terminal amino acid sequences, which increase the predicted molecular weight of the Flt4 ligand subunit in comparison with other ligands of this family, show a pattern of spacing of cysteine residues reminiscent of the Balbiani ring 3 protein (BR3P) sequence (Dignam et al., Gene, 88:133-140 (1990)), as depicted schematically in Fig. 9. Such a sequence may encode an independently folded domain present in a Flt4 ligand precursor and it may be involved, for example, in 15 the regulation of secretion, solubility, stability, cell surface localization or activity of the Flt4 ligand.

S. Interestingly, at least one cysteine motif of the BR3P type is also found in the VEGF carboxy terminal amino acid sequences.

20 Thus, the Flt4 ligand mRNA appears first to be translated into a precursor from the mRNA corresponding to the cDNA insert of plasmid FLT4-L, from which the mature ligand is derived by proteolytic cleavage. To define the mature Flt4 ligand polypeptide, one first expresses the cDNA clone (which is deposited in the pcDNAI expression vector) in cells, such as COS cells.

One uses antibodies generated against encoded polypeptides, fragments thereof, or bacterial Flt4 fusion proteins, such as a GST-fusion protein, to raise antibodies against the VEGF-homologous domain and the amino- and carboxyl-terminal propeptides of Flt4 ligand.

One then follows the biosynthesis and processing of the Flt4 ligand in the transfected cells by pulse-chase analysis using radioactive cysteine for labelling of the cells, immunoprecipitation and gel electrophoresis.

Using antibodies against the three domains of the product encoded by the cDNA insert of plasmid FLT4-L, material

S

52 for radioactive or nonradioactive amino-terminal sequence analysis is isolated. The determination of the aminoterminal sequence of the mature VEGF-C polypeptide allows for identification of the amino-terminal proteolytic processing site. The determination of the amino-terminal sequence of the carboxyl-terminal propeptide will give the carboxyl-terminal processing site. This is confirmed by site-directed mutagenesis of the amino acid residues adjacent to the cleavage sites, which would prevent the cleavage.

The Flt4 ligand is further characterizeable by progressive 3' deletions in the 3' coding sequences of the Flt4 ligand precursor clone, introducing a stop codon resulting in carboxy-terminal truncations of its protein 15 product. The activities of such truncated forms are assayed by, for example, studying Flt4 autophosphorylation induced by the truncated proteins when applied to cultures of cells, such as NIH 3T3 cells expressing LTRFlt4. By extrapolation from studies of the structure of the related platelet derived growth factor (PDGF, Heldin et al., Growth Factors, 8:245-252 (1993)) one determines that the region critical for receptor activation by the Flt4 ligand is contained within the first approximately 180 amino acid residues of the secreted VEGF-C protein lacking the putative 102 amino acid prepro leader (SEQ ID NO: 33, residues 103-282), and apparently within the first approximately 120 amino acid residues (SEQ ID NO: 33, residues 103-223).

On the other hand, the difference between the molecular weights observed for the purified ligand and deduced from the open reading frame of the Flt4 ligand clone may be due to the fact that the soluble ligand was produced from an alternatively spliced mRNA which would also be present in the PC-3 cells, from which the isolated ligand was derived. To isolate such alternative cDNA clones one uses cDNA fragments of the deposited clone and PCR primers made according to the sequence 53 provided as well as techniques standard in the art to isolate or amplify alternative cDNAs from the PC-3 cell cDNA library. One may also amplify using reverse transcription (RT)-PCR directly from the PC-3 mRNA using the primers provided in the sequence of the cDNA insert of plasmid FLT4-L. Alternative cDNA sequences are determined from the resulting cDNA clones. One can also isolate genomic clones corresponding to the Flt4 ligand mRNA transcript from a human genomic DNA library using methods standard in the art and to sequence such clones or their subcloned fragments to reveal the corresponding exons. Alternative exons can then be identified by a number of methods standard in the art, such as heteroduplex analysis of cDNA and genomic DNA, which are 15 subsequently characterized.

EXAMPLE 12 Expression of the Gene Encoding VEGF-C in Human Tumor Cell Lines Expression of transcripts corresponding to the 20 Flt4 ligand (VEGF-C) was analyzed by hybridization of Northern blots containing isolated poly(A) RNA from HT- 1080 and PC-3 human tumor cell lines. The probe was the radioactively labelled insert of the 2.1 kb cDNA clone (pFlt4-L/VEGF-C, specific activity 108-109 cpm/mg of DNA).

The blot was hybridized overnight at 42 C using formamide, 5x SSPE buffer, 2% SDS, 10 x Denhardt's solution, 100 mg/ml salmon sperm DNA and 1 x 106 cpm of the labelled probe/ml. The blot was washed at room temperature for 2 x 30 minutes in 2x SSC containing 0.05% SDS, and then for 2 x 20 minutes at 52"C in 0.1x SSC containing 0.1% SDS. The blot was then exposed at -70 C for three days using intensifying screens and Kodak XAR film. Both cell lines expressed an Flt4 ligand mRNA of about 2.4 kb, as well as VEGF and VEGF-B mRNAs (Fig. 12).

54 EXAMPLE 13 VEGF-C Chains Are Proteolytically Processed after Biosynthesis and Disulfide Linked The predicted molecular mass of a secreted human VEGF-C polypeptide, as deduced from the VEGF-C open reading frame, is 46,883 kD, suggesting that VEGF-C mRNA may be first translated into a precursor, from which the ligands of 21/23 kD and 29/32 kD are derived by proteolytic cleavage.

This possibility was explored by metabolic labelling of 293 EBNA cells expressing

VEGF-C.

Initially, 293 EBNA cells were transfected with the VEGF-C construct. Expression products were labeled by the addition of 100 pCi/ml of Pro-mix" L-[3 5 S] in vitro 15 cell labelling mix ((containing 3S-methionine and cysteine) Amersham, Buckinghamshire, England) to the culture medium devoid of cysteine and methionine. After two hours, the cell layers were washed twice with PBS and the medium was then replaced with DMEM-0.2% BSA. After 20 1, 3, 6, 12 and 24 hours of subsequent incubation, the culture medium was collected, clarified by centrifugation, and concentrated, and human VEGF-C was bound to 30 Il of a slurry of Flt4EC-Sepharose overnight at +4 C, followed by three washes in PBS, two washes in mM Tris-HC1 (pH alkylation, SDS-PAGE and autoradiography. Alkylation was carried out by treatment of the samples with 10mM 1,4 Dithiothreitol (Boehringer- Mannheim, Mannheim, Germany) for one hour at 25 C, and subsequently with 30 mM iodoacetamide (Fluka, Buchs, Switzerland).

These experiments demonstrated that a putative precursor polypeptide of 32 kD apparent molecular mass was bound to the Flt4EC affinity matrix from the conditioned medium of metabolically labelled cells transfected with the human VEGF-C expression vector (Fig.

13A), but not from mock transfected cells. Increased amounts of a 23 kD receptor binding polypeptide

J

55 accumulated in the culture medium of VEGF-C transfected cells during a subsequent chase period of three hours, but not thereafter (lanes 2-4 and data not shown), suggesting that the 23 kD form is produced by proteolytic processing, which is incomplete, at least in the transiently transfected cells. The arrows in Fig. 13A indicate the 32 kD and 23 kD polypeptides of secreted VEGF-C. Subsequent experiments showed that the 32kD VEGF-C form contains two components migrating in the absence of alkylation as polypeptides of 29 and 32 kD (Figs. 21-23).

In a related experiment, human VEGF-C isolated using Flt4EC-Sepharose after a 4 h continuous metabolic labelling was analyzed by polyacrylamide gel 15 electrophoresis in nonreducing conditions (Fig. 13B).

Higher molecular mass forms were observed under nonreducing conditions, suggesting that the VEGF-C polypeptides can form disulfide-linked dimers and/or multimers (arrows in Fig. 13B).

20 EXAMPLE 14 Stimulation Of VEGFR-2 Autophosphorylation By VEGF-C Conditioned medium (CM) from 293 EBNA cells .transfected with the human VEGF-C vector also was used to stimulate porcine aortic endothelial (PAE) cells 25 expressing VEGFR-2 (Kdr). Pajusola et al., Oncogene, 9:3545-55 (1994); Waltenberger et al., J. Biol. Chem., 269:26988-26995 (1994). The cells were lysed and immunoprecipitated using VEGFR-2 specific antiserum (Waltenberger et al., 1994).

PAE-KDR cells (Waltenberger et al., 1994) were grown in Ham's F12 medium-10% fetal calf serum (FCS).

Confluent NIH 3T3-Flt4 cells or PAE-KDR cells were starved overnight in DMEM or Ham's F12 medium, respectively, supplemented with 0.2% bovine serum albumin (BSA), and then incubated for 5 minutes with the analyzed media. Recombinant human VEGF (R&D Systems) and PDGF-BB, 56 O functional as stimulating agents, were used as controls.

The cells were washed twice with ice-cold Tris-Buffered Saline (TBS) containing 100 mM sodium orthovanadate and lysed in RIPA buffer containing 1 mM phenylmethylsulfonyl fluoride,(PMSF), 0.1 U/ml aprotinin and 1 mM sodium orthovanadate. The lysates were sonicated, clarified by centrifugation at 16,000 x g for 20 minutes and incubated for 3-6 hours on ice with 3-5 gl of antisera specific for Flt4 (Pajusola et al., 1993), VEGFR-2 or PDGFR-f (Claesson-Welsh et al., J. Biol. Chem., 264:1742-1747 (1989); Waltenberger et al., 1994). Immunoprecipitates were bound to protein A-Sepharose, washed three times with RIPA buffer containing imM PMSF, imM sodium orthovanadate, washed twice with 10 mM Tris-HC1 (pH 7.4), 15 and subjected to SDS-PAGE using a 7% gel. Polypeptides were transferred to nitrocellulose by Western blotting and analyzed using PY20 phosphotyrosine-specific monoclonal antibodies (Transduction Laboratories) or receptor-specific antiserum and the ECL detection method (Amersham Corp.).

*The results of the experiment are presented in Figs. 14A and 14B. As shown in Fig. 14A, PAE cells expressing VEGFR-2 were stimulated with 10- or 2-fold concentrated medium from mock-transfected 293-EBNA cells (lanes 1 and or with 5- or 10-fold concentrated medium from 293-EBNA cell cultures expressing the recombinant VEGF-C (lanes VEGFR-2 was immunoprecipitated with specific antibodies and analyzed by SDS-PAGE and Western blotting using phosphotyrosine antibodies. For comparison, the stimulations were carried out with non-conditioned medium containing ng/ml of purified recombinant VEGF (lanes 7 and 8).

Lanes 6 and 7 show stimulation with VEGF-C- or VEGFcontaining media pretreated with Flt4EC. As depicted in Fig. 14B, PDGFR-f-expressing NIH 3T3 cells were stimulated with non-conditioned medium (lane concentrated CM from mock-transfected (lane 2) or VEGF-C 57 transfected (lanes 3 and 4) cells, or with non-conditioned medium containing 50 ng/ml of recombinant human PDGF-BB (lane Medium containing VEGF-C was also pretreated with recombinant Flt4EC (lane

PDGFR-

B was immunoprecipitated with specific antibodies and analyzed by SDS-PAGE and Western blotting using phosphotyrosine antibodies with subsequent stripping and reprobing of the membrane with antibodies specific for

PDGFR-.

Referring again to Fig. 14A, a basal level of tyrosine phosphorylation of VEGFR-2 was detected in cells stimulated by CM from the mock-transfected cells.

A

further concentration of this medium resulted in only a slight enhancement of VEGFR-2 phosphorylation (lanes 1 15 and CM containing recombinant VEGF-C stimulated tyrosine autophosphorylation of VEGFR-2 and the intensity of the autophosphorylated polypeptide band was increased upon concentration of the VEGF-C CM (lanes Furthermore, the stimulating effect was abolished after .9 20 pretreatment of the medium with the Flt4EC affinity matrix (compare lanes 1, 5 and The maximal effect of VEGF-C in this assay was comparable to the effect of recombinant VEGF added to unconditioned medium at 9 concentration of 50 ng/ml (lane Pretreatment of the medium containing VEGF with Flt4EC did not abolish its stimulating effect on VEGFR-2 (compare lanes 7 and 8).

These results suggest that the VEGF-C expression vector encodes a ligand not only for Flt4 (VEGFR-3), but also for VEGFR-2 (Kdr).

In order to further confirm that the stimulating effect of VEGF-C on tyrosine phosphorylation of VEGFR-3 and VEGFR-2 was receptor-specific, we analyzed the effect of VEGF-C on tyrosine phosphorylation of PDGF receptor f (PDGFR-f) which is abundantly expressed on fibroblastic cells. As can be seen from Fig. 14B, a weak tyrosine phosphorylation of PDGFR-3 was detected upon stimulation of Flt4-expressing NIH 3T3 cells with CM from 58 the mock-transfected cells (compare lanes 1 and

A

similar low level of PDGFR-f phosphorylation was observed when the cells were incubated with CM from the VEGF-C transfected cells, with or without prior treatment with Flt4EC (lanes 3 and In contrast, the addition of ng/ml of PDGF-BB induced a prominent tyrosine autophosphorylation of PDGFR-0 (lane EXAMPLE 1 VEGF-C Stimulates Endothelial Cell Migration In Collagen Gel Conditioned media (CM) from cell cultures transfected with the VEGF-C expression vector was placed in a well made in collagen gel and used to stimulate the migration of bovine capillary endothelial (BCE) cells in 15 the three-dimensional collagen gel as follows.

BCE cells (Folkman et al., Proc. Natl. Acad.

Sci. (USA), 76:5217-5221 (1979) were cultured as described in Pertovaara et al., J. Biol. Chem., 269:6271- 74 (1994). The collagen gels were prepared by mixing 20 type I collagen stock solution (5 mg/ml in 1 mM HC1) with an equal volume of 2x MEM and 2 volumes of MEM containing 10% newborn calf serum to give a final collagen concentration of 1.25 mg/ml. The tissue culture plates cm diameter) were coated with about 1 mm thick layer 25 of the solution, which was allowed to polymerize at 37 C.

BCE cells were seeded on top of this layer. For the migration assays, the cells were allowed to attach inside a plastic ring (1 cm diameter) placed on top of the first collagen layer. After 30 minutes, the ring was removed and unattached cells were rinsed away. A second layer of collagen and a layer of growth medium newborn calf serum solidified by 0.75% low melting point agar (FMC BioProducts, Rockland, ME), were added. A well (3 mm diameter) was punched through all the layers on both sides of the cell spot at a distance of 4 mm, and the sample or control media were pipetted daily into the 59 wells. Photomicrographs of the cells migrating out from the spot edge were taken after six days through an Olympus CK 2 inverted microscope equipped with phase-contrast optics. The migrating cells were counted after nuclear staining with the fluorescent dye bisbenzimide (1 mg/ml, Hoechst 33258, Sigma).

Fig. 15 depicts a comparison of the number of cells migrating at different distances from the original area of attachment towards wells containing media conditioned by the non-transfected (control) or transfected (mock; VEGF-C; VEGF) cells, 6 days after addition of the media. The number of cells migrating out from the original ring of attachment was counted in five adjacent 0.5 mm x 0.5 mm squares using a microscope 15 ocular lens grid and 10x magnification with a 'fluorescence microscope. Cells migrating further than 0.5 mm were counted in a similar way by moving the grid in 0.5 mm steps. The experiments were carried out twice with similar results, and medium values from the one of 20 the experiments are presented with standard error bars.

As can be seen from the columns, VEGF-C-containing

CM

stimulated cell migration more than medium conditioned by the non-transfected or mock-transfected cells but less than medium from cells transfected with a VEGF expression vector. Daily addition of 1 ng of FGF2 into the wells resulted in the migration of approximately twice the number of cells when compared to the stimulation by CM from VEGF-transfected cells.

EXAMPLE 16 VEGF-C Is Expressed In Multiple Tissues Northern blots containing 2 micrograms of isolated poly(A) RNA from multiple human tissues (blot from Clontech Laboratories, Inc., Palo Alto, CA) were probed with radioactively labelled insert of the 2.1 kb VEGF-C cDNA clone. Northern blotting and hybridization analysis showed that the 2.4 kb RNA and smaller amounts 60 of a 2.0 kb mRNA are expressed in multiple human tissues most prominently in the heart, placenta, muscle, ovary and small intestine (Fig. 16A). Very little VEGF-C

RNA

was seen in the brain, liver or thymus and peripheral blood leukocytes (PBL) appeared negative. A similar analysis of RNA from human fetal tissues (Fig. 16B) shows that VEGF-C is highly expressed in the kidney and lung and to a lesser degree in the liver, while essentially no expression is detected in the brain. Interestingly,

VEGF

expression correlates with VEGF-C expression in these tissues, whereas VEGF-B is highly expressed in all tissues analyzed.

EXAMPLE 17 The VEGF-C Gene Localizes To Chromosome 4q34 15 A DNA panel of 24 interspecies somatic cell hybrids, which had retained one or two human chromosomes, was used for the chromosomal localization of the VEGF-C gene (Bios Laboratories, Inc., New Haven, CT). Primers were designed to amplify an about 250 bp fragment of the 20 VEGF-C gene from somatic cell hybrid DNA. The primers and conditions for polymerase chain reaction (PCR) were 5'-TGAGTGATTTGTAGCTGCTGTG-3, (forward) [SEQ ID NO: 34] and 5'-TATTGCAGCAACCCCCACATCT-3, (reverse) [SEQ ID NO: for VEGF-C (94"C, 60s/62"C, 45s/72"C, 60s). The PCR 25 products were evaluated by electrophoresis in 1% agarose gels and visualized by ethidium bromide staining in ultraviolet light. [a- 32 P]-dCTP- labelled cDNA inserts of a plasmid representing the complete VEGF-C coding domain was used as a probe in Southern blotting and hybridization analysis of the somatic cell hybrid DNAs as instructed by the supplier (Bios Laboratories).

The cell lines for fluorescence in situ hybridization (FISH) were obtained from the American Type Culture Collection (Rockville, MD). Purified DNA from P1 clones 7660 and 7661 (VEGF-C) (Genome Systems, Inc., St.

Louis, MO) were confirmed positive by Southern blotting 61 of EcoRI- digested DNA followed by hybridization with the VEGF-C cDNA. The P1 clones were then labelled by nick translation either with biotin-ll-dUTP, biotin-14-ATP (Sigma Chemical Co., St. Louis, MO) or digoxigenin 11-dUTP (Boehringer Mannheim GmbH, Mannheim, Germany) according to standard protocols. PHA-stimulated peripheral blood lymphocyte cultures were treated with (BrdU) at an early replicating phase to induce G-banding. See Takahashi et al., Human Genet., 86:14-16 (1995); Lemieux et al., Cytogenet. Cell Genet., 59:311-12 (1992). The FISH procedure was carried out in formamide, 10% dextran sulphate in 2x SSC using wellknown procedures. See Rytk6nnen et al., Cytogenet Cell Genet., 68:61-63 (1995); Lichter et al., Proc. Natl.

15 Acad. Sci. (USA), 85:9664-68 (1988). Repetitive sequences were suppressed with 50-fold excess of Cot-1 DNA (BRL, Gaithersburg, MD) compared with the labeled probe. Specific hybridization signals were detected by incubating the hybridized slides in labelled 20 antidigoxigenin antibodies, followed by counterstaining with 0.lmmol/L 4 6 -diamino-2-phenylindole. Probe detection for two-color experiments was accomplished by incubating the slides in fluorescein isothiocyanate (FITC)-anti-digoxigenin antibodies (Sigma Chemical Co.) and Texas red-avidin (Vector Laboratories, Burlingame, CA) or rhodamine-anti-digoxigenin and FITC-avidin.

Multi-color digital image analysis was used for acquisition, display and quantification of hybridization signals of metaphase chromosomes. The system contains a PXL camera (Photometrics Inc., Tucson, AZ) attached to a PowerMac 7100/Av workstation. IPLab software controls the camera operation, image acquisition and Ludl Filter wheel. At least 50 nuclei were scored. Overlapping nuclei and clusters of cells were ignored. A slide containing normal lymphocyte metaphase spreads and interphase nuclei was included in each experiment to control for the efficiency and specificity of the -62 hybridization.

0 In order to determine the chromosomal localization of the human VEGF-C gene, DNAs from human rodent somatic cell hybrids containing defined sets of human chromosomes were analyzed by Southern blotting and hybridization with the VEGF-C cDNA probe. Among 24 DNA samples on the hybrid panel, representing different human chromosomes, human-specific signals were observed only in hybrids which contained human chromosome 4. The results were confirmed by PCR of somatic cell hybrid DNAs using VEGF-C specific primers, where amplified bands were obtained only from DNAs containing human chromosome 4.

A genomic P1 plasmid for VEGF-C was isolated .using specific primers and PCR and verified by Southern 15 blotting and hybridization using a VEGF-C specific cDNA probe. The chromosomal localization of VEGF-C was further studied using metaphase FISH. Using the Pl probe for VEGF-C in FISH a specific hybridization to the 4q34 chromosomal band was detected in 40 out of 44 metaphases.

20 Double-fluorochrome hybridization using a cosmid probe specific for the aspartylglucosaminidase (AGA) gene showed that VEGF-C is located just proximal to the AGA gene previously mapped to the 4q34-35 chromosomal band.

Biotin labelled VEGF-C P1 and digoxigenin 25 labeled AGA cosmid probes were hybridized simultaneously Sto metaphase chromosomes. This experiment demonstrated that the AGA gene is more telomerically located than the VEGF-C gene. The foregoing example demonstrates the utility of polynucleotides of the invention as chromosomal markers and for the presence or absence of the VEGF-C gene region in normal or diseased cells. The VEGF-C locus at 4q34 is a candidate target for mutations leading to vascular malformations or cardiovascular diseases.

63 SEXAMPLE 18 Effect of glucose concentration and hypoxia on VEGF, VEGF-B and VEGF-C mRNA levels in C6 glioblastoma cells Confluent cultures of C6 cells (ATCC CCL 107) were grown on 10 cm diameter tissue culture plates containing 2.5 ml of DMEM and 5% fetal calf serum plus antibiotics. The cultures were exposed for 16 hours to normoxia in a normal cell culture incubator containing 5% CO 2 or hypoxia by closing the culture plates in an airtight glass chamber and burning a piece of wood inside until the flame was extinguished due to lack of oxygen.

Polyadenylated RNA was isolated (as in the other examples), and 8 micrograms of the RNA was 15 electrophoresed and blot-hybridized with a mixture of the VEGF, VEGF-B and VEGF-C probes (see Fig. 12). The results show that hypoxia strongly induces VEGF mRNA oo. expression, both in low and high glucose, but has no significant effect on the VEGF-B mRNA levels. The VEGF-C mRNA isolated from hypoxic cells runs slightly faster in gel electrophoresis and an extra band of faster mobility can be seen below the upper mRNA band. This observation suggests that hypoxia affects VEGF-C RNA processing. One explanation for this observation is that VEGF-C mRNA 25 splicing is altered, affecting the VEGF-C open reading frame and resulting in an alternative VEGF-C protein being produced by hypoxic cells. Such alternative forms of VEGF-C and VEGF-C-encoding polynucleotides are contemplated as an aspect of the invention. This data indicates screening and diagnostic utilities for polynucleotides and polypeptides of the invention, such as methods whereby a biological sample is screened for the hypoxia-induced form of VEGF-C and/or VEGF-C mRNA.

The data further suggests a therapeutic indication for antibodies and/or other inhibitors of the hypoxia-induced form of VEGF-C or the normal form of VEGF-C.

64 EXAMPLE 19 Pulse-chase labeling and immunoprecipitation of VEGF-C polypeptides from 293 EBNA cells transfected with VEGF-C expression vector.

The following VEGF-C branched amino-terminal peptide, designated PAM126, was synthesized for production of anti-VEGF-C antiserum:

NH

2 -E-E-T-I-K-F-A-A-A-H-Y-N-T-E-I-L-K-COOH (SEQ ID NO: 39).

In particular, PAM126 was synthesized as a branched polylysine structure K3PA4 having four peptide acid (PA) chains attached to two available lysine residues.

The synthesis was performed on a 433A Peptide Synthesizer (Applied Biosystems) using Fmoc-chemistry and TentaGel

S

15 MAP RAM10 resin mix (RAPP Polymere GmbH, Tubingen, Germany), yielding both cleavable and resin-bound eptides. The cleavable peptide was purified via reverse phase HPLC and was used together with the resin-bound peptide in immunizations. The correctness of the 20 synthesis products were confirmed using mass-spectroscopy (Lasermatt).

The PAM126 peptide was dissolved in phosphate buffered saline (PBS), mixed with Freund's adjuvant, and used for immunization of rabbits at bi-weekly intervals using methods standard in the art (Harlow and Lane, Antibodies, a laboratory manual, Cold Spring Harbor Laboratory Press (1988)). Antisera obtained after the fourth booster immunization was used for immunoprecipitation of VEGF-C in pulse-chase experiments, as described below.

For pulse-chase analysis, 293 EBNA cells transfected with a VEGF-C expression vector the FLT4-L cDNA inserted into the pREP7 expression vector as described above) were incubated for 30 minutes in methionine-free, cysteine-free, serum-free DMEM culture medium at 37'C. The medium was then changed, and 200 ACi of Pro-mix" (Amersham), was added. The cell layers were incubated in this labeling medium for two hours, washed a 65 with PBS, and incubated for 0, 15, 30, 60, 90, 120, or 180 minutes in serum-free DMEM (chase). After the various chase periods, the medium was collected, the cells were again washed two times in PBS, and lysed in immunoprecipitation buffer. The VEGF-C polypeptides were analyzed from both the culture medium and from the cell lysates by immunoprecipitation, using the VEGF-C-specific antiserum raised against the NH 2 -terminal peptide (PAM126) of the 23 kD VEGF-C form. Immunoprecipitated polypeptides were analyzed via SDS-PAGE followed by autoradiography.

Referring to Fig. 19, the resultant :autoradiograms demonstrate that immediately after a 2 hour labeling (chase time the VEGF-C vector- V 15 transfected cells contained a radioactive 55 kD polypeptide band, which is not seen in mock-transfected cells This 55 kD polypeptide band gradually diminishes in intensity with increasing chase periods, and is no longer detected in the cells by 180 minutes of 20 chase. A 32 kD polypeptide band also is observed in VEGF-C transfected cells (and not mock-transfected cells). This 32 kD band disappears with similar kinetics to that of the 55 kD band. Simultaneously, increasing amounts of 32 kD (arrow) and subsequently 23 kD (arrow) 25 and 14 kD polypeptides appear in the medium.

Collectively, the data from the pulse-chase experiments indicate that the 55 kD intracellular polypeptide represents a pro-VEGF-C polypeptide, which is not secreted from cells, but rather is first proteolytically cleaved into the 32 kD form. The 32 kD form is secreted and simultaneously further processed by proteolysis into the 23 kD and 14 kD forms. Without intending to be limited to a particular theory, it is believed that processing of the VEGF-C precursor occurs as removal of a signal sequence, removal of the COOH-terminal domain (BR3P), and removal of an amino terminal polypeptide, resulting in a VEGF-C polypeptide

A

66 having the TEE... amino terminus.

At high resolution, the 23 kD polypeptide band appears as a closely-spaced polypeptide doublet, suggesting heterogeneity in cleavage or glycosylation.

EXAMPLE Isolation of Mouse and Quail cDNA Clones Encoding

VEGF-C

To clone a mouse variant of VEGF-C, approximately 1 x 106 bacteriophage lambda clones of a commercially-available 12 day mouse embryonal cDNA library (lambda EXlox library, Novagen, catalog number 69632-1) were screened with a radiolabeled fragment of human VEGF-C cDNA containing nucleotides 495 to 1661 of SEQ ID NO: 32. One positive clone was isolated.

15 A 1323 bp EcoRI/HindIII fragment of the insert of the isolated mouse cDNA clone was subcloned into the corresponding sites of the pBluescript SK+ vector (Stratagene) and sequenced. The cDNA sequence of this clone was homologous to the human VEGF-C sequence reported herein, except that about 710 bp of sequence present in the human clone was not present in the mouse clone.

For further screening of mouse cDNA libraries, a HindIII-BstXI (HindIII site is from the pBluescript

SK+

S. 25 polylinker) fragment of 881 bp from the coding region of *j the mouse cDNA clone was radiolabeled and used as a probe to screen two additional mouse cDNA libraries. Two additional cDNA clones from an adult mouse heart ZAP II cDNA library (Stratagene, catalog number 936306) were identified. Three additional clones also were isolated from a mouse heart 5'-stretch-plus cDNA library in Xgtll (Clontech Laboratories, Inc., catalog number ML5002b).

Of the latter three clones, one was found to contain an insert of about 1.9 kb. The insert of this cDNA clone was subcloned into EcoRI sites of pBluescript SK+ vector and both strands of this clone were completely sequenced, resulting in the nucleotide and deduced amino acid.

a 67 sequences shown in SEQ ID NOs: 40 and 41.

It is contemplated that the polypeptide corresponding to SEQ ID NO: 41 is processed into a mature mouse VEGF-C protein, in a manner analogous to the processing of the human VEGF-C prepropeptide. Putative cleavage sites for the mouse protein are identified using procedures outlined above for identification of cleavage sites for the human VEGF-C polypeptide.

The foregoing results demonstrate the utility of polynucleotides of the invention for identifying and isolating polynucleotides encoding other non-human mammalian variants of VEGF-C. Such identified and isolated polynucleotides, in turn, can be expressed (using procedures similar to those described in preceding 15 examples) to produce recombinant polypeptides corresponding to non-human mammalian variants of VEGF-C.

The mouse and human VEGF-C sequences were used to design probes for isolating a quail VEGF-C cDNA from a quail cDNA library. A fragment of the human VEGF-C cDNA comprising nucleotides 495-1670 of SEQ ID NO: 32 was obtained by PCR amplification, cloned into the pCRII vector (Invitrogen) according to the manufacturer's instructions, and amplified. The insert was isolated by Eco RI digestion and preparative gel electrophoresis and 25 then labelled using radioactive dCTP and random priming.

A cDNA library made from quail embryos of stage E-4 in pcDNA-1 vector (Invitrogen) was then screened using this probe. About 200,000 colonies were plated and filter replicas were hybridized with the radioactive probe.

Nine positive clones were identified and secondarily plated. Two of the nine clones hybridized in secondary screening. The purified clones (clones 1 and 14) had approximately 2.7 kb Eco RI inserts. Both clones were amplified and then sequenced using the T7 and SP6 primers (annealing to the vector). In addition, an internal Sph I restriction endonuclease cleavage site was identified 68 about 1.9 kb from the T7 primer side of the vector and used for subcloning and Sph I fragments, followed by sequencing from the Sph I end of the subclones. The sequences obtained were identical from both clones and showed a. high degree of similarity to the human VEGF-C coding region. Subsequently, walking primers were made in both directions and double-stranded sequencing was completed for 1743 base pairs, including the full-length open reading frame.

The cDNA sequence obtained includes a long open reading frame and 5' untranslated region. The DNA and deduced amino acid sequences for the quail cDNA are set forth in SEQ ID NOs: 52 and 53, respectively. As shown Sin Fig. 8, the human, murine, and avian (quail) VEGF-C 15 precursor amino acid sequences share a significant degree ee" of conservation. This high degree of homology permits the isolation of VEGF-C encoding sequences from other species, especially vertebrate species, and more particularly mammalian and avian species, using polynucleotides of the present invention as probes and using standard molecular biological techniques such as those described herein.

EXAMPLE 21 N-terminal peptide sequence analyses of recombinant

VEGF-C

"o *Cells (293 EBNA) transfected with VEGF-C cDNA (see Example 13) secrete several forms of recombinant VEGF-C (Fig. 21A, lane IP). In the absence of alkylation, the three major, proteolytically-processed forms of VEGF-C migrate in SDS-PAGE as proteins with apparent molecular masses of 32/29 kD (doublet), 21 kD and 15 kD. Two minor polypeptides exhibit approximate molecular masses of 63 and 52 kD, respectively. One of these polypeptides is presumably a glycosylated and nonprocessed form; the other polypeptide is presumably glycosylated and partially processed.

To determine sites of proteolytic cleavage of 69 the VEGF-C precursor, an immunoaffinity column was used to purify VEGF-C polypeptides from the conditioned medium of 293 EBNA cells transfected with VEGF-C cDNA. To prepare the immunoaffinity column, a rabbit was immunized with a synthetic peptide corresponding to amino acids 104-120 of SEQ ID NO: 33: H 2 N-EETIKFAAAHYNTEILK (see PAM126 in Example 19). The IgG fraction was isolated from the serum of the immunized rabbit using protein A Sepharose (Pharmacia). The isolated IgG fraction was covalently bound to CNBr-activated Sepharose CL-4B (Pharmacia) using standard techniques at a concentration of 5 mg IgG/ml of Sepharose. This immunoaffinity matrix was used to isolate processed VEGF-C from 1.2 liters of the conditioned medium (CM).

The purified material eluted from the column was analyzed by gel electrophoresis and Western blotting.

Fractions containing VEGF-C polypeptides were combined, dialyzed against 10 mM Tris HC1, vacuum-dried, electrotransferred to Immobilon-P (polyvinylidene S 20 difluoride or PVDF) transfer membrane (Millipore, Marlborough, MA) and subjected to N-terminal amino acid sequence analysis.

The polypeptide band of 32 kD yielded two distinct sequences:

NH

2 -FESGLDLSDA... and NH 2 25 AVVMTQTPAS... (SEQ ID NO: 51), the former corresponding to the N-terminal part of VEGF-C after cleavage of the signal peptide, starting from amino acid 32 (SEQ ID NO: 33), and the latter corresponding to the kappa-chain of IgG, which was present in the purified material due to "leakage" of the affinity matrix during the elution procedure.

In order to obtain the N-terminal peptide sequence of the 29 kD form of VEGF-C, a construct (VEGF-C NHis) encoding a VEGF-C variant was generated. In particular, the construct encoded a VEGF-C variant that fused a 6xHis tag to the N-terminus of the secreted precursor between amino acids 31 and 33 in SEQ ID 70 NO: 33). The phenylalanine at position 32 was removed to prevent possible cleavage of the tag sequence during secretion of VEGF-C. The VEGF-C NHis construct was cloned into pREP7 as a vector; the construction is described more fully in Example 28, below.

The calcium phosphate co-precipitation technique was used to transfect VEGF-C NHis into 293 EBNA cells. Cells were incubated in DMEM/10% fetal calf serum in 15 cm cell culture dishes (a total of 25 plates). The following day, the cells were reseeded into fresh culture dishes (75 plates) containing the same medium and incubated for 48 hours. Cell layers were then washed once with PBS and DMEM medium lacking FCS was added.

Cells were incubated in this medium for 48 hours and the 15 medium was collected, cleared by centrifugation at 5000 x and concentrated 500X using an Ultrasette Tangential Flow Device (Filtron, Northborough, MA), as described in Example 5 above. VEGF-C NHis was purified from the concentrated conditioned medium using TALON" Metal 20 Affinity Resin (Clontech Laboratories, Inc.) and the *""manufacturer's protocol for native protein purification using imidazole-containing buffers. The protein was eluted with a solution containing 20 mM Tris-HCl (pH 100 mM NaC1, and 200 mM imidazole. The eluted fractions containing purified VEGF-C NHis were detected by immunoblotting with Antiserum 882 (antiserum from rabbit 882, immunized with the PAM-126 polypeptide).

Fractions containing VEGF-C NHis were combined, dialyzed and vacuum-dried. As can be seen in Fig. 27, due to the presence of the 6xHis tag at the N-terminus of this form of VEGF-C, the upper component of the major doublet of the VEGF-CNHis migrates slightly slower than the 32 kD form of wild type VEGF-C, thereby improving the separation of the VEGF-CNHis 32 kD variant from the 29 kD band using SDS-PAGE. Approximately 15 Mg of the purified VEGF-C were subjected to SDS-PAGE under reducing conditions, electrotransferred to Immobilon-P

(PVDF)

71 transfer membrane (Millipore, Inc., Marlborough, MA) and the band at 29 kD was subjected to N-terminal amino acid sequence analysis. This sequence analysis revealed an Nterminal sequence of H 2 N-SLPAT corresponding to amino acids 228-232 of VEGF-C (SEQ ID NO: 33).

The polypeptide band of 21 kD yielded the sequence

H

2 N-AHYNTEILKS corresponding to an amino-terminus starting at amino acid 112 of SEQ ID NO: 33. Thus, the proteolytic processing site which results in the 21 kD form of VEGF-C produced by transfected 293 EBNA cells apparently occurs nine amino acid residues downstream of the cleavage site which results in the 23 kD form of VEGF-C secreted by PC-3 cells.

S" The N-terminus of the 15 kD form was identical 15 to the N-terminus of the 32 kD form (NH 2

-FESGLDLSDA...).

The 15 kD form was not detected when recombinant

VEGF-C

was produced by COS cells. This suggests that production of this form is cell lineage specific.

Example 22 Dimeric and monomeric forms of VEGF-C The composition of VEGF-C dimers was analyzed as follows. Cells (293 EBNA cells), transfected with the pREP7 VEGF-C vector as described in Example 11, were metabolically labelled with Pro-mix L-[ 3 5 S] labelling mix 25 (Amersham Corp.) to a final concentration of 100 pCi/ml.

In parallel, a VEGF-C mutant, designated "R102S", was prepared and analyzed. To prepare the DNA encoding VEGF-C-R102S, the arginine codon at position 102 of SEQ ID NO: 33 was replaced with a serine codon. This VEGF-C-R102S-encoding DNA, in a pREP7 vector, was transfected into 293 EBNA cells and expressed as described above. VEGF-C polypeptides were immunoprecipitated using antisera 882 (obtained by immunization of a rabbit with a polypeptide corresponding to residues 104-120 of SEQ ID NO: 33 (see previous Example)) and antisera 905 (obtained by immunization of a j 72 rabbit with a polypeptide corresponding to a portion of the prepro- VEGF-C leader: H2N-ESGLDLSDAEPDAGEATAYASK (residues 33 to 54 of SEQ ID NO: 33).

The immunoprecipitates from each cell culture were subjected to SDS-PAGE under non-denaturing conditions (Fig. 21B). Bands 1-6 were cut out from the gel, soaked for 30 minutes in ix gel-loading buffer containing 200 mM B-mercaptoethanol, and individually subjected to SDS-PAGE under denaturing conditions (Figs.

21A and 21C, lanes 1-6).

As can be seen from Figures 21A-C, each high molecular weight form of VEGF-C (Fig. 21B, bands 1-4) consists of at least two monomers bound by disulfide bonds (Compare Figs. 21A and 21C, lanes 1-4, in the 15 reducing gels). The main component of bands 1-3 is the doublet of 32/29 kD, where both proteins are present in an equimolar ratio. The main fraction of the 21 kD form is secreted as either a monomer or as a homodimer connected by means other than disulfide bonds (bands 6 20 and lanes 6 in Figs. 21A-C).

The R102S mutation creates an additional site for N-linked glycosylation in VEGF-C at the asparagine residue at position 100 in SEQ ID NO: 33. Glycosylation at this additional glycosylation site increases the 25 apparent molecular weight of polypeptides containing the site, as confirmed in Figures 21A-C and Figures 22A-B.

The additional glycosylation lowers the mobility of forms of VEGF-C-R102S that contain the additional glycosylation site, when compared to polypeptides of similar primary structure corresponding to VEGF-C. Figures 21A-C and Figures 22A-B reveal that the VEGF-C-R102S polypeptides corresponding to the 32 kD and 15 kD forms of wt VEGF-C exhibit increased apparent molecular weights, indicating that each of these polypeptides contains the newly introduced glycosylation site. In particular, the VEGF- C-R102S polypeptide corresponding to the 15 kD polypeptide from VEGF-C comigrates on a gel with the 21 73 kD form of the wild type (wt) VEGF-C, reflecting a shift on the gel to a position corresponding to a greater apparent molecular weight. (Compare lanes 4 in Figures 21A and 21C).

In a related experiment, another VEGF-C mutant, designated "R226,227S," was prepared and analyzed. To prepare a DNA encoding VEGF-C-R226,227S, the arginine codons at positions 226 and 227 of SEQ ID NO: 33 were replaced with serine codons by site-directed mutagenesis.

The resultant DNA was transfected into 293 EBNA cells as described above and expressed and analyzed under the same conditions as described for VEGF-C and VEGF-C-R102S. In the conditioned medium from the cells expressing

VEGF-C-

R226,227S, no 32 kD form of VEGF-C was detected. These 15 results indicate that a C-terminal cleavage site of wildtype VEGF-C is adjacent to residues 226 and 227 of SEQ ID NO: 33, and is destroyed by the mutation of the arginines to serines. Again, the mobility of the 29 kD component of the doublet was unchanged (Figures 22A-B).

c 20 Taken together, these data indicate that the major form of the processed VEGF-C is a heterodimer consisting of a polypeptide of 32 kD containing amino acids 32-227 of the prepro-VEGF-C (amino acids 32 to 227 in SEQ ID NO: 33) attached by disulfide bonds to 25 a polypeptide of 29 kD beginning with amino acid 228 in SEQ ID NO: 33. These data are also supported by a comparison of the pattern of immunoprecipitated, labelled VEGF-C forms using antisera 882 and antisera 905.

When VEGF-C immunoprecipitation was carried out using conditioned medium, both antisera (882 and 905) recognized some or all of the three major processed forms of VEGF-C (32/29 kD, 21 kD and 15 kD). When the conditioned medium was reduced by incubation in the presence of 10 mM dithiothreitol for two hours at room temperature with subsequent alkylation by additional incubation with 25 mM iodoacetamide for 20 minutes at room temperature, neither antibody precipitated the 29 kD

AW

VM

74 component, although antibody 882 still recognized polypeptides of 32 kD, 21 kD and 15 kD. These results are consistent with the nature of the oligopeptide antigen used to elicit the antibodies contained in antisera 882, an oligopeptide containing amino acid residues 104-120 of SEQ ID NO: 33. On the other hand, antisera 905 recognized only the 32 kD and 15 kD polypeptides, which include sequence of the oligopeptide (amino acids 33 to 54 of SEQ ID NO: 33) used for immunization to obtain antisera 905. Taking into account the mobility shift of the 32 kD and 15 kD forms, the immunoprecipitation results with the R102S mutant were similar (Figs. 23A-B). The specificity of antibody 905 is confirmed by the fact that it did not recognize a 15 VEGF-C AN variant form wherein the N-terminal propeptide spanning residues 32-102 of the unprocessed polypeptide had been deleted (Fig. 23B).

The results of these experiments also demonstrate that the 21 kD polypeptide is found in 20 heterodimers with other molecular forms (see Figs. 21A-C and Figs. 22A-B), and secreted as a monomer or a homodimer held by bonds other than disulfide bonds (Figs.

21A and 21B, lanes 6).

The experiments disclosed in this example demonstrate that several forms of VEGF-C exist.

A

variety of VEGF-C monomers were observed and these monomers can vary depending on the level and pattern of glycosylation. In addition, VEGF-C was observed as a multimer, for example a homodimer or a heterodimer. The processing of VEGF-C is schematically presented in Fig.

18 (disulfide bonds not shown). All forms of VEGF-C are within the scope of the present invention.

Example 23 In situ Hybridization of Mouse Embryos To analyze VEGF-C mRNA distribution in different cells and tissues, sections of 12.5 and 14.5-

A

75 O day post-coitus mouse embryos were prepared and analyzed via in situ hybridization using labeled

VEGF-C

probes. In situ hybridization of tissue sections was performed as described in Vastrik et al., J. Cell Biol., 128:1197-1208 (1995). A mouse VEGF-C antisense RNA probe was generated from linearized pBluescript II SK+ plasmid (Stratagene Inc., La Jolla, CA), containing a cDNA fragment corresponding to nucleotides 499-979 of a mouse VEGF-C cDNA (SEQ ID NO: 40). Radiolabeled RNA was synthesized using T7 polymerase and 35 S]-UTP (Amersham).

Mouse VEGF-B antisense and sense RNA probes were synthesized in a similar manner from linearized pCRII plasmid containing the mouse VEGF-B cDNA insert as described Olofsson et al., Proc. Natl. Acad. Sci. (USA), 15 93:2576-2581 (1996). The high stringency wash was for minutes at 65C in a solution containing 30 mM dithiothreitol (DTT) and 4 x SSC. The slides were exposed for 28 days, developed and stained with hematoxylin. For comparison, similar sections were 20 hybridized with a VEGFR-3 probe and the 12.5-day p.c.

embryos were also probed for VEGF-B mRNA.

Figures 34A-D show darkfield (Figures 34A-C) and lightfield (Figure 34D) photomicrographs of 12.5 day p.c. embryo sections probed with the antisense (Fig. 34A) 25 and sense (Figs. 34C-D) VEGF-C probes. Fig. 34A illustrates a parasagittal section, where VEGF-C mRNA is particularly prominent in the mesenchyme around the vessels surrounding the developing metanephros In addition, hybridization signals were observed between the developing vertebrae in the developing lung mesenchyme in the neck region and developing forehead. The specificity of these signals is evident from the comparison with VEGF-B expression in an adjacent section (Fig. 34B), where the myocardium gives a very strong signal and lower levels of VEGF-B mRNA are detected in several other tissues. Both genes appear to be expressed in between the developing vertebrae in 76 the developing lung (lu) and forehead. Hybridization of the VEGF-C sense probe showed no specific expression within these structures (Fig. 34C).

Figs. 35A-D show a comparison of the expression patterns of VEGF-C and VEGFR-3 in 12.5 day p.c. mouse embryos in the jugular region, where the developing dorsal aorta and cardinal vein are located. This is the area where the first lymphatic vessels sprout from venous sac-like structures according to the long-standing theory of Sabin, Am. J. Anat., 9:43-91 (1909). As can be seen from Figs. 35A-D, an intense VEGF-C signal is detected in the mesenchyme surrounding the developing venous sacs (Figs. 35A and 35C) which are positive for VEGFR-3 (Figs.

35B and 15 The mesenterium supplies the developing gut with blood and contains developing lymphatic vessels.

The developing 14.5 day p.c. mesenterium is positive for i VEGF-C mRNA, with particularly high expression in connective tissue surrounding certain vessels (arrowheads 20 in Figs. 35E-H). This signal in Fig. 35E should be distinguished from the false positive reflection of light from.red blood cells within the vessel. The adjacent mesenterial VEGFR-3 signals shown in Fig. 35F originate from small capillaries of the mesenterium (arrowhead).

Therefore, there appears to be a paracrine relationship between the production of the mRNAs for VEGF-C and its receptor. This data indicates that VEGF-C is expressed in a variety of tissues. Moreover, the pattern of expression is consistent with a role for VEGF-C in venous and lymphatic vessel development. Further, the data reveals that VEGF-C is expressed in non-human animals.

Example 24 Analysis of VEGF, VEGF-B, and VEGF-C mRNA Expression in Fetal and Adult Tissues A human fetal tissue Northern blot containing 2 pg of polyadenylated RNAs from brain, lung, liver and kidney (Clontech Inc.) was hybridized with a pool of the

A

77 O following probes: a human full-length VEGF-C cDNA insert (Genbank Acc. No. X94216), a human

VEGF-B

67 cDNA fragment (nucleotides 1-382, Genbank Acc. No. U48800) obtained by PCR amplification; and a human VEGF 581 bp cDNA fragment covering base pairs 57-638 (Genbank Acc. No. X15997).

Blots were washed under stringent conditions, using techniques standard in the art.

Mouse embryo multiple tissue Northern blot (Clontech Inc.) containing 2 Ag of polyadenylated RNAs from 7, 11, 15 and 17 day postcoital embryos was hybridized with mouse VEGF-C cDNA fragment (base pairs 499-656). A mouse adult tissue Northern blot was hybridized with the probes for human VEGF, VEGF-B 167 VEGF-C and with a VEGFR-3 cDNA fragment (nucleotides 1- 15 595; Genbank Acc. No. X68203).

In adult mouse tissues, both 2.4 kb and 2.0 kb mRNA signals were observed with the VEGF-C probe, at an approximately 4:1 ratio. The most conspicuous signals were obtained from lung and heart RNA, while kidney, liver, brain, and skeletal muscle had lower levels, and spleen and testis had barely visible levels. As in the human tissues, VEGF mRNA expression in adult mice was o most abundant in lung and heart RNA, whereas the other samples showed less coordinate regulation with VEGF-C 25 expression. Skeletal muscle and heart tissues gave the .highest VEGF-B mRNA levels from adult mice, as previously reported Olofsson et al., Proc. Natl. Acad. Sci. (USA), 93:2576-2581 (1996). Comparison with VEGFR-3 expression showed that the tissues where VEGF-C is expressed also contain mRNA for its cognate receptor tyrosine kinase, although in the adult liver VEGFR-3 mRNA was disproportionally abundant.

To provide a better insight into the regulation of the VEGF-C mRNA during embryonic development, polyadenylated RNA isolated from mouse embryos of various gestational ages 11, 15, and 17 day was hybridized with the mouse VEGF-C probe. These analyses 78 showed that the amount of 2.4 kb VEGF-C mRNA is relatively constant throughout the gestational period.

Example Regulation of mRNAs for VEGF family members by serum, interleukin-1 and dexamethasone in human fibroblasts in culture Human IMR-90 fibroblasts were grown in DMEM medium containing 10% FCS and antibiotics. The cells were grown to 80% confluence, then starved for 48 hours in 0.5 FCS in DMEM. Thereafter, the growth medium was changed to DMEM containing 5% FCS, with or without ng/ml interleukin-1 (IL-1) and with or without 1 mM dexamethasone, as indicated in Figs. 24A-B. The culture plates were incubated with these additions for the times 15 indicated, and total cellular RNA was isolated using the TRIZOL kit (GIBCO-BRL). About 20 ig of total RNA from each sample was electrophoresed in 1.5% formaldehydeagarose gels as described in Sambrook et al., supra (1989). The gel was used for Northern blotting and hybridization with radiolabeled insert DNA from the human VEGF clone (a 581 bp cDNA covering bps 57-638, Genbank Acc. No. 15997) and a human VEGF-B 167 cDNA fragment .(nucleotides 1-382, Genbank Acc. No. U48800) (Fig. Subsequently, the Northern blots were probed with radiolabelled insert from the VEGF-C cDNA plasmid (Fig.

S.24A). Primers were labelled using a standard technique involving enzymatic extension reactions of random primers, as would be understood by one of ordinary skill in the art. The mobilities of the 28S and 18S ribosomal RNA bands are indicated, based on UV photography of ethidium bromide stained RNA before the transfer.

As can be seen in Figs. 24A-B, very low levels of VEGF-C and VEGF are expressed by the starved cells as well as cells after 1 hour of stimulation. In contrast, abundant VEGF-B mRNA signal is visible under these conditions. After a 4 hours of serum stimulation, there is a strong induction of VEGF-C and VEGF mRNAs, 79 O which are further increased in the IL-1 treated sample.

The effect of IL-1 seems to be abolished in the presence of dexamethasone. A similar pattern of enhancement is maintained in the 8 hour sample, but a gradual downregulation of all signals occurs for both RNAs in the 24 hour and 48 hour samples. In contrast, VEGF-B mRNA levels remain constant and thus show remarkable stability throughout the time period. The results are useful in guiding efforts to use VEGF-C and its fragments, its antagonists, and anti-VEGF-C antibodies in methods for treating a variety of disorders.

Example 26 Expression and analysis of recombinant murine VEGF-C The mouse VEGF-C cDNA was expressed as a 15 recombinant protein and the secreted protein was analyzed for its receptor binding properties. The binding of mouse VEGF-C to the human VEGFR-3 extracellular domain was studied by using media from Bosc23 cells transfected with mouse VEGF-C cDNA in a retroviral expression vector.

The 1.8 kb mouse VEGF-C cDNA was cloned as an EcoRI fragment into the retroviral expression vector pBabe-puro containing the SV40 early promoter region [Morgenstern et al., Nucl. Acids Res., 18:3587-3595 (1990)], and transfected into the Bosc23 packaging cell 25 line [Pearet et al., Proc. Natl. Acad. Sci. (USA), 90:8392-8396 (1994)] by the calcium-phosphate precipitation method. For comparison, Bosc23 cells also were transfected with the previously- described human VEGF-C construct in the pREP7 expression vector. The transfected cells were cultured for 48 hours prior to metabolic labelling. Cells were changed into DMEM medium devoid of cysteine and methionine, and, after 45 minutes of preincubation and medium change, Pro-mix™ L-[ 35 S] in vitro cell labelling mix (Amersham Corp.), in the same medium, was added to a final concentration of about 120 yCi/ml. After 6 hours of incubation, the culture medium 80 was collected and clarified by centrifugation.

For immunoprecipitation, 1 ml aliquots of the media from metabolically-labelled Bosc23 cells transfected with empty vector or mouse or human recombinant VEGF-C, respectively, were incubated overnight on ice with 2 ~l of rabbit polyclonal antiserum raised against an N-terminal 17 amino acid oligopeptide of mature human VEGF-C

(H

2 N-EETIKFAAAHYNTEILK) (SEQ ID NO: 33, residues 104-120). Thereafter, the samples were incubated with protein A sepharose for 40 minutes at 4 C with gentle agitation. The sepharose beads were then washed twice with immunoprecipitation buffer and four times with 20 mM Tris-HCl, pH 7.4. Samples were boiled in Laemmli buffer and analyzed by 12.5% sodium dodecyl 15 sulfate polyacrylamide gel electrophoresis

(SDS-PAGE).

S"Immunoprecipitation of VEGF-C from media of transfected and metabolically-labelled cells revealed bands of approximately 3 0-32x10 3 Mr (a doublet) and 22- 23x10 3 Mr in 12.5% SDS-PAGE. These bands were not 20 detected in samples from nontransfected or mocktransfected cells as shown in Fig. 32 lanes marked "vector"). These results show that antibodies raised against human VEGF-C recognize the corresponding mouse ligand.

25 For receptor binding experiments, 1 ml aliquots of media from metabolically-labelled Bosc23 cells were incubated with VEGFR-3 extracellular domain (see Example covalently coupled to sepharose, for 4 hours at 4 C with gentle mixing. The sepharose beads were washed four times with ice-cold phosphate buffered saline

(PBS),

and the samples were analyzed by gel electrophoresis as described in Joukov et al., EMBO 15:290-298 (1996).

As can be seen from Fig. 32, similar 30-32 x 103 M, doublet and 22-23 x 103 Mr polypeptide bands were obtained in the receptor binding assay as compared to the immunoprecipitation assay. Thus, mouse VEGF-C binds to human VEGFR-3. The slightly faster mobility of the mouse 81 VEGF-C polypeptides may be caused by the four amino acid residue difference observed in sequence analysis (residues H88-E91, Fig. 31).

The capacity of mouse recombinant VEGF-C to induce VEGFR-3 autophosphorylation was also investigated.

For the VEGFR-3 receptor stimulation experiments, subconfluent NIH 3T3-Flt4 cells, Pajusola et al., Oncogene, 9:3545-3555 (1994), were starved overnight in serum-free medium containing 0.2% BSA. In general, the cells were stimulated with the conditioned medium from VEGF-C vector-transfected cells for 5 minutes, washed three times with cold PBS containing 200 AM vanadate, and lysed in RIPA buffer for immunoprecipitation analysis.

The lysates were centrifuged for 25 minutes at 16000 x g 15 and the resulting supernatants were incubated for 2 hours on ice with the specific antisera, followed by immunoprecipitation using protein A-sepharose and analysis in 7% SDS-PAGE. Polypeptides were transferred to nitrocellulose and analyzed by immunoblotting using 20 anti-phosphotyrosine (Transduction Laboratories) and :anti-receptor antibodies, as described by Pajusola et al., Oncogene, 9:3545-3555 (1994). Filter stripping was S:carried out at 50'C for 30 minutes in 100 mM 2mercaptoethanol, 2% SDS, 62.5 mM Tris-HCl, pH 6.7, with occasional agitation. The results of the experiment are shown in Fig. 33. The results demonstrate that culture medium containing mouse VEGF-C stimulates the autophosphorylation of VEGFR-3 to a similar extent as human baculoviral VEGF-C or the tyrosyl phosphatase inhibitor pervanadate.

VEGFR-2 stimulation was studied in subconfluent porcine aortic endothelial (PAE) cells expressing Kdr (VEGFR-2) (PAE-VEGFR-2) [Waltenberger et al., J. Biol.

Chem., 269:26988-26995 (1994)], which were starved overnight in serum-free medium containing 0.2% BSA.

Stimulation was carried out and the lysates prepared as described above. For receptor immunoprecipitation, a 82 specific antiserum for VEGFR-2 [Waltenberger et al., J.

Biol. Chem., 269:26988-26995 (1994)] was used. The immunoprecipitates were analyzed as described for VEGFR-3 in 7% SDS-PAGE followed by Western blotting with antiphosphotyrosine antibodies, stripping of the filter, and re-probing it with anti-VEGFR-2 antibodies (Santa Cruz).

Mouse VEGF-C appeared to be a potent inducer of VEGFR-3 autophosphorylation, with the 195x10 3 M, precursor and proteolytically cleaved 125x10 3 Mr tyrosine kinase polypeptides of the receptor (Pajusola et al., Oncogene, 9:3545-3555 (1994)), being phosphorylated. VEGFR-2 stimulation was first tried with unconcentrated medium from cells expressing recombinant VEGF-C, but immunoblotting analysis did not reveal any receptor 15 autophosphorylation.

To further determine whether mouse recombinant VEGF-C can also induce VEGFR-2 autophosphorylation as observed for human VEGF-C, PAE cells expressing VEGFR-2 were stimulated with tenfold concentrated medium from 20 cultures transfected with mouse VEGF-C expression vector .and autophosphorylation was analyzed. For comparison, cells treated with tenfold concentrated medium containing human recombinant VEGF-C (Joukov et al., (1996)), unconcentrated medium from human VEGF-C baculovirus infected insect cells, or pervanadate (a tyrosyl phosphatase inhibitor) were used. As can be seen from Fig. 33, in response to human baculoviral VEGF-C as well as pervanadate treatment, VEGFR-2 was prominently phosphorylated, whereas human and mouse recombinant

VEGF-

C gave a weak and barely detectable enhancement of autophosphorylation, respectively. Media from cell cultures transfected with empty vector or VEGF-C cloned in the antisense orientation did not induce autophosphorylation of VEGFR-2. Therefore, mouse VEGF-C binds to VEGFR-3 and activates this receptor at a much lower concentration than needed for the activation of VEGFR-2. Nevertheless, the invention comprehends methods 83 for using the materials of the invention to take advantage of the interaction of VEGF-C with VEGFR-2, in addition to the interaction between VEGF-C and VEGFR-3.

Example 27 VEGF-C E104-8213 fragment expressed in Pichia yeast stimulates autophosphorylation of Flt4 (VEGFR-3) and KDR (VEGFR-2) A truncated form of human VEGF-C cDNA was constructed wherein the sequence encoding residues of a putative mature VEGF-C amino terminus

H

2 N-E(104)ETIK (SEQ ID NO: 33, residues 104 et seq.) was fused in-frame to the yeast PHO1 signal sequence (Invitrogen Pichia Expression Kit, Catalog #K1710-01), and a stop codon Swas introduced after amino acid 213 (H 2 N- RCMS; i.e., 15 after codon 213 of SEQ ID NO: 32). The resultant truncated cDNA construct was then inserted into the Pichia pastoris expression vector pHIL-SI (Invitrogen).

For the cloning, an internal BglII site in the VEGF-C coding sequence was mutated without change of the encoded polypeptide sequence.

This VEGF-C expression vector was then transfected into Pichia cells and positive clones were identified by screening for the expression of VEGF-C protein in the culture medium by Western blotting. One 25 positive clone was grown in a 50 ml culture, and induced with methanol for various periods of time from 0 to hours. About 10 Al of medium was analyzed by gel electrophoresis, followed by Western blotting and detection with anti-VEGF-C antiserum, as described above.

As can be seen in Figure 25, an approximately 24 kD polypeptide (note the band spreading due to glycosylation) accumulates in the culture medium of cells transfected with the recombinant VEGF-C construct, but not in the medium of mock-transfected cells or cells transfected with the vector alone.

The medium containing the recombinant

VEGF-C

protein was concentrated by Centricon 30 kD cutoff 84 ultrafiltration and used to stimulate NIH 3T3 cells expressing Flt4 (VEGFR-3) and porcine aortic endothelial (PAE) cells expressing KDR (VEGFR-2). The stimulated cells were lysed and immunoprecipitated using VEGFRspecific.antisera and the immunoprecipitates were analyzed by Western blotting using anti-phosphotyrosine antibodies, chemiluminescence, and fluorography. As a positive control for maximal autophosphorylation of the VEGFRs, vanadate

(VO

4 treatment of the cells for minutes was used. As can be seen from the results shown in Fig. 26, medium from Pichia cultures secreting the recombinant VEGF-C polypeptide induces autophosphorylation of both Flt41 polypeptides of 195 kD and 125 kD as well as the KDR polypeptide of about 200 15 kD. Vanadate, on the other hand, induces heavy tyrosyl phosphorylation of the receptor bands in addition to other bands probably coprecipitating with the receptors.

These results demonstrate that a VEGFhomologous domain of VEGF-C consisting of amino acid 20 residues 104E 213S (SEQ ID NO: 33, residues 104-213) can be recombinantly produced in yeast and is capable of stimulating the autophosphorylation of Flt4 (VEGFR-3) and KDR (VEGFR-2). Recombinant VEGF-C fragments such as the fragment described herein, which are capable of stimulating Flt4 or KDR autophosphorylation are intended as aspects of the invention; methods of using these fragments are also within the scope of the invention.

Example 28 Properties of the differentially processed forms of VEGF-C The following oligonucleotides were used to generate a set of VEGF-C variants: TCTCTTCTGTGCTTGAGTTGAG (SEQ ID NO: 42), used to generate VEGF-C R102S (arginine mutated to serine at position 102 (SEQ ID NO: 33)); (SEQ ID NO: 43), used to generate VEGF-C R102G (arginine mutated to glycine at 85 position 102 (SEQ ID NO: 33)); GGCGGCGGGCGCCTCGCGAGGACC (SEQ ID NO: 44), used to generate VEGF-C AN (deletion of N-terminal propeptide corresponding to amino acids 32-102 (SEQ ID NO: 33)); CTGGCAGGGAACTGCTAATAATGGAATGAA 3' (SEQ ID NO: used to generate VEGF-C R226,227S (arginine codons mutated to serines at positions 226 and 227 (SEQ ID NO: 33)); GATGGTGATGGGCGGCGGCGGCGGCGGGCGCCTCGCGAGGACC (SEQ ID NO: 46), used to generate VEGF-C NHis (this construct encodes a polypeptide with a 6xHis tag fused to the Nterminus of the secreted precursor (amino acid 32 of SEQ 15 ID NO: 33)).

Some of the foregoing VEGF-C variant constructs were further modified to obtain additional constructs.

For example, VEGF-C R102G in pALTER (Promega) and oligonucleotide 5'-GTATTATAATGTCCTCCACCAAATTTTATAG -3' 20 (SEQ ID NO: 47) were used to generate VEGF-C 4G, which encodes a polypeptide with four point mutations: R102G, A110G, A111G, and A112G (alanines mutated to glycines at positions 110-112 (SEQ ID NO: 33). These four mutations are adjacent to predicted sites of cleavage of VEGF-C expressed in PC-3 and recombinantly expressed in 293 EBNA cells.

Another construct was created using VEGF-C AN and oligonucleotide

GGCCGCTAGTGATGGTGATGGTGATGAATAATGGAATGAACTTGTCTGTAAACATCC

AG (SEQ ID NO: 48) to generate VEGF-C ANACHis. This construct encodes a polypeptide with a deleted N-terminal propeptide (amino acids 32-102); a deleted C-terminal propeptide (amino acids 226-419 of SEQ ID NO: 33); and an added 6xHis tag at the C-terminus.

All constructs were further digested with HindIII and NotI, subcloned into HindIII/NotI digested pREP7 vector, and used to transfect 293 EBNA cells.

86 About 48 hours after transfection, the cells were either metabolically labelled with Pro-mix m as described above, or starved in serum-free medium for 2 days. Media were then collected and used in subsequent experiments. As can be seen from Figs. 27A-B, wild type (wt) VEGF-C, VEGF-C NHis and VEGF-C ANACHis were expressed to similar levels in 293 EBNA cells. At the same time, expression of the VEGF-C 4G polypeptide was considerably lower, possibly due to the changed conformation and decreased stability of the translated product. However, all the above VEGF-C variants were secreted from the cells (compare Figs. 27A and 27B). The conditioned media from the transfected and starved cells were concentrated fold and used to assess their ability to stimulate 15 tyrosine phosphorylation of Flt4 (VEGFR-3) expressed in NIH 3T3 cells and KDR (VEGFR-2) expressed in PAE cells.

Figs. 28A-B show that wild type (wt) VEGF-C, as well as all three mutant polypeptides, stimulate tyrosine phosphorylation of VEGFR-3. The most prominent stimulation is by the short mature VEGF-C ANACHis. This mutant, as well as VEGF-C NHis, also stimulated tyrosine phosphorylation of VEGFR-2. Thus, despite the fact that a major component of secreted recombinant VEGF-C is a dimer of 32/29 kD, the active part of VEGF-C responsible for its binding to VEGFR-3 and VEGFR-2 is localized between amino acids 102 and 226 (SEQ ID NO: 33) of the VEGF-C precursor. Analysis and comparison of binding properties and biological activities of these VEGF-C proteins and variants, using assays such as those described herein, will provide data concerning the significance of the observed major 32/29 kD and 21-23 kD VEGF-C processed forms. The data indicate that constructs encoding amino acid residues 103-225 of the VEGF-C precursor (SEQ ID NO: 33) generate a recombinant ligand that is functional for both VEGFR-3 and VEGFR-2.

The data from this and preceding examples demonstrate that numerous fragments of the VEGF-C 87 O polypeptide retain biological activity. A naturally occurring VEGF-C polypeptide spanning amino acids 103-226 (or 103-227) of SEQ ID NO: 33, produced by a natural processing cleavage defining the C-terminus, has been shown to be active. Example 27 demonstrates that a fragment with residues 104-213 of SEQ ID NO: 33 retains biological activity.

In addition, data from Example 21 demonstrates that a VEGF-C polypeptide having its amino terminus at position 112 of SEQ ID NO: 33 retains activity.

Additional experiments have shown that a fragment lacking residues 1-112 of SEQ ID NO: 33 retains biological activity.

In a related experiment, a stop codon was 15 substituted for the lysine at position 214 of SEQ ID NO: 33 (SEQ ID NO: 32, nucleotides 991-993). The resulting recombinant polypeptide still was capable of inducing i Flt4 autophosphorylation, indicating that a polypeptide spanning amino acid residues 113-213 of SEQ ID NO: 33 is 20 biologically active.

Sequence comparisons of members of the VEGF family of polypeptides provides an indication that still smaller fragments of the polypeptide depicted in SEQ ID NO: 33 will retain biological activity. In particular, eight highly conserved cysteine residues of the VEGF family of polypeptides define a region from residues 131 211 of SEQ ID NO: 33 (see Figure 31) of evolutionary signficance; therefore, a polypeptide spanning from about residue 131 to about residue 211 is expected to retain VEGF-C biological activity. In fact, a polypeptide which retains the conserved motif RCXXCC a polypeptide comprising from about residue 161 to about residue 211 of SEQ ID NO: 33 is postulated to retain VEGF-C biological activity. To maintain native conformation of these fragments, it may be preferred to retain about 1-2 additional amino acids at the carboxy-terminus and 1-2 or more amino acids at the amino terminus.

s 88 Beyond the preceding considerations, evidence exists that smaller fragments and/or fragment variants which lack the conserved cysteines nonetheless will retain VEGF-C biological activity. Consequently, the materials and methods of the invention include all VEGF-C fragments and variants that retain at least one biological activity of VEGF-C, regardless of the presence or absence of members of the conserved set of cysteine residues.

Example 29 Expression of human VEGF-C under the human K14 keratin promoter in transgenic mice induces abundant growth of lymphatic vessels in the skin The Flt4 receptor tyrosine kinase is relatively 4 15 specifically expressed in the endothelia of lymphatic o: vessels. Kaipainen et al., Proc. Natl. Acad. Sci. (USA), 92: 3566-3570 (1995). Furthermore, the VEGF-C growth factor stimulates the Flt4 receptor, showing less activity towards the KDR receptor of blood vessels 20 (Joukov et al., EMBO 15: 290-298 (1996); See Example 26).

Experiments were conducted in transgenic mice to analyze the specific effects of VEGF-C overexpression in tissues. The human K14 keratin promoter is active in 25 the basal cells of stratified.squamous epithelia (Vassar Set al., Proc. Natl. Acad. Sci. (USA), 86:1563-1567 (1989)) and was used as the expression control element in the recombinant VEGF-C transgene. The vector containing the K14 keratin promoter is described in Vassar et al., Genes Dev., 5:714-727 (1991) and Nelson et al., J. Cell Biol. 97:244-251 (1983).

The recombinant VEGF-C transgene was constructed using the human full length VEGF-C cDNA (GenBank Acc. No. X94216). This sequence was excised from a pCI-neo vector (Promega) with XhoI/NotI, and the resulting 2027 base pair fragment containing the open reading frame and stop codon (nucleotides 352-1611 of SEQ 89 g ID NO: 32) was isolated. The isolated fragment was then subjected to an end-filling reaction using the Klenow fragment of DNA polymerase. The blunted fragment was then ligated to a similarly opened BamHI restriction site in the K14 vector. The resulting construct contained the EcoRI site derived from the polylinker of the pCI-neo vector. This EcoRI site was removed using standard techniques (a Klenow-mediated fill-in reaction following partial digestion of the recombinant intermediate with EcoRI) to facilitate the subsequent excision of the DNA fragment to be injected into fertilized mouse oocytes.

The resulting clone, designated K14-VEGF-C, is illustrated in Fig. The EcoRI-HindIII fragment from clone K14 VEGF- C containing the K14 promoter, VEGF-C cDNA, and K14 ooo polyadenylation signal was isolated and injected into o fertilized oocytes of the FVB-NIH mouse strain. The injected zygotes were transplanted to oviducts of pseudopregnant C57BL/6 x DBA/2J hybrid mice. The 20 resulting founder mice were analyzed for the presence of the transgene by polymerase chain reaction of tail DNA using the primers: 5'-CATGTACGAACCGCCAG-3' (SEQ ID NO: 49) and 5'-AATGACCAGAGAGAGGCGAG-3' (SEQ ID NO: 50). In addition, the tail DNAs were subjected to EcoRV digestion 25 and subsequent Southern analysis using the EcoRI-HindIII fragment injected into the mice. Out of 8 pups analyzed at 3 weeks of age, 2 were positive, having approximately 40-50 copies and 4-6 copies of the transgene in their respective genomes.

The mouse with the high copy number transgene was small, developed more slowly than its litter mates and had difficulty eating suckling). Further examination showed a swollen, red snout and poor fur.

Although fed with a special liquid diet, it suffered from edema of the upper respiratory and digestive tracts after feeding and had breathing difficulties. This mouse died eight weeks after birth and was immediately processed for 90 histology, immunohistochemistry, and in situ hybridization.

Histological examination showed that in comparison to the skin of littermates, the dorsal dermis of Kt4-VEGF-C transgenic mice was atrophic and connective tissue was replaced by large lacunae devoid of red cells, but lined with a thin endothelial layer (white arrows in Figs. 29A-D). These distended vessel-like structures resembled those seen in human lymphangiomas. The number 0 of skin adnexal organs and hair follicles were reduced.

In the snout region, an increased number of vessels was also seen. Therefore, VEGF-C overexpression in the basal epidermis is capable of promoting the growth of extensive .vessel structure in the underlying skin, including large 15 vessel lacunae. The endothelial cells surrounding these lacunae contained abundant Flt4 mRNA in in situ hybridization (see Examples 23 and 30 for methodology).

SThe vessel morphology indicates that VEGF-C stimulates the growth of vessels having features of lymphatic vessels. The other K14-VEGF-C transgenic mouse had a similar skin histopathology. r a n s g n c Sorhe foregoing in vivo data indicates utilities for both VEGF-C polypeptides and polypeptide variants having VEGF-C biological t i S having VEGF-C biological activity, and (ii) anti-VEGF-C antibodies and VEGF-C antagonists that inhibit

VEGF-C

activity by binding VEGF-C or interfering with VEGF-C/receptor interactions. For example, the data indicates a therapeutic utility for VEGF-C polypeptides in patients wherein growth of lymphatic tissue may be desirable in patients following breast cancer or other surgery where lymphatic tissue has been removed and where lymphatic drainage has therefore been compromised, resulting in swelling; or in patients suffering from elephantiasis). The data indicates a therapeutic utility for anti-VEGF-C antibody substances and VEGF-C antagonists for conditions wherein growth-inhibition of lymphatic tissue may be desirable treatment of

A

91 Slymphangiomas). Accordingly, methods of administering VEGF-C and VEGF-C variants and antagonists are contemplated as methods and materials of the invention.

Example Expression of VEGF-C and Flt4 in the Developing Mouse Embryos from a 16-day post-coitus pregnant mouse were prepared and fixed in 4% paraformaldehyde (PFA), embedded in paraffin, and sectioned at 6 cm. The sections were placed on silanated microscope slides and treated with xylene, rehydrated, fixed for 20 minutes in 4% PFA, treated with proteinase K (7mg/ml; Merck, Darmstadt, Germany) for 5 minutes at room temperature, again fixed in 4% PFA and treated with acetic anhydride, 15 dehydrated in solutions with increasing ethanol concentrations, dried and used for in situ hybridization.

In situ hybridization of sections was performed as described (Vdstrik et al., J. Cell Biol., 128:1197-1208 (1995)). A mouse VEGF-C antisense RNA 20 probe was generated from linearized pBluescript II SK+ plasmid (Stratagene Inc.), containing a fragment corresponding to nucleotides 499-979 of mouse VEGF-C S* cDNA, where the noncoding region and the BR3P repeat were removed by Exonuclease III treatment. The fragment had 25 been cloned into the EcoRI and HindIII sites of pBluescript II SK+. Radiolabeled RNA was synthesized using T7 RNA Polymerase and 35 S]-UTP (Amersham, Little Chalfont, UK). About two million cpm of the VEGF-C probe was applied per slide. After an overnight hybridization, the slides were washed first in 2x SSC and 20-30 mM DDT for 1 hour at 50'C. Treatment continued with a high stringency wash, 4x SSC and 20 mM DTT and 50% deionized formamide for 30 minutes at 65C followed by RNase A treatment (20 pg/ml) for 30 minutes at 37C. The high stringency wash was repeated for 45 minutes. Finally, the slides were dehydrated and dried for 30 minutes at 92 I room temperature. The slides were dipped into photography emulsion and exposed for 4 weeks. Slides were developed using Kodak D-16 developer, counterstained with hematoxylin and mounted with Permount (FisherChemical).

For in situ hybridizations of Flt4 sequences, a mouse Flt4 cDNA fragment covering bp 1-192 of the published sequence (Finnerty et al., Oncogene, 8:2293- 2298 (1993)) was used, and the above-described protocol was followed, with the following exceptions.

Approximately one million cpm of the Flt4 probe were applied to each slide. The stringent washes following hybridization were performed in Ix SSC and 30 mM DTT for 105 minutes.

o 15 The figure shows photomicrographs of the hybridized sections in dark field microscopy (Figs. 36A- C) and light field microscopy (Fig. 36D). Magnifications used for photography were 4x for Figs. 36A-B and 10x for Figs. 36C-D. The transverse sections shown are from the cephalic region and the area shown for VEGF-C and FLT4 are about 14 sections apart, Flt4 being more cranially located in the embryo. In Fig. 36A (Flt4 probe), the developing nasopharyngeal cavity is in the midline in the upper, posterior part; in the anterior part of Fig. 36A 25 is the snout with emerging fibrissal follicles and, in the midline, the forming nasal cavity. On both sides, the retinal pigment gives a false positive signal in dark field microscopy. The most prominently Flt4-hybridizing structures appear to correspond to the developing lymphatic and venous endothelium. Note that a plexus-like endothelial vascular structure surrounds the developing nasopharyngeal mucous membrane. In Fig. 36B, the most prominent signal is obtained from the posterior part of the developing nasal conchae, which in higher magnification (Figs. 36C-D) show the epithelium surrounding loose connective tissue/forming cartilage.

This structure gives a strong in situ hybridization 93 Ssignal for VEGF-C. Also in Fig. 36B, more weakly hybridizing areas can be seen around the snout, although this signal is much more homogeneous in appearance.

Thus, the expression of VEGF-C is strikingly high in the developing nasal conchae.

The conchae are surrounded with a rich vascular plexus, important in nasal physiology as a source for the mucus produced by the epithelial cells and for warming inhaled air. It is suggested that VEGF-C is important in the formation of the concheal venous plexus at the mucous membranes, and that it may also regulate the permeability of the vessels needed for the secretion of nasal mucus.

Possibly, VEGF-C and its derivatives, and antagonists, could be used in the regulation of the turgor of the 15 conchal tissue and mucous membranes and therefore the diameter of the upper respiratory tract, as well as the quantity and quality of mucus produced. These factors are of great clinical significance in inflammatory (including allergic) and infectious diseases of the upper 20 respiratory tract. Accordingly, the invention contemplates the use of the materials of the invention, including VEGF-C, Flt4, and their derivatives, in methods of diagnosing and treating inflammatory and infectious conditions affecting the upper respiratory tract, 25 including nasal structures.

Example 31 Characterization of the exon-intron organization of the human VEGF-C gene Two genomic DNA clones covering exons 1, 2, and 3 of the human VEGF-C gene were isolated from a human genomic DNA library using VEGF-C cDNA fragments as probes. In particular, a human genomic library in bacteriophage EMBL-3 lambda (Clontech) was screened using a PCR-generated fragment corresponding to nucleotides 629-746 of the human VEGF-C cDNA (SEQ ID NO: 32). One positive clone, designated "lambda was identified, and the insert was subcloned as a 14 kb XhoI fragment 94 W into the pBSK II vector (Stratagene). The genomic library also was screened with a labeled 130 bp NotI-SacI fragment from the 5'-noncoding region of the VEGF-C cDNA (the NotI site is in the polylinker of the cloning vector; the SacI site corresponds to nucleotides 92-97 of SEQ ID NO: 32). Two positive clones, designated "lambda and "lambda were obtained. Restriction mapping analysis showed that clone lambda 3 contains exons 2 and 3, while clone lambda 5 contains exon 1 and the putative promoter region.

Three genomic fragments containing exons 4, 6 and 7 were subcloned from a genomic VEGF-C P1 plasmid clone. In particular, purified DNA from a genomic P1 plasmid clone 7660 (Paavonen et al., Circulation, 93: 1079-1082 (1996)) was used. EcoRI fragments of the P1 insert DNA were ligated into pBSK II vector. Subclones of clone 7660 which contained human VEGF-C cDNA homologous sequences were identified by colony hybridization, using the full-length VEGF-C cDNA as a 20 probe. Three different genomic fragments were identified and isolated, which contained the remaining exons 4-7.

To determine the genomic organization, the clones were mapped using restriction endonuclease cleavage. Also, the coding regions and exon-intron 25 junctions were partially sequenced. The result of this analysis is depicted in Figures 11 and 17. The sequences of all intron-exon boundaries (Fig. 17, SEQ ID NOs: 57- 68) conformed to the consensus splicing signals (Mount, Nucl. Acids Res., 10: 459-472 (1982)). The length of the intron between exon 5 and 6 was determined directly by nucleotide sequencing and found to be 301 bp. The length of the intron between exons 2 and 3 was determined by restriction mapping and Southern hybridization and was found to be about 1.6 kb. Each of the other introns was over 10 kb in length.

A similar analysis was performed for the murine genomic VEGF-C gene. The sequences of murine VEGF-C 95 W intron-exon boundaries are depicted in Figure 17 and SEQ ID NOs: 69-80.

The restriction mapping and sequencing data indicated that the signal sequence and the first residues of the N-terminal propeptide are encoded by exon 1. The second exon encodes the carboxy-terminal portion of the N-terminal propeptide and the amino terminus of the VEGF homology domain. The most conserved sequences of the VEGF homology domain are distributed in exons 3 (containing 6 conserved cysteine residues) and 4 (containing 2 cys residues). The remaining exons encode cysteine-rich motifs of the type C-6X-C-10X-CRC (exons and 7) and a fivefold repeated motif of type C-6X-B-3X-C-C-C, which is typical of a silk protein.

15 To further characterize the VEGF-C gene promoter, the lambda 5 clone was further analyzed.

Restriction mapping of this clone using a combination of single- and double-digestions and Southern hybridizations .indicated that it includes: an approximately 5 kb 20 region upstream of the putative initiator ATG codon, (2) exon 1, and part of intron I of the VEGF-C gene.

A 3.7 kb Xba I fragment of clone lambda containing exon 1 and 5' and 3' flanking sequences, was subcloned and further analyzed. As reported previously, 25 a major VEGF-C mRNA band migrates at a position of about 2.4 kb. Calculating from the VEGF-C coding sequence of 1257 bp and a 391 bp 3' noncoding sequence plus a polyA sequence of about 50-200 bp, the mRNA start site should be located about 550-700 bp upstream of the translation initiation codon.

To further characterize the promoter of the human VEGF-C gene, a genomic clone encompassing about 1.4 kb upstream of the translation initiation site was isolated, and the 5' noncoding cDNA sequence and putative promoter region were sequenced. The sequence obtained is set forth in SEQ ID NO: 54. Similar to what has been observed with the VEGF gene, the VEGF-C promoter is rich a 96 in G and C residues and lacks consensus TATA and CCAAT sequences. Instead, it has numerous putative binding sites for Spl, a ubiquitous nuclear protein that can initiate transcription of TATA-less genes. See Pugh and Tjian, Genes and Dev., 5:105-119 (1991). In addition, sequences upstream of the VEGF-C translation start site were found to contain frequent consensus binding sites for the AP-2 factor. This suggests that the cAMP-dependent protein kinase and protein kinase C, as activators of AP-2 transcription factor [Curran and Franza, Cell, 55:395-397 (1988)], mediate

VEGF-C

transcriptional regulation.

The VEGF-C gene is abundantly expressed in adult human tissues, such as heart, placenta, ovary and 15 small intestine, and is induced by a variety of factors.

Indeed, several potential binding sites for regulators of tissue-specific gene expression, like NFkB and GATA, are located in the distal part of the VEGF-C promoter. For example, NFkB is known to regulate the expression of 20 tissue factor in endothelial cells. Also, transcription factors of the GATA family are thought to regulate cell-type specific gene expression.

Unlike VEGF, the VEGF-C gene does not contain a binding site for the hypoxia-inducible factor, HIF-1 25 (Levy et al., J. Biol. Chem., 270: 13333-13340 (1995)).

This finding suggests that if the VEGF-C mRNA is regulated by hypoxia, the mechanism would be based mainly on the regulation of mRNA stability. In this regard, numerous studies have shown that the major control point for the hypoxic induction of the VEGF gene is the regulation of the steady-state level of mRNA. See Levy et al., J. Biol. Chem., 271: 2746-2753 (1996). The relative rate of VEGF mRNA stability and decay is considered to be determined by the presence of specific sequence motifs in its 3' untranslated region

(UTR),

which have been demonstrated to regulate mRNA stability (Chen and Shyu, Mol. Cell Biol., 14: 8471-8482 (1994)).

The 3'-UTR of the VEGF-C gene also contains a putative 97 W motif of this type (TTATTT), at positions 1873-1878 of SEQ ID NO: 32.

Example 32 Identification of a VEGF-C splice variant As reported in Example 16, a major 2.4 kb VEGF- C mRNA and smaller amounts of a 2.0 kb mRNA are observable. To clarify the origin of these RNAs, several additional VEGF-C cDNAs were isolated and characterized.

A human fibrosarcoma cDNA library from HT1080 cells in the lambda gtll vector (Clontech, product #HL1048b) was screened using a 153 bp human VEGF-C cDNA fragment as a probe as described in Example 10. See also Joukov et al., EMBO 15:290-298 (1996). Nine positive clones were picked and analyzed by PCR amplification using 15 oligonucleotides 5'-CACGGCTTATGCAAGCAAAG-3' (SEQ ID NO: 55) and 5'-AACACAGTTTTCCATAATAG-3' (SEQ ID NO: 56) These oligonucleotides were selected to amplify the portion of the VEGF-C cDNA corresponding to nucleotides 495-1661 of SEQ ID NO: 32. PCR was performed using an annealing temperature of 55C and 25 cycles.

.The resultant PCR products were electrophoresed on agarose gels. Five clones out of the nine analyzed generated PCR fragments of the expected length of 1147 base pairs, whereas one was slightly shorter. The 25 shorter fragment and one of the fragments of expected length were cloned into the pCRTMII vector (Invitrogen) and analyzed by sequencing. The sequence revealed that the shorter PCR fragment had a deletion of 153 base pairs, corresponding to nucleotides 904 to 1055 of SEQ ID NO: 32. These deleted bases correspond to exon 4 of the human and mouse VEGF-C genes, schematically depicted in Fig. 17. Deletion of exon 4 results in a frameshift, which in turn results in a C-terminal truncation of the full-length VEGF-C precursor, with fifteen amino acid residues translated from exon 5 in a different frame than the frame used to express the full-length protein. Thus, 98 the C-terminal amino acid sequence of the resulting truncated polypeptide would be Leu 18 1)-Ser-Lys-Thr- Val-Ser-Gly-Ser-Glu-Gln-Asp-Leu-Pro-His-Glu-Leu-His-Val- Glu(199) (SEQ ID NO: 81). The VEGF-C variant encoded by this splice variant would not contain the C-terminal cleavage site of the VEGF-C precursor. Thus, a putative alternatively spliced RNA form lacking conserved exon 4 was identified in HT-1080 fibrosarcoma cells and this form is predicted to encode a protein of 199 amino acid residues, which could be an antagonist of VEGF-C.

Example 33 VEGF-C is similarly processed in different cell cultures in vitro 15 To study whether VEGF-C is similarly processed in different cell types, 293 EBNA cells, COS-1 cells and HT-1080 cells were transfected with wild type human VEGF- C cDNA and labelled with Pro-MixTM as described in Example 22. The conditioned media from the cultures were col- 20 lected and subjected to immunoprecipitation using antiserum 882 (described in Example 21, recognizing a peptide corresponding to amino acids 104-120 of SEQ ID NO: 33). The immunoprecipitated polypeptides were separated via SDS-PAGE, and detected via autoradiography. The major form of secreted recombinant VEGF-C observed from all cell lines tested is a 29/32 kD doublet. These two polypeptides are bound to each other by disulfide bonds, as described in Example 22. A less prominent band of approximately 21 kD also was detected in the culture media.

Additionally, a non-processed VEGF-C precursor of 63 kDa was observed. This form was more prominent in the COS-1 cells, suggesting that proteolytic processing of VEGF-C in COS cells is less efficient than in 293 EBNA cells.

Endogenous VEGF-C (in non-transfected cells) was not detectable under these experimental conditions in the HT-1080 cells, but was readily detected in the conditioned medium of the PC-3 cells. Analysis of the subunit 99 polypeptide sizes and ratios in PC-3 cells and 293 EBNA cells revealed strikingly similar results: the most prominent form was a doublet of 29/32 kDa and a less prominent form the 21 kD polypeptide. The 21 kD form produced by 293 EBNA cells was not recognized by the 882 antibody in'the Western blot, although it is recognized when the same antibody is used for immunoprecipitation (see data in previous examples). As reported in Example 21, cleavage of the 32 kD form in 293 EBNA cells occurs between amino acid residues 111 and 112 (SEQ ID NO: 33), downstream of the cleavage site in PC-3 cells (between residues 102 and 103). Therefore, the 21 kD form produced in 293 EBNA cells does not contain the complete N-terminal peptide used to generate antiserum 882.

15 In a related experiment, PC-3 cells were cultured in serum-free medium for varying periods of time (1 8 days) prior to isolation of the conditioned medium. The conditioned medium was concentrated using a Centricon device (Amicon, Beverly, USA) and subjected to 20 Western blotting analysis using antiserum 882. After one day of culturing, a prominent 32 kD band was detected.

Increasing amounts of a 21-23 kD form were detected in the conditioned media from 4 day and 8 day cultures. The diffuse nature of this polypeptide band, which is simply called the 23 kD polypeptide in example 5 and several o: subsequent examples is most likely due to a heterogenous and variable amount of glycosylation. These results indicate that, initially, the cells secrete a 32 kD polypeptide, which is further processed or cleaved in the medium to yield the 21-23 kD form. The microheterogeneity of this polypeptide band would then arise from the variable glycosylation degree and, from microheterogeneity of the processing cleavage sites, such as obtained for the amino terminus in PC-3 and 293 EBNA cell cultures. The carboxyl terminal cleavage site could also vary, examples of possible cleavage sites would be between residues 225-226, 226-227 and 227-228 as well as between residues 216-217.

s= 100 Taken together, these data suggest the possibility that secreted cellular protease(s) are responsible for the generation of the 21-23 kD form of VEGF-C from the 32 kD polypeptide. Such proteases could be used in vitro to cleave VEGF-C precursor proteins in solution during the production of VEGF-C, or used in cell culture and in vivo to release biologically active VEGF-C.

Example 34 Differential binding of VEGF-C forms by the extracellular domains of VEGFR-3 and VEGFR-2 In two parallel experiments, 293 EBNA cells were transfected with a construct encoding recombinant wild type VEGF-C or a construct encoding VEGF-C DNDCHis 15 (Example 28) and about 48 hours after transfection, metabolically labelled with Pro-MixTM as described in ~previous examples. The media were collected from mocktransfected and transfected cells and used for receptor binding analyses.

20 Receptor binding was carried out in binding buffer (PBS, 0.5% BSA, 0.02% Tween 20, 1 microgram/ml heparin) containing approximately 0.2 microgram of either a fusion protein comprising a VEGFR-3 extracellular domain fused to an immunoglobulin sequence (VEGFR-3-Ig) or a fusion protein comprising VEGFR-2 extracellular domain fused to an alkaline phosphatase sequence (VEGF- R-2-AP; Cao et al., J. Biol. Chem. 271:3154-62 (1996)).

As a control, similar aliquots of the 293 EBNA conditioned media were mixed with 2 pl of anti-VEGF-C antiserum (VEGF-C IP).

After incubation for 2 hours at room temperature, anti-VEGF-C antibodies and VEGFR-3-Ig protein were adsorbed to protein A-sepharose (PAS) and VEGFR-2-AP was immunoprecipitated using anti-AP monoclonal antibodies (Medix Biotech, Genzyme Diagnostics, San Carlos, CA, USA) and protein G-sepharose. Complexes containing

VEGF-C

bound to VEGFR-3-Ig or VEGFR-2-AP were washed three times 101 in binding buffer, twice in 20 mM Tris-HCl (pH 7.4) and VEGF-C immunoprecipitates were washed three times in RIPA buffer and twice in 20 mM tris-HCl (pH 7.4) and analyzed via SDS-PAGE under reducing and nonreducing conditions.

As a control, the same media were precipitated with anti- AP and protein G-sepharose (PGS) or with PAS to control for possible nonspecific adsorption.

These experiments revealed that VEGFR-3 bound to both the 32/29 kD and 21-23 kD forms of recombinant VEGF-C, whereas VEGFR-2 bound preferentially to the 21-23 kD component from the conditioned media. In addition, small amounts of 63 kD and 52 kD VEGF-C forms were observed binding with VEGFR-3. Further analysis under nonreducing conditions indicates that a great proportion of the 15 21-23 kD VEGF-C bound to either receptor does not contain interchain disulfide bonds. These findings reinforce the results that VEGF-C binds VEGFR-2. This data suggests a utility for recombinant forms of VEGF-C which are active towards VEGFR-3 only or which are active towards both 20 VEGFR-3 and VEGFR-2. On the other hand, these results, S. together with the results in Example 28, do not eliminate the possibility that the 32/29 kD dimer binds VEGFR-3 but does not activate it. The failure of the 32/29 kD dimer to activate VEGFR-3 could explain the finding that conditioned medium from the N-His VEGF-C transfected cells induced a less prominent tyrosine phosphorylation of o VEGFR-3 than medium from VEGF-C DNDCHis transfected cells, even though expression of the former polypeptide was much higher (see Figs. 27 and 28). Stable VEGF-C polypeptide variants that bind to a VEGF-C receptor but fail to activate the receptor are useful as VEGF-C antagonists.

102 Deposit of Biological Materials: Plasmid FLT4- L has been deposited with the American Type Culture Collection (ATCC), 12301 Parklawn Dr., Rockville MD 20952 (USA), pursuant to the provisions of the Budapest Treaty, and has been assigned a deposit date of 24 July 1995 and ATCC accession number 97231.

While the present invention has been described in terms of specific embodiments, it is understood that variations and modifications will occur to those in the art. Accordingly, only such limitations as appear in the appended claims should be placed on the invention.

e *o*oo* o* *e ;'2b EDITORIAL NOTE No.10072/00 This following sequence listing form part of the specification.

Claims commence on page 135 103 SEQUENCE LISTING GENERAL INFORMATION: APPLICANT: Helsinki University Licensing Ltd Oy (ii) TITLE OF INVENTION: Receptor Ligand VEGF-C (iii) NUMBER OF SEQUENCES: 81 (iv) CORRESPONDENCE

ADDRESS:

ADDRESSEE: Oy Jalo Ant-Wuorinen Ab STREET: Iso Roobertinkatu 4-6 A CITY: Helsinki COUNTRY: Finland ZIP: 00120 COMPUTER READABLE FORM: MEDIUM TYPE: Floppy disk COMPUTER: IBM PC compatible OPERATING SYSTEM: PC-DOS/MS-DOS SOFTWARE: PatentIn Release Version #1.30 (vi) CURRENT APPLICATION DATA: APPLICATION NUMBER: FILING DATE:

CLASSIFICATION:

•(vii) PRIOR APPLICATION

DATA:

APPLICATION NUMBER: 08/671,573 FILING DATE: 28-JUN-1996 (vii) PRIOR APPLICATION

DATA:

APPLICATION NUMBER: 08/601,132 FILING DATE: 14-FEB-1996 (vii) PRIOR APPLICATION DATA: APPLICATION NUMBER: 08/585,895 FILING DATE: 12-JAN-1996 (vii) PRIOR APPLICATION

DATA:

APPLICATION NUMBER: 08/510,133 FILING DATE: 01-AUG-1995 (viii) ATTORNEY/AGENT

INFORMATION:

NAME: Karvinen, Leena REFERENCE/DOCKET NUMBER: 28999 (ix) TELECOMMUNICATION

INFORMATION:

TELEPHONE: +358 0 648 606 TELEFAX: +358 0 640 575 TELEX: 123505 JALO FI INFORMATION FOR SEQ ID NO:l: SEQUENCE CHARACTERISTICS: LENGTH: 20 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA (xi) SEQUENCE DESCRIPTION: SEQ ID NO:1: TGTCCTCGCT GTCCTTGTCT a 104- INFORMATION FOR SEQ ID NO:2: SEQUENCE

CHARACTERISTICS:

LENGTH: 70 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA (xi) SEQUENCE DESCRIPTION: SEQ ID NO:2: ACATGCATGC CACCATGCAG CGGGGCGCCG CGCTGTGCCT GCGACTGTGG CTCTGCCTGG

GACTCCTGGA

INFORMATION FOR SEQ ID NO:3: SEQUENCE CHARACTERISTICS: LENGTH: 24 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA (xi) SEQUENCE DESCRIPTION: SEQ ID NO:3: ACATGCATGC CCCGCCGGTC ATCC 24 INFORMATION FOR SEQ ID NO:4: SEQUENCE CHARACTERISTICS: LENGTH: 22 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA (xi) SEQUENCE DESCRIPTION: SEQ ID NO:4: CGGAATTCCC CATGACCCCA AC 22 INFORMATION FOR SEQ ID SEQUENCE CHARACTERISTICS: LENGTH: 33 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA (xi) SEQUENCE DESCRIPTION: SEQ ID CCATCGATGG ATCCTACCTG AAGCCGCTTT CTT 33 INFORMATION FOR SEQ ID NO:6: SEQUENCE CHARACTERISTICS: LENGTH: 17 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA SEQUENCE DESCRIPTION: SEQ ID NO:6: 105 ATTTAGGTGA CACTATA 17 INFORMATION FOR SEQ ID NO:7: SEQUENCE CHARACTERISTICS: LENGTH: 34 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA (xi) SEQUENCE DESCRIPTION: SEQ ID NO:7: CCATCGATGG ATCCCGATGC TGCTTAGTAG CTGT 34 INFORMATION FOR SEQ ID NO:8: SEQUENCE CHARACTERISTICS: LENGTH: 40 amino acids TYPE: amino acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: peptide (xi) SEQUENCE DESCRIPTION: SEQ ID NO:8: Pro Met Thr Pro Thr Thr Tyr Lys Gly Ser Val Asp Asn Gln Thr Asp 1 5 10 Ser Gly Met Val Leu Ala Ser Glu Glu Phe Glu Gln Ile Glu Ser Arg 25 His Arg Gln Glu Ser Gly Phe Arg INFORMATION FOR SEQ ID NO:9: SEQUENCE CHARACTERISTICS: LENGTH: 21 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear MOLECULE TYPE: cDNA (xi) SEQUENCE DESCRIPTION: SEQ ID NO:9: CTGGAGTCGA CTTGGCGGAC T 21 INFORMATION FOR SEQ ID SEQUENCE CHARACTERISTICS: LENGTH: 60 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA (xi) SEQUENCE DESCRIPTION: SEQ ID CGCGGATCCC TAGTGATGGT GATGGTGATG TCTACCTTCG ATCATGCTGC CCTTATCCTC INFORMATION FOR SEQ ID NO:11: S(i) SEQUENCE CHARACTERISTICS: LENGTH: 34 base pairs TYPE: nucleic acid 106 STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA (xi) SEQUENCE DESCRIPTION: SEQ ID NO:11: CCCAAGCTTG GATCCAAGTG GCTACTCCAT GACC 34 INFORMATION FOR SEQ ID NO:12: SEQUENCE CHARACTERISTICS: LENGTH: 20 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA (xi) SEQUENCE DESCRIPTION: SEQ ID NO:12: GTTGCCTGTG ATGTGCACCA INFORMATION FOR SEQ ID NO:13: SEQUENCE CHARACTERISTICS: LENGTH: 18 amino acids TYPE: amino acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: peptide (xi) SEQUENCE DESCRIPTION: SEQ ID NO:13: Xaa Glu Glu Thr Ile Lys Phe Ala Ala Ala His Tyr Asn Thr Glu Ile 1 5 10 Leu Lys S(2) INFORMATION FOR SEQ ID NO:14: SEQUENCE CHARACTERISTICS: LENGTH: 17 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA (xi) SEQUENCE DESCRIPTION: SEQ ID NO:14: GCAGARGARA CNATHAA 17 INFORMATION FOR SEQ ID SEQUENCE CHARACTERISTICS: LENGTH: 5 amino acids TYPE: amino acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: peptide (xi) SEQUENCE DESCRIPTION: SEQ ID Glu Glu Thr Ile Lys -1 107 0 INFORMATION FOR SEQ ID NO:16: SEQUENCE CHARACTERISTICS: LENGTH: 18 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA (xi) SEQUENCE DESCRIPTION: SEQ ID NO:16: GCAYTTNARD ATYTCNGT INFORMATION FOR SEQ ID NO:17: SEQUENCE CHARACTERISTICS: LENGTH: 5 amino acids TYPE: amino acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: peptide (xi) SEQUENCE DESCRIPTION: SEQ ID NO:17: Thr Glu Ile Leu Lys 1 INFORMATION FOR SEQ ID NO:18: SEQUENCE CHARACTERISTICS: LENGTH: 22 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA (xi) SEQUENCE DESCRIPTION: SEQ ID NO:18: ATTCGCTGCA GCACACTACA AC INFORMATION FOR SEQ ID NO:19: SEQUENCE CHARACTERISTICS: LENGTH: 19 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA (xi) SEQUENCE DESCRIPTION: SEQ ID NO:19: TCNGTGTTGT AGTGTGCTG INFORMATION FOR SEQ ID SEQUENCE CHARACTERISTICS: LENGTH: 7 amino acids TYPE: amino acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: peptide SEQUENCE DESCRIPTION: SEQ ID *-Ala Ala His Tyr Asn Thr Glu 108 9 INFORMATION FOR SEQ ID NO:21: SEQUENCE CHARACTERISTICS: LENGTH: 20 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA (xi) SEQUENCE DESCRIPTION: SEQ ID NO:21: TAATACGACT CACTATAGGG INFORMATION FOR SEQ ID NO:22: SEQUENCE CHARACTERISTICS: LENGTH: 24 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA (xi) SEQUENCE DESCRIPTION: SEQ ID NO:22: GTTGTAGTGT GCTGCAGCGA ATTT INFORMATION FOR SEQ ID NO:23: SEQUENCE CHARACTERISTICS: LENGTH: 8 amino acids TYPE: amino acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: peptide (xi) SEQUENCE DESCRIPTION: SEQ ID NO:23: Lys Phe Ala Ala Ala His Tyr Asn 1 INFORMATION FOR SEQ ID NO:24: SEQUENCE CHARACTERISTICS: LENGTH: 21 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA (xi) SEQUENCE DESCRIPTION: SEQ ID NO:24: TCACTATAGG GAGACCCAAG C INFORMATION FOR SEQ ID SEQUENCE CHARACTERISTICS: LENGTH: 219 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA Jxi) SEQUENCE DESCRIPTION: SEQ ID 109 TCACTATAGG GAGACCCAAG CTTGGTACCG AGCTCGGATC CACTAGTAAC GGCCGCCAGT GTGGTGGAAT TCGACGAACT CATGACTGTA CTCTACCCAG AATATTGGAA AATGTACAAG 120 TGTCAGCTAA GGCAAGGAGG CTGGCAACAT AACAGAGAAC AGGCCAACCT CAACTCAAGG 180 ACAGAAGAGA CTATAAAATT CGCTGCAGCA CACTACAAC 219 INFORMATION FOR SEQ ID NO:26: SEQUENCE CHARACTERISTICS: LENGTH: 18 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA (xi) SEQUENCE DESCRIPTION: SEQ ID NO:26: ACAGAGAACA GGCCAACC 18 INFORMATION FOR SEQ ID NO:27: SEQUENCE CHARACTERISTICS: LENGTH: 19 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA (xi) SEQUENCE DESCRIPTION: SEQ ID NO:27: TCTAGCATTT AGGTGACAC 19 INFORMATION FOR SEQ ID NO:28: SEQUENCE CHARACTERISTICS: LENGTH: 25 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA (xi) SEQUENCE DESCRIPTION: SEQ ID NO:28: AAGAGACTAT AAAATTCGCT GCAGC INFORMATION FOR SEQ ID NO:29: SEQUENCE CHARACTERISTICS: LENGTH: 20 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA (xi) SEQUENCE DESCRIPTION: SEQ ID NO:29: CCCTCTAGAT GCATGCTCGA INFORMATION FOR SEQ ID SEQUENCE CHARACTERISTICS: LENGTH: 24 base pairs TYPE: nucleic acid STRANDEDNESS: single 110

S

TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA (xi) SEQUENCE DESCRIPTION: SEQ ID GTTGTAGTGT GCTGCAGCGA

ATTT

INFORMATION FOR SEQ ID NO:31: SEQUENCE CHARACTERISTICS: LENGTH: 21 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA (xi) SEQUENCE DESCRIPTION: SEQ ID NO:31: TCACTATAGG GAGACCCAAG

C

INFORMATION FOR SEQ ID NO:32: SEQUENCE CHARACTERISTICS: LENGTH: 1997 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA (ix) FEATURE: NAME/KEY: CDS LOCATION: 352..1608 (xi) SEQUENCE DESCRIPTION: SEQ ID NO:32: CCCGCCCCGC CTCTCCAAAA AGCTACACCG ACGCGGACCG CGGCGGCGTC CTCGCTTCAC CTCGCGGGCT CCGAATGCGG GGAGCTCGGA

TGTCCGGTTT

TTTTACCTGA CACCCGCCGC CTTTCCCCGG CACTGGCTGG GAGGGCGCCC GGAACGCGGA GCCCCGGACC CGCTCCCGCC GCCTCCGGCT

CGCCCAGGGG

GAGGAGCCCG GGGGAGAGGG ACCAGGAGGG GCCCGCGGCC TCGCAGGGGC CCACCCCTGC CCCCGCCAGC GGACCGGTCC CCCACCCCCG GTCCTTCCAC

S

S

S

CTCCCTCGCC

CCTGTGAGGC

TGCAAAGTTG

GGGTCGCCGG

GCCCGCGCCC

C ATG CAC Met His 1 120 180 240 300 357 405 453 501 549 TTG CTG GGC TTC TTC TCT GTG GCG TGT TCT CTG CTC GCC GCT GCG CTG Leu Leu Gly Phe Phe Ser Val Ala Cys Ser Leu Leu Ala Ala Ala Leu 10 CTC CCG GGT CCT CGC GAG GCG CCC GCC GCC GCC GCC GCC TTC GAG TCC Leu Pro Gly Pro Arg Glu Ala Pro Ala Ala Ala Ala Ala Phe Glu Ser 25 GGA CTC GAC CTC TCG GAC GCG GAG CCC GAC GCG GGC GAG GCC ACG GCT Gly Leu Asp Leu Ser Asp Ala Glu Pro Asp Ala Gly Glu Ala Thr Ala 40 45 TAT GCA AGC AAA GAT CTG GAG GAG CAG TTA CGG TCT GTG TCC AGT GTA Tyr Ala Ser Lys Asp Leu Glu Glu Gln Leu Arg Ser Val Ser Ser Val 60 ill GAT GAA CTC Asp Glu Leu ACT GTA CTC TAC Thr Val Leu Tyr CCA GAA TAT TGG AAA ATG TAC AAG Pro Giu Tyr Trp Lys Met Tyr Lys 0 TGT CAG Cys Gin CTC AAC Leu Asn 100 CTA AGO AAA GGA GGC Leu Arg Lys Gly Gly

TGG

Trp 90 CAA CAT AAC AGA Gin His Asn Arg

GAA

Glu CAG GCC AAC Gin Ala Asn TCA AGO ACA GAA Ser Arg Thr Giu

GAG

Giu 105 ACT ATA AAA Thr Ile Lys TTT GCT Phe Ala 110 OCA GCA CAT TAT Ala Ala His Tyr

AAT

Asn 115 ACA GAG ATC TTG Thr Giu Ile Leu

AAA

Lys 120 AGT ATT GAT AAT Ser Ile Asp Asn

GAG

Glu 125 TGG AGA AAG ACT Trp Arg Lys Thr

CAA

Gin 130 TGC ATG CCA CGO Cys Met Pro Arg

GAG

Giu 135 GTG TGT ATA GAT Val Cys Ile Asp

GTG

Val 140 GGO AAG GAG TTT Gly Lys Oiu Phe GGA GTC Oly Val 145 GCG ACA AAC Ala Thr Asn GOG GOT TGC Gly Gly Cys 165

ACC

Thr 150 TTC TTT AAA CCT Phe Phe Lys Pro

CCA

Pro 155 TGT GTG TCC GTC Cys Val Ser Val TAC AGA TOT Tyr Arg Cys 160 ACC AGC ACG Thr Ser Thr 4 4

S.

6.@S 0 *550 5055.5 0 TGC AAT AGT GAG Cys Asn Ser Giu CTG CAG TGC ATG Leu Gin Cys Met

AAC

Asn 175 597 645 693 741 789 837 885 933 981 1029 1077 1125 1173 AGC TAC Ser Tyr 180 CTC AGC AAG ACG Leu Ser Lys Thr

TTA

Leu 185 TTT GAA ATT ACA Phe Giu Ile Thr CCT CTC TCT CAA Pro Leu Ser Gin

GGC

Oly 195 CCC AAA CCA GTA Pro Lys Pro Vai

ACA

Thr 200 ATC AGT TTT GCC Ile Ser Phe Ala

AAT

Asn 205 CAC ACT TCC TOC His Thr Ser Cys

CGA

Arg 210 TGC ATG TCT AAA Cys Met Ser Lys CTG GAT GTT TAC AGA CAA GTT CAT TCC ATT ATT AGA Leu Asp Val Tyr Arg Gin Val His Ser Ile Ile Arg 215 220 225 *SSSSq

SSS*

5* S 5 4 5 COT TCC CTG Arg Ser Leu TGC CCC ACC Cys Pro Thr 245

CCA

Pro 230 GCA ACA CTA CCA Ala Thr Leu Pro

CAG

Gin 235 TGT CAG GCA GCG Cys Gin Ala Ala AAC AAG ACC Asn Lys Thr 240 TGC CTG OCT Cys Leu Ala AAT TAC ATG TGG Asn Tyr Met Trp

AAT

Asn 250 AAT CAC ATC TGC Asn His Ile Cys

AGA

Arg 255 CAG GAA Gin Giu 260 GAT TTT ATG TTT Asp Phe Met Phe

TCC

Ser 265 TCG GAT OCT OGA Ser Asp Ala Gly GAC TCA ACA OAT Asp Ser Thr Asp

OGA

Gly 275 TTC CAT GAC ATC Phe His Asp Ile TOT GOA CCA AAC AAG GAG CTG OAT OAA GAG ACC Cys Gly Pro Asn Lys Giu Leu Asp Oiu Oiu Thr 280 285 290 TOT CAG TOT GTC TGC AGA OCO GOG CTT CG Cys Gin Cys Val Cys Arg Ala Gly Leu Arg 295 300 CCT 0CC AGC TOT Pro Ala Ser Cys OGA CCC Gly Pro 305 1221 1269 1317 1365 CAC AAA GAA His Lys Giu CTC TTC CCC Leu Phe Pro 325

CTA

Leu 310 GAC AGA AAC TCA Asp Arg Asn Ser

TOC

Cys 315 CAG TGT OTC TOT Gin Cys Val Cys AAA AAC AAA Lys Asn Lys 320 OAA AAC ACA Olu Asn Thr AGC CAA TGT 000 Ser Gin Cys Gly

GCC

Ala 330 AAC CGA GAA TTT Asn Arg Giu Phe

OAT

Asp 335 -112- TGC CAG TGT GTA TGT AAA AGA ACC TGC CCC AGA AAT CAA CCC CTA AAT 1413 Cys Gin Cys Val Cys Lys Arg Thr Cys Pro Arg Asn Gin Pro Leu Asn 340 345 350 CCT GGA AAA TGT GCC TGT GAA TGT ACA GAA AGT CCA CAG AAA TGC TTG 1461 Pro Gly Lys Cys Ala Cys Glu Cys Thr Glu Ser Pro Gin Lys Cys Leu 355 360 365 370 TTA AAA GGA AAG AAG TTC CAC CAC CAA ACA TGC AGC TGT TAC AGA CGG 1509 Leu Lys Gly Lys Lys Phe His His Gin Thr Cys Ser Cys Tyr Arg Arg 375 380 385 CCA TGT ACG AAC CGC CAG AAG GCT TGT GAG CCA GGA TTT TCA TAT AGT 1557 Pro Cys Thr Asn Arg Gin Lys Ala Cys Glu Pro Gly Phe Ser Tyr Ser 390 395 400 GAA GAA GTG TGT CGT TGT GTC CCT TCA TAT TGG AAA AGA CCA CAA ATG 1605 Glu Glu Val Cys Arg Cys Val Pro Ser Tyr Trp Lys Arg Pro Gin Met 405 410 415 AGC TAAGATTGTA CTGTTTTCCA GTTCATCGAT TTTCTATTAT GGAAAACTGT 1658 Ser GTTGCCACAG TAGAACTGTC TGTGAACAGA GAGACCCTTG TGGGTCCATG CTAACAAAGA 1718 :CAAAAGTCTG TCTTTCCTGA ACCATGTGGA TAACTTTACA GAAATGGACT GGAGCTCATC 1778 TGCAAAAGGC CTCTTGTAAA GACTGGTTTT CTGCCAATGA CCAAACAGCC AAGATTTTCC 1838 TCTTGTGATT TCTTTAAAAG AATGACTATA TAATTTATTT CCACTAAAAA TATTGTTTCT 1898 505GCATTCATTT TTATAGCAAC AACAATTGGT AAAACTCACT GTGATCAATA TTTTTATATC 1958 ATGCAAAATA TGTTTAAAAT AAAATGAAAA TTGTATTAT 1997 INFORMATION FOR SEQ ID NO:33: SEQUENCE CHARACTERISTICS: LENGTH: 419 amino acids TYPE: amino acid TOPOLOGY: linear (ii) MOLECULE TYPE: protein (xi) SEQUENCE DESCRIPTION: SEQ ID NO:33: *so Met His Leu Leu Giy k-e k-ne Ser Val Ala Cys Ser Leu Leu Ala Ala 0 1 5 10 Ala Leu Leu Pro Gly Pro Arg Glu Ala Pro Ala Ala Ala Ala Ala Phe 25 Glu Ser Gly Leu Asp Leu Ser Asp Ala Glu Pro Asp Ala Gly Glu Ala 40 Thr Ala Tyr Ala Ser Lys Asp Leu Glu Glu Gin Leu Arg Ser Val Ser 55 Ser Val Asp Glu Leu Met Thr Val Leu Tyr Pro Glu Tyr Trp Lys Met 70 75 Tyr Lye Cys Gin Leu Arg Lye Gly Gly Trp Gin His Asn Arg Glu Gin 90 Ala Asn Leu Asn Ser Arg Thr Glu Glu Thr Ile Lye Phe Ala Ala Ala 100 105 110 His Tyr Asn Thr Glu Ile Leu Lye Ser Ile Asp Asn Glu Trp Arg Lye 115 120 125 113 Thr Gin Cys Met Pro Arg 130 Glu Val Cys Ile Asp Val Gly 145 Arg Ser Ser Cys Ile 225 Lys Leu Thr Giu Gly 305 Asn Asn Leu Cys Arg 385 Tyr Gin Vai Cys Thr Gin Arg 210 Arg Thr Aia Asp Thr 290 Pro Lys Thr Asn Leu 370 Arg Ser Met Al a Giy Ser Gly 195 Cys Arg Cys Gin Giy 275 Cys His Leu Cys Pro 355 Leu Pro Giu Ser Thr Gly Tyr 180 Pro Met Ser Pro Giu 260 Phe Gin Lys Phe Gin 340 Gly Lys Cys Giu Asn Cys 165 Leu Lys Ser Leu Thr 245 Asp His Cys Giu Pro 325 Cys Lys Gly Thr Val 405 Thr Phe iso Cys Asn Ser Lys Pro Val Lys Leu 215 Pro Aia 230 Asn Tyr Phe Met Asp Ile Val Cys 295 Leu Asp 310 Ser Gin Val Cys Cys Ala Lys Lys 375 Asn Arg 390 Cys Arg Phe Ser Thr Thr 200 Asp Thr Met Phe Cys 280 Arg Arg Cys Lys Cys 360 Phe Gin Cys Lys Giu Leu 185 Ile Vai Leu Trp Ser 265 Giy Ala Asn Gly Arg 345 Giu His Lys Val Pro Gly 170 Phe Ser Tyr Pro Asn 250 Ser Pro Gly Ser Al a 330 Thr Cys His Al a Pro 410 Pro 155 Leu Giu Phe Arg Gin 235 Asn Asp Asn Leu Cys 315 Asn Cys Thr Gin Cys 395 Ser 140 Cys Gin Ile Ala Gin 220 Cys His Al a Lys Arg 300 Gin Arg Pro Glu Thr 380 Giu Tyr Gly Lys Val Ser Cys Met Thr Val 190 Asn His 205 Val His Gin Ala Ile Cys Gly Asp 270 Giu Leu 285 Pro Ala Cys Vai Glu Phe Arg Asn 350 Ser Pro 365 Cys Ser Pro Gly Trp Lys Giu Phe Val Tyr 160 Asn Thr 175 Pro Leu Thr Ser Ser Ile Ala Asn 240 Arg Cys 255 Asp Ser Asp Giu Ser Cys Cys Lys 320 Asp Glu 335 Gin Pro Gin. Lys Cys Tyr Phe Ser 400 Arg Pro 415 INFORMATION FOR SEQ ID NO:34: Wi SEQUENCE CHARACTERISTICS: LENGTH: 22 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA (xi) SEQUENCE DESCRIPTION: SEQ ID NO:34:

TGAGTGATTTGTAGCTGCTGTG

114 INFORMATION FOR SEQ ID SEQUENCE CHARACTERISTICS: LENGTH: 22 base pairs TYPE: nucleic acid STRAN'DEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA (xi) SEQUENCE DESCRIPTION: SEQ ID

TATTGCAGCAACCCCCACATCT

INFORMATION FOR SEQ ID NO:36: SEQUENCE CHARACTERISTICS: LENGTH: 4416 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA (xi) SEQUENCE DESCRIPTION: SEQ ID NO:36: CCACGCGCAG CGGCCGGAGA TGCAGCGGGG CGCCGCGCTG TGCCTGCGAC

CCTGGGACTC

CACGGAGGAG

GCACCCCCTC

CAGCGAGGAC

GGTGTTGCTG

GTACATCAAG

CTTTGAGCAG

GTGGGTGCCC

GGTGCTGTGG

CACGCCACTG

CTTCCTTTCC

GTTGCCCAGG

GTGGGCTGAG

GCGGGGTAAG

CCTGACCATC

CGGCATCCAG

CGTCGAGTGG

GCCCGTGAAG

ACTGTCCGGG

AGGCACCTAC

GGAGCTGGTG

CTGGACGGCC

TCACACGTCA

GAGTGGGCTT

ACGGGGGTGG

CTGCACGAGG

GCACGCATCG

CCATTCATCA

TGTCTGGTGT

CCAGACGGGC

CTGCACGATG

AACCCCTTCC

AAGTCGCTGG

TTTAACTCAG

TGGGTGCCCG

CACAACGTCA

CGATTTCGGG

CTCAAAGGAC

CTGGCAGCGT

CGCCACAGTC

ACCCTCGCCC

GTGAATGTGC

TGGTGAGTGG

TCGACACCGG

GGCCAGGAGC

TGCGAGACTG

TACATGCCAA

AGGGCACCAC

ACAAGCCTGA

CCATCCCCGG

AGGAGGTGGT

CCCTGTACCT

TGGTGCACAT

AGCTGCTGGT

GTGTCACCTT

AGCGACGCTC

GCCAGCACGA

AGAGCACCGA

CCATCCTGGA

ACCCCCCGCC

CACATGCCCT

TGTGGAACTC

CCCCCCAGAT

CTACTCCATG

TGACAGCCTG

TCAGGAGGCG

CGAGGGCACA

CGACACAGGC

GGCCGCCAGC

CACGCTCTTG

CCTCAATGTC

GTGGGATGAC

GCAGTGCGAG

CACAGGCAAC

AGGGGAGAAG

TGACTGGGAC

CCAGCAGACC

CCTGGGCTCG

GGTCATTGTG

GGCCACGGCA

CGAGTTCCAG

GGTGCTCAAG

CGCTGCTGGC

ACATGAGAAG

ACCCCCCCGA

TCCATCTCCT

CCAGCCACCG

GACGCCAGGC

AGCTACGTCT

TCCTACGTGT

GTCAACAGGA

ACGCTGCGCT

CGGCGGGGCA

ACCACCTGGG

GAGCTCTATG

CTGGTCCTGA.

TACCCAGGGA

CACACAGAAC

TATGTGTGCA

CATGAAAATC

GGAGACGAGC

TGGTACAAGG

GAGGTGACAG

CTGAGGCGCA

GAGGCCTCCT

TGTGGCTCTG

CCTTGAACAT

GCAGGGGACA

GAGACAAGGA

CCTACTGCAA

GCTACTACAA

TCGTGAGAGA

AGGACGCCAT

CGCAAAGCTC

TGCTCGTGTC

GAGACCAGGA

ACATCCAGCT

ACTGCACCGT

AGCAGGCAGA

TCTCCAGCAT

AGGCCAACAA

CCTTCATCAG

TGGTGAAGCT

ATGGAAAGGC

AGGCCAGCAC

ACATCAGCCT

CCCCCAGCAT

120 180 240 300 360 420 480 540 600 660 720 780 840 900 960 1020 1080 1140 1200 1260 1320 115 AGGCCCTCAC CTGCACGGCC CTACTCGCGT CACAGCCGCC TACGGGGTGC CCCTGCCTCT

CAGCATCCAG

CCGGCGGCGG

GCAGGATGCC

GAATAAGACT

TGTGGTCTCC

CCCCGACGGC

GCTCCTGAGC

CCTGTCCACG

TCTGTTCGCC

CACGCTCAGC

AGTGCAAGAC

CCTGGAAGCC

GCTGGAGATG

CGAGAGGCTG

CATCCAGCGC

GGGCTGCGTC

GGAGATCGTG

CCTCATCTTC

CATCATCATG

TGCCAGCCAG

CGCCTTCGGG

CACCGTGGCC

GTCGGAGCTC

GGCGTGCACC

CCTCTCCAAC

CGAGCAGCGC

GGGGAGCAGC

GGCTTCTCCA

TGTCTGCTAC

CCACAGAGAC

TGACTTTGGC

CCGGCTGCCC

GAGTGACGTG

GTACCCTGGG

TGGCACTGGC

CAGCAGCAAG

GTGAACCCCA

GTGAGCAAGC

'AACAAGGTGG

TTCACCATCG

TGCCAAGCCG

CTGCACGATG

ACCCCTCTGG

CTGAGTATCC

CGGCGCAGCC

CCTCGGCTCA

CAGTGCTTGG

CTGGAGGAAA

GTGCGCGAGG

AACTCCTCCG

ATCCTTGTCG

TGTAACATGA

GACCCCGGGG

TGGGAATTCC

AAGGTGGTGG

GTGAAAATGC

AAGATCCTCA

AAGCCGCAGG

TTCCTGCGCG

GGACGCTTCC

GACAGGGTCC

GACCAAGAAG

AGCTTCCAGG

CTGGCTGCTC

CTTGCCCGGG

CTGAAGTGGA

TGGTCCTTTG

GTGCAGATCA

GGCCCTGGAC

ACCTCATGCC

TCGAGAGCCT

TGGTGATCCA

GCCAGGATGA

AATCCAAGCC

ACAGCTACAA

CGCACGGGAA

CCGCCAGCCT

CCCGCGTCGC

ATGACAAGCA

CGCAGAACTT

TGGCCGGAGC

AGTCTGGAGT

AGGATGCGGG

CCAGCGTGGC

GTACCGGCGT

GGAGGCCGGC

AGGTGCCTCT

CCCGAGAGCG

AAGCCTCCGC

TGAAAGAGGG

TTCACATCGG

GCCCCCTCAT

CCAAGCGGGA

GCGCCATGGT

TCTTCGCGCG

CTGAGGACCT

TGGCCAGAGG

GGAACATTCT

ACATCTACAA

TGGCCCCTGA

GGGTGCTTCT

ATGAGGAGTT

ACCCTGCAAG

ACAGTGCCGT

GGACACCTGG

GAATGCCAAC

Gi2GGCTCATC

ATCCGAGGAG

GTACGAGCAT

CCCGCTTCTG

GGAGGAGGTG

GCCCGAGCAC

CTGCCACAAG

GACCGACCTC

GCACGCGCCC

CGACTTGGCG

ACGCTATCTG

CGTGGAAGGC

CATCGCTGTC

CCACGCAGAC

GGAGGAGCAA

GCTGCACCTG

TTTCGGCATC

CGCCACGGCC

CAACCACCTC

GGTGATCGTG

CGCCTTCAGC

GGAGCTCGCC

GTTCTCGAAG

GTGGCTGAGC

GATGGAGTTC

GCTGTCGGAA

AGACCCTGAC

AAGCATCTTC

CTGGGAGATC

CTGCCAGCGG

ATGTTTGCCC

GACTGGAGGG

ACCGAGTTTG

GTGTCTGCCA

TACTTCTATG

CTACTAGAGG

CTGCGCTGGT

CTCGACTGCA

GCACCTGGGG

GAGGGCCACT

AAGTACCTGT

CTGGTGAACG

AGCATCGTGT

GACTCCAACC

TGCAGCGTGT

TCCGAGGATA

TTCTTCTGGG

ATCAAGACGG

TGCGAATACC

GGGAGAGTGC

CACAAGGGCA

AGCGAGCACC

AACGTGGTCA

GAGTTCTGCA

CCCTGCGCGG

AGGCTGGATC

ACCGAGGGCG

CCGCTGACCA

CTGGCTTCCC

PGCGACGTGG

TACGTCCGCA

GACAAGGTGT

TTCTCTCTGG

CTGAGAGACG

AGCGTAGTCT

CGGTGACCAC

TGGAGGGAAA

TGTACAAGTG

TGACCACCAT

GCCAGCCGGT

ACCGCCTCAA

AGAACGTGCA

CGCGCCACGC

ATGTGTGCGA

CGGTGCAGGC

TGAGCGACTC

GGTACAAAGA

AGAAGCTGAG

GCAACGCCAA

AGGGCAGCAT

TCCTCCTCCT

GCTACCTGTC

TGTCCTACGA

TCGGCTACGG

GCAGCTGTGA

GCGCGCTGAT

ACCTCCTCGG

AGTACGGCAA

AGAAGTCTCC

GGAGGCGGCC

GAGCGAGGCG

TGGAAGATCT

GAAAGTGCAT

TGAAGATCTG

AGGGCAGTGC

ACACCACGCA

GGGCCTCCCC

GCACAAGGAT

1380 1440 1500 1560 1620 1680 1740 1800 1860 1920 1980 2040 2100 2160 2220 2280 2340 2400 2460 2520 2580 2640 2700 2760 2820 2880 2940 3000 3060 3120 3180 3240 3300 3360 3420 116 GAGGGCCCCG GAGcTGGCCA CTCCCGCCAT ACGCCGCATC ATGCTGAACT

GCTGGTCCGG

AGACCCCAAG GCGAGACCTG CATTCTCGGA GCTGGTGGAG

GGGCAGGGGC

AGAAGAGGGC

TGAGGACAGC

GTCCTTTCCC

ATTTGAGGAA

CAGTGGGATG

AAGCGGCTTC

TCTGCACTTA

TACTACAAAC

GTGACCACTG

GATAATATCC

GAAAGGGAGA

TACTGCTTGA

CTTGCCTTCT

GTTATTCCTT

CTGCAAGAGG

AGCTTCTCGC

CCGCCAAGCC

'GGGTGCCTGG

TTCCCCATGA

GTGCTGGCCT

AGGTAGCTGA

TAAGAAAGAT

TTCAAAGAGG

AAGCACCACA

AGCCTCCCAC

CGCCCTTTCA

CCAAAGAGCC

AGGTCACTTC

GGTAATATGA

AAGAGGAGGT CTGCATGGCC

AGGTGTCCAC

TGCAGCGCCA

CCAGAGGGGC

CCCCAACGAC

CGGAGGAGTT

AGCAGAGAGA

CAAAGACTTT

.AACCAGGAGG,

GGGAAGGGGT

AAGAAGCTGG

TGGTCTGCTG

CTCAAGCGGC

TCACACAATG

GTAATACATC

CATGGCCCTA

CAGCCTGGCC

TGAGACCCGT

CTACAAAGGC

TGAGCAGATA

GAGAAGGCAG

AAGACTTTCG

ACAAGAGGAG

TAGGCCTCCG

TGGAGCAGAG

AGTAACAGGT

CCTTATGCCA.

TCCCTTCAGC

AAAGAG

ATCCTGGGGG

CCGCGC-AGCT

CACATCGCCC

GCCAGGTATT

GGTTCCTCCA

TCTGTGGACA

GAGAGCAGGC

CATACGTCAG

CTATTTCTTC

CATGAAAGTG

GATGACTGCG

TGTTCCCTGA

GCNTTCCCAG

GCGTGACAGA

ACCTGACCCT

ACCTGCTCCA

CTCAGAGCTC

AGGCTGACGC

ACAACTGGGT

GGATGAAGAC

ACCAGACAGA

ATAGACAAGA

CATTTTCTTC

TACTGCTATC

GACAAGGAGT

GGCAGGCCTG

CTCCTCCAAG

ACACTGGCGT

GGGCTCACCT

GTGCCCGCCA

3480 3540 3600 3660 3720 3780 3840 3900 3960 4020 4080 4140 4200 4260 4320 4380 4416 INFORMATION FOR SEQ ID NO:37: i)SEQUENCE CHARACTERISTICS: LENGTH: 4273 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA (xi) SEQUENCE DESCRIPTION: SEQ ID NO:37: AAGCTTATCG ATTTCGAACC CGGGGGTACC GAATTCCTCG AGTCTAGAGG AGCATGCCTG

CAGGTCGACC

CCTCGCGGAG

TCCGCGCATC

GGATCTTTGT

AGATTTAAAG

TTCTAATTGT

GGA.ATGCCTT

AGGCTACTGC

CCAAGGACTT

CTCTTGCTTG

TTATGGAAAA

GGGCTCGATC

TTCTACCGGC

CATGCCCCCG

GAAGGAACCT

CTCTAAGGT-A

TTGTGTATTT

TAATGAGGAA

TGACTCTCAA

TCCTTCAGAA

CTTTGCTATT

ATATTCTGTA

CCCTCGCGAG

AGTGCAAATC

AACTGCAGGA

TACTTCTGTG

AATATAAAAT

TAGATTCCAA

AACCTGTTTT

CATTCTACTC

TTGCTAAGTT

TACACCACAA

ACCTTTATAA

TTGGTTCAGC

CGTCGGCATC

GTGGGGAGGC

GTGTGACATA

TTTTAAGTGT

CCTATGGAAC

GCTCAGAAGA

CTCCAAAAAA

TTTTGAGTCA

AGGAAAAAGC

GTAGGCATAA

TGCTGCCTGA

CAGGAAACCA.

ACGATGGCCG

ATTGGACAAA

ATAATGTGTT

TGATGAATGG

AATGCCATCT

GAAGAGAAAG

TGCTGTGTTT

TGCACTGCTA

CAGTTATAAT

GGCTGGACGA

GCAGCGGCTA

CTTTGGTCCC

CTACCTACAG

AAACTACTGA

GAGCAGTGGT

AGTGATGATG

GTAGAAGACC

AGTAATAGAA

TACAAGAAAA

CATAACATAC

120 180 240 300 360 420 480 540 600 660 720

TGTTTTTTCI

TGTGTACCTI

CCTTGACTAG

AACCTCCCAC

TTGTTTATTG

AAAGCATTTT

CATGTCTGGA

ACCTGCATTA

CCGCTTCCTC

CTCACTCAAA

TGTGAGCAAA

TCCATAGGCT

GAAACCCGAC

CTCCTGTTCC

TGGCGCTTTC

AGCTGGGCTG

ATCGTCTTGA

ACAGGATTAG

ACTACGGCTA

TCGGAA'AAAG

TTTTTGTTTG

TCTTTTCTAC

TGAGATTATC

CAATCTAAAG

CACCTATCTC

AGATAACTAC

ACCCACGCTC

GCAGAAGTGG

CTAGAGTAAG

TCGTGGTGTC

GGCGAGTTAC

TCGTTGTCAG

ATTCTCTTAC

AGTCATTCTG

ATAATACCGC

*TACTCCACAC

TAGCTTTTTA

AGATCATAAT

ACCTCCCCCT

CAGCTTATAA

2'TTCACTGCA

TCTGCCGGTC

ATGAATCGGC

GCTCACTGAC

GGCGGTAATA

AGGCCAGCAA

CCGCCCCCCT

AGGACTATAA

GACCCTGCCG

TCAATGCTCA

TGTGCACGAA

GTCCAACCCG

CAGAGCGAGG

CACTAGAAGG

AGTTGGTAGC

CAAGCAGCAG

GGGGTCTGAC

AAAAAGGATC

TATATATGAG

AGCGATCTGT

GATACGGGAG

ACCGGCTCCA

TCCTGCAACT

TAGTTCGCCA

ACGCTCGTCG

ATGATCCCCC

AAGTAAGTTG

TGTCATGCCA

*AGGCATAGAG

ATTTGTAAAG

CAGCCATACC

GAACCTGAAA

TGGTTACAAA

TTCTAGTTGT

TCCCTATAGT

CAACGCGCGG

TCGCTGCGCT

CGGTTATCCA

AAGGCCAGGA

GACGAGCATC

AGATACCAGG

CTTACCGGAT

CGCTGTAGGT

CCCCCCGTTC

GTAAGACACG

TATGTAGGCG

ACAGTATTTG

TCTTGATCCG

ATTACGCGCA

GCTCAGTGGA

TTCACCTAGA

TAAACTTGGT

CTATTTCGTT

GGCTTACCAT

GATTTATCAG

TTATCCGCCT

GTTAATAGTT

TTTGGTATGG

ATGTTGTGCA.

GCCGCAGTGT

TCCGTAAGAT

117 TGTCTrGCTAT

GGGTTAATAA

ACATTTGTAG

CATAAAATGA

TAAAGCAATA

GGTTTGTCCA

GAGTCGTATT

GGAGAGGCGG

CGGTCGTTCG

CAGAATCAGG

ACCGTAAAAA

ACAAAAATCG

CGTTTCCCCC

ACCTGTCCGC

ATcTC-AGTTc

AGCCCGACCG

ACTTATCGCC

GTGCTACAGA

GTATCTGCGC

GCAAACAAAC

GAAAAAAAGG

ACGAAAACTC

TCCTTTTAAA

CTGACAGTTA

CATCCATAGT

CTGGCCCCAG

CAATAAACCA

CCATCCAGTC

TGCGCAACGT

CTTCATTCAG

AAAAAGCGGT

TATCACTCAT

GCT'rTTCTGT

CGAGTTGCTC

AAGTGCTCAT

TAATAACTAT

GGAATATTTG

AGGTTTTACT

ATGCAATTGT

GCATCACAAA

AACTCATCAA

AATTTCGATA

TTTGCGTATT

GCTGCGGCGA

GGATAACGCA

GGACGCGTTG

ACGCTCAAGT

TGGAAGCTCC

CTTTCTCCCT

GGTGTAGGTC

CTGCGCCTTA

ACTGGCAGCA

GTTCTTGAAG

TCTGCTGAAG

CACCGCTGGT

ATCTCAAGAA

ACGTTAAGGG

TTAAAAATGA

CCAATGCTTA

TGCCTGACTC

TGCTGCAATG

GCCAGCCGGA

TATTAATTGT

TGTTGCCATT

CTCCGGTTCC

TAGCTCCTTC

37GTTATGGCA

GACTGGTGAG

TTGCCCGGCG

CATTGGAAAA

GCTCAAAAAT

ATGTATAGTG

TGCTTTAAAA

TGTTGTTAAC

TTTCACAAAT

TGTATCTTAT

AGCCAGGTTA

GGGCGCTCTT

GCGGTATCAG

GGAAAGAACA

CTGGCGTTTT

CAGAGGTGGC

CTCGTGCGCT

TCGGGAAGCG

GTTCGCTCCA

TCCGGTAACT

GCCACTGGTA

TGGTGGCCTA

CCAGTTACCT

AGCGGTGGTT

GATCCTTTGA

ATTTTGGTCA

AGTTTTAAAT

ATCAGTGAGG

CCCGTCGTGT

ATACCGCGAG

AGGGCCGAGC

TGCCGGGAAG

GCTACAGGCA

CAACGATCAA

GGTCCTCCGA

GCACTGCATA

TACTCAACCA

TCAATACGGG

CGTTCTTCGG

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 2700 2760 2820 AGAATAGTGT ATGCGGCGAC GCCACATAGC AGAACTTTAA GGCGAAAACT CTCAAGGATC TTACCGCTGT CACCCAACTG ATCTTCAGCA

TCTTTTACTT

GAAGGCAAAA TGCCGCAAAA AAGGGAATAA TCTTCCTTTT TCAATATTAT

TATTTGAATG

TGCCACCTGA

TCACGAGGCC

AGCTCCCGGA

AGGGCGCGTC

AGATTGTACT

ACATAACCTT

TTCCAAATTG

GTTTGAGGGA

ACCCCTCATT

CCAATTTGTT

CCAGCTGAAG

GAAAAAGGGG

ATTTTGCAAG

TATTTAGAAA

CGTCTAAGAA

CTTTCGTCTC

GACGGTCACA

AGCGGGTGTT

GAGAGTGCAC

ATGTATCATA

AGAGAGAGGC

CTGTTTAACA

GTACTCCTAA

AAAGACAGGA.

CCTATAGAGT

TGAAGCATTT

AATAAACAAA

ACCATTATTA

GCGCGTTTCG

GCTTGTCTGT

GGCGGGTGTC

CATATGGACA

CACATACGAT

TTAATCAGAG

GATCCCCTTG

TGATTTTGCT

TATCAGTGGT

ACGAGCCATA

118 TGAGATccAG

TCACCAGCGT

GGGCGACACG

ATCAGGGTTA

TAGGGGTTCC

TCATGACATT

GTGATGACGG

AAGCGGATGC

GGGGCTGGCT

TATTGTCGTT

TTAGGTGACA

ACAGAAACTG

GTTTACCAc

CTTCGGACCC

CCAGGCTCTA

GATAAAATAA.

TAGGTTTGGC.

GAGAATAGAG

TTGTCTCATG

GCGCACATTT

AACCTATAAA

TGAAAACCTC

CGGGAGCAGA

TAACTATGCG

AGAACGCGGC

CTATAGAACT

TTTGAGTCAA

TTGATATCTA

TGCATTCTTA

GTTTTGACTC

AAGATTTTAT

A~AGCTAGCTT

AAGTTCAGAT

TTCGATGTAA CCCACTCGTG TTCTGGGTGA. GCAAAAACAG GAAATGTTGA ATACTCATAC

AGCGGATACA

CCCCGAAAAG

AATAGGCGTA

TGACACATGc

CAAGCCCGTC

GCATCAGAGC

TACAATTAAT

CGAGCAGAGc

CTCAAGGATG

CCATTATGGG

ATCGATTAGT

AACAATATCA

TTAGTCTCCA

AAGTAACGCC

CAAGGTCAGG

GGAATGAAAG ACCCCACCTG GCATGGAAAA ATACATAACT 2880 2940 3000 3060 3120 3180 3240 3300 3360 3420 3480 3540 3600 3660 3720 3780 3840 3900~ 3960 4020 4080 4140 4200 4260 4273 120 180 216 AACAGATGGA ACAGCTGAAT ATGGGCCAAA CAGGA TATCT GTGGTAAGCA

GTTCCTGCCC

CGGCTCAGGG CCAAGAACAG ATGGAACAGC TGAATATGGG CCAAACAGGA

TATCTGTGGT

AAGCAGTTCC TGCCCCGGCT CAGGGCCAAG AACAGATGGT CCCCAGATGC

GGTCCAGCCC

TCAGCAGTTT CTAGAGAACC ATCAGATGTT TCCAGGGTGC CCCAAGGACC

TGAAATGACC

CTGTGCCTTA TTTGAACTAA CCAATCAGTT CGCTTCTCGC TTCTGTTCGC

GCGCTTCTGC

TCCCCGAGCT CAATAAAAGA GCCCACAACC CCTCACTCGG GGCGCCAGTC

CTCCGATTGA

CTGAGTCGCC

CGG

INFORMATION FOR SEQ ID NO:38: Wi SEQUENCE CHARACTERISTICS: LENGTH: 216 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA (xi) SEQUENCE DESCRIPTION: SEQ ID NO:38: CAAGAAAGCG GCTTCAGCTG TAAAGGACCT GGCCAGAATG TGGCTGTGAC CAGGGCACAC CCTGACTCCC AAGGGAGGCG GCGGCGGCCT GAGCGGGGGG CCCGAGGAGG CCAGGTGTTT TACAACAGCG AGTATGGGGA GCTGTCGGAG CCAAGCGAGG AGGACCACTG

CTCCCCGTCT

GCCCGCGTGA CTTTCTTCAC AGACAACAGC TACTAA 119 INFORMATION FOR SEQ ID NO:39: SEQUENCE CHARACTERISTICS: LENGTH: 17 amino acids TYPE: amino acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: peptide (xi) SEQUENCE DESCRIPTION: SEQ ID NO:39: Glu Glu Thr Ile Lys Phe Ala Ala Ala His Tyr Asn Thr Glu Ile Leu 1 5 10 Lys INFORMATION FOR SEQ ID SEQUENCE CHARACTERISTICS: LENGTH: 1836 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA (ix) FEATURE: NAME/KEY: CDS LOCATION: 168..1412 (xi) SEQUENCE DESCRIPTION: SEQ ID GCGGCCGCGT CGACGCAAAA GTTGCGAGCC GCCGAGTCCC GGGAGACGCT

CGCCCAGGGG

GGTCCCCGGG AGGAAACCAC GGGACAGGGA CCAGGAGAGG ACCTCAGCCT CACGCCCCAG CCTGCGCCAG CCAACGGACC GGCCTCCCTG CTCCCGGTCC ATCCACC ATG CAC TTG Met His Leu 1 120 176 224 e CTG TGC TTC Leu Cys Phe TTG TCT CTG GCG Leu Ser Leu Ala 10 TGT TCC CTG CTC Cys Ser Leu Leu

GCC

Ala GCT GCG CTG ATC Ala Ala Leu Ile

CCC

Pro AGT CCG CGC GAG Ser Pro Arg Glu

GCG

Ala CCC GCC ACC GTC Pro Ala Thr Val GCC TTC GAG TCG Ala Phe Glu Ser 4* CTG GGC TTC TCG GAA GCG GAG CCC GAC Leu Gly Phe Ser Glu Ala Glu Pro Asp 40

GGG

Gly 45 GGC GAG GTC AAG Gly Glu Val Lys GCT TTT Ala Phe GAA GGC AAA Glu Gly Lys GAG CTG ATG Glu Leu Met

GAC

Asp CTG GAG GAG CAG Leu Glu Glu Gin

TTG

Leu 60 CGG TCT GTG TCC Arg Ser Val Ser AGC GTA GAT Ser Val Asp TAC AAG TGC Tyr Lys Cys 368 416 TCT GTC CTG TAC Ser Val Leu Tyr

CCA

Pro GAC TAC TGG AAA Asp Tyr Trp Lys CAG CTG Gin Leu CGG AAA GGC GGC Arg Lys Gly Gly

TGG

Trp 90 CAG CAG CCC ACC Gin Gin Pro Thr AAT ACC AGG ACA Asn Thr Arg Thr

GGG

Gly 100 GAC AGT GTA AAA Asp Ser Val Lys TTT GCT Phe Ala 105 GCT GCA CAT TAT AAC ACA GAG ATC Ala Ala His Tyr Asn Thr Glu Ile

CTG

Leu 115 120 AAA AGT ATT GAT AAT Lys Ser Ile Asp Asn 120 GAG TGG AGA AAG ACT CAA TGC ATG CCA CGT GAG Glu Trp Arg Lys Thr Gin Cys Met Pro Arg Giu 125 130 GTG TGT ATA Val Cys Ile TTT AAA CCT Phe Lys Pro 150 GAT GTG Asp Val 135 GGAAG GAG TTT GGA GCA Giy Lys Giu Phe Gly Ala GCC ACA AAC ACC TTC Ala Thr Asn Thr Phe 145 CCA TGT GTG TCC Pro Cys Val Ser

GTC

Vai 155 TAC AGA TGT Tyr Arg Cys GGG GGT Ciy.Giy TGC TGC AAC Cys Cys Asn AGG GAG Arg Giu 165 GGG CTG GAG TGC Gly Leu Gin Cys

ATG

Met 170 AAC ACC AGC ACA Asn Thr Ser Thr

GT

Giy TAC CTC AGC AAG Tyr Leu Ser Lys

ACO

Thr 180 TTG TTT GAA ATT Leu Phe Giu Ile

ACA

Thr 185 GTG CCT CTC TCA Val Pro Leu Ser

CAA

Gin 190 CCC CCC AAA CCA Gly Pro Lys Pro

GTC

Val ACA ATC ACT TTT Thr Ile Ser Phe

CC

Ala 200 AAT CAC ACT TCC Asn His Thr Ser

TGC

Cys CGG TGC ATG TCT Arg Cys Met Ser AAA CTG Lys Leu CAT GTT TAC Asp Val Tyr ACA TTA CCA Thr Leu Pro 230

AGA

Arg 215 CAA CTT CAT TCA Gin Val His Ser

ATT

Ile 220 ATT AGA CGT TCT le Arg Axg Ser CTC CCA GCA Leu Pro Ala 225 ACA AAC TAT Thr Asn Tyr GAG TGT GAG GCA Gin Cys Gin Ala GCT AAC Ala Asn 235 AAC ACA TGT Lys Thr Cl's

CCA

Pro GTG TGG Val Trp 245 AAT AAC TAC ATG Asn Asn Tyr Met

TGC

Cl's 250 CCA TGC CTC CCT Arg Cl's Leu Ala

CAG

Gin CAG CAT TTT ATC Gin Asp Phe Ile 560 608 656 704 752 800 848 896 944 992 1040 1088 1136 1184 1232 1280 1328

T

Phe 260 TAT TCA AAT GTT Tyr Ser Asn Val

GAA

Giu 265 GAT GAC TCA ACC Asp Asp Ser Thr

AAT

Asn 270 GGA TTC CAT GAT Cly Phe His Asp

CTC

Val TGT GGA CCC AAC Cys Giy Pro Asn

AAC

Lys 280 GAG CTG GAT GAA Giu Leu Asp Giu

GAC

Asp 285 ACC TGT GAG TGT Thr Cys Gin Cys GTC TGC Val Cys AAG CCC GGG Lys Gil' Cly AGA GAC TCA Arg Asp Ser 310

CTT

Leu 295 CGG CCA TCT AGT Arg Pro Ser Ser

TGT

Cl's 300 GGA CCC CAC AAA Giy Pro His Lys GAA CTA GAT Giu Leu Asp 305 CCT AAT TCA Pro Asn Ser a TGT GAG TGT GTC Cl's Gin Cl's Val

TGT

Cys 315 AAA AAC AAA CTT Lys Asn Lys Leu

TTC

Phe TGT OGA Cys Gil' 325 AAA AGA Lys Arg 340 GCC AAC AGG GAA Ala Asn Arg Ciu

TTT

Phe 330 CAT GAG AAT AGA Asp Giu Asn Thr

TGT

Cl's GAG TGT GTA TGT Gin Cys Vai Cys ACG TGT CGA Thr Cys Pro

AGA

Arg 345 AAT GAG CCC CTG Asn Gin Pro Leu CCT CGG AAA TGT Pro Cly Lys Cys

CC

Ala TGT GAA TGT ACA Cl's Ciu Cys Thr

GAA

Giu 360 AAC ACA GAG AAG Asn Thr Gin Lys

TGC

Cl's 365 TTC CTT AAA CCC Phe Leu Lys Gly AAG AAG Lys Lys TTC GAC CAT Phe His His

CAA

Gin 375 ACA TGC AGT TCT Thr Cys Ser CyS

TAC

Tyr 380 AGA AGA CCG TGT Arg Arg Pro Cys GCG AAT CGA Ala Asn Arg 385 121 CTG AAG CAT TGT GAT CCA GGA CTG TCC TTT AGT GAA GAA GTA TGC CGC Leu Lys His Cys Asp Pro Gly Leu Ser Phe Ser Glu Glu Val Cys Arg 390 395 n TGT GTC CCA TCG TAT TGG AAA AGG CCA CAT CTG Cys Val Pro Ser Tyr Trp, Lys Arg Pro His Leu 405 410 CCAGTTTTCA GTCAGTCACA GTCATTTACT CTCTTGAAGA ACTGTCTATG t2ACAGAAAGA CTCTGTGGGA CCACATGGTA TTATTGAACC ATGTGGATTA CTGCGGGAGA

GGACTGGCAC

TTCAAAGACT GGTTTTCTGC CAGGGACCAG

ACAGCTGAGG

AAAAGAATGA CTATATAATT TATTTCCACT

AAAAATATTG

GCAATAACAA~ TTGGTAAAGC TCACTGTGAT CAGTATTTTT AAC TAAGATCATA Asn 415 CTGTTGGAAc

ACAGAGGCCC

TCATGTGCAA

TTTTTCTCTT

TTCCTGCATT

ATAACATGCA

AGCACTTAGC

AAGTCTGTGT

AAAAAACCTC

GTGATTTAAA

CATTTTTATA

AAACTATGTT

1376 1422 1482 1542 1602 1662 1722 1782 1836 TAAAATAAAA TGAAAATTGT ATTATAAAAA AAAAAA AAAAA

GCTT

INFORMATION FOR SEQ ID NO:41: SEQUENCE CHARACTERISTICS: LENGTH: 415 amino acids TYPE: amino acid TOPOLOGY: linear (ii) MOLECULE TYPE: protein (xi) SEQUENCE DESCRIPTION: SEQ ID NO:41: Met His Leu Leu Cys Phe Leu Ser Leu Ala Cys Ser Leu Leu Ala Ala Pro Arg Glu Ala Pro a Ala Leu Ile Giu Ser Giy 35 Lys Ala Phe Pro Ser Ala Thr Val Ala Ala Phe Gly Giu Val Ser Val Ser Leu Glu Gly Phe Ser Giu 40 Gly Lys Asp Leu 55 Leu Met Ser Val Ala Glu Pro Asp Gly Arg Giu Glu Gin Ser Val Leu Asp Asp Glu Leu Tyr Tyr Tyr Trp Lys Lys Cys Gin a.

S a Leu 85 Asp Arg Lys Gly Gly Trp 90 Ala Gin Gin Pro Thr Leu Asn Thr Arg Thr Glu Ile Leu 115 Gly 100 Lys Ser Val Lys Phe 105 Glu Ala Ala His Ser Ile Asp Asn 120 Val Trp Arg Lys Tyr Asn Thr 110 Gin Cys Met Ala Ala Thr Pro Arg 130 Asn Thr 145 Cys Cys GlU Val Cys Ile Asp 135 Pro Gly Lys Glu Phe 140 Phe Phe Lys Pro 150 Asn Arg Glu Gly 165 Lys Thr Leu Phe 180 Cys Val Ser Vai 155 Tyr Arg Cys Gly Leu Gin Cys Glu Ile Thr 185 Met Asn 170 Val Pro Thr Ser Thr Leu Ser Gin 190 Gly Tyr 175 Gly Pro Leu Ser

S

122 Lys Pro Val Thr Ile Ser Phe Ala Asn His Th 195 200 Ser Lys Leu Asp Val Tyr Arg Gin Val His Se: 210 215 Leu Pro Ala Thr Leu Pro Gin Cys Gin Ala Al 225 230 23! Thr Asn Tyr Val Trp Asn Asn Tyr Met Cys Arc 245 250 Asp Phe Ile Phe Tyr Ser Asn Val Glu Asp As, 260 265 His Asp Val Cys Gly Pro Asn Lys Glu Leu Asp 275 280 Cys Val Cys Lys Gly Gly Leu Arg Pro Ser Ser 290 295 Glu Leu Asp Arg Asp Ser Cys Gin Cys Val Cys 305 310 315 Pro Asn Ser Cys Gly Ala Asn Arg Glu Phe Asp 325 330 Cys Val Cys Lys Arg Thr Cys Pro Arg Asn Gln 340 345 Lys Cys Ala Cys Glu Cys Thr Glu Asn Thr Gin 355 360 Gly Lys Lys Phe His His Gin Thr Cys Ser Cys 370 375 Ala Asn Arg Leu Lys His Cys Asp Pro Gly Leu 385 390 395 Val Cys Arg Cys Val Pro Ser Tyr Trp Lys Arg 405 410 INFORMATION FOR SEQ ID NO:42: SEQUENCE

CHARACTERISTICS:

LENGTH: 22 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA (Xi) SEQUENCE DESCRIPTION: SEQ ID NO:42: TCTCTTCTGT GCTTGAGTTG

AG

INFORMATION FOR SEQ ID NO:43: SEQUENCE

CHARACTERISTICS:

LENGTH: 22 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA (xi) SEQUENCE DESCRIPTION: SEQ ID NO:43: TCTCT-TCTGT CCCTGAGTTG

AG

r Ser Cy 20 r Ile Ili 220 a Asn Lyi SCys Lei SSer Thr Glu Asp 285 Cys Gly 300 Lys Asn Glu Asn Pro Leu Lys Cys 365 Tyr Arg 380 Ser Phe Pro His s Arg Cys Met 5 e Arg Arg Ser 3 Thr Cys Pro 240 1 Ala Gin Gin 255 Asn Gly Phe 270 SThr Cys Gin Pro His Lys Lys Leu Phe 320 Thr Cys Gin 335 Asn Pro Gly 350 Phe Leu Lys Arg Pro Cys Ser Glu Glu 400 Leu Asn 415 123 INFORMATION FOR SEQ ID NO:44: SEQUENCE CHARACTERISTICS: LENGTH: 65 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA (xi) SEQUENCE DESCRIPTION: SEQ ID NO:44: TGTGCTGCAG CAAATTTTAT AGTCTCTTCT GTGGCGGCGG CGGCGGCGGG CGCCTCGCGA

GGACC

INFORMATION FOR SEQ ID SEQUENCE CHARACTERISTICS: LENGTH: 30 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA (xi) SEQUENCE DESCRIPTION: SEQ ID CTGGCAGGGA ACTGCTAATA ATGGAATGAA INFORMATION FOR SEQ ID NO:46: SEQUENCE

CHARACTERISTICS:

LENGTH: 84 base pairs r" TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA T* (xi) SEQUENCE DESCRIPTION: SEQ ID NO:46: GGGCTCCGCG TCCGAGAGGT CGAGTCCGGA CTCGTGATGG TGATGGTGAT GGGCGGCGGC oooo GGCGGCGGGC GCCTCGCGAG GACC 84 84 INFORMATION FOR SEQ ID NO:47: SEQUENCE CHARACTERISTICS: LENGTH: 31 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA (xi) SEQUENCE DESCRIPTION: SEQ ID NO:47: GTATTATAAT GTCCTCCACC AAATTTTATA G 31 INFORMATION FOR SEQ ID NO:48: SEQUENCE CHARACTERISTICS: LENGTH: 93 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA .dt.; 124 (xi) SEQUENCE DESCRIPTION: SEQ ID NO:48: GTTCGCTGCC TGACACTGTG GTAGTGTTGC TGGCGGCCGC TAGTGATGGT GATGGTGATG AATAATGGAA TGAACTTGTC TGTAAACATC

CAG

INFORMATION FOR SEQ ID NO:49: SEQUENCE

CHARACTERISTICS:

LENGTH: 18 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA (xi) SEQUENCE DESCRIPTION: SEQ ID NO:49: CATGTACGAA CCGCCAGG 18 INFORMATION FOR SEQ ID SEQUENCE

CHARACTERISTICS:

LENGTH: 20 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA (xi) SEQUENCE DESCRIPTION: SEQ ID oool AATGACCAGA

GAGAGGCGAG

INFORMATION FOR SEQ ID NO:51: SEQUENCE

CHARACTERISTICS:

LENGTH: 10 amino acids TYPE: amino acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: peptide (xi) SEQUENCE DESCRIPTION: SEQ ID NO:51: Ala Val Val Met Thr Gln Thr Pro Ala Ser 1 5 INFORMATION FOR SEQ ID NO:52: SEQUENCE

CHARACTERISTICS:

LENGTH: 1741 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA (ix) FEATURE: NAME/KEY:

CDS

LOCATION: 453..1706 (xi) SEQUENCE DESCRIPTION: SEQ ID NO:52: GCCCCCGCCG AGCGCTCCGC GCGCAGCCGC CGGGCCGGGC CGGCCCGCGG AGGGCGCGCT GCGAGCGGCC ACTGGGTCCT GCTTCCCTCC TTCCTCTCCC TCCTCCTCCT CCTCCTTCTC 120 TCTGQGCTTT CCACCGCTCC CGAGCGAGCG CACGCTCGGA TGTCCGGTTT CCTGGTGGGT 180 -125 TTTTTACCTG GCAAAGTCCG GATAACTTCG

GTGAGAATTT

GCAAAGAGGC TGGGAGCTCC 0 CCTGCAGGCG TCTGGGAGCT GCTGCCGCCG TCGCATCTTC TCCATCCCGC

GGATTTTACT

GCCTTGGATA TTGCGAGGGG AGGGAGGGGG

GTGAGGACAG

GGGGGAGAGA AAAGGAAAAG AAGGAGCCTC

GGAATTGTGC

GCGGCCCCCC TCCGCTCTGC CATCTCCGCA CA ATG CAC

CAAAAAGAAA

CCGCATTCCT

GGGGTGGGGG

GCGCTGCCCC

240 300 360 420 473 TTG CTG GAG ATG Met His Leu Leu Glu Met

CTC

*0 4

C

S

54*

S

S

S. 55 S S

S

S

S

TCC

Ser

CAG

Gin

GAG

Giu

GAA

Glu

CTT

Leu

GGT

Gly

GAT

Asp

AGT.

Ser 120

TGT

Cys AAAi Lys

GAA

Glu

TTG

Leu GTC2 Val 200

CTG

Leu

CCG

Pro

GAG

Glu

GAG

Giu

TAC

Tyr

TGG

Trp

TCA

Ser 105

ATT

Ile

GTG

V'al :cc Pro

GGA

31 y rTT Phe 185 kGT 3er

GGC

Gly

ICCC

Pro

CCC

Pro

CAG

Gin

CCA

Pro

CAA

Gin 90

TTG

Leu

GAT

Asp

GAT

Asp

CCG

Pro

CTC

Leu 170

GAG

Giu

TTT

Phe

TGC

Cys

GTC

Val

GGT

Gly

TTG

Leu

GAA

Giu 75

CAC

His

AAA

Lys

ACT

Thr

TTG

Leu

TGT

Cys 155

CAG

Gln

PITT

Ile a3cc kla TGC CTC Cys Leu GCC GCC Ala Ala GCC GGG Ala Gly 45 CGA TCT Arg Ser TAC TGG Tyr Trp AAC AGG Asn Arg TTT GCC Phe Ala GAA TGG Glu Trp 125 GGG AAA Gly Lys 140 GTA TCC Val Ser TGT ATG Cys Met ACA GTG Thr Val AAT CAC Asn His 205 GC1 Ala

GCC

Ala 30

GAA

Giu

GTG

Val

AAA

Lys

GAA

Glu

GCA

Ala 110

AGA

Axg

GAG

Giu

ATC

Ile Asn

CCT

Pro 1.90 kCG rhr

GCT

Ala 15

TAC

Tlyr

*CCC

*Pro

TCC

Ser

ATG

Met

CAC

His 95

GCA

Ala

AAA

Lys

TTT

Phe

TAC

Tyr

ATC

Ile 175

CTC

Leu

TCC

Ser

GGC

Gly

GAG

Glu

AAG

Lys

AGT

Ser

TTC

Phe 80

TCC

Ser

CAT

His

ACC

Thr

GGA

Gly

AGA

Arg 160

ALGC

Ser

TCT

Ser

TGC

-l's

GCC

Ala

TCC

Ser

GCT

Ala

GTG

Val 65

AAA

Lys

AGC

Ser

TAT

Tyr

CAG

Gin

GCA

Ala 145

TGT

Cys

ACA

Thr

CAT

His

CGA

Arg

GTG

Val

GGG

Gly

CAT

His 50

GAT

Asp

TGT

Cys

TCT

Ser

AAT

Asn

GGC

Gly 130

ACT

Thr

GGA

G

1 y

P.AT

Asn

GGC

Gly

TGC

Cys 210

CTC

*Leu

CAC

*His 35

GCA

Ala

GAA

Glu

CAG

Gin

GAT

Asp

GCA

Ala 115

ATG

Met

ACA

Thr

GGT

Gly

TAC

Tyr

CCC

Pro 195

ATG

Met

CTG

Leu 20

GGC

Gly

AGC

Ser

CTC

Leu

TTG

Leu

ACA

Thr 100

GAG.

Giu

CCA

Pro

AAC

Asn

TGC

Cys

ATC

Ile 180

~AA

Lys TCT2 Ser

GGA

Gly

TAC

Tyr

AAA

Lys

ATG

Met

AGG

Arg

AGA

Arg

ATC

Ile

CGT

Arg AiCC rhr

TGC

Cys 1.65 kGC 3er

CT

?ro

!LAG

ys

CC(

Prc

TAC

Tyr

GAC

Asp

ACA

Thr

AAA

Lys

TCA

Ser

CTG

Leu

GAA

Giu

TTC

Phe 150

AAT

Asn

AAG

Lys

GTA

Val

TTG

Leu

CGG

Arg

GAG

Giu

CTG

ILeu

GTA

Val

GGA

Gly

GAT

Asp

AAA

Lys

GTG

Val 135

TTT

Phe

AGT

Ser

ACA

Thr

ACA

Thr

GAT

Asp 215 521 569 617 665 713 761 809 857 905 953 1001 1049 1097 1145

GTT

Val TAC AGA CAA GTT CAT TCT ATC ATA AGA CGT TCC TTG, CCA GCA ACA Tryr Arg (.in V/al 220 His Ser Ile Ile Arg 225 Arg Ser Leu Pro Ala Thr 126 CAA ACT CAG Gin Thr Gin TGG AAT AAT Trp Asn Asn 250

TGT

Cys 235 CAT GTG GCA AAC His Val Ala Asn

AAG

Lys 240 ACC TOT CCA Thr Cys Pro AAA AAT CAT GTC Lys Asn His Val 245 CAG ATT TGC AGA Gin Ile Cys Arg

TGC

Cys 255 TTA GCA CAG CAC Leu Ala Gin His

GAT

Asp 260 TTT GOT TTC Phe Gly Phe 1193 1241 1289 TCT TCT Ser Ser 265 CAC CTT OGA GAT His Leu Gly Asp

TCT

Ser 270 GAC ACA TCT GAA Asp Thr Ser Glu

GGA

Gly 275 TTC CAT ATT TGT Phe His Ile Cys

GGG

Gly 280 CCC AAC AAA GAG Pro Asn Lys Glu

CTG

Leu 285 OAT GAA GAA ACC Asp Glu Glu Thr

TGT

Cys 290 CAA TGC OTC TGC Gin Cys Val Cys

AAA

Lys 295 GGA GGT GTG CGG Oly Oly Vai Arg

CCC

Pro 300 ATA AOC TOT GGC Ile Ser Cys Gly CAC AAA OAA CTA His Lys Glu Leu GAC AGO Asp Arg 310 OCA TCA TGT Ala Ser Cys GGG CCT AAC Gly Pro Asn 330

CAG

Gin 315 TGC ATO TOC AAA Cys Met Cys Lys

AAC

Asn 320 AAA CTO CTC CCC Lys Leu Leu Pro AGT TCC TGT Ser Ser Cys 325 OTA TOT AAA Val Cys Lys AAA GAA TTT OAT Lys Glu Phe Asp

OAA

Olu 335 OAA AAO TOC CAG Olu Lys Cys Gin

TGT

Cys 340 1337 1385 1433 1481 1529 1577 1625 1673 S. S 0O 0

S.

S

0

S

5

S

*S*0 0@ 5

S

0 AAO ACC Lys Thr 345 TGT CCC AAA CAT Cys Pro Lys His

CAT

His 350 CCA CTA AAT CCT Pro Leu Asn Pro

OCA

Ala 355 AAA TGC ATC TGC Lys Cys Ile Cys

GAA

Glu 360 TGT ACA GAA TCT Cys Thr Glu Ser

CCC

Pro 365 AAT AAA TOT TTC Asn Lys Cys Phe

TTA

Leu 370 AAA OGA AAA AGA Lys Giy Lys Arg

TTT

Phe 375 CAT CAC CAG ACA His His Gin Thr AGT TOT TAC AGA Ser Cys Tyr Arg

CCA

Pro 385 CCA TOT ACA OTC Pro Cys Thr Val CGA ACO Arg Thr 390 AAA COC TGT Lys Arg Cys GTA COC ACA Vai Arg Thr 410

GAT

Asp 395 OCT OGA TTT CTO Ala Oly Phe Leu

TTA

Leu 400 OCT GAA OAA GTG Ala Giu Glu Val TGC CGC TGT Cys Arg Cys 405 TCT TGG AAA AGA Ser Trp Lys Arg CCA CTT Pro Leu 415 ATO AAT TAAGCGAAGA

AAOCACTACT

Met Asn 1726 CGCTATATAG

TGTCG

INFORMATION FOR SEQ ID NO:53: SEQUENCE

CHARACTERISTICS:

LENGTH: 418 amino acids TYPE: amino acid TOPOLOGY: linear (ii) MOLECULE TYPE: protein (xi) SEQUENCE DESCRIPTION: SEQ ID NO:53: Met His Leu Leu Olu Met Leu Ser Leu Oly Cys Cys 1 5 10 Ala Val Leu Leu Oly Pro Arg Gin Pro Pro Val Ala 25 1741 Leu Ala Ala Gly 15 Ala Ala Tyr Glu 30 Ser Oly His Gly Tyr Tyr Giu Glu Glu Pro Gly Ala Gly Olu Pro Lys 40 127 Ala His Ala Ser Lys Asp Leu Giu Giu Gin Leu Arg 55 Ser Val Ser Ser

S

0 0 0 00I 0 0 0 00 0 .0.0 *.0 .0.0 0 0 0:00.

Val Lys Ser Tyr Gin Ala 145 Cys Thr His Arg Arg 225 Thr Ala Ser Thr Pro 305 Lys Lys Asn Phe Pro 385 Ala 4 Met Asp Cys Ser Asn Gly 130 Thr Gly Asn Gly Cys 210 Arg Cys Gin Glu Cys 290 His Leu Cys Pro Leu 370 Pro 31u Rsn Glu Gin Asp Ala 115 Met Thr Gly Tyr Pro 195 Met Ser Pro His Gly 275 Gin Lys Leu Gin Ala 355 Lys Cys Glu Leu Leu Thr 100 Glu Pro Asn Cys Ile 180 Lys Ser Leu Lys Asp 260 Phe Cys lu Pro :ys 340 Lys fly rhr lal Met Arg Arg Ile Arg Thr Cys 165 Ser Pro Lys Pro Asn 245 Phe His Val Leu Ser 325 Val Cys Lys Val Cys 405 Thr 70 Lys Ser Leu Glu Phe 150 Asn Lys Val Leu Ala 230 His Gly Ile Cys Asp 310 Ser Cys Ile Arg Arg 390 Arg Val Gly Asp Lys Val 135 Phe Ser Thr Thr Asp 215 Thr Val Phe Cys Lys 295 Arg Cys Lys Cys Phe I 375 Thr I Cys Leu Gly Asp Ser 120 Cys Lys Glu Leu Val 200 Val Gin Trp Ser Gly 280 Gly kia fly Lys flu 360 iis -ys lai Tyr Trp Ser 105 Ile Val Pro Gly Phe 185 Ser Tyr Thr Asn Ser 265 Pro Gly Ser Pro Thr 345 Cys His Arg Arg Pro Gin 90 Leu Asp Asp Pro Leu 170 Glu Phe Arg Gin Asn 250 His Asn Vai Cys 4 Asn 330 Cys Thr Gin Cys Thr 410 Glu 75 His Lys Thr Leu Cys 155 Gin Ile Ala Gin Cys 235 Gin Leu Lys Arg Gln 315 Lys Pro flu rhr ksp 395 Ser Tyr Asn Phe Glu Gly 140 Val Cys Thr Asn Val 220 His Ile Gly.

Glu Pro 300 Cys Glu Lys Ser Cys 380 Ala Trp, Trr Arg Ala Trp 125 Lys Ser Met Val His 205 His Val Cys Asp Leu 285 Ile Met Phe His Pro 365 Ser Gly Lys Lys Glu Ala 110 Arg Glu Ile Asn Pro 190 Thr Ser Ala Arg Ser 270 Asp Ser Cys Asp His 350 Asn Cys Phe Arg Met His Ala Lys Phe Tyr Ile 175 Leu Ser Ile Asn Cys 255 Asp Glu Cys Lys Glu 335 Pro Lys yr Leu Pro 415 Phe Ser His Thr Gly Arg 160 Ser Ser Cys Ile Lys 240 Leu Thr Glu Gly Asn 320 Glu Leu Cys Arg Leu 400 Leu 128 INFORMATION FOR SEQ ID NO:54: Wi SEQUENCE

CHARACTERISTICS:

LENGTH: 1582 base pairs TYPE: nucleic acid STRANDEDNEsS: single TOPOLOGY: linear (ii) MOLECULIE TYPE: DNA (genornic) (xi) SEQUENCE DESCRIPTION: SEQ ID NO:54: CTAGAGTTGA ACCAGATAAG AAAGTCTCTT CTTCCGGTAA

GATATTATGG

TCTGTGTACT TAAAAGTAGA TTGGGAGTGA AAGGCAGACT

TTTGATGTTC

ACCTATAACA

TGAAACCCCT TAGCGTGGTC CTCTGTAACC TGCTCACCCT GCCCCAAGGA GGAr.,, 9*

CAATGCCAC,

TCTTTGGATi

CCTTTTTCAX

AAGATTCTG(

TCCATCC=~

TTCAACTAO

CATAGTGATC

GAGGACAAG;

CCAGTTTCTC

CCAACCGCCA

GGGGCGGGGA

CGAGGGAAAC

CCCGGGCTCG

CGCGTCAGTC

CGCTCTGGAG

GGCCCTCCTC

CTCCTCTCAC

GGGCTGAACA

GACGCGGACC

GGGAGCTCGG

GCACTGGCTG

CGCCTCCGGC

GGCCCGCGC

CCCCACCCCC

C AGCCCAACG( 3 CTACACATCC k~ GTAAACCAAC 3CCCTTCTCT'I I CAGCCTGCAC k CAGACCAAGG

;ACCTTTTTCC

LACTCGGGAGT

CCCGCTGCAC

GCCCCGGGAC

GGGAGAGATC

GGGGAGCTCC

GCACGCTCCC

ATGCCCTGCC

GATCCTGCGC

CCGCCCCCGG

TTCGGGGAAG

TCGCGGGGTG

GCGGCGGCGT

ATGTCCGGTT

GGAGGGCGCC

TCGCCCAGGG

CTCGCAGGGG

;AAACCCCAG'

TTTCTGGAA1

TTCTTGCAW~

CACCTGTCAC

TGCATccTGP,

GAGAGAGGGG

AAACTTTGAG

GGCCGAGGAT

GTGGTCCAGG

TGAACTTGCC

CAGAGGGGGG

AGGGAGACGG

TCCCTCGGCC

CCTGCGCCCG

CGCGGCGCTC

CACCGCCGCC

GGGAGGGAGG

TTCTGGTGTC

CCTCCCTCGC

TCCTGTGAGG

CTGCAAAGTT

GGGGTCGCCG

CGCCCGCGCC

r GCTTTTCCAJ rATGTCTCAC) AGCTGACAAj

AAGTTTAGG;

LGAACTGCGG1

ACCCCTCCG.A

CAGGGCGCTG

AAAGCGGGGG

GTGGTCGCAT

CCTCCGGCCG

CTGGGGGAGG

CTTCCGAGGG

GCTTTCTCTC

CCGCCGCCGC

CCGGGCCCCG

AGCGCCCCCG

AGGGGGACGA

CCCCGCCCCG

CCTCGCTTCA

CTTTTACCTG

GGGAACGCGG

GGAGGAGCCC

CCCACCCCTG

TGGGGAAATG CAGTCACTTT kCACATcTCT( 7GTGTCTCTT1

LTTCCAAAGGC.

TCTTGGATC;

LGGTCCCATAG.

GGGGCCAGGc

CTCCCTCCAC

CACCTCTAAA

CCCGCTCCCC

TGGGNccGCC

AGAGTGAGAG

ACATAAGCGC

CGCCGCCGCT

CCGCCGCCAG

CCGCAGCGCC

GGGCTCTGGC

CCTCTCCAAA

CCTCGCGGGC

ACACCCGCCG

AGCCCCGGAC

GGGGGAGAGG

CCCCCGCCAG

:TTTATCACCC

7ACTCTCCACG

;ATCATTAGA

LTCAGGCAACT

GGTTCTCTGA

GTGCGGGAGG

CCCACGGTGC

GCCGGTCCCG

GCAGGGGACA

GGGGAGGAGN

GGGAGGGCAG

AGGCAGAGGG

CAGCCCGGCG

CCGCCCCGGC

CGCGGCCCGG

GGGTTTGGAG

AAGCTACACC

TCCGAATGCG

CCTTTCCCCG

CCGCTCCCGC

GACCAGGAGG

CGGACCGGTC

120 180 240 300 360 420 480 540 600 660 720 780 840 900 960 1020 1080 1140 1200 1260 1320 1380 1440 1500 GGTCCTTCCA CC 1582 INFORMATION FOR SEQ ID W) SEQUENCE CHARACTERISTICS: 129 LENGTH: 20 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (xi) SEQUENCE DESCRIPTION: SEQ ID CACGGCTTAT GCAAGCAAAG INFORMATION FOR SEQ ID NO:56: SEQUENCE CHARACTERISTICS: LENGTH: 20 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (xi) SEQUENCE DESCRIPTION: SEQ ID NO:56: AACACAGTTT TCCATAATAG INFORMATION FOR SEQ ID NO:57: SEQUENCE CHARACTERISTICS: LENGTH: 18 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (xi) SEQUENCE DESCRIPTION: SEQ ID NO:57: GCCACGGTAG GTCTGCGT 18 INFORMATION FOR SEQ ID NO:58: SEQUENCE CHARACTERISTICS: LENGTH: 18 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (xi) SEQUENCE DESCRIPTION: SEQ ID NO:58: TTTCTTTGAC AGGCTTAT 18 INFORMATION FOR SEQ ID NO:59: SEQUENCE CHARACTERISTICS: LENGTH: 21 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (xi) SEQUENCE DESCRIPTION: SEQ ID NO:59: ATCTTGAAAA GTAAGTATGG G 21 130 INFORMATION FOR SEQ ID SEQUENCE

CHARACTERISTICS:

LENGTH: 20 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (xi) SEQUENCE DESCRIPTION: SEQ ID ATGACTTGAC

AGGTATTGAT

INFORMATION FOR SEQ ID NO:61: 2 0 SEQUENCE

CHARACTERISTICS:

LENGTH: 20 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (xi) SEQUENCE DESCRIPTION: SEQ ID NO:61: AGCAAGACGG

TGGGTATTGT

INFORMATION FOR SEQ ID NO:62: SEQUENCE

CHARACTERISTICS:

LENGTH: 22 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (xi) SEQUENCE DESCRIPTION: SEQ ID NO:62: CCCTTCTTTG TAGTTATTTG AA 22 INFORMATION FOR SEQ ID NO:63: SEQUENCE

CHARACTERISTICS:

LENGTH: 20 base pairs TYPE: nucleic acid STRANDEDNESS: single S* TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (xi) SEQUENCE DESCRIPTION: SEQ ID NO:63: CCACAGTGAG

TATGAATTAA

INFORMATION FOR SEQ ID NO:64: SEQUENCE

CHARACTERISTICS:

LENGTH: 18 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (xi) SEQUENCE DESCRIPTION: SEQ ID NO:64: TTCTTCCAAA

GGTGTCAG

a. 18 131 INFORMATION FOR SEQ ID SEQUENCE CHARACTERISTICS: LENGTH: 18 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (xi) SEQUENCE DESCRIPTION: SEQ ID GGAGATGGTA GCAGAATG 18 INFORMATION FOR SEQ ID NO:66: SEQUENCE CHARACTERISTICS: LENGTH: 23 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (xi) SEQUENCE DESCRIPTION: SEQ ID NO:66: CTATTTGTCT AGACTCAACA GAT 23 INFORMATION FOR SEQ ID NO:67: SEQUENCE CHARACTERISTICS: LENGTH: 22 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (xi) SEQUENCE DESCRIPTION: SEQ ID NO:67: ooo° CAAACATGCA GGTAAGAGAT CC 22 INFORMATION FOR SEQ ID NO:68: SEQUENCE CHARACTERISTICS: LENGTH: 21 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (xi) SEQUENCE DESCRIPTION: SEQ ID NO:68: TGTTCTCCTA GCTGTTACAG A 21 INFORMATION FOR SEQ ID NO:69: SEQUENCE CHARACTERISTICS: LENGTH: 24 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (xi) SEQUENCE DESCRIPTION: SEQ ID NO:69: GGCGAGGTCA AGGTAGGTGC AAGG 24 132 INFORMATION FOR SEQ ID SEQUENCE

CHARACTERISTICS:

LENGTH: 26 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (xi) SEQUENCE DESCRIPTION: SEQ ID ATTGTCTTTG ACAGGCTTTT TGAAGG 26 INFORMATION FOR SEQ ID NO:71: SEQUENCE

CHARACTERISTICS:

LENGTH: 21 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (xi) SEQUENCE DESCRIPTION: SEQ ID NO:71: GAGATCCTGA AAAGTAAGTA G 21 INFORMATION FOR SEQ ID NO:72: SEQUENCE

CHARACTERISTICS:

LENGTH: 24 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (xi) SEQUENCE DESCRIPTION: SEQ ID NO:72: TGTGACTCGA CAGGTATTGA

TAAT

24 INFORMATION FOR SEQ ID NO:73: SEQUENCE

CHARACTERISTICS:

LENGTH: 20 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (xi) SEQUENCE DESCRIPTION: SEQ ID NO:73: CTCAGCAAGA CGGTAGGTAT INFORMATION FOR SEQ ID NO:74: SEQUENCE CHARACTERISTICS: LENGTH: 25 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (xi) SEQUENCE DESCRIPTION: SEQ ID NO:74: TTGTCCCTTG TAGTTGTTTG AAATT ag, 133 INFORMATION FOR SEQ ID SEQUENCE CHARACTERISTICS: LENGTH: 20 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (xi) SEQUENCE DESCRIPTION: SEQ ID ACATTACCAC AGTGAGTATG INFORMATION FOR SEQ ID NO:76: SEQUENCE CHARACTERISTICS: LENGTH: 26 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear S(ii) MOLECULE TYPE: DNA (xi) SEQUENCE DESCRIPTION: SEQ ID NO:76: GTCTCCCCAA AAGGTGTCAG GCAGCT 26 INFORMATION FOR SEQ ID NO:77: SEQUENCE CHARACTERISTICS: LENGTH: 23 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (xi) SEQUENCE DESCRIPTION: SEQ ID NO:77: AATGTTGAAG ATGGTAAGTA AAA 23 INFORMATION FOR SEQ ID NO:78: SEQUENCE CHARACTERISTICS: LENGTH: 16 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (xi) SEQUENCE DESCRIPTION: SEQ ID NO:78: TCTAGACTCA ACCAAT 16 INFORMATION FOR SEQ ID NO:79: SEQUENCE CHARACTERISTICS: LENGTH: 22 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (xi) SEQUENCE DESCRIPTION: SEQ ID NO:79: CAAACATGCA GGTAAGGAGT GT 22 134 INFORMATION FOR SEQ ID SEQUENCE CHARACTERISTICS: LENGTH: 24 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (xi) SEQUENCE DESCRIPTION: SEQ ID TTTTCCCCTA GTTGTTACAG AAGA 24 INFORMATION FOR SEQ ID NO:81: SEQUENCE CHARACTERISTICS: LENGTH: 19 amino acids TYPE: amino acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: peptide (xi) SEQUENCE DESCRIPTION: SEQ ID NO:81: Leu Ser Lys Thr Val Ser Gly Ser Glu Gin Asp Leu Pro His Glu Leu 1 5 10 ~His Val Glu e

Claims (130)

1. A purified and isolated polypeptide comprising an amino acid sequence encoded by plasmid pFLT4-L, deposited as ATCC Accession Number 97231, wherein the polypeptide binds to the extracellular domain of Flt4 receptor tyrosine kinase (Flt4). s 2. A purified and isolated polypeptide comprising an amino acid sequence encoded by plasmid pFLT4-L, deposited as ATCC Accession Number 97231, wherein said polypeptide lacks the carboxyl terminal portion of the amino acid sequence encoded by plasmid pFLT4-L that contains cysteine motifs of a Balbiani ring 3 protein (BR3P).

3. A purified and isolated polypeptide that binds to the extracellular domain of human Flt4 receptor tyrosine kinase, said polypeptide comprising an amino acid sequence that includes a contiguous portion of the amino acid sequence in SEQ ID NO:33 effective Sto permit said binding, wherein said polypeptide lacks any portion of SEQ ID NO:33 that has one or more cysteine motifs of a Balbiani ring 3 protein (BR3P).

4. A polypeptide according to claim 3, wherein said contiguous portion includes eight cysteine residues that are conserved in human vascular endothelial growth factor (VEGF), human platelet derived growth factor A (PDGF-A), and human platelet derived growth factor B (PDGF-B).

5. A purified and isolated polypeptide that binds to the extracellular domain of 20 human Flt4 receptor tyrosine kinase, said polypeptide comprising a portion of the amino S acid sequence in SEQ ID NO:33 effective to permit said binding, said polypeptide lacking at least carboxy-terminal residues of SEQ ID NO:33 beyond residue 227.

6. A purified polypeptide that binds to the extracellular domain of human Flt4 receptor tyrosine kinase, said polypeptide produced by a method comprising the steps of: expressing a nucleic acid in a host cell, wherein said nucleic acid comprises a nucleotide sequence encoding a portion of the amino acid sequence in SEQ ID NO:33 effective to permit such binding; and purifying a polypeptide that binds to the extracellular domain of human Flt4 from said host cell or from a growth medium of said host cell, wherein said polypeptide lacks at least the carboxy-terminal portion of SEQ ID NO:33 that is characterized by cysteine motifs of a Balbiani ring 3 protein.

7. A purified and isolated polypeptide according to any one of claims 3-6 wherein said portion of the amino acid sequence in SEQ ID NO:33 includes residues 161 to 211 of SEQ ID NO:33. [R:\LIBZZ]04999.doc:mr 136

8. A purified and isolated polypeptide according to any one of claims 3-6 wherein said portion of the amino acid sequence in SEQ ID NO:33 includes residues 131 to 211 of SEQ ID NO:33.

9. A purified and isolated polypeptide according to any one of claims 3-6, wherein said portion of the amino acid sequence in SEQ ID NO:33 includes residues 113 to 213 of SEQ ID NO:33. A purified and isolated polypeptide according to any one of claims 3-6, wherein said portion of the amino acid sequence in SEQ ID NO:33 includes residues 113 to 227 of SEQ ID NO:33. 1o 11. A purified and isolated polypeptide according to any one of claims 3-6, wherein said portion of the amino acid sequence in SEQ ID NO:33 includes amino acids 103 to 217 of SEQ ID NO:33.

12. A purified and isolated polypeptide according to any one of claims 3-6, wherein said portion of the amino acid sequence in SEQ ID NO:33 includes amino acids 103 to 225 of SEQ ID NO:33.

13. A purified and isolated polypeptide according to any one of claims 3-6, wherein said portion of the amino acid sequence in SEQ ID NO:33 includes amino acids 32 to 211 of SEQ ID NO:33.

14. A purified and isolated polypeptide according to any one of claims 3-6, S 20 wherein said portion of the amino acid sequence in SEQ ID NO:33 includes amino acids S 32 to 227 of SEQ ID NO:33.

15. A purified and isolated polypeptide according to any one of claims 3-6, .wherein said portion of the amino acid sequence in SEQ ID NO:33 is a continuous portion having as its amino terminal residue an amino acid between residues 30 and 162 of SEQ ID NO:33 and having as its carboxy terminal residue an amino acid between residues 210 and 228 of SEQ ID NO:33.

16. A purified and isolated polypeptide according to any one of claims 3-6, wherein said portion of the amino acid sequence in SEQ ID NO:33 is a continuous portion having as its amino terminal residue an amino acid between residues 102 and 132 of SEQ ID NO:33 and having as its carboxy terminal residue an amino acid between residues 210 and 228 of SEQ ID NO:33.

17. A purified and isolated polypeptide according to any one of claims 3-6, wherein said portion of the amino acid sequence in SEQ ID NO:33 is a continuous Si portion having, as its amino terminal residue, residue 103 of SEQ ID NO:33, and having, 1O hi [R:\LIBZZ]04999.doc: mrr 137 as its carboxy terminal residue an amino acid between residues 216 and 228 of SEQ ID NO:33.

18. A purified and isolated polypeptide that binds to the extracellular domain of Flt4 receptor tyrosine kinase (Flt4), wherein the polypeptide comprises a portion of SEQ s ID NO:33 effective to permit such binding, and wherein the polypeptide has an apparent molecular weight of approximately 23 kD as assessed by SDS-PAGE under reducing conditions.

19. A purified and isolated polypeptide according to any one of claims 3-18, wherein the polypeptide has an amino acid sequence consisting of a portion of the amino o0 acid sequence in SEQ ID NO:33. A purified and isolated polypeptide comprising a human polypeptide that Sbinds to the extracellular domain of Flt4 receptor tyrosine kinase and has an apparent molecular weight of approximately 23 kD as assessed by SDS-PAGE under reducing conditions, wherein amino terminal amino acids 2 through 18 of said human polypeptide is have an amino acid sequence corresponding to amino acids 2 through 18 set forth in SEQ ID NO:13.

21. A purified and isolated polypeptide that binds to the extracellular domain of Flt4 receptor tyrosine kinase, wherein said polypeptide has an apparent molecular weight of approximately 23 kD as assessed by SDS-PAGE under reducing conditions and is 20 purifyable from conditioned media from a PC-3 prostatic adenocarcinoma cell line, said cell line having ATCC CRL No. 1435, using an affinity purification procedure wherein the afffinity purification matrix comprises a polypeptide comprising the extracellular domain of Flt4 receptor tyrosine kinase.

22. A purified and isolated polypeptide that binds to the extracellular domain of human Flt4 receptor tyrosine kinase (Flt4EC), wherein said polypeptide comprises an amino acid sequence encoded by a DNA which hybridizes to a non-coding strand complementary to nucleotides 352 to 1611 of SEQ ID NO:32 under the following hybridization conditions: hybridization at 42°C in a hybridization solution comprising 50% formamide, 5 X SSC, 20 mM Na*P0 4 pH 6.8; and washing in 0.2 X SSC at 55 0 C, and wherein said polypeptide lacks any cysteine motifs of a Balbiani ring 3 protein (BR3P).

23. A purified and isolated mammalian polypeptide that binds to the extracellular s domain of human Flt4 receptor tyrosine kinase (Flt4EC), [R:\UBZZ]04999.doc: nv 138 said polypeptide including a contiguous portion of a mammalian Flt4 ligand precursor polypeptide that is sufficient to bind human Flt4EC, wherein said mammalian Flt4 ligand precursor polypeptide comprises an amino acid sequence that is encoded by a DNA which hybridizes to a non-coding strand s complementary to nucleotides 352 to 1611 of SEQ ID NO: 32 under the following hybridization conditions: hybridization at 42 0 C in a hybridization solution comprising formamide, 5 X SSC, 20 mM Na*P0 4 pH 6.8; and washing in 0.2 X SSC at 55 0 C, wherein said contiguous portion includes eight cysteine residues that are conserved in human vascular endothelial growth factor (VEGF), human platelet derived growth factors A and B (PDGF-A, PDGF-B), human placenta growth factor (PIGF-1), and human vascular endothelial growth factor B (VEGF-B), and wherein said polypeptide lacks any portion of the mammalian Flt4 ligand precursor polypeptide that has one or more cysteine motifs of a Balbiani ring 3 protein (BR3P). S 15 24. A purified and isolated polypeptide according to any one of claims 2-12, 16- 17, and 22-23 having an apparent molecular weight of about 23 kD as assessed by SDS PAGE under reducing conditions. A purified and isolated polypeptide according to any one of claims 2-15 and 22-23 having an apparent molecular weight of about 32 kD as assessed by SDS PAGE 20 under reducing conditions. S 26. A purified and isolated polypeptide according to any one of claims 1-25, said "i polypeptide having an amino acid sequence consisting of a portion of the amino acid sequence set forth in SEQ ID NO:33.

27. A non-human mammalian polypeptide according to claim 22 or 23.

28. A human polypeptide according to claim 22 or 23.

29. A polypeptide according to any one of claims 1-28, wherein said polypeptide is capable of stimulating tyrosine phosphorylation of Flt4 in a host cell expressing said Flt4. A purified and isolated polypeptide which is capable of binding the extracellular domain of Kdr receptor tyrosine kinase (VEGFR-2), said polypeptide having an amino acid sequence comprising a portion of SEQ ID NO:33 effective to permit such binding.

31. A polypeptide according to any one of claims 1-30 further comprising a detectable label. (R:\LIBZZ]04999.doc:rn 139

32. A composition comprising a polypeptide according to any one of claims 1-31 in a pharmaceutically acceptable diluent, adjuvant, excipient, or carrier.

33. A purified protein comprising a first polypeptide linked to a second polypeptide, wherein at least one of said first polypeptide and said second polypeptide is a polypeptide according to any one of claims 1-31, and wherein said protein is capable of binding to Flt4 receptor tyrosine kinase.

34. A purified protein according to claim 33, wherein said first polypeptide is covalently linked to said second polypeptide. A purified protein comprising a first polypeptide linked to a second o0 polypeptide, wherein each of said first polypeptide and said second polypeptide is a polypeptide according to any one of claims 1-31.

36. A pharmaceutical composition comprising a protein according to any one of claims 33-35 in a pharmaceutically-acceptable diluent, adjuvant, excipient, or carrier.

37. A purified and isolated nucleic acid comprising a nucleotide sequence is encoding a polypeptide according to any one of claims 1-31.

38. A nucleic acid according to claim 37 having the nucleotide sequence of nucleotides 352 to 1608 of SEQ ID NO:32.

39. A nucleic acid according to claim 37 comprising a VEGF-C encoding insert of plasmid pFLT4-L, deposited as ATCC Accession Number 97231. 20 40. A purified and isolated nucleic acid comprising a nucleotide sequence that encodes a polypeptide that binds to human Flt4 receptor tyrosine kinase, said polypeptide having an amino acid sequence comprising a portion of the amino acid sequence shown in SEQ ID NO:33 effective to permit such binding, said nucleic acid lacking a nucleotide sequence that encodes the portion of the amino acid sequence shown in SEQ ID NO:33 that has cysteine motifs of a Balbiani ring 3 protein.

41. A purified and isolated nucleic acid according to claim 40 wherein said polypeptide stimulates tyrosine phosphorylation of human Flt4.

42. A nucleic acid according to claim 40, wherein said polypeptide has an apparent molecular weight of about 23 kD as assessed by SDS polyacrylamide gel electrophoresis under reducing conditions.

43. A nucleic acid according to any one of claims 40-42, wherein said polypeptide comprises an amino-terminal amino acid sequence set forth in SEQ ID NO:13. [R:\LIBZZ]04999.doc:mr 140

44. A purified and isolated nucleic acid according to any one of claims 40-42, wherein amino terminal amino acids 2 through 18 of said polypeptide have an amino acid sequence identical to amino acids 2 through 18 set forth in SEQ ID NO: 13. A purified and isolated nucleic acid according to any one of claims 40-44, wherein said polypeptide comprises approximately 120 amino acids.

46. A purified and isolated nucleic acid according to any one of claims 40-44, wherein said polypeptide comprises amino acids 103 to 222 of SEQ ID NO:33.

47. A nucleic acid according to claim 40 or 41, wherein said polypeptide has an apparent molecular weight of about 32 kDa as assessed by SDS polyacrylamide gel electrophoresis under reducing conditions.

48. A nucleic acid according to claim 40 or 41, wherein said portion of the amino acid sequence shown in SEQ ID NO:33 is a continuous portion that includes eight Scysteines of SEQ ID NO:33 that are conserved in human vascular endothelial growth factor (VEGF), human platelet derived growth factor A (PDGF-A), and human platelet 15 derived growth factor B (PDGF-B), and excludes the carboxyl terminal portion of SEQ ID NO:33 that contains cysteine motifs ofa Balbiani ring 3 protein.

49. A nucleic acid according to claim 48 wherein said continuous portion has amino acid 103 of SEQ ID NO:33 as its amino terminus.

50. A nucleic acid according to claim 40 or 41, wherein said portion of the amino acid sequence shown in SEQ ID NO:33 is a continuous portion having amino acid 103 of SEQ ID NO:33 as its amino terminal residue, and having as its carboxy terminal residue an amino acid between residues 221 and 228 of SEQ ID NO:33.

51. A purified and isolated nucleic acid encoding a polypeptide that binds the extracellular domain of human Flt4 receptor tyrosine kinase and stimulates tyrosine phosphorylation of Flt4 receptor tyrosine kinase, said polypeptide having an amino acid sequence consisting of a continuous portion of the sequence shown in SEQ ID NO:33, said continuous portion commencing at residue number 103 of SEQ ID NO:33 and lacking at least carboxy terminal residues of SEQ ID NO:33 beyond residue 227.

52. A purified and isolated nucleic acid comprising a nucleotide sequence that encodes a polypeptide that binds human Flt4 receptor tyrosine kinase, said polypeptide having an amino acid sequence comprising a continuous portion of the amino acid sequence shown in SEQ ID NO:33 effective to permit such binding, said nucleic acid lacking a nucleotide sequence that encodes the carboxy-terminal portion of the amino L acid sequence shown in SEQ ID NO:33 beyond residue 227. [RLIUBZZ]04999.doc:mrr 141

53. A purified and isolated nucleic acid according to claim 52, wherein said encoded polypeptide stimulates tyrosine phosphorylation of human Flt4.

54. A nucleic acid according to claim 51 or 52, wherein said nucleic acid lacks a nucleotide sequence that encodes amino acids 32-102 of SEQ ID NO:33.

55. A purified and isolated nucleic acid according to claim 51 or 52, wherein said nucleic acid lacks a nucleotide sequence that encodes the amino terminal portion of the amino acid sequence shown in SEQ ID NO:33 that precedes residue 103.

56. A nucleic acid according to any one of claims 37-55, wherein the nucleic acid further comprises an expression control sequence operatively linked to the encoding sequence.

57. An expression construct comprising the nucleic acid according to any one of claims 37-55 operatively linked to an expression control sequence, said expression construct lacking a nucleotide sequence that encodes the carboxy-terminal portion of the amino acid sequence shown in SEQ ID NO:33 beyond residue 227. 15 58. A vector comprising a nucleic acid according to any one of claims 37-55, wherein said vector lacks a nucleotide sequence that encodes the portion of the amino acid sequence shown in SEQ ID NO:33 that has cysteine motifs of a Balbiani ring 3 protein.

59. A host cell transformed or transfected with a nucleic acid according to any one of claims 37-56. A host cell transformed or transfected with an expression construct or vector *according to claim 57 or 58.

61. A host cell transformed or transfected with a polynucleotide, wherein said polynucleotide includes a strand that hybridizes to a DNA comprising the non-coding strand complementary to SEQ ID NO:32, under the following hybridization conditions: hybridization at 42 0 C for 20 hours in a soltion containing formamide, 5 x SSPE, 5 x Denhardt's solution, 0.1% SDS and 0.1 mg/ml denatured salmon sperm DNA; and washing the filter twice for thirty minutes at room temperature and twice for thirty minutes at 65 0 C with a wash solution containing 1 x SSC, and 0.1% SDS; and wherein said host cell expresses a polypeptide encoded by said polynucleotide, Swherein said polypeptide includes a domain defined by eight cysteine residues that S 3' are conserved in human vascular endothelial growth factor (VEGF), human platelet [R:\LIBZZ]04999.doc:mr 142 derived growth factor A (PDGF-A), and human platelet derived growth factor B (PDGF- B), wherein said polypeptide lacks any domain that has one or more cysteine motifs of a Balbiani ring 3 protein (BR3P), and wherein said polypeptide binds to the extracellular domain of human Flt4 receptor tyrosine kinase.

62. A host cell transformed or transfected with a polynucleotide comprising a nucleotide sequence that encodes a polypeptide that is capable of binding to the extracellular domain of human Flt4 receptor tyrosine kinase, wherein said polynucleotide includes a strand that hybridizes to a DNA comprising the non-coding strand complementary to SEQ ID NO:32, under the following hybridization conditions: hybridization at 42 0 C for 20 hours in a solution containing formamide, 5 x SSPE, 5 x Denhardt's solution, 0.1% SDS and 0.1 mg/ml denatured 15 salmon sperm DNA; and washing the filter twice for thirty minutes at room temperature and twice for thirty minutes at 65 0 C with a wash solution containing 1 x SSC, and 0.1% SDS; and wherein said host cell expresses and secretes a polypeptide encoded by said 20 polynucleotide, and wherein said polypeptide binds the extracellular domain of human Flt4 receptor tyrosine kinase and has a molecular weight of about 23 kD or about 32 kD as assessed by SDS-PAGE under reducing conditions.

63. A host cell according to claim 61 or 62 that expresses a naturally occurring Flt4 ligand protein encoded by said polynucleotide.

64. A host cell according to claim 61 or 62 that expresses a human Flt4 ligand protein encoded by said polynucleotide. A host cell transformed or transfected with a polynucleotide comprising a nucleotide sequence that encodes the amino acid sequence shown in SEQ ID NO:33, wherein said host cell expresses a polypeptide encoded by said polynucleotide, said polypeptide including a contiguous portion of SEQ ID NO:33 that is sufficient to bind to the extracellular domain of human Flt4 receptor tyrosine kinase (Flt4EC), wherein said contiguous portion includes eight cysteine residues that are conserved iLI, in human vascular endothelial growth factor (VEGF), human platelet derived growth factor A (PDGF-A), and human platelet derived growth factor B (PDGF-B), (R:\LIBZZ]04999.doc:mrr 143 wherein said polypeptide lacks any portion of SEQ ID NO:33 that has one or more cysteine motifs of a Balbiani ring 3 portein (BR3P), and wherein said polypeptide binds to Flt4EC.

66. A host cell comprising a polynucleotide that includes the insert of plasmid pFLT4-L, deposited as ATCC accession No. 97231, wherein said host cell expresses and secretes a polypeptide encoded by said insert, wherein said secreted polypeptide binds to human Flt4 receptor tyrosine kinase and includes a domain defined by eight cysteine residues that are conserved in human vascular endothelial growth factor (VEGF), human platelet derived growth factor A (PDGF-A), and human platelet derived growth factor B (PDGF-B), and wherein said secreted polypeptide lacks any domain that has one or more cysteine motifs of a Balbiani ring 3 protein (BR3P).

67. A host cell according to claim 66, wherein said host cell expresses said polynucleotide and produces a human protein that is capable of binding to the i: s15 extracellular domain of human Flt4 receptor tyrosine kinase, said protein having a molecular weight of about 23 kD as assessed by SDS-PAGE under reducing conditions. 0

68. A host cell according to claim 67, wherein said host cell expresses said polynucleotide and produces a human protein that is capable of binding to the extracellular domain of human Flt4 receptor tyrosine kinase, said protein having a 20 molecular weight of about 32 kD assessed by SDS-PAGE under reducing conditions.

69. A host cell transformed or transfected with a polynucleotide comprising a nucleotide sequence that encodes the amino acid sequence shown in SEQ ID NO:33, wherein said host cell expresses a polypeptide encoded by said polynucleotide, said polypeptide including a contiguous portion of SEQ ID NO:33 that is sufficient to bind to the extracellular domain of human Flt4 receptor tyrosine kinase (Flt4EC), wherein said contiguous portion includes eight cysteine residues that are conserved in human vascular endothelial growth factor (VEGF), human platelet derived growth factor A (PDGF-A), and human platelet derived growth factor B (PDGF-B), wherein said polypeptide lacks any portion of SEQ ID NO:33 that precedes position 103 and lacks any portion of SEQ ID NO:33 that has one or more cysteine motifs of a Balbiani ring 3 protein (BR3P), and wherein said polypeptide has a molecular weight of about 23 kD as assessed by SDS PAGE under reducing conditions and binds to Flt4EC. [R:\LIBZZ]04999.doc:mrr 144 A host cell according to any one of claims 61-69, wherein said polynucleotide is an expression vector, said expression vector including an expression control sequence operatively linked to sequence that encodes said polypeptide.

71. A eukaryotic host cell according to any one of claims 61-70 that secretes said polypeptide.

72. A method of making a polypeptide that binds to the extracellular domain of Flt4 receptor tyrosine kinase, said method comprising the steps of: growing a host cell transformed or transfected with a nucleic acid according to any one of claims 37-56 under conditions in which the cell expresses a polypeptide encoded by the nucleic acid; and purifying the polypeptide from said host cell or from a growth medium of said host cell, wherein the polypeptide binds to the extracellular domain of Flt4 receptor tyrosine kinase.

73. A method for producing a polypeptide that is capable of binding the 15 extracellular domain of human Flt4 receptor tyrosine kinase and stimulating tyrosine phosphorylation of Flt4 receptor tyrosine kinase, comprising the steps of: growing a host cell according to any one of claims 59-71 under conditions which permit expression in said host cell of a polypeptide encoded by said polynucleotide; and isolating said polypeptide from the host cell or the growth medium of the host cell, 20 wherein said polypeptide binds to the extracellular domain of human Flt4 receptor tyrosine kinase and stimulates phosphorylation of Flt4 receptor tyrosine kinase.

74. A method according to claim 72 or 73, wherein said host cell is a mammalian host cell that secretes said polypeptide and wherein said isolating step comprises isolating S said polypeptide from said growth medium.

75. A method for producing a polypeptide that binds to the extracellular domain (EC) of human Flt4 receptor tyrosine kinase (Flt4), comprising steps of: growing a host cell comprising a polynucleotide that comprises a nucleotide sequence that encodes the amino acid sequence set forth in SEQ ID NO:33, under conditions in which the host cell expresses and secretes a polypeptide encoded by the polynucleotide; and isolating a polypeptide that binds Flt4 EC from the growth medium of the host cell, said polypeptide having a molecular weight of approximately 23 kD as assessed by SDS- PAGE under reducing conditions and having an amino acid sequence comprising a portion of SEQ ID NO:33 effective to bind Flt4 EC. [R:\LIBZZ]04999.doc: rr 145

76. A method according to claim 75 wherein said polynucleotide comprises an expression vector that comprises a nucleotide sequence that encodes the amino acid set forth in SEQ ID NO:33.

77. A method according to claim 75 wherein said host cell comprises a PC-3 prostatic adenocarcinoma cell (ATCC CRL1435).

78. A method according to claim 75 wherein said polynucleotide comprises the insert of plasmid pFLT4-L, deposited as ATCC Accession No. 97231.

79. A purified polypeptide that binds to the extracellular domain of Flt4 receptor tyrosine kinase, said polypeptide produced by a method according to any one of claims 72-78. A polypeptide according to any one of claims 1-31 or 79, wherein said polypeptide further includes a peptide tag to facilitate purification of the polypeptide.

81. A polypeptide according to claim 80, wherein said tag comprises a polyhistidine amino acid sequence. 15 82. A polypeptide according to claim 22 or 23 that is a murine polypeptide.

83. A purified polypeptide that binds Flt4 receptor tyrosine kinase, said polypeptide comprising the amino acid sequence set forth in SEQ ID NO:41 or a portion thereof effective to permit such binding.

84. A purified and isolated murine Flt4 ligand precursor polypeptide comprising 20 the amino acid sequence set forth in SEQ ID NO:41. A composition comprising a polypeptide according to any one of claims 79- **o 84, and further comprising a pharmaceutically acceptable diluent, adjuvant, excipient, or carrier.

86. A purified and isolated nucleic acid comprising a nucleotide sequence encoding the polypeptide according to claim 82, 83 or 84.

87. A purified polypeptide capable of binding to the extracellular domain of human Flt4 receptor tyrosine kinase, said polypeptide produced by a method comprising the steps of: expressing a nucleic acid in a host cell, said nucleic acid comprising a nucleotide sequence encoding the amino acid sequence in SEQ ID NO:41 or a portion thereof effective to permit such binding; and purifying a polypeptide that binds to the extracellular domain of human Flt4 receptor tyrosine kinase from said host cell or from a growth medium of said host cell, wherein said polypeptide lacks at least the carboxy-terminal portion of SEQ ID NO:41 S ,that is characterized by cysteine motifs of a Balbiani ring 3 protein. 1/' R:\LIBZZ)O4999.docm r 146

88. A composition comprising polyclonal antibodies raised against a polypeptide according to any one of claims 1-31 and 79-84.

89. A purified antibody that binds to a peptide consisting of the amino acid sequence set forth in SEQ ID NO:39.

90. An antibody according to claim 89 which is a monoclonal antibody.

91. A polyclonal antiserum produced by a process comprising the steps of: immunizing a mammal with a composition comprising a polypeptide according to any one of claims 1-31 and 79-84; and obtaining antiserum from said mammal after said immunizing step, said antiserum 0o containing antibodies that bind to said polypeptide.

92. Polyclonal antibodies purified from an antiserum according to claim 91, said polyclonal antibodies including antibodies that bind to said polypeptide.

93. A method for detecting endothelial cells in a biological tissue sample comprising the steps of: 15 exposing a biological tissue sample comprising endothelial cells to a polypeptide according to any one of claims 1-31 and 79-84, under conditions wherein said polypeptide binds to endothelial cells; and detecting said polypeptide bound to endothelial cells in said biological tissue, thereby detecting said endothelial cells. 20 94. The method according to claim 93, further comprising the step of washing said biological tissue, said washing step being performed after said exposing step and before said detecting step. A method of modulating the growth of mammalian endothelial cells, comprising the step of: contacting mammalian endothelial cells with a polypeptide according to any one of claims 1-31 and 79-84, in an amount effective to modulate the growth of the mammalian endothelial cells.

96. Use of a polypeptide according to any one of claims 1-31 and 79-84 in the preparation of a medicament for modulating the growth of mammalian endothelial cells.

97. A method of modulating the activity of human Flt4 receptor tyrosine kinase comprising administering to a person in need of modulation of Flt4 receptor tyrosine kinase activity a composition according to claim 32 or

98. A method of modulating the activity of human Flt4 receptor tyrosine kinase comprising contacting cells that express human Flt4 receptor tyrosine kinase with a ''lypeptide according to any one of claims 1-31 and 79-84. 147

99. Use of a polypeptide according to any one of claims 1-31 and 79-84 in the preparation of a medicament for modulating the activity of Flt4 receptor tyrosine kinase.

100. A method of modulating the growth of lymphatic endothelia, comprising steps of: identifying a patient in need of modulation of the growth of lymphatic endothelia; and administering to the patient a composition comprising a polypeptide in an amount effective to modulate the growth of lymphatic endothelial cells, wherein said polypeptide is a polypeptide according to any one of claims 1-31 and 79-84.

101. A method according to claim 100, wherein the identifying step comprises selecting a patient having lymphatic vessel loss or damage due to surgical intervention, wherein the polypeptide binds and stimulates phosphorylation of Flt4 expressed in endothelial cells, and wherein the polypeptide is administered in an amount effective to stimulate growth 15 of lymphatic endothelia. S..102. A method according to claim 101, wherein the polypeptide is administered in an amount effective to stimulate regrowth of lymphatic vessels.

103. A method according to claim 100, wherein the identifying step comprises identifying a patient having or at risk for developing edema, 20 wherein the polypeptide binds and stimulates phosphorylation of Flt4 expressed in endothelial cells, and wherein the polypeptide is administered in an amount effective to stimulate growth of lymphatic endothelia.

104. A method according to claim 100, wherein the identifying step comprises identifying a patient having lymphatic vessel aplasia, wherein the polypeptide binds and stimulates phosphorylation of Flt4 expressed in endothelial cells, and wherein the polypeptide is administered in an amount effective to stimulate growth of lymphatic endothelia.

105. A method according to claim 100, wherein the identifying step comprises selecting an organ transplant patient, wherein the polypeptide binds and stimulates phosphorylation of Flt4 expressed in endothelial cells, and wherein the polypeptide is administered in an amount effective to stimulate growth %S p lymphatic endothelia. 148

106. A method according to claim 100, wherein the identifying step comprises identifying a patient having reduced lymphatic vessel function due to lymphatic vessel blockage, wherein the polypeptide binds and stimulates phosphorylation of Flt4 expressed in endothelial cells, and wherein the polypeptide is administered in an amount effective to stimulate growth of lymphatic endothelia.

107. A method according to claim 100, wherein the identifying step comprises identifying a patient having lymphatic vessel occlusion, wherein the polypeptide binds and stimulates phosphorylation of Flt4 expressed in endothelial cells, and wherein the polypeptide is administered in an amount effective to stimulate growth of lymphatic endothelia.

108. A method according to claim 100, wherein the identifying step comprises 15 identifying a patient having elephantiasis, wherein the polypeptide binds and stimulates phosphorylation of Flt4 expressed in endothelial cells, and wherein the polypeptide is administered in an amount effective to stimulate growth S. of lymphatic endothelia. 20

109. A method according to claim 100, wherein the identifying step comprises identifying a patient having a lymphangioma, wherein the polypeptide binds and inhibits phosphorylation of Flt4 expressed in endothelial cells.

110. A method according to claim 100, wherein the administering step comprises administering a composition that comprises the polypeptide and a diluent, adjuvant, excipient, or carrier.

111. The use of a polypeptide according to any one of claims 1-31 and 79-84 in the manufacture of a medicament for modulating the growth of lymphatic endothelia.

112. The use of a polypeptide according to any one of claims 1-31 and 79-84 in the manufacture of a medicament for treatment of a disease or condition selected from the group consisting of lymphatic vessel loss or damage due to surgical intervention, edema, lymphatic vessel aplasia, lymphatic vessel occlusion, and elephantiasis.

113. A method of modulating the activity of Flt4 receptor tyrosine kinase (Flt4) in Flt4-expressing cells, comprising steps of: 35 providing a nucleic acid according to any one of claims 37-56; 149 transforming or transfecting a cell with the nucleic acid such that the cell expresses and secretes a polypeptide encoded by said nucleic acid, wherein said secreted polypeptide binds the extracellular domain of human Flt4 and has a molecular weight of about 23 kD as assessed by SDS-PAGE under reducing conditions; and contacting Flt4-expressing cells with the secreted 23 kD polypeptide.

114. Use of a nucleic acid according to any one of claims 37-56 in the manufacture of a medicament for the modulation of the growth of lymphatic endothelia.

115. A purified nucleic acid comprising a vertebrate VEGF-C promoter nucleotide sequence. 1 0

116. A purified and isolated nucleic acid comprising a portion of SEQ ID NO:54, wherein said portion is capable of promoting expression of a protein encoding gene operatively linked thereto.

117. A purified and isolated mammalian nucleic acid which hybridizes to a nucleic acid complementary to SEQ ID NO:54 under the following hybridization conditions: 15 hybridization at 42 0 C in a hybridization solution comprising 50% formamide, 5 X SSC, 20 mM Na*P0 4 pH 6.8; and washing in 0.2 x SSC at 55 0 C, S* wherein said nucleic acid is capable of promoting expression of a protein encoding gene operatively linked thereto. 2 118. A polypeptide according to any one of claims 1 to 30, substantially as S 20 hereinbefore described with reference to any one of the examples.

119. A method of producing a polypeptide according to any one of claims 1 to said method substantially as hereinbefore described with reference to any one of the examples.

120. A polypeptide produced according to the method of claim 119.

121. A pharmaceutical composition comprising a polypeptide according to claim 118 or 120 together with a pharmaceutically acceptable diluent, adjuvant, excipient or carrier.

122. A purified and isolated nucleic acid sequence according to any one of claims 37 to 56, said nucleic acid sequence substantially as hereinbefore described with reference to any one of the examples.

123. An expression construct comprising a nucleic acid sequence according to claim 122.

124. A host cell transformed or transfected with a nucleic acid sequence according ^^TRGo claim 122 or an expression construct according to claim 123. [R:\LIBZZ]04999.doc: rr 150

125. A method for detecting endothelial cells in a biological tissue sample comprising the steps of: exposing a biological tissue sample comprising endothelial cells to a polypeptide according to claim 118 or 120, under conditions wherein said polypeptide binds to endothelial cells; and detecting said polypeptide bound to endothelial cells in said biological tissue, thereby detecting said endothelial cells.

126. A polypeptide according to any one of claims 1 to 31, 79 to 84, 118 and 120 when used for detecting endothelial cells in a biological tissue sample.

127. A method of modulating the growth of mammalian endothelial cells, comprising the step of: contacting mammalian endothelial cells with a polypeptide according to claim 118 or 120, in an amount effective to modulate the growth of the mammalian endothelial cells. 15 128. A polypeptide according to any one of claims 1 to 31, 79 to 84, 118 and 120 when used for modulating the growth of mammalian cells.

129. Use of a polypeptide according to claim 118 or 120 in the preparation of a medicament for modulating the growth of mammalian endothelial cells.

130. A method of modulating the activity of human Flt4 receptor tyrosine kinase c. comprising contacting cells that express human Flt4 receptor tyrosine kinase with a polypeptide according to claim 118 to 120. S 131. A polypeptide according to any one of claims 1 to 31, 79 to 84, 118 and 120 when used for modulating the activity of human Flt4 receptor tyrosine kinase.

132. Use of a polypeptide according to claim 118 or 120 in the preparation of a medicament for modulating the activity of Flt4 receptor tyrosine kinase.

133. A method of modulating the growth of lymphatic endothelia, comprising steps of: identifying a patient in need of modulation of the growth of lymphatic endothelia; and administering to the patient a composition comprising a polypeptide in an amount effective to modulate the growth of lymphatic endothelial cells, wherein said polypeptide is a polypeptide according to claim 118 or 120.

134. A polypeptide according to any one of claims 1 to 31, 79 to 84, 118 and 120 S when used for modulating the growth of lymphatic endothelia. [R:\LIBZZ]04999.doc: rr 151

135. Use of a polypeptide according to claim 118 or 120 in the preparation of a medicament for modulating the growth of lymphatic endothelia.

136. A polypeptide according to any one of claims 1 to 31, 79 to 84, 118 and 120 when used for treatment of a disease or condition selected from the group consisting of lymphatic vessel loss or damage due to surgical intervention, edema, lymphatic vessel aplasia, lymphatic vessel occlusion, and elephantiasis.

137. Use of a polypeptide according to claim 118 or 120 in the manufacture of a medicament for treatment of a disease or condition selected from the group consisting of lymphatic vessel loss or damage due to surgical intervention, edema, lymphatic vessel aplasia, lymphatic vessel occlusion, and elephantiasis.

138. A method of modulating the activity of Flt4 receptor tyrosine kinase (Flt4) in Flt4-expressing cells, comprising steps of: providing a nucleic acid according to claim 122; transforming or transfecting a cell with the nucleic acid such that the cell expresses and secretes a polypeptide encoded by said nucleic acid, wherein said secreted polypeptide binds the extracellular domain of human Flt4 and has a molecular weight of about 23 kD as assessed by SDS-PAGE under reducing conditions; and contacting Flt4-expressing cells with the secreted 23 kD polypeptide.

139. A nucleic acid according to any one of claims 37 to 56 and 122, when used for modulating the activity of Flt4 receptor tyrosine kinase (Flt4) in Flt4-expressing cells.

140. Use of a nucleic acid according to claim 122 in the manufacture of a medicament for the modulation of the growth of lymphatic endothelia.

141. A purified and isolated polypeptide according to any one of claims 11, 12, 17 and 18, wherein said polypeptide is capable of binding the extracellular domain of Kdr receptor tyrosine kinase (VEGFR-2).

142. A purified and isolated nucleic acid sequence comprising a nucleotide sequence that encodes a polypeptide according to claim 141.

143. A host cell transformed or transfected with a nucleic acid sequence according to claim 142.

144. A pharmaceutical composition comprising a polypeptide according to claim 141 together with a pharmaceutically-acceptable diluent, adjuvant, excipient, or carrier.

145. Use of a polypeptide according to claim 141 in the manufacture of a medicament for modulating the growth of mammalian endothelial cells. [R:\LIBZZ]04999.doc:mrr 152

146. A method of modulating the growth of mammalian endothelial cells comprising the step of: contacting mammalian endothelial cells with a polypeptide according to claim 141, in an amount effective to modulate the growth of the mammalian endothelial cells.

147. A polypeptide according to claim 141 when used for modulating the growth of mammalian endothelial cells.

148. A purified protein according to claim 33, wherein said second polypeptide has an amino acid sequence corresponding to amino acid residues 228 to 419 of SEQ ID NO:33. to 149. A purified protein according to claim 33 or 148, wherein said first polypeptide is a polypeptide according to claim 14 or

150. A purified protein according to any one of claims 33, 148 or 149, wherein said protein is a heterodimer.

151. A protein according to any one of claims 33 and 148 to 150, wherein said 15 protein is a tetramer.

152. A pharmaceutical composition comprising a protein according to any one of claims 33 and 148 or 151 together with a pharmaceutically-acceptable diluent, adjuvant, excipient, or carrier.

153. Use of a protein according to any one of claims 33 and 148 or 151 in the S 20 manufacture of a medicament for modulating the growth of mammalian endothelial cells.

154. A method of modulating the growth of mammalian endothelial cells comprising the step of: contacting mammalian endothelial cells with a protein according to any one of claims 33 and 148 or 151, in an amount effective to modulate the growth of the mammalian endothelial cells.

155. A protein according to any one of claims 33 and 148 or 151 when used for modulating the growth of mammalian endothelial cells. Dated 4 October, 2002 Ludwig Institute For Cancer Research Helsinki University Licensing Ltd. OY Patent Attorneys for the Applicant/Nominated Person SSPRUSON FERGUSON [R:\LIBZZ]04999.doc:mr