HK1065806A - Flt4(vegfr-3) as a target for tumor imaging and anti-tumor therapy - Google Patents

Description

Flt4(VEGFR-3) as a target for tumor imaging and anti-tumor therapy

This application is a continuation-in-progress application claiming united states patent application serial No. 09/169,079 filed on 9/10/1998; U.S. patent application serial No. 08/901,710, filed on 1997, 7/28/6,107,046; now U.S. patent No.5,776,755, U.S. patent application serial No. 08/340,011 filed on 14/11/1994; priority of the currently abandoned U.S. patent application serial No. 08/257,754 filed on 9/6 in 1994; the latter two applications are in turn part of a now-filed, U.S. patent application serial No. 07/959,951 filed on 10/9 1992. All of these applications are incorporated herein by reference in their entirety.

Technical Field

The present invention relates generally to receptor genes, particularly receptor tyrosine kinases, which are inserted into recombinant DNA vectors and the resulting proteins produced in host microbial strains and host eukaryotic cells. More particularly, the invention relates to Flt4, a receptor tyrosine kinase; to Flt 4-encoding nucleotide sequences; to methods for the production of Flt 4-encoding DNAs and gene products thereof; to nucleic acid probes that specifically recognize (hybridize) to nucleic acids encoding the receptor; to antibodies that specifically recognize the receptor; and to methods of using such probes and antibodies and other Flt4 binding compounds, e.g., for identifying lymphatic and post-capillary venules (HEVs) in animal and human tissue, and for promoting or preventing growth of Flt 4-expressing cells under pathological conditions.

Background

The behavior of cells responsible for the development, maintenance and repair of differentiated cells and tissues is largely regulated by intercellular signals conducted through growth factors and similar ligands and their receptors. Receptors are located on the cell surface of effector cells and they bind peptides or polypeptides that are thought to be growth factors and other hormone-like ligands. The result of this interaction is a rapid biochemical change in the effector cell, and a rapid and long-term readjustment of cellular gene expression. Some receptors associated with various cell surfaces can bind specific growth factors.

Tyrosine phosphorylation is a key mode of signal transduction across the plasma membrane. Several tyrosine kinase genes encode transmembrane receptors for polypeptide growth factors and hormones such as Epidermal Growth Factor (EGF), insulin-like growth factor-I (IGF-I), platelet derived growth factors (PDGF-A and-B) and Fibroblast Growth Factors (FGFs) [ Heldin et al, Cell Regulation, 1: 555-566 (1990); ullrich et al, Cell, 61: 243-54(1990)]. Some receptors for hematopoietic growth factors are tyrosine kinases; they include c-fms, the colony stimulating factor 1 receptor [ Sherr et al, Cell, 41: 665-: 225-33(1990)].

These receptors differ in their specificity and affinity. In general, receptor tyrosine kinases are glycoproteins that consist of an extracellular domain capable of binding a specific growth factor, a transmembrane domain that is usually the alpha-helical portion of the protein, a juxtamembrane domain (where the receptor may be regulated, for example, by protein phosphorylation), a tyrosine kinase domain (which is the enzymatic component of the receptor), and a carboxy-terminal tail that is involved in the recognition and binding of tyrosine kinase substrates in many receptors.

In some receptor tyrosine kinases, alternative splicing processes and alternative polyadenylation can produce several different polypeptides from the same gene. They may or may not contain the various domains listed above. Thus, some extracellular domains may be expressed as isolated proteins secreted by cells and some forms of the receptor may lack a tyrosine kinase domain and contain only the extracellular domain inserted into the plasma membrane through a transmembrane domain plus a short carboxy-terminal tail.

The physiology of the vascular system, embryonic angiogenesis and vasculogenesis, blood clotting, wound healing and regeneration, and several diseases are associated with the vascular endothelium lining blood vessels. The formation of the vascular tree occurs through angiogenesis and, according to some theories, the formation of the lymphatic system begins shortly after the formation of arteries and veins by sprouting from the veins. See Sabin, f.r., am.j.anat., 9: 43 (1909); and van der push, s.c.j, adv.anat.embryol.cell biol., 51: 3(1975).

After the fetal stage, endothelial cells proliferate very slowly except during angiogenesis associated with the formation of new vessels. Growth factor-stimulated angiogenesis exerts its effects through specific endothelial cell surface receptor tyrosine kinases.

Among the ligands for receptor tyrosine kinases, Platelet Derived Growth Factor (PDGF) has been shown to be angiogenic, albeit weakly, in the chick chorioallantoic membrane. Transforming growth factor alpha (TGF α) is an angiogenic factor secreted by some tumor cell types and by macrophages. Hepatocyte Growth Factor (HGF), a ligand for the receptor encoded by the c-met proto-oncogene, also has strong angiogenic properties, inducing a response similar to TGF α in cultured endothelial cells.

Evidence suggests that there are endothelial cell-specific growth factors and receptors primarily responsible for stimulating endothelial cell growth, differentiation, and certain differentiation functions. The most widely studied growth factor is Vascular Endothelial Growth Factor (VEGF), a member of the PDGF family. Vascular endothelial growth factor is a dimeric glycoprotein of disulfide-linked 23kDa subunits, discovered for its mitogenic activity on endothelial cells and its ability to induce vascular permeability (hence its alternative name vascular permeability factor). Other reported effects of VEGF include the transfer of intracellular Ca2+, the induction of plasminogen activator and plasminogen activator inhibitor-1 synthesis, the stimulation of hexose transport in endothelial cells, and the promotion of monocyte migration in vitro. The 4 VEGF isoforms encoded by the different mRNA splice variants appear to be equally capable of stimulating mitosis in endothelial cells. The 121 and 165 amino acid isoforms of VEGF are secreted in soluble form, while the isoforms of 189 and 206 amino acid residues remain associated with the cell surface and have strong affinity for heparin. Soluble non-heparin-binding and heparin-binding forms of placental growth factor have also been described (PlGF; 131 and 152 amino acids, respectively) and are expressed in placenta, trophoblastic tumors, and cultured human endothelial cells.

The expression pattern of VEGF suggests that it is involved 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 neuroectodermal chamber produces VEGF during the capillary ingrowth stage. On day two of quail development, the vascularized area of the yolk sac as well as the whole embryo appeared to express VEGF. In addition, epithelial cells near the curtain endothelial membrane showed sustained VEGF expression in adult mice, suggesting a role in the maintenance of this particular endothelial phenotype and function.

Two high affinity receptors for VEGF have been identified, VEGFR-1/Flt1 (fms-like tyrosine kinase-1) and VEGFR-2/Kdr/Flk-1 (kinase insert domain containing receptor/fetal liver kinase-1). These receptors belong to the PDGF-receptor family. However, the VEGF receptor has 7 immunoglobulin-like loops (relative to 5 in the other members of the PDGF family) and a longer kinase insert in its extracellular domain. Expression of VEGF receptors occurs primarily in vascular endothelial cells, although some are also present in monocytes and melanoma cell lines. 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 by VEGFR-1 and VEGFR-2 appear to be cell type specific.

VEGFR-1 and VEGFR-2 bind VEGF165 (K) with high affinity_dApproximately 20pM and 200pM, respectively). Flk-1 receptor has also been shown to autophosphorylate in response to VEGF, but phosphorylation of Flt1 is barely detectable. VEGFR-2 mediated signals cause a significant change in morphology, actin reorganization and membrane ruffling of porcine aortic endothelial cells overexpressing this receptor. In these cells, VEGFR-2 also mediates ligand-induced chemotaxis and mitogenicity; whereas VEGFR-1 transfected cells lack mitogenic response to VEGF. In contrast, VEGF has a strong growth stimulating effect on rat sinusoidal endothelial cells expressing VEGFR-1. Phosphoproteins co-precipitate differently with VEGFR-1 and VEGFR-2, suggesting that different signaling molecules interact with receptor-specific intracellular sequences.

In situ hybridization experiments, mouse VEGFR-2mRNA expression was found in the yolk sac and the intraembryonic mesoderm (estimated as 7.5 day p.c. (p.c.) embryos from which endothelium was produced), and later in putative angioblasts, the endocardium and large and small vascular endothelium (12.5 days p.c.). The abundance of VEGFR-2mRNA in vascular sprouts and endothelial cells proliferating in the branches of the embryonic and early postnatal brains and the reduced expression in adult brains suggests that VEGFR-2 is a major regulator of angiogenesis and vasculogenesis. VEGFR-1 expression was also associated with early angiogenesis in mouse embryos and with the formation of new vessels in healing skin wounds. However, high levels of VEGFR-1 expression were detected in adult organs, suggesting that VEGFR-1 plays a role in quiescent endothelium of mature vessels not involved in cell growth. The avian homolog of VEGFR-2 was observed in the mesoderm from the onset of gastrulation, whereas the VEGFR-1 homolog was first found in cells co-expressing endothelial markers. FGF-2, required for angiogenic differentiation of these cells, upregulates VEGFR-2 expression in quail ectodermal cultures in vitro. It was found that the expression of both VEGF receptors becomes more limited late in development. VEGFR-1 and VEGFR-2 appear to overlap, but to be expressed in slightly different patterns, in human fetal tissues. These data suggest that VEGF and its receptors act in a paracrine fashion to regulate endothelial cell differentiation and tissue neovascularization.

Recently, it has been shown that VEGF is a hypoxia-inducible stimulator of endothelial cell growth and angiogenesis, and inhibition of VEGF activity using specific monoclonal antibodies has been shown to reduce the growth of experimental tumors and the density of their blood vessels. [ Ferrara et al, Endocrine Reviews, 18: 4-25 (1997); shibuya et al, adv. 281 and 316 (1995); kim et al, Nature, 362: 841-844(1993)].

Growth of solid tumors in sizes exceeding a few cubic millimeters is dependent on vascular supply, making angiogenesis an attractive target for anticancer therapy. Encouraging results have been reported with endogenous angiogenesis inhibitors or "statins" including angiostatin, fragments of plasminogen, and endostatin, fragments of collagen 18. [ O' Reilly et al, Cell, 79: 315-; o' Reilly et al, Cell, 88: 277-85(1997). ]. Both factors are normally produced by the primary tumor and remain dormant for metastasis. Systemic administration of either statin was also shown to induce and maintain dormancy of primary tumors in animal models. Receptors and signaling through inhibin, as well as proteases that activate them, remain to be identified. A need still exists for additional therapeutic molecules that control angiogenesis in the treatment of cancer and other pathological disease states.

Primary breast cancers have been shown to express several angiogenic polypeptides, of which VEGF is most abundant. [ see, e.g., Relf et al, Cancer Res., 57: 963-969(1997)]. In invasive and non-invasive ductal (in situ) breast cancer, tumor cells contain high levels of VEGF mRNA. [ Brown et al, hum. Pathol., 26: 86-91(1995)]. Endothelial cells adjacent to the carcinoma in situ expressed VEGFR-1 and VEGFR-2 mRNAs. VEGF and its receptors are responsible for the angiogenic progression of malignant breast tumors, since in several independent studies, a correlation between tumor vascular density and the prognosis of the disease was found. [ Weidner et al, J.Natl.cancer Inst., 84: 1875-1887(1992)]. There is a need for additional markers for breast cancer and breast cancer-associated angiogenesis in order to improve diagnosis and screening, and to serve as targets for therapeutic intervention.

The primary function of the lymphatic system is to provide fluid return to the tissues and to transport many extravascular substances back to the blood. In addition, during the suppurative process, lymphocytes leave the blood, migrate through lymphoid organs and other tissues, and enter the lymphatic vessels, returning the blood through the thoracic duct. Specialized venules, i.e., postcapillary venules (HEVs), re-bind lymphocytes and cause their extravasation into tissues. Thus, lymphatic vessels, and in particular lymph nodes, play an important role in immunology and in the development of metastases of different tumors.

Since the beginning of the 20 th century, three different theories have been proposed as to the embryonic origin of the lymphatic system. However, lymphatic vessels are difficult to identify because there are no known specific markers available for them.

Lymphatic vessels are most commonly studied with the aid of lymphography. In lymphography, an X-ray contrast agent is injected directly into the lymphatic vessels. The contrast agent is distributed along the output catheter of the lymphatic system. The contrast agent is concentrated in the lymph nodes where it stays for up to half a year, during which time X-ray analysis allows the size and structure of the lymph nodes to be tracked. This diagnostic method is particularly important in cancer patients with lymph node metastases and lymphoid malignancies, such as lymphoma. However, there remains a need in the art for improved materials and methods for lymphatic tissue imaging.

Summary of the invention

The present invention describes a novel receptor tyrosine kinase gene located on chromosome 5, identified as an unknown tyrosine kinase-homologous PCR-cDNA fragment from human leukemia cells [ Aprelikova et al, Cancer Res., 52.746-748(1992) ]. This gene and its encoded protein are referred to as Flt 4. The abbreviation comes from the phrase fms-like tyrosine kinase 4.

Flt4 is a receptor tyrosine kinase closely related in structure to the VEGFR-1 and VEGFR-2 gene products. Due to this similarity and the subsequent similarities found between ligands for these three receptors, the Flt4 receptor is also known as VEGFR-3. The names Flt4 and VEGFR-3 are used interchangeably herein. Despite the similarity between these three receptors, the mature form of Flt4 differs from VEGFRs in that it is cleaved proteolytically in the extracellular domain into two disulfide-linked 125/120kD and 75kD polypeptides. The Flt4 gene encodes 4.5 and 5.8kb mRNAs, exhibits alternative 3' exons and encodes polypeptides of 190kD and 195kD, respectively.

Further evidence of differentiation is that VEGF does not exhibit specific binding to Flt4 and does not induce its autophosphorylation.

Comparison of Flt4, Flt1, and KDR/Flk-1 receptor mRNA signals in tissue studies showed overlapping, but distinct expression patterns. Kaipain et al, j.exp.med., 178: 2077(1993). Flt4 gene expression appeared to be more restricted than VEGFR-1 or VEGFR-2 expression. Expression of Flt4 was first detectable by in situ hybridization in the first place in head mesenchymal hemangioblasts, the major vein and in the extraembryonic area of the allantois of 8.5 day post-coital mouse embryos. In 12.5 day p.c. embryos, Flt4 signals were observed on developing venous and putative lymphatic endothelia, while arterial endothelia appeared negative. During late stages of development, Flt4mRNA became localized in developing lymphatic vessels. Flt4mRNA was expressed in adult human tissues only in lymphatic endothelium and some retrocapillary venules and increased expression occurred in the lymphatic sinuses of lymph node metastatic cancers and in lymphangiomas. This result supports the theory of venous origin of lymphatic vessels.

The protein product of the Flt4 receptor tyrosine kinase cDNA cloned from the human erythroleukemia cell line is N-glycosylated and contains 7 immunoglobulin-like loops in its extracellular domain. The cytoplasmic tyrosine kinase domain of Flt4 is approximately 80% identical at the amino acid level to the corresponding domains of Flt1 and KDR and is approximately 60% identical to the receptors for platelet derived growth factor, colony stimulating factor-1, stem cell factor, and Flt3 receptor. See Pajusola et al, Cancer res, 52: 5738(1992).

The present invention provides isolated polynucleotides (e.g., structurally defined DNA or RNA fragments) encoding Flt4 receptor tyrosine kinase useful in the production of Flt4 protein and peptide fragments thereof and for the recovery of related genes from other sources.

The present invention provides recombinant DNA vectors containing heterologous fragments encoding Flt4 receptor tyrosine kinase or related proteins which can be inserted into microorganisms or eukaryotic cells and which are capable of expressing the encoded proteins.

The present invention provides eukaryotic cells capable of producing useful amounts of Flt4 receptor tyrosine kinase and functionally similar proteins from many species.

The present invention provides peptides that can be synthesized in the laboratory or produced by microorganisms that mimic the activity of the native Flt4 receptor tyrosine kinase protein. In another embodiment, the invention relates to peptides that inhibit the activity of the Flt4 receptor tyrosine kinase protein.

Particularly preferred peptides are selected from the group consisting of: (a) flt 4-short chain forms having the nucleotide and deduced amino acid sequences shown in SEQ ID NOs.1 and 2; and (b) a second form having different nucleotides and corresponding amino acid residues at its carboxy terminus, i.e., the Flt 4-long form, having the nucleotide and putative amino acid sequences shown in SEQ. ID NOs.3 and 4. The Flt4 long chain form has a length of 1363 amino acid residues.

DNA and RNA molecules, recombinant DNA vectors, and modified microorganisms or eukaryotic cells comprising nucleotide sequences encoding any of the above proteins or peptides are also part of the invention. In particular, sequences containing all or part of the following two DNA sequences, complementary DNA or RNA sequences, or corresponding RNA sequences are particularly preferred: (a) such as SEQ ID NO: 1, encoding Flt 4-short chain form [ SEQ ID NO: 2], and (b) a nucleic acid sequence such as SEQ ID NO: 3, encoding the DNA sequence of seq id NO: 1, nucleotide 3913 and 4416, encoding the Flt 4-long form [ SEQ ID NO: 4].

DNA and RNA molecules containing fragments of longer sequences are also provided for use in practicing preferred aspects of the invention involving the production of the peptides and the production of oligonucleotide probes by genetic engineering techniques.

As the DNA sequence encoding the Flt4 protein is identified herein, DNA encoding the Flt4 protein may be produced, for example, by polymerase chain reaction or by chemical synthesis using commercially available equipment, after which the gene is inserted into any of a number of available DNA vectors using known techniques of recombinant DNA technology. In addition, automated equipment is also available, making direct synthesis of any of the peptides disclosed herein easy to achieve.

The invention also relates to Flt4 peptides and other constructs, and to the use of Flt4 as specific markers for lymphatic endothelial cells.

In one embodiment, the invention relates to nucleic acid probes and antibodies, particularly monoclonal antibodies, that recognize Flt4, and compositions containing the antibodies.

In yet another embodiment, the invention relates to a method of monitoring lymphatic vessels in tissue samples and organisms. It is also an object of the present invention to provide a clinical test for describing lymphatic tissue, and in particular lymphatic vessel status (inflammation, infection, injury, growth, etc.), and to provide a method for detecting lymphatic vessels, and thus lymphatic vascularization in an organism.

It is another object of the present invention to provide monoclonal antibodies that specifically recognize the Flt4 receptor protein or various epitopes thereof. Diagnostic purposes using these monoclonal antibodies for detecting and measuring the amount of Flt4 receptor in tissues, and particularly lymphoid tissues, are also an object of the present invention. In the context of anti-Flt 4 antibodies, the terms "specifically recognize Flt 4", "specifically bind to Flt 4", "specific for Flt 4", and the like, refer to antibodies that preferentially bind to Flt4 (immunoreaction) relative to other endothelial cell surface receptors, including VEGFR-2/Kdr/Flk-1 and VEGFR-1/Flt 1. Thus, anti-Flt 4 antibodies or other Flt4 binding compounds specific for Flt4 may be useful for identifying and/or labeling Flt4 (e.g., medical imaging, detection, screening, or targeted therapy) in tissues or biological samples according to the methods of the invention described herein, as they either are unable to bind epitopes of other antigens at all, or bind other antigens only with an affinity that is significantly lower than their Flt4 binding affinity so as to be unobvious in these practical settings.

Another aspect of the present invention relates to a method for determining the presence of Flt 4-receptor in a cell sample comprising the steps of: (a) contacting a cell sample with an antibody of the invention, particularly a monoclonal antibody; and (b) detecting binding of said monoclonal antibody to Flt4 receptor. As will be apparent from the detailed description below, information regarding the presence, quantity, density, and location of Flt4 receptors in a tissue sample has diagnostic and prognostic relevance to the type and severity of the disease state; and if necessary, to specifically adapt an anti-Flt 4-based therapeutic regimen to only those patients with a disease characterized by expression of Flt4 in the tumor or in vessels or lymphatic vessels and tissues surrounding, serving or supplying the tumor. Screening for the presence of Flt4 receptor may therefore constitute a first step in a treatment regimen, and/or a monitoring step during a course of treatment.

The invention also relates to methods of modulating (e.g., antagonizing or potentiating) Flt4 function in lymphatic vascularization and in inflammatory, infectious, and immunological conditions. For example, in one embodiment, the method comprises inhibiting Flt-4 mediated lymphatic vascularization by providing a sufficient amount of a Flt 4-binding compound to inhibit the Flt4 endothelial cell sites involved in the response, particularly where Flt4 function is associated with diseases such as metastatic cancer, lymphoma, inflammation (chronic or acute), infection, and immunological diseases. Since many tumors metastasize through lymphatic vessels, therapies involving inhibition of the interaction between Flt4 ligand and Flt4 are expected to have broad application as part of an anti-cancer treatment regimen for inhibiting tumor metastasis.

The invention also relates to specific Flt 4-stimulating ligands and monoclonal antibodies and their use for stimulating lymphatic endothelium, and fragments and peptides and antibodies that inhibit Flt4 function when desired from studies with such ligands, e.g., in various disease states associated with Flt4 function.

The present invention provides a cell line source of ligands for the Flt4 receptor tyrosine kinase. Using conditioned media from these cells, the Flt4 ligand can be purified and cloned by using methods standard in the art. Using this conditioned medium or purified ligand, an assay system for Flt4 ligand and dimerization inhibitors as well as Flt4 signaling inhibitors is available that allows for the identification and preparation of such inhibitors.

In a preferred embodiment of the invention, the conditioned medium of the PC-3 cell line comprises a protein or fragment thereof capable of stimulating the Flt4 receptor and modulating the growth and differentiation functions of certain endothelial cells. Flt4 ligand or a peptide or derivative thereof is useful for modulating endothelial cell growth, differentiation and its differentiation function and for producing agonists and antagonists of the ligand. In particular, Flt4 ligand is useful for regulating lymphatic endothelia. However, Flt4 ligand may also stimulate the relevant KDR/Flk-1 receptor when purified or produced from recombinant sources.

The identification of Flt 4-stimulating ligands makes it possible directly to determine inhibitors of this ligand or inhibitors of Flt4 function. The inhibitors may simply be added to conditioned media containing Flt4 ligand, and if they inhibit autophosphorylation, they act as inhibitors of Flt4 signaling. For example, recombinant or synthetic peptides (including but not limited to fragments of the extracellular domain of Flt4) may be assayed for inhibition of Flt 4-ligand interaction or Flt4 dimerization. Such putative inhibitors of Flt4 and additional antibodies against Flt4 ligand, peptides or other compounds that inhibit Flt4 receptor-ligand interactions, and antisense oligonucleotides complementary to mRNA sequences encoding Flt4 ligand are useful for modulating endothelial cells and for treating diseases associated with endothelial cell function.

Detailed identification of the Flt4 ligand, designated VEGF-C, is described in PCT patent application No. PCT/US98/01973, filed 2.2.1998 and published as International publication No. WO 98/33917; in PCT patent application PCT/FI96/00427, published as International publication No. WO97/05250, 1996, 8/1; and in U.S. patent application priority documents, on which priority is claimed, all of which are incorporated herein by reference. The putative amino acid sequence of pro-VEGF-C is set forth herein as SEQ ID NO: 21, description.

A detailed description of a second Flt4 ligand, designated VEGF-D, is described in Achen et al, Proc.nat' l Acad.Sci.U.S.A., 95 (2): 548-. The putative amino acid sequence of pro-VEGF-D is set forth herein as SEQ ID NO: 22.

The invention also relates to methods of treating a mammalian organism having a disease characterized by expression of Flt4 tyrosine kinase (Flt4) in cells comprising the step of administering to the mammalian organism a composition comprising a compound effective to inhibit binding of a Flt4 ligand protein to Flt4 expressed in cells of the organism, thereby inhibiting Flt4 function. The disease may be a disease as already mentioned above, for example a disease characterized by undesired lymphatic vascularization. In addition, it has been found that Flt4 expression also occurs in the vasculature associated with at least some breast cancers, and possibly other cancers (i.e., the expression level is well beyond the barely detectable or undetectable expression level in the vasculature of the corresponding normal (healthy) tissue). Thus, in a preferred embodiment, the cells comprise endothelial cells (endothelial cells of lymphatic or blood vessels). In another embodiment, the cell comprises a tumor cell, such as certain lymphomas that express Flt 4. Particularly including treatment of humans.

By "a compound effective to inhibit the binding of Flt4 ligand protein to Flt4 expressed in biological cells" is meant any compound that inhibits the binding of Flt4 ligand, described herein as vascular endothelial growth factor C, which may be isolated from PC-3 conditioned media. The compound is also expected to be effective in inhibiting the binding of vascular endothelial growth factor D to Flt 4. Exemplary compounds include the following polypeptides: (a) a polypeptide comprising an antigen-binding fragment of an anti-Flt 4 antibody; (b) a polypeptide comprising a soluble Flt4 fragment (e.g., an extracellular domain fragment), wherein the fragment and polypeptide are capable of binding to a Flt4 ligand; (c) a polypeptide comprising a fragment or analog of a vertebrate vascular endothelial growth factor C (VEGF-C) polypeptide, wherein the polypeptide and fragment or analog bind to, but are unable to activate, Flt4 expressed on native host cells (i.e., biological cells expressing native Flt4 protein on their surface); and (D) a polypeptide comprising a fragment or analog of a vertebrate vascular endothelial growth factor D (VEGF-D) polypeptide, wherein the polypeptide and fragment or analog bind, but do not activate, Flt4 expressed on the native host cell. Small molecule inhibitors identifiable in standard in vitro screening assays using, for example, VEGF-C and recombinantly expressed Flt4 are also contemplated. Polypeptides comprising antigen-binding fragments of anti-Flt 4 antibodies are highly preferred. Such polypeptides include, for example, polyclonal and monoclonal antibodies that specifically bind Flt 4; a fragment of the antibody; chimeric and humanized antibodies; bispecific antibodies that specifically bind Flt4 and also specifically bind another antigen, and the like. The use of compounds that bind circulating Flt4 ligand and thereby inhibit the binding of the ligand to Flt4 is also contemplated. The compounds include anti-VEGF-C or anti-VEGF-D antibodies or polypeptides comprising antigen-binding fragments thereof. In related variations, the invention encompasses therapeutic methods for disrupting downstream intracellular Flt4 signaling, thereby inhibiting Flt4 function.

In a preferred variation, the compound further comprises a detectable label, as described elsewhere herein, or a cytotoxic agent. Exemplary cytotoxic agents include plant toxins (e.g., ricin, saporin), bacterial or fungal toxins, radioisotopes (e.g., 211-astatine, 212-bismuth, 90-yttrium, 131-iodine, 99 m-technetium, and other elements described herein), anti-metabolic drugs (e.g., methotrexate, 5-fluorodeoxyuridine), alkylating agents (e.g., chlorambucil), antimitotic agents (e.g., vinca alkaloids), and DNA intercalating agents (e.g., doxorubicin). Other exemplary agents include compounds or treatments that induce DNA damage when applied to cells. The agents and factors include radiation and waves that induce DNA damage, such as-radiation, X-rays, ultraviolet radiation, microwaves, electron emission, and the like. Various compounds, also described as "chemotherapeutic agents," function to induce DNA damage, all of which are intended for use in the combination therapy methods disclosed herein. Chemotherapeutic agents contemplated for use include, for example, doxorubicin, 5-fluorouracil (5FU), epipodophyllotoxin glucopyranoside (VP-16), camptothecin, actinomycin-D, mitomycin C, cis-platinum (CDDP) and even hydrogen peroxide. The invention also encompasses the use of one or more DNA damaging agents in combination, whether radiation-based or actual compounds, for example, using X-rays with cisplatin or using cisplatin with etoposide. Still other agents are doxorubicin (also known as doxorubicin), VP-16 (also known as epipodophyllotoxin glucopyranoside), and the like, daunorubicin (which is inserted into DNA, inhibits DNA-mediated RNA polymerase and inhibits DNA synthesis); mitomycin (also known as mutamycin and/or mitomycin-C); actinomycin D; vincristine and cyclophosphamide; bleomycin; VP16 (epipodophyllotoxin glucopyranoside); tumor necrosis factor [ TNF ]; paclitaxel; melphalan; cyclophosphamide, chlorambucil. Administration of the peptides of the invention may be carried out before or after treatment with other agents, with intervals ranging from a few minutes to a few weeks. In embodiments where the other agent and expression construct are administered separately, it is generally ensured that a significant time period does not expire between the time of each administration, so that the agent and peptide-based therapeutic agent are still able to exert a superior binding effect. In such a case, it is contemplated that the two modalities may be administered within about 12-24 hours of each other, more preferably within about 6-12 hours of each other, and most preferably with a delay time of only about 12 hours. In some cases, an extended period of time is required for the treatment to be effective, although several days (2, 3, 4,5, 6, or 7) to several weeks (1, 2, 3, 4,5, 6,7, or 8) elapse between administrations. Particularly including repeated treatments with one or both agents.

Likewise, for improved administration, the composition preferably further comprises a pharmaceutically acceptable diluent, adjuvant, or carrier medium.

As detailed herein, Flt4 expression, although largely restricted to lymphatic endothelium in healthy adults, has been identified as Flt4 expression in the vascular system surrounding at least some tumors. Accordingly, the present invention also includes a method of treating a mammalian organism having a neoplastic disease characterized by expression of Flt4 tyrosine kinase (Flt4) in vascular endothelial cells, comprising the steps of: administering to a mammalian organism in need of such treatment a composition comprising a compound effective to inhibit the binding of Flt4 ligand protein to Flt4 expressed in vascular endothelial cells of the organism, thereby inhibiting Flt 4-mediated proliferation of the vascular endothelial cells. Specifically contemplated is the treatment of a neoplastic disease selected from the group consisting of carcinoma (e.g., breast cancer), squamous cell carcinoma, lymphoma, melanoma, and sarcoma. However, it will be apparent that the screening techniques detailed herein can identify other tumors characterized by Flt4 expression in vascular endothelial cells that are candidate tumors for susceptibility to the anti-Flt 4 treatment regimens described herein. Particularly contemplated are the treatment of breast cancer characterized by expression of Flt4 in vascular endothelial cells. A neoplastic disease characterized by expression of Flt4 tyrosine kinase in vascular endothelial cells refers to a disease in which Flt4 is identifiable at a much higher level in the vascular system than the undetectable or barely detectable levels normally observed in the blood vessels of healthy tissue, as exemplified herein.

A therapeutically effective amount of a compound may be determined empirically using art recognized dose escalation and dose response assays. A therapeutically effective amount for tumor treatment refers to an amount effective to reduce tumor growth, or to stop tumor growth, or to shrink or eliminate tumors altogether without unacceptable levels of side effects for the patient being treated for cancer. If the compound comprises an antibody or other polypeptide, it is particularly comprised in a dose scale of 0.1 to 100mg of antibody per kilogram of body weight, and more preferably 1 to 10 mg/kg. The humanized antibody which generally exhibits a long circulating half-life includes, in particular, administration at intervals ranging from every day to every 1 month, more preferably at weekly intervals, or every 1 week, or every 3 weeks. Monitoring the progress of the treatment, the patient's side effects, and circulating antibody levels will provide further guidance for an optimal dosing regimen. Data from published and ongoing clinical trials for other antibody-based cancer treatments (e.g., anti-HER 2, anti-EGF receptor) also provide useful dosing regimen guidance.

For the methods of treatment described herein, preferred compounds include polypeptides comprising an antigen binding fragment of an anti-Flt 4 antibody, polypeptides comprising a soluble Flt4 extracellular domain fragment. Human and humanized anti-Flt 4 antibodies are highly preferred. A highly preferred Flt4 extracellular domain fragment contains a ligand binding portion of the Flt4 extracellular domain. For example, soluble fragments containing the first 3 immunoglobulin-like domains of the Flt4 extracellular domain are highly preferred. Smaller fragments that bind Flt4 ligand alone, or when fused to other peptides (e.g., immunoglobulin-like domains of VEGFR-1 or VEGFR-2) are also contemplated. Likewise, improvements that increase solubility and/or stability, serum half-life, or other characteristics to increase therapeutic efficacy are contemplated. For example, polypeptides comprising a fusion between the Flt4 extracellular domain and an immunoglobulin Fc peptide (particularly the IgG1 Fc isotype) to increase solubility and serum half-life are contemplated. [ Compare Achen et al, Proc.Natl.Acad.Sci.USA, 95: 548-553(1998)].

An expected advantage of the treatment methods of the present invention lies in the fact that Flt4 is not generally expressed at any significant level in the vascular system of healthy tissue. In a highly preferred embodiment, the therapeutic compound comprises a bispecific antibody, or fragment thereof, wherein the antibody or fragment specifically binds Flt4 and specifically binds a vascular endothelial marker antigen. By "vascular endothelial marker antigen" is meant any cell surface antigen expressed on proliferating vascular endothelial cells, and preferably not on lymphatic endothelial cells. Exemplary vascular endothelial markers include PAL-E [ deWaal et al, am.j.pathol., 150: 1951-1957(1994) ], VEGFR-1 and VEGFR-2[ Ferrara et al, Endocrine Reviews, 18: 4-25(1997), Tie [ Partanen et al, mol.cell.biol., 12: 1698-1707(1992) ], endoglin [ U.S. Pat. No.5,776,427, incorporated herein by reference in its entirety ], and von Willebrandt factor. Bispecific antibodies are expected to preferentially localize to tumor-associated vasculature expressing Flt4 and vascular endothelial markers. In a highly preferred embodiment, the compound further comprises an anti-neoplastic or cytotoxic agent coupled to the bispecific antibody for killing tumor cells and/or killing the vasculature supplying tumor cells. Exemplary agents include those described above, as well as therapeutic proteins that stimulate an immune response in a host against the tumor, such as statins, cytokines, chemokines, and the like.

In alternative embodiments, the compound comprises an antibody (or bispecific antibody) that recognizes one (or more) epitopes comprised of the Flt4/Flt4 ligand complex (e.g., a complex comprised of Flt4 bound to VEGF-C or VEGF-D).

Therapeutic compounds coupled to or co-administered with broad spectrum agents having the potential to inhibit angiogenic factors are also contemplated. Such agents include, for example, heparin binding drugs that bind heparin-inhibiting angiogenic factors (e.g., pentosan and suramin analogs); chemical agents that inhibit endothelial cell growth and migration, such as fumagillin analogs. Other agents currently under investigation include Marimastat (british biotech, Annapolis MD; indicative of non-small cell lung cancer, small cell lung cancer and breast cancer); AG3340(Agouron, LaJolla, Calif.; for glioblastoma multiforme); COL-3(Collagenex, Newtown PA; for brain tumors); neovastat (Aetema, Quebec, Canada; for renal and non-small cell lung cancers); BMS-275291(Bristol-Myers Squibb, Wallingford CT; for metastatic non-small cell lung cancer); thalidomide (Celgen; for melanoma, head and neck cancer, ovarian cancer, metastatic prostate cancer, and Kaposi's sarcoma; recurrent or metastatic colorectal cancer (with adjuvant); gynecological sarcoma, liver cancer, multiple myeloma; CLL, recurrent or progressive brain cancer, multiple myeloma, non-small cell lung cancer, non-metastatic prostate cancer, refractory multiple myeloma, and renal cancer); squalamine (Magainin Pharmaceuticals plus meouth Meeting, PA; non-small cell and ovarian cancers); endostatin (EntreMed, Rockville, Md.; for solid tumors); SU5416(Sugen, San Francisco, CA; recurrent head and neck cancer, advanced solid tumors, stage IIIB or IV breast cancer; recurrent or progressive brain cancer (pediatric); ovarian cancer, AML; glioma, advanced malignancy, advanced colorectal cancer, von-Hippel Lindau disease, advanced soft tissue cancer, prostate cancer, colorectal cancer, metastatic melanoma, multiple myeloma, malignant mesothelioma; metastatic renal cancer, advanced or recurrent head and neck cancer, metastatic colorectal cancer); SU6668(Sugen San Francisco, CA; advanced tumor); interferon- α; anti-VEGF antibodies (national cancer institute, Bethesda MD; Genentech san Fransciosco, Calif.; refractory solid tumors; metastatic renal cell carcinoma, untreated advanced colorectal cancer); EMD121974(Merck KCgaA, Darmstadt, Germany; HIV-associated Kaposi sarcoma, progressive or recurrent degenerative glioma); interleukin 12(Genetics Institute, Cambridge, MA; Kaposi's sarcoma) and IM862(Cytran, Kirkland, WA; ovarian cancer, untreated metastatic cancers of colon and rectal origin and Kaposi's sarcoma).

It is also contemplated that the coupling of an anti-Flt 4 compound to a prodrug may be through targeting of the anti-Flt 4 compound to tumor blood vessels and subsequent directed activation (e.g., by irradiation) at the site of tumor growth. The strategy of using this prodrug has the expected advantage of minimizing the side effects of the drug on healthy lymphatic vessels expressing Flt 4.

Likewise, the present invention includes a method of treating a mammalian organism having a neoplastic disease characterized by expression of Flt4 tyrosine kinase (Flt4) in vascular endothelial cells, comprising the steps of: identifying a mammalian organism having a neoplastic disease state characterized by expression of Flt4 in vascular endothelial cells, and administering to the mammalian organism in need of such treatment a composition comprising a compound effective to inhibit binding of a Flt4 ligand protein to Flt4 expressed in vascular endothelial cells of the organism, thereby inhibiting Flt 4-mediated proliferation of vascular endothelial cells.

The present invention also provides a method of screening a biological sample for the presence of Flt4 receptor tyrosine kinase protein (Flt4), comprising the steps of: (a) contacting a biological sample suspected of containing Flt4 with a composition comprising a Flt4 binding compound under conditions wherein the compound binds to Flt4 in the biological sample; (b) washing the biological sample under conditions which remove Flt4 binding compound from the sample which is not bound to Flt 4; and (c) screening the sample for the presence of Flt4 by detecting Flt4 binding compounds that bind to Flt4 receptor tyrosine kinase in the sample after the washing step. Preferably, the compound comprises a polypeptide selected from the group consisting of: (a) a polypeptide comprising an antigen binding fragment of an anti-Flt 4 antibody; and (b) a polypeptide comprising Flt4 ligand or a Flt4 binding fragment or analog thereof. Antibodies that specifically bind Flt4 and also contain a detectable label are highly preferred.

The present invention also relates to a method of imaging vertebrate tissue suspected of containing cells expressing Flt4 receptor tyrosine kinase protein (Flt4), comprising the steps of: (a) contacting vertebrate tissue with a composition comprising a Flt4 binding compound; and (b) imaging the tissue by detecting Flt4 binding compound bound to the tissue. Preferably, the tissue is human tissue, and the method further comprises the step of washing the tissue after the contacting step and before the imaging step under conditions which remove from the tissue the Flt4 compound which does not bind to Flt4 in the tissue.

In a related variation, the invention provides a method of imaging a tumor in a tissue of a vertebrate organism, comprising the steps of: (a) contacting vertebrate tissue suspected of containing a tumor with a composition comprising a Flt4 binding compound; (b) detecting the Flt4 binding compound bound to cells in the tissue; and (c) imaging the solid tumor by identifying vascular endothelial cells to which the Flt4 binding compound binds, wherein blood vessels expressing Flt4 are associated with the presence and location of the tumor in the tissue. In a preferred embodiment, the method further comprises contacting the tissue with a second compound (e.g., an antibody) that specifically binds to a vascular endothelial marker (e.g., PAL-E, VEGFR-1, VEGFR-2) that is substantially absent from lymphatic endothelium; and a step of detecting a second compound bound to cells in the tissue; wherein the imaging step comprises identifying blood vessels labeled with the Flt4 binding compound and the second compound, and wherein the blood vessels labeled with the Flt4 binding compound and the second compound are correlated with the presence and location of a tumor in the tissue. It is contemplated that the use of the second compound may help physicians more rapidly distinguish between blood vessels expressing Flt4 and normal lymphatic vessels expressing Flt4 on their surface.

The present invention also relates to a method of screening for a neoplastic disease state comprising the steps of: (a) contacting a tissue of a mammalian organism suspected of having a neoplastic disease state with a composition comprising an antibody or antibody fragment that specifically binds to Flt4 receptor tyrosine kinase; (b) detecting the antibody or antibody fragment bound to cells in the mammalian organism; and (c) screening for a neoplastic disease from the amount or distribution of antibodies that bind to cells of the mammalian organism. As described herein, Flt4 (which is generally undetectable or barely detectable in the vascular system) is strongly stained in the vascular system of at least some tumors. Thus, in one embodiment, the detection of the antibody or antibody fragment bound to vascular endothelial cells in the screening step is correlated with the presence of a neoplastic disease. In this method, it is understood that "detecting" refers to detecting at a level that is significantly higher than the barely detectable or undetectable level present in the corresponding normal (healthy) tissue, as described herein. This differential expression can be confirmed by comparison with a control with tissue from a healthy organism. Particularly comprising screening mammalian tissue for tumors. As described above, the method can be further facilitated by administering to said mammal a second compound that specifically binds to a vascular endothelial marker, wherein the step of detecting comprises detecting binding of said first and second compounds to neovascular endothelial cells.

It is also contemplated from the foregoing that the various compounds described for use in the methods of the invention are also contemplated as aspects of the invention. Such compounds include, for example, anti-Flt 4 antibodies and bispecific antibodies as described above. Likewise, the use of any of the compounds described herein (alone or in combination) in the manufacture of a medicament for therapeutic or diagnostic or imaging purposes as described herein is also intended as an aspect of the invention. The medicament may further comprise pharmaceutically acceptable diluents, adjuvants, carriers and the like.

Likewise, the invention includes kits containing the compounds or compositions of the invention packaged in a manner that facilitates their use in practicing the methods of the invention. In its simplest embodiment, the kit comprises a compound or composition of the invention packaged in a container, such as a sealed bottle or tube, with a label affixed to the container or contained in the package describing the use of the compound or composition in practicing the methods of the invention. Preferably, the compound or composition is packaged in unit dosage form. In another embodiment, the kits of the invention comprise the Flt4 binding compound packaged with a second compound that binds to a marker (antigen) that is expressed on the surface of vascular endothelial cells but is substantially absent from lymphatic endothelium.

In addition, many aspects of the invention are described in the context of peptides or polypeptides for imaging or therapy, and/or the use of antibodies to use Flt4 protein expression on cell surfaces as targets for detection, screening, imaging, and the like. Therapeutic delivery of protein therapeutics, such as polypeptides containing ligand-binding soluble Flt4 fragments, can also be accomplished using materials and methods for gene therapy. For example, a naked DNA construct or gene therapy expression vector construct containing a polynucleotide encoding a therapeutic peptide of interest is delivered to a mammalian subject in need of such treatment. Preferably, the construct contains a promoter or other expression control sequence operably linked to the sequence encoding the therapeutic peptide to drive expression of the therapeutic peptide in vivo. In one variation, the nucleic acid is encapsulated in a liposome. In another variation, the nucleic acid is a viral vector such as a retrovirus, adenovirus, adeno-associated virus, vaccinia virus, herpes virus, or other vector developed for use in gene therapy protocols in mammals. Exemplary methods of treatment include the step of administering to a patient a pharmaceutical composition containing a gene therapy construct, or the steps of transforming or transfecting cells ex vivo and introducing the transformed cells into the patient. Similarly, detection of Flt4 expression in cells or tissues may use polynucleotide probes that specifically hybridize to Flt4mRNA sequences in Northern hybridization or in situ hybridization assays; or by performing quantitative PCR or other techniques for measuring Flt4mRNA in a sample.

Other features and variations of the present invention will be apparent to those skilled in the art from the entirety of this application, including the detailed description, and all such features are intended as aspects of the present invention. Likewise, features of the invention described herein may be re-combined into other embodiments also intended as aspects of the invention, whether or not that combination of features is specifically mentioned above as an aspect or embodiment of the invention. In addition, only the defining features described herein as being critical to the invention may be considered as such; variations of the invention that do not contain the limiting features not described herein as critical are intended as aspects of the invention.

In addition to the foregoing, the present invention includes as a further aspect all embodiments of the invention narrower in any way than the variations specifically mentioned above. Although the full scope of the appended claims has been invented by the applicant, the appended claims are not intended to be construed as encompassing other persons' prior art efforts. Thus, where a patent office or other entity or individual reminds the applicant of legal prior art included within the scope of a claim, the applicant reserves the right to exercise amendment rights under applicable patent laws to redefine the subject entity of the claim to expressly exclude such legal prior art or obvious variations of legal prior art from the scope of the claim. Variations of the invention as defined in the appended claims are also intended as aspects of the invention.

Brief description of the drawings

FIG. 1A is a schematic depiction of the structure of the Flt4cDNA clone;

FIG. 1B is a photographic reproduction of a Northern hybridization gel;

FIGS. 2A-F provide a graphical depiction of the structural features of Flt4 and a comparison with the Flt1 tyrosine kinase sequence;

FIG. 3A is a schematic depiction of the 3' end of the cDNA insert for clones J.1.1 and I.1.1;

FIG. 3B is a reproduction of an autoradiogram of hybridization of long and short forms of Flt4 RNA with an antisense RNA probe;

FIG. 3C is a reproduction of an autoradiogram of hybridization of long and short forms of Flt4 RNA with an antisense RNA probe;

FIG. 4 is a photographic copy of a gel showing hybridization analysis of Flt4 sequences in DNA samples from different species;

FIGS. 5A-5H show immunohistochemical identification of VEGFR-3 expressing vessels in intraductal carcinomas. In adjacent sections (FIG. 5A, B), VEGFR-3 and PAL-E decorate similar patterns of "necklace" vessels (arrows) around cancer cell filled ducts. Another set of adjacent sections stained for VEGFR-3 (FIG. 5C), laminin (FIG. 5D), collagen XVIII (FIG. 5E) and SMA (FIG. 5F) was compared. Double staining for PAL-E and VEGFR-3 (FIG. 5G) and comparison with adjacent sections stained for VEGFR-3 only (FIG. 5H). The vessels adjacent to the diseased vessels were double positive (arrows), while the VEGFR-3 positive vessels were present in the stroma between the vessels at a close distance from the diseased vessels (arrows). Note that the basal layer was positive for PAL-E in the double staining procedure. Magnification: fig. 5A, B are 400 times. Fig. 5C, D, E, F are 320 times. In FIG. 5E, F is 480 times.

Detailed Description

The cloning, sequencing and expression of a novel receptor tyrosine kinase designated Flt4 are described below. The Flt4 gene is located on the 5q35 chromosomal region where many growth factors and growth factor receptors are located. The extracellular domain of Flt4 consists of 7 immunoglobulin-like loops including 12 potential glycosylation sites. Based on structural similarity, Flt4 and the previously known Flt1 and KDR/FLK1 receptors constitute a subfamily of type III tyrosine kinases. The Flt4 gene was expressed as 5.8kb and 4.5kb mRNAs that were found to differ in their 3' terminal sequences and to be differentially expressed in HEL and DAMI leukemia cells.

The Wilm's tumor cell line, retinoblastoma cell line, and undifferentiated teratocarcinoma cell line express Flt 4; while differentiated teratocarcinoma cells are negative. Most fetal tissues also expressed Flt4mRNA, with the spleen, brain intermediate zone and lung showing the highest levels. In adult tissues, the highest expression level was found in placenta, lung, kidney, heart and liver in order of decreasing expression. In situ hybridization, Flt4 autoradiographic particles were interspersed with endothelial cells of fetal lungs. Immunohistochemical staining of Flt4 in fetal tissues confirmed staining of endothelial cells. The pattern of Flt4 expression compared to Flt1 and KDR was very different in 18-week-old human fetal tissues. See kaipain et al, j.exp.med., 178: 2077(1993).

Expression vectors containing Flt4cDNA have been generated and expressed in COS and NIH3T3 cells as described in examples 4 and 11.

The Flt4 DNAs and polypeptides of the invention are useful for purifying Flt4 ligands, and for modulating the growth and differentiation of endothelial cells in various organs. They may also prove valuable in the diagnosis/treatment of certain diseases.

In the description that follows, many terms used in recombinant DNA (rDNA) technology are widely used. In order to provide a clear and consistent understanding of the specification and claims, including the scope to be given to such terms, the following definitions are provided.

DNA sequence containing RNA polymerase template. The RNA transcribed from a gene may or may not encode a protein. The RNA that encodes the protein is called messenger RNA (mrna), and in eukaryotes it is transcribed by RNA polymerase II. However, it is also known to construct genes containing RNA polymerase II templates that transcribe RNA sequences, which have sequences complementary to a particular mRNA but are not generally translated. This gene construct is referred to herein as an "antisense RNA gene" and the RNA transcript is referred to as an "antisense RNA". Antisense RNA is generally not translatable due to the presence of a translation stop codon in the antisense RNA sequence.

"complementary DNA" or "cDNA" genes include recombinant genes synthesized by reverse transcription of mRNA without intervening sequences (introns).

Cloning vectorIt is a plasmid or phage DNA or other DNA sequence which is capable of autonomous replication in a host cell, and is characterized by one or a few endonuclease recognition sites at which the DNA sequence can be cut in a determinable fashion without loss of essential biological function of the vector, and into which DNA can be spliced in order to bring about its replication and cloning. The cloning vector may further comprise a marker suitable for identifying cells transformed with the cloning vector. For example, the marker may be tetracycline resistance or ampicillin resistance. The term "vector" is sometimes used to refer to a "cloning vector".

Expression vectorIt is a vector similar to the cloning vector and capable of expressing the gene cloned therein after transformation into a host. The cloned gene is typically placed under the control of (i.e., operably linked to) certain regulatory sequences, such as a promoter sequence. Expression control sequences may vary depending on whether the vector is designed to express an operably linked gene in a prokaryotic or eukaryotic host, and may also contain transcriptional elements, such as enhancer elements, termination sequences, tissue-specific elements, and/or translation initiation and termination sites.

The present invention relates to the expression of recombinant Flt4 protein (both short and long chain forms) and functional derivatives of these proteins.

Functional derivativesA "functional derivative" of Flt4 protein is a protein having biological activity (function or structure) substantially similar to that of non-recombinant Flt4 protein. Functional derivatives of Flt4 protein may or may not contain post-translational modifications such as covalently linked carbohydrates, depending on the necessity of such modifications for the effect of a particular function. The term "functional derivative" is intended to include "fragments", "variants", "analogues", and "chemical derivatives" of a molecule.

As used herein, a molecule is said to be a "chemical derivative" of another molecule when it contains other chemical moieties that are not normally part of the molecule. The half-molecule can improve the solubility, absorption rate, biological half-life, etc. of the molecule. Alternatively, the moiety may reduce the toxicity of the molecule and eliminate or attenuate any undesirable side effects of the molecule, etc. Half molecules capable of mediating this effect are disclosed in Remington's pharmaceutical Sciences (1980). Methods for coupling such moieties to a molecule are well known in the art.

Segment, method and apparatus for producing the sameA "fragment" of a molecule such as Flt4 protein refers to any part of the molecule, e.g. the peptide core region, or a variant of the peptide core region.

Variants"variants" of a molecule such as Flt4 protein refers to molecules that are substantially similar in structure and biological activity to the whole molecule or fragments thereof. Thus, two molecules may be considered variants as the term is used herein, provided that they have similar activity, even if the composition or secondary, tertiary, or quaternary structure of one molecule differs from that found in the other, or even if the sequence of amino acid residues differs.

Analogues"analogs" of Flt4 protein or genetic sequence refer to proteins or genetic sequences that are substantially similar in function to Flt4 protein or genetic sequences herein.

Description of the preferred embodiments

The present invention relates to what applicants call "Flt 4", receptors for tyrosine kinases, Flt4 encoding nucleic acid molecules (e.g., cDNAs, genomic DNAs, RNAs, antisense RNAs, etc.), the production of Flt4 peptides or Flt4 proteins from Flt4 gene sequences and products thereof, recombinant Flt4 expression vectors, Flt4 analogs and derivatives, and diagnostic and/or therapeutic uses of Flt4 and related proteins, Flt4 ligands, Flt4 antagonists and anti-Flt 4 antibodies.

Production of recombinant Flt4

Biologically active Flt4 may be produced by cloning and expressing the Flt4 encoding sequence or a functional equivalent thereof in a suitable host cell.

The production of Flt4 using recombinant DNA techniques is described as a step-by-step process: (1) isolating or producing the coding sequence (gene) of the desired Flt 4; (2) constructing an expression vector capable of directing the synthesis of the desired Flt 4; (3) transfection or transformation of a suitable host cell capable of replicating and expressing the Flt4 gene and/or processing the gene product to produce the desired Flt 4; and (4) identification and purification of the desired Flt4 product.

Isolation or production of the Flt4 Gene

The nucleotide coding sequence for Flt4 or a functional equivalent thereof may be used to construct recombinant expression vectors capable of directing the expression of the desired Flt4 product. In the practice of this method of the invention, the nucleotide sequences described herein, or fragments or functional equivalents thereof, may be used to produce recombinant molecules capable of directing the expression of recombinant Flt4 product in a suitable host cell. The Flt 4-encoding nucleotide sequences may be obtained from a variety of cell sources that produce Flt 4-like activity and/or express Flt 4-encoding mRNA. Applicants have identified a number of suitable human cell sources for Flt4, including human placenta, leukemia cells, and certain tumor cell lines.

The Flt4 encoding sequence may be obtained from RNA isolated and purified from this cellular source by cDNA cloning or by genomic cloning. For example, the Flt4 sequence may be amplified by polymerase chain reaction from cDNA or genomic DNA material using techniques well known in the art. Cloned cDNA or genomic libraries may be prepared using techniques well known in the art and may be screened for particular Flt4 DNAs using nucleotide probes that are substantially complementary to any portion of the Flt4 gene. Full-length clones, i.e., those containing the entire coding region of the desired Flt4, may be selected for use in constructing expression vectors. Alternatively, Flt 4-encoding DNAs may be synthesized in whole or in part by chemical synthesis using techniques standard in the art. Due to the genetic degeneracy of nucleotide coding sequences, other DNA sequences encoding substantially the same or functionally equivalent amino acid sequences may be used in the practice of the methods of the invention. Such alterations of the Flt4 nucleotide sequence include deletions, additions or substitutions of different nucleotides, resulting in the sequence encoding the same or a functionally equivalent gene product. The gene product may contain deletions, additions or substitutions of amino acid residues within the sequence that result in silent changes, thereby producing a biologically active product. Such amino acid substitutions may be made on the basis of similarity in polarity, charge, solubility, hydrophobicity, hydrophilicity and/or the amphipathic nature of the residues involved. For example, negatively charged amino acids include aspartic acid and glutamic acid; positively charged amino acids include lysine and arginine; amino acids having uncharged polar or non-polar head groups with similar hydrophilicity values include the following amino acids: leucine, isoleucine, valine; glycine, alanine; asparagine, glutamine; serine, threonine; phenylalanine, tyrosine.

Construction of Flt4 expression vectors

Using this information, various recombinant DNA vectors are provided which are capable of providing Flt4 receptor tyrosine kinase in reasonable quantities. Additional recombinant DNA vectors encoding related structures for synthetic proteins having the key structural features identified herein, as well as proteins of the same family from other sources, may be generated from the Flt4 receptor tyrosine kinase cDNA using standard techniques of recombinant DNA technology. Transformants expressing Flt4 receptor tyrosine kinase were generated as an example of this technique (see examples 3 and 4). The newly discovered sequence and structural information may be used to prepare the Flt4 receptor tyrosine kinase and its various domains for biological purposes by transfecting eukaryotic cells.

Identification of transfectants or transformants expressing the Flt4 Gene product

Host cells containing recombinant coding sequences and expressing mature products with biological activity can be identified by at least 4 common methods: (a) DNA-DNA, DNA-RNA or RNA-antisense RNA hybridization; (b) with or without "marker" gene function; (c) assessing the level of transcription by measuring the expression of Flt4mRNA transcripts in the host cell; and (d) detection of the mature gene product by immunoassay and ultimately by measurement of its biological activity.

In the first method, the presence of the Flt4 coding sequence inserted into an expression vector can be detected by DNA-DNA hybridization using a probe containing a nucleotide sequence homologous to the Flt4 coding sequence.

In the second approach, recombinant expression vector/host systems can be identified and selected based on the presence or absence of certain "marker" gene functions (e.g., thymidine kinase activity, antibiotic resistance, methotrexate resistance, transformation phenotype, inclusion body formation in baculoviruses, etc.). For example, if the Flt4 coding sequence is inserted into a marker gene sequence of a vector, recombinants containing the coding sequence can be identified by the lack of marker gene function. Alternatively, a marker gene may be placed in tandem with the Flt4 sequence under the control of the same or a different promoter used to control expression of the Flt4 coding sequence. Expression of the marker in response to induction or selection indicates expression of the Flt4 coding sequence.

In a third approach, the transcriptional activity of the Flt4 coding region may be assessed by hybridization assays. For example, polyadenylated RNA can be isolated and analyzed by Northern blotting using a probe homologous to the Flt4 coding sequence or a specific portion thereof. Alternatively, the total nucleic acid of the host cell can be extracted and hybridization to the probe determined.

In a fourth method, the expression of Flt4 may be assessed immunologically, for example, by Western blotting, immunoassays such as radioimmunoprecipitation, enzyme-linked immunoassays, and the like. However, the final test for the success of the expression system involved the detection of the biologically active Flt4 gene product. If the host cell secretes the gene product, the cell-free medium obtained from culturing the transfected host cell may be assayed for Flt4 activity. If the gene product is not secreted, the activity of the cell lysate can be determined. In each case, assays measuring ligand binding to Flt4 or other biological activity of Flt4 may be used.

Flt4 derivatives, analogs and peptides

The production and use of derivatives, analogs and peptides related to Flt4 are also contemplated and are within the scope of the invention. Depending on the particular application, the biological activity of the derivative, analog, or peptide may be enhanced or decreased as compared to native Flt 4. The Flt 4-related derivatives, analogs and peptides of the invention can be produced by various methods known in the art. Methods and procedures at the genetic and protein levels are also within the scope of the invention. Flt4 peptide may be obtained using standard peptide synthesis methods in the art. At the protein level, Flt 4-like derivatives, analogs, or peptides can be generated using a number of chemical modifications by techniques known in the art, including, but not limited to, specific chemical cleavage, acetylation, formylation, oxidation, etc., of endopeptidases (e.g., cyanogen bromide, trypsin, chymotrypsin, V8 protease, etc.) or exopeptidases.

Preferred derivatives, analogs, and peptides are those that retain Flt4 ligand binding activity. Those derivatives, analogs and peptides that bind Flt4 ligand but do not transduce a signal in response thereto are useful as Flt4 inhibitors. Those derivatives, analogs and peptides that bind Flt4 ligand and transduce a signal in response thereto, for example, by a process involving intracellular Flt4 autophosphorylation, can be used in the same manner as native Flt 4. A preferred Flt4 ligand for use in this binding and/or autophosphorylation assay is one that contains an approximately 23kd polypeptide that can be isolated from PC-3 conditioned media as described herein. This ligand, designated vascular endothelial growth factor-C (VEGF-C), has been identified in detail in PCT patent application PCT/FI96/00427, published as 1996, 8/1 and published as International publication WO97/05250, and in the U.S. patent application priority documents on which priority is claimed, all of which are incorporated herein by reference in their entirety.

anti-Flt 4 antibodies

The production of polyclonal and monoclonal antibodies that recognize Flt4 or related proteins is also within the scope of the invention.

Polyclonal antibodies against the Flt4 epitope may be produced using various methods known in the art. For the production of antibodies, various host animals (including but not limited to rabbits, mice, rats, etc.) may be immunized by injection with Flt4 or synthetic Flt4 peptides. Various adjuvants may be used to enhance the immune response, depending on the host species, including but not limited to Freund's (complete and incomplete) adjuvants, mineral gels such as aluminum hydroxide, surface active substances such as lysolecithin, polyols, polyanions, oil emulsions, keyhole limpet hemocyanin, dinitrophenol, and potentially useful human adjuvants such as BCG (Bacillus Calmette-Guerin) and Corynebacterium parvum.

Monoclonal antibodies against the Flt4 epitope may be prepared by using any of the techniques provided for the production of antibody molecules in culture by continuous cell lines. These techniques include, but are not limited to, those originally manufactured by Kohler et al, Nature, 256: 495-497(1975), and more recently the human B-cell hybridoma technology [ Kosbor et al, Immunology Today, 4: 72(1983) and EBV-hybridoma technology [ Cole et al, Monoclonal Antibodies and Cancer Therapy, Alan R Liss, Inc., pp 77-96 (1985) ]. Antibodies against Flt4 may also be produced in bacteria from cloned immunoglobulin cDNAs. Using recombinant phage antibody systems, antibodies can be rapidly produced and selected in bacterial culture and their structures can be genetically engineered.

Antibody fragments containing the idiotype of the molecule can be generated by known techniques. For example, the segments include, but are not limited to: egg with stomach-passing functionDigestion of antibody molecules with Baise to yield F (ab')₂Fragmenting; by reduction of F (ab')₂Disulfide bonds of the fragments, and two Fab fragments generated by treating the antibody molecule with papain and a reducing agent.

Antibodies against Flt4 are useful for the qualitative and quantitative detection of mature Flt4 and Flt4 precursor and subcomponent forms in the affinity purification of Flt4 polypeptides and in the interpretation of Flt4 biosynthesis, metabolism and function. Detection of Flt4 tyrosine kinase activity may be used as an enzymatic means of generating and amplifying Flt4 specific signals in this assay. anti-Flt 4 antibodies are also useful as diagnostic and therapeutic agents.

Use of Flt4, nucleic acid molecules encoding Flt4 and antibodies against-Flt 4

Applicants contemplate that compositions of the present invention have a wide variety of uses, including diagnostic and/or therapeutic uses for Flt4, Flt4 analogs and derivatives, Flt 4-encoding nucleic acid molecules, antisense nucleic acid molecules, and anti-Flt 4 antibodies.

Flt 4-encoding nucleic acid molecules or fragments thereof may be used as probes for the detection and quantification of Flt 4-encoding mRNAs. Assays that utilize nucleic acid probes to detect sequences containing all or part of a known gene sequence are well known in the art. Flt4mRNA levels may indicate the presence and/or presence of neoplasia and the onset and/or progression of other human diseases. Thus, assays capable of detecting and quantifying Flt4mRNA provide valuable diagnostic tools.

Antisense Flt4 RNA molecules are therapeutically useful for inhibiting the translation of Flt 4-encoding mRNAs, where the therapeutic objective involves the need to remove or down-regulate the levels of Flt4 present. For example, Flt4 antisense RNA may be useful as Flt4 antagonists in the treatment of diseases in which Flt4 is implicated in etiology, e.g., due to its overexpression.

In addition, Flt4 antisense RNAs are useful in explaining the functional mechanisms of Flt 4. Flt 4-encoding nucleic acid molecules may be used to produce recombinant Flt4 protein and related molecules as described separately in this application.

anti-Flt 4 antibodies are useful for diagnosis and quantification of Flt4 in a variety of contexts. For example, antibodies against various domains of Flt4 may be used as the basis for Flt4 immunoassays or Flt4 immunohistochemical evaluations. The tyrosine kinase activity of Flt4 may be used in these assays as an enzyme amplification reaction to generate the Flt4 signal. anti-Flt 4 antibodies may also be used to study the amount of Flt4 on the cell surface.

Antibodies may be produced that act as agonists or antagonists of Flt4 ligand, thereby making it possible to modulate the activity of Flt 4. Alternatively, random peptides may be produced synthetically or by recombinant means from random oligonucleotides and peptides exhibiting specific binding to the Flt4 receptor selected by means of the Flt4 extracellular domain. The Flt4 extracellular domain may also be used to select the peptide fragments from phage display libraries using methods standard in the art. The peptide may have antagonistic or antagonistic activity. Flt4 antibodies may also be conjugated to various compounds to provide valuable diagnostic tools for in vivo imaging of Flt4 expressing cells and tissues or tumors.

Monoclonal antibodies against Flt4 may be covalently or non-covalently coupled to suitable supermagnetic, paramagnetic, electron-dense, sonogenic or radioactive agents to produce targeted contrast agents. Antibody fragments produced by proteolytic or chemical treatment or molecules produced by using epitope binding domains of monoclonal antibodies can be used in place of intact antibodies. The contrast agent can then be used for diagnostic purposes as a contrast agent for human X-ray, magnetic resonance, sonogram or scintigraphy.

Molecular biology of Flt4

The complete sequence of the Flt4cDNA clone is shown in SEQ ID NOs: 1 and 3, stretch open reading frames of 4195 or 4795 nucleotides in length and containing 1298 or 1363 amino acids, depending on alternative splicing. Nucleotides and the putative Flt4 amino acid sequence (short form) are shown in SEQ ID NOs: 1 and 2. FIG. 2 shows a comparison of the Flt4 amino acid sequence with the Flt1 tyrosine kinase amino acid sequence. See Shibuya et al, Oncogene, 5: 519-524(1990).

The putative signal peptide sequence, which is mostly a hydrophobic amino acid, follows the initiator methionine. The sequence surrounding the corresponding ATG is identical to the consensus translational start sequence [ Kozak, nucleic acids res, 15: 8125-8135(1987)]. The putative extracellular portions of both Flt4 polypeptides were 775 amino acids long and contained 12 potential asparagine-linked glycosylation (NXS/T) sites. It also contains some amino acid residues that exhibit the spacing pattern described for proteins that are members of the immunoglobulin superfamily [ Williams et al, annu. rev. immunol., 6: 381-405(1988)]. It has 12 cysteine residues and can organize into 7 immunoglobulin-like domains. As shown in fig. 2, 7 immunoglobulin-like domains were identified approximately as follows: Ig-I (SEQ ID NO: 1, position 158-364; SEQ ID NO: 2, amino acids 47-115); Ig-II (SEQ ID NO: 1, position 479-; Ig-III (SEQ ID NO: 1, position 761-961; SEQ ID NO: 2, amino acid 248-314); Ig-IV (SEQ ID NO: 1, position 1070-1228; SEQ ID NO: 2, amino acids 351-403); Ig-V (SEQ ID NO: 1, position 1340-1633; SEQ ID NO: 2, amino acids 441-538); Ig-VI (SEQ ID NO: 1, position 1739 and 1990; SEQ ID NO: 2, amino acids 574 and 657); and Ig-VII (SEQ ID NO: 1, position 2102-2275; SEQ ID NO: 2, amino acids 695-752). The putative Ig-like domain IV lacks cysteine residues. FIG. 2 also shows the extracellular domain of Flt1 (SEQ. ID No.5), which is the closest human homolog to Flt 4. From this figure, it can be seen that the cysteine residues of the Ig-like regions compare and closely resemble the composition.

The cytoplasmic domain of Flt4 is separated from the extracellular portion by a putative transmembrane region of 23 hydrophobic amino acid residues. The sequence is flanked on the cytoplasmic side by a basic region, which is considered to be the junction between the transmembrane and cytoplasmic domains. The tyrosine kinase homology domain begins at residue 843 and contains an ATP-binding pocket and a putative autophosphorylation site homologous to Y416 of c-src at Y1068 (fig. 2). The tyrosine kinase catalytic domain of Flt4 is divided into two subdomains by a 65 amino acid sequence (amino acid 944-1008) which is largely hydrophobic and shows no homology to Flt 1. Unlike Flt1, Flt4 contained no tyrosine residues in its kinase insert.

A second form of Flt4mRNA has an alternative 3' end encoding the long form of the Flt4 protein.

In FIGS. 3A-C, the generation of short and long forms of Flt4mRNA by alternative splicing is shown. FIG. 3A shows a schematic of the structure of the 3' end of the cDNA insert for clones J.1.1 and I.1.1. The TAG stop codon and the polyadenylation site (poly A) of clone J.1.1 are indicated. Clone I.1.1 differs from clone J.1.1 by the shaded segment (long and short forms of Flt4mRNA, respectively). TAA and polyA represent the stop codon and polyadenylation site of clone I.1.1. In addition, the cleavage sites for restriction endonucleases for EcoRI and Aval are indicated. The 256bp EcoRl-AvaI insert of clone I.1.1 used for the cRNA protection assay is indicated below. The most heavily shaded segment represents the polylinker sequence on the linearized sense RNA template for in vitro transcription of the antisense strand. Also shown is a schematic representation of the structure of the protected fragment after RNAse protection analysis. FIGS. 3B and 3C show 256bp³⁵Autoradiograms of S-labeled antisense RNA probes, and 211 and 124bp digested fragments represent short and long forms of Flt4 RNA protected by polyadenylated RNA from the indicated cell line (Tera-2 is a teratocarcinoma cell line, analyzed here with or without Retinoic Acid (RA) treatment for 10 days). The negative control lane shows the results of protection with transfer RNA. Note the downregulation of Flt4mRNAs during the differentiation of Tera-2 cells. The Tera-2 cells of clone 13 were provided by Dr.C.F.Graham (department of animals, Oxford university, England). Cells from passage 18-40 were used for this experiment. Cells were maintained in Eagle's Minimal Essential Medium (MEM) supplemented with 10% fetal bovine serum and antibiotics. To induce differentiation, cells were plated at 1.5X 10³Individual cell/cm²Coated on gelatin-coated tissue culture grade dishes. The next day 2X 10 medium was added^-6RA of M. Cells were cultured in the presence of RA for 10 days.

The results shown in FIGS. 3A-C show the generation of the carboxy termini of the two Flt4 (short and long chain) forms resulting from alternative splicing.

Flt4 belongs to the class III RTKs based on its putative amino acid sequence. More specifically, Flt4 belongs to the RTKs superfamily, which contains 7 Ig-rings in its extracellular portion and is therefore different from other members of class III RTKs which contain 5 Ig-rings. Flt4 is related to a prototype receptor of the FLT family as a clone of v-ros-associated DNA from a human genomic DNA library, i.e., Flt1[ Shibuya et al, Oncogene, 5: 519-524(1990) and the mouse FLK1 receptor cloned from hematopoietic stem Cell-rich regions of mouse liver [ Matthews et al, Cell, 65: 1143 1152 (1991); matthews et al, proc.natl.acad. sci.usa, 88: 9026-. The extracellular domain of Flt4 shows 33% and 37% amino acid sequence identity with human Flt1 and mouse FLK1, respectively. Flt1 and FLK1 are widely expressed in various normal tissues such as lung, heart and kidney as well as Flt 4. In addition, the recently identified human endothelial cell receptor tyrosine kinase KDR [ Terman et al, Oncogene, 6: 1677. sup. 1683(1991) ] show significant homology with Flt4 and Flt1 family members. From available sequence data, it was calculated that KDR shares 81% identity with Flt4 in the Tyrosine Kinase (TK) domain. In addition, the extracellular domain of KDR also has 7 Ig-ring structures and its TK1 and TK2 domains are 95% and 97% identical to the corresponding domains of the mouse FLK1 receptor. This indicates that KDR is a human homolog of mouse FLK 1.

Although the Flt4 TK domain is approximately 80% identical to the TK domains of Flt1 and FLK1/KDR, it is only approximately 60% identical to the TK domains of the other receptors of the type III RTK. Since these other receptors also have only 5 Ig-like domains in the extracellular region, Flt4, Flt1 and FLK1/KDR can be classified into an independent FLT subfamily within type III RTKs.

The kinase insert in PDGFRs, c-fms and c-kit is located in the sequence D/E-D/E-y-M/V-P/D/E-M [ Cantley, et al, Cell, 64: the tyrosine residue in 281-302(1991) ] (SEQ. ID NO.6) is an autophosphorylation site that, when phosphorylated, binds to the SH2 domain of phosphatidylinositol 3' -kinase (PI-3K) [ Reediijk et al, EMBO J., 11: 1365-1372(1992)]. Interestingly, unlike these type III RTKs, members of the FLT subfamily or the Flt3/FLK2 receptor do not contain this consensus motif.

8 human type III RTK genes are clustered on 3 different chromosomes. Chromosome 4 contains the c-kit, PDGFR-alpha and KDR genes [ Yarden et al, EMBO J., 6: 3341-3351 (1987); stenman et al, Genes, Chromosomes, Cancer, 1: 155-158 (1989); terman et al, Oncogene, 6: 1677-1683(1991)]. The Flt1 and Flt3 genes are located on chromosome 13q12 [ Satoh et al, jpn.j. cancer res., 78: 772-; rosnet et al, Genomics, 9: 380- & gt 385(1991) ], whereas Flt4 is located on the q35 band of chromosome 5 [ Aprelikova et al, Cancer Res., 52: 746-748(1992) ]; close to the fms and PDGFR-beta genes [ Warrington et al, Genomics, 11: 701-708(1991)]. The long arm of chromosome 5 is found to contain a translocation in leukemia cells. Deletion of the long arm portion of chromosome 5 was found in bone marrow cells of patients with refractory anemia and macrocytosis [ VanDen Berghe et al, Nature, 251: 437-439(1974)]. In some other myeloproliferative diseases, such as refractory anemia with excess blasts [ Swolin et al, Blood, 58: 986-: 41-48(1978) ], chronic myeloleukemia [ Tomiyasu et al, Cancer Genet.Cytogene, 2: 309-315(1980) ], polycythemia vera [ Van Den Berghe et al, Cancer gene. cytogene, 1: 157 (1979) and primary thrombocytosis [ Nowell et al, Cancer, 42: 2254-2260(1978) ].

The findings on Flt4mRNA expression indicate that its protein product is characteristic for certain leukemia cells. Several distinct antigens have been shown to be shared between megakaryoblasts and endothelial cells, an example being platelet glycoprotein IIIa [ Ylanne et al, Blood, 72: 1478-1486 (1988); kieffer et al, Blood, 72: 1209-1215 (1988); berridge et al, Blood, 66: 76-85(1985)]. In addition, certain endothelial cells, such as those of the lungs and kidneys during fetal life, express Flt 4.

To further understand the role of Flt4 during development, partial cDNAs for mouse Flt4 were cloned. The expression of Flt4mRNA during mouse development was analyzed using these probes in situ hybridization. It was determined that Flt4 is expressed during angiogenesis and angiogenesis of the lymphatic system. The relevance of these findings was also confirmed in normal and pathological adult tissues, as Flt4 was found in lymphatic endothelial cells of adult tissues, as well as in some post-capillary venules (HEVs), under both normal and pathological conditions.

Cloning of the mouse Flt4cDNA fragment showed that its putative amino acid sequence was nearly identical to the corresponding human sequence (about 96% amino acid identity in the two fragments studied). Further evidence of the identity of the mouse Flt4cDNA was obtained from Northern hybridization experiments in which probes from both species generated a typical 5.8kb mRNA signal from mouse tissue. Analysis of RNA isolated from various tissues of adult mice showed Flt4 expression in liver, lung, heart, spleen and kidney with no or minimal hybridization in brain and testis. This expression pattern is similar to that described by Galland et al, Oncogene, 8: 1233(1993) in an earlier reported manner. The results of RNase protection indicate that the Flt4 gene is required during mouse development from 8.5 day p.c. embryos and that the relative expression levels appear to be quite stable.

For in situ hybridization, two fragments of mouse Flt4cDNA encoding the extracellular domain sequences were selected. This allows a clear distinction between the relevant FLK-1 hybridization pattern and the Flt1 receptor pattern, Flt1 and Flt4 showing only a very low degree of sequence identity in the extracellular region. See Millauer et al, Cell, 72: 835 (1993); yamaguchi et al, Development, 118: 489 (1993); peters et al, proc.natl.acad.sci.usa, 90: 8915 (1993); finnerty et al, Oncogene, 8: 2293(1993).

Similar to the FLK-1, Flt1, Tie and Tek endothelial receptor tyrosine kinase genes, Flt4 is not expressed in 7.5 day p.c. (p.c.). In 8.5 day p.c. embryos, there was a strong Flt4 signal located in the allantois, angioblasts of the head mesenchyme, the dorsal aorta, and the major veins. Faint signals were visible in the endocardium. In contrast, the yolk sac was negative for hemangioblasts, unlike FLK-1 and Flt1, Tie and Tek. See Korhonen et al, Oncogene, 8: 395 (1993); and Peters et al, proc.natl.acad.sci.usa, 90: 8915(1993). The restriction of Flt4 expression was even clearer in mouse embryo samples from day 11.5 mice, where the venous system was restricted, while Tie mRNA was also expressed in arteries. In 12.5 day p.c. embryos, the Flt4 signal patched developing venous and putative lymphatic endothelia, but arterial endothelia were negative unlike endothelial Tie receptor tyrosine kinase. In the later stages of development, Flt4mRNA became restricted to the vascular plexus devoid of blood cells, which represents the developing lymphatic vessels. Only lymphatic endothelium and some postcapillary venules expressed Flt4mRNA in adult tissues. Increased expression occurs in lymphatic sinuses and in the posterior capillary venules, metastatic lymph nodes, and in lymphangiomas.

Because of the difficulty in interpreting data from mouse embryos, human endothelium was studied because the human lymphatic system is more defined. In addition, cells established from various endothelia may be studied in cell culture to see if the specificity of Flt4 expression is maintained under in vitro conditions. Endothelial cell lines are known to lose differentiation characteristics when cultured in vitro. Therefore, they cannot be unexpectedly expected to be negative for Flt4 mRNA. Cultured aortic endothelial cells were also free of Flt4 mRNA. However, signals are available from the microvasculature and from human endothelial cells grown from the femoral and umbilical veins. Thus, at least some specificity of Flt4 expression was retained in cell culture.

In situ hybridization analysis of adult tissues confirmed the results that Flt4 was localized to the lymphatic system observed in developing mouse embryos. Flt4 expression was seen in lymphatic endothelium and in human lymph node sinuses. Interestingly, some HEVs, which have cubic endothelium and exhibit the function of transporting leukocytes to lymph nodes, are also Flt 4-positive. In addition, parallel hybridization analysis showed that Flt4mRNA levels were enhanced in these structures in metastatic cancer compared to normal lymph nodes. Flt4 was also very significant in lymphangiomas, which are benign tumors consisting of a connective tissue matrix and growing endothelial-lined lymphatic vessels. Flt4mRNA was localized to the lymphatic endothelium of these tumors and was absent from their arteries, veins and capillaries. In the human lung, lymphatic structure was the only Flt 4-positive tube identified.

The above results indicate that Flt4 is a novel marker for lymphatic vessels and some of the posterior capillary venules in adult tissues. This result also supports the theory on the venous origin of lymphatic vessels. Flt4, a receptor for growth factor, may be involved in the differentiation and function of these blood vessels. Detailed identification of Flt4 by a ligand of Flt4, namely the biological effect mediated by VEGF-C, is provided in PCT patent application PCT/FI96/00427, published as International publication No. WO97/05250, 1996, 8/1.

These results, in combination with the Flt 4-binding compounds according to the invention, allow selective labelling of lymphatic endothelia, in particular by using antibodies of the invention coupled to radioactive, electron-dense or other visible reporter substances. A substance containing a Flt4 receptor internalization-inducing monoclonal antibody or ligand may be injected into the lymphatic system, thereby transporting the predetermined molecule to the lymphatic endothelium. Flt 4-binding compounds according to the invention may also be used to detect postcapillary venules, in particular activated HEVs which express enhanced levels of the Flt4 receptor. To our knowledge, no such specific marker is currently available for lymphatic endothelia.

Gene therapy

The invention also relates to gene therapy methods. In particular, the vasculature of a cancer cell or the cancer cell itself can be contacted with an expression construct capable of providing a therapeutic peptide, such as a soluble VEGFR-3 fragment of the present invention, to the vasculature of the cell in a manner that achieves a therapeutic effect. The therapeutic effect may be, for example, inhibition of VEGFR-3 in tumor vasculature, inhibition of angiogenesis, inhibition of lymphangiogenesis, elimination, regression or inhibition of tumor growth, induction of apoptosis of the tumor or even the tumor cell's own vascular or lymphatic vasculature.

For these embodiments, exemplary expression constructs comprise viruses or engineered constructs produced from viral genomes. The expression construct generally comprises a nucleic acid encoding the gene to be expressed and also contains other regulatory regions which cause expression of the gene in the cell to which it is administered. Such regulatory regions include, for example, promoters, enhancers, polyadenylation signals, and the like.

It is now generally recognized that DNA can be introduced into cells using a variety of viral vectors. In this embodiment, the expression construct comprising the viral vector containing the gene of interest can be an adenovirus (see, e.g., U.S. Pat. No.5,824,544; U.S. Pat. No.5,707,618; U.S. Pat. No.5,693,509; U.S. Pat. No.5,670,488; U.S. Pat. No.5,585,362; each incorporated by reference herein), a retrovirus (see, e.g., U.S. Pat. No.5,888,502; U.S. Pat. No.5,830,725; U.S. Pat. No.5,770,414; U.S. Pat. No.5,686,278; U.S. Pat. No. 4,861,719; each incorporated by reference herein), an adeno-associated virus (see, e.g., U.S. Pat. No.5,474,935; U.S. Pat. No.5,139,941; U.S. Pat. No.5,856; U.S. 5,658,776; U.S. Pat. No.5,773,289; U.S. 5,789,390; U.S. Pat. No.5,834,441; U., U.S. patent No.5,856,152, incorporated herein by reference) or vaccinia virus or herpes virus (see, e.g., U.S. patent No.5,879,934; U.S. patent nos. 5,849,571; U.S. patent nos. 5,830,727; U.S. patent nos. 5,661,033; U.S. patent No.5,328,688, each incorporated herein by reference).

In other embodiments, non-viral delivery is included. They include calcium phosphate precipitation (Graham and Van Der Eb, Virology, 52: 456-467, 1973; Chen and Okayama, mol.Celbiol., 7: 2745-2752, 1987; Rippe et al, mol.Celbiol., 10: 689-695, 1990), DEAE-dextran (Gopal, mol.Cell biol., 5: 1188-1190, 1985), electroporation (Tur-Kaspa et al, mol.Cell biol., 6: 716-718, 1986; Potter et al, Proc.Nat.Acad.Sci.USA, 81: 7161-7165, 1984), direct microinjection (Hareand and Weintraub, J.biol., Biol., 109101, 1985), liposomes (Liposome, USA, Vol.92, Liposome, Vol, Vol.185, USA, Vol, Vol.32, Vol.Vol.Vol.Vol, gene bombardment with high-velocity microparticles (Yang et al, Proc. Natl. Acad. SciUSA, 87: 9568-9572, 1990), and receptor-mediated transfection (Wu and Wu, J. biol. chem., 262: 4429-4432, 1987; Wu and Wu, Biochemistry, 27: 887-892, 1988; Wu and Wu, adv. drug Delivery Rev., 12: 159-167, 1993).

In a particular embodiment of the invention, the expression construct (or even the peptide described above) may be embedded in liposomes. Liposomes are vesicular structures characterized by a phospholipid bilayer membrane and an internal aqueous medium. Multilamellar liposomes have multiple lipid layers separated by an aqueous medium. They form spontaneously when phospholipids are suspended in excess aqueous solution. The lipid components rearrange themselves before forming a closed structure and entrap water and dissolved solutes between lipid bilayers (Ghosh and Bachhawat, see: Liver diseases, targeted diagnosis and therapy using specific receptors and ligands, Wu G, Wu C eds., New York: Marcel Dekker, pp. 87-104, 1991). The addition of DNA to cationic liposomes causes a topological transformation from liposomes to optically birefringent liquid crystal compressed spheres (Radler et al, Science, 275 (5301): 810-4, 1997). These DNA-lipid complexes are potential non-viral vectors for gene therapy and delivery.

Liposome-mediated nucleic acid delivery and in vitro expression of exogenous DNA have been very successful. The present invention also encompasses various commercial methods involving "lipofection" techniques. In certain embodiments of the invention, the liposome can be complexed with Hemagglutinating Virus (HVJ). This has been shown to facilitate fusion with the cell membrane and to facilitate entry of liposome-encapsulated DNA into the cell (Kaneda et al, Science, 243: 375-. In other embodiments, liposomes can be complexed or linked to nuclear non-histone chromosomal proteins (HMG-1) for use (Kato et al, J.biol.chem., 266: 3361-3364, 1991). In another embodiment, the liposomes can be complexed or linked to both HVJ and HMG-1 for use. Given that such expression constructs have been successfully used for the transfer and expression of nucleic acids in vitro and in vivo, they are applicable to the present invention.

Other vector delivery systems that can be used to deliver nucleic acids encoding therapeutic genes into cells include receptor-mediated delivery vectors. They take advantage of the selective uptake of macromolecules in almost all eukaryotic cells by receptor-mediated endocytosis. This delivery can be highly specific due to the cell type specific distribution of the various receptors (Wu and Wu, 1993, supra).

Receptor-mediated gene targeting vectors generally consist of two components: cell receptor specific ligands and DNA binding reagents. Several ligands have been used for receptor-mediated gene transfer. The most widely identified ligands are asialo orosomucoid (ASOR) (Wu and Wu, 1987, supra) and transferrin (Wagner et al, Proc. nat' l. Acad Sci. USA, 87 (9): 3410-. Recently, synthetic nascent glycoproteins that recognize the same receptor as ASOR have been used as gene delivery vectors (Ferkol et al, FASEB J., 7: 1081- & 1091, 1993; Perales et al, Proc. Natl. Acad. Sci., USA 91: 4086- & 4090, 1994) and Epidermal Growth Factor (EGF) has also been used to deliver genes to squamous carcinoma cells (Myers, EPO 3085).

In other embodiments, the delivery vehicle may comprise a ligand and a liposome. For example, Nicolau et al (Methods enzymol., 149: 157-176, 1987) used lactosyl ceramide, asialoglycoside at the end of galactose incorporated into liposomes and observed increased hepatocyte uptake of the insulin gene. Thus, it is possible that nucleic acids encoding therapeutic genes may also be specifically delivered into specific cell types through a variety of receptor-ligand systems with or without liposomes.

In another embodiment of the invention, the expression construct may simply consist of naked recombinant DNA or plasmid. The transfer of the construct may be through the physical or chemical penetration of the cell membrane mentioned aboveEither method is performed. It is particularly suitable for in vitro transfer, however, it may also be suitable for in vivo use. Dubensky et al (Proc. Nat. Acad. Sci. USA, 81: 7529-₄The injection of polyomaviral DNA in pellet form into the liver and spleen of adult and neonatal mice confirmed active viral replication and acute infection. Benveninsty and Neshif (Proc. Nat. Acad. Sci. USA, 83: 9551-₄The precipitated plasmid results in expression of the transfected gene.

Another embodiment of the invention for transferring naked DNA expression constructs into cells may comprise particle bombardment. This method relies on the ability to accelerate DNA-coated microparticles to a high velocity that allows them to penetrate the cell membrane and enter the cell without killing them (Klein et al, Nature, 327: 70-73, 1987). Some devices have been developed for accelerating small particles. One such device relies on a high voltage discharge to generate an electric current which in turn powers (Yang et al, Proc. Natl. Acad. Sci USA, 87: 9568-. The microparticles used consist of biologically inert substances such as tungsten or gold beads.

Those skilled in the art are familiar with how to apply gene delivery to in vivo and ex vivo situations. For viral vectors, typically a viral vector stock is prepared. Depending on the type of virus and the titer achievable, a 1X 10 can be delivered to the patient⁴，1×10⁵，1×10⁶，1×10⁷，1×10⁸，1×10⁹，1×10¹⁰，1×10¹¹Or 1X 10¹²And (c) infectious particles. Similar values can be inferred for liposomes or other non-viral formulations by comparing relative absorption efficiencies. Formulations as pharmaceutically acceptable compositions are described below.

Various delivery pathways are contemplated for various tumor types. The following sections on delivery routes encompass a broad list of possible routes. For almost any tumor, systemic delivery is contemplated. This proves particularly important for attacking microscopic or metastatic cancers. If discrete tumor masses can be identified, various direct, local and regional methods can be employed. For example, tumors can be injected directly with expression vectors or proteins. The tumor basal layer may be treated before, during or after resection. After resection, the vector is typically delivered through a catheter left in place after surgery. The vector can be introduced into the tumor by injection into a supporting vein or artery using the tumor vasculature. More peripheral vascular supply routes may also be utilized.

In a different embodiment, ex vivo gene therapy is contemplated. In an ex vivo embodiment, cells of the patient are removed and maintained in vitro for at least a period of time. During this time, delivering the treatment, followed by reintroducing the cells into the patient; preferably, any tumor cells in the sample have been killed.

The following examples are provided only to illustrate the present invention and do not limit its scope in any way.

Example 1

Isolation and characterization of Flt 4-encoding cDNA clones

Materials and methods

oligo-dT primer was generated at bacteriophage lambda gtl 1[ gifted by Dr. Mortimerponcz, Philippi, Philippines, Pa; poncz et al, Blood, 69: 219- "223 (1987) ] was screened with cDNA fragments PCR-amplified from the same library [ Aprelikova et al, cancer Res., 52: 746-748(1992)]. Positive plaques were identified and purified as described [ Sambrook et al, molecular cloning-A Laboratory Manual, Cold spring harbor Laboratory Press, (1989) ]. The cDNA insert of bacteriophage lambda was isolated as an EcoRI fragment and subcloned into the GEM3Zf (+) plasmid (Promega). The entire Flt4 protein coding region was isolated. 3 overlapping clones isolated from the HEL-library were sequenced using the dideoxy chain termination method with oligonucleotide primers designed based on the sequence obtained (shown in FIG. 1). All portions of the cDNAs were sequenced on both strands. Using the GCG software package program [ Devereux et al, Nucleic Acids res, 12: 387-395(1984) and the Prosite program of Apple MacIntosh ].

FIG. 1A shows a schematic of the structure of the Flt4cDNA clones analyzed. The arrow indicates the subcloned restriction fragment (whose size is in kb) used to probe the Northern blot shown in FIG. 1B. E ═ EcoRI site, S ═ SphI site. FIG. 1B shows Northern hybridization analysis of DAMI and HEL leukemia cell RNAs using the probes shown in FIG. 1A.

Results

A210 bp long Flt4cDNA fragment isolated from a HEL cell cDNA library by PCR cloning method was used as a molecular probe to screen a human erythroleukemia cell cDNA library generated with oligo-dT-primers.

Nucleotide sequence analysis of the clones revealed an open reading frame of 1298 amino acid (aa) residues (SEQ ID NO: 2, FIG. 2). The translation initiator methionine marked in the figure is surrounded by typical consensus sequences [ Kozak, Nucleic Acids res., 12: 857-.

The extracellular domain of Flt4 was arranged into 7 immunoglobulin-like loops (FIG. 2). This figure also shows a comparison of Flt4 with Flt1, which contain very similar structures. The amino acid sequence of Flt1 is shown as seq.id no: and 5, providing.

Amino acid residues 775-798 form a sequence of a hydrophobic chain that is likely to function as the transmembrane domain of the receptor, followed by some basic residues on the putative cytoplasmic side of the polypeptide. The juxtamembrane domain is 44 residues in length, before the beginning of tyrosine kinase sequence homology at amino acid 842. Since homology is interrupted over the 65 amino acid kinase insert sequence, the homology is first lost at amino acid 1175 of the carboxy terminal tail of the receptor. Searches of the relevant tyrosine kinase domains in the amino acid sequence databases (Swissprot and NBRF) identified Flt1 and PDGFRB tyrosine kinases with approximately 80% and 60% homology, respectively, in the catalytic tyrosine kinase region.

Example 2

Preparation of anti-Flt 4 antiserum

A657 base pair EcoRI fragment encoding the putative C-terminus of the Flt4 short chain form was cloned into the reading frame of the pGEX-1. lamda.T bacterial expression vector (Pharmacia) containing the glutathione-S-transferase coding region to produce the GST-Flt4 fusion protein in E.coli. The resulting fusion protein was produced in bacteria and partially purified by glutathione affinity chromatography according to the manufacturer's instructions. Rabbits were immunized with this protein to generate polyclonal antibodies against Flt 4. Antisera after the third booster immunization were used.

Example 3

Expression of Flt4 in COS cells

Materials and methods

The coding sequence of the full-length Flt4 protein (assembled from 3 clones, fig. 1) was inserted into an SVpoly mammalian expression vector [ stabey et al, Nucleic Acids res., 18: 2829(1990) ] construction of SV14-2 at the HindIII-BamHI site. The expression vector (SV-FLT4 short chain and SV-FLT4 long chain, containing the respective forms of Flt4 cDNA) was introduced into COS cells by DEAE-dextran transfection [ McCutchannetal, J.Natl.cancer Inst., 41.351-357(1968) ]. 2 days after transfection, cells were washed with Phosphate Buffered Saline (PBS) and scraped into immunoprecipitation buffer (10mM Tris pH7.5, 50mM NaCl, 0.5% sodium deoxycholate, 0.5% Nonidet P40, 0.1% SDS, 0.1TIU/ml aprotinin). The lysate was sonicated, centrifuged at 10,000Xg for 15 minutes and incubated with 3ml of antiserum on ice overnight. Protein A Sepharose (Pharmacia) was added and incubation continued for 30 minutes under spinning conditions. The pellet was washed 4 times with immunoprecipitation buffer, once with PBS and once with water before SDS-PAGE analysis.

Results

The expected structure of the Flt4cDNA sequence was examined by cloning the coding regions for the full-length Flt4 short and long chain proteins into the HindIII-BamHI sites of the pSVpoly expression vector and transfecting these expression vectors into COS cells. The proteins produced by these two constructs differ in their C-terminal: the long chain form contains an additional 65 amino acids. 2 days after transfection, cells were lysed and immunoprecipitated with antibodies raised against GST-Flt4 fusion proteins containing the 40 carboxy-terminal amino acid residues of the short form of the expected Flt4 protein (i.e., a portion common to both the short and long forms of Flt 4). The immunoprecipitated polypeptides were analyzed by SDS-polyacrylamide gel electrophoresis. Preimmune sera did not show any specific bands, whereas the Flt 4-specific antibody recognized two bands of approximately 170 and 190 kD. These two bands represent the different glycosylated forms of Flt4 protein.

Example 4

Expression of Flt4 in NIH3T3 cells

Materials and methods

The full-length Flt4cDNA (short form) was subcloned into an LTRpoly vector containing the Moloney murine leukemia virus long terminal repeat promoter (see Makela, et al, Gene, 118: 293-294(1992), disclosing plasmid vector pLTRpoly, having ATCC accession number 77109 and GeneBank accession number X60280). The LTR-FLT4 expression vector was used to co-transfect NIH3T3 cells with pSV2neo marker plasmid and G418 resistant clones were analyzed for Flt4 expression.

For Western immunoblot analysis, cells on one confluent large plate were lysed in 2.5% SDS, 125mM Tris, pH 6.5. Cell lysates were electrophoresed on SDS-PAGE and electroblotted onto nitrocellulose membranes. The membranes were incubated with antiserum raised against the Flt4 carboxy-terminal peptide and bound antibodies were visualized using horseradish peroxidase conjugated porcine anti-rabbit antiserum (Dako) and ECL reagent (Amersham). For metabolic labeling, 100. mu. Ci/ml was used³⁵S-methionine labeled cultures for 1 hour. After labeling, cells were washed twice and cultured in their growth medium for 1 or 2 hours, lysed, immunoprecipitated with anti-Flt 4 antibody, and analyzed by SDS-PAGE and autofluorescence.

Results

Polypeptides of 170 and 190KD were detectable in NIH3T3 cells transfected with the short-chain form of Flt4, but not in cells transfected with pSV2neo alone. In addition to these two bands, a major band of approximately 120Kd was observed in the Flt 4-producing clone. Metabolic labeling and pulse-chase experiments indicated that the protein was produced as a result of post-translational processing of the short form of the Flt4 polypeptide.

Example 5

Chromosomal mapping of the Flt4 locus

Since aggregation of some type III receptor genes has been observed, it is of great interest to determine the chromosomal location of Flt 4. Thus, rodent-human hybrids were analyzed, indicating linkage of Flt4 to human chromosome 5.

The Flt4 gene was identified to be located in the 5q33- > 5qter region using hybrid cells carrying part of chromosome 5. These hybrids were tested for the presence of the Flt4 locus by filter hybridization. The region of chromosome 5 that is common to Flt 4-positive hybrids and is not present in Flt 4-negative hybrids is 5q 33.1-qter. Thus, human chromosome 5q33-qter was associated with the presence of the Flt4 sequence in the hybrid cells. This regional mapping result indicates that the Flt4 locus is clustered distally (telomelic) with the CSF 1R/platelet-derived growth factor receptor-beta (PDGFRB) locus and with the beta-adrenergic receptor (ADRBR) locus, as these loci are present in Flt 4-negative hybrid cell GB 13.

Example 6

Expression of Flt4mRNA in tumor cell lines and endothelial cells

The leukemia cell line used in this assay (K562) has been reported in some previous literature; [ Lozzio et al, Blood, 45: 321- & ltSUB & gt 334(1975) ], HL-60[ Collins et al, Nature, 270: 347-349(1977) ], HEL [ Martin et al, Science, 216: 1233-1235(1982) ], DAMI [ Greenberg et al, Blood, 72: 1968-: 891-895(1972) ], Jurkat [ Schwenk et al, Blut, 31: 299-charge 306(1975) ], U937[ Sundstrom et al, int.J. cancer, 17: 565-: 1153-. The following tumor cell lines obtained from the american type culture collection were also analyzed: JEG-3, choriocarcinoma; a204, rhabdomyosarcoma; SK-NEP-1, nephroblastoma; BT-474, breast cancer; y79, retinoblastoma. Leukemia cells were grown in RPMI containing 10% Fetal Calf Serum (FCS) and antibiotics. Dami cells were cultured in Iscove's modified DMEM containing 10% horse serum. A permanent endothelial hybrid cell line (EAhy926) [ Edgell et al, proc.natl.acad.sci.usa, 50: 3734-3737(1983) were cultured in DMEM-HAT medium containing 10% FCS and antibiotics.

According to [ Sambrook et al, supra]Said extracting Poly (A) from the cell line⁺RNA. 5 μ g of Poly (A)⁺RNA samples were electrophoresed in agarose gels containing formaldehyde and blotted using standard conditions [ Sambrook et al, supra]. The insert of the Flt4cDNA clone was labeled by the random primer method and hybridized to the blot. Hybridization was performed in 50% formamide, 5 XDenhardt's solution (100 XDenhardt's solution is Ficoll, polyvinylpyrrolidone and bovine serum albumin 2% each), 5 XSSPE (3M NaCl, 200mM NaH)₂PO₄·H₂O, 20mM EDTA, pH7.0), 0.1% SDS (sodium dodecyl sulfate), and 0.1mg/ml sonicated salmon sperm DNA at 42 ℃ for 18-24 hours. The filters were washed in 1 XSSC (150mM NaCl, 15mM sodium citrate, pH7.0), 0.1% SDS at 65 ℃ and exposed to Kodak XAR-5 film.

Poly (A) extracted from 8 leukemia cell lines (HEL, K562, DAMI, U937, MOLT4, HL60, Jurkat, and KG-1) and endothelial hybrid cell line (EAhy926) was used⁺RNA for NorAnd (4) carrying out thermal analysis. Hybridization with the GAPDH probe was used as an internal control for loading equal amounts of RNA for analysis. Only HEL erythroleukemia cells, and DAMI megakaryoblastic leukemia cells expressed 5.8kb and 4.5kb Flt4 mRNA. K562 erythroleukemia, Jurkat and MOLT-4T-cell leukemia, as well as HL-60 promyelocytic leukemia, U937 monocytic leukemia, and KG-1 myeloid leukemia cells were negative for Flt4 mRNA.

Using poly (A) extracted from 5 tumor cell lines (JEG-3, A-204, SK-NEP-1, BT-474, and Y79) and two of the above leukemia cell lines (JOK-1, MOLT4)⁺Northern analysis was performed on the RNA. The labeled S2.5 cDNA clone (see FIG. 1) was used as a hybridization probe. Hybridization with the β -actin probe served as an internal control for loading equal amounts of RNA for analysis. Only the SK-NEP-1 nefroplastoma and Y79 retinoblastoma cells were observed to contain the Flt4 transcript.

Tera-2 teratocarcinoma cells were analyzed 10 days after treatment with vehicle (-) or retinoic acid (+) to induce neuronal differentiation [ Thompson et al, j.cell sci., 72: 37-64(1984)]. In the case of poly (A) isolated from the cell⁺In Northern blot analysis of RNA, undifferentiated cells were found to express 5.8kb and 4.7kb Flt4mRNAs, but after 10 days of differentiation, Flt4mRNA was not detected in Northern blots and hybridizations. These results indicate that Flt4 was downregulated during the differentiation of these cells.

Flt4mRNA expression was also analyzed in undifferentiated and TPA-differentiated HEL cells. Both HEL and DAMI cell lines have dual erythroblast/megakaryoblast phenotypes and can induce further expression of megakaryoblast markers by treatment with the tumor promoter 12-O-tetradecanoylphosphol-13-acetate (TPA). We analyzed whether Flt4 expression was stimulated in these cells during their differentiation. HEL cells were analyzed 2 days after treatment with TPA or DMSO to dissolve it. After stripping the Flt4 signal, the filters were probed with Rb-1 and β -actin cDNAs to confirm equal loading of the lanes. Based on densitometric scanning analysis of the autoradiogram and normalization to constitutive expression of the GAPDH gene (normalization), it was determined that Flt4mRNA levels increased approximately 3.4-fold in TPA-induced HEL cells when the cells underwent megakaryoblastic differentiation.

Example 7

Flt4 expression in fetal lungs

In situ hybridization: human fetal lung tissue at the age of 15 weeks was obtained under the approval of the joint ethics committee at the university of central hospital finland and at the university of Turku. The samples were fixed in 10% formalin for 18 hours at 4 ℃, dehydrated, embedded in wax, and cut into 6 μm sections. Using SP6 and T7 polymerase and [ 2]³⁵S]UTP produces RNA probes of 206 and 157 bases (antisense and sense) from linearized plasmid DNA. According to Wilkinson et al, Development, 99: 493-500 (1987); wilkinson, Cell, 50: 79-88(1987) in situ hybridization of the sections using the following modifications: 1) xylene was used instead of toluene before paraffin embedding; 2) cut into 6 μm sections and place on a layer of diethyl pyrocarbonate-treated water on the surface of a glass slide pretreated with 2% 3-aminopropyl-triethoxysilane (Sigma); 3) omitting alkaline hydrolysis of the probe; 4) the hybridization mixture contained 60% deionized formamide; 5) highly stringent condition washes are 80 minutes at 65 ℃ in a solution containing 50mM DTT and 1 XSSC; 6) the sections were covered with NTB-2 emulsion (Kodak) and stored at 4 ℃. After 14 days of exposure, the slides were developed in Kodak D-19 developer for 2.5 minutes and fixed with Unifix for 5 minutes. Sections were stained with hematoxylin dissolved in water.

In hybridization experiments using antisense probes, Flt4mRNA was observed predominantly in certain endothelial cells of the lungs of fetuses aged 15 weeks. Control hybridizations with the sense strand probe and sections treated with RNAse A-did not yield a signal above background.

For immunoperoxidase staining, 1: 100 dilution of anti-Flt 4 antibody, peroxidase-conjugated porcine anti-rabbit antibody and standard methods in the art were used. Control staining with preimmune serum or immunogen blocked serum produced no signal. Lung tissue from a human fetus at age 17 weeks was analyzed and the results were consistent with those of mRNA in situ hybridization experiments: the endothelium of some pulmonary large vessels stained positive with rabbit anti-Flt 4 antiserum.

Example 8

Identification of the Flt4 Gene in a non-human mammalian species

In FIG. 4, the results of an experiment to examine the presence of Flt4 sequences in DNA of different species are shown. To reveal the degree of conservation of the Flt4 gene during evolution, 2.5kb cDNA fragments (see FIG. 1) were hybridized with genomic DNAs purified from different animals and yeast and digested with EcoRI. The hybridization solution contained 50% formamide, 5 XDenhardt's solution (100 XDenhardt's solution is Ficoll, polyvinylpyrrolidone and bovine serum albumin each 2%), 5 XDenhardt phosphate solution-EDTA (3M NaCl, 200mM NaH)₂PO₄-H₂O, 20mM EDTA, pH7.0), 0.1% sodium dodecyl sulfate, and 0.1mg/ml sonicated salmon sperm DNA. Hybridization was carried out at 42 ℃ for 24 hours. The filters were washed in 1 × standard citrate solution (150mM NaCl, 15mM sodium citrate, pH7.0) and 0.1% sodium dodecyl sulfate at 65 deg.C and exposed to Kodak XAR-5 film. Specific bands were found in monkey, rat, mouse, dog, cow, rabbit, and chicken DNAs, but no signal was produced by yeast DNA. Flt4cDNA has been isolated from quails. See Eichmann et al, Gene, 174 (1): 3-8(1996, 9/26) and Genbank accession number X83287.

Example 9

Expression of Flt4 Gene in adult human tissues

2 μ g of poly (A) using heart, brain, placenta, lung, liver, skeletal muscle, kidney and pancreatic tissue⁺RNA (Multiple Tissue Northern Blot, Clontech Inc.) was analyzed for Flt4mRNA expression in adult tissues by hybridization to a Flt4cDNA probe. Control hybridizations with probes for constitutively expressed genes showed uniform loading of the lanes.

Poly (A) of various human tissues⁺Hybridization of RNA with Flt4cDNA fragments showedmRNA bands with 5.8 and 4.5kb mobility and a faint marker band of 6.2kb were noted in placenta, lung, heart and kidney. Faint mRNA bands were visible in liver and skeletal muscle, while the pancreas and brain appeared to contain very little if any Flt4 RNA.

Example 10

Flt4 expression in human fetal tissues

To examine Flt4mRNA expression in human fetal tissues, Northern blots containing total RNA from the following tissues from 16-19 week human fetuses were hybridized to a 1.9kb Flt4cDNA fragment (see FIG. 1) and the resulting autoradiographs were scanned with a densitometer. The results were normalized by estimating the amount of RNA from UV photographs of the corresponding ethidium bromide (EtBr) -stained gels. The following symbols represent sequentially increasing mRNA levels: -,+,++,+++.

TABLE 1

Fetal tissue mRNA

Brain

Meninges calcium carbonate

Bark plate + C

Mid band ++

Room pipe film zone +

Small brain +

Mailuo Bu

Liver disease

Pancreas splash

Small intestine-

Heart disease

Lung + + +

Kidney +

Adrenal gland +

Skin +

Spleen + + ++

Thymus gland

Analysis of human fetal tissues showed Flt4 transcripts contained in addition to the thymus and small intestine. The highest expression levels were found in lung and spleen.

Example 11

Flt4 expression vectors

Full-length Flt4cDNA (short form) was generated as follows: a) the SphI-cleaved Flt4 PCR fragment [ amplified from the s2.5kb clone (see fig. 1) using primer oligonucleotides 5'-ACATGCATGCCACCATGCAGCGGGGCGCCGCGCTGTGCCTGCGACTGTGGCTCTGCCTGGGACTCCTGGA-3' (seq. id No.7) (forward) and 5'-ACATGCATGCCCCGCCGGTCATCC-3' (reverse) (seq. id No.8) ] was ligated to the 5 ' end of the s2.5kb fragment, subcloned into the pSP73 vector (Promega) using two SphI sites; b) the PCR fragment containing the last 138 bases amplified from the 0.6kb EcoRI fragment (see FIG. 1) with oligonucleotide primers 5'-CGGAATTCCCCATGACCCCAAC-3' (SEQ. ID No.9) (forward) and 5'-CCATCGATGGATCCTACCTG AAGCCGCTTTCTT-3' (SEQ. ID No.10) (reverse) was ligated to the 3 ' end of construct a) using EcoRI and BamHI sites; c) the EcoRI site of the construct b) is connected with a 1.2kb EcoRI fragment; d) the resulting full-length HindIII-BamHI fragment was ligated into a HindIII-BamHI cleaved SV-poly expression vector [ Stacey et al, Nucl. acids Res., 18: 2829 (1990).

Example 12

Identification of Flt4 ligand

Conditioned media obtained from a 7 day culture of the PC-3 prostate cancer cell line (ATCC CRL 1435) in F12 medium without Fetal Bovine Serum (FBS) were clarified and screened for the ability to induce tyrosine phosphorylation of Flt4 by centrifugation at 16,000Xg for 20 minutes.

NIH3T 3-cells recombinantly expressing Flt4 (see example 13) were reseeded on 5cm diameter cell culture dishes and grown to confluence in Dulbecco's modified minimal essential medium (DMEM) containing 10% fetal bovine serum and antibiotics. Confluent cells were washed twice in Phosphate Buffered Saline (PBS) and starved in DMEM/0.2% bovine serum albumin overnight. For stimulation, starvation medium was replaced with 1ml of conditioned medium and cells were cultured at 37 ℃ for 5 minutes.

After stimulation with PC-3 conditioned medium, the plates containing the cells were placed on ice and washed twice with Tris-HCl, pH7.4, 150mM NaCl containing 100mM NaVO 4. The wash solution was removed from the dishes and the cells were lysed in RIPA buffer [10mM Tris-HCl pH7.5, 50mM NaCl, 0.5% sodium deoxycholate, 0.5% Nonidet P40, 0.1% Sodium Dodecyl Sulfate (SDS) ] containing aprotinin, 1mM PMSF and 1mM NaVO4, and the lysates were sonicated twice for 10 seconds. The lysate was then centrifuged at 16,000Xg for 30 minutes and the supernatant was transferred to a new tube and used for immunoprecipitation.

Polyclonal antibodies against the C-terminus of Flt4 (described above) may be used for immunoprecipitation. Supernatants from cell lysates were incubated with 2 to 4 μ l of rabbit polyclonal anti-Flt 4 antiserum for 2 hours on ice. Approximately 30. mu.l of 50% (vol/vol) protein A-Sepharose (pharmacia) in PBS was added and incubation continued for 45 minutes at +4 ℃ with rotation. The immunoprecipitates were washed 3 times with RIPA buffer and once with PBS. And then at 7.The immunoprecipitates were subjected to SDS-polyacrylamide gel electrophoresis (SDS-PAGE) in a 5% gel and blotted onto nitrocellulose membranes. Western blots were incubated with monoclonal anti-phosphotyrosine antibody (anti-P-Tyr) (PT-66 Sigma, cat. P-3300 diluted 1: 2000) followed by detection with peroxidase-conjugated rabbit anti-mouse antibody (1: 1000 dilution, Dako, cat. P161) using a chemiluminescent detection system (Amersham). In some cases, the blot was stripped for 30 minutes at 50 ℃ under occasional stirring in 100mM 2-mercaptoethanol, 2% SDS, 62.5mM Tris-HCl pH6.7 to clear the previous signal and re-stained with anti-Flt 4 antibody (1: 1000 dilution) followed by peroxidase conjugated porcine anti-rabbit antibody (1: 1000 dilution, Dako, P217). As a positive control for tyrosine phosphorylation of Flt4, sodium perovanadate (PerVO) as a 100mM tyrosyl phosphatase inhibitor was used₄) anti-Flt 4 immunoprecipitates from NIH3T3 cells expressing Flt4 were treated for 20 minutes. By adding 100mM (final concentration) sodium orthovanadate and 2mM (final concentration) hydrogen peroxide to the cell culture medium and 5% CO at 37 ℃₂The cells were cultured for 20 minutes for sodium perborate treatment of the cells. This procedure results in the production of a peroxidized form of vanadate (vanadyl hydroperoxide), which is a very potent inhibitor of protein tyrosine phosphatases in living cells.

Conditioned media from PC-3 cells stimulate tyrosine phosphorylation of a 120kD polypeptide that co-migrates with the tyrosine phosphorylated, processed mature form of Flt 4. This co-migration was confirmed after re-staining the blot with anti-Flt 4 antibody.

To confirm that the 120kD polypeptide is not a non-specific component of the conditioned medium, 15ml of conditioned medium was separated by SDS-PAGE, blotted onto nitrocellulose, and the blots stained with anti-P-Tyr antibody. Some polypeptides were detected, but none of them migrated with Flt4, indicating that the 120kD band is indeed a tyrosine phosphorylated protein immunoprecipitated from stimulated cells. Stimulation analysis of PC-3 conditioned media pretreated with heparin Sepharose CL-6B (pharmacia) at room temperature for 2 hours (lane 3) indicated that Flt4 ligand does not bind to heparin.

Unconditioned media did not induce Flt4 autophosphorylation. In addition, both untransfected NIH3T3 cells and NIH3T3 cells transfected with FGFR-4 did not exhibit tyrosine phosphorylation of the 120KD polypeptide when stimulated with conditioned medium from PC-3 cells. The stimulatory activity was greatly increased when PC-3 conditioned media was concentrated 4-fold using a Centricon-10 concentrator (Amicon). In addition, the flow-off obtained after concentration contained proteins with a molecular weight of not more than 10,000 (< 10,000) and failed to stimulate phosphorylation of Flt 4. The concentrated conditioned medium of PC-3 cells pretreated with 50ml of Flt4 extracellular domain (Flt4EC-6XHis, see below) (1mg/ml) coupled to CNBr-activated agarose completely abolished tyrosine phosphorylation of Flt4 as directed by the manufacturer. The conditioned medium was also pretreated with agarose CL-4B without affecting its stimulatory activity.

These data confirm that PC-3 cells produce soluble ligands for Flt 4. The above experiments confirmed that the ligand binds to the recombinant Flt4EC domain. Thus, the ligand can be purified in affinity chromatography using the recombinant Flt4EC domain. The purified protein can be electrophoresed in SDS-PAGE, blotted onto polyvinylidene fluoride (PVDF) membranes and its amino terminal sequence determined by standard methods in the art. Alternatively, the purified ligand may be digested into peptides for determination of its amino terminal sequence. Peptide sequences obtained from the purified proteins were used to synthesize a mixture of oligonucleotides encoding the sequences. The oligonucleotide and its complementary DNA strand counterpart can be radiolabeled and used to screen cDNA libraries prepared from PC-3 cells to obtain cDNA encoding the ligand, by methods standard in the art (Wen et al, Cell 69: 559-572 (1992)). Alternatively, the oligonucleotides and their counterparts can be used as primers in Polymerase Chain Reaction (PCR) to amplify sequences encoding the ligand using cDNA prepared from PC-3 cell RNA as a template. The methods for cDNA synthesis and PCR (RT-PCR) are standard in the art (Innis et al, 1990, PCR protocols, Academic Press; McPherson, M.J. et al, 1991, PCR, a practical prophacach, IRL Press; Partanen et al, Proc. Natl.Acad.Sci., USA, 87: 8913-. Another option is to clone the ligand 4 from PC-3 cells by using cDNAs cloned into eukaryotic expression vectors (e.g., using the Invitrogen Librarian cloning kit and vectors provided, e.g., pcDNA I or pcDNA III) and screening the library for Flt 4-alkaline phosphatase (Cheng and Flanagan, Cell, 79: 157-168, (1994)), Flt 4-immunoglobulin (Flt4-Ig) (Lyman et al, Cell, 75: 1157-1167(1993)), or similar affinity reagents, by standard methods in the art.

Example 13

Cell lines and transfections

NIH3T3 cells and 293-EBNA cells (Invitrogen) were cultured in DMEM containing 10% FCS. For stable expression, NIH3T3 cells were transfected with LTR-FLT41 vector together with pSV-neo vector (see example 4 above) by lipofection using DOTAP transfection reagent (Boehringer-Mannheim), in which the Flt4cDNA was expressed under the control of the Moloney murine leukemia virus LTR promoter. COS-1 cells were transfected by the DEAE dextran method (McClutchan and Pagano, J.Natl.cancer Inst., 41: 351-35 (1968)). Transfected cells were selected in 500mg/ml neomycin.

Example 14

Construction and expression of Flt4 fusion proteins

pVTBac-FLT4EC-6XHis fusion construct. The end of the cDNA encoding Flt4 was modified as follows: the Flt4cDNA sequence encoding the extracellular domain (EC) was 3 'terminated using the oligonucleotide 5' -CTGGAGTCGACTTGGCGGACT-3 '(SEQ ID NO: 13, SalI site underlined, containing the sequence corresponding to nucleotides 2184-2204 of SEQ ID NO: 1) and 5' CGC encoding 6 histidine residues for binding to Ni-NTA columns (Qiagen, Hilden, Germany) followed by a stop codonGGATCCCTAGTGATGGTGATGGTGATGTCTACCTTCGATCATGCTGCCCTTATCCTC-3' (SEQ ID NO: 14, the BamHI site underlined, containing the sequence complementary to nt 2341-2324 of SEQ ID NO: 1) was amplified. The amplified fragment was digested with SalI and BamHI and ligated as a SalI-BamHI fragment into the LTR-FLT41 vector (see example 4) in place of the vector containing the transmembrane and cytoplasmic domains encoding Flt4A single SalI-BamHI fragment of the sequence.

The 5 'end of the Flt4cDNA without the Flt4 signal sequence coding region was used with oligonucleotide 5' -CCCAAGCTTGGATCCAAGTGGCTACTCCATGACC-3 ' (SEQ ID NO: 11, with HindIII and BamHI sites underlined and containing the sequence corresponding to nucleotides 86-103 of SEQ ID NO: 1) and 5'-GTTGCCTGTGATGTGCACCA-3' (SEQ ID NO: 12, containing the sequence complementary to nucleotide 700 and 681 of SEQ ID NO: 1) were amplified by PCR. The amplified fragment (containing nucleotides 86-700 of SEQ ID NO: 1) was digested with HindIII and SphI (the SphI site corresponding to nucleotide 588-593 of SEQ ID NO: 1 is within the amplified region of the Flt4 cDNA).

The resulting HindIII-SphI fragment was used to replace the HindIII-SphI fragment in the modified LTR-FLT41 vector described immediately above (the HindIII site was 5' to the junction of the Flt4 insert and the pLTRpoly portion of the vector, the SphI site was in the Flt4cDNA and corresponds to nucleotide 588-593 of SEQ ID NO: 1). The resulting Flt4EC-6XHis insert was then ligated as a BamHI fragment into the BamHI site of the pVTBac plasmid (Tessier et al, Gene 98: 177-183 (1991)). This construct was transfected into SF-9 cells by lipofection together with baculovirus genomic DNA. Recombinant viruses were generated and used to infect High-Five cells (Invitrogen).

Flt4-AP fusion constructs. The 3 ' end of the sequence encoding the Flt4EC domain was amplified using oligonucleotides 5'-CTGGAGTCGACTTGGCGGACT-3' (SEQ ID NO: 15) and 5'-CGGGATCCCTCCATGCTGCCCTTATCCT-3' (SEQ ID NO: 16) and ligated into the LTR-FLT41 vector as a SalI-BamHI fragment, replacing the sequences encoding the transmembrane and cytoplasmic domains. The resulting insert was then ligated as a HindIII-BamHI fragment into the HindIII-BglII site of plasmid APtag-1, in reading frame with the coding region for alkaline phosphatase (Flanagan and Leder, 1990, Cell63, 185-. NIH3T3 cells were co-transfected with the Flt4-AP construct and pSV2neo (Southern and Berg, J.mol.appl.Genet.1: 327-341(1982)) by lipofection using DOTAP transfection reagent (Boehringer) and transfected cells were selected in the presence of 500mg/ml neomycin. Recombinant protein produced in this medium was detected by a colorimetric reaction that stained for alkaline phosphatase activity (Cheng and Flanagan, Cell 79.157-168 (1994)).

The Flt4-Ig construct. Recombinant DNA encoding the Flt 4-immunoglobulin chimera was constructed as follows. The 5 ' end of the cDNA encoding Flt4, including the Flt4 nucleotide encoding the signal sequence, was amplified by PCR using primers 5'-GGCAAGCTTGAATTCGCCACCATGCA GCGGGGCGCC-3' (SEQ ID NO: 17) and 5'-GTTGCCTGTGAT GTGCACCA-3' (SEQ ID NO: 18) and ligated into the LTR-FLT41 vector as a HindIII-SphI fragment. The 3 ' end of the Flt4 EC-coding sequence was amplified using oligonucleotides 5'-CTGGAGTCGACTTGGCGGACT-3' (SEQ ID NO: 19) and 5'-CGCGGATCCAAGCTTACTTACCTTCCATGCT GCCCTTATCCTCG-3' (SEQ ID NO: 20) and ligated as a SalI-BamHI fragment into the LTR-FLT41 vector in place of the sequences encoding the transmembrane and cytoplasmic domains. This Flt4EC insert containing the splice donor site was first ligated into pH γ CE2(Karjalainen, K., TIBTECH, 9: 109-113(1991)) containing exons encoding the hinge of the heavy chain of human immunoglobulin and the constant region exons. The EcoRI-BamHI insert containing the Flt4-Ig chimera was then blunt-ended by standard methods in the art (Klenow) and ligated into the blunt-ended HindIII site of pREP7 (Invitrogen). This construct was transfected into 293-EBNA cells by calcium phosphate precipitation and the conditioned medium was used for isolation of the Flt4-Ig protein by protein A-Sepharose affinity chromatography.

Examples 15 to 17

Purification and sequencing of Flt4 ligand

Cell culture supernatants produced from PC-3 cells under serum-depleted conditions were concentrated 30-50 fold using Centriprep filter cartridges and loaded onto a column that immobilized the extracellular domain of Flt 4. Two affinity matrices were prepared using alternative constructs and methods. In the first case, the Flt4EC-6XHis fusion protein was cross-linked to CNBr-activated Sepharose 4B (pharmacia), and in the second case, the Flt4-Ig fusion protein was coupled to protein A Sepharose using dimethylpimelic acid (dimethylpimelidate) (Schneider et al, 1982, J.biol.chem.257: 10766-10769). Eluting from an affinity columnThe material was further purified using ion exchange and reverse phase high pressure chromatography and SDS-polyacrylamide gel electrophoresis. The chromatographic fractions were tested for their ability to stimulate tyrosine phosphorylation of Flt 4. Microsequencing of purified biologically active ligand proteins and preparation of degenerate oligonucleotides based on the amino acid sequences obtained for isolation of poly (A) from, e.g., PC-3 cells⁺cDNA encoding the ligand is isolated and cloned from a cDNA library generated from RNA.

Detailed identification of Flt4 ligand, designated vascular endothelial growth factor C (VEGF-C), and the natural human, non-human mammalian, and avian polynucleotide sequences encoding VEGF-C, and International patent application No. PCT/US98/01973, filed on 2/1998 as VEGF-C variants and analogs (published as International publication No. WO 98/33917 on 6/8/1998); joukov et al, j.biol.chem., 273 (12): 6599 and 6602 (1998); joukov et al, EMBO J., 16 (13): 3898-3911 (1997); and International patent application No. PCT/FI96/00427 (published as International publication No. WO 97/05250), filed on 1/8/1996, all of which are incorporated herein by reference in their entirety. As detailed therein, human VEGF-C is initially produced in human cells as a prepro-VEGF-C polypeptide of 419 amino acids. The amino acid sequence of human prepro-VEGF-C is set forth in SEQ ID NO: 21 and the cDNA encoding human VEGF-C was deposited under the Budapest treaty at the American Type Culture Collection (ATCC), 10801 University Blvd, Manassas, VA 20110-. VEGF-C sequences from other species have also been reported. See, for example, Genbank accession number MMU73620(Mus musculus); and CCY15837 (Coturnix), incorporated herein by reference.

The prepro-VEGF-C polypeptide is processed in multiple stages to produce the mature and most active VEGF-C polypeptide of approximately 21-23kD (as estimated by SDS-PAGE under reducing conditions). This processing involves cleavage of the signal peptide (SEQ ID NO: 21, residues 1-31); cleavage of the carboxy terminal peptide (pattern of spaced cysteine residue traces [ Dignam et al, Gene, 88: 133-40 (1990); Paulsson et al, J.mol.biol., 211: 331-49(1990) ] corresponding approximately to amino acids 228-419 of SEQ ID NO: 21 and having the sequence of the Baldiani Loop 3 protein (BR3P) to yield a partially processed form of approximately 29 kD); cleavage (apparently extracellular) of the amino-terminal peptide (approximately corresponding to amino acids 32-103 of SEQ ID NO: 21) to yield a fully processed mature form of approximately 21-23 kD. Experimental evidence demonstrates that partially processed forms of VEGF-C (e.g., the 29kD form) bind to the Flt4(VEGFR-3) receptor, while high affinity binding to VEGFR-2 occurs only with fully processed forms of VEGF-C. It appears that VEGF-C polypeptides are naturally associated as non-disulfide linked dimers.

In addition, SEQ ID NO: amino acids 103-227 of 2 are not all critical for maintaining VEGF-C function. Consisting of SEQ ID NO: 2 (and not containing residues 103-112 and 214-227) retains the ability to bind and stimulate the VEGF-C receptor, it is expected that a polypeptide spanning from about residue 131 to about residue 211 will retain the biological activity of VEGF-C. The cysteine residue at position 156 has been shown to be important for the binding ability of VEGFR-2. However, VEGF-C Δ C₁₅₆The polypeptide (i.e., the analog lacking this cysteine due to deletion or substitution) remains a potent activator of VEGFR-3. SEQ ID NO: 2 is required for binding to either receptor, whereas analogs lacking a cysteine at position 83 or 137 compete with native VEGF-C for binding to and stimulation of both receptors.

Sequence alignment of human VEGF-C with VEGF-C from other species (using any of the commonly accepted sequence alignment algorithms) shows additional residues into which modifications (e.g., insertions, substitutions, and/or deletions) can be introduced without disrupting the biological activity of VEGF-C. Any position of a different amino acid, in particular a different amino acid with a side chain of different chemical nature, on the VEGF-C polypeptides of two or more species in alignment is likely to be a position that allows modification without abolishing function. An exemplary alignment of human, murine and quail VEGF-C is provided in FIG. 5 of PCT/US 98/01973.

In addition to the above factors, it should be understood that numerous conservative amino acid substitutions may be made in the wild-type VEGF-C sequence, which, particularly if the number of such substitutions is small, will likely result in the polypeptide retaining the biological activity of VEGF-C. "conservative amino acid substitution" refers to an amino acid substitution with an amino acid having a side chain with similar chemical properties. Similar amino acids with conservative substitutions include those with acidic side chains (glutamic acid, aspartic acid); basic side chains (arginine, lysine, histidine); polar amide side chains (glutamine, asparagine); hydrophobic aliphatic side chains (leucine, isoleucine, valine, alanine, glycine); aromatic side chains (phenylalanine, tryptophan, tyrosine); small side chains (glycine, alanine, serine, threonine, methionine); or aliphatic hydroxyl side chains (serine, threonine). Also included are one or several internal amino acid additions or deletions that do not disrupt the biological activity of VEGF-C.

From the foregoing, it is expected that many VEGF-C polypeptides and variants bind Flt4(VEGFR-3) with high affinity and, therefore, may be useful as Flt4 binding compounds in aspects of the invention involving imaging or screening tissue samples using Flt4 binding compounds. Of particular interest are forms of VEGF-C that contain alterations that reduce or eliminate VEGFR-2 binding affinity such that the resulting polypeptide has increased binding specificity for VEGFR-3. As described above, the alteration includes Cys₁₅₆That substantially eliminates VEGFR-3 binding affinity, or that disrupts the amino acid sequence alteration of the native prepro-VEGF-C proteolytic processing site (since fully processed VEGF-C has the highest VEGFR-2 affinity). In addition, VEGF-C molecules modified to retain Flt4 binding affinity but not to activate Flt4 autophosphorylation are useful Flt4 antagonists in the therapeutic methods described herein. It is also apparent from the above teachings that Flt4 ligand described herein may be used in assays as an additional marker to confirm the identity of human Flt4 allelic variants, and to confirm that non-human gene sequences having homology to the Flt4 sequences taught herein (see, e.g., example 8 and FIG. 4) are in fact the non-human counterparts to Flt 4. The predicted amino acid sequence of prepro-VEGF-C is set forth herein as SEQ ID NO: 21 are provided.

A detailed description of a second Flt4 ligand, designated vascular endothelial growth factor D (VEGF-D), as well as human polynucleotide sequences encoding VEGF-D, and International patent application No. PCT/US97/14696, published as International publication No. WO 98/07832, 8/21 of 1997, and as VEGF-D variants and analogs, respectively; and Achen, et al, proc.nat' l acad. scu.u.s.a., 95 (2): 548-553(1998), also incorporated herein by reference. As described therein in detail, human VEGF-D is first produced in human cells as a prepro-VEGF-D polypeptide of 354 amino acids. The cDNA and putative amino acid sequence of prepro-VEGF-D are described herein in SEQ ID NO: 22. VEGF-D sequences from other species have also been reported. See, e.g., Genbank accession No. D89628(Mus musculus); and AF014827(Rattus norvegicus), incorporated herein by reference.

The prepro-VEGF-D polypeptide has a putative signal peptide of 21 amino acids and is apparently proteolytically processed in a manner similar to the processing of prepro-VEGF-C. Lacks SEQ ID NO: residues 1-92 and 202-354 of 22 retain the ability to activate the receptors VEGFR-2 and VEGFR-3, and appear to associate as a non-covalently linked dimer. The use of VEGF-D polypeptides as Flt4 binding compounds of the invention is similar to that described above for VEGF-C. Similarly, similar changes to VEGF-D are expected (removal of the second of the 8 conserved cysteines in the VEGF homology domain, Cys₁₃₆Or removal of the proteolytic processing site) will result in a reduction or abolition of the VEGFR-2 binding affinity of the polypeptide, thereby increasing the specificity of Flt 4. VEGF-D molecules modified to retain Flt4 binding affinity but not to activate Flt4 autophosphorylation are useful Flt4 antagonists in the methods of treatment described herein.

Example 18

Cloning of mouse Flt4cDNA Probe

Lambda FlX from 129SV mice^II genomic library (Stratagene) about 10⁶Individual plaques were screened with the S2.5 human Flt4 receptor cDNA fragment described above covering the extracellular domain. See also Pajusola et al, Cancer Res.,52: 5738(1992). A2.5 kb BamHI fragment was subcloned from positive plaques and sequenced from both ends. From this subclone, an exon fragment covering nucleotide 1745-. See Finnerty et al, Oncogene, 8: 2293(1993).

A second fragment covering nucleotides 1 to 192 was also cloned.

Example 19

Analysis of Flt4mRNA in mouse tissues

According to Chomczynski et al, anal. biochem, 162: 156(1987) isolation of total RNA from developing embryos (8-18 days post-mating and 1 day old mice). Samples from 8 day p.c. embryos also included placenta.

For the RNase protection assay, [ 2]³²P]UTP and T7 polymerase for antisense orientation RNA probes were generated from the linearized murine Flt4 plasmid obtained in example 18. The β -actin probe used corresponded to nucleotide 1188-. See Tokunaga, et al, nucleic.acid.res., 14: 2829(1986). After purification in a 6% polyacrylamide/7M urea gel, the labeled transcripts were hybridized with 30. mu.g of total RNA overnight at 52 ℃. Unhybridized RNA was digested with RNase A (10U/ml) and T1(1mg/ml) at 37 ℃ for 1 hour at pH 7.5. Rnases were inactivated by proteinase K digestion at 37 ℃ for 15 minutes and the samples were analyzed on a 6% polyacrylamide/7M urea gel.

The expression pattern of Flt4 analyzed in this experiment showed that very weak mRNA signals were obtained from lung, liver, heart, kidney, skeletal muscle and spleen, whereas testis and brain apparently did not have specific signals. Analysis of a series of RNAs collected at different stages of mouse development by the RNase protection assay showed that Flt4mRNA was expressed throughout embryogenesis from day 8 post-coital to neonatal mice with no major change in signal intensity.

Example 20

In situ hybridization of Flt4 in mouse embryos

To better localize Flt4 transcripts to cells and tissues, 7.5 and 8.5 day p.c. mouse embryo sections were crossed with labeled Flt4 RNAs. Mouse embryos were generated from the mating of CBA and NMRI mice. Pregnant mice were sacrificed by cervical dislocation and embryos were immediately frozen or transferred into 4% paraformaldehyde by phosphate buffer. Embryos and isolated mouse organs were fixed at 4 ℃ for 18 hours, dehydrated, embedded in paraffin, and cut into 6 μm sections.

The RNA probes (antisense and sense) of 192 nucleotides and 305 nucleotides (see example 18) were used³⁵S]-UTP is generated from linearized plasmids. In situ hybridization of sections was performed according to Wilkinson et al, Development, 99: 493 (1987); and Wilkinson et al, Cell, 50: 79(1987), which are incorporated herein by reference, with the following modifications: 1) xylene was used instead of toluene before paraffin embedding; 2) cut into 6 μm sections and placed on a layer of diethyl pyrocarbonate-treated water on the surface of a glass slide pretreated with 2% 3-triethoxysilylpropylamine; 3) omitting alkaline hydrolysis of the probe; and 4) highly stringent washes are at 65 ℃ for 80 minutes in a solution containing 30mM DTT and 1 XSSC. The sections were covered with NTB-2 emulsion (Kodak) and stored at 4 ℃. Slides were exposed for 14 days, developed, and stained with hematoxylin. Control hybridizations with the sense strand and RNAse A-treated sections did not produce a specific signal above background.

Flt4mRNA expression was not detected in mouse embryos 7.5 days post-coital, but a bright signal was detected in the developing aorta on day 8.5 of development. In contrast, the developing yolk sac was Flt 4-negative. In extraembryonic tissues, Flt4 was significantly expressed in the allantois, while the developing yolk sac blood island was negative. On the other hand, angioblasts of head mesenchyme were strongly Flt 4-positive. In the developing placenta, Flt4 signals were first visible in the peripheral venous antrum. In the 9.5 day p.c. placenta, the venous sinus endothelium and giant cells fused to the Reichert's membrane fraction expressed Flt4 mRNA.

Thus, although Flt4 expression was very significant in the earliest endothelial cell precursors, i.e., angioblasts, it appeared to be restricted to only certain vessels in 8.5 day p.c. embryos. It is known that Tie receptors are expressed in all endothelial cells of developing mouse embryos and thus provides a marker for these cells. See Korhonen, et al, Oncogene, 8: 395 (1993); and Korhonen et al, blood.80: 2548-2555(1992). Clearly, in contrast to the Tie probe, the Flt4 probe hybridized very weakly to the arterial endothelium of 11.5 day p.c. embryos, e.g., to the developing dorsal aortic or carotid endothelium. In contrast, Flt4 signal was much more pronounced in developing veins. For example, Flt4 signal was detected in veins surrounding the developing metanephros, while the Tie probe primarily recognized capillaries within the metanephros.

Flt4mRNA distribution was observed to be particularly pronounced in some regions of 12.5 day p.c. mouse embryos, particularly in the dilated vessels in the axillary region. Similar Flt 4-positive vascular structures were seen in mid sagittal sections of the neck region (data not shown). Vascular plexus-like patterns of Flt 4-expressing vessels occur in the periorbital region and around the developing spine. In addition, located directly beneath the developing skin, there was a clear Flt 4-positive vascular network. Weaker capillary signals were obtained in some areas including the developing brain. Flt4mRNA was also detected in small vessels in the cervical region, in small vessels in the developing nasal orifices and at the base of the developing tongue, as well as in the caudal region. In addition, the liver was strongly positive for Flt4mRNA in a punctate fashion.

During further development, Flt4mRNA appeared to become more restricted to certain blood vessels of the embryo. Embryos at 14.5 days post-mating show this restricted expression pattern. In the midsagittal section of this embryo, the most significant Flt4 signal was observed along the developing spine in its anterior portion. This signal is believed to originate from endothelial cells of the thoracic duct, which is the largest lymphatic vessel formed during this developmental period. In contrast, the dorsal aorta and inferior vena cava were negative. Dilated vessels in the mesenteric region were also strongly positive for Flt 4. In addition, as with 12.5 day p.c. embryos, the vascular network along the anatomical boundaries of the periorbital, mandibular, and cervical regions contained Flt4 positive endothelium. Similar structures exist in the pericardial cavity and in all subcutaneous tissues. Apparently, all Flt 4-positive vessels had no blood cells in their lumen, in contrast to Flt 4-negative vessels. These expression patterns indicate that Flt4 becomes restricted to lymphatic endothelia during this developmental period. We observed that another site of Flt4 expression is in the developing bone marrow sinusoids.

Cross sections of the upper thorax of 16.5 day p.c. embryos were also analyzed for hybridization to the Flt4 probe. Hematoxylin-eosin staining was performed to visualize the different vessel types in this area. They include the carotid and brachiocephalic arteries, the vena cava, and the thoracic duct, which is small and free of surrounding muscle and connective tissue. Hybridization of endothelial cells of the thoracic duct and nearby small vessels to the Flt4 probe was observed at higher magnification.

Example 21

Analysis of Flt4mRNA in cultured endothelial cells

The in situ hybridization results described in example 20 indicate that Flt4 was expressed in venous endothelial cells and later in lymphatic vessels and some venous endothelial cells, but not in arterial endothelium. To determine whether this modulation was maintained in vitro, we investigated cultured endothelial cells using Northern blot and hybridization analysis.

Endothelial cells were isolated from human aorta, femoral vein, umbilical vein, and from foreskin microvasculature, cultured and identified as previously described in the art. See Van Hinsberg et al, ariteriosclerosis, 7: 389 (1987); and Van Hinsberg, et al, Thromb. Haemostas, 57: 148(1987). They were used to isolate polyadenylated RNA when confluent densities were reached after 5 to 8 generations (split ratio 1: 3).

The endothelial cell lines EA hy926(Edgel et al, Proc. Natl. Acad. Sci., 80: 3734-5237 (1983)), BCE (Folkman et al, Proc. Natl. Acad. Sci., 76: 5217-5221(1979)) and LEII (Schreiber et al, Proc. Natl. Acad. Sci., 82: 6138(1985)) do not express Flt 4. However, cultured human microvascular, venous and umbilical vein endothelial cells were positive for Flt 4-specific 5.8 and 4.5kb mRNAs, while aortic endothelial cells were negative. In contrast, another endothelial receptor tyrosine kinase gene, Tie, was expressed as a 4.4kb mRNA in all endothelial cell types tested.

Example 22

Flt4mRNA in adult human tissues

The results obtained in example 20 indicate that Flt4mRNA became largely restricted to lymphatic endothelium during development. Because of the potential significance of this finding in humans, we also studied Flt4 expression in adult tissues using the human Flt4 probe. The human Flt4 probe used was an EcoRI-SphI fragment covering base pairs 1-595(SEQ ID NO: 1) of the cDNA. See also Pajusola et al, Cancer res, 52: 5738(1992). The Von Willebrand factor probe is an EcoRI-HindIII fragment covering base pairs 1-2334. Bonthron, et al, Nucleic Acids res, 141: 7125(1986).

We performed histopathological diagnosis using conventional immobilized materials. Normal lung tissue was obtained from a left lower lobectomy with epidermoid carcinoma. Mesenteric and mesenteric lymph nodes were obtained from patients with colon adenocarcinoma. Normal lymph nodes adjacent to salivary glands were removed due to their abnormal size. The tonsils and the two appendices from two patients had no diagnostic changes. Two lymphangiomas and three gall bladder lymphangiomas were studied with similar results.

For human tissues, normal samples fixed with 10% formalin were used for histopathological diagnosis, normal in situ hybridization generated just the background, and microwave treatment instead of proteinase K allowed specific hybridization. Shi, et al, j.biol.chem., 266: 5774 (1991); catoretti, et al, j.of pathol, 168: 357(1992).

Flt4 signal was produced in the mesenteric, pulmonary and appendiceal lymphatic endothelia, while veins, arteries and capillaries were negative. To investigate whether Flt4 was expressed in HEVs, tonsils were investigated. Indeed, in the tonsils, Flt 4-specific autoradiographic particles were detected in some HEVs.

Example 23

Analysis of Flt4mRNA in Normal and metastatic lymph nodes and lymphangiomas

A portion of human mesenteric lymph nodes (see example 22) were analyzed for Flt4 expression. Flt4 expression was observed in the lymphatic sinuses and afferent and efferent lymphatic vessels. The same pattern was observed in lymph nodes containing adenocarcinoma metastases. Positive also in some HEVs of normal and metastatic lymph nodes. In comparison with the in situ signal of von Willebrandt factor in all blood vessels, it is evident that Flt4 expression is characteristic of lymphatic endothelium in gallbladder lymphangiomas.

Consistent with these results, immunostaining for Flt4 was strongly positive in the endothelium of cutaneous lymphangioma, a rare disease characterized by presumed hyperplasia of the lymphatic endothelium. See Lymboussaki et al, am.j.pathol., 153 (2): 395-403 (month 8 1998), which is incorporated herein by reference in its entirety.

In addition, immunostaining for Flt4 identified spindle-shaped cells in Kaposi's sarcoma cutaneous nodular lesion tissue samples. See Jussila et al, Cancer res, 58: 1599 step 1604 (4 months 1998). In view of the apparent lymphatic specificity of Flt4, these results are believed to be consistent with the notion that certain cells in Kaposi's sarcoma have lymphatic endothelial origin. See, e.g., beckstad et al, Am j. pathol., 119: 294-300 (1985); and Dictor et al, Am j. pathol., 130: 411-417(1988).

Example 24

Flt4 localisation to foetal endothelial cells

As described in example 2, the Flt4cDNA fragment encoding the 40 carboxy terminal amino acids in short chain form was cloned as a 657bp EcoRI fragment into the coding region of the glutathione-S-transferase of the pGEX-1. lamda.T bacterial expression vector (Pharmacia). The resulting GST-Flt4 fusion protein was produced in E.coli and purified by affinity chromatography using a glutathione-Sepharose 4B column. The purified protein was lyophilized, dissolved in PBS, mixed with Freund's adjuvant, and used to immunize rabbits. Antisera after the third booster immunization were used.

Human fetal tissues of 17 and 20 weeks of age were obtained from legal abortions induced with prostaglandins. The study was approved by the ethical committee of the hoelkikyu university central hospital. Gestational age was estimated from fetal foot length. Fetal Tissue was embedded in Tissue-Tek (Miles), immediately frozen, and stored at-70 ℃.

anti-Flt 4 antiserum was cross-adsorbed to a GST-agarose column to remove anti-GST-antibodies and then purified by GST-Flt4 affinity chromatography. Some 6 μm thick cryostat tissue sections were fixed with acetone and treated with 0.3% H in methanol₂O₂Treatment for 30 minutes to inhibit endogenous peroxidase activity. After washing, the sections were incubated with 5% normal pig serum. The sections were then incubated with anti-Flt 4 antibody and washed. Bound antibodies were detected with peroxidase-conjugated porcine anti-rabbit IgG followed by staining for peroxidase activity using 0.2% 3, 3-diaminobenzidine (Amersham) as substrate. Sections were counterstained in Meyer's hematoxylin.

anti-Flt 4 immunoperoxidase staining of human fetal mesentery indicated the presence of Flt4 protein on the endothelium of some blood vessels, whereas control staining with antigen-blocking anti-Flt 4 antibody and preimmune serum was negative. For comparison, sections were stained with antiserum against the factor VIII-related antigen, which is specific for vascular endothelial cells. Immunoperoxidase staining for Flt4 was observed in vascular endothelial cells without red blood cells, while the vessels were negative. Blood vessels without red blood cells are likely lymphatic endothelial cells; this blood vessel is particularly common in mesentery. Antibodies against factor VIII-related antigens stained endothelial cells in all blood vessels.

Example 25

anti-Flt 4 aloneProduction of cloned antibodies

Fusion I:

recombinant Flt4 extracellular domain protein was produced by expressing the Flt4EC-6XHis-pVTBac plasmid construct (example 14) in High-Five cells. The Flt4 extracellular domain (Flt4EC) was purified from the culture medium of infected High-Five cells using Ni-NTA affinity chromatography following the manufacturer's instructions (Qiagen) to bind and elute the 6XHis tag encoded by the COOH-terminus of the recombinant Flt4 extracellular domain.

Balb/c male mice 4 months of age were immunized by intraperitoneal injection of purified recombinantly produced Flt4 extracellular domain protein (150. mu.g/mouse) emulsified with Freund's complete adjuvant. 150 μ g of boost injections were given at 3 to 4 week intervals and the final boost was given after another 3 week interval (10 μ g Flt4EC in PBS, intraperitoneal administration). 4 days after the final booster dose, mice were sacrificed and mouse spleen lymphocytes were fused with SP 2/0 plasmacytoma cells at a ratio of 2: 1, respectively.

Fused cells were harvested in Ex-Cell 320 medium (SERALAB) containing 20% fetal bovine serum and HAT supplement (hypoxanthine-aminopterin-thymidine; GIBCO, 043-01060H; diluted 50-fold) in 96-well culture plates (NUNC). Cells were cultured at +37 ℃ in air at 5% CO 2. After 10 days, the HAT supplemented medium was replaced with HT supplemented cell culture medium (GIBCO; 043-. HT medium is the same as HAT medium, but lacks aminopterin.

At 3 weeks, the production of specific antibodies was determined by the antigen specific ImmunoFluoroMetric assay (IFMA) described in example 26 below. The method is described by Staszewski et al, Yale Journal of Biologyand Medicine, 57: 865-868(1984) by limiting dilution. Positive clones were passaged onto 24-well tissue culture plates (NUNC), recloned, and re-tested in the same manner. Positive clones were detected by Fluorescence Activated Cell Sorting (FACS).

The immunoglobulins secreted by the stable clones belong to the Ig class, except that one clone produces an Ig which is likely to belong to the IgA classG₁Type (b). The subclass of monoclonal antibodies was determined using rat monoclonal antibodies directed against a mouse subclass as biotin conjugates (SEROTEC) in IFMA.

Balb/c mice were used to produce monoclonal antibodies in ascites fluid. The hybridomas were injected intraperitoneally into mice pretreated with pristane (98% 2, 6,10, 14-tetramethylpentadecane, ALDRICH-CHEMIE D7924Steinheim, Cat. No. T2, 280-2). Approximately two weeks prior to hybridoma cells, 0.5ml of pristanane (i.v.) was injected. The amount of cells injected is about 7.5 to 9X 10 per mouse⁶And (4) respectively. Ascites was collected 10 to 14 days after hybridoma injection.

And (2) fusion II:

two month old Balb/c mice (females) were immunized by intraperitoneal injection of recombinantly produced Flt4 extracellular domain protein (20. mu.g/mouse) emulsified with Freund's complete adjuvant. 20 μ g of boost injections were given at 3 to 4 week intervals and the final boost was given after another 3 week interval (10 μ g Flt4 in PBS, i.v. dosing). 4 days after the last booster dose, mice were sacrificed and mouse spleen lymphocytes were fused with SP 2/0 plasmacytomas at a ratio of 2: 1, respectively.

Fused cells were harvested in 96-well culture plates (FALCON) in OptiMEM 1 (containing Glutamax, 1, 51985-. Cells were cultured at 37 ℃ in air with 5% CO 2. After 10 days, the HAT supplemented medium was replaced with HT supplemented cell culture medium (GIBCO BRL; 41065-012, diluted 1: 50 fold).

At 3 weeks, the production of specific antibodies was determined by the antigen specific ImmunoFluoroMetric assay (IFMA) described in example 26 below. The main clone was cloned by limiting dilution as described in Staszewski et al (1984). Positive clones were passaged onto 24-well tissue culture plates (FALCON), recloned, and re-tested in the same manner. Positive clones were detected by FACS.

2E1The 1 and 6B2 clones secreted IgG₁Immunoglobulin of type 2B12 clone produces Ig belonging to subclass IgM. The mouse subclass IgG1 was determined using a rat monoclonal antibody against the mouse subclass heavy chain as biotin conjugate (SEROTEC) in IFMA and the mouse subclass IgM was determined using the mouse monoclonal antibody isotype classification kit (Dipstick Format) (19663-012, Life Technologies Inc.).

Example 26

Specificity of anti-Flt 4 monoclonal antibody

Purified, recombinant Flt4 extracellular domain-6 XHis fusion product (produced as described in examples 14 and 25) was prepared according to Mukkala et al, anal. biochem, 176 (2): 319-325(1989) with europium and modified as follows: a250-fold molar excess of isothiocyanates DTTA-Eu (N1 chelate, WALLAC, Finland) was added to the Flt4 solution (0.5mg/ml in PBS) and the pH was adjusted to about 9 by the addition of 0.5M sodium carbonate buffer, pH 9.8. Labeling was performed overnight at +4 ℃. Unbound label was removed using PD-10(PHARMACIA, Sweden) with TSA buffer (50mM Tris-HCl, pH7.8, containing 0.15M NaCl) as eluent.

After purification, 1mg/ml Bovine Serum Albumin (BSA) was added to the labeled Flt4 and the label was stored at +4 ℃. By measuring the fluorescence with known EuCl₃The standard ratio (Hemmila et al, anal. biochem., 137: 335-343(1984)) was determined to have an average number of europium ions incorporated per Flt4 molecule of 1.9.

The antibodies produced in example 25 were screened using a sandwich immunofluorescence assay using microtiter strip wells (NUNC, polysorb) covered with rabbit anti-mouse Ig (Z259, dakopats). The pre-coated wells were washed once with DELFIA wash solution by platwash 1296-024 (WALLAC). DELFIA assay buffer was used as a dilution buffer (at a dilution of 1: 1000 to 1: 100,000) for cell culture supernatants and splenectomized mouse sera, which were used as positive controls for the primary screening assay.

Incubation at +4 ℃ overnight (or at room temperature for 2 hours) was started by shaking on a Plateshake shaker (1296-001, WALLAC) for 5 minutes, followed by 4 washes with washing solution as described above.

Europium-labeled Flt4 was added at a dilution of 1: 500 in 100. mu.l of assay buffer. After 5 minutes on a Plateshake shaker and 1 hour incubation at room temperature, the titration bars were washed as described above.

Enhancement solution (DELFIA) was added at 200. mu.l/well. The plate was then shaken on a Plateshake shaker for 5 minutes and the intensity of fluorescence was measured by ARCUS-1230(WALLAC) for 10-15 minutes. (Lovgren et al, ed.: Collins W.P. (eds.) Alternative Immunoaslabs, John Wiley & Sons Ltd. (1985), p. 203-216). DELFIA results indicate that all monoclonal antibodies tested bind the Flt4EC antigen. Monoclonal antibodies reactive with Flt4 (and hybridomas that produce the antibodies) were selected for further screening.

The resulting monoclonal antibodies were used for double antibody immunofluorescence staining of NIH3T3 cells expressing the LTR-FLT41 construct and neomycin resistant transfected NIH3T3 cells. The cells were detached from the plates using EDTA, stained, and analyzed on a Fluorescence Activated Cell Sorter (FACS). The results of the FACS analysis are provided as the percentage of cells staining positive for the indicated monoclonal antibody (see table 2 below).

TABLE 2
				Cloning of Mab	LTR％a)	NEO％b)	DELFIA-count
1B1	67.3	1	20625
				1B1D11	75	1.2	19694
1B1F8	76.1	1.4	18580
				4F6	69.9	1.2	23229
4F6B8G12	75	0.3	24374
				4F6B8H11	75.9	0.3	28281
4F6B8E12	74.8	0.4	27097
				4F6B8G10	75.3	0.4	26063
9D9	45.1	0.75	17316
				9D9D10	71.7	2.3	18230
9D9F9	73	1.8	11904
				9D9G6	74.3	2.9	16743
9D9G7	70.7	1.3	17009
				10E4	24.2	1.4	39202
10E4B10E12	32.3	0.3	42490
				10E4B10G10	36.5	0.3	54815
10E4B10F12	45.6	0.4	43909
				10E4B10G12	45.7	0.5	35576
11G2	30.2	1.6	11304
				11G2D12	74.4	1.5	14660
11G2G9	74.2	0.9	10283
				11G2H7	74.4	2.1	25382

a) FACS results of LTR transfected cells

b) FACS results of NEO cells (control)

FACS results of LTR-FLT 41-transfected cells indicated that this antibody recognized Flt 4-expressing cells efficiently. The same antibody produced only background staining of neomycin phosphotransferase transfected NIH3T3 cells. Thus, the antibody specifically recognized Flt4 tyrosine kinase on the cell surface.

It was found that one clone, designated anti-Flt 4 hybridoma 9D9F9, stably secreted as IgG of the immunoglobulin class determined by IFMA₁The monoclonal antibody of (1). Hybridoma 9D9F9 was deposited at the german collection of microorganisms and cell cultures, department of human and animal cell cultures and viruses, massachoder Weg lb, 3300 Braunschweig, Germany on 23.3.1995 and is given the accession number ACC 2210.

Fusion II antibodies

The europium-labeled Flt4 extracellular domain protein described above was also used to screen fusion II antibodies described in example 25. The antibodies were screened using Flt 4-specific IFMA using microtiter wells (Nunc, Polysorb) covered with rabbit anti-mouse Ig (Z259, DAKO). The pre-coated wells were washed once with a washing solution (Wallac) by using DELFIA Plate wash.

DELFIA assay buffer used as cell culture supernatant: (Dilution 1: 2 in the primary screen) and splenectomized mouse serum (dilution from 1: 1000 to 1: 100,000) as a positive control. As a standard, purified anti-Flt 49D 9F9 (mouse subclass IgG) was used at a concentration between 1.0ng/ml and 250ng/ml₁). The samples were first shaken on a Plateshake shaker for 5 minutes at room temperature and then incubated at +4 ℃ for approximately 18 hours. The scaffolds were first washed 4 times, then Eu-labeled Flt4 (1: 2000 in 100. mu.l assay buffer) was added, and finally the scaffolds were incubated at room temperature for 1 hour. After washing as described, the enhancing solution (200. mu.l/well, Wallac) was added and the rack was shaken on a Plateshake shaker for 5 minutes. Fluorescence intensity was measured by ARCUS-1230 (Wallac). Monoclonal antibodies reactive with Flt4 were selected for further screening in a two-antibody immunofluorescence staining assay using Flt 4-expressing NIH3T3 cells as described above.

The resulting anti-Flt 4 monoclonal antibody of fusion II and the corresponding results of FACS analysis (expressed as the percentage of cells staining positive with the indicated monoclonal antibody) are summarized in Table 3.

A standard curve for quantification of anti-Flt 4 antibodies was prepared using affinity purified anti-Flt 49D 9F 9. The linear range reaches from 1.0ng/ml to 250 ng/ml.

Lysates of NIH3T3 cells co-transfected with the pLTRFLT41 construct expressing full-length Flt4 on the surface were electrophoresed on 6.5% SDS-PAGE to transfer proteins to nitrocellulose nitrate membrane (0.45 μm, SCHLEICHER)&SCHUELL) and immunoblotted with hybridoma cell culture supernatant containing monoclonal antibody (1: 10 in 50mM TRIS-40mM glycine buffer containing 4% methanol, 0.04% SDS). The specificity of the monoclonal antibodies was detected using HRP-conjugated rabbit anti-mouse Ig (P161, DAKO, 1: 1000 dilution in 20mM TRIS buffer containing 150mM saline, 5% milk powder, pH 7.5) and ECL (enhanced chemiluminescence, AMERSHAM) incubation. Is based on^a

TABLE 3
					Cloning of Mab	LTR％a)	NEOb)	Approximate Mab yield ng/ml/106Individual cellc)	WB
2B12E10	39.5	6.0	440	+
					2E11D11	44.6	8.8	110	+
2E11F9	49.5	4.5	100	+
					2E11F12	46.0	4.1	180	+
2E11G8	41.2	7.8	160	+
					6B2E12	NF	NF	1390	+
6B2F8	NF	NF	470	+
					6B2G6	NF	NF	630	+
6B2H5	NF	NF	740	+
					6B2H8	NF	NF	1800	+

a) FACS results of LTR transfected cells

b) FACS results of NEO cells (control)

c) anti-FLT 9D9F9 antibody based on affinity purification for standard quantitation

Mab yield

NF not functioning in FACS

WB successfully used in Western immunoblotting

Example 27

Identification in cell lysates using anti-Flt 4 antibodies

And Flt4 expressed in lymphatic endothelial cells of human tissues

The monoclonal antibody produced by hybridoma 9D9 described in the previous examples was used for immunoprecipitation and Western blotting of lysates of HEL cells. As reported in example 6, Flt4mRNA expression was previously observed in HEL cells. About 2X 10⁷Individual cultured HEL cells were lysed in RIPA buffer as defined in example 11 and immunoprecipitated with approximately 2 μ g of 9D9 antibody (as described for polyclonal antibody in example 11). For Western analysis, immunoprecipitates were electrophoresed by SDS-PAGE (6% gel) and electroblotted onto nitrocellulose membranes. Polypeptide bands corresponding to 175kD and 125kD of the Flt4 polypeptide were detected in Western blot analysis of immunoprecipitates using a dilution of 1 microgram/ml 9D9 antibody.

Immunostaining of human skin tissue was performed using the 9D9 monoclonal antibody and the alkaline phosphatase ABC-AP kit (Dako). Briefly, slides containing 6 μm adult skin samples were dried at Room Temperature (RT) for 30 minutes, fixed with cold acetone for 10 minutes, and then washed once with Phosphate Buffered Saline (PBS) for 5 minutes. The samples were then incubated with 2% horse serum for 30 minutes at RT and washed 3 times for 5 minutes in PBS.

For immunostaining, samples were incubated with 9D9 primary antibody for 1 hour at room temperature and washed 3 times for 5 minutes with PBS. After washing, the samples were incubated with biotin-labeled rabbit anti-mouse secondary antibody for 30 minutes at room temperature and washed 3 times with PBS for 5 minutes.

Bound antibody was detected by incubating the sample with ABC-AP complex for 30 minutes at room temperature, washing 3 times with PBS, incubating for 15 minutes at room temperature with AP-substrate (Sigma Fast Red TR/Naphtol AS-MX (Cat. No. F-4648)), and rinsing with water. The samples were then counterstained with Mayer's hematoxylin for 30 seconds and rinsed with water. The sample was analyzed under a microscope using water mounted and coverslipped. The lymphatic endothelial cells in these human skin sections were observed for staining with the 9D9 antibody. The vascular endothelium showed very weak or no staining. Further analysis was used to confirm the apparent specificity of lymphatic endothelia. See Lymboussaki et al, am.j.pathol., 153 (2): 395-403 (month 8 1998); and Jussila et al, Cancer res, 58: 1599-.

These results also demonstrate the utility of Flt4 as a useful marker of lymphatic endothelium and the use of anti-Flt 4 antibodies for identifying and observing Flt4 expressed in tissue samples in these cells.

Example 28

VEGF-C/VEGFR-3 signaling pathway

Up-regulation in breast cancer angiogenesis

The above examples demonstrate that Flt4(VEGFR-3) acts as a specific antigenic marker for the lymphatic endothelium of normal tissues. The following methods also demonstrate the use of VEGFR-3 as an antigenic marker (e.g., for diagnosis and screening) and as a therapeutic target for malignant breast tumors. VEGFR-3 positive vessels were found to be greatly elevated in invasive breast cancers compared to histologically normal breast tissue (P < 0.0001).

Materials and methods

Freshly frozen breast tissue samples were retrieved from the case of the department of pathology, university of helsinki. The samples consisted of ductal carcinoma (n ═ 6), lobular carcinoma (n ═ 6), intraductal carcinoma (n ═ 8), fibroadenoma (n ═ 4), and histologically normal breast tissue (n ═ 12). All samples were frozen in liquid nitrogen immediately after surgical resection and stored at-70 ℃.

Mouse monoclonal antibodies (Mabs) against human Flt4(VEGFR-3) were produced essentially as described in the previous examples, e.g., example 25. VEGFR-3 ectodomain proteins (VEGFR-3EC) were expressed by recombinant baculovirus in insect cells and purified from the culture medium. Mouse monoclonal antibodies against VEGFR-3EC were then produced using standard methods and purified from hybridoma ascites fluid or Tecnomouse by protein A affinity chromatography^The culture supernatant was purified from the immunoglobulin fraction.

Tissue samples of 5 μm were air dried, frozen sections and fixed in cold acetone for 10 minutes. Sections were rehydrated in Phosphate Buffered Saline (PBS) and incubated in 5% normal horse serum for 30 minutes at room temperature. The sections were then incubated with Mabs 9D9F9 (example 26) at a concentration of 1.0. mu.g/ml for 2 hours at room temperature in a humid atmosphere. Other anti-VEGFR-3 mabs against different epitopes of VEGFR-3EC have also been studied; clones 2E11D11 (example 26) and 7B8F9 (prepared essentially as described in example 26) were used at concentrations of 9.5 and 8.5. mu.g/ml, respectively. This was followed by incubation in biotin-labeled anti-Mouse serum for 30 minutes followed by incubation for 60 minutes using the reagents of the Vectastain Elite Mouse IgG ABC kit (Vector laboratories, Burlingame, USA). Peroxidase activity was developed with 3-amino-9-ethylcarbazole (AEC, Sigma, St.Louis, USA) for 10 min. Finally, the sections were stained with hematoxylin for 20 seconds. Negative controls were performed by omitting the primary antibody, or by using an unrelated primary antibody of the same isotype. The purified baculovirus immunogen was used to block the binding of the 9D9 antibody as another negative control. In these experiments, the antibodies were incubated overnight with a 10-fold molar excess of VEGFR-3EC protein in PBS. After centrifugation at 4000rpm for 4 minutes at +4 ℃, the supernatant was carefully collected and then used as the primary antibody. The 5 μm frozen sections adjacent to sections stained with anti-VEGFR-3 antibody were used to immunostain the vascular endothelial marker PAL-E (0.15 μ g/ml, Monosan, Uden, the Netherlands), laminin (1: 4000 diluted clone LAM-89 supernatant, Sigma, St Louis, MO), collagen XVIII (1.9 μ g/ml), alpha-smooth muscle actin (SMA, 0.5 μ g/ml, clone 1A4, Sigma), VEGFR-1 (1: 200 diluted clone 19 supernatant) or VEGFR-2 (1: 100 dilution).

All samples were pathologically examined after the staining procedure. According to Gasparini and Harris, [ Gasparini G, and Harris a, J clin. 765-782(1995) ] recommended guidance for staining slides from PAL-E [ de Waal et al, am.J.Pathol., 150: 1951-1957(1997) to obtain the density of the blood vessels. The density of VEGFR-3 positive vessels was tested in the same manner. The slides were first viewed at low magnification and then the vascular density within the tumor was estimated by counting the number of stained vessels in the region of highest vascular density ("vascular hot spots") or in the region with the highest VEGFR-3 positive vascular density in each 400x magnification high-power field (hpf). A minimum of 5 fields were counted per slide followed by an average of the 3 highest counts.

Double staining was performed in two intraductal carcinomas to distinguish immunohistochemical staining of lymphatic and blood vessels. Acetone-fixed 5 μm frozen sections were incubated with anti-PAL-E antibody for 1 hour, biotin-labeled horse anti-Mouse antibody (Vectastain Elite Mouse IgG ABC kit, Vector laboratories, Burlingame, USA) for 30 minutes, ABC-peroxidase (Vectastain, 1: 100) for 45 minutes, and finally developed with AEC for 10 minutes. For the second step, the sections were incubated with anti-VEGFR-3 antibody for 1 hour (0.14. mu.g/ml), followed by a biotin-labeled anti-mouse antibody for 30 minutes (1: 200 diluted clone supernatant), ABC-peroxidase for 30 minutes (1: 100), biotin-labeled tyramine (tyramin) solution containing 0.01% peroxide (1: 2.000) for 5 minutes, ABC-alkaline phosphatase (1: 100) for 20 minutes, and developed with fast blue (Sigma, St.Louis, USA) for 20 minutes according to the ISH signal enhancement method described in the previous literature. [ Kerstens et al, J.Histochem.Cytochem., 43: 347-352(1995)]. Frozen sections (5 μm) adjacent to the double stained sections were also immunostained with VEGFR-3 antibody alone as described above.

Polyclonal antibodies were produced in rabbits against a synthetic peptide corresponding to amino acid residues 2-18 of the N-terminal end of mature secreted human vascular endothelial growth factor C (VEGF-C), residues 104-120 of the VEGF-C prepro-VEGF-C polypeptide, as described in the literature. [ Joukov et al, EMBO J., 16: 3898-3911(1997), which is incorporated herein by reference in its entirety. The antiserum was affinity purified using immunogenic polypeptides coupled to an epoxy-activated agarose-6B column and tested for specific staining for VEGF-C using cells infected with adenoviral vectors expressing either VEGF-C or control β -galactosidase.

8 intraductal carcinomas were selected and all invasive carcinomas used to analyze VEGFR-3 were further analyzed for VEGF-C expression. The 5 micron frozen sections adjacent to the sections stained with anti-VEGFR-3 antibody were air dried and fixed in cold acetone for 10 minutes. The sections were rehydrated in PBS and incubated in 5% normal goat serum for 30 minutes, then incubated in humid air at room temperature for 2 hours with rabbit anti-human VEGF-C polyclonal antibody diluted 1: 200 in PBS. This was followed by incubation in biotin-labeled anti-Rabbit serum for 30 minutes followed by incubation for 60 minutes using the reagents of the Vectastain Elite Rabbit IgG ABC kit (Vector laboratories, Burlingame, USA). The slices were further processed as described above. As a negative control, purified immunogen was used to block binding of VEGF-C antibody. In these experiments, VEGF-C antibodies were incubated overnight with a 10-fold molar excess of VEGF-C protein in PBS. After centrifugation at 4000rpm for 4 minutes at +4 ℃, the supernatant was carefully collected and used for immunostaining.

The collagen production was carried out by contacting the collagen with a recombinant polypeptide QH48.18 corresponding to the consensus region of the N-terminal NC1 domain of human type XVIII collagen [ Saarela et al, Matrix Biology, 16: 319-28(1998) ] mice are immunized with a monoclonal antibody against human collagen type XVIII produced by DiaBor Ltd. (Oulu, Finland). The clones were screened by ELISA assay and Western analysis using the polypeptide QH48.18 and also by immunofluorescence staining of frozen human tissue sections. Screening of hybridoma clones yielded 3 monoclonal antibodies, positive in all three mentioned assays (ELISA, Western, immunofluorescence staining). One antibody, DB 144-N2, with the strongest signal was used in subsequent experiments. It produced the same staining pattern as polyclonal anti-all hu (XVIII) (e.g., in adult skin and kidney samples).

Results

A. In histologically normal breast tissue and in benign fibroadenomas

VEGFR-3 of (1)

Immunohistochemical staining of VEGFR-3 in normal breast tissue showed very weak staining in capillaries of the intervascular stroma. These vessels do not form any specific pattern, but are dispersed throughout the matrix. The density of VEGFR-3 positive vessels in normal breast tissue samples ranged from 6 to 17 per hpf with a median of 9 (n-12). Most of these vessels were strongly stained for the vascular endothelial marker PAL-E and the basal lamina component collagen XVIII, indicating that VEGFR-3 is weakly expressed in the vessels of normal breast tissue. However, some thin vessels in the stroma that clearly stained VEGFR-3 were PAL-E negative and XVIII type collagen weakly positive, indicating that they are lymphatic vessels. VEGFR-3 positive vessels were also found in benign fibroadenomas, but their density (median of 8 vessels per hpf; range 3-19; n ═ 4) was indistinguishable from histologically normal breast tissue (median 8 vs 9; P > 0.1, Mann-Whitney test).

B. VEGFR-3 positive blood vessels in intraductal carcinomas

In intraductal carcinomas, different patterns of strongly stained VEGFR-3 positive vessels were observed. The vessel forms an arch around the diseased catheter (fig. 5A). This "necklace" pattern was also observed in staining of the vascular endothelial marker PAL-E of adjacent sections (FIG. 5B), indicating enhanced VEGFR-3 expression in capillary endothelium. To more clearly distinguish between vessels and lymphatic vessels and to search for smooth muscle cells and adventitial cells present in the vessel wall, further staining was performed using antibodies against smooth muscle alpha-actin (SMA) and the basal lamina components laminin and type XVIII collagen. According to this staining, small vessels near the intraductal carcinoma expressed both VEGFR-3 and basal lamina proteins, but SMA stained less strongly, indicating that they were not completely covered by adventitial/smooth muscle cells in the vessel wall (black arrows in fig. 5C-5F). In contrast, larger vessels some distance from the intraluminal lesion were generally negative for VEGFR-3, and positive for laminin, collagen XVIII and SMA (red arrows). In addition, blood vessels were found that were positive for VEGFR-3, but only weakly stained for the basal lamina proteins laminin and type XVIII collagen and not stained at all for SMA (green arrows). They are considered to represent lymphatic vessels.

C. Differential double staining of blood and lymphatic vessels

Two intraductal carcinomas were selected for immunohistochemical double staining procedures to more clearly distinguish lymphatic vessels from blood vessels. [ see de Waal et al, am.J.Pathol., 150: 1951-1957(1997)]. Using this method, VEGFR-3 positive vessels were stained blue, while PAL-E positive vessels and basal lamina were stained brown. The samples from both tests showed similar staining patterns: blood vessels filling the intraductal lining of the tumor were predominantly PAL E positive (arrows in fig. 5G and 5H) while VEGFR-3 positive vessels, presumed to be lymphatic vessels, a short distance apart in the interductal stroma were PAL E negative (black arrows in fig. 5G and 5H). To exclude false positives due to possible double staining artifacts, adjacent 5 μm sections were stained separately with anti-VEGFR-3. This staining confirmed that some PAL-E positive vessels were also VEGFR-3 positive.

D. VEGF-C, VEGFR-1, and VEGFR-2 in intraductal carcinoma cells

And receptors in their adjacent vessels

Polyclonal affinity purified antibodies against human VEGF-C were used to stain 8 intraductal carcinoma samples. All samples tested contained at least some VEGF-C, but considerable heterogeneity in staining intensity and expression pattern was observed. In some cases, most cancer cells were strongly positive for VEGF-C, while in other cases only some cancer cells produced staining signals. In contrast, little or no staining was observed in normal tissue surrounding the diseased catheter, although weak signals were also obtained in the non-diseased normal ductal epithelium. Antigen blocking experiments showed that VEGF-C staining was specific. Other VEGF-C receptors, VEGFR-2, and other VEGF receptors (VEGFR-1) are all expressed in the same "necklace" vessels adjacent to intraductal carcinoma cells.

E. VEGF-C in VEGFR-3 positive vascular and invasive breast cancers

Strongly stained VEGFR-3 positive vessels were also present in all invasive ductal and lobular carcinomas tested. VEGFR-3 positive vessels do not appear to form any specific pattern of distribution; most of these vessels also immunoreact with the PAL-E antigen. The intratumoral VEGFR-3 positive vascular density (median 21, ranging from 9-56 vessels per hpf; n ═ 12) was significantly increased in invasive breast cancer compared to normal breast tissue (median 21 to 9; P < 0.0001, Mann-Whitney test). Sometimes, cancer cells were observed to invade into VEGFR-3 positive lymphatic vessels.

The immunostaining for VEGF-C was very different in the aggressive cancers tested (n-12). Some cancer cells were strongly positive for VEGF-C, while others stained very weakly, or in some cases no staining was observed. As with intraductal carcinomas, little or no staining was observed in the connective tissue of these sections.

The above data revealed that VEGFR-3, which appears to be primarily a lymphatic endothelial marker in most adult tissues, is also very weakly expressed in the capillary endothelium of normal breast tissues. More significantly, in intraductal carcinomas, strongly staining VEGFR-3 positive vessels in the form of "necklaces" were detected in the inner layer of the tumor-filled vessels. Most of these vessels expressed the vascular endothelial markers PAL-E and basal lamina components laminin and collagen XVIH, but had significantly fewer adventitial/smooth muscle cells than vessels located further away from tumor cells, as shown by staining with anti-SMA antibodies. These features indicate that "necklace" vessels are undergoing angiogenesis. A second group of vessels, some distance from the diseased vessels, were positive for VEGFR-3, but very weakly positive for basal lamina components and negative for PAL-E, indicating that they are lymphatic vessels. These vessels also lack SMA-positive adventitial cellular components. Also in invasive breast cancers, PAL-E positive vessels up-regulate VEGFR-3, although the vascular patterns seen are organized more randomly in the connective tissue matrix surrounding the tumor cells. The results indicate that VEGFR-3 expression is upregulated in breast cancer during angiogenesis associated with tumor growth. The large increase in the number of VEGFR-3 positive vessels found in carcinoma in situ is consistent with the hypothesis that cancer cells produce factors that activate the growth of blood vessels in the immediate vicinity of the cancer cells.

Since VEGF-C binds VEGFR-3 and VEGFR-2 with high affinity, and since intraductal and invasive cancer cells usually stain positive for VEGF-C protein, this growth factor is a candidate growth factor for VEGFR-3 and VEGFR2 positive vessels in cancer. These data are consistent with another study in which nearly half of 35 random malignant invasive tumors (including breast cancer, squamous cell carcinoma, lymphoma, melanoma, and sarcoma) contained VEGF-C mRNA in Northern blot analysis. [ see Salven et al, am. R Pathol, 153 (1): 103-108 (month 7 of 1998), which is incorporated herein by reference in its entirety. In summary, the data reported herein provide an indication of treatment of breast cancer and possibly other non-lymphoid cancers with agents that inhibit VEGF-C mediated stimulation of VEGFR-3 and/or VEGFR-2. Contemplated inhibitors include: an anti-VEGF-C antibody; anti-VEGFR-3 antibodies; anti-VEGFR-2 antibodies; a bispecific antibody that binds to VEGFR-3 and one of VEGFR-2 or VEGFR-1; a soluble ectodomain fragment of VEGFR-3 that binds circulating VEGF-C; VEGF-C fragments and analogs that bind to VEGFR-3 and/or VEGFR-2 and inhibit activation of the receptor; VEGF-C polypeptides, fragments, and analogs that bind VEGFR-3 and/or VEGFR-2 and are conjugated to a suitable therapeutic agent; VEGFR-3 tyrosine kinase inhibitors; and small molecules that bind to and inhibit these receptors. In addition, since VEGF-D binds to VEGFR-3 and VEGFR-2, anti-VEGF-D antibodies and inhibitory VEGF-D fragments and analogs are expected to be suitable inhibitors. Human or humanized antibodies and fragments thereof are preferred for the case where antibody agents are selected for human therapy. In addition, as another aspect of the invention, it is contemplated that any of the above-described agents may be used to evaluate mammalian tissue in vitro or in vivo, for example, for diagnostic purposes and to screen for malignancy and spread of malignancy.

For any of the above agents, it is contemplated that the detectable label may be attached by attachment of a detectable label, including but not limited to a radioisotope (e.g.,¹⁴C，¹³³i and¹²⁵I) chromophores (e.g., fluorescein, phycobiliprotein; tetraethyl rhodamine; an enzyme that produces a fluorescent or colored product for detection by fluorescence, absorbance, visible color, or agglutination, produces an electron-dense product for detection by electron microscopy); or electron dense molecules such as ferritin, peroxidase, or gold particles further improve the reagent for diagnosis and screening. Also, the compounds can be prepared by reaction with molecules having anti-tumor properties such as toxins of plant, animal, microbial or fungal origin; a radioactive isotope; drugs, enzymes; and/or the linking (e.g., coupling) or co-administration of cytokines and other therapeutic proteins further improves the use of the agents for therapeutic purposes. (see, e.g., Pietersz&McKenzie, "antibody conjugates for cancer therapy", Immunological Reviews, 129: 57-80(1992), incorporated herein by reference.

Example 29

anti-Flt 4 antibodies for administration as human therapeutics

A. Humanization of anti-Flt 4 monoclonal antibodies

The biology of Flt4 reported herein, for example in example 28, suggests therapeutic uses of Flt4 inhibitors (antagonists) that inhibit ligand-mediated Flt4 receptor signaling. Flt 4-neutralizing antibodies comprise a class of therapeutic agents useful as Flt4 antagonists. The following methods are used to improve the utility of anti-Flt 4 monoclonal antibodies as human therapeutics by "humanizing" the monoclonal antibodies to increase their serum half-life and render them less immunogenic in a human host (i.e., preventing human antibody responses to non-human anti-Flt 4 antibodies).

The principle of humanization has been described in the literature and is achieved by modular assembly of antibody proteins. To minimize the potential for binding complement, humanized antibodies of the IgG4 isotype are preferred.

For example, a level of humanization is achieved by generating chimeric antibodies comprising the variable regions of a non-human antibody protein of interest, such as the anti-Flt 4 monoclonal antibody described herein, and the constant regions of a human antibody molecule. (see, e.g., Morrison and Oi, adv. Immunol., 44: 65-92 (1989)). Flt4 neutralizes the variable region of anti-Flt 4 antibodies were cloned from the genomic DNA of B-cell hybridomas or cDNA produced from mRNA isolated from the hybridoma of interest. The V region gene fragments are ligated to exons encoding human antibody constant regions, and the resulting construct is expressed in a suitable mammalian host cell (e.g., myeloma or CHO cells).

To achieve a higher level of humanization, only that portion of the variable region gene fragment encoding the antigen binding complementarity determining regions ("CDRs") of the non-human monoclonal antibody gene is cloned into a human antibody sequence. [ see, e.g., Jones et al, Nature, 321: 522-525 (1986); riechmann et al, Nature, 332: 323-327 (1988); verhoeyen et al, Science, 239: 1534-36 (1988); and Tempest et al, biol technology, 9: 266-71(1991)]. If necessary, the beta-sheet structure of the human antibody surrounding the CDR3 region is also modified to more closely reflect the three-dimensional structure of the antigen-binding region of the original monoclonal antibody. (see, Kettleborough et al, Protein Engin, 4: 773-.

In another alternative method, the surface of a monoclonal antibody of non-human interest can be humanized by altering selected surface residues of the non-human antibody, e.g., by site-directed mutagenesis, while retaining all internal and contact residues of the non-human antibody. See Padlan, Molecular immunol, 28 (4/5): 489-98(1991).

The use of Flt 4-neutralizing anti-Flt 4 monoclonal antibodies and hybridomas which produce them, such as antibody 9D9F9, to produce humanized Flt 4-neutralizing antibodies for use in treating or alleviating disorders in which Flt4 expression is detrimental, is carried out using the methods described above.

B. Human Flt 4-neutralizing antibodies from phage display

Human Flt 4-neutralizing antibodies are produced by phage display technology, for example as described by Aujam et al, human antibodies, 8 (4): 155-; hoogenboom, TIBTECH, 15: 62-70 (1997); and Rader et al, curr. opin. biotechnol, 8: 503-508(1997), all of which are incorporated herein by reference. For example, the antibody variable region of a Fab fragment format or a linked single chain Fv fragment is fused to the filamentous phage secondary coat protein pIII. Expression of the fusion protein and its incorporation into the mature phage coat generates phage particles that have antibodies present on their surface and contain genetic material encoding the antibodies. Phage libraries containing this construct were expressed in bacteria and the libraries were panned (screened) for Flt 4-specific phage antibodies using labeled or immobilized Flt4 as an antigen probe.

C. Human Flt 4-neutralizing antibodies from transgenic mice

Substantially as described by Bruggemann and Neuberger, immunol.today, 17 (8): 391-97(1996), and Bruggemann and tausig, curr. opin biotechnol, 8: 455-58(1997) production of human Flt 4-neutralizing antibodies in transgenic mice. Transgenic mice carrying human V-gene fragments in germline configuration and expressing the transgene in their lymphoid tissues were immunized with the Flt4 composition using conventional immunization protocols. Hybridomas are generated using B cells from immunized mice using conventional methods and screened to identify hybridomas secreting anti-Flt 4 human antibodies (e.g., as described above).

D. Bispecific antibodies

Bispecific antibodies that specifically bind to Flt4 and that specifically bind to other antigens associated with pathology and/or therapy are generated, isolated, and tested using standard methods that have been described in the literature. See, e.g., Pluckthun & Pack, immunology, 3: 83-105 (1997); carter et al, j.hemartherapy, 4: 463-470 (1995); renner & Pfreundischuh, immunologicalReviewss, 1995, 145 th, 179 th page 209; U.S. patent No.5,643,759 to Pfreundschuh; segal et al, j.hemartherapy, 4: 377-382 (1995); segal et al, immunology, 185: 390-402 (1992); and Bolhuis et al, Cancer immunol. 1-8(1991), all of which are incorporated herein by reference in their entirety.

Example 30

Animal models demonstrating the efficacy of anti-Flt 4 therapy for cancer treatment

It is contemplated that any animal acceptable for cancer treatment may be used to demonstrate the efficacy of anti-Flt 4 therapy for cancer treatment. Experimental models for demonstrating the efficacy of breast Cancer treatment using standard dose-response assays include those described in Tekmal and Durgam, Cancer lett, 118 (1): 21-28 (1997); moshakis et al, br.j. cancer, 43: 575-580 (1981); and Williams et al, j.nat. cancer inst, 66: 147-155 (1981). In addition to murine models, dog and pig models are also included, as at least some anti-human Flt4 antibodies (e.g., 9D9 antibodies) also recognize Flt4 from dogs and pigs. Tumor size and side effects were monitored to confirm the effect of treatment relative to controls.

Example 31

Soluble FLT4 inhibits VEGF-C mediated tumor growth and metastasis

To further confirm the in vivo role of VEGF-C in tumorigenesis, MCF-7 human breast cancer cells overexpressing recombinant VEGF-C were orthotopically transplanted into SCID mice. Overexpression of VEGF-C enhances tumor growth, but unlike overexpression of VEGF-A, it has little effect on tumor angiogenesis. On the other hand, VEGF-C strongly promotes the growth of tumor-associated lymphatic vessels that surround the tumor, often infiltrated with tumor cells. These effects of VEGF-C can be inhibited by soluble VEGFR-3 fusion proteins. These data indicate that VEGF-C can upregulate tumor growth and/or metastasis through lymphatic vessels, and that these effects can be inhibited by inhibiting the interaction between VEGF-C and its receptors. In particular, soluble VEGFR-3/Flt4 may be used to inhibit this interaction.

Materials and methods

A. Preparation of plasmid expression vectors

Will encode human VEGF-C or VEGF₁₆₅The cDNAs of (1) were introduced into pEBS7 plasmid (Peterson and Legerski, Gene, 107: 279-84, 1991). The same vector was used to express the soluble receptor chimera VEGFR-3-Ig containing the first 3 immunoglobulin homeodomains of VEGFR-3 fused to the Fc-domain of the human immunoglobulin gamma chain, and VEGFR-1-Ig containing the first 5 Ig homeodomains of VEGFR-1 in a similar construct (Achen, et al, Proc Natl Acad Sci USA, 95: 548-53, 1998).

B. Generation and analysis of transfected cells

The MCF-7S 1 subclone of the human MCF-7 breast Cancer cell line was transfected with plasmid DNA by electroporation and stable cell colonies were selected and cultured as previously described (Egeblad and Jaattela, Int J Cancer, 86: 617-25, 2000). Is supplemented with 100. mu. Ci/ml³⁵S]-methionine and [ alpha ], [ alpha ]³⁵S]Cysteine (Redivue Pro-Mix, Amersham Pharmacia Biotech) in methionine and cysteine free MEM (Gibco). Immunoprecipitation of the labeled growth factor (Joukov, et al, EMBO J, 16: 3898-&D Systems). The immune complexes and VEGFR-Ig fusion proteins were precipitated using protein A Sepharose (Amersham pharmacia Biotech), washed twice in PBS with 0.5% BSA, 0.02% Tween 20 and once in PBS, and analyzed in SDS-PAGE under reducing conditions.

C. Cell proliferation and tumorigenesis assays

Cells (20,000/well) were plated in quadruplicate in 24-well plates, trypsinized on parallel plates after 1, 4, 6, or 8 days and counted using a hemocytometer. Fresh medium was provided after 4 and 6 days. For the tumorigenesis experiments, sub-confluent cultures were harvested by pancreatin, washed twice and 10 in PBS⁷The individual cells were inoculated into the fat pad of the second (axillary) mammary gland of an ovariectomized SCID mouse carrying a subcutaneous 60-day sustained release pellet containing 0.72mg of 17 β -estradiol (Innovative Research of America). Ovariectomy and pellet transplantation were performed 4-8 days prior to tumor cell inoculation. Tumor length and width were measured twice weekly in a blind fashion, with tumor volume calculated as length X width X depth X0.5, assuming the tumor is hemiellipsoidal and the depth and width are the same (Benz et al, Breast Cancer Restreat, 24: 85-95, 1993).

D. Histology and quantification of blood vessels

Tumors were excised, fixed in 4% paraformaldehyde (ph7.0) for 24 hours, and embedded in paraffin. Sections (7 μm) were immunostained according to published methods (Partanen et al, Cancer, 86: 2406-12, 1999) with monoclonal antibodies against PECAM-1(Pharmingen), VEGFR-3(Kubo et al, Blood, 96: 546-553, 2000) or PCNA (zymed laboratories) or polyclonal antibodies against LYVE-1(Banerji et al, J cell biol, 144: 789-801, 1999), VEGF-C (Joukov et al, EMBO J, 16: 3898-911, 1997) or laminin. The mean number of PECAM-1 positive vessels was determined from 3 regions (60X magnification) of the highest vessel density (vessel hot spot) in the section. All histological analyses were performed using blinded tumor samples.

E. Adenovirus expression and Evans blue exclusion assay for soluble VEGFR-3

The cDNA encoding the VEGFR-3-Ig fusion protein was subcloned into the pAdBglII plasmid and adenovirus generated as described previously (Laitinen et al, Hum Gene ther., 9: 1481-6, 1998). VEGFR-3-Ig or LacZ control (Laitinen et al, Hum Gene ther., 9: 1481-6, 1998) adenoviruses were treated at 10 h before tumor cell inoculation⁹pfu/mouse was injected intravenously into SCID mice. After 3 weeks, four mice in each group were anesthetized, the abdominal skin was incised and a few microliters of 3% evans blue dye (Sigma) in PBS was injected into the tumor. The dye expulsion from the tumor was visually observed.

Results

A. Expression of VEGF-C or VEGFR-3-Ig did not affect

MCF-7 cell growth in vitro

MCF-7 human breast cancer cells were transfected with expression plasmids encoding full-length human VEGF-C or soluble VEGFR-3 fusion protein (VEGFR-3-Ig) and stable cell colonies were selected as described above. For comparison, human VEGF was expressed in the same cells₁₆₅Or VEGFR-1-Ig. Immunoprecipitation was used to analyze the conditioned media of these cells for efficient production and secretion of the protein. Immunoprecipitates of VEGF-C, VEGF or soluble receptor proteins from metabolically labeled MCF-7 cells were analyzed in PAGE under reducing conditions.

This study revealed that overexpression of VEGF-C, VEGF, soluble VEGFR-3 fusion protein or soluble VEGFR-1 fusion protein did not affect the in vitro proliferation of MCF-7 breast cancer cells. When the cells were seeded into 24-well plates and their growth was measured using a hemacytometer, it was found that the growth rate of transfected cells was not affected.

B. VEGF-C proliferation into tumor growth without affecting tumor angiogenesis

To determine the in vitro potency of VEGF-C, MCF-7 cell colonies were implanted into ovariectomized ovaries

In the mammary fat pad of SCID mice carrying a slow-release estrogen pill to provide support

MCF-7 constant hormone levels required for tumor growth.

Overexpression of VEGF-C significantly increased tumor growth (VEGF-C: 545 mm)³±110mm³And comparison: 268mm³±69mm³On day 13, n is 8, p < 0.0001, student t-test). However, the effects of over-expression of VEGF-C on tumor growth were much less significant than VEGF-A (VEGF-A: 1136 mm)³±339mm³And comparison: 189mm³±57mm³On day 15, n is 6, p < 0.0001, student t-test). Increased tumor growth can be counteracted by mixing VEGF-C or VEGF over-expressed MCF-7 cells with cells expressing soluble VEGFR-3 or VEGFR-1 fusion proteins, respectively. In addition, circulating soluble VEGFR-3-Ig expressed in the liver by intravenous injection of recombinant adenovirus was found to also inhibit the increase in VEGF-C over-expressed tumor growth.

To investigate the effect of VEGF-C on tumor angiogenesis, tumor sections were stained for PECAM-1, an endothelial antigen that is expressed predominantly in blood vessels and weakly in lymphatic vessels. Quantification of PECAM-1 positive vessels in tumors revealed that overexpression of VEGF-C had a minimal effect on tumor vessel density (40.2 ± 12.2 vessels per microscopic field for VEGF-C tumors, n ═ 18 and 36.6 ± 11.6 for control tumors, n ═ 23, mean of three different experiments). In contrast, overexpression of VEGF-A increased vascular density by approximately two-fold.

C. Overexpression of VEGF-C and lymphangiogenesis

Associated with the internal lymphatic growth of tumor cells

The effect of VEGF-C on tumor-associated lymphatic vessels was analyzed by immunostaining for the lymphatic specific marker LYVE-1(Banerji et al, J Cell Biol, 144: 789-801, 1999). This marker revealed a high degree of lymphatic proliferation around the VEGF-C overexpressing tumor. Proliferating Cell Nuclear Antigen (PCNA) was detected in many LYVE-1 day endothelial cells, indicating that these lymphatic vessels are actively proliferating. Lymphatic structure was identified by VEGFR-3 staining and lack of laminin staining for the basal lamina component. There are also thin lymphatic vessels inside some VEGF-C overexpressing tumors.

Lymphatic vessels in the periphery of tumors are usually infiltrated by VEGF-C positive tumor cells. In stark contrast, VEGF-over-expressed and control tumors contained no or only a few lymphatic vessels.

D. Circulating soluble VEGFR-3 fusion protein inhibition

VEGF-C induced lymphangiogenesis

In human breast cancer, the centinel node method is used to track lymphatic drainage and metastatic cancer spread (for review, see Parker et al, radio Clin North Am, 38: 809-23, 2000). To follow lymphatic drainage of MCF-7 tumors, Evans blue dye was injected into VEGF-C overexpressing or control tumors in mice infected with VEGFR-3-Ig or control adenovirus. Control experiments showed that infection of cultured human embryonic kidney cells with VEGFR-3-Ig adenovirus resulted in large secretion of soluble VEGFR-3-Ig fusion protein and that intravenous infection of mice resulted in high systemic levels of VEGFR-3-Ig fusion protein in the serum. Injection of evans blue dye into tumors resulted in staining of lymphatic vessels but not blood vessels, and revealed an increased number of dilated lymphatic vessels surrounding the VEGF-C overexpressing tumors compared to control tumors. Most of the expanded lymphatic vessels were absent in VEGF-C overexpressing tumors in mice treated with VEGFR-3-Ig adenovirus.

The above data confirm that VEGF-C overexpression strongly and specifically induces tumor-associated lymphatic vessel growth in MCF-7 breast tumors, but has no significant effect on tumor angiogenesis. In addition, it was demonstrated that VEGF-C-mediated tumor growth increase and tumor-associated lymphatic formation were inhibited by the soluble VEGFR-3 fusion protein (i.e., an agent selected to inhibit VEGF-C-mediated stimulation of VEGFR-3 expressing endothelial cells).

Due to the lack of specific markers, it has been difficult in the past to determine whether a tumor actively induces lymphatic formation or simply surrounds existing lymphatic vessels by overgrowth and stresses them due to high interstitial fluid pressure within the tumor. Recent data from various experimental models indicate the latter (Leu et al, Cancer Res, 60: 4324-. Here it is shown for the first time that overexpression of VEGF-C induces lymphatic growth associated with experimental tumors. VEGF-C induced lymphatic vessels in the periphery of the tumor were highly hyperplastic and mostly filled with tumor cells, while lymphatic vessels within the tumor were flattened and lacuna. Unlike lymphatic endothelial cells in normal adult tissues, lymphatic endothelial cells associated with the MCF-7 tumor are actively proliferating. Thus, it appears that most lymphatic vessels in the periphery of and within tumors can be produced by the proliferation of existing lymphatic endothelial cells.

Cancer spread through lymph into regional lymph nodes has long been an important prognostic indicator in clinical use. The growth of tumor cells inside the expanded lymphatic vessels associated with VEGF-C overexpressing tumors demonstrated in this example was very similar to the lymphatic invasion in the periphery of tumors associated with metastatic spread into lymph nodes and low survival in human breast Cancer (Lauria et al, Cancer, 76: 1772-8, 1995). Thus, the data reported herein provide evidence that VEGF-C expression promotes tumor metastasis through the lymphatic system. Thus, given that example 28 and other data indicate that VEGFR-3 is upregulated in many types of solid tumor vessels (Valtola et al, Am J Pathol, 154: 1381-90, 1999; Partanen et al, Cancer, 86: 2406-12, 1999; Kubo et al, Blood, 96: 546-553, 2000), this example demonstrates that most tumor models that achieve VEGF-C overexpression have lymphangiogenesis.

As demonstrated by the ability of the VEGFR-3 fusion protein to inhibit the growth of VEGF-C overexpressing tumors, the effects of VEGF-C on tumor growth are not solely due to inter-colony variation. By injecting evans blue dye into tumors, it was demonstrated that an increase in the number of large draining lymphatic vessels was associated with tumors overexpressing VEGF-C. It is likely that a higher number of functional lymphatic vessels leads to better lymphatic drainage and thus lower interstitial pressure and enhanced blood perfusion in VEGF-C overexpressing tumors. Whether VEGF-C/D-VEGFR-3 mediated progressive tumors achieve specific tumor processes through angiogenic lymphangiogenesis, or both, the therapeutic methods of the present invention will inhibit these processes.

Finally, the above results indicate that VEGF-C produced by tumor cells can induce the growth of lymphatic vessels around the tumor, thereby promoting the intralymphatic spread of cancer. This data suggests that inhibition of tumor-associated lymphangiogenesis by, for example, gene therapy using soluble VEGFR-3 proteins is a valuable approach to inhibiting tumor metastasis.

Example 32

Inhibition of lymphangiogenesis in mice expressing soluble VEGFR-3/FLT4

The previous examples demonstrate that VEGF-C increases tumor growth in vivo and that promotion of this tumor growth is associated with lymphatic vessels. These effects of VEGF-C are inhibited by soluble VEGFR-3 fusion proteins. This example provides further evidence that soluble VEGFR-3 is a potent inhibitor of VEGF-C/VEGF-D signaling, which, when expressed in the skin of transgenic mice, inhibits lymphangiogenesis and induces regression of already formed lymphatic vessels while the vascular system remains intact.

More specifically, this example demonstrates that chimeric proteins (VEGFR-3-Ig), consisting of a ligand-binding portion of the extracellular portion of VEGFR-3 linked to the Fc region of the immunoglobulin (Ig) γ -chain, neutralize the activity of VEGF-C and VEGF-D and inhibit the formation of the cutaneous lymphatic vasculature when expressed in the mouse epidermis under the control of the keratin-14 (K14) promoter. Since the vascular network remained normal in these mice, this inhibition appeared to be specific for lymphatic vessels. VEGFR-3-Ig induced regression of lymphatic vessels during embryonic development, suggesting that continuous signaling of this receptor is essential for the maintenance of the lymphatic vasculature.

Materials and methods

A. Production of VEGF-C, VEGF-D and VEGFR-Ig fusion proteins

The mature forms of human VEGF-C are described above in example 31 and Joukov et al, (EMBO J.16: 3898-. VEGF-D was obtained from R & D Systems (Minneapolis, Minnesota). VEGFR-1-Ig and VEGFR-3-Ig proteins, consisting of the ligand binding regions of human VEGFRs fused to the Fc region of human IgGI, were produced in Drosophila S2 cells (Invitrogen, Carlsbad, California).

B. VEGFR-3 bioassay

Ba/F3 cells expressing the VEGFR-3/EpoR chimera (Achen, Eur. J biochem.267, 2505-2515, 2000) were seeded in triplicate at 15,000 cells/well in 96-well microtiter plates supplemented with 100ng/ml VEGF-C and the VEGFR-Ig protein at the indicated concentrations. After 48 hours, cell viability was determined by addition of MTT (3- (4, 5-dimethylthiazol-2-yl) -2, 5-diphenyltetrazolium bromide (Sigma), 0.5mg/ml), followed by incubation for an additional 2 hours, addition of an equal volume of cell lysate solution (10% SDS, 10nM HCl) and incubation at 37 ℃ overnight. The absorbance was measured at 540 nm.

C. Generation of transgenic mice

PCR was used to amplify the sequences encoding human VEGFR-3 Ig-homology domains 1-3. The primers used for this purpose were: 5'-TACAAAGCTTTTCGCCACCATGCAG-3' (SEQ ID NO: 23) and 5 '-IACAGGATCCI ℃ ATGCACAATGACCTC-3' (SEQ ID NO: 24).

The PCR product was cloned into the reading frame of the plg-plus vector (Ingenius, R & D Systems) containing the Fc tail of human IgGI. The VEGFR-3-Ig construct was then transferred into a human keratin-14 promoter expression vector. The expression cassette fragment was injected into fertilized mouse oocytes of FVB/NIH and DBAxBalbC hybrids to generate 7 lines of K14-VEGFR-3-Ig mice. Transgene expression was analyzed and the phenotype confirmed from all 3 established lines expressing the transgene as described below.

D. Analysis of transgene expression

For northern blotting, 10. mu.g of total RNA extracted from the skin in 1% agarose was electrophoresed, transferred onto a nylon membrane (Nytran), and used as a template³²P]-hybridizing the labeled cDNA probes and exposing by autoradiography. For western blotting, skin biopsies were homogenized and supplemented with 1mM PMSF, 1mU/ml aprotinin, 1mM Na₃VO₄And 10. mu.g/ml leupeptin lysis buffer (20mM Tris, pH7.6, 1mM EDTA, 50mM NaCl, 50mM NaF, 1% Triton-X100). The Ig-fusion proteins were precipitated from 1mg of total protein and separated in SDS-PAGE, transferred onto nitrocellulose and detected using horseradish peroxidase conjugated rabbit anti-human IgG antibody (DAKO, Carpinteria, California) and an enhanced chemiluminescence detection system.

E. Immunohistochemistry and TUNEL staining

Paraffin sections (5 μm) from 4% Paraformaldehyde (PFA) fixed tissues were stained with either rat monoclonal antibodies against mouse VEGFR-3(Kubo et al, Blood 96: 546-553, 2000) or CD31/PECAM-1(PharMingen, San Diego, California), rabbit polyclonal antibodies against mouse LYVE-1(Banerji et al, J Cell Biol, 144: 789-801, 1999), or biotin-labeled mouse monoclonal antibodies against human IgG Fc region (Zymed, San Diego, California). For TUNEL staining, detection of DNA fragments was performed using an IN situ cell death detection kit (fluorescein; Roche, Indianapolis, Ind.).

F. Observation of blood and lymph vessels

To visualize blood vessels (Thurston et al, Science 286, 2511-2514(1999)), 100. mu.l of 1mg/ml biotin-labeled tomato (Lycopersicon esculentum) lectin (Sigma) was injected through the femoral vein (IV) and allowed to circulate for 2 minutes. Bound lectin was visualized by ABC-3, 3' -diaminobenzidine peroxidase reaction after fixation by perfusion with 1% PFA/0.5% glutaraldehyde in PBS. Lymphatic vessels in VEGFR-3+/LacZ mice were then stained by the beta-galactosidase substrate X-Gal (Sigma, St. Louis, Mo.). To visualize functional lymphatic vessels, Evans blue dye (5 mg/ml; Sigma, St. Louis, Mo.) was injected into the lower limb paw pad or TRITC-dextran (Sigma, 8mg/ml) was injected into the ear or tail and lymphatic vessels were analyzed by light or fluorescence microscopy, respectively.

G. Detection of VEGFR-3-Ig protein in serum

ELISA plates (Nunc Maxisorp, Copenhagen, Denmark) were coated with mouse antibodies against human IgG (Zymed, 2. mu.g/ml in PBS) or human VEGFR-3 (clone 7B8, 4. mu.g/ml). Mouse sera were diluted into incubation buffer (5mg/ml BSA, 0.05% Tween 20 in PBS) and allowed to bind for 2 hours at room temperature. The plates were then washed 3 times with incubation buffer 1 hour before addition of mouse anti-human IgG1(Zymed, 1: 500). Streptavidin conjugated alkaline phosphatase (Zymed, 1: 5000) was then incubated in the wells for 30 min, followed by addition of substrate (1mg/ml p-nitrophenyl phosphate in 0.1M diethanolamine, pH 10.3) and absorbance reading at 405 nm.

H. Magnetic resonance imaging

MRI data was obtained using an s.m.i.s. console (Surrey Medical Imaging Systems, Guildford, UK) connected to a 9.4T vertical magnet (Oxford Instruments, Oxford, UK). A single loop surface coil (35 mm diameter) was used for signal transmission and detection. T is₂FOV used for the weighted (TR2000ms, TE 40ms, 4 scans/line) multislice spin-echo sequence is 25.6mm²(matrix size: 256X 128) and a slice thickness of 1.3mm in the transverse direction. A saturation pulse centered at 1.2ppm was used to reduce the fat signal in the T2-image. Using a unipolar diffusion gradient (b-value ≈ 800 s/mm) along the slice axis in a spin-echo sequence (TR2000ms, TE 40ms)²) Diffusion weighted MRIs are obtained and the water surface diffusion coefficient (ADC) is calculated by fitting the MRI data to a single exponential function of the b-value.

Results

A. Soluble VEGFR-3 inhibits VEGF-C-mediated in vitro signaling

To inhibit VEGF-C signaling through VEGFR-3, a fusion protein consisting of the first 3 Ig-homeodomain of VEGFR-3 and an IgG Fc domain was used. VEGFR-3-Ig bound VEGF-C and VEGF-D with the same efficiency as the full-length extracellular domain and inhibited VEGF-C-induced VEGFR-3 phosphorylation and subsequent p42/p44 mitogen-activated protein kinase (MAPK) activation in VEGFR-3 expressing endothelial cells. In contrast, a similar VEGFR-1-Ig fusion protein that did not bind VEGF-C did not affect p42/p44 MAPK activation.

The effect of soluble VEGFR-3 on VEGF-C signaling was also determined in biological assays using chimeric VEGFR-3/erythropoietin (Epo) receptors capable of transmitting VEGF-C dependent survival and proliferation signals of IL-3 dependent Ba/F3 cells in the absence of IL-3 (Achen et al, Eur. J. biochem., 267: 2505-propan 2515, 2000). In this cell assay, VEGF-C-dependent cell survival was completely inhibited at a 0.5: 1 molar ratio (VEGFR-3-Ig: VEGF-C), whereas VEGFR-1-Ig had no effect. Similarly, VEGFR-3-Ig also abolished VEGF-D-induced survival of VEGFR-3/EpoR cells. In contrast, even a 10-fold molar excess of VEGFR-2-Ig only partially abolished VEGF-C dependent activity, probably due to the lower affinity of VEGF-C for VEGFR-2.

B. Soluble VEGFR-3 inhibits the formation of lymphatic vessels in vivo

To determine the inhibitory effect of VEGFR-3-Ig in vivo, the fusion protein was expressed under the control of the K14 promoter which directs transgene expression into basal epidermal cells of the skin. VEGFR-3-Ig expression was detected in mice by northern blotting of skin RNA and western blotting of skin protein extracts. These mice appeared healthy and fertile and had a normal lifespan. Histological examination of the skin revealed thickening of the dermis and subcutaneous layers. Antibody staining confirmed the expression of VEGFR-3-Ig in basal keratinocytes. Lymphatic endothelial markers VEGFR-3(Jussila et al, cancer Res.58: 1599-1604, 1998; Kubo et al, Blood, 96: 546-553, 2000) and LYVE-1(Banerji et al, J Cell Biol, 144: 789-801, 1999) staining of skin sections no lymphatic vessels were observed in the transgenic mice, even though they were stained in the skin of the control mice. In contrast, the vascular panendothelial marker PECAM-1/CD31 was stained in both transgenic and wild-type skin.

C. Soluble VEGFR-3 inhibits lymphangiogenesis but not angiogenesis

To better visualize lymphatic vessels, K14-VEGFR-3-Ig mice were mated with a heterozygote VEGFR-3+/LacZ mouse expressing β -gal at the Flt4 locus (Dumont et al, Science 282, 946 949, 1998). When the substrate X-gal was used to stain whole fixed tissue specimens of the ear skin, no lymphatic vessels were detected, whereas in control mice blue-stained lymphatic vessels were observed. In vascular perfusion staining with biotin-labeled lectin (Thurston et al, Science 286, 2511-2514, 1999), blood vessels appeared normal in K14-VEGFR-3-Ig mice.

The absence of lymphatic vessels was also confirmed using a functional assay to monitor the fate of evans blue dye or TRITC-dextran injected into the skin. After injection into the lower limb paw pad of wild type mice, the dye rapidly concentrated to the lymphatic vessels surrounding the ischial veins, whereas no dye was seen in the lymphatic vessels in transgenic mice where the collecting lymphatic vessels were absent or degenerated. Lymphatic vessels in control mice were also observed using fluorescence microscopy on TRITC dextran injected intradermally into the ear or tail, but not in transgenic mice.

D. Circulating soluble VEGFR-3 and internal organs

Associated with transient loss of lymphoid tissue

At two weeks of age, the VEGFR-3-Ig/VEGFR-3+/LacZ mice had only a few, thin and degenerating, if any, lymphatic vessels within organs such as the septum, heart, lung, caecum, pancreas, mesentery, and esophagus when compared to control VEGFR-3+/LacZ littermate mice. This finding obtained by X-Gal staining was confirmed by immunostaining VEGFR-3 and LYVE-1. In addition, the lack of lymphatic vessels in the pericardium is associated with accumulation of pericardial fluid in at least some mice. At 3 weeks of age, regrowth of lymphatic vessels was evident. In adult transgenic mice, only some organs such as the heart and diaphragm have abnormal forms and incompletely developed lymphatic vessels.

This effect, seen in internal organs, suggests that the soluble VEGFR-3-Ig protein circulates in the bloodstream. The fusion protein is actually detected in the serum of the transgenic mice by using a specific enzyme-linked immunosorbent assay; the levels varied between 100 and 200ng/ml, with the highest levels in the young mice. According to our in vitro experiments, this concentration neutralizes approximately 20-40ng/ml of VEGF-C. The VEGFR-3-Ig protein is fairly stable in the bloodstream because the intravenously injected recombinant VEGFR-3-Ig is present in the serum for at least 9 hours.

E. The transgenic phenotype is characterized by human lymphedema

K14-VEGFR-3-Ig mice were distinguished from their wild type littermate mice by swelling of their feet and were already visible at birth. Older mice showed increased skin thickening, dermal fibrosis and increased subcutaneous fat deposition. Magnetic Resonance Imaging (MRI) revealed significant T in the foot skin and subcutaneous tissue of transgenic mice₂High intensity zone, indicating increased fluid accumulation, whereas this zone was absent in littermate control mice. The surface diffusion coefficient (ADC) of these high intensity regions was 1.99[0.60 ]]×10^-3mm²(s) ratio ADC of 1.32[ 0.21%]×10^-3mm²Normal tissue is higher and about 1-2 orders of magnitude higher than this value for fat (Thurston, et al, Science 286, 2511-2514 (1999). additionally, there is a change in the size and appearance of lymph nodes, particularly in large periaortic lymph nodes surrounding the inferior vena cava, however, mesenteric lymph nodes and Peyer's patches are visible in VEGFR-3-Ig mice.

F. Apoptosis of developing lymphatic vessels by endothelial cells

Is subject to degeneration

During embryogenesis, a significant increase in K14-initiated transgene expression occurred at E14.5, and by E16.5, the expression surrounded the entire embryonic skin (Byrne, et al, Development 120, 2369-2383, 1994). There was no difference in the lymphatic network of the skin between the transgene of E15 and the wild-type embryo when analyzed in the VEGFR-3+/LacZ background by X-Gal staining. At E15.5-16.5, the lymphatic vessels of the transgenic embryos regressed in certain areas. At E17.5, lymphatic vessels still formed a continuous network but were finer than in the control embryos. At E18.5, the entire cutaneous lymphatic network was disrupted in the transgenic embryos, and there were no or only a few single disrupted lymphatic vessels in the skin after birth, mainly accompanied by large cutaneous blood vessels. Thus, lymphatic vessels begin to form in the skin during embryogenesis, but degenerate when the transgene begins to be expressed. However, formation of the dermal vasculature was not inhibited in K14-VEGFR-3-Ig embryos, as shown by X-Gal staining in the Tie-promoter-LacZ background (Korhonen et al, Blood86, 1828-1835 (1995)).

TUNEL staining was used to detect apoptosis in endothelial cells, which can be identified by simultaneous staining for PECAM-1. Apoptotic endothelial cells were first seen in the dermis of the transgenic embryos at E17.5 and E18.5. No apoptosis of endothelial cells was seen in wild type embryos. TUNEL-positive cells were detected in transgenic skin almost exclusively in VEGFR-3 positive endothelium, suggesting that VEGFR-3-Ig mediated apoptosis targets lymphatic endothelium.

This example demonstrates that soluble VEGFR-3 fusion proteins inhibit lymphangiogenesis and cause regression of existing fetal lymphatic vessels in vivo. Continuous VEGFR-3 signaling is therefore essential for fetal development and maintenance of the lymphatic vasculature.

The absence of lymphatic vessels in the skin of K14-VEGFR-3-Ig mice is associated with a thickening of the dermis and especially the subcutaneous fat layer, such as human lymphedema (a disease caused by lymphatic insufficiency and characterized by swollen extremities of increasing severity) (Witte et al, lymphangiogenesis: mechanism, meaning and clinical meaning. see Regulation of angiogenisis (eds., Goldberg, I.D. & Rosen, E.M.)65-112(Birkhauser Verlag, Basel, Switzerland) 1997; Morttimer, Cancer 83, 2798-. In primary lymphedema as a genetic disease, superficial or subcutaneous lymphatic vessels are often underdeveloped or have regeneration disorders that do not transport lymph fluid into the venous circulation. Non-hereditary secondary or acquired lymphedema develops when lymphatic vessels are damaged by surgery, radiation, infection or trauma. In lymphedema, accumulation of protein-rich fluid in the interstitial space leads to tissue fibrosis and steatosis, interferes with wound healing, and is susceptible to infection. In K14-VEGFR-3-Ig mice, there was a lack of macromolecular trafficking in the dermis and, in particular, there was evidence of dermal fibrosis in older mice. In addition, foot swelling and increased fluid accumulation in skin and subcutaneous tissues in transgenic mice are similar to the symptoms of human lymphedema. Thus the skin phenotype of K14-VEGFR-3-Ig mice shares some of the characteristics of human lymphedema. In some studies of the lymphedema family, missense mutations were detected in the tyrosine kinase coding region of Flt4 in heterozygous inactivation (Karkkanen et al, Nature Genet., 25: 153-159, 2000; Irrthum et al, am.J.hum.Gen.67: 259-3012001). At least some lymphedema patients have lymphatic dysfunction due to defective VEGFR-3 signaling. Consistent with these observations, the results in this example indicate that disruption of VEGFR-3 signaling by soluble VEGFR-3 proteins can completely disrupt the lymphatic network and lead to a lymphedema-like phenotype. In addition, in some cases of lymphedema, certain regional lymph nodes may vary in size and appearance, indicating that lymphatic flow and a functioning lymphatic vasculature are essential for normal lymph node formation.

VEGFR-3-Ig also induced regression of already formed lymphatic vessels. Thus, inhibition of VEGF-C and/or VEGF-D binding to VEGFR-3 during development resulted in apoptosis of lymphatic endothelial cells and disruption of the lymphatic network, suggesting that continuous VEGFR-3 signaling is essential for lymphatic endothelial cell survival. In cell culture, VEGFR-3 activates a biochemical signaling cascade associated with endothelial cell survival. Although transgenic mice overexpressing VEGF-C (Jeltsch et al, Science, 276: 1423-1425, 1997) or VEGF-D in the skin developed a hyperplastic dermal lymphatic vasculature, their dermal lymphatic vessels also regressed when mated with K14-VEGFR-3-Ig mice. Since both VEGFR-3 ligands are also expressed in the skin, the phenotype observed in K14-VEGFR-3-Ig mice is likely due to simultaneous inhibition of VEGF-C and VEGF-D.

Although VEGF-C and VEGF-D have mitogenic activity on vascular endothelial cells both in vitro and in vivo (Joukov et al, EMBO J.16, 3898-3911, 1997; Achen et al, Proc. Natl.Acad.Sci.USA 95, 548-553, 1998; Cao et al, Proc. Natl.Acad.Sci.USA 95, 14389-143921998; Witzenbichler et al, am.J. Pathol.153, 381-394, 1998; Marconcini et al, Proc. Natl.Acad.Sci.USA 96, 9671-9676, 1999), VEGFR-3-Ig does not appear to affect blood vessels. The delayed onset of K14-promoter expression may explain the lymphatic specificity of the VEGFR-3-Ig protein. No substantial increase in K14-promoter activity was seen until approximately E14.5-16.5 (Byrne et al, Development 120: 2369-2383, 1994). Although endogenous VEGFR-3 expression was first detected in developing blood vessels at E8.5, by E14.5-16.5, it was down-regulated to a large extent in healthy vascular endothelium (Dumont et al, Science 282, 946-9491998; Kaipain et al, Proc. Natl. Acad. Sci. USA 92, 3566-3570, 1995). Thus, during development, VEGFR-3 signaling plays a lesser role in angiogenesis in the skin than other receptors when VEGFR-3 is no longer normally present in the vascular endothelium of healthy tissue.

In young VEGFR-3-Ig mice, some internal organs were almost completely devoid of lymphatic vessels, but they regenerated in adult mice, albeit into abnormal forms in some organs. Thus growth and maintenance of the lymphatic vasculature can be reactivated in adult organs. A decrease in VEGFR-3 inhibition levels or independent signals from, for example, the mature connective tissue matrix, could reactivate lymphangiogenesis, but no evidence was obtained that would suggest that increased levels of VEGF-C or VEGF-D are responsible for it. However, administration of VEGF-C by adenoviral vectors in amounts exceeding those typically found in interstitial fluid can lead to lymphatic vessel growth in adult tissues. The use of modified VEGF-C that no longer binds VEGFR-2 in gene therapy (U.S. Pat. No.6,130,071; Joukev et al, J.biol.chem., 273: 6599-6602) prevents its potential impact on vascular endothelium.

Thus, soluble VEGFR-3 is a potent and specific inhibitor of lymphangiogenesis in vivo. Soluble VEGFR-3 comprises the extracellular fragment of Flt4 described elsewhere in this specification. As seen above, the preferred soluble VEGFR-3 is VEGFR-3 which comprises the first 3 domains of VEGFR-3, however, it is understood that the soluble VEGFR-3 may be a fragment of VEGFR-3 which contains more or less of the wild-type sequence of VEGFR-3 as shown in FIG. 2. For example, the soluble peptide may also comprise one or more of IgIV, IgV, IgVI, IgVII. Alternatively, soluble VEGFR-3 may comprise only IgI in any combination with one or more domains selected from the group consisting of IgII, IgIII, IgIV, IgV, IgVI and IgVII.

In addition, this example also establishes a mouse model with human lymphedema characteristics. Since lymphedema is usually associated with the skin, this mouse model is useful for understanding and identifying the disease and for testing new therapies applicable to patients.

All documents, including patents and journal articles, cited in the summary and detailed description of the invention are hereby incorporated by reference in their entirety.

While the invention has been described herein in connection with specific embodiments thereof, it will be understood that it is capable of further modifications and this application is intended to cover any variations, uses, or adaptations of the invention following, in general, the principles of the invention and including such departures from the present disclosure as come within known or customary practice within the art to which the invention pertains and as may be applied to the essential features hereinbefore set forth and as follows in the scope of the appended claims.

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415 420 425

gag gcc tcc tcc ccc agc atc tac tcg cgt cac agc cgc cag gcc ctc 1348

Glu Ala Ser Ser Pro Ser Ile Tyr Ser Arg His Ser Arg Gln Ala Leu

430 435 440

acc tgc acg gcc tac ggg gtg ccc ctg cct ctc agc atc cag tgg cac 1396

Thr Cys Thr Ala Tyr Gly Val Pro Leu Pro Leu Ser Ile Gln Trp His

445 450 455

tgg cgg ccc tgg aca ccc tgc aag atg ttt gcc cag cgt agt ctc cgg 1444

Trp Arg Pro Trp Thr Pro Cys Lys Met Phe Ala Gln Arg Ser Leu Arg

460 465 470 475

cgg cgg cag cag caa gac ctc atg cca cag tgc cgt gac tgg agg gcg 1492

Arg Arg Gln Gln Gln Asp Leu Met Pro Gln Cys Arg Asp Trp Arg Ala

480 485 490

gtg acc acg cag gat gcc gtg aac ccc atc gag agc ctg gac acc tgg 1540

Val Thr Thr Gln Asp Ala Val Asn Pro Ile Glu Ser Leu Asp Thr Trp

495 500 505

acc gag ttt gtg gag gga aag aat aag act gtg agc aag ctg gtg atc 1588

Thr Glu Phe Val Glu Gly Lys Asn Lys Thr Val Ser Lys Leu Val Ile

510 515 520

cag aat gcc aac gtg tct gcc atg tac aag tgt gtg gtc tcc aac aag 1636

Gln Asn Ala Asn Val Ser Ala Met Tyr Lys Cys Val Val Ser Asn Lys

525 530 535

gtg ggc cag gat gag cgg ctc atc tac ttc tat gtg acc acc atc ccc 1684

Val Gly Gln Asp Glu Arg Leu Ile Tyr Phe Tyr Val Thr Thr Ile Pro

540 545 550 555

gac ggc ttc acc atc gaa tcc aag cca tcc gag gag cta cta gag ggc 1732

Asp Gly Phe Thr Ile Glu Ser Lys Pro Ser Glu Glu Leu Leu Glu Gly

560 565 570

cag ccg gtg ctc ctg agc tgc caa gcc gac agc tac aag tac gag cat 1780

Gln Pro Val Leu Leu Ser Cys Gln Ala Asp Ser Tyr Lys Tyr Glu His

575 580 585

ctg cgc tgg tac cgc ctc aac ctg tcc acg ctg cac gat gcg cac ggg 1828

Leu Arg Trp Tyr Arg Leu Asn Leu Ser Thr Leu His Asp Ala His Gly

590 595 600

aac ccg ctt ctg ctc gac tgc aag aac gtg cat ctg ttc gcc acc cct 1876

Asn Pro Leu Leu Leu Asp Cys Lys Asn Val His Leu Phe Ala Thr Pro

605 610 615

ctg gcc gcc agc ctg gag gag gtg gca cct ggg gcg cgc cac gcc acg 1924

Leu Ala Ala Ser Leu Glu Glu Val Ala Pro Gly Ala Arg His Ala Thr

620 625 630 635

ctc agc ctg agt atc ccc cgc gtc gcg ccc gag cac gag ggc cac tat 1972

Leu Ser Leu Ser Ile Pro Arg Val Ala Pro Glu His Glu Gly His Tyr

640 645 650

gtg tgc gaa gtg caa gac cgg cgc agc cat gac aag cac tgc cac aag 2020

Val Cys Glu Val Gln Asp Arg Arg Ser His Asp Lys His Cys His Lys

655 660 665

aag tac ctg tcg gtg cag gcc ctg gaa gcc cct cgg ctc acg cag aac 2068

Lys Tyr Leu Ser Val Gln Ala Leu Glu Ala Pro Arg Leu Thr Gln Asn

670 675 680

ttg acc gac ctc ctg gtg aac gtg agc gac tcg ctg gag atg cag tgc 2116

Leu Thr Asp Leu Leu Val Asn Val Ser Asp Ser Leu Glu Met Gln Cys

685 690 695

ttg gtg gcc gga gcg cac gcg ccc agc atc gtg tgg tac aaa gac gag 2164

Leu Val Ala Gly Ala His Ala Pro Ser Ile Val Trp Tyr Lys Asp Glu

700 705 710 715

agg ctg ctg gag gaa aag tct gga gtc gac ttg gcg gac tcc aac cag 2212

Arg Leu Leu Glu Glu Lys Ser Gly Val Asp Leu Ala Asp Ser Asn Gln

720 725 730

aag ctg agc atc cag cgc gtg cgc gag gag gat gcg gga cgc tat ctg 2260

Lys Leu Ser Ile Gln Arg Val Arg Glu Glu Asp Ala Gly Arg Tyr Leu

735 740 745

tgc agc gtg tgc aac gcc aag ggc tgc gtc aac tcc tcc gcc agc gtg 2308

Cys Ser Val Cys Asn Ala Lys Gly Cys Val Asn Ser Ser Ala Ser Val

750 755 760

gcc gtg gaa ggc tcc gag gat aag ggc agc atg gag atc gtg atc ctt 2356

Ala Val Glu Gly Ser Glu Asp Lys Gly Ser Met Glu Ile Val Ile Leu

765 770 775

gtc ggt acc ggc gtc atc gct gtc ttc ttc tgg gtc ctc ctc ctc ctc 2404

Val Gly Thr Gly Val Ile Ala Val Phe Phe Trp Val Leu Leu Leu Leu

780 785 790 795

atc ttc tgt aac atg agg agg ccg gcc cac gca gac atc aag acg ggc 2452

Ile Phe Cys Asn Met Arg Arg Pro Ala His Ala Asp Ile Lys Thr Gly

800 805 810

tac ctg tcc atc atc atg gac ccc ggg gag gtg cct ctg gag gag caa 2500

Tyr Leu Ser Ile Ile Met Asp Pro Gly Glu Val Pro Leu Glu Glu Gln

815 820 825

tgc gaa tac ctg tcc tac gat gcc agc cag tgg gaa ttc ccc cga gag 2548

Cys Glu Tyr Leu Ser Tyr Asp Ala Ser Gln Trp Glu Phe Pro Arg Glu

830 835 840

cgg ctg cac ctg ggg aga gtg ctc ggc tac ggc gcc ttc ggg aag gtg 2596

Arg Leu His Leu Gly Arg Val Leu Gly Tyr Gly Ala Phe Gly Lys Val

845 850 855

gtg gaa gcc tcc gct ttc ggc atc cac aag ggc agc agc tgt gac acc 2644

Val Glu Ala Ser Ala Phe Gly Ile His Lys Gly Ser Ser Cys Asp Thr

860 865 870 875

gtg gcc gtg aaa atg ctg aaa gag ggc gcc acg gcc agc gag cac cgc 2692

Val Ala Val Lys Met Leu Lys Glu Gly Ala Thr Ala Ser Glu His Arg

880 885 890

gcg ctg atg tcg gag ctc aag atc ctc att cac atc ggc aac cac ctc 2740

Ala Leu Met Ser Glu Leu Lys Ile Leu Ile His Ile Gly Asn His Leu

895 900 905

aac gtg gtc aac ctc ctc ggg gcg tgc acc aag ccg cag ggc ccc ctc 2788

Asn Val Val Asn Leu Leu Gly Ala Cys Thr Lys Pro Gln Gly Pro Leu

910 915 920

atg gtg atc gtg gag ttc tgc aag tac ggc aac ctc tcc aac ttc ctg 2836

Met Val Ile Val Glu Phe Cys Lys Tyr Gly Asn Leu Ser Asn Phe Leu

925 930 935

cgc gcc aag cgg gac gcc ttc agc ccc tgc gcg gag aag tct ccc gag 2884

Arg Ala Lys Arg Asp Ala Phe Ser Pro Cys Ala Glu Lys Ser Pro Glu

940 945 950 955

cag cgc gga cgc ttc cgc gcc atg gtg gag ctc gcc agg ctg gat cgg 2932

Gln Arg Gly Arg Phe Arg Ala Met Val Glu Leu Ala Arg Leu Asp Arg

960 965 970

agg cgg ccg ggg agc agc gac agg gtc ctc ttc gcg cgg ttc tcg aag 2980

Arg Arg Pro Gly Ser Ser Asp Arg Val Leu Phe Ala Arg Phe Ser Lys

975 980 985

acc gag ggc gga gcg agg cgg gct tct cca gac caa gaa gct gag gac 3028

Thr Glu Gly Gly Ala Arg Arg Ala Ser Pro Asp Gln Glu Ala Glu Asp

990 995 1000

ctg tgg ctg agc ccg ctg acc atg gaa gat ctt gtc tgc tac agc ttc 3076

Leu Trp Leu Ser Pro Leu Thr Met Glu Asp Leu Val Cys Tyr Ser Phe

1005 1010 1015

cag gtg gcc aga ggg atg gag ttc ctg gct tcc cga aag tgc atc cac 3124

Gln Val Ala Arg Gly Met Glu Phe Leu Ala Ser Arg Lys Cys Ile His

1020 1025 1030 1035

aga gac ctg gct gct cgg aac att ctg ctg tcg gaa agc gac gtg gtg 3172

Arg Asp Leu Ala Ala Arg Asn Ile Leu Leu Ser Glu Ser Asp Val Val

1040 1045 1050

aag atc tgt gac ttt ggc ctt gcc cgg gac atc tac aaa gac cct gac 3220

Lys Ile Cys Asp Phe Gly Leu Ala Arg Asp Ile Tyr Lys Asp Pro Asp

1055 1060 1065

tac gtc cgc aag ggc agt gcc cgg ctg ccc ctg aag tgg atg gcc cct 3268

Tyr Val Arg Lys Gly Ser Ala Arg Leu Pro Leu Lys Trp Met Ala Pro

1070 1075 1080

gaa agc atc ttc gac aag gtg tac acc acg cag agt gac gtg tgg tcc 3316

Glu Ser Ile Phe Asp Lys Val Tyr Thr Thr Gln Ser Asp Val Trp Ser

1085 1090 1095

ttt ggg gtg ctt ctc tgg gag atc ttc tct ctg ggg gcc tcc ccg tac 3364

Phe Gly Val Leu Leu Trp Glu Ile Phe Ser Leu Gly Ala Ser Pro Tyr

1100 1105 1110 1115

cct ggg gtg cag atc aat gag gag ttc tgc cag cgg ctg aga gac ggc 3412

Pro Gly Val Gln Ile Asn Glu Glu Phe Cys Gln Arg Leu Arg Asp Gly

1120 1125 1130

aca agg atg agg gcc ccg gag ctg gcc act ccc gcc ata cgc cgc atc 3460

Thr Arg Met Arg Ala Pro Glu Leu Ala Thr Pro Ala Ile Arg Arg Ile

1135 1140 1145

atg ctg aac tgc tgg tcc gga gac ccc aag gcg aga cct gca ttc tcg 3508

Met Leu Asn Cys Trp Ser Gly Asp Pro Lys Ala Arg Pro Ala Phe Ser

1150 1155 1160

gag ctg gtg gag atc ctg ggg gac ctg ctc cag ggc agg ggc ctg caa 3556

Glu Leu Val Glu Ile Leu Gly Asp Leu Leu Gln Gly Arg Gly Leu Gln

1165 1170 1175

gag gaa gag gag gtc tgc atg gcc ccg cgc agc tct cag agc tca gaa 3604

Glu Glu Glu Glu Val Cys Met Ala Pro Arg Ser Ser Gln Ser Ser Glu

1180 1185 1190 1195

gag ggc agc ttc tcg cag gtg tcc acc atg gcc cta cac atc gcc cag 3652

Glu Gly Ser Phe Ser Gln Val Ser Thr Met Ala Leu His Ile Ala Gln

1200 1205 1210

gct gac gct gag gac agc ccg cca agc ctg cag cgc cac agc ctg gcc 3700

Ala Asp Ala Glu Asp Ser Pro Pro Ser Leu Gln Arg His Ser Leu Ala

1215 1220 1225

gcc agg tat tac aac tgg gtg tcc ttt ccc ggg tgc ctg gcc aga ggg 3748

Ala Arg Tyr Tyr Asn Trp Val Ser Phe Pro Gly Cys Leu Ala Arg Gly

1230 1235 1240

gct gag acc cgt ggt tcc tcc agg atg aag aca ttt gag gaa ttc ccc 3796

Ala Glu Thr Arg Gly Ser Ser Arg Met Lys Thr Phe Glu Glu Phe Pro

1245 1250 1255

atg acc cca acg acc tac aaa ggc tct gtg gac aac cag aca gac agt 3844

Met Thr Pro Thr Thr Tyr Lys Gly Ser Val Asp Asn Gln Thr Asp Ser

1260 1265 1270 1275

ggg atg gtg ctg gcc tcg gag gag ttt gag cag ata gag agc agg cat 3892

Gly Met Val Leu Ala Ser Glu Glu Phe Glu Gln Ile Glu Ser Arg His

1280 1285 1290

aga caa gaa agc ggc ttc agg tagctgaagc agagagagag aaggcagcat 3943

Arg Gln Glu Ser Gly Phe Arg

1295

acgtcagcat tttcttctct gcacttataa gaaagatcaa agactttaag actttcgcta 4003

tttcttctac tgctatctac tacaaacttc aaagaggaac caggaggaca agaggagcat 4063

gaaagtggac aaggagtgtg accactgaag caccacaggg aaggggttag gcctccggat 4123

gactgcgggc aggcctggat aatatccagc ctcccacaag aagctggtgg agcagagtgt 4183

tccctgactc ct 4195

<210>2

<211>1298

<212>PRT

<213> human (Homo sapiens)

<400>2

Met Gln Arg Gly Ala Ala Leu Cys Leu Arg Leu Trp Leu Cys Leu Gly

1 5 10 15

Leu Leu Asp Gly Leu Val Ser Gly Tyr Ser Met Thr Pro Pro Thr Leu

20 25 30

Ash Ile Thr Glu Glu Ser His Val Ile Asp Thr Gly Asp Ser Leu Ser

35 40 45

Ile Ser Cys Arg Gly Gln His Pro Leu Glu Trp Ala Trp Pro Gly Ala

50 55 60

Gln Glu Ala Pro Ala Thr Gly Asp Lys Asp Ser Glu Asp Thr Gly Val

65 70 75 80

Val Arg Asp Cys Glu Gly Thr Asp Ala Arg Pro Tyr Cys Lys Val Leu

85 90 95

Leu Leu His Glu Val His Ala Asn Asp Thr Gly Ser Tyr Val Cys Tyr

100 105 110

Tyr Lys Tyr Ile Lys Ala Arg Ile Glu Gly Thr Thr Ala Ala Ser Ser

115 120 125

Tyr Val Phe Val Arg Asp Phe Glu Gln Pro Phe Ile Asn Lys Pro Asp

130 135 140

Thr Leu Leu Val Asn Arg Lys Asp Ala Met Trp Val Pro Cys Leu Val

145 150 155 160

Ser Ile Pro Gly Leu Asn Val Thr Leu Arg Ser Gln Ser Ser Val Leu

165 170 175

Trp Pro Asp Gly Gln Glu Val Val Trp Asp Asp Arg Arg Gly Met Leu

180 185 190

Val Ser Thr Pro Leu Leu His Asp Ala Leu Tyr Leu Gln Cys Glu Thr

195 200 205

Thr Trp Gly Asp Gln Asp Phe Leu Ser Asn Pro Phe Leu Val His Ile

210 215 220

Thr Gly Asn Glu Leu Tyr Asp Ile Gln Leu Leu Pro Arg Lys Ser Leu

225 230 235 240

Glu Leu Leu Val Gly Glu Lys Leu Val Leu Asn Cys Thr Val Trp Ala

245 250 255

Glu Phe Asn Ser Gly Val Thr Phe Asp Trp Asp Tyr Pro Gly Lys Gln

260 265 270

Ala Glu Arg Gly Lys Trp Val Pro Glu Arg Arg Ser Gln Gln Thr His

275 280 285

Thr Glu Leu Ser Ser Ile Leu Thr Ile His Asn Val Ser Gln His Asp

290 295 300

Leu Gly Ser Tyr Val Cys Lys Ala Asn Asn Gly Ile Gln Arg Phe Arg

305 310 315 320

Glu Ser Thr Glu Val Ile Val His Glu Asn Pro Phe Ile Ser Val Glu

325 330 335

Trp Leu Lys Gly Pro Ile Leu Glu Ala Thr Ala Gly Asp Glu Leu Val

340 345 350

Lys Leu Pro Val Lys Leu Ala Ala Tyr Pro Pro Pro Glu Phe Gln Trp

355 360 365

Tyr Lys Asp Gly Lys Ala Leu Ser Gly Arg His Ser Pro His Ala Leu

370 375 380

Val Leu Lys Glu Val Thr Glu Ala Ser Thr Gly Thr Tyr Thr Leu Ala

385 390 395 400

Leu Trp Asn Ser Ala Ala Gly Leu Arg Arg Asn Ile Ser Leu Glu Leu

405 410 415

Val Val Asn Val Pro Pro Gln Ile His Glu Lys Glu Ala Ser Ser Pro

420 425 430

Ser Ile Tyr Ser Arg His Ser Arg Gln Ala Leu Thr Cys Thr Ala Tyr

435 440 445

Gly Val Pro Leu Pro Leu Ser Ile Gln Trp His Trp Arg Pro Trp Thr

450 455 460

Pro Cys Lys Met Phe Ala Gln Arg Ser Leu Arg Arg Arg Gln Gln Gln

465 470 475 480

Asp Leu Met Pro Gln Cys Arg Asp Trp Arg Ala Val Thr Thr Gln Asp

485 490 495

Ala Val Asn Pro Ile Glu Ser Leu Asp Thr Trp Thr Glu Phe Val Glu

500 505 510

Gly Lys Asn Lys Thr Val Ser Lys Leu Val Ile Gln Asn Ala Asn Val

515 520 525

Ser Ala Met Tyr Lys Cys Val Val Ser Asn Lys Val Gly Gln Asp Glu

530 535 540

Arg Leu Ile Tyr Phe Tyr Val Thr Thr Ile Pro Asp Gly Phe Thr Ile

545 550 555 560

Glu Ser Lys Pro Ser Glu Glu Leu Leu Glu Gly Gln Pro Val Leu Leu

565 570 575

Ser Cys Gln Ala Asp Ser Tyr Lys Tyr Glu His Leu Arg Trp Tyr Arg

580 585 590

Leu Asn Leu Ser Thr Leu His Asp Ala His Gly Asn Pro Leu Leu Leu

595 600 605

Asp Cys Lys Asn Val His Leu Phe Ala Thr Pro Leu Ala Ala Ser Leu

610 615 620

Glu Glu Val Ala Pro Gly Ala Arg His Ala Thr Leu Ser Leu Ser Ile

625 630 635 640

Pro Arg Val Ala Pro Glu His Glu Gly His Tyr Val Cys Glu Val Gln

645 650 655

Asp Arg Arg Ser His Asp Lys His Cys His Lys Lys Tyr Leu Ser Val

660 665 670

Gln Ala Leu Glu Ala Pro Arg Leu Thr Gln Asn Leu Thr Asp Leu Leu

675 680 685

Val Asn Val Ser Asp Ser Leu Glu Met Gln Cys Leu Val Ala Gly Ala

690 695 700

His Ala Pro Ser Ile Val Trp Tyr Lys Asp Glu Arg Leu Leu Glu Glu

705 710 715 720

Lys Ser Gly Val Asp Leu Ala Asp Ser Asn Gln Lys Leu Ser Ile Gln

725 730 735

Arg Val Arg Glu Glu Asp Ala Gly Arg Tyr Leu Cys Ser Val Cys Asn

740 745 750

Ala Lys Gly Cys Val Asn Ser Ser Ala Ser Val Ala Val Glu Gly Ser

755 760 765

Glu Asp Lys Gly Ser Met Glu Ile Val Ile Leu Val Gly Thr Gly Val

770 775 780

Ile Ala Val Phe Phe Trp Val Leu Leu Leu Leu Ile Phe Cys Asn Met

785 790 795 800

Arg Arg Pro Ala His Ala Asp Ile Lys Thr Gly Tyr Leu Ser Ile Ile

805 810 815

Met Asp Pro Gly Glu Val Pro Leu Glu Glu Gln Cys Glu Tyr Leu Ser

820 825 830

Tyr Asp Ala Ser Gln Trp Glu Phe Pro Arg Glu Arg Leu His Leu Gly

835 840 845

Arg Val Leu Gly Tyr Gly Ala Phe Gly Lys Val Val Glu Ala Ser Ala

850 855 860

Phe Gly Ile His Lys Gly Ser Ser Cys Asp Thr Val Ala Val Lys Met

865 870 875 880

Leu Lys Glu Gly Ala Thr Ala Ser Glu His Arg Ala Leu Met Ser Glu

885 890 895

Leu Lys Ile Leu Ile His Ile Gly Asn His Leu Asn Val Val Asn Leu

900 905 910

Leu Gly Ala Cys Thr Lys Pro Gln Gly Pro Leu Met Val Ile Val Glu

915 920 925

Phe Cys Lys Tyr Gly Asn Leu Ser Asn Phe Leu Arg Ala Lys Arg Asp

930 935 940

Ala Phe Ser Pro Cys Ala Glu Lys Ser Pro Glu Gln Arg Gly Arg Phe

945 950 955 960

Arg Ala Met Val Glu Leu Ala Arg Leu Asp Arg Arg Arg Pro Gly Ser

965 970 975

Ser Asp Arg Val Leu Phe Ala Arg Phe Ser Lys Thr Glu Gly Gly Ala

980 985 990

Arg Arg Ala Ser Pro Asp Gln Glu Ala Glu Asp Leu Trp Leu Ser Pro

995 1000 1005

Leu Thr Met Glu Asp Leu Val Cys Tyr Ser Phe Gln Val Ala Arg Gly

1010 1015 1020

Met Glu Phe Leu Ala Ser Arg Lys Cys Ile His Arg Asp Leu Ala Ala

025 1030 1035 1040

Arg Asn Ile Leu Leu Ser Glu Ser Asp Val Val Lys Ile Cys Asp Phe

1045 1050 1055

Gly Leu Ala Arg Asp Ile Tyr Lys Asp Pro Asp Tyr Val Arg Lys Gly

1060 1065 1070

Ser Ala Arg Leu Pro Leu Lys Trp Met Ala Pro Glu Ser Ile Phe Asp

1075 1080 1085

Lys Val Tyr Thr Thr Gln Ser Asp Val Trp Ser Phe Gly Val Leu Leu

1090 1095 1100

Trp Glu Ile Phe Ser Leu Gly Ala Ser Pro Tyr Pro Gly Val Gln Ile

105 1110 1115 1120

Asn Glu Glu Phe Cys Gln Arg Leu Arg Asp Gly Thr Arg Met Arg Ala

1125 1130 1135

Pro Glu Leu Ala Thr Pro Ala Ile Arg Arg Ile Met Leu Asn Cys Trp

1140 1145 1150

Ser Gly Asp Pro Lys Ala Arg Pro Ala Phe Ser Glu Leu Val Glu Ile

1155 1160 1165

Leu Gly Asp Leu Leu Gln Gly Arg Gly Leu Gln Glu Glu Glu Glu Val

1170 1175 1180

Cys Met Ala Pro Arg Ser Ser Gln Ser Ser Glu Glu Gly Ser Phe Ser185 1190 1195 1200

Gln Val Ser Thr Met Ala Leu His Ile Ala Gln Ala Asp Ala Glu Asp

1205 1210 1215

Ser Pro Pro Ser Leu Gln Arg His Ser Leu Ala Ala Arg Tyr Tyr Asn

1220 1225 1230

Trp Val Ser Phe Pro Gly Cys Leu Ala Arg Gly Ala Glu Thr Arg Gly

1235 1240 1245

Ser Ser Arg Met Lys Thr Phe Glu Glu Phe Pro Met Thr Pro Thr Thr

1250 1255 1260

Tyr Lys Gly Ser Val Asp Asn Gln Thr Asp Ser Gly Met Val Leu Ala

265 1270 1275 1280

Ser Glu Glu Phe Glu Gln Ile Glu Ser Arg His Arg Gln Glu Ser Gly

1285 1290 1295

Phe Arg

<210>3

<211>4795

<212>DNA

<213> human (Homo sapiens)

<220>

<221>CDS

<222>(20)..(4108)

<400>3

ccacgcgcag cggccggag atg cag cgg ggc gcc gcg ctg tgc ctg cga ctg 52

Met Gln Arg Gly Ala Ala Leu Cys Leu Arg Leu

1 5 10

tgg ctc tgc ctg gga ctc ctg gac ggc ctg gtg agt ggc tac tcc atg 100

Trp Leu Cys Leu Gly Leu Leu Asp Gly Leu Val Ser Gly Tyr Ser Met

15 20 25

acc ccc ccg acc ttg aac atc acg gag gag tca cac gtc atc gac acc 148

Thr Pro Pro Thr Leu Asn Ile Thr Glu Glu Ser His Val Ile Asp Thr

30 35 40

ggt gac agc ctg tcc atc tcc tgc agg gga cag cac ccc ctc gag tgg 196

Gly Asp Ser Leu Ser Ile Ser Cys Arg Gly Gln His Pro Leu Glu Trp

45 50 55

gct tgg cca gga gct cag gag gcg cca gcc acc gga gac aag gac agc 244

Ala Trp Pro Gly Ala Gln Glu Ala Pro Ala Thr Gly Asp Lys Asp Ser

60 65 70 75

gag gac acg ggg gtg gtg cga gac tgc gag ggc aca gac gcc agg ccc 292

Glu Asp Thr Gly Val Val Arg Asp Cys Glu Gly Thr Asp Ala Arg Pro

80 85 90

tac tgc aag gtg ttg ctg ctg cac gag gta cat gcc aac gac aca ggc 340

Tyr Cys Lys Val Leu Leu Leu His Glu Val His Ala Asn Asp Thr Gly

95 100 105

agc tac gtc tgc tac tac aag tac atc aag gca cgc atc gag ggc acc 388

Ser Tyr Val Cys Tyr Tyr Lys Tyr Ile Lys Ala Arg Ile Glu Gly Thr

110 115 120

acg gcc gcc agc tcc tac gtg ttc gtg aga gac ttt gag cag cca ttc 436

Thr Ala Ala Ser Ser Tyr Val Phe Val Arg Asp Phe Glu Gln Pro Phe

125 130 135

atc aac aag cct gac acg ctc ttg gtc aac agg aag gac gcc atg tgg 484

Ile Asn Lys Pro Asp Thr Leu Leu Val Asn Arg Lys Asp Ala Met Trp

140 145 150 155

gtg ccc tgt ctg gtg tcc atc ccc ggc ctc aat gtc acg ctg cgc tcg 532

Val Pro Cys Leu Val Ser Ile Pro Gly Leu Asn Val Thr Leu Arg Ser

160 165 170

caa agc tcg gtg ctg tgg cca gac ggg cag gag gtg gtg tgg gat gac 580

Gln Ser Ser Val Leu Trp Pro Asp Gly Gln Glu Val Val Trp Asp Asp

175 180 185

cgg cgg ggc atg ctc gtg tcc acg cca ctg ctg cac gat gcc ctg tac 628

Arg Arg Gly Met Leu Val Ser Thr Pro Leu Leu His Asp Ala Leu Tyr

190 195 200

ctg cag tgc gag acc acc tgg gga gac cag gac ttc ctt tcc aac ccc 676

Leu Gln Cys Glu Thr Thr Trp Gly Asp Gln Asp Phe Leu Ser Asn Pro

205 210 215

ttc ctg gtg cac atc aca ggc aac gag ctc tat gac atc cag ctg ttg 724

Phe Leu Val His Ile Thr Gly Asn Glu Leu Tyr Asp Ile Gln Leu Leu

220 225 230 235

ccc agg aag tcg ctg gag ctg ctg gta ggg gag aag ctg gtc ctg aac 772

Pro Arg Lys Ser Leu Glu Leu Leu Val Gly Glu Lys Leu Val Leu Asn

240 245 250

tgc acc gtg tgg gct gag ttt aac tca ggt gtc acc ttt gac tgg gac 820

Cys Thr Val Trp Ala Glu Phe Asn Ser Gly Val Thr Phe Asp Trp Asp

255 260 265

tac cca ggg aag cag gca gag cgg ggt aag tgg gtg ccc gag cga cgc 868

Tyr Pro Gly Lys Gln Ala Glu Arg Gly Lys Trp Val Pro Glu Arg Arg

270 275 280

tcc cag cag acc cac aca gaa ctc tcc agc atc ctg acc atc cac aac 916

Ser Gln Gln Thr His Thr Glu Leu Ser Ser Ile Leu Thr Ile His Asn

285 290 295

gtc agc cag cac gac ctg ggc tcg tat gtg tgc aag gcc aac aac ggc 964

Val Ser Gln His Asp Leu Gly Ser Tyr Val Cys Lys Ala Asn Asn Gly

300 305 310 315

atc cag cga ttt cgg gag agc acc gag gtc att gtg cat gaa aat ccc 1012

Ile Gln Arg Phe Arg Glu Ser Thr Glu Val Ile Val His Glu Asn Pro

320 325 330

ttc atc agc gtc gag tgg ctc aaa gga ccc atc ctg gag gcc acg gca 1060

Phe Ile Ser Val Glu Trp Leu Lys Gly Pro Ile Leu Glu Ala Thr Ala

335 340 345

gga gac gag ctg gtg aag ctg ccc gtg aag ctg gca gcg tac ccc ccg 1108

Gly Asp Glu Leu Val Lys Leu Pro Val Lys Leu Ala Ala Tyr Pro Pro

350 355 360

ccc gag ttc cag tgg tac aag gat gga aag gca ctg tcc ggg cgc cac 1156

Pro Glu Phe Gln Trp Tyr Lys Asp Gly Lys Ala Leu Ser Gly Arg His

365 370 375

agt cca cat gcc ctg gtg ctc aag gag gtg aca gag gcc agc aca ggc 1204

Ser Pro His Ala Leu Val Leu Lys Glu Val Thr Glu Ala Ser Thr Gly

380 385 390 395

acc tac acc ctc gcc ctg tgg aac tcc gct gct ggc ctg agg cgc aac 1252

Thr Tyr Thr Leu Ala Leu Trp Asn Ser Ala Ala Gly Leu Arg Arg Asn

400 405 410

atc agc ctg gag ctg gtg gtg aat gtg ccc ccc cag ata cat gag aag 1300

Ile Ser Leu Glu Leu Val Val Asn Val Pro Pro Gln Ile His Glu Lys

415 420 425

gag gcc tcc tcc ccc agc atc tac tcg cgt cac agc cgc cag gcc ctc 1348

Glu Ala Ser Ser Pro Ser Ile Tyr Ser Arg His Ser Arg Gln Ala Leu

430 435 440

acc tgc acg gcc tac ggg gtg ccc ctg cct ctc agc atc cag tgg cac 1396

Thr Cys Thr Ala Tyr Gly Val Pro Leu Pro Leu Ser Ile Gln Trp His

445 450 455

tgg cgg ccc tgg aca ccc tgc aag atg ttt gcc cag cgt agt ctc cgg 1444

Trp Arg Pro Trp Thr Pro Cys Lys Met Phe Ala Gln Arg Ser Leu Arg

460 465 470 475

cgg cgg cag cag caa gac ctc atg cca cag tgc cgt gac tgg agg gcg 1492

Arg Arg Gln Gln Gln Asp Leu Met Pro Gln Cys Arg Asp Trp Arg Ala

480 485 490

gtg acc acg cag gat gcc gtg aac ccc atc gag agc ctg gac acc tgg 1540

Val Thr Thr Gln Asp Ala Val Asn Pro Ile Glu Ser Leu Asp Thr Trp

495 500 505

acc gag ttt gtg gag gga aag aat aag act gtg agc aag ctg gtg atc 1588

Thr Glu Phe Val Glu Gly Lys Asn Lys Thr Val Ser Lys Leu Val Ile

510 515 520

cag aat gcc aac gtg tct gcc atg tac aag tgt gtg gtc tcc aac aag 1636

Gln Asn Ala Asn Val Ser Ala Met Tyr Lys Cys Val Val Ser Asn Lys

525 530 535

gtg ggc cag gat gag cgg ctc atc tac ttc tat gtg acc acc atc ccc 1684

Val Gly Gln Asp Glu Arg Leu Ile Tyr Phe Tyr Val Thr Thr Ile Pro

540 545 550 555

gac ggc ttc acc atc gaa tcc aag cca tcc gag gag cta cta gag ggc 1732

Asp Gly Phe Thr Ile Glu Ser Lys Pro Ser Glu Glu Leu Leu Glu Gly

560 565 570

cag ccg gtg ctc ctg agc tgc caa gcc gac agc tac aag tac gag cat 1780

Gln Pro Val Leu Leu Ser Cys Gln Ala Asp Ser Tyr Lys Tyr Glu His

575 580 585

ctg cgc tgg tac cgc ctc aac ctg tcc acg ctg cac gat gcg cac ggg 1828

Leu Arg Trp Tyr Arg Leu Asn Leu Ser Thr Leu His Asp Ala His Gly

590 595 600

aac ccg ctt ctg ctc gac tgc aag aac gtg cat ctg ttc gcc acc cct 1876

Asn Pro Leu Leu Leu Asp Cys Lys Asn Val His Leu Phe Ala Thr Pro

605 610 615

ctg gcc gcc agc ctg gag gag gtg gca cct ggg gcg cgc cac gcc acg 1924

Leu Ala Ala Ser Leu Glu Glu Val Ala Pro Gly Ala Arg His Ala Thr

620 625 630 635

ctc agc ctg agt atc ccc cgc gtc gcg ccc gag cac gag ggc cac tat 1972

Leu Ser Leu Ser Ile Pro Arg Val Ala Pro Glu His Glu Gly His Tyr

640 645 650

gtg tgc gaa gtg caa gac cgg cgc agc cat gac aag cac tgc cac aag 2020

Val Cys Glu Val Gln Asp Arg Arg Ser His Asp Lys His Cys His Lys

655 660 665

aag tac ctg tcg gtg cag gcc ctg gaa gcc cct cgg ctc acg cag aac 2068

Lys Tyr Leu Ser Val Gln Ala Leu Glu Ala Pro Arg Leu Thr Gln Asn

670 675 680

ttg acc gac ctc ctg gtg aac gtg agc gac tcg ctg gag atg cag tgc 2116

Leu Thr Asp Leu Leu Val Asn Val Ser Asp Ser Leu Glu Met Gln Cys

685 690 695

ttg gtg gcc gga gcg cac gcg ccc agc atc gtg tgg tac aaa gac gag 2164

Leu Val Ala Gly Ala His Ala Pro Ser Ile Val Trp Tyr Lys Asp Glu

700 705 710 715

agg ctg ctg gag gaa aag tct gga gtc gac ttg gcg gac tcc aac cag 2212

Arg Leu Leu Glu Glu Lys Ser Gly Val Asp Leu Ala Asp Ser Asn Gln

720 725 730

aag ctg agc atc cag cgc gtg cgc gag gag gat gcg gga cgc tat ctg 2260

Lys Leu Ser Ile Gln Arg Val Arg Glu Glu Asp Ala Gly Arg Tyr Leu

735 740 745

tgc agc gtg tgc aac gcc aag ggc tgc gtc aac tcc tcc gcc agc gtg 2308

Cys Ser Val Cys Asn Ala Lys Gly Cys Val Asn Ser Ser Ala Ser Val

750 755 760

gcc gtg gaa ggc tcc gag gat aag ggc agc atg gag atc gtg atc ctt 2356

Ala Val Glu Gly Ser Glu Asp Lys Gly Ser Met Glu Ile Val Ile Leu

765 770 775

gtc ggt acc ggc gtc atc gct gtc ttc ttc tgg gtc ctc ctc ctc ctc 2404

Val Gly Thr Gly Val Ile Ala Val Phe Phe Trp Val Leu Leu Leu Leu

780 785 790 795

atc ttc tgt aac atg agg agg ccg gcc cac gca gac atc aag acg ggc 2452

Ile Phe Cys Asn Met Arg Arg Pro Ala His Ala Asp Ile Lys Thr Gly

800 805 810

tac ctg tcc atc atc atg gac ccc ggg gag gtg cct ctg gag gag caa 2500

Tyr Leu Ser Ile Ile Met Asp Pro Gly Glu Val Pro Leu Glu Glu Gln

815 820 825

tgc gaa tac ctg tcc tac gat gcc agc cag tgg gaa ttc ccc cga gag 2548

Cys Glu Tyr Leu Ser Tyr Asp Ala Ser Gln Trp Glu Phe Pro Arg Glu

830 835 840

cgg ctg cac ctg ggg aga gtg ctc ggc tac ggc gcc ttc ggg aag gtg 2596

Arg Leu His Leu Gly Arg Val Leu Gly Tyr Gly Ala Phe Gly Lys Val

845 850 855

gtg gaa gcc tcc gct ttc ggc atc cac aag ggc agc agc tgt gac acc 2644

Val Glu Ala Ser Ala Phe Gly Ile His Lys Gly Ser Ser Cys Asp Thr

860 865 870 875

gtg gcc gtg aaa atg ctg aaa gag ggc gcc acg gcc agc gag cac cgc 2692

Val Ala Val Lys Met Leu Lys Glu Gly Ala Thr Ala Ser Glu His Arg

880 885 890

gcg ctg atg tcg gag ctc aag atc ctc att cac atc ggc aac cac ctc 2740

Ala Leu Met Ser Glu Leu Lys Ile Leu Ile His Ile Gly Asn His Leu

895 900 905

aac gtg gtc aac ctc ctc ggg gcg tgc acc aag ccg cag ggc ccc ctc 2788

Asn Val Val Asn Leu Leu Gly Ala Cys Thr Lys Pro Gln Gly Pro Leu

910 915 920

atg gtg atc gtg gag ttc tgc aag tac ggc aac ctc tcc aac ttc ctg 2836

Met Val Ile Val Glu Phe Cys Lys Tyr Gly Asn Leu Ser Asn Phe Leu

925 930 935

cgc gcc aag cgg gac gcc ttc agc ccc tgc gcg gag aag tct ccc gag 2884

Arg Ala Lys Arg Asp Ala Phe Ser Pro Cys Ala Glu Lys Ser Pro Glu

940 945 950 955

cag cgc gga cgc ttc cgc gcc atg gtg gag ctc gcc agg ctg gat cgg 2932

Gln Arg Gly Arg Phe Arg Ala Met Val Glu Leu Ala Arg Leu Asp Arg

960 965 970

agg cgg ccg ggg agc agc gac agg gtc ctc ttc gcg cgg ttc tcg aag 2980

Arg Arg Pro Gly Ser Ser Asp Arg Val Leu Phe Ala Arg Phe Ser Lys

975 980 985

acc gag ggc gga gcg agg cgg gct tct cca gac caa gaa gct gag gac 3028

Thr Glu Gly Gly Ala Arg Arg Ala Ser Pro Asp Gln Glu Ala Glu Asp

990 995 1000

ctg tgg ctg agc ccg ctg acc atg gaa gat ctt gtc tgc tac agc ttc 3076

Leu Trp Leu Ser Pro Leu Thr Met Glu Asp Leu Val Cys Tyr Ser Phe

1005 1010 1015

cag gtg gcc aga ggg atg gag ttc ctg gct tcc cga aag tgc atc cac 3124

Gln Val Ala Arg Gly Met Glu Phe Leu Ala Ser Arg Lys Cys Ile His

1020 1025 1030 1035

aga gac ctg gct gct cgg aac att ctg ctg tcg gaa agc gac gtg gtg 3172

Arg Asp Leu Ala Ala Arg Asn Ile Leu Leu Ser Glu Ser Asp Val Val

1040 1045 1050

aag atc tgt gac ttt ggc ctt gcc cgg gac atc tac aaa gac cct gac 3220

Lys Ile Cys Asp Phe Gly Leu Ala Arg Asp Ile Tyr Lys Asp Pro Asp

1055 1060 1065

tac gtc cgc aag ggc agt gcc cgg ctg ccc ctg aag tgg atg gcc cct 3268

Tyr Val Arg Lys Gly Ser Ala Arg Leu Pro Leu Lys Trp Met Ala Pro

1070 1075 1080

gaa agc atc ttc gac aag gtg tac acc acg cag agt gac gtg tgg tcc 3316

Glu Ser Ile Phe Asp Lys Val Tyr Thr Thr Gln Ser Asp Val Trp Ser

1085 1090 1095

ttt ggg gtg ctt ctc tgg gag atc ttc tct ctg ggg gcc tcc ccg tac 3364

Phe Gly Val Leu Leu Trp Glu Ile Phe Ser Leu Gly Ala Ser Pro Tyr

1100 1105 1110 1115

cct ggg gtg cag atc aat gag gag ttc tgc cag cgg ctg aga gac ggc 3412

Pro Gly Val Gln Ile Asn Glu Glu Phe Cys Gln Arg Leu Arg Asp Gly

1120 1125 1130

aca agg atg agg gcc ccg gag ctg gcc act ccc gcc ata cgc cgc atc 3460

Thr Arg Met Arg Ala Pro Glu Leu Ala Thr Pro Ala Ile Arg Arg Ile

1135 1140 1145

atg ctg aac tgc tgg tcc gga gac ccc aag gcg aga cct gca ttc tcg 3508

Met Leu Asn Cys Trp Ser Gly Asp Pro Lys Ala Arg Pro Ala Phe Ser

1150 1155 1160

gag ctg gtg gag atc ctg ggg gac ctg ctc cag ggc agg ggc ctg caa 3556

Glu Leu Val Glu Ile Leu Gly Asp Leu Leu Gln Gly Arg Gly Leu Gln

1165 1170 1175

gag gaa gag gag gtc tgc atg gcc ccg cgc agc tct cag agc tca gaa 3604

Glu Glu Glu Glu Val Cys Met Ala Pro Arg Ser Ser Gln Ser Ser Glu

1180 1185 1190 1195

gag ggc agc ttc tcg cag gtg tcc acc atg gcc cta cac atc gcc cag 3652

Glu Gly Ser Phe Ser Gln Val Ser Thr Met Ala Leu His Ile Ala Gln

1200 1205 1210

gct gac gct gag gac agc ccg cca agc ctg cag cgc cac agc ctg gcc 3700

Ala Asp Ala Glu Asp Ser Pro Pro Ser Leu Gln Arg His Ser Leu Ala

1215 1220 1225

gcc agg tat tac aac tgg gtg tcc ttt ccc ggg tgc ctg gcc aga ggg 3748

Ala Arg Tyr Tyr Asn Trp Val Ser Phe Pro Gly Cys Leu Ala Arg Gly

1230 1235 1240

gct gag acc cgt ggt tcc tcc agg atg aag aca ttt gag gaa ttc ccc 3796

Ala Glu Thr Arg Gly Ser Ser Arg Met Lys Thr Phe Glu Glu Phe Pro

1245 1250 1255

atg acc cca acg acc tac aaa ggc tct gtg gac aac cag aca gac agt 3844

Met Thr Pro Thr Thr Tyr Lys Gly Ser Val Asp Asn Gln Thr Asp Ser

1260 1265 1270 1275

ggg atg gtg ctg gcc tcg gag gag ttt gag cag ata gag agc agg cat 3892

Gly Met Val Leu Ala Ser Glu Glu Phe Glu Gln Ile Glu Ser Arg His

1280 1285 1290

aga caa gaa agc ggc ttc agc tgt aaa gga cct ggc cag aat gtg gct 3940

Arg Gln Glu Ser Gly Phe Ser Cys Lys Gly Pro Gly Gln Asn Val Ala

1295 1300 1305

gtg acc agg gca cac cct gac tcc caa ggg agg cgg cgg cgg cct gag 3988

Val Thr Arg Ala His Pro Asp Ser Gln Gly Arg Arg Arg Arg Pro Glu

1310 1315 1320

cgg ggg gcc cga gga ggc cag gtg ttt tac aac agc gag tat ggg gag 4036

Arg Gly Ala Arg Gly Gly Gln Val Phe Tyr Asn Ser Glu Tyr Gly Glu

1325 1330 1335

ctg tcg gag cca agc gag gag gac cac tgc tcc ccg tct gcc cgc gtg 4084

Leu Ser Glu Pro Ser Glu Glu Asp His Cys Ser Pro Ser Ala Arg Val

1340 1345 1350 1355

act ttc ttc aca gac aac agc tac taagcagcat cggacaagac ccccagcact 4138

Thr Phe Phe Thr Asp Asn Ser Tyr

1360

tgggggttca ggcccggcag ggcgggcaga gggctggagg cccaggctgg gaactcatct 4198

ggttgaactc tggtggcaca ggagtgtcct cttccctctc tgcagacttc ccagctagga 4258

agagcaggac tccaggccca aggctcccgg aattccgtca ccacgactgg ccagggcacg 4318

ctccagctgc cccggcccct ccccctgaga ttcagatgtc atttagttca gcatccgcag 4378

gtgctggtcc cggggccagc acttccatgg gaatgtctct ttggcgacct cctttcatca 4438

cactgggtgg tggcctggtc cctgttttcc cacgaggaat ctgtgggtct gggagtcaca 4498

cagtgttgga ggttaaggca tacgagagca gaggtctccc aaacgccctt tcctcctcag 4558

gcacacagct actctcccca cgagggctgg ctggcctcac ccacccctgc acagttgaag 4618

ggaggggctg tgtttccatc tcaaagaagg catttgcagg gtcctcttct gggcctgacc 4678

aaacagccaa ctagcccctg gggtggccac cagtatgaca gtattatacg ctggcaacac 4738

agaggcagcc cgcacacctg cgcctgggtg ttgagagcca tcctgcaagt ctttttc 4795

<210>4

<211>1363

<212>PRT

<213> human (Homo sapiens)

<400>4

Met Gln Arg Gly Ala Ala Leu Cys Leu Arg Leu Trp Leu Cys Leu Gly

1 5 10 15

Leu Leu Asp Gly Leu Val Ser Gly Tyr Ser Met Thr Pro Pro Thr Leu

20 25 30

Asn Ile Thr Glu Glu Ser His Val Ile Asp Thr Gly Asp Ser Leu Ser

35 40 45

Ile Ser Cys Arg Gly Gln His Pro Leu Glu Trp Ala Trp Pro Gly Ala

50 55 60

Gln Glu Ala Pro Ala Thr Gly Asp Lys Asp Ser Glu Asp Thr Gly Val

65 70 75 80

Val Arg Asp Cys Glu Gly Thr Asp Ala Arg Pro Tyr Cys Lys Val Leu

85 90 95

Leu Leu His Glu Val His Ala Asn Asp Thr Gly Ser Tyr Val Cys Tyr

100 105 110

Tyr Lys Tyr Ile Lys Ala Arg Ile Glu Gly Thr Thr Ala Ala Ser Ser

115 120 125

Tyr Val Phe Val Arg Asp Phe Glu Gln Pro Phe Ile Asn Lys Pro Asp

130 135 140

Thr Leu Leu Val Asn Arg Lys Asp Ala Met Trp Val Pro Cys Leu Val

145 150 155 160

Ser Ile Pro Gly Leu Asn Val Thr Leu Arg Ser Gln Ser Ser Val Leu

165 170 175

Trp Pro Asp Gly Gln Glu Val Val Trp Asp Asp Arg Arg Gly Met Leu

180 185 190

Val Ser Thr Pro Leu Leu His Asp Ala Leu Tyr Leu Gln Cys Glu Thr

195 200 205

Thr Trp Gly Asp Gln Asp Phe Leu Ser Asn Pro Phe Leu Val His Ile

210 215 220

Thr Gly Asn Glu Leu Tyr Asp Ile Gln Leu Leu Pro Arg Lys Ser Leu

225 230 235 240

Glu Leu Leu Val Gly Glu Lys Leu Val Leu Asn Cys Thr Val Trp Ala

245 250 255

Glu Phe Asn Ser Gly Val Thr Phe Asp Trp Asp Tyr Pro Gly Lys Gln

260 265 270

Ala Glu Arg Gly Lys Trp Val Pro Glu Arg Arg Ser Gln Gln Thr His

275 280 285

Thr Glu Leu Ser Ser Ile Leu Thr Ile His Asn Val Ser Gln His Asp

290 295 300

Leu Gly Ser Tyr Val Cys Lys Ala Asn Asn Gly Ile Gln Arg Phe Arg

305 310 315 320

Glu Ser Thr Glu Val Ile Val His Glu Asn Pro Phe Ile Ser Val Glu

325 330 335

Trp Leu Lys Gly Pro Ile Leu Glu Ala Thr Ala Gly Asp Glu Leu Val

340 345 350

Lys Leu Pro Val Lys Leu Ala Ala Tyr Pro Pro Pro Glu Phe Gln Trp

355 360 365

Tyr Lys Asp Gly Lys Ala Leu Ser Gly Arg His Ser Pro His Ala Leu

370 375 380

Val Leu Lys Glu Val Thr Glu Ala Ser Thr Gly Thr Tyr Thr Leu Ala

385 390 395 400

Leu Trp Asn Ser Ala Ala Gly Leu Arg Arg Asn Ile Ser Leu Glu Leu

405 410 415

Val Val Asn Val Pro Pro Gln Ile His Glu Lys Glu Ala Ser Ser Pro

420 425 430

Ser Ile Tyr Ser Arg His Ser Arg Gln Ala Leu Thr Cys Thr Ala Tyr

435 440 445

Gly Val Pro Leu Pro Leu Ser Ile Gln Trp His Trp Arg Pro Trp Thr

450 455 460

Pro Cys Lys Met Phe Ala Gln Arg Ser Leu Arg Arg Arg Gln Gln Gln

465 470 475 480

Asp Leu Met Pro Gln Cys Arg Asp Trp Arg Ala Val Thr Thr Gln Asp

485 490 495

Ala Val Asn Pro Ile Glu Ser Leu Asp Thr Trp Thr Glu Phe Val Glu

500 505 510

Gly Lys Asn Lys Thr Val Ser Lys Leu Val Ile Gln Asn Ala Asn Val

515 520 525

Ser Ala Met Tyr Lys Cys Val Val Ser Asn Lys Val Gly Gln Asp Glu

530 535 540

Arg Leu Ile Tyr Phe Tyr Val Thr Thr Ile Pro Asp Gly Phe Thr Ile

545 550 555 560

Glu Ser Lys Pro Ser Glu Glu Leu Leu Glu Gly Gln Pro Val Leu Leu

565 570 575

Ser Cys Gln Ala Asp Ser Tyr Lys Tyr Glu His Leu Arg Trp Tyr Arg

580 585 590

Leu Asn Leu Ser Thr Leu His Asp Ala His Gly Asn Pro Leu Leu Leu

595 600 605

Asp Cys Lys Asn Val His Leu Phe Ala Thr Pro Leu Ala Ala Ser Leu

610 615 620

Glu Glu Val Ala Pro Gly Ala Arg His Ala Thr Leu Ser Leu Ser Ile

625 630 635 640

Pro Arg Val Ala Pro Glu His Glu Gly His Tyr Val Cys Glu Val Gln

645 650 655

Asp Arg Arg Ser His Asp Lys His Cys His Lys Lys Tyr Leu Ser Val

660 665 670

Gln Ala Leu Glu Ala Pro Arg Leu Thr Gln Asn Leu Thr Asp Leu Leu

675 680 685

Val Asn Val Ser Asp Ser Leu Glu Met Gln Cys Leu Val Ala Gly Ala

690 695 700

His Ala Pro Ser Ile Val Trp Tyr Lys Asp Glu Arg Leu Leu Glu Glu

705 710 715 720

Lys Ser Gly Val Asp Leu Ala Asp Ser Asn Gln Lys Leu Ser Ile Gln

725 730 735

Arg Val Arg Glu Glu Asp Ala Gly Arg Tyr Leu Cys Ser Val Cys Asn

740 745 750

Ala Lys Gly Cys Val Asn Ser Ser Ala Ser Val Ala Val Glu Gly Ser

755 760 765

Glu Asp Lys Gly Ser Met Glu Ile Val Ile Leu Val Gly Thr Gly Val

770 775 780

Ile Ala Val Phe Phe Trp Val Leu Leu Leu Leu Ile Phe Cys Asn Met

785 790 795 800

Arg Arg Pro Ala His Ala Asp Ile Lys Thr Gly Tyr Leu Ser Ile Ile

805 810 815

Met Asp Pro Gly Glu Val Pro Leu Glu Glu Gln Cys Glu Tyr Leu Ser

820 825 830

Tyr Asp Ala Ser Gln Trp Glu Phe Pro Arg Glu Arg Leu His Leu Gly

835 840 845

Arg Val Leu Gly Tyr Gly Ala Phe Gly Lys Val Val Glu Ala Ser Ala

850 855 860

Phe Gly Ile His Lys Gly Ser Ser Cys Asp Thr Val Ala Val Lys Met

865 870 875 880

Leu Lys Glu Gly Ala Thr Ala Ser Glu His Arg Ala Leu Met Ser Glu

885 890 895

Leu Lys Ile Leu Ile His Ile Gly Asn His Leu Asn Val Val Asn Leu

900 905 910

Leu Gly Ala Cys Thr Lys Pro Gln Gly Pro Leu Met Val Ile Val Glu

915 920 925

Phe Cys Lys Tyr Gly Asn Leu Ser Asn Phe Leu Arg Ala Lys Arg Asp

930 935 940

Ala Phe Ser Pro Cys Ala Glu Lys Ser Pro Glu Gln Arg Gly Arg Phe

945 950 955 960

Arg Ala Met Val Glu Leu Ala Arg Leu Asp Arg Arg Arg Pro Gly Ser

965 970 975

Ser Asp Arg Val Leu Phe Ala Arg Phe Ser Lys Thr Glu Gly Gly Ala

980 985 990

Arg Arg Ala Ser Pro Asp Gln Glu Ala Glu Asp Leu Trp Leu Ser Pro

995 1000 1005

Leu Thr Met Glu Asp Leu Val Cys Tyr Ser Phe Gln Val Ala Arg Gly

1010 1015 1020

Met Glu Phe Leu Ala Ser Arg Lys Cys Ile His Arg Asp Leu Ala Ala

025 1030 1035 1040

Arg Asn Ile Leu Leu Ser Glu Ser Asp Val Val Lys Ile Cys Asp Phe

1045 1050 1055

Gly Leu Ala Arg Asp Ile Tyr Lys Asp Pro Asp Tyr Val Arg Lys Gly

1060 1065 1070

Ser Ala Arg Leu Pro Leu Lys Trp Met Ala Pro Glu Ser Ile Phe Asp

1075 1080 1085

Lys Val Tyr Thr Thr Gln Ser Asp Val Trp Ser Phe Gly Val Leu Leu

1090 1095 1100

Trp Glu Ile Phe Ser Leu Gly Ala Ser Pro Tyr Pro Gly Val Gln Ile

105 1110 1115 1120

Asn Glu Glu Phe Cys Gln Arg Leu Arg Asp Gly Thr Arg Met Arg Ala

1125 1130 1135

Pro Glu Leu Ala Thr Pro Ala Ile Arg Arg Ile Met Leu Asn Cys Trp

1140 1145 1150

Ser Gly Asp Pro Lys Ala Arg Pro Ala Phe Ser Glu Leu Val Glu Ile

1155 1160 1165

Leu Gly Asp Leu Leu Gln Gly Arg Gly Leu Gln Glu Glu Glu Glu Val

1170 1175 1180

Cys Met Ala Pro Arg Ser Ser Gln Ser Ser Glu Glu Gly Ser Phe Ser

185 1190 1195 1200

Gln Val Ser Thr Met Ala Leu His Ile Ala Gln Ala Asp Ala Glu Asp

1205 1210 1215

Ser Pro Pro Ser Leu Gln Arg His Ser Leu Ala Ala Arg Tyr Tyr Asn

1220 1225 1230

Trp Val Ser Phe Pro Gly Cys Leu Ala Arg Gly Ala Glu Thr Arg Gly

1235 1240 1245

Ser Ser Arg Met Lys Thr Phe Glu Glu Phe Pro Met Thr Pro Thr Thr

1250 1255 1260

Tyr Lys Gly Ser Val Asp Asn Gln Thr Asp Ser Gly Met Val Leu Ala

265 1270 1275 1280

Ser Glu Glu Phe Glu Gln Ile Glu Ser Arg His Arg Gln Glu Ser Gly

1285 1290 1295

Phe Ser Cys Lys Gly Pro Gly Gln Asn Val Ala Val Thr Arg Ala His

1300 1305 1310

Pro Asp Ser Gln Gly Arg Arg Arg Arg Pro Glu Arg Gly Ala Arg Gly

1315 1320 1325

Gly Gln Val Phe Tyr Asn Ser Glu Tyr Gly Glu Leu Ser Glu Pro Ser

1330 1335 1340

Glu Glu Asp His Cys Ser Pro Ser Ala Arg Val Thr Phe Phe Thr Asp

345 1350 1355 1360

Asn Ser Tyr

<210>5

<211>1311

<212>PRT

<213> human (Homo sapiens) (FLT1)

<400>5

Met Val Ser Tyr Trp Asp Thr Gly Val Leu Leu Cys Ala Leu Leu Ser

1 5 10 15

Cys Leu Leu Leu Thr Gly Ser Ser Ser Gly Ser Lys Leu Lys Asp Pro

20 25 30

Glu Leu Ser Leu Lys Gly Thr Gln His Ile Met Gln Ala Gly Gln Thr

35 40 45

Leu His Leu Gln Cys Arg Gly Glu Ala Ala His Lys Trp Ser Leu Pro

50 55 60

Glu Asn Asn Asn Asn Asn Asn Met Val Ser Lys Glu Ser Glu Arg Leu

65 70 75 80

Ser Ile Thr Lys Ser Ala Cys Gly Arg Asn Gly Lys Gln Phe Cys Ser

85 90 95

Thr Leu Thr Leu Asn Thr Ala Gln Ala Asn His Thr Gly Phe Tyr Ser

100 105 110

Cys Lys Tyr Leu Ala Val Pro Thr Ser Lys Lys Lys Glu Thr Glu Ser

115 120 125

Ala Ile Tyr Ile Phe Ile Ser Asp Thr Gly Arg Pro Phe Val Glu Met

130 135 140

Tyr Ser Glu Ile Pro Glu Ile Ile His Met Thr Glu Gly Arg Glu Leu

145 150 155 160

Val Ile Pro Cys Arg Val Thr Ser Pro Asn Ile Thr Val Thr Leu Lys

165 170 175

Lys Phe Pro Leu Asp Thr Leu Ile Pro Asp Gly Lys Arg Ile Ile Trp

180 185 190

Asp Ser Arg Lys Gly Phe Ile Ile Ser Asn Ala Thr Tyr Lys Glu Ile

195 200 205

Gly Leu Leu Thr Cys Glu Ala Thr Val Asn Gly His Leu Tyr Lys Thr

210 215 220

Asn Asn Tyr Leu Thr His Arg Gln Thr Asn Thr Ile Ile Asp Val Gln

225 230 235 240

Ile Ser Thr Pro Arg Pro Val Lys Leu Leu Arg Gly His Thr Leu Val

245 250 255

Leu Asn Cys Thr Ala Thr Thr Pro Leu Asn Thr Arg Val Gln Met Thr

260 265 270

Trp Ser Tyr Pro Asp Asn Asn Asn Glu Lys Asn Lys Arg Ala Ser Val

275 280 285

Arg Arg Arg Ile Asp Gln Ser Asn Ser His Ala Asn Ile Phe Tyr Ser

290 295 300

Val Leu Thr Ile Asp Lys Met Gln Asn Lys Asp Lys Gly Leu Tyr Thr

305 310 315 320

Cys Arg Val Arg Ser Gly Pro Ser Phe Lys Ser Val Asn Thr Ser Val

325 330 335

His Ile Tyr Asp Lys Ala Phe Ile Thr Val Lys His Arg Lys Gln Gln

340 345 350

Val Leu Glu Thr Val Ala Gly Lys Arg Ser Tyr Arg Leu Ser Met Lys

355 360 365

Val Lys Ala Phe Pro Ser Pro Glu Val Val Trp Leu Lys Asp Gly Leu

370 375 380

Pro Ala Thr Glu Lys Ser Ala Arg Tyr Leu Thr Arg Gly Tyr Ser Leu

385 390 395 400

Ile Ile Lys Asp Val Thr Glu Glu Asp Ala Gly Asn Tyr Thr Ile Leu

405 410 415

Leu Ser Ile Lys Gln Ser Asn Val Phe Lys Asn Leu Thr Ala Thr Leu

420 425 430

Ile Val Asn Val Lys Pro Gln Ile Tyr Glu Lys Ala Val Ser Ser Phe

435 440 445

Pro Asp Pro Ala Leu Tyr Pro Leu Gly Ser Arg Gln Ile Leu Thr Cys

450 455 460

Thr Ala Tyr Gly Ile Pro Gln Pro Asn Thr Ile Lys Trp Phe Trp His

465 470 475 480

Pro Cys Asn His Asn His Ser Glu Ala Arg Cys Asp Phe Cys Ser Asn

485 490 495

Asn Glu Glu Ser Phe Ile Leu Asp Asn Asn Asn Asn Asn Asn Asn Ala

500 505 510

Asp Ser Asn Met Gly Asn Arg Ile Glu Ser Ile Thr Gln Arg Met Ala

515 520 525

Ile Ile Glu Gly Lys Asn Lys Met Ala Ser Thr Leu Val Val Ala Asp

530 535 540

Ser Arg Ile Ser Gly Ile Tyr Ile Cys Ile Ala Ser Asn Lys Val Gly

545 550 555 560

Thr Val Gly Arg Asn Ile Ser Phe Tyr Ile Thr Asp Val Pro Asn Gly

565 570 575

Phe His Val Asn Leu Glu Lys Met Pro Thr Asn Asn Glu Gly Glu Asp

580 585 590

Leu Lys Leu Ser Cys Thr Val Asn Lys Phe Leu Tyr Arg Asp Val Thr

595 600 605

Trp Ile Leu Leu Asn Asn Asn Asn Asn Asn Asn Asn Asn Asn Asn Asn

610 615 620

Asn Asn Asn Asn Asn Arg Thr Val Asn Asn Arg Thr Met His Tyr Ser

625 630 635 640

Ile Ser Lys Gln Lys Met Ala Ile Thr Lys Glu His Ser Ile Thr Leu

645 650 655

Asn Leu Thr Ile Met Asn Val Ser Leu Gln Asp Ser Gly Thr Tyr Ala

660 665 670

Cys Arg Ala Arg Asn Val Tyr Thr Gly Glu Glu Ile Leu Gln Lys Lys

675 680 685

Glu Ile Thr Ile Arg Asp Gln Glu Ala Pro Tyr Leu Leu Arg Asn Leu

690 695 700

Ser Asp His Thr Val Ala Ile Ser Ser Ser Thr Thr Leu Asp Cys His

705 710 715 720

Ala Asn Gly Val Pro Glu Pro Gln Ile Thr Trp Phe Lys Asn Asn His

725 730 735

Lys Ile Gln Gln Glu Pro Gly Ile Ile Leu Gly Pro Gly Ser Ser Thr

740 745 750

Leu Phe Ile Glu Arg Val Thr Glu Glu Asp Glu Gly Val Tyr His Cys

755 760 765

Lys Ala Thr Asn Gln Lys Gly Ser Val Glu Ser Ser Ala Tyr Leu Thr

770 775 780

Val Gln Gly Thr Ser Asp Lys Ser Asn Leu Glu Leu Ile Thr Leu Thr

785 790 795 800

Cys Thr Cys Val Ala Ala Thr Leu Phe Trp Leu Leu Leu Thr Leu Leu

805 810 815

Ile Arg Lys Met Lys Arg Ser Ser Asn Ser Glu Ile Lys Thr Asp Tyr

820 825 830

Leu Ser Ile Ile Met Asp Pro Asp Glu Val Pro Leu Asp Glu Gln Cys

835 840 845

Glu Arg Leu Pro Tyr Asp Ala Ser Lys Trp Glu Phe Ala Arg Glu Arg

850 855 860

Leu Lys Leu Gly Lys Ser Leu Gly Arg Gly Ala Phe Gly Lys Val Val

865 870 875 880

Gln Ala Ser Ala Phe Gly Ile Lys Lys Ser Pro Thr Cys Arg Thr Val

885 890 895

Ala Val Lys Met Leu Lys Glu Gly Ala Thr Ala Ser Glu Tyr Lys Ala

900 905 910

Leu Met Thr Glu Leu Lys Ile Leu Thr His Ile Gly His His Leu Asn

915 920 925

Val Val Asn Leu Leu Gly Ala Cys Thr Lys Gln Gly Gly Pro Leu Met

930 935 940

Val Ile Val Glu Tyr Cys Lys Tyr Gly Asn Leu Ser Asn Tyr Leu Lys

945 950 955 960

Ser Lys Arg Asp Leu Phe Phe Leu Asn Lys Asp Ala Ala Leu His Met

965 970 975

Glu Pro Lys Lys Glu Lys Met Glu Pro Gly Leu Glu Gln Gly Lys Lys

980 985 990

Pro Arg Leu Asp Ser Val Thr Ser Ser Glu Ser Phe Ala Ser Ser Gly

995 1000 1005

Phe Gln Glu Asp Lys Ser Leu Ser Asp Val Glu Glu Glu Glu Asp Ser

1010 1015 1020

Asp Gly Phe Tyr Lys Glu Pro Ile Thr Met Glu Asp Leu Ile Ser Tyr

1025 1030 1035 1040

Ser Phe Gln Val Ala Arg Gly Met Glu Phe Leu Ser Ser Arg Lys Cys

1045 1050 1055

Ile His Arg Asp Leu Ala Ala Arg Asn Ile Leu Leu Ser Glu Asn Asn

1060 1065 1070

Val Val Lys Ile Cys Asp Phe Gly Leu Ala Arg Asp Ile Tyr Lys Asn

1075 1080 1085

Pro Asp Tyr Val Arg Lys Gly Asp Thr Arg Leu Pro Leu Lys Trp Met

1090 1095 1100

Ala Pro Glu Ser Ile Phe Asp Lys Ile Tyr Ser Thr Lys Ser Asp Val

1105 1110 1115 1120

Trp Ser Tyr Gly Val Leu Leu Trp Glu Ile Phe Ser Leu Gly Gly Ser

1125 1130 1135

Pro Tyr Pro Gly Val Gln Met Asp Glu Asp Phe Cys Ser Arg Leu Arg

1140 1145 1150

Glu Gly Met Arg Met Arg Ala Pro Glu Tyr Ser Thr Pro Glu Ile Tyr

1155 1160 1165

Gln Ile Met Leu Asp Cys Trp His Arg Asp Pro Lys Glu Arg Pro Arg

1170 1175 1180

Phe Ala Glu Leu Val Glu Lys Leu Gly Asp Leu Leu Gln Ala Asn Val

1185 1190 1195 1200

Gln Gln Asp Gly Lys Asp Tyr Ile Pro Ile Asn Ala Ile Leu Thr Gly

1205 1210 1215

Asn Ser Gly Phe Thr Tyr Ser Thr Pro Ala Phe Ser Glu Asp Phe Phe

1220 1225 1230

Lys Glu Ser Ile Ser Ala Pro Lys Phe Asn Ser Gly Ser Ser Asp Asp

1235 1240 1245

Val Arg Tyr Val Asn Ala Phe Lys Phe Met Ser Leu Glu Arg Ile Lys

1250 1255 1260

Thr Phe Glu Glu Leu Leu Pro Asn Ala Thr Ser Met Phe Asp Asp Tyr

1265 1270 1275 1280

Gln Gly Asp Ser Ser Thr Leu Leu Ala Ser Pro Met Leu Lys Arg Phe

1285 1290 1295

Thr Trp Thr Asp Ser Lys Pro Lys Ala Ser Leu Lys Ile Glu Val

1300 1305 1310

<210>6

<211>6

<212>PRT

<213> Artificial sequence

<220>

<223> the amino acids at the 1 st and 2 nd positions are independently selected from aspartic acid or glutamic acid

<220>

<223> the amino acid at position 4 is independently selected from methionine or valine

<220>

<223> the amino acid at position 5 is independently selected from proline or aspartic acid or glutamic acid

<220>

<223> description of artificial sequences: consensus sequences

<400>6

Xaa Xaa Tyr Xaa Xaa Met

1 5

<210>7

<211>70

<212>DNA

<213> Artificial sequence

<220>

<223> description of artificial sequences: oligonucleotide probe

<400>7

acatgcatgc caccatgcag cggggcgccg cgctgtgcct gcgactgtgg ctctgcctgg 60

gactcctgga 70

<210>8

<211>24

<212>DNA

<213> Artificial sequence

<220>

<223> description of artificial sequences: oligonucleotide probe

<400>8

acatgcatgc cccgccggtc atcc 24

<210>9

<211>22

<212>DNA

<213> Artificial sequence

<220>

<223> description of artificial sequences: oligonucleotide probe

<400>9

cggaattccc catgacccca ac 22

<210>10

<211>33

<212>DNA

<213> Artificial sequence

<220>

<223> description of artificial sequences: oligonucleotide probe

<400>10

ccatcgatgg atcctacctg aagccgcttt ctt 33

<210>11

<211>34

<212>DNA

<213> Artificial sequence

<220>

<223> description of artificial sequences: oligonucleotide probe

<400>11

cccaagcttg gatccaagtg gctactccat gacc 34

<210>12

<211>20

<212>DNA

<213> Artificial sequence

<220>

<223> description of artificial sequences: oligonucleotide probe

<400>12

gttgcctgtg atgtgcacca 20

<210>13

<211>21

<212>DNA

<213> Artificial sequence

<220>

<223> description of artificial sequences: oligonucleotide probe

<400>13

ctggagtcga cttggcggac t 21

<210>14

<211>60

<212>DNA

<213> Artificial sequence

<220>

<223> description of artificial sequences: oligonucleotide probe

<400>14

cgcggatccc tagtgatggt gatggtgatg tctaccttcg atcatgctgc ccttatcctc 60

<210>15

<211>21

<212>DNA

<213> Artificial sequence

<220>

<223> description of artificial sequences: oligonucleotide probe

<400>15

ctggagtcga cttggcggac t 21

<210>16

<211>28

<212>DNA

<213> Artificial sequence

<220>

<223> description of artificial sequences: oligonucleotide probe

<400>16

cgggatccct ccatgctgcc cttatcct 28

<210>17

<211>36

<212>DNA

<213> Artificial sequence

<220>

<223> description of artificial sequences: oligonucleotide probe

<400>17

ggcaagcttg aattcgccac catgcagcgg ggcgcc 36

<210>18

<211>20

<212>DNA

<213> Artificial sequence

<220>

<223> description of artificial sequences: oligonucleotide probe

<400>18

gttgcctgtg atgtgcacca 20

<210>19

<211>21

<212>DNA

<213> Artificial sequence

<220>

<223> description of artificial sequences: oligonucleotide probe

<400>19

ctggagtcga cttggcggac t 21

<210>20

<211>44

<212>DNA

<213> Artificial sequence

<220>

<223> description of artificial sequences: oligonucleotide probe

<400>20

cgcggatcca agcttactta ccttccatgc tgcccttatc ctcg 44

<210>21

<211>419

<212>PRT

<213> human (Homo sapiens)

<400>21

Met His Leu Leu Gly Phe Phe Ser Val Ala Cys Ser Leu Leu Ala Ala

1 5 10 15

Ala Leu Leu Pro Gly Pro Arg Glu Ala Pro Ala Ala Ala Ala Ala Phe

20 25 30

Glu Ser Gly Leu Asp Leu Ser Asp Ala Glu Pro Asp Ala Gly Glu Ala

35 40 45

Thr Ala Tyr Ala Ser Lys Asp Leu Glu Glu Gln Leu Arg Ser Val Ser

50 55 60

Ser Val Asp Glu Leu Met Thr Val Leu Tyr Pro Glu Tyr Trp Lys Met

65 70 75 80

Tyr Lys Cys Gln Leu Arg Lys Gly Gly Trp Gln His Asn Arg Glu Gln

85 90 95

Ala Asn Leu Asn Ser Arg Thr Glu Glu Thr Ile Lys Phe Ala Ala Ala

100 105 110

His Tyr Asn Thr Glu Ile Leu Lys Ser Ile Asp Asn Glu Trp Arg Lys

115 120 125

Thr Gln Cys Met Pro Arg Glu Val Cys Ile Asp Val Gly Lys Glu Phe

130 135 140

Gly Val Ala Thr Asn Thr Phe Phe Lys Pro Pro Cys Val Ser Val Tyr

145 150 155 160

Arg Cys Gly Gly Cys Cys Asn Ser Glu Gly Leu Gln Cys Met Asn Thr

165 170 175

Ser Thr Ser Tyr Leu Ser Lys Thr Leu Phe Glu Ile Thr Val Pro Leu

180 185 190

Ser Gln Gly Pro Lys Pro Val Thr Ile Ser Phe Ala Asn His Thr Ser

195 200 205

Cys Arg Cys Met Ser Lys Leu Asp Val Tyr Arg Gln Val His Ser Ile

210 215 220

Ile Arg Arg Ser Leu Pro Ala Thr Leu Pro Gln Cys Gln Ala Ala Asn

225 230 235 240

Lys Thr Cys Pro Thr Asn Tyr Met Trp Asn Asn His Ile Cys Arg Cys

245 250 255

Leu Ala Gln Glu Asp Phe Met Phe Ser Ser Asp Ala Gly Asp Asp Ser

260 265 270

Thr Asp Gly Phe His Asp Ile Cys Gly Pro Asn Lys Glu Leu Asp Glu

275 280 285

Glu Thr Cys Gln Cys Val Cys Arg Ala Gly Leu Arg Pro Ala Ser Cys

290 295 300

Gly Pro His Lys Glu Leu Asp Arg Asn Ser Cys Gln Cys Val Cys Lys

305 310 315 320

Asn Lys Leu Phe Pro Ser Gln Cys Gly Ala Asn Arg Glu Phe Asp Glu

325 330 335

Asn Thr Cys Gln Cys Val Cys Lys Arg Thr Cys Pro Arg Asn Gln Pro

340 345 350

Leu Asn Pro Gly Lys Cys Ala Cys Glu Cys Thr Glu Ser Pro Gln Lys

355 360 365

Cys Leu Leu Lys Gly Lys Lys Phe His His Gln Thr Cys Ser Cys Tyr

370 375 380

Arg Arg Pro Cys Thr Asn Arg Gln Lys Ala Cys Glu Pro Gly Phe Ser

385 390 395 400

Tyr Ser Glu Glu Val Cys Arg Cys Val Pro Ser Tyr Trp Lys Arg Pro

405 410 415

Gln Met Ser

<210>22

<211>354

<212>PRT

<213> human (Homo sapiens)

<400>22

Met Tyr Arg Glu Trp Val Val Val Asn Val Phe Met Met Leu Tyr Val

1 5 10 15

Gln Leu Val Gln Gly Ser Ser Asn Glu His Gly Pro Val Lys Arg Ser

20 25 30

Ser Gln Ser Thr Leu Glu Arg Ser Glu Gln Gln Ile Arg Ala Ala Ser

35 40 45

Ser Leu Glu Glu Leu Leu Arg Ile Thr His Ser Glu Asp Trp Lys Leu

50 55 60

Trp Arg Cys Arg Leu Arg Leu Lys Ser Phe Thr Ser Met Asp Ser Arg

65 70 75 80

Ser Ala Ser His Arg Ser Thr Arg Phe Ala Ala Thr Phe Tyr Asp Ile

85 90 95

Glu Thr Leu Lys Val Ile Asp Glu Glu Trp Gln Arg Thr Gln Cys Ser

100 105 110

Pro Arg Glu Thr Cys Val Glu Val Ala Ser Glu Leu Gly Lys Ser Thr

115 120 125

Asn Thr Phe Phe Lys Pro Pro Cys Val Asn Val Phe Arg Cys Gly Gly

130 135 140

Cys Cys Asn Glu Glu Ser Leu Ile Cys Met Asn Thr Ser Thr Ser Tyr

145 150 155 160

Ile Ser Lys Gln Leu Phe Glu Ile Ser Val Pro Leu Thr Ser Val Pro

165 170 175

Glu Leu Val Pro Val Lys Val Ala Asn His Thr Gly Cys Lys Cys Leu

180 185 190

Pro Thr Ala Pro Arg His Pro Tyr Ser Ile Ile Arg Arg Ser Ile Gln

195 200 205

Ile Pro Glu Glu Asp Arg Cys Ser His Ser Lys Lys Leu Cys Pro Ile

210 215 220

Asp Met Leu Trp Asp Ser Asn Lys Cys Lys Cys Val Leu Gln Glu Glu

225 230 235 240

Asn Pro Leu Ala Gly Thr Glu Asp His Ser His Leu Gln Glu Pro Ala

245 250 255

Leu Cys Gly Pro His Met Met Phe Asp Glu Asp Arg Cys Glu Cys Val

260 265 270

Cys Lys Thr Pro Cys Pro Lys Asp Leu Ile Gln His Pro Lys Asn Cys

275 280 285

Ser Cys Phe Glu Cys Lys Glu Ser Leu Glu Thr Cys Cys Gln Lys His

290 295 300

Lys Leu Phe His Pro Asp Thr Cys Ser Cys Glu Asp Arg Cys Pro Phe

305 310 315 320

His Thr Arg Pro Cys Ala Ser Gly Lys Thr Ala Cys Ala Lys His Cys

325 330 335

Arg Phe Pro Lys Glu Lys Arg Ala Ala Gln Gly Pro His Ser Arg Lys

340 345 350

Asn Pro

Claims (63)

1. A purified polypeptide comprising a portion of the extracellular domain (EC) of mammalian vascular endothelial growth factor receptor-3 (VEGFR-3),

wherein said portion binds at least one VEGFR-3 ligand and consists of at least a first, second, and third immunoglobulin-like domain of VEGFR-3EC, and wherein the polypeptide lacks VEGFR-3 immunoglobulin domains IV-VII,

wherein the polypeptide is a soluble polypeptide lacking any transmembrane domain.

2. The purified polypeptide according to claim 1, wherein the portion consists essentially of the first, second, and third immunoglobulin domains of VEGFR-3-EC.

3. The purified polypeptide according to any of claims 1-2, wherein VEGFR-3 is human VEGFR-3.

4. The purified polypeptide according to any of claims 1-3, wherein the polypeptide further comprises an immunoglobulin-like domain of VEGFR-2 or VEGFR-3.

5. The purified polypeptide according to any of claims 1-4, wherein the VEGFR-3 comprises a sequence selected from the group consisting of SEQ ID NOs: 2 and SEQ ID NO: 4.

6. The purified polypeptide according to any of claims 1-4, wherein the VEGFR-3 comprises an amino acid sequence encoded by a polynucleotide or oligonucleotide that hybridizes to a human gene encoding VEGFR-3 under hybridization conditions in which said polynucleotide or oligonucleotide does not hybridize to the human gene encoding VEGFR-1, said hybridization conditions comprising:

(a) the hybridization solution contained 50% formamide, 5 XDenhardt's solution, 5 XSSPE, 0.1% SDS, and 0.1mg/ml sonicated salmon sperm DNA;

(b) hybridization at a temperature of 42 ℃ for 18 to 24 hours; and

(c) after hybridization, washing was carried out at a washing temperature of 65 ℃ using a washing solution containing 1 XSSC and 0.1% SDS.

7. A fusion protein comprising a polypeptide according to any one of claims 1-6 fused to an immunoglobulin Fc peptide.

8. The polypeptide or fusion protein according to any one of claims 1-7, further comprising a cytotoxic agent attached to the polypeptide.

9. The polypeptide or fusion protein according to claim 8, wherein the cytotoxic agent is selected from the group consisting of a plant toxin, a bacterial toxin, a fungal toxin, a radioisotope, an anti-metabolite, an alkylating agent, an anti-mitotic agent, and a DNA intercalating agent.

10. A composition comprising a polypeptide or fusion protein according to any one of claims 1-9 and a pharmaceutically acceptable carrier.

11. A polynucleotide comprising a nucleotide sequence encoding a polypeptide or fusion protein according to any one of claims 1-7.

12. A polynucleotide according to claim 11, further comprising an expression control sequence operably linked to the sequence encoding the polypeptide.

13. An expression vector comprising an expression control sequence operably linked to a polynucleotide according to claim 11.

14. The expression vector according to claim 13, wherein the expression control sequence comprises a promoter that promotes expression in mammalian cells.

15. The expression vector according to claim 14, which is a viral vector selected from the group consisting of retrovirus, adenovirus, adeno-associated virus, vaccinia virus and herpes virus.

16. A composition comprising a polynucleotide or vector according to any one of claims 11-15 and a pharmaceutically acceptable carrier.

17. A cell transformed or transfected with a polynucleotide or vector according to any of claims 11-15.

18. A polypeptide, fusion protein, or composition according to any one of claims 1-10 for use in a method of diagnosing a neoplastic disease.

19. A polypeptide, fusion protein, or composition according to any one of claims 1-10 for use in a method of treating a neoplastic disease.

20. The polypeptide, fusion protein, or composition of any of claims 18-19, wherein the neoplastic disease is a tumor characterized by neovascularization of blood or lymph vessels, and wherein the neovascularization comprises endothelial cells that express VEGFR-3.

21. The polypeptide, fusion protein, or composition according to any one of claims 18-19, wherein the neoplastic disease is breast cancer.

22. The polypeptide, fusion protein, or composition according to any one of claims 1-10 for use in a method of diagnosing tumor metastasis.

23. The polypeptide, fusion protein, or composition according to any one of claims 1-10 for use in a method of preventing tumor metastasis.

24. A polynucleotide, vector, cell, or composition according to any one of claims 11-17 for use in a method of diagnosing a neoplastic disease.

25. A polynucleotide, vector, cell, or composition according to any one of claims 11-17 for use in a method of treating a neoplastic disease.

26. A polynucleotide, vector, cell, or composition according to any one of claims 24-25, wherein said neoplastic disease is a tumor characterized by neovascularization of blood or lymph vessels, and wherein the neovascularization comprises endothelial cells that express VEGFR-3.

27. A polynucleotide, vector, cell, or composition according to any one of claims 24-25, wherein the neoplastic disease is breast cancer.

28. A polynucleotide, vector, cell, or composition according to any one of claims 11-17 for use in a method of diagnosing tumor metastasis.

29. A polynucleotide, vector, cell, or composition according to any one of claims 11-17 for use in a method of preventing metastasis of a tumor.

30. A method of inhibiting the proliferation or metastatic spread of cancer cells, comprising administering to a mammalian subject having cancer a polynucleotide comprising a nucleotide sequence encoding a polypeptide comprising a portion of a mammalian VEGFR-3-EC domain,

wherein said portion binds to at least one VEGFR-3 ligand and consists of at least one immunoglobulin-like domain of VEGFR-3-EC,

wherein the encoded polypeptide is a soluble polypeptide lacking any transmembrane domain,

and wherein the administration is under conditions that allow the polynucleotide to be taken up and thereby the encoded polypeptide to be expressed by the cells in the mammalian subject, and wherein the polynucleotide, vector, or composition is administered in an amount effective to inhibit proliferation of said cells.

31. The method of claim 30, wherein the administering comprises administering a vector comprising the polynucleotide.

32. The method according to claim 30, wherein said administering comprises administering said polynucleotide in a polynucleotide, vector, or composition according to any one of claims 11-17.

33. The method of claims 30-32, wherein the polynucleotide, vector or composition is encapsulated in a liposome.

34. The method of claim 31, wherein said viral vector is selected from the group consisting of a retrovirus, an adenovirus, an adeno-associated virus, a vaccinia virus, and a herpes virus.

35. The method according to any of claims 30-34, wherein said mammalian subject is diagnosed with a cancer characterized by proliferation of endothelial cells that express VEGFR-3 and wherein said administering inhibits at least one of angiogenesis and lymphangiogenesis in said subject.

36. The method of claim 35, wherein the cancer comprises a tumor characterized by neovascularization of blood vessels or lymphatic vessels, and wherein the neovascularization comprises endothelial cells that express VEGFR-3 and wherein said administering inhibits at least one of angiogenesis and lymphangiogenesis in said subject.

37. The method according to any one of claims 30-33, wherein the cancer cells express at least one polypeptide selected from the group consisting of VEGFR-3, VEGF-C and VEGF-D.

38. The method according to any one of claims 30-37, wherein the subject is a human.

39. A method according to any one of claims 30 to 37 wherein the cancer is breast cancer.

40. The method of any one of claims 30-37, further comprising the step of administering to the subject a second cancer therapeutic.

41. The method of claim 40, wherein said second cancer therapeutic comprises a chemotherapeutic agent, a radioactive agent, a nucleic acid encoding a cancer therapeutic and an anti-lymphangiogenic or anti-angiogenic agent.

42. The method according to any one of claims 30-41, wherein the cell is in a tumor that is operable, and wherein the administering step is performed before, during, or after the tumor is resected.

43. The method according to claim 30, wherein the subject has a cancer of a tissue, organ, or cell selected from the group consisting of brain, lung, liver, spleen, kidney, lymph node, small intestine, blood cell, pancreas, colon, stomach, breast, endometrium, prostate, testis, ovary, skin, head and neck, esophagus, bone marrow, and blood.

44. A method of inhibiting metastatic spread of cancer in a mammalian subject, comprising administering to a mammalian subject suspected of having cancer an effective amount of a polypeptide, fusion protein or composition according to any one of claims 1 to 10 to inhibit metastatic spread of said cancer.

45. A method of inhibiting metastatic spread of a cancer in a mammalian subject, comprising administering to a mammalian subject suspected of having a cancer an effective amount of a polypeptide, fusion protein or composition according to any one of claims 1 to 10 to inhibit or reduce the growth of said cancer.

46. A method of treating cancer comprising administering to a mammalian subject diagnosed with cancer a composition comprising a polypeptide, fusion protein, or composition according to any one of claims 1-10 in an amount effective to inhibit or reduce the growth or tumor spread of the cancer.

47. Use of a polynucleotide comprising a nucleotide sequence encoding a polypeptide comprising a portion of a mammalian VEGFR-3-EC domain wherein said portion binds to at least one VEGFR-3 ligand and consists of at least one immunoglobulin-like domain of VEGFR-3-EC in the manufacture of a medicament for inhibiting cell proliferation of a cancer cell, wherein the encoded polypeptide is a soluble polypeptide lacking any transmembrane domain.

48. The use according to claim 47, wherein said polynucleotide is in a vector.

49. Use according to claim 47, wherein said polynucleotide is a polynucleotide, vector, cell or composition according to any one of claims 11-17.

50. The use according to claims 47-49, wherein said polynucleotide is encapsulated in a liposome.

51. The use according to claim 48, wherein said vector is a viral vector comprising a member selected from the group consisting of a retrovirus, an adenovirus, an adeno-associated virus, a vaccinia virus and a herpes virus.

52. The use according to claim 47, wherein said medicament is for a mammalian subject diagnosed with a disease characterized by proliferation of endothelial cells that express VEGFR-3 and wherein said medicament inhibits at least one of angiogenesis and lymphangiogenesis in said subject.

53. The use according to claim 52, wherein the disease comprises a tumor characterized by neovascularization of blood vessels or lymphatic vessels, and wherein the neovascularization comprises endothelial cells that express VEGFR-3 and wherein said medicament inhibits at least one of angiogenesis and lymphangiogenesis in said subject.

54. The use according to claim 52, wherein the cancer cells express a polypeptide selected from the group consisting of VEGFR-3, VEGF-C and VEGF-D.

55. The use according to any one of claims 47 to 54, wherein the cancer is breast cancer.

56. The use according to any one of claims 47-54, wherein the medicament further comprises a second cancer therapeutic agent.

57. The use of claim 56, wherein said second cancer therapeutic comprises a chemotherapeutic agent, a nucleic acid encoding a cancer therapeutic and an anti-lymphangiogenic or anti-angiogenic agent.

58. The use according to claim 47, wherein the cell is in a tumor that is operable, and wherein the administering step is performed before, during, or after resection of the tumor.

59. The use according to claim 47, wherein said medicament is for use in a subject having a tissue, organ, or cellular cancer selected from the group consisting of brain, lung, liver, spleen, kidney, lymph node, small intestine, blood cell, pancreas, colon, stomach, breast, endometrium, prostate, testis, ovary, skin, head and neck, esophagus, bone marrow, and blood.

60. Use of a polypeptide, fusion protein or composition according to any one of claims 1-10 in the manufacture of a medicament for inhibiting the spread of metastasis of a cancer in a mammalian subject.

61. Use of a polypeptide, fusion protein or composition according to any one of claims 1-10 in the manufacture of a medicament for inhibiting or reducing cancer growth in a mammalian subject.

62. Use of a polypeptide, fusion protein or composition according to any one of claims 1-10 in the manufacture of a medicament for treating cancer in a mammalian subject.

63. Use of a polypeptide, fusion protein or composition according to any one of claims 1-10 in the manufacture of a medicament for treating cancer in a mammalian subject.