WO2008134445A2 - Platelet activation receptor clec-2: compositions and uses thereof - Google Patents

Abstract

Disclosed are compositions comprising the platelet activation receptor C-type Lectin- like Receptor 2 (CLEC-2), and compositions comprising anti-CLEC-2 antibodies or antibodies that inhibit the interaction of CLEC-2 with podoplanin. Also disclosed are and methods of treating subjects in need thereof, comprising administering to the subject an effective amount of the disclosed compositions. Disclosed are recombinant CLEC-2 polypeptides, nucleic acids, antibodies, and compositions comprising recombinant CLEC-2 polypeptides, nucleic acids and antibodies. CLEC-2, has been found to interact with podoplanin. Recombinant CLEC-2 inhibits platelet aggregation induced by podoplanin- expressing tumor cells or lymphatic endothelial cells. Thus, CLEC-2 is responsible for platelet aggregation induced by endogenously expressed podoplanin on cell surfaces. CLEC- 2 is therefore a physiological target protein of podoplanin and is involved in podoplanin- induced platelet aggregation, tumor metastasis, and other cellular responses related to podoplanin. Disclosed are methods of treating, for instance, platelet aggregation-related disorders by administering effective amounts of the disclosed compositions to subjects in need thereof.

Description

PLATELET ACTIVATION RECEPTOR CLEC-2: COMPOSITIONS AND USES THEREOF

[0001] Disclosed are C-type Lectήi-like Receptor 2 (CLEC-2) polypeptides, and compositions comprising CLEC-2 polypeptides. Methods of treating diseases comprising administering the compositions are also disclosed. CLEC-2, functionally interacts with podoplanin. Recombinant CLEC-2 is shown to inhibit platelet aggregation induced by podoplanin-expressing tumor cells or lymphatic endothelial cells. Thus, CLEC-2 is responsible for platelet aggregation induced by endogenously expressed podoplanin on cell surfaces. CLEC-2 is therefore a physiological target protein of podoplanin and is involved in podoplanin-induced platelet aggregation, tumor metastasis, and other cellular responses related to the functioning of podoplanin. To this end, disclosed are methods of treating, for instance, platelet aggregation-related disorders, by administering effective amounts of the disclosed compositions to subjects in need thereof. The present invention includes compositions comprising platelet activation receptor, CLEC-2, and compositions comprising anti-CLEC-2 antibodies. The present invention also includes methods of treating subjects in need thereof, comprising administering to the subject an effective amount of the disclosed compositions.

[0002] There is an increasing body of evidence showing that platelets are involved in cancer metastasis and/or cancer progression (1, 2). Several studies on tumor cell-induced platelet activation suggest that aggregation contributes to tumor cell nestling, while released growth factors contribute to angiogenesis, or tumor growth. Podoplanin, a sialoglycoprotein on the surface of cancer cells, induces platelet aggregation (3-5). Increased expression of podoplanin has been observed in various tumor cells, including squamous cell carcinomas (6, 7), testicular seminoma (8), and brain tumors (9, 10). Recent investigations have reinforced the notion that podoplanin expression is associated with tumor metastasis or progression of malignancies (10, 11).

[0003] Podoplanin is expressed abundantly on glomerular epithelial cells (podocytes), type I lung alveolar cells, and lymphatic endothelial cells (6, 12). While the physiologic roles of podoplanin in the functioning of these cells are not well characterized, podoplanin is nonetheless considered to be one of the most useful markers for lymphatic vessels. Furthermore, several investigations using podoplanin knockout mice suggest that podoplanin is crucially involved in lymphatic vessel formation (13). However, podoplanin regulation of the formation of lymphatic vessels or tumor-platelet interaction is not well-characterized. A goal is the identification of the pathophysiological targets upon which podoplanin impinges. [0004] In the process of physiologic thrombus formation with platelet aggregation, the first step is platelet interaction with exposed collagen fibers at sites of vessel injury (14). Platelet adhesion and aggregation on collagen fibers and the subsequent stable clot formation is an integrated process that involves several platelet receptors and agonists such as adenosine diphosphate (ADP), thromboxane A2 and coagulation factors including thrombin. One of the major receptors involved in this process is a collagen receptor, glycoprotein VI/FcRγ-chain complex (GPVI). The signal transduction pathway related to GPVI encompasses a number of intracellular signaling molecules, such as tyrosine kinases Src, spleen tyrosine kinase (Syk), an adapter protein, SH2 domain containing leukocyte protein of 76kDa (SLP-76), and phospholipase Cγ2 (PLCγ2) (14).

[0005] The platelet activation receptor, C-type lectin-like receptor 2 (CLEC-2), is a non- classical C-type lectin and acts as a receptor located on the platelet membrane for a platelet- aggregating snake venom, rhodocytin (15). CLEC-2 generates activation signals depending on protein tyrosine phosphorylation, including a tyrosine kinase Src, Syk, an adapter protein SLP-76, and PLCγ2 in platelets, in a manner similar to GPVI (15, 16). Although the powerful stimulatory action of CLEC-2 on platelets suggests that CLEC-2 plays an important role in vivo, a physiological ligand of the novel signaling receptor has thus far not been identified.

SUMMARY OF THE INVENTION

[0006] The present invention includes, but is not limited to, various nucleic acids and polypeptides encoding various fonns of CLEC-2, and compositions comprising these nucleic acids and polypeptides for the treatment of diseases effected by podoplanin interaction. For instance, the present invention is directed to the use of polypeptides comprising CLEC-2 or an amino acid sequence 95% identical thereto, nucleic acids encoding the same or nucleic acids comprising a sequence 95% identical thereto, polypeptides comprising an extra-cellular domain of CLEC-2 or comprising a sequence 95% identical thereto, and nucleic acids encoding such polypeptides. [0007] The present invention also includes pharmaceutical compositions comprising the polypeptides, nucleic acids and antibodies of the present invention and pharmaceutically acceptable carriers, diluents and/or excipients for use in the treatment of diseases effected by podoplanin interaction.

[0008] The presently claimed invention also includes antibodies which inhibits the interaction between CLEC-2 and podoplanin, such as, but not limited to, anti-CLEC-2 antibodies. These antibodies may be included in the compositions of the present invention. [0009] The presently claimed invention also includes compounds other than antibodies which block the interaction between CLEC-2 and podoplanin, and compositions comprising such compounds.

[0010] Many methods may be executed utilizing the peptides, nucleic acids, antibodies and compositions of the present invention. For instance, some non-limiting examples of methods of treatment that may be achieved by administering the presently claimed peptides, nucleic acids, antibodies and compositions thereof include methods of inhibiting tumor growth or metastasis, methods of inhibiting or decreasing platelet aggregation, and methods of inhibiting or decreasing thrombus formation in kidneys, all of which comprise administering to a subject in need thereof an effective amount of peptides, nucleic acids, antibodies and/or compositions thereof according to the present invention to a subject in need of such treatment. Subjects in need of treatment may include those suffering from hemolytic uremic syndrome or thrombotic thrombocytopenic purpura.

[0011] Based on the properties of CLEC-2 disclosed, one of skill in the art will be able to devise additional peptides, nucleic acids, antibodies, compositions thereof and methods taking into consideration the biochemical interactions of CLEC-2 disclosed herein and known mechanisms of various diseases and disorders which are caused by dysfunction of the interactions of the molecules disclosed.

BRIEF DESCRIPTION OF THE FIGURES

[0012] Figure 1 : Src kinase dependent platelet aggregation induced by podoplanin. [0013] A) Washed human platelets (1 x 10⁹/ml) were preincubated with dimethylsulfoxide (DMSO), 10 μM 4-amino-7-phenylpyrazolol[3,4]pyrimidine (PP3), or 10 μM 4-amino-5-(4-chlorophenyl)-7-(t-butyl)pyrazolo[3,4-d]pyriraidine (PP2) for 5 min at 37°C. The platelets were stimulated by addition of 1.5 x 10⁶/inl of Chinese Hamster Ovary (CHO) cells transiently-transfected with mouse podoplanin (CHO/mPod) and platelet aggregation was monitored, using an aggregometer, for 15 min.

[0014] B) Washed human platelets (I x 10⁹/ml) were pretreated with 1 mM GIy- Arg-

Gly-Asp-Ser (GRGDS) peptide (SEQ ID NO:9) to inhibit platelet aggregation. Then, the platelets were stimulated by addition of 1.5 x lOVml of CHO/mPod for the indicated duration. Reactions were terminated by addition of 2x ice-cold lysis buffer. Platelet lysates were dissolved with sodium dodecylsulfate (SDS) sample buffer, separated by 10% SDS-

PAGE, and Western blotted using anti-phospho tyrosine antibody (4G10).

[0015] C) Washed murine platelets (2.5 x 10⁸/ml) were stimulated by addition of 20 μg/ml of mPod-rFc2. Platelet aggregation was monitored using an AG- 10 aggregation analyzer for 15 min. The data are representative of at least two experiments.

[0016] Figure 2: PLCγ2-dependent, GPVI-independent platelet aggregation induced by podoplanin.

[0017] A) Washed murine platelets (2.5 x 10⁸/ml) from wild type (WT) or GPVI/FcRγ- deficient (GPVI/FcRγ KO) mice were stimulated by addition of 1.5 x 10⁶/ml of CHO/mPod

(pod), 10 nM rhodocytin (rhod), 2 μg/ml of collagen (col), and platelet aggregation was monitored, using an aggregometer, for 15 min.

[0018] B) Washed murine platelets (2.5 X loVml) from WT or PLCγ2-deficient (PLCγ2

KO) mice were stimulated by addition of 1.5 x 10⁶/ml of CHO/mPod (pod), 10 nM rhodocytin (rhod), 0.5 U/ml of thrombin (thr) and platelet aggregation was monitored, using an aggregometer, for 15 min. The data are representative of at least two experiments.

[0019] Figure 3: Platelet aggregation stimulated by association between CLEC-2 and podoplanin.

[0020] A) CHO cells transiently-transfected with MOCK (CHO/MOCK) or mouse podoplanin (CHO/mPod) were incubated with anti-mouse podoplanin antibody (i), hCLEC-2- hJFc2 (ii), or liGPVI-hFc2 (iii) for 20 min at room temperature. 293T-REx cells, which express CLEC-2 under a tet repressor, were incubated with vehicle (CLEC-2 (-) 293T-REx) or 1 μg/ml of doxycycline for 48 hours (CLEC-2 (+) 293T-REx). Cells were incubated with anti-human CLEC-2 antibody (iv), mPod-rFc2 (v), or mCLEC-2-rFc2 (vi) for 20 min at room temperature. After unbound antibodies or proteins were removed by centrifugation, cells were stained with fluorescein isothiocyanate (FITC)-conjugated anti-hamster IgG (i), anti- human IgG (ii, iii), anti-goat IgG (iv), or anti-rabbit IgG antibody (v, vi) for 15 min and analyzed by fluorescence-activated cell sorting using a FACScalibur.

[0021] B) CHO/mPod were incubated with phosphate-buffered saline (PBS), hCLEC-2- hFc2, or hGPVI-hFc2, for 10 min at room temperature. The cell-recombinant protein mixture was added to washed human platelets (I x 10⁹/ml, final concentration of CHO/mPod: 1.5 x 10⁶/ml). Platelet aggregation was monitored using an AG- 10 aggregometer for 15 min. [0022] C) A thromboxane A2 mimetic, U46619 (1 mM, 1.5 μl) was incubated with PBS (9 μl) or hCLEC-2-hFc2 (0.5 mg/ml, 9 μl) for 10 min at room temperature. The peptide- recombinant protein mixture was added to washed human platelets (I x 10⁹/ml, 300 μl, final concentration of U46619: 5 μM). Platelet aggregation was monitored using an AG- 10 aggregometer for 15 min. The data are representative of at least two experiments. [0023] Figure 4: Sialic acid-dependent binding of podoplanin to CLEC-2. [0024] A) CHO cells (CHO/hPod) or a CHO cell mutant, Lec2 (Lec2/hPod, stably- transfected with human podoplanin) were incubated with anti-human podoplanin antibody, or control rat IgG, for 20 min at room temperature. After unbound antibodies were removed by centrifugation, cells were stained with FITC-conjugated anti-rat IgG antibody and analyzed by a FACScalibur.

[0025] B) Washed human platelets (1 x 10⁹/ml) were stimulated by addition of 1.5 x 10⁶/ml of CHO/hPod or Lec2/hPod. Aggregation was monitored, by an AG-10 aggregometer, for 15 min.

[0026] C) CHO/hPod or Lec2/hPod were incubated with PBS, or hCLEC-2-hFc2, for 20 min at room temperature. After unbound proteins were removed by centrifugation, cells were stained with FITC-coηjugated anti-human IgG antibody and analyzed by a FACScalibur.

The data are representative of at least two experiments.

[0027] Figure 5: Platelet aggregation induced by podoplanin-expressing tumor cell lines or lymphatic endothelial cells through association between CLEC-2 and podoplanin.

[0028] A) i) A human glioblastoma cell line, LN319, was incubated with anti-human podoplanin antibody (line), or control rat IgG (fill), for 20 min at room temperature. After unbound antibodies were removed by centrifugation, cells were stained with FtTC- conjugated anti-rat antibody and analyzed by a FACScalibur. ii) LN319 was incubated with

PBS, hCLEC-2-hFc2, or hGPVI-hFc2, for 10 min at room temperature. The cell- recombinant protein mixture was added to human washed platelets (1 * 10⁹/ml, final cell concentration of LN319: 1.5 x 10⁶/ml). Platelet aggregation was monitored, using an AG- 10 aggregometer, for 15 min.

[0029] B) i) Colon-26 cells (a mouse colon carcinoma cell line) were incubated with anti-mouse podoplanin antibody (line), or control hamster IgG (fill), for 20 min at room temperature. After unbound antibodies were removed by centrifugation, the cells were stained with FITC-conjugated anti-hamster IgG antibody and analyzed by a FACScalibur. ii)

Colon-26 cells were incubated with PBS, hCLEC-2-hFc2, or hGPVI-hFc2, for 10 min at room temperature. The procedure followed is as described for Figure 5A)ii).

[0030] C) i) Human lymphatic endothelial cells were incubated with anti-human podoplanin antibody (line), or control rat IgG (fill), for 20 min at room temperature. After unbound antibodies were removed from the cells by centrifugation, the cells were stained with FITC-conjugated anti-rat antibody and analyzed by a FACScalibur. ii) LECs were incubated with PBS, hCLEC-2-hFc2, or hGPVI-hFc2, for 10 min at room temperature. The procedure followed is as described in Figure 5A)ii). The data are representative of at least two experiments.

[0031] Figure 6: Inhibition of podoplanin-induced metastasis by hPod-hFc2 or mCLEC-

2-hFc2.

[0032] A) CHO/hPod cells were harvested, washed, and resuspended in HBSS (2.5xlO⁶ cells/ml). Then, the cells were incubated with 100 μg of hPod-hFc, mCLEC-hFc, or control

PBS, and inoculated intravenously (5x10 cells/head) into lateral tail vein of female BALB/c- nu/nu mice (three or four mice in each group). After 17 days, the mice were euthanized, and surface lung metastatic foci were counted. [0033] B) Detailed numbers of the lung metastasis nodules in each mouse are shown. Data are means ± SD. *, P < 0.05, by t-test.

DETAILED DESCRIPTION

[0034] It is reported herein the identity of the target molecule for podoplanin on the platelet membrane. Platelet aggregation can be used as a marker for the interaction of podoplanin. To this end, the present Inventors investigated the characteristics of podoplanin- induced platelet aggregation, and compared these interactions with those of well-defined platelet stimulators.

[0035] Disclosed herein is the finding that podoplanin, located on the surface of tumor cells, induces platelet aggregation by interacting with CLEC-2. Podoplanin, also known as aggrus, is a transmembrane sialoglycoprotein which is involved in tumor cell-induced platelet aggregation, tumor metastasis, and lymphatic vessel formation. This discovery was confirmed by flow cytometry. Furthermore, this association is confirmed to be dependent on sialic acid on O-glycans of podoplanin.

[0036] As a result of the present characterization of podoplanin-induced platelet aggregation, the present Inventors discovered that the mode of platelet activation induced by podoplanin is reminiscent of the same activation by rhodocytin. Reported herein are results showing that CLEC-2 serves as a physiological counterpart for podoplanin. Recombinant

CLEC-2 has been found to inhibit platelet aggregation induced by podoplanin-expressing tumor cells or lymphatic endothelial cells.

[0037] These findings show that podoplanin stimulates platelet aggregation by interacting with CLEC-2 and that the interaction between podoplanin and CLEC-2 regulates various cellular responses related to podoplanin.

[0038] Therefore, a goal of the present invention is to formulate compositions of CLEC-

2 and utilize these compositions to treat diseases caused by cellular responses triggered through podoplanin interactions.

[0039] The role of podoplanin has been implicated in tumor metastasis/progression (10) and lymphatic vessel formation (13). Although the identification of the podoplanin receptor should provide a clue to understanding of podoplanin-related cellular responses, until now, search for its physiologic counter has not been successful.

[0040] Platelets are activated by the imrnunogloblin (Ig) superfamily and by G protein- coupled receptor (GPCR). CLEC-2 is a non-classical C-type lectin, which is a new class of platelet-activating receptor. The powerful stimulatory action of CLEC-2 on platelets shows that CLEC-2 is physiologically important in vivo. However, the CLEC-2 ϊigand, until now, was not identified.

[0041] Disclosed herein is the identification of the physiological counterparts of podoplanin and CLEC-2. It has been discovered that these molecules are related as being a receptor and a ligand for each other. This has paved the way for new research fields concerning the role(s) of the interaction between CLEC-2 and podoplanin. [0042] Specific interaction of the recombinant extracellular domain of CLEC-2 to podoplanin-expressing cells was confirmed by flow cytometory. (See, Figure 3A)ii). Inversely, the recombinant extracellular domain of podoplanin is reported herein to specifically associate with CLEC-2-expressing cell lines. (See, Figure 3)A)v). Moreover, it is presently disclosed that pretreatment of podoplanin-expressing CHO cells with recombinant CLEC-2 completely inhibits podoplanin-induced platelet aggregation. (See, Figure 3)B). Thus, the interaction between CLEC-2 and podoplanin is responsible for podoplanin-induced platelet aggregation.

[0043] The present Inventors sought to elucidate the mode of podoplanm-CLEC-2 interaction and the biochemical mechanism of podoplanin-induced platelet aggregation mediated through CLEC-2. It was found that podoplanin expressed in cytidine monophosphate (CMP)-sialic acid transporter-deficient Lec2 CHO cells (Lec2), which lack 90% of the common sialic acid moieties in both glycoproteins and glycolipids, did not associate with CLEC-2 and did not induce platelet aggregation. (See, Figure 4). These results indicate that sialic acid on the O-glycans of podoplanin molecules is involved in interacting with CLEC-2.

[0044] A previous report on the structure and mutational binding analysis of CLEC-2 also indicates that an endogenous ligand may be a protein with a predominantly negatively- charged binding surface (25). The data disclosed herein are consistent with this report, since sialic acid is negatively charged.

[0045] A recent study on the crystal structure and mutational binding analysis of CLEC-2 revealed that ligand interaction with CLEC-2 may not transmit signals by inducing a major conformational change of CLEC-2 upon ligand binding (25). However, without wishing to be bound by any specific teleological theory, the interaction of podoplanin with CLEC-2 may bring the cytoplasmic signaling domains of several CLEC-2 molecules into closer proximity. [0046] It is disclosed herein that recombinant podoplanin, expressed as a dimeric IgG Fc fusion protein (mPod-rFc2), induced more powerful platelet aggregation and protein tyrosine phosphorylation than podoplanin expressed on the surface of CHO cells. {See, Figure 1). Conceivably, without wishing to be bound by any specific teleological theory, a small dimeric recombinant protein, such as mPod-rFc2, may more effectively cluster CLEC-2 than podoplanin expressed on the surfaces of CHO cells. Such podoplanin may not be located close enough to form dimers. Alternatively, the difference may be due to podoplanin concentration.

[0047] The discoveries reported herein clearly demonstrate that podoplanin expressed on the surface of tumor cells induces platelet aggregation by interacting with CLEC-2. [0048] As further support for this conclusion, a study is reported in which the metastatic abilities of several clones from a mouse colon adenocarcinoma 26 cell line revealed that a highly metastatic clone expressed more podoplanin and induces platelet aggregation to a greater extent (24). Moreover, anti-podoplanin antibody that inhibits podoplanin-induced platelet aggregation successfully suppressed lung colonization of colon adenocarcinoma (26). These findings, taken together, indicate that podoplanin-induced platelet activation through CLEC-2 is one of the mechanisms of tumor metastasis.

[0049] Furthermore, the role of podoplanin-CLEC-2 interaction may not be confined to tumor metastasis. Activated platelets release several angiogenic factors that are stored in platelets, such as vascular endothelial growth factor, platelet-derived growth factor, and sphingosine-1 -phosphate (27). Angiogenic factors released from tumor- activated platelets, along with adhesive molecules on the platelet surfaces, may contribute to the process of tumor angiogenesis, thereby facilitating tumor growth or metastasis (28). [0050] Thus, inhibition of the interaction between CLEC-2 and podoplanin is a realistic therapeutic target which will prevent tumor growth and metastasis.

[0051] Furthermore, it may be possible that podoplanin is expressed in atherosclerotic lesions and upon plaque rupture, podoplanin may contribute to pathological thrombus formation by activating platelets through CLEC-2 interaction. If this is the case, blocking the interaction between CLEC-2 and podoplanin is a therapeutic target for treatment of atherosclerotic lesions, as this would inhibit only pathological thrombus formation without substantially affecting physiological hemostasis. [0052] As reported herein, it has been discovered that podoplanin expressed on the surface of lymphatic endothelial cells also induces platelet aggregation. Such expression is important during organ development and during pathological conditions. Podoplanin- deficient mice have defects in lymphatic vessel pattern formation (13). Intracellular signaling molecules, Syk and SLP-76, regulate blood and lymphatic vascular separation (29), although they are not detected in endothelium, which suggest that Syk and SLP work by way of blood cells. Syk and SLP-76 in platelets are requisites for podoplanin-induced platelet activation mediated by CLEC-2 (15). Thus, podoplanin-induced platelet activation, through CLEC-2, may also regulate proper formation of lymphatic vessels.

[0053] Therefore, the novel platelet activation receptor, CLEC-2, is the physiological counterpart for podoplanin, and the interaction of these two molecules induces platelet aggregation. The sialic acid residue on O-glycans of podoplanin is important for this recognition by CLEC-2. The interaction between CLEC-2 and podoplanin therefore is believed to regulate tumor growth/metastasis, and furthermore, is related to the formation of lymphatic vessels.

[0054] Thus, the present invention includes methods for treating diseases which operate through interaction of CLEC-2 and podoplanin, such as tumor growth or metastasis, or thrombus formation in kidneys. Subjects amenable to such treatment may include, but are not limited to, for instance, subjects suffering from hemolytic uremic syndrome or thrombotic thrombocytopenic purpura. Thus, methods of the present invention include, but are not limited to, methods for inhibiting tumor growth or metastasis, or for inhibiting or decreasing platelet aggregation, or for inhibiting or decreasing thrombus formation in kidneys. [0055] Embodiments of the present invention include, but are not limited to, recombinant CLEC-2, compositions comprising recombinant CLEC-2, antibodies to CLEC-2 and methods of treatment comprising administering compositions comprising recombinant or isolated CLEC-2.

[0056] "Recombinant CLEC-2" is meant to refer to CLEC-2 protein expressed from a vector encoding the CLEC-2 protein or functional mutants thereof. Functional mutants include any mutation which does not destroy the activity of CLEC-2 and its ability to interact with podoplanin. Such mutants may have 90% sequence identity to CLEC-2, 91% sequence identity to CLEC-2, 92% sequence identity to CLEC-2, 93% sequence identity to CLEC-2, 94% sequence identity to CLEC-2, 95% sequence identity to CLEC-2, 96% sequence identity to CLEC-2, 97% sequence identity to CLEC-2, 98% sequence identity to CLEC-2, and 99% sequence identity to CLEC-2. Such mutants include, but are not limited to, CLEC-2 in which any transmembrane and/or intracellular domain is removed from the protein. Mutants may also include fusion proteins in which CLEC-2, or mutants thereof, are genetically engineered to be expressed as a fusion protein, fused to any other known polypeptide. Recombinant CLEC-2 also encompasses CLEC-2 expressed from a CLEC-2 nucleic acid sequence integrated into a genome and expressed therefrom or transiently expressed from a genome. Recombinant CLEC-2 also encompasses CLEC-2 overexpressed from a cell in which it naturally occurs (so-called gene activation).

[0057] The present invention also encompasses nucleic acids encoding the polypeptides of the present invention. The present invention includes vectors comprising nucleic acids of the present invention, including shuttle vectors, expression vectors, and any other cloning vector known in the art. The present invention also encompasses transfection or transformation of any type of cell with the nucleic acids of the present invention and cells so transformed or transfected. Transformation and transfection methods are commonly known to one of skill in the art, as disclosed hereinbelow.

[0058] Compositions comprising polypeptides of the present invention, encoding CLEC- 2 and/or mutants thereof, may also include any one or more pharmaceutically acceptable carriers, diluents and/or excipients. By "pharmaceutically acceptable" it is meant that the carrier, diluent or excipient must be compatible with CLEC-2 as provided according to the present invention, and not be deleterious to a recipient thereof. Suitable carriers, diluents or excipients are well known in the art.

[0059] Aqueous suspensions containing CLEC-2 may be in admixture with excipients suitable for the manufacture of aqueous suspensions. Such excipients may be suspending agents known in the art, for example, sodium carboxymethylcellulose, methylcellulose, hydroxypropyl methylcellulose, sodium alginate, polyvinylpyrrolidone, gum tragacanth and gum acacia; dispersing or wetting agents may be a naturally occurring phosphatide, for example, lecithin, or condensation products or an alkylene oxide with fatty acids, for example polyoxyethylene stearate, or condensation products of ethylene oxide with long chain aliphatic alcohols, for example heptadecaethylene oxycetanol, or condensation products of ethylene oxide with partial esters derived from fatty acids and hexitol such as polyoxyethylene sorbitol monooleate, or condensation products of ethylene oxide with partial esters derived from fatty acids and hexitol anhydrides, for example polyethylene sorbitan monooleate. The aqueous suspensions may also contain one or more preservatives, for example ethyl, or n-propyl p-hydroxybenzoate, one or more coloring agents, one or more flavoring agents, and one or more sweetening agents, such as sucrose or saccharin. [0060] The compositions of the present invention can be formulated according to known methods for preparing pharmaceutically useful compositions. Formulations are described in detail in a number of sources which are well known and readily available to those skilled in the art. For example, Remington's Pharmaceutical Science by E. W. Martin describes formulations which can be used in connection with the subject invention, hi general, the compositions of the subject invention are formulated such that an effective amount of the CLEC-2 peptides, nucleic acids, antibodies or compositions is combined with a suitable carrier in order to facilitate effective administration of the composition. [0061] Further, acceptable carriers can be either solid or liquid. Solid form preparations include powders, tablets, pills, capsules, cachets, suppositories and dispersible granules. A solid carrier can be one or more substances that may act as diluents, flavoring agents, solubilizers, lubricants, suspending agents, binders, preservatives, tablet disintegrating agents or encapsulating materials.

[0062] The disclosed pharmaceutical compositions may be subdivided into unit doses containing appropriate quantities of the active component. The unit dosage form can be a packaged preparation, such as packeted tablets, capsules, and powders in paper or plastic containers or in vials or ampoules. Also, the unit dosage can be a liquid based preparation or formulated to be incorporated into solid food products, chewing gum, or lozenges. [0063] In effecting treatment of a patient, CLEC-2 as provided according to the present invention can be administered in any form or mode which makes the compound bioavailable in effective amounts, including oral and parenteral routes. For example, the polypeptides according to the present invention may be administered orally, subcutaneously, intramuscularly, intravenously, transdermally, intranasally, rectally, and the like. One skilled in the art of preparing formulations can readily select the proper form and mode of administration depending upon the disease state to be treated and the stage of the disease. [0064] The tablets, pills, capsules, and the like of the present invention may also contain one or more of the following adjuvants: binders, such as microcrystalline cellulose, gum tragacanth or gelatin; excipients, such as starch or lactose; disintegrating agents such as alginic acid, corn starch and the like; lubricants, such as magnesium stearate; glidants, such as colloidal silicon dioxide; and sweetening agents, such as sucrose or saccharin. When the dosage unit form is a capsule, it may contain, in addition to materials of the above type, a liquid carrier such as polyethylene glycol or a fatty oil. Other dosage unit forms may contain other various materials which modify the physical form of the dosage unit, for example, as coatings. Thus, tablets or pills may be coated with sugar, shellac, or other enteric coating agents. A syrup may contain, in addition to the active ingredient, sucrose as a sweetening agent and certain preservatives. Materials used in preparing these various compositions should be pharmaceutically pure and non-toxic in the amounts used.

[0065] For the purpose of parenteral administration, polypeptides according to the present invention may be incorporated into a solution or suspension. The solutions or suspensions may also include one or more of the following adjuvants: sterile diluents such as water for injection, saline solution, fixed oils, polyethylene glycols, glycerine, propylene glycol or other synthetic solvents; antibacterial agents such as benzyl alcohol or methyl paraben; antioxidants such as ascorbic acid or sodium bisulfite; chelating agents such as ethylene diaminetetraacetic acid; and buffers such as acetates, citrates or phosphates. The parenteral preparation can be enclosed in ampoules, disposable syringes or multiple dose vials made of glass or plastic.

[0066] The present invention also contemplates the generation and use of antibodies which specifically interact or bind to CLEC-2. That is, one of skill in the art knows how to generate both monoclonal and polyclonal antibodies. Such procedures are routine in the art. One of skill in the art can generate both polyclonal and monoclonal antibodies against CLEC- 2 in light of the present disclosure. Such antibodies may also include humanized antibodies. The process of humanizing antibodies is also known to one of skill in the art. Antibodies may be obtained from various animals, such as rabbits, mice, hamsters, dogs, cats, monkeys, goats, horse, etc. Furthermore, one of skill in the art may general antibodies of various classes, such as, but not limited to, IgG, IgM, IgE, IgA and IgD.

[0067] Antibodies also include diabodies, microbodies, Fab antibodies and other fragments. Generation of such antibody fragments is also routine in the art and within the capabilities of one of skill in the art. Antibodies may also include other polypeptides or compounds fused to the antibody such as reporter molecules, affinity labels, and other enzymes or proteins having unrelated functions, as needed. Obtaining such antibody fusion proteins is also routine in the art.

[0068] Methods of the present invention include, but are not limited to, methods of treating any disease which is caused by or acts through any pathway requiring the interaction of CLEC-2 with any other molecule, such as podoplanin. It is disclosed herein that CLEC-2 interacts with or binds to podoplanin, a molecule involved in platelet aggregation. Therefore, any disease known to act through or require platelet aggregation may be treated or alleviated utilizing the presently disclosed compositions. Non-limiting examples of such diseases or conditions include tumor metastasis, thrombus formation in kidneys and other platelet aggregation-related disorders.

[0069] Methods of the present invention may be performed on a subject in need thereof by administering an effective amount of CLEC-2 peptides, nucleic acids, antibodies and/or compositions of the present invention. Subjects in need thereof may include, but are not limited to, mammals, such as humans. Effective amounts of the presently disclosed compositions are determinable by routine experimentation within the capabilities of one of skill in the art. Common treatment regimens may be used in the presently disclosed methods, such as treatment regimens commonly used to administer effective amounts of other biologically active peptides and antiserum. Formulation of such compositions is within the capabilities of one of skill in the art, as discussed above.

EXAMPLES

[0070] The present invention is exemplified by the following examples. The examples are illustrative only and are not intended to in any way limit the scope of the present invention.

Example 1 - Experimental Procedures

[0071] Animals and Materials - The examples utilized genetically modified mice deficient in PLCγ2 (RIKEN Research Center for Allergy and Immunology, Yokohama, Japan) and FcRγ-chain-deficient mice (RTKEN Research Center for Allergy and Immunology, Yokohama, lapan). Rhodocytin was purified from the venom of Calloselasma rhodostoma (Meiji Pharmaceutical University, Tokyo, Japan). Experiments also used the dimeric form of soluble GPVI (hGPVI-hFc2) (Kurume University, Fukuoka, Japan) (18). The 293T-REx cell line expresses CLEC-2 under a tet repressor protein (University of Erlangen-Nϋrnberg, Erlangen, Germany) (19). Anti-human podoplanin antibody, NZ-I was generated as described previously (20). Anti-mouse podoplanin monoclonal antibody was from AngioBio Co. (D er Mar, CA). Polyclonal anti-CLEC-2 antibody was from R&D Systems (Minneapolis, MN). Anti-phospho tyrosine antibody (4Gl 0) was purchased from Upstate Biotechnology. Src kinase inhibitor PP2, and its inactive control PP3 were purchased from Calbiochem (San Diego, CA). Horm collagen (predominately Type I; derived from equine tendon) was from Nycomed (Munich, Germany). Thrombin was purchased from Sigma-Ardrich. Gly-Arg-Gly-Asp-Ser (GRGDS) peptide (SEQ ID NO:9) was obtained from the Peptide Institute (Osaka, Japan).

[0072] Platelet preparation - Venous blood from healthy drug-free volunteers was collected into 10% sodium citrate. Murine blood was drawn from mice, which were terminally anesthetized by diethyl ether, by portal vein puncture and taken into 100 μl of acid citrate dextrose. Washed human and murine platelets were obtained by centrifugation as previously described, using prostacyclin to prevent activation during the isolation procedure (21). Both sets of washed platelets were re-suspended in modified Tyrode buffer (21) at a cell density of 2.5 x 10⁸ and 1 x 10⁹/ml.

[0073] Transient transfection - Chinese hamster ovary (CHO) cells were obtained from The Health Science Research Resources Bank (Osaka, Japan).

[0074] Mouse podoplanin cDNA (AJ297944) incorporated with FLAG tag sequence was subcloned into the vector pcDNA3 (Invitrogen, mouse pod pcDNA). Cell culture and transient transfection was performed as described previously (22). Briefly, 1 x 10⁷ CHO cells were placed into an electroporation cuvette with 40 μg of mouse pod pcDNA or pcDNA only (see "Cell lines and stable transfection" below), followed by electroporation. Surface expression of mouse podoplanin was confirmed by flow cytometry using anti-mouse podoplanin monoclonal antibody as shown in Figure 3)A)i.

[0075] Cell lines and stable transfection - CLEC-2 was expressed under a tet repressor protein in the 293T-REx cell line (Invitrogen, Carlsbad, CA) and grown as described (19). CLEC-2 expression was induced by addition of 1 μg/mL doxycycline to the cell culture medium 24 to 48 hours before experimentation. Vehicle-added cells were used as control samples. A CHO cell mutant, Lec2 cells, was obtained from ATCC. Cell culture and stable transfection of human podoplanin into these cells was performed as described previously (4). Briefly, human podoplanin cDNA (AB 127958), incorporated with FLAG tag sequence, was subcloned into the vector pcDNA3 (Invitrogen, pcDNA-hpod). CHO and Lec2 cells were then transfected with pcDNA-hpod by a procedure using LipofectAMINE reagent (Invitrogen). Stable transfectants were obtained using a selective culture in a medium containing 1 mg/ml Geneticin (G418; Sigma- Aldrich). A glioblastoma cell line, LN319 (Ludwig Institute for Cancer Research, San Diego, CA), was cultured as described (20). The murine colon carcinoma cell line, Colon-26, was obtained from the Cell Resource Center for Biomedical Research, Tohoku University (Sendai, Japan). Colon-26 cells were grown in RPMI 1640 medium containing 100 units/ml penicillin, 100 μg/ral streptomycin, and 10% heat-inactivated fetal bovine serum. Lymphatic endothelial cells were purchased from Sanko Junyaku Co., Ltd (Tokyo, Japan) and cultured using the EGM-2-MV BulletKit (Takara Bio Inc., Shiga, Japan) according to the manufacturer's instructions.

[0076] Expression and purification of soluble podoplanin - The cDNA of human CLEC- 2 (AF124841), mouse CLEC-2 (AF201457), human podoplanin (AB 127958), mouse podoplanin (AJ297944), containing the extracellular domains of these proteins, were obtained by PCR using their cDNA as templates. PCR was performed using PfU turbo DNA polymerase (Stratagene, La Jolla, CA). The following oligonucleotides were used as primers (the EcoRV in the forward primer and the BgHI site in the reverse primer were underlined): human CLEC-2

(forward: CCGATTACACAGCGCAATTACCT (SEQ ID NO:1), reverse: GAAGATCTAGGTAGTTGGTCCAC (SEQ ID NO:2)), mouse CLEC-2

(forward: CCGATTACCCAGCAAAAGTATCTA (SEQ ID NO:3), reverse: GAAGATCTAAGC AGTTGGTC CAC (SEQ ID NO:4)), human podoplanin

(forward: GCGATATCAGAAGGAGCCAGCACAGG (SEQ ID NO:5), reverse: GGCAGATCTTGTTGACAAACCATCTTTC (SEQ ID NO:6)), mouse podoplanin

(forward: GTGATATCTGGGAGCGTTTGGTTCTG (SEQ ID NO:7), reverse: GGCAGATCTTGGCAAGCCATCTTTC (SEQ ID NO:8)).

[0077] The PCR products were purified by using a QIAquick gel Extraction Kit (Qiagen K.K., Tokyo, Japan), digested with EcoRV and BgHI, purified again, and then ligated to the pFUSE-hFc2 (IL2ss) vector or the pFUSE-mFc2 (IL2ss) vector (Invivogen, CA, USA), which contains interleukin 2 signal sequence (IL2ss) before the ligation site to allow secretion of Fc-Fusion proteins. pFUSE-hFc2 (IL2ss) and pFUSE-rFc2 (IL2ss) contain human IgG Fc and rabbit IgG Fc after the ligation sites, respectively. The digested PCR product of human CLEC-2 and human podoplanin were ligated to the pFUSE-hFc2 (IL2ss) vector {hCLEC-2-hFc2, hPod-hFc2, respectively). The digested PCR products of the mouse CLEC-2 and the mouse podoplanin were ligated to the pFUSE-rFc2 (IL2ss) vector (mCLEC- 2-rFc2, mPod-rFc2, respectively). The ligation mixture was transformed into Escherichia coli DH5α cells. The obtained construct was verified by restriction enzyme digestion and DNA sequencing. COS-7 cells were transfected with hCLEC-2-hFc, hPod-hFc2, mCLEC-2- rFc, and mPod-rFc2 by electroporation (22). The day following electroporation, the culture medium was replaced by reduced-serum medium, Opti-MEM (Invitrogen). For the purification of the fusion proteins, the medium was centrifuged and the obtained supernatant was applied to a column of protein A-Sepharose (Amersham Biosciences). After extensive washing with phosphate-buffered saline (PBS), the fusion proteins were eluted by 0.2 M glycin pH 2.0, followed by neutralization using IM Tris pH 10.0. The proteins were dialyzed against PBS.

[0078] Platelet aggregation - Washed human or murine platelets at the indicated concentrations were stimulated by addition of 1.5 x 10⁷/ml of the indicated cell lines, 20 μg/ml of mPod-rFc2, 10 nM rhodocytin, 2 μg/ml of Horm collagen, 0.5 U/ml of thrombin, or 5 μM U46619. Platelet aggregation was monitored by measuring light transmission with the use of an AG-IO aggregation analyzer (Kowa, Tokyo, Japan) for 15 min under constant stirring at 1000 rpm at 37°C. The instrument was calibrated with buffer for 100% transmission. Where indicated, DMSO, 10 μM PP3, 10 μM PP2, or 1 mM GRGDS were incubated with the washed platelets for 5 min at 37°C, before stimulation, hi some experiments, 2.5 x 10⁷/ml of cell lines expressing podoplanin were incubated with 2.5 mg/ml of the indicated recombinant proteins for 10 min at room temperature before addition to washed platelets. The final concentrations of podoplanin expressing cell lines and that of the recombinant proteins in the platelet suspension were 1.5 ^χ 10⁹/ml and 10 μg/ml, respectively. [0079] Western blot analysis - Western blot was performed as described previously (21). Briefly, washed human platelets (1 x 10⁹/ml) were pretreated with 1 mM GRGDS peptide to inhibit platelet aggregation. Then, the platelets were stimulated by addition of 1.5 x 10⁹/ml of CHO cells transiently-transfected with mouse podoplanin, for the indicated duration. Reactions were terminated by addition of 2x ice-cold lysis buffer. Platelet lysates were precleared and detergent-insoluble debris was clarified as described. The supernatant was dissolved with SDS sample buffer, separated by 10% SDS-PAGE, electrotransferred, and Western blotted using anti-phospho tyrosine antibody (4G10). Washed murine platelets (2.5 x 10⁸/ml) pretreated with 1 mM GRGDS were stimulated by 20 μg/ml of mPod-rFc2. Reactions were terminated by addition of 4x SDS sample buffer, separated by 10% SDS- PAGE, electrotransferred, and Western blotted using anti-phospho tyrosine antibody (4Gl 0). [0080] Flow Cytometry Studies - Cells suspended in PBS (5 x 10⁶/ml, 100 μl) were incubated with 5 μg/ml of the indicated first antibodies or 100 μg/ml of the indicated recombinant proteins, for 20 min at room temperature. After washing with PBS, the cells were re-suspended with 100 μl PBS and stained using 3 μl of the secondary antibodies conjugated with FITC, for 15 min. Stained cells were analyzed immediately using a FACScalibur (Becton Dickinson). Data were recorded and analyzed using CellQuest software.

[0081] Experimental lung metastasis — CHO/hPod cells were harvested, washed, and resuspended in Hanks' Balanced Solutions (HBSS; 2.5xlO⁶ cells/ml). Next, the cells were incubated with 100 μg of hPod-hFc (hPod-Fc), mCLEC-2-hFc (mCL2-Fc), or control PBS, and inoculated intravenously (2.5x10⁵ cells/mouse) into the lateral tail vein of femaile BALB/c-nu/nu mice. After 17 days, the mice were euthanized and the surface lung metastatic foci were counted.

Example 2 - Platelet aggregation induced by podoplanin is dependent on a Src kinase and PLCΎ2, but not on GPVI

[0082] The characteristics of podoplanin-induced platelet aggregation were investigated and compared with those of well-defined platelet stimulators. CHO cells transiently transfected with mouse podoplanin (CHO/mPod) stimulates aggregation of human platelets with a long lag phase, which was completely inhibited by the Src kinase inhibitor PP2, but not by its inactive enentiomer PP3. (See, Figure IA). It was previously reported that both human and mouse podoplanin stimulates aggregation of murine platelets and that non- transfected CHO cells do not stimulate platelet aggregation (7). It was confirmed that CHO cells stably transfected with human podoplanin stimulate aggregation of human platelets, which was completely inhibited by PP2, but not by PP3 (data not shown). Consistent with the susceptibility to PP2₅ protein tyrosine phosphorylation was observed during podoplanin- induced platelet aggregation. (See, Figure IB).

[0083] Whether recombinant podoplanin stimulates platelet aggregation was also examined. The recombinant extracellular domain of mouse podoplanin was expressed as a dimeric rabbit immunoglobin Fc domain fusion protein (mPod-rFc2) and murine platelets were stimulated with the protein. To exclude the possibility that the Fc portion of the fusion protein interacts a stimulatory Fc receptor, FcγRUA, murine platelets were used which lack FcγRIIA. mPod-rFc2 also stimulated aggregation. (See, Figure 1C). This indicates that podoplanin, but not other cellular components, is responsible for platelet aggregation. [0084] The long lag phase before the initiation of aggregate formation and Src kinase- dependent platelet activation are similar to platelet activation induced by collagen through GPVI or by the snake venom rhodocytin (16), but not through GPCR, wherein platelet aggregation is not dependent on Src kinase. Therefore, the possibility that podoplanin activation of platelets by binding to GPVI was investigated using murine platelets deficient in GPVI/FcRγ-charn complex.

[0085] Podoplanin or rhodocytin, but not collagen, induced platelet aggregation in mice deficient in GPVI/FcRγ-chain complex. (See, Figure 2A). This indictaes that GPVI is not a target protein of podoplanin.

[0086] It was then determined whether podoplanin activates platelets by a mechanism similar to rhodocytin. A novel class of platelet activation receptor, C-type lectin-like receptor 2 (CLEC-2), was discovered, which belongs to a non-classical C-type lectin family and which is a receptor on the platelet membrane for rhodocytin (15). Since PLCγ2 is a crucial signaling molecule for rhodocytin-induced platelet aggregation through CLEC-2 (15), it was determined whether PLCγ2 is required for podoplanin-induced platelet aggregation. Podoplanin, as well as rhodocytin, was found to induce platelet aggregation in wild type mice, but not in PLCγ2-deficient mice. (See, Figure 2B). On the other hand, thrombin invariably induced platelet aggregation in both mice. (See, Id). Since all the data hitherto have suggested that podoplanin-induced platelet aggregation has a profile quite similar to that of rhodocytin, it seemed likely that CLEC-2 is a physiological counterpart for podoplanin.

Example 3 - Podoplanin stimulates platelet aggregation by interacting with CLEC-2

[0087] The interaction of recombinant CLEC-2 with podoplanin-expressing CHO cells was investigated. These studies made use of the recombinant extracellular domain of human CLEC-2, expressed as a dimeric human immunoglobin Fc domain fusion protein (hCLEC-2- hPc2). A fusion protein of human GPVI and human IgG Fc (hGPVI-hFc2) were used as negative controls. CHO cells were transiently-transfected with pcDNA alone, or pcDNA encoding mouse podoplanin. Flow cytometric analysis using a specific anti-podoplanin antibody confirmed podoplanin expression on the surface of CHO cells. (See, Figure 3A-i). Mock- (CHO/MOCK), or mouse podoplanin-transfected CHO cells (CHO/mPod), were incubated with hCLEC-2-hFc2, or hGPVI-hFc2, and then specific binding was detected with FITC-labeled anti-human IgG hCLEC-2-hFc2. (See, Figure 3A-ii). However, no specific binding was detected for hGPVI-hFc2 bound to podoplanin-expressing CHO cells. (See, Figure 3 A-iii). This indicates that CLEC-2 associates with podoplanrn. [0088] Inversely, specific binding or interaction of recombinant podoplanin with CLEC- 2-expressing cells was also examined. CLEC-2 expressing cells were 293T-REx cells in which CLEC-2 was expressed under the control of a tet repressor protein. The addition of doxycycline to transfected 293T-REx cells induced surface expression of CLEC-2, as assessed with a specific antibody to the lectin receptor. (See, Figure 2A-iv). As shown in Figure 3A-v and Figure 3A-vi, mPod-rFc2, but not its negative control, mCLEC-2-rFc2 (a fusion protein of mouse CLEC-2 and rabbit IgG Fc), bound to the CLEC-2-expressing 293T- REx cell line. This further confirms that podoplanin is a physiological ligand of CLEC-2. [0089] It was also confirmed that the extracellular domain of human podoplanin, expressed as a dimeric human immunoglobin Fc domain fusion protein (hPod-hFc2), but not its negative control, hGPVI-hFc2, bound to the human CLEC-2-expressing 293T-REx cell line (data not shown). Moreover, pretreatment of human podoplanin-expressing CHO cells with hCLEC-2-hFc2, but not with hGPVI-hFc2, completely inhibited podoplanin-induced platelet aggregation (data not shown). Furthermore, hCLEC-2-hFc2 did not inhibit platelet aggregation stimulated by agonists other than podoplanin or rhodocytin. (See, Figure 3C). These findings confirm that podoplanin stimulates platelet aggregation by interacting with CLEC-2.

Example 4 - Sialic acid on O-glycans in podoplanin is necessary for its interaction with

CLEC-2

[0090] CLEC-2 belongs to a family of non-classical C-type lectin-like proteins which contain a C-type lectin-like domain (CTLD). The CTLD is homologous to a carbohydrate recognition domain, but lacks the consensus sequence for binding sugars and calcium (23). To assess the attribution of glycosylation to podoplanin interaction with CLEC-2, recombinant podoplanin was stably expressed on wild type CHO cells (CHO) or CMP -sialic acid transporter-deficient Lec2 CHO cells (Lec2), which lack 90% of the common sialic acid decoration in both glycoproteins and glycolipids (4, 5).

[0091] As reported previously (4), podoplanin expressed on Lec2 cells (Lec2/hPod) fails to induce platelet aggregation, although equal expression of the recombinant molecules in both CHO/hPod and Lec2/hPod was confirmed by flow cytometry using a specific antibody. {See, Figures 4A and 4B). Consistent with these findings, recombinant hCLEC-2-hFc2 associates with podoplanin expressed on CHO/hPod, but not podoplanin expressed on Lec2/hPod. (See, Figure 4C). These observations indicate that the presence of sialic acid on O-glycans of podoplanin is critical for interaction between CLEC-2 and podoplanin.

Example 5 - Endogenous podoplanin expressed on the surface of tumor cells or lymphatic endothelial cells stimulates platelet aggregation

[0092] Since platelet aggregation, shown in Figure 3B, was induced by CHO cells that were forced to express podoplanin by transfection, it was investigated whether tumor cell lines, which express endogenous podoplanin, also stimulate platelet aggregation by interacting with CLEC-2. The human glioblastoma cell line, LN319, and the murine colon carcinoma cell line, Colon-26, highly express podoplanin and induce platelet aggregation (20, 24). The surface expression of podoplanin in LN319 cells, Colon-26 cells, and platelet aggregation, stimulated by the cell lines, was confirmed. (See, Figures 5 A and 5B). [0093] Pretreatment of LN319 or Colon-26 with hCLEC-2-hFc2, but not with hGPVI- hFc2, completely inhibited platelet aggregation induced by the cell lines. (See, Figures 5 A and 5B). These findings indicate that endogenously expressed podoplanin in tumor cells also induces platelet activation through interaction with CLEC-2.

[0094] Podoplanin is highly expressed in lymphatic endothelial cells, but not in vascular endothelial cells. Podoplanin is used as a specific marker of lymphatic endothelium (6, 12). It was therefore investigated whether lymphatic endothelial cells (LECs), which endogenously express podoplanin, stimulate platelets through interacting with CLEC-2. It was confirmed that LECs express podoplanin and stimulate platelet aggregation (4). (See, Figure 5C). The LEC-induced platelet aggregation was completely inhibited by hCLEC-2- hFc2, but not by hGPVI-hFc2. {See, Figure 5C). This indicates that podoplanin expressed on LECs is also able to stimulate platelet aggregation by interacting with CLEC-2,

Example 6 - Inhibition of podoplanin-induced metastasis by hPod-hFc2 or mCLEC-2-hFc2 [0095] It was determined whether human hPod-hFc2 and mCLEC-2-hFc2 could suppress podoplanin-induced pulmonary metastasis in an experimental metastasis model. Injection of CHO/hPod cells led to development of multiple lung metastatic foci. {See, Figure 6). The number of metastatic lung nodules in mice injected with hPod-hFc2 or mCLEC-2-F2 was lower than that observed in CHO/hPod with vehicle. {See, Figure 6B). These results indicate that hPod-hFc2 and mCLEC-2-hF2 inhibit CLEC-2-podoplanin interaction by saturating their counterparts on the surface of platelets and CHO/hPod, respectively, and inhibit pulmonary metastasis. These results further show that the podoplanin-CLEC-2 interaction is pathophysiologically important in podoplanin-induced platelet aggregation and metastasis. [0096] All references, including publications, patents, and patent applications, cited herein are hereby incorporated by reference to the same extent as if the disclosure each reference were individually and specifically indicated to be incorporated by reference and were set forth in its entirety herein. The present application also specifically incorporates herein by reference the entirety of the disclosure of U.S. Provisional Patent Application Serial No. 60/913,909.

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Claims

What is claimed is:

L An isolated polypeptide for inhibiting tumor growth or metastasis, or inhibiting or decreasing platelet aggregation, or inhibiting or decreasing thrombus formation in kidneys, wherein the polypeptide comprises C-type Lectin-like Receptor 2 (CLEC-2) or an amino acid sequence which is 95% identical thereto.

2. An isolated nucleic acid encoding the polypeptide according to claim 1.

3. An isolated polypeptide for inhibiting tumor growth or metastasis, or for inhibiting or decreasing platelet aggregation, or for inhibiting or decreasing thrombus formation in kidneys, wherein the isolated polypeptide comprises at least an extra-cellular domain of C- type Lectin-like Receptor 2 (CLEC-2) or an amino acid sequence which is 95% identical thereto.

4. An isolated nucleic acid encoding the polypeptide according to claim 3.

5. A composition comprising the polypeptide according to claim 1, for inhibiting tumor growth or metastasis, or for inhibiting or decreasing platelet aggregation, or for inhibiting or decreasing thrombus formation in kidneys.

6. A composition comprising the polypeptide according to claim 3, for inhibiting tumor growth or metastasis, or for inhibiting or decreasing platelet aggregation, or for inhibiting or decreasing thrombus formation in kidneys.

7. A pharmaceutical composition comprising the polypeptide according to claim 1 and a pharmaceutically acceptable carrier, diluent and/or excipient, for inhibiting tumor growth or metastasis, or for inhibiting or decreasing platelet aggregation, or for inhibiting or decreasing thrombus formation in kidneys.

8. A pharmaceutical composition comprising the polypeptide according to claim 3 and a pharmaceutically acceptable carrier, diluent and/or excipient, for inhibiting tumor growth or metastasis, or for inhibiting or decreasing platelet aggregation, or for inhibiting or decreasing thrombus formation in kidneys.

9. An isolated antibody for inhibiting tumor growth or metastasis, or for inhibiting or decreasing platelet aggregation, or for inhibiting or decreasing thrombus formation in kidneys, wherein the isolated antibody inhibits the interaction between C-type Lectin-like Receptor 2 (CLEC-2) and podoplanin.

10. A pharmaceutical composition comprising the antibody according to claim 9 and a pharmaceutically acceptable carrier, diluent and/or excipient, for inhibiting tumor growth or metastasis, or for inhibiting or decreasing platelet aggregation, or for inhibiting or decreasing thrombus formation in kidneys.

11. A method of inhibiting tumor growth or metastasis, which comprises administering to a subject in need thereof an effective amount of a polypeptide comprising C- type Lectin-like Receptor 2 (CLEC-2) or an amino acid sequence which is 95% identical thereto.

12. A method of inhibiting tumor growth or metastasis, which comprises administering to a subject in need thereof an effective amount of an antibody, wherein the antibody inhibits the interaction of C-type Lectin-like Receptor 2 (CLEC-2) with podoplanin.

13. A method of inhibiting tumor growth or metastasis, which comprises administering to a subject in need thereof an effective amount of a compound which inhibits the interaction of C-type Lectin-like Receptor 2 (CLEC-2) with podoplanin.

14. A method of inhibiting or decreasing platelet aggregation, which comprises administering to a subject in need thereof an effective amount of a polypeptide comprising C- type Lectin-like Receptor 2 (CLEC-2) or an amino acid sequence which is 95% identical thereto.

15. A method of inhibiting or decreasing platelet aggregation, which comprises administering to a subject in need thereof an effective amount of an antibody, wherein the antibody inhibits the interaction of C-type Lectin-like Receptor 2 (CLEC-2) with podoplanin.

16. A method of inhibiting or decreasing platelet aggregation, which comprises administering to a subject in need thereof an effective amount of a compound which inhibits the interaction of C-type Lectin-like Receptor 2 (CLEC-2) with podoplanin.

17. A method of inhibiting or decreasing thrombus formation in kidneys, which comprises administering to a subject in need thereof an effective amount of a polypeptide comprising C-type Lectin-like Receptor 2 (CLEC-2) or an amino acid sequence which is 95% identical thereto.

18. A method of inhibiting or decreasing thrombus formation in kidneys, which comprises administering to a subject in need thereof an effective amount of an antibody, wherein the antibody inhibits the interaction of C-type Lectin-like Receptor 2 (CLEC-2) with podoplanin.

19. A method of inhibiting or decreasing thrombus formation in kidneys, which comprises administering to a subject in need thereof an effective amount of a compound which inhibits the interaction of C-type Lectin-like Receptor 2 (CLEC-2) with podoplanin.

20. The method according to any one of claims 17-19, wherein the subject suffers from hemolytic uremic syndrome or thrombotic thrombocytopenic purpura.

21. Use of any one of the polypeptides, nucleic acids, compositions or antibodies of claims 1-10 in preparation of a medicament for inhibiting tumor growth or metastasis, or inhibiting or decreasing platelet aggregation, or inhibiting or decreasing thrombus formation in kidneys.