CA2803254A1 - Slit and roundabount (robo) mediated lymph vessel formation and uses thereof - Google Patents

Abstract

The invention provides a method for preventing or treating a disorder mediated by a Slit protein, which comprises administering to a subject a therapeutically effective amount of an agent for modulating or preventing interactions between the Slit protein and a Robo protein, wherein the disorder involves lymph vessel formation. The invention further discloses a composition for preventing or treating a disorder mediated by the Slit protein and a method for prognosing or diagnosing a disease or disorder mediated by the Slit protein.

Description

SLIT AND ROUNDABOUNT (ROBO) MEDIATED LYMPH VESSEL FORMATION
AND USES THEREOF

BACKGROUND OF INVENTION
Field of the Invention [0001] The invention relates generally to reagents and methods for preventing, treating, or diagnosing a disease or a disorder associated with Slit-Robo signaling pathways;
particularly, the invention relates to methods and reagents for treating cancer progression and cancer metastasis.

Background Art [0002] Cancer metastasis in advanced cancers is a main cause for mortality in cancer patients.
A great deal of efforts have been directed to cancer research and therapy, particularly the mechanisms underlying cancer growth and metastasis. However, the molecular mechanism responsible for cancer metastasis is still not well understood.
Generally, cancer cells metastasize by invasion of local tissues. Once the cancer cells break away from their primary sites, they may enter the circulation system and lymph system, by which the cancer cells then travel to different sites, where they may establish new growth.
The fact that cancer cells often metastasize to the lymph nodes suggests that the lymph system plays an important role in the metastasis of cancers..

[0003] Lymphatic system is important for maintaining fluid balance in the body, immune response, and absorption of fatty acids. In addition to these normal physiological functions, lymphatic system is also involved in lymphatic fluid swelling and inflammatory reactions. In patients with metastatic tumors, significant increases in lymph vessel formation are frequently observed. These observations suggest the importance of the lymphatic system in facilitating cancer metastasis. However, it is not entirely clear how the lymphatic system plays a role in cancer metastasis.

[0004] Because the lymphoid systems are involved in cancer metastasis, targeting tumor lymphatic metastasis, therefore, represents a rational approach to cancer treatment.
SUMMARY OF INVENTION

[0005] One aspect of the present invention relates to methods for preventing or treating a disorder mediated by a Slit protein. A method in accordance with one embodiment of the invention for preventing or treating a disorder mediated by a Slit protein includes administering to a subject a therapeutically effective amount of an agent that modulates or prevents interactions between the Slit protein and a Robo protein, wherein the disorder involves lymph vessel formation. The agent may be an antibody against the Slit protein, an antibody against the Robo protein, a fragment of the extracellular domain of the Robo protein, or a fusion protein of a fragment of the extracellular domain of the Robo protein.
The Slit protein may be Slit2. The Robo protein may be Robol or Robo4.

[0006] Another aspect of the invention relates to compositions for preventing, or treating a disorder mediated by a Slit protein. A composition for preventing, or treating a disorder mediated by a Slit protein includes an agent that can modulate or interfere with interactions between the Slit protein and a Robo protein; and a pharmaceutically acceptable excipient, carrier, or solvent, wherein the disorder involves lymph vessel formation. The agent may be an antibody against the Slit protein, an antibody against the Robo protein, a fragment of the extracellular domain of the Robo protein, or a fusion protein of a fragment of the extracellular domain of the Robo protein. The Slit protein may be Slit2. The Robo protein may be Robol or Robo4.

[0007] Another aspect of the invention relate to methods for prognosis or diagnosis of a disease or disorder mediated by a Slit protein. A method for prognosis or diagnosis of a disease or disorder mediated by a Slit protein includes: (a) obtaining a test sample from a test subject and assessing expression levels of a Slit protein, a Robo protein, or both the Slit protein and the Robo protein in the test sample; (b) obtaining a control sample from a subject not having the disease or disorder and assessing the expression levels of the Slit protein, the Robo protein, or both the Slit protein and the Robo protein in the control sample; and (c) comparing the expression levels assessed in (a) and (b) to obtain a result about the disease or disorder mediated by the Slit protein. The Slit protein may be Slit2. The Robo protein may be Robol or Robo4. The expression levels may be DNA, RNA, and/or protein expression levels.

[0008] Other aspects and advantages of the invention will be apparent from the following description and the appended claims.

BRIEF DESCRIPTION OF DRAWINGS

[0009] FIG. 1 shows the expressions of Slit2 in cancers. (a) Immunoblotting of cell lysates from various cancer cell lines with the anti-Slit2 Ab. The Slit2/293 cells are used as the positive control while the V/293 cells are used as the negative control. (b) The immunohistochemical staining of human cancers with the anti-Slit2 Ab. Arrows indicated the expression of Slit2 on tumor cells. Bar scale, 100 .im for the upper panel and 10 .im for the lower panel, respectively. (c) The Slit2 gradients on human breast invasive carcinoma visualized by the staining with the anti-Slit2 Ab. Arrows indicated the microvessels within tumors. Bar scales, 100 .im for the upper panel and 40 .im for the lower panel, respectively.

[0010] FIG. 2 shows the construction of Slit2 transgenic mice. (a) A diagram of the human Slit2 (hSlit2) expression vector used for the construction of transgenic mice.
(b) Spot hybridization of the newly generated hSlit2 transgenic mice, indicating hSlit2-positive in #9 mouse. (c) Southern blot analysis of the newly generated hSlit2 transgenic mice, indicating hSlit2-positive in #9 mouse. (d) Immunoblotting analysis for the hSli2 expression in the pancreas tissues of the hSlit2 transgenic mice using 5A5 monoclonal antibody. The isotypic IgG is used as a negative control. hSlit2/293 cells are used as a positive control. (e) Immunoblotting analysis of hSlit2 expression using M2 monoclonal antibody specific for the FLAG protein. (f) PCR analysis for the FLAG
expression in the Slit2-positive transgenic mice. C57 mice are used as a negative control.
(g) No hSlit2 expression in the pancreas tissue of a normal mouse (upper left panel), the rest of the panels show the positive expression of hSlit2, insulin, robol, and vWF in the pancreas tissues of Ripl-Tag2 transgenic mice using immunohistochemical analysis. (h) The expression of hSlit2 protein determined by immunohistochemical analysis is higher in the pancreas tissues of hSlit2/Ripl-Tag2 transgenic mice than that of the Ripl-Tag2 mice. * p < 0.05; scale bar: 10 m.

[0011] FIG. 3 shows that Slit2 promotes the growth of pancreatic tumor.
Comparison between Ripl-Tag2 and Slit2/Ripl-Tag2 transgenic mice in terms of (a) number of angiogenic pancreatic islets, (b) tumor volume, (c) tumor number, (d) leukocyte count in pancreas and blood. (e) Florescence staining of the pancreatic tumors in the transgenic mice. (f) Hematoxylin and eosin (HE) staining of the pancreatic tumors in the transgenic mice. Both florescence staining and HE staining show the larger tumor size and more blood vessels in the Sli2/Ripl-Tag2 transgenic mice than the Ripl-Tag2 transgenic mice. Comparison between the Sli2/Ripl-Tag2 transgenic mice and the Ripl-Tag2 transgenic mice in (g) the blood vessel density, (h) proliferation index, and (i) apoptosis index as determined by immunohistochemical analysis, indicating the Sli2/Ripl-Tag2 transgenic mice have more angiogenesis, higher proliferation, and reduced apoptosis than the Ripl-Tag2 transgenic mice. Scale bar: 50 m; *: p <
0.05;
**:p<0.01.

[0012] FIG. 4 shows the lymph vessel formation and tumor lymphatic metastasis in the Slit2/Rip1-Tag2 transgenic mice. The Slit2-overexpression Slit2/Ripl-Tag2 transgenic mice show (a) the tumor intestinal lymphatic neovascularization (indicated by arrows), (b) tumors inside the lymph vessels by HE staining (indicated by arrows), and (c) staining of the intestinal lymphoid nodes. LYVE-1 staining is for detecting the endothelial cells of the lymph vessels. There are more lymphatic endothelial cells in the peripheral tissues of the pancreas in the Slit2/Rip-l-Tag2 transgenic mice (d, e, f) than that of the Ripl-Tag2 transgenic mice as determined by immunohistochemical analysis (g, h, i). The lymphatic endothelial cell proliferation in the pancreas is higher in the Slit2/Ripl-Tag2 transgenic mice (1) than that in the wild type C57 mice (j) and the Ripl-Tag2 transgenic mice (k) as determined by the immunofluorescence staining using anti-LYVE-1 antibody. The expression of Robol and LYVE-1 in the lymphatic endothelial cells in the pancreas are determined by immunofluorescence analysis using anti-Robol antibody (m) and anti-LYVE-1 antibody (n). The overlay of Robol and LYVE-1 expression is shown (o). LN: lymphoid node; C: tumor tissue; *: p <
0.05;
Scale bars: 1 mm (j-1) and 100 m (the rest of panels).

[0013] FIG. 5 shows that Slit2 recombinant protein promotes migration and vessel formation of human lymphatic endothelial cells. Robol expression on the cell membrane of lymphatic endothelial cells (a, left panel (Rb7); right panel (R4)). (b) The density of lymph vessels in the peripheral of pancreas in the Slit2/Ripl-Tag2 transgenic mice is 1.5-fold higher than that in the Ripl-Tag2 mice. The recombinant Slit2.1 protein promotes migration of lymphatic endothelial cells (c) and vessel formation (d, e). the migration and vessel formation can be specifically inhibited by R5 (c, d, and e). "None"
indicates a negative control. VEGF-C serves as a position control. Bar: 25 m;
*: p <
0.05; **: p < 0.01.

[0014] FIG. 6 shows (A) hRobo-Fc and hSlit2 protein interaction; (B) R5 (a monoclonal antibody binds to the first Immunoglobulin-like motif of Robol protein) inhibits the hRobo-Fc and hSlit2 protein interaction.

[0015] FIG. 7 shows the expression of a - 85 kD hRobo-Fc recombinant protein detected by a rabbit anti-human IgG conjugated with horse radish peroxidase.

[0016] FIG. 8 shows a functional analysis of the anti-Slit2 monoclonal antibody and its use in various human tumor tissues. Immunoblotting is used to test the ability of S 1 and S3 antibodies to detect full-length or fragments of Slit2 protein. (a) S 1 detects the full-length Slit2 protein by binding to the N-terminal of the protein. 9E10 and S3 antibodies detect the full-length Slit2 by binding to the C-terminal of the protein. S 1 and S3 antibodies specifically detect human Slit2 protein and S 1 antibody also detects human Slitl and Slit3 proteins. 9E10 serves as a positive control. Si monoclonal antibody is used to detect Slit2 expression in colorectal cancer and breast cancer (b), primary lung cancer and its metastatic lung cancer (c), and colorectal cancer with and without lymphatic metastasis (d). mIgG serves as a negative control (immunohistochemical analysis). Scale bar: 10 m.

DETAILED DESCRIPTION

[0017] Embodiments of the invention relate to methods, compositions, or agents for preventing, treating, or diagnosing a disease or disorder mediated by Slit proteins, particularly those disease or disorders associated with lymph vessel formations. The diseases or disorders mediated by Slit protein involve the interactions between Slit proteins and their receptors, Robo proteins. These disorders or diseases include tumor growth and/or tumor metastasis, such as tumor lymphatic metastasis.

[0018] Inventors of the present invention have unexpectedly found that interactions between Slit proteins and Robo proteins (which are receptors of Slit proteins) result in signal transduction that leads to lymph vessel formation. Therefore, reagents that can modulate or interfere with the interactions between the Slit proteins and their receptors (Robo proteins) may be used to diagnose, treat or control undesired lymph vessel formation, such as those involved in cancer metastasis.

[0019] Some embodiments of the invention relate to reagents for modulating or interfering with the interactions between Slit proteins and their receptors (Robo proteins). These reagents may be protein fragments or analogs of Slit or Robo proteins (including fusion proteins or fragments of these proteins), or antibodies (polyclonal, monoclonal, or humanized) against Slit or Robo proteins. Some embodiments of the invention relate to methods for diagnosing, preventing, or treating disorders medicated by Slit-Robo interactions, particularly those involving cancer progression and cancer metastasis.

[0020] The following examples illustrate embodiments of the invention using Slit2 as an example of the Slit proteins and Robo 1 or Robo4 as an example of the Robo proteins.
However, embodiments of the invention may also include other Slit or Robo proteins that are involved in signal transductions related to lymph vessel formations.

[0021] The Slit genes encode secreted proteins essential for neural development. These proteins function as "neurological" migratory cues. In mammals, there are three known Slit genes, i.e., Slitl, Slit2, and Slit3. These genes also express in non-nervous system (Holmes, G. P. et al. Mech. Dev. 79:57-72 (1998)). For example, mRNAs for Slit2 and Slit3 (but not for Slitl) are found in rat endothelial cells (Wu, J. Y. et al.
Nature 410:948-952 (2001)).

[0022] Structurally, Slit proteins each possess 4 leucine-rich repeat sequences, 9 epidermal growth factor (EGF) like functional domains, one ALPS element located between the sixth and seventh EGF functional domains, and one cysteine knot at the C-terminal.
Although Slit proteins are soluble proteins, most Slit2 proteins are associated with cell surface.

[0023] Slit proteins bind to their receptors, Roundabout (Robo) proteins.
Together, Slit and Robo proteins may function as repellents in axon guidance (Kidd, T. et al.
Cell 92:201-215 (1998)) and branching (Wang, K.-H. et al. Cell 96:771-784 (1999)), and in neuronal migration (Wu, W. et al. Nature 400:331-336 (1999)). In addition, they also function as endogenous inhibitors for leukocyte chemotaxis (Wu, J. Y. et al.
Nature 410:948-952 (2001)).

[0024] Being an endogenous inhibitor for leukocyte chemotaxis, Slit proteins (e.g., Slit2) play a role in inflammation (Wu, J. Y. et al. Nature 410:948-952 (2001)). In inflammation processes, leukocytes attach to the endothelial cells in blood vessels, penetrate capillary wall, and arrive at the tissues of damage or infection. It is generally believed that leukocyte attachment and penetration are well coordinated in time and location by the actions of many molecules, such as chemotactic factors, cell adhesion molecules, cytoskeleton proteins, and metalloenzymes. Wu et al. show that Slit2 can inhibit leukocyte chemotaxis induced by chemotactic factors in vitro.
Moreover, inhibition of leukocyte chemotaxis by Slit2 can be blocked by a soluble extracellular fragment of Robo protein (RoboN) (Wu et al., Nature, 410:948, 2001), proving that inhibition of leukocyte chemotaxis by Slit2 is mediated by Robo receptor-mediated signal pathways.

[0025] Robo proteins are transmembrane proteins with a single transmembrane domain.
They function as receptors of Slit proteins (e.g., Slit2). There are four Robo genes, robol, robo2, rig-1 and robo4, in mammals. Robo proteins also express outside the nervous system (Piper, M. et al. Mech. Dev. 94: 213-217 (2000)). For example, Robol RNA is found in mouse leukocytes (Wu, J. Y. et al. Nature 410:948-952 (2001)).
Further, human endothelial cells express Robo4 (Huminiecki, L. et al.
Genomics.
79:547-552 (2002)).

[0026] Upon binding by the ligands (i.e., Slit proteins), the Robo receptors transduce intracellular signals that eventually lead to inhibitory actions in various biological processes, including axon guidance and branching, neuronal migration, and leukocyte chemotaxis.

[0027] As noted above, Robo family proteins are transmembrane proteins, having a single transmembrane domain. Structurally, the extracellular domains of Robo 1, Robo2, and Robo3 each have 5 immunoglobulin-like (Ig-like) domains and 3 fibronectin type III
sequences. The intracellular domains contain many structural sub-domains, including CCO, CC1, CC2, and CC3 sequences. Robo4 is smaller and has 2 immunoglobulin-like domains in its extracellular domain, and it contains CCO and CC2 sequences in its intracellular domain.

[0028] It has been shown that Slit2 binds to the first immunoglobulin-like domain of Robol through multiple interactions (Chen et al., J Neurosci. 2001 21:1548-1556). In addition, a monoclonal antibody R5, which binds to the first immunoglobulin-like domain of Robol protein, is shown to neutralize the Slit2-mediated angiogenesis (Wang et al., Cancer Cell. 2003 4:19-29), suggesting the specific binding of Slit2 to Robol is essential for the Slit2-mediated angiogenesis.

[0029] Slit and Robo proteins have been shown to be involved in various biological activities, including angiogenesis. However, inventors of the present invention have unexpectedly found that Slit-Robo interactions are also involved in lymphogenesis (i.e., lymph vessel formation). More importantly, inventors found that such lymph vessel formation is involved in cancer metastasis. Therefore, agents that can interrupt Slit-Robo interactions can be useful agents in the prevention and treatments of cancers, particularly in the prevention and treatment of cancer metastasis.

[0030] Slit2 is expressed at high levels in various cancer tissues (see FIG.
1). In contrast, Slit2 is expressed at low or undetectable levels in normal tissues or atypical proliferating tissues. The fact that Slit2 expression is up-regulated in various cancers suggests that Slit2 may play an important role in cancer progression and/or metastasis. As described in the following sections, Slit2 indeed plays a rile in cancer progression and metastasis.
One possible mechanism by which Slit2 may contribute to cancer progression and metastasis is by promoting tumor lymph vessel formation.

[0031] The possibility that Slit2 plays a role in tumor progression and metastasis has been tested in animal models. For example, mice expressing high level Slit2 were generated, as illustrated in FIG.2. Crossing the Slit2 transgenic mice with Ripl-Tag2 transgenic mice, which can spontaneously develop pancreatic cancers, produce progenitor mice (Slit2/Ripl-Tag2 mice). Details regarding the construction of the Slit2/Ripl-Tag2 transgenic mouse strain are described later in the EXAMPLES section. These progenitor mice (Slit2/Rpil-Tag2) produce significantly higher levels of Slit2, as determined by immunohistochemical analysis (FIG. 2h). In addition, these progenitor mice exhibit substantial increase in new lymph vessel formation, increased tumor sizes, tumor metastasis to intestine membrane lymph nodes (FIG. 3). These results indicate that increased expression of Slit2 is associated with increased cancer growth and increased cancer metastasis.

[0032] Slit2 promotes the growth and metastasis of pancreatic cancer.
Comparison between Ripl-Tag2 and Slit2/Ripl-Tag2 transgenic mice reveals that Slit2 increases the number of angiogenic pancreatic islets (FIG. 3a), tumor volume (FIG. 3b), and tumor number (FIG. 3c), but not leukocytes count in pancreas and blood (FIG. 6d).
FIG. 3e shows florescence staining of the pancreatic cancers in the transgenic mice, while FIG. 3f shows hematoxylin and eosin (HE) staining of the pancreatic cancers in the transgenic mice. Both florescence staining and HE staining show the larger tumor size and more blood vessels in the S1i2/Ripl-Tag2 transgenic mice, as compare with the Ripl-Tag2 transgenic mice.

[0033] Comparison between the S1i2/Ripl-Tag2 transgenic mice and the Ripl-Tag2 transgenic mice reveals that Slit2 expression increases the blood vessel density (FIG. 3g), enhances the proliferation index (FIG. 3h), and reduces the apoptosis index (FIG. 3i), as determined by immunohistochemical analysis. These results indicate that the S1i2/Ripl-Tag2 transgenic mice have more angiogenesis, higher proliferation, and reduced apoptosis potential, as compared with the Rip1-Tag2 transgenic mice.

[0034] The observation that Slit2 expression increases tumor number suggests a possibility that it might also be involved in tumor metastasis. This possibility is tested with respect to the effects of Slit2 on lymph vessel formation because lymphatic systems is believed to be involved in most tumor metastasis.

[0035] FIG. 4 shows that Slit2 overexpression in the S1i2/Ripl-Tag2 mice indeed results in increased lymph vessel formation and tumor lymphatic metastasis. The Slit2/Ripl-Tag2 transgenic mice exhibit increased tumor intestinal lymph vessel formation (FIG. 4a;
indicated by arrows), as well as increased number of tumor cells inside the lymph vessels, as revealed by HE staining (FIG. 4b; indicated by arrows). FIG. 4c shows the intestinal lymphoid nodes, as revealed by positive LYVE-1 immunostaining, on the endothelial cells of the lymph vessels.

-g-[0036] Furthermore, the Slit2/Rip-l-Tag2 transgenic mice have more lymphatic endothelial cells in the peripheral tissues of the pancreas, as compared with the Rip1-Tag2 transgenic mice (FIGs. 4g, 4h, and 4i). The lymphatic endothelial cell proliferation in the pancreas, as determined by the immunofluorescence staining using anti-LYVE-1 antibody, is also higher in the Slit2/Rip 1-Tag2 transgenic mice (FIG. 41), as compared with that in the wild type C57 mice (FIG. 4j) and the Ripl-Tag2 transgenic mice (FIG. 4k). These results indicate that overexpression of Slit2 can induce lymph vessel formation and promote cancer lymphatic metastasis.

[0037] The roles of Slit2 in inducing tumor lymph vessel formation are further supported by the observation that the Slit2-induced migration and vessel formation of lymphatic endothelial cells can be specifically inhibited by R5, a monoclonal antibody against Robol (see, FIGs. 5c-e), or by RoboN, an extracellular domain fragment of Robol capable of binding to Slit2 (data not shown). FIG 6 shows R5 can specifically disrupt the interactions between a recombinant Robol (hRobo-Fc) (FIG. 7) and Slit2 (FIGs. 6A
and 6B).

[0038] The above observations clearly show the involvement of Slit-Robo interactions in the tumor-induced lymph vessel formation and tumor lymphatic metastasis.
Therefore, reagents that can interfere with or modulate the interactions between Slit proteins and Robo proteins can be used as therapeutic agents to prevent or treat metastatic cancers or as diagnostic agents to identify cancer patients suitable for certain cancer treatments. In accordance with embodiments of the invention, such reagents may include antibodies against the Slit proteins, antibodies against the Robo proteins, protein fragments containing the extracellular domains of the Robo proteins, and the like.
Antibodies for use with embodiments of the invention may be polyclonal or monoclonal antibodies.
Furthermore, such antibodies may be humanized to reduce immune rejections or complications.

[0039] In accordance with some embodiments of the invention, Robo specific antibodies or extracellular active site fragment of Robo proteins may be used to competitively inhibit Slit-Robo interactions, resulting in the inhibition of Slit-Robo signal transduction mediated biological functions. Similarly, antibodies against the Slit proteins can also be used to diagnose, prevent or treat disorders mediated by the Slit-Robo signal transduction.

[0040] In accordance with some embodiments of the invention, antibodies for use in treating Slit-mediated disorders may be humanized antibodies, which may be used in the clinics with minimum immune response. The methods for humanization of antibodies are well known in the art. Some embodiments of the invention relate to methods for mass producing these recombinant proteins as therapeutic agents. These methods and reagents are illustrated in examples described below.

[0041] In addition to preventing or treating disorders mediated by Slit proteins, some embodiments of the invention relate to methods for diagnosing such disorders.
As noted above, expression levels of Slit proteins are enhanced in various cancers.
Therefore, detection of increased expression of Slit proteins may indicate the presence of Slit-mediated disorders.

[0042] FIG. 8 shows functional analyses of the anti-Slit2 monoclonal antibodies and their uses in the detection of various human tumors. Three antibodies, S1, S3, and 9E10 are used in these experiments. Immunoblotting is used to assess the ability of S 1 and S3 antibodies to detect full-length or fragments of Slit2 protein. As shown in FIG. 8a, S 1 detects the full-length Slit2 protein by binding to the N-terminal of the protein. 9E10 and S3 antibodies detect the full-length Slit2 by binding to the C-terminal of the protein.
S 1 and S3 antibodies specifically detect human Slit2 protein and S 1 antibody also detects human Slitl and Slit3 proteins. The 9E10 antibody (obtained from ATCC) serves as a positive control.

[0043] As shown in FIG. 8b, 8c, and 8d, Si monoclonal antibody can be used to detect Slit2 expression in colorectal cancer and breast cancer (FIG. 8b), primary lung cancer and its metastatic lung cancer (FIG. 8c), and colorectal cancer with and without lymphatic metastasis (FIG. 8d). The above examples clearly show that antibodies against Slit proteins may be used as diagnostic tools to detect or predict the presence or outcome of various tumors.

[0044] The following examples further illustrate embodiments of the invention.

Methods for Preventing or Treating a Disease or Disorder Associated with Slit2 Mediated lymph vessel formation [0045] As noted above, some embodiments of the present invention relate to methods for preventing or treating a disease or disorder associated with Slit2 mediated lymph vessel formation in a subject. A method in accordance with one embodiment of the invention, for example, may comprise reducing or preventing Slit2-Robo interactions in a subject to a level sufficient to prevent or treat a disease or disorder associated with Slit2 mediated lymph vessel formation in said subject. Such diseases or disorders may be related to cancer progression or cancer metastasis.

[0046] The Slit2-Robo interactions (e.g., Slit2-Robol or Slit2-Robo4 interactions) can be reduced by any suitable methods. For example, the Slit2-Robo interactions can be reduced via administering to the subject an effective amount of a substance that reduces replication of Slit2 gene, replication of Slit2 receptor gene, transcription of Slit2 gene, transcription of Slit2 receptor gene, splicing or translation of Slit2 mRNA, splicing or translation of Slit2 receptor mRNA, maturation or cellular trafficking of Slit2 precursor, or maturation or cellular trafficking of Slit2 receptor precursor.

[0047] In accordance with other embodiments of the invention, the Slit2-Robo interactions can be reduced or prevented via administering to a subject an effective amount of a substance that reduces or blocks Slit2-Robo protein-protein interactions. Any suitable substance can be used to reduce or block the Slit2-Robo protein-protein interactions.
Such substances, for example, may include anti-Slit2 antibodies, anti-Robo antibodies, or a Robo protein fragment derived from its extracellular domain that is capable of binding to Slit2. Any suitable anti-Slit2 antibody or anti-Robo antibody can be used to reduce or prevent the Slit2-Robo protein-protein interactions, including polyclonal antibodies, monoclonal antibodies, Fab fragments or F(ab')2 fragments, and such antibodies may be humanized. In addition, Robo receptor antagonists, which can bind to the Robo receptor directly or indirectly, may also be used. Examples of receptor antagonists may include small molecules or analogs of Slit2 that can bind to the Robo receptors without eliciting the normal signal transduction.

[0048] The methods of the invention can be used to prevent or treat a disease or disorder associated with any Slit2 receptor mediated lymph vessel formation. For example, the methods of the invention can be used to prevent or treat a disease or disorder associated with any Slit2-Robol or Slit2-Robo4 mediated lymph vessel formation. The Slit2-Robol or Slit2-Robo4 interactions may be reduced by any suitable methods.
Preferably, the Slit2-Robol or Slit2-Robo4 interactions may be reduced or prevented via administering to a subject an effective amount of a substance that reduces or prevents Slit2-Robol or Slit2-Robo4 protein-protein interactions. Such exemplary substance can be an anti-Slit2 antibody, an anti-Robol antibody, an anti-Robo4 antibody, or a Robol or Robo4 fragment derived from extracellular domain of the Robol or Robo4 that is capable of binding to Slit2.

[0049] Any suitable anti-Slit2 antibody can be used, including the anti-Slit2 antibody disclosed in Hu, Neuron August 1999; 23(4):703-11. Similarly, any suitable anti-Robol antibody may be used, including the anti-Robol antibody disclosed in Hivert et al., Mol Cell Neurosci December 2002;21(4):534-45. Preferably, the anti-Robol antibody is an antibody against the first immunoglobulin domain of Robo 1. More preferably, the antibody against the first immunoglobulin domain of Robol is R5. Any suitable Robol fragment derived from the extracellular domain of Robol may be used, e.g., RoboN.

[0050] A substance used in a method of the invention may be administered by itself.
Preferably, the substance may be administered with a pharmaceutically acceptable carrier or excipient.

[0051] Methods of the invention may be used to prevent or treat any disease or disorder associated with Slit2 mediated lymph vessel formation. Such disorders, for example, include cancer. Preferably, the cancer is metastatic. Cancers, for example, may be malignant melanoma, bladder squamous carcinoma, neuroblastoma, small cell lung cancer, colon adenocarcinoma, bladder transitional cell carcinoma, breast cancer, adenoid cystic carcinoma of salivary gland, hepatocellular carcinoma or rhabdomyosarcoma.

[0052] Methods of the invention may be used to prevent or treat any disease or disorder associated with Slit2 mediated lymph vessel formation in any subject.
Preferably, the subject is a mammal. More preferably, the mammal is a human.

[0053] In some embodiments of the invention, the subject is a human and the substance to be administered to the human may be a humanized monoclonal antibody.

Pharmaceutical Compositions and Combinations for Preventing or Treating a Disease or Disorder Associated with Slit2 Mediated Lymph vessel Formation [0054] In another aspect, embodiments of the present invention relate to pharmaceutical compositions for preventing or treating a disease or disorder associated with Slit2 mediated lymph vessel formation in a subject. Such pharmaceutical compositions, for example, may comprise an effective amount of a substance that reduces or prevents Slit2-Robo interactions. The pharmaceutical compositions may further comprise a pharmaceutically acceptable carrier or excipient.

[0055] The formulations, dosages and routes of administration may be determined according to methods known in the art (see e.g., Remington: The Science and Practice of Pharmacy, Alfonso R. Gennaro (Editor) Mack Publishing Company, April 1997; Therapeutic Peptides and Proteins: Formulation, Processing, and Delivery Systems, Banga, 1999; and Pharmaceutical Formulation Development of Peptides and Proteins, Hovgaard and Frkjr (Ed.), Taylor & Francis, Inc., 2000; Biopharmaceutical Drug Design and Development, Wu-Pong and Rojanasakul (Ed.), Humana Press, 1999).

[0056] In the treatment or prevention of a disease or disorder associated with Slit2 mediated lymph vessel formation, an appropriate dosage level will generally be about 0.01 to 500 mg per kg body weight per day. Preferably, the dosage level will be about 0.1 to about 250 mg/kg per day. In more preferred embodiments, the dosage level will range from about 0.1 to about 20 mg/kg per day. The appropriate dosage can be administered in single or multiple doses. It will be understood that the specific dose level and frequency of dosage for any particular subject may be varied and will depend upon a variety of factors, including the activity of the specific compound used, the metabolic stability and length of action of that compound, the age, body weight, general health, sex, diet, mode and time of administration, rate of excretion, drug combination, the severity of the particular condition, and the patient undergoing therapy.

[0057] The pharmaceutical compositions of this invention may be administered orally, parenterally, by inhalation spray, topically, rectally, nasally, buccally, vaginally, via an implanted reservoir, or any suitable form of administration.

[0058] In still another aspect, some embodiments of the present invention relate to combinations for preventing or treating a disease or disorder associated with Slit2 mediated lymph vessel formation in a subject, which combinations may comprise:
a) an effective amount of a therapeutic agent of the invention that can reduce or block Slit2-Robo interactions; and b) an effective amount of another agent that can reduce or block lymph vessel formation.

[0059] For treating cancer, any anti-neoplasm agent can be used in a combination of the present invention. Examples of anti-neoplasm agents that can be used in the compositions and methods of the present invention are described in U.S. patent application Ser. No.
2002/044,919. In one embodiment, the anti-neoplasm agent used is an anti-angiogenic agent. The anti-angiogenic agent can be an inhibitor of basement membrane degradation, an inhibitor of cell migration, an inhibitor of endothelial cell proliferation, and an inhibitor of three-dimensional organization and establishment of potency.
Examples of such anti-angiogenic agent are illustrated in Auerbach and Auerbach, Pharmacol. Ther., 63: 265-311 (1994); O'Reilly, Investigational New Drugs, 15: 5-13 (1997); J.
Nat'l Cancer Instit., 88: 786-788 (1996); and U.S. Pat. Nos. 5,593,990; 5,629,327 and 5,712,291. In another embodiment, the anti-neoplasm agent used is an alkylating agent, an antimetabolite, a natural product, a platinum coordination complex, an anthracenedione, a substituted urea, a methylhydrazine derivative, an adrenocortical suppressant, a hormone, and an antagonist.

Methods and Kits for Predicting or Diagnosing a Disease or Disorder Associated with Slit2 Mediated Lymph vessel Formation [0060] Some embodiments of the invention relate to methods for predicting or diagnosing a disease or disorder associated with Slit2 mediated lymph vessel formation in a subject.
A method in accordance with embodiments of the invention, for example, may comprise:
a) obtaining a test sample from a test subject and assessing Slit2 and/or Slit2 receptor (Robo) level in said test sample; b) obtaining a control sample from a control subject not having a disease or disorder associated with Slit2-mediated lymph vessel formation and assessing Slit2 and/or Slit2 receptor (Robo) level in said control sample; and c) comparing Slit2 and/or Slit2 receptor (Robo) levels assessed in a) and b), whereby an elevated Slit2 and/or Slit2 receptor level in said test subject relative to Slit2 and/or Slit2 receptor level in said control subject indicates that said test subject has said disease or disorder associated with Slit2 mediated lymph vessel formation. In accordance with some embodiments of the invention, the Slit2 receptor tested in these methods may be Robol or Robo4. The Slit2 and/or Slit2 receptor level can be assessed at suitable levels, e.g., at nucleic acid or protein level.

[0061] Some embodiments of the present invention relate to kits for predicting or diagnosing a disease or disorder associated with Slit2 mediated lymph vessel formation in a subject, which kit comprises: a) a means for obtaining a test sample from a test subject and a control sample from a control subject; b) a means for assessing Slit2 and/or Slit2 receptor level in said test and control samples; and, optionally, c) a means for assessing and/or comparing Slit2 and/or Slit2 receptor levels in said test and control samples. In accordance with some embodiments of the invention, the Slit2 receptor tested may be Robo 1 or Robo4.

EXAMPLES
(1) The Expression of Slit2 in Human Cancers [0062] To explore the implications of our findings, we examine whether human cancer cell lines originated from different tissues and organs express Slit2. FIG. la shows that Slit2 is expressed in A375 cells (malignant melanoma), SCaBER cells (bladder squamous carcinoma), SK-N-SH cells (neuroblastoma), NCI-H446 cells (small cell lung cancer), LoVo cells (colon adenocarcinoma), T24 cells (bladder transitional cell carcinoma), ZR-75-30 cells (breast cancer), Acc-2 and Acc-M cells (adenoid cystic carcinoma of salivary gland), SMMC-7721 cells (hepatocellular carcinoma) and A673 cells (rhabdomyosarcoma). But Slit2 is apparently absent in A549 cells (lung cancer), HeLa cells (cervical epithelial adenocarcinoma), MCF-7 cells (breast adenocarcinoma) and 786-0 cells (primary renal cell adenocarcinoma).

[0063] The finding of Slit2 expression in a variety of cancer cell lines is consistent with the observation that Slit2 is expressed in prostate cancers detected by RT-PCR. In addition, FIG. lb shows that Slit 2 is expressed in human malignant melanoma, rectal mucinous adenocarcinoma, breast invasive carcinoma, and hepatocellular carcinoma.
Notably, within the sections of breast carcinoma, it appears that more Slit2 staining is detected in locations having more cancer cells and blood vessels (FIG. lc). In contrast, less Slit2 staining is detected in locations having fewer cancer cells and blood vessels.
Similar patterns can be observed for human hepatocellular carcinoma, but not in malignant melanoma or rectal mucinous adenocarcinoma (data not shown). These direct correlations between Slit2 expression and cancer/blood vessel cell proliferation suggest a significant role of the Slit2-induced tumor lymph vessel formation in the pathogenesis of various human cancers.

(2) Construction of Slit2/Ripl-Tag2 Transgenic Mice [0064] FIG. 2a shows an expression vector harboring human Slit2 (hSlit2) gene fused with a DNA sequence coding for a Flag fragment driven by CMV promoter. This expression vector is used to generate 3 strains of Slit2 transgenic mouse lines. The presence of hSlit2 gene in these transgenic mice are confirmed by spot hybridization (FIG.
2b) and Southern blot (FIG. 2c), and expression of hSlit2 protein in these transgenic mice is verified by immunoblotting analysis (FIG. 2d). The expression of Flag tag (FIG. 2e) and the presence of Flag DNA sequence (FIG. 2f) are detected in the pancreas tissue extracts by Western blot using an anti-Flag monoclonal antibody (M2) and PCR analysis, respectively. These results confirm that the transgenic mouse strains expressing hSlit2 have been successfully established.

[0065] FIG. 2g (upper left panel) shows that pancreas of normal mice do not express Slit2 gene, whereas Slit2 is expressed in 12-week old Ripl-Tag2 transgenic mice (FIG. 2h, upper right panel). In addition, the expression of Slit2 overlaps with the expression of insulin (FIG. 2g, upper middle panel). These results indicate that Slit2 expression is up-regulated by pancreatic tumorgenesis in pancreatic 0 cells. Further, while Robol and vWF are both expressed in endothelial cells, these proteins are not expressed in pancreatic 0 cells in Rip1-Tag2 transgenic mice (FIG. 2g, bottom left and bottom right panels).

[0066] To further assess the role of Slit2 in tumorigenesis, the Slit2 transgenic mice are crossed with Ripl-Tag2 transgenic mice to create Slit2/Ripl-Tag2 transgenic mice.
Compared with Ripl-Tag2 transgenic mice at the same age, the Slit2/Ripl-Tag2 transgenic mice express higher Slit2 protein levels at 10 and 13 week old (FIG. 2h), indicating a successful overexpression of Slit2 in the Slit2/Ripl-Tag2 transgenic mice.
The results show that the R cells of the pancreas can be induced to secret Slit2 protein via a paracrine mechanism. Slit2, in turn, may interact with Robol, which is stably expressed on the endothelial cells of the pancreatic blood vessels, to activate cellular signaling pathways during pancreatic tumorigenesis.

(3) Slit2 Promotes Pancreatic Tumor Growth [0067] FIG. 3a shows that the number of angiogenic islets in pancreas significantly increases in Slit2/Ripl-Tag2 transgenic mice at 10 and 12 week old. FIG. 3b and 3c show that both the volume and the number of pancreatic tumors are greater in the Slit2/Ripl-Tag2 transgenic mice than those of the Ripl-Tag2 transgenic mice. However, there is no significant difference in leukocyte count between the Slit2/Ripl-Tag2 and the Ripl-Tag2 transgenic mice (FIG. 3d). In addition, FIG. 3e shows that the Slit2/Ripl-Tag2 transgenic mice have enlarged blood vessels, whereas the Ripl-Tag2 transgenic mice exhibit no change or slightly enlarged blood vessels, as determined after injecting a fluorescent dye into blood vessels. FIG. 3f shows that the Slit2/Ripl-Tag2 transgenic mice have obvious blood vessel enlargement and bleeding. In contrast, the Ripl-Tag2 transgenic mice show little bleeding. Further, the Slit2/Ripl-Tag2 transgenic mice have more new blood vessels (FIG. 3g), higher proliferation index (FIG. 3h), and lower apoptosis index (FIG. 3i), as compared with the Ripl-Tag2 transgenic mice.
These results indicate that Slit2 overexpression promotes angiogenesis and growth of pancreatic tumors in the Rip1-Tag2 transgenic mice.

(4) Slit2 Overexpression Promotes Lymphatic Metastasis Of Pancreatic Tumors [0068] The Ripl-Tag2 transgenic mice develop pancreatic tumors, which go through 4 stages: angiogenic islet stage, atypical growth stage, pancreatic tumor stage, and invasive pancreatic tumor stage. Majority of these mice eventually die of hyperglycemia at 15-week old. Although no metastasis of pancreatic tumors is observed in the Rip1-Tag2 transgenic mice during their life time, high incidence (24.32%) of intestinal membrane metastasis is observed in the Slit2/Ripl-Tag2 transgenic mice (FIG. 4a and 4b). Tumor nodules are also observed inside the lymph vessels of the Slit2/Ripl-Tag2 transgenic mice (FIG. 4b). No difference in tumor weight is observed between the Ripl-Tag2 and the Slit2/Ripl-Tag2 transgenic mice.

[0069] Using LYVE-1 as a lymphatic endothelial cell marker, the endothelial cells in the lymph vessels are assessed by immunohistochemical analysis. FIG. 4c, 4d, and 4e show that the Slit2/Ripl-Tag2 transgenic mice have more new lymph vessels, which are distributed mainly in the peripheral regions of the pancreas and inside the lymphoid nodes (FIG. 4f). In contrast, the Ripl-Tag2 transgenic mice contain fewer newly developed lymph vessels (FIG. 4g, 4h, and 4i). FIG. 4j, 4k, 41 show that when compared with the wild type C57 mice and the Ripl-Tag2 transgenic mice, the Slit2/Ripl-Tag2 transgenic mice have more newly developed lymph vessels, as determined by immunofluorescence staining analysis. FIG. 4m, 4n, and 4o show that Robol is expressed in the lymphatic endothelial cells of the Slit2/Ripl-Tag2 transgenic mice. There is about 1.5-fold increase in the number of lymph vessels, as determined by a semi-quantitative immunohistochemical analysis, in the pancreas of Slit2/Ripl-Tag2 transgenic mice, as compared with the Ripl-Tag2 transgenic mice (FIG. 5b). Together, these results indicate that Slit2 overexpression can induce lymph vessel formation and tumor lymphatic metastasis.

(5) Slit2 Recombinant Protein Promotes Lymphatic Endothelial Cell Migration And Vessel Formation In Vitro [0070] FIG. 5a shows the expression of Robol in the primary human lymphatic endothelial cells is assessed by immunofluorescence analysis using polyclonal antibody (Rb7) and monoclonal antibody (R4). R4 specifically recognizes Robol, but not Robo2-4.
FIG.
5a shows that Robol is expressed on the cell membrane of human lymphatic endothelial cells. FIG. 5b shows that the density of lymph vessels in the pancreas of the Slit2/Rip1-Tag2 transgenic mice is about 1.5-fold higher than that of the Ripl-Tag2 transgenic mice.

[0071] To determine whether Slit2.1 recombinant protein can affect the migration of human lymphatic endothelial cells, Boyden Chamber Assay is used. VEGF-C is used as a positive control in these experiments. FIG. 5c shows that Slit2.1 promotes human lymphatic endothelial cell migration at > 200 pM. Excess amount of R5 (anti-Robol antibody) prevents Robol from binding to Slit2, resulting in an inhibition of Slit2-mediated lymphatic endothelial cell migration.

[0072] To determine whether Slit2.1 affects vessel formation of human lymphatic endothelial cells in vitro, Matrigel assay is performed. Again, VEGF-C as a positive control. FIG. 5d and 5e show that Slit2.1 induces vessel formation at 10 nM.
Excess amount of R5 inhibits the S1i2-mediated lymphatic endothelial cell vessel formation.
Together, these results indicate that Slit2 protein binds to its receptor and promotes lymphatic endothelial cell migration and lymph vessel formation.

(6) Slit2 Overexpression Is A Predictor Of Tumor Lymphatic Metastasis In Cancer Patients [0073] The results described above suggest that Slit2 expression may be a useful marker for cancer metastasis to lymphatic system. This notion is supported by the immunochemistry staining data from human tumor specimens using anti-Slit2 antibodies.
The properties of various antibodies used in these experiments are illustrated in FIG. 8a.
S1, an anti-Slit2 monoclonal antibody, can recognize both the full-length and N-terminal fragments of Slit2 protein. 9E10 recognizes the c-myc tag fused to the Slit2 protein.
S3, another anti-Slit2 monoclonal antibody, recognizes both the full-length and C-terminal fragments of Slit2 protein. Si also recognizes human Slitl and Slit3 (FIG.
8a). Therefore, S 1 can be used to detect different Slit proteins. Using Si immunostaining, 742 out of 955 human tumor samples are found to be positive for Slit expression (Table 1; FIG. 8b-d). Importantly, Slit expressions in colorectal cancer, stomach cancer, and lung cancer are significantly higher in lymphatic metastasis than in those without metastasis (Table 2; FIG. 8c and 8d).

[0074] Table 1: Slit protein expression in various human tumor tissues, as determined by immunohistochemical analysis using Si monoclonal antibody Total Number Of Number Of % Of Slit-Positive Cancer Type Samples Tested Slit-Positive Samples Samples Colorectal Cancer 314 256 81.53 Stomach Cancer 326 237 72.7 Esophageal Cancer 89 86 96.63 Squamous Cell Lung 43 38 88.37 Carcinoma Lung 9 9 100 Adenocarcinoma hepatocellular 53 45 84.91 carcinoma Breast Invasive 95 55 57.89 Ductal Carcinoma Bladder Cancer 26 16 61.54 [0075] Table 2: Slit protein expression in various human tumor tissues, as determined by immunohistochemical analysis using S 1 monoclonal antibody % Positive % Positive (Slit-Positive Samples (Slit-Positive Samples Total Number Of With Lymphatic Without Lymphatic Cancer Type Samples Tested Metastasis / Total Metastasis / Total Number Of Samples Number Of Samples With Lymphatic Without Lymphatic Metastasis) Metastasis) Colorectal Cancer 155 56.06 (37/66) 25.84 (23/89) Stomach Cancer 69 44.64 (25/56) 23.08 (3/13) Lung Cancer 49 45.45 (10/22) 29.63 (8/27) [0076] The examples presented above clearly indicate that the Slit-Robo signal pathways play an important role in regulating lymph vessel formation and tumor lymphatic metastasis. Therefore, agents, techniques, and methods that can modulate or interfere with the interaction between Slit proteins and their Robo receptors can be used for treating lymph vessel formation and/or tumor lymphatic metastasis-related diseases mediated by Slit-Robo signal transduction pathways.

[0077] For example, Robo protein fragments (e.g., Robo extracellular domains or its fusion proteins such as hRobo-Fc fusion proteins) or antibodies targeting Slit or Robo proteins may be used to modulate or interfere with Slit-Robo protein interactions, thereby providing treatments for the Slit-Robo signal transduction pathway-mediated diseases and disorders. In this regard, Robo proteins fragments, such as extracellular fragments, may be used as decoys to bind with Slit ligands, thus preventing or reducing the binding of Slit proteins to Robo receptors. In addition to therapeutic applications, the Slit or Robo receptor proteins or fragments or antibodies against Slit or Robo proteins can be used as diagnostic or prognostic markers. For example, antibodies against Slit or Robo proteins may be used to assess tumor lymphatic metastasis by comparing the expression levels of Slit and/or Robo proteins in patients.

[0078] Some embodiments of the invention relate to Slit and/or Robo protein based regents and methods of their use in the treatment, diagnosis, prognosis of diseases or disorders related to the Slit-Robo signaling pathways. Some examples of such embodiments are described below.

(7) Generation of a recombinant protein containing an extracellular IgG domain of human Robol protein fused with the Fc region of human immunoglobulin (hRobo-Fc) (7a) Cloning of the Fc region of human immunoglobulin [0079] Amplify the 687 base pairs (bp) region located at the C-terminal of immunoglobulin heavy chain by PCR using pAc-k-CH3 plasmid (which harbors the constant regions of a human immunoglobulin gene) (US Biological Co.) as a template. The sequences of the hFc forward and reverse primers used in the PCR amplification are as follows:

hFc forward: 5'-AACCGTGCGGCCGCTGTTGTGACAAAACTCACAC-3' [SEQ ID NO: 1]
hFc reverse: 5'-CGCGGAGATCTTCATTTACCCGGAGACAGGGAGAGGC-3' [SEQ ID NO: 2 ]

The PCR product is cleaved with Notl and BglII restriction enzymes, followed by ligation of the NOTI/BglII fragment with a pVL1393 plasmid (BD Pharmingen) that has been digested with Notl and BglII. This produces plasmid pVL1393-hFc, which carries the Fc region of human immunoglobulin sequence. The sequence of the Fc region is confirmed by DNA sequencing. No mutation is found.

(7b) Cloning of Robo 1 gene fragments [0080] A Robol cDNA clone (Origene, DNA sequence No. NM_002941.2, Product No.
SC109739) is used as a template. The extracellular domain containing 5 immunoglobulin-like domains (1-1621 bp) is selected for PCR amplification. The sequences of he Robol forward and reverse primers used in PCR amplification are as follows:

Robol forward: 5'-GGCGGCCTCTAGAATGAAATGGAAACATGTTCC-3' [SEQ ID
NO: 3]

Robol reverse: 5'-CTATAAGCGGCCGCCAATGTAAGCACTCCATGTT-3' [SEQ ID
NO: 4]

The PCR product is then cleaved with Xbal and Notl restriction enzymes. The XbaI/NotI
fragment is ligated to pVL1393-hFc, which had been previously digested with Xbal and NotI, to generate an expression vector, pVL1393-hRobo-Fc. The pVL1393-hRobo-Fc expression vector encodes a protein containing the extracellular domain of Robol (containing 5 immunoglobulin-like (Ig-like) domains) fused with the human Fc region of the immunoglobulin. The sequence of the Robo Ig-like domains in this expression vector is confirmed by DNA sequencing. No mutation is found. An open reading frame of the Robol Ig-like domains fused with the hFc region is established properly with an initiation codon and a stop codon at the correct positions.

(7c) Expression and purification of hRobo-Fc fusion protein [0081] EXAMPLE I: Large-scale plasmid production of pVL1393-hRobo-Fc is performed and purified using a plasmid isolation kit (QIAGEN plasmid Midi Kit) according to the protocols provided in the kit. The purified pVL1393-hRobo-Fc is used to generate hRobo-Fc recombinant baculovirus according to the protocol (BD Pharmingen, BaculoGoldTM Starter Package and Transfection Kit).

[0082] After screening, the recombinant virus is amplified and used to infect Sf9 insect cells (grown in IPL-41 media (Invitrogen) in a 27 C incubator) to express hRobo-Fc fusion protein. The fusion protein is detected as a - 85 kD monomer, under reducing conditions, using a horse radish peroxidase-conjugated rabbit anti-human IgG
antibody (Santa Cruz Biotech) (FIG. 7). The hRobo-Fc fusion protein can be expressed on a large scale by infecting SF9 cells with the hRobo-Fc recombinant baculovirus. The recombinant protein thus produced can be purified using aProtein A affinity column (e.g., GE Healthcare, nProtein A SepharoseTM 4 Fast Flow). In general, milligram quantities of hRobo-Fc fusion protein may be obtained from one liter of sf9 cell culture.
The hRobo-Fc fusion protein expressed in the baculovirus system is functional, as evidenced by its ability to specifically bind hSlit2 protein (FIG. 6A). In addition, R5 (a monoclonal antibody recognizing the first immunoglobulin-like domain of Robo 1 protein) can inhibit the interactions between the hRobo-Fc fusion protein and hSlit2 (FIG. 6B).
These data confirm the specificity and biological activity of the hRobo-Fc fusion protein.

[0083] In accordance with some embodiments of the invention, similar or variant Robo fusion proteins may be generated using other systems, such as using different extracellular domains of the Robo gene fragment (e.g., those containing one to four immunoglobulin-like domains), using different fusion proteins (e.g., glutathione S-transferase (GST)), and/or using different protein expression system (e.g., CHO-dhfr:
Chinese hamster ovary cells deficient of dehydrofolate reductase). Some of these embodiments are described below.

[0084] EXAMPLE II: Large-scale production of the extracellular domain of mouse Robol and the Fc region of mouse immunoglobulin fusion protein (mRobo-Fc) using a Baculovirus protein expression system.

1. Selection and cloning of the Fc region of mouse immunoglobulin:

[0085] Using mouse IgG2b monoclonal antibody gene as a template, a 720-nucleotide sequence encoding the C-terminal of the heavy chain is selected for PCR
amplification.
The PCR primers are shown as follows:

mFc forward: 5'-GCACTCTAGACTTGAGCCCAGCGGGCCCAT-3';
[SEQ ID NO: 5]
mFc reverse: 5'-CTGAGGATCCTCATTTACCCGGAGACCGGG-3'; [SEQ
ID NO: 6]

[0086] The PCR products are digested by Xbal and BamHI. The Xbal and BamHI
fragments are subcloned into pVL1392 vector (BD Pharmingen), pre-digested by Xbal and BamHI to generate pVL1392-mFc. DNA sequencing verifies the correct Fc sequence.

2. Selection and cloning of the mouse Robol gene fragments [0087] Using mouse Robol gene (NM4019413) as a template, a sequence, from ATG
to 2445 encompassing five Ig-like regions and three Fibronectin type 3 structural domains (FN3 domain), is amplified using the following primers:

mRobol forward: 5'-ATCGAGATCTATGATCGCGGAGCCTGCTCACT-3' [SEQ ID NO: 7]
mRobol reverse: 5'- GCTCTCTAGACGCTGCCACCTCCACACTGTA-3' [SEQ ID NO: 8]
The PCR products are digested by Bg1II and XbaI. The Bg1II and XbaI
fragments are subcloned into pVL1392-mFc, pre-digested by Bg1II and XbaI to generate pVL1392-mRobo-Fc. DNA sequencing confirms that the mouse Robo 1 gene fragment and mFc form the same open reading frame. The start and stop codons are confirmed as being located at the correct positions.

[0088] Large-scale isolation and purification of pVL1392-mRobo-Fc plasmids is performed using the QIAGEN Plasmid Midi Kit according to the user manual (Qiagen). The purified pVL1392-mRobo-Fc plasmids are used to generate recombinant Baculoviruses expressing mRobo-Fc fusion protein according to the user manual for the Baculovirus expression system (BD Pharmingen). The recombinant Baculoviruses are used to infect sf9 insect cells to determine infection efficiency and identify high expressers for the fusion proteins. The mRobo-Fc proteins are purified using Protein A affinity chromatography (GE Healthcare) according to the nProtein A SepharoseTM 4 Fast Flow user manual (GE Healthcare).

[0089] EXAMPLE III: Large-scale production of the extracellular domain (with different lengths) of human Robol and the Fc region of human immunoglobulin fusion proteins using a Baculovirus protein expression system.

1. Selection and cloning of the Fc region of human immunoglobulin:

[0090] Using the constant region of human immunoglobulin gene (pAc-k-CH3) (US
Biological) as a template, the 687-nucleotide sequence encoding the C-terminal of the heavy chain is selected for PCR amplification. The PCR primers are shown as follows:
hFc forward:
5'-AACCGTGCGGCCGCTGTTGTGACAAAACTCACAC-3' [SEQ ID NO: 9]
hFc reverse:
5'-CGCGGAGATCTTCATTTACCCGGAGACAGGGAGAGGC-3' [SEQ ID
NO: 10]
The PCR products are digested by Notl and BagII. The Notl and BagII
fragments are subcloned into pVL1392 vector (BD Pharmingen), pre-digested by Notl and BagII to generate pVL1392-hFc. DNA sequencing verifies the correct Fc sequence.
2. Selection and cloning of the human Robo 1 gene fragments:

[0091] Using human Robol gene (NM4002941.2, Origene Product No. SC109739) as a template, a sequence, from ATG to 1342 encompassing four Ig-like regions, is amplified using the following primers:

Robol forward: 5'-GGCGGCCTCTAGAATGAAATGGAAACATGTTCC-3' [SEQ ID
NO: 11]

Robo reverse2:
5'-CATTATGCGGCCGCCATCTGTAACTTCCAAATAT-3' [SEQ ID NO:
12]

[0092] Using human Robol gene (NM4002941.2, Origene Product No. SC109739) as a template, a sequence, from ATG to 1051 encompassing three Ig-like regions, is amplified using the following primers:

Robo 1 forward:
5'-GGCGGCCTCTAGAATGAAATGGAAACATGTTCC-3' [SEQ ID NO:
13]
Robo 1 reverse3:
5'-ATAATAGCGGCCGCGAGGTTCTTGAACAGTCAGAGTA-3' [SEQ ID
NO: 14]

[0093] Using human Robol gene (NM4002941.2, Origene Product No. SC109739) as a template, a sequence, from ATG to 829 encompassing two Ig-like regions, is amplified using the following primers:

Robo 1 forward:
5'-GGCGGCCTCTAGAATGAAATGGAAACATGTTCC-3' [SEQ ID NO:
15]
Robo 1 reverse4:
5'-ATAATTGCGGCCGCCATCCACAGTTACTGCCAAGTTACT-3' [SEQ ID
NO: 16]

[0094] Using human Robol gene (NM4002941.2, Origene Product No. SC109739) as a template, a sequence, from ATG to 529 encompassing one Ig-like regions, is amplified using the following primers:

Robo 1 forward:
5'-GGCGGCCTCTAGAATGAAATGGAAACATGTTCC-3' [SEQ ID NO: 17]
Robot reverses: 5'-TTCTATGCGGCCGCAAGGGTTTTGTCTGAAGTCAT-3' [SEQ ID NO: 18]

[0095] PCR products obtained from the steps described above are digested by Xba1 and Not1.
The Xba1 and Not1 fragments are subcloned into pVL1393-hFc, pre-digested by Xba1 and Not1 to generate the vectors that express proteins having different lengths of extracellular domain of human Robol fused with the Fc region of human immunoglobulin. DNA
sequencing confirms that the human Robo 1 gene fragments and hFc form the same open reading frame. The start and stop codons are confirmed as being located at the correct positions.

[0096] Large-scale isolation and purification of pVL1393-hRobo-Fc plasmids is performed using the QIAGEN Plasmid Midi Kit according to the user manual (Qiagen). The purified pVL1392-hRobo-Fc plasmids are used to generate recombinant Baculoviruses expressing hRobo-Fc fusion protein according to the user manual for the Baculovirus expression system (BD Pharmingen). The recombinant Baculoviruses are used to infect sf9 insect cells to determine infection efficiency and identify high expressers. The hRobo-Fc fusion proteins are purified using Protein A affinity chromatography (GE
Healthcare) according to the nProtein A SepharoseTM 4 Fast Flow user manual (GE
Healthcare).

[0097] EXAMPLE IV: Large-scale production of the extracellular domain (with different lengths) of human Robol and the Fc region of human immunoglobulin fusion proteins using a Chinese hamster ovary cells deficient of dehydrofolate reductase (CHO-dhfr) system.

1. Selection and cloning of the human Robol gene fragments:

[0098] Using human Robol gene (NM4002941.2, Origene Product No. SC109739) as a template, a sequence, from ATG to 1760 encompassing five Ig-like regions, is amplified using the following primers:

Robol f1760: 5'-GGCCAAGCTTATGAAATGGAAACATGTTCC-3';
[SEQ ID NO: 19]
Robol r1760: 5'- TCCACGGAATTCAAATTTGGTTGCC-3' [SEQ ID NO:
20]

[0099] The PCR products are digested by HindI11 and Ecoki. The HindI11 and Ecoki fragments encompassing five Ig-like regions of the extracellular domain of human Robo 1 are subcloned into pEGFP-N1 vector (BD Pharmingen), pre-digested by HindI11 and EcoRl to generate pEGFP-N1-hRobo. DNA sequencing confirms the human Robo sequence is correct.

2. Selection and cloning of the Fc region of human immunoglobulin:

[00100] Using the constant region of human immunoglobulin gene (pAc-k-CH3) (US
Biological) as a template, the 714-nucleotide sequence encoding the C-terminal of the heavy chain is selected for PCR amplification. The PCR primers are shown as follows:

hFc forward2: 5'-AACCGTGAATTCCGTGGACAAGAGAGTTGAGCC
-3'; [SEQ ID NO: 21]
hFc reverse2: 5'-TACGGGTCGACTCATTTACCCGGAGACAGGG-3' [SEQ ID NO: 22]

[00101] PCR products are digested by Ecoki and Sall. The Ecoki and Sall fragments are subcloned into pEGFP-N1 pre-digested by Ecoki and Sall to generate the vector (pEGFP-N1-hRobo-Fc) that express proteins that contain five Ig-like regions of the extracellular domain of human Robot fused with the Fc region of human immunoglobulin.
DNA sequencing confirms that the human Robo 1 gene fragments and hFc form the same open reading frame. The start and stop codons are confirmed as being located at the correct positions.

[00102] The pEGFP-N1-hRobo-Fc vectors are digested by Hindlll and Sall. The Hindlll and Sall fragments are subcloned into p3CI-dhfr pre-digested by Hindlll and Sall to generate p3CI-dhfr-hRobo-Fc that express proteins that contain five Ig-like regions of the extracellular domain of human Robol fused with the Fc region of human immunoglobulin. DNA sequencing confirms that the human Robo 1 gene fragments and hFc form the same open reading frame. The start and stop codons are confirmed as being located at the correct positions.

[00103] Large-scale isolation and purification of p3CI-dhfr-hRobo-Fc plasmids is performed using the QIAGEN Plasmid Midi Kit according to the user manual (Qiagen).
The purified pVL1392-hRobo-Fc plasmids are used to transfect CHO-dhfr cells to screen for high expression clones according to the Lipofectin user manual (Invitrogen). The media obtained from the high expressing clones are used to purify the hRobo-Fc proteins having different lengths using Protein A affinity chromatography (GE
Healthcare) according to the nProtein A SepharoseTM 4 Fast Flow user manual (GE
Healthcare).

[00104] EXAMPLE V: Large-scale production of a fusion protein between the extracellular IgG-like region of human Robol and the Fc region of human immunoglobulin using a Baculovirus protein expression system.

1. Construction ofpVL1393-hRobol-Fc:

[00105] Using the p3CI-dhfr-hRobo-Fc plasmids obtained from EXAMPLE IV as a template, PCR is performed to amplify the Fc region of human immunoglobulin gene using the following primers:

Robo 1 forward' :
5'-AGGCGGCCTCTAGAATGAAATGGAAACATGTTCC-3'; [SEQ ID NO:
23]
hFc reverse3: 5'-TACGGGCGGCCGCTCATTTACCCGGAGACAG -3' [SEQ ID NO: 24]

[00106] The PCR products are digested by Xba1 and Not1. The Xba1 and Not1 fragments are subcloned into pVL1393 vector (BD Pharmaingen) pre-digested by Xba1 and Not1 to generate pVL1393-hRobo-Fc that express proteins containing five Ig-like regions of the extracellular domain of human Robol fused with the Fc region of human immunoglobulin. DNA sequencing confirms that the human Robo 1 gene fragments and hFc form the same open reading frame. The start and stop codons are confirmed as being located at the correct positions.

2. Expression and purification of hRobo-Fc fusion protein:

[00107] Large-scale isolation and purification of pVL1393-hRobo-Fc plasmids is performed using the QIAGEN Plasmid Midi Kit according to the user manual (Qiagen).
The purified pVL1393-hRobo-Fc plasmids are used to generate recombinant Baculoviruses expressing hRobo-Fc fusion protein according to the user manual for the Baculovirus expression system (BD Pharmingen). The recombinant Baculoviruses are used to infect sf9 insect cells to determine infection efficiency and identify high expressers. The hRobo-Fc fusion proteins are purified using Protein A affinity chromatography (GE Healthcare) according to the nProtein A SepharoseTM 4 Fast Flow user manual (GE Healthcare).

METHOD S

Methods of RT-PCR and Northern Blotting [00108] Primary HUVECs were cultured as previously described (Geng, J.-G. et al.
Nature 343:757-760 (1990)). Semi-quantitative RT-PCR was performed as before (Ma, Y.-Q. and Geng, J.-G. J. Immunol. 165:558-565 (2000)). Primers used were human Robol sense (+4440) 5'-CCT ACA CAG ATG ATC TTC C-3' (SEQ ID NO: 25) and antisense (-4956) 5'-CAG AGG AGC CTG CAG CTC AGC TTT CAG TTT CCT C-3' (SEQ ID NO: 26); human Slit2 sense (+3611) 5'-GGT GAC GGA TCC CAT ATC GCG
GTA GAA CTC-3' (SEQ ID NO: 27) and antisense (-4574) 5'-GGA CAC CTC GAG

CGT ACA GCC GCA CTT CAC-3' (SEQ ID NO: 28); human (3-actin sense (+1) 5'-ATG
GAT GAT GAT ATC GCC GC-3' (SEQ ID NO: 29) and antisense (-1127) 5'-CTA GAA
GCA TTT GCG GTG G-3' (SEQ ID NO: 30). Total RNAs from A375 cells and the primary culture of HIJVECs were also probed with the 32P-labeled Slit2 or Robo 1 cDNA
fragments.

Methods of Antibody Generation, Immunoblotting and Immunostaining [00109] Slit2-GST (encoding amino acids 57-207 of human Slit2) and Robol-GST
fusion proteins (encoding either amino acids 1-168 or amino acids 961-1217 of rat Robol) were constructed using a pGEX-4T-1 vector (Amersham Pharmacia Biotech). The fusion proteins were used as antigens to immunize rabbits or mice for generation of anti-Slit2 and anti-Robol polyclonal or monoclonal antibodies. Immunohistochemical examinations were performed as described before (Liu, L.-P. et al. Biochem.
Biophys.
Res. Commun. 286:281-291 (2001)).

Methods of Isolation of Slit2 and RoboN

[00110] A stable human embryonic kidney 293 cell line secreting full-length human Slit2 with a myc tag at its carboxyl terminus (Slit2/293 cells) was established as previously described (Li, H. S. et al. Cell 96:807-818 (1999); Wang, K.-H. et al. Cell 96:771-784 (1999)). The highly purified Slit2 protein was obtained from the conditioned medium by affinity chromatography using 9E10 mAb against the myc tag (-1 mg/ml of Affi-Gel 10; Bio-Rad). The silver staining and immunoblotting of the purified Slit2 was carried out as described (Ma, L. et al. J. Biol. Chem. 269:27739-27746 (1994)).

[00111] A stable 293 cell line expressing an extracellular fragment of Robol with a hemoagglutinin tag at its carboxyl terminus (RoboN) was established as previously described (Li, H. S. et al. Cell 96:807-818 (1999); Whitford, K. L. et al.
Neuron 33:47-61 (2002)). Affinity beads coupled with an mAb against hemoagglutinin, HA11 (BAbCO), were used to purify RoboN from the conditioned medium.

Boyden Chamber Assay [00112] Cell migration assay was conducted in a 48-well micro-chemotaxis chamber (or similar devices) (Neuro Probe, Inc.) (Terranova, V. P. et al. J. Cell. Biol.
101:2330-2334 (1985)). PVP-free polycarbonate membranes (8 m pores) were coated with 1%
gelatin.
The bottom chambers were loaded with or without Slit2 or bFGF (Sigma), while the upper chambers was seeded with HIJVECs, Robol/293 cells or V/293 cells resuspended in M199 medium supplemented with 1% heat inactivated fetal calf serum (FCS) (all at 5x105 cells/ml). They were incubated at 37 C for 4 h. The filters were then fixed, stained with 0.5% crystal violet, and the cells migrated through the filters were counted.

Directional Migration Assay [00113] Microscopic gradients of proteins were produced as described (Hopker, V. H. et al. Nature 401:69-73 (1999)). Briefly, a repetitive pressure injection of picolitre volumes of 0.15 M Slit2 or 1 M bFGF was applied through a micropipette having a tip opening of -1 m. The 24-well culture plate was coated with a thin layer of Matrigel (Becton Dickinson Labware) and the testing cells were allowed to settle down and to loosely attach to the Matrigel. The experiments were carried out at 37 C in the presence of 5%
CO2. The pipette tip was placed 100 m away from the centre of any given cell [should this be well?] under test. Microscopic images were recorded with a CCD camera (JVC) attached to a phase contrast microscope (Olympus IX70) and stored in a computer for the detailed analysis using NIH Image or any suitable software. The migration distances of the cells (e.g., HUVECs) at an appropriate time point (e.g., 2 h) were analyzed.

Tube Formation Assay [00114] The 96-well cell culture plates were coated with 100 l/well of Matrigel and incubated at 37 C for 30 min to promote gelling (Malinda, K. M. et al., Identification of laminin al and 01 chain peptides active for endothelial cell adhesion, tube formation and aortic sprouting, FASEB J. 13:53-62 (1999)). HUVECs (passages 2 to 3) were resuspended at 1.3x10 5 cells per well in M199 medium supplemented with 2%
heat inactivated fetal calf serum. Aliquots of the cells (0.1 ml per aliquot) were added to each Matrigel-containing well. The tubular structures were recorded and photographed at 12 to 18 h.

Methods of Xenografted Tumor Growth Model [00115] A375 cells were transfected using LipofectinTM (Gibco) and selected by 400 .ig ml-1 hygromycin B (Sigma). RoboN/A375_C1, C2 and C3 cells, as well as V/A375 cells, were verified by immunoblotting with the antibodies against Robo 1, Slit2 or tubulin (as the loading control). They were resuspended in 0.2 ml DME medium and injected subcutaneously into athymic nude mice (O'Reilly, M. S. et al., Endostatin: An endogenous inhibitor of angiogenesis and tumor growth, Cell 88:277-285 (1997)). For antibody inhibition experiments, mice bearing A375 cell tumors were treated with intraperitoneal injections of R5 or control IgG2b twice per week (1 mg per injection) (Muller, A. et al. Nature 410:50-56 (2001)). Mice were sacrificed 30-35 days later. Mouse bloods were collected before sacrifice for leukocyte counts. Myeloperoxidase assays for measurements of neutrophils within tumor solids were performed as previously described (Wang, J.-G. et al. Inflamm. Res. 51:435-43 (2002)).

Cell Proliferation Assay [00116] All transfectants were grown to exponential phases and detached by trypsin treatment. Viable cells (5x103 cells/ml) were plated into 96-well tissue culture plates in 100 l complete medium and cultured at 37 C in 5% CO2 atmosphere. At different time points, tetrazolium salt was added (20 l per well) and incubated at 37 C for 4 h. The insoluble blue formazan product was solubilized by addition of 100 l/well 10%
SDS/5%
isobutanol. The plates were read on a microtiter plate reader using a test wavelength of 570 nm and a reference wavelength of 630 nm.

[00117] Other methods such as immunoprecipitation, lectin perfusion assay, HE
staining, and immunofluorescence staining are well known in the art.

[00118] Embodiments of the invention may include one or more of the following advantages. By targeting the signaling pathways that mediate angiogenesis, lymph vessel formation, and/or tumor lymphatic metastasis, novel therapeutic agents may be developed to treat or diagnose diseases or disorders such as cancers, which are associated with angiogenesis, lymphatic genesis (vessel formation), and/or tumor lymphatic metastasis. Embodiments of the present invention include agents that can be used to target such signal transduction pathways, i.e., the Slit-Robo signaling pathways. These agents can disrupt the interactions between the ligands, Slit proteins, and their receptors, Robo proteins, resulting in inhibition of angiogenesis, lymph vessel formation, and/or tumor lymphatic metastasis. Furthermore, other agents are developed in accordance with embodiments of the invention to provide diagnostic tools to predict the presence (and/or prognosis) of diseases or disorders associated with angiogenesis, lymph vessel formation, and/or tumor lymphatic metastasis.

[00119] While the invention has been described with respect to a limited number of embodiments, those skilled in the art, having benefit of this disclosure, will appreciate that other embodiments can be devised which do not depart from the scope of the invention as disclosed herein. Accordingly, the scope of the invention should be limited only by the attached claims.

Claims (27)

1. A method for preventing or treating a disorder mediated by a Slit protein, comprising:
administering to a subject a therapeutically effective amount of an agent that modulates or prevents interactions between the Slit protein and a Robo protein, wherein the disorder involves lymph vessel formation.

2. The method of claim 1, wherein the Slit protein is Slit2.

3. The method of claim 1, wherein the Robo protein is Robo 1 or Robo4.

4. The method of claim 1, wherein the agent is an antibody against the Slit protein.

5. The method of claim 1, wherein the agent is an antibody against the Robo protein.

6. The method of claim 4 or 5, wherein the antibody is a monoclonal antibody.

7. The method of claim 6, wherein the antibody is a humanized antibody.

8. The method of claim 1, wherein the agent is a fragment of an extracellular domain of the Robo protein.

9. The method of claim 8, wherein the fragment is derived from the first immunoglobulin-like domain of the Robo protein.

10. The method of claim 1, wherein the agent is a Robo fusion protein.

11. The method of claim 10, wherein the Robo fusion protein is a Robo-Fc fusion protein.

12. A composition for preventing or treating a disorder mediated by a Slit protein, comprising:
an agent that can modulate or interfere with interactions between the Slit protein and a Robo protein; and a pharmaceutically acceptable excipient, carrier, or solvent, wherein the disorder involves lymph vessel formation.

13. The composition of claim 12, wherein the Slit protein is Slit2.

14. The composition of claim 12, wherein the agent is an antibody against a Slit protein or a Robo protein.

15. The composition of claim 14, wherein the antibody is a monoclonal antibody.

16. The composition of claim 15, wherein the antibody is a humanized antibody.

17. The composition of claim 12, wherein the agent is a fragment of an extracellular domain of the Robo protein.

18. The composition of claim 17, wherein the fragment is derived from the first immunoglobulin-like domain of the Robo protein.

19. The composition of claim 12, wherein the agent is a Robo fusion protein.

20. The composition of claim 19, wherein the Robo fusion protein is a Robo-Fc fusion protein.

21. A method for prognosis or diagnosis of a disease or disorder mediated by a Slit protein, comprising:
(a) obtaining a test sample from a test subject and assessing expression levels of a Slit protein, a Robo protein, or both the Slit protein and the Robo protein in the test sample;
(b) obtaining a control sample from a subject not having the disease or disorder and assessing the expression levels of the Slit protein, the Robo protein, or both the Slit protein and the Robo protein in the control sample; and (c) comparing the expression levels assessed in (a) and (b) to obtain a result about the disease or disorder mediated by the Slit protein.

22. The method of claim 21, wherein the Slit protein is Slit2.

23. The method of claim 21, wherein the Robo protein is Robo 1 or Robo4.

24. The method of claim 21, wherein the expression levels are DNA expression levels, RNA
expression levels, protein expression levels, or a combination thereof.

25. The method of claim 1, 12, or 21, wherein the disorder is cancer.

26. The method of claim 25, wherein the cancer is metastatic.

27. The composition of claim 25, wherein the cancer is one selected from the group consisting of colorectal cancer, stomach cancer, esophageal cancer, lung cancer, hepatocellular carcinoma, breast cancer, and bladder cancer.