KR20130023262A - Detection and modulation of slit and roundabount(robo) mediated lymph vessel formation and uses thereof - Google Patents

Abstract

The present invention provides a method for preventing or treating a disorder mediated by a slit protein, comprising administering to the subject a therapeutically effective amount of a substance that modulates or interferes with the interaction between the slit protein and the Robo protein. As such, the disorder provides for a method that is involved in the formation of lymph vessels. The invention also provides compositions for preventing or treating disorders mediated by slit proteins and methods for prognosis or diagnosis of diseases or disorders mediated by slit proteins.

Description

Diagnosis and modulation of Slit and Roundabount (Robo) mediated lymph vessel formation and uses approximately}

FIELD OF THE INVENTION The present invention relates generally to reagents and methods for the prevention, treatment or diagnosis of a disease or disorder associated with the Slit-Robo signaling pathway, and the present invention particularly relates to the treatment of cancer progression and metastasis. And to reagents.

In advanced cancer, cancer metastasis is the leading cause of death of cancer patients. Much effort has been directed to cancer research and treatment, particularly basic cancer growth and metastasis mechanisms. However, the molecular mechanisms leading to cancer metastasis are still poorly understood. In general, cancer cells are metastasized by invasion of local tissues. Once the cancer cells are out of their initial position, they can enter the circulation system and the lymph system and move to other locations where they can begin new growth. The fact that cancer cells often metastasize to lymph nodes suggests that the lymphatic system plays an important role in the metastasis of cancer.

The lymphatic system is important for maintaining body fluid equilibrium, immune response and absorption of fatty acids. The lymphatic system is involved in these normal physiological functions as well as lymphatic fluid edema and inflammatory responses. In patients with metastatic tumors, a marked increase in lymphatic vessel formation is frequently observed. These observations suggest the importance of the lymphatic system in promoting cancer metastasis. However, the role of the lymphatic system in cancer metastasis is not entirely clear.

Since the lymphatic system is involved in cancer metastasis, targeting of tumor lymphoid metastasis thus represents a reasonable approach to cancer treatment.

Summary of the Invention

One aspect of the invention relates to a method for preventing or treating a disorder mediated by slit protein. A method according to one embodiment of the present invention for the prevention or treatment of disorders mediated by slit proteins is directed to a therapeutic of a substance that modulates or interferes with the interaction between the slit protein and the Robo protein. Administering an effective amount to the subject, wherein the disorder is involved in the formation of a lymph vessel. The substance can be an antibody against a slit protein, an antibody against a robo protein, a fragment of an extracellular domain of a robo protein or a fusion protein of an extracellular domain fragment of a robo protein. The slit protein may be Slit2. Robo protein may be Robo1 or Robo4.

Another aspect of the invention relates to a composition for the prevention or treatment of disorders mediated by slit proteins. Compositions for the prevention or treatment of disorders mediated by slit proteins include substances that can modulate or interfere with the interaction between the Slit protein and the Robo protein; And pharmaceutically acceptable excipients, carriers or solvents, wherein the disorder is involved in the formation of lymph vessels. The substance may be an antibody against a slit protein, an antibody against a robo protein, a fragment of an extracellular domain of a robo protein, or a fusion protein of an extracellular domain fragment of a robo protein. The slit protein may be slit2. Robo protein may be Robo1 or Robo4.

Another aspect of the invention relates to a method for prognosis or diagnosis of a disease or disorder mediated by slit proteins. Methods for prognosis or diagnosis of a disease or disorder mediated by slit proteins include (a) obtaining a test sample from a test subject and evaluating the expression level of slit protein, robo protein or both slit protein and robo protein in the test sample. step; (b) obtaining a control sample from the subject without disease or disorder and evaluating the expression level of slit protein, robo protein or both slit protein and robo protein in the control sample; And (c) comparing the expression levels evaluated in (a) and (b) to obtain a result regarding a disease or disorder mediated by the slit protein. The slit protein may be Slit2. Robo protein may be Robo1 or Robo4. The expression level can be a DNA, RNA and / or protein expression level.

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

1 shows the expression of Slit2 in cancer. (a) Immunoblotting of cell lysates of various cancer cell lines with anti-Slit2 antibodies. Slit2 / 293 cells were used as positive controls and V / 293 cells were used as negative controls. (b) Immunohistochemical staining of human cancers with anti-Slit2 antibody. Arrows indicate the expression of Slit2 in tumor cells. The rod size is 100 μm for the top panel and 10 μm for the bottom panel, respectively. (c) Slit2 gradient in human invasive breast cancer visualized by staining with anti-Slit2 antibody. Arrows indicate microvessels within the tumor. The rod size is 100 μm for the top panel and 40 μm for the bottom panel, respectively.
2 shows the construction of Slit2 transgenic mice. (a) Diagram of human slit2 (hSlit2) expression vector used for construction of transgenic mice. (b) Spot hybridization of newly constructed hSlit2 transgenic mice showing hSlit2-positive in # 9 mice. (c) Southern blot analysis of newly constructed hSlit2 transgenic mice showing hSlit2-positive in # 9 mice. (d) Immunblotting analysis for hSlit2 expression in pancreatic tissue of hSlit2 transgenic mice using 5A5 monoclonal antibody. Isotypic IgG was used as a negative control. hSlit2 / 293 cells were used as positive controls. (e) Immunoblotting analysis of hSlit2 expression using M2 monoclonal antibody specific for FLAG protein. (f) PCR analysis for FLAG expression in slit2-positive transgenic mice. C57 mice were used as negative controls. (g) No immunohistochemical analysis showed hSlit2 expression in pancreatic tissue of normal mice (top left panel), and the other panel showed positive expression of hSlit2, insulin, Robo1 and vWF in pancreatic tissue of Rip1-Tag2 transgenic mice. Shows. (h) The expression of hSlit2 protein measured by immunohistochemical analysis was higher in pancreatic tissue of hSlit2 / Rip1-Tag2 transgenic mice than pancreatic tissue of Rip1-Tag2 mice. * p <0.05; Scale bar: 10 μιη.
3 shows that slit2 promotes the growth of pancreatic tumors. Between (a) the number of angiogenic pancreatic islets, (b) tumor size, (c) tumor number, and (d) leukocyte count in pancreas and blood, between Rip1-Tag2 and Slit2 / Rip1-Tag2 transgenic mice. compare. (e) Fluorescent staining of pancreatic tumors in transgenic mice. (f) Hematoxylin and eosin (HE) staining of pancreatic tumors in transgenic mice. Fluorescence staining and HE staining show larger tumor size and more blood vessels in Slit2 / Rip1-Tag2 transgenic mice than Rip1-Tag2 transgenic mice. Comparison between Slit2 / Rip1-Tag2 transgenic mice and Rip1-Tag2 transgenic mice of (g) blood vessel density, (h) proliferation index and (i) apoptosis index measured by immunohistochemical analysis Shows that Slit2 / Rip1-Tag2 transgenic mice result in more angiogenesis, higher proliferation and reduced apoptosis than Rip1-Tag2 transgenic mice. Scale bar: 50 μm; *: p <0.05; **: p <0.01.
4 shows lymphatic tube formation and tumor lymphoid metastasis in Slit2 / Rip1-Tag2 transgenic mice. Slit2-overexpressing Slit / Rip1-Tag2 transgenic mice were subjected to (a) lymphovascular neovascularization (indicated by arrows) of intestinal tumors, (b) tumors within the lymphatic vessels (indicated by arrows) by HE staining, and ( c) show LYVE-1 staining of intestinal lymph nodes. LYVE-1 staining is for detecting endothelial cells of lymphatic vessels. Peripheral pancreatic tissue of Slit2 / Rip-1-Tag2 transgenic mice (d, e, f) measured by immunohistochemical analysis had more lymphatic endothelial cells than Rip1-Tag2 transgenic mice (g, h, i) . Lymphatic endothelial cell proliferation in the pancreas measured by immunofluorescence staining with anti-LYVE-1 antibody was greater in Slit2 / Rip1-Tag2 transgenic mice (l) than in wild type C57 mice (j) and Rip1-Tag2 transgenic mice (k). Higher in Expression of Robo1 and LYVE-1 in lymphatic endothelial cells in the pancreas was measured by immunofluorescence analysis using (m) anti-Robo1 antibody and (n) anti-LYVE-1 antibody. The overlay of Robo1 and LYVE-1 expression is shown in (o). LN: lymph node; C: tumor tissue; *: p <0.05; Scale bar: 1 mm (j-1) and 100 μm (rest panel).
5 shows a Slit2 recombinant protein that promotes migration and angiogenesis of human lymphatic endothelial cells. (a) Robo1 expression of lymphatic endothelial cell membranes (left panel: Rb7, right panel: R4). (b) The density of peripancreatic lymphatic vessels in Slit2 / Rip1-Tag2 transgenic mice is 1.5 times higher than that of Rip1-Tag2 mice. Recombinant Slit2.1 protein promotes (c) lymphoid endothelial cell migration and (d, e) angiogenesis. Migration and angiogenesis can be specifically inhibited by R5 (c, d and e). "None" represents negative control. VEGF-C was used as position control. Bar: 25 μιη; *: p <0.05; **: p <0.01.
6 (A) hRobo-Fc and hSlit2 protein interactions; (B) shows R5 (monoclonal antibody binding to the first immunoglobulin-like motif of Robo1 protein) which inhibits the interaction of hRobo-Fc with hSlit2 protein.
FIG. 7 shows expression of about 85 kD hRobo-Fc recombinant protein detected with rabbit anti-human IgG bound to horse radish peroxidase (HRP).
8 shows the functional analysis of antislit2 monoclonal antibodies and their use in various human tumor tissues. Immunoblots were used to verify the ability of S1 and S3 antibodies to detect full length or fragments of Slit2 protein. (a) S1 binds to the N-terminus of the protein to detect the full length slit2 protein. The 9E10 and S3 antibodies bind to the C-terminus of the protein to detect full length slit2. S1 and S3 antibodies specifically detect human Slit2 proteins, while S1 antibodies also detect human Slit1 and Slit3 proteins. 9E10 was used as a positive control. S1 monoclonal antibodies have been used to detect Slit2 expression in (b) colorectal cancer and breast cancer, (c) early lung cancer and metastasized lung cancer thereof, and (d) rectal cancer with and without lymphoid metastasis. mIgG was used as negative control (immunochemical analysis). Scale bar: 10 μιη.

Embodiments of the invention relate to methods, compositions or materials for the prevention, treatment or diagnosis of diseases or disorders mediated by slit proteins, in particular diseases or disorders related to lymphatic vessel formation. Diseases or disorders mediated by slit proteins are involved in the interaction between slit proteins and their receptor, robo protein. The disorder or disease includes tumor growth and / or tumor metastasis, such as tumor lymphoid metastasis.

The inventors of the present invention unexpectedly found that the interaction between the slit protein and the robo protein (receptor of the slit protein) causes signal transduction leading to lymphatic vessel formation. Thus, reagents that can modulate or interfere with the interaction between slit proteins and their receptors (robotic proteins) can be used to diagnose, treat or regulate unwanted lymphatic vessel formation, such as those involved in cancer metastasis.

Some embodiments of the invention relate to reagents for modulating or interfering interactions between slit proteins and their receptors (robotic proteins). The reagent may be a protein fragment or analog of a slit or robo protein (including a fusion protein or fragment of these proteins), or an antibody against a slit or robo protein (polyclonal, monoclonal or humanized antibody). Some embodiments of the present invention are directed to methods for diagnosing, preventing or treating a disorder mediated by slit-robot interactions, in particular interactions related to cancer progression and cancer metastasis.

The following examples illustrate embodiments of the invention using Slit2 as an example of slit protein and Robo1 or Robo4 as an example of robo protein. However, embodiments of the present invention may also include other slit or robo proteins involved in signaling related to lymphatic vessel formation.

The slit gene encodes secretory proteins essential for neural development. The protein acts as a "neurological" mobility signal. Mammals have three known slit genes: slit 1, slit 2 and slit 3. The genes are also expressed in a non-nervous system (Holmes, G. P. et al. Mech. Dev. 79: 57-72 (1998)). For example, mRNAs for slit 2 and slit 3 (except slit 1) are found in rat endothelial cells (Wu, J. Y. et al. Nature 410: 948-952 (2001)).

Structurally, the slit protein consists of four leucine-rich repeat sequences, nine epithelial growth factor (EGF) -like functional domains, one ALPS element located between the sixth and seventh domains of the EGF functional domain, and It has one cysteine knot at the C-terminus. Slit proteins are soluble proteins, but most of the slit2 proteins are linked to the cell surface.

Slit proteins bind to their receptor, Roundabout (Robo) protein. At the same time, the slit and robo protein were axon guided (Kidd, T. et al. Cell 92: 201-215 (1998)) and branch (Wang, K.-H. et al. Cell 96: 771-784 (1999)) and neuronal migration (Wu, W. et al. Nature 400: 331-336 (1999)). In addition, they also act as endogenous inhibitors for leukocyte chemotaxis (Wu, J. Y. et al. Nature 410: 948-952 (2001)).

Slit proteins (eg, Slit2) play a role in the inflammatory response as endogenous inhibitors of leukocyte chemotaxis (Wu, J. Y. et al. Nature 410: 948-952 (2001)). In the course of the inflammatory response, leukocytes attach to endothelial cells in blood vessels, penetrate the capillary walls and reach damaged or infected tissues. Leukocyte adhesion and infiltration are generally believed to be well matched in time and position by the action of many molecules such as chemotactic factors, cell adhesion molecules, cytoskeletal proteins and metalloenzymes. Wu et al. Suggested that slit2 could inhibit leukocyte chemotaxis induced by chemotaxis factors in vitro. In addition, inhibition of leukocyte chemotaxis by slit 2 can be blocked by soluble extracellular fragments of Robo protein (RoboN) (Wu et al, Nature, 410: 948, 2001), which is caused by slit 2 It demonstrates that inhibition of leukocyte chemotaxis is mediated by robo receptor-mediated signaling pathways.

Robo proteins are transmembrane proteins with one transmembrane domain. They act as receptors for slit proteins (eg slit2). Mammals have four Robo genes, Robo1, Robo2, Rig-1 and Robo4. Robo proteins are also expressed outside of the nervous system (Piper, M. et al. Mech. Dev. 94: 213-217 (2000)). For example, Robo1 RNA is found in mouse leukocytes (Wu, J. Y. et al. Nature 410: 948-952 (2001)). In addition, human endothelial cells express Robo4 (Huminiecki, L. et al. Genomics. 79: 547-552 (2002)).

Upon binding by a ligand (ie, slit protein), the robo receptor carries axon guidance and intracellular signals that eventually induce inhibitory action in various biological processes including branching, neuronal migration and leukocyte chemotaxis. (transduction)

As mentioned above, robo family proteins are transmembrane proteins with one transmembrane domain. Structurally, the extracellular domains of Robo1, Robo2, and Robo3 each have five immunoglobulin-like (Ig-like) domains and three fibronectin type III sequences. Intracellular domains include many structural sub-domains comprising the CC0, CC1, CC2 and CC3 sequences. Robo4 is smaller and has two immunoglobulin-like domains in its extracellular domain, and the intracellular domain comprises the CC0 and CC2 sequences.

Slit2 was found to bind to the first immunoglobulin-like domain of Robo1 through a complex interaction (Chen et al, J Neurosci. 2001 21: 1548-1556). In addition, monoclonal antibody R5 binding to the first immunoglobulin-like domain of Robo1 protein has been shown to neutralize slit2-mediated angiogenesis (Wang et al, Cancer Cell. 2003 4: 19-29), It suggests that the specific binding of slit2 to Robo1 is essential for slit2-mediated angiogenesis.

Slits and robo proteins have been shown to be involved in a variety of biological activities, including angiogenesis. However, the inventors of the present invention unexpectedly found that slit-robot protein interaction is also involved in lymphangiogenesis (ie, lymphatic vessel formation). More importantly, the inventors found that the lymphatic vessel formation is involved in cancer metastasis. Thus, materials that can interfere with slit-robot interaction may be useful materials for the prevention and treatment of cancer, in particular for the prevention and treatment of cancer metastasis.

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 below, slit 2 actually plays a role in cancer progression and metastasis. One possible mechanism by which Slit2 may contribute to cancer progression and metastasis is to promote tumor lymphatic vessel formation.

The possibility that slit2 plays a role in tumor progression and metastasis has been tested in animal models. For example, mice expressing high levels of Slit 2 were constructed as shown in FIG. 2. Crossing between Rip1-Tag2 transgenic mice and slit2 transgenic mice in which pancreatic cancer may occur naturally produces progenitor mice (Slit2 / Rip1-Tag2 mice). Details on the construction of the Slit2 / Rip1-Tag2 transgenic mouse strain are described later in the Examples section. These progenitor mice (Slit2 / Rpil-Tag2) produce significantly higher levels of Slit2 as measured by immunohistochemical analysis (FIG. 2H). In addition, these progenitor mice show new lymphatic vessel formation, increased tumor size, and marked increase in 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.

Slit2 promotes the growth and metastasis of pancreatic cancer. Comparison between Rip1-Tag2 and Slit2 / Rip1-Tag2 transgenic mice showed that Slit2 increased the number of angiogenic pancreatic Langerhans islets (FIG. 3A), tumor size (FIG. 3B), and tumor number (FIG. 3C), but the pancreas And did not increase the white blood cell count in the blood (FIG. 6D). FIG. 3E shows fluorescence staining of pancreatic cancer in transgenic mice, and FIG. 3F shows hematoxylin and eosin (HE) staining of pancreatic cancer in transgenic mice. Both fluorescence and HE staining show larger tumor size and more blood vessels in Slit2 / Rip1-Tag2 transgenic mice, compared to Rip1-Tag2 transgenic mice.

Comparison between Slit2 / Rip1-Tag2 transgenic mice and Rip1-Tag2 transgenic mice, as determined by immunohistochemical analysis, showed that slit2 expression increased vascular density (FIG. 3G), increased proliferation index (FIG. 3H), and cells Decreases the death index (FIG. 3I). These results indicate that Slit2 / Rip1-Tag2 transgenic mice have more angiogenesis, higher proliferation, and reduced apoptosis potential compared to Rip1-Tag2 transgenic mice.

The observation that slit2 expression increases the number of tumors suggests the possibility that it may also be involved in tumor metastasis. Since the lymphatic system is thought to be involved in most tumor metastases, this possibility has been tested for the effect of slit2 on lymphatic vessel formation.

4 shows that slit2 overexpression in Slit2 / Rip1-Tag2 mice actually results in increased lymphatic formation and tumor lymphoid metastasis. Slit2 / Rip1-Tag2 transgenic mice show increased tumor cell number inside the lymphatic vessels (Fig. 4b; indicated by arrows) as well as increased lymphatic tube formation of intestinal tumors (Fig. 4a; indicated by arrows) as indicated by HE staining. . 4C shows intestinal lymph nodes shown by positive LYVE-1 immunostaining for endothelial cells of lymphatic vessels.

Moreover, Slit2 / Rip-1-Tag2 transgenic mice have more lymphatic endothelial cells in peripancreatic tissue compared to Rip1-Tag2 transgenic mice (FIGS. 4G, 4H and 4I). Lymphatic endothelial cell proliferation of the pancreas, measured by immunofluorescence staining with anti-LYVE-1 antibody, was also compared to the proliferation of Slit2 / Rip1-Tag2 in wild-type C57 mice (FIG. 4J) and Rip1-Tag2 transgenic mice (FIG. 4K). Higher in transgenic mice (FIG. 4L). These results indicate that overexpression of Slit2 can induce lymphatic tube formation and promote cancer lymphoid metastasis.

Slit2's role in the induction of tumor lymphatic vessel formation may be attributed to slit2-induced migration and tube formation of lymphatic endothelial cells, R5, which is a monoclonal antibody to Robo1 (see FIGS. 5C-E), or Robo1 that can bind to Slit2. It is further supported by the observation that it can be specifically inhibited by the extracellular domain fragment of LoboN (data not shown). 6 shows that R5 can specifically interfere with the interaction between recombinant Robo1 (hRobo-Fc) (FIG. 7) and Slit 2 (FIGS. 6A and 6B).

The observation clearly shows the involvement of the slit-robot interaction in tumor-induced lymphatic formation and tumor lymphoid metastasis. Therefore, reagents that can prevent or control the interaction between the slit protein and the robo protein can be used as a therapeutic agent for preventing or treating metastatic cancer or as a diagnostic agent for identifying cancer patients suitable for treating a specific cancer. According to an embodiment of the present invention, the reagent may include an antibody against a slit protein, an antibody against a robo protein, a protein fragment including an extracellular domain of the robo protein, and the like. Antibodies for use in embodiments of the invention may be polyclonal or monoclonal antibodies. Moreover, the antibodies can be humanized to reduce immune rejection or complications.

According to some embodiments of the invention, extracellular active site fragments of robo specific antibodies or robo proteins can be used to competitively inhibit slit-robot interactions resulting in inhibition of slit-robot signaling mediated biological function. . Similarly, antibodies against slit proteins can also be used for the diagnosis, prevention or treatment of disorders mediated by slit-robotic signal transduction.

According to some embodiments of the invention, the antibody for use in the treatment of slit-mediated disorders may be a humanized antibody that may be used for the treatment of minimizing the immune response. Methods for humanization of antibodies are well known in the art. Some embodiments of the invention relate to a method for mass production of said recombinant protein as a therapeutic agent. The methods and reagents are described in the Examples described below.

In addition to the prevention or treatment of disorders mediated by slit proteins, some embodiments of the invention relate to methods for diagnosing such disorders. As mentioned above, the expression level of slit protein is increased in various cancers. Thus, detection of increased expression of slit proteins may indicate the presence of slit-mediated disorders.

8 shows functional analysis and use of anti-Slit2 monoclonal antibodies in the detection of various human tumors. Three antibodies, S1, S3 and 9E10, were used for this experiment. Immunoblots were used to assess the ability of S1 and S3 antibodies to detect full length or fragments of Slit2 protein. As shown in FIG. 8A, S1 binds to the N-terminus of the protein to detect the full-length slit2 protein. 9E10 and S3 antibodies bind to the C-terminus of the protein to detect full length slit2. S1 and S3 antibodies specifically detect human Slit2 proteins, and S1 antibodies also detect human Slit1 and Slit3 proteins. 9E10 antibody (obtained from ATCC) was used as a positive control.

As shown in Figures 8B, 8C and 8D, the S1 monoclonal antibody is slit2 in rectal and breast cancer (Figure 8b), early lung cancer and its metastatic lung cancer (Figure 8c) and rectal cancer with and without lymphoid metastasis (Figure 8d). It can be used to detect expression. The example clearly shows that antibodies against slit proteins can be used as a diagnostic tool for detecting or predicting the presence or outcome of various tumors.

The following examples further illustrate embodiments of the present invention.

Slit2  Methods for preventing or treating a disease or disorder associated with mediated lymphatic vessel formation

As mentioned above, some embodiments of the present invention relate to methods for preventing or treating diseases or disorders associated with slit2 mediated lymphatic vessel formation in a subject. The method according to one embodiment of the present invention reduces or hinders the slit2-robo interaction of the subject to a level sufficient to prevent or treat a disease or disorder related to slit2 mediated lymphatic formation in the subject, for example. It may include. The disease or disorder may be related to cancer progression or cancer metastasis.

Slit2-Robo interaction (eg, Slit2-Robo1 or Slit2-Robo4 interaction) can be reduced by any suitable method. For example, slit2-robot interactions may include replication of the Slit2 gene, replication of the Slit2 receptor gene, transcription of the Slit2 gene, transcription of the Slit2 receptor gene, splicing or translation of the Slit2 mRNA, By administering to the subject an effective amount of a substance that reduces splicing or translation of Slit2 receptor mRNA, maturation or cellular trafficking of the Slit2 precursor precursor, or maturation or cellular transport of the Slit2 receptor precursor Can be reduced.

According to another embodiment of the present invention, slit2-robot interaction may be reduced or prevented by administering to the subject an effective amount of a substance that reduces or inhibits slit2-robot protein-protein interaction. Any suitable material can be used to reduce or inhibit slit2-robot protein-protein interactions. The material may include, for example, an anti-slit2 antibody, an anti-robotic antibody or a robo protein fragment from an extracellular domain of a robo protein capable of binding to slit2. Any suitable anti-slit2 antibody or anti-robot antibody, including polyclonal antibodies, monoclonal antibodies, Fab fragments or F (ab ') ₂ fragments, may be used to reduce or interfere with slit2-robobo protein-protein interactions. Can be used, and the antibody can be humanized. In addition, robo receptor antagonists that can bind directly or indirectly to robo receptors can also be used. Examples of receptor antagonists may include small molecules or analogs of slit2 that can bind to robo receptors without inducing normal signal transduction.

The methods of the invention can be used to prevent or treat diseases or disorders associated with any Slit2 receptor mediated lymphatic vessel formation. For example, the methods of the present invention can be used to prevent or treat diseases or disorders associated with any slit2-Robo1 or slit2-Robo4-mediated lymphatic vessel formation. Slit2-Robo1 or Slit2-Robo4 interaction can be reduced by any suitable method. Preferably, the slit2-robo1 or slit2-robo4 interaction is achieved by administering to the subject an effective amount of a substance that reduces or interferes with the slit2-robo1 or slit2-robo4 protein-protein interaction. Can be reduced or prevented. The preferred substance may be an anti-Slit2 antibody, an anti-Robo1 antibody, an anti-Robo4 antibody, or a Robo1 or Robo1 fragment from the extracellular domain of Robo1 or Robo4 capable of binding to Slit2. .

Any suitable anti-slit2 antibody can be used, including the anti-slit2 antibody disclosed in Hu (Neuron August 1999; 23 (4): 703-l 1). Similarly, any suitable anti-Robo1 antibody can be used, including the anti-Robo1 antibody disclosed in Hivert et al. (Mol Cell Neurosci December 2002; 21 (4): 534-45). Preferably, the anti-Robo1 antibody is an antibody against the first immunoglobulin domain of Robo1. More preferably, the antibody against the first immunoglobulin domain of Robo1 is R5. Any suitable Robo1 fragment, such as RoboN, from the extracellular domain of Robo1 can be used.

The substances used in the methods of the invention may be administered alone. Preferably, the substance may be administered with a pharmaceutically acceptable carrier or excipient.

The methods of the present invention can be used to prevent or treat any disease or disorder associated with slit2 mediated lymphatic vessel formation. The disorder includes, for example, cancer. Preferably, the cancer is metastatic. Cancers include, for example, malignant melanoma, bladder squamous carcinoma, neuroblastoma, small cell lung cancer, colon adenocarcinoma, bladder transitional epithelium It may be a blade transitional cell carcinoma, breast cancer, adenoid cystic carcinoma of the salivary glands, hepatocellular carcinoma or rhabdomyosarcoma.

The methods of the present invention can be used to prevent or treat any disease or disorder associated with the formation of slit2 mediated lymphatic vessels in any subject. Preferably, the subject is a mammal. More preferably, the mammal is a human.

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

Slit2  Pharmaceutical compositions and combinations for preventing or treating diseases or disorders associated with mediated lymphatic vessel formation

In another aspect, embodiments of the invention are directed to pharmaceutical compositions for preventing or treating a disease or disorder associated with slit2 mediated lymphatic formation in a subject. The pharmaceutical composition may comprise, for example, an effective amount of a substance that reduces or interferes with slit2-robot interaction. The pharmaceutical composition may further comprise a pharmaceutically acceptable carrier or excipient.

Formulation, dosage and route of administration can be determined according to methods known in the art (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).

In the treatment or prevention of diseases or disorders associated with slit2 mediated lymphatic vessel formation, appropriate dosage levels may generally be from about 0.01 to 500 mg / kg body weight per day. Preferably, the dosage level may be about 0.1 to about 250 mg / kg per day. In a more preferred embodiment, the dosage level can be about 0.1 to about 20 mg / kg per day. Appropriate dosages can be administered in single or multiple doses. Specific dosage levels and frequency of administration for any particular subject can vary and include the activity of the particular compound employed, the metabolic stability and duration of action of the compound, age, weight, general health, sex, diet, mode of administration, and It will be appreciated that it may depend on various factors including time, rate of excretion, drug combination, severity of the particular condition, and the patient being treated.

The pharmaceutical compositions of the present invention can be administered orally, parenterally, by inhalation spray, locally, rectally, nose, orally, vaginally, via implanted reservoir, or any It may be administered in an appropriate dosage form.

In another aspect, some embodiments of the present invention are directed to combinations for preventing or treating a disease or disorder associated with slit2 mediated lymphatic vessel formation in a subject. The combination may be a) an effective amount of a therapeutic agent of the invention that can reduce or inhibit slit2-robot interaction; And b) effective amounts of other substances that can reduce or arrest lymphatic vessel formation.

For the treatment of cancer, any anti-neoplasmic substance can be used in the combination of the present invention. Examples of anti-tumor materials that can be used in the compositions and methods of the present invention are described in US Patent Application 2002 / 044,919. In one embodiment, the anti-tumor material used may be an anti-angiogenic material. Anti-angiogenic materials can be basement membrane degradation inhibitors, cell migration inhibitors, endothelial cell proliferation inhibitors, and three-dimensional organization and inhibitors of effects. Examples of such anti-angiogenic substances are described in Auerbach and Auerbach, Pharmacol. Ther, 63: 265-311 (1994); O'Reilly, Investigational New Drugs, 15: 5-13 (1997); J. Nat'l Cancer Instil, 88: 786-788 (1996); And US Pat. Nos. 5,593,990, 5,629,327 and 5,712,291. In another embodiment, the anti-tumor material used is an alkylating agent, antimetabolite, natural product, platinum coordination complex, anthracenedione, substituted element ( substituted urea), methylhydrazine derivatives, adrenocortical suppressants, hormones and antagonists.

Slit2  Methods for predicting or diagnosing diseases or disorders associated with mediated lymphatic vessel formation and Kit

Some embodiments of the present invention are directed to methods for predicting or diagnosing a disease or disorder associated with slit2 mediated lymphatic formation in a subject. The method according to an embodiment of the present invention comprises, for example, a) obtaining a test sample from a test subject and evaluating slit2 and / or slit2 receptor (robot) levels in the test sample; b) obtaining a control sample from a control subject without a disease or disorder associated with slit2 mediated lymphatic formation and evaluating slit2 and / or slit2 receptor (robot) levels of the control sample; And c) comparing the slit 2 and / or slit 2 receptor (robot) levels assessed in a) and b) above, wherein the slit 2 and / or slit 2 receptor levels of the control subject are compared. Increased Slit2 and / or Slit2 receptor levels in a test subject indicate that the test subject has a disease or disorder related to slit2 mediated lymphatic vessel formation. According to some embodiments of the invention, the Slit2 receptor tested in the above method may be Robo1 or Robo4. Slit2 and / or slit2 receptor levels can be assessed at appropriate levels, such as nucleic acid or protein levels.

Some embodiments of the invention relate to kits for predicting or diagnosing a disease or disorder associated with slit2 mediated lymphatic formation in a subject. The kit comprises a) means for obtaining a test sample from the test subject and a control sample from the control subject; b) means for assessing slit2 and / or slit2 receptor levels in the test sample and the control sample; And optionally c) means for evaluating and / or comparing slit2 and / or slit2 receptor levels of said test sample and control sample. According to some embodiments of the invention, the slit2 receptor tested may be Robo1 or Robo4.

Example

(1) in human cancer Slit2  Expression

To investigate the results of the present invention, we examined whether slit2 is expressed in human cancer cell lines derived from various tissues and organs. 1A shows slit divalent A375 cells (malignant melanoma), SCaBER cells (bladder squamous cell carcinoma), SK-N-SH cells (neuroblastoma), NCI-H446 cells (small cell lung cancer), LoVo cells (colon adenocarcinoma), T24 cells (bladder transitional epithelial carcinoma), ZR-75-30 cells (breast cancer), Acc-2 and Acc-M cells (adenocarcinoma of the salivary glands), SMMC-7721 cells (hepatocellular carcinoma) and A673 cells ( Rhabdomyosarcoma). However, Slit2 was superficially absent in A549 cells (lung cancer), HeLa cells (cervical epithelial adenocarcinoma), MCF-7 cells (breast adenocarcinoma) and 786-0 cells (primary renal cell adenocarcinoma).

The discovery of slit2 expression in various cancer cell lines is consistent with the observations detected by RT-PCR that slit2 is expressed in prostate cancer. In addition, FIG. 1B shows that Slit2 is expressed in human malignant melanoma, rectal mucinous adenocarcinoma, invasive breast carcinoma and hepatocellular carcinoma. Clearly, at the site of breast carcinoma, it appears that more Slit2 staining is detected at locations with more cancer cells and blood vessels (FIG. 1C). In contrast, less Slit2 staining is detected at locations with fewer cancer cells and blood vessels. Similar behavior can be observed in human hepatocellular carcinoma, but not in malignant melanoma or mucosal adenocarcinoma (data not shown). This direct correlation between slit2 expression and cancer / vascular cell proliferation suggests an important role of slit2-induced tumor lymphatic vessel formation in the pathogenesis of various human cancers.

(2) Slit2 Of Rip1 - Tag2  Construction of Transgenic Mice

2A shows an expression vector comprising a human Slit2 (hSlit2) gene fused with a Flag fragment coding DNA sequence operated by a CMV promoter. The expression vectors were used to make three lines of Slit2 transgenic mouse lines. The presence of hSlit2 gene in the transgenic mice was confirmed by spot hybridization (FIG. 2B) and Southern blot (FIG. 2C), and expression of hSlit2 protein in the transgenic mice was determined by immunoblot analysis (FIG. 2D). Confirmed. Expression of the Flag tag (FIG. 2E) and the presence of the Flag DNA sequence (FIG. 2F) were detected in pancreatic tissue extracts by Western blot and PCR analysis using anti-Flag monoclonal antibody (M2), respectively. These results demonstrate that transgenic mouse lines expressing hSlit2 have been successfully constructed.

2G shows that pancreas in normal mice does not express the Slit2 gene (upper left panel), whereas Slit2 is expressed in 12 week old Rip1-Tag2 transgenic mice (FIG. 2H, top right panel). In addition, the expression of Slit 2 overlaps with the expression of insulin (FIG. 2G, top middle panel). These results indicate that Slit2 expression is upregulated by pancreatic tumorigenesis in pancreatic β cells. In addition, while both Robol and vWF are expressed in endothelial cells, these proteins are not expressed in pancreatic β cells of Rip1-Tag2 transgenic mice (FIG. 2G, bottom left and right panels).

To further evaluate the role of Slit2 in tumorigenesis, Slit2 transgenic mice were crossed with Rip1-Tag2 transgenic mice to generate Slit2 / Rip1-Tag transgenic mice. Compared to Rip1-Tag2 transgenic mice of the same age, 10 and 13 week old Slit2 / Rip1-Tag2 transgenic mice express higher levels of Slit2 protein (FIG. 2H), which is a Slit2 / Rip1-Tag2 transgenic mouse. Shows successful overexpression of slit2. The results show that pancreatic β cells can induce the secretion of Slit2 protein through a paracrine mechanism. Eventually, slit2 can interact with Robo1, which is stably expressed in endothelial cells of pancreatic blood vessels and activates the signaling pathways of cells during the formation of tumors of the pancreas.

(3) Slit 2  Promotes tumor growth in the pancreas

3A shows a significant increase in the number of angiogenic Langerhans islets of the pancreas in 10 and 12 week old Slit2 / Rip1-Tag2 transgenic mice. 3B and 3C show that tumor volume and number of pancreas are higher in Slit2 / Rip1-Tag2 transgenic mice than Rip1-Tag2 transgenic mice. However, there was no significant difference in leukocyte count between Slit2 / Rip1-Tag2 and Rip1-Tag2 transgenic mice (FIG. 3D). In addition, FIG. 3E shows that Slit2 / Rip1-Tag2 transgenic mice have enlarged blood vessels, as measured after injecting fluorescent dye into the blood vessel, while Rip1-Tag2 transgenic mice show no change or slightly enlarged blood vessels. Shows. 3F shows apparent vasodilation and bleeding in Slit2 / Rip1-Tag2 transgenic mice. In contrast, Rip1-Tag2 transgenic mice had little bleeding. Moreover, Slit2 / Rip1-Tag2 transgenic mice have more neovascularization (FIG. 3G), higher proliferation index (FIG. 3H), and lower apoptosis index (FIG. 3I) than Rip1-Tag2 transgenic mice. These results indicate that slit2 overexpression promotes angiogenesis and growth of pancreatic tumors in Rip1-Tag2 transgenic mice.

(4) Slit2  Overexpression promotes lymphoid metastasis of pancreatic tumors

Rip1-Tag2 transgenic mice develop pancreatic tumors through four stages: angiogenic islet stage, atypical growth stage, pancreatic tumor stage, and invasive pancreatic tumor stage. The majority of these mice eventually die of hyperglycemia at 15 weeks of age. Pancreatic tumor metastasis was not observed during their lifetime in Rip1-Tag2 transgenic mice, but high incidence (24.32%) of intestinal membrane metastasis was observed in Slit2 / Rip1-Tag2 transgenic mice (FIGS. 4A and 4B). ). Tumor nodules are also observed inside the lymphatic vessels of Slit2 / Rip1-Tag2 transgenic mice (FIG. 4B). No tumor weight difference was observed between Rip1-Tag2 and Slit2 / Rip1-Tag2 transgenic mice.

Lymphatic endothelial cells were evaluated by immunohistochemical analysis using LYVE-1 as a lymphatic endothelial cell marker. 4C, 4D and 4E show that Slit2 / Rip1-Tag2 transgenic mice have more new lymphatic vessels distributed mainly in the peripheral region of the pancreas and inside the lymph nodes (FIG. 4F). In contrast, Rip1-Tag2 transgenic mice contain fewer newly developed lymphatic vessels (FIGS. 4G, 4H and 4I). As measured by immunofluorescence staining assay, Figures 4J, 4K and 4L show that Slit2 / Rip1-Tag2 transgenic mice have more newly developed lymphatic vessels compared to wild type C57 mice and Rip1-Tag2 transgenic mice. Shows. 4m, 4n and 4o show that Robo1 is expressed in lymphatic endothelial cells of Slit2 / Rip1-Tag2 transgenic mice. Lymphocyte counts determined by semiquantitative immunohistochemical analysis increased about 1.5-fold in Slit2 / Rip1-Tag2 transgenic mouse pancreas compared to Rip1-Tag2 transgenic mice (FIG. 5B). At the same time, these results indicate that Slit2 overexpression can induce lymphatic tube formation and tumor lymphoid metastasis.

(5) Slit2  Recombinant protein promotes lymphatic endothelial cell migration and tube formation in vitro

5A shows that Robo1 expression of primary human lymphatic endothelial cells was assessed by immunofluorescence analysis with polyclonal antibody (Rb7) and monoclonal antibody (R4). R4 specifically recognizes Robo1, but does not recognize Robo2-4. 5A shows that Robo1 is expressed on the cell membrane of human lymphatic endothelial cells. 5B shows that the lymphatic density of the Slit2 / Rip1-Tag2 transgenic mouse pancreas is about 1.5 times higher than the lymphatic density of Rip1-Tag2 transgenic mice.

To determine if the slit2.1 recombinant protein could affect the migration of human lymphatic endothelial cells, Boyden chamber analysis was used. VEGF-C was used as a positive control in the experiment. 5C shows that slit 2.1 promotes human lymphatic endothelial cell migration above 200 pM. Excess of R5 (anti-Robo1 antibody) interferes with binding of Robo1 to Slit2, resulting in inhibition of Slit2 mediated lymphatic endothelial cell migration.

In vitro, Matrigel analysis was performed to determine whether slit2.1 affects tube formation of human lymphatic endothelial cells. Again, VEGF-C was used as a positive control. 5D and 5E show that 10 nM of slit 2.1 induces tube formation. Excess of R5 inhibits slit2 mediated lymphatic endothelial cell tube formation. At the same time, these results indicate that Slit2 protein binds to its receptor and promotes lymphatic endothelial cell migration and lymphatic tube formation.

(6) Slit2  Overexpression is a precursor to tumor lymphoid metastasis in cancer patients

The results described above suggest that slit2 expression may be a useful marker for cancer metastasis to the lymphatic system. This view is supported by immunochemical staining data of human tumor specimens with anti-Slit2 antibodies. The properties of the various antibodies used in this experiment are shown in FIG. 8A. S1, an anti-Slit2 monoclonal antibody, can recognize both full-length and N-terminal fragments of Slit2 protein. 9E10 recognizes a c-myc tag fused to a Slit2 protein. Another anti-Slit2 monoclonal antibody, S3, recognizes both full-length and C-terminal fragments of the Slit2 protein. S1 also recognizes human slit 1 and slit 3 (FIG. 8A). Thus, S1 can be used to detect various slit proteins. Using S1 immunostaining, 742 out of 955 human tumor samples were shown to be positive for slit expression (Table 1; FIGS. 8B-D). Importantly, slit expression in rectal cancer, gastric cancer and lung cancer is significantly higher in lymphoid metastasis than cancer without metastasis (Table 2; FIGS. 8C and 8D).

Slit protein expression in various human tumor tissues measured by immunohistochemical analysis using S1 monoclonal antibody Cancer type Tested  gun
Number of samples Slit -positivity
Number of samples Slit -positivity
% Of sample Rectal cancer 314 256 81.53 Stomach cancer 326 237 72.7 Esophageal Cancer 89 86 96.63 Lung squamous cell carcinoma 43 38 88.37 Lung adenocarcinoma 9 9 100 Hepatocellular Carcinoma 53 45 84.91 Invasive ductal carcinoma 95 55 57.89 Bladder cancer 26 16 61.54

Slit protein expression in various human tumor tissues measured by immunohistochemical analysis using S1 monoclonal antibody Cancer type Tested  gun
Number of samples Positive rate (%)
(Slit-positive samples with lymphatic transitions /
Total number of samples with lymphoid metastasis) Positive rate (%)
(Slit-positive sample / without lymphoid transfer
Total number of samples without lymphoid metastasis) Rectal 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)

The examples presented above clearly show that the slit-robotic signaling pathway plays an important role in regulating lymphatic vessel formation and tumor lymphoid metastasis. Thus, materials, techniques, and methods that may modulate or interfere with the interaction between slit proteins and robo receptors are directed to treating lymphatic vessel formation and / or tumor lymphoid metastasis-related diseases mediated by slit-robotic signaling pathways. Can be used.

For example, a robo protein fragment (eg, a fusion protein of a robo extracellular domain such as a robo extracellular domain or a hRobo-Fc fusion protein) or an antibody targeting a slit or robo protein modulates slit-robot protein interactions. Or to interfere, it provides treatment for a slit-robot signaling pathway-mediated disease or disorder. In this regard, robo protein fragments such as extracellular domains can be used as decoys for binding to slit ligands, thus preventing or reducing the binding of slit proteins to robo receptors. In addition to therapeutic applications, slit or robo receptor proteins or fragments or antibodies against slit or robo protein can be used as diagnostic or prognostic markers. For example, antibodies against slit or robo protein can be used to assess tumor lymphoid metastasis by comparing the expression levels of slit and / or robo protein in a patient.

Some embodiments of the present invention relate to methods of their use in the treatment, diagnosis, and prognosis of diseases or disorders related to slit and / or robo protein and slit-robot signaling pathways. Some examples of such embodiments are described below.

(7) human immunoglobulin Fc  Human fused with the realm Robo 1  protein( hRobo - Fc Extracellular IgG  Preparation of Recombinant Proteins Containing Domains

(7a) of human immunoglobulins Fc  Area Cloning

PCR using the pAc-k-CH3 plasmid (including the constant region of the human immunoglobulin gene) (US Biological Co.) as a template revealed a 687 bp region located at the C-terminus of the immunoglobulin heavy chain. Amplified. The sequences of the hFc forward and reverse primers used for PCR amplification are as follows:

hFc forward: 5'-AACCGTGCGGCCGCTGTTGTGACAAAACTCACAC-3 '(SEQ ID NO: 1)

hFc reverse: 5'-CGCGGAGATCTTCATTTACCCGGAGACAGGGAGAGGC-3 '(SEQ ID NO: 2)

After PCR products were cut with NotI and BglII restriction enzymes, NotI / BglII fragments were linked with pVL1393 plasmid (BD Pharmingen) truncated with NotI and BglII to construct a pVL1393-hFc plasmid with the Fc region of human immunoglobulin sequences. The sequence of the Fc region was confirmed by DNA sequencing, and no mutation was found.

(7b) Robo 1  Gene fragment Cloning

Robo1 cDNA clone (Origene, DNA SEQ ID NO: NM_002941.2, Article No. SC109739) was used as template. Extracellular domains containing five immunoglobulin-like domains (1-1621 bp) were selected for PCR amplification. The sequences of Robo1 forward and reverse primers used for PCR amplification are as follows:

Robo1 forward: 5'-GGCGGCCTCTAGAATGAAATGGAAACATGTTCC-3 '(SEQ ID NO: 3)

Robo1 reverse: 5'-CTATAAGCGGCCGCCAATGTAAGCACTCCATGTT-3 '(SEQ ID NO: 4)

PCR products were then cut with XbaI and NotI restriction enzymes. The XbaI / NotI fragment was linked to pVL1393-hFc, previously cut with XbaI and NotI, to construct the expression vector pVL1393-hRobo-Fc. The pVL1393-hRobo-Fc expression vector encodes a protein comprising the extracellular domain of Robo 1 (including five immunoglobulin-like (Ig-like) domains) fused with the Fc region of human immunoglobulin. The sequence of the Robo Ig-like domain of the expression vector was confirmed by DNA sequencing, and no mutation was found. The open reading frame (ORF) of the Robo1 Ig-like domain fused with the hFc region was properly constructed with start codons and stop codons at the correct locations.

(7c) hRobo - Fc  Expression and Purification of Fusion Proteins

Example I Mass plasmid production of pVL1393-hRobo-Fc was carried out and purified using the plasmid separation kit (QIAGEN plasmid Midi Kit) according to the protocol provided in the kit. Purified pVL1393-hRobo-Fc was used to construct hRobo-Fc recombinant baculovirus (BD Pharmingen, BaculoGold ™ Starter Package and Transfection Kit) according to the protocol.

After selection, the recombinant virus was amplified and used to infect Sf9 insect cells (grown in IPL-41 medium (Invitrogen) in a 27 ° C. incubator) to express the hRobo-Fc fusion protein. The fusion protein was detected with a monomer of about 85 kD under reducing conditions using a horse radish peroxidase (HRP) -binding rabbit anti-human IgG antibody (Santa Cruz Biotech) (FIG. 7). The hRobo-Fc fusion protein can be expressed in large quantities by infecting Sf9 cells with hRobo-Fc recombinant baculovirus. The recombinant protein produced can thus be purified using a Protein A affinity column (eg, GE Healthcare, nProtein A Sepharose ™ 4 Fast Flow). In general, mg amount of hRobo-Fc fusion protein can be obtained with 1 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 (monoclonal antibody recognizing the first immunoglobulin-like domain of Robo1 protein) can inhibit the interaction between hRobo-Fc fusion protein and hSlit2 (FIG. 6B). The data demonstrates the specificity and biological activity of the hRobo-Fc fusion protein.

According to some embodiments of the invention, the similar or modified robo fusion protein may comprise other extracellular domains of the robo gene fragment (eg, comprising 1 to 4 immunoglobulin-like domains), other fusion proteins (eg For example, it may be prepared using other systems such as glutathione S-transferase (GST) and / or other protein expression systems (eg, CHO-dhfr: dehydrofolate reductase deficient CHO cells). have. Some of these embodiments are described below.

Example II : Baculovirus  Mice Using Protein Expression Systems Robo 1  Extracellular Domain and Mouse Immunoglobulin Fusion Proteins Fc  Area ( mRobo - Fc Mass production

1. Mouse Immunoglobulins Fc  Selection of areas and Cloning

Using a mouse IgG _2b monoclonal antibody as a template, 720-nucleotide sequences encoding the C-terminus of the heavy chain were selected for PCR amplification. PCR primers are presented below:

mFc forward: 5'-GCACTCTAGACTTGAGCCCAGCGGGCCCAT-3 '(SEQ ID NO: 5)

mFc reverse: 5'-CTGAGGATCCTCATTTACCCGGAGACCGGG-3 '(SEQ ID NO: 6)

PCR products were cut with XbaI and BamHI, and pVL1392-mFc was constructed by subcloning the XbaI and BamHI fragments into pVL1392 vector (BD Pharmingen) previously cut with XbaI and BamHI. DNA sequencing confirmed the correct Fc sequence.

2. Mouse Robo 1  Selection of gene fragments and Cloning

Using the mouse Robo1 gene (NM_019413) as a template, sequences from ATG to 2445 comprising five Ig-like regions and three fibronectin type 3 structural domains (FN3 domains) were 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 product was cut with BglII and XbaI and pVL1392-mRobo-Fc was constructed by subcloning BglII and XbaI fragments into pVL1392-mFc previously cut with BglII and XbaI. DNA sequencing confirmed that the mouse Robo1 gene fragment and mFc formed the same ORF, and that the initiation and termination codons were located in the correct position.

Mass separation and purification of the pVL1392-mRobo-Fc plasmid was performed using the QIAGEN Plasmid Midi Kit (Qiagen) according to the User's Guide. Purified pVL1392-mRobo-Fc plasmid was used to construct recombinant baculoviruses expressing the mRobo-Fc fusion protein according to the User's Guide to the Baculovirus Expression System (BD Pharmingen). Recombinant baculovirus was used to infect Sf9 insect cells to determine infection efficiency and to identify high expressers for fusion proteins. mRobo-Fc protein was purified using Protein A affinity chromatography (GE Healthcare) according to the nProtein A Sepharose ™ 4 Fast Flow User Guide (GE Healthcare).

Example III : Baculovirus  Human using protein expression system Robo 1  Extracellular domains (having different lengths) and of human immunoglobulin fusion proteins Fc  Mass production of areas

1. Human Immunoglobulins Fc  Selection of areas and Cloning

Using the constant region of the human immunoglobulin gene (pAc-k-CH3) (US Biological) as a template, 687-nucleotide sequences encoding the C-terminus of the heavy chain were selected for PCR amplification. PCR primers are presented below:

hFc forward: 5'-AACCGTGCGGCCGCTGTTGTGACAAAACTCACAC-3 '(SEQ ID NO: 9)

hFc reverse: 5'-CGCGGAGATCTTCATTTACCCGGAGACAGGGAGAGGC-3 '(SEQ ID NO: 10)

PCR products were cut into NotI and BagII, and pVL1392-hFc was constructed by subcloning NotI and BagII fragments into pVL1392 vector (BD Pharmingen) previously cut into NotI and BagII. DNA sequencing confirmed the correct Fc sequence.

2. Human Robo 1  Selection of gene fragments and Cloning

Using the human Robo1 gene (NM_002941.2, Origene Product No. SC109739) as a template, sequences from ATG to 1342 containing four Ig-like regions were amplified using the following primers:

Robo1 forward: 5'-GGCGGCCTCTAGAATGAAATGGAAACATGTTCC-3 '(SEQ ID NO: 11)

Robo1 Reverse 2: 5'-CATTATGCGGCCGCCATCTGTAACTTCCAAATAT-3 '(SEQ ID NO: 12)

Using the human Robo1 gene (NM_002941.2, Origene Product No. SC109739) as a template, sequences from ATG to 1051 containing three Ig-like regions were amplified using the following primers:

Robol forward: 5'-GGCGGCCTCTAGAATGAAATGGAAACATGTTCC-3 '(SEQ ID NO: 13)

Robol reverse 3: 5'-ATAATAGCGGCCGCGAGGTTCTTGAACAGTCAGAGTA-3 '(SEQ ID NO: 14)

Using the human Robo1 gene (NM_002941.2, Origene Product No. SC109739) as a template, sequences from ATG to 829 containing two Ig-like regions were amplified using the following primers:

Robo1 forward: 5'-GGCGGCCTCTAGAATGAAATGGAAACATGTTCC-3 '(SEQ ID NO: 15)

Robo1 Reverse 4: 5'-ATAATTGCGGCCGCCATCCACAGTTACTGCCAAGTTACT-3 '(SEQ ID NO: 16)

Using the human Robo1 gene (NM_002941.2, Origene Product No. SC109739) as a template, sequences from ATG to 529 containing one Ig-like region were amplified using the following primers:

Robo1 forward: 5'-GGCGGCCTCTAGAATGAAATGGAAACATGTTCC-3 '(SEQ ID NO: 17)

Robo1 reverse 5: 5'-TTCTATGCGGCCGCAAGGGTTTTGTCTGAAGTCAT-3 '(SEQ ID NO: 18)

PCR products obtained in the steps described above were cut with XbaI and NotI. XbaI and NotI fragments were subcloned into pVL1392-hFc previously cut into XbaI and NotI to construct vectors expressing proteins with extracellular domains of human Robo1 of various lengths fused with the Fc region of human immunoglobulins. DNA sequencing confirmed that the human Robo1 gene fragment and hFc formed the same ORF. Initiation and termination codons were found to be in place.

Mass separation and purification of the pVL1392-hRobo-Fc plasmid was performed using the QIAGEN Plasmid Midi Kit (Qiagen) according to the User's Guide. Purified pVL1392-hRobo-Fc plasmid was used to construct recombinant baculovirus expressing the hRobo-Fc fusion protein according to the user guide for the Baculovirus Expression System (BD Pharmingen). Recombinant baculovirus was used to infect Sf9 insect cells to determine infection efficiency and to identify high expressers. hRobo-Fc fusion proteins were purified using Protein A affinity chromatography (GE Healthcare) according to the nProtein A Sepharose ™ 4 Fast Flow User Guide (GE Healthcare).

Example IV: Human Using CHO-dhfr (Dihydrofolate Reductase Deficient CHO Cells) System Robo 1  Extracellular domains (having different lengths) and of human immunoglobulin fusion proteins Fc  Mass production of areas

1. Human Robo 1  Selection of gene fragments and Cloning

Using the human Robo1 gene (NM_002941.2, Origene Product No. SC109739) as a template, sequences from ATG to 1760 containing five Ig-like regions were amplified using the following primers:

Robo1 f1760: 5'-GGCCAAGCTTATGAAATGGAAACATGTTCC-3 '(SEQ ID NO: 19)

Robo1 rl760: 5'-TCCACGGAATTCAAATTTGGTTGCC-3 '(SEQ ID NO: 20)

PCR products were cut with HindIII and EcoRI. HindIII and EcoRI fragments comprising the five Ig-like regions of the extracellular domain of human Robo1 were subcloned into pEGFP-N1 vectors (BD Pharmingen) previously cut with HindIII and EcoRI to construct pEGFP-N1-hRobo. DNA sequencing confirmed that the human robo sequence was correct.

2. Human Immunoglobulins Fc  Selection of areas and Cloning

Using the constant region of the human immunoglobulin gene (pAc-k-CH3) (US Biological) as a template, 714-nucleotide sequences encoding the C-terminus of the heavy chain were selected for PCR amplification. PCR primers are presented below:

hFc Forward 2: 5'-AACCGTGAATTCCGTGGACAAGAGAGTTGAGCC-3 '(SEQ ID NO: 21)

hFc reverse 2: 5'-TACGGGTCGACTCATTTACCCGGAGACAGGG-3 '(SEQ ID NO: 22)

PCR products were cut with EcoRI and SalI. Vectors expressing proteins comprising five Ig-like regions of the extracellular domain of human Robo1 fused to the Fc region of human immunoglobulin by subcloning the EcoRI and SalI fragments into pEGFP-N1 previously truncated with EcoRI and SalI pEGFP-N1-hRobo-Fc) was prepared. DNA sequencing confirmed that the human Robo1 gene fragment and hFc formed the same ORF. Initiation and termination codons were found to be in place.

The pEGFP-N1-hRobo-Fc vector was cut with HindIII and SalI. HindIII and SalI fragments were subcloned into p3CI-dhfr previously cut with HindIII and SalI to express a protein comprising five Ig-like regions of the extracellular domain of human robo1 fused with the Fc region of human immunoglobulin dhfr-hRobo-Fc was produced. DNA sequencing confirmed that the human Robo1 gene fragment and hFc formed the same ORF. Initiation and termination codons were found to be in place.

Mass separation and purification of the p3CI-dhfr-hRobo-Fc plasmid was performed using the QIAGEN Plasmid Midi Kit (Qiagen) according to the User's Guide. Purified p3CI-dhfr-hRobo-Fc plasmid was used to infect CHO-dhfr cells to select high expressing clones according to the Lipofectin User Guide (Invitrogen). Medium from high expressing clones was used to purify hRobo-Fc proteins of various lengths using Protein A affinity chromatography (GE Healthcare) according to the nProtein A Sepharose ™ 4 Fast Flow User Guide (GE Healthcare).

Example  V: Baculovirus  Human using protein expression system Robo 1  Extracellular IgG -Like regions and of human immunoglobulins Fc  Mass Production of Fusion Proteins Between Regions

One. pVL1393 - hRobo1 - Fc Build

Using the p3CI-dhfr-hRobo-Fc plasmid obtained in Example IV as a template, PCR was performed with the following primers to amplify the Fc region of the human immunoglobulin gene:

Robol forward ': 5'-AGGCGGCCTCTAGAATGAAATGGAAACATGTTCC-3' (SEQ ID NO: 23)

hFc reverse 3: 5'-TACGGGCGGCCGCTCATTTACCCGGAGACAG-3 '(SEQ ID NO: 24)

PCR products were cut with XbaI and NotI. The XbaI and NotI fragments were subcloned into the pVL1393 vector (BD Pharmaingen) previously cut into XbaI and NotI to express a protein comprising five Ig-like regions of the extracellular domain of human robo1 fused to the Fc region of human immunoglobulin. PVL1393-hRobo-Fc was prepared. DNA sequencing confirmed that the human Robo1 gene fragment and hFc formed the same ORF. Initiation and termination codons were found to be in place.

2. hRobo - Fc  Expression and Purification of Fusion Proteins

Mass separation and purification of the pVL1393-hRobo-Fc plasmid was performed using the QIAGEN Plasmid Midi Kit (Qiagen) according to the User's Guide. Purified pVL1393-hRobo-Fc plasmid was used to construct recombinant baculovirus expressing the hRobo-Fc fusion protein according to the user guide for the Baculovirus Expression System (BD Pharmingen). Recombinant baculovirus was used to infect Sf9 insect cells to determine infection efficiency and to identify high expressers. hRobo-Fc fusion proteins were purified using Protein A affinity chromatography (GE Healthcare) according to the nProtein A Sepharose ™ 4 Fast Flow User Guide (GE Healthcare).

Way

RT - PCR  And northern Blot  ( Northern blotting ) Way

Primary HUVECs were cultured in the manner previously described (Geng, J.-G. et al. Nature 343: 757-760 (1990)). Semiquantitative RT-PCR was performed in the same manner as before (Ma, Y.-Q. and Geng, J.-GJ Immunol. 165: 558-565 (2000)). Primers used were human Robo1 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 β-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 Was. Total RNA of A375 cells and HUVEC primary culture cells were also probed with ³² P-labeled slit2 or Robo1 cDNA fragments.

Antibody Fabrication, Immunity Blot  And immunostaining method

Slit2-GST (encoding 57-207 amino acids of human slit2) and Robo1-GST fusion protein (coding 1-168 amino acids or 961-1217 amino acids of rat Robo1) are pGEX-4T-1 vectors (Amersham Pharmacia Biotech). Fusion proteins were used as antigens to immunize rabbits or mice to produce anti-Slit2 and anti-Robo1 polyclonal antibodies or monoclonal antibodies. Immunohistochemical examination was performed by the method previously described (Liu, L.-P. et al. Biochem. Biophys. Res. Commun. 286: 281-291 (2001)).

Slit2  And Robo Separation method of N

A stable human embryonic kidney 293 cell line (Slit2 / 293 cells) secreting full length human slit2 with a myc tag at the carboxy terminus was constructed by the method described previously (Li, HS et al. Cell 96: 807-818 (1999). Wang, K.-H. et al. Cell 96: 771-784 (1999)). Highly purified Slit2 protein was obtained in a conditioned medium by affinity chromatography using 9E10 mAb for myc tag (Affi-Gel 10; ˜1 mg / ml of Bio-Rad). Silver staining and immunoblot of purified slit2 were performed by the method previously described (Ma, L. et al. J. Biol. Chem. 269: 27739-27746 (1994)).

Stable 293 cell lines expressing the extracellular fragment of Robo 1 (roboN) with a hemagglutinin tag at the carboxy terminus were constructed by the previously described method (Li, HS et al. Cell 96: 807- 818 (1999); Whitford, KL et al. Neuron 33: 47-61 (2002)). HA11 (BAbCO), an affinity bead that binds mAb against hemagglutinin, was used to purify roboN in the control medium.

Boyden chamber  analysis

Cell migration assays were performed in 48-well micro-cheatogenic chambers (or similar devices) (Neuro Probe, Inc.) (Terranova, VP et al. J. Cell. Biol. 101: 2330-2334 (1985)). PVP-free polycarbonate membrane (8 μm pore) was coated with 1% gelatin. HUVEC, Robo1 / 293 cells or V / 293 suspended in M199 medium with or without slit2 or bFgF (Sigma) in the lower chamber and 1% heat inactivated fetal calf serum (FCS) in the upper chamber. Cells (all 5 × 10 ⁵ cells / ml) were inoculated. They were incubated at 37 ° C. for 4 hours. After fixing the filter, it was stained with 0.5% crystal violet and the cells moved through the filter were counted.

Directional movement analysis

Microscopic gradients of proteins were made by the methods previously described (Hopker, VH et al. Nature 401: 69-73 (1999)). Briefly, repetitive pressure injection of 0.15 μΜ Slit2 or 1 μΜ bFGF of picolitre volume was applied via a micropipette with a tip opening of about 1 μm. 24-well culture dishes were coated with a thin layer of Matrigel (Becton Dickinson Labware) and test cells settled on Matrigel and loosely attached. The experiment was performed at 37 ° C. in the presence of 5% CO ₂ . The pipette tip was located 100 μm away from the center of any given cell under experiment (this is fine?). Microscope images were recorded with a CCD camera (JVC) attached to a phase contrast microscope (Olympus IX70) and stored on a computer for detailed analysis using NIH images or suitable software. At appropriate time points (eg 2 hours), the migration distance of the cells (eg HUVEC) was analyzed.

Tube Formation Analysis

96-well cell culture dishes were coated with 100 μl of Matrigel per well and incubated for 30 minutes at 37 ° C. to facilitate gelling (Malinda, KM et al, Identification of laminin al and β1 chain peptides active for endothelial cell adhesion, tube formation and aortic sprouting, FASEB J. 13: 53-62 (1999)). HUVECs (2-3 passages) were suspended at 1.3 × 10 ⁵ cells per well in M199 medium supplemented with 2% heat inactivated FCS. Cell Aliquots (0.1 ml per aliquot) were added to Matrigel-containing wells respectively. The tubular structure was recorded and photographed at 12-18 hours.

Xenograft ( xenograft A) method of tumor growth model

A375 cells were transfected using Lipofectin ™ (Gibco) and selected with 400 μg / ml of hygromycin B (Sigma). RoboN / A375_C1, C2 and C3 cells as well as V / A375 cells were identified by immunoblot with antibodies against Robo1, Slit2 or tubulin (loading control). They were suspended in 0.2 ml DME medium and injected subcutaneously in athymic nude mice (O'Reilly, MS et al, Endostatin: An endogenous inhibitor of angiogenesis and tumor growth, Cell 88: 277-285 ( 1997)). For antibody inhibition experiments, R5 or control IgG _2b was injected twice per week (1 mg per injection) into the peritoneum of mice with A375 cell tumors (Muller, A. et al. Nature 410: 50-56 (2001) ). Mice were sacrificed after 30-35 days. Mouse blood was collected before sacrifice for leukocyte counting. Myeloperoxidase assays for the determination of neutrophils in solid tumors were performed by the previously described method (Wang, J.-G. et al. Inflamm. Res. 51: 435- 43 (2002)).

Cell proliferation assay

All transfectants were incubated up to the exponential phase and isolated by trypsin treatment. Living cells (5 × 10 ³ cells / ml) were plated in 100 μl complete medium in 96-well tissue culture dishes and incubated at 37 ° C. in 5% CO ₂ atmosphere. At various time points, tetrazolium salt was added (20 μl per well) and incubated at 37 ° C. for 4 hours. Insoluble blue formazan product was dissolved by adding 100 μl / well of 10% SDS / 5% isobutanol. The dish was read on a microtiter plate reader using a test wavelength of 570 nm and a reference wavelength of 630 nm.

Other methods such as immunoprecipitation, lectin perfusion analysis, HE staining and immunofluorescence staining are well known in the art.

Embodiments of the invention may include one or more of the following advantages. By targeting signaling pathways that mediate angiogenesis, lymphatic formation, and / or tumor lymphoid metastasis, the new therapeutic agent may be used to treat diseases such as cancer related to angiogenesis, lymphangogenesis (vascular formation) and / or tumor lymphoid metastasis, or It can be developed to treat or diagnose a disorder. Embodiments of the present invention may include a material that can be used to target the signal transduction pathway, ie, the slit-robot signaling pathway. The agent may interfere with the interaction between the slit protein as a ligand and the robo protein as their receptor, resulting in inhibition of angiogenesis, lymphatic formation and / or tumor lymphoid metastasis. Moreover, other agents may be developed in accordance with embodiments of the present invention to provide diagnostic tools for predicting the presence (and / or prognosis) of diseases or disorders related to angiogenesis, lymphangiogenesis and / or tumor lymphoid metastasis. have.

Although the present invention has been described by a limited number of embodiments, those skilled in the art having the benefit of this disclosure should appreciate that other embodiments may be devised without departing from the scope of the present invention disclosed herein. Accordingly, the scope of the invention may be limited only by the appended claims.

<110> Geng, Jianguo        Chen, Ming        Yang, Xiaomei        Chi, Shan   <120> DETECTION AND MODULATION OF SLIT AND ROUNDABOUT (ROBO) MEDIATED LYMPH VESSEL FORMATION AND USES THEREOF <130> 17257/021001 <150> PCT / CN2011 / 073505 <151> 2011-04-29 <150> CN 201010169208.8 <151> 2010-04-30 <160> 30 <170> PatentIn version 3.3 <210> 1 <211> 34 <212> DNA <213> Primer <400> 1 aaccgtgcgg ccgctgttgt gacaaaactc acac 34 <210> 2 <211> 37 <212> DNA <213> Primer <400> 2 cgcggagatc ttcatttacc cggagacagg gagaggc 37 <210> 3 <211> 33 <212> DNA <213> Primer <400> 3 ggcggcctct agaatgaaat ggaaacatgt tcc 33 <210> 4 <211> 34 <212> DNA <213> Primer <400> 4 ctataagcgg ccgccaatgt aagcactcca tgtt 34 <210> 5 <211> 30 <212> DNA <213> Primer <400> 5 gcactctaga cttgagccca gcgggcccat 30 <210> 6 <211> 30 <212> DNA <213> Primer <400> 6 ctgaggatcc tcatttaccc ggagaccggg 30 <210> 7 <211> 32 <212> DNA <213> Primer <400> 7 atcgagatct atgatcgcgg agcctgctca ct 32 <210> 8 <211> 31 <212> DNA <213> Primer <400> 8 gctctctaga cgctgccacc tccacactgt a 31 <210> 9 <211> 34 <212> DNA <213> Primers <400> 9 aaccgtgcgg ccgctgttgt gacaaaactc acac 34 <210> 10 <211> 37 <212> DNA <213> Primer <400> 10 cgcggagatc ttcatttacc cggagacagg gagaggc 37 <210> 11 <211> 33 <212> DNA <213> Primer <400> 11 ggcggcctct agaatgaaat ggaaacatgt tcc 33 <210> 12 <211> 34 <212> DNA <213> Primer <400> 12 cattatgcgg ccgccatctg taacttccaa atat 34 <210> 13 <211> 33 <212> DNA <213> Primer <400> 13 ggcggcctct agaatgaaat ggaaacatgt tcc 33 <210> 14 <211> 37 <212> DNA <213> Primer <400> 14 ataatagcgg ccgcgaggtt cttgaacagt cagagta 37 <210> 15 <211> 33 <212> DNA <213> Primer <400> 15 ggcggcctct agaatgaaat ggaaacatgt tcc 33 <210> 16 <211> 39 <212> DNA <213> Primer <400> 16 ataattgcgg ccgccatcca cagttactgc caagttact 39 <210> 17 <211> 33 <212> DNA <213> Primer <400> 17 ggcggcctct agaatgaaat ggaaacatgt tcc 33 <210> 18 <211> 35 <212> DNA <213> Primer <400> 18 ttctatgcgg ccgcaagggt tttgtctgaa gtcat 35 <210> 19 <211> 30 <212> DNA <213> Primer <400> 19 ggccaagctt atgaaatgga aacatgttcc 30 <210> 20 <211> 25 <212> DNA <213> Primer <400> 20 tccacggaat tcaaatttgg ttgcc 25 <210> 21 <211> 33 <212> DNA <213> Primer <400> 21 aaccgtgaat tccgtggaca agagagttga gcc 33 <210> 22 <211> 31 <212> DNA <213> Primer <400> 22 tacgggtcga ctcatttacc cggagacagg g 31 <210> 23 <211> 34 <212> DNA <213> Primer <400> 23 aggcggcctc tagaatgaaa tggaaacatg ttcc 34 <210> 24 <211> 31 <212> DNA <213> Primer <400> 24 tacgggcggc cgctcattta cccggagaca g 31 <210> 25 <211> 19 <212> DNA <213> Primer <400> 25 cctacacaga tgatcttcc 19 <210> 26 <211> 34 <212> DNA <213> Primer <400> 26 cagaggagcc tgcagctcag ctttcagttt cctc 34 <210> 27 <211> 30 <212> DNA <213> Primer <400> 27 ggtgacggat cccatatcgc ggtagaactc 30 <210> 28 <211> 30 <212> DNA <213> Primer <400> 28 ggacacctcg agcgtacagc cgcacttcac 30 <210> 29 <211> 20 <212> DNA <213> Primer <400> 29 atggatgatg atatcgccgc 20 <210> 30 <211> 19 <212> DNA <213> Primer <400> 30 ctagaagcat ttgcggtgg 19

Claims (27)

A method of preventing or treating a disorder mediated by a slit protein, comprising administering to the subject a therapeutically effective amount of a substance that modulates or interferes with the interaction between a slit protein and a Robo protein. The disorder is characterized in that it is involved in the formation of lymph vessels. The method of claim 1, wherein the slit protein is Slit2. The method of claim 1, wherein the Robo protein is Robo1 or Robo4. The method of claim 1, wherein the substance is an antibody against slit protein. The method of claim 1, wherein the substance is an antibody against robo protein. The method of claim 4 or 5, wherein the antibody is a monoclonal antibody. The method of claim 6, wherein the antibody is a humanized antibody. The method of claim 1, wherein the substance is a fragment of an extracellular domain of a robo protein. The method of claim 8, wherein the fragment is derived from a first immunoglobulin-like domain of robo protein. The method of claim 1, wherein the substance is a robo fusion protein. The method of claim 10, wherein the robo fusion protein is a Robo-Fc fusion protein. Substances that modulate or interfere with the interaction between the Slit protein and the Robo protein; And a pharmaceutically acceptable excipient, carrier or solvent comprising a composition for preventing or treating a disorder mediated by a slit protein, wherein the disorder is involved in the formation of a lymph vessel. The composition of claim 12, wherein the slit protein is slit2. The composition of claim 12, wherein the substance is an antibody against slit protein or robo protein. The composition of claim 14, wherein the antibody is a monoclonal antibody. The composition of claim 15, wherein the antibody is a humanized antibody. The composition of claim 12, wherein the substance is a fragment of an extracellular domain of a robo protein. 18. The composition of claim 17, wherein the fragment is derived from the first immunoglobulin-like domain of robo protein. The composition of claim 12, wherein the substance is a robo fusion protein. 20. The composition of claim 19, wherein the robo fusion protein is a Robo-Fc fusion protein. A prognosis or diagnostic method of a disease or disorder mediated by a slit protein, comprising the following steps:
(a) obtaining a test sample from a test subject and evaluating the expression level of slit protein, robo protein or both slit protein and robo protein in the test sample;
(b) obtaining a control sample from the subject without disease or disorder and evaluating the expression level of slit protein, robo protein or both slit protein and robo protein in the control sample; And
(c) comparing the expression levels evaluated in (a) and (b) to obtain a result regarding a disease or disorder mediated by the slit protein. The method of claim 21, wherein the slit protein is slit2. The method of claim 21, wherein the robo protein is Robo 1 or Robo 4. The method of claim 21, wherein the expression level is a DNA expression level, an RNA expression level, a protein expression level, or a combination thereof. 22. The method of any one of claims 1, 12 or 21, wherein the disorder is cancer. The method of claim 25, wherein the cancer is metastatic. The composition of claim 25, wherein the cancer is selected from the group consisting of rectal cancer, gastric cancer, esophageal cancer, lung cancer, hepatocellular carcinoma, breast cancer and bladder cancer.

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