# Patent

## Title
Common lymphatic endothelial and vascular endothelial receptor-1 (CLEVER-1) and uses thereof

## Abstract

A novel protein Common Lymphatic Endothelial and Vascular Endothelial Receptor-1 (CLEVER-1) is described. CLEVER-1 mediates leukocyte and malignant cell binding to vascular and lymphoid endothelial cells. CLEVER-1 is the first protein that has been reported to mediate both influx into and efflux from the lymph nodes. Also provided are methods of treating inflammation and preventing metastasis of malignant cells by providing an inhibitor of CLEVER-1 binding.

## Background
BACKGROUND OF THE INVENTION
1. Field of the Invention
The invention is in the field of cell adhesion proteins. Specifically, the invention is in the field of CLEVER-1, a novel protein that facilitates the influx of leukocytes and malignant cells into the lymphatic system, and also the efflux of the same out of the lymph nodes.
2. Background Art
Leukocytes are the major cellular components of inflammatory and immune responses. Leukocytes include lymphocytes, natural killer (NK) cells, monocytes, dendritic cells and granulocytes (neutrophils, eosinophils and basophils). See, HARRISON'S PRINCIPLES OF INTERNAL MEDICINE, Fauci, A. S. et al. eds. (14th ed. 1998). Lymphocytes are composed of B cells and T cells. B cells provide humoral immunity and are the precursors of plasma cells. T cells provide cell mediated immunity. In tissues monocytes differentiate further into macrophages. At sites of inflammation, blood monocytes can attach to inflamed endothelia. Macrophages recognize and take up a wide range of exogenous materials such as bacteria. Granulocytes also have critical roles in inflammation. They are needed to clear infections with extracellular bacteria. The immune response has an important role in the growth, differentiation, and mobilization of granulocytes.
Continuous lymphocyte recirculation between blood and lymphoid tissues forms a basis for the function of the immune system. However such lymphocyte recirculation inadvertently also facilitates at least two medical conditions: inflammation and metastasis.
Lymphocytes enter the lymphoid tissues by binding to vascular endothelial cells. Lymphocyte adherence to endothelial cells is mediated by complementary, surface expressed molecules on both cell types. The adhesion molecules and mechanisms of lymphocyte entrance into the tissues from the blood have been thoroughly characterized, but mechanisms controlling lymphocyte exit from the non-lymphoid and lymphoid tissues via lymphatics have remained unknown.
The majority of lymphocytes extravasate into the lymph nodes via specialized vessels called high endothelial venules, or HEV. The rest of the incoming lymphocytes enter the nodes via afferent lymphatics together with antigens and other types of hematopoietic cells such as dendritic cells, macrophages and granulocytes. However, only lymphocytes are able to leave the nodes via the efferent lymphatic system by first traversing the sinusoidal endothelium and then entering the efferent lymphatic vessel. To maintain the homeostasis in the lymph node the numbers of entering and exiting lymphocytes need to be well balanced. The molecular mechanisms involved in lymphocyte exit are unknown.
In addition to being of fundamental importance in normal lymphocyte recirculation, the lymphatics also regulate seeding of metastasizing cells in approximately 50% of cancers that use this type of vessel for spreading. Lymph nodes are often the first organ to develop metastases, especially in the case of carcinomas. The design of the lymphatic system makes it relatively easy for malignant tumor cells to enter (Sleeman, J. P., Recent Results Cancer Res. 157:55-81 (2000)), and thus compounds that prevent the entry and exit of malignant tumor cells from the lymphatics have tremendous therapeutic potential.
BRIEF SUMMARY OF THE INVENTION
Recognizing the need to control lymphocyte recirculation, the inventors initiated a study of the proteins of the efferent lymphatic vessels. These studies have culminated in the discovery of a novel protein, Common Lymphatic Endothelial and Vascular Endothelial Receptor-1 (CLEVER-1), a binding protein that mediates adhesion of lymphocytes (and malignant tumor cells) to endothelium in both the systemic vasculature and in the lymphatics. The inventors have discovered that by blocking the interaction of CLEVER-1 and its lymphocyte substrate, the artisan can, for the first time simultaneously, control lymphocyte recirculation and lymphocyte migration, and related conditions such as inflammation, at the site of lymphocyte influx into, and efflux from, the tissues. The inventors have also discovered that CLEVER-1 also mediates binding of other types of leukocytes such as monocytes and granulocytes to HEV-like vessels. Further, by blocking the interaction of CLEVER-1 and malignant tumor cells, the artisan can also, for the first time, control metastasis by preventing malignant cells that bind to CLEVER-1 from being taken up by the lymphatic vessels, and thus preventing spread of the malignancy into the lymph nodes.
Accordingly, in a first embodiment, the invention is directed to cellular and subcellular extracts that contain CLEVER-1.
In a further embodiment, the invention is directed to purified or isolated CLEVER-1.
The invention further provides a method for inhibiting CLEVER-1 mediated leukocyte (such as lymphocyte, monocyte, and granulocyte) adhesion in a subject in need of the same, such method comprising administering a CLEVER-1 binding agent, or soluble CLEVER-1, to such subject.
The invention further provides a method for preventing or reducing CLEVER-1 mediated inflammation in a subject in need of the same, such method comprising administering a CLEVER-1 binding agent, or soluble CLEVER-1, to such subject.
The invention further provides a method for inhibiting CLEVER-1 mediated metastasis of malignant cells in a subject in need of the same, especially metastasis into the lymph nodes, such method comprising blocking the interaction of CLEVER-1 and the metastasizing cell in such subject with a CLEVER-1 binding agent, or by administering soluble CLEVER-1, to such subject.
The invention further provides a method for extracting cells that bind to CLEVER-1 from a population of cells, such method comprising mixing a population of cells with a preparation that contains CLEVER-1 and purifying the CLEVER-1-cell complex away from the rest of the non-binding cells in the population. Such method can be used to enrich for a subpopulation of CLEVER-1 binding cells.
The invention further provides a method for identifying an agent that binds to CLEVER-1, such method comprising determining whether a substance will successfully or unsuccessfully compete with leukocytes, malignant cells or with CLEVER-1 antibodies for binding to CLEVER-1.
The invention further provides a method for identifying an agent that is capable of inhibiting CLEVER-1 mediated cell migration, such method comprising assaying CLEVER-1 trafficking of lymphocytes into the afferent lymphatics or HEV, and/or out of the lymph nodes, in the presence of such agent, and identifying such agent on the basis of its ability to inhibit such trafficking. In another embodiment, CLEVER-1 trafficking of malignant cells is assayed.
The present invention also provides a method of stimulating CLEVER-1 binding, for example, in immunocompromised hosts to facilitate leukocyte (such as lymphocyte, monocyte and granulocyte) trafficking and the function of immune defense systems.



BRIEF DESCRIPTION OF THE DRAWINGS
FIGS. 1a-1i. Indirect immunoperoxidase staining showing that monoclonal antibodies 3-266 and 3-372 recognize endothelium both in afferent and efferent lymphatic systems and on HEV. FIGS. 1a-1c are from the skin, FIGS. 1d-1i are from a lymph node. FIGS. 1a, 1d and 1g show the staining with monoclonal antibody 3-266, FIGS. 1b, 1e and 1h show the staining with monoclonal antibody 3-372 and FIGS. 1c, 1f and 1i show the staining with a negative control antibody, 3G6. In FIGS. 1a, 1b and 1c, the arrows point to the epithelium and arrowheads to afferent lymphatics. In FIGS. 1d and 1e, the arrows point to the lymphatic vessles (lymphatic sinusoids that belong to the efferent lymphatic system) within the lymph node. In FIGS. 1g and 1h, the arrows point to HEV.
FIG. 2. Monoclonal antibodies 3-266 and 3-372 recognize an about 270-300 kDa molecule. Molecules in lymph node lysates were separated by SDS-PAGE, blotted to nitrocellulose sheets and probed with monoclonal antibody 3-266 and 3-372 or with a negative control antibody (3G6).
FIGS. 3A and 3B. CLEVER-1 is involved in lymphocyte binding to endothelial ells both in HEV and lymphatics. An adhesion assay was performed to measure lymphocyte binding to HEV (FIG. 3A) and to lymphatic endothelium (FIG. 3B). The sections were pre-incubated with monoclonal antibody 3-266 or 3-372, or negative control antibody anti-HLA ABC or 3G6 (“neg co”) after which the sections were overlaid with normal lymphocytes. The results of three to four independent inhibition experiments are shown as mean percentage of maximal binding±SEM.
FIGS. 4A and 4B. CLEVER-1 is involved in binding of tumor cells to endothelial cells both in HEV and lymphatics. An adhesion assay was performed to measure binding of different tumor cell lines to HEV (FIG. 4A) and to lymphatic endothelium (FIG. 4B). The sections were pre-incubated with 3-372 or negative control antibody (anti-HLA ABC) after which the sections were overlaid with different tumor cells: Nu, NA, IBW4, KCA and CRL 1648. The results of three to four independent inhibition experiments are shown as mean percentage of maximal binding±SEM.
FIG. 5. CLEVER-1 is induced on HEV-like vessels at sites of inflammation in connection to infiltrations of inflammatory cells (FIGS. 5a-5c, synovium; FIGS. 5d-5f, skin). FIG. 5a. Fibrotic type of inflamed synovium without any marked infiltrations of inflammatory cells. Only the afferent lymphatics expressed CLEVER-1 (arrows). FIG. 5b. CLEVER-1 was upregulated on a HEV-like vessel (marked by a dashed line) within a heavy lymphocytic infiltration. FIG. 5c. Staining with a negative control antibody (3G6). FIG. 5d. In normal skin afferent lymphatics expressed CLEVER-1 (arrows), but in inflamed skin HEV-like vessels (dashed line) also expressed CLEVER-1 (FIG. 5e). Negative control staining (FIG. 5f). Arrowheads point to epidermis (FIGS. 5d-5f).
FIG. 6. CLEVER-1 mediates binding of monocytes and granulocytes to HEV-like vessels at sites of inflammation. Contribution of CLEVER-1 in binding of monocytes and granulocytes to inflamed synovial vessels and binding of granulocytes to tonsil was tested using Stamper-Woodruff type of binding assay. 3-372 and 3-266 (pooled) but not the class-matched control antibody (3G6) significantly inhibited binding of granulocytes and monocytes to HEV-like vessels in the organs tested. The results of four independent assays are shown as mean percentage of maximal binding (=100% in the presence of the control antibody)±SEM.
FIG. 7. Molecular characterization of CLEVER-1. (a) Antibodies 3-266 and 3-372 recognize a 270-300 kDa molecule in immunoblotting. 3G6 is a negative control antibody. (b) In gels run 48 hr for better resolution, at least three different isoforms of CLEVER-1 are seen, and enzymatic digestions with neuraminadase and O-glycans reveal the sialoglycoprotein nature of CLEVER-1. (c) Relative contribution of different isoforms of CLEVER-1 is different in tonsil, lymph nodes and synovium. (d) An alternative spliced form missing exon 27 is present in 1. lung, 2. brain, 3. placenta, 4. heart, 5. liver, 6. skeletal muscle, 7. kidney, 8. pancreas, 9. spleen, 10. thymus, 11. prostata, 512. testis, 13. ovary, 14. small testine, 15 colon, 16. lymph nodes. Water control negaive (lane 17). The upper band represents the standard form and the lower one is the splice variant of CLEVER-1.
FIG. 8. CLEVER-1 is expressed on the surface of endothelium in vivo and inhibition of its function blocks lymphocyte trafficking. Intravenously given 3-372 antibody (a) but not a negative class-matched control antibody (b) localized on the surface of HEV in lymph nodes after a 5 min circulation. HEV is pointed out by arrows in a. (c) Anti-CLEVER antibody treatment significantly inhibits the increase of the size of the lymph nodes draining the footpads. (One lymph node of a 3-372 treated rabbit was not found). (d) Lymphatic sinusoids of 3-372 treated animals contained less lymphocytes than those of control treated rabbits (e).
FIG. 9. The nucleotide sequence (7879 nt) of CLEVER-1 (SEQ ID NO:1). Boxed in grey are the translation initiation codon, translation stop codon, the two RGDs, the potential polyadenylation signal and the four nucleotide differences compared to Genebank entry AJ 2752213 (stabilin-1), i.e., nucleotides 1131, 2767, 6629 and 6969. Underlined are the nucleotides corresponding to the alternatively spliced exons.



DETAILED DESCRIPTION OF THE INVENTION
The term “ameliorate” denotes a lessening of an effect. To ameliorate a condition or disease refers to a lessening of the symptoms of the condition or disease.
The term “modulate” means to control in a predictable fashion, either by increasing or by decreasing the targeted parameter, as indicated from the context.
The term “effective amount” refers to that amount of the indicated agent that is sufficient to achieve the desired effect.
The term “inflammatory condition” refers to a physiological or pathological condition that is accompanied by an inflammatory response in a subject, which includes, inter alia, an undesired accumulation of leukocytes at one or more sites in such subject. The inflammatory condition can be hyperacute, acute, subacute or chronic. The inflammatory condition can be localized at the site of the inflammatory lesion or diffuse throughout the subject.
The term “drug” denotes any pharmaceutical or physiological agent, composition, bioactive compound, or combinations thereof, useful in the diagnosis, cure, mitigation, treatment, or prevention of a disease, or for any other medical purpose. The term “drug” is intended to be interpreted broadly and is not limited in terms of chemical composition or biological activity.
The term “essentially free of contaminants” refers to a substance that is of, undesired or unnecessary substances that had been present during the in vitro or in vivo synthesis of the desired substance.
The term “treatment” or “treating” refers to the administration of an agent to a subject for purposes which can include prophylaxis, amelioration, prevention or cure of an undesired disorder. Such treatment need not necessarily completely ameliorate the disorder, for example, inflammation; it is sufficient that such treatment ameliorates the disorder to a degree that is beneficial to the subject to which it is administered. Further, such treatment can be used in conjunction with other traditional treatments, for example, alternative treatments for reducing the inflammatory condition, known to those of skill in the art and as desired by the practitioner.
By “systemic vasculature” is meant the vascular network of blood vessels throughout the body of an animal or human.
By “lymphatic system” is meant the specialized part of the circulatory system that consists of lymph, the lymphatics, and the lymph nodes. The lymph nodes are located along the paths of the lymph collecting vessels and in isolated nodules of lymphatic patches in the intestinal wall. Additionally, there are specialized lymphatic organs such as the tonsils, thymus and spleen. B lymphocytes begin their final stages of maturation within the germinal centers of the lymph nodes' cortical nodules. Maturing lymphocytes are then pushed to the more densely packed outer layers as they mature, before being released into the efferent lymphatics. The lymph nodes that are located in the floor of the mouth are called the submental and submaxillary lymph nodes. The superficial cervical lymph nodes are located in the neck. The superficial cubital or supratroclear lymph nodes are located just above the bend in the elbow. The axiliary lymph nodes are clustered deep within the underarm and upper chest region. Inguinal lymph nodes are located in the groin. By “lymphatics” is meant the vessels that return lymph to the blood. Lymph is the clear fluid that flow in the lymphatics. Lymph arises from plasma that filters into the interstitial spaces from blood flowing through the capillaries. Although most of this plasma is taken up and absorbed by cells or the blood, a small amount is not absorbed. The lymphatics act as drains to collect this excess fluid and return it to the venous blood just before it reaches the heart. The lymph nodes act as filters that collect the lymph from several different lymphatics and “percolate” the lymph through spaces termed sinuses before draining into a single efferent draining vessel.
By “afferent lymphatics” is meant the vessels through which antigens enter the lymph nodes. Lymphocytes can enter the lymph nodes via the afferent lymphatics or via the high endothelial venules (HEV).
By “high endothelial venules” (HEV) is meant a specialized cortical postcapillary venules whose endothelium is simple cuboidal to columnar instead of simple squamous. HEVs are located mainly in the paracortex of the lymph nodes. Lymphocytes cross the HEV, and thus “traffic” into the lymph nodes by diapedesis, that is, the lymphocytes stick to the luminal surface of the HEV, and then squeeze into the space between two or more HEV cells.
By “efferent lymphatics” meant the vessels that drain the lymph nodules (nodes).
By “lymphocyte recirculation” is meant the continuous movement of lymphocytes throughout the circulatory and lymph system. Lymphocytes leave the lymph node and are first delivered via the lymph to venous system draining into the heart.
The lymphocytes then circulate throughout the body in the blood-stream. Most of the lymphocytes are redelivered to the spleen or to another lymph node. About 10% go to non-lymphoid organs. Lymphocytes that have never been activated cannot enter non-lymphoid organs.
Lymphocyte “trafficking” refers to lymphocyte cell movement to specific locations. Outside of the lymph nodes, the trafficking of circulating lymphocytes allows the lymphocyte to accumulate at sites of inflammation. Activated effector lymphocytes tend to home to areas of inflammation, resulting in a large influx of lymphocytes in areas of inflammation. At the inflamed site, lymphocytes attach to the endothelial cells that line the blood vessels. This attachment localizes the lymphocyte at the site of inflammation and allows for subsequent emigration of the cells into the surrounding tissues (extravasation).
The Identification and Purification of CLEVER-1
The basis of the invention is the discovery of a new molecule, a novel protein herein designated “Common Lymphatic Endothelial and Vascular Endothelial Receptor-1 in the systemic vasculature, and in the afferent and efferent lymphatics. It has been found that leukocytes such as lymphocytes, monocytes, and granulocytes, and malignant cells specifically bind to this protein. It has also been found that this protein acts as a receptor that facilitates entry of bound leukocytes and malignant cells through the walls of the systemic vasculature, into the lymph nodes and out of the lymph nodes.
To search for a protein that played a role in lymphocyte lymphatic efflux, the inventors first identified cell migration-associated lymphatic structures from isolated efferent lymphatic vessels of human lymph nodes. These structures were used to produce monoclonal antibodies. Hybridomas were screened on frozen sections of human lymph nodes using immunoperoxidase staining.
Two of the hybridomas produced antibodies (designated 3-266 and 3-372) tat clearly stained lymphatic endothelium both in afferent and efferent lymphatic systems and vascular endothelium on HEV, while other structures remained unstained. This is consistent with the expected pattern for an antibody that recognizes a lymphocyte migration-associated structure. Cell culture of 3-266 (DSM ACC2519) and cell culture of 3-372 (DSM ACC2590) were both deposited under the terms of the Budapest Treaty on the International Recognition of the Deposit of Micro-organisms for the Purposes of Patent Procedure on Aug. 21, 2001, with DSMZ-Deutsche Sammlung von Mikroorganismen und Zelikulturen GmbH, Mascheroder Weg 1b, D-38124 Braunschweig.
In molecular weight determinations by immunoblotting, both anti-bodies recognized a molecule of the same size, about 270-300 kDa. Due to this and an identical staining pattern, these antibodies were considered to recognize the same antigen; that antigen was named Common Lymphatic Endothelial and Vascular Endothelial Receptor-1 (CLEVER-1).
CLEVER-1 was purified from CLEVER-1 containing lymph node preparations using affinity chromatography with the 3-372 antibody. The eluted material was subjected to SDS-PAGE analysis and silver staining. The specific band was excised, reduced, alkylated and digested with trypsin.
After cleavage with trypsin, mass spectrometric analyses yielded 27 peptides. Twenty-one (77%) of those had identical sequences with stabilin. These sequences covered altogether 268 amino acids (10% of the 2570 amino acids of stabilin-1) and spanned the amino acids between 53 and 2301 (Table 1). The peptide data suggest that CLEVER-1 has some homology with stabilin-i at the structural level. No functional information regarding stabilin-1 can be found in the literature.
Peptide analysis of CLEVER-1 indicates no significant homology with any of the known endothelial homing-associated molecules, such as ICAM-1 (Intercellular Adhesion Molecule), ICAM-2, or VAP-1 (Vascular Adhesion Protein).
CLEVER-1 has several structural motifs that are associated with adhesive functions in other molecules. They include a proteoglycan link homology region important in CD44 for hyaluronan binding and two RGD motifs known to serve as integrin ligands in certain molecules, such as in fibornectin. In addition, CLEVER-1 has seven fascicling domains also present in several molecules such as priostin, fasciclin and transforming growth factor-β-induced gene, big-h3 and in all of these cases it is essential for adhesive function of these molecules. Interestingly, twenty-two epidermal growth factor (EGF) repeats are also found in CLEVER-1. This structural domain is also present in all members of the selectin family. Although the lectin domains of selectins are of utmost importance for the transient interaction with their sialomucin ligands, EGF-repeats have been reported to functionally contribute with lectin domains to binding between leukocytes and endothelium. Based on the structural complexity of CLEVER-1 it may turn out have several ligand molecules and be multifunctional in its nature.
It has been discovered that CLEVER-1 is involved in the process of lymphocyte recirculation. CLEVER-1 is present on the endothelium of the systemic vasculature, especially on the HEV and also on the endothelium of both afferent and efferent lymphatic systems. CLEVER-1 is a protein adhesion molecule, and especially, a cell adhesion molecule (CAM), that mediates adhesion of lymphocytes and of malignant tumor cells to CLEVER-1 in the systemic vasculature and the lymphatic system. These sites are of utmost importance as control points in lymphocyte trafficking.
CLEVER-1 is the first molecule that has been identified to facilitate lymphocyte and malignant cell exit from the lymph nodes. Additionally, CLEVER-1 is the first molecule that has been identified that regulates both entrance of lymphocytes and tumor cells into the lymph nodes and exit of lymphocytes and tumor cells from the lymph nodes. CLEVER-1 has been found to also mediate binding of other leukocytes such as monocytes and granulocytes to HEV-like vessels.
By “CLEVER-1 mediated cell binding” is meant the specific association of CLEVER-1 with either a leukocyte, such as a lymphocyte, monocyte, or granulocyte, or a CLEVER-1-binding malignant cell. CLEVER-1 mediated cell binding can occur with CLEVER-1 in a soluble form or in a particulate form (for example, when CLEVER-1 is present in a form that is membrane associated).
CLEVER-1 Mediated Binding of Leukocytes
According to the invention, adhesion of leukocytes, such as lymphocytes, monocytes, and granulocytes, to the endothelium (that is, to an endothelial cell(s)) in the systemic vasculature, especially the HEV, and to the endothelium in the afferent and efferent lymphatics can be blocked by blocking the binding between such endothelial cell's CLEVER-1 and leukocyte.
In the systemic vasculature, inhibiting or preventing endothelial cell CLEVER-1 mediated lymphocyte binding will inhibit or prevent lymphocytes, especially activated lymphocytes, from accumulating at such sites, and thus prevent or lessen inflammation at such sites. Thus, the invention provides a method of treating inflammation, by administering an agent that inhibits or prevents CLEVER-1 mediated endothelial cell binding to lymphocytes.
In the afferent lymphatics, inhibiting or preventing lymphocytes from binding to endothelial cell CLEVER-1 will inhibit or prevent such lymphocytes from entering the afferent lymphatics and thus the lymph nodes. Thus, the invention provides a method of treating inflammation, by administering an agent that inhibits or prevents CLEVER-1 mediated endothelial cell binding to lymphocytes in the afferent lymphatics and especially at HEV in lymph nodes or HEV-like venules at sites of inflammation. The invention also provides a method of inhibiting lymphocyte trafficking into the lymph nodes, by administering an agent that inhibits or prevents afferent lymphatic CLEVER-1 mediated endothelial cell binding, and especially HEV binding, to lymphocytes and other leukocytes.
In the efferent lymphatics, inhibiting or preventing lymphocytes from binding to endothelial cell CLEVER-1 will prevent the lymphocytes from exiting the lymph node and entering the blood. Thus, the invention provides a method of treating inflammation, by administering an agent that inhibits or prevents CLEVER-1 mediated endothelial cell binding to lymphocytes in the efferent lymphatics. The invention also provides a method of inhibiting lymphocyte trafficking out of the lymph nodes, by administering an agent that inhibits or prevents efferent lymphatic CLEVER-1 mediated endothelial cell binding to lymphocytes.
Therefore, CLEVER-1 binding with lymphocytes presents a unique, three-prong approach to treat diseases or conditions characterized by an undesired lymphocyte accumulation or trafficking in which the artisan can target lymphocyte entry into the lymph nodes, lymphocyte exit from the lymph nodes, and lymphocyte binding to the systemic vasculature, with the same agent.
The discovery of CLEVER-1 and its role has thus resulted in a new method to control lymphocyte migration by inhibiting CLEVER-1 mediated cell binding to such cells. Thus, the present invention provides a method of inhibiting undesired CLEVER-1 mediated lymphocyte trafficking, and thus blocking harmful or otherwise undesired lymphocyte migration, by preventing the association of CLEVER-1 with lymphocytes. Similarly, the invention provides a method of inhibiting undesired CLEVER-1 mediated binding of other leukocytes by preventing association of CLEVER-1 with the leukocytes.
The present invention also provides a method of stimulating CLEVER-1 binding, for example, in immunocompromised hosts to facilitate lymphocyte trafficking and other leukocyte binding and the function of immune defense systems.
CLEVER-1 Mediated Cell Binding to Malignant Cells
Because cancer cells often break away from a malignant tumor and enter the lymphatics, cancer cells travel to and establish themselves in the lymph nodes. According to the invention, the ability of a malignant tumor cell to establish itself in a lymph node can be inhibited or prevented by inhibiting or preventing CLEVER-1 binding to such malignant tumor cell.
The term “tumor” refers to a neoplasm, a tissue mass that is characteristic of a neoplasia. Neoplasia is distinguished from other forms of tissue growth, first, by the formation of a tissue mass, a neoplasm, or tumor. Second, neoplasia is considered to be an irreversible process. Third, neoplastic tissue tends to morphologically resemble its tissue of origin. Fourth, neoplastic tissue tends to functionally resemble its tissue of origin. Fifth, neoplasms grow and function somewhat independently of the homeostatic mechanisms that control normal tissue growth and function.
A neoplasm can be benign or malignant. A benign neoplasm consists of a discrete tissue mass that continues to grow. A benign neoplasm will simply push adjacent tissues out of its way as it grows.
The definitive features of a malignant neoplasm, a malignancy, are invasion and metastasis, that is, the spread of the neoplasm to a distant site. A malignant neoplasm will grow into the adjacent tissue, rather than pushing it away. The terms “malignant neoplasm,” “malignant tumor,” and “cancer” are synonymous.
Cancer cells typically invade thin-walled vessels such as small veins, venules, capillaries and lymphatics. The passage of cancer cells via lymphatics to lymph nodes, and via blood vessels to other organs and structures, and the subsequent implantation and growth of the cancer cells in those sites is referred to as “metastasis.” The lymph nodes are common sites for metastasis.
Cancer cells can also spread by seeding—shedding into, for example, the peritoneal fluid. The cells can be carried by the fluid to a distant site on the peritoneal surface where they can implant and form new foci of cancer growth.
Most neoplasms are one of four types: epithelial, non-epithelial, blastomas and teratomas. Malignant epithelial neoplasms are termed carcinomas. An adenocarcinoma is a carcinoma in which gland-like structures are present. Carcinomas can be papillary or cystic. Benign epithelial neoplasms are generally adenomas, polyps or papillomas.
Non-epithelial tumors can also be benign or malignant. They are generally named by a prefix that indicates the histologic type and a suffix. The suffix -oma generally means benign while the suffix -sarcoma means malignant. However, several malignant neoplasms have traditional names ending in -oma: for example, melanoma, hepatoma, and lymphoma.
Lymphomas are malignant neoplasms arising from cells of the lymphoid series. Blastomas and teratomas contain more than one type of tissue. Malignant teratomas are often termed teratocarcinomas.
A “leukemia” is a tumor of white blood cells that is present in the bone marrow and blood. A “lymphoma” is a tumor of white blood cells that is present in the lymph nodes and tissues.
According to the invention, the binding of CLEVER-1-binding malignant cells to the endothelium in the systemic vasculature, especially the HEV, and to the endothelium in the afferent and efferent lymphatics can be inhibited or prevented by inhibiting or preventing the binding between such endothelial cell's CLEVER-1 and such malignant cell. Thus, the invention provides a method of treating cancer, and especially, a method of preventing metastasis, by administration of an agent that inhibits or prevents CLEVER-1 mediated malignant cell binding to the endothelium.
In the systemic vasculature, inhibiting or preventing CLEVER-1 mediated cell binding will inhibit or prevent the establishment of CLEVER-1 binding malignant cells at such sites, and thus lessen or prevent metastasis of such malignant cells. Thus, the invention provides a method of treating cancer, and especially, a method for preventing metastasis of a CLEVER-1 binding malignant cell, by administering an agent that inhibits or prevents CLEVER-1 mediated endothelial cell binding to CLEVER-1-binding tumor cells in the systemic vasculature. The invention also provides a method of inhibiting metastasis, by administering an agent that inhibits or prevents systemic vasculature CLEVER-1 mediated endothelial cell binding to such malignant cells.
In the afferent lymphatics, inhibiting or preventing CLEVER-1 binding malignant cells from binding to endothelial cell CLEVER-1 will inhibit or prevent such CLEVER-1 binding malignant cell from entering and establishing in the lymph node, and thus lessen or prevent metastasis of such cell to the lymph node or thus to other sites in the body. In this context it is worth to note that a metastasizing malignant cell cannot survive long times without matrix support—a condition present for example in blood. Thus, the invention provides a method of treating cancer, and especially, a method for preventing metastasis of a CLEVER-1 binding malignant cell, by administering an agent that inhibits or prevents CLEVER-1 mediated endothelial cell binding to CLEVER-1-binding malignant cells in the afferent lymphatics and at HEV in systemic vasculature. The invention also provides a method of inhibiting metastasis, by administering an agent that inhibits or prevents afferent lymphatic CLEVER-1 mediated endothelial cell binding, and especially HEV binding, to such malignant cells.
In the efferent lymphatics, inhibiting or preventing CLEVER-1 binding malignant cells from binding to endothelial cell CLEVER-1 will inhibit or prevent such CLEVER-1 binding malignant cell from leaving the lymph node, and thus lessen or prevent metastasis of such cell from the lymph node to other sites in the body. Thus, the invention provides a method of treating cancer, and especially, a method for preventing metastasis of a CLEVER-1 binding malignant cell, by administering an agent that inhibits or prevents CLEVER-1 mediated endothelial cell binding to CLEVER-1-binding malignant cells in the efferent lymphatics. The invention also provides a method of inhibiting metastasis, by administering an agent that inhibits or prevents efferent lymphatic CLEVER-1 mediated endothelial cell binding to such malignant cells.
CLEVER-1 interaction with CLEVER-1 binding malignant cells thus presents a unique, three-prong approach to inhibit or prevent metastasis in which not only can the artisan block such malignant cells from entering into and exiting from the lymph system, but also, the artisan can block association of such malignant cell with CLEVER-1 in the vascular endothelium.
Agents that Block or Inhibit CLEVER-1 Mediated Cell Binding
Soluble CLEVER-1 and antibodies to CLEVER-1 can be provided to the host cell to block or inhibit CLEVER-1 mediated cell binding. Soluble CLEVER-1 can be used to “coat” the CLEVER-1 binding sites on the leukocyte, such as lymphocyte, monocyte, or granulocyte, or tumor cell and thus prevent the coated cell from association with the native CLEVER-1 on the HEV or afferent or efferent lymphatics.
CLEVER-1 antibodies can be administered to a patient in need of the same to coat CLEVER-1 that is present on the vascular endothelium or lymphatics, of such patient, especially the afferent lymphatics so as to prevent leukocyte or malignant cell binding to such CLEVER-1 in the patient. CLEVER-1 antibody producing cells can be administered directly to the patient so as to provide a source of the same.
Moreover, the present invention provides a method of identifying an agent that inhibits the binding of CLEVER-1 to cells by providing an agent to cells in the presence of CLEVER-1 and comparing the binding of CLEVER-1 to cells provided with the agent to binding of CLEVER-1 in the absence of the agent. Similarly, the invention provides a method of identifying an agent that stimulates the binding of CLEVER-1 to cells by providing an agent to cells in the presence of CLEVER-1 and comparing the binding of CLEVER-1 to cells provided with the agent to binding of CLEVER-1 in the absence of said agent.
The term “antibody” is used in the broadest sense and specifically covers single monoclonal antibodies (including agonist and antagonist antibodies), polyclonal antibodies, as well as antibody fragments and single chain antibodies (e.g., Fab, F(ab′)2, Fv), so long as they exhibit the desired biological activity.
Papain digestion of antibodies produces two identical antigen binding fragments, called Fab fragments, each with a single antigen binding site, and a residual “Fc” fragment, whose name reflects its ability to crystallize readily. Pepsin treatment yields an F(ab′)2 fragment that has two antigen combining sites and is still capable of cross-linking antigen.
Single chain “Fv” is the minimum antibody fragment which contains a complete antigen recognition and binding site. This region consists of a dimer of one heavy and one light chain variable domain in tight, noncovalent association. It is in this configuration that the three CDRs of each variable domain interact to define an antigen binding site on the surface of the VH-VL dimer. Collectively, the six CDRs confer antigen binding specificity to the antibody. However, even a single variable domain (or half of an Fv comprising only three CDRs specific for an antigen) has the ability to recognize and bind antigen, although at a lower affinity than the entire binding site. See, Ladner et al., U.S. Pat. No. 4,946,778, and Bird, R. E. et al., Science, 242:423-426 (1988).
The term “monoclonal antibody” as used herein refers to an antibody obtained from a population of substantially homogeneous antibodies, i.e., the individual antibodies comprising the population are identical except for possible naturally occurring mutations that may be present in minor amounts. Monoclonal antibodies are highly specific, being directed against a single antigenic site. Furthermore, in contrast to conventional (polyclonal) antibody preparations which typically include different antibodies directed against different determinants (epitopes), each monoclonal antibody is directed against a single determinant on the antigen. In addition to their specificity, the monoclonal antibodies are advantageous in that they are synthesized by a hybridoma culture, uncontaminated by other immunoglobulins. The modifier “monoclonal” indicates the character of the antibody as being obtained from a substantially homogeneous population of antibodies, and is not to be construed as requiring production of the antibody by any particular method. For example, the monoclonal antibodies to be used in accordance with the present invention may be made by the hybridoma method first described by Kohler and Milstein, Nature 256:495 (1975), or may be made by recombinant DNA methods (e.g., Cabilly et al., U.S. Pat. No. 4,816,567).
Preparation of immunizing antigen, and polyclonal and monoclonal antibody production can be performed as described herein, or using other suitable techniques. A variety of methods have been described (see e.g., Kohler et al., Nature 256:495-497 (1975), and Eur. J. Immunol. 6:511-519 (1976); Milstein et al., Nature 266:550-552 (1977); Koprowski et al., U.S. Pat. No. 4,172,124; Harlow, E. and D. Lane, Antibodies: A Laboratory Manual (Cold Spring Harbor Laboratory: Cold Spring Harbor, N.Y., 1988); CURRENT PROTOCOLS IN MOLECULAR BIOLOGY, Vol. 2 (Supplement 27, 1994), Ausubel, F. M. et al., John Wiley & Sons, eds., New York, N.Y.), Chapter 11, (1991)). Generally, a hybridoma can be produced by fusing a suitable immortal cell line (e.g., a myeloma cell line such as SP2/0) with antibody producing cells. The antibody producing cell, preferably those of the spleen or lymph nodes, are obtained from animals immunized with the antigen of interest. The fused cells (hybridomas) can be isolated using selective culture conditions, and cloned by limiting dilution. Cells which produce antibodies with the desired binding properties can be selected by a suitable assay (e.g., ELISA).
The term “antibody” also includes chimeric, humanized or primatized (CDR-grafted) antibodies, as well as chimeric or CDR-grafted single chain antibodies, and the like, comprising portions derived from different species. “Chimeric” antibodies (immunoglobulins) have a portion of the heavy and/or light chain is identical with or homologous to corresponding sequences in antibodies derived from a particular species or belonging to a particular antibody class or subclass, while the remainder of the chain(s) is identical with or homologous to corresponding sequences in antibodies derived from another species or belonging to another antibody class or subclass, as well as fragments of such antibodies, so long as they exhibit the desired biological activity (Cabilly et al., U.S. Pat. No. 4,816,567; Morrison et al., Proc. Natl. Acad. Sci. USA 81:6851-6855 (1984). The various portions of these antibodies can be joined together chemically by conventional techniques, or can be prepared as a contiguous protein using genetic engineering techniques. For example, nucleic acids encoding a chimeric or humanized chain can be expressed to produce a contiguous protein. See, e.g., Cabilly et al., U.S. Pat. No. 4,816,567; Cabilly et al., EP 0 125 023 B1; Boss et al., U.S. Pat. No. 4,816,397; Boss et al., EP 0120694 B1; Neuberger, M. S. et al., WO 86/01533; Neuberger, M. S. et al., EP 0194276 B1; Winter, U.S. Pat. No. 5,225,539; Winter, EP 0239400 B1; and Queen et al., U.S. Pat. Nos. 5,585,089, 5,698,761 and 5,698,762. See also, Newman, R. et al., Bio-Technology 10: 1455-1460 (1992), regarding primatized antibody.
By “agonist antibody” is meant an antibody which is able to bind to CLEVER-1 and facilitate adhesion of lymphocytes (and malignant tumor cells) to endothelium. By “antagonist antibody” is meant an antibody that is able to bind to CLEVER-1 and inhibit adhesion of lymphocytes (and malignant tumor cells) to endothelium.
Anti-idiotypic antibodies are also provided. Anti-idiotypic antibodies recognize antigenic determinants associated with the antigen-binding site of another antibody. Anti-idiotypic antibodies can be prepared against second antibody by immunizing an animal of the same species, and preferably of the same strain, as the animal used to produce the second antibody. See e.g., U.S. Pat. No. 4,699,880.
In Vitro Adhesion Assay and Diagnostic Uses Thereof
In a further embodiment, the present invention is directed to an adhesion assay in which CLEVER-1 binding is used to assay for the presence of leukocytes or malignant cells that bind to HEV and lymphatic endothelium. Both static and non-static assays are possible. The adhesion assay is exemplified in Example 4. Both static and non-static assays can be used to study leukocyte binding to systemic vasculature.
In the static assay, a tissue section is exposed to leukocytes or malignant cells for a desired period of time, without continuous agitation or rotation of the preparation during the exposure. Static assays are preferred for examining the ability of leukocytes and malignant cells to bind to lymphatic endothelium, especially the efferent lymph vessels.
In the non-static assay, the CLEVER-1 containing tissue sample and leukocytes are constantly rotated during the time period in which the leukocytes are given to adhere to the CLEVER-1. Non-static assays are preferred for studying leukocyte and malignant cell binding to CLEVER-1 in the HEV. The non-static assay mimics adhesion to the systemic vasculature.
In another embodiment, the present invention relates to a method for detection of malignant tumor cells. As explained with detail in Example 1, CLEVER-1 antibodies reduce the binding of malignant tumor cells to the vascular and lymphoid endothelium, demonstrating CLEVER-1 is a receptor for such malignant tumor cells. CLEVER-1 protein, or fragments thereof, or CLEVER-1 binding compounds, including but not restricted to, antibodies against CLEVER-1, both monoclonal and polyclonal, antibodies against antigenic fragments of CLEVER-1, both monoclonal and polyclonal, antigenic polypeptides, small molecule inhibitors or drugs, can be used in both quantitative and qualitative assays to detect the presence of malignant tumor cells in a sample, said sample being tissue or blood from a human or animal.
CLEVER-1 protein, or fragments thereof, or the above-mentioned CLEVER-1 binding compounds can be attached to a solid support matrix, including but not limited to microtiter plates, agarose columns, or magnetic beads. The above-mentioned sample can then be exposed to said solid support matrix, and the percentage of cells retained by said solid support matrix determined. For example, a sample from a normal, healthy individual, said individual being either a human or an animal, would have a statistically predicted number of leukocytes that bind to CLEVER-1. A sample that contains both leukocytes and malignant tumor cells would have a detectably higher number of cells that bind CLEVER-1.
In a preferred embodiment, a blood or tissue sample that has been taken from a patient who is in need of treatment, especially treatment for cancer, is used as the source of the CLEVER-1 binding cells in the in vitro adhesion assay. Such patient can be a patient being treated for a previously diagnosed malignancy, or a patient suspected of having a malignant tumor, or a patient who appears to be cured of such malignant tumor but is in need of monitoring for the reoccurrence of the same. Preferably, such blood or tissue sample is from a patient who is to be tested for the presence of malignant cells that bind to CLEVER-1 in such sample.
The blood or tissue sample that is to be examined in the in vitro assay of the invention can be processed, if desired, by methods known in the art so as to further extract or concentrate any CLEVER-1 binding cells that might be present in the sample, prior to the sample's being used in the in vitro adhesion assay of the invention.
Additionally, once the in vitro adhesion assay is complete, the adherent cells can be studied using other methods. For example, in the static assay, where the bound cells have been fixed, ability of the adherent cells to be recognized by a monoclonal antibody that is diagnostic for the type of tumor can be performed.
Detecting the binding of malignant cells to the CLEVER-1 containing lymphatic endothelium indicates that the patient is in need of treatment for such malignant cells, and especially to prevent the metastasis of such malignant cells
The present invention provides in this aspect a novel, efficient, and convenient assay for identifying antagonists, including but not limited to, monoclonal and polyclonal antibodies, peptides, protein fragments, small molecular inhibitors, drugs, and other agents, which can inhibit the adhesion of leukocytes and malignant tumor cells to the vascular and lymphatic endothelium.
For example, CLEVER-1 containing samples of lymph node sections can be incubated with and without the agent, and the number of bound lymphocytes and/or malignant cells determined. The antagonists can be preincubated with lymph node sections (a non-competitive assay) or simultaneously added with lymphocytes to the lymph node sections (a competitive assay).
Such a screen can also be used to customize an anti-metastasis treatment to an individual patient, and allows the practitioner to identify and select those agents or combinations thereof that have the best ability to inhibit CLEVER-1 malignant cell binding to vascular and/or lymphatic endothelium in such patient, and thus maximize the benefit of the treatment with such agents for such patient.
Additionally, such in vitro assay allows the practitioner to select for agents that provide a beneficial effect on disrupting malignant cell: CLEVER-1 containing endothelium interactions, nevertheless, minimize, if possible, the effect of such treatment on CLEVER-1 mediated leukocyte binding,
An antagonist can inhibit malignant cell or lymphocyte cell migration into or out of the lymph nodes. In a preferred embodiment, antagonists would inhibit both entrance and exit of an undesired cell into and out of the lymph nodes, respectively. As such, malignant tumor cells would preferably be prevented from entering a lymph node, and establishing there, and any that did enter the lymph nodes via an afferent lymph vessel independent mechanisms would be contained, thus slowing metastasis.
This assay can be used further to monitor the efficacy of chemotherapy treatments administered to an individual, said individual being a human or an animal, in need thereof. Samples can be analyzed before, during, and after chemotherapy for the presence of malignant tumor cells that bind to CLEVER-1 or antigenic fragments thereof, or CLEVER-1 binding compounds.
In a further embodiment, purified CLEVER-1 protein, or fragments thereof can be used for high volume screening of antagonists that are capable of preventing or lowering the ability of a leukocyte or malignant cell to adhere to endothelial cell CLEVER-1. CLEVER-1 protein, or fragments thereof can be attached to a solid support matrix, including but not limited to a microtiter plate, an agarose column, or magnetic beads, using standard methods well known in the art. Antagonists can be screened for interaction with CLEVER-1 or fragments thereof, either in the absence or presence of leukocytes. Leukocytes or malignant cells can be labeled with fluorescent dyes such as, for example, bis-carboxyethyl carboxyfluorescein or fluorescein isothiocyanate and the number of bound cells in presence or absence of the antagonists can be analyzed by a fluoroimager.
The high volume screen assay of this aspect of the invention can be used to screen combinatorial libraries for molecules that inhibit the binding of leukocytes and malignant tumor cells to CLEVER-1 or a fragments thereof. Antagonists that show strong affinity for purified CLEVER-1 protein or fragments thereof can be screened further using the in vitro adhesion assay described above.
Antibodies used in the methods of the invention as CLEVER-1 binding compounds are preferably antibodies with a specificity against CLEVER-1, or an antigenic fragment thereof. Such antibodies can be polyclonal or monoclonal.
Another potential CLEVER-1 antagonist is a peptide derivative of the CLEVER-1 polypeptide that are naturally or synthetically modified analogs of the polypeptides that have lost biological function yet still recognize and bind to the ligand of the polypeptides to thereby effectively block the interaction of said ligand with CLEVER-1. Examples of peptide derivatives include, but are not limited to, small peptides or peptide-like molecules.
Another potential human CLEVER-1 antagonist is a peptide derivative of the ligand polypeptides which are naturally or synthetically modified analogs of the polypeptides that have lost biological function yet still recognize and bind to CLEVER-1 to thereby effectively block CLEVER-1. Examples of peptide derivatives include, but are not limited to, small peptides or peptide-like molecules.
The present invention relates to a diagnostic method for the detection of cells that contain CLEVER-1, that is, CLEVER-1 positive cells, in samples taken from the human or animal body. Such a method would involve the use of CLEVER-1 binding compounds, including but not limited to, monoclonal and polyclonal antibodies, with specificity for CLEVER-1. Such compounds can be labeled with a substance, such as a colorimetric dye or radioactive molecule, to permit rapid and easy detection of binding of the compound to cells that express CLEVER-1.
Therapeutic Uses of CLEVER-1 Antagonists
In another embodiment, the present invention relates to a method of treating malignant carcinomas. It is common for carcinomas to metastasize first to the regional lymph nodes (Sleeman, J. P., Recent Results Cancer Res. 157:55-81 (2000)). As described herein, CLEVER-1 is involved in the entrance and exit of malignant tumor cells to and from the lymph nodes. As such, antagonists that inhibit malignant tumor cell binding to CLEVER-1, including but not limited to, monoclonal and polyclonal antibodies, peptides, small molecule inhibitors, drugs, and other such agents can reduce metastasis and serve as effective chemotherapeutic agents.
In another aspect, the present invention relates to a method of treating disorders where the leukocyte-endothelial cell adhesion reaction is associated with acute or chronic inflammatory diseases such as skin inflammations, diabetes, connective tissue diseases (such as lupus, rheumatoid arthritis, ostheoarthritis), obstructive and restrictive lung diseases (such as asthma, ARDS, sarcoidosis, idiopathic pulmonary fibrosis), inflammatory bowel diseases (such as ulcerative colitis and Crohn's disease), various nephritides, non-viral hepatitis, cirrhosis, cholangitis, atherosclerosis, vasculitis, thyroiditis, multiple sclerosis, myositis, ischemia reperfusion injury, transplantation rejection.
The antagonists can also be employed to treat histamine-mediated allergic reactions and immunological disorders including late phase allergic reactions, chronic urticaria, and atopic dermatitis. IgE-mediated allergic reactions such as allergic asthma, rhinitis, and eczema can also be treated.
The antagonists can also be employed to treat chronic and acute inflammation by preventing the extravasation of leukocytes to a wound area. They can also be employed to regulate normal pulmonary macrophage populations, since chronic and acute inflammatory pulmonary diseases are associated with sequestration of mononuclear phagocytes in the lung.
Antagonists can also be employed to treat rheumatoid arthritis by preventing the extravasation of leukocytes into synovial fluid in the joints of patients. Monocyte influx and activation plays a significant role in the pathogenesis of both degenerative and inflammatory arthropathies.
The antagonists can also be employed to treat asthma and allergy by preventing eosinophil accumulation in the lung. The antagonists can also be employed to treat subepithelial basement membrane fibrosis which is a prominent feature of the asthmatic lung.
The antagonists can also be employed for treating atherosclerosis, by preventing monocyte infiltration in the artery wall.
The antagonists can be employed in a composition with a pharmaceutically acceptable carrier, e.g., as hereinafter described.
Formulations of Compounds
The antagonists of CLEVER-1 can be used as therapeutic compositions. The antagonists of CLEVER-1 can be administered as a single dose or in multiple doses. The antagonists of the present invention can be administered either as an independent therapeutic regime or in combination with other therapeutic agents. The antagonists can be combined with conventional therapies, which can be administered simultaneously or sequentially.
Such therapeutic compositions can consist solely of the antagonist of CLEVER-1 although, preferably, the compositions will contain the antagonist of CLEVER-1 combined in admixture with a pharmaceutically acceptable carrier vehicle. Suitable vehicles and their formulation, inclusive of other human proteins, e.g., human serum albumin, are described for example in Remington: The Science and Practice of Pharmacy, Gennaro, Alfonso, 20th ed. (2000). In order to form a pharmaceutically acceptable composition that is suitable for effective administration to a patient in need of such composition, such compositions will contain an effective amount of the antagonist of CLEVER-1 together with a suitable amount of carrier vehicle.
Compositions containing antagonists of CLEVER-1 can be administered perorally, intravenously, intramuscularly, or sub-cutaneously at the appropriate dosages, which will depend upon the severity of the condition of the patient and upon such criteria as the patient's height, weight, sex, age, and medical history. The dose will also depend upon whether the compound of the invention is being administered to a human patient or in a veterinary setting to an animal, in need thereof.
For the purpose of parenteral administration, compositions containing the antagonists of CLEVER-1 are preferably dissolved in distilled water and the pH-value is preferably adjusted to about 6 to 8. In order to facilitate the lyophilization process resulting in a suitable product, lactose can be added to the solution. Preferably, the solution is then filtered sterilized, introduced into vials, and lyophilized. In a preferred embodiment, the compound of the invention is administered orally to a patient, at the time of eating or shortly thereafter. The concentration of the antagonists of CLEVER-1 in these composition, whether oral or parenteral, can vary, e.g., from 10−12 M to 10−3 M.
Additional pharmaceutical methods can be employed to control the duration of action. Controlled release preparations can be achieved by the use of polymers to complex or adsorb the antagonists of CLEVER-1. The controlled delivery can be exercised by selecting appropriate macromolecules (for example, polyesters, polyamino acids, polyvinyl pyrrolidone, ethylenevinylacetate, methylcellulose, carboxymethylcellulose, and protamine sulfate) and the concentration of macromolecules as well as the methods of incorporation in order to control release. Another possible method to control the duration of action by controlled release preparations is to incorporate the antagonists of CLEVER-1 into particles of a polymeric material such as polyesters, polyamino acids, hydrogels, poly (lactic acid) or ethylene vinylacetate copolymers. Alternatively, instead of incorporating the CLEVER-1 antagonists into these polymeric particles, it is possible to entrap these derivatives in microcapsules prepared, for example, by coacervation techniques or by interfacial polymerization, for example, bydroxymethylcellulose or gelatin-microcapsules and poly (methylmethacrylate) microcapsules, respectively, or in colloidal drug delivery systems, for example, liposomes, albumin microspheres, microemulsions, nanoparticles, and nanocapsules or in macroemulsions. Such teachings are disclosed in Remington: The Science and Practice of Pharmacy, Gennaro, Alfonso, 20th ed. (2000).
The following Example serves only to illustrate the invention, and is not to be construed as in any way limiting the invention.
EXAMPLE 1 
Production of Monoclonal Antibodies
Balb/c mice were immunized to footpads four times at one week intervals, with incomplete Freund's adjuvant containing suspension made from lymphatic vessels excised from human lymph nodes under stereo microscope. The suspension was made by cutting the vessels to small pieces by scissors and the pieces in phosphate buffered saline were then drawn back and forth into a syringe connected to a 21 q needle. The popliteal lymph node lymphocytes from the immunized mice were isolated by a glass homogenizer. The popliteal lymph node lymphocytes of the immunized mice were fused with Sp2/0 myeloma cells. Hybridoma supernatants were primarily tested on frozen sections of human lymph nodes using immunoperoxidase staining. The testing conditions were the same for antibodies 3-266 and 3-372 generated by two of the hybridomas.
Immunoperoxidase stainings were performed as described (Salmi, Science 257:1407-1409 (1992)). Briefly, acetone fixed 6 μm frozen sections from different human tissues (lymph nodes, appendix, bronchus, cerebellum, epididymis, esophagus, heart, small and large intestine, kidney, liver, lung, normal and psoriatic skin, synovium, testis and tonsil) were stained with antibody 3-266, 3-372 or 3G6, a negative class matched control antibody for 3-266 and 3-372 (mouse IgG1) and 3,3-diaminobenzidine was used as a substrate. Procedures for tissue collection were approved by the Local and National Boards of Medicolegal Affairs in Finland.
Two of the hybridomas produced antibodies (3-266 (DSM ACC2519) and 3-372 (DSM ACC2590)) that clearly stained lymphatic endothelium both in afferent and efferent lymphatic systems and vascular endothelium on HEV, while the other structures remained unstained. The staining of the lymphatic endothelium is shown in FIG. 1. FIG. 1 is an indirect immunoperoxidase staining that shows that monoclonal antibodies 3-266 and 3-372 recognize endothelium both in afferent and efferent lymphatic systems and on HEV. FIGS. 1a-1c are from the skin. FIGS. 1d-1i are from a lymph node. FIGS. 1a, 1d and 1g show the staining with monoclonal antibody 3-266, FIGS. 1b, 1e and 1h show the staining with monoclonal antibody 3-372 and FIGS. 1c, 1f and 1i show the staining with a negative control antibody, 3G6. In FIGS. 1a, 1b and 1c, the arrows point to the epithelium and arrowheads to afferent lymphatics. In FIGS. 1d and 1e, the arrows point to the lymphatic vessels (lymphatic sinusoids that belong to the efferent lymphatic system) within the lymph node. In FIGS. 1g and 1h, the arrows point to HEV.
EXAMPLE 2 
Determination of Molecular Weight of CLEVER-1
Molecular weight determination was performed by immunoblotting. One percent NP-40 lysates containing of human lymph nodes was analyzed using 5-12.5% sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE). SDS-PAGE was run in non-reducing conditions. The molecules in the gel were blotted overnight to nitrocellulose sheets and probed with 3-266, 3-372 or a negative control antibody (3G6) (Salmi, M. et al., J. Exp. Med. 183:569-579 (1996). Peroxidase conjugated rabbit anti-mouse Ig was used as the second stage reagent. Detection was performed using enhanced chemiluminescence system according to the instructions of the manufacturer (Amersham).
Both antibodies recognized a molecule of the same size (about 270-300 kDa; FIG. 2). Due to this and an identical staining pattern, these antibodies were assumed to recognize the same antigen and this antigen was named CLEVER-1.
EXAMPLE 3 
Purification and Molecular Characterization of CLEVER-1
The molecule recognized by 3-372 antibody was purified from human lymph node lysate overnight (lysis buffer: 150 mM NaCl, 10 mM Trisbase, pH 7.2, 1.5 mM MgCl2, 1% NP-40, 1% aprotinin, and 1 mM PMSF) as described in Smith, D. J. et al., J. Exp. Med. 188:17-27 (1998). After centrifugation, the lysate supernatant was applied sequentially to immunoaffinity columns containing CnBr-activated Sepharose beads armed with irrelevant mAbs and 3-372 (3 mg/ml beads). After washing with lysis buffer, the antigens recognized by 3-372 were eluted with 50 mM triethylamine, frozen and subsequently lyophilized. The eluted material was then subjected to SDS-PAGE analysis and silver staining (O'Connell, K. L. and Stults, J. T., Electrophoresis 18:349-359 (1997)). The specific band was excised, reduced, alkylated and digested with trypsin (Promega) overnight at +37° C. as described (Shevchenko, A. et al., Anal. Chem. 68:850-855 (1996); O'Connell, K. L. and Stults, J. T., Electrophoresis 18:349-359 (1997)). The peptides were analyzed using PerSeptive BioSystems Voyager DE-PRO mass spectrometer operated in the reflectron delayed-extraction mode. Calibration of the spectrum was performed internally by using autolysis products of trypsin or with added calibration mixture 2 (PerSeptive BioSystems). Database search was performed by MS-Fit algorithm (prospector.ucsf.edu/ucsfhtml3.2/msfit.htm) of the University of California, San Francisco mass spectrometry facility.
After cleavage with trypsin, mass spectrometric analyses yielded 27 peptides. 21 (77%) of those had identical nucleotide sequences with two Genebank entries: AJ 275213, a submission for a cDNA clone called stabilin-1, and D87433, a cDNA clone KIAA0246 isolated from the cell line KG-1. The peptide sequences covered altogether 268 amino acids (100% of the 2570 amino acids of stabilin-1) and spanned the amino acids between 53 an 2301.
Next we designed primers based on the peptide sequences, the 5′ end of the cDNA for stabilin-1 and the 3′ end of the cDNA of KIAA0246 and used them to make several RT-PCR fragments that were then ligated together to clone the full-length 7879 bp cDNA (SEQ ID NO:1). Sequencing of the whole construct revealed a high homology with the existing 3′ end KIAA0246 sequence available in the data bank. However, it contained 4 nucleotide differences, when compared to the Stabilin sequence. They all cause a change at the amino acid level. Two of these changes are identical with the genomic sequence data available from the HUGO project (AC 006208), but since the genomic clone only covers about half of the gene for this cDNA, the nature of the two other changes remains to be determined.
Sequencing of several different CLEVER-1 cDNA-clones also revealed the existence of at least two alternatively spliced isoforms of the molecule: the regions covered by exons 23 (nucleotides 2377-2562) and 27 (nucleotides 2914-3009) can be spliced out. We could confirm the existence of one of those splice variants (lacking exon 27) also at the mRNA level (FIG. 7) but the second one (lacking exon 23) that we cloned from a human peripheral lymph node library was not visible in the system suggesting a low abundance/turnover of the mRNA encoding this form.
The sequence comparisons revealed significant homologies to proteoglycan link protein-like sequence, epidermal growth factor-like repeats and two RGD motifs being well in line with the adhesive properties of CLEVER-1.
EXAMPLE 4 
In Vitro Adhesion Assay
Lymph node sections were first incubated with 3-266, 3-372 or control antibodies against human HLA ABC (HB-95, ATCC and 3G6 (against chicken T cells) and then overlaid with Ficoll gradient (Pharmacia) purified peripheral blood mononuclear cells or different human tumor cell lines (lymphoblastoid cell lines, KCA and IBW4; a Burkitt lymphoma CRL-1648; squamocellular carcinoma lines NA and NU). Thereafter, the sections were subjected to two different types of assays: 1. A non-static assay, which optimally measures binding of cells to HEV and is performed under rotatory conditions (60 rpm on orbital shaker for 30 min at +7° C.). 2. A static assay, in which the sections overlaid with cells are let to stay in static conditions for 15 min, followed by 5 min of rotation at 60 rpm and then again 15 min without rotation at 7° C. (Static conditions were needed to optimal binding to lymphatic endothelium). The adherent cells were fixed in 1% glutaraldehyde. The number of lymphocytes bound to HEV and to sinusoidal (lymphatic) endothelium was counted single blind under dark-field illumination in which setting the sinusoidal vessels are easily recognizable. The results of the inhibition assays are presented as percentage of control binding (the number of adherent cells/vessel in the presence of control mAb defines 100% adherence).
When the assay was performed in non-static conditions, mimicking the blood flow, lymphocyte binding to HEV was reduced 43.6% and 45.2% by 3-266 and 3-372, respectively (FIG. 3A). To mimic the conditions at sites of lymphocyte exit, the assay was performed in static conditions. In these assays lymphocyte binding to lymphatic endothelium was decreased 46.4% and 64% by 3-266 and 3-372, respectively (FIG. 3B). These data indicate that the molecule recognized by 3-266 and 3-372 indeed mediate lymphocyte binding both to HEV and to lymphatic endothelium.
To study the role of this molecule in migration of malignant cells, the assays were performed using three lymphoma cell lines (CRL 1648, KCA and IBW4) and two squamocellular carcinoma cell lines (NA and NU). For these assays 3-372 antibody was chosen because of its higher inhibitory capacity (FIG. 3). The results of these experiments clearly demonstrated that CLEVER-1 is also involved in binding of malignant cells to endothelium both at entrance and exit sites within the lymph nodes (FIGS. 4A and 3).
EXAMPLE 5 
CLEVER-1 is Upregulated at Sites of Inflammation on HEV-like Vessels
Synovial samples from 18 patients suffering from chronic arthritis and undergoing synovectomies, skin samples from diseased skin of patients suffering from psoriasis (n=5), lichen (n=1), mycosis fungoides (n=1), erythrodermia (n=2), exanthema (n=1), folliculitis (n=3) and normal skin samples from 15 individuals were studied for expression of CLEVER-1 using immunoperoxidase method as described above. Like in normal non-lymphoid tissues CLEVER-1 was present in afferent lymphatic vessels in inflamed synovial and both in normal and diseased skin samples. In addition, CLEVER-1 expression was induced on HEV-like vessels that appear at sites of inflammation and are surrounded by heavy infiltrations of inflammatory cells (FIG. 5). Table 2 illustrates complete correlation between the extent of inflammatory infiltration and upregulation of CLEVER-1 expression in synovial samples. The same phenomenon was observed in skin samples: all diseased skin samples had inflammatory infiltrations that contained CLEVER-1 positive HEV-like vessels. Those vessels were absent in normal skin samples.
EXAMPLE 6 
Clever-1 Also Mediates Binding of Monocytes and Granulocytes to HEV-like Vessels
Human monocytes from peripheral blood were purified from Ficoll-gradient (Pharmacia) isolated mononuclear cells by letting them to adhere to plastic surfaces for an hour at +37° C. Granulocytes were purified from leukocyte rich buffy coats from human blood using Percoll-gradient (Pharmacia) centrifugation. Their binding was tested to HEV-like vessels in inflamed synovium. In addition, granulocyte binding was tested to tonsil HEV that brightly express CLEVER-1. (When tonsils are removed they always have variable extent of inflammation, although they as a lymphoid organ have HEV without any inflammation). Both granulocytes and monocytes bound efficiently to HEV-like vessels in inflamed synovium and granulocytes adhered avidly to HEV in tonsils. Their binding to these organs was significantly inhibited by the antibody pool containing 3-372 and 3-266 but not with the control antibody (FIG. 6).
EXAMPLE 7 
CLEVER-1 Controls Lymphocyte Trafficking in Vivo
In order to verify that CLEVER-1 has a functional role in vivo, it was at first confirmed by intravenous injection of 3-372 antibody that rabbits express CLEVER-1 on the surface of endothelium in vivo. The presence of CLEVER-1 on HEV was detected after the 3-372 antibody had circulated 5 minutes in vivo using frozen sections and FITC labelled anti-mouse IgG second stage antibody after sacrification (FIG. 8a). In this time frame the intravenously given 180 kDa immunoglobulin molecule does not have a possibility to leak and diffuse into the tissue. Based on these results antibody 3-372 (and a class-matched negative control antibody) was given to the rabbits immunized with keyhole limpet hemocyanin to footpads and the effects of the antibody treatment on the size and cellularity of the lymph nodes draining the footpads was analyzed.
Antibody treatment against CLEVER-1 significantly prevented increase of the size of the popliteal lymph nodes (FIG. 8c) indicating that CLEVER-1 has a functional role in lymphocyte traffic in vivo. Most likely it exerts its effects both at lymphocyte entrance in HEV and their exit in lymphatic sinusoids, because the rabbits treated with 3-372 antibody had only few lymphocytes in their lymphatic sinusoids when analyzed using histological section (FIG. 8d) Intravenously given 3-372 antibody was also detected to bind CLEVER-1 on lymphatic sinuses when tested at sacrification 3 days after the final 3-372 dose. No signal was detected in rabbits which received a control antibody (data not shown).
All documents, e.g., scientific publications, patents and patent publications recited herein are hereby incorporated by reference in their entirety to the same extent as if each individual document was specifically and individually indicated to be incorporated by reference in its entirety. Where the document cited only provides the first page of the document, the entire document is intended, including the remaining pages of the document.







TABLE 1







Matches with CLEVER-1 and stabilin-1


1. 21/27 matches (77%). 275350.0 Da, pI = 6.04. Acc. #6469374. HOMO


SAPIENS. (AJ275213) stabilin-1.














m/z
MH+
Delta


Peptide Sequence




submitted
matched
ppm
start
end
(Click for Fragment Ions)
Modifications

















775.488
775.483
6.4034
372
377
(R)VFLQLR(V)









(SEQ ID NO:2)


 


787.36
787.3739
−17.6253
1299
1305
(R)SGFSFSR(G)







(SEQ ID NO:3)


 


799.495
799.5042
−11.4617
1585
1591
(R)VGLELLR(D)







(SEQ ID NO:4)


 


812.495
812.4994
−5.4309
1047
1053
(R)TLPNLVR(A)







(SEQ ID NO:5)


 


917.502
917.4997
2.4556
1040
1046
(R)AFWLQPR(T)







(SEQ ID NO:6)


 


1017.44
1017.425
14.4862
2295
2301
(R)WDAYCFR(V)







(SEQ ID NO:7)


 


1104.54
1104.526
12.6406
53
61
(K)OTCPSGWLR(E)







(SEQ ID NO:8)


 


1212.7
1212.695
3.9482
1021
1032
(R)VTALVPSEAAVR(Q)







(SEQ ID NO:9)


 


1284.65
1284.622
21.4530
1678
1688
(R)EGSIYLNDFAR(V)







(SEQ ID NO:10)


 


1291.79
1291.774
12.5434
613
624
(R)ILLGPEGVPLQR(V)







(SEQ ID NO:11)


 


1330.63
1330.575
41.7401
953
965
(R)AGNGGCHGLATCR(A)







(SEQ ID NO:12)


 


1330.63
1330.633
−1.9754
1873
1812
(R)CDHFBTRPLR(L)







(SEQ ID NO:13)


 


1374.66
1374.632
20.1117
62
72
(R)ELPDQITQDCR(Y)







(SEQ ID NO:14)


 


1456.79
1456.776
9.6229
1069
1082
(R)LGGQEVATLNPTTR(W)







(SEQ ID NO:15)


 


1493.8
1493.796
2.4215
508
521
(R)TIGQILASTEAPSR(F)







(SEQ ID NO:16)


 


1555.7
1555.663
23.5678
219
231
(R)CLPGYTQQGSECR(A)







(SEQ ID NO:17)


 


1678.94
1678.913
16.1953
1802
1817
(R)NVEALASDLPNLGPLR(T)







(SEQ ID NO:18)


 


1730.89
1730.887
1.9674
1054
1068
(R)AHPLQGALFEEELAR(L)







(SEQ ID NO:19)


 


1912.82
1912.832
−6.3742
936
952
(K)LGEAGDGYQCSPIDPCR(A)







(SEQ ID NO:20)


 


2057.05
2057.03
9.5484
1655
1673
(R)SEDLLEQGYATALSGHPLR(F)







(SEQ ID NO:21)


 


2165.11
2165.14
−13.6320
1707
1725
(R)VLLPPEALHWEPDDAPIPR(R)







(SEQ ID NO:22)


 


2295.22
2295.224
−1.5853
389
410
(R)EILTTAGPFTVLVPSVSSFSSR(T)







(SEQ ID NO:23)





6 unmatched masses: 871.5410 949.4800 1360.6500 1538.6900 1787.9200 2008.1400


The matched peptides cover 10% (268/2570 AA's) of the protein.


Coverage Map for This Hit (MS-Digest index #): 427477













TABLE 2







CLEVER-1 Expression Is Induced Mainly on Vessels Sur-


rounded by Lymphocytic Infiltrations in Inflamed Synovia










The degree of




inflammatory


Expression of CLEVER-1
infiltration2









on HEV-like vessels1
0/1 (n = 9)
2/3 (n = 9)





−/+ 
100%
0


++/+++
0
100%





1Intensity was scored as −, ±, + negative or weak, ++, +++ moderate or strong.


2Degree of the inflammatory cell infiltration in 18 synovial samples was scored as: 0/1, none or few lymphocytes around the vessels, 2/3 marked or massive lymphocytic infiltrations.

## Claims

1. A method of treating inflammation in a patient in need of the same, said method comprising administering, to said patient, an agent that inhibits CLEVER-1 mediated leukocyte binding, wherein said agent is a CLEVER-1 antagonist antibody or a fragment thereof that contains an antigen binding site and wherein said CLEVER-1 antagonist antibody is antibody 3-266 produced by the hybridoma DSM ACC2519 or antibody 3-372 produced by the hybridoma DSM ACC2590, or said fragment thereof, or chimeric, humanized or primitized antibody thereof.

2. A method of treating inflammation in a patient in need of the same, said method comprising administering an agent that inhibits CLEVER-1 mediated leukocyte binding to said patient, wherein said inhibiting agent is antibody 3-266 produced by the hybridoma DSM ACC2519, or fragment thereof that contains an antigen binding site.

3. A method of treating inflammation in a patient in need of the same, said method comprising administering an agent that inhibits CLEVER-1 mediated leukocyte binding to said patient, wherein said inhibiting agent is antibody 3-372 produced by the hybridoma DSM ACC2590, or fragment thereof that contains an antigen binding site.

4. The method of claim 1, wherein said antibody is a monoclonal antibody.

5. Antibody is said chimeric, humanized or primatized antibody.

6. The method of claim 1, wherein said antibody is said antibody fragment.

7. The method of claim 6, wherein said fragment is a Fab, F(ab′)2 or Fv antibody fragment.