ES2352497T3 - METHOD AND COMPOSITIONS FOR THE TREATMENT OF INFLAMMATORY DISEASE THROUGH THE USE OF MODULATING AGENTS OF CADHERINA-11. - Google Patents

Abstract

Use of a therapeutically effective amount of a cadherin-11 inhibitory agent that inhibits the binding of cadherin-11 to a cadherin-11 counter-receptor that is cadherin-11, in the manufacture of a medicament for treating a subject having an inflammatory disorder of the joints, wherein the cadherin-11 inhibitor is an antibody or fragment thereof, a cadherin-11 polypeptide or fragment thereof, or an Fc fusion protein comprising an extracellular domain of cadherin-11, wherein the cadherin-11 inhibitor selectively binds cadherin-11.

Description

Field of the Invention

This invention relates to methods and compositions for the treatment of inflammatory diseases of the joints, such as those involving synovial hyperplasia and overproduction of biologically active factors. The methods involve administering a cadherin-11 modulating agent to a subject to modulate the function of cadherin-11 in areas of joint injury. Screening assays are also provided for the identification of 10 cadherin-11 modulating agents.

Background of the Invention

It is believed that adhesive interactions between cells and between cells and the extracellular matrix play critical roles in a wide variety of processes, including, for example, immune system modulation, regulation of developmental processes and tumor progression and metastasis. These interactions are mediated by 15 adhesion molecules that transduce information from the extracellular matrix to the intracellular one.

In WO 99/35166, modulating agents that enhance or inhibit ocludin-mediated cell adhesion have been disclosed.

Four families of adhesion molecules that mediate these interactions have been identified: integrins, cadherins, selectins, and immunoglobulin-related molecules. In general, adhesion molecules 20 are transmembrane proteins that contain an extracellular domain to interact with an extracellular matrix or cellular component, a transmembrane domain that spans the cell membrane, and a cytoplasmic domain to interact with one or more cytoskeletal components or cytoplasmic

Cadherins play an important role in establishing and maintaining intercellular connections between cells of the same type (reviewed in Geiger B. et al. (1992) Annual Review of Cell Biology 8: 307; Kemler R. (1993) 25 Trends in Gastroenterology 9: 317; Takeichi M. (1990) Annual Review of Biochem. 59: 237; Takeichi M. (1991) Science 251: 1451). Cadherins are a superfamily of structurally related molecules that function in homophilic Ca + 2 dependent adhesion. Cadherins are expressed in cells that form solid tissues, and are responsible for segregating and classifying cells during embryogenesis, establishing cell polarity, and maintaining tissue morphology. Structurally, cadherins are single stranded polypeptides that are synthesized as precursors and cleaved during post-translational processing. They have large extracellular regions formed by 5 homologous domains, an individual transmembrane segment and a cytoplasmic tail.

Cadherins are synthesized as precursors, which are cleaved during post-translational processing. Mature cadherins are single stranded molecules that include a relatively large extracellular domain (typically divided into five sections or "ectodomain"), a single transmembrane region and a cytoplasmic tail. Among the classic cadherins (i.e., the cadherin P (of the placenta), E (epithelial) and N (neural)), the cytoplasmic domain contains the highest degree of homology. The high degree of homology observed for the cytoplasmic domain disclosed is a reflection of the association of cadherins with a group of intracellular proteins, called catenins, which stabilize the active conformation of cadherin (Kemler R. (1993) Trends in Gastroenterology 9: 317). It is generally believed that sequences in the extracellular domain are necessary to mediate homophilic binding (i.e., cadherin to cadherin). A review of the literature indicates that research aimed at understanding cadherin-mediated adhesion has focused on efforts to elucidate the mechanism underlying homophilic cell adhesion mediated by cadherins. Little attention has been given to understanding what role, if any, cadherins play in heterophilic adhesion. While it has been known for some time that integrins and other adhesion molecules work in modulating the immune system, for example by playing a role in the adhesion of peripheral lymphocytes to the endothelium and returning them to the lymph nodes, relatively little is known with respect to the mechanism by which lymphocytes return and transmigrate through the vascular endothelium to specifically target certain tissue locations, such as the synovium.

The most highly conserved sequence shared by cadherins is found in the cytoplasmic domain. It is this region that mediates interaction with cytoplasmic cateninic proteins (Hirano S. et al. Cell 70: 293-301, 1992). 50 The presence of the cytoplasmic domain is essential for the functioning of cadherin, since deletions in this region suppress catenin binding as well as cell-to-cell adhesion (Hulsken J, et al. J Cell Biol 127: 1375-80, 1994). Catenins (α, 102 kDa; β, 88-93 kDa, and γ, 80-83 kDa) begin to associate with cadherins almost immediately with biosynthesis in a stable manner that is not interrupted in TX-100 detergent (Takeichi M. Curr Opin Cell Biol 7: 619-27, 1995), and is thought to mediate the anchoring of cadherins to the cytoskeleton (Yap AS. Et al. 55 Annu Rev Cell Dev Biol 13: 11946, 1997). Functionally, α-catenin is necessary for homophilic adhesion mediated by cadherins. Tumor cells expressing cadherin E on the cell surface, but lacking the expression of α-catenin, do not form cell-to-cell contacts unless the expression of α-catenin is restored through transfection (Chen H. et. al. J Cell Sci 1141345-56, 1997; and Knudsen
KA et al. J Cell Biol 130: 67-77, 1995). Β-Catenin is homologous to the Drosophila armadillo segment polarity protein, as well as to the cadherin-associated protein placoglobin. Placoglobin, also called γ-catenin, interacts weaker with the cadherin / catenin complex, and is not always observed in cadherin precipitates.

A number of newly identified cadherin cDNA clones have been isolated using consensus oligonucleotides that correspond to cytoplasmic domain sequences that are highly conserved between cadherins and cloned by PCR (Suzuki S. et al. Cell Reg 2: 261-70, 1991).

Although the role of cadherins classically involves homophilic cell-to-cell adhesion (i.e., cadherin E binds to cadherin E typically in another cell of the same type), however murine cadherin E expressed in epidermal keratinocytes also mediates the adhesion to cadherin E of Langerhans cells (Tang A. et al. Nature 361: 82-5, 1993). Recently, another counter-receptor for cadherin E has been identified, namely, integrin αEβ7, which is expressed in intraepithelial T cells (Cepek
KL et al. Nature 372: 190-3, 1994 and US Patent No. 5,610,281). This represents an example of heterophile binding of cadherin E to the integrin counterreceptor αEβ7.

Rheumatoid arthritis (RA) is a chronic systemic autoimmune disease of unknown etiology that affects 1-2% of the population. It is dominated by progressive joint destruction, which can result in a notable disability. Histologically, the joints reveal synovial hyperplasia mainly of synoviocytes type A, but also of synoviocytes type B, lymphocellular infiltration of T and B cells, and neovascularization. These processes lead to the secretion of destructive factors and the invasive growth of the synovial membrane (cloth) in the adjacent cartilage and bone, with the consequent destruction of the joint.

Summary of the Invention

The invention is based, in part, on the discovery of mRNA and cadherin-11 protein expression in 20 cells, namely human type B synoviocytes, recovered from the synovium of patients with inflammatory joint disorders. Before the present discovery, the expression of cadherin-11 in the synovium had not been disclosed. Although it is not intended to be bound by any particular theory, it is postulated that the expression of cadherin-11 in synovial cells is involved in the accommodation, retention and activation of cells (such as T and B cells) in this area. In addition, it is also postulated that cadherin-11 mediated adhesion mediates the invasion of synovium in cartilage and bone during some inflammatory joint disorders. Synoviocyte-synoviocyte contact may also be involved in some of these disorders. Cadherin-11 mediated adhesion can be characterized as homophilic (binding of cadherin-11 to cadherin-11) or heterophile (binding of cadherin-11 to a non-cadherin-11 counterreceptor). Consequently, some of the compositions and methods of the present invention are directed to inhibit cadherin-11 mediated adhesion that occurs between these and other cell types. Still other methods and compositions provided by the invention involve the modulation of cellular functions that are mediated by cadherin-11, such as, for example, cell signaling, proliferation, apoptosis and secretion of factors.

The present invention provides the use of a therapeutically effective amount of a cadherin-11 inhibitor, which inhibits the binding of cadherin-11 to a cadherin-11 counterreceptor that is cadherin-11, in the manufacture of a medicament for treating a subject having an inflammatory joint disorder, in which the cadherin-11 inhibitor is an antibody or its fragment, a cadherin-11 polypeptide or fragment thereof, or an Fc fusion protein comprising an extracellular domain of cadherin-11, wherein the cadherin-11 inhibitor selectively binds cadherin-11.

A method of treating a subject who has an inflammatory joint disorder is also described herein. The method involves administering to a subject in need of such treatment a therapeutically effective amount of a cadherin-11 modulating agent. A cadherin-11 modulating agent is an agent that modulates (for example, potency, inhibits, changes, etc.) a function of cadherin-11. In a particular embodiment, the cadherin-11 modulating agent is a cadherin-11 inhibiting agent. A cadherin-11 inhibitor is an agent that inhibits the binding of cadherin-11 to a cadherin-11 counterreceptor. In preferred embodiments, the subject is a human being. In more preferred embodiments, the subject does not have an abnormal adhesion mediated by cadherin-11 that occurs in the liver or brain. Cadherin-11 mediated adhesion is the binding of cadherin-11 to its counter receptor.

In one embodiment, the inflammatory joint disorder is chronic synovitis. In another embodiment, the inflammatory joint disorder is an autoimmune disease. In a preferred embodiment, the autoimmune disease is rheumatoid arthritis. fifty

The cadherin-11 inhibitor can be administered systemically. In preferred embodiments, the cadherin-11 inhibitory agent is administered locally to a synovium or synovial fluid of a subject.

The cadherin-11 inhibitor can be cadherin-11 that binds to a cadherin-11 counterreceptor in many ways. In one embodiment, the cadherin-11 inhibitor selectively binds cadherin-11. In another embodiment, the cadherin-11 inhibitor selectively binds to a cadherin-11 counterreceptor. The exemplary inhibitors are described in the detailed description. In all these embodiments, the cadherin-11 inhibiting agent works by inhibiting cadherin-11 mediated adhesion.

Cadherin-11 and the cadherin-11 counterreceptor can be expressed by the same cell type or by different cell types. The cadherin-11 counterreceptor does not need to be on the cell surface, but rather it can be part of an interstitial material, or it can be a segregated material that binds to any other molecule or surface. As an example of this last embodiment, the cadherin-11 counterreceptor is a component of an extracellular matrix of a tissue, a cartilage or a bone. The cadherin-11 counterreceptor can also be a molecule secreted by a cell. In certain embodiments, cadherin-11 is expressed by a cell, and the cadherin-11 counterreceptor is expressed by a different cell. Cells expressing cadherin-11 can be selected from the group consisting of a synovium type A or synovium type B, a fibroblast derived from synovium, a lining of the synovial membrane, an osteoblast, a cell derived from cartilage, and a cell derived from the invasive cloth. Cells expressing the cadherin-11 counterreceptor may be selected from the group consisting of a T lymphocyte, a B lymphocyte, a plasma cell, a macrophage, a dendritic cell, a natural killer cell (NK), a mast cell, a type A synoviocyte or a type B synoviocyte, a synovium-derived fibroblast, an osteoblast, a cartilage derived cell, a synovial membrane lining cell, and a cell derived from the invasive cloth.

In some embodiments, cadherin-11 modulating agents, such as, for example, cadherin-11 inhibitors, are not antibodies, such as, for example, monoclonal antibodies. In some embodiments, the agents described herein do not include the antibodies described in U.S. Pat. 5,597,725 and in PCT Application PCT / US93 / 03691 (document WO / 93/21302). Thus, in certain related embodiments, the agents described herein do not encompass the monoclonal antibodies produced by the hybridomas designated 30Q8A (HB11316), 30Q4H (HB11317), 45A5G (HB 11318), 30S2F (HB11319), 45C6A (HB 11320) , 30T11G (HB 11324), 20 64G11F (HB 11527).

A method for screening a molecular library to identify an agent (eg, a pharmaceutical leader compound) that modulates cadherin-11 mediated adhesion between a first cell expressing cadherin-11 and a second cell expressing a counter receptor is also described herein. of cadherin-11. The agent can inhibit cadherin-11 mediated adhesion, in which case it is a cadherin-11 inhibitor. The method involves carrying out a first adhesion test between the first cell and the second cell, to obtain a first adhesion test result, carrying out a second adhesion test between the first cell and the second cell in the presence of at least one member of the molecular library, to obtain a second result of the adhesion test, and compare the first and second results of the adhesion test to determine whether the at least one member of the molecular library modulates cadherin-11 mediated adhesion between the first cell and the second cell. Cell types, cadherin molecules and inhibitory agents are as described above and in the detailed description.

According to one embodiment, the first cell is selected from the group consisting of a synoviocyte, such as a synoviocyte type A, a synovium type B, a fibroblast derived from the synovium, a lining cell of the synovial membrane, and an osteoblast. The second cell can be selected from the group consisting of a type A synoviocyte, a type B synoviocyte, a synovium-derived fibroblast, a synovial membrane lining cell, an osteoblast, a T lymphocyte, a B lymphocyte, a cell of plasma, a macrophage, a dendritic cell, a natural killer cell, and a mast cell. In yet another embodiment, the first cell derives from the invasive cloth, and the second cell derives from cartilage. In a further embodiment, the first cell is derived from the invasive cloth, and the second cell is an osteoblast.

According to the screening method provided, a difference in the first and second results of the adhesion test 40 indicates the presence of at least one element of the molecular library that modulates cadherin-11 mediated adhesion between the first cell and the second cell. The assay can be designed to identify agents that inhibit or enhance cadherin-11 mediated adhesion.

The molecular library can be produced recombinantly, or it can be chemically synthesized. In still other embodiments, the molecular library is a peptide library. Four. Five

In yet another aspect of the invention, another method is provided for screening a molecular library to identify a leading pharmaceutical compound that modulates cadherin-11 mediated adhesion. The method involves carrying out a first adhesion test between cadherin-11 and a counter-receptor of cadherin-11, to obtain a first adhesion test result, carrying out a second adhesion test between cadherin-11 and the cadherin counterreceptor -11 in the presence of at least one member of the molecular library, to obtain a second result of the adhesion test, and compare the first and second results of the adhesion tests to determine whether the at least one member of the molecular library modulates Cadherin-11 mediated adhesion.

In certain embodiments of the invention, the cadherin-11 counterreceptor is selected from the group consisting of a cadherin, an integrin, an integrin subunit, a member of the immunoglobulin family, and a carbohydrate. In preferred embodiments, the cadherin is cadherin-11. In one embodiment, the cadherin-55 11 counterreceptor is a cadherin-11 fusion polypeptide. In yet another embodiment, the cadherin-11 counterreceptor is an antibody that selectively binds cadherin-11.

Cadherin-11 and / or the cadherin-11 counterreceptor can be isolated. Cadherin-11 and / or the cadherin-11 counterreceptor may also be soluble.

In yet another embodiment, cadherin-11 is presented by a cell. In still a further embodiment, the cadherin-11 counterreceptor is presented by a cell. The cell expressing cadherin-11 can be selected from the group consisting of a synovium of type A or a synoviocyte of type B, a fibroblast derived from synovium, a lining cell of the synovial membrane, an osteoblast, a cell derived from cartilage , and a cell derived from the invasive cloth. The cell expressing the cadherin-11 counterreceptor can be selected from the group consisting of a synoviocyte, a synovium-derived fibroblast, a synovial membrane lining cell, an osteoblast, a cartilage-derived cell, a cell derived from the invasive cloth, a T lymphocyte, a B lymphocyte, a plasma cell, a macrophage, a dendritic cell, a mast cell, and a natural killer cell. In still other embodiments, the cadherin-11 counterreceptor may be a component of an extracellular matrix of tissue, cartilage or bone, or it may be a molecule secreted by a cell and found anywhere in a tissue.

Also described herein is a method of treating a subject having an inflammatory joint disorder, which comprises administering to a subject in need of such treatment a therapeutically effective amount of an agent that modulates a cellular function in a cell expressing cadherin-11. , preferably a cellular function other than cadherin-11 mediated adhesion. The agent can modulate the activity by directly interacting with cadherin-11 (for example, a binding agent that selectively binds cadherin-11 and thereby modulates its activity), or indirectly interacting with another cellular component, which then modulates the Cadherin-11 activity. Cellular function can be selected from the group consisting of cell proliferation, factor secretion, apoptosis, migration, and / or binding. In important embodiments, the agent that modulates a cellular function in a cell expressing cadherin-11 does not inhibit the binding of cadherin-11 to a cadherin-11 counterreceptor. Thus, in some embodiments, cellular function is not the binding of cadherin-11 to a counter-receptor of cadherin-11 (ie, cellular function is not adhesion mediated by cadherin-11).

A method for screening a molecular library to identify a pharmaceutical leader compound that modulates a cellular function in a cell expressing cadherin-11 (preferably, a cellular function other than adhesion mediated by cadherin-11) is also described herein. The method comprises determining a first value of cellular function for a cell expressing cadherin-11 in the absence of a member of the molecular library, determining a second value of cellular function for a cell expressing cadherin-11 in the presence of al least one member of the molecular library, and compare the first value and the second value to determine whether the at least one member of the molecular library modulates a cellular function in a cell expressing cadherin-11. Cellular function can be selected from the group consisting of cell proliferation, factor secretion, apoptosis, migration and / or binding. 30

A similar method for screening a molecular library to identify a leading pharmaceutical compound that modulates the secretion of factors in a cell expressing cadherin-11 is also described herein. In one embodiment of this last aspect, the secretion of factors is selected from the group consisting of stromelysin secretion, collagen secretion, collagenase secretion and IL-6 secretion.

Similarly, the screening method can be used to identify leading pharmaceutical compounds that modulate a cellular function in a cell that expresses the cadherin-11 counterreceptor. In important embodiments, the pharmaceutical leader compound is screened for its ability to inhibit the binding of cadherin-11 to a cadherin-11 counterreceptor. Preferably, in certain embodiments, the pharmaceutical leader compound does not inhibit the binding of cadherin-11 to its counter receptor. Thus, in some embodiments, the cellular function that is modulated is not the binding of cadherin-11 to a counter-receptor of cadherin-11. 40

The invention also provides, in another aspect, a pharmaceutical composition (i.e., a pharmaceutical preparation) comprising an effective amount of a cadherin-11 modulating agent (eg, a cadherin-11 inhibiting agent) and a pharmaceutically acceptable carrier. , as well as a method to obtain such a composition. The cadherin-11 modulating agent is present in an amount effective to modulate the function of cadherin-11. If the agent is a cadherin-11 inhibitor, then it may be present in an amount effective to inhibit cadherin-11 mediated adhesion (such as, for example, that which occurs in the synovium). In one embodiment, the composition is contained in a syringe for injection locally into the synovium, synovial fluid, joint or joint capsule of a subject.

Also described herein is a method for obtaining a pharmaceutical composition, a pharmaceutical preparation and / or a medicament, that is, by placing the cadherin-11 modulating agent (for example, a cadherin-50 11 inhibiting agent) of the invention in a pharmaceutically acceptable vehicle. The pharmaceutical composition or preparation may contain one or more cadherin-11 modulating agents, and, optionally, other therapeutic agents that are useful in the treatment of the disorder described herein (eg, NSAID).

These and other aspects of the invention, as well as various advantages and utilities, will be more apparent with reference to the detailed description of the preferred embodiments and the accompanying drawings. 55

Brief Description of the Drawings

The Examples refer to and include a brief description of various figures. It will be understood that the drawings or figures are illustrative only and are not required to allow the inventions described herein.

Figure 1 illustrates PCR products separated by gel electrophoresis. The negative control is shown in line 1, and the expected 385 bp size product obtained in type B synoviocyte cDNA is shown in line 2.

Figure 2 illustrates results from a Northern mRNA analysis from line 16E6.A5 EpC (line 1), type B synoviocytes (line 2), Jurkat cells (line 3) and SKNSH neuroblastoma cell line (line 4). ), hybridized with the 385 bp fragment of cadherin-11 (upper panel), and a glyceraldehyde-3-phosphate dehydrogenase (Clontech) probe (lower panel) as a control. The molecular mass markers are indicated on the left.

Figure 3 is a schematic of the structure of the human fusion protein of cadherin-11 with Fc. The sequence of the wild-type cadherin-11 juxtamembrane region and the alterations resulting from fusion with the human Fc region is shown. The regions that correspond to the Fc portion are shown in bold.

Figure 4 is a series of flow cytometry histograms showing the expression of cadherin-11 on the cell surface in L cells transfected with cadherin-11 cDNA. L cells transfected with pLK-neo (left column) or with pLK-neo / C11 (right column) are shown. Staining with the control mAb P3 is shown not shaded. Staining with aCad-11, 3H10, 5H6, and 2G4 is shaded.

Figure 5 is a photocopy of a photograph illustrating the immunohistochemical staining of synovial lining cells and some cells below the lining in RA synovitis, using the 2G4 mAb. Frozen tissue sections of human RA synovium (panel A and B) were stained using the indirect immunoperoxidase method, using 2G4 mAb, and stained with hematoxylin (20x [panel A] and 60x [panel B] magnification). The majority of the 20 cells in the lining, and a few in the undercoating, were 2G4 + (panel B).

Figure 6 illustrates a flow cytometric analysis of cadherin-11 expression by line 5 of synovial T cells (left panel) and CP-B cells (right panel).

Figure 7 is a graph of the percentage of cells in line 5 of synovial T cells and CP-B cells bound to control proteins or cadherin-11. 25

Sequence Listing

SEQ ID NO: 1 is the nucleotide sequence of human cadherin-11 cDNA.

SEQ ID NO: 2 is the amino acid sequence of human cadherin-11 protein.

SEQ ID NO: 3 is the amino acid sequence of residues 753-762 of human cadherin E.

SEQ ID NO: 4 is the amino acid sequence of residues 840-847 of human cadherin E. 30

SEQ ID NO: 5 is the amino acid sequence of residues 853-859 of human cadherin E.

SEQ ID NO: 6 is the amino acid sequence of residues 865-875 of human cadherin E.

SEQ ID NO: 7 is the nucleotide sequence of a degenerate sense primer.

SEQ ID NO: 8 is the nucleotide sequence of a degenerated antisense primer.

SEQ ID NO: 9 is the nucleotide sequence of primer XV14. 35

SEQ ID NO: 10 is the nucleotide sequence of primer XV15.

SEQ ID NO: 11 is the nucleotide sequence of primer XVCad11A.

SEQ ID NO: 12 is the nucleotide sequence of primer XVCad11E.

Detailed description of the invention

The invention is based, in part, on the discovery that mRNA and cadherin-11 protein are expressed in 40 synovial cells of patients with inflammatory joint disorders. The discovery was made by coprecipitating cadherin-11 with an antiserum for catenin in human synoviocytes type B. The demonstration of the expression of a cadherin, namely cadherin-11, in the synovium of a patient with rheumatoid arthritis provides a previously unrecognized opportunity. to select therapies that improve rheumatoid arthritis as well as other inflammatory arthritis in which synovial hyperplasia and overproduction of toxic and biologically active factors 45 mediate joint damage.

Cadherin-11 is a transmembrane molecule that, among others, mediates the union of cells with each other through interaction with themselves or their counterreceptors. Like other cadherins, it is proposed that cadherin-11 mediates the adhesion of similar cells to each other, as well as the adhesion of cells of different strains to each other. In accordance with the discovery on which the present invention is based, it is proposed that cadherin-11, among others, mediates adhesion between similar cells, such as synoviocytes, as well as between different cells, such as synoviocytes and lymphocytes (by example, T and B cells).

Human and mouse cadherin-11 genes have been previously isolated and sequenced Suzuki S. et al. Cell Reg 2: 261-70, 1991). See also Genbank accession number NM_001797, (SEQ ID NO: 1 and SEQ ID NO: 2) for the human cadherin-11 cDNA and predicted amino acid sequences, respectively.

Lymphocytes have the ability to circulate along blood vessels and then detect antigens or provide immune protection in all tissues. It is believed that specific adhesion molecules (e.g., selectins and integrins) and their counterreceptors mediate lymphocyte accommodation and transmigration through the vascular endothelium to sites of inflammation (Springer TA. Cell 76: 301-314, 1994). Each integrin is a non-covalently associated αβ heterodimer complex. Subfamilies of integrins have been defined by the use of particular β chains that are often paired with one of several different α chains. Only selected integrins are expressed in 15 T cells. Members of the β1 and β3 subfamily (a1β1, α2β1, α4β1, α5β1 and αvβ3) are mainly involved in the adhesion of lymphocytes to the extracellular matrix, except for α4β1, heterodimer (VLA -4) that binds to vascular cell adhesion molecule 1 (VCAM-1), a counter-receptor in activated vascular endothelium (Elices
MJ. et al. Cell 60: 577-584, 1990). From the α subfamily, LFA-1 (αLβ2) is expressed in T cells, and mediates binding to ICAM-1 counter receptors (Marlin SD. Et al. Cell 51: 813-819, 1987), ICAM-2 (deFourgerolles AR. et al. J Exp Med 174: 253-267, 1991) and 20 ICAM-3 (deFourgerolles AR. et al. J Exp Med 175: 185-190, 1992; Fawcett J. et al. Nature 360: 481-484, 1992; and Vazeux R. et al. Nature 1992; 360: 481-488). LFA-1 also mediates binding to inflamed vascular endothelium, allowing firm binding of T lymphocytes before their transmigration out of the bloodstream. Lymphocyte migration to inflammatory sites involves a multi-stage process in which selectins (such as selectin L in T cells) mediate rolling, after which activation by integrins results in firm binding mediated by LFA-1. and 25 VLA-4, followed by transmigration to tissues. This lymphocyte trafficking is a requirement for the accumulation of lymphocytes at sites of infection, or abnormally at sites of chronic inflammation, such as rheumatoid synovium.

Thus, the invention encompasses the use of cadherin-11 modulating agents in the treatment of inflammatory joint disorders. Additionally, screening methods are provided for the identification of cadherin-11 modulating agents that possess the characteristics described herein. 30

The methods and compositions of the invention relate to cadherin-11 modulating agents. A cadherin-11 modulating agent, as used herein, encompasses agents that inhibit the binding of cadherin-11 to its counter-receptor (i.e., cadherin-11 inhibiting agents), and agents that modulate cellular functions in cadherin-expressing cells. 11 (for example, agents that trigger, or those that inhibit, the signaling associated with cadherin-11). Cadherin-11 modulating agents are capable of modulating (1) cell proliferation, (2) the secretion of molecules such as, but not limited to, stromelysin, collagen, collagenase and IL-6, (3) apoptosis, (4) the migration, and / or (5) the binding, of cells expressing cadherin-11. In important embodiments, such agents lead to a decrease in cell proliferation, a decrease in the secretion of molecules such as, but not limited to, cytokines, and an increase in apoptosis in cells expressing cadherin-11. Indeed, these agents preferably function to slow the growth rate of, or ultimately kill, cadherin-11 cells 40 such as synoviocytes. It has been previously disclosed in other cell types that the loss of cadherin E can lead to a cancerous or metastatic phenotype.

The invention thus also encompasses cadherin-11 inhibitors, and their method of use. Unless stated otherwise, it will be understood that these two categories of agents, that is, agents that modulate cadherin-11 mediated adhesion (i.e., cadherin-11 inhibitors) and agents that modulate other functions of cadherin -11, such as proliferation, factor secretion, and apoptosis, do not need to be mutually exclusive or need to overlap completely. For example, it is expected that some agents identified in the screening assays of the invention will function only as cadherin-11 inhibitors (i.e., that have the ability to block the binding of cadherin-11 to their counter-receptor), others will function by modulating (for example, enhancing or inhibiting) cellular functions, such as proliferation, secretion, apoptosis, binding and migration, while still other agents will be capable of both functions. Thus, some agents will be able to inhibit the binding of cadherin-11 to their counter-receptor, although they will not have any impact on other functions mediated by cadherin-11, such as cell signaling. Similarly, other agents will enhance or inhibit cadherin-11 mediated functions, such as proliferation, for example, although they will have no effect on the binding of cadherin-11 to its counter receptor. 55

In one aspect, the invention provides the use of a therapeutically effective amount of a cadherin-11 inhibitor that inhibits the binding of cadherin-11 to a cadherin-11 counterreceptor that is cadherin-11, in the manufacture of a medicament for treating a subject having an inflammatory joint disorder, in which the cadherin-11 inhibitor is an antibody or fragment thereof, a cadherin-11 polypeptide or fragment thereof, or an Fc fusion protein comprising a extracellular domain of cadherin-11, in which the cadherin-11 inhibitor selectively binds cadherin-11.

As used herein, an inflammatory joint disorder is one in which synovial hyperplasia and / or overproduction of biologically active factors mediates joint damage. In inflammatory disorders of the joints, the synovium becomes inflamed and thicker, and, in advanced cases, this is followed by an invasion of the synovium in the cartilage and bone. The synovium is the membrane that covers the capsule of a joint. Synovitis (i.e., inflammation of the synovium) can manifest itself in acute or chronic forms. In acute synovitis, the onset of pain and discomfort is usually sudden and short-lived, as is also the case in pigmented villonodular synovitis. On the contrary, in chronic synovitis, pain and discomfort are recurrent and persistent. In any case, the symptoms can be caused by arthritis, injury, joint overuse, and infection. 10

The modulating agent, such as the inhibiting agent, is preferably administered locally to the subject's synovium. The affected joints that can be treated are those that exist throughout the body, in areas such as the shoulders, back, wrists, hands, elbows, knees, hips, ankles and feet, and are lined by a synovial membrane.

Exemplary conditions that result from or cause inflammatory joint disorders include chronic synovitis, autoimmune diseases, psoriatic arthritis, arthritis of chronic Lyme disease, arthritis associated with inflammatory bowel disease, arthritis associated with ankylosing spondylitis, Reiter's syndrome, arthritis associated with systemic lupus erythematosus, arthritis associated with juvenile chronic arthritis, arthritis associated with infection, arthritis associated with immune response to infectious agents and nonspecific synovitis of unknown etiology. An autoimmune disorder is one in which the body's immune system reacts against one or more body tissues and thus attacks the tissue as if it were a foreign antigen or pathogen. Rheumatoid arthritis is an example of an autoimmune disorder, in which the components of the immune system perpetuate the inflammation of a joint, resulting in damage to synovium, cartilage, bone, and associated joint tissues.

A method provided by the invention involves administering to a subject in need of such treatment a cadherin-11 inhibitor. A cadherin-11 inhibitor is an agent that inhibits the binding of cadherin-11 to a cadherin-11 counterreceptor. The cadherin-11 inhibitor is administered to the subject in a therapeutically effective amount. A therapeutically effective amount is a dose of cadherin-11 inhibitor sufficient to provide a medically desirable result. In the method of treatment of the invention, the therapeutically effective amount of the cadherin-11 inhibiting agent may be that amount that is sufficient to reduce inflammation or swelling of the joint, or to relieve joint pain. As used herein, 30 treatment encompasses the use of cadherin-11 inhibitors to reduce the adverse medical condition that is mediated by the binding of cadherin-11 to its counter-receptor in vivo, as well as prophylactic treatment of subjects at risk of develop an inflammatory disorder of the joints.

As mentioned earlier, the treatment methods described herein encompass the use of cadherin-11 inhibitors or agents that modulate cellular functions (eg, signaling, proliferation, apoptosis, etc.) of cadherin-11 other than the binding of cadherin-11 to a counter-receptor of cadherin-11, or the use of both. Thus, as described herein, the method of treating a subject having an inflammatory joint disorder, may alternatively comprise administering to the subject an effective amount of an agent that modulates one or more cellular functions of cadherin-11. In important embodiments, the one or more cellular functions do not include the binding of cadherin-11 to its counter receptor. 40

A subject, as used herein, refers to any mammal that is likely to have or that currently has an inflammatory joint disorder. Preferably, the subject is one who has an inflammatory joint disorder. In more preferred embodiments, the subject is a human being. In certain embodiments, the subject has no abnormal cadherin-11 mediated adhesion in the brain and / or liver.

In general, cadherin-11 inhibitors are agents that inhibit cadherin-11 mediated adhesion. Cadherin-11 mediated adhesion, as used herein, refers to the binding of a cadherin-11 polypeptide, hereinafter referred to as cadherin-11, to a cadherin-11 counterreceptor. A cadherin-11 counterreceptor is a molecule that selectively binds cadherin-11, and, in some cases, is expressed on the surface of a cell. In other cases, the cadherin-11 counterreceptor is not bound to the cell but rather is a component of an interstitial matrix, such as an extracellular matrix of tissue, cartilage or bone. The cadherin-11 counterreceptor can also be a molecule secreted by a cell. Exemplary cells expressing a cadherin-11 counterreceptor include synovial type A and type B, synovial-derived fibroblasts, cartilage-derived cells, osteoblasts, T and B lymphocytes, plasma cells, macrophages, dendritic cells, NK cells, mast cells, synovial lining cells, and cells derived from the invasive cloth. A cadherin-11 counterreceptor encompasses cadherin-11, members of the immunoglobulin superfamily, carbohydrate epitopes of a glycoprotein or glycolipid, a selectin, a lectin-containing ligand, and an integrin. At least 21 different integrins composed of eight different β chains associated with 15 different α chains have been identified. The integrins useful in the invention include α1β1, α2β1, α3β1, α4β1, α5β1, α6β1, α7β1, α8β1, α9β1, αLβ2, αMβ2, αxβ2, αIIbβ3, αvβ3, α6β1, αvβ5, α4β7, α7β7, α7β7, α7β7, α7β7, α7β7, α7β2 In some embodiments, the preferred integrins are α1β1, α2β1, α3β1, α4β1, α5β1, α6β1, αLβ2, αMβ2, αvβ3, α4β7 and αEβ7.

The invention is based, in part, on the observation that T cells and B cells, which have not previously been disclosed that both express cadherin-11, are capable of binding in vitro to immobilized recombinant cadherin-11. Thus, this finding suggests that T and B cells express a cadherin-11 counterreceptor that is not cadherin-11. This is the first report of a cadherin-11 counterreceptor that is not cadherin-11 expressed by T and B cells. Comparing cells that bind purified or isolated cadherin-11 (and known to not express cadherin-11) with cells that are incapable of such binding, can be identified as cadherin-11 counter-receptors that are not cadherin-11. Exemplary methods for comparing these two cell types include, for example, genetic screening using techniques such as differential presentation, as well as subtractive hybridization to identify transcripts that are expressed by cells that bind cadherin-11 and are not expressed by cells that do not bind cadherin-11. These techniques are usually practiced by those of ordinary skill in the art. These techniques allow rapid isolation of nucleic acid encoding a cadherin-11 counterreceptor. Once the cells that bind cadherin-11 but do not express cadherin-11 have been identified, cell membrane preparations can be analyzed, for example, by affinity purification, to isolate the cadherin-11 counter receptor. For example, a GST fusion protein with cadherin-11, immobilized on a column, can be used to extract cadherin-11 counterreceptors from a crude cell preparation. In a preferred embodiment, monoclonal antibodies are generated against cells expressing a cadherin-11 counter receptor, and the resulting antibodies are screened to determine their ability to block the binding of cadherin-11 to such cells. twenty

The cadherin-11 modulating agents of the invention, including cadherin-11 inhibitors, encompass nucleic acid molecules, polypeptides, carbohydrates, and synthetically produced and recombinantly produced molecules.

In certain other embodiments of the invention, the cadherin-11 modulating agent is a polypeptide (ie, a cadherin-11 modulating polypeptide). As such, the modulating agent, including cadherin-11, 25 inhibitor can be (1) a cadherin-11 polypeptide or a (preferably unique) fragment thereof, or (2) a cadherin-11 counterreceptor polypeptide or a (preferably unique) fragment thereof; (3) a "binding peptide" to cadherin-11 (other than a cadherin-11 counterreceptor); and (4) a "binding peptide" to a cadherin-11 counterreceptor (other than a cadherin-11 polypeptide). As used herein, a cadherin-11 binding peptide refers to peptides that selectively bind cadherin-11. Similarly, a cadherin-11 counterreceptor-binding peptide 30 refers to a peptide that selectively binds to a cadherin-11 counter-receptor. In some important embodiments, the cadherin-11 binding peptide does not inhibit the binding of cadherin-11 to a cadherin-11 counterreceptor. That is, in certain embodiments, the binding peptide is not a cadherin-11 inhibitor, but can still modulate other functions mediated by cadherin-11, such as proliferation. The same may be true for cadherin-11 counterreceptor binding peptides. The invention encompasses the use of these last two classes of binding peptides in the treatment of subjects in which it is not necessary to inhibit the binding of cadherin-11 to a cadherin-11 counterreceptor in order to effect treatment.

The binding peptides may be antibodies or antibody fragments ("binding polypeptides"), which have the ability to selectively bind cadherin-11 polypeptides or a cadherin-11 counterreceptor. The antibodies include polyclonal and monoclonal antibodies that can be prepared according to conventional methodology. 40 Binding peptides and binding polypeptides can also be derived from sources other than antibody technology. For example, such binding peptides can be provided by degenerated peptide libraries that can be readily prepared in solution, in immobilized form, as bacterial flagella peptide presentation libraries, or as phage display libraries. Combinatorial libraries of peptides containing one or more amino acids can also be synthesized. In addition, peptide libraries and synthetic non-peptide moieties can be synthesized. Exemplary cadherin-11 binding peptides that are antibodies and fusion proteins are described in the Examples. (See also USP Patent No. 5,639,634 and 5,597,725 and PCT Patent Application WO 93/21302).

Cadherin-11 inhibitor polypeptides can be cell adhesion molecules such as integrins and cadherins. fifty

Cadherin-11 inhibitor peptides are useful for inhibiting the binding of cadherin-11 to its counter receptor. As used herein, a fragment of a polypeptide refers to one that is capable of binding cadherin-11 or the cadherin-11 counterreceptor, as the case may be. Preferred cadherin-11 inhibitor polypeptides of the invention have the amino acid sequence of SEQ ID NO. 2, or a functionally equivalent fragment of SEQ ID NO. 2. Thus, cadherin-11 inhibitor peptides encompass functionally equivalent fragments, variants, and analogs of SEQ ID NO. 2, provided that the fragments, variants, and the like bind to cadherin-11 or a cadherin-11 counterreceptor and thereby reduce or prevent the adhesion of cadherin-11 to its counter-receptor. The invention also encompasses proteins and peptides encoded by any of the above cadherin-11 inhibitor nucleic acid molecules.

A single fragment of a cadherin-11 polypeptide has, in general, the features and characteristics of unique fragments as described herein in relation to nucleic acids. As those skilled in the art will recognize, the size of the single fragment will depend on factors such as whether the fragment constitutes a portion of a conserved protein domain. Thus, some regions of SEQ ID NO: 2 will require longer segments to be unique, while others will require only short segments, typically between 5 and 12 amino acids (e.g., 5, 6, 7, 8, 9, 5 10 , 11 and 12 amino acids in length or more, including each integer up to the full length polypeptide). Virtually, any segment of SEQ ID NO: 2 that is 9 or more amino acids in length and that is not common to other different polypeptides will be unique. The unique fragments of a polypeptide are preferably those fragments that retain a functional capacity other than the polypeptide. Functional capabilities that can be retained in a single fragment of the cadherin-11 polypeptide include the ability to bind to a counter-receptor of 10 cadherin-11. Similarly, a single fragment of a cadherin-11 counterreceptor polypeptide will possess the ability to bind to a cadherin-11 polypeptide.

As used herein with respect to polypeptides, the term "isolated" means separated from its native environment in a sufficiently pure form so that they can be manipulated or used for any one of the purposes of the invention. Thus, isolated means pure enough to be used (i) to generate and / or isolate antibodies, (ii) as a reagent in an assay, or (iii) for sequencing, etc.

Phage display can be particularly effective in identifying useful binding peptides according to the invention. Briefly, a phage library is prepared (using for example phage m13, fl, or lambda), which has inserts of 4 to about 80 amino acid residues, using conventional procedures. The inserts can represent, for example, a completely degenerated or skewed matrix. Then 20 inserts possessing the phage can be selected, which bind to the cadherin-11 polypeptide or a cadherin-11 counterreceptor. This process can be repeated through several cycles of phage reselection that binds to the cadherin-11 polypeptide or a cadherin-11 counterreceptor. Repeated rounds lead to phage enrichment that has particular sequences. The peptide sequence presented may vary in size. As the size increases, the complexity of the library increases. It is preferred that the total size of the presented peptide sequence (random amino acids 25 plus any spacer amino acids) should not be greater than about 100 amino acids in length, more preferably not more than about 50 amino acids in length, and most preferably no greater than about 25 amino acids in length. In certain embodiments, the libraries may have at least one restriction imposed on the presented peptide sequence. A restriction includes, but is not limited to, a positive or negative charge, hydrophobia, hydrophilicity, a cleavable link and the necessary moieties surrounding that link, and their combinations. In certain embodiments, more than one restriction is present in each of the peptide sequences of the library.

DNA sequence analysis can be carried out to identify the sequences of the expressed polypeptides. The minimum linear portion of the sequence that binds to the cadherin-11 polypeptide or a cadherin-11 counterreceptor can be determined. The procedure can be repeated using a biased library containing inserts that contain part or all of the minimum linear portion plus one or more additional degenerate debris in the 5 ’or 3 ′ direction thereof. The screening methods of two yeast hybrids can also be used to identify polypeptides that bind cadherin-11 polypeptides or a cadherin-11 counterreceptor. Thus, the cadherin-11 polypeptides or a cadherin-11 counterreceptor of the invention, or a fragment thereof, can be used to screen peptide libraries, including phage display libraries, to identify and select pairs of peptide binding of cadherin-11 polypeptides or a cadherin-11 counter receptor of the invention. Such molecules can be used, as described, for screening assays, for purification protocols, to directly interfere with the functioning of cadherin-11 or the cadherin-11 counterreceptor, and for other purposes that will be apparent to normal experts in The technique.

A cadherin-11 polypeptide or a cadherin-11 counterreceptor, or a fragment thereof, can also be used to isolate other binding partners (eg, naturally occurring cadherin-11 counterreceptors). The isolation of the binding partners can be carried out according to well known methods. For example, isolated polypeptides of cadherin-11 or a cadherin-11 counterreceptor can be attached to a substrate, and then a solution suspected of containing a cadherin-11 binding partner or a counter-receptor can be applied to the substrate of cadherin-11. If the binding partner for the cadherin-11 polypeptides or a cadherin-11 counterreceptor is present in the solution, then it will bind to the cadherin-11 polypeptide or a 50-cadherin-11 counterreceptor attached to the substrate. Then the binding partner can be isolated.

In some important embodiments, the cadherin-11 inhibitor is a functionally equivalent peptide analog of cadherin-11 or a cadherin-11 counterreceptor. As used herein, the term "functionally equivalent peptide analog" refers to a peptide analog that is capable of inhibiting the binding of cadherin-11 to the cadherin-11 counter-receptor competing with cadherin-11 for the binding to a cadherin-11 counter-receptor, or with a counter-receptor of cadherin-11 by binding to cadherin-11. Functionally equivalent peptide analogs of cadherin-11 are identified, for example, in in vitro adhesion assays, as described below and in the Examples, which measure the ability of the peptide analogue to inhibit adhesion, mediated by cadherin-11, between cells expressing cadherin-11 and its counter-receptor, or between isolated cadherin-11 and an isolated cadherin-11 counter-receptor, or some combination thereof. Accordingly, functionally equivalent equivalent peptide analogs of cadherin-11 include the extracellular domain of cadherin-11, fragments of the extracellular domain and peptide analogs of the extracellular domain (e.g., peptides containing conservative amino acid substitutions), with the proviso that Peptide fragments and analogs are capable of inhibiting adhesion between cadherin-11 and its counter receptor when these molecules are present in isolation or in the context of a cell membrane in vivo and / or in vitro. Similarly, a functionally equivalent peptide analog of cadherin-11 receptor includes the extracellular domain of the cadherin-11 receptor, 5 extracellular domain fragments and peptide analogs of the extracellular domain (eg, peptides containing conservative amino acid substitutions), with the provided that the peptide fragments and analogs are capable of inhibiting adhesion between cadherin-11 and its counter-receptor when these molecules are present in isolation or in the context of a cell membrane in vivo and / or in vitro. Preferably, the peptide fragments and / or the like contain between about three and about one hundred amino acids. More preferably, the peptide analogs contain between about ten and about twenty-five amino acids.

Inhibitors of the invention include antibodies and fusion proteins that inhibit the binding of cadherin-11 to its counter receptor. Thus, the peptides of the invention can be specifically reactive with an antibody (preferably a monoclonal antibody) that selectively binds cadherin-11, or an antibody that selectively binds to a cadherin-11 counterreceptor, and thereby inhibits adhesion between cadherin-11 and a cadherin-11 counterreceptor. Thus, the cadherin-11 fusion polypeptides and the cadherin-11 counterreceptor are also encompassed by the present invention, as is their use in the methods described herein. A fusion polypeptide, as used herein, is a polypeptide that contains peptide regions from at least two different proteins. For example, a fusion cadherin-11 polypeptide can be formed by fusing, usually at the nucleotide level, the cadherin-11 coding sequence to the coding sequence of another distinct protein. As described below, a cadherin-11 fusion protein can be synthesized with GST, and is useful for screening agents that bind cadherin-11. Also as described in the Examples, a fusion protein of cadherin-11 with Fc has been synthesized by binding the extracellular domains of cadherin-11 to the Fc portion of immunoglobulin. Depending on their nature, some fusion proteins are suitable for in vitro use, while others are more suitable for in vivo use. 25

In certain preferred embodiments, the cadherin-11 modulating agent is an antibody or a fragment of an antibody that has the ability to selectively bind cadherin-11 polypeptides or a cadherin-11 counterreceptor. The antibodies include polyclonal and monoclonal antibodies, prepared according to conventional methodology.

Significantly, as is well known in the art, only a small portion of an antibody molecule, the paratope, is involved in the binding of the antibody to its epitope (see, in general, Clark, WR (1986) The Experimental Foundations of Modern Immunology Wiley & Sons, Inc., New York; Roitt, I. (1991) Essential Immunology, 7th ed., Blackwell Scientific Publications, Oxford). The pFc ’and Fc regions, for example, are effectors of the complement cascade, but are not involved in antigen binding. An antibody from which the pFc ’region has been enzymatically cleaved, or that has been produced without the pFc’ region, called an F (ab ’) 2 fragment, retains the two antigen binding sites of an intact antibody. Similarly, an antibody from which the Fc region has been enzymatically cleaved, or that has been produced without the Fc region, called a Fab fragment, retains one of the antigen-binding sites of an intact antibody molecule. Further on, the Fab fragments consist of a covalently linked antibody light chain and a portion of the antibody heavy chain represented by Fd. Fd fragments are the main determinant of antibody specificity (a single Fd fragment can be associated with up to ten different light chains without altering the specificity of the antibody), and Fd fragments retain epitope binding capacity in isolation.

Within the antigen-binding portion of an antibody, as is well known in the art, there are complementarity determining regions (CDR), which interact directly with the epitope of the antigen, and framework regions (FR), which maintain the tertiary structure of the paratope (see, in general, Clark, 1986; Roitt, 1991). Both in the heavy chain Fd fragment and the IgG immunoglobulin light chain, there are four frame regions (FR1 to FR4) respectively separated by three regions that determine complementarity (CDR1 to CDR3). The CDRs, and in particular the CDR3 regions, and more particularly the heavy chain CDR3, are tremendously responsible for the specificity of the antibody.

It is now well established in the art that non-CDR regions of a mammalian antibody can be replaced by similar regions of conspecific or hetero-specific antibodies while retaining the epitope specificity of the original antibody. This is most clearly manifested in the development and use of “humanized” antibodies, in which non-human CDRs covalently bind to human FR and / or Fc / pFc ’regions, to produce a functional antibody. Thus, for example, PCT International Publication No. WO 92/04381 teaches the production and use of murine anti-RSV humanized antibodies, in which at least a portion of the murine FR regions have been replaced by FR regions of origin. human. Such antibodies, including fragments of intact antibodies with the ability to bind antigens, are often referred to as "chimeric" antibodies.

Thus, as will be apparent to one of ordinary skill in the art, the present invention also provides F (ab ’) 2, Fab, Fv and Fd fragments; chimeric antibodies, in which the Fc and / or FR and / or CDR1 and / or CDR2 and / or CDR3 light chain regions have been replaced by homologous human or non-human sequences; chimeric 60 F (ab ’) 2 fragment antibodies, in which the FR and / or CDR1 and / or CDR2 and / or CDR3 light chain regions have been replaced by homologous human or non-human sequences; chimeric Fab fragment antibodies, in which the FR and / or CDR1 and / or CDR2 and / or CDR3 light chain regions have been replaced by homologous human or non-human sequences; and chimeric Fd fragment antibodies, in which the FR and / or CDR1 and / or CDR2 regions have been replaced by homologous human or non-human sequences. The present invention also includes so-called single chain antibodies.

For inoculation or prophylactic uses, the antibodies of the present invention are preferably intact antibody molecules that include the Fc region. Such intact antibodies will have longer half-lives than antibodies from smaller fragments (eg Fab), and are more suitable for intravenous, intraperitoneal, intramuscular, intracavity, subcutaneous, or transdermal administration. 10

Fab fragments, including chimeric Fab fragments, are preferred in methods in which the peptides of the invention are administered directly to a local tissue environment. For example, Fab fragments are preferred when the peptide of the invention is administered directly to the afflicted joint or to the inflamed synovium. Fabs offer several advantages over F (ab ’) 2 and all immunoglobulin molecules for this therapeutic modality. First, because Fabs have only one binding site for their cognate antigen, the formation of immune complexes is excluded as such complexes can be generated when bivalent F (ab ') 2 and all immunoglobulin molecules They find their target antigen. This is of some importance because the deposition of immune complexes in tissues can cause adverse inflammatory reactions. Second, because the Fabs lack an Fc region, they cannot cause adverse inflammatory reactions that are activated by Fc, such as complement cascade activation. Third, it is likely that the tissue penetration of the small Fab molecule is much better than that of the larger complete antibody. Fourth, Fabs can be easily and cheaply produced in bacteria, such as E. coli, while complete immunoglobulin antibody molecules require mammalian cells for production in useful quantities. The production of Fab in E. coli makes it possible to produce these antibody fragments in large fermenters, which are cheaper than products derived from cell culture. 25

A cadherin-11 modulating agent can also be a nucleic acid molecule (i.e., a nucleic acid molecule that modulates cadherin-11). A nucleic acid molecule that modulates cadherin-11 is a nucleic acid molecule that functions to modulate a cellular function of cadherin-11, such as, for example, cell proliferation, factor secretion, apoptosis, migration and Union. A cadherin-11 inhibitor nucleic acid molecule is a nucleic acid molecule that functions to inhibit cadherin-11 mediated adhesion. These 30 nucleic acid molecules can function directly (for example, as antisense nucleic acid molecules) or indirectly (for example, via the peptides and / or polypeptides they encode). A nucleic acid molecule that modulates cadherin-11 and a cadherin-11 inhibitor nucleic acid molecule can be selected from the group consisting of nucleic acid molecules that (1) encode a cadherin-11 polypeptide or a fragment (preferably unique ) of the same; (2) encode a polypeptide of a cadherin-11 counterreceptor or a fragment (preferably unique) thereof; or (3) are cadherin-11 or cadherin-11 antisense molecules that inhibit transcription or translation of the above nucleic acid molecules.

In one embodiment, a nucleic acid that modulates cadherin-11, such as, for example, a cadherin-11 inhibitor nucleic acid, (1) hybridizes under restrictive conditions to a nucleic acid having a sequence of SEQ ID NO: 1 , and (2) encodes a cadherin-11 polypeptide or a (preferably unique) fragment thereof that is capable of specifically binding to cadherin-11 or a cadherin-11 counterreceptor. In one embodiment, the cadherin-11 polypeptide, or its fragment, binds to a cadherin-11 counterreceptor on the surface of another cell, and thereby inhibits the binding of cadherin-11 to its counterreceptor, and optionally inhibits the union of one cell to another. Inhibition of the binding of a cell to the extracellular matrix is also provided. The preferred cadherin-11 inhibitor nucleic acid molecule has a nucleotide sequence of SEQ ID NO: 1. The cadherin-11 modulating nucleic acid molecules described herein also include homologs and alleles of a nucleic acid molecule having a sequence. of SEQ ID NO: 1.

Cadherin-11 modulating nucleic acid molecules, and preferably cadherin-11 inhibiting nucleic acid molecules, can encode polypeptides that are soluble cadherin-11 polypeptides or membrane bound polypeptides. Soluble cadherin-11 polypeptides lack a transmembrane domain, and, optimally, also contain amino acids that make the polypeptide soluble (e.g., fusion proteins, which contain all or part of cadherin-11, which inhibit the binding of cadherin-11 to its counter receptor). Cadherin-11 modulating agents that are membrane bound (or membrane associated) preferably contain a transmembrane domain.

Cadherin-11 modulating nucleic acid molecules, and preferably cadherin-11 inhibiting nucleic acid molecules, further encompass nucleic acid molecules encoding a cadherin-11 polypeptide having the amino acid sequence of SEQ ID NO: 2, but which may differ from the sequence of SEQ ID NO: 1, due to the degeneracy of the genetic code. Similarly, the cadherin-11 modulating nucleic acid molecule, and preferably the cadherin-11 inhibiting nucleic acid molecule, can encode polypeptides and fragments thereof of the cadherin-11 counterreceptor that are soluble or membrane bound . 60

In some important embodiments, the nucleic acids that modulate the cellular function of cadherin-11 preferably do not encode a polypeptide or a fragment thereof of cadherin-11 or of the counter-receptor of cadherin-11 that is capable of inhibiting the binding of cadherin-11 to a cadherin-11 counterreceptor, nor are they antisense molecules that inhibit the transcription or translation of the above nucleic acid molecules.

The cadherin-11 modulating nucleic acid molecules described herein can be identified by conventional techniques, for example by identifying nucleic acid sequences encoding the cadherin-11 polypeptide and hybridizing to a nucleic acid molecule having the sequence of SEQ ID NO: 1, in restrictive conditions. The term "restrictive conditions", as used herein, refers to parameters with which the technique is familiar. More specifically, the restrictive conditions, as used herein, refer to hybridization at 65 ° C in hybridization buffer (3.5 x SSC, 0.02% formamide, 0.02% polyvinylpyrrolidone, 0.02% bovine serum albumin , 10 2.5 mM NaH2PO4 (pH 7), 0.5% SDS, 2 mM EDTA). SSC is 0.15 M sodium chloride / 0.15 M sodium citrate, pH 7; SDS is sodium dodecyl sulfate; and EDTA is ethylenediaminetetraacetic acid. After hybridization, the membrane to which the DNA is transferred is washed at 2 x SSC at room temperature, and then at 0.1 x SSC / 0.1 x SDS at 65 ° C.

There are other conditions, reagents, and others that can be used, which result in a similar degree of restriction. The expert will be familiar with such conditions, and, thus, are not given here. It will be understood, however, that the expert will be able to manipulate the conditions so as to allow clear identification of homologs and alleles of the nucleic acid molecules of the invention. The expert is also familiar with the methodology for screening cells and libraries for the expression of molecules, such as cadherin-11 inhibitor nucleic acid molecules, which can be isolated and sequenced. For example, in screening for cadherin-11 sequences, a Southern blot can be carried out using the above conditions, together with a radioactive probe. After washing the membrane to which the DNA is finally transferred, the membrane can be placed in front of an X-ray film to detect the radioactive signal.

In general, homologs and alleles of cadherin-11 will typically share at least 70% nucleotide identity with SEC. ID. NO: 1; and in some cases, they will share at least 75% of nucleotide identity; and still in other cases, they will share at least 80% of nucleotide identity. Watson-Crick complements of the above 25 nucleic acids are also contemplated. Preferred cadherin-11 homologues have at least 85% sequence homology with SEC. ID. NO: 1. More preferably, the cadherin-11 homologues have at least 90%, and more preferably at least 95% sequence homology with SEC. ID. NO: 1. Homology can be calculated using various publicly available software tools, developed by NCBI (Bethesda, Maryland), which can be obtained through the Internet (http: /
ncbi.nlm.nih.gov/pub/). Exemplary tools include the 30 BLAST system, available at http: //
www.ncbi.nlm.nih.gov. Alignments in pairs and ClustalW (matrix adjustment BLOSUM30), as well as Kyte-Doolittle hydropathic analysis, can be obtained using MacVetor sequence analysis software (Oxford Molecular Group).

Degenerate nucleic acids that include alternative codons to those present in the naturally occurring nucleic acid encoding, for example, the human cadherin-11 polypeptide are also contemplated. As is well known in the art, and as an example, serine residues are encoded by codons TCA, AGT, TCC, TCG, TCT and AGC. Each of the six codons is equivalent for the purposes of coding a serine residue. Thus, it will be apparent to a person skilled in the art that any of the nucleotide codons encoding serine can be used to direct the protein synthesis apparatus, in vitro or in vivo, to incorporate a serine moiety. Similarly, nucleotide sequence triplets encoding other amino acid residues include, but are not limited to, CCA, CCC, CCG and CCT (proline codons); CGA, CGC, CGG, CGT, AGA and AGG (arginine codons); HERE. ACC, ACG and ACT (threonine codons); AAC and AAT (asparagine codons); and ATA, ATC and ATT (isoleucine codons). Other amino acid residues can be similarly encoded by multiple nucleotide sequences. It should be understood that homologues and alleles and degenerates of nucleic acid molecules encoding a cadherin-11 counter receptor are also contemplated, and can be identified using the same types of techniques and procedures described herein with reference to homologs and cadherin alleles. 11 and degenerates of nucleic acid molecules encoding a cadherin-11.

As used herein with respect to nucleic acids, the term "isolated" means: (i) amplified in vitro by, for example, polymerase chain reaction (PCR); (ii) recombinantly produced by cloning; (iii) purified, such as by cleavage and gel separation; or (iv) synthesized, for example, by chemical synthesis. An isolated nucleic acid is one that is easily manipulated by recombinant DNA techniques well known in the art. Thus, a nucleotide sequence contained in a vector in which the 5 'and 3' restriction sites are known or for which polymerase chain reaction (PCR) primer sequences have been described is considered isolated, but a nucleic acid sequence that exists in its native state in its natural host no. An isolated nucleic acid can be substantially purified, but it does not need to be. For example, a nucleic acid that is isolated in a cloning or expression vector is not pure in that it can comprise only a small percentage of the material in the cell in which it resides. However, such a nucleic acid is isolated, as the term is used herein, because it is easily manipulated by standard techniques known to those of ordinary skill in the art.

The nucleic acid molecule described herein (for example, the cadherin-11 inhibitor nucleic acid molecule), in one embodiment, is operably linked to a gene expression sequence that directs the expression of the cadherin-inhibitor nucleic acid molecule. 11 in a eukaryotic cell. The "gene expression sequence" is any regulatory nucleotide sequence, such as a promoter sequence or a promoter-enhancer combination that facilitates the effective transcription and translation of the cadherin-5 11 inhibitor nucleic acid molecule to which it is operably linked. . The gene expression sequence can be, for example, a mammalian or viral promoter, such as a constitutive or inducible promoter. Constituent mammalian promoters include, but are not limited to, promoters for the following genes: hypoxanthine phosphoribosyl transferase (HPTR), adenosine deaminase, pyruvate kinase, the β-actin promoter and other constitutive promoters. Exemplary viral promoters that function constitutively in eukaryotic cells include, for example, simian virus promoters, 10 papillomavirus, adenovirus, human immunodeficiency virus (HIV), Rous sarcoma virus, cytomegalovirus, long terminal repeats ( LTR) of Moloney leukemia virus and other retroviruses, and the thymidine kinase promoter of herpes simplex virus. Other constitutive promoters are known to those of ordinary skill in the art. Promoters useful as gene expression sequences of the invention also include inducible promoters. Inducible promoters are expressed in the presence of an inducing agent. For example, the metallothionein promoter is induced to promote transcription and translation in the presence of certain metal ions. Other inducible promoters are known to those of ordinary skill in the art.

In general, the gene expression sequence should include, as necessary, non-transcriber sequences in 5 'and non-translator in 5', involved with the initiation of transcription and translation, respectively, such as a TATA box, protection sequence of the ends, CAAT sequence, and the like. Especially, such non-5 'transcription sequences will include a promoter region that includes a promoter sequence for the transcriptional control of the operably linked cadherin-11 inhibitor nucleic acid molecule. Gene expression sequences optionally include enhancer sequences, or 5 'direction activator sequences, as desired.

Nucleic acid molecules encoding cadherin-11 polypeptides and fragments thereof, cadherin-11 counter-receptor polypeptides and fragments thereof, and cadherin-11 inhibitors are also contemplated as described herein. These nucleic acid molecules can be used in both in vivo treatment methods and in vitro screening assays. When nucleic acid expression is preferred (as opposed to some embodiments of antisense treatment methods), the nucleic acid molecule is linked to a gene expression sequence that allows the expression of the nucleic acid molecule. The nucleic acid molecules of the invention can be introduced into a cell in vitro, followed by the transfer of the cell to the site of inflammation. The cell into which the nucleic acid molecule is introduced can be collected from the site of inflammation (for example, a lymphocyte, such as a T cell, a synoviocyte, such as a type B synoviocyte, a mast cell, a macrophage, a cell dendritic, a plasma cell, a synovial-derived fibroblast, a synovial membrane lining cell, an osteoblast, a cartilage-derived cell, or a cell derived from the invasive cloth), or it can be a cell that is not normally present at the site of inflammation. A sequence that allows the expression of the nucleic acid in a cell located in a joint, such as, for example, a synoviocyte, is one that is selectively active transcriptionally in the cell and thereby causes the expression of the nucleic acid in such a cell Those of ordinary skill in the art will be able to easily identify alternative promoters that are capable of expressing such a nucleic acid molecule in a synoviocyte, an osteoblast, a cell derived from cartilage, a lymphocyte or any other cell type mentioned herein. Alternatively, the transduced cell 40 can be cultured in vitro to produce a cadherin-11 modulating agent (eg, cadherin-11 inhibitor), or it can be used in in vitro screening assays. For example, the gene expression sequence can be used to express a cadherin-11 nucleic acid molecule in a cell that does not inherently express cadherin-11. The cadherin-11 nucleic acid molecule can also be expressed in a cell that does not inherently express neither cadherin-11 nor the cadherin-11 counterreceptor. It is claimed that the sequences of the nucleic acid molecules described herein (for example, cadherin-11 inhibitor nucleic acid molecules) and the gene expression sequence are "operably linked" when they are covalently linked in such a way to position the transcription and / or translation of the sequence of nucleic acid molecules (for example, a sequence encoding cadherin-11) under the influence or control of the gene expression sequence. If it is desired that the nucleic acid molecule be translated into a functional protein, it is claimed that two DNA sequences are operably linked if the induction of a promoter in the 5 'gene expression sequence results in the transcription of the molecule of nucleic acid and if the nature of the link between the two DNA sequences does not (1) result in the introduction of a frame shift mutation, (2) interferes with the ability of the promoter region to direct the transcription of the molecule of nucleic acid, or (3) interferes with the ability of the corresponding RNA transcript to be translated into a polypeptide. Thus, a gene expression sequence would be operably linked to a nucleic acid molecule if the gene expression sequence were capable of transcribing that nucleic acid molecule so that the resulting transcript can be translated into the desired polypeptide. .

In still other embodiments, the cadherin-11 modulating agent is a nucleic acid molecule that, instead of encoding a polypeptide, functions as an antisense molecule. Also described herein are antisense oligonucleotides that selectively bind to a nucleic acid molecule encoding a cadherin-11 polypeptide, or a fragment thereof, or a cadherin-11 counterreceptor (eg, an integrin or one or more of its subunits), or a fragment thereof, to decrease adhesion mediated by cadherin-11. When antisense preparations are used, local administration to synovium or synovial fluid is preferred.

As used herein, the term "antisense oligonucleotide" or "antisense" describes an oligonucleotide that is an oligoribonucleotide, oligodeoxyribonucleotide, modified oligoribonucleotide, or modified oligodeoxyribonucleotide that hybridizes under physiological conditions to DNA comprising a particular gene, or to an RNA transcript. of that gene and, thus, inhibits the transcription of that gene and / or the translation of that mRNA. Antisense molecules are designed to interfere with the transcription or translation of a target gene by hybridizing with the target gene or transcript. Those skilled in the art will recognize that the exact length of the antisense oligonucleotide and its degree of complementarity with its target will depend on the specific target selected, including the sequence of the target and the particular bases comprising that sequence. It is preferred that the antisense oligonucleotide be constructed and arranged to selectively bind to the target under physiological conditions, that is, to hybridize substantially more to the target sequence than to any other sequence in the target cell under physiological conditions. Based on SEQ ID NO: 1 or on genomic and / or cDNA allelic or homologous sequences, one skilled in the art can easily choose and synthesize any of a number of appropriate antisense molecules, for use according to the present invention. In order to be sufficiently selective and potent for inhibition, such antisense oligonucleotides 15 should comprise at least 10, and more preferably at least 15, consecutive bases, which are complementary to the target, although in certain cases they have been used successfully as antisense oligonucleotides modified oligonucleotides with a length as short as 7 bases (Wagner et al., Nat. Med 1 (11): 1116-1118, 1995). Most preferably, the antisense oligonucleotides comprise a complementary sequence of 20-30 bases.

Although oligonucleotides that are antisense to any region of the gene or mRNA transcripts can be chosen, in 20 preferred embodiments the antisense oligonucleotides correspond to N-terminal sites or in the 5 'direction, such as translation initiation sites, transcription initiation sites. or promoters. In addition, 3 ’untranslated regions can be selected by antisense oligonucleotides. Selection of mRNA splice sites has also been used in the art, but may be less preferred if alternative mRNA splicing occurs. In addition, the antisense is directed, preferably to sites where secondary mRNA structure 25 is not expected (see, for example, Sainio et al., Cell Mol. Neurobiol. 14 (5): 439-457, 1994), and in which proteins are not expected to bind. Finally, although SEQ ID NO: 1 describes a cDNA sequence, the person skilled in the art can easily derive the genomic DNA corresponding to this sequence. Thus, antisense oligonucleotides that are complementary to genomic DNA corresponding to SEQ ID NO: 1 are also contemplated. Similarly, antisense to allelic or homologous cadherin-11 cDNAs, or, alternatively, the cadherin-11 counterreceptor, and genomic DNAs are also allowed without undue experimentation.

In a set of embodiments, the antisense oligonucleotides of the invention may be composed of deoxyribonucleotides, "natural" ribonucleotides, or any combination thereof. That is, the 5 ′ end of a native nucleotide, and the 3 ′ end of another native nucleotide, can be covalently linked, as in natural systems, via an internucleoside phosphodiester bond. These oligonucleotides can be prepared by methods recognized in the art, which can be carried out manually or by an automated synthesizer. They can also be produced recombinantly by vectors.

In preferred embodiments, however, the antisense oligonucleotides described herein may also include "modified" oligonucleotides. That is, oligonucleotides can be modified in many ways that do not prevent them from hybridizing to their target, but that enhance their stability or target selection, or that otherwise potentiate their therapeutic efficacy.

The term "modified oligonucleotide", as used herein, describes an oligonucleotide in which (1) at least two of its nucleotides are covalently linked via a synthetic internucleoside bond (ie, a link other than a phosphodiester bond between the end 5 'of a nucleotide and the 3' end of another nucleotide), and / or (2) a chemical group not normally associated with nucleic acids has been covalently bound to the oligonucleotide. Preferred synthetic internucleoside linkages are phosphorothioates, alkylphosphonates, phosphorodithioates, phosphate esters, alkylphosphonothioates, phosphoramidates, carbamates, carbonates, phosphate esters, acetamidates, carboxymethyl esters and peptides.

The term "modified oligonucleotide" also encompasses oligonucleotides with a covalently modified base and / or sugar. For example, modified oligonucleotides include oligonucleotides having main chain sugars that are covalently linked to low molecular weight organic groups other than a hydroxyl group at the 3 ′ position, and distinct from a phosphate group at the 5 ′ position. Thus, the modified oligonucleotides can include a 2′-O-alkylated ribose group. In addition, modified oligonucleotides may include sugars such as arabinose, instead of ribose.

The present description thus contemplates pharmaceutical preparations containing modified antisense molecules 55 that are complementary to and that can hybridize with, under physiological conditions, nucleic acids encoding cadherin-11 polypeptides or cadherin-11 counterreceptor, together with pharmaceutically acceptable carriers. Antisense oligonucleotides can be administered as part of a pharmaceutical composition. Such pharmaceutical composition may include antisense oligonucleotides in combination with standard physiologically and / or pharmaceutically acceptable carriers, which are known in the art. The compositions should be sterile, 60 and should contain a therapeutically effective amount of the antisense oligonucleotides in a unit of weight or volume suitable for administration to a patient.

The nucleic acids described herein can be delivered, for example, to synovium and joint cells alone or in association with a vector. In the broadest sense, a "vector" is any vehicle capable of facilitating: (1) the delivery of a nucleic acid molecule to a target cell, and / or (2) the uptake of a nucleic acid molecule 5 with a target cell Preferably, the vectors transport the cadherin-11 modulating nucleic acid molecule into the target cell with reduced degradation relative to the degree of degradation that would result in the absence of the vector. Optionally, a "target selection ligand" may be attached to the vector, to selectively deliver the vector to a cell that expresses on its surface the cognate receptor for the target selection ligand. In this way, the vector (containing, for example, a cadherin-11 inhibitor nucleic acid molecule 10) can be selectively delivered to the synovial lining in an articular capsule. Methodologies for selecting targets include conjugates, such as those described in U.S. Pat. 5,391,723. Preferably, the nucleic acid molecules described herein are selected for delivery to a cell located in an articular capsule, such as a synoviocyte or a lymphocyte.

In general, vectors useful in such methods are divided into two classes: biological vectors and chemical / physical vectors. Biological vectors are useful for the delivery / uptake of nucleic acids to / by a target cell. Biological vectors include, but are not limited to, plasmids, phagemids, viruses, other vehicles derived from viral or bacterial sources that have been manipulated by insertion or incorporation of the nucleic acid sequences described herein and additional nucleic acid fragments (by example, enhancers, promoters) that can be linked to the nucleic acid sequences described herein. Viral vectors are a preferred type of biological vector, and include, but are not limited to, nucleic acid sequences from the following viruses: adenovirus; adeno-associated viruses; retroviruses, such as Moloney murine leukemia virus; Harvey's murine sarcoma virus; murine mammary tumor virus; Rous sarcoma virus; virus type SV40; polyoma viruses; Epstein-Barr virus; papillomavirus; herpes virus; vaccine virus, polio virus; and RNA virus, such as a retrovirus. Other vectors not named but known in the art can easily be employed. 25

A particularly preferred virus for certain applications is the adeno-associated virus, a double-stranded DNA virus. The adeno-associated virus is capable of infecting a wide range of cell types and species, and can be engineered to be replication deficient. It also has advantages such as thermal stability and lipid solvents, high transduction frequencies in cells of various strains, including hematopoietic cells, and lacks superinfection inhibition, thus allowing multiple series of transductions. As disclosed, adeno-associated viruses can be integrated into human cell DNA in a site-specific manner, thereby minimizing the possibility of insertion mutagenesis and expression variability of the inserted gene. In addition, infections by wild-type adeno-associated viruses have been followed in tissue culture for more than 100 passes in the absence of selective pressure, implying that the genomic integration of adeno-associated viruses is a relatively stable event. The adeno-associated virus can also function in an extrachromosomal manner.

In general, other preferred viral vectors are based on non-cytopathic eukaryotic viruses, in which the nonessential genes have been replaced by the gene of interest. Non-cytopathic viruses include retroviruses, whose life cycle involves reverse transcription of genomic viral RNA in DNA, with subsequent proviric integration into host cell DNA. Adenoviruses and retroviruses have been approved for human gene therapy assays. In general, retroviruses are replication deficient (ie, capable of directing the synthesis of the desired proteins, but unable to make an infectious particle). Such genetically altered retroviral expression vectors have general utility for transduction of genes in vivo with high efficiency. Standard protocols for producing replication-deficient retroviruses (including the steps of incorporating exogenous genetic material into a plasmid, transfection of a plasmid packaging cell line, production of recombinant retroviruses by the packaging cell line, collection of viral particles from tissue culture media, and infection of the target cells with viral particles) are provided in Kriegler, M., "Gene Transfer and Expression, A Laboratory Manual", WH Freeman C.O., New York (1990) and Murry, E.J. Ed. "Methods in Molecular Biology", vol. 7, Human Press, Inc., Clifton, New Jersey (1991). Another preferred retroviral vector is the vector derived from Moloney murine leukemia virus, as described in Nabel, E.G., et al., Science, v. 249, p. 1285-1288 (1990). fifty

In addition to biological vectors, chemical / physical vectors are useful for the delivery / uptake of nucleic acids or polypeptides to / by a target cell. As used herein, a "chemical / physical vector" refers to a natural or synthetic molecule, other than those derived from bacteriological or viral sources, capable of delivering the cadherin-11 modulating agent to a cell.

A preferred chemical / physical vector is a colloidal dispersion system. Colloidal dispersion systems include 55 lipid-based systems, which include oil-in-water emulsions, micelles, mixed micelles, and liposomes. A preferred colloidal system of the invention is a liposome. Liposomes are artificial membranous vesicles that are useful as a delivery vector in vivo or in vitro. It has been shown that large unilamellar vesicles (LUV), whose size ranges from 0.2 to 4.0 µm, can encapsulate large macromolecules. RNA, DNA, and intact virions can be encapsulated in the aqueous interior, and can be delivered to cells in a biologically active form 60 (Fraley, et al., Trends Biochem. Sci., V. 6, p. 77 ( 1981)). In order for a liposome to be an efficient gene transfer vector, one or more of the following characteristics should be present: (1) encapsulation of the gene of interest at high efficiency with retention of biological activity; (2) preferential and substantial binding to a target cell, compared to non-target cells; (3) supply of the aqueous contents of the vesicle to the cytoplasm of the target cell at a high efficiency; and (4) the exact and effective expression of genetic information. 5

Liposomes can be directed against a particular tissue, by coupling the liposome to a specific ligand, such as a monoclonal antibody, sugar, glycolipid, or specific protein for the particular tissue or cell type (eg, synovial-specific surface proteins) . Additionally, the vector can be coupled to a target selection nuclear peptide, which will direct the cadherin-11 modulating nucleic acid molecule to the nucleus of the host cell. 10

Liposomes are commercially available from Gibco BRL, for example, as LIPOFECTINTM and LIPOFECTACETM, which are formed of cationic lipids such as N- [1- (2,3-dioleyloxy) -propyl] -N, N, N-trimethylammonium chloride (DOTMA) and dimethyldioctadecylammonium bromide (DDAB). Methods for obtaining liposomes are well known in the art, and have been described in many publications. Liposomes have been reviewed by Gregoriadis, G. in Trends in Biotechnology, V. 3, p. 235-241 (1985). fifteen

In general, cadherin-11 modulating nucleic acid molecules can be administered to the subject (any mammalian receptor) using the same modes of administration that are currently used for gene therapy in humans (eg, adenovirus-mediated gene therapy). . In U.S. Patent 5,399,346, and in the documents presented in the documented history of that patent, all of which are publicly available documents, a patented procedure for carrying out ex vivo gene therapy is outlined. In general, ex vivo gene therapy involves introducing in vitro a functional copy of a gene or fragment thereof into a cell or cells of a subject, and returning the genetically engineered cell or cells to the subject. The functional copy of the gene or fragment thereof is under the operable control of regulatory elements that allow the expression of the gene in the genetically engineered cell or cells. Accordingly, the nucleic acids described herein, which include cadherin-11 inhibitor nucleic acid molecules, can be delivered to synoviocytes or lymphocytes or other cells of the joint capsule ex vivo or in vivo, to treat an inflammatory disorder of the joints Numerous transfection and transduction techniques, as well as appropriate expression vectors, are well known to those of ordinary skill in the art, some of which are described in PCT Application WO 95/00654.

As an illustrative example, a vector containing a nucleic acid molecule is supplied to a site of joint inflammation in a subject that is a candidate for such gene therapy. Then, the vector genetically modifies the synoviocytes, osteoblasts, hematopoietic cells such as macrophages and NK cells, and / or lymphocytes in vivo with DNA encoding, for example, a cadherin-11 inhibitor polypeptide of the invention. Such genetically modified joint cells are expected to interfere in vivo with the binding of cadherin-11 to their counter receptor.

In an alternative embodiment, primary human synoviocytes can be obtained from a subject that is a candidate for such gene therapy. Then, such cells can be genetically manipulated ex vivo with DNA encoding, for example, a cadherin-11 inhibitor polypeptide of the invention. Such recombinant cells are expected to inhibit cadherin-11 mediated adhesion in vivo. In yet another example, another cell type that does not express either cadherin-11 or the cadherin-11 counterreceptor can be genetically engineered in vitro to express a cadherin-11 inhibitor, and then introduced into the site of inflammation.

Exemplary compositions that can be used to facilitate in vitro uptake of nucleic acid molecules 40 by a target cell include calcium phosphate and other chemical mediators of intracellular transport, compositions for microinjection, compositions for electroporation and homologous recombination (e.g., to integrate a nucleic acid into a preselected location within the chromosome of the target cell).

Unique isolated fragments of an isolated nucleic acid molecule selected from the group consisting of SEQ ID NO: 1 are also provided. A single nucleic acid fragment is one that is a "distinctive feature" of the larger nucleic acid. For example, the single fragment is long enough to ensure that its precise sequence is not found in molecules within the human genome outside the cadherin-11 nucleic acid molecules defined herein. Those of ordinary skill in the art can apply procedures that do not go beyond usual to determine if a fragment is unique within the human genome.

The invention further provides a pharmaceutical composition (ie, a pharmaceutical preparation) for modulating a cadherin-11 mediated function in a subject. The composition includes a pharmaceutically acceptable carrier and a cadherin-11 modulating agent (for example, a cadherin-11 inhibiting agent).

Pharmaceutical preparations, as described above, are administered in effective amounts. For therapeutic applications, it is generally that amount sufficient to achieve a medically desirable result. In general, a therapeutically effective amount is that amount necessary to delay the onset of, inhibit the progression of, or stop all along with the particular condition being treated. As an example, the effective amount is generally that amount that relieves the symptoms (eg, pain, inflammation, etc.) of the disorders described herein. The effective amount will depend on the mode of administration, the particular condition being treated, and the desired result. It will also depend on the stage of the condition, the severity of the condition, the age and physical condition of the subject to be treated, the nature of the concurrent therapy, if any, the duration of the treatment, the specific route of administration, and similar factors. within the knowledge and expertise of the doctor. For prophylactic applications, it is that amount sufficient to delay the onset of, inhibit the progression of, or stop all along with the particular condition that is prevented, and can be measured by the amount required to prevent the onset of symptoms. 5

Generally, the doses of the active compounds of the present invention would be from about 0.01 mg / kg per day to 1000 mg / kg per day, preferably from about 0.1 mg / kg to 200 mg / kg, and more preferably from about 0.2 mg / kg to about 20 mg / kg, in one or more daily administrations of the dose, for one or more days. It is expected that doses ranging from 1-500 mg / kg, and preferably doses ranging from 1-100 mg / kg, and even more preferably doses ranging from 1-50 mg / kg will be suitable. The preferred amount 10 can be determined by a person with normal skill in the art according to standard practice to determine optimal dosage levels of the agent. It is generally preferred to use a maximum dose of a cadherin-11 modulating agent which is the highest safe dose according to medical judgment.

The cadherin-11 modulating agents of the invention can be administered to a subject in need of such treatment in combination with concurrent therapy to treat an inflammatory joint disorder. Concurrent therapy may be invasive, such as the removal of fluid from the joint capsule, or it may involve drug therapy, such as the administration of non-steroidal anti-inflammatory drugs, anti-rheumatic drugs (e.g., gold and pencylamine), modulatory drugs of the immune system (for example, cyclosporine) and corticosteroid drugs. These drug therapies are well known to people with normal skill in the art, and are administered in ways known to those skilled in the art. Drug therapies are administered in 20 amounts that are effective in achieving physiological objectives (for example, to reduce inflammation in a joint), in combination with, for example, the cadherin-11 inhibitor of the invention. Thus, it is contemplated that drug therapies can be administered in amounts that are not able to prevent or reduce the physiological consequences of an inflammatory joint disorder when drug therapies are administered alone, but which are capable of reducing Consequences when administered in combination with the cadherin-11 modulating agents of the invention.

The cadherin-11 modulating agent can be administered alone or in combination with the drug therapies described above as part of a pharmaceutical composition. Such pharmaceutical composition may include the cadherin-11 modulating agent in combination with any standard physiological and / or pharmaceutically acceptable carrier known in the art. The compositions should be sterile and contain a therapeutically effective amount of the cadherin-11 modulating agent in a unit of weight or volume suitable for administration to a patient.

The term "pharmaceutically acceptable carrier", as used herein, means one or more compatible solid or liquid fillers, diluents or encapsulating substances, which are suitable for administration to a human or other animal. The term "pharmaceutically acceptable" means a non-toxic material that does not interfere with the efficacy of the biological activity of the active ingredients. Pharmaceutically acceptable also means a non-toxic material that is compatible with a biological system such as a cell, cell culture, tissue, or organism. The term "vehicle" represents an organic or inorganic ingredient, natural or synthetic, with which the active ingredient is combined to facilitate application. The characteristics of the vehicle will depend on the route of administration. The components of the pharmaceutical compositions can also be thoroughly mixed with the agents of the present invention, and among themselves, so that there is no interaction that substantially alters the desired pharmaceutical efficacy. The pharmaceutically acceptable carrier must be sterile for in vivo administration. Physiologically and pharmaceutically acceptable carriers include diluents, fillers, salts, buffers, stabilizers, solubilizers, and other materials that are well known in the art.

Compositions suitable for parenteral administration conveniently comprise a sterile aqueous preparation of cadherin-11 modulating agents, which is preferably isotonic with the blood of the recipient. This aqueous preparation can be formulated according to known methods using suitable dispersing or wetting agents, and suspending agents. The sterile injectable preparation may also be a sterile injectable solution or suspension in a nontoxic parenterally acceptable diluent or solvent, for example as a solution in 1,3-butanediol. Among the acceptable vehicles and solvents that can be employed are water, Ringer's solution, and isotonic sodium chloride solution. In addition, sterile fixed oils are conventionally used as solvent or suspension medium. For this purpose, any soft fixed oil, including synthetic mono- or diglycerides, can be used. In addition, in the preparation of injectables, fatty acids such as oleic acid can be used. Vehicle formulations suitable for oral, subcutaneous, intravenous, intramuscular, etc. administrations, can be found at Remington’s Pharmaceutical Sciences, Mack Publishing Co., 55 Easton, PA.

There are a variety of routes of administration. The particular mode selected will, of course, depend on the particular drug selected, the severity of the condition being treated, and the dose required for therapeutic efficacy. The methods of the invention, generally speaking, can be practiced using any mode of administration that is medically acceptable, meaning any mode that produces effective levels of the active compounds without causing clinically unacceptable side effects. Such modes of administration include oral, rectal, topical, nasal, intradermal, or parenteral routes. The term "parenteral" includes subcutaneous, intravenous, intramuscular, or by infusion. Intravenous or intramuscular routes are not particularly suitable for long-term therapy and prophylaxis. However, they may be preferred in emergency situations. For prophylactic treatment, oral administration will be preferred, for the convenience of the patient as well as the dosing schedule. In preferred embodiments, the pharmaceutical composition is administered directly to the synovium, synovial fluid or joint capsule by injection, preferably with a syringe.

Formulations for use according to the methods of the invention include a syringe containing a cadherin-11 modulating agent, such as a cadherin-11 inhibiting agent, and a pharmaceutically acceptable carrier that is suitable for injection into synovial fluid. 10

The pharmaceutical compositions may conveniently be presented in unit dosage form, and may be prepared by any of the methods well known in the art of pharmacy. All methods include the step of associating cadherin-11 modulating agents with a vehicle that constitutes one or more accessory ingredients. In general, the compositions are prepared by uniformly and intimately associating the cadherin-11 modulating agents with a liquid vehicle, a finely divided solid vehicle, or both, and then, if necessary, forming the product. Compositions suitable for oral administration may be presented as discrete units, such as capsules, tablets, tablets, each containing a predetermined amount of the cadherin-11 modulating agent. Other compositions include suspensions in aqueous liquids or non-aqueous liquids, such as a syrup, elixir or an emulsion.

In a particular embodiment, the preferred vehicle for delivery of the cadherin-11 modulating agents of the invention is a biocompatible microparticle or implant that is suitable for implantation in a joint or in the vicinity of a joint afflicted in the recipient. Exemplary bioerodible implants that are useful in accordance with this method are described in PCT International Application No. PCT / US / 03307 (Publication No. WO 95/24929, entitled "Polymeric Gene Supply System", which claims the priority of the Application for US Patent No. 213,668, filed March 15, 1994). PCT / US / 0307 describes a biocompatible polymer matrix, preferably biodegradable, to contain an exogenous gene under the control of an appropriate promoter. The polymer matrix is used to achieve sustained release of the exogenous gene in the subject. In accordance with the present invention, the cadherin-11 modulating agents described herein are encapsulated or dispersed within the biocompatible, preferably biodegradable, polymer matrix described in PCT / US / 03307. The polymeric matrix is preferably in the form of a microparticle, such as a microsphere (in which, for example, the inhibitor of cadherin-11 is dispersed along a solid polymeric matrix) or a microcapsule (in which, for example, the cadherin-11 inhibitory agent is stored in the core of a polymeric cortex). Other forms of the polymer matrix to contain the cadherin-11 modulating agent include films, coatings, gels, implants, and vascular stents. The size and composition of the polymer matrix device are selected to result in a favorable release kinetics in the tissue in which the matrix device is implanted. The size of the polymer matrix device is further selected according to the method of delivery to be used, typically injection into a tissue, such as the joint, joint capsule, synovial membrane or synovial fluid, or administration of an aerosol suspension. in the nasal and / or pulmonary areas. The polymer matrix composition can be selected to have both favorable degradation rates and also to be formed of a bioadhesive material, to further increase the transfer efficiency when the device is administered to the synovium of a joint. The matrix composition can also be selected not to degrade but rather to be released by diffusion over a prolonged period of time.

Both non-biodegradable and biodegradable polymer matrices can be used to deliver the cadherin-11 modulating agents, including the cadherin-11 inhibitors of the invention, to the subject. Biodegradable matrices are preferred. Such polymers can be natural or synthetic polymers. Synthetic polymers are preferred. The polymer is selected based on the period of time over which the release is desired, generally of the order of a few hours to a year or more. Typically, release over a period ranging from a few hours to three to twelve months is very desirable. The polymer is optionally in the form of a hydrogel, which can absorb up to about 90% of its weight in water, and is also optionally crosslinked with multivalent ions or with other polymers. fifty

In general, the cadherin-11 modulating agents of the invention are supplied using the bioerodible implant by means of diffusion, or more preferably by degradation of the polymer matrix. Exemplary synthetic polymers that can be used to form the biodegradable delivery system include: polyamides, polycarbonates, polyalkylenes, polyalkylene glycols, alkylene polyoxides, alkylene polyterephthalates, vinyl polyalcohols, vinyl polyethers, vinyl polyesters, vinyl polyhalides, polyvinyl polyglycosides, polyvinyl polydisyl polyhydrosides , 55 polyurethanes and their copolymers, alkyl cellulose, hydroxyalkyl celluloses, cellulose ethers, cellulose esters, nitrocelluloses, polymers of acrylic and methacrylic esters, methyl cellulose, ethyl cellulose, hydroxypropyl cellulose, hydroxypropyl methyl cellulose, hydroxybutyl methyl cellulose, cellulose acetate, cellulose acetate, cellulose acetate, cellulose acetate, cellulose acetate, cellulose acetate cellulose, cellulose acetate phthalate, carboxy ethyl cellulose, cellulose triacetate, sodium salt of cellulose sulfate, poly (methyl methacrylate), poly (ethyl methacrylate), poly (butyl methacrylate), poly (isobutyl methacrylate), 60 poly (met hexyl acrylate), poly (isodecyl methacrylate), poly (lauryl methacrylate), poly (phenyl methacrylate), poly (methyl acrylate), poly (isopropyl acrylate), poly (isobutyl acrylate), poly (acrylate) octadecyl), polyethylene, polypropylene, poly (ethylene glycol), poly (ethylene oxide), poly (ethylene terephthalate), poly (vinyl alcohols), vinyl polyacetate, polyvinyl chloride, polystyrene and polyvinyl pyrrolidone.

Examples of biodegradable polymers include synthetic polymers such as polymers of lactic acid and glycolic acid, polyanhydrides, poly (ortho) esters, polyurethanes, poly (butyl acid), poly (valeric acid), and poly (lactide-co-5 caprolactone) , and natural polymers such as alginate and other polysaccharides, including dextran and cellulose, collagen, chemical derivatives thereof (substitutions, additions of chemical groups, for example alkyl, alkylene, hydroxylations, oxidations, and other modifications customarily performed by skilled in the art), albumin and other hydrophilic proteins, ceine and other prolamines and hydrophobic proteins, copolymers and mixtures thereof. In general, these materials are degraded either by enzymatic hydrolysis or by exposure to water 10 in vivo, by surface or massive erosion.

Bioadhesive polymers of particular interest include bioerodible hydrogels (described by HS Sawhney, CP Pathak and JA Hubell in Macromolecules, 1993, 26, 581-587), hyaluronic polyacids, gelatin, glutin, polyanhydrides, acrylic polyacid, alginate, chitosan, poly ( methyl methacrylates), poly (ethyl methacrylates), poly (butyl methacrylate), poly (isobutyl methacrylate), poly (hexyl methacrylate), poly (isodecyl methacrylate), poly (lauryl methacrylate), 15 poly ( phenyl methacrylate), poly (methyl acrylate), poly (isopropyl acrylate), poly (isobutyl acrylate), and poly (octadecyl acrylate).

Examples of non-biodegradable polymers include ethylene-vinyl acetate, (meth) acrylic polyacid, polyamides, copolymers and mixtures thereof.

Other delivery systems may include time-release, delayed-release or sustained-release delivery systems. Such systems can avoid repeated administrations of the cadherin-11 modulating agents described above, increasing comfort for the patient and the physician. There are many types of delivery delivery systems, and they are known to people of ordinary skill in the art. They include the polymer systems described above, as well as systems based on polymers such as poly (lactide-glycolide), copolyloxalates, polycaprolactones, polyesteramides, polyorthoesters, hydroxybutyric polyacid, and polyanhydrides. The microcapsules of the above polymers containing drugs are described, for example, in U.S. Pat. 5,075,109. The delivery systems also include non-polymeric systems that are: lipids, including sterols such as cholesterol, cholesterol esters, and neutral fatty acids or fats, such as mono-, di- and triglycerides; hydrogel release systems; silastic systems; peptide based systems; wax coatings; pressed tablets using conventional binders and excipients; partially fused implants; and 30 similar. Specific examples include, but are not limited to: (a) eroding systems, in which the cadherin-11 modulating agent is contained in form within a matrix such as those described in U.S. Pat. Nos. 4,452,775, 4,675,189 and 5,736,152, and (b) diffusion systems, in which an active component permeates at a controlled rate from a polymer, as described in U.S. Pat. Nos. 3,854,480, 5,133,974 and 5,407,686. In addition, supply systems with pump-based hardware can be used, some of which are adapted for implantation.

The use of a long-term sustained-release implant may be particularly suitable for the treatment of chronic conditions. Long-term release, as used herein, means that the implant is constructed and mounted to deliver therapeutic levels of the active ingredient for at least 30 days, and preferably 60 days. Long-term sustained-release implants are well known to those of ordinary skill in the art, and 40 include some of the release systems described above.

Another aspect of the invention includes a screening test method to determine if a putative cadherin-11 inhibitor modulates cadherin-11 mediated adhesion. In certain embodiments, an in vitro adhesion assay is used as a screening assay to measure the ability of an agent, for example a pharmaceutical leader compound, an antibody or a fragment thereof, a member of a library or a peptide analog, for inhibit in vitro adhesion mediated by cadherin-11 between a first cell expressing cadherin-11 and a second cell expressing a counter-receptor of cadherin-11. The assay serves as a prognosis of the agent's ability to inhibit cadherin-11 mediated activity in vivo.

The binding partners in the adhesion assays are the particular ligands and receptors involved in cadherin-11 mediated adhesion. Accordingly, adhesion assays can be carried out in which the 50 binding partners are: (1) a cell expressing a cadherin, such as cadherin-11 (eg, synoviocytes, synovial-derived fibroblasts, osteoblasts and cells transfected with cadherin-11), and a cell that expresses a counter-receptor of cadherin-11 (for example, synoviocytes, synovial-derived fibroblasts, osteoblasts, T and B lymphocytes, plasma cells, macrophages, dendritic cells, mast cells, NK cells and cells transfected with the cadherin-11 counterreceptor); (2) an isolated cadherin-11 and a cell that expresses the counter receptor; (3) an isolated cadherin-11 counter receptor and a cell expressing cadherin-11; and (4) an isolated cadherin-11 polypeptide and its isolated counterreceptor. When isolated cadherin-11 or an isolated cadherin-11 counterreceptor are used, they may be present in immobilized form (for example, immobilized on a solid surface), or in soluble form.

As used herein with respect to adhesion assays, a cadherin-11 counterreceptor may be present as an isolated cadherin-11 counter-receptor, a functionally equivalent peptide fragment or analogue of the isolated cadherin-11 counter-receptor, or a cell that expresses extracellularly the counterreceptor or its functionally equivalent peptide fragment or analog. Similarly, cadherin-11 may be present as an isolated cadherin-11 polypeptide, a functionally equivalent peptide fragment or analog of cadherin-11, or an extracellularly expressing cadherin-11 cell or its functionally equivalent peptide fragment or analog . Cadherin-11 or its counter receptor can be immobilized on supports, such as microtiter plates or beads, using procedures known to the person of ordinary skill in the art.

In this way, a high-performance screening assay can be carried out to select pharmaceutical leader compounds, in which, for example, (1) cadherin-11 or the cadherin-11 counterreceptor is immobilized on the surface of a microtiter well, (2) aliquots of a molecular library containing library members are added to the wells, (3) cells expressing a cadherin-11 or cadherin-11 counterreceptor are added to the wells (as may be the case), and (4) the components of the wells are allowed to incubate for a period of time that is sufficient for the cells to bind to immobilized cadherin-11. Preferably, the cells are labeled (for example, pre-incubated with 51 Cr or a fluorescent dye) before being added to the 15 microtiter well. After the incubation period, the wells are washed to remove non-adherent cells, and the signal is determined (attributable to the marker in the adherent cells). To establish the maximum adhesion value, a positive control (for example, no library member is present) is used on the same microtiter plate. To establish the maximum levels of adhesion inhibition, a negative control (for example, soluble cadherin-11 added to the microtiter well) is used in the same microtiter plate. As an example, cadherin-11 can be immobilized to the surface, and the added cells can be T-cells. Consistent with other embodiments of the invention, the high-performance screening assay can also use a cadherin-11 and / or a counter-receptor. of isolated cadherin-11, which may be in immobilized or soluble form.

The agents to be screened can be pharmaceutical leader compounds synthesized in molecular libraries. These libraries can produce peptides (ie, peptide libraries) or small organic or inorganic molecules. Agents to be screened may also be peptide analogs of cadherin-11 or the cadherin-11 counterreceptor. Preferably, the cadherin-11 peptide or peptide analogue of the cadherin-11 counterreceptor corresponds to the portion of the polypeptide responsible for binding with its binding partner. For both polypeptides, this portion is extracellular.

According to yet another aspect of the invention, there is provided a method for screening a molecular library to identify pharmaceutical leader molecules that inhibit in vitro adhesion between a first cell expressing cadherin-11 and a second cell expressing a cadherin-11 counterreceptor . For example, as a screening assay, the ability of a molecule to inhibit the binding of a synovial cell to a T lymphocyte, or the binding of a synovial cell to another synovial cell in vitro, can be used to identify leader compounds that inhibit binding of cadherin-11 to its counter receptor. Such adhesion assays are well known in the art, and are illustrated by the assay provided in the Examples. 35

A preferred screening method involves carrying out an adhesion test between a first cell and a second cell in the presence or absence of at least one member of the molecular library, to determine whether the library member modulates adhesion between the first cell. and the second cell in vitro. Preferably, the first cell expresses cadherin-11, and the second cell expresses a counter-receptor of cadherin-11. This embodiment involves: (1) performing a first adhesion test between the first cell and the second cell, to obtain a first adhesion test result 40; (2) carry out a second adhesion test between the first cell and the second cell in the presence of the library member, to obtain a second adhesion test result; and (3) compare the first and second results of the adhesion test, to determine whether the library member modulates the adhesion between the first cell and the second cell. A difference between the first and second results of the adhesion test indicates the ability of the library member to modulate the junction between the first cell and the second cell, and thus the binding of cadherin-11 to its counter receptor. Thus, for example, an adhesion test result showing a reduced bond between the first cell and the second cell when the test is performed in the presence of a library member, compared to the result of the test obtained when the Assay is performed in the absence of the library member, indicates that the library member inhibits the binding of the first cell and the second cell. An exemplary adhesion test is provided in the Examples. Other such adhesion assays are well known in the art and can be developed and carried out using experimentation no more than usual. Thus, for example, the adhesion test can be carried out by replacing the first cell and the second cell with an isolated cadherin-11 and its isolated counterreceptor.

In yet another aspect, the invention provides a screening assay for the search for leading pharmaceutical compounds that modulate cell function through cadherin-11. These screening assays involve determining a first value for a cellular parameter (for example, cell proliferation) of a cell expressing cadherin-11 (for example, a synoviocyte) in the absence of a member of the molecular library, determining a second value for the same cellular parameter in a cell expressing cadherin-11 in the presence of a member of the molecular library, and comparing the first and second value of the cellular parameter as a measure of the effect of the molecular library member on that particular cellular parameter . As an example, a second value that is less than the first value 60 indicates a reduction in cell proliferation. Cell proliferation can be measured in many ways, well known to the normal expert, including, but not limited to, the incorporation of radioactive nucleotides (eg, thymidine uptake assays) and counting the cells. In U.S. Patent 6,077,833 describes an example of a T cell proliferation assay. In these assays, the cells to be used may be adhered to a solid surface, or they may be present in a suspension.

Likewise, the assay can also be carried out by measuring other cellular parameters, such as apoptosis, migration, binding and / or secretion of factors (eg, inflammatory factor). For apoptosis screening assays, cell counts can be used as a reading of the amount of apoptosis. In U.S. Patent No. 6,107,088 other apoptosis assays are described. For factor secretion assays, the supernatant in which cells exist can be tested for the presence of factors, using other functional assays or immunological assays (e.g., ELISA assays, bioassays, Western blots, RIA assays, etc. , to detect 10 particular cytokines). For example, IL-6 secretion can be measured using the supernatant in an assay that measures the cell growth of IL-6 dependent cells. The activation of general immune cells can also be measured, using assays such as those described in U.S. Pat. No. 5,569,585.

In some preferred embodiments, cellular function is a function other than the binding of cadherin-11 to a cadherin-11 counter receptor. Thus, the preferable pharmaceutical leader compound, according to this aspect of the invention, is one that enhances or inhibits a cellular function mediated by cadherin-11, such as proliferation, signaling, apoptosis, production or secretion of factors, migration or binding. , although it does not inhibit the binding of cadherin-11 to a counter-receptor of cadherin-11. Preferably, cadherin-11 modulating agents that do not inhibit the binding of cadherin-11 to its counter receptor are selected from libraries of small molecules, such as combinatorial chemistry libraries. twenty

In all the aforementioned tests, a preliminary binding screening is optionally initially carried out before the screening tests described herein. Such preliminary binding screening would enrich and in some cases identify leading pharmaceutical compounds that bind cadherin-11 or its counterreceptor. These preliminary tests would not necessarily measure the inhibitory or modulating capacity of the signal of the identified compounds, but rather would serve to reduce the number of compounds to be screened later. Thus, as an example, initially a molecular library can be contacted with cadherin-11 or a cadherin-11 counter-receptor (preferably immobilized), and library members that bind cadherin-11 or a counter-receptor of Cadherin-11 would subsequently be screened for its ability to inhibit the binding of cadherin-11 to its counter-receptor, or to modulate the cellular function of cells expressing cadherin-11. Since the agents that modulate cell function do so in cells expressing cadherin-11, the preliminary screening appropriate for these agents would be the binding to cadherin-11. For inhibitors of the invention, preliminary screening can test for binding to cadherin-11 or a cadherin-11 counterreceptor.

The cadherin-11 modulating agents of the invention can be synthesized from peptides or other biomolecules, including but not limited to, saccharides, fatty acids, sterols, isoprenoids, purines, pyrimidines, derivatives or structural analogs of the foregoing, or combinations of the same and similar. 35 phage display libraries and chemical combinatorial libraries can be used to develop and select synthetic compounds that are modulating agents and / or inhibitors of cadherin-11. The use of agents obtained from peptoids, random bio-oligomers (US Patent 5,650,489), benzodiazepines, diversomers such as didantoins, benzodiazepines and dipeptides, non-peptide peptidomimetics with a beta-D-glucose framework is also contemplated in the invention. , oligocarbamates or peptidyl phosphonates. 40

The agents of the invention can be mass produced using library technology. In some aspects, the methods of the invention use this library technology to subsequently generate and identify small molecules, including small peptides, that bind to a cadherin-11 or a cadherin-11 counterreceptor. An advantage of the use of libraries is the easy manipulation of millions of different putative candidates of small size in small reaction volumes (ie, in synthesis and screening reactions). Another advantage of the libraries is the ability to synthesize agents that otherwise may not be obtainable using sources of natural origin, particularly in the case of non-peptide moieties.

A "molecular library" refers to a collection of structurally diverse molecules. Molecular libraries can be chemically synthesized or can be produced recombinantly. As used herein, a "member of a molecular library" refers to a molecule that is present in the molecular library. In general, a molecular library 50 contains from two to 1012 molecules, and any integer between them, for example 2, 3, 4, 5, 10, 102, 103, 104, 105, 106, 107, 108, 109, 1010 , 1011, 1012, etc., as if each and every integer has been cited here.

Methods for preparing molecule libraries are well known in the art, and many libraries exist commercially. The libraries of interest in the invention include peptide libraries, randomized oligonucleotide libraries, synthetic organic combinatorial libraries, and the like. Degenerated peptide libraries can easily be prepared in solution, in immobilized form as peptide presentation libraries of bacterial flagella, or as phage display libraries. Peptide ligands can be selected from combinatorial libraries of peptides containing at least one amino acid. Libraries can be synthesized from peptoids and synthetic non-peptide moieties. In addition, such libraries containing synthetic non-peptide moieties that are less subject to enzymatic degradation can be synthesized compared to their naturally occurring counterparts. The libraries 60 also include, for example, but are not limited to, peptide in plasmid libraries, polysomal libraries, aptamer libraries, synthetic peptide libraries, libraries of small synthetic molecules and chemical libraries. The libraries may also comprise a cyclic carbon structure or a heterocyclic structure and / or aromatic or polyaromatic structures substituted with one or more of the functional groups identified above.

Many of these agents of the invention can be synthesized using recombinant or chemical library approaches. 5 A vast set of agents can be generated from libraries of synthetic or natural compounds. There are, or can be easily produced, libraries of natural compounds in the form of bacterial, fungal, vegetable and animal extracts. Naturally and synthetically produced libraries and compounds can be easily modified through conventional chemical, physical, and biochemical means. Known cadherin-11 binding partners can be subjected to random or directed chemical modifications such as acylation, alkylation, esterification, amidification, etc. 10 to produce structural analogues of these binding partners, which can function as agonists or antagonists.

Libraries include recombinantly produced libraries of fusion proteins. An exemplary recombinantly produced library is prepared by ligating cadherin-11 cDNA fragments into, for example, the pGEX-2T vector (Pharmacia, Piscataway, NJ). This vector contains the carboxy terminus of glutathione S-transferase (GST) from Schistosoma japonicum. The use of the GST-containing vector facilitates the purification of GST fusion proteins with cadherin-11 from bacterial lysates by affinity chromatography on glutathione-sepharose. After elution from the affinity column, cadherin-11 fusion proteins with GST are evaluated for activity, for example by contacting at least one fusion protein with a cell expressing cadherin-11 before (or concurrently ) of contacting the cell expressing the cadherin-11 counterreceptor with a cell expressing cadherin-11. Fusion proteins that inhibit binding between cells expressing the cadherin-11 counterreceptor and cells expressing cadherin-11 are selected as pharmaceutical leader compounds. These proteins are also useful in the further characterization of the portion of cadherin-11 to which the counterreceptor binds. See, for example, Koivunen E. et al. (1993) J. Biol. Chem. 268 (27): 20205 describing the selection of peptides that bind to integrin α5β1 from a phage display library.

Synthetic DNA and RNA libraries are also commonly used in the art. For example, Ellington and Szostak 25 describe the use of random nucleotide libraries to identify new ligands (Ellington and Szostak, Nature, 346, 818-822 (1990)). As an example, modifications can be made that create variants of cadherin-11 at the level of the nucleic acid sequence encoding a cadherin-11 polypeptide. Amino acid substitutions can be made by PCR-directed mutation, site-directed mutagenesis according to the Kunkel method (Kunkel, Proc. Nat. Acad. Sci. USA 82: 488-492, 1985), or by chemical synthesis of the molecules of nucleic acid encoding cadherin-11 or a counter-receptor of cadherin-11.

As described in U.S. 5,908,609, exemplary library compounds also include, but are not limited to, peptides such as, for example, soluble peptides, including but not limited to members of random peptide libraries (see, for example, Lam, KS et al. 1991, Nature 354: 82-84; Houghten, R et al. 1991, Nature 354: 84-86), and molecular libraries derived from combinatorial chemistry obtained from amino acids of configuration D and / or 35 L, phosphopeptides (including , but not limited to, members of randomized or partially degenerated, directed phosphopeptide libraries (see, for example, Songyang, Z. et al. 1993, Cell 72: 767-778), antibodies (including, but not limited to, polyclonal, monoclonal, humanized, anti-idiotypic, chimeric or single-chain antibodies, or fragments of Fab, F (ab ') 2 and Fab expression libraries (and their epitope-binding fragments), and small organic or inorganic molecules Other compounds that can be n screening according to the invention includes but is not limited to small organic molecules.

Compounds of the invention that can be designed to meet the above criteria include polypeptides and peptidomimetics. The peptidomimetic may be a hybrid molecule that includes both amino acid and non-amino acid components, for example the mimetic may include amino acid components for positively charged and negatively charged regions, and a non-amino acid (e.g., piperidine) having the same Approximate size and dimension of a hydrophobic amino acid (eg, phenylalanine) as a hydrophobic component.

Combinatorial libraries of small molecules can also be generated. A combinatorial library of small organic compounds is a collection of closely related analogs that differ from each other at one or more points of diversity, and are synthesized by organic techniques using multistage processes. Combinatorial libraries include a vast number of small organic compounds. A type of combinatorial library is prepared by means of parallel synthesis methods, to produce a set of compounds. A "set of compounds," as used herein, is a collection of compounds identifiable by their spatial directions in Cartesian coordinates and arranged so that each compound has a common molecular core and one or more elements of varying structural diversity. The compounds in such a set of compounds are produced in parallel in separate reaction vessels, each compound being identified and followed by its spatial direction. The 55 examples of parallel synthesis mixtures and parallel synthesis methods are provided in U.S.S.N. 08 / 177,497, filed on January 5, 1994, and its corresponding PCT Published Patent Application WO95 / 18972, published on July 13, 1995, and U.S. Pat. No. 5,712,171, granted on January 27, 1998, and the corresponding PCT Published Patent Application WO 96/22529.

Thus, the invention provides, in part, low molecular weight compounds that modulate cadherin-11 mediated functions. These compounds can be used to modulate the function of cadherin-11, or they can be used as the leading compounds for the design of better compounds using computer-based rational drug design methods.

One skilled in the art will be familiar with methods for predicting the effect on protein conformation of a change in the protein sequence, and can thus "design" a variant that functions as a modulating agent according to known methods. An example of such a method is described by Dahiyat and Mayo in Science 278: 82-87, 1997, which describe de novo protein design. The method can be applied to a known protein to vary only a portion of the polypeptide sequence. By applying the computational methods of Dahiyat and Mayo, specific variants of cadherin-11 or a counter-receptor of cadherin-11 can be proposed and tested, to determine whether variant 10 retains a desired conformation. Similarly, Blake (U.S. Patent No. 5,565,325) teaches the use of known ligand structures to predict and synthesize variants with a similar or modified function.

Other methods for preparing or identifying peptides that bind to a particular target are known in the art. For example, molecular printing can be used for de novo construction of macromolecular structures, such as peptides, that bind to a particular molecule. See, for example, Kenneth J. Shea, Molecular Imprinting of 15 Synthetic Network Polymers: The De Novo synthesis of Macromolecular Binding and Catalytic Sites, TRIP Vol. 2, No. 5, May 1994; Klaus Mosbach, Molecular Imprinting, Trends in Biochem. Sci., 19 (9) January 1994; and Wulff, G., in Polymeric Reagents and Catalysts (Ford, W. T., Ed.) ACS Symposium Series No. 308, p. 186-230, American Chemical Society (1986). As an example, a method for preparing cadherin-11 counterreceptor mimetics involves the steps of (i) polymerizing functional monomers around a known substrate (the mold, or, in this case, the cadherin-11 counter-receptor binding domain ) that shows a desired activity; (ii) remove the template molecule; and then (iii) polymerize a second class of monomers in the empty space left by the mold, to provide a new molecule that shows one or more desired properties that are similar to those of the mold. In addition to preparing peptides in this manner, other binding molecules such as polysaccharides, nucleosides, drugs, nucleoproteins, lipoproteins, carbohydrates, glycoproteins, steroids, lipids, and other biologically active materials can also be prepared. This method is useful for designing a wide variety of biological mimetics that are more stable than their natural counterparts, because they are typically prepared by polymerization of functional monomers by free radicals, resulting in a compound with a non-biodegradable main chain. Other methods for designing such molecules include, for example, the design of drugs based on structure activity relationships, which require the synthesis and evaluation of a number of compounds and the formation of molecular models.

As mentioned above, the invention further encompasses agents comprising peptidomimetic moieties, including amino acids that are not naturally occurring. Such variants can be synthesized by substituting amino acid residues involved in functions mediated by cadherin-11 with peptidomimetic moieties. For example, glutamine (Glu) moieties can be substituted for α-aminoadipate molecules, and tyrosine positions can be substituted for 4-carboxymethyl-Phe. In the variants, phosphorus-based and non-phosphorus analogs, such as phosphorotyrosine mimetics, can be used. Tyrosine analogs that can be used instead of tyrosine residues include phenylalanine (Phe), pentafluorophenylalanine (PfPhe), 4-carboxymethyl-L-phenylalanine (cmPhe), 4-carboxydifluoromethyl-L-phenylalanine (F2cmPhe), 4- phosphonomethylphenylalanine (Pmp), (difluorophosphonomethyl) phenylalanine (F2Pmp), O-malonyl-L-tyrosine (malTyr or OMT), and fluoro-O-malonyl tyrosine (FOMT). Synthetic phosphonate-based mimetic agonists and antagonists can also be incorporated into substituents, which replace a methylene unit with the tyrosyl phosphate ester linkage. Additionally, the glutamic acid moieties can be modified to have an additional methylene group, or they can simply be replaced by α-amino-adipate (Adi). Other moieties that can be used include the non-naturally occurring amino acid 1-aminocyclohexylcarboxylic acid (Ac6c) and 3- (2-hydroxynaphthalen-1-yl) -propyl acid, or 2-azetidinecarboxylic acid or pipecolic acid (which have annular structures of 6 members and 4 members, 45 respectively) for proline residues, S-ethylisothiourea, 2-NH2-thiazoline and 2-NH2-thiazole. Also useful in the synthesis of variants is the use of substitutes for asparagine residues, such as 3-indolyl-propyl. It will be apparent to a person of ordinary skill in the art that the invention encompasses the synthesis of a wide variety of variants that have any combination of amino acid analogs and / or peptidomimetic moieties as described above and as are known in the art. Other potential modifications provided for by the invention include modifications of 50 cysteines, histidines, lysines, arginines, tyrosines, glutamines, asparagine, prolines, and carboxylic groups, as are well known in the art and are described in US 6,037,134. The synthesis of the variants mentioned above is described in the cited references, and they are perfectly within the field of the person of normal skill in the art.

Variants can also be modified to introduce or stabilize certain structural features. As an example, β flexures can be incorporated into the variants, preferably peptides, or the variants can be synthesized as cyclic peptides, for example by incorporating thioether bonds.

The screening methods of the invention provide useful information for the rational pharmaceutical design of new agents that are capable, for example, of modulating an immune system response. In Saragovi, H. et al., (1992) Biotechnology 10: 773; Haber E., (1983) Biochem. Pharmacol 32 (13): 1967; and Connolly Y., (1991) Methods of 60 Enzymology 203, Cap. 29 “Computer-Assisted Rational Drug Design” p. 587-616, exemplary procedures for rational pharmaceutical design are provided.

In this way, knowledge of the primary, secondary or tertiary structures of naturally occurring ligands and receptors can be used to rationally choose or design molecules that will bind with the ligand or receptor. In particular, knowledge of the ligand and receptor binding regions can be used to rationally choose and design compounds that are more potent than naturally occurring ligands in causing their normal response, or that are competitive inhibitors of the ligand-receptor interaction.

Once chosen or rationally designed and selected, library members can be altered, for example in the primary sequence, to produce new and different peptides. These fragments can be produced by site-directed mutagenesis, or they can be synthesized in vitro. These new fragments can then be tested for their ability to bind to the receptor or ligand, and, by varying their primary sequences and observing the effects, peptides with an increased binding or inhibitory capacity can be produced.

Alternatively, the nucleotide and amino acid sequences of the cadherin-11 modulating agents of the present invention, for example those corresponding to the extracellular domain of cadherin-11, can be used in computer-based model formation systems, for predict the secondary and tertiary structure of the extracellular domain 15. Such computer-based systems are well known to those of normal skill in the art of rational drug design. Based on the tertiary structure of a receptor protein, it is often possible to identify a binding region that is involved in its biological activity. From this information, peptides or other compounds that include or mimic this structure, and / or are capable of binding to it can be rationally designed. In this way, new compounds can be designed that mimic the activity of the receptor or ligand, or that will act as competitive inhibitors of the receptor or ligand.

Those skilled in the art will appreciate that various modifications of the above peptide analogs can be made without departing from the essential nature of the invention. Accordingly, it is intended that peptides that include conservative substitutions and coupled proteins in which a peptide of the invention is coupled to a solid support, such as a polymeric bead, a carrier molecule, such as Californian limpet hemocyanin, or a group The informant, such as a radiolabel or other tag, is also encompassed within the teachings of the invention.

As used herein, "conservative amino acid substitution" refers to an amino acid substitution that does not alter the relative loading or size characteristics of the peptide in which the amino acid substitution is performed. Conservative amino acid substitutions include substitutions made between amino acids within the following 30 groups: (a) MILV; (b) FYW; (c) KRH; (d) AG; (its T; (f) QN; and (g) ED.

Monoclonal antibodies of the invention that inhibit the binding of cadherin-11 to its counterreceptor are also useful in screening assays to identify pharmaceutical leader compounds in molecular libraries. The antibodies of the invention specifically bind a cadherin or a cadherin counterreceptor, and thereby inhibit the binding of these two molecules to each other. Thus, screening assays using monoclonal antibodies 35 are also useful for assessing the ability of a library molecule to inhibit the binding of cadherin-11 to its counter receptor. Such antibody-based screening assays are performed by contacting an antibody (which specifically binds cadherin-11 and, for example, inhibits adhesion between a T lymphocyte and a cell expressing cadherin-11 with cadherin-11 in the presence and absence of at least one member of the molecular library, and determining whether the library member modulates the binding between the antibody and cadherin-11 In a particularly preferred embodiment, cadherin-11 is presented as a cell expressing cadherin -11, an isolated cadherin-11, or an isolated peptide related to, or derived from, the extracellular domain of cadherin-11. Similarly, the cadherin-11 counterreceptor can be presented in any one of these forms.

In a particularly preferred embodiment in which the antibody is an anti-cadherin-11 antibody, the antibody screening method involves: (1) conducting a first antibody test in the presence of the antibody and cadherin-11 and in the absence of a library molecule, to obtain a first test result with antibodies; (2) carry out a second antibody test in the presence of the antibody, cadherin-11 and the library molecule, to obtain a second antibody test result; and (3) compare the first and second results of the antibody assays, to determine if the molecular library member modulates the binding between the antibody and cadherin-11. According to this embodiment, a reduced binding between the antibody and cadherin-11 in the presence of the library member indicates that the library member has inhibited the binding of the antibody to cadherin-11. Antibody binding assays can also be used to assess the relative ability of a member of the molecular library to block the binding between a specific antibody for the cadherin-11 counterreceptor and the counterreceptor, using experimentation not beyond usual. .

These and other screening assays can also be used to identify the functionally equivalent cadherin-11 peptide analogs of the invention that are useful for inhibiting the binding of cadherin-11 to its counter receptor.

Cadherin-11 modulating agents can also be used, for example, to direct a toxin (e.g., ricin) or a detectable agent (e.g., a radiolabel, a fluorescent marker, an enzymatic marker) to cells that express counterreceptors of cadherin-11 or cadherin-11. Methods for coupling such toxins and / or agents to proteins and / or antibodies for in vivo and in vitro applications are described, for example, in Killen and Lindstrom (1984), "Specific killing of lymphocytes that cause experimental Autoimmune Myestenia Gravis by toxin -acetylcholine receptor conjugates ”, J. Immun. 133: 1335; Jansen, F.K., et al. (1982), "Immunotoxins: Hybrid molecules combining high specificity and potent cytotoxicity", Immunolog. Rev. 62: 185-216, see also U.S. Pat. Nos. 3,652,761; 5 4,478,946 and 4,554,088.

The invention will be more fully understood by reference to the following examples. However, these examples are simply intended to illustrate the embodiments of the invention and should not be construed as limiting the scope of the invention. It is also understood that the reference figures are illustrative only and are not essential for carrying out the claimed invention. 10

Examples

The importance of catenins in the mediation of cell-to-cell adhesion dependent on cadherins suggests that they can be used as probes to identify new cadherins. Thus, for these studies, antisera were obtained against both α- and human β-catenin, and were used to identify coprecipitating proteins in 15 human type B synoviocytes, since it was not known that this cell type expressed a cadherin. In addition, based on the alignments of human E, P and N cadherin, four regions of identity could be seen. Using these regions, sense and antisense oligonucleotides were synthesized, and PCR was carried out under normal restriction conditions using as RNA template derived from human type B synoviocytes. The clones derived from the PCR were then sequenced to identify those that were sequences of Authentic cadherin Such candidate clones were then used as probes in Northern blot analysis using synoviocyte RNA.

Prior to the present invention, it had not been reported that cadherins were expressed in synoviocytes. The present invention is based, at least in part, on the discovery that synovial-derived fibroblast-like cells and synovial membrane lining cells express a cadherin. The gene coding for this cadherin was cloned, monoclonal antibodies against it were obtained, and its expression was demonstrated in transfectant cells, cultured human synoviocytes and in recently isolated human rheumatoid synovium.

Example 1

Material and methods:

Antibodies Antisera for catenin P were previously described (Cepek
KL et al. Proc Natl Acad Sci USA 93: 6567-71, 1996). The pan-cadherin antiserum directed against the 24 C-terminal amino acids of 30-chicken N-cadherin was previously described (Geiger B. et al. J Cell Sci 97: 607-14, 1990) and was obtained from Sigma (St. Louis, MO). The specific mouse monoclonal antibodies (mAbs) against human cadherin E (E4.6, IgG1) were obtained in this laboratory (Cepek
KL et al. Nature 372: 190-3, 1994), the human anti-cadherin N (13A9, IgG1) was kindly supplied by M. Wheelock (Department of Biology, Toledo University, Toledo OH), the human anti-cadherin VE (BV9, IgG1 ) was a gift from MG Lampugnani (Laboratory of Vascular Biology, Mario Negri Institute, Milano, Italy), and the human anti-cadherin P (NCC-CAD-299, IgG1) was provided by S. Hirohashi (Division of Pathology, National Cancer Research Institute, Tokyo, Japan), Leu 4 (anti-CD3) from Becton Dickinson (Mountain View, CA), OKT4 (anti-CD4), OKT8 (anti-CD8) from Ortho Pharmaceutical Corp. (Raritan, NJ), and anti-CD68 from Dako (Carpenteira, CA).

Cell culture. Synovial membranes from patients with RA diagnosed based on current criteria (Arnett
FC. et al. Arthritis Rheum 31: 315-324, 1988) were obtained during surgical procedures of synovectomy of the hand and wrist, and joint replacement. Synovial tissue was prepared by itching, and treated with 1 mg / ml collagenase (type 1, Worthington Biochemicals, Frehold, NJ), 0.15 mg / ml Dnase I (Sigma, St. Louis, MO) and 5 nM of CaCl2 in phosphate buffered saline (PBS) (Gibco, Grand Island, NY), and stirred at 37 ° C for 1 hour. The cell suspension was passed through a 40 mesh metal sieve, and placed in a 75 cm 2 tissue culture flask (BD Labware, Lincoln Park, NJ). Cells released from synovial tissue were seeded in flasks in 45 DME supplemented with 10% fetal calf serum and 10% human serum, 2 mM L-glutamine, 1 mM sodium pyruvate, 100 U / ml penicillin, 100 µg / ml streptomycin sulfate, and 50 µM of 2-mercaptoethanol, and were maintained in 10% CO2. It was found that the confluent monolayers were composed mainly of type II synoviocytes (similar to fibroblasts) after the third pass.

The murine fibroblastic L cell line (ATCC CCL1.3) was grown in DMEM, high in glucose with 10% of 50 bovine calf serum (Hyclone), 10 mM HEPES, 2 mM L-glutamine, 1 mM sodium pyruvate, 10 µM of non-essential amino acids (Gibco BRL, Gaithersburg, MD), 100 U / ml of penicillin, 100 µg / ml of streptomycin sulfate, and 50 µM of 2-mercaptoethanol, and kept at 10% CO2 . L cell transfectants were grown in the anterior medium with G418 (Gibco), at 1 mg / ml.

Human embryonic kidney cells HEK293 (obtained from ATCC) were maintained in 10% (vol / vol) of heat-inactivated FBS 55 (Hyclone Labs Inc., Logan, UT) and DME medium (Gibco BRL) at 37 ° C in CO2 at 10%

Cos-7 cells were grown in DMEM, high glucose with 10% Nu-Serum serum (Collaborative Research, Inc., Bedford, MA), 10 mM HEPES, 2 mM L-glutamine, 1 mM pyruvate sodium, 100 U / ml penicillin, 100 µg / ml streptomycin sulfate, and 50 µM 2-mercaptoethanol, and were maintained in 10% CO2.

Human breast epithelial cells 16E6.A5 were maintained as described (Cepek
KL et al. Proc Natl Acad Sci USA 93: 6567-71, 1996). 5

T lymphocytes were isolated from PMBC derived from Ficoll-Hypaque density gradient, using anti-CD3 (or anti-CD2 mAb) and magnetic beads. They can be used directly after release using Detach-a-bead, or they can be stimulated with PHA and cultured in medium containing IL-2, such as long-term T-cell lines. Recent isolated lymphocytes will be used in adhesion assays to synoviocyte monolayers. Adhesion will be examined in static cell-to-cell assays in 96-well plates using fluorescently labeled lymphocytes. The percentage of fluorescently labeled T cells that bind to synoviocyte monolayers is determined using a plate reader by fluorescence, and the effects of specific mAb blocking can be determined.

Marking and Immunoprecipitation. 1 x 107 confluent synoviocyte monolayers were labeled on the surface with 2 mCi of Na125I (Du Pont-New England Nuclear, Boston, MA) using lactoperoxidase and hydrogen peroxide in 0.5 ml of PBS as previously described (Brenner, et al. 1987). The cells were solubilized in lysis buffer containing 15 Tris buffered saline (TBS, 50 mM Tris base, pH 7.6, 140 mM NaCl) with 1% Triton X-100, 8 mM iodoacetamide (IAA ) and 1 mM phenylmethylsulfonyl fluoride (PMSF, Sigma, St. Louis, MO) 1 mM CaCl 2, for 1 h at 4 ° C. The material insoluble in the detergent was removed by centrifugation for 20 minutes at 8,000 g, at 4 ° C. The supernatant was pre-rinsed with 6 µl of normal rabbit serum and 300 µl of mAb 187.1 as a culture supernatant for 30 minutes, followed by two rounds (1 h and 12 h) of 200 µl of a 10% cell suspension 20 (weight / volume) of Cowphy strain I of Staphylococcus aureus fix (PANSCORBIN, Calbiochem, San Diego, CA). Lysates containing the equivalent of 2 x 106 cells were immunoprecipitated with 10-15 µl of rabbit antiserum or 15 µl of β-catenin antiserum or 1 µl of E4.6 ascites, and 100 µl of each of the supernatants of mAb 13A9, BV9, Cad-299, 2G4, 5H6, 3H10 plus 100 µl of rat mouse mouse mAb 187.1 culture supernatant, for optimal protein binding A. Then, the immune complexes were incubated with protein A-Sepharose (Pharmacia 25 Biotechnology Inc., Piscataway, NJ) for 1 h at 4 ° C with stirring. The immunoprecipitates were washed three times with 0.5% (vol / vol) Triton X-100 in 0.5 M NaCl with 50 mM, 1 mM CaCl2. The resin pellet was boiled in sample buffer (10% glycerol, 3% SDS, 0.5 M Tris, pH 6.8) containing 2-mercaptoethanol (5% final concentration, reducing conditions), and It was analyzed by 7.5% SDS-PAGE as described (Laemmli UK. Nature 227: 680-5, 1970), and subjected to standard fluorographic procedures. 30

Molecular Cloning of the Gene Encoding the Synovial Cadherin. A number of new cadherin cDNA clones have been isolated using PCR-based consensus oligonucleotides that correspond to cytoplasmic domain sequences that are highly conserved among cadherins (Suzuki S. et al. Cell Reg 2: 261-70, 1991) . Based on the alignments of human cadherins, four identity regions can be seen, corresponding to residues 753-762 (EEGGGEEDQD) (SEQ ID NO: 3), remains 840-847 (SLSSLNSS) (SEQ ID NO: 4) , residues 853-859 35 (QDYDYLN) (SEQ ID NO: 5), and remains 865-875 (FKKLADMYGGG) (SEQ ID NO: 6) of human cadherin E, which in each case are identical to cadherins E, P and N. Using these regions, the following sense degenerate oligonucleotides were designed: 5'-GCGGGATCCGAIGARGGIGGNGGNGA-3 '(SEQ ID NO: 7) and antisense: 5'GGGGAGCTCTCIGCIARYTTYTIRAA-3' (N = A, T, G, C; I = Inosine; Y = C, T and R = A, G) (SEQ ID NO: 8). Rheumatoid synoviocyte mRNA was extracted, and reverse transcribed and used as a template for PCR amplification using 0.5 U / Taq polymerase reaction and the following conditions: denaturation at 95 ° C for 30 seconds, hybridization at 60 ° C for 1 minute, extension at 72 ° C for 1 minute, for 30 cycles. PCR products of the expected size of 385 bp were cloned into plasmid pCRII using the TA cloning system (Invitrogen Corp., Carlsbad, CA), and subsequently sequenced with the Sequenase kit (United Stated Biochemical Corp., Cleveland, OH) to identify those that appear to be authentic cadherin sequences. 45 The GenBank database was searched using the FASTA / BLAST program.

Northern transfer. To confirm the expression in synoviocytes of the cloned cadherin, a Northern blot analysis was carried out. MRNA, isolated from type B synoviocytes, 16A5 cells, Jurkat cells and a SKNSH, was hybridized with the 385 bp fragment of the cloned cadherin by PCR, and with glyceraldehyde-3-phosphate dehydrogenase as a control, both labeled with [32P] dCTP / dGTP by random priming. Hybridization was carried out at 43 ° C for 50 16 h. After hybridization, the membrane was washed at a final restriction of 0.1 x SSC with 2% (w / v) SDS at 56 ° C, and autoradiographed on a Kodak MS film at -70 ° C.

Construction of the Full Length Clone. Full length cadherin-11 was constructed by PCR using the following primers XV14 (5'-CCAAAAATGAAGGAGAACTACT-3 ') (SEQ ID NO: 9) and XV15 (5'-GGGGGATCCATTGTTAAGAATCGTCATCAAA-3') (SEQ ID NO: 10), comprising the coding region of cadherin-11. The ,42.4 kb product was subcloned into the Hind III / Bam HI sites of pBluescript SK, and sequenced to confirm that no mutations were introduced during PCR.

Construction of the Cadherina-11-Fc Expression Vector. A double stranded DNA adapter was produced, containing a blunt end in Msc I in 5 ', and the final five codons of the extracellular region of human cadherin-11, and a cohesive end Xho I in 3', hybridizing the complementary oligonucleotides XVCad11A (5'-GCTGGCACCGTGGTTGGGAGAGT-3 ') (SEQ ID NO: 11) and XVCad11 E (5'-GGGGGGCTCGAGGTAGGCCTCTGCGTTGCAGG-3') (SEQ ID NO: 12) using Pyrococcus furiostage (PFU J) according to Strat (PFU J) Manufacturer's recommendations. This adapter was then ligated to the 3 ′ end of a Hind III-Msc I fragment that encodes the rest of the extracellular region of human cadherin-11 from the full length clone of previously generated cadherin-11. The resulting Hind III-Xho I fragment was introduced into the 5 'direction of the coding of the hinge and Fc region of human IgG1, in a derivative of pCDM8 (pCDMBFc), also cleaved with Hind III-Xho I. The sequence of the binding region is shown in Figure 3. The construct was sequenced to confirm its integrity in the binding region using an automated DNA sequencer (Perkin Elmer). Finally, the cadherin-11-Fc cDNA was cut from pCDM8 using Hind III and Not 10 I, and inserted into the expression vector pCEP4 (Invitrogen Corp. Carlsbad, CA), cleaved with Hind III and Not I.

Cadherin-11-Fc protein production. HEK293 cells (106 cells per 75 cm2 flask) were stably transfected with 20 µg of plasmid DNA, using the mammalian transfection kit (Stratagene). After growing for 24 h in non-selective medium, the cells were transferred to 96-well tissue culture plates and incubated in selective medium containing 200 µg / ml of hygromycin B. After 15 days, the supernatants from Wells containing resistant colonies were evaluated for fusion proteins by ELISA.

To produce cadherin-11-Fc protein, positive clones were grown in 500 cm2 triple layer flasks (Nunc, Roskilde, Denmark) in 10% (v / v) Ultralow Ig FBS (GIBCO BRL), 200 µg / ml of hygromycin B and DMEM. After 7-10 days of culture, the supernatant was harvested and filtered through a 0.2 µm membrane. The cadherin-11-Fc fusion protein was then purified on a previously unused GammaBind G-Sepharose column (Pharmacia Biotech, Piscataway, NJ). The column was washed with TBS and 1 mM CaCl2, pH 7.4, and then eluted with 0.2 M glycine and 1 mM CaCl2, pH 2.3. Fractions containing purified fusion protein were dialyzed in TBS and 1 mM CaCl2, pH 7.4, and then stored at -20 ° C. The purity of the Coomassie blue staining, and the concentration was determined by the Bradford assay using BSA as a standard (BioRad Labs., 25 Hercules, CA).

Generation of Stable Cadherin-11 Transfectants with L. Cells To produce cells expressing cadherin-11 on the surface, L cells were transfected with 20 µg of pLK / Cad11 or with the neo pLK vector alone, using the mammalian transfection kit (Stratagene) Then, the transfected cells were selected by culture in 1 mg / ml of G418. 30

Generation of Mouse Monoclonal Antibodies vs. Cadherina-11. 2G4, 5H6 and 3H10 mAbs were produced by immunizing BALB / c mice with three intraperitoneal injections of 20 µg of purified cadherin-11-Fc. The initial injection was in CFA, and the subsequent 2 were in IFA, at intervals of 2 weeks, followed by a final IV revaccination of 30 µg. Three days after intravenous immunization, splenocytes were isolated and fused with murine myeloma NS1 cells, in the presence of PEG (molecular weight 1450), as previously described (Lerner EA. Yale J Biol Med 54: 387-402 . 1981). Hybridomas were selected with medium containing aminopterin, and hybridoma supernatants were screened by differential ELISA on plates coated with 301-Fc, E-cad-Fc and human IgG1. Subsequent screening was performed in the clones selected by FACS on cells expressing cadherin-11, compared to cells positive for cadherin E. The selected hybridomas were subcloned three times by limiting dilution. The 2G4 (IgG1), 5H6 (IgG1) and 3H10 (IgG1) isotypes were determined by ELISA using murine isotype specific mAb (Jackson Immunoresearch Lab).

Immunohistochemistry Normal human tissue samples were obtained and quickly frozen in OCT compound (Ames Co., Elkart, IN) cooled with liquid nitrogen at about -140 ° C. The frozen tissue sections were selected (6 µm) with a CM 10800 cryostat (Leica Inc. Deerfield, IL), and then fixed in acetone for 10 minutes, dried briefly in air, and stained using an immunoperoxidase method indirect, using the avidin-biotin-peroxidase complex (Vector Labs, Burlingame, CA) and 3-amino-9-ethylcarbazole (Sigma, St. Louis, MO) as a chromogen.

Results

Cloning of the Cadinoin of Synoviocytes. PCR-based cloning of degenerated oligonucleotides of a cadherin into cDNA of type B synoviocytes showed a specific synoviocyte product that was absent in the negative control (Figure 1). This product was identified as a cadherin-11 fragment when sequenced and compared to the GenBank database. The expression of cadherin-11 in synovium-derived RNA was then confirmed by Northern blotting, using as a probe the 385 bp fragment cloned by PCR. Figure 2 shows the positive hybridization of the cad-11 probe in type B synoviocytes and in a positive control, a neuroblastoma cell line (SKNSH), and was absent in epithelial cells (16EA5) and Jurkat cells. In order to obtain the full length gene, a clone comprising the entire human cadherin-11 coding sequence was generated by PCR. The PCR product obtained was sequenced to confirm the lack of mutations.

Production of Human Cadherin-11-Fc Fusion Protein. A construct encoding the extracellular portion of human cadherin-11 was linked in the framework to a construct encoding the Fc region of IgG1 (including hinge domains, CH2, and CH3). The transfection of HEK293 cells and the selection with hygromycin B led to the generation of stable strains expressing the soluble fusion protein of cadherin-11-Fc. Cadherin-11-Fc fusion protein is expected to be dimer, due to the presence of disulfide bonds in (Figure 3), possibly similar to the 5 dimer of cadherin on the cell surface (Shapiro L. et al. Nature 374: 327-37, 1995; Nagar BM. Et al. 380: 360-4, 1996). After purification in G-Sepharose protein, SDS-PAGE revealed the presence of a protein of the expected size (∼123 kD), after reduction.

Generation of Anti-Cadherin-11 Monoclonal Antibodies. To produce mAbs that specifically recognize the extracellular region of cadherin-11, BALB / c mice were immunized with the purified fusion protein of cadherin-10 11-Fc. MAbs that reacted preferentially with cadherin-11-Fc but not with human IgG1 or with cadherin E-Fc by ELISA were selected for further study. Three of these mAb antibodies 3H10, 5H6 and 2G4 specifically recognized cadherin-11-Fc in ELISA. Also, these mAbs stain cadherin-11 expressed on the surface of full-length cadherin-11 transfected L cells, as shown in Figure 4. These three mAbs also precipitated a 123 kD protein from the labeled synoviocytes in the surface. fifteen

In order to study the pattern of protein distribution in the tissue, a direct immunohistochemistry was performed on frozen tissue sections. The remarkable staining pattern was observed, in which anti-cadherin-11 mAb preferentially stained the lining cells in the RA synovium. This suggests that cadherin-11 plays an important role in determining the adhesion of the synovial lining cells to each other, and thus in determining the tissue architecture of the synovium. This role can be critical for growth and proliferation as well as for the activation of synovial membrane cells. Note that synovial membranes lack an epithelial layer, and instead have the synovial lining layer. Interestingly, these anti-cadherin-11 mAbs also recognized a few cells in the subcoat adjacent to the areas of T cells that infiltrate the synovium (Figure 6, panels A and B).

Example 2: Adhesion Tests 25

Monolayers of adherent cells (ie, human synoviocytes) were grown in flat-bottom 96-well Linbro tissue culture plates. 104 adherent cells in 100 µl of complete medium were added per well, and allowed to grow for two to three days until they reached confluence. Just before the addition of T cells or COS-7 cells transfected with the cadherin-11 counterreceptor, the adherent cell monolayers were washed with assay medium. To label the T-cells or COS-7 cells, 25 µg of 2 ', 7'-bis- (2-carboxyethyl) -5 (and 6) -30 carboxyfluorescein (BCECF-AM, Molecular Probes, Inc., Eugene) was diluted , OR) in 5 µl of DMSO, and were added to a 5 X 10 6 / ml suspension of T cells or COS-7 cells in complete medium. The cells were incubated at 37 ° C for 25 minutes, and then washed twice in assay medium (PBS containing 1 mM CaCl 2, 2 mM MgCl 2 and 10 mM HEPES). After washing, 50,000 T cells or labeled COS-7 cells, in 100 µl of test medium, were added to the adherent cell monolayers. T cells or COS-7 cells were allowed to settle on 35 adherent cell monolayers for 25 or 40 minutes at 37 ° C. Unbound cells were removed by changing the plate medium. Bound cells were detected using a fluorescence plate reader (IDEXX Co., Portland, ME). If blocking with the antibody, T cells, COS-7 cells, or synoviocyte cell monolayers were pre-incubated with a 1: 250 dilution of ascites fluid or 10 µg / ml of purified mAb for five minutes at 37 ° C before the encounter with the second type of cells. At least four aftershocks were carried out. The% 40 of bound cells is calculated by reading the fluorescence units obtained after unbound cells were washed off, dividing this number among the input fluorescence units, and multiplying by 100. Serial dilutions of labeled cells showed as few as 1000 cells are detected in the linear range.

Example 3: An adhesion assay to select members of the library as pharmaceutical leader compounds.

The adhesion assay described here is based on the assay described by Cepek, K., et al., In J. Immunol. 150 (8): 3459-3470 (1993).

To screen a molecular library or other mixture to determine the presence of a functionally equivalent peptide analog or a member of the library capable of inhibiting cadherin-11 mediated adhesion, T cells are washed with 50 HBSS (Hank buffered saline, Gibco ) and are pre-equilibrated with serial dilutions, containing HBSS, of the library solution or of another solution that contains peptides or that contains small molecules (over a wide range of concentrations (for example, 1 ng / ml a 100 ug / ml) for selected times (for example, 30 min., 1 hour, 2 hours, 6 hours) at 37 ° C before incubation with synovial monolayers that have been washed with HBSS. Functionally equivalent peptide analogs or inhibitory agents of cadherin-11 are identified in search of their ability to inhibit the binding of T cells to the synovial monolayer.

Example 4: Cadherin-11 counterreceptor in T and B cells

A flow cytometric assay of the synovial T cell line 5 and CP-B cells was carried out with a 2G4 anti-cadherin-11 monoclonal antibody. Control staining was carried out with the P3 control monoclonal antibody. Figure 6 demonstrates the lack of staining of the T or B cell line with the cadherin-11 specific monoclonal antibody.

To test the binding of cadherin-11 to T and B cells, line 5 of synovial T cells and CP-B cells were used. The 5 plates were treated with cadherin-11-Fc or ICAM-1-Fc and IgG1. Both cell lines were labeled with a fluorescent dye and added to the plates, and the input fluorescence in a plate reader was determined by fluorescence. The cells were allowed to adhere for 30 minutes at 37 ° C, washed subsequently, and the ratio of fluorescence bound cells was determined. The percentage of bound cells was determined using the equation: fluorescence after washing / input fluorescence x 100. The binding to cadherin-11-Fc but not to the control proteins ICAM-1-FC and IgG1 is blocked by the antibody monoclonal 7D3 anti-cadherin-11. The results are expressed as the mean + 1 SD (n = 4), as shown in Figure 7.

Several test lines support the possibility that cadherin-11 binds to a counterreceptor expressed in B and T lymphocytes. First, the immunohistochemical analysis of synovium RNA demonstrated the expression of cadherin-11 in cells of the sub-coating in close contact with lymphocytes Secondly, some T 15 cell lines derived from synovium with RA and peripheral blood derived B cells specifically bind cadherin-11-Fc, and this interaction is blocked by anti-cadherin-11 mAbs. Third, cadherin-11 was not expressed in these leukocyte strains, suggesting that the interaction is not mediated by homophilic binding but by a heterophile binding to a cadherin-11 receptor (C11CR) expressed in B and T cells.

Discussion 20

The presence of a cadherin expressed in synoviocytes is described here for the first time. Cadherins are expressed in all cells that form solid tissues, and are responsible for segregating and classifying cells during tissue morphogenesis. They play a role in establishing cell polarity and maintaining tissue morphology in adult tissues. Although cadherins function classically to mediate homophilic cell-to-cell adhesion, they sometimes bind to non-identical cadherins or integrins, such as integrin αEβ7. 25

The findings of the preferred expression of cadherin-11 in the synovial lining with RA suggests that this adhesion molecule could be the key molecule used by the invasive cloth to bind to the cartilage and eventually erode the bone, particularly since it has been given to Know that this cadherin is expressed by osteoblasts. This is of particular relevance because it can interfere with the chronic destructive process, characteristic of RA, if the adhesive function of cadherin-11 can be modulated. 30

The identification of cadherin-11 from human type B synoviocytes is considered an important finding, and its tissue distribution in the synovium with RA supports this relevant role in the invasion and eventually erosion of the adjacent bone.

  SEQUENCE LIST

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Claims (26)

1.-Use of a therapeutically effective amount of a cadherin-11 inhibitor that inhibits the binding of cadherin-11 to a cadherin-11 counter-receptor that is cadherin-11, in the manufacture of a medicament for treating a subject having an inflammatory joint disorder, wherein the cadherin-11 inhibitor is an antibody or fragment thereof, a cadherin-11 polypeptide or fragment thereof, or an Fc fusion protein comprising an extracellular domain of cadherin-11, in which the cadherin-11 inhibitor selectively binds cadherin-11. 2. Use according to claim 1, wherein the inflammatory joint disorder is either chronic synovitis or an autoimmune disease such as rheumatoid arthritis. 10 3. Use according to claim 1, wherein the cadherin-11 inhibitor agent is administered locally to a synovium of the subject. 4. Use according to claim 1, wherein the cadherin-11 inhibitor is an antibody. 5. Use according to claim 4, wherein the antibody is a monoclonal antibody directed against the extracellular domain of cadherin-11. fifteen 6. Use according to claim 1, wherein the cadherin-11 inhibitor is a soluble cadherin-11 polypeptide. 7. Use according to claim 1, wherein the cadherin-11 is expressed by a cell selected from the group consisting of a synoviocyte type A, a synoviocyte type B, a fibroblast derived from synovium, a cell lining the membrane synovial, an osteoblast, a cell derived from cartilage, and a cell derived from the invasive cloth. twenty 8.-A method for screening a molecular library to identify a leading pharmaceutical compound that modulates cadherin-11 mediated adhesion, the method comprising carry out a first in vitro adhesion test between cadherin-11 and a cadherin-11 counterreceptor, to obtain a first result of the in vitro adhesion test, carrying out a second in vitro adhesion test between cadherin-11 and the cadherin-11 counterreceptor in the presence of at least one member of the molecular library, to obtain a second in vitro adhesion result, and compare the first and second results of the in vitro adhesion test to determine whether the at least one member of the molecular library modulates cadherin-11 mediated adhesion, in which one or both of cadherin-11 and the cadherin-11 counterreceptor they are isolated, and in which the cadherin-11 counterreceptor is cadherin-11, a fusion protein of cadherin-11 with Fc, or an extracellular domain of cadherin-11. 30 9. The method of claim 8, wherein cadherin-11 is isolated. 10. The method of claim 8, wherein cadherin-11 is presented by a cell. 11. The method of claim 10, wherein the cell is selected from the group consisting of a type A synoviocyte, a type B synoviocyte, a synovium-derived fibroblast, a synovial membrane lining cell, an osteoblast, a cell derived from cartilage, and a cell derived from the invasive cloth. 35 12. The method of claim 8, wherein the cadherin-11 counterreceptor is isolated. 13. The method of claim 8, wherein the cadherin-11 counterreceptor is presented by a cell. 14. The method of claim 8, wherein cadherin-11 is soluble. 15. The method of claim 8, wherein the cadherin-11 counterreceptor is soluble. 16. The method of claim 8, wherein cadherin-11 is immobilized. 40 17. The method of claim 8, wherein the cadherin-11 counterreceptor is immobilized. 18. The method of claim 8, wherein the molecular library is recombinantly produced or chemically synthesized. 19. The method of claim 8, wherein the molecular library is a peptide library. 20.-A composition comprising a fusion protein of cadherin-11 with Fc, which comprises an extracellular domain of human cadherin-11 from SEQ ID No. 2 fused to a human Fc domain, for use as a medicament. 21.-A pharmaceutical composition for use in the treatment of an inflammatory joint disorder, which comprises a cadherin-11 inhibitor that inhibits the binding of cadherin-11 to a cadherin-11 counter-receptor that is cadherin-11, in wherein the cadherin-11 inhibitor is an antibody or fragment thereof, a cadherin-11 polypeptide or fragment thereof, or an Fc fusion protein comprising an extracellular domain of cadherin-11, in which the agent Cadherin-11 inhibitor selectively binds cadherin-11. 5 22. The pharmaceutical composition of claim 21, wherein the inflammatory joint disorder is chronic synovitis or an autoimmune disease such as rheumatoid arthritis. 23. The pharmaceutical composition of claim 21, wherein the treatment comprises administering the cadherin-11 inhibitor agent locally to a synovium of a subject having inflammatory joint disorder. 24. The pharmaceutical composition of claim 21, wherein the cadherin-11 polypeptide or fragment thereof is a soluble cadherin-11 polypeptide. 25. The pharmaceutical composition of claim 21, wherein the cadherin-11 inhibitor is an antibody. 26. The pharmaceutical composition of claim 25, wherein the antibody is a monoclonal antibody directed against an extracellular domain of cadherin-11.