Compounds and methods for modulating junctional adhesion molecule-mediated functions

Abstract

Methods for using modulating agents to enhance or inhibit junctional adhesion molecule (JAM)-mediated cell adhesion in a variety of in vivo and in vitro contexts are provided. The modulating agents comprise at least one JAM cell adhesion recognition sequence or an antibody or fragment thereof that specifically binds thee JAM cell adhesion recognition sequence. Modulating agents may additionally comprise one or more cell adhesion recognition sequences recognized by other adhesion molecules. Such modulating agents may, but need not, be linked to a targeting agent, drug and/or support material.

What is claimed is:  
     
         1 . A cell adhesion modulating agent that: 
 (a) comprises a JAM CAR sequence; and    (b) contains 3-16 amino acid residues linked by peptide bonds.    
     
     
         2 . A modulating agent that: 
 (a) comprises at least five consecutive amino acid residues of a JAM CAR sequence having the formula:    Ser—Phe—Thr—Ile—Asp—Pro—Lys—Ser—Gly (SEQ ID NO:1); and    (b) contains no more than 50 consecutive amino acid residues present within the JAM.    
     
     
         3 . A modulating agent that: 
 (a) comprises at least seven consecutive amino acid residues of a JAM CAR sequence having the formula:    Ser—Phe—Thr—Ile—Asp—Pro—Lys—Ser—Gly (SEQ ID NO:1); and    (b) contains no more than 50 consecutive amino acid residues present within the JAM.    
     
     
         4 . A modulating agent that: 
 (a) comprises at least eight consecutive amino acid residues of a JAM CAR sequence having the formula;    Ser—Phe—Thr—Ile—Asp—Pro—Lys—Ser—Gly (SEQ ID NO:1); and    (b) contains no more than 50 consecutive amino acid residues present within the JAM.    
     
     
         5 . A modulating agent according to  claim 2 , wherein the agent is a peptide ranging in-size from 3 to 50 amino acid residues.  
     
     
         6 . A modulating agent according-to  claim 1  or  claim 2 , wherein the agent is a peptide ranging in size from 4 to 16 amino acid residues.  
     
     
         7 . A modulating agent according to  claim 1  or  claim 2 , wherein the agent comprises an N-terminal acetyl group.  
     
     
         8 . A modulating agent according to  claim 1  or  claim 2 , wherein the CAR sequence is present within a cyclic peptide.  
     
     
         9 . A modulating agent according to  claim 8 , wherein the cyclic peptide has the formula:  
       
         
           
           
               
               
           
         
         wherein X 1 , and X 2  are optional, and if present, are independently selected from the group consisting of amino acid residues and combinations thereof in which the residues are linked by peptide bonds, and wherein X 1  and X 2  independently range in size from 0 to 10 residues, such that the sum of residues contained within X 1  and X 2  ranges from 1 to 12;  
         wherein Y 1  and Y 2  are independently selected from the group consisting of amino acid residues, and wherein a covalent bond is formed between residues Y 1  and Y 2 ; and  
         wherein Z 1  and Z 2  are optional, and if present, are independently selected from the group consisting of amino acid residues and combinations thereof in which the residues are linked by peptide bonds.  
       
     
     
         10 . A modulating agent according to  claim 9 , wherein Y 1  and Y 2  are covalently linked via a disulfide bond.  
     
     
         11 . A modulating agent according to  claim 10 , wherein Y 1  and Y 2  are each independently selected from the group consisting of penicillamine, β,β-tetramethylene cysteine, β,β-pentamethylene cysteine, β-mercaptopropionic acid, β,β-pentamethylene-β-mercaptopropionic acid, 2-mercaptobenzene, 2-mercaptoaniline and 2-mercaptoproline.  
     
     
         12 . A modulating agent according to  claim 10 , wherein Y 1  and Y 2  are cysteine residues.  
     
     
         13 . A modulating agent according to  claim 9 , wherein Y 1  and Y 2  are covalently linked via an amide bond.  
     
     
         14 . A modulating agent according to  claim 13 , wherein the amide bond is formed between terminal functional groups.  
     
     
         15 . A modulating agent according to  claim 13 , wherein the amide bond is formed between amino acid residue side-chains.  
     
     
         16 . A modulating agent according to  claim 13 , wherein the amide bond is formed between one terminal functional group and one amino acid residue side chain.  
     
     
         17 . A modulating agent according to  claim 13 , wherein: 
 (a) Y 1  is lysine or ofnithine and Y 2  is aspartate or glutamate; or    (b) Y 2  is lysine or ornithine and Y 1  is aspartate or glutamate.    
     
     
         18 . A modulating agent according to  claim 9 , wherein Y 1  and Y 2  are covalently linked via a thioether bond.  
     
     
         19 . A modulating agent according to  claim 9 , wherein Y 1  and Y 2  are each tryptophan or a derivative thereof, such that the covalent bond generates a δ 1 δ 1 -ditryptophan, or a derivative thereof.  
     
     
         20 . A polynucleotide encoding a modulating agent according to  claim 1  or  claim 2 .  
     
     
         21 . An expression vector comprising a polynucleotide according to  claim 20 .  
     
     
         22 . A host cell transformed or transfected with an expression vector according to  claim 21 .  
     
     
         23 . A modulating agent comprising an antibody or antigen-binding fragment thereof that specifically binds to a JAM CAR sequence and modulates a JAM-mediated function, wherein the JAM CAR sequence comprises the sequence:  
       Ser—Phe—Thr—Ile—Asp—Pro—Lys—Ser—Gly (SEQ ID NO:1).  
     
     
         24 . A modulating agent comprising a mimetic of a JAM CAR sequence that comprises at least three consecutive amino acid residues of a JAM CAR sequence having the formula:  
       Ser—Phe—Thr—Ile—Asp—Pro—Lys—Ser—Gly (SEQ ID NO:1);  wherein the mimetic is capable of modulating a JAM-mediated function.    
     
     
         25 . A modulating agent comprising a mimetic of a JAM CAR sequence that comprises at least five consecutive amino acid residues of a JAM CAR sequence having the formula:  
       Ser—Phe—Thr—Ile—Asp—Pro—Lys—Ser—Gly (SEQ ID NO:1);  wherein the mimetic is capable of modulating a JAM-mediated function.    
     
     
         26 . A modulating agent according to any one of claims  1 ,  2  or  23  linked to a drug.  
     
     
         27 . A modulating agent according to any one of claims  1 ,  2  or  23  linked to a detectable marker.  
     
     
         28 . A modulating agent according to any one of claims  1 ,  2  or  23  linked to a targeting agent.  
     
     
         29 . A modulating agent according to any one of claims  1 ,  2  or  23  linked to a support material.  
     
     
         30 . A modulating agent according to  claim 29 , wherein the support material is a polymeric matrix.  
     
     
         31 . A modulating agent according to  claim 29 , wherein the support material is selected from the group consisting of plastic dishes, plastic tubes, sutures, membranes, ultra thin films, bioreactors and microparticles.  
     
     
         32 . A cell adhesion modulating agent according to any one of claims  1 ,  2  or  23 , further comprising one or more of: 
 (a) a cell adhesion recognition sequence that is bound by an adhesion molecule other than a JAM, wherein the cell adhesion recognition sequence is separated from any JAM CAR sequence(s) by a linker; and/or  
 (b) an antibody or antigen-binding fragment thereof that specifically binds to a cell adhesion recognition sequence bound by an adhesion molecule other than a JAM.  
 
     
     
         33 . A cell adhesion modulating agent according to  claim 32 , wherein the adhesion molecule is selected from the group consisting of integrins, cadherins, occludin, A claudins; members of the immunoglobulin family such as N-CAM and PECAM; fibronectin, laminin and other extracellular matrix proteins.  
     
     
         34 . A pharmaceutical composition comprising a adhesion modulating agent according to any one of claims  1 ,  2  or  23 , in combination with a pharmaceutically acceptable carrier.  
     
     
         35 . A composition according to  claim 34 , further comprising a drug.  
     
     
         36 . A composition, according to  claim 34 , wherein the cell adhesion modulating agent is present within a sustained-release formulation.  
     
     
         37 . A composition according to  claim 34 , further comprising one or more of: 
 (a) a peptide comprising a cell adhesion recognition sequence that is bound by an adhesion molecule other than a JAM; and/or    (b) an antibody or antigen-binding fragment thereof that specifically binds to a cell adhesion recognition sequence bound by an adhesion molecule other than a JAM.    
     
     
         38 . A composition according to  claim 37 , wherein the adhesion molecule is selected from the group consisting of integrins, cadherins, occludin, claudins, members of the immunoglobulin family such as N-CAM, PECAM, fibronectin, laminin and other extracellular matrix proteins.  
     
     
         39 . A modulating agent according to  claim 1  or  claim 2 , wherein the agent comprises a linear peptide having the sequence SFTIDPKSG (SEQ ID NO:1).  
     
     
         40 . A method for modulating cell adhesion, comprising contacting a JAM-expressing cell with a cell adhesion modulating agent according to  claim 1  or  claim 2 .  
     
     
         41 . A method for increasing vasopermeability in a mammal, comprising administering to a mammal a cell adhesion modulating agent according to  claim 1  or  claim 2 , wherein the modulating agent inhibits JAM-mediated cell adhesion.  
     
     
         42 . A method for reducing unwanted cellular adhesion in a mammal, comprising administering to a mammal a cell adhesion modulating agent according to  claim 1  or  claim 2 , wherein the modulating agent inhibits JAM-mediated cell adhesion.  
     
     
         43 . A method for enhancing the delivery of a drug through the skin of a mammal, comprising contacting epithelial cells of a mammal with a cell adhesion modulating agent according to  claim 1  or  claim 2  and a drug, wherein the modulating agent inhibits JAM-mediated cell adhesion, and wherein the step of contacting is performed under conditions and for a time sufficient to allow passage of the drug across the epithelial cells.  
     
     
         44 . A method for enhancing the delivery of a drug to a tumor in a mammal, comprising administering to a mammal a cell adhesion modulating agent according to  claim 1  or  claim 2  and a drug, wherein the modulating agent inhibits JAM-mediated cell adhesion.  
     
     
         45 . A method for treating cancer in a mammal, comprising administering to a mammal a cell adhesion modulating agent according to  claim 1  or  claim 2 , wherein the modulating agent inhibits JAM-mediated cell adhesion.  
     
     
         46 . A method for inhibiting angiogenesis in a mammal, comprising administering to a mammal a cell adhesion modulating agent according to  claim 1  or  claim 2 , wherein the modulating agent inhibits JAM-mediated cell adhesion.  
     
     
         47 . A method for enhancing drug delivery to the central nervous system of a mammal, comprising administering to a mammal a cell adhesion modulating agent according to  claim 1  or  claim 2 , wherein the modulating agent inhibits JAM-mediated cell adhesion.  
     
     
         48 . A method for enhancing wound healing in a mammal, comprising contacting a wound in a mammal with a cell adhesion modulating agent according to  claim 1  or  claim 2 , wherein the modulating agent, enhances JAM-mediated cell adhesion.  
     
     
         49 . A method for enhancing adhesion of foreign tissue implanted within a mammal, comprising contacting a site of implantation of foreign tissue in a mammal with a cell adhesion modulating agent according to  claim 1  or  claim 2 , wherein the modulating agent enhances JAM-mediated cell adhesion.  
     
     
         50 . A method for inducing apoptosis in a JAM-expressing cell, comprising contacting a JAM-expressing cell with a cell adhesion modulating agent according to  claim 1  or  claim 2 , wherein the modulating agent inhibits JAM mediated cell adhesion.  
     
     
         51 . A method for modulating monocyte traffic in a mammal, comprising administering to a mammal a cell adhesion modulating agent according to  claim 1  or  claim 2 , and thereby modulating monocyte traffic in the mammal.  
     
     
         52 . A method for detecting the presence of JAM-expressing cells in a sample, comprising: (a) contacting a sample with a modulating agent according to  claim 23  under conditions and for a time sufficient to allow formation of a modulating agent-JAM complex; and (b) detecting the level of modulating agent-JAM complex, and therefrom detecting the presence of JAM-expressing cells in the sample.  
     
     
         53 . A kit for detecting the presence of JAM-expressing cells in a sample, comprising: (a) a modulating agent according to  claim 23;  and (b) a detection reagent.  
     
     
         54 . A kit for enhancing transdermal drug delivery, comprising: (a) a skin patch; and (b) a cell adhesion modulating agent-according to  claim 1  or  claim 2 , wherein the modulating agent inhibits JAM-mediated cell adhesion.