Anti-tumour agents and methods of identifying anti-tumour agents

Abstract

The FNIII13 domain of fibronectin and smaller fragments thereof have a tumour cell proliferation inhibitory effect. Compositions are provided comprising fragments of fibronectin having the FNIII 13 domain and fragments thereof. A system comprising cells exposed to fibronectin and caused to proliferate by the presence of tenascin are used as an in vitro method for screening possible anti-tumour agents. Cell-free systems comprising a fibronectin ligand and tenascin are also employed for screening potential anti-tumour or anti-cancer agents. Test compounds are assayed for the ability to disrupt binding of the fibronectin ligand to tenascin. A further cell-free system additionally includes a syndecan molecule.

1 . A composition comprising a polypeptide as set forth in SEQ ID NO:1 or a portion or variant thereof, wherein said portion interferes with tenascin binding to fibronectin, for use as a pharmaceutical.  
     
     
         2 . A composition as claimed in  claim 1  for the treatment or prophylactic treatment of tumourigenesis or cancer.  
     
     
         3 . A composition as claimed in  claim 1  for the treatment or prophylactic treatment of an immune related diseases, e.g. rheumatism, asthma, allergic diseases, autoimmune diseases, and/or transplant rejection.  
     
     
         4 . A composition as claimed in  claim 1  for the treatment or prophylactic treatment of thrombosis.  
     
     
         5 . A composition as claimed in  claim 1  for the treatment or prophylactic treatment of atherosclerosis.  
     
     
         6 . A composition as claimed in  claim 1  for use in wound healing.  
     
     
         7 . A composition as claimed in  claim 1  for the treatment or prophylactic treatment of any disease or condition dependent on the interaction of tenascin with fibronectin.  
     
     
         8 . A composition as claimed in  claim 1 , comprising said portion, said portion comprising the first five amino acids of the amino acid sequence set forth in SEQ ID NO:3, wherein Xaa is any amino acid.  
     
     
         9 . A composition as claimed in  claim 8 , wherein said portion comprises the amino acid sequence set forth in SEQ ID NO:3 or a fragment or variant thereof, wherein Xaa is any amino acid.  
     
     
         10 . A composition as claimed in  claim 8 , wherein said portion is a polypeptide having the amino acid sequence set forth in SEQ ID NO:4 or a fragment or variant thereof.  
     
     
         11 . A composition as claimed in  claim 8 , wherein said portion is a polypeptide having the amino acid sequence set forth in SEQ ID NO:2 or a fragment or variant thereof.  
     
     
         12 . A composition as claimed in  claim 8 , wherein said portion is free of any other fibronectin domain or module.  
     
     
         13 . A composition as claimed in  claim 1 , further comprising a pharmaceutically acceptable excipient, diluent or carrier.  
     
     
         14 . A composition as claimed in  claim 1 , wherein said portion is capable of restoring syndecan signalling.  
     
     
         15 . A composition as claimed in  claim 14 , wherein the syndecan signalling is mediated by syndecan-4.  
     
     
         16 . A composition as claimed in  claim 1 , wherein said portion binds to tenascin.  
     
     
         17 . A composition as claimed in  claim 1 , wherein said portion binds to tenascin C.  
     
     
         18 . A composition as claimed in  claim 1  wherein said portion further binds to syndecan, preferably syndecan 4.  
     
     
         19 . A composition as claimed in  claim 1 , wherein said portion competes with the 13th fibronectin type III repeat of the native fibronectin protein for tenascin binding.  
     
     
         20 . A composition as claimed in  claim 1 , wherein said portion is recombinant.  
     
     
         21 . A polypeptide as set forth in SEQ ID NO:1 or a portion or variant thereof, wherein said portion interferes with tenascin binding to fibronectin, for use as a pharmaceutical.  
     
     
         22 . A portion of the polypeptide as set forth in SEQ ID NO: 1, said portion having any one or more of the features of said portions referred to  claim 8 , for use as a pharmaceutical.  
     
     
         23 . A use of the polypeptide of  claim 21  or a portion or variant thereof for the manufacture of a medicament for the treatment or prophylactic treatment of tumourigenesis or cancer.  
     
     
         24 . The use of the polypeptide of  claim 21  or a portion or variant thereof, for the manufacture of a medicament for the treatment or prophylactic treatment of an immune related disease, e.g. rheumatism, asthma, allergic diseases, autoimmune diseases, or transplant rejection.  
     
     
         25 . The use of the polypeptide of  claim 21  or a portion or variant thereof, for the manufacture of a medicament for the treatment or prophylactic treatment of thrombosis.  
     
     
         26 . The use of the polypeptide of  claim 21  or a portion or variant thereof, for the manufacture of a medicament for the treatment or prophylactic treatment of atherosclerosis.  
     
     
         27 . The use of the polypeptide of  claim 21  or a portion or variant thereof, for the manufacture of a medicament for use in wound healing.  
     
     
         28 . The use of the polypeptide of  claim 21  or a portion or variant thereof, for the manufacture of a medicament for the treatment or prophylactic treatment of any disease or condition dependent on the interaction of tenascin with fibronectin.  
     
     
         29 . The use as claimed in  claim 23 , wherein the tumour or tumour cells express tenascin, preferably tenascin C.  
     
     
         30 . The use as claimed in  claim 29 , wherein the expression of tenascin is at least two-fold greater than that in non-tumour tissue or cells.  
     
     
         31 . The use as claimed in  claim 23 , wherein the tumour is a solid tumour.  
     
     
         32 . The use as claimed in  claim 23 , wherein the tumour is mesenchymal or epithelial cancer.  
     
     
         33 . The use as claimed in  claim 23 , wherein the tumour is a glioblastoma or breast carcinoma.  
     
     
         34 . A method for the treatment or prophylactic treatment of tumourigenesis or cancer, comprising administering an effective amount of a polypeptide as set forth in SEQ ID NO:1 or a portion or variant thereof, wherein said portion interferes with tenascin binding to fibronectin.  
     
     
         35 . The method as claimed in  claim 34  for the treatment or prophylactic treatment of an immune related disease, e.g. rheumatism, asthma, allergic diseases, autoimmune diseases, or transplant rejection.  
     
     
         36 . The method as claimed in  claim 34  for the treatment or prophylactic treatment of thrombosis.  
     
     
         37 . The method as claimed in  claim 34  for the treatment or prophylactic treatment of atherosclerosis.  
     
     
         38 . The method as claimed in  claim 34  for use in wound healing.  
     
     
         39 . The method as claimed in  claim 34  for the treatment or prophylactic treatment of any disease or condition dependent on the interaction of tenascin with fibronectin.  
     
     
         40 . The method as claimed in  claim 35 , wherein said portion has the amino acid sequence set out in SEQ ID NO:2, SEQ ID NO:3 or SEQ ID NO:4.  
     
     
         41 . The method as claimed in  claim 34 , wherein said cancer expresses tenascin, preferably tenascin C.  
     
     
         42 . The method as claimed in  claim 41 , wherein the expression of tenascin is at least two-fold greater than in non-tumour tissue or cells.  
     
     
         43 . A method as claimed in  claim 41 , wherein the cancer is a solid tumour.  
     
     
         44 . A method as claimed in  claim 41 , wherein the cancer is mesenchymal or epithelial cancer.  
     
     
         45 . A method as claimed in  claim 41 , wherein the cancer is a glioblastoma or breast carcinoma.  
     
     
         46 . An antibody specifically reactive against the polypeptide as set forth in SEQ ID NO:2 or SEQ ID NO:4.  
     
     
         47 . An antibody as claimed in  claim 46  for use as a pharmaceutical.  
     
     
         48 . A method for identifying agents for the treatment or prophylactic treatment of tumourigenesis or cancer, comprising contacting a test compound with cells exposed to a polypeptide as set forth in SEQ ID NO:1 or a portion or variant thereof, wherein said portion interferes with tenascin binding to fibronectin, and tenascin or a portion thereof and then measuring one or more of: 
 a) cell proliferation;    b) DNA synthesis;    c) cell adhesion;    d) cell spreading;    e) focal adhesion and actin stress fiber formation on fibronectin;    f) the proportion of cells entering S-phase of the cell cycle;    g) the binding of cells to extracellular matrix (ECM), preferably wherein the ECM is or comprises fibronectin; or    h) any other output from the syndecan signalling pathway.    
     
     
         49 . A method as claimed in  claim 48 , wherein the cells express syndecan, preferably syndecan-4.  
     
     
         50 . A method as claimed in  claim 48 , further comprising measuring (a), (b), (c), (d), (e), (f), (g) and/or (h) in control of cells grown in the absence of test compound.  
     
     
         51 . A method as claimed in  claim 48 , wherein the cells are cultured on a solid substrate.  
     
     
         52 . A method for identifying agents effective against any disease or condition dependent on the interaction of tenascin with fibronectin, comprising contacting a test compound with a system comprising: 
 i) a polypeptide as set forth in SEQ ID NO:1 or a portion or variant thereof, wherein said portion interferes with tenascin binding to fibronectin,    ii) tenascin or a portion thereof capable of binding (i), and/or    iii) a syndecan molecule capable of binding (i);    and then measuring the binding of (i) and (ii) and/or the binding of (i) and (iii).    
     
     
         53 . A method for identifying agents as claimed in  claim 52 , further comprising correlating a decrease in the binding of (i) and (ii) with an anti-proliferative or anti-tumour agent.  
     
     
         54 . A method for identifying agents as claimed in  claim 52 , further comprising correlating an increase in the binding of (i) and (iii) with an anti-proliferative or anti-tumour agent.  
     
     
         55 . A method as claimed in  claim 52 , wherein the system further comprises measuring the binding of (i) and (ii) and/or the binding of (i) and (iii) in the absence of a test compound.  
     
     
         56 . A method as claimed in  claim 52 , wherein said system comprises said portion, said portion comprising the amino acid sequence as set forth in SEQ ID NO:2, SEQ ID NO:3 or SEQ ID NO:4.  
     
     
         57 . A method as claimed in  claim 52 , wherein tenascin is intact tenascin, preferably tenascin C.  
     
     
         58 . A method as claimed in  claim 54 , wherein the syndecan molecule is the ectodomain of syndecan, preferably that of syndecan 4.  
     
     
         59 . A method as claimed in  claim 54 , wherein said polypeptide, portion or variant thereof is attached to a solid phase.  
     
     
         60 . A method as claimed in  claim 54 , wherein tenascin bound to said polypeptide, portion or variant thereof is measured using an antibody reactive against tenascin.  
     
     
         61 . A method as claimed in  claim 54 , wherein tenascin is attached to a solid phase.  
     
     
         62 . A method as claimed in  claim 54 , wherein syndecan is attached to a solid phase.  
     
     
         63 . A method as claimed in  claim 54 , wherein one or more of the components of fibronectin, tenascin, syndecan, or a variant or fragment thereof is labeled.  
     
     
         64 . A method as  claim 63 , wherein said label comprises radiolabels, fluorescent dyes, electron dense reagents, enzymes, biotin/avidin, dioxigenin, or haptens.  
     
     
         65 . An agent identified by  claim 48 .  
     
     
         66 . A method of diagnosing or prognosing cancer comprising: 
 i) obtaining a sample from an individual;    j) analysing said sample for the presence of accessible FNIII13 or a portion or variant thereof; and    k) correlating the presence of accessible FNIII13 or a portion or variant thereof with a favourable prognosis or diagnosis.    
     
     
         67 . A method of diagnosing or prognosing cancer as claimed in  claim 66  wherein said accessible FNIII13 or a portion or variant thereof is detected using an antibody specific for FNIII13 or a portion or variant thereof, optionally wherein a control antibody specific to a part of fibronectin other than FNIII13 is used in a control reaction.  
     
     
         68 . A method of diagnosing or prognosing cancer as claimed in  claim 66  or in  claim 67  wherein said sample comprises cells grown in culture.