Expression of herstatin, an alternative HER-2/neu product, in cells that express either p185HER-2 or the EGF receptor inhibits receptor activity and cell growth

Abstract

An alternative HER-2/neu product, herstatin, consists of subdomains I and II from the ectodomain of p185HER-2 and a unique 79 amino acid C-terminus encoded by intron 8. Recombinant herstatin added to cells was found to bind to and inhibit p185HER-2. The effects of ectopic expression of herstatin in combination with either p185HER-2 or with its homolog, the EGF receptor, in several cell lines was studied. Cotransfection of herstatin with HER-2 inhibited p185HER-2 levels and caused an approximate 8-fold reduction in p185 tyrosine phosphorylation. Inhibition ofp185HER-2 tyrosine phosphorylation corresponded to a dramatic decline in colony formation by cells that coexpressed p185HER-2 and herstatin. Herstatin also interferred with EGF activation of the EGF receptor in cotransfected cells as demonstrated by impaired receptor tyrosine phosphorylation, reduced receptor down-regulation, and growth suppression. For both p185HER-2 and the EGF receptor, the extent of inhibition was affected by the expression levels of herstatin relative to the receptor. Herstatin is an autoinhibitor of p185HER-2 and expands its inhibitory activity to another member of the group I family of receptor tyrosine kinases, the EGF receptor. Herstatin blocked the activated Akt-mediated EGF survival signal, as well as transforming growth factor alpha (TGFα)-mediated EGF receptor activation, survival signal and proliferation signal. Purified recombinant herstatin specifically inhibited human carcinoma cells that over-express HER-2, and was effectively absorbed into the blood of intraperitoneally injected mice, where it was not proteolytically degraded and was present for between one and three hours. Herstatin was shown to be efficacious in rat and mouse xenograft models of human cancer.

1 . A method for treating a solid tumor characterized by expression of EGF receptor, comprising administering an agent that binds to the extracellular domain of EGF receptor, wherein the agent is selected from the group consisting of (a) an isolated polypeptide having from about 50 to 79 amino acids taken from the sequence of SEQ ID NO: 1, wherein the polypeptide binds to the extracellular domain of EGF receptor at an affinity of at least 10 8 , (b) an isolated and glycosylated polypeptide having from about 80 to 419 amino acids taken from the sequence of SEQ ID NO: 2, wherein the C-terminal 79 amino acids are present, and wherein at least three N-linked glycosylation sites are present, (c) a monoclonal antibody that binds to the extracellular domain of EGF receptor, and (d) combinations thereof, with the proviso that the agent cannot be the monoclonal antibody alone.  
     
     
         2 . The method of  claim 1 , wherein the solid tumor that expresses EGF receptor is selected from the group consisting of squamous cell carcinoma, lung carcinoma, colon carcinoma, and glial cell tumors.  
     
     
         3 . The method of  claim 1 , wherein the agent is the isolated polypeptide having from about 50 to 79 amino acids taken from the sequence of SEQ ID NO: 1.  
     
     
         4 . The method of  claim 1 , wherein the agent is a combination of the isolated polypeptide having from about 50 to 79 amino acids taken from the sequence of SEQ ID NO: 1 and the monoclonal antibody that binds to the extracellular domain of EGF receptor.  
     
     
         5 . The method of  claim 1 , wherein the agent is an isolated and glycosylated polypeptide having from about 80 to 419 amino acids taken from the sequence of SEQ ID NO: 2, wherein the C-terminal 79 amino acids are present, and wherein at least three N-linked glycosylation sites are present.  
     
     
         6 . The method of  claim 1 , wherein the agent is a combination of an isolated and glycosylated polypeptide having from about 80 to 419 amino acids taken from the sequence of SEQ ID NO: 2, wherein the C-terminal 79 amino acids are present, and wherein at least three N-linked glycosylation sites are present and the monoclonal antibody that binds to the extracellular domain of EGF receptor.  
     
     
         7 . A pharmaceutical composition for treating solid tumors that express EGF receptor, comprising an agent selected from the group consisting of (a) an isolated polypeptide having from about 50 to 79 amino acids taken from the sequence of SEQ ID NO: 1, wherein the polypeptide binds to the extracellular domain of EGF receptor at an affinity of at least 10 8 , (b) an isolated and glycosylated polypeptide having from about 80 to 419 amino acids taken from the sequence of SEQ ID NO: 2, wherein the C-terminal 79 amino acids are present, and wherein at least three N-linked glycosylation sites are present, (c) a monoclonal antibody that binds to the extracellular domain of EGF receptor, and (d) combinations thereof, with the proviso that the agent cannot be the monoclonal antibody alone, and a pharmaceutically acceptable carrier.  
     
     
         8 . The pharmaceutical composition for treating solid tumors that express EGF receptor of  claim 7 , wherein the agent is the isolated polypeptide having from about 50 to 79 amino acids taken from the sequence of SEQ ID NO: 1.  
     
     
         9 . The pharmaceutical composition for treating solid tumors that express EGF receptor of  claim 7 , wherein the agent is a combination of the isolated polypeptide having from about 50 to 79 amino acids taken from the sequence of SEQ ID NO: 1 and the monoclonal antibody that binds to the extracellular domain of EGF receptor.  
     
     
         10 . The pharmaceutical composition for treating solid tumors that express EGF receptor of  claim 7 , wherein the agent is an isolated and glycosylated polypeptide having from about 80 to 419 amino acids taken from the sequence of SEQ ID NO: 2, wherein the C-terminal 79 amino acids are present, and wherein at least three N-linked glycosylation sites are present.  
     
     
         11 . The pharmaceutical composition for treating solid tumors that express EGF receptor of  claim 7 , wherein the agent is a combination of an isolated and glycosylated polypeptide having from about 80 to 419 amino acids taken from the sequence of SEQ ID NO: 2, wherein the C-terminal 79 amino acids are present, and wherein at least three N-linked glycosylation sites are present and the monoclonal antibody that binds to the extracellular domain of EGF receptor.  
     
     
         12 . A method for targeting a therapeutic agent to solid tumor tissue, wherein the solid tumor tissue is characterized by expression of EGF receptor, comprising attaching the therapeutic agent to an isolated polypeptide having from about 50 to 79 amino acids taken from the sequence of SEQ ID NO: 1, wherein the polypeptide binds to the extracellular domain of EGF receptor at an affinity of at least 10 8 .  
     
     
         13 . The method for targeting a therapeutic agent to solid tumor tissue of  claim 12 , wherein the isolated polypeptide is from about 69 to 79 amino acids taken from the sequence of SEQ ID NO: 1.  
     
     
         14 . A method for determining the prognosis of tumor treatment in a patient for a tumor that expresses EGF receptor, comprising: (a) obtaining a bodily fluid sample from a patient, wherein the bodily fluid is selected from the group consisting blood, serum, urine, lymph, saliva, tumor tissue, placental tissue, umbilical cord tissue, amniotic fluid, chorionic villi tissue and combinations thereof; (b) measuring the amount of p68HER-2 expressed using an anti-p68HER-2 antibody-based assay, wherein the assay is selected from the group consisting of ELISA, immunoprecipitation, immunohistocytochemistry, and Western analysis, (c) measuring the amount of EGF receptor extracellular domain in the bodily fluid, and (d) determining a ratio between the amount of p68HER-2 and EGF receptor extracellular domain, whereby the higher the p68HER-2 to EGF receptor extracellular domain ratio, the better the prognosis of the patient.