Since metastatic cancer is a systemic disease, ultimately systemic (blood-borne) therapies must be developed that can reach tumor cells wherever in the body they have spread. The blood-brain-barrier (BBB) poses a formidable challenge to designing such therapies for cancer spread to the CNS. The overall goal of this project will be to develop an effective means of delivering systemically administered drugs to the brain where they would be used to diagnose and/or treat brain malignancies. The therapies must be able both to target across the BBB and once within the brain target the malignant cells. The strategy will be to use the growth factor receptors on the BBB as transport vehicles for gaining access to the brain. In earlier studies, the investigators have shown that both transferrin and antibodies to the transferrin receptor can be used to transport fusion proteins across the BBB. They will use tumor specific antibodies recognizing HER2/neu and would target cell lines expressing HER2/neu that have been injected into the brain. Proteins that they will produce include an anti-TfR-avidin fusion protein which will be used to deliver a biotinylated anti-tumor antibody, an anti-tumor- Tf fusion protein, and a tetra-valent, bispecific molecule with recognition both for the Tar and tumor associated antigens. These initial proteins will not be inherently cytotoxic but would be used to target radioactivity as either a diagnostic or cytotoxic agent to tumors. Proteins will be evaluated in vitro for their size, assembly, stability and ability to bind their respective targets (the Tar or tumor antigen) and in vivo for their pharmacokinetic properties including their transport across the BBB and ability to secondarily target tumors. The investigators will also develop potential therapeutic molecules using the Antibody Directed Enzyme Prodrug Therapy (ADEPT) approach. In this approach, the antibody will be used to target an enzyme to the tumor; the targeted enzyme will then convert a non-toxic prodrug to a toxic agent only at the site of the tumor. Fusion proteins will be constructed using herpes virus thymidine kinase (HSV-TK) with ganciclovir as the prodrug and using cytosine deamidase (CD) with 5-fluorocytosine (5-FC) as the prodrug. These fusion proteins will be evaluated in vitro for enzymatic activity and ability to bind antigen and the TfR and in vivo for their transport across the BBB and ability to target tumors and kill them in the presence of the prodrug.
