Gene dependent enzyme prodrug therapy for head and neck cancer.

Abstract

5-fluorouracil (5-FU) and methotrexate (MTX) are two of the most active single agents in the treatment of head and neck cancer (HNC). Therefore, we evaluated two g&barbelow;ene d&barbelow;ependent e&barbelow;nzyme p&barbelow;rodrug t&barbelow;herapies (GDEPT) to localize high-doses of MTX or 5-FU to the tumor site through the <italic> in situ</italic> activation of systemically administered non-toxic prodrugs. Soluble and cell-surface localized forms of carboxypeptidase A (CPA) were developed that are activated by subtilisn-like propeptidases in the absence of trypsin dependent zymogen-cleavage. Enzymatic assays and protein sequencing revealed that these endogenously activated peptides were structurally and functionally identical to trypsin activated CPA. Further, retroviral infection of tumor cell-lines <italic>in vitro</italic> with endogenously active CPA sensitized cells to MTX-alpha-peptide prodrugs in a dose and time-dependent manner. Finally, cell-surface expression of CPA through fusion with a glycophospholipid membrane anchor resulted in an enhanced therapeutic index and an increased bystander effect compared to expression of the soluble enzyme. To study the cytosine deaminase (CD)/5-fluorocytosine (5-FC) GDEPT an orthotopic animal model of head and neck cancer was utilized. Treatment of animals bearing CD-expressing tumors with the maximum tolerated dose of either 5-FU or radiotherapy (RT) had no impact upon tumor growth or animal survival even when 5-FU/RT were administered concomitantly. In contrast, treatment with 5-FC resulted in a greater than 2-fold increase in time of survival which was enhanced by the addition of concurrent RT. In order to improve upon the CD/5-FC GDEPT we next compared the bacterial and yeast CD enzymes for their ability to convert 5-FC to 5-FU. CD derived from <italic>S. cerivisiea</italic> had a 250-fold higher capacity to convert 5-FC to 5-FU than its bacterial counterpart, and this resulted in both a 30-fold lower IC₅₀ for 5-FC <italic>in vitro</italic> and a markedly enhanced bystander effect. <italic> In vivo</italic> the yeast enzyme resulted in a significant decrease in tumor growth rate following treatment with 5-FC and a 60% rate of long-term survivors, while there were no long-term survivors for the animals bearing tumors expressing bacterial CD. These studies suggest a role for GDEPT in the treatment of locally advanced head and neck cancer.