Chimeric TNT-3/Human β -Glucuronidase Fusion Proteins for Antibody-Directed Enzyme Prodrug Therapy (ADEPT)

Abstract

ADEPT (antibody-directed enzyme prodrug therapy) is a novel therapeutic approach that targets an enzyme into tumors to convert a relatively nontoxic prodrug into an active cytotoxic agent. This method has a number of advantages, including the reduction of systemic toxicity, but to date it has not realized its full potential. A critical component of ADEPT is the choice of the monoclonal antibody (MAb) to target the enzyme into the tumor mass. Prior studies have utilized MAbs directed against tumor cell surface antigens which are oftentimes labile and heterogeneous in nature and do not provide an ideal site for the enzyme. As an alternative approach, we now describe the use of Tumor Necrosis Therapy (TNT) MAbs to deliver the enzyme to necrotic regions of tumors in order to enhance the effectiveness of ADEPT. Biodistribution and autoradiographic studies performed using TNT MAbs have shown that localization of these antibodies occurs in degenerating cells and necrotic regions of tumors and that binding is retained within the tumor mass for extended periods of time. Since necrotic regions are often located in the center of tumors, are universal in nature, and constitute between 30 and 80% of the tumor mass, TNT MAbs may be ideal targeting agents for ADEPT.

To test this hypothesis, fusion proteins consisting of single chain Fv (scFv), Fab, or F(ab′)2 fragments of chTNT-3 and the human β-glucuronidase(βG) enzyme were constructed for ADEPT. Each of these reagents was tested to assess specificity and avidity of antigen binding as compared to the parental antibody. In addition, studies were performed to demonstrate enzymatic function of the fusion proteins and retention of catalytic activity in circulating blood, specific tissues, and tumor after in vivo administration. Pharmacokinetic and biodistribution studies of radiolabeled fusion proteins were conducted over time to evaluate the characteristics of the fusion proteins. Finally, one of the constructs (chTNT-3 Fab/βG) was used in a pilot treatment study with a glucuronide prodrug of doxorubicin to demonstrate the anti-tumor activity of ADEPT using the chemoresistant MAD109 murine lung carcinoma tumor model transplanted into BALB/c mice. The results of these experiments show that all three constructs retained their antigen binding capability and demonstrated active enzymatic function against substrate in vitro. Moreover, after in vivo administration, the βG enzyme was shown to localize to tumor and remain active for up to 9 days demonstrating a key characteristic of TNT targeting. Pharmacokinetic and biodistribution studies confirmed specific localization of the fusion proteins and rapid clearance from blood and normal tissues over time. Finally, therapeutic studies using only two doses of fusion protein followed by prodrug administration demonstrated active cytotoxicity against established tumors without systemic toxicity. These preliminary studies show that the use of TNT MAbs to target the enzyme to the tumor may be a significant advance in ADEPT and that further studies are warranted to test this novel therapeutic approach in the treatment of solid tumors.