Chimeric XXXX -3/ @entity1 beta-glucuronidase fusion proteins for antibody-directed enzyme prodrug therapy (ADEPT).

@entity1 @entity632 @entity137 @entity39 @entity5 @entity19 @entity1334 @entity10390
ADEPT (antibody-directed enzyme prodrug therapy) is a novel therapeutic approach that targets an enzyme into @entity5 to convert a relatively nontoxic prodrug into an active cytotoxic agent. This method has a number of advantages, including the @entity137 , 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 @entity5 mass. Prior studies have utilized MAbs directed against @entity5 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 @entity39 ( @entity39 ) MAbs to deliver the enzyme to @entity39 regions of @entity5 in order to enhance the effectiveness of ADEPT. Biodistribution and autoradiographic studies performed using @entity39 MAbs have shown that localization of these antibodies occurs in @entity5 and that binding is retained within the @entity5 mass for extended periods of time. Since @entity39 regions are often located in the center of @entity5 , are universal in nature, and constitute between 30 and 80% of the @entity5 mass, @entity39 MAbs may be ideal targeting agents for ADEPT. To test this hypothesis, fusion proteins consisting of single chain Fv ( @entity10390 ), @entity1334 , or F(ab')2 fragments of chTNT-3 and the @entity1 beta-glucuronidase (betaG) 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 @entity5 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 @entity1334 /betaG) was used in a pilot treatment study with a glucuronide prodrug of @entity632 to demonstrate the anti- @entity5 activity of ADEPT using the chemoresistant MAD109 @entity19 lung carcinoma @entity5 model transplanted into BALB/c @entity19 . 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 betaG enzyme was shown to localize to @entity5 and remain active for up to 9 days demonstrating a key characteristic of @entity39 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 @entity137 against @entity5 without systemic @entity137 . These preliminary studies show that the use of @entity39 MAbs to target the enzyme to the @entity5 may be a significant advance in ADEPT and that further studies are warranted to test this novel therapeutic approach in the treatment of solid @entity5 .
