PEGylated carboxypeptidase A for solid tumor targeting

This thesis project has focused primarily on exploring the use of poly(ethylene glycol) [PEG] to deliver prod rug-activating enzymes to solid tumors. We hypothesized that tumor-to-blood ratios of a targeted enzyme would be improved if an appropriate number of PEG chains were attached to the conjugate in order to tailor the balance between the tumor accumulation rate and the circulatory retention of the PEG-enzyme conjugates. The prototype for our series of targeted enzymes was bovine carboxypeptidase A (CPA) chemically coupled to methoxy-PEG (mPEG) to form conjugates that have molecular weights ranging from 40 to 50 kDa. Preparative scale quantities of relatively pure conjugates were obtained through controlled PEGylation followed by fractionation using size-exclusion chromatography. Attachment of up to three mPEG chains to the surface of the enzyme improved both the stability and the catalytic properties towards hippuryl-L -phenylalanine.;It is clearly demonstrated that mPEG-modified derivatives of CPA containing 2--3 polymer moieties exhibited a prolonged circulatory half-life yet displayed minimal uptake in normal tissues. The strong linear correlation of pharmacokinetic parameters, such as the systemic clearance, to the hydrodynamic radius of mPEG-CPA conjugates allows for desirable pharmacokinetic properties to be predicted on the basis of effective size, which can be modified through the use of mPEG modification. An increase in tumor retention as a result of reduced systemic clearance observed in mPEG2-CPA and mPEG3-CPA led to enhanced tumor-to-blood ratios.;To further improve the selective delivery of CPA to tumor sites, a pretargeting approach utilizing the strong interaction between biotin and avidin (AV) was explored. Low levels of biotinylation of CPA using the activated bPEG retained substrate binding affinity yet enhanced catalytic efficiency for hippl- L-phe. The bPEG-CPA analogs demonstrated enhanced stability with respect to the specific peptidase activity. In addition, the ability for bPEG 1-CPA to interact strongly with avidin offers a rationale for the development of an active avidin-based clearance mechanism to achieve high a tumor-to-blood ratio. The formation of stable trimolecular bPEG1-CPA/AV/b-Fab demonstrated the feasibility of the alternative coupling strategy for preparing mAb-enzyme conjugates.