| Photosensitizer immunoconjugates (PICs) offer improved photosensitizer delivery specificity and could broaden the applicability of photodynamic therapy (PDT) in the treatment of various lesions (e.g., tumors, bacterial infections, atherosclerotic lesions). Over the past two decades, efforts to develop clinically useful PICs failed primarily due to the hydrophobic and aggregative properties of porphyrin-type photosensitizers. Another major shortcoming was the lack of a proper target antigen. The target antigen must allow delivery of sufficient amounts of photosensitizer to the lesion such that phototoxicity can be achieved using clinically practical light doses. In this thesis, several measures were taken to overcome these problems. First utilization of a novel 50% DMSO/50% aqueous two-solvent system greatly diminished the tendency of photosensitizer to aggregate and bind noncovalently to the PIC, thereby allowing for (1) efficient direct Covalent linkage of photosensitizer-activated ester to antibody lysine residues while maintaining biological activity of the antibody and (2) thorough purification of the PICs by gel filtration. Second, PEGylation of a small number of antibody lysine residues dramatically enhanced the solubility of the PICs, and an albumin-assisted exchange process permitted transfer of the PICs from 50% DMSO to purely aqueous buffer without forming large, insoluble aggregates. Third, a chimeric anti epidermal growth factor receptor (EGFR) antibody was chosen as the targeting vehicle because various tumor cells overexpress EGFR, and BPD Verteporfin (BPD) was chosen as the photosensitizer because of its exceptional phototoxic potency and nearly optimal photophysical properties. Gel electrophoresis (SDS-PAGE) of the PICs verified purity and covalent linkages. Studies using EGFR-positive, EGFR-negative, and macrophage cells demonstrated that PIC phototoxicity is very specific and predominantly mediated through binding of the PIC to EGFR. Confocal microscopy indicated that PICs undergo endocytosis and localize in lysosomes, whereas free BPD diffusely stains the cytosol and perinuclear membranes. Spectroscopic studies showed that intact PICs are less photoactive than free BPD due to static concentration quenching. Time-coursed incubations revealed that PICs lysosomally breakdown and increase in potency as the incubation period is increased. The results suggest that PICs may be useful in PDT applications requiting precise targeting, provided the targeted cells highly express and/or rapidly replenish target antigen. |
