Hemocyanins in the Immunotherapy of Superficial Bladder Cancer

Chemoand immunotherapeutic approaches have been used to prevent recurrence of transitional cell carcinoma (TCC), the most common type of superficial bladder cancer (SBC). The bacillus Calmette-Guérin (BCG) vaccine for tuberculosis, which consists of an attenuated form of Mycobacterium bovis, is the most commonly used immunotherapeutic agent (Morales et al., 1976). Despite the successful results achieved with BCG, its serious side effects have led researchers to investigate other immunostimulatory substances. In the early 1970s, Olsson and collaborators reported that subcutaneous stimulation with keyhole limpet hemocyanin (KLH) from the Californian marine gastropod Megathura crenulata significantly reduced SBC recurrence frequency in TCC patients without any toxic side effects, making it ideal for long-term repetitive treatments (Olsson et al., 1974). These results provided promising support for the use of mollusk hemocyanins as alternative agents in SBC immunotherapy. Hemocyanins, blue respiratory glycoproteins that were discovered in 1878 by Léon Fredericq (Ghiretti-Magaldi & Ghiretti, 1992), are found freely dissolved in the blood of some mollusks and arthropods. These proteins are giant structures with molecular weights between 4 and 8 MDa, and they exhibit some of the most complex and sophisticated quaternary structures known. Hemocyanins are part of the type-3 group of copper proteins that includes phenoloxidases and tyrosinases (Decker & Tuczek, 2000). These proteins contain active copper-containing sites in which the Cu(I,I) state is oxidized to the Cu(II,II) state, thus accounting for their distinctive deep blue color. Because of these properties, the biochemistry of hemocyanins has been intensively studied (van Holde & Miller, 1995). The pioneering work of Weigle in the 1960s on the immunochemical properties of KLH demonstrated its remarkable immunostimulatory properties in an experimental animal model (Weigle, 1964). These results were quickly incorporated into clinical studies to evaluate its immunological effects. Because the primary amino acid sequences of mollusk hemocyanins are highly divergent from mammalian sequences, they are strongly recognized by the immune system, resulting in potent immunogenicity; these proteins can be used therapeutically as non-specific immunostimulants with beneficial clinical outcomes. Moreover, hemocyanins have been extensively used as carriers to generate antibodies against diverse hapten molecules and