Photodynamic therapy (PDT) involves a two-stage process. A light-absorbing photosensitiser (Ps) is endocytosed and then stimulated by light, inducing transfer of energy to a cytoplasmic acceptor molecule and the generation of reactive oxygen species that initiate damage to cellular membrane components and cytolysis. The expanded use of PDT in the clinic is hindered by the lack of Ps targetcell specificity and the limited tissue penetration by external light radiation. This study demonstrates that bioconjugates composed of transferrin and haematoporphyrin (Tf -Hp), significantly improve the specificity and efficiency of PDT for erythroleukemic cells by a factor of almost seven-fold. Fluorescence microscopy showed that the conjugates accumulate in intracellular vesicles whereas free Hp was mostly membrane bound. Experiments with cells deliberately exposed to Tf -Hp at oLD 100 doses showed that surviving cells did not develop resistance to subsequent treatments with the conjugate. Furthermore, we show that the compound luminol induces intracellular chemiluminescence. This strategy was then used to obviate the use of external radiation for Ps activation by incubating the cells with luminol either before or together with Tf -Hp. This novel chemical means of PDT activation induced cytotoxicity in 95% of cells. These combined approaches provide an opportunity to develop broader and more effective applications of PDT.

Many types of leukemia including multiple myeloma remain essentially incurable despite recent developments in immuno- and chemotherapy. The effectiveness of these therapies might be greatly enhanced by targeting cell surface proteins unique to the malignant clone, which for leukemias of the B cell lineage means clonotypic surface immunoglobulin (sIg). As this immunoglobulin (Ig) is necessarily epitope specific, we are developing ligand-toxin conjugates (LTCs) as a strategy for delivering toxins and other drugs to clonotypic tumor cells. Here we report in vitro studies that illustrate the effectiveness of this approach. LTC comprising the DNP hapten conjugated to ricin A toxin (DNP-RTA) were shown to specifically and effectively kill anti-DNP secreting murine hybridoma (U7.6) cells but not other hybridoma cells (1B12), a murine erythroleukemia cell line (Friend's Leukemia or) normal mouse spleen cells. In addition to direct toxicity, LTC treatment negatively affected the growth characteristics of the few surviving cells as reflected in decreased growth index and an increase in growth inhibition over 72 h post treatment. Interestingly, U7.6 cells that survived one or two LD90 dose(s) of LTC showed no alteration in their dose response to a subsequent attack of LTC indicating that this treatment strategy may not induce drug resistance. These data suggest that LTC therapy may be a new and effective strategy for specific destruction of tumor cells such as myeloma plasma cells and could be extended to other tumors where clonotypic receptors can be identified.

Targeted Intracellular Photodynamic Therapy (TIP) is a novel advance on traditional photodynamic therapy (PDT) in which photosensitizer activation is afforded by chemiluminescence rather than by laser beam. This advance allows PDT access to target cells essentially anywhere in the body. TIP also has advantages over bioluminescence as there is no need for transfection of cells with enzymes such as luciferase. It can therefore be more easily applied both in vitro and in vivo and has potential as a new targeted cancer therapeutic modality that could be applied to a wide variety of cancers. We have previously described in vitro the efficacy and specificity of TIP toward murine leukemic cells (Brit. J. Cancer, 95; 189-196, 2006). We now report that TIP is effective in vivo using a murine model of cancer. To target the photosensitizer, Protoporphyrin IX was conjugated to Transferrin (Trnsf-PrP). Balb/c mice were injected subcutaneously with the mouse hybridomas U13.6 or U7.6, both of which overexpress the Transferrin receptor CD71. The use of Trnsf-PrP resulted in rapid uptake of photosensitizer into cells and conjugate was retained for over 48hrs. Animals were treated with TIP by injecting Trnsf-PrP and the chemiluminescent agent Luminol (LM) under reduced light conditions. Experiments were performed whereby different parameters of the system were varied, including: time of treatment initiation after tumor cell inoculation; dose and number of Trnsf-PrP and luminol treatments; and time intervals between treatments. Initial experiments in which TIP was performed at the time of tumor inoculation showed that this procedure completely abolished tumor growth. In subsequent experiments where tumors were palpable before treatment began, various treatment protocols were tested. TIP reduced tumor mass by over 40% and increased survival by 50%. These results demonstrate that TIP is active against cancer cells in vivo and encourages the further development of the modality as a targeted cancer therapy. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 5483.