Photochemical internalization (PCI) of the recombinant melanoma-targeting immunotoxin scFvMEL/rGel causes rapid tumor regression in vivo Free

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Intracellular sequestration of therapeutic agents and the subsequent degradation in endocytic vesicles is a major obstacle for several drug-based therapies of cancer. The purpose of the present study was to establish in vivo photochemical internalization (PCI) of a tumor-targeting immunotoxin (IT) for enhanced tumor selectivity, cytosolic delivery and anti-tumor activity. PCI is based on photodynamic therapy (PDT), a photochemical method generating reactive oxygen species (ROS) after light activation of a photosensitizer.¹ The main PDT-induced ROS product is singlet oxygen, which can destroy a number of biomolecules including membrane lipids and proteins of endosomes and lysosomes.The anti-gp240 antibody fusion toxin scFvMEL/rGel² containing the powerful ribosome-inactivating plant toxin(rGel) was tested against human non-pigmented melanoma (A-375, antigen positive) and gp-240 negative bladder carcinoma T-24 cells in vitro, and in a xenograft with A-375 injected s.c. PCI in vitro was performed by light activation of cells co-incubated with scFvMEL/rGel and the endo-lysosomal targeting photosensitizers AlPcS_2a or TPPS_2a. Cells were chased in drug-free medium 4 h prior to light exposure. Cytotoxicity was evaluated by the MTT assay 48 h post light. Mice with 50-100 mm³ A375 tumors got one i.p. injection of 5 mg/kg AlPcS2a 48 hrs and 2 mg/kg scFvMEL/rGel administered once i.v. 24 h prior to 670 nm laser light exposure. Fluence rate of light was 100 mW/cm² and the total fluency given was 20 J/cm². Tumor growth was measured twice per week until tumors reached 1000 mm³. PCI of scFvMEL/rGel demonstrated synergistic cytotoxic effects in A-375 cells in vitro, while PCI of the non-conjugated rGel was 20-fold less toxic (IC₅₀) than the IT. There were no differences in toxicity between the IT and the toxin in gp-240 negative T-24 cells. Preliminary results in vivo show that 2 of 3 mice demonstrated a complete response after PCI of MEL scFv-rGel on day 1, while no mice receiving either light exposure of tumors with IT alone (n=5) or animals that received IT treatment alone (n=5) did not acquire a CR. Light exposure of tumors with AlPcS_2a alone (photodynamic therapy, PDT) resulted in 1 CR of 8 mice (day 21). This is the first in vivo demonstration of PCI of a tumor cell-targeting macromolecule based agent. PCI of immunotoxins may be a potent drug delivery strategy which warrant further pre-clinical evaluation. Research conducted, in part, by the Clayton Foundation for Research.

1. Berg, K.; Selbo, P. K. et al., Cancer Res. 1999, 59, 1180-1183.

2. Rosenblum, M. G.; Cheung, L. H. et al., Cancer Res. 2003, 63, 3995-4002.