Multi-Modality Therapeutics with Potent Anti-Tumor Effects: Photochemical Internalization Enhances Delivery of the Fusion Toxin scFvMEL/rGel

BACKGROUND: There is a need for drug delivery systems (DDS) that can enhance cytosolic delivery of anti-cancer drugs trapped in the endo-lysosomal compartments. Exposure of cells to specific photosensitizers followed by light exposure (photochemical internalization, PCI) results in transfer of agents from the endocytic compartment into the cytosol. METHODOLOGY AND PRINCIPAL FINDINGS: The recombinant single-chain fusion construct scFvMEL/rGel is composed of an antibody targeting the progenitor marker HMW-MAA/NG2/MGP/gp240 and the highly effective toxin gelonin (rGel). Here we demonstrate enhanced tumor cell selectivity, cytosolic delivery and anti-tumor activity by applying PCI of scFvMEL/rGel. PCI performed by light activation of cells co-incubated with scFvMEL/rGel and the endo-lysosomal targeting photosensitizers AlPcS(2a) or TPPS(2a) resulted in enhanced cytotoxic effects against antigen-positive cell lines, while no differences in cytotoxicity between the scFvMEL/rGel and rGel were observed in antigen-negative cells. Mice bearing well-developed melanoma (A-375) xenografts (50-100 mm(3)) were treated with PCI of scFvMEL/rGel. By 30 days after injection, approximately 100% of mice in the control groups had tumors>800 mm(3). In contrast, by day 40, 50% of mice in the PCI of scFvMEL/rGel combination group had tumors<800 mm(3) with no increase in tumor size up to 110 days. PCI of scFvMEL/rGel resulted in a synergistic effect (p<0.05) and complete regression (CR) in 33% of tumor-bearing mice (n = 12). CONCLUSIONS/SIGNIFICANCE: This is a unique demonstration that a non-invasive multi-modality approach combining a recombinant, targeted therapeutic such as scFvMEL/rGel and PCI act in concert to provide potent in vivo efficacy without sacrificing selectivity or enhancing toxicity. The present DDS warrants further evaluation of its clinical potential

Photosensitisation facilitates cross-priming of adjuvant-free protein vaccines and stimulation of tumour-suppressing CD8 T cells

Cancer vaccines aim to induce CD8 T cells infiltrating the tumour. For protein-based vaccines, the main biological barrier to overcome is the default MHC class-II-pathway, with activation of CD4 T cells rather than CD8 T cells. The latter requires antigens to access the cytosol and MHC class I antigen presentation. We applied photosensitiser and light to trigger disruption of antigen-containing endosomes and thereby MHC class I cross-presentation of a model cancer vaccine. This "photochemical internalisation" resulted in activation, proliferation, and IFN-γ production of cytotoxic CD8 T cells, which suppressed tumour growth by infiltrating CD8 T cells and caspase-3-dependent apoptosis. The process was independent of MHC class II, MyD88, and TLR4 signalling, but dependent on trypsin- and caspase-like proteasome activity and partly also on chloroquine. This novel method of vaccination may find applications in cancer immunotherapy where the activation of CD8 T cells is important

Photochemical Internalization of Peptide Antigens Provides a Novel Strategy to Realize Therapeutic Cancer Vaccination

Effective priming and activation of tumor-specific CD8+ cytotoxic T lymphocytes (CTLs) is crucial for realizing the potential of therapeutic cancer vaccination. This requires cytosolic antigens that feed into the MHC class I presentation pathway, which is not efficiently achieved with most current vaccination technologies. Photochemical internalization (PCI) provides an emerging technology to route endocytosed material to the cytosol of cells, based on light-induced disruption of endosomal membranes using a photosensitizing compound. Here we investigated the potential of PCI as a novel, minimally invasive and well-tolerated vaccination technology to induce priming of cancer-specific CTL responses to peptide antigens. We show that PCI effectively promotes delivery of peptide antigens to the cytosol of antigen presenting cells (APCs) in vitro. This resulted in a 30-fold increase in MHC class I/peptide complex formation and surface presentation, and a subsequent 30-100-fold more efficient activation of antigen-specific CTLs compared to using the peptide alone. The effect was found to be highly dependent on the dose of the PCI treatment, where optimal doses promoted maturation of immature dendritic cells, thus also providing an adjuvant effect. The effect of PCI was confirmed in vivo by the successful induction of antigen-specific CTL responses to cancer antigens in C57BL/6 mice following intradermal peptide vaccination using PCI technology. We thus show new and strong evidence that PCI technology holds great potential as a novel strategy for improving the outcome of peptide vaccines aimed at triggering cancer-specific CD8+ CTL responses

Photochemical Internalization of Peptide Antigens Provides a Novel Strategy to Realize Therapeutic Cancer Vaccination

Effective priming and activation of tumor-specific CD8+ cytotoxic T lymphocytes (CTLs) is crucial for realizing the potential of therapeutic cancer vaccination. This requires cytosolic antigens that feed into the MHC class I presentation pathway, which is not efficiently achieved with most current vaccination technologies. Photochemical internalization (PCI) provides an emerging technology to route endocytosed material to the cytosol of cells, based on light-induced disruption of endosomal membranes using a photosensitizing compound. Here, we investigated the potential of PCI as a novel, minimally invasive, and well-tolerated vaccination technology to induce priming of cancer-specific CTL responses to peptide antigens. We show that PCI effectively promotes delivery of peptide antigens to the cytosol of antigen-presenting cells (APCs) in vitro. This resulted in a 30-fold increase in MHC class I/peptide complex formation and surface presentation, and a subsequent 30- to 100-fold more efficient activation of antigen-specific CTLs compared to using the peptide alone. The effect was found to be highly dependent on the dose of the PCI treatment, where optimal doses promoted maturation of immature dendritic cells, thus also providing an adjuvant effect. The effect of PCI was confirmed in vivo by the successful induction of antigen-specific CTL responses to cancer antigens in C57BL/6 mice following intradermal peptide vaccination using PCI technology. We thus show new and strong evidence that PCI technology holds great potential as a novel strategy for improving the outcome of peptide vaccines aimed at triggering cancer-specific CD8+ CTL responses