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Targeting cancer vasculature is an emerging field in cancer treatment. Photochemical internalization (PCI) is a drug delivery technology based on photochemical lysis of drug-bearing endocytic vesicles originally designed to target cancer cells. Recent investigations have revealed a lower PCI efficacy in vascular endothelial cells (HUVECs) in vitro than in HT1080 fibrosarcoma cells. This manuscript aims to explore the limiting factor for the PCI effect in HUVECs. Cellular uptake of the photosensitizers AlPcS(2a) and TPPS(2a), and a model compound for macromolecular drugs taken up by fluid phase endocytosis, Alexa⁴⁸⁸-dextran, was explored by flow cytometry. The uptake of AlPcS(2a) and TPPS(2a) was 3.8-fold and 37-fold higher in HUVECs than in HT1080 cells, respectively, while the Alexa⁴⁸⁸-dextran uptake was 50% lower. AlPcS(2a) (but not TPPS(2a)) was shown to reduce Alexa⁴⁸⁸-dextran uptake in a concentration-dependent manner, resulting in 66% and 33% attenuation of Alexa⁴⁸⁸-dextran uptake at 20 μg/ml AlPcS(2a) in HUVECs and HT1080 cells respectively. Studies of intracellular localization of Alexa⁴⁸⁸-dextran and AlPcS(2a) by confocal microscopy in HUVECs uncovered a concentration-dependent AlPcS(2a)-induced inhibition of Alexa⁴⁸⁸-dextran trafficking into AlPcS(2a)-stained and acidic vesicles. The localization of Alexa⁴⁸⁸-dextran to AlPcS(2a)-localizing compartments was reduced by 40% when the AlPcS(2a) concentration was increased from 5 to 20 μg/ml. The treatment dose of AlPcS(2a) was found to influence on the efficacy of PCI of saporin, but to a lesser extent than expected considering the data from cellular uptake and intracellular trafficking of Alexa⁴⁸⁸-dextran. The implications of these results for further development of vascular targeting-PCI are discussed.

Immunotherapy (IT) with the anti-CD20 monoclonal antibody rituximab in combination with chemotherapy has resulted in significantly improved response rate and survival in patients with various types of CD20 positive B-cell lymphoproliferative disorders. To be effective, rituximab depends on selective expression of a sufficient number of CD20 antigens per cell. Treatment with rituximab alone or in combination with chemotherapy can, however, result in disappearance of the CD20 expression, which may result in reduced clinical effect of subsequent CD20 targeted treatments. We have discovered that treatment of NHL in vitro and in vivo with the anti-CD37 antibody radionuclide conjugate (ARC) 177Lu-DOTA-HH1 (177Lu-HH1 or Betalutin™) results in an upregulation of the CD20 antigen expression, and therefore represents a rationale for a combination treatment with both agents. The in vitro expression of CD20 in Burkitt's Lymphoma, Daudi, cells 1-7 days after treatment with 177Lu-HH1 increased up to 120 % when compared with cells treated with unlabeled mAb, while Ramos (Burkitt's Lymphoma) and Rec-1 (Mantle Cell Lymphoma) cells showed 10 to 30 % increase, indicating a variation of the antigen upregulation in vitro with different cell lines. An upregulation of CD20 at the same order of magnitude was observed when cells where treated with similar absorbed radiation doses of external beam radiation. Treatment of nude mice with Ramos xenografts with 177Lu-HH1 resulted in a 3 times higher uptake of radiolabeled rituximab in tumor xenografts 5 days after start of treatment than in mice treated with unlabeled HH1 (p < 0.05) while uptake in normal organs was similar in both treatment groups (p > 0.05). SCID mice with intravenously injected Rec-1 cells were treated with NaCl, 100 mg rituximab, 40 MBq/kg 177Lu-HH1 or with the combination of 40 MBq/kg 177Lu-HH1 followed with 100 mg rituximab 5 days later. The combination of 177Lu-HH1 and rituximab resulted in significantly improved survival as compared with NaCl or rituximab alone, and a strong therapeutic gain as compared with 177Lu-HH1 alone (Table 1). In conclusion, 177Lu-HH1 treatment seems to improve uptake of rituximab and increase tumor suppression when used prior to anti-CD20 monoclonal antibody targeting in preclinical models. The reason for the upregulation of CD20 is probably related to the oxidative stress induced by the ARC-treatment, which will be evaluated in further studies. If the upregultation of CD20 is confirmed in clinical studies this effect could affect the way ARC and CD20 immunotherapy would be used in the future. Table 1. Therapy experiment groups and result Group Median ± SD Surviving fraction at the end of the study % Increase in symptom free survival compared to control NaCl + NaCl 64 ± 2 0.1 ---- NaCl + Rituximab 75 ± 10 0.3 15.4 177 Lu-HH1 + NaCl 92 ± 14 * 0.3 43.8 177 Lu-HH1 + Rituximab > 132 * 0.7 > 106.3 *Significantly different from NaCl + NaCl group (p < 0.01) Disclosures Repetto-Llamazares: Nordic Nanovector ASA: Employment, Equity Ownership. Larsen:Nordic Nanovector ASA: Equity Ownership, Membership on an entity's Board of Directors or advisory committees. Stokke:Nordic nanovector ASA: Equity Ownership. Generalov:Nordic Nanovector ASA: Employment. Dahle:Nordic Nanovector ASA: Employment, Equity Ownership.

Aims: Polysaccharide nanoparticles were studied as drug delivery vehicles for chemopreventive agents. Materials & methods: Green tea polyphenol epigallocatechin-3-gallate (EGCG) was incorporated into a carbohydrate matrix of gum arabic and maltodextrin with an encapsulation efficiency of approximately 85%. Results: Encapsulated EGCG retained its biological activity, reducing the cell viability and inducing apoptosis of Du145 prostate cancer cells. Clonogenic assay demonstrated that encapsulation of EGCG enhanced its inhibitory effect on cell proliferation (10–20%) at lower concentrations (1–2 µM), compared with free EGCG. Conclusion: This study highlights the use of polysaccharide nanoparticles in chemoprevention as they can be used to deliver natural antioxidants capable of inhibiting steps of the tumorigenesis process.