Selective occlusion of tumor blood vessels by targeted delivery of an antibody-photosensitizer conjugate Monica Fabbrini 1 , Eveline Trachsel 2 , Patrizia Soldani 1 , Stefano Bindi 1 , Patrizia Alessi 2 , Luisa Bracci 1 , Hartwig Kosmehl 3 , Luciano Zardi 4 , Dario Neri 2 * and Paolo Neri 1 1 Department of Molecular Biology, University of Siena, Siena, Italy 2 Department of Chemistry and Applied Biosciences, Swiss Federal Institute of Technology Zurich, Zurich, Switzerland 3 Institute of Pathology, Helios Klinikum Erfurt, Erfurt, Germany 4 Unit of Innovative Therapies, Department of Experimental and Clinical Immunology, Istituto Giannina Gaslini, Centro di Biotecnologie Avanzate, Genoa, Italy The irregular vasculature and high interstitial pressure of solid tumors hinder the delivery of cytotoxic agents to cancer cells. As a consequence, the doses of chemotherapy necessary to achieve com- plete tumor eradication are associated with unacceptably high tox- icities. The selective thrombosis of tumor blood vessels has been postulated as an alternative avenue for combating cancer, depriv- ing tumors of nutrients and oxygen and causing an avalanche of tumor cell deaths. The human antibody L19, specific to the EDB domain of fibronectin, a marker of angiogenesis, is capable of selective in vivo localization around tumor blood vessels and is thus a suitable agent for delivering toxic payloads to the tumor neovasculature. Here we show that a chemical conjugate of the L19 antibody with the photosensitizer bis(triethanolamine)Sn(IV) chlorin e 6 , after intravenous injection and irradiation with red light, caused an arrest of tumor growth in mice with subcutaneous tumors. By contrast, a photosensitizer conjugate obtained with an antibody of identical pharmacokinetic properties but irrelevant specificity did not exhibit a significant therapeutic effect. These results confirm that vascular targeting strategies, aimed at the selective occlusion/disruption of tumor blood vessels, have a signif- icant anticancer therapeutic potential and encourage the use of antibody-photosensitizer conjugates for the therapy of superficial tumors and possibly other angiogenesis-related pathologies. ' 2005 Wiley-Liss, Inc. Key words: photodynamic therapy; angiogenesis; photosensitizer; EDB domain of fibronectin; vascular targeting The majority of pharmacologic approaches for the treatment of solid tumors suffer from poor selectivity, thus limiting dose esca- lation (i.e., the doses of drug that are required to kill tumor cells cause unacceptable toxicities to normal tissues). 1–3 The situation is made more dramatic by the fact that most anticancer drugs accumulate preferentially in normal tissues rather than at neoplas- tic sites due to the irregular vasculature 4 and the high interstitial pressure of solid tumors. 5 The selective thrombosis of tumor blood vessels 6 appears to be a promising strategy for circumventing the problem of poor drug delivery to neoplastic cells and for achieving an indirect tumor cell killing. Aggressive solid tumors typically exhibit an exuberant proliferation of new blood vessels, 7 which provide neoplastic cells with oxygen and nutrients. An avalanche of cell deaths may result as a consequence of the selective occlusion of individual blood vessels. This strategy is attractive, since tumor endothelial cells are genetically stable and thus unlikely to develop multidrug resistance. 8 Furthermore, the accessibility of tumor blood vessels for intravenously administered agents may facilitate the develop- ment of targeted therapeutic strategies. Following the pioneering work of Ran et al. 9 and Huang et al. 10 on the targeted delivery of the procoagulant protein truncated tissue factor (tTF) to the tumor neovasculature, antivascular strat- egies gained momentum with the observation of a synergistic effect between tTF and low-dose lipopolysaccharides 11 and with the availability of monoclonal antibodies capable of selective in vivo tumor vascular targeting. 12 Furthermore, the selective occlusion of tumor blood vessels can be achieved with the tumor destabilizing agent combretastatin 13 and with tumor necrosis factor (TNF), either in high-dose isolated limb perfusion proto- cols 14 or with TNF derivatives capable of preferential tumor targeting. 15–17 The development of new antivascular tumor therapeutic strat- egies requires the identification of suitable molecular structures capable of inducing thrombosis of blood vessels. Additionally, it is important to clarify to which extent tumor cells may survive a vascular insult. This requires the availability of therapeutic agents, which selectively act at the level of the tumor endothelium, and not on the tumor cells. The targeted delivery of photosensitizers is ideally suited for this purpose, as these compounds act locally (< 0.1 lm) by generating toxic oxygen species (e.g., singlet oxy- gen) after irradiation. 18 In the past, we have shown that the photosensitizer bis(trietha- nolamine)Sn(IV) chlorin e 6 (SnChe 6 ), coupled to the scFv(L19) monoclonal antibody fragment, mediated the selective occlusion of new blood vessels induced in the rabbit cornea, after irradiation with red light. 19 The human antibody fragment scFv(L19), 20 spe- cific to the EDB domain of fibronectin, a marker of angiogene- sis, 21–24 has been shown to localize to the tumor neovasculature after intravenous administration, both in animal models of can- cer 25–28 and in patients with solid tumors. 29 Fusion proteins, based on scFv(L19), are capable of selective localization in tumors and display a potent anticancer activity in rodents. 12,16,17,30,31 Also, antibody-photosensitizer conjugates specific for tumor cell mem- brane antigens have been used before in animal models. 32 A comparative biodistribution analysis of different formats of the L19 antibody [scFv, small immune protein (SIP) and IgG] has shown that the SIP format 33 may be preferable for a number of tumor targeting applications on account of an excellent in vivo stability, long residence times in tumors and good tumor:blood and tumor:organ ratios. 28 The SIP format consists of a fusion between scFv and a CH4 domain of human IgE, which mediates the homodimerization of the recombinant antibody, further Grant sponsor: the Italian Ministry of Health; Grant sponsor: the Euro- pean Commission Project; Grant sponsor: the Swiss National Science Foundation; Grant sponsor: the Krebsforschung; Grant sponsor: the Asso- ciazione Italiana Ricerca sul Cancro. *Correspondence to: Department of Chemistry and Applied Biosci- ences, Swiss Federal Institute of Technology Zurich, Wolfgang-Pauli-Strasse 10, ETH H€ onggerberg, HCI G396, CH-8093 Zurich, Switzerland. Fax: 141- 44633-1358. E-mail: neri@pharma.ethz.ch Received 5 October 2004; Accepted after revision 1 June 2005 DOI 10.1002/ijc.21412 Published online 10 October 2005 in Wiley InterScience (www.interscience. wiley.com). Abbreviations: EDB, extra domain B of fibronectin; NHS, N-hydroxy- succinimide; PBS, phosphate-buffered saline; PDT, photodynamic therapy; PS, photosensitizer; RBC, red blood cell; scFv, single-chain Fv antibody fragment; SDS-PAGE, sodium dodecyl sulfate-polyacrylamide gel electro- phoresis; SIP, small immunoprotein (also called miniantibody); SnChe 6 , bis (triethanolamine)Sn(IV) chlorin e 6 . Int. J. Cancer: 118, 1805–1813 (2006) ' 2005 Wiley-Liss, Inc. Publication of the International Union Against Cancer