Angiogenic processes depend on the precise coordination of different cell types and a complex exchange of signals, many of which derive from new specific components of the provisional, angiogenesisrelated, extracellular matrix (ECM). Angiogenesis-associated ECM components thus represent appealing targets for the selective delivery of therapeutic molecules to newly forming tumor vessels. Results of a previous study indicated that a high affinity recombinant antibody (L19) to ED-B, a domain contained in the angiogenesis-associated isoform of fibronectin (B-FN), selectively and efficiently targets tumor vessels. The present study shows that a fusion protein between L19 and interleukin 2 (L19-IL-2) mediates the selective delivery and concentration of IL-2 to tumor vasculature, thereby leading to a dramatic enhancement of the therapeutic properties of the cytokine. By contrast, IL-2 fused to an irrelevant recombinant antibody used as a control fusion protein showed neither accumulation in tumors nor therapeutic efficacy. Tumors in mice treated with L19-IL-2 were significantly smaller compared to those in animals treated with saline, the control fusion protein, or IL-2 alone (P ‫؍‬ .003, .003, and .002, respectively). Moreover, no significant differences in size were observed among the tumors from the different control groups (using the control fusion pro- IntroductionDuring tumor progression, the extracellular matrix (ECM) of the normal tissues in which the tumor grows is remodeled through 2 different processes: proteolytic degradation and neosynthesis of ECM components by both neoplastic and stromal cells. These processes generate a "tumoral ECM" that differs quantitatively and qualitatively from the normal tissue ECM and that apparently gives rise to a more suitable environment (inductive or instructive) for tumor progression. [1][2][3] In particular, ECM components modulate vascular cell behavior and angiogenic processes. 4,5 This observation is upheld by the recent report that the majority of messenger RNAs (mRNAs) newly expressed by tumoral endothelial cells encode for ECM proteins. 6 Thus, these provisional ECM components that appear during the angiogenic processes represent an appealing target for the selective delivery of therapeutic molecules to newly forming blood vessels. 7 Furthermore, because new vessel formation is common to all solid tumors, the angiogenesisassociated ECM components can be regarded as pan-tumoral antigens. [8][9][10][11][12] One such ECM component is a fibronectin (FN) isoform, B-FN, which contains an extra FN type III repeat of 91 amino acids, the domain B (ED-B). 13 Because the amino acid sequence of ED-B is identical in mouse, humans, and other mammals, antibodies to this domain react equally well with mouse, human, and other species B-FN. B-FN is detectable only in the stroma of fetal and neoplastic tissues and around newly forming blood vessels, but not in mature vessels. 14,15 Using a radioiodinated human recombinant single-chain Fv (scFv; L19) to the ED-B domain of FN, we demon...

We sought to enhance the selective toxicity of tumor necrosis factor ␣ (TNF␣) to permit its systemic use in cancer therapy. Because ligand-targeted therapeutics have proven successful in improving the selective toxicity of drugs, we prepared a fusion protein (L19mTNF␣) composed of mouse TNF␣ and a high-affinity antibody fragment (L19 scFv) to the extradomain B (ED-B) domain of fibronectin, a marker of angiogenesis. L19mTNF␣ was expressed in mammalian cells, purified, and charac- IntroductionDuring tumor progression the microenvironment surrounding tumor cells undergoes extensive modifications that generate a "tumoral environment" which could ultimately represent a suitable target for antibody-based tumor therapy. 1 In fact, the concept that the altered tumor microenvironment is itself a carcinogen that can be targeted is increasingly gaining consensus. Molecules that are able to effectively deliver therapeutic agents to the tumor microenvironment thus represent promising and important new tools for cancer therapy. [1][2][3] Fibronectin is an extracellular matrix (ECM) component that is widely expressed in a variety of healthy tissues and body fluids. Different fibronectin (FN) isoforms can be generated by the alternative splicing of the FN pre-mRNA, a process modulated by cytokines and extracellular pH. [4][5][6][7] The complete type III repeat extradomain B (ED-B) may be entirely included or omitted in the FN molecule. 8 ED-B is highly conserved in different species, having 100% homology in all mammalians thus far studied (human, rat, mouse) and 96% homology with a similar domain in chicken. The FN isoform containing ED-B (B-FN) is undetectable immunohistochemically in healthy adult tissues, with the exception of tissues undergoing physiologic remodeling (eg, endometrium and ovary) and during wound healing. 5,9 By contrast, its expression in tumors and fetal tissues is high. 5 Furthermore, it was demonstrated that B-FN is a marker of angiogenesis 10,11 and that endothelial cells invading tumor tissues migrate along ECM fibers containing B-FN. 12 We reported on the possibility to selectively target tumoral vasculature, both in experimental tumor models and in patients with cancer, using a human recombinant antibody, L19 scFv, specific for B-FN. [12][13][14][15][16][17][18][19] This observation paved the way for the antibody's use in both in vivo diagnostic (immunoscintigraphy) and therapeutic approaches entailing the selective delivery of radionuclides or toxic agents to tumoral vasculature. In addition, Birchler et al 20 showed that L19, chemically coupled to a photosensitizer, selectively accumulates in the newly formed blood vessels of the angiogenic rabbit cornea model and, after irradiation with near infrared light, mediates the complete and selective occlusion of ocular neovasculature. More recently, Nilsson et al 21 reported that the immunoconjugate of L19 with the extracellular domain of tissue factor mediates selective infarction in different types of murine tumor models. Furthermore, the cytokines int...

Interleukin-12 (IL-12) is a heterodimeric cytokine with potent immunostimulatory activity and anti-angiogenic properties. Its clinical applications are limited, however, by severe side-effects. Here we report that an IL-12 fusion protein, consisting of IL-12 fused to a human antibody fragment specific to the oncofetal ED-B domain of fibronectin, markedly enhances the antitumor activity of this cytokine, as demonstrated in a mouse lung-metastasis model and in two models of mice bearing different aggressive murine tumors. The residual small tumor masses seen in the treated mice were infiltrated with lymphocytes, macrophages, and natural killer cells and had elevated interferon gamma (IFN-gamma). These results are of therapeutic relevance as the ED-B domain of fibronectin, a naturally occurring marker of angiogenesis identical in mouse and man, is expressed in the majority of aggressive solid tumors but is not detectable in normal vessels and tissues.