The alternatively spliced extra-domain B of fibronectin is one of the best characterized markers of tumor angiogenesis. Similarly, the extra-domain A (EDA), which can also be inserted in the fibronectin transcript by a mechanism of alternative splicing, has been shown to preferentially accumulate around new blood vessels in certain tumors, but this antigen has not been investigated so far as a target for antibody-based biomolecular intervention. We here describe the generation of 3 human monoclonal antibodies (named F8, B7 and D5), which recognize the same epitope of EDA, but which differ in terms of their dissociation constant to the human antigen (K D 5 3.1, 16 and 17 nM, measured for monomeric preparations of the F8, B7 and D5 antibodies, respectively, in recombinant scFv format). When the 3 antibody fragments were cloned and expressed with a 5 amino acid linker, the 3 resulting homodimeric antibody preparations displayed comparable tumor: organ ratios in quantitative biodistribution studies, performed in immunocompetent 129SvEv mice, bearing subcutaneous syngeneic F9 murine tumors. The percent injected dose per gram (%ID/g) values in tumors 24 hr after intravenous injection were 9.3, 10.2 and 13 for F8, B7 and D5, respectively. The F8 antibody may serve as useful building block for the development of antibody-based targeted anti-cancer therapeutics. Preclinical and clinical investigations are facilitated by the fact that F8 recognizes the human and mouse antigen with comparable affinity, and by the observation that EDA over-expression is detectable not only in solid tumors, but also in hematological malignancies.

One of the most promising new avenues for the development of more selective and efficacious cancer therapies relies on the antibody-mediated targeted delivery of bioactive agents (e.g., cytokines) to the tumor environment. The identification of quantitative differences in the expression of accessible vascular proteins in metastatic lesions and host organs facilitate the development of antibody-based strategies, which should be highly efficient and selective, considering the fact that an over-exuberant neovasculature is a characteristic feature of aggressive cancers, and that tumor blood vessels are readily accessible for i.v. administered therapeutic agents. Metastasis is the main cause of death in cancer. The availability of metastasis-specific antigens accessible from the bloodstream will allow a selective delivery of therapeutic agents to metastatic lesions using antibodies as vehicles. Using a combination of vascular biotinylation of 129Sv mice bearing F9 liver metastases and mass spectrometry, we have identified 435 accessible proteins in metastasis and host organ specimens, of which 117 were exclusively detected in metastases. In particular, we found that the alternatively spliced extradomain A (EDA) of fibronectin is strongly expressed in the neovasculature of liver metastases, while being undetectable in most normal organs. A human antibody to EDA was used to show EDA expression in the neovasculature of metastases and primary tumors of human cancer patients and to target mouse liver metastases and subcutaneous tumors in vivo. Human antibody fragments specific to the EDA domain of fibronectin promise to serve as general vehicles for the efficient and selective delivery of imaging agents or therapeutic molecules to metastatic sites. [Cancer Res 2007;67(22):10948-57]

The antibody-mediated delivery of therapeutic agents to sites of angiogenesis is an attractive strategy for anticancer therapy, but is largely unexplored in hematologic malignancies. In the present study, we show that the extra domain B (EDB) of fibronectin, a marker of angiogenesis, is expressed in B-cell non-Hodgkin lymphoma (NHL) and that the human monoclonal anti-EDB antibody L19 can selectively localize to the lymphoma-associated subendothelial extracellular matrix. In vivo, the preferential accumulation of the antibody at the tumor site was confirmed by quantitative biodistribution analyses with radioiodinated antibody preparations. The fusion protein L19-IL2, which mediates the delivery of interleukin-2 (IL-2) to the neovasculature, displayed a superior antilymphoma activity compared with unconjugated IL-2 in localized and systemic xenograft models of NHL. When coadministered with rituximab, L19-IL2 induced complete remissions of estab- IntroductionConventional cytotoxic therapies of cancer often do not discriminate between tumor and normal tissues. To achieve therapeutically relevant concentrations in the tumor mass, large drug doses have to be administered to the patient, leading to a poor therapeutic index and unacceptable toxicities to healthy tissues. The selective delivery of therapeutic agents to the tumor site using antibodies against tumor-associated antigens represents a promising strategy to overcome the disadvantages of conventional cancer therapies. 1-3 Antigens expressed in the tumoral neovasculature are especially attractive targets for antibody-based pharmacodelivery applications due to their inherent accessibility for blood-borne agents. [4][5][6] The efficacy of targeting either tumor endothelial cells or the modified subendothelial extracellular matrix has been demonstrated in a variety of animal models of solid cancers using antibodies functionalized with different effector moieties, 4,[7][8][9][10][11][12][13][14] leading to the clinical development of immunocytokines and radioimmunoconjugates for the therapy of solid tumors. 6,15 Tumortargeting strategies based on the preferential accumulation of biopharmaceuticals around new blood vessels could also be conceivable for the therapy of leukemias and lymphomas, since the dependence of hematologic malignancies on a functional neovasculature has been highlighted already a decade ago. 16,17 Non-Hodgkin lymphoma (NHL) is the most common hematologic malignancy, with now more than 60 000 new cases being diagnosed each year in the United States. 18 The approval of rituximab, a chimeric monoclonal immunoglobulin G1 (IgG1) antibody specific to CD20, represented a major step toward a more selective and effective therapy of NHLs of B cell origin. While first shown to be effective in relapsed follicular lymphoma, anti-CD20 immunotherapy is nowadays incorporated in front-line therapy schemes of follicular and diffuse large B-cell lymphoma. 18 However, in spite of the unquestionable clinical effectiveness of rituximab, a high percentage of patients ...