Transforming growth factor-β1 (TGFβ1), a major promoter of myofibroblast differentiation, induces α-smooth muscle (sn) actin, modulates the expression of adhesive receptors, and enhances the synthesis of extracellular matrix (ECM) molecules including ED-A fibronectin (FN), an isoform de novo expressed during wound healing and fibrotic changes. We report here that ED-A FN deposition precedes α-SM actin expression by fibroblasts during granulation tissue evolution in vivo and after TGFβ1 stimulation in vitro. Moreover, there is a correlation between in vitro expression of α-SM actin and ED-A FN in different fibroblastic populations. Seeding fibroblasts on ED-A FN does not elicit per se α-SM actin expression; however, incubation of fibroblasts with the anti-ED-A monoclonal antibody IST-9 specifically blocks the TGFβ1-triggered enhancement of α-SM actin and collagen type I, but not that of plasminogen activator inhibitor-1 mRNA. Interestingly, the same inhibiting action is exerted by the soluble recombinant domain ED-A, but neither of these inhibitory agents alter FN matrix assembly. Our findings indicate that ED-A–containing polymerized FN is necessary for the induction of the myofibroblastic phenotype by TGFβ1 and identify a hitherto unknown mechanism of cytokine-determined gene stimulation based on the generation of an ECM-derived permissive outside in signaling, under the control of the cytokine itself.

We recently demonstrated that a human recombinant scFv, L19, reacting with the ED-B domain of fibronectin, a marker of angiogenesis, selectively targets tumoral vasculature in vivo. Using the variable regions of L19, we constructed and expressed a human "small immunoprotein" (SIP) and a complete human IgG1 and performed biodistribution studies in tumor-bearing mice to compare the blood clearance rate, in vivo stability and performance in tumor targeting of the 3 L19 formats [dimeric scFv (scFv) 2 , SIP and IgG1]. The accumulation of the different antibody formats in the tumors studied was a consequence of the clearance rate and in vivo stability of the molecules. Using the SIP, the %ID/g in tumors was 2-5 times higher than that of the (scFv) 2 , reaching a maximum 4 -6 hr after injection. By contrast, the accumulation of IgG1 in tumors constantly rose during the experiments. However, due to its slow clearance, the tumor-blood ratio of the %ID/g after 144 hr was only about 3 compared to a ratio of 10 for the (scFv) 2 and 70 for the SIP after the same period of time. The different in vivo behavior of these 3 completely human L19 formats could be exploited for different diagnostic and/or therapeutic purposes, depending on clinical needs and disease. Furthermore, the fact that ED-B is 100% homologous in human and mouse, which ensures that L19 reacts equally well with the human and the murine antigen, should expedite the transfer of these reagents to clinical trials. © 2002 Wiley-Liss, Inc. Key words: antibody formats; tumor vasculature; tumor targeting; clinical applications; cancer diagnosis and therapyDespite their enormous potential as therapeutic agents, monoclonal antibodies (mAbs) of nonhuman origin have not performed as well as expected in clinical trials as a result of their immunogenicity, 1,2 poor pharmacokinetic properties 3,4 and inefficiency in recruiting effector functions. 5,6 The recent prospect of isolating human antibody fragments from phage display libraries 7-10 transcends these problems, revitalizing studies and rekindling hopes of using these reagents to treat major diseases. Indeed, these molecules should serve as ideal building blocks for novel diagnostic and therapeutic tools. 11,12 Furthermore, these antibodies can be "matured" to reach affinities in the picomolar range, 13 desirable, if not necessary, for their clinical use. 14,15 Clinical applications of human antibody fragments for the selective delivery of diagnostic or therapeutic agents nonetheless require highly specific targets. In the case of tumors, the most popular targets are cell-surface antigens, which are usually neither abundant nor stable. On the other hand, during tumor progression the microenvironment surrounding tumor cells undergoes extensive modification that generates a "tumoral environment" that could ultimately represent a suitable target for antibody-based tumor therapy. 16 In fact, the concept that the altered tumor microenvironment is itself a carcinogen that can be targeted is increasingly gaining consensus. Mol...

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.

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...