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...
Abstract. Fibronectin (FN) represents the mixture of a number of structurally different molecules (isoforms) whose make-up varies depending on the FN sources. FN from cultured transformed human cells has a very different isoform composition with respect to its normal counterpart. In fact, SV-40-transformed WI-38VAI3 human fibroblasts produce high levels of a FN isoform (B-FN) which is very poorly expressed in their normal, WI-38, counterpart. We have recently demonstrated that the B-FN isoform derives from a differential splicing pattern of the FN primary transcript which leads, in transformed cells, to a high level expression of the exon ED-B (Zardi, L., B. Carnemolla, A. Siri, T. E. Petersen, G. Paolella, G. Sebastio, and F. E. . EMBO (Eur. Mol. Biol. Organ.) J. 6:2337-2342). Here we report on the production and characterization of a monoclonal antibody (BC-1) which recognizes an epitope within the protein sequence coded for by the ED-B exon. This monoclonal antibody makes it possible to carry out immunohistochemical analysis of the distribution of the ED-B-containing FN isoform (B-FN) in human tissues. The results show that while in normal, adult, human tissues total FN has a widespread distribution, the B-FN isoform is restricted only to synovial cells, to some vessels and areas of the interstitium of the ovary, and to the myometrium. On the contrary, the B-FN isoform has a much greater expression in fetal and tumor tissues. These results demonstrate that, in vivo, different FN isoforms have a differential distribution and indicate that the B-FN isoform may play a role in ontogenesis and oncogenetic processes. It has been previously demonstrated that FN polymorphism is at least partially caused by alternative splicing schemes in three regions of the primary transcript of a single gene which may generate 20 different FN subunit isoforms (18,19,29). One of these regions (IIICS, see Fig. 1 A) is between the last two type lZI homology repeats; a single exon is subdivided to yield five alternative patterns of splicing. Hynes and co-workers (34) showed that inclusion of the IIICS sequence contributed to the differences in size between the larger and smaller subunit of plasma FN. Humphries et At the second region of variation (ED-A), a single exon encoding a complete type III repeat is either included or omitted from the mature mRNA. This variation is tissue specific and the ED-A sequence is absent in the mRNA of liver (10,20,21) which is the source of plasma FN (38). Using a rabbit antiserum to the rat ED-A segment, Paul et al. (31) At the third region of variation (ED-B), a single exon encoding a complete type III repeat is either included or omitted from the mature mRNA (14,33,44). The ED-B sequence presents two interesting peculiarities: (a) it is the more conserved FN region, 100 and 96% homology with rat and chicken FN, respectively (28,44); and (b) this exon is highly expressed in transformed human cells, while it is barely de-
Purification and amino acid sequence analysis of a proteolytic fragment of fibronectin (FN) from transformed human cells demonstrated that a high percentage of these FN molecules contains an extra amino acid sequence which is present only in a very low percentage of FN molecules from normal fibroblasts and is undetectable in plasma FN. This new amino acid sequence introduces into the FN molecule a site very sensitive to a number of proteolytic enzymes. By analyzing the cellular mRNA and genomic clones, we have demonstrated that this sequence derives from a differential splicing pattern of the FN mRNA precursors, which leads in transformed cells to a high‐level expression of an extra type III homology repeat (ED‐B) coded for by a previously unobserved exon. Here we also report the complete sequence of this new exon. These results demonstrate that in malignant cells the mechanisms regulating the splicing of FN mRNA precursors are altered.
Two major tenascin-C (TN-C) isoforms are generated by the alternative splicing of the pre-mRNA. The large isoform contains seven extra type three repeats that, by contrast, are omitted in the small TN-C isoform. The large TN-C isoform is mainly expressed at the onset of cellular processes that entail active cell migration, proliferation, or tissue remodeling such as occur in neoplasia, wound healing, and during development. Thus, the large TN-C isoform seems to be a specific component of the provisional extracellular matrix. Here we have studied the degradation of the large and small TN-C isoforms by matrix metalloproteinases (MMPs) 2, 3, 7, and 9. Among these proteolytic enzymes only MMP-7 can degrade the small TN-C isoform removing the NH2-terminal knob. The large TN-C isoform shows the same MMP-7-sensitive site adjacent to the NH2-terminal sequence, but is further degraded in the splicing area where three fibronectin-like type III repeats are completely digested. Moreover, the large TN-C isoform is degraded by MMP-2 and MMP-3 which completely digest a single type III repeat inside the splicing area. By contrast, the large TN-C isoform is resistant to MMP-9 digestion. The results show that the presence of the spliced sequence introduces new protease-sensitive sites in the large TN-C isoform.
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