Purpose: ED-B fibronectin is expressed only during angiogenic processes and in tissues undergoing growth and/or extensive remodeling.
We demonstrated previously the possibility to target and ...selectively deliver therapeutic substances to tumor vasculature in
experimental animal models using a human recombinant antibody fragment, L19, specific for the ED-B domain of fibronectin.
Here we evaluate the possibility of targeting primary tumors and metastatic lesions in cancer patients through immunoscintigraphy
using 123 I-labeled dimeric L19 L19(scFv) 2 .
Experimental Design: Twenty patients (34–79 years of age) with lung, colorectal, or brain cancer, whose tumors had been confirmed by imaging techniques
and/or histologically, were admitted to the immunoscintigraphic investigation.
Results: The dimeric L19 antibody selectively localized in tumor lesions in aggressive types of lung cancer and colorectal cancer.
Because ED-B fibronectin is expressed only during angiogenic processes and in tissues undergoing growth and/or extensive remodeling,
L19(scFv) 2 is able to distinguish between quiescent and actively growing lesions. No side effects were observed.
Conclusions: The ability of L19(scFv) 2 to target tumors in patients provides the foundations for new therapeutic applications, in which the L19 antibody is engineered
to selectively deliver bioactive molecules to primary tumors as well as to metastases.
We have previously reported an anti-fibronectin monoclonal antibody (mAb) (BC-1) which reacts with an ED-B-containing beta-galactosidase-fibronectin
fusion protein but not with an identical ...beta-galactosidase-fibronectin fusion protein in which the ED-B sequence is omitted.
In further experiments aimed at localizing more precisely the epitope recognized by this mAb, we demonstrate that 1) the mAb
BC-1 is indeed specific for ED-B-containing fibronectin (FN) molecules even though the epitope recognized by this mAb is localized
on the type III homology repeat 7 (the one which precedes the ED-B sequence) and 2) in fibronectin molecules lacking the ED-B
sequence, this epitope is masked. We further demonstrate that, to mask the epitope recognized by the mAb BC-1, the presence
of at least half of the FN type III homology repeat 9 is necessary. We also report the production of the mAb IST-6 which recognizes
only FN molecules in which the ED-B sequence is lacking. These data clearly demonstrate that the presence of the ED-B sequence
within FN molecules generates conformational modification in the central part of the molecules that unmasks previously cryptic
sequences and masks others.
The oncofetal fibronectin (B-FN) isoform is present in vessels of neoplastic tissues during angiogenesis but not in mature vessels. B-FN could therefore provide a target for diagnostic imaging and ...therapy of cancer. Phage display libraries have been used to isolate human antibody fragments with pan-species recognition of this isoform. We describe the use of these fragments in nude mice to target an aggressive tumor (grafted F9 murine teratocarcinoma). Imaging in real time was done by infrared photodetection of a chemically coupled fluorophore. The targeting was improved by use of affinity-matured fragments with low kinetic dissociation rates (koff = 1.5 x 10(-4) s-1) and also by engineering dimeric fragments via a C-terminal amphipathic helix.
Tenascin-R is an extracellular matrix protein expressed exclusively in the central nervous system where it is thought to play a relevant role in regulating neurite outgrowth. We have i) cloned the ...cDNA of the rat tenascin-R 5' region; ii) defined its genomic organization, obtaining the sequence of two novel untranslated exons; iii) mapped the gene to rat chromosome 13q23 and suggested a previously unreported synteny between rat chromosome 13q23, human chromosome 1q24, and mouse chromosome 4E; and iv) sequenced and characterized the elements responsible for its neural cell-restricted transcription. We found that two discrete regions of the rat gene (the first in the proximal promoter, the second in the first exon) are independently able to activate to a high degree the transcription of a reporter gene in either human or rat neuroblastoma cell lines but not in other cell lines. Based on this observation, we re-evaluated the arrangement of transcriptionally active regions in the human tenascin-R gene we recently cloned and found that the human gene also contains an exon sequence able to initiate and sustain transcription independently of promoter sequences.
Large granular lymphocytes (LGL) are defined as nonadherent mononuclear cells with cytoplasmic azurophilic granules, avid receptors for the Fc portion of IgG, and cytotoxic functions (NK or ADCC ...activities). In the present study, the granules of LGL isolated from human peripheral blood have been analyzed by enzyme cytochemistry and electron microscopy. It had been found that: (1) in the single cells, granules at different stages of maturation could be detected: in addition, packaging of the granules took place in the proximity of the Golgi apparatus, which is similar to that seen in secretory cell types. (2) Acid phosphatase (AP) was observed within the granules and the vesicles located in the Golgi area: the Golgi apparatus identified through its thiamine pyrophosphatase-positivity was consistently negative for AP. (3) Alpha naphthyl-acetate esterase (ANAE) activity was localized in the granules as well as on the membrane of LGL and monocytes. (4) The ANAE activity of LGL was of the monocytic and not of the lymphocytic type, as shown by NaF inhibition. (5) The LGL granules, although identifiable as primary lysosomes, were not involved in the process of phagocytosis, since LGL failed consistently to ingest latex particles or opsonized red cells.
Functionally different tenascin (TN) isoforms containing varying numbers of III homology repeats are generated by alternative splicing of a single TN primary transcript. It has recently been reported ...that the larger TN isoform is, in general, more expressed in neoplastic tissues than in the normal tissues from which the tumor originates. This is due, at least in breast lesions, to the high proliferative activity of stromal elements. In fact, TN splicing is cell-cycle dependent, thus offering a viable system to study the molecular mechanisms that regulate alternative splicing and suggesting that cell-cycle dependent modifications in the splicing pattern of primary transcripts (which very likely are not limited to the TN pre-mRNA) may also be a cell-cycle regulatory mechanism. Furthermore, the very high accumulation of the larger TN isoform in neoplasia allows wider diagnostic and therapeutic monoclonal antibodies specific for the larger TN isoforms be considered for a number of tumors.
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