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) = 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.