Increased binding affinity and valence of recombinant antibody fragments lead to improved targeting of tumoral angiogenesis

The formation of new blood vessels (angiogenesis) is an important step in t umor progression. Molecules capable of selectively targeting markers of ang iogenesis may offer opportunities for the in vivo imaging of aggressive tum ors and for the delivery of toxic agents to the tumoral vasculature, Using antibody phage display libraries and combinatorial mutagenesis, Ne isolated single-chain Fv antibody fragments, which recognize with different affinit ies the same epitope of the ED-B domain of fibronectin, a marker of angioge nesis, Two single-chain Fv fragments, El and L19,,vith dissociation constan ts of 41 nM and 0.054 nM, respectively, were investigated for their ability to target F9 murine teratocarcinoma grafted s,c. in nude mice when injecte d i,v, in either monomeric or homodimeric form (M-r 27,000 and 54,000, resp ectively). Biodistribution studies, performed at two time points (4 h and 2 4 h) with radiolabeled samples, showed that the higher affinity antibody ta rgets the tumor significantly better than the lower affinity one, in terms both of tumor:organ ratios and of the amounts of antibody delivered to the tumor. In particular, more than 20% of the injected dose of dimeric L19 acc umulated per gram of tumor at 4 h; the tumor:organ ratios at 4 h and 24 h w ere in the (2.1-8.6):1 and (10.3-29.4):1 range, respectively. This study de monstrates that, although vasculature represents only a small fraction of t he total tumor mass, anti-ED-B antibodies can selectively target tumors in vivo and that this process is particularly efficient if very high-affinity binders are used.