A good-quality marker for both tumoral and non-tumoral neovasculature is the extra-domain B (ED-B) of fibronectin, a sequence of 91 amino acids that can be inserted into the fibronectin molecule by a mechanism of alternative splicing.
To date, the production of monoclonal antibodies directly recognizing the ED-B domain has not been possible using hybridoma technology, because of tolerance. In collaboration with Prof. Dr. Luciano Zardi (Istituto Nazionale per la Ricerca sul Cancro, Genova, Italy), we have overcome this problem using large synthetic antibody phage libraries, generating several high-affinity antibody fragments specific for fibronectin containing the ED-B domain, and mapping their epitope onto the three-dimensional structure of the antigen. These antibodies stain vascular structures in tumor sections and selectively target tumor neovasculature, as shown in tumor-bearing mice using infrared fluorescence and radioactive techniques. Increased binding affinity leads to improved targeting of tumoral angiogenesis, as demonstrated by biodistribution studies performed using the L19 antibody fragment with affinity for the ED-B domain in the picomolar range and L19 mutants with reduced affinity.
In this lecture, I will present therapy results obtained in tumor-bearing mice treated with a number of L19-based fusion proteins. Fusion partners for the L19 antibody include tTF (a pro-coagulant factor) and anti-tumor cytokines.
The results obtained are of therapeutic relevance, since the ED-B domain of fibronectin, a naturally occurring marker of angiogenesis identical in mouse and man, is expressed in the majority of aggressive solid tumors, but is undetectable in normal vessels and tissues.
References:
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B. Carnemolla, L. Borsi, E. Balza, P. Castellani, R. Meazza, A. Berndt, S. Ferrini, H. Kosmehl, D. Neri, L. Zardi. (2002) Enhancement of the anti-tumor properties of interleukin-2 by its targeted delivery to the tumor blood vessel extracellular matrix. Blood, in press.
C. Halin, S. Rondini, F. Nilsson, A. Berndt, H. Kosmehl, L. Zardi, D. Neri (2002) Enhancement of the anti-tumor activity of interleukin-12 by targeted delivery to neo-vasculature. Nature Biotechnol., in press.