HIGHLY SPECIFIC IN-VIVO TUMOR TARGETING BY MONOVALENT AND DIVALENT FORMS OF 741F8 ANTI-C-ERBB-2 SINGLE-CHAIN FV

The in vivo properties of monovalent and divalent single-chain Fv (sFv )-based molecules with the specificity of the anti-c-erbB-2 monoclonal antibody 741F8 were examined in scid mice bearing SK-OV-3 tumor xenog rafts. 741F8 sFv monomers exhibited rapid, biphasic clearance from blo od, while a slightly slower clearance was observed with the divalent 7 41F8 (sFv')2 comprising a pair of 741F8 sFv' with a C-terminal Gly4Cys joined by a disulfide bond. Following i.v. injection, the 741F8 sFv m onomer was selectively retained in c-erbB-2-overexpressing SK-OV-3 tum or, with excellent tumor:normal organ ratios uniformly exceeding 10:1 by 24 h. The specificity of this effect was demonstrated by the lack o f retention of the anti-digoxin 26-10 sFv monomer, as evaluated by bio distribution studies, gamma camera imaging, and cryomacroautoradiograp hy studies. The specificity index (741F8 sFv retention/26-10 sFv reten tion) of 741F8 monomer binding, measured by the percentage of injected dose per g of tissue, was 13.2:1 for tumor, and 0.8 to 2.1 for all te sted normal organs, with specificity indices for tumor:organ ratios ra nging from 7.0 (kidneys) to 16.7 (intestines). Comparing divalent 741F 8 (sFv')2 with the 26-10 (sFv')2, similar patterns emerged, with speci ficity indices for retention in tumor of 16.9 for the Gly4Cys-linked ( sFv')2. These data demonstrate that, following their i.v. administrati on, both monovalent and divalent forms of 741F8 sFv are specifically r etained by SK-OV-3 tumors. This antigen-specific binding, in conjuncti on with the 26-10 sFv controls, precludes the possibility that passive diffusion and pooling in the tumor interstitium contributes significa ntly to long-term tumor localization. 741F8 (sFv')2 species with pepti de spacers exhibited divalent binding and increased retention in tumor s as compared with 741F8 sFv monomers. Since the blood retention of th e (sFv')2 is slightly more prolonged than that of the monomer, it was necessary to demonstrate that the increased tumor localization of the peptide-linked (sFv')2 was due to its divalent nature. The significant ly greater localization of the divalent bismalimidohexane-linked 741F8 (sFv')2 as compared with a monovalent 741F8 Fab fragment of approxima tely the same size suggests that the increased avidity of the (sFv')2 is a factor in its improved tumor retention. This is the first report of successful specific in vivo targeting of tumors by divalent forms o f sFv molecules. The improved retention of specific divalent (sFv')2 b y tumors may have important consequences for targeted diagnostic or th erapeutic strategies.