Targeting of bivalent anti-ErbB2 diabody antibody fragments to tumor cellsis independent of the intrinsic antibody affinity

In immunodeficient mice antitumor single-chain Fv (scFv) molecules penetrat e tumors rapidly and have rapid serum clearance, leading to excellent tumor :normal organ ratios. However, the absolute quantity of scFv retained in th e tumor is love due to rapid serum clearance and monovalent scFv binding. W e previously demonstrated that the presence of an additional binding site p rolongs in vitro and in vivo association of scFv-based molecules with tumor cells expressing relevant antigen. The contribution of the intrinsic affin ity of each component scFv to the association between a dimeric scFv and it s target antigen is largely unknown. Here, we have constructed bivalent dia body molecules From three affinity mutants of the human anti-ErbB2 (HER2/ne u) scFv molecule C6.5 by shortening the peptide linker between the heavy (V -H) and light (V-L) chains variable domains from 15 to 5 amino acids. The s horter linker prevents intramolecular pairing of V-H and V-L, resulting in intermolecular pairing and creation of a dimeric M-r 50,000 molecule with t wo antigen-binding sites. The scFv used to create the diabodies span a 133- fold range of affinity fur the same epitope of ErbB2 [133 nM (C6G98A), 25 n n (C6.5), and 1 nhl (C6ML3-9)] and differ by only one to three amino acids. Diabody binding kinetics were determined by surface plasmon resonance on t he immobilized ErbB2 extracellular domain. The association rate constants o btained for each diabody molecule were similar to that of the parental (com ponent) scFv. However, the dissociation rate constants obtained for the biv alent diabodies were up to 15-fold slower. The magnitude of the decrease in the bivalent dissociation rate constant was inversely proportional to the monovalent interaction, ranging from only 3-fold fur that of the C6ML-9 dia body to 15-fold for the C6G98A diabody, This resulted in only a 22-fold dif ference in bivalent affinity, compared with a 133-fold difference in affini ty fur the respective scFv. Equilibrium-binding constants obtained by surfa ce plasmon resonance correlated well with the equilibrium-binding constants determined in vitro on ErbB2 overexpressing cells. Biodistribution studies were performed in scid mice bearing established SKOV3 tumors. At 24 h, 3-3 7-fold more diabody was retained in tumor compared with the parental scFv m onomers. This likely results from a higher apparent affinity, because of bi valent binding, and a slower serum clearance. Surprisingly, the differences in affinity between diabodies did not result in differences in quantitativ e tumor retention or tumor to blood ratios. In fact, the diabody constructe d from the lowest affinity scFv exhibited the best tumor-targeting properti es. We conclude that, above a threshold affinity, other factors regulate qu antitative tumor retention. In addition, straightforward dimerization of a low-affinity scFv leads to significantly greater tumor localization than do es exhaustive scFv affinity maturation.