Summary Single-chain Fv (scFv) molecules exhibit highly specific tumour-targeting properties in tumour-bearing mice. However, because of their smaller size and monovalent binding, the quantities of radiolabelled scFv retained in tumours limit their therapeutic applications. Diabodies are dimeric antibody-based molecules composed of two non-covalently associated scFv that bind to antigen in a divalent manner. In vitro, diabodies produced from the anti-HER2/neu (c-erbB-2) scFv C6.5 displayed approximately 40-fold greater affinity for HER2Ineu by surface plasmon resonance biosensor measurements and significantly prolonged association with antigen on the surface of SK-OV-3 cells (tl,2 cell surface retention of > 5 h vs 5 min) compared with C6.5 scFv. In SK-OV-3 tumour-bearing scid mice, radioiodinated C6.5 diabody displayed a highly favourable balance of quantitative tumour retention and specificity. By as early as 4 h after i.v. administration, significantly more diabody was retained in tumour (10 %ID g-1) than in blood (6.7 %ID ml-') or normal tissue (liver, 2.8 %ID g-'; lung, 7.1 %ID g-1; kidney, 5.2 %ID g-1). Over the next 20 h, the quantity present in blood and most tissues dropped approximately tenfold, while the tumour retained 6.5 %ID g-1 or about two-thirds of its 4-h value. In contrast, the 24-h tumour retention of radioiodinated C6.5 scFv monomer was only 1 %ID g-1. When diabody retentions were examined over the course of a 72-h study and cumulative area under the curve (AUC) values were determined, the resulting tumor-organ AUC ratios were found to be superior to those previously reported for other monovalent or divalent scFv molecules. In conclusion, the diabody format provides the C6.5 molecule with a distinct in vitro and in vivo targeting advantage and has promise as a delivery vehicle for therapeutic agents.

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We previously reported the initial characterization of five human single-chain Fv (scFv) antibody fragments specific for the blood group antigens B, D(Rh), E(Rh), Kpb and HI. The scFvs were isolated from a phage-antibody library constructed from the variable region genes of two non-immunized donors. In this paper we report the specificity, affinity and kinetics of antigen binding of these scFv fragments. All five scFvs agglutinated the appropriate red cell phenotype following the addition of a monoclonal antibody which recognizes a peptide tag incorporated into the scFv. The anti-B and anti-HI scFv molecules, which recognize high density carbohydrate antigens, spontaneously polymerized and agglutinated red cells directly. None of the antibody fragments showed cross-reactivity with other red cell antigens, with the exception of the anti-E which reacted weakly with E-negative cells. Specific scFv binding was confirmed by ELISA, flow cytometry and radioactive labelling. The anti-D scFv recognized 17,600 sites on cDE/cDE red cells with an association constant (Ka), of 5.2 x 10(7) M-1 and a rate constant for dissociation (koff) of 1.9 x 10(-2) s-1. The anti-E scFv recognized 29,800 and 39,800 sites on cDE/cDE red cells in two experiments with Kas of 8.4 x 10(6) and 4.4 x 10(7) M-1. The koff for this antibody was 2.7 x 10(-2) s-1. The results demonstrate that scFv antibody fragments specific for cell surface antigens and possessing affinities typical of the primary immune response can be obtained from a phage-display library.

The domain organization of the botulinum neurotoxin serotype A was studied by using antibody mapping of 44 monoclonal single-chain variable fragments. The analysis was carried out on (i) the individual domains of botulinum neurotoxin holotoxin (binding, translocation, and catalytic), (ii) botulinum neurotoxin holotoxin, (iii) the botulinum neurotoxin holotoxin in complex with the nontoxic portion, and (iv) botulinum neurotoxin holotoxin and nontoxic portion of the complex recombined in vitro. All 44 antibodies mapped to individual domains of botulinum neurotoxin. Forty of the 44 single-chain variable fragments bound the botulinum neurotoxin holotoxin relative to the isolated domains, suggesting that 4 epitopes are covered when the individual domains are in the holotoxin form. Only 20 of the antibodies showed a positive reaction to the toxin while in complex with the nontoxic portion. All of the covered epitopes were mapped to the binding domain of botulinum neurotoxin, which suggested that the binding domain is in direct contact with the nontoxic portion in the complex. Based on the antibody mapping to the different domains of the botulinum neurotoxin holotoxin and the entire complex, a model of the botulinum neurotoxin complex is proposed.