Characterization of engineered anti-p185HER-2 (scFv-CH3)2 antibody fragments (minibodies) for tumor targeting

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Abstract
An engineered antibody fragment (minibody; scFv-CH3γ1 dimer, Mr 80 000) specific for carcinoembryonic antigen (CEA) has previously demonstrated excellent tumor targeting coupled with rapid clearance in vivo. In this study, variable (V) genes from the anti- p185HER-2 10H8 antibody were similarly assembled and expressed. Four constructs were made: first, the V genes were assembled in both orientations (VL-linker-VH and VH-linker-VL) as single chain Fvs (scFvs). Then each scFv was fused to the human IgG1 CH3 domain, either by a two amino acid linker (ValGlu) that resulted in a non-covalent, hingeless minibody, or by IgG1 hinge and a GlySer linker peptide to produce a covalent, hinge-minibody. The constructs, expressed in NS0 mouse myeloma cells at levels of 20–60 mg/l, demonstrated binding to the human p185HER-2 overexpressing breast cancer cell line, MCF7/HER2. Binding affinities (KD ∼ 2–4 nM) were equivalent to that for the parental 10H8 mAb (KD ∼ 1.6 nM). Radioiodinated 10H8 hinge-minibody was evaluated in athymic mice, bearing MCF7/HER2 xenografts. Maximum tumor uptake was 5.6 (±1.65)% injected dose/g (ID/g) at 12 h, which was lower than that of the anti-CEA minibody, whereas the blood clearance (β-phase, 5.62 h) was similar. Thus, minibodies with different specificities display similar pharmacokinetics, while tumor uptake may vary depending on the antigen–antibody system.

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