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PEDS Advance Access originally published online on June 8, 2004
Protein Engineering Design and Selection 2004 17(4):315-323; doi:10.1093/protein/gzh040
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Protein Engineering, Design & Selection vol. 17 no. 4 © Oxford University Press 2004; all rights reserved

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

Tove Olafsen1,2, Giselle J. Tan3, Chia-wei Cheung3, Paul J. Yazaki3, Jinha M. Park1, John E. Shively4, Lawrence E. Williams5, Andrew A. Raubitschek6, Michael F. Press7 and Anna M. Wu1,3

1Crump Institute for Molecular Imaging, Department of Molecular and Medical Pharmacology, David Geffen School of Medicine at UCLA, 700 Westwood Plaza, Los Angeles, CA 90095, 3Division of Molecular Biology and 4Division of Immunology, Beckman Research Institute of the City of Hope, 1450 East Duarte Road, Duarte, CA 91010, 5Division of Radiology and 6Department of Radioimmunotherapy, City of Hope National Medical Center, 1500 East Duarte Road, Duarte, CA 91010 and 7Department of Pathology, Norris Comprehensive Cancer Center School of Medicine at USC, 1441 Eastlake Avenue, Mailstop 73, Los Angeles, CA 90033, USA

2 To whom correspondence should be addressed. E-mail: tolafsen{at}mednet.ucla.edu

An engineered antibody fragment (minibody; scFv-CH3{gamma}1 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 (ß-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.

Received December 19, 2003; revised March 18, 2004; accepted April 21, 2004.

Edited by Paul Carter


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