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PEDS Advance Access published online on June 8, 2004
Protein Engineering Design and Selection, doi:10.1093/protein/gzh040
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Received December 19, 2003
Revised March 18, 2004
Accepted April 21, 2004

Article

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

Tove Olafsen 1*, Giselle J. Tan 2, Chia-Wei Cheung 2, Paul J. Yazaki 2, Jinha M. Park 1, John E. Shively 3, Lawrence E. Williams 4, Andrew A. Raubitschek 5, Michael F. Press 6, Anna M. Wu 7

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

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


  Abstract

An engineered antibody fragment (minibody; scFv-CH3{gamma}1 dimer, Mr 80,000) specific for 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 µg/ml, 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 hingeminibody was evaluated in athymic mice, bearing MCF7/HER2 xenografts. Maximum tumor uptake was 5.6 (±1.65)% injected dose/g (ID/g) at 12 hrs, which was lower than that of the anti-CEA minibody, whereas the blood clearance ({beta}-phase, 5.62 hrs) was similar. Thus, minibodies with different specificities display similar pharmacokinetics, while tumor uptake may vary depending on the antigen-antibody system.

Keywords: antibody fragments, HER2, minibody, breast cancer, biodistribution


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