Characterization of engineered anti-p185HER-2 (scFv-CH3)2 antibody fragments (minibodies) for tumor targeting.
An engineered antibody fragment (minibody; scFv-C(H)3gamma(1) dimer, M(r) 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- p185(HER-2) 10H8 antibody were similarly assembled and expressed. Four constructs were made: first, the V genes were assembled in both orientations (V(L)-linker-V(H) and V(H)-linker-V(L)) as single chain Fvs (scFvs). Then each scFv was fused to the human IgG1 C(H)3 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 p185(HER-2) overexpressing breast cancer cell line, MCF7/HER2. Binding affinities (K(D) approximately 2-4 nM) were equivalent to that for the parental 10H8 mAb (K(D) approximately 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 (beta-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.
Olafsen
Tove
T
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, USA. tolafsen@mednet.ucla.edu
Tan
Giselle J
GJ
Cheung
Chia-Wei
CW
Yazaki
Paul J
PJ
Park
Jinha M
JM
Shively
John E
JE
Williams
Lawrence E
LE
Raubitschek
Andrew A
AA
Press
Michael F
MF
Wu
Anna M
AM
D000595:Amino Acid Sequence; D000818:Animals; D045744:Cell Line, Tumor; D005434:Flow Cytometry; D006801:Humans; D007128:Immunoglobulin Fragments / Q000737:chemistry* / Q000627:therapeutic use*; D007150:Immunohistochemistry; D008325:Mammary Neoplasms, Experimental / Q000628:therapy*; D051379:Mice; D008819:Mice, Nude; D008969:Molecular Sequence Data; D015202:Protein Engineering; D018719:Erb-b2 Receptor Tyrosine Kinases / Q000276:immunology*
D007128:Immunoglobulin Fragments; D018719:Erb-b2 Receptor Tyrosine Kinases