Affibody molecules for in vivo characterization of HER2-positive tumors by near-infrared imaging.
OBJECTIVE
HER2 overexpression has been associated with a poor prognosis and resistance to therapy in breast cancer patients. We are developing molecular probes for in vivo quantitative imaging of HER2 receptors using near-infrared (NIR) optical imaging. The goal is to provide probes that will minimally interfere with the studied system, that is, whose binding does not interfere with the binding of the therapeutic agents and whose effect on the target cells is minimal.


METHODS
We used three different types of HER2-specific Affibody molecules [monomer ZHER2:342, dimer (ZHER2:477)2, and albumin-binding domain-fused-(ZHER2:342)2] as targeting agents and labeled them with Alexa Fluor dyes. Trastuzumab was also conjugated, using commercially available kits, as a standard control. The resulting conjugates were characterized in vitro by toxicity assays, Biacore affinity measurements, flow cytometry, and confocal microscopy. Semiquantitative in vivo NIR optical imaging studies were carried out using mice with s.c. xenografts of HER2-positive tumors.


RESULTS
The HER2-specific Affibody molecules were not toxic to HER2-overexpressing cells and their binding to HER2 did interfere with neither binding nor effectives of trastuzumab. The binding affinities and specificities of the Affibody-Alexa Fluor fluorescent conjugates to HER2 were unchanged or minimally affected by the modifications. Pharmacokinetics and biodistribution studies showed the albumin-binding domain-fused-(ZHER2:342)2-Alexa Fluor 750 conjugate to be an optimal probe for optical imaging of HER2 in vivo.


CONCLUSIONS
Our results suggest that Affibody-Alexa Fluor conjugates may be used as a specific NIR probe for the noninvasive semiquantitative imaging of HER2 expression in vivo.
Lee
Sang Bong
SB
Radiation Oncology Branch, Center for Cancer Research, National Cancer Institute, National Institute of Child Health and Human Development, NIH, Bethesda, Maryland, USA.
Hassan
Moinuddin
M
Fisher
Robert
R
Chertov
Oleg
O
Chernomordik
Victor
V
Kramer-Marek
Gabriela
G
Gandjbakhche
Amir
A
Capala
Jacek
J
D000418:Albumins / Q000378:metabolism; D000818:Animals; D000911:Antibodies, Monoclonal / Q000627:therapeutic use; D061067:Antibodies, Monoclonal, Humanized; D000970:Antineoplastic Agents / Q000627:therapeutic use; D005260:Female; D005456:Fluorescent Dyes; D018734:Genes, erbB-2; D006801:Humans; D051379:Mice; D008807:Mice, Inbred BALB C; D008819:Mice, Nude; D009369:Neoplasms / Q000175:diagnosis* / Q000235:genetics / Q000628:therapy; D017434:Protein Structure, Tertiary; D011993:Recombinant Fusion Proteins / Q000737:chemistry; D019265:Spectroscopy, Near-Infrared / Q000379:methods*; D000068878:Trastuzumab; D014407:Tumor Cells, Cultured; D023041:Xenograft Model Antitumor Assays
D000418:Albumins; D000911:Antibodies, Monoclonal; D061067:Antibodies, Monoclonal, Humanized; D000970:Antineoplastic Agents; D005456:Fluorescent Dyes; D011993:Recombinant Fusion Proteins; C515865:Z(HER2.4)2 affibody; D000068878:Trastuzumab