Affibody Molecules for In vivo Characterization of HER2-Positive Tumors by Near-Infrared Imaging

Abstract

Purpose: 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. Experimental Design: We used three different types of HER2-specific Affibody molecules [monomer Z HER2:342 , dimer (Z HER2:477 ) 2 , and albumin-binding domain-fused-(Z HER2:342 ) 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-(Z HER2:342 ) 2 -Alexa Fluor 750 conjugate to be an optimal probe for optical imaging of HER2 in vivo. Conclusion: 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.

Dual-Modality Image-Guided Surgery of Prostate Cancer with a Radiolabeled Fluorescent Anti-PSMA Monoclonal Antibody

“…The use of targeted imaging approaches could allow more sensitive and real-time detection of cancerous foci and positive surgical margins in the submillimeter range and would permit patient-tailored surgical interventions. Several groups so far have reported the use of fluorophores conjugated to monoclonal antibodies targeting receptors such as the epidermal growth factor receptor (16), HER2/neu receptor (17), and vascular endothelial growth factor (18).…”

Affibody molecules: Engineered proteins for therapeutic, diagnostic and biotechnological applications

“…Should the affinity of the primary leads not suffice, affinity maturation generally results in improved binders and may be achieved by either helix shuffling or sequence alignment combined with directed combinatorial mutagenesis [9,10]. The newly identified molecules with their altered binding surface generally keep PET 68 Ga, 76 Br, 64 Cu 18 F, 124 I [27] SPECT 111 In, 99m Tc, 125 I 57 Co [26] Bacterial toxin PE38 [50] Fc fusion RSV [84] Optical MRI Conjugated fluorescence [47,48] Fused fluorescent protein [90] …”

“…A disadvantage with optical imaging is that the technique is at best semi-quantitative and that the sensitivity is lower. The lower sensitivity may be an explanation for a somewhat surprising finding by Capala and co-workers [47]. They compared a monomeric HER2-specific Affibody molecule, alone or as fused to an albumin binding domain (ABD), with the antibody trastuzumab, all labeled with Alexa Fluor 750 (a near infrared probe).…”

PET-CT Imaging in Breast Cancer Patients: New Tracers, Future Directions

“…The preliminary results of this study are expected to be published in the autumn of 2013, the end of the study will be in 2015 [46]. [47][48][49][50]. However other randomized studies with larger number of enrolled patients will also be required to further analyze the significance of HER2-imaging in treatment planning and daily oncological practice.…”