Comparison of the Biodistribution and Tumor Targeting of Two ^99mTc-Labeled Anti-EGFR Nanobodies in Mice, Using Pinhole SPECT/Micro-CT

Abstract

Camelidae possess an unusual class of antibodies devoid of light chains. Nanobodies are intact antigen-binding fragments that are stable, easily generated against different targets, and fully functional. Their rapid clearance from the blood circulation favors their use as imaging agents. We compared the in vivo tumor uptake and biodistribution of 2 anti-epidermal growth factor receptor (anti-EGFR) Nanobodies, 99m Tc-7C12 and 99m Tc-7D12. Methods: Nanobodies were labeled via their hexahistidine tail with 99m Tc-tricarbonyl ( 99m Tc(CO) 3 ) generated from a kit. Mice bearing subcutaneous A431 (EGFR-positive) and R1M (EGFRnegative) xenografts were intravenously injected with 99m Tc-7C12 and 99m Tc-7D12 on separate days. Pinhole SPECT/ micro-CT images were acquired at 1 h after administration to assess noninvasively the biodistribution and tumor targeting of the labeled compounds. Pinhole SPECT and micro-CT images from the same mouse were automatically fused on the basis of a mathematic rigid-body-transformation algorithm using six 57 Co sources. Images were quantified, and tracer uptake was expressed as percentage injected activity per gram per cubic centimeter (%IA/cm 3 ) of tissue. Ex vivo biodistribution of mice bearing A431 injected with either 99m Tc-7C12 or 99m Tc-7D12 was also assessed; activity in the tumor and organs was recorded and expressed as percentage injected activity per gram (%IA/g). Results: Binding of both tracers was receptorspecific. Image analysis showed high and similar tumor uptake values for both 99m Tc-7C12 and 99m Tc-7D12 (4.55 6 0.24 %IA/ cm 3 and 4.62 6 0.36 %IA/cm 3 , respectively) in A431 xenografts, whereas the uptake in the negative tumor (R1M) was low (1.16 6 0.14 for 99m Tc-7C12 and 1.49 6 0.60 for 99m Tc-7D12). 99m Tc-7C12 showed significantly higher kidney uptake (63.48 6 2.36 vs. 56.25 6 2.46 %IA/cm 3 ) and lower liver uptake (2.55 6 0.26 vs. 4.88 6 0.86 %IA/cm 3 ) than did 99m Tc-7D12. The ex vivo analysis confirmed the image quantification with high tumor-tobackground ratio; however, 99m Tc-7C12 showed higher tumor uptake (9.11 6 1.12 %IA/g) than did 99m Tc-7D12 (6.09 6 0.77 %IA/g). 99m Tc-7D12 demonstrated significantly higher blood activity than did 99m Tc-7C12, but both showed short plasma half-lives (,10 min).Conclusion: The Nanobody fragments used here show high tumor uptake, low liver uptake, and rapid blood clearance. Nanobodies are promising probes for noninvasive radioimmunodetection of specific targets early after administration. On the basis of its favorable biodistribution, 99m Tc-7C12 was selected for further studies. Epi dermal growth factor receptor (EGFR or ErbB1) is a member of a receptor tyrosine kinase family together with Her-2-neu/ErbB2, HER-3/ErbB3, and HER-4/ErbB4. EGFR is implicated in many cellular processes such as proliferation, differentiation, and survival (1). Several reports have shown that EGFR signaling is abnormal in many tumors of epithelial origin, such as cancer of the breast, head and neck, prostate, lung, and skin. Aberrant signaling of...

Localization, mechanism and reduction of renal retention of technetium‐99m labeled epidermal growth factor receptor‐specific nanobody in mice

“…This requires relatively hydrophilic molecules with a molecular size that is inferior to 60 kDa threshold for glomerular filtration. Nanobodies fit this description: they are hydrophilic, have a molecular size of 15 kDa, have a fast blood clearance and show low liver uptake (10). Together with their characteristic of specific targeting with affinities in the nanomolar range, they represent an interesting class of probes for the generic development of molecular imaging agents.…”

“…With the recent identification of a universal humanized nanobody scaffold, these probes are now also ready for clinical translation (8,9). In our previous work we identified 99m Tc-7C12 nanobody (Ablynx NV) as the lead compound for monitoring EGFR expression of cancer based on its high tumor uptake, fast blood clearance and low liver uptake (10,11). High-contrast images of EGFR-expressing lesions can be obtained as early as 1 h post injection (p.i.).…”

“…mice were anesthetized with a mixture of 18.75 mg kg À1 ketamine hydrochloride (Ketamine 1000 1 , CEVA, Brussels, Belgium) and 0.5 mg kg À1 medetomidin hydrochloride (Domitor 1 , Pfizer, Brussels, Belgium). Total body pinhole SPECT/microCT was performed as described previously (10). Briefly, microCT imaging (Skyscan 1178, Aartselaar, Belgium) was followed by pinhole SPECT (e.cam180 Siemens Medical Solutions, IL, USA) on separate imaging systems and images were fused automatically using six 57 Co sources based on a mathematical software (23).…”

Single Domain Antibodies and Bioengineering Drugs on Their Basis: New Opportunities for Diagnostics and Therapy

“…Физико-химические свойства и небольшие размеры наноантител приводят к чрезвычайно быстрому распределению молекул по органам и тканям [47,121].…”

Nanobodies Targeting the Hepatocyte Growth Factor: Potential New Drugs for Molecular Cancer Therapy

“…Initial biodistribution studies with monospecific anti-EGFR (aEGFR)-Nanobodies in tumor-bearing mice showed, as expected, rapid blood clearance with a serum half-life time of less than an hour and low tumor uptake (15)(16)(17). Therefore, monospecific Nanobodies do not seem to be qualified for long-term blockage of growth factors and their receptors.…”