: A novel, promising strategy for cancer diagnosis and therapy is the use of a pretargeting approach. For this purpose, the non-natural DNA/RNA analogues Peptide Nucleic Acids (PNAs) are ideal candidates as in vivo recognition units due to their high metabolic stability and lack of unspecific accumulation. In the pretargeting approach, an unlabeled, highly specific antibody–PNA conjugate has sufficient time to target a tumor before administration of a small fast-clearing radiolabeled complementary PNA that hybridizes with the antibody–PNA conjugate at the tumor site. Herein, we report the first successful application of this multistep process using a PNA-modified epidermal growth factor receptor (EGFR) specific antibody (cetuximab) and a complementary 99mTc-labeled PNA. In vivo studies on tumor bearing mice demonstrated a rapid and efficient in vivo hybridization of the radiolabeled PNA with the antibody–PNA conjugate. Decisively, a high specific tumor accumulation was observed with a tumor-to-muscle ratio of >8, resulting in a clear visualization of the tumor by single photon emission computed tomography (SPECT) A novel, promising strategy for cancer diagnosis and therapy is the use of a pretargeting approach. For this purpose, the non-natural DNA/RNA analogues Peptide Nucleic Acids (PNAs) are ideal candidates as in vivo recognition units due to their high metabolic stability and lack of unspeci fi c accumulation. In the pretargeting approach, an unlabeled, highly speci fi c antibody – PNA conjugate has su ffi cient time to target a tumor before administration of a small fast-clearing radiolabeled complementary PNA that hybridizes with the antibody – PNA conjugate at the tumor site. Herein, we report the fi rst successful application of this multistep process using a PNA-modi fi ed epidermal growth factor receptor (EGFR) speci fi c antibody (cetuximab) and a complementary 99m Tc-labeled PNA. In vivo studies on tumor bearing mice demonstrated a rapid and e ffi cient in vivo hybridization of the radiolabeled PNA with the antibody – PNA conjugate. Decisively, a high speci fi c tumor accumulation was observed with a tumor-to-muscle ratio of >8, resulting in a clear visualization of the tumor by single photon emission computed tomography (SPECT).

BACKGROUND AND PURPOSE:Systemic molecular radiotherapy utilizes internal irradiation by radionuclide-labeled tumor-targeting agents with the potential to destroy (micro-)metastases. However, doses that are applicable in solid tumors do not reach the levels nessecary for tumor control. Thus, the combination of molecular and external radiotherapy is a promising treatment strategy, as enhanced tumor doses can be delivered with and without minor overlapping toxicities. Here, we combined a 90Y-labeled anti-EGFR antibody (Cetuximab) with clinically relevant fractionated radiotherapy in a preclinical trial using head and neck squamous cell carcinoma xenograft tumors.MATERIALS AND METHODS:To model 90Y-Cetuximab uptake for treatment schedule optimization, FaDu-bearing mice were injected with near-infrared-labeled-Cetuximab at different time points during radiotherapy with differing doses. Cetuximab uptake was longitudinally followed by in vivo-optical imaging. Tumor control probability experiments with fractionated radiotherapy (30 fx, 6 weeks, 8 dose groups/ arm) in combination with 90Y-Cetuximab were performed to test the curative potential.RESULTS:Imaging of near-infrared-labeled-Cetuximab uptake revealed that low to moderate external beam doses can enhance antibody uptake. Using the optimized schedule, combination of molecular and external radiotherapy using 90Y-Cetuximab at a dose that did not result in permanent tumor inactivation in previous experiments, led to substantially increased tumor control compared to radiotherapy alone.CONCLUSION:Our results indicate that combination of radiolabeled therapeutics with clinically relevant fractionated radiotherapy has a remarkable potential to improve curative treatment outcome. Application of some radiation dose prior to injection may improve drug uptake and enable patient stratification and treatment personalization via a corresponding PET-tracer during therapy.

Unnatural mirror image l-configured oligonucleotides (L-ONs) are a convenient substance class for the application as complementary in vivo recognition system between a tumor specific antibody and a smaller radiolabeled effector molecule in pretargeting approaches. The high hybridization velocity and defined melting conditions are excellent preconditions of the L-ON based methodology. Their high metabolic stability and negligible unspecific binding to endogenous targets are superior characteristics in comparison to their d-configured analogs. In this study, a radiopharmacological evaluation of a new l-ONs based pretargeting system using the epidermal growth factor receptor (EGFR) specific antibody cetuximab (C225) as target-seeking component is presented. An optimized PEGylated 17mer-L-DNA was conjugated with p-SCN-Bn-NOTA (NOTA') to permit radiolabeling with the radionuclide 64Cu. C225 was modified with the complementary 17mer-L-DNA (c-L-DNA) strand as well as with NOTA' for radiolabeling and use for positron emission tomography (PET). Two C225 conjugates were coupled with 1.5 and 5.0 c-L-DNA molecules, respectively. In vitro characterization was done with respect to hybridization studies, competition and saturation binding assays in EGFR expressing squamous cell carcinoma cell lines A431 and FaDu. The modified C225 derivatives exhibited high binding affinities in the low nanomolar range to the EGFR. PET and biodistribution experiments on FaDu tumor bearing mice with directly 64Cu-labeled NOTA'3-C225-(c-L-DNA)1.5 conjugate revealed that a pretargeting interval of 24 h might be a good compromise between tumor accumulation, internalization, blood background, and liver uptake of the antibody. Despite internalization of the antibody in vivo pretargeting experiments showed an adequate hybridization of 64Cu-radiolabeled NOTA'-L-DNA to the tumor located antibody and a good tumor-to-muscle ratio of about 11 resulting in a clearly visible image of the tumor after 24 h up to 72 h. Furthermore, low accumulation of radioactivity in organs responsible for metabolism and excretion was determined. The presented results indicate a high potential of complementary L-ONs for the pretargeting approach which can also be applied to therapeutic radionuclides such as 177Lu, 90Y, 186Re, or 188Re.

The first successful application of a pretargeting approach using a PNA-modified epidermal growth factor receptor specific antibody and a complementary 99mTc-labeled PNA is presented.

Department of Chemistry, University of Zu Zurich, Switzerland. E-mail: gilles.ga gassergroup.com; Tel: +41 44 635 46 30 Helmholtz-Zentrum Dresden – Rossendorf, Research, Bautzner Landstraße 400, Dstephan@hzdr.de; Web: http://www.hzdr.d 3091 Lehrstuhl für Anorganische Chemie I – Bioa und Biochemie, Ruhr-Universität Bochum, U Germany † Electronic supplementary information PNAs used this study (Fig [Tc](Tc-Dpa)-(Cys-PEG10kDa)-PNA (Fig. three complementary 17-mer PNA sys different PNAs (Fig. S16), radio HPLC of t (original), in rat arterial blood plasma (Fi PNAs in Wistar rats (%ID mean radiolabeled PNAs in Wistar rats (SUV comparison of the biodistribution of [ and mouse (Fig. S18). See DOI: 10.1039/c5 ‡ These authors have contributed equally § Current address: Department of On University Avenue, Edmonton, Alberta, T6 Cite this: Chem. Sci., 2015, 6, 5601

Two basic and simple synthetic routes for mono- and bis-maleimide bearing 1,4,7-triazacyclononane-N,N',N''-triacetic acid (NOTA) chelators as new bifunctional chelators are described. The syntheses are characterized by their simplicity and short reaction times, as well as practical purification methods and acceptable to very good chemical yields. The usefulness of these two synthetic pathways is demonstrated by the preparation of a set of mono- and bis-maleimide functionalized NOTA derivatives. In conclusion, these two methods can easily be expanded to the syntheses of further tailored maleimide-NOTA chelators for diverse applications.