The human epidermal growth factor receptor 3 (HER3) is overexpressed in several cancers, being linked to a more resistant phenotype and hence leading to poor patient prognosis. Imaging HER3 is challenging owing to the modest receptor number (<50000 receptors/cell) in overexpressing cancer cells. Therefore, to image HER3 in vivo, high target affinity PET probes need to be developed. This work describes two different [(18)F]AlF radiolabeling strategies of the ZHER3:8698 affibody molecule specifically targeting HER3. The one-pot radiolabeling of ZHER3:8698 performed at 100 °C and using 1,4,7-triazanonane-1,4,7-triacetate (NOTA) as chelator resulted in radiolabeled products with variable purity attributed to radioconjugate thermolysis. An alternative approach based on the inverse electron demand Diels-Alder (IEDDA) reaction between a novel tetrazine functionalized 1,4,7-triazacyclononane-1,4-diacetate (NODA) chelator and the trans-cyclooctene (TCO) functionalized affibody molecule was also investigated. This method enabled the radiolabeling of the protein at room temperature. The [(18)F]AlF-NOTA-ZHER3:8698 and [(18)F]AlF-NODA-ZHER3:8698 conjugates showed a specific uptake at 1 h after injection in high HER3-expressing MCF-7 tumors of 4.36 ± 0.92% ID/g and 4.96 ± 0.65% ID/g, respectively. The current results are encouraging for further investigation of [(18)F]AlF-NOTA-ZHER3:8698 as a HER3 imaging agent.

2 Translational RelevanceCurrently, inter-and intra-tumor heterogeneity is a clinical challenge, as histological techniques can fail to provide a representative indication of molecular variation, due to dependence on the section of tumor that is chosen for sampling. This underscores the need to introduce novel imaging biomarkers that allow the examination of the whole tumor mass and may significantly help to better understand and treat cancer. Therefore, we have developed a novel PET radiotracer that will provide information on heterogeneous HER3 expression and receptor expression changes due to downstream signaling inhibition, which is increasingly being recognized as a key player in therapeutic resistance. This would aid in the selection of patients for novel HER3 targeted therapies and potentially enable patients to be spared ineffective therapies, and, if necessary, being switched sooner to more effective therapeutic regimens.Research. Zr-DFO-Z HER3:8698 can track changes in receptor expression in HER3-positive xenograft models and monitor the outcome of AUY922 treatment. Our in vitro findings showed that MCF-7 cells, which are phenotypically different from BT474, develop resistance to AUY922 through HER3/IGF-1R-mediated signaling. Of note, the lack of response in vitro due to HER3 recovery was confirmed in vivo using 89 Zr-DFO-Z HER3:8698 -based imaging. Upon AUY922 treatment, higher radioconjugate uptake was detected in treated MCF-7 xenografts, correlating with an AUY922-induced HER3 up-regulation concomitant with an increase in IGF-1R expression. Conclusion: These data underline the potential of HER3-based PET imaging to noninvasively provide information about HER3 expression and to identify patients not-responding to targeted therapies due to HER3 recovery.

In head and neck squamous cell carcinoma (HNSCC), the human epidermal growth factor receptor 1 (EGFR) is the dominant signaling molecule among all members of the family. So far, cetuximab is the only approved anti-EGFR mAb used for the treatment of HNSCC, but despite the benefits of adding it to standard treatment regimens, attempts to define a predictive biomarker to stratify patients for cetuximab treatment have been unsuccessful. We hypothesized that imaging with EGFR-specific radioligands may facilitate non-invasive measurement of EGFR expression across the entire tumor burden and also allow for dynamic monitoring of cetuximab-mediated changes in receptor expression. EGFR-specific Affibody molecule (ZEGFR:03115) was radiolabeled with zirconium-89 (Zr) and fluorine-18 (F). The radioligands were characterized in vitro and in mice bearing subcutaneous tumors with varying levels of EGFR expression. The protein dose for imaging studies was assessed by injecting Zr-DFO-ZEGFR:03115 (2.4-3.6 MBq, 2 µg) either together with or 30 mins after increasing amounts of unlabeled ZEGFR:03115 (1, 5, 10, 15 and 20 µg). PET images were acquired at 3, 24 and 48 h post-injection and the image quantification data were correlated with the biodistribution results. The EGFR expression and biodistribution of the tracer were assessed ex vivo by immunohistochemistry, Western blot and autoradiography. To downregulate the EGFR level, treatment with cetuximab was performed andF-AlF-NOTA-ZEGFR:03115 (12 µg, 1.5-2 MBq/mouse) used to monitor receptor changes. In vivo studies demonstrated that co-injecting 10 µg of non-labeled molecules withZr-DFO-ZEGFR:03115 allows for clear tumor visualization 3 h post-injection. The radioconjugate tumor accumulation was EGFR-specific and PET imaging data showed a clear differentiation between xenografts with varying EGFR expression levels. A strong correlation was observed between PET analysis, ex vivo estimates of tracer concentration and receptor expression in tumor tissues. Additionally, F-AlF-NOTA-ZEGFR:03115 could measure receptor downregulation in response to EGFR inhibition. ZEGFR03115-based radioconjugates can assess different levels of EGFR level in vivo and measure receptor expression changes in response to cetuximab, indicating a potential for assessment of adequate treatment dosing with anti-EGFR antibodies.

Radioimmunotherapy (RIT) with monoclonal antibodies and their fragments labelled with radionuclides emitting α -particles, β-particles or Auger electrons have been used for many years in the development of anticancer strategies. While RIT has resulted in approved radiopharmaceuticals for the treatment of hematological malignancies, its use in solid tumors still remains challenging. Areas covered: In this review, we discuss the exciting progress towards elucidating the potential of current and novel radioimmunoconjugates and address the challenges for translation into clinical practice. Expert opinion: There are still technical and logistical challenges associated with the use of RIT in routine clinical practice, including development of novel and more specific targeting moieties, broader access α to α-emitters and better tailoring of pre-targeting approaches. Moreover, improved understanding of the heterogeneous nature of solid tumors and the critical role of tumor microenvironments will help to optimize clinical response to RIT by delivering sufficient radiation doses to even more radioresistant tumor cells.

A critical need has emerged for volumetric thermometry to visualize 3D temperature distributions in real time during deep hyperthermia treatments used as an adjuvant to radiation or chemotherapy for cancer. For the current effort, magnetic resonance thermal imaging (MRTI) is used to measure 2D temperature rise distributions in four cross sections of large extremity soft tissue sarcomas during hyperthermia treatments. Novel hardware and software techniques are described which improve the signal to noise ratio of MR images, minimize motion artifact from circulating coupling fluids, and provide accurate high resolution volumetric thermal dosimetry. For the first 10 extremity sarcoma patients, the mean difference between MRTI region of interest and adjacent interstitial point measurements during the period of steady state temperature was 0.85°C. With 1min temporal resolution of measurements in four image planes, this non-invasive MRTI approach has demonstrated its utility for accurate monitoring and realtime steering of heat into tumors at depth in the body.