Suzanne E. Lapi

A historical perspective on the specific activity of radiopharmaceuticals: What have we learned in the 35 years of the ISRC?

Specific activity (SA), defined as the amount of radioactivity per unit mass of a compound, is arguably one of the most important parameters in radiopharmaceutical development, particularly in quality control of carbon-11- and fluorine-18-labeled compounds. This review article will outline the progression of improvements in SA over the last few decades. The International Symposium of Radiopharmaceutical Chemistry abstracts were an excellent source of materials for this review and will be referenced throughout.

Designing the Magic Bullet? The Advancement of Immuno-PET into Clinical Use

The development of noninvasive imaging techniques using monoclonal antibodies (mAbs) is a quickly evolving field. Immuno-PET uses positron-emitting isotopes to track the localization of mAbs with excellent image quality. Procedures for labeling mAbs with 89Zr or 124I using good manufacturing procedures have been established, and therefore these radiopharmaceuticals are being investigated for clinical use. This short review will focus on immuno-PET with full mAbs using long-lived positron-emitting isotopes (89Zr and 124I) over the past 5 y and discuss their progress into clinical use.

89Zr-Radiolabeled Trastuzumab Imaging in Orthotopic and Metastatic Breast Tumors.

The human epidermal growth factor receptor 2 (HER2/neu) is overexpressed in 20–30% of breast cancers and is associated with tumor growth, angiogenesis, and development of distant metastases. Trastuzumab, an anti-HER2 monoclonal antibody, is used for the treatment of HER2 positive breast cancer and clinical efficacy of this agent is dependent on HER2 expression. Targeted PET imaging of HER2 with radiolabeled trastuzumab may be used to determine HER2 expression levels and guide therapy selection. The purpose of the current study was to evaluate a facile 89Zr-trastuzumab preparation method that can be efficiently applied for clinical grade production. Also, relative HER2 expression levels in orthotopic and metastatic breast cancer models were assessed by PET imaging using the 89Zr-trastuzumab produced by this simpler method.

A semi-automated system for the routine production of copper-64.

An automated system for the production of high specific activity (64)Cu via the irradiation of electroplated enriched (64)Ni targets has been developed. We have been operating this system continually on a biweekly or weekly basis for more than two years. Since the inception of this automated production system, (October 1, 2008), we have had 145 productions, produced 53562 mCi and shipped out 25629 mCi of this isotope to external users. We routinely produce over 400 mCi of this isotope per batch with a specific activity of 14,000 ± 7600 mCi/μmol for distribution to some 12-15 centers each production.

Development and characterization of 89Zr-labeled panitumumab for immuno-positron emission tomographic imaging of the epidermal growth factor receptor.

The epidermal growth factor receptor (EGFR) is overexpressed in the majority of malignancies and has been associated with poor outcomes. Panitumumab, an anti-EGFR monoclonal antibody that binds to the extracellular binding domain of EGFR, is increasingly used with radiotherapy and chemotherapy but has associated toxicities. The purpose of this study was to develop and characterize a novel targeted imaging agent for the EGFR using radiolabeled panitumumab. Flow cytometry studies were performed to evaluate EGFR expression in several cell lines. Desferrioxamine-Bz-NCS (DFO) was conjugated to panitumumab and labeled with (89)Zr. Cell uptake studies were performed in four cell lines. For biodistribution studies and micro-positron emission tomography/computed tomography (PET/CT), mouse xenograft models were generated using the same cell lines. PET was performed, and tumors and select organs were harvested for biodistribution studies. Panitumumab was radiolabeled with (89)Zr with high radiochemical purity and specific activity and was found to be stable in serum. Cell binding studies demonstrated that radiotracer uptake in cells correlated with the degree of EGFR expression. MicroPET/CT imaging studies demonstrated a high intensity of (89)Zr-panitumumab in A431 and HCT 116 tumors in comparison with the EGFR-negative tumors. Biodistribution studies confirmed the results from the imaging studies. (89)Zr-panitumumab imaging of EGFR-positive tumors demonstrated levels of radiotracer uptake associated with EGFR expression.

Evaluation of 89Zr-pertuzumab in Breast Cancer Xenografts

Pertuzumab is a monoclonal antibody that binds to HER2 and is used in combination with another HER2–specific monoclonal antibody, trastuzumab, for the treatment of HER2+ metastatic breast cancer. Pertuzumab binds to an HER2 binding site distinct from that of trastuzumab, and its affinity is enhanced when trastuzumab is present. We aim to exploit this enhanced affinity of pertuzumab for its HER2 binding epitope and adapt this antibody as a PET imaging agent by radiolabeling with 89Zr to increase the sensitivity of HER2 detection in vivo. Here, we investigate the biodistribution of 89Zr-pertuzumab in HER2–expressing BT-474 and HER2–nonexpressing MDA-MB-231 xenografts to quantitatively assess HER2 expression in vivo. In vitro cell binding studies were performed resulting in retained immunoreactivity and specificity for HER2–expressing cells. In vivo evaluation of 89Zr-pertuzumab was conducted in severely combined immunodeficient mice, subcutaneously inoculated with BT-474 and MDA-MB-231 cells. 89Zr-pertuzumab was systemically administered and imaged at 7 days postinjection (p.i.) followed by terminal biodistribution studies. Higher tumor uptake was observed in BT-474 compared to MDA-MB-231 xenografts with 47.5 ± 32.9 and 9.5 ± 1.7% ID/g, respectively at 7 days p.i (P = 0.0009) and blocking studies with excess unlabeled pertuzumab showed a 5-fold decrease in BT-474 tumor uptake (P = 0.0006), confirming the in vivo specificity of this radiotracer. Importantly, we observed that the tumor accumulation of 89Zr-pertuzumab was increased in the presence of unlabeled trastuzumab, at 173 ± 74.5% ID/g (P = 0.01). Biodistribution studies correlate with PET imaging quantification using max SUV (r = 0.98, P = 0.01). Collectively, these results illustrate that 89Zr-pertuzumab as a PET imaging agent may be beneficial for the quantitative and noninvasive assessment of HER2 expression in vivo especially for patients undergoing trastuzumab therapy.

Detection of Rapalog-Mediated Therapeutic Response in Renal Cancer Xenografts Using 64Cu-bevacizumab ImmunoPET

The importance of neovascularization for primary and metastatic tumor growth fostered numerous clinical trials of angiogenesis inhibitors either alone or in combination with conventional antineoplastic therapies. One challenge with the use of molecularly targeted agents has been the disconnection between size reduction and tumor biologic behavior, either when the drug is efficacious or when tumor resistance emerges. Here, we report the synthesis and characterization of 64Cu-NOTA-bevacizumab as a PET imaging agent for imaging intratumoral VEGF content in vivo. 64Cu-NOTA-bevacizumab avidly accumulated in 786-O renal carcinoma xenografts with lower levels in host organs. RAD001 (everolimus) markedly attenuated 64Cu-NOTA-bevacizumab accumulation within 786-O renal carcinoma xenografts. Tumor tissue and cellular molecular analysis validated PET imaging, demonstrating decreases in total and secreted VEGF content and VEGFR2 activation. Notably, 64Cu-NOTA-bevacizumab PET imaging was concordant with the growth arrest of RAD001 tumors. These data suggest that immunoPET targeting of angiogenic factors such as VEGF could be a new class of surrogate markers complementing the RECIST criteria in patients receiving molecularly targeted therapies.

THE EFFECTS OF IRON AND COPPER AVAILABILITY ON THE COPPER STOICHIOMETRY OF MARINE PHYTOPLANKTON 1

We studied the interactive effects of iron (Fe) and copper (Cu) availability on the growth rates, Cu quotas, and steady‐state Cu‐uptake rates (ρssCu) of 12 phytoplankton (from four classes and two marine environments). A mixed‐effect statistical model indicated that low Fe significantly decreased phytoplankton growth rates. In contrast, lowering Cu levels only decreased the growth rates of the oceanic phytoplankton. Under Fe/Cu sufficiency, the Cu quotas ranged from 0.36 to 3.8 μmol Cu · mol−1 C. Copper levels in the growth medium had a significant positive effect on the Cu quotas, and this effect was dependent on the algal class. Under Fe/Cu sufficiency, the highest average Cu quotas were observed for the Bacillariophyceae, followed by the Cyanophyceae, Prymnesiophyceae, and lastly the Dinophyceae. Similar taxonomic trends were observed for the ρssCu. Although the Cu:C ratios were not significantly higher in oceanic strains, there are five independent lines of evidence supporting a more important role of Cu in the physiology of the oceanic phytoplankton. The mixed‐effect model indicated a significant Cu effect on the growth rates and ρssCu of the oceanic strains, but not the coastal strains. In addition, lowering the Cu concentration in the media decreased the Cu quotas and ρssCu of the oceanic strains to a greater extent (5.5‐ and 5.4‐fold, respectively) than those of the coastals (3.8‐ and 4.7‐fold, respectively). Iron limitation only had a significant effect on the Cu quotas of the oceanic strains, and this effect was dependent on Cu level and taxonomic class. Our results highlight a complex physiological interaction between Fe and Cu in marine phytoplankton.

Long-term evaluation of TiO2-based 68Ge/68Ga generators and optimized automation of [68Ga]DOTATOC radiosynthesis

Interest in using (68)Ga is rapidly increasing for clinical PET applications due to its favorable imaging characteristics and increased accessibility. The focus of this study was to provide our long-term evaluations of the two TiO(2)-based (68)Ge/(68)Ga generators and develop an optimized automation strategy to synthesize [(68)Ga]DOTATOC by using HEPES as a buffer system. This data will be useful in standardizing the evaluation of (68)Ge/(68)Ga generators and automation strategies to comply with regulatory issues for clinical use.

Imaging the L-Type Amino Acid Transporter-1 (LAT1) with Zr-89 ImmunoPET

The L-type amino acid transporter-1 (LAT1, SLC7A5) is upregulated in a wide range of human cancers, positively correlated with the biological aggressiveness of tumors, and a promising target for both imaging and therapy. Radiolabeled amino acids such as O-(2-[18F]fluoroethyl)-L-tyrosine (FET) that are transport substrates for system L amino acid transporters including LAT1 have met limited success for oncologic imaging outside of the brain, and thus new strategies are needed for imaging LAT1 in systemic cancers. Here, we describe the development and biological evaluation of a novel zirconium-89 labeled antibody, [89Zr]DFO-Ab2, targeting the extracellular domain of LAT1 in a preclinical model of colorectal cancer. This tracer demonstrated specificity for LAT1 in vitro and in vivo with excellent tumor imaging properties in mice with xenograft tumors. PET imaging studies showed high tumor uptake, with optimal tumor-to-non target contrast achieved at 7 days post administration. Biodistribution studies demonstrated tumor uptake of 10.5 ± 1.8 percent injected dose per gram (%ID/g) at 7 days with a tumor to muscle ratio of 13 to 1. In contrast, the peak tumor uptake of the radiolabeled amino acid [18F]FET was 4.4 ± 0.5 %ID/g at 30 min after injection with a tumor to muscle ratio of 1.4 to 1. Blocking studies with unlabeled anti-LAT1 antibody demonstrated a 55% reduction of [89Zr]DFO-Ab2 accumulation in the tumor at 7 days. These results are the first report of direct PET imaging of LAT1 and demonstrate the potential of immunoPET agents for imaging specific amino acid transporters.