HER3 PET Imaging: 68Ga-Labeled Affibody Molecules Provide Superior HER3 Contrast to 89Zr-Labeled Antibody and Antibody-Fragment-Based Tracers

HER3 (human epidermal growth factor receptor type 3) is a challenging target for diagnostic radionuclide molecular imaging due to the relatively modest overexpression in tumors and substantial expression in healthy organs. In this study, we compared four HER3-targeting PET tracers based on different types of targeting molecules in a preclinical model: the 89Zr-labeled therapeutic antibody seribantumab, a seribantumab-derived F(ab)2-fragment labeled with 89Zr and 68Ga, and the 68Ga-labeled affibody molecule [68Ga]Ga-ZHER3. The novel conjugates were radiolabeled and characterized in vitro using HER3-expressing BxPC-3 and DU145 human cancer cells. Biodistribution was studied using Balb/c nu/nu mice bearing BxPC-3 xenografts. HER3-negative RAMOS xenografts were used to demonstrate binding specificity in vivo. Autoradiography was conducted on the excised tumors. nanoPET/CT imaging was performed. New conjugates specifically bound to HER3 in vitro and in vivo. [68Ga]Ga-DFO-seribantumab-F(ab’)2 was considered unsuitable for imaging due to the low stability and high uptake in normal organs. The highest tumor-to-non-tumor contrast with [89Zr]Zr-DFO-seribantumab and [89Zr]Zr-DFO-seribantumab-F(ab’)2 was achieved at 96 h and 48 h pi, respectively. Despite lower tumor uptake, [68Ga]Ga-ZHER3 provided the best imaging contrast due to the fastest clearance from blood and normal organs. The results of our study suggest that affibody-based tracers are more suitable for PET imaging of HER3 expression than antibody- and antibody-fragment-based tracers.

In vivo regulation of AT1a receptor-mediated intracellular uptake of [125I]Val5-ANG II in the kidneys and adrenals of AT1a receptor-deficient mice

Using type 1a angiotensin receptor (AT1a) receptor-deficient (Agtr1a−/−) mice and in vivo autoradiography, we tested the hypothesis that intracellular uptake of ANG II in the kidney and adrenal glands is primarily mediated by AT1a receptors and that the response is regulated by prevailing endogenous ANG II. After pretreatment of wild-type (Agtr1a+/+) and Agtr1a−/− mice ( n = 6–9 each group) with or without captopril (25 mg·kg−1·day−1) or losartan (10 mg·kg−1·day−1) for 2 wk, [125I]Val5-ANG II was infused for 60 min. Intracellular uptake of [125I]Val5-ANG II was determined by quantitative in vivo autoradiography after washout of circulating [125I]Val5-ANG II. Basal intracellular ANG II levels were 65% lower in the kidney ( P < 0.001), but plasma ANG II levels were threefold higher, in Agtr1a−/− than wild-type mice ( P < 0.01). Although plasma [125I]Val5-ANG II levels were similar, urinary excretion of [125I]Val5-ANG II was fourfold higher in Agtr1a−/− mice ( P < 0.001). By contrast, intracellular [125I]Val5-ANG II levels were ∼80% lower in the kidney and adrenal glands of Agtr1a−/− mice ( P < 0.01). Captopril decreased endogenous plasma and renal ANG II levels ( P < 0.01) but increased intracellular uptake of [125I]Val5-ANG II in the kidney and adrenal glands of wild-type and Agtr1a−/− mice ( P < 0.01). Losartan largely blocked renal and adrenal uptake of [125I]Val5-ANG II in wild-type and Agtr1a−/− mice. Thus 80% of intracellular ANG II uptake in the kidney and adrenal glands is mediated by AT1a receptors, whereas AT1b receptor- and other non-receptor-mediated mechanisms account for 20% of the response. Our results suggest that AT1a receptor-mediated uptake of extracellular ANG II may play a physiological role in the kidney and adrenal glands.

GABA-A Function and Receptor Binding in the Paraventricular Nucleus in Chronic Renal Wrap Hypertension

P48 The onset of renal wrap hypertension is associated with a reduced tonic GABA inhibition in the paraventricular nucleus (PVN) on the sympathetic nervous system. This reduced functional inhibition occurs without a change in GABA-A receptor binding in the PVN. The goal of the present study was to determine if GABAergic transmission and GABA binding is altered in chronic renal wrap hypertensive rats. Sprague-Dawley rats were made hypertensive or sham-operated. Four weeks later, animals were prepared with femoral artery catheters for the measurement of arterial pressure. Subgroups were also prepared with bilateral cannulae directed at the PVN. The renal wrap rats had higher mean arterial pressure (MAP): 139±4 mmHg vs.113±2 mmHg, but heart rate (HR) was not different (354±12 bpm vs. 369±6 bpm) as compared to control animals. Administration of the GABA-A antagonist, bicuculline, into the PVN caused a greater increase in MAP and HR in wrap animals (25±2 mmHg and 150±30 bpm) compared to sham operated rats (16±2 mmHg and 89±12 bpm). GABA-A binding sites in the PVN were estimated using in vivo autoradiography. [3H]-Flunitrazepam was used as the receptor ligand. Magnocellular neurons of the PVN showed a higher density of receptors than other areas of the nucleus. However, the number of binding sites was not different between normotensive and hypertensive rats in either the high density (1825±56 vs. 1756±41 fmol/mg protein) or low density (1454±26 vs. 1433±57 fmol/mg protein) regions of PVN. These data indicate that the inhibition by GABA in the PVN is augmented in the chronic stage of hypertension, and appears to be unrelated to a change in the number of GABA binding sites. The increased GABAergic inhibition is in contrast to the reduced inhibition that has been observed during the onset of hypertension.

The Cerebral Metabolic Consequences of Nitric Oxide Synthase Deficiency: Glucose Utilization in Endothelial and Neuronal Nitric Oxide Synthase Null Mice

Nitric oxide has multiple physiologic roles in the CNS. Inhibiting nitric oxide synthesis might therefore alter functional activity within the brain. We used [14C]-2-deoxyglucose in vivo autoradiography to measure local CMRglc in “knockout” mice lacking the genes for either the endothelial (eNOS) or neuronal (nNOS) isoforms of nitric oxide synthase, and in the progenitor strains (SV129, CS7B1/6). Glucose utilization levels did not significantly differ between nNOS and eNOS knockout mice and C57B1/6 mice in any of the 48 brain regions examined, but were relatively lower in some subcortical regions in SV129 mice.