In vivo trafficking of a tumor-targeting IgE antibody: molecular imaging demonstrates rapid hepatobiliary clearance compared to IgG counterpart.

IgE antibodies elicit powerful immune responses, recruiting effector cells to tumors more efficiently and with greater cytotoxicity than IgG antibodies. Consequently, IgE antibodies are a promising alternative to conventional IgG-based therapies in oncology (AllergoOncology). As the pharmacokinetics of IgE antibodies are less well understood, we used molecular imaging in mice to compare the distribution and elimination of IgE and IgG antibodies targeting the human tumor-associated antigen chondroitin sulfate proteoglycan 4 (CSPG4). Anti-CSPG4 IgE and IgG1 antibodies with human Fc domains were radiolabeled with 111In. CSPG4-expressing A375 human melanoma xenografts implanted in NOD-scid IL2rg-/- mice were also engrafted with human immune cells by intravenous administration. 111In-anti-CSPG4 antibodies were administered intravenously. Their distribution was determined by single-photon emission computed tomography (SPECT) and ex vivo gamma-counting over 120 h. SPECT imaging was conducted from 0 to 60 min after antibody administration to precisely measure the early phase of IgE distribution. 111In-labeled anti-CSPG4 IgG and IgE showed serum stability in vitro of >92% after 5 days. In A375 xenograft-bearing mice, anti-CSPG4 IgE showed much faster blood clearance and higher accumulation in the liver compared to anti-CSPG4 IgG. However, tumor-to-blood and tumor-to-muscle ratios were similar between the antibody isotypes and higher compared with a non-tumor-targeting isotype control IgE. IgE excretion was much faster than IgG. In non-tumor-bearing animals, early SPECT imaging revealed a blood clearance half-life of 10 min for IgE. Using image-based quantification, we demonstrated that the blood clearance of IgE is much faster than that of IgG while the two isotypes showed comparable tumor-to-blood ratios.

An overview of nuclear medicine research in the UK and the landscape for clinical adoption.

BACKGROUND AND OBJECTIVES: Nuclear medicine contributes greatly to the clinical management of patients and experimental medicine. This report aims to (1) outline the current landscape of nuclear medicine research in the UK, including current facilities and recent or ongoing clinical studies and (2) provide information about the available pathways for clinical adoption and NHS funding (commissioning) of radiopharmaceuticals. METHODS: Evidence was obtained through database searches for UK-based nuclear medicine clinical studies and by conducting a questionnaire-based survey of UK radiopharmaceutical production facilities. A recent history of clinical commissioning, either through recommendations from the National Institute for Health and Care Excellence (NICE) or through NHS specialised services commissioning, was compiled from publicly available documents and policies. RESULTS: The collected data highlighted the UK's active nuclear medicine research community and recent investment in new facilities and upgrades. All commissioning routes favour radiopharmaceuticals that have marketing authorisation and since 2017 there has been a requirement to demonstrate both clinical and cost-effectiveness. Whilst radiopharmaceuticals for molecular radiotherapy are well suited to these commissioning pathways, diagnostic radiotracers have not historically been assessed in this manner. CONCLUSIONS: We hope that by collating this information we will provide stimulus for future discussion and consensus statements around this topic.

Optimal His-Tag Design for Efficient [99mTc(CO)3]+ and [188Re(CO)3]+ Labeling of Proteins for Molecular Imaging and Radionuclide Therapy by Analysis of Peptide Arrays.

Hexahistidine tags (His-tags), incorporated into recombinant proteins to facilitate purification using metal-affinity chromatography, are useful binding sites for radiolabeling with [99mTc(CO)3]+ and [188Re(CO)3]+ for molecular imaging and radionuclide therapy. Labeling efficiencies vary unpredictably, and the method is therefore not universally useful. To overcome this, we have made quantitative comparisons of radiolabeling of a bespoke Celluspots array library of 382 His-tag-containing peptide sequences with [99mTc(CO)3]+ and [188Re(CO)3]+ to identify key features that enhance labeling. A selected sequence with 10-fold enhanced labeling efficiency compared to the most effective literature-reported sequences was incorporated into an exemplar protein and compared biologically with non-optimized analogues, in vitro and in vivo. Optimal labeling with either [99mTc(CO)3]+ or [188Re(CO)3]+ required six consecutive His residues in the protein sequence, surrounded by several positively charged residues (Arg or Lys), and the presence of phosphate in the buffer. Cys or Met residues in the sequence were beneficial, to a lesser extent. Negatively charged residues were deleterious to labeling. His-tags with adjacent positively charged residues could be labeled as much as 40 times more efficiently than those with adjacent negatively charged residues. 31P NMR of [Re(CO)3(H2O)3]+ and electrophoresis of solutions of [99mTc(CO)3(H2O)3]+ suggest that phosphate bridges form between cationic residues and the cationic metal synthon during labeling. The trial optimized protein, a scFv targeted to the PSMA antigen expressed in prostate cancer, was readily labeled in >95% radiochemical yield, without the need for subsequent purification. Labeling occurred more quickly and to higher specific activity than comparable non-optimized proteins, while retaining specific binding to PSMA and prostate cancer in vivo. Thus, optimized His-tags greatly simplify radiolabeling of recombinant proteins making them potentially more widely and economically available for imaging and treating patients.

In vitro cytotoxicity of Auger electron-emitting [67Ga]Ga-trastuzumab.

INTRODUCTION: Molecular radiotherapy exploiting short-range Auger electron-emitting radionuclides has potential for targeted cancer treatment and, in particular, is an attractive option for managing micrometastatic disease. Here, an approach using chelator-trastuzumab conjugates to target radioactivity to breast cancer cells was evaluated as a proof-of-concept to assess the suitability of 67Ga as a therapeutic radionuclide. METHODS: THP-trastuzumab and DOTA-trastuzumab were synthesised and radiolabelled with Auger electron-emitters 67Ga and 111In, respectively. Radiopharmaceuticals were tested for HER2-specific binding and internalisation, and their effects on viability (dye exclusion) and clonogenicity of HER2-positive HCC1954 and HER2-negative MDA-MB-231 cell lines was measured. Labelled cell populations were studied by microautoradiography. RESULTS: Labelling efficiencies for [67Ga]Ga-THP-trastuzumab and [111In]In-DOTA-trastuzumab were 90% and 98%, respectively, giving specific activities 0.52 ± 0.16 and 0.61 ± 0.11 MBq/µg (78-92 GBq/µmol). At 4 nM total antibody concentration and 200 × 103 cells/mL, [67Ga]Ga-THP-trastuzumab showed higher percentage of cell association (10.7 ± 1.3%) than [111In]In-DOTA-trastuzumab (6.2 ± 1.6%; p = 0.01). The proportion of bound activity that was internalised did not differ significantly for the two tracers (62.1 ± 1.4% and 60.8 ± 15.5%, respectively). At 100 nM, percentage cell binding of both radiopharmaceuticals was greatly reduced compared to 4 nM and did not differ significantly between the two (1.2 ± 1.0% [67Ga]Ga-THP-trastuzumab and 0.8 ± 0.9% for [111In]In-DOTA-trastuzumab). Viability and clonogenicity of HER2-positive cells decreased when each radionuclide was incorporated into cells by conjugation with trastuzumab, but not when the same level of radioactivity was confined to the medium by omitting the antibody conjugation, suggesting that 67Ga needs to be cell-bound or internalised for a therapeutic effect. Microautoradiography showed that radioactivity bound to individual cells varied considerably within the population. CONCLUSIONS: [67Ga]Ga-THP-trastuzumab reduced cell viability and clonogenicity only when cell-bound, suggesting 67Ga holds promise as a therapeutic radionuclide as part of a targeted radiopharmaceutical. The causes and consequences of non-homogeneous uptake among the cell population should be explored.

The effects of trace metal impurities on Ga-68-radiolabelling with a tris(3-hydroxy-1,6-dimethylpyridin-4-one) (THP) chelator.

GMP-grade 68Ge/68Ga generators provide access to positron-emitting 68Ga, enabling preparation of Positron Emission Tomography (PET) tracers and PET imaging at sites that do not have access to cyclotron-produced radionuclides. Radiotracers based on tris(3-hydroxy-1,6-dimethylpyridin-4-one) (THP) chelators enable simple one-step preparations of 68Ga PET radiopharmaceuticals from pre-fabricated kits without pre-processing of generator eluate or post-purification. However, trace metal impurities eluted along with 68Ga could compete for THP and reduce radiochemical yields (RCY). We have quantified trace metal impurities in 68Ga eluate from an Eckert & Ziegler (E&Z) generator using ICP-MS. The metals Al, Fe, natGa, Pb, Ti and natZn were present in generator eluate in significantly higher concentrations than in the starting eluent solution. Concentrations of Fe and natGa in eluate were in the range of 0.01-0.1 µM, Al, Zn and Pb in the range of 0.1-1 µM, and Ti in the range of 0.9-1.5 µM. To assess the ability of THP to chelate 68Ga in the presence of such metal ions, radiolabelling reactions were undertaken in which selected metal ions were added to make them equimolar with THP, or higher. Al3+, Fe3+, natGa3+ and Ti4+ reduced RCY at concentrations equimolar with THP and higher, but at lower concentrations they did not affect RCY. Pb2+, Zn2+, Ni2+ and Cr3+ had no effect on RCY (even under conditions in which each metal ion was present in 100-fold molar excess over THP). The multi-sample ICP-MS analysis reported here is (to date) the most comprehensive and robust quantification of metal impurities in the widely used E&Z 68Ga generator. 68Ga from an E&Z generator enables near-quantitative radiolabelling of THP at chelator concentrations as low as 5 µM (lower than other common gallium chelators) without pre-processing. The combination of Al3+, Fe3+, natGa3+ and Ti4+ in unprocessed 68Ga eluate is likely to decrease RCY of 68Ga radiolabelling if a lower amount of THP chelator is used, and future kit design should take this into account. To increase specific activities by using even lower THP concentrations, purification of 68Ga from trace metal ions will likely be required.

Visualization of Tumor-Immune Interaction - Target-Specific Imaging of S100A8/A9 Reveals Pre-Metastatic Niche Establishment.

Background Systemic cancer spread is preceded by the establishment of a permissive microenvironment in the target tissue of metastasis - the premetastatic niche. As crucial players in establishment of the pre-metastatic niche, myeloid derived suppressor cells (MDSC) release S100A8/A9, an exosomal protein that contributes to metastasis, angiogenesis, and immune suppression. We report the application of antibody-based single-photon emission computed tomography (SPECT) for detection of S100A8/A9 in vivo as an imaging marker for pre-metastatic tissue priming. Methods A syngeneic model system for invasive breast cancer with (4T1.2) or without (67NR) the tendency to form lung metastasis was established in BALB/c mice. A SPECT-probe has been generated and tested for visualization of S100A9 release. Tumor-associated changes in numbers and fuction of immune cells in pre-metastatic tissue were evaluated by flow cytometry and confocal microscopy. Results S100A8/A9 imaging reflected MDSC abundance and the establishment of an immunosuppressive environment in pre-metastatic lung tissue (activity 4T1.2 vs. healthy control: 0.95 vs. 0.45 %ID; p<0.001). The S100A8/A9 imaging signal in the pre-metastatic lung correlated with the subsequent metastatic tumor burden in the same organ (r2=0.788; p<0.0001). CCL2 blockade and the consecutive inhibition of premetastatic niche establishment was clearly depicted by S100A9-SPECT (lung activity untreated vs. treated: 2 vs, 1.4 %ID). Conclusion We report S100A8/A9 as a potent imaging biomarker for tumor-mediated immune remodeling with potential applications in basic research and clinical oncology.

In-Cell Activation of Organo-Osmium(II) Anticancer Complexes.

The family of iodido OsII arene phenylazopyridine complexes [Os(η6 -p-cym)(5-R1 -pyridylazo-4-R2 -phenyl))I]+ (where p-cym=para-cymene) exhibit potent sub-micromolar antiproliferative activity towards human cancer cells and are active in vivo. Their chemical behavior is distinct from that of cisplatin: they do not readily hydrolyze, nor bind to DNA bases. We report here a mechanism by which they are activated in cancer cells, involving release of the I- ligand in the presence of glutathione (GSH). The X-ray crystal structures of two active complexes are reported, 1-I (R1 =OEt, R2 =H) and 2-I (R1 =H, R2 =NMe2 ). They were labelled with the radionuclide 131 I (ß- /γ emitter, t1/2 8.02 d), and their activity in MCF-7 human breast cancer cells was studied. 1-[131 I] and 2-[131 I] exhibit good stability in both phosphate-buffered saline and blood serum. In contrast, once taken up by MCF-7 cells, the iodide ligand is rapidly pumped out. Intriguingly, GSH catalyzes their hydrolysis. The resulting hydroxido complexes can form thiolato and sulfenato adducts with GSH, and react with H2 O2 generating hydroxyl radicals. These findings shed new light on the mechanism of action of these organo-osmium complexes.

PET/CT and MR imaging biomarker of lipid-rich plaques using [64Cu]-labeled scavenger receptor (CD68-Fc).

Continued uptake of modified low-density lipoproteins (LDL) by the scavenger receptor, CD68, of activated macrophages is a crucial process in the development of atherosclerotic plaques and leads to the formation of foam cells. Eight-weeks-old male Apolipoprotein E-deficient (ApoE(-/-)) mice (n = 6) were fed a high-fat diet for 12 weeks. C57BL/6J wildtype (WT) mice served as controls (n = 6). Positron emission tomography (PET) with an acquisition time of 1800 s (NanoPET/CT scanner; Mediso, Hungary & Bioscan, USA) was carried out 24h after intravenous tail vein administration of 50 µl (64)Cu-CD68-Fc (~20-30 µg labeled protein/mouse containing approximately 10-12 MBq (64)Cu-CD68-Fc per mouse). Three days after PET/CT, all mice received an intravenous administration of 0.2 mmol/kg body weight of a gadolinium-based elastin-binding contrast agent to assess plaque burden and vessel wall remodeling. Two hours after injection, mice were imaged in a 3T clinical MR scanner (Philips Healthcare, Best, NL) using a dedicated single loop surface coil (23 mm). Enhanced (64)Cu-CD68-Fc uptake was found in the aortic arches of ApoE(-/-) compared to WT mice (ApoE(-/-) mice:10.5 ± 1.5 Bq/cm(3) vs. WT mice: 2.1 ± 0.3 Bq/cm(3); P = 0.002). Higher gadolinium-based elastin-binding contrast agent uptake was also detected in the aortic arch of ApoE(-/-) compared to WT mice using R(1) maps (R(1) = 1.47 ± 0.06 s(-1) vs. 0.92 ± 0.05 s(-1); P <0.001). Radiolabeled scavenger receptor ((64)Cu-CD68-Fc) may help to target foam cell rich plaques with high content of oxidized LDL. This novel imaging biomarker tool may have potential to identify unstable plaques and for risk stratification.

Positron emission tomography/computed tomographic and magnetic resonance imaging in a murine model of progressive atherosclerosis using (64)Cu-labeled glycoprotein VI-Fc.

BACKGROUND: Plaque erosion leads to exposure of subendothelial collagen, which may be targeted by glycoprotein VI (GPVI). We aimed to detect plaque erosion using (64)Cu-labeled GPVI-Fc (fragment crystallized). METHODS AND RESULTS: Four-week-old male apolipoprotein E-deficient (ApoE(-/-)) mice (n=6) were fed a high-fat diet for 12 weeks. C57BL/6J wild-type (WT) mice served as controls (n=6). Another group of WT mice received a ligation injury of the left carotid artery (n=6) or sham procedure (n=4). All mice received a total activity of ≈12 MBq (64)Cu-GPVI-Fc by tail vein injection followed by delayed (24 hours) positron emission tomography using a NanoPET/computed tomographic scanner (Mediso, Hungary; Bioscan, USA) with an acquisition time of 1800 seconds. Seventy-two hours after positron emission tomography/computed tomography, all mice were scanned 2 hours after intravenous administration of 0.2 mmol/kg body weight of a gadolinium-based elastin-specific MR contrast agent. MRI was performed on a 3-T clinical scanner (Philips Healthcare, Best, The Netherlands). In ApoE(-/-) mice, the (64)Cu-GPVI-Fc uptake in the aortic arch was significantly higher compared with WT mice (ApoE(-/-): 13.2±1.5 Bq/cm(3) versus WT mice: 5.1±0.5 Bq/cm(3); P=0.028). (64)Cu-GPVI-Fc uptake was also higher in the injured left carotid artery wall compared with the intact right carotid artery of WT mice and as a trend compared with sham procedure (injured: 20.7±1.3 Bq/cm(3) versus intact: 2.3±0.5 Bq/cm(3); P=0.028 versus sham: 12.7±1.7 Bq/cm(3); P=0.068). Results were confirmed by ex vivo histology and in vivo MRI with elastin-specific MR contrast agent that measures plaque burden and vessel wall remodeling. Higher R1 relaxation rates were found in the injured carotid wall with a T1 mapping sequence (injured: 1.44±0.08 s(-1) versus intact: 0.91±0.02 s(-1); P=0.028 versus sham: 0.97±0.05 s(-1); P=0.068) and in the aortic arch of ApoE(-/-) mice compared with WT mice (ApoE(-/-): 1.49±0.05 s(-1) versus WT: 0.92±0.04 s(-1); P=0.028). CONCLUSIONS: (64)Cu-GPVI-Fc positron emission tomographic imaging allows identification of exposed subendothelial collagen in injured WT and high-fat diet-fed ApoE(-/-) mice.

Incorporation of paramagnetic, fluorescent and PET/SPECT contrast agents into liposomes for multimodal imaging.

A series of metal-chelating lipid conjugates has been designed and synthesized. Each member of the series bears a 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid (DOTA) macrocycle attached to the lipid head group, using short n-ethylene glycol (n-EG) spacers of varying length. Liposomes incorporating these lipids, chelated to Gd(3+), (64)Cu(2+), or (111)In(3+), and also incorporating fluorescent lipids, have been prepared, and their application in optical, magnetic resonance (MR) and single-photon emission tomography (SPECT) imaging of cellular uptake and distribution investigated in vitro and in vivo. We have shown that these multimodal liposomes can be used as functional MR contrast agents as well as radionuclide tracers for SPECT, and that they can be optimized for each application. When shielded liposomes were formulated incorporating 50% of a lipid with a short n-EG spacer, to give nanoparticles with a shallow but even coverage of n-EG, they showed good cellular internalization in a range of tumour cells, compared to the limited cellular uptake of conventional shielded liposomes formulated with 7% 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[carboxy(polyethyleneglycol)(2000)] (DSPE-PEG2000). Moreover, by matching the depth of n-EG coverage to the length of the n-EG spacers of the DOTA lipids, we have shown that similar distributions and blood half lives to DSPE-PEG2000-stabilized liposomes can be achieved. The ability to tune the imaging properties and distribution of these liposomes allows for the future development of a flexible tri-modal imaging agent.

Development and validation of the 57Co assay for determining the ligand to antibody ratio in bifunctional chelate/antibody conjugates for use in radioimmunotherapy.

INTRODUCTION: The ligand to antibody ratio is an important characteristic of a chelate/antibody conjugate. It has been widely reported that if the ratio is too high, there will be detrimental effects on immunoreactivity and biodistribution; conversely, if the ratio is too low, the radionuclide may not bind efficiently, and the stability and the specific activity will be reduced. There are little published data on the accuracy or precision of the (57)Co assay. The UK Clinical Trials Regulations state that "systems with procedures that assure the quality of every aspect of the trial should be implemented". The aims of this study were to assess the reliability and accuracy of the (57)Co binding assay and validate it against defined criteria. METHOD: Thirty-two serial assays were assessed for reliability. Two batches of conjugated antibody were also analysed by matrix-assisted laser desorption/ionisation time of flight (MALDI-TOF) mass spectrometry (MS) to allow the comparison of the functional test with a physical method. RESULTS: Reliability: The coefficient of variation was 0.13. Accuracy: There was 9% variation between the (57)Co binding assay and MALDI-TOF MS results. CONCLUSION: A detailed method for the (57)Co ligand to antibody test is described that allows a discrete value to be obtained. The assay was validated as fit for purpose against target values of coefficient of variation <0.20, accuracy±10%, over a permissive range of 0.5-3.0 ligand to antibody ratio.

Macrobicyclic cage amine ligands for copper radiopharmaceuticals: a single bivalent cage amine containing two Lys3-bombesin targeting peptides.

The synthesis of new cage amine macrobicyclic ligands with pendent carboxylate functional groups designed for application in copper radiopharmaceuticals is described. Reaction of [Cu((NH(2))(2)sar)](2+) (sar = 3,6,10,13,16,19-hexaazabicyclo[6.6.6]icosane) with either succinic or glutaric anhydride results in selective acylation of the primary amine atoms of [Cu((NH(2))(2)sar)](2+) to give derivatives with either one or two aliphatic carboxylate functional groups separated from the cage amine framework by either a four- or five-atom linker. The Cu(II) serves to protect the secondary amine nitrogen atoms from acylation, and can be removed to give the free ligands. The newly appended carboxylate functional groups can be used as sites of attachment for cancer-targeting peptides such as Lys(3)-bombesin. The synthesis of the first dimeric sarcophagine-peptide conjugate, possessing two Lys(3)-bombesin peptides tethered to a single cage amine, is presented. This species has been radiolabeled with copper-64 at ambient temperature and there is minimal dissociation of Cu(II) from the conjugate even after two days of incubation in human serum.

Selective apoptotic killing of malignant hemopoietic cells by antibody-targeted delivery of an amphipathic peptide.

The alpha-helical amphipathic peptide D-(KLAKLAK)2 is toxic to eukaryotic cells if internalized by a suitable targeting mechanism. We have targeted this peptide to malignant hemopoietic cells via conjugation to monoclonal antibodies, which recognize lineage-specific cell surface molecules. An anti-CD19/peptide conjugate efficiently killed 3/3 B lymphoid lines. However, an anti-CD33/peptide conjugate was cytotoxic to only one of three CD33-positive myeloid leukemia lines. The IC50 towards susceptible lines were in the low nanomolar range. Conjugates were highly selective and did not kill cells that did not express the appropriate cell surface cognate of the antibody moiety. Anti-CD19/peptide conjugates efficiently killed cells from patients with chronic lymphocytic leukemia but anti-CD33/peptide reagents were less effective against fresh acute myeloid leukemia cells. We therefore suggest that amphipathic peptides may be of value as targeted therapeutic agents for the treatment of a subset of hematologic malignancies.