In vivo biodistribution and pharmacokinetics of sotrovimab, a SARS-CoV-2 monoclonal antibody, in healthy cynomolgus monkeys.

PURPOSE: Sotrovimab (VIR-7831), a human IgG1κ monoclonal antibody (mAb), binds to a conserved epitope on the SARS-CoV-2 spike protein receptor binding domain (RBD). The Fc region of VIR-7831 contains an LS modification to promote neonatal Fc receptor (FcRn)-mediated recycling and extend its serum half-life. Here, we aimed to evaluate the impact of the LS modification on tissue biodistribution, by comparing VIR-7831 to its non-LS-modified equivalent, VIR-7831-WT, in cynomolgus monkeys. METHODS: 89Zr-based PET/CT imaging of VIR-7831 and VIR-7831-WT was performed up to 14 days post injection. All major organs were analyzed for absolute concentration as well as tissue:blood ratios, with the focus on the respiratory tract, and a physiologically based pharmacokinetics (PBPK) model was used to evaluate the tissue biodistribution kinetics. Radiomics features were also extracted from the PET images and SUV values. RESULTS: SUVmean uptake in the pulmonary bronchi for 89Zr-VIR-7831 was statistically higher than for 89Zr-VIR-7831-WT at days 6 (3.43 ± 0.55 and 2.59 ± 0.38, respectively) and 10 (2.66 ± 0.32 and 2.15 ± 0.18, respectively), while the reverse was observed in the liver at days 6 (5.14 ± 0.80 and 8.63 ± 0.89, respectively), 10 (4.52 ± 0.59 and 7.73 ± 0.66, respectively), and 14 (4.95 ± 0.65 and 7.94 ± 0.54, respectively). Though the calculated terminal half-life was 21.3 ± 3.0 days for VIR-7831 and 16.5 ± 1.1 days for VIR-7831-WT, no consistent differences were observed in the tissue:blood ratios between the antibodies except in the liver. While the lung:blood SUVmean uptake ratio for both mAbs was 0.25 on day 3, the PBPK model predicted the total lung tissue and the interstitial space to serum ratio to be 0.31 and 0.55, respectively. Radiomics analysis showed VIR-7831 had mean-centralized PET SUV distribution in the lung and liver, indicating more uniform uptake than VIR-7831-WT. CONCLUSION: The half-life extended VIR-7831 remained in circulation longer than VIR-7831-WT, consistent with enhanced FcRn binding, while the tissue:blood concentration ratios in most tissues for both drugs remained statistically indistinguishable throughout the course of the experiment. In the bronchiolar region, a higher concentration of 89Zr-VIR-7831 was detected. The data also allow unparalleled insight into tissue distribution and elimination kinetics of mAbs that can guide future biologic drug discovery efforts, while the residualizing nature of the 89Zr label sheds light on the sites of antibody catabolism.

Novel multimodal molecular imaging of Vitamin H (Biotin) transporter activity in the murine placenta.

Vitamin H (biotin) is delivered to the fetus transplacentally by an active biotin-transport mechanism and is critical for fetal development. Our objective was to develop a comprehensive MRI technique for mapping biotin transporter activity in the murine placenta. Visualization of transporter activity can employ MRI's unique T2*-dependent signal 'off-switch', which is triggered by transporter mediated aggregation of biotinylated contrast agent (b-BSA-Gd-DTPA). MRI data were collected from pregnant mice after administration of b-BSA-Gd-DTPA and analyzed using a new sub-voxel biophysical signal model. Validation experiments included competition with native biotin, comparative tests using PET, histology, and ICPMS. MRI signal was governed by binding, aggregation, and clearance of biotin (confirmed by histology). Signal dynamics reflected the placenta's perfusion pattern modulated by biotin transporter activity and trophoblast mediated retention, and were in congruence with a three-compartment sub-voxel model. Pre-saturation of the transporters with free biotin suppressed b-BSA-Gd-DTPA uptake. The results were confirmed by PET, histology and ICPMS. The presented MRI-based platform allows to track activity of essential molecular transporters in the placenta, reflecting a transporter-mediated uptake, followed by retention and aggregation, and recycling associated with the large b-BSA-Gd-DTPA conjugate. The presented DCE-MRI technique can furthermore be used to map and characterize microstructural compartmentation and transporter activity without exposing the fetus to contrast media.

Imaging of HER2 with [89Zr]pertuzumab in Response to T-DM1 Therapy.

Background: The success of human epidermal growth factor receptor 2 (HER2)-targeted therapy depends on accurate characterization of HER2 expression, but current methods available have several limitations. This study aims to investigate the feasibility of [89Zr]pertuzumab imaging to monitor early response to Ado-trastuzumab emtansine (T-DM1) therapy in mice bearing xenografts of HER2-positive breast cancer (BCa). Materials and Methods: Pertuzumab was conjugated to DFO-Bz-NCS and labeled with 89Zr. Mice bearing BT-474 tumors were imaged with [89Zr]pertuzumab and [18F]FDG before and after T-DM1 therapy. Results: Pertuzumab was successfully labeled with 89Zr with a specific activity of 0.740 MBq/µg. Overall [18F]FDG images showed poor delineation of tumors. Using [18F]FDG-PET to measure tumor volume, the volume remained unchanged from 107.6 ± 20.7 mm3 before treatment to 89.87 ± 66.55 mm3 after treatment. In contrast, [89Zr]pertuzumab images showed good delineation of HER2-positive tumors, allowing accurate detection of changes in tumor volume (from 243.80 ± 40.91 mm3 before treatment to 78.4 ± 40.43 mm3 after treatment). Conclusion: [89Zr]pertuzumab may be an imaging probe for monitoring the response of HER2-positive BCa patients to T-DM1 therapy.

Radiolabeled Cationic Peptides for Targeted Imaging of Infection.

Molecular probes targeting bacteria provide opportunities to target bacterial infections in vivo for both imaging and therapy. In the current study, we report the development of positron emission tomography (PET) probes for imaging of live bacterial infection based on the small molecules HLys-DOTA, a polycationic peptide synthesized as the D-isomer (RYWVAWRNRG) conjugated to 1, 4, 7, 10-tetraazacyclododecane-N',N″,N‴,N-tetraacetic acid (DOTA) and AB1-HLys-DOTA, which includes an unnatural amino acid AB1 that preferentially binds to bacteria membrane lipids with amine groups via formation of iminoboronates. HLys-DOTA and AB1-HLys-DOTA peptides were radiolabeled with 64Cu and investigated as PET imaging agents to track bacterial infection in vitro and in intramuscularly infected (IM) mice models. Cell uptake studies at 37°C in Staphylococcus aureus (SA) show higher uptake of 64Cu-AB1-HLys-DOTA; 98.47 ± 3.54% vs 64Cu-HLys-DOTA; 39.12 ± 3.27% at 24 h. Standard uptake values (SUV) analysis of the PET images resulted in mean SUV of 0.70 ± 0.08, 0.49 ± 0.04, and 0.31 ± 0.01 for 64Cu-AB1-HLys-DOTA and 0.17 ± 0.06, 0.16 ± 0.02, and 0.13 ± 0.01 for 64Cu-HLys-DOTA at 1, 4, and 24 h post injection, respectively, in the infected muscles. Similarly, in the biodistribution studies, dose uptake in the infected muscles was 4 times higher in the targeted 64Cu-AB1-HLys-DOTA group than in the 64Cu-HLys-DOTA group and 2-3 times higher than in the PBS control group at 1, 4, and 24 h post injection. 64Cu-AB1-HLys-DOTA was able to distinguish between SA-infected muscle and Pseudomonas aeruginosa (PA) infected muscle with lower mean SUV of 0.28 ± 0.10 at 1 h post injection. This illustrates the utility of the AB1 covalently targeting group in synergy with the HLys peptide, which noncovalently binds to bacterial membranes. These results suggest that 64Cu-labeled AB1-HLys-DOTA peptide could be used as an imaging probe for detection of bacterial infection in vivo with specificity for Gram-positive bacteria.

Biodistribution and PET Imaging of pharmacokinetics of manganese in mice using Manganese-52.

Manganese is essential to life, and humans typically absorb sufficient quantities of this element from a normal healthy diet; however, chronic, elevated ingestion or inhalation of manganese can be neurotoxic, potentially leading to manganism. Although imaging of large amounts of accumulated Mn(II) is possible by MRI, quantitative measurement of the biodistribution of manganese, particularly at the trace level, can be challenging. In this study, we produced the positron-emitting radionuclide 52Mn (t1/2 = 5.6 d) by proton bombardment (Ep<15 MeV) of chromium metal, followed by solid-phase isolation by cation-exchange chromatography. An aqueous solution of [52Mn]MnCl2 was nebulized into a closed chamber with openings through which mice inhaled the aerosol, and a separate cohort of mice received intravenous (IV) injections of [52Mn]MnCl2. Ex vivo biodistribution was performed at 1 h and 1 d post-injection/inhalation (p.i.). In both trials, we observed uptake in lungs and thyroid at 1 d p.i. Manganese is known to cross the blood-brain barrier, as confirmed in our studies following IV injection (0.86%ID/g, 1 d p.i.) and following inhalation of aerosol, (0.31%ID/g, 1 d p.i.). Uptake in salivary gland and pancreas were observed at 1 d p.i. (0.5 and 0.8%ID/g), but to a much greater degree from IV injection (6.8 and 10%ID/g). In a separate study, mice received IV injection of an imaging dose of [52Mn]MnCl2, followed by in vivo imaging by positron emission tomography (PET) and ex vivo biodistribution. The results from this study supported many of the results from the biodistribution-only studies. In this work, we have confirmed results in the literature and contributed new results for the biodistribution of inhaled radiomanganese for several organs. Our results could serve as supporting information for environmental and occupational regulations, for designing PET studies utilizing 52Mn, and/or for predicting the biodistribution of manganese-based MR contrast agents.

Investigating the pharmacokinetics and biological distribution of silver-loaded polyphosphoester-based nanoparticles using (111) Ag as a radiotracer.

Purified (111) Ag was used as a radiotracer to investigate silver loading and release, pharmacokinetics, and biodistribution of polyphosphoester-based degradable shell crosslinked knedel-like (SCK) nanoparticles as a comparison to the previously reported small molecule, N-heterocyclic silver carbene complex analog (SCC1) for the delivery of therapeutic silver ions in mouse models. Biodistribution studies were conducted by aerosol administration of (111) Ag acetate, [(111) Ag]SCC1, and [(111) Ag]SCK doses directly into the lungs of C57BL/6 mice. Nebulization of the (111) Ag antimicrobials resulted in an average uptake of 1.07 ± 0.12% of the total aerosolized dose given per mouse. The average dose taken into the lungs of mice was estimated to be 2.6 ± 0.3% of the dose inhaled per mouse for [(111) Ag]SCC1 and twice as much dose was observed for the [(111) Ag]SCKs (5.0 ± 0.3% and 5.9 ± 0.8% for [(111) Ag]aSCK and [(111) Ag]zSCK, respectively) at 1 h post administration (p.a.). [(111) Ag]SCKs also exhibited higher dose retention in the lungs; 62-68% for [(111) Ag]SCKs and 43% for [(111) Ag]SCC1 of the initial 1 h dose were observed in the lungs at 24 h p.a.. This study demonstrates the utility of (111) Ag as a useful tool for monitoring the pharmacokinetics of silver-loaded antimicrobials in vivo.

The use of 111Ag as a tool for studying biological distribution of silver-based antimicrobials.

Recently, there has been an emergence of significant interest in silver-based antimicrobials. Our goal was to develop a radioactive tracer for investigating the biological fate of such compounds. Purified 111Ag was incorporated into the methylated caffeine analogue, IC1 to yield the silver carbene complex designated as [111Ag]SCC1 and investigated in biodistribution studies.

Enhancing peptide ligand binding to vascular endothelial growth factor by covalent bond formation.

Formation of a stable covalent bond between a synthetic probe molecule and a specific site on a target protein has many potential applications in biomedical science. For example, the properties of probes used as receptor-imaging ligands may be improved by increasing their residence time on the targeted receptor. Among the more interesting cases are peptide ligands, the strongest of which typically bind to receptors with micromolar dissociation constants, and which may depend on processes other than simple binding to provide images. The side chains of cysteine, histidine, or lysine are attractive for chemical attachment to improve binding to a receptor protein, and a system based on acryloyl probes attaching to engineered cysteine provides excellent positron emission tomographic images in animal models (Wei et al. (2008) J. Nucl. Med. 49, 1828-1835). In nature, lysine is a more common but less reactive residue than cysteine, making it an interesting challenge to modify. To seek practically useful cross-linking yields with naturally occurring lysine side chains, we have explored not only acryloyl but also other reactive linkers with different chemical properties. We employed a peptide-VEGF model system to discover that a 19mer peptide ligand, which carried a lysine-tagged dinitrofluorobenzene group, became attached stably and with good yield to a unique lysine residue on human vascular endothelial growth factor (VEGF), even in the presence of 70% fetal bovine serum. The same peptide carrying acryloyl and related Michael acceptors gave low yields of attachment to VEGF, as did the chloroacetyl peptide.

Combination of isothermal titration calorimetry and time-resolved luminescence for high affinity antibody-ligand interaction thermodynamics and kinetics.

For experiments using synthetic ligands as probes for biological experiments, it is useful to determine the specificity and affinity of the ligands for their receptors. As ligands with higher affinities are developed (K(A)>10(8)M(-1); K(D)<10(-8)M), a new challenge arises: to measure these values accurately. Isothermal titration calorimetry measures heat produced or consumed during ligand binding, and also provides the equilibrium binding constant. However, as normally practiced, its range is limited. Displacement titration, where a competing weaker ligand is used to lower the apparent affinity of the stronger ligand, can be used to determine the binding affinity as well as the complete thermodynamic data for ligand-antibody complexes with very high affinity. These equilibrium data have been combined with kinetic measurements to yield the rate constants as well. We describe this methodology, using as an example antibody 2D12.5, which captures yttrium S-2-(4-aminobenzyl)-1, 4, 7, 10-tetraazacyclododecanetetraacetate.

New covalent capture probes for imaging and therapy, based on a combination of binding affinity and disulfide bond formation.

We describe the synthesis and development of new reactive DOTA-metal complexes for covalently targeting engineered receptors in vivo, which have superior tumor uptake and clearance properties for biomedical applications. These probes are found to clear efficiently through the kidneys and minimally through other routes, but bind persistently in the tumor target. We also explore the new technique of Cerenkov luminescence imaging to optically monitor radiolabeled probe distribution and kinetics in vivo. Cerenkov luminescence imaging uniquely enables sensitive noninvasive in vivo imaging of a ß(-) emitter such as (90)Y with an optical imager.

Chemically modified antibodies as diagnostic imaging agents.

Notable new applications of antibodies for imaging involve genetically extracting the essential molecular recognition properties of an antibody, and in some cases enhancing them by mutation, before protein expression. The classic paradigm of intravenous administration of a labeled antibody to image not only its target but also its metabolism can be improved on. Protocols involving molecular targeting with an engineered unlabeled protein derived from an antibody, followed by capture of a small probe molecule that provides a signal, are being developed to a high level of utility. This is accompanied by new strategies for probe capture such as irreversible binding, incorporation of engineered enzyme active sites, and antibody-ligand systems that generate a signal only upon binding or uptake.

Rates and equilibria for probe capture by an antibody with infinite affinity.

Probe-capture systems based on proteins and synthetic ligands have become important for new analytical and imaging applications. We have used kinetic measurements of luminescence and measurements of binding by isothermal calorimetry to determine essential rate and equilibrium constants for a system that permanently captures modified DOTA chelates for positron imaging. We used that information along with previous results to quantitatively characterize the behavior of this system in vitro and in vivo. Under physiological conditions at 37 degrees C, the equilibrium dissociation constant for yttrium S-2-(4-aminobenzyl)-1,4,7,10-tetraazacyclododecanetetraacetate from antibody 2D12.5 is 2.0 (+/- 0.4) x 10(-9) M and the dissociation rate constant is 7.0 (+/- 0.7) x 10(-3) s(-1), leading to an inferred association rate constant of 3.5 x 10(6) M(-1) s(-1). Using these values to interpret data from earlier experiments leads to the rate constant 2.5 x 10(-2) s(-1) for covalent attachment of bound yttrium S-2-(4-acrylamidobenzyl)-1,4,7,10-tetraazacyclododecanetetraacetate to the G54C mutant of antibody 2D12.5. These values lead to a model for the detailed behavior of the latter system for tumor imaging in vivo that is consistent with experimental observations.