Immuno-PET Monitoring of CD8+ T Cell Infiltration Post ICOS Agonist Antibody Treatment Alone and in Combination with PD-1 Blocking Antibody Using a 89Zr Anti-CD8+ Mouse Minibody in EMT6 Syngeneic Tumor Mouse.

PURPOSE: The presence and functional competence of intratumoral CD8+ T cells is often a barometer for successful immunotherapeutic responses in cancer. Despite this understanding and the extensive number of clinical-stage immunotherapies focused on potentiation (co-stimulation) or rescue (checkpoint blockade) of CD8+ T cell antitumor activity, dynamic biomarker strategies are often lacking. To help fill this gap, immuno-PET nuclear imaging has emerged as a powerful tool for in vivo molecular imaging of antibody targeting. Here, we took advantage of immuno-PET imaging using 89Zr-IAB42M1-14, anti-mouse CD8 minibody, to characterize CD8+ T-cell tumor infiltration dynamics following ICOS (inducible T-cell co-stimulator) agonist antibody treatment alone and in combination with PD-1 blocking antibody in a model of mammary carcinoma. PROCEDURES: Female BALB/c mice with established EMT6 tumors received 10 µg, IP of either IgG control antibodies, ICOS agonist monotherapy, or ICOS/PD-1 combination therapy on days 0, 3, 5, 7, 9, 10, or 14. Imaging was performed at 24 and 48 h post IV dose of 89Zr IAB42M1-14. In addition to 89Zr-IAB42M1-14 uptake in tumor and tumor-draining lymph node (TDLN), 3D radiomic features were extracted from PET/CT images to identify treatment effects. Imaging mass cytometry (IMC) and immunohistochemistry (IHC) was performed at end of study. RESULTS: 89Zr-IAB42M1-14 uptake in the tumor was observed by day 11 and was preceded by an increase in the TDLN as early as day 4. The spatial distribution of 89Zr-IAB42M1-14 was more uniform in the drug treated vs. control tumors, which had spatially distinct tracer uptake in the periphery relative to the core of the tumor. IMC analysis showed an increased percentage of cytotoxic T cells in the ICOS monotherapy and ICOS/PD-1 combination group compared to IgG controls. Additionally, temporal radiomics analysis demonstrated early predictiveness of imaging features. CONCLUSION: To our knowledge, this is the first detailed description of the use of a novel immune-PET imaging technique to assess the kinetics of CD8+ T-cell infiltration into tumor and lymphoid tissues following ICOS agonist and PD-1 blocking antibody therapy. By demonstrating the capacity for increased spatial and temporal resolution of CD8+ T-cell infiltration across tumors and lymphoid tissues, these observations underscore the widespread potential clinical utility of non-invasive PET imaging for T-cell-based immunotherapy in cancer.

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.

Multimodal imaging distribution assessment of a liposomal antibiotic in an infectious disease model.

Liposomes are promising targeted drug delivery systems with the potential to improve the efficacy and safety profile of certain classes of drugs. Though attractive, there are unique analytical challenges associated with the development of liposomal drugs including human dose prediction given these are multi-component drug delivery systems. In this study, we developed a multimodal imaging approach to provide a comprehensive distribution assessment for an antibacterial drug, GSK2485680, delivered as a liposomal formulation (Lipo680) in a mouse thigh model of bacterial infection to support human dose prediction. Positron emission tomography (PET) imaging was used to track the in vivo biodistribution of Lipo680 over 48 h post-injection providing a clear assessment of the uptake in various tissues and, importantly, the selective accumulation at the site of infection. In addition, a pharmacokinetic model was created to evaluate the kinetics of Lipo680 in different tissues. Matrix-assisted laser desorption/ionization (MALDI) imaging mass spectrometry (IMS) was then used to quantify the distribution of GSK2485680 and to qualitatively assess the distribution of a liposomal lipid throughout sections of infected and non-infected hindlimb tissues at high spatial resolution. Through the combination of both PET and MALDI IMS, we observed excellent correlation between the Lipo680-radionuclide signal detected by PET with the GSK2485680 and lipid component signals detected by MALDI IMS. This multimodal translational method can reduce drug attrition by generating comprehensive biodistribution profiles of drug delivery systems to provide mechanistic insight and elucidate safety concerns. Liposomal formulations have potential to deliver therapeutics across a broad array of different indications, and this work serves as a template to aid in delivering future liposomal drugs to the clinic.

The use of micro-CT imaging to examine and illustrate fetal skeletal abnormalities in Dutch Belted rabbits and to prove concordance with Alizarin Red stained skeletal examination.

OBJECTIVES: In our laboratory we evaluated the use of micro-computed tomography (micro-CT) using a high resolution acquisition protocol and fetuses obtained on Gestation Day (GD) 29 (mating = GD 0). METHODS: To show concordance between traditional Alizarin Red S stain and micro-CT skeletal examination methods, 103 fetuses from 19 untreated Dutch belted rabbits were obtained by cesarean section and stored frozen. The fetuses were thawed, imaged and examined digitally by micro-CT, then stained and re-examined using traditional methods. RESULTS: A total of 12 individual malformations and 35 unique variations were detected by both methods. Differences in the extent of ossification were found in only 51 of 26,196 bones while 99.8% of the observations were identical. Of the 51 differences, 31 were an unossified fifth medial phalanx of the forepaw indicating that very low-density skeletal bones may be visible by Alizarin Red stain but not by micro-CT scan. To establish this methodology under pharmaceutical testing conditions, we obtained and imaged by micro-CT Alizarin Red S stained abnormal fetal rabbit skeletons previously exposed to a drug candidate associated with craniofacial malformations in humans. All of the types of skeletal abnormalities first identified by staining were also detected by micro-CT examination. Representative images of these 66 different fetal skeletal abnormalities were characterized, and compiled to illustrate visual concordance between micro-CT scanned and traditional Alizarin Red S stained skeletons. CONCLUSION: Micro-CT imaging is an accurate, reliable and robust method that can be used as an alternative to stain when examining fetal rabbit skeletons in developmental toxicity studies.

Non invasive imaging assessment of the biodistribution of GSK2849330, an ADCC and CDC optimized anti HER3 mAb, and its role in tumor macrophage recruitment in human tumor-bearing mice.

The purpose of this work was to use various molecular imaging techniques to non-invasively assess GSK2849330 (anti HER3 ADCC and CDC enhanced 'AccretaMab' monoclonal antibody) pharmacokinetics and pharmacodynamics in human xenograft tumor-bearing mice. Immuno-PET biodistribution imaging of radiolabeled 89Zr-GSK2849330 was assessed in mice with HER3 negative (MIA-PaCa-2) and positive (CHL-1) human xenograft tumors. Dose dependency of GSK2849330 disposition was assessed using varying doses of unlabeled GSK2849330 co-injected with 89Zr-GSK2849330. In-vivo NIRF optical imaging and ex-vivo confocal microscopy were used to assess the biodistribution of GSK2849330 and the HER3 receptor occupancy in HER3 positive xenograft tumors (BxPC3, and CHL-1). Ferumoxytol (USPIO) contrast-enhanced MRI was used to investigate the effects of GSK2849330 on tumor macrophage content in CHL-1 xenograft bearing mice. Immuno-PET imaging was used to monitor the whole body drug biodistribution and CHL-1 xenograft tumor uptake up to 144 hours post injection of 89Zr-GSK2849330. Both hepatic and tumor uptake were dose dependent and saturable. The optical imaging data in the BxPC3 xenograft tumor confirmed the tumor dose response finding in the Immuno-PET study. Confocal microscopy showed a distinguished cytoplasmic punctate staining pattern within individual CHL-1 cells. GSK2849330 inhibited tumor growth and this was associated with a significant decrease in MRI signal to noise ratio after USPIO injection and with a significant increase in tumor macrophages as confirmed by a quantitative immunohistochemistry analysis. By providing both dose response and time course data from both 89Zr and fluorescently labeled GSK2849330, complementary imaging studies were used to characterize GSK2849330 biodistribution and tumor uptake in vivo. Ferumoxytol-enhanced MRI was used to monitor aspects of the immune system response to GSK2849330. Together these approaches potentially provide clinically translatable, non-invasive techniques to support dose optimization, and assess immune activation and anti-tumor responses.