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Advances in antibody engineering have enabled the construction of novel molecular formats in diverse shapes and sizes, providing new opportunities for cancer immunotherapeutic drug discovery while also revealing limitations in knowledge of structure–activity relationships. The current understanding of renal filtration originates largely from data reported for dextrans, IgG, albumin, and selected globular proteins. For a one-armed IgG-based T-cell imaging agent, we observed higher renal signal than typically observed for bivalent IgGs, prompting us to explore the factors governing renal filtration of biologics. We constructed a small representative library of IgG-like formats with varied shapes and hinge flexibilities falling broadly into two categories: branched molecules including bivalent IgG and (scFv)2Fc, and nonbranched molecules including one-armed IgG, one-armed IgG with stacked Fab, and one-armed IgG with a rigid IgA2 hinge. Transmission electron microscopy revealed Y-shaped structures for the branched molecules and pseudo-linear structures for the nonbranched molecules. Single-photon emission CT imaging, autoradiography, and tissue harvest studies demonstrated higher renal uptake and catabolism for nonbranched molecules relative to branched molecules. Among the nonbranched molecules, the one-armed IgG with rigid IgA2 hinge molecule demonstrated higher kidney uptake and decreased systemic exposure relative to molecules with a more flexible hinge. Our results show that differences in shape and hinge flexibility drive the increased glomerular filtration of one-armed relative to bivalent antibodies and highlight the practical advantages of using imaging to assess renal filtration properties. These findings are particularly relevant for T-cell–dependent bispecific molecules, many of which have nonstandard antibody structures.

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A. PET uptake in the circulation per cohort, as determined by region of interest drawn in left ventricle; cohort 1 (in blue) contained 2 patients (0 mg additional MMOT0530A), cohort 2 (in green) contained 9 patients (10 mg additional MMOT0530A). On X axis the different PET moments: 2, 4 and 7 days post injection. On the Y axis the mean SUVmax per cohort.

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Purpose: Mesothelin (MSLN) is frequently overexpressed in pancreatic and ovarian cancers, making it a potential drug target. We performed an 89Zr-PET imaging study with MMOT0530A, a MSLN antibody, in conjunction with a phase I study with the antibody–drug conjugate DMOT4039A, containing MMOT0530A bound to MMAE. The aim was to study antibody tumor uptake, whole-body distribution, and relation between uptake, response to treatment, and MSLN expression.Experimental Design: Before DMOT4039A treatment, patients received 37 MBq 89Zr-MMOT0530A followed by PET/CT imaging 2, 4, and 7 days postinjection. Tracer uptake was expressed as standardized uptake value (SUV). MSLN expression was determined with immunohistochemistry (IHC) on archival tumor tissue.Results: Eleven patients were included, 7 with pancreatic and 4 with ovarian cancer. IHC MSLN expression varied from absent to strong. Suitable tracer antibody dose was 10 mg MMOT0530A and optimal imaging time was 4 and 7 days postinjection. Tumor tracer uptake occurred in 37 lesions with mean SUVmax of 13.1 (±7.5) on PET 4 days postinjection, with 11.5 (±7.5) in (N = 17) pancreatic and 14.5 (±8.7) in (N = 20) ovarian cancer lesions. Within patients, a mean 2.4-fold (±1.10) difference in uptake between tumor lesions existed. Uptake in blood, liver, kidneys, spleen, and intestine reflected normal antibody distribution. Tracer tumor uptake was correlated to IHC. Best response to DMOT4039A was partial response in one patient.Conclusions: With 89Zr-MMOT0530A-PET, pancreatic and ovarian cancer lesions as well as antibody biodistribution could be visualized. This technique can potentially guide individualized antibody-based treatment. Clin Cancer Res; 22(7); 1642–52. ©2015 AACR.

Immune checkpoint inhibitors (ICIs), by reinvigorating CD8+ T cell mediated immunity, have revolutionized cancer therapy. Yet, the systemic CD8+ T cell distribution, a potential biomarker of ICI response, remains poorly characterized. We assessed safety, imaging dose and timing, pharmacokinetics and immunogenicity of zirconium-89-labeled, CD8-specific, one-armed antibody positron emission tomography tracer 89ZED88082A in patients with solid tumors before and ~30 days after starting ICI therapy (NCT04029181). No tracer-related side effects occurred. Positron emission tomography imaging with 10 mg antibody revealed 89ZED88082A uptake in normal lymphoid tissues, and tumor lesions across the body varying within and between patients two days after tracer injection (n = 38, median patient maximum standard uptake value (SUVmax) 5.2, IQI 4.0–7.4). Higher SUVmax was associated with mismatch repair deficiency and longer overall survival. Uptake was higher in lesions with stromal/inflamed than desert immunophenotype. Tissue radioactivity was localized to areas with immunohistochemically confirmed CD8 expression. Re-imaging patients on treatment showed no change in average (geometric mean) tumor tracer uptake compared to baseline, but individual lesions showed diverse changes independent of tumor response. The imaging data suggest enormous heterogeneity in CD8+ T cell distribution and pharmacodynamics within and between patients. In conclusion, 89ZED88082A can characterize the complex dynamics of CD8+ T cells in the context of ICIs, and may inform immunotherapeutic treatments.

Immune checkpoint inhibitors (ICIs), by reinvigorating CD8