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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

Summary Limitations to the application of molecularly targeted cancer therapies are the inability to accurately match patient with effective treatment and the absence of a prompt readout of posttreatment response. Noninvasive agents that rapidly report vascular endothelial growth factor (VEGF) levels using positron emission tomography (PET) have the potential to enhance anti-angiogenesis therapies. Using phage display, two distinct classes of peptides were identified that bind to VEGF with nanomolar affinity and high selectivity. Co-crystal structures of these different peptide classes demonstrate that both bind to the receptor-binding region of VEGF. 18 F-radiolabelling of these peptides facilitated the acquisition of PET images of tumor VEGF levels in a HM7 xenograph model. The images obtained from one 59-residue probe, 18 F-Z-3B, 2 hr postinjection are comparable to those obtained with anti-VEGF antibody B20 72 hr postinjection. Furthermore, VEGF levels in growing SKOV3 tumors were followed using 18 F-Z-3B as a PET probe with VEGF levels increasing with tumor size.

Three thiol reactive reagents were developed for the chemoselective conjugation of desferrioxamine (Df) to a monoclonal antibody via engineered cysteine residues (thio-trastuzumab). The in vitro stability and in vivo imaging properties of site-specifically radiolabeled 89 Zr-Df-thio-trastuzumab conjugates were investigated. Methods The amino group of desferrioxamine B was acylated by bromoacetyl bromide, N -hydroxysuccinimidyl iodoacetate, or N -hydroxysuccinimidyl 4-[ N -maleimidomethyl]cyclohexane-1-carboxylate to obtain thiol reactive reagents bromoacetyl-desferrioxamine (Df-Bac), iodoacetyl-desferrioxamine (Df-Iac) and maleimidocyclohexyl-desferrioxamine (Df-Chx-Mal), respectively. Df-Bac and Df-Iac alkylated the free thiol groups of thio-trastuzumab by nucleophilic substitution forming Df-Ac-thio-trastuzumab, while the maleimide reagent Df-Chx-Mal reacted via Michael addition to provide Df-Chx-Mal-thio-trastuzumab. The conjugates were radiolabeled with 89 Zr and evaluated for serum stability, and their positron emission tomography (PET) imaging properties were investigated in a BT474M1 (HER2-positive) breast tumor mouse model. Results The chemoselective reagents were obtained in 14% (Df-Bac), 53% (Df-Iac) and 45% (Df-Chx-Mal) yields. Site-specific conjugation of Df-Chx-Mal to thio-trastuzumab was complete within 1 h at pH 7.5, while Df-Iac and Df-Bac respectively required 2 and 5 h at pH 9. Each Df modified thio-trastuzumab was chelated with 89 Zr in yields exceeding 75%. 89 Zr-Df-Ac-thio-trastuzumab and 89 Zr-Df-Chx-Mal-thio-trastuzumab were stable in mouse serum and exhibited comparable PET imaging capabilities in a BT474M1 (HER2-positive) breast cancer model reaching 20–25 %ID/g of tumor uptake and a tumor to blood ratio of 6.1–7.1. Conclusions The new reagents demonstrated good reactivity with engineered thiol groups of trastuzumab and very good chelation properties with 89 Zr. The site-specifically 89 Zr-labeled thio-antibodies were stable in serum and showed PET imaging properties comparable to lysine conjugates.

Receptor-specific proteins produced by genetic engineering are attractive as PET imaging agents, but labeling with conventional (18)F-based prosthetic groups is problematic due to long synthesis times, poor radiochemical yields, and low specific activities. Therefore, we developed a modular platform for the rapid preparation of water-soluble prosthetic groups capable of efficiently introducing (18)F into proteins. The utility of this platform is demonstrated by the thiol-specific prosthetic group, [(18)F]FPEGMA, which was used to produce site-specifically (18)F-labeled protein ((18)F-trastuzumab-ThioFab) in 82 min with a total radiochemical yield of 13 +/- 3% and a specific activity of 2.2 +/- 0.2 Ci/micromol. (18)F-trastuzumab-ThioFab retained the biological activity of native protein and was successfully validated in vivo with microPET imaging of Her2 expression in a xenograft tumor-bearing murine model modulated by the Hsp90 inhibitor, 17-(allylamino)-17-demethoxygeldanamycin.

The purpose of this study was to examine relationships between aerobic fitness (AF), fatness, and coronary-heart-disease (CHD) risk factors in 12- to 13-year-olds. The data were obtained from 208 schoolchildren (100 boys; 108 girls) ages 12.9 ± 0.3 years. Measurements included AF, indices of obesity, blood pressure, blood lipids and lipoproteins, fibrinogen, homocysteine, and C-reactive protein. An inverse relationship was found between AF and fatness (p < .05). Fatness was related to a greater number of CHD risk factors than fitness was (p < .05). Further analysis revealed fatness to be an independent predictor of triglyceride and blood-pressure levels (p < .05). Our findings indicate that, for young people, fatness rather than fitness is independently related to CHD risk factors.

Mesothelin is a tumor differentiation antigen expressed by epithelial tumors, including pancreatic cancer. Currently, mesothelin is being targeted with an antibody-drug conjugate (ADC) consisting of a mesothelin-specific antibody coupled to a highly potent chemotherapeutic drug. Considering the toxicity of the ADC and reduced accessibility of pancreatic tumors, non-invasive imaging could provide necessary information. We therefore developed a zirconium-89 (89Zr) labeled anti-mesothelin antibody (89Zr-AMA) to study its biodistribution in human pancreatic tumor bearing mice. Biodistribution and dose-finding of 89Zr-AMA were studied 144 h after tracer injection in mice with subcutaneously xenografted HPAC. MicroPET imaging was performed 24, 72 and 144 h after tracer injection in mice bearing HPAC or Capan-2. Tumor uptake and organ distribution of 89Zr-AMA were compared with nonspecific 111In-IgG. Biodistribution analyses revealed a dose-dependent 89Zr-AMA tumor uptake. Tumor uptake of 89Zr-AMA was higher than 111In-IgG using the lowest tracer dose. MicroPET showed increased tumor uptake over 6 days, whereas activity in blood pool and other tissues decreased. Immunohistochemistry showed that mesothelin was expressed by the HPAC and CAPAN-2 tumors and fluorescence microscopy revealed that AMA-800CW was present in tumor cell cytoplasm. 89Zr-AMA tumor uptake is antigen-specific in mesothelin-expressing tumors. 89Zr-AMA PET provides non-invasive, real-time information about AMA distribution and tumor targeting.

This study determined the prevalence of coronary heart disease (CHD) risk factors in young people1of differing socio-economic status (SES). A cohort of 100 boys and 108 girls, aged 12.9, SD 0.3 years drawn of differing SES were assessed for CHD risk factors. Measurements included indices of obesity, blood pressure, aerobic fitness, diet, blood lipids and lipoproteins. Significant differences ( P ≤ 0.05) were detected with respect to SES, for systolic blood pressure, total cholesterol and low density lipopro tein cholesterol, but in boys only. Significant sex differences ( P ≤ 0.05) were identified for systolic blood pressure, diastolic blood pressure, aerobic fitness, waist to hip ratio, summation of four skinfold thicknesses, average daily kilocalorie intake, total choles terol and low density lipoprotein cholesterol. Moreover, increased body fatness, low aerobic fitness, a high fat diet and elevated lipoprotein (a), were the most frequently identified CHD risk factors in this cohort. This study reveals a high incidence of CHD risk factors in young people irrespective of SES and sex, and highlights the need for vigilance in identifying such risk factors from a young age.