89Zr-3,2-HOPO-Mesothelin Antibody PET Imaging Reflects Tumor Uptake of Mesothelin-Targeted 227Th-Conjugate Therapy in Mice.

The mesothelin (MSLN)-targeted 227Th conjugate is a novel α-therapy developed to treat MSLN-overexpressing cancers. We radiolabeled the same antibody-chelator conjugate with 89Zr to evaluate whether PET imaging with 89Zr-MSLN matches 227Th-MSLN tumor uptake, biodistribution, and antitumor activity. Methods: Serial PET imaging with protein doses of 4, 20, or 40 µg of 89Zr-MSLN and 89Zr-control was performed up to 168 h after tracer injection in human tumor-bearing nude mice with high (HT29-MSLN) and low (BxPc3) MSLN expression. 89Zr-MSLN and 227Th-MSLN ex vivo tumor uptake and biodistribution were compared at 6 time points in HT29-MSLN and in medium-MSLN-expressing (OVCAR-3) tumor-bearing mice. 89Zr-MSLN PET imaging was performed before 227Th-MSLN treatment in HT29-MSLN and BxPc3 tumor-bearing mice. Results: 89Zr-MSLN PET imaging showed an SUVmean of 2.2 ± 0.5 in HT29-MSLN tumors. Ex vivo tumor uptake was 10.6% ± 2.4% injected dose per gram at 168 h. 89Zr-MSLN tumor uptake was higher than uptake of 89Zr-control (P = 0.0043). 89Zr-MSLN and 227Th-MSLN showed comparable tumor uptake and biodistribution in OVCAR-3 and HT29-MSLN tumor-bearing mice. Pretreatment SUVmean was 2.2 ± 0.2 in HT29-MSLN tumors, which decreased in volume on 227Th-MSLN treatment. BxPc3 tumors showed an SUVmean of 1.2 ± 0.3 and remained similar in size after 227Th-MSLN treatment. Conclusion: 89Zr-MSLN PET imaging reflected MSLN expression and matched 227Th-MSLN tumor uptake and biodistribution. Our data support the clinical exploration of 89Zr-MSLN PET imaging together with 227Th-MSLN therapy, both using the same antibody-chelator conjugate.

Mesothelin/CD3 half-life extended bispecific T-cell engager molecule shows specific tumor uptake and distributes to mesothelin and CD3 expressing tissues.

BiTE ® (bispecific T-cell engager) molecules exert antitumor activity by binding one arm to CD3 on cytotoxic T-cells and the other arm to a tumor-associated antigen. We generated a fully mouse cross-reactive mesothelin (MSLN)-targeted BiTE molecule that is genetically fused to a Fc-domain for half-life extension, and evaluated biodistribution and tumor targeting of a zirconium-89 (89Zr)-labeled MSLN HLE BiTE molecule in 4T1 breast cancer bearing syngeneic mice with positron emission tomography (PET). Biodistribution of 50 µg 89Zr-MLSN HLE BiTE was studied over time by PET imaging in BALB/c mice and revealed uptake in tumor and lymphoid tissues with an elimination half-life of 63.4 hours. Compared to a non-targeting 89Zr-control HLE BiTE, the 89Zr-MLSN HLE BiTE showed a 2-fold higher tumor uptake and higher uptake in lymphoid tissues. Uptake in the tumor colocalized with mesothelin expression, while uptake in the spleen colocalized with CD3 expression. Evaluation of the effect of protein doses on the biodistribution and tumor targeting of 89Zr-MSLN HLE BiTE revealed for all dose groups that uptake in the spleen was faster than in the tumor (day 1 vs day 5). The lowest dose of 10 µg 89Zr-MSLN HLE BiTE had higher spleen uptake and faster blood clearance compared to higher doses of 50 µg and 200 µg. 89Zr-MSLN HLE BiTE tumor uptake was similar at all doses. Conclusion: The MSLN HLE BiTE showed specific tumor uptake and both arms contributed to the biodistribution profile. These findings support the potential for clinical translation of HLE BiTE molecules.

Radiolabeled Monoclonal Antibody Against Colony-Stimulating Factor 1 Receptor Specifically Distributes to the Spleen and Liver in Immunocompetent Mice.

Macrophages can promote tumor development. Preclinically, targeting macrophages by colony-stimulating factor 1 (CSF1)/CSF1 receptor (CSF1R) monoclonal antibodies (mAbs) enhances conventional therapeutics in combination treatments. The physiological distribution and tumor uptake of CSF1R mAbs are unknown. Therefore, we radiolabeled a murine CSF1R mAb and preclinically visualized its biodistribution by PET. CSF1R mAb was conjugated to N-succinyl-desferrioxamine (N-suc-DFO) and subsequently radiolabeled with zirconium-89 (89Zr). Optimal protein antibody dose was first determined in non-tumor-bearing mice to assess physiological distribution. Next, biodistribution of optimal protein dose and 89Zr-labeled isotype control was compared with PET and ex vivo biodistribution after 24 and 72 h in mammary tumor-bearing mice. Tissue autoradiography and immunohistochemistry determined radioactivity distribution and tissue macrophage presence, respectively. [89Zr]Zr-DFO-N-suc-CSF1R-mAb optimal protein dose was 10 mg/kg, with blood pool levels of 10 ± 2% injected dose per gram tissue (ID/g) and spleen and liver uptake of 17 ± 4 and 11 ± 4%ID/g at 72 h. In contrast, 0.4 mg/kg of [89Zr]Zr-DFO-N-suc-CSF1R mAb was eliminated from circulation within 24 h; spleen and liver uptake was 126 ± 44% and 34 ± 7%ID/g, respectively. Tumor-bearing mice showed higher uptake of [89Zr]Zr-DFO-N-suc-CSF1R-mAb in the liver, lymphoid tissues, duodenum, and ileum, but not in the tumor than did 89Zr-labeled control at 72 h. Immunohistochemistry and autoradiography showed that 89Zr was localized to macrophages within lymphoid tissues. Following [89Zr]Zr-DFO-N-suc-CSF1R-mAb administration, tumor macrophages were almost absent, whereas isotype-group tumors contained over 500 cells/mm2. We hypothesize that intratumoral macrophage depletion by [89Zr]Zr-DFO-N-suc-CSF1R-mAb precluded tumor uptake higher than 89Zr-labeled control. Translation of molecular imaging of macrophage-targeting therapeutics to humans may support macrophage-directed therapeutic development.

Fluorescent image-guided surgery in breast cancer by intravenous application of a quenched fluorescence activity-based probe for cysteine cathepsins in a syngeneic mouse model.

PURPOSE: The reoperation rate for breast-conserving surgery is as high as 15-30% due to residual tumor in the surgical cavity after surgery. In vivo tumor-targeted optical molecular imaging may serve as a red-flag technique to improve intraoperative surgical margin assessment and to reduce reoperation rates. Cysteine cathepsins are overexpressed in most solid tumor types, including breast cancer. We developed a cathepsin-targeted, quenched fluorescent activity-based probe, VGT-309, and evaluated whether it could be used for tumor detection and image-guided surgery in syngeneic tumor-bearing mice. METHODS: Binding specificity of the developed probe was evaluated in vitro. Next, fluorescent imaging in BALB/c mice bearing a murine breast tumor was performed at different time points after VGT-309 administration. Biodistribution of VGT-309 after 24 h in tumor-bearing mice was compared to control mice. Image-guided surgery was performed at multiple time points tumors with different clinical fluorescent camera systems and followed by ex vivo analysis. RESULTS: The probe was specifically activated by cathepsins X, B/L, and S. Fluorescent imaging revealed an increased tumor-to-background contrast over time up to 15.1 24 h post probe injection. In addition, VGT-309 delineated tumor tissue during image-guided surgery with different optical fluorescent imaging camera systems. CONCLUSION: These results indicate that optical fluorescent molecular imaging using the cathepsin-targeted probe, VGT-309, may improve intraoperative tumor detection, which could translate to more complete tumor resection when coupled with commercially available surgical tools and techniques.

The Biodistribution of a CD3 and EpCAM Bispecific T-Cell Engager Is Driven by the CD3 Arm.

Bispecific T-cell engager (BiTE) molecules are designed to engage and activate cytotoxic T cells to kill tumor cells. Little is known about their biodistribution in immunocompetent settings. Methods: To explore their pharmacokinetics and the role of the immune cells, BiTE molecules were radiolabeled with the PET isotope 89Zr and studied in immunocompetent and immunodeficient mouse models. Results: PET images and ex vivo biodistribution in immunocompetent mice with [89Zr]Zr-DFO-N-suc-muS110, targeting mouse CD3 (dissociation constant [KD], 2.9 nM) and mouse epithelial cell adhesion molecule (EpCAM; KD, 21 nM), and with [89Zr]Zr-DFO-N-suc-hyS110, targeting only mouse CD3 (KD, 2.9 nM), showed uptake in the tumor, spleen, and other lymphoid organs, whereas the human-specific control BiTE [89Zr]Zr-DFO-N-suc-AMG 110 showed similar tumor uptake but lacked spleen uptake. [89Zr]Zr-DFO-N-suc-muS110 spleen uptake was lower in immunodeficient than in immunocompetent mice. After repeated administration of nonradiolabeled muS110 to immunocompetent mice, 89Zr-muS110 uptake in the spleen and other lymphoid tissues decreased and was comparable to uptake in immunodeficient mice, indicating saturation of CD3 binding sites. Autoradiography and immunohistochemistry demonstrated colocalization of [89Zr]Zr-DFO-N-suc-muS110 and [89Zr]Zr-DFO-N-suc-hyS110 with CD3-positive T cells in the tumor and spleen but not with EpCAM expression. Also, uptake in the duodenum correlated with a high incidence of T cells. Conclusion: [89Zr]Zr-DFO-N-suc-muS110 biodistribution is dependent mainly on the T-cell-targeting arm, with a limited contribution from its second arm, targeting EpCAM. These findings highlight the need for extensive biodistribution studies of novel bispecific constructs, as the results might have implications for their respective drug development and clinical translation.

Development and Evaluation of Interleukin-2-Derived Radiotracers for PET Imaging of T Cells in Mice.

Recently, N-(4-18F-fluorobenzoyl)-interleukin-2 (18F-FB-IL2) was introduced as a PET tracer for T cell imaging. However, production is complex and time-consuming. Therefore, we developed 2 radiolabeled IL2 variants, namely aluminum 18F-fluoride-(restrained complexing agent)-IL2 (18F-AlF-RESCA-IL2) and 68Ga-gallium-(1,4,7-triazacyclononane-4,7-diacetic acid-1-glutaric acid)-IL2 (68Ga-Ga-NODAGA-IL2), and compared their in vitro and in vivo characteristics with 18F-FB-IL2. Methods: Radiolabeling of 18F-AlF-RESCA-IL2 and 68Ga-Ga-NODAGA-IL2 was optimized, and stability was evaluated in human serum. Receptor binding was studied with activated human peripheral blood mononuclear cells (hPBMCs). Ex vivo tracer biodistribution in immunocompetent BALB/cOlaHsd (BALB/c) mice was performed at 15, 60, and 90 min after tracer injection. In vivo binding characteristics were studied in severe combined immunodeficient (SCID) mice inoculated with activated hPBMCs in Matrigel. Tracer was injected 15 min after hPBMC inoculation, and a 60-min dynamic PET scan was acquired, followed by ex vivo biodistribution studies. Specific uptake was determined by coinjection of tracer with unlabeled IL2 and by evaluating uptake in a control group inoculated with Matrigel only. Results:68Ga-Ga-NODAGA-IL2 and 18F-AlF-RESCA-IL2 were produced with radiochemical purity of more than 95% and radiochemical yield of 13.1% ± 4.7% and 2.4% ± 1.6% within 60 and 90 min, respectively. Both tracers were stable in serum, with more than 90% being intact tracer after 1 h. In vitro, both tracers displayed preferential binding to activated hPBMCs. Ex vivo biodistribution studies on BALB/c mice showed higher uptake of 18F-AlF-RESCA-IL2 than of 18F-FB-IL2 in liver, kidney, spleen, bone, and bone marrow. 68Ga-Ga-NODAGA-IL2 uptake in liver and kidney was higher than 18F-FB-IL2 uptake. In vivo, all tracers revealed uptake in activated hPBMCs in SCID mice. Low uptake was seen after a blocking dose of IL2 and in the Matrigel control group. In addition, 18F-AlF-RESCA-IL2 yielded the highest-contrast PET images of target lymph nodes. Conclusion: Production of 18F-AlF-RESCA-IL2 and 68Ga-Ga-NODAGA-IL2 is simpler and faster than that of 18F-FB-IL2. Both tracers showed good in vitro and in vivo characteristics, with high uptake in lymphoid tissue and hPBMC xenografts.

A review of bispecific antibodies and antibody constructs in oncology and clinical challenges.

Bispecific antibodies (bsAbs) are antibodies that bind two distinct epitopes to cancer.. For use in oncology, one bsAb has been approved and 57 bsAbs are in clinical trials, none of which has reached phase 3. These bsAbs show great variability in design and mechanism of action. The various designs are often linked to the mechanisms of actions. The majority of bsAbs engage immune cells to destroy tumor cells. However, some bsAbs are also used to deliver payloads to tumors or to block tumor signaling pathways. This review provides insight into the choice of construct for bsAbs, summarizes the clinical development of bsAbs in oncology and identifies subsequent challenges.

89Zr-labeled Bispecific T-cell Engager AMG 211 PET Shows AMG 211 Accumulation in CD3-rich Tissues and Clear, Heterogeneous Tumor Uptake.

PURPOSE: Biodistribution of bispecific antibodies in patients is largely unknown. We therefore performed a feasibility study in 9 patients with advanced gastrointestinal adenocarcinomas to explore AMG 211 biodistribution (also known as MEDI-565), an approximately 55 kDa bispecific T-cell engager (BiTE®) directed against carcinoembryonic antigen (CEA) on tumor cells and cluster of differentiation 3 (CD3) on T-cells. EXPERIMENTAL DESIGN: 89Zr-labeled AMG 211 as tracer was administered alone or with cold AMG 211, for PET imaging before and/or during AMG 211 treatment. RESULTS: Before AMG 211 treatment, the optimal imaging dose was 200-µg 89Zr-AMG 211 + 1,800-µg cold AMG 211. At 3 hours, the highest blood pool standardized uptake value (SUV)mean was 4.0, and tracer serum half-life was 3.3 hours. CD3-mediated uptake was clearly observed in CD3-rich lymphoid tissues including spleen and bone marrow (SUVmean 3.2 and 1.8, respectively), and the SUVmean decreased more slowly than in other healthy tissues. 89Zr-AMG 211 remained intact in plasma and was excreted predominantly via the kidneys in degraded forms. Of 43 visible tumor lesions, 37 were PET quantifiable, with a SUVmax of 4.0 [interquartile range (IQR) 2.7-4.4] at 3 hours using the optimal imaging dose. The tracer uptake differed between tumor lesions 5-fold within and 9-fold between patients. During AMG 211 treatment, tracer was present in the blood pool, whereas tumor lesions were not visualized, possibly reflecting target saturation. CONCLUSIONS: This first-in-human study shows high, specific 89Zr-AMG 211 accumulation in CD3-rich lymphoid tissues, as well as a clear, inter- and intraindividual heterogeneous tumor uptake.