Phase I Study of [68Ga]Ga-Anti-CD206-sdAb for PET/CT Assessment of Protumorigenic Macrophage Presence in Solid Tumors (MMR Phase I).

Macrophages play an important role throughout the body. Antiinflammatory macrophages expressing the macrophage mannose receptor (MMR, CD206) are involved in disease development, ranging from oncology to atherosclerosis and rheumatoid arthritis. [68Ga]Ga-NOTA-anti-CD206 single-domain antibody (sdAb) is a PET tracer targeting CD206. This first-in-human study, as its primary objective, evaluated the safety, biodistribution, and dosimetry of this tracer. The secondary objective was to assess its tumor uptake. Methods: Seven patients with a solid tumor of at least 10 mm, an Eastern Cooperative Oncology Group score of 0 or 1, and good renal and hepatic function were included. Safety was evaluated using clinical examination and blood sampling before and after injection. For biodistribution and dosimetry, PET/CT was performed at 11, 90, and 150 min after injection; organs showing tracer uptake were delineated, and dosimetry was evaluated. Blood samples were obtained at selected time points for blood clearance. Metabolites in blood and urine were assessed. Results: Seven patients were injected with, on average, 191 MBq of [68Ga]Ga-NOTA-anti-CD206-sdAb. Only 1 transient adverse event of mild severity was considered to be possibly, although unlikely, related to the study drug (headache, Common Terminology Criteria for Adverse Events grade 1). The blood clearance was fast, with less than 20% of the injected activity remaining after 80 min. There was uptake in the liver, kidneys, spleen, adrenals, and red bone marrow. The average effective dose from the radiopharmaceutical was 4.2 mSv for males and 5.2 mSv for females. No metabolites were detected. Preliminary data of tumor uptake in cancer lesions showed higher uptake in the 3 patients who subsequently progressed than in the 3 patients without progression. One patient could not be evaluated because of technical failure. Conclusion: [68Ga]Ga-NOTA-anti-CD206-sdAb is safe and well tolerated. It shows rapid blood clearance and renal excretion, enabling high contrast-to-noise imaging at 90 min after injection. The radiation dose is comparable to that of routinely used PET tracers. These findings and the preliminary results in cancer patients warrant further investigation of this tracer in phase II clinical trials.

Phase I Trial of 131I-GMIB-Anti-HER2-VHH1, a New Promising Candidate for HER2-Targeted Radionuclide Therapy in Breast Cancer Patients.

131I-GMIB-anti-human epidermal growth factor receptor type 2 (HER2)-VHH1 is a targeted radionuclide theranostic agent directed at HER2-expressing cancers. VHH1 is a single-domain antibody covalently linked to therapeutic 131I via the linker N-succinimidyl 4-guanidino-methyl-3-iodobenzoate (SGMIB). The phase I study was aimed at evaluating the safety, biodistribution, radiation dosimetry, and tumor-imaging potential of 131I-GMIB-anti-HER2-VHH1 in healthy volunteers and breast cancer patients. Methods: In a first cohort, 6 healthy volunteers were included. The biodistribution of 131I-GMIB-anti-HER2-VHH1 was assessed using whole-body (anterior and posterior) planar images obtained at 40 min and at 2, 4, 24, and 72 h after intravenously administered (38 ± 9 MBq) 131I-GMIB-anti-HER2-VHH1. Imaging data were analyzed using OLINDA/EXM software to determine the dosimetry. Blood and urine samples were obtained over 72 h. In the second cohort, 3 patients with metastatic HER2-positive breast cancer were included. Planar whole-body imaging was performed at 2 and 24 h after injection. Additional SPECT/CT images were obtained after the whole-body images at 2 and 24 h if there was relevant uptake in known cancer lesions. Results: No drug-related adverse events were observed throughout the study. The biologic half-life of 131I-GMIB-anti-HER2-VHH1 in healthy subjects was about 8 h. After intravenous administration, the compound was eliminated from the blood with a 2.5-h half-life. The drug was eliminated primarily via the kidneys. The drug was stable in circulation, and there was no increased accumulation in the thyroid or stomach. The absorbed dose to the kidneys was 1.54 ± 0.25 mGy/MBq, and to bone marrow it was 0.03 ± 0.01 mGy/MBq. SPECT/CT imaging in patients with advanced breast cancer showed focal uptake of 131I-GMIB-anti-HER2-VHH1 in metastatic lesions. Conclusion: Because of its favorable toxicity profile and its uptake in HER2-positive lesions, this radiopharmaceutical can offer new therapeutic options to patients who have progressed on trastuzumab, pertuzumab, or trastuzmab emtansine, given its difference in mode-of-action. A dose escalation is planned in a subsequent phase I/II study to assess the therapeutic window of this compound (NCT04467515).

Population Pharmacokinetic Approach Applied to Positron Emission Tomography: Computed Tomography for Tumor Tissue Identification in Patients with Glioma.

BACKGROUND AND AIMS: 18F-fluoro-ethyl-tyrosine (FET) is a radiopharmaceutical used in positron emission tomography (PET)-computed tomography in patients with glioma. We propose an original approach combining a radiotracer-pharmacokinetic exploration performed at the voxel level (three-dimensional pixel) and voxel classification to identify tumor tissue. Our methodology was validated using the standard FET-PET approach and magnetic resonance imaging (MRI) data acquired according to the current clinical practices. METHODS: FET-PET and MRI data were retrospectively analyzed in ten patients presenting with progressive high-grade glioma. For FET-PET exploration, radioactivity acquisition started 15 min after radiotracer injection, and was measured each 5 min during 40 min. The tissue segmentation relies on population pharmacokinetic modeling with dependent individuals (voxels). This model can be approximated by a linear mixed-effects model. The tumor volumes estimated by our approach were compared with those determined with the current clinical techniques, FET-PET standard approach (i.e., a cumulated value of FET signal is computed during a time interval) and MRI sequences (T1 and T2/fluid-attenuated inversion recovery [FLAIR]), used as references. The T1 sequence is useful to identify highly vascular tumor and necrotic tissues, while the T2/FLAIR sequence is useful to isolate infiltration and edema tissue located around the tumor. RESULTS: With our kinetic approach, the volumes of tumor tissue were larger than the tissues identified by the standard FET-PET and MRI T1, while they were smaller than those determined with MRI T2/FLAIR. CONCLUSION: Our results revealed the presence of suspected tumor voxels not identified by the standard PET approach.

Phase I Study of 68Ga-HER2-Nanobody for PET/CT Assessment of HER2 Expression in Breast Carcinoma.

UNLABELLED: Human epidermal growth factor receptor 2 (HER2) status is one of the major tumor characteristics in breast cancer to guide therapy. Anti-HER2 treatment has clear survival advantages in HER2-positive breast carcinoma patients. Heterogeneity in HER2 expression between primary tumor and metastasis has repeatedly been described, resulting in the need to reassess HER2 status during the disease course. To avoid repeated biopsy with potential bias due to tumor heterogeneity, Nanobodies directed against HER2 have been developed as probes for molecular imaging. Nanobodies, which are derived from unique heavy-chain-only antibodies, are the smallest antigen-binding antibody fragments and have ideal characteristics for PET imaging. The primary aims were assessment of safety, biodistribution, and dosimetry. The secondary aim was to investigate tumor-targeting potential. METHODS: In total, 20 women with primary or metastatic breast carcinoma (score of 2+ or 3+ on HER2 immunohistochemical assessment) were included. Anti-HER2-Nanobody was labeled with (68)Ga via a NOTA derivative. Administered activities were 53-174 MBq (average, 107 MBq). PET/CT scans for dosimetry assessment were obtained at 10, 60, and 90 min after administration. Physical evaluation and blood analysis were performed for safety evaluation. Biodistribution was analyzed for 11 organs using MIM software; dosimetry was assessed using OLINDA/EXM. Tumor-targeting potential was assessed in primary and metastatic lesions. RESULTS: No adverse reactions occurred. A fast blood clearance was observed, with only 10% of injected activity remaining in the blood at 1 h after injection. Uptake was seen mainly in the kidneys, liver, and intestines. The effective dose was 0.043 mSv/MBq, resulting in an average of 4.6 mSv per patient. The critical organ was the urinary bladder wall, with a dose of 0.406 mGy/MBq. In patients with metastatic disease, tracer accumulation well above the background level was demonstrated in most identified sites of disease. Primary lesions were more variable in tracer accumulation. CONCLUSION: (68)Ga-HER2-Nanobody PET/CT is a safe procedure with a radiation dose comparable to other routinely used PET tracers. Its biodistribution is favorable, with the highest uptake in the kidneys, liver, and intestines but very low background levels in all other organs that typically house primary breast carcinoma or tumor metastasis. Tracer accumulation in HER2-positive metastases is high, compared with normal surrounding tissues, and warrants further assessment in a phase II trial.

PET Imaging of Macrophage Mannose Receptor-Expressing Macrophages in Tumor Stroma Using 18F-Radiolabeled Camelid Single-Domain Antibody Fragments.

UNLABELLED: Tumor-associated macrophages constitute a major component of the stroma of solid tumors, encompassing distinct subpopulations with different characteristics and functions. We aimed to identify M2-oriented tumor-supporting macrophages within the tumor microenvironment as indicators of cancer progression and prognosis, using PET imaging. This can be realized by designing (18)F-labeled camelid single-domain antibody fragments (sdAbs) specifically targeting the macrophage mannose receptor (MMR), which has been identified as an important biomarker on this cell population. METHODS: Cross-reactive anti-MMR sdAbs were generated after immunization of an alpaca with the extracellular domains of both human and mouse MMR. The lead binder was chosen on the basis of comparisons of binding affinity and in vivo pharmacokinetics. The PET tracer (18)F-fluorobenzoate (FB)-anti-MMR sdAb was developed using the prosthetic group N-succinimidyl-4-(18)F-fluorobenzoate ((18)F-SFB), and its biodistribution, tumor-targeting potential, and specificity in terms of macrophage and MMR targeting were evaluated in mouse tumor models. RESULTS: Four sdAbs were selected after affinity screening, but only 2 were found to be cross-reactive for human and mouse MMR. The lead anti-MMR 3.49 sdAb, bearing an affinity of 12 and 1.8 nM for mouse and human MMR, respectively, was chosen for its favorable in vivo biodistribution profile and tumor-targeting capacity. (18)F-FB-anti-MMR 3.49 sdAb was synthesized with a 5%-10% radiochemical yield using an automated and optimized protocol. In vivo biodistribution analyses showed fast clearance via the kidneys and retention in MMR-expressing organs and tumor. The kidney retention of the fluorinated sdAb was 20-fold lower than a (99m)Tc-labeled counterpart. Compared with MMR- and C-C chemokine receptor 2-deficient mice, significantly higher uptake was observed in tumors grown in wild-type mice, demonstrating the specificity of the (18)F tracer for MMR and macrophages, respectively. CONCLUSION: Anti-MMR 3.49 was denoted as the lead cross-reactive MMR-targeting sdAb. (18)F radiosynthesis was optimized, providing an optimal probe for PET imaging of the tumor-promoting macrophage subpopulation in the tumor stroma.

Radiolabeled mannosylated dextran derivatives bearing an NIR-fluorophore for sentinel lymph node imaging.

Current methods for sentinel lymph node (SLN) mapping involve the use of radioactivity detection with technetium-99m sulfur colloid and/or visually guided identification using a blue dye. To overcome the kinetic variations of two individual imaging agents through the lymphatic system, we report herein on two multifunctional macromolecules, 5a and 6a, that contain a radionuclide ((99m)Tc or (68)Ga) and a near-infrared (NIR) reporter for pre- and/or intraoperative SLN mapping by nuclear and NIR optical imaging techniques. Both bimodal probes are dextran-based polymers (10 kDa) functionalized with pyrazole-diamine (Pz) or 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid (DOTA) chelating units for labeling with fac-[(99m)Tc(CO)3](+) or (68)Ga(III), respectively, mannose units for receptor targeting, and NIR fluorophore units for optical imaging. The probes allowed a clear visualization of the popliteal node by single-photon emission computed tomography (SPECT/CT) or positron emission tomography (PET/CT), as well as real-time optically guided excision. Biodistribution studies confirmed that both macromolecules present a significant accumulation in the popliteal node (5a: 3.87 ± 0.63% IA/organ; 6a: 1.04 ± 0.26% IA/organ), with minimal spread to other organs. The multifunctional nanoplatforms display a popliteal extraction efficiency >90%, highlighting their potential to be further explored as dual imaging agents.

Synthesis, preclinical validation, dosimetry, and toxicity of 68Ga-NOTA-anti-HER2 Nanobodies for iPET imaging of HER2 receptor expression in cancer.

UNLABELLED: Nanobodies are the smallest fully functional antigen-binding antibody fragments possessing ideal properties as probes for molecular imaging. In this study we labeled the anti-human epidermal growth factor receptor type 2 (HER2) Nanobody with (68)Ga via a 1,4,7-triazacyclononane-1,4,7-triacetic acid (NOTA) derivative and assessed its use for HER2 iPET imaging. METHODS: The 2Rs15dHis6 Nanobody and the lead optimized current-good-manufacturing-practice grade analog 2Rs15d were conjugated with S-2-(4-isothiocyanatobenzyl)-1,4,7-triazacyclononane-1,4,7-triacetic acid (p-SCN-Bn-NOTA) to enable fast and efficient (68)Ga labeling. Biodistribution and PET/CT studies were performed on HER2-positive and -negative tumor xenografts. The effect of injected mass on biodistribution was evaluated. The biodistribution data were extrapolated to calculate radiation dose estimates for the adult female using OLINDA software. A single-dose extended-toxicity study for NOTA-2Rs15d was performed on healthy mice up to a dose of 10 mg/kg. RESULTS: Radiolabeling was quantitative (>97%) after 5 min of incubation at room temperature; specific activity was 55-200 MBq/nmol. Biodistribution studies showed fast and specific uptake (percentage injected activity [%IA]) in HER2-positive tumors (3.13 ± 0.06 and 4.34 ± 0.90 %IA/g for (68)Ga-NOTA-2Rs15dHis6 and (68)Ga-NOTA-2Rs15d, respectively, at 1 h after injection) and high tumor-to-blood and tumor-to-muscle ratios at 1 h after injection, resulting in high-contrast PET/CT images with high specific tumor uptake. A remarkable finding of the biodistribution studies was that kidney uptake was reduced by 60% for the Nanobody lacking the C-terminal His6 tag. The injected mass showed an effect on the general biodistribution: a 100-fold increase in NOTA-2Rs15d mass decreased liver uptake from 7.43 ± 1.89 to 2.90 ± 0.26 %IA/g whereas tumor uptake increased from 2.49 ± 0.68 to 4.23 ± 0.99 %IA/g. The calculated effective dose, based on extrapolation of mouse data, was 0.0218 mSv/MBq, which would yield a radiation dose of 4 mSv to a patient after injection of 185 MBq of (68)Ga-NOTA-2Rs15d. In the toxicity study, no adverse effects were observed after injection of a 10 mg/kg dose of NOTA-2Rs15d. CONCLUSION: A new anti-HER2 PET tracer, (68)Ga-NOTA-2Rs15d, was synthesized via a rapid procedure under mild conditions. Preclinical validation showed high-specific-contrast imaging of HER2-positive tumors with no observed toxicity. (68)Ga-NOTA-2Rs15d is ready for first-in-human clinical trials.