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Current immunotherapies have brought major progress in cancer treatments, but not all patients benefit. Therefore, insight into reasons for treatment failure and optimal biomarkers for patient selection are warranted. Current approved biomarkers for cancer immunotherapy do not provide insight into characteristics across tumor lesions in a patient or their heterogeneity. Here, whole-body PET imaging with specific tracers may provide support. Moreover, the biodistribution of cell therapies and complex molecules, such as bispecific antibodies, can be visualized by PET imaging, and repeat PET imaging allows to study the whole-body kinetics of the immune response. In this review, we present the status of using PET imaging-derived biomarkers for patients with cancer receiving immunotherapy. Next, the hopes and scientific challenges ahead to optimize current PET imaging biomarker development and to discover novel PET-derived baseline and dynamic biomarkers to potentially guide us in drug development and more precise patient and therapy selection will be discussed., (©2024 American Association for Cancer Research.)

Multispecific antibodies are engineered antibody derivatives that can bind to two or more distinct epitopes or antigens. Unlike mixtures of monospecific antibodies, the binding properties of multispecific antibodies enable two specific molecules to be physically linked, a characteristic with important applications in cancer therapy. The field of multispecific antibodies is highly dynamic and expanding rapidly; to date, 15 multispecific antibodies have been approved for clinical use, of which 11 were approved for oncological indications, and more than 100 new antibodies are currently in clinical development. Nevertheless, substantial challenges limit the applications of multispecific antibodies in cancer therapy, particularly inefficient targeting of solid tumours and substantial adverse effects. Both PET and single photon emission CT imaging can reveal the biodistribution and complex pharmacology of radiolabelled multispecific antibodies. This Review summarizes the insights obtained from preclinical and clinical molecular imaging studies of multispecific antibodies, focusing on their structural properties, such as molecular weight, shape, target specificity, affinity and avidity. The opportunities associated with use of molecular imaging studies to support the clinical development of multispecific antibody therapies are also highlighted., Competing Interests: Competing interests D.J.A.d.G. declares that he received institutional financial support for clinical trials or contracted research from Amgen, Bayer, GE Healthcare, Hoffmann La Roche and Siemens. E.G.E.d.V. declares that she received institutional financial support for acting as a member of advisory boards or consultancy services from Crescendo Biologics, Daiichi Sankyo and NSABP, as well as institutional financial support for clinical trials or contracted research grants from Amgen, Bayer, Crescendo Biologics, Genentech, Regeneron, Roche and Servier. M.N.L.-d.H. declares that she received institutional financial support for acting as a member of advisory boards or consultancy services from Merck and institutional financial support for conducting clinical and preclinical studies from Amgen, Bayer and Servier. The other authors declare no competing interests., (© 2024. Springer Nature Limited.)

Understanding which patients with human epidermal growth factor receptor 2 (HER2)-negative or -low metastatic breast cancer (MBC) benefit from HER2-targeted strategies is urgently needed. We assessed the whole-body heterogeneity of HER2 expression on 89 Zr-trastuzumab PET (HER2 PET) and the diagnostic performance of HER2 PET in a large series of patients, including HER2-negative and -low MBC. Methods: In the IMPACT-MBC study, patients with newly diagnosed and nonrapidly progressive MBC of all subtypes were included. Metastasis HER2 status was determined by immunohistochemistry and in situ hybridization. 89 Zr-trastuzumab uptake was quantified as SUV max and SUV mean HER2 immunohistochemistry was related to the quantitative 89 Zr-trastuzumab uptake of all metastases and corresponding biopsied metastasis, uptake heterogeneity, and qualitative scan evaluation. A prediction algorithm for HER2 immunohistochemistry positivity based on uptake was developed. Results: In 200 patients, 89 Zr-trastuzumab uptake was quantified in 5,163 metastases, including 186 biopsied metastases. With increasing HER2 immunohistochemistry status, uptake was higher (geometric mean SUV max of 7.0, 7.6, 7.3, and 17.4 for a HER2 immunohistochemistry score of 0, 1, 2, or 3+, respectively; P < 0.001). High uptake exceeding 14.6 (90th percentile) was observed in one third of patients with a HER2-negative or -low metastasis biopsy. The algorithm performed best when lesion site and size were incorporated (area under the curve, 0.86; 95% CI, 0.79-0.93). Conclusion: HER2 PET had good diagnostic performance in MBC, showing considerable whole-body HER2 heterogeneity and uptake above background in HER2-negative and -low MBC. This provides novel insights into HER2-negative and -low MBC compared with standard HER2 immunohistochemistry on a single biopsy., (© 2024 by the Society of Nuclear Medicine and Molecular Imaging.)

Objective: Improving patient selection and development of biological therapies such as vedolizumab in IBD requires a thorough understanding of the mechanism of action and target binding, thereby providing individualised treatment strategies. We aimed to visualise the macroscopic and microscopic distribution of intravenous injected fluorescently labelled vedolizumab, vedo-800CW, and identify its target cells using fluorescence molecular imaging (FMI)., Design: Forty three FMI procedures were performed, which consisted of macroscopic in vivo assessment during endoscopy, followed by macroscopic and microscopic ex vivo imaging. In phase A, patients received an intravenous dose of 4.5 mg, 15 mg vedo-800CW or no tracer prior to endoscopy. In phase B, patients received 15 mg vedo-800CW preceded by an unlabelled (sub)therapeutic dose of vedolizumab., Results: FMI quantification showed a dose-dependent increase in vedo-800CW fluorescence intensity in inflamed tissues, with 15 mg (153.7 au (132.3-163.7)) as the most suitable tracer dose compared with 4.5 mg (55.3 au (33.6-78.2)) (p=0.0002). Moreover, the fluorescence signal decreased by 61% when vedo-800CW was administered after a therapeutic dose of unlabelled vedolizumab, suggesting target saturation in the inflamed tissue. Fluorescence microscopy and immunostaining showed that vedolizumab penetrated the inflamed mucosa and was associated with several immune cell types, most prominently with plasma cells., Conclusion: These results indicate the potential of FMI to determine the local distribution of drugs in the inflamed target tissue and identify drug target cells, providing new insights into targeted agents for their use in IBD., Trial Registration Number: NCT04112212., Competing Interests: Competing interests: GD received research grants from Royal DSM, Takeda and Janssen Pharmaceuticals and speaker fees from AbbVie, Pfizer, Takeda and Janssen Pharmaceuticals. EAMF is supported by a ZonMW Clinical Fellowship grant (project number 90719075) and has received an unrestricted research grant from Takeda. MD and SJ are employees and shareholders of Regeneron Pharmaceuticals., (© Author(s) (or their employer(s)) 2024. Re-use permitted under CC BY-NC. No commercial re-use. See rights and permissions. Published by BMJ.)

Monoclonal antibodies are important cancer medicines. The European Medicines Agency (EMA) approved 48 and the Food and Drug Administration (FDA) 56 anticancer monoclonal antibody-based therapies. Their high prices burden healthcare systems and hamper global drug access. Biosimilars could retain costs and expand the availability of monoclonal antibodies. In Europe, five rituximab biosimilars, six trastuzumab biosimilars, and eight bevacizumab biosimilars are available as anti-cancer drugs. To gain insight into the biosimilar landscape for cancer treatment, we performed a literature search and analysis. In this review, we summarize cancer monoclonal antibodies' properties crucial for the desired pharmacology and point out sources of variability. The analytical assessment of all EMA-approved bevacizumab biosimilars is highlighted to illustrate this variability. The global landscape of investigational and approved biosimilars is mapped, and the challenges for access to cancer biosimilars are identified., Competing Interests: E.G.E.d.V. reports institutional financial support for advisory boards/consultancy from NSABP, Daiichi Sankyo, and Crescendo Biologics and institutional financial support for clinical trials or contracted research grants from Amgen, Genentech, Roche, Bayer, Servier, Regeneron, and Crescendo Biologics, all outside this work., (© 2024 The Authors.)

Immuno-positron emission tomography (immunoPET) enables imaging of specific targets that play a role in targeted therapy and immunotherapy, such as antigens on cell membranes, targets in the disease microenvironment, or immune cells. The most common immunoPET applications use a monoclonal antibody labeled with a relatively long-lived positron emitter such as 89 Zr ( T 1/2  = 78.4 h), but smaller antibody-based constructs labeled with various other positron emitting radionuclides are also being investigated. This molecular imaging technique can thus guide the development of new drugs and may have a pivotal role in selecting patients for a particular therapy. In early phase immunoPET trials, multiple imaging time points are used to examine the time-dependent biodistribution and to determine the optimal imaging time point, which may be several days after tracer injection due to the slow kinetics of larger molecules. Once this has been established, usually only one static scan is performed and semi-quantitative values are reported. However, total PET uptake of a tracer is the sum of specific and nonspecific uptake. In addition, uptake may be affected by other factors such as perfusion, pre-/co-administration of the unlabeled molecule, and the treatment schedule. This article reviews imaging methodologies used in immunoPET studies and is divided into two parts. The first part summarizes the vast majority of clinical immunoPET studies applying semi-quantitative methodologies. The second part focuses on a handful of studies applying pharmacokinetic models and includes preclinical and simulation studies. Finally, the potential and challenges of immunoPET quantification methodologies are discussed within the context of the recent technological advancements provided by long axial field of view PET/CT scanners., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest. The author(s) declared that they were an editorial board member of Frontiers, at the time of submission. This had no impact on the peer review process and the final decision., (© 2024 Mohr, van Sluis, Lub-de Hooge, Lammertsma, Brouwers and Tsoumpas.)

The purpose of this study was to quantify any differences between the SUVs of 89 Zr immuno-PET scans obtained using a PET/CT system with a long axial field of view (LAFOV; Biograph Vision Quadra) compared to a PET/CT system with a short axial field of view (SAFOV; Biograph Vision) and to evaluate how LAFOV PET scan duration affects image noise and SUV metrics. Methods: Five metastatic breast cancer patients were scanned consecutively on SAFOV and LAFOV PET/CT scanners. Four additional patients were scanned using only LAFOV PET/CT. Scans on both systems lasted approximately 30 min and were acquired 4 d after injection of 37 MBq of 89 Zr-trastuzumab. LAFOV list-mode data were reprocessed to obtain images acquired using shorter scan durations (15, 10, 7.5, 5, and 3 min). Volumes of interest were placed in healthy tissues, and tumors were segmented semiautomatically to compare coefficients of variation and to perform Bland-Altman analysis on SUV metrics (SUV max , SUV peak , and SUV mean ). Results: Using 30-min images, 2 commonly used lesion SUV metrics were higher for SAFOV than for LAFOV PET (SUV max , 16.2% ± 13.4%, and SUV peak , 10.1% ± 7.2%), whereas the SUV mean of healthy tissues showed minimal differences (0.7% ± 5.8%). Coefficients of variation in the liver derived from 30-min SAFOV PET were between those of 3- and 5-min LAFOV PET. The smallest SUV max and SUV peak differences between SAFOV and LAFOV were found for 3-min LAFOV PET. Conclusion: LAFOV 89 Zr immuno-PET showed a lower SUV max and SUV peak than SAFOV because of lower image noise. LAFOV PET scan duration may be reduced at the expense of increasing image noise and bias in SUV metrics. Nevertheless, SUV peak showed only minimal bias when reducing scan duration from 30 to 10 min., (© 2023 by the Society of Nuclear Medicine and Molecular Imaging.)

Purpose: As novel tracers are continuously under development, it is important to obtain reliable radiation dose estimates to optimize the amount of activity that can be administered while keeping radiation burden within acceptable limits. Organ segmentation is required for quantification of specific uptake in organs of interest and whole-body dosimetry but is a time-consuming task which induces high interobserver variability. Therefore, we explored using manual segmentations versus an artificial intelligence (AI)-based automated segmentation tool as a pre-processing step for calculating whole-body effective doses to determine the influence of variability in volumetric whole-organ segmentations on dosimetry., Procedures: PET/CT data of six patients undergoing imaging with 89 Zr-labelled pembrolizumab were included. Manual organ segmentations were performed, using in-house developed software, and biodistribution information was obtained. Based on the activity biodistribution information, residence times were calculated. The residence times served as input for OLINDA/EXM version 1.0 (Vanderbilt University, 2003) to calculate the whole-body effective dose (mSv/MBq). Subsequently, organ segmentations were performed using RECOMIA, a cloud-based AI platform for nuclear medicine and radiology research. The workflow for calculating residence times and whole-body effective doses, as described above, was repeated., Results: Data were acquired on days 2, 4, and 7 post-injection, resulting in 18 scans. Overall analysis time per scan was approximately 4 h for manual segmentations compared to ≤ 30 min using AI-based segmentations. Median Jaccard similarity coefficients between manual and AI-based segmentations varied from 0.05 (range 0.00-0.14) for the pancreas to 0.78 (range 0.74-0.82) for the lungs. Whole-body effective doses differed minimally for the six patients with a median difference in received mSv/MBq of 0.52% (range 0.15-1.95%)., Conclusion: This pilot study suggests that whole-body dosimetry calculations can benefit from fast, automated AI-based whole organ segmentations., (© 2022. The Author(s).)

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 89 ZED88082A 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 89 ZED88082A 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 (SUV max ) 5.2, IQI 4.0-7.4). Higher SUV max 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, 89 ZED88082A can characterize the complex dynamics of CD8 + T cells in the context of ICIs, and may inform immunotherapeutic treatments., (© 2022. The Author(s).)

The mesothelin (MSLN)-targeted 227 Th conjugate is a novel α-therapy developed to treat MSLN-overexpressing cancers. We radiolabeled the same antibody-chelator conjugate with 89 Zr to evaluate whether PET imaging with 89 Zr-MSLN matches 227 Th-MSLN tumor uptake, biodistribution, and antitumor activity. Methods: Serial PET imaging with protein doses of 4, 20, or 40 μg of 89 Zr-MSLN and 89 Zr-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. 89 Zr-MSLN and 227 Th-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. 89 Zr-MSLN PET imaging was performed before 227 Th-MSLN treatment in HT29-MSLN and BxPc3 tumor-bearing mice. Results: 89 Zr-MSLN PET imaging showed an SUV mean 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. 89 Zr-MSLN tumor uptake was higher than uptake of 89 Zr-control ( P = 0.0043). 89 Zr-MSLN and 227 Th-MSLN showed comparable tumor uptake and biodistribution in OVCAR-3 and HT29-MSLN tumor-bearing mice. Pretreatment SUV mean was 2.2 ± 0.2 in HT29-MSLN tumors, which decreased in volume on 227 Th-MSLN treatment. BxPc3 tumors showed an SUV mean of 1.2 ± 0.3 and remained similar in size after 227 Th-MSLN treatment. Conclusion: 89 Zr-MSLN PET imaging reflected MSLN expression and matched 227 Th-MSLN tumor uptake and biodistribution. Our data support the clinical exploration of 89 Zr-MSLN PET imaging together with 227 Th-MSLN therapy, both using the same antibody-chelator conjugate., (© 2022 by the Society of Nuclear Medicine and Molecular Imaging.)

The advent of immune checkpoint inhibitors has reinvigorated the field of immuno-oncology. These monoclonal antibody-based therapies allow the immune system to recognize and eliminate malignant cells. This has resulted in improved survival of patients across several tumor types. However, not all patients respond to immunotherapy therefore predictive biomarkers are important. There are only a few Food and Drug Administration-approved biomarkers to select patients for immunotherapy. These biomarkers do not consider the heterogeneity of tumor characteristics across lesions within a patient. New molecular imaging tracers allow for whole-body visualization with positron emission tomography (PET) of tumor and immune cell characteristics, and drug distribution, which might guide treatment decision making. Here, we summarize recent developments in molecular imaging of immune checkpoint molecules, such as PD-L1, PD-1, CTLA-4, and LAG-3. We discuss several molecular imaging approaches of immune cell subsets and briefly summarize the role of FDG-PET for evaluating cancer immunotherapy. The main focus is on developments in clinical molecular imaging studies, next to preclinical studies of interest given their potential translation to the clinic., Competing Interests: Competing interests: EDV reports an advisory role at Daiichi Sankyo, NSABP, and Sanofi and research funding from Amgen, AstraZeneca, Bayer, Chugai Pharma, Crescendo, CytomX Therapeutics, G1 Therapeutics, Genentech, Nordic Nanovector, Radius Health, Regeneron, Roche, Servier, and Synthon (paid to UMCG). AvdW reports an advisory role at Janssen, Takeda, and Boehringer-Ingelheim (paid to UMCG) and research funding from AstraZeneca, Boehringer-Ingelheim, Pfizer, Roche, and Takeda. ML-dH reports research funding from Merck, Bayer, and Amgen (paid to UMCG). SO reports research funding from Novartis, Pfizer and Celldex Therapeutics (paid to UMCG) and an advisory role at Bristol Myers Squibb (paid to the UMCG)., (© Author(s) (or their employer(s)) 2022. Re-use permitted under CC BY-NC. No commercial re-use. See rights and permissions. Published by BMJ.)

Due to ethical and practical reasons, a knowledge gap exists on the pharmacokinetics (PK) of inflammatory bowel disease (IBD)-related drugs in pregnant women with IBD. Before evidence-based dosing can be proposed, insight into the PK has to be gained to optimize drug therapy for both mother and fetus. This systematic review aimed to describe the effect of pregnancy and IBD on the PK of drugs used for IBD. One aminosalicylate study, two thiopurine studies and twelve studies with biologicals were included. Most drugs within these groups presented data over multiple moments before, during and after pregnancy, except for mesalazine, ustekinumab and golimumab. The studies for mesalazine, ustekinumab and golimumab did not provide enough data to demonstrate an effect of pregnancy on concentration and PK parameters. Therefore, no evidence-based dosing advice was given. The 6-thioguanine nucleotide levels decreased during pregnancy to 61% compared to pre-pregnancy levels. The potentially toxic metabolite 6-methylmercaptopurine (6-MMP) increased to maximal 209% of the pre-pregnancy levels. Although the PK of the thiopurines changed throughout pregnancy, no evidence-based dosing advice was provided. One study suggested that caution should be exercised when the thiopurine dose is adjusted, due to shunting 6-MMP levels. For the biologicals, infliximab levels increased, adalimumab stayed relatively stable and vedolizumab levels tended to decrease during pregnancy. Although the PK of the biologicals changed throughout pregnancy, no evidence-based dosing advice for biologicals was provided. Other drugs retrieved from the literature search were mesalazine, ustekinumab and golimumab. We conclude that limited studies have been performed on PK parameters during pregnancy for drugs used in IBD. Therefore, more extensive research to determine the values of PK parameters is warranted. After gathering the PK data, evidence-based dosing regimens can be developed.

Background: Programmed cell death protein 1 (PD-1) antibody treatment is standard of care for melanoma and non-small-cell lung cancer (NSCLC). Accurately predicting which patients will benefit is currently not possible. Tumor uptake and biodistribution of the PD-1 antibody might play a role. Therefore, we carried out a positron emission tomography (PET) imaging study with zirconium-89 ( 89 Zr)-labeled pembrolizumab before PD-1 antibody treatment., Patients and Methods: Patients with advanced or metastatic melanoma or NSCLC received 37 MBq (1 mCi) 89 Zr-pembrolizumab (∼2.5 mg antibody) intravenously plus 2.5 or 7.5 mg unlabeled pembrolizumab. After that, up to three PET scans were carried out on days 2, 4, and 7. Next, PD-1 antibody treatment was initiated. 89 Zr-pembrolizumab tumor uptake was calculated as maximum standardized uptake value (SUV max ) and expressed as geometric mean. Normal organ uptake was calculated as SUV mean and expressed as a mean. Tumor response was assessed according to (i)RECIST v1.1., Results: Eighteen patients, 11 with melanoma and 7 with NSCLC, were included. The optimal dose was 5 mg pembrolizumab, and the optimal time point for PET scanning was day 7. The tumor SUV max did not differ between melanoma and NSCLC (4.9 and 6.5, P = 0.49). Tumor 89 Zr-pembrolizumab uptake correlated with tumor response (P trend = 0.014) and progression-free (P = 0.0025) and overall survival (P = 0.026). 89 Zr-pembrolizumab uptake at 5 mg was highest in the spleen with a mean SUV mean of 5.8 (standard deviation ±1.8). There was also 89 Zr-pembrolizumab uptake in Waldeyer's ring, in normal lymph nodes, and at sites of inflammation., Conclusion: 89 Zr-pembrolizumab uptake in tumor lesions correlated with treatment response and patient survival. 89 Zr-pembrolizumab also showed uptake in lymphoid tissues and at sites of inflammation., Competing Interests: Disclosure MJ reports consultancy fees from AstraZeneca (paid to UMCG). JBH reports consultancy roles for Achilles Therapeutics, BioNTech, BMS, GSK, Immunocore, Instil Bio, Molecular Partners, MSD, Merck Serono, Neogene Therapeutics, Novartis, Pfizer, PokeAcel, Roche/Genentech, Sanofi, T-Knife, and Third Rock Ventures and research grants from Amgen, Asher-Bio, BMS, BioNTech, MSD, Novartis, and Neogene Therapeutics (paid to the Netherlands Cancer Institute). WT reports fees from Merck, Sharp, Dohme, and Bristol-Myers-Squibb (paid to UMCG). EGEdV reports an advisory role at Daiichi Sankyo, NSABP, and Sanofi and research funding from Amgen, AstraZeneca, Bayer, Chugai Pharma, Crescendo, CytomX Therapeutics, G1 Therapeutics, Genentech, Nordic Nanovector, Radius Health, Regeneron, Roche, Servier, and Synthon (paid to UMCG). GAPH reports consulting and advisory role at Amgen, Roche, MSD, BMS, Pfizer, Novartis, and Pierre Fabry and research funding from BMS and Seerave (paid to UMCG). TJNH reports consultancy fees (paid to UMCG) from BMS, MSD, Merck, Boehringer, AstraZeneca, and Roche. AJvdW reports an advisory role at Janssen, Takeda, and Boehringer-Ingelheim (paid to UMCG) and research funding from AstraZeneca, Boehringer-Ingelheim, Pfizer, Roche, and Takeda. MNL-dH reports research funding from Merck, Bayer, and Amgen (paid to UMCG). HJMG reports an advisory role at Eli Lilly, MSD, BMS, and Novartis. All other authors have declared no conflicts of interest., (Copyright © 2021. Published by Elsevier Ltd.)

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 ( 89 Zr). Optimal protein antibody dose was first determined in non-tumor-bearing mice to assess physiological distribution. Next, biodistribution of optimal protein dose and 89 Zr-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. [ 89 Zr]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 [ 89 Zr]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 [ 89 Zr]Zr-DFO- N -suc-CSF1R-mAb in the liver, lymphoid tissues, duodenum, and ileum, but not in the tumor than did 89 Zr-labeled control at 72 h. Immunohistochemistry and autoradiography showed that 89 Zr was localized to macrophages within lymphoid tissues. Following [ 89 Zr]Zr-DFO- N -suc-CSF1R-mAb administration, tumor macrophages were almost absent, whereas isotype-group tumors contained over 500 cells/mm 2 . We hypothesize that intratumoral macrophage depletion by [ 89 Zr]Zr-DFO- N -suc-CSF1R-mAb precluded tumor uptake higher than 89 Zr-labeled control. Translation of molecular imaging of macrophage-targeting therapeutics to humans may support macrophage-directed therapeutic development., Competing Interests: EV reports institutional financial support for her advisory role from Daiichi Sankyo, Merck, NSABP, Pfizer, Sanofi, and Synthon and institutional financial support for clinical trials or contracted research from Amgen, AstraZeneca, Bayer, Chugai Pharma, CytomX Therapeutics, G1 Therapeutics, Genentech, Nordic Nanovector, Radius Health, Roche, Synthon, and Servier. CS reports receiving unrestricted research grants from Novartis, Roche, Genentech, Pfizer, SNS Oncology, and G1 Therapeutics that were made available to UMCG. KdV reports receiving research grants from Roche and is a consultant for Third Rock Ventures. The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2021 Waaijer, Suurs, Hau, Vrijland, de Visser, de Groot, de Vries, Lub-de Hooge and Schröder.)