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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.)

Purpose: Programmed cell death-1 receptor (PD-1) and its ligand (PD-L1) are the targets for immunotherapy in many cancer types. Although PD-1 blockade has therapeutic effects, the efficacy differs between patients. Factors contributing to this variability are PD-L1 expression levels and immune cells present in tumors. However, it is not well understood how PD-1 expression in the tumor microenvironment impacts immunotherapy response. Thus, imaging of PD-1-expressing immune cells is of interest. This study aims to evaluate the biodistribution of Zirconium-89 ( 89 Zr)-labeled pembrolizumab, a humanized IgG4 kappa monoclonal antibody targeting PD-1, in healthy cynomolgus monkeys as a translational model of tracking PD-1-positive immune cells., Procedures: Pembrolizumab was conjugated with the tetrafluorophenol-N-succinyl desferal-Fe(III) ester (TFP-N-sucDf) and subsequently radiolabeled with 89 Zr. Four cynomolgus monkeys with no previous exposure to humanized monoclonal antibodies received tracer only or tracer co-injected with pembrolizumab intravenously over 5 min. Thereafter, a static whole-body positron emission tomography (PET) scan was acquired with 10 min per bed position on days 0, 2, 5, and 7. Image-derived standardized uptake values (SUV mean ) were quantified by region of interest (ROI) analysis., Results: 89 Zr-N-sucDf-pembrolizumab was synthesized with high radiochemical purity (> 99 %) and acceptable molar activity (> 7 MBq/nmol). In animals dosed with tracer only, 89 Zr-N-sucDf-pembrolizumab distribution in lymphoid tissues such as mesenteric lymph nodes, spleen, and tonsils increased over time. Except for the liver, low radiotracer distribution was observed in all non-lymphoid tissue including the lung, muscle, brain, heart, and kidney. When a large excess of pembrolizumab was co-administered with a radiotracer, accumulation in the lymph nodes, spleen, and tonsils was reduced, suggestive of target-mediated accumulation., Conclusions: 89 Zr-N-sucDf-pembrolizumab shows preferential uptake in the lymphoid tissues including the lymph nodes, spleen, and tonsils. 89 Zr-N-sucDf-pembrolizumab may be useful in tracking the distribution of a subset of immune cells in non-human primates and humans., Trial Registration: ClinicalTrials.gov Identifier: NCT02760225.

18 F-BMS-986192, an adnectin-based human programmed cell death ligand 1 (PD-L1) tracer, was developed to noninvasively determine whole-body PD-L1 expression by PET. We evaluated the usability of 18 F-BMS-986192 PET to detect different PD-L1 expression levels and therapy-induced changes in PD-L1 expression in tumors. Methods: In vitro binding assays with 18 F-BMS-986192 were performed on human tumor cell lines with different total cellular and membrane PD-L1 protein expression levels. Subsequently, PET imaging was performed on immunodeficient mice xenografted with these cell lines. The mice were treated with interferon γ (IFNγ) intraperitoneally for 3 d or with the mitogen-activated protein kinase kinase inhibitor selumetinib by oral gavage for 24 h. Afterward, 18 F-BMS-986192 was administered intravenously, followed by a 60-min dynamic PET scan. Tracer uptake was expressed as percentage injected dose per gram of tissue. Tissues were collected to evaluate ex vivo tracer biodistribution and to perform flow cytometric, Western blot, and immunohistochemical tumor analyses. Results: 18 F-BMS-986192 uptake reflected PD-L1 membrane levels in tumor cell lines, and tumor tracer uptake in mice was associated with PD-L1 expression measured immunohistochemically. In vitro IFNγ treatment increased PD-L1 expression in the tumor cell lines and caused up to a 12-fold increase in tracer binding. In vivo, IFNγ affected neither PD-L1 tumor expression measured immunohistochemically nor 18 F-BMS-986192 tumor uptake. In vitro, selumetinib downregulated cellular and membrane levels of PD-L1 in tumor cells by 50% as measured by Western blotting and flow cytometry. In mice, selumetinib lowered cellular, but not membrane, PD-L1 levels of tumors, and consequently, no treatment-induced change in 18 F-BMS-986192 tumor uptake was observed. Conclusion: 18 F-BMS-986192 PET imaging allows detection of membrane-expressed PD-L1 as soon as 60 min after tracer injection. The tracer can discriminate a range of tumor cell PD-L1 membrane expression levels., (© 2020 by the Society of Nuclear Medicine and Molecular Imaging.)

Mature lymphoproliferative diseases are a heterogeneous group of neoplasms arising from different stages of B-cell and T-cell development. With improved understanding of the molecular processes in lymphoma and novel treatment options, arises a growing need for the molecular characterisation of tumours. Molecular imaging with single-photon-emission CT and PET using specific radionuclide tracers can provide whole-body information to investigate cancer biology, to evaluate phenotypic heterogeneity, to identify resistance to targeted therapy, and to assess the biodistribution of drugs in patients. In this Review, we evaluate the existing literature on molecular imaging in lymphoma, other than 18 F-fluordeoxyglucose molecular imaging. The aim is to examine the contribution of molecular imaging to the understanding of the biology of lymphoma and to discuss potential implications for the diagnostics and therapy of this disease. Finally, we discuss possible applications for molecular imaging of patients with lymphoma in the clinical context., (Copyright © 2020 Elsevier Ltd. All rights reserved.)

Immune checkpoint inhibitors (ICIs) have substantially changed the field of oncology over the past few years. ICIs offer an alternative treatment strategy by exploiting the patients' immune system, resulting in a T cell mediated anti-tumor response. These therapies are effective in multiple different tumor types. Unfortunately, a substantial group of patients do not respond to ICIs. Molecular imaging, using single-photon emission computed tomography (SPECT) and positron emission tomography (PET), can provide non-invasive whole-body visualization of tumor and immune cell characteristics and might support patient selection or response evaluations for ICI therapies. In this review, recent studies with 18 F-fluorodeoxyglucose-PET imaging, imaging of immune checkpoints and imaging of immune cells will be discussed. These studies are until now mainly exploratory, but the first results suggest that molecular imaging biomarkers could have a role in the evaluation of ICI therapy., Competing Interests: Competing Interests: E.G.E. de Vries reports institutional financial support for her advisory role from Daiichi Sankyo, Merck, NSABP, Pfizer, Sanofi, Synthon and for clinical trials or contracted research from Amgen, AstraZeneca, Bayer, Chugai Pharma, CytomX Therapeutics, G1 Therapeutics, Genentech, Nordic Nanovector, Radius Health, Roche, Synthon. S.F. Oosting reports institutional financial support for clinical trials or contracted research from Celldex, Pfizer, Novartis, Bristol Myers Squibb, Kura Oncology, MedImmune, Roche, Merck Sharp & Dohme. A.J. van der Wekken reports institutional financial support for his advisory role from Pfizer, Boehringer-Ingelheim, Roche (diagnostics), Astra Zeneca and institutional financial support for clinical trials from AstraZeneca. All remaining authors have declared no conflicts of interest., (© The author(s).)

Effective patient selection before or early during treatment is important to increasing the therapeutic benefits of anticancer treatments. This selection process is often predicated on biomarkers, predominantly biospecimen biomarkers derived from blood or tumour tissue; however, such biomarkers provide limited information about the true extent of disease or about the characteristics of different, potentially heterogeneous tumours present in an individual patient. Molecular imaging can also produce quantitative outputs; such imaging biomarkers can help to fill these knowledge gaps by providing complementary information on tumour characteristics, including heterogeneity and the microenvironment, as well as on pharmacokinetic parameters, drug-target engagement and responses to treatment. This integrative approach could therefore streamline biomarker and drug development, although a range of issues need to be overcome in order to enable a broader use of molecular imaging in clinical trials. In this Perspective article, we outline the multistage process of developing novel molecular imaging biomarkers. We discuss the challenges that have restricted the use of molecular imaging in clinical oncology research to date and outline future opportunities in this area.

Cancer immunotherapy has led to impressive antitumor effects. However, not all patients respond to immunotherapy, serious toxicity can occur and combination therapy may be warranted. Strategies for rational early treatment choices are urgently required. In the absence of ideal accompanying biomarkers it remains challenging to capture the dynamic, heterogeneous and complex tumor behavior. Tumor immune response involves next to tumor cells, numerous other cells and molecules in the tumor microenvironment. We review research to identify potential novel imaging biomarkers by non-invasive whole body molecular imaging with positron emission tomography and single-photon emission computed tomography for cancer immunotherapy. Firstly, imaging with radiolabeled immune checkpoint targeting molecules. Secondly, imaging of immune cells with ex vivo or in vivo radiolabeled tracers and thirdly, imaging extracellular matrix components, including adhesion molecules, growth factors and cytokines. These molecular imaging strategies - used alone, in combination or serially - could potentially contribute to patient selection upfront or early during immunotherapy., (Copyright © 2018 Elsevier Ltd. All rights reserved.)

Purpose: AMG 211, a bispecific T-cell engager (BiTE) antibody construct, targets carcinoembryonic antigen (CEA) and the CD3 epsilon subunit of the human T-cell receptor. AMG 211 was labeled with zirconium-89 ( 89 Zr) or fluorescent dye to evaluate the tumor-targeting properties. Experimental Design: 89 Zr-AMG211 was administered to mice bearing CEA-positive xenograft tumors of LS174T colorectal adenocarcinoma or BT474 breast cancer cells, as well as CEA-negative HL-60 promyelocytic leukemia xenografts. Biodistribution studies with 2- to 10-μg 89 Zr-AMG211 supplemented with unlabeled AMG 211 up to 500-μg protein dose were performed. A BiTE that does not bind CEA, 89 Zr-Mec14, served as a negative control. 89 Zr-AMG211 integrity was determined in tumor lysates ex vivo Intratumoral distribution was studied with IRDye800CW-AMG211. Moreover, 89 Zr-AMG211 was manufactured according to Good Manufacturing Practice (GMP) guidelines for clinical trial NCT02760199 Results: 89 Zr-AMG211 demonstrated dose-dependent tumor uptake at 6 hours. The highest tumor uptake was observed with a 2-μg dose, and the lowest tumor uptake was observed with a 500-μg dose. After 24 hours, higher uptake of 10-μg 89 Zr-AMG211 occurred in CEA-positive xenografts, compared with CEA-negative xenografts. Although the blood half-life of 89 Zr-AMG211 was approximately 1 hour, tumor retention persisted for at least 24 hours. 89 Zr-Mec14 showed no tumor accumulation beyond background level. Ex vivo autoradiography revealed time-dependent disintegration of 89 Zr-AMG211. 800CW-AMG211 was specifically localized in CEA-expressing viable tumor tissue. GMP-manufactured 89 Zr-AMG211 fulfilled release specifications. Conclusions: 89 Zr-AMG211 showed dose-dependent CEA-specific tumor targeting and localization in viable tumor tissue. Our data enabled its use to clinically evaluate AMG 211 in vivo behavior. Clin Cancer Res; 24(20); 4988-96. ©2018 AACR ., (©2018 American Association for Cancer Research.)

Development of new oncology drugs has increased since the improved understanding of cancer's complex biology. The oncology field has become the top therapeutic research area for new drugs. However, only a limited number of drugs entering clinical trials will be approved for use as the standard of care for cancer patients. Molecular imaging is increasingly perceived as a tool to support go/no-go decisions early during drug development. It encompasses a wide range of techniques that include radiolabeling a compound of interest followed by visualization with SPECT or PET. Radiolabeling can be performed using a variety of radionuclides, which are preferably matched to the compound on the basis of size and half-life. Imaging can provide information on drug behavior in vivo, whole-body drug target visualization, and heterogeneity in drug target expression. This review focuses on current applications of molecular imaging in the development of small molecules, antibodies, and antihormonal anticancer drugs., (© 2018 by the Society of Nuclear Medicine and Molecular Imaging.)

In vivo tumor labeling with fluorescent agents may assist endoscopic and surgical guidance for cancer therapy as well as create opportunities to directly observe cancer biology in patients. However, malignant and nonmalignant tissues are usually distinguished on fluorescence images by applying empirically determined fluorescence intensity thresholds. Here, we report the development of fSTREAM, a set of analytic methods designed to streamline the analysis of surgically excised breast tissues by collecting and statistically processing hybrid multiscale fluorescence, color, and histology readouts toward precision fluorescence imaging. fSTREAM addresses core questions of how to relate fluorescence intensity to tumor tissue and how to quantitatively assign a normalized threshold that sufficiently differentiates tumor tissue from healthy tissue. Using fSTREAM we assessed human breast tumors stained in vivo with fluorescent bevacizumab at microdose levels. Showing that detection of such levels is achievable, we validated fSTREAM for high-resolution mapping of the spatial pattern of labeled antibody and its relation to the underlying cancer pathophysiology and tumor border on a per patient basis. We demonstrated a 98% sensitivity and 79% specificity when using labeled bevacizumab to outline the tumor mass. Overall, our results illustrate a quantitative approach to relate fluorescence signals to malignant tissues and improve the theranostic application of fluorescence molecular imaging. Cancer Res; 77(3); 623-31. ©2016 AACR., (©2016 American Association for Cancer Research.)

Molecular imaging with PET is a rapidly emerging technique. In breast cancer patients, more than 45 different PET tracers have been or are presently being tested. With a good rationale, after development of the tracer and proven feasibility, it is of interest to evaluate whether there is a potential meaningful role for the tracer in the clinical setting-such as in staging, in the (early) prediction of a treatment response, or in supporting drug choices. So far, only (18)F-FDG PET has been incorporated into breast cancer guidelines. For proof of the clinical relevance of tracers, especially for analysis in a multicenter setting, standardization of the technology and access to the novel PET tracer are required. However, resources for PET implementation research are limited. Therefore, next to randomized studies, novel approaches are required for proving the clinical value of PET tracers with the smallest possible number of patients. The aim of this review is to describe the process of the development of PET tracers and the level of evidence needed for the use of these tracers in breast cancer. Several breast cancer trials have been performed with the PET tracers (18)F-FDG, 3'-deoxy-3'-(18)F-fluorothymidine ((18)F-FLT), and (18)F-fluoroestradiol ((18)F-FES). We studied them to learn lessons for the implementation of novel tracers. After defining the gap between a good rationale for a tracer and implementation in the clinical setting, we propose solutions to fill the gap to try to bring more PET tracers to daily clinical practice., (© 2016 by the Society of Nuclear Medicine and Molecular Imaging, Inc.)

Molecular imaging of breast cancer can potentially be used for breast cancer screening, staging, restaging, response evaluation and guiding therapies. Techniques for molecular breast cancer imaging include magnetic resonance imaging (MRI), optical imaging, and radionuclide imaging with positron emission tomography (PET) or single photon emission computed tomography (SPECT). This review focuses on PET and SPECT imaging which can provide sensitive serial non invasive information of tumor characteristics. Most clinical data are gathered on the visualization of general processes such as glucose metabolism with the PET-tracer [(18)F]fluorodeoxyglucose (FDG) and DNA synthesis with [18F]fluoro-L-thymidine (FLT). Increasingly more breast cancer specific targets are imaged such as the estrogen receptor (ER), growth factors and growth factor receptors. Imaging of the ER with the PET tracer 16-alpha-[(18)F]fluoro-17-beta-estradiol (FES) has shown a good correlation between FES tumor uptake and ER density. (111)In-trastuzumab SPECT to image the human epidermal growth factor receptor 2 (HER2) showed that in most patients with metastatic HER2 overexpressing disease more lesions were detected than with conventional staging procedures. The PET tracer (89)Zr-trastuzumab showed excellent, quantifiable, and specific tumor uptake. (111)In-bevacizumab for SPECT and (89)Zr-bevacizumab for PET-imaging have been developed for vascular endothelial growth factor (VEGF) imaging as an angiogenic marker. Lastly, tracers for the receptors EGFR, IGF-1R, PDGF-betaR and the ligand TGFbeta are under development. Although molecular imaging of breast cancer is still not commonly used in daily clinical practice, its application portfolio is expanding rapidly.