Chapter 2 Molecular Imaging to Enlighten Cancer Immunotherapies and Underlying Involved Processes

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Radiopharmaceuticals for translational imaging studies in the field of cancer immunotherapy van der Veen, Elly

DOI: 10.33612/diss.128579303

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Download date: 03-10-2021 Chapter 2 Molecular imaging to enlighten cancer immunotherapies and underlying involved processes

Elly L. van der Veen1, Frederike Bensch1, Andor W.J.M. Glaudemans2, Marjolijn N. Lub-de Hooge3, Elisabeth G.E. de Vries1

1Department of Medical Oncology, 2Nuclear Medicine and Molecular Imaging, 3Clinical Pharmacy and Pharmacology, University of Groningen, Groningen, University Medical Center Groningen, The Netherlands.

Cancer Treat Rev. 2018;70:232-244. Chapter 2

ABSTRACT

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.

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INTRODUCTION

Cancer immunotherapy is increasingly becoming an important treatment strategy across a broad spectrum of tumor types.1 Over 2000 different immunotherapeutics are in the 2 development pipeline and several monoclonal antibody (mAb) immune checkpoint inhibitors have already been approved for use in the clinic.2 Moreover, combinations of these immune checkpoint inhibitors with chemotherapeutic drugs and targeted agents can enhance their antitumor effect, while radiotherapy can also induce immunomodulatory effects.3 However, not all patients benefit from immunotherapy, serious toxicity can occur and most immunotherapeutic drugs are expensive. Moreover, the rapidly increasing number of immunotherapy combinations, which are currently evaluated in over 3000 ongoing clinical trials, require an unprecedented number of patients and major financial investments.

Therefore, strategies to improve patient selection and accelerate immuno-oncology clinical development are urgently needed.2 In this respect the development and implementation of biomarkers is critical, but this has been slowed by the complexity and dynamics of the tumor immune response. Next to serum or peripheral blood biomarkers, which would be convenient for clinical use, analyses of tumor tissues have been expanded.4 There are now two FDA approved biomarkers, the programmed death-ligand 1 (PD-L1) measured with immunohistochemistry (IHC), and microsatellite instability-high and mismatch repair deficient status measurement by IHC and polymerase-chain-reaction (PCR)-based assays. Interest in mutational tumor load as a predictive biomarker is also increasing, with growing evidence that a higher mutational load leads to a higher probability response chance.5,6 However immune checkpoint inhibitors can sometimes induce responses in tumors without these biomarkers or fail to induce responses despite presence of these biomarkers.7 A possible explanation is that a single biopsy may not capture the dynamics of the complex immune response and the heterogeneity across various tumor lesions in a patient and even within a single lesion.8,9 Moreover, the available PD-L1 IHC assays show differences in PD-L1 detection, especially in immune cells.10,11

Based on the abovementioned biomarkers, prediction of response to immunotherapy is obviously a challenge. Moreover during immunotherapy, routine anatomic tumor response measurement can be difficult due to pseudoprogression. Therefore special immune-related response criteria have been developed and finally, guidelines were defined for response (iRECIST) criteria to be used in trials testing immunotherapies.12-14 These measurements, however, only provide information about tumor size, and therefore do not specify other characteristics of the tumor.

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To overcome some of these issues, additional information can be obtained using whole body molecular imaging modalities, positron emission tomography (PET) and single-photon emission computed tomography (SPECT), with specific radiopharmaceuticals to capture a more detailed, dynamic picture of characteristics of all tumor lesions within the body of an individual patient. These techniques can provide non-invasive information about the biodistribution of immunomodulatory drugs in the body, heterogeneity of target expression, effects of immunotherapy on immune cells, and therapy effects on other cells in the tumor microenvironment (TME). Several tracers, earlier studied clinically for infectious inflammatory disease, can be used in oncology to provide information about the TME. Moreover, numerous novel tracers are being developed. We therefore performed a literature search (for search strategy see appendix A).

This review summarizes current preclinical and clinical research and molecular imaging approaches under clinical development, to support immunotherapy decision making.

CELLS INVOLVED IN TUMOR IMMUNOLOGY AND IMMUNOTHERAPEUTICS

Besides tumor cells, multiple non-malignant cells are recruited to the tumor site where they settle in the TME.15,16 These include tumor infiltrating lymphocytes (TILs), such as T-cells, B-cells and Natural Killer (NK) cells, as well as macrophages, dendritic cells (DCs) and granulocytes, and their precursors. These cells can create an inflammatory environment that enhances tumor growth.17-19 Moreover the TME is characterized by the extracellular matrix (ECM), which contains components such as cell adhesion molecules, growth factors and cytokines. The three distinct tumor phenotypes relevant for response to immunotherapy are an inflamed phenotype, genomically unstable with a high presence of TILs, the immune- excluded phenotype with immune cells in the tumor surrounding stroma, and the immune- desert phenotype, genomically stable with fewer TILs, and containing highly proliferating tumor cells.20,21

Immunotherapeutics target cells and components in the TME to improve the tumor immune response. Tumors can escape the immune response due to a dominance of inhibitory immune signaling pathways: the immune checkpoints. Inhibiting these pathways by mAbs leads to (re)activation of the immune response, enabling immune cells to attack cancer cells. Immune checkpoint inhibitors now have a prominent role in clinical practice, with several mAbs targeting cytotoxic T-lymphocyte-associated protein 4 (CTLA-4) and programmed cell death protein (PD-1)/PD-L1 FDA/EMA approved. To enhance their immune-mediated effector functions advanced modifications are made, including changed amino acid sequence

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or glycoengineering, potentially leading to induced antibody-dependent cell-mediated cytotoxicity (ADCC) and cell-mediated cytotoxicity (CMC).22 In addition, numerous bispecific antibodies (BsAbs) are being developed that recognize two different epitopes, with one arm targeting host effector cells, such as CD3 on T-cells, and the other arm targeting cancer 2 cells.23 In this way effector cells are directed to cancer cells.

Cancer vaccines, preventive as well as therapeutically administered are another group of immunotherapeutics. Moreover, adoptive cell transfer (ACT) is performed; in this approach the patient’s own ex vivo-activated effector immune cells are re-injected. These T-cells can be genetically engineered to produce receptors on their surface, called chimeric antigen receptors (CARs), which recognize specific tumor antigens. Especially in lymphoid malignancies treatment with CAR-T cells has antitumor effects.24 Another strategy was tested preclinically with in situ vaccination. Here immune enhancing agents are injected locally into the tumor, thereby triggering a T-cell immune response.25 Approved immunotherapeutics for clinical use and their indications are summarized in Supplementary Table 1.

MOLECULAR IMAGING AND IMMUNOTHERAPY

Molecular imaging techniques, including SPECT and PET imaging, are widely used in the clinic. PET is increasingly performed given its better temporal and spatial resolution and the possibility for absolute quantification. Extensively used radioisotopes for SPECT are technetium-99m (99mTc) and indium-111 (111In), with half-lives of 6 hours and 2.8 days, respectively, as well as iodine isotopes (123I, 125I, and 131I). For PET imaging, shorter half- life radioisotopes can be used, such as fluor-18 (18F), gallium-68 (68Ga), and carbon-11 (11C) with half-lives of 109.7, 67.7 and 20.3 minutes, respectively, while zirconium-89 (89Zr) and copper-64 (64Cu) have longer half-lives of 78.4 and 12.7 hours.

The various radioisotopes require different labeling methods. Iodines can be labeled directly, whereas for radiometal ions, such as 89Zr, the molecule is first conjugated to a chelator, followed by chelating the metal ion. These radiometals have residualizing properties, meaning that after internalization of the target by the tumor cells, the radioisotope is trapped inside cells, leading to an accumulation of PET signal over time.26 In contrast, non- residualizing radioisotopes, such as the iodines, are rapidly detached from tumor cells. The most optimal imaging technique and tracer depends on the intended aim for tracer use, for instance on which cells have to be targeted, the properties of the drug targets, and the tumor type and localization.

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Molecular imaging could provide a biomarker for immunotherapy (Fig. 1). Firstly, targeting tumor cells, for instance using drug-based tracers such as radiolabeled immune checkpoint inhibitors, might be a tool for evaluating drug biodistribution and target expression. Secondly, as the effect of immunotherapy is driven by activation of immune cells, serial imaging of immune cells might give information on immune cell migration and can detect specific immune cell populations. When used upfront and during immunotherapy, this might be a tool for response prediction. Thirdly, molecular imaging of components in the ECM could increase insight into their role in immunotherapy efficacy.

Targeting tumor cells for molecular imaging Targeting immune checkpoint proteins for molecular imaging Immune checkpoint receptors and their ligands are expressed by tumor and immune cells (Supplementary Fig. 1). SPECT or PET imaging, using radiolabeled mAbs or smaller molecules targeting these immune checkpoints, can provide information on the biodistribution of these molecules and indicate whether they reach the tumor. Imaging studies with radiolabeled mAbs for other tumor targets have shown intra- and interpatient heterogeneity in tumor uptake. Moreover, the drug does not always reach the tumor, even though the target is present. Low tumor uptake was seen in 29% of the patients with 89Zr-trastuzumab (anti- HER2) PET and in 37% of the patients with 111In-labeled anti-human death receptor 5 antibody tigatuzumab SPECT, even though based on IHC the tracer target was considered to be positive.27,28

Preclinical studies (Table 1) have shown the feasibility of visualizing biodistribution of immune checkpoint inhibitors and immune checkpoint targeting molecules. 64Cu-DOTA-anti-CTLA PET has visualized CTLA-4 positive mouse tumors.29 In vitro studies showed that T-cells were responsible for the CTLA-4 expression within these tumors. 64Cu-DOTA-ipilimumab tumor uptake was demonstrated in mice xenografted with different human non-small cell lung cancer (NSCLC) cell lines.30 For PD-L1, radiolabeled and fluorescently labeled PD-L1 targeting antibodies accumulated only in PD-L1-positive tumors. Moreover, high and low PD-L1 tumor expression could be discriminated (Fig. 2A).31-37 Modulation of PD-L1 expression was visualized, as interferon-γ treatment radiotherapy and paclitaxel increased uptake while doxorubicin treatment lowered uptake.36-38

PD-L1 imaging has not only visualized PD-L1-positive tumors, but also normal lymphoid organs in immune competent mouse models. Substantial uptake of PD-L1 targeting mAbs was seen in spleen, thymus, lymph nodes and brown adipose tissue (BAT), which consists of brown adipocytes expressing PD-L1.32-35,38 Co-administration of unlabeled mAb resulted in blocking of tracer uptake in the spleen, lymph nodes and BAT.32,38

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Figure 1. Targets for molecular imaging strategies to visualize 1) tumor cells 2) immune cells and 3) extracellular matrix components. Targets for radiotracers are shown. Both ex vivo and in vivo labeling strategies have been performed. Abbreviations: FDG: 18F-fluorodeoxyglucose; PD-L1: programmed death-ligand 1; NCA: nonspecific crossreacting antigen; LTB4: leukotriene B4; FPR: formyl peptide receptor; CD: cluster of differentiation; CXCR: C-X-C chemokine receptor; CTLA- 4: cytotoxic T-lymphocyte-associated protein 4; PD-1: programmed cell death protein; Arag: arabinofuranosylguanine; SSTR: somatostatin receptor; MMR: macrophage mannose receptor; TSPO: translocator protein; MDSC: myeloid-derived suppressor cells; VCAM: vascular cell adhesion molecule-1; VAP: vascular adhesion protein-1; ICAM: intercellular adhesion molecule-1; ILs: interleukins; TNF: tumor necrosis factor; IDO: indolamine 2,3-dioxygenase.

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Besides full antibodies, smaller molecules targeting PD-L1 have been used. Their faster clearance, allows injection of the tracer and imaging on the same day. Imaging with radiolabeled PD-L1 targeting small molecules could efficiently distinguish between PD-L1-positive and PD-L1- negative tumors in mice.39-41 In mice, a 64Cu-labeled anti-PD-L1 small molecule showed higher spleen uptake compared to other organs.42 In cynomolgus monkeys, a 18F-labeled anti-PD-L1 small molecule showed high uptake in PD-L1 positive tissue, such as the spleen.43 99mTc-labeled anti-PD-L1 nanobodies detected not only spleen and lymph nodes, but also BAT.41 Modification of anti-PD-L1 targeting small molecules showed that chelate, glycosylation and the type of radiometal can strongly influence uptake in PD-L1-positive tumors and other organs. For example, aglycosylated variants showed lower aspecific uptake and increased tumor uptake.44

In contrast to broad expression of PD-L1 by tumor and various immune cells, PD-1 is mainly expressed on T-cells, B-cells and macrophages.45 64Cu-DOTA-anti-PD-1, 89Zr and 64Cu-labeled pembrolizumab, as well as 89Zr-nivolumab have all been studied preclinically. In melanoma- bearing mice, prominent uptake was seen in tumor and spleen (Fig. 2B).46-48 This uptake was lower when blocked with a pre-injection of cold mAb. Bioluminescence imaging of CD4+ T-regulatory cells (Tregs), which express PD-1, confirmed the presence of TILs at the time of PET imaging. 64Cu-anti-PD-1 mAb PET imaging was used to detect PD-1-positive tumors. Flow cytometry confirmed PD-1 expression levels on TILs.38 Lymph nodes and spleen also showed high uptake of 64Cu-anti-PD-1 although less than 64Cu-anti-PD-L1 tracer, probably due to the fact that PD-L1 is expressed by a broader range of immune cells than PD-1. High spleen uptake of 89Zr-nivolumab in non-human primates, was blocked by co-administration of excess nivolumab.49 Moreover 89Zr-nivolumab showed high tumor uptake in mice engrafted with human peripheral blood mononuclear cells (PBMCs), corresponding with T-cell infiltration confirmed with IHC.50 The mice in all these studies were in general young, which could influence the results, as ageing of the lymphoid system leads to less active immune cells.51

Several clinical studies are ongoing with radiolabeled immune checkpoint targeting molecules. Anti-PD-L1 89Zr-atezolizumab PET in patients with locally advanced or metastatic NSCLC, bladder cancer or triple-negative breast cancer showed high uptake in tumor lesions, with large heterogeneity in uptake within and between patients. Moreover, even IHC PD-L1-negative tumor lesions showed tracer uptake. Uptake was also found in lymph nodes and tonsils, and high uptake in normal spleen.52 Other ongoing clinical imaging studies are using 89Zr-pembrolizumab (anti-PD-1), 89Zr-nivolumab or 18F-BMS986192 (anti-PD-L1 targeting small molecule) (ClinicalTtrials. gov identifier NCT02760225, EudraCT numbers 2015-004260-10 and 2015-004760-11). Results obtained in 10 NSCLC patients with both 89Zr-nivolumab and 18F-BMS986192 PET imaging showed that uptake of both tracers was heterogeneous within and between patients. Patients with ≥50% PD-L1 tumor expression, determined by IHC, showed higher 18F-BMS986192 uptake, whereas patients with high PD-1 tumor expression showed higher 89Zr-nivolumab uptake.53

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Figure 2. Examples of molecular imaging strategies in cancer immunotherapy: preclinical studies. (A) 64Cu-NOTA-PD-L1 PET/CT in immune competent mice bearing sc. wild-type mouse melanoma B16F10 (Tu WT, left) and PD-L1 knockout (Tu KO, right) tumors.38 Image shows higher 64Cu-NOTA- PD-L1 uptake 24 hours after injection in WT tumors compared to KO tumors. Also shown is uptake in spleen (Sp), cervical lymph node (cLN), axillary lymph node (aLN) and brown adipose tissue (BAT). (B) 89Zr-pembrolizumab anti-PD-1 PET/CT in immune deficient NSG mice (NSG-ctl) and humanized NSG (hNSG-nblk, engrafted with human PBMCs) mice, bearing A375 human melanoma.47 Higher uptake of 89Zr-pembrolizumab is shown in tumor (T), spleen (S) and liver (L) 144 hours after injection in hNSG mice compared to control NSG mice. (C) 89Zr-malDFO-169 cDb (anti-CD8) PET in immune competent mice bearing sc. CT26 mouse melanoma tumors, treated with anti-PDL1.66 Image shows higher 89Zr-malDFO-169 cDb uptake 22 hours post-injection in anti-PDL1 responding (right) compared to non-responding (left) mice. (D) [18F]FB-IL2 PET image of the inflammatory lesion of a rat inoculated with CD25+ cells in the shoulder (red arrow), 0 to 60 minutes after injection of [18F]FB-IL2.77 All figures are reprinted with permission.

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Table 1. Preclinical in vivo imaging studies with radiolabeled immune checkpoint targeting molecules Target Type of imaging Tracer Origin and reactivity Tumor model Animal model Results Ref CTLA-4 PET 64Cu-DOTA-CTLA-4 antibody Murine anti-mouse CT26 (mouse colon cancer) Balb/c mice High uptake in CT26 tumor 29 4-6 weeks old Immune competent PET 64Cu-DOTA-ipilimumab Humanized anti-human A549, H460, H358 (human Athymic nude mice Uptake in CTLA-4 expressing 30 NSCLC) 4-7 weeks old tumors Immune deficient PD-L1 SPECT/CT 111In-PD-L1.3.1 antibody Murine anti-human MDA-MB-231, SK-Br-3, MCF-7 Balb/c nude Detected PD-L1-positive tumor 31 (human breast cancer) mice xenografts 6-8 weeks old Discriminated between high and Immune deficient low PD-L1 tumor expression SPECT 111In-DTPA-PD-L1 antibody Hamster anti-mouse NT2.5 (mouse mammary neu-N transgenic mice Substantial uptake in tumor, 32 tumor) 8-12 weeks old spleen and thymus Immune competent Co-administration and excess unlabeled mAb blocked PD-L1 sites in spleen SPECT 111In-PD-L1 antibody Humanized anti-human Human cell lines NSG mice Specific tumor uptake and higher 33 (cross-reactivity with mouse) 6-8 weeks old uptake in spleen, lungs and liver Immune deficient PET 64Cu-PD-L1 antibody Humanized anti-human (cross- Human cell lines NSG mice Specific uptake in tumors with 34 reactivity with mouse) 6-8 weeks old high PD-L1 expression Immune deficient 4T1 (mouse mammary Balb/c mice Uptake in PD-L1 expressing tumors carcinoma) 4-6 weeks old Immune competent SPECT 111In-DTPA-PD-L1 antibody Rat anti-mouse B16F10 (murine melanoma) C57BL/6 mice Uptake in tumor, spleen, lungs, 35 and 6-8 weeks old liver, thymus and brown adipose EL4 (murine lymphoma) Immune competent tissue Increasing protein concentration reduces uptake in spleen and lungs PET 89Zr-anti-PD-L1 mAb Rat anti-mouse HPV+ HNSCC or B16F10 C57BL/6 mice Increased tracer uptake in 36 melanoma 6-8 weeks old irradiated versus non-irradiated Immune competent tumors

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Table 1. Preclinical in vivo imaging studies with radiolabeled immune checkpoint targeting molecules Target Type of imaging Tracer Origin and reactivity Tumor model Animal model Results Ref CTLA-4 PET 64Cu-DOTA-CTLA-4 antibody Murine anti-mouse CT26 (mouse colon cancer) Balb/c mice High uptake in CT26 tumor 29 4-6 weeks old Immune competent 2 PET 64Cu-DOTA-ipilimumab Humanized anti-human A549, H460, H358 (human Athymic nude mice Uptake in CTLA-4 expressing 30 NSCLC) 4-7 weeks old tumors Immune deficient PD-L1 SPECT/CT 111In-PD-L1.3.1 antibody Murine anti-human MDA-MB-231, SK-Br-3, MCF-7 Balb/c nude Detected PD-L1-positive tumor 31 (human breast cancer) mice xenografts 6-8 weeks old Discriminated between high and Immune deficient low PD-L1 tumor expression SPECT 111In-DTPA-PD-L1 antibody Hamster anti-mouse NT2.5 (mouse mammary neu-N transgenic mice Substantial uptake in tumor, 32 tumor) 8-12 weeks old spleen and thymus Immune competent Co-administration and excess unlabeled mAb blocked PD-L1 sites in spleen SPECT 111In-PD-L1 antibody Humanized anti-human Human cell lines NSG mice Specific tumor uptake and higher 33 (cross-reactivity with mouse) 6-8 weeks old uptake in spleen, lungs and liver Immune deficient PET 64Cu-PD-L1 antibody Humanized anti-human (cross- Human cell lines NSG mice Specific uptake in tumors with 34 reactivity with mouse) 6-8 weeks old high PD-L1 expression Immune deficient 4T1 (mouse mammary Balb/c mice Uptake in PD-L1 expressing tumors carcinoma) 4-6 weeks old Immune competent SPECT 111In-DTPA-PD-L1 antibody Rat anti-mouse B16F10 (murine melanoma) C57BL/6 mice Uptake in tumor, spleen, lungs, 35 and 6-8 weeks old liver, thymus and brown adipose EL4 (murine lymphoma) Immune competent tissue Increasing protein concentration reduces uptake in spleen and lungs PET 89Zr-anti-PD-L1 mAb Rat anti-mouse HPV+ HNSCC or B16F10 C57BL/6 mice Increased tracer uptake in 36 melanoma 6-8 weeks old irradiated versus non-irradiated Immune competent tumors

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Table 1. Continued Target Type of imaging Tracer Origin and reactivity Tumor model Animal model Results Ref PD-L1 PET 89Zr-C4 (recombinant IgG1 Engineered anti-mouse an anti- H1975 and A549 (human Nu/nu mice Tracer uptake in tumor, spleen 37 mAb) human NSCLC), PC3 (human prostatic 3-5 weeks old and liver small cell carcinoma) Immune deficient Detect increase or decrease in B16F10 (mouse melanoma) C57BL/6 mice PD-L1 expression after treatment 3-5 weeks old Immune deficient PDX model of EGFR mutant Tumor uptake in PDX model (L858R) NSCLC PET 64Cu-WL12 (PD-L1 binding Engineered anti-human CHO cell line expressing NSG mice Specific uptake in PD-L1-positive 39 peptide) hPD-L1 6-8 weeks old tumors. Low uptake in PD-L1- Immune deficient negative tumors

18 PET F-AlF-NOTA-ZPD-L1_1 (anti PD- Engineered anti-human affibody LOX-IMVI (human melanoma) SCID beige mice Detected PD-L1-positive tumor 40 L1 small molecule, affibody) and SUDHL6 (human B-cell 6-8 weeks old (LOX). Blocking reduced uptake lymphoma) Immune deficient No uptake in PD-L1-negative tumor (SUDHL6) SPECT 99mTC-anti-PD-L1 nanobodies Engineered anti-mouse TC-1 (mouse lung epithelial), C57BL/6 mice and PD- Detected PD-L1-positive, but not 41 nanobodies PD-L1 knock-in and knock-out L1 knockout mice PD-L1-negative tumors 6 weeks old In naïve mice, uptake in lungs, Immune competent heart, thymus, spleen, lymph nodes and brown adipose tissue PET 64Cu-PD-1 Engineered anti-human CT26 (mouse colon cancer) NSG mice Imaging distinguished between 42 ectodomain targeting PD-L1 expressing hPD-L1 Immune deficient PD-L1-positive and PD-L1-negative tumors PET 18F-BMS-986192 (anti-PD-L1 Engineered anti-human Human L2987 (PD-L1+) and Immune deficient mice Uptake in PD-L1+ > PD-L1- tumors 43 small molecule) HT-29 (PD-L1-) in mice Cynomolgus monkeys Uptake in PD-L1 expressing tissue, such as spleen, in cynomolgus monkeys PET 64Cu-PD-1 Engineered anti-human CT26 (mouse colon cancer) NSG mice All tracers variants distinguished 44 ectodomains expressing hPD-L1 6-8 weeks old PD-L1 positive and negative Immune deficient tumors Tracer modification changed tracer uptake, specificity and clearance

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Table 1. Continued Target Type of imaging Tracer Origin and reactivity Tumor model Animal model Results Ref PD-L1 PET 89Zr-C4 (recombinant IgG1 Engineered anti-mouse an anti- H1975 and A549 (human Nu/nu mice Tracer uptake in tumor, spleen 37 mAb) human NSCLC), PC3 (human prostatic 3-5 weeks old and liver small cell carcinoma) Immune deficient Detect increase or decrease in 2 B16F10 (mouse melanoma) C57BL/6 mice PD-L1 expression after treatment 3-5 weeks old Immune deficient PDX model of EGFR mutant Tumor uptake in PDX model (L858R) NSCLC PET 64Cu-WL12 (PD-L1 binding Engineered anti-human CHO cell line expressing NSG mice Specific uptake in PD-L1-positive 39 peptide) hPD-L1 6-8 weeks old tumors. Low uptake in PD-L1- Immune deficient negative tumors

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Table 1. Continued Target Type of imaging Tracer Origin and reactivity Tumor model Animal model Results Ref PD-1 PET 64Cu-PD-1 antibody Hamster anti-mouse B16F10 (mouse melanoma) Treg+ transgenic mice PD-1 expression in tumor and 46 (Foxp3+.LuciDTR) spleen detected. Blocking with Immune competent cold mAb indicated specific uptake PET 64Cu- and 89Zr-pembrolizumab Humanized anti-human A375 (human melanoma) NSG and humanized Uptake in spleen and tumor 47 NSG mice PET 89Zr-pembrolizumab Humanized anti-human No tumor model; PBMC ICR (CD-1) mice and Highest uptake in blood, liver and 48 engraftment Hsd Sprague-Dawley spleen rats, 5 weeks old Immune competent NSG mice and Highest uptake in spleen, kidney humanized NSG mice and liver for both mice models engrafted with human High uptake in salivary glands of PBMCs (hu-PBL-SCID) hu-PBL-SCID mice 5-8 weeks old Immune deficient PET 89Zr-Df-nivolumab Humanized anti-human A549 (human lung cancer) NSG mice and Uptake in tumor, corresponding 50 humanized NSG mice with infiltrated T-cells engrafted with human High uptake in salivary glands of PBMCs (hu-PBL-SCID hu-PBL-SCID 3-5 weeks old mice Immune deficient PET 89Zr-nivolumab Humanized anti-human - Healthy non-human High uptake in spleen, blocked by 49 primates excess nivolumab PD-1 and PET 64Cu-PD-1 and 64Cu-PD-L1 Murine anti-mouse B16F10 (mouse melanoma) C57BL/6N mice 64Cu-anti-PD-1 detected 38 PD-L antibody PD-1-deficient mice PD-1 positive TILs after PD-L1-deficient mice immunoradiotherapy, 64Cu-PD-L1 Immune competent antibody uptake in PD-L1 knockout < wild-type mice and PD-L1 upregulation by IFN-γ treatment visualized Abbreviations: DOTA: 1,4,7,10-tetraazacyclododecane- N,N’,N″,N’″-tetraacetic acid; NSCLC: non- small cell lung cancer; DTPA: diethylenetriaminepentaacetic acid; AlF: aluminiumfluoride; NOTA: 1,4,7-triazacyclononane-N,N’,N’’-triacetic acid; HPV: human papillomavirus ; HNSCC: head and neck squamous-cell carcinoma; PDX: patient-derived xenograft; EGFR: epidermal growth factor receptor; TILs: tumor-infiltrating lymphocytes; IFN-γ: interferon-gamma; PBMC: peripheral blood mononuclear cell.

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Table 1. Continued Target Type of imaging Tracer Origin and reactivity Tumor model Animal model Results Ref PD-1 PET 64Cu-PD-1 antibody Hamster anti-mouse B16F10 (mouse melanoma) Treg+ transgenic mice PD-1 expression in tumor and 46 (Foxp3+.LuciDTR) spleen detected. Blocking with Immune competent cold mAb indicated specific 2 uptake PET 64Cu- and 89Zr-pembrolizumab Humanized anti-human A375 (human melanoma) NSG and humanized Uptake in spleen and tumor 47 NSG mice PET 89Zr-pembrolizumab Humanized anti-human No tumor model; PBMC ICR (CD-1) mice and Highest uptake in blood, liver and 48 engraftment Hsd Sprague-Dawley spleen rats, 5 weeks old Immune competent NSG mice and Highest uptake in spleen, kidney humanized NSG mice and liver for both mice models engrafted with human High uptake in salivary glands of PBMCs (hu-PBL-SCID) hu-PBL-SCID mice 5-8 weeks old Immune deficient PET 89Zr-Df-nivolumab Humanized anti-human A549 (human lung cancer) NSG mice and Uptake in tumor, corresponding 50 humanized NSG mice with infiltrated T-cells engrafted with human High uptake in salivary glands of PBMCs (hu-PBL-SCID hu-PBL-SCID 3-5 weeks old mice Immune deficient PET 89Zr-nivolumab Humanized anti-human - Healthy non-human High uptake in spleen, blocked by 49 primates excess nivolumab PD-1 and PET 64Cu-PD-1 and 64Cu-PD-L1 Murine anti-mouse B16F10 (mouse melanoma) C57BL/6N mice 64Cu-anti-PD-1 detected 38 PD-L antibody PD-1-deficient mice PD-1 positive TILs after PD-L1-deficient mice immunoradiotherapy, 64Cu-PD-L1 Immune competent antibody uptake in PD-L1 knockout < wild-type mice and PD-L1 upregulation by IFN-γ treatment visualized Abbreviations: DOTA: 1,4,7,10-tetraazacyclododecane- N,N’,N″,N’″-tetraacetic acid; NSCLC: non- small cell lung cancer; DTPA: diethylenetriaminepentaacetic acid; AlF: aluminiumfluoride; NOTA: 1,4,7-triazacyclononane-N,N’,N’’-triacetic acid; HPV: human papillomavirus ; HNSCC: head and neck squamous-cell carcinoma; PDX: patient-derived xenograft; EGFR: epidermal growth factor receptor; TILs: tumor-infiltrating lymphocytes; IFN-γ: interferon-gamma; PBMC: peripheral blood mononuclear cell.

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Targeting metabolic processes within the tumor for molecular imaging The PET tracer 18F-fluorodeoxyglucose 18( F-FDG) is used in daily practice in several tumor types to detect tumor lesions (Fig. 1). This glucose analogue is incorporated in cancer cells with an increased glycolysis rate. For immunotherapy follow-up purposes, however, detecting tumor lesions with 18F-FDG PET might be affected by the fact that activated immune cells in and around the tumor also show uptake of this tracer.

Two retrospective studies analyzed the relation between metabolic parameters and immune related markers. In a study in patients with NSCLC the metabolic parameters on 18F-FDG PET correlated with intratumoral immune-related markers, such as PD-1 and CD8 expressing TILs in the surgical resection specimens.54 In contrast, a study in head and neck squamous-cell carcinoma patients found that 18F-FDG uptake was inversely correlated with a gene co-expression network accounting for immune cell aggregation and activation function.55

A few studies investigated the role of 18F-FDG PET imaging to provide information with regards to response to immunotherapy (Fig. 3). Two small studies studied the effect of immunotherapy early during the treatment. Reduction in 18F-FDG accumulation in 24 patients after the first cycle of nivolumab treatment in NSCLC patients seemed to distinguish responders and non-responders earlier compared to morphological changes in CT scans.56 18F-FDG imaging was also used to evaluate response to ipilimumab treatment in 25 patients with metastatic melanoma.57,58 Changes in standardized uptake values (SUV) in single lesions did not differ between patients with clinical benefit and patients without clinical benefit.57

Larger studies will be required to understand the precise role of 18F-FDG PET in this setting. Moreover it is to be anticipated that pseudoprogression could lead to false positive results, due to an increased inflammatory response, thereby increasing 18F-FDG PET uptake. A larger study comprising 104 patients with metastatic melanoma 18F-FDG PET imaging at 1 year revealed that the majority of patients in partial response by conventional CT response evaluation had a complete metabolic response, with excellent medium-term outcomes, mirroring those with complete response on CT. However those with residual FDG avidity fared less well.59

32 Molecular imaging to enlighten cancer immunotherapies

Figure 3. Examples of 18F-FDG PET imaging. Baseline FDG-PET, patient with NSCLC. (A) Coronal MIP image showing the primary lung tumor in the left upper lobe and multiple lymph node metastases. (B) Fused transversal image showing the primary lung tumor and mediastinal and hilar lymph node metastases. (C) Fused transversal image showing bilateral lymph node metastases in the upper mediastinum; FDG-PET after 2 cycles of immunotherapy with nivolumab. (D) Coronal MIP image showing an overall decrease in metabolic activity of the lesions, but high uptake in especially the left lobe of the thyroid. (E) Fused transversal image showing decrease in metabolic activity of the primary tumor and the mediastinal and hilar lymph nodes. (F) Fused transversal image showing high uptake in the thyroid (especially the left lobe) due to thyroiditis as side effect of the therapy, no uptake anymore at the bilateral lymph node metastases in the upper mediastinum. Courtesy Dr. B.I. Hiddinga, University Medical Center Groningen.

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Targeting immune cells for molecular imaging Molecular imaging of immune cells has been investigated for decades to diagnose and evaluate therapy in inflammatory and infectious diseases with ex vivo and in vivo labeling approaches. For ex vivo labeling, leukocytes of a patient are isolated and radiolabeled ex vivo. The labeled leukocytes are then re-administered intravenously (iv). The SPECT tracers 99mTc-HMPAO and 111In-oxine are still widely used in the clinic for white blood cell (WBC) scintigraphy.60,61 WBC scintigraphy can provide high diagnostic accuracy in detecting infections, especially in the extremities, but clearly does not provide information about which subgroup of leukocytes is involved. Several studies used ex vivo radiolabeled specific subgroups of leukocytes, such as granulocytes, T-cells, NK-cells and macrophages (Supplementary Table 2).

Besides the ex vivo labeling approaches, non-invasive PET and SPECT imaging with tracers targeting in vivo specific leukocytes or immune cell progenitor cells can be performed to target subpopulations of cells via cell-type specific characteristics. Additionally, molecular imaging techniques with labeled immune checkpoint targeting molecules have been used to visualize immune cells, which are addressed above.

Granulocyte imaging Granulocytes, the largest fraction of the leukocytes, include neutrophils, basophils, eosinophils and mast cells. These cells are important for tumor immune responses; for instance, neutrophils play a role in directing adaptive immune responses.62 The potential of non-invasive granulocyte SPECT imaging was shown for inflammation and infections with radiolabeled antibodies and antibody fragments directed against targets on human granulocytes (Supplementary Table 3). Granulocyte tracers have not yet been used in the context of cancer immunotherapy.

Lymphocyte imaging T-cell-specific imaging approaches – indirect labeling In addition to ex vivo direct labeling methods, indirect cell labeling approaches have also been tested for T-cells (Table 2). T-cells are ex vivo transfected with a reporter gene expressing a certain protein or enzyme, which can be targeted by a specific radiopharmaceutical. An example of an enzyme-based strategy is transfection with the reporter gene herpes simplex virus type 1 thymidine kinase (HSV1-TK), which can be targeted by 18F-FHBG, 18F-FEAU and 124I-FIAU.63 These tracers are phosphorylated by HSV-TK and incorporated into the cellular DNA of proliferating cells. Recently, 18F-FHBG imaging was performed in patients to track CAR-engineered cytotoxic T-cells (CTLs) in gliomas.64 The CTLs expressed both a tumor- targeting CAR and the HSV1-tk gene reporter. 18F-FHBG PET scans prior to and after intracranial infusion administration of CTLs in seven patients showed increased tumor uptake of 18F-FHBG (Fig. 4A).

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Figure 4. Examples of molecular imaging strategies in cancer immunotherapy: clinical studies. (A) 18F-FHBG PET imaging in a glioblastoma patient before and 1 week after infusion of CAR cytotoxic T-cells (CTL), infusion site indicated by red arrow.64 Image shows higher uptake after CTL infusion. (B) 99mTc-IL2 SPECT image of a melanoma patient.81 Image shows uptake in tumor (green arrow) and lymph nodes (red arrow). (C) 89Zr-RG7356 (anti-CD44) PET imaging in a head and neck cancer patient 5 days after tracer injection.122 Upper panel shows 89Zr-RG7356 uptake in lymph node metastases, lower panel shows 18F-FDG scan. All figures are reprinted with permission.

T-cell-specific imaging approaches – targeting subtypes T-cells contain several subpopulations, such as CD8+ cytotoxic T-cells, CD4+ T-helper (Th) or CD4+ regulatory T-cells (Tregs). CD8+ cells attack tumor cells after antigen recognition on the tumor cells. Th cells stimulate proliferation and differentiation of other T-cells and activate B-cells and macrophages, while Tregs inhibit immune responses.65 For immunotherapeutic approaches T-cell subpopulations are increasingly targeted using direct in vivo approaches (Table 2). The T-cell marker CD3 has been targeted with 89Zr-anti-CD3 antibody and used to detect T-cells 2 weeks after immune checkpoint therapy with PET imaging in immune competent mice bearing subcutaneously (sc) mouse colon tumors.66 Mice with high tracer tumor uptake after 2 weeks of anti-CTLA4 therapy showed stronger tumor growth inhibition. Tracer uptake was related to CD3+ T-cell infiltration measured with IHC.

PET imaging of CD8+ cells in mice with 64Cu-labeled CD8+ targeting antibody fragments showed specific uptake in spleen and lymph nodes.67 After hematopoietic stem cell therapy in mice, 89Zr–labeled anti-CD4 and anti-CD8 cys-diabodies (cDbs) detected CD4+ and CD8+ T-cells in spleen and lymph nodes, indicating T-cell repopulation.68 89Zr-labeled anti-CD8+ cDb detected tumor accumulation of adoptively transferred mouse CD8+ T-cells in murine tumor-bearing mice.69

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89 CD3+ T-cells Direct in vivo Zr-DFO-CD3 Preclinical • Detect CD3+ T-cells in tumors 66 • 89Zr-DFO-CD3 uptake in liver, spleen, lymph nodes and thymus • Tumor volume of mice with high uptake of 89Zr-DFO-CD3 after anti-CTLA4 treatment < mice with low uptake/control CD4+ or CD8+ Direct in vivo 64Cu-labeled anti CD8+ Preclinical/mice Detect CD8+ T-cells, specific uptake in spleen and lymph nodes 67 T-cells antibody fragments 89Zr-labeled anti CD4+ Preclinical/mice • Detect CD4+ or CD8+ T-cells after hematopoietic stem cell therapy 68,69 or CD8+ cDb (89Zr- • Detect antigen-specific tumor targeting of CD4+ T-cells malDFO-169 cDb) • Higher tumor uptake of 89Zr-malDFO-169 cDb after anti-CD137 therapy. Tumor uptake in anti-PD-L1 responders>non-responders 89Zr-labeled PEGylated Preclinical/mice • Detected intratumoral CD+ T-cells. Uptake in thymus, spleen and lymph nodes. 70 anti-CD8 single-domain • Homogeneous tumor uptake correlated with strong response to CTLA4 therapy, antibody fragments (VHH) heterogeneous uptake with partial response. Activated Direct in vivo 123I-labeled IL2, 99mTc-IL2, Preclinical and clinical studies • 123I-IL2 uptake associated with extent of lymphocyte infiltration in mice 72-82 T-cells [18F]FB-IL2 • Higher uptake of 99mTc-IL2 in kidneys, liver and spleen in mice • Detect T cell infiltration in patients with atherosclerotic plaques, melanoma and SCCHN • Detect CD25+ human T cells • Binding potential [18F]FB-IL2 correlates with number of CD25+ T-cells Activated Indirect in vivo 18F-AraG Preclinical and clinical studies Uptake in activated mouse T cells > naïve T cells in cell assays 83-85 T-cells High uptake in lymphoid organs in aGVHD mouse model In healthy volunteers higher uptake in clearance organs, heart and spleen In RA mice model higher uptake in RA affected joints compared to control B-cells general Direct in vivo 99mTc- or 124I-labeled anti- Preclinical and clinical studies Detection of B cells in several autoimmune diseases 86-88 CD20 rituximab 89Zr- anti-CD20 rituximab Preclinical and clinical studies • Detect CD20+ B cells in patients with B cell lymphoma 89 • Tumor uptake reduced by pre-dose unlabeled rituximab in B cell-depleted patients. With preserved B cells higher tracer uptake with the pre-dose in less accessible tumor lesions with lower spleen uptake 89Zr- anti-CD45 Preclinical Detect B cells in mice models 90 NK-cells Direct in vivo 99mTc-anti-CD56 mAb Preclinical Detect human NK cells injected in mice 93 MDSCs Direct in vivo 99mTc anti-CD11b EP1345Y Preclinical studies Uptake in tumor, bone marrow and spleen 102 Dendritic cells Indirect ex vivo 18F-tetrafluoroborate Preclinical studies Detected migration of DC cells to draining lymph nodes in mice 104 Abbrevations: 18F-FHBG: 18F-9-[4-fluoro-3-(hydromethyl)butyl]guanine; 18F-FEAU: 18F-2-fluoro-2- deoxyarabinofuranosyl-5-ethyluracil; 124I-FIAU: 124I-5-iodo-2-fluoro-2-deoxy-1-β-D-arabino-furanosyl- uracil; CAR: chimeric antigen receptor; DFO: desferrioxamine; SCCHN: squamous cell carcinoma of the head and neck; 18F-AraG: 2’-deoxy-2’-[18F]fluoro-9-β-D-arabinofuranosylguanine; aGVHD: acute graft- versus-host disease; RA: rheumatoid arthritis; NK: natural killer; MDSCs: myeloid-derived-suppressor cells; DC: dendritic cell.

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Table 2. Tracers to image T-cells, B-cells, NK-cells, MDSCs and dendritic cells Cell type Method Tracers Preclinical/clinical Studies Application/results Ref T-cell general Indirect ex vivo 18F-FHBG, 18F-FEAU, Preclinical and clinical • Infection and inflammation imaging 63-64 124I-FIAU • Track CAR T-cells in glioma patients 2 89 CD3+ T-cells Direct in vivo Zr-DFO-CD3 Preclinical • Detect CD3+ T-cells in tumors 66 • 89Zr-DFO-CD3 uptake in liver, spleen, lymph nodes and thymus • Tumor volume of mice with high uptake of 89Zr-DFO-CD3 after anti-CTLA4 treatment < mice with low uptake/control CD4+ or CD8+ Direct in vivo 64Cu-labeled anti CD8+ Preclinical/mice Detect CD8+ T-cells, specific uptake in spleen and lymph nodes 67 T-cells antibody fragments 89Zr-labeled anti CD4+ Preclinical/mice • Detect CD4+ or CD8+ T-cells after hematopoietic stem cell therapy 68,69 or CD8+ cDb (89Zr- • Detect antigen-specific tumor targeting of CD4+ T-cells malDFO-169 cDb) • Higher tumor uptake of 89Zr-malDFO-169 cDb after anti-CD137 therapy. Tumor uptake in anti-PD-L1 responders>non-responders 89Zr-labeled PEGylated Preclinical/mice • Detected intratumoral CD+ T-cells. Uptake in thymus, spleen and lymph nodes. 70 anti-CD8 single-domain • Homogeneous tumor uptake correlated with strong response to CTLA4 therapy, antibody fragments (VHH) heterogeneous uptake with partial response. Activated Direct in vivo 123I-labeled IL2, 99mTc-IL2, Preclinical and clinical studies • 123I-IL2 uptake associated with extent of lymphocyte infiltration in mice 72-82 T-cells [18F]FB-IL2 • Higher uptake of 99mTc-IL2 in kidneys, liver and spleen in mice • Detect T cell infiltration in patients with atherosclerotic plaques, melanoma and SCCHN • Detect CD25+ human T cells • Binding potential [18F]FB-IL2 correlates with number of CD25+ T-cells Activated Indirect in vivo 18F-AraG Preclinical and clinical studies Uptake in activated mouse T cells > naïve T cells in cell assays 83-85 T-cells High uptake in lymphoid organs in aGVHD mouse model In healthy volunteers higher uptake in clearance organs, heart and spleen In RA mice model higher uptake in RA affected joints compared to control B-cells general Direct in vivo 99mTc- or 124I-labeled anti- Preclinical and clinical studies Detection of B cells in several autoimmune diseases 86-88 CD20 rituximab 89Zr- anti-CD20 rituximab Preclinical and clinical studies • Detect CD20+ B cells in patients with B cell lymphoma 89 • Tumor uptake reduced by pre-dose unlabeled rituximab in B cell-depleted patients. With preserved B cells higher tracer uptake with the pre-dose in less accessible tumor lesions with lower spleen uptake 89Zr- anti-CD45 Preclinical Detect B cells in mice models 90 NK-cells Direct in vivo 99mTc-anti-CD56 mAb Preclinical Detect human NK cells injected in mice 93 MDSCs Direct in vivo 99mTc anti-CD11b EP1345Y Preclinical studies Uptake in tumor, bone marrow and spleen 102 Dendritic cells Indirect ex vivo 18F-tetrafluoroborate Preclinical studies Detected migration of DC cells to draining lymph nodes in mice 104 Abbrevations: 18F-FHBG: 18F-9-[4-fluoro-3-(hydromethyl)butyl]guanine; 18F-FEAU: 18F-2-fluoro-2- deoxyarabinofuranosyl-5-ethyluracil; 124I-FIAU: 124I-5-iodo-2-fluoro-2-deoxy-1-β-D-arabino-furanosyl- uracil; CAR: chimeric antigen receptor; DFO: desferrioxamine; SCCHN: squamous cell carcinoma of the head and neck; 18F-AraG: 2’-deoxy-2’-[18F]fluoro-9-β-D-arabinofuranosylguanine; aGVHD: acute graft- versus-host disease; RA: rheumatoid arthritis; NK: natural killer; MDSCs: myeloid-derived-suppressor cells; DC: dendritic cell.

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Table 3. Tracers for imaging components of the tumor microenvironment Cell type Target Method Tracers Preclinical/clinical Application/results Refs Cell Integrins Direct in vivo Radiolabeled RGD peptides (targeting integrin Preclinical and clinical studies Imaging in inflammation and oncology 113,114 adhesion ανβ3) molecules VCAM-1, VAP-1, Direct in vivo Radiolabeled VCAM-1, VAP-1, ICAM-1 targeting Preclinical and clinical studies Imaging in inflammation and oncology 115-119 ICAM-1 molecules CD44 Direct in vivo 89Zr-RG7356 Preclinical and clinical studies Detected CD44 expressing tumors in mice and human 121,122 Uptake in spleen, salivary glands and bone marrow in cynomolgus monkeys Signaling Interleukins Direct in vivo Radiolabeled IL-2 Preclinical and clinical studies Imaging in inflammation and oncology 72-82 molecules Radiolabeled IL-18 Preclinical studies • 64Cu-DOTA-IL-18bp-Fc-specific tumor uptake in 124,125 (64Cu-DOTA-IL-18bp-Fc, 99mTc-IL-18bp-Fc-IL-1ra) mice • 99mTc-IL-18bp-Fc-IL-1ra detected inflammatory lesions in mice TNF-α Direct in vivo 64Cu-DOTA-etanercept Preclinical studies Detected ear inflammation in mice, low uptake in 126 other organs 99mTc-labeled TNFR2-Fc-IL-1ra Preclinical studies Detected inflammatory regions in ischemic-reperfused 127 rat heart model 99mTc-labeled infliximab Preclinical and clinical studies Imaging in inflammatory diseases 128-131 IDO Direct in vivo 11C-1MTrp Preclinical studies In healthy rats rapid clearance and low accumulation 135 in normal organs Abbreviations: RGD: arginylglycylaspartic acid; VCAM-1: vascular cell adhesion molecule-1; VAP-1: vascular adhesion protein-1; ICAM-1: intercellular adhesion molecule-1; IL: interleukin; TNF-α: tumor necrosis factor α; IDO: indolamine 2,3-dioxygenase; 1MTrp: 1-methyl-tryptophan.

The potential of these imaging approaches to obtain information about immunotherapy treatment effects, like tumor immune cell infiltration, was illustrated in immune competent mice bearing mouse colon tumors that received immune activating anti-CD137 antibody therapy. 89Zr-labeled anti-CD8+ cDb tumor uptake was higher in anti-CD137 treated mice than in untreated ones. This reflected an increase of CD8+ TILs after anti-CD137 therapy, as confirmed by IHC. Moreover, in mice treated with mouse anti-PD-L1 antibody,89 Zr- labeled anti-CD8+ cDb tumor uptake was higher and corresponded with more tumor CD8+ cells as measured by flow cytometry in tumor tissue in case of response (Fig. 2C).69 89Zr-labeled anti-CD8 single-domain antibody fragments imaging in mice bearing melanoma or breast tumors showed a homogenous uptake in tumors responding to anti-CTLA4 therapy, while heterogeneous uptake correlated with faster tumor growth and worse response.70

38 Molecular imaging to enlighten cancer immunotherapies

Table 3. Tracers for imaging components of the tumor microenvironment Cell type Target Method Tracers Preclinical/clinical Application/results Refs Cell Integrins Direct in vivo Radiolabeled RGD peptides (targeting integrin Preclinical and clinical studies Imaging in inflammation and oncology 113,114 adhesion ανβ3) 2 molecules VCAM-1, VAP-1, Direct in vivo Radiolabeled VCAM-1, VAP-1, ICAM-1 targeting Preclinical and clinical studies Imaging in inflammation and oncology 115-119 ICAM-1 molecules CD44 Direct in vivo 89Zr-RG7356 Preclinical and clinical studies Detected CD44 expressing tumors in mice and human 121,122 Uptake in spleen, salivary glands and bone marrow in cynomolgus monkeys Signaling Interleukins Direct in vivo Radiolabeled IL-2 Preclinical and clinical studies Imaging in inflammation and oncology 72-82 molecules Radiolabeled IL-18 Preclinical studies • 64Cu-DOTA-IL-18bp-Fc-specific tumor uptake in 124,125 (64Cu-DOTA-IL-18bp-Fc, 99mTc-IL-18bp-Fc-IL-1ra) mice • 99mTc-IL-18bp-Fc-IL-1ra detected inflammatory lesions in mice TNF-α Direct in vivo 64Cu-DOTA-etanercept Preclinical studies Detected ear inflammation in mice, low uptake in 126 other organs 99mTc-labeled TNFR2-Fc-IL-1ra Preclinical studies Detected inflammatory regions in ischemic-reperfused 127 rat heart model 99mTc-labeled infliximab Preclinical and clinical studies Imaging in inflammatory diseases 128-131 IDO Direct in vivo 11C-1MTrp Preclinical studies In healthy rats rapid clearance and low accumulation 135 in normal organs Abbreviations: RGD: arginylglycylaspartic acid; VCAM-1: vascular cell adhesion molecule-1; VAP-1: vascular adhesion protein-1; ICAM-1: intercellular adhesion molecule-1; IL: interleukin; TNF-α: tumor necrosis factor α; IDO: indolamine 2,3-dioxygenase; 1MTrp: 1-methyl-tryptophan.

The potential of these imaging approaches to obtain information about immunotherapy These methods illustrate the possibility of visualizing specific subgroups of T-cells by treatment effects, like tumor immune cell infiltration, was illustrated in immune competent molecular imaging, and the potential to use this for immunotherapy evaluation. For instance, mice bearing mouse colon tumors that received immune activating anti-CD137 antibody CD8-targeting might be relevant as recently heterogeneity in CD8-cell infiltration in different therapy. 89Zr-labeled anti-CD8+ cDb tumor uptake was higher in anti-CD137 treated mice tumor lesions of one patient was shown with IHC.71 In an ongoing clinical study the potential than in untreated ones. This reflected an increase of CD8+ TILs after anti-CD137 therapy, of a 89Zr-radiolabeled CD8-targeting tracer is evaluated in patients with different tumor types as confirmed by IHC. Moreover, in mice treated with mouse anti-PD-L1 antibody,89 Zr- (ClinicalTrials.gov identifier NCT03107663). labeled anti-CD8+ cDb tumor uptake was higher and corresponded with more tumor CD8+ cells as measured by flow cytometry in tumor tissue in case of response (Fig. T-cell-specific imaging approaches – targeting activated T-cells 2C).69 89Zr-labeled anti-CD8 single-domain antibody fragments imaging in mice bearing The main actors that induce tumor cell death are activated effector T-cells. Specific targeting melanoma or breast tumors showed a homogenous uptake in tumors responding to of these cells might provide additional information on immunotherapy effects. The IL2 anti-CTLA4 therapy, while heterogeneous uptake correlated with faster tumor growth receptor CD25 is expressed on the surface membrane of activated T-cells. Recombinant and worse response.70 human IL2, which binds human and mouse IL2 receptors, has been labeled with 99mTc, 123I

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and 18F (Table 2). In rodent models, lymphocyte infiltration in inflammation was detected with these tracers.72-75 Moreover, [18F]FB-IL2 PET imaging detected sc-injected CD25+ activated T-cells, confirmed by hematoxylin and eosin staining (Fig. 2D).76,77 In patients, 99mTc-IL2 and 123I-IL2 visualized T-cell infiltration in atherosclerotic plaques, melanoma and squamous cell head and neck cancers (Fig. 4B).78-82 Trials are ongoing with serial 99mTc-IL2 and [18F]FB-IL2 scanning in patients with melanoma receiving immune checkpoint inhibitors (ClinicalTrials. gov identifiers NCT02922283 and NCT01789827).

Besides these direct approaches, 2’-deoxy-2’-[18F]fluoro-9-β-D-arabinofuranosylguanine (18F-AraG) is an indirect tracer for activated T-cells. The AraG prodrug nelarabine is a FDA approved guanosine analog for treatment of relapsed T-cell acute lymphoblastic leukemia and T-cell lymphoblastic lymphomas. In vitro 18F-AraG uptake by activated mouse T-cells was higher than uptake by naive T-cells.83 In mice with acute graft-versus-host disease (aGVHD), 18F-AraG uptake was higher in the spleen and lymph nodes compared to control mice. In healthy volunteers 18F-AraG PET showed highest presence in clearance organs, moreover uptake was seen in the spleen.84 In a rheumatoid arthritis mouse model higher 18F-AraG uptake was found in affected joints compared to non-affected joints.85 18F-AraG PET is currently being investigated in patients with bladder cancer before and during anti-PD-L1 atezolizumab treatment (ClinicalTrials.gov identifier NCT03007719).

B-cell-specific imaging approaches B-cells regulate immune responses by influencing other immune cells. They exert tumor promoting and suppressing effects. Molecular imaging has been used to visualize B-cell infiltration in patients with a variety of autoimmune diseases using radiolabeled mAb rituximab targeting the cell surface protein CD20 (Table 2). In patients, specific tracer uptake of99m Tc or 124I labeled rituximab was seen in inflamed tissues such as in rheumatoid arthritis.86-88 In relapsed CD20+ B-cell lymphoma patients, the biodistribution of 89Zr-labeled rituximab with and without a standard pre-dose of unlabeled rituximab was evaluated, showing that a pre- dose impaired tumor tracer uptake in patients with a B-cell depletion.89

Besides CD20, other proteins upregulated on B-cells, such as CD45 (BB220), have been used as targets for molecular imaging. A mouse-specific 89Zr-labeled anti-CD45 served to visualize B-cell trafficking in mice.90 Tissue hot spots for B-cells were detected in normal spleen and lymph nodes at 24-48 hours after tracer injection. Flow cytometry confirmed a high frequency of B-cells at these sites. After blocking binding sites by administration of a pre-dose unlabeled anti-CD45 mAb, no signal was seen in these lymphoid organs.

40 Molecular imaging to enlighten cancer immunotherapies

Natural Killer cells specific imaging approaches Activated NK-cells can kill tumor cells directly by cytoplasmic granule release, death receptor- induced apoptosis, effector molecule production or ADCC. In addition, NK-cells can improve antigen uptake and presentation of DCs, thereby promoting antigen-specific responses of 2 CTLs. NK-cells also produce cytokines, such as interferon-γ, which stimulate CD8+ T-cells.91 Adoptive transfer therapy of NK-cells, in which the patient’s own NK-cells are stimulated in vitro and re-injected, is in clinical development for both solid and hematologic malignancies. In addition, immune checkpoint inhibitors also exert their anti-tumor effect by means of NK- cell activation.92

Direct NK-cell-specific targeting imaging methods are being investigated, for instance by targeting the NK-cell surface protein CD56 (Table 2). A 99mTc labeled CD56 antibody was used to visualize injected human CD56+ NK-cells in the thigh in immune deficient mice. In mice xenografted with a human thyroid tumor, 99mTc-anti-CD56 tumor uptake was visible 24 hours after human NK-cell injection, which reflected presence of NK-cells as verified with IHC.93 This tracer has not yet been evaluated in humans but might provide meaningful insight into NK-cell distribution during immunotherapy.

Imaging macrophages and macrophage-like cells Monocytes are the third group of leukocytes and comprise macrophages and myeloid DCs. Macrophages located near or within the tumor are tumor-associated macrophages, which promote tumor progression and metastasis via several mechanisms, such as facilitating angiogenesis by the production of angiogenic factors or matrix breakdown.94 Two phenotypes, M1 and M2 macrophages, play complementary roles in tumor progression. M1 macrophages are involved in the inflammatory response, pathogen clearance and antitumor immunity, while M2 macrophages play an anti-inflammatory role and are involved in tumor initiation and progression.95,96 Antibodies targeting M2 macrophages are currently evaluated in trials in cancer patients.

Several tracers have been developed to assess migration and accumulation of macrophages and macrophage-like cells in the context of infection and inflammation imaging, as well as in oncology, in preclinical and clinical studies (Supplementary Table 4).

Myeloid-derived-suppressor cell imaging Leukocytes arise from myeloid and lymphoid progenitor cells. Myeloid-derived suppressor cells (MDCSs), a heterogeneous population of myeloid-cell progenitors, play a role in tumor progression by their immunosuppressive activity in the TME.97 Tumor-bearing mice contained more MDSCs in the spleen than non-tumor bearing mice. Moreover, more MDSCs were found in the peripheral blood of cancer patients and in the spleen, isolated after splenectomy, compared to patients with benign pancreatic cysts.98-101

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MDSCs have been targeted by 99mTc-labeled anti-CD11b antibody EP1345Y. CD11b is expressed by immature myeloid cells and MDSCs. In a murine colon cancer model, 99mTc-labeled anti- CD11b specific uptake was seen in tumor tissue, bone marrow and spleen. Anti-CD11b immunofluorescence staining and flow cytometry confirmed infiltration of CD11b positive cells in tumor regions and an increase of CD11b positive immune cells in peripheral blood and bone marrow.102

Dendritic cell imaging DCs present antigens to naïve T-cells in lymphoid organs, thereby activating cytotoxic T-cells. DC- based cancer immunotherapy, mainly DC cancer vaccines, has generated anti-tumor responses in various tumor types, both preclinically and clinically.103 Moreover, DCs express a variety of immune checkpoints, such as PD-L1. These cells have been tracked with molecular imaging using an indirect labeling method via the human sodium/iodide symporter (hNIS) reporter gene (Table 2). Murine DCs co-expressing this gene were injected in the footpad of mice, and their migration to the lymph nodes could be tracked using the radiotracer 18F-tetrafluoroborate.104

Targeting tumor and immune cells with bispecific antibodies Biodistribution of BsAbs, when developed to target both tumor cells and immune cells, might be affected by the different binding affinity for their two targets. Molecular imaging could be used to gain insight in the biodistribution. There are a few studies performed. 89Zr-labeled BsAb targeting carcinoembryonic antigen (CEA) on tumor cells and the IL2 receptor on immune cells showed CEA mediated accumulation in tumors and uptake in lymph nodes and spleen in patients with solid tumors.105 89Zr-labeled AMG 211, targeting CEA and CD3, showed accumulation in CEA positive tumors.106 Currently, 89Zr-AMG 211 PET imaging is performed in patients with gastrointestinal adenocarcinoma (ClinicalTrials.gov identifier NCT02760199).

Targeting extracellular matrix components for molecular imaging The ECM of the tumor is composed of a complex network of macromolecules. Abnormalities in the ECM can promote cancer progression, not only by inducing cellular transformation and metastasis, but also by deregulation of stromal cells, leading to tumor-associated angiogenesis and inflammation.107 To increase the effect of immune checkpoint inhibitor therapy, several ongoing clinical trials are focusing on the combination with drugs influencing components of the ECM, such as chemotherapeutics, cytokine-based therapies including interferon-α or IL2 or vascular endothelial growth factor-A (VEGF-A) directed therapy.108,109 Just recently increased tumor growth factor (TGF)-β in the TME was shown to represent a mechanism of immune evasion.110,111 With the TGF-β antibody 89Zr-fresolimumab specific uptake in recurrent high-grade gliomas was shown.112 This example shows that molecular imaging might serve as a tool to gain insight into these ECM components before and during immunotherapy. For other indications imaging with tracers targeting several ECM components has already been performed (Table 3).

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Imaging cell adhesion molecules Cell adhesion molecules, such as integrins, are important in tumor-induced angiogenesis and tumor metastasis. Integrins are upregulated on activated endothelial cells, and integrin expression is a potential molecular marker for angiogenesis.113 Integrins are also 2 expressed by tumor cells and a variety of non-malignant cells, such as smooth muscle cells and macrophages and osteoclasts. Therefore, PET imaging with tracers targeting integrins should be able to visualize expression on vasculature and tumor cells as well as inflammatory processes.114 In inflammation and oncology studies especially the integrin αvβ3 tracer has been used (Table 3).

Integrin-binding molecules, like vascular cell adhesion molecule-1 (VCAM-1), vascular adhesion protein-1 (VAP-1) and intercellular adhesion molecule-1 (ICAM-1), play a role in adhesion and migration of leukocytes. Tracers targeting these molecules have been used mainly to detect inflammation or atherosclerosis.115-118 111In-labeled VCAM-1 targeting peptide detected VCAM expression and response to platinum-based agents in an ovarian cancer mouse model.119

CD44 is another cell surface molecule, expressed on a variety of cells and involved in cell interactions, migration and adhesion.120 CD44 can be targeted by specific mAbs, such as RG7356. CD44 expression and mAb biodistribution has been explored with 89Zr-RG7356 in human tumor xenograft-bearing mice and non-tumor-bearing cynomolgus monkeys. 89Zr-RG7356 could selectively target CD44+ tumors in mice and CD44+ organs in monkeys, such as the spleen, bone marrow and salivary glands.121 89Zr-RG7356 PET imaging of patients with advanced, CD44-expressing solid tumors showed highest tracer uptake in liver, spleen and bone marrow as well as malignant lymph nodes (Fig. 4C).122

Cytokine imaging Cytokines, signaling molecules within the ECM, are responsible for the induction of intracellular pathways and regulation of processes, such as cell proliferation, activation, differentiation and migration. They are mainly secreted by immune cells to initiate an immune response. A large part of cytokines are ILs, which elicit a wide variety of immunomodulatory effects in cells and tissues.123 Several recombinant ILs have been developed for cancer treatment, including human (rh)-IL2. For molecular imaging of inflammatory lesions and activated immune cells, IL-receptor-targeting drugs have been radiolabeled. Besides radiolabeled IL2, previously discussed under lymphocyte imaging, radiolabeled IL18 binding protein (64Cu-DOTA-IL-18bp-Fc) also showed specific tumor uptake in mice models.124 A 99mTc-labeled dual cytokine ligand targeting IL18 and IL1 detected inflammatory lesions in mice and rats.125

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The cytokine tumor necrosis factor α (TNF-α) is involved in inflammation. Radiolabeled TNF-α antibodies or TNF-receptor ligands were used to detect inflammatory lesions in preclinical models.126,127 99mTc-infliximab, a chimeric TNF-α mAb, detected inflammatory sites in patients with rheumatoid arthritis. 99mTc-infliximab uptake was higher in the affected joints than in non-affected joints. Responders showed a higher increase in pretherapy uptake (from 6-20 hours) in the affected joints than non-responders, indicating that more TNF-α may be present in these joints.128 But in patients with inflammatory bowel disease and pulmonary sarcoidosis 99mTc-infliximab imaging did not correlate with therapy response.129,130 However, 99mTc might not be the best radionuclide for this purpose, since its half-life of 6 h does not match the long half-life of 9.5 days of the mAb. Another clinical study in pulmonary sarcoidosis patients showed differences in uptake between patients, suggesting interpatient variability in TNF-α levels.131

Indolamine 2,3-dioxygenase (IDO) imaging The enzyme indolamine 2,3-dioxygenase (IDO) catalyzes the degradation of the essential amino acid L-tryptophan. When IDO levels are increased in the TME, tryptophan is depleted and pro-apoptotic metabolites are generated, inhibiting the effector T-cells. IDO inhibitors, such as 1-methyl-tryptophan (1MTrp), show antitumor potential by competitively blocking the activity of IDO in tumor and tumor infiltrating myeloid cells. In preclinical studies, IDO inhibitors favor the immune response and several clinical trials are ongoing to evaluate these drugs in cancer patients.132-134

For molecular imaging purposes, both stereoisomers of the IDO inhibitor 1MTrp have been radiolabeled with 11C. They were both rapidly excreted in rats via liver and kidneys with low accumulation in normal organs.135 A next step would be to translate this 1MTrp PET imaging to IDO-positive tumor models and pharmacodynamics studies in which IDO inhibitors are evaluated.

CONCLUDING REMARKS

With regard to immunogenicity and biomarkers that predict response to immunotherapy, accurate insight into all tumor lesions in individual patients is urgently needed. The tumor immune response is a highly dynamic interplay between tumor cells, immune cells and other molecules with marked heterogeneity between and even within tumor lesions. This review shows that molecular imaging strategies can enable visualization of various components that are important in immunotherapy, especially tumor cells, immune cells, and ECM components. This could potentially guide treatment decisions, which are becoming increasingly complex as shown by the numerous combination trials that are currently ongoing in immunotherapy.

44 Molecular imaging to enlighten cancer immunotherapies

The various molecular imaging strategies could contribute to patient selection upfront or early during immunotherapy. These strategies might answer questions about what immune cells and specific immune cell populations are present before and during immunotherapy. Moreover, these imaging strategies could provide insight into the mechanisms underlying 2 cancer immunotherapy. For example, immune cell targeting tracers, with short-lived tracers such as [18F]FB-IL2, could be used shortly before imaging with radiolabeled immune checkpoint targeting molecules, with isotopes with a long half-life such as 89Zr. This would provide non-invasive, whole body information, not only about the presence of immune cells, but also about the biodistribution of immune checkpoint targeting drugs. Together with analysis of tumor biopsy, providing information on DNA, RNA and protein, a complete data set can be assembled. Moreover, combinations with other imaging modalities, such as optical imaging using fluorescent-labeled immune checkpoint targeting molecules, would provide even more information on tumor characteristics.

Funding: this work was supported by ERC Advanced Grant: OnQview, and Dutch Cancer Society Grant: RUG 2016-10034.

Appendix A. Supplementary materials 1. Search strategy, selection method and full search 2. Supplementary Figure 1 3. Supplementary Table 1 4. Supplementary Table 2 5. Supplementary Table 3 6. Supplementary Table 4 7. Supplementary References

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of integrin αvβ3. Theranostics. 2016;6:78-92. 115. Patel N, Duffy BA, Badar A, Lythgoe MF, Arstad E. Bimodal imaging of Inflammation with SPECT/ CT and MRI using iodine-125 labeled VCAM-1 targeting microparticle conjugates. Bioconjug Chem. 2015;26:1542-1549. 116. Autio A, Vainio PJ, Suilamo S, Mali A, Vainio J, Saanijoki T, et al. Preclinical evaluation of a radioiodinated fully human antibody for in vivo imaging of vascular adhesion protein-1-positive vasculature in inflammation. J Nucl Med. 2013;54:1315-1319. 117. Zern BJ, Chacko AM, Liu J, Greineder CF, Blankemeyer ER, Radhakrishnan R, et al. Reduction of nanoparticle avidity enhances the selectivity of vascular targeting and PET detection of pulmonary inflammation. ACS Nano. 2013;7:2461-2469. 118. Nahrendorf M, Keliher E, Panizzi P, Zhang H, Hembrador S, Figueiredo J, et al. 18F-4V for PET–CT imaging of VCAM-1 expression in atherosclerosis. JACC Cardiovasc Imaging. 2009;2:1213-1222. 119. Scalici JM, Thomas S, Harrer C, Raines TA, Curran J, Atkins KA, et al. Imaging VCAM-1 as an indicator of treatment efficacy in metastatic ovarian cancer. J Nucl Med. 2013;54:1883-1889. 120. Ponta H, Sherman L, Herrlich PA. CD44: from adhesion molecules to signalling regulators. Nat Rev Mol Cell Biol. 2003;4:33-45. 121. Vugts DJ, Heuveling DA, Stigter-van Walsum M, Weigand S, Bergstrom M, van Dongen GA, et al. Preclinical evaluation of 89Zr-labeled anti-CD44 monoclonal antibody RG7356 in mice and cynomolgus monkeys: prelude to phase 1 clinical studies. MAbs. 2014;6:567-575. 122. Menke-van der Houven van Oordt CW, Gomez-Roca C, Herpen CV, Coveler AL, Mahalingam D, Verheul HM, et al. First-in-human phase I clinical trial of RG7356, an anti-CD44 humanized antibody, in patients with advanced, CD44-expressing solid tumors. Oncotarget. 2016;7:80046-80058.

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123. Akdis M, Burgler S, Crameri R, Eiwegger T, Fujita H, Gomez E, et al. Interleukins, from 1 to 37, and interferon-gamma: receptors, functions, and roles in diseases. J Allergy Clin Immunol. 2011;127:701- 21.e1-70. 124. Cao Q, Cai W, Niu G, He L, Chen X. Multimodality imaging of IL-18-binding protein-Fc therapy of experimental lung metastasis. Clin Cancer Res. 2008;14:6137-6145. 2 125. Liu Z, Wyffels L, Barbera C, Hui MM, Woolfenden JM. A99m Tc-Labeled dual-domain cytokine ligand for imaging of inflammation. Nucl Med Biol. 2011;38:795-805. 126. Cao Q, Cai W, Li ZB, Chen K, He L, Li HC, et al. PET imaging of acute and chronic inflammation in living mice. Eur J Nucl Med Mol Imaging. 2007;34:1832-1842. 127. Liu Z, Barber C, Wan L, Liu S, Hui MM, Furenlid LR, et al. SPECT imaging of inflammatory response in ischemic-reperfused rat hearts using a 99mTc-labeled dual-domain cytokine ligand. J Nucl Med. 2013;54:2139-2145. 128. Conti F, Malviya G, Ceccarelli F, Priori R, Iagnocco A, Valesini G, et al. Role of scintigraphy with 99mTc- infliximab in predicting the response of intraarticular infliximab treatment in patients with refractory monoarthritis. Eur J Nucl Med Mol Imaging. 2012;39:1339-1347. 129. D’alessandria C, Malviya G, Viscido A, Aratari A, Maccioni F, Amato A, et al. Use of a 99mTc labeled anti-TNF-α monoclonal antibody in Crohn’s disease: in vitro and in vivo studies. Q J Nucl Med Mol Imaging. 2007;51:334-342. 130. Galli F, Lanzolla T, Pietrangeli V, Malviya G, Ricci A, Bruno P, et al. In vivo evaluation of TNF-alpha in the lungs of patients affected by sarcoidosis. BioMed Res Int. 2015;2015:401341. 131. Vis R, Malviya G, Signore A, Grutters JC, Meek B, van de Garde EM, et al. 99mTc-anti-TNF-alpha antibody for the imaging of disease activity in pulmonary sarcoidosis. Eur Respir J. 2016;47:1198- 1207. 132. Soliman H, Mediavilla-Varela M, Antonia S. Indoleamine 2,3-dioxygenase: is it an immune suppressor? Cancer J. 2010;16:354-359. 133. Katz JB, Muller AJ, Prendergast GC. Indoleamine 2, 3-dioxygenase in T-cell tolerance and tumoral immune escape. Immunol Rev. 2008;222:206-221. 134. Vacchelli E, Aranda F, Eggermont A, Sautes-Fridman C, Tartour E, Kennedy EP, et al. Trial watch: IDO inhibitors in cancer therapy. Oncoimmunology. 2014;3:e957994. 135. Xie L, Maeda J, Kumata K, Yui J, Zhang Y, Hatori A, et al. Development of 1-N-11C-methyl-L- and -D-tryptophan for pharmacokinetic imaging of the immune checkpoint inhibitor 1-methyl-tryptophan. Sci Rep. 2015;5:16417.

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APPENDIX A. SUPPLEMENTARY MATERIAL

1. Search strategy, selection method and full search 2. Supplementary Figure 1 3. Supplementary Table 1 4. Supplementary Table 2 5. Supplementary Table 3 6. Supplementary Table 4 7. Supplementary References

Search strategy, selection method and full search The MEDLINE (PubMed) data base was searched using the search terms: immunomodulation, immunotherapy, inflammation, lymphocyte, T-cell, radiolabeled antibody, radionuclide imaging, molecular imaging, positron emission tomography (PET) and single-photon emission computed tomography (SPECT). The search terms were tested against initial manual searches. The retrieved output was screened by two authors, and articles were selected that described molecular imaging techniques assessing drug targets and cellular effects potentially useful as biomarkers in cancer immunotherapy. The subsequent list was complemented with relevant articles identified by reviewing the reference lists of already retrieved publications and articles not yet MEDLINE indexed that were known from conferences. The database ClinicalTrials.gov was searched for active trials in the area of molecular imaging and immunotherapy, and the mAb database IMGT/mAB-DB (www. imgt.org) for therapeutic immune checkpoint modulating antibodies was searched up to November 2017. Abstracts of recent conferences, the American Society of Clinical Oncology (ASCO) annual meeting 2017, the European Society for Medical Oncology (ESMO) annual meeting 2017 and the American Association of Cancer Research (AACR) were searched using the above mentioned keywords.

Full search (“Immunomodulation”[Mesh] OR immunotherap*[tiab] OR immuno therap*[tiab] OR immune therap*[tiab] OR immune therap*[tiab] OR lymphocyt*[ti] OR inflammat*[ti] OR T-cell*[ti] OR (antibod*[ti] AND radiolab*[ti]) OR “Inflammation/radionuclide imaging”[Majr] OR “Lymphocytes/radionuclide imaging”[Majr]) AND (“Tomography, Emission-Computed”[Mesh] OR (positron[tiab] AND tomography[tiab]) OR PET[tiab] OR SPECT[tiab] OR PET CT[tiab]))

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Supplementary Figure 1. Immune checkpoint receptors and ligands as a target for molecular imaging on tumor and immune cells. Figure shows targets for currently registered immune checkpoint inhibitors against CTLA-4 and PD1/PDL1. More drugs are in development for other immune checkpoint receptors and ligands. Abbreviations: PD-L1 programmed death-ligand 1; PD-1: programmed cell death protein; CTLA-4: cytotoxic T-lymphocyte-associated protein 4.

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Supplementary Table 1. FDA/EMA approved immunotherapeutics Type Target Drug Approved for Approval date - FDA Approval date - EMA Immune checkpoint inhibitors CTLA-4 Ipilimumab Metastatic melanoma March 2011 November 2012 Adjuvant treatment of patients with stage III October 2015 melanoma Unresectable or metastatic melanoma in pediatric July 2017 patients 12 years of age and older Intermediate- and poor-risk advanced renal cell April 2018 carcinoma – in combination with nivolumab Previously treated MSI-H/dMMR metastatic July 2018 colorectal cancer – in combination with nivolumab PD-1 Nivolumab Metastatic melanoma December 2014 April 2015 Advanced or metastatic NSCLC March 2015 April 2016 Metastatic renal cell carcinoma November 2015 April 2016 Hodgkin’s lymphoma May 2016 November 2016 Head and neck cancer November 2016 April 2017 Locally advanced or metastatic urothelial February 2017 June 2017 carcinoma MSI-H or dMMR metastatic colorectal cancer that August 2017 has progressed following treatment Hepatocellular carcinoma September 2017 Completely resected melanoma with lymph node December 2017 involvement or metastatic disease Intermediate- and poor-risk advanced renal cell April 2018 carcinoma – in combination with ipilimumab Pembrolizumab Advanced melanoma September 2014 July 2015 Advanced NSCLC October 2015 August 2016 Recurrent or metastatic head and neck cancer August 2016 Hodgkin’s lymphoma March 2017 March 2017 First-line combination with pemetrexed May 2017 and carboplatin for patients with metastatic nonsquamous NSCLC, irrespective of PD-L1 expression Advanced or metastatic urothelial carcinoma May 2017 September 2017 Unresectable or metastatic solid tumors having a May 2017 MSI-H or dMMR) Recurrent locally advanced or metastatic gastric or September 2017 gastroesophageal junction cancer whose tumors express PD-L1 Recurrent or metastatic cervical cancer whose June 2018 tumors express PD-L1

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Supplementary Table 1. FDA/EMA approved immunotherapeutics Type Target Drug Approved for Approval date - FDA Approval date - EMA Immune checkpoint inhibitors CTLA-4 Ipilimumab Metastatic melanoma March 2011 November 2012 Adjuvant treatment of patients with stage III October 2015 2 melanoma Unresectable or metastatic melanoma in pediatric July 2017 patients 12 years of age and older Intermediate- and poor-risk advanced renal cell April 2018 carcinoma – in combination with nivolumab Previously treated MSI-H/dMMR metastatic July 2018 colorectal cancer – in combination with nivolumab PD-1 Nivolumab Metastatic melanoma December 2014 April 2015 Advanced or metastatic NSCLC March 2015 April 2016 Metastatic renal cell carcinoma November 2015 April 2016 Hodgkin’s lymphoma May 2016 November 2016 Head and neck cancer November 2016 April 2017 Locally advanced or metastatic urothelial February 2017 June 2017 carcinoma MSI-H or dMMR metastatic colorectal cancer that August 2017 has progressed following treatment Hepatocellular carcinoma September 2017 Completely resected melanoma with lymph node December 2017 involvement or metastatic disease Intermediate- and poor-risk advanced renal cell April 2018 carcinoma – in combination with ipilimumab Pembrolizumab Advanced melanoma September 2014 July 2015 Advanced NSCLC October 2015 August 2016 Recurrent or metastatic head and neck cancer August 2016 Hodgkin’s lymphoma March 2017 March 2017 First-line combination with pemetrexed May 2017 and carboplatin for patients with metastatic nonsquamous NSCLC, irrespective of PD-L1 expression Advanced or metastatic urothelial carcinoma May 2017 September 2017 Unresectable or metastatic solid tumors having a May 2017 MSI-H or dMMR) Recurrent locally advanced or metastatic gastric or September 2017 gastroesophageal junction cancer whose tumors express PD-L1 Recurrent or metastatic cervical cancer whose June 2018 tumors express PD-L1

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Supplementary Table 1. Continued Type Target Drug Approved for Approval date - FDA Approval date - EMA Immune checkpoint inhibitors PD-1 Pembrolizumab Refractory or relapsed primary mediastinal large June 2018 B-cell lymphoma (PMBCL) Metastatic nonsquamous NSCLC with no EGFR or August 2018 ALK genomic tumor aberrations PD-L1 Atezolizumab Urothelial carcinoma May 2016 September 2017 Metastatic NSCLC October 2016 September 2017 Advanced bladder cancer April 2017 Avelumab MCC March 2017 September 2017 Urothelial carcinoma May 2017 Durvalumab Locally advanced or metastatic urothelial May 2017 carcinoma Stage III unresectable NSCLC February 2018 Bispecific T-cell engagers CD19 and CD3 Blinatumomab Philadelphia chromosome-negative relapsed or December 2014 November 2015 refractory B-cell precursor acute lymphoblastic leukemia Cancer vaccines Vaccination with ex vivo Sipuleucel-T Metastatic, asymptomatic, hormone-refractory April 2010 Approved September generated DCs prostate cancer 2013 Withdrawal 2015 Oncolytic virus therapy Genetically engineered Talimogene laherparepvec Melanoma October 2015 December 2015 herpes virus (T-Vec) CAR T-cell therapy CD19 Tisagenlecleucel Acute lymphoblastic leukemia Augustus 2017 August 2018 Large B-cell lymphoma May 2018 Axicabtagene ciloleucel Relapsed or refractory large B-cell October 2017 August 2018 lymphoma Abbreviations: CAR: chimeric antigen receptor; NSCLC: non-small cell lung cancer; MSI-H: microsatellite instability-high; dMMR: mismatch repair deficient; MCC: Merkel cell carcinoma.

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Supplementary Table 1. Continued Type Target Drug Approved for Approval date - FDA Approval date - EMA Immune checkpoint inhibitors PD-1 Pembrolizumab Refractory or relapsed primary mediastinal large June 2018 B-cell lymphoma (PMBCL) 2 Metastatic nonsquamous NSCLC with no EGFR or August 2018 ALK genomic tumor aberrations PD-L1 Atezolizumab Urothelial carcinoma May 2016 September 2017 Metastatic NSCLC October 2016 September 2017 Advanced bladder cancer April 2017 Avelumab MCC March 2017 September 2017 Urothelial carcinoma May 2017 Durvalumab Locally advanced or metastatic urothelial May 2017 carcinoma Stage III unresectable NSCLC February 2018 Bispecific T-cell engagers CD19 and CD3 Blinatumomab Philadelphia chromosome-negative relapsed or December 2014 November 2015 refractory B-cell precursor acute lymphoblastic leukemia Cancer vaccines Vaccination with ex vivo Sipuleucel-T Metastatic, asymptomatic, hormone-refractory April 2010 Approved September generated DCs prostate cancer 2013 Withdrawal 2015 Oncolytic virus therapy Genetically engineered Talimogene laherparepvec Melanoma October 2015 December 2015 herpes virus (T-Vec) CAR T-cell therapy CD19 Tisagenlecleucel Acute lymphoblastic leukemia Augustus 2017 August 2018 Large B-cell lymphoma May 2018 Axicabtagene ciloleucel Relapsed or refractory large B-cell October 2017 August 2018 lymphoma Abbreviations: CAR: chimeric antigen receptor; NSCLC: non-small cell lung cancer; MSI-H: microsatellite instability-high; dMMR: mismatch repair deficient; MCC: Merkel cell carcinoma.

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Supplementary Table 2. Tracers for direct ex vivo labeling of immune cells Cell type Method Tracers Preclinical/clinical Application/results Suppl Ref Leukocytes general Direct ex vivo 99mTc-HMPAO, 111In-oxine, 18F-FDG, 64Cu-PTSM Preclinical and clinical studies. Most Infection and inflammation imaging 136-140 used 99mTc-HMPAO and 111In-oxine Granulocytes Direct ex vivo 111In-labeled neutrophils, 99mTc-labeled eosinophils Preclinical and clinical studies Detect neutrophil or eosinophil infiltration in 141,142 inflammatory diseases T-cells Direct ex vivo 99mTc-HMPAO, 111In-oxine, 18F-FDG, 64Cu-PTSM Preclinical • Detect accumulation of 111In-oxine labeled 143-148 CD8+ T-cells into tumor site. • High uptake of 111In-oxine labeled CD4+ T-cells in liver and spleen compared to other organs. • Detect 99mTc-HMPAO labeled T-cells after DC vaccination. Specific T-cell Direct ex vivo 64Cu-labeled cOVA-TCR–specific mAbs 64( Cu-TCR Preclinical/mice • 64Cu-TCR mAbs internalized in T-cells in 149 receptors mAbs) vitro < 24 hours • 64Cu-TCR detected T-cells NK-cells Direct ex vivo 111In-oxine, 18F-FDG and 11C methyl iodide Preclinical and clinical studies Infection and inflammation imaging 150-152

Abbreviations: 99mTc-HMPAO: 99mTc-hexamethyl propylene amine oxime; 18F-FDG: 18F-fluorodeoxyglucose; 64Cu-PTSM: 64Cu-pyruvaldehyde-bis(N4-methylthiosemicarbazone); cOVA: chicken ovalbumin; TCR: T-cell receptor.

Supplementary Table 3. Tracers to image granulocytes Cell type Method Tracers Preclinical/clinical Application/results Suppl Ref Granulocytes general Direct in vivo 99mTc labeled sulesomab (anti-NCA-90), 99mTc labeled Clinical Infection and inflammation imaging 153-155 besilesomab (anti-NCA-95) 99mTc-fanolesomab (anti-CD15) Clinical use suspended Imaging CD15 expression on activated 156 neutrophils in inflammatory diseases FPR targeting imaging tracers Preclinical and clinical studies Infection and inflammation imaging 157-159 111In-DPC11870 (anti-leukotriene B4 antibody) Preclinical studies Imaging leukotriene B4 160-162

Radiolabeled chemotactic factors; 99mTc-NAP2 Preclinical and clinical studies Detection neutrophil activity in inflammation 163-168 (CXCL-7), 99mTc-IL8 (CXCL-8) and infection Abbreviations: NCA-90: nonspecific crossreacting antigen; FPR: formyl peptide receptor; NAP2: neutrophil-activating peptide-2; CXCL: chemokine (C-X-C motif) ligand; IL8: interleukin-8.

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Supplementary Table 2. Tracers for direct ex vivo labeling of immune cells Cell type Method Tracers Preclinical/clinical Application/results Suppl Ref Leukocytes general Direct ex vivo 99mTc-HMPAO, 111In-oxine, 18F-FDG, 64Cu-PTSM Preclinical and clinical studies. Most Infection and inflammation imaging 136-140 used 99mTc-HMPAO and 111In-oxine 2 Granulocytes Direct ex vivo 111In-labeled neutrophils, 99mTc-labeled eosinophils Preclinical and clinical studies Detect neutrophil or eosinophil infiltration in 141,142 inflammatory diseases T-cells Direct ex vivo 99mTc-HMPAO, 111In-oxine, 18F-FDG, 64Cu-PTSM Preclinical • Detect accumulation of 111In-oxine labeled 143-148 CD8+ T-cells into tumor site. • High uptake of 111In-oxine labeled CD4+ T-cells in liver and spleen compared to other organs. • Detect 99mTc-HMPAO labeled T-cells after DC vaccination. Specific T-cell Direct ex vivo 64Cu-labeled cOVA-TCR–specific mAbs 64( Cu-TCR Preclinical/mice • 64Cu-TCR mAbs internalized in T-cells in 149 receptors mAbs) vitro < 24 hours • 64Cu-TCR detected T-cells NK-cells Direct ex vivo 111In-oxine, 18F-FDG and 11C methyl iodide Preclinical and clinical studies Infection and inflammation imaging 150-152

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Supplementary Table 4. Tracers to image macrophages Cell type Target Method Tracer examples Preclinical/clinical Application/results Suppl Ref Macrophages Somatostatin Direct in vivo 68Ga-DOTATE, 68Ga-DOTATOC, 68Ga- Clinical studies Detect inflammatory lesions in acute myocardial 169-171 receptor DOTANOC infarction, acute peri-/myocarditis, idiopathic pulmonary fibrosis, NETs

TSPO Direct in vivo 11C-PK11195, 18F-DPA-714, 11C-PBR28, Preclinical and clinical studies Infection and inflammation imaging 172-176 18F-PBR111, 18F-FEDAC, 125I-CLINDE

Sialoadhesin Direct in vivo 99mTc-SER-4 Preclinical studies In heart transplantation models 99mTc-SER-4 levels in 177 allogeneic grafts > syngeneic grafts

Macrophage Direct in vivo 99mTc-labeled nanobodies, Preclinical studies Detected inflammatory cells in rheumatoid arthritis 178-180 mannose 18F-labeled single-domain antibody model, tumor-promoting macrophages tumor receptor fragments (e.g. 18F-FB-anti-MMR stroma of tumor models and macrophages in 3.49 sdAb) atherosclerotic plaques

F4/80 receptor Direct in vivo 111In-anti-F4/80-A3-1 Preclinical studies Uptake in tissues infiltrated by macrophages in 181 tumor and rheumatoid arthritis models Folic acid Direct in vivo 68Ga–DOTA–PEG–FA Preclinical studies Implant-associated macrophages and associated 182 receptor foreign body reactions Phagocytosis by Direct in vivo 64Cu-TNP Preclinical studies Uptake by macrophages in atherosclerotic plaques 183 macrophages Abbreviations: DOTA: 1,4,7,10-tetraazacyclododecane- N,N’,N″,N’″-tetraacetic acid; 68Ga-DOTATE: 68Ga- DOTA-d-Phe1,Tyr3-octreotate; 68Ga-DOTATOC: 68Ga-DOTA-d-Phe1,Tyr3-octreotide; 68Ga-DOTANOC: 68Ga-DOTA-1-Nal3-octreotide; NETs: neuroendocrine tumors; TSPO: translocator protein; DPA-714: N,N-diethyl-2-[4-(2-fluoroethoxy)phenyl]-5,7-dimethylpyrazolo[1,5-a]pyrimidine-3-acetamide; PK11195: N-butan-2-yl-1-(2-chlorophenyl)-N-methylisoquinoline-3-carboxamide; PBR: peripheral benzodiazepine receptor; 18F-FEDAC: N-benzyl-N-methyl-2-[7,8-dihydro-7-(2-[18F]fluoroethyl)-8-oxo-2-phenyl-9H-purin- 9-yl]acetamide; CLINDE: 6-chloro-2-(4’iodophenyl)-3-(N,N-diethyl)-imidazo[1,2-a]pyridine-3-acetamide; SER: sheep erythrocyte receptor; MMR: macrophage mannose receptor; PEG: polyethylene glycol; FA: folic acid; 64Cu-TNP: 64Cu-labeled trireporter nanoparticle.

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TSPO Direct in vivo 11C-PK11195, 18F-DPA-714, 11C-PBR28, Preclinical and clinical studies Infection and inflammation imaging 172-176 18F-PBR111, 18F-FEDAC, 125I-CLINDE

Sialoadhesin Direct in vivo 99mTc-SER-4 Preclinical studies In heart transplantation models 99mTc-SER-4 levels in 177 allogeneic grafts > syngeneic grafts

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