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Targeted radionuclide therapy (TRT) using probes labeled with Lutetium-177 (177Lu) represents a new and growing type of cancer therapy. We studied immunologic changes in response to TRT with 177Lu labeled anti-human CD20 camelid single domain antibodies (sdAb) in a B16-melanoma model transfected to express human CD20, the target antigen, and ovalbumin, a surrogate tumor antigen. High-dose TRT induced melanoma cell death, calreticulin exposure, and ATP-release in vitro. Melanoma-bearing mice received fractionated low and high-dose TRT via tumor targeting anti-human CD20 sdAbs, as opposed to control sdAbs. Tumor growth was delayed with both doses. Low- and high-dose TRT increased IL10 serum levels. Low-dose TRT also decreased CCL5 serum levels. At the tumor, high-dose TRT induced a type I IFN gene signature, while low-dose TRT induced a proinflammatory gene signature. Low- and high-dose TRT increased the percentage of PD-L1pos and PD-L2pos myeloid cells in tumors with a marked increase in alternatively activated macrophages after high-dose TRT. The percentage of tumor-infiltrating T cells was not changed, yet a modest increase in ovalbumin-specific CD8pos T-cells was observed after low-dose TRT. Contradictory, low and high-dose TRT decreased CD4pos Th1 cells in addition to double negative T cells. In conclusion, these data suggest that low and high-dose TRT induce distinct immunologic changes, which might serve as an anchoring point for combination therapy.

To this day, multiple myeloma remains an incurable cancer. For many patients, recurrence is unavoidably a result of lacking treatment options in the minimal residual disease stage. This is due to residual and treatment-resistant myeloma cells that can cause disease relapse. However, patient-specific membrane-expressed paraproteins could hold the key to target these residual cells responsible for disease recurrence. Here, we describe the therapeutic potential of radiolabeled, anti-idiotypic camelid single-domain antibody fragments (sdAbs) as tumor-restrictive vehicles against a membrane-bound paraprotein in the syngeneic mouse 5T33 myeloma model and analogously assess the feasibility of sdAb-based personalized medicine for patients with multiple myeloma. Llamas were immunized using extracts containing paraprotein from either murine or human sera, and selective sdAbs were retrieved using competitive phage display selections of immune libraries. An anti-5T33 idiotype sdAb was selected for targeted radionuclide therapy with the β−-particle emitter 177Lu and the α-particle emitter 225Ac. sdAb-based radionuclide therapy in syngeneic mice with a low 5T33 myeloma lesion load significantly delayed tumor progression. In five of seven patients with newly diagnosed myeloma, membrane expression of the paraprotein was confirmed. Starting from serum-isolated paraprotein, for two of three selected patients anti-idiotype sdAbs were successfully generated.

Supplementary Data

IntroductionImmune checkpoint therapy (ICT) has recently changed the treatment landscape of multiple cancers. However, the highly dynamic tumor environment has resulted in intrinsic and acquired resistance against current ICTs in many patients. Therefore, ICTs against next-generation immune checkpoints, including TIGIT, are being developed. To better predict the treatment outcome of these ICTs, patient stratification using reliable non-invasive diagnostic methods are required. Small antigen binding moieties such as nanobodies (Nbs), are promising candidates for such tracer development. MethodsLlamas were immunized with recombinant TIGIT proteins, phage-display libraries were constructed and screened for TIGIT specific Nbs. Binding to TIGIT was confirmed by flow cytometry (FC) and ELISA. The affinity of the produced and purified Nbs was determined using surface plasmon resonance (SPR) and FC. Thermostability was assessed by thermofluor assays. Radiochemical purity (RCP) after 99mTc labeling was assessed by iTLC. The 99mTc-Nbs were injected intravenously (i.v.) in naive C57BL/6 mice, in TIGIT overexpressing (TIGIT+) - or wild type (WT) - TC-1 tumor xenografted nude mice and in mice lacking mouse TIGIT. SPECT/CT imaging was performed 1h post-injection (p.i.), followed by ex vivo biodistribution analysis by gamma counting. Results/DiscussionWe obtained 154 different clones after ELISA screening. Three Nbs with sub-nanomolar binding affinity to mouse but not human TIGIT were produced recombinantly and demonstrated binding to TIGIT expressing CD3+ splenocytes (Fig. 1a,b). The Nbs were evaluated for their potential as a non-invasive imaging tracer upon 99mTc labeling. All 99mTc-Nbs exhibited >98% RCP. Biodistribution data of naive C57BL/6 mice showed significant higher uptake of 99mTc-Nbs in different organs (e.g., spleen, lymph nodes, intestines) compared to mice lacking mouse TIGIT (Fig. 2a). Expected high uptake is seen in the kidneys and the bladder due to renal clearance. In tumor bearing mice, uptake (%IA/g ± SD) of Nb16988 was significantly higher in the TIGIT+ tumor (4.317 ± 1.021) compared to the WT tumor (0.709 ± 0.237) and to the uptake of a control Nb R3B23 in both tumors (0.282 ± 0.065) (Fig. 2b,c). All Nbs showed significant higher TIGIT+-to-WT-tumor ratio compared to the control Nb (Fig. 2d). ConclusionsThis is the first report to non-invasively detect mouse TIGIT in vivo upon same-day nuclear imaging with radiolabeled Nbs, with Nb16988 as lead supported by the high signal-to-noise ratios. Ongoing experiments with Nb 68Ga or 18F labeling and the development of a human TIGIT specific PET tracer hold promise toward phenotyping tumors in patients who would be eligible for TIGIT-targeted therapies. AcknowledgementThe authors would like to thank Petra Roman and Elsy Vaeremans for the production of the plasmids and Jan De Jonge for his support with the purification of the Nbs.

Lyophilization is commonly used in the production of pharmaceutical compounds to increase the stability of the Active Pharmaceutical Ingredient (API) by removing solvents. This study investigates the possibility to lyophilize an anti-HER2 and an anti-MMR single-domain antibody fragment (sdAb)-based precursor as a first step in the development of a diagnostic kit for PET imaging. Methods NOTA-sdAb precursors have been lyophilized with the following formulation: 100 µg NOTA-sdAb in 0.1 M NaOAc (NaOAc), 5% (w/v%) mannitol-sucrose mix at a 2:1 ratio and 0.1 mg/mL polysorbate 80. During development of the formulation and drying cycle, factors such as cake appearance, glass transition temperature and residual moisture were analyzed to ensure qualitative and stable lyophilized samples. Stability studies of lyophilized precursor were conducted up to 18 months after storage at 2–8 °C by evaluating the precursor integrity, aggregation, functionality and 68Ga-labeling efficiency. A comparative biodistribution study (lyophilized vs non-lyophilized precursor) was conducted in wild type mice (n = 3) and in tumor bearing mice (n = 6). Results The lyophilized NOTA-anti-HER2 precursor shows consistent stability data in vitro for up to 12 months at 2–8 °C in three separate batches, with results indicating stability even for up to T18m. No aggregation, degradation or activity loss was observed. Radiochemical purity after 68Ga-labeling is consistent over a period of 12 months (RCP ≥ 95% at T12m). In vivo biodistribution analyses show a typical [68Ga]Ga-NOTA-anti-HER2 sdAb distribution profile and a comparable tumor uptake for the lyophilized compound vs non-lyophilized (5.5% vs 5.7 %IA/g, respectively). In vitro results of lyophilized NOTA-anti-MMR precursor indicates stability for up to 18 months, while in vivo data show a comparable tumor uptake (2.5% vs 2.8 %IA/g, respectively) and no significant difference in kidney retention (49.4% vs 47.5 %IA/g, respectively). Conclusion A formulation and specific freeze-drying cycle were successfully developed to lyophilize NOTA-sdAb precursors for long-term storage at 2–8 °C. In vivo data show no negative impact of the lyophilization process on the in vivo behavior or functionality of the lyophilized precursor. These results highlight the potential to develop a kit for the preparation of 68Ga-sdAb-based radiopharmaceuticals.

There are multiple imaging modalities in primary hyperparathyroidism. Ultrasound examination and subtraction scintigraphy are usually the first-line imaging techniques. When these results are negative or inconsistent, additional [11C]-methionine PET/CT (MET-PET/CT) or 4-dimensional computed tomography can be performed. ​This study aims to evaluate MET-PET/CT in comparison with other imaging techniques in primary hyperparathyroidism. This is a retrospective cohort study. Eighty-four patients with primary hyperparathyroidism, who underwent parathyroid surgery, were included. ​Imaging results have been correlated to the perioperative drop in parathyroid hormone level and to the pathological analysis. ​Descriptive statistics are used, supplemented with 95% Clopper–Pearson confidence intervals for sensitivity and specificity and a sub-analysis with the McNemar test on paired data only. The per-lesion sensitivity of MET-PET/CT seems higher than that of [99mTc]-sestamibi or [99mTc]-tetrofosmin and [99mTc]-pertechnetate subtraction scintigraphy. The McNemar test, on paired data only, shows significantly higher sensitivity of MET-PET/CT compared to ultrasound (p=0.039) and significantly higher specificity of ultrasound compared to subtraction scintigraphy (p=0.035).​ MET-PET/CT after inconclusive or negative ultrasound and/or subtraction scintigraphy has an additional value in 70% of the cases.​ Preoperative parathyroid hormone levels were higher in patients in whom MET-PET/CT correctly predicted the pathological parathyroid glands, compared to those where MET-PET/CT missed at least one adenoma. The same trend was seen for 4-dimensional computed tomography. In conclusion, MET-PET/CT seems a valuable imaging modality in primary hyperparathyroidism, at least as second line imaging approach, with a higher per-lesion sensitivity than ultrasound in such setting. Especially when ultrasound and/or subtraction scintigraphy are inconclusive or negative, MET-PET/CT directs the surgeon to the correct localization of the parathyroid adenoma.

ObjectivesTumour cells use immune checkpoints, such as Programmed Death Ligand-1 (PD-L1), to escape the anti-tumour immune response by limiting the T-cell activity. Cancer immunotherapy and more specifically Programmed Death-1 (PD-1): PD-L1 checkpoint blockade therapy has proven to be effective against multiple types of cancer. The expression of PD-L1 in the tumour microenvironment has proven to be a positive indicator for the responsiveness to therapy but its assessment is limited by the inherent limitations of immunohistochemistry assays.1 Non-invasive imaging techniques, such as Positron Emission Tomography (PET)-imaging could overcome these limitations as they depict the whole picture of PD-L1 expression within the body. This study aims to develop a radiofluorinated Nanobody (Nb)-based tracer targeting human PD-L1 (hPD-L1) for PET-imaging.MethodsThe anti-hPD-L1 Nb was labelled with N-succinimidyl-4-[18F]fluorobenzoate ([18F]-SFB) prosthetic group. [18F]-SFB is produced using disposable cassettes on an AllinOne® synthesizer module (Trasis) in a 3-step, 1-pot reaction, previously described.2 Radiochemical purity (RCP) was assessed by reverse phase chromatography (RPC) on a diisopropylcyanopropylsilane column. The dried [18F]SFB was incubated with the anti-hPD-L1 Nb (4.34 mg/mL) for 30 min at room temperature in 0.2 M phosphate buffer pH 8.5-8 containing 20% V/V% ethanol. The radiolabelled Nb was purified using a disposable PD-10 size exclusion column (SEC). RCP was assessed by SEC and RPC on a dissopropylcyanopropylsilane column. The affinity and specificity towards PD-L1 were assessed on PD-L1-positive (pos.) or -negative (neg.) 624-MEL cells as control and biodistribution was evaluated in female C57BL/6 mice (N = 3, 6 weeks old). The biodistribution study was compared to previously obtained data from [68Ga]-Ga-NOTA-(hPD-L1) Nb.3Results[18F]-SFB was obtained after the automated production with a RCP > 95% and decay corrected radiochemical yield of 31 ± 0.04% (N = 8), in a procedure time of 48 minutes. [18F]-FB-(hPD-L1) Nb was obtained with > 95% RCP. In vitro characterisation showed that the radiofluorinated Nb retained its affinity (KD= 2.5 nM) (figure 1a) and specificity (2.37 ± 0.01% of total well activity on pos. cells vs. 0.32 ± 0.00% on neg cells, p

PD-1 Immune checkpoint inhibitors, such as Pembrolizumab, can have a durable beneficial therapeutic effect in patients with advanced melanoma. However, not all patients will benefit equally from these therapies, and (potentially life-threatening) immune-related adverse events may occur. In this study, we investigate the value of early response assessment by FDG-PET/CT as a biomarker for predicting survival. We identified all patients with advanced melanoma who were treated with Pembrolizumab in our medical center and underwent a baseline and at least one follow-up FDG-PET/CT. The total metabolic tumor volume (TMTV) was calculated, and the evolution was compared to survival parameters. A total of 77 patients underwent a baseline and at least one follow-up FDG-PET/CT, 36 patients had follow-up imaging within 2–4 months, and 21 patients an FDG-PET/CT 5–6 months after baseline. When the TMTV evolution was categorized into two subgroups (stable/decrease versus increase), an association was found between stability or decrease in TMTV and better PFS and OS. A similar trend, however non-significant, was observed at 5–6 months. The evolution in TMTV as assessed by FDG-PET/CT 2–4 months after treatment initiation is associated with long-term outcomes in patients with advanced melanoma treated with Pembrolizumab.

To this day, multiple myeloma remains an incurable cancer. For many patients, recurrence is unavoidably a result of lacking treatment options in the minimal residual disease stage. This is due to residual and treatment-resistant myeloma cells that can cause disease relapse. However, patient-specific membrane-expressed paraproteins could hold the key to target these residual cells responsible for disease recurrence. Here, we describe the therapeutic potential of radiolabeled, anti-idiotypic camelid single-domain antibody fragments (sdAbs) as tumor-restrictive vehicles against a membrane-bound paraprotein in the syngeneic mouse 5T33 myeloma model and analogously assess the feasibility of sdAb-based personalized medicine for patients with multiple myeloma. Llamas were immunized using extracts containing paraprotein from either murine or human sera, and selective sdAbs were retrieved using competitive phage display selections of immune libraries. An anti-5T33 idiotype sdAb was selected for targeted radionuclide therapy with the β−-particle emitter 177Lu and the α-particle emitter 225Ac. sdAb-based radionuclide therapy in syngeneic mice with a low 5T33 myeloma lesion load significantly delayed tumor progression. In five of seven patients with newly diagnosed myeloma, membrane expression of the paraprotein was confirmed. Starting from serum-isolated paraprotein, for two of three selected patients anti-idiotype sdAbs were successfully generated.

Immune checkpoint blockade (ICB) of the PD-1 pathway revolutionized the survival forecast for advanced non-small cell lung cancer (NSCLC). Yet, the majority of PD-L1+ NSCLC patients are refractory to anti-PD-L1 therapy. Recent observations indicate a pivotal role for the PD-L1+ tumor-infiltrating myeloid cells in therapy failure. As the latter comprise a heterogenous population in the lung tumor microenvironment, we applied an orthotopic Lewis Lung Carcinoma (LLC) model to evaluate 11 different tumor-residing myeloid subsets in response to anti-PD-L1 therapy. While we observed significantly reduced fractions of tumor-infiltrating MHC-IIlow macrophages and monocytes, serological levels of TNF-a restored in lung tumor-bearing mice. Notably, we demonstrated in vivo and in vitro that anti-PD-L1 therapy mediated a monocytespecific production of, and response to TNF-a, further accompanied by their significant upregulation of CD80, VISTA, LAG-3, SIRP-a and TIM-3. Nevertheless, co-blockade of PD-L1 and TNF-a did not reduce LLC tumor growth. A phenomenon that was partly explained by the observation that monocytes and TNF-a play a Janus-faced role in anti-PD-L1 therapy-mediated CTL stimulation. This was endorsed by the observation that monocytes appeared crucial to effectively boost T cell-mediated LLC killing in vitro upon combined PD-L1 with LAG-3 or SIRP-a blockade. Hence, this study enlightens the biomarker potential of lung tumor-infiltrated monocytes to define more effective ICB combination strategies. This research was performed with the financial support of the Research Foundation-Flanders (FWO-V, grant 1515718N, FWO-SBO, grant S004317N and FWO Junior Research, grant G041721N), Wetenschappelijk Fonds Willy Gepts of the UZ Brussel, and Kom op tegen Kanker, the Flemish Cancer Society. RMA, YDV and TE received an FWO-SB fellowship. LDB received an FWO-V fellowship. MK is senior clinical investigator at FWO-V. MIZ was funded by a PhD scholarship from Universidad Pública de Navarra, Pamplona (Spain) and DE is funded by Gobierno de Navarra (project BMED 050-2019), ISCIII (FEDER-PI17/02119, FEDER-PI20/00010) and AECC (PROYE16001ESCO). CG is funded by the Research Council of the Vrije Universiteit Brussel (OZR).

In oncology, 18-fluorodeoxyglucose (This work proposes a fully automated system for lesion detection and segmentation on whole-bodyOn an unseen dataset of 53 PET/CT images, a median F1-score of 0.7500 was achieved with, on average, 1.566 false positive lesions per scan. Metabolically-active tumours were segmented with a median dice score of 0.8493 and absolute volume difference of 0.2986 ml.The proposed fully automated method for the segmentation and detection of metabolically-active lesions on whole-body

The blockade of immune checkpoints (ICPs), such as cytotoxic T lymphocyte associated protein-4 (CTLA-4) and programmed death-1 (PD-1) and its ligand (PD-L1), has propelled the field of immuno-oncology into its current era. Drugs targeting these ICPs have improved clinical outcome in a number of patients with solid and hematological cancers. Nonetheless, some patients have no benefit from these ICP-blocking therapies. This observation has instigated research into alternative pathways that are responsible for the escape of cancer cells from anti-cancer immune responses. From this research, a number of molecules have emerged as promising therapeutic targets, including lymphocyte activating gene-3 (LAG-3), a next-generation ICP. We will review the current knowledge on the biological activity of LAG-3 and linked herewith its expression on activated immune cells. Moreover, we will discuss the prognostic value of LAG-3 and how LAG-3 expression in tumors can be monitored, which is an aspect that is of utmost importance, as the blockade of LAG-3 is actively pursued in clinical trials.

Recent advancements in the field of immune-oncology have led to a significant increase in life expectancy of patients with diverse forms of cancer, such as hematologic malignancies, melanoma and lung cancer. Unfortunately, these encouraging results are not observed in the majority of patients, who remain unresponsive and/or encounter adverse events. Currently, researchers are collecting more insight into the cellular and molecular mechanisms that underlie these variable responses. As an example, the human lymphocyte activation gene-3 (huLAG-3), an inhibitory immune checkpoint receptor, is increasingly studied as a therapeutic target in immune-oncology. Noninvasive molecular imaging of the immune checkpoint programmed death protein-1 (PD-1) or its ligand PD-L1 has shown its value as a strategy to guide and monitor PD-1/PD-L1-targeted immune checkpoint therapy. Yet, radiotracers that allow dynamic, whole body imaging of huLAG-3 expression are not yet described. We here developed single-domain antibodies (sdAbs) that bind huLAG-3 and showed that these sdAbs can image huLAG-3 in tumors, therefore representing promising tools for further development into clinically applicable radiotracers. Supplementary Information The online version contains supplementary material available at 10.1186/s13550-021-00857-9.

Recent advances in the field of immune-oncology led to the discovery of next-generation immune checkpoints (ICPs). Lymphocyte activation gene-3 (LAG-3), being the most widely studied among them, is being explored as a target for the treatment of cancer patients. Several antagonistic anti-LAG-3 antibodies are being developed and are prime candidates for clinical application. Furthermore, validated therapies targeting cytotoxic T-lymphocyte-associated protein-4, programmed cell-death protein-1, or programmed cell-death ligand-1 showed that only subsets of patients respond. This finding highlights the need for better tools for patient selection and monitoring. The potential of molecular imaging to detect ICPs noninvasively in cancer is supported by several preclinical and clinical studies. Here, we report on a single-domain antibody to evaluate whole-body LAG-3 expression in various syngeneic mouse cancer models using nuclear imaging. Methods: SPECT/CT scans of tumor-bearing mice were performed 1 h after injection with radiolabeled single-domain antibody. Organs and tumors of mice were isolated and evaluated for the presence of the radiolabeled tracer and LAG-3-expressing immune cells using a γ-counter and flow cytometry respectively. PD-1/LAG-3-blocking antibodies were injected in MC38-bearing mice. Results: The radiolabeled single-domain antibody detected LAG-3 expression on tumor-infiltrating lymphocytes (TILs) as soon as 1 h after injection in MC38, MO4, and TC-1 cancer models. The single-domain antibody tracer visualized a compensatory upregulation of LAG-3 on TILs in MC38 tumors of mice treated with PD-1-blocking antibodies. When PD-1 blockade was combined with LAG-3 blockade, a synergistic effect on tumor growth delay was observed. Conclusion: These findings consolidate LAG-3 as a next-generation ICP and support the use of single-domain antibodies as tools to noninvasively monitor the dynamic evolution of LAG-3 expression by TILs, which could be exploited to predict therapy outcome.

Monoclonal antibodies that target the inhibitory immune checkpoint axis consisting of programmed cell death protein 1 (PD-1) and its ligand, PD-L1, have changed the immune-oncology field. We identified K2, an anti-human PD-L1 single-domain antibody fragment, that can enhance T cell activation and tumor cell killing. In this study, the potential of different K2 formats as immune checkpoint blocking medicines was evaluated using a gene-based delivery approach. We showed that 2K2 and 3K2, a bivalent and trivalent K2 format generated using a 12 GS (glycine-serine) linker, were 313- and 135-fold more potent in enhancing T cell receptor (TCR) signaling in PD-1POS cells than was monovalent K2. We further showed that bivalent constructs generated using a 30 GS linker or disulfide bond were 169- and 35-fold less potent in enhancing TCR signaling than was 2K2. 2K2 enhanced tumor cell killing in a 3D melanoma model, albeit to a lesser extent than avelumab. Therefore, an immunoglobulin (Ig)G1 antibody-like fusion protein was generated, referred to as K2-Fc. K2-Fc was significantly better than avelumab in enhancing tumor cell killing in the 3D melanoma model. Overall, this study describes K2-based immune checkpoint medicines, and it highlights the benefit of an IgG1 Fc fusion to K2 that gains bivalency, effector functions, and efficacy., Graphical abstract, Awad et al. describe how formatting of a human PD-L1 sdAb impacts the efficiency of immune checkpoint blockade upon lentiviral-mediated sdAb delivery. The authors systematically study how an increase in avidity, choice of linkage, and gain of Fc functions affect the sdAb performance when delivered either genetically or in the protein form.

Introduction:131I-GMIB-Anti-HER2-VHH1 is a targeted radionuclide theranostic agent directed at HER2 expressing cancers. VHH1 is a single domain antibody fragment covalently linked to therapeutic radio-iodine 131I via the linker SGMIB. The Phase I study presented was aimed at evaluating the safety, biodistribution, radiation dosimetry and tumor imaging potential of 131I-GMIB-Anti-HER2-VHH1 in healthy volunteers and breast cancer patients. Methods: In a first cohort, six healthy volunteers were included. The biodistribution of 131I-GMIB-Anti-HER2-VHH1 was assessed using whole body (anterior and posterior) planar images obtained at 40 min., 2, 4, 24 and 72 h following i.v. administered (38 ± 9 MBq) 131I-GMIB-VHH1. Imaging data were analyzed using OLINDA/EXM software 1.0 to determine the dosimetry. Blood and urine samples were obtained over 72h. In the second cohort, three patients with metastatic HER2 positive breast cancer were included. Planar whole-body imaging was performed at 2 h and 24 h after injection. Additional SPECT/CT images were obtained following the whole body images at 2 and 24 h in case of relevant uptake in known cancer lesions Results: No drug related adverse events (AEs) were observed throughout the study. The biological half-life of 131I-GMIB-Anti-HER2-VHH1 in healthy subjects was about 8 h. After i.v. administration, the compound is eliminated from the blood with a 2.5 h half-life. The drug is primarily eliminated via the kidneys. The drug was stable in circulation and there was no increased accumulation in thyroid or stomach. The absorbed dose to the kidneys was 1.54 ± 0.25 mGy/MBq, while to bone marrow 0.03 ± 0.01 mGy/MBq. SPECT/CT imaging in patients with advanced breast cancer showed focal uptake of 131I-GMIB-Anti-HER2-VHH1 in metastatic lesions. Conclusion: No AEs were observed after iv administration of 131I-GMIB-Anti-HER2-VHH1 at low activity. Unbound drug is rapidly eliminated via the kidneys. In patients with stage IV HER2 positive breast cancer accumulation of 131I-GMIB-Anti-HER2-VHH1in metastatic sites was observed. Dosimetry predicts kidneys as the dose limiting organ upon dose escalation, but kidney toxicity should only occur at very high injected activities. Dose escalation is planned in a subsequent phase I/II study to assess the therapeutic window of this compound (NCT04467515).

Background Differentiated thyroid cancer (DTC) is a common malignancy with increasing incidence. Follow-up care for DTC includes thyroglobulin (Tg) measurement and ultrasound (US) of the neck, combined with 131I remnant ablation when indicated. Diagnostic precision has evolved with the introduction of the new high-sensitive Tg-assays (sensitivity ≤0.1 ng/mL). The aim of the study was to determine the prognostic utility of high-sensitive Tg and the need for other diagnostic tests in DTC. Methods This was a retrospective, observational study. Patients with pathologically confirmed DTC, treated with total thyroidectomy and 131I remnant ablation, who had their complete follow-up care in our institution were selected (October 2013–December 2018). Subjects with possible thyroglobulin autoantibody interference were excluded. Statistical analysis was performed using the IBM SPSS® Statistics 24 software package. Results Forty patients were eligible for analysis. A total of 24 out of the 40 patients (60%) had an undetectable high-sensitive Tg 6 months after total thyroidectomy. None of these patients had a stimulated Tg above 1 ng/mL, or remnant on the 123I Whole-Body Scan (WBS) after 1 year of follow-up. Ultrasound of the neck, performed between 6 and 12 months postoperative, was negative in 21 out of the 24 patients. Conclusions This study shows that an undetectable high-sensitive Tg can change the management of patients with DTC and decrease the use and need of stimulated Tg and 123I WBS.

During the preparation of [68Ga]Ga-NOTA-sdAb at high activity, degradation of the tracers was observed, impacting the radiochemical purity (RCP). Increasing starting activities in radiolabelings is often paired with increased degradation of the tracer due to the formation of free radical species, a process known as radiolysis. Radical scavengers and antioxidants can act as radioprotectant due to their fast interaction with formed radicals and can therefore reduce the degree of radiolysis. This study aims to optimize a formulation to prevent radiolysis during the labeling of NOTA derivatized single domain antibody (sdAbs) with 68Ga. Gentisic acid, ascorbic acid, ethanol and polyvinylpyrrolidone were tested individually or in combination to find an optimal mix able to prevent radiolysis without adversely influencing the radiochemical purity (RCP) or the functionality of the tracer. RCP and degree of radiolysis were assessed via thin layer chromatography and size exclusion chromatography for up to three hours after radiolabeling. Individually, the radioprotectants showed insufficient efficacy in reducing radiolysis when using high activities of 68Ga, while being limited in amount due to negative impact on radiolabeling of the tracer. A combination of 20% ethanol (VEtOH/VBuffer%) and 5 mg ascorbic acid proved successful in preventing radiolysis during labeling with starting activities up to 1–1.2 GBq of 68Ga, and is able to keep the tracer stable for up to at least 3 h after labeling at room temperature. The prevention of radiolysis by the combination of ethanol and ascorbic acid potentially allows radiolabeling compatibility of NOTA-sdAbs with all currently available 68Ge/68Ga generators. Additionally, a design is proposed to allow the incorporation of the radioprotectant in an ongoing diagnostic kit development for 68Ga labeling of NOTA-sdAbs.

Lyophilization is commonly used in the production of pharmaceutical compounds to increase the stability of the Active Pharmaceutical Ingredient (API) by removing solvents. This study investigates the possibility to lyophilize an anti-HER2 and an anti-MMR single-domain antibody fragment (sdAb)-based precursor as a first step in the development of a diagnostic kit for PET imaging.NOTA-sdAb precursors have been lyophilized with the following formulation: 100 µg NOTA-sdAb in 0.1 M NaOAc (NaOAc), 5% (w/v%) mannitol-sucrose mix at a 2:1 ratio and 0.1 mg/mL polysorbate 80. During development of the formulation and drying cycle, factors such as cake appearance, glass transition temperature and residual moisture were analyzed to ensure qualitative and stable lyophilized samples. Stability studies of lyophilized precursor were conducted up to 18 months after storage at 2-8 °C by evaluating the precursor integrity, aggregation, functionality andThe lyophilized NOTA-anti-HER2 precursor shows consistent stability data in vitro for up to 12 months at 2-8 °C in three separate batches, with results indicating stability even for up to T18m. No aggregation, degradation or activity loss was observed. Radiochemical purity afterA formulation and specific freeze-drying cycle were successfully developed to lyophilize NOTA-sdAb precursors for long-term storage at 2-8 °C. In vivo data show no negative impact of the lyophilization process on the in vivo behavior or functionality of the lyophilized precursor. These results highlight the potential to develop a kit for the preparation of

Background: Pembrolizumab improves the survival of patients with advanced melanoma. A comprehensive analysis of baseline variables that predict the benefit of pembrolizumab monotherapy has not been conducted. Methods: Survival data of patients with advanced melanoma who were treated with pembrolizumab in a single university hospital were collected. A multivariate Cox regression analysis was performed to correlate baseline clinical, laboratory, and radiologic characteristics and NanoString IO360 gene expression profiling (GEP) with survival. Results: 183 patients were included (stage IV 85.2%, WHO performance status &ge, 1 31.1%, pembrolizumab first-line 25.7%), of whom 112 underwent baseline 18F-FDG-PET/CT imaging, 58 had circulating tumor DNA (ctDNA) assessments, and GEP was available in 27 patients. Active brain metastases, a higher number of metastatic sites, lower albumin and absolute lymphocyte count (ALC), higher C-reactive protein (CRP) and neutrophil-to-lymphocyte ratio, higher total metabolic tumor volume (TMTV), and higher ctDNA levels were associated with worse survival. Elevated lactate dehydrogenase (LDH) &ge, 2ULN (upper limit of normal), CRP &ge, 10ULN, or ALC &lt, 750/mm3 delineate a subpopulation where treatment with pembrolizumab is futile. A TMTV &ge, 80 mL encompassed 17/21 patients with LDH &ge, 2ULN, CRP &ge, 750/mm3. No significant associations were observed between baseline GEP scores and survival. Conclusion: Multiple baseline variables correlate with survival on pembrolizumab. TMTV is a more comprehensive baseline biomarker than CRP, LDH, or ALC in predicting the futility of pembrolizumab.

Simple Summary The role of innate immune cells in the tumor microenvironment (TME), more specifically the presence of the tumor associated macrophages (TAMs), is becoming more important in the prognosis and treatment of patients diagnosed with malignancies. The aim of this systematic review and meta-analysis was to assess the potential prognostic value of CD206-expressing TAMs, a subclass of macrophages, which were previously proposed to negatively impact the patient’s prognosis. We identified 27 manuscripts describing the role of CD206 in patient prognosis for 14 different tumor types. Despite a large heterogeneity in the results, we identified a significantly worse overall and disease-free survival for patients with increased CD206-expressing TAMs in the TME. The use of CD206-expressing TAMs could therefore be used as a prognostic marker in patients diagnosed with solid malignancies. Abstract An increased presence of CD206-expressing tumor associated macrophages in solid cancers was proposed to be associated with worse outcomes in multiple types of malignancies, but contradictory results are published. We performed a reproducible systematic review and meta-analysis to provide increased evidence to confirm or reject this hypothesis following the Preferred Reporting Items for Systematic Reviews and Meta-analyses statement. The Embase, Web of Science, and MEDLINE-databases were systematically searched for eligible manuscripts. A total of 27 papers studying the prognostic impact of CD206 in 14 different tumor types were identified. Meta-analyses showed a significant impact on the overall survival (OS) and disease-free survival (DFS). While no significant differences were revealed in progression-free survival (PFS) and disease-specific survival (DSS), a shift towards negative survival was correlated with increased CD206-expresion. As a result of the different tumor types, large heterogeneity was present between the different tumor types. Subgroup analysis of hepatocellular carcinoma and gastric cancers revealed no heterogeneity, associated with a significant negative impact on OS in both groups. The current systematic review displays the increased presence CD206-expressing macrophages as a significant negative prognostic biomarker for both OS and DFS in patients diagnosed with solid cancers. Because a heterogenous group of tumor types was included in the meta-analysis, the results cannot be generalized. These results can, however, be used to further lead follow-up research to validate the specific prognostic value of CD206 in individual tumor types and therapeutic approaches.

Background: Human epidermal growth factor receptor 2 (HER2) status is an important predictive biomarker in breast cancer (BC). Tumor heterogeneity has been described, with changes in HER2 expression levels between lesions and over the disease course. HER2 expression is assessed on tissue biopsies, at primary diagnosis and in metastatic lesions. A whole-body imaging technique such as PET/CT could help understand expression levels in different lesions. A 68Ga-labeled single domain antibody (sdAb) targeting the HER2 receptor has been developed and proven safe (Keyaerts et al., 2016). Imaging is performed at 90 min post-injection (pi). We report results of a phase II trial to assess the repeatability of the technique in 20 patients and the correlation of tracer uptake with HER2 tissue expression of the lesions present at the time of imaging. Methods: Twenty patients (pts) with a locally advanced or metastatic BC with at least one lesion of minimum 12 mm were included. Pts were injected intravenously with a typical protein mass of 100 µg and a radioactive dose ranging from 98-168 MBq 68GaNOTA-anti-HER2 sdAb. PET/CT images were obtained at 90 min pi. A second tracer injection followed by PET/CT was done with a maximal interval of 8 days. To assess repeatability, up to 5 lesions per pt were selected, with no more than 2 in a single organ. Peak Standard Uptake Values (SUVpeak) of the lesions were measured on both scans and compared with a t-test and Bland-Altman Plots. Images were compared to other available medical or imaging data and interpreted considering the subject’s disease course. Serum and plasma samples were collected before injection and between 60 and 365 days pi and stored for future detection of anti-drug antibodies (ADA) and liquid biopsies analysis for the presence of HER2 amplification. Tissue samples were assessed by central labs using mass spectrometry, immunohistochemistry and in fluorescence situ hybridization. Results: Twenty women with BC (6 HER2+, 14 HER2-) with a mean age of 58.6 y (37-81) were included. Three pts were scanned only once (2 due to withdrawal of consent, 1 due to covid pandemic). Repeatability of the technique was visually scored as excellent. For quantification, 50 lesions were compared on both scans in 17 pts without significant differences between the two measurements (p=0.40). The repeatability coefficient (RC) was 38.2%. The mean absolute percentage difference (MAPD) was 13.6%, comparable to repeat values reported for 18F-FDG. In 3 out of 6 HER2-positive (HER2+) patients, lesions showed high uptake, even better visible than using 18F-FDG in 2 of them. In 2 HER2+ subjects with a negative scan, lesions were confirmed to be true negatives: one patient did not relapse from BC but had tuberculosis; the other was confirmed to have a radiopneumonitis after radiotherapy and no relapse. In 1 HER2+ patient, the uptake was unexpectedly low. However, the HER2 status was also not reconfirmed in the metastatic setting for this subject. In 1 HER2-negative patient, the tumor HER2 status was changed from negative to positive based on a subsequent image-guided biopsy performed in this study. High tracer uptake was also seen in many of the patients presenting with HER2-low BC (IHC 1+ or 2+), indicating the potential of the tracer to detect low-level HER2 expression. Additional correlation to centrally performed tissue and blood analysis is ongoing. Conclusion: 68GaNOTA-Anti-HER2 PET/CT shows high uptake in HER2-expressing BC lesions but also in HER2-low lesions. The technique shows good repeatability and, in some cases, even better sensitivity than 18F-FDG PET/CT. Specificity was confirmed in relapse-free lesions such as tuberculosis and radiopneumonitis. Its sensitivity makes it a promising technique to assess HER2+ and HER2-low lesions in BC patients. Citation Format: Odrade Gondry, Catarina Xavier, Wim Waelput, Omar Al Dabssi, Marian Vanhoeij, Sandrine Aspeslagh, Sofie Joris, Christel Fontaine, Guy Verfaillie, Jacques De Grève, Katrien Glorieus, Ine Luyten, Frederik Vandenbroucke, Sophie Bourgeois, Laurens Raes, Sheeno Thyparambil, Nick Devoogdt, Ilse Vaneycken, Julie Cousaert, Vicky Caveliers, Hendrik Everaert, Tony Lahoutte, Marleen Keyaerts. Assessment of repeatability and uptake quantification of 68GaNOTA-anti-HER2 sdAb PET/CT in patients with locally advanced or metastatic breast cancer [abstract]. In: Proceedings of the 2021 San Antonio Breast Cancer Symposium; 2021 Dec 7-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2022;82(4 Suppl):Abstract nr P3-02-05.

Immune checkpoints, such as programmed death-ligand 1 (PD-L1), limit T-cell function and tumor cells use this ligand to escape the anti-tumor immune response. Treatments with monoclonal antibodies blocking these checkpoints have shown long-lasting responses, but only in a subset of patients. This study aims to develop a Nanobody (Nb)-based probe in order to assess human PD-L1 (hPD-L1) expression using positron emission tomography imaging, and to compare the influence of two different radiolabeling strategies, since the Nb has a lysine in its complementarity determining region (CDR), which may impact its affinity upon functionalization. The Nb has been conjugated with the NOTA chelator site-specifically via the Sortase-A enzyme or randomly on its lysines. [68Ga]Ga-NOTA-(hPD-L1) Nbs were obtained in &gt, 95% radiochemical purity. In vivo tumor targeting studies at 1 h 20 post-injection revealed specific tumor uptake of 1.89 &plusmn, 0.40%IA/g for the site-specific conjugate, 1.77 &plusmn, 0.29%IA/g for the random conjugate, no nonspecific organ targeting, and excretion via the kidneys and bladder. Both strategies allowed for easily obtaining 68Ga-labeled hPD-L1 Nbs in high yields. The two conjugates were stable and showed excellent in vivo targeting. Moreover, we proved that the random lysine-conjugation is a valid strategy for clinical translation of the hPD-L1 Nb, despite the lysine present in the CDR.

Background Immune checkpoint blockade such as ipilimumab and anti-PD1 monoclonal antibodies have significantly improved survival in advanced melanoma. Biomarkers are urgently needed as a majority of patients do not respond, despite treatment-related toxicities. We analysed pre-treatment 18F-fluorodeoxyglucose positron emission tomography/computerised tomography (FDG PET/CT) parameters to assess its correlation with patient outcome. Methods This retrospective study evaluated pre-treatment FDG PET/CT scans in a discovery cohort of patients with advanced melanoma treated with ipilimumab or anti-PD1. Pre-treatment scans were assessed for maximum tumoral standardised uptake value (SUVmax), metabolic tumour volume (MTV) and spleen to liver ratio (SLR). Progression-free survival (PFS) and overall survival (OS) were characterised and modelled using univariable and multivariable analyses. Correlation of SLR and OS was validated in an independent cohort. Blood parameters and stored sera of patients from the discovery cohort was analysed to investigate biological correlates with SLR. Results Of the 90 evaluable patients in the discovery cohort: 50 received ipilimumab monotherapy, 20 received anti-PD1 monotherapy, and 20 patients received ipilimumab followed by anti-PD1 upon disease progression. High SLR > 1.1 was associated with poor PFS (median 1 vs 3 months; HR 3.14, p = 0.008) for patients treated with ipilimumab. High SLR was associated with poor OS after ipilimumab (median 1 vs 21 months; HR 5.83, p = 0.0001); as well as poor OS after first line immunotherapy of either ipilimumab or anti-PD1 (median 1 vs 14 months; HR 3.92, p = 0.003). The association of high SLR and poor OS after ipilimumab was validated in an independent cohort of 110 patients (median 2.3 months versus 11.9 months, HR 3.74). SLR was associated with poor OS in a multi-variable model independent of stage, LDH, absolute lymphocyte count and MTV. Conclusions Pre-treatment Spleen to liver ratio (SLR) > 1.1 was associated with poor outcome after ipilimumab in advanced melanoma. This parameter warrants prospective evaluation.

HER2-targeted therapies have drastically improved the outcome for breast cancer patients. However, when metastasis to the brain is involved, current strategies fail to hold up to the same promise. Camelid single-domain antibody-fragments (sdAbs) have been demonstrated to possess favorable properties for detecting and treating cancerous lesions in vivo using different radiolabeling methods. Here we evaluate the anti-HER2 sdAb 2Rs15d, coupled to diagnostic &gamma, and therapeutic &alpha, and &beta, &minus, emitting radionuclides for the detection and treatment of HER2pos brain lesions in a preclinical setting. 2Rs15d was radiolabeled with 111In, 225Ac and 131I using DTPA- and DOTA-based bifunctional chelators and Sn-precursor of SGMIB respectively and evaluated in orthotopic tumor-bearing athymic nude mice. Therapeutic efficacy as well as systemic toxicity were determined for 131I- and 225Ac-labeled sdAbs and compared to anti-HER2 monoclonal antibody (mAb) trastuzumab in two different HER2pos tumor models. Radiolabeled 2Rs15d showed high and specific tumor uptake in both HER2pos SK-OV-3-Luc-IP1 and HER2pos MDA-MB-231Br brain lesions, whereas radiolabeled trastuzumab was unable to accumulate in intracranial SK-OV-3-Luc-IP1 tumors. Administration of [131I]-2Rs15d and [225Ac]-2Rs15d alone and in combination with trastuzumab showed a significant increase in median survival in 2 tumor models that remained largely unresponsive to trastuzumab treatment alone. Histopathological analysis revealed no significant early toxicity. Radiolabeled sdAbs prove to be promising vehicles for molecular imaging and targeted radionuclide therapy of metastatic lesions in the brain. These data demonstrate the potential of radiolabeled sdAbs as a valuable add-on treatment option for patients with difficult-to-treat HER2pos metastatic cancer.