The Design and Preclinical Evaluation of a Single-Label Bimodal Nanobody Tracer for Image-Guided Surgery

Intraoperative guidance using targeted fluorescent tracers can potentially provide surgeons with real-time feedback on the presence of tumor tissue in resection margins. To overcome the limited depth penetration of fluorescent light, combining fluorescence with SPECT/CT imaging and/or gamma-ray tracing has been proposed. Here, we describe the design and preclinical validation of a novel bimodal nanobody-tracer, labeled using a “multifunctional single attachment point” (MSAP) label, integrating a Cy5 fluorophore and a diethylenetriaminepentaacetic acid (DTPA) chelator into a single structure. After conjugation of the bimodal MSAP to primary amines of the anti-HER2 nanobody 2Rs15d and 111In-labeling of DTPA, the tracer’s characteristics were evaluated in vitro. Subsequently, its biodistribution and tumor targeting were assessed by SPECT/CT and fluorescence imaging over 24 h. Finally, the tracer’s ability to identify small, disseminated tumor lesions was investigated in mice bearing HER2-overexpressing SKOV3.IP1 peritoneal lesions. [111In]In-MSAP.2Rs15d retained its affinity following conjugation and remained stable for 24 h. In vivo SPECT/CT and fluorescence images showed specific uptake in HER2-overexpressing tumors with low background. High tumor-to-muscle ratios were obtained at 1h p.i. and remained 19-fold on SPECT/CT and 3-fold on fluorescence images over 24 h. In the intraperitoneally disseminated model, the tracer allowed detection of larger lesions via nuclear imaging, while fluorescence enabled accurate removal of submillimeter lesions. Bimodal nuclear/fluorescent nanobody-tracers can thus be conveniently designed by conjugation of a single-molecule MSAP-reagent carrying a fluorophore and chelator for radioactive labeling. Such tracers hold promise for clinical applications.


Introduction
Intraoperative guidance with targeted fluorescent contrast agents is an emerging tool to achieve more complete removal of malignant tissue in oncologic surgery due to its unique capability to provide real-time visual feedback to the surgeon about tumor margins and local invasion [1]. Nevertheless, the technique is limited to the detection of superficial lesions or lesions lying at most a centimeter deep in tissue, despite the deeper tissue penetration of fluorescent signals with excitation and emission in the far-red and near-infrared region as compared to a lower wavelength visible light [2]. Therefore, hybrid imaging combining fluorescence imaging with diagnostic nuclear medicine techniques has Figure 1. Schematic representation of the anti-HER2 Nanobody 2Rs15d being randomly labeled on its lysines via NHSchemistry (blue) with the MSAP containing a Cy5 dye (red) and a DTPA-chelator complexed with an Indium-111 ( 111 In) radioisotope (yellow). The MSAP analogue's backbone is displayed in black.

Production and MSAP-Conjugation of Nanobodies
The used HER2-specific Nanobody 2Rs15d has been generated in a previous study [32] and was expressed and purified without a C-terminal hexahistidine-tag [33]. The NHS-ester functionalized MSAP molecule (Figure 1), which consists of a peptide scaffold bearing a trisulphonated Cy5 fluorophore (wavelength of maximal absorption/emission: 646/662 nm [34]) and a pentetic acid (diethylenetriaminepentaacetic acid, DTPA) chelator, was synthetized as previously described [17]. For MSAP conjugation to the primary amines on the Nanobody, 1 mg of 2Rs15d was incubated with a 1.2× molar excess of MSAP in 1 mL of 0.1 M K2HPO4 (Merck KGaA, Darmstadt, Germany) buffer at a final pH of 8.3-8.5. The conjugated Nanobody MSAP.2Rs15d was then purified using size-exclusion chromatography (SEC) on a Superdex 75 10/300GL column (GE Healthcare, Chicago, IL, USA) with 0.1 M NH4OAc (Acros Organics, Fair Lawn, New Yersey) pH 7 as running buffer (0.5 mL/min). Quality control runs of the purified MSAP.2Rs15d were performed on a Superdex 75 5/150GL column with PBS pH 7.4 as running buffer (0.3 mL/min). All buffers were treated with 2 g/L chelex (Merck KGaA) before use.
One MSAP molecule contains exactly one DTPA chelator and one fluorophore, thus we can determine the MSAP degree of labeling (DOL) of the compound using the fluorophore's concentration. Therefore, the DOL was calculated as the ratio of the fluorophore's to the Nanobody's concentration. This was determined by absorbance measurement at 650 and 280 nm, respectively (Nanodrop 2000 spectrophotometer, Thermo Fisher Scientific). To account for the absorbance contribution of the fluorophore and the chelator at 280 nm, the measured absorbance at 280 nm was corrected by subtracting it with 5% of the absorbance value at 650 nm.

Production and MSAP-Conjugation of Nanobodies
The used HER2-specific Nanobody 2Rs15d has been generated in a previous study [32] and was expressed and purified without a C-terminal hexahistidine-tag [33]. The NHSester functionalized MSAP molecule (Figure 1), which consists of a peptide scaffold bearing a trisulphonated Cy5 fluorophore (wavelength of maximal absorption/emission: 646/662 nm [34]) and a pentetic acid (diethylenetriaminepentaacetic acid, DTPA) chelator, was synthetized as previously described [17]. For MSAP conjugation to the primary amines on the Nanobody, 1 mg of 2Rs15d was incubated with a 1.2× molar excess of MSAP in 1 mL of 0.1 M K 2 HPO 4 (Merck KGaA, Darmstadt, Germany) buffer at a final pH of 8.3-8.5. The conjugated Nanobody MSAP.2Rs15d was then purified using size-exclusion chromatography (SEC) on a Superdex 75 10/300GL column (GE Healthcare, Chicago, IL, USA) with 0.1 M NH 4 OAc (Acros Organics, Fair Lawn, NJ, USA) pH 7 as running buffer (0.5 mL/min). Quality control runs of the purified MSAP.2Rs15d were performed on a Superdex 75 5/150GL column with PBS pH 7.4 as running buffer (0.3 mL/min). All buffers were treated with 2 g/L chelex (Merck KGaA) before use.
One MSAP molecule contains exactly one DTPA chelator and one fluorophore, thus we can determine the MSAP degree of labeling (DOL) of the compound using the fluorophore's concentration. Therefore, the DOL was calculated as the ratio of the fluorophore's to the Nanobody's concentration. This was determined by absorbance measurement at 650 and 280 nm, respectively (Nanodrop 2000 spectrophotometer, Thermo Fisher Scientific). To account for the absorbance contribution of the fluorophore and the chelator at 280 nm, the measured absorbance at 280 nm was corrected by subtracting it with 5% of the absorbance value at 650 nm. of 35-500 µL at pH 4.5. The reaction mixture was incubated for 30 min at 50 • C [35]. After radiolabeling, the product was filtered using a 0.2 µm filter (PALL Corporation, Port Washington, NY, USA). Radiochemical purity was determined by instant thin layer chromatography (iTLC) using 0.1 M sodium citrate as mobile phase. Chemical identity of [ 111 In]In-MSAP.2Rs15d (5 µg diluted in PBS with 0.1% Tween ® 80 detergent) was confirmed by analytical SEC on a Superdex 75 5/150GL column using PBS pH 7.4 as running buffer. Radioactivity was detected online using a Gabi detector (Elysia-Raytest, Angleur, Belgium) and was compared to the absorbance measured at 280 (Nanobody) and 650 (Cy5) nm. Stability of the bimodal tracer was furthermore evaluated by SEC after 24 h incubation in PBS at room temperature or in human serum at 37 • C.
The specificity of the compound was further validated by a fluorescence-based cellbinding study, using HER2-overexpressing SKOV3 and HER2-negative control Chinese hamster ovarian (CHO) cells. SKOV3 and CHO cells were seeded in 24-well plates at 100,000 cells/well and incubated at 37 • C and 5% CO 2 for 48 h. MSAP.2Rs15d was added at a final concentration of 1.1 × 10 −8 M to wells with SKOV3 cells, CHO cells, or coincubated on SKOV3 cells with a 100-fold molar excess of unmodified 2Rs15d Nanobody. The cells were then incubated for 2 h at 4 • C, washed and remaining fluorescence in the wells was measured in the 700 nm channel of a flatbed fluorescence scanner (Odyssey, LI-COR Biosciences, Lincoln, Nebraska). For each condition three replicates were included.
To determine the affinity (K D ) of [ 111 In]In-MSAP.2Rs15d after radiolabeling, a saturation cell-binding study was performed using HER2 overexpressing SKOV3 cells. Cells were seeded in 24-well plates at 100,000 cells/well and incubated at 37 • C with 5% CO 2 for 48 h. After 111 In-labeling, a 1/3 dilution series (ranging from 1.0 × 10 −7 M to 4.6 × 10 −11 M, in triplicate) of [ 111 In]In-MSAP.2Rs15d was added to the cells, either with or without a 100× molar excess of unlabeled 2Rs15d. After 1 h of incubation at 4 • C, the wells were washed, and cells were detached using 1 M NaOH. The bound activity was then measured using a gamma counter (Wizard2 2480, Perkin Elmer, Waltham, MA, USA).

In Vivo Biodistribution and Tumor Targeting Potential Using Bimodal Imaging
All animal experiments were approved by the Ethical Committee for Animal Experiments of the Vrije Universiteit Brussel (project nr. 15-272-5). The mice were housed in individually ventilated cages at 19-24 • C in 40-60% humidity with a light/dark cycle of 14/10 h. Low fluorescence food pellets (Teklad 2016, Envigo, Indianapolis, IN, USA) and water were provided ad libitum.

Longitudinal Biodistribution Study in Subcutaneous Xenografts
SKOV3 (10 × 10 6 cells) or HER2-negative MDA-MB-435S (2 × 10 6 cells) subcutaneous tumors were implanted on the right flank of female athymic nude Crl:NU-Foxn1 nu mice (Charles River, n = 3 per group), and grown until the tumors for all animals within a group reached a volume between 100 and 500 mm 3 .
Of [ 111 In]In-MSAP.2Rs15d 7.5 µg (12.3 ± 0.5 MBq, corresponding to 1 nmol MSAP, and apparent molar specific activity of 13.5 ± 0.6 GBq/µmol) was injected via the tail vein of either SKOV3 or MDA-MB-435S xenograft bearing mice. Consecutive single photon emission computed tomography/computed tomography (SPECT/CT) scans were performed at 1, 4, and 24 h post-injection, and after each scan, the same animal was subjected to fluorescence imaging. After the final timepoint, animals were killed by cervical dislocation for further ex vivo biodistribution studies. Fluorescence imaging of individual organs and tissues was performed, whereafter the organs and tissues were weighed, and their radioactive signal measured using a gamma counter (Wizard2 2480, Perkin Elmer). Results were decay-corrected and expressed as percentage of injected dose per cm 3 (%ID/cm 3 ).

Imaging Protocols
During all imaging procedures, for intravenous injections and for cervical dislocation, mice were anaesthetized with isoflurane gas (5% for induction, 2% for maintenance during the scan, 0.5-1.0 mL/min oxygen flow rate). MicroSPECT/CT imaging was performed using a Vector + system (Milabs) equipped with a general-purpose rat/mouse 1.5 mm 75 pinhole collimator. Scans were performed in spiral mode with 6 bed positions and an acquisition time of 200 s per bed position. For image reconstruction, 2 subsets and 4 iterations were used, with a voxel size of 0.4 mm in U-SPECT-Rec software (Milabs). The CT scan was made in 1 bed position, with a duration of 146 s at 60 kV and a pixel size of 80 µm. Further quantitative image analysis was performed with AMIDE software (calculation of percent injected dose per cm 3 (%ID/cm 3 ) in region-of-interests (ROIs)), and 3D images were prepared in Osirix software (Pixmeo, Bernex, Switzerland). Radioactive tumor-to-muscle (TMR rad ) ratios were determined by dividing the tumor's %ID/cm 3 by the muscle's %ID/cm 3 .
Fluorescence imaging was performed using a KIS700 camera (Kaer Labs, Nantes, France), an open surgical system with resolution of 1920 × 1200, excitation wavelength of 640 nm and emission light collection above 665 nm (high pass). Background fluorescence (measured without excitation) was subtracted from the images and analysis was performed using ImageJ. For the different ROIs, mean fluorescent intensity (MFI) was calculated. Fluorescent TMR (TMR fluo ) ratios were determined by dividing the tumor's MFI values by the muscle's MFI values.

Statistical Analysis
For the fluorescence in vitro functionality assay, results were compared using an ordinary one-way ANOVA, corrected for multiple comparisons. Uptake between HER2expressing and HER2-negative tumors was compared using an unpaired Student's t-test. The correlation between nuclear and fluorescent signals was investigated by linear regression. Statistical analyses were performed using Prism 7 (Graphpad Software, San Diego, CA, USA). All data on the graphs is displayed as the mean ± SD.

Production, MSAP Conjugation, and Radiolabeling of Nanobodies
Following MSAP conjugation to the 2Rs15d Nanobody, SEC analysis confirmed the purity of MSAP.2Rs15d. The compound eluted as a single peak with a retention time of 6-7 min (as expected for a Nanobody), and absorbing at both 280 and 650 nm. The average DOL was determined to be 1.1 MSAP molecules per Nanobody.
MSAP.2Rs15d could be successfully 111 In-labeled, yielding a radiochemical purity of >95% as determined by iTLC. Further quality controls (QC) performed by radio-SEC, confirmed that the radioactive signal matched the retention times of MSAP.2Rs15d (absorbance at 280 and 650 nm) (Figure 2a), and that the tracer remained stable up to at least 24 h incubation in PBS or serum as no degradation of the protein, fluorescent, and/or radioactive signal was seen (Figure 2b,c) biomolecules 2021, 17, x. https://doi.org/10.3390/biomleculesxxxxx 6 of 13 7 min (as expected for a Nanobody), and absorbing at both 280 and 650 nm. The average DOL was determined to be 1.1 MSAP molecules per Nanobody. MSAP.2Rs15d could be successfully 111 In-labeled, yielding a radiochemical purity of > 95% as determined by iTLC. Further quality controls (QC) performed by radio-SEC, confirmed that the radioactive signal matched the retention times of MSAP.2Rs15d (absorbance at 280 and 650 nm) (Figure 2a), and that the tracer remained stable up to at least 24 h incubation in PBS or serum as no degradation of the protein, fluorescent, and/or radioactive signal was seen (Figure 2b,c)

In Vitro Functionality
SPR measurements revealed the dissociation constant (KD) of the MSAP.2Rs15d construct to be 5.0 ± 0.1 × 10 −9 M (Figure 3a). Specificity of the construct was further demonstrated by the significantly higher binding to HER2-expressing SKOV3 cells than to non-HER2-expressing CHO cells, or SKOV3 cells incubated with an excess of unlabeled 2Rs15d (Figure 3b). With a KD of 1.6 ± 0.2 × 10 −9 M as determined via a saturation binding assay on SKOV3 cells, it was confirmed that also after 111 In-labeling, the affinity of the Nanobody was not negatively affected (Figure 3c). The small difference in KD measured using SPR and cell-binding studies can be explained by the use of purified recombinant HER2 protein in the case of SPR, and high HER2-overexpressing cells for the radioligandbinding assay. Nevertheless, both values are in the low nanomolar range and comparable to that of the unconjugated 2Rs15d (3.2 ± 0.1 × 10 −9 M) ( Figure S1). In-MSAP.2Rs15d is dissolved in PBS 0.05% Tween ® 80; the additional peak seen around 4 min is attributed to the absorbance of Tween 80 at 280 nm. In panel (c), the absorbance of the Nanobody at 280 nm is negligible compared to the intense signal at 280 nm of the serum proteins. Therefore, the stability of the compound is determined based on the presence of the radioactive signal peak and the MSAP absorption peak at 650 nm at 6-7 min, comparable to the profiles in panel (a,b). Furthermore, serum proteins with a retention time of 3-5 min exhibit some autofluorescence at 650 nm, explaining the peak in 650 nm absorbance at 3-5 min.

In Vitro Functionality
SPR measurements revealed the dissociation constant (K D ) of the MSAP.2Rs15d construct to be 5.0 ± 0.1 × 10 −9 M (Figure 3a). Specificity of the construct was further demonstrated by the significantly higher binding to HER2-expressing SKOV3 cells than to non-HER2-expressing CHO cells, or SKOV3 cells incubated with an excess of unlabeled 2Rs15d (Figure 3b). With a K D of 1.6 ± 0.2 × 10 −9 M as determined via a saturation binding assay on SKOV3 cells, it was confirmed that also after 111 In-labeling, the affinity of the Nanobody was not negatively affected (Figure 3c). The small difference in K D measured using SPR and cell-binding studies can be explained by the use of purified recombinant HER2 protein in the case of SPR, and high HER2-overexpressing cells for the radioligandbinding assay. Nevertheless, both values are in the low nanomolar range and comparable to that of the unconjugated 2Rs15d (3.2 ± 0.1 × 10 −9 M) ( Figure S1).

Longitudinal Biodistribution in Subcutaneous Xenografts
As shown in Figure 4, SPECT/CT and fluorescence images coincided, and revealed an analogous biodistribution pattern for [ 111 In]In-MSAP.2Rs15d at 1 h, 4 h, and 24 h postinjection, with low non-specific uptake in untargeted organs, except for the kidneys due to renal retention of the tracer. SKOV3 tumors could be clearly distinguished as soon as 1 h post-injection, while no uptake in control MDA-MB-435S xenografts was seen (Figures  4a and S2). This was confirmed by quantification of the tumor uptake and tumor-to-muscle ratios (TMRs) based on the SPECT/CT images, yielding significant differences between SKOV3 and MDA-MB-435S at all timepoints (Figure 4b,c). More specifically, in SKOV3 xenografted mice a TMR rad of 19.8 ± 3.8 was achieved within 1 h and preserved over 24 h (21.1 ± 10.5). The absolute specific uptake did gradually decrease over time (Figure 4b) from 2.2 ± 0.5 %ID/cm 3 at 1 h to 1.0 ± 0.4 %ID/cm 3 at 24 h post-injection. The TMR rad for MDA-MB-435S control tumors was only 1.1 ± 1.0 (p < 0.05). On the fluorescent images, tumor uptake in SKOV3 xenografted mice could be clearly visualized with TMR fluo of 4.6 ± 1.5 and 4.5 ± 1.6 at respectively 1 and 4 h. These values were lower than for SPECT/CT imaging in consequence of signal attenuation by tissue and background autofluorescence.

Longitudinal Biodistribution in Subcutaneous Xenografts
As shown in Figure 4, SPECT/CT and fluorescence images coincided, and revealed an analogous biodistribution pattern for [ 111 In]In-MSAP.2Rs15d at 1 h, 4 h, and 24 h postinjection, with low non-specific uptake in untargeted organs, except for the kidneys due to renal retention of the tracer. SKOV3 tumors could be clearly distinguished as soon as 1 h post-injection, while no uptake in control MDA-MB-435S xenografts was seen (Figure 4a and Figure S2). This was confirmed by quantification of the tumor uptake and tumor-tomuscle ratios (TMRs) based on the SPECT/CT images, yielding significant differences between SKOV3 and MDA-MB-435S at all timepoints (Figure 4b,c). More specifically, in SKOV3 xenografted mice a TMR rad of 19.8 ± 3.8 was achieved within 1 h and preserved over 24 h (21.1 ± 10.5). The absolute specific uptake did gradually decrease over time (Figure 4b) from 2.2 ± 0.5 %ID/cm 3 at 1 h to 1.0 ± 0.4 %ID/cm 3 at 24 h post-injection. The TMR rad for MDA-MB-435S control tumors was only 1.1 ± 1.0 (p < 0.05). On the fluorescent images, tumor uptake in SKOV3 xenografted mice could be clearly visualized with TMR fluo of 4.6 ± 1.5 and 4.5 ± 1.6 at respectively 1 and 4 h. These values were lower than for SPECT/CT imaging in consequence of signal attenuation by tissue and background autofluorescence. The in vivo findings were further confirmed by the ex vivo biodistribution analysis after 24 h (Figure 4d, Tables S1 and S2). Indeed, the semiquantitative fluorescent data reflects the trend seen in the quantitative data obtained through gamma counting, with exception of the elevated fluorescent signals in the intestines and stomach (due to mouse chow [37]). The overall tumor uptake after 24 h in SKOV3 xenografts was measured to be 1.5 ± 0.2 %ID/g, which is similar to the in vivo measured data. Table S1 further illustrates the high tumor-to-organ ratios.

Image-Guided Resection of Intraperitoneally Disseminated Tumor Lesions
SPECT/CT scans (1 h post-injection) could be used to visualize the largest tumor masses (Figure 5a), however, smaller tumor nodules could not be visualized because of the inherent limited resolution and sensitivity of the imaging technique and shine through of the nearby kidneys. Subsequent opening of the abdominal cavity allowed in situ fluorescence-based visualization and fluorescence-guided removal of even submillimeter tumor lesions that were spread at the surface of the animal's peritoneum (38 lesions were removed in total, Figure 5a-c). Ex vivo analysis of BLI-confirmed tumor lesions was then used to correlate the uptake of fluorescence and radioactive signals in the resected lesions. The %ID of radioactivity in resected tumor lesions was plotted against their total fluorescence signal (Figure 5d), and after fitting a line with linear regression, an R 2 value of 0.97 was obtained, which indicates the good correlation between both modalities. The biodistribution and tumor-to-organ ratio's obtained through ex vivo fluorescence imaging and gamma counting is displayed in Figure S3 and Table S3.    used to correlate the uptake of fluorescence and radioactive signals in the resected lesio The %ID of radioactivity in resected tumor lesions was plotted against their total fluor cence signal (Figure 5d), and after fitting a line with linear regression, an R 2 value of 0 was obtained, which indicates the good correlation between both modalities. The biod tribution and tumor-to-organ ratio's obtained through ex vivo fluorescence imaging a gamma counting is displayed in Figure S3 and Table S3.

Discussion
Fluorescence surgical guidance has potential to improve the detection and removal of cancerous tissue, however, the technique is held back by unfavorable interaction of light and tissue (attenuation and scattering). Therefore, the use of bimodal labels such as the MSAP analogues, combining fluorescence and nuclear imaging may help overcome this limitation. Such a combination has been shown to allow pre/intraoperative detection of disease in a depth-independent manner and thus facilitates surgical navigation [3]. We here demonstrated that this concept can also be used to advance the use of Nanobodies in molecular imaging towards such bimodal applications.
We showed that an MSAP analogue carrying both a Cy5 fluorescent dye and a DTPA chelator for 111 In-labeling could be readily conjugated to a HER2-specific Nanobody without impacting its affinity or its pharmacokinetics. Administration of a single dose of the tracer resulted in high-contrast and specific visualization of HER2-expressing tumors via both SPECT/CT and fluorescence imaging (1-24 h p.i.), and with the exception of the kidneys, little to no non-specific uptake in HER2-negative tumors or non-targeted tissues. This corroborates with previous biodistribution profiles obtained after labeling of the same Nanobody with various PET, SPECT, or therapeutic radioisotopes [32,33,[38][39][40][41], and is in stark contrast with the drastic effect random labeling with the heptamethine fluorophore IRDye800CW has on the biodistribution of Nanobodies (high non-specific uptake and hepatic clearance [29]). The high fluorescent signal in the kidneys as observed in mice will most likely pose no problems in patients due to the attenuation of the signal by perinephric fat. However, the radioactive signal originating from the kidneys may create a significant background signal and affect adjacent tumor lesion detection. The use of Gelofusin or positively charged amino acids have been shown to reduce kidney retention in previous studies and could possibly be applied to counteract this limitation [42]. In the future, site-specific conjugation methods using cysteine-maleimide chemistry [41] or using the enzyme Sortase [40] could be considered to further standardize the tracer's composition if needed. Additional improvements can be found in minimizing the bimodal labels and using chelates that allow radiolabeling with 99m Tc, a radioisotope with more translational potential [43].
The proof-of-concept study in a murine model of intraperitoneally disseminated cancer showed that addition of a nuclear modality to the fluorescent component can further extend the potential of Nanobody-based imaging. Besides improvement of the characterization process and enabling a more exact determination of the tracer's biodistribution, [ 111 In]In-MSAP.2Rs15d could be used for both preoperative planning, and for precise and sensitive guidance during the actual surgical intervention. Indeed, preoperative nuclear medicine imaging can provide useful information on the anatomical localization of the tumor lesions and lymph node metastases [44]. Radioguided surgery solutions in the form of intraoperative gamma tracing can help to further guide the surgeon towards cancerous lesions, a concept that is currently being explored for prostate cancer [45]. A limitation of the study is certainly the choice of HER2 as biomarker, given its overexpression in a restricted number of cancer types and its intratumoral heterogeneity [46].

Conclusions
In this study we described the preparation of a bimodal nuclear/fluorescent Nanobodytracer through the convenient conjugation of a single-molecule MSAP-reagent carrying both a fluorophore and chelator for radioactive labeling. The Nanobody-tracer possessed an adequate biodistribution profile enabling fast and high-contrast nuclear and fluorescent imaging with low background. Such a tracer holds promise for clinical application in the context of image-guided surgery as was demonstrated by a proof-of-concept study in which intraperitoneally tumor lesions could be localized preoperatively using SPECT/CT, and then precisely excised via intraoperative fluorescence imaging.

Informed Consent Statement: Not applicable.
Data Availability Statement: Data sharing is not applicable for this article. All data is contained within the article or supplementary material. The data presented in this study are available in 'The Design and Preclinical Evaluation of a Single-Label Bi-modal Nanobody Tracer for Image-Guided Surgery-Supplementary data'.