Sustained release of a human PD-L1 single-domain antibody using peptide-based hydrogels.

Monoclonal antibodies (mAbs) targeting the immune checkpoint axis, which contains the programmed cell death protein-1 (PD-1) and its ligand PD-L1, revolutionized the field of oncology. Unfortunately, the large size of mAbs and the presence of an Fc fraction limit their tumor penetrative capacities and support off-target effects, potentially resulting in unresponsive patients and immune-related adverse events (irAEs) respectively. Single-domain antibodies (sdAbs) are ten times smaller than conventional mAbs and represent an emerging antibody subclass that has been proposed as next generation immune checkpoint inhibitor (ICI) therapeutics. They demonstrate favorable characteristics, such as an excellent stability, high antigen-binding affinity and an enhanced tumor penetration. Because sdAbs have a short half-life, methods to prolong their presence in the circulation and at the target site might be necessary in some cases to unfold their full therapeutic potential. In this study, we investigated a peptide-based hydrogel as an injectable biomaterial depot formulation for the sustained release of the human PD-L1 sdAb K2. We showed that a hydrogel composed of the amphipathic hexapeptide hydrogelator H-FQFQFK-NH2 prolonged the in vivo release of K2 after subcutaneous (s.c.) injection, up to at least 72 h, as monitored by SPECT/CT and fluorescence imaging. Additionally, after encapsulation in the hydrogel and s.c. administration, a significantly extended systemic presence and tumor uptake of K2 was observed in mice bearing a melanoma tumor expressing human PD-L1. Altogether, this study describes how peptide hydrogels can be exploited to provide the sustained release of sdAbs, thereby potentially enhancing its clinical and therapeutic effects.

The GEM-handle as convenient labeling strategy for bimodal single-domain antibody-based tracers carrying 99mTc and a near-infrared fluorescent dye for intra-operative decision-making.

Intra-operative fluorescence imaging has demonstrated its ability to improve tumor lesion identification. However, the limited tissue penetration of the fluorescent signals hinders the detection of deep-lying or occult lesions. Integrating fluorescence imaging with SPECT and/or intra-operative gamma-probing synergistically combines the deep tissue penetration of gamma rays for tumor localization with the precision of fluorescence imaging for precise tumor resection. In this study, we detail the use of a genetically encoded multifunctional handle, henceforth referred to as a GEM-handle, for the development of fluorescent/radioactive bimodal single-domain antibody (sdAb)-based tracers. A sdAb that targets the urokinase plasminogen activator receptor (uPAR) was engineered to carry a GEM-handle containing a carboxy-terminal hexahistidine-tag and cysteine-tag. A two-step labeling strategy was optimized and applied to site-specifically label IRDye800CW and 99mTc to the sdAb. Bimodal labeling of the sdAbs proved straightforward and successful. 99mTc activity was however restricted to 18.5 MBq per nmol fluorescently-labeled sdAb to prevent radiobleaching of IRDye800CW without impeding SPECT/CT imaging. Subsequently, the in vivo biodistribution and tumor-targeting capacity of the bimodal tracer were evaluated in uPAR-positive tumor-bearing mice using SPECT/CT and fluorescence imaging. The bimodal sdAb showed expected renal background signals due to tracer clearance, along with slightly elevated non-specific liver signals. Four hours post-injection, both SPECT/CT and fluorescent images achieved satisfactory tumor uptake and contrast, with significantly higher values observed for the anti-uPAR bimodal sdAb compared to a control non-targeting sdAb. In conclusion, the GEM-handle is a convenient method for designing and producing bimodal sdAb-based tracers with adequate in vivo characteristics.

Safety assessment of fluorescently labeled anti-EGFR Nanobodies in healthy dogs.

Introduction: Surgical resection is one of the main treatment options for several types of cancer, the desired outcome being complete removal of the primary tumor and its local metastases. Any malignant tissue that remains after surgery may lead to relapsing disease, negatively impacting the patient's quality of life and overall survival. Fluorescence imaging in surgical oncology aims to facilitate full resection of solid tumors through the visualization of malignant tissue during surgery, following the administration of a fluorescent contrast agent. An important class of targeting molecules are Nanobodies® (Nbs), small antigen-binding fragments derived from camelid heavy chain only antibodies. When coupled with a fluorophore, Nbs can bind to a specific receptor and demarcate tumor margins through a fluorescence camera, improving the accuracy of surgical intervention. A widely investigated target for fluorescence-guided surgery is the epidermal growth factor receptor (EGFR), which is overexpressed in several types of tumors. Promising results with the fluorescently labeled anti-EGFR Nb 7D12-s775z in murine models motivated a project employing the compound in a pioneering study in dogs with spontaneous cancer. Methods: To determine the safety profile of the study drug, three healthy purpose-bred dogs received an intravenous injection of the tracer at 5.83, 11.66, and 19.47 mg/m2, separated by a 14-day wash-out period. Physical examination and fluorescence imaging were performed at established time points, and the animals were closely monitored between doses. Blood and urine values were analyzed pre- and 24 h post administration. Results: No adverse effects were observed, and blood and urine values stayed within the reference range. Images of the oral mucosa, acquired with a fluorescence imaging device (Fluobeam®), suggest rapid clearance, which was in accordance with previous in vivo studies. Discussion: These are the first results to indicate that 7D12-s775z is well tolerated in dogs and paves the way to conduct clinical trials in canine patients with EGFR-overexpressing spontaneous tumors.

Severity Classification of Laboratory Animal Procedures in Two Belgian Academic Institutions.

According to the EU Directive 2010/63, all animal procedures must be classified as non-recovery, mild, moderate or severe. Several examples are included in the Directive to help in severity classification. Since the implementation of the Directive, different publications and guidelines have been disseminated on the topic. However, due to the large variety of disease models and animal procedures carried out in many different animal species, guidance on the severity classification of specific procedures or models is often lacking or not specific enough. The latter is especially the case in disease models where the level of pain, suffering, distress and lasting harm depends on the duration of the study (for progressive disease models) or the dosage given (for infectious or chemically induced disease models). This, in turn, may lead to inconsistencies in severity classification between countries, within countries and even within institutions. To overcome this, two Belgian academic institutions with a focus on biomedical research collaborated to develop a severity classification for all the procedures performed. This work started with listing all in-house procedures and assigning them to 16 (sub)categories. First, we determined which parameters, such as clinical signs, dosage or duration, were crucial for severity classification within a specific (sub)category. Next, a severity classification was assigned to the different procedures, which was based on professional judgment by the designated veterinarians, members of the animal welfare body (AWB) and institutional animal ethics committee (AEC), integrating the available literature and guidelines. During the classification process, the use of vague terminology, such as 'minor impact', was avoided as much as possible. Instead, well-defined cut-offs between severity levels were used. Furthermore, we sought to define common denominators to group procedures and to be able to classify new procedures more easily. Although the primary aim is to address prospective severity, this can also be used to assess actual severity. In summary, we developed a severity classification for all procedures performed in two academic, biomedical institutions. These include many procedures and disease models in a variety of animal species for which a severity classification was not reported so far, or the terms that assign them to a different severity were too vague.

Method To Diversify Cyanine Chromophore Functionality Enables Improved Biomolecule Tracking and Intracellular Imaging.

Heptamethine indocyanines are invaluable probes for near-infrared (NIR) imaging. Despite broad use, there are only a few synthetic methods to assemble these molecules, and each has significant limitations. Here, we report the use of pyridinium benzoxazole (PyBox) salts as heptamethine indocyanine precursors. This method is high yielding, simple to implement, and provides access to previously unknown chromophore functionality. We applied this method to create molecules to address two outstanding objectives in NIR fluorescence imaging. First, we used an iterative approach to develop molecules for protein-targeted tumor imaging. When compared to common NIR fluorophores, the optimized probe increases the tumor specificity of monoclonal antibody (mAb) and nanobody conjugates. Second, we developed cyclizing heptamethine indocyanines with the goal of improving cellular uptake and fluorogenic properties. By modifying both the electrophilic and nucleophilic components, we demonstrate that the solvent sensitivity of the ring-open/ring-closed equilibrium can be modified over a wide range. We then show that a chloroalkane derivative of a compound with tuned cyclization properties undergoes particularly efficient no-wash live cell imaging using organelle-targeted HaloTag self-labeling proteins. Overall, the chemistry reported here broadens the scope of accessible chromophore functionality, and, in turn, enables the discovery of NIR probes with promising properties for advanced imaging applications.

Near-Infrared Fluorescence Imaging of Pancreatic Cancer Using a Fluorescently Labelled Anti-CEA Nanobody Probe: A Preclinical Study.

Molecular fluorescence-guided surgery using near-infrared light has the potential to improve the rate of complete resection of cancer. Typically, monoclonal antibodies are being used as targeting moieties, however smaller fragments, such as single-domain antibodies (i.e., Nanobodies®) improve tumor specificity and enable tracer injection on the same day as surgery. In this study, the feasibility of a carcinoembryonic antigen-targeting Nanobody (NbCEA5) conjugated to two zwitterionic dyes (ZW800-1 Forte [ZW800F] and ZW800-1) for visualization of pancreatic ductal adenocarcinoma (PDAC) was investigated. After site-specific conjugation of NbCEA5 to the zwitterionic dyes, binding specificity was evaluated on human PDAC cell lines with flow cytometry. A dose escalation study was performed for both NbCEA5-ZW800F and NbCEA5-ZW800-1 in mice with subcutaneously implanted pancreatic tumors. Fluorescence imaging was performed up to 24 h after intravenous injection. Furthermore, the optimal dose for NbCEA5-ZW800-1 was injected in mice with orthotopically implanted pancreatic tumors. A dose-escalation study showed superior mean fluorescence intensities for NbCEA5-ZW800-1 compared to NbCEA5-ZW800F. In the orthotopic tumor models, NbCEA5-ZW800-1 accumulated specifically in pancreatic tumors with a mean in vivo tumor-to-background ratio of 2.4 (SD = 0.23). This study demonstrated the feasibility and potential advantages of using a CEA-targeted Nanobody conjugated to ZW800-1 for intraoperative PDAC imaging.

Size-advantage of monovalent nanobodies against the macrophage mannose receptor for deep tumor penetration and tumor-associated macrophage targeting.

Rationale: Nanobodies (Nbs) have emerged as an elegant alternative to the use of conventional monoclonal antibodies in cancer therapy, but a detailed microscopic insight into the in vivo pharmacokinetics of different Nb formats in tumor-bearers is lacking. This is especially relevant for the recognition and targeting of pro-tumoral tumor-associated macrophages (TAMs), which may be located in less penetrable tumor regions. Methods: We employed anti-Macrophage Mannose Receptor (MMR) Nbs, in a monovalent (m) or bivalent (biv) format, to assess in vivo TAM targeting. Intravital and confocal microscopy were used to analyse the blood clearance rate and targeting kinetics of anti-MMR Nbs in tumor tissue, healthy muscle tissue and liver. Fluorescence Molecular Tomography was applied to confirm anti-MMR Nb accumulation in the primary tumor and in metastatic lesions. Results: Intravital microscopy demonstrated significant differences in the blood clearance rate and macrophage targeting kinetics of (m) and (biv)anti-MMR Nbs, both in tumoral and extra-tumoral tissue. Importantly, (m)anti-MMR Nbs are superior in reaching tissue macrophages, an advantage that is especially prominent in tumor tissue. The administration of a molar excess of unlabelled (biv)anti-MMR Nbs increased the (m)anti-MMR Nb bioavailability and impacted on its macrophage targeting kinetics, preventing their accumulation in extra-tumoral tissue (especially in the liver) but only partially influencing their interaction with TAMs. Finally, anti-MMR Nb administration not only allowed the visualization of TAMs in primary tumors, but also at a distant metastatic site. Conclusions: These data describe, for the first time, a microscopic analysis of (m) and (biv)anti-MMR Nb pharmacokinetics in tumor and healthy tissues. The concepts proposed in this study provide important knowledge for the future use of Nbs as diagnostic and therapeutic agents, especially for the targeting of tumor-infiltrating immune cells.

Impact of doubling peptide length on in vivo hydrogel stability and sustained drug release.

Peptide-based hydrogels represent promising systems for the sustained release of different types of drugs, ranging from small molecules to biologicals. Aiming at subcutaneous injection, which is a desirable parenteral administration route, especially for biologicals, we herein focus on physically crosslinked systems possessing thixotropic behaviour. The purpose of this study was to evaluate the in vitro and in vivo properties of hydrogels based on the amphipathic hexapeptide H-FQFQFK-NH2, which served as the lead sequence. Upon doubling the length of this peptide, the dodecapeptide H-FQFQFKFQFQFK-NH2 gave a significant improvement in terms of in vivo stability of the hydrogel post-injection, as monitored by nuclear SPECT/CT imaging. This increased hydrogel stability also led to a more prolonged in vivo release of encapsulated peptide cargoes. Even though no direct link with the mechanical properties of the hydrogels before injection could be made, an important effect of the subcutaneous medium was noticed on the rheological properties of the hydrogels in post in vivo injection measurements. The results were validated in vivo for a therapeutically relevant analgesic peptide using the hot-plate test as an acute pain model. It was confirmed that elongation of the hydrogelator sequence induced more extended antinociceptive effects. Altogether, this simple structural modification of the hydrogelating peptide could provide a basis for reaching longer durations of action upon use of these soft biomaterials.

Emerging applications of nanobodies in cancer therapy.

Cancer is a heterogeneous disease, requiring treatment tailored to the unique phenotype of the patient's tumor. Monoclonal antibodies (mAbs) and variants thereof have enabled targeted therapies to selectively target cancer cells. Cancer cell-specific mAbs have been used for image-guided surgery and targeted delivery of radionuclides or toxic agents, improving classical treatment strategies. Cancer cell-specific mAbs can further inhibit tumor cell growth or can stimulate immune-mediated destruction of cancer cells, a feature that has also been achieved through mAb-mediated manipulation of immune cells and pathways. Drawbacks of mAbs and their variants, together with the discovery of camelid heavy chain-only antibodies and the many advantageous features of their variable domains, referred to as VHHs, single domain antibodies or nanobodies (Nbs), resulted in the exploration of Nbs as an alternative targeting moiety. We therefore review the state-of-the-art as well as novel exploitation strategies of Nbs for targeted cancer therapy.

In vivo Visualization of M2 Macrophages in the Myocardium After Myocardial Infarction (MI) Using 68 Ga-NOTA-Anti-MMR Nb: Targeting Mannose Receptor (MR, CD206) on M2 Macrophages.

Introduction and Objectives: Wound healing after myocardial infarction (MI) is a dynamic and complex multiple phase process, and a coordinated cellular response is required for proper scar formation. The current paradigm suggests that pro-inflammatory monocytes infiltrate the MI zone during the initial pro-inflammatory phase and differentiate into inflammatory macrophages, and then switch their phenotypes to anti-inflammatory during the reparative phase. Visualization of the reparative phase post-MI is of great interest because it may reveal delayed resolution of inflammation, which in turn predicts adverse cardiac remodeling. Imaging of anti-inflammatory macrophages may also be used to assess therapy approaches aiming to modulate the inflammatory response in order to limit MI size. Reparative macrophages can be distinguished from inflammatory macrophages by the surface marker mannose receptor (MR, CD206). In this study we evaluated the feasibility of 68Ga-NOTA-anti-MMR Nb for imaging of MR on alternatively activated macrophages in murine MI models. Methods: Wildtype and MR-knockout mice and Wistar rats were subjected to MI via permanent ligation of the left coronary artery. Non-operated or sham-operated animals were used as controls. MR expression kinetics on cardiac macrophages was measured in mice using flow cytometry. PET/CT scans were performed 1 h after intravenous injection of 68Ga-NOTA-anti-MMR Nb. Mice and rats were euthanized and hearts harvested for ex vivo PET/MRI, autoradiography, and staining. As a non-targeting negative control, 68Ga-NOTA-BCII10 was used. Results: In vivo-PET/CT scans showed focal radioactivity signals in the infarcted myocardium for 68Ga-NOTA-anti-MMR Nb which were confirmed by ex vivo-PET/MRI scans. In autoradiography images, augmented uptake of the tracer was observed in infarcts, as verified by the histochemistry analysis. Immunofluorescence staining demonstrated the presence and co-localization of CD206- and CD68-positive cells, in accordance to infarct zone. No in vivo or ex vivo signal was observed in the animals injected with control Nb or in the sham-operated animals. 68Ga-NOTA-anti-MMR Nb uptake in the infarcts of MR-knockout mice was negligibly low, confirming the specificity of 68Ga-NOTA-anti-MMR Nb to MR. Conclusion: This exploratory study highlights the potential of 68Ga-NOTA-anti-MMR Nb to image MR-positive macrophages that are known to play a pivotal role in wound healing that follows acute MI.

Fluorescent Anti-CEA Nanobody for Rapid Tumor-Targeting and Imaging in Mouse Models of Pancreatic Cancer.

Tumor-specific targeting with fluorescent probes can enhance contrast for identification of cancer during surgical resection and visualize otherwise invisible tumor margins. Nanobodies are the smallest naturally-occurring antigen-binding molecules with rapid pharmacokinetics. The present work demonstrates the efficacy of a fluorescent anti-CEA nanobody conjugated to an IR800 dye to target and label patient derived pancreatic cancer xenografts. After intravenous administration, the probe rapidly localized to the pancreatic cancer tumors within an hour and had a tumor-to-background ratio of 2.0 by 3 h. The fluorescence signal was durable over a prolonged period of time. With the rapid kinetics afforded by fluorescent nanobodies, both targeting and imaging can be performed on the same day as surgery.

Group sequential designs for in vivo studies: Minimizing animal numbers and handling uncertainty in power analysis.

Interim analysis is the practice of performing a statistical analysis when the data have only been partially collected, for example, to save resources or to handle the uncertainty of the true effect size. Most statistical designs featuring interim analysis have been developed either in a general statistical setting or for application in clinical trials. As a result, most of them make assumptions and have conditions that in a preclinical setting are usually not met. In this paper, we present necessary changes to the most common forms of interim analysis enhanced for animal experiments, specifically for the t-test and the one-way ANOVA. Finally, we present software that allows freeware use to serve the research community to facilitate the design of experiments featuring interim analyses. The app can be found at icds.be/gsdesigner. It is in the public domain and its code can be found on github.com/ICDS-vubUZ/gsd-designer. In this GitHub folder, one can also find a tutorial for the app.

Design and Validation of Site-Specifically Labeled Single-Domain Antibody-Based Tracers for in Vivo Fluorescence Imaging and Image-Guided Surgery.

Near-infrared fluorescence molecular imaging has become an established preclinical technique to investigate molecular processes in vivo and to study novel therapies. Furthermore, fluorescence molecular imaging is gaining significant interest from clinicians as an intra-operative guidance tool. This technique makes use of targeted fluorescent tracers as contrast agents that recognize specific biomarkers expressed at the site of disease. Single-domain antibodies have shown to possess excellent properties for in vivo imaging in comparison to conventional antibodies. In this chapter, we describe a method for site-specific conjugation of a near-infrared fluorophore to single-domain antibodies by exploiting cysteine-maleimide chemistry. As opposed to random conjugation, site-specific conjugation results in a homogenously labeled fluorescent tracer and avoids inference with antigen binding.

Reducing the renal retention of low- to moderate-molecular-weight radiopharmaceuticals.

The field of nuclear imaging and therapy is rapidly progressing with the development of targeted radiopharmaceuticals that show rapid targeting and rapid clearance with minimal background. Unfortunately, they are often reabsorbed in the kidneys, leading to possible nephrotoxicity, limiting the therapeutic dose, and/or reducing imaging quality. The blocking of endocytic receptors has been extensively used as a strategy to reduce kidney radiation. Alternatively, the physicochemical properties of radiotracers can be modulated to either prevent their reuptake or promote the excretion of radiometabolites. Other interesting strategies focus on the insertion of a cleavable linker between the radiolabel and the targeting moiety or pretargeting approaches in which the targeting moiety and radiolabel are administered separately. In the context of this review, we will discuss the latest advances and insights on strategies used to reduce renal retention of low- to moderate-molecular-weight radiopharmaceuticals.

Rapid tumor-labeling kinetics with a site-specific near-infrared anti-CEA nanobody in a patient-derived orthotopic xenograft mouse model of colon cancer.

BACKGROUND/OBJECTIVES: Nanobodies are the smallest biologic antigen-binding fragments derived from camelid-derived antibodies. Nanobodies effect a peak tumor signal within minutes of injection and present a novel opportunity for fluorescence-guided surgery (FGS). The present study demonstrates the efficacy of an anti-CEA nanobody conjugated to near-infrared fluorophore LICOR-IRDye800CW for rapid intraoperative tumor labeling of colon cancer. METHODS: LS174T human colon cancer cells or fragments of patient-derived colon cancer were implanted subcutaneously or orthotopically in nude mice. Anti-CEA nanobodies were conjugated with IRDye800CW and 1-3 nmol were injected intravenously. Mice were serially imaged over time. Peak fluorescence signal and tumor-to-background ratio (TBR) were recorded. RESULTS: Colon cancer tumors were detectable using fluorescent anti-CEA nanobody within 5 min of injection at all three doses. Maximal fluorescence intensity was observed within 15 min-3 h for all three doses with TBR values ranging from 1.3 to 2.3. In the patient-derived model of colon cancer, fluorescence was detectable with a TBR of 4.6 at 3 h. CONCLUSIONS: Fluorescent anti-CEA nanobodies rapidly and specifically labeled colon cancer in cell-line-based and patient-derived orthotopic xenograft (PDOX) models. The kinetics of nanobodies allow for same day administration and imaging. Anti-CEA-nb-800 is a promising and practical molecule for FGS of colon cancer.

Targeted Repolarization of Tumor-Associated Macrophages via Imidazoquinoline-Linked Nanobodies.

Tumor-associated macrophages (TAMs) promote the immune suppressive microenvironment inside tumors and are, therefore, considered as a promising target for the next generation of cancer immunotherapies. To repolarize their phenotype into a tumoricidal state, the Toll-like receptor 7/8 agonist imidazoquinoline IMDQ is site-specifically and quantitatively coupled to single chain antibody fragments, so-called nanobodies, targeting the macrophage mannose receptor (MMR) on TAMs. Intravenous injection of these conjugates result in a tumor- and cell-specific delivery of IMDQ into MMRhigh TAMs, causing a significant decline in tumor growth. This is accompanied by a repolarization of TAMs towards a pro-inflammatory phenotype and an increase in anti-tumor T cell responses. Therefore, the therapeutic benefit of such nanobody-drug conjugates may pave the road towards effective macrophage re-educating cancer immunotherapies.

Decorating sdAbs with Chelators: Effect of Conjugation on Biodistribution and Functionality.

Single domain antibodies (sdAbs) have proven to be valuable probes for molecular imaging. In order to produce such probes, one strategy is the functionalization of the reactive amine side chain of lysines with a chelator, resulting in a mixture of compounds with a different degree of conjugation. In this study, we implemented anion exchange chromatography (AEX) to separate the different compounds or fractions that were further characterized and evaluated to study the impact of the conjugation degree on pharmacokinetic properties and functionality. Anti-HER2 and anti-MMR sdAbs were functionalized with NOTA or DTPA chelator. Anion exchange chromatography was performed using 0.02 mol/L Tris pH 7.5 as the first solvent and 0.25 M or 0.4 M NaCl (in case of NOTA chelator or DTPA chelator, respectively) as the second solvent applied as a gradient. The fractions were characterized via mass spectrometry (MS), surface plasmon resonance (SPR), and isoelectric focusing gel electrophoresis (IEF), while in vivo studies were performed after radiolabeling with either 68Ga (NOTA) or 111In (DTPA) to assess the impact of the conjugation degree on pharmacokinetics. AEX could successfully be applied to separate fractions of (chelator)n-anti-HER2 and (chelator)n-anti-MMR sdAb constructs. MS confirmed the identity of different peaks obtained in the separation process. SPR measurement suggests a small loss of affinity for (chelator)3-anti-sdAb, while IEF revealed a correlated decrease in isoelectric point (pI) with the number of conjugated chelators. Interestingly, both the reduction in affinity and in pI was stronger with the DTPA chelator than with NOTA for both sdAbs. In vivo data showed no significant differences in organ uptake for any construct, except for (DTPA)n-anti-MMR, which showed a significantly higher liver uptake for (DTPA)1-anti-MMR compared to (DTPA)2-anti-MMR and (DTPA)3-anti-MMR. For all constructs in general, high kidney uptake was observed, due to the typical renal clearance of sdAb-based tracers. The kidney uptake showed significant differences between fractions of a same construct and indicates that a higher conjugation degree improves kidney clearance. AEX allows the separation of sdAbs with a different degree of conjugation and provides the opportunity to further characterize individual fractions. The conjugation of a chelator to sdAbs can alter some properties of the tracers, such as pI; however, the impact on the general biodistribution profile and tumor targeting was minimal.

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.

Tumor-specific near-infrared nanobody probe rapidly labels tumors in an orthotopic mouse model of pancreatic cancer.

BACKGROUND: Nanobodies, derived from camelid antibodies made of only heavy chains, are the smallest, biologic, antigen-binding fragments (~15kDa) with faster pharmacokinetics and better tumor penetration efficiency than standard antibodies. The present study evaluates the efficacy of a fluorescent, anti-carcinoembryonic antigen (CEA) nanobody for rapid tumor labeling in an orthotopic mouse model of pancreatic cancer. METHODS: Anti-CEA or control nanobodies were conjugated with the near-infrared fluorophore IRDye 800CW. Fragments of BxPC-3 (high-CEA expressing) or MiaPACA-2 (low-CEA expressing) human pancreatic cancer cell lines were orthotopically implanted into the pancreatic tail of nude mice. After tumors reached 7 to 10 mm in size, 2 nmol anti-CEA or control nanobody-IRDye800CW were injected intravenously. Mice were imaged at various time points hours post-injection. RESULTS: Anti-CEA nanobodies clearly labeled BxPC3 orthotopic pancreatic tumors 3 hours after injection. The signal was present as early as 15 minutes after injection and was robust at 1 to 3 hours after injection with a tumor-to-background ratio of 2.66. In contrast, there was very low accumulation in the low CEA-expressing, MiaPACA2 pancreatic orthotopic tumors. The fluorophore-conjugated nanobody was specific for CEA-expressing tumors, while the control nanobody did not show any tumor-specific signal. Both nanobodies had strong kidney uptake as expected for small-molecule probes. The fluorescence signal was detectable using 2 clinical, Food and Drug Administration-approved, 800 nm imaging devices as well as small animal imaging systems. CONCLUSION: This anti-CEA, nanobody-based, fluorescent probe labeled pancreatic orthotopic tumors within 15 minutes of intravenous injection. Fluorescent anti-CEA nanobodies have labeling kinetics that approach the speed of nonspecific dyes such as indocyanine green but with the specificity of antibodies. The use of fluorescently-labeled, intact antibodies leads to a labeling delay of 48 to 96 hours between probe administration and the necessarily delayed time of operation, which can be avoided with nanobodies. The kinetics of a nanobody-based probe makes it a practical agent for same-day, patient administration and fluorescence-guided surgery.