RESUMO
Fluorescence molecular imaging plays a vital role in image-guided surgery. In this context, the urokinase plasminogen activator receptor (uPAR) is an interesting biomarker enabling the detection and delineation of various tumor types due to its elevated expression on both tumor cells and the tumor microenvironment. In this study, anti-uPAR Nanobodies (Nbs) are generated through llama immunization with human and murine uPAR protein. Extensive in vitro characterization and in vivo testing with radiolabeled variants are conducted to assess their pharmacokinetics and select lead compounds. Subsequently, the selected Nbs are converted into fluorescent agents, and their application for fluorescence-guided surgery is evaluated in various subcutaneous and orthotopic tumor models. The study yields a panel of high-affinity anti-uPAR Nbs, showing specific binding across multiple types of cancer cells in vitro and in vivo. Lead fluorescently-labeled compounds exhibit high tumor uptake with high contrast at 1 h after intravenous injection across all assessed uPAR-expressing tumor models, outperforming a non-targeting control Nb. Additionally, rapid and accurate tumor localization and demarcation are demonstrated in an orthotopic human glioma model. Utilizing these Nbs can potentially enhance the precision of surgical tumor resection and, consequently, improve survival rates in the clinic.
Assuntos
Receptores de Ativador de Plasminogênio Tipo Uroquinase , Anticorpos de Domínio Único , Cirurgia Assistida por Computador , Animais , Anticorpos de Domínio Único/imunologia , Camundongos , Receptores de Ativador de Plasminogênio Tipo Uroquinase/metabolismo , Cirurgia Assistida por Computador/métodos , Humanos , Modelos Animais de Doenças , Linhagem Celular Tumoral , Imagem Óptica/métodos , Neoplasias/imunologia , Neoplasias/diagnóstico por imagem , Neoplasias/cirurgia , Corantes Fluorescentes , Camelídeos AmericanosRESUMO
Introduction: Multiple myeloma (MM) remains incurable, despite the advent of chimeric antigen receptor (CAR)-T cell therapy. This unfulfilled potential can be attributed to two untackled issues: the lack of suitable CAR targets and formats. In relation to the former, the target should be highly expressed and reluctant to shedding; two characteristics that are attributed to the CS1-antigen. Furthermore, conventional CARs rely on scFvs for antigen recognition, yet this withholds disadvantages, mainly caused by the intrinsic instability of this format. VHHs have been proposed as valid scFv alternatives. We therefore intended to develop VHH-based CAR-T cells, targeting CS1, and to identify VHHs that induce optimal CAR-T cell activation together with the VHH parameters required to achieve this. Methods: CS1-specific VHHs were generated, identified and fully characterized, in vitro and in vivo. Next, they were incorporated into second-generation CARs that only differ in their antigen-binding moiety. Reporter T-cell lines were lentivirally transduced with the different VHH-CARs and CAR-T cell activation kinetics were evaluated side-by-side. Affinity, cell-binding capacity, epitope location, in vivo behavior, binding distance, and orientation of the CAR-T:MM cell interaction pair were investigated as predictive parameters for CAR-T cell activation. Results: Our data show that the VHHs affinity for its target antigen is relatively predictive for its in vivo tumor-tracing capacity, as tumor uptake generally decreased with decreasing affinity in an in vivo model of MM. This does not hold true for their CAR-T cell activation potential, as some intermediate affinity-binding VHHs proved surprisingly potent, while some higher affinity VHHs failed to induce equal levels of T-cell activation. This could not be attributed to cell-binding capacity, in vivo VHH behavior, epitope location, cell-to-cell distance or binding orientation. Hence, none of the investigated parameters proved to have significant predictive value for the extent of CAR-T cell activation. Conclusions: We gained insight into the predictive parameters of VHHs in the CAR-context using a VHH library against CS1, a highly relevant MM antigen. As none of the studied VHH parameters had predictive value, defining VHHs for optimal CAR-T cell activation remains bound to serendipity. These findings highlight the importance of screening multiple candidates.
Assuntos
Imunoterapia Adotiva , Mieloma Múltiplo , Receptores de Antígenos Quiméricos , Anticorpos de Domínio Único , Mieloma Múltiplo/imunologia , Mieloma Múltiplo/terapia , Humanos , Receptores de Antígenos Quiméricos/imunologia , Receptores de Antígenos Quiméricos/genética , Receptores de Antígenos Quiméricos/metabolismo , Anticorpos de Domínio Único/imunologia , Imunoterapia Adotiva/métodos , Animais , Linhagem Celular Tumoral , Camundongos , Ativação Linfocitária/imunologia , Linfócitos T/imunologia , Linfócitos T/metabolismo , Família de Moléculas de Sinalização da Ativação Linfocitária/imunologia , Família de Moléculas de Sinalização da Ativação Linfocitária/metabolismo , Anticorpos de Cadeia Única/imunologia , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
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.
Assuntos
Carcinoma Ductal Pancreático , Neoplasias Pancreáticas , Humanos , Animais , Camundongos , Antígeno Carcinoembrionário , Neoplasias Pancreáticas/diagnóstico por imagem , Neoplasias Pancreáticas/patologia , Imagem Óptica/métodos , Carcinoma Ductal Pancreático/diagnóstico por imagem , Corantes , Linhagem Celular Tumoral , Neoplasias PancreáticasRESUMO
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.
Assuntos
Neoplasias , Anticorpos de Domínio Único , Humanos , Receptor de Manose , Lectinas Tipo C , Lectinas de Ligação a Manose , Receptores de Superfície Celular , Macrófagos Associados a Tumor , Neoplasias/tratamento farmacológicoRESUMO
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 ß--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.
Assuntos
Mieloma Múltiplo/tratamento farmacológico , Mieloma Múltiplo/radioterapia , Medicina de Precisão/métodos , Radioisótopos/uso terapêutico , Anticorpos de Domínio Único/uso terapêutico , Animais , Feminino , Humanos , Camundongos , Radioisótopos/farmacologia , Anticorpos de Domínio Único/farmacologiaRESUMO
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.
Assuntos
Antígeno Carcinoembrionário/imunologia , Neoplasias do Colo/diagnóstico por imagem , Imagem Óptica , Anticorpos de Domínio Único , Animais , Modelos Animais de Doenças , Corantes Fluorescentes , Xenoenxertos , Humanos , Camundongos Nus , Neoplasias ExperimentaisRESUMO
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.
RESUMO
PURPOSE: The combination of standard-of-care radiation therapy (RT) with immunotherapy is moving to the mainstream of non-small cell lung cancer treatment. Multiple preclinical studies reported on the CD8+ T cell stimulating properties of RT, resulting in abscopal therapeutic effects. A literature search demonstrates that most preclinical lung cancer studies applied subcutaneous lung tumor models. Hence, in-depth immunologic evaluation of clinically relevant RT in orthotopic lung cancer models is lacking. METHODS AND MATERIALS: We studied the therapeutic and immunologic effects of low-dose fractionated RT on lungs from C57BL/6 mice, challenged 2 weeks before with firefly luciferase expressing Lewis lung carcinoma cells via the tail vein. Low-dose fractionation was represented by 4 consecutive daily fractions of image guided RT at 3.2 Gy. RESULTS: We showed reduced lung tumor growth upon irradiation using in vivo bioluminescence imaging and immunohistochemistry. Moreover, significant immunologic RT-induced changes were observed in irradiated lungs and in the periphery (spleen and blood). First, a significant decrease in the number of CD8+ T cells and trends toward more CD4+ and regulatory T cells were seen after RT in all evaluated tissues. Notably, only in the periphery did the remaining CD8+ T cells show a more activated phenotype. In addition, a significant expansion of neutrophils and monocytes was observed upon RT locally and systemically. Locally, RT increased the influx of tumor-associated macrophages and conventional type 2 dendritic cells, whereas the alveolar macrophages and conventional type 1 DCs dramatically decreased. Functionally, these antigen-presenting cells severely reduced their CD86 expression, suggesting a reduced capacity to induce potent immunity. CONCLUSIONS: Our results imply that low-dose fractionated RT of tumor-bearing lung tissue shifts the immune cell balance toward an immature myeloid cell dominating profile. These data argue for myeloid cell repolarizing strategies to enhance the abscopal effects in patients with non-small cell lung cancer treated with fractionated RT.
Assuntos
Células Apresentadoras de Antígenos/efeitos da radiação , Linfócitos T CD8-Positivos/efeitos da radiação , Fracionamento da Dose de Radiação , Neoplasias Pulmonares/radioterapia , Animais , Feminino , Neoplasias Pulmonares/imunologia , Neoplasias Pulmonares/patologia , Camundongos , Camundongos Endogâmicos C57BLRESUMO
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.
Assuntos
Anticorpos de Domínio Único/química , Cirurgia Assistida por Computador , Animais , Células CHO , Linhagem Celular Tumoral , Cricetulus , Humanos , Camundongos , Neoplasias/diagnóstico por imagem , Neoplasias/patologia , Compostos Radiofarmacêuticos/química , Distribuição Tecidual , Tomografia Computadorizada de Emissão de Fóton Único , Tomografia Computadorizada por Raios X , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
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 >95% radiochemical purity. In vivo tumor targeting studies at 1 h 20 post-injection revealed specific tumor uptake of 1.89 ± 0.40%IA/g for the site-specific conjugate, 1.77 ± 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.
Assuntos
Anticorpos Anti-Idiotípicos/imunologia , Antígeno B7-H1/imunologia , Neoplasias/diagnóstico por imagem , Tomografia por Emissão de Pósitrons , Anticorpos Monoclonais/farmacologia , Antígeno B7-H1/farmacologia , Linhagem Celular Tumoral , Humanos , Marcação por Isótopo , Neoplasias/imunologia , Neoplasias/patologia , Compostos Radiofarmacêuticos/farmacologia , Distribuição Tecidual/efeitos dos fármacosRESUMO
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.
Assuntos
Corantes Fluorescentes , Neoplasias Experimentais , Neoplasias Pancreáticas , Anticorpos de Domínio Único , AnimaisRESUMO
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 γ- and therapeutic α- and ß--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.
RESUMO
A compound's intratumoural distribution is an important determinant for the effectiveness of molecular therapy or imaging. Antibodies (Abs), though often used in the design of targeted compounds, struggle to achieve a homogenous distribution due to their large size and bivalent binding mechanism. In contrast, smaller compounds like nanobodies (Nbs) are expected to distribute more homogenously, though this has yet to be demonstrated in vivo at the microscopic level. We propose an intravital approach to evaluate the intratumoural distribution of different fluorescently labeled monomeric and dimeric Nb tracers and compare this with a monoclonal antibody (mAb). Monomeric and dimeric formats of the anti-HER2 (2Rb17c and 2Rb17c-2Rb17c) and control (R3B23 and R3B23-R3B23) Nb, as well as the dimeric monovalent Nb 2Rb17c-R3B23 were generated and fluorescently labeled with a Cy5 fluorophore. The mAb trastuzumab-Cy5 was also prepared. Whole-body biodistribution of all constructs was investigated in mice bearing subcutaneous xenografts (HER2+ SKOV3) using in vivo epi-fluorescence imaging. Next, for intravital experiments, GFP-expressing SKOV3 cells were grown under dorsal window chambers on athymic nude mice (n = 3/group), and imaged under a fluorescence stereo microscope immediately after intravenous injection of the tracers. Consecutive fluorescence images within the tumour were acquired over the initial 20 min after injection and later, single images were taken at 1, 3 and 24 h post-injection. Additionally, two-photon microscopy was used to investigate the colocalization of GFP (tumour cells) and Cy5 fluorescence (tracers) at higher resolution. Whole-body images showed rapid renal clearance of all Nbs, and fast tumour targeting for the specific Nbs. Specific tumour uptake of the mAb could only be clearly distinguished from background after several hours. Intravital imaging revealed that monomeric Nb tracers accumulated rapidly and distributed homogenously in the tumour mere minutes after intravenous injection. The dimeric compounds initially achieved lower fluorescence intensities than the monomeric. Furthermore, whereas the HER2-specific dimeric bivalent compound remained closely associated to the blood vessels over 24 h, the HER2-specific dimeric monovalent tracer achieved a more homogenous tumour distribution from 1 h post-injection onwards. Non-specific tracers were not retained in the tumour. Trastuzumab had the most heterogenous intratumoural distribution of all evaluated compounds, while -due to the long blood retention- achieving the highest overall tumour uptake at 24 h post-injection. In conclusion, monomeric Nbs very quickly and homogenously distribute through tumour tissue, at a rate significantly greater than dimeric Nbs and mAbs. This underlines the potential of monomeric Nb tracers and therapeutics in molecular imaging and targeted therapies.
Assuntos
Anticorpos de Domínio Único , Animais , Linhagem Celular Tumoral , Cinética , Camundongos , Camundongos Nus , Receptor ErbB-2/metabolismo , Anticorpos de Domínio Único/metabolismo , Distribuição TecidualRESUMO
Molecular imaging is paving the way towards noninvasive detection, staging, and treatment follow-up of diseases such as cancer and inflammation-related conditions. Monoclonal antibodies have long been one of the staples of molecular imaging tracer design, although their long blood circulation and high nonspecific background limits their applicability. Nanobodies, unique antibody-binding fragments derived from camelid heavy-chain antibodies, have excellent properties for molecular imaging as they are able to specifically find their target early after injection, with little to no nonspecific background. Nanobody-based tracers using either nuclear or fluorescent labels have been heavily investigated preclinically and are currently making their way into the clinic. In this review, we will discuss different important factors in nanobody-tracer design, as well as the current state of the art regarding their application for nuclear and fluorescent imaging purposes. Furthermore, we will discuss how nanobodies can also be exploited for molecular therapy applications such as targeted radionuclide therapy and photodynamic therapy.
RESUMO
Real-time intraoperative guidance is essential during oncological surgery for complete and safe tumour resection. Fluorescence imaging in the near-infrared spectrum has shown potential for guiding surgeons during complex interventions. Recently, there has been a shift towards the use of fluorescence contrast agents for molecular imaging. The first targeted fluorescent agents, of which most consist of approved therapeutic antibodies conjugated to a fluorescent dye, have been evaluated in several early-phase clinical trials. Moreover, advances in protein engineering and drug design have led to the development of a variety of tracers suitable for molecular fluorescence image-guided surgery. In this Review, we discuss preclinical and clinical evidence, ongoing clinical trials, and the latest developments in the field of molecular near-infrared tracers for fluorescence-guided cancer surgery.
Assuntos
Fluorescência , Corantes Fluorescentes , Cuidados Intraoperatórios/métodos , Imagem Molecular/métodos , Neoplasias/diagnóstico por imagem , Neoplasias/cirurgia , Cirurgia Assistida por Computador , Ensaios Clínicos como Assunto , Previsões , HumanosRESUMO
Fluorescence imaging is an emerging technology that can provide real-time information about the operating field during cancer surgery. Non-specific fluorescent agents, used for the assessment of blood flow and sentinel lymph node detection, have so far dominated this field. However, over the last decade, several clinical studies have demonstrated the great potential of targeted fluorescent tracers to visualize tumor lesions in a more specific way. This has led to an exponential growth in the development of novel molecular fluorescent contrast agents. In this review, the design of fluorescent molecular tracers will be discussed, with particular attention for agents and approaches that are of interest for clinical translation.
RESUMO
PURPOSE: Macrophage accumulation characterizes the development of atherosclerotic plaques, and the presence of certain macrophage subsets might be an indicator of plaque phenotype and (in)stability. The macrophage mannose receptor (MMR) is expressed on alternatively activated macrophages and found at sites of intraplaque hemorrhage and neovascularization. It has been proposed as target to identify vulnerable plaques. Therefore, we aimed to assess the feasibility of using anti-MMR nanobodies (Nbs) as molecular tracers for nuclear imaging in an animal model of atherosclerosis. PROCEDURE: Anti-MMR and control Nb, radiolabeled with Tc-99m, were injected in ApoE-/- and/or C57Bl/6 mice (n = 6). In vivo competition studies involving pre-injection of excess of unlabeled anti-MMR Nb (n = 3) and injection of anti-MMR Nb in MMR-/- mice (n = 3) were performed to demonstrate specificity. At 3 h p.i. radioactive uptake in organs, tissues and aorta segments were evaluated. Autoradiography and immunofluorescence were performed on aortic sections. RESULTS: Significantly higher uptake was observed in all aortic segments of ApoE-/- mice injected with anti-MMR Nb compared to control Nb (1.36 ± 0.67 vs 0.38 ± 0.13 percent of injected dose per gram (%ID/g), p ≤ 0.001). Surprisingly, high aortic uptake was also observed in C57Bl/6 mice (1.50 ± 0.43%ID/g, p ≥ 0.05 compared to ApoE-/-), while aortic uptake was reduced to background levels in the case of competition and in MMR-/- mice (0.46 ± 0.10 and 0.22 ± 0.06%ID/g, respectively; p ≤ 0.001). Therefore, expression of MMR along healthy aortas was suggested. Autoradiography showed no specific radioactive signal within atherosclerotic plaques, but rather localization of the signal along the aorta, correlating with MMR expression in perivascular tissue as demonstrated by immunofluorescence. CONCLUSIONS: No significant uptake of MMR-specific Nb could be observed in atherosclerotic lesions of ApoE-/- mice in this study. A specific perivascular signal causing a non-negligible background level was demonstrated. This observation should be considered when using MMR as a target in molecular imaging of atherosclerosis, as well as use of translational animal models with vulnerable plaques.
Assuntos
Lectinas Tipo C/metabolismo , Macrófagos/metabolismo , Lectinas de Ligação a Manose/metabolismo , Placa Aterosclerótica/diagnóstico por imagem , Compostos Radiofarmacêuticos/química , Receptores de Superfície Celular/metabolismo , Anticorpos de Domínio Único/metabolismo , Tecnécio/química , Animais , Aorta/diagnóstico por imagem , Aorta/patologia , Autorradiografia , Feminino , Humanos , Receptor de Manose , Camundongos Endogâmicos C57BL , Coloração e Rotulagem , Distribuição TecidualRESUMO
PURPOSE: Debulking followed by combination chemotherapy is currently regarded as the most effective treatment for advanced ovarian cancer. Prognosis depends drastically on the degree of debulking. Accordingly, near-infrared (NIR) fluorescence imaging has been proposed to revolutionize cancer surgery by acting as a sensitive, specific, and real-time tool enabling visualization of cancer lesions. We have previously developed a NIR-labeled nanobody that allows fast, specific, and high-contrast imaging of HER2-positive tumors. In this study, we applied this tracer during fluorescence-guided surgery in a mouse model and investigated the effect on surgical efficiency. PROCEDURES: 0.5 × 106 SKOV3.IP1-Luc+ cells were inoculated intraperitoneally in athymic mice and were allowed to grow for 30 days. Two nanomoles of IRDye800CW-anti-HER2 nanobody was injected intravenously. After 1h30, mice were killed, randomized in two groups, and subjected to surgery. In the first animal group (n = 7), lesions were removed by a conventional surgical protocol, followed by excision of remaining fluorescent tissue using a NIR camera. The second group of mice (n = 6) underwent directly fluorescence-guided surgery. Bioluminescence imaging was performed before and after surgery. Resected tissue was categorized as visualized during conventional surgery or not, fluorescent or not, and bioluminescent positive or negative. RESULTS: Fluorescence imaging allowed clear visualization of tumor nodules within the abdomen, up to submillimeter-sized lesions. Fluorescence guidance resulted in significantly reduced residual tumor as compared to conventional surgery. Moreover, sensitivity increased from 59.3 to 99.0 %, and the percentage of false positive lesions detected decreased from 19.6 to 7.1 %. CONCLUSIONS: This study demonstrates the advantage of intraoperative fluorescence imaging using nanobody-based tracers on the efficiency of debulking surgery.
Assuntos
Procedimentos Cirúrgicos de Citorredução , Implantes Experimentais , Raios Infravermelhos , Imagem Óptica , Neoplasias Peritoneais/diagnóstico por imagem , Neoplasias Peritoneais/cirurgia , Anticorpos de Domínio Único/química , Animais , Linhagem Celular Tumoral , Modelos Animais de Doenças , Feminino , Fluorescência , Humanos , CamundongosRESUMO
Advances in optical imaging technologies have stimulated the development of near-infrared (NIR) fluorescently labeled targeted probes for use in image-guided surgery. As nanobodies have already proven to be excellent candidates for molecular imaging, we aimed in this project to design NIR-conjugated nanobodies targeting the tumor biomarker HER2 for future applications in this field and to evaluate the effect of dye and dye conjugation chemistry on their pharmacokinetics during development. IRDye800CW or IRdye680RD were conjugated either randomly (via lysines) or site-specifically (via C-terminal cysteine) to the anti-HER2 nanobody 2Rs15d. After verification of purity and functionality, the biodistribution and tumor targeting of the NIR-nanobodies were assessed in HER2-positive and -negative xenografted mice. Site-specifically IRDye800CW- and IRdye680RD-labeled 2Rs15d as well as randomly labeled 2Rs15d-IRDye680RD showed rapid tumor accumulation and low nonspecific uptake, resulting in high tumor-to-muscle ratios at early time points (respectively 6.6 ± 1.0, 3.4 ± 1.6, and 3.5 ± 0.9 for HER2-postive tumors at 3 h p.i., while <1.0 for HER2-negative tumors at 3 h p.i., p < 0.05). Contrarily, using the randomly labeled 2Rs15d-IRDye800CW, HER2-positive and -negative tumors could only be distinguished after 24 h due to high nonspecific signals. Moreover, both randomly labeled 2Rs15d nanobodies were not only cleared via the kidneys but also partially via the hepatobiliary route. In conclusion, near-infrared fluorescent labeling of nanobodies allows rapid, specific, and high contrast in vivo tumor imaging. Nevertheless, the fluorescent dye as well as the chosen conjugation strategy can affect the nanobodies' properties and consequently have a major impact on their pharmacokinetics.