RESUMEN
Fibroblast activation protein α (FAP) is highly expressed on cancer-associated fibroblasts of epithelial-derived cancers. Breast, colon, and pancreatic tumors often show strong desmoplastic reactions, which result in a dominant presence of stromal cells. FAP has gained interest as a target for molecular imaging and targeted therapies. Single-domain antibodies (sdAbs) are the smallest antibody-derived fragments with beneficial pharmacokinetic properties for molecular imaging and targeted therapy. Methods: We describe the generation, selection, and characterization of a sdAb against FAP. In mice, we assessed its imaging and therapeutic potential after radiolabeling with tracer-dose 131I and 68Ga for SPECT and PET imaging, respectively, and with 131I and 225Ac for targeted radionuclide therapy. Results: The lead sdAb, 4AH29, exhibiting picomolar affinity for a distinct FAP epitope, recognized both purified and membrane-bound FAP protein. Radiolabeled versions, including [68Ga]Ga-DOTA-4AH29, [225Ac]Ac-DOTA-4AH29, and [131I]I-guanidinomethyl iodobenzoate (GMIB)-4AH29, displayed radiochemical purities exceeding 95% and effectively bound to recombinant human FAP protein and FAP-positive GM05389 human fibroblasts. These radiolabeled compounds exhibited rapid and specific accumulation in human FAP-positive U87-MG glioblastoma tumors, with low but specific uptake in lymph nodes, uterus, bone, and skin (â¼2-3 percentage injected activity per gram of tissue [%IA/g]). Kidney clearance of unbound [131I]I-GMIB-4AH29 was fast (<1 %IA/g after 24 h), whereas [225Ac]Ac-DOTA-4AH29 exhibited slower clearance (8.07 ± 1.39 %IA/g after 24 h and 2.47 ± 0.18 %IA/g after 96 h). Mice treated with [225Ac]Ac-DOTA-4AH29 and [131I]I-GMIB-4AH29 demonstrated prolonged survival compared with those receiving vehicle solution. Conclusion: [68Ga]Ga-DOTA-4AH29 and [131I]I-GMIB-4AH29 enable precise FAP-positive tumor detection in mice. Therapeutic [225Ac]Ac-DOTA-4AH29 and [131I]I-GMIB-4AH29 exhibit strong and sustained tumor targeting, resulting in dose-dependent therapeutic effects in FAP-positive tumor-bearing mice, albeit with kidney toxicity observed later for [225Ac]Ac-DOTA-4AH29. This study confirms the potential of radiolabeled sdAb 4AH29 as a radiotheranostic agent for FAP-positive cancers, warranting clinical evaluation.
Asunto(s)
Neoplasias Pancreáticas , Anticuerpos de Dominio Único , Femenino , Humanos , Animales , Ratones , Anticuerpos de Dominio Único/metabolismo , Radioisótopos de Galio , Neoplasias Pancreáticas/patología , Radiofármacos/química , Línea Celular TumoralRESUMEN
Immune checkpoint inhibition (ICI) is a promising cancer therapy, which has progressed rapidly from a preclinical concept to clinical implementation. Commonly considered targets in ICI are CTLA-4, PD-1/PD-L1, and LAG-3, and the list grows. As ICI is generally only beneficial for a subset of patients, there is a need to select patients that are eligible for therapy as well as to monitor therapy response. There is growing interest to do this noninvasively, by molecular imaging with target-specific tracers. To this day, noninvasive imaging has focused on CTLA-4 and PD-1/PD-L1, while there is no noninvasive tool available to accurately assess LAG-3 expression in vivo. In this proof-of-concept study, we developed nanobodies, the smallest functional fragments from camelid heavy chain-only antibodies, to noninvasively evaluate mouse LAG-3 expression using single photon emission computed tomography (SPECT)/CT imaging. The in vitro characterization of 114 nanobodies led to the selection of nine nanobodies binding to mouse LAG-3. The injection of 99mTechnetium-labeled nanobodies in healthy mice showed specific uptake in immune peripheral organs like the spleen and lymph nodes, which was not observed in LAG-3 gene knock-out mice. Moreover, nanobody uptake could be visualized using SPECT/CT and correlated to the presence of LAG-3 as assessed in flow cytometry and immunohistochemistry. SPECT/CT scans of tumor bearing mice further confirmed the diagnostic potential of the nanobodies. These findings substantiate the approach to use nanobodies as a tool to image inhibitory immune checkpoints in the tumor environment.
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Anticuerpos/química , Antígenos CD/análisis , Imagen Molecular , Anticuerpos de Dominio Único/química , Animales , Anticuerpos/inmunología , Anticuerpos/farmacología , Antígenos CD/inmunología , Camelidae , Ratones , Proteínas Recombinantes/química , Proteínas Recombinantes/inmunología , Anticuerpos de Dominio Único/inmunología , Anticuerpos de Dominio Único/farmacología , Proteína del Gen 3 de Activación de LinfocitosRESUMEN
Current cancer therapeutics suffer from a lack of specificity in targeting tumor cells and cause severe side effects. Therefore, the design of highly specialized drugs comprising antibody derivatives inducing apoptosis in targeted cancer cells is considered to be a promising strategy. Drugs acting on death receptor 5 (DR5) such as DR5 agonist antibodies replacing "TNF-related apoptosis-inducing ligand" (TRAIL) offer feasible opportunities in this direction. Although such agonists provided good antitumor activity in preclinical studies, they were less effective in clinical studies, possibly due to a disturbed Fc interaction with Fc-γ receptors. Thus, multimerized antigen binding fragments without Fc have been proposed to increase their efficacy. We generated nanobodies (Nbs), recombinant variable domains of heavy chain-only antibodies of camelids, against the DR5 ectodomain. Nb24 and Nb28 had an affinity in the nM and sub-nM range, but only Nb28 competes with TRAIL for binding to DR5. Bivalent, trivalent, and tetravalent constructs were generated, as well as an innovative pentameric Nb complex, to provoke avidity effects. In our cellular assays, these trimeric, tetrameric, and pentameric Nbs have a higher apoptotic capacity than monomeric Nbs and seem to mimic the activity of the natural TRAIL ligand on various cancer cells.
Asunto(s)
Antineoplásicos Inmunológicos/farmacología , Receptores del Ligando Inductor de Apoptosis Relacionado con TNF/agonistas , Anticuerpos de Dominio Único/farmacología , Animales , Antineoplásicos Inmunológicos/química , Apoptosis/efectos de los fármacos , Línea Celular Tumoral , Modelos Animales de Enfermedad , Epítopos/química , Epítopos/inmunología , Epítopos/metabolismo , Humanos , Ratones , Unión Proteica , Receptores de IgG/química , Receptores de IgG/metabolismo , Receptores del Ligando Inductor de Apoptosis Relacionado con TNF/química , Proteínas Recombinantes , Anticuerpos de Dominio Único/química , Ligando Inductor de Apoptosis Relacionado con TNF/farmacología , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
The tumor microenvironment of numerous prevalent cancer types is abundantly infiltrated with tumor-associated macrophages (TAMs). Macrophage mannose receptor (MMR or CD206) expressing TAMs have been shown to be key promoters of tumor progression and major opponents of successful cancer therapy. Therefore, depleting MMR+ TAMs is an interesting approach to synergize with current antitumor therapies. We studied the potential of single-domain antibodies (sdAbs) specific for MMR to target proteins to MMR+ TAMs. Anti-MMR sdAbs were genetically coupled to a reporter protein, mWasabi (wasabi green, WG), generating sdAb "drug" fusion proteins (SFPs), referred to as WG-SFPs. The resulting WG-SFPs were highly efficient in targeting MMR+ macrophages both in vitro and in vivo. As we showed that second mitochondria-derived activator of caspase (SMAC) mimetics modulate MMR+ macrophages, we further coupled the anti-MMR sdAb to an active form of SMAC, referred to as tSMAC. The resulting tSMAC-SFPs were able to bind and upregulate caspase3/7 activity in MMR+ macrophages in vitro. In conclusion, we report the proof-of-concept of an elegant approach to conjugate anti-MMR sdAbs to proteins, which opens new avenues for targeted manipulation of MMR+ tumor-promoting TAMs.
Asunto(s)
Sistemas de Liberación de Medicamentos , Lectinas Tipo C/metabolismo , Macrófagos/efectos de los fármacos , Lectinas de Unión a Manosa/metabolismo , Receptores de Superficie Celular/metabolismo , Anticuerpos de Dominio Único/administración & dosificación , Animales , Proteínas Reguladoras de la Apoptosis/administración & dosificación , Proteínas Reguladoras de la Apoptosis/farmacología , Femenino , Células HEK293 , Humanos , Macrófagos/metabolismo , Receptor de Manosa , Ratones Endogámicos C57BL , Proteínas Mitocondriales/administración & dosificación , Proteínas Mitocondriales/farmacología , Modelos Moleculares , Neoplasias/tratamiento farmacológico , Neoplasias/metabolismo , Proteínas Recombinantes de Fusión/administración & dosificación , Proteínas Recombinantes de Fusión/farmacología , Anticuerpos de Dominio Único/farmacología , Microambiente Tumoral/efectos de los fármacosRESUMEN
Single-domain antibody fragments, also called nanobodies (Nbs), are increasingly being used as targeting molecular tools for imaging and/or targeted radionuclide therapy. To translate these tools to the clinic, it is preferred to obtain a homogeneous, well-defined, and well-characterized product. It has been shown that Sortase A, a transpeptidase found in Staphylococcus aureus, catalyzes the site-specific conjugation between a recognition oligopeptide (LPXTG, known as sortag) and an oligoglycine functionalized probe. This versatile technique manages to couple various molecular reagents, such as biotin, fluorophores, bifunctional chelators, etc., to the target protein containing the sortag. This chapter focuses on the site-specific coupling of a bifunctional chelator (e.g., CHX-A"-DTPA) to a Nb equipped with a C-terminal sortag. The chelator conjugated to the Nb can be radiolabeled with 111In or 177Lu for SPECT imaging or targeted radionuclide therapy, respectively.
Asunto(s)
Radiactividad , Anticuerpos de Dominio Único/metabolismo , Coloración y Etiquetado/métodos , Aminoaciltransferasas/metabolismo , Proteínas Bacterianas/metabolismo , Secuencia de Bases , Cisteína Endopeptidasas/metabolismo , Escherichia coli/genética , Escherichia coli/metabolismo , Genes Bacterianos , Vectores Genéticos/metabolismo , Transformación GenéticaRESUMEN
Tumor-associated macrophages (TAMs) with high expression levels of the Macrophage Mannose Receptor (MMR, CD206) exhibit a strong angiogenic and immune suppressive activity. Thus, they are a highly attractive target in cancer immunotherapy, with the aim to modulate their protumoral behavior. Here, we introduce polymer nanogels as potential drug nanocarriers which were site-specifically decorated with a Nanobody (Nb) specific for the MMR. Using azide-functionalized RAFT chain transfer agents, they provide access to amphiphilic reactive ester block copolymers that self-assemble into micelles and are afterwards core-cross-linked toward fully hydrophilic nanogels with terminal azide groups on their surface. MMR-targeting Nb can site-selectively be functionalized with one single cyclooctyne moiety by maleimide-cysteine chemistry under mildly reducing conditions which enables successful chemoorthogonal conjugation to the nanogels. The resulting Nb-functionalized nanogels were highly efficient in targeting MMR-expressing cells and TAMs both in vitro and in vivo. We believe that these findings pave the road for targeted eradication or modulation of pro-tumoral MMRhigh TAMs.
Asunto(s)
Anticuerpos Monoclonales/inmunología , Portadores de Fármacos/síntesis química , Inmunoterapia/métodos , Lectinas Tipo C/inmunología , Macrófagos/efectos de los fármacos , Lectinas de Unión a Manosa/inmunología , Neoplasias/terapia , Receptores de Superficie Celular/inmunología , Alquinos , Azidas , Reacción de Cicloadición , Humanos , Receptor de Manosa , Micelas , Neoplasias/inmunología , PolímerosRESUMEN
There are presently no reliable ways to quantify endocrine cell mass (ECM) in vivo, which prevents an accurate understanding of the progressive beta cell loss in diabetes or following islet transplantation. To address this unmet need, we coupled RNA sequencing of human pancreatic islets to a systems biology approach to identify new biomarkers of the endocrine pancreas. Dipeptidyl-Peptidase 6 (DPP6) was identified as a target whose mRNA expression is at least 25-fold higher in human pancreatic islets as compared to surrounding tissues and is not changed by proinflammatory cytokines. At the protein level, DPP6 localizes only in beta and alpha cells within the pancreas. We next generated a high-affinity camelid single-domain antibody (nanobody) targeting human DPP6. The nanobody was radiolabelled and in vivo SPECT/CT imaging and biodistribution studies were performed in immunodeficient mice that were either transplanted with DPP6-expressing Kelly neuroblastoma cells or insulin-producing human EndoC-ßH1 cells. The human DPP6-expressing cells were clearly visualized in both models. In conclusion, we have identified a novel beta and alpha cell biomarker and developed a tracer for in vivo imaging of human insulin secreting cells. This provides a useful tool to non-invasively follow up intramuscularly implanted insulin secreting cells.
Asunto(s)
Dipeptidil-Peptidasas y Tripeptidil-Peptidasas/metabolismo , Células Secretoras de Insulina/citología , Proteínas del Tejido Nervioso/metabolismo , Canales de Potasio/metabolismo , Transporte de Proteínas , Tomografía Computarizada por Tomografía Computarizada de Emisión de Fotón Único/métodos , Anticuerpos de Dominio Único/metabolismo , Coloración y Etiquetado/métodos , Animales , Humanos , Ratones , Análisis de Secuencia de ARNRESUMEN
Purpose: Camelid single-domain antibody-fragments (sdAb) have beneficial pharmacokinetic properties, and those targeted to HER2 can be used for imaging of HER2-overexpressing cancer. Labeled with a therapeutic radionuclide, they may be used for HER2-targeted therapy. Here, we describe the generation of a 131I-labeled sdAb as a theranostic drug to treat HER2-overexpressing cancer.Experimental Design: Anti-HER2 sdAb 2Rs15d was labeled with 131I using [131I]SGMIB and evaluated in vitro Biodistribution was evaluated in two HER2+ murine xenograft models by micro-SPECT/CT imaging and at necropsy, and under challenge with trastuzumab and pertuzumab. The therapeutic potential of [131I]SGMIB-2Rs15d was investigated in two HER2+ tumor mouse models. A single-dose toxicity study was performed in mice using unlabeled [127I]SGMIB-sdAb at 1.4 mg/kg. The structure of the 2Rs15d-HER2 complex was determined by X-ray crystallography.Results: [131I]SGMIB-2Rs15d bound specifically to HER2+ cells (Kd = 4.74 ± 0.39 nmol/L). High and specific tumor uptake was observed in both BT474/M1 and SKOV-3 tumor xenografted mice and surpassed kidney levels by 3 hours. Extremely low uptake values were observed in other normal tissues at all time points. The crystal structure revealed that 2Rs15d recognizes HER2 Domain 1, consistent with the lack of competition with trastuzumab and pertuzumab observed in vivo [131I]SGMIB-2Rs15d alone, or in combination with trastuzumab, extended median survival significantly. No toxicity was observed after injecting [127I]SGMIB-2Rs15d.Conclusions: These findings demonstrate the theranostic potential of [131I]SGMIB-2Rs15d. An initial scan using low radioactive [*I]SGMIB-2Rs15d allows patient selection and dosimetry calculations for subsequent therapeutic [131I]SGMIB-2Rs15d and could thereby impact therapy outcome on HER2+ breast cancer patients. Clin Cancer Res; 23(21); 6616-28. ©2017 AACR.
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Neoplasias de la Mama/radioterapia , Receptor ErbB-2/antagonistas & inhibidores , Anticuerpos de Dominio Único/administración & dosificación , Nanomedicina Teranóstica , Animales , Neoplasias de la Mama/tratamiento farmacológico , Neoplasias de la Mama/genética , Neoplasias de la Mama/patología , Línea Celular Tumoral , Cristalografía por Rayos X , Femenino , Humanos , Radioisótopos de Yodo/administración & dosificación , Radioisótopos de Yodo/química , Ratones , Radiometría , Receptor ErbB-2/química , Receptor ErbB-2/genética , Tomografía Computarizada por Tomografía Computarizada de Emisión de Fotón Único , Anticuerpos de Dominio Único/química , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
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.
Asunto(s)
Bencenosulfonatos/administración & dosificación , Indoles/administración & dosificación , Nanopartículas/metabolismo , Neoplasias/diagnóstico , Anticuerpos de Dominio Único/metabolismo , Distribución Tisular/efectos de los fármacos , Animales , Células CHO , Línea Celular , Línea Celular Tumoral , Cricetulus , Femenino , Ratones , Ratones Desnudos , Imagen Molecular/métodos , Neoplasias/metabolismo , Receptor ErbB-2/metabolismo , Espectroscopía Infrarroja Corta/métodos , Cirugía Asistida por Computador/métodosRESUMEN
Gold nanoparticles hold great promise as anti-cancer theranostic agents against cancer by actively targeting the tumor cells. As this potential has been supported numerously during in vitro experiments, the effective application is hampered by our limited understanding and control of the interactions within complex in vivo biological systems. When these nanoparticles are exposed to a biological environment, their surfaces become covered with proteins and biomolecules, referred to as the protein corona, reducing the active targeting capabilities. We demonstrate a chemical strategy to overcome this issue by reducing the protein corona's thickness by blocking the active groups of the self-assembled monolayer on gold nanostars. An optimal blocking agent, 2-mercapto ethanol, has been selected based on charge and length of the carbon chain. By using a nanobody as a biological ligand of the human epidermal growth factor 2 receptor (HER2), the active targeting is demonstrated in vitro and in vivo in an experimental tumor model by using darkfield microscopy and photoacoustic imaging. In this study, we have established gold nanostars as a conceivable theranostic agent with a specificity for HER2-positive tumors.
Asunto(s)
Sistemas de Liberación de Medicamentos/métodos , Mercaptoetanol/química , Neoplasias Ováricas/inmunología , Corona de Proteínas/química , Receptor ErbB-2/inmunología , Anticuerpos de Dominio Único/química , Anticuerpos de Dominio Único/inmunología , Animales , Células CHO , Línea Celular Tumoral , Cricetulus , Femenino , Oro/química , Humanos , Nanopartículas del Metal/química , Neoplasias Ováricas/patología , Neoplasias Ováricas/terapia , Anticuerpos de Dominio Único/ultraestructura , Nanomedicina Teranóstica/métodos , Resultado del TratamientoRESUMEN
A generic site-specific conjugation method that generates a homogeneous product is of utmost importance in tracer development for molecular imaging and therapy. We explored the protein-ligation capacity of the enzyme Sortase A to label camelid single-domain antibody-fragments, also known as nanobodies. The versatility of the approach was demonstrated by conjugating independently three different imaging probes: the chelating agents CHX-A"-DTPA and NOTA for single-photon emission computed tomography (SPECT) with indium-111 and positron emission tomography (PET) with gallium-68, respectively, and the fluorescent dye Cy5 for fluorescence reflectance imaging (FRI). After a straightforward purification process, homogeneous single-conjugated tracer populations were obtained in high yield (30-50%). The enzymatic conjugation did not affect the affinity of the tracers, nor the radiolabeling efficiency or spectral characteristics. In vivo, the tracers enabled the visualization of human epidermal growth factor receptor 2 (HER2) expressing BT474M1-tumors with high contrast and specificity as soon as 1 h post injection in all three imaging modalities. These data demonstrate Sortase A-mediated conjugation as a valuable strategy for the development of site-specifically labeled camelid single-domain antibody-fragments for use in multiple molecular imaging modalities. Copyright © 2016 John Wiley & Sons, Ltd.
Asunto(s)
Aminoaciltransferasas/química , Proteínas Bacterianas/química , Cisteína Endopeptidasas/química , Imagen Multimodal/métodos , Anticuerpos de Dominio Único/química , Coloración y Etiquetado/métodos , Animales , Línea Celular Tumoral , Xenoinjertos , Humanos , Ratones , Neoplasias/diagnóstico por imagen , Imagen Óptica/métodos , Tomografía de Emisión de Positrones/métodos , Receptor ErbB-2/análisis , Receptor ErbB-2/química , Receptor ErbB-2/inmunología , Anticuerpos de Dominio Único/inmunología , Tomografía Computarizada de Emisión de Fotón Único/métodosRESUMEN
INTRODUCTION: Radioimmunotracers are a promising class of companion diagnostics for precision medicine. They are composed of an antibody-based targeting agent and a radiolabeled imaging probe. Together with the tendency towards the use of small antibody-derived fragments, the employed conjugation method is gaining increasing attention. Conventional bioconjugation methods result in heterogeneous tracer populations of which the single elements can differ in immunoreactivity, pharmacokinetic behavior and stability. Site-specific conjugation strategies try to overcome these shortcomings and facilitate radioimmunotracer delivery, characterization and manufacturing. AREAS COVERED: An overview is provided of site-specific conjugation strategies for use in radioimmunotracer development. Currently applied strategies are discussed, together with other emerging site-specific conjugation methods that are applicable to diabodies, single-chain variable fragments (scFvs) and camelid single-domain antibody-fragments (sdAbs or nanobodies). EXPERT OPINION: The ultimate goal of site-specific bioconjugation strategies is to allow precise control over the conjugation site, to result in homogenous tracer populations, and to be versatile in use with different imaging probes. Chemoenzymatic methods appear to be promising in this respect.
Asunto(s)
Anticuerpos/administración & dosificación , Anticuerpos de Dominio Único/administración & dosificación , Humanos , Fragmentos de Inmunoglobulinas/administración & dosificaciónRESUMEN
Site-specific labeling of molecular imaging probes allows the development of a homogeneous tracer population. The resulting batch-to-batch reproducible pharmacokinetic and pharmacodynamic properties are of great importance for clinical translation. Camelid single-domain antibody-fragments (sdAbs)-the recombinantly produced antigen-binding domains of heavy-chain antibodies, also called Nanobodies-are proficient probes for molecular imaging. To safeguard their intrinsically high binding specificity and affinity and to ensure the tracer's homogeneity, we developed a generic strategy for the site-specific labeling of sdAbs via a thio-ether bond. The unpaired cysteine was introduced at the carboxyl-terminal end of the sdAb to eliminate the risk of antigen binding interference. The spontaneous dimerization and capping of the unpaired cysteine required a reduction step prior to conjugation. This was optimized with the mild reducing agent 2-mercaptoethylamine in order to preserve the domain's stability. As a proof-of-concept the reduced probe was subsequently conjugated to maleimide-DTPA, for labeling with indium-111. A single conjugated tracer was obtained and confirmed via mass spectrometry. The specificity and affinity of the new sdAb-based imaging probe was validated in a mouse xenograft tumor model using a modified clinical lead compound targeting the human epidermal growth factor receptor 2 (HER2) cancer biomarker. These data provide a versatile and standardized strategy for the site-specific labeling of sdAbs. The conjugation to the unpaired cysteine results in the production of a homogeneous group of tracers and is a multimodal alternative to the technetium-99m labeling of sdAbs.
Asunto(s)
Camelus/inmunología , Cisteína/química , Imagen Molecular , Neoplasias Experimentales/diagnóstico , Anticuerpos de Dominio Único/química , Anticuerpos de Dominio Único/inmunología , Animales , Reacciones Antígeno-Anticuerpo , Biomarcadores de Tumor/análisis , Cisteína/inmunología , Femenino , Humanos , Ratones , Ratones Desnudos , Modelos Moleculares , Sondas Moleculares/química , Sondas Moleculares/inmunología , Ingeniería de Proteínas , Receptor ErbB-2/análisis , Distribución Tisular , Células Tumorales CultivadasRESUMEN
PURPOSE: Molecular imaging has the potential to provide quantitative information about specific biological aspects of developing atherosclerotic lesions. This requires the generation of reliable, highly specific plaque tracers. This study reports a new camelid single-domain antibody fragment (sdAb) targeting the Lectin-like oxidized low-density lipoprotein receptor (LOX-1), a biomarker for the detection and molecular phenotyping of vulnerable atherosclerotic plaques. PROCEDURES: A camelid sdAb was generated and selected for high affinity binding to LOX-1. Ex vivo biodistribution and in vivo single photon emission computed tomography (SPECT)/computed tomography (CT) imaging studies were performed in wild-type mice and in fat-fed atherosclerotic apolipoprotein E-deficient mice with (99m)Tc-labeled sdAbs. Gamma-counting and autoradiography analyses were performed on dissected aorta segments with different degrees of plaque burden. The specificity of the LOX-1-targeting sdAb was evaluated by blocking with unlabeled sdAb or by comparison with a nontargeting (99m)Tc-labeled control sdAb. RESULTS: We generated a sdAb binding LOX-1 with a KD of 280 pM ± 62 pM affinity. After (99m)Tc-labeling, the tracer had radiochemical purity higher then 99 % and retained specificity in in vitro binding studies. Tracer blood clearance was fast with concomitant high kidney retention. At 3 h after injection, uptake in tissues other than plaques was low and not different than background, suggesting a restricted expression pattern of LOX-1. Conversely, uptake in aortic segments increased with plaque content and was due to specific LOX-1 binding. In vivo SPECT/CT imaging 160 min after injection in atherosclerotic mice confirmed specific targeting of LOX-1-expressing aortic plaques. CONCLUSIONS: The LOX-sdAb specifically targets LOX-1-expressing atherosclerotic plaques within hours after injection. The possibility to image LOX-1 rapidly after administration combined with the favourable biodistribution of a sdAb are beneficial for molecular phenotyping of atherosclerotic plaques and the generation of a future prognostic tracer.
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Apolipoproteínas E/deficiencia , Placa Aterosclerótica/metabolismo , Receptores Depuradores de Clase E/metabolismo , Anticuerpos de Dominio Único/inmunología , Secuencia de Aminoácidos , Animales , Aorta/metabolismo , Aorta/patología , Biomarcadores/metabolismo , Células CHO , Camelus , Cricetinae , Cricetulus , Femenino , Ratones Endogámicos C57BL , Datos de Secuencia Molecular , Placa Aterosclerótica/patología , Unión Proteica , Anticuerpos de Dominio Único/química , Tecnecio , Distribución TisularRESUMEN
99mTc-tricarbonyl chemistry provides an elegant technology to site-specifically radiolabel histidine-tagged biomolecules. Considering their unique biochemical properties, this straightforward technology is particularly suited for Nanobodies. This chapter gives a detailed guide to generate highly specific Nanobody-derived radiotracers for both in vitro binding studies and in vivo molecular imaging.
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Histidina/química , Marcaje Isotópico/métodos , Anticuerpos de Dominio Único/química , Tecnecio/químicaRESUMEN
Molecular imaging is a noninvasive method to measure specific biological processes in animal models and patients using imaging. In recent years there has been a tremendous evolution in hardware and software for imaging purposes. This progress has created an urgent need for new labeled targeted molecular probes. The unique physicochemical and pharmacokinetic properties of Nanobodies match the requirements of the ideal molecular imaging tracer. Preclinical studies show strong and specific targeting in vivo with rapid clearance of unbound probe resulting in high contrasted images at early time points after intravenous administration. These data suggest that the Nanobody platform might become a generic method for the development of next generation molecular imaging probes.