RESUMEN
PURPOSE: Cadherin-17 (CDH17) is a calcium-dependent cell adhesion protein that is overexpressed in several adenocarcinomas, including gastric, colorectal, and pancreatic adenocarcinoma. High levels of CDH17 have been linked to metastatic disease and poor prognoses in patients with these malignancies, fueling interest in the protein as a target for diagnostics and therapeutics. Herein, we report the synthesis, in vitro validation, and in vivo evaluation of a CDH17-targeted 89Zr-labeled immunoPET probe. METHODS: The CDH17-targeting mAb D2101 was modified with an isothiocyanate-bearing derivative of desferrioxamine (DFO) to produce a chelator-bearing immunoconjugate - DFO-D2101 - and flow cytometry and surface plasmon resonance (SPR) were used to interrogate its antigen-binding properties. The immunoconjugate was then radiolabeled with zirconium-89 (t1/2 ~ 3.3 days), and the serum stability and immunoreactive fraction of [89Zr]Zr-DFO-D2101 were determined. Finally, [89Zr]Zr-DFO-D2101's performance was evaluated in a trio of murine models of pancreatic ductal adenocarcinoma (PDAC): subcutaneous, orthotopic, and patient-derived xenografts (PDX). PET images were acquired over the course of 5 days, and terminal biodistribution data were collected after the final imaging time point. RESULTS: DFO-D2101 was produced with a degree of labeling of ~ 1.1 DFO/mAb. Flow cytometry with CDH17-expressing AsPC-1 cells demonstrated that the immunoconjugate binds to its target in a manner similar to its parent mAb, while SPR with recombinant CDH17 revealed that D2101 and DFO-D2101 exhibit nearly identical KD values: 8.2 × 10-9 and 6.7 × 10-9 M, respectively. [89Zr]Zr-DFO-D2101 was produced with a specific activity of 185 MBq/mg (5.0 mCi/mg), remained >80% stable in human serum over the course of 5 days, and boasted an immunoreactive fraction of >0.85. In all three murine models of PDAC, the radioimmunoconjugate yielded high contrast images, with high activity concentrations in tumor tissue and low uptake in non-target organs. Tumoral activity concentrations reached as high as >60 %ID/g in two of the cohorts bearing PDXs. CONCLUSION: Taken together, these data underscore that [89Zr]Zr-DFO-D2101 is a highly promising probe for the non-invasive visualization of CDH17 expression in PDAC. We contend that this radioimmunoconjugate could have a significant impact on the clinical management of patients with both PDAC and gastrointestinal adenocarcinoma, most likely as a theranostic imaging tool in support of CDH17-targeted therapies.
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Cadherinas , Radioisótopos , Circonio , Animales , Humanos , Ratones , Cadherinas/metabolismo , Línea Celular Tumoral , Neoplasias Pancreáticas/diagnóstico por imagen , Neoplasias Pancreáticas/metabolismo , Deferoxamina/química , Adenocarcinoma/diagnóstico por imagen , Inmunoconjugados/farmacocinética , Anticuerpos Monoclonales/farmacocinética , Distribución Tisular , Tomografía de Emisión de PositronesRESUMEN
Despite decades of work, small-cell lung cancer (SCLC) remains a frustratingly recalcitrant disease. Both diagnosis and treatment are challenges: low-dose computed tomography (the approved method used for lung cancer screening) is unable to reliably detect early SCLC, and the malignancy's 5 year survival rate stands at a paltry 7%. Clearly, the development of novel diagnostic and therapeutic tools for SCLC is an urgent, unmet need. CD133 is a transmembrane protein that is expressed at low levels in normal tissue but is overexpressed by a variety of tumors, including SCLC. We previously explored CD133 as a biomarker for a novel autoantibody-to-immunopositron emission tomography (PET) strategy for the diagnosis of SCLC, work that first suggested the promise of the antigen as a radiotheranostic target in the disease. Herein, we report the in vivo validation of a pair of CD133-targeted radioimmunoconjugates for the PET imaging and radioimmunotherapy of SCLC. To this end, [89Zr]Zr-DFO-αCD133 was first interrogated in a trio of advanced murine models of SCLCâi.e., orthotopic, metastatic, and patient-derived xenograftsâwith the PET probe consistently producing high activity concentrations (>%ID/g) in tumor lesions combined with low uptake in healthy tissues. Subsequently, a variant of αCD133 labeled with the ß-emitting radiometal 177Luâ[177Lu]Lu-DTPA-Aâ³-CHX-αCD133âwas synthesized and evaluated in a longitudinal therapy study in a subcutaneous xenograft model of SCLC, ultimately revealing that treatment with a dose of 9.6 MBq of the radioimmunoconjugate produced a significant increase in median survival compared to a control cohort. Taken together, these data establish CD133 as a viable target for the nuclear imaging and radiopharmaceutical therapy of SCLC.
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Neoplasias Pulmonares , Carcinoma Pulmonar de Células Pequeñas , Humanos , Animales , Ratones , Neoplasias Pulmonares/diagnóstico por imagen , Neoplasias Pulmonares/radioterapia , Detección Precoz del Cáncer , Línea Celular Tumoral , Carcinoma Pulmonar de Células Pequeñas/diagnóstico por imagen , Carcinoma Pulmonar de Células Pequeñas/radioterapia , Tomografía de Emisión de Positrones/métodosRESUMEN
Microplastics and nanoplastics have become ubiquitous environmental pollutants. The threat these plastics pose to human health has fueled research focused on their pathophysiology and toxicology, yet many of their fundamental properties - for example, their in vivo pharmacokinetics - remain poorly understood. In this investigation, we have harnessed positron emission tomography (PET) to track the in vivo fate of micro- and nanoplastics administered to mice intratracheally and intravenously. To this end, 1 µm and 20 nm diameter amine-functionalized polystyrene particles were modified with an isothiocyanate-bearing variant of desferrioxamine (DFO) and radiolabeled with the positron-emitting radiometal [89Zr]Zr4+. Both radioplastics - [89Zr]Zr-DFO-PS1000 and [89Zr]Zr-DFO-PS20 - were produced in â¼95% radiochemical yield and found to be >85% stable to demetallation over one week at 37 °C in human serum and simulated lung fluid. The incubation of [89Zr]Zr-DFO-PS1000 and [89Zr]Zr-DFO-PS20 with MH-S cells revealed that the majority of the former were phagocytosed by alveolar macrophages within 4 h, while the latter largely evaded consumption. Finally, the in vivo behavior of the radioplastics was interrogated in mice upon intravenous and intratracheal administration. PET imaging and biodistribution experiments revealed that the intravenously injected plastics accumulated primarily in the liver and spleen, yielding hepatic radioactivity concentrations of 101 ± 48 %ID/g and 92 ± 22 %ID/g at 168 h post-injection for [89Zr]Zr-DFO-PS1000 and [89Zr]Zr-DFO-PS20, respectively. In contrast, the mice that received the radioplastics via intratracheal installation displayed the highest uptake in the lungs at the end of one week: 4 ± 2 %ID/g for [89Zr]Zr-DFO-PS1000 and 32 ± 6 %ID/g for [89Zr]Zr-DFO-PS20. Ultimately, this work illustrates the critical role that the route of exposure plays in the bioaccumulation of plastic particles, reveals that size dramatically influences the pulmonary retention of inhaled particles, and underscores the value of PET imaging as a tool for studying the pharmacokinetics of environmental pollutants.
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Contaminantes Ambientales , Radioisótopos , Humanos , Animales , Ratones , Microplásticos , Distribución Tisular , Plásticos , Deferoxamina , Tomografía de Emisión de Positrones/métodos , Circonio , Línea Celular TumoralRESUMEN
The worldwide proliferation of persistent environmental pollutants is accelerating at an alarming rate. Not surprisingly, many of these pollutants pose a risk to human health. In this review, we examine recent literature in which molecular imaging and radiochemistry have been harnessed to study environmental pollutants. Specifically, these techniques offer unique ways to interrogate the pharmacokinetic profiles and bioaccumulation patterns of pollutants at environmentally relevant concentrations, thereby helping to determine their potential health risks.
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Contaminantes Ambientales , Humanos , Contaminantes Ambientales/análisis , Radioquímica , Imagen MolecularRESUMEN
Galectin-3 binding protein (Gal-3BP) is a glycoprotein that is overexpressed and secreted by several cancers and has been implicated as a marker of both tumor progression and poor prognosis in melanoma, non-small cell lung cancer, head and neck squamous cell carcinoma, and breast cancer. The expression of Gal-3BP by a variety of neoplasms makes it an enticing target for both diagnostics and therapeutics, including immuno-positron emission tomography (immunoPET) probes and antibody-drug conjugates (ADCs). Herein, we report the development, in vitro characterization, and in vivo evaluation of a pair of Gal-3BP-targeting radioimmunoconjugates for 89Zr-immunoPET. A humanized anti-Gal-3BP antibody, 1959, and its corresponding ADC, 1959-sss/DM4 (DM4 = ravtansine), were modified with desferrioxamine (DFO) to yield DFO-1959 and DFO-1959-sss/DM4 immunoconjugates bearing 1-2 DFO/monoclonal antibody. Both DFO-modified immunoconjugates retained their affinity for Gal-3BP in enzyme-linked immunosorbent assay experiments. The chelator-bearing antibodies were radiolabeled with zirconium-89 (t1/2 ≈ 3.3 d) to produce radioimmunoconjugates â [89Zr]Zr-DFO-1959 and [89Zr]Zr-DFO-1959-sss/DM4 â with high specific activity (>444 MBq/mg, >12 mCi/mg) and stability (>80% intact after 168 h in human serum at 37 °C). In mice bearing subcutaneous Gal-3BP-secreting A375-MA1 xenografts, [89Zr]Zr-DFO-1959 clearly delineated tumor tissue, reaching a maximum tumoral activity concentration (54.8 ± 15.8%ID/g) and tumor-to-background contrast (tumor-to-blood = 8.0 ± 4.6) at 120 h post-injection. The administration of [89Zr]Zr-DFO-1959 to mice bearing subcutaneous Gal-3BP-expressing melanoma patient-derived xenografts produced similarly promising results. [89Zr]Zr-DFO-1959 and [89Zr]Zr-DFO-1959-sss/DM4 exhibited nearly identical pharmacokinetic profiles in the mice bearing A375-MA1 tumors, though the latter produced higher uptake in the spleen and kidneys. Both [89Zr]Zr-DFO-1959 and [89Zr]Zr-DFO-1959-sss/DM4 effectively visualized Gal-3BP-secreting tumors in murine models of melanoma. These results suggest that both probes could play a role in the clinical imaging of Gal-3BP-expressing malignancies, particularly as companion theranostics for the identification of patients likely to respond to Gal-3BP-targeted therapeutics such as 1959-sss/DM4.
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Carcinoma de Pulmón de Células no Pequeñas , Inmunoconjugados , Neoplasias Pulmonares , Melanoma , Animales , Humanos , Ratones , Línea Celular Tumoral , Deferoxamina/química , Galectina 3 , Inmunoconjugados/química , Tomografía de Emisión de Positrones/métodos , Circonio/químicaRESUMEN
Site-specifically modified radioimmunoconjugates exhibit superior in vitro and in vivo behavior compared to analogues synthesized via traditional stochastic methods. However, the development of approaches to site-specific bioconjugation that combine high levels of selectivity, simple reaction conditions, and clinical translatability remains a challenge. Herein, we describe a novel solution to this problem: the use of dual-variable domain immunoglobulins (DVD-IgG). More specifically, we report the synthesis, in vitro evaluation, and in vivo validation of a 177Lu-labeled radioimmunoconjugate based on HER2DVD, a DVD-IgG containing the HER2-targeting variable domains of trastuzumab and the catalytic variable domains of IgG h38C2. To this end, we first modified HER2DVD with a phenyloxadiazolyl methlysulfone-modified variant of the chelator CHX-Aâ³-DTPA (PODS-CHX-A''-DTPA) and verified the site-specificity of the conjugation for the reactive lysines within the catalytic domains via chemical assay, MALDI-ToF mass spectrometry, and SDS-PAGE. The chelator-bearing immunoconjugate was subsequently labeled with [177Lu]Lu3+ to produce the completed radioimmunoconjugate, [177Lu]Lu-CHX-Aâ³-DTPAPODS-HER2DVD, in >80% radiochemical conversion and a specific activity of 29.5 ± 7.1 GBq/µmol. [177Lu]Lu-CHX-Aâ³-DTPAPODS-HER2DVD did not form aggregates upon prolonged incubation in human serum, displayed 87% stability to demetalation over a 7 days of incubation in serum, and exhibited an immunoreactive fraction of 0.95 with HER2-coated beads. Finally, we compared the pharmacokinetic profile of [177Lu]Lu-CHX-Aâ³-DTPAPODS-HER2DVD to that of a 177Lu-labeled variant of trastuzumab in mice bearing subcutaneous HER2-expressing BT-474 human breast cancer xenografts. The in vivo performance of [177Lu]Lu-CHX-Aâ³-DTPAPODS-HER2DVD matched that of 177Lu-labeled trastuzumab, with the former producing a tumoral activity concentration of 34.1 ± 12.1 %ID/g at 168 h and tumor-to-blood, tumor-to-liver, and tumor-to-kidney activity concentration ratios of 10.5, 9.6, and 21.8, respectively, at the same time point. Importantly, the DVD-IgG did not exhibit a substantially longer serum half-life than the traditional IgG despite its significantly larger size (202 kDa for the former vs 148 kDa for the latter). Taken together, these data suggest that DVD-IgGs represent a viable platform for the future development of highly effective site-specifically labeled radioimmunoconjugates for diagnostic imaging, theranostic imaging, and radioimmunotherapy.
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Neoplasias de la Mama , Inmunoconjugados , Humanos , Animales , Ratones , Femenino , Inmunoconjugados/uso terapéutico , Línea Celular Tumoral , Trastuzumab/uso terapéutico , Trastuzumab/farmacocinética , Quelantes/química , Neoplasias de la Mama/tratamiento farmacológico , Ácido Pentético/química , Inmunoglobulina G/uso terapéuticoRESUMEN
The synthesis of radioimmunoconjugates via the stochastic attachment of bifunctional chelators to lysines can yield heterogeneous products with suboptimal in vitro and in vivo behavior. In response to this, several site-selective approaches to bioconjugation have been developed, yet each has intrinsic drawbacks, such as the need for expensive reagents or the complexity of incorporating unnatural amino acids into IgGs. Herein, we describe the use of a simple and facile approach to lysine-directed site-selective bioconjugation for the generation of radioimmunoconjugates. This strategy relies upon on the selective modification of single lysine residues within each light chain of the monoclonal antibody (mAb) with a branched azide-bearing perfluorophenyl ester (PFP-bisN3) followed by the ligation of dibenzocyclooctyne (DBCO)-bearing payloads to these bioorthogonal handles via the strain-promoted azide-alkyne cycloaddition. This methodology was used to create [89Zr]Zr-SSKDFO-pertuzumab, a radioimmunoconjugate of the HER2-targeting mAb pertuzumab labeled with desferrioxamine (DFO) and the positron-emitting radiometal zirconium-89 (89Zr). [89Zr]Zr-SSKDFO-pertuzumab was compared to a pair of analogous probes: one synthesized via random lysine modification ([89Zr]Zr-DFO-pertuzumab) and another via thiol-maleimide chemistry ([89Zr]Zr-malDFO-pertuzumab). The bioconjugation strategy was assessed using ESI mass spectrometry, SDS-PAGE, and autoradiography. All three immunoconjugates demonstrated comparable binding to HER2 via flow cytometry and surface plasmon resonance (SPR), and 89Zr-labeled variants of each were synthesized in >99% radiochemical yield and molar activities of up to â¼55.5 GBq/µmol (10 mCi/mg). Subsequently, the in vivo behavior of this trio of 89Zr-immunoPET probes was interrogated in athymic nude mice bearing subcutaneous HER2-expressing BT-474 human breast cancer xenografts. [89Zr]Zr-SSKDFO-pertuzumab, [89Zr]Zr-malDFO-pertuzumab, and [89Zr]Zr-DFO-pertuzumab produced positron emission tomography (PET) images with high tumoral uptake and high tumor-to-healthy organ activity concentration ratios. A terminal biodistribution study complemented the PET results, revealing tumoral activity concentrations of 126.9 ± 50.3%ID/g, 86.9 ± 53.2%ID/g, and 92.5 ± 27.2%ID/g at 144 h post-injection for [89Zr]Zr-SSKDFO-pertuzumab, [89Zr]Zr-malDFO-pertuzumab, and [89Zr]Zr-DFO-pertuzumab, respectively. Taken together, the data clearly illustrate that this highly modular and facile approach to site-selective bioconjugation produces radioimmunoconjugates that are better-defined and more homogeneous than stochastically modified constructs and also exhibit excellent in vitro and in vivo performance. Furthermore, we contend that this lysine-directed strategy holds several key advantages over extant approaches to site-selective bioconjugation, especially in the context of production for the clinic.
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Neoplasias de la Mama , Inmunoconjugados , Alquinos , Animales , Anticuerpos Monoclonales/química , Azidas , Línea Celular Tumoral , Quelantes , Deferoxamina/química , Ésteres , Femenino , Humanos , Inmunoconjugados/química , Lisina , Maleimidas , Ratones , Ratones Desnudos , Tomografía de Emisión de Positrones/métodos , Compuestos de Sulfhidrilo , Distribución Tisular , Circonio/químicaRESUMEN
Radiolabeled antibodies have become indispensable tools in nuclear medicine. However, the natural roles of antibodies within the immune system mean that they have several intrinsic limitations as a platform for radiopharmaceuticals. In recent years, the field has increasingly turned to antibody engineering to circumvent these issues while retaining the manifold benefits of the immunoglobulin framework. In this "Focus on Molecular Imaging" review, we cover recent advances in the application of antibody engineering to immunoPET, immunoSPECT, and radioimmunotherapy. Specifically, we address how antibody engineering has been used to improve radioimmunoconjugates on four fronts: optimizing pharmacokinetics, facilitating site-specific bioconjugation, modulating Fc interactions, and creating bispecific constructs.
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Inmunoconjugados , Radioinmunoterapia , Anticuerpos , Inmunoconjugados/uso terapéutico , Imagen Molecular/métodos , Radioinmunoterapia/métodos , Radiofármacos/uso terapéuticoRESUMEN
Small cell lung cancer (SCLC) is a deadly neuroendocrine tumor for which there are no screening methods sensitive enough to facilitate early, effective intervention. We propose targeting the neuroendocrine tumor neoantigen CD133 via antibody-based early detection and PET (immunoPET) to facilitate earlier and more accurate detection of SCLC. Methods: RNA sequencing datasets, immunohistochemistry, flow cytometry, and Western blots were used to quantify CD133 expression in healthy and SCLC patients. CD133 was imaged in vivo using near-infrared fluorescence (NIRF) immunoimaging, and 89Zr immunoPET. Anti(α)-CD133 autoantibody levels were measured in SCLC patient plasma using antibody microarrays. Results: Across 6 publicly available datasets, CD133 messenger RNA was found to be higher in SCLC tumors than in other tissues, including healthy or normal adjacent lung and non-SCLC samples. Critically, the upregulation of CD133 messenger RNA in SCLC was associated with a significant increase (hazard ratio, 2.62) in death. CD133 protein was expressed in primary human SCLC, in SCLC patient-derived xenografts, and in both SCLC cell lines tested (H82 and H69). Using an H82 xenograft mouse model, we first imaged CD133 expression with NIRF. Both in vivo and ex vivo NIRF clearly showed that a fluorophore-tagged αCD133 homed to lung tumors. Next, we validated the noninvasive visualization of subcutaneous and orthotopic H82 xenografts via immunoPET. An αCD133 antibody labeled with the positron-emitting radiometal 89Zr demonstrated significant accumulation in tumor tissue while producing minimal uptake in healthy organs. Finally, plasma αCD133 autoantibodies were found in subjects from cohort studies up to 1 year before SCLC diagnosis. Conclusion: In light of these findings, we conclude that the presence of αCD133 autoantibodies in a blood sample followed by CD133-targeted 89Zr-immunoPET could be an effective early detection screening strategy for SCLC.
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Neoplasias Pulmonares , Tumores Neuroendocrinos , Carcinoma Pulmonar de Células Pequeñas , Animales , Humanos , Ratones , Carcinoma Pulmonar de Células Pequeñas/metabolismo , Tomografía de Emisión de Positrones/métodos , Ratones Desnudos , Detección Precoz del Cáncer , Neoplasias Pulmonares/metabolismo , Modelos Animales de Enfermedad , Biomarcadores , Autoanticuerpos , ARN Mensajero , Línea Celular TumoralRESUMEN
PURPOSE: Advances in our understanding of the contribution of aberrant glycosylation to the pro-oncogenic signaling and metastasis of tumor cells have reinvigorated the development of mucin-targeted therapies. Here, we validate the tumor-targeting ability of a novel monoclonal antibody (mAb), AR9.6, that binds MUC16 and abrogates downstream oncogenic signaling to confer a therapeutic response. EXPERIMENTAL DESIGN: The in vitro and ex vivo validation of the binding of AR9.6 to MUC16 was achieved via flow cytometry, radioligand binding assay (RBA), and immunohistochemistry (IHC). The in vivo MUC16 targeting of AR9.6 was validated by creating a 89Zr-labeled radioimmunoconjugate of the mAb and utilizing immunoPET and ex vivo biodistribution studies in xenograft models of human ovarian and pancreatic cancer. RESULTS: Flow cytometry, RBA, and IHC revealed that AR9.6 binds to ovarian and pancreatic cancer cells in an MUC16-dependent manner. The in vivo radiopharmacologic profile of 89Zr-labeled AR9.6 in mice bearing ovarian and pancreatic cancer xenografts confirmed the MUC16-dependent tumor targeting by the radioimmunoconjugate. Radioactivity uptake was also observed in the distant lymph nodes (LNs) of mice bearing xenografts with high levels of MUC16 expression (i.e., OVCAR3 and Capan-2). IHC analyses of these PET-positive LNs highlighted the presence of shed antigen as well as necrotic, phagocytized, and actively infiltrating neoplastic cells. The humanization of AR9.6 did not compromise its ability to target MUC16-expressing tumors. CONCLUSIONS: The unique therapeutic mechanism of AR9.6 combined with its excellent in vivo tumor targeting makes it a highly promising theranostic agent. huAR9.6 is poised for clinical translation to impact the management of metastatic ovarian and pancreatic cancers.
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Inmunoconjugados , Neoplasias Ováricas , Neoplasias Pancreáticas , Animales , Anticuerpos Monoclonales/farmacología , Apoptosis , Antígeno Ca-125 , Carcinogénesis , Línea Celular Tumoral , Femenino , Humanos , Inmunoconjugados/uso terapéutico , Proteínas de la Membrana/metabolismo , Ratones , Mucinas/metabolismo , Neoplasias Ováricas/patología , Neoplasias Pancreáticas/tratamiento farmacológico , Neoplasias Pancreáticas/patología , Radioisótopos/uso terapéutico , Distribución Tisular , Circonio , Neoplasias PancreáticasRESUMEN
The proliferation of plastics in the environment continues at an alarming rate. Plastic particles have been found to be persistent and ubiquitous pollutants in a variety of environments, including sea water, fresh water, soil, and air. In light of this phenomenon, the scientific and medical communities have become increasingly wary of the dangers posed to human health by chronic exposure to microplastics (< 5 mm diameter) and nanoplastics (< 100 nm diameter). A critical component of the study of the health effects of these pollutants is the accurate determination of their pharmacokinetic behavior in vivo. Herein, we report the first use of molecular imaging to track polystyrene (PS) micro- and nanoplastic particles in mammals. To this end, we have modified PS particles of several sizes-diameters of 20 nm, 220 nm, 1 µm, and 6 µm-with the chelator desferrioxamine (DFO) and radiolabeled these DFO-bearing particles with the positron-emitting radiometal zirconium-89 (89Zr; t1/2 ~ 3.3 d). Subsequently, positron emission tomography (PET) was used to visualize the biodistribution of these radioplastics in C57BL/6J mice at 6, 12, 24, and 48 h after ingestion. The imaging data reveal that the majority of the radioplastics remain in the gastrointestinal tract and are eliminated through the feces by 48 h post-ingestion, a result reinforced by acute biodistribution studies. Ultimately, this work suggests that nuclear imaging-and PET in particular-can be a sensitive and effective tool in the urgent and rapidly growing effort to study the in vivo behavior and potential toxicity of micro- and nanoplastics.
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Microplásticos , Nanopartículas/toxicidad , Poliestirenos , Tomografía de Emisión de Positrones , Animales , Femenino , Humanos , Ratones , Microplásticos/farmacocinética , Microplásticos/toxicidad , Poliestirenos/farmacocinética , Poliestirenos/toxicidadRESUMEN
Radiolabeled antibodies have shown promise as tools for both the nuclear imaging and endoradiotherapy of cancer, but the protracted circulation time of radioimmunoconjugates can lead to high radiation doses to healthy tissues. To circumvent this issue, we have developed an approach to positron emission tomography (PET) imaging and radioimmunotherapy (RIT) predicated on radiolabeling the antibody after it has reached its target within the body. This in vivo pretargeting strategy is based on the rapid and bio-orthogonal inverse electron demand Diels-Alder reaction between tetrazine (Tz) and trans-cyclooctene (TCO). Pretargeted PET imaging and RIT using TCO-modified antibodies in conjunction with Tz-bearing radioligands produce high activity concentrations in target tissues as well as reduced radiation doses to healthy organs compared to directly labeled radioimmunoconjugates. Herein, we describe how to prepare a TCO-modified antibody (humanized A33-TCO) as well as how to synthesize two Tz-bearing radioligands: one labeled with the positron-emitting radiometal copper-64 ([64Cu]Cu-SarAr-Tz) and one labeled with the ß-emitting radiolanthanide lutetium-177 ([177Lu]Lu-DOTA-PEG7-Tz). We also provide a detailed description of pretargeted PET and pretargeted RIT experiments in a murine model of human colorectal carcinoma. Proper training in both radiation safety and the handling of laboratory mice is required for the successful execution of this protocol.
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Química Clic/métodos , Reacción de Cicloadición , Electrones , Tomografía de Emisión de Positrones , Radioinmunoterapia , Animales , Ciclooctanos/química , Ratones Desnudos , Reproducibilidad de los ResultadosRESUMEN
Delta-like ligand 3 (DLL3) is a therapeutic target for the treatment of small cell lung cancer, neuroendocrine prostate cancer, and isocitrate dehydrogenase mutant glioma. In the clinic, DLL3-targeted 89Zr-immunoPET has the potential to aid in the assessment of disease burden and facilitate the selection of patients suitable for therapies that target the antigen. The overwhelming majority of 89Zr-labeled radioimmunoconjugates are synthesized via the random conjugation of desferrioxamine (DFO) to lysine residues within the immunoglobulin. While this approach is admittedly facile, it can produce heterogeneous constructs with suboptimal in vitro and in vivo behavior. In an effort to circumvent these issues, we report the development and preclinical evaluation of site-specifically labeled radioimmunoconjugates for DLL3-targeted immunoPET. To this end, we modified a cysteine-engineered variant of the DLL3-targeting antibody SC16-MB1 with two thiol-reactive variants of DFO: one bearing a maleimide moiety (Mal-DFO) and the other containing a phenyloxadiazolyl methyl sulfone group (PODS-DFO). In an effort to obtain immunoconjugates with a DFO-to-antibody ratio (DAR) of 2, we explored both the reduction of the antibody with tris(2-carboxyethyl) phosphine (TCEP) as well as the use of a combination of glutathione and arginine as reducing and stabilizing agents, respectively. While exerting control over the DAR of the immunoconjugate proved cumbersome using TCEP, the use of glutathione and arginine enabled the selective reduction of the engineered cysteines and thus the formation of homogeneous immunoconjugates. A head-to-head comparison of the resulting 89Zr-radioimmunoconjugates in mice bearing DLL3-expressing H82 xenografts revealed no significant differences in tumoral uptake and showed comparable radioactivity concentrations in most healthy nontarget organs. However, 89Zr-DFOPODS-DAR2SC16-MB1 produced 30% lower uptake (3.3 ± 0.5 %ID/g) in the kidneys compared to 89Zr-DFOMal-DAR2SC16-MB1 (4.7 ± 0.5 %ID/g). In addition, H82-bearing mice injected with a 89Zr-labeled isotype-control radioimmunoconjugate synthesized using PODS exhibited â¼40% lower radioactivity in the kidneys compared to mice administered its maleimide-based counterpart. Taken together, these results demonstrate the improved in vivo performance of the PODS-based radioimmunoconjugate and suggest that a stable, well-defined DAR2 radiopharmaceutical may be suitable for the clinical immunoPET of DLL3-expressing cancers.
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Inmunoconjugados/administración & dosificación , Inmunoconjugados/química , Péptidos y Proteínas de Señalización Intracelular/química , Proteínas de la Membrana/química , Neoplasias/diagnóstico por imagen , Tomografía de Emisión de Positrones/métodos , Animales , Línea Celular Tumoral , Humanos , Ratones , Ensayos Antitumor por Modelo de Xenoinjerto , Circonio/químicaRESUMEN
Metastatic melanoma can be difficult to detect until at the advanced state that decreases the survival rate of patients. Several FDA-approved BRAF inhibitors have been used for treatment of metastatic melanoma, but overall therapeutic efficacy has been limited. Lutetium-177 (177 Lu) enables simultaneous tracking of tracer accumulation with single-photon emission computed tomography and radiotherapy. Therefore, the codelivery of 177 Lu alongside chemotherapeutic agents using nanoparticles (NPs) might improve the therapeutic outcome in metastatic melanoma. Cellulose nanocrystals (CNC NPs) can particularly deliver payloads to lung capillaries in vivo. Herein, 177 Lu-labeled CNC NPs loaded with vemurafenib ([177 Lu]Lu-CNC-V NPs) is developed and the therapeutic effect in BRAF V600E mutation-harboring YUMM1.G1 murine model of lung metastatic melanoma is investigated. The [177 Lu]Lu-CNC-V NPs demonstrate favorable radiolabel stability, drug release profile, cellular uptake, and cell growth inhibition in vitro. In vivo biodistribution reveals significant retention of the [177 Lu]Lu-CNC-V NPs in the lung, liver, and spleen. Ultimately, the median survival time of animals is doubly increased after treatment with [177 Lu]Lu-CNC-V NPs compared to control groups. The enhanced therapeutic efficacy of [177 Lu]Lu-CNC-V NPs in the lung metastatic melanoma animal model provides convincing evidence for the potential of clinical translation for theranostic CNC NP-based drug delivery systems after intravenous administration.
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Melanoma , Nanopartículas , Animales , Línea Celular Tumoral , Celulosa , Sistemas de Liberación de Medicamentos , Humanos , Melanoma/tratamiento farmacológico , Ratones , Medicina de Precisión , Distribución TisularRESUMEN
Triple negative breast cancer (TNBC) has no targeted detection or treatment method. Mutant p53 (mtp53) is overexpressed in >80% of TNBCs, and the stability of mtp53 compared to the instability of wild-type p53 (wtp53) in normal cells makes mtp53 a promising TNBC target for diagnostic and theranostic imaging. We generated Cy5p53Tet, a novel nucleus-penetrating mtp53-oligomerization-domain peptide (mtp53ODP) to the tetramerization domain (TD) of mtp53. This mtp53ODP contains the p53 TD sequence conjugated to a Cy5 fluorophore for near-infrared fluorescence imaging (NIRF). In vitro co-immunoprecipitation and glutaraldehyde cross-linking showed a direct interaction between mtp53 and Cy5p53Tet. Confocal microscopy and flow cytometry demonstrated higher uptake of Cy5p53Tet in the nuclei of TNBC MDA-MB-468 cells with mtp53 R273H than in ER-positive MCF7 cells with wtp53. Furthermore, depletion of mtp53 R273H caused a decrease in the uptake of Cy5p53Tet in nuclei. In vivo analysis of the peptide in mice bearing MDA-MB-468 xenografts showed that Cy5p53Tet could be detected in tumor tissue 12 min after injection. In these in vivo experiments, significantly higher uptake of Cy5p53Tet was observed in mtp53-expressing MDA-MB-468 xenografts compared with the wtp53-expressing MCF7 tumors. Cy5p53Tet has clinical potential as an intraoperative imaging agent for fluorescence-guided surgery, and the mtp53ODP scaffold shows promise for modification in the future to enable the delivery of a wide variety of payloads including radionuclides and toxins to mtp53-expressing TNBC tumors.
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Antineoplásicos/farmacología , Núcleo Celular/efectos de los fármacos , Péptidos/farmacología , Neoplasias de la Mama Triple Negativas/tratamiento farmacológico , Proteína p53 Supresora de Tumor/metabolismo , Animales , Apoptosis/efectos de los fármacos , Línea Celular Tumoral , Proliferación Celular/efectos de los fármacos , Femenino , Humanos , Células MCF-7 , Ratones , Ratones Desnudos , Mutación/efectos de los fármacos , Neoplasias de la Mama Triple Negativas/metabolismoRESUMEN
Over the past decade, theranostic imaging has emerged as a powerful clinical tool in oncology for identifying patients likely to respond to targeted therapies and for monitoring the response of patients to treatment. Herein, we report a theranostic approach to pretargeted radioimmunotherapy (PRIT) based on a pair of radioisotopes of copper: positron-emitting copper-64 (64Cu, t1/2 = 12.7 h) and beta particle-emitting copper-67 (67Cu, t1/2 = 61.8 h). This strategy is predicated on the in vivo ligation between a trans-cyclooctene (TCO)-bearing antibody and a tetrazine (Tz)-based radioligand via the rapid and bioorthogonal inverse electron-demand Diels-Alder reaction. Longitudinal therapy studies were conducted in a murine model of human colorectal carcinoma using an immunoconjugate of the huA33 antibody modified with TCO (huA33-TCO) and a 67Cu-labeled Tz radioligand ([67Cu]Cu-MeCOSar-Tz). The injection of huA33-TCO followed 72 h later by the administration of 18.5, 37.0, or 55.5 MBq of [67Cu]Cu-MeCOSar-Tz produced a dose-dependent therapeutic response, with the median survival time increasing from 68 d for the lowest dose to >200 d for the highest. Furthermore, we observed that mice that received the highest dose of [67Cu]Cu-MeCOSar-Tz in a fractionated manner exhibited improved hematological values without sacrificing therapeutic efficacy. Dual radionuclide experiments in which a single administration of huA33-TCO was followed by separate injections of [64Cu]Cu-MeCOSar-Tz and [67Cu]Cu-MeCOSar-Tz revealed that the positron emission tomography images produced by the former accurately predicted the efficacy of the latter. In these experiments, a correlation was observed between the tumoral uptake of [64Cu]Cu-MeCOSar-Tz and the subsequent therapeutic response to [67Cu]Cu-MeCOSar-Tz.
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Radioisótopos de Cobre/farmacología , Radioisótopos de Cobre/uso terapéutico , Medicina de Precisión/métodos , Radioinmunoterapia/métodos , Animales , Anticuerpos , Línea Celular Tumoral , Neoplasias Colorrectales/tratamiento farmacológico , Reacción de Cicloadición , Relación Dosis-Respuesta a Droga , Femenino , Humanos , Inmunoconjugados , Ratones , Ratones Desnudos , Tomografía de Emisión de Positrones/métodos , Radioisótopos/farmacología , Radioisótopos/uso terapéutico , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
Ovarian cancer is the fifth leading cause of cancer deaths among women, accounting for more deaths than any other cancer of the female reproductive system. The foundation of its management consists of cytoreductive surgery (CRS) followed by systemic chemotherapy, with the completeness of surgical resection consistently identified as one of the most important prognostic factors for the disease. The goal of our investigation is the development of a near-infrared fluorescence (NIRF) imaging agent for the intraoperative imaging of high-grade serous ovarian cancer (HGSOC). As surgeons are currently limited to the visual and manual assessment of tumor tissue during CRS, this technology could facilitate more complete resections as well as serve important functions at other points in the surgical management of the disease. Elevated levels of cancer antigen 125 (CA125) have proven a useful biomarker of HGSOC, and the CA125-targeting antibody B43.13 has shown potential as a platform for immunoPET imaging in murine models of ovarian cancer. Herein, we report the development of a NIRF imaging agent based on B43.13: ssB43.13-IR800. We site-specifically modified the heavy chain glycans of B43.13 with the near-infrared dye IRDye 800CW using a chemoenzymatic approach developed in our laboratories. SDS-PAGE analysis confirmed the specificity of the conjugation reaction, and flow cytometry, immunostaining, and fluorescence microscopy verified the specific binding of ssB43.13-IR800 to CA125-expressing OVCAR3 human ovarian cancer cells. NIRF imaging studies demonstrated that ssB43.13-IR800 can be used to image CA125-expressing HGSOC tumors in subcutaneous, orthotopic, and patient-derived xenograft mouse models. Finally, ex vivo analyses confirmed that ssB43.13-IR800 can bind and identify CA125-expressing cells in primary tumor and metastatic lymph node samples from human patients with HGSOC.
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Microscopía Fluorescente/métodos , Imagen Óptica/métodos , Neoplasias Ováricas/diagnóstico por imagen , Neoplasias Ováricas/patología , Espectroscopía Infrarroja Corta/métodos , Animales , Biomarcadores de Tumor/metabolismo , Antígeno Ca-125/metabolismo , Línea Celular Tumoral , Femenino , Humanos , Ratones , Neoplasias Ováricas/metabolismoRESUMEN
High expression levels of the tumor-associated antigen MUC1 have been correlated with tumor aggressiveness, poor response to therapy, and poor survival in several tumor types, including breast, pancreatic, and epithelial ovarian cancer. Herein, we report the synthesis, characterization, and in vivo evaluation of a novel radioimmunoconjugate for the immuno-positron emission tomography (immunoPET) imaging of MUC1 expression based on the AR20.5 antibody. To this end, we modified AR20.5 with the chelator desferrioxamine (DFO) and labeled it with the positron-emitting radiometal zirconium-89 (t1/2 ~3.3 d) to produce [89Zr]Zr-DFO-AR20.5. In subsequent in vivo experiments in athymic nude mice bearing subcutaneous MUC1-expressing ovarian cancer xenografts, [89Zr]Zr-DFO-AR20.5 clearly delineated tumor tissue, producing a tumoral activity concentration of 19.1 ± 6.4 percent injected dose per gram (%ID/g) at 120 h post-injection and a tumor-to-muscle activity concentration ratio of 42.4 ± 10.6 at the same time point. Additional PET imaging experiments in mice bearing orthotopic MUC1-expressing ovarian cancer xenografts likewise demonstrated that [89Zr]Zr-DFO-AR20.5 enables the visualization of tumor tissue-including metastatic lesions-with promising tumor-to-background contrast.
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Anticuerpos Monoclonales/química , Anticuerpos Antineoplásicos/química , Inmunoconjugados/química , Metástasis Linfática/diagnóstico por imagen , Mucina-1/metabolismo , Neoplasias/diagnóstico por imagen , Radioisótopos/química , Circonio/química , Animales , Anticuerpos Monoclonales/metabolismo , Anticuerpos Monoclonales/farmacocinética , Anticuerpos Antineoplásicos/metabolismo , Disponibilidad Biológica , Deferoxamina/química , Femenino , Expresión Génica , Xenoinjertos , Humanos , Inmunoconjugados/metabolismo , Inmunoconjugados/farmacocinética , Metástasis Linfática/patología , Ratones , Ratones Desnudos , Mucina-1/genética , Neoplasias/metabolismo , Neoplasias/patología , Tomografía de Emisión de Positrones/métodos , Unión Proteica , Relación Señal-Ruido , Distribución TisularRESUMEN
The N-linked biantennary glycans on the heavy chain of immunoglobulin G (IgG) antibodies (mAbs) are instrumental in the recognition of the Fc region by Fc-gamma receptors (FcγR). In the case of full-length mAb-based imaging tracers targeting immune cell populations, these Fc:FcγR interactions can potentially deplete effector cells responsible for tumor clearance. To bypass this problem, we hypothesize that the enzymatic removal of the Fc glycans will disrupt Fc:FcγR interactions and spare tracer-targeted immune cells from depletion during immunopositron emission tomography (immunoPET) imaging. Herein, we compared the in vitro and in vivo properties of 89Zr-radiolabeled CD8-specific murine mAb (anti-CD8wt, clone 2.43), a well-known depleting mAb, and its deglycosylated counterpart (anti-CD8degly). Deglycosylation was achieved via enzymatic treatment with the peptide: N-glycosidase F (PNGaseF). Both anti-CD8wt and anti-CD8degly mAbs were conjugated to p-SCN-Bn-desferrioxamine (DFO) and labeled with 89Zr. Bindings of both DFO-conjugated mAbs to FcγR and CD8+ splenocytes were compared. In vivo imaging and distribution studies were conducted to examine the specificity and pharmacokinetics of the radioimmunoconjugates in tumor-naive and CT26 colorectal tumor-bearing mice. Ex vivo analysis of CD8+ T cell population in spleens and tumors obtained postimaging were measured via flow cytometry and qRT-PCR. The removal of the Fc glycans from anti-CD8wt was confirmed via SDS-PAGE. A reduction in FcγR interaction was exhibited by DFO-anti-CD8degly, while its binding to CD8 remained unchanged. Tissue distribution showed similar pharmacokinetics of [89Zr]Zr-DFO-anti-CD8degly and the wt radioimmunoconjugate. In vivo blocking studies further demonstrated retained specificity of the deglycosylated radiotracer for CD8. From the imaging studies, no difference in accumulation in both spleens and tumors was observed between both radiotracers. Results from the flow cytometry analysis confirmed depletion of CD8+ T cells in spleens of mice administered with DFO-anti-CD8wt, whereas an increase in CD8+ T cells was shown with DFO-anti-CD8degly. No statistically significant difference in tumor infiltrating CD8+ T cells was observed in cohorts administered with the probes when compared to control unmodulated mice. CD8 mRNA levels from excised tumors showed increased transcripts of the antigen in mice administered with [89Zr]Zr-DFO-anti-CD8degly compared to mice imaged with [89Zr]Zr-DFO-anti-CD8wt. In conclusion, the removal of Fc glycans offers a straightforward approach to develop full length antibody-based imaging probes specifically for detecting CD8+ immune molecules with no consequential depletion of their target cell population in peripheral tissues.
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Linfocitos T CD8-positivos/metabolismo , Animales , Línea Celular , Citometría de Flujo , Humanos , Inmunoconjugados/química , Masculino , Ratones , Ratones Endogámicos BALB C , Tomografía de Emisión de Positrones , RadioquímicaRESUMEN
Recent years have played witness to the advent of nuclear theranostics: the synergistic use of "matched pair" radiopharmaceuticals for diagnostic imaging and targeted radiotherapy. In this investigation, we report the extension of this concept to in vivo pretargeting based on the rapid and bioorthogonal inverse electron demand Diels-Alder reaction between tetrazine (Tz) and trans-cyclooctene (TCO). We demonstrate that a single injection of a TCO-modified immunoconjugate can be used as a platform for pretargeted PET imaging and radiotherapy via the sequential administration of a pair of Tz-bearing radioligands labeled with the positron-emitting radiometal copper-64 (t1/2 ≈ 12.7 h) and the beta-emitting radiometal lutetium-177 (t1/2 ≈ 6.7 days). More specifically, a mouse model of human colorectal carcinoma received a dose of the A33 antigen-targeting immunoconjugate huA33-TCO, followed 24 and 48 h later by injections of [64Cu]Cu-SarAr-Tz and [177Lu]Lu-DOTA-PEG7-Tz, respectively. This approach produces high activity concentrations of both radioligands in tumor tissue (16.4 ± 2.7 %ID/g for [64Cu]Cu-SarAr-Tz at 48 h post-injection and 18.1 ± 2.1 %ID/g for [177Lu]Lu-DOTA-PEG7-Tz at 120 h post-injection) as well as promising tumor-to-healthy organ activity concentration ratios. Ultimately, we believe that this work could not only have important implications in nuclear theranostics-most excitingly with isotopologue-based radioligand pairs such as [64Cu]Cu-SarAr-Tz and [67Cu]Cu-SarAr-Tz-but also in the delivery of fractionated doses during pretargeted radioimmunotherapy.