RESUMEN
Detection of useful cellular targets has strongly stimulated personalized tumor-targeted imaging and therapy approaches, also involving synthesis and evaluation of nuclear imaging probes with potential for clinical applications. Reviews of preclinical and translational studies concerning such probes, including radiolabeled antibodies, nanobodies, affibodies, peptides, small molecule inhibitors, and nanoparticles, are presented in this issue. As most tracers described in these articles have been developed for the field of cancer imaging and radionuclide therapy, the current article on preclinical studies will focus on cancer research as well. The main steps in developing a nuclear probe for clinical application for radionuclide imaging and therapy, after identification of a suitable molecular target on tumor cells, comprise: 1) synthesis and radiolabeling of the probe; 2) in vitro characterization, such as the evaluation of target binding affinity; 3) in vivo evaluation to assess the biodistribution and tumor targeting capability, for radionuclide therapy purposes also dosimetry studies to determine the absorbed doses and efficacy; 4) radiolabeled probes that successfully pass such tests as well as toxicological studies may enter clinical evaluation. For preclinical testing of radiolabeled probes various relevant in vitro and in vivo models dedicated to oncological research have been developed along with preclinical imaging platforms, including positron emission tomography (PET) and single photon emission computed tomography (SPECT) systems, in combination with magnetic resonance imaging (MRI) or computed tomography (CT). These developments hold great promise for fast translation of new candidate probes from preclinical validation into the clinic. This overview article describes preclinical studies typically being performed to bring a new radiopharmaceutical into clinical oncology practice. It also aims to raise awareness of confounding factors during translation of preclinical studies and ways to overcome them.
Asunto(s)
Imagen Molecular/métodos , Neoplasias/diagnóstico por imagen , Neoplasias/radioterapia , Radiofármacos , Radioterapia/métodos , Animales , Evaluación Preclínica de Medicamentos , Humanos , Técnicas In VitroRESUMEN
Adoptive immunotherapy using γδ T cells harnesses their natural role in tumor immunosurveillance. The efficacy of this approach is enhanced by aminobisphosphonates such as zoledronic acid and alendronic acid, both of which promote the accumulation of stimulatory phosphoantigens in target cells. However, the inefficient and nonselective uptake of these agents by tumor cells compromises the effective clinical exploitation of this principle. To overcome this, we have encapsulated aminobisphosphonates within liposomes. Expanded Vγ9Vδ2 T cells from patients and healthy donors displayed similar phenotype and destroyed autologous and immortalized ovarian tumor cells, following earlier pulsing with either free or liposome-encapsulated aminobisphosphonates. However, liposomal zoledronic acid proved highly toxic to SCID Beige mice. By contrast, the maximum tolerated dose of liposomal alendronic acid was 150-fold higher, rendering it much more suited to in vivo use. When injected into the peritoneal cavity, free and liposomal alendronic acid were both highly effective as sensitizing agents, enabling infused γδ T cells to promote the regression of established ovarian tumors by over one order of magnitude. Importantly however, liposomal alendronic acid proved markedly superior compared with free drug following i.v. delivery, exploiting the "enhanced permeability and retention effect" to render advanced tumors susceptible to γδ T cell-mediated shrinkage. Although folate targeting of liposomes enhanced the sensitization of folate receptor-α(+) ovarian tumor cells in vitro, this did not confer further therapeutic advantage in vivo. These findings support the development of an immunotherapeutic approach for ovarian and other tumors in which adoptively infused γδ T cells are targeted using liposomal alendronic acid.
Asunto(s)
Alendronato/administración & dosificación , Carcinoma/terapia , Inmunoterapia Adoptiva/métodos , Neoplasias Ováricas/terapia , Linfocitos T/efectos de los fármacos , Alendronato/química , Animales , Carcinoma/inmunología , Línea Celular Tumoral , Citotoxicidad Inmunológica , Femenino , Humanos , Inmunización , Liposomas/química , Ratones , Ratones SCID , Neoplasias Ováricas/inmunología , Receptores de Antígenos de Linfocitos T gamma-delta/metabolismo , Linfocitos T/inmunología , Linfocitos T/trasplante , Carga Tumoral/efectos de los fármacos , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
PURPOSE: Human epidermal growth factor receptor-2 (HER2) overexpression is a predictor of response to anti-HER2 therapy in breast and gastric cancer. Currently, HER2 status is assessed by tumour biopsy, but this may not be representative of the larger tumour mass or other metastatic sites, risking misclassification and selection of suboptimal therapy. The designed ankyrin repeat protein (DARPin) G3 binds HER2 with high affinity at an epitope that does not overlap with trastuzumab and is biologically inert. We hypothesized that radiolabelled DARPin G3 would be capable of selectively imaging HER2-positive tumours, and aimed to identify a suitable format for clinical application. METHODS: G3 DARPins tagged with hexahistidine (His6) or with histidine glutamate (HE)3 and untagged G3 DARPins were manufactured using a GMP-compatible Pichia pastoris protocol and radiolabelled with (125)I, or with (111)In via DOTA linked to a C-terminal cysteine. BALB/c mice were injected with radiolabelled G3 and tissue biodistribution was evaluated by gamma counting. The lead construct ((HE)3-G3) was assessed in mice bearing HER2-positive human breast tumour (BT474) xenografts. RESULTS: For both isotopes, (HE)3-G3 had significantly lower liver uptake than His6-G3 and untagged G3 counterparts in non-tumour-bearing mice, and there was no significantly different liver uptake between His6-G3 and untagged G3. (HE)3-G3 was taken forward for evaluation in mice bearing HER2-positive tumour xenografts. The results demonstrated that radioactivity from (111)In-(HE)3-G3 was better maintained in tumours and cleared faster from serum than radioactivity from (125)I-(HE)3-G3, achieving superior tumour-to-blood ratios (343.7 ± 161.3 vs. 22.0 ± 11.3 at 24 h, respectively). On microSPECT/CT, (111)In-labelled and (125)I-labelled (HE)3-G3 could image HER2-positive tumours at 4 h after administration, but there was less normal tissue uptake of radioactivity with (111)In-(HE)3-G3. Preadministration of trastuzumab did not affect the uptake of (HE)3-G3 by HER2-positive tumours. CONCLUSION: Radiolabelled DARPin (HE)3-G3 is a versatile radioligand with potential to allow the acquisition of whole-body HER2 scans on the day of administration.
Asunto(s)
Repetición de Anquirina , Complejos de Coordinación/farmacocinética , Radiofármacos/farmacocinética , Receptor ErbB-2/metabolismo , Tomografía Computarizada de Emisión de Fotón Único , Animales , Línea Celular Tumoral , Femenino , Humanos , Radioisótopos de Indio/farmacocinética , Neoplasias Mamarias Experimentales/diagnóstico por imagen , Ratones , Ratones Endogámicos BALB C , Unión Proteica , Proteínas Recombinantes de Fusión/farmacocinética , Distribución TisularRESUMEN
Genomic changes affecting tumour suppressor genes are fundamental to cancer. We applied SNP array analysis to a panel of testicular germ cell tumours to search for novel tumour suppressor genes and identified a frequent small deletion on 6q25.3 affecting just one gene, ZDHHC14. The expression of ZDHHC14, a putative protein palmitoyltransferase with unknown cellular function, was decreased at both RNA and protein levels in testicular germ cell tumours. ZDHHC14 expression was also significantly decreased in a panel of prostate cancer samples and cell lines. In addition to our findings of genetic and protein expression changes in clinical samples, inducible overexpression of ZDHHC14 led to reduced cell viability and increased apoptosis through the classic caspase-dependent apoptotic pathway and heterozygous knockout of ZDHHC14 increased [CORRECTED] cell colony formation ability. Finally, we confirmed our in vitro findings of the tumour suppressor role of ZDHHC14 in a mouse xenograft model, showing that overexpression of ZDHHC14 inhibits tumourigenesis. Thus, we have identified a novel tumour suppressor gene that is commonly down-regulated in testicular germ cell tumours and prostate cancer, as well as given insight into the cellular functional role of ZDHHC14, a potential protein palmitoyltransferase that may play a key protective role in cancer.
Asunto(s)
Aciltransferasas/genética , Genes Supresores de Tumor , Neoplasias de Células Germinales y Embrionarias/genética , Neoplasias de la Próstata/genética , Neoplasias Testiculares/genética , Aciltransferasas/metabolismo , Animales , Apoptosis , Línea Celular Tumoral , Proliferación Celular , Supervivencia Celular , Regulación hacia Abajo , Eliminación de Gen , Perfilación de la Expresión Génica/métodos , Regulación Enzimológica de la Expresión Génica , Regulación Neoplásica de la Expresión Génica , Células HEK293 , Humanos , Masculino , Ratones , Ratones Desnudos , Neoplasias de Células Germinales y Embrionarias/enzimología , Neoplasias de Células Germinales y Embrionarias/patología , Análisis de Secuencia por Matrices de Oligonucleótidos , Polimorfismo de Nucleótido Simple , Neoplasias de la Próstata/enzimología , Neoplasias de la Próstata/patología , Neoplasias de la Próstata/prevención & control , Interferencia de ARN , Neoplasias Testiculares/enzimología , Neoplasias Testiculares/patología , Factores de Tiempo , Transfección , Carga TumoralRESUMEN
The ErbB network is dysregulated in many solid tumors. To exploit this, we have developed a chimeric Ag receptor (CAR) named T1E28z that targets several pathogenetically relevant ErbB dimers. T1E28z is coexpressed with a chimeric cytokine receptor named 4αß (combination termed T4), enabling the selective expansion of engineered T cells using IL-4. Human T4(+) T cells exhibit antitumor activity against several ErbB(+) cancer types. However, ErbB receptors are also expressed in several healthy tissues, raising concerns about toxic potential. In this study, we have evaluated safety of T4 immunotherapy in vivo using a SCID beige mouse model. We show that the human T1E28z CAR efficiently recognizes mouse ErbB(+) cells, rendering this species suitable to evaluate preclinical toxicity. Administration of T4(+) T cells using the i.v. or intratumoral routes achieves partial tumor regression without clinical or histopathologic toxicity. In contrast, when delivered i.p., tumor reduction is accompanied by dose-dependent side effects. Toxicity mediated by T4(+) T cells results from target recognition in both tumor and healthy tissues, leading to release of both human (IL-2/IFN-γ) and murine (IL-6) cytokines. In extreme cases, outcome is lethal. Both toxicity and IL-6 release can be ameliorated by prior macrophage depletion, consistent with clinical data that implicate IL-6 in this pathogenic event. These data demonstrate that CAR-induced cytokine release syndrome can be modeled in mice that express target Ag in an appropriate distribution. Furthermore, our findings argue that ErbB-retargeted T cells can achieve therapeutic benefit in the absence of unacceptable toxicity, providing that route of administration and dose are carefully optimized.
Asunto(s)
Inmunoterapia Adoptiva , Neoplasias/inmunología , Proteínas Oncogénicas v-erbB/metabolismo , Proteínas Recombinantes de Fusión/uso terapéutico , Linfocitos T/metabolismo , Animales , Línea Celular , Humanos , Interferón gamma/biosíntesis , Interferón gamma/metabolismo , Interleucina-2/biosíntesis , Interleucina-2/metabolismo , Interleucina-4 , Interleucina-6/biosíntesis , Interleucina-6/metabolismo , Macrófagos , Ratones , Ratones SCID , Neoplasias/terapia , Transducción de SeñalRESUMEN
Carbon nanotubes (CNTs) have been proposed as one of the most promising nanomaterials to be used in biomedicine for their applications in drug/gene delivery as well as biomedical imaging. The present study developed radio-labeled iron oxide decorated multi-walled CNTs (MWNT) as dual magnetic resonance (MR) and single photon emission computed tomography (SPECT) imaging agents. Hybrids containing different amounts of iron oxide were synthesized by in situ generation. Physicochemical characterisations revealed the presence of superparamagnetic iron oxide nanoparticles (SPION) granted the magnetic properties of the hybrids. Further comprehensive examinations including high resolution transmission electron microscopy (HRTEM), fast Fourier transform simulations (FFT), X-ray diffraction (XRD) and X-ray photoelectron spectroscopy (XPS) assured the conformation of prepared SPION as γ-Fe2O3. High r2 relaxivities were obtained in both phantom and in vivo MRI compared to the clinically approved SPION Endorem®. The hybrids were successfully radio-labeled with technetium-99m through a functionalized bisphosphonate and enabled SPECT/CT imaging and γ-scintigraphy to quantitatively analyze the biodistribution in mice. No abnormality was found by histological examination and the presence of SPION and MWNT were identified by Perls stain and Neutral Red stain, respectively. TEM images of liver and spleen tissues showed the co-localization of SPION and MWNT within the same intracellular vesicles, indicating the in vivo stability of the hybrids after intravenous injection. The results demonstrated the capability of the present SPION-MWNT hybrids as dual MRI and SPECT contrast agents for in vivo use.
RESUMEN
OBJECTIVES: The synovial endothelium targeting peptide (SyETP) CKSTHDRLC has been identified previously and was shown to preferentially localise to synovial xenografts in the human/severe combined immunodeficient (SCID) mouse chimera model of rheumatoid arthritis (RA). The objective of the current work was to generate SyETP-anti-inflammatory-cytokine fusion proteins that would deliver bioactive cytokines specifically to human synovial tissue. METHODS: Fusion proteins consisting of human interleukin (IL)-4 linked via a matrix metalloproteinase (MMP)-cleavable sequence to multiple copies of either SyETP or scrambled control peptide were expressed in insect cells, purified by Ni-chelate chromatography and bioactivity tested in vitro. The ability of SyETP to retain bioactive cytokine in synovial but not control skin xenografts in SCID mice was determined by in vivo imaging using nano-single-photon emission computed tomography-computed tomography (nano-SPECT-CT) and measuring signal transducer and activator of transcription 6 (STAT6) phosphorylation in synovial grafts following intravenous administration of the fusion protein. RESULTS: In vitro assays confirmed that IL-4 and the MMP-cleavable sequence were functional. IL-4-SyETP augmented production of IL-1 receptor antagonist (IL-1ra) by fibroblast-like synoviocytes (FLS) stimulated with IL-1ß in a dose-dependent manner. In vivo imaging showed that IL-4-SyETP was retained in synovial but not in skin tissue grafts and the period of retention was significantly enhanced through increasing the number of SyETP copies from one to three. Finally, retention correlated with increased bioactivity of the cytokine as quantified by STAT6 phosphorylation in synovial grafts. CONCLUSIONS: The present work demonstrates that SyETP specifically delivers fused IL-4 to human rheumatoid synovium transplanted into SCID mice, thus providing a proof of concept for peptide-targeted tissue-specific immunotherapy in RA. This technology is potentially applicable to other biological treatments providing enhanced potency to inflammatory sites and reducing systemic toxicity.
Asunto(s)
Artritis Reumatoide , Citocinas/administración & dosificación , Sistemas de Liberación de Medicamentos/métodos , Inmunoterapia/métodos , Interleucina-4/administración & dosificación , Proteínas Recombinantes de Fusión/administración & dosificación , Animales , Artritis Reumatoide/tratamiento farmacológico , Modelos Animales de Enfermedad , Humanos , Ratones , Ratones SCID , Imagen Multimodal , Péptidos/administración & dosificación , Tomografía de Emisión de Positrones , Membrana Sinovial/efectos de los fármacos , Membrana Sinovial/metabolismo , Tomografía Computarizada por Rayos X , Trasplante HeterólogoRESUMEN
Radiolabeled neuropeptides are widely investigated to diagnose and therapy of tumors. These peptides get internalization after binding with particular receptors at the surface of cells and finally move to lysosome. Internalization into tumor cells helps in mapping the infected site. Minigastrin peptide analogues (MG-CL1-4) were synthesised and labeled with 111-In radioisotope under different sets of conditions for imaging CCk-2 receptor bearing tumors. Different parameters such as temperature (80-100°C), pH (4-12), incubation time (5-30 minutes) and dilution effect were investigated to get the maximum labeling yield and stability. The results indicated that MG-CL1-4 is successfully labeled with indium-111 at pH 4.5 with heating at 98°C for 15 minute. At these conditions i.e. heating, pH and incubation minimum oxidized and maximum labeling yield, more than 94 %, was obtained. The labeling stability was studied by incubating the radiolabeled complex for predefined time points in PBSA and blood serum. Results show that more than 90% radiolabeled MG-CL1-4 remained intact.
Asunto(s)
Gastrinas/síntesis química , Radioisótopos de Indio , Marcaje Isotópico , Gastrinas/sangre , Gastrinas/normas , Humanos , Concentración de Iones de Hidrógeno , Marcaje Isotópico/normas , Oxidación-Reducción , Estabilidad Proteica , Control de Calidad , Temperatura , Factores de TiempoRESUMEN
Sunitinib is a potent and clinically approved tyrosine kinase inhibitor that can suppress tumour growth by inhibiting angiogenesis. However, conflicting data exist regarding the effects of this drug on the growth of metastases in preclinical models. Here we use 4T1 and RENCA tumour cells, which both form lung metastases in Balb/c mice, to re-address the effects of sunitinib on the progression of metastatic disease in mice. We show that treatment of mice with sunitinib prior to intravenous injection of tumour cells can promote the seeding and growth of 4T1 lung metastases, but not RENCA lung metastases, showing that this effect is cell line dependent. However, increased metastasis occurred only upon administration of a very high sunitinib dose, but not when lower, clinically relevant doses were used. Mechanistically, high dose sunitinib led to a pericyte depletion effect in the lung vasculature that correlated with increased seeding of metastasis. By administering sunitinib to mice after intravenous injection of tumour cells, we demonstrate that while sunitinib does not inhibit the growth of 4T1 lung tumour nodules, it does block the growth of RENCA lung tumour nodules. This contrasting response was correlated with increased myeloid cell recruitment and persistent vascularisation in 4T1 tumours, whereas RENCA tumours recruited less myeloid cells and were more profoundly devascularised upon sunitinib treatment. Finally, we show that progression of 4T1 tumours in sunitinib treated mice results in increased hypoxia and increased glucose metabolism in these tumours and that this is associated with a poor outcome. Taken together, these data suggest that the effects of sunitinib on tumour progression are dose-dependent and tumour model-dependent. These findings have relevance for understanding how anti-angiogenic agents may influence disease progression when used in the adjuvant or metastatic setting in cancer patients.
Asunto(s)
Antineoplásicos/uso terapéutico , Indoles/uso terapéutico , Metástasis de la Neoplasia/tratamiento farmacológico , Pirroles/uso terapéutico , Animales , Ratones , Ratones Endogámicos BALB C , Tomografía de Emisión de Positrones , Sunitinib , Tomografía Computarizada por Rayos XRESUMEN
Pharmacological targeting of individual ErbB receptors elicits antitumor activity, but is frequently compromised by resistance leading to therapeutic failure. Here, we describe an immunotherapeutic approach that exploits prevalent and fundamental mechanisms by which aberrant upregulation of the ErbB network drives tumorigenesis. A chimeric antigen receptor named T1E28z was engineered, in which the promiscuous ErbB ligand, T1E, is fused to a CD28 + CD3ζ endodomain. Using a panel of ErbB-engineered 32D hematopoietic cells, we found that human T1E28z⺠T cells are selectively activated by all ErbB1-based homodimers and heterodimers and by the potently mitogenic ErbB2/3 heterodimer. Owing to this flexible targeting capability, recognition and destruction of several tumor cell lines was achieved by T1E28⺠T cells in vitro, comprising a wide diversity of ErbB receptor profiles and tumor origins. Furthermore, compelling antitumor activity was observed in mice bearing established xenografts, characterized either by ErbB1/2 or ErbB2/3 overexpression and representative of insidious or rapidly progressive tumor types. Together, these findings support the clinical development of a broadly applicable immunotherapeutic approach in which the propensity of solid tumors to dysregulate the extended ErbB network is targeted for therapeutic gain.
Asunto(s)
Transformación Celular Neoplásica/genética , Multimerización de Proteína/efectos de los fármacos , Receptor ErbB-2/genética , Linfocitos T/metabolismo , Animales , Neoplasias de la Mama/inmunología , Neoplasias de la Mama/terapia , Línea Celular Tumoral , Femenino , Ingeniería Genética , Neoplasias de Cabeza y Cuello/inmunología , Neoplasias de Cabeza y Cuello/terapia , Humanos , Interleucina-4/inmunología , Interleucina-4/metabolismo , Ratones , Ratones SCID , Receptor ErbB-2/química , Receptor ErbB-2/metabolismo , Receptores de Antígenos/genética , Receptores de Antígenos/metabolismo , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/metabolismo , Linfocitos T/inmunología , Transducción Genética , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
The fibrous shape of carbon nanotubes (CNTs) raises concern that they may pose an asbestos-like inhalation hazard, leading to the development of diseases, especially mesothelioma. Direct instillation of long and short CNTs into the pleural cavity, the site of mesothelioma development, produced asbestos-like length-dependent responses. The response to long CNTs and long asbestos was characterized by acute inflammation, leading to progressive fibrosis on the parietal pleura, where stomata of strictly defined size limit the egress of long, but not short, fibers. This was confirmed by demonstrating clearance of short, but not long, CNT and nickel nanowires and by visualizing the migration of short CNTs from the pleural space by single-photon emission computed tomographic imaging. Our data confirm the hypothesis that, although a proportion of all deposited particles passes through the pleura, the pathogenicity of long CNTs and other fibers arises as a result of length-dependent retention at the stomata on the parietal pleura.
Asunto(s)
Progresión de la Enfermedad , Inflamación/complicaciones , Inflamación/patología , Nanotubos de Carbono/química , Pleura/patología , Cavidad Pleural/patología , Animales , Proliferación Celular , Epitelio/patología , Fibrosis , Ganglios Linfáticos/patología , Mediastino/patología , Ratones , Nanotubos de Carbono/ultraestructura , Nanocables/ultraestructura , Tamaño de la Partícula , Pleura/ultraestructura , Cavidad Pleural/ultraestructura , Factores de Tiempo , Tomografía Computarizada de Emisión de Fotón Único , Tomografía Computarizada por Rayos XRESUMEN
OBJECTIVE: To isolate recombinant antibodies with specificity for human arthritic synovium and to develop targeting reagents with joint-specific delivery capacity for therapeutic and/or diagnostic applications. METHODS: In vivo single-chain Fv (scFv) antibody phage display screening using a human synovial xenograft model was used to isolate antibodies specific to the microvasculature of human arthritic synovium. Single-chain Fv antibody tissue-specific reactivity was assessed by immunostaining of synovial tissues from normal controls and from patients with rheumatoid arthritis and osteoarthritis, normal human tissue arrays, and tissues from other patients with inflammatory diseases displaying neovasculogenesis. In vivo scFv antibody tissue-specific targeting capacity was examined in the human synovial xenograft model using both (125)I-labeled and biotinylated antibody. RESULTS: We isolated a novel recombinant human antibody, scFv A7, with specificity for the microvasculature of human arthritic synovium. We showed that in vivo, this antibody could efficiently target human synovial microvasculature in SCID mice transplanted with human arthritic synovial xenografts. Our results demonstrated that scFv A7 antibody had no reactivity with the microvasculature or with other cellular components found in a comprehensive range of normal human tissues including normal human synovium. Further, we showed that the reactivity of the scFv A7 antibody was not a common feature of neovasculogenesis associated with chronic inflammatory conditions. CONCLUSION: Here we report for the first time the identification of an scFv antibody, A7, that specifically recognizes an epitope expressed in the microvasculature of human arthritic synovium and that has the potential to be developed as a joint-specific pharmaceutical.
Asunto(s)
Especificidad de Anticuerpos/inmunología , Artritis Reumatoide/tratamiento farmacológico , Osteoartritis/tratamiento farmacológico , Anticuerpos de Cadena Única/uso terapéutico , Animales , Artritis Reumatoide/inmunología , Modelos Animales de Enfermedad , Epítopos/inmunología , Humanos , Ratones , Ratones SCID , Microvasos , Osteoartritis/inmunología , Proteínas Recombinantes/inmunología , Proteínas Recombinantes/uso terapéutico , Anticuerpos de Cadena Única/inmunología , Membrana Sinovial/irrigación sanguínea , Membrana Sinovial/inmunología , Trasplante HeterólogoRESUMEN
Getting rid of the tubes: An assessment of the retention of functionalized multi-walled carbon nanotubes (MWNTs) in the organs of mice was carried out using single photon emission computed tomography and quantitative scintigraphy (see scheme). Increasing the degree of functionalization on MWNTs enhanced renal clearance, while lower functionalization promoted reticuloendethelial system accumulation.
Asunto(s)
Aminas/química , Nanotubos de Carbono/química , Aminas/farmacocinética , Animales , Ratones , Microscopía Electrónica de Transmisión , Modelos Moleculares , Estructura Molecular , Nanotubos de Carbono/ultraestructura , Especificidad de ÓrganosRESUMEN
Adoptive immunotherapy using chimeric antigen receptor-engrafted T cells is a promising emerging therapy for cancer. Prior to clinical testing, it is mandatory to evaluate human therapeutic cell products in meaningful in vivo pre-clinical models. Here, we describe the use of fused single-photon emission CT-CT imaging to monitor real-time migration of chimeric antigen receptor-engineered T cells in immune compromised (SCID Beige) mice. Following intravenous administration, human T cells migrate in a highly similar manner to that reported in man, but penetrate poorly into established tumors. By contrast, when delivered via intraperitoneal or subcutaneous routes, T cells remain at the site of inoculation with minimal systemic absorption-irrespective of the presence or absence of tumor. Together, these data support the validity of pre-clinical testing of human T-cell immunotherapy in SCID Beige mice. In light of their established efficacy, regional administration of engineered human T cells represents an attractive therapeutic option to minimize toxicity in the treatment of selected malignancies.
Asunto(s)
Movimiento Celular , Inmunoterapia Adoptiva/efectos adversos , Inmunoterapia Adoptiva/métodos , Receptores de Antígenos de Linfocitos T/genética , Linfocitos T/inmunología , Traslado Adoptivo/métodos , Animales , Carcinoma de Células Escamosas/inmunología , Carcinoma de Células Escamosas/terapia , Línea Celular Tumoral , Humanos , Ratones , Ratones SCID , Mucina-1/inmunología , Proteínas Recombinantes de Fusión , Linfocitos T/citología , Linfocitos T/trasplante , Trasplante Heterólogo , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
Functionalization of nanomaterials for precise biomedical function is an emerging trend in nanotechnology. Carbon nanotubes are attractive as multifunctional carrier systems because payload can be encapsulated in internal space whilst outer surfaces can be chemically modified. Yet, despite potential as drug delivery systems and radiotracers, such filled-and-functionalized carbon nanotubes have not been previously investigated in vivo. Here we report covalent functionalization of radionuclide-filled single-walled carbon nanotubes and their use as radioprobes. Metal halides, including Na(125)I, were sealed inside single-walled carbon nanotubes to create high-density radioemitting crystals and then surfaces of these filled-sealed nanotubes were covalently modified with biantennary carbohydrates, improving dispersibility and biocompatibility. Intravenous administration of Na(125)I-filled glyco-single-walled carbon nanotubes in mice was tracked in vivo using single-photon emission computed tomography. Specific tissue accumulation (here lung) coupled with high in vivo stability prevented leakage of radionuclide to high-affinity organs (thyroid/stomach) or excretion, and resulted in ultrasensitive imaging and delivery of unprecedented radiodose density. Nanoencapsulation of iodide within single-walled carbon nanotubes enabled its biodistribution to be completely redirected from tissue with innate affinity (thyroid) to lung. Surface functionalization of (125)I-filled single-walled carbon nanotubes offers versatility towards modulation of biodistribution of these radioemitting crystals in a manner determined by the capsule that delivers them. We envisage that organ-specific therapeutics and diagnostics can be developed on the basis of the nanocapsule model described here.
Asunto(s)
Nanotecnología/tendencias , Nanotubos de Carbono/química , Acetilglucosamina/metabolismo , Metabolismo de los Hidratos de Carbono , Glicosilación , Humanos , Marcaje Isotópico/métodos , Microscopía Electrónica de Transmisión de Rastreo/métodos , Nanotecnología/métodos , Oxidación-Reducción , Radioisótopos/metabolismo , Radioisótopos/farmacocinética , Estómago/diagnóstico por imagen , Glándula Tiroides/diagnóstico por imagen , Distribución Tisular , Tomografía Computarizada por Rayos X/métodosRESUMEN
The integrin alphavbeta6 is expressed only on epithelia and then usually only during processes of tissue remodelling including cancer, where its high expression correlates with reduced survival. Thus, alphavbeta6 represents an important target for imaging and therapy of cancer and new molecular-specific targeting agents are required. We have developed A20FMDV2, a peptide derived from the VP1 coat protein of foot-and-mouth-disease virus that binds specifically and stably to alphavbeta6. Using a newly generated pair of isogenic human cell lines that differ only in alphavbeta6 expression, it was shown, using biodistribution and SPECT imaging, that indium-111-labelled A20FMDV2 locates specifically to alphavbeta6-expressing tissues in vivo, achieving at least seven-times higher retention in alphavbeta6-positive than in alphavbeta6-negative tumours. In further studies with MCF10.DCIS.COM and MCF10A.CA1a breast carcinoma cell lines, which express alphavbeta6 endogenously, the radiopeptide achieved similar levels of tumour retention and permitted excellent discriminatory imaging of tumours. Thus, A20FMDV2 can be used for molecular-specific targeting of alphavbeta6 for imaging in vivo the often more aggressive, alphavbeta6-positive cancers. In the future, A20FMDV2 could serve also to deliver therapy to these same cancers.
Asunto(s)
Antígenos de Neoplasias/metabolismo , Integrinas/metabolismo , Neoplasias Mamarias Experimentales/diagnóstico por imagen , Animales , Femenino , Humanos , Radioisótopos de Indio/metabolismo , Radioisótopos de Indio/farmacocinética , Neoplasias Mamarias Experimentales/metabolismo , Ratones , Ratones Endogámicos , Trasplante de Neoplasias , Ácido Pentético/farmacocinética , Fragmentos de Péptidos/metabolismo , Fragmentos de Péptidos/farmacocinética , Tomografía de Emisión de Positrones/métodos , Radiofármacos/farmacocinética , Distribución Tisular , Tomografía Computarizada de Emisión de Fotón Único/métodos , Trasplante Heterólogo , Células Tumorales CultivadasRESUMEN
The palliation of pain due to bone metastases using targeted compounds containing beta-emitters such as rhenium-188 ((188)Re) is an accepted and effective form of treatment. Here, we describe the efficient synthesis and preclinical evaluation of (188)Re(CO)(3)-dipicolylamine(DPA)-alendronate, a novel bifunctional bisphosphonate for the palliative treatment of bone metastases. (188)Re(CO)(3)-DPA-alendronate can be easily synthesized with high specific activities and yields (18.8 GBq/mg, radiochemical yield > or =96%) in two steps using kit-based methodology, and in contrast with the clinically approved bisphosphonate (186/188)Re-HEDP, it forms inert, single species that have been well-characterized. In vivo imaging and biodistribution studies demonstrate that (188)Re(CO)(3)-DPA-alendronate is superior to (188)Re-HEDP in targeting and accumulating in areas of high metabolic bone activity while having low soft-tissue uptake. In addition to these studies, a simple and convenient new method for purifying its precursor, fac-[(188)Re(CO)(3)(H(2)O)(3)](+), is described.
Asunto(s)
Alendronato/farmacocinética , Antineoplásicos/farmacocinética , Neoplasias Óseas/radioterapia , Neoplasias Óseas/secundario , Difosfonatos/farmacocinética , Radiofármacos/farmacocinética , Renio/farmacocinética , Alendronato/síntesis química , Alendronato/química , Animales , Antineoplásicos/síntesis química , Antineoplásicos/química , Difosfonatos/síntesis química , Difosfonatos/química , Femenino , Humanos , Ratones , Ratones Endogámicos BALB C , Radioisótopos/química , Radioisótopos/farmacocinética , Radiofármacos/síntesis química , Radiofármacos/química , Renio/químicaRESUMEN
Somatic mutations in skin cancers and other ultraviolet (UV)-exposed cells are typified by C>T and CC>TT substitutions at dipyrimidine sequences; however, many oncogenic "driver" mutations in melanoma do not fit this UV signature. Here, we use genome sequencing to characterize mutations in yeast repeatedly irradiated with UV light. Analysis of ~50,000 UV-induced mutations reveals abundant non-canonical mutations, including T>C, T>A, and AC>TT substitutions. These mutations display transcriptional asymmetry that is modulated by nucleotide excision repair (NER), indicating that they are caused by UV photoproducts. Using a sequencing method called UV DNA endonuclease sequencing (UVDE-seq), we confirm the existence of an atypical thymine-adenine photoproduct likely responsible for UV-induced T>A substitutions. Similar non-canonical mutations are present in skin cancers, which also display transcriptional asymmetry and dependence on NER. These include multiple driver mutations, most prominently the recurrent BRAF V600E and V600K substitutions, suggesting that mutations arising from rare, atypical UV photoproducts may play a role in melanomagenesis.
Asunto(s)
Melanoma/genética , Mutación/efectos de la radiación , Rayos Ultravioleta/efectos adversos , Secuencia de Bases/genética , Daño del ADN/genética , Reparación del ADN/genética , Melanoma/metabolismo , Mutación/genética , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismo , Análisis de Secuencia de ADN/métodosRESUMEN
UNLABELLED: The aim of this study was to determine the effects of assisted coordination by amino acids such as histidine and glutamic acid on the function of (99m)Tc-labeled gastrin peptide-hydrazinonicotinamide (HYNIC) conjugates and their ability to target cholecystokinin-R in small-animal models. METHODS: Three peptide-HYNIC conjugates containing the -AYGWMDF-NH2 C-terminal sequence and combinations of histidine, glutamic acid, and glycine were synthesized, radiolabeled with (99m)Tc/(99)Tc using either tricine or ethylenediaminediacetic acid as a coligand, and analyzed by the high-performance liquid chromatography and liquid chromatography-mass spectrometric techniques. Stability, receptor binding, and internalization and in vivo targeting in AR42J-bearing mice were assessed. RESULTS: When radiolabeling was performed using tricine as a coligand, the insertion of a histidine residue near the HYNIC residue resulted in the displacement of one molecule of tricine from the coordination sphere, a reduction in the number of radiolabeled species formed, an improvement in the in vitro stability, an increase in the rate of radiopeptide internalization, and a significant improvement in tumor uptake in vivo. When radiolabeling was performed using ethylenediaminediacetic acid as a coligand, no effect on coligand binding, homogeneity, or in vitro stability was observed but a significant improvement in the internalization in vitro and tumor uptake in vivo was again found. All of the complexes formed showed similar receptor affinity in competitive radioligand binding assays. CONCLUSION: The insertion of histidine into the sequence of peptide-HYNIC conjugates can result in more stable, more homogeneous complexes that show improvements in tumor-targeting performance both in vitro and in vivo.
Asunto(s)
Aumento de la Imagen/métodos , Neoplasias Pancreáticas/diagnóstico por imagen , Neoplasias Pancreáticas/metabolismo , Radiofármacos/farmacocinética , Aminoácidos/química , Animales , Marcaje Isotópico/métodos , Tasa de Depuración Metabólica , Ratones , Ratones Desnudos , Oligopéptidos/síntesis química , Oligopéptidos/farmacocinética , Especificidad de Órganos , Compuestos Organometálicos/síntesis química , Compuestos Organometálicos/farmacocinética , Compuestos de Organotecnecio/síntesis química , Compuestos de Organotecnecio/farmacocinética , Cintigrafía , Radiofármacos/síntesis química , Distribución TisularRESUMEN
OBJECTIVE: The purpose of this study was to explore the development of a pre-clinical nuclear imaging model as a tool for testing novel radiopharmaceutical agents for imaging and/or delivery systems to human tissues. Here we report for the first time the imaging of human synovial tissue transplanted into SCID mice using a radiolabelled anti-E-selectin antibody and NanoSPECT/CT technology. METHODS: Human synovium was transplanted into SCID mice. Two to three weeks post-transplantation tissue vasculature was stimulated with TNF-alpha by intra-graft injection 5 h prior to intravenous injection of (111)In-labelled anti-E-selectin or isotype control antibody. At 1, 4, 24 and 48 h animals were imaged and transplant activity quantified. RESULTS: Activity was detectable in the grafts at all time points, with clear delineation of the transplants in the reconstructed images. A significant difference in graft radioactivity was observed at 4 and 24 h with a significantly higher uptake (P < 0.05) of (111)In-anti-E-selectin compared with isotype control antibody. CONCLUSIONS: This article highlights NanoSPECT/CT imaging in the SCID mouse chimeric model as a powerful tool for the pre-clinical development of radiopharmaceutical and delivery agents targeting human synovial tissue in vivo.