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1.
ACS Nano ; 14(5): 5818-5835, 2020 05 26.
Artigo em Inglês | MEDLINE | ID: mdl-32347709

RESUMO

Staphylococcus aureus (S. aureus) is a highly pathogenic facultative anaerobe that in some instances resides as an intracellular bacterium within macrophages and cancer cells. This pathogen can establish secondary infection foci, resulting in recurrent systemic infections that are difficult to treat using systemic antibiotics. Here, we use reconstructed apoptotic bodies (ReApoBds) derived from cancer cells as "nano decoys" to deliver vancomycin intracellularly to kill S. aureus by targeting inherent "eat me" signaling of ApoBds. We prepared ReApoBds from different cancer cells (SKBR3, MDA-MB-231, HepG2, U87-MG, and LN229) and used them for vancomycin delivery. Physicochemical characterization showed ReApoBds size ranges from 80 to 150 nm and vancomycin encapsulation efficiency of 60 ± 2.56%. We demonstrate that the loaded vancomycin was able to kill intracellular S. aureus efficiently in an in vitro model of S. aureus infected RAW-264.7 macrophage cells, and U87-MG (p53-wt) and LN229 (p53-mt) cancer cells, compared to free-vancomycin treatment (P < 0.001). The vancomycin loaded ReApoBds treatment in S. aureus infected macrophages showed a two-log-order higher CFU reduction than the free-vancomycin treatment group. In vivo studies revealed that ReApoBds can specifically target macrophages and cancer cells. Vancomycin loaded ReApoBds have the potential to kill intracellular S. aureus infection in vivo in macrophages and cancer cells.

2.
Chem Commun (Camb) ; 56(2): 281-284, 2019 Dec 19.
Artigo em Inglês | MEDLINE | ID: mdl-31807738

RESUMO

Bioluminescence resonance energy transfer (BRET) is a commonly used assay system for studying protein-protein interactions and protein folding in vivo. Conventional BRET systems have solely depended on an overlap of the energy donor and acceptor spectra. In this study, we engineered a conceptually unique ligand-activatable BRET system (termed BRET9), where a full-length Artificial Luciferase variant 23 (ALuc23), acting as the energy donor, is sandwiched between a protein pair of interest, FRB and FKBP12, and linked to a fluorescent protein as the energy acceptor. A specific ligand, rapamycin, then activates inter- and intramolecular interactions of FRB and FKBP12, which develop molecular strain in the sandwiched ALuc23 to accelerate further folding. We found that this system greatly enhanced both the total bioluminescence spectrum and the BRET signal in the far-red (FR) region. We characterized the molecular construct by studying 18 different designs categorized into four groups. The best BRET system design allowed an approximately 5-fold enhancement of the bioluminescence intensities in the FR region. This new BRET system provides a robust ligand-activatable platform that efficiently reports FR bioluminescence signals in cells and living animal models.


Assuntos
Luciferases/química , Serina-Treonina Quinases TOR/metabolismo , Proteína 1A de Ligação a Tacrolimo/metabolismo , Animais , Técnicas de Transferência de Energia por Ressonância de Bioluminescência/métodos , Linhagem Celular Tumoral , Humanos , Ligantes , Limite de Detecção , Luciferases/genética , Proteínas Luminescentes/química , Camundongos Endogâmicos BALB C , Camundongos Nus , Mutação , Ligação Proteica , Sirolimo/química , Sirolimo/metabolismo
3.
Nat Commun ; 10(1): 4673, 2019 10 14.
Artigo em Inglês | MEDLINE | ID: mdl-31611594

RESUMO

Advances in precision molecular imaging promise to transform our ability to detect, diagnose and treat disease. Here, we describe the engineering and validation of a new cystine knot peptide (knottin) that selectively recognizes human integrin αvß6 with single-digit nanomolar affinity. We solve its 3D structure by NMR and x-ray crystallography and validate leads with 3 different radiolabels in pre-clinical models of cancer. We evaluate the lead tracer's safety, biodistribution and pharmacokinetics in healthy human volunteers, and show its ability to detect multiple cancers (pancreatic, cervical and lung) in patients at two study locations. Additionally, we demonstrate that the knottin PET tracers can also detect fibrotic lung disease in idiopathic pulmonary fibrosis patients. Our results indicate that these cystine knot PET tracers may have potential utility in multiple disease states that are associated with upregulation of integrin αvß6.


Assuntos
Antígenos de Neoplasias/metabolismo , Fibrose Pulmonar Idiopática/diagnóstico , Integrinas/metabolismo , Neoplasias/diagnóstico , Cristalografia por Raios X , Voluntários Saudáveis , Humanos , Imagem por Ressonância Magnética , Tomografia por Emissão de Pósitrons
4.
Protein Eng Des Sel ; 32(5): 231-240, 2019 12 31.
Artigo em Inglês | MEDLINE | ID: mdl-31612217

RESUMO

The programmed death-ligand 1 (PD-L1) is a major checkpoint protein that helps cancer cells evade the immune system. A non-invasive imaging agent with rapid clearance rate would be an ideal tool to predict and monitor the efficacy of anti-PD-L1 therapy. The aim of this research was to engineer a subnanomolar, high-affinity fibronectin type 3 domain (FN3)-based small binder targeted against human PD-L1 (hPD-L1) present on tumor cells. A naive yeast G4 library containing the FN3 gene with three binding loop sequences was used to isolate high-affinity binders targeted to purified full-length hPD-L1. The selected binder clones displayed several mutations in the loop regions of the FN3 domain. One unique clone (FN3hPD-L1-01) with a 6x His-tag at the C-terminus had a protein yield of >5 mg/L and a protein mass of 12 kDa. In vitro binding assays on six different human cancer cell lines (MDA-MB-231, DLD1, U87, 293 T, Raji and Jurkat) and murine CT26 colon carcinoma cells stably expressing hPD-L1 showed that CT26/hPD-L1 cells had the highest expression of hPD-L1 in both basal and IFN-γ-induced states, with a binding affinity of 2.38 ± 0.26 nM for FN3hPD-L1-01. The binding ability of FN3hPD-L1-01 was further confirmed by immunofluorescence staining on ex vivo CT26/hPD-L1 tumors sections. The FN3hPD-L1-01 binder represents a novel, small, high-affinity binder for imaging hPD-L1 expression on tumor cells and would aid in earlier imaging of tumors. Future clinical validation studies of the labeled FN3hPD-L1 binder(s) have the potential to monitor immune checkpoint inhibitors therapy and predict responders.

5.
Methods Mol Biol ; 2033: 301-313, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31332762

RESUMO

Bioconjugation of biologically useful proteins is in great demand (e.g., conjugation to biotins, metal chelators, and drug carriers to target specific tissues for both in vitro and in vivo use). These conjugates provide widespread opportunities for various biological and biomedical applications. Evolving state-of-the-art protein conjugation strategies have led to the development of many affinity ligands, including for cancer imaging and diagnosis. However, to achieve the desirable protein conjugates, there are many challenges that remain to be addressed in order to obtain a reproducible procedure for all proteins and ligands. These include a control over the protein modification and the efficiency of the conjugation while retaining the original biological protein affinity postmodification. Here we present detailed conjugation methods for the human fibronectin tenth type III domain (FN3) protein scaffold for use in preclinical PET imaging. More specifically, this chapter provides detailed methods to produce a FN3 and a FN3-chelator-conjugate, its labeling with the radionuclide 64-Cu, and its use for noninvasive PET imaging in mice.


Assuntos
Portadores de Fármacos/química , Domínio de Fibronectina Tipo III/genética , Imagem Molecular/métodos , Neoplasias/diagnóstico , Animais , Biotina/química , Linhagem Celular Tumoral , Quelantes/química , Radioisótopos de Cobre/química , Humanos , Ligantes , Camundongos , Neoplasias/genética
6.
Theranostics ; 9(9): 2646-2661, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31131059

RESUMO

Background: Bioluminescence imaging (BLI) is one of the most widely used optical platforms in molecular imaging, but it suffers from severe tissue attenuation and autoluminescence in vivo. Methods: Here, we developed a novel BLI platform on the basis of bioluminescence resonance energy transfer (BRET) for achieving a ~300 nm blue-to-near infrared shift of the emission (NIR-BRET) by synthesizing an array of 18 novel coelenterazine (CTZ) derivatives, named "Bottle Blue (BBlue)" and a unique iRFP-linked RLuc8.6-535SG fusion protein as a probe. Results: The best NIR-BRET was achieved by tuning the emission peaks of the CTZ derivatives to a Soret band of the iRFP. In mammalian cells, BBlue2.3, one of the CTZ derivatives, emits light that is ~50-fold brighter than DBlueC when combined with RLuc8.6-535SG, which shows stable BL kinetics. When we used a caged version of BBLue2.3, it showed a BL half decay time of over 60 minutes while maintaining the higher signal sensitivity. This NIR BL is sufficiently brighter to be used for imaging live mammalian cells at single cell level, and also for imaging metastases in deep tissues in live mice without generating considerable autoluminescence. A single-chain probe developed based on this BLI platform allowed us to sensitively image ligand antagonist-specific activation of estrogen receptor in the NIR region. Conclusion: This unique optical platform provides the brightest NIR BLI template that can be used for imaging a diverse group of cellular events in living subjects including protein‒protein interactions and cancer metastasis.

7.
Clin Cancer Res ; 25(6): 1774-1785, 2019 03 15.
Artigo em Inglês | MEDLINE | ID: mdl-30373750

RESUMO

PURPOSE: To design and evaluate a small engineered protein binder targeting human programmed death-1 ligand (hPD-L1) in vivo for PET imaging in four mouse tumor models, and in situ in human cancer specimens.Experimental Design: The hPD-L1 protein binder, FN3hPD-L1, was engineered using a 12-kDa human fibronectin type-3 domain (FN3) scaffold. The binder's affinity was assayed in CT26 mouse colon carcinoma cells stably expressing hPD-L1 (CT26/hPD-L1). 64Cu-FN3hPD-L1 was assayed for purity, specific activity, and immunoreactivity. Four groups of NSG mice (n = 3-5/group) were imaged with 64Cu-FN3hPD-L1 PET imaging (1-24 hours postinjection of 3.7 MBq/7 µg of Do-FN3 in 200 µL PBS): Nod SCID Gamma (NSG) mice bearing (i) syngeneic CT26/hPD-L1tumors, (ii) CT26/hPD-L1 tumors blocked (blk) by preinjected nonradioactive FN3hPD-L1 binder, (iii) hPD-L1-negative Raji xenografts, and (iv) MDA-MB-231 xenografts. The FN3hPD-L1 binder staining was evaluated against validated hPD-L1 antibodies by immunostaining in human cancer specimens. RESULTS: FN3hPD-L1 bound hPD-L1 with 1.4 ± 0.3 nmol/L affinity in CT26/hPD-L1 cells. 64Cu-FN3hPD-L1 radiotracer showed >70% yield and >95% purity. 64Cu-FN3hPD-L1 PET imaging of mice bearing CT26/hPD-L1 tumors showed tumor-to-muscle ratios of 5.6 ± 0.9 and 13.1 ± 2.3 at 1 and 4 hours postinjection, respectively. The FN3hPD-L1 binder detected hPD-L1 expression in human tissues with known hPD-L1 expression status based on two validated antibodies. CONCLUSIONS: The 64Cu-FN3hPD-L1 radiotracer represents a novel, small, and high-affinity binder for imaging hPD-L1 in tumors. Our data support further exploration and clinical translation of this binder for noninvasive identification of cancer patients who may respond to immune checkpoint blockade therapies.

8.
Cell Death Discov ; 4: 113, 2018.
Artigo em Inglês | MEDLINE | ID: mdl-30534421

RESUMO

Glioblastoma is the most common yet most lethal of primary brain cancers with a one-year post-diagnosis survival rate of 65% and a five-year survival rate of barely 5%. Recently the U.S. Food and Drug Administration approved a novel fourth approach (in addition to surgery, radiation therapy, and chemotherapy) to treating glioblastoma; namely, tumor treating fields (TTFields). TTFields involves the delivery of alternating electric fields to the tumor but its mechanisms of action are not fully understood. Current theories involve TTFields disrupting mitosis due to interference with proper mitotic spindle assembly. We show that TTFields also alters cellular membrane structure thus rendering it more permeant to chemotherapeutics. Increased membrane permeability through the imposition of TTFields was shown by several approaches. For example, increased permeability was indicated through increased bioluminescence with TTFields exposure or with the increased binding and ingress of membrane-associating reagents such as Dextran-FITC or ethidium D or with the demonstration by scanning electron microscopy of augmented number and sizes of holes on the cellular membrane. Further investigations showed that increases in bioluminescence and membrane hole production with TTFields exposure disappeared by 24 h after cessation of alternating electric fields thus demonstrating that this phenomenom is reversible. Preliminary investigations showed that TTFields did not induce membrane holes in normal human fibroblasts thus suggesting that the phenomenom was specific to cancer cells. With TTFields, we present evidence showing augmented membrane accessibility by compounds such as 5-aminolevulinic acid, a reagent used intraoperatively to delineate tumor from normal tissue in glioblastoma patients. In addition, this mechanism helps to explain previous reports of additive and synergistic effects between TTFields and other chemotherapies. These findings have implications for the design of combination therapies in glioblastoma and other cancers and may significantly alter standard of care strategies for these diseases.

9.
Mol Imaging ; 17: 1536012118788637, 2018.
Artigo em Inglês | MEDLINE | ID: mdl-30043654

RESUMO

Cerenkov luminescence imaging (CLI) is commonly performed using two-dimensional (2-D) conventional optical imaging systems for its cost-effective solution. However, quantification of CLI comparable to conventional three-dimensional positron emission tomography (PET) is challenging using these systems due to both the high attenuation of Cerenkov radiation (CR) on mouse tissue and nonexisting depth resolution of CLI using 2-D imaging systems (2-D CLI). In this study, we developed a model that estimates effective tissue attenuation coefficient and corrects the tissue attenuation of CLI signal intensity independent of tissue depth and size. To evaluate this model, we used several thin slices of ham as a phantom and placed a radionuclide (89Zr and 64Cu) inside the phantom at different tissue depths and sizes (2, 7, and 12 mm). We performed 2-D CLI and MicroPET/CT (Combined small animal PET and Computed Tomography (CT)) imaging of the phantom and in vivo mouse model after administration of 89Zr tracer. Estimates of the effective tissue attenuation coefficient (µeff) for 89Zr and 64Cu were ∼2.4 and ∼2.6 cm-1, respectively. The computed unit conversion factor to %ID/g from 2-D CLI signal was 2.74 × 10-3 µCi/radiance estimated from phantom study. After applying tissue attenuation correction and unit conversion to the in vivo animal study, an average quantification difference of 10% for spleen and 35% for liver was obtained compared to PET measurements. The proposed model provides comparable quantification accuracy to standard PET system independent of deep tissue CLI signal attenuation.


Assuntos
Luminescência , Medições Luminescentes/métodos , Tomografia por Emissão de Pósitrons/métodos , Animais , Fígado/diagnóstico por imagem , Camundongos , Imagens de Fantasmas , Reprodutibilidade dos Testes , Baço/diagnóstico por imagem
10.
Cancer Biomark ; 22(2): 333-344, 2018.
Artigo em Inglês | MEDLINE | ID: mdl-29689709

RESUMO

BACKGROUND AND OBJECTIVE: To monitor therapies targeted to epidermal growth factor receptors (EGFR) in non-small cell lung cancer (NSCLC), we investigated Peroxiredoxin 6 (PRDX6) as a biomarker of response to anti-EGFR agents. METHODS: We studied cells that are sensitive (H3255, HCC827) or resistant (H1975, H460) to gefitinib. PRDX6 was examined with either gefitinib or vehicle treatment using enzyme-linked immunosorbent assays. We created xenograft models from one sensitive (HCC827) and one resistant cell line (H1975) and monitored serum PRDX6 levels during treatment. RESULTS: PRDX6 levels in cell media from sensitive cell lines increased significantly after gefitinib treatment vs. vehicle, whereas there was no significant difference for resistant lines. PRDX6 accumulation over time correlated positively with gefitinib sensitivity. Serum PRDX6 levels in gefitinib-sensitive xenograft models increased markedly during the first 24 hours of treatment and then decreased dramatically during the following 48 hours. Differences in serum PRDX6 levels between vehicle and gefitinib-treated animals could not be explained by differences in tumor burden. CONCLUSIONS: Our results show that changes in serum PRDX6 during the course of gefitinib treatment of xenograft models provide insight into tumor response and such an approach offers several advantages over imaging-based strategies for monitoring response to anti-EGFR agents.


Assuntos
Antineoplásicos/farmacologia , Carcinoma Pulmonar de Células não Pequenas/sangue , Receptores ErbB/antagonistas & inibidores , Neoplasias Pulmonares/sangue , Inibidores de Proteínas Quinases/farmacologia , Animais , Antineoplásicos/uso terapêutico , Biomarcadores , Carcinoma Pulmonar de Células não Pequenas/tratamento farmacológico , Linhagem Celular Tumoral , Modelos Animais de Doenças , Ensaio de Imunoadsorção Enzimática , Feminino , Gefitinibe , Humanos , Neoplasias Pulmonares/tratamento farmacológico , Camundongos , Peroxirredoxina VI/sangue , Peroxirredoxina VI/genética , Peroxirredoxina VI/metabolismo , Inibidores de Proteínas Quinases/uso terapêutico , Quinazolinas/farmacologia , Quinazolinas/uso terapêutico , Resultado do Tratamento , Ensaios Antitumorais Modelo de Xenoenxerto
11.
Sci Rep ; 8(1): 633, 2018 01 12.
Artigo em Inglês | MEDLINE | ID: mdl-29330552

RESUMO

The immune checkpoint programmed death 1 receptor (PD-1) expressed on some tumor-infiltrating lymphocytes, and its ligand (PD-L1) expressed on tumor cells, enable cancers to evade the immune system. Blocking PD-1 with the monoclonal antibody pembrolizumab is a promising immunotherapy strategy. Thus, noninvasively quantifying the presence of PD-1 expression in the tumor microenvironment prior to initiation of immune checkpoint blockade may identify the patients likely to respond to therapy. We have developed a 64Cu-pembrolizumab radiotracer and evaluated human dosimetry. The tracer was utilized to image hPD-1 levels in two subcutaneous mouse models: (a) 293 T/hPD-1 cells xenografted into NOD-scid IL-2Rγnull mice (NSG/293 T/hPD-1) and (b) human peripheral blood mononuclear cells engrafted into NSG bearing A375 human melanoma tumors (hNSG/A375). In each mouse model two cohorts were evaluated (hPD-1 blockade with pembrolizumab [blk] and non-blocked [nblk]), for a total of four groups (n = 3-5/group). The xenograft-to-muscle ratio in the NSG/293 T/hPD-1 model at 24 h was significantly increased in the nblk group (7.0 ± 0.5) compared to the blk group (3.4 ± 0.9), p = 0.01. The radiotracer dosimetry evaluation (PET/CT ROI-based and ex vivo) in the hNSG/A375 model revealed the highest radiation burden to the liver. In summary, we validated the 64Cu-pembrolizumab tracer's specific hPD-1 receptor targeting and predicted human dosimetry.


Assuntos
Anticorpos Monoclonais Humanizados/administração & dosagem , Radioisótopos de Cobre/química , Melanoma/diagnóstico por imagem , Receptor de Morte Celular Programada 1/antagonistas & inibidores , Animais , Anticorpos Monoclonais Humanizados/química , Anticorpos Monoclonais Humanizados/farmacologia , Linhagem Celular Tumoral , Humanos , Melanoma/metabolismo , Camundongos , Transplante de Neoplasias , Tomografia Computadorizada com Tomografia por Emissão de Pósitrons , Traçadores Radioativos , Radiometria , Pesquisa Médica Translacional , Microambiente Tumoral
12.
J Nucl Med ; 58(11): 1845-1851, 2017 11.
Artigo em Inglês | MEDLINE | ID: mdl-28687602

RESUMO

B lymphocytes are a key pathologic feature of multiple sclerosis (MS) and are becoming an important therapeutic target for this condition. Currently, there is no approved technique to noninvasively visualize B cells in the central nervous system (CNS) to monitor MS disease progression and response to therapies. Here, we evaluated 64Cu-rituximab, a radiolabeled antibody specifically targeting the human B cell marker CD20, for its ability to image B cells in a mouse model of MS using PET. Methods: To model CNS infiltration by B cells, experimental autoimmune encephalomyelitis (EAE) was induced in transgenic mice that express human CD20 on B cells. EAE mice were given subcutaneous injections of myelin oligodendrocyte glycoprotein fragment1-125 emulsified in complete Freund adjuvant. Control mice received complete Freund adjuvant alone. PET imaging of EAE and control mice was performed 1, 4, and 19 h after 64Cu-rituximab administration. Mice were perfused and sacrificed after the final PET scan, and radioactivity in dissected tissues was measured with a γ-counter. CNS tissues from these mice were immunostained to quantify B cells or were further analyzed via digital autoradiography. Results: Lumbar spinal cord PET signal was significantly higher in EAE mice than in controls at all evaluated time points (e.g., 1 h after injection: 5.44 ± 0.37 vs. 3.33 ± 0.20 percentage injected dose [%ID]/g, P < 0.05). 64Cu-rituximab PET signal in brain regions ranged between 1.74 ± 0.11 and 2.93 ± 0.15 %ID/g for EAE mice, compared with 1.25 ± 0.08 and 2.24 ± 0.11 %ID/g for controls (P < 0.05 for all regions except striatum and thalamus at 1 h after injection). Similarly, ex vivo biodistribution results revealed notably higher 64Cu-rituximab uptake in the brain and spinal cord of huCD20tg EAE, and B220 immunostaining verified that increased 64Cu-rituximab uptake in CNS tissues corresponded with elevated B cells. Conclusion: B cells can be detected in the CNS of EAE mice using 64Cu-rituximab PET. Results from these studies warrant further investigation of 64Cu-rituximab in EAE models and consideration of use in MS patients to evaluate its potential for detecting and monitoring B cells in the progression and treatment of this disease. These results represent an initial step toward generating a platform to evaluate B cell-targeted therapeutics en route to the clinic.


Assuntos
Linfócitos B , Esclerose Múltipla/diagnóstico por imagem , Tomografia por Emissão de Pósitrons/métodos , Compostos Radiofarmacêuticos , Animais , Autorradiografia , Encéfalo/diagnóstico por imagem , Radioisótopos de Cobre , Encefalomielite Autoimune Experimental/diagnóstico por imagem , Humanos , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Compostos Radiofarmacêuticos/farmacocinética , Rituximab/farmacocinética , Medula Espinal/diagnóstico por imagem , Distribuição Tecidual
13.
Mol Imaging Biol ; 19(6): 903-914, 2017 12.
Artigo em Inglês | MEDLINE | ID: mdl-28247187

RESUMO

PURPOSE: It is well known that cancers exploit immune checkpoints (programmed death 1 receptor (PD-1) and its ligand (PD-L1)) to evade anti-tumor immune responses. Although immune checkpoint (IC) blockade is a promising approach, not all patients respond. Hence, imaging of tumor-infiltrating lymphocytes (TILs) is of high specific interest, as they are known to express PD-1 during activation and subsequent exhaustion in the tumor microenvironment and are thought to be potentially predictive of therapeutic responses to IC blockade. PROCEDURES: We developed immune-tracers for positron emission tomography (PET) to image hPD-1 status of human peripheral blood mononuclear cells (hPBMCs) adoptively transferred to NOD-scid IL-2Rγnull (NSG) mice (hNSG) bearing A375 human skin melanoma tumors. The anti-PD-1 human antibody (IgG; keytruda) was labeled with either Zr-89 or Cu-64 radiometals to image PD-1-expressing human TILs in vivo. RESULTS: [89Zr] Keytruda (groups = 2; NSG-ctl (control) and hNSG-nblk (non-blocking), n = 3-5, 3.2 ± 0.4 MBq/15-16 µg/200 µl) and [64Cu] Keytruda (groups = 3; NSG-ctl, NSG-blk (blocking), and hNSG-nblk; n = 4, 7.4 ± 0.4 MBq /20-25 µg/200 µl) were administered in mice. PET-CT scans were performed over 1-144 h ([89Zr] Keytruda) and 1-48 h ([64Cu] Keytruda) on mice. hNSG mice exhibited a high tracer uptake in the spleen, lymphoid organs and tumors. At 24 h, human TILs homing into melanoma of hNSG-nblk mice exhibited high signal (mean %ID/g ± SD) of 3.8 ± 0.4 ([89Zr] Keytruda), and 6.4 ± 0.7 ([64Cu] Keytruda), which was 1.5- and 3-fold higher uptake compared to NSG-ctl mice (p = 0.01), respectively. Biodistribution measurements of hNSG-nblk mice performed at 144 h ([89Zr] Keytruda) and 48 h ([64Cu] Keytruda) p.i. revealed tumor to muscle ratios as high as 45- and 12-fold, respectively. CONCLUSIONS: Our immunoPET study clearly demonstrates specific imaging of human PD-1-expressing TILs within the tumor and lymphoid tissues. This suggests these anti-human-PD-1 tracers could be clinically translatable to monitor cancer treatment response to IC blockade therapy.


Assuntos
Anticorpos Monoclonais Humanizados/química , Antígeno B7-H1/metabolismo , Linfócitos do Interstício Tumoral/metabolismo , Linfócitos do Interstício Tumoral/patologia , Tomografia por Emissão de Pósitrons , Animais , Linhagem Celular Tumoral , Cromatografia Líquida de Alta Pressão , Modelos Animais de Doenças , Humanos , Camundongos , Tomografia Computadorizada com Tomografia por Emissão de Pósitrons , Compostos Radiofarmacêuticos , Distribuição Tecidual
14.
J Nucl Med ; 58(4): 538-546, 2017 04.
Artigo em Inglês | MEDLINE | ID: mdl-27980047

RESUMO

Immune checkpoint blockade has emerged as a promising cancer treatment paradigm. Unfortunately, there are still a large number of patients and malignancies that do not respond to therapy. A major barrier to validating biomarkers for the prediction and monitoring of responders to clinical checkpoint blockade has been the lack of imaging tools to accurately assess dynamic immune checkpoint expression. Here, we sought to optimize noninvasive immuno-PET imaging of human programmed death-ligand 1 (PD-L1) expression, in a preclinical model, using a small high-affinity engineered protein scaffold (HAC-PD1). Six HAC-PD1 radiotracer variants were developed and used in preclinical imaging and biodistribution studies to assess their ability to detect human PD-L1 expression in vivo. Radiotracer design modifications included chelate, glycosylation, and radiometal. HACA-PD1 was adopted as the naming convention for aglycosylated tracer variants. NOD scid γ-(NSG) mice were inoculated with subcutaneous tumors engineered to either be constitutively positive (CT26 hPD-L1) or be negative (ΔmPD-L1 CT26) for human PD-L1 expression. When the tumors had grown to an average size of 1 cm in diameter, mice were injected with 0.75-2.25 MBq (∼10 µg) of an engineered radiotracer variant and imaged. At 1 h after injection, organs were harvested for biodistribution. Of the practical immuno-PET tracer modifications considered, glycosylation was the most prominent design factor affecting tracer uptake, specificity, and clearance. In imaging studies, aglycosylated 64Cu-NOTA-HACA-PD1 most accurately visualized human PD-L1 expression in vivo. We reasoned that because of the scaffold's small size (14 kDa), its pharmacokinetics may be suitable for labeling with the short-lived and widely clinically available radiometal 68Ga. At 1 h after injection, 68Ga-NOTA-HACA-PD1 and 68Ga-DOTA-HACA-PD1 exhibited promising target-to-background ratios in ex vivo biodistribution studies (12.3 and 15.2 tumor-to-muscle ratios, respectively). Notably, all HAC-PD1 radiotracer variants enabled much earlier detection of human PD-L1 expression (1 h after injection) than previously reported radiolabeled antibodies (>24 h after injection). This work provides a template for assessing immuno-PET tracer design parameters and supports the translation of small engineered protein radiotracers for imaging human immune checkpoints.


Assuntos
Desenho de Fármacos , Imunoconjugados , Tomografia por Emissão de Pósitrons/métodos , Animais , Antígeno B7-H1/química , Antígeno B7-H1/genética , Antígeno B7-H1/imunologia , Antígeno B7-H1/metabolismo , Linhagem Celular Tumoral , Radioisótopos de Cobre , Regulação Neoplásica da Expressão Gênica , Glicosilação , Compostos Heterocíclicos com 1 Anel/química , Humanos , Imunoconjugados/química , Imunoconjugados/farmacocinética , Camundongos , Modelos Moleculares , Conformação Proteica , Engenharia de Proteínas , Traçadores Radioativos , Distribuição Tecidual
15.
J Neurooncol ; 126(2): 253-64, 2016 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-26650066

RESUMO

Glioblastoma multiforme (GBM) is an aggressive, malignant cancer Johnson and O'Neill (J Neurooncol 107: 359-364, 2012). An extract from the winter cherry plant (Withania somnifera ), AshwaMAX, is concentrated (4.3 %) for Withaferin A; a steroidal lactone that inhibits cancer cells Vanden Berghe et al. (Cancer Epidemiol Biomark Prev 23: 1985-1996, 2014). We hypothesized that AshwaMAX could treat GBM and that bioluminescence imaging (BLI) could track oral therapy in orthotopic murine models of glioblastoma. Human parietal-cortical glioblastoma cells (GBM2, GBM39) were isolated from primary tumors while U87-MG was obtained commercially. GBM2 was transduced with lentiviral vectors that express Green Fluorescent Protein (GFP)/firefly luciferase fusion proteins. Mutational, expression and proliferative status of GBMs were studied. Intracranial xenografts of glioblastomas were grown in the right frontal regions of female, nude mice (n = 3-5 per experiment). Tumor growth was followed through BLI. Neurosphere cultures (U87-MG, GBM2 and GBM39) were inhibited by AshwaMAX at IC50 of 1.4, 0.19 and 0.22 µM equivalent respectively and by Withaferin A with IC50 of 0.31, 0.28 and 0.25 µM respectively. Oral gavage, every other day, of AshwaMAX (40 mg/kg per day) significantly reduced bioluminescence signal (n = 3 mice, p < 0.02, four parameter non-linear regression analysis) in preclinical models. After 30 days of treatment, bioluminescent signal increased suggesting onset of resistance. BLI signal for control, vehicle-treated mice increased and then plateaued. Bioluminescent imaging revealed diffuse growth of GBM2 xenografts. With AshwaMAX, GBM neurospheres collapsed at nanomolar concentrations. Oral treatment studies on murine models confirmed that AshwaMAX is effective against orthotopic GBM. AshwaMAX is thus a promising candidate for future clinical translation in patients with GBM.


Assuntos
Antineoplásicos/administração & dosagem , Neoplasias Encefálicas/tratamento farmacológico , Glioblastoma/tratamento farmacológico , Extratos Vegetais/administração & dosagem , Withania/química , Vitanolídeos/administração & dosagem , Animais , Antineoplásicos/farmacologia , Neoplasias Encefálicas/metabolismo , Neoplasias Encefálicas/patologia , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Modelos Animais de Doenças , Relação Dose-Resposta a Droga , Receptores ErbB/metabolismo , Feminino , Glioblastoma/metabolismo , Glioblastoma/patologia , Humanos , Medições Luminescentes , Camundongos , Camundongos Nus , Células-Tronco Neurais/efeitos dos fármacos , Extratos Vegetais/química , Vitanolídeos/farmacologia , Ensaios Antitumorais Modelo de Xenoenxerto
16.
Proc Natl Acad Sci U S A ; 112(47): E6506-14, 2015 Nov 24.
Artigo em Inglês | MEDLINE | ID: mdl-26604307

RESUMO

Signaling through the immune checkpoint programmed cell death protein-1 (PD-1) enables tumor progression by dampening antitumor immune responses. Therapeutic blockade of the signaling axis between PD-1 and its ligand programmed cell death ligand-1 (PD-L1) with monoclonal antibodies has shown remarkable clinical success in the treatment of cancer. However, antibodies have inherent limitations that can curtail their efficacy in this setting, including poor tissue/tumor penetrance and detrimental Fc-effector functions that deplete immune cells. To determine if PD-1:PD-L1-directed immunotherapy could be improved with smaller, nonantibody therapeutics, we used directed evolution by yeast-surface display to engineer the PD-1 ectodomain as a high-affinity (110 pM) competitive antagonist of PD-L1. In contrast to anti-PD-L1 monoclonal antibodies, high-affinity PD-1 demonstrated superior tumor penetration without inducing depletion of peripheral effector T cells. Consistent with these advantages, in syngeneic CT26 tumor models, high-affinity PD-1 was effective in treating both small (50 mm(3)) and large tumors (150 mm(3)), whereas the activity of anti-PD-L1 antibodies was completely abrogated against large tumors. Furthermore, we found that high-affinity PD-1 could be radiolabeled and applied as a PET imaging tracer to efficiently distinguish between PD-L1-positive and PD-L1-negative tumors in living mice, providing an alternative to invasive biopsy and histological analysis. These results thus highlight the favorable pharmacology of small, nonantibody therapeutics for enhanced cancer immunotherapy and immune diagnostics.


Assuntos
Imunoterapia , Proteínas Mutantes/uso terapêutico , Neoplasias/diagnóstico por imagem , Neoplasias/terapia , Tomografia por Emissão de Pósitrons , Receptor de Morte Celular Programada 1/uso terapêutico , Engenharia de Proteínas , Sequência de Aminoácidos , Animais , Linhagem Celular Tumoral , Evolução Molecular Direcionada , Modelos Animais de Doenças , Humanos , Depleção Linfocítica , Camundongos Endogâmicos BALB C , Dados de Sequência Molecular , Neoplasias/imunologia , Receptor de Morte Celular Programada 1/química , Ligação Proteica , Linfócitos T/metabolismo
17.
Mol Pharm ; 12(12): 4554-60, 2015 Dec 07.
Artigo em Inglês | MEDLINE | ID: mdl-26460685

RESUMO

The resistance of a tumor to a drug is the result of bulk properties of the tumor tissue as well as phenotypic variations displayed by single cells. Here, we show that radioisotopic detection methods, commonly used for tracking the tissue distribution of drug compounds, can be extended to the single-cell level to image the same molecule over a range of physical scales. The anticancer drug rituximab was labeled with short-lived radionuclides ((89)Zr/(64)Cu) and its accumulation at the organ level was imaged using PET in a humanized transgenic mouse model of non-Hodgkin's lymphoma. To capture the distribution of the drug at a finer scale, tissue sections and single living cells were imaged using radioluminescence microscopy (RLM), a novel method that can detect radionuclides with single-cell resolution. In vivo PET images (24 h postinjection) showed that [(89)Zr]rituximab targeted the intended site of human CD20 expression, the spleen. Within this organ, RLM was used to resolve radiotracer accumulation in the splenic red pulp. In a separate study, RLM highlighted marked differences between single cells, with binding of the radiolabeled antibody ranging from background levels to 1200 radionuclides per cell. Overall, RLM images demonstrated significantly higher spatial resolution and sensitivity than conventional storage-phosphor autoradiography. In conclusion, this combination of PET and RLM provides a unique opportunity for exploring the molecular mechanism of drugs by tracking the same molecule over multiple physical scales, ranging from single living cells to organs substructures and entire living subjects.


Assuntos
Antineoplásicos/farmacologia , Linfoma não Hodgkin/tratamento farmacológico , Radioisótopos/metabolismo , Compostos Radiofarmacêuticos/metabolismo , Rituximab/metabolismo , Rituximab/farmacologia , Animais , Antígenos CD20/metabolismo , Autorradiografia/métodos , Modelos Animais de Doenças , Humanos , Camundongos , Camundongos Transgênicos , Tomografia por Emissão de Pósitrons/métodos , Radioisótopos/administração & dosagem , Compostos Radiofarmacêuticos/administração & dosagem , Distribuição Tecidual
18.
Bioconjug Chem ; 26(10): 2062-9, 2015 Oct 21.
Artigo em Inglês | MEDLINE | ID: mdl-26307602

RESUMO

Immune checkpoint signaling through the programmed death 1 (PD-1) axis to its ligand (PD-L1) significantly dampens anti-tumor immune responses. Cancer patients treated with checkpoint inhibitors that block this suppressive signaling have exhibited objective response rates of 20-40% for advanced solid tumors, lymphomas, and malignant melanomas. This represents a tremendous advance in cancer treatment. Unfortunately, all patients do not respond to immune checkpoint blockade. Recent findings suggest that patients with tumor infiltrating lymphocytes (TILs) expressing PD-1 may be most likely to respond to αPD-1/PD-L1 checkpoint inhibitors. There is a compelling need for diagnostic and prognostic imaging tools to assess the PD-1 status of TILs in vivo. Here we have developed a novel immunoPET tracer to image PD-1 expressing TILs in a transgenic mouse model bearing melanoma. A (64)Cu labeled anti-mouse antibody (IgG) PD-1 immuno positron emission tomography (PET) tracer was developed to detect PD-1 expressing murine TILs. Quality control of the tracer showed >95% purity by HPLC and >70% immunoreactivity in an in vitro cell binding assay. ImmunoPET scans were performed over 1-48 h on Foxp3+.LuciDTR4 mice bearing B16-F10 melanoma tumors. Mice receiving anti-PD-1 tracer (200 ± 10 µCi/10-12 µg/200 µL) revealed high tracer uptake in lymphoid organs and tumors. BLI images of FoxP3(+) CD4(+) Tregs known to express PD-1 confirmed lymphocyte infiltration of tumors at the time of PET imaging. Biodistribution measurements performed at 48 h revealed a high (11×) tumor to muscle uptake ratio of the PET tracer (p < 0.05). PD-1 tumors exhibited 7.4 ± 0.7%ID/g tracer uptake and showed a 2× fold signal decrease when binding was blocked by unlabeled antibody. To the best of our knowledge this data is the first report to image PD-1 expression in living subjects with PET. This radiotracer has the potential to assess the prognostic value of PD-1 in preclinical models of immunotherapy and may ultimately aid in predicting response to therapies targeting immune checkpoints.


Assuntos
Linfócitos do Interstício Tumoral/patologia , Tomografia por Emissão de Pósitrons/métodos , Receptor de Morte Celular Programada 1/metabolismo , Compostos Radiofarmacêuticos/farmacocinética , Animais , Anticorpos Monoclonais/química , Morte Celular/imunologia , Radioisótopos de Cobre/química , Compostos Heterocíclicos/química , Imunoconjugados/química , Imunoconjugados/farmacocinética , Isotiocianatos/química , Linfócitos do Interstício Tumoral/imunologia , Melanoma/patologia , Camundongos Transgênicos , Receptor de Morte Celular Programada 1/imunologia , Distribuição Tecidual , Ensaios Antitumorais Modelo de Xenoenxerto
19.
Radiology ; 276(1): 191-8, 2015 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-25734548

RESUMO

PURPOSE: To develop and compare three copper 64 ((64)Cu)-labeled antibody fragments derived from a CA6-targeting antibody (huDS6) as immuno-positron emission tomography (immuno-PET)-based companion diagnostic agents for an antibody-drug conjugate by using huDS6. MATERIALS AND METHODS: Three antibody fragments derived from huDS6 were produced, purified, conjugated to 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid (DOTA), and evaluated in the following ways: (a) the affinity of the fragments and the DOTA conjugates was measured via flow cytometry, (b) the stability of the labeled fragments was determined ex vivo in human serum over 24 hours, and (c) comparison of the in vivo imaging potential of the fragments was evaluated in mice bearing subcutaneous CA6-positive and CA6-negative xenografts by using serial PET imaging and biodistribution. Isotype controls with antilysozyme and anti-DM4 B-Fabs and blocking experiments with an excess of either B-Fab or huDS6 were used to determine the extent of the antibody fragment (64)Cu-DOTA-B-Fab binding specificity. Immunoreactivity and tracer kinetics were evaluated by using cellular uptake and 48-hour imaging experiments, respectively. Statistical analyses were performed by using t tests, one-way analysis of variance, and Wilcoxon and Mann-Whitney tests. RESULTS: The antibody fragment (64)Cu-DOTA-B-Fab was more than 95% stable after 24 hours in human serum, had an immunoreactivity of more than 70%, and allowed differentiation between CA6-positive and CA6-negative tumors in vivo as early as 6 hours after injection, with a 1.7-fold uptake ratio between tumors. Isotype and blocking studies experiments showed tracer-specific uptake in antigen-positive tumors, despite some nonspecific uptake in both tumor models. CONCLUSION: Three antibody fragments were produced and examined as potential companion diagnostic agents. (64)Cu-DOTA-B-Fab is a stable and effective immuno-PET tracer for CA6 imaging in vivo.


Assuntos
Radioisótopos de Cobre , Fragmentos de Imunoglobulinas , Tomografia por Emissão de Pósitrons/métodos , Animais , Células Cultivadas , Tratamento Farmacológico , Epitopos , Humanos , Testes Imunológicos , Camundongos , Traçadores Radioativos
20.
Mol Imaging ; 142015.
Artigo em Inglês | MEDLINE | ID: mdl-25762106

RESUMO

Manufacturing of 64Cu-1,4,7,10-tetraazacyclododecane-N,N',N'',N'''-tetraacetic acid (DOTA)-rituximab injection under good manufacturing practices (GMP) was validated for imaging of patients with CD20+ B-cell non-Hodgkin lymphoma. Rituximab was purified by size exclusion high performance liquid chromatography (HPLC) and conjugated to DOTA-mono-(N-hydroxysuccinimidyl) ester. 64CuCl2, buffers, reagents, and other raw materials were obtained as high-grade quality. Following a semi-automated synthesis of 64Cu-DOTA-rituximab, a series of quality control tests was performed. The product was further tested in vivo using micro-positron emission tomography/computed tomography (PET/CT) to assess targeting ability towards human CD20 in transgenic mice. Three batches of 64Cu-DOTA-rituximab final product were prepared as per GMP specifications. The radiolabeling yield from these batches was 93.1 ± 5.8%; these provided final product with radiopharmaceutical yield, purity, and specific activity of 59.2 ± 5.1% (0.9 ± 0.1 GBq of 64Cu), > 95% (by HPLC and radio-thin layer chromatography), and 229.4 ± 43.3 GBq/µmol (or 1.5 ± 0.3 MBq/µg), respectively. The doses passed apyrogenicity and human serum stability specifications, were sterile up to 14 days, and retained > 60% immunoreactivity. In vivo micro-PET/CT mouse images at 24 hours postinjection showed that the tracer targeted the intended sites of human CD20 expression. Thus, we have validated the manufacturing of GMP grade 64Cu-DOTA-rituximab for injection in the clinical setting.


Assuntos
Radioisótopos de Cobre/química , Compostos Heterocíclicos com 1 Anel/química , Linfoma de Células B/diagnóstico por imagem , Linfoma não Hodgkin/diagnóstico por imagem , Tomografia por Emissão de Pósitrons , Rituximab/química , Animais , Anticorpos Monoclonais/química , Antígenos CD20/química , Humanos , Camundongos , Camundongos Transgênicos , Projetos Piloto , Compostos Radiofarmacêuticos/química , Tomografia Computadorizada por Raios X
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