Cellular and molecular‐level interactions of nanoparticles with biological systems are a rapidly evolving field requiring an improved understanding of endocytic trafficking as the principal driver ...and regulator of signaling events and cellular responses. An understanding of these processes is vital to nanomedicine applications. Studies investigating the complex interplay of these processes and their relationship to targeted nanoparticles exploiting endocytic pathways are notably lacking. It is known that integrins traffic through the endosomal pathway and participate in diverse roles controlling signal transduction, cell migration, and proliferation. Here, it is shown that ultrasmall, nontoxic, core–shell silica nanoparticles (C‐dots), surface‐functionalized with cRGDY peptides, modestly activate integrin‐signaling pathways, in turn, promoting the enhancement of cellular functions. First, nanomolar concentrations, two orders of magnitude higher than clinical trial doses, internalize within αvβ3 integrin‐expressing melanoma and endothelial cells, predominantly through an integrin receptor‐dependent endocytic route. Second, integrin‐mediated activation of focal adhesion kinase (FAK) and downstream signaling pathways occurs, in turn, upregulating phosphorylated protein expression levels and promoting concentration‐dependent cellular migration and proliferative activity. Inhibiting FAK catalytic activity leads to decreased phosphorylation levels and cellular migration rates. These findings may inform the design of more effectively‐targeted nanomedicines and provide insights into endocytic regulation of signal transduction.
The interactions of nanoparticles with biological systems require a better understanding of endocytic trafficking as a driver and regulator of signal transduction pathways and cellular events. Exposure of integrin‐expressing human melanoma and endothelial cells to a sub‐10‐nm fluorescent silica nanoparticle displaying cRGD peptides modestly activates integrin‐signaling pathways, promoting migration and proliferation.
Bispecific antibodies (BsAb) have proven to be useful targeting vectors for pretargeted radioimmunotherapy (PRIT). We sought to overcome key PRIT limitations such as high renal radiation exposure and ...immunogenicity (e.g., of streptavidin-antibody fusions), to advance clinical translation of this PRIT strategy for diasialoganglioside GD2-positive GD2(+) tumors. For this purpose, an IgG-scFv BsAb was engineered using the sequences for the anti-GD2 humanized monoclonal antibody hu3F8 and C825, a murine scFv antibody with high affinity for the chelator 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid (DOTA) complexed with β-particle-emitting radiometals such as (177)Lu and (90)Y. A three-step regimen, including hu3F8-C825, a dextran-based clearing agent, and p-aminobenzyl-DOTA radiolabeled with (177)Lu (as (177)Lu-DOTA-Bn; t1/2 = 6.71 days), was optimized in immunocompromised mice carrying subcutaneous human GD2(+) neuroblastoma (NB) xenografts. Absorbed doses for tumor and normal tissues were approximately 85 cGy/MBq and ≤3.7 cGy/MBq, respectively, with therapeutic indices (TI) of 142 for blood and 23 for kidney. A therapy study (n = 5/group; tumor volume, 240 ± 160 mm(3)) with three successive PRIT cycles (total (177)Lu: ∼33 MBq; tumor dose ∼3,400 cGy), revealed complete tumor response in 5 of 5 animals, with no recurrence up to 28 days after treatment. Tumor ablation was confirmed histologically in 4 of 5 mice, and normal organs showed minimal overall toxicities. All nontreated mice required sacrifice within 12 days (>1.0-cm(3) tumor volume). We conclude that this novel anti-GD2 PRIT approach has sufficient TI to successfully ablate subcutaneous GD2(+)-NB in mice while sparing kidney and bone marrow.
I-PU-H71 is an investigational first-in-class radiologic agent specific for imaging tumor epichaperome formations. The intracellular epichaperome forms under cellular stress and is a clinically ...validated oncotherapeutic target. We conducted a first-in-human study of microdose
I-PU-H71 for PET to study
biodistribution, pharmacokinetics, metabolism, and safety; and the feasibility of epichaperome-targeted tumor imaging.
Adult patients with cancer (
= 30) received
I-PU-H71 tracer (201±12 MBq, <25 μg) intravenous bolus followed by PET/CT scans and blood radioassays.
I-PU-H71 PET detected tumors of different cancer types (breast, lymphoma, neuroblastoma, genitourinary, gynecologic, sarcoma, and pancreas).
I-PU-H71 was retained by tumors for several days while it cleared rapidly from bones, healthy soft tissues, and blood. Radiation dosimetry is favorable and patients suffered no adverse effects.
Our first-in-human results demonstrate the safety and feasibility of noninvasive
detection of tumor epichaperomes using
I-PU-H71 PET, supporting clinical development of PU-H71 and other epichaperome-targeted therapeutics.
Abstract
Diseases are a manifestation of how thousands of proteins interact. In several diseases, such as cancer and Alzheimer’s disease, proteome-wide disturbances in protein-protein interactions ...are caused by alterations to chaperome scaffolds termed epichaperomes. Epichaperome-directed chemical probes may be useful for detecting and reversing defective chaperomes. Here we provide structural, biochemical, and functional insights into the discovery of epichaperome probes, with a focus on their use in central nervous system diseases. We demonstrate on-target activity and kinetic selectivity of a radiolabeled epichaperome probe in both cells and mice, together with a proof-of-principle in human patients in an exploratory single group assignment diagnostic study (ClinicalTrials.gov Identifier: NCT03371420). The clinical study is designed to determine the pharmacokinetic parameters and the incidence of adverse events in patients receiving a single microdose of the radiolabeled probe administered by intravenous injection. In sum, we introduce a discovery platform for brain-directed chemical probes that specifically modulate epichaperomes and provide proof-of-principle applications in their use in the detection, quantification, and modulation of the target in complex biological systems.
Epithelial ovarian cancer (EOC) is often asymptomatic and presents clinically in an advanced stage as widespread peritoneal microscopic disease that is generally considered to be surgically ...incurable. Targeted α-therapy with the α-particle-emitting radionuclide
Ac (half-life, 9.92 d) is a high-linear-energy-transfer treatment approach effective for small-volume disease and even single cells. Here, we report the use of human epidermal growth factor receptor 2 (HER2)
Ac-pretargeted radioimmunotherapy (PRIT) to treat a mouse model of human EOC SKOV3 xenografts growing as peritoneal carcinomatosis (PC).
On day 0, 10
SKOV3 cells transduced with a luciferase reporter gene were implanted intraperitoneally in nude mice, and tumor engraftment was verified by bioluminescent imaging (BLI). On day 15, treatment was started using 1 or 2 cycles of 3-step anti-HER2
Ac-PRIT (37 kBq/cycle as
Ac-
DOTA), separated by a 1-wk interval. Efficacy and toxicity were monitored for up to 154 d.
Untreated PC-tumor-bearing nude mice showed a median survival of 112 d. We used 2 independent measures of response to evaluate the efficacy of
Ac-PRIT. First, a greater proportion of the treated mice (9/10 1-cycle and 8/10 2-cycle; total, 17/20; 85%) survived long-term compared with controls (9/27, 33%), and significantly prolonged survival was documented (log-rank Mantel-Cox
= 0.0042). Second, using BLI, a significant difference in the integrated BLI signal area to 98 d was noted between controls and treated groups (
= 0.0354). Of a total of 8 mice from the 2-cycle treatment group (74 kBq total) that were evaluated by necropsy, kidney radiotoxicity was mild and did not manifest itself clinically (normal serum blood urea nitrogen and creatinine). Dosimetry estimates (relative biological effectiveness-weighted dose, where relative biological effectiveness = 5) per 37 kBq administered for tumors and kidneys were 56.9 and 16.1 Gy, respectively. One-cycle and 2-cycle treatments were equally effective. With immunohistology, mild tubular changes attributable to α-toxicity were observed in both therapeutic groups.
Treatment of EOC PC-tumor-bearing mice with anti-HER2
Ac-PRIT resulted in histologic cures and prolonged survival with minimal toxicity. Targeted α-therapy using the anti-HER2
Ac-PRIT system is a potential treatment for otherwise incurable EOC.
Background The purpose of this work was to examine the ability of an oncolytic vaccinia virus expressing the human sodium iodine transporter (hNIS) to provide real time monitoring of viral therapy ...and effective treatment of malignant pleural mesothelioma (MPM). Methods Infectivity and cytotoxic effects of GLV-1h153 on mesothelioma cell lines of all histologic subtypes were assayed in vitro. Viral replication was examined by standard viral plaque assay. Orthotopic MPM xenografts were generated in athymic nude mice, treated with intrapleural GLV-1h153, and assessed for effect on tumor burden and survival. Orthotopic tumors were also imaged on single photon emission computed tomography (SPECT)/computed tomography (CT) after131 I administration. Results GLV-1h153–infected and killed all cell lines in a time- and concentration-dependent manner. Viral replication demonstrated a >2.5-log increase in titer over 4 days. Intrapleural treatment of orthotopic MPM xenografts resulted in a significant decrease in tumor burden 1 week after treatment and an improvement in survival. Infection of orthotopic xenografts was both therapeutic and facilitated monitoring by131 I-SPECT/CT via expression of hNIS in infected tissue. Conclusion Our results suggest that GLV-1h153 may be a promising therapeutic agent for MPM and warrants further investigation.
Hypoxia is one of the main causes of the failure to achieve local control using radiotherapy. This is due to the increased radioresistance of hypoxic cells. (18)F-fluoromisonidazole ((18)F-FMISO) ...positron emission tomography (PET) is a noninvasive imaging technique that can assist in the identification of intratumor regions of hypoxia. The aim of this study was to evaluate the reproducibility of (18)F-FMISO intratumor distribution using two pretreatment PET scans.
We enrolled 20 head and neck cancer patients in this study. Of these, 6 were excluded from the analysis for technical reasons. All patients underwent an (18)F-fluorodeoxyglucose study, followed by two (18)F-FMISO studies 3 days apart. The hypoxic volumes were delineated according to a tumor/blood ratio >or=1.2. The (18)F-FMISO tracer distributions from the two (18)F-FMISO studies were co-registered on a voxel-by-voxel basis using the computed tomography images from the PET/computed tomography examinations. A correlation between the (18)F-FMISO intensities of the corresponding spatial voxels was derived.
A voxel-by-voxel analysis of the (18)F-FMISO distributions in the entire tumor volume showed a strong correlation in 71% of the patients. Restraining the correlation to putatively hypoxic zones reduced the number of patients exhibiting a strong correlation to 46%.
Variability in spatial uptake can occur between repeat (18)F-FMISO PET scans in patients with head and neck cancer. Blood data for one patient was not available. Of 13 patients, 6 had well-correlated intratumor distributions of (18)F-FMISO-suggestive of chronic hypoxia. More work is required to identify the underlying causes of changes in intratumor distribution before single-time-point (18)F-FMISO PET images can be used as the basis of hypoxia-targeting intensity-modulated radiotherapy.
Purpose
Glycolysis is increased by hypoxia, suggesting a possible correlation between the accumulation of
2-18Ffluoro-2-deoxy-D-glucose
(FDG) in malignant tumors and regional hypoxia defined by ...1H-1-(3-18Ffluoro-2-hydroxypropyl)-2-nitroimidazole (FMISO) PET. The aim of this study is to investigate the intra-tumoral spatial distribution and quantitative relationship between FDG and FMISO in a cohort of head and neck squamous cell cancer (HNSCC) patients.
Methods
Twenty HNSCC patients with 20 primary tumors and 19 metastatic lymph nodes (LNs) underwent FDG and FMISO PET within 1 week. The metabolic target volume (MTV) was defined on the FDG PET images using a region growing algorithm. The hypoxic volume (HV) was defined by the volume of voxels in an FMISO image within the MTV that satisfy a tumor-to-blood ratio (T/B) greater than 1.2. FDG and FMISO lesions were co-registered, and a voxel-by-voxel correlation between the two datasets was performed. FDG and FMISO TVs’ SUVs were also compared as well as the intra-tumoral homogeneity of the two radiotracers. Separate analysis was performed for the primary tumors and LNs.
Results
Twenty-six percent of the primary tumors and 15% of LNs showed a strong correlation (R > 0.7) between FDG and FMISO intra-tumor distributions when considering the MTV. For the HV, only 19% of primary tumors and 12% of LN were strongly correlated. A weak and moderate correlation existed between the two markers SUV
avg
, and SUV
max
in the case of the primary tumors, respectively. However, this was not the case for the LNs. Good concordances were also observed between the primary tumor’s and LNs HV SUV
avg
s as well as between the corresponding hypoxic fractions (HF’s).
Conclusions
A moderate correlation between FDG and hypoxia radiotracer distribution, as measured by FMISO, seems to exist for primary tumors. However, discordant results were found in the case of LNs. Hypoxia appears to be the dominant driver of high FDG uptake in selected tumors only, and therefore FDG PET images cannot be used as a universal surrogate to identify or predict intra-tumor hypoxia.
Most patients with prostate cancer treated with androgen receptor (AR) signaling inhibitors develop therapeutic resistance due to restoration of AR functionality. Thus, there is a critical need for ...novel treatment approaches. Here we investigate the theranostic potential of hu5A10, a humanized mAb specifically targeting free PSA (
).
LNCaP-AR (LNCaP with overexpression of wildtype AR) xenografts (NSG mice) and
_Hi-
transgenic mice were imaged with
Zr- or treated with
Y- or
Ac-labeled hu5A10; biodistribution and subcellular localization were analyzed by gamma counting, PET, autoradiography, and microscopy. Therapeutic efficacy of
Achu5A10 and
Yhu5A10 in LNCaP-AR tumors was assessed by tumor volume measurements, time to nadir (TTN), time to progression (TTP), and survival. Pharmacokinetics of
Zrhu5A10 in nonhuman primates (NHP) were determined using PET.
Biodistribution of radiolabeled hu5A10 constructs was comparable in different mouse models. Specific tumor uptake increased over time and correlated with PSA expression. Treatment with
Y/
Achu5A10 effectively reduced tumor burden and prolonged survival (
≤ 0.0054). Effects of
Yhu5A10 were more immediate than
Achu5A10 (TTN,
< 0.0001) but less sustained (TTP,
< 0.0001). Complete responses were observed in 7 of 18
Achu5A10 and 1 of 9 mice
Yhu5A10. Pharmacokinetics of
Zrhu5A10 were consistent between NHPs and comparable with those in mice.
Zrhu5A10-PET visualized the NHP-prostate over the 2-week observation period.
We present a complete preclinical evaluation of radiolabeled hu5A10 in mouse prostate cancer models and NHPs, and establish hu5A10 as a new theranostic agent that allows highly specific and effective downstream targeting of AR in PSA-expressing tissue. Our data support the clinical translation of radiolabeled hu5A10 for treating prostate cancer.
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