Near-Room-Temperature Magnetoelectric Coupling via Spin Crossover in an Iron(II) Complex.

Magnetoelectric coupling is achieved near room temperature in a spin crossover FeII molecule-based compound, [Fe(1bpp)2 ](BF4 )2 . Large atomic displacements resulting from Jahn-Teller distortions induce a change in the molecule dipole moment when switching between high-spin and low-spin states leading to a step-wise change in the electric polarization and dielectric constant. For temperatures in the region of bistability, the changes in magnetic and electrical properties are induced with a remarkably low magnetic field of 3 T. This result represents a successful expansion of magnetoelectric spin crossovers towards ambient conditions. Moreover, the observed 0.3-0.4 mC m-2 changes in the H-induced electric polarization suggest that the high strength of the coupling obtained via this route is accessible not just at cryogenic temperatures but also near room temperature, a feature that is especially appealing in the light of practical applications.

Bifunctional Elastin-like Polypeptide Nanoparticles Bind Rapamycin and Integrins and Suppress Tumor Growth in Vivo.

Recombinant protein-polymer scaffolds such as elastin-like polypeptides (ELPs) offer drug-delivery opportunities including biocompatibility, monodispersity, and multifunctionality. We recently reported that the fusion of FK-506 binding protein 12 (FKBP) to an ELP nanoparticle (FSI) increases rapamycin (Rapa) solubility, suppresses tumor growth in breast cancer xenografts, and reduces side effects observed with free-drug controls. This new report significantly advances this carrier strategy by demonstrating the coassembly of two different ELP diblock copolymers containing drug-loading and tumor-targeting domains. A new ELP nanoparticle (ISR) was synthesized that includes the canonical integrin-targeting ligand (Arg-Gly-Asp, RGD). FSI and ISR mixed in a 1:1 molar ratio coassemble into bifunctional nanoparticles containing both the FKBP domain for Rapa loading and the RGD ligand for integrin binding. Coassembled nanoparticles were evaluated for bifunctionality by performing in vitro cell-binding and drug-retention assays and in vivo MDA-MB-468 breast tumor regression and tumor-accumulation studies. The bifunctional nanoparticle demonstrated superior cell target binding and similar drug retention to FSI; however, it enhanced the formulation potency, such that tumor growth was suppressed at a 3-fold lower dose compared to an untargeted FSI-Rapa control. This data suggests that ELP-mediated scaffolds are useful tools for generating multifunctional nanomedicines with potential activity in cancer.

A potent immunotoxin targeting fibroblast activation protein for treatment of breast cancer in mice.

Fibroblast activation protein (FAP) is highly expressed in the tumor-associated fibroblasts (TAFs) of most human epithelial cancers. FAP plays a critical role in tumorigenesis and cancer progression, which makes it a promising target for novel anticancer therapy. However, mere abrogation of FAP enzymatic activity by small molecules is not very effective in inhibiting tumor growth. In this study, we have evaluated a novel immune-based approach to specifically deplete FAP-expressing TAFs in a mouse 4T1 metastatic breast cancer model. Depletion of FAP-positive stromal cells by FAP-targeting immunotoxin αFAP-PE38 altered levels of various growth factors, cytokines, chemokines and matrix metalloproteinases, decreased the recruitment of tumor-infiltrating immune cells in the tumor microenvironment and suppressed tumor growth. In addition, combined treatment with αFAP-PE38 and paclitaxel potently inhibited tumor growth in vivo. Our findings highlight the potential use of immunotoxin αFAP-PE38 to deplete FAP-expressing TAFs and thus provide a rationale for the use of this immunotoxin in cancer therapy.

Small-Animal PET Imaging of Pancreatic Cancer Xenografts Using a 64Cu-Labeled Monoclonal Antibody, MAb159.

UNLABELLED: Overexpression of the GRP78 receptor on cell surfaces has been linked with tumor growth, metastasis, and resistance to therapy. We developed a (64)Cu-labeled probe for PET imaging of tumor GRP78 expression based on a novel anti-GRP78 monoclonal antibody, MAb159. METHODS: MAb159 was conjugated with the (64)Cu-chelator DOTA through lysines on the antibody. DOTA-human IgG was also prepared as a control that did not bind to GRP78. The resulting PET probes were evaluated in BXPC3 pancreatic cancer xenografts in athymic nude mice. RESULTS: The radiotracer was synthesized with a specific activity of 0.8 MBq/µg of antibody. In BXPC3 xenografts, (64)Cu-DOTA-MAb159 demonstrated prominent tumor accumulation (4.3 ± 1.2, 15.4 ± 2.6, and 18.3 ± 1.0 percentage injected dose per gram at 1, 17, and 48 after injection, respectively). In contrast, (64)Cu-DOTA-human IgG had low BXPC3 tumor accumulation (4.8 ± 0.5, 7.5 ± 0.7, and 4.6 ± 0.8 percentage injected dose per gram at 1, 17, and 48 h after injection, respectively). CONCLUSION: We demonstrated that GRP78 can serve as a valid target for pancreatic cancer imaging. The success of this approach will be valuable for evaluating disease course and therapeutic efficacy at the earliest stages of anti-GRP78 treatment. Moreover, these newly developed probes may have important applications in other types of cancer overexpressing GRP78.

Improved metabolic stability for 18F PET probes rapidly constructed via tetrazine trans-cyclooctene ligation.

The fast kinetics and bioorthogonal nature of the tetrazine trans-cyclooctene (TCO) ligation makes it a unique tool for PET probe construction. In this study, we report the development of an (18)F-labeling system based on a CF3-substituted diphenyl-s-tetrazine derivative with the aim of maintaining high reactivity while increasing in vivo stability. c(RGDyK) was tagged by a CF3-substituted diphenyl-s-tetrazine derivative via EDC-mediated coupling. The resulting tetrazine-RGD conjugate was combined with a (19)F-labeled TCO derivative to give HPLC standards. The analogous (18)F-labeled TCO derivative was combined with the diphenyl-s-tetrazine-RGD at µM concentration. The resulting tracer was subjected to in vivo metabolic stability assessment, and microPET studies in murine U87MG xenograft models. The diphenyl-s-tetrazine-RGD combines with an (18)F-labeled TCO in high yields (>97% decay-corrected on the basis of TCO) using only 4 equiv of tetrazine-RGD relative to the (18)F-labeled TCO (concentration calculated based on product's specific activity). The radiochemical purity of the (18)F-RGD peptides was >95% and the specific activity was 111 GBq/µmol. Noninvasive microPET experiments demonstrated that (18)F-RGD had integrin-specific tumor uptake in subcutaneous U87MG glioma. In vivo metabolic stability of (18)F-RGD in blood, urine, and major organs showed two major peaks: one corresponded to the Diels-Alder conjugate and the other was identified as the aromatized analog. A CF3-substituted diphenyl-s-tetrazine displays excellent speed and efficiency in (18)F-PET probe construction, providing nearly quantitative (18)F labeling within minutes at low micromolar concentrations. The resulting conjugates display improved in vivo metabolic stability relative to our previously described system.

18F-FAZA PET imaging response tracks the reoxygenation of tumors in mice upon treatment with the mitochondrial complex I inhibitor BAY 87-2243.

PURPOSE: We describe a noninvasive PET imaging method that monitors early therapeutic efficacy of BAY 87-2243, a novel small-molecule inhibitor of mitochondrial complex I as a function of hypoxia-inducible factor-1α (HIF1α) activity. EXPERIMENTAL DESIGN: Four PET tracers [(18)F-FDG, (18)F-Fpp(RGD)2, (18)F-FLT, and (18)F-FAZA] were assessed for uptake into tumor xenografts of drug-responsive (H460, PC3) or drug-resistant (786-0) carcinoma cells. Mice were treated with BAY 87-2243 or vehicle. At each point, RNA from treated and vehicle H460 tumor xenografts (n = 3 each) was isolated and analyzed for target genes. RESULTS: Significant changes in uptake of (18)F-FAZA, (18)F-FLT, and (18)F-Fpp(RGD)2 (P < 0.01) occurred with BAY 87-2243 treatment with (18)F-FAZA being the most prominent. (18)F-FDG uptake was unaffected. (18)F-FAZA tumor uptake declined by 55% to 70% (1.21% ± 0.10%ID/g to 0.35 ± 0.1%ID/g; n = 6, vehicle vs. treatment) in both H460 (P < 0.001) and PC3 (P < 0.05) xenografts 1 to 3 days after drug administration. (18)F-FAZA uptake in 786-0 xenografts was unaffected. Decline occurred before significant differences in tumor volume, thus suggesting (18)F-FAZA decrease reflected early changes in tumor metabolism. BAY 87-2243 reduced expression of hypoxia-regulated genes CA IX, ANGPTL4, and EGLN-3 by 99%, 93%, and 83%, respectively (P < 0.001 for all), which corresponds with reduced (18)F-FAZA uptake upon drug treatment. Heterogeneous expression of genes associated with glucose metabolism, vessel density, and proliferation was observed. CONCLUSIONS: Our studies suggest suitability of (18)F-FAZA-PET as an early pharmacodynamic monitor on the efficacy of anticancer agents that target the mitochondrial complex I and intratumor oxygen levels (e.g., BAY 87-2243).

Integrin α2ß1 targeted GdVO4:Eu ultrathin nanosheet for multimodal PET/MR imaging.

Multifunctional nanoprobes open exciting possibilities for accurate diagnosis and therapy. In this research, we developed a (64)Cu-labeled GdVO4:4%Eu two-dimension (2D) tetragonal ultrathin nanosheets (NSs) that simultaneously possess radioactivity, fluorescence, and paramagnetic properties for multimodal imaging. The carboxyl-functionalized Eu(3+)-doped GdVO4 NSs were synthesized by a facile solvothermal reaction, followed by ligand exchange with polyacrylic acid (PAA). With ultrathin thickness of ∼5 nm and width of ∼150 nm, the carboxyl-functionalized NSs were further modified by DOTA chelator for (64)Cu labeling and Asp-Gly-Glu-Ala (DGEA) peptide for integrin α2ß1 targeting. After initial evaluation of the cytotoxicity and targeting capability with PC-3 cells, the obtained multifunctional nanoprobes ((64)Cu-DOTA-GdVO4:4%Eu-DGEA) were further explored for targeted positron emission tomography (PET) and T1-weighted magnetic resonance imaging (MRI) of PC-3 tumor (prostate cancer, high integrin α2ß1 expression) in vivo. Based on the strong fluorescence of the NSs, the particle distribution in mouse tissues was also determined by fluorescent microscopy. In summary, GdVO4:4%Eu NS is a potential multimodal multiscale nanoprobe that could not only be used for in vivo imaging, but also be tracked in cellular scale and ex vivo due to its fluorescent property.

Design, synthesis, and validation of Axl-targeted monoclonal antibody probe for microPET imaging in human lung cancer xenograft.

Accumulating experimental evidence indicates that overexpression of the oncogenic receptor tyrosine kinase, Axl, plays a key role in the tumorigenesis and metastasis of various types of cancer. The objective of this study is to design a novel imaging probe based on the monoclonal antibody, h173, for microPET imaging of Axl expression in human lung cancer. A bifunctional chelator, DOTA, was conjugated to h173, followed by radiolabeling with (64)Cu. The binding of DOTA-h173 to the Axl receptor was first evaluated by a cell uptake assay and flow cytometry analysis using human lung cancer cell lines. The probe (64)Cu-DOTA-h173 was further evaluated by microPET imaging, and ex vivo histology studies in the Axl-positive A549 tumors. In vitro cellular study showed that Axl probe, (64)Cu-DOTA-h173, was highly immuno-reactive with A549 cells. Western blot analysis confirmed that Axl is highly expressed in the A549 cell line. For microPET imaging, the A549 xenografts demonstrated a significantly higher (64)Cu-DOTA-h173 uptake compared to the NCI-H249 xenograft (a negative control model). Furthermore, (64)Cu-DOTA-h173 uptake in A549 is significantly higher than that of (64)Cu-DOTA-hIgG. Immuno-fluorescence staining was consistent with the in vivo micro-PET imaging results. In conclusion, (64)Cu-DOTA-h173 could be potentially used as a probe for noninvasive imaging of Axl expression, which could collect important information regarding tumor response to Axl-targeted therapeutic interventions.

(64)Cu labeled sarcophagine exendin-4 for microPET imaging of glucagon like peptide-1 receptor expression.

The Glucagon-like peptide 1 receptor (GLP-1R) has become an important target for imaging due to its elevated expression profile in pancreatic islets, insulinoma, and the cardiovascular system. Because native GLP-1 is degraded rapidly by dipeptidyl peptidase-IV (DPP-IV), several studies have conjugated different chelators to a more stable analog of GLP-1 (such as exendin-4) as PET or SPECT imaging agents with various advantages and disadvantages. Based on the recently developed Sarcophagin chelator, here, we describe the construction of GLP-1R targeted PET probes containing monomeric and dimeric exendin-4 subunit. The in vitro binding affinity of BarMalSar-exendin-4 and Mal2Sar-(exendin-4)2 was evaluated in INS-1 cells, which over-express GLP-1R. Mal2Sar-(exendin-4)2 demonstrated around 3 times higher binding affinity compared with BaMalSar-exendin-4. After (64)Cu labeling, microPET imaging of (64)Cu-BaMalSar-exendin-4 and (64)Cu-Mal2Sar-(exendin-4)2 were performed on subcutaneous INS-1 tumors, which were clearly visualized with both probes. The tumor uptake of (64)Cu-Mal2Sar-(exendin-4)2 was significantly higher than that of (64)Cu-BaMaSarl-exendin-4, which could be caused by polyvalency effect. The receptor specificity of these probes was confirmed by effective blocking of the uptake in both tumor and normal positive organs with 20-fold excess of unlabeled exendin-4. In conclusion, sarcophagine cage conjugated exendin-4 demonstrated persistent and specific uptake in INS-1 insulinoma model. Dimerization of exendin-4 could successfully lead to increased tumor uptake in vivo. Both (64)Cu-BaMalSar-exendin-4 and (64)Cu-Mal2Sar-(exendin-4)2 hold a great potential for GLP-1R targeted imaging.

Multimeric disintegrin protein polymer fusions that target tumor vasculature.

Recombinant protein therapeutics have increased in number and frequency since the introduction of human insulin, 25 years ago. Presently, proteins and peptides are commonly used in the clinic. However, the incorporation of peptides into clinically approved nanomedicines has been limited. Reasons for this include the challenges of decorating pharmaceutical-grade nanoparticles with proteins by a process that is robust, scalable, and cost-effective. As an alternative to covalent bioconjugation between a protein and nanoparticle, we report that biologically active proteins may themselves mediate the formation of small multimers through steric stabilization by large protein polymers. Unlike multistep purification and bioconjugation, this approach is completed during biosynthesis. As proof-of-principle, the disintegrin protein called vicrostatin (VCN) was fused to an elastin-like polypeptide (A192). A significant fraction of fusion proteins self-assembled into multimers with a hydrodynamic radius of 15.9 nm. The A192-VCN fusion proteins compete specifically for cell-surface integrins on human umbilical vein endothelial cells (HUVECs) and two breast cancer cell lines, MDA-MB-231 and MDA-MB-435. Confocal microscopy revealed that, unlike linear RGD-containing protein polymers, the disintegrin fusion protein undergoes rapid cellular internalization. To explore their potential clinical applications, fusion proteins were characterized using small animal positron emission tomography (microPET). Passive tumor accumulation was observed for control protein polymers; however, the tumor accumulation of A192-VCN was saturable, which is consistent with integrin-mediated binding. The fusion of a protein polymer and disintegrin results in a higher intratumoral contrast compared to free VCN or A192 alone. Given the diversity of disintegrin proteins with specificity for various cell-surface integrins, disintegrin fusions are a new source of biomaterials with potential diagnostic and therapeutic applications.

Facile Preparation of a Thiol-Reactive (18)F-Labeling Agent and Synthesis of (18)F-DEG-VS-NT for PET Imaging of a Neurotensin Receptor-Positive Tumor.

UNLABELLED: Accumulating evidence suggests that neurotensin receptors (NTRs) play key roles in cancer growth and survival. In this study, we developed a simple and efficient method to radiolabel neurotensin peptide with (18)F for NTR-targeted imaging. METHODS: The thiol-reactive reagent (18)F-(2-(2-(2-fluoroethoxy)ethoxy)ethylsulfonyl)ethane ((18)F-DEG-VS) was facilely prepared through 1-step radiofluorination. After high-pressure liquid chromatography purification, (18)F-DEG-VS was incubated with the c(RGDyC) and c(RGDyK) peptide mixture to evaluate its specificity toward the reactive thiol. Thiolated neurotensin peptide was then labeled with (18)F using this novel synthon, and the resulting imaging probe was subjected to receptor-binding assay and small-animal PET studies in a murine xenograft model. The imaging results and metabolic stability of (18)F-DEG-VS-NT were compared with the thiol-specific maleimide derivative N-[2-(4-(18)F-fluorobenzamido)ethyl]maleimide-neurotensin ((18)F-FBEM-NT). RESULTS: (18)F-DEG-VS was obtained in high labeling yield. The reaction of (19)F-DEG-VS was highly specific for thiols at neutral pH, whereas the lysine of c(RGDyK) reacted at a pH greater than 8.5. (18)F-DEG-VS-c(RGDyC) was the preferred product when both c(RGDyK) and c(RGDyC) were incubated together with (18)F-DEG-VS. Thiolated neurotensin peptide (Cys-NT) efficiently reacted with (18)F-DEG-VS, with a 95% labeling yield (decay-corrected). The radiochemical purity of the (18)F-DEG-VS-NT was greater than 98%, and the specific activity was about 19.2 ± 4.3 TBq/mmol. Noninvasive small-animal PET demonstrated that (18)F-DEG-VS-NT had an NTR-specific tumor uptake in subcutaneous HT-29 xenografts. The tumor-to-muscle, tumor-to-liver, and tumor-to-kidney ratios reached 30.65 ± 22.31, 11.86 ± 1.98, and 1.91 ± 0.43 at 2 h after injection, respectively, based on the biodistribution study. Receptor specificity was demonstrated by blocking experiment. Compared with (18)F-FBEM-NT, (18)F-DEG-VS-NT was synthesized with fewer steps and provided significantly improved imaging quality in vivo. CONCLUSION: We have established a facile (18)F-labeling method for site-specific labeling of the Cys-NT. Using this method, we synthesized an NTR-targeted PET agent, which demonstrated high tumor-to-background contrast.

A hybrid protein-polymer nanoworm potentiates apoptosis better than a monoclonal antibody.

B-cell lymphomas continue to occur with a high incidence. The chimeric antibody known as Rituximab (Rituxan) has become a vital therapy for these patients. Rituximab induces cell death via binding and clustering of the CD20 receptor by Fcγ expressing effector cells. Because of the limited mobility of effector cells, it may be advantageous to cluster CD20 directly using multivalent nanostructures. To explore this strategy, this manuscript introduces a nanoparticle that assembles from a fusion between a single chain antibody and a soluble protein polymer. These hybrid proteins express in Escherichia coli and do not require bioconjugation between the antibody and a substrate. Surprisingly a fusion between an anti-CD20 single chain antibody and a soluble protein polymer assemble worm-like nanostructures, which were characterized using light scattering and cryogenic transmission electron microscopy. These nanoworms competitively bind CD20 on two B-cell lymphoma cell lines, exhibit concentration-dependent induction of apoptosis, and induce apoptosis better than Rituximab alone. Similar activity was observed in vivo using a non-Hodgkin lymphoma xenograft model. In comparison to Rituximab, systemic nanoworms significantly slowed tumor growth. These findings suggest that hybrid nanoworms targeted at CD20 may be useful treatments for B-cell related malignancies. Because of the ubiquity of antibody therapeutics, related nanoworms may have uses against other molecular targets.

Axl-targeted cancer imaging with humanized antibody h173.

PURPOSE: The tyrosine kinase receptor Axl is overexpressed in various types of cancer and correlated with cancer malignancy. Selective Axl blockade reduces tumor growth and metastasis. The purpose of this study was to examine whether the humanized anti-Axl antibody humanized 173 (h173) labeled with near-infrared fluorescence (NIRF) dye Cy5.5 could be applied as a molecular imaging probe for NIRF imaging of Axl expression in tumor models. PROCEDURES: NIRF dye Cy5.5 was conjugated to h173 or human normal immunoglobulin G (hIgG) control through amino groups. The resulting probes were evaluated in both A549 (Axl positive) and NCI-H249 (Axl negative) lung cancer xenografts through in vivo NIRF imaging. Ex vivo imaging and probe distribution assay were also carried out to confirm the in vivo imaging results. RESULTS: After conjugation, binding activity of h173-Cy5.5 was determined to be 97.75 % ± 2.09 % of the unmodified h173. In vitro fluorescence-activated cell sorting (FACS) and fluorescence microscopy analysis validated the specific binding of h173 toward Axl-positive A549 cells. h173-Cy5.5 was then applied to image Axl expression in vivo. In A549 (Axl positive) cancer xenografts, the tumor uptake of h173-Cy5.5 was significantly higher than that of the hIgG-Cy5.5 control (P < 0.05) at late time points (1, 2, 3, 4, and 7 days). On the contrary, in NCI-H249 (Axl negative) cancer xenografts, the tumor uptake of both hIgG-Cy5.5 and h173-Cy5.5 was low and showed no significant difference (P > 0.05) at all time points examined. Ex vivo imaging and immunofluorescence staining analysis further validated the in vivo imaging results. CONCLUSIONS: Collectively, all in vitro, in vivo, and ex vivo data suggested that h173-Cy5.5 could serve as a valid probe for Axl-targeted cancer imaging, which could therefore aid in tumor diagnosis, prognosis, and treatment monitoring.

Characterization and evaluation of (64)Cu-labeled A20FMDV2 conjugates for imaging the integrin αvß 6.

PURPOSE: The integrin αvß6 is overexpressed in a variety of aggressive cancers and serves as a prognosis marker. This study describes the conjugation, radiolabeling, and in vitro and in vivo evaluation of four chelators to determine the best candidate for (64)Cu radiolabeling of A20FMDV2, an αvß6 targeting peptide. PROCEDURES: Four chelators were conjugated onto PEG28-A20FMDV2 (1): 11-carboxymethyl-1,4,8,11-tetraazabicyclo[6.6.2]hexadecane-4-methanephosphonic acid (CB-TE1A1P), 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid (DOTA), 1,4,7-triazacyclononane-1,4,7-triacetic acid (NOTA), and 4,4'-((3,6,10,13,16,19-hexazazbicyclo[6.6.6]ico-sane-1,8-diylbis(aza-nediyl))bis(methylene)dibenzoic acid (BaBaSar). All peptides were radiolabeled with (64)Cu in ammonium acetate buffer at pH 6 and formulated to pH 7.2 in PBS for use. The radiotracers were evaluated using in vitro cell binding and internalization assays and serum stability assays. In vivo studies conducted include blocking, biodistribution, and small animal PET imaging. Autoradiography and histology were also conducted. RESULTS: All radiotracers were radiolabeled in good radiochemical purity (>95 %) under mild conditions (37-50 °C for 15 min) with high specific activity (0.58-0.60 Ci/µmol). All radiotracers demonstrated αvß6-directed cell binding (>46 %) with similar internalization levels (>23 %). The radiotracers (64)Cu-CB-TE1A1P-1 and (64)Cu-BaBaSar-1 showed improved specificity for the αvß6 positive tumor in vivo over (64)Cu-DOTA-1 and (64)Cu-NOTA-1 (+/- tumor uptake ratios-3.82 +/- 0.44, 3.82 ± 0.41, 2.58 ± 0.58, and 1.29 ± 0.14, respectively). Of the four radiotracers, (64)Cu-NOTA-1 exhibited the highest liver uptake (10.83 ± 0.1 % ID/g at 4 h). CONCLUSIONS: We have successfully conjugated, radiolabeled, and assessed the four chelates CB-TE1A1P, DOTA, NOTA, and BaBaSar both in vitro and in vivo. However, the data suggests no clear "best candidate" for the (64)Cu-radiolabeling of A20FMDV2, but instead a trade-off between the different properties (e.g., stability, selectivity, pharmacokinetics, etc.) with no obvious effects of the individual chelators.

The synthesis of lanthanide-doped GdVO4 ultrathin nanosheets with great optical and paramagnetic properties for FRET biodetection and in vivo MR imaging.

The development of two dimension (2D) inorganic nanosheets (NSs) with unique physical and chemical properties has been the focus of intense study in recent years. Herein, we report the synthesis of lanthanide ion (Ln3+ = Eu3+, Dy3+)-doped GdVO4 2D tetragonal NSs (with a thickness of ∼5 nm and a width of ∼150 nm) using a facile solvothermal reaction. After the ligand exchange reaction with polyacrylic acid (PAA), the hydrophilic 2D NSs demonstrated a high fluorescence (absolute quantum yield (QY) of 24%) and excellent paramagnetic properties (longitudinal relaxivity reached 37.8 mM-1 s-1). Moreover, this carboxyl functionalized NS could be easily modified for bio-application and exhibited a low toxicity towards cells. Based on the bright fluorescence of the GdVO4:Dy NSs, a sensitive Förster resonance energy transfer (FRET) bioprobe was developed to quantitatively detect the presence of streptavidin. In parallel, the paramagnetic properties of the NSs allows the magnetic resonance imaging (MRI) of integrin α2ß1 expression in human prostate cancer. Collectively, multifunctional NSs with a 2D nanostructure demonstrated unique fluorescence and paramagnetic properties, which could be used to construct high performance nanoprobes for biomedical applications.

EphB4-targeted imaging with antibody h131, h131-F(ab')2 and h131-Fab.

Accumulating evidence suggests that overexpression of the tyrosine kinase receptor EphB4, a mediator of vascular development, is a novel target for tumor diagnosis, prognosis and therapy. Noninvasive imaging of EphB4 expression could therefore be valuable for evaluating disease course and therapeutic efficacy at the earliest stages of anti-EphB4 treatment. In this study, we systematically investigated the use of anti-EphB4 antibody h131 (150 kDa) and its fragments (h131-F(ab')2, 110 kDa; h131-Fab, 50 kDa) for near-infrared fluorescence (NIRF) imaging of EphB4 expression in vivo. h131-F(ab')2 and h131-Fab were produced through pepsin and papain digestion of h131 respectively, whose purity was confirmed by FPLC and SDS-PAGE. After conjugation with Cy5.5, in vivo characteristics of h131, h131-F(ab')2 and h131-Fab were evaluated in EphB4-positive HT29 tumor model. Although h131-Cy5.5 demonstrated highest tumor uptake among these probes, its optimal tumor uptake level was obtained at 2 days post injection (p.i.). For h131-Fab-Cy5.5, maximum tumor uptake was achieved at 4 h p.i. However, no significant difference was observed between h131-Fab-Cy5.5 and hIgG-Fab-Cy5.5, indicating the tumor accumulation was mainly caused by passive targeting. In contrast, h131-F(ab')2-Cy5.5 demonstrated prominent tumor uptake at 6 h p.i. The target specificity was confirmed by hIgG-F(ab')2-Cy5.5 control and immunofluorescent staining. Collectively, h131-F(ab')2 exhibited prominent and specific tumor uptake at early time points, which suggests it is a promising agent for EphB4-targeted imaging.

Monoclonal antibody against cell surface GRP78 as a novel agent in suppressing PI3K/AKT signaling, tumor growth, and metastasis.

PURPOSE: The ER chaperone GRP78 translocates to the surface of tumor cells and promotes survival, metastasis, and resistance to therapy. An oncogenic function of cell surface GRP78 has been attributed to the activation of the phosphoinositide 3-kinase (PI3K) pathway. We intend to use a novel anti-GRP78 monoclonal antibody (MAb159) to attenuate PI3K signaling and inhibit tumor growth and metastasis. EXPERIMENTAL DESIGN: MAb159 was characterized biochemically. Antitumor activity was tested in cancer cell culture, tumor xenograft models, tumor metastasis models, and spontaneous tumor models. Cancer cells and tumor tissues were analyzed for PI3K activity. MAb159 was humanized and validated for diagnostic and therapeutic application. RESULTS: MAb159 specifically recognized surface GRP78, triggered GRP78 endocytosis, and localized to tumors but not to normal organs in vivo. MAb159 inhibited tumor cell proliferation and enhanced tumor cell death both in vitro and in vivo. In MAb159-treated tumors, PI3K signaling was inhibited without compensatory MAPK pathway activation. Furthermore, MAb159 halted or reversed tumor progression in the spontaneous PTEN-loss-driven prostate and leukemia tumor models, and inhibited tumor growth and metastasis in xenograft models. Humanized MAb159, which retains high affinity, tumor specific localization, and the antitumor activity, was nontoxic in mice, and had desirable pharmacokinetics. CONCLUSIONS: GRP78-specific antibody MAb159 modulates the PI3K pathway and inhibits tumor growth and metastasis. Humanized MAb159 will enter human trials shortly.

A novel aliphatic 18F-labeled probe for PET imaging of melanoma.

Radiofluorinated benzamide and nicotinamide analogues are promising molecular probes for the positron emission tomography (PET) imaging of melanoma. Compounds containing aromatic (benzene or pyridine) and N,N-diethylethylenediamine groups have been successfully used for development of melanin targeted PET and single-photon emission computed tomography (SPECT) imaging agents for melanoma. The objective of this study was to determine the feasibility of using aliphatic compounds as a molecular platform for the development of a new generation of PET probes for melanoma detection. An aliphatic N,N-diethylethylenediamine precursor was directly coupled to a radiofluorination synthon, p-nitrophenyl 2-(18)F-fluoropropionate ((18)F-NFP), to produce the probe N-(2-(diethylamino)ethyl)-2-(18)F-fluoropropanamide ((18)F-FPDA). The melanoma-targeting ability of (18)F-FPDA was further evaluated both in vitro and in vivo through cell uptake assays, biodistribution studies, and small animal PET imaging in C57BL/6 mice bearing B16F10 murine melanoma tumors. Beginning with the precursor (18)F-NFP, the total preparation time for (18)F-FPDA, including the final high-performance liquid chromatography purification step, was approximately 30 min, with a decay-corrected radiochemical yield of 79.8%. The melanin-targeting specificity of (18)F-FPDA was demonstrated by significantly different uptake rates in tyrosine-treated and untreated B16F10 cells in vitro. The tumor uptake of (18)F-FPDA in vivo reached 2.65 ± 0.48 %ID/g at 2 h postinjection (p.i.) in pigment-enriched B16F10 xenografts, whereas the tumor uptake of (18)F-FPDA was close to the background levels, with rates of only 0.37 ± 0.07 %ID/g at 2 h p.i. in the nonpigmented U87MG tumor mouse model. Furthermore, small animal PET imaging studies revealed that (18)F-FPDA specifically targeted the melanotic B16F10 tumor, yielding a tumor-to-muscle ratio of approximately 4:1 at 1 h p.i. and 7:1 at 2 h p.i. In summary, we report the development of a novel (18)F-labeled aliphatic compound for melanoma imaging that can be easily synthesized in high yields using the radiosynthon (18)F-NFP. The PET probe (18)F-FPDA exhibits high B16F10 tumor-targeting efficacy and favorable in vivo pharmacokinetics. Our study demonstrates that aliphatic compounds can be used as a new generation molecular platform for the development of novel melanoma targeting agents. Further evaluation and optimization of (18)F-FPDA for melanin targeted molecular imaging are therefore warranted.

Urokinase plasminogen activator receptor (uPAR) targeted nuclear imaging and radionuclide therapy.

Urokinase-type plasminogen activator receptor (uPAR) is a glycosylphosphatidylinositol (GPI)-anchored protein. Besides regulating proteolysis, uPAR could also activate many intracellular signaling pathways that promote cell motility, invasion, proliferation, and survival through cooperating with transmembrane receptors. uPAR is overexpressed across a variety of tumors and is associated with cancer invasion and metastasis. In order to meet the demand for a rapid development and potential clinical application of anti-cancer therapy based on uPA/uPAR system, it is desirable to develop non-invasive imaging methods to visualize and quantify uPAR expression in vivo. In this review, we will discuss recent advances in the development of uPAR-targeted nuclear imaging and radionuclide therapy agents. The successful development of molecular imaging probes to visualize uPAR expression in vivo would not only assist preclinical researches on uPAR function, but also eventually impact patient management.

PET imaging of colorectal and breast cancer by targeting EphB4 receptor with 64Cu-labeled hAb47 and hAb131 antibodies.

UNLABELLED: Accumulating evidence suggests that ephrin type B receptor 4 (EphB4) plays a key role in the progression of numerous cancer types. In this study, we developed a series of (64)Cu-labeled antibodies for PET imaging of tumor EphB4 expression. METHODS: Anti-EphB4 antibodies (hAb47 and hAb131) were conjugated with the (64)Cu-chelator DOTA through lysine, cysteine, or oligosaccharide on the antibody. DOTA-human IgG (hIgG) was also prepared as a control, which did not bind to EphB4. The EphB4 binding activity of these probes was evaluated through the bead-based binding assay with EphB4-alkaline phosphatase. The resulting PET probes were further evaluated in both HT29 (colorectal cancer) and MDA-MB-231 (breast cancer) xenografts. RESULTS: All 3 conjugation methods retained most of the EphB4 binding activity of the antibodies (83.85% ± 3.82%, 76.25% ± 5.90%, 98.93% ± 3.75%, and 82.09% ± 4.14% for DOTA-Lys-hAb47, DOTA-Cys-hAb47, DOTA-Sug-hAb47, and DOTA-Lys-hAb131, respectively). Although DOTA-Sug-hAb47 demonstrated the highest receptor binding activity based on a EphB4 binding assay, the corresponding PET probe was trapped in the liver quickly in vivo. In HT29 xenografts, both (64)Cu-DOTA-Lys-hAb47 and (64)Cu-DOTA-Cys-hAb47 demonstrated prominent tumor accumulation, which reached a maximum at 48 h after injection (18.13 ± 1.73 percentage injected dose [%ID]/g and 11.81 ± 2.05 %ID/g, respectively). In contrast, (64)Cu-DOTA-Lys-hIgG had a low tumor accumulation, thus demonstrating the target specificity of EphB4-antibody-based probes. Moreover, (64)Cu-DOTA-Lys-hAb131 (29.48 ± 2.60 %ID/g) demonstrated significantly higher HT29 tumor accumulation than (64)Cu-DOTA-Lys-hAb47. (64)Cu-DOTA-Lys-hAb131 was also found to specifically accumulate in the MDA-MB-231 tumor model (12.96 ± 2.31 %ID/g). CONCLUSION: We have demonstrated that EphB4 can serve as a valid target for colorectal and breast cancer imaging. This approach would be valuable for evaluating disease course and therapeutic efficacy at the earliest stages of anti-EphB4 treatment. Moreover, these newly developed probes may have important applications in other cancer types overexpressing EphB4.