Targeting renal cell carcinoma with a HIF-2 antagonist.

Clear cell renal cell carcinoma (ccRCC) is characterized by inactivation of the von Hippel-Lindau tumour suppressor gene (VHL). Because no other gene is mutated as frequently in ccRCC and VHL mutations are truncal, VHL inactivation is regarded as the governing event. VHL loss activates the HIF-2 transcription factor, and constitutive HIF-2 activity restores tumorigenesis in VHL-reconstituted ccRCC cells. HIF-2 has been implicated in angiogenesis and multiple other processes, but angiogenesis is the main target of drugs such as the tyrosine kinase inhibitor sunitinib. HIF-2 has been regarded as undruggable. Here we use a tumourgraft/patient-derived xenograft platform to evaluate PT2399, a selective HIF-2 antagonist that was identified using a structure-based design approach. PT2399 dissociated HIF-2 (an obligatory heterodimer of HIF-2α-HIF-1ß) in human ccRCC cells and suppressed tumorigenesis in 56% (10 out of 18) of such lines. PT2399 had greater activity than sunitinib, was active in sunitinib-progressing tumours, and was better tolerated. Unexpectedly, some VHL-mutant ccRCCs were resistant to PT2399. Resistance occurred despite HIF-2 dissociation in tumours and evidence of Hif-2 inhibition in the mouse, as determined by suppression of circulating erythropoietin, a HIF-2 target and possible pharmacodynamic marker. We identified a HIF-2-dependent gene signature in sensitive tumours. Gene expression was largely unaffected by PT2399 in resistant tumours, illustrating the specificity of the drug. Sensitive tumours exhibited a distinguishing gene expression signature and generally higher levels of HIF-2α. Prolonged PT2399 treatment led to resistance. We identified binding site and second site suppressor mutations in HIF-2α and HIF-1ß, respectively. Both mutations preserved HIF-2 dimers despite treatment with PT2399. Finally, an extensively pretreated patient whose tumour had given rise to a sensitive tumourgraft showed disease control for more than 11 months when treated with a close analogue of PT2399, PT2385. We validate HIF-2 as a target in ccRCC, show that some ccRCCs are HIF-2 independent, and set the stage for biomarker-driven clinical trials.

Theranostic Small-Molecule Prodrug Conjugates for Targeted Delivery and Controlled Release of Toll-like Receptor 7 Agonists.

We previously reported the design and synthesis of a small-molecule drug conjugate (SMDC) platform that demonstrated several advantages over antibody-drug conjugates (ADCs) in terms of in vivo pharmacokinetics, solid tumor penetration, definitive chemical structure, and adaptability for modular synthesis. Constructed on a tri-modal SMDC platform derived from 1,3,5-triazine (TZ) that consists of a targeting moiety (Lys-Urea-Glu) for prostate-specific membrane antigen (PSMA), here we report a novel class of chemically identical theranostic small-molecule prodrug conjugates (T-SMPDCs), [18/19F]F-TZ(PSMA)-LEGU-TLR7, for PSMA-targeted delivery and controlled release of toll-like receptor 7 (TLR7) agonists to elicit de novo immune response for cancer immunotherapy. In vitro competitive binding assay of [19F]F-TZ(PSMA)-LEGU-TLR7 showed that the chemical modification of Lys-Urea-Glu did not compromise its binding affinity to PSMA. Receptor-mediated cell internalization upon the PSMA binding of [18F]F-TZ(PSMA)-LEGU-TLR7 showed a time-dependent increase, indicative of targeted intracellular delivery of the theranostic prodrug conjugate. The designed controlled release of gardiquimod, a TLR7 agonist, was realized by a legumain cleavable linker. We further performed an in vivo PET/CT imaging study that showed significantly higher uptake of [18F]F-TZ(PSMA)-LEGU-TLR7 in PSMA+ PC3-PIP tumors (1.9 ± 0.4% ID/g) than in PSMA- PC3-Flu tumors (0.8 ± 0.3% ID/g) at 1 h post-injection. In addition, the conjugate showed a one-compartment kinetic profile and in vivo stability. Taken together, our proof-of-concept biological evaluation demonstrated the potential of our T-SMPDCs for cancer immunomodulatory therapies.

PSMA-Targeting Imaging and Theranostic Agents-Current Status and Future Perspective.

In the past two decades, extensive efforts have been made to develop agents targeting prostate-specific membrane antigen (PSMA) for prostate cancer imaging and therapy. To date, represented by two recent approvals of [68Ga]Ga-PSMA-11 and [18F]F-DCFPyL by the United States Food and Drug Administration (US-FDA) for positron emission tomography (PET) imaging to identify suspected metastases or recurrence in patients with prostate cancer, PSMA-targeting imaging and theranostic agents derived from small molecule PSMA inhibitors have advanced to clinical practice and trials of prostate cancer. The focus of current development of new PSMA-targeting agents has thus shifted to the improvement of in vivo pharmacokinetics and higher specific binding affinity with the aims to further increase the detection sensitivity and specificity and minimize the toxicity to non-target tissues, particularly the kidneys. The main strategies involve systematic chemical modifications of the linkage between the targeting moiety and imaging/therapy payloads. In addition to a summary of the development history of PSMA-targeting agents, this review provides an overview of current advances and future promise of PSMA-targeted imaging and theranostics with focuses on the structural determinants of the chemical modification towards the next generation of PSMA-targeting agents.

Comparison of BMIPP-SPECT/CT to 18FDG-PET/CT for Imaging Brown or Browning Fat in a Preclinical Model.

Obesity is a leading cause of preventable death and morbidity. To elucidate the mechanisms connecting metabolically active brown adipose tissue (BAT) and metabolic health may provide insights into methods of treatment for obesity-related conditions. 18F-fluorodeoxyglucose positron emission tomography/computed tomography (18FDG-PET/CT) is traditionally used to image human BAT activity. However, the primary energy source of BAT is derived from intracellular fatty acids and not glucose. Beta-methyl-p-iodophenylpentadecanoic acid (BMIPP) is a fatty acid analogue amenable to in vivo imaging by single photon emission computed tomography/CT (SPECT/CT) when radiolabeled with iodine isotopes. In this study, we compare the use of 18FDG-PET/CT and 125I-BMIPP-SPECT/CT for fat imaging to ascertain whether BMIPP is a more robust candidate for the non-invasive evaluation of metabolically active adipose depots. Interscapular BAT, inguinal white adipose tissue (iWAT), and gonadal white adipose tissue (gWAT) uptake of 18FDG and 125I-BMIPP was quantified in mice following treatment with the BAT-stimulating drug CL-316,243 or saline vehicle control. After CL-316,243 treatment, uptake of both radiotracers increased in BAT and iWAT. The standard uptake value (SUVmean) for 18FDG and 125I-BMIPP significantly correlated in these depots, although uptake of 125I-BMIPP in BAT and iWAT more closely mimicked the fold-change in metabolic rate as measured by an extracellular flux analyzer. Herein, we find that imaging BAT with the radioiodinated fatty acid analogue BMIPP yields more physiologically relevant data than 18FDG-PET/CT, and its conventional use may be a pivotal tool for evaluating BAT in both mice and humans.

Validation of SV2A-Targeted PET Imaging for Noninvasive Assessment of Neuroendocrine Differentiation in Prostate Cancer.

Neuroendocrine prostate cancer (NEPC) is an aggressive and lethal variant of prostate cancer (PCa), and it remains a diagnostic challenge. Herein we report our findings of using synaptic vesicle glycoprotein 2 isoform A (SV2A) as a promising marker for positron emission tomography (PET) imaging of neuroendocrine differentiation (NED). The bioinformatic analyses revealed an amplified SV2A gene expression in clinical samples of NEPC versus castration-resistant PCa with adenocarcinoma characteristics (CRPC-Adeno). Importantly, significantly upregulated SV2A protein levels were found in both NEPC cell lines and tumor tissues. PET imaging studies were carried out in NEPC xenograft models with 18F-SynVesT-1. Although 18F-SynVesT-1 is not a cancer imaging agent, it showed a significant uptake level in the SV2A+ tumor (NCI-H660: 0.70 ± 0.14 %ID/g at 50-60 min p.i.). The SV2A blockade resulted in a significant reduction of tumor uptake (0.25 ± 0.03 %ID/g, p = 0.025), indicating the desired SV2A imaging specificity. Moreover, the comparative PET imaging study showed that the DU145 tumors could be clearly visualized by 18F-SynVesT-1 but not 68Ga-PSMA-11 nor 68Ga-DOTATATE, further validating the role of SV2A-targeted imaging for noninvasive assessment of NED in PCa. In conclusion, we demonstrated that SV2A, highly expressed in NEPC, can serve as a promising target for noninvasive imaging evaluation of NED.

Functional imaging of RAS pathway targeting in malignant peripheral nerve sheath tumor cells and xenografts.

BACKGROUND: Malignant peripheral nerve sheath tumor (MPNST) is an aggressive form of soft-tissue sarcoma (STS) in children. Despite intensive therapy, relatively few children with metastatic and unresectable disease survive beyond three years. RAS pathway activation is common in MPNST, suggesting MEK pathway inhibition as a targeted therapy, but the impact on clinical outcome has been small to date. PROCEDURE: We conducted preclinical pharmacokinetic (PK) and pharmacodynamic studies of two MEK inhibitors, trametinib and selumetinib, in two MPNST models and analyzed tumors for intratumor drug levels. We then investigated 3'-deoxy-3'-[18 F]fluorothymidine (18 F-FLT) PET imaging followed by 18 F-FDG PET/CT imaging of MPNST xenografts coupled to short-term or longer-term treatment with selumetinib focusing on PET-based imaging as a biomarker of MEK inhibition. RESULTS: Trametinib decreased pERK expression in MPNST xenografts but did not prolong survival or decrease Ki67 expression. In contrast, selumetinib prolonged survival of animals bearing MPNST xenografts, and this correlated with decreased pERK and Ki67 staining. PK studies revealed a significantly higher fraction of unbound selumetinib within a responsive MPNST xenograft model. Thymidine uptake, assessed by 18 F-FLT PET/CT, positively correlated with Ki67 expression in different xenograft models and in response to selumetinib. CONCLUSION: The ability of MEK inhibitors to control MPNST growth cannot simply be predicted by serum drug levels or drug-induced changes in pERK expression. Tumor cell proliferation assessed by 18 F-FLT PET imaging might be useful as an early response marker to targeted therapies, including MEK inhibition, where a primary effect is cell-cycle arrest.

Design of a Small-Molecule Drug Conjugate for Prostate Cancer Targeted Theranostics.

Targeted therapy has become an effective strategy of precision medicine for cancer treatment. Based on the success of antibody-drug conjugates (ADCs), here we report a theranostic design of small-molecule drug conjugates (T-SMDCs) for targeted imaging and chemotherapy of prostate cancer. The structure of T-SMDCs built upon a polyethylene glycol (PEG) scaffold consists of (i) a chelating moiety for positron emission tomography (PET) imaging when labeled with (68)Ga, a positron-emitting radioisotope; (ii) a prostate specific membrane antigen (PSMA) specific ligand for prostate cancer targeting; and (iii) a cytotoxic drug (DM1) for chemotherapy. For proof-of-concept, such a T-SMDC, NO3A-DM1-Lys-Urea-Glu, was synthesized and evaluated. The chemical modification of Lys-Urea-Glu for the construction of the conjugate did not compromise its specific binding affinity to PSMA. The PSMA-mediated internalization of (68)Ga-labeled NO3A-DM1-Lys-Urea-Glu displayed a time-dependent manner, allowing the desired drug delivery and release within tumor cells. The antiproliferative activity of the T-SMDC showed a positive correlation with the PSMA expression level. Small animal PET imaging with (68)Ga-labeled NO3A-DM1-Lys-Urea-Glu exhibited significantly higher uptake (p < 0.01) in the PSMA positive PC3-PIP tumors (4.30 ± 0.20%ID/g) at 1 h postinjection than in the PSMA negative PC3-Flu tumors (1.12 ± 0.42%ID/g). Taken together, we have successfully designed and synthesized a T-SMDC system for prostate cancer targeted imaging and therapy.

Renal clearance and degradation of glutathione-coated copper nanoparticles.

Degradation of inorganic nanoparticles (NPs) into small molecular complexes is often observed in the physiological environment; however, how this process influences renal clearance of inorganic NPs is largely unknown. By systematically comparing renal clearance of degradable luminescent glutathione coated copper NPs (GS-CuNPs) and their dissociated products, Cu(II)-glutathione disulfide (GSSG) complexes (Cu(II)-GSSG), we found that GS-CuNPs were eliminated through the urinary system surprisingly faster and accumulated in the liver much less than their smaller dissociation counterparts. With assistance of radiochemistry and positron emission tomography (PET) imaging, we found that the observed "nano size" effect in enhancing renal clearance is attributed to the fact that GS-CuNPs are more resistant to serum protein adsorption than Cu(II)-GSSG. In addition, since dissociation of GS-CuNPs follows zero-order chemical kinetics, their renal clearance and biodistribution also depend on initial injection doses and their dissociation processes. Quantitative understanding of size effect and other factors involved in renal clearance and biodistribution of degradable inorganic NPs will lay down a foundation for further development of renal-clearable inorganic NPs with minimized nanotoxicity.

Click-chemistry strategy for labeling antibodies with copper-64 via a cross-bridged tetraazamacrocyclic chelator scaffold.

We report a click-chemistry based modular strategy for antibody labeling with (64)Cu (t1/2 = 12.7 h; ß(+) 0.656 MeV, 17.4%; ß(-) 0.573 MeV, 39%; EC 43%) under ambient condition utilizing a cross-bridged tetraazamacrocyclic (CB-TE2A) analogue, which otherwise requires harsh conditions that make the CB-TE2A analogues under-utilized for protein labeling despite the fact that they form kinetically inert copper complexes with high in vivo stability. Our strategy involves prelabeling a CB-TE2A based scaffold (CB-TE2A-1C) with (64)Cu and its subsequent reaction with an antibody via the tetrazine-norbornene mediated click chemistry. The effectiveness of this strategy was demonstrated by labeling two monoclonal antibodies, an anti-PSMA antibody (YPSMA-1) and a chimeric anti-phosphatidylserine antibody (Bavituximab). The immunoreactivity of the antibodies remained unchanged after the tetrazine modification and click-chemistry (64)Cu labeling. To further demonstrate the practicality of the modular (64)Cu labeling strategy, we tested positron emission tomography (PET) imaging of tumor with the (64)Cu-labeled bavituximab in a mouse xenograft model. The tumor visualization and uptake of the labeled antibody exhibited the versatility of the click-chemistry strategy.

Molecular platform for design and synthesis of targeted dual-modality imaging probes.

We report a versatile dendritic structure based platform for construction of targeted dual-modality imaging probes. The platform contains multiple copies of 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid (DOTA) branching out from a 1,4,7-triazacyclononane-N,N',N″-triacetic acid (NOTA) core. The specific coordination chemistries of the NOTA and DOTA moieties offer specific loading of (68/67)Ga(3+) and Gd(3+), respectively, into a common molecular scaffold. The platform also contains three amino groups which can potentiate targeted dual-modality imaging of PET/MRI or SPECT/MRI (PET: positron emission tomography; SPECT: single photon emission computed tomography; MRI: magnetic resonance imaging) when further functionalized by targeting vectors of interest. To validate this design concept, a bimetallic complex was synthesized with six peripheral Gd-DOTA units and one Ga-NOTA core at the center, whose ion T1 relaxivity per gadolinium atom was measured to be 15.99 mM(-1) s(-1) at 20 MHz. Further, the bimetallic agent demonstrated its anticipated in vivo stability, tissue distribution, and pharmacokinetic profile when labeled with (67)Ga. When conjugated with a model targeting peptide sequence, the trivalent construct was able to visualize tumors in a mouse xenograft model by both PET and MRI via a single dose injection.

A comparative study of trans- and cis-isomers of a bone-seeking agent, DO2A2P.

The macrocyclic bone-seeking agent, DO2A2P, bears a cyclen core and two pairs of peripheral phosphonate and carboxylate groups. The geometric disposition of the peripheral functionalities gives arise to a pair of geometric isomers: cis-DO2A2P and trans-DO2A2P. In order to compare the biological behavior of the isomer pair, cis-DO2A2P was synthesized. Both isomers were successfully radiolabeled with (177)Lu, which might potentiate their applications in both radiotherapy and imaging of bone diseases. Through a set of biological assays including the hydroxyapatite binding, in vitro stability, and in vivo distribution, we demonstrated that the geometric pair of DO2A2P had virtually identical biological properties.

A multivalent approach of imaging probe design to overcome an endogenous anion binding competition for noninvasive assessment of prostate specific membrane antigen.

2[(3-Amino-3-carboxypropyl)(hydroxy)(phosphinyl)methyl]pentane-1,5-dioic acid) (GPI) is a highly potent inhibitor of prostate specific membrane antigen (PSMA) with a rapid in vivo clearance profile from nontarget organs including kidneys, but its use for imaging of PSMA is impeded by an endogenous anion (serum phosphate) competition, which compromises its specific binding to the antigen. Multipresentation of a targeting molecule on a single entity has been recognized as a practical way for imaging sensitivity enhancement. Herein, we demonstrate a multivalent approach based on a (64)Cu-specific bifunctional chelator scaffold to overcome the endogenous phosphate competition thus enabling the utility of GPI conjugates for in vivo detection of PSMA and imaging quantification. Both monomeric (H2CBT1G) and dimeric (H2CBT2G) conjugates were synthesized and labeled with (64)Cu for in vitro and in vivo evaluations. A 4-fold enhancement of PSMA binding affinity was observed for H2CBT2G as compared to H2CBT1G from the PSMA competitive binding assays performed on LNCaP cells. In vivo PET imaging studies were conducted on mouse xenograft models established with a PSMA(+) cell line, LNCaP, and PSMA(-) PC3 and H2009 cell lines. (64)Cu-CBT2G showed significantly higher LNCaP tumor uptake than (64)Cu-CBT1G at 1, 4, and 24 h postinjection (p.i.) (p < 0.05). In addition, tumor uptake of (64)Cu-CBT2G remained steady out to 24 h p.i. (1.46 ± 0.54, 1.12 ± 0.56, and 1.00 ± 0.50% ID/g at 1, 4, and 24 h p.i., respectively), while (64)Cu-CBT1G showed a great decrease from 1 to 4 h p.i. The PSMA imaging specificity of both H2CBT1G and H2CBT2G was demonstrated by their low uptake in PSMA(-) tumors (PC3 and H2009) and further confirmed by a significant signal reduction in PSMA(+) LNCaP tumors in the blockade study. In addition, the LNCaP tumor uptake (% ID/g) of (64)Cu-CBT2G was found to be in a positive linear correlation with the tumor size (R(2) = 0.92, 0.94, and 0.93 for 1 h, 4 h, and 24 h p.i.). This may render the probe with potential application in the management of patients with prostate cancer.

In vivo evaluation of medical device-associated inflammation using a macrophage-specific positron emission tomography (PET) imaging probe.

To image implant-surrounding activated macrophages, a macrophage-specific PET probe was prepared by conjugating folic acid (FA) and 2,2',2″,2‴-(1,4,7,10-tetraazacyclododecane-1,4,7,10-tetrayl)tetracetic acid (DOTA) to polyethylene glycol (PEG) and then labeling the conjugate with Ga-68. In vivo PET imaging evaluations demonstrate that the probe is able to detect foreign body reactions, and more importantly, quantify the degree of inflammatory responses to an implanted medical device. These results were further validated by histological analysis.

Role of DAB2IP in modulating epithelial-to-mesenchymal transition and prostate cancer metastasis.

A single nucleotide polymorphism in the DAB2IP gene is associated with risk of aggressive prostate cancer (PCa), and loss of DAB2IP expression is frequently detected in metastatic PCa. However, the functional role of DAB2IP in PCa remains unknown. Here, we show that the loss of DAB2IP expression initiates epithelial-to-mesenchymal transition (EMT), which is visualized by repression of E-cadherin and up-regulation of vimentin in both human normal prostate epithelial and prostate carcinoma cells as well as in clinical prostate-cancer specimens. Conversely, restoring DAB2IP in metastatic PCa cells reversed EMT. In DAB2IP knockout mice, prostate epithelial cells exhibited elevated mesenchymal markers, which is characteristic of EMT. Using a human prostate xenograft-mouse model, we observed that knocking down endogenous DAB2IP in human carcinoma cells led to the development of multiple lymph node and distant organ metastases. Moreover, we showed that DAB2IP functions as a scaffold protein in regulating EMT by modulating nuclear beta-catenin/T-cell factor activity. These results show the mechanism of DAB2IP in EMT and suggest that assessment of DAB2IP may provide a prognostic biomarker and potential therapeutic target for PCa metastasis.

Analysis of oligo-arginine cell-permeable peptides uptake by prostate cells.

Recently, we have shown that oligo-arginine peptide (i.e., R11), a unique cell-permeable peptide (CPP), can be used as an imaging probe for prostate cancer detection. In this study, the mechanism(s) of oligo-arginine peptide in prostate cells was further analyzed. The length of the oligo-arginine peptide appears to be critical for the efficiency of uptake by prostate cells: poly (11)-arginine (R11) > poly (9)-arginine (R9) > poly (13)-arginine peptide (R13). The uptake of R11 peptide by prostate cells is mediated by macropinocytosis as evidenced by the fact that uptake can be blocked by a macropinocytosis inhibitor. However, the use of an inhibitor for carbohydrate chain elongation of glycosaminoglycan or inhibitors for carbohydrate synthesis of glycoprotein via either O-link or N-link showed minimal effects on R11 uptake. Nevertheless, pentosan sulfate (PentS) or dextran sulfate (DS) exhibited the highest inhibitory effect on R11 uptake in several prostate cells treated with various soluble glycosaminoglycans (GAGs) or anionic polymers. It is known that laminin receptor has been characterized as a PentS binding partner. Knocking down 37LRP (laminin receptor precursor) expression in prostate cells showed a reduction in their ability to uptake R11 peptides. In conclusion, laminin receptor is one of the initial binding site(s) responsible for R11 peptide uptake in prostate cells.

ImmunoPET Imaging with 89Zr-Labeled Atezolizumab Enables In Vivo Evaluation of PD-L1 in Tumorgraft Models of Renal Cell Carcinoma.

PURPOSE: Immune checkpoint inhibitors (ICI) targeting the programmed cell death protein 1 and its ligand (PD-1/PD-L1) have transformed the treatment paradigm for metastatic renal cell carcinoma (RCC). However, response rates to ICIs as single agents or in combination vary widely and predictive biomarkers are lacking. Possibly related to the heterogeneity and dynamic nature of PD-L1 expression, tissue-based methods have shown limited value. Immuno-positron emission tomography (immunoPET) may enable noninvasive, comprehensive, and real-time PD-L1 detection. Herein, we systematically examined the performance of immunoPET for PD-L1 detection relative to IHC in an RCC patient-derived tumorgraft (TG) platform. EXPERIMENTAL DESIGN: Eight independent RCC TGs with a wide range of PD-L1 expression (0%-85%) were evaluated by immunoPET. Uptake of 89Zr-labeled atezolizumab ([89Zr]Zr-DFO-ATZ) was compared with PD-L1 expression in tumors by IHC through double-blind analyses. Clinical outcomes of ICI-treated patients whose TGs were examined were analyzed to evaluate the clinical role of immunoPET in RCC. RESULTS: ImmunoPET with [89Zr]Zr-DFO-ATZ (day 6/7 postinjection) revealed a statistically significant association with PD-L1 IHC assays (P = 0.0014; correlation ρXY = 0.78). Furthermore, immunoPET can be used to assess the heterogeneous distribution of PD-L1 expression. Finally, studies in the corresponding patients (n = 4) suggest that PD-L1 signal may influence ICI responsiveness. CONCLUSIONS: ImmunoPET with [89Zr]Zr-DFO-ATZ may enable a thorough and dynamic assessment of PD-L1 across sites of disease. The power of immunoPET to predict ICI response in RCC is being explored in an ongoing clinical trial (NCT04006522).

Multivalent bifunctional chelator scaffolds for gallium-68 based positron emission tomography imaging probe design: signal amplification via multivalency.

The role of the multivalent effect has been well recognized in the design of molecular imaging probes toward the desired imaging signal amplification. Recently, we reported a bifunctional chelator (BFC) scaffold design, which provides a simple and versatile approach to impart multivalency to radiometal based nuclear imaging probes. In this work, we report a series of BFC scaffolds ((t)Bu(3)-1-COOH, (t)Bu(3)-2-(COOH)(2), and (t)Bu(3)-3-(COOH)(3)) constructed on the framework of 1,4,7-triazacyclononane-1,4,7-triacetic acid (NOTA) for (68)Ga-based PET probe design and signal amplification via the multivalent effect. For proof of principle, a known integrin α(v)ß(3) specific ligand (c(RGDyK)) was used to build the corresponding NOTA conjugates (H(3)1, H(3)2, and H(3)3), which present 1-3 copies of c(RGDyK) peptide, respectively, in a systematic manner. Using the integrin α(v)ß(3) binding affinities (IC(50) values), enhanced specific binding was observed for multivalent conjugates (H(3)2: 43.9 ± 16.1 nM; H(3)3: 14.7 ± 5.0 nM) as compared to their monovalent counterpart (H(3)1: 171 ± 60 nM) and the intact c(RGDyK) peptide (204 ± 76 nM). The obtained conjugates were efficiently labeled with (68)Ga(3+) within 30 min at room temperature in high radiochemical yields (>95%). The in vivo evaluation of the labeled conjugates, (68)Ga-1, (68)Ga-2, and (68)Ga-3, was performed using male severe combined immunodeficiency (SCID) mice bearing integrin α(v)ß(3) positive PC-3 tumor xenografts (n = 3). All (68)Ga-labeled conjugates showed high in vivo stability with no detectable metabolites found by radio-HPLC within 2 h postinjection (p.i.). The PET signal amplification in PC-3 tumor by the multivalent effect was clearly displayed by the tumor uptake of the (68)Ga-labeled conjugates ((68)Ga-3: 2.55 ± 0.50%ID/g; (68)Ga-2: 1.90 ± 0.10%ID/g; (68)Ga-1: 1.66 ± 0.15%ID/g) at 2 h p.i. In summary, we have designed and synthesized a series of NOTA-based BFC scaffolds with signal amplification properties, which may find potential applications as diagnostic gallium radiopharmaceuticals.

A cell permeable peptide analog as a potential-specific PET imaging probe for prostate cancer detection.

Non-invasive detection of prostate cancer or metastases still remains a challenge in the field of molecular imaging. In our recent work of screening arginine- or lysine-rich peptides for intracellular delivery of a therapeutic agent into prostate cancer cells, an arginine-rich cell permeable peptide (NH(2)GR(11)) was found with an unexpectedly preferential uptake in prostate cancer cell lines. The goal of this work was to develop this peptide as a positron emission tomography (PET) imaging probe for specific detection of distant prostate cancer metastases. The optimal length of arginine-rich peptides was evaluated by the cell uptake efficiency of three fluorescein isothiocyanate (FITC)-tagged oligoarginines (NHGR(9), NHGR(11), and NHGR(13)) in four human prostate cell lines (LNCaP, PZ-HPV-7, DU145, and PC3). Of the three oligoarginines, NH(2)GR(11) showed the highest cell uptake and internalization efficiency with its subcellular localization in cytosol. The biodistribution of FITC-NHGR(9), FITC-NHGR(11), and FITC-NHGR(13) performed in control nude mice displayed the unique preferential accumulation of FITC-NHGR(11) in the prostate tissue. Further in vivo evaluation of FITC-NHGR(11) in PC3 tumor-bearing nude mice revealed elevated uptake of this peptide in tumors as compared to other organs. In vivo pharmacokinetics evaluated with (64)Cu-labeled NH(2)GR(11) showed that the peptide was rapidly cleared from the blood (t(1/2) = 10.7 min) and its elimination half-life was 17.2 h. The PET imaging specificity of (64)Cu-labled NH(2)GR(11) was demonstrated for the detection of prostate cancer in a comparative imaging experiment using two different human cancer xenograft models.

Copper-67 radioimmunotheranostics for simultaneous immunotherapy and immuno-SPECT.

Copper-67 (t1/2 = 2.58 days) decays by ß- ([Formula: see text]: 562 keV) and γ-rays (93 keV and 185 keV) rendering it with potential for both radionuclide therapy and single-photon emission computed tomography (SPECT) imaging. Prompted by the recent breakthrough of 67Cu production with high specific activity, high radionuclidic purity, and sufficient quantities, the interest in the theranostic potential of 67Cu has been rekindled. This work addresses the practicability of developing 67Cu-labeled antibodies with substantially improved quality for cancer radioimmunotheranostics. Proof of concept is demonstrated with pertuzumab, a US-FDA-approved monoclonal antibody for combination therapies of HER2-positive breast cancer. With an average number of 1.9 chelators coupled to each antibody, we achieved a two-order of magnitude increase in radiolabeling efficiency compared to literature reports. In a preclinical therapeutic study, mice (n = 4-7/group) bearing HER2+ xenografts exhibited a 67Cu-dose dependent tumor-growth inhibition from 67Cu-labeled-Pertuzumab co-administered with trastuzumab. Furthermore, greater tumor size reduction was observed with 67Cu-labeled-pertuzumab formulations of higher specific activity. The potential of SPECT imaging with 67Cu radiopharmaceuticals was tested after 67Cu-labeled-Pertuzumab administration. Impressively, all tumors were clearly visualized by SPECT imaging with 67Cu-labeled-Pertuzumab even at day 5 post injection. This work demonstrates it is practical to use 67Cu radioimmunoconjugates for cancer radioimmunotheranostics.

Selective depletion of radiolabeled HER2-specific antibody for contrast improvement during PET.

The prolonged in vivo persistence of antibodies results in high background and poor contrast during their use as molecular imaging agents for positron emission tomography (PET). We have recently described a class of engineered Fc fusion proteins that selectively deplete antigen-specific antibodies without affecting the levels of antibodies of other specificities. Here, we demonstrate that these Fc fusions (called Seldegs, for selective degradation) can be used to clear circulating, radiolabeled HER2-specific antibody during diagnostic imaging of HER2-positive tumors in mice. The analyses show that Seldegs have considerable promise for the reduction of whole-body exposure to radiolabel and improvement of contrast during PET.