Tracking of NK Cells by Positron Emission Tomography Using 89Zr-Oxine Ex Vivo Cell Labeling.

A 89Zr-oxine ex vivo cell labeling method for tracking various cells by positron emission tomography (PET) imaging has recently been developed. 89Zr-oxine is synthesized from oxine and 89Zr-chloride, which was converted from 89Zr-oxalate, with neutralization. To track migration of natural killer (NK) cells in vivo in real time by PET imaging, NK cells are labeled with 89Zr-oxine ex vivo and infused to a recipient. The labeling is performed by mixing 89Zr-oxine solution to NK cell suspension at room temperature, followed by washing. Care should be taken to label the cells at optimal radioactivity doses that maintain their viability and functionality. 89Zr-oxine labeled NK cells can be tracked for their migration and distribution by PET/computed tomography imaging for at least 7 days. Of note, this protocol is applicable to other types of cells.

Reply to the 'Comment on "Investigation of Zr(iv) and 89Zr(iv) complexation with hydroxamates: progress towards designing a better chelator than desferrioxamine B for immuno-PET imaging"' by A. Bianchi and M. Savastano, Chem. Commun., 2020, 56, D0CC01189D.

The alternative analysis of A. Bianchi and M. Savastano is a valuable contribution to the understanding of the complex systems at stake in the complexation chemistry of Zr4+ by considering polynuclear species. Placed in the context of nuclear medicine where such aggregates are unlikely and considering recent literature data, this however points out that no clear agreement exists to describe such complex formation.

Immuno-PET Imaging of the Programmed Cell Death-1 Ligand (PD-L1) Using a Zirconium-89 Labeled Therapeutic Antibody, Avelumab.

OBJECTIVE: The goal is to evaluate avelumab, an anti-PD-L1 monoclonal immunoglobulin G antibody labeled with zirconium-89 in human PD-L1-expressing cancer cells and mouse xenografts for clinical translation. METHODS: [89Zr]Zr-DFO-PD-L1 monoclonal antibody (mAb) was synthesized using avelumab conjugated to desferrioxamine. In vitro binding studies and biodistribution studies were performed with PD-L1+MDA-MB231 cells and MDA-MB231 xenograft mouse models, respectively. Biodistributions were determined at 1, 2, 3, 5, and 7 days post coinjection of [89Zr]Zr-DFO-PD-L1 mAb without or with unlabeled avelumab (10, 20, 40, and 400 µg). RESULTS: [89Zr]Zr-DFO-PD-L1 mAb exhibited high affinity (Kd ∼ 0.3 nM) and detected moderate PD-L1 expression levels in MDA-MB231 cells. The spleen and lymph nodes exhibited the highest [89Zr]Zr-DFO-PD-L1 mAb uptakes in all time points, while MDA-MB231 tumor uptakes were lower but highly retained. In the unlabeled avelumab dose escalation studies, spleen tissue-muscle ratios decreased in a dose-dependent manner indicating specific [89Zr]Zr-DFO-PD-L1 mAb binding to PD-L1. In contrast, lymph node and tumor tissue-muscle ratios increased 4- to 5-fold at 20 and 40 µg avelumab doses. CONCLUSIONS: [89Zr]Zr-DFO-PD-L1 mAb exhibited specific and high affinity for PD-L1 in vitro and had target tissue uptakes correlating with PD-L1 expression levels in vivo. [89Zr]Zr-DFO-PD-L1 mAb uptake in PD-L1+tumors increased with escalating doses of avelumab.

Single Low-Dose Injection of Evans Blue Modified PSMA-617 Radioligand Therapy Eliminates Prostate-Specific Membrane Antigen Positive Tumors.

Prostate cancer is the most frequently diagnosed malignant tumor in men worldwide. Prostate-specific membrane antigen (PSMA) is a surface molecule specifically expressed by prostate tumors that has been shown to be a valid target for internal radionuclide therapy in both preclinical and clinical settings. The most common radiotherapeutic agent is the small molecule 177Lu-PSMA-617, which is under clinical evaluation in multiple countries. Nevertheless, its efficacy in causing tumor regression is still suboptimal, even when administered in several cycles per patient, perhaps due to poor pharmacokinetics (PK), which limits uptake by the tumor cells. We postulated that the addition of the Evans blue (EB) moiety to PSMA-617 would improve the PK by extending circulation half-life, which would increase tumor uptake and improve radiotherapeutic efficacy. PSMA-617 was modified by conjugation of a 2-thiol acetate group onto the primary amine and thereafter reacted with a maleimide functional group of an EB derivative, to give EB-PSMA-617. The PK and radiotherapeutic efficacy of 90Y- or 177Lu-EB-PSMA-617 was compared to the clinically used radiopharmaceutical 90Y- or 177Lu- PSMA-617 in PC3-PIP tumor-bearing mice. EB-PSMA-617 retained binding to serum albumin as well as a high internalization rate by tumor cells. Upon injection, metal-labeled EB-PSMA-617 demonstrated an extended blood half-life compared to PSMA-617 and, thereby, prolonged the time window for binding to PSMA. The improved PK of EB-PSMA-617 resulted in significantly higher accumulation in PSMA+ tumors and highly effective radiotherapeutic efficacy. Remarkably, a single dose of 1.85 MBq of 90Y- or 177Lu-EB-PSMA-617 was sufficient to eradicate established PMSA+ tumors in mice. No significant body weight loss was observed, suggesting little to no gross toxicity. The construct described here, EB-PSMA-617, may improve the radiotherapeutic efficacy for patients with PSMA-positive tumors by reducing both the amount of activity needed for therapy as well as the frequency of administration, as compared to PSMA-617.

Tumor-Shed Antigen Affects Antibody Tumor Targeting: Comparison of Two 89Zr-Labeled Antibodies Directed against Shed or Nonshed Antigens.

We investigated the effect of shed antigen mesothelin on the tumor uptake of amatuximab, a therapeutic anti-mesothelin mAb clinically tested in mesothelioma patients. The B3 mAb targeting a nonshed antigen was also analyzed for comparison. The mouse model implanted with A431/H9 tumor, which expresses both shed mesothelin and nonshed Lewis-Y antigen, provided an ideal system to compare the biodistribution and PET imaging profiles of the two mAbs. Our study demonstrated that the tumor and organ uptakes of 89Zr-B3 were dose-independent when 3 doses, 2, 15, and 60 µg B3, were compared at 24 h after injection. In contrast, tumor and organ uptakes of 89Zr-amatuximab were dose-dependent, whereby a high dose (60 µg) was needed to achieve tumor targeting comparable to the low dose (2 µg) of 89Zr-B3, suggesting that shed mesothelin may affect amatuximab tumor targeting as well as serum half-life. The autoradiography analysis showed that the distribution of 89Zr-B3 was nonuniform with the radioactivity primarily localized at the tumor periphery independent of the B3 dose. However, the autoradiography analysis for 89Zr-amatuximab showed dose-dependent distribution profiles of the radiolabel; at 10 µg dose, the radiolabel penetrated toward the tumor core with its activity comparable to that at the tumor periphery, whereas at 60 µg dose, the distribution profile became similar to those of 89Zr-B3. These results suggest that shed antigen in blood may act as a decoy requiring higher doses of mAb to improve serum half-life as well as tumor targeting. Systemic mAb concentration should be at a severalfold molar excess to the shed Ag in blood to overcome the hepatic processing of mAb-Ag complexes. On the other hand, mAb concentration should remain lower than the shed Ag concentration in the tumor ECS to maximize tumor penetration by passing binding site barriers.

Anatomy, Functionality, and Neuronal Connectivity with Manganese Radiotracers for Positron Emission Tomography.

PURPOSE: Manganese ion has been extensively used as a magnetic resonance imaging (MRI) contrast agent in preclinical studies to assess tissue anatomy, function, and neuronal connectivity. Unfortunately, its use in human studies has been limited by cellular toxicity and the need to use a very low dose. The much higher sensitivity of positron emission tomography (PET) over MRI enables the use of lower concentrations of manganese, potentially expanding the methodology to humans. PROCEDURES: PET tracers manganese-51 (Mn-51, t1/2 = 46 min) and manganese-52 (Mn-52, t1/2 = 5.6 days) were used in this study. The biodistribution of manganese in animals in the brain and other tissues was studied as well as the uptake in the pancreas after glucose stimulation as a functional assay. Finally, neuronal connectivity in the olfactory pathway following nasal administration of the divalent radioactive Mn-52 ([52Mn]Mn2+) was imaged. RESULTS: PET imaging with the divalent radioactive Mn-51 ([51Mn]Mn2+) and [52Mn]Mn2+ in both rodents and monkeys demonstrates that the accumulation of activity in different organs is similar to that observed in rodent MRI studies following systemic administration. Furthermore, we demonstrated the ability of manganese to enter excitable cells. We followed activity-induced [51Mn]Mn2+ accumulation in the pancreas after glucose stimulation and showed that [52Mn]Mn2+ can be used to trace neuronal connections analogous to manganese-enhanced MRI neuronal tracing studies. CONCLUSIONS: The results were consistent with manganese-enhanced MRI studies, despite the much lower manganese concentration used for PET (100 mM Mn2+ for MRI compared to ~ 0.05 mM for PET). This indicates that uptake and transport mechanisms are comparable even at low PET doses. This helps establish the use of manganese-based radiotracers in both preclinical and clinical studies to assess anatomy, function, and connectivity.

Bone Marrow Cell Trafficking Analyzed by 89Zr-oxine Positron Emission Tomography in a Murine Transplantation Model.

Purpose: The success of hematopoietic stem cell transplantation (HSCT) depends on donor cell homing to the bone marrow. However, there is no reliable method of noninvasively monitoring the kinetics and distribution of transferred cells. Using zirconium-89 (89Zr)-oxine cell labeling combined with PET imaging, we sought to visualize and quantify donor cell homing in a mouse bone marrow transplantation model.Experimental Design: The effect of 89Zr-oxine labeling on bone marrow cell viability and differentiation was evaluated in vitro89Zr-labeled bone marrow cells (2 × 107 cells, 16.6 kBq/106 cells) were transferred intravenously, and serial microPET images were obtained (n = 5). The effect of a CXCR4 inhibitor, plerixafor (5 mg/kg) and G-CSF (2.5 µg) on bone marrow homing and mobilization were examined (n = 4). Engraftment of the transferred 89Zr-labeled cells was evaluated (n = 3).Results:89Zr-oxine-labeled bone marrow cells showed delayed proliferation, but differentiated normally. Transferred bone marrow cells rapidly migrated to the bone marrow, spleen, and liver (n = 5). Approximately 36% of donor cells homed to the bone marrow within 4 hours, irrespective of prior bone marrow ablation. Inhibition of CXCR4 by plerixafor alone or with G-CSF significantly blocked the bone marrow homing (P < 0.0001, vs. nontreated, at 2 hours), confirming a crucial role of the CXCR4-CXCL12 system. Mobilization of approximately 0.64% of pretransplanted bone marrow cells induced a 3.8-fold increase of circulating bone marrow cells. 89Zr-labeled donor cells engrafted as well as nonlabeled cells.Conclusions:89Zr-oxine PET imaging reveals rapid bone marrow homing of transferred bone marrow cells without impairment of their stem cell functions, and thus, could provide useful information for optimizing HSCT. Clin Cancer Res; 23(11); 2759-68. ©2016 AACR.

Imaging the Met Receptor Tyrosine Kinase (Met) and Assessing Tumor Responses to a Met Tyrosine Kinase Inhibitor in Human Xenograft Mouse Models with a [99mTc] (AH-113018) or Cy 5** (AH-112543) Labeled Peptide.

Developing an imaging agent targeting the hepatocyte growth factor receptor protein (Met) status of cancerous lesions would aid in the diagnosis and monitoring of Met-targeted tyrosine kinase inhibitors (TKIs). A peptide targeting Met labeled with [(99m)Tc] had high affinity in vitro (Kd = 3.3 nM) and detected relative changes in Met in human cancer cell lines. In vivo [(99m)Tc]-Met peptide (AH-113018) was retained in Met-expressing tumors, and high-expressing Met tumors (MKN-45) were easily visualized and quantitated using single-photon emission computed tomography or optical imaging. In further studies, MKN-45 mouse xenografts treated with PHA 665752 (Met TKI) or vehicle were monitored weekly for tumor responses by [(99m)Tc]-Met peptide imaging and measurement of tumor volumes. Tumor uptake of [(99m)Tc]-Met peptide was significantly decreased as early as 1 week after PHA 665752 treatment, corresponding to decreases in tumor volumes. These results were comparable to Cy5**-Met peptide (AH-112543) fluorescence imaging using the same treatment model. [(99m)Tc] or Cy5**-Met peptide tumor uptake was further validated by histologic (necrosis, apoptosis) and immunoassay (total Met, p Met, and plasma shed Met) assessments in imaged and nonimaged cohorts. These data suggest that [(99m)Tc] or Cy5**-Met peptide imaging may have clinical diagnostic, prognostic, and therapeutic monitoring applications.

Tumor and organ uptake of (64)Cu-labeled MORAb-009 (amatuximab), an anti-mesothelin antibody, by PET imaging and biodistribution studies.

OBJECTIVES: To investigate the effect of the injection dose of MORAb-009 (amatuximab, an anti-mesothelin monoclonal antibody), the tumor size and the level of shed mesothelin on the uptake of the antibody in mesothelin-positive tumor and organs by biodistribution (BD) and positron emission tomography (PET) imaging studies. METHODS: 2-S-(4-Isothiocyanatobenzyl)-1,4,7-triazacyclononane-1,4,7-triacetic acid (p-SCN-Bn-NOTA) was conjugated to amatuximab and labeled with (64)CuCl2 in 0.25 M acetate buffer, pH4.2. The resulting (64)Cu-NOTA-amatuximab was purified with a PD 10 column. To investigate the dose effect or the effect of tumor size, the BD was performed in groups of nude mice (n=5) with mesothelin-expressing A431/H9 tumors (range, 80-300 mm(3)) one day after iv injection of (64)Cu-NOTA-amatuximab (10 µCi) containing a total amatuximab dose of 2, 30, or 60 µg. The BD and PET imaging were also investigated 3, 24 and 48 h after injecting a total dose of 30 µg (10 µCi for BD), and 2 or 60 µg (300 µCi for PET), respectively. RESULTS: Comparing the results of the BDs from three different injection doses, the major difference was shown in the uptake (%ID/g) of the radiolabel in tumor, liver and blood. The tumor uptake and blood retention from 30 and 60 µg doses were greater than those from 2 µg dose, whereas the liver uptake was smaller. The BD studies also demonstrated a positive correlation between tumor size (or the level of shed mesothelin in blood) and liver uptake. However, there was a negative correlation between tumor size (or the shed mesothelin level) and tumor uptake and between tumor size and blood retention. These findings were confirmed by the PET imaging study, which clearly visualized the tumor uptake with the radiolabel concentrated in the tumor core and produced a tumor to liver ratio of 1.2 at 24h post-injection with 60 µg amatuximab, whereas the injection of 2 µg amatuximab produced a tumor to liver ratio of 0.4 at 24h post-injection. CONCLUSION: Our studies using a nude mouse model of A431/H9 tumor demonstrated that the injection of a high amatuximab dose (30 to 60 µg) could provide a beneficial effect in maximizing tumor uptake while maintaining minimum liver and spleen uptakes of the radiolabel, and in facilitating its penetration into the tumor core.

PET imaging of tenascin-C with a radiolabeled single-stranded DNA aptamer.

UNLABELLED: Tenascin-C is an extracellular matrix glycoprotein that is expressed by injured tissues and by various cancers. Recent publications showed that tenascin-C expression by cancer lesions predicts tumor growth, metastasis, and angiogenesis, suggesting tenascin-C as a potential therapeutic target. Currently there is no noninvasive method to determine tumoral tenascin-C expression in vivo. To address the need for an agent to image and quantify tenascin-C, we report the development of a radioactive PET tracer based on a tenascin-C-specific single-stranded DNA aptamer (tenascin-C aptamer). METHODS: Tenascin-C aptamer was radiolabeled with (18)F and (64)Cu. PET imaging studies for the evaluation of tumor uptake and pharmacokinetics of tenascin-C aptamer were performed in comparison to a nonspecific scrambled aptamer (Sc aptamer). RESULTS: The labeled tenascin-C aptamer provided clear visualization of tenascin-C-positive but not tenascin-C-negative tumors. The uptake of tenascin-C aptamer was significantly higher than that of Sc aptamer in tenascin-C-positive tumors. The labeled tenascin-C aptamer had fast clearance from the blood and other nonspecific organs through the kidneys, resulting in high tumor contrast. CONCLUSION: Our data suggest that suitably labeled tenascin-C aptamer can be used as a PET tracer to image tumor expression of tenascin-C with a high tumor-to-background ratio and might provide insightful and personalized medical data that will help determine appropriate treatment and monitoring.

(89)Zr-Oxine Complex PET Cell Imaging in Monitoring Cell-based Therapies.

PURPOSE: To develop a clinically translatable method of cell labeling with zirconium 89 ((89)Zr) and oxine to track cells with positron emission tomography (PET) in mouse models of cell-based therapy. MATERIALS AND METHODS: This study was approved by the institutional animal care committee. (89)Zr-oxine complex was synthesized in an aqueous solution. Cell labeling conditions were optimized by using EL4 mouse lymphoma cells, and labeling efficiency was examined by using dendritic cells (DCs) (n = 4), naïve (n = 3) and activated (n = 3) cytotoxic T cells (CTLs), and natural killer (NK) (n = 4), bone marrow (n = 4), and EL4 (n = 4) cells. The effect of (89)Zr labeling on cell survival, proliferation, and function were evaluated by using DCs (n = 3) and CTLs (n = 3). Labeled DCs (444-555 kBq/[5 × 10(6)] cells, n = 5) and CTLs (185 kBq/[5 × 10(6)] cells, n = 3) transferred to mice were tracked with microPET/CT. In a melanoma immunotherapy model, tumor targeting and cytotoxic function of labeled CTLs were evaluated with imaging (248.5 kBq/[7.7 × 10(6)] cells, n = 4) and by measuring the tumor size (n = 6). Two-way analysis of variance was used to compare labeling conditions, the Wilcoxon test was used to assess cell survival and proliferation, and Holm-Sidak multiple tests were used to assess tumor growth and perform biodistribution analyses. RESULTS: (89)Zr-oxine complex was synthesized at a mean yield of 97.3% ± 2.8 (standard deviation). It readily labeled cells at room temperature or 4°C in phosphate-buffered saline (labeling efficiency range, 13.0%-43.9%) and was stably retained (83.5% ± 1.8 retention on day 5 in DCs). Labeling did not affect the viability of DCs and CTLs when compared with nonlabeled control mice (P > .05), nor did it affect functionality. (89)Zr-oxine complex enabled extended cell tracking for 7 days. Labeled tumor-specific CTLs accumulated in the tumor (4.6% on day 7) and induced tumor regression (P < .05 on day 7). CONCLUSION: We have developed a (89)Zr-oxine complex cell tracking technique for use with PET that is applicable to a broad range of cell types and could be a valuable tool with which to evaluate various cell-based therapies.

Chelator-free (64)Cu-integrated gold nanomaterials for positron emission tomography imaging guided photothermal cancer therapy.

Using positron emission tomography (PET) imaging to monitor and quantitatively analyze the delivery and localization of Au nanomaterials (NMs), a widely used photothermal agent, is essential to optimize therapeutic protocols to achieve individualized medicine and avoid side effects. Coupling radiometals to Au NMs via a chelator faces the challenges of possible detachment of the radiometals as well as surface property changes of the NMs. In this study, we reported a simple and general chelator-free (64)Cu radiolabeling method by chemically reducing (64)Cu on the surface of polyethylene glycol (PEG)-stabilized Au NMs regardless of their shape and size. Our (64)Cu-integrated NMs are proved to be radiochemically stable and can provide an accurate and sensitive localization of NMs through noninvasive PET imaging. We further integrated (64)Cu onto arginine-glycine-aspartic acid (RGD) peptide modified Au nanorods (NRs) for tumor theranostic application. These NRs showed high tumor targeting ability in a U87MG glioblastoma xenograft model and were successfully used for PET image-guided photothermal therapy.

Immuno-PET imaging of tumor endothelial marker 8 (TEM8).

Tumor endothelial marker 8 (TEM8) is a cell surface receptor that is highly expressed in a variety of human tumors and promotes tumor angiogenesis and cell growth. Antibodies targeting TEM8 block tumor angiogenesis in a manner distinct from the VEGF receptor pathway. Development of a TEM8 imaging agent could aid in patient selection for specific antiangiogenic therapies and for response monitoring. In these studies, L2, a therapeutic anti-TEM8 monoclonal IgG antibody (L2mAb), was labeled with (89)Zr and evaluated in vitro and in vivo in TEM8 expressing cells and mouse xenografts (NCI-H460, DLD-1) as a potential TEM8 immuno-PET imaging agent. (89)Zr-df-L2mAb was synthesized using a desferioxamine-L2mAb conjugate (df-L2mAb); (125)I-L2mAb was labeled directly. In vitro binding studies were performed using human derived cell lines with high, moderate, and low/undetectable TEM8 expression. (89)Zr-df-L2mAb in vitro autoradiography studies and CD31 IHC staining were performed with cryosections from human tumor xenografts (NCI-H460, DLD-1, MKN-45, U87-MG, T-47D, and A-431). Confirmatory TEM8 Western blots were performed with the same tumor types and cells. (89)Zr-df-L2mAb biodistribution and PET imaging studies were performed in NCI-H460 and DLD-1 xenografts in nude mice. (125)I-L2mAb and (89)Zr-df-L2mAb exhibited specific and high affinity binding to TEM8 that was consistent with TEM8 expression levels. In NCI-H460 and DLD-1 mouse xenografts nontarget tissue uptake of (89)Zr-df-L2mAb was similar; the liver and spleen exhibited the highest uptake at all time points. (89)Zr-L2mAb was highly retained in NCI-H460 tumors with <10% losses from day 1 to day 3 with the highest tumor to muscle ratios (T:M) occurring at day 3. DLD-1 tumors exhibited similar pharmacokinetics, but tumor uptake and T:M ratios were reduced ∼2-fold in comparison to NCI-H460 at all time points. NCI-H460 and DLD-1 tumors were easily visualized in PET imaging studies despite low in vitro TEM8 expression in DLD-1 cells indicating that in vivo expression might be higher in DLD-1 tumors. From in vitro autoradiography studies (89)Zr-df-L2mAb specific binding was found in 6 tumor types (U87-MG, NCI-H460, T-47D MKN-45, A-431, and DLD-1) which highly correlated to vessel density (CD31 IHC). Westerns blots confirmed the presence of TEM8 in the 6 tumor types but found undetectable TEM8 levels in DLD-1 and MKN-45 cells. This data would indicate that TEM8 is associated with the tumor vasculature rather than the tumor tissue, thus explaining the increased TEM8 expression in DLD-1 tumors compared to DLD-1 cell cultures. (89)Zr-df-L2mAb specifically targeted TEM8 in vitro and in vivo although the in vitro expression was not necessarily predictive of in vivo expression which seemed to be associated with the tumor vasculature. In mouse models, (89)Zr-df-L2mAb tumor uptakes and T:M ratios were sufficient for visualization during PET imaging. These results would suggest that a TEM8 targeted PET imaging agent, such as (89)Zr-df-L2mAb, may have potential clinical, diagnostic, and prognostic applications by providing a quantitative measure of tumor angiogenesis and patient selection for future TEM8 directed therapies.

Investigation of Zr(IV) and 89Zr(IV) complexation with hydroxamates: progress towards designing a better chelator than desferrioxamine B for immuno-PET imaging.

Single crystal X-ray diffraction shows that Zr(iv) forms an octa-coordinated complex with 4 bidentate hydroxamates whose solution structures were investigated by utilizing density functional theory at the level of B3LYP/DGDZVP. Stability constants obtained by potentiometry were in accordance with the tendency observed when radiolabeling with (89)Zr.

Improvement of CXCR4 tracer specificity for PET imaging.

Tumors expressing the chemokine receptor CXCR4 have been reported to be more aggressive and to produce more metastatic seeding in specific organs, such as the bone marrow. However, evaluation of tumors for CXCR4 expression requires testing of ex vivo biopsy samples, and is not routinely done in cancer management. In prior work to address this issue, we and others have developed tracers for positron emission tomography (PET) that targeted CXCR4, but in addition to binding to CXCR4 these tracers also bound to red blood cells (and to other unrelated targets) in vivo. Here we report two new tracers based on the CXCR4 peptide antagonist 4F-benzoyl-TN14003 (T140) that bind to CXCR4, but not to undesired targets. These tracers, NOTA-NFB and DOTA-NFB, show slight reductions in both 1) binding affinities for CXCR4 and 2) inhibition of CXCL12 induced migration, compared to T140, in vitro. Both NOTA-NFB and DOTA-NFB specifically accumulate in CXCR4-positive, but not CXCR4-negative, tumor xenografts in mice and allow clear visualization of CXCR4 expression by PET. Evaluation of NOTA-NFB and DOTA-NFB for their potential to mobilize immune cells and progenitor cells from the bone marrow to the peripheral blood revealed slightly reduced, but still comparable, results to the parent molecule T140. The tracers reported here may allow the evaluation of CXCR4 expression in primary tumors and metastatic nodules, and enable better informed, more personalized treatment for patients with cancer.

Positron emission tomography imaging of tumors expressing the human chemokine receptor CXCR4 in mice with the use of 64Cu-AMD3100.

PURPOSE: Expression of CXCR4 in cancers has been correlated with poor prognosis and increased metastasis. Quantifying CXCR4 expression non-invasively might aid in prognostication and monitoring therapy. We evaluated a radiolabeled antagonist of CXCR4, 64Cu-AMD3100, as a positron-emitting imaging agent. PROCEDURES: CXCR4-transfected or non-transfected cell lines were injected into mice to form xenografts. Accumulation of 64Cu-AMD3100 in tumors was analyzed by small-animal PET and biodistribution assays. RESULTS: 64Cu-AMD3100 accumulated in CXCR4-expressing, but not CXCR4-negative, tumors. For CXCR4-expressing tumors, tumor-to-blood and tumor-to-muscle ratios were 23-41 and 50-59, respectively, depending on tumor type. Excess of unlabeled Cu-AMD3100 or AMD3100 significantly reduced 64Cu-AMD3100 accumulation in CXCR4-expressing tumors. Human-absorbed dose calculations predicted a dose limit of 444 MBq. CONCLUSIONS: CXCR4 can be imaged in tumors using 64Cu-AMD3100. Dosimetry studies suggest that imaging in humans is feasible. We conclude that 64Cu-AMD3100 should be investigated as a potential agent for imaging and quantifying CXCR4 in tumors.

PET imaging of CXCR4 using copper-64 labeled peptide antagonist.

Expression of CXCR4 in cancer has been found to correlate with poor prognosis and resistance to chemotherapy. In this study we developed a derivative of the CXCR4 peptide antagonist, T140-2D, that can be labeled easily with the PET isotope copper-64, and thereby enable in vivo visualization of CXCR4 in tumors. T140 was conjugated to 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid mono (N-hydroxysuccinimide ester) (DOTA-NHS) to give T140-2D, which contains a DOTA molecule on each of the two lysine residues. (64)Cu-T140-2D was evaluated in vitro by migration and binding experiments, and in vivo by microPET imaging and biodistribution, in mice bearing CXCR4-positive and CXCR4-negative tumor xenografts. T140-2D was labeled with copper-64 to give (64)Cu-T140-2D in a high radiochemical yield of 86 ± 3% (not decay-corrected) and a specific activity of 0.28 - 0.30 mCi/µg (10.36 - 11.1 MBq/µg). (64)Cu-T140-2D had antagonistic and binding characteristics to CXCR4 that were similar to those of T140. In vivo, (64)Cu-T140-2D tended to bind to red blood cells and had to be used in a low specific activity form. In this new form (64)Cu-T140-2D enabled specific imaging of CXCR4-positive, but not CXCR4-negative tumors. Undesirably, however, (64)Cu-T140-2D also displayed high accumulation in the liver and kidneys. In conclusion, (64)Cu-T140-2D was easily labeled and, in its low activity form, enabled imaging of CXCR4 in tumors. It had high uptake, however, in metabolic organs. Further research with imaging tracers targeting CXCR4 is required.

[(76) Br]BMK-152, a nonpeptide analogue, with high affinity and low nonspecific binding for the corticotropin-releasing factor type 1 receptor.

Corticotropin-releasing factor (CRF), a neuropeptide, regulates endocrine and autonomic responses to stress through G-protein coupled receptors, CRF(1) or CRF(2) . A PET ligand able to monitor changes in CRF(1) receptor occupancy in vivo would aid in understanding the pathophysiology of stress-related diseases as well as in the clinical development of nonpeptide antagonists with therapeutic value. We have radiolabeled the CRF(1) receptor ligand, [8-(4-bromo-2,6-dimethoxyphenyl)-2,7-dimethylpyrazolo[1,5-α][1,3,5]triazin-4-yl]-N,N-bis-(2-methoxyethyl)amine (BMK-152) (ClogP = 2.6), at both the 3 and 4 position with [(76) Br]. Using in vitro autoradiography saturation studies the 4-[(76) Br]BMK-152 exhibited high affinity binding to both rat (K(d) = 0.23 ± 0.07 nM; n = 3) and monkey frontal cortex (K(d) = 0.31 ± 0.08 nM; n = 3) consistent with CRF(1) receptor regional distribution whereas with the 3-[(76) Br]BMK-152, the K(d) s could not be determined due to high nonspecific binding. In vitro autoradiography competition studies using [(125) I]Tyr(0) -o-CRF confirmed that 3-Br-BMK-152 (K(i) = 24.4 ± 4.9 nM; n = 3) had lower affinity (70-fold) than 4-Br-BMK-152 (K(i) = 0.35 ± 0.07 nM; n = 3) in monkey frontal cortex and similiar studies using [(125) I]Sauvagine confirmed CRF(1) receptor selectivity. In vivo studies with P-glycoprotein (PGP) knockout mice (KO) and their wild-type littermates (WT) showed that the brain uptake of 3-[(76) Br]BMK/4-[(76) Br]BMK was increased less than twofold in KO versus WT indicating that 3-[(76) Br]BMK-152/4-[(76) Br]BMK was not a Pgp substrate. Rat brain uptakes of 4-[(76) Br] BMK-152 from ex vivo autoradiography studies showed regional localization consistent with known published CRF(1) receptor distribution and potential as a PET ligand for in vivo imaging of CRF(1) receptors.

MicroPET imaging of integrin αvß3 expressing tumors using 89Zr-RGD peptides.

PURPOSE: The dimeric transmembrane integrin, α(v)ß(3), is a well-investigated target by different imaging modalities through suitably labeled arginine-glycine-aspartic acid (RGD) containing peptides. In this study, we labeled four cyclic RGD peptides with or without PEG functional groups: c(RGDfK) (denoted as FK), PEG(3)-c(RGDfK) (denoted as FK-PEG(3)), E[c(RGDfK)](2) (denoted as [FK](2)), and PEG(4)-E[PEG(4)-c(RGDfK)](2) (denoted as [FK](2)-3PEG(4)), with (89)Zr (t(1/2) = 78.4 h), using the chelator desferrioxamine-p-SCN (Df) for imaging tumor integrin α(v)ß(3). METHODS: The Df conjugated RGD peptides were subjected to integrin α(v)ß(3) binding assay in vitro using MDA-MB-435 breast cancer cells. The (89)Zr-labeled RGD peptides were then subjected to small animal positron emission tomography (PET) and direct tissue sampling biodistribution studies in an orthotopic MDA-MB-435 breast cancer xenograft model. RESULTS: All four tracers, (89)Zr-Df-FK, (89)Zr-Df-FK-PEG(3), (89)Zr-Df-[FK](2), and (89)Zr-Df-[FK](2)-3PEG(4), were labeled in high radiochemical yield (89 ± 4%) and high specific activity (4.07-6 MBq/µg). Competitive binding assay with (125)I-echistatin showed that conjugation of the RGD peptides to the Df chelator did not have significant impact on their integrin α(v)ß(3) binding affinity and the dimeric peptides were shown to be more potent than the monomers. In agreement with binding results, tumor uptake of (89)Zr-Df-[FK](2) and (89)Zr-Df-[FK](2)-3PEG(4) was significantly higher (4.32 ± 1.73%ID/g and 4.72 ± 0.66%ID/g, respectively, at 2 h post-injection) than the monomers (89)Zr-Df-FK and (89)Zr-Df-FK-PEG(3) (1.97 ± 0.38%ID/g and 1.57 ± 0.49%ID/g, respectively, at 2 h post-injection). Out of the four labeled peptides, (89)Zr-Df-[FK](2)-3PEG(4) gave the highest tumor-to-background ratio (18.21 ± 2.52 at 2 h post-injection and 19.69 ± 3.99 at 4 h post-injection), with the lowest uptake in metabolic organs. Analysis of late time points biodistribution data revealed that the uptake in the tumor was decreased, along with increase in the bone, which implies decomplexation of (89)Zr-Df. CONCLUSION: Efficient radiolabeling of peptides with an appropriate chelator such as Df-RGD with (89)Zr was observed. The (89)Zr radiolabeled peptides provided high-quality and high-resolution microPET images in xenograft models. (89)Zr-Df-[FK](2)-3PEG(4) demonstrated the highest tumor-to-background ratio of the compounds tested. Preparation of (89)Zr peptides to take advantage of the longer half-life is unwarranted due to the relatively rapid clearance from the tumor region of peptide tracers prepared for this study and the increased uptake in the bone of transchelated (89)Zr with time (2.0 ± 0.36%ID/g, 24 h post-injection).

PET imaging of HER1-expressing xenografts in mice with 86Y-CHX-A''-DTPA-cetuximab.

PURPOSE: Cetuximab is a recombinant, human/mouse chimeric IgG(1) monoclonal antibody that binds to the epidermal growth factor receptor (EGFR/HER1). Cetuximab is approved for the treatment of patients with HER1-expressing metastatic colorectal cancer. Limitations in currently reported radiolabeled cetuximab for PET applications prompted the development of (86)Y-CHX-A''-DTPA-cetuximab as an alternative for imaging HER1-expressing cancer. (86)Y-CHX-A''-DTPA-cetuximab can also serve as a surrogate marker for (90)Y therapy. METHODS: Bifunctional chelate, CHX-A''-DTPA was conjugated to cetuximab and radiolabeled with (86)Y. In vitro immunoreactivity was assessed in HER1-expressing A431 cells. In vivo biodistribution, PET imaging and noncompartmental pharmacokinetics were performed in mice bearing HER1-expressing human colorectal (LS-174T and HT29), prostate (PC-3 and DU145), ovarian (SKOV3) and pancreatic (SHAW) tumor xenografts. Receptor blockage was demonstrated by coinjection of either 0.1 or 0.2 mg cetuximab. RESULTS: (86)Y-CHX-A''-DTPA-cetuximab was routinely prepared with a specific activity of 1.5-2 GBq/mg and in vitro cell-binding in the range 65-75%. Biodistribution and PET imaging studies demonstrated high HER1-specific tumor uptake of the radiotracer and clearance from nonspecific organs. In LS-174T tumor-bearing mice injected with (86)Y-CHX-A''-DTPA-cetuximab alone, (86)Y-CHX-A''-DTPA-cetuximab plus 0.1 mg cetuximab or 0.2 mg cetuximab, the tumor uptake values at 3 days were 29.3 +/- 4.2, 10.4 +/- 0.5 and 6.4 +/- 0.3%ID/g, respectively, demonstrating dose-dependent blockage of the target. Tumors were clearly visualized 1 day after injecting 3.8-4.0 MBq (86)Y-CHX-A''-DTPA-cetuximab. Quantitative PET revealed the highest tumor uptake in LS-174T (29.55 +/- 2.67%ID/cm(3)) and the lowest tumor uptake in PC-3 (15.92 +/- 1.55%ID/cm(3)) xenografts at 3 days after injection. Tumor uptake values quantified by PET were closely correlated (r (2) = 0.9, n = 18) with values determined by biodistribution studies. CONCLUSION: This study demonstrated the feasibility of preparation of high specific activity (86)Y-CHX-A''-DTPA-cetuximab and its application for quantitative noninvasive PET imaging of HER1-expressing tumors. (86)Y-CHX-A''-DTPA-cetuximab offers an attractive alternative to previously labeled cetuximab for PET and further investigation for clinical translation is warranted.