Radiochemistry and In Vivo Imaging of [45Ti]Ti-THP-PSMA.

Titanium-45 (45Ti) is a radionuclide with excellent physical characteristics for use in positron emission tomography (PET) imaging, including a moderate half-life (3.08 h), decay by positron emission (85%), and a low mean positron energy of 0.439 MeV. However, challenges associated with titanium chemistry have led to the underdevelopment of this radionuclide for incorporation into radiopharmaceuticals. Expanding on our recent studies, which showed promising results for the complexation of 45Ti with the tris hydroxypyridinone (THPMe) chelator, the current work aimed to optimize the chemistry and imaging attributes of [45Ti]Ti-THP-PSMA as a new PET radiopharmaceutical. Methods. Radiolabeling of THP-PSMA was optimized with [45Ti]Ti-citrate at varying pHs and masses of the precursor. The stability of the radiolabeled complex was assessed in mouse serum for up to 6 h. The affinity of [45Ti]Ti-THP-PSMA for prostate-specific membrane antigen (PSMA) was assessed using LNCaP (PSMA +) and PC3 (PSMA -) cell lines. In vivo imaging and biodistribution analysis were performed in tumor-bearing xenograft mouse models to confirm the specificity of the tumor uptake. Results. > 95% of radiolabeling was achieved with a high specific activity of 5.6 MBq/nmol under mild conditions. In vitro cell binding studies showed significant binding of the radiolabeled complex with the PSMA-expressing LNCaP cell line (11.9 ± 1.5%/mg protein-bound activity) compared to that with the nonexpressing PC3 cells (1.9 ± 0.4%/mg protein-bound activity). In vivo imaging and biodistribution studies confirmed specific uptake in LNCaP tumors (1.6 ± 0.27% ID/g) compared to that in PC3 tumors (0.39 ± 0.2% ID/g). Conclusion. This study showed a simple one-step radiolabeling method for 45Ti with THP-PSMA under mild conditions (pH 8 and 37 °C). In vitro cell studies showed promise, but in vivo tumor xenograft studies indicated low tumor uptake. Overall, this study shows the need for more chelators for 45Ti for the development of a PET radiopharmaceutical for cancer imaging.

Optimal His-Tag Design for Efficient [99mTc(CO)3]+ and [188Re(CO)3]+ Labeling of Proteins for Molecular Imaging and Radionuclide Therapy by Analysis of Peptide Arrays.

Hexahistidine tags (His-tags), incorporated into recombinant proteins to facilitate purification using metal-affinity chromatography, are useful binding sites for radiolabeling with [99mTc(CO)3]+ and [188Re(CO)3]+ for molecular imaging and radionuclide therapy. Labeling efficiencies vary unpredictably, and the method is therefore not universally useful. To overcome this, we have made quantitative comparisons of radiolabeling of a bespoke Celluspots array library of 382 His-tag-containing peptide sequences with [99mTc(CO)3]+ and [188Re(CO)3]+ to identify key features that enhance labeling. A selected sequence with 10-fold enhanced labeling efficiency compared to the most effective literature-reported sequences was incorporated into an exemplar protein and compared biologically with non-optimized analogues, in vitro and in vivo. Optimal labeling with either [99mTc(CO)3]+ or [188Re(CO)3]+ required six consecutive His residues in the protein sequence, surrounded by several positively charged residues (Arg or Lys), and the presence of phosphate in the buffer. Cys or Met residues in the sequence were beneficial, to a lesser extent. Negatively charged residues were deleterious to labeling. His-tags with adjacent positively charged residues could be labeled as much as 40 times more efficiently than those with adjacent negatively charged residues. 31P NMR of [Re(CO)3(H2O)3]+ and electrophoresis of solutions of [99mTc(CO)3(H2O)3]+ suggest that phosphate bridges form between cationic residues and the cationic metal synthon during labeling. The trial optimized protein, a scFv targeted to the PSMA antigen expressed in prostate cancer, was readily labeled in >95% radiochemical yield, without the need for subsequent purification. Labeling occurred more quickly and to higher specific activity than comparable non-optimized proteins, while retaining specific binding to PSMA and prostate cancer in vivo. Thus, optimized His-tags greatly simplify radiolabeling of recombinant proteins making them potentially more widely and economically available for imaging and treating patients.

Synthesis, Characterization, and Application of Core-Shell Co0.16Fe2.84O4@NaYF4(Yb, Er) and Fe3O4@NaYF4(Yb, Tm) Nanoparticle as Trimodal (MRI, PET/SPECT, and Optical) Imaging Agents.

Multimodal nanoparticulate materials are described, offering magnetic, radionuclide, and fluorescent imaging capabilities to exploit the complementary advantages of magnetic resonance imaging (MRI), positron emission tomography/single-photon emission commuted tomography (PET/SPECT), and optical imaging. They comprise Fe3O4@NaYF4 core/shell nanoparticles (NPs) with different cation dopants in the shell or core, including Co0.16Fe2.84O4@NaYF4(Yb, Er) and Fe3O4@NaYF4(Yb, Tm). These NPs are stabilized by bisphosphonate polyethylene glycol conjugates (BP-PEG), and then show a high transverse relaxivity (r2) up to 326 mM(-1) s(-1) at 3T, a high affinity to [(18)F]-fluoride or radiometal-bisphosphonate conjugates (e.g., (64)Cu and (99m)Tc), and fluorescent emissions from 500 to 800 nm under excitation at 980 nm. The biodistribution of intravenously administered particles determined by PET/MR imaging suggests that negatively charged Co0.16Fe2.84O4@NaYF4(Yb, Er)-BP-PEG (10K) NPs cleared from the blood pool more slowly than positively charged NPs Fe3O4@NaYF4(Yb, Tm)-BP-PEG (2K). Preliminary results in sentinel lymph node imaging in mice indicate the advantages of multimodal imaging.

Monitoring the efficacy of dendritic cell vaccination by early detection of (99m) Tc-HMPAO-labelled CD4(+) T cells.

DC vaccines have been used to induce tumour-specific cytotoxic T cells . However, this approach to cancer immunotherapy has had limited success. To be successful, injected DCs need to migrate to the LNs where they can stimulate effector T cells . We and others have previously demonstrated by MRI that tumour antigen-pulsed-DCs labelled ex vivo with superparamagnetic iron oxide nanoparticles migrated to the draining LNs and are capable of activating antigen-specific T cells . The results from our study demonstrated that ex vivo superparamagnetic iron oxide nanoparticles-labelled and OVA-pulsed DCs prime cytotoxic CD8(+) T-cell responses to protect against a B16-OVA tumour challenge. In the clinic, a possible noninvasive surrogate marker for efficacy of DC vaccination is to image the specific migration and accumulation of T cells following DC vaccination.

[(89)Zr]oxinate4 for long-term in vivo cell tracking by positron emission tomography.

PURPOSE: (111)In (typically as [(111)In]oxinate3) is a gold standard radiolabel for cell tracking in humans by scintigraphy. A long half-life positron-emitting radiolabel to serve the same purpose using positron emission tomography (PET) has long been sought. We aimed to develop an (89)Zr PET tracer for cell labelling and compare it with [(111)In]oxinate3 single photon emission computed tomography (SPECT). METHODS: [(89)Zr]Oxinate4 was synthesised and its uptake and efflux were measured in vitro in three cell lines and in human leukocytes. The in vivo biodistribution of eGFP-5T33 murine myeloma cells labelled using [(89)Zr]oxinate4 or [(111)In]oxinate3 was monitored for up to 14 days. (89)Zr retention by living radiolabelled eGFP-positive cells in vivo was monitored by FACS sorting of liver, spleen and bone marrow cells followed by gamma counting. RESULTS: Zr labelling was effective in all cell types with yields comparable with (111)In labelling. Retention of (89)Zr in cells in vitro after 24 h was significantly better (range 71 to >90%) than (111)In (43-52%). eGFP-5T33 cells in vivo showed the same early biodistribution whether labelled with (111)In or (89)Zr (initial pulmonary accumulation followed by migration to liver, spleen and bone marrow), but later translocation of radioactivity to kidneys was much greater for (111)In. In liver, spleen and bone marrow at least 92% of (89)Zr remained associated with eGFP-positive cells after 7 days in vivo. CONCLUSION: [(89)Zr]Oxinate4 offers a potential solution to the emerging need for a long half-life PET tracer for cell tracking in vivo and deserves further evaluation of its effects on survival and behaviour of different cell types.

Real-time differential tracking of human neutrophil and eosinophil migration in vivo.

BACKGROUND: Hitherto, in vivo studies of human granulocyte migration have been based on indiscriminate labeling of total granulocyte populations. We hypothesized that the kinetics of isolated human neutrophil and eosinophil migration through major organs in vivo are fundamentally different, with the corollary that studying unseparated populations distorts measurement of both. METHODS: Blood neutrophils and eosinophils were isolated on 2 separate occasions from human volunteers by using Current Good Manufacturing Practice CD16 CliniMACS isolation, labeled with technetium 99m-hexamethylpropyleneamine oxime, and then reinfused intravenously. The kinetics of cellular efflux were imaged over 4 hours. RESULTS: Neutrophils and eosinophils were isolated to a mean purity of greater than 97% and greater than 95%, respectively. Activation of neutrophils measured as an increase in their CD11b mean fluorescence intensity in whole blood and after isolation and radiolabeling was 25.98 ± 7.59 and 51.82 ± 17.44, respectively, and was not significant (P = .052), but the mean fluorescence intensity of CD69 increased significantly on eosinophils. Analysis of the scintigraphic profile of lung efflux revealed exponential clearance of eosinophils, with a mean half-life of 4.16 ± 0.11 minutes. Neutrophil efflux was at a significantly slower half-life of 13.72 ± 4.14 minutes (P = .009). The migration of neutrophils and eosinophils was significantly different in the spleen at all time points (P = .014), in the liver at 15 minutes (P = .001), and in the bone marrow at 4 hours (P = .003). CONCLUSIONS: The kinetics of migration of neutrophils and eosinophils through the lung, spleen, and bone marrow of human volunteers are significantly different. Study of mixed populations might be misleading.

Sialoadhesin - a macrophage-restricted marker of immunoregulation and inflammation.

Sialoadhesin (Sn, also known as Siglec-1 and CD169) is a macrophage-restricted cell surface receptor that is conserved across mammals. Sn is a member of the sialic acid-binding IgG-like lectin (Siglec) family of proteins characterized by affinity to specifically sialylated ligands, and under normal conditions is expressed on subsets of macrophages in secondary lymphoid tissues, such as lymph node and spleen. However, Sn-positive macrophages can also be found in a variety of pathological conditions, including (autoimmune) inflammatory infiltrates and tumours. Sn has been shown to contribute to sialylated pathogen uptake, antigen presentation and lymphocyte proliferation, and to influence both immunity and tolerance. This review presents Sn as a macrophage-specific marker of inflammation and immunoregulation with the potential to becoming an important biomarker for immunologically active macrophages and a target for therapy.

An indium-111-labelled membrane-targeted peptide for cell tracking with radionuclide imaging.

Cell labelling agents that enable longitudinal in vivo tracking of administered cells will support the clinical development of cell-based therapies. Radionuclide imaging with gamma and positron-emitting radioisotopes can provide quantitative and longitudinal mapping of cells in vivo. To make this widely accessible and adaptable to a range of cell types, new, versatile and simple methods for directly radiolabelling cells are required. We have developed [111In]In-DTPA-CTP, the first example of a radiolabelled peptide that binds to the extracellular membrane of cells, for tracking cell distribution in vivo using Single Photon Emission Computed Tomography (SPECT). [111In]In-DTPA-CTP consists of (i) myristoyl groups for insertion into the phospholipid bilayer, (ii) positively charged lysine residues for electrostatic association with negatively charged phospholipid groups at the cell surface and (iii) a diethylenetriamine pentaacetate derivative that coordinates the γ-emitting radiometal, [111In]In3+. [111In]In-DTPA-CTP binds to 5T33 murine myeloma cells, enabling qualitative SPECT tracking of myeloma cells' accumulation in lungs immediately after intravenous administration. This is the first report of a radiolabelled cell-membrane binding peptide for use in cell tracking.

Correlation of hypoxia PET tracer uptake with hypoxic radioresistance in cancer cells: PET biomarkers of resistance to stereotactic radiation therapy?

PURPOSE: The pO2 threshold of an ideal PET hypoxia tracer for radiotherapy planning in cancer would match those observed in clinically and biologically relevant processes such as radioresistance and HIF1α expression. To identify such tracers, we directly compared uptake in vitro of hypoxia PET tracers ([18F]FMISO, [64Cu]CuATSM, and analogues [64Cu]CuATS, [64Cu]CuATSE, [64Cu]CuCTS, [64Cu]CuDTS, [64Cu]CuDTSE, [64Cu]CuDTSM) with levels of radioresistance and HIF1α expression in cultured cancer cells under identical hypoxic conditions ranging from extreme hypoxia to normoxia. Pimonidazole uptake was also compared as a marker of hypoxia. METHODS: A custom-built hypoxia apparatus enabled all experiments to be performed under identical hypoxic conditions with constant measurement of pO2 in media using an OxyLab pO2™ probe. HCT116 human colonic carcinoma and MCF-7 human Caucasian breast adenocarcinoma cells were irradiated using a cobalt teletherapy unit. Clonogenic assays were used to assess survival. HIF1α expression was determined by western blotting, tracer uptake by gamma counting and pimonidazole binding by flow cytometry. RESULTS: Radioresistance, pimonidazole binding and HIF1α expression increased gradually as pO2 decreased between 25 mmHg and 0 mmHg. In contrast, all the PET hypoxia tracers showed a sharp increase in uptake only when pO2 levels fell below 1 mmHg. Above this threshold, tracer uptake was not elevated above that in normoxic cells. CONCLUSION: This study highlights an important mismatch in pO2 thresholds between these PET tracers and other markers of hypoxia: tracer uptake only occurred at oxygen levels that were well below levels that induced radioresistance, pimonidazole uptake and HIF1α expression. Although their pO2 thresholds do not match the threshold for resistance to conventionally fractionated radiotherapy (pO2 2.5-10 mmHg), their specificity for extreme hypoxia (pO2 ≪ 1 mmHg) suggests these PET tracers may be of particular use to predict outcomes in stereotactic radiation therapy where these maximally resistant cells play a key role in determining the biological effect.

The TLR9 ligand CpG promotes the acquisition of Plasmodium falciparum-specific memory B cells in malaria-naive individuals.

Despite the central role of memory B cells (MBC) in protective immune responses, little is understood about how they are acquired in naive individuals in response to Ag exposure, and how this process is influenced by concurrent activation of the innate immune system's TLR. In this longitudinal study of malaria-naive individuals, we examined the MBC response to two candidate malaria vaccines administered with or without CpG, a TLR9 ligand. We show that the acquisition of MBC is a dynamic process in which the vaccine-specific MBC pool rapidly expands and then contracts, and that CpG enhances the kinetics, magnitude, and longevity of this response. We observed that the percentage of vaccine-specific MBC present at the time of reimmunization predicts vaccine-specific Ab levels 14 days later; and that at steady-state, there is a positive correlation between vaccine-specific MBC and Ab levels. An examination of the total circulating MBC and plasma cell pools also suggests that MBC differentiate into plasma cells through polyclonal activation, independent of Ag specificity. These results provide important insights into the human MBC response, which can inform the development of vaccines against malaria and other pathogens that disrupt immunological memory.

Comparison of biological activity of human anti-apical membrane antigen-1 antibodies induced by natural infection and vaccination.

Vaccines represent a significant potential means of decreasing global morbidity and mortality due to malaria. Clinical trials in the United States with Plasmodium falciparum Apical Membrane Antigen 1 (AMA1) showed that the vaccine induced biologically active Abs judged by an in vitro parasite growth inhibition assay (GIA). However, the same vaccine in Malian adults did not increase biological activity, although it elevated ELISA titers. Because GIA has been used to evaluate the biological activity of Abs induced by blood stage malarial vaccine candidates, we explored this discrepancy in this study. We affinity purified AMA1-specific Abs from both U.S. vaccinees and nonvaccinated individuals living in a malaria-endemic area of Mali and performed ELISA and GIA. Both AMA1-specifc Abs induced by vaccination (U.S.) and by natural infection (Mali) have comparable biological activity in GIA when the ELISA titer is normalized. However, a fraction of Malians' IgG that did not bind to AMA1 protein (Mali-non-AMA1 IgG) reduced the biological activity of the AMA1 Abs from U.S. vaccinees; in contrast, U.S.-non-AMA1 IgGs did not show a reduction of the biological activity. Further investigation revealed that the reduction was due to malaria-specific IgGs in the Mali-non-AMA1 IgGs. The fact that both U.S.- and Mali-AMA1-specific Abs showed comparable biological activity supports further development of AMA1-based vaccines. However, the reduction of biological activity of AMA1-specific Ab by other malaria-specific IgGs likely explains the limited effect on growth-inhibitory activity of Abs induced by AMA1 vaccination in Malian adults and may complicate efforts to develop a blood stage malaria vaccine.

Efficient site-specific radiolabeling of a modified C2A domain of synaptotagmin I with [99mTc(CO)3]+: a new radiopharmaceutical for imaging cell death.

We describe the design and synthesis of a new Tc-99m labeled bioconjugate for cell-death imaging, based on C2A, the phosphatidylserine (PS)-binding domain of rat synaptotagmin I. Since several lysine residues in this protein are critical for PS binding, we engineered a new protein, C2AcH, to include the C-terminal sequence CKLAAALEHHHHHH, incorporating a free cysteine (for site-specific covalent modification) and a hexahistidine tag (for site-specific radiolabeling with [99mTc(CO)3(OH2)3]+). We also engineered a second derivative, C2Ac, in which the C-terminal sequence included only the C-terminal cysteine. These proteins were characterized by electrospray mass spectrometry, SDS/PAGE, and size exclusion chromatography and radiolabeled with [99mTc(CO)3(OH2)3]+. Conjugates of the proteins with the rhenium analogue [Re(CO)3(OH2)3]+ were also synthesized. Site-specific labeling was confirmed by performing a tryptic digest of rhenium tricarbonyl-labeled C2AcH, and only peptides containing the His-tag contained the [Re(CO)3]+. The labeled proteins were tested for binding to red blood cells (RBC) with exposed PS in a calcium dependent manner. Labeling 100 microg of C2AcH with [99mTc(CO)3(OH2)3]+ at 37 degrees C for 30 min gave a radiochemical yield of > 96%. However, C2AcH that had first been conjugated with fluorescein maleimide or iodoacetamide via the Cys residue gave only 50% and 83% radiochemical yield, respectively, after incubation for 30 min at 37 degrees C. Serum stability results indicated that >95% of radiolabeled C2AcH remained stable for at least 18 h at 37 degrees C. Site-specifically labeled C2AcH exhibited calcium-dependent binding to the PS on the RBC, whereas a nonspecifically modified derivative, C2AcH-B, in which lysines had been modified with benzyloxycarbonyloxy, did not. We conclude that (i) the combination of Cys and a His-tag greatly enhances the rate and efficiency of labeling with [99mTc(CO)3(OH2)3]+ compared to either the His-tag or the Cys alone, and this sequence deserves further evaluation as a radiolabeling tag; (ii) non-site-specific modification of C2A via lysine residues impairs target binding affinity; (iii) 99mTc-C2AcH has excellent radiolabeling, stability and PS binding characteristics and warrants in vivo evaluation as a cell-death imaging agent.

Cold Kit for Prostate-Specific Membrane Antigen (PSMA) PET Imaging: Phase 1 Study of 68Ga-Tris(Hydroxypyridinone)-PSMA PET/CT in Patients with Prostate Cancer.

68Ga-labeled urea-based inhibitors of the prostate-specific membrane antigen (PSMA), such as 68Ga-labeled N,N'-bis(2-hydroxybenzyl)ethylenediamine-N,N'-diacetic acid (HBED)-PSMA-11, are promising small molecules for targeting prostate cancer. A new radiopharmaceutical, 68Ga-labeled tris(hydroxypyridinone) (THP)-PSMA, has a simplified design for single-step kit-based radiolabeling. It features the THP ligand, which forms complexes with 68Ga3+ rapidly at a low concentration, at room temperature, and over a wide pH range, enabling direct elution from a 68Ge/68Ga generator into a lyophilized radiopharmaceutical kit in 1 step without manipulation. The aim of this phase 1 study was to assess the safety and biodistribution of 68Ga-THP-PSMA. Methods: Cohort A comprised 8 patients who had proven prostate cancer and were scheduled to undergo prostatectomy; they had Gleason scores of 7-10 and a mean prostate-specific antigen level of 7.8 µg/L (range, 5.4-10.6 µg/L). They underwent PET/CT after the administration of 68Ga-THP-PSMA. All patients proceeded to prostatectomy (7 with pelvic nodal dissection). Dosimetry from multi-time-point PET imaging was performed with OLINDA/EXM. Cohort B comprised 6 patients who had positive 68Ga-HBED-PSMA-11 PET/CT scanning results and underwent comparative 68Ga-THP-PSMA scanning. All patients were monitored for adverse events. Results: No adverse events occurred. In cohort A, 6 of 8 patients had focal uptake in the prostate (at 2 h: average SUVmax, 5.1; range, 2.4-9.2) and correlative 3+ staining of prostatectomy specimens on PSMA immunohistochemistry. The 2 68Ga-THP-PSMA scans with negative results had only 1+/2+ staining. The mean effective dose was 2.07E-02 mSv/MBq. In cohort B, 68Ga-THP-PSMA had lower physiologic background uptake than 68Ga-HBED-PSMA-11 (in the parotid glands, the mean SUVmax for 68Ga-THP-PSMA was 3.6 [compared with 19.2 for 68Ga-HBED-PSMA-11]; the respective corresponding values in the liver were 2.7 and 6.3, and those in the spleen were 2.7 and 10.5; P < 0.001 for all). In 5 of 6 patients, there was concordance in the number of metastases identified with 68Ga-HBED-PSMA-11 and 68Ga-THP-PSMA. Thirteen of 15 nodal abnormalities were subcentimeter. In 22 malignant lesions, the tumor-to-liver contrast with 68Ga-THP-PSMA was similar to that with 68Ga-HBED-PSMA (4.7 and 5.4, respectively; P = 0.15), despite a higher SUVmax for 68Ga-HBED-PSMA than for 68Ga-THP-PSMA (30.3 and 10.7, respectively; P < 0.01). Conclusion:68Ga-THP-PSMA is safe and has a favorable biodistribution for clinical imaging. Observed focal uptake in the prostate was localized to PSMA-expressing malignant tissue on histopathology. Metastatic PSMA-avid foci were also visualized with 68Ga-THP-PSMA PET. Single-step production from a Good Manufacturing Practice cold kit may enable rapid adoption.

Author Correction: Non-Invasive whole-body detection of complement activation using radionuclide imaging in a mouse model of myocardial ischaemia-reperfusion injury.

A correction to this article has been published and is linked from the HTML and PDF versions of this paper. The error has not been fixed in the paper.

Formulation of vaccines containing CpG oligonucleotides and alum.

CpG oligodeoxynucleotides are potent immunostimulants. For parenterally delivered alum-based vaccines, the immunostimulatory effect of CpG depends on the association of the CpG and antigen to the alum. We describe effects of buffer components on the binding of CPG 7909 to aluminum hydroxide (Alhydrogel), assays for measuring binding of CPG 7909 to alum and CPG 7909 induced dissociation of antigen from the alum. Free CPG 7909 is a potent inducer of IP-10 in mice. However the lack of IP-10 production from formulations containing bound CPG 7909 suggested that CPG 7909 does not rapidly dissociate from the alum after injection. It also suggests that IP-10 assays are not a good basis for potency assays for alum-based vaccines containing CPG 7909.

A 99mTc-labelled scFv antibody fragment that binds to prostate-specific membrane antigen.

INTRODUCTION: Prostate-specific membrane antigen (PSMA) is an extensively studied antigen for imaging prostate cancer. We prepared a single-chain variable fragment (scFv) of J591, a monoclonal antibody that recognises an external epitope of PSMA, incorporating a His-tag for labelling with Tc tricarbonyl, and evaluated its binding using human PCa cell lines. METHODS: J591(scFv) was expressed in HEK-293T cells and purified by metal ion affinity chromatography, followed by size exclusion chromatography. Stability and monomer/dimer ratios of purified scFv under different storage conditions were analysed by SDS-PAGE and analytical size exclusion chromatography. J591(scFv) was labelled with (Equation is included in full-text article.)at 37°C for 60 min. The stability of Tc-scFv in human serum was analysed by SDS-PAGE with autoradiography. Cell-binding studies were carried out using PC3LN3 (PSMA negative) and PC3LN3-PSMA (a variant engineered to express PSMA) cell lines. RESULTS: J591(scFv) was most stable to dimerisation on storage at -80°C compared with -20 and 4°C. Radiochemical yields of 85-90% were obtained with the final radiochemical purity of more than 99% after purification by gel filtration. In these small-scale studies, the maximum specific activity achieved was 7 MBq/µg. Liquid chromatography-mass spectrometry showed the formation of Tc-J591(scFv), which was radiochemically stable in serum, with no dissociation of Tc over 24 h. Cell-binding assays showed specific binding to PSMA-positive cells. CONCLUSION: J591(scFv) can be radiolabelled with (Equation is included in full-text article.)conveniently and efficiently. The labelled product was stable in serum. It showed selective binding to PSMA-positive cells compared with PSMA-negative cells. This potential radiotracer warrants evaluation in PCa xenograft models.

Simple, mild, one-step labelling of proteins with gallium-68 using a tris(hydroxypyridinone) bifunctional chelator: a 68Ga-THP-scFv targeting the prostate-specific membrane antigen.

BACKGROUND: Labelling proteins with gallium-68 using bifunctional chelators is often problematic because of unsuitably harsh labelling conditions such as low pH or high temperature and may entail post-labelling purification. To determine whether tris(hydroxypyridinone) (THP) bifunctional chelators offer a potential solution to this problem, we have evaluated the labelling and biodistribution of a THP conjugate with a new single-chain antibody against the prostate-specific membrane antigen (PSMA), an attractive target for staging prostate cancer (PCa). A single-chain variable fragment (scFv) of J591, a monoclonal antibody that recognises an external epitope of PSMA, was prepared in order to achieve biokinetics matched to the half-life of gallium-68. The scFv, J591c-scFv, was engineered with a C-terminal cysteine. RESULTS: J591c-scFv was produced in HEK293T cells and purified by size-exclusion chromatography. A maleimide THP derivative (THP-mal) was coupled site-specifically to the C-terminal cysteine residue. The THP-mal-J591c-scFv conjugate was labelled with ammonium acetate-buffered gallium-68 from a 68Ge/68Ga generator at room temperature and neutral pH. The labelled conjugate was evaluated in the PCa cell line DU145 and its PSMA-overexpressing variant in vitro and xenografted in SCID mice. J591c-scFv was produced in yields of 4-6 mg/l culture supernatant and efficiently coupled with the THP-mal bifunctional chelator. Labelling yields > 95% were achieved at room temperature following incubation of 5 µg conjugate with gallium-68 for 5 min without post-labelling purification. 68Ga-THP-mal-J591c-scFv was stable in serum and showed selective binding to the DU145-PSMA cell line, allowing an IC50 value of 31.5 nM to be determined for unmodified J591c-scFv. Serial PET/CT imaging showed rapid, specific tumour uptake and clearance via renal elimination. Accumulation in DU145-PSMA xenografts at 90 min post-injection was 5.4 ± 0.5%ID/g compared with 0.5 ± 0.2%ID/g in DU145 tumours (n = 4). CONCLUSIONS: The bifunctional chelator THP-mal enabled simple, rapid, quantitative, one-step room temperature radiolabelling of a protein with gallium-68 at neutral pH without a need for post-labelling purification. The resultant gallium-68 complex shows high affinity for PSMA and favourable in vivo targeting properties in a xenograft model of PCa.

Non-Invasive whole-body detection of complement activation using radionuclide imaging in a mouse model of myocardial ischaemia-reperfusion injury.

Complement activation is a recognised mediator of myocardial ischaemia-reperfusion-injury (IRI) and cardiomyocytes are a known source of complement proteins including the central component C3, whose activation products can mediate tissue inflammation, cell death and profibrotic signalling. We investigated the potential to detect and quantify the stable covalently bound product C3d by external body imaging, as a marker of complement activation in heart muscle in a murine model of myocardial IRI. We used single-photon-emission-computed-tomography (SPECT) in conjunction with 99mTechnecium-labelled recombinant complement receptor 2 (99mTc-rCR2), which specifically detects C3d at the site of complement activation. Compared to control imaging with an inactive CR2 mutant (99mTc-K41E CR2) or an irrelevant protein (99mTc-PSMA) or using 99mTc-rCR2 in C3-deficient mice, the use of 99mTc-rCR2 in complement-intact mice gave specific uptake in the reperfused myocardium. The heart to skeletal muscle ratio of 99mTc-rCR2 was significantly higher than in the three control groups. Histological analysis confirmed specific uptake of 99mTc-rCR2. Following therapeutic inhibition of complement C3 activation, we found reduced myocardial uptake of 99mTc-rCR2. We conclude, therefore that 99mTc-rCR2 imaging can be used for non-invasive detection of activated complement and in future could be exploited to quantify the severity of myocardial damage due to complement activation.

Critical evaluation of different methods for measuring the functional activity of antibodies against malaria blood stage antigens.

Antibodies are thought to be the primary immune effectors in the defense against erythrocytic stage Plasmodium falciparum. Thus, malaria vaccines directed to blood stages of infection are evaluated based on their ability to induce antibodies with anti-parasite activity. Such antibodies may have different effector functions (e.g., inhibition of invasion or inhibition of parasite growth/development) depending on the target antigen. We evaluated four methods with regards to their ability to differentiate between invasion and/or growth inhibitory activities of antibodies specific for two distinct blood stage antigens: AMA1 and MSP1(42). We conclude that antibodies induced by these vaccine candidates have different modes of action that vary not only by the antigen, but also by the strain of parasite being tested. Analysis based on parasitemia and viability was essential for defining the full range of anti-parasite activities in immune sera.

Opportunities and challenges for metal chemistry in molecular imaging: from gamma camera imaging to PET and multimodality imaging.

The development of medical imaging is a highly multidisciplinary endeavor requiring the close cooperation of clinicians, physicists, engineers, biologists and chemists to identify capabilities, conceive challenges and solutions and apply them in the clinic. The chemistry described in this article illustrates how synergistic advances in these areas drive the technology and its applications forward, with each discipline producing innovations that in turn drive innovations in the others. The main thread running through the article is the shift from single photon radionuclide imaging towards PET, and in turn the emerging shift from PET/CT towards PET/MRI and further, combination of these with optical imaging. Chemistry to support these transitions is exemplified by building on a summary of the status quo, and recent developments, in technetium-99m chemistry for SPECT imaging, followed by a report of recent developments to support clinical application of short lived (Ga-68) and long-lived (Zr-89) positron emitting isotopes, copper isotopes for PET imaging, and combined modality imaging agents based on radiolabelled iron oxide based nanoparticles.