Positron emission tomography imaging of CD105 expression with a 64Cu-labeled monoclonal antibody: NOTA is superior to DOTA

Optimizing the in vivo stability of positron emission tomography (PET) tracers is of critical importance to cancer diagnosis. In the case of (64)Cu-labeled monoclonal antibodies (mAb), in vivo behavior and biodistribution is critically dependent on the performance of the bifunctional chelator used to conjugate the mAb to the radiolabel. This study compared the in vivo characteristics of (64)Cu-labeled TRC105 (a chimeric mAb that binds to both human and murine CD105), through two commonly used chelators: 1,4,7-triazacyclononane-1,4,7-triacetic acid (NOTA) and 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid (DOTA). Flow cytometry analysis confirmed that chelator conjugation of TRC105 did not affect its CD105 binding affinity or specificity. PET imaging and biodistribution studies in 4T1 murine breast tumor-bearing mice revealed that (64)Cu-NOTA-TRC105 exhibited better stability than (64)Cu-DOTA-TRC105 in vivo, which resulted in significantly lower liver uptake without compromising the tumor targeting efficiency. In conclusion, this study confirmed that NOTA is a superior chelator to DOTA for PET imaging with (64)Cu-labeled TRC105.     

Evaluation of 64Cu radiolabeled anti-hPD-L1 Nb6 for positron emission tomography imaging in lung cancer tumor mice model

Recently, we selected a novel anti-hPD-L1-specific HCAb named Nb6 with high affinity (EC₅₀ = 0.65 ng/mL) for potential hPD-L1 targeted non-invasive PET imaging. In this research, Nb6 was conjugated with the bifunctional chelator NCS-Bz-NOTA ((2-[(4-Isothiocyanophenyl) methyl]-1,4,7-triazacy-clononane-1,4,7-triacetic acid)) and further labeled with radio-nuclide ⁶⁴Cu. ⁶⁴Cu-NOTA-Nb6 was prepared with over 95% labeling yield, over 99% radiochemical purity and 14–16 GBq/μmol specific activity after PD-10 column purification. It shows good stability in 0.01 M PBS and 5% HSA solutions. ⁶⁴Cu-NOTA-Nb6 has a high binding affinity to 3.60 nM which was tested by human lung adenocarcinoma A549 cell lines. Tumor lesion can be clearly observed from 20 h to 38 h by Micro-PET equipment after ⁶⁴Cu-NOTA-Nb6 administration. The study revealed that ⁶⁴Cu-NOTA-Nb6 has good lesion detection ability, high ratios between tumor and non-tumor signal and can specifically target A549 xenografted tumor model. Taken together of good stability, high binding affinity, and tumor detection ability, ⁶⁴Cu labeled Nb6 is a promising radio-tracer in diagnosing of hPD-L1 overexpression tumor, supposed to monitor PD-L1overexpression tumor progression and guide targeted therapy with PET molecular imaging.     

Evaluation of 64Cu-labeled bifunctional chelate-bombesin conjugates

Several bifunctional chelates (BFCs) were investigated as carriers of (64)Cu for PET imaging. The most widely used chelator for (64)Cu labeling of BFCs is DOTA (1,4,7,10-tetraazacyclododecane-N,N',N″,N'-tretraacetic acid), even though this complex exhibits only moderate in vivo stability. In this study, we prepared a series of alternative chelator-peptide conjugates labeled with (64)Cu, measured in vitro receptor binding affinities in human breast cancer T47D cells expressing the gastrin-releasing peptide receptor (GRPR) and compared their in vivo stability in mice. DOTA-, NOTA-(1,4,7-triazacyclononane-1,4,7-triacetic acid), PCTA-(3,6,9,15-tetraazabicyclo[9.3.1]pentadeca-1(15),11,13-triene-3,6,9-triacetic acid), and Oxo-DO3A-(1-oxa-4,7,10-triazacyclododecane-4,7,10-triacetic acid) peptide conjugates were prepared using H(2)N-Aoc-[d-Tyr(6),βAla(11),Thi(13),Nle(14)]bombesin(6-14) (BBN) as a peptide template. The BBN moiety was selected since it binds with high affinity to the GRPR, which is overexpressed on human breast cancer cells. A convenient synthetic approach for the attachment of aniline-BFC to peptides on solid support is also presented. To facilitate the attachment of the aniline-PCTA and aniline-Oxo-DO3A to the peptide via an amide bond, a succinyl spacer was introduced at the N-terminus of BBN. The partially protected aniline-BFC (p-H(2)N-Bn-PCTA(Ot-Bu)(3) or p-H(2)N-Bn-DO3A(Ot-Bu)(3)) was then coupled to the resulting N-terminal carboxylic acid preactivated with DEPBT/ClHOBt on resin. After cleavage and purification, the peptide-conjugates were labeled with (64)Cu using [(64)Cu]Cu(OAc)(2) in 0.1 M ammonium acetate buffer at 100 °C for 15 min. Labeling efficacy was >90% for all peptides; Oxo-DO3A-BBN was incubated an additional 150 min at 100 °C to achieve this high yield. Specific activities varied from 76 to 101 TBq/mmol. Competition assays on T47D cells showed that all BFC-BBN complexes retained high affinity for the GRPR. All BFC-BBN (64)Cu-conjugates were stable for over 20 h when incubated at 37 °C in mouse plasma samples. However, in vivo, only 37% of the (64)Cu/Oxo-DO3A complex remained intact after 20 h while the (64)Cu/DOTA-BBN complex was completely demetalated. In contrast, both (64)Cu/NOTA- and (64)Cu/PCTA-BBN conjugates remained stable during the 20 h time period. Our results indicate that it is possible to successfully conjugate aniline-BFC with peptide on solid support. Our data also show that (64)Cu-labeled NOTA- and PCTA-BBN peptide conjugates are promising radiotracers for PET imaging of many human cancers overexpressing the GRP receptor.     

NODAPA-OH and NODAPA-(NCS)n: synthesis, 68Ga-radiolabelling and in vitro characterisation of novel versatile bifunctional chelators for molecular imaging

This report concerns synthesis, (68)Ga-radiolabelling and stability data of 1,4,7-triazacyclononane-1,4-diacetic acid-7-p-isothio-cyanatophenyl-acetic acid (NODAPA-NCS), 1,4,7-triazacyclononane-1-acetic acid-4,7-di-p-isothiocyanatophenyl-acetic acid (NODAPA-(NCS)(2)) and 1,4,7-triazacyclononane-1,4-diacetic acid-7-p-hydroxyphenyl-acetic acid (NODAPA-OH), versatile bifunctional chelators with potential for molecular imaging. Protein binding and exemplified conjugation are also reported.     

In vivo behavior of copper-64-labeled methanephosphonate tetraaza macrocyclic ligands

Copper-64 ( T(1/2)=12.7 h; beta(+): 0.653 MeV, 17.4%; beta(-): 0.578 MeV, 39%) is produced in a biomedical cyclotron and has applications in both imaging and therapy. Macrocyclic chelators are widely used as bifunctional chelators to bind copper radionuclides to antibodies and peptides owing to their relatively high kinetic stability. In this paper, we evaluated three tetraaza macrocyclic ligands with two, three, and four pendant methanephosphonate functional groups. DO2P [1,4,7,10-tetraazacyclododecane-1,7-di(methanephosphonic acid)], DO3P [1,4,7,10-tetraazacyclododecane-1,4,7-tri(methanephosphonic acid)], and DOTP [1,4,7,10-tetraazacyclododecane-1,4,7,10-tetra(methanephosphonic acid)] were all radiolabeled with (64)Cu in high radiochemical yields. Copper-64-labeled DO2P and DOTP were highly stable in rat serum out to 24 h, while (64)Cu-DO3P remained 73% intact, with the remainder possibly forming a (64)Cu(.)2DO3P dimer by 24 h. The biodistribution experiments were performed in normal Sprague-Dawley rats. Of the three complexes, (64)Cu-DO2P demonstrated the most optimal clearance through the blood and liver. Copper-64-DO3P and (64)Cu-DOTP exhibited higher liver uptake and longer retention of liver activity, possibly because of the large negative charge of the complexes under physiological conditions. All three (64)Cu-labeled complexes showed high accumulation in bone, likely due to the binding of the methanephosphonate groups to hydroxyapatite. These results suggest that this series of methanephosphonate macrocyclic ligands may be useful as potential bone-imaging agents. The thermodynamic stability constants of the Cu(II) complexes with these three ligands were determined, and were found to be significantly higher than those of their acetate analogues. The Cu(II)-DO2P complex exhibited the highest stability constant among divalent transition metal ion DO2P complexes. Metabolism studies of (64)Cu-DO2P in rat liver suggest that the DO2P ligand may be used as a bifunctional chelator for copper radionuclides in radiodiagnostic or radiotherapeutic studies.     

A novel side-bridged hybrid phosphonate/acetate pendant cyclam: Synthesis,characterization, and 64Cu small animal PET imaging

Copper-64 (t_1/2 = 12.7 h; β⁺: 0.653 MeV, 17.4%; β^?: 0.578 MeV, 39%) is produced in a biomedical cyclotron and has applications in both imaging and therapy. Macrocyclic chelators are widely used as bifunctional chelators to bind copper radionuclides to antibodies and peptides owing to their relatively high kinetic stability. A novel side-bridged cyclam featuring both pendant acetate and phosphonate groups was synthesized using a Kabachnik–Fields approach followed by hydrobromic acid deprotection. The Cu(II) complex of the novel ligand was synthesized, radiolabeling with ⁶⁴Cu was demonstrated, and in vitro (serum) stability was performed. In addition, in vivo distribution and clearance of the ⁶⁴Cu-labeled complex was visualized by positron emission tomography (PET) imaging. This novel chelate may be useful in ⁶⁴Cu-mediated diagnostic positron emission tomography (PET) imaging as well as targeted radiotherapeutic applications.     

A versatile bifunctional chelate for radiolabeling humanized anti-CEA antibody with In-111 and Cu-64 at either thiol or amino groups: PET imaging of CEA-positive tumors with whole antibodies

Radiolabeled anti-carcinoembryonic antigen (CEA) antibodies have the potential to give excellent images of a wide variety of human tumors, including tumors of the colon, breast, lung, and medullar thyroid. In order to realize the goals of routine and repetitive clinical imaging with anti-CEA antibodies, it is necessary that the antibodies have a high affinity for CEA, low cross reactivity and uptake in normal tissues, and low immunogenicity. The humanized anti-CEA antibody hT84.66-M5A (M5A) fulfills these criteria with an affinity constant of >10 (10) M (-1), no reactivity with CEA cross-reacting antigens found in normal tissues, and >90% human protein sequence. A further requirement for routine clinical use of radiolabeled antibodies is a versatile method of radiolabeling that allows the use of multiple radionuclides that differ in their radioemissions and half-lives. We describe a versatile bifunctional chelator, DO3A-VS (1,4,7-tris(carboxymethyl)-10-(vinylsulfone)-1,4,7,10-tetraazacyclododecane) that binds a range of radiometals including 111 In for gamma-ray imaging and 64Cu for positron emission tomography (PET), and which can be conjugated with negligible loss of immunoreactivity either to sulfhydryls (SH) in the hinge region of lightly reduced immunoglobulins or surface lysines (NH) of immunoglobulins. Based on our correlative studies comparing the kinetics of radiolabeled anti-CEA antibodies in murine models with those in man, we predict that 64Cu-labeled intact, humanized antibodies can be used to image CEA positive tumors in the clinic.     

New macrobicyclic chelator for the development of ultrastable 64Cu-radiolabeled bioconjugate

Ethylene cross-bridged cyclam with two acetate pendant arms, ECB-TE2A, is known to form the most kinetically stable (64)Cu complexes. However, its usefulness as a bifunctional chelator is limited because of its harsh radiolabeling conditions. Herein, we report new cross-bridged cyclam chelator for the development of ultrastable (64)Cu-radiolabeled bioconjugates. Propylene cross-bridged TE2A (PCB-TE2A) was successfully synthesized in an efficient way. The Cu(II) complex of PCB-TE2A exhibited much higher kinetic stability than ECB-TE2A in acid decomplexation studies, and also showed high resistance to reduction-mediated demetalation. Furthermore, the quantitative radiolabeling of PCB-TE2A with (64)Cu was achieved under milder conditions compared to ECB-TE2A. Biodistribution studies strongly indicate that the (64)Cu complexes of PCB-TE2A cleared out rapidly from the body with minimum decomplexation.     

Convenient preparation of 68Ga-based PET-radiopharmaceuticals at room temperature

A straightforward labeling using generator produced positron emitting (68)Ga, which provides high quality images, may result in kit type production of PET radiopharmaceuticals and make PET examinations possible also at centers lacking accelerators. The introduction of macrocyclic bifunctional chelators that would provide fast (68)Ga-complexation at room temperature would simplify even further tracer preparation and open wide possibilities for (68)Ga-labeling of fragile and potent macromolecules. Gallium-68 has the potential to facilitate development of clinically practical PET and to promote PET technique for individualized medicine. The macrocyclic chelator, 1,4,7-triazacyclononanetriacetic acid (NOTA), and its derivative coupled to an eight amino acid residue peptide (NODAGA-TATE, [NODAGA (0), Tyr(3)]Octreotate) were labeled with (68)Ge/(68)Ga-generator produced positron emitting (68)Ga. Formation kinetics of (68)Ga-NOTA was studied as a function of pH and formation kinetics of (68)Ga-NODAGA-TATE was studied as a function of the bioconjugate concentration. The nearly quantitative radioactivity incorporation (RAI>95%) for (68)Ga-NOTA was achieved within less than 10 min at room temperature and pH 3.5. The concentrations of NODAGA-TATE required for RAI of >90% and >95% were, respectively, 2-5 and 10 microM. In both cases the purification of the (68)Ga-labeled products was not necessary since the radiochemical purity was >95% and the preparation buffer, 4-(2-hydroxyethyl) piperazine-1-ethanesulfonic acid (HEPES) is suitable for human use. In order to confirm the identity of the products, complexes comprising (nat)Ga were synthesized and analyzed by mass spectrometry. The complex was found to be stable in the reaction mixture, phosphate buffer, and human plasma during 4.5 h incubation. Free and peptide conjugated NOTA formed stable complexes with (68)Ga at room temperature within 10 min. This might be of special interest for the labeling of fragile and potent macromolecules and allow for kit type preparation of (68)Ga-based radiopharmaceuticals.     

Novel method to label solid lipid nanoparticles with 64cu for positron emission tomography imaging

Solid lipid nanoparticles (SLNs) are submicrometer (1-1000 nm) colloidal carriers developed in the past decade as an alternative system to traditional carriers (emulsions, liposomes, and polymeric nanoparticles) for intravenous applications. Because of their potential as drug carriers, there is much interest in understanding the in vivo biodistribution of SLNs following intravenous (i.v.) injection. Positron emission tomography (PET) is an attractive method for investigating biodistribution but requires a radiolabeled compound. In this work, we describe a method to radiolabel SLN for in vivo PET studies. A copper specific chelator, 6-[p-(bromoacetamido)benzyl]-1,4,8,11-tetraazacyclotetradecane-N,N',N',N'-tetraacetic acid (BAT), conjugated with a synthetic lipid, was incorporated into the SLN. Following incubation with (64)CuCl(2) for 1 h at 25 °C in 0.1 M NH(4)OAc buffer (pH 5.5), the SLNs (～150 nm) were successfully radiolabeled with (64)Cu (66.5% radiolabeling yield), exhibiting >95% radiolabeled particles following purification. The (64)Cu-SLNs were delivered intravenously to mice and imaged with PET at 0.5, 3, 20, and 48 h post injection. Gamma counting was utilized post imaging to confirm organ distributions. Tissue radioactivity (% injected dose/gram, %ID/g), obtained by quantitative analysis of the images, suggests that the (64)Cu-SLNs are circulating in the bloodstream after 3 h (blood half-life ～1.4 h), but are almost entirely cleared by 48 h. PET and gamma counting demonstrate that approximately 5-7%ID/g (64)Cu-SLNs remain in the liver at 48 h post injection. Stability assays confirm that copper remains associated with the SLN over the 48 h time period and that the biodistribution patterns observed are not from free, dissociated copper. Our results indicate that SLNs can be radiolabeled with (64)Cu, and their biodistribution can be quantitatively evaluated by in vivo PET imaging and ex vivo gamma counting.     

Synthetic Strategy to Prepare DOTA-Based Bifunctional Chelating Agent Ready to Bind Biomolecular Probes

Due to the continued interest in new bifunctional chelating agents (BFCA), we focused on the development of a convenient synthesis of 1,4,7,10-tetraazacyclododecane-1,4,7-tris(acetic acid)-10-butyrate mono (N-hydroxysuccinimidyl ester). It consists in the macrocycle DO3A derivatized with an aliphatic linker containing an active ester that requires selective and mild conditions to react with the targeting biomolecule. It is important to underlay the versatility of the obtained BFCA, which can be conjugated both to a biomolecule (protein, Fab fragment) or to a synthetic molecule. In a subsequent step, the developed chelator was successfully conjugated to a peptide sequence.     

Preparation and biological evaluation of (64)Cu labeled Tyr(3)-octreotate using a phosphonic acid-based cross-bridged macrocyclic chelator

Somatostatin receptors (SSTr) are overexpressed in a wide range of neuroendocrine tumors, making them excellent targets for nuclear imaging and therapy, and radiolabeled somatostatin analogues have been investigated for positron emission tomography imaging and radionuclide therapy of SSTr-positive tumors, especially of the subtype-2 (SSTr2). The aim of this study was to develop a somatostatin analogue, Tyr(3)-octreotate (Y3-TATE), conjugated to a novel cross-bridged macrocyclic chelator, 11-carboxymethyl-1,4,8,11-tetraazabicyclo[6.6.2]hexadecane-4-methanephosphonic acid (CB-TE1A1P). Unlike traditional cross-bridged macrocycles, such as 4,11-bis(carboxymethyl)-1,4,8,11-tetraazabicyclo[6.6.2]hexadecane (CB-TE2A), CB-TE1A1P-Y3-TATE was radiolabeled with (64)Cu in high purity and high specific activity using mild conditions. Saturation binding assays revealed that (64)Cu-CB-TE1A1P-Y3-TATE had comparable binding affinity but bound to more binding sites in AR42J rat pancreatic tumor cell membranes than (64)Cu-CB-TE2A-Y3-TATE. Both radiopharmaceuticals showed comparable uptake in SSTr2 positive tissues in AR42J tumor-bearing rats. (64)Cu-CB-TE1A1P-Y3-TATE demonstrated improved blood clearance compared to (64)Cu-CB-TE2A-Y3-TATE, as the tumor/blood ratios of (64)Cu-CB-TE1A1P-Y3-TATE were shown to be significantly higher than those of (64)Cu-CB-TE2A-Y3-TATE at 4 and 24 h postinjection. (64)Cu-CB-TE1A1P-Y3-TATE, in spite of a relatively high kidney uptake, accumulated less in nontarget organs such as liver, lung, and bone. Small animal PET/CT imaging of (64)Cu-CB-TE1A1P-Y3-TATE in AR42J tumor bearing rats validated significant uptake and good contrast in the tumor. This study suggests that CB-TE1A1P is a promising bifunctional chelator for (64)Cu-labeled for Y3-TATE, owing to high binding affinity and target tissue uptake, the ability to radiolabel the agent at lower temperatures, and improved tumor/nontarget organ ratios over (64)Cu-CB-TE2A-Y3-TATE.     

Synthesis of a cross-bridged cyclam derivative for peptide conjugation and 64Cu radiolabeling

The increased use of copper radioisotopes in radiopharmaceutical applications has created a need for bifunctional chelators (BFCs) that form stable radiocopper complexes and allow covalent attachment to biological molecules. Previous studies have established that 4,11-bis-(carbo- tert-butoxymethyl)-1,4,8,11-tetraazabicyclo[6.6.2]hexadecane (H 2CB-TE2A), a member of the ethylene "cross-bridged" cyclam (CB-cyclam) class of bicyclic tetraaza macrocycles, forms highly kinetically stable complexes with Cu(II) and is less susceptible to in vivo transchelation than its nonbridged analogue, 1,4,8,11-tetraazacyclotetradecane-1,4,8,11-tetraacetic acid (TETA). Herein, we report a convenient synthesis of a novel cross-bridged BFC that is structurally analogous to CB-TE2A in that it possesses two coordinating acetate arms, but in addition possesses a third orthogonally protected arm for conjugation to peptides and other targeting agents. Application of this strategy to cross-bridged chelators may also enable the development of even further improved agents for (64)Cu-mediated diagnostic positron emission tomography (PET) imaging as well as for targeted radiotherapeutic applications.     

Design, evaluation, and comparison of ghrelin receptor agonists and inverse agonists as suitable radiotracers for PET imaging

Ghrelin agonist and inverse agonist radiotracers, suitable for positron emission tomography (PET), were developed to study the behavior of ghrelin receptor ligands in vivo and for further design of druggable peptides. The target peptides were synthesized on solid support and conjugated to the bifunctional chelator 1,4,7-triazacyclononane,1-glutaric acid-4,7-acetic acid (NODAGA), which is known to form a stable complex with Ga(3+). Complexation with (68)Ga could be achieved under mild conditions and led to radiotracers with high radiochemical purity and specific activity. The biological activity of the radiotracers was evaluated in vitro by an inositol phosphate turnover assay. Pharmacokinetic profile and metabolic stability of the (68)Ga-NODAGA-radiotracers were investigated by small animal PET in rodent. Ghrelin derived agonists presented very high kidney accumulation, negligible tissue distribution, fast blood clearance, and poor stability in blood. Contrarily, the inverse agonist radiotracer exhibited very high stability in blood, large diffusion in tissues, reasonable kidney and liver metabolism, and slow blood clearance. This pharmacokinetic profile makes the ghrelin inverse agonist motif KwFwLL-CONH(2) suitable for further development of radiotracers and a promising lead to design peptide-based therapeutics against obesity.     

De novo synthesis of a new diethylenetriaminepentaacetic acid (DTPA) bifunctional chelating agent

Diethylene triamine pentaacetic acid (DTPA) has been in extensive use as a metal chelator in the development of radiopharmaceuticals and contrast agents. The former application uses DTPA mostly as a bifunctional chelating agent (BCA) conjugated to tumor-targeting vehicles (TTVs) such as monoclonal antibodies (MAbs) and receptor-directed peptides. A new bifunctional DTPA derivative was synthesized by a fully organic scheme. This compound, N(4),N(alpha),N(alpha),N(epsilon),N(epsilon)-[pentakis(carboxymethyl)]-N(4)-(carboxymethyl)-2,6-diamino-4-azahexanoic hydrazide (20) was prepared by a convergent synthesis strategy using N(alpha)-benzyloxycarbonyl-2,3-diaminopropionic acid as the starting compound. This commercially available material was used to build a functionalized triamine which served as the molecular core template for assembling the target molecule. To evaluate the conjugation and radiolabeling capabilities of this new molecule, it was covalently attached to the anti-TAG-72 MAb, Delta CH2HuCC49, and the conjugate was radiolabeled in near-quantitative yields with yttrium-90 ((90)Y) and lutetium-177 ((177)Lu). Biodistribution of the (177)Lu-labeled DTPA-Delta CH2HuCC49 in tumor-bearing nude mice demonstrated preservation of the immunoreactivity of the MAb as indicated by high tumor uptake. In addition to the introduction of a new bifunctional DTPA, this work reports on a novel synthetic approach for preparation of this useful metal chelator and introduces a new conjugation protocol.     

Synthesis of multi-galactose-conjugated 2′-O-methyl oligoribonucleotides and their in vivo imaging with positron emission tomography

⁶⁸Ga labelled 2′-O-methyl oligoribonucleotides (anti-miR-15b) bearing one, three or seven d-galactopyranoside residues have been prepared and their distribution in healthy rats has been studied by positron emission tomography (PET). To obtain the heptavalent conjugate, an appropriately protected 1,4,7-triazacyclononane-1,4,7-triacetic acid (NOTA) precursor bearing a 4-[4-(4,4′-dimethoxytrityloxy)butoxy]phenyl side arm was first immobilized via a base labile linker to the support and the oligonucleotide was assembled on the detritylated hydroxyl function of this handle. A phosphoramidite building block bearing two phthaloyl protected aminooxy groups and one protected hydroxyl function was introduced into the 5′-terminus. One acetylated galactopyranoside was coupled as a phosphoramidite to the hydroxyl function, the phthaloyl protections were removed on-support and two trivalent galactopyranoside clusters were attached as aldehydes by on-support oximation. A two-step cleavage with aqueous alkali and ammonia released the conjugate in a fully deprotected form, allowing radiolabelling with ⁶⁸Ga in solution. The mono- and tri-galactose conjugates were obtained in a closely related manner. In vivo imaging in rats with PET showed remarkable galactose-dependent liver targeting of the conjugates.     

Influence of Macrocyclic Chelators on the Targeting Properties of 68Ga-Labeled Synthetic Affibody Molecules: Comparison with 111In-Labeled Counterparts

Affibody molecules are a class of small (7 kDa) non-immunoglobulin scaffold-based affinity proteins, which have demonstrated substantial potential as probes for radionuclide molecular imaging. The use of positron emission tomography (PET) would further increase the resolution and quantification accuracy of Affibody-based imaging. The rapid in vivo kinetics of Affibody molecules permit the use of the generator-produced radionuclide ⁶⁸Ga (T_1/2 = 67.6 min). Earlier studies have demonstrated that the chemical nature of chelators has a substantial influence on the biodistribution properties of Affibody molecules. To determine an optimal labeling approach, the macrocyclic chelators 1,4,7,10-tetraazacylododecane-1,4,7,10-tetraacetic acid (DOTA), 1,4,7-triazacyclononane-N,N,N-triacetic acid (NOTA) and 1-(1,3-carboxypropyl)-1,4,7- triazacyclononane-4,7-diacetic acid (NODAGA) were conjugated to the N-terminus of the synthetic Affibody molecule Z_HER2:S1 targeting HER2. Affibody molecules were labeled with ⁶⁸Ga, and their binding specificity and cellular processing were evaluated. The biodistribution of ⁶⁸Ga-DOTA-Z_HER2:S1, ⁶⁸Ga-NOTA-Z_HER2:S1 and ⁶⁸Ga-NODAGA-Z_HER2:S1, as well as that of their ¹¹¹In-labeled counterparts, was evaluated in BALB/C nu/nu mice bearing HER2-expressing SKOV3 xenografts. The tumor uptake for ⁶⁸Ga-DOTA-Z_HER2:S1 (17.9±0.7%IA/g) was significantly higher than for both ⁶⁸Ga-NODAGA-Z_HER2:S1 (16.13±0.67%IA/g) and ⁶⁸Ga-NOTA-Z_HER2:S1 (13±3%IA/g) at 2 h after injection. ⁶⁸Ga-NODAGA-Z_HER2:S1 had the highest tumor-to-blood ratio (60±10) in comparison with both ⁶⁸Ga-DOTA-Z_HER2:S1 (28±4) and ⁶⁸Ga-NOTA-Z_HER2:S1 (42±11). The tumor-to-liver ratio was also higher for ⁶⁸Ga-NODAGA-Z_HER2:S1 (7±2) than the DOTA and NOTA conjugates (5.5±0.6 vs.3.3±0.6). The influence of chelator on the biodistribution and targeting properties was less pronounced for ⁶⁸Ga than for ¹¹¹In. The results of this study demonstrate that macrocyclic chelators conjugated to the N-terminus have a substantial influence on the biodistribution of HER2-targeting Affibody molecules labeled with ⁶⁸Ga.This can be utilized to enhance the imaging contrast of PET imaging using Affibody molecules and improve the sensitivity of molecular imaging. The study demonstrated an appreciable difference of chelator influence for ⁶⁸Ga and ¹¹¹In.     

Synthesis of 64Cu-labeled magnetic nanoparticles for multimodal imaging

Complementary imaging modalities provide more information than either method alone can yield and we have developed a dual-mode imaging probe for combined magnetic resonance (MR) and positron emission tomography (PET) imaging. We have developed dual-mode PET/MRI active probes targeted to vascular inflammation and present synthesis of (1) an aliphatic amine polystyrene bead and (2) a novel superparamagnetic iron oxide nanoparticle targeted to macrophages that were both coupled to positron-emitting copper-64 isotopes. The amine groups of the polystyrene beads were directly conjugated with an amine-reactive form (isothiocyanate) of aza-macrocycle 1,4,7,10-tetraazacyclo-dodecane-1,4,7,10-tetraacetic acid (DOTA). Iron oxide nanoparticles are dextran sulfate coated, and the surface was modified to contain aldehyde groups to conjugate to an amine-activated DOTA. Incorporation of chelated Cu-64 to nanoparticles under these conditions, which is routinely used to couple DOTA to macromolecules, was unexpectedly difficult and illustrates that traditional conjugation methods do not always work in a nanoparticle environment. Therefore, we developed new methods to couple Cu-64 to nanoparticles and demonstrate successful labeling to a range of nanoparticle types. We obtained labeling yields of 24% for the amine polystyrene beads and 21% radiolabeling yield for the anionic dextran sulfate iron oxide nanoparticles. The new coupling chemistry can be generalized for attaching chelated metals to other nanoparticle platforms.     

Solid-Supported NOTA and DOTA Chelators Useful for the Synthesis of 3'-Radiometalated Oligonucleotides

Esterified precursors of 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid (DOTA; 18) and 1,4,7-triazacyclononane-1,4,7-trisacetic acid (NOTA; 17,19) ligands bearing a dimethoxytritylated hydroxyl side arm were prepared and immobilized via an ester linkage to long chain alkyl amine derivatized controlled pore glass (LCAA-CPG). Oligonucleotide chains were then assembled on the hydroxyl function and conjugates were released and deprotected by a two-step cleavage with aqueous alkali and ammonia. The 3'-DOTA and 3'-NOTA conjugated oligonucleotides were converted to (68)Ga chelates by a brief treatment with [(68)Ga]Cl(3) at elevated temperature. Applicability of the conjugates for in vivo imaging with positron emission tomography (PET) was verified.     

Development of a Novel PET Tracer [18F]AlF-NOTA-C6 Targeting MMP2 for Tumor Imaging

Background and Objective

The overexpression of gelatinases, that is, matrix metalloproteinase MMP2 and MMP9, has been associated with tumor progression, invasion, and metastasis. To image MMP2 in tumors, we developed a novel ligand termed [¹⁸F]AlF-NOTA-C6, with consideration that: c(KAHWGFTLD)NH₂ (herein, C6) is a selective gelatinase inhibitor; Cy5.5-C6 has been visualized in many in vivo tumor models; positron emission tomography (PET) has a higher detection sensitivity and a wider field of view than optical imaging; fluorine-18 (¹⁸F) is the optimal PET radioisotope, and the creation of a [¹⁸F]AlF-peptide complex is a simple procedure.

Methods

C6 was conjugated to the bifunctional chelator NOTA (1, 4, 7-triazacyclononanetriacetic acid) for radiolabeling [¹⁸F]AlF conjugation. The MMP2-binding characteristics and tumor-targeting efficacy of [¹⁸F]AlF-NOTA-C6 were tested in vitro and in vivo.

Results

The non-decay corrected yield of [¹⁸F]AlF-NOTA-C6 was 46.2–64.2%, and the radiochemical purity exceeded 95%. [¹⁸F]AlF-NOTA-C6 was favorably retained in SKOV3 and PC3 cells, determined by cell uptake. Using NOTA-C6 as a competitive ligand, the uptake of [¹⁸F]AlF-NOTA-C6 in SKOV3 cells decreased in a dose-dependent manner. In biodistribution and PET imaging studies, higher radioactivity concentrations were observed in tumors. Pre-injection of C6 caused a marked reduction in tumor tissue uptake. Immunohistochemistry showed MMP2 in tumor tissues.

Conclusions

[¹⁸F]AlF-NOTA-C6 was easy to synthesize and has substantial potential as an imaging agent that targets MMP2 in tumors.