High-yielding aqueous 18F-labeling of peptides via Al18F chelation

The coordination chemistry of a new pentadentate bifunctional chelator (BFC), NODA-MPAA 1, containing the 1,4,7-triazacyclononane-1,4-diacetate (NODA) motif with a methylphenylacetic acid (MPAA) backbone, and its ability to form stable Al(18)F chelates were investigated. The organofluoroaluminates were easily accessible from the reaction of 1 and AlF(3). X-ray diffraction studies revealed aluminum at the center of a slightly distorted octahedron, with fluorine occupying one of the axial positions. The tert-butyl protected prochelator 7, which can be synthesized in one step, is useful for coupling to biomolecules on solid phase or in solution. High yield (55-89%) aqueous (18)F-labeling was achieved in 10-15 min with a tumor-targeting peptide 4 covalently linked to 1. Defluorination was not observed for at least 4 h in human serum at 37 °C. These results demonstrate the facile application of Al(18)F chelation using BFC 1 as a versatile labeling method for radiofluorinating other heat-stable peptides for positron emission imaging.     

Preparation of 18F-labeled peptides using the copper(I)-catalyzed azide-alkyne 1,3-dipolar cycloaddition

An optimized procedure for preparing fluorine-18 ((18)F)-labeled peptides by the copper-catalyzed azide-alkyne 1,3-dipolar cyloaddition (CuAAC) is presented here. The two-step radiosynthesis begins with the microwave-assisted nucleophilic (18)F-fluorination of a precursor containing a terminal p-toluenesulfonyl, terminal azide and polyethylene glycol backbone. The resulting (18)F-fluorinated azide-containing building block is coupled to an alkyne-decorated peptide by the CuAAC. The reaction is accelerated by the copper(I)-stabilizing ligand bathophenanthroline disulfonate and can be performed in either reducing or nonreducing conditions (e.g., to preserve disulfide bonds). After an HPLC purification, (18)F-labeled peptide can be obtained with a 31 ± 6% radiochemical yield (n = 4, decay-corrected from (18)F-fluoride elution) and a specific activity of 39.0 ± 12.4 Ci μmol(-1) within 77 ± 4 min.     

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

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

In vivo and in vitro (27)Al NMR studies of aluminum(III) chelates of triazacyclononane polycarboxylate ligands.

The metallic radioisotope of a known radiopharmaceutical chelate, (67)Ga(NOTA) (NOTA=1,4,7-triazacyclonane-1,4,7-triacetic acid), used for tumor detection, was substituted by the chemically similar but non radioactive aluminum ion. Our aim was to detect and evaluate the in vivo behavior of the chelate. For this purpose, Al(NOTA) and the related chelate Al(NODASA) (NODASA=1,4,7-triazacyclononane-1-succinic acid-4,7-diacetic acid) were studied using in vitro and in vivo (27)Al NMR spectroscopy in rats. Both chelates showed high stability towards acid catalyzed dissociation and their (27)Al NMR resonances are characteristic of highly symmetrical species, with chemical shifts within the range for octahedral or pseudo-octahedral geometries. The thermodynamic stability constant of the novel chelate Al(NODASA) was estimated using (27)Al NMR. The value obtained suggested that the chelate does not undergo in vivo demetalation by transferrin. The in vivo spectroscopic studies and the analysis of blood and urine samples for Al(III) concentrations indicated that the chelates remain intact under physiological conditions and that they are mainly eliminated from the body through the kidneys.     

Contribution of the 37-kDa laminin receptor precursor in the anti-metastatic PSP94-derived peptide PCK3145 cell surface binding

PURPOSE: PCK3145 is an anti-metastatic synthetic peptide with promising therapeutic efficacy against hormone-refractory prostate cancer. The characterization of the PCK3145 peptide cell surface binding/internalization mechanisms and of the receptors involved remained to be explored. RESULTS: [(14)C]PCK3145 cell surface binding assays showed rapid and transient kinetic profile, that was inhibited by RGD peptides, laminin, hyaluronan, and type-I collagen. RGD peptides were however unable to inhibit PCK3145 intracellular uptake. Far-Western ligand binding studies enabled the identification of the 37-kDa laminin receptor precursor (37LRP) as a potential ligand for PCK3145. Overexpression of the recombinant 37LRP indeed led to an increase in PCK3145 binding but unexpectedly not to its uptake. CONCLUSIONS: Our data support the implication of laminin receptors in cell surface binding and in transducing PCK3145 anti-metastatic effects, and provide a rational for targeting cancers that express high levels of such laminin receptors.     

F-18 Labeling Protocol of Peptides Based on Chemically Orthogonal Strain-Promoted Cycloaddition under Physiologically Friendly Reaction Conditions

We introduce the high-throughput synthesis of various (18)F-labeled peptide tracers by a straightforward (18)F-labeling protocol based on a chemo-orthogonal strain-promoted alkyne azide cycloaddition (SPAAC) using aza-dibenzocyclootyne-substituted peptides as precursors with (18)F-azide synthon to develop peptide based positron emission tomography (PET) molecular imaging probes. The SPAAC reaction and subsequent chemo-orthogonal purification reaction with azide resin proceeded quickly and selectively under physiologically friendly reaction conditions (i.e., toxic chemical reagents-free, aqueous medium, room temperature, and pH ≈7), and provided four (18)F-labeled tumor targetable bioactive peptides such as cyclic Arg-Gly-Asp (cRGD) peptide, bombesin (BBN), c-Met binding peptide (cMBP), and apoptosis targeting peptide (ApoPep) in high radiochemical yields as direct injectable solutions without any HPLC purification and/or formulation processes. In vitro binding assay and in vivo PET molecular imaging study using the (18)F-labeled cRGD peptide also demonstrated a successful application of our (18)F-labeling protocol.     

Structural and in vivo studies of metal chelates of Ga(III) relevant to biomedical imaging

The solution chemistry and structure of the complex of the triazamacrocyclic ligand NOTP (1,4,7-triazacyclononane-1,4,7-tris(methylenephosphonate)) with Ga3+ in D2O have been investigated by 1H, 71Ga and 31P NMR spectroscopy. These NMR results show the presence of a 1:1 Ga(NOTP)3- complex, with a highly symmetrical, pseudo-octahedral geometry, possibly with a C3 axis. The 1H spectrum shows that the triazamacrocyclic chelate ring is very rigid, with all the ring protons non-equivalent. The complex is stable in aqueous solution in a wide pH range. Its high thermodynamic stability agrees well with previous results from biodistribution and gamma imaging studies in Wistar rats with 67Ga3+ chelates of triaza macrocyclic ligands, which showed that the neutral chelates 67Ga(NOTA) (where NOTA is 1,4,7-triazacyclononane-1,4,7-triacetate) and 67Ga(NOTPME) (where NOTPME is 1,4,7-triazacyclononane-1,4,7-tris(methylenephosphonate monoethylester)) have similar in vivo behaviour, with high stability and rapid renal excretion, but the high negatively charged 67Ga(NOTP)3- has a considerably slower kidney uptake and elimination.     

Efficient (18)F-Labeling of Large 37-Amino-Acid pHLIP Peptide Analogues and Their Biological Evaluation

Solid tumors often develop an acidic microenvironment, which plays a critical role in tumor progression and is associated with increased level of invasion and metastasis. The 37-residue pH (low) insertion peptide (pHLIP) is under study as an imaging platform because of its unique ability to insert into cell membranes at a low extracellular pH (pH(e) < 7). Labeling of peptides with [(18)F]-fluorine is usually performed via prosthetic groups using chemoselective coupling reactions. One of the most successful procedures involves the alkyne-azide copper(I) catalyzed cycloaddition (CuAAC). However, none of the known "click" methods have been applied to peptides as large as pHLIP. We designed a novel prosthetic group and extended the use of the CuAAC "click chemistry" for the simple and efficient (18)F-labeling of large peptides. For the evaluation of this labeling approach, a d-amino acid analogue of WT-pHLIP and an l-amino acid control peptide K-pHLIP, both functionalized at the N-terminus with 6-azidohexanoic acid, were used. The novel 6-[(18)F]fluoro-2-ethynylpyridine prosthetic group, was obtained via nucleophilic substitution on the corresponding bromo-precursor after 10 min at 130 °C with a radiochemical yield of 27.5 ± 6.6% (decay corrected) with high radiochemical purity ≥98%. The subsequent Cu(I)-catalyzed "click" reaction with the azido functionalized pHLIP peptides was quantitative within 5 min at 70 °C in a mixture of water and ethanol using Cu-acetate and sodium l-ascorbate. [(18)F]-d-WT-pHLIP and [(18)F]-l-K-pHLIP were obtained with total radiochemical yields of 5-20% after HPLC purification. The total reaction time was 85 min including formulation. In vitro stability tests revealed high stability of the [(18)F]-d-WT-pHLIP in human and mouse plasma after 120 min, with the parent tracer remaining intact at 65% and 85%, respectively. PET imaging and biodistribution studies in LNCaP and PC-3 xenografted mice with the [(18)F]-d-WT-pHLIP and the negative control [(18)F]-l-K-pHLIP revealed pH-dependent tumor retention. This reliable and efficient protocol promises to be useful for the (18)F-labeling of large peptides such as pHLIP and will accelerate the evaluation of numerous [(18)F]-pHLIP analogues as potential PET tracers.     

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.     

The Choice of HLA‐Associated Peptide Enrichment and Purification Strategy Affects Peptide Yields and Creates a Bias in Detected Sequence Repertoire

Understanding the most appropriate workflow for biochemical human leukocyte antigen (HLA)‐associated peptide enrichment prior to ligand sequencing is essential to achieve optimal sensitivity in immunopeptidomics experiments. The use of different detergents for HLA solubilization as well as complementary workflows to separate HLA‐bound peptides from HLA protein complex components after their immunoprecipitation including HPLC, C18 cartridge, and 5 kDa filter are described. It is observed that all solubilization approaches tested led to similar peptide ligand identification rates; however, a higher number of peptides are identified in samples lysed with CHAPS compared with other methods. The HPLC method is superior in terms of HLA‐I peptide recovery compared with 5 kDa filter and C18 cartridge peptide purification methods. Most importantly, it is observed that both the choice of detergent and peptide purification strategy creates a significant bias for the identified peptide sequences, and that allele‐specific peptide repertoires are affected depending on the workflow of choice. The results highlight the importance of employing a suitable strategy for HLA peptide enrichment and that the obtained peptide repertoires do not necessarily reflect the true distributions of peptide sequences in the sample.     

Uptake, transport and regulation of JBP485 by PEPT1 in vitro and in vivo

Cyclo-trans-4-l-hydroxyprolyl-l-serine (JBP485) is a dipeptide with anti-hepatitis activity that has been chemically synthesized. Previous experiments in rats showed that JBP485 was well absorbed by the intestine after oral administration. The human peptide transporter (PEPT1) is expressed in the intestine and recognizes compounds such as dipeptides and tripeptides. The purposes of this study were to determine if JBP485 acted as a substrate for intestinal PEPT1, and to investigate the characteristics of JBP485 uptake and transepithelial transport by PEPT1. The uptake of JBP485 was pH dependent in human intestinal epithelial cells Caco-2. And JBP485 uptake was also significantly inhibited by glycylsarcosine (Gly-Sar, a typical substrate for PEPT1 transporters), JBP923 (a derivative of JBP485), and cephalexin (CEX, a β-lactam antibiotic and a known substrate of PEPT1) in Caco-2 cells. The rate of apical-to-basolateral transepithelial transport of JBP485 was 1.84 times higher than that for basolateral-to-apical transport. JBP485 transport was obviously inhibited by Gly-Sar, JBP923 and CEX in Caco-2 cells. The uptake of JBP485 was increased by verapamil but not by cyclosporin A (CsA) and inhibited by the presence of Zn²⁺ or the toxic metabolite of ethanol, acetaldehyde (AcH) in Caco-2 cells. The in vivo uptake of JBP485 was increased by verapamil and decreased by ethanol in vivo, which was consisted with the in vitro study. PEPT1 mRNA levels were enhanced after exposure of the cells to JBP485 for 24 h, compared to control. In conclusion, JBP485 was actively transported by the intestinal oligopeptide transporter PEPT1. This mechanism is likely to contribute to the rapid absorption of JBP485 by the gastrointestinal tract after oral administration.     

Evaluation of a maleimido derivative of NOTA for site-specific labeling of affibody molecules

Radionuclide molecular imaging has the potential to improve cancer treatment by selection of patients for targeted therapy. Affibody molecules are a class of small (7 kDa) high-affinity targeting proteins with appreciable potential as molecular imaging probes. The NOTA chelator forms stable complexes with a number of radionuclides suitable for SPECT or PET imaging. A maleimidoethylmonoamide NOTA (MMA-NOTA) has been prepared for site-specific labeling of Affibody molecules having a unique C-terminal cysteine. Coupling of the MMA-NOTA to the anti-HER2 Affibody molecule Z(HER2:2395) resulted in a conjugate with an affinity (dissociation constant) to HER2 of 72 pM. Labeling of [MMA-NOTA-Cys(61)]-Z(HER2:2395) with (111)In gave a yield of >95% after 20 min at 60 °C. In vitro cell tests demonstrated specific binding of [(111)In-MMA-NOTA-Cys(61)]-Z(HER2:2395) to HER2-expressing cell lines. In mice bearing prostate cancer DU-145 xenografts, the tumor uptake of [(111)In-MMA-NOTA-Cys(61)]-Z(HER2:2395) was 8.2 ± 0.9% IA/g and the tumor-to-blood ratio was 31 ± 1 (4 h postinjection). DU-145 xenografts were clearly visualized by a gamma camera. Direct in vivo comparison of [(111)In-MMA-NOTA-Cys(61)]-Z(HER2:2395) and [(111)In-MMA-DOTA-Cys(61)]-Z(HER2:2395) demonstrated that both conjugates provided equal radioactivity uptake in tumors, but the tumor-to-organ ratios were better for [(111)In-MMA-NOTA-Cys(61)]-Z(HER2:2395) due to more efficient clearance from normal tissues. In conclusion, coupling of MMA-NOTA to a cysteine-containing Affibody molecule resulted in a site-specifically labeled conjugate, which retains high affinity, can be efficiently labeled, and allows for high-contrast imaging.     

Development of a bifunctional chelating agent containing isothiocyanate residue for one step F-18 labeling of peptides and application for RGD labeling

We report herein a novel isothiocyanate active ligand for fluorine-18 labeling prepared by four step synthesis. It can be conjugated to a target molecule containing an amino functional group under weak basic conditions by way of thiourea bond formation. We explored the application of synthesized ligand by conjugating to well known α_vβ₃ integrin targeting peptide, c(RGDyK). The conjugated peptide showed good radiochemical yield and efficiency with an excellent radiochemical purity (97.1 ± 1.2%) in a short reaction time (10 min). Labeled peptide showed excellent in vitro and in vivo stability (>95%). α_vβ₃ integrin specific tumor uptake was observed both in biodistribution and small animal microPET studies on α_vβ₃-positive U87MG (human glioma cells) xenograft bearing mice. In general, successful application of synthesized ligand for labeling of RGD peptide could facilitate the possibility of using this ligand for labeling peptides containing an amino functional group.     

Distinct uptake mechanisms but similar intracellular processing of two different toll-like receptor ligand-peptide conjugates in dendritic cells

Covalent conjugation of Toll-like receptor ligands (TLR-L) to synthetic antigenic peptides strongly improves antigen presentation in vitro and T lymphocyte priming in vivo. These molecularly well defined TLR-L-peptide conjugates, constitute an attractive vaccination modality, sharing the peptide antigen and a defined adjuvant in one single molecule. We have analyzed the intracellular trafficking and processing of two TLR-L conjugates in dendritic cells (DCs). Long synthetic peptides containing an ovalbumin cytotoxic T-cell epitope were chemically conjugated to two different TLR-Ls the TLR2 ligand, Pam(3)CysSK(4) (Pam) or the TLR9 ligand CpG. Rapid and enhanced uptake of both types of TLR-L-conjugated peptide occurred in DCs. Moreover, TLR-L conjugation greatly enhanced antigen presentation, a process that was dependent on endosomal acidification, proteasomal cleavage, and TAP translocation. The uptake of the CpG approximately conjugate was independent of endosomally-expressed TLR9 as reported previously. Unexpectedly, we found that Pam approximately conjugated peptides were likewise internalized independently of the expression of cell surface-expressed TLR2. Further characterization of the uptake mechanisms revealed that TLR2-L employed a different uptake route than TLR9-L. Inhibition of clathrin- or caveolin-dependent endocytosis greatly reduced uptake and antigen presentation of the Pam-conjugate. In contrast, internalization and antigen presentation of CpG approximately conjugates was independent of clathrin-coated pits but partly dependent on caveolae formation. Importantly, in contrast to the TLR-independent uptake of the conjugates, TLR expression and downstream TLR signaling was required for dendritic cell maturation and for priming of na?ve CD8(+) T-cells. Together, our data show that targeting to two distinct TLRs requires distinct uptake mechanism but follows similar trafficking and intracellular processing pathways leading to optimal antigen presentation and T-cell priming.     

Structure-activity relationship study of the cell-penetrating peptide pVEC

The peptide pVEC is a recently described cell-penetrating peptide, derived from the murine vascular endothelial-cadherin protein. In order to define which part of this 18-amino acid long peptide is important for the cellular translocation, we performed a structure-activity relationship study of pVEC. Together with the l-alanine substituted peptides, the retro-pVEC, D-pVEC and the scramble pVEC are studied for comparison. The peptide analogues are labeled with carboxyfluorescein at the N-terminus for monitoring the cellular uptake into human Bowes melanoma cells with different efficacy. We show that all the Fl-pVEC analogues internalize in live Bowes melanoma cells. l-Alanine substitution of the five respective N-terminal hydrophobic amino acids significantly decreases the translocation property, while replacing of Arg(6), Arg(8) or Ser(17) by alanine enhances the uptake. The uptake of pVEC is significantly reduced by treatment with an endocytosis inhibitor wortmannin. Treatment with heparinase III, nystatin and EIPA had no effect on the peptide uptake. The data presented here show that the N-terminal hydrophobic part of pVEC is crucial for efficient cellular translocation.     

Construction, identification and application of HeLa cells stably transfected with human PEPT1 and PEPT2

The purpose of this study was to construct stably transfected HeLa cells with human peptide transporters (hPEPT1/hPEPT2) and to identify the function of the transfected cells using the substrate JBP485 (a dipeptide) and a typical substrate for PEPTs, glycylsarcosine (Gly-Sar). An efficient and rapid method was established for the preparation and transformation of competent cells of Escherichia coli. After extraction and purification, hPEPT1/hPEPT2-pcDNA3 was transfected into HeLa cells by the liposome transfection method, respectively. HeLa-hPEPT1/hPEPT2 cells were selected by measuring the protein expression and the uptake activities of JBP485 and Gly-Sar. A simple and rapid chromatography-tandem mass spectrometry (LC-MS/MS) method was developed for the simultaneous determination of JBP485 and Gly-Sar in biological samples. The Michaelis-Menten constant (K(m)) values of Gly-Sar uptake by the hPEPT1 and hPEPT2-expressing transfectants were 1.03 mM and 0.0965 mM, respectively, and the K(m) values of JBP485 uptake were 1.33 mM for PEPT1 and 0.144 mM for PEPT2. The uptake of Gly-Sar was significantly inhibited by JBP485 with a K(i) value of 8.11 mM (for PEPT1) and 1.05 mM (for PEPT2). Maximal uptake of Gly-Sar were detected at pH 5.8 (for PEPT1) and pH 6.5 (for PEPT2), suggesting that both HeLa-hPEPT1 and HeLa-hPEPT2 were H(+) dependent transporters. Stably transfected HeLa-hPEPT1/HeLa-hPEPT2 cells were constructed successfully, and the functions of hPEPT1/hPEPT2 were identified using their substrates, JBP485 and Gly-Sar. The transfected cells with transporters were used to investigate drug-drug interactions (DDIs) between JBP485 and other substrates (cephalexin or lisinopril) of PEPT1 and PEPT2.     

Enhanced targeting specificity to tumor cells by simultaneous recognition of two antigens

Radioimmunotherapy recently afforded convincing results for B-cell non-Hodgkin's lymphoma treatment with antibody specific for B-cell differentiation antigens. High doses of unlabeled or labeled antibodies are necessary to saturate specific sites on normal B-cells. We thus developed a new targeting strategy, taking advantage of dual binding cooperativity, to enhance the specificity of the radioactive uptake by tumor cells. This approach was evaluated using human Burkitt lymphoma cells (Ramos) which express both CD10 and CD20 antigens. Most normal cells express at most one of these two differentiation antigens but many hematological tumors, including most human B type acute lymphoblastic leukemia cells, express both. Cells pretargeted with two bispecific antibodies, one recognizing CD10 and a histamine derivative (HSG), the other recognizing CD20 and the DTPA-indium complex, bind cooperatively radiolabeled mixed-haptens (DTPA-HSG). Increased binding (about 5-fold compared to binding to only one of CD10 or CD20 antigens) is observed at 37 degrees C, demonstrating the feasibility of the technique. This binding enhancement is a slow process, not observed at 4 degrees C. Such a binding enhancement will increase specificity for targeting isotopes to double antigen positive tumor cells compared to nontumor tissue cells bearing only one of them. This approach might be used to increase tumor irradiation with minimal irradiation of normal cells.     

Rapid and simple one-step F-18 labeling of peptides

Labeling biomolecules with 1?F is usually done through coupling with prosthetic groups, which requires several time-consuming radiosynthesis steps and therefore in low labeling yield. In this study, we designed a simple one-step 1?F-labeling strategy to replace the conventional complex and the long process of multiple-step radiolabeling procedure. Both monomeric and dimeric cyclic RGD peptides were modified to contain 4-NO?-3-CF? arene as precursors for direct 1?F labeling. Binding of the two functionalized peptides to integrin α(v)β? was tested in vitro using the MDA-MB-435 human breast cell line. The most promising functionalized peptide, the dimeric cyclic RGD, was further evaluated in vivo in an orthotopic MDA-MB-435 tumor xenograft model. The use of relatively low amount of precursor (~0.5 μmol) gave reasonable yield, ranging from 7 to 23% (decay corrected, calculated from the start of synthesis) after HPLC purification. Overall reaction time was 40 min, and the specific activity of the labeled peptide was high. Modification of RGD peptides did not significantly change the biological binding affinities of the modified peptides. Small animal PET and biodistribution studies revealed integrin specific tumor uptake and favorable biokinetics. We have developed a novel one-step 1?F radiolabeling strategy for peptides that contain a specific arene group, which shortens reaction time and labor significantly, requires low amount of precursor, and results in specific activity of 79 ± 13 GBq/μmol. Successful introduction of 4-fluoro-3-trifluoromethylbenzamide into RGD peptides may be a general strategy applicable to other biologically active peptides and proteins.     

99mTc-bioorthogonal click chemistry reagent for in vivo pretargeted imaging

Metal-free click chemistry has become an important tool for pretargeted approaches in the molecular imaging field. The application of bioorthogonal click chemistry between a pretargeted trans-cyclooctene (TCO) derivatized monoclonal antibody (mAb) and a ^99mTc-modified 1,2,4,5-tetrazine for tumor imaging was examined in vitro and in vivo. The HYNIC tetrazine compound was synthesized and structurally characterized, confirming its identity. Radiolabeling studies demonstrated that the HYNIC tetrazine was labeled with ^99mTc at an efficiency of >95% and was radiochemically stable. ^99mTc–HYNIC tetrazine reacted with the TCO–CC49 mAb in vitro demonstrating its selective reactivity. In vivo biodistribution studies revealed non-specific liver and GI uptake due to the hydrophobic property of the compound, however pretargeted SPECT imaging studies demonstrated tumor visualization confirming the success of the cycloaddition reaction in vivo. These results demonstrated the potential of ^99mTc–HYNIC–tetrazine for tumor imaging with pretargeted mAbs.     

New F-18 prosthetic group via oxime coupling

A novel fluorine-18 prosthetic ligand, 5-(1,3-dioxolan-2-yl)-2-(2-(2-(2-fluoroethoxy)ethoxy)ethoxy)pyridine [(18)F]2, has been synthesized. The prosthetic ligand is formed in high radiochemical yield (rcy = 71 ± 2%, n = 3) with excellent radiochemical purity (rcp = 99 ± 1%, n = 3) in a short reaction time (10 min). [(18)F]2 is a small, neutral, organic complex, easily synthesized in four steps from a readily available starting material. It can be anchored onto a target molecule containing an aminooxy functional group under acidic conditions by way of an oxime bond. We report herein two examples [(18)F]23 and [(18)F]24, potential imaging agents for β-amyloid plaques, which were labeled with this prosthetic group. This approach could be used for labeling proteins and peptides containing an aminooxy group. Biodistribution in male ICR mice for both oxime labeled complexes [(18)F]23 and [(18)F]24 were compared to that of the known β-amyloid plaque indicator, [(18)F]-AV-45, florbetapir 1. Oximes [(18)F]23 and [(18)F]24 are larger in size and therefore should reduce the blood-brain barrier (BBB) penetration. The brain uptake for oxime [(18)F]23 appeared to be reduced, but still retained some capability to cross the BBB. Oxime [(18)F]24 showed promising results after 2 min post injection (0.48% dose/gram); however, the uptake increased after 30 min post injection (0.92% dose/gram) suggesting an in vivo decomposition/metabolism of compound [(18)F]24. We have demonstrated a general protocol for the fluoride-18 labeling with a new prosthetic ligand [(18)F]2 that is tolerant toward several functional groups and is formed via chemoselective oxime coupling.