Investigations into the Signaling Pathways Involving Glucose-Stimulated Zinc Secretion (GSZS) from Prostate Epithelial Cells In Vitro and In Vivo.

PURPOSE: Recently, we reported that exposure of prostate cells in vitro or the in vivo prostate to high glucose results in release of Zn2+ ions, a process now referred to as glucose-stimulated zinc secretion (GSZS). To our knowledge, the metabolic event(s) that trigger GSZS remain largely unknown. Here, we explore several signaling pathways both in vitro using a prostate epithelial cell line and in vivo from the rat prostate. METHODS: PNT1A cells grown to confluence were washed and tagged with ZIMIR to monitor zinc secretion by optical methods. The expression levels of GLUT1, GLUT4, and Akt in cells cultured in either zinc-rich or zinc-poor media and after exposure to high versus low glucose were determined. Zinc secretion from the rat prostate in vivo as detected by MRI was compared in control animals after injection of glucose, deoxyglucose, or pyruvate to initiate zinc secretion and in animals pre-treated with WZB-117 (a GLUT1 inhibitor) or S961 (a peripheral insulin receptor inhibitor). RESULTS: PNT1A cells exposed to high levels of glucose secrete zinc whereas cells exposed to an equivalent amount of deoxyglucose or pyruvate do not. Expression of Akt was dramatically altered by zinc supplementation of the culture media but not after exposure to glucose while GLUT1 and GLUT4 levels were less affected. Rats pre-treated with WZB-117 prior to imaging showed a reduction in GSZS from the prostate compared to controls whereas rats pre-treated with S961 showed no difference. Interestingly, in comparison to PNT1A cells, pyruvate and deoxyglucose also stimulate zinc secretion in vivo likely through indirect mechanisms. CONCLUSIONS: GSZS requires metabolism of glucose both in vitro (PNT1A cells) and in vivo (rat prostate). Pyruvate also stimulates zinc secretion in vivo but likely via an indirect pathway involving rapid production of glucose via gluconeogenesis. These combined results support the conclusion that glycolytic flux is required to trigger GSZS in vivo.

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

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

The Roles of ZnT1 and ZnT4 in Glucose-Stimulated Zinc Secretion in Prostate Epithelial Cells.

PURPOSE: We have previously demonstrated by MRI that high glucose stimulates efflux of zinc ions from the prostate. To our knowledge, this phenomena had not been reported previously and the mechanism remains unknown. Here, we report some initial observations that provide new insights into zinc processing during glucose-stimulated zinc secretion (GSZS) in the immortalized human prostate epithelial cell line, PNT1A. Additionally, we identified the subtypes of zinc-containing cells in human benign prostatic hyperplasia (BPH) tissue to further identify which cell types are likely responsible for zinc release in vivo. PROCEDURE: An intracellular fluorescence marker, FluoZin-1-AM, was used to assess the different roles of ZnT1 and ZnT4 in zinc homeostasis in wild type (WT) and mRNA knockdown PNT1A cell lines. Additionally, Bafilomycin A1 (Baf) was used to disrupt lysosomes and assess the role of lysosomal storage during GSZS. ZIMIR, an extracellular zinc-responsive fluorescent marker, was used to assess dynamic zinc efflux of WT and ZnT1 mRNA knockdown cells exposed to high glucose. Electron microscopy was used to assess intracellular zinc storage in response to high glucose and evaluate how Bafilomycin A1 affects zinc trafficking. BPH cells were harvested from transurtheral prostatectomy tissue and stained with fluorescent zinc granule indicator (ZIGIR), an intracellular zinc-responsive fluorescent marker, before being sorted for cell types using flow cytometry. RESULTS: Fluorescent studies demonstrate that ZnT1 is the major zinc efflux transporter in prostate epithelial cells and that loss of ZnT1 via mRNA knockdown combined with lysosomal storage disruption results in a nearly 4-fold increase in cytosolic zinc. Knockdown of ZnT1 dramatically reduces zinc efflux during GSZS. Electron microscopy (EM) reveals that glucose stimulation significantly increases lysosomal storage of zinc; disruption of lysosomes via Baf or ZnT4 mRNA knockdown increases multi-vesicular body (MVB) formation and cytosolic zinc levels. In human BPH tissue, only the luminal epithelial cells contained significant amounts of zinc storage granules. CONCLUSIONS: Exposure of prostate epithelial cells to high glucose alters zinc homeostasis by inducing efflux of zinc ions via ZnT1 channels and increasing lysosomal storage via ZnT4. Given that prostate cancer cells undergo profound metabolic changes that result in reduced levels of total zinc, understanding the complex interplay between glucose exposure and zinc homeostasis in the prostate may provide new insights into the development of prostate carcinogenesis.

Grafting Islets to a Dissected Peritoneal Pouch to Improve Transplant Survival and Function.

BACKGROUND: Although the liver is the primary site for clinical islet transplantation, it poses several restrictions, especially limited tissue volume due to portal vein pressure. We evaluated the preperitoneal space as an extrahepatic islet transplant site to deliver high tissue volumes and sustain long-term graft function. METHODS: A peritoneal pouch was formed by dissecting the parietal peritoneum from the transversalis fascia of mice. Syngeneic C57BL/6 donor islets were transplanted into the peritoneal pouch of diabetic mouse recipients. Blood glucose was monitored for islet function, and miR-375 was analyzed for islet damage. Islet graft morphology and vascularization were evaluated by immunohistochemistry. [F] fluoro-D-glucose positron emission tomography/computed tomography was used to image islet grafts. RESULTS: Transplantation of 300 syngeneic islets into the peritoneal pouch of recipients reversed hyperglycemia for >60 days. Serum miR-375 was significantly lower in the peritoneal pouch group than in the peritoneal cavity group. Peritoneal pouch islet grafts showed high neovascularization and sustained insulin and glucagon expression up to 80 days posttransplantation. A peritoneal pouch graft with high tissue volume (1000 islets) could be visualized by positron emission tomography/computed tomography imaging. Human islets transplanted into the peritoneal pouch of diabetic nude mice also reversed hyperglycemia successfully. CONCLUSIONS: Islets transplanted into a dissected peritoneal pouch show high efficiency to reverse diabetes and sustain islet graft function. The preperitoneal site has the advantages of capacity for high tissue volume, enriched revascularization and minimal inflammatory damage. It can also serve as an extrahepatic site for transplanting large volume of islets necessitated in islet autotransplantation.

Imaging Insulin Secretion from Mouse Pancreas by MRI Is Improved by Use of a Zinc-Responsive MRI Sensor with Lower Affinity for Zn2+ Ions.

It has been demonstrated that divalent zinc ions packaged with insulin in ß-cell granules can be detected by MRI during glucose-stimulated insulin secretion using a gadolinium-based Zn2+-sensitive agent. This study was designed to evaluate whether a simpler agent design having single Zn2+-sensing moieties but with variable Zn2+ binding affinities might also detect insulin secretion from the pancreas. Using an implanted MR-compatible window designed to hold the pancreas in a fixed position for imaging, we now demonstrate that focally intense "hot spots" can be detected in the tail of the pancreas using these agents after administration of glucose to stimulate insulin secretion. Histological staining of the same tissue verified that the hot spots identified by imaging correspond to clusters of islets, perhaps reflecting first-responder islets that are most responsive to a sudden increase in glucose. A comparison of images obtained when using a high-affinity Zn2+ sensor versus a lower-affinity sensor showed that the lower-affinity sensors produced the best image contrast. An equilibrium model that considers all possible complexes formed between Zn2+, the GdL sensor, and HSA predicts that a GdL sensor with lower affinity for Zn2+ generates a lower background signal from endogenous Zn2+ prior to glucose-stimulated insulin secretion (GSIS) and that the weaker binding affinity agent is more responsive to a further increase in Zn2+ concentration near ß-cells after GSIS. These model predictions are consistent with the in vivo imaging observations.

Zinc as an Imaging Biomarker of Prostate Cancer.

Zinc has long been the focus of many biological investigations because of its essential role in biology including a catalytic role in many enzymes, a structural role in the many zinc finger proteins, and a physiological role in many secretory cell processes. Divalent zinc is known to be highly abundant in healthy prostate tissues and lower in prostate cancer (PCa). Given the need for newer diagnostic methods for detection of prostate cancer, zinc-responsive probes of various types have been considered as imaging tools for detecting tissue levels of zinc. Among them, recent zinc-responsive MRI probes show great promise for non-invasive detection of zinc ion secretion from the prostate and other tissues in vivo. In this review, we summarize the need for new diagnostic tools and demonstrate how responsive zinc probes and MRI could satisfy this unmet need.

Presentation of underglycosylated mucin 1 in pancreatic adenocarcinoma (PDAC) at early stages.

Underglycosylated mucin 1 antigen (uMUC1) is a proven biomarker of cancer progression relevant to many malignancies including pancreatic ductal adenocarcinoma (PDAC). However, while ample evidence exists of the expression of total MUC1, little is known about the abundance of the underglycolsylated form of the antigen and its significance in disease progression. Such knowledge is important because the underglycosylated form of MUC1 is intimately linked to metastatic potential. Here, we investigated the expression uMUC1 at various stages of PDAC including pancreatic intraepithelial neoplasia (PanIN). Immunohistochemical analysis was performed on human tissue microarrays (TMAs) containing PDAC and PanIN using monoclonal antibody specific to uMUC1. uMUC1 expression was analyzed by a traditional pathological scoring system and using automatic imaging analysis software. Our results demonstrated low uMUC1 abundance in PanIN lesions and a transient increase in antigen availability in stage I PDAC, followed by decreased expression in later stages of the disease. An additional finding was that there was intermediate expression of uMUC1 in adjacent normal tissues from PDAC irrespective of the stage. These studies suggest the intriguing possibility that a pro-metastatic uMUC1 expression signature may appear at early stages of PDAC, providing an additional clue about the aggressive nature of pancreatic cancer.

Zinc-sensitive MRI contrast agent detects differential release of Zn(II) ions from the healthy vs. malignant mouse prostate.

Many secretory tissues release Zn(II) ions along with other molecules in response to external stimuli. Here we demonstrate that secretion of Zn(II) ions from normal, healthy prostate tissue is stimulated by glucose in fasted mice and that release of Zn(II) can be monitored by MRI. An ∼50% increase in water proton signal enhancement is observed in T1-weighted images of the healthy mouse prostate after infusion of a Gd-based Zn(II) sensor and an i.p. bolus of glucose. Release of Zn(II) from intracellular stores was validated in human epithelial prostate cells in vitro and in surgically exposed prostate tissue in vivo using a Zn(II)-sensitive fluorescent probe known to bind to the extracellular surface of cells. Given the known differences in intracellular Zn(II) stores in healthy versus malignant prostate tissues, the Zn(II) sensor was then evaluated in a transgenic adenocarcinoma of the mouse prostate (TRAMP) model in vivo. The agent proved successful in detecting small malignant lesions as early as 11 wk of age, making this noninvasive MR imaging method potentially useful for identifying prostate cancer in situations where it may be difficult to detect using current multiparametric MRI protocols.

Antigen dynamics govern the induction of CD4(+) T cell tolerance during autoimmunity.

Antigen-specific T cell tolerance holds great promise for the treatment of autoimmune diseases. However, strategies to induce durable tolerance using high doses of soluble antigen have to date been unsuccessful, due to lack of efficacy and the risk of hypersensitivity. In the current study we have overcome these limitations by developing a platform for tolerance induction based on engineering the immunoglobulin Fc region to modulate the dynamic properties of low doses (1 µg/mouse; ∼50 µg/kg) of Fc-antigen fusions. Using this approach, we demonstrate that antigen persistence is a dominant factor governing the elicitation of tolerance in the model of multiple sclerosis (MS), experimental autoimmune encephalomyelitis (EAE), induced by immunizing B10.PL mice with the N-terminal epitope of myelin basic protein. Unexpectedly, our analyses reveal a stringent threshold of antigen persistence for both prophylactic and therapeutic treatments, although distinct mechanisms lead to tolerance in these two settings. Importantly, the delivery of tolerogenic Fc-antigen fusions during ongoing disease results in the downregulation of T-bet and CD40L combined with amplification of Foxp3(+) T cell numbers. The generation of effective, low dose tolerogens using Fc engineering has potential for the regulation of autoreactive T cells.

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

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

Derivatization of (±) dihydrotetrabenazine for copper-64 labeling towards long-lived radiotracers for PET imaging of the vesicular monoamine transporter 2.

Dihydrotetrabenazine (DTBZ) derivatized from (+) Tetrabenazine (TBZ) has been used for imaging the expression of VMAT2 when labeled with (11)C (t1/2=20.3 min) or (18)F (t1/2=110 min) in neurodegenerative diseases or pancreatic beta-cell. Because (11)C or (18)F radiolabels are only available in the proximity of a biomedical cyclotron facility, here we report our work of derivatizing (+) and (-) DTBZ using a (64)Cu-specific bifunctional chelator scaffold ((64)Cu: t1/2=12.7 h) for the preparation of long-lived VMAT2 targeted radiotracers, (64)Cu-CB-TE2A-(+)-DTBZ and (64)Cu-CB-TE2A-(-)-DTBZ. The specific VMAT2 binding affinity of (64)Cu-CB-TE2A-(+)-DTBZ measured using rat brain homogenate or porcine islets was not compromised by our chemical modifications while that of its (-) counterpart remained low as in (11)C or (18)F labeled (±) DTBZ.

Design, synthesis and biological assessment of a triazine dendrimer with approximately 16 Paclitaxel groups and 8 PEG groups.

The synthesis and characterization of a generation three triazine dendrimer that displays a phenolic group at the core for labeling, up to eight 5 kDa PEG chains for solubility, and 16 paclitaxel groups is described. Three different diamine linkers--dipiperidine trismethylene, piperazine, and aminomethylpiperidine--were used within the dendrimer. To generate the desired stoichiometric ratio of 8 PEG chains to 16 paclitaxel groups, a monochlorotriazine was prepared with two paclitaxel groups attached through their 2'-hydroxyls using a linker containing a labile disulfide. This monochlorotriazine was linked to a dichlorotriazine with aminomethylpiperidine. The resulting dichlorotriazine bearing two paclitaxel groups could be reacted with the eight amines of the dendrimer. NMR and MALDI-TOF confirm successful reaction. The eight monochlorotriazines of the resulting material are used as the site for PEGylation affording the desired 2:1 stoichiometry. The target and intermediates were amenable to characterization by (1)H and (13)C NMR, and mass spectrometry. Analysis revealed that 16 paclitaxel groups were installed along with 5-8 PEG chains. The final construct is 63% PEG, 22% paclitaxel, and 15% triazine dendrimer. Consistent with previous efforts and computational models, 5 kDa PEG groups were essential for making the target water-soluble. Molecular dynamics simulations showed a high degree of hydration of the core, and a radius of gyration of 2.8 ± 0.2 nm. The hydrodynamic radius of the target was found to be 15.8 nm by dynamic light scattering, an observation indicative of aggregation. Drug release studies performed in plasma showed slow and identical release in mouse and rat plasma (8%, respectively). SPECT/CT imaging was used to follow biodistribution and tumor uptake. Using a two component model, the elimination and distribution half-lives were 2.65 h and 38.2 h, respectively. Compared with previous constructs, this dendrimer persists in the vasculature longer (17.33 ± 0.88% ID/g at 48 h postinjection), and showed higher tumor uptake. Low levels of dendrimer were observed in lung, liver, and spleen (~6% ID/g). Tumor saturation studies of small prostate cancer tumors (PC3) suggest that saturation occurs at a dose between 23.2 mg/kg and 70.9 mg/kg.

In vitro and in vivo evaluation of lactoferrin-conjugated liposomes as a novel carrier to improve the brain delivery.

In this study, lactoferrin-conjugated PEGylated liposomes (PL), a potential drug carrier for brain delivery, was loaded with radioisotope complex, 99mTc labeled N,N-bis(2-mercaptoethyl)-N',N'-diethylethylenediamine (99mTc-BMEDA) for in vitro and in vivo evaluations. The hydrophilicity of liposomes was enhanced by PEGylation which was not an ideal brain delivery system for crossing the blood brain barrier (BBB). With the modification of a brain-targeting ligand, lactoferrin (Lf), the PEGylated liposome (PL) might become a potential brain delivery vehicle. In order to test the hypothesis in vitro and in vivo, 99mTc-BMEDA was loaded into the liposomes as a reporter with or without Lf-conjugation. The mouse brain endothelia cell line, bEnd.3 cells, was cultured to investigate the potential uptake of liposomes in vitro. The in vivo uptake by the mouse brain of the liposomes was detected by tissue biodistribution study. The results indicated that Lf-conjugated PEGylated liposome showed more than three times better uptake efficiency in vitro and two-fold higher of brain uptake in vivo than PEGlyated liposome. With the success of loading the potential Single Photon Emission Tomography (SPECT) imaging probe, 99mTc-BMEDA, Lf-PL might serve as a promising brain delivery system for loading diagnostics or therapeutics of various brain disorders.

Dendrimer nanoscaffolds for potential theranostics of prostate cancer with a focus on radiochemistry.

Dendrimers are a class of structurally defined macromolecules featured with a central core, a low-density interior formed by repetitive branching units, and a high-density exterior terminated with surface functional groups. In contrast to their polymeric counterparts, dendrimers are nanosized and symmetrically shaped, which can be reproducibly synthesized on a large scale with monodispersity. These unique features have made dendrimers of increasing interest for drug delivery and other biomedical applications as nanoscaffold systems. Intended to address the potential use of dendrimers for the development of theranostic agents, which combines therapeutics and diagnostics in a single entity for personalized medicine, this review focuses on the reported methodologies of using dendrimer nanoscaffolds for targeted imaging and therapy of prostate cancer. Of particular interest, relevant chemistry strategies are discussed due to their important roles in the design and synthesis of diagnostic and therapeutic dendrimer-based nanoconjugates and potential theranostic agents, targeted or nontargeted. Given the developing status of nanoscaffolded theranostics, major challenges and potential hurdles are discussed along with the examples representing current advances.

Antitumor activity and molecular dynamics simulations of paclitaxel-laden triazine dendrimers.

The antitumor activities of triazine dendrimers bearing paclitaxel, a well-known mitotic inhibitor, are evaluated in SCID mice bearing human prostate cancer xenografts. To increase the activity of a first generation prodrug 1 that contained twelve paclitaxel molecules tethered via an ester linkage, the new construct described here, prodrug 2, tethers paclitaxel with linkers containing both an ester and disulfide. While PEGylation is necessary for solubility, and may improve biocompatibility and increase plasma half-life, it increases the heterogeneity of the sample with an average of eight to nine PEG chains (2 kDa each) incorporated. The heterogeneous population of PEGylated materials was used without fractionation based on models obtained from molecular dynamics simulations. Three models were examined; hexaPEGylated, nonaPEGylated, and dodecaPEGylated constructs. Intravenous delivery of prodrug 2 was performed by single, double or triple dosing regimes with doses spaced by one week. The doses varied from 50 mg of paclitaxel/kg to 200 mg of paclitaxel/kg. Tumor growth arrest and regression was observed over the 10-week treatment period without mortality for mice treated with the 50 mg of paclitaxel/kg treated three times.

Biological assessment of triazine dendrimer: toxicological profiles, solution behavior, biodistribution, drug release and efficacy in a PEGylated, paclitaxel construct.

The physicochemical characteristics, in vitro properties, and in vivo toxicity and efficacy of a third generation triazine dendrimer bearing approximately nine 2 kDa polyethylene glycol chains and twelve ester linked paclitaxel groups are reported. The hydrodynamic diameter of the neutral construct varies slightly with aqueous solvent ranging from 15.6 to 19.4 nm. Mass spectrometry and light scattering suggest radically different molecular weights with the former approximately 40 kDa mass consistent with expectation, and the latter 400 kDa mass consistent with a decameric structure and the observed hydrodynamic radii. HPLC can be used to assess purity as well as paclitaxel release, which is insignificant in organic solvents or aqueous solutions at neutral and low pH. Paclitaxel release occurs in vitro in human, rat, and mouse plasma and is nonlinear, ranging from 7 to 20% cumulative release over a 48 h incubation period. The construct is 2-3 orders of magnitude less toxic than Taxol by weight in human hepatocarcinoma (Hep G2), porcine renal proximal tubule (LLC-PK1), and human colon carcinoma (LS174T) cells, but shows similar cytotoxicity to Abraxane in LS174T cells. Both Taxol and the construct appear to induce caspase 3-dependent apoptosis. The construct shows a low level of endotoxin, is not hemolytic and does not induce platelet aggregation in vitro, but does appear to reduce collagen-induced platelet aggregation in vitro. Furthermore, the dendrimer formulation slightly activates the complement system in vitro due most likely to the presence of trace amounts (<1%) of free paclitaxel. An animal study provided insight into the maximum tolerated dose (MTD) wherein 10, 25, 50, and 100 mg of paclitaxel/kg of construct or Abraxane were administered once per week for three consecutive weeks to non tumor bearing athymic nude mice. The construct showed in vivo toxicity comparable to that of Abraxane. Both formulations were found to be nontoxic at the administered doses, and the dendrimer had an acute MTD greater than the highest dose administered. In a prostate tumor model (PC-3-h-luc), efficacy was observed over 70 days with an arrest of tumor growth and lack of luciferase activity observed in the twice treated cohort.

Design, synthesis, characterization, and biological evaluation of triazine dendrimers bearing paclitaxel using ester and ester/disulfide linkages.

The design, synthesis, characterization, and preliminary biological assessment of three dendrimers are reported. All three dendrimers, 1-3, present twelve paclitaxel groups linked by acylation of the 2'-hydroxyl group. The linker for dendrimers 2 and 3 also includes a disulfide. Installation of the paclitaxel group relies on reacting twelve primary amines of a second generation triazine dendrimer, a scaffold available on kilogram scale, with a dichlorotriazine bearing the drug. This dichlorotriazine is available in four steps by (i) reacting paclitaxel with glutaric anhydride, (ii) activating with N-hydroxysuccinimide (NHS), (iii) treating the resulting ester with either 1,3-diaminopropane (for 1) or cystamine (for 2 and 3), and (iv), finally, reacting with cyanuric chloride. After reaction with the dendrimer, the resulting monochlorotriazine groups are reacted with 4-aminomethylpiperidine (AMP) and then a poly(ethylene glycol) (PEG) group of molecular weight 2 kDa. Two different PEG-NHS esters are employed that differ in lability. For 1 and 2, the PEG incorporates an ester-linked succinic acid group. For 3, the PEG incorporates an ether-linked acetic acid group. Both mass spectrometry and 1H NMR spectroscopy prove valuable for determining the final ratios of dendrimer:paclitaxel:AMP:PEG. These values are typically 1:12:12:9. Cytotoxicity of these constructs using an MTT-based assay and PC-3 cells reveals IC(50) values in the low nanomolar range with dithiothreitol and glutathione enhancing the toxicity of the disulfide-containing constructs 2 and 3. Preliminary toxicology assessment of 1 suggests that it is well tolerated in vivo with preferential renal clearance. The elimination half-lives of all of the dendrimers appear shorter than predicted from the previous results. Tumor localization is observed for all the three dendrimers.

Targeting the neonatal fc receptor for antigen delivery using engineered fc fragments.

The development of approaches for Ag delivery to the appropriate subcellular compartments of APCs and the optimization of Ag persistence are both of central relevance for the induction of protective immunity or tolerance. The expression of the neonatal Fc receptor, FcRn, in APCs and its localization to the endosomal system suggest that it might serve as a target for Ag delivery using engineered Fc fragment-epitope fusions. The impact of FcRn binding characteristics of an Fc fragment on in vivo persistence allows this property to also be modulated. We have therefore generated recombinant Fc (mouse IgG1-derived) fusions containing the N-terminal epitope of myelin basic protein that is associated with experimental autoimmune encephalomyelitis in H-2(u) mice. The Fc fragments have distinct binding properties for FcRn that result in differences in intracellular trafficking and in vivo half-lives, allowing the impact of these characteristics on CD4(+) T cell responses to be evaluated. To dissect the relative roles of FcRn and the "classical" FcgammaRs in Ag delivery, analogous aglycosylated Fc-MBP fusions have been generated. We show that engineered Fc fragments with increased affinities for FcRn at pH 6.0-7.4 are more effective in delivering Ag to FcRn-expressing APCs in vitro relative to their lower affinity counterparts. However, higher affinity of the FcRn-Fc interaction at near neutral pH results in decreased in vivo persistence. The trade-off between improved FcRn targeting efficiency and lower half-life becomes apparent during analyses of T cell proliferative responses in mice, particularly when Fc-MBP fusions with both FcRn and FcgammaR binding activity are used.

Direct 99m Tc labeling of Herceptin (trastuzumab) by 99m Tc(I) tricarbonyl ion.

By simply incubating Herceptin (trastuzumab) with [99m Tc(CO)3(OH2)3]+ ion in saline, a significant yield of 99m Tc-labeled trastuzumab was found to be achievable. The effective labeling may be based on that trastuzumab is inherent with endogenous histidine group to which 99m Tc(I) tricarbonyl ion can be strongly bound. For practical 99m Tc labeling processing, trastuzumab was purified beforehand from the commercial product, Herceptin (Genentech) via size exclusion chromatography to remove the excipient, alpha-histidine and a high-labeled yield could be obtained by incubating the purified trastuzumab with [99m Tc(CO)3(OH2)3]+. Retention of bioactivity of the 99m Tc(I)-labeled trastuzumab was validated using a cell binding test.