Aptamer-Aptamer Chimera for Targeted Delivery and ATP-Responsive Release of Doxorubicin into Cancer Cells.

Aptamers offer a great opportunity to develop innovative drug delivery systems that can deliver cargos specifically into targeted cells. In this study, a chimera consisting of two aptamers was developed to deliver doxorubicin into cancer cells and release the drug in cytoplasm in response to adenosine-5'-triphosphate (ATP) binding. The chimera was composed of the AS1411 anti-nucleolin aptamer for cancer cell targeting and the ATP aptamer for loading and triggering the release of doxorubicin in cells. The chimera was first produced by hybridizing the ATP aptamer with its complementary DNA sequence, which is linked with the AS1411 aptamer via a poly-thymine linker. Doxorubicin was then loaded inside the hybridized DNA region of the chimera. Our results show that the AS1411-ATP aptamer chimera was able to release loaded doxorubicin in cells in response to ATP. In addition, selective uptake of the chimera into cancer cells was demonstrated using flow cytometry. Furthermore, confocal laser scanning microscopy showed the successful delivery of the doxorubicin loaded in chimeras to the nuclei of targeted cells. Moreover, the doxorubicin-loaded chimeras effectively inhibited the growth of cancer cell lines and reduced the cytotoxic effect on the normal cells. Overall, the results of this study show that the AS1411-ATP aptamer chimera could be used as an innovative approach for the selective delivery of doxorubicin to cancer cells, which may improve the therapeutic potency and decrease the off-target cytotoxicity of doxorubicin.

4'-C-Trifluoromethyl modified oligodeoxynucleotides: synthesis, biochemical studies, and cellular uptake properties.

Herein, we report the synthesis of 4'-C-trifluoromethyl (4'-CF3) thymidine (T4'-CF3) and its incorporation into oligodeoxynucleotides (ODNs) through solid-supported DNA synthesis. The 4'-CF3 modification leads to a marginal effect on the deoxyribose conformation and a local helical structure perturbation for ODN/RNA duplexes. This type of modification slightly decreases the thermal stability of ODN/RNA duplexes (-1 °C/modification) and leads to improved nuclease resistance. Like the well-known phosphorothioate (PS) modification, heavy 4'-CF3 modifications enable direct cellular uptake of the modified ODNs without any delivery reagents. This work highlights that 4'-CF3 modified ODNs are promising candidates for antisense-based therapeutics, which will, in turn, inspire us to develop more potent modifications for antisense ODNs and siRNAs.

Inhibition of cerebral vascular inflammation by brain endothelium-targeted oligodeoxynucleotide complex.

The present study generated a novel DNA complex to specifically target endothelial NF-κB to inhibit cerebral vascular inflammation. This DNA complex (GS24-NFκB) contains a DNA decoy which inhibits NF-κB activity, and a DNA aptamer (GS-24), a ligand of transferrin receptor (TfR), which allows for targeted delivery of the DNA decoy into cells. The results indicate that GS24-NFκB was successfully delivered into a murine brain-derived endothelial cell line, bEND5, and inhibited inflammatory responses induced by tumor necrosis factor α (TNF-α) or oxygen-glucose deprivation/re-oxygenation (OGD/R) via down-regulation of the nuclear NF-κB subunit, p65, as well as its downstream inflammatory cytokines, inter-cellular adhesion molecule-1 (ICAM-1) and vascular cell adhesion molecule (VCAM-1). The inhibitory effect of the GS24-NFκB was demonstrated by a significant reduction in TNF-α or OGD/R induced monocyte adhesion to the bEND5 cells after GS24-NFκB treatment. Intravenous (i.v.) injection of GS24-'NFκB (15mg/kg) was able to inhibit the levels of phoseph-p65 and VCAM-1 in brain endothelial cells in a mouse lipopolysaccharide (LPS)-induced inflammatory model in vivo. In conclusion, our approach using DNA nanotechnology for DNA decoy delivery could potentially be utilized for inhibition of inflammation in ischemic stroke and other neuro-inflammatory diseases affecting cerebral vasculature.

Refilling drug delivery depots through the blood.

Local drug delivery depots have significant clinical utility, but there is currently no noninvasive technique to refill these systems once their payload is exhausted. Inspired by the ability of nanotherapeutics to target specific tissues, we hypothesized that blood-borne drug payloads could be modified to home to and refill hydrogel drug delivery systems. To address this possibility, hydrogels were modified with oligodeoxynucleotides (ODNs) that provide a target for drug payloads in the form of free alginate strands carrying complementary ODNs. Coupling ODNs to alginate strands led to specific binding to complementary-ODN-carrying alginate gels in vitro and to injected gels in vivo. When coupled to a drug payload, sequence-targeted refilling of a delivery depot consisting of intratumor hydrogels completely abrogated tumor growth. These results suggest a new paradigm for nanotherapeutic drug delivery, and this concept is expected to have applications in refilling drug depots in cancer therapy, wound healing, and drug-eluting vascular grafts and stents.

Quantification of oligonucleotides by LC-MS/MS: the challenges of quantifying a phosphorothioate oligonucleotide and multiple metabolites.

BACKGROUND: LC-MS/MS allows quantification of therapeutic oligonucleotides in biological fluids at low ng/ml concentrations. Achieving selectivity between metabolites and parent molecules in a single assay is one of the biggest challenges when developing a method. We present a strategy that allows quantification of an 18-mer antisense therapeutic, trabedersen, and six metabolites in human plasma. RESULTS/METHODOLOGY: The method utilizes phenol-chloroform and SPE with UHPLC-MS/MS to independently quantify trabedersen and the 5´n-1, 5´n-2, 5´n-3, 3´n-1, 3´n-2 and 3´n-3 metabolites in a single assay. The qualification data indicate that if the method was validated it would meet regulatory expectations for precision, accuracy and selectivity. CONCLUSION: We show that quantification of an oligonucleotide and multiple metabolites, including isobaric 3´ and 5´ metabolites, is achievable in a single assay through good sample clean-up and careful optimization of the LC-MS/MS parameters. The strategy presented here can be applied elsewhere and may be useful for other oligonucleotides and their metabolites.

A phase II trial of AS1411 (a novel nucleolin-targeted DNA aptamer) in metastatic renal cell carcinoma.

BACKGROUND: DNA aptamers represent a novel strategy in anti-cancer medicine. AS1411, a DNA aptamer targeting nucleolin (a protein which is overexpressed in many tumor types), was evaluated in patients with metastatic, clear-cell, renal cell carcinoma (RCC) who had failed treatment with ≥1 prior tyrosine kinase inhibitor. METHODS: In this phase II, single-arm study, AS1411 was administered at 40 mg/kg/day by continuous intravenous infusion on days 1-4 of a 28-day cycle, for two cycles. Primary endpoint was overall response rate; progression-free survival (PFS) and safety were secondary endpoints. RESULTS: 35 patients were enrolled and treated. One patient (2.9 %) had a response to treatment. The response was dramatic (84 % reduction in tumor burden by RECIST 1.0 criteria) and durable (patient remains free of progression 2 years after completing therapy). Whole exome sequencing of this patient's tumor revealed missense mutations in the mTOR and FGFR2 genes which is of interest because nucleolin is known to upregulate mTOR pathway activity by enhancing AKT1 mRNA translation. No other responses were seen. Thirty-four percent of patients had an AS1411-related adverse event, all of which were mild or moderate. CONCLUSIONS: AS1411 appears to have minimal activity in unselected patients with metastatic RCC. However, rare, dramatic and durable responses can be observed and toxicity is low. One patient in this study had an excellent response and was found to have FGFR2 and mTOR mutations which will be of interest in future efforts to discover and validate predictive biomarkers of response to nucleolin targeted compounds. DNA aptamers represent a novel way to target cancer cells at a molecular level and continue to be developed with a view to improving treatment and imaging in cancer medicine.

The CpG ODNs enriched diets enhance the immuno-protection efficiency and growth rate of Chinese mitten crab, Eriocheir sinensis.

CpG oligodeoxynucleotides (ODNs), the well-known vaccine adjuvant in mammals, have been proved to mount innate immune responses in crustaceans. In the present study, CpG ODNs was employed as supplements in diets to fed crab Eriocheir sinensis, and the changes of immune parameters as well as weight gain were investigated to evaluate its possible application in crab farming. After the crabs were fed with 40 mg/kg and 100 mg/kg CpG ODNs containing diets (designated as C40 and C100 group) for four weeks, the lysozyme activities were significantly enhanced (p < 0.01) in both groups, while the catalase activity was only increased (p < 0.01) in the C40 group. When those crabs were subsequently challenged with Aeromonas hydrophila, the cumulative mortalities in C40 and C100 groups were declined by 10.4% and 10.8% (p < 0.05) compared with that of control group, respectively. Interestingly, the final weights of crabs were increased after four weeks' feeding of CpG ODNs, and the percentage of weight gain in C40 group reached 124.5 ± 14.2%, which was significantly higher (p < 0.05) than that of control group (78.1 ± 19.2%) and C100 group (107.3 ± 28.2%). The uptake of CpG ODNs by haemocytes and the possible mechanism of CpG ODNs to active the immune response were investigated by using the laser scanning confocal microscope. CpG ODNs (labeled with 5'-end-FAM) could be internalized by the haemocytes after incubation of 20 min, with strong signals detected at the cell membrane and in the cytoplasm. In the cytoplasm, most of the CpG ODNs were localized in lysosome, and some of them escaped from the lysosomal compartments and aggregated around the nuclear. The results clearly demonstrated that CpG ODNs could be internalized directly by crab haemocytes and mostly located in the late endosome. The enhancements of immuno-protection efficiency and growth rate from CpG ODNs as supplements in diets might depend on the uptaking and locating processes, and they could be used as a potential immunostimulant for the crab aquaculture.

Development and validation of LC-MS/MS method for the detection and quantification of CpG oligonucleotides 107 (CpG ODN107) and its metabolites in mice plasma.

CpG oligodeoxynucleotide 107 (CpG ODN107) could be used as a novel radiosensitizer for glioma. Herein, a novel and sensitive reversed-phase HPLC coupled with electrospray triple quadrupole mass spectrometry (LC-MS/MS) following a one-step C18 solid-phase extraction (SPE) for biological matrix removal was developed and fully validated for the determination of CpG ODN107 and its metabolites such as 5'N-1, 3'N-1, 3'N-2, and 3'N-3 in mouse plasma. The analytes were separated on an Extend-C18 analytical column (150 mm × 2.1 mm, 3.5 µm) using an eluent of acetonitrile-0.05% aqueous NH(3) (20:80, v/v) and detected by electrospray ionization (ESI) mass spectrometry in the negative multiple reaction monitoring mode (MRM). The assay was specific, and it showed a good linearity with a determination coefficient (r(2)) that was greater than or equal to 0.998 for CpG ODN107 and its metabolites in the biological matrices. The precision, accuracy, and relative recovery values were found to be <15%, ±15%, and 95-105%, respectively. This method was successfully applied to measure the concentrations of CpG ODN107 and its metabolites in the plasma following the intravenous administration of 15.0 mg/kg of CpG ODN107 in mice; therefore, the method was suitable for preclinical pharmacokinetic studies on CpG ODN107 and its metabolites.

2'-O,4'-C-aminomethylene-bridged nucleic acid modification with enhancement of nuclease resistance promotes pyrimidine motif triplex nucleic acid formation at physiological pH.

Due to the instability of pyrimidine motif triplex DNA at physiological pH, triplex stabilization at physiological pH is crucial in improving its potential in various triplex-formation-based strategies in vivo, such as gene expression regulation, genomic DNA mapping, and gene-targeted mutagenesis. To this end, we investigated the thermodynamic and kinetic effects of our previously reported chemical modification, 2'-O,4'-C-aminomethylene-bridged nucleic acid (2',4'-BNA(NC)) modification of triplex-forming oligonucleotide (TFO), on triplex formation at physiological pH. The thermodynamic analyses indicated that the 2',4'-BNA(NC) modification of TFO increased the binding constant of the triplex formation at physiological pH by more than 10-fold. The number and position of the 2',4'-BNA(NC) modification in TFO did not significantly affect the magnitude of the increase in the binding constant. The consideration of the observed thermodynamic parameters suggested that the increased rigidity and the increased degree of hydration of the 2',4'-BNA(NC)-modified TFO in the free state relative to the unmodified TFO may enable the significant increase in the binding constant. Kinetic data demonstrated that the observed increase in the binding constant by the 2',4'-BNA(NC) modification resulted mainly from the considerable decrease in the dissociation rate constant. The TFO stability in human serum showed that the 2',4'-BNA(NC) modification significantly increased the nuclease resistance of TFO. Our results support the idea that the 2',4'-BNA(NC) modification of TFO could be a key chemical modification to achieve higher binding affinity and higher nuclease resistance in the triplex formation under physiological conditions, and may lead to progress in various triplex-formation-based strategies in vivo.

A combination of Flt3 ligand cDNA and CpG oligodeoxynucleotide as nasal adjuvant elicits protective secretory-IgA immunity to Streptococcus pneumoniae in aged mice.

Our previous study showed that a combination of a plasmid-expressing Flt3 ligand (pFL) and CpG oligodeoxynucleotides (CpG ODN) as a combined nasal adjuvant elicited mucosal immune responses in aged (2-y-old) mice. In this study, we investigated whether a combination of pFL and CpG ODN as a nasal adjuvant for a pneumococcal surface protein A (PspA) would enhance PspA-specific secretory-IgA Ab responses, which could provide protective mucosal immunity against Streptococcus pneumoniae infection in aged mice. Nasal immunization with PspA plus a combination of pFL and CpG ODN elicited elevated levels of PspA-specific secretory-IgA Ab responses in external secretions and plasma in both young adult and aged mice. Significant levels of PspA-specific CD4(+) T cell proliferative and PspA-induced Th1- and Th2- type cytokine responses were noted in nasopharyngeal-associated lymphoreticular tissue, cervical lymph nodes, and spleen of aged mice, which were equivalent to those in young adult mice. Additionally, increased numbers of mature-type CD8, CD11b-expressing dendritic cells were detected in mucosal inductive and effector lymphoid tissues of aged mice. Importantly, aged mice given PspA plus a combination of pFL and CpG ODN showed protective immunity against nasal S. pneumoniae colonization. These results demonstrate that nasal delivery of a combined DNA adjuvant offers an attractive possibility for protection against S. pneumoniae in the elderly.

Preclinical toxicity and toxicokinetics of GTI-2040, a phosphorothioate oligonucleotide targeting ribonucleotide reductase R2.

PURPOSE: GTI-2040, a 20-mer phosphorothioate oligonucleotide, was designed to hybridize to the mRNA sequence of human ribonucleotide reductase R2. GTI-2040 has been shown to inhibit human cancer cell proliferation by downregulation of R2 expression in vitro and to significantly inhibit tumor growth in xenograft models of human cancer in mice. As part of the safety evaluation for human clinical trials, the toxicity and toxicokinetics of GTI-2040 were determined in Sprague-Dawley rats and rhesus monkeys. METHODS: GTI-2040 was administered to rats at 2, 10, and 50 mg/kg/day by bolus intravenous injection every second day for 21 days with a 21-day recovery. In monkeys, an acute study was performed with single, escalating doses of GTI-2040 ranging from 10 to 80 mg/kg given as a 24-h continuous intravenous infusion. As well, a 21-day, continuous intravenous infusion study with GTI-2040 was conducted in monkeys at 2, 10, and 50 mg/kg/day, with a 3-week recovery. Blood sampling was done to measure GTI-2040 plasma concentrations, metabolites, and pharmacokinetic parameters, and tissues were collected to assess the distribution of GTI-2040 and/or metabolites. RESULTS: The toxicities of GTI-2040 in both rats and monkeys were typical for the phosphorothioate oligonucleotide class of compounds. In monkeys, there was a dose-related increase in GTI-2040 plasma levels with concomitant increase in complement activation and prolongation of activated partial thromboplastin time. In both rats and monkeys, the tissues having the highest concentrations of GTI-2040 (kidney, liver, spleen) had the largest dose-related toxic effects. Adverse effects were diminished or absent in the recovery animals. CONCLUSIONS: GTI-2040 was well tolerated when infused over 24 h at doses up to 80 mg/kg in monkeys. In rats and monkeys, GTI-2040 was reasonably well tolerated and showed reversible toxicities when administered at doses up to 50 mg/kg/day for 21 days. The no observed adverse effect dose level for GTI-2040 in both animal species was 2 mg/kg/day. There were no apparent sequence-specific effects related to the interaction of GTI-2040 with the R2 component of the mRNA expressing ribonucleotide reductase.

CpG-ODN 2006 and human parvovirus B19 genome consensus sequences selectively inhibit growth and development of erythroid progenitor cells.

Recent studies have shown that anemia is commonly observed after exposure to pathogens or pathogen-derived products, which are recognized via Toll-like receptor 9 (TLR9). In the current study, we demonstrate that CpG oligodeoxynucleotide-2006, a TLR9 ligand with phosphodiester (PO; 2006-PO) but not with the phosphorothioate backbone, selectively inhibits the erythroid growth derived from human CD34(+) cells. The 2006-PO was internalized by the erythroid progenitors within 30 minutes; however, expression of TLR9 mRNA was not detected in these cells. The 2006-PO directly inhibited burst-forming unit-erythroid growth, resulted in the accumulation of cells in S and G(2)/M phases, and increased cell size and frequency of apoptotic cells. These features were similar to those observed in erythroid progenitors infected with human parvovirus B19 that causes pure red cell aplasia. The consensus sequence of 2006-PO was defined as 5'-GTTTTGT-3', which was located in the P6-promoter region of B19 and inhibited erythroid growth in a sequence-specific manner and down-regulated expression of erythropoietin receptor (EPOR) mRNA and EPOR. B19 genome extracted from serum also inhibited erythroid growth and down-regulated expression of EPOR on glycophorin A(+) cells. These results provide a possible insight into our understanding of the mechanisms of human parvovirus B19-mediated inhibition of erythropoiesis.

LL-37 promotes rapid sensing of CpG oligodeoxynucleotides by B lymphocytes and plasmacytoid dendritic cells.

LL-37 is a cationic antimicrobial peptide derived from neutrophils and keratinocytes. It plays an important role in protection against bacterial infection in the skin and mucosal surfaces. However, its role within the blood compartment remains unclear given that serum inhibits its bactericidal property. In this study, we show that LL-37 promotes very rapid and highly efficient sensing of CpG motifs in bacterial DNA by human B lymphocytes and plasmacytoid dendritic cells (pDCs) in serum-containing media and in whole blood. LL-37 allowed detection of CpG oligodeoxynucleotide (ODN) within minutes of exposure. Without LL-37, 20-30 times more CpG was required to produce the same effect. The promotion of CpG detection by LL-37 was independent of the backbone of the ODN, as the effect was observed not only in ODNs with modified phosphorothioate backbone, but also in ODNs with natural phosphodiester backbone, as found in genomic DNA. Unmethylated CpG motifs within the phosphodiester ODN and LL-37-mediated delivery are required for pDCs to respond. In keeping with the above, cells responded to CpG-rich bacterial DNA and LL-37, but not to human DNA and LL-37. The ability of LL-37 to enhance delivery of CpG to stimulate immune cells is independent of its amphipathic structure and its bactericidal property. LL-37 aids the delivery of CpG to B cells and pDCs, but not T cells. These findings are pertinent to rapid recognition of microbial DNA and are highly relevant to contemporary studies of CpG/TLR9 agonists in vaccines and cancer therapy.

[Haemodynamic role of blood-plasma circulating cell-free DNA and contained therein high-molecular-weight CpG-rich fraction in pathogenesis of arterial hypertension and atherosclerosis obliterans of carotid arteries].

The hydrodynamic resistance (HR) of blood is one of the components of the total peripheral resistance. High-molecular-weight DNA appears to decrease the HR in accordance with the Toms's effect. The present study was undertaken to investigate the HR and properties of cell-free DNA circulating in the blood plasma (hereinafter referred to as pDNA) of the control donors, patients suffering from either arterial hypertension (AH) alone or that combined with atherosclerotic lesions of the carotid arteries (CAs). Within the normal concentrations of pDNA, we revealed an inverse dependence of the HR thereupon and upon the content in pDNA of the high-molecular-weight CpG-rich fraction (CpG-DNA), i. e., a transcribed region of the ribosomal repeat (rDNA). A decrease or an increase in the pDNA concentration in all the patients examined was accompanied by an elevation of the rDNA concentration in the blood plasma. Exceeding a certain level thereof appeared to give rise to an increase in both the HR and arterial pressure (AP). Patients presenting with degree I essential AH were found to have a decreased endonuclease activity of the blood plasma, with the pDNA concentration being more than two-fold higher with no change in the rDNA content. Their HR appeared to be increased (p<0.01). Patients diagnosed as having degree II AH were characterized by a normal or decreased level of pDNA and an elevated content of pDNA, with the HR being slightly lowered. In patients presenting with atherosclerosis obliterans of the ACs, the initial manifestations of the lesions of the carotid arteries were typically revealed on the background of a lowered HR (p<0.05). All patients suffering from atherosclerotic lesions of the ACs could be subdivided into two groups, which in our opinion is probably associated with different various mechanisms of destructive damage to the arterial intima. In some of them, the pDNA concentration does not differ from the normal values, but in its composition, there is an increased content of rDNA, elevating as obliteration of the vessels' lumen increases, with the HR being decreased. The majority of them have degree II AH. In others, the pDNA concentration is by an order of magnitude higher than the normal values, while the rDNA content in pDNA is decreased, with the HR being elevated. Most of them have degree III AH. Pronounced and rough stenoses take an asymptomatic course in patients with decreased values of the HR and a slightly elevated level of pDNA and/or rDNA in the blood plasma. A higher level thereof leads to a rise in the HR and to the appearance of neurological symptomatology. Hence, CpG-DNA circulating in the composition of pDNA is a constantly acting endogenous blood factor decreasing the HR (the Toms's effect) and normalizing AP under physiological conditions, being however a cause of their increase and impairment of blood circulation in the pathogenesis of AH and atherosclerosis obliterans of the CAs.

The effect of circulation lifetime and drug-to-lipid ratio of intravenously administered lipid nanoparticles on the biodistribution and immunostimulatory activity of encapsulated CpG-ODN.

The encapsulation of conventional drugs in lipid nanoparticles (LNs) has been extensively utilized to enhance therapeutic activity by altering their pharmacokinetic (PK) and biodistribution (BD) properties. We have previously shown that the immunostimulatory activity of unmethylated cytidine-guanosine (CpG)-containing immunostimulatory oligodeoxynucleotides (ODN) is greatly enhanced when encapsulated in an LN (LN CpG-ODN). Here, we investigate the effect of circulation lifetime (determined by lipid composition) and drug-to-lipid (D/L) ratio of intravenously (i.v.) administered LN CpG-ODN on PK, BD, and cellular uptake and correlate these parameters with the immunostimulatory activity. Results from these studies show that despite significant differences in the circulation lifetime and the D/L ratio, the immune response is similar with respect to immune cell activation and cytolytic activity in the spleen and the blood compartments. Our findings indicate that the benefits of liposomal nanoparticles for the delivery of immunomodulatory drugs such as CpG-ODN are defined by a different paradigm than that for conventional drugs.

Improved systemic pharmacokinetics, biodistribution, and antitumor activity of CpG oligodeoxynucleotides complexed to endogenous antibodies in vivo.

CpG oligodeoxynucleotides (CpG-ODNs) fail to elicit antitumor immunity after intravenous administration presumably due to their rapid renal clearance and low tumor accumulation. To address this issue, we tested the hypothesis that endogenous IgG can be used as systemic drug carriers to improve the pharmacokinetics, tumor accumulation, and antitumor activity of intravenously administered CpG-ODNs. To this end, tritium-labeled CpG-ODNs conjugated with one or two dinitrophenyl (DNP) haptens (DNP- and DNP(2)-[(3)H]-CpG-ODN) were intravenously dosed into DNP-immunized Balb/c mice bearing subcutaneous CT26 colorectal tumors. Serum and tissue samples for pharmacokinetic and biodistribution profiling were collected at predetermined timepoints and analyzed by liquid scintillation. In antitumor efficacy studies, DNP-immunized, CT26 tumor-bearing mice were intravenously dosed with PBS, CpG-ODN, or DNP-CpG-ODN every five days. Tumor volumes and macroscopic and histological examination of resected solid tumors were used to quantitatively and qualitatively assess tumor growth inhibition. Relative to [(3)H]-CpG-ODN, dinitrophenylated [(3)H]-CpG-ODNs displayed substantial increases in systemic exposure (900-1650 fold) and half-life (100-300 fold), marked decreases in systemic clearance (750-1500 fold) and volume of tissue distribution (13-37 fold), as well as substantial and sustained tumor accumulation (approximately 30% vs. <2% injected dose/g). Antitumor efficacy studies demonstrated that DNP-CpG-ODN inhibited tumor growth by up to 60% relative to PBS control whereas CpG-ODN treatment had no apparent effect. Macroscopic and histological examination of harvested tumors at various timepoints revealed the presence of regions of necrotic tissue only in tumors from mice treated with DNP-CpG-ODN. Collectively, these results show the potential of endogenous IgG to mediate the systemic delivery of CpG-ODN to solid tumors and to enhance their antitumor activity following intravenous administration.

Vaccination with NY-ESO-1 protein and CpG in Montanide induces integrated antibody/Th1 responses and CD8 T cells through cross-priming.

The use of recombinant tumor antigen proteins is a realistic approach for the development of generic cancer vaccines, but the potential of this type of vaccines to induce specific CD8(+) T cell responses, through in vivo cross-priming, has remained unclear. In this article, we report that repeated vaccination of cancer patients with recombinant NY-ESO-1 protein, Montanide ISA-51, and CpG ODN 7909, a potent stimulator of B cells and T helper type 1 (Th1)-type immunity, resulted in the early induction of specific integrated CD4(+) Th cells and antibody responses in most vaccinated patients, followed by the development of later CD8(+) T cell responses in a fraction of them. The correlation between antibody and T cell responses, together with the ability of vaccine-induced antibodies to promote in vitro cross-presentation of NY-ESO-1 by dendritic cells to vaccine-induced CD8(+) T cells, indicated that elicitation of NY-ESO-1-specific CD8(+) T cell responses by cross-priming in vivo was associated with the induction of adequate levels of specific antibodies. Together, our data provide clear evidence of in vivo cross-priming of specific cytotoxic T lymphocytes by a recombinant tumor antigen vaccine, underline the importance of specific antibody induction for the cross-priming to occur, and support the use of this type of formulation for the further development of efficient cancer vaccines.

Identification and quantification of GC-rich oligodeoxynucleotides in tissue extracts by capillary gel electrophoresis.

Capillary gel electrophoresis (CGE) is a widely used method for quantification of oligonucleotide-based drugs, such as CpG oligodeoxynucleotides (CpG ODN), aptamers and small interfering ribonucleic acids (siRNAs) that allows accurate quantification of parent compound as well as metabolites. Stable secondary structure formation of these molecules frequently prevents analysis by conventional CGE methods and impedes pharmacokinetic assessment. Herein, we describe development of a CGE method for identification and quantification of complex mixtures of secondary structure forming GC-rich ODN in biological samples at dose levels of 0.5mg/kg and above. Samples containing GC-rich CpG ODN and metabolite markers were treated by solid-phase-extraction (SPE) and subsequently analyzed by CGE using a 50cm neutrally coated capillary at 60 degrees C together with a 7M urea buffer system containing 30% dimethylsulfoxide (DMSO). Peak resolutions >or=1 were typically achieved, enabling pharmacokinetic assessment of secondary structure forming oligonucleotides in biological samples that hitherto were unsusceptible to quantitative analysis.

Binding with serum components favorably affects cellular uptake of 111In-oligonucleotide in a leukemia cell line.

PURPOSE: The influence of serum components on the intracellular uptake of an 111In-oligonucleotide (ODN) against mdr1 mRNA was investigated in the murine leukemia cell line, P388/S, and its mdr1-overexpressed P388/R. METHODS: 111In-ODNs naked and vectorized with lipids were analyzed for binding with serum components using high-performance liquid chromatography (HPLC). 111In-ODN was incubated in albumin and transferrin solutions. 111In-DTPA and 111In-mononucleotide were incubated in serum. Degradation of naked 111In-ODN was detected in phosphate buffered saline (PBS) and serum containing endonuclease S1. Cellular uptakes of naked and vectorized 111In-ODN in the above cells were examined with and without fetal calf serum (FCS). RESULTS: Time-dependent binding of naked and vectorized 111In- ODN with serum components was observed throughout 24 hours. Transchelation of 111In to transferrin was not detected. HPLC profiles of 111In-DTPA and 111In-mononucleotide did not change in serum. Degradation of 111In-ODN by S1 was less remarkable in serum than in PBS. Specific accumulation of vectorized 111In-ODN in P388/R cells was achieved in culture with and without 10% FCS. CONCLUSIONS: This study verified the intense binding of ODN with serum components, leading to no inhibition on ODN intracellular specific uptake. Binding with serum components protects 111In-ODN from degradation by endonuclease and thus may facilitate ODN transmembrane delivery.

Quantification of oligodeoxynucleotides in human plasma with a novel hybridization assay offers greatly enhanced sensitivity over capillary gel electrophoresis.

Capillary gel electrophoresis using UV detection (CGE-UV) has been used to quantify oligodeoxynucleotides (ODN) in human plasma. Although the sensitivity of this method is adequate to detect antisense ODN, which are administered in daily doses up to 10 mg/kg, CGE-UV is not sensitive enough to detect the much lower quantities of ODN administered for other purposes, such as immune stimulation by CpG ODN. We have developed a very sensitive colorimetric hybridization assay that increases the sensitivity of detection by more than four logs compared with CGE-UV. The hybridization assay uses sequence-specific capture and detection ODN probes complementary to portions of the ODN sequence. Herein we provide a prototype for assay development and validation using a 24- mer immunostimulatory phosphorothioate ODN. Probes were locked nucleic acids (LNA), resulting in increased sensitivity and specificity. The linear range of the assay is 7.8-1000 pg/ml, with a 7.8 pg/ml lower limit of quantification (LLOQ) and a detection limit of 2.8 pg/ml. This translates to detection of 40 attamoles. Intraassay and interassay precision were < or =5.0% CV and < or =12.9% CV, respectively, for quality control samples. The assay is suitable for a variety of matrices, including monkey and rat plasma, allowing application to toxicokinetic samples. The methodology is highly specific, with the ability to distinguish almost all single-base mismatched ODN. The assay detects 100% of the parent as well as some metabolites up to N-4, which are known to be the primary metabolites forming in the first hours after in vivo administration and are physiologically active with in vitro assays.