Sustained release of granulocyte-macrophage colony-stimulating factor from a modular peptide-based cancer vaccine alters vaccine microenvironment and enhances the antigen-specific T-cell response.

The recent identification and molecular characterization of tumor antigens provides the opportunity to explore the rational development of peptide-based cancer vaccines. However, the response to these vaccines remains variable, and peptide-based cancer vaccines may even produce tolerance induction and enhanced tumor growth. The authors have developed a unique method for the isolation of a polysaccharide polymer of chemically pure poly- N -acetyl glucosamine (p-GlcNAc). This highly purified polysaccharide can be formulated into a stable gel matrix (designated F2 gel matrix) with unique properties of a sustained-release delivery system that has previously been shown to be an effective immune adjuvant. F2 gel matrix is capable of providing sustained release of antigenic peptide and cytokine in vitro. The purposes of this study were to characterize the ability of F2 gel matrix to provide sustained local release of cytokines in vivo and to test the hypothesis that such sustained release can enhance the microenvironment for antigen presentation, leading to a more effective antitumor response. Subcutaneous administration of F2 gel/cytokine matrix resulted in sustained release of cytokine at the vaccine site for up to 120 hours. Sustained release of granulocyte-macrophage colony-stimulating factor (GM-CSF) was associated with an increased inflammatory infiltrate at the vaccine site and enhanced dendritic cell activation. Further, accination with F2 gel/SIINFEKL/GM-CSF matrix resulted in enhanced antigen-specific immunity. Addition of GM-CSF to the F2 gel matrix resulted in an increase in the percentage of antigen-specific T cells in the draining lymph nodes, enhanced cytotoxicity, a sustained presence of antigen-specific T cells in the peripheral blood, and protection from E.G7 tumor challenge. These results support the potential of an F2 gel matrix modular vaccine delivery system that can provide sustained local release of cytokine in vivo, and confirm the powerful effects of GM-CSF as an immune adjuvant.

CaSm/gemcitabine chemo-gene therapy leads to prolonged survival in a murine model of pancreatic cancer.

BACKGROUND: CaSm, the cancer-associated Sm-like oncogene, is overexpressed in greater than 80% of pancreatic tumors. We previously reported that an adenovirus expressing antisense RNA to CaSm (Ad-alpha CaSm) can decrease pancreatic tumor growth in vivo but is not curative. In the current study we investigated the mechanism of Ad-alpha CaSm's antitumor effect to rationally approach combinatorial therapy for improved efficacy. METHODS: AsPC-1 and Panc-1 human pancreatic cancer cells were treated with Ad-alpha CaSm and examined by MTT assay for in vitro proliferation changes. Flow cytometry determined the effect of CaSm down-regulation on the cell cycle, and then cells treated with Ad-alpha CaSm in combination with cisplatin, etoposide, or gemcitabine chemotherapies were reexamined by MTT assay. SCID-Bg mice bearing subcutaneous AsPC-1 tumors were treated with Ad-alpha CaSm, gemcitabine, or the combination and monitored for tumor growth and survival. RESULTS: Treatment with Ad-alpha CaSm reduced the proliferation of AsPC-1 and Panc-1 cells (59% and 44%, respectively; P <.05). The cell cycle revealed a cytostatic block with decreased G(1) phase and increased DNA content in treated cells. The combination of Ad-alpha CaSm with gemcitabine significantly reduced in vitro proliferation (66% vs 39% and 48% for controls), decreased in vivo AsPC-1 tumor growth by 71% (n = 10), and extended survival time from 57 to 100 days. CONCLUSIONS: Down-regulation of CaSm reduces the growth of pancreatic cancer cells by altering the cell cycle in a cytostatic manner. The combination of Ad-alpha CaSm with gemcitabine is more effective than either agent used separately.

The cancer-associated Sm-like oncogene: a novel target for the gene therapy of pancreatic cancer.

BACKGROUND: The prognosis for pancreatic cancer (PC) remains dismal, providing a clear need for the development of novel therapies. We have previously shown that the cancer-associated Sm-like (CaSm) oncogene is overexpressed in the great majority of pancreatic tumors and is required to maintain the transformed phenotype. The purpose of this study was to determine whether the application of CaSm antisense gene therapy would generate a significant antitumor effect against PC. METHODS: An adenoviral vector (Ad-alphaCaSm) expressing a 900-base pair antisense RNA to CaSm was created. The PC cell lines AsPC-1 and Capan-1 were infected with this vector and examined for changes in in vitro proliferation by using methyl thiazol tetrazolium and soft agar assays. SCID-Bg mice bearing subcutaneous AsPC-1 tumors were treated with Ad-alphaCaSm (1 x 10(9) plaque-forming units) as a single intratumor injection with tumor growth and survival monitored. RESULTS: AsPC-1 and Capan-1 cells showed decreased in vitro proliferation (93%, P =.0041, and 70%, P =. 0038, respectively) and anchorage independent growth (55%, P =.02, and 45%, P =.03, respectively) after treatment. Ad-alphaCaSm reduced in vivo AsPC-1 tumor growth by 40% (n = 10), extending median survival time from 35 to 60 days. CONCLUSIONS: Ad-alphaCaSm demonstrates a significant antitumor effect against pancreatic cancer both in vitro and in vivo. These results support the role of CaSm as a significant gene involved in the neoplastic transformation of pancreatic tumors. Thus CaSm represents a novel gene target in PC and holds potential as a new treatment approach either alone or in combination with existing therapies.

Endoscopic injection of bleeding esophageal varices with a poly-N-acetyl glucosamine gel formulation in the canine portal hypertension model.

BACKGROUND: It has been shown that poly-N-acetyl glucosamine produces rapid hemostasis by stimulating erythrocyte aggregation. Endoscopic injection of this substance may be effective in the treatment of bleeding varices. METHODS: In eight heparinized dogs with a bleeding esophageal varix greater than 2 mm in diameter, 2.5% to 3.5% poly-N-acetyl glucosamine gel was injected intravariceally and paravariceally. Endoscopy, endosonography, and histopathology were performed at 1, 7, 21, and 90 days after injection. RESULTS: In all cases, the variceal hemorrhage was stopped with three to four injections of a mean total gel volume of 1.9 mL. No recurrence of bleeding, ulceration, or stricture formation occurred. Through replacement of the gel by connective tissue, the varix was permanently obliterated in its whole course in five cases and in more than 70% of its length in three cases. No embolization and no poly-N-acetyl glucosamine antibodies were detected. CONCLUSIONS: Endoscopic injection of bleeding esophageal varices in this animal model with the use of poly-N-acetyl glucosamine gel was an effective and safe method for stopping the hemorrhage and inducing permanent varix obliteration.

Characterization of a sustained-release delivery system for combined cytokine/peptide vaccination using a poly-N-acetyl glucosamine-based polymer matrix.

Identification of tumor-associated antigens (TAAs) and their class I MHC-restricted epitopes now allows for the rational design of peptide-based cancer vaccines. A biocompatible system capable of sustained release of biologically relevant levels of cytokine and TAA peptide could provide a more effective microenvironment for antigen presentation. Our goal was to test a sustained-release cytokine/TAA peptide-based formulation using a highly purified polysaccharide [poly-N-acetyl glucosamine (p-GlcNAc)] polymer. Granulocyte-macrophage colony-stimulating factor (GM-CSF; 100 microgram) and MART-1(27-35) peptide (128 microgram in DMSO) were formulated into p-GlcNAc. Peptide release was assayed in vitro using interleukin 2 production from previously characterized MART-1(27-35)-specific Jurkat T cells (JRT22). GM-CSF release was assayed via ELISA and proliferation of M-07e (GM-CSF-dependent) cells. Local bioavailability of MART-1(27-35) peptide for uptake and presentation by antigen-presenting cells was demonstrated for up to 6 days (>0.5 microgram/ml). More than 1.0 microgram/ml GM-CSF was concomitantly released over the same period. Biocompatibility and local tissue response to p-GlcNAc releasing murine GM-CSF was determined in C57BL/6 mice via s.c. injection using murine GM-CSF (0. 2 microgram/ml) in 200 microliter of a 2.5% polymer gel. Significant lymphocytic and eosinophilic infiltration was observed 2-7 days after injection with polymer containing murine GM-CSF. The results of our studies show that this biocompatible system is capable of a sustained concomitant release of biologically active peptide and cytokine into the local microenvironment. These findings support further studies to validate a p-GlcNAc delivery system vehicle for a cytokine/TAA peptide-based cancer vaccine.

The primary structure of a fungal chitin deacetylase reveals the function for two bacterial gene products.

Chitin deacetylase (EC 3.5.1.41) hydrolyzes the N-acetamido groups of N-acetyl-D-glucosamine residues in chitin. A cDNA to the Mucor rouxii mRNA encoding chitin deacetylase was isolated, characterized, and sequenced. Protein sequence comparisons revealed significant similarities of the fungal chitin deacetylase to rhizobial nodB proteins and to an uncharacterized protein encoded by a Bacillus stearothermophilus open reading frame. These data suggest the functional homology of these evolutionarily distant proteins. NodB is a chitooligosaccharide deacetylase essential for the biosynthesis of the bacterial nodulation signals, termed Nod factors. The observed similarity of chitin deacetylase to the B. stearothermophilus gene product suggests that this gene encodes a polysaccharide deacetylase.

An efficient technique for obtaining sequences flanking inserted retroviruses.

Genomic mapping studies frequently employ retrovirus-mediated transfer of dominant selectable markers to specific target chromosomes. DNA probes containing sequences adjacent to inserted proviruses are valuable mapping tools in such studies. We have implemented a strategy for amplification of chromosomal sequences flanking the 5' LTR of MoMuLV-based vectors. Probes derived from these amplification products successfully differentiated murine versus human proviral localization in retrovirus-infected mouse-human chromosome 17q hybrid cells.

Optimization of the microenvironment for mammalian cell culture in flexible collagen microspheres in a fluidized-bed bioreactor.

Flexible, three-dimensional, collagen Microspheres have been developed to actively promote a natural, optimal microenvironment for large-scale tissue culture of mammalian cells. The transport of nutrients into and cell products out of the Microspheres is enhanced by forced convective flow, which is the result of the tumbling of Microspheres and the dynamic properties of media flow in the fluidized-bed bioreactor. The collagen Microspheres have important characteristics of composition and morphology essential for optimal cell-matrix and cell-cell interactions. These interactions lead to high cell density and productivity through the dynamic modification of the microenvironment by cell-derived extracellular constituents. The collagen and Microsphere/fluidized-bed system provides the means to control and optimize the diffusive and contact components of the cells' microenvironment. Adaptation of cells to this microenvironment often results in dramatic increases in cell-specific productivity. Production of biotherapeutics in this process can be routinely performed in serum-free media, often leading to high productivity and product quality.

Determination of equilibrium binding affinity of distamycin and netropsin to the synthetic deoxyoligonucleotide sequence d(GGTATACC)2 by quantitative DNase I footprinting.

A new method for determining the equilibrium binding constant of antitumor drugs to specific DNA sequences by quantitative DNase I footprinting is presented. The use of a short synthetic DNA oligomer to define a homogeneous population of DNA binding sites enables the calculation of the free drug concentration and the fraction of DNA sites complexed with drug in solution and is described for the first time. Since a 1:1 stoichiometry is observed for each drug-oligomer DNA complex, it becomes possible to calculate equilibrium binding constants in solution. By use of this technique, the binding affinities of the nonintercalating drugs netropsin and distamycin to the synthetic oligonucleotide d(GGTATACC)2 are determined to be Ka (25 degrees C) = 1.0 X 10(5) and 2.0 X 10(5) M-1, respectively. Quantitation of the temperature dependence associated with complex formation results in a determination of standard enthalpies of -3.75 and -8.48 kcal mol-1 for the binding of netropsin and distamycin, respectively. Calculation of other thermodynamic parameters are found to be in agreement with previous studies and indicate that the DNA binding process for these compounds is predominantly enthalpy driven. This method of quantitative DNase I footprinting is demonstrated to be a useful technique for the measurement of drug affinities to specific binding sites on DNA oligomers which are designed and synthesized expressly for this purpose. Applications of the technique to the determination of drug binding affinities at specific sites within native DNA sequences are discussed.

Properties of DNase I digestion of the deoxyoligonucleotide: 5'd(ATCGTACGAT)2(3').

Deoxyribonuclease I digestion of the deoxyoligodecamer 5'd(ATCGTACGAT)2(3') has been examined in detail to study the kinetic and structural properties of this enzyme substrate system in solution. In addition, these studies have defined, in general, those DNase I conditions to be used in future drug-DNA footprinting experiments. Special attention has been taken of those properties of DNase I that are critical for quantitation of ligand binding to small DNA fragments, and that aid in designing oligomers to be used in footprinting experiments. Enzyme activity was observed at all phosphodiester bonds in the decamer studied with varying affinity, except for the first four bonds at the 5' end of the oligomer. The DNA substrate concentration is always in excess, in order to achieve conditions of no more than one DNase I cleavage per DNA molecule. Reactions were controlled so that 65% or more of the initial amount of decamer substrate remained after DNase I digestion. It was observed that the rate of enzyme reactivity decreases with digestion time and is sensitive to the experimental conditions.

Computer assisted microdensitometric analysis of footprinting autoradiographic DATA.

A system comprised of a linear scanning microdensitometer interfaced to a personal computer has been developed to facilitate analysis of ligand-DNA footprinting autoradiograms. The system, which can be used to record density and sequence information from autoradiographic films, enables the user to relate the area under an autoradiographic band to the concentration of radiolabeled molecules present in the electrophoresis gel. This report describes the computer program which performs the calculations and discusses the ability of the system to accurately determine oligonucleotide concentration, as a function of band separation, photographic response, and the computational algorithm used to calculate band areas.

RNA structure analysis using methidiumpropyl-EDTA.Fe(II): a base-pair-specific RNA structure probe.

Methidiumpropyl-EDTA.Fe(II) [MPE.Fe(II)] in the presence of dithiothreitol, is shown to cleave phenylalanine-accepting tRNA (tRNAPhe) in a structure-specific fashion. Molar ratios of MPE.Fe(II) to tRNAPhe of less than 1 preferentially cleave phosphodiester bonds known to occur in double-stranded regions of the tRNAPhe molecule. Microdensitometric analysis of autoradiograms of MPE.Fe(II) cleavage products following gel electrophoresis reveals a correspondence between preferred sites of MPE.Fe(II) cleavage and sites in tRNAPhe most sensitive to cobra venom ribonuclease, a double-strand-specific endoribonuclease. Conversely, sites of cleavage by the single-strand-specific S1 nuclease correspond to those nucleotides that are least susceptible to MPE.Fe(II) hydrolysis. Sensitive helical regions in tRNAPhe include the dihydrouracil and the "T psi C" stems, which cannot be detected by cobra venom ribonuclease because of steric constraints. Phosphodiester bonds within the T psi C and dihydrouracil loop regions, which are not detected by S1 nuclease under rigorously controlled digestion conditions, are revealed by inference from their relative insensitivity to MPE.Fe(II). These results demonstrate the utility of MPE.Fe(II) as a general small molecular weight probe of RNA structure, having a greater accessibility to base-paired regions than do the more bulky enzymic probes.

RNA structure analysis using T2 ribonuclease: detection of pH and metal ion induced conformational changes in yeast tRNAPhe.

We describe the use of an enzymic probe of RNA structure, T2 ribonuclease, to detect alterations of RNA conformation induced by changes in Mg2+ ion concentration and pH. T2 RNase is shown to possess single-strand specificity similar to S1 nuclease. In contrast to S1 nuclease, T2 RNase does not require divalent cations for activity. We have used this enzyme to investigate the role of Mg2+ ions in the stabilization of RNA conformation. We find that, at neutral pH, drastic reduction of the available divalent metal ions results in a decrease in the ability of T2 RNase to cleave the anticodon loop of tRNAPhe. This change accompanies an increase in the cleavage of the molecule in the T psi C and in the dihydrouracil loops. Similar treatment of Tetrahymena thermophila 5S ribosomal RNA shows that changes in magnesium ion concentration does not have a pronounced effect on the cleavage pattern produced by T2 RNase. T2 RNase activity has a broader pH range than S1 nuclease and can be used to study pH induced conformational shifts in RNA structure. We find that upon lowering the pH from 7.0 to 4.5, nucleotide D16 in the dihydrouracil loop of tRNAPhe becomes highly sensitive to T2 RNase hydrolysis. This change accompanies a decrease in the relative sensitivity of the anticodon loop to the enzyme. The role of metal ion and proton concentrations in maintenance of the functional conformation of tRNAPhe is discussed.

RNase H-catalyzed site-specific deadenylylation of rabbit alpha- and beta- globin mRNAs. Secondary structure of 3'-noncoding regions.

A general method is described for the removal of 3'-terminal polyadenylate tracts from eukaryotic messenger RNA to generate essentially homogeneous length products that can be 3'-end labeled and sequenced. Hybridization of specific oligodeoxyribonucleotides was used to direct ribonuclease H to the junction of the 3'-noncoding region and the polyadenylate sequence of rabbit alpha and beta globin mRNAs. Site-specific deadenylylation of both globin mRNAs is demonstrated by partial enzymatic sequence analysis following 3'-terminal labeling with 5'-pCp (where p indicates the labeled phosphate group). The secondary structure of the 3'-noncoding region is studied by enzymatic digestion with S1 nuclease and cobra venom ribonuclease. Structural features of the 3'-noncoding regions of these mRNAs are described, including the protein synthesis termination and the poly(A) addition recognition sites.

Quantitation of base substitutions in eukaryotic 5S rRNA: selection for the maintenance of RNA secondary structure.

Eukaryotic 5S rRNA sequences from 34 diverse species were compared by the following method: (1) The sequences were aligned; (2) the positions of substitutions were located by comparison of all possible pairs of sequences; (3) the substitution sites were mapped to an assumed general base pairing model; and (4) the R-Y model of base stacking was used to study stacking pattern relationships in the structure. An analysis of the sequence and structure variability in each region of the molecule is presented. It was found that the degree of base substitution varies over a wide range, from absolute conservation to occurrence of over 90% of the possible observable substitutions. The substitutions are located primarily in stem regions of the 5S rRNA secondary structure. More than 88% of the substitutions in helical regions maintain base pairing. The disruptive substitutions are primarily located at the edges of helical regions, resulting in shortening of the helical regions and lengthening of the adjacent nonpaired regions. Base stacking patterns determined by the R-Y model are mapped onto the general secondary structure. Intrastrand and interstrand stacking could stabilize alternative coaxial structures and limit the conformational flexibility of nonpaired regions. Two short contiguous regions are 100% conserved in all species. This may reflect evolutionary constraints imposed at the DNA level by the requirement for binding of a 5S gene transcription initiation factor during gene expression.

Sequence specificity of actinomycin D and Netropsin binding to pBR322 DNA analyzed by protection from DNase I.

A direct approach to determining the sequence specificities of equilibrium binding drugs by using the DNase protection technique is described. The method utilizes singly end-labeled restriction fragments and partial digestion of the drug fragment complex with DNase I. Microdensitometry of autoradiograms produced after electrophoretic separation of digestion products allows determination of sequences that are affected by drug binding. The feasibility of the technique for locating small ligands bound to DNA and its eventual use as a quantitative thermodynamic approach to studying ligand binding to heterogeneous DNA as a function of sequence is illustrated by using actinomycin D and Netropsin.

Genetic manipulation of antibiotic-producing microorganisms.

The application of directed selection techniques and genetic engineering methods for manipulation of antibiotic-producing microorganisms is generating a new era in industrial microbiology. Modern methods, based on advances in the knowledge of the biosynthetic pathways and regulatory mechanisms involved in the induction and repression of genes involved in antibiotic synthesis, provide a means of increasing antibiotic activity. Hence, recombinant DNA and protoplast fusion methods are used to alter the genetics of antibiotic producers in a semirandom fashion for the development of novel hybrid antibiotics. Directed mutation and selection, protoplast fusion, and both semirandom and specific recombinant DNA methods are examples of alternative procedures for manipulating the biosynthetic pathways of microorganisms for strain improvement and for new hybrid antibiotic synthesis.