Patent Abstract:
the invention proves a method of producing mature dendritic cells in vitro , which comprises the step of culturing the immature dendritic cells in the presence of a specifically configured high molecular weight double stranded rna polymer . the specifically configured high molecular weight dsrna polymer is typically selected from the group comprising poly : poly ; poly : poly ; poly : poly ; poly : poly ; poly : poly ; poly : poly ; poly : poly ; poly : poly ; poly : poly ; poly : poly and ampligen ® : poly ), where x is on average a number from 3 to 40 . the immature dendritic cells may be exposed to an antigen before they are matured , and a vaccine including the antigen - presenting mature dendritic cells can then be prepared . a method of treating cancer , a virus , parasite and microorganism is also disclosed .

Detailed Description:
the invention will now be described in more detail with reference to fig1 and 2 . fig1 shows the uv wavelength spectrum for poly [ c ]: poly [ i 12 u ]; fig2 shows the uv wavelength spectrum for poly [ i ]: poly [ c 6 u ]; fig3 shows the uv wavelength spectrum for poly [ i ]: poly [ c 24 u ]; fig4 shows the effect of poly [ i ]: poly [ c ] and ampligen ® ( poly [ i ]: poly [ c 12 u ]) on immature monocyte - derived dendritic cells as determined by class ii and cd83 ; fig5 shows the effects on cd83 and class ii expression of dendritic cells from healthy individuals treated with poly [ i ]: poly [ c 6 u ], poly [ i ]: poly [ c 24 u ] and poly [ c ]: poly [ i 12 u ]; and fig6 shows the influence of maturation agents poly [ i ]: poly [ c ] and ampligen ® ( poly [ i ]: poly [ c 12 u ]) on dendritic cells and the time course of il 12 p70 production by dendritic cells . peripheral blood mononuclear cells were obtained using leukophoresis . the dendritic cells were cultured from adherent peripheral blood mononuclear cells ( using modified romani &# 39 ; s method ) in serum - free medium aim - v ( gibco ) in the presence of 1000 iu / ml gm - csf ( novartis ) and 1000 iu / ml il 4 ( pharmingen ) for 6 to 7 days . an immature dendritic cell population was harvested and split into two equal aliquots . one aliquot was treated with a maturation agent cells were left in culture at 37 ° c . for a further two days . flow cytometric analysis of dendritic cell differentiation was performed on facscan ( becton dickinson ) using fluorescein conjugated monoclonal antibodies to cd14 , cd1a , cd80 , cd86 , cd40 , cd54 , cd83 , class i & amp ; class ii . a ) il 12 production was assayed using an il 12 p70 elisa kit assay ( r & amp ; d systems ). the assay employs the quantitative sandwich enzyme immunoassay designed to measure il 12 in cell culture supematants . b ) cd83 & amp ; upregulation of class ii & amp ; cd86 expression was determined using flow cytometry . a ) poly [ i ]: poly [ c ] was obtained from sigma ltd ( uk ); b ) ampligen ® ( poly [ i ]: poly [ c 12 ]) was obtained from bioclones ( pty ) limited , south africa ; c ) other specifically configured high molecular weight dsrna polymers were prepared as described below . single - stranded rna ( ssrna ) molecules were synthesized by enzymatic polymerization of nucleotide di - phosphates using the enzyme polynucleotide phosphorylase ( ec 2 . 7 . 7 . 8 ) isolated from microccocus luteus . in the case of specifically configured single - stranded rna &# 39 ; s , containing more than one nucleotide , the polymers were synthesized using the stated molar ratio &# 39 ; s of the respective nucleotide - diphosphates . the exact ratios in the final polymer were then determined the single - stranded rna molecules which were synthesized ( poly [ c 6 u ], poly [ c 24 u ] and poly [ i 12 u ]) are listed in table 1 . the single - stranded rna &# 39 ; s were synthesized using polynucleotide phosphorylase at 0 . 4 units / ml in a buffer containing 0 . 1 m tris ph 9 . 0 , 0 . 3 m urea , 7 . 5 mm mgcl 2 and 0 . 5 mm edta . na 2 and the respective nucleoside di - phosphates ( ndp &# 39 ; s ) in the correct proportions to a final concentration of 23 . 5 mm . the polymerization was carried out at 22 - 24 ° c . for 22 to 40 hours , depending on the polymer . during this time , in - process samples were removed to determine the incorporation of ndp into the polymer by hplc . viscosity measurements were also taken at specific times . at the end of the polymerization ( normally determined by the degree of incorporation of nucleotide di - phosphate ), the polymer - containing solution was concentrated 3 fold using a millipore minitan apparatus containing a 100 000 nominal molecular weight cutoff membrane . tris , sds and phenol were then added to the solution and agitated for 3 × 60 second intervals . the solution was then centrifuged at 5 000 rpm in a beckman ja10 rotor and the lower phenol layer removed . phenol , tris and sds were then added and the procedure was repeated 3 times . following the phenol extraction , the polymer was then precipitated with chilled ethanol after adding kcl to a 0 . 5 m concentration . the polymer precipitate was re - dissolved and precipitated a second time . the second polymer precipitate was dissolved in water and diafiltered , first against an edta buffer to remove any heavy metals , and then against potassium acetate , and finally against 10 volumes of water , before being filtered through a 0 . 22 μm filter and lyophilized . the molecules were run on agarose gel electrophoresis to provide an estimate of size for the purposes of matching them with the complimentary strand . all samples were compared to a standard poly c 12 u material ( batch number ru040105 ) with a mean sedimentation coefficient of 6 . 8 s and a number averaged molecular weight of 500 000 as determined by multi - angle light scattering . based on the mobility of the samples on agarose gel electrophoresis , the largest and smallest ssrna &# 39 ; s were selected for analytical ultracentrifugation on a beckman xla analytical ultracentrifuge . the largest polymer , ooly c , had a mean sedimentation coefficient of 8 . 9 s and the smallest , poly i 12 u , had a mean sedimentation coefficient of 5 . 3 s . the remaining polymers are all expected to be within this size range . molar equivalents were determined by measuring total phosphorous . this value was used to determine the mass of polymer required in order to achieve an 8 mm polymer concentration during annealing . ( i . e . 8 mm with respect to phosphorous [ 1 phosphorous = 1 monomer )). inorganic phosphorous was also determined in order to ensure accurate determination of organic phosphorous . a uv wavelength plot was recorded as a means of identification and also as an assessment of hypochromicity upon annealing of the double stranded polymer . the polymers were enzymatically digested to the incorporated nucleotides and run on hplc to demonstrate base purity and also base ratios of those polymers with a deliberate incorporation of more than one base in the single - strand the endotoxin was determined using the cape cod lal methods ( as endotoxin itself can induce maturation of dendritic cells , it was important that the polymers were essentially endotoxin free ). annealing was performed in ampligen ® ( poly [ i ]: poly [ c 12 u ]) buffer containing 10 mm sodium phosphate ( ph 7 . 4 ), 150 mm sodium chloride and 1 mm magnesium chloride . polymers were dissolved to 8 mm with regard to total organic phosphorous at 50 ° c . and annealed by mixing equal volumes of the respective complimentary ssrna solutions , heating to 65 ° c . for 10 minutes and then cooling to room temperature . the material was then filtered through a 0 . 22 pm filter and vialed under laminar flow . samples of the two ssrna solutions prepared for annealing as well as the annealed dsrna were taken and the uv wavelength profile assessed ( fig1 to 3 ). the hypochromic shift ( generally at the peak absorbance of the ssrna ) was evaluated as an indication of annealing . endotoxin of the dsrna was assessed using the cape cod lal assay . concentration was assumed to be equivalent to the starting concentration of the ssrna entities . the reduced hypochromic shift obtained for poly [ i ]: poly [ c 6 u ] can be explained by the increased frequency of the uridine mismatch in the double stranded rna , which will yield a higher proportion of single stranded rna in the dsrna chain when compared to a more fully base - paired polymer such as poly [ i ]: poly [ c 12 u ] or poly [ i ]: poly [ c 24 u ]. ( a ) comparison of dendritic cell maturation effects of ampligen ® ( poly [ i ]: poly [ c 12 u ]) and other high molecular weight specifically configured dsrna polymers and poly [ i ]: poly [ c ] on immature monocyte - derived dendritic cells : a direct comparison of poly [ i ]: poly [ c 12 u ], poly [ c ]: poly [ i 12 u ], poly [ i ]: poly [ c 6 u ], poly [ i ]: poly [ c 24 u ] and poly [ i ]: poly [ c ] as the maturation agents for human monocyte derived dendritic cells , as determined by changes in cell surface phenotype , was conducted using facs analysis . immature dendritic cells were generated by culturing leucophoresed peripheral blood mononuclear cells in the presence of gm - csf and il 4 for seven days . the maturation stimulus was introduced in culture for 48 hours in a uniform culture condition , utilizing poly [ i ]: poly [ c ] in one test and poly [ i ]: poly [ c 12 u ], poly [ c ]: poly [ i 12 u ], poly [ i ]: poly [ c 8 u ] and poly [ i ]: poly [ c 24 u ] in other tests . using facs analysis , the phenotype of the mature dendritic cells was determined ( fig4 and 5 ). the mature dendritic cells were identified as strongly positive for cd83 ( a glycoprotein expressed predominately on the surface of mature dendritic cells ). class ii molecules were also up - regulated compared with immature dendritic cells ( untreated ). no evidence of cellular toxicity was seen at the dose levels tested . ( b ) comparison of time course of poly [ i ]: poly [ c ] and ampligen ® ( polyl [ i ]: poly [ c 12 u ]) ( and other high molecular weight specifically configured dsrna polymers ) on dendritic cell maturation and il 12 production : mononuclear cells were isolated by density gradient centrifugation . adherent cells were cultured for six days in the presence of il 4 and gm - csf . cells were harvested after immunophenotypic characterisation ( immature ) and recultured in serum - free medium in the presence of il 4 and gm - csf . three separate plates of the cells were prepared as follows : plate one — no treatment ; plate two — poly [ i ]: poly [ c ] ( 100 μg / ml ); plate three — poly [ i ]: poly [ c 12 u ] ( 250 μg / ml ). seven hours after the addition of the maturation agent , the culture medium was removed from each plate and fresh medium without the maturation agent was added . this was repeated at time intervals and the il 12 production was determined on each culture supematant collected . the facs analysis results comparing poly [ i ]: poly [ c 12 u ], poly [ c ]: poly [ i 12 u ], poly [ i ]: poly [ c 6 u ], poly [ i ]: poly [ c 24 u ] and poly [ i ]: poly [ c ] as maturation agents for human monocyte - derived dendritic cells are summarized and illustrated in fig4 and 5 . compared to the untreated dendritic cells , poly [ i ]: poly [ c ], ampligen ® ( poly [ i ]: poly [ c 12 u ]), poly [ c ]: poly [ i 12 u ], poly [ i ]: poly [ c 6 u ] and poly [ i ]: poly [ c 24 u ] produced a significantly greater level of expression of the two markers , with a higher amplitude response being associated with ampligen ® ( poly [ i ]: poly [ c 12 u ]), poly [ c ]: poly [ i 12 u ], poly [ i ]: poly [ c 6 u ] and poly [ i ]: poly [ c 24 u ]. over the time course studied , the untreated dendritic cells did not produce any il 12 as detectable by the specific elisa technique . the poly [ i ]: poly [ c ] and ampligen ® ( poly [ i ]: poly [ c 12 u ]) did not show any il 12 production at 4 hours . however , they both produced significant and equally high levels of il 12 at 19 hours . the il 12 level was subsequently lower at 27 hours and continued to drop at 43 hours . the overall fall in production level at each time point was more marked with poly [ i ]: poly [ c ] compared with ampligen ® ( poly [ i ]: poly [ c 12 u ]) the results show for the first time the capacity of ampligen ® ( poly [ i ]: poly [ c 12 u ]) to cause both phenotypic maturation of dendritic cells and activation of il 12 production in these cells . ampligen ® ( poly [ i ]: poly [ c 12 u ]) is also shown to have a greater capacity to mature dendritic cells compared to poly [ i ]: poly [ c ]. furthermore , the il 12 production induced by ampligen ® ( poly [ i ]: poly [ c 12 u ]) appears to be sustained for a longer period compared with that associated with poly [ i ]: poly [ c ]. the overall findings indicate that ampligen ® ( poly [ i ]: poly [ c 12 u ]), with its non toxic clinical profile , possesses significant potential as an agent for causing dendritic cell maturation and activation of il 12 production , two attributes which are believed to be important in optimal priming and induction of antigen - specific cytotoxic t - cell response by antigen - primed dendritic cells . furthermore , as ampligen ® ( poly [ i ]: poly [ c 12 u ]) is manufactured to a clinical grade and has a non toxic clinical profile , it is suitable for use together with dendritic cells which are intended for use in a vaccine . a vaccine for stimulating the cellular immune response in patient &# 39 ; s diagnosed with cancer can be produced . in the method of producing the vaccine , immature dendritic cells are exposed to the patient &# 39 ; s tumour - associated antigens to produce tumour antigen presenting immature dendritic cells . the antigen presenting dendritic cells are then matured in the presence of ampligen ® ( poly [ i ]: poly [ c 12 u ]) by the method described above , and then included in a pharmaceutical formulation in the form of a vaccine . the vaccine can then be injected into the patient , whereupon it is expected that the mature dendritic cells will migrate to the patient &# 39 ; s regional lymph nodes to induce ctl response . the applicant believes that poly [ i ]: poly [ c 12 u ], poly [ c ]: poly [ i 12 u ), poly [ i ]: poly [ c 6 u ] and poly [ i ]: poly [ c 24 u ] are representative of the class of specifically configured high molecular weight dsrna polymers , and from the results shown it is to be expected that other specifically configured high molecular weight dsrna polymers will also be suitable for maturing dendritic cells . examples of such specifically configured high molecular weight dsrna polymers are poly [ i ]: poly [ c x u ]; 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