Patent Application: US-201113876103-A

Abstract:
the present invention concerns a nucleotide aptamer having the sequence : 5 ′- augaucaaucgccucaauucgacaggaggcucac - 3 ′ for use in the treatment and / or prevention and / or diagnosis of an axl receptor tyrosine kinase induced disorder and a pharmaceutical composition comprising the same . the invention also relates to a method for the diagnosis of an axl receptor tyrosine kinase induced disorder in a patient from which a sample is obtained and related diagnostic kit .

Description:
gl21 52 - 85 is a 2 ′- fluoropyrimidine ( 2 ′ f - py ) nuclease - resistant rna aptamer consisting of 34 nt : 5 ′ augaucaaucgccucaauucgacaggaggcucac3 ′ ( seq id no : 1 ) gl21 52 - 85 and an unrelated sequence used as a negative control were purchased from sigma ( sigma , st . louis , mo .). human glioma u87mg , human breast skbr3 , mcf7 , mda - mb - 231 cells and epidermoid carcinoma a431 ( all from american type culture collection , manassas , va . ), were grown in dulbecco &# 39 ; s modified eagle medium ( dmem ) supplemented with 10 % fetal bovine serum ( fbs ) and 2 mm l - glutamine ( invitrogen , carlsbad , calif .). axl gene silencing in glioma u87mg cells was established by transfection of an high performance short hairpin rna ( shrna ) specifically targeting axl ( from expression arrest ™ human shrna collection , open biosystems , huntsville , ala .). controls were performed using a non - related shrna ( shrnactrl ) that do not lead to the specific degradation of axl mrna open biosystem ( cat . number rhs1707 ). axl expression in human breast skbr3 cells was obtained by transfection of axl truclone ( origene , rockville , md .). cells ( 3 . 5 × 10 5 cells per 6 cm plate ) were grown and overlaid with the transfection mixtures containing the shrna against axl or axl truclone ( 6 μg ) and lipofectamine 2000 ( invitrogen , carlsbad , calif .) in opti - mem i reduced serum medium ( invitrogen ). after 5 hours incubation , complete culture medium was added to the cells and incubation was prolonged up to 72 hs . for binding assays transfected cells were plated in 24 well plates after 24 hs from transfection . binding experiments were performed with 5 ′-[ 32 p ]- labeled rna . for labeling 2 ′- f - py rnas were 5 ′- end dephosphorylated using bacterial alcaline phosphatase ( invitrogen , carlsbad , calif .) before [ 32 p ]- 5 ′- end - labeled using t4 kinase ( invitrogen ) and y -[ 32 p ]- atp ( 6 × 10 3 ci / mmol , ge healthcare bio - sciences , uppsala , sweden ) according to the supplier &# 39 ; s instructions . for binding on cells , 3 . 5 × 10 4 cells were plated in 24 - well plates in triplicate and were incubated with gl21 52 - 85 aptamer or unrelated sequence used as a negative control at 50 nm concentration in 200 μl of dmem serum free for 20 min at rt in the presence of 100 μg / ml polyinosine as a nonspecific competitor ( sigma , st . louis , mo .). after five washings with 500 μl dmem , bound sequences were recovered in 300 μl of sds 1 %, and the amount of radioactivity recovered was counted . the aptamers ability to bind axl , dtk and mer soluble extracellular domain was investigated by filter binding by plotting the fraction of rna bound to the nitrocellulose filter as a function of protein concentration , using the following equation : where bmax is the extrapolated maximal amount of rna protein complex that will be bound . 1 nm of radiolabelled aptamers ( gl21 52 - 85 or unrelated ) were incubated with 1 , 3 . 2 , 10 , 32 , 100 , 320 and 1000 nm of axl , dtk , mer soluble extracellular domain ( all from r & amp ; d systems , minneapolis , minn .) for 15 min at 37 ° in phosphate - buffered saline ( pbs ) supplemented with 0 . 01 % bovine serum albumin . after incubation , the aptamer - protein mix was passed through nitrocellulose membrane filter ( millipore co ., bedford , mass .) and filters were counted . in all binding assays the background values obtained with the unrelated rna aptamer were subtracted from the values obtained with the gl21 52 - 85 specific aptamer . to assess the effects of aptamers on axl activity , u87mg cells ( 1 . 5 × 10 5 cells per 3 . 5 - cm plate ) were serum - starved overnight , pretreated with 200 nm gl21 52 - 85 aptamer or the unrelated aptamer used as a negative control for 3h and then stimulated for 30 min with 400 ng / ml gas6 ( r & amp ; d systems , minneapolis , minn .) either alone or in presence of each aptamer . the aptamers were subjected to a short denaturation - renaturation step ( 85 ° c . for 5 min , snap - cooled on ice for 2 min , and allowed to warm up to 37 ° c .) before each treatments . to prepare cell extracts , cells were washed twice in ice - cold pbs , and lysed in buffer a ( 50 mmtris - hcl ph 8 . 0 buffer containing 150 mmnacl , 1 % nonidet p - 40 , 2 mg / ml aprotinin , 1 mg / ml pepstatin , 2 mg / ml leupeptin , 1 mm na 3 vo 4 ). protein concentration was determined by the bradford assay using bovine serum albumin as the standard . the cell lysates were subjected to sds - page . gels were electroblotted into polyvinylidene difluoride membranes ( millipore co ., bedford , mass . ), and filters were probed with the indicated primary antibodies : anti - axl and anti - phospho - axl ( r & amp ; d systems ); anti - erk1 ( c - 16 ) ( santa cruz biotechnology , calif ., usa ); anti - phospho - 44 / 42 map kinase ( e10 ) ( also indicated as p - erk ) ( cell signaling , beverly , mass . ); anti - α - αtubulin ( dm 1a ) ( sigma , st . louis , mo .). proteins were visualized with peroxidase - conjugated secondary antibodies using the enhanced chemiluminescence system ( ge healthcare bio - sciences , uppsala , sweden ). where indicated , filters were stripped in 62 . 5 mm tris - hcl ph 6 . 8 with 100 mm 2 - mercaptoethanol and 2 % sds for 30 min at 54 ° c ., and reprobed . cell viability was assessed with celltiter 96 ® aqueous one solution cell proliferation assay ( promega , madison , wis .) according to according to the supplier &# 39 ; s instructions . cells ( 4 × 10 3 cells / well ) were plated in 96 - well plates in triplicate and were treated for 24 hs with heat denatured gl21 52 - 85 or the unrelated aptamer at 3 μm concentration . rna concentrations were determined to ensure the continuous presence of a concentration of at least 200 nm , which takes into account the 6 hs - half life of the aptamer in 10 % serum . the optical density ( od ) was measured using a multilabel counter ( bio - rad ) at a wavelength of 490 nm and cell viability was calculated by the following formula : a549 or u87 mg cells were pre - treated with 200 nmgl21 52 - 85 aptamer or unrelated aptamer and following 3 h trypsinized , re - suspended in dmem serum free , and counted . cells ( 10 × 10 4 in 100 μl serum - free medium per well ) were then plated into the upper chamber of a 24 - well transwell ( corning ). cells were exposed in the presence of gas6 ( 400 ng / ml ) as an inducer of migration in serum - free medium ( 0 . 6 ml ) in the lower chamber , or were exposed in the presence of 10 % fbs . after incubation at 37 ° c . in humidified 5 % co2 for 24 hs , cells were visualized by crystal violet staining . 10 4 u87mg or a549 cells were plated in 60mm dishes in a solution containing dulbecco &# 39 ; s modified eagle &# 39 ; s medium 2 × ( sigma , st louis , mo ., usa ), tryptose phosphate broth ( difco , bd , franklin lakes , n . j ., usa ), and 1 . 25 % of noble agar ( difco ). briefly , cells were harvested and counted then a layer of 7 ml with the solution containing noble agar were left to polymerize on the bottom of the dishes . then cells were re - suspended in 2 ml of same solution and plated . cells were left grown for 2 weeks in the incubator . cells were grown in dmem - f12 supplemented with 1 % b - 27 , human recombinant bfgf ( 10 ng / ml ), and egf ( 20 ng / ml ). the number of spheroid - forming colonies was counted after 10 days . athymic nude mice ( nu / nu ) were maintained in a sterile environment according to guidelines established by the us department of agriculture and the american association for accreditation of laboratory animal care ( aaalac ). mice were inoculated with either 3 × 10 6 ( in 100 μl ) in vitro propagated mda - mb - 231 or a549 cells subcutaneously injected into each flank . approximately 24 non - necrotic tumors for each tumor type , of about 1 cm in diameter , were randomly divided into three groups of eight mice per treatment group as follows : group 1 , no treatment ; group 2 , treated with unrelated rna as a negative control ( 200 pmols / injection ); group 3 , treated with gl21 52 - 85 ( 200 pmols / injection ). compounds were injected intra - tumorally in 100 - μl volumes three times a week for two weeks . day 0 marks the first day of injection . aptamers may also be administrated systemically , in particular when optimized by addition of polyethyleneglycol ( peg ). the volume injections are small enough to preclude the compounds being forced inside the cells due to a nonspecific high - pressure injection . tumors were measured every 3 days with calipers in three dimensions . the following formula was used to calculate tumor volume : v t =( w × l × h )× 0 . 5236 ( w , the shortest dimension ; l , the longest dimension , h , intermediate dimension ). the growth curves are plotted as the means tumor volume ± s . e . m . statistical analysis of tumor size data was conducted using a one - way anova . a p - value of 0 . 05 or less was considered to indicate a statistically significant difference . gl21 52 - 85 is a 2 ′- fluoropyrimidine ( 2 ′ f - py ), nuclease - resistant rna aptamer consisting of 34 nt ( fig1 ). it was obtained by reducing the length of the aptamer gl21 ( 92 mer ) that the authors have previously generated by a differential cell - selex approach on tumorigenic glioblastoma u87mg cells . the adopted selection strategy has been published and is disclosed in the international patent application wo 2010 / 023327 . the gl21 52 - 85 aptamer binds to u87mg target cells with a kd of 90 nm . the authors have identified the axl receptor as the target of gl21 52 - 85 aptamer . indeed , the ability of gl21 52 - 85 aptamer to bind to u87mg cells is significantly reduced upon decreased axl expression by means of a specific shrna ( fig2 a ). conversely , gl21 52 - 85 aptamer binds breast skbr3 cells transfected with human axl while it shows no binding on parental skbr3 cells that do not express axl receptor ( fig2 b ). accordingly , binding analyses with the gl21 52 - 85 aptamer on different cancer cell lines that display a different expression of axl show a correlation between the binding of the aptamer and axl expression ( fig3 ). indeed , among the cells tested gl21 52 - 85 aptamer binds axl - positive human glioma u87mg , breast mda - mb - 231 and epidermoid carcinoma a431 cells , whereas it does not bind to breast mcf7 and skbr3 cells that do not express the receptor . the gl21 52 - 85 aptamer recognizes specifically the axl receptor expressed on the surface of cancer cells as well as the purified soluble extracellular domain of the receptor ( fig4 ). indeed , a filter binding analysis performed with the soluble extracellular domain of axl ( indicated as ec - axl ) confirmed a strong affinity of gl21 52 - 85 for ec - axl ( kd of 13 nm ) whereas a lower affinity ( kd of 47 nm ) was obtained for the extracellular domain of dtk ( indicated as ec - dtk ). regarding the binding to the extracellular domain of mer ( ec - mer ), under the protein concentration used , no kd value could be calculated indicating that the aptamer does not bind to ec - mer or binds to the protein but with an affinity of a magnitude at least 10 3 lower . for comparison , r428 exhibits an ec50 / ic50 of 14 nmol / l in in vitro biochemical kinase assays using recombinant axl protein ( holland et al ., 2010 ). the kd value of axl mab yw327 . 6s2 vs human axl is of about 1 nm ( ye et al , 2010 ). ski - 606 and na80x1 inhibit axl kinase activity with an ic50 of 0 . 56 ± 0 . 08 micromol / l and 12 . 67 ± 0 . 45 micromol / l , respectively ( zhang et al , 2008and wo2009127417 ). the identification of an aptamer that binds to axl receptor raises the obvious question of whether this aptamer may interfere with the receptor activity . thus , the authors analysed whether gl21 52 - 85could inhibit axl phosphorylation both in basal condition ( fig5 a ) and following gas6 stimulation of axl - positive u87mg cells ( fig5 b ). as shown in fig5 a , the gl21 52 - 85aptamer , at a concentration of 200 nm , strongly inhibits phosphorylation of axl and of the downstream effector erk . furthermore , it drastically reduced the gas6 - dependent phosphorylation of axl and erk ( fig5 b ). as next step , the authors checked the effect of gl21 52 - 85 on cell growth of axl - positive cells by mtt assay . u87mg , mda - mb - 231 or a431 cells were treated for 24 hrs with gl21 52 - 85 aptamer or unrelated rna as negative control . gl21 52 - 85 aptamer reduces cell viability of all cell lines by comparison with untreated cells or treated with the control rna ( fig5 c ). the authors further examined the effects of gl21 52 - 85 aptamer on u87mg ( left panels ) and a549 ( right panels ) cell migration , and found that cells treated with the gl21 52 - 85 aptamer have a decreased capacity in motility in comparison with the cells treated with the unrelated sequence in transwell migration assay either in the presence of 10 % fbs ( fig6 a ) or in the presence of gas6 as chemo - attractant ( fig6 b ). accordingly , gl21 52 - 85 aptamer strongly reduces u87mg cell invasion in the presence of 10 % fbs ( fig6 c ). furthermore , gl21 52 - 85 reduces the number of colonies in soft agar colony formation assay ( fig6 d ) in comparison with the unrelated sequence , either in u87mg ( left panels ) or in a549 cells ( right panels ). then , the authors demonstrated that gl21 52 - 85 aptamer interferes with spheroid formation of u87mg cells ( b ), in comparison with the unrelated sequence , reducing the spheroid size ( fig7 a ) and the migration of treated u87mg - treated cells from the initial spheroids ( fig7 b ). it has been reported that inhibition of axl significantly attenuated tumor growth ( li et al ., 2009 ; ye et al ., 2010 ; holland et al ., 2010 ). thus , the authors evaluated gl21 52 - 85 aptamer on xenograft tumor growth . to this aim , nude mice were inoculated with the human breast tumor line mda - mb - 231 ( fig8 a ) or a549 ( fig8 b ) cells expressing high levels of axl and tumors were allowed to grow until they reached about 1 cm in diameter in the longest dimension . tumors were then injected ( day 0 ) with 100 μl ( 200 pmoles - final concentration ) of gl21 52 - 85 aptamer or the unrelated rna used as negative control . the injections were administered three times a week for the following two weeks . tumors were measured every 3 days . as shown in fig8 , a pronounced reduction in tumor volume is observed in the presence of the gl21 52 - 85 aptamer . indeed , from day 9 to day 15 the gl21 52 - 85 - treated mda - mb - 231 tumors stopped to grow and had a reduction in volume . accordingly , in a549 - mouse xenografts a pronounced reduction in tumor volume was observed in the presence of gl21 52 - 85 - treatment , leading at day 16 to 55 % inhibition with respect to the negative control . suppression of tumor volume was specific to the gl21 52 - 85 - treated group and was not observed with the unrelated rna . the receptor tyrosine kinase axl is expressed in various types of cancer and is involved in multiple processes of tumorigenesis , including promoting tumor cell growth , migration , invasion , metastasis as well as angiogenesis , resulting as an attractive target for therapeutic strategies . in the present invention , the authors have identified a synthetic nuclease resistant rna aptamer named gl21 52 - 85 , directed to the extracellular domain of the human axl receptor . the authors have demonstrated that the gl21 52 - 85 aptamer recognizes specifically the axl receptor expressed on the surface of cancer cells ( non small cell lung cancer , breast , glioma ) as well as the purified soluble extracellular domain of the receptor . on the other hand , it does not bind to cell lines that do not express axl . the treatment of axl - positive cancer cells with the aptamer strongly inhibits basal and ligand - mediated axl activation , leading to inactivation of axl downstream signaling with a reduction of erk phosphorylation . further , gl21 52 - 85 inhibits proliferation of cancer cells in vitro . previous studies have established the role of axl in promoting tumor cell growth ( shieh et al ., 2005 ; sainaghiet al ., 2005 ; zhang et al ., 2008 ; koorstra et al ., 2009 ; hutterer et al ., 2008 ; li et al ., 2009 ; ye et al ., 2010 ; holland et al ., 2010 ). the authors have tested the anti - tumoral efficacy of gl21 52 - 85 aptamer in xenograft model of human breast mda - mb - 231 cancer cells . remarkably , as a consequence of the axl inhibition , gl21 52 - 85 aptamer resulted able to strongly inhibit tumor growth . the data of the present invention are persuasive evidence for the clinical development of gl21 52 - 85 aptamer as an innovative inhibitor of axl for both therapeutic and diagnosis scopes . in conclusion , the identification of a neutralizing rna - aptamer specifically targeting the axl receptor opens the ways to the development of innovative cancer diagnostic and therapeutic strategies . cerchia l , de franciscis v , condorelli g . “ method for obtaining oligonucleotide aptamers and uses thereof ”, international patent application wo 2010 / 023327 . chung b i , et al ., ( 2003 ) dna cell biol 22 : 533 - 540 . graham d k , sarher s l . “ axl tyrosine kinase inhibitors and methods of making and using the same ”, international patent application wo2008098139 . holland s j , pan a , franci c , hu y et al . 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