Patent Application: US-73959908-A

Abstract:
the invention relates to a novel peptide , polypeptide or monoclonal antibody for use in the treatment of conditions characterized by elevated levels of β - amyloid in a subject , especially alzheimer &# 39 ; s disease . the invention particularly relates to an amyloid precursor protein - binding polypeptide which , when administered outside an app - expressing cell , reduces the release of aβ 39 - 43 by the cell .

Description:
monoclonal antibodies were raised against an immunogenic peptide ( eeisevkmdaefrhd ; seq id no : 3 ), termed ka , which represented the amino acid sequence spanning the β - secretase cleavage site in human app . the peptide was synthesised using a poly - lysine , multiple antigenic peptide ( map ) core and then used to immunise female balb / c mice ( 20 - 25 g ). all experiments on mice were performed in accordance with the animals ( scientific procedures ) act 1986 administered by the u . k . government home office and with ethical approval from cardiff university . mice were immunized and hybridomas were generated by standard methods as first developed by kohler and milstein ( 1975 ) and detailed elsewhere , including liddell and cryer ( 1991 ). the hybridoma supernatants were screened for high - affinity mabs by indirect elisa using the immunising peptide , prior to more complete cross - reactivity screenings . the 2b3 and 2b 12 clones were chosen for further screening based on preliminary experiments . a human cell line , astrocytoma mog - g - uvw ( mog ), which constitutively expresses app , was used to investigate the characteristics of 2b3 and 2b12 . the cells were used as a source of app and to characterise the binding properties of 2b3 and 2b12 . they were also used as a model system to investigate whether 2b3 and 2b12 could reduce aβ40 levels and for cell viability studies . mog were grown in a 1 : 1 solution of ham &# 39 ; s f10 and dulbecco &# 39 ; s modified eagle &# 39 ; s medium supplemented with 10 % foetal bovine serum ( fbs ) ( perbio science u . k . ltd , cramlington , u . k .) and 2 mm glutamine . cell lines were incubated at 37 ° c . in 5 % co 2 in air . the 2b3 and 2b12 antibodies were concentrated from culture medium using amicon centriplus ym - 100 filters ( millipore , billerica , mass ., usa ) with a nominal molecular weight cut off of 100 kda and the isotype determined using the isostrip mouse monoclonal antibody isotyping kit ( serotec , oxford , u . k .). detection of 2b3 and 2b12 binding to app by western blotting western blotting was performed using standard methods ( kidd et al 1998 ). the samples and molecular weight marker ( precision plus protein standards marker , bio - rad laboratories , hercules , calif .) were loaded onto 10 % polyacrylamide gels and separated by sodium dodecyl sulphate - polyacrylamide gel electrophoresis ( sds - page ) in running buffer ( 25 mm tris base , 190 mm glycine , 0 . 05 % sds , ph 8 . 3 ). the separated proteins were then blotted on to 0 . 2 μm nitrocellulose membranes ( amersham biosciences , little chalfont , uk ), washed in tris - buffered saline with tween 20 ( tbst , 2 mm tris , 15 mm nacl , 0 . 1 % tween - 20 , ph 7 . 5 ) and blocked for 1 hour , at room temperature ( rt ), in tbst supplemented with 5 % w / v fat - free dried milk ( blotto ). blots were then incubated with the relevant antibody in 1 % blotto at 4 ° c . overnight . membranes were washed five times in tbst and incubated with the relevant horseradish peroxidise ( hrp )- conjugated secondary antibody ( 1 : 20 , 000 , vector laboratories , burlingame , calif .) for 1 hour at rt . the membranes were washed as above in tbst , the bands visualised using enhanced chemiluminescent detection ( super signal ®, west dura , perbio science u . k .) and exposed to high performance chemiluminescent x - ray film ( amersham biosciences ). we used a competition assay in conjunction with a sandwich elisa which recognised an epitope in the region of the c - terminus of app ( app duoset , r & amp ; d systems , abingdon , u . k .). lysates were prepared from mog cells using lysis buffer ( 50 mm tris , 5 mm edta , 150 mm nacl , 1 % triton , 0 . 4 mm navo 4 , 50 mm naf , 1 mm pmsf , 20 μm phenylarsine oxide , 10 mm sodium molybdate , 10 μg / ml leupeptin , 10 μg / ml aprotinin ) and then concentrated through amicon centriplus ym - 100 centrifugal filters with a nominal molecular weight cut - off of 100 kda . recovered app was quantified using a sandwich elisa by comparing values with standard recombinant human βpp from the app duoset . the elisa followed the manufacturer &# 39 ; s guidelines . briefly , the capture antibody was used at 4 μg / ml in phosphate - buffered saline ( 137 mm nacl , 1 . 5 mm kh 2 po 4 , 8 mm na 2 hpo 4 . 12h 2 o , 2 . 5 mm kcl , ph 7 . 4 ) ( pbs ) to coat 96 - well microplates ( greiner bio - one , stonehouse , u . k .) and incubated overnight at room temperature ( rt ). plates were blocked with 1 % bovine serum albumin ( bsa ) and 5 % sucrose in pbs for a minimum of 30 minutes . samples and standards were prepared in 1 % bsa in pbs and incubated on the plate for 2 hours . a six point standard curve was prepared with a highest concentration of 20 ng / ml . biotinylated detection antibody , 300 ng / ml in 1 % bsa in pbs , was incubated for 1 . 5 hours and detected using streptavidin - hrp , diluted 1 : 200 in pbs with 0 . 05 % tween 20 ( pbst ) for 20 minutes , followed by the enzyme substrate , o - phenylenediamine ( opd ), in 0 . 1m citrate - phosphate buffer , ph 5 . 0 , incubated for 20 minutes . the reaction was stopped with 2 . 5m h 2 so 4 and the absorbance determined , at 492 nm . all incubations were performed at room temperature ( rt ), unless otherwise stated and wells were aspirated and washed four times with pbst between each stage . the competition assay for app followed the same protocol except for the following modifications . mog cell lysate was used to provide app at a concentration of 30 ng / ml . prior to incubating with the detection antibody , the samples were incubated with 2b3 , b12 or an irrelevant mouse igg antibody ( perbio science ) from 1 to 20 μg / ml for 1 hour . binding of these antibodies was then inferred by a decrease in binding of the detection antibody compared to the pbst control alone . affinity ranking of the two antibodies was accomplished by comparing their binding properties to a peptide , kb , which spans the β - secretase cleavage site on app , in an indirect elisa . this peptide represents a 15 amino acid sequence ( sevkmdaefrhdsgy ; seq id no : 4 ), slightly further into the aβ region of app than ka , the immunising peptide . kb was adsorbed to a 96 well microtitre plate at a concentration of 10 ug / ml in carbonate / bicarbonate buffer ( 15 mm na 2 co 3 , 35 mm nahco 3 , ph9 . 8 ) overnight at 4 ° c . plates were then blocked with 1 % ( w / v ) non - fat milk powder in pbst for 1 hour . 2b3 or 2b 12 were incubated for 2 hours at concentrations ranging from 0 . 00001 to 30 μg / ml and detected with a secondary anti - mouse antibody conjugated to hrp , 1 : 2500 ( pierce ) for 1 hour , followed by opd and detection as above . all results were expressed as a proportion of a standard antibody ( 6e10 , chemicon , chandlers ford , u . k .) at 0 . 05 ug / ml . curve fitting was performed using graphpad prism ® version 4 . persistence of 2b3 and 2b12 binding in a range of ph buffers the binding persistence of 2b3 and 2b12 to kb was investigated at a range of ph values using an indirect elisa . methods followed those detailed above with the following modifications . kb was adsorbed and blocked as above . 2b3 and 2b12 ( 5 μg / ml ) were incubated with kb for 1 hour in pest ph 7 . 4 and the antibody solution was aspirated . the immunocomplex was then incubated for up to a further hour in pbst at either ph 7 . 4 , 6 , 5 or 4 for 0 , 15 , 30 or 60 minutes . binding of both antibodies was detected as above and all results were expressed as a proportion of a standard antibody ( 6e10 ) at 0 . 05 μg / ml . pbs was adjusted to the correct ph with h 3 po 4 . the epitopes of 2b3 and 2b12 on app were investigated by comparing the relative binding profiles of the antibodies to cleavage products of app in an indirect elisa . methods 20 , followed those detailed above with the following modifications . recombinant sappα and sappβ ( sigma ) were adsorbed to a 96 well microtitre plate at a concentration of 5 ug / ml ( in carbonate / bicarbonate buffer ) overnight at 4 ° c . plates were blocked with 1 % non - fat milk powder for 1 hour and 2b3 and 2b12 subsequently incubated at 1 ug / ml for 2 hours . binding of both antibodies was detected as above and binding to sappβ was expressed as a percentage of the binding of the respective antibody to sappα . viability studies were performed on mog cells after incubation with 2b3 or 2b12 using the mts ( 3 -( 4 , 5 - dimethylthiazol - 2 - yl )- 5 -( 3 - carboxymethoxyphenyl )- 2 -( 4 - sulfophenyl )- 2h - tetrazolium inner salt ) assay ( celltiter 96 ® aqueous one solution cell proliferation assay , promega ). mog cells were added to 96 - well cluster plates at a concentration of 2000 per well in 100 μl , allowed to adhere overnight and then incubated with 100 μl of either 2b3 , 2b12 ( 10 ug / ml ) or media control for 48 hours . 20 μl celltiter 96 ® aqueous one solution reagent was added directly to the culture wells and incubated in the dark for 1 . 5 hours . the resulting formazan product was quantified by measuring absorbance at 490 nm and results were expressed as a percentage of media control values . all experiments were performed in 24 well cluster plates . 1 . 08 × 10 4 mog cells were plated per well . all samples were done in triplicate on each plate . cells were allowed to attach overnight then incubated with control media , 2b3 , 2b12 , an anti - n - terminal app antibody ( millipore , watford , u . k .) or the irrelevant mouse igg ( all at 10 μg / ml ) in relevant culture media for 2 days at 37 ° c . for subsequent analysis of aβ40 , 450 μl of media were collected from each well and subjected to combined immunoprecipitation and elisa . media was first immunoprecipitated with 1 : 2 , 000 bam401s ( autogen bioclear , calne , u . k , specific to the c - terminus of human aβ40 , overnight at 4 ° c . samples were then incubated with protein a ( santa cruz biotechnology , santa cruz , calif ., usa ) for 2 hours and then washed with chaps buffer ( 150 mm nacl , 50 mm tris , 1 mm edta , 10 mm chaps ) and centrifuged at 3275 g , repeated 3 times . this was followed by a final wash in pbs . samples were then boiled at 95 ° c . for 5 minutes to dissociate the protein a before being centrifuged for 1 minute at 3275 g . the supernatant was subsequently removed and tested in an elisa for aβ40 following the methods above , except for the following . the elisa employed the n - terminal aβ mab 6e10 ( chemicon ), as the capture antibody and affinity - purified bam401ap ( autogen bioclear ), specific to the c - terminus of human aβ40 , as the detection antibody . 6e10 , 5 μg / ml in carbonate / bicarbonate buffer , was incubated overnight at 4 ° c . plates were then blocked with 0 . 1 % milk powder in pbs for 1 hour . samples and standards were added to the plates and incubated for 1 . 5 hours followed by the detection antibody , 0 . 45 μg / ml in pbst for 1 . 5 hours . the plates were then blocked for 30 minutes and a secondary anti - rabbit antibody conjugated to hrp , 1 : 2500 ( vector laboratories ), was incubated for 1 hour . binding was detected with opd as above . this resulted in an elisa with a lower limit of sensitivity of approximately 0 . 1 ng / ml giving good differentiation within the range of aβ40 immunoprecipitated from the cell lines . the concentration of aβ40 in samples from the cells was calculated from an aβ40 standard curve ( 0 - 12 . 5 ng / ml ) run on the same plates and generated in graphpad prism and normalised to the total cell protein content . aβ40 concentrations were then expressed as a percentage of the relevant control values . the competition assay data were analysed by two methods . differences from media controls at 100 % were analysed with student &# 39 ; s t - tests while differences between 2b3 and 2b12 were analysed using anova followed by bonferroni post - hoc tests . affinity ranking of 2b3 and 2b12 was performed using graphpad prism ® 4 . log antibody concentration was plotted against % absorbance and a sigmoidal dose - response curve fitted to the data . curve parameters were compared using the f - test . differences between 2b3 and 2b12 at each concentration were compared using a student &# 39 ; s t - test . the persistence in binding of the two antibodies , at a range of ph values , was investigated separately , using glm univariate analysis , with absorbance as the dependent variable and ph and time as factors . this was followed by bonferroni post - hoc tests . antibody ( either 2b3 or 2b12 ) binding to sappβ was expressed as a % of binding to sappα ( 100 %). this value was compared to 100 % with a student &# 39 ; s t - test . the % change in binding to the two fragments for each antibody was also compared using a student &# 39 ; s t - test . the absorbance obtained at 490 nm in the mts assay , after incubation with either 2b3 or 2b12 , was expressed as a % of media control ( 100 %) and this value was again compared to 100 % with a student &# 39 ; s t - test . the data generated in the elisa to measure aβ40 levels were analysed using a student &# 39 ; s t - test to determine if aβ concentrations were significantly lower than media controls ( 100 %) or if there was a significant difference between 2b3 and 2b12 . unless specified , data analyses were performed using spss 12 . all comparisons were made at the two - tailed significance level . 2b3 was generated with the same peptide , ka ( eeisevkmdaefrhd ; seq id no : 3 ) conjugated to a map core , used to make 2b12 . however , the two antibodies are different as 2b12 is of the igg2b class while 2b3 is an igg1 . importantly , the two antibodies also bind to different epitopes as the pattern of binding revealed by western blotting for app from cell lysates of mog cells which constitutively express app is different ( fig1 ). both antibodies recognise app ( 106 kda band ) but the binding of 2b12 to this band is stronger than that to the 56 kda band thought to be the thrombin cleavage product . however , the reverse is seen with 2b3 which binds more strongly to the 56 kda fragment than to full - length app . furthermore , additional bands , probably other fragments of app , were detected with 2b3 but were not seen with 2b12 . therefore the antibodies are clearly not the same . 2b3 binds more effectively to app than 2b12 as demonstrated by a competition assay to detect binding to app ( fig2 ). a commercial elisa kit to measure binding to app was used to quantify the binding of both antibodies . here , one antibody from the kit was used to capture app from mog cell lysate and media or 1 - 20 μg / ml of 2b12 , 2b3 or an irrelevant mouse igg was added to bind to a different epitope on app . finally , the second antibody from the kit was used which binds to a third epitope on app . any reduction in the binding of the second antibody seen with 2b12 , 2b3 or the irrelevant mouse igg was expressed as a percentage of the media control . fig2 clearly shows that , even at a very high concentration of the irrelevant igg ( 20 μg / ml ), the binding of the second commercial antibody was only reduced non - significantly by 3 . 3 %, probably due to non - specific binding arising from the high concentration of protein present . at and 20 μg / ml 2b12 significantly reduced the binding of the second commercial antibody by 10 . 9 and 17 . 1 %, respectively , similar to the levels we had seen previously ( thomas et al ., 2006 ). however , at 10 and 20 μg / ml 2b3 significantly reduced the binding of the second commercial antibody by 17 . 7 % and 27 . 1 %, respectively . these were significantly greater reductions than those seen with 2b12 . therefore we can conclude that 2b3 binds significantly more efficiently to app than 2b12 . this experiment also provides further confirmation that the two antibodies are not the same and bind to different epitopes . the comparison of the half - maximal binding of 2b3 and 2b12 to the peptide kb ( sevkmdaefrhdsgy ; seq id no : 4 ), which spans the 13 - secretase cleavage site , confirmed these results . the half - maximal concentration for binding of 2b3 to kb , 1 . 279 μg / ml ( 95 % confidence interval ( ci ) 1 . 153 to 1 . 418 ), was significantly lower than that of 2b12 , 2 . 963 μg / ml ( ci - 1 . 696 to 5 . 177 ) ( p & lt ; 0 . 0001 ). in addition , the entire curve for 2b3 binding to kb was significantly different to that for 2b12 ( p & lt ; 0 . 0001 ) and the binding of 2b3 to kb was significantly higher than 2b12 at all concentrations greater than 0 . 1 μg / ml ( p & lt ; 0 . 05 , fig3 ). these data further support our contention that the two antibodies are not the same . 2b3 and 2b12 will bind to app outside the cell at ph 7 . 4 but , once internalised into endosomes / lysosomes , they will be subjected to a much lower ph environment around ph 4 . 5 . therefore we tested the persistence of binding of a single concentration ( 5 μg / ml ) of 2b3 and 2b12 to kb at different ph values . the antibodies were bound to the peptide at physiological ph 7 . 4 and then incubated for different periods of time at ph 4 , 5 , 6 or 7 . 4 . we showed that the binding of 2b3 to kb was significantly affected by ph with a reduction in binding compared to ph 6 and ph 7 . 4 seen with ph 4 ( p & lt ; 0 . 01 , p & lt ; 0 . 0001 , respectively ) and 5 ( p & lt ; 0 . 05 , p & lt ; 0 . 0001 , respectively ) ( fig4 ). however , the time of incubation at different ph values did not significantly affect the binding . the binding of 2b12 to kb was not significantly affected by ph or time of incubation ( fig4 ). these data are important as they show that , although 2b3 binding was reduced at lower ph values , it still had higher binding than 2b12 at all ph values and time points . furthermore the lack of effect of time suggests that the antibodies will continue to work to prevent β - secretase access inside the cell for a biologically relevant period of time . finally , these findings support earlier evidence that 2b3 and 2b12 are different antibodies . additional evidence to support our contention that 2b3 and 2b12 are different antibodies with different epitopes comes from studies looking at the binding of these two antibodies to sappα and sappβ , produced from app by the cleavage of α - and β - secretases , respectively . the % of antibody bound to sappβ compared to sappα was 76 . 2 ± 1 . 7 % for 2b3 and 105 . 6 ± 2 . 1 % for 2b12 . the % bound for 2b3 was both significantly different from 100 % ( p & lt ; 0 . 01 ) and from 2b12 ( p & lt ; 0 . 001 ) while the % bound for 2b12 was not different from 100 %. these data show that 2b3 recognises sappα better than sappβ while 2b12 recognises both app fragments to a similar extent . this is probably due to differences in binding sites with the likely epitope of 2b3 being more in aβ than that for 2b12 ( fig5 ), although importantly neither antibody binds to aβ per se . we have previously show that 2b12 was not toxic to cells in culture ( thomas et al ., 2006 ) and have now extended this data to compare the effect of 2b3 and 2b12 on the growth of mog cells . cells were incubated with either antibody at 10 μg / ml for 2 days ( same time course as for the inhibition of aβ production ) and the numbers of viable cells were then measured using an mts assay . neither antibody had any significant effect compared to the media control on the number of viable cells measured after 48 hours ( fig6 ). these data demonstrate that 2b3 is not toxic to cells in culture and that the reductions seen in aβ40 levels are due to another mechanism besides cell death . having demonstrated that 2b3 bound more efficiently to app than 2b12 , we then investigated whether it reduced the production of aβ40 in a similar manner to 2b12 . aβ40 levels from mog cells incubated with either media alone or media containing 10 μg / ml 2b12 , 2b3 , the anti - n - terminal app antibody or the irrelevant mouse igg for 2 days were measured . the results were expressed as a percentage of the media control which was taken as 100 %. similarly to our previous results ( thomas et al ., 2006 ), we found that the irrelevant igg reduced the production of aβ40 by 10 . 5 % which was not significant ( fig7 ). the anti - n - terminal app antibody increased the production of aβ40 by 6 . 8 % which was also not significant ( fig7 ). 2b12 significantly inhibited the production of aβ40 by mog cells by 34 . 7 % compared to control , a similar result to that we had obtained before ( thomas et al ., 2006 ). however , 2b3 produced a much greater inhibition of aβ40 production by mog cells of 63 . 2 % which was significantly different to both control and 2b12 ( fig7 ). these data clearly demonstrate that 2b3 can block the access of β - secretase to its cleavage site more effectively than 2b12 and thus produce a greater reduction in the production of aβ40 . it is likely that this is due to increased binding of 2b3 to app shown above ( fig3 ). furthermore , we have now demonstrated that neither incubation with a large protein ( irrelevant igg ) nor with another antibody which binds to a different region of app upstream of 2b3 and 2b12 ( anti - n - terminal app antibody ), produces steric hindrance of the action of β - secretase . these data suggest that our findings are due to the specific effect of our antibodies on β - secretase activity . our data have shown that 2b3 is a significantly better antibody than 2b12 with regard to its binding to app , its half - maximal binding to kb and its ability to inhibit the production of aβ40 . in addition , 2b3 is different to the antibody produced by arbel et al . ( 2005 ) as it is raised to the native peptide spanning the β - secretase cleavage site rather than to the half - swedish mutation peptide used by arbel and co - workers ( 2005 ). this is advantageous as the majority of people with ad do not express this mutation so our antibody will recognise the constitutively expressed app found in the majority of ad patients very efficiently . in addition , unlike arbel et al . ( 2005 ), 2b3 was characterised using a cell line which expresses endogenous app , not one which over - expresses this protein . again this reflects the situation seen in the majority of ad sufferers who do not express the very high levels of app found in transfected cells . the very large decreases in aβ40 seen in this system suggest that 2b3 might be effective in man while there is no comparable data available for the antibody made by arbel et al . ( 2005 ). arbel , m ., yacoby , i . and solomon , b . 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