Patent Application: US-201214239354-A

Abstract:
the present invention relates to a multi - functional protease inhibitor , which may be conjugated to various molecules . the present invention also relates to uses of the protease inhibitor and conjugates thereof .

Description:
the present invention will now be further described with reference to the following figures which show : fig1 a shows a representation of a cystatin - pepstatin conjugate as a potential inhibitor ( cpi ) of all 3 major endosomal protease families : the papain - like cysteine proteases ( plcp ), aspartyl proteases ( cat - d / e ) and asparagine endopeptidase ; fig1 b identifies three different cystatin mutants which were tested . mutation sites were picked for solvent accessibility , as well as for their location , with residues which are part of the inhibitory motifs ( as highlighted by the dashed circles ), excluded . three mutations where chosen , one on a b - sheet ( r77c ), one on an a - helix ( l117c ) and on a non - structured loop region of the protein ( t102c ). fig1 c shows relative inhibitory rates of the three mutants as determined by serial dilution of the various cystatin c mutants ( concentration in mm ) in presence of 5 ng of cathepsin l / well . fig2 a shows 4 preferred cystatin - pepstatin conjugates as a potential inhibitor ( cpi ) of all 3 major endosomal protease families : the papain - like cysteine proteases ( plcp ), aspartyl proteases ( cat - d / e ) and asparagine endopeptidase . fig2 b shows the mass spectrometric data of conjugates 4 , 5 , and 6 , as well as a western blot analysis with anti - flag 27 antibody of conjugate 7 . fig3 shows the results of inhibition of proteases : 1 - 3 . inhibition of recombinant cath l ( plcp ), aep , and cath d ( aspartyl protease ) by cystatin c ( red ), pepstatin ( blue ) or the cpi ( black ). 4 - 6 . inhibition of these same enzymes in the lysates of dendritic cells as measured by turnover of fluorescent substrates . 7 : test of residual post - lysis protease activity after feeding of the inhibitors to live dendritic cells . 8 : residual protease activity in live immune cells after feeding of the constructs as measured using a quenched fluorophoric casein substrate . fig4 : shows inhibition of degradation of apo - hrp by macrophage lysosomal proteins . pbs , nor pepstatin a warrant protection . cystatin c appears to protect a fragment only . the cpi protects the unstable antigen back to the level of wild - type hrp . fig5 a : cpi arrests egf receptor downregulation and sustains signalling . ( a ) cost cells were stimulated with egf for up to 90 mins following a pre - incubation with or without either cpi or human cystatin c (˜ 0 . 35 mg / ml ). the downregulation of the egf receptor ( top panel ) was assayed by western blotting along with levels of phospho - erk1 / 2 ( p42 / 44 map kinase ; middle panel ) and rsk2 to assess total cellular protein loading . ( b ) quantitation of data shown in a fig5 b : shows the effect of the cpi on the lifetime of the egf - egfr complex in cells upon stimulation with egf . it can be observed by confocal microscopy that the egfr receptor breakdown is retarded in presence of the inhibitor . fig6 a : shows the determination of the loading levels of ovalbumin and cystatin c on iron oxide adjuvant . equal amounts of modified iron oxide were analysed by western blot against cystatin c and ovalbumin . three different concentrations of ovalbumin were loaded together with a fixed amount of cystatin c , pepstatin a or a cystatin - peptstatin inhibitor . fig6 b : shows the responses of ot - i ( top ) and ot - ii ( bottom ) t - cells to dendritic cells incubated with constructs carrying three different amounts of ovalbumin , as determined in fig6 a with or without protease inhibitor . fig6 c shows the improved antigen presentation on class i mhc ( cross - presentation ) by co - encapsulation of antigen with cpi . plga microspheres were titrated in 96 - well plates and ovalbumin presentation measured by the addition of murine bone marrow derived dendritic cells (˜ 5 × 10 4 / well ) and oti t cells (˜ 5 × 10 2 / well ). ( i ) after ˜ 72 hours t cell proliferation was measured by addition of 1 μci 3h - thymidine . cells were harvested 16 hours later and 3 h incorporation measured by scintillation counting . alternatively ( ii ) an aliquot of the supernatant was removed after 72 hours and il - 2 production measured by standard elisa assay . fig6 d shows the improved antigen presentation on class ii mhc by co - capsulation of antigen with cpi . conditions similar to those in fig6 c except that otii ( class ii mhc restricted ) t cells were used instead of oti . fig6 e shows the improved presentation of the ovalbumin antigen in vivo by co - encapsulation with cpi . c57bl / 6 mice were immunised with different varieties of ovalbumin - containing plga microspheres and were additionally loaded with cfse labelled t cells ( see methods outline ). as a positive control ovalbumin was admixed with the strong adjuvant alum . t cell proliferation was subsequently measured by cfse dilution in the lymph nodes draining the site of injection ( sub - cut . base of tail ). t cells were also stained with anti - cd8 antibodies to distinguish ot1 from otii cells . ( i ) leftmost panels show raw facs data while centre and right panels show histograms of t cell numbers at different cell doublings for oti ( centre ) and otii ( right ). ( ii ) total accumulated t cells by integration of cell doubling data . the data reveal a hierarchy of expansion for both ot1 and otii . as expected ova / alum promoted the strongest expansion while ova / cpi plga promoted stronger expansion than ova plg . fig7 : inhibition of cysteine protease activity and aspartyl protease activity in t . brucei cell lysates as determined using substrates specific for either cathepsin b / l / s like activity or cathepsin d / e like activity . fig8 : shows some constructs that can be used for linking the protease inhibitor to another moiety , for example a targeting group , or a complex with a targeting group . ovalbumin was purchased from sigma - aldrich ( a5503 ), or worthingtons ( ls3056 ; in vitro digest ), cystatin c from genway ( 11 - 511 - 248839 ). bovine serum albumin and myoglobin ( from horse - heart ) were purchased from sigma - aldrich . anti - ovalbumin was obtained from polysciences ( 2344 - 5 ) anti - 5h is was purchased from quiagen ( 34660 ), anti - cystatin c ( mab1196 ) and recombinant cathepsins came from r & amp ; d systems . pepstatin a and fluorescent substrates were purchased from bachem . nboc - 1 - cysteine methanethiosulfonate was purchased from toronto research chemicals ( b646250 ). the cystatin c was amplified as described previously &# 39 ; using the following primers : cysc for ( caggattacaattggtaccatggccgggcccc ) and cyscrev ( gcctactcgagcttaatgatgatgatgatgatggtcctgacag ) to introduce a c - terminal 6 - histidine tag . the amplification product was cloned into a pcdna - dhfr vector used previously 2 between xhoi and kpni restriction sites . threonine 102 was determined to be solvent accessible using the naccess software 3 based on the crystal structure of human cystatin c 4 and domain - swapped human cystatin c 5 . it was also a residue away from the inhibitory motifs of both the papain - like cathepsins and asparagine endopeptidase 6 . the mutations were introduced with primers cysct102cfor ( caggtgtaccaagtgccagcccaacttgg ) and cysct102crev ( ccaagttgggctggcacttggtacacgtg ). dhfr - negative cho cells were grown in dmem - based medium containing 10 % dialysed fcs , 5 mm glutamine and 0 . 1 mm hypoxanthine and 0 . 01 mm thymidine . following transfection using lipofectamine with dhfr - cysc - t102c - 6h is plasmid , the hypoxanthine and thymidine supplement were removed and the cells were cultured at low density in 15 cm dishes ( 104 cells per dish ) in medium containing 20 nm methotrexate ( mtx ). after 2 days the medium was replaced by medium containing 50 mm mtx . the cells were grown at 5 % co 2 at 37 ° c . for a further 2 days upon which single colonies had begun to form . these were picked and placed in a 96 - well plate ( tissue culture treated ) in medium containing 100 nm methotrexate . the colonies were assessed for cystatin c expression levels using an anti - cystatin c antibody mab1196 ( r & amp ; d systems , mouse anti - human cysc ; 1 : 3000 dilutions ) and the highest producing clones were harvested and transferred to a 24 - well dish . here they were allowed to grow to 80 % confluency prior to the addition of medium containing increasing amounts of mtx ( up to 2 mm ). at the final mtx - concentration one clone ( svkd2 - 25 - a7 ) was selected for large - scale production of cystatin c . this clone was grown to confluency in 10 225 cm 2 tissue culture flasks in medium containing 2 mm mtx . the cells were incubated at 37 degrees for 2 weeks prior to harvest of the supernatant . the ph of the supernatant ( 2 l ) was adjusted to 8 . 0 and nacl was added to a final concentration of 250 mm . the supernatant was filtered through a 0 . 22 μm filter prior to passing it over 6 ml of ninta agarose ( quiagen ) at 4 ° c . the resin was washed with 10 column volumes of 50 mm napo 4 , 300 mm nacl ( ph 8 . 3 ) and 5 column volumes of the same buffer containing 5 mm imidazole . the bound protein was eluted by gently shaking the agarose with 2 × 8 ml of 500 mm imidazole containing buffer followed by elution . the eluent was passed over a superdex g75 column ( ge healthcare , xk26 / 60 ) in batches of 1 . 5 ml . the fractions containing pure cystatin c were pooled and concentrated by ultrafiltration ( mwco 6 - 8000 ) to yield 21 ml of a 0 . 8 mg / ml protein solution ( as determined by uv absorbance ). mass spectrometry ( after reduction ): pepstatin ( 147 mg , 0 . 21 mmol ) was dissolved in dmf ( 15 ml ). n - hydroxysuccinimide ( 217 mg , 1 . 4 mmol ) and 1 - ethyl - 3 -( 3 - dimethylaminopropyl ) carbodiimide ( 323 mg , 1 . 7 mmol ) were added and the mixture was stirred at room temperature for 23 hours . the mixture was concentrated under high vacuum to yield a glass - like solid , which was washed with water ( 3 × 15 ml ) and diethyl ether ( 3 × 15 ml ) to yield a white powder ( 153 mg , 93 % yield ) of pepstatin a - n - hydroxysuccinimidyl ester . m / z ( esi +) observed 783 . 4 ; calculated : 783 . 5 ; 1 h nmr ( 300 mhz , dmso ) δ 7 . 91 ( d , j = 7 . 3 hz , 1h , nh ), 7 . 80 ( dd , j = 11 . 3 , 9 . 0 hz , 2h , 2 × nh ), 7 . 46 ( t , j = 9 . 2 hz , 2h2 × nh ), 5 . 27 ( d , j = 5 . 8 hz , 1h , oh ), 4 . 82 ( d , j = 5 . 0 hz , 1h , oh ), 4 . 33 - 4 . 07 ( m , 3h ), 3 . 83 - 3 . 94 ( m , 4h ), 2 . 80 ( s , 4h , hosu ), 2 . 71 ( dd , j = 16 . 5 , 2 . 2 hz , 2h ), 2 . 21 - 1 . 84 ( m , 5h ), 1 . 67 - 1 . 46 ( m , 2h ), 1 . 47 - 1 . 23 ( m , 4h ), 1 . 19 ( d , j = 7 . 1 hz , 3h , ch3 ), 0 . 97 - 0 . 72 ( m , 30h , 10 × ch3 ). 13 c nmr ( 75 mhz , dmso ) δ 172 . 34 , 171 . 54 , 171 . 09 , 170 . 72 , 170 . 64 , 170 . 05 , 167 . 38 , 68 . 96 , 68 . 37 , 57 . 95 , 57 . 80 , 50 . 64 , 50 . 53 , 48 . 26 , 44 . 38 , 40 . 36 , 40 . 08 , 39 . 80 , 39 . 52 , 39 . 24 , 38 . 96 , 38 . 68 , 38 . 49 , 35 . 27 , 30 . 28 , 30 . 03 , 25 . 62 , 25 . 39 , 24 . 16 , 23 . 37 , 23 . 24 , 22 . 22 , 21 . 76 , 21 . 62 , 19 . 26 , 19 . 22 , 18 . 31 , 18 . 27 , 18 . 09 . pepstatin ( 164 mg , 0 . 21 mmol ) was dissolved in dmf ( 20 ml ). fmoc - lysine 77 . 2 mg , 0 . 21 mmol ) was added and shaken for 2 days at room temperature . the reaction mixture was concentrated under vacuum and washed with 0 . 1m hcl solution ( 2 × 2 × 50 ml ) and water ( 6 × 50 ml ) before being lyophilised to yield 148 mg of a white solid , which could be further purified by hplc . m / z ( esi +) observed : 1036 . 6 ; calculated 1036 . 6 . 1 h nmr ( 300 mhz , dmso ) δ 7 . 91 - 7 . 29 ( m , 15h , 7 × nh & amp ; ar — h ), 4 . 84 ( s , 2h , 2 × oh ), 4 . 33 - 4 . 05 ( m , 6h ), 3 . 98 - 3 . 67 ( m , 5h ), 3 . 02 ( dt , j = 11 . 8 , 6 . 5 hz , 2h ), 2 . 20 - 1 . 81 ( m , 9h ), 1 . 77 - 1 . 22 ( m , 15h ), 1 . 20 ( d , j = 7 . 0 hz , 3h ), 0 . 95 - 0 . 66 ( m , 30h , 10 × ch3 ). 13 c nmr ( 75 mhz , dmso ) δ 173 . 92 , 172 . 14 , 171 . 54 , 171 . 07 , 170 . 82 , 170 . 67 , 170 . 62 , 156 . 11 , 143 . 81 , 143 . 77 , 140 . 67 , 127 . 60 , 127 . 03 , 125 . 24 , 120 . 06 , 69 . 15 , 69 . 01 , 65 . 57 , 57 . 96 , 57 . 78 , 53 . 74 , 50 . 71 , 50 . 42 , 48 . 33 , 46 . 63 , 44 . 38 , 40 . 35 , 40 . 07 , 30 . 41 , 30 . 28 , 30 . 05 , 28 . 68 , 25 . 62 , 24 . 14 , 23 . 40 , 23 . 22 , 23 . 05 , 22 . 21 , 21 . 89 , 21 . 61 , 19 . 26 , 19 . 21 , 18 . 34 , 18 . 26 , 18 . 13 . 3 was synthesised by standard fmoc - solid phase peptide chemistry using pybop as a coupling agent on a syro i peptide synthesiser . the coupling of pepstatin - lysine - fmoc was reacted for 24 hours . boc - cysteine mts ( trc research chemicals ) was introduced manually using standard pybop coupling conditions in the last step of the synthesis ( 4 - fold excess of amino acid and coupling reagent ). m / z ( esi +) observed : 1036 . 6 ; calculated 1036 . 6 three further conjugates 5 , 6 and 7 were made in a similar fashion , showing that a variety of peptide linker moities are suitable . dtt was added to a solution of cystatin c - t102c - 6his ( 1 mg / ml in pbs , 2 . 5 ml ) to a final concentration of 20 mm . the mixture was gently shaken at room temperature for 15 minutes and buffer exchanged into phosphate buffer ( ph 8 . 5 , 100 mm phosphate , 300 mm nacl ) by sephadex g - 25 resin ( ge healthcare ). to the reduced protein ( 50 μm 3 . 5 μl ) was added a solution of 3 in dmso ( 2 mm , 4 × 50 μl ) in 4 portions at 1 hour intervals . prior to characterization , the modified protein was purified by 6his - affinity chromatography , followed by dialysis ( pbs , 6000 - 8000 mwco , 3 × 4l ). all protease inhibition studies were carried out as described previously 7 - 9 on a fluostar optima fluorimeter ( bmg ) with 360 nm excitation and 460 nm emission wavelength filters as described previously . 1 , 2 , 9 - 10 recombinant cathepsin d and cathepsin s were purchased from r & amp ; d systems . recombinant asparagine to a aep ( 100 ng / well ) in assay buffer ( 50 mm naoac , 300 mm nacl , ph 4 . 5 , 50 μl ) was added 10 nl of the inhibitors ( in pbs ). the plate was then incubated at room temperature for 20 minutes . zala - ala - asp - mec was then added ( 100 μm , bachem , 60 μl ) and 7 - amino - 4 - methylcoumarin release was measured by fluorescence spectroscopy over time . initial rates were plotted against inhibitor concentration . 10 cathepsin d inhibition studies were performed as previously described 9 . to a solution of activated mouse cathepsin l ( r & amp ; d systems , 0 . 25 ng / μl ) in assay buffer ( 25 mm mes , ph 5 . 5 , 30 μl ) were added various serial dilutions of cystatin c ( from 1 . 4 μm ), cystatin - pepstatin and pepstatin alone . the mixture was gently shaken at room temperature for 5 minutes prior to the addition of z - leu - arg - amc ( 40 μm in assay buffer , made from a 1 mm dmso stock solution ). 7 - amino - 4 - methylcoumarin release was measured by fluorescence spectroscopy over time . initial rates were plotted against inhibitor concentration 11 . inhibition of proteases in dendritic cell or t . brucei lysates three spleens from c57 / b - 6 mice or from 10 9 t . brucei promastigotes were homogenised in a glass homogeniser in 16 ml of citrate buffer ( 200 mm , ph 5 . 5 ). the cells were lysed by repeated freeze / thaw cycles ( 6 cycles ). the supernatant was cleared by centrifugation at 18 , 000 g for 30 minutes . the cleared supernatant had a protein concentration of 5 mg / ml . 20 μg of lysate was used as protease source in the same inhibition assays as for the recombinant proteases . inhibition of endo - lysosomal protease activity in live bone - marrow derived dendritic cells to a suspension of dendritic cells , derived from bone marrow precursors as described previously 12 , ( 107 cells , 100 μl ) the inhibitors were added ( 70 μm ; 100 μl in pbs ). the cells were incubated at 37 ° c ., 5 % co2 , for 3 hours . 1 ml of cold crpmi with 10 % fcs was added to each tube and the cells were collected by centrifugation . the cells were washed 3 times with medium prior to lysis ( 50 μl ; 50 mm citrate , 1 % triton x - 100 , ph 5 . 0 ). 5 μg of this protein mixture was used to determine residual proteolytic activity as described previously 1 . lysosomes isolated from bone - marrow derived macrophages using a percoll density - gradient fractionation ( 400 ng / μl , 10 μl ) were resuspended in assay buffer ( 100 mm citrate , 2 mm dtt , ph 4 . 5 , 0 . 5 % v / v triton x - 100 ; 590 μl ). 50 μl of this diluted lysosomal suspension was plated in triplicates in a 96 - well flat - bottomed plate . 10 μl of either cystatin , pepstatin of cpi ( 3 μm final concentration ) was added to each set of three wells with pbs as a control . the mixtures were incubated at room temperature for 20 minutes prior to the addition of enzchek substrate ( invitrogen , catalog number e6638 , 20 ng / ml in assay buffer ). fluorescence emergence ( excitation 485 nm ; emission 530 nm ) was measured every 5 minutes on a fluorescent plate reader at 37 ° c . initial rates of fluorescence emergence were plotted for each of the inhibitors . a20 cells ( 5 × 10 6 / ml ; 100 μl / well ) were plated in a 96 - well plate inhibitors ( 28 μm in pbs + 1 % dmso ; 50 μl ) were added to each wells and the cells were incubated at 37 ° c ., 5 % co 2 for 30 minutes . after this time enzchek ( invitrogen , catalog number e6638 , 20 μg / ml in dmem + 10 % fcs ) was added and the fluorescence emergence ( excitation 485 nm ; emission 530 nm ) was measured every hour on a fluorescent plate reader at 37 ° c . rates of fluorescence emergence were plotted for each of the inhibitors . lysosomes isolated from bone - marrow derived macrophages using a percoll density - gradient fractionation ( 400 ng / μl , 3 . 6 μl ) were resuspended in assay buffer ( 50 mm citrate , ph 4 . 5 , 0 . 5 % triton x - 100 ; 59 μl ) inhibitor ( 70 μm ; 12 μl in pbs + 5 % dmso ) was added and the mixture incubated for 15 minutes . protein substrate ( 1 mg / ml in pbs , 9 μl ) was added to the mixture and the reaction was incubated at 37 ° c . at the indicated timepoints 20 μl of the reaction mixture was removed and boiled with lds - sample buffer and analysed by sds - page . cost cells growing in 12 - well tissue culture plates were preincubated for 1 hour with or without cystatin c ( 0 . 35 mg / ml ) or cpi ( 0 . 32 mg / ml ) and then stimulated with 100 ng / ml egf for the times shown . the cells were scraped from the well in 50 μl lysis buffer containing 50 mm tris , 150 mm nacl , 1 mm egta , 1 mm edta , 1 % np40 plus protease inhibitors ( roche , miniprotease tablet ). aliquots were heated in sds sample buffer and run on a 4 × 12 % mops % gel ( invitrogen ). after transfer to hybond ecl membrane egf receptor was revealed with a rabbit anti - egf receptor antibody ( santa cruz ) followed by peroxidase conjugated donkey anti - rabbit ( jackson ) and a standard ecl protocol ( millipore ). the blot was then stripped and reprobed for phosphor - erk ( p42 / 44 map kinase ; cell signalling ) and as as loading control , rsk2 ( santa cruz ) with appropriate secondary antibodies . the signals were quantitated using imagej analysis software . if was performed according to standard protocols . for visualization of fluorescent substrate processing ( enzchek , vide infra ), murine a20 b - cell blasts were grown on coverslips and incubated with enzchek ( 1 μg / ml final concentration ) in presence or absence of protease inhibitors as indicated ( 20 μm ) for 5 hours under standard growth conditions . following treatment , cells were fixed with 3 . 7 % paraformaldehyde in pbs , triton x - 100 permeabilized ( 0 . 1 % in pbs ), and immunostained against cd63 , visualized using an anti - mouse secondary antibody - alexa 647 conjugate on a leica sp - 2 confocal microscope using a 63 × magnification objective . analysis of egfr endocytosis and trafficking was performed as previously described with the following modifications . hela cells were grown on coverslips and serum starved in the absence or presence of cpi ( 20 μm ; 0 . 1 % fcs in dmem - pbs 1 : 1 ) and stimulated with egf ( 100 ng / ml final concentration ) for 5 or 90 minutes . samples were prepared as previously described . all images were collected using a leica sp - 2 confocal microscope equipped with a 63 × magnification objective . post - image processing and data analysis were performed using imagej . image quantification was based on 25 - 50 cells per condition per experiment and significance was calculated using a standard student &# 39 ; s t - test . synthesis and analysis of a model vaccine consisting of model antigen and protease inhibitor a vaccine construct was synthesized by conjugating both the model antigen ovalbumin and an inhibitor of all three classes of protease to a solid phase adjuvant . talon - modified iron oxide beads were dissolved in 500 μl of 100 mm phosphate buffer , 600 mm nacl ; ph 8 . 0 with 0 . 02 % tween 20 . ovalbumin - 6his was added ( 0 . 3 nmol ; 20 μl of a 0 . 23 mg / ml solution , serially diluted 1 : 2 ) was added and the mixture was shaken ( 1000 rpm ) at room temperature for 2 hours . the sample was then split into 2 equal portions . one portion was left shaking . to the other portion was added 0 . 3 nmol of cystatin - pepstatin - conjugate - 6h is . the solutions were then left shaking for a further 2 hours . after this period the particles were collected in a dynal - particle concentrator magnet ( mpc - s ) and resuspended in pbs ( 1 . 5 ml ). the particles were reconcentrated and washed with a further 3 portions of pbs . the purified particles were resuspended in 50 μl of pbs . iron content of modified particles was determined using a bathophenantroline assay , based on that reported by perry et al . 31 150 μg of particles ( 30 μl ) were diluted with lds - sample buffer ( invitrogen ; nupage ) and analysed on a 12 % nupage sds - gel ( invitrogen ). the protein content was transferred to a nitrocellulose membrane by semi - dry transfer . the membrane was blocked with 5 % w / v skimmed milk powder in pbs with 0 . 1 % tween 20 for 1 hour at room temperature . after this time a solution of anti - ovalbumin ( polysciences , 2344 - 5 , 5 mg / ml , 1 : 5000 dilution ) was added in 5 % skimmed milk powder in pbs - tween 20 and the membrane was gently shaken at 4 ° c . for 17 hours . after this period the gel was washed 3 times with pbs - tween and incubated with secondary pig - anti - rabbit - horseradish peroxidase conjugate ( 1 : 3000 in 5 % skimmed milk powder in pbs - tween 20 at room temperature for 1 hour . the membrane was washed 4 times with pbs - tween before visualising the presence of hrp with ecl western blotting detection reagent ( ge healthcare ) and exposure to photographic film . the density of the bands was normalised against a standard curve of ovalbumin of known concentrations using totallab gel analysis software ( nonlinear dynamics , newcastle upon tyne , uk ) to approximate the loading of ovalbumin on the particles . cystatin - levels and cystatin - pepstatin levels were determined using the mouse anti - human cystatin c antibody ( mouse anti - human cystatin c , mab1196 , r & amp ; d systems , 1 : 3000 dilution ) using the above western blot protocol . 10 , 000 mouse bone marrow - derived dendritic cells were incubated with 0 . 1 mg of the ovalbumin / inhibitor modified particles for 2 hours in a 96 - well round - bottom plate . the cells were washed prior to the addition of 100 , 000 ot - i or ot - ii t - cells ( purified by negative selection ) to each of the wells . after 3 days 3 h - labeled thymidine was added and incorporation thereof measured . in vitro studies : dendritic cells are incubated for 48 - 72 hours with the 3 varieties of plga microspheres ( cpi only , ovalbumin only and cpi / ovalbumin ) in the presence of t cells able to detect the presentation of peptides derived from processed and presented antigen ( ot1 and otii ). t cell activation is then measured by incorporation of 3h - thymidine during a 12 - 16 hour period . in vivo studies : c57bl / 6 mice were injected sub - cutaneously with the same plga microspheres ( 1 . 75 mg plga containing approx . 5 % w / w ovalbumin ). the injected volumes additionally contained 1 μg / ml lps to activate antigen presenting cells at the site of injection . all mice previously ( 30 - 60 mins earlier ) received 10 6 cfse labeled ot1 and otii t cells . after 24 - 48 hours t cell proliferation in draining lymph nodes was measured by cfse dilution . in one embodiment , the present invention provides a molecule which displays tight control over the stoichiometry and localization of the introduced pepstatin ; with no more than one pepstatin molecule per cystatin at a site away from the inhibitory domains of cystatin c ( see for example fig1 b ) this has been achieved through introduction of a free cysteine into the protein backbone of cystatin c by site - directed mutagenesis 25 , as it can be selectively modified in presence of other nucleophilic residues . issues associated with disulfide scrambling with the two existing disulfide bridges in cystatin c were avoided by using a mammalian expression system . various mutants were tested ( see table 1 and fig1 b ), and t102c was found to have the most favourable inhibitory properties . a c - terminal 6his - tag was also introduced , for facilitating purification and possible conjugation of the inhibitor to a solid phase carrier . in accordance with a preferred embodiment methanethiosulfonate chemistry was used to introduce the pepstatin onto the free cysteine of cystatin c 25b , 26 , due to its high selectivity for sulfhydryls and its facile introduction into the peptide backbone through a mts - boc - cysteine building block . furthermore there is the potential for endosomal release of the pepstatin by reduction of disulfides by the lysosomal thiol reductase gilt 27 . as it has been reported that conjugation of the c - terminal end of pepstatin to lysine residues did not reduce its inhibitory potential significantly 23 , the inventors decided to introduce a charged peptide between the pepstatin and the mts group ( fig2 ) to increase solubility of the conjugate . after mild reduction with , for example , 5 mm dtt ( conditions to which the disulfide - bridges of cysatin c have been shown to be stable ) of the free cysteine residue prior to coupling , the inventors obtained & gt ; 95 % protein recovery levels and & gt ; 80 % modification as determined by mass spectrometry ( fig2 ). any unreacted cystatin c could be readily separated from the cpi using hplc purification . next , the inhibitory capacity was analyzed against recombinant members of each of the three target protease families the cpi showed similar ic50 values against representative members of the three classes of endosomal protease even without reduction of the disulfide bond between cystatin c and pepstatin a ( fig3 , panel 1 - 3 ). moreover , the cpi was able to inhibit the same 3 classes of protease activity present in dendritic cell lysates ( fig3 , panel 4 - 6 ). most importantly , when the cpi conjugate was incubated with a20 cells and their protease activity determined using the enzcheck substrate , the inventors found that the probe could simultaneously abolish cathepsin d / e activity as well as reduce plcp and aep activity by ( fig3 , panel 7 ) with no cell death occurring ( as determined by trypan blue assay ). one of the potential therapeutic applications of the cpi is as a modulator of antigen processing . it has been reported that unstable antigens can be over - processed in the endo - lysosomal pathway leading to a reduction in antigen presentation 28 and that protease resistant antigens frequently make for better immunogens . the inventors tested whether these unstable antigens could be ‘ protected ’ from lysosomal over - degradation by the cpi with the eventual aim of improving antigen presentation of such unstable antigens in vaccine preparations . in recent studies by delamarre et al it was demonstrated that a destabilized variant of horseradish peroxidase ( apo - hrp ) from which the heme - group had been removed was more sensitive than heme containing hrp to proteolysis in vitro and gave a much weaker immune response in vivo . the authors suggested that heme - free hrp was too rapidly degraded by the antigen processing machinery , preventing efficient loading of mhc - complexes . the inventors tested whether it was possible to protect unstable apo - hrp from lysosomal degradation in vitro by adding a cpi to the invention . as a source of endo - lysosomal proteases the inventors used purified lysosomes from mouse macrophages as they express high levels of all these enzymes . indeed , even before the first measurement apo - hrp was fully degraded by macrophage endo / lysosomes ( fig4 ). addition of cystatin alone or pepstatin alone caused little or no stabilization of apo - hrp , indicating a functional redundancy between the lysosomal enzymes in these macrophages . when a cpi was added , however , apo - hrp was as stable to lysosomal digestion as wt - hrp ( fig4 ). these results suggest that incorporation of a cpi into immunological adjuvants for unstable antigens may be worthwhile and is currently under investigation . the inventors next assessed the capacity of cpi to inhibit endo / lysosomal proteases in live cells and whether a cpi could successfully modulate the biological functions of this compartmental system . one important role of the endocytic pathway is to degrade activated growth factor receptors following their ligand - stimulated endocytosis . for example , following the egf receptor ( egfr ) being ubiquitinated , it is clustered in clathrin coated pits and delivered to the endosome system where it becomes sequestered on the internal vesicles of multivesicular bodies ( mvbs ) preventing recycling and shutting down its capacity to signal 29 . mvbs then fuse with lysososomes and the egf receptor is degraded ; the specific lysosomal proteases remain to be fully defined . 11 , 35 . the inventors precincubated the egfr positive kidney cell line cos - 7 in the presence or absence of a cpi or cystatin c ( the insolubility of pepstatin a prevented this compound from giving meaningful data in this experiment ) and then challenged with egf . at different times the level of egfr remaining was monitored by western blotting ( fig5 ). in control cells downregulation of egfr was evident after 40 minutes and virtually complete after 90 minutes ( fig5 a ). in contrast levels of egfr were much more persistent in cos7 cells preincubated with cpi demonstrating a block in receptor degradation as quantified in fig5 b . preincubation with cystatin also suppressed egfr downregulation but not to the same extent as cpi indicating that both cysteine and aspartyl proteases are involved in egfr processing . the arrest in receptor processing was not due to inhibitor toxicity since the map kinases erk1 / 2 were activated 5 normally in cpi and cystatin treated cells . in fact , there was more sustained erk activation in cpi treated cells consistent with the persistence of egfr ( fig5 a , 2nd panel ). thus , cpi is taken up by cells and can suppress key proteolytic events within the endo / lysosomal system . the inventors also tested the protease inhibitor in a model vaccine . first , defined amounts of antigen ( ovalbumin ) and inhibitor ( fig6 a ) were loaded onto a solid - phase carrier not unlike those used in commercial vaccine preparations . next these vaccine preparates were fed to mouse dendritic cells for 2 hours . after washing , purified ot - i and ot - ii t - cells specific for epitopes on ovalbumin were added and an improved response of the ot - i and ot - ii t - cells could be observed ( fig6 b ). fig6 c and d show that the inclusion of cpi substantially improves presentation of the ovalbumin antigen . beads containing cpi alone produced no t cell proliferation ( not shown . moreover , a ova / cpi plga (+ lps ) formulation produces more potent antigen presentation than the ova plga (+ lps ) formulation in vivo ( see fig6 e ). this shows that , in principle , the pan - endosomal protease inhibitor can improve antigen presentation in relevant immune cells . it was also tested whether the pan protease inhibitor could inhibit the proteolytic activity of pathogenic species , more specifically that of the trypanosomes . it was indeed observed that all three families of protease activities from t . brucei could be powerfully inhibited ( fig7 ). in summary , the inventors have presented the construction of a single molecular entity that inhibits all three major families of endo / lysosomal proteases . this broad inhibition has been shown to attenuate destructive processing of labile proteins in vitro and to attenuate proteolytic events within the endo / lysosomal pathway in vivo . 1 . leung , d . ; 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