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:
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 fig2 ) 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 exisiting disulfide bridges in cystatin c were avoided by using a mammalian expression system . various mutants were tested ( see table 1 and fig2 ), 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 ( fig4 a - 4 d - 7 ( a )- 7 ( c )) 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 ( fig4 a - 4 d - 7 ( a )- 7 ( c )). 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 ( fig8 , panel ( a )-( c )). moreover , the cpi was able to inhibit the same 3 classes of protease activity present in dendritic cell lysates ( fig8 , panel ( d )-( f )). 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 ( fig8 , panel g ) 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 ( fig9 ). 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 ( fig9 ). 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 ( fig1 ). in control cells downregulation of egfr was evident after 40 minutes and virtually complete after 90 minutes ( fig1 a - 10b ). in contrast levels of egfr were much more persistent in cos7 cells preincubated with cpi demonstrating a block in receptor degradation as quantified in fig1 a - 11c . 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 ( fig1 a - 10b , 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 ( fig1 ) 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 ( fig1 ( a -) 13 ( 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 fig1 ( a )- 16 ( b ) ). 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 ( fig1 ). 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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