Patent Application: US-72237600-A

Abstract:
a method for the prevention or treatment of human cytomegalovirus is described . aminopeptidases , preferably in soluble form is administered exogenously to the patient . the invention involves the discovery that aminopeptidases are a cmv surface protein involved in post - binding events in the cmv infection process . the invention includes the discovery that ap , including specifically apn , on the surface of the virion and on the surface of the cell is involved in the cmv infection process .

Description:
this invention relates to the involvement of aminopeptidases in human cmv infection . antibodies to ape not only inhibit infection , but also block binding of cmv virions to susceptible cells by binding to and thus blocking cmv virions . compounds which inhibit aminopeptidase activity completely block cmv infection . cmv - resistant murine fibroblasts became susceptible to cmv infection after transfection with complementary dna encoding human apn . however , murine fibroblasts transfected with mutant apn , lacking the enzymatic domain , remain susceptible to cmv infection . thus , apn appears to mediate cmv infection but its enzymatic domain is not necessary for infection . to investigate the role that apn plays in the early interaction of cmv with human fibroblasts , cell strains hl734 and mrc - 5 were exposed to virus in the presence of apn - specific monoclonal antibodies u71 , u81 , wm47 , or anti - leu m7 . as shown in fig1 u71 protected hl734 from infection with cmv rc256 , a recombinant form of cmv towne strain that encodes the β - galactosidase ( β - gal )- gene linked to a cmv early promoter ( 22 ). no protection was observed in cells treated with control mouse ascites , mouse iqg or with antibodies directed against the epidermal growth factor - receptor ( anti - egfr ) known to be present on the surface of these fibroblasts . the dose response curve of cmv inhibition by u71 indicated that inhibition was directly proportional to the amount of anti - apn antibody used ( see fig2 a ). similarly , u 81 , wm47 , and anti - leu m7 all inhibited cmv infection in a dose - dependent fashion . u81 and u71 inhibited infection to a greater extent than anti - leu m7 or wm47 . see table 1 . d chemical inhibitors were tested at the concentrations that gave maximum inhibition without toxicity to the cells : actinonin , 5 . 45 mm ; bestatin , 4 . 35 mm ; 1 , 10 - phenathroline , 0 . 45 mm ; 2 , 2 ′- dipyridyl , 3 . 74 mm . the 50 % inhibitory dose given in millimolar concentration is listed in parenthesis . antibodies were tested at a concentration that gave maximum effect : u71 , 1 . 8 mg / ml ; u81 , 2 mg / ml ; wm47 , 20 ug / ml ; anti - leum7 , 3 . 7 ug / ml . + = & gt ; 50 inhibition compared to mouse igg control ; a the inhibition of cmv infection was determined as described in the description of fig2 . b the inhibition of semliki forest virus ( sfv ) infection was determined as described in the description of fig2 . c the inhibition of enzymatic activity was measured using the substrate alanine - p - nitroanilide as described in the description of fig2 . d chemical inhibitors were tested at the concentrations that gave maximum inhibition without toxicity to the cells : actinonin , 5 . 45 mm ; bestatin , 4 . 35 mm ; 1 , 10 - phenathroline , 0 . 45 em ; 2 , 2 ′- dipyridyl , 3 . 74 mm . the 50 % inhibitory dose given in millimolar concentration is listed in parenthesis . antibodies were tested at a concentration that gave maximum effect : u71 , 1 . 8 mg / ml ; u81 , 2 mg / ml ; wm47 , 20 ug / ml ; anti - leum7 , 3 . 7 ug / ml . +=& gt ; 50 inhibition compared to mouse igg control ; −= no inhibition . to investigate whether the enzymatic activity of apn is necessary for cmv infection , four different aminopeptidase inhibitors , actinonin , bestatin , 2 , 2 ′- dipyridyl and 1 , 10 - phenanthroline , were evaluated for their effect on enzymatic activity and cmv infection bestatin and actinonin are competitive inhibitors of arinoneptidases , ard 1 , 10 - phenanthroline and 2 , 2 ′- dipyridyl inhibit aminopeptidase activity by chelating zinc required for enzyme function . these compounds were used at concentrations which were demonstrated to inhibit apn in cells using alanine - p - nitronailide as substrate ( see table 1 ). toxicity of these substances , measured by trypan blue uptake in cells treated with the inhibitors , was less than 10 % in the highest concentrations used ( data not shown ). as shown in fig2 b , bestatin inhibited cmv infection at a 50 % inhibitory concentration of 2 . 5 mm . as shown in table 1 , actinonin , 1 , 10 - phenanthroline and 2 , 3 ′- dipyridyl inhibited cmv infection with 50 % inhibition doses of 3 . 3 , 0 . 25 , and 2 . 8 mm , respectively , further suggesting that apn is involved in cmv infection . the effect of apn - specific antibodies on binding of 35 s - labeled cmv virions to mrc - 5 cells was analyzed . incubation of cells with increasing concentration of virions resulted in increased binding of cmv to the cells ( see fig2 c ). if binding was done in the presence of control mouse igg , there was no effect on binding ( fig2 c ). however , binding in the presence of apn - specific antibodies ( anti - leum7 ) resulted in a decrease in cmv binding ( see fig2 c ). to further investigate the importance of apn and its enzymatic activity during cmv infection , murine cells ( nth - 3t3 ) were transfected with dna for hapn ( hapn - 3t3 ) or for a truncated form of the protein lacking 39 amino acids from a region containing the active site ( hapmut - 3t3 ). the presence of surface hapn was confirmed on the human cells and on the transfected nih - 3t3 cells by flow cytometric analyses using apn - specific monoclonal antibodies ( data not shown ). to confirm the level of apn activity in these cells , they were tested for ability to cleave aminopeptidase substrate alanine - p - nitroanilide . enzymatic activity was present in the hapn - 3t3 and hl734 cells but not in the parent nih - 3t3 or in hapmut - 3t3 cells ( see fig2 d ). all cells were then challenged with cmv rc256 , β - gal , the marker for virus infection , was observed on a greater number of the cells which expressed hapn ( see fig3 ). parental cells showed little or no expression of cmv encoded genes compared to the apn - expressing cells ( compare fig3 b and 3 d / 3 f ). similarly , when evaluated for expression of the 72k major immediate early ( mie ) protein of cmv , there was greatly enhanced expression in the transfected cells ( data not shown ). thus , expression of hapn on murine cells permits infection of the cells by hcmv . importantly , virus infection was observed in a greater number of cells expressing the truncated form of apn than in those with unmodified hapn . ( see table 2 ) infection of these cells expressing the truncated form of hapn with cmv resulted in a 50 - fold increase of cmv - infected cells compared to the hapn - nonexpressing nih 3t3 cells which is a 5 - fold increase over cells transfected with native hapn . a percent cells expressing the 72 - kda mie antigen of hcmv were determined 1 , 3 or 7 days postinfection with ad169 . monolayers of hl734 cells were inoculated at moi = 1 - 10 for 2 hours at 4 ° c ., washed and incubated in fresh media . at day 1 , 3 and 7 postinfection , the cells were fixed and assayed for the expression of hcmv mie antigen . the expression of mie nuclear antigen was detected after fixation in ethanol / acetone by incubation with a mouse monoclonal directed against the # 72 - kda hcmv mie protein ( nen - dupont , boston , ma or biosoft , paris , france ) followed by a fitc - labelled ( f ( ab 1 ) 2 fragment of rabbit - anti mouse igg ( dakopatts ). infected cells were detected by using a nikon microscope fitted with epi - illumination optics . the number of hcmv - antigen positive cells were counted and presented as percent of total cells . a percent cells expressing the 72 - kda mie antigen of hmcv were determined 1 , 3 or 7 days postinfection with ad169 . monolayers of hl734 cells were inoculated at moi = 1 - 10 for 2 hours at 4 ° c ., washed and incubated in fresh media . at day 1 , 3 and 7 postinfection , the cells were fixed and assayed for the expression of hcmv mie antigen . the expression of mie nuclear antigen was detected after fixation in ethanol / acetone by incubation with a mouse monoclonal directed against the 72 - kda hclc mie protein ( nen - dupont , boston , mass . or biosoft , paris , france ) followed by a fitc - labelled ( f ( ab ′) 2 fragment of rabbit - anti mouse igg ( dakopatts ). infected cells were detected by using a nikon microscope fitted with epi - illumination optics . the number of hcmv - antigen positive cells were counted and presented as percent of total cells . these results suggest that the enzymatic activity of this molecule is not essential for infection and that the truncated form of apn expressed in the hapn - mut 3t3 cells enhances a cells susceptibility to cmv infection to a greater extent than the native form ( compare fig3 d and 3 f and see table 3 ). the mutant form of hapn , hapnmut - 3t3 , was obtained as follows . the full length human hapn cdna subcloned in the pbluescript plasmid was digested with the bsteii restriction endonuclease , which excised an internal 117 base pair ( bp ) restriction fragment from the cdna , but did not cut elsewhere in the dna or in the vector . the deleted fragment of 39 amino acids ( positions 360 - 399 included the coding sequence for the zinc binding motif ( hexxh ) at amino acid positions ( 388 - 392 ) known to be necessary for the enzymatic activity . dna , lacking the deleted sequences , was religated correctly and transfected into nih 3t3 cells by calcium - phosphate precipitation ( 21 ). the presence of hapn on the cell surface was confirmed by immunostaining by monoclonal antibodies and flow cytometric analysis . the invention this particularly comprises the use in the method of the invention of an aminopeptidase which lacks the site responsible for aminopeptidase activity while retaining hcmv binding activity . thus , human aminopeptidase n lacking at least the amino acids at positions 388 to 392 , may be used , more particularly human aminopeptidase n lacking the amino acids at positions 360 to 399 . these experiments have shown that cmv infection of fibroblasts , as well as binding of virions to these cells , can be inhibited by different monoclonal antibodies directed against apn as well as by inhibitors of apn activity . however , certain of these apn - specific monoclonal antibodies do not inhibit hapn activity . furthermore , enhanced cmv infection occurred in murine cells expressing hapn and in cells in which the enzymatic site had beer , deleted as compared to the parental mouse cells . it appears the , that the enzymatic active site is not essential either for anti - apn inhibition of cmv infection or for the infection of apn - expressing murine cells . the apn - inhibiting compounds which blocked infection may act by changing the conformation of the apn molecule , and thus altering a region which is important for infection . these data suggest that a non - enzyme domain of apn is important for cmv infection of fibroblasts . soluble ap is preferred for use in the practice of this invention and may be prepared in known manner . for example , soluble aminopeptidase n are obtained by purification from mouse cells expressing human apn . this may be done by a modification of the methodology of danielsen and cowell ( danielsen , e . m . and cowell , g . m . j . biochem . biophys . methods 8 : 41 - 47 ( 1983 )). peptides corresponding to various domains of human apn are derived by ( 1 ) proteolytic digests of soluble hapn followed by hplc purification of the peptides , ( 2 ) expression in e . coli , by recombinant dna methodologies of various regions of hapn . these e . coli expressed peptides may be purified by hplc , and ( 3 ) synthesis of synthetic peptides . table 3 demonstrates that soluble porcine - leucine aminopeptidases block cmv infection in vitro . a mrc - 5 cells , plated in 96 well plates were infected with rc256 in the absence or presence of soluble porcine aminopeptidases ( rap ). cells ware incubated with virus plus pap for two hours at 4 ° c ., washed and incubated 18 hours with fresh media at 37 ° c . cells were fixed with 1 % glutaraldehyde and assayed for expression of β - galactosidase by incubation with blue - gal ( 430 ug / ml ) for 16 hours at 37 ° c . infected cells were detected by presence of precipitated blue substrate # observed by light microscopy . the number of cells infected under each condition was normalized to cells infected in absence of any aminopeptidase . b cytosolic pap = cytosolic porcine leucine aminopeptidase obtained from sigma chemical co ., inc . ( catalogue no . l - 9875 ). c microsomal pap = microsomal porcine leucine aminopeptidase obtained from sigma chemical co ., inc . ( catalogue no . l - 5006 ). a mrc - 5 cells , plated in 96 well plates were infected with rc256 in the absence or presence of soluble porcine aminopeptidases ( rap ). cells ware incubated with virus plus pap for two hours at 4 ° c ., washed and incubated 18 hours with fresh media at 37 ° c . cells were fixed with 1 % glutaraldehyde and assayed for expression of β - galactosidase by incubation with blue - gal ( 430 ug / ml ) for 16 hours at 37 ° c . infected cells were detected by presence of precipitated blue substrate observed by light microscopy . the number of cells infected under each condition was normalized to cells infected in absence of any aninopeptidase . b cytosolic pap = cytosolic porcine leucine aminopeptidase obtained from sigma chemical co ., inc . ( catalogue no . l - 9875 ). c microsonal pap = microsomal porcine leucine aminopeptidase obtained from sigma chemical co ., inc . ( catalogue no . l - 5006 ). as table 3 shows , sapn is an antiviral agent useful for the prevention or treatment of cmv associated diseases . it has also been determined that preincubation of cells with antibodies to hapn followed by excessive washing does not block cmv infection , whereas preincubation of the virus with antibodies to hapn , but not antibodies directed toward other cell surface markers , does block infection . further , hapn negative human cells can be infected with cmv and the infection blocked with antibody to hapn . this suggests that hapn is not a simple receptor for cmv as suggested by soderberg , et al . ( 11 - 13 ) and , at least in part , the hapn on the surface of the virion is , important for cmv infection . however , as indicated by the expression of hapn in mouse cells and the effect of sap on cmv infection in fibroblasts , cell associated aminopeptidases play an important role in cmv infection . 1 . zaia , j . a ., et al ., hematology / oncology clinics n . a . 4 : 603 - 623 ( 1990 ). 2 . rubin , r . h ., et al ., transplantation 24 : 458 - 464 ( 1977 ). 3 . jacobson , m . a ., et al ., ann . intern . med . 108 : 585 - 594 ( 1988 ). 4 . griffiths , p . d ., et al ., biochemistry 241 ; 313 - 325 ( 1987 ). 5 . taylor , h . p ., et al ., j . virol 63 : 3991 - 3098 ( 1990 ). 6 . addish , j . d ., et al ., virology 176 : 337 - 345 ( 19 ). 7 . nowlin , d . m ., et al ., j . virol 65 : 3114 - 3121 ( 1991 ). 8 . kaay , s ., et al ., proc . natl . acad . sci . usa 86 : 10100 - 10103 ( 1989 ). 9 . kari , b . et al ., j . virol . 66 : 1761 - 1764 ( 1992 ). 10 . grindy , d . e ., et al ., gen . virol . 68 : 793 - 803 ( 1987 ). 11 . soderberg , c . et al ., transpl . proc . 25 : 1416 - 1418 ( 1993 ). 12 . soderberg , c . et al ., p . 222 , 17th international herpes virus workshop , edinburgh , scotland , aug . 1 - 7 , ( 1992 ). 13 . soderberg , c . et al ., j . virol . 67 : 3166 - 3175 ( 1993 ). 14 . kenny , a . j . et al . ; in kenny , a . j . and turner , a . j . 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