Patent Application: US-46624204-A

Abstract:
the present invention relates to materials and methods for treatment of hepatitis c . more closely , the invention relates to human monoclonal antibodies against hcv e1 antigen , to a reagent comprising such antibodies , and to vaccine compositions comprising such antibodies . futhermore , the invention relates to a method of treating or preventing hcv infection by administration of a vaccine composition comprising the monoclonal antibodies of te invention .

Description:
monoclonal mouse anti - human - myc from invitrogen was used in 1 : 1 . 50 - 1 : 75 dilution ( 9 - 19 μg / ml ). ascites produced mouse anti - e1 monoclonal antibody was diluted 1 : 250 . a fitc conjugated rabbit - anti - mouse immunoglobulin from dako was used as secondary antibody in 1 : 15 - 1 : 10 dilution ( 67 - 100 μg / ml ). the library was derived from a bone marrow donation from a patient infected with hcv genotype 1a . construction of the library was performed as described in allander et al ., 2000 . in brief , lymphocytes were isolated from bone marrow using ficoll - paque ( pharmacia ), and total rna was extracted by the acid guanidium thiocyanate - phenol method . first strand cdna synthesis was performed using an oligo - dt primer and the “ first strand cdna synthesis ” kit ( amersham pharmacia ). the cdna was subsequently used as template for for pcr amplification of γ1 fd and κ light chains ( kang et al ., 1991 ), and ligated into the phagemid vector pcomb3h ( barbas and wagner , 1995 ). the signal sequence and echtodomain of e1 ( aa 174 to 359 ) was inserted into the vector pdisplay ( invitrogen ). the pdisplay vector has a mouse igκ signal sequence upstream of its cloning cassette and a pdgf membrane anchor sequence downstream of the cassette for cell - surface expression . in addition , the vector contains two tag sequences so that the expressed protein will have a n - terminal hemagglutinin a epitope tag , and a c - terminal myc epitope tag before the membrane anchor . the signal sequence and echtodomain fragment of e1 was pcr amplified from a full - length hcv clone ( pcv h77c , genotype 1a ), which was a generous gift from dr j bucht , nih , usa ( yanagi et al ., 1997 ). since the hemagglutinin a tag would be localized n - terminal of the e1 fragment and possibly disturb recognition by anti - e1 or anti - myc antibodies , the signal sequence and the hemagglutinin tag were deleted from the vector using eco ri and pst i ( life technologies ). the e1 fragment was subsequently inserted with the 3 ′ end in the cloning cassette and the 5 ′ end replacing the removed sequences . the pcr was performed as follows : first 94 ° c . 5 min ; then 94 ° c . 1 min , 52 ° c . 0 . 5 min , and 72 ° c . 10 min for 35 cycles ; and finally 72 ° c . 10 min . primers ( symbion , denmark ) were designed to contain specific restriction enzyme sites for directional cloning and an atg start codon in the sense primers : sense primer ( h77c - s1b ) 5 ′- gg aac ctt cct gaa ttc ggc ttg ggg atg ttc tct atc - 3 ′ ( restriction site for eco ri underlined ), antisense primer ( h77c - as1 ) 5 ′- cat gga gaa ata cgc ctg cag cgc cag - 3 ′ ( restriction site for pst i underlined ). the pcr fragment was cleaved with the mentioned restriction endonucleases , and gel purified on 1 % agarose . the band of correct size ( approx . 600 bp ) was cut out from the gel , and dna was eluted using concert dna purification kit ( life technologies ). the pdisplay vector ( invitrogen ) was cut using the same restriction enzymes and likewise gel purified . the fragment was ligated to the vector in 1 : 1 , 1 : 3 and 1 : 6 ratios at + 4 ° c . o . n . using t4 dna ligase ( life technologies ). the 1 : 1 ligation product was linearized , self - religated and used to transform e - coli ( xl - 1 blue , stratagene ) by electroporation . single ampicillin resistant clones were picked and cultured , and dna was extracted using wizards plus midipreps dna purification system ( promega ). to distinguish which clones contained correct insert , dna from single clones was analyzed both by pcr and by restriction enzyme digestion . expression of the e1 - myc construct was tested in cho and hela cells . cells were grown in 6 - well plates to near confluence in rmpi 1640 ( cho cells ) or dmem ( hela cells ) supplemented with 10 % fetal bovine serum ( fbs ) and 100 u / ml penicillin , streptomycin 100 μg / ml ( life technologies ). transfection was performed using fugene 6 ( boehringer - mannheim ) with a fugene 6 to dna ratio of 3 - 6 μl to 1 - 2 μg per well . geneticin ( life technologies ) was added to the culture media , in a final concentration of 200 μg / ml , 48 h after transfection . analysis with immunofluorescence microscope ( leitz dmrbe , leica ) and flowcytometry ( facsort , becton dickinson ) were carried out on solubilized cells . cells were loosened using a rubber policeman and washed twice in wash buffer dulbeccos &# 39 ; pbs , 0 . 5 % fbs , 0 . 1 % sodium azide ( life technologies , usa ). cells were centrifuged and resuspended in a small volume of primary antibody ( anti - myc or anti - e1 ) and incubated at rt for 60 min . after two to three washes in wash buffer , cells were incubated in the dark at rt for 30 min in secondary rabbit - anti - mouse ig - fitc . finally cells were briefly incubated in hoerst 1 : 1000 , washed 3 times and mounted on glass slides in 5 - 10 μl vectashield ( vector ). cells to be analysed with flowcytometry were redisolved in 250 μl washbuffer . anti - e1 clones were selected from three different sets of selections . for the first two sets , hela cells were grown to semi confluence in t75 culture flasks . in the first set of selections , cells were transfected with 8 - 9 μg e1 dna ( 47 - 53 μl fugene6 ). in the second set , hela cells were transfected twice with the e1 dna to further increase the surface expression prior to selection . cells were first transfected ( 5 . 5 μg dna ) in a t25 culture flask for two days , grown under geneticin selection pressure for 5 days , and then moved and transfected in a t75 flask ( 16 μg dna ). in the third set , cho cells were used because of their higher surface expression even after the first transfection . cells were transfected with 16 - 30 μg dna in t75 flasks . one to four days after transfection cells were harvested using a rubber policeman , and suspended in dpbs — 4 % nonfat milk — 0 . 02 % sodium azide . to deplete the phage library of non - specific binders , the phages were incubated with non - transfected cells for one hour on an orbit shaker 100 rpm at rt or incubated overnight at + 4 ° c . and then on a turning - wheel ( 7 rpm ) for one hour at rt . cells were removed by centrifugation , and the depleted phages were incubated with transfected cells for 1 . 5 - 2 h on a turning - wheel at rt . cells were then washed three times in wash - buffer ( dulbeccos pbs , 0 . 5 % fbs , 0 . 02 % sodium azide ). cells were resuspended in 100 μl anti - myc 1 : 75 dilution and incubated at rt for 2 h . after two washes the cells were resuspended in fitc - anti - mouse — igg 1 : 15 dilution and incubated in the dark for 1 h . the cells were washed twice before resuspended ( 10 6 cells / ml ) in wash - buffer . sorting was performed in a facsvantage se ( becton dickinson ). phages were eluted from the sorted cells by adding 200 μl 0 . 1 m hcl - glycine ph 2 . 2 . eluted phages were used to infect freshly cultured xl - 1 blue . the infected bacteria were then plated out on la - amp plates . the next day , colonies were harvested in sb and grown to a 50 - 100 ml culture . phages were induced and harvested as described ( barbas et al ., 1991 ), and used for a next round of selection . colonies were picked , propagated and analyzed as single clones . fab production was induced and a periplasmic fraction was prepared by freeze thawing ( allander et al 2000 ). fab production was determined in an elisa using anti - fd ( the binding site , uk ) and ap conjugated anti - fab ( pierce , usa ). specificity for the antigen was initially tested in an elisa against an recombinant e1 / e2 protein , or against recombinant e1 protein . the recombinant e1 / e2 heterodimer protein ( genotype 1a ) expressed in cho cells was generously provided by dr m . houghton , chiron corp . and has been described elsewhere ( spaete et al ., 1992 ). the soluble e1 protein was expressed and secreted into the medium from cos cells , and was generously provided by dr . a . patel , mrc virology unit , glasgow , u . k . the elisa assays with these antigens were performed as described below . elisa assay for determination of specific binding of the antibodies to hcv proteins elisa for e1 reactivity : gna lectin ( sigma ) was diluted to 2 . 5 μg / ml in pbs and coated to microtiter wells ( costar 3690 ) over night at room temperature . the wells were washed once in pbs with 0 . 05 % tween 20 ( pbs - t ) and the wells were blocked with 4 % non - fat dry milk in pbs for 2 hours at room temperature . a : fter discarding the blocking solution , 50 μl of recombinant e1 ( approx . concentration 50 μg / ml ) was added and incubated for two hours at room temperature . after the e1 solution had been discarded , the antibody preparations were added and bound antibodies detected as described below . recombinant e1 / e2 heterodimer ( genotype 1a ) was diluted to 1 μg / ml in pbs , and coated to microtitre wells overnight at + 4 ° c . unbound antigen was discarded , and the wells were blocked with 5 % non - fat dry milk in pbs for 60 minutes at room temperature . blocking solution was discarded , and antibody solutions to be tested added in 1 : 3 - 1 : 24 dilutions ( diluent : pbs with 0 . 05 % tween 20 ). the plates were incubated at 37 ° c . temperature for 2 hours , washed four times with pbs - t , and alkaline phosphatase ( ap ) coupled - goat anti - human f ( ab ′) 2 ( pierce , rocherford , ill .) antibodies in a 1 : 1000 dilution were added . after 60 minutes at 37 ° and subsequent washes , substrate solution ( p - nitrophenyl phosphate , sigma , st . louis , mo .) was added and absorbency measured at 405 nm . for control purposes , recombinant e2 ( genotype 1a ), or bsa ( sigma ) coated at 1 μg / ml were used in corresponding elisas to control for unspecific reactivity . the fd and the light chain gene segments were transferred from the phagernide vector pcomb3h to the eukaryotic vector pcigg1 as previously reported ( samuelsson et al ., 1996 ). plasmid dna was transfected into cho cells using lipofectamine plus ( life technologies ) according to the manufacturer &# 39 ; s instructions . medium containing secreted igg was harvested every second day and frozen until analyzed . the elisa to determine igg concentration used a rabbit anti - human igg and an ap conjugated rabbit anti - human igg , and a purified human igg standard as reference ( dako ) ( samuelsson et al ., 1996 ). ligation of γ1 fd genes into the pcomb3h vector gave a library of 7 . 8 × 10 6 cfu / μg , and ligation of κ light chain genes into the pcomb3h gave a library of 1 . 6 × 10 7 cfu / μg . the resulting combinatorial library comprised 3 . 7 × 10 7 members . the first ligation of pdisplay vector and e1 fragment resulted in similar number of clones independent of ligation ratios . plasmid dna extracted from cultures of the 1 : 1 ligation was linearized and separated on a gel . two bands of approximately 5 . 5 and 6 . 0 appeared on the gel . since the expected correct sized band was 5 . 8 kb , both bands were purified and religated separately . single clones were grown and insertion of the fragment was demonstrated by pcr using the same sense and antisense primers as in the cloning step ; 9 clones out of 20 showed the correct fragment length . subsequent restriction enzyme digestion showed that the correct sized fragment also could be cleaved from all 9 clones . nucleic acid sequencing of the clones showed that they all differed from the original e1 sequence by a few nucleotides or more . initially hela cells were preferred . to determine optimal expression of the myc - tag , expression was investigated by fluorescence microscopy and flow cytometry on day 1 , 2 , 4 , and 7 after transfection . this time study indicated that immunofluorescence detection of the myc tag was optimal 4 days after transfection . for the second set of selection surface expression was increased slightly by transfecting the hela cells twice . for the third set cho cells were chosen since they appear to be more tolerant to transfection as well as show clear e1 expression already after one day of transfection . in the first round of each selection series , approximately 10 11 cfiu of phage library was depleted against 10 6 - 10 7 non - transfected cells . unbound , depleted phages were subsequently incubated with 3 - 8 × 10 6 transfected , e1 expressing cells . after sorting for myc positive cells , phages were eluted from the sorted cells and re - propagated in fresh xl1 blue . 2 - 8 × 10 10 cfu of re - propagated phages were used in the next selection round . in the first series , two rounds of selection were performed . after the second round , only 108 colonies were formed , 98 of which were tested in elisas for fab expression and binding to recombinant e1 / e2 antigen . sixteen clones expressed fab , of which nine were positive for binding to e1 / e2 . after nucleic acid sequencing , two clones were judged not to be proper fab fragments . two clones ( clones 13 and 98 ) from this selection series were further characterised and included in the present collection of antibodies ( table 1 , seq id no . 1 - 4 ). in the second series , six selection rounds were performed . after the fourth round , 42 single clones were picked and assayed for fab expression . twentytwo clones produced fab , while only two were positive when tested for e1 reactivity . subsequent sequence analysis revealed that the clones were identical ( clone 4 : 6 ; seq id no . 13 - 14 ). an additional clone , isolated from the sixth panning round in this series , was also characterised ( clone 6a : 5 ; table 1 and seq id no . 15 - 16 ). in the third series , two rounds of selection were made and the second round was repeated once . 45 - 75 % of propagated clones expressed fab . the majority of our e1 specific fab clones were isolated from this selection series ( clones with prefix 1 :, 2a : and 2b :). the reactivity of the different fab clones to the hcv proteins e1 , e1 / e2 in complex , free e2 or bsa was determined by elisa . all clones showed a significantly higher reactivity to e1 and / or e1 / e2 than against e2 or bsa ( negative control antigens ) ( table 1 ). from these data , it seems that some clones may be particularly efficient binders : clones 1 . 4 , 1 : 8 , 2a : 13 , 2a : 23 , 2a : 30 , 2b : 5 and 4 : 6 allander t , drakenberg k , beyene a , rosa d , abrignani s , houghton m , widell a , grillner l , persson maa . recombinant human monoclonal antibodies against different conformational epitopes of the e2 envelope glycoprotein of hepatitis c virus that inhibit its interactions with cd81 . j gen virol 2000 ; 81 : 2451 - 2459 . barbas iii c f , kang a s , lerner r a , benkovic s j . assembly of combinatorial antibody libraries on phage surfaces : the gene iii site . proc natl acad sci u s a 1991 ; 88 : 7978 - 82 . barbas iii c f , wagner j . synthetic human antibodies : selecting and evolving finctional proteins . methods 1995 ; 8 : 94 - 103 . burioni r , plaisant p , manzin a , rosa d , delli carri v , bugli f , solforosi l , abrignani s , varaldo p e , fadda g , clementi m . dissection of human humoral immune response against hepatitis c virus e2 glycoprotein by repertoire cloning and generation of recombinant fab fragments . hepatology 1998 ; 28 : 810 - 814 . houghton m . hepatitis c virus . in fields virology , ( eds b n fields , d m knipe , p m howley ) lippincott - raven publishers , philadelphia , 1996 pp1035 - 1058 . kang a s , burton d r , lemer r a . combinatorial inimunoglobulin libraries in phage λ . methods : comp . methods in enzymol . 1991 ; 2 : 111 - 8 . maertens g , priem s , ducatteeuw a , verschoorl e , verstrepen b , roskams t , desmet v , fuller s , van hoek k , vandeponseele p , bosman f , buyse m a , van doom l j , heeney j , kos a , depla e . improvement of chronic active hepatitis c in chronically infected chimpanzees after therapeutic vaccination with the hcv e1 protein . acta gastroenterologica belgica . 2000 ; 63 : 203 . samuelsson a , yari f , hinkula j , ersoy o , norrby e , persson m a a . human antibodies from phage libraries : neutralizing activity against human immunodeficiency virus type 1 equally improved after expression as fab and igg in mammalian cells . eur j immunol 1996 ; 26 : 3029 - 34 . spaete r r , alexander d , rugroden m e , choo q l , berger k , crawford k , kuo c , leng s , lee c , ralston r , and others . characterization of the hepatitis c virus e2 / ns 1 gene product expressed mammalian cells . virology 1992 ; 188 : 819 - 830 . yanagi m , purcell r h , emerson s u , bukh j . transcripts from a single full - length cdna clone of hepatitis c virus are infectious when directly transfected into the liver of a chimpanzee . proc natl acad sci usa 1997 ; 94 : 8738 - 8743 . tyr gly met his trp val arg gln ala pro gly lys gly leu glu trp val lys gly arg phe thr ile ser arg asp asn ala lys asn ser 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