RS60929B1 - Kompozicije za supresiju stvaranja inhibitora protiv faktora viii kod pacijenata sa hemofilijom a - Google Patents

Description

Opis

OBLAST PRONALASKA

[0001] Predmetni pronalazak se odnosi na oblasti rekombinantnih biljaka i lečenja poremećaja kod kojih je poželjna indukcija oralne tolerancije na terapeutski isporučene antigene. Preciznije, pronalazak obezbeđuje kompozicije i postupke za indukovanje oralne tolerancije na faktor VIII i druge faktore koagulacije, čime se poboljšavaju terapijski ishodi.

OSNOV PRONALASKA

[0002] Hemofilija je poremećaj krvarenja koagulacije vezan za X hromozom koga uzrokuju mutacije faktora koagulacije IX (FIX, hemofilija B) ili njegovog kofaktora, faktora VIII (FVIII, hemofilija A). Pošto serinska proteaza FIX ima vrlo nisku aktivnost u odsustvu FVIII, mutacije bilo kog proteina mogu prouzrokovati koagulacioni defekt. Ova bolest pogađa 1 od 7.500 muške novorođenčadi širom sveta kad je u pitanju hemofilija A i 1 od 30.000 kad je u pitanju hemofilija B.<1-3>. Otuda većina pacijenata ima nedostatak FVIII. Trenutno se standardni tretman zasniva na intravenskoj (IV) infuziji koncentrata ovog faktora dobijenog iz plazme ili koncentrata rekombinantnog faktora. Glavna komplikacija ove terapije je stvaranje inhibitornih antitela („inhibitora“), koja se javlja kod 20-30% pacijenata sa teškom hemofilijom A (što je definisano koagulacionom aktivnošću od <1%) i u ∼5% pacijenata sa teškom hemofilijom B.<1,4-6>Inhibitori ozbiljno komplikuju lečenje i povećavaju morbiditet i mortalitet usled ove bolesti. Povećane doze faktora mogu biti u stanju da obnove hemostazu kod pacijenata sa inhibitorima sa niskim titrom (< 5 jedinica Bethesda, BU), dok su faktori premošćivanja potrebni za lečenje krvarenja u prisustvu inhibitora sa visokim titrom. Međutim, ovi tretmani su skupi i moraju da se doziraju pažljivo. Klinički protokoli za reversal poništavanje odgovora antitela putem indukcije imune tolerancije (ITI) sastoje se od čestih primena faktora u visokim dozama tokom dužeg perioda (meseci do> 1 godine), vrlo su skupi (> 1.000.000 USD), a kod 30% pacijenata sa inhibitorima FVIII ne dolazi do odgovora.4

[0003] U publikaciji Lei TC et al, Blood. 2005 Jun 15;105(12):4865-70 opisuje se tolerancija na inhibitore faktora VIII putem genske terapije imunodominantnim domenima A2 i C2 koje u vidu Ig funzionih proteina prezentuju B ćelije.

KRATAK PREGLED PRONALASKA

[0004] U skladu sa predmetnim pronalaskom obezbeđena je kompozicija koja sadrži liofilizovani biljni materijal koji sadrži najmanje jedan fragment FVIII konjugovan sa subjedinicom kolera toksina B (CTB), pri čemu se pomenuti fragment sastoji od FVIII domena odabranog iz grupe koju čine C2 i teški lanac (HC), i pri čemu je pomenuti fragment konjugovan sa CTB proizveden u hloroplastima unutar pomenute biljke i zadržava imunogenost u liofilizovanom obliku, koji pri oralnoj primeni kod sisaru kome je to potrebno efikasno dovodi do oralne tolerancije na FVIII.

[0005] U nekim primerima izvođenja, biljka je odabrana iz grupe koja se sastoji od zelene salate, paradajza, jabuke, bobica kao što su jagode i maline, agruma, banane, šargarepe, celera, karfiola; brokolija, raštana, krastavca, dinje, lubenice, parike, kruške, grožđa, breskve, rotkvica i kejl kupusa.

[0006] Takođe je u skladu sa pronalaskom obezbeđena i kompozicija koji sadrži liofilizovani biljni materijal koji sadrži najmanje jedan fragment FVIII konjugovan sa subjedinicom kolera toksina B (CTB), pri čemu se navedeni fragment sastoji od domena FVIII odabranog iz grupe koja se sastoji od C2 i teškog lanca (HC), gde se navedeni fragment konjugovan sa CTB proizvodi u hloroplastima unutar navedene biljke i zadržava imunogenost u liofilizovanom obliku, koji je pri oralnoj primeni kod sisara kome je to potrebno, efikasan u postizanju oralne tolerancije na FVIII, pri čemu je kompozicija namenjena za upotrebu u postupku za razvoj oralne tolerancije za lečenje hemofilije A.

[0007] U određenim primerima izvođenja, jedan, dva, tri, četiri, pet ili šest domena se primenjuju zajedno. Primeri kompozicija i metoda uključuju one gde se fuzioni proteini C2-CTB i HC-CTB primenuju zajedno. Pronalazači predmetnog pronalaska otkrili su da su ovde prikazane kompozicije efikasne u smanjivanju stvaranja inhibitora protiv FVIII kod subjekata sa hemofilijom A. Prema tome, pronalazak takođe obezbeđuje kompozicije prema pronalasku za upotrebu u postupku za lečenje hemofilije A kod subjekta kome je to potrebno, što uključuje primenu fuzionih proteina C2-CTB i HC-CTB, pri čemu je navedena kompozicija efikasna u indukovanju ekspresije u slezini CD4 CD25- LAP regulatornih T ćelija koje proizvode TGF-β.

KRATAK OPIS SLIKA

[0008]

Slike 1A i 1B: Vektori za transformaciju hloroplasta i integraciju transgena u genom hloroplasta. A. Eskpresioni vektori hloroplasta duvana. Homologe prirubne sekvence genoma hloroplasta koje sadrže sekvence gena 16S 3,’ trnI, trnA; Prrn, promotor ribozomalnog RNK operona sa GGAGG mestom za vezivanje ribozoma; aadA, gen za aminoglikozid 3’-adenililtransferazu koji obezbeđuje rezistenciju na spektinomicin; 5’ UTR , promotor i 5’ UTR psbA gena duvana; 3’ UTR, 3’ UTR psbA gena duvana; CTB, subjedinica kolera toksina B. U oba vektora vectors a Gly Pro Gly Pro (GPGP) hinge and furin cleavage site (RRKR) is included između sekvence CTB i sekvence FVIII domena. WT, netransformisani divlji tip. Nt, Nicotiana tabacum. Naznačeno je restrikciono mesto enzima AflIII i veličine fragmenata dobijenih Southern blotingom.

B. Southern blot, CTB-HC duvana, WT (netransformisan), 1-3 transplastomic linije. Ukupna genomska DNK duvana podvrgnuta je digestiji sa AflIII i detektovana pomoću 0.81kb trnI/trnA flanking region fragment. C. Southern blot, CTB-C2 duvana, WT (netransformisan wild type), 1-4 transplastomic lines.

Figures 2A -2F. Characterization of CTB-HC and CTB-C2 expression in tobacco chloroplasts. A. Detection of heavy chain fusion protein probed with the CTB antibody. CTB standard: 6.25ng, 12.5ng, 25ng. WT: netransformisan wild type. 1-4, transplastomic lines. Five mg total protein of homogenate fraction per lane was loaded. B. Detection of heavy chain probed with the A2 antibody. CTB: 25ng. 1-4, transplastomic lines. Five mg total protein of homogenate fraction per lane was loaded. C. Detection of C2 fusion protein probed with the CTB antibody. CTB standard: 5ng, lOng, 20ng. S: supernatant fraction; H: homogenate fraction. Two mg total protein of supernatant or homogenate fraction per lane was loaded. D. Quantitation of CTB-HC and CTB-C2 expression in tobacco chloroplasts. Proteini su ekstrahovani iz zrelih listova u različitim vremenskim trenucima istoga dana. TLP, ukupni proteini iz lista. E. ELISA test vezivanja gangliozida DM1. CTB standard (0.1ng); CTB-HC iz duvana (5mg); CTB-C2 (1mg); netransformisani divlji tip duvana iz duvana (5mg); BSA, bovine serum albumin (5mg). F. Blue Native Gel Electrophoresis and western blot analysis to evaluate pentamer assembly. Pentamer sizes: CTB: 57.5 kDa; CTB-C2: 155 kDa; CTB-HC: 490 kDa. Samples loaded: CTB standard, 100 ng; WT, 40 mg; CTB-HC, 40 mg; CTB-C2, 10 mg.

Slike 3A - 3F. Supresija stvaranja inhibitora protiv FVIII kod miševa C57BL6/129 sa hemofilijom A putem oralne primene 1:1 smeše bioenkapsuliranih CTB-C2 i CTB-HC FVIII antigena. A.

Vremenska linija oralne primene antigena i intravenskog lečenja pomoću BDD-FVIII. Broj u krugu označava vremensku tačku uzorkovanja krvi iz repa.

B. Inhibitor titers (in BU/ml) after 4 weekly i.v. injections of FVIII in non-fed animals ("no plant") or mice fed with WT or FVIII containing plant material. IgG1 (C), IgG2a (D), IgG2b (E) titers against FVIII for the same experimental groups. Data in B-E are shown for individual mice and as averages 6 SEM.

Titri inhibitora (u BU/ml) nakon 4 nedelj i.v. injekcije FVIII kod neohranjenih životinja („bez biljaka“) ili miševa hranjenih biljnim materijalom koji sadrži VT ili FVIII. Titri IgG1 (C), IgG2a (D), IgG2b (E) protiv FVIII za iste eksperimentalne grupe. Podaci u B-E prikazani su za pojedinačne miševe i u proseku 6 SEM. F. Nakon izvlačenja krvi, miševi su žrtvovani I sakupljen su slezine. Kulture ćelija slezine pojedinačnih miševa (n= 3 to 5 per group) stimulisane su in vitro pomoću 10 mg/ml BDD-FVIII tokom 48 sati. nakon toga su ćelije sakupljene I podvrgnute kvantitativnoj , cells were harvested and subjected to quantitative RT-PCR analizi. "Stopa povećanja" je promena RNK transkripata u kulturama stimulisanim sa FVIII nasuprot kulturama kod kojih je stimulacija simulirana. Isprekidana horizontalna linija pokazuje minimalan neophdan porast od 2.5 puta za statistički značajnu razliku. G: Ćelije slezine dobijene iz istih eksperimentalnih miševa podvrgnuti su ELISPOT analizi radi utvrđivanja učestalosti ćelijske populacije koja luči IL-10. Svi podaci su prikazani za pojedinačne miševe i as averages 6 SEM. Za računanje P-vrednosti(* ** P<0.01) korišćeni su neupareni dvostrani Studentovi T-testovi

Slike 4A - 4E. Supresija stvaranja inhibitora protiv FVIII kod BALB/c miševa sa hemofilijom A by oralne primene administration of a 1:1 mixture of bioencapsulated CTB-C2 and CTB-HC FVIII antigens. A: Feeding and FVIII treatment schedule. Number in circle indicates timepoint for tail bleed. B: Inhibitor titers (in BU/ml) after 4 weekly i.v. injections of BDD-FVIII in "No plant", "WT plant", and "FVIII plant" fed groups. IgG1 (C), IgG2a (D), IgG2b (E) titers against FVIII for the same experimental groups. All data are shown for individual mice and as averages 6 SEM. Unpaired two-tailed Student’s T-tests were used to calculate P values (* P<0.05, ** P<0.01).

Figures 5A -5F. Long-term control and reversal of inhibitor formation in hemophilia A BALB/c mice. A: Feeding (HC and C2 material) and FVIII administration schedule for prevention of inhibitor formation. Numbers in circles indicate time-point for blood collection. Inhibitor titers in BU/ml (B) and IgG1 titers against FVIII (C) at weeks 8, 12, and 21 of the experiment for FVIII fed mice (n=5, back square symbols) are compared to control mice (which were fed with WT plant material, n=7, gray diamonds). Statistically significant differences between these groups for specific time points are indicated

Hranjenje (HC i C2 materijal) i raspored primene FVIII za sprečavanje stvaranja inhibitora. Brojevi u krugovima označavaju vremensku tačku za prikupljanje krvi. Titri inhibitora u titrima BU / ml (B) i IgG1 protiv FVIII (C) u 8., 12. i 21. nedelji eksperimenta za miševe hranjene FVIII (n = 5, zadnji kvadratni simboli) upoređuju se sa kontrolnim miševima (koji su hranjeni sa VT biljnim materijalom, n = 7, sivi dijamanti). Ukazane su statistički značajne razlike između ovih grupa za određene vremenske tačke (* P<0.05, ** P<0.01, and *** P<0.001, as calculated by unpaired two tailed student’s T-test; data are averages 6 SEM). A third group of mice (n=7) was also fed with FVIII material, and FVIII was administered IV once/week starting one month after initiation of the oral tolerance regimen. However, FVIII feeding and treatment was continued for the remaining duration of the experiment (i.e. 20 weeks of FVIII feeding; these mice are labeled as ("FVIII continuously fed" and graphed with black triangle symbols and dotted line in panels B and C; data are averages 6 SEM). D: FVIII administration and feeding schedule for reversal of inhibitor formation. Inhibitor formation was induced by repeated weekly IV injections of FVIII as indicated. Mice were divided into 2 groups with similar average inhibitor titers. Control mice (n=5) did not receive any further treatment. The second group ("FVIII fed", n=4) was fed with FVIII plant material twice per week for the following 3 months. Inhibitor titers in BU/ml (E) and IgG1 titers against FVIII (F) are graphed for weeks 5, 9, 13, and 17 of the experiment as explained above.

Figures 6A and 6B. Active suppression of antibody formation against FVIII by induction of regulatory T cells. A. Adoptive transfer experiments. CD4-, CD4+CD25-, and CD4+CD25+ cells were purified via magnetic sorting from spleens and mesenteric lymph nodes (MLN) of FVIII fed mice (n=3) at time-point 3 indicated in Sl. 5A and pooled (with a final ratio of approximately 30% spleen and 70% MLN-derived CD4+ T cells). Cells (106 per mouse) were adoptively transferred into naive BALB/c mice via tail vein injection. Control cells were from unchallenged naïve mice of the same strain. Twenty-four hours later, all recipient mice (n=5 per group) were challenged with 1 IU FVIII in adjuvant via subcutaneous injection. IgG titers against FVIII were determined 3 weeks later. All data are shown as averages 6 SEM; * P<0.05, ** P<0.01. B. Frequencies of Treg subsets in FVIII fed and control hemophilia A BALB/c mice. Cells derived from spleens, mesenteric lymph nodes (MLN), inguinal lymph nodes (ILN), and Peyer’s patches (PP) were isolated from mice that had either been fed with FVIII (HC+C2, "FVIII fed") or WT plant material ("control") followed by IV treatment with FVIII ("FVIII fed"). Stained cells were first gated for live CD4+ cells (positive CD4-eFluor 450 and negative viability dye eFluor 506 staining). The frequencies of CD4+CD25 LAP+ cells, CD4+CD25+Foxp3+ cells, and Tr1 cells (CD4+LAG-3+CD49b+) were calculated using flow cytometric analysis. Data for individual animals as well as averages 6 SEM are shown (n=3-5/group). Unpaired two-tailed Student’s T tests were used to calculate P values for all panels.

Figures. 7A-7D. Delivery of FVIII antigen to the GALT and into circulation. A-C:

Immunostains (original magnification 200X) of ileum cryo-sections from unfed (A, negative control) or CTB-C2 fed (B: lamina propria, C: Peyer’s patch) BALB/c hemophilia A mice. Stains are for C2 domain of FVIII (green), CD11c (red), and nuclei (DAPI; blue). D: Human FVIII antigen levels were measured in plasma or liver protein extract of the CTB-HC fed C57BL6/129 and BALB/c hemophilia A mice and WT fed control mice of the same strain using HC-specific ELISA. All data are shown for individual mice and as averages 6 SEM.

Figures 8A-8C: Antibody responses against CTB protein in hemophilia A mice fed with FVIII chloroplast transgenic plant material. IgG1 (A), IgG2a (B), and IgG2b (C) serum titers against CTB following 8 weeks of feeding with a mixture of CTB-HC and CTB-C2 transgenic tobacco material. Sera from mice of the same strain 2 weeks after challenging with 20 mg CTB in adjuvant served as positive controls. Unchallenged naive mice of the same strain served as negative controls. The dotted horizontal line indicates the background of the assay (average readings for negative controls). All data are shown for individual mice and as averages 6 SEM.

Figures 9A - 9G Generation and characterization of CTB-C2 transplastomic lettuce plants A: Lettuce CTBC2 expression vector and a portion of native chloroplast genome. 16S trnI and trnA 23S, lettuce homologous chloroplast genome flanking sequences comprising of 16S 3’ (or 23 5’) end sequences and complete trnl, trnA genes; Prrn, ribosomal RNA operon promoter with GGAGG ribosome binding site; aadA, aminoglycoside 3’-adenylytransferase gene for spectinomycin resistance; 5’ UTR, promoter and 5’ UTR of psbA gene from lettuce; 3’ UTR, 3’ UTR of psbA gene from lettuce; CTB, cholera toxin B subunit. A Gly Pro Gly Pro (GPGP) hinge and furin cleavage site (RRKR) is included between CTB and the C2 sequence. WT, wild type. The restriction site of AflIII and the sizes of Southern blot positive bands are indicated. B: Southern blot. WT (netransformisan wild type), 1-5: transplastomic lines, lettuce genomic DNA was digested with AflIII and probed with 1.12-kb lettuce flanking region. The 7.4-kb positive band contains the CTB-C2 insertion fragment in the transplastomic lines. The absence of WT fragment (4.8-kb) in transplastomic lines 1 and 4 clearly demonstrate homoplasmy. C: Western blot. Probe, anti-CTB pAb. CTB standard: 5ng, lOng, 20ng. S: supernatant fraction; H: homogenate fraction. Molecuar weight of CTB-C2: 31 kDa. CTB: 12 kDa. 2.5 mg total protein of supernatant or homogenate fraction per lane was loaded. D: Ganglioside GM1 ELISA binding assay. CTB standard (0.1ng); CTB-C2 (1 mg) ; netransformisan lettuce wild type (1 mg); BSA, bovine serum albumin (5 mg). E: CTB-C2 expression levels (Average6STDEV) in mg/g of fresh and lyophilized leaves.

Lyophilized leaf materials from T0 plants: 20046136; Fresh: 9466. F: Comparision of protein concentrations between the lyophilized leaf and fresh leaf samples. Equal amount of lyophilized or fresh leaf material was used for this analysis . 1, undiluted CTB-C2 extract (60 mg leaf powder); 1:10, 10 times dilution (6 mg leaf powder); 1:20, 20 times dilution (3 mg leaf powder). WT, wild type lettuce. CTB standard: 7.5 ng and 15 ng per lane loaded for quantitation. G: Preparation of capsules: lyophilized and powdered lettuce leaves expressing CTB-C2 fusion protein for use as oral antigen.

Slike 10A i 10B. Skrining za CTB-HC biljke zelene salate (T1) (A); western blot analizom (B). Proba: anti-CTB ; CTB-HC = 97.7 kDa. Sve pojedinačne biljke, izuzev biljke br.14, pokazale su ekspresiju CTB-HC fuzionog proteina.

Slika 11. Skrining za CTB-C2 biljke zelene salate (T1) western blot analizom. Sve pojedinačne biljke su pokazale ekspresiju CTB-HC fuzionog proteina.

DETALJAN OPIS PRONALASKA

[0009] Hemophilia A is the X-linked bleeding disorder uslovljena nedostatkom deficiency of coagulation factor VIII (FVIII). To address serious complications of inhibitory antibody formation in current replacement therapy, we created tobacco transplastomic lines expressing FVIII antigens, heavy chain (HC) and C2, fused with the transmucosal carrier, cholera toxin B subunit (CTB). CTB-HC and CTB-C2 fusion proteins expressed up to 80 or 370 mg/g in fresh leaves, assembled into pentameric forms, and bound to GM1 receptors. Protection of FVIII antigen through bioencapsulation in plant cells and oral delivery to the gut immune system was confirmed by immunostaining. Feeding of HC/C2 mixture substantially suppressed T helper cell responses and inhibitor formation against FVIII in hemophilia A mice of two different strain backgrounds. Prolonged oral delivery was required to control inhibitor formation long-term. Substantial reduction of inhibitor titers in pre-immune mice demonstrated that the protocol could also reverse inhibitor formation. Gene expression and flow cytometry analyses showed up-regulation of immune suppressive cytokines (TGF-β/LAP and IL 10). Adoptive transfer experiments confirmed an active suppression mechanism and revealed induction of CD4+CD25+ and CD4+CD25- T cells that potently suppressed anti-FVIII formation. In sum, these data support plant cell-based oral tolerance for suppression of inhibitor formation against FVIII.

[0010] Osim ako nisu drugačije definisani, svi tehnički i naučni izrazi koji se ovde koriste imaju isto značenje kao što to uobičajeno podrazumeva stručnjak u oblasti molekularne biologije. Iako se metode i materijali slični ili ekvivalentni ovde opisanim mogu koristiti u praktikovanju ili testiranju ovog pronalaska, ovde su opisani pogodni postupci i materijali.

[0011] U slučaju neslaganja, odlučujuća će biti predmetna specifikacija, uključujući definicije. Osim toga, materijali, postupci i primeri su samo ilustrativni, i nije predviđeno da budu ograničavajući.

[0012] U ovom dokumentu su citirani standardni udžbenici za molekularnu biologiju koji sadrže definicije i metode i sredstva za izvođenje osnovnih tehnika, obuhvaćenih predmetnim pronalaskom. Videti, na primer, Maniatis et al., Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory Press, New York (1982) i Sambrook et al., Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory Press, New York (1989); Methods in Plant Molecular Biology, Maliga et al, Eds., Cold Spring Harbor Laboratory Press, New York (1995); Arabidopsis, Meyerowitz et al, Eds., Cold Spring Harbor Laboratory Press, New York (1994) i različite reference koje se u njima navode.

[0013] Methods, vectors, and compositions for transforming plants and plant cells are taught for example in WO 01/72959; WO 03/057834; and WO 04/005467. WO 01/64023 discusses use of marker free gene constructs.

Metode, vektori i kompozicije za transformisanje biljaka i biljnih ćelija predaju se, na primer, u VO 01/72959; VO 03/057834; i VO 04/005467. U WO 01/64023 se razmatra upotreba genskih konstrukata bez markera.

[0014] Proteini eksprimirani u skladu sa određenim primerima izvođenja za koje su uputstva navedena u ovom dokumentu, mogu da se koriste in vivo primenom kod subjekta, čoveka ili životinje, na različite načine. Farmaceutske kompozicije mogu da se primenjuju oralno ili parenteralno, tj. subkutano, intramuskularno ili intravenski. Prema tome, ovaj pronalazak obezbeđuje kompozicije za parenteralnu primenu koje sadrže rastvor fuzionog fuzionisanog proteina (ili njegovog derivata) ili njegov koktel rastvoren u prihvatljivom nosaču, poželjno vodenom. Mogu se koristiti različiti vodeni nosači, npr. voda, puferovana voda, 0,4% fiziološki rastvor, 0,3% glicerin i slično. Ovi rastvori su sterilni i uglavnom ne sadrže čestični materijal. Ove smeše mogu da se sterilišu uobičajenim, dobro poznatim tehnikama sterilizacije. Kompozicije mogu da sadrže farmaceutski prihvatljive pomoćne supstance potrebne kako bi se postigli uslovi približni fiziološkim uslovima, kao što su sredstva za podešavanje pH i puferovanje, sredstva za podešavanje toksičnosti i slično, na primer natrijum acetat, natrijum hlorid, kalijum hlorid, kalcijum hlorid, natrijum laktat, itd. Koncentracija fuzionog proteina (ili njegovog dela) u ovim formulacijama može da varira u velikoj meri u zavisnosti od specifične amino kiselinske sekvence predmetnih proteina i željene biološke aktivnosti, npr. od manje od oko 0,5%, obično ili najmanje od oko 1%, pa do čak 15 ili 20% po težini, i biće odabrani prvenstveno na osnovu zapremine fluida, viskoznosti itd., u skladu sa konkretnim odabranim načinom primene.

[0015] U nekim primerima izvođenja, kompozicije prema predmetnom pronalasku mogu da se primenjuju putem konzumiranja namirnica koje su proizvedene sa transgenom biljkom koja proizvodi terapeutski protein. Jestivi deo biljke koristi se kao komponenta ishrane, pri čemu se u tom postupku procesu primenjuje terapeutski protein.

[0016] Tako, u jednom primeru izvođenja, pronalazak se odnosi na primenjivu kompoziciju koja indukuje toleranciju koja sadrži faktor oralne tolerancije, kao što je faktor koagulacije, koji je prethodno eksprimiran u biljci ili biljnom ostatku. Biljni ostatak može da sadrži jedan ili više molekula (kao što su, ali ne i ograničeno na, proteini i njihovi fragmenti, minerali, nukleotidi i njihovi fragmenti, biljne strukturne komponente (kao što su ćelijski odeljci) itd.), dobijeni iz biljke u kojoj je antigen eksprimiran. Shodno tome, vakcina koja se odnosi na ceo biljni materijal (npr. celinu ili delove biljnih listova, stabljika, plodova itd.) ili sirovi biljni ekstrakt, sigurno bi sadržala visoku koncentraciju biljnih ostataka, kao i kompoziciju koja sadrži prečišćeni antigen tog i jednog ili većeg broja biljnih ostataka koji mogu da se detektuju.

[0017] Kompozicije koje indukuju toleranciju prema nekim primerima izvođenja predmetnog pronalaska mogu da se formulišu sa farmaceutskim vehikulumom ili razblaživačem za oralnu, intravensku, subkutanu, intranazalnu, intrabronhijalnu ili rektalnu primenu. Farmaceutska kompozicija može da se formuliše na klasičan način upotrebom čvrstih ili tečnih vehikuluma, razblaživača i aditiva koji su pogodni za željeni način primene. Oralno, kompozicija može da se primeni u obliku tableta, kapsula, granula, praškova i slično, sa najmanje jednim vehikulumom, npr. skrobom, kalcijum karbonatom, saharozom, laktozom, želatinom itd. Aktivni imunogeni sastojak se često meša sa ekscipijensima koji su farmaceutski prihvatljivi i kompatibilni sa aktivnim sastojkom. Pogodni ekscipijensi su, npr., voda, fiziološki rastvor, dekstroza, glicerol, etanol ili slično, i njihove kombinacije. Pored toga, po želji, vakcina može da sadrži neznatne količine pomoćnih supstanci kao što su sredstva za vlaženje ili emulgovanje, sredstva za puferovanje pH ili adjuvanse koja pojačavaju efikasnost vakcina. Preparacija za parenteralnu primenu uključuje sterilisanu vodu, suspenziju, emulziju i supozitorije. Za emulgatore mogu da se koriste propilen glikol, polietilen glikol, maslinovo ulje, etiloleat itd. Tradicionalna vezivna sredstva i nosači koji se koriste za supozitorije mogu da uključuju polialken glikol, trigliceride, vitepsol, makrogol, tween 61, kakao puter, gliceroželatin, itd. Pored toga, kao ekscipijensi mogu da se koriste manitol, laktoza, skrob, magnezijum stearat, natrijum saharin, celuloza, magnezijum karbonat i slično, sa farmaceutskim stepenom čistoće.

[0018] Oral tolerance factors may be administered by the consumption of the foodstuff that has been manufactured with the transgenic plant and the edible part of the plant expressing the antigen is used directly as a dietary component while the vaccine is administrated in the process.

Faktori oralne tolerancije mogu da se primenjuju putem konzumiranja namirnica koje su proizvedene sa transgenom biljkom, a jestivi deo biljke koji eksprimira antigen koristi se direktno kao komponenta ishrane, dok se vakcina primenjuje u postupku. VIDI uporedi 0015

[0019] Primeri lako jestivih biljaka koje mogu da se transformišu tako da eksprimiraju ovde opisane konstrukte uključuju, ali nisu ograničeni na, jabuku, bobice kao što su jagode i maline, agrume, paradajz, bananu, šargarepu, celer, karfiol; brokoli, raštan, krastavac, dinju, lubenicu, papriku, krušku, grožđe, breskvu, rotkvice i kejl kupus.

[0020] Proteini koji indukuju toleranciju mogu da budu obezbeđeni putem soka transgenih biljaka radi pogodnosti primene. U navedenu svrhu biljke koje se transformišu poželjno se biraju od jestivih biljaka koje se sastoje od paradajza, šargarepe i jabuke, a koje se obično konzumiraju u obliku soka.

[0021] Stručnjaci u ovoj oblasti razumeće da aktivne varijante gena koji su ovde konkretno prikazani mogu da se koriste za proizvodnju terapeutskih kompozicija dobijenih iz biljaka. U publikaciji J Exp Med. 1997 May 19;185(10):1793-801 su obezbeđeni neki specifični primeri fragmenata poznatih antigenih proteina i gena koji ih kodiraju.

[0022] Takođe je opisan transformisani genom hloroplasta koji je transformisan pomoću vektora koji sadrži heterologi gen koji eksprimira faktor koagulacije. U srodnom primeru izvođenja, predmetni pronalazak se odnosi na biljku koja sadrži najmanje jednu ćeliju transformisanu tako da eksprimira faktor koagulacije.

[0023] U jednom primeru izvođenja, polipeptidi faktora koagulacije prema pronalasku sadrže najmanje 12, 15, 25, 50, 75, 100, 125, 150, 175, 200, 225, 250 ili 265 susednih aminokiselina sa sekvencom poznatog faktora koagulacije. Polipeptid faktora koagulacije prema pronalasku stoga može da bude deo (ili fragment) faktora koagulacije, protein koagulacionog faktora sa punom dužinom ili fuzijski protein koji sadrži ceo ili deo proteina faktora koagulacije. Stručnjacima u ovoj oblasti, uz poznavanje instrukcija iz ovog dokumenta, biće moguće da eksprimiraju i koriste druge poznate faktore koagulacije. Primeri drugih faktora koagulacije koji mogu da se koriste sa predmetnim pronalaskom uključuju, ali nisu ograničeni na, polipeptidne sekvence povezane sa sledećim pristupnim brojevima. NG_009258.1; NG_008953.1; NG_008107.1; NG_008051.1; NM_001993.3 i NM_00128.3, kao što je dato u relevantnim bazama podataka na datum podnošenja predmetnog pronalaska. Takođe, Dodatak A prikazuje reprezentativne primere sekvenci koje se odnose na navedene faktore koagulacije. Interaktivni regioni i proteazni regioni ovih sekvenci su poznati.

[0024] Polipeptidne varijante faktora koagulacije koje su biološki aktivne, tj. obezbeđuju sposobnost povećanja tolerancije prema odgovarajućem faktoru pri oralnoj primeni, takođe se smatraju polipeptidima faktora koagulacije u smislu ove patentne prijave. Poželjno, polipeptidne varijante faktora koagulacije koje se prirodno pojavljuju ili ne pojavljuju, imaju aminokiselinske sekvence koje su najmanje oko 55, 60, 65 ili 70, poželjno oko 75, 80, 85, 90, 96, 96 ili 98% identične amino kiselinskoj sekvenci poznate sekvence faktora koagulacije. Procenat identiteta pretpostavljene polipeptidne varijante koagulacionog faktora i poznate amino kiselinske sekvence može da se odredi, na primer, pomoću programa za upoređivanje sekvenci Blast2 (Blosum62, Ekpect 10, standardni genetski kodovi).

[0025] Varijacije u procentnu identiteta mogu da postoje usled, na primer, amino kiselinskih supstitucija, insercija ili delecija. Amino kiselinske supstitucije se definišu kao zamena jedne aminokiseline drugom. U prirodi su konzervativne kada amino kiselina kojom je izvršena supstitucija ima slična strukturna i/ili hemijska svojstva. Primeri konzervativnih zamena su supstitucija leucina izoleucinom ili valinom, aspartata glutamatom ili treonina serinom.

[0026] Amino kiselinske insercije ili delecije su promene amino kiselinske sekvence ili unutar aminokiselinske sekvence. Tipično spadaju u opseg od oko 1 do 5 amino kiselina. Smernice za određivanje koji amino kiselinski ostaci mogu da budu supstituisani, inserirani ili deletirani, a da ne dođe do gubitka biološke ili imunološke aktivnosti polipeptida faktora koagulacije mogu se naći upotrebom računarskih programa koji su dobro poznati u tehnici, kao što je softver DNASTAR. Da li neka izmena amino kiseline dovodi do nastanka biološki aktivnog polipeptidnog faktora koagulacije, može lako da se utvrdi tako što se testira aktivnost faktora koagulacije, kao što je opisano, na primer, u konkretnim Primerima, kasnije u tekstu.

[0027] Polinukleotid faktora koagulacije može biti jednolančani ili dvolančani i sadrži kodirajuću sekvencu ili komplement kodirajuće sekvence za polipeptid faktora koagulacije. Primeri drugih polinukleotidnih faktora koagulacije koji mogu da se koriste sa predmetnim pronalaskom uključuju, ali nisu ograničeni na, one polinukleotidne sekvence povezane sa sledećim pristupnim brojevima. NG_009258.1; NG_008953.1; NG_008107.1; NG_008051.1; NM_001993.3 i NM_00128.3, kao što je dato u relevantnim bazama podataka na datum podnošenja predmetnog pronalaska.

[0028] FVIII je visoko imunogeni molekul koji pri malim dozama antigena može da dovede do intenzivnih odgovora antitela kod pacijenata sa hemofilijom A i kod eksperimentalnih životinja. U nekim okolnostima, auto-antitela protiv FVIII mogu da se formiraju kod osoba koje nemaju hemofiliju, što dovodi do stečene hemofilije A. Većina inhibitora se vezuje za domen A2, A3 ili C2. Ove visoko imunogene sekvence takođe sadrže nekoliko CD4 epitopa T ćelija. Studije na životinjama ukazuju na to da stvaranje inhibitora protiv FVIII može da se spreči stvaranjem tolerancije na delove molekula, kao što je kombinacija A2 i C2 domena, dok samo jedan domen može da bude nedovoljan. Ovde je prikazano da teški lanac FVIII i C2 domen mogu da se eksprimiraju kao fuzioni proteini sub jedinice kolera toksina B (CTB) u duvanu. Oralno isporučivanje smeše ovih bioinkapsuliranih antigena suprimira stvaranje inhibitora kod miševa sa hemofilijom A.

[0029] Shodno tome, imajući u vidu instrukcije date u ovom tekstu, treba imati na umu da je moguća upotreba sekvence faktora koagulacije sa punom dužinom ili njenih delova u kojima su očuvani epitopni peptidi. Delovi mogu da uključuju polipeptidne fragmente od najmanje 15 aminokiselina s ciljem da se uključi i očuva imunogeni potencijal najmanje jednog peptidnog epitopa ove sekvence. Međutim, u idealnom slučaju, primenjena sekvenca uključuje što je moguće više epitopa, jer bi to povećalo verovatnoću uspešnog stvaranja tolerancije. Na primer, kad je u pitanju FVIII sekvenca, cela sekvenca može da bude eksprimirana, ili njeni delovi, tipično kao fuzioni proteini sa CTB. U publikaciji Van Haren et al., Mol Cell Proteomics, 2011)Jun;10(6): M110.002246, dato je nekoliko sekvenci koje se odnose na epitope (vidi, npr., Sl.2B).

Druge reference u kojima se navode lokacije različitih epitopa FVIII sekvence uključuju Jones et al., Journal of Thrombosis and Haemostasis 2005, 3:991- 1000; Reding et al., Journal of Thrombosis and Haemostasis, 2003, 1:1777:1784; Pratt et al., Thromb Haemost 2004: 92:522-8; i Hu et al., Journal of Thrombosis and Haemostasis 2004, 2:1908-1917. FVIII sadrži više domena: A1, A2, A3, B, C1 i C2. U nekim primerima izvođenja, sekvenca se odnosi na celu sekvencu najmanje jednog od domena i opciono najmanje deo ili sve od dva, tri, četiri, pet ili šest ovih domena. Sledi aminokiselinska sekvenca FVIII (homo sapiens), koja može da se koristi u šemi za ekspresiju u plastidima (na primer, primenom polinukleotida koji kodira celu amino kiselinsku sekvencu ili njene delove) ili kompozicije na bazi plastida kako su ovde navedene:

[0030] Degenerate nucleotide sequences encoding coagulation factor polypeptides, as well as homologous nucleotide sequences which are at least about 50, 55, 60, 65, 60, preferably about 75, 90, 96, or 98% identical to the nucleotide sequence encoding a coagulation factor also are coagulation factor polynucleotides. Percent sequence identity between the sequences of two polynucleotides is determined using computer programs such as ALIGN which employ the FASTA algorithm, using an affine gap search with a gap open penalty of -12 and a gap extension penalty of 2. Complementary DNA (cDNA) molecules, species homologs, and variants of coagulation factor polynucleotides which encode biologically active coagulation factor polypeptides also are coagulation factor polynucleotides.

Izrođene nukleotidne sekvence koje kodiraju polipeptide faktora koagulacije, kao i homologe nukleotidne sekvence koje su najmanje oko 50, 55, 60, 65, 60, poželjno oko 75, 90, 96 ili 98% identične nukleotidnoj sekvenci koja kodira faktor koagulacije, takođe su polinukleotidi faktora koagulacije. Procentualna identičnost sekvence između sekvenci dva polinukleotida određuje se pomoću računarskih programa kao što je ALIGN koji koriste FASTA algoritam, korišćenjem afinnog pretraživanja jaza sa kaznama otvorenog jaza od -12 i kaznom produženja jaza od 2. Molekuli komplementarne DNK (cDNK) , homolozi vrsta i varijante polinukleotida faktora koagulacije koji kodiraju biološki aktivne polipeptide faktora koagulacije takođe su polinukleotidi faktora koagulacije.

[0031] Stručnjacima u ovoj oblasti biće očigledno da se takve supstitucije mogu izvršiti van regiona kritičnih za funkcionisanje molekula, a da se i dalje dobija aktivni polipeptid. Amino kiselinski ostaci esencijalni za aktivnost polipeptida kodiranog izolovanim polinukleotidom prema pronalasku, i koji stoga poželjno da ne podležu supstituciji, mogu se identifikovati u skladu sa postupcima poznatim u oblasti, kao što je mesto-usmerena mutageneza ili alanin-skenirajuća mutageneza. (videti, npr., Cunningham and Wells, 1989, Science 244: 1081-1085). U ovoj drugoj tehnici, mutacije se uvode na svakom pozitivno naelektrisanom ostatku u molekulu, a aktivnost dobijenih mutiranih molekula se testira kako bi se identifikovali amino kiselinski ostaci koji su kritični za aktivnost molekula. Mesta interakcije supstrat-enzim takođe se mogu odrediti analizom trodimenzionalne strukture kako se određuje tehnikama kao što su analiza nuklearne magnetne rezonance, kristalografija ili fotoafinitetno obeležavanja (videti, npr., de Vos et al., 1992, Science 255: 306-312; Smith et al., 1992, Journal of Molecular Biology 224: 899-904; Wlodaver et al., 1992, FEBS Letters 309: 59-64).

TABELA 2: Amino kiselinske supstitucije

[0032] Značajne promene u funkciji ili imunološkom identitetu se sprovode tako što se biraju supstitucije koje su manje konzervativne od supstitucija u Tabeli 2, tj. tako što se biraju ostaci koji se značajnije razlikuju po svom uticaju na održavanje (a) strukture polipeptidne kičme u oblasti supstitucije , na primer u vidu ploče ili spiralne konformacije, (b) naelektrisanja ili hidrofobnosti molekula na ciljnom mestu ili (c) glavnine bočnog lanca. Supstitucije za koje se generalno očekuje da dovedu do najvećih promena u svojstvima proteina biće one supstitucije u kojima (a) hidrofilni ostatak, npr. seril ili treonil, je supstituisan (ili se supstituiše) hidrofobnim ostatkom, npr. leucil, izoleucil, fenilalanil, valil ili alanil; (b) cistein ili prolin su supstituisani (ili se supstituiše) bilo kojim drugim ostatkom; (c) ostatak koji ima elektropozitivni bočni lanac, npr. lizil, arginil ili histidil, je supstituisan (ili se supstituiše) elektronegativnim ostatkom, npr. glutamilom ili aspartilom; ili kada je (d) ostatak koji ima glomazan bočni lanac, npr. fenilalanin, supstituisan (ili se supstituiše) nekim ostatkom koji nema bočni lanac, npr. glicinom, u ovom slučaju, (e) broj mesta za sulfataciju i/ili glikozilaciju povećan.

[0033] Na primer, zamena jednog amino kiselinskog ostatka drugim koji je biološki i/ili hemijski sličan poznat je stručnjacima kao konzervativna supstitucija. Na primer, konzervativna supstitucija bi bila kada se jednan hidrofobni ostatak zameni drugim, ili jedan polarni ostatak drugim. Supstitucije uključuju kombinacije kao što su, na primer, Gly, Ala; Val, Ile, Leu; Asp, Glu; Asn, Gln; Ser, Thr; Lis, Arg; i Phe, Tir. Takve konzervativno supstituisane varijacije svake eksplicitno prikazane sekvence uključene su u ovde obezbeđene mozaične polipeptide.

[0034] Mutageneza supstitucijom ili delecijom može da se koristi za inserciju mesta za N-glikozilaciju (Asn-X-Thr/Ser) ili O-glikozilaciju (Ser ili Thr). Delecije cisteina ili drugih labilnih ostataka takođe mogu da budu poželjne. Delecije ili supstitucije potencijalnih mesta proteolize, npr. Arg, postiže se na primer delecijom jednog od baznih ostataka ili supstitucijom nekog od njih glutaminilom ili histidilom.

[0035] Neke post-translacione derivatizacije su posledica delovanja rekombinantnih ćelija domaćina na eksprimirani polipeptid. Glutaminil i asparaginil ostaci često se posle translacije deamiduju do odgovarajućih glutamil i asparil ostataka. Alternativno, ovi ostaci se deamiduju u blago kiselim uslovima. Ostale posttranslacione modifikacije uključuju hidroksilaciju prolina i lizina, fosforilaciju hidroksilnih grupa seril ili treonil ostataka, metilaciju o-amino grupa lizinskih, argininskih i histidinskih bočnih lanaca (TE Creighton, Proteins: Structure and Molecular Properties, VH Freeman & Co., San Francisco str. 79-86 [1983]), acetilaciju N-terminalnog amina i, u nekim slučajevima, amidaciju C-terminalnog karboksila.

[0036] Pošto se u ovoj specifikaciji razmatraju različiti proteini i proteinske sekvence, podrazumeva se da su takođe prikazane nukleinske kiseline koje mogu da kodiraju te proteinske sekvence. To bi obuhvatilo sve izrođene sekvence srodne specifičnoj proteinskoj sekvenci, tj. sve nukleinske kiseline koje imaju sekvencu koja kodira jednu konkretnu proteinsku sekvencu, kao i sve nukleinske kiseline, uključujući izrođene nukleinske kiseline, koje kodiraju prikazane varijante i derivate proteinskih sekvenci. Prema tome, iako ovde ne mora da bude napisana svaka pojedinačna sekvenca nukleinske kiseline, podrazumeva se da je svaka sekvenca ovde zapravo otkrivena i opisana kroz prikazanu sekvencu proteina.

[0037] Varijante i homolozi polinukleotida faktora koagulacije koji su prethodno u tekstu opisani, takođe su polinukleotidi faktora koagulacije. Homologe polinukleotidne sekvence faktora koagulacije uobičajeno mogu da se identifikuju hibridizacijom polinukleotida kandidata sa poznatim polinukleotidima faktora koagulacije pod restriktivnim uslovima, kao što je poznato u struci. Na primer, svaka homologa sekvenca koja sadrži najviše oko 25-30% pogrešno sparenih baza može da se identifikuje upotrebom sledećih uslova ispiranja: 2 X SSC (0.3 M NaCl, 0.03 M natrijum citrat, pH 7.0), 0.1% SDS, sobna temperatura dva puta, po 30 minuta; zatim 2 X SSC, 0.1% SDS, 50 ° C jednom, 30 minuta; zatim 2 X SSC, dva puta na sobnoj temperaturi, svaki put po 10 minuta. Još poželjnije, lanci homologih nukleinskih kiselina sadrže 15-25% pogrešno sparenih baza, još poželjnije 5-15% pogrešno sparenih baza.

[0038] Homolozi drugih vrsta za polinukleotide faktora koagulacije koji su ovde prikazani takođe se mogu identifikovati pravljenjem pogodnih proba ili prajmera i skriningom ekspresionih cDNK biblioteka. Dobro je poznato da se Tm dvolančane DNK smanjuje za 1-1,5OC sa svakih 1% smanjenja homologije (Bonner i sar., J. Mol. Biol. 81, 123 (1973). Varijante polinukleotida faktora koagulacije ili polinukleotida drugih vrsta mogu se stoga identifikovati hibridizacijom pretpostavljenog homologog polinukleotida faktora koagulacije sa polinukleotidom koji ima nukleotidnu sekvencu SEK ID NO: 1 ili sa njegovim komplementom, kako bi se obrazovao test hibrid. Temperatura topljenja test hibrida se upoređuje sa izračunava se temperatura topljenja hibrida koji sadrži polinukleotide koji imaju savršeno komplementarne nukleotidne sekvence i izračunava se broj ili procenat pogrešno sparenih baza u okviru test hibrida.

[0039] Nukleotidne sekvence koje hibridizuju sa polinukleotidima faktora koagulacije ili njihovim komplementima nakon restriktivnih uslova hibridizacije i/ili ispiranja takođe su polinukleotidi faktora koagulacije. Restriktivni uslovi ispianja su dobro poznati i proučeni u stanju tehnike i prikazani su, na primer, u Sambrook et al., MOLECULAR CLONING: A LABORATORY MANUAL, 2nd ed., 1989, na stranama 9.50-9.51.

[0040] Uobičajeno je da se za restriktivne uslove hibridizacije bira kombinacija temperature i koncentracije soli koja je približno 12-20°C niža od izračunate Tm hibrida koji se proučava. Tm hibrida nastalog od polinukleotida faktora koagulacije koji ima nukleotidnu sekvencu prikazanu u SEK ID NO: 1 ili njenog komplementa i polinukleotidne sekvence koja je najmanje oko 50, poželjno oko 75, 90, 96 ili 98% identična jednoj od tih nukleotidnih sekvenci, mogu da se izračunaju, na primer, koristeći jednačinu po Bolton-u i McCarthi-ju, Proc. Natl. Acad. Sci. SAD 48, 1390 (1962): Tm = 81,5°C-16,6 (log10[Na ]) 0.41 (% G C) 0.63 (% formamida) -600/l), gde je l = dužina hibrida u baznim parovima.

[0041] Restriktivni uslovi ispiranja uključuju, na primer, 4 X SSC na 65° C, ili 50% formamid, 4 X SSC na 42°C, ili 0.5 X SSC, 0.1% SDS na 65°C. Veoma restriktivni uslovi ispiranja uključuju, na primer, 0.2 X SSC na 65°C.

[0042] Also described is a method of treating a subject having a genetic disease prone and at risk to experiencing an anaphylactic reaction responsive to protein replacement therapy. The method comprises administering to the subject an effective amount of a composition comprising a tolerance factor and a plant remnant, and administering a therapeutically effective amount of a protein corresponding to a defect or deficiency associated with said disease.

Takođe je opisan postupak za lečenje subjekta koji ima genetsku bolest sklonu riziku da doživi anafilaktičku reakciju koja reaguje na terapiju zamenom proteina. Postupak sadrži primenu kod subjekta efikasne količine kompozicije koja sadrži faktor tolerancije i biljni ostatak i davanje terapeutski efikasne količine proteina koja odgovara defektu ili nedostatku koji je u vezi sa navedenom bolešću. Uobičajeno, faktor tolerancije se odnosi na faktor koagulacije, (videti prethodno u tekstu), kiselu α-glukozidazu (pristupni br. NM_001079803.1, NM_001079804.1, NM_0001152.3), α-galaktozidazu A, (pristupni br. NM_000169.2) Glukocerebrozidazu (pristupni br. J03059, J03060), α-L-iduronidase (accession no. NM_000203.3), or sphingomyelinase (pristupni br. NM_000543.3, NM_001007593.1. The principles described above with respect to coagulation factor polypeptides and polynucleotides and variants in the preceding eleven paragraphs also apply to the sequences asscociated with the accession nos. provided in this paragraph. Also, the disease treated typically pertains to Hemophilia A, Hemophilia B, Pompe disease, Fabry disease, Gaucher disease, Mucopolysaccharidosis I, or Niemann-Pick disease. The tolerance factors may be conjugated to a CTB protein (see, e.g., Lai, CY, Journal of Biological Chemistry, (1977) 252:7249-7256, or accession no. DQ523223, also see ref 39) to enhance oral tolerance potential.

[0043] Sledeći primeri su obezbeđeni kako bi se ilustrovali određeni primeri izvođenja prema pronalasku. Nije predviđeno da oni na bilo koji način ograniče pronalazak.

PRIMER I

[0044] While there are currently no prophylactic protocols against inhibitor formation in patients, preclinical experiments in murine models of hemophilia A have provided proof-of-principle that preventive immune tolerance to FVIII can be established.6-11 However, such protocols utilize genetic manipulation or immune suppressive drugs, raising safety concerns for translation to human treatment. In contrast, oral tolerance could be a more readily acceptable form of prophylactic tolerance induction and may be more readily tested in clinical trials.12,13

Iako trenutno ne postoje profilaktički protokoli protiv stvaranja inhibitora kod pacijenata, pretklinički eksperimenti na mišjim modelima hemofilije A pružili su dokaz principa da se može uspostaviti preventivna imunološka tolerancija na FVIII.6-11 Međutim, takvi protokoli koriste genetsku manipulaciju ili imunološki supresivne lekove, izazivajući zabrinutost za bezbednost prevođenja na lečenje ljudi. Nasuprot tome, oralna tolerancija može biti lakše prihvatljiv oblik profilaktičke indukcije tolerancije i može se lakše testirati u kliničkim ispitivanjima.12,13However, effective tolerogenic delivery of coagulation factor antigen to the gut-associated lymphoid tissue (GALT) is a challenge.14 We have developed a costeffective system for production of high levels of protein in chloroplasts of transplastomic plant cells, which provide bioencapsulation of the antigen through the cellulose containing cell walls.15,16 Because of the high number of chloroplast genomes per cell and our optimized expression system, transgenic proteins can accumulate in green leaves at much higher levels than this is the case for more traditional transgenic plant technologies.17,18 Oral delivery of transplastomic plant cells has been effective in prevention of insulitis in non-obese diabetic mice and of inhibitor formation in hemophilia B mice.19,20

3Međutim, efikasna tolerogena isporuka antigena faktora koagulacionog limfoidnog tkiva povezanog sa crevima (GALT) predstavlja izazov.14 Razvili smo kofektivni sistem za proizvodnju visokog nivoa proteina u hloroplastima transplastomskih biljnih ćelija, koji obezbeđuju bioenkapsulaciju antigena kroz ćelijske zidove koji sadrže celulozu.15,16 Zbog velikog broja genoma hloroplasta po ćeliji i našeg optimizovanog sistema ekspresije, transgeni proteini se mogu akumulirati u zelenom lišću na mnogo višim nivoima nego što je to slučaj kod tradicionalnijih tehnologija transgenih biljaka.17 , 18 Oralna isporuka transplastomnih biljnih ćelija bila je efikasna u prevenciji insulitisa kod ne-gojaznih dijabetičkih miševa i stvaranja inhibitora kod miševa hemofilije B.19,20

[0045] For FIX inhibitors, ITI is often not sustainable because of anaphylactic reactions and development of nephrotic syndrome. In hemophilia B mice, we demonstrated that repeated oral delivery of bioencapsulated FIX prevented inhibitor formation and fatal anaphylaxis in subsequent replacement therapy.20 Encouraged by these results, we sought to develop a protocol for hemophilia A. FVIII is a large protein, comprised of a signal peptide and a 2332 amino acid polypeptide. Structurally, FVIII contains 6 distinct domains, which are organized in the following order: A1-A2 B-A3-C1-C2.21 The large, central B domain is highly glycosylated and aids in secretion of the molecule.22-24

Za FIKS inhibitore, ITI često nije održiv zbog anafilaktičkih reakcija i razvoja nefrotskog sindroma. Kod miševa hemofilije B pokazali smo da ponovljena oralna primena bioenkapsuliranog FIKS sprečava stvaranje inhibitora i fatalnu anafilaksiju u naknadnoj zamenskoj terapiji.20 Ohrabreni ovim rezultatima, pokušali smo da razvijemo protokol za hemofiliju A. FVIII je veliki protein, koji se sastoji od signalni peptid i 2332 aminokiselinski polipeptid. Strukturno, FVIII sadrži 6 različitih domena, koji su organizovani sledećim redosledom: A1-A2 B-A3-C1-C2.21 Veliki, centralni B domen je visoko glikoziliran i pomaže u sekreciji molekula. 22-24

Međutim, FVIII sa delecijom rekombinantnog B domena (BDD) biološki je aktivan i predstavlja jedan od proizvoda koji su trenutno u kliničkoj upotrebi. FVIII se izlučuje kao heterodimer nakon najmanje dva unutarćelijske degradacije unutar B domena. Usled toga, FVIII u cirkulaciji se sastoji od teškog lanca (koji sadrži domene A1-A2-B) i lakog lanca (domeni A3-C1-C2), koji su nekovalentno vezani.21,23

[0046] FVIII is a highly immunogenic molecule that can cause potent antibody responses in hemophilia A patients and in experimental animals at low antigen doses.6,25 The majority of inhibitors bind to A2, A3, or C2 domain.6,26-28 These highly immunogenic sequences also contain several CD4+ T cell epitopes.6,29,30 Animal studies suggest that inhibitor formation against FVIII can be prevented by tolerization to parts of the molecule, such as combination of A2 and C2 domains, while a single domain may not be sufficient.14,31 Here, we demonstrate that FVIII heavy chain and C2 domain can be expressed as cholera toxin B subunit (CTB) fusion proteins in tobacco chloroplasts. Oral delivery of a mixture of these bioencapsulated antigens suppressed and also reversed inhibitor formation in a mouse model of hemophilia A.

[0047] Sledeći materijali i metode su obezbeđeni kako bi se olakšalo praktikovanje primena predmetnog pronalaska.

Dizajn i konstruisanje ekspresionih vektora za hloroplaste

[0048] Because efficient delivery of bioencapsulated antigen to the GALT is required for tolerance induction and is facilitated by transmucosal carriers, human FVIII antigens were expressed as CTB fusions, a successful strategy for tolerogenic delivery of FIX and proinsulin.20,32 Because of the large size of the FVIII molecule and the need for CTB fusions to form pentamers to bind to the GM1 receptor on gut epithelial cells, two separate FVIII chloroplast transformation vectors were constructed to include either the heavy chain (abbreviated as HC, with identical amino acid sequence as in recombinant BDD-FVIII and therefore containing A1 and A2 domains and 5 amino acids of B domain) or the C2 domain. The cDNA fragment of human FVIII-HC was amplified by PCR. PCR products, flanked with a furin cleavage and suitable restriction sites, were cloned into the pCR BluntII Topo vector (Invitrogen), and the sequence was verified. Then, the HC DNA fragment was ligated with pLD-Ctv-5CP chloroplast transformation vector containing the CTB and GPGP hinge sequences to create the pLD-CTB-HC expression vector.19,33 An analogous pLD-CTB-C2 expression vector was also constructed. Chloroplast vectors pLD-CTB-HC and pLD-CTB-C2 (Sl.

1A) contain homologous flanking sequences 16S/trnI and trnA/23S from tobacco chloroplast genome to facilitate recombination with the native chloroplast genome. Expression of CTB-HC and CTB-C2 is regulated by the highly expressed tobacco chloroplast psbA 5’UTRpromoter and 3’UTR. The CTB-HC and CTB-C2 expression cassettes contain a glycine-proline-glycine-proline (GPGP) hinge between CTB and the HC or C2 element to prevent steric hindrance of the fusion proteins. In addition, a furin cleavage site, Arg-Arg-Lys-Arg, was created at the junction region of the fusion proteins to efficiently release the FVIII domains after internalization by epithelial cells.34 The expression cassettes include the aadA (aminoglycoside 3’ adenylyltransferase) selection marker gene with a GGAG ribosome binding site, driven by a tobacco plastid ribosomal operon promoter (Prrn), to confer spectinomycin resistance. The final chloroplast transformation vectors pLD-CTB HC and pLD-CTB-C2 (Sl.1A) were sequenced and used for transformation.33

Regeneracija biljaka duvana sa transformisanim hloroplastima

[0049] Vektori za transformaciju hloroplasta duvana, pLD-CTB-HC i pLD-CTB-C2 (Sl. 1A) upotrebljeni su kako bi se duvan (Nicotiana tabacum) transformisao putem bombardovanja česticama zlata obloženim plazmidnom DNK.<33>Bombardovani listovi su zatim prebačeni u medijum za selekciju/regeneraciju. Regeneracija of FVIII transplastomic tobacco plants was performed as described earlier.33,34,35

Characterization of FVIII expression in leaf tissues of transplastomic plants

[0050] Immunoblot analysis and quantitation of the CTB-HC and CTB-C2 fusion proteins were performed by previously reported protocols.18,34 GM1-ganglioside receptor binding assay was performed as reported earlier.34 The Bis-Tris 3-12% gradient native gel electrophoresis followed by immunoblot analysis was carried out by following the instruction manual of the NativePage Novex Bis-Tris Gel System (Life Technologies).

[0051] The cDNA fragment of human FVIII heavy chain (A1-A2 domains plus first 5 amino acids of B domain) is amplified by PCR. PCR products, flanked with a furin cleavage and suitable restriction sites, are cloned into the pCR BluntII Topo vector (Invitrogen), and the sequence is verified. Lettuce expression vector pLsDV-CTB.C2 containing the CTB-C2 DNA fragment was amplified from pLD-CTB.C2 by PCR, and the sequence of the PCR product was confirmed after cloning into pCR BluntII Topo vector. Lettuce expression vector pLs-CTB.C2 was created by subcloning the Ls 5’ UTR/CTB-C2/Ls 3’ UTR cassette into the pLsDV vector.34,35 Regeneration of lettuce plants was performed as described earlier.33,35

Characterization of the F.VIII transplastomic lettuce plant

[0052] To evaluate transgene integration and homoplasmy of the transplastomic lines, PCR and Southern blot analyses were carried out. Immunoblot analysis and quantitation of the CTB-HC and CTB-C2 fusion proteins were performed by previously reported protocols. GM1-ganglioside receptor binding assay was performed as reported earlier. The Bis-Tris 3-12% gradient native gel electrophoresis followed by immunoblot analysis was carried out by following the instruction manual of the NativePage Novex Bis-Tris Gel System (Life Technologies).

Lyophilization and storage

[0053] Lettuce leaves expressing CTB-C2 was lyophilized in Freezone Benchtop Freeze Dry Systems (Labconco) as previously described.36 Lyophilized leaves were stored at room temperature under vacuum for a few weeks and ground to fine powder in Warring blender. The lyophilized lettuce fine powder was stored dry at room temperature for several months.

Mouse strains and experiments

[0054] Male hemophilia A mice with targeted deletion of F8 exon 16 (F8e16-/-) on a mixed C57BL6/129 or on a pure BALB/c background were housed under special pathogen-free conditions and were ∼2 months of age at the onset of experiments.9,36 Leaf material was ground in liquid nitrogen and stored at -80°C. A mixture of CTB-HC and CTB-C2 material (total of 125 mg per mouse per dose) was suspended in sterile PBS (200 ml/dose), homogenized, and delivered via oral gavage using a 20-G bulb-tipped gastric gavage needle. For antigen challenge, mice were administrated 1 IU BBD-human FVIII (Xyntha, Pfizer, New York, NY) into the tail vein once a week. Plasma samples were obtained by tail bleed was as published.37 ELISA for FVIII antigen and anti-FVIII, Bethesda assays, and lymphocyte assays were as published.9,36,37 Characterization and quantitation of the CTB-HC and CTB-C2 fusion proteins Tobacco leaves were collected from mature plants at different time points (10 am, 2 pm and 6 pm) on the same day.1,2 In brief, the leaf samples were frozen and ground in liquid nitrogen. The ground leaf powder was suspended in an extraction buffer (200 mM Tris-HCl, pH 8.0, 100 mM NaCl, 100 mM DTT, 0.1% SDS, 400 mM sucrose, 0.05% Tween 20, 2 mM PMSF and proteinase inhibitor cocktail). The leaf suspension (i.e., homogenate) was either directly used for western blot or underwent an additional centrifugation to separate the supernatant portion. Bradford assay was performed using protein assay dye reagent (Bio-Rad) to determine the protein concentration of the total leaf protein in homogenate fraction and the protein concentration of the total soluble protein in the supernatant portion. To detect the fusion protein expression level in both homogenate fraction and supernatant portion, western blot analysis was performed with anti-CTB antibody as a probe using known concentration of purified CTB protein (Sigma) as the standard. Then, the concentration of CTB-HC or CTBC2 fusion protein was quantitatively measured by densitometry analysis using Alpha imager 2000. The percentage of fusion protein and amount of transgenic protein (mg/g or mg/g) is calculated according to the formula published earlier.3

Lymphocyte assays

[0055] Murine splenocytes were isolated by standard methods and cultured in RPMI 1640 media containing 5 mM β-mercaptoethanol, 100 mM insulin / transferrin / selenium, glutamine and pencillin / streptomycin with or without 10 mg/ml of FVIII for 48 hours at 37 °C and 5% CO2. Cells were then harvested and RNA was extracted using Qiagen Rneasy isolation kit (Valencia, CA, USA) according to manufacturer’s protocol. Quantitative RT-PCR was performed as previously described using SABiosciences kit.4 Frequencies of IL-10 secreting cells in hemophilia A C57BL6/129 mice in each group were determined by ELISpot assay (R&D System, Minneapolis, MN, USA) according to manufacturer’s protocol. Spots were counted using the CTL-ImmunoSpotH S5 UV analyzer (Cellullar Technology, Shaker Heights, OH, USA). Each sample was run in duplicate. CD4-, CD4+CD25-, and CD4+CD25- cells were purified from spleens and MLN using a magnetic isolation kit (Miltenyi Biotech, Bergisch Gladbach, Germany). The cells were then pooled for each experimental group and adoptively transferred to naive BALB/c mice at 106 cells per mouse via tail vein injection. Recipient mice were challenged 24 hours later via subcutaneous injection of 1 IU FVIII in adjuvant (Sigma Adjuvant System, Sigma, St. Louis, MO, USA).

Antibody and antigen assays

[0056] Antibody responses to FVIII were measured via ELISA for anti-FVIII IgG1, IgG2a, and IgG2b , and by Bethesda assay for inhibitor formation as described before.4-6 Human FVIII HC antigen in circulation was detected via ELISA. Mice were fed with CTB-HC (250 mg) twice per day for 2 days, and bled and sacrificed 5 hours after the last gavage. Harvested liver was washed with cold PBS and homogenized in RIPA buffer (PBS with 1% Nonidet P-40, 0.5% sodium deoxycholate, and 0.1% SDS, pH 7.5) with complete protease inhibitor mixture (Roche Diagnostics, Indianapolis, IN, USA) for 30 min on ice. After centrifugation for 20 min at 14,0003 g at 4°C, the supernatant was collected. For Hcspecific ELISA, plate was coated with 2 mg/ml GMA-012 (Green Mountain Antibodies, Burlington, VT, USA) overnight. BBD-FVIII (Xyntha, Pfizer, New York, NY, USA) was used as the standard. Sheep anti-human FVIII (Haematologic Technologies Inc, Essex Junction, VT, USA) was diluted to 0.3 mg/ml, and HRP-rabbit anti-sheep IgG (Invitrogen, Camarillo, CA, USA0 was used at 1:2000 dilution. Antibody responses to CTB were measured via ELISA as published.7

Flow cytometry

[0057] Spleens, mesenteric lymph nodes, Peyer’s patches, and inguinal lymph nodes were harvested, and single cell suspensions were prepared using 70-nm cell strainer in cold PBS buffer, followed by Fc blocker for 5 min at room temperature. Cells were surface stained for CD4-eFluor 450, CD25-PE, LAP-Perp at 4°C for 30 min in PBS, followed by viability dye eFluor 506 stain at 4°C for 30 min in PBS. Fixation and Foxp3 Alexa Fluor 647 stain was performed using the transcriptional factor staining buffer set. Other cells were surface stained with CD4-eFluor 450, CD49b-APC, and LAG3-PE (Tr1 staining) at 4°C for 30 min in PBS, followed by viability dye eFluor 506. Controls included isotype control, single positive, and unstained cells. All kits and antibodies were purchased form eBiosciences (San Diego, CA). Flow cytometry was performed on a LSRII system (BD Bioscience, San Jose, CA), and data were analyzed with FCSExpress software (De Novo Software, Los Angeles, CA).

Immunohistochemistry

[0058] Mice were fed with CTB-C2 (250 mg) twice per day for 2 days and sacrificed 5 hours after the last gavage. Tissue was collected, frozen, cryo-sectioned, and stained as described.19 The following antibodies were used: rabbit anti-FVIII light chain (1:200, Santa Cruz Biotechnology , Dallas, TX); biotin anti-mouse CD11c (1:200, BD Biosciences, San Jose, CA; Alexa Fluor-488 donkey anti-rabbit IgG (1:400, Jackson ImmunoResearch Laboratories, West Grove, PA), and streptavidin Alexa Fluor-568 (1:400, Invitrogen, Grand Island, NY). Images were captured using Nikon Eclipse 80i fluorescence microscope, Retiga 2000R digital camera (Qlmaing, Surrey, BC, Canada), and Nikon Elements software.

RESULTS

Characterization of FVIII-transplastomic lines

[0059] Putative transplastomic tobacco lines obtained after bombardment of chloroplast vectors were first screened by PCR analysis. Site-specific transgene integration into the chloroplast genome was confirmed with two specific primer sets 3P/3M and 5P/2M, which anneal specifically to complementary sequences of transgene cassette and the chloroplast genome.33 Three independent tobacco lines from pLD-CTB-HC transformation and four independent lines from pLDCTB- C2 transformation showed positive PCR products of correct sizes (data not shown). The CTB-HC- and CTB-C2-transplastomic tobacco lines were further examined by Southern blot analysis for site specific stable integration and homoplasmy. Homoplasmy is achieved when all copies of the chloroplast genomes have stably integrated transgenes. The results showed that all 3 tested lines of CTB-HC transplastomic lines had integrated transgenes at specific sites and were homoplasmic, showing only the larger genome fragment (8.6kb) with the transgene insert when compared with the 4.4kb fragment in the netransformisan control genome (Sl. 1B). The CTB-C2-transplastomic tobacco lines also showed integration of transgenes into the chloroplast genome and homoplasmy (Sl. 1C).

[0060] Expression of CTB-HC and CTB-C2 fusion proteins in protein extracts from leaves of transplastomic tobacco plants was evaluated by western blot analysis. Under fully denatured and reducing conditions, blots probed with anti-CTB polyclonal antibody revealed full-length CTB-HC fusion protein with the expected molecular mass of 98 kDa (Sl.2A) in all transplastomic lines. No cleaved products were observed even after solubilization of pentamers and destabilization of disulfide bonds with reducing agents. A similar banding pattern was observed in a parallel blot probed with an anti-A2 domain specific monoclonal antibody. In addition, there was no cross reactivity of CTB standard protein or any other plant protein in netransformisan leaf extracts (both used as negative controls) with the anti-A2 antibody (Sl.2B). Quantitation of the fusion protein was performed by densitometry on western blots of leaf extracts using known amounts of purified CTB protein as the standard. The CTB-HC fusion protein was found to accumulate up to 0.8% total leaf protein or 80 mg/g fresh leaf tissue (Sl. 2A and D). The CTB-C2 fusion protein was similarly analyzed with anti-CTB polyclonal antibody in tobacco plants. As shown in Sl. 2C, a 31 kDa polypeptide representing the correct size of CTB-C2 fusion protein was detected in both fractions (supernatant and homogenate) of independent transplastomic lines. The CTB-C2 fusion protein accumulated up to 4.2% in the homogenate (i.e., 4.2% TLP or 370 mg per g of fresh leaf, Sl.2D). Pentamer assembly of CTB-HC and CTB-C2 in transgenic chloroplasts

[0061] A plasma membrane receptor (GM1-ganglioside) binds CTB in vivo, and a pentameric structure is required for binding to GM1 receptor.38-40 To evaluate receptor binding ability of CTB-HC and CTB-C2 fusion proteins produced in tobacco chloroplasts, GM1-binding ELISA was performed. As observed in Sl.2E, CTB-HC and CTB-C2 fusion protein extracts along with purified CTB protein showed strong binding affinity to GM1. Therefore, CTB-HC and CTB-C2 fusion proteins assembled properly to form pentameric structures within transformed chloroplasts. To further evaluate the pentamer assembly directly, we ran blue native gels, and the blots were probed with anti-CTB polyclonal antibody. These results indicate that the pentameric structure (CTB-HC, 490 kDa; CTB-C2, 155 kDa) was formed in both CTB-HC and CTB-C2-transformed tobacco chloroplasts. In addition, other oligomeric forms larger than pentamers were also observed (Sl.2F). Lack of cleaved products confirmed stability of assembled pentamers or multimers within transformed chloroplasts.

Oral delivery of bioencapsulated FVIII suppresses inhibitor formation in hemophilic mice [0062] Plant leaf materials were ground in liquid nitrogen as published.19 CTB-HC and CTB-C2 materials were mixed and suspended in PBS buffer so that the final product contained approximately equal amounts of both fusion proteins (∼5mg HC/6mg C2 per dose/mouse). Male hemophilia A mice (F8e16-/-) on C57BL6/129 genetic background received oral gavage of 125 mg mixed material per dose, twice per week for 2 months (Sl. 3A). During the second month, FVIII concentrate (recombinant BDD-FVIII) was given IV once per week at 1 IU/mouse. As expected based on prior findings, control mice that received no gavage (n=9) or were fed with WT plant material (n=6) formed very high-titer inhibitors (50-391 BU/ml; Sl. 3B).9,36 These were predominantly IgG1 with substantially less IgG2a and IgG2b formation (Sl. 3C-E). In contrast, inhibitor formation was significantly suppressed (on average 7-fold) in those mice that had been fed with FVIII plant material (n=6). These differences in BU correlated with the level of suppression of FVIII-specific IgG1 formation (Sl.3C). IgG2a and IgG2b anti-FVIII became undetectable in FVIII-fed mice (Sl.3D-E).

[0063] To address the effect of oral antigen delivery on T cell responses to FVIII, we harvested splenocytes from C57BL6/129 F8e16-/- mice that had been fed with WT or FVIII expressing plant material and treated with FVIII. In vitro re-stimulation with FVIII induced expression of several cytokines associated with different T helper cell responses in cultures from WT fed mice (Sl. 3F). IL-6 was the most highly and consistently expressed cytokine, which we have previously shown to be expressed by CD4+ T cells of this strain in response to FVIII.9 These control mice lacked expression of immune suppressive cytokines or Treg markers. In contrast, splenocytes from FVIII fed did not show expression of IL-6 or other cytokines associated with Th1 (IL-2, IFN-γ), Th2 (IL-4, IL-13), or Th17 (IL-17) responses. Instead, up-regulation of Treg markers (CD25, FoxP3, CTLA-4) and, more markedly, of suppressive cytokines IL-10 and TGF-β was observed. Hence, the response was shifted from an effector to a suppressive/regulated response. These results were further supported by an increase in IL-10 producing splenocytes in ELISpot assay (Sl.3G).

Suppression of inhibitor formation is successful in different strain backgrounds

[0064] The identical experiment was performed in hemophilia A mice with the same F8 mutation but backcrossed on a BALB/c background. Inhibitor formation in this strain is not as brisk.36,41 Nonetheless, control mice (n=8-11/group) invariably formed high-titer inhibitors (8-200 BU/ml) after 4 weekly IV injections of FVIII (Sl.4A-B). The response was again dominated by IgG1, albeit that IgG2a and IgG2b responses were also observed at substantial titers in some of the animals (Sl.

4C-E). Among FVIII-fed mice, 7 had undetectable inhibitors and 4 formed low-titer inhibitors (1-4 BU/ml), indicating more complete suppression in this strain by oral antigen administration (Sl. 4B). Total IgG formation was suppressed by approximately 1 log, with absent IgG2a and IgG2b and IgG1 reduced to low-titer (Sl.4C-E). Next, we extended weekly IV administration of FVIII (without additional feeding) for another month in 5 animals previously fed with FVIII plant material (3 of which had initially undetectable inhibitors). All 5 mice showed an increase in Bethesda titers to 35-138 BU/ml after 1 month (Fig 5B). As expected, inhibitor and anti-FVIII IgG titers further increased in control animals treated with FVIII in parallel, reaching levels substantially higher (on average 9fold) than those in initially FVIIIfed mice (445-998 BU/ml, Sl.5B). Control mice were subsequently fed with WT plant material for 2 months without further exposure to FVIII (Sl. 5A). These animals maintained their Bethesda titers and showed a modest decline in IgG1 anti-FVIII (Sl. 5B-C). In animals initially tolerized to FVIII, further oral delivery of FVIII plant material for 2 more months reversed inhibitor titers to an average of 11 BU/ml, ranging from undetectable to 20 BU/ml, which correlated with a reversal of IgG1 formation (Sl. 5A-C). An additional experimental group (n=7) was orally tolerized, again followed by weekly IV injections of FVIII starting 1 month after initiation of oral tolerance. However, in this case the oral tolerance regimen was continued along with replacement therapy ("FVIII continuously fed" group in Sl. 5B,C), which resulted in further suppression of the average inhibitor titer at time point #2, (1.7-fold compared to mice with discontinued oral delivery and 15-fold compared to control mice), and suppression was again sustained (Sl. 5B). Reminiscent of our published data on FIX, binding antibodies against FVIII remained detectable by ELISA in this group (Sl.5C).20

Reversal of inhibitor formation

[0065] To test whether the oral protocol is effective in pre-immune mice, we treated hemophilia A BALB/c mice with FVIII and divided them into two groups (n=4-5) with similar average Bethesda titers (∼60 BU). One group (control) was not further exposed to FVIII antigen, while the other group was subjected to the oral tolerance regimen (Sl. 5D). Inhibitor titers in control animals spontaneously rose further to an average of nearly 150 BU and eventually contracted to the original titer of ∼60 BU (Sl. 5E). IgG1 anti-FVIII titers showed a substantial further increase to a level that was subsequently maintained (Sl. 5F). In contrast, oral FVIII delivery slowed and then reversed inhibitor formation, resulting in a 3- to 7-fold decrease compared to controls after 2-3 months of feeding (Sl.5E). IgG1 formation was also significantly decreased (by ∼2.5-fold, Sl.5F).

Oral antigen delivery induces a Treg response against FVIII

[0066] Lymphocyte assays in the C57BL6/129 strain suggested Treg induction. We sought to obtain more direct evidence for induction of active immune suppression using adoptive transfer studies, which was possible in the pure BALB/c background. Lymphocytes were isolated from spleens and mesenteric lymph nodes (MLN) of FVIII-fed hemophilic BALB/c mice at the end of the experiment outlined in Sl. 5A. Upon adoptive transfer to naive mice of the same strain, CD4+CD25+ T cells, and even more so CD4+CD25- T cells (but not CD4- cells) were able to significantly suppress antibody formation to FVIII (Sl. 6A). From previous studies, it has become clear that CD4+CD2S+FoxP3+ Treg are critical in tolerance induction to coagulation factors.8,42 In order to identify potential suppressor cells in the CD4+CD25- T cell population, we performed flow cytometric analyses of various lymphatic tissues in tolerized vs control mice (Sl. 6B). We found significant induction of CD4+CD25-LAP+ T cells (which express high levels of TGF-β) in spleens, MLN, and Peyer’s patches, but no induction of type 1 regulatory T (Tr1) cells (which express high levels of IL-10 and are LAG- 3+CD49b+).12,13,43 Consistent with in vitro RT-PCR array data and our previous findings, overall frequencies of CD4+CD2S+FoxP3+ Treg showed only a subtle increase as antigen-specific cells of this subset function at low cell numbers.42,44

Local and systemic delivery of bioencapsulated FVIII

[0067] Delivery of FVIII antigen to the GALT was demonstrated by immunostaining, which showed presence of fed FVIII antigen in epithelial cells and delivery to dendritic cells (DC) in the lamina propria and Peyer’s patches of the small intestine (Sl. 7A-C). Presence of a furin cleavage site between CTB and FVIII sequences should facilitate systemic delivery of FVIII antigen following uptake in the gut. Indeed, we found HC antigen in plasma samples and liver protein extracts from samples obtained from hemophilia A mice 5 hrs following the last gavage (Sl. 7D). Delivery of CTBHC/ CTB-C2 only infrequently elicited systemic antibody responses to CTB, and there was no correlation between anti-CTB and FVIII inhibitor titers (Figure 8 and data not shown). The reason for anti-CTB formation in a subset of C57BL6/129 mice is unclear but may relate to processing of the receptor-bound CTB antigen (that is cleaved off FVII sequences) or strength of B or T cell epitopes for this antigen/strain combination. [0068] In order to facilitate clinical translational studies, antigens should be developed in an edible crop. Therefore, we first generated lettuce (Lactuca sativa) CTB-C2 transplastomic plants. The lettuce chloroplast expression vector pLs-CTB.C2 was created by introducing lettuce flanking sequences 16S/trnI and trnA/23S, and 5’ UTR and promoter of psbA gene from lettuce (Sl. 9A). Transplastomic plants were obtained after 3 – 4 months of selection and regeneration following bombardment of lettuce leaves. Southern blot analysis indicated that homoplasmy was achieved in most transplastomic lines (Sl.9B). Up to 1.7% TLP (or 102 mg/g fresh leaves) of CTB-C2 fusion protein was expressed in lettuce chloroplasts as observed in western blot analysis (Sl. 9C). The pentamer form of lettuce-made CTB-C2 was also verified by GM1 binding assay and by blue native gel electrophoresis/immunoblot analysis (Sl.9D-E). No cleaved products were observed even after dissolution of pentamers and destabilization of disulfide bonds by reducing agents (Sl. 9C). The C2 antigen concentration per gram of leaf tissue increased 20 fold after lyophilization. Up to 2004 mg CTB-C2 protein per gram of lyophilized leaf materials was observed (Sl.9F-G) when compared to 102 mg/g in fresh leaves, which was detected with a parallel immunoblot assay. The lyophilized and ground lettuce CTB-C2 powder can be prepared as capsules (or in a different formulation more suitable for pediatric patients) and stored at room temperature for long periods to facilitate oral delivery in clinical studies (Sl.9H).

[0069] In order to scale up biomass of CTB-HC leaf materials, more than 200 T1 plants from the CTB-HC transplastomics were transplanted into soils in the greenhouse. Western blot analysis to screen the positive plants was first performed (Figure 10). Lettuce leaves with high-level expression of CTB-HC fusion protein were harvested. More than 10 kg of fresh CTB-HC lettuce leaves have been collected. Approximately 200 g (dry weight) of lyophilized CTB-HC lettuce leaf powders were obtained from 4 kg of fresh CTB-HC leaves. The concentration of CTB-HC protein in the lyophilized leaf powders was 101 mg per g of dry weight.

[0070] In the same way (Figure 11), approximately 5 kg of CTB-C2 lettuce fresh leaves have been harvested. A total of 150 g of lyophilized CTB-C2 leaf materials have been obtained From 3 kg of fresh leaves. The CTB-C2 concentration in the lyophilized leaf materials was 3.32 mg per g of dry weight.

Advantages of the plant-based platform for oral tolerance in hemophilia

[0071] An oral tolerance protocol would be ideal for induction of antigen-specific tolerance while avoiding use of genetic manipulation of patient cells or of immune suppressive drugs, which have undesired side effects, increase the risk of infection, and may impact development of the immune system.12,14,16,20 Therefore, oral delivery of FVIII antigen may be an acceptable form of prophylactic tolerance induction in pediatric patients. Our current study demonstrates that multiple domains of FVIII can be expressed in plant chloroplasts. Moreover, oral administration of a mixture of bioencapsulated HC and C2 domain antigens substantially suppressed inhibitor formation in subsequent replacement therapy or animals with pre-existing response to FVIII infusion.

[0072] Oral delivery of plant-made pharmaceutical proteins is emerging as an effective approach. Bioencapsulation of therapeutic proteins within plant cells protects them from harsh environment of the gastrointestinal tract.15,45,46 In addition, elimination of highly expensive purification, cold storage, transportation and sterile injections significantly reduces their costs. Although we have not optimized the codons for FVIII expression in the current investigation, it has been reported that protein expression can reach up to 70% of the total leaf protein, under optimal conditions.18 Thus, the constructs described herein can be constructed using the humanized versions of FVIII, thereby further increasing efficacy. Multigene engineering is especially relevant for studies with FVIII because all domains may be simultaneously expressed in a single transformation cassette.47 Clinical implications

[0073] We provide proof-of-principle for suppression of FVIII inhibitors by oral delivery of transplastomic plant material. Transgene codon optimization can be employed to increase expression, as observed for several other human genes in chloroplasts.18,32 The C2 domain is expressed 3.6-fold higher than the heavy chain because of higher codon compatibility, underscoring the need for codon optimization to achieve higher levels of expression in chloroplasts. Using a combination of FVIII domains, their ratio becomes important. For example, we observed only a 3 fold reduction in inhibitor titers in hemophilic C57BL6/129 mice when a ratio of HC-C2 of 1:3 (instead of 1:1) was used (data not shown). The tolerogenic antigen mix may be further optimized by preparing different ratios of domains or subunits or by addition of more domains such as A3. The new data with FVIII demonstrate that the approach can also be applied to reversal of pre-existing inhibitors that have formed during replacement therapy. Thus, new oral tolerance protocols for prevention of inhibitor formation in high-risk patients and as an alternative or an addition to current ITI have been developed.

Induction of suppressive CD4+ T cell responses

[0074] Adoptive transfer studies demonstrate that oral FVIII delivery induced multiple subsets of CD4+ T cells that actively suppress antibody formation. Therefore, this mechanism is distinct from immune tolerance induced by hepatocyte- derived antigen, which primarily induces CD4+CD2S+FoxP3+ Treg.44,48,49 Antigen presented in the GALT additionally induced a strongly suppressive CD4+CD25- T cell response. We do not believe this reflects memory effector T cell activity, as transfer of such FVIII-experienced cells from mice that had not received oral delivery increases rather than suppresses anti-FVIII formation (X Wang, unpublished observations). Rather, flow cytometric analyses of CD4+ T cells suggests induction of CD4+CD25-LAP+ Treg, which are known to be inducible by antigen presentation in the gut and suppress by expression of large amounts of TGF-β, a cytokine that is also required for peripheral induction of CD4+CD2S+FoxP3+ Treg. Consistent with tologenic oral antigen delivery, induction of CD4+CD25-LAP+ was observed in Peyer’s patches and MLN, which drain the gut, but not non-draining lymph nodes. Increased frequency in the spleen is consistent with suppression of a systemic response, which is required to control inhibitor formation against IV delivered FVIII antigen. We found no evidence for induction of Trl cells. Nonetheless, there was induction of IL-10, a critical antiinflammatory cytokine in the GALT. Both FoxP3+ Treg and LAP+ Treg are potential sources of IL-10 expression. Codelivery of HC and C2 domain was sufficient to suppress inhibitor formation against the entire FVIII molecule in the BALB/c strain. In humans, additional T cell epitopes in other domains likely exist. However, efficient induction of Treg may provide sufficient suppression so that not all epitopes have to be covered by the orally delivered antigens.

[0075] U zaključku se može reći da domeni FVIII mogu da se proizvode u visokim nivoima u listovima biljaka sa transformisanim hloroplastima. Oralno isporučivanje modifikovanih biljnih ćelija indukuje Treg koji suprimira obrazovanje antitela na FVIII koji je isporučen intravenski, te stoga predstavlja obećavajući pristup za kontrolu stvaranja inhibitora.

LITERATURA

[0076]

1. Berntorp E, Shapiro AD. Modern haemophilia care. Lancet.2012;379(9824):1447-1456.

2. Graw J, Brackmann HH, Oldenburg J, Schneppenheim R, Spannagl M, Schwaab R. Haemophilia A: from mutation analysis to new therapies. Nat Rev Genet.2005;6(6):488-501.

3. Jayandharan GR, Srivastava A. Hemophilia: Disease, Diagnosis and Treatment. J Genet Syndr Gene Ther 2011;S1005.

4. DiMichele DM. Immune tolerance in haemophilia: the long journey to the fork in the road. Br J Haematol. 2012;159(2):123-134.

5. Ehrenforth S, Kreuz W, Scharrer I, et al. Incidence of development of factor VIII and factor IX inhibitors in haemophiliacs. Lancet.1992;339(8793):594-598.

6. Scott DW, Pratt KP, Miao CH. Progress toward inducing immunologic tolerance to factor VIII. Blood. 2013;121(22):4449-4456.

7. Adair P, Su Y, Scott DW. Tolerance induction in hemophilia A animal models: battling inhibitors with antigenspecific immunotherapies. Discov Med.2013;15(84):275-282.

8. Miao CH. Immunomodulation for inhibitors in hemophilia A: the important role of Treg cells. Expert Rev Hematol. 2010;3(4):469-483.

9. Moghimi B, Sack BK, Nayak S, Markusic DM, Mah CS, Herzog RW. Induction of tolerance to factor VIII by transient co-administration with rapamycin. J Thromb Haemost. 2011;9(8):1524 1533.

10. Oliveira VG, Agua-Doce A, Curotto de Lafaille MA, Lafaille JJ, Graca L. Adjuvant facilitates tolerance induction to factor VIII in hemophilic mice through a Foxp3-independent mechanism that relies on IL-10. Blood.2013;121(19):3936-3945, S3931.

11. Sabatino DE, Nichols TC, Merricks E, Bellinger DA, Herzog RW, Monahan PE. Animal models of hemophilia. Prog Mol Biol Transl Sci.2012;105151-209.

12. Wang X, Sherman A, Liao G, et al. Mechanism of oral tolerance induction to therapeutic proteins. Adv Drug Deliv Rev.2013;65(6):759-773.

13. Weiner HL, da Cunha AP, Quintana F, Wu H. Oral tolerance. Immunol Rev. 2011;241(1):241 259.

14. Rawle FE, Pratt KP, Labelle A, Weiner HL, Hough C, Lillicrap D. Induction of partial immune tolerance to factor VIII through prior mucosal exposure to the factor VIII C2 domain. J Thromb Haemost. 2006;4(10):2172-2179.

15. Daniell H, Singh ND, Mason H, Streatfield SJ. Plant-made vaccine antigens and biopharmaceuticals. Trends Plant Sci.2009;14(12):669-679.

16. Kwon KC, Verma D, Singh ND, Herzog R, Daniell H. Oral delivery of human biopharmaceuticals, autoantigens and vaccine antigens bioencapsulated in plant cells. Adv Drug Deliv Rev.2013;65(6):782-799.

17. Clarke JL, Daniell H. Plastid biotechnology for crop production: present status and future perspectives. Plant Mol Biol.2011;76(3-5):211-220.

18. Ruhlman T, Verma D, Samson N, Daniell H. The role of heterologous chloroplast sequence elements in transgene

integration and expression. Plant Physiol. 2010;152(4):2088-2104.

19. Ruhlman T, Ahangari R, Devine A, Samsam M, Daniell H. Expression of cholera toxin B-proinsulin fusion protein

in lettuce and tobacco chloroplasts--oral administration protects against development of insulitis in non-obese diabetic

mice. Plant Biotechnol J.2007;5(4):495-510.

20. Verma D, Moghimi B, LoDuca PA, et al. Oral delivery of bioencapsulated coagulation factor IX prevents inhibitor

formation and fatal anaphylaxis in hemophilia B mice. Proc Natl Acad Sci U S A.

2010;107(15):7101-7106.

21. Vehar GA, Keyt B, Eaton D, et al. Structure of human factor VIII. Nature.1984;312(5992):337-342.

22. Dorner AJ, Bole DG, Kaufman RJ. The relationship of N-linked glycosylation and heavy chainbinding protein

association with the secretion of glycoproteins. J Cell Biol.1987;105(6 Pt 1):2665-2674.

23. Pipe SW. Functional roles of the factor VIII B domain. Haemophilia. 2009;15(6):1187-1196.

24. Roberts SA, Dong B, Firrman JA, Moore AR, Sang N, Xiao W. Engineering factor VIII for hemophilia gene

therapy.. J Genet Syndr Gene Ther.2011;S1006.

25. Wroblewska A, Reipert BM, Pratt KP, Voorberg J. Dangerous liaisons: how the immune system deals with factor

VIII. J Thromb Haemost. 2013;1 1(1):47-55.

26. Markovitz RC, Healey JF, Parker ET, Meeks SL, Lollar P. The diversity of the immune response to the A2 domain

of human factor VIII. Blood.2013;121(14):2785-2795.

27. Meeks SL, Healey JF, Parker ET, Barrow RT, Lollar P. Antihuman factor VIII C2 domain antibodies in hemophilia

A mice recognize a functionally complex continuous spectrum of epitopes dominated by inhibitors of factor VIII

activation. Blood.2007;110(13):4234-4242.

28. Pratt KP. Inhibitory antibodies in hemophilia A. Curr Opin Hematol.2012;19(5):399-405.

29. Pratt KP, Thompson AR. B-cell and T-cell epitopes in anti-factor VIII immune responses. Clin Rev Allergy

Immunol. 2009;37(2):80-95.

30. Steinitz KN, van Helden PM, Binder B, et al. CD4+ T-cell epitopes associated with antibody responses after

intravenously and subcutaneously applied human FVIII in humanized hemophilic E17 HLA-DRB1*1501 mice. Blood.

2012;119(17):4073-4082.

31. Lei TC, Scott DW. Induction of tolerance to factor VIII inhibitors by gene therapy with immunodominant A2 and

C2 domains presented by B cells as Ig fusion proteins. Blood.2005;105(12):4865-4870.

32. Boyhan D, Daniell H. Low-cost production of proinsulin in tobacco and lettuce chloroplasts for injectable or oral

delivery of functional insulin and C-peptide. Plant Biotechnol J.2011;9(5):585-598.

33. Verma D, Samson NP, Koya V, Daniell H. A protocol for expression of foreign genes in chloroplasts. Nat Protoc.

2008;3(4):739-758.

34. Kwon KC, Nityanandam R, New JS, Daniell H. Oral delivery of bioencapsulated exendin-4 expressed in chloroplasts

lowers blood glucose level in mice and stimulates insulin secretion in beta-TC6 cells. Plant Biotechnol J.

2013;11(1):77-86.

35. Lakshmi PS, Verma D, Yang X, Lloyd B, Daniell H. Low cost tuberculosis vaccine antigens in capsules: expression

in chloroplasts, bio-encapsulation, stability and functional evaluation in vitro. PLoS One.

2013;8(1):e54708.

36. Sack BK, Merchant S, Markusic DM, et al. Transient B cell depletion or improved transgene expression by codon

optimization promote tolerance to factor VIII in gene therapy. PLoS One.2012;7(5):e37671.

37. Cao O, Loduca PA, Hoffman BE, et al. Impact of the underlying mutation and the route of vector administration

on immune responses to factor IX in gene therapy for hemophilia B. Mol Ther. 2009;17(10):1733-1742.

38. Daniell H, Lee SB, Panchal T, Wiebe PO. Expression of the native cholera toxin B subunit gene and assembly

as functional oligomers in transgenic tobacco chloroplasts. J Mol Biol.2001;311(5):1001-1009.

39. de Haan L, Verweij WR, Feil IK, et al. Role of GM1 binding in the mucosal immunogenicity and adjuvant activity

of the Escherichia coli heat-labile enterotoxin and its B subunit. Immunology. 1998;94(3):424-430.

40. Tsuji T, Watanabe K, Miyama A. Monomer of the B subunit of heat-labile enterotoxin from enterotoxigenic

Escherichia coli has little ability to bind to GM1 ganglioside compared to its coligenoid. Microbiol Immunol.

1995;39(10):817-819.

41. Qadura M, Waters B, Burnett E, et al. Immunoglobulin isotypes and functional anti-FVIII antibodies in response

to FVIII treatment in Balb/c and C57BL/6 haemophilia A mice. Haemophilia.2011;17(2):288-295.

42. Markusic DM, Hoffman BE, Perrin GQ, et al. Effective gene therapy for haemophilic mice with pathogenic factor

IX antibodies. EMBO Mol Med.2013;5(11):1698-1709.

43. Gagliani N, Magnani CF, Huber S, et al. Coexpression of CD49b and LAG-3 identifies human and mouse T

regulatory type 1 cells. Nat Med.2013;19(6):739-746.

44. Hoffman BE, Martino AT, Sack BK, et al. Nonredundant roles of IL-10 and TGF-beta in suppression of immune

responses to hepatic AAV-factor IX gene transfer. Mol Ther.2011;19(7): 1263-1272.

45. Kohli N, Westerveld DR, Ayache AC, et al. Oral delivery of bioencapsulated proteins across blood-brain and

blood-retinal barriers. Mol Ther.2014;22(3):535-546.

46. Limaye A, Koya V, Samsam M, Daniell H. Receptor-mediated oral delivery of a bioencapsulated green fluorescent

protein expressed in transgenic chloroplasts into the mouse circulatory system. FASEB J.

2006;20(7):959-961.

47. Kumar S, Hahn FM, Baidoo E, et al. Remodeling the isoprenoid pathway in tobacco by expressing the cytoplasmic

mevalonate pathway in chloroplasts. Metab Eng. 2012;14(1):19-28.

48. Cao O, Dobrzynski E, Wang L, et al. Induction and role of regulatory CD4+CD25+ T cells in tolerance to the

transgene product following hepatic in vivo gene transfer. Blood.2007;110(4):1132-1140.

49. Cao O, Armstrong E, Schlachterman A, et al. Immune deviation by mucosal antigen administration suppresses

gene-transfer-induced inhibitor formation to factor IX. Blood.2006;108(2):480-486.

Claims (6)

Patentni zahtevi

1. Kompozicija koja sadrži liofilizirani biljni materijal koji sadrži najmanje jedan fragment FVIII konjugovan sa subjedinicom kolera toksina B (CTB), gde se navedeni fragment sastoji od FVIII domena odabranog iz grupe koja se sastoji od C2 i teškog lanca (HC), pri čemu je navedeni fragment konjugovan sa CTB proizveden u hloroplastima unutar navedene biljke i zadržava imunogenost u liofiliziranom obliku, koja je pri oralnoj primeni kod sisara kome je to potrebno efikasna u indukovanju oralne tolerancije na FVIII.

2. Kompozicija prema patentnom zahtevu 1, za upotrebu u postupku za indukciju oralne tolerancije za lečenje hemofilije A.

3. Kompozicija prema patentnom zahtevu 1, naznačena time što je navedena biljka odabrana iz grupe koja se sastoji od zelene salate, paradajza, jabuke, bobica kao što su jagode i maline, agruma, banane, šargarepe, celera, karfiola; brokolija, raštana, krastavca, dinje, lubenice, parike, kruške, grožđa, breskve, rotkvica i kejl kupusa.

4. Kompozicija prema patentnom zahtevu 1, naznačena time što između navedenog CTB i navedenog najmanje jednog fragmenta FVIII postoji zglobni peptid, GPGP, i mesto za sečenje furinom, RRKR.

5. Kompozicija za upotrebu u postupku za indukciju oralne tolerancije za lečenje hemofilije A prema patentnom zahtevu 2, naznačena time što navedeni postupak sadrži primenu fuzionih proteina C2-CTB i HC-CTB zajedno, pri čemu je navedena kompozicija efikasna u indukciji ekspresije CD4+CD25-LAP+ regulatornih T-ćelija koje proizvode TGF-β u slezini.

6. Kompozicija za upotrebu u postupku za indukciju oralne tolerancije za lečenje hemofilije A prema patentnom zahtevu 2, naznačena time što navedenu oralnu toleranciju izaziva supresija stvaranja inhibitora ili inverzija već postojećih inhibitora obrazovanih tokom lečenja nadoknađivanjem FVIII.