Patent Application: US-201213458083-A

Abstract:
the invention relates to the field of glycoprotein processing in transgenic plants used as cost efficient and contamination safe factories for the production of recombinant biopharmaceutical proteins or pharmaceutical compositions comprising these . the invention provides a plant comprising functional mammalian β1 , 4 - galactosyltransferase and mammalian sialyl transferase for producing complex n - linked glycans that are galactosylated and sialylated , said plant additionally comprising at least a second mammalian protein or functional fragment thereof that is normally not present in plants .

Description:
one important enzyme involved in mammalian n - glycan biosynthesis that is not present in plants is β1 , 4 - galactosyltransferase . here , for one , the stable expression of β1 , 4 - galactosyltransferase in tobacco plants is described . the physiology of these plants is not obviously changed by introducing β1 , 4 - galactosyltransferase and the feature is inheritable . crossings of a tobacco plant expressing β1 , 4 - galactosyltransferase with a plant expressing the heavy and light chain of a mouse antibody produced antibody having terminal galactose in similar amounts as hybridoma produced antibodies . herein it is thus shown that the foreign enzyme can be successfully introduced in plants . a clear increase in galactose containing glycoproteins is observed . moreover , this feature is inheritable and there is no visible phenotypical difference between the galactosyltransferase plants and wild type . a mouse monoclonal antibody produced in these plants has a degree of terminal galactoses comparable to hybridoma produced antibody . this shows that not only endogenous proteins become galactosylated but also a recombinantly expressed mammalian protein . a plant transformation vector containing human β1 , 4 - galactosyltransferase was constructed as follows : a 1 . 4 kb bamhi / xbai fragment of pcdnai - galt ( aoki et al ., 1992 ; yamaguchi and fukuda , 1995 ) was ligated in the corresponding sites of puc19 . subsequently , this fragment was re - isolated using surrounding kpni and hincii sites and cloned into the kpni and smai site of prap33 ( named prap33 - hgalt ). using asci and paci sites the camv35s promotor - cdna - nos terminator cassette of prap33 - hgalt was cloned in the binary vector pbinplus ( van engelen et al ., 1995 ). modifications to the published protocol are : after incubation with a . tum ., leaf discs were incubated for three days in medium containing 1 mg / ml of naa and 0 . 2 mg / ml bap and the use of 0 . 25 mg / ml cefotaxime and vancomycine to inhibit bacterial growth in the callus and shoot inducing medium . 25 rooted shoots were transformed from in vitro medium to soil and , after several weeks , leaf material of these plants was analysed . the β1 , 4 - galactosyltransferase rna level in the transgenic plants was analyzed by northern blotting ( sambrook et al ., 1989 ) rna was isolated from leafs of transgenic and control plants as described ( de vries et al ., 1991 ). ten μg of total rna was used per sample . the blot was probed with a [ 32 p ] datp labeled ssti / xhoi fragment , containing the whole galt cdna , isolated from pbinplus - hgalt . total protein extracts of tobacco were prepared by grinding leafs in liquid nitrogen . ground material was diluted 10 times in sds page loading buffer ( 20 mm of this - hcl ph 6 . 8 , 6 % glycerol , 0 . 4 % sds , 20 mm dtt , 2 . 5 ig / ml bromophenol blue ). after incubation at 100 ° c . for 5 min insoluble material was pelleted . supernatants ( 12 . 5 μl / sample ) were run on 10 % sds - page and blotted to nitrocellulose . blots were blocked overnight in 0 . 5 % tween - 20 in tbs and incubated for 2 hours with peroxidase conjugated rca 120 ( ricinus communis agglutinin , sigma ) ( 1 μg / ml ) in tbs - 0 . 1 % tween - 20 . blots were washed 4 times 10 minutes in tbs - 0 . 1 % tween - 20 and incubated with lumi - light western blotting substrate ( roche ) and analysed in a lumianalyst ( roche ). a rabbit polyclonal antibody directed against horseradish peroxidase ( hrp , rockland immunochemicals ) was split in reactivity against the xylose and fucose of complex plant glycans by affinity chromatography with bee venom phospholipase according to ( faye et al ., 1993 ). a rabbit anti lewisa antibody was prepared as described ( fitchette laine et al ., 1997 ). blots were blocked with 2 % milkpowder in tbs and incubated in the same buffer with anti - hrp , anti - xylose , anti - fucose or anti - lewis - a . as secondary antibody alkaline hrp - conjugated sheep - anti - mouse was used and detection was as described above . mgr48 ( smant et al ., 1997 ) is a mouse monoclonal igg that has been expressed in tobacco plants . the construct used for transformation was identical to monoclonal antibody 21c5 expressed in tobacco ( van engelen et al ., 1994 ). flowers of selected tobacco plants with high expression of β1 , 4 - galactosyltransferase were pollinated with plants expressing mgr48 antibody . the f1 generation was seeded and plants were screened for leaf expression of antibody by western blots probed hrp - conjugated sheep - anti - mouse and for galactosyltransferase expression by rca as described above . freshly harvested tobacco leaves were ground in liquid nitrogen . to 50 g of powdered plant material , 250 ml of pbs , containing 10 mm na 2 s 2 o 5 , 0 . 5 mm edta , 0 . 5 mm pmsf and 5 g polyvinylpolypyrrolid , was added . after soaking for 1 hour ( rotating at 4 ° c . ), insoluble material was removed by centrifugation ( 15 min , 15 , 000 g , 4 ° c .). the supernatant was incubated overnight ( rotating at 4 ° c .) with 1 ml of proteing - agarose beads . the beads were collected in a column and washed with 10 volumes of pbs . bound protein was eluted with 0 . 1 m glycine ph 2 . 7 and immediately brought to neutral ph by mixing with 1 m tris ph 9 . 0 ( 50 μl per ml of eluate ). purified antibody was quantified by comparison of the binding of hrp - conjugated sheep - anti - mouse to the heavy chain on a western blot with mgr48 of known concentration purified from hybridoma medium ( smant et al ., 1997 ). hybridoma mgr48 and plant produced mgr48 was run on 10 % sds - page and blotted as described above . detection with rca was as described above . for antibody detection , blots were probed with hrp - conjugated sheep - anti - mouse and detected with lumi - light western blotting substrate as described above . human β1 , 4 - galactosyltransferase ( masri et al ., 1988 ) was introduced in tobacco plants by agrobacterium mediated leaf disk transformation of plasmid pbinplus - hgalt containing a cdna that includes a complete coding sequence . twenty - five plants selected for kanamicin resistance were analysed for mrna levels by northern hybridization ( fig2 a ). the same plants were analyzed by the galactose binding lectin rca 120 ( ricinus cummunis agglutinin ). rca binds to the reaction product of β1 , 4 - galt ( galβ1 , 4glcnac ) but also to other terminal β linked galactose residues . rca binds to one or more high molecular weight proteins isolated from non transgenic control tobacco plants ( fig2 b ). probably these are arabinogalactan or similar proteins . rca is known to bind to arabinogalactan proteins ( schindler et al ., 1995 ). in a number of the plant transformed with human β1 , 4 - galactosyltransferase , in addition , binding of rca to a smear of proteins is observed . this indicates that in these plants many proteins contain terminal β linked galactose residues . there is a good correlation between the galactosyltransferase rna expression level and the rca reactivity of the trangenic plants . human β1 , 4 - galactosyltransferase expressed in transgenic plants is therefor able to galactosylate endogenous glycoproteins in tobacco plants . as it is known that galactosylated n - glycans are poor acceptors for plant xylosyl - and fucosyltransferase ( johnson and chrispeels , 1987 ), the influence of expression of β1 , 4 - galactosyltransferase on the occurrence of the xylose and fucose epitope was investigated by specific antibodies . a polyclonal rabbit anti - hrp antibody that reacts with both the xylose and fucose epitope shows a clear difference in binding to isolated protein from both control and transgenic plants ( fig3 ). the effect of expression of β1 , 4 - galactosyltransferase on a recombinantly expressed protein was investigated . three tobacco plants expressing β1 , 4 - galactosyltransferase ( no . galt6 , galt8 and galt15 from fig2 ) were selected to cross with a tobacco plant expressing a mouse monoclonal antibody . this plant , expressing monoclonal mgr48 ( smant et al ., 1997 ), was previously generated in our laboratory . flowers of the three plants were pollinated with mgr48 . of the f1 generation 12 progeny plants of each crossing were analysed for the expression of both antibody and β1 , 4 - galactosyltransferase by the method described in materials and methods . of crossing galt6 × mgr48 and galt15 × mgr48 no plants were found with both mgr48 and galt expression . several were found in crossing galt8 × mgr48 . two of these plants ( no . 11 and 12 ), were selected for further analysis . using proteing affinity , antibody was isolated from tobacco plants expressing mgr48 and from the two selected plants expressing both mgr48 and β1 , 4 - galactosyltransferase . equal amounts of isolated antibody was run on a protein gel and blotted . the binding of sheep - anti - mouse - igg and rca to mgr48 from hybridoma cells , tobacco and crossings galt8 × mgr48 - 11 and 12 was compared ( fig4 ). sheep - anti - mouse - igg bound to both heavy and light chain of all four antibodies isolated . rca , in contrast , bound to hybridoma and galt plant produced antibody but not to the antibody produced in plants expressing only mgr48 . when the binding of sheep - anti - mouse - igg and rca to the heavy chain of the antibody is quantified , the relative reaction of rca ( rca binding / sheep - anti - mouse - igg binding ) to galt8 × mgr48 - 11 and 12 is respectively 1 . 27 and 1 . 63 times higher than the ratio of hybridoma produced antibody . this shows that rca binding to the glycans of antibody produced in galt plants is even higher than to hybridoma produced antibody . although the galactosylation mgr48 from hybridoma is not quantified , this is a strong indication that the galactosylation of antibody produced in these plants is very efficient . construction of plant expression vectors with cdna &# 39 ; s encoding α2 , 6 sialytransferase , β1 , 3 - glucuronyltransferase and β1 , 4 - galactosyltransferase . the available β1 , 4 - galactosyltransferase vector was not in a suitable format to easily combine with α2 , 6 - sialyltransferase and β1 , 3 - glucuronyltransferase clones . therefore , by using pcr , the coding region of β1 , 4 - galactosyl - transferase cdna , α2 , 6 - sialyltransferase cdna and β 1 , 3 - glucuronyl - transferase cdna have been cloned in plant expression vectors . constructs are made in which galactosyltransferase is combined with either sialyltransferase or glucuronyltransferase in one vector , in order to enable simultaneous expression of the enzymes in transgenic plants after only one transformation . the galactosyltransferase expression is controlled by the 35s promoter , whereas expression of sialyltransferase and glucuronyltransferase is controlled by the 2 ′ promoter . there is a need for an accessible and standardised source of fsh for therapeutic and diagnostic purposes , which is guaranteed to be free of lh activity . fsh preparations normally are derived from ovine or porcine pituitaries , which always implies the presence of ( traces of ) lh , and the risk of contamination with prion - like proteins . substitution of brain derived fsh for plant produced recombinant fsh may be a good method of eliminating these problems . however , production of bioactive animal glycoproteins in plants , especially for therapeutic purposes , requires modification of plant - specific sugar sidechains into a mammalian type of glycans . the invention provides recombinant bfsh by infecting stably transformed tobaccoplants capable of forming mammalian type of glycans , with recombinant tobacco mosaic virus tmv containing the genes for bfsh or bfshr . construction of single chain ( sc ) bfsh into pks (+) bluescript vector , construction of sc - bfsh - tmv and sc - bfsh - his - tmv in order to circumvent the need of simultaneous expression of the two separate genes of bfsh - alpha and bfsh - beta subunits in plants , we decided to construct a bfsh fusion gene . by overlap pcr we fused the carboxyl end of the beta subunit to the amino end of the alpha subunit ( without a linker ). in addition , we constructed a second sc - bfsh version carrying a 6 × his tag at the c - terminus of the alpha subunit , which will allow us to purify the recombinant protein from the plant . both , sc - bfsh and sc - bfsh - his constructs were subcloned into the cloning vector pks (+) bluescript . the correctness of the clones was confirmed by sequence analysis . sc - bfsh was subcloned into the tmv vector . two positive clones were chosen to make in vitro transcripts and inoculate n . bentahamiana plants . after a few days , plants showed typical viral infection symptoms , which suggested the infective capacity of the recombinant tmv clones . in order to test whether the sc - bfsh rna is stably expressed in systemically infected leaves , 8 days post inoculation rna was isolated from infected n . benthamiana leaves and a reverse transcriptase polymerase chain reactions using bfsh specific primers was performed . in all cases we obtained a pcr fragment of the expected size , indicating the stability of our sc - bfsh - tmv construct . extracts of infected plants are used for western blot analyses and elisa to determine whether sc - bfsh is expressed and folded properly . comparison of rna levels and product of β1 , 4 - galactosyltransferase . upper panel : northern blot of total rna isolated from 25 transgenic plants , including a not transformed control plant ( 0 ), detected with a human β1 , 4 - galactosyltransferase probe . lower panel : western blot of the same plant probed with rca to detect terminal galactose residues on glycoproteins . m . indicates the molecular weight marker . western blot showing the binding of lectin and antibody to protein isolated from wild - type and a β1 , 4 - galactosyltransferase plant ( no . 8 from fig2 ). a : rca as in fig2 , b : anti hrp ( detecting both xylose and fucose ) antibody , c : anti xylose antibody , d : anti fucose antibody .) western blot showing rca and sheep - anti - mouse - igg binding to purified antibody produced in hybridoma culture ( hyb ), tobacco plants ( plant ) and tobacco plants co - expressing β1 , 4 - galactosyltransferase ( galt11 and galt12 ). h . c . : heavy chain , l . c . light chain . tobacco cell cultures expressing galactosyltransferase produce unnatural hybrid n - glycans while tobacco plants expressing galactosyltransferase have natural , mammalian like galactosylation . to get natural galactosylation , galactosyltransferase should act after mannosidase ii and glcnactransferase ii . western blot showing the expression of glcaβ1 , 3gal structure in transgenic tobacco by binding of an antibody ( 412 ) directed against the glucuronic acid - galactose ( glcaβ1 , 3gal ) structure to protein isolated from 8 plants expressing human β1 , 4 galactosyltransferase and rat β1 , 3 glucuronyltransferase and a wildtype control plant (−). aoki , d ., lee , n ., yamaguchi , n ., dubois , c ., and fukuda , m . n . 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