Patent Application: US-51752506-A

Abstract:
the invention concerns a method for obtaining and selecting monoclonal antibodies by an addc - type test , said antibodies capable of activating type iii fcy receptors and having a particular glycan structure . the inventive anti - d antibodies can be used for preventing rhesus isoimmunisation in rh negative persons , in particular for haemolytic disease in a new - born baby of for uses such as idiopathic thrombocytopenic pupura .

Description:
the b lymphocyte donor is selected from anti - rh ( d ) donors undergoing plasmapheresis , based on the activity of his or her anti - rh ( d ) serum antibodies in the adcc activity assay described in § 33 . after a whole blood donation , in 1998 , the “ buffy coat ” fraction ( leukocyte concentrate ) was recovered . the peripheral blood mononuclear cells are separated from the other elements by centrifugation on ficoll plus ( pharmacia ). they are then diluted to 10 6 cell / ml in imdm containing 20 % ( v / v ) of fetal calf serum ( fcs ), to which 20 % of culture supernatant of the b95 - 8 line ( atcc - crl1612 ), 0 . 1 μg / ml of cyclosporin a ( sandoz ), 50 μg / ml of gentamycin sulfate ( life technologies ) are added , and distributed into round - bottomed 96 - well plates or 24 - well plates ( p24 greiner ). they are then placed in an incubator at 37 ° c ., 7 % co 2 . after 3 weeks , the presence of anti - rh ( d ) antibodies is sought by adcc . each one of the 16 microwells of a positive p24 plate well is transferred into a new p24 well . this enrichment is repeated after 10 to 15 days of culturing and each microwell is amplified in a p96 and then in a p24 . the positive p96 wells are taken up and amplified in a flat - bottomed p24 ( nunc ). after a few days of culturing , the presence of anti - rh ( d ) antibodies is sought by adcc . the cells derived from one or more p24 wells are enriched in specific cells by the formation and separation of rosettes with papain - treated rh ( d )- positive red blood cells : one volume of red blood cells washed in 0 . 9 % nacl is incubated for 10 minutes at 37 ° c . with 1 volume of papain ( merck ) solution at 1 / 1 000th ( m / v ), and then washed 3 times in 0 . 9 % nacl . the cells were then washed once in hanks solution , suspended in fcs and mixed with the papain - treated red blood cells in a ratio of 1 cell to 33 red blood cells . the mixture is placed in a cone - bottomed centrifuged tube , centrifuged for 5 minutes at 80 g and incubated for one hour in melting ice . the mixture is then carefully agitated and ficoll is deposited at the bottom of the tube for separation at 900 g for 20 minutes . the pellet containing the rosettes is hemolyzed in a solution of nh 4 cl for 5 , minutes and the cells are placed in culture again in a p24 containing irradiated human mononuclear cells . after approximately 1 week , the supernatants are evaluated in cela ( paragraph 3 . 2 ) and adcc assays for the presence of anti - rh ( d ) antibodies having good activity . a further cycle of enrichment is carried out if the percentage of cells forming rosettes significantly increases compared to the preceding cycle . the ir - enriched cells are distributed at 5 and 0 . 5 cells per well in round - bottomed 96 - well plates containing irradiated human mononuclear cells . after approximately 4 weeks of culturing , the supernatants from the wells containing cell aggregates are evaluated by adcc assay . the wells from cloning the ebv - transformed cells exhibiting an advantageous adcc activity are amplified in culture and then fused with the heteromyeloma k6h6 - b5 ( atcc crl - 1823 ) according to the standard peg technique . after fusion , the cells are distributed , in a proportion of 2 × 10 4 cells / well , into flat - bottomed p96s containing murine intraperitoneal macrophages and in a selective medium containing aminopterin and ouabain ( sigma ). after 3 to 4 weeks of culturing , the supernatants of the wells containing cell aggregates are evaluated by adcc assay . cloning by limiting dilution is carried out at 4 , 2 and 1 cell / well in flat - bottomed p96s . after 2 weeks , the microscopic appearance of the wells is examined in order to identify the single clones , and the medium is then renewed . after approximately 2 weeks , the supernatants of the wells containing cell aggregates are evaluated by adcc assay . ebv transformation of the cells of donor d13 made it possible to select a well , designated t125 2a2 , on which the following were successively carried out : 2 enrichments , 3 cycles of ir , and cloning at 5 cells / well to give 2 clones : 1 ) t125 2a2 ( 5 / 1 ) a2 from which the dna was extracted in order to prepare the recombinant vector ; 2 ) t125 ( 5 / 1 ) a2 which was fused with k6h6 - b5 to give f60 2f6 and then , after 5 rounds of cloning , f60 2f6 ( 5 ) 4c4 , a clone selected for constituting a cell stock prior to preparing libraries . a line producing an igg3 was prepared according to the same method as that used to prepare the antibody of igg1 isotype . the cells of origin originate from a donation of whole blood , from a designated donor , from which the “ buffy coat ” fraction ( leukocyte concentrate ) was recovered . after purification by affinity chromatography on protein a sepharose ( pharmacia ) and dialysis in 25 mm tris buffer , 150 mm nacl , ph 7 . 4 , the concentration of the antibody t125 is determined by the elisa technique . the biological activity in vitro is then measured by the adcc technique . coating : anti - igg ( calbiochem ) at 2 μg / ml in 0 . 05m carbonate buffer , ph 9 . 5 , overnight at 4 ° c . saturation : dilution buffer ( pbs + 1 % bsa + 0 . 05 % tween 20 , ph 7 . 2 ), 1 h at ambient temperature . washing ( to be renewed at each step ): h 2 o + 150 mm nacl + 0 . 05 % tween 20 . dilution of the samples , in dilution buffer to approximately 100 ng / ml and of the control range made up of lfb polyvalent human iggs prediluted to 100 ng / ml . incubation for 2 h at ambient temperature . conjugate : anti - igg ( diagnostic pasteur ) diluted to 1 / 5 000 , 2 hours at ambient temperature . substrate : opd at 0 . 5 mg / ml ( sigma ) in phosphate - citrate buffer containing sodium perborate ( sigma ), 10 minutes in the dark . reaction stopped with 1n hcl , and read at 492 nm . coating : anti - kappa ( caltag lab ) at 5 μg / ml in 0 . 05m carbonate buffer , ph 9 . 5 , overnight at 4 ° c . saturation : dilution buffer ( pbs + 1 % bsa + 0 . 05 % tween 20 , ph 7 . 2 ), 1 h at ambient temperature . the washing ( to be renewed at each step ): h 2 o + 150 mm nacl + 0 . 05 % tween 20 . dilution of the samples , in dilution buffer , to approximately 100 ng / ml and of the control range made up of the lfb monoclonal antibody ad3t1 ( kappa / gamma 3 ) prediluted to 100 ng / ml . incubation for 2 h at ambient temperature . conjugate : biotinylated anti - kappa ( pierce ) diluted to 1 / 1 000 in the presence of streptavidin - peroxidase ( pierce ) diluted to 1 / 1 500 , 2 hours at ambient temperature . substrate : opd at 0 . 5 mg / ml ( sigma ) in phosphate - citrate buffer containing sodium perborate ( sigma ), 10 minutes in the dark . the reaction is stopped with 1n hcl , and read at 492 nm . 3 . 2 - specific assaying of anti - d by the cela ( cellular enzyme linked assay ) technique : this method is used for specifically assaying the anti - d antibodies in particular when this involves a culture supernatant at culturing stages at which other non - anti - d immunoglobulins are present in the solution ( early stages after ebv transformation ). principle : the anti - d antibody is incubated with rhesus - positive red blood cells and then revealed with an alkaline phosphatase - labeled anti - human ig . 100 μl of rh + red blood cells at 10 % diluted in liss - 1 % bsa dilution buffer . dilution of the samples , in dilution buffer , to approximately 500 ng / ml and of the control range made up of a purified monoclonal human anti - d igg ( df5 , lfb ) prediluted to 500 ng / ml . incubation for 45 min at ambient temperature . washing ( to be renewed at each step ): h 2 o + 150 mm nacl . conjugate : anti - igg alkaline phosphatase ( jackson ) diluted to 1 / 4 000 in pbs + 1 % bsa , 1 h 30 at ambient temperature . substrate : pnpp at 1 mg / ml ( sigma ) in 1m diethanolamine , 0 . 5 mm mgcl 2 , ph 9 . 8 . the reaction is stopped with 1n naoh , and read at 405 nm . the adcc ( antibody - dependent cellular cytotoxicity ) technique makes it possible to evaluate the ability of the ( anti - d ) antibodies to induce lysis of rh - positive red blood cells , in the presence of effector cells ( mononuclear cells or lymphocytes ). briefly , the red blood cells of an rh - positive cell concentrate are treated with papain ( 1 mg / ml , 10 min at 37 ° c .) and then washed in 0 . 9 % nacl . the effector cells are isolated from a pool of at least 3 buffy - coats , by centrifugation on ficoll ( pharmacia ), followed by a step of adhesion in the presence of 25 % fcs , so as to obtain a lymphocyte / monocyte ratio of the order of 9 . the following are deposited , per well , into a microtitration plate ( 96 well ): 100 μl of purified anti - d antibody at 200 ng / ml , 25 μl of rh + papain - treated red blood cells ( i . e . 1 × 10 6 ), 25 μl of effector cells ( i . e . 2 × 10 6 ) and 50 μl of polyvalent igg ( tegeline , lfb , for example ) at the usual concentrations of 10 and 2 mg / ml . the dilutions are made in imdm containing 0 . 25 % fcs . after overnight incubation at 37 ° c ., the plates are centrifuged , and the hemoglobin released into the supernatant is then measured in the presence of a substrate specific for peroxidase activity ( 2 , 7 - diaminofluorene , daf ). the results are expressed as percentage lysis , 100 % corresponding to total red blood cell lysis in nh 4 cl ( 100 % control ), and 0 % to the reaction mixture without antibody ( 0 % control ). the specific lysis is calculated as a percentage according to the following formula : the results given in fig1 show the activity of the antibody produced by the heterohybrid f60 compared to those of the reference antibodies : the anti - rh ( d ) polyclonal antibodies poly - d lfb 51 and winrho w03 ( cangene )= positive controls the monoclonal antibody df5 ( inactive in vivo on clearance of rh ( d )- positive red blood cells ( brossard / fnts , 1990 , not published ))= negative control the igg1s purified ( separated from the igg3s ) from the polyclonal winrho w03 . two concentrations of human iggs ( tegeline lfb ) are used to show that inhibition of activity of the negative control is linked to the binding of competing iggs to the fcγ type i receptors . this assay makes it possible to assess the binding of the anti - rh ( d ) antibodies of igg1 isotype to fcγriii , and in particular to differentiate igg3 antibodies . given the - low affinity of this receptor for monomeric iggs , prior binding of the antibodies to the d antigen is necessary . principle : the antibody to be tested ( anti - d ) is added to membranes of rh + red blood cells coated with a microtitration plate , followed by transfected jurkat cells expressing the fcγriii receptor at their surface . after centrifugation , the “ rh + membrane / anti - d / cd 16 jurkat ” interaction is visualized by a homogeneous plating of the cd16 jurkats in the well . in the absence of interaction , the cells are , on the contrary , grouped at the center of the well . the intensity of the reaction is expressed as numbers of +. method : 1 ) incubation for 1 h at 37 ° c . of the anti - d antibody ( 50 μl at 1 μg / ml in imdm ) on a capture r plate ( immunochim ), and then washes in water + 0 . 9 % nacl . addition of cd16 jurkat ( 2 × 10 6 cells / ml ) in imdm + 10 % fcs . incubation for 20 min at 37 ° c . and then centrifugation and evaluation of cell adhesion ( against a control range ). 2 ) revelation of the anti - d bound to the capture r plates by an elisa - type technique using anti - human igg - peroxidase at 1 / 5 000 ( sanofi diagnostics pasteur ) after having lysed the cd16 jurkat cells with 0 . 2m tris - hcl , 6m urea , ph 5 . 3 - 5 . 5 . opd revelation and then reading of optical density ( o . d .) at 492 nm . expression of results : an arbitrary value of 0 to 3 is allotted as a function of the binding and of the plating of the cd 16 jurkat cells . these values are allotted at each od interval defined ( increments of 0 . 1 ). the following are plotted : either a curve : adhesion of the jurkat cells ( y ) as a function of the amount of anti - d bound to the red blood cell membranes ( x ). or a histogram of the “ binding indices ” corresponding , for each antibody , to the sum of each jurkat cell binding value ( 0 to 3 ) allotted per od interval ( over a portion common to all the antibodies tested ). an example of a histogram is given in fig2 . the anti - rh ( d ) antibodies of igg1 isotype ( f60 and t125 yb2 / 0 ) show a binding index close to that of the polyclonal igg1s ( winrho ), whereas the negative control antibodies df5 and ad1 do not bind . similarly , the antibody of igg3 isotype ( f41 ) exhibits a good binding index , slightly less than that of the igg3s purified from the polyclonal winrho and greater than that of the antibody ad3 ( other igg3 tested and ineffective in clinical trial , in a mixture with ad1 ( biotest / lfb , 1997 , not published ). 1 - isolation and amplification of the cdnas encoding the heavy and light chains of the ab the total rnas were extracted from an anti - d ab - producing clone ( igg g1 / kappa ) obtained by ebv transformation : t125 a2 ( 5 / 1 ) a2 ( see paragraph 2 , example 1 ). the corresponding cdnas were synthesized by reverse transcription of the total rnas using oligo dt primers . the vh / t125 - a2 sequence is obtained by amplification of the t125 - a2 cdnas using the following primers : primer a2vh5 , located 5 ′ of the leader region of the vh gene of t125 - a2 , introduces a consensus leader sequence ( in bold ) deduced from leader sequences already published and associated with vh genes belonging to the same vh3 - 30 family as the vh gene of t125 - a2 ; this sequence also comprises an eco ri restriction site ( in italics ) and a kozak sequence ( underlined ): antisense primer gsp2anp , located 5 ′ of the constant region ( ch ) of t125 - a2 : the ch / t125 - a2 sequence is obtained by amplification of the t125 - a2 cdnas using the following primers : the first g base of the ch sequence is here replaced with a c ( underlined ) in order to recreate , after cloning , an eco ri site ( see paragraph 2 . 1 . 1 ). antisense primer h3 ′ xba , located 3 ′ of the ch of t125 - a2 , introduces an xba i site ( underlined ) 3 ′ of the amplified sequence : the entire kappa chain of t125 - a2 ( k / t125 - a2 sequence ) is amplified from the t125 - a2 cdnas using the following primers : primer a2vk3 , located 5 ′ of the leader region of the vk gene of t125 - a2 , introduces a consensus sequence ( in bold ) deduced from the sequence of several leader regions of vk vh genes belonging to the same vk1 subgroup as the vk gene of t125 - a2 ; this sequence also comprises an eco ri restriction site ( in italics ) and a kozak sequence ( underlined ): antisense primer kse1 , located 3 ′ of kappa , introduces an eco ri site ( underlined ): fig1 gives a diagrammatic illustration of the strategies for amplifying the heavy and light chains of t125 - a2 . the construction of t125 - h26 is summarized in fig2 . it is carried out in two stages : first of all , construction of the intermediate vector v51 - ch / t125 - a2 by insertion of the constant region of t125 - a2 into the expression vector v51 derived from pci - neo ( fig3 ) and then cloning of the variable region into v51 - ch / t125 - a2 . the amplified ch / t125 - a2 sequence is inserted , after phosphorylation , at the eco ri site of the vector v51 ( fig3 ). the ligation is performed after prior treatment of the eco ri sticky ends of v51 with the klenow polymerase in order to make them “ blunt - ended .” the primer g1 used for amplifying ch / t125 - a2 makes it possible to recreate , after its insertion into v51 , an eco ri site 5 ′ of ch / t125 - a2 . the vh / t125 - a2 sequence obtained by amplification is digested with eco ri and apa i and then inserted at the eco ri and apa i sites of the vector v51 - g1 / t125 - a2 . the construction of t125 - k47 is given in fig4 . the k / t125 - a2 sequence obtained by pcr is digested with eco ri and inserted at the eco ri site of the expression vector v47 derived from pci - neo ( fig5 ). the construction of t125 - ig24 is illustrated diagrammatically in fig6 . this vector , which contains the two transcription units for the heavy and kappa chains of t125 - a2 , is obtained by inserting the sal i - xho i fragment of t125 - k47 , containing the transcription unit for k / t125 - a2 , at the xho i and sal i sites of t125 - h26 . thus , the heavy and light chains of t125 - a2 are expressed under the control of the cmv promoter ; other promoters may be used : rsv , igg heavy chain promoter , mmlv ltr , hiv , β - actin , etc . a second vector for expressing t125 - a2 is also constructed , in which the consensus leader sequence of the kappa chain is replaced with the real sequence of the leader region of t125 - a2 determined beforehand by sequencing products from “ pcr 5 ′- race ” ( rapid amplification of cdna 5 ′ ends ). the construction of this t125 - ls4 vector is described in fig7 . it is carried out in two stages : first of all , construction of a new vector for expressing the t125 - a2 kappa chain , t125 - kls18 , and then assembly of the final expression vector , t125 - ls4 , containing the two heavy chain and modified light chain transcription units . the 5 ′ portion of the kappa consensus leader sequence of the vector t125 - k47 is replaced with the specific leader sequence of t125 ( kls / t125 - a2 ) during a step of amplification of the k / t125 - a2 sequence carried out using the following primers : primer a2vk9 , modifies the 5 ′ portion of the leader region ( in bold ) and introduces an eco ri site ( underlined ) and also a kozak sequence ( in italics ): the vector t125 - kls18 is then obtained by replacing the eco ri fragment of t125 - k47 , containing the k / t125 - a2 sequence of origin , with the new sequence kls / t125 - a2 digested via eco ri . the sal i - xho i fragment of t125 - kls18 , containing the modified kls / t125 - a2 sequence , is inserted into t125 - h26 at the xho i and sal i sites . the two expression vectors t125 - ig24 and t125 - ls4 were used to transfect cells of the yb2 / 0 line ( rat myeloma , atcc line no . 1662 ). after transfection by electroporation and selection of transformants in the presence of g418 ( neo selection ), several clones were isolated . the production of recombinant anti - d abs is approximately 0 . 2 μg / 10 6 cells / 24 h ( value obtained for clone 3b2 of r270 ). the adcc activity of this recombinant ab is greater than or equal to that of the poly - d controls ( fig1 ). the abs produced using the two expression vectors are not significantly different in terms of level of production or of adcc activity . the gene amplification system used is based on the selection of transformants resistant to methotrexate ( mtx ). it requires the prior introduction of a transcription unit encoding the dhfr ( dihydrofolate reductase ) enzyme into the vector for expressing the recombinant ab ( shitari et al ., 1994 ). the scheme shown in fig8 describes the construction of the vector for expressing t125 - a2 , containing the murine dhfr gene . a first vector ( v64 ) was constructed from a vector derived from pci - neo , v43 ( fig9 ), by replacing , 3 ′ of the sv40 promoter and 5 ′ of a synthetic polyadenylation sequence , the neo gene ( hind iii - csp 45 i fragment ) with the cdna of the murine dhfr gene ( obtained by amplification from the plasmid pmt2 ). this vector is then modified so as to create a cla i site 5 ′ of the dhfr transcription unit . the cla i fragment containing the dhfr transcription unit is then inserted at the cla i site of t125 - ls4 . yb2 / 0 cells transfected by electroporation with the vector t125 - dhfr13 are selected in the presence of g418 . the recombinant ab - producing transformants are then subjected to selection in the presence of increasing doses of mtx ( from 25 nm to 25 μm ). the progression of the recombinant ab production , reflecting the gene amplification process , is followed during the mtx selection steps . the mtx - resistant transformants are then cloned by limiting dilution . the level and the stability of the recombinant ab production are evaluated for each clone obtained . the anti - d antibody productivity after gene amplification is approximately 13 (± 7 ) μg / 10 6 cells / 24 h . yb2 / 0 cells transfected by electroporation with vector t125 - dhfr13 are selected in the presence of g418 . the best recombinant ab - producing transformants are cloned by limiting dilution before selection in the presence of increasing doses of mtx . the progression of the production by each clone , reflecting the gene amplification process , is followed during the mtx selection steps . the level and the stability of the recombinant ab production are evaluated for each mtx - resistant clone obtained . after purification by affinity chromatography on protein a sepharose ( pharmacia ) and dialysis into 25 mm tris buffer , 150 mm nacl , ph 7 . 4 , the concentration of the t125 antibody is determined by the elisa technique . the biological activity in vitro is then measured by the adcc assay described above . the results are given in fig1 . several studies describe the effect of enzymatic inhibitors on the glycosylation of immunoglobulins and on their biological activity . an increase in adcc activity is reported by rothman et al ., 1989 , this being an increase which cannot be attributed to an enhancement of the affinity of the antibody for its target . the modification of glycosylation caused by adding dmm consists of inhibition of the α - 1 , 2 mannosidase i present in le golgi . it leads to the production of a greater proportion of polymannosylated , nonfucosylated structures . various anti - rh ( d ) antibody - producing lines were brought into contact with dmm and the functional activity of the monoclonal antibodies produced was evaluated in the form of culture supernatants or after purification . the cells ( heterohybrid or lymphoblastoid cells ) are seeded at between 1 and 3 × 10 5 cell / ml , and cultured in imdm culture medium ( life technologies ) with 10 % of fcs and in the presence of 20 μg / ml of dmm ( sigma , boehringer ). after having renewed the medium 3 times , the culture supernatants are assayed by human igg elisa and then by adcc . the addition of dmm may make it possible to restore the adcc activity of an antibody derived from the cloid t125 = t125 ri ( 3 ) ( described in example 1 ) and which has lost this activity through sustained culturing . the strong activity of the antibody produced by the heterohybridoma f60 ( the production of which is described in example 1 ) is not modified by culturing in the presence of dmm . the nucleotide sequence of the antibody df5 , a negative control in the adcc assay , is used to study the transfection of this antibody into some lines , in parallel to transfection of the antibody t125 . the sequences encoding the ab df5 are isolated and amplified according to the same techniques used for the recombinant ab t125 - a2 . the corresponding cdnas are first of all synthesized from total rna extracted from the anti - d ab -( igg g1 / lambda )- producing clone 2mdf5 obtained by ebv transformation . amplification of the heavy and light chains is then carried out from these cdnas using the primers presented below . amplification of the variable region of the heavy chain of df5 ( vh / df5 sequence ): primer df5vh1 , located 5 ′ of the leader region ( in bold ) of the vh gene of df5 ( sequence published : l . chouchane et al . ); this primer also comprises an eco ri restriction site ( in italics ) and a kozak sequence ( underlined ): antisense primer gsp2anp , located 5 ′ of the constant region ( ch ) already described in paragraph 1 . 2 ( example 2 ). amplification of the constant region ch of df5 ( ch / df5 sequence ): primers g1 and h3 ′ xba already described in paragraph 1 . 3 ( example 2 ). primer df5vlbd1 , located 5 ′ of the leader region of the vl gene of df5 , introduces a consensus sequence ( in bold ) deduced from the sequence of several leader regions of vl genes belonging to the same vl1 subgroup as the vl gene of 2mdf5 ; this sequence also comprises an eco ri restriction site ( in italics ) and a kozak sequence ( underlined ): antisense primer lse1 , located 3 ′ of lambda , introduces an eco ri site ( underlined ): the construction of the vectors for expressing the heavy chain ( df5 - h31 ), light chain ( df5 - l10 ) and heavy and light chains ( df5 - ig1 ) of the ab df5 is carried out according to a construction scheme similar to vectors expressing the ab t125 - a2 . all the leader sequences of origin ( introduced in the amplification primers ) are conserved in these various vectors . 2 . 2 - transfection of various cell lines with the antibodies t125 and df5 the three expression vectors t125 - ig24 , t125 - ls4 and df5 - igg1 are used to transfect cells of various lines : stable or transient transfections are performed by electroporation or using a transfection reagent . the modification of effector activity of a humanized monoclonal antibody as a function of the expressing cell has been described by crowe et al . ( 1992 ), with the cho , nso and yb2 / 0 cell lines . the results obtained here confirm the importance of the expressing cell line with respect to the functional characteristics of the antibody to be produced . among the cells tested , only the vero , yb2 / 0 and cho lec - 1 lines make it possible to express recombinant anti - rh ( d ) monoclonal antibodies with strong lytic activity in the adcc assay ( see example 1 and table 4 ). characterization of the glycan structures of the anti - rh - d antibody was carried out on four purified products having an adcc activity ( f60 , and three recombinant proteins derived from t125 ) in comparison with two purified products inactive or very weakly active in the adcc assay according to the invention ( d31 and df5 ). in practice , the oligosaccharides are separated from the protein by specific enzymatic deglycosylation with pngase f at asn 297 . the oligosaccharides thus released are labeled with a fluorophore , separated and identified by various complementary techniques which allow : fine characterization of the glycan structures by matrix - assisted laser desorption ionization ( maldi ) mass spectrometry by comparison of the experimental masses with the theoretical masses . determination of the degree of sialylation by ion exchange hplc ( glycosep c ) separation and quantification of the oligosacharride forms according to hydrophilicity criteria by normal - phase hplc ( glycosep n ) separation and quantification of the oligosaccharides by high performance capillary electrophoresis - laser induced fluorescence ( hpce - lif ). the various active forms studied are f60 and three recombinant antibodies , r 290 , r 297 and r 270 , derived from t125 and produced in yb2 / 0 . fine characterization of the glycan structures by mass spectrometry ( fig7 ) shows that these forms are all of the bi - antennary type . in the case of r 270 , the major form is of the agalactosylated , nonfucosylated type ( g0 , exp . mass 1459 . 37 da , fig1 ). three other structures are identified : agalactosylated , fucosylated ( g0f at 1605 . 41 da ), monogalactosylated , nonfucosylated ( g1 at 1621 . 26 da ) and monogalactosylated , fucosylated ( g1f at 1767 . 43 da ) in minor amount . these same four structures are characteristic of r 290 , f 60 and r 297 ( fig1 ). these four antibodies which are active in adcc are also characterized by the absence of oligosaccharides having a bisecting n - acetylglucosamine residue . quantification of the glycan structures by the various techniques of hplc and hpce - lif ( table 1 ) confirms the presence of the four forms identified by mass : g0 , g0f , g1 and g1f . the degree of sialylation is very low , in particular for the recombinant products , from 1 to 9 . 4 %, which is confirmed by the similarity of the mass spectra obtained before and after enzymatic desialylation . the degree of fucosylation ranges from 34 to 59 %. the various inactive forms studied are d31 and df5 . quantification of the glycan structures by the various chromatographic and capillary electrophoresis techniques ( table 1 ) reveals , for these two antibodies , a degree of sialylation close to 50 %, and a degree of fucosylation of 88 and 100 % for d31 and df5 , respectively . these degrees of sialylation and fucosylation are much higher than those obtained from the active forms . characterization of the glycan structures shows that the major form is , for the two antibodies , of the bi - antennary , monosialylated , digalactosylated , fucosylated type ( g2s1f , table 1 ). the characterization by mass spectrometry of d31 ( fig7 ) reveals that the neutral forms are mainly of the monogalactosylated , fucosylated type ( g1f at 1767 . 43 da ) and digalactosylated , fucosylated type ( g2f at 1929 . 66 da ). the inactive antibody df5 is characterized by the presence of oligosaccharides having an intercalated gl cnac residue . in particular , the mass analysis ( fig8 ) reveals the presence of a major neutral form of the monogalactosylated , fucosylated , bisecting , intercalated g1cnac type ( g1fb at 1851 . 03 da ). on the other hand , these structural forms are undetectable or present in trace amounts on the active antibodies studied . the adcc activity of d31 after the action of dmm increases from 10 % to 60 %. the glycan structures of dmm d31 differ from those of d31 by the presence of oligomannose forms ( man 5 , man 6 and man 7 ) ( see fig9 ). the various active antibodies are modified on asn 297 with n - glycosylations of the bi - antennary and / or oligomannoside type . for the bi - antennary forms , this involves short structures with a very low degree of sialylation , a low degree of fucosylation , a low degree of galactosylation and no intercalated glcnac . boylston , j . m ., gardner , b ., anderson , r . l ., and hughes - jones , n . c . production of human igm anti - d in tissue culture by eb virus - transformed lymphocytes . scand . j . immunol . 12 : 355 - 358 ( 1980 ). bron , d ., feinberg , m . b ., teng , n . n . h . and kaplan , h . s . production of human monoclonal igg antibodies against rhesus ( d ) antigen . proc . nat . acad . sci . usa 81 : 3214 - 3217 ( 1984 ). chouchane , l ., van spronsen , a ., breyer , j ., guglielmi , p ., and strosberg , a d . molecular characterization of a human anti - rh ( d ) antibody with a dii segment encoded by a germ - line sequence . eur . j . biochem . 1 ; 207 ( 3 ): 1115 - 1121 ( 1992 ). crawford , d . h ., barlow , m . j ., harrison , j . f ., winger , l . and huehns , e . r . production of human monoclonal antibody to rhesus d antigen . lancet , i : 386 - 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